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https://openalex.org/W2077634623
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https://bmcresnotes.biomedcentral.com/counter/pdf/10.1186/1756-0500-4-344
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English
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Pharmacokinetic and technical comparison of Sandostatin® LAR® and other formulations of long-acting octreotide
|
BMC research notes
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| 5,332
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SHORT REPORT Open Access Petersen et al. BMC Research Notes 2011, 4:344
http://www.biomedcentral.com/1756-0500/4/344 Petersen et al. BMC Research Notes 2011, 4:344
http://www.biomedcentral.com/1756-0500/4/344 Petersen et al. BMC Research Notes 2011, 4:344 © 2011 Petersen 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. Findings maintaining the efficacy of Sandostatin immediate-
release whilst significantly reducing the number of injec-
tions administered [1,2]. Based on a well defined and
consistent pharmacokinetic (PK) profile, the efficacy and
safety of Sandostatin LAR have been established over
more than a decade of clinical experience [3]. Abstract Background: Sandostatin® LAR® (Novartis Pharma AG) is a long-acting repeatable formulation of the somatostatin
analogue octreotide, the safety and efficacy of which has been established through 15 years of clinical experience. Recently, other formulations of octreotide using polymer poly(lactic-co-glycolic acid) technology have been
developed. This study compares the composition and pharmacokinetic (PK) profile of Sandostatin LAR with three
other versions of the depot delivery system (formulations A, B and C, available in selected countries). Findings: Sandostatin LAR exhibited a characteristic concentration-time profile with a limited initial release of
octreotide (’burst’), an erosion phase from weeks 3-5, and a slowly declining concentration to day 52. The PK
profiles of formulations A and B were characterized by a large initial burst during days 0-2, with up to 41% of the
overall area under the plasma-concentration time curve achieved. Low and variable octreotide concentrations were
observed during the microparticle erosion phase (days 2-62 [day 82 formulation C]) for formulations A, B and C. Sandostatin LAR microparticles are spherical in shape with an average diameter of approximately 50 μm,
determined by scanning electron microscopy evaluation. Formulation A had smaller, irregular microparticles, and
formulations B and C exhibited a large range of particle diameters (< 20 to > 100 μm). Inductively coupled plasma-
optical emission spectroscopy detected a high tin content of 104 mg/kg in formulation B, the presence of which
may suggest inadequate purification following polymer synthesis using tin(II)-octoate as catalyst. PK profiles for
formulations A, B and C after a single intramuscular injection of 4 mg/kg in male New Zealand rabbits differed
markedly from the PK profile of Sandostatin LAR. Conclusions: Clear differences were seen between Sandostatin LAR and formulations A, B and C, including
variations in microparticle size, shape and impurity content. Considering the significant differences in the octreotide
release profile between Sandostatin LAR and the other formulations, the safety and efficacy of the other
formulations cannot be inferred from the Sandostatin LAR efficacy and safety profile; each of these other
formulations should be assessed accordingly. Pharmacokinetic and technical comparison of
Sandostatin® LAR® and other formulations of
long-acting octreotide Holger Petersen*, Jean-Claude Bizec, Helmut Schuetz and Marie-Laure Delporte Holger Petersen*, Jean-Claude Bizec, Helmut Schuetz and Marie-Laure Delporte * Correspondence: holger.petersen@novartis.com
Novartis Pharma AG, Basel, Switzerland Background and aims
®
® Sandostatin® LAR® is a long-acting octreotide formula-
tion for the treatment of patients with acromegaly and
symptoms associated with certain types of neuroendo-
crine tumors. Approved at doses of 10, 20 and 30 mg
(and up to 40 mg for patients with acromegaly in cer-
tain countries such as the US and Japan), Sandostatin
LAR
allows
for
once-monthly
administration, Recently, other long-acting octreotide formulations
have become available for clinical use in selected mar-
kets. Evidence regarding bioequivalence or product
property equivalence between these new formulations
and Sandostatin LAR is not available. Such information
is important as clinical guidelines recommending the * Correspondence: holger.petersen@novartis.com
Novartis Pharma AG, Basel, Switzerland Petersen et al. BMC Research Notes 2011, 4:344
http://www.biomedcentral.com/1756-0500/4/344 Page 2 of 8 Page 2 of 8 the vials supplied was stated by the manufacturer to be
20 mg. As such, concentrations were calculated assum-
ing 20 mg of octreotide per vial; verification of the
octreotide content was not performed. For formulation
C, rabbits were given a target dose of 10 mg/rabbit. Actual doses are reported in Table 1. Rabbits were
housed in single cages with elevated sitting boards, and
allowed free access to standard rabbit and guinea pig
chow and water. Environmental conditions were 19 ± 2°
C with 55 ± 15% humidity. use of sustained-release octreotide are based on experi-
ence with Sandostatin LAR, and assumes that other
long-acting octreotide formulations would be of a simi-
lar quality, uniformity and reliability. Here we report
findings from a series of assessments performed to com-
pare Sandostatin LAR with three other formulations of
long-acting octreotide manufactured by companies other
than Novartis. The properties of Sandostatin® LAR®, and those of
three other long-acting octreotide products, were quan-
titatively and qualitatively assessed. Formulations A, B
and C were manufactured from 2008-2009. The evalua-
tions aimed to compare the composition and physico-
chemical properties of the other formulations with those
of Sandostatin LAR. PK data were compared using a
rabbit model. The different formulations were tested in
three separate animal studies of similar study design
that allowed for comparison of data. Blood collection was taken pre-dose, and then up to
52, 62 or 82 days post-dose. Plasma concentrations of
octreotide were determined by radioimmunoassay with a
lower limit of quantification of 0.05 ng/mL. Area under
the plasma concentration-time curve (AUC), maximum
plasma concentration (Cmax) and time to Cmax (tmax)
were evaluated. Statistical methods All PK parameters were calculated with WinNonlin soft-
ware version 5. PK parameters were calculated using a
non-compartmental model. AUC from days 0 to the last
sampling time (AUClast, i.e. AUC0-52d, AUC0-62d and
AUC0-82d) was calculated using the linear trapezoidal
rule. Percentage of burst from days 0-2 (AUC0-2d) was
calculated as: 100 × AUC0-2d/AUClast. One-way analysis
of AUC and Cmax was performed using the Kruskal-
Wallis method when data were non-parametric. Background and aims
®
® Cmax and tmax values were calculated in
both the burst phase from days 0-2 (Cmax-burst; tmax-burst)
and erosion phase from days 2-last sampling time (Cmax-
erosion; tmax-erosion). Microparticle appearance and composition Microparticle appearance and composition
Samples were analyzed according to cGLP in an
unblinded manner. Scanning electron microscopy
(Zeiss Supra 40) was used to evaluate microparticle
size, shape, porosity and surface appearance. Samples
were sputtered with gold-palladium prior to analysis. For evaluation of cross-sections, microparticles were
embedded in an epoxy resin, polymerized and frac-
tured in liquid nitrogen before sputtering with gold-
palladium. Proton nuclear magnetic resonance (1H-
NMR) was used to assess the composition of each for-
mulation and to determine the ratio of lactide to gly-
colide within the poly(lactic-co-glycolic acid; PLGA)
polymer. Samples of 1.1-1.3 mg dissolved in 0.5 mL
dimethyl-d6 sulfoxide (DMSO-d6) were analyzed with
500-600 MHz 1H-NMR for 12 hours at ambient tem-
perature. Gel-permeation chromatography with IR
detection was used to determine the molecular weight
of the PLGA polymer against polystyrene standards. Inductively coupled plasma-optical emission spectro-
scopy (ICP-OES; limit of detection 1 mg/kg) was used
to detect tin levels in the polymer by measuring any
residual presence of the tin(II)-octoate catalyst. The
sample was decomposed at 250°C in a closed pressur-
ized system and signals were quantified versus an
external calibration function. Microparticle size, shape, porosity and surface
appearance Sandostatin® LAR® microparticles were spherical in
shape with approximately 50 μm diameter on average
(Figure 1; Overview). In contrast, formulation A micro-
particles had a much smaller diameter and were of irre-
gular shape. The microparticles in formulations B and C
were mostly spherical in shape and had a similar mean
diameter to the Sandostatin LAR microparticles, how-
ever, a larger range of particle diameters was observed
(< 20 to > 100 μm). Furthermore, some microparticles
in formulation C had an irregular shape and appeared
to be damaged (Figure 1; Overview). Sample cross-sec-
tions of the microparticles revealed that the Sandostatin
LAR microparticles were very compact with only minor
pores, microparticles in formulation A had no pores and
those in formulation B had a very high porosity
(approximately 1-3 μm diameters: Figure 1; Cross- Methods Animal research was performed in accordance with
international guidelines and follows the Swiss law for
animal experimentation. Microparticle appearance and composition Analysis of PK parameters Sandostatin LAR, as well as formulations A, B and C,
were administered as single intramuscular injections at a
nominal dose of 4 mg/kg to male New Zealand white
rabbits aged 3.5-4 months and weighing 3.00-3.46 kg
(four cohorts, n = 3, 4 or 7 rabbits per group). For for-
mulations A and B, the absolute octreotide content in Petersen et al. BMC Research Notes 2011, 4:344
http://www.biomedcentral.com/1756-0500/4/344 Petersen et al. BMC Research Notes 2011, 4:344
http://www.biomedcentral.com/1756-0500/4/344 Page 3 of 8 Table 1 PK parameters of Sandostatin® LAR®, and formulations A, B and C
PK parameters
Sandostatin® LAR®
Formulation A
Formulation B
Formulation C
Number of rabbits
7
4
3
3
Actual dose, mg/kg (± SD)
3.34 ± 0.236
3.42 ± 0.410
3.00 ± 1.84
1.27 ± 0.426
Burst phase, days 0-2
tmax-burst, hours, median
2
1.0
0.5
0.02
Cmax-burst, ng/mL, mean (± SD)
3.49 ± 4.21
167 ± 31.1†
22.8 ± 18.0
20.5 ± 6.64
Erosion phase, days 2-62*
tmax-erosion, days, median
20
14 and 34
20.0
12
Cmax-erosion, ng/mL, mean (± SD)
9.97 ± 4.21
3.89 ± 1.73, and 2.42 ± 2.11
4.83 ± 3.91
13.3 ± 3.52
AUC0-last, d·ng/mL (± SD)*
179 ± 62.0
163 ± 33.7
86.1 ± 73.2
247 ± 33.4
AUC0-2d, d·ng/mL (± SD)
4.42 ± 5.70
65.3 ± 4.57
2.61 ± 0.680
2.39 ± 0.592
Burst, % (± SD)
2.19 ± 1.97
41.0 ± 6.32
22.2 ± 35.0
1
*The AUC0-last was measured over 52 days (AUC0-52d) for Sandostatin LAR, over 62 days (AUC0-62d) for formulations A and B, and over 82 days (AUC0-82d) for
formulation C. †Cmax-burst of formulation A is significantly different to that of Sandostatin LAR. AUC area under the plasma concentration time curve; C
maximum plasma concentration; SD standard deviation; t
time to C Table 1 PK parameters of Sandostatin® LAR®, and formulations A, B and C Cmax-burst of formulation A is significantly different to that of Sandostatin LAR. AUC, area under the plasma concentration-time curve; Cmax, maximum plasma concentration; SD, standard deviation; tmax, time to Cmax. max burst
g
y
AUC, area under the plasma concentration-time curve; Cmax, maximum plasma concentration; SD, standard deviation; tmax, time to C y
AUC, area under the plasma concentration-time curve; Cmax, maximum plasma concentration; SD, typically had a non-crystalline shape in formulations A
and B. Analysis of PK parameters The mannitol in formulation A was also more
porous and of a smaller size than that in Sandostatin
LAR. In formulation C, mannitol particles were very
large and irregularly shaped (Figure 1; Surface). section). There was an insufficient quantity of formula-
tion C available for a cross-sectional analysis. Sandostatin LAR and formulations A, B and C incor-
porate mannitol as a bulk excipient. NMR evaluation
confirmed the presence of mannitol (Figure 2). Inspec-
tion of the microparticle surface of lyophilized samples
revealed differences in the mannitol appearance among
the formulations. Mannitol was found in a crystalline
shape loosely connected to the microparticles in the
Sandostatin LAR formulation. In contrast, mannitol Molecular composition The prescribing information for Sandostatin LAR, and
formulations A and C, lists acetate as the octreotide salt;
the salt type was not stated in the prescribing Figure 1 Scanning electron microscopy comparisons of Sandostatin® LAR®, and formulations A, B and C. Figure 1 Scanning electron microscopy comparisons of Sandostatin® LAR®, and formulations A, B and C. Figure 1 Scanning electron microscopy comparisons of Sandostatin® LAR®, and formulations A, B and C Page 4 of 8 Petersen et al. BMC Research Notes 2011, 4:344
http://www.biomedcentral.com/1756-0500/4/344 Petersen et al. BMC Research Notes 2011, 4:344
http://www.biomedcentral.com/1756-0500/4/344 Chemical shift (ppm)
5.0
74.03
54.00
4.5
4.0
3.5
Chemical shift (ppm)
5.0
55.55
90.00
4.5
4.0
3.5 Chemical shift (ppm)
5.0
50.29
96.00
4.5
4.0
3.5
Chemical shift (ppm)
5.0
74.03
54.00
4.5
4.0
3.5
Chemical shift (ppm)
5.0
55.55
90.00
4.5
4.0
3.5
Figure 2 1H-NMR of Sandostatin® LAR®, formulation A and formulation B.Upper spectrum: Sandostatin LAR, molar ratio actide 55, glycolide 45. Middle spectrum: formulation A, molar ratio lactide 73, glycolide 27. Lower spectrum: formulation B, molar ratio lactide 50, glycolide 50. Signals at 5.2
ppm indicate the single methine proton of the lactide monomer. Signals at 4.8 ppm are assigned to the two methylene protons of the glycolide
monomer. Signals at lower shifts than 4.5 ppm indicate mannitol (mannitol-OH and -CH/CH2). Chemical shift (ppm)
5.0
55.55
90.00
4.5
4.0
3.5 Chemical shift (ppm)
5.0
74.03
54.00
4.5
4.0
3.5
(pp
) Chemical shift (ppm) Chemical shift (ppm)
5.0
50.29
96.00
4.5
4.0
3.5
Chemical shift (ppm) Figure 2 1H-NMR of Sandostatin® LAR®, formulation A and formulation B.Upper spectrum: Sandostatin LAR, molar ratio actide 55, glycolide 45. Middle spectrum: formulation A, molar ratio lactide 73, glycolide 27. Lower spectrum: formulation B, molar ratio lactide 50, glycolide 50. Signals at 5.2
ppm indicate the single methine proton of the lactide monomer. Signals at 4.8 ppm are assigned to the two methylene protons of the glycolide
monomer. Signals at lower shifts than 4.5 ppm indicate mannitol (mannitol-OH and -CH/CH2). Petersen et al. BMC Research Notes 2011, 4:344
http://www.biomedcentral.com/1756-0500/4/344 Page 5 of 8 formulation B. ICP-OES analysis was not performed on
formulation C due to an insufficient quantity of sample. information for formulation B. 1H-NMR analysis of for-
mulations A and B suggested the presence of an acetate
component. Comparison of PK profile in rabbits Sandostatin LAR demonstrated controlled release of
octreotide. During the burst phase (days 0-2) the mean
Cmax-burst value was 3.49 ng/mL, corresponding to 1.9%
of the overall AUC0-52d. The octreotide concentration
increased during the erosion phase to reach mean peak
levels of 9.97 ng/mL (Figure 3; Table 1). The release pat-
tern of Sandostatin LAR demonstrated an erosion phase
in octreotide concentrations during weeks 3-5, similar to
the concentration-time profile observed in humans [4]. Molecular weight of the polymer Differences were observed between the molecular weight
and composition of the polymer used in Sandostatin
LAR, and formulations A, B and C. Whereas the mole-
cular weight of the Sandostatin LAR polymer was 52
kDa, formulations A, B and C had lower molecular
weights of approximately 16, 32 and 14.5 kDa, respec-
tively. The ratio of the lactide:glycolide co-monomers
was 55:45 in Sandostatin LAR, 73:27 for formulation A
and 50:50 for formulation B. For formulation C, the
supplier claimed a ratio of 62.5:37.5 based on a 1:1
blend of 50:50 and 75:25 PLGA polymers. Compared with the PK profile of Sandostatin LAR, for-
mulations A, B and C achieved a much higher octreotide
concentration over the first 2 days post injection, display-
ing mean Cmax-burst of 167, 22.8 and 20.5 ng/mL, respec-
tively. Although Cmax-burst was high for formulation C,
AUC0-2d was relatively low. During the first 2 days after
injection, 41.0% and 22.2% of the overall AUC0-62d was
observed with formulations A and B, respectively. Conse-
quently, during the erosion phase, octreotide concentra-
tions in these formulations were much lower and more
variable compared with those of Sandostatin LAR; these
formulations had no obvious plateau phase (Figure 3). Overall octreotide concentrations were also found to be Molecular composition Compared with Sandostatin LAR, formula-
tions A and B exhibited a lower molar ratio between the
acid and base of the octreotide salt (16% and 75% of
that ratio determined for Sandostatin LAR [100%],
respectively). 1H-NMR analysis was not performed on
formulation C due to an insufficient quantity of sample. Impurities No heavy metals or other potentially toxic substances
were detected in Sandostatin LAR, or in formulation A. A high tin content of 104 mg/kg was found in –7
0
7
14
21
28
35
42
49
56
63
70
77
200000
100000
Sandostatin® LAR
15000
10000
5000
0
Time (days)
Octreotide concentration (pg/mL)
84
Formulation A
Formulation B
Formulation C
Figure 3 Plasma concentration-time profiles ± SD of Sandostatin® LAR®, and formulations A, B and C. Sandostatin® LAR
Formulation A
Formulation B
Formulation C Octreotide concentration (pg/mL) Figure 3 Plasma concentration-time profiles ± SD of Sandostatin® LAR®, and formulations A, B and C. Petersen et al. BMC Research Notes 2011, 4:344
http://www.biomedcentral.com/1756-0500/4/344 Page 6 of 8 lower with formulations A and B. Whereas the Sandosta-
tin LAR samples demonstrated an AUC0-52d of 179 d·ng/
mL, formulation A and B demonstrated AUC0-62d of 163
and 86.1 d·ng/mL, respectively, and formulation C
demonstrated AUC0-82d of 247 d·ng/mL. Additionally,
formulation B was found to be very difficult to inject due
to needle clogging and, as a result, one rabbit was
excluded as no octreotide concentration was detected
after an unsuccessful injection. A second rabbit had an
octreotide concentration considerably lower than the
mean dose exhibited in the other cohorts (23.2% of the
intended dose, individual data not shown). Concerning
formulation C, early erosion was observed with tmax-ero-
sion of 12 days with a high Cmax-burst of 20.5 ng/mL. from the appearance of the microparticles, and poses
potential safety risks. This finding was particularly evi-
dent in formulation A, with 41% of the overall AUC
achieved within the first 2 days after injection. Formula-
tions A and B also demonstrated much lower concentra-
tions
of
octreotide,
while
formulation
C
was
characterized by an early and narrow erosion phase with
no discernable plateau. As such, in addition to potential
safety concerns related to the large burst phase, the for-
mulations may also fail to consistently deliver therapeu-
tic concentrations of octreotide to patients throughout
the interval between injections. It is important to note the constraints of our study
that limit the interpretation of our findings. First, in
vivo rabbit PK data do not always accurately reflect, and
cannot replace, clinical PK studies in humans. A rabbit
PK profile similar to that of Sandostatin LAR is not
proof of clinical bioequivalence to Sandostatin LAR and
cannot replace demonstrating human bioequivalence. Impurities This underlines the importance of performing clinical PK
studies in all new depot delivery systems of octreotide. Second, clinical studies have to demonstrate equivalent
safety and efficacy in specific indications; target patient
populations include those with acromegaly or neuroen-
docrine tumors. In addition, although the in vivo study
described here was designed to evaluate the formulations
in an equal number of rabbits per cohort, needle clogging
in formulation B caused one animal to be excluded from
analysis and one animal to receive part of the intended
sample amount. Furthermore, only a small quantity of
formulation C was available and, therefore, this sample
could be evaluated only in three rabbits. A further study
with a larger sample size would strengthen the evidence
presented here. Finally, disparities in study design should
be taken into account: differences in serum sample time
points between formulations occurred because of
resource availability and the fact that the in vivo evalua-
tions of different formulations were performed on differ-
ent calendar dates. Nevertheless, it is reasonable to
compare the kinetic profile of the formulations because
the serum sample times covered the long in vivo release
profile expected in these products. Discussion
® BMC Research Notes 2011, 4:344
http://www.biomedcentral.com/1756-0500/4/344 Page 7 of 8 Page 7 of 8 that the other formulations do not share the same pro-
duct characteristics as Sandostatin LAR. manufacturing process. Variations in the diameter of the
microspheres and the thickness of the polymer coat in
formulations B and C have the potential to affect the
drug-release profile [9], with possible failure to deliver
continuous therapeutic drug concentrations, and/or can
potentially cause adverse events related to excessive
drug release during the initial burst phase. In addition,
differences in the mannitol appearance were observed
between the formulations. Since mannitol is used to
improve flow and dispersability and to improve stability
in drug delivery systems, it could be postulated that
changes in its appearance could affect the pharmaceuti-
cal processability of PLGA-based drug delivery systems
as well as the preparation of the drug for administration. In conclusion, clear differences were seen between
Sandostatin LAR and formulations A, B and C, includ-
ing significant differences in the PK profile, and varia-
tions in microparticle size, shape molecular weight, acid:
base ratio, and impurity content. These findings suggest
that other long-acting octreotide formulations may have
a different drug-release pattern to that of Sandostatin
LAR despite similar composition. Considering these dif-
ferences, formulations A, B and C are most likely not
bioequivalent to Sandostatin LAR in humans. Conse-
quently, the safety and efficacy of these new formula-
tions cannot be inferred from the Sandostatin LAR
clinical and safety profile. Each of these other formula-
tions should be assessed by appropriate clinical studies
to determine their clinical benefit and safety profiles. as we
as t e p epa at o
o t e d ug o ad
st at o . Factors such as the molecular weight and composition
of the PLGA polymer also affect drug release, with low
molecular weight accelerating the rate of drug release
and a high lactide:glycolide ratio causing the polymer to
degrade more slowly because the lactide monomer is
more hydrophobic than the glycolide monomer. In pre-
vious studies of octreotide release from PLGA polymers
of various molecular weights and lactide:glycolide ratios,
pH and impurity content also influenced the percentage
of octreotide release [10,11]. Discussion
® Sandostatin® LAR® is a long-acting repeatable formula-
tion of octreotide. Its development required extensive
analytical support to ensure the quality and consistency
of the formulation. Clinical PK studies have established
that Sandostatin LAR produces a reliable, sustained
release of octreotide [4,5], with proven therapeutic uti-
lity in patients [3,6,7]. More recently, other long-acting
formulations of octreotide have been introduced in
selected markets. In humans, Sandostatin LAR has a well-character-
ized consistent and predictable PK profile, which can
be described as exhibiting three distinct phases: (1)
release of surface-absorbed octreotide (burst); (2) pore
diffusion, biodegradation, osmotic swelling and ionic
interactions (erosion phase leading to a drug concen-
tration plateau); and (3) fragmentation and complete
biodegradation of the polymer (erosion phase leading
to complete drug release) [8]. This tripartite pattern
has been regularly observed and is evident with var-
ious Sandostatin LAR doses. Octreotide concentra-
tions exhibited an initial peak on day 1, followed by a
decline over the following 3-5 days, before slowly
increasing and reaching a plateau 2-3 weeks post
injection before declining [4]. The steady-state PK
simulation of Sandostatin LAR 20 mg suggested a
mean concentration of 1216 pg/mL (range, 1065-1585
pg/mL) with a fluctuation index of 43%. Additionally,
inter-subject variability in mean Cmax was 32% for
Sandostatin LAR 20 mg [4]. Sandostatin LAR consists of octreotide acetate encap-
sulated and uniformly distributed within PLGA D-(+)
glucose microspheres. Slow release of the drug occurs as
the polymer biodegrades, primarily through hydrolysis. The polymer has an average molecular weight of ~52
kDa and the microparticles exhibit a mean diameter of
~50 μm [8]. During the in vivo rabbit PK evaluations in the present
study, differences in the concentration-time profile
between formulations A, B and C, and Sandostatin LAR,
were observed. The Sandostatin LAR concentration-time
profiles in these in vivo investigations were similar to
those observed in humans [4]. During the burst phase,
the three other formulations displayed AUC0-2d values
ranging from 2.39-65.3 d·ng/mL, compared with 4.42
d·ng/mL for Sandostatin LAR. This variability may result Compared with the established characteristics of San-
dostatin LAR, formulations A and C exhibited greater
irregularity in microparticle shape and size. This is sug-
gestive of inadequately encapsulated octreotide mole-
cules and may indicate a lack of quality control in the Petersen et al. Acknowledgements and Funding Financial support for medical editorial assistance was provided by Novartis
Pharmaceuticals Corporation. We thank Helene Darmofal for medical
editorial assistance with this manuscript. List of abbreviations
AUC
d
h
l AUC: area under the plasma concentration-time curve; AUClast: AUC from
days 0 to the last sampling time; Cmax: maximum plasma concentration;
Cmax-burst: maximum octreotide concentration during the burst phase from
days 0-2; Cmax-erosion: maximum octreotide concentration during the erosion
phase from days 2-62; DMSO-d6: dimethyl-d6 sulfoxide; 1H-NMR: proton
nuclear magnetic resonance; ICP-OES: inductively-coupled plasma-optical
emission spectroscopy; kDa: kilodalton; LAR: long-acting repeatable; PLGA:
poly(lactic-co-glycolic acid); PK: pharmacokinetic; tmax: time to Cmax; tmax-burst:
time to Cmax-burst; tmax-erosion: time to Cmax-erosion Authors’ contributions J-CB, HS and M-LD participated in performance and interpretation of the PK
analysis, and HS and M-LD wrote the PK analysis report on which this
manuscript is based. HP designed and performed the technical quality
assessment tests and wrote the report on which the non-PK aspects of this
manuscript are based. All authors helped to critically revise manuscript
drafts. All authors read and approved the final manuscript. Discussion
® Although the very low
molecular weight in formulations A and C may be in
part offset by a higher amount of lactide monomer, the
differences in molecular weights and lactide:glycolide
ratios between the three formulations are likely to cause
different octreotide release patterns. As PLGA polymers
are routinely used in sustained-release formulations and
can be manufactured to a much higher purity than that
present in formulation B, the polymer can be considered
to be of poor quality. Variability was further evident in
the porosity of microparticles in formulations A and B. Previous studies have found that biodegradation and
drug release are dependent on the porosity, with varia-
tions affecting the rate of drug mobility [12]. Competing interests The high tin concentration found in formulation B
may indicate that high amounts of tin(II)-octoate were
used in the polymer synthesis without proper purifica-
tion, likely to be due to residual product from the cata-
lyst used during production of the polymer. As tin(II)-
octoate has been reported to be highly cytotoxic,[13]
this may affect patient safety. This impurity was not
observed in Sandostatin LAR or formulation A and no
arsenic content was found in any sample. Quality con-
trol to guarantee these characteristics is paramount to
LAR formulations. In addition, formulations A and B
had a low acid component to the octreotide salt. Theo-
retically, in the case of an acid-base pair, the ratio of
acetate molecules to octreotide molecules should be 2:1. In these formulations, octreotide is likely to be present
as a free base rather than as an acetate salt indicating This study was funded by Novartis Pharma AG. HP, J-CB, HS and M-LD are
employees of Novartis Pharma AG. Received: 23 February 2011 Accepted: 9 September 2011
Published: 9 September 2011 1.
Grass P, Marbach P, Bruns C, Lancranjan I: Sandostatin LAR
(microencapsulated octreotide acetate) in acromegaly: pharmacokinetic
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Rubin J, Ajani J, Schirmer W, Venook AP, Bukowski R, Pommier R, Saltz L,
Dandona P, Anthony L: Octreotide acetate long-acting formulation versus
open-label subcutaneous octreotide acetate in malignant carcinoid
syndrome. J Clin Oncol 1999, 17:600-606.
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Anthony L, Freda PU: From somatostatin to octreotide LAR: evolution of
a somatostatin analogue. Curr Med Res Opin 2009, 25:2989-2999.
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Astruc B, Marbach P, Bouterfa H, Denot C, Safari M, Vitaliti A, Sheppard M:
Long-acting octreotide and prolonged-release lanreotide formulations
have different pharmacokinetic profiles. J Clin Pharmacol 2005,
45:836-844. References 1. Grass P, Marbach P, Bruns C, Lancranjan I: Sandostatin LAR
(microencapsulated octreotide acetate) in acromegaly: pharmacokinetic
and pharmacodynamic relationships. Metabolism 1996, 45:27-30. 1. Grass P, Marbach P, Bruns C, Lancranjan I: Sandostatin LAR
(microencapsulated octreotide acetate) in acromegaly: pharmacokinetic
and pharmacodynamic relationships. Metabolism 1996, 45:27-30. 2. Rubin J, Ajani J, Schirmer W, Venook AP, Bukowski R, Pommier R, Saltz L,
Dandona P, Anthony L: Octreotide acetate long-acting formulation versus
open-label subcutaneous octreotide acetate in malignant carcinoid
syndrome. J Clin Oncol 1999, 17:600-606. 3. Anthony L, Freda PU: From somatostatin to octreotide LAR: evolution of
a somatostatin analogue. Curr Med Res Opin 2009, 25:2989-2999. 4. Astruc B, Marbach P, Bouterfa H, Denot C, Safari M, Vitaliti A, Sheppard M:
Long-acting octreotide and prolonged-release lanreotide formulations
have different pharmacokinetic profiles. J Clin Pharmacol 2005,
45:836-844. 2. Rubin J, Ajani J, Schirmer W, Venook AP, Bukowski R, Pommier R, Saltz L,
Dandona P, Anthony L: Octreotide acetate long-acting formulation versus
open-label subcutaneous octreotide acetate in malignant carcinoid
syndrome. J Clin Oncol 1999, 17:600-606. 3. Anthony L, Freda PU: From somatostatin to octreotide LAR: evolution of
a somatostatin analogue. Curr Med Res Opin 2009, 25:2989-2999. 4. Astruc B, Marbach P, Bouterfa H, Denot C, Safari M, Vitaliti A, Sheppard M:
Long-acting octreotide and prolonged-release lanreotide formulations
have different pharmacokinetic profiles. J Clin Pharmacol 2005,
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http://www.biomedcentral.com/1756-0500/4/344 Petersen et al. BMC Research Notes 2011, 4:344
http://www.biomedcentral.com/1756-0500/4/344 5. Chen T, Miller TF, Prasad P, Lee J, Krauss J, Miscik K, Kalafsky G, McLeod JF:
Pharmacokinetics, pharmacodynamics, and safety of microencapsulated
octreotide acetate in healthy subjects. J Clin Pharmacol 2000, 40:475-481. 5. Chen T, Miller TF, Prasad P, Lee J, Krauss J, Miscik K, Kalafsky G, McLeod JF:
Pharmacokinetics, pharmacodynamics, and safety of microencapsulated
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Beneficial effect of dose escalation of Octreotide-LAR as first-line
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Cytotoxicity of some catalysts commonly used in the synthesis of
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Medicine 1994, 5:393-396. doi:10.1186/1756-0500-4-344
Cite this article as: Petersen et al.: Pharmacokinetic and technical
comparison of Sandostatin®® LAR®® and other formulations of long-
acting octreotide. BMC Research Notes 2011 4:344. doi:10.1186/1756-0500-4-344
Cite this article as: Petersen et al.: Pharmacokinetic and technical
comparison of Sandostatin®® LAR®® and other formulations of long-
acting octreotide. BMC Research Notes 2011 4:344. doi:10.1186/1756-0500-4-344
Cite this article as: Petersen et al.: Pharmacokinetic and technical
comparison of Sandostatin®® LAR®® and other formulations of long-
acting octreotide. BMC Research Notes 2011 4:344. Petersen et al. BMC Research Notes 2011, 4:344
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Setting the research agenda for living with and beyond cancer with comorbid illness: reflections on a research prioritisation exercise
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Research involvement and engagement
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RESEARCH ARTICLE Open Access Setting the research agenda for living with
and beyond cancer with comorbid illness:
reflections on a research prioritisation
exercise D. Cavers1*, S. Cunningham-Burley1, E. Watson2, E. Banks3 and C. Campbell1 Abstract Background: People living with and beyond cancer are more likely to have comorbid conditions and poorer
mental and physical health, but there is a dearth of in-depth research exploring the psychosocial needs of people
experiencing cancer and comorbid chronic conditions. A patient partnership approach to research prioritisation and
planning can ensure outcomes meaningful to those affected and can inform policy and practice accordingly, but
can be challenging. Methods: We aimed to inform priorities for qualitative inquiry into the experiences and support needs of people
living with and beyond cancer with comorbid illness using a partnership approach. A three-step process including
a patient workshop to develop a consultation document, online consultation with patients, and academic expert
consultation was carried out. The research prioritisation process was also appraised and reflected upon. Results: Six people attended the workshop, ten responded online and eight academic experts commented on the
consultation document. Five key priorities were identified for exploration in subsequent qualitative studies,
including the diagnostic journey, the burden of symptoms, managing medications, addressing the needs of
informal carers, and service provision. Limitations of patient involvement and reflections on procedural ethics, and
the challenge of making measurable differences to patient outcomes were discussed. Conclusions: Findings from this research prioritisation exercise will inform planned qualitative work to explore
patients’ experiences of living with and beyond cancer with comorbid illness. Including patient partners in the
research prioritisation process adds focus and relevance, and feeds into future work and recommendations to
improve health and social care for this group of patients. Reflections on the consultation process contribute to a
broadening of understanding the field of patient involvement. Keywords: Patient and public involvement, Research prioritisation, Qualitative, Living with and beyond cancer,
Comorbid illness, Multi-morbidity * Correspondence: Debbie.Cavers@ed.ac.uk
1Usher Institute, University of Edinburgh, Medical School, Rm 123, Doorway
1, Teviot Place, Edinburgh EH8 9AG, UK
Full list of author information is available at the end of the article * Correspondence: Debbie.Cavers@ed.ac.uk
1Usher Institute, University of Edinburgh, Medical School, Rm 123, Doorway
1, Teviot Place, Edinburgh EH8 9AG, UK
Full list of author information is available at the end of the article (2020) 6:17 (2020) 6:17 Cavers et al. Research Involvement and Engagement
https://doi.org/10.1186/s40900-020-00191-9 * Correspondence: Debbie.Cavers@ed.ac.uk
1Usher Institute, University of Edinburgh, Medical School, Rm 123, Doorway
1, Teviot Place, Edinburgh EH8 9AG, UK
Full list of author information is available at the end of the article Plain English summary area with the National Cancer Research Institute (NCRI)
and the James Lind Alliance (www.jameslindalliance.co. uk) recently reporting on the ‘Top 10 research priorities
for people living with and beyond cancer’ (https://www. ncri.org.uk/lwbc/#lwbc_questions). Better coordination
of care for people with complex health needs (to include
multiple conditions) was identified as priority number
three. Little qualitative research has been done to find out
what life is like for people living with and beyond cancer
alongside other long term illnesses (such as heart disease
or diabetes). It makes sense to ask people in this situ-
ation to have a say in issues that affect them. This paper
reports on a patient involvement exercise to help iden-
tify the main things people living with and beyond can-
cer with other long term illness would like researchers
to find out more about in order to improve their experi-
ences. People were asked to give their views at a work-
shop and via an online survey. Other researchers
interested in this area were also asked for their views. Five key priorities were identified: the diagnostic journey,
the burden of symptoms, managing medications, ad-
dressing the needs of informal carers, and service
provision. The challenges of this kind of patient involve-
ment exercise are also discussed, focussing on ethical
concerns and making discernible changes to people’s
lives. One important dimension of this field of study is the
psychosocial experience of living with existing comorbid
conditions alongside cancer, across the illness trajectory
and beyond. At present, little has been published focus-
ing on the experience of cancer and comorbid condi-
tions and its implications for coordinated, quality care. A recent systematic review and evidence synthesis con-
ducted by the authors (following PRISMA guidance) has
highlighted the gaps in this field of research [16]. Themes for further exploration identified in the evidence
synthesis included: Interaction between cancer and co-
morbid conditions; symptom experience; illness identities
and ageing; self-management, and the role of primary
and secondary care. The need for further research to
understand experiences of people with cancer and other
multi-morbidity has also been identified elsewhere in the
literature c.f [17–20]. Further in-depth work is needed
to explore what cancer means to people in relation to
their other health conditions, and whether these ill-
nesses are experienced separately or in a more holis-
tic way. © The Author(s). 2020 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 9 Cavers et al. Research Involvement and Engagement (2020) 6:17 Plain English summary This may be less obvious to clinicians and
those researching illness narratives, where the focus
tends to remain on one condition. As new models of
survivorship care continue to shift and accommodate
patients with a complex picture of health, there are
also implications for the changing roles of primary
and secondary care [10, 17, 20, 21], care quality [22]
and self-management [23]. Introduction Approximately 363,000 people are diagnosed with a new
cancer each year in the UK and the incidence is pre-
dicted to rise by a further 2% by 2035 [1]. Cancer sur-
vival has also more than doubled in the last 40 years
from 24 to 50% of people living disease-free for 10 years
or more as early detection and treatment improves [1]. Cancer survival is couched in the context of an ageing
society, with the number of UK residents over 65 pre-
dicted to rise to 26% of the total population by 2041,
compared with 15.8% in 1991. Therefore, more people
are living with and beyond cancer [2]. A range of health
and social care needs of people living with and beyond
cancer have been described [3, 4]. However, these are
often complicated by the presence of comorbid condi-
tions. As many as 78% of cancer patients are living with
at least one other condition, and the number of condi-
tions increases as people get older [5, 6]. Cancer patients
are more likely to have more comorbid conditions and
therefore experience poorer physical and mental health
than those without cancer [7]. There is, therefore, a need
for a better understanding of the support needs of this
patient group, and for service developments to better
meet these needs [8]. Background to patient partnership and rationale Background to patient partnership and rationale
A patient partnership model - defined here as working
in collaboration with relevant patient groups as partners
to develop, inform and be involved in health research,
rather than a unidirectional research model with patients
as participants whom data is collected on [24] - ensures
that objectives and findings of subsequent studies are
meaningful and responsive to patients’ needs, ultimately
translating
into
relevant
policy
and
practice
[25]. Methods of stakeholder involvement in research priori-
tisation, including patients, is a fast developing field. In
addition to more established techniques such as the Del-
phi method, recently applied to health care research [26,
27], a proliferation of approaches are being suggested in
the literature to provide structure and transparency to
the process [28]. More structured methods, such as
those of the James Lind Alliance and used by the Child The research agenda related to living beyond cancer
has received increased attention in recent years [9], with
the development of the National Survivorship Initiative
[10] in England and Wales. Survivorship is also a key
focus of Scotland’s Better Cancer Care [11]. A growing
body of research is being developed around understand-
ing the psychosocial dimensions of living with and be-
yond cancer (c.f [3, 12–15].. There have also been
significant efforts to set priorities for research in this Page 3 of 9 Cavers et al. Research Involvement and Engagement (2020) 6:17 Cavers et al. Research Involvement and Engagement The consultation process asked: Health Nutrition and Research Initiative [28, 29], are ex-
pected to take precedence over the coming years. A tai-
lored partnership approach that is patient-centred was
favoured here, adapting aspects of the existing ap-
proaches to reflect the small scale of the exercise and
the exploratory aims of the study [24, 30]. The approach
drew on tenets of the outlined approaches, particularly
the iterative workshop and consultation method used in
the early phase of the Delphi approach (round one) and
subsequent online questionnaires modelling the eDelphi
approach (rounds two and three), also resonating with
crowdsourcing techniques of consulting relevant audi-
ences and using online methods [31] to consult with pa-
tients, their informal carers and academic experts in the
field of cancer survivorship, multi-morbidity and follow-
up care. A patient partnership approach is not without
its challenges, however, and poses questions around pur-
pose, effective processes and ethics that need to be ad-
dressed [32, 33]. Background to patient partnership and rationale What are the key topics and priorities identified by
individuals with a diagnosis of cancer and a
comorbid illness and their informal carers? How can these priorities be further developed and
refined with online academic expert input? p
p
What are the key methodological challenges posed
by this research prioritisation process and what are
its strengths and shortfalls? The paper follows GRIPP2 reporting guidelines (see
Additional file 1). Research prioritisation exercise approach The RPE phase of the wider project involved three com-
ponents: a patient and public consultation event result-
ing in the development of a consultation document,
online social media and email consultation with con-
sumers on the document, and finally, consultation with
academic experts to refine and finalise the document
(see Fig. 1). As patient partnership engagement, this
work did not require NHS ethical approval but did go
through institutional ethics to review the ethical integrity
of the planned approach. This paper reports on small-scale research prioritisa-
tion work. It involved consulting with patients, informal
carers and academic experts and combining the findings
with our prior systematic review [16], to inform a follow
on in-depth qualitative study exploring patient and in-
formal carer experiences of living with and beyond can-
cer with comorbid illness, and health care provider
experiences of caring for this patient group. This paper
presents the outcomes of the research prioritisation ex-
ercise and reflects on the methodology and challenges
posed by the process. Social media and email consultation of patients and
carers Social media and email consultation of patients and
carers Social media and email consultation of patients and
carers views on key topics for research exploring the experience
of living with and beyond cancer with comorbid illness. The workshop was modelled on the consultation ap-
proach in the first round of the Delphi process [28], and is
a similar approach to that used previously [28, 34]. Rele-
vant quality markers developed as part of the Delphi and
CHNRI methods informed the planning and implementa-
tion of the workshop, such as adequate planning and
preparation, inclusiveness of relevant stakeholders and
transparency in reporting [29, 35]. views on key topics for research exploring the experience
of living with and beyond cancer with comorbid illness. The workshop was modelled on the consultation ap-
proach in the first round of the Delphi process [28], and is
a similar approach to that used previously [28, 34]. Rele-
vant quality markers developed as part of the Delphi and
CHNRI methods informed the planning and implementa-
tion of the workshop, such as adequate planning and
preparation, inclusiveness of relevant stakeholders and
transparency in reporting [29, 35]. The summary patient priorities document, presenting
the identified priorities with free text comment boxes,
(modelled on the second round of an online eDelphi
questionnaire approach) was shared with a group of on-
line service users and their family members using Twit-
ter as a platform in addition to online Maggie’s Centre
and, with permission, local patient support groups, to
encourage further engagement and consultation on what
is relevant as a priority in the current research and pol-
icy climate. A brief outline and link to the full document
(via Survey Monkey) was posted on Twitter. To reach a
wide audience, relevant organisations such as the Na-
tional Institute for Health Research’s INVOLVE, Our
Voice Scotland, and the Alliance were tagged in order to
invite their feedback. It was anticipated that the Twitter
post would be shared by tagged online groups to high-
light awareness of the consultation. Interested parties
could post a comment with their views, or use the online
link or contact the research team to request a paper
copy of the consultation document with FREEPOST re-
turn envelope. Attendees at the consultation workshop
were also sent the consultation document by email or
post and asked for comments and feedback on each
priority. Social media and email consultation of patients and
carers Potential workshop attendees were invited via a poster
advertised (either displayed in print or sent electronic-
ally) on Twitter and through the Edinburgh Cancer
Centre, Edinburgh Maggie’s Centre, local GP surgeries,
and consumer panels of such groups as the South East
Scotland Cancer Network (SCAN) Patient Involvement
Group and local charity support groups. People had the
opportunity to register their interest and receive an in-
formation sheet to aid their decision to attend. At-
tendees were offered support with travel expenses to the
central Edinburgh venue. Patient and carer consultation event People with a diagnosis of cancer and one or more co-
morbid illnesses and their informal carers were invited
to attend a facilitated discussion workshop to share their Fig. 1 The research prioritisation process Fig. 1 The research prioritisation process Cavers et al. Research Involvement and Engagement (2020) 6:17 Cavers et al. Research Involvement and Engagement (2020) 6:17 Cavers et al. Research Involvement and Engagement (2020) 6:17 Page 4 of 9 Cavers et al. Research Involvement and Engagement Page 4 of 9 The workshop p
As attendees arrived, they were offered lunch and there
was informal discussion to build rapport among the
group. The researcher, Debbie Cavers (DC), and con-
sumer member of the research team Elspeth Banks (EB)
then gave an introduction on the format for the day and
some insights into patient and public involvement in re-
search. It was planned that the room would then be split
into small groups for in-depth discussion of ideas for re-
search, with each group facilitated by experienced mem-
bers of staff working in the field of cancer experience, as
well as EB, who has considerable experience of con-
sumer involvement and has supported similar events in
the past. However, due to small numbers of attendees,
only one group was formed with the facilitators. At-
tendees were prompted with suggested topics and ques-
tions. Post-it notes and marker pens were used to
annotate ideas. With consent, the discussion was re-
corded using a small digital recorder to capture all
thoughts for further consideration after the event. This
culminated in a set of consensus priorities being docu-
mented. DC conducted further analysis of these prior-
ities
to
collate
and
combine
similar
issues
and
summarise key patient priorities (further details below),
validated by the audio recordings of the event and in the
context of existing research or gaps in the field (identi-
fied by the systematic review), to form a summary of the
key issues. The summary formed the basis for the con-
sultation document shared with patients and carers on-
line in the second part of the patient consultation
process. Academic expert consultation Academic expert consultation
In order to further broaden the reach of the consult-
ation, the document was also emailed separately to
twenty professional stakeholders –academics (including
clinical academics) who have published research on can-
cer survivorship and multimorbidity -for their input, re-
flective
of
the
third
round
of
an
eDelphi
online
questionnaire [26]. Feedback from this exercise con-
firmed and added to the themes identified in the first
component. These were contrasted and compared with
the identified gaps in the literature from the systematic
review and evidence synthesis [16]. Close parallels were
drawn between the findings from the evidence synthesis
and patient priorities identified via the prioritisation ex-
ercise. These were combined to inform the development
of an empirical study; in particular, the research ques-
tions and the interview schedule for in-depth patient
and carer interviews to be carried out by the authors. The issues raised through the consultation were not
subject to formal qualitative data analysis but did
undergo an analytic process to group and summarise
shared patient and academic expert priorities and link
them with existing gaps in the literature identified in a
separately published systematic review and evidence syn-
thesis to produce priorities for research [16]. Issues iden-
tified at the workshop were initially added to a list. After
the
event,
DC
reviewed
the
list
and
looked
for Cavers et al. Research Involvement and Engagement (2020) 6:17 Page 5 of 9 Cavers et al. Research Involvement and Engagement Page 5 of 9 commonalities or overlap and merged points where
deemed appropriate. These merged points were given
overarching labels, thus generating the topics identified
as priority issues. Further input from the online consult-
ation with patients and academic stakeholders were used
to refine or expand the priorities established as a result
of combining the workshop and patient consultation
with outputs from the systematic review, loosely follow-
ing an online eDelphi approach (without explicitly rank-
ing priorities) [26, 28]. This added to topics being
expanded or new topics generated e.g. support for
carers. appropriate support; seeking some semblance of normal-
ity, routine and functioning in everyday life; and prac-
tical and financial concerns. The online consultation
identified supported self-management, including advice
and support on lifestyle and nutrition after cancer as a
patient and stakeholder priority. Self-management for
those with cancer and comorbid conditions was also
identified as a research gap in the literature review. The diagnostic journey The diagnosis of cancer was viewed as being a crucial
moment in the health care trajectory and cancer journey,
associated for the most part with fear, shock and dis-
tress; although in some cases possibly with relief also. Those consulted talk about their lives being turned up-
side down in an instant. Potential questions identified
for research included a comparison of the experience of
diagnosis of cancer versus another condition, with issues
such as cancer and other disease type, severity and the
sequencing of the illnesses being pertinent. Further con-
sultation and analysis of feedback highlights the meaning
ascribed to cancer in comparison to other conditions as
being a relevant line of enquiry, with implications for ill-
ness identity. The burden of symptoms Those consulted discussed the importance of looking at
the impact on people’s bodies and emotions of having to
deal with complex and varied symptoms of more than
one condition as well as treatment side effects. It was The online consultation generated a total of ten re-
sponses, again a smaller number of responses than antic-
ipated. Of a total of 20 international academic experts
contacted, eight responded or asked a member of their
research team to respond on their behalf, representing
countries including the UK, USA, Holland, Denmark
and Australia. Outputs generated from the research
prioritisation process There were six attendees at the initial workshop to high-
light topics for research, feeding into the consultation
document, a much smaller number than anticipated. Due to the nature of their involvement, personal demo-
graphic information or details of their health were not
recorded. However, all attendees self-reported at least
one diagnosis of cancer (including bowel, breast and
prostate) and one other long term condition (including
heart disease, diabetes and chronic pain). Patient and public involvement Patient and public involvement
Dr. Allison Worth (PPI advisor for the Wellcome Trust
Clinical Research Facility) and G.W., a Wellcome Trust
consumer panel member read and commented on the
proposed consultation and supporting documents (ad-
vertising poster, attendee information sheet, agenda and
proposed content of discussion). The consumer member
of the study advisory group, EB, supported the design
and planning of the consultation event and its outcomes,
including commenting on all documentation. An add-
itional attendee at the consultation workshop was in-
vited to join the research team as a consumer advisor
for the remainder of the project. Academic expert consultation Re-
search exploring the experience of cancer and comorbid
illness will necessarily feature these topics and consider-
ation is needed of how they extend to and influence liv-
ing with additional chronic illness. The key issues arising that applied to the experience of
cancer and additional long term illnesses are set out
below and summarised in Table 1. They have been ar-
ranged according to broad themes although many of the
issues are cross-cutting. Table 1 Issues identified for further qualitative inquiry • Explore the complex symptom experience, blurring of symptoms,
and the intersection with perceptions and expectations of ageing. • Explore the complex symptom experience, blurring of symptoms,
and the intersection with perceptions and expectations of ageing. • Understand help-seeking and negotiating health services for more
than one illness. • Explore illness identity and the meaning of cancer in comparison to
the other illness, with attention to issues such as cancer and other
illness type, severity and sequencing. Issues that can be attributed to the experience of can-
cer in general were given emphasis by those consulted,
without reference to comorbid illnesses. Topics identi-
fied included: issues related to the burden of cancer-
related and treatment side effects; psychological distress,
anxiety, uncertainty and fear of recurrence; lack of infor-
mation provision and poor communication between pa-
tient and doctors and between hospital and primary care
doctors; social isolation, support and the timing of • Ascertain views on the notion of holistic care and continuity of care
between specialists and other health and social care providers and
the challenges of providing this form of care. • Explore preferences for treatment decision-making, professional sup-
port and follow-up, including views on specialist versus primary care. • Examine views on self-management and supported self-
management Cavers et al. Research Involvement and Engagement (2020) 6:17 Cavers et al. Research Involvement and Engagement Page 6 of 9 Page 6 of 9 qualitative investigation of this issue is required to
understand
more
fully
the
experience
of
service
provision for those with multiple conditions. Other re-
lated points identified included communication between
primary and secondary care or between secondary care
providers (potentially influenced by complex health con-
ditions and an increased number of people involved in a
patient’s care); the role of specialist support and reassur-
ance (having a ‘hotline to the specialist’ versus GP care);
management of follow-up for multi-morbid cancer pa-
tients; and the impact of comorbid conditions on cancer
treatment decision making (as discussed in relation to
managing medications). Self-management and supported
self-management were also highlighted as key areas for
research on patient, carer and professional views to help
manage the challenges of survivorship care for people
with complex health needs. viewed as key to explore the impact on managing daily
living. Table 1 Issues identified for further qualitative inquiry Another issue identified was the ability to make a dis-
tinction between the symptoms from the cancer or side
effects of cancer treatment versus the symptoms from
the other illness. A qualitative inquiry could explore any
potential difference in the types of symptoms experi-
enced and varying severity. Related to this are the vary-
ing
kinds
of
symptoms
people
may
experience
depending on the type of cancer and the nature of their
other illness. For example, for some the cancer may be
treated but they are left with enduring (and/or exacer-
bated) symptoms from their other condition(s) or vice
versa, which changes the experience of moving on from
cancer. Research in this area would need to consider this
carefully at the design stage. Finally, the potential blur-
ring of symptoms - when it was not possible to know
which illness was causing them; especially for general
symptoms such as tiredness or dizziness – and discern-
ing who, where and when to seek help was highlighted
as a priority. Addressing the needs of informal carers Exploration of the impact on carers was also identified
as a priority in the online and academic expert consulta-
tions. This was identified as a broad and general topic to
be qualitatively explored to identify carers’ unique sup-
port needs related to helping patients manage the multi-
dimensional aspects of complex illness. Managing medications A
number
of
areas
related
to
medications
were
highlighted as of potential interest for research, includ-
ing managing multiple drug regimes. Management issues
such as drug interactions, contraindications and their
potential for unwanted side effects and emergencies (re-
lated to communication between health professionals
about medications) were also raised. The impact of
existing medication on side effects, treatment and trial
participation were also identified in the second stage of
the consultation to warrant research attention. A one-off workshop and an online consultation have
combined with a systematic literature review to identify
pervasive issues related to living with and beyond cancer
as well as those associated with complex combinations
of disease. The diagnostic journey (comparing pathways;
issues of severity and sequencing of conditions), the bur-
den of symptoms (complexity, comparison and blurring
of symptoms), managing medications (polypharmacy
and drug interactions), addressing carer needs (helping
to
manage
complex
illness)
and
issues
of
service
provision (negotiating health services, holistic care, pro-
fessional support and follow-up and supported self-
management) have all been identified as areas where
more exploratory research is needed to understand peo-
ple’s views and experiences in these circumstances. Discussion
Summary Living with and beyond cancer has become a key topic
of interest in cancer experience research. The added
complexity of comorbid conditions among cancer survi-
vors is one important dimension prioritised by a recent
NCRI and James Lind Alliance initiative (https://www. ncri.org.uk/lwbc/#lwbc_questions). This report of a re-
search prioritisation exercise focuses on issues relating
to living with and beyond cancer with comorbid illness,
tying in with priority three of the NCRI ‘top ten’. Reflections on the research prioritisation process A patient partnership and stakeholder involvement ap-
proach was adopted to plan meaningful and relevant re-
search to ultimately influence policy and practice and
improve care for patients and their families. However, it
is important to consider one’s own assumptions in influ-
encing interpretations of priorities in order to fit in with
planned research. Practicing reflexivity is key and keep-
ing an open agenda for topics to be added to can help to
address these concerns [36]. Going into this exercise, the research team held cer-
tain assumptions that is it important to surface here and
consider the extent to which they were challenged. The
team are interested in psychosocial aspects of living with
and beyond cancer and so cancer was the index condi-
tion in relation to other comorbid illness. Therefore,
cancer has already been implicitly prioritised as the
centre of patients’ stories. Hearing suggestions of illness
identity and prioritisation of conditions as being more
complex, and cancer not always being at the centre of
people’s lives (depending on the type and severity of the
cancer
and
comorbidities),
has
challenged
this
assumption. There are also limitations to the number of people
taking part in the consultation and reflections to be
made on the planning process to ensure a broader scope
and diversity of consultants, speaking to existing debates
around whether or not patient involvement includes the
voices of a wide enough group of people, including those
from marginalised groups and those less able to expertly
self-advocate and articulate their views and beliefs [32]. This brings into question whether formal training should
be offered to more people in order to empower and en-
able people of all backgrounds to engage with the in-
volvement process more fully [40]. This sits within a
wider critical debate about the role of and spaces occu-
pied by participatory research [33, 40] and potential
power imbalances in the context of citizen science and
consumer accountability [41–43]. The importance of
bringing in evidence relating to gaps in the literature
identified in the aforementioned systematic review car-
ried out by the authors, as well as subsequent empirical
research to capture the diverse needs of patients living
with and beyond cancer with comorbid conditions, is
also heightened, to contribute to a robust evidence base
with transferable findings. Reflections on the research prioritisation process p
There can also be a mismatch between patients’, clini-
cians’ and research community priorities for research, so
taking a partnership approach involving the views of all
of these groups was important to inform the priorities
set and shape future work [37]. There is an additional
danger of a ‘policy disconnect’ [as described by Alan Ir-
win in his critical review of public involvement in rela-
tion to science and technology research [38]], where
there is a lack of evidence that patient partnership work
is taken forward and leads to tangible changes in care
provision. It can often be difficult to see the thread from
prioritisation and then research through to improved
care and experience, particularly in studies exploring
more conceptual dimensions of living with illness. The
steps in providing the evidence base are often slow from
early consultation and exploratory work, to more fo-
cused topic work before reaching practical interventions
that may be shown to improve patient care. Embedding
these in sustained provision of care in a resource con-
strained
National
Health
Service
is
yet
another
challenge. g
DC is also a relative novice in doing this form of pa-
tient involvement work and so there were lessons to be
learned in terms of publicising the event and online con-
sultation, as well as a lack of power and influence in
reaching certain groups and being heard. Approaches
where invitations came from a trusted source, e.g. post-
ers in clinic rooms or personal invitations endorsed by
online patient group coordinators, appeared to be more
successful than social media appeals with endorsement
of trusted sources. Employing a wider range of strategies
for finding people in order to invite them to be involved
in this consultation, such as via community groups and
councils, may have been a better approach [44]. Service provision The final area highlighted as being a relevant area for re-
search exploring the experience of cancer and comorbid
illness is that of service provision. Issues of receiving
good quality of care were raised and exploration of the
potential tensions between the need for holistic thinking
when addressing individual needs versus fragmented
care were identified as a gap in the evidence base. Those
consulted suggested that individuals are being followed
up separately by more than one specialist and their pri-
mary care providers, and described being ‘passed from
pillar to post’ with a lack of joined up care. Further This exercise, combined with a systematic review of the
literature that has identified gaps in the qualitative evi-
dence base that map onto patient-identified priorities [16],
has highlighted key issues (as listed above) that appear to
be central to the experience of cancer with additional con-
ditions. This approach, working in partnership with
people living with and beyond cancer with comorbid con-
ditions, has identified priorities for future empirical
research. Cavers et al. Research Involvement and Engagement (2020) 6:17 Page 7 of 9 Page 7 of 9 Cavers et al. Research Involvement and Engagement (2020) 6:17 Cavers et al. Research Involvement and Engagement approach [28]. People’s motivations for patient involve-
ment are complex [39]. Upon reflection, it is possible
that ill health and limited mobility prevented people
from becoming involved, which will have included travel
for some. There are also possible social or structural
barriers: perhaps people did not feel they qualified to
represent others in this way or comfortable working in
an academic environment on such a project? It certainly
felt as though there was more groundwork to be done in
terms of building productive relationships and allowing
people time to share their personal stories before mov-
ing forward to a shared research agenda, suggesting that
a longer term process may have generated different, and
possibly better, outcomes. To this end, a second work-
shop after the online consultation would have helped to
consolidate or expand on the priorities identified in the
initial workshop and reach a clearer consensus, but re-
source and time constraints did not permit this. Limitations of the exercise Ultimately, this consultation did not generate as much
involvement
and
feedback
as
was
anticipated
and
planned for by the research team, impacting on confi-
dence in the consensus reached. Although limited by
time and financial resources, there are important lessons
to be learned about mitigation planning and a flexible
approach to enable expansion on consultation methods
in response to low interest, as set out in the Delphi Cavers et al. Research Involvement and Engagement (2020) 6:17 Page 8 of 9 Cavers et al. Research Involvement and Engagement (2020) 6:17 Page 8 of 9 Cavers et al. Research Involvement and Engagement (2020) 6:17 This research prioritisation exercise did pose some
challenging questions around the blurring of the bound-
aries between patient involvement work and research. It
problematises what counts as research data and there-
fore how the study should be designed and the ethical
considerations managed, including the role of formal
ethical review (procedural ethics). These issues seem
particularly pertinent in relation to scenarios such as this
exercise where information is being generated that will
be used to support research and policy impact. The line
between treating the discussion as informing research
and as research data can be blurred: this paper is an ex-
ample of that ambiguity. However, important steps have
been taken to clarify this distinction, both here and in
the broader literature, and a call for transparency and
clarity in reporting of engagement work [45]. The en-
gagement process employed a sensitive and considered
ethical approach and went through required institutional
approval (Usher Research Ethics Group). It was clear
that people were being asked to join the researchers as
partners in a process of collaborative information collec-
tion, and the discussions were summarised and key is-
sues identified, rather than analysed as they would be in
a qualitative research process. However, a social con-
structionist approach to intersubjective data generation
is arguably not that different; neither is a thematic ana-
lysis. While we feel confident in our own ethical pos-
ition,
we
are
nonetheless
uncomfortable
operating
within the ambiguous space of patient involvement in
research that is, if it is to be effective, generative of rele-
vant information in a research context. Funding
h This consultation did not undergo formal evaluation
but it was clear that those involved helped to validate
the thinking of the research team and contributed topics
that would not otherwise have been identified without
consulting stakeholders. We would argue that more at-
tention needs to be paid to evaluation, including by self-
reflection, and for this to contribute to the evidence base
for PPI. This project forms part of a post-doctoral fellowship funded by the Chief
Scientist Office of the Scottish Government (Ref: PDF/15/06). Abbreviations
l NCRI: National Cancer Research Institute; PPI: Patient and Public Involvement;
DC: Debbie Cavers; EB: Elspeth Banks; GP: General Practitioner; NHS: National
Health Service; CSO: Chief Scientist’s Office Authors’ contributions DC conceived of the idea for this study and all authors contributed to its
development. All authors contributed to the development of study materials
and commented on study outcomes. DC drafted the manuscript and all
authors helped to develop the final version. The authors read and approved
the final manuscript. Ethics approval and consent to participate This research prioritisation work was discussed with the South East Scotland
Research Ethics Service (12/08/2016) and did not require NHS ethical review. All components of the consultation were reviewed and approved by the
Usher Institute Research Ethics Committee (Component 1: 14/10/2016,
Component 2: 17/07/2017). Acknowledgements We thank Emily Ross for her help in facilitating the consultation workshop. We thank Allison Worth and Grahame White for commenting on the
proposed consultation and supporting documents. We thank academic
experts in the field who contributed to the research prioritisation
consultation document: Marjan van den Akker, Camilla Hoffman-Merrild, Gill
Hubbard, Diane Sartafi, Larissa Nekhlyudov, Jeannine Stairmand, Peter Lewis
and Annette Berendsen. We would also like to thank the peer reviewers for
their detailed insight and feedback to help strengthen the manuscript. Additional file 1. Additional file 1. Conclusion Including patients in the consultation in addition to aca-
demics and clinicians with a broad view of the research
landscape on an international stage aims to lead to more
meaningful
and
relevant
in-depth
research
and, Availability of data and materials This paper does not include research data and this statement is therefore
not applicable. Limitations of the exercise We would argue
that a new approach to ethics is needed in this environ-
ment to work with this tension and to reassure both re-
searchers and patient participants that an appropriate
form of ethical review has been undertaken and that eth-
ical issues will continue to be reflected upon during the
process of the engagement and subsequent research. This is all the more important as PPI is increasingly a
core component of research in the UK [46]. ultimately, to corresponding interventions that will im-
prove patient, carer and provider experience. Findings from this research prioritisation exercise will
directly inform further in-depth research exploring ex-
perience of cancer and other comorbid conditions as
topics identified will be integrated into a planned inter-
view study with patients and carers. On a broader scale,
reflections on the consultation will contribute to under-
standings of the role of patient partners in the research
process. It can provide the foundations for future work
to provide outputs that are relevant and meaningful, not
only to patients, but to policy makers, in order to make
recommendations for health and social care services in
Scotland, as elsewhere, and improve patient experience,
to meet the targets set out by Better Cancer Care [11]. Supplementary information
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complexity; marshalling resources: a systematic review and qualitative meta- Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. 23. Coventry PA, Small N, Panagioti M, Adeyemi I, Bee P. Living with
complexity; marshalling resources: a systematic review and qualitative meta-
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Self-concept as a significant determinant of brand choice and consumer buying behaviour
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1 U domaćoj literaturi se malo pisalo o konceptu „ličnosti brenda“. Međutim, simultano se
koriste termini „personalitet brenda“ i „ličnost brenda“. U ovom radu smo se opredelili
za isključivo korišćenje termina „ličnost brenda“, posebno iz razloga što se vrlo često
poredi sa ličnošću i ličnim karakteristikama osobe, te bi stoga bilo neadekvatno da se
koristi termin „personalitet“. UDK 658.626+366.1, Pregledni rad Lični koncept kao značajna
determinanta izbora brendova i
ponašanja potrošača u kupovini Slađana Starčević Uvod Apstrakt: Potreba da se predvidi pona-
šanje potrošača ima veliki značaj u mar-
ketingu. Danas jednu od najpopularnijih
psiholoških konstrukcija u društvenim
naukama i marketingu predstavlja lični
koncept, koji se definiše kao suma ideja,
mišljenja i osećanja koje pojedinac ima
o samom sebi, u odnosu na druge osobe
i objekte u društveno determinisanom
okviru. Brojni istraživači su prepoznali
značaj ličnog koncepta za predviđanje
ponašanja potrošača i izbor brendova. Ljudi nastoje da održavaju i pojačavaju
sopstveni lični koncept potrošnjom onih
brendova čiji su imidž i lične karakteristike
u skladu sa njihovim imidžom i ličnim
karakteristikama. Mnoge studije su po-
tvrdile da visok nivo podudarnosti između
ličnog kocepta potrošača i imidža/lično-
sti brenda pozitivno utiče na stavove o
brendu, preferencije, evaluaciju, kupovne
namere, satisfakciju i lojalnost brendu. Ovim radom smo nastojali da istražimo
način na koji je jedan psihološki koncept
dobio svoje značajno mesto u istraživanji-
ma i praksi iz oblasti marketinga. Koncept
je paralelno sagledan iz ugla psihologije i
marketinga. Poseban akcenat je stavljen
na način na koji lični koncept potrošača,
posredstvom podudarnosti sa imidžom/
ličnošću brenda, utiče na izbor brendova
i kupovno ponašanje potrošača. Takođe
smo ukazali na značaj poznavanja ovog
koncepta i preporuke vezane za njegovu
upotrebu u praktične svrhe u okviru brend
menadžmenta. Dobar deo ljudskog ponašanja je odraz ličnosti tj. ličnih karakteristika
pojedinca. Allport, ličnost definiše kao „dinamičku organizaciju onih
psiho-fizičkih sistema unutar pojedinca, koji određuju njeno karakteri-
stično ponašanje i razmišljanje (prema: Mischel, W., 1999, str. 212)“. U
suštini, ličnost se može definisati na razne načine i gotovo da nije bilo
istaknutijeg istraživača i teoretičara iz ove oblasti koji nije pokušao da lič-
nost definiše na svoj način. Isto tako, postoji veliki broj znatno različitih
teorija o ljudskoj ličnosti. U zavisnosti od teorije, razlikuje se i definicija,
kao i elementi ličnosti koji se stavljaju u prvi plan, a samim tim se razli-
kuju i tehnike koje se koriste u izučavanju ličnosti. Međutim, sve defi-
nicije ličnosti uvažavaju činjenicu da se radi o jedinstvenom profilu ili
kombinaciji osobina koje izdvajaju ličnost od ostalih jedinki u okruženju. Sumarno, osnovne karakteristike ličnosti su: da odražava individualne
razlike, da je konzistentna i trajna, te da se pod određenim okolnostima
može promeniti (Schiffman, Kanuk, 2000, str. 114). Ljudi nastoje da održavaju i pojačavaju
sopstveni lični koncept potrošnjom onih
brendova čiji su imidž i lične karakteristike
u skladu sa njihovim imidžom i ličnim
karakteristikama. Uvod Celovit lični koncept je u osnovi pozi-
tivan, mada u nekim delovima to ne mora biti slučaj
(Maričić, B., 2008, str. 388). Više od četiri decenije lič-
ni koncept privlači pažnju teoretičara i praktičara iz
oblasti marketinga, iako je u psihologiji počeo da se
izučava mnogo ranije. Naime, do toga je dovelo za-
pažanje da svoj lični koncept ljudi najviše održavaju i
pojačavaju potrošnjom određenih brendova. Pokaza-
lo se da potrošači upravo biraju one brendove koji naj-
bolje odražavaju njihov lični koncept, tj. čiji su imidž i
lične karakteristike u skladu sa njihovom sopstvenom
imidžom i ličnim karakteristikama (Webb, Gountas,
2006, str. 3). Veliki broj studija je pokazao da visok
nivo podudarnosti između ličnog koncepta potrošača
i imidža/ličnosti brenda ima pozitivan uticaj na stavo-
ve o brendu, preferencije, evaluaciju, kupovne name-
re, stvarno ponašanje, satisfakciju i lojalnost potroša-
ča brendu. Zbog svog značaja, ovo područje se danas
smatra posebnim aspektom brend menadžmenta. Teorije o ponašanju i odlučivanju potrošača su gene-
ralno zasnovane na ideji da se marketing stimulusi
interno procesiraju od strane potrošača u kontekstu
delovanja eksternih faktora, što dovodi do toga da se
kupuje ili ne kupuje određeni proizvod (Milisavlje-
vić et al, 2009, str. 213). Ovim radom smo nastojali
da istražimo način na koji je jedan psihološki koncept
dobio svoje značajno mesto u istraživanjima i praksi
iz oblasti marketinga. U prvom delu rada se govori
prirodi koncepta iz ugla psihologije, da bi se zatim isti
koncept obradio iz ugla marketinga. Prikazano je na
koji način lični koncept, poredstvom podudarnosti sa
imidžom i ličnošću brenda utiče na pojedine oblike
ponašanja potrošača i izbor brendova na tržištu. Ta-
kođe se ukazuje na značaj poznavanja ovog koncepta sti brenda2, da bi se razgraničile unutrašnje karakteri-
stike (koje čine ličnost u skladu sa definicijom ličnosti
u psihologiji) i ostali elementi identiteta brenda (de-
mografske karakteristike, fizička pojava itd.), imajući
u vidu da je identitet brenda mnogo širi pojam od lič-
nosti brenda (Azoulay, Kapferer, 2003, str. 143). (
y
p
)
Mnogi teoretičari i praktičari marketinga se slažu
oko toga da ličnost brenda predstavlja veoma značajnu
formu diferenciranja u savremenim uslovima, kada su
tradicionalne forme diferenciranja brendova izgubile
na značaju. (Sweeney, Brandon, 2006, str. 639). Uvod Mnoge studije su po-
tvrdile da visok nivo podudarnosti između
ličnog kocepta potrošača i imidža/lično-
sti brenda pozitivno utiče na stavove o
brendu, preferencije, evaluaciju, kupovne
namere, satisfakciju i lojalnost brendu. f
Ličnost značajno utiče na ponašanje pojedinca kao potrošača, kao i na
izbor brendova (Maričić, B., 2008, str. 387). Naime, uočeno je da potro-
šači brendove posmatraju, nekad potpuno nesvesno, kao živa bića. Pri-
pisuju im pozitivne i negativne karakteristike svojstvene ljudima (hariz-
matičan, odlučan, bojažljiv, neprilagođen itd.) i ostale odrednice kao što
su pol, godine, status itd. Jednostavnije rečeno, oni biraju brendove kao
prijatelje i pomoću njih žele da izraze svoj sopstveni identitet ili lični kon-
cept. Prema teoriji animizma, potrošači antropomotorizuju objekte da bi
olakšali komunikaciju sa okolinom. Ova i slična zapažanja su i dovela do
uvođenja pojma „ličnost/personalitet brenda“1 (eng. brand personality) u
marketing. U literaturi iz oblasti marketinga se najčešće susreće definicija
ličnosti brenda koju je dala J. Aaker 1997. godine, prema kojoj „ličnost
brenda čini set ljudskih karakteristika koje se povezuju sa brendom (Aa-
ker, J.,1997, str. 347)“. Međutim, veoma je bitno istaći da se za potrebe
istraživanja u ovoj oblasti mora koristiti dosta preciznija definicija lično- Ovim radom smo nastojali da istražimo
način na koji je jedan psihološki koncept
dobio svoje značajno mesto u istraživanji-
ma i praksi iz oblasti marketinga. Koncept
je paralelno sagledan iz ugla psihologije i
marketinga. Poseban akcenat je stavljen
na način na koji lični koncept potrošača,
posredstvom podudarnosti sa imidžom/
ličnošću brenda, utiče na izbor brendova
i kupovno ponašanje potrošača. Takođe
smo ukazali na značaj poznavanja ovog
koncepta i preporuke vezane za njegovu
upotrebu u praktične svrhe u okviru brend
menadžmenta. Ključne reči: lični koncept, imidž bren-
da, ličnost brenda, lična podudarnost,
ponašanje potrošača 106 Dugi niz godina teoretičari su nastojali da katego-
rizuju potrošače na određene tipove ličnosti, te da ih
povežu sa ponašanjem pri kupovini i nizom drugih
marketinških varijabli. Međutim, pokazalo se da se u
predviđanju ponašanja potrošača, posebno što se tiče
izbora brendova na tržištu, ne sme izostaviti razma-
tranje jednog blisko povezanog koncepta. Reč je o
ličnom konceptu ili ličnom imidžu potrošača, koji se
definiše kao „suma ideja, mišljenja i osećanja pojedin-
ca o samom sebi u odnosu na druge osobe i objekte
u društveno determinisanom okviru” (Ataman, Ul-
engin, 2003, str. 238). Drugačije rečeno, to je lična
percepcija pojedinca o sopstvenim sposobnostima,
ograničenjima, fizičkoj pojavi i osobinama u odnosu
na druge ljude. • Persepektive pojedinačnih društvenih grupa • Persepektive pojedinačnih društvenih grupa. To znači da postoji nekoliko tipova ličnog identi-
teta, u zavisnosti od uloge koju određena društvena
grupa igra u životu pojedinca. U skladu sa tim po-
smatranjem, Tajfel dalje razrađuje pojam „društve-
nog ličnog identiteta“, koji je povezan sa percepcijom
pojedinca o tome da pripada određenim društvenim
grupama koje su za njega značajne, u smislu emocio-
nalnih veza i vrednosnih sistema koje te grupe nose sa
sobom. Prema teoriji samo-kategorizacije, pojedinci
sami sebe kategorizuju u određene društvene grupe,
prihvatanjem njihovih normi u ponašanju (Abrams,
Hogg, 1990, str. 195). • Materijalni identitet (prošireni identitet) – koji
čine telo pojedinca, njegova familija i imovina; • Društveni identitet – koji predstavlja način na koji
druge osobe vide pojedica; • Duhovni identitet – koji predstavlja „unutrašnje
biće“ pojedinca (stavove, motive, emocije, intere-
sovanja, mišljenja, želje itd.). Dok se James u svojim istraživanjima fokusirao na
lični identitet kao strukturu koja pojedinca „gura“ na
akciju, Cooley je u prvi plan stavio društvenu deter-
minisanost ličnog identiteta. On je kreiranje ličnog
identiteta opisao kao „refleksiju u ogledalu“, u smislu
da pojedinac formira sopstveni identitet na osnovu
poruka koje prima od drugih ljudi. U tom kontekstu,
Cooley je izdvojio tri komponente ličnog identiteta
(Brown, 1998, str. 78): U suštini, postoji onoliko definicija ličnog identite-
ta, koliko postoji različitih škola mišljenja i psihološ-
kih teorija, što je slučaj i sa definicijom ličnosti. Pored
definicije navedene u uvodnom izlaganju, u literaturi
se vrlo često susreće definicija Rosenberga, prema či-
jem mišljenju se lični identitet definiše kao „ukupna
suma razmišljanja i osećanja putem kojih pojedinac
može opisati sebe kao objekat“ (Achouri, Bouslama,
2010, str. 34). Na osnovu toga, Brunel (prema: Achou-
ri, Bouslama, 2010, str. 35)razmatra lični identitet kao
konstrukciju koja istovremeno obuhvata: • Percepciju pojedica o tome kako on deluje drugim
ljudima; • Percepciju toga kako drugi ljudi sude o njegovoj
pojavi; • Kognitivnu komponentu – ideje i mišljenja koje
pojedinac ima o sebi; • Osećanja pojedinca koja se stvaraju na osnovu
percipirane procene drugih ljudi o njemu samom. • Emocionalnu komponentu – impresije i osećanja
koja pojedinac ima prema sebi; Osećanja deluju kao pojačivač pri formiranju lič-
nog identiteta. Ako pojedinac ima pozitivna osećanja,
on održava svoju percipiranu pojavu tj. identitet, a u
suprotnom slučaju je modifikuje. U suštini, nije toliko
bitno na koji način drugi ljudi procenjuju pojedinca,
koliko je bitno kako to pojedinac lično doživljava. Uvod Smatra
se da kreiranje adekvatne ličnosti brenda ima potenci-
jal stvaranja jakog odnosa između potrošača i brenda,
koji vodi jačim preferencijama i stavovima ka kon-
kretnom brendu, podsticanju namera za kupovinu,
stvaranju baze lojalnih potrošača i uvećanju vredno-
sti brenda kao neopipljive imovine. Doživljaj ličnosti
brenda, kao sastavnog dela ukupnog imidža brenda,
jeste ustvari kupčev omocionalni odgovor kompani-
ji i njenom proizvodu, te se njome mora adekvatno
upravljati (Veljković, S., 2010, str. 91) . Iz navedenih
razloga, ličnost brenda predstavlja veoma atraktivnu
tematiku savremenog marketinga. Ona se smatra jed-
nom od ključnih diferencirajućih dimenzija identiteta
brenda, koja je možda i najbliža potrošaču. Međutim,
izučavanje ličnosti brenda i ličnosti potrošača svoje
polazište ima u psihologiji ličnosti čoveka, teorijama
i metodima koji pripadaju ovoj disciplini. Stoga je pri
analizi ovih koncepata bitno sagledati njihove psiho-
loške korene. 2 Iako ličnost brenda predstavlja veoma star koncept, prvi put
pomenut 1958. godine, veliki talas istraživanja u ovoj oblasti je
pokrenut studijom koju je sprovela J. Aaker 1997. godine. Ona
je predložila teoretski model ličnosti brenda, definisala osnovne
dimenzije i pod-dimenzije ličnosti brenda i konstruisala mernu
skalu za merenje ovog koncepta. Međutim, gotovo sve naredne
studije (čak i savremene) su bez preispitivanja koristile njenu
originalnu definiciju i konstrukciju ličnosti brenda, što je dovelo
do niza nepravilnosti u izvođenju zaključaka pri poređenju
ličnosti potrošača i ličnosti brenda. Mnogi psiholozi se ne bi
složili sa ovako širokom definicijom ličnosti, jer ona pored
psiholoških procesa obuhvata demografske karakteristike i
fizičku pojavu (pojavni oblik) tj. elemente identiteta brenda,
što predstavlja mnogo širi koncept od ličnosti brenda. Iako
su drugi istraživači, kao npr. Allen i Olson, usvojili mnogo
precizniju definiciju, te povezali ličnost brenda sa „unutrašnjim
karakteristikama“ u skladu sa definicijom iz psihologije, mnogi
drugi su ostali potpuno nesvesni problema konceptualizacije
ličnosti brenda i koristili definiciju J. Aaker pri istraživanjima
i konstruisanju sopstvenih skala, kao što su npr. Caprara,
d’Astous i Levesque, Sung i Tinkham i dr. Kako se ličnost brenda
danas smatra jednom od ključnih dimenzija identiteta brenda
i značajnim diferencirajućim sredstvom u dugom roku, mnogi
istraživači se u poslednjim godinama intenzivno bave ovom
tematikom. Lični koncept kao značajna determinanta izbora brendova i ponašanja potrošača u kupovini 10 108 Slađana Starčević 1. Priroda ličnog koncepta 1. Priroda ličnog koncepta pira evaluaciju od strane drugih ljudi – kao povoljnu
ili nepovoljnu. Ovaj model je u određenoj meri kriti-
kovan, jer pojedinci ipak ne mogu precizno da proce-
ne šta drugi ljudi misle o njima, već stvaraju generalno
mišljenje o sudu drugih ljudi. Termin „lični koncept“ se danas intenzivno koristi u
literaturi iz oblasti marketinga i ponašanja potrošača,
a potiče iz psiholoških disciplina, tačnije, od termi-
na „lični identitet“. Treba imati u vidu da ovi termini
imaju isto značenje, samo što se u psihologiji češće
koristi temin „lični identitet“ (eng. self-identity), a u
marketingu termin „lični koncept“ (eng. self-concept)
ili čak „lični imidž“ (eng. self-image) potrošača. Mead (prema: Roberts, Donahue, 1994, str. 201) je
posebno istakao ulogu društvene interakcije u razvoju
ličnog identiteta, jer se lični identitet razvija usklađi-
vanjem perspektive i iznošenja mišljenja drugih ljudi
sa svojom ličnom percepcijom o tome kako ga drugi
vide . Ovaj autor ističe da pojedinac pravi razliku iz-
među: Prvo objašnjenje značenja „ličnog identita“ je dao
James još 1890. godine, kada su publikovana dva toma
čuvenih „Principa psihologije“. On je tvrdio da se lič-
ni identitet pojedinca bazira na sećanjima, navikama
i poimanju samog sebe kao ličnosti, pri čemu je veo-
ma bitna svesnost pojedinca o samom sebi. James je
identifikovao tri tipa ličnog identiteta (prema: Huang,
2008, str. 78): • Perspektive drugih ljudi generalno; • Persepektive pojedinačnih društvenih grupa Na
bazi ovog razmatranja, Kinch (prema: Kenny, Bella,
1993, str. 158) razvija „cirkularni model identiteta“
u kojem ističe da se lični identitet pojedinca održava,
pojačava ili modifikuje na osnovu toga kako on perci- • Društvenu komponentu – percepciju projekcije
drugih ljudi o njemu samom. U najširem smislu, lični identitet ili lični koncept se
odnosi na percepciju pojedinca o samom sebi, koja je
konstruisana na osnovu društvene interakcije, steče-
nog iskustva i osećanja. To je ustvari organizacija kva-
liteta koje pojedinac sam sebi pripisuje. Ti kvaliteti se
odnose na izraze koje pojedinac koristi da opiše sebe,
kao i uloge u kojima se on prepoznaje u društvu. Ovi kvaliteti se kreiraju, modifikuju i održavaju ponaša-
njem koje je vezano za ispoljavanje identiteta. Kako
ističe Jamal, pojam ličnog identiteta je blisko povezan
sa pojmom ličnog znanja, jer definisanje ličnog iden-
titeta od strane pojedinca zahteva određeni nivo zna-
nja o samom sebi (Jamal, Goode, 2001, str. 482). brojni istraživači i vrlo često je izučavan u kontekstu
formiranja ciljeva i uticaja na ponašanje potrošača
u kupovini. Bez sumnje, istraživanja vezana za lični
koncept potrošača i ličnost brenda (koja se vrlo če-
sto integrišu) su znatno obogatila saznanja o odnosu
između potrošača i brendova. Međutim, potrebno je
napomenuti da se u području marketinga i istraživa-
nja potrošača lični koncept posmatra na nešto uži na-
čin nego u psihologiji, tj. najviše se bavi razmatranjem
ličnog imidža potrošača. Stoga se vrlo često nailazi
na simultano korišćenje termina lični koncept i lični
imidž potrošača. Michel i Morf (2003, str. 15) ističu da lični identitet
uključuje kognitivnu, afektivnu i konativnu kompo-
nentu (skolonost da se preduzme određena akcija). Kako navode ovi autori, lični identitet je kognitiv-
no-afektivno-aktivni sistem, a istovremeno društve-
no-interaktivni i samo-konstruktivni sistem. Ovakvo
razmatranje ličnog identiteta je u skladu sa humani-
stičkim teorijama ličnosti i teorijama društvenog uče-
nja. Postoje razlozi zašto je toliko veliki značaj istraži-
vanja ličnog koncepta u kontekstu ponašanja potro-
šača u kupovini. Naime, lični koncept nije statička
kategorija već se vremenom razvija u skladu sa ra-
zvojem mišljenja pojedinca o samom sebi, te mišlje-
nja i reakcija drugih ljudi. Čovek svojim ponašanjem
stalno teži da ga održi i poboljša. Oblik ponašanja u
kojem to najviše dolazi do izražaja jeste potrošnja, jer
ljudi nastoje da kupuju one proizvode za koje smatra-
ju da najbolje odražavaju i ako je moguće, pojačavaju
njihov lični imidž. • Persepektive pojedinačnih društvenih grupa Jedna od tehnika merenja imidža
brenda kojom se traži od ispitanika da nacrtaju nje-
nog korisnika je ukazala na to da potrošači za odre-
đene brendove vezuju određene tipove ličnosti. To su
upravo ličnosti u kojima potrošači pronalaze sebe ili
čijem imidžu teže. Kako je napisao jedan autor, “Svet
biznisa je kao jedan veliki teatar, svako traži scenu na
kojoj će da igra svoju ulogu. Svako od nas ulogu bira
u skladu sa svojim dugoročnim, ali uglavnom prilično
nejasnim životnim ciljevima. Brendovi pomažu poje-
dincima da ojačaju svoju ulogu i da im potvrde da je
njihov izbor zaista pravi” (Bergval, 2002, str. 2). Tako
npr. muškarci koji sebe smatraju snažnim i uopšte na-
glašavaju muškost, često preferiraju Marlboro cigarete,
dok je moguće da žene koje sebe smatraju atraktivnim
i modernim preferiraju Virginia Slims cigarete. Postoje i brojne teorije o konstrukciji ličnog kon-
cepta u smislu dimenzija koje uključuje, počevši od
onih koje tvrde da je to jedinstveni koncept, do onih
koje tvrde da ima nekoliko dimenzija. Prema teoriji
o jedinstvom ličnom konceptu, koju je izneo Allport
još 1943. godine, pojedinci poseduju jedinstveni lični
imidž koji je konzistentan sa svim aspektima života,
nasuprot teorijama o višedimenzionalnom ličnom
konceptu, prema kojima pojedinici sebe vide različito
u zavisnosti od situacionih faktora (Gountas, Mavon-
do, 2003, str. 3). Primera radi, jedan predavač marke-
tinga ima nekoliko različitih ličnih koncepata, jer je
istovremeno predavač, konsultant, roditelj, sportista,
politički aktivista, obožavalac savremene umetnosti
itd. Ideja o jedinstvenom ličnom konceptu teško može
da bude argumentovana, jer se svako od nas nalazi u
različitim uslovima okruženja i izložen je različitim
situacijama. Stoga se smatra da je posmatranje ličnog
koncepta kao multidimenzionalnog konstrukta mno-
go adekvatnije, između ostalog i u razumevanju po-
našanja potrošača u različitim kupovnim situacijama. 2. Lični koncept i potrošnja g
g
Sirgy takođe tvrdi da postoji više dimenzija ličnog
koncepta, a svaku od njih potrošač izražava u različi-
tim situacijama: • Stvarni lični koncept - kako osoba stvarno vidi
sebe; • Idealni lični koncept - kako bi osoba želela da
vidi sebe; • Stvari društveni lični koncept - kako osoba oseća
da je drugi vide; • Idealni društveni lični koncept - kako bi osoba
želela da je drugi vide (Jamal, Goode, 2001, str. 482). Osnovna pretpostavka Sirgy-eve teorije poduda-
nosti jeste da je ponašanje potrošača delom determi-
nisano podudarnošću koja proizilazi iz psihološkog
poređenja imidža brenda ili tipičnog korisnika brenda
i ličnog koncepta potrošača. Ovo psihološko pore-
đenje može pokazati da je lična podudarnost visoka,
kada potrošač percipira da je imidž brenda tj. tipičnog
korisnika prilično usklađen sa njegovim sopstvenim
imidžom. U suprotnom slučaju je reč o niskoj ličnoj
podudarnosti. Ova teorija je i oformljena da bi se na
osnovu nje predviđale varijable ponašanja potrošača,
kao što su stavovi prema brendu, preferencije, na-
mere pri kupovini, stvarno ponašanje, satisfakcija i
lojalnost. S obzirom na to da je dobro postavljena i
zasnovana na dokazima, teorija o ličnoj podudarnosti
je postala sastavni deo psihologije potrošača. Posledično, istraživači su povezali ponašanje po-
trošača u kupovini sa ličnim identitetom potrošača,
koji se u marketingu prvenstveno izražava terminom
„lični koncept“, kao što je već navedeno. Pre toga,
istraživanjima se nastojala utvrditi veza između tipo-
va ličnosti potrošača i ponašanja u kupovini, ali bez
razmatranja ličnog koncepta, što je rezultovalo utvr-
đivanjem veoma slabe korelacije. Značaj uključivanja
ličnog koncepta su potvrdile brojne studije. Primera
radi, Grubb i Hupp su još 1968. godine sproveli istra-
živanje na kategoriji automobila za brendove Pontiac
i Volkswagen. Pokazalo se da vlasnici automobila
percipiraju veliku sličnost između svog ličnog imidža
i imidža vlasnika istog brenda automobila. Sa druge
strane, sebe lično su doživeli kao veoma različite u od-
nosu na vlasnike konkurentskog brenda automobila
(Grubb, Hupp, 1968, str. 58). j p
p
g j p
Izbor brendova koji su konzistentni sa ličnim
konceptom je vezan za motive samokonzistentnosti
i samopoštovanja (Sirgy, 1997, str. 229). Rosenberg
definiše motiv samo-konzistentnosti kao motiv da se
dejstvuje u skladu sa ličnim konceptom da bi se isti
održao, bez obzira na to što se javljaju izazovi. 2. Lični koncept i potrošnja p
j
g
g
Potrošači kupuju proizvode i brendove zbog znače-
nja koja oni nose sa sobom u društvenom okviru. „Po-
sedovati nešto“ danas ima značenje u društvu. Razlog
zbog kojeg se vrednuje ono što ljudi poseduju leži u
značenjima koje vlasnici pripisuju imovini, a ta zna-
čenja su oformljena na osnovu društvene interkacije. Gardner i Levy su bili među prvim istraživačima koji
su koncept „značenja“ primenili na proizvode i bren-
dove u marketingu. Oni su istakli da su ta značenja
potrošačeve vrednosti i procene o brendovima, takva
da marketari imaju ulogu da kreiraju željena znače-
nja brendova za potrošača, koja se koriste da se pojača
njihov lični identitet (Levy, 1959, str. 117). Lični koncept je u marketingu počeo da se izučava
1960-ih godina, a do pravog zamaha je došlo 1970-ih
godina. Studije ponašanja potrošača su se sve više po-
čele baviti utvrđivanjem veza koje potrošači pronalaze
između imidža određenog brenda i svog ličnog imid-
ža. Ove studije su uglavnom bile povezane sa teorija-
ma o ličnom konceptu koje su postavili motivacioni
psiholozi K. Rogers i A. Maslov, kao i sa Frojdovom
psihoanalitičkom teorijom i poimanjem „ega“. Lični koncept je danas veoma popularna psihološ-
ka konstrukcija u društvenim naukama. Značaj lič-
nog koncepta u istraživanju potrošača su prepoznali Lični koncept kao značajna determinanta izbora brendova i ponašanja potrošača u kupovini 109 Iako danas postoji veliki broj proizvoda kod kojih
je pri kupovini veoma bitan funkcionalni aspekt, iz-
bor i potrošnja se danas mnogo više baziraju na sim-
boličkim značenjima koje proizvodi nose sa sobom. Tzv. simbolička potrošnja je već duže vreme u fokusu
marketinških istraživanja. Dokazano je da se značenja
tj. simbolika koju nose sa sobom različiti brendovi pr-
venstveno koristi da bi prezentovala svoje vlasnike i
izrazila njihov lični identitet. Prema Thompsonu, po-
jedinci su kao „simbolički projekti“, oni koriste raspo-
ložive simboličke materijale (kao što su npr. brendovi)
da bi aktivno konstruisali priču o sopstvenom ličnom
identitetu. Ti simbolički materijali nose ona značenja
za koja pojedinci misle da su u skladu sa kvalitetima
koji čine njihov sopstveni identitet (Thompson, Hir-
chman, 1995, str. 139). Interpretacija tih simboličkih
značenja brendova nije racionalna, već prvenstveno
emocionalna. riše da je radi preciznosti rezultata adekvatnije vršiti
poređenje sa percipiranim ličnim karakteristikama
brenda, nego imidžom brenda generalno. 110 Slađana Starčević 2. Lični koncept i potrošnja Stoga se mar-
ketari suočavaju sa velikim izazovom, kada postoji i
najmanja mogućnost za „stvaranje veze sa potroša-
čem“. Na današnjem velikom homogenom tržištu, čak
i male fluktuacije u preferencijama ka brendu mogu
determinisati uspeh kompanije. Iako neki istraživači
tvrde da potrošači mogu kreirati „privatni identitet“
korišćenjem proizvoda koji nisu društveno vidljivi,
značenje tog privatnog identiteta je takođe društve-
no determinisano, iako potrošači nekad nisu ni svesni
toga (Van de Rijdt, 2008, str. 13). nelagodno da ga stvarno poseduje i vozi po komšiluku
stvarajući buku. Ovo ukazuje na izbor između poja-
čavanja ili održavanja ličnog koncepta (Van de Rijdt,
2008, str. 9). S obzirom na višedimenzionalnost ličnog koncep-
ta, u istraživanjima se izdvajaju četiri tipa ocene po-
dudarnosti između imidža potrošača i brenda (Sirgy,
1982, str. 287): • Podudarnost sa stvarnim ličnim konceptom –
odnosi se na nivo sličnosti između stvarnog ličnog
imidža potrošača i tipičnog imidža koji potrošač
dodeljuje proizvodu. Vođeni motivacijom da
zaštite sopstveni identitet, potrošači nastoje da
konzumiraju proizvode koji reflektuju njihov
istinski lični koncept. • Podudarnost sa idealnim ličnim konceptom –
odnosi se na stepen sličnosti između potrošačevog
idealnog ličnog imidža i imidža tipičnog potro-
šača proizvoda. Kako navodi Galan, ostvarenje
određenog idealnog ličnog imidža kroz potrošnju
ili posedovanje proizvoda zadovoljava potrošače-
vu potrebu za samopoštovanjem. • Podudarnost sa stvarnim društvenim ličnim
konceptom – odnosi se na stepen sličnosti iz-
među potrošačevog društvenog ličnog imidža i
imidža tipičnog potrošača proizvoda. Ova po-
dudarnost zadovoljava potrebu za društvenom po-
vezanošću, što je ustvari motivacija da se razviju
ili održe stavovi i ponašanje koji su usklađeni sa
načinom na koji drugi ljudi vide pojedinca. g
j
Stvarni lični koncept nije statička percepcija, već je
pod uticajem prošlog iskustva i događaja, kao i per-
cepcije toga kakav bi budući lični imidž mogao da
bude i šta pojedinac smatra idealnim imidžom koji
treba da dostigne (Schiffman, Kanuk, 2000, str. 212.). Lični koncept je posebno dinamičan u vreme pro-
mena uloga osobe, kao npr. student-zaposlena oso-
ba (Maričić, 2008, str. 389). Istraživanja su potvrdila
značaj iskustva u oblikovanju podudarnosti između
imidža potrošača i brenda. Pokazalo se da ova podu-
darnost raste sa povećavanjem iskustva sa brendom,
tj. postoji jasan linearan odnos. Ukoliko ispitanici po-
seduju brend, pokazalo se da postoji veća podudar-
nost između njihovog imidža i imidža brenda (Leary,
Tangey, 2003, str. 112). 2. Lični koncept i potrošnja Kako je
naveo Markin, proizvod koji potrošač vidi kao nekon-
zistentan sa sopstvenim imidžom stvara kognitivnu
disonancu koju on nastoji da reši dovođenjem ovih
nekonzistentnosti u balans (Hamilton, Sun, 2009, str. 12). U tržišnom kontekstu, potreba za ličnom konzi-
stentnošću je posrednički faktor koji utiče na stavove,
satisfakciju i i lojalnost prema brendu (Kressman et. al, 2006, str. 295). Motiv samopoštovanja predstavlja
tendenciju da se dostigne određeno željeno stanje ili
standard vezan za ličnost. Motivi samokonzistentno-
sti i samopoštovanja mogu biti saglasni, ali i konflik-
tni. Primera radi, određeni muškarac može imati veli-
ku želju za posedovanjem brzog sportskog automobila
u skladu sa motivom samopoštovanja, ali da se oseća Sirgy (et al, 1991, str. 363) je ovu podudarnost
između ličnog koncepta potrošača i imidža brenda
nazvao „lična podudarnost“ (eng. self-congruency),
na osnovu čega je oblikovan čuveni „model lične po-
dudarnosti“, koji je od svoje pojave u fokusu mnogo-
brojnih marketinških istraživanja”. Pored studije koju
je realizovao Sirgy, mnoge studije replike su pružile
dokaze za potvrdu hipoteze podudarnosti između lič-
nog koncepta/imidža potrošača i imidža brenda, koju
je Sirgy i postavio, a prema kojoj „potrošači imaju po-
zitivne stavove i kupovne namere prema brendovima
koje percipiraju kao podudarne sa njihovim ličnim
konceptom tj. imidžom, i obrnuto, imaju manje po-
voljne stavove i kupovne namere prema brendovi-
ma kod kojih je ova podudarnost mala ili je nema“
(Graeff, 1996, str. 28). Sirgy-ev model je dodatno ra-
zrađen, posebno u novijm studijama kojima se suge- 2002, str. 67). To se dešava zbog toga što je potrošnja
jedan od načina na koji ljudi sebe iskazuju, te su su
skloni da modifikuju svoj stvarni lični koncept i da ga
idealizuju. Ovo je naročito naglašeno kod tzv. statu-
sne i javno upadljive potrošnje, kojom nastoje da se
impresioniraju drugi ljudi. Sa druge strane, periferni
proizvodi (npr. prašak za posuđe, šećer, imalin itd.)
se veoma retko kupuju na osnovu imidža. Međutim i
način potrošnje perifernih proizvoda je vrlo diskuta-
bilan, u zavisnosti od toga koliko je tu kategoriju za-
hvatio trend brendiranja. Naime, “...marketing može
u svaki proizvod da utisne simboličke vrednosti, bez
obzira da li se radi o perifernim proizvodima ili ne. Npr. kukuruznim pahuljicama je pomoću simbolič-
kog oglašavanja kreiran imidž, a nemaju ništa više od
običnog hleba koji ima višestruko nižu cenu, ali ga
nije toliko zahvatio trend brendiranja”. 2. Lični koncept i potrošnja Naime, i u ranijim
istraživanjima je utvrđeno da sklad između ličnih ka-
rakteristika brenda i ličnih karakteristika potrošača
može imati značajne implikacije na stavove o brendu
i ponašanje potrošača (Aaker, 1999, str. 45). U zavi-
snosti od situacionih uslova, specifična ličnost bren-
da može biti efektivnija nego neka drugačija ličnost
brenda (Kromher et al, 2009). Konkurentska prednost
je na strani onih kompanija koje uspeju da kreiraju
veću vrednost za svoje ciljne potrošače (Stanković,
2009, str. 97). Zbog jasnih dokaza da ličnost brenda
može predstavljati održivo diferencirajuće sredstvo na
zasićenom tržištu, ovoj temi se danas posvećuje velika
pažnja, posebno u istraživanjima. Marketari moraju
da pronađu načine da adekvatno povežu brend sa lič-
nim karakteristikam potrošača ili ličnim konceptom. Cilj je da se naučni rezultati pretoče u metode koji će
služiti u praktične svrhe i omogućiti efektivno uprav-
ljanje ličnošću brenda, sa mogućnostima predviđanja 2. Lični koncept i potrošnja Pokazalo se da
su ljudi koji imaju izražen BESC više materijalistički
nastrojeni nego u suprotnom slučaju, imaju izražen
sopstveni monitoring i više su pod uticajem situaci-
onih faktora. Isto tako, ispoljavaju povoljnije stavove
prema brendovima koji snažno doprinose izgradnji
jakog ličnog identiteta (npr. nacionalni brendovi i do-
bro pozicionirani lokalni brendovi), nasuprot bren-
dovima koji imaju slabije diferenciranu marketing
strategiju (npr. privatni brendovi). Osobe sa visokim
nivoom BESC ispoljavaju znatno povoljnije stavove
i evaluaciju omiljenih brendova, jer je kod njih jače
izražena tendencija da te brendove uključe u lični
koncept. Ne samo da su stavovi pod uticajem BESC
koncepta, već i lojalnost prema brendovima. Rezul-
tati istraživanja sprovedeni na elektronskim kućnim
uređajima su pokazali da su osobe sa visokim nivoom
BESC sklone da čekaju duže da bi kupili novi proizvod
koji nosi njihov omiljeni brend, nego da kupe alterna-
tivni brend sa željenom inovacijom. Mnogi istraživači su se bavili efektima uticaja lič-
ne podudarnosti na ponašanje potrošača. Interakcija
imidža/ličnosti brenda i ličnog koncepta potrošača,
kao i uticaj te interakcije na percipirane performanse
brenda i kupovno ponašanje potrošača predstavlja po-
seban aspekt brend menadžmenta. Veliki broj studija
je pokazao da visok nivo podudarnosti ima pozitivan
efekat na stavove prema brendu, preferencije, evalua-
ciju, kupovne namere, stvarno ponašanje, satisfakciju i
lojalnost prema brendu. Poslednji trend u modernom
brendingu i jeste primena rezultata istraživanja koja
se bave otkrivanjem neopipljive i apstraktne prirode
brendova, te primena ovog znanja na razvoj strategije
pozicioniranja brendova (Keller, 2008, str. 392). p
j
(
)
Treba istaći da se veliki broj studija fokusirao na
podudarnost između ličnog koncepta potrošača i op-
šteg imidža brenda, a ne podudarnost sa ličnim karak-
teristikama brenda. Neka istraživanja su imala relativ-
no mali emprijiski dokaz o uticaju na performanse,
što može da se objasni time što podudarnost izme-
đu opšteg imidža brenda i ličnosti potrošača možda
i nije toliko značajna za formiranje stavova potrošača
i kupovnih odluka. Ističe se da je za istraživanja ovog
tipa ličnost brenda mnogo relevantnija konstrukcija
od opšteg imidža, s obzirom na to da se imidž bren-
da odnosi i na funkcionalne atribute koje potrošači
toliko ne dovode u korelaciju sa sopstvenim koncep-
tom kao simboliku. Ličnost brenda se odnosi na lične
karakteristike, što je bliža veza sa ličnošću potrošača
nego funkcionalni atributi brenda. 2. Lični koncept i potrošnja Bez
sumnje, ljudi imaju sklonost da stvaraju konekciju sa
više brendova, isto kao što pojedinac ima više različi-
tih važnih osoba u sopstvenom životu. Pokazalo se da
su ljudi koji imaju izražen BESC više materijalistički
nastrojeni nego u suprotnom slučaju, imaju izražen
sopstveni monitoring i više su pod uticajem situaci-
onih faktora. Isto tako, ispoljavaju povoljnije stavove
prema brendovima koji snažno doprinose izgradnji
jakog ličnog identiteta (npr. nacionalni brendovi i do-
bro pozicionirani lokalni brendovi), nasuprot bren-
dovima koji imaju slabije diferenciranu marketing
strategiju (npr. privatni brendovi). Osobe sa visokim
nivoom BESC ispoljavaju znatno povoljnije stavove
i evaluaciju omiljenih brendova, jer je kod njih jače
izražena tendencija da te brendove uključe u lični
koncept. Ne samo da su stavovi pod uticajem BESC
koncepta, već i lojalnost prema brendovima. Rezul-
tati istraživanja sprovedeni na elektronskim kućnim
uređajima su pokazali da su osobe sa visokim nivoom
BESC sklone da čekaju duže da bi kupili novi proizvod
koji nosi njihov omiljeni brend, nego da kupe alterna-
tivni brend sa željenom inovacijom. određenih akcija, a brojna istraživanja su pokazala da
lična podudarnost utiče na ponašanje i izbor brendo-
va potrošača direktno i indirektno (Sirgy, et al, 1997,
str. 229). postati sastavni deo nečijeg ličnog koncepta (prema:
Sprot et al, 2006, str. 7). Ovu ideju je detaljnije obja-
snio Belk (1998, str. 139), baveći se proširenim ličnim
konceptom (eng. extended-self), oformljenim na bazi
posedovanja stvari. Sprott i sar. su proširili ovu bazič-
nu ideju uključujući brendove u potrošačev lični kon-
cept. Oni definišu BESC kao opštu sklonost osobe da
inkorporira važne brendove u lični koncept. Drugim
rečima, ta varijabla odražava individualne razlike u
sklonosti potrošača da uključe brendove u razmatra-
nje samih sebe. Njihov rad se takođe bazirao na istra-
živanjima koja su sproveli Cross (et al, 2000, str. 791)
čime je ustanovljeno da ljudi u manjem ili većem ste-
penu uključuju druge važne osobe tj. referentne grupe
u njihov lični koncept. Treba napomenuti da se ova
konstrukcija odnosi na utvrđivanje opšte sklonosti
osobe da uključi brendove u lični koncept, za razliku
od bliskih koncepata koji se uglavnom bave istraživa-
njem povezanosti osobe sa specifičnim brendom. Bez
sumnje, ljudi imaju sklonost da stvaraju konekciju sa
više brendova, isto kao što pojedinac ima više različi-
tih važnih osoba u sopstvenom životu. 112 Slađana Starčević 2. Lični koncept i potrošnja Na taj način je potvrđeno da
je lični identitet zaista iskustveni koncept, kao što je
navedeno u uvodnom delu izlaganja. • Podudarnost sa idealnim društvenim ličnim
konceptom – odnosi se na stepen sličnosti iz-
među potrošačevog idealnog društvenog ličnog
imidža i imidža tipičnog potrošača. Takva podu-
darnost je u skladu sa potrošačevom potrebom za
društvenim odobravanjem. Lična podudarnost ne mora biti značajan faktor
pri kupovini i evaluaciji svih kategorija proizvoda. Potrošnja nekih proizvoda je znatno više upadljiva tj. društveno vidljiva, te je vrlo važno kakav imidž nose
sa sobom ti proizvodi i na koji način prezentuju njiho-
ve korisnike. Stoga se uočavaju razlike pri ispoljavanja
ličnog koncepta kod: Sprott (et al, 2006, str. 4). su pokazali da se poje-
dinci razlikuju prema tendenciji da uključe važne
brendove u njihov lični koncept. Isti autori su razvili
merilo ove diferencirajuće varijable koja je nazvana
„brand-extended self-construal“ (BESC), u kontek-
stu proširenja sopstvenog ličnog koncepta pomoću
brendova. James je još 1980. godine u svojoj knjizi
„Principi psihologije“ istakao da „posedovanje“ može • Privatno konzumiranih brendova; • Javno konzumiranih brendova. Pokazalo se da je evaluacija javno konzumiranih
brendova mnogo više pod uticajem podudarnosti iz-
među idealnog ličnog koncepta i imidža/ličnosti bren-
da, dok kod privatno konzumiranih brendova idealni
i stvarni lični koncept imaju isti značaj (O’Cass, Frost, Lični koncept kao značajna determinanta izbora brendova i ponašanja potrošača u kupovini 111 111 postati sastavni deo nečijeg ličnog koncepta (prema:
Sprot et al, 2006, str. 7). Ovu ideju je detaljnije obja-
snio Belk (1998, str. 139), baveći se proširenim ličnim
konceptom (eng. extended-self), oformljenim na bazi
posedovanja stvari. Sprott i sar. su proširili ovu bazič-
nu ideju uključujući brendove u potrošačev lični kon-
cept. Oni definišu BESC kao opštu sklonost osobe da
inkorporira važne brendove u lični koncept. Drugim
rečima, ta varijabla odražava individualne razlike u
sklonosti potrošača da uključe brendove u razmatra-
nje samih sebe. Njihov rad se takođe bazirao na istra-
živanjima koja su sproveli Cross (et al, 2000, str. 791)
čime je ustanovljeno da ljudi u manjem ili većem ste-
penu uključuju druge važne osobe tj. referentne grupe
u njihov lični koncept. Treba napomenuti da se ova
konstrukcija odnosi na utvrđivanje opšte sklonosti
osobe da uključi brendove u lični koncept, za razliku
od bliskih koncepata koji se uglavnom bave istraživa-
njem povezanosti osobe sa specifičnim brendom. 3. Uticaj lične podudarnosti na
izbor brendova i ponašanje
potrošača u kupovini Predviđanje ponašanja potrošača je veoma važna
tema istraživanja u marketingu. Costa i McCrae (pre-
ma: Gountas, Mavondo, 2003, str. 3) su istakli da je
lični koncept veoma uticajna komponenta u dinamič-
kom procesu odlučivanja potrošača i preduzimanja tržišnih rezultata. Međutim, za sada je mali broj studi-
ja emprijski istražio uticaj interakcije između ličnosti
brenda i ličnosti potrošača na performanse, što znači
da je ovo dosta otvoreno područje za istraživanje. ti društveni identiteti bitni u različitim situacijama i
sve zavisi od njihove istaknutosti u tom trenutku, što
opredeljuje i koji su „preferirani brendovi“ u datoj si-
tuaciji (Reed, 2002, str. 1087). j
p
j
j
Istraživanja su pokazala da je lični koncept uzroč-
nik pozitivnih i negativnih stavova koji značajno uti-
ču na akciju i kupovno ponašanje potrošača. Naime,
mnogi modeli o stavovima su nastojali da objasne
izvore i uticaje na akcije pojedinca kombinovanjem
afektivne, kognitivne i konativne komponente (Goun-
tas, Mavondo, 2003, str. 3). Međutim, utvrđeno je da u
formiranju stavova prethodno ulogu odigraju ličnost i
lični koncept, sa čime se povezuje sklonost ka odbra-
ni ega, potreba za spoznajom, želja za akcijom, dosti-
gnućima i sigurnošću (Claibourne, Sigry, 1990, str. 5). O’Brien i Sanchez (1976, str. 608) u svojoj studiji su-
gerišu da se analiza ličnog koncepta može koristiti za
identifikovanje motiva potrošača pri izboru proizvoda
i usluga. Oni su svojim istraživanjem pružili značajne
dokaze o povezanosti između stvarnog/idealnog lič-
nog koncepta i percepcije imidža različitih proizvoda
i usluga (O’Brien, T. and Sanchez, H., 1976, str. 608). U suštini, veliki broj istraživanja se bavio ispitivanjem
veze između motivacije i ličnog koncepta, jer se po-
kazalo da kombinacija ova dva konstrukta mnogo
sadržajnije objašnjava ponašanje potrošača. Smatra
se da motivacija da se postigne određeni cilj utiče na
percepciju ličnog koncepta od strane potrošača. Veći
broj autora je potvrdio pretpostavku da su motivi i ra-
skorak između stvarnog i idealnog ličnog koncepta u
pozitivnoj korelaciji sa preduzimanjem određene ak-
cije (Gountas,J. and Mavondo, F., 2003, str. 3). Jedna od čestih tema istraživanja jeste uticaj lične
podudarnosti na satisfakciju potrošača, a studije su se
uglavnom fokusirale na ponašanje nakon kupovine. Primera radi, Chon, Jamal i Goode su istraživali pod-
ručje turističkih usluga, polazeći od Sirgy-eve teorije
podudarnosti (Chon, 1990, str. 123). Oni su ustanovili
da postoji značajna pozitivna korelacija između ličnog
koncepta potrošača i ličnosti brenda, kao i evidentan
uticaj ove podudarnosti na satisafakciju potrošača. Park i Lee (2005, str. 3. Uticaj lične podudarnosti na
izbor brendova i ponašanje
potrošača u kupovini 39) su sproveli istraživanja na ve-
likom broju proizvoda široke potrošnje i takođe došli
do istih rezultata. Isto tako, dokazano je da ova varijabla pozitivno
utiče i na lojalnost potrošača (Yi, La, 2002, str. 21). Kromher i sar. su se bavili ispitivanjem uticaja podu-
darnosti ličnosti potrošača i ličnosti brenda na lojal-
nost prema brendu, uključujući poverenje kao mo-
deracionu varijablu. Njihovi rezultati su pokazali da
stvarna lična podudarnost generalno ima jači efekat
na lojalnost prema brendu, nego idealna lična po-
dudarnost. Ovakvi nalazi se mogu objasniti time što
potrošači stavljaju poseban akcenat na familijarne
brendove i iskustva koja potvrđuju njihov lični kon-
cept, a nastoje da izbegavaju iskustva koja taj koncept
ugrožavaju. g
j
Međutim, neizbežan je zaključak da su u istraživa-
njima često izostavljene moderacione varijable koje
bolje razjašnjavaju pod kojim uslovima podudarnost
između imidža/ličnosti brenda i ličnog koncepta
potrošača ima pozitivan uticaj na evaluaciju perfor-
mansi brenda i stvarno ponašanje. Tako se pokazalo
da uključenost potrošača ima negativni moderacioni
efekat na odnos između stvarne/idealne lične podu-
darnosti i poverenja prema brendu. Drugačije rečeno,
za visoko uključene potrošače, podudarnost između
sopstvene ličnosti i ličnosti brenda je manje važna za
izgradnju njihovog poverenja u brend. Kao dalji za-
klučak se nametnulo shvatanje da idealna lična podu-
darnost potpuno gubi značaj kod uticaja na perfor-
manse brenda u slučaju visoke uključenosti potrošača,
dok je u ovom slučaju stvarna podudarnost mnogo
relevantnija (Kromher, H. et al., 2009). j
Dokazan je i pozitivan uticaj lične podudarnosti
na preferencije i buduće namere prema brendu. Sa-
svim je logična ideja da potrošači preferiraju i kupuju
brendove koji su slični tome kako oni vide sami sebe
ili kako bi voleli da se vide. Konzumiranje proizvoda
koji mogu poboljšati naročito društveni lični identi-
tet, može čak izmeniti potrošačeve percepcije o atri-
butima proizvoda. Efekat može biti toliko jak da eva-
luacija atributa tog proizvoda bude mnogo povoljnija
nego evaluacija atributa konkurentskih proizvoda koji
su realno kvalitetniji. Jedno od skorijih istraživanja na
ovu temu su realizovali Marchlewski i sar (2006, str. 12). na snažno pozicioniranim regionalnim brendo-
vima piva u Nemačkoj, a čija konzumacija snažno
utiče na pojačavanje društvenog identiteta i isticanje
pripadnosi pojedinca određenom regionu. Jasno se
pokazalo da je ocena atributa preferiranog piva koje
pojačava društveni identitet neuporedivo povoljnija u
uslovima kada potrošači znaju o kojem je pivu reč, u
odnosu na slepi test. Treba takođe istaći da su različi- Sirgy i sar. Lični koncept kao značajna determinanta izbora brendova i ponašanja potrošača u kupovini 113 Influence“, European Review of Social Psychology,
No.1, str. 195-228. hf 3. Uticaj lične podudarnosti na
izbor brendova i ponašanje
potrošača u kupovini su pokazali da lična podudarnost uti-
če na različite aspekte ponašanja potrošača (stavovi
prema brendu, kupovne namere, lojalnost prodajnom
mestu itd.) direktno i indirektno putem funkcionalne
podudarnosti, dok su Kressman i sar. demonstrirali
efekat lične podudarnosti na lojalnost prema brendu, Lični koncept kao značajna determinanta izbora brendova i ponašanja potrošača u kupovini 11 113 takođe direktno i indirektno putem funkcionalne po-
dudarnosti (Sirgy et al, 1991, str. 252; Kressman et al,
2006, str. 995). Funkcionalna podudarnost predstavlja
sklad između potrošačevih verovanja o atributima/ko-
ristima brenda i referentnih atributa ili idealnih atri-
buta koji čine kriterijum za ocenu određenog brenda. Sirgy tvrdi da iako i lična i funkcionalna podudarnost
utiču na stavove o brendu, lična podudarnost ima
suptilniju ulogu. To se dešava jer se lična podudar-
nost odvija na mnogo nižem nivou svesnosti, utičući
suptilno na evaluaciju funkcionalnih atributa, koja
se odvija na mnogo svesnijem nivou. Samim tim je
uticaj funkcionalne podudarnosti direktniji od uticaja
lične podudarnosti, te je na osnovu nje lakše predvi-
deti stavove potrošača. Međutim, kako je navedeno,
postoji veliki broj brendova koji se kupuju na osno-
vu simboličke komponente, bez preterane evaluacije
funkcionalnih atributa, tako da je svakako primaran
uticaj lične podudarnosti na izbor brendova. Istim
istraživanjima Sirgy i sar. potrvrđuju hipoteze da se na
osnovu lične podudarnosti mnogo bolje predviđaju
stavovi kada je reč o brendovima upadljive potrošnje
i kada je jedinstvenost brenda na visokom nivou, dok
se na osnovu funkcionalne podudarnosti stavovi bolje
predviđaju kada je visoka uključenost potrošača, što i
jeste logično (Sirgy et al, 1991, str. 258). U kontekstu
tipova proizvoda za koje se vrše istraživanja, pokazalo
se da skale koje se koriste za utvrđivanje lične podu-
darnosti imaju veću mogućnost utvrđivanja efekata
kada je reč o proizvodima upadljive potrošnje i gene-
ralno simboličkim kategorijama proizvoda. Vernett je
utvrdio da kategorija proizvoda veoma važan faktor u
procesu povezivanja/nepovezivanja ličnosti potrošača
i brenda, kao i pri postizanju efekta na preferencije
brenda (Van de Rijdt, 2008, str. 4). Do sličnih zaklju-
čaka se došlo na osnovu većeg broja istraživanja koje je obuhvatilo različite kategorije proizvoda kao što su
npr. automobili, nakit, putne destinacije, pića, modna
odeća itd. Zaključak Pregled istraživanja koja su se bavila ličnom podu-
darnošću ukazuje na to da su istraživači ovaj koncept
počeli da ispituju u brojnim područjima marketinga,
među kojima su sponzorstvo, potencijali ekstenzije
brenda, kobrendiranje, oglašavanje itd. što je dovelo
do veoma interesantnih zaključaka. Međutim, ovo je
područje u kojem postoji još dosta otvorenih tema za
istraživanje, posebno imajući u vidu da njegovo do-
bro poznavanje može značno poboljšati upravljanje
brendovima, posebno iz ugla njihovog pozicioniranja
i kreiranja strategije tržišnih komunikacija. U nared-
nim istraživanjima je veoma bitno voditi računa o
konceptualnim nedostacima prethodnih istraživanja,
posebno imajući u vidu da se utvrđivanje lične podu-
darnosti uglavnom sprovodi metodom samo-ocenji-
vanja, što neminovno unosi subjektivnu komponentu
u istraživanja i umanjuje preciznost rezultata. Sa dru-
ge strane, uočljiv je nedostatak praktičnih modela u
oblasti marketinga koji bi brend menadžerima omo-
gućili lakše profilisanje brenda i primenu rezultata
istraživanja u praktične svhe. Kada se generalno sa-
gledaju praktični modeli koji se koriste u svrhu pozi-
cioniranja brendova i kreiranja tržišnih komunikacija,
neminovno se nameće zaključak da se lični koncept,
ličnost brenda, pa čak i identitet brenda posmatraju
kao isuviše opšte kategorije. To znatno ograničava nji-
hovu pravu ulogu u oblikovanju strategije pozicioni-
ranja brenda, što znači da je ovo područje koje dodat-
no treba istraživati i dobijene rezultate na adekvatan
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Integrated Model of Self-Congruity and Functional 116 Slađana Starčević Summary Self-Concept as a Significant
Determinant of Brand Choice and
Consumer Buying Behaviour Slađana Starčević ation of brands, buying intentions, satisfaction and brand
loyalty. In this study, we have researched how has self-con-
cept, as a psychological construct, gained in importance in
the field of marketing research and practice. The concept
is analyzed simultaneously as a psychological and market-
ing construct. By presenting literature review, we have also
analysed the consequences of congruence between brand
image/brand personality and consumer self-concept on
consumer behavior and choice of brands. We have also
pointed out the significance and references connected with
the use of this concept for practical purposes in the realm
of brand management. The need to predict consumer behavior outcomes is con-
sidered to be a very important issue for marketers. Today,
one of the most popular psychological constructs in social
sciences and marketing is the self-concept, as the total sum
of ideas, thoughts and feelings through which individual
can describe themselves in regards to other individuals
in socially determined environment. The importance of
self-concept in predicting consumer behavior and choice
of brands has been recognized by many researchers. People tend to maintain and reinforce their self-concept by
consumption of brands that have an image and personal-
ity in accordance to their own self-concept. Many studies
have confirmed that high level of congruency between
brand image/personality and consumer self-concept have
positive influence on brand attitudes, preferences, evalu- Key words: self-concept, brand image, brand personality,
self-congruity, consumer behavior Kontakt: Slađana Starčević
Profile South and Eastern Europe
Petra Lekovića 24a, Beograd Lični koncept kao značajna determinanta izbora brendova i ponašanja potrošača u kupovini 117 Lični koncept kao značajna determinanta izbora brendova i ponašanja potrošača u kupovini 117
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Prevalence of human papillomavirus genotypes among women with cervical cancer in Ghana
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Infectious agents and cancer
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cc-by
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© 2016 Awua 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. Awua et al. Infectious Agents and Cancer (2016) 11:4
DOI 10.1186/s13027-016-0050-4 Awua et al. Infectious Agents and Cancer (2016) 11:4
DOI 10.1186/s13027-016-0050-4 Abstract Background: Human Papillomavirus (HPV) infections have been shown to be a necessary risk factor for the
development of cervical cancer. However, HPV genotype distribution varies geographically, both in type and
relative prevalence. In order to ensure a successful introduction of available vaccines, there is the need to identify
pre-vaccination HPV genotype prevalence in Ghana and the extent of single and multiple-infections. Methods: Paraffin-embedded cervical tissues of 256 confirmed cervical cancer cases diagnosed at the Korle-Bu
Teaching Hospital during the period January 2004 to December 2006 were selected after hematoxylin and eosin
staining and confirmation. Following a heat-proteinase K-based tissue lysis, HPV was detected and typed by a
nested-multiplex PCR assay using an E6/E7 consensus primer and type-specific primers. Results: Of the 256 cases, 230 (89.8 %, 95 % CI 85.7–93.4 %) were positive for HPV DNA. HPV18 (47.4 %), HPV59
(42.2 %), HPV45 (37.4 %) and HPV16 (9.0 %) were the four common HPV genotypes detected. A total of 110 (47.8 %)
of the 230 HPV DNA positive tissues, were infected by a single HPV genotype while the other 120 (52.2 %) were
infected by multiple HPV genotypes. A significant association was determined between each of the following HPV
genotypes and multiple-infection; HPV18 (OR = 6.97; 95 % CI, 3.89–12.50), HPV59 (OR = 9.56; 95 % CI, 5.57–20.02) and
HPV45 (OR = 1.94; 95 % CI, 1.12–3.35). Conclusion: The prevalence of the following high risk HPV genotypes (HPV18, HPV59, HPV45) were relatively high
among the cases of cervical cancers reported at this hospital in Ghana during the study period. Additionally, there
was a high frequency of HPV multiple-infections among these cases. Keywords: Nested PCR, Multiplex PCR, Human Papillomavirus, Cervical cancer, Paraffin-embedded tissues, Ghana [2]. At the same hospital, a 10 year (1991–2000) study of
cancer mortality pattern revealed that cervical cancer
was one of the four leading causes of cancer deaths (Age
Standardized Cancer Ratio of 8.74 %) in females [3]. Prevalence of human papillomavirus
genotypes among women with cervical
cancer in Ghana A. K. Awua1,2*, S. T. Sackey1, Y. D. Osei1, R. H. Asmah3 and E. K. Wiredu3,4, A. K. Awua1,2*, S. T. Sackey1, Y. D. Osei1, R. H. Asmah3 and E. K. Wiredu3,4,5 * Correspondence: a_awua@yahoo.com
1Department of Biochemistry, Cell and Molecular Biology, University of
Ghana, Legon, Accra, Ghana
2Cellular and Clinical Research Centre, Radiological and Medical Science
Research Institute, Ghana Atomic Energy Commission, Accra, Ghana
Full list of author information is available at the end of the article Background The West African region was estimated to bear the sec-
ond highest burden of cervical cancer, with a mortality
to incidence ratio of 81.2 % and cervical cancer was esti-
mated to be the most frequent cancer among women in
Ghana as at the time of this study [1]. The age standar-
dised ratio (ASR) of 39.5 per 100,000 women [1]. An
earlier study at the Korle-Bu Teaching Hospital also re-
ported that 58.3 % of gynaecological cancers seen at the
hospital in the year 2000 were cases of cervical cancer A number of studies have shown that persistent infec-
tions with certain human Papillomavirus (HPV) geno-
types known as high risk HPV type (HPV-16, -18, -31,
-35, -39, -45, -51, -52, -56, -58, -59 and -68) are neces-
sary in the aetiology of cervical cancer. These high risk
HPV genotypes have been shown to be associated with
greater than 99.0 % of all cervical cancer cases [4–7]. Of
these high risk HPV genotypes HPV16 and HPV18 are
the two prevalent HPV types in cervical carcinoma glo-
bally and are associated with approximately 60.0 % and
10.0 % of cervical cancer cases respectively [8–10]. * Correspondence: a_awua@yahoo.com
1Department of Biochemistry, Cell and Molecular Biology, University of
Ghana, Legon, Accra, Ghana
2Cellular and Clinical Research Centre, Radiological and Medical Science
Research Institute, Ghana Atomic Energy Commission, Accra, Ghana
Full list of author information is available at the end of the article Page 2 of 9 Awua et al. Infectious Agents and Cancer (2016) 11:4 Page 2 of 9 However, epidemiological studies have shown a global
geographical variation in the type specific and relative
HPV prevalence among different populations; these
range between 2 and 44 % [11]. Fortunately, there are
indications of high efficacy of available vaccines against
HPV infections and a greater possibility of preventing
cervical cancer. However, it not clear how the population
specific HPV genotype distribution, extent of multiple-
infections
and
the
HPV
types
involved
in
these
multiple-infections will change following a successful
implementation of these efficacious vaccines. Evidence
is building on the occurrence of cross protection against
non-vaccine HPVs and the possibility of the occurrence of
HPV genotype replacements among vaccinated popula-
tions. In order to determine the occurrence of such
changes, knowledge of the pre- and post-vaccination HPV
genotype distribution is very important. microfuge tube for DNA extraction. Background The first and third
sections were stained with hematoxylin and eosin (H/E)
and examined for cancerous tissues by 2 Consultant
Pathologist. The presence of cancerous tissue in the
stained sections confirmed that the middle section used
for DNA extraction actually contained cancer tissue. 256
samples, consisting of 10 cases of carcinoma in situ
(CIS), 19 cases of invasive adenocarcinoma (IAC) and
227 cases of invasive squamous cell carcinoma (ISCC)
were found to have contain cancerous tissues and so
were used for subsequent analysis. y
DNA was extracted from the 256 samples, as described
by Dabic’ et al., [13] with slight modifications. In brief,
10 μm section was incubated in 250 μL of extraction
buffer (consisting of 1 mg/mL Proteinase K in 50 mM
Tris-HCl pH 8, 1 mM EDTA and 0.5 % Tween-20) for
16 h at 56OC. The proteinase K was thereafter inactivated
by heating at 100OC for 5 min. The tubes were allowed to
cool to room temperature after which the tubes were
centrifuged at 5000xg for 5 min. The tissue lysates were
transferred for storage at -20 °C as aliquots of 70 μL. Positive and negative controls (previously confirmed can-
cerous and non-cancerous cervical tissues respectively),
were used for the DNA extraction. However, empirical data on the overall and HPV geno-
type specific prevalence in cases of cervical cancers in
Ghana were very few at the time of this study. Addition-
ally, and in order to evaluate the potential benefits of an
introduction of HPV vaccination in Ghana, this study
was designed to detect, genotype and determine the
extent of multiple HPV infection using archival formalin-
fixed
paraffin-embedded
cervical
tumour
specimen
collected from the Pathology Department of Korle-Bu
Teaching Hospital during the years 2004–2006. For the
detection of HPV, a nested-multiplex PCR method that
had been shown to be very sensitive and specific, with de-
tection rates of between 91.8 % for cervical intraepithelial
neoplasia (CIN I) and 99.3 % for CIN III was used [12]. Detection and genotyping of HPV by nested-multiplex
PCR The method and primer sets used for the nested-multiplex
PCR were as described by Soltar et al., [14] with slight
modification. Its sensitivity was as higher as that of the
MY/GP primer assay (102–101 viral copy detection) [14]
and an initial adoption of this method in our laboratory
showed it was effective for the purpose of genotyping the
high risk HPVs [15]. This assay targets the detection of 18
HPV genotypes (6/11, 16, 18, 31, 33, 35, 39, 42, 43, 44, 45,
51, 52, 56, 58, 59, 66 and 68) with a sensitivity comparable
to that of the MY/GP assay but higher than those of the
MY09-MY11 and GP5 + -GP6+ assays which amplifies the
L1 region of the HPV genome. Briefly, HPV DNA in the
lysate was amplified using a single consensus forward pri-
mer, GP-E6-3 F [GGGWG KKACT GAAAT CGGT], and
two
consensus
reverse
primers
GP-E7-5B
[CTGAG
CTGTC ARNTA ATTGC TCA] and GP-E7-6B – [TCCTC
TGAGT YGYCT AATTG CTC]. The four second round
primer-cocktail-sets, as shown in Table 1, were used for the
genotyping. Both first and second round PCRs were per-
formed in a final volume of 25 μL and each PCR mixture
contained 50 mM KCl, 9 mM Tris-HCl (pH 9.0), 1 %
Triton-X100, 2.0 mM MgCl2, 0.2 mM of each dNTP, 320
nM of each of the primers and 1.25 U of Taq polymerase. The amplifications were carried out using a thermal cycler
(Applied Biosystems 1720) with the following parameters: Method Sample collection and DNA extraction The Korle-Bu Teaching Hospital is one of the tertiary re-
ferral centre for persons or specimens with any form of
suspected
malignancy
in
Ghana. Paraffin-embedded
formalin-fixed cervical tissues of women who were re-
ferred to the Pathology Department of the Korle-Bu
Teaching Hospital, Accra, between January 2004 and
December 2006 and diagnosed histologically with high
grade precursor lesion (CIN III, high grade squamous
Intraepithelial lesion (HSIL), or carcinoma in situ) or
cervical cancer (adenocarcinoma, adenosquamous car-
cinoma or any form of squamous cell carcinoma), were
selected for this study. Cases diagnosed as adenocarcin-
oma and adenosquamous were grouped together as
invasive adenocarcinoma (IAC), any form of squamous
cell carcinoma were grouped as invasive squamous cell
carcinoma (ISCC) and all forms of carcinoma in situ
were grouped as carcinoma in situ (CIS). Each tissue block was sectioned to obtain three 9-μm
sections, each with a separate and sterile microtone
blade. The middle section was stored in a 2 mL sterile For the first round PCR, using 5.0 μL or 1 μL of lysate,
an initial denaturation at 94 °C for 4 min was followed Awua et al. Infectious Agents and Cancer (2016) 11:4 Page 3 of 9 by 40 cycles of denaturation at 94 °C for 1 min, anneal-
ing at 40 °C for 2 min and an extension at 72 °C for
2 min. A single final extension at 72 °C for 9 min was
roun
used
tails
Table 1 Sequences of synthetic oligonucleotide primers used for HPV ge
Primer cocktail
HPV genotype primer
Size of a
Consensus primers
GP-E6-3 F
GP-E7-5B
GP-E7-6B
Cocktail 1
HPV16f
457
HPV16r
HPV18f
323
HPV18r
HPV31f
263
HPV31r
HPV59f
215
HPV59r
HPV45f
151
HPV45r
Cocktail 2
HPV33f
398
HPV33r
HPV6_11f
334
HPV6_11r
HPV58f
274
HPV58r
HPV52f
229
HPV52r
HPV56f
181
HPV56r
Cocktail 3
HPV35f
358
HPV35r
HPV42f
277
HPV42r
HPV43f
219
HPV43r
HPV44f
163
HPV44r
Cocktail 4
HPV68f
333
HPV68r
HPV39f
280
HPV39r
HPV51f
223
HPV51r
HPV66f
172
HPV66r
F, f forward, r reverse, B back
Single letter code: W = A/T; K = G/T; R = A/G; Y = C/T; N = A/C/G/T; X = unknown. Method (Adapted Table 1 Sequences of synthetic oligonucleotide primers used for HPV genotyping
Primer cocktail
HPV genotype primer
Size of amplicon (bp)
Sequence 5′to 3′)
Consensus primers
GP-E6-3 F
GGGWG KKACT GAAAT CGGT
GP-E7-5B
CTGAG CTGTC ARNTA ATTGC TCA
GP-E7-6B
TCCTC TGAGT YGYCT AATTG CTC
Cocktail 1
HPV16f
457
CACAG TTATG CACAG AGCTGC
HPV16r
CATAT ATTCA TGCAA TGTAG GTGTA
HPV18f
323
CACTT CACTG CAAGA CATAG A
HPV18r
GTTGT GAAAT CGTC GTTTT TCA
HPV31f
263
GAAAT TGCAT GAACT AAGCT CG
HPV31r
CACAT ATACC TTTGT TTGTC AA
HPV59f
215
CAAAG GGGAA CTGCA AGAAA G
HPV59r
TATAA CAGCG TATCA GCACC
HPV45f
151
GTGGA AAAGT GCATT ACAGG
HPV45r
ACCTC TGTGC GTTCC AATGT
Cocktail 2
HPV33f
398
ACTAT ACACAACATT GAACT A
HPV33r
GTTTT TACAC GTCAC AGTGC A
HPV6_11f
334
TGCAA GAATG CACTG ACCAC
HPV6_11r
TGCAT GTTGT CCAGC AGTGT
HPV58f
274
GTAAA GTGTG CTTAC GATTG C
HPV58r
GTTGT TACAG GTTAC ACTTG T
HPV52f
229
TAAGG CTGCA GTGTG TGCAG
HPV52r
CTAA TAGTT ATTTCA CTTAA TGGT
HPV56f
181
GTGTG CAGAG TATGT TTATT G
HPV56r
TTTCT GTCAC AATGC AATTG C
Cocktail 3
HPV35f
358
CAACG AGGTA GAAGA AAGCA TC
HPV35r
CCGAC CTGTC CACCG TCCAC CG
HPV42f
277
CCCAA AGTAG TGGTC CCAGT TA
HPV42r
GATCT TTCGT AGTGT CGCAG TG
HPV43f
219
GCATA ATGTC TGCAC GTAGC TG
HPV43r
CATGAAACTG TAGAC AGGCC AAG
HPV44f
163
TAAAC AGTTA TATGT AGTGT ACCG
HPV44r
TATCA GCACG TCCAG AATTG AC
Cocktail 4
HPV68f
333
GCAGAAGGCA ACTAC AACGG
HPV68r
GTTTA CTGGT CCAGC AGTGG
HPV39f
280
GACGACCACT ACAGC AAACC
HPV39r
TTATG AAATC TTCGT TTGCT
HPV51f
223
GAGTA TAGAC GTTAT AGCAG G
HPV51r
TTTCG TTACG TTGTC GTGTA CG
HPV66f
172
TTCAG TGTAT GGGGC AACAT
HPV66r
AAACA TGACC CGGTC CATGC
F, f forward, r reverse, B back
Single letter code: W = A/T; K = G/T; R = A/G; Y = C/T; N = A/C/G/T; X = unknown. (Adapted from Soltar et al., [14]) F, f forward, r reverse, B back
Single letter code: W = A/T; K = G/T; R = A/G; Y = C/T; N = A/C/G/T; X = unknown. (Adapted from Soltar et al., [14]) by 40 cycles of denaturation at 94 °C for 1 min, anneal-
ing at 40 °C for 2 min and an extension at 72 °C for
2 min. A single final extension at 72 °C for 9 min was
performed before a soaking step at 4 °C. Method For the second by 40 cycles of denaturation at 94 °C for 1 min, anneal-
ing at 40 °C for 2 min and an extension at 72 °C for
2 min. A single final extension at 72 °C for 9 min was
performed before a soaking step at 4 °C. For the second round PCR, 2.0 μL of the first round PCR product were
used as the template DNA with each of the four cock-
tails of type specific primers for genotyping (Table 1). The cycling parameters were as follows: an initial Awua et al. Infectious Agents and Cancer (2016) 11:4 Page 4 of 9 Fig. 1 Distribution of diagnosed cervical cancer cases denaturation at 94 °C for 4 min was followed by 35 cycles
of denaturation at 94 °C for 30 s, annealing at 56 °C for
30 s, and an extension at 72 °C for 45 s. This was
followed by a single final extension at 72 °C of 4 min,
before a soaking step at 4 °C. For each round of PCR
reaction, HPV16 or/and HPV18 genome in plasmid
DNA as well as a sample positive for HPV used in the
optimisation of PCR were used as positive controls while
nuclease free water and DNA extracts form the non-
cancerous tissues was used as a negative control. A sample
each, found to be HPV negative or positive, were subse-
quently used as additional negative and positive control,
respectively. Following the second round PCR, 8.0 μL of the prod-
ucts were resolved on a 2 % agarose gel stained with
0.001 mg/mL ethidium bromide. The electrophoresis
was carried out in 1X Tris Acetate EDTA (TAE) buffer
at 100 V for 1 h and the gel photographed over a UV
trans-illuminator. The molecular weights of the resolved
PCR products were used to determine the genotypes of
HPV detected for each sample according to the expected
weight for each primer (Table 1). Statistical analysis Data were managed and analysed with the Epi Info stat-
istical software, version 3.3 (CDC, Atlanta, USA). The
number and proportion of cases of carcinoma in situ
(CIS), invasive adenocarcinoma (IAC) and invasive squa-
mous cell carcinoma (ISCC) were determined. Further-
more, the number and proportions of detected HPV
genotypes, stratified by mode of infection (overall, single
and multiple infections), type of cervical cancer case and
age of the women were determined and presented in
frequency tables. The association between each HPV
genotype and multiple infection status was determined
by an analysis of odds ratio. HPV18 (47.4 %), HPV59 (42.2 %), HPV45 (37.4 %) and
HPV16 (10.0 %) (Table 2). Single infections were ob-
served among 110 (47.8 %) of the 230 HPV DNA posi-
tive cases and the same four HPV genotypes were the
commonest single infecting genotypes but in a different
order. This was as follows, HPV45 (29.4 %), HPV18
(23.95 %), HPV59 (15.6 %) and HPV16 (9.2 %). Eight of
the single infections were solely of low-risk HPV types
(Table 2), these were; 4 ISCC cases with HPV44, 1 CIS
and 1 ISCC case with HPV6_11 and 2 ISCC cases with
HPV42. Multiple-infections occurred in 120 (52.2 %) of
the 230 DNA positive cases. Double infections were
observed in 90 (39.1 %), triple infection in 28 (12.0 %)
and quadruple infections in 5 (2.2 %) of the 230 HPV
positive cases (Table 3). Double infections involving
HPV18 were the commonest. Those with HPV18 and
HPV59 occurred in 30 (13.0 %) cases, those with HPV18
and HPV16 occurred in 3 (1.3 %) and those with HPV18
and HPV45 occurred in 2 (0.9 %) cases. Furthermore,
the commonest triple HPV infections were detected in
17 (7.3 %) cases and these involved HPV-18, -45 and
-59. All the other 9 triple infections were each detected
once while all the 5 quadruple infection involved HPV-
16, -18, -45 and -59 (Table 3). Results Round 2 amplification products were resolved at 100 V for 1 h on a 2 % agarose
gel stained with 0.001 mg/mL of ethidium bromide and photographed under UV illumination. The HPVs were identified based on the molecular
weight of the amplification products for each are shown below the gel. Lane -ve ⇒HPV negative control; Lane MW ⇒Bands of the 100 base pair
molecular weight maker; Lane + ve ⇒HPV16 and HPV18 DNA positive control Fig. 2 Detected type specific HPV DNA in multiple infections. Round 2 amplification products were resolved at 100 V for 1 h on a 2 % agarose
gel stained with 0.001 mg/mL of ethidium bromide and photographed under UV illumination. The HPVs were identified based on the molecular
weight of the amplification products for each are shown below the gel. Lane -ve ⇒HPV negative control; Lane MW ⇒Bands of the 100 base pair
molecular weight maker; Lane + ve ⇒HPV16 and HPV18 DNA positive control Fig. 2 Detected type specific HPV DNA in multiple infections. Round 2 amplification products were resolved at 100 V for 1 h on a 2 % agarose
gel stained with 0.001 mg/mL of ethidium bromide and photographed under UV illumination. The HPVs were identified based on the molecular
weight of the amplification products for each are shown below the gel. Lane -ve ⇒HPV negative control; Lane MW ⇒Bands of the 100 base pair
molecular weight maker; Lane + ve ⇒HPV16 and HPV18 DNA positive control and 44.5 % of the IAC, CIS and ISSC cases were ob-
served among the age range of 40–59 years. Further-
more, 46.5 % of all HPV infections, 51.4 % of single
infections and 42.4 % of multiple infections (represent-
ing the highest proportion in category) were detected
among cases of this age range. Specific genotype distributions among the diagnosis
categories were presented in Table 5. HPV16 was ob-
served in 3 (33.3 %) of the 9 CIS cases, 3 (15.8 %) of the
19 IAC cases and 17 (7.5 %) of the 227 ISCC cases. HPV18 was observed in 3 (33.3 %) of the 9 CIS cases, 7
(36.8 %) of the 19 IAC cases and 99 (43.6 %) of the 227 and 44.5 % of the IAC, CIS and ISSC cases were ob-
served among the age range of 40–59 years. OR = Measures the odds of the association between being positive for each type HPV and the state of multiple-infection
apercentage of total infections
b UD undeterminable, as a result of less than five detections of each HPV for each state of infection
OR = Measures the odds of the association between being positive for each type HPV and the state of multiple-infection UD undeterminable, as a result of less than five detections of each HPV for each state of infection
OR = Measures the odds of the association between being positive for each type HPV and the state of multiple-infection
a
t
f t t l i f
ti p
g
g
dSignificantly associated with multiple-infections because odds ratio is not = 1.0 and a 95 % CI does not overlapping 1.0 bpercentage of total multiple-infections
cpercentage of total single infections
d Results After the hematoxylin and eosin staining of the second
10 μm section of each of the 342 paraffin embedded
formalin fixed cervical cancerous tissue blocks, 256 were
determined to have been adequate for PCR testing;
These consisted of 10 (3.9 %) cases of carcinoma in situ
(CIS), 19 (7.4 %) cases of invasive adenocarcinoma (IAC)
and 227 (88.7 %) cases of invasive squamous cell carcin-
oma (ISCC), as shown in Fig. 1. HPV-specific DNA was detected in 230 samples
(Fig. 2), which is a prevalence of 89.8 % (95 % CI 85.7–
93.4 %). These positive samples were distributed among
the diagnosis categories as follows, 90.0 % (9 of 10)
Carcinoma in situ (CIS) cases, 89.5 % (17 of 19) invasive
adenocarcinoma (IAC) cases and 89.9 % (204 of 227)
invasive squamous cell carcinoma (ISCC). The four
commonly detected (overall prevalence) HPVs were An age stratified distributions of the data showed that
the age groups 40–59 years and 60–79 years carried the
highest burden of each diagnosis categories of cervical
cancer, overall HPV infection positivity, single and mul-
tiple HPV infections (Table 4). Specifically, 63.2, 60.0 Awua et al. Infectious Agents and Cancer (2016) 11:4 Page 5 of 9 Fig. 2 Detected type specific HPV DNA in multiple infections. Round 2 amplification products were resolved at 100 V for 1 h on a 2 % agarose
gel stained with 0.001 mg/mL of ethidium bromide and photographed under UV illumination. The HPVs were identified based on the molecular
weight of the amplification products for each are shown below the gel. Lane -ve ⇒HPV negative control; Lane MW ⇒Bands of the 100 base pair
molecular weight maker; Lane + ve ⇒HPV16 and HPV18 DNA positive control Fig. 2 Detected type specific HPV DNA in multiple infections. Round 2 amplification products were resolved at 100 V for 1 h on a 2 % agarose
gel stained with 0.001 mg/mL of ethidium bromide and photographed under UV illumination. The HPVs were identified based on the molecular
weight of the amplification products for each are shown below the gel. Lane -ve ⇒HPV negative control; Lane MW ⇒Bands of the 100 base pair
molecular weight maker; Lane + ve ⇒HPV16 and HPV18 DNA positive control Fig. 2 Detected type specific HPV DNA in multiple infections. Results Further-
more, 46.5 % of all HPV infections, 51.4 % of single
infections and 42.4 % of multiple infections (represent-
ing the highest proportion in category) were detected
among cases of this age range. Table 2 Distribution of specific HPV genotypes detected in cancerous cervical tissues, occurring either as single infections or
alongside others in multiple-infections
HPV type
Overall, (%)a (N = 230)
Multiple-infections (%)b (N = 120)
Single infections (%)c (N = 110)
Odds ratio (OR)
95 % CI
18
109 (47.4)
83 (68.6)
26 (23.9)
6.97d
3.89–12.50
59
97 (42.2)
80 (66.1)
17 (15.6)
10.56d
5.57–20.02
45
86 (37.4)
54 (44.6)
32 (29.4)
1.94d
1.12–3.35
16
23 (10.0)
13 (10.7)
10 (9.2)
1.19
0.50–2.84
31
20 (8.7)
14 (11.6)
6 (5.5)
2.50
0.83–6.07
56
14 (6.1)
9 (7.4)
5 (4.6)
1.67
0.54–5.15
6_11
10 (4.3)
6 (5.0)
2 (1.8)
1.40
0.37–1.99
35
8 (3.5)
6 (5.0)
2 (1.8)
2.79
0.55–14.13
42
7 (3.0)
4 (3.3)
2 (1.8)
1.21
0.26–5.52
44
7 (3.0)
3 (2.5)
4 (3.7)
0.66
0.15–3.05
58
5 (2.2)
3 (2.5)
2 (1.8)
1.36
0.22–8.30
33
3 (1.3)
3 (2.5)
0 (0.0)
UD
UD
66
2 (0.9)
1 (0.8)
1 (0.9)
0.90
0.06–14.57
43
1 (0.4)
1 (0.8)
0 (0.0)
UD
UD
51
1 (0.4)
0 (0.0)
1 (0.9)
UD
UD Table 2 Distribution of specific HPV genotypes detected in cancerous cervical tissues, occurring either as single infections or
alongside others in multiple-infections Awua et al. Results Infectious Agents and Cancer (2016) 11:4 Page 6 of 9 Table 3 Distribution of multiple- and double infections involving
HPV-18 and the other most detected HPV genotypes
Multiple-infection
Number (% of total HPV infections)
Double infections
90 (39.1)
HPV-18 and HPV-59
30
HPV 18 and HPV 31
4
HPV-18 and HPV-16
3
HPV-18 and HPV 45
2
Triple infections
25 (10.8)
HPV-18,-45 and -59
17
HPV-18,-31 and 6_11
1
HPV-18, -35 and -42
1
HPV-18, -35 and -58
1
HPV-18, -42 and -6_11
1
HPV -18, -45 and -56
1
HPV -18, -59 and -6_11
1
HPV -16, -18 and -31
1
HPV-16, -18 and -59
1
HPV-16, -42 and -66
1
HPV-31, -45 and 6_11
1
HPV-45, -56 and 6_11
1
Quadruple infections
5 (2.2)
HPV-16, -18, -45 and -59
5 Table 5 Distribution of the common HPV genotypes among
the types of cancers Table 5 Distribution of the common HPV genotypes among
the types of cancers
HPV genotype
Number (%)
CIS
IAC
ISCC
18
3 (30.0)
7 (36.8)
99 (43.6)
59
4 (40.0)
8 (42.1)
85 (37.4)
16
3 (30.0)
3 (15.8)
17 (7.5)
45
0 (0.0)
7 (36.8)
79 (34.8)
35
1 (9.0)
0 (0.0)
7 (3.1)
6_11
2 (20.0)
0 (0.0)
8 (3.5)
56
0 (0.0)
1 (5.3)
y5.7)
Total tested
10
19
227
% are of the total of each type of cancer (because of multiple-infections, the
percentage may add up to more than 100) consistent with global data on cervical cancer (Table 4). Similarly, the prevalence of HPV DNA (89.8 %, 95 % CI
85.7–93.4 %) among these cases of cervical cancer,
which was based on the amplification of the viral E6 and
E7 oncogenes, was comparable to that (89.4 %) reported
by a study of cervical tumour samples in the neighbour-
ing Cote d’Ivoire [17]. Additionally, a comparable HPV
prevalence of 93.9 % was reported among cases diag-
nosed as CIN II or higher in the study from which the
HPV detection and genotyping methods were adapted
[14]. However, in an earlier study of 50 similarly proc-
essed samples collected between January and December
2003 at the same hospital and using the same HPV
detection methods, an HPV positivity of 98.0 % was
reported [15]. Results Additionally, data reported for Ghana in a
multi-centre study, which used biopsy samples collected
between October 2007 and March 2010 and a different
HPV detection and genotyping methods, indicated an
overall HPV prevalence of 93.9 % [18]. In respect of the
genotype specific prevalence (Table 2), HPV18 was the
commonest genotype detected in this study as was the
case the earlier study in Ghana [15], however, HPV16
was the commonest for the Ghanaian data reported in
the multi-centre study [18]. ISCC cases. HPV 6_11was observed in 2 (22.2 %) of
the 9 CIS cases, 8 of the 227 ISCC cases but was not
observed in IAC cases. Furthermore, Table 6 shows
the age distribution of the commonly detected HPV
genotype. The age groups 60–79 years harbored a
high proportion of HPV-18, 59 and 45 although the
age group 40–59 years harboured the highest overall
HPV infection (Table 4). aAge group with significantly higher proportions of cases and HPV infection Discussion These suggest
that a phylogenetic dependency in the colonization of
cervical epithelium might contribute to HPV prevalence,
as was observed by Conesa-Zamora et al., [24] for HPV18
and HPV45. Therefore, these may suggest a phylogenetic
related HPV prevalence in Ghana, although the bases for
such specificities are still not clear. reports of within country variation in HPV genotype
specific prevalence [11], the differences between these
studies may have been influenced by the following facts. The first is the difference in the type of specimen
used in these studies. Specifically, this and the study
by Attoh et al., [15] used archived formalin-fixed
paraffin-embedded tissue blocks while the study by
Denny et al., [18] used freshly collected biopsy sam-
ples. Also, the processing of the archived tissue blocks
used in this studyand that by Attoh et al., [15] were
not standardized and therefore different levels of in-
hibitors may have been present in the samples and in
extracted DNA. The second was that the cases in
these studies were received from different locations
across the country and therefore the differences in
prevalence may be a reflection of the inter-country varia-
tions. Thirdly, the variations in the times of samples
collection, without overlaps, (2003, 2004–2006 and 2007–
2010) and the relatively small number (n = 50) of samples
used in the study by Attoh et al., [15] may have contrib-
uted to the differences in the data of these studies. Although, the frequency of multiple-infections varies
with the type of HPV detection method used [25], the
52.2 % multiple-infections observed in this study as
compared to that of the earlier Ghanaian study, 19.6 %
[18], are discussed in light of the fact that most (about
96 %) of the cases in this study (Fig. 1) were invasive
cancers (IAC and ISSC) which are known to be associ-
ated with high multiple-infections [25]. Also, data from
neighbouring countries have shown similar high frequen-
cies of multiple-infections. A 52.9 % rate of multiple-
infection was observed in a study in Burkina Faso [20],
while a 40.2 % rate was reported by a in Benin [19]. These
may suggest a high rate of multiple-infection in the West
African region. Discussion % are of the total number of HPV positive cases (presented as n) in
each age range Table 6 Distribution of the most frequent HPV genotype
infection among cervical cancer patient according to age HPV16 (17.6 %), and HPV18 (14.8 %) as the common
HPV genotypes detected [15]. Also, a study in Cote
d’Ivoire reported HPV16 (45.0 %), HPV18 (21.0 %),
HPV45 (9.0 %), HPV35 (8.0 %), and HPV31 (3.0 %) as the
common genotypes [17] while in a study in Burkina Faso,
HPV52 (14.7 %), HPV35 (9.4 %), HPV58 (9.4 %), and
HPV51 (8.6 %) were the common genotypes [20]. Further-
more, study form other regions in and outside Africa
confirm the assertion that although HPV16 and HPV18
are the commonest HPVs in cervical cancers globally, they
are not always the two commonest HPVs in every country. For instance, in Tanzania, HPV16 and HPV58 were the
first two prevalent genotypes while HPV18 was the fifth
[21]; In Mozambique, HPV35 was the most prevalent
HPV genotype while no HPV18 genotype was detected
among women diagnosed with HSIL or carcinoma [22]. Liaw et al., [23] reported HPV52 and HPV58 as the most
prevalent type in parts of China. The number HPV risk types may add up to more than the total number of
cancer patients for each age group due to multiple-infection of some patients
or may be less than reported in Table 2 because some participants’ age was not
available. % are of the total number of HPV positive cases (presented as n) in
each age range The number HPV risk types may add up to more than the total number of
cancer patients for each age group due to multiple-infection of some patients
or may be less than reported in Table 2 because some participants’ age was not
available. % are of the total number of HPV positive cases (presented as n) in
each age range p
yp
p
Interestingly, HPV18, HPV59 and HPV45, which are
of the same phylogenetic family [7], as expected were
the common genotypes in adenocarcinoma (Table 5). On the other hand, HPV16 and its phylogenetic related
family members, HPV31 and HPV35 were respectively
the fourth, fifth and eighth prevalent HPV in this study. Furthermore, most of the multiple-infections observed
in this study involved HPV18 and HPV59. Discussion As shown in Fig. 1, the observed distribution of cervical
cancer cases was in line with global observations re-
ported for the study period [16] and the age group with
the highest burden of cervical cancer in this study was y
Although these overall HPV prevalence were within
the expected range of 90–100 % [19] and that there are Table 4 Age stratified distribution of histologic type and HPV infection among the cervical cancer cases
Age group
(years)
Histological type, n (%)
HPV status, n (%)
Type of Infection, n (%)
CIS
IAC
ISCC
HPV (−)
HPV (+)
Multiple
Single
20–39
1 (10.0)
1 (5.3)
17 (7.5)
4 (15.4)
15 (6.5)
7 (5.8)
8 (7.2)
40–59a
6 (60.0)
12 (63.2)
101 (44.5)
12 (46.2)
107 (46.5)
51 (42.5)
56 (50.9)
60–79
3 (30.0)
5 (26.3)
89 (39.2)
10 (38.5)
88 (38.3)
50 (41.6)
38 (34.5)
>79
0 (0.0)
0 (0.0)
18 (7.9)
0 (0.0)
18 (7.8)
10 (8.3)
8 (7.3)
-
0 (0.0)
0 (0.0)
2 (0.9)
0 (0.0)
2 (0.9)
2 (1.6)
0 (0.0)
Total
10
19
227
26
230
120
110
aAge group with significantly higher proportions of cases and HPV infection Awua et al. Infectious Agents and Cancer (2016) 11:4 Page 7 of 9 Page 7 of 9 Table 6 Distribution of the most frequent HPV genotype
infection among cervical cancer patient according to age
HPV type
Number (%) of cancer patients
20–39 years
(n = 15)
40–59 years
(n = 107)
60–79 years
(n = 88)
>79 years
(n = 18)
18
7 (46.7)
48 (44.9)
47 (53.4)
7 (38.8)
59
6 (40.0)
40 (37.4)
42 (47.7)
9 (50.0)
16
1 (6.6)
13 (12.1)
7 (8.0)
2 (11.1)
45
6 (40.0)
36 (33.6)
37 (42.0)
7 (38.8)
35
1 (6.6)
3 (2.8)
3 (3.4)
1 (5.5)
42
0
4 (3.7)
2 (2.3)
1 (5.3)
6_11
0
6 (5.6)
4 (4.5)
0 (0.0)
44
0
2 (1.9)
3 (3.4)
2 (11.0)
The number HPV risk types may add up to more than the total number of
cancer patients for each age group due to multiple-infection of some patients
or may be less than reported in Table 2 because some participants’ age was not
available. References
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h
h The authors are very grateful for the assistance of Prof. Y. Tettey, Prof. R. Gyasi, Dr. S. Attoh and Mr. M. K. Ametepe and the staff of the Cytology
Laboratory, Pathology Department, Korle-Bu Teaching Hospital. Also, we are
grateful to Prof. W. Ampofu, Dr. Evelyn Bonney, Prof. G Armah, Dr. Susana
Damanka, Dr. Berthel Ekow Bentum all of the Noguchi Memorial Institute for
Medical Research, Legon for making their laboratories facilities available to us
when different rooms were needed for the bench work. Authors’ contributions AKA: contributed to the development of the proposal, molecular analysis,
data analysis, writing of the draft manuscript and review of the manuscript. YDO: contributed to the development of the proposal and molecular
laboratory analysis, review of the manuscript. STS: contributed to the
development of the proposal, molecular laboratory analysis and review of
the manuscript. RHA: Contributed to the development of the proposal and
molecular laboratory analysis and review of the manuscript. EKW:
Contributed to the conception of the study, to the development of the
proposal and the histology, molecular analysis and review of the manuscript. All authors read and approved the final manuscript. Another finding worth commenting on was the obser-
vation that low risk HPV types (HPV-6/11, -42 and -44)
were solely detected as single infections in 8 cervical
cancer cases (1 CIS, 7 ISCC). These were least expected
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type HPVs are rarely observed as single infections in
invasive cancers [7]. However, due to the limitation
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embedded paraffin-embedded specimen (presence of
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risk
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be present at varying concentrations, the concentration
of the target DNA and its quality may have been greatly
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ous tissues [29–31]. The limitation of this study includes
the fact that it was not powered to determine the as-
sociations between HPV genotypes and the diagnosis
categories of cervical cancer. Also, the HIV statuses
of the patients, which may influence HPV prevalence,
were not determined. Author details
1 1Department of Biochemistry, Cell and Molecular Biology, University of
Ghana, Legon, Accra, Ghana. 2Cellular and Clinical Research Centre,
Radiological and Medical Science Research Institute, Ghana Atomic Energy
Commission, Accra, Ghana. 3Department of Medical Laboratory Sciences,
School of Biomedical and Allied Health Science, College of Health Sciences,
University of Ghana, Korle-Bu, Accra, Ghana. 4Department of Pathology,
School of Biomedical and Allied Health Science, College of Health Sciences,
University of Ghana, Korle-Bu, Accra, Ghana. 5University of Health and Allied
Sciences, Ho, Ghana. 9.
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cific observation as was shown by two studies in Spain, a
25.6 % rate of multiple-infection among HSIL cases in
Southern Spain [24], while a 34.0 % was observed in a
cohort of women in Madrid [24, 26]. Inrespect of the genotype specific prevalence, the high
prevalence of HPV59 and its frequency in multiple infec-
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of the variability in the HPV prevalence in the Ghanaian
population and therefore there is the need for a well-
controlled randomized population based HPV prevalence
study in Ghana. On the other hand, a comparison of these
HPV distributions with those of Ghana’s neighbouring
populations strongly supports the existence of geograph-
ical difference in the prevalence of HPV genotypes and the
possibility that HPV16 may not be the most prevalent
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in Benin reported HPV59 (24.6 %), HPV35 (22.5 %), Since both cross-protection of the available HPV16/18
vaccines and its clinical relevance determined with the
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cervical cancers in Ghana may be further increased. Specifically, if the infection by HPV59 depends on a
prior infection by HPV18 [24] since they most oc-
curred together in this study, then a lower prevalence
of both HPV18, HPV 59 and lower frequency of mul-
tiple infection may result after the introduction of the
HPV16/18 vaccines. 10.
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HPV profile for Ghana, which has important implication
for the introduction of HPV vaccination and forms part
of a growing body of evidence of the pre-vaccination
HPV prevalence for Ghana. However, rigorous epidemi-
ologic data and well-controlled randomized trials are
needed in order to estimate the extent of protection or
prevention of cervical cancer that may be achieved with
the introduction of HPV vaccination in Ghana; particu-
larly in light of the high multiple-infection prevalence
observed in this study. These also underscore the need
for community based screening of women for both cer-
vical cancer and HPV infection, which should include
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g
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Human Papillomavirus Prevalence in 5 Continents: Meta‐Analysis of 1 Million
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Neoplasia. N Engl J Med. 2009;361(3):271–8. 27. Kahn JA. HPV Vaccination for the Prevention of Cervical Intraepithelial
Neoplasia. N Engl J Med. 2009;361(3):271–8. 28. Dunne EF, Datta SD, Markowitz EL. A review of prophylactic human
papillomavirus vaccines: Recommendations and monitoring in the US. Cancer. 2008;113(S10):2995–3003. 28. Dunne EF, Datta SD, Markowitz EL. A review of prophylactic human
papillomavirus vaccines: Recommendations and monitoring in the US. Cancer. 2008;113(S10):2995–3003. 29. Zsikla V, Baumann M, Cathomas G. Effect of buffered formalin on
amplification of DNA from paraffin wax embedded small biopsies using
real-time PCR. J Clin Pathol. 2004;57(6):654–6. 29. Zsikla V, Baumann M, Cathomas G. Effect of buffered formalin on
amplification of DNA from paraffin wax embedded small biopsies using
real-time PCR. J Clin Pathol. 2004;57(6):654–6. Submit your next manuscript to BioMed Central
and we will help you at every step: Submit your next manuscript to BioMed Central
and we will help you at every step: 30. Unger ER, Vernon SD, Lee DR, Miller DL, Reeves WC. Detection of human
papillomavirus in archival tissues. Comparison of in situ hybridization and
polymerase chain reaction. J Histochem Cytochem. 1998;46(4):535–40. • We accept pre-submission inquiries
• Our selector tool helps you to find the most relevant journal
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and we will help you at every step: 31. An SF, Fleming KA. Removal of inhibitor(s) of the polymerase chain reaction
from formalin fixed, paraffin wax embedded tissues. J Clin Pathol. 1991;
44(11):924–7.
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ESCALA DE MENSURAÇÃO SUBJETIVA: ESTUDO COM FOCO NA PERCEPÇÃO DOS MATERIAIS EM CADEIRAS DE JANTAR
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Blucher Design Proceedings
Novembro de 2014, Número 4, Volume 1
www.proceedings.blucher.com.br/evento/11ped
Gramado – RS
De 30 de setembro a 2 de outubro de 2014
ESCALA DE MENSURAÇÃO SUBJETIVA: estudo com foco na percepção
dos materiais em cadeiras de jantar
Maria Regina Álvares Dias
Escola de Design – Universidade do Estado de Minas Gerais – UEMG
regina.alvares@gmail.com
Gilberto Almeida Jr.
Escola de Design – Universidade do Estado de Minas Gerais – UEMG
gajunior@gmail.com
Resumo: Este artigo apresenta um método de avaliação de produtos em
relação à percepção de seus materiais pelos usuários, a partir de estudos
realizados com cadeiras de jantar. O método de estudo é composto de
diferentes testes e um deles será apresentado nesse artigo mostrando a
avaliação de diferentes atributos associados a materiais de quatro
diferentes famílias ‐ metálicos, cerâmicos, poliméricos e naturais –
utilizados na fabricação de cadeiras para mesa de jantar. O estudo é parte
de um trabalho desenvolvido como pesquisa de mestrado e seus
resultados mostram que o produto incorpora parte das propriedades de
seus materiais e interferem consequentemente, nos mecanismos de
percepção do usuário, refletindo no julgamento das cadeiras, mesmo que
essas apresentem modelo semelhante. O método pode ainda ser aplicado
em outras tipologias de produtos, juntamente com diferentes ferramentas
do design.
Palavras‐chave: Design, Materiais, Percepção, Mobiliário
Abstract: This paper presents a method of product evaluation in relation to
the perception of their material by users from studies with dining chairs.
The method of study is composed of different tests and one of them will be
presented in this paper showing the evaluation of different attributes
associated with four different families of materials ‐ metals, ceramics,
polymers and natural ‐ used in the manufacture of chairs for the dining
table. The study is part of a work as master research and their results show
that the product incorporates some properties of its material and
consequently interfere in the user’s perception mechanisms, reflecting the
judgment of seats, even those presenting similar model. The method can
also be applied to other types of products, along with different design tools.
Keywords: Design, Materials, Perception, Furniture
2
1. INTRODUÇÃO
O foco do presente estudo está na relação entre os materiais empregados para
a fabricação de cadeiras domésticas para mesa de jantar e sua influência nos aspectos
de preferência do usuário. O estudo é mais amplo em outros aspectos e propõe um
método composto por escalas de mensuração de questões subjetivas, referentes à
preferência do usuário em relação ao material aplicado no produto (Almeida Jr, 2013).
O método em si apresentava diferentes etapas de avaliação dos materiais:
percepção de texturas de material polimérico, identificação dos materiais, adequação
do material para aplicação no produto e por último, conheceu‐se a percepção dos
atributos associados a quatro tipos de materiais, aplicados em um mesmo modelo de
cadeira para mesa de jantar através de análise semântica e emitindo sua preferência
em relação aos modelos apresentados na pesquisa.
Do ponto de vista do design, o produto é composto a partir de vários
elementos, como forma, escala, volume, cor, material, textura, brilho, som, cheiro e
outros que, de maneira indivisíveis, conformam o artefato final. Um dos elementos
mais importantes é o material, que “permite a interface imediata entre os artefatos e
o homem” (Manzini, 1993). Os materiais desempenham papel essencial no processo
de concepção do produto: eles podem definir seu leque de funções, a durabilidade, os
custos e sua aparência final. Da mesma forma, a experiência dos usuários tem papel
importante nesse processo, uma vez que, ao interagirem com o produto, estabelecem
relações sensoriais ‐ táteis, visuais, auditivas, olfativas ou gustativas ‐ que podem ser
determinantes em sua concepção. Nesse sentido, o material da cadeira, bem como as
características de sua superfície e sua conformação são elementos preponderantes na
percepção de questões subjetivas ligadas ao bem‐estar dos usuários.
Nessa perspectiva, o estudo experimental realizado com usuários para avaliar
distintos materiais e cadeiras de jantar de diferentes materiais serviu para validar o
método proposto por Almeida Jr (2013) “Avaliação dos aspectos subjetivos
relacionados aos materiais: proposta de método de mensuração aplicado ao setor
moveleiro”. Considera‐se que as avaliações subjetivas resultantes da pesquisa podem
ser revertidas em informações objetivas, como, por exemplo, na definição das
características do produto, na especificação técnica dos materiais, na definição de
texturas e acabamentos, bem como em inúmeras possibilidades aplicativas.
2. CADEIRAS E MATERIAIS
As cadeiras são os objetos mais explorados pelos designers e arquitetos em
seus projetos, sendo difícil identificar algum desses profissionais que não tenha
projetado um exemplar. A diversidade de formas, cores, acabamentos e materiais
empregados, representam bem a infinidade de possibilidades para sua construção. Os
materiais utilizados na fabricação das cadeiras são os mais diversos ‐ dos mais rústicos
e tradicionais como a madeira maciça e fibras naturais, até os materiais processados
através de grande tecnologia como polímeros e fibras de alta tecnologia que
imprimem características particulares a cada objeto.
Ao longo da história foi atribuído a este produto simbolismo de posição social e
status, sendo considerado um dos objetos mais importantes do mobiliário. As cadeiras
conhecidas, datadas como mais antigas, foram as egípcias, que demonstravam grande
riqueza e esplendor, facilmente percebidos pela utilização de materiais nobres na sua
construção. Eram feitas de ébano, marfim, madeira dourada e cobertas com metais
3
preciosos (GALI, 1988).
Atualmente as cadeiras e poltronas são fabricadas de inúmeros materiais e
processos de fabricação, os quais influenciam na sua forma e significado. Abreu et al
(2001) relatam que a indústria moveleira utiliza tanto materiais de natureza sintética
quanto artificial, como os polímeros reforçados com fibras de vidro ou na forma de
laminados plásticos para acabamentos em chapas de madeira.
São inúmeros os processos existentes e variadas as formas de se trabalhar com
os incontáveis materiais, que atualmente ultrapassam 100 mil tipos diferentes. O uso
dos materiais não se limita, pelo contrário, à medida que novos materiais são
desenvolvidos e apresentados aos profissionais, um campo enorme de atuação do
profissional de design se amplia.
3. O MÉTODO
A seleção do material é complexa e não se baseia apenas em exigências
funcionais, na tecnologia de fabricação, sustentabilidade, economia, mas, igualmente,
em seus valores estéticos, nas propriedades sensoriais, nos aspectos ergonômicos, em
seus significados culturais e simbólicos.
Compreender como as pessoas respondem às propriedades sensoriais dos
materiais, bem como saber quais são as reações afetivas e emocionais diante destes,
ajuda os designers e engenheiros a selecioná‐los, levando‐se em conta a capacidade de
proporcionar uma experiência positiva ao usuário, que em consequência, aumenta o
valor percebido do produto.
O estudo objetivou estudar a percepção de materiais poliméricos,
vitrocerâmicos, naturais e metálicos, empregados em quatro cadeiras. O teste de
percepção foi aplicado com participantes voluntários. A amostra total foi de 30
indivíduos, sendo 20 homens e 10 mulheres. Como os testes propuseram diferentes
tipos de análises, o perfil do usuário pôde ter caráter bastante abrangente. Preferiu‐se
os participantes que apresentaram maior grau de escolaridade, sem se restringir
qualquer participação.
A montagem dos testes e seus momentos de avaliação considerou que os
atributos estéticos do material têm relação direta com a impressão estética que
sentimos sobre um objeto por meio dos sentidos, equivale ao prazer fisiológico
(Jordan, 2002) e ao nível visceral do design, Norman (2005).
Os atributos práticos do material se relacionam diretamente ao uso, manuseio
e experiência dos usuários com os objetos, resultando no prazer e efetividade,
equivalem ao prazer psicológico (Jordan, 2002), o qual tem relação com as reações
cognitivas, mentais e emocionais dos indivíduos e ao design comportamental de
Norman (2005). Os atributos simbólicos dos materiais têm relação com os aspectos de
estima, psíquicos e sociais, e correspondem ao prazer social e ideológico propostos por
Jordan (2002) e ao design reflexivo definido por Norman (2005).
O método é composto por três testes, sendo que os dois primeiros tratam das
questões relacionadas aos materiais com possível aplicação em cadeiras e o terceiro
(FIG. 1) avalia o produto construído com diferentes materiais.
Qualquer um dos três testes pode ser aplicado isoladamente, dependendo do
que se pretende analisar. Todos os registros foram feitos pelo avaliador dos testes,
garantindo que todos os campos fossem devidamente preenchidos, no tempo
adequado permitindo que o teste fosse conduzido de forma confortável e garantindo
4
liberdade de interação dos avaliados com os materiais. Apenas a etapa que consistia
da autoavaliação emocional foi preenchida pelo próprio usuário.
Teste 3
Momento 1 e 2:
Objetivo:
Tipo de avaliação:
Avaliar a preferência pelas
cadeiras e a percepção de
diferentes atributos
associados a quatro
materiais escolhidos para a
fabricação de um modelo
de cadeira para mesa de
jantar. Outro propósito é a
avaliação emocional dos
modelos apresentados.
Momento 1 e 2: avaliar e
escolher entre 4 cadeiras a
mais e menos adequada de
acordo com seu material.
Cadeiras apresentadas na
forma de imagens coloridas
com suas respectivas
amostras de materiais:
metal, acrílico, madeira e
vidro.
Momento 3:
Obs.: Não foi testada a
cadeira de vidro pela
dificuldade apresentada em
construir protótipo com o
material.
Momento 3: avaliar, testar
e escolher a cadeira mais e
menos adequada entre 3
cadeiras apresentadas na
forma de protótipos nos
materiais: metal, acrílico e
madeira.
Figura 1 – Detalhamento do teste 3 e seus respectivos momentos de avaliação.
Fonte: Elaborado pelo autor, com base na pesquisa realizada.
Esse artigo discute a terceira etapa do método, avaliando a percepção de
diferentes atributos, associados a quatro materiais escolhidos para a fabricação de um
mesmo modelo de cadeira para mesa de jantar. Além da avaliação semântica, o
usuário emitiu sua preferência em relação aos modelos apresentados na pesquisa,
através de visualização de imagens representativas dos produtos e experimentação
dos protótipos.
4. TESTE 3: PERCEPÇÃO DOS MATERIAIS DAS CADEIRAS, PREFERÊNCIA,
EXPERIMENTAÇÃO E ESCOLHA DO PRODUTO
A avaliação de materiais quando aplicados em produtos exige mais esforço do
aparelho cognitivo do indivíduo se comparado à análise de amostras de materiais sem
o contexto do produto. Segundo Rouvray (2006), a forma de avaliar e interagir com o
produto produz níveis de percepção distintos. Existem diferentes formas de se avaliar
a resposta emocional da interação humano‐produto, desde a mais simples e menos
custosa, através de fotografias, a intermediária, através de prototipagem virtual, até a
interação completa com produtos reais inseridos no ambiente de uso indicado.
Comprovando os estudos do autor, o nível de interação ou informação sobre o que é
avaliado, interfere na percepção dos indivíduos e nas suas escolhas. Existe diferença
no resultado quando só se pode ver, quando se pode tocar e mais ainda quando a
interação é possível.
Com a intenção de conhecer quais os atributos possuem maior e menor
importância em relação a uma cadeira de jantar, e como a decisão de compra do
5
produto é influenciada por eles, os usuários indicaram subjetivamente o grau de
importância de vários critérios. A FIG. 2 mostra a ocorrência e distribuição das
atribuições de valor para cada item. A análise dos dados mostrou que os critérios de
design, conforto, material e acabamento, e experimentação e teste da cadeira antes
da compra foram indicados como de maior importância. Vale ressaltar que o critério
design abrange questões relacionadas ao estilo do produto, inovação, qualidade de
projeto, entre outras. Os atributos peso, moda e tendências, aspectos de status, luxo e
marca e aspectos ligados à sustentabilidade, receberam notas variadas com grande
percentual de notas de menor ou pouca importância. Os atributos ergonomia, preço,
facilidade de limpeza e segurança de uso, apresentaram notas intermediárias com
valores indicando serem de maior importância.
Figura 2 – Detalhamento dos três testes aplicados e seus respectivos momentos de avaliação.
Fonte: Elaborado pelo autor, com base na pesquisa realizada.
O teste 3 deu‐se em três momentos de avaliação. No primeiro foi realizada a
avaliação visual dos quatro modelos de cadeiras construídas em quatro famílias de
materiais como já mencionado, escolhendo a cadeira preferida e a que não escolheria
considerando o material. Juntamente com a imagem de cada cadeira foi
disponibilizada uma amostra do material correspondente para que houvesse
interação.
4.1 Primeiro Momento de Avaliação
Iniciou‐se o teste 3 com a seguinte instrução: “simulando o momento de
aquisição, selecione a cadeira de sua preferência considerando os materiais,
independente da ergonomia, cores e preço.” Para sua realização, era importante olhar
as imagens das cadeiras e tocar nas amostras dos materiais localizados próximos à
imagem correspondente. Na finalização, os usuários deveriam indicar uma segunda
cadeira como sendo a que não escolheriam. A figura 3 indica os índices de preferência
6
e rejeição para cada uma das quatro cadeiras.
Figura 3 – Percentual de escolha das cadeiras através das fotos e contato com o material.
Fonte: Elaborado pelo autor, com base na pesquisa realizada.
Observa‐se que tanto a cadeira de acrílico quanto a de madeira, tiveram cada
uma 43% das escolhas dos usuários. A cadeira que não seria escolhida indicando 50 de
rejeição seria a de vidro. Outro modelo que teve alto índice de recusa foi a de metal,
recebendo 40% dos votos.
4.2 Segundo Momento de Avaliação
No segundo momento do teste foi apresentado um grupo de descritores e era
avaliado o grau de importância de cada um deles. Foram listados 12 pares de atributos
com escala de valores de 6 graus (muito, médio e pouco) para cada atributo (FIG. 4).
Figura 4 – Diferencial semântico com grupo de descritores para avaliar o grau de importância de cada
atributo.
Fonte: Elaborado pelo autor, com base na pesquisa realizada.
7
Os atributos correspondiam a: som, peso, temperatura, durabilidade,
estabilidade, conforto, limpabilidade, inovação, ecologia, personalidade, preço,
naturalidade do material. Foi dada a instrução para “assinalar o grau de importância
dos atributos para cada uma das cadeiras” apresentadas através de imagem que
indicava o material aplicado. Como o procedimento teve registro de voz e vídeo, o
formulário possuía ícones específico abaixo das imagens das cadeiras para que o
avaliador indicasse se usuário apresentou algum movimento ou disse algo relevante
para análise posterior.
Os dados obtidos através dessa avaliação passaram por tratamento estatístico
para análise de variância pela técnica ANOVA. Este método considera conjuntamente
as avaliações de todos os avaliados e assume que todos apresentam o mesmo
comportamento, considerando suas individualidades. É possível identificar ainda a
média para cada atributo sensorial avaliado (Minim, 2010).
A cadeira de metal foi considerada a
mais silenciosa e a de acrílico a mais
barulhenta. A cadeira de madeira e a
de vidro apresentaram mesma
classificação, indicando que são pouco
barulhentas.
O atributo temperatura revelou que a
cadeira de metal e a de vidro foram
ambas consideradas muito frias,
enquanto a de madeira foi avaliada
como médio quente, e a de acrílico a
mais quente de todas.
A cadeira de vidro foi a mais pesada na
opinião dos usuários do teste, seguida
pela cadeira de metal. De acordo com
a escala utilizada, a cadeira de madeira
foi considerada médio leve e a de
acrílico a mais leve entre todas as
quatro testadas.
A cadeira de metal foi considerada a
mais durável. Todas as demais
indicaram possuir valores positivos de
durabilidade.
Quanto a estabilidade, as cadeiras de
madeira e de metal, são as mais
estáveis. A cadeira de vidro indicou ser
a mais instável.
Em relação ao conforto, a cadeira de
madeira foi indicada como a mais
confortável e a de vidro a mais
desconfortável.
8
A cadeira de metal foi considerada a
mais durável. Todas as demais
indicaram possuir valores positivos de
durabilidade.
Quanto a estabilidade, as cadeiras de
madeira e de metal, são as mais
estáveis. A cadeira de vidro indicou ser
a mais instável.
Em relação ao conforto, a cadeira de
madeira foi indicada como a mais
confortável e a de vidro a mais
desconfortável.
Quanto a limpabilidade apenas a
cadeiras de madeira foi considerada
pouco fácil de limpar, todas as outras
foram consideradas muito fáceis de
serem limpas.
O critério inovação apresenta todas as
cadeiras como mais inovadoras,
apenas a de madeira indicada como
mais conservadora entre as quatro.
No aspecto ecologia, a cadeira de
acrílico foi considerada a menos
ecológica e a de madeira a mais
ecológica. As cadeira de metal e vidro
atingiram classificação neutra.
Materiais, processos, forma, peso e
cor, contribuem para a criação de
personalidade. Nessa avaliação foi
medido o caráter masculino e feminino
do produto.
Portanto, em relação à personalidade,
a cadeira de acrílico e a de vidro
apresentaram caráter feminino. Já a de
metal e a de madeira apresentaram
caráter masculino. A cadeira de acrílico
foi a mais feminina e a de metal a mais
masculina.
Em relação ao preço, a cadeira de
madeira foi considerada a mais barata
de todas e a de vidro a mais cara. A
cadeira de metal foi considerada
médio cara e a de acrílico pouco cara.
9
Quanto à naturalidade do material, a
cadeira de madeira foi considerada
como sendo de origem natural e tanto
as cadeira de metal, acrílico e vidro
foram consideradas artificiais.
Figura 5 – Resultado da ANOVA para cada a dos 12 pares de atributos avaliados.
Fonte: Elaborado pelo autor, com base na pesquisa realizada.
Ainda no segundo momento, o usuário respondia uma escala para
autoavaliação emocional, apresentando quais sensações eram atribuídas a cada uma
das quatro cadeiras. Aplicou‐se um método utilizado por Desmet (2004) e Dias (2009),
por ter sido empregado em estudos anteriores que comprovaram sua eficiência.
Define‐se como uma escala circular com 7 emoções positivas e 7 negativas,
sendo as positivas localizadas no lado direito e as negativas no lado esquerdo, com
intensidade variando de muito forte, médio ou fraco, com a parte externa do círculo
indicando mais intensidade e a interna menos intensidade.
Figura 6 – Escala utilizada para autoavaliação emocional utilizada no estudo.
Fonte: Dias (2009).
O resultado da análise de autoavaliação emocional das cadeiras foi organizado
de acordo com os atributos avaliados. A porcentagem do total de escolhas visto na
figura 7 representa a amostra de 30 usuários.
10
Figura 7 – Distribuição das emoções de acordo com a cadeira avaliada.
Fonte: Elaborado pelo autor, com base na pesquisa realizada.
A cadeira com maiores emoções positivas foi a cadeira de acrílico, seguida pela
cadeira de madeira. As cadeiras de metal e vidro foram respectivamente as que
receberam mais indicações negativas. Como o preenchimento não implicava na
obrigatoriedade em indicar todas as emoções apresentadas na escala circular, a soma
das opiniões referentes às emoções positivas e negativas não precisa representar
numericamente 100%, pois elas não aconteceram a todos os indivíduos de forma
similar. O modelo utilizado com preenchimento dos dados da autoavaliação da cadeira
de metal é apresentado na figura 8 com caráter ilustrativo.
Figura 8 – Resultado da autoavaliação emocional.
Fonte: Elaborado pelo autor, com base na pesquisa realizada.
Além das emoções do círculo de emoções os usuários poderiam indicar outras
sensações que sentiam durante a interação e que julgavam ausentes na escala. Em
relação a cadeira de metal foram indicadas as sensação de frio, estranheza,
desinteresse, aflição ao toque. Quanto a cadeira de acrílico foram relatados bom gosto
e curiosidade. A cadeira de madeira instigou indiferença e curiosidade. Já a cadeira de
vidro refletiu medo, receio, desconforto, perigo, intranquilidade (frágil), desconfiança
e estranheza/inquietação, todos valores considerando negativos em relação a uma
cadeira.
11
4.3 Terceiro Momento de Avaliação
No terceiro momento, os usuários realizavam a avaliação das cadeiras reais e
novamente escolhiam a mais e menos preferida após interação, conforme figura 9. Foi
dada a orientação para que os usuários tocassem, levantassem e sentassem nas
cadeiras.
Figura 9 – Momento de interação com as cadeiras.
Fonte: Elaborado pelo autor, com base na pesquisa realizada.
Após interação com as cadeiras, os usuários foram avaliados quanto a sua
preferência inicial quando viram as imagens das cadeiras. A intenção foi examinar se,
após interação com o produto real, alguma preferência tinha mudado e
consequentemente, a escolha feita anteriormente.
Os dados obtidos indicaram que ouve mudança no julgamento após a
interação: a cadeira de metal teve diminuição de rejeição de 3% após contato com o
produto; a cadeira de acrílico aumentou em 10% sua aceitação; a de madeira
apresentou diminuição de sua aceitação de 6%; já a cadeira de vidro indicou
diminuição de 4% em sua aceitação e aumento de 3% de rejeição.
O teste 3 permitiu identificar o perfil subjetivo do material representado por
suas características intangíveis, ou seja, os significados atribuídos e as emoções
evocadas, que não podem ser puramente identificadas com valores numéricos ou
quantificadas.
5. CONSIDERAÇÕES FINAIS
Os estudos apresentaram‐se como importante meio para conhecimento sobre
materiais, produtos e usuários. O conhecimento dos atributos mais valorizados por um
grupo de indivíduos permite a construção de um mapa semântico acerca dos
materiais, comprovando se suas características técnicas correspondem com a forma
como são percebidos pelas pessoas.
A avaliação de materiais, quando aplicados em produtos, exige mais esforço do
aparelho cognitivo do indivíduo se comparado à análise de amostras de materiais sem
o contexto do produto. Comprovando estudos de Rouvray (2006), o nível de interação
ou informação sobre o que é avaliado, interfere na percepção dos indivíduos e nas
suas escolhas. Foi possível comprovar também a afirmativa de Ashby e Johnson (2011)
que “o produto incorpora parte das propriedades de seus materiais”, pois houve
reflexo das características deles no julgamento das cadeiras, já que todas eram
similares em formato. A personalidade do material influencia nas escolhas individuais.
Em alguns casos, os materiais podem transmitir interpretações distorcidas quanto aos
aspectos de sustentabilidade, ecologia, durabilidade entre outros.
12
A escala de autoavaliação subjetiva teve sua eficiência comprovada assim como
descrito por Desmet (2004) e Dias (2009), fornecendo parâmetros de julgamento
quanto à intensidade dos atributos sentidos, bem como permitindo classificá‐los com
valores negativos e positivos. Configura‐se, portanto, como uma importante
ferramenta de avaliação subjetiva entre materiais, produtos e usuários.
Através das escalas do modelo proposto, foi possível conhecer o
comportamento dos indivíduos em relação às cadeiras e seus materiais, compreender
que as pessoas repetem padrões preestabelecidos, construídos através de
experiências anteriores e que são por sua vez comuns à grande parte das pessoas.
Caso não fosse assim, diferentes perfis de usuários não apresentariam possuir um
mesmo julgamento em relação a determinado material.
As cadeiras mostraram‐se bons objetos de estudo, uma vez que são produtos
simples, de uso intuitivo e autoexplicativo, conhecidos e presentes nas casas das
pessoas e que, dentro da indústria de mobiliário, mostram‐se de relativa significância
pelo grande volume de vendas que representam.
REFERÊNCIAS
ABREU, Aline; CÂNDIDO, G. Ataíde; TEIXEIRA, Joselena de A.. A utilização dos materiais
no design e a competitividade da indústria moveleira da Região Metropolitana de
Curitiba: um estudo de caso. Produção. São Paulo. v. 11, n. 1, p. 17‐30, 2001.
ALMEIDA JR, G. Avaliação dos aspectos subjetivos relacionados aos materiais:
proposta de método de mensuração aplicado ao setor moveleiro. 2013. 184 f.
Dissertação (Mestrado em Design) ‐ Escola de Design, Universidade do Estado de
Minas Gerais, UEMG, Belo Horizonte,2013.
ASHBY, Mike F.; JOHNSON, Kara. Materiais e design: arte e ciência da seleção de
materiais no design de produto. São Paulo: Elsevier, 2011.
DESMET, P. M. A. A multilayered model of product emotions. The Design Journal, v. 6,
n. 2, p. 04‐13, 2004.
DIAS, M.R.A.C. Percepção dos materiais pelos usuários: modelo de avaliação
Permatus. Florianópolis: UFSC, 2009. 352 p. (Doutorado em Engenharia e Gestão do
Conhecimento), UFSC, Florianópolis, 2009.
GALI, Vera. Mobiliário brasileiro: a cadeira no Brasil. São Paulo: Empresa das Artes,
1988.
JORDAN, P. W. Designing pleasurable products: an introduction to the new human
factors. London: Taylor & Francis, 2002.
MANZINI, Ezio. A matéria da invenção. Lisboa: Centro Português de Design, 1993.
MINIM, Valéria P. R. Análise sensorial: estudos com consumidores. 2.ed. Viçosa:
Editora UFV, 2010.
NORMAN, D. A. El diseño emocional: por qué nos gustan (o no) los objetos cotidianos.
Barcelona, Ediciones Paidós Ibérica, 2005.
ROUVRAY, Alexandre de. Integration des preferences emotionnelles et sensorielles
dans la conception de produits d' amaeublent. 2006. Tese (Doutorado). L’ École
Nationale Supériere d' Arts et Métiers, Paris, 2006.
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Sociology of Rights: "I Am Therefore I Have Rights": Human Rights in Islam between Universalistic and Communalistic Perspectives
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Muslim World Journal of Human
Rights
Volume 2, Issue 1
2005
Article 11
SPECIAL “FROM THE FIELD” ISSUE: ISLAM AND HUMAN
RIGHTS ADVOCACY FOR SOCIAL CHANGE IN LOCAL
CONTEXTS
Sociology of Rights: “I Am Therefore I Have
Rights”: Human Rights in Islam between
Universalistic and Communalistic Perspectives
Recep Senturk∗ Volume 2, Issue 1
2005
Article 11
SPECIAL “FROM THE FIELD” ISSUE: ISLAM AND HUMAN
RIGHTS ADVOCACY FOR SOCIAL CHANGE IN LOCAL
CONTEXTS Sociology of Rights: “I Am Therefore I Have
Rights”: Human Rights in Islam between
Universalistic and Communalistic Perspectives Recep Senturk∗ ∗Emory Islam and Human Rights Program, ras13@tnn.net ∗Emory Islam and Human Rights Program, ras13@tnn.net Copyright c⃝2005 by The Berkeley Electronic Press. All rights reserved. No part of this pub-
lication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any
means, electronic, mechanical, photocopying, recording, or otherwise, without the prior written
permission of the publisher, bepress, which has been given certain exclusive rights by the au-
thor. Muslim World Journal of Human Rights is produced by Berkeley Electronic Press (bepress). http://www.bepress.com/mwjhr Sociology of Rights: “I Am Therefore I Have
Rights”: Human Rights in Islam between
Universalistic and Communalistic
Perspectives∗ Recep Senturk INTRODUCTION The age of Descartes was characterized by puzzlement with human
existence: How can we rationally prove that we really exist? In response, he
offered the famous postulation: I think therefore I am. I ask, “then what?” because
Descartes’ query appears to culminate at that point, without exploring the social
and moral implications of human existence. However, because of the change in
the Zeitgeist, by the shift from the intellectual primacy of metaphysical quest to the
pinnacle of the search for human well being on earth, our generation is
characterized by puzzlement with human rights: How can we justify that we have
human rights? Tackling this question, ideologies compete to patronize or
monopolize the justification of human rights, an exclusionist position I challenge
below. Exclusionist groups who subscribe to secular or religious ideologies claim
that only their world-view can justify and provide human rights. However, I
argue, along with the inclusionists from these groups, that all universalistic
worldviews in the world do so. Islam is one of them. I postulate my response to this puzzle as I am therefore I have rights. My very
existence suffices as a substantiation of my rights, irrespective of my innate,
inherited, gained or ascribed qualities. By tying human existence to human rights,
I explore the prerequisites and the inevitable implications of our existence in
society at the universal level. My approach to this question derives from both the
Islamic and the modern secular notions of justice, freedom and human rights. Using the example of Islamic legal tradition, I will demonstrate below how both
religious (divinely inspired, deriving from scriptures) and secular (rationally
inspired, deriving from human mind) world views, may justify human rights in
their own terms, yet arriving at parallel conclusions. y
g
p
There is a gap between two approaches on the universal human rights:
legal perspective with an emphasis on universalism and social scientific
perspective with an emphasis on relativism. The gap became manifest during the
preparation of the UN Declaration in 1948. The American Anthropological
Association (AAA) publicly opposed the entire project of the universal human
rights declaration. In contrast, the legal approach has triumphantly claimed that
the universal human rights can be codified and justified at the universal level, yet
they did so within the parameters of a particular culture, namely secular and
Western. Abstract “I am therefore I have rights,” argues this paper. Mere existence qualifies a human being for
universal human rights. Yet human beings do not live in solitude; they are always embedded in
a network of social relations which determines their rights and duties in its own terms. Conse-
quently, the debate about the universality and relativism of human rights can be best understood
by combining legal and sociological perspectives. Such an approach is used in this article to ex-
plore the tensions and contests around the universality of human rights in Islamic law. Whether
all human beings or just citizens are qualified for the inviolability of human rights is a question
which divided Muslim jurists into two schools: Universalistic School, emanating from Abu Han-
ifa, advocated for the universality of human rights, while Communalistic School, originating from
Malik, Shafii and Ibn Hanbal, advocated for civil rights. Universalistic School was adopted by
such great cosmopolitan empires as Umayyads, Abbasids, Mughals and Ottomans. It was also re-
formed by the Ottomans during the nineteenth century in the light of the new notions of universal
human rights in Europe to purge remaining discriminatory practices against non-Muslim citizens
and to justify constitutionalism and democracy. Yet the universalistic tradition in Islamic law has
been forgotten as the chain of memory was broken after the collapse of Ottoman Empire. This ar-
ticle briefly unearths the forgotten universalistic approach in Islamic law to build upon it a modern
universalistic human rights theory for which there is a pressing need at this age of globalization. KEYWORDS: human rights, Islam, culture, globalization ∗Associate Professor of Sociology at the Center for Islamic Research (ISAM), Istanbul, Turkey. He specializes in social network analysis, social theory, political sociology, sociology of religion,
sociology of law and human rights with a focus on Islam, Ottoman Empire, modern Turkey and
Egypt. He is the author of several books on modernization in Islam, sociology of religion in
the West, social memory and networks of elite intellectuals in Islam. His most recent book is
Narrative Social Structure: Anatomy of the Hadith Transmission Network 610-1505 (Stanford
University Press, Forthcoming 2005). His current book project is tentatively titled Sociology of
Rights: Human Rights between Universalism and Communalism. His books and articles are in
English, Turkish and Arabic. He received his Ph.D. from Columbia University. Senturk: Sociology of Rights INTRODUCTION The anthropological approach has claimed that the universal human
rights are impossible to define because of the irreconcilable social and cultural
diversity of the people in the world; hence the Western and secular definition and
justification is ethnocentric. I argue that, combining the legal and cultural approaches will allow us to
reconcile the tension between these two contesting paradigms. The global
cultural diversity does not preclude the possibility of a number of common
denominators or universal values; cultures with diverse languages and dialects
may justify and interpret human rights differently but can still meet at a common Published by Berkeley Electronic Press, 2005 1 Muslim World Journal of Human Rights, Vol. 2, Iss. 1 [2005], Art. 11 ground. Anthropologists who initially objected the possibility of universal human
rights have also come to this point1. One can concur with the universalist and relativist claims on human
rights, yet only on a particular level. The problem emerges when these claims are
generalized. In my view, law operates at two levels, universal and communal. Both levels have conceptual and sociological dimensions. The former is
characterized by uniformity while the latter is characterized by diversity. In other
words, there are certain principles on which there is universal consensus while
certain issues vary from culture to culture2. Global society requires universal consensus on the rules of exchange,
such as reciprocity, for international trade, sports, law and politics to be possible. I call these rules, following traditional Muslim jurists, “axioms of law.3” These
principles are unanimously accepted worldwide and taken as given. This is true
not only for the field of law and the Science of Law but also for all fields of social
and academic life. In every domain of life a consensus is needed on certain
principles for this domain to operate well. Such a consensus already exists in the
world, most visibly in the area of trade and sports, as it had existed from the very
beginning of human history. Therefore, without these axiomatic principles,
neither law nor the Science of Law is possible. Furthermore, axioms make
diversity and change possible, in an ordered manner, without causing anarchy and
disorder, as they draw the line between what is fixed and what is not4. y
I argue that all universal cultures, be they religious or secular, ancient or
modern, commonly agree on the inviolability of all human beings5. 4 For instance, Ten Commandments played this role for many centuries. For similar rules in
the Quran, see Surah Isra, 17: 22-39. The UN Declaration of Human Rights also emerged
from a need to regulate global relations after WW II in accordance with universally accepted
rules. 3 See Abu Hamid al-Ghazzali, al-Mustasfa fi ‘Ilm al-Usul, Beirut: Dar al-Arqam 1414/1994,
vol. I, p. 633-637.
4 5 Although it is not our subject here, I should add here that all universal cultures also agree on
due process and reciprocity. 1 A recent declaration by the American Anthropological Association reflects this tension:
“Thus, the AAA founds its approach on anthropological principles of respect for concrete
human differences, both collective and individual, rather than the abstract legal uniformity of
Western tradition.” Declaration on Anthropology and Human Rights, Committee for Human
Rights, American Anthropological Association, Adopted by the AAA membership June 1999.
2 See An-Na’im, Abdullahi A. and Francis M. Deng (eds.), Human Rights in Africa: Cross-
Cultural Perspectives, Washington, D.C. 1990.
3 See Abu Hamid al-Ghazzali, al-Mustasfa fi ‘Ilm al-Usul, Beirut: Dar al-Arqam 1414/1994,
vol. I, p. 633-637.
4 For instance, Ten Commandments played this role for many centuries. For similar rules in
the Quran, see Surah Isra, 17: 22-39. The UN Declaration of Human Rights also emerged
from a need to regulate global relations after WW II in accordance with universally accepted
rules.
5 Although it is not our subject here, I should add here that all universal cultures also agree on
due process and reciprocity. 1 A recent declaration by the American Anthropological Association reflects this tension:
“Thus, the AAA founds its approach on anthropological principles of respect for concrete
human differences, both collective and individual, rather than the abstract legal uniformity of
Western tradition.” Declaration on Anthropology and Human Rights, Committee for Human
Rights, American Anthropological Association, Adopted by the AAA membership June 1999.
2 See An-Na’im, Abdullahi A. and Francis M. Deng (eds.), Human Rights in Africa: Cross-
Cultural Perspectives, Washington, D.C. 1990.
3 INTRODUCTION Yet they do
so in their own terms, which is an inevitable outcome of social and cultural
diversity. Consequently, there is not a single universalism, which is unanimously
accepted by humanity as a whole, instead, there are various universalisms
emanating from different cultures. They affirm each other. Acknowledging such diversity in ways human sanctity is justified brings
more strength to human rights cause, instead of undermining it. There is not only
a single way to justify and talk about human rights, or any other matter in the
world. There are in the world multiple discourses to talk about human rights and
multiple grounds to justify human rights, reflecting the diversity of cultures on 1 A recent declaration by the American Anthropological Association reflects this tension:
“Thus, the AAA founds its approach on anthropological principles of respect for concrete
human differences, both collective and individual, rather than the abstract legal uniformity of
Western tradition.” Declaration on Anthropology and Human Rights, Committee for Human
Rights, American Anthropological Association, Adopted by the AAA membership June 1999. 2 See An-Na’im, Abdullahi A. and Francis M. Deng (eds.), Human Rights in Africa: Cross-
Cultural Perspectives, Washington, D.C. 1990. 3 5 Although it is not our subject here, I should add here that all universal cultures also agree on
due process and reciprocity. 2 http://www.bepress.com/mwjhr/vol2/iss1/art11
DOI: 10.2202/1554-4419.1030 Senturk: Sociology of Rights the globe. Universalist legal cultures share a common ground which unites them
but also there is a great diversity among them. Yet the points of agreement are
sufficient to serve as the axioms of a global dialogue among them. These
discourses may, however, vary in content, scope and mechanisms of
implementation. It would also be a clear anachronism to treat Islamic and
modern discourses on human rights as the same because they reflect the different
historical circumstances in which they emerged and put in use. Consequently,
rather than subscribing to the current blanket generalizations in the academic and
popular literature, this study explores similarities and differences between them. However, currently, some of the representatives of these cultures
compete with each other to monopolize the cause of human rights. Each one
claims that only her culture grants and protects human rights6. Or they claim that
human rights had initially originated in their culture. This rivalry is unnecessary,
counterproductive and inconsistent with the universalism each ideology claims to
represent. 6 Donnelly, Jack, Universal Human Rights in Theory and Practice, Ithaca, N.Y. : Cornell
University Press, 1989.
7 An-Na‘im, Abdullahi A., Toward an Islamic Reformation: Civil Liberties, Human Rights,
and International Law, Syracuse: SUNY Press 1990. IS UNIVERSAL HUMAN RIGHTS POSSIBLE? INTRODUCTION I challenge this exclusivist position, regardless of which culture it stems
from, and offer an inclusive alternative from a sociological perspective which
takes into account the diversity of cultures in the world and their right to produce
and maintain their distinct discourse on human inviolability. We, as humanity, had in our history an ongoing and evolving discourse
on human inviolability all along, yet in diverse conceptual and institutional forms. From this perspective, a truly universalistic position on human rights is
characterized by three features: accepting the inviolability of all human beings;
doing so by virtue of their humanity; acknowledging that other universal cultures
also respect the inviolability of all humanity. The claim that only we, as a group,
nation or civilization, respect human rights, defies itself inadvertently and turns
into an exclusionist ideology, with a claim for superiority, and instigates backlash. g
p
g
From this perspective, since there is a common ground, it is possible to
relate Islamic and modern secular discourse on human rights to each other in the
present world. There is a gap which needs to be bridged here.7 Historians of
religion see Islam as a Western religion. Yet, strikingly, scholars of law are not
aware of this history and its implications on legal thought and practice. The
classical Islamic discourse on human rights may serve as an antecedent or a
significant source, for Muslims and others, to develop or reinvigorate human
rights discourse which would more effectively respond to the needs of the
modern world in the age of globalization. I think Islamic legacy is important to
take into account while re-thinking about human rights at the beginning of the
21st century. Muslims ruled the most troubled areas of the present world for so
many centuries in peace under cosmopolitan empires from India to the Middle
East and to Balkans. 8 For the contrast between “human rights” and “civil rights” see Rex Martin, A System of
Rights, Oxford: Oxford University Press 1997, pp. 73-126. John Dickinson summarized it as
follows: “The term “civil rights” is sometimes used by the courts in the broad sense of rights
enjoyed and protected under positive municipal law in contrast with so-called “inherent
rights” vesting in the individual by virtue of a supposed “natural law”; more frequently it is
used in the United States in a narrower technical sense acquired in constitutional discussion
concerning the legal rights of free Negroes in the years before and immediately following the
Civil War. It was often coupled by way of contrast with the term “political rights”…” John
Dickinson, “Civil Rights” in Encyclopedia of Social Sciences, New York: Macmillan
Company [1930] 1935, vol. 2, p. 513.
9 S
D
ll
ibid
37 45 Muslim World Journal of Human Rights, Vol. 2, Iss. 1 [2005], Art. 11 Muslim World Journal of Human Rights, Vol. 2, Iss. 1 [2005], Art. 11 Scholars from a variety of disciplines disagree on whether universal
human rights are possible or not. Those who agree that universal human rights
are possible also disagree on how. These cleavages in the scholarly community
became manifest during the preparation of the UN Declaration in 1948. The
politicians and scholars were divided on the issue of whether this was a feasible
project. Those who believed that it was feasible were also divided on whether a
single culture should patronize the cause of universal human rights or a
consensus should be sought among all cultures in the world. Some cultures still
express discontent that their voice was not incorporated fully in the UN
Declaration. Hence they prepare alternative human rights declarations. A
considerable number of Muslims are also among them. In contrast, some
Muslims have expressed contentment with the UN Declaration since the
beginning. How to approach to the UN Declaration divided academicians and policy
makers from all religious communities and nations thereby brining to the surface
a deeper cleavage between universalists and communalists or inclusivists and
exclusivists in those religions and nations. Among the politicians, the supporters
of the UN Declaration advocated for the universal human rights while those who
opposed it advocated for the Civil Rights, that is the rights of the citizens in their
state8. Relativist academicians, in particular from Anthropology, accused the UN
for being ethnocentric and imposing Western values on the rest of the world. Pious people from different religions tended to perceive the UN Charter as a
secular attempt to create a new religion for humanity. There is also another gap among scholars concerning whether human
rights are exclusively modern, Western and secular; in other words, whether
universal human rights exist in religious and particularly non-Western cultures. Some argue that religious and non-Western cultures also promote human rights
while some argue that these cultures are incompatible with human rights. The
latter group aims to universalize a particular kind of approach to human rights
which is derived from a certain ideological tradition in the West9. Before any attempt to answer this question, we should ask: what makes
human rights possible? IS UNIVERSAL HUMAN RIGHTS POSSIBLE? IS UNIVERSAL HUMAN RIGHTS POSSIBLE? Published by Berkeley Electronic Press, 2005 3 3 p
y [
]
,
, p
9 See Donnelly, ibid, pp. 37-45. 12 Rawls, John, A Theory of Justice, Cambridge: Harvard University Press 1971; Political
Liberalism, New York: Columbia University Press 1993. 11 See the works of Michael Perry, in particular his recent book, Under God? Religious Faith
and Liberal Democracy, Cambridge University Press, 2003. On the relationship between
religion, morality and law, contrast the works of H.L. Hart (The Concept of Law, Oxford,
[1961] 1997) and his American critics such as Lon Fuller (Morality of Law, 1964) and R. M.
Dworkin (Law’s Empire, 1986). The recent edition of Hart’s The Concept of Law has a
Postscript where he responds to the critiques leveled by Fuller and Dworkin, pp. 238-276.
Also see, Berman, Harold, Faith and Law: the Reconciliation of Law and Religion, Atlanta,
Ga.: Scholars Press, 1993; Berman, Harold, Law and Revolution: the Formation of Western
Legal Tradition, Cambridge, Mass.: Harvard University Press, 1983; Witte, John, Jr. and
Johan D. van der Vyver (eds.), Religious Human Rights in Global Perspective I-II, The Hague
/ Boston / London: Martinus Nijhoff Publishers 1996; An-Na’im, Abdullahi A., Jerald D.
Gort, Henry Jansen, Hendrik M. Vroom (eds.), Human Rights and Religious Values: An
Uneasy Relationship?, Grand Rapids, Michigan 1995; Audi, Robert and Nicholas
Wolterstorff, Religion in the Public Square: The Place of Religious Convictions in Political
Debate, Rowan and Littlefield, 1997; Eberle, Christopher, Religious Convictions in Liberal
Politics, Cambridge University Press 2002; Stout, Jeffrey, Democracy and Tradition,
Princeton Univerity Press 2004. 10 Rorty, Richard, Truth and Progress: Philosophical Books, vol. 3, Cambridge: Cambridge
University Press 1998. In one of his essays Rorty describes how the Bosnians were
dehumanized by the Serbs before the war against them.
11 13 Ann Elizabeth Mayer draws attention the inconsistency between Islamic law in theory and
its actual practice today. See her works, “Universal Versus Islamic Human Rights: A Class of
Cultures or a Clash with a Construct?”, Michigan Journal of International Law, vol. 15, no 2,
Winter 1994, pp. 304-404; also by the same author, Islam and Human Rights: Tradition and
Politics, London 1991. Richard Bulliet draws attention to the contribution of the Western
countries to the oppression in the Muslim world—which means diversion from both Islamic
and universal ideals—by supporting authoritarian governments. See Richard Bulliet, The
Case for Islamo-Christian Civilization, Columbia University Press 2004, p. 120. He writes,
“The people we supported as agents of modernity became tyrants.” Muslim World Journal of Human Rights, Vol. 2, Iss. 1 [2005], Art. 11 Muslim World Journal of Human Rights, Vol. 2, Iss. 1 [2005], Art. 11 There are actual constraints, however, for the realization of the ideal of
universal human rights on the ground on a permanent basis. The absence of due
process and civil society are among these constraints. Therefore even if we have
the universal human rights on the conceptual level, it does not ensure their
existence on the ground. Nor does it ensure their implementation on a
sustainable manner. One of the crucial prerequisites for the implementation of human rights
in a society is the existence of due process. If due process exists in a society, it is
very much likely that human rights will be implemented in that society. Existence
of due process is usually a reflection of the rule of law and formally defined
principles of justice. If there is no due process, we cannot expect human rights to
exist effectively on the ground. Presently, majority of the developing societies
suffer from serious problems in the due process. This observation includes
Muslim countries with authoritarian regimes, even though due process is required
by Islamic law13. The existence of a middle class and civil society, educated about and
committed to human rights, is another crucial prerequisite for the steady
enforcement and endurance of human rights. Even if human rights exist in a
culture on the conceptual level, if there is no civil society to vigilantly defend
them for all, they will be violated by the governments on the ground. In countries
where there is no middle class, we cannot expect human rights to be
implemented in a continuous manner because there will be no deterrent and
punishment if the state violates them. Today, the so-called third world countries,
including Muslim countries, lack a middle class and hence civil society. Consequently, authoritarian regimes prevail in these countries. Society cannot
resist them because they are poor and therefore preoccupied with meeting their
most basic needs. Furthermore, they are not organized to defend their rights. In
such cases, it would be wrong to blame the culture or religion of these societies
for the lack of human rights. In the absence of a middle class, civil society and the democratic
mechanisms to defend human rights, resistance against abusive governments
takes the form of rebellion, insurgence and revolution which may bring about
more abuse of human rights, and eventually turn into a vicious circle. Muslim World Journal of Human Rights, Vol. 2, Iss. 1 [2005], Art. 11 I argue that the existence of an inclusive concept of the
universal human being, detached from innate, acquired and ascribed qualities,
makes the existence of universal human rights likely while its absence makes it
impossible. The universal human is a decontextualized conceptualization of the
human being, which is constructed by methodologically discarding the inherited, 4 http://www.bepress.com/mwjhr/vol2/iss1/art11
DOI: 10.2202/1554-4419.1030 Senturk: Sociology of Rights gained and ascribed physical, cultural, racial, geographical, national and religious
qualities an individual may have. The existence of a concept of the universal human is the primary
prerequisite for the universal human rights to be possible because it is the subject
to which rights are accorded. If the subject is absent, the rights will also be
absent. Therefore, prior to posing the question on whether there are universal
human rights inherent within a culture, we should first ask whether there is a
concept of universal human in the this particular culture. The lack of the latter
(abstract concept of a universal human being) is the cause for the absence of the
former (universal human rights). In the absence of universal recognition of a
human within a society, the legal and political culture relies on the religiously,
culturally, racially or geographically determined exclusive categories, which
forestalls the rise or appropriation of universal human rights within a culture10. pp
p
g
All universal cultures have fostered a concept of human being at the
universal level and the due process to achieve justice in society. Here lies the
common ground universal cultures share. The examples include Buddhism,
Judaism, Christianity, Islam and modern secular ideologies after the European
Enlightenment such as liberalism and socialism. It is possible that some did so
more forcefully in some aspects or in some periods. Secular approaches to
human rights tend to neglect the metaphysical dimension in their justification of
human rights. The lack of metaphysical foundation in the secular discourses may
be seen as a weakness in advocating human rights11. Religious discourses, on the
other hand, tend to focus exclusively on the co-believers. The emphasis on the
religious community based on brotherhood in the true faith may also be seen as a
source of weakness of religious discourses from a secularist perspective12. 5 Published by Berkeley Electronic Press, 2005 Senturk: Sociology of Rights The above mentioned social constraints can only be removed through a
struggle at different levels. The process through which human rights are gained
by social groups or nations requires cultural, social and political struggle. States
usually resist granting human rights to their citizens and other human beings out
of benevolence, except when they are compelled to do so under social and
political pressure. Nor do the states continue respecting human rights if these
pressures seize to exist. These pressures might be internal or international. Yet,
without a solid internal pressure on the state, the international pressure would be
a futile effort because it cannot be sustained for a long time. For the ideal of human rights to be put in practice and protected on the
ground, those who demand these rights and wish to enjoy them on a permanent
base must struggle for them against the authoritarian state. A social group, society
or nation cannot depend on the benevolence of others for the protection of its
human rights. Such clientalism, as all other forms of dependency, is an outcome
of discrepancy of power between client and patron groups and states. The
interference of foreign powers to promote human rights in a society against their
existing state may play a delegitimizing role by turning the internal struggle for
human rights into an illegitimate cause in the eye of citizens who give priority to
freedom of their nation. The lives of great human rights thinkers in the West and East testify that,
besides their theoretical contributions, they also fought for them and a significant
number of them risked or sacrificed their lives for that cause. In Islamic legal
history, the great jurists, mujtahids, not only produced doctrines, ijtihad, to
advocate civil or human rights but at the same time fought for them. For
instance, the father of the doctrine of universal human rights in Islam, Abu
Hanifa, sacrificed his life for the cause of human rights as he was put in prison
and tortured to death at a very old age merely for refusing to cooperate with the
authority. Similarly, Ibn Hanbal, another mujtahid and founder of Hanbali School
of Law, did not compromise on his ijtihad under pressure from state and
demonstrated legendary resistance renown in Islamic history as mihna. Muslim World Journal of Human Rights, Vol. 2, Iss. 1 [2005], Art. 11 Ironically in
such cases human rights of the citizens are not violated only by the authoritarian
states but also at the hands of the groups whose initial goal has indeed been
defending human rights. Only on rare occasions did such movements succeed. http://www.bepress.com/mwjhr/vol2/iss1/art11
DOI: 10.2202/1554-4419.1030 6 Senturk: Sociology of Rights Another
legendary example that deserves mentioning here is Sarakhsi, a great Hanafi
jurist, who advocated universal human rights in his books which he dictated to
his students while in prison. The theoretical contributions of these scholars will
be discussed below. For law to be free from the control of an authoritarian state, minds of
subjects, in particular jurists, must be free. The historical survey below in this
paper demonstrates that the most significant contributions to the theory of
human rights in Islamic law came from liberal minded mujtahids in earlier
generations, who worked independently of the state and refused official posts or
payments. Likewise, there is a correlation between the lack of theoretical
contribution to the theory of human rights in the modern era and the lack of
mujtahids on the model of earlier ones in Muslim societies. The relationship
between advocacy of human rights and ijtihad in Islam may not be accidental; it
may even be seen as an indication that the march to freedom from violation of
human rights begins with freedom of thought which classical scholars called
“inviolability of mind,” ‘ismah al-‘aql. Inner freedom of the agency thus precedes 7 Published by Berkeley Electronic Press, 2005 Muslim World Journal of Human Rights, Vol. 2, Iss. 1 [2005], Art. 11 the struggle for liberation from political oppression and violation of human
rights. Islamic law sanctifies struggle and sacrifice for human rights at the
highest level possible by granting them the honorary title of martyrdom. As I will
explain below, Prophet Muhammad is reported to have repeatedly said that those
who die while protecting their basic human rights such as right to life, property,
religion, honor and family against tyrants or violators are martyrs whom God will
reward with Paradise in the Hereafter. This is because Islamic law assumes that
human rights cannot be protected unless those who have them are ready to
struggle and make sacrifice for them. In the subsequent generations after
Prophet Muhammad from Abu Hanifa to Malcom X many Muslims acted on
that principle and sacrificed their lives. The fight for human rights—since Moses
and Socrates—may easily turn into a bloody struggle for people, prophets and
thinkers. Senturk: Sociology of Rights Consequently, expecting that the ideals Islamic law presents concerning
universal human rights will be realized on the ground without Muslims, who wish
to put them in practice, struggle for them, as other nations did so, would defy the
sociological patterns in the Western and Islamic history of human rights. These social and institutional prerequisites demonstrate that the issue of
human rights is not a merely legal issue which can be solved by reforming law. Instead it requires comprehensive reforms in the legal system and social and
economic structure. Human rights emerge when there is a balance of power in
the society. Otherwise, in the case of absolute imbalance in power relations
between society and the state, the implementation of human rights is left to the
benevolence of the ruling class and international pressures, if there is any, which
never produce sustainable results and reforms. The case of human rights in Islam can be understood better against this
broader backdrop, rather than exclusively concentrating whether Islam has
offered universal human rights at the ideal and conceptual level, which is
necessary but not sufficient for their existence and sustained implementation on
the ground. The present study will contribute to this broad discussion by
providing a balanced and historically well-grounded answer to the following
question: Is there a concept of universal human rights in Islam? Following the above
perspective, the answer is that such a concept is contingent on the existence of
the basis for the universal human being in Islamic law: If the basic conceptual
groundwork exists, then it is likely that Islamic law does feature universal human
rights. Otherwise, without such a framework, it would be impossible to justify
universal human rights. Therefore, we should first ask: Do the fundamental elements
of a universal human being already exist in Islamic law? Senturk: Sociology of Rights Senturk: Sociology of Rights relevant classical and modern literature demonstrates, this is a long and widely
debated issue in the juridical and theological discourse since the early history of
Islam14. Briefly put, there are rival universalistic and communalistic views
represented by a network of Sunni and Shiite scholars, both supported by a rich
literature and sophisticated arguments and counterarguments. The universal
perspective advocates equal human rights for all. In contrast, the communalistic
perspective advocates equal rights only for the citizens of the Islamic state, be
they Muslims or non-Muslims. Yet this contest in the Islamic legal tradition is not
currently known to most scholars in the field of human rights. The lack of
modern literature and research underscores this void in the current discourse. I argue that the latent tension in Islamic law between the advocates of
universalistic and communalistic perspectives, which has so far been
circumvented by the researchers, is analogous to the tension between the
advocates of the civil rights and human rights paradigms in modern Western legal
thought. The recent political debates in the US testify that the advocates of civil
rights, concentrated exclusively on the rights of the citizenry, still hold, despite
the declaration and ratification of the UN declarations since 1948. This may be
attributed to the fact that the European constitutions incorporate the human
rights paradigm while the US constitution incorporates the civil rights paradigm. Hence emerges occasional tensions between the UN and the European
perspectives, on the one hand, and US policies, on the other. The recent debates
on the International Criminal Court (ICC) may be viewed as a manifestation of
this tension. The current US policy concerning the ICC has been to forcefully call
for exemption of US citizens. p
The two rival paradigms in Islamic law have been advocated by two
separate networks of scholars. I will briefly present the views of the two schools
of thought to highlight the existence of the universalistic approach to human
rights—initially formulated by Abu Hanifa, the founder of Hanafite School. Abu
Hanifa’s universalistic paradigm had been adopted by a wide network of scholars
affiliated with different schools of law. However, Abu Hanifa’s ideas have yet to
be fully explored by modern researchers in the West and the Islamic world. 14 For the concept of right (haq pl. huqûq) in Islamic law, see 'Abd al-Razzaq al-Sanhuri,
Masadir al-Haq fi al-Fiqh al-Islami, Beirut: al-Majma' al-'ilmi al-'Arabi al-Islami, 1953-1954,
I, 13-99. HUMAN BEING AND HUMAN RIGHTS IN ISLAM: A CONTESTED
RELATIONSHIP The answer to this much-debated puzzle is not plainly positive or
negative, unlike the monolithic arguments found in the majority of the current
literature based on sweeping and one-sided generalizations. As the survey of the http://www.bepress.com/mwjhr/vol2/iss1/art11
DOI: 10.2202/1554-4419.1030 8 Senturk: Sociology of Rights y
p
y
Most of the concerns and theological arguments of the Muslim jurists
who lived during the middle ages no longer have a ground in the present world,
characterized as it is with radically different and secular national and international
legal concepts and structures. Likewise, before proceeding further, it is worth noting that it would be a stark
anachronism to project modern notions of human rights which emerged during
the peculiar conditions following WWII to the writings of the Muslim jurists who
lived centuries ago. Consequently, the most appropriate way to approach Islamic
legal culture—as it is required for any culture—to try to understand it from
within in its own terms. Therefore, we need, for such a daunting task, to adopt Published by Berkeley Electronic Press, 2005 9 Muslim World Journal of Human Rights, Vol. 2, Iss. 1 [2005], Art. 11 the method of thick description, as Geerts put it15, to avoid the mistakes of the thin
descriptions produced by Muslims and non-Muslims in friendly or adverse terms. The word “Adam” is one such concept that requires careful treatment. It
is commonly used in the Bible and the Quran for “human being,” and in
particular for the first one, with rich connotations. Furthermore, it is one of the
commonly used words which found their way to languages used by Jews,
Christians and Muslims worldwide. It is the Hebrew word for man, deriving
from adama, “earth,” just as the Latin humanus, “human,” is related to the Latin
humus, earth16. In Arabic, âdamî stands for “man” while âdamiyya stands for
“woman.” The legal term âdamiyyah denotes “humanity,” possessing the quality of a
human being, or more technically “personhood.” Islam, like many other
religions, views every human being a perfect creation of God, His representative
on the earth, superior to the creation as a whole including angels, and blessed
with intellect and free-will to be tried by the Creator. The term huquq al- âdamiyyin
is used in the classical literature to indicate the rights of human beings. The term ‘ismah means “inviolability” of a human being which includes
the rights to inviolability of her life, property, religion, reason, family and
honor17. It is a synonym to hurmah, “legal protection”. These rights are
hierarchically ordered; for instance, right to life has overriding power over others. Muslim jurists derived them through a survey of the Quran and hadith along with
rational inquiry. 15 Geertz, Clifford, The Interpretation of Cultures, New York: Basic 1973, p. 14; Shankman,
P., “The Thick and the Thin: On the interpretive Theoretical Program of Clifford Geertz”,
Current Anthropology, 25 (1984), p. 69; Wuthnow, Robert, Meaning and Moral Order:
Explorations in Cultural Analysis. University of California Press 1987.
16 15 Geertz, Clifford, The Interpretation of Cultures, New York: Basic 1973, p. 14; Shankman,
P., “The Thick and the Thin: On the interpretive Theoretical Program of Clifford Geertz”,
Current Anthropology, 25 (1984), p. 69; Wuthnow, Robert, Meaning and Moral Order:
Explorations in Cultural Analysis. University of California Press 1987.
16 Hendrickson, Robert, QBP Encyclopedia of Word and Phrase Origins, 2004, p. 7.
17 In Arabic: ‘ismah al-nafs or ‘ismah al-dam, ‘ismah al-mal, ‘ismah al-din, ‘ismah al-‘aql,
‘ismah al-nasl, and ‘ismah al-‘ird.
18 In the lexicon, the verb ‘asama means “he protected” which is considered synonymous to
waqâ and mana‘a. For instance, ‘asamahu al-ta‘am as a sentence means “the food protected
him from hunger.” The infinitive al-‘ismah means protection. See, al-Fayruzabadi, al-Qamus
al-Muhit, Beirut: Muessese al-Risala 1419/1998, p. 1198; Ibn al-Manzur, Lisan al-‘Arab,
Beirut: Dar al-Ihya al-Turath al-‘Arabi 1419/1999, pp. 244-247. In Islamic theology, the term
‘ismah corresponds to “infallibility” which does not concern us in this article. For the legal
concept al-‘ismah, see Muhammad Rawwas Qal‘aji, al-Mawsu‘ah al-Fiqhiyya al-Muyassara,
Beirut: Dar al-Nafais, 2000/1421, vol. I, p. 1401; for the equivalent term hurmah, see ibid,
vol. I, p. 745-747; For the usage of ‘ismah in Islamic law, see Recep Şentürk, “İsmet” TDV
İslam Ansiklopedisi, vol. 23, p. 137-138; “‘ismah,” in al-Mawsu‘ah al-Fiqhiyye, vol. 30, pp.
137-140. p
y
y
16 Hendrickson, Robert, QBP Encyclopedia of Word and Phrase Origins, 2004, p. 7.
17 18 In the lexicon, the verb ‘asama means “he protected” which is considered synonymous to
waqâ and mana‘a. For instance, ‘asamahu al-ta‘am as a sentence means “the food protected
him from hunger.” The infinitive al-‘ismah means protection. See, al-Fayruzabadi, al-Qamus
al-Muhit, Beirut: Muessese al-Risala 1419/1998, p. 1198; Ibn al-Manzur, Lisan al-‘Arab,
Beirut: Dar al-Ihya al-Turath al-‘Arabi 1419/1999, pp. 244-247. In Islamic theology, the term
‘ismah corresponds to “infallibility” which does not concern us in this article. For the legal
concept al-‘ismah, see Muhammad Rawwas Qal‘aji, al-Mawsu‘ah al-Fiqhiyya al-Muyassara,
Beirut: Dar al-Nafais, 2000/1421, vol. I, p. 1401; for the equivalent term hurmah, see ibid,
vol. I, p. 745-747; For the usage of ‘ismah in Islamic law, see Recep Şentürk, “İsmet” TDV
İslam Ansiklopedisi, vol. 23, p. 137-138; “‘ismah,” in al-Mawsu‘ah al-Fiqhiyye, vol. 30, pp.
137-140. 7 In Arabic: ‘ismah al-nafs or ‘ismah al-dam, ‘ismah al-mal, ‘ismah al-din, ‘ismah al-‘aql,
‘ismah al-nasl, and ‘ismah al-‘ird.
8 Senturk: Sociology of Rights They are justified by evidence from the Quran and Hadith (dalil
manqul) and also by purely rational arguments (dalil manqul). q )
y p
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(
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The issue of universal human rights in Islamic law cannot be
comprehended without fully exploring the rise, evolution and the key role played
by these two concepts. Yet it is a broad subject for which the present article may
serve only as a preliminary introduction. Muslim jurists from the classical period
disagreed on the relationship between ‘ismah18 (inviolability) and âdamiyyah
(humanity). More plainly put, the debate revolved around who possessed the six 10 http://www.bepress.com/mwjhr/vol2/iss1/art11
DOI: 10.2202/1554-4419.1030 Senturk: Sociology of Rights basic rights covered under the title of ‘ismah19. Abu Hanifa and his followers from
Hanafite and other schools attached ‘ismah with âdamiyyah, while al-Shâfii and his
followers from his own and other schools attached it to iman (declaration of
Islamic faith) or amân (making a treaty of security). I call the former Universalistic
School and the latter Communalistic School. Muslim jurists in the classical era unanimously agreed on what rights
should be protected under the coverage of ‘ismah, but there was a question that
divided them: Who has the right to ‘ismah? Is it the entirety of humanity or a
segment of it? Can Islamic law legislate for non-citizens to grant them human
rights? Does all of humanity or the citizenry of the Islamic state alone,
composed of Muslims and non-Muslims, fall under the jurisdiction of Islamic
law? To what extent are Muslims allowed to intervene on legal traditions under
their rule and on what grounds? In brief, there has been consensus about what
constitute basic human rights but there has also been an enduring contest about
who is entitled to them20. Both sides developed arguments to defend their
positions. This issue is far from being resolved even today. Below I will briefly
survey the arguments advanced by both positions. A. The Universalistic View: Basic Rights Are Accorded
By Virtue Of Being A Human Abu Hanifa and his followers21 advanced the cause of universal human
rights – universally and unconditionally granted to all by birth, on a permanent
and equal basis, by virtue of being a human - which cannot be taken away by any
authority. Abu Hanifa established an unbreakable relationship between the
concept of âdamiyyah (personhood, humanity) and the concept of ‘ismah
(inviolability)22. 23 See for instance, al-Marghinani, Burhanaddin ‘Ali ibn Abi Bakr (d. 593 H), al-Hidayah
Sharh Bidayah al-Mubtadi, (eds. Muhammad Muhammad Tamir, Hafiz ‘Ashur Hafiz), Cairo:
Dar al-Salam, 1420/2000, II, 852. The author states that "al-'ismah al-muaththimah bi al-
adamiyyah." This will be discussed below in greater detail. 22 For the concept of ‘ismah in the Hanafi tradition, see “Der ‘isma-Begriff im hanafitischen
Recht” in Johansen, Baber, Contingency in a Sacred Law: Legal and Ethical Norms in the
Muslim Fiqh, Leiden: Brill 1999, pp. 238-262. ,
f
,
( ), pp
21 On the history of the Hanafi School of Law, see “Hanefi Mezhebi” in TDV İslam
Ansiklopedisi, XVI, 1-12.
22 q
20 For the emergence and evolution of these concepts, see Recep Senturk, “Adamiyyah and
‘Ismah: The Contested Relationship between Humanity and Human Rights in the Classical
Islamic Law”, Turkish Journal of Islamic Studies, 2002 (8), pp. 39-70. 19 On the concept of ‘ismah, see Johansen, Baber, Contingency in a Sacred Law: Legal and
Ethical Norms in the Muslim Fiqh, Leiden: Brill 1999. 19 On the concept of ‘ismah, see Johansen, Baber, Contingency in a Sacred Law: Legal and
Ethical Norms in the Muslim Fiqh, Leiden: Brill 1999.
20 For the emergence and evolution of these concepts, see Recep Senturk, “Adamiyyah and
‘Ismah: The Contested Relationship between Humanity and Human Rights in the Classical
Islamic Law”, Turkish Journal of Islamic Studies, 2002 (8), pp. 39-70.
21 On the history of the Hanafi School of Law, see “Hanefi Mezhebi” in TDV İslam
Ansiklopedisi, XVI, 1-12.
22 For the concept of ‘ismah in the Hanafi tradition, see “Der ‘isma-Begriff im hanafitischen
Recht” in Johansen, Baber, Contingency in a Sacred Law: Legal and Ethical Norms in the
Muslim Fiqh, Leiden: Brill 1999, pp. 238-262.
23 See for instance, al-Marghinani, Burhanaddin ‘Ali ibn Abi Bakr (d. 593 H), al-Hidayah
Sharh Bidayah al-Mubtadi, (eds. Muhammad Muhammad Tamir, Hafiz ‘Ashur Hafiz), Cairo:
Dar al-Salam, 1420/2000, II, 852. The author states that "al-'ismah al-muaththimah bi al-
adamiyyah." This will be discussed below in greater detail. Senturk: Sociology of Rights Based on this relationship, he argued that being a child of Adam
or a human, whether Muslim or not, serves as the legal ground for possessing
basic rights (al-’ismah bi al-âdamiyyah)23. Although the concepts of ‘ismah and
“âdamiyyah” require a more thorough explanation, we can phrase this principle in
plain English as follows: Basic human rights are granted to all human beings for
the sake of their humanity. The Hanafites such as Sarakhsi, Zaylai, Dabusi, 11 Published by Berkeley Electronic Press, 2005 Muslim World Journal of Human Rights, Vol. 2, Iss. 1 [2005], Art. 11 Marghinani, Ibn Humam, Bâbartî, Kâsânî and Timurtâshi, to name a few, are of
this opinion24. The universalistic jurists used mainly the following arguments while
defending their doctrine: (1) God’s purpose in creating humanity, the trial (ibtila)
and holding them responsible (taklif) for their actions, cannot be achieved unless
all human beings are granted sanctity and freedom. (2) A human being must be
protected because God does not want His creation to be destroyed, which is
possible only by granting sanctity to each one of them. (3) God in the Quran and
Prophet Muhammad in his sayings strictly prohibited assaulting and slaying any
human being25. They ordered protecting non-Muslim women, children and clergy
even during war. (4) Disbelief (kufr) is not normally harmful to Muslims unless
the disbelievers engage in a war against Muslims. So it must be tolerated. (5) Jihad
is a defensive, but not an offensive, war. Therefore, when non-Muslims do not
assault other people they should enjoy sanctity. (6) The objective of war is not to
exterminate the enemies but to force them to make peace and, if required, pay
tribute. (7) The justifying reason for war is protecting sanctity against those who
assault it. The disbelief of the enemies is not a valid reason to make war against
them. Therefore when peace prevails everyone must enjoy sanctity. (8) The non-
Muslims must be given chance to learn about Islam which they cannot do unless
they are granted sanctity. (9) Compulsion in religion is forbidden in the Quran26. Below we will turn to these arguments in greater detail. g
g
The evidence in the above arguments is either grounded on purely
rational thinking (dalil ma’qul) or on excerpts from the Quran and Hadith (dalil al-
manqul). 27 The Mogul rulers of India outlawed the sati practice although they could not completely
exterminate it. See Sri Ram Sharma, The Religious Policy of the Mughal Emperors, Bombay:
Asia Publishing House 1972, p. 42-44; Zulfaqar Mubed (d. approx. 1670 AD), Hinduism
During the Mughal India of the 17th Century, (tr. David Shea and Antony Troyer), Patna:
Khuda Bakhsh Oriental Public Library [1843] 1993, p. 77. Senturk: Sociology of Rights These arguments are all based on the notion of a universal human and
her place in the network of social relations with other people worldwide. It also
aims to establish peaceful relations not only between Muslims and non-Muslims
but also among non-Muslims from different religions. 24 See for instance, al-Marghinani, al-Hidaya, II, 852; Abu Muhammed Badraddin Mahmud
ibn Ahmad ibn Musa al-Hanafi al-‘Ayni (855/1451) al-Binaya fi Sharh al-Hidaya; (ed. Muhammad ‘Umar). n.p. : Dar al-Fikr, 1980/1400, vol. V, p. 830-831, al-Kâsânî, Alâ’ al-Din
Abi Bakr ibn Mustafa, Bedâi’ al-Sanâi’ fi Tartîb al-Sharâi’, Beirut 1406/1986, vol. VII, p. 233-241. 25 25 “ Nor take life - which Allah has made sacred - except for just cause. And if anyone is slain
wrongfully, we have given his heir authority (to demand retaliation or to forgive): but let him
not exceed bounds in the matter of taking life; for he is helped (by the Law)” (The Quran
17:33). "O believers, be you securers of justice, witness for God. Let not detestation for a
people move you not to be equitable; be equitable - that is nearer to God-fearing" (The Quran
5:8). "...Whoso slays a soul not to retaliate for a soul slain, nor for corruption done in the
land, should be as if he had slain humankind altogether" (The Quran 5:32). In the address
which the Prophet delivered on the occasion of the Farewell Hajj, he said: "Your lives and
properties are forbidden to one another till you meet your Lord on the Day of Resurrection."
The Prophet has also said about the dhimmis (the non-Muslim citizens of the Muslim state):
"One who kills a man under covenant (i.e., dhimmi) will not even smell the fragrance of
Paradise." 26 “Let there be no compulsion in religion: Truth stands out clear from Error: whoever rejects
evil and believes in Allah hath grasped the most trustworthy hand-hold that never breaks. And
Allah heareth and knoweth all things” (The Quran 2:256). 12 http://www.bepress.com/mwjhr/vol2/iss1/art11
DOI: 10.2202/1554-4419.1030 28 It is stated in the best-known Hanafi handbook on Islamic Jurisprudence, al-Manar by Ibn
Habib al-Halabi, that “al-Kuffar Mukhatabun” (Non-Muslims are addressed and held
responsible by God.) See for a commentary on al-Manar, Ibn Qutlubugha (802-879 AH)
Sharh Mukhtasar al-Manar, (ed. Zuhair ibn Nasir al-Nasir), Dimashq: Dar Ibn al-Kathir
1413/1993, p. 66-67. The author explains that the jurists disagreed on whether God required
non-Muslims to fulfill all His commands or to accept the Islamic faith first as a prerequisite
for the rest of the requirements. The Hanafi jurists from Iraq advocated the first view, while
scholars from Central Asia defended the second. See also 'Ala al-Din Abu Bakr Muhammad
b. Ahmad al-Samarqandi (d. 539/1144), Mizan al-Usul fi Nataij al-'Uqul (ed. Muhammad
Zaki 'Abd al-Barr), Qatar 1404/1984, p. 194; Abu al-Barakat Hafızuddin Abdullah ibn Ahmad
ibn Mahmud al-Nasafi (710/1310), Kashf al-Asrar Sharh al-Musannif ‘ala al-Manar, Beirut:
Dar al-Kutub al-‘Ilmiyye 1986. 27 The Mogul rulers of India outlawed the sati practice although they could not completely
exterminate it. See Sri Ram Sharma, The Religious Policy of the Mughal Emperors, Bombay:
Asia Publishing House 1972, p. 42-44; Zulfaqar Mubed (d. approx. 1670 AD), Hinduism
During the Mughal India of the 17th Century, (tr. David Shea and Antony Troyer), Patna:
Khuda Bakhsh Oriental Public Library [1843] 1993, p. 77.
28 It is stated in the best-known Hanafi handbook on Islamic Jurisprudence, al-Manar by Ibn
Habib al-Halabi, that “al-Kuffar Mukhatabun” (Non-Muslims are addressed and held
responsible by God.) See for a commentary on al-Manar, Ibn Qutlubugha (802-879 AH)
Sharh Mukhtasar al-Manar, (ed. Zuhair ibn Nasir al-Nasir), Dimashq: Dar Ibn al-Kathir
1413/1993, p. 66-67. The author explains that the jurists disagreed on whether God required
non-Muslims to fulfill all His commands or to accept the Islamic faith first as a prerequisite
for the rest of the requirements. The Hanafi jurists from Iraq advocated the first view, while
scholars from Central Asia defended the second. See also 'Ala al-Din Abu Bakr Muhammad
b. Ahmad al-Samarqandi (d. 539/1144), Mizan al-Usul fi Nataij al-'Uqul (ed. Muhammad
Zaki 'Abd al-Barr), Qatar 1404/1984, p. 194; Abu al-Barakat Hafızuddin Abdullah ibn Ahmad
ibn Mahmud al-Nasafi (710/1310), Kashf al-Asrar Sharh al-Musannif ‘ala al-Manar, Beirut:
Dar al-Kutub al-‘Ilmiyye 1986. 29 Abi Bakr Muhammad b. Ahmad b. Abi Sahl al-Sarakhsi (d. 490 AH), Usul al-Sarakhsi,
(ed. Abu al-Wafa al-Afghani), Istanbul: Kahraman yay. 1984, p. 86-88.
30 Sarakhsi, Usul, p. 333-334. “Li anna Allah ta’ala lemma khalaqa al-insan li haml amanatih
akramahu bi al-‘aql wa al-dhimmah li yakuna biha ahlan li wujub huquqillah ta’alah alayhi.
Thumma athbata lahu al-‘ismah wa al-hurriyyah wa al-malikiyyah li yabqa fa yatamakkana
min ada’i ma hummila min al-amanati. Thumma hazihi al-amanah wa al-hurriyyah wa al-
malikiyyah thabitah li al-mar’i min hinin yuladu, al-mumayyiz wa ghayr al-mumayyiz fihi
sawaun. Fakazalika al-dhimmah al-saliha li wujub al-huquq fiha thabit lahu min hinin yulad
yastawi fihi al-mumayyiz wa ghayr al-mumayyiz.” Senturk: Sociology of Rights A 13 Published by Berkeley Electronic Press, 2005 Muslim World Journal of Human Rights, Vol. 2, Iss. 1 [2005], Art. 11 human’s religious choice must be honored even if it is in contradiction with the
Islamic teaching. Her life must be protected because this is the only way he can
respond to the divine call29. Her reason must also be honored since reason is the
mechanism by which moral choices of right and wrong are made. Reason is also
the only way through which humans understand the divine message and
implement it. The mind of everyone must be honored and protected even if they
oppose the way we think. The classical doctors of Islamic law used these
theological arguments to justify the six basic rights. For instance, Sarakhsi (d. 1090) wrote: Upon creating human beings, God graciously bestowed upon
them intelligence and the capability to carry responsibilities
and rights (person-hood). This was to make them ready for
duties and rights determined by God. Then He granted them
the right to inviolability, freedom and property to let them
continue their lives so that they can perform the duties they
have shouldered. Then these rights to carry responsibility and
enjoy rights, freedom and property exist with a human being
when he is born. The insane/child and the sane/adult are the
same concerning these rights. This is how the proper
person-hood is given to him when he is born for God to
charge him with the rights and duties when he is born. In this
regard, the insane/child and sane/adult are equal30. The idol-worshippers and polytheists, who lived outside Arabia, had been
allowed to practice their religions freely under Islamic rule. This is because, in
practice, Islamic law extended the status of the “People of the Book” (ahl al-kitab)
to all religions, including such religions as Zoroastrianism, Buddhism and
Hinduism. Therefore these religious communities survived for centuries under
Islamic rule until today. They had been seen as adami and therefore given basic
human rights. g
To illustrate this issue further, we may also briefly look at the Hanafite
view on war. From the Hanafite perspective, denial of Islam (kufr) does not
justify war and deprivation from the six basic rights (‘ismah). For Abu Hanifa,
war, not disbelief, is the cause of war. Senturk: Sociology of Rights The protection of six basic rights is also considered the common ground
of all religions, which provides a juridical ground for religious pluralism. For this
reason they are called “the objectives of the law” (maqasid al-shariah). It is
apparent that Islamic law assumes that people would always belong to a religion,
which is not the case today. According to Islamic theology and jurisprudence,
these six principles constitute the unchangeable core of all religions and the legal
systems in the world. Islam defines its mission as to re-affirm these eternal and
universal principles of law and morality. It is agreed by all Muslims that the creed
(‘aqidah) does not accept alteration but law (shariah) accepts it because societies
evolve and undergo change. Therefore the faiths taught by all the Prophets have
been the same but the laws issued by them changed over time. Yet the main
purpose of all religious legal systems across history--formulated as the protection
of six basic rights--remained unchanged. g
g
One consequence of this approach is that Muslims allowed the non-
Muslim populations they ruled to practice their laws unless it harmed one of the
protected basic rights. For instance, narrative has it that when Egypt was
conquered, ‘Amr ibn ‘Âs allowed the Egyptians to practice their conventional
laws except the custom of sacrificing a girl to the Nile for more water. Likewise,
it is also reported that, in India, the Hindus were allowed to practice their law
except the custom of sati, burning the widow with the body of her late husband27. These two customs in Egypt and India were outlawed by Muslim rulers of the
time because they contradicted the right to life. It was argued that these customs
could not originate from the practice of the founders of these religions because
they normally would respect the six protected basic rights. Similarly, the marriage
between brothers and sisters were outlawed among Zoroastrians in Iran because
it was seen as violating the protection of the family. The above named scholars considered the protection of the six basic
rights necessary based on the argument that the purpose of God in creating the
human family on this earth is “trial” (taklif), which cannot be achieved unless the
human is free and protected. Otherwise, if human beings were not granted basic
freedoms and protections, their purpose on earth would be unrealizable28. Senturk: Sociology of Rights In other words, non-Muslims are protected
during peaceful times since they are human beings (âdamî), and difference of faith
is not a cause for war. Even in the case of war, the enemy side must be granted 14 http://www.bepress.com/mwjhr/vol2/iss1/art11
DOI: 10.2202/1554-4419.1030 Senturk: Sociology of Rights certain rights because âdamiyyah never ceases to exist; however, certain constraints
emerging from the conflict situation apply. Yet violating the ‘ismah of others result in the termination of one’s own
‘ismah, but neither as a whole nor forever. An official court, but not individuals,
determines the consequential punishment based on objective rules. Yet, if the
public authorities fail to protect the ‘ismah of the citizenry, or if they are the ones
who violate the ‘ismah of their own citizens, then, the individual is entitled and
obliged to protect his or her ‘ismah. If people die during the struggle to protect
their ‘ismah, they are revered as martyrs31. In other words, the struggle to protect
basic human rights, such as protecting religion, reason, life, family and property,
which are necessary for a free and just society, is considered to be equally
important as the struggle in the battle to protect the abode of Islam against
outside enemies. This is because the ‘ismah is indivisible and cannot be suspended under
any condition for all humans who are in principle granted the same basic rights
on the equal and permanent basis. However, as far as the criminals who deserve
a punishment are concerned, the ‘ismah becomes divisible according to the
Hanafites and thus during the punishment, it is suspended only in part and for a
limited period of time. The Hanafites claim that only the relevant part (mahall al-
jaza) from the ‘ismah of the criminals, which is legally determined, is suspended
during punishment while the rest remains intact. For instance, the property of a
burglar should still be protected even if he is punished for burglary. The Hanafite School has been strongly influential in the Indian
Subcontinent, Central Asia, Asia Minor and the Balkans, particularly during the
life of the Ottoman State. The discourse of the Ottoman scholars of law
confirms the Hanafite perspective, briefly outlined above. Yet, presently, the
research is lacking to determine the extent to which the Ottoman State actually
followed the Hanafite principles in their seven-centuries-long history. 31 The Prophet Muhammad repeatedly stated that the one who is killed in the struggle to
protect his or her life, property and family or to recover a loan, or to defend himself against
any
kind
of
aggression
is
a
martyr”
(Man
qutila
dûne
malihi/ahlihi/damihi/daynihi/mazlamatin fa huwa shahîd). For numerous narrations on this
issue, see Bukhari, Mazalim 33; Muslim, Iman 226; Abu Dawud, Sunnah 29; Tirmidhi, Diyat
21; Nasai, Tahrim 22-24; Ibn Majah, Hudud 21; Ahmad b. Hanbal, I, 79, 187, 188, 189, 190,
305 and II, 163, 193, 194, 205, 206, 210, 215, 217, 221, 324. Muslim World Journal of Human Rights, Vol. 2, Iss. 1 [2005], Art. 11 Muslim World Journal of Human Rights, Vol. 2, Iss. 1 [2005], Art. 11 The modern concepts of citizenship and rights are based on different
philosophical grounds than the way they were viewed by classical scholars of
Islamic law. Yet despite the manifest differences between the pre-modern-
universalistic approach in Islamic law and the modern legal thought, which I do
not need to enumerate here for our present purpose, a similarity is striking
concerning the concept of a universal human, which serves in both legal cultures
as the philosophical foundation of universal human rights. Abu Hanifa’s influence continued until the beginning of the 20th century. For instance, Al-Miydani (d. 1881), a Syrian scholar from Damascus, wrote at the
end of the 19th century that the person has sanctity by virtue of her existence (al-
Hurr ma‘sum bi nafsihi)32. By the fall of the Ottoman State, the Hanafi view
suffered from an eclipse until today. The so-called contemporary “Islamic” states
disinherited the Ottoman legacy and disowned the universalistic view in Islamic
law in favor of the communalistic legal doctrine on human rights, which will be
outlined below. B. The Communalistic View: Basic Rights Are Accorded By
Virtue Of Islamic Faith Or A Treaty The competing discourse network, emanating from al-Shâfii and crossing
the conventional school borders, also gained followers from other schools of
thought. This discourse lacks the abstract concept of human qua human as the
possessor of rights. Instead, it relies on the religiously defined categories, such as
disbeliever (kafir) and believer (mu’min). f
Non-Shâfi‘ite scholars such as Imam Mâlik (712-795), Ahmad ibn Hanbal
(780-855), and the majority of their followers (e.g. Dawud al-Zahiri, Ibn Hajar al-
Haythami, Shirbini, Kurtubi, Karafi, Bujayrimi, Ibn Arabi, Khallaf) also defend
the same perspective. Although its first renowned advocate was al-Shâfii, an
inter-school network of scholars defends this perspective. The majority of the
classical Shiite scholars also adopted the same approach (e.g. Tûsî, and Hilli). These scholars generally use the following arguments: (1) The injunction
on fighting against infidels in the Quran33 is a general commandment. (2) The
Prophet said: “I am ordered to fight against people until they say: there is no
deity but Allah.” (3) Disbelief (kufr), they argue, is the worst sin and cannot be
allowed. 32 Al-Miydani, al-Lubab fi al-Sharh al-Kitab, (ed. Muhammad Muhyiddin Abdulhamid),
Cairo 1383/1963, vol. IV, p.128.
33 Tawba 9:5, 12; Anfal 8:39. Senturk: Sociology of Rights At this
moment, the only observation we can make with certainty is that they gave
primacy, at least in the official discourse of the Millet System, to the Hanafite law
in their effort to rule a multi-national and multi-religious state on a vast
geography for an exceptionally longer period of time. The Ottoman legal
discourse on the Millet System and the debated rights of non-Muslims under
Ottoman rule can be followed in the writings of the Ottoman Shaikhulislams and
Ulama on Fiqh. The Ottoman example is one among many parallel examples
from Andalusia to India. Therefore, although it should not be seen as the only or
the authentic practice of Islam, Ottoman experience provides a significant and
relatively recent Islamic example for a noticeably plural society under Islamic
rule. 15 Published by Berkeley Electronic Press, 2005 15 Muslim World Journal of Human Rights, Vol. 2, Iss. 1 [2005], Art. 11 Based on my initial research, the category of a universal human as the
subject of law, comparable to the Hanafite concept of âdamiyyah, does not exist in
the legal thought of the scholars who subscribe to the communalistic doctrine. Instead, their legal thought relies on the religiously defined categories of
“Muslims” and “non-Muslims.” For them, Muslims are qualified for the ‘ismah
by virtue of their faith (iman). However, non-Muslims are not qualified for the
‘ismah unless they make a treaty with the Muslim state and secure their protection 16 http://www.bepress.com/mwjhr/vol2/iss1/art11
DOI: 10.2202/1554-4419.1030 16 Senturk: Sociology of Rights in exchange for the taxes they pay. This treaty is called dhimmah and the tax paid
for it is called jizya. According to Hanafites, the treaty of dhimmah is not a reason
for ‘ismah (which is already universally present), rather it is an alliance against the
third parties and allegiance to the state. Likewise, according to the majority of the
Shâfi‘ites, being a non-Muslim, with the exception of dhimmis, is a cause for war. From the communalistic perspective, since non-Muslims do not have ‘ismah, the
relationship between Muslims and non-Muslims is considered to be a continuous
state of war unless there is a treaty of peace. Yet, according to the Hanafites,
non-Muslims who are not the citizens of the Islamic state are also protected
because they have ‘ismah as humans. Likewise, the apostate (murtadd) is
punishable because of his—but not her—apostasy (kufr), according to the
Shâfi‘ites. For Hanafites, apostasy is punishable, not because it is a denial of
Islam as a true religion, but because of the conspiracy it involves against the
community and the confusion of faith it causes.( This issue will be further
explored below.) These points can be seen as just some implications of the lack
of a concept of the universal human and the rights attributed to it in the Shâfi‘ite
doctrine. The Shâfi‘ite view, which is also shared by a significant number of
scholars from the Mâlikite, the Hanbalite and Shiite schools, has been influential
in Hijaz, Egypt, North Africa, Spain and Iran in varying degrees until the
Ottoman rule took over. The Jews and Christians residing in these regions
maintained their life as dhimmis who possessed ‘ismah due to their treaty with the
Islamic rulers who followed the Shâfi‘ite doctrine. Muslim World Journal of Human Rights, Vol. 2, Iss. 1 [2005], Art. 11 A method of historical and contextual interpretation of the legal evidence
is needed to critically examine the arguments. This approach has already been
used by the scholars who adopted the universalistic approach to basic human
rights in their counterarguments against those who called for a communalistic
view. The communalistic arguments, as briefly presented above, are criticized as
follows: Regarding the first and second arguments, it is claimed that the various
orders in the Quran and Hadith to fight against non-Muslims apply to the times
of war and to a particular group of Arab polytheists living in Hijaz. Therefore,
these orders cannot be generalized to the times of peace and to other people
outside Arabia. Against the third communalistic argument mentioned above, it is
argued that the non-Muslims must be given chance to learn about Islam. Besides,
Islamic law does not punishes all sins against God unless they harm other
members of the society. Punishing disobedience against God is not the duty of
people unless their well-being is affected by it. Furthermore, the compulsion to
accept Islam is forbidden. On a more philosophical level, the prominent Hanafi
scholar Marghinani (d. 1197) criticized the Shâfi‘ite view as follows: With respect to the arguments of al-Shâfii, we reply that his assertion,
that the “sin-creating protection (al-’ismah al-mu’thimah) is attached to Islam”
is not admitted; for, the sin-creating protection is attached, not to Islam,
but to the person; because man is created with an intent that he should
bear the burdens imposed by the LAW, which men would be unable to
do unless the molestation or slaying of them were prohibited, since if Published by Berkeley Electronic Press, 2005 17 Muslim World Journal of Human Rights, Vol. 2, Iss. 1 [2005], Art. 11 the slaying of a person were not illegal, he would be incapable of
performing the duties required of him. The person therefore is the original
subject of protection, and property follows as the dependant thereof,
since property is, in its original state, neutral, and created for the use of
mankind, and is protected only on account of the right of the
proprietor, to the end that each may be enabled to enjoy that which is
his own….34 The universalistic approach crossed the boundaries of the Hanafite
school and gained followers from other schools of thought (madhhab) in Islam,
which gave rise to an inter-school discourse network. Muslim World Journal of Human Rights, Vol. 2, Iss. 1 [2005], Art. 11 A brief survey of other like-
minded scholars and their intellectual affiliation will demonstrate this structure. Non-Hanafite scholars such as Ghazzali from the Shâfi‘ite school, Ibn Taymiyya
and Ibn al-Qayyim al-Jawziyya from the Hanbali school, Ibn Rushd, Shâtibî and
Ibn al-‘Âshûr from the Mâlikite school, and Maghniyya from the Jafari Shiite
School also share the universalistic view initially advanced by the Hanafites. Therefore, it would be misleading to take the universalistic view on human rights
as an exclusively Hanafite perspective—despite the fact that its originator was
Abu Hanifa. 34 Ebü'l-Hasan Burhaneddin Ali b. Ebi Bekr Merghinani, The Hedaya or Guide: a
Commentary on the Mussulman Laws, (tr. Charles Hamilton), Karachi: Daru'l-Ishaat, 1989, II,
pp. 201-2. IMPLICATIONS OF THE CONTEST BETWEEN UNIVERSALISTIC
AND COMMUNALISTIC DOCTRINES The preceding cleavage shaped many issues in the Islamic legal tradition
as the advocates of the contesting paradigms systematically and persistently took
their views to their logical ends. They projected their perspectives on all the
relevant practical questions in social and international relations. Therefore, there
are numerous political and legal issues emanating from it. Fully recovering all the
implications is not our purpose here, which may require a painstaking survey of
all classical literature. The purpose here is to demonstrate the wide ranging
impact of the contest on the relationship between the human and human rights. The examples, presented below, will suffice for this purpose. 36 For the views of Muslim jurists on other legal systems, see Fakhr al-Islam Bazdawi (d.
482/1089), Kanz al-Wusul ila Ma’rifat al-Usul, Karachi: Mir Muhammad Kutuphana Markaz
‘Ilm wa Adab n.d. For a commentary on it, see ‘Alauddin ‘Abdulaziz ibn Ahmad al-Bukhari
(d. 730), Kashf al-Asrar ‘an Usul Fakhr al-Islam al-Bazdawi (ed. Muhammad al-Mu’tasim
Billah al-Baghdadi), Beirut: Dar al-Kitab al-Arabi 1418/1997, pp. 397-405. 35 Ömer Nasuhi Bilmen, Hukuk-ı İslamiyye Kamusu, İstanbul: Bilmen Yayınevi, nd, III, p.
356. Muhammad al-Shaibani authored two important books on the international law, which he
termed al-Siyar, to help regulate international and inter-communal relations between Muslims
and non-Muslims. Al-Shaibani’s book on international relations, al-Siyar al-Kabir, was
among the first books translated into Turkish and published after the Ottomans opened a
printing house in Istanbul. (see Ebu Bekr Şemsüleimme Muhammed b. Ahmed b. Sehl Serahsi
(483/1090), Tercüme-i Şerh-i Siyeri'l-Kebir (trans. Mehmed Münib Ayıntabi) İstanbul :
Matbaa-i Amire, 1825.) This evidence demonstrates the significant role of al-Shaibani’s
legacy in shaping the Ottoman practice. For English translation of al-Shaibani’s work, see
Muhammad Hasan al-Shaibani, The Islamic Law of Nations: Shaybani’s Siyar, (tr. Majid
Khadduri) Baltimore: John Hopkins University Press 1966. Shaibani’s work was also
translated to French by Muhammad Hamidullah (Le Grand Livre de la Conduite de l'Etat =
Siyerü'l-kebir, Ankara : Türkiye Diyanet Vakfı, 1990). 1.
What is the subject of law, humans or citizens? Abu Hanifa and his followers made “human being” or “person” the
subject of law to which rights and responsibilities are accorded. This
universalistic approach is evident in the Hanafi definition of law: “knowledge of
the self about her rights and duties” (ma’rifah al-nafs ma laha wa ma alaiha). It
should be noted that, in this definition, law is not restricted to Muslims or http://www.bepress.com/mwjhr/vol2/iss1/art11
DOI: 10.2202/1554-4419.1030 18 18 Senturk: Sociology of Rights citizens alone; nor a reference is made to a particular way of knowing rights and
duties, be it religious or secular. In contrast, the subject of law from the communalistic perspective is the
citizenry, or more precisely the subjects of the Islamic state, be they Muslims or
non-Muslims. Therefore, rights and duties can only be accorded to the citizenry. From this perspective, non-citizens are not seen eligible for “personhood” which
would enable them to bear rights and duties. The compact of dhimmah, which
literally means “liability,” entitles them to the right for personhood. It is a
prerequisite for the entitlement to all other rights and duties. 3.
Whose ‘ismah are we required to protect? 35 Ömer Nasuhi Bilmen, Hukuk-ı İslamiyye Kamusu, İstanbul: Bilmen Yayınevi, nd, III, p.
356. Muhammad al-Shaibani authored two important books on the international law, which he
termed al-Siyar, to help regulate international and inter-communal relations between Muslims
and non-Muslims. Al-Shaibani’s book on international relations, al-Siyar al-Kabir, was
among the first books translated into Turkish and published after the Ottomans opened a
printing house in Istanbul. (see Ebu Bekr Şemsüleimme Muhammed b. Ahmed b. Sehl Serahsi
(483/1090), Tercüme-i Şerh-i Siyeri'l-Kebir (trans. Mehmed Münib Ayıntabi) İstanbul :
Matbaa-i Amire, 1825.) This evidence demonstrates the significant role of al-Shaibani’s
legacy in shaping the Ottoman practice. For English translation of al-Shaibani’s work, see
Muhammad Hasan al-Shaibani, The Islamic Law of Nations: Shaybani’s Siyar, (tr. Majid
Khadduri) Baltimore: John Hopkins University Press 1966. Shaibani’s work was also
translated to French by Muhammad Hamidullah (Le Grand Livre de la Conduite de l'Etat =
Siyerü'l-kebir, Ankara : Türkiye Diyanet Vakfı, 1990).
36 For the views of Muslim jurists on other legal systems, see Fakhr al-Islam Bazdawi (d.
482/1089), Kanz al-Wusul ila Ma’rifat al-Usul, Karachi: Mir Muhammad Kutuphana Markaz
‘Ilm wa Adab n.d. For a commentary on it, see ‘Alauddin ‘Abdulaziz ibn Ahmad al-Bukhari
(d. 730), Kashf al-Asrar ‘an Usul Fakhr al-Islam al-Bazdawi (ed. Muhammad al-Mu’tasim
Billah al-Baghdadi), Beirut: Dar al-Kitab al-Arabi 1418/1997, pp. 397-405. 4.
Is apostasy a crime and why? The universalistic perspective argues that apostasy alone is not a
punishable crime unless it is accompanied by a conspiracy to harm the sanctity of
Islam as a religion. This may happen through discrediting Islamic religion with
propaganda. The verse in the Quran on apostasy was revealed after a group of
conspirators decided to enter Islam as a group and leave it after a short while to
persuade others that they could not find what they looked for in Islam39. Therefore, for the stipulation about the punishment to be applicable to a
particular case of apostasy, it must be carried on with the purpose of conspiracy
against Islam, but not out of mere conviction. From this perspective, a non-
Muslim, even if he is an apostate, is not by default an enemy. 37 Hence comes the principle that “a human being is honored, even if he is a non-Muslim.”
(al-Adamiy mukarram wa law kafiran.) Ibn ‘Abidin, Hashiya, V, 58. Ibn ‘Abidin also notes
that slavery contradicts with this principle. 38 Ahmet Özel, İslam Hukukunda Ülke Kavramı: Darulislam, Darulharb, İstanbul: İz
Yayıncılık, 1998, p. 57. 39 39 “A section of the People of the Book say: "Believe in the morning what is revealed to the
believers [Muslims], but reject it at the end of the day; perchance they may (themselves) turn
back; and believe no one unless he follows your religion." Say: "True guidance is the
Guidance of God. (Fear ye) Lest a revelation be sent to someone (else) Like unto that which
was sent unto you? or that those (Receiving such revelation) should engage you in argument
before your Lord?" Say: "All bounties are in the hand of God. He granteth them to whom He
pleaseth: And God careth for all, and He knoweth all things." For His Mercy He specially
chooseth whom He pleaseth; for God is the Lord of bounties unbounded. Among the People
of the Book are some who, if entrusted with a hoard of gold, will (readily) pay it back; others,
who, if entrusted with a single silver coin, will not repay it unless thou constantly stoodest
demanding, because, they say, "there is no call on us (to keep faith) with these ignorant
(Pagans)." but they tell a lie against God, and (well) they know it. Nay.- Those that keep their
plighted faith and act aright,-verily God loves those who act aright. Muslim World Journal of Human Rights, Vol. 2, Iss. 1 [2005], Art. 11 We are required to protect the sanctity of all humanity, argue the
universalistic jurists. For them, all human beings fall under the jurisdiction of
Islam. Therefore, Muslims must stand even for the human rights of non-
Muslims. Each individual, community and state is responsible for the entirety of
humanity. Failing to act makes each one of them accountable, legally and
religiously37. In contrast, the communalist jurists claim that we are responsible only to
protect the sanctity of the citizenry. Disbelief disqualifies non-Muslims who did
not make a treaty with the Islamic state to have human rights. Consequently,
Muslims are not responsible for the rights of non-Muslims. Also, they argue,
only the citizenry falls under the jurisdiction of Islamic state38. 37 Hence comes the principle that “a human being is honored, even if he is a non-Muslim.”
(al-Adamiy mukarram wa law kafiran.) Ibn ‘Abidin, Hashiya, V, 58. Ibn ‘Abidin also notes
that slavery contradicts with this principle.
38 Ahmet Özel, İslam Hukukunda Ülke Kavramı: Darulislam, Darulharb, İstanbul: İz
Yayıncılık, 1998, p. 57.
39 “A section of the People of the Book say: "Believe in the morning what is revealed to the
believers [Muslims], but reject it at the end of the day; perchance they may (themselves) turn
back; and believe no one unless he follows your religion." Say: "True guidance is the
Guidance of God. (Fear ye) Lest a revelation be sent to someone (else) Like unto that which
was sent unto you? or that those (Receiving such revelation) should engage you in argument
before your Lord?" Say: "All bounties are in the hand of God. He granteth them to whom He
pleaseth: And God careth for all, and He knoweth all things." For His Mercy He specially
chooseth whom He pleaseth; for God is the Lord of bounties unbounded. Among the People
of the Book are some who, if entrusted with a hoard of gold, will (readily) pay it back; others,
who, if entrusted with a single silver coin, will not repay it unless thou constantly stoodest
demanding, because, they say, "there is no call on us (to keep faith) with these ignorant
(Pagans)." but they tell a lie against God, and (well) they know it. Nay.- Those that keep their
plighted faith and act aright,-verily God loves those who act aright. As for those who sell the
faith they owe to God and their own plighted word for a small price, they shall have no
portion in the Hereafter: Nor will God (Deign to) speak to them or look at them on the Day of
Judgment, nor will He cleans them (of sin): They shall have a grievous penalty. There is
among them a section who distort the Book with their tongues: (As they read) you would
think it is a part of the Book, but it is no part of the Book; and they say, "That is from God,"
but it is not from God. It is they who tell a lie against God, and (well) they know it!” (The
Quran, 3:73-78). 37 Hence comes the principle that “a human being is honored, even if he is a non-Muslim.”
(al-Adamiy mukarram wa law kafiran.) Ibn ‘Abidin, Hashiya, V, 58. Ibn ‘Abidin also notes
that slavery contradicts with this principle.
38 Ahmet Özel, İslam Hukukunda Ülke Kavramı: Darulislam, Darulharb, İstanbul: İz
Yayıncılık, 1998, p. 57.
39 2.
What is the de facto state of international relations? Peace, the universalistic approach argues, is the de facto state of
international relations between Muslims and non-Muslims, unless otherwise
proven. By default, non-Muslims are friends. If there are indications proving the
contrary, then they are considered enemies. For them “the cause of war is war.”
In other words, if non-Muslims initiate war, Muslims also engage a defensive war
against them. The universalistic scholars carefully distinguished between adversity
(harb) and infidelity (kufr): all enemies may be infidels but not all the infidels are
enemies35. War, argues the communalist perspective, is the de facto state of relations
between Muslims and non-Muslims36, unless otherwise proven, on the grounds
that the cause of war is infidelity (kufr). Consequently, non-Muslims are by
default enemies (harbi); if there is an indication to the contrary, then, they are
considered friends. Therefore, non-Muslims, with no treaty of peace, have no
sanctity. 3. Whose ‘ismah are we required to protect? 35 Ömer Nasuhi Bilmen, Hukuk-ı İslamiyye Kamusu, İstanbul: Bilmen Yayınevi, nd, III, p. 356. Muhammad al-Shaibani authored two important books on the international law, which he
termed al-Siyar, to help regulate international and inter-communal relations between Muslims
and non-Muslims. Al-Shaibani’s book on international relations, al-Siyar al-Kabir, was
among the first books translated into Turkish and published after the Ottomans opened a
printing house in Istanbul. (see Ebu Bekr Şemsüleimme Muhammed b. Ahmed b. Sehl Serahsi
(483/1090), Tercüme-i Şerh-i Siyeri'l-Kebir (trans. Mehmed Münib Ayıntabi) İstanbul :
Matbaa-i Amire, 1825.) This evidence demonstrates the significant role of al-Shaibani’s
legacy in shaping the Ottoman practice. For English translation of al-Shaibani’s work, see
Muhammad Hasan al-Shaibani, The Islamic Law of Nations: Shaybani’s Siyar, (tr. Majid
Khadduri) Baltimore: John Hopkins University Press 1966. Shaibani’s work was also
translated to French by Muhammad Hamidullah (Le Grand Livre de la Conduite de l'Etat =
Siyerü'l-kebir, Ankara : Türkiye Diyanet Vakfı, 1990). 19 Published by Berkeley Electronic Press, 2005 4.
Is apostasy a crime and why? As for those who sell the
faith they owe to God and their own plighted word for a small price, they shall have no
portion in the Hereafter: Nor will God (Deign to) speak to them or look at them on the Day of
Judgment, nor will He cleans them (of sin): They shall have a grievous penalty. There is
among them a section who distort the Book with their tongues: (As they read) you would
think it is a part of the Book, but it is no part of the Book; and they say, "That is from God,"
but it is not from God. It is they who tell a lie against God, and (well) they know it!” (The
Quran, 3:73-78). 20 http://www.bepress.com/mwjhr/vol2/iss1/art11
DOI: 10.2202/1554-4419.1030 40 “Those who believe, then reject faith, then believe (again) and (again) reject faith, and go on
increasing in unbelief,- Allah will not forgive them nor guide them nor guide them on the
way. To the Hypocrites give the glad tidings that there is for them (but) a grievous penalty”
(The Quran 4:137). “They swear by Allah that they said nothing (evil), but indeed they
uttered blasphemy, and they did it after accepting Islam; and they meditated a plot which they
were unable to carry out: this revenge of theirs was (their) only return for the bounty with
which Allah and His Messenger had enriched them! If they repent, it will be best for them; but
if they turn back (to their evil ways), Allah will punish them with a grievous penalty in this
life and in the Hereafter: They shall have none on earth to protect or help them” (The Quran
9:74). “And there are those who put up a mosque by way of mischief and infidelity - to
disunite the Believers - and in preparation for one who warred against Allah and His
Messenger aforetime. They will indeed swear that their intention is nothing but good; But
Allah doth declare that they are certainly liars” (The Quran 9:107). Senturk: Sociology of Rights The communalist perspective, however, argues that apostasy in itself is a
punishable crime. This argument is based on the verse from the Quran40; it does
not take into account the historical circumstances in which the stipulation was
made. The apostate looses his citizenship by loosing his religion because, for the
communalist jurists, citizenship is granted to him by virtue of faith. Consequently, he also looses his sanctity. From this perspective, since the
apostate is a non-Muslim who is not a citizen, by default he is considered an
enemy. I should also note that none of the schools in Islamic law requires
punishment of an apostate woman who has left Islam. They also unanimously
agree that non-Muslim women must not be killed during war because of the utter
prohibition by the Prophet Muhammad. Hanafi scholars used this unanimously
accepted practice to support their claim that the apostate is not punished for
leaving Islam or converting to another religion but for plotting against Islamic
community. They argued that had the apostasy was the ground for the
punishment of apostate, the female apostate would also be punished the same
way as the male apostate. Therefore, they conclude, it is not apostasy, but
engaging in a war or conspiracy against Muslims is the reason why the apostate is
punished. 5.
What are the implications on woman’s human rights? Since the protection of family is a basic human right, some of the
disagreements in the Islamic family law may be traced back to the contesting
positions on the human rights. The universalistic perspective does not make a
distinction in principle between man and woman; both are considered human,
adami, and are entitled to the same human rights. However, outside the basic
human rights, one can discern, looking back from a modern perspective, that
women are treated differently than men in such areas as inheritance, testimony
and family law. These practices were not traditionally considered unequal
treatment. Yet, the notion of equality and the gender roles have undergone a
great change during modernization. These changes may be attributed to the
prevailing customs and structures that influence law. Islamic jurisprudence states
that custom always changes; so are the rules grounded on them. There is a heated 40 “Those who believe, then reject faith, then believe (again) and (again) reject faith, and go on
increasing in unbelief,- Allah will not forgive them nor guide them nor guide them on the
way. To the Hypocrites give the glad tidings that there is for them (but) a grievous penalty”
(The Quran 4:137). “They swear by Allah that they said nothing (evil), but indeed they
uttered blasphemy, and they did it after accepting Islam; and they meditated a plot which they
were unable to carry out: this revenge of theirs was (their) only return for the bounty with
which Allah and His Messenger had enriched them! If they repent, it will be best for them; but
if they turn back (to their evil ways), Allah will punish them with a grievous penalty in this
life and in the Hereafter: They shall have none on earth to protect or help them” (The Quran
9:74). “And there are those who put up a mosque by way of mischief and infidelity - to
disunite the Believers - and in preparation for one who warred against Allah and His
Messenger aforetime. They will indeed swear that their intention is nothing but good; But
Allah doth declare that they are certainly liars” (The Quran 9:107). Published by Berkeley Electronic Press, 2005 21 Muslim World Journal of Human Rights, Vol. 2, Iss. 1 [2005], Art. 11 debate currently going on in this area, which falls beyond the scope of this
paper41. 7.
What is the Jizya for? 41 See, Abdullahi A. an-Na’im (ed.), Islamic Family Law in a Changing World : a Global
Resource Book, London: Zed Books, 2002. 5.
What are the implications on woman’s human rights? According to Hanafi scholars, a woman, be she a virgin or a widow, can
marry herself independently. In all schools, a marriage contract is invalid without
the consent of woman. Yet, the communalistic perspective gives greater authority
to the family over a woman’s marriage; a marriage contract is invalid without the
consent of a guardian from the family of the woman. The consent of both the
bride and her guardian are among the prerequisites for a valid marriage contract. A virgin cannot conduct the marriage act by herself without the permission of a
guardian; only a widow can marry herself independently. Communalist jurists
argued that the requirement of consensus concerning marriage contract between
the virgin bride and her family serves the interests of the woman better because
the guardians are more experienced in the intricacies of marriage than the
inexperienced young woman. The Hanafis object to this approach by arguing that
if she is allowed to make sales contract—which is unanimously accepted by all
schools, she should also be allowed to make a marriage contract because sales
contract also involves risks for her interests. Islamic law has produced a complicated system of ending marriage,
involving methods and concepts that may have no parallels in modern law. Marriage may be conducted and dissolved independently, by the consent of the
parties involved, without authorization from state or religious authorities. Without going into details, it suffices us to say that the Hanafi law grants equal
rights to a unilateral dissolution of marriage (talaq); both parties are entitled to
negotiate on the three rights of unilateral divorce without the court’s decision
(tawfid al-talaq). According to the Shafii School, however, a woman is not entitled
to the unilateral dissolution of marriage (talaq). Both schools accept that she is
entitled to file a divorce with the court, in which case the dissolution is produced
by a court decision (tafriq). 42 For the rights of non-Muslims under Islamic rule, see Senturk, Recep, “Minority Rights in
Islam: From Dhimmi to Citizen” in Islam and Human Rights: Advancing a U.S.-Muslim
Dialogue (Shireen T. Hunter, with Huma Malik, ed., Washington, D.C.: CSIS, forthcoming
2005); Zaydan, ‘Abd al-Karim, Ahkam al-Dhimmiyyin wa al-Müsta'manin fi Dar al-Islam,
Baghdad : Maktabat al-Quds, 1982.
43 On this discussion see, Ibn Qayyim al-Jawziyya, Ahkam Ahl al-Dhimmah, (ed. By Subhi
Salih), Beirut: Dar al-‘Ilm li al-Malayin [1961] 1983, pp. 18-25. Ibn Qayyim refutes the
Shafii view.
44 Mehmet Yıldız, “1856 Islahat Fermanına Giden Yolda Meşruiyet Arayışları: Uluslararası
Baskılar ve Cizye Sorununa Bulunan Çözümün İslami Temelleri,” Türk Kültürü İncelemeleri
Dergisi 7, İstanbul 2002, 75-117. 6.
Does indictment cause ‘ismah to fall entirely or in part? This issue is related to the rights of the criminals and prisoners. Indictment does not cause the ‘ismah to fall completely in any school of law; all
agree that an indicted person still enjoys basic human rights. However, the
Hanafi jurists are more attentive to keep it as intact as possible. Consequently,
they refuse coupling reparation with punishment. For instance, from the Hanafi
perspective, either punishment or reparation is required to punish theft (sirqat). However, the Shafii scholars argue that both punishment and reparation apply in
the case of theft. 22 http://www.bepress.com/mwjhr/vol2/iss1/art11
DOI: 10.2202/1554-4419.1030 Senturk: Sociology of Rights Non-Muslim citizens are required to pay tax to the Muslim state which is
termed jizya42. Scholars disagreed on why such a tax was imposed on non-
Muslims. The Communalist School sees the jizya as a fee for the security
provided by the state to its non-Muslim subjects. Yet the Universalistic School
objects to this view. According to the Universalistic school, the jizya is merely a
tax on non-Muslim citizens, comparable to zakat which Muslim citizens are
required to pay. For them jizya cannot be seen as a fee for security, because
security is the natural right of all human beings regardless of their citizenship,
who they are and where they live43. Ottomans abolished jizya as part of the late
nineteenth century reforms in Islamic law because of its discriminatory
approach44. These examples are sufficient to observe how the different positions on
the issue of ‘ismah led to different legislations. A wide array of judgments
emanated from the contest between rival paradigms on human rights. Exploring
the tension between universalistic and communalistic perspectives thus allows us
better understand the diversity in Islamic law and appreciate the logic behind it. 44 Mehmet Yıldız, “1856 Islahat Fermanına Giden Yolda Meşruiyet Arayışları: Uluslararası
Baskılar ve Cizye Sorununa Bulunan Çözümün İslami Temelleri,” Türk Kültürü İncelemeleri
Dergisi 7, İstanbul 2002, 75-117. MUSLIMS VIS-À-VIS UNIVERSALISTIC HUMAN RIGHTS IN THE MODERN ERA The dichotomy between the Universalistic and Communalistic Schools in
Islamic law played a decisive role in determining the approach Muslims adopted
toward modern universal human rights discourse. The reaction of the Muslims to
the rise and spread of universal human rights in the era of modernization is also
characterized by their earlier familiarity with the abstract concept of the universal
human. The contemporary impact of the time-honored conflicting views on who
has the ‘ismah is also observable in a survey of the diverse Muslim responses to
the evolution of human rights in the West and their penetration in the Islamic
world. The first concrete action on the state level begins with the Declaration of
the Tanzimat (Royal Charter of Regulations) in 1839 in the Ottoman State by
Sultan Mahmud II. The latest significant example may be “the Cairo Declaration
on Human Rights in Islam” by the Organization of the Islamic Conference
(OIC) in 1990. The Ottoman Caliph, advised mostly by the Hanafite Ulama, granted
equal rights to non-Muslims for the protection of life, property, honor and
religion in the 1839 declaration of Tanzimat. Later, other declarations concerning
human rights had also been issued in the reforming Ottoman State, which, in 23 23 Published by Berkeley Electronic Press, 2005 Muslim World Journal of Human Rights, Vol. 2, Iss. 1 [2005], Art. 11 some aspects, resembled the decrees by earlier sultans known as Adalatname45 or
Kanunname. Faced with Western ideological and cultural influence, the Ottomans
had to compete with the European powers in extending rights to their citizens on
equal basis. They had Hanafite law at their disposal to achieve this objective. The
universalistic approach to human rights made it possible for them to reform
Islamic law, parallel to changing legal customs. The major debate, carried on by the Ottoman bureaucrats, Ulama and the
intellectuals, during the second half of the nineteenth century, revolved around
whether rights should be given equally to all citizens and how to limit the power
of the Ottoman sultan. The Charter of Allaince in 1808, between the Sultan and
the Dignitaries, ignited this trend. The Ottomans had framed the declaration of
Tanzimat as a public reiteration of the rights already granted by the classical
Islamic law, Shari’ah. Consequently, these legal reforms did not get any
significant opposition from the conservative Ulama. 45 İnalcık, Halil, “Adâletnâmeler”, Belgeler, vol. III, no 3-4, 1965, Ankara 1967. MUSLIMS VIS-À-VIS UNIVERSALISTIC HUMAN RIGHTS IN THE MODERN ERA The execution of the
reformist Pertev Pasha in 1837 prompted bureaucrats like Rifat Pasha to take
measures for their own protection, which should also be viewed as another major
reason behind the declaration of the first human rights charter by a Muslim state. This was coupled by the considerable pressure from European allies for reforms
concerning the rights of minority Christians. When Europe was shaken by the French Revolution in 1789, Selim III
(1789-1807) ascended the Ottoman throne as the Sultan-Caliph. Ruling from
1789 until 1807, Selim III also initiated a highly radical reform project. With the
purpose of getting feedback from the public, he issued a decree to civil, military
and religious dignitaries requesting them to submit their views on the possible
causes of the weakness of the Ottoman society and the state as well as their
proposals for their reform. Following the Ottoman tradition, the dignitaries,
from a wide ranging social spectrum, presented their ideas in the form of
memorials. Three distinct perspectives emerged from these reform proposals: (1)
Conservative: recover the glories of the Ottoman golden age by reverting to its
traditional methods. (2) Eclectic: reconcile the European system with the existing
order. (2) Radical: replace the traditional system with a modern one. ( )
p
y
The Sultan adopted the third and the most radical of the perspectives,
which was also maintained by his successors persistently until the collapse of the
Ottoman State. He promulgated, in 1792 and 1793, a whole series of new
instructions and regulations which came to be known collectively as the New
Order (Nizam-i Cedid). He established a new corps of regular infantry, trained and
equipped on the modern European model, and a special new treasury to fund it. He also took some disciplinary measures to reform the administration. He
improved diplomatic relations with the European states. For this purpose, he
established regular and permanent Ottoman embassies in major European
capitals such as London, Paris, Vienna and Berlin. Prior to him, Ottomans did
not have embassies in European capitals. Mahmud II, who ascended the Ottoman throne in 1808, rigorously
maintained the reform program of Selim III. The first outcome was the above 45 İnalcık, Halil, “Adâletnâmeler”, Belgeler, vol. III, no 3-4, 1965, Ankara 1967. 24 24 http://www.bepress.com/mwjhr/vol2/iss1/art11
DOI: 10.2202/1554-4419.1030 Senturk: Sociology of Rights mentioned Charter of Alliance (Sened-i İttifak), which was an agreement between
the Sultan and the dignitaries (Ayan). 47 Tanzimat (tän´zemät) , [Turkish, reorganization], the name referring to a period of
modernizing reforms instituted under the Ottoman State from 1839 to 1876. In 1839, under
the rule of Sultan Abdulmecid , the edict entitled Hatti-i Sharif of Gülhane laid out the
fundamental principles of Tanzimat reform. Foremost among the laws was the equal
citizenship, security of honor, life, and property for all Ottoman subjects, regardless of race or
religion. Other reforms, which sought to reduce theological dominance, included the lifting of
monopolies, fairer taxation, secularized schools, a changed judicial system, and new rules
regarding military service. Tanzimat is commonly considered to have ended in 1876 during
the reign Abdulhamid II, when the ideas for a Turkish constitution and parliament were first
implemented and then rejected by the same sultan. The constitution and parliament were
reintroduced after Abdulhamid II was dethroned by the Young Turks in 1908. 46 The history of Ottoman reforms in Islamic law during the 19th century has yet to fully
documented in English. For a general history of this period, see Stanford Shaw, Between Old
and New, The Ottoman State Under Sultan Selim 1789-1807, Cambridge, 1971. For “Sened-i
İttifak” see; Akyıldız, Ali, “Sened-i İttifak’ın İlk Tam Metni” İslam Araştırmaları Dergisi,
1998 (2), pp. 209-222; Also see, İnalcık, Halil, "The Nature of Traditional Society: Turkey, "
in Robert E. Ward ve Dankward A. Rustow, Political Modernization in Japan and Turkey
(Princeton, N.J.: Princeton University Press, 1964), pp. 13-14. Muslim World Journal of Human Rights, Vol. 2, Iss. 1 [2005], Art. 11 Muslim World Journal of Human Rights, Vol. 2, Iss. 1 [2005], Art. 11 The 1876 Constitution marks the most important step along the road to
the rule of law, initiating the First Constitutional Period, which continued for
only a year under the rule of Abdulhamid II. The first Ottoman constitution is
seen somewhat restrictive in the exercise of powers, but nevertheless for the first
time it recognized a parliamentary system. This constitution has provisions
covering basic rights and privileges and the independence of courts and the safety
of judges, among other aspects. In 1908, the Young Turks who dethroned
Abdulhamid II launched the Second Constitutional Period and laid the
foundations of a parliamentary system, which continued until the fall of the
Ottoman State48. The Ottoman efforts to establish universal human rights through legal
reforms had been promoted by the rising Ottoman civil society and middle class
which operated within the parameters of a religious paradigm. Intellectuals
played a decisive role in the process. Yet, by the fall of the Ottoman state, the
activities of this middle class seized. Under the newly established Turkish
Republic, there was a total state control on all fields of social, economic, legal and
cultural life to ensure the paradigm shift from Islamic to a secular worldview. Yet
the new Turkish Republic with a strictly secular and anti-Ottoman ideology paid
little attention to philosophically grounding human rights in the Turkish and
Islamic culture. Instead, human rights have been copied and translated verbatim
from the West by the State officials. The Turkish Muslims had no objection to
these rights in their new secular dress but they remained for the most part on
paper with limited implementation on the ground due to lack of democracy, due
process and civil society. Therefore it was impossible to promote human rights
until the emergence of a middle class and civil society was allowed after the
democratic reforms around 1950 as pressures from inside and the West
amounted on the ruling elite. As the theory of ‘ismah went into an eclipse, the period after the fall of the
Ottoman State may be described as “human rights dependency,” during which
human rights came to Muslim world through the efforts of international
organizations. Indigenous efforts, in the absence of a civil society and due
process, hardly bared any fruit. 48 For the intellectual and political history of this period, see for istance, Mardin, Şerif, The
Genesis of Young Ottoman Thought, Princeton: Princeton University Press, 1962; Hanioğlu,
M. Şükrü, The Young Turks in Opposition, New York : Oxford University Press, 1995.
49 For a survey of the Islamic discourse on human rights discourse in Arabic see, Silmi al-
Khadra al-Jayusi (ed.) Huquq al-Insan fi al-Fikr al-'Arabi, Beirut: Markaz al-Dirasat al-
Wahdah al-'Arabiyya 2002. MUSLIMS VIS-À-VIS UNIVERSALISTIC HUMAN RIGHTS IN THE MODERN ERA With this document, the Sultan's sovereign
power was limited for the first time in the Ottoman history; therefore historians
consider it an important document46, signaling the move toward more political
representation of people’s will, broader political inclusion, democratic reforms,
limits to the power of the state and the Sultan, and more rights for citizens. This
document is also seen as the first step towards a constitution. Constitutional movements during the Ottoman period commenced
toward the end of the 18th century. Sultan Selim III (1789-1808) set up the
Advisory Assembly (Meclis-i Meshveret), within the context of the New System
(Nizam-i Cedid), initiating the march towards a constitutional government system. His successor Mahmud II, who was also a radical reformer, signed the Charter of
Alliance (Sened-i İttifak) in 1808, which is seen as the first important document
from the point of view of a constitutional order. It restricted Sultan's power and
delegated some authority to the senate body, called the Ayan. The Royal Decree of the Rose Garden (Gülhane Hatt-ı Hümayunu) was
launched in 1839, during the Tanzimat Reforms47. This declaration, which may be
seen as the first declaration of human rights by a Muslim state, assured all citizens
their basic rights: right to life, property, freedom of religion, protection of honor,
education, employment and due process. The Tanzimat declaration was grounded
on the doctrine of ‘ismah in Islamic law. The document is especially significant for
its recognition of equal rights in education and in government administration for
those of Christian persuasion, exemplifying egalitarian principles. The Ferman
declared: “All Muslim or non-Muslim subjects shall benefit from these rights. Everyone's life, chastity, honor and property is under the guarantee of the state
according to the Shari‘ah laws.” Representatives of all religious groups and the
ambassadors of European states were present in the declaration ceremony, which
was closed by the prayer of Shaikh al-Islam. In 1875, the Imperial Edict on
Justice (Ferman-i Adalet) provided for independence of the judicial courts and
ensured the safety of judges. 25 Published by Berkeley Electronic Press, 2005 Muslim World Journal of Human Rights, Vol. 2, Iss. 1 [2005], Art. 11 Muslims became recipients of human rights but
they were no longer contributors to the human rights cause49. The doctrine of
universal human rights was no longer rooted in the native Islamic or Turkish
culture. The contemporary Turkish Muslim scholars have displayed a favorable
approach towards the universal human rights during the twentieth century
although these rights were presented within the parameters of a secular paradigm
and discourse. The fact that universal human rights were codified by a secular
Western institution did not pose a problem for them. They were struck by the http://www.bepress.com/mwjhr/vol2/iss1/art11
DOI: 10.2202/1554-4419.1030 26 Senturk: Sociology of Rights convergence of the Islamic universalist understanding of human rights with the
Western paradigm. In 1949, Huseyin Kazim Kadri, a renowned author on Islam, wrote an
Islamic commentary on the UN Charter where he concluded that it is in
complete conformity with Islamic law50. After the declaration of the UN Charter,
Ali Fuad Başgil, a religiously oriented professor of law from Istanbul University,
strongly supported the concept of universal human rights in his public lectures51. But the state followed an “a-religious” approach to human rights, completely
banning religious education in the country for decades. Ali Fuad Basgil could
barely save his life from the military rule and was not allowed to participate in
politics to implement his views on human rights. He was accused for being a
“reactionary” because of his belief in and advocacy for the freedom of
expression. The first school of theology was opened in Ankara, the capital of Turkey,
under pressure from NATO during the early sixties and it remained the only one
until 1982. The religious life and education have been under strict control of the
state. The irony is that this secular Tsarian (or Caesarianist) system, which had no
parallel in the Western world, except the USSR, was introduced as a part of
Westernization and modernization project. Consequently, it may pose a great
problem for the prospective integration of Turkey in the EU. The recent efforts
of integration with the EU unmasked this phenomenon. The authoritarian
Turkish ruling elite claims defending Western values but there is a great
divergence between the European models of secularism and the Turkish system,
which was instituted allegedly as Western. g
y
The EU has been pressuring Turkey, as did NATO, for more human
rights, freedom of religion and the separation of religion and state. 50 Kadri, Hüseyin Kazım, İnsan Hakları Beyannamesi’nin İslâm Hukukuna Göre İzahı, (Yay.
Osman Ergin) İstanbul 1949.
51 Başgil, Ali Fuat, Cihan Sulhu ve İnsan Hakları, İstanbul n.d.; Vatandaş Hak ve
Hürriyetlerinin Korunması Meselesi ve Anayasamız, İstanbul 1956; Vatandaş Hürriyeti ve
Bunun Teminatı, İstanbul 1948.
52 Juxtaposing the cases of Ali Fuad Basgil in Turkey and Mahmoud M. Taha in Sudan
demonstrates how. Muslim World Journal of Human Rights, Vol. 2, Iss. 1 [2005], Art. 11 The Turkish
government is also under pressure from major human rights organizations for its
negative record. Ironically, the conservative Islamist wing rigorously supports
integration with the EU, for gaining better human rights and particularly freedom
of religion. Whether they will use more freedom of religion, if they ever get it, to
undermine and abolish the democratic system which provides it remains
unknown. This is the worry their opponents highlight to discredit them. The authoritarian governments, be they secular or religious, tend to
misuse both religion and secularism to silence voices for better human rights,
which they expediently frame as political opposition52. The absence of a viable
middle class, civil society and public sphere cripples all the efforts in the struggle
for better human rights. The Muslim population, who sees no internal solution
after exhausting all the potential strategies, eventually turns to international 27 Published by Berkeley Electronic Press, 2005 27 Muslim World Journal of Human Rights, Vol. 2, Iss. 1 [2005], Art. 11 organizations to bring more pressure on their own governments, which signed
international documents on human rights. During the preparation of universal human rights documents, the
delegations from Muslim countries confused the world by displaying inconsistent
attitudes, particularly on the rights of workers and women. Some Muslim states
expressed objections against some of the provisions of the UN Declaration in
1948. Yet, some voted for it without any objection. Those who signed have not
taken them as seriously as their Western counterparts have done; it has usually
remained as a lip service. The so-called Muslim states and some intellectuals which have displayed
discontent with the UN Declaration produced two alternative international
human rights declarations: UNESCO Declaration (1981) and the ICO
Declaration (1990), known as the “Cairo Declaration on Human Rights in
Islam.”53 All Muslim states signed the latter document, which took around two
decades to prepare. The Islamic Conference Organization (ICO) seems to be the
most significant platform and vehicle for the advancement of human rights in the
Muslim world. Yet its voice is hardly heard as Muslim societies face great
challenges and grave human rights violations today. Without putting the issue into an historical and sociological perspective,
the confusion on and deprivation from human rights cannot be understood and
solved in the Muslim world. 53 Adopted and issued at the Nineteenth Islamic Conference of Foreign Ministries in Cairo on
August 5, 1990. CONCLUSION: I AM THEREFORE I HAVE DUTIES I conclude by reiterating my claim that I am therefore I have rights. My very
existence justifies my rights. They are indivisible and inalienable. Yet, this means
at the same time that I am therefore I have duties. My rights are best justified by my
duties. I am charged to stand for my own rights and for the rights of the entire
humanity. It is a duty for me to recognize all my fellow human beings as equal
persons and protect their rights the same way I do for my own rights. My
community and state must also do the same. Law is not about rights alone but
also about duties. Since society is a network of interdependent relations, duties
of one are the rights of the other. Therefore, fulfilling duties is the best way to
assure one’s own rights. Otherwise, rights remain as mere abstractions. Protecting human rights must be the objective of all legal systems, if it is
already not. The legitimacy of the political authority and the law should be judged
by their conformation with basic human rights. Individuals should not defer their
moral capacity to their superiors and therefore always judge the judgments and
the judges from the perspective of human rights. j
g
p
p
g
These are some of the principles one may also derive from the classical
Islamic law. They are ancient yet still speak to us. Nevertheless, the universalistic
view, represented by a branch of the classical Islamic law, is curiously neglected
in the Modern Islamic discourse on human rights. Today, the Turkish and
Arabic discourse on human rights occasionally utilize the term ‘ismah (sanctity)
but rarely do they utilize the category âdemiyyah (humanity), the absence of which
cripples any attempt to philosophically ground human rights on the universal
level. Unfortunately, with the break in the chain of memory, the modern
Islamic legal discourse has lost the universal dimension that characterized the
discourse of some jurists in the classical era. Most of the modern Islamic
discourse on human rights revolves around religiously defined social categories
such as muslim and kafir (non-Muslim), rather than a universally inclusive concept
of humanity (adamiyyah). Unearthing and reintroducing the classical Islamic
concept of universal human can transcend this communalistic approach. Muslim World Journal of Human Rights, Vol. 2, Iss. 1 [2005], Art. 11 Nor can the human rights dependency, on the part
of Muslims who believe in human rights, be overcome without linking the chain
of memory to the past cultural reservoir. Human rights discourse in the Muslim
world needs philosophical, moral and historical roots to grow on, gain strength
and bear fruits. Otherwise, its defenders will remain dependent on the Western
discourse and consequently will get easily dismissed by the conservative
population, traditional Ulama and the authoritarian rulers. The power of
precedence, on the theoretical and historical levels, must be put in use in
justifying human rights in Islam today. j
g
g
The over all Western attitudes are also confusing for the observers inside
and outside the Muslim world. On the one hand, they carry on a rhetoric which
champions human rights in the Muslim world yet on the other hand they ally
themselves with the oppressive regimes. This confusion is usually guided by
“instrumental rationality” which gives priority to short-term political and
economical interests. We have to wait until “value rationality” with an emphasis
on the “ideal interests” such as human rights will prevail in the Western approach
to Muslim world. Those who adopt value rationality among Muslims and
Westerners need to coordinate their efforts until such a major paradigm shift
occurs on the international level. This is required to overcome the constraints in
implementing and maintaining the universalistic legal tradition on human rights
in Islam. Otherwise, the lack of international support combined with the lack of
due process, civil society and middle class to promote human rights, the
universalistic tradition in Islamic law may remain in eclipse forever. 28 http://www.bepress.com/mwjhr/vol2/iss1/art11
DOI: 10.2202/1554-4419.1030 Senturk: Sociology of Rights On a broader level, a global coordination is needed among the civil
communities and intellectuals from around the globe, acting with the “value
rationality,” to cure the human rights dependency of the non-Western world. Reinvigorating the universalistic approaches in all cultures and combining them
with the modern notions will provide a remedy to human rights dependency of
the non-Western world for the benefit of all around the globe. CONCLUSION: I AM THEREFORE I HAVE DUTIES Even though the traditional-Islamic and modern-Western approaches to
universal human rights share a common ground, they cannot be expected to
completely converge due to the historical, cultural and religious reasons. Therefore it would be wrong to jump to the conclusion that what the UN
constructed after the World War II in the second half of the 20th century had
already existed in the Islamic culture. At best, this would be a great anachronism. Yet it would also be wrong, as it is presently done by some of the leading figures Published by Berkeley Electronic Press, 2005 29 Muslim World Journal of Human Rights, Vol. 2, Iss. 1 [2005], Art. 11 in the field, to claim that universal human rights are alien to or incompatible with
Islam. The segments of Muslim society who have welcomed the rise of universal
human rights in the West, culminating in the UN declaration, have been those
who have already found in their cultural repository some of the abstract
constructions on which the Declaration was based, the most important one being
the abstract concept of a universal human. This concept exists in one strand of
Islamic law and needs to be unearthed to provide a solid philosophical
foundation for universal human rights in Islam, toward which this paper is a
preliminary step. The theory of âdamiyyah and ‘ismah needs to be researched
further and explained in modern human rights language to both Muslims and
non-Muslims. Deriving from this theoretical ground, we can extend this venue
until a full-fledged theory of universal human rights is developed and expressed
in modern language to meet the present needs of the Muslim society in its
internal relations with other fellow Muslims and external relations with other
fellow humans—a pressing need in the present globalized world. Muslims lagged behind the modern world regarding the universal human
rights despite the classical universalistic tradition in Islamic law and the rigorous
reforms by the Ottomans during the 19th century to bring traditional Islamic
polity in line with its modern counterparts. Yet, with the collapse of the
Ottoman State, the chain of memory has been broken in the Islamic civilization. Presently, some Muslim states and intellectuals try to start over in producing and
justifying rights. I offer an alternative strategy which exploits the authority of
precedence, on theory and practice of law. Published by Berkeley Electronic Press, 2005 CONCLUSION: I AM THEREFORE I HAVE DUTIES p
y
p
I also recognize the need for each culture and religion to do so. This is an
obligation and a right for each culture today. This approach contradicts with the
dominant ideological approaches characterized by an exclusivist claim for the
justification of human rights. Diverse ways of justifying human rights by different
cultures in their own terms will empower human rights cause and increase
compliance globally. A historical precedence for this claim comes from the
Islamic tradition, which is by no means an exception to the rule. y
p
A strategy needs to be adopted to indiscriminately combine the ideas and
notions from different cultures, past and present, East and West, on the meaning,
prerequisites and implications of human existence in society. There is a room in
this perspective for the universality and relativity. Universalism cannot be
monopolized or patronized by a particular ideology. Nor can it be precluded
because of the social and cultural diversity on the globe. Globalization helps us
increasingly discover the commonality of human experience from different
cultures, times and places. Yet we need to make an effort to discover the links,
and to fill the gaps, among them to demonstrate how they bear upon each other. Such an integrative view makes human rights paradigm multi-potential and fluid,
rather than exclusivist and static. Diverse cultures of the world, be they religious
or secular, may thus variably reaffirm the universality of human rights in their
own terms, adding to the power of each other and to the power of the human
rights paradigm and cause. 30 30 http://www.bepress.com/mwjhr/vol2/iss1/art11
DOI: 10.2202/1554-4419.1030 Senturk: Sociology of Rights Senturk: Sociology of Rights 31 Published by Berkeley Electronic Press, 2005 31
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DUBBELE BELASTINGHEFFING BIJ OVERDRACHT VAN EEN BEDRIJF
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Inleiding. In het Besluit op de inkomstenbelasting 1941 wordt een onderscheid
gemaakt tussen twee categorieën belastingplichtigen, die wij kortheidshalve
de ,,bedrijfsman” en de „particulier” zullen noemen. De eerste is degene,
die een bedrijf voert of een zelfstandig beroep uitoefent. Aangenomen, dat
hij geen andere inkomstenbronnen heeft dan zijn bedrijf of beroep, moet
hij zijn winst als inkomen opgeven, de bepaling waarvan is geregeld in de
artt. 6/12 en 22 van het besluit. De particulier geeft zijn zuivere inkomsten
op, dat is het netto bedrag van zijn verschillende bronnen van inkomen, de
algebraïsche som waarvan het onzuivere inkomen oplevert. De bepaling
hiervan is geregeld in de artt. 13 e.v. van het besluit. Voor beide categorieën
geldt, dat ter bepaling van het zuivere inkomen, de winst, resp. het on
zuivere inkomen, wordt verminderd met de onverrekende verliezen en de
persoonlijke verplichtingen. p
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Zowel voor de particulier als voor de bedrijfsman geldt art. 48, regelende
de gevallen, waarin het bijzondere tarief zal worden toegepast. Dit is door
gaans het geval als baten, die in een reeks van jaren zijn ontstaan, in één
jaar worden gerealiseerd. Terecht is de wetgever van oordeel geweest, dat
hier een matiging van de progressie moet plaatsvinden, omdat het marginale
percentage der heffing over zulk een gerealiseerde bate veel hoger zou zijn,
dan indien die bate in kleine gedeelten aan de jaarlijkse winst zou zijn
toegevoegd geweest. M.b.t. de bedrijfsman wordt dat bijzondere tarief
toegepast bij liquidatie of overdracht van zijn bedrijf (art. 20) of van zijn
zelfstandig uitgeoefende beroep (art. 24), waarbij de eerste ƒ 7.500,—
liquidatiewinst buiten de heffing blijven. In welke gevallen art. 48 voor de
particulier van toepassing is, is voor onze beschouwing van geen betekenis
en wordt daarom niet besproken. DUBBELE BELASTINGHEFFING BIJ OVERDRACHT
VAN EEN BEDRIJF door ]. P. H. Smits Winst. „Winst is het bedrag van de gezamenlijke voordelen die. onder welke
naam of in welke vorm ook, worden verkregen uit bedrijf of zelfstandig
uitgeoefend beroep” luidt art. 6 I. B. Het is niet onaardig, de herkomst van
deze definitie na te gaan. Een rederij besloot tot liquidatie — het was een
N.V. doch dat doet niet ter zake, aangezien de N.V. haar winst op de
zelfde wijze bepaalt als de bedrijfsman, daar de artt. 6/11 en 22 I. B. ook
van toepassing zijn bij de winstbepaling door lichamen — en maakte winst
bij de verkoop van haar schepen. Deze gaf ze niet op en, daarvoor ter ver
antwoording geroepen door de Inspecteur stelde zij, dat volgens de statuten
het doel van de N.V. was (geweest) „het exploiteren van één of meer
schepen in de ruimste zin”, doch, aldus de belanghebbende, hoe ruim het
woord „exploiteren” ook wordt genomen, nóóit kan daar verkoop mee zijn
bedoeld. De Hoge Raad was het hiermede nochtans niet eens en defin
ieerde het winstbegrip als bovenstaand, daaraan nog toevoegende: „zelfs
als het behalen van die winst niet in de bedoeling van de bedrijfsuitoefening
heeft gelegen. g
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Liquidatiewinst is derhalve een voordeel, dat wordt verkregen uit een
bedrijf of zelfstandig uitgeoefend begrip, en als zodanig winst. m a b blz. 222 m a b Art. 36 I. B. Dit artikel, dat voor onze beschouwing van grote betekenis is, geven wij
letterlijk. Het luidt: Dit artikel, dat voor onze beschouwing van grote betekenis is, geven wij
letterlijk. Het luidt: „1. Tot de bestanddelen van het in art. 5 lid 1, omschreven onzuiver
inkomen behoren mede: „1. Tot de bestanddelen van het in art. 5 lid 1, omschreven onzuiver
inkomen behoren mede: le. afkoopsommen, schadeloosstellingen en tegemoetkomingen,
welke toegekend zijn: a. ter vervanging van gederfde of te derven beloningen,
al dan niet ingegane periodieke uitkeringen en andere
opbrengsten en inkomsten, dan wel b. terzake van het staken of het niet verrichten van werk
zaamheden in de ruimste zin, dan wel wegens het prijs
geven van een uitzicht op deelgerechtigdheid in een
bedrijf of zelfstandig uitgeoefend beroep: j
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2e. enz. voor ons artikel van geen betekenis. j
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enz. voor ons artikel van geen betekenis. 2. Bestaat de afkoopsom, schadeloosstelling of tegemoetkoming,
dan wel het in lid 1, 2e, bedoelde bedrag in de toekenning van
een recht, dat periodieke opbrengsten oplevert (zoals het recht
op een lijfrente of op een winstaandeel) dan worden slechts deze
periodieke opbrengsten, doch niet het eraan ten grondslag lig
gende recht (stamrecht) tot het inkomen gerekend. 3. De onder lid 1 vallende bedragen worden, voor zover zij niet
onder lid 2 vallen, op daartoe gedaan verzoek afzonderlijk belast
op de voet van art. 48.” Arrest van de Hoge Raad dd. 28 juni 1944. De hoofdzaken van dit uiterst belangrijke arrest, dat de voorloper is
geweest van het arrest dd. 17 oktober 1956, dat de directe aanleiding is
geweest voor deze beschouwing, laten wij hier volgen. De casuspositie was aldus: een accountant had met een N.V. een overeenkomst gesloten, waarbij hij
voor t.b.v. de N.V. te verrichten werkzaamheden, levenslang een jaarlijkse
vergoeding van ƒ 4.000,— had bedongen. In 1941 werd deze overeenkomst
teniet gedaan en als schadeloosstelling ontving de accountant een bedrag
ad. ƒ 6.000, — , dat hij, ex art. 36, 3e lid, wilde zien belast op de voet van
art. 48. Met de Inspecteur was de Raad van Beroep van mening, dat de
accountant de bedoelde werkzaamheden niet had verricht in dienstbetrek
king maar in zelfstandig uitgeoefend beroep van accountant, doch trok
hieruit, anders dan de Inspecteur, niet de conclusie, dat nu art. 36 niet van
toepassing zou zijn. Weliswaar, aldus de R.v.B., geldt art. 36 volgens de
leidraad niet voor winst uit een zelfstandig uitgeoefend beroep, doch deze
mededeling vindt geen steun in enige bepaling van het besluit. Dit klemt
temeer, nu in par. 41 van de toelichting en leidraad wordt gezegd, dat de
bedoeling van art. 48 is, de progressie te matigen t.a.v. bestanddelen van
het onzuivere inkomen, welke eigenlijk op een reeks van — reeds verstreken
of toekomstige .—■ jaren betrekking hebben. g
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Op grond hiervan vernietigde de R.v.B. de beschikking van de Inspecteur
en belastte de bewuste ƒ 6.000,— op de voet van art. 48. De waarnemende secretaris-generaal van het Departement van Finan
ciën tekende cassatie aan en stelde natuurlijk, dat de ƒ 6.000.— behoorden m a b blz. 223 m a b tot de winst van de belastingplichtige ingevolge het —- toen nog geldige —
art. 8 van het besluit en dat die winst moest worden berekend als een on
deelbaar geheel in die zin, dat niet wordt onderscheiden naar de aard der
baten, die haar samenstellen. Slechts bij het overdragen van het beroeps
vermogen of bij het staken of overdragen van de beroepsuitoefening wordt
eventueel een gedeelte van de winst afzonderlijk in beschouwing genomen
en art. 36 maakt op de ondeelbaarheid van de winst geen verdere inbreuk. Niet aldus de Hoge Raad. Wij laten zijn overwegingen hier in extenso
volgen: „dat weliswaar een door de belastingplichtige ontvangen schadeloos
stelling als waarvan ten deze sprake is ingevolge art. Arrest van de Hoge Raad dd. 28 juni 1944. 8 van het Be
sluit op de inkomstenbelasting 1941 behoort tot zijn winst en dus
ook buiten art. 36, lid 1, om tot de bestanddelen van zijn onzuiver
inkomen moeten worden gerekend, doch hieruit nog niet volgt, dat
een dergelijke schadeloosstelling niet zou behoren tot de in dat lid 1
bedoelde schadeloosstellingen, toegekend ter vervanging van te der
ven beloningen; g
dat het immers vooreerst geen twijfel kan lijden, dat onder „belonin
gen” in dat lid 1, onder a, vermeld het loon voor in dienstbetrekking
verrichte arbeid is begrepen en dat dus de afkoopsom, schadeloosstel
ling of tegemoetkoming, welke ter vervanging van gederfd of te der
ven arbeidsloon is toegekend, is een afkoopsom, schadeloosstelling
of tegemoetkoming als in dat eerste lid bedoeld, doch zulks niet weg
neemt, dat een zodanige afkoopsom, schadeloosstelling of tegemoet
koming omdat zij uit dienstbetrekking wordt genoten, ingevolge ar
tikel 26 van het besluit mede als „loon” wordt aangemerkt en dus
reeds ingevolge dit laatste artikel, dus eveneens buiten artikel 36 om,
tot de bestanddelen van het onzuiver inkomen moeten worden ge
rekend; dat naar het geldende spraakgebruik onder „beloningen” niet slechts
is te verstaan het (arbeids)loon van artikel 1637a van het Burgerlijk
Wetboek, maar evengoed een in geld en geldswaarde bestaande ver
goeding voor in de zelfstandige uitoefening van een beroep verrichte
werkzaamheden en dus een uitlegging, volgens welke een schade
loosstelling, toegekend wegens het derven van een dergelijke ver
goeding, niet onder het eerste lid van artikel 36 zou vallen, met de
tekst der bepalingen niet in overeenstemming is; p
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dat verder in dat eerste lid, onder le sub a, mede, al weer geheel in
het algemeen, van „verdere opbrengsten of inkomsten” waaronder
volgens artikel 5 ook winst uit bedrijf of zelfstandig uitgeoefend
beroep is te verstaan, sprake is, terwijl voorts gewezen wordt op de
omstandigheid dat artikel 36 voorkomt in de afdeling welke het op
schrift draagt: „Bepalingen, aan alle bestanddelen van het onzuiver
inkomen gemeen”, hetgeen aan de opvatting, dat dit artikel in zijn
eerste lid op afkoopsommen, schadeloosstellingen en tegemoetko
mingen welke als vergoeding voor gederfde of te derven inkomsten
uit bedrijf of zelfstandig uitgeoefend beroep zijn toegekend geen be
trekking zou hebben, allerminst steun geeft.” Men zou zo zeggen dat er, fiscaal-juridisch gezien, tegen deze argumen
tering weinig kan worden ingebracht! Het beroep van de waarnemende
secretaris-generaal werd dan ook verworpen. Arrest van de Hoge Raad dd. 28 juni 1944. In een reeks van resoluties, m a b
blz. 224 die thans hun betekenis hebben verloren, werd de opvatting van de Hoge
Raad ten faveure van de belastingplichtige uitgebreid in dier voege dat,
als goodwill en stille reserves in één complex werden gerealiseerd, art. 36,
2e lid, óók t.a.v. de stille reserves kon worden toegepast — een praktische
maatregel ter voorkoming van een moeilijke splitsing van de overdrachts-
winst in goodwill en stille reserves. In de vakpers werd deze ministeriële
opvatting te vrijgevig gevonden. Zo betoogden de Hoofdinspecteurs Fray
en Sleddering in het ,.Weekblad der Belastingen”, dat de lijfrente-con-
structie wel kon worden toegepast op de goodwill, doch niet op de stille
reserves. t) Uit de hieronder besproken resolutie van 14 november 1956 blijkt, dat de Inspecteur
inderdaad machtiging had gekregen, een proefproces te beginnen. De salto-mortale van de Hoge Raad. Als een bom viel het arrest van de Hoge Raad dd. 17 oktober 1956
(B.N.B. 1956/313) in de, tengevolge van het bovenbesproken arrest dd. 28 juni 1944 en de daarop gevolgde resoluties, jarenlang bestaande fiscale
praktijk. W e beginnen weer met het geven van de casuspositie. Een belanghebbende oefende met zijn broer een vennootschap onder
firma uit, welke zij in 1952 inbrachten in een N.V. Door deskundigen was
het verschil tussen de bedrijfswaarde der ingebrachte zaken en de fiscale
boekwaarde getaxeerd op ƒ 72.242.68 en de goodwill op ƒ 22.000,—•. Beide firmanten kregen van de N.V. een lijfrente groot ƒ 47.000,—, terwijl
zij beiden in rekening-courant met de N.V. werden gecrediteerd voor
ƒ 121.34. De Inspecteur beschouwde het volle bedrag ad ƒ 47.121.34 als
overdrachtswinst, waarvan ƒ 11.000,— (de goodwill derhalve) werd vrij
gesteld ex art. 36, 2e lid .—■ hierbij kennelijk de opvatting van Fray en
Sleddering volgende. Natuurlijk beriep de requirant in cassatie zich op de uitspraak van de
Hoge Raad dd. 28 juni 1944 en de reeks van daarop gevolgde ministeriële
resoluties, aan welke het hoogste rechtscollege echter geredelijk voorbij
kon gaan, omdat die geen rechtskracht bezitten. De Inspecteur betoogde
dat het hem bekend was, dat het thans door hem ingestelde standpunt door
het Ministerie van Financiën wordt gedeeld 1) (wij cursiveren) en dat
art. 36 volgens de leidraad niet van toepassing is op inkomsten uit bedrijf. Hij stelde vervolgens, dat bij vervreemding van opbrengstgevende ver
mogensbestanddelen de prijsvorming mede wordt beïnvloed door de ver
wachtingen omtrent het rendement en dat, daar dit niet meer door de
vervreemder zal worden genoten, de verkoopprijs een afkoopsom -— aldus
de Inspecteur -— bij een bedrijf alleen de tegenwaarde van de goodwill
betreft, omdat de verkoopwaarde hiervan alleen wordt bepaald door de
opbrengstverwachtingen. M.b.t. de opbrengstwaarde van de bedrijfs
middelen kan niet worden gezegd, dat hierin de invloed van de opbrengst-
factor kan worden berekend op het bedrag van de fiscale overdrachtswinst. De salto-mortale van de Hoge Raad. 36 overeenstem
mend voorschrift bevat, weshalve voor vrijstelling van de belasting
van overdrachtswinst, op grond, dat zij wordt genoten in de vorm van
een toegekend recht op lijfrente, geen plaats is; g
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dat het middel derhalve geen doel treft; dat uit het bovenstaande volgt, dat bij de regeling van de aanslag
ten onrechte een gedeelte, groot ƒ 11.000,—, van de door de belang
hebbende behaalde winst niet is belast; dat uit het bovenstaande volgt, dat bij de regeling van de aanslag
ten onrechte een gedeelte, groot ƒ 11.000,—, van de door de belang
hebbende behaalde winst niet is belast;
dat de Hoge Raad echter deze onjuistheid niet kan herstellen,
daar zulks zou leiden tot een verhoging van de aanslag waartoe de dat de Hoge Raad echter deze onjuistheid niet kan herstellen,
daar zulks zou leiden tot een verhoging van de aanslag, waartoe de
rechter in belastingzaken niet bevoegd is.” In zijn arrest dd. 19 maart 1952 (B. 9204), dat wij, terwille van de
plaatsruimte, niet zullen bespreken, had de Hoge Raad er weliswaar tegen
gewaarschuwd, aan art. 36 een te ruime betekenis toe te kennen en gecon
stateerd, dat het mogelijk zou zijn, dat inkomstenbelasting zou worden
geheven over het stamrecht zowel als over de daaruit voortvloeiende in
komsten, doch hij achtte toen nog deze dubbele heffing onjuist, als „ver
vroegde inkomsten” bestaande in een stamrecht, zouden worden genoten. De salto-mortale van de Hoge Raad. De verdere motivering is ons niet geheel duidelijk, doch wij interpreteren
die als volgt: g
bij waardering van bedrijfsmiddelen op de kostprijs minus redelijke af
schrijvingen kan niet worden gezegd, dat de opbrengstverwachtingen zijn
verwaarloosd, doch, door de wijziging van de koopkracht van de gulden
kan desniettemin een discrepantie tussen geldswaarde en fiscale boek
waarde ontstaan, waardoor bij overdracht stille reserves worden ge- t) Uit de hieronder besproken resolutie van 14 november 1956 blijkt, dat de Inspecteur
inderdaad machtiging had gekregen, een proefproces te beginnen. m a b
blz. 225 realiseerd. M.b.t. de jarenlang bestaande heffingspraktijk voegde de In
specteur hieraan toe, dat het oordeel van de Minister van invloed kan
zijn op hetgeen goed koopmansgebruik vordert, doch geen gewicht in de
schaal kan leggen bij de uitlegging van wetsartikelen, die niet naar goed
koopmansgebruik verwijzen. Ten overvloede wijzen wij erop, dat in art. 7,
derhalve geldende voor de bedrijfsman, naar goed koopmansgebruik wordt
verwezen. Aan de cassatiemiddelen, die de belanghebbende aanvoerde, gaan wij
voorbij, omdat ze, naar het ons voorkomt, vanzelf spreken. Maar wel geven
wij de letterlijke tekst van de salto-mortale van de Hoge Raad: „overwegende:
dat het middel ervan uitgaat, dat art. 36 van het Besluit op de In
komstenbelasting 1941 toepasselijk is ten aanzien van de belasting
van winst, behaald met of bij het overdragen of liquideren van een
bedrijf of een gedeelte ervan, doch ten onrechte; „overwegende:
dat het middel ervan uitgaat, dat art. 36 van het Besluit op de In
komstenbelasting 1941 toepasselijk is ten aanzien van de belasting
van winst, behaald met of bij het overdragen of liquideren van een
bedrijf of een gedeelte ervan, doch ten onrechte; j
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dat met betrekking tot het belasten van de bedoelde winst uitslui
tend van toepassing is de daaromtrent in art. 20 van het besluit opge
nomen bijzondere regeling; dat die regeling geen met het tweede lid van art. Is het arrest van de Hoge Raad dd. 17 oktober 1956 juist? Uit de overweging blijkt, dat de Hoge Raad toepassing van art. 36
categorisch van de hand wijst, indien art. 20 van toepassing is. Hij ging
hierbij nog verder dan de Inspecteur, die althans de goodwill wilde uit
sluiten. Wij vragen ons af, of de opvatting van de Hoge Raad juist is en
nemen daarom art. 36 nogmaals in beschouwing. In het algemeen wordt art. 36, wegens zijn derde lid, beschouwd als een
voor de belastingplichtige gunstig artikel. Maar. ... is het dat wel? Met
name het eerste lid, le, letter b) wekt te dien aanzien ernstige twijfel. Hier
vinden wij een aantal baten opgesomd: „het staken of het niet verrichten
van werkzaamheden in de ruimste zin” en „het prijsgeven van een uitzicht
op deelgerechtigdheid in een bedrijf of zelfstandig uitgeoefend beroep”, die,
zonder art. 36, buiten de inkomstenbelasting zouden vallen, doch daar nu
in worden betrokken. Wij geven hiervan een voorbeeld: m a b blz. 226 Een amateur-accordeonist hinderde zijn overnerveuze bovenbuurvrouw
mateloos met de bespeling van zijn instrument, waarmede hij echter prompt
om elf uur ’s avonds ophield. Juridisch stond hij derhalve volkomen in zijn
recht. De bovenbuurman nochtans, bedacht op de huiselijke vrede, bood de
amateur-musicus een som ineens aan op de voorwaarde, dat hij tussen
's avonds acht uur en ’s morgens acht uur niet op zijn accordeon zou spelen,
hetgeen werd aanvaard. De accordeonist had er geen flauwe notie van, dat
hij inkomen ex art. 36 had genoten, wat toch het geval was, aangezien hij
een vergoeding had gekregen terzake van het niet verrichten van werk
zaamheden. Schijnbaar misleidend is ook de tirade: ,,dan wel wegens het prijsgeven
van een uitzicht op deelgerechtigdheid in een bedrijf of zelfstandig uit
geoefend beroep” waardoor het lijkt, alsof de liquidatiewinst onder art. 36
wordt gebracht —- en de overwegingen van de Hoge Raad dd. 28 juni 1944
geven ook wel aanleiding om dit te denken. Toch komt het ons voor, dat dit
niet juist is. W ant een uitzicht op deelgerechtigdheid impliceert immers,
dat er nog geen deelgerechtigdheid is, het zou, bij het bestaan van een deel
gerechtigdheid, zéér gewrongen zijn, de ook in de toekomst bestaande
deelgerechtigdheid met een uitzicht daarop te betitelen. 2) Een zinsnede, die herhaaldelijk voorkomt in de arresten van de Hoge Raad: „wat ook
zij van de overwegingen van de Raad van Beroep, is zijn beslissing juist”. Is het arrest van de Hoge Raad dd. 17 oktober 1956 juist? Als iemand een
dienstbetrekking heeft, dan heeft hij geen uitzicht op een dienstbetrekking,
maar wel uitzicht op uit die dienstbetrekking voortvloeiende beloningen -—
het uitzicht op een bron van inkomen is éénmalig, het uitzicht op de op
brengst daarvan periodiek. Hieruit volgt dus, dat art. 36, le lid letter b) niet de bedrijfsman, maar de
aspirant-bedrijfsman in de inkomstenbelasting betrekt en dat de eerste zijn
inkomen uitsluitend bepaalt op de voet van de artt. 6/12 en 22 I.B., waarbij
de artt. 20 en 24 een tariefaangelegenheid regelen doch, anders dan vóór
1950, niet meer bepalen, wat onder overdrachtswinst moet worden ver
staan. Onze conclusie moet dan ook luiden ■— en enigszins ondeugend citeren
wij nu de Hoge Raad zelf in zo menig arrest: ,,wat ook zij van de over
wegingen van de Hoge Raad in 1944” —• is zijn arrest dd. 17 oktober 1956
juist. 2) j
)
Waar ligt de fout? Al
d g
f
Als de „gewone man er het zijne van zou zeggen”, zou hij eenvoudig
constateren, dat dubbele heffing van hetzelfde inkomen een dwaasheid zou
zijn. Maar -— helaas ■— de wetgevende en rechtsprekende colleges zijn
geen „gewone mannen". Toch moeten wij de gewone man volkomen gelijk
geven •—- er ligt dus ergens in de wet of in de rechtspraak een fout ■—■ waar
ligt die fout? Ligt die bij de Hoge Raad? Naar wij menen te hebben aan
getoond, stellig niet. Meer dan in 1944 heeft het hoogste rechtscollege zich
gehouden aan de tekst van het besluit en dat kan niet anders dan worden
toegejuicht. W ant te vaak reeds heeft de Hoge Raad rechtgesproken naar
hetgeen de wetgever had bedoeld maar niet gezegd en, met alle respect
voor de deskundigheid en de integriteit van dit college, hiermede toch een
element van onzekerheid geschapen, die de beoordeling van de vraag, of
een cassatieprocedure aanhangig moest worden gemaakt, in de sfeer van de
speculatie bracht. Dat de Hoge Raad hierop is teruggekomen, kan niet
anders dan worden toegejuicht. g j
Ook ligt de fout niet in de voorschriften omtrent de bepaling van de Ook ligt de fout niet in de voorschriften omtrent de bepaling van de m ab blz. 227 winst, 3) ook niet in artikel 36 doch in art. 34 volgens hetwelk periodieke
uitkeringen integraal door de inkomstenbelasting worden getroffen. Is het arrest van de Hoge Raad dd. 17 oktober 1956 juist? Iedere
levensverzekering, en lijfrente-uitkering bestaat uit een spaardeel, de pre
mie, vermeerderd met de samengestelde intrest daarover. M.b.t. de levens
verzekering is de premie niet aftrekbaar van het inkomen en de belasting
wordt bij uitkering alleen berekend over het rentebestanddeel (art. 33). Bij een lijfrente-uitkering is echter zowel het spaardeel als de intrest belast. Als de belastingplichtige er nu maar voor zorgt, dat de premie het in art. 16
genoemde maximum van ƒ 3.600,— niet overschrijdt, zal hij eerder vóór
dan nadeel van art. 34 hebben, omdat doorgaans het arbeidsinkomen bij
het ingaan van een lijfrente aanzienlijk is verminderd en het marginale
percentage daarom lager zal zijn. Maar als de verzekerde nu een koopsom
heeft gestort? Elke levensverzekeringsmaatschappij zal hem dat, vanwege
de fiscale consequenties, ernstig afraden, want van die koopsom zal, ex art. 16, slechts éénmaal ƒ 3.600,— als persoonlijke verplichting in aanmerking
worden genomen. Door het storten van een koopsom zou de verzekerde
zich sterk benadelen, omdat hij, op ƒ 3.600,—- na zowel over het stamrecht
als over de uitkeringen inkomstenbelasting zou moeten betalen. Over het
stamrecht zou hij immers betalen, omdat de koopsom zou worden voldaan
uit besparingen op zijn inkomen, waarover in het verleden reeds inkomsten
belasting was betaald. Maar bij realisering van stille reserves en goodwill bij overdracht of
liquidatie van een beroep of bedrijf tegen beding van een lijfrente, doet de
belastingplichtige in wezen niet anders, dan een lijfrente kopen tegen be
taling van een koopsom. 4) Met alle nare fiscale consequenties, daaraan
verbonden. Het is ons nooit duidelijk geweest, waarom de lijfrente-uitkering in haar
geheel wordt belast en de levensverzekeringsuitkering niet, m.a.w., waarom
de lijfrentepremie wel in mindering van het inkomen mag worden gebracht
en de levensverzekeringspremie niet. Maar uit het bovenstaande volgt wel
dat het belasten van de volle lijfrentetermijn in bepaalde gevallen — met
name bij het storten van een koopsom of het bedingen van een lijfrente bij
liquidatie, een onbillijke dubbele belastingheffing meebrengt. Daar —- en
daar alleen — ligt de fout. g
Een fout overigens, die gemakkelijk zou kunnen worden hersteld. Aan
genomen, dat ook de wetgever dubbele belastingheffing van hetzelfde in
komen uit den boze vindt, kunnen alternatief twee wijzigingen in het be
sluit worden aangebracht en wel: a. voor lijfrenteuitkeringen wordt dezelfde regeling gegeven als voor
levensverzekeringsuitkeringen, n.1., dat alleen het rentebestanddeel is
belast. De vrijstelling van de lijfrentepremie ex art. 16 zou dan dienen
te vervallen. *) Fiscaal-technisch gezien! Bedrijfseconomisch is op de fiscale winstbepaling wel het
een en ander aan te merken, getuige o.a. de publicaties van Prof. Dr. J. L. Meij.
4) Uit het arrest blijkt niet, of die ƒ 3.600,— als lijfrentepremie — en dus als persoon
lijke verplichting — in aanmerking zijn genomen. Wij nemen aan, dat dit wel het geval
is geweest. *) Fiscaal-technisch gezien! Bedrijfseconomisch is op de fiscale winstbepaling wel
een en ander aan te merken, getuige o.a. de publicaties van Prof. Dr. J. L. Meij. *) Fiscaal-technisch gezien! Bedrijfseconomisch is op de fiscale winstbepaling wel het
een en ander aan te merken, getuige o.a. de publicaties van Prof. Dr. J. L. Meij.
4) Uit het arrest blijkt niet, of die ƒ 3.600,— als lijfrentepremie — en dus als persoon
lijke verplichting — in aanmerking zijn genomen. Wij nemen aan, dat dit wel het geval g
g
p
j
4) Uit het arrest blijkt niet, of die ƒ 3.600,— als lijfrentepremie — en dus als persoon
ke verplichting — in aanmerking zijn genomen. Wij nemen aan, dat dit wel het geval
geweest. De opvatting van Prof. Smeets. De opvatting van Prof. Smeets. Prof. Smeets heeft een uitvoerige annotatie gegeven bij het gerucht
makende arrest van 17 oktober 1956, waaraan wij het volgende ontlenen. In beginsel is de opvatting van Fray en Sleddering juist, doch zij maken
onvoldoende onderscheid tussen stille en geheime reserves. Prof. Smeets
merkt dan op: „het bestaan van de eerste kan uit de gepubliceerde jaarstuk
ken blijken, zonder dat nochtans haar omvang bekend is; bij geheime
reserves blijven zowel het bestaan als de omvang verborgen.” Deze geheime
reserves — aldus Z.H .G ..— welke b.v. het gevolg zijn van in de balans niet
voorkomende, nochtans aanwezige, activa, zullen behoren tot de normale
winst van het jaar, waarin zij aan de dag treden ■—■ maar, voegt hij eraan
toe „praktisch zijn geheime reserves niet te achterhalen, zij zullen zich
voordoen als overdrachts- of liquidatiewinst.” M.a.w. de fiscus zal on
machtig zijn, de geheime reserves op de juiste wijze te belasten. W at de
stille reserves betreft, ligt de zaak anders — Prof. Smeets wijst op het
lifo-systeem, waarbij het bestaan van fiscaal-toegestane reserves uit de
balans blijkt, maar goed koopmansgebruik toelaat, dat de winst hierop
wordt gerekend tot de winst van het jaar, waarin die voorraad geheel of ten
dele wordt gerealiseerd. g
Z.H.G. geeft dan een interessante analsye van de overdrachtswinst die
hij theoretisch (wij cursiveren) onderscheidt in drie elementen t.w.: een vergoeding voor in het verleden gederfde winst, a. een vergoeding voor in het verleden gederfde winst, a. een vergoeding voor in het verleden gederfde winst, g
g
g
b. idem voor in de toekomst te derven winst, b. idem voor in de toekomst te derven winst, c. winst, die fiscaalrechtelijk ontstaat en dus behoort tot het jaar, waarin
de overdracht of liquidatie plaats vindt en waarvoor art. 36, le lid, niet
geschreven behoefde te worden (of... . is? S ), omdat deze reeds onder
art. 7, le lid valt en wordt belast volgens art. 20 le en 3e lid en waarop
de lijfrenteconstructie niet kan worden toegepast. En hij concludeert
dan: „dat de Hoge Raad hiervoor art. 36, 2e lid, niet van toepassing
acht, lijkt mij juist.” c. winst, die fiscaalrechtelijk ontstaat en dus behoort tot het jaar, waarin
de overdracht of liquidatie plaats vindt en waarvoor art. 36, le lid, niet
geschreven behoefde te worden (of... . is? S ), omdat deze reeds onder
art. Is het arrest van de Hoge Raad dd. 17 oktober 1956 juist? Art. 31, le lid, 4e zou moeten worden gewijzigd in „in
teressen, begrepen in levensverzekering- en lijfrente-uitkeringen” en
aan art. 34 zou een derde lid moeten worden toegevoegd: „het in de
vorige leden bepaalde is niet van toepassing op lijfrenteuitkeringen”,
waaraan dan, ter voorkoming van misverstand, een zinsnede zou kun
nen worden toegevoegd: „onder een lijfrente wordt verstaan een perio- a. voor lijfrenteuitkeringen wordt dezelfde regeling gegeven als voor
levensverzekeringsuitkeringen, n.1., dat alleen het rentebestanddeel is
belast. De vrijstelling van de lijfrentepremie ex art. 16 zou dan dienen
te vervallen. Art. 31, le lid, 4e zou moeten worden gewijzigd in „in
teressen, begrepen in levensverzekering- en lijfrente-uitkeringen” en
aan art. 34 zou een derde lid moeten worden toegevoegd: „het in de
vorige leden bepaalde is niet van toepassing op lijfrenteuitkeringen”,
waaraan dan, ter voorkoming van misverstand, een zinsnede zou kun
nen worden toegevoegd: „onder een lijfrente wordt verstaan een perio- ni a b blz, 228 ni a b blz, 228 dieke uitkering, die afhankelijk is van het leven van degene(n) die
haar geniet (en). b. door aan art. 20 een vierde lid toe te voegen: „bestaat de liquidatiewinst
of overdrachtswinst in de toekenning van een recht, dat periodieke
uitkeringen oplevert, dan worden slechts deze periodieke uitkeringen
en niet het daaraan ten grondslag liggende recht (stamrecht) tot het
inkomen gerekend.” Art. 36, 2e lid zou dan kunnen (doch niet be
hoeven) te worden gewijzigd in: „met betrekking tot afkoopsommen,
schadeloosstellingen en tegemoetkomingen, als in het eerste lid bedoeld,
vindt art.20, 4e lid overeenkomstige toepassing.” Het Ministeriële standpunt. Nog even terugkomende op de annotatie van Prof. Smeets, halen wij de
aanvang daarvan aan, luidende: ,,het bovenstaande arrest zet met één klap
alles op losse schroeven, wat sinds de res. van 15 april 1947, B. Nr. 8338
aan lijfrenteconstructies, soms zeer vernuftig, is tot stand gebracht; natuur
lijk zal de Minister wel overgangsbepalingen moeten maken om alles wat,
mede met zijn sanctie, op dit gebied tot stand is gekomen, zo veilig mogelijk
te stellen.” En dat heeft de Minister ook gedaan in de resolutie van 14 november
1956, nr. 193, waaraan wij het volgende ontlenen. Met instemming van de
Minister is dit proces aanhangig gemaakt, omdat de vrijstelling van
ƒ 7.500,— liquidatiewinst en de verhoging van de aftrekbare lijfrentepremie
tot ƒ 3.600,—■ per jaar, de situatie dermate had veranderd, dat dit tot
nadere bezinning op de desbetreffende wetsbepalingen en de praktische
toepassing daarvan noopte. Allereerst was daartoe nodig te weten, welke
interpretatie de Hoge Raad aan de desbetreffende bepalingen gaf. Dit is
thans bekend — de verschillende aanschrijvingen van de jaren 1944 t/m
1953 zijn hiermede achterhaald. Nochtans heeft hij zich bereid verklaard,
van geval tot geval te onderzoeken, of er geen termen aanwezig zijn, de
z.g. „hardheidsclausule” toe te passen. In twee artikelen van het Besluit is
deze te vinden, n.1. in art. 55, le lid 3e en in art. 59, le lid 4e. Naar de
laatste, waarvan de redaktie luidt: „. . . .om in bepaalde gevallen of groe
pen van gevallen tegemoet te komen aan onbillijkheden van overwegende
aard, die zich bij de toepassing van dit besluit mochten voordoen” wordt in
de resolutie verwezen. De Minister zal derhalve van geval tot geval be
kijken, of, indien een bedrijf of beroepsuitoefening wordt gestaakt of over
gedragen tegen een contraprestatie, welke geheel of ten dele uit een stam-
recht bestaat, dan wel, indien in onmiddellijke samenhang met de over
dracht of liquidatie van een bedrijf of beroep een lijfrente wordt gekocht,
sprake zal zijn van een bijzondere hardheid. De Minister voegt hieraan toe:
„ik heb hierbij in het bijzonder die gevallen op het oog, waarin de belasting
plichtige genoodzaakt is een lijfrente te bedingen of te kopen, teneinde
op deze wijze in zijn levensonderhoud en dat van zijn echtgenote te voor
zien”. Naar het ons voorkomt, zijn door deze departementale tegemoetkoming
aan het arrest de ergste giftanden uitgetrokken. Toch rijst de vraag: „wat
moet worden verstaan onder levensonderhoud? De opvatting van Prof. Smeets. 7, le lid valt en wordt belast volgens art. 20 le en 3e lid en waarop
de lijfrenteconstructie niet kan worden toegepast. En hij concludeert
dan: „dat de Hoge Raad hiervoor art. 36, 2e lid, niet van toepassing
acht, lijkt mij juist.” Hieruit volgt o.i., dat Prof. Smeets de lijfrenteconstructie wel van toe
passing acht op de elementen sub a. en b. van de liquidatiewinst en dat hij
dus, impliciet, de uitspraak van de Hoge Raad verwerpt. Zeer duidelijk
blijkt dit uit de zinsnede: „naar mijn mening mag althans voor het goodwill
bestanddeel zeer wel een beroep worden gedaan op art. 36, 2e lid juncto
het lelid, letter a — zoniet ook letter b.”. Met alle respect, aan deze fiscale autoriteit verschuldigd, menen wij toch,
van inzicht met hem te moeten verschillen. Prof. Smeets redeneert, naar het
ons voorkomt, op de eerste plaats als econoom — de leden van de Hoge
Raad zijn op de eerste plaats juristen. Prof. Smeets beoordeelt het probleem m ab
blz. 229 blz. 229 m ab naar de billijkheid m.b.t. de belastingheffing en daar ook hij kennelijk van
mening is, dat de wetgever geen dubbele belasting over hetzelfde inkomen
heeft gewild, komt hij tot zijn boven weergegeven conclusie. Maar ook
Prof. Smeets redeneert hier vanuit hetgeen de wetgever heeft bedoeld maar
niet gezegd. De Hoge Raad daarentegen heeft zich gehouden aan hetgeen
de wetgever wel heeft gezegd en op deze grond achten wij zijn standpunt
— met alle waardering voor het betoog van Prof. Smeets, toch juister dan
dat van Z.H.G. 5) Het laatste gedeelte hebben wij vrijwel letterlijk ontleend aan ons artikel in het
„Algemeen Handelsblad'' 8 juni 1957. Het Ministeriële standpunt. Zoals bekend is, gaat de
Hoge Raad m.b.t. de buitengewone last zeer ver — zal de Minister hem
hierin volgen? Zal, als iemand een vermogen heeft, van welks opbrengst
hij niet kan leven, de hardheidsclausule worden toegepast of zal het stand
punt worden ingenomen, dat alsdan de lijfrente niet noodzakelijk is voor
levensonderhoud van de belanghebbende en zijn echtgenote? m a b
blz. 230 m a b Uiteraard zijn deze vragen voor de praktijk van groot belang. Het komt
in de praktijk immers herhaaldelijk voor, dat een vennoot wil uittreden uit
een vennootschap onder een firma en dan een bepaald percentage van de
winst bedingt voor zichzelf en zijn echtgenote. Dit is dan een lijfrente ■—
dat het bedrag der jaarlijkse uitkering niet vaststaat, doet niet er zake. Door het arrest van de Hoge Raad zouden dan niet alleen de jaarlijkse
winstuitkeringen zijn belast, doch ook de contante waarde daarvan -— het
stamrecht. Het is te begrijpen, dat het arrest bij vele belastingplichtigen, die voor
nemens zijn, hun bedrijf of beroepsuitoefening te staken of op een of andere
wijze over te dragen, een gevoel van onzekerheid en groot onbehagen te
weeg heeft gebracht — vanwege de fiscale consequenties, die, als de
overdracht heeft plaatsgevonden, onafwendbaar zijn geworden. Gezien
de grote belangen, die hiermede zijn gemoeid, zal de belastingplichtige,
ingeval van twijfel, er goed aan doen, het Ministerie van Financiën te
raadplegen, alvorens de overeenkomst definitief aan te gaan. Een wellicht
tijdrovende, doch zekere weg, waardoor veel narigheid achteraf kan wor
den voorkomen. 5) m a b blz. 231
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Subsampled Data Based Alternative Regularized Estimators
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Journal of Data Science, 18(2). P. 238 - 256,2020
DOI:10.6339/JDS.202004 18(2).0002
SUBSAMPLED DATA BASED ALTERNATIVE REGULARIZED
ESTIMATORS
Subir Ghosh1* , Gabriel Ruiz2 , and Brandon Wales 3
1,2,3
Department of Statistics, University of California Riverside USA .
Abstract
Subsampling the data is used in this paper as a learning method about the influence of the data points for drawing inference on the parameters of a fitted logistic
regression model. The alternative, alternative regularized, alternative regularized
lasso, and alternative regularized ridge estimators are proposed for the parameter
estimation of logistic regression models and are then compared with the maximum
likelihood estimators. The proposed alternative regularized estimators are obtained
by using a tuning parameter but the proposed alternative estimators are not regularized. The proposed alternative regularized lasso estimators are the averaged
standard lasso estimators and the alternative regularized ridge estimators are also
the averaged standard ridge estimators over subsets of groups where the number of
subsets could be smaller than the number of parameters. The values of the tuning
parameters are obtained to make the alternative regularized estimators very close to
the maximum likelihood estimators and the process is explained with two real data
as well as a simulated study. The alternative and alternative regularized estimators
always have the closed form expressions in terms of observations that the maximum
likelihood estimators do not have. When the maximum likelihood estimators do
not have the closed form expressions, the alternative regularized estimators thus
obtained provide the approximate closed form expressions for them.
Key words and phrases: Item response, lasso, logistic regression, maximum likelihood, regularized, ridge, subsampling, tuning parameter.
SUBSAMPLED DATA BASED ALTERNATIVE REGULARIZED ESTIMATORS 239
1.
Introduction
Scientific investigations often involve fitting a logistic regression model to the binary response
data. Subsampling the data is implemented as a learning tool for enhancing the understanding of the estimation process in fitting a logistic regression model by harnessing the strength of
information from some special subsampled data. The logistic regression model is popular for describing the binary response data (Agresti, 2012; Bishop, Fienberg, & Holland, 1975; Chaloner
& Larntz, 1989; Cox, 1958; Efron, 1975; Engelhardt, 1975; Khan & Shaw, 2011; Webb, Wilson
& Chong, 2004). In computer age statistical inference (Efron & Hastie, 2016), the computationally intensive statistical methods play an important role in paving the pathways for statistical
learning and discoveries (James, Witten, Hastie, & Tibshirani, 2013).
The item response theory (IRT) refers to a family of mathematical models for describing
the relationship between latent traits (unobservable variables) and their realizations (observed
outcome variables). A link is established between the properties of items on an instrument, subjects responding to these items and the underlying trait being measured. IRT assumes that the
latent construct and items of a measure are organized in an unobservable continuum. The main
goal is to establish the subject position on that continuum. Since the subjects are different,
the positions on the continuum are not identical and are determined by the subjects included
in the research study and item parameters. An item should discriminate the subjects on the
basis of their responses on the continuum. Item response theory psychometrics are frequently
based upon logistic regression model (Lord (1983a,b& c, 1986), Rasch (1960), Stone (1992),
Baker and Kim (2017)). The item response function of the two parameter logistic model for a
dichotomous item is defined as
Pij (θj , bi , ai ) =
e[ai (θj −bi )]
,
1 + e[ai (θj −bi )]
where θj is the ability parameter of the individual j, bi is the difficulty parameter of the item i
or the item parameter, ai is the discrimination parameter and Pij (θj , bi , ai ) is the probability of
correct response for the individual j on the item i.
For the convenience of presentation, the following abbreviations are used for the estimators
throughout the paper.
Abbreviation
AE
ARE
ARLE
ARRE
MLE
Estimator
Alternative Estimator
Alternative Regularized Estimator
Alternative Regularized Lasso Estimator
Alternative Regularized Ridge Estimator
Maximum Likelihood Estimator
Consider a response variable Y generated from a binary random variable taking two realized
values 1 (success) and 0 (failure). One of the most popular models for studying the dependence
of the response variable Y on q explanatory variables X1 , ... , Xq in N groups, is the logistic
regression model. Denote the vector of q explanatory variables by (X1 , ... , Xq )0 . The response
variable Y for the ith group is denoted by Yi and the corresponding vector of explanatory
variables by Xi = (Xi1 , ... , Xiq )0 for i = 1, ..., N. For the ith group, the probability that the binary
random variable taking the value 1 is pi and the value 0 is (1 − pi ). The total number of realized
values of the binary random variable is ni , i = 1, ..., N. The Yi takes the value yi as the number
of observed value 1 and (ni − yi ) as the number of observed value 0. The random variables Y1 ,
... , YN are independent binomial random variables having
E(Yi ) = ni pi , Var(Yi ) = ni pi (1 − pi ), and Cov(Yi , Yi0 ) = 0, for i 6= i0 .
240
Subir Ghosh, Gabriel Ruiz, Brandon Wales
The realized value of the vector Xi is xi = (xi1 , ... , xiq )0 , for i = 1, ..., N. In the logistic regression
model
0
pi
eα+β xi
def
def
0
loge
= α + β xi , pi =
,
(1.1)
1 − pi
1 + eα+β 0 xi
where α and β 0 = (β1 , ..., βq ) are the unknown parameters. The likelihood function is denoted
by L(α, β). The score functions of the first derivatives of the log–likelihood function `(α, β)(=
logL(α, β)) with respect to α and the elements of
β are
denoted by U(α, β). The MLEs of α
b
b
and β, denoted by α
b and β, satisfy the MLEEs U α
b, β = 0. The Newton–Raphson and Fisher
scoring iterative methods provide the fast numerical values of α
b and βb using the computational
b Consequently, the exact
softwares, they do not provide any closed form expressions of α
b and β.
statistical inferences about them are not possible with their numerical values particularly in
small samples.
Section 2 considers two cases N = (q + 1) and N ≥ (q + 1). For the first case N = (q + 1),
the closed form expressions of MLEs are obtained when the observed values of yi are not equal
to 0 and ni , i = 1, ..., N. The AEs are then proposed for the second case N ≥ (q + 1) when
the closed form expressions of MLEs are not available. The AREs are introduced in Section
3 as an approximation for MLEs. The closed form expressions of AREs are available and are
dependent on a tuning parameter. Two real data examples are also presented in this section
to illustrate the proposed AREs. In Section 4, the ARLEs and the ARREs are introduced for
N ≥ or ≤ or = (q + 1) and compared for the second illustrative example. A simulation study
is given in Section 5. The closing Section 6 draws conclusions.
2.
MLEs and AEs
Denote an (N × (q + 1)) matrix X and a (1 × N) vector y by
1 ... 1 ... 1
0
X =
, y = (y1 , . . . , yN )0 .
x1 . . . x i . . . x N
Although the closed form expressions of MLEs α
b and βb are not available for the general case
N ≥ (q + 1), they are available for the special situation N = (q + 1) = Rank (X) and yi 6= 0
and yi 6= ni for i = 1, ..., N . Alternative estimators (AEs) of α and β are proposed for the general case N ≥ (q + 1) using the subsampled data and harnessing the learning from the situation
N = (q + 1) = Rank (X).
2.2. N = (q + 1) = Rank (X), yi 6= 0, ni , i =
1, ...,N
For the special situation N = q + 1, the MLEEs U α̂, β̂ = 0 become
b.
X0 y = X0 y
(2.1)
b. In addition, when yi 6= 0 and
When Rank (X) = N = (q + 1), it follows from (2.1) that y = y
yi 6= ni for i = 1, ..., (N = (q + 1)), it can be seen
yi
ni eαb+β xi
, α
b + βb0 xi = log
.
yi = b
yi =
b0 xi
α
b
+
β
ni − yi
1+e
b0
(2.2)
SUBSAMPLED DATA BASED ALTERNATIVE REGULARIZED ESTIMATORS 241
Denote
β = (α, β1 , . . . , βq ) , β = (b
α, βb1 , . . . , βbq ) , y = log
∗
0
b∗
0
∗
b
y1
b
yN
, . . . , log
n1 − b
y1
nN − b
yN
0
.
It can be seen from (2.3) that
Xβb∗ = y∗ , βb∗ = X−1 y∗ .
(2.3)
The (2.4) provides the closed form expression of βb∗ , the MLE of β ∗ , for the special situation
N = q + 1 = Rank (X) and yi 6= 0 and yi 6= ni for i = 1, ..., (N = (q + 1)).
2.2. Subsampled data when N ≥ (q + 1)
Consider the general situation when N ≥ (q + 1). The class S of all possible v (= q +N 1 )
subsamples of (q + 1) groups are considered to use (2.3) to find the closed form estimators
of β ∗ . Suppose that the closed form estimators of β ∗ can be found for the subclass S∗ of S,
consisting of v∗ subsamples out of v subsamples. The (v − v∗ ) subsamples in S but not in S∗
do not provide the closed form estimators because the rank condition Rank (X) = N = (q +
1) may not hold or the yi value could be ni or 0. For the jth subsample of S∗ , the closed form
estimator of β ∗ is denoted by βbj∗ . The proposed alternative estimator of β ∗ is
X
∗ def 1
βbj∗ .
βbAE
= ∗
v j∈S∗
(2.4)
∗
in (2.4) is different from the MLE βb∗ when N > (q + 1).
The proposed alternative estimator βbAE
∗
is identical to the MLE βb∗ in (3.9) when
However, the proposed alternative estimator βbAE
N = (q + 1).
2.3. Example 1
The Heart Disease and Blood Pressure Data given on page 217 in Table 6.5 of Agresti (2013)
consist of a sample of male residents of Framingham, Massachusetts, aged 40 to 59, having the
binary response variable Y as whether the residents developed coronary heart disease (Yes = 1)
during a six year follow up period and the explanatory variable (the risk factor) as the systolic
blood pressure X1 obtained from the range. Thus, q = 1 for this example.
Table 1 summarizes the data in the format of the
of this paper. Note that
presentation
8
∗
N = 8, q + 1 = 2, and N > (q + 1). Moreover, v = 2 = 28 = v . The MLE of β ∗ = (α, β1 )0 is
βb∗ = (b
α, βb1 )0 = (−6.082, 0.024)0 . The log–likelihood function `(α, β1 ) at α = α
b and β1 = βb1 is
∗
−19.305. On the other hand, the proposed alternative estimate βbAE
in (2.4) is (−6.514, 0.028)0
and the log–likelihood function `(α, β1 ) at α = −6.514 and β1 = 0.028 is −19.558.
2.4. Example 2
Smoking, obesity, and snoring were investigated for hypertension in 433 men aged 40 or over
and the data are given in Altman (1991). Table 2 summarizes the data in the format of this
paper. Note that N = 8, q + 1 = 4, and N > (q + 1). The MLE of β ∗ = (α, β1 , β2 , β3 )0 is
βb∗ = (b
α, βb1 , βb2 , βb3 )0 = (−2.378, −0.068, 0.695, 0.872)0
and the log–likelihood function is `(βb∗ ) = −13.269.
The numerical value of v becomes v = q +8 1 = 84 = 70. Excluding the 35 sub–samples
including Group 4 having y4 = 0 and observing that the rank condition Rank (X) = 4 does not
242
Subir Ghosh, Gabriel Ruiz, Brandon Wales
Table 1: The Heart Disease and Blood Pressure Data (Agresti(2013)))
Group (i)
Range
xi1
yi
ni
1
2
3
4
5
6
7
8
< 117
117 − 126
127 − 136
137 − 146
147 − 156
157 − 166
167 − 186
> 186
111.5
121.5
131.5
141.5
151.5
161.5
176.5
191.5
3
17
12
16
12
8
16
8
156
252
284
271
139
85
99
43
hold for 6 subsamples out of the remaining (70 − 35) = 35 subsamples, the numerical value of
∗
= (−2.399, 0.173, 0.665, 0.796)0
v∗ is (35 − 6) = 29. The proposed alternative estimate is βbAE
∗
) = −13.683.
and the log–likelihood function is `(βbAE
Table 2: The Hypertension Data (Altman (1991)))
Group
i
1
2
3
4
5
6
7
8
3.
Smoking
xi1
0
1
0
1
0
1
0
1
Obesity
xi2
0
0
1
1
0
0
1
1
Snoring
xi3
0
0
0
0
1
1
1
1
Hypertensive
yi
5
2
1
0
35
13
15
8
Mean
ni
60
17
8
2
187
85
51
23
AREs
The proposed alternative regularized estimators (AREs) depend on a non–negative integer
valued tuning (regularization) parameter λ where
0 ≤ λ ≤ (v∗ −1). For a fixed λ, in the class S∗
∗
of v∗ subsamples of (q + 1) groups, consider vλ possible subclasses, each consisting of (v∗ − λ)
∗
subsamples. For the jth subsample within the g(λ)th subclass S∗g(λ) , g(λ) = 1, ..., vλ , denote the
closed form estimator of β ∗ by βb∗ using (4) and obtain the alternative estimator following
jg(λ)
SUBSAMPLED DATA BASED ALTERNATIVE REGULARIZED ESTIMATORS 243
(2.4) as
b∗
βAE(λ)
∗
X
1
v
∗
= ∗
.
βbjg(λ) , g(λ) = 1, ...,
λ
(v − λ) j∈S∗
def
(3.5)
g(λ)
∗
Denote LL(λ) = ` βbAE
(λ) . Find a λ (= λM , say) maximizing the log−likelihood, LL(λ), for
∗
λ = 1, ..., (v − 1). In other words, λM is the value of λ satisfying
def
LL(λM ) = max [LL(λ)] .
λ
(3.6)
Define the alternative regularized estimator (ARE) of β ∗ as
def ∗
∗
(λM ) = βbAE
βbARE
(λM ) .
(3.7)
∗
∗
(λM ) depends on the tuning parameter λ through βbAE
Notice that βbARE
(λ) , for all values of λ,
∗
0 ≤ λ ≤ (v − 1). The (2.4) and (3.5) become equal for λ = 0 or equivalently,
∗
b∗
βbAE
(0) = βAE .
(3.8)
The value λM is the choice for the tuning parameter λ to satisfy
βb∗ (λM ) ' βb∗ , LL(λM ) = ` βb∗ (λM ) ' ` βb∗ .
ARE
ARE
(3.9)
Consequently, the MLE is approximately equal to the ARE for the choice of tuning parameter
λ = λM .
In Example 1 of Section 2.3, it can be seen that λM = 20. Define two Transformed LogLikelihood (TLL)s from LL(λ) as
TLL1(λ) = (LL(λ) + 19) , TLL2(λ) = (LL(λ) + 19.305) ×109 .
Figure 1 displays the plot of TLL1(λ) against λ (= 0, 1, ... , 27). Figure 2 highlighting the
maximum (MAX) of Figure 1 presents the plot of TLL2(λ) against λ (= 8, 9, ... , 21). From
Table 3 and Figure 2,
∗
βb∗ = (−6.082, 0.024)0 and βbARE
(20) = (−6.082, 0.024)0
are identical up to 4 (after truncation) decimal places for the first component and 6 decimal
places
for the
second component. Moreover,
∗
b
` βARE (20) = −19.30519373 and ` βb∗ = −19.30519372
are identical up to 7 decimal places.
Hence
it follows from (3.9) that
∗
∗
∗
∗
βbARE (20) ' βb , ` βbARE (20) ' ` βb .
Figure 3 displays the plot of LL(λ) against λ = 0, 1, ..., 28 for N = 8 and q = 3. On the
other hand, Figure 4 displays the plot of LL(λ) against λ = 0, 1, ..., 19 for N = 7 and q = 2.
Subir Ghosh, Gabriel Ruiz, Brandon Wales
−0.40
−0.45
−0.55
−0.50
Transformed Log−Likelihood (TLL)
−0.35
−0.30
244
0
5
10
15
20
25
30
Lambda
Figure 1: Plot of TLL1 against λ.
For N = 8 and q = 3, it can be seen that
∗
0
b
β = (−2.378, −0.068, 0.695, 0.872) and ` βb∗ = −13.269.
For λ = 22,
0
∗
(22)
βbARE
0.143, 0.651, 0.814)
= (−2.377,
∗
and ` βbARE
(22) = −13.277,
which is the maximum value of LL(λ) for λ = 0, 1, ..., 28. The λM = 22 is then approximating
∗
βb∗ by βbARE
(22).
Some aspects of the fitting statistics for logistic regression model to the data in Table 2 are
given in Table 4. The explanatory variable “smoking” is not significant in having the P value
as 0.808. Deleting the explanatory variable
smoking from the model, q + 1 becomes 3. The
7
7
numerical value of v becomes v = q + 1 = 3 = 35. The rank condition, Rank (X) = 4, holds
for only 20 subsamples and does not hold for 15 out of 35 subsamples. Therefore, v∗ = 20.
For N = 7 and q = 2 excluding “smoking” as an explanatory variable, it can be seen from
Figure 4 that the maximum value -13.302061150 of LL(λ) for λ = 0, 1, ..., 19 is attained at
∗
λM = 3 for approximating βb∗ by βbARE
(3).
SUBSAMPLED DATA BASED ALTERNATIVE REGULARIZED ESTIMATORS 245
−800
−850
−900
Transformed Log−Likelihood (TLL)
−750
MAX
10
15
20
Lambda[=8,...,21]
Figure 2: Plot of TLL2 against the partial values of λ[= 8, ..., 21].
4.
ARLE and ARRE for N > or < or = (q+1)
In this section, N can be equal to, greater or even less than (q + 1). Groups having yi = 0
(Group 4 in Example 2) or ni = yi are ignored to reduce the value of N. Choose a number γ,
0 < γ < N, when N ≥ (q + 1) or a number γ, 0 < γ ≤ N, when N < (q + 1). For the lasso and
ridge estimations, the tuning parameters are denoted by δL and δR respectively. The optimum
values of δL and δR are determined by minimizing the Generalized
Cross Validations (GCVs)
(Eq. (7.46), p. 217, Hastie et al. (2001)) for all possible Nγ subsamples of groups. Then the
Lasso estimates βbL∗ (δL ) and the Ridge estimates βbR∗ (δR ) are then calculated for Nγ subsamples
∗
∗
of groups. The average of Nγ both Lasso and Ridge estimates provide βbARLE
and βbARRE
similar
∗
b
to βAE in (5). In Example 2 excluding
Group 4, N = 7 and (q + 1) = 4. Taking γ = 4, the
∗
∗
Lasso estimates βb (δL ) and ` βb (δL ) for 35 subsamples of groups are displayed in Table 5 for
L
L
γ = 4. It can be seen from Table 5 that
∗
∗
βbARLE
= (−1.977, 0.074, 0.362, 0.370)0 and therefore ` βbARLE
= −15.221.
∗
∗
b
b
Again taking γ = 4, the Ridge estimates βR (δR ) and ` βR (δR ) for 35 subsamples of groups
are displayed in Table 6. Moreover, it can be seen from
Table
6 that
∗
0
∗
b
b
βARRE = (−2.013, 0.094, 0.365, 0.428) and therefore ` βARRE = −14.948.
Taking γ = 3, the Lasso estimates βbL∗ (δL ) and ` βbL∗ (δL ) for 35 subsamples of groups are displayed in Table 7. It can be seen from Table 7 that
∗
∗
βbARLE
= (−1.751, 0.067, 0.191, 0.124)0 and therefore ` βbARLE
= −17.506.
246
Subir Ghosh, Gabriel Ruiz, Brandon Wales
Table 3: Comparison among MLE, AE, and ARE for λ = 20
MLE
β∗ :
α
b
βb
1
∗
`(β )
ARE for λ = 20
βb∗
− 6.082
AE
≡ (ARE for λ = 0)
∗
βbAE
− 6.514
0.024
`(βb∗ )
− 19.305
0.028
∗
)
`(βbAE
− 19.558
0.024
∗
b
(20))
`(βARE
− 19.305
∗
βbARE
(20)
− 6.082
Table 4: Fitting statistics for logistic regression model to the Hypertension Data (Altman
(1991))
Explanatory
Variable
Parameter
MLE
Std. Error
z value
P value
Intercept
Smoking
Obesity
Snoring
α
β1
β2
β3
- 2.378
- 0.068
0.695
0.872
0.381
0.278
0.285
0.398
- 6.254
- 0.244
2.439
2.193
4 e−10
0.808
0.015
0.028
−13.5
−13.6
Log−Likelihood (LL)
−13.4
−13.3
MAX
0
5
10
15
20
25
Lambda
Figure 3: Plot of LL(λ) against λ for N = 8 and q = 3.
30
−13.30
SUBSAMPLED DATA BASED ALTERNATIVE REGULARIZED ESTIMATORS 247
−13.34
−13.38
−13.36
Log−Likelihood
−13.32
MAX
0
5
10
15
20
Lambda
Figure 4: Plot of LL(λ) against λ for N = 8 and q = 2.
Again taking γ = 3, the Ridge estimates βbR∗ (δR ) and ` βbR∗ (δR ) for 35 subsamples of groups
are displayed in Table 6. Moreover, it can be seen from
8 that
Table
∗
0
∗
b
b
βARRE = (−1.600, 0.005, 0.020, 0.021) and therefore ` βARRE = −19.382.
The results in Table 9 demonstrate that ARE (λ = 22) is closest to MLE. Lasso(1,5,7),
Lasso(1,5,6,7), AE, and Ridge(1,5,6,7) are also closer to MLE but behind ARE (λ = 22).
ARLE(γ = 4), ARLE(γ = 3), ARRE(γ = 4), and ARRE(γ = 3) are a bit away from MLE.
5.
A Simulation Study
The 1,000 data sets are generated on yi , i = 1, ..., 8 under the logistic regression model in (1)
for q = 1 having the assumed values of α = −6.08 and β1 = 0.025 and keeping the (xi1 , ni ) values
∗
the same as in Table 1. For the ith data set the numerical values of MLE βb∗ , AE βbAE
, and ARE
(
i
)
∗(
i
)
(i)
( i)
∗
∗(i)
(i) b
0 b
b
b
βARE (λ), 0 ≤ λ ≤ 27 are obtained and denote them by β
= (b
α , β1 ) , βAE = (b
αAE , βb1AE )0 ,
∗(i)
(i)
(i)
and βbARE (λ) = (b
αARE (λ), βb1ARE (λ))0 . Define
1000
1 X (i)
α
b=
α
b ,
1000 i=1
1000
1000
α
bAE
1 X (i)
=
α
bAE ,
1000 i=1
1000
1000
1 X (i)
α
bARE (λ) =
α
bARE (λ),
1000 i=1
1000
(5.10)
1 X b(i) b
1 X b(i) b
1 X b(i)
βb1 =
β1 , β 1AE =
β1AE , β 1ARE (λ) =
β1ARE (λ).
1000 i=1
1000 i=1
1000 i=1
∗
∗
The log likelihoods `(βb∗ ), `(βbAE
), and `(βbARE
(λ)), 0 ≤ λ ≤ 27 are also obtained. Then their
averages are found over 1,000 data sets.
248
Subir Ghosh, Gabriel Ruiz, Brandon Wales
1000
¯ βb∗ ) =
`(
1000
1000
1 X b∗(i) ¯ b∗
1 X b∗(i) ¯ b∗
1 X b∗(i)
`(β ), `(βAE ) =
`(βAE ), `(βARE (λ)) =
`(βARE (λ)).
1000 i=1
1000 i=1
1000 i=1
(5.11)
Table 10 presents the averages in (5.10) and (5.11) with the exceptions on βb1ARE (λ) and
¯ β (λ)) only for the λ values: 6, 21, and 25 to save the space.
`(
ARE
b∗
∗
The value of λM satisfying (3.9) is determined from Table 10 as λM = 21 giving βb ARE (21) =
∗
¯ βb∗ (21)) = −19.714012 in contrast to βb = (−6.083941, 0.025019))0
(−6.083899, 0.025019)0 and `(
ARE
and `(βb∗ ) = −19.714009. The true value of β ∗ = (α, β1 )0 = (−6.08, 0.025)0 . The value of λT
satisfying
∗
βbARE
(λT ) ' β ∗ ,
(5.12)
∗
is also obtained from Table 10. A choice for λT is 25 having βb ARE (25) = (−6.083519, 0.025016)0
closest to β ∗ = (−6.080, 0.025)0 but giving `(βb∗ ARE (25)) = − 19.716031. Denote the mean
square error by MSE and the mean absolute difference by MAD. Define
1000
1000
2
1 X (i)
1 X (i)
MSE(b
α) =
α
b + 6.080 ,
α
b + 6.080 , MAD(b
α) =
1000 i=1
1000 i=1
1000
1000
2
1 X b(i)
1 X b(i)
b
b
MSE(β1 ) =
β − 0.025 , MAD(β1 ) =
β − 0.025 .
1000 i=1 1
1000 i=1 1
(5.13)
Table 11 presents the numerical values of MSE(b
α), MSE(βb1 ), MAD(b
α), and MAD(βb1 ) for
∗
∗
(λ) when λ = 6, 21, and 25. It is clear from Table 11 that the best choice for λT
, βbARE
βb∗ , βbAE
is 21 with respect to the criterion functions MSE and MAD. Note that
−6.080000 < α
b = −6.083941 < −6.083899 = α
bARE (21),
¯ βb∗
`(
ARE
0.025000 < 0.025019 = βb1 = βb1ARE (21),
¯ βb∗ ),
(21)) = −19.714012 < −19.714009 = `(
1000
1 X (i)
α
bARE (21) − α
b(i) = 0.000812,
1000 i=1
(5.14)
1000
1 X b(i)
β1ARE (21) − βb1(i) = 0.000005,
1000 i=1
demonstrating the closeness of the true value of β ∗ = (α, β1 )0 = (−6.08, 0.025)0 to the av∗
∗
eraged MLEs βb = (−6.083941, 0.025019)0 as well as to the averaged AREs βb ARE (21) =
(−6.083899, 0.025019)0 from the 1000 simulated data sets. In other words, the performance
of ARE for the choice of the tuning parameter λ = 21 is almost equivalent to MLE.
SUBSAMPLED DATA BASED ALTERNATIVE REGULARIZED ESTIMATORS 249
6.
Conclusions
Subsampling methods are implemented to obtain the AE, AREs, ARLEs, ARREs. By using
these estimators, new insights are gained for fitting the logistic models to the data. The proposed AREs depend on the tuning parameter λ and AEs. The tuning parameter value can be
obtained to make the ARE and its likelihood approximately equal to the corresponding values
of MLE. Two real data one with an explanatory variable and the other with three explanatory
variables are presented to illustrate the proposed methods. Example 2 demonstrates the closeness of ARE for a choice of the tuning parameter λ relative to AE, ARLE, ARRE, Lasso and
Ridge for specific sub-sampled data. A simulated data example is also presented to demonstrate
the almost equivalent performance of ARE and MLE for a choice of tuning parameter. The
AREs have closed form expressions helpful to study their statistical properties even for small
samples and are good closed form approximate representations for MLEs particularly when they
do not exist.
The future investigation of this work will include the detailed theoretical properties of the
AE, AREs, ARLEs and ARREs in comparison with the MLEs. More detailed simulation studies
will be beneficial. The investigation for the high dimensional data will be another important
direction. For the longitudinal data (Chan, 2014; Ghosh & Chakravartty, 2009), the investigation remains to be performed. In the area of quantitatively estimate the accuracy of artificial
intelligence in machine learning (Hastie, T. et al., 2001; James, G. et al., 2013), IRT could be
very useful and our proposed average estimators could be applicable.
Acknowledgements
The authors are grateful to the Editor and the entire review team for their constructive
comments on the earlier version of this paper.
References
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[19] Rao, C. R. (1973). Linear Statistical Inference and Its Applications, Wiley: New York,2nd Edition.
[20] Rasch, G. (1960). Probabilistic Models for Some Intelligence and Attainment Tests,Danish Institute for Educational Research: Copenhagen. (Expanded edition (1980) with foreword and afterword
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[21] Stone C.A. (1992). Recovery of Marginal Maximum Likelihood Estimates in the Two-Parameter
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SUBSAMPLED DATA BASED ALTERNATIVE REGULARIZED ESTIMATORS 251
∗
∗
b
b
Table 5: Lasso estimates βL (δL ) and ` βL (δL ) for 35 subsamples of groups
Subset
1,
1,
1,
1,
1,
1,
1,
1,
1,
1,
1,
1,
1,
1,
1,
1,
1,
1,
1,
1,
2,
2,
2,
2,
2,
2,
2,
2,
2,
2,
3,
3,
3,
3,
5,
2,
2,
2,
2,
2,
2,
2,
2,
2,
2,
3,
3,
3,
3,
3,
3,
5,
5,
5,
6,
3,
3,
3,
3,
3,
3,
5,
5,
5,
6,
5,
5,
5,
6,
6,
3,
3,
3,
3,
5,
5,
5,
6,
6,
7,
5,
5,
5,
6,
6,
7,
6,
6,
7,
7,
5,
5,
5,
6,
6,
7,
6,
6,
7,
7,
6,
6,
7,
7,
7,
5
6
7
8
6
7
8
7
8
8
6
7
8
7
8
8
7
8
8
8
6
7
8
7
8
8
7
8
8
8
7
8
8
8
8
δL
0.058
0.077
0.213
0.010
0.128
0.044
0.010
0.228
0.010
0.011
0.121
0.008
0.010
0.008
0.010
0.010
0.008
0.231
0.010
0.010
0.003
0.202
0.233
0.172
0.203
0.008
0.006
0.008
0.081
0.027
0.111
0.134
0.008
0.164
0.075
βbL∗ (δL )
-2.042, 0.000,
-2.018, 0.000,
-1.836, 0.000,
-2.359, 0.344,
-1.951, 0.000,
-2.118, 0.000,
-2.357, 0.342,
-1.750, 0.000,
-2.357, 0.342,
-2.322, 0.273,
-1.930, 0.000.
-2.401, 0.000,
-2.358, 0.389,
-2.398,-0.260,
-2.517, 0.963,
-2.357, 0.206,
-2.365,-0.177,
-1.552, 0.000,
-2.356, 0.205,
-2.356, 0.206,
-1.809,-0.206,
-1.576, 0.000,
-1.515, 0.000,
-1.637, 0.000,
-1.575, 0.000,
-2.132, 0.149,
-1.747,-0.243,
-1.804,-0.179,
-1.691, 0.000,
-1.906, 0.000,
-1.500, 0.000,
-1.439, 0.000,
-2.448, 0.247,
-1.290, 0.000,
-1.440, 0.000,
0.000, 0.342
0.000, 0.000
0.000, 0.111
0.413, 0.934
0.000, 0.105
0.417, 0.650
0.457, 0.888
0.000, 0.000
1.042, 0.303
1.412, 0.000
0.000, 0.099
0.491, 0.968
0.412, 0.890
0.483, 0.978
0.729, 0.000
0.411, 1.070
0.593, 0.863
0.000, 0.000
0.593, 0.888
1.042, 0.439
0.124, 0.316
0.000, 0.000
0.000, 0.000
0.000, 0.000
0.000, 0.000
0.186, 1.103
0.569, 0.279
1.019, 0.303
0.555, 0.222
0.905, 0.195
0.000, 0.000
0.000, 0.000
0.532, 1.010
0.000, 0.000
0.537, 0.000
` βbL∗ (δL )
-19.524
-27.259
-20.588
-15.479
-22.763
-13.976
-15.096
-21.437
-17.707
-23.117
-22.485
-13.600
-15.549
-13.833
-29.954
-15.822
-13.500
-19.608
-14.107
-15.864
-19.400
-19.708
-19.529
-20.112
-19.703
-20.541
-15.019
-15.727
-15.420
-15.128
-19.522
-19.645
-14.537
-21.002
-17.480
252
Subir Ghosh, Gabriel Ruiz, Brandon Wales
∗
∗
b
b
Table 6: Ridge estimates βR (δR ) and ` βR (δR ) for 35 subsamples of groups
Subset
1,
1,
1,
1,
1,
1,
1,
1,
1,
1,
1,
1,
1,
1,
1,
1,
1,
1,
1,
1,
2,
2,
2,
2,
2,
2,
2,
2,
2,
2,
3,
3,
3,
3,
5,
2,
2,
2,
2,
2,
2,
2,
2,
2,
2,
3,
3,
3,
3,
3,
3,
5,
5,
5,
6,
3,
3,
3,
3,
3,
3,
5,
5,
5,
6,
5,
5,
5,
6,
6,
3,
3,
3,
3,
5,
5,
5,
6,
6,
7,
5,
5,
5,
6,
6,
7,
6,
6,
7,
7,
5,
5,
5,
6,
6,
7,
6,
6,
7,
7,
6,
6,
7,
7,
7,
5
6
7
8
6
7
8
7
8
8
6
7
8
7
8
8
7
8
8
8
6
7
8
7
8
8
7
8
8
8
7
8
8
8
8
δR
βbR∗ (δR )
` βbR∗ (δR )
57.993
53.175
0.031
0.037
0.131
0.031
0.038
0.256
0.038
0.040
0.163
0.030
0.037
0.029
0.036
0.039
0.029
0.085
0.039
0.039
0.012
0.074
232.959
0.337
0.362
0.031
0.023
0.030
0.030
0.031
111.376
134.265
0.029
163.871
0.030
-1.957,-0.000, 0.000, 0.001
-2.018, 0.000, 0.000, 0.0004
-2.265, 0.0205, 0.406, 0.807
-2.360, 0.409, 0.466, 0.701
-2.035, 0.028, 0.000, 0.246
-2.211, 0.161, 0.520, 0.703
-2.270, 0.299, 0.488, 0.743
-1.941,-0.031, 0.280, 0.273
-2.323, 0.322, 0.800, 0.385
-2.266, 0.259, 0.657, 0.656
-1.991, 0.028, 0.016, 0.198
-2.300, 0.000, 0.431, 0.824
-2.259, 0.458, 0.340, 0.729
-2.327,-0.139, 0.475, 0.783
-2.435, 0.451, 0.627, 0.450
-2.303, 0.302, 0.446, 0.863
-2.249,-0.147, 0.545, 0.714
-2.136, 0.155, 0.562, 0.488
-2.232, 0.258, 0.561, 0.726
-2.290, 0.185, 0.868, 0.480
-1.805,-0.176,-0.116, 0.273
-1.874,-0.085, 0.175, 0.464
-1.515, 0.000, 0.000, 0.001
-1.724,-0.091, 0.091, 0.173
-1.847, 0.105, 0.175, 0.211
-2.161, 0.211, 0.326, 0.889
-1.683,-0.277, 0.455, 0.253
-1.837,-0.097, 0.779, 0.346
-1.993, 0.0970, 0.591, 0.537
-1.998, 0.054, 0.801, 0.332
-1.501,-0.000, 0.000, 0.000
-1.439, 0.001, 0.000, 0.001
-2.131, 0.338, 0.349, 0.740
-1.291, 0.000, 0.000, 0.001
-1.500, 0.001, 0.656, 0.000
-25.615
-27.253
-14.427
-15.248
-21.131
-13.951
-14.574
-16.687
-16.406
-14.114
-21.121
-13.742
-16.143
-13.984
-17.850
-15.020
-13.714
-14.302
-14.363
-14.789
-19.670
-15.649
-19.523
-17.873
-17.256
-16.289
-15.577
-14.604
-14.313
-14.393
-19.518
-19.638
-16.132
-20.991
-16.912
SUBSAMPLED DATA BASED ALTERNATIVE REGULARIZED ESTIMATORS 253
∗
∗
b
b
Table 7: Lasso estimates βL (δL ) and ` βL (δL ) for 35 subsamples of groups
Subset
1,
1,
1,
1,
1,
1,
1,
1,
1,
1,
1,
1,
1,
1,
1,
2,
2,
2,
2,
2,
2,
2,
2,
2,
2,
3,
3,
3,
3,
3,
3,
5,
5,
5,
6,
2,
2,
2,
2,
2,
3,
3,
3,
3,
5,
5,
5,
6,
6,
7,
3,
3,
3,
3,
5,
5,
5,
6,
6,
7,
5,
5,
5,
6,
6,
7,
6,
6,
7,
7,
3
5
6
7
8
5
6
7
8
6
7
8
7
8
8
5
6
7
8
6
7
8
7
8
8
6
7
8
7
8
8
7
8
8
8
δL
βbL∗ (δL )
` βbL∗ (δL )
0.058
0.164
0.006
0.296
0.014
0.156
0.103
0.012
0.015
0.180
0.013
0.014
0.262
0.015
0.333
0.114
0.060
0.246
0.300
0.004
0.009
0.247
0.009
0.012
0.281
0.079
0.172
0.125
0.145
0.173
0.201
0.007
0.214
0.007
0.213
-2.120, 0.000, 0.000, 0.000
-1.960, 0.000, 0.000, 0.000
-2.382, 0.367, 0.000, 0.287
-1.763, 0.000, 0.000, 0.000
-2.357, 0.343, 1.346, 0.000
-1.938, 0.000, 0.000, 0.000
-2.019, 0.000, 0.000, 0.000
-2.361, 0.000, 0.416, 1.034
-2.354, 1.274, 0.409, 0.000
-1.859, 0.000, 0.000, 0.000
-2.360, 0.0000, 0.555, 0.891
-2.356, 0.798, 0.000, 0.888
-1.662, 0.000, 0.000, 0.000
-2.354, 0.642, 1.039, 0.000
-1.398, 0.000, 0.146, 0.000
-1.810, 0.000, 0.000, 0.000
-1.891, 0.000, 0.000, 0.000
-1.612, 0.000, 0.000, 0.000
-1.530, 0.000, 0.000, 0.000
-1.772,-0.230, 0.0000, 0.290
-1.469,-0.520, 0.566, 0.000
-1.371, 0.000, 0.000, 0.000
-1.179,-0.808, 0.000, 0.275
-1.980, 0.000, 1.048, 0.268
-1.173, 0.000, 0.000, 0.000
-1.709, 0.000, 0.000, 0.000
-1.430, 0.000, 0.000, 0.000
-1.571, 0.465, 0.000, 0.103
-1.511, 0.000, 0.000, 0.000
-1.429, 0.000, 0.000, 0.000
-1.344, 0.000, 0.000, 0.291
-1.469,-0.223, 0.573, 0.000
-1.270, 0.000, 0.000, 0.000
-1.448, 0.227, 0.573, 0.000
-1.072, 0.000, 0.000, 0.000
-30.360
-25.723
-26.833
-21.632
-23.613
-25.148
-27.285
-14.215
-31.109
-23.385
-13.429
-22.293
-20.340
-23.747
-18.935
-22.424
-24.059
-19.920
-19.551
-18.225
-17.955
-20.076
-25.545
-15.196
-23.213
-20.870
-19.682
-21.271
-19.526
-19.688
-23.039
-16.908
-21.318
-18.803
-25.977
254
Subir Ghosh, Gabriel Ruiz, Brandon Wales
∗
∗
b
b
Table 8: Ridge estimates βR (δR ) and ` βR (δR ) for 35 subsamples of groups
Subset
1,
1,
1,
1,
1,
1,
1,
1,
1,
1,
1,
1,
1,
1,
1,
2,
2,
2,
2,
2,
2,
2,
2,
2,
2,
3,
3,
3,
3,
3,
3,
5,
5,
5,
6,
2,
2,
2,
2,
2,
3,
3,
3,
3,
5,
5,
5,
6,
6,
7,
3,
3,
3,
3,
5,
5,
5,
6,
6,
7,
5,
5,
5,
6,
6,
7,
6,
6,
7,
7,
3
5
6
7
8
5
6
7
8
6
7
8
7
8
8
5
6
7
8
6
7
8
7
8
8
6
7
8
7
8
8
7
8
8
8
δL
57.886
102.978
1.806
4.096
4.424
118.239
102.264
3.990
4.750
2.486
4.141
4.558
3.983
4.817
4.615
113.750
59.709
3.098
300.400
1.435
2.926
3.760
3.040
3.800
3.541
79.064
171.980
3.320
144.959
172.388
3.348
2.199
2.959
2.205
2.953
` βbL∗ (δL )
βbL∗ (δL )
-2.120, 0.000, 0.000,
-1.961,-0.000, 0.000,
-2.059, 0.018, 0.000,
-1.788,-0.011, 0.043,
-1.742, 0.038, 0.055,
-1.938, 0.000,-0.000,
-2.019, 0.000, 0.000,
-1.777, 0.000, 0.033,
-1.715, 0.050, 0.036,
-1.880, 0.007, 0.000,
-1.618, 0.000, 0.034,
-1.556, 0.042, 0.042,
-1.698,-0.003, 0.039,
-1.645, 0.038, 0.045,
-1.388, 0.035, 0.057,
-1.810,-0.000,-0.000,
-1.891, 0.000,-0.000,
-1.632,-0.020, 0.020,
-1.531, 0.000, 0.000,
-1.732,-0.013, 0.000,
-1.472,-0.027, 0.029,
-1.406, 0.005, 0.036,
-1.539,-0.032, 0.032,
-1.484, 0.000, 0.041,
-1.222,-0.015, 0.044,
-1.709, 0.000,-0.000,
-1.430, 0.000, 0.000,
-1.385, 0.037, 0.006,
-1.511,-0.000, 0.000,
-1.430, 0.001, 0.000,
-1.188, 0.028, 0.000,
-1.354,-0.018, 0.024,
-1.287, 0.010, 0.032,
-1.013, 0.018, 0.024,
-1.087,-0.009, 0.032,
0.000
0.001
0.016
0.043
0.055
0.001
0.000
0.044
0.050
0.027
0.039
0.045
0.037
0.038
0.057
0.000
0.000
0.039
0.001
0.014
0.027
0.031
0.024
0.028
0.044
0.000
0.000
0.031
0.001
0.001
0.043
0.000
0.000
0.000
0.000
-30.359
-25.711
-27.794
-20.707
-19.624
-25.138
-27.279
-20.601
-19.596
-23.106
-19.139
-18.848
-19.734
-19.153
-19.703
-22.421
-24.058
-19.380
-19.543
-20.972
-19.141
-19.514
-19.086
-19.031
-22.287
-20.869
-19.679
-20.016
-19.521
-19.680
-23.572
-20.018
-20.921
-28.138
-25.341
SUBSAMPLED DATA BASED ALTERNATIVE REGULARIZED ESTIMATORS 255
Table 9: Comparison of MLE, AE, ARE, ARLE (γ = 4), ARRE (γ = 4), ARLE (γ = 3) and
ARRE (γ = 3) for Example 2
Method (u)
βbu∗
` βbu∗
MLE
AE
ARE(λ = 22)
ARLE(γ = 4)
ARRE(γ = 4)
ARLE(γ = 3)
ARRE(γ = 3)
Lasso(1, 5, 6, 7)
δL = 0.00830
Ridge(1, 5, 6, 7)
δR = 0.02845
Lasso(1, 5, 7)
δL = 0.01264
Ridge(1, 5, 7)
δR = 4.14056
Ridge (1,5,8)
δR = 4.55745
-2.378, -0.068, 0.695, 0.872
-2.399, 0.173, 0.665, 0.796
-2.377, 0.143, 0.651, 0.814
-1.977, 0.074, 0.362, 0.370
-2.013, 0.094, 0.365, 0.428
-1.751, 0.067, 0.191, 0.124
-1.600, 0.005, 0.020, 0.021
-2.364, -0.177, 0.593, 0.863
-13.269
-13.683
-13.277
-15.221
-14.948
-17.506
-19.382
-13.500
-2.249, -0.147, 0.545, 0.714
-13.714
-2.360, 0.000, 0.555, 0.891
-13.429
-1.618, 0.000, 0.034, 0.039
-19.139
-1.556, 0.042, 0.042, 0.045
-18.848
¯ βb∗ ), `(
¯ βb∗ ), and `(
¯ βb∗ (λ)) calculated
Table 10: The average values βb∗ , βb∗ AE , βb∗ ARE (λ), `(
AE
ARE
over 1,000 simulated data for λ = 6, 21, and 25
∗
∗
∗
∗
∗
β∗ :
βb
α
β1
− 6.084
0.025
− 6.183
0.026
− 6.084
0.025
− 6.084
0.025
− 6.084
0.025
l(β ∗ )
l̄(βb∗ )
¯ βb∗ )
`(
AE
∗
l̄(βbARE
(6))
¯ βb∗ (21))
`(
ARE
¯ βb∗ (25))
`(
ARE
− 19.714
− 20.458
− 19.725
− 19.714
− 19.716
βb
AE
βb
ARE
(6)
βb
ARE
(21)
βb
ARE
(25)
256
Subir Ghosh, Gabriel Ruiz, Brandon Wales
∗
∗
Table 11: The MSE(b
α), MSE(βb1 ), MAD(b
α), and MAD(βb1 ) for βb∗ , βbAE
, βbARE
(λ) when
λ = 6, 21, and 25
MSE(b
α)
MSE(βb1 )
MAD(b
α)
MAD(βb1 )
βb∗
0.444
0.000
0.530
0.004
∗
βbAE
0.712
0.000
0.654
0.005
∗
(6)
βbARE
0.447
0.000
0.532
0.004
∗
βbARE
(21)
0.444
0.000
0.530
0.004
∗
(25)
βbARE
0.446
0.000
0.531
0.004
|
|
https://openalex.org/W4312947397
|
https://wnus.edu.pl/ais/file/article/view/19842.pdf
|
Polish
| null |
Legal definition of a “beneficiary” of public funds transferred on the basis of a project financing agreement under the cohesion policy
|
Acta Iuris Stetinensis
| 2,022
|
cc-by-sa
| 8,582
|
Robert Talaga
dr
Wojewódzki Sąd Administracyjny w Poznaniu
e-mail: r.talaga@wsa.poznan.pl
ORCID: 0000-0002-5281-2188 4
S. Prutis, Dobór instrumentów prawnych służących wsparciu rozwoju obszarów wiejskich, „Studia
Iuridica Agraria” 2009, t. VII, s. 198; S. Prutis, Mechanizmy prawne wsparcia rozwoju obszarów
wiejskich ze środków Unii Europejskiej (dobór metody regulacji prawnej), w: A. Doliwa, S. Pru-
tis (red.), Tom III – Wypieranie prawa administracyjnego przez prawo cywilne, w: D.R. Kijowski,
P.J. Suwaj (red.), Kryzys prawa administracyjnego?, Warszawa 2012, s. 122. 1
Zob. W. Stankiewicz, Budżet Unii Europejskiej – kierunki ewolucji systemu finansowania w aspekcie
nowej perspektywy finansowej 2007–2013, „Rocznik Integracji Europejskiej” 2007, nr 1, s. 219–235;
K. Brysiewicz, System wyboru i oceny projektów współfinansowanych ze środków europejskich w per-
spektywie finansowej 2014–2020, „Przegląd Prawa Publicznego” 2014, nr 10, s. 7–24; R. Poździk,
Zasady wdrażania funduszy unijnych w latach 2014–2020, „Europejski Przegląd Sądowy” 2014,
nr 12, s. 4–11. 3
Zob. A. Gajda, Spójność gospodarcza i społeczna, w: J. Barcz (red.), Prawo Unii Europejskiej, Tom
II Prawo materialne i polityki, Warszawa 2006, s. II, 651–668; M. Świstak, Polityka regionalna Unii
Europejskiej jako polityka publiczna, Kraków 2018, s. 33 i n. 2
Zob. M. Świstak, J.W. Tkaczyński, Polityki publiczne – zagadnienia teoretyczne, w: M. Świstak,
J.W. Tkaczyński (red.), Wybrane polityki publiczne Unii Europejskiej. Stan prawny i perspektywy,
Kraków 2015, s. 11–28. Streszczenie Streszczenie Zdefiniowanie określonego pojęcia najczęściej ma zastosowanie do konkretnego aktu praw-
nego. W praktyce jednak wprowadzona definicja może mieć szersze znaczenie systemowe. Z taką sytuacją mamy do czynienia w przypadku definicji beneficjenta, która – została wpro-
wadzona krajowymi przepisami prawnymi. Z jednej strony wymaga ona uwzględnienia pojęć
o charakterze uniwersalnym dla całego systemu prawnego. Z drugiej jednak jest ogranicza-
na regulacjami stosowanymi przy realizacji określonych celów strategicznych wyznaczonych
przez poszczególne programy operacyjne. Zasygnalizowane uwarunkowania powinny być
brane pod uwagę w procesie wykładni pojęcia podmiotu uprawnionego do skorzystania ze
środków unijnych. Celem niniejszego artykułu jest analiza definicji beneficjenta środków
unijnych przeznaczonych na realizację polityki spójności. Z uwagi na obszerność tematyki
przeprowadzoną analizę ograniczono do ustawy o Narodowym Planie Rozwoju oraz ustawy
o zasadach prowadzenia polityki rozwoju, co wiązało się z uwzględnieniem pojęcia benefi-
cjenta stosowanego jeszcze przed dniem akcesji, a także z zastosowaniem podobnych, ale
bardziej rozbudowanych konstrukcji, które zostały wprowadzone później do krajowego sys-
temu prawnego. W konsekwencji w opracowaniu zastosowano zarówno metodę historyczną,
jak i metodę dogmatyczno-analityczną. W pewnej mierze uwzględniono również metodę
analizy pojęciowej w zakresie odnoszącym się do definicji beneficjenta oraz poszczególnych
jej części składowych. Na tej podstawie możliwe było sformułowanie ogólnych twierdzeń 126 Robert Talaga dotyczących zarówno podmiotowych, jak i przedmiotowych ograniczeń związanych z moż-
liwością uzyskania statusu beneficjenta. Słowa kluczowe: polityka spójności, umowa o dofinansowanie projektu, beneficjent,
definicja legalna, wykładnia prawa Słowa kluczowe: polityka spójności, umowa o dofinansowanie projektu, beneficjent,
definicja legalna, wykładnia prawa 5
Zob. A. Błaś, Formy aktów administracyjnych w polskim ustawodawstwie, w: J. Boć (red.), Prawo
administracyjne, Wrocław 2001, s. 344; M. Stahl, Prawne formy działania administracji publicz-
nej – ugody, porozumienia administracyjne, umowy publicznoprawne, publiczne, administracyjne,
umowy cywilne, w: M. Stahl (red.), Prawo administracyjne. Pojęcia, instytucje, zasady w teorii
i orzecznictwie, Warszawa 2013, s. 477 i n.; M. Baran, Przekształcenia w systemie prawnych form
działania administracji publicznej, w: R. Hauser, Z. Niewiadomski, A. Wróbel (red.), System Prawa
Administracyjnego. Tom 3. Europeizacja prawa administracyjnego, Warszawa 2014, s. 652 i n. 8
W ustawie z dnia 11 lipca 2014 r. o zasadach realizacji programów w zakresie polityki spójności
finansowanych w perspektywie finansowej 2014–2020 (Dz.U. z 2020 r. poz. 818), która w art. 2
pkt 1) przez określenie „beneficjent” nakazuje rozumieć podmiot, o którym mowa w art. 2 pkt 10
rozporządzenia ogólnego, oraz podmiot, o którym mowa w art. 63 rozporządzenia ogólnego,
a w art. 2 pkt 22) określa, że rozporządzenie ogólne to rozporządzenie Parlamentu Europejskiego
i Rady (UE) nr 1303/2013 z dnia 17 grudnia 2013 r. ustanawiające wspólne przepisy dotyczące
Europejskiego Funduszu Rozwoju Regionalnego, Europejskiego Funduszu Społecznego, Fun-
duszu Spójności, Europejskiego Funduszu Rolnego na rzecz Rozwoju Obszarów Wiejskich oraz
Europejskiego Funduszu Morskiego i Rybackiego oraz ustanawiające przepisy ogólne dotyczące 6
Ustawa z dnia 20 kwietnia 2004 r. o Narodowym Planie Rozwoju (Dz.U. nr 116, poz. 1206 ze zm.),
dalej jako ustawa o NPR. 7
Ustawa z dnia 6 grudnia 2006 r. o zasadach prowadzenia polityki rozwoju (Dz.U. z 2019 r.,
poz. 1295), dalej jako u.z.p.p.r. Wprowadzenie Transfer funduszy unijnych do poszczególnych państw członkowskich jest konse-
kwencją realizacji zasad wydatkowania budżetu przyjętego na określone siedmio-
letnie okresy jego programowania1. W ten sposób w Unii Europejskiej realizowane
są różne polityki prowadzone dla osiągnięcia wspólnych celów zmierzających do
urzeczywistnienia traktatowych założeń przyjętych przez państwa członkowskie2. Jedną z nich jest polityka spójności ukierunkowana na niwelowanie różnic w roz-
woju poszczególnych regionów wchodzących w skład państw członkowskich3.i Środki finansowe w ramach realizowanej polityki mogą być przekazywane
zarówno na podstawie umowy o dofinansowanie, jak i decyzji o przyznaniu okre-
ślonej kwoty dofinansowania. Stosowanie decyzji administracyjnych w odniesie-
niu do transferu środków unijnych było uznawane za dopuszczalne z uwagi na
schematyczność udzielanej pomocy „bez potrzeby zindywidualizowania sposobu
wykorzystania środków finansowych”4. W tym względzie najczęściej dochodziło
jednak do zawierania umów o dofinansowanie określonego projektu w ramach Definicja legalna beneficjenta środków publicznych… 127 określonego programu operacyjnego. Taka konstrukcja stanowiła „kombinację
umów i aktów administracyjnych” uznanych za część składową „reżimu kontrak-
towego”5. Zawierane umowy o dofinansowanie projektów określały szczegółowo
zasady udzielania finansowego wsparcia oraz finansowego rozliczenia planowa-
nego projektu (przedsięwzięcia). Ich istotą jest specyficzne zobowiązanie dotyczące
realizacji strategicznych celów określonych w programach operacyjnych (zarówno
krajowych, jak i regionalnych). Stosowny kontrakt był zawierany z podmiotem,
który ubiegał się o dofinansowanie. Tym samym uzyskiwał on status beneficjenta
przekazywanych środków, zdefiniowany zarówno przez ustawodawcę krajowego,
jak i unijnego, co będzie przedmiotem dalszych rozważań. Precyzując, dalsza analiza zostanie zawężona do zakresu obowiązywania
podobnej konstrukcji definicji beneficjenta wprowadzonej w ustawie o Narodo-
wym Planie Rozwoju6 oraz w ustawie o zasadach prowadzenia polityki rozwoju7. Obie regulacje stworzyły w istocie podstawy do strategicznego planowania rozwoju
z uwzględnieniem środków krajowych oraz środków przeznaczonych na realizację
unijnej polityki spójności. Na potrzeby przeprowadzonej analizy uwzględniono
regulacje, które zmieniały się w ewolucyjny sposób, aby uzyskać w efekcie zbliżoną
formę definicji beneficjenta. Warto w tym względzie zaznaczyć, że zupełnie inną
konstrukcję definicji tego pojęcia wprowadzono w ustawie o zasadach realizacji
programów w zakresie polityki spójności finansowanych w perspektywie finanso-
wej 2014–20208. 8
W ustawie z dnia 11 lipca 2014 r. o zasadach realizacji programów w zakresie polityki spójności
finansowanych w perspektywie finansowej 2014–2020 (Dz.U. z 2020 r. poz. 818), która w art. 2
pkt 1) przez określenie „beneficjent” nakazuje rozumieć podmiot, o którym mowa w art. 2 pkt 10
rozporządzenia ogólnego, oraz podmiot, o którym mowa w art. 63 rozporządzenia ogólnego,
a w art. Wprowadzenie 2 pkt 22) określa, że rozporządzenie ogólne to rozporządzenie Parlamentu Europejskiego
i Rady (UE) nr 1303/2013 z dnia 17 grudnia 2013 r. ustanawiające wspólne przepisy dotyczące
Europejskiego Funduszu Rozwoju Regionalnego, Europejskiego Funduszu Społecznego, Fun-
duszu Spójności, Europejskiego Funduszu Rolnego na rzecz Rozwoju Obszarów Wiejskich oraz
Europejskiego Funduszu Morskiego i Rybackiego oraz ustanawiające przepisy ogólne dotyczące 128 Robert Talaga 10 Zob. E. Waśkowski, Teoria wykładni prawa cywilnego, metodologia dogmaty tyki cywilistycznej
w zarysie, Warszawa 1936, s. 29; L. Seidler, Teoria państwa i prawa (część analityczna), Kraków 1951,
s. 44; Z. Ziembiński, O zwrotach definicyjnych w ustawodawstwie PRL, „Zeszyty Naukowe Uniwer-
sytetu im. Adama Mickiewicza w Poznaniu. Zeszyt Specjalny”, Poznań 1956, s. 61; L. Nowak, Spór
o definicje legalne a pojmowanie „prawodawcy”, „Państwo i Prawo” 1963, nr 3, s. 515; J. Pieńkoś,
Podstawy juryslingwistyki. Język w prawie – prawo w języku, Warszawa 1999, s. 89. 9
Zob. B. Wróblewski, Język prawny i prawniczy, Kraków 1948, s. 88; J. Lande, Nauka o normie praw-
nej, Lublin 1956, s. 12–14; J. Gregorowicz, Definicje w prawie i nauce prawa, Łódź 1962, s. 51. Europejskiego Funduszu Rozwoju Regionalnego, Europejskiego Funduszu Społecznego, Fundu-
szu Spójności i Europejskiego Funduszu Morskiego i Rybackiego oraz uchylające rozporządzenie
Rady (WE) nr 1083/2006 (Dz. Urz. UE L 347 z 20.12.2013, str. 320). 17 S. Grzybowski, Wypowiedź normatywna oraz jej struktura formalna, „Zeszyty Naukowe Uniwer-
sytetu Jagiellońskiego” 1961, nr 39, s. 38. Definicje legalne jako dyrektywy interpretacyjne W literaturze prezentowane są zarówno poglądy negujące charakter normatywny
definicji legalnych9, jak i go aprobujące10. Zwolennicy teorii normatywnego
charakteru definicji pojęć prawnych przede wszystkim wskazują, że dzięki nim
ustawodawca narzuca adresatowi tekstu prawnego sposób rozumienia danego
określenia, w związku z tym definicje terminów prawnych są normami nakazu-
jącymi11. W istocie chodzi zatem o zachowanie adekwatności rozumienia wpro-
wadzanego zwrotu do zamysłów ustawodawcy poprzez wprowadzenie narracji
narzucającej, w jaki sposób rozumieć „określone znaczenie używanych terminów
odróżniające od funkcjonującego w języku potocznym”12. Z tych względów defini-
cje legalne można traktować jako „nakazy rozumienia w określony sposób termi-
nów w tekście prawnym”13. W konsekwencji przyjmuje się, że definicje ustawowe są
„wypowiedziami prawa pozytywnego”, które ustanawiają określoną normę prawną14. W związku z tym definicje pojęć prawnych bywają uznawane za normy nakazu-
jące15. J. Wróblewski uznawał, że definicje legalne określonego pojęcia stanowią
część interpretowanej i stosowanej normy prawnej16. Podobnie uważał S. Grzybow-
ski, stwierdzając, że definicje legalne określonego wyrażenia stanowią tylko cząstkę
ujawnionej przez ustawodawcę dyrektywy określonego postępowania w określo-
nych warunkach17. Także A. Malinowski podzielił pogląd, że definicje legalne to 9
Zob. B. Wróblewski, Język prawny i prawniczy, Kraków 1948, s. 88; J. Lande, Nauka o normie praw-
nej, Lublin 1956, s. 12–14; J. Gregorowicz, Definicje w prawie i nauce prawa, Łódź 1962, s. 51. 10 Zob. E. Waśkowski, Teoria wykładni prawa cywilnego, metodologia dogmaty tyki cywilistycznej
w zarysie, Warszawa 1936, s. 29; L. Seidler, Teoria państwa i prawa (część analityczna), Kraków 1951,
s. 44; Z. Ziembiński, O zwrotach definicyjnych w ustawodawstwie PRL, „Zeszyty Naukowe Uniwer-
sytetu im. Adama Mickiewicza w Poznaniu. Zeszyt Specjalny”, Poznań 1956, s. 61; L. Nowak, Spór
o definicje legalne a pojmowanie „prawodawcy”, „Państwo i Prawo” 1963, nr 3, s. 515; J. Pieńkoś,
Podstawy juryslingwistyki. Język w prawie – prawo w języku, Warszawa 1999, s. 89. 11 Zob. M. Zieliński, Wykładnia prawa. Zasady. Reguły. Wskazówki, Warszawa 2010, s. 215. 12 T. Chauvin, T. Stawecki, P. Winczorek, Wstęp do prawoznawstwa, Warszawa 2017, s. 82–83.i 13 S. Kaźmierczyk, Definicja legalna, w: A. Bator (red.), Wprowadzenie do nauk prawnych. Leksykon
tematyczny, Warszawa 2012, s. 203. 14 J. Pieńkoś, op. cit., Warszawa 1999, s. 88–89. 15 Zob. M. Zieliński, op. cit., s. 215. 16 J. Wróblewski, Zagadnienia…, s. 247. 17 S. Grzybowski, Wypowiedź normatywna oraz jej struktura formalna, „Zeszyty Naukowe Uniwer-
sytetu Jagiellońskiego” 1961, nr 39, s. 38. 21 Zgodnie z poglądem wyrażonym przez Naczelny Sąd Administracyjny „wykładnią prawa jest ope-
racja myślowa (…) w toku której dokonuje się przekładu zbioru przepisów ogłoszonych w aktach
prawodawczych na zbiór norm postępowania równoznaczny jako całość z danym zbiorem przepi-
sów” – por. wyrok NSA z dnia 18 grudnia 2013 r., I OSK 2320/12, Legalis nr 810366. Definicje legalne jako dyrektywy interpretacyjne Definicja legalna beneficjenta środków publicznych… 129 uproszczenia redakcyjne w tekście prawnym, które „są jedynie podstawą do ustala-
nia norm zachowania językowego”18. W tym sensie określone zwroty, które zostały
zdefiniowane przez ustawodawcę, stają się co najmniej elementami składowymi
norm w zależności od konstrukcji przyjętej przez ustawodawcę. Końcowe znacze-
nie wyinterpretowanych norm prawnych jest wynikiem nakładania się znaczenia
definicyjnego pojęć zawartych w jednostkach redakcyjnych tekstu, w tym rów-
nież definicji legalnych, które mogą prowadzić do uzyskania zamierzonego przez
ustawodawcę celu odbiegającego od powszechnie przyjętego (tzw. celu zmanipu-
lowanego)19. W tym względzie definicje terminologiczne mają co najmniej walor
użytkowy, pozostając regułami interpretacyjnymi20. 22 Zob. W. Jakimowicz, Wykładnia w prawie administracyjnym, Kraków–Warszawa 2006, s. 485 i n. 19 L. Lindhl, Deduction and Justification in the Law. The Role of Legal Terms and Concepts, „Ratio
Iuris” 2004, t. 17, nr 2, s. 182 i n. 18 A. Malinowski, Polski język prawny. Wybrane zagadnienia, Warszawa 2006, s. 157. 20 J. Pieńkoś, op. cit., s. 89. Interpretacja definicji legalnych pojęcia beneficjenta Zdefiniowane przez ustawodawcę pojęcie beneficjenta oznaczające jedną ze stron
umowy o dofinansowanie projektu stanowiło kluczową wskazówkę w procesie
dekodowania prawidłowej normy prawnej będącej podstawą stosowania przepisów
prawa w ramach wdrażania polityki spójności. W tym przypadku dotyczy to spe-
cjalistycznego sposobu rozumienia stron umowy o dofinansowanie projektu. Natu-
ralne pozostaje zatem pytanie nie tylko o wykładnię przepisów prawa, które dotyczą
beneficjenta, lecz także o wykładnię samej definicji legalnej beneficjenta21. Mimo
jednoznacznego charakteru wprowadzonej definicji zasadna była ich wykładnia. Wprowadzone definicje beneficjenta nie były bowiem kompletne w obu aktach
prawnych. W przypadku gdy do interpretowanej definicji nie odnosiły się inne
definicje ani pojęcia funkcjonujące w systemie prawnym na potrzeby wdrażania
polityki spójności, za dopuszczalne można było uznać odwołanie się do definicji
zaczerpniętych z innych gałęzi powszechnie obowiązującego prawa22. 130 Robert Talaga Interpretacja wprowadzonej definicji beneficjenta wymagała równocześnie
uwzględnienia obowiązujących definicji i pojęć o charakterze uniwersalnym,
które funkcjonują w całym systemie prawnym23. Co więcej, takie regulacje mogły
zawierać odesłania do innych przepisów, które należało też uwzględnić w procesie
wykładni pojęcia beneficjenta. Ewentualnego uzupełnienia definicji beneficjenta
należało poszukiwać również w przepisach prawa finansowego oraz prawa dotyczą-
cego realizacji polityki spójności. Przepisy tych ustaw musiały być stosowane jako
ewentualne uzupełnienie wprowadzonych definicji beneficjenta w pierwszej kolej-
ności. W tym względzie nadrzędny charakter miały przepisy ustawy o finansach
publicznych, która określała szeroki zakres zastosowania umowy o dofinansowanie
projektu w całym systemie prawnym. Dopiero regulacje wprowadzone w poszcze-
gólnych ustawach związanych z wydatkowaniem funduszy unijnych uszczegóło-
wiało definicję beneficjenta do potrzeb realizowanej polityki spójności. 23 Wyrok NSA z dnia 12 marca 2014 r., II GSK 2024/12, LEX nr 1488083.
24 K. Borowicz, Zasady prowadzenia polityki rozwoju, Warszawa 2008, s. 21. 23 Wyrok NSA z dnia 12 marca 2014 r., II GSK 2024/12, LEX nr 1488083. Wyrok NSA z dnia 12 marca 2014 r., II GSK 2024/12, LEX nr 1488083.
24 K. Borowicz, Zasady prowadzenia polityki rozwoju, Warszawa 2008, s. 21. 25 Zgodnie z art. 2 pkt 10 ustawy o NPR wprowadzono definicję legalną uzupełniająca, że publiczne
środki krajowe to „środki finansowe pochodzące z budżetu państwa oraz państwowych funduszy
celowych, środki budżetów jednostek samorządu terytorialnego, środki innych jednostek sektora
finansów publicznych, a także inne środki jednostek oraz form organizacyjnoprawnych sektora
finansów publicznych”. Definicja beneficjenta zawarta w ustawie o Narodowym Planie Rozwoju
oraz w ustawie o zasadach prowadzenia polityki rozwoju Definicje beneficjenta wprowadzone w ustawie o Narodowym Planie Rozwoju oraz
w ustawie o zasadach prowadzenia polityki rozwoju mają podobną konstrukcję
legislacyjną24. W tekstach obu aktów prawnych mogły jednocześnie znajdować się
(i znajdowały się) przepisy odnoszące się do pojęcia beneficjenta. Wymagały one
uwzględnienia jako uzupełnienie zakresu rozumienia definicji legalnej wprowa-
dzonej przez ustawodawcę. Mimo podobnej konstrukcji ustawodawca posłużył się
rożnymi sformułowaniami przy definiowaniu pojęcia beneficjenta. Istniały zatem
pewne różnice, które należy dostrzec dla precyzji prowadzonej analizy. Definicja
beneficjenta zawarta w ustawie o NPR odnosiła się mianowicie do podmiotów
„korzystających z publicznych środków wspólnotowych i publicznych środków
krajowych”, podczas gdy definicja zawarta w u.z.p.p.r. odnosiła się do podmiotów
„realizujących projekty finansowane z budżetu państwa lub ze źródeł zagranicz-
nych”. Oba pojęcia, choć zostały odmiennie sformułowane, w istocie miały podobny
zakres zastosowania. Ustawodawca krajowy na potrzeby NPR zaznaczył, że bene-
ficjent to w pierwszej kolejności podmiot korzystający ze środków wspólnotowych,
a dopiero w dalszej kolejności ze środków krajowych. Takie rozwiązanie było zgodne
z przepisami przyjętymi w ustawie o finansach publicznych. Oprócz tego w ustawie Definicja legalna beneficjenta środków publicznych… 131 ujęto także przepisy zawierające definicje ustawowe odnoszone do sformułowań
cząstkowych definiujących beneficjenta. W praktyce prowadziły one do dalszego
rozbudowania wprowadzonej definicji ustawowej. Przykładowo konieczne było
uwzględnienie, że ustawodawca wprowadził definicje legalne „publicznych środ-
ków wspólnotowych”25 oraz „publicznych środków krajowych”26. Niewątpliwie
znalazły one pomocnicze zastosowanie w przypadku definicji beneficjenta. Nato-
miast w przypadku beneficjenta zdefiniowanego w u.z.p.p.r. ustawodawca zastoso-
wał prostsze rozwiązanie, wskazując, że beneficjentem pozostaje osoba „realizująca
projekty finansowane z budżetu państwa lub ze źródeł zagranicznych”. Na potrzeby
realizacji tej ustawy kolejność pochodzenia środków publicznych transferowanych
jednostkom została odwrócona – w pierwszej kolejności wskazano środki pocho-
dzące z budżetu państwa, a dopiero następnie ze źródeł zagranicznych. W tym
względzie posłużono się pojęciami „budżetu państwa” oraz „źródeł zagranicznych”,
które z uwagi na jednoznaczne ich odniesienie nie wymagały korzystania z innych
definicji legalnych. Pomocne było w dalszej kolejności poszukiwanie uzupełnień
stosownych definicji w ustawie o finansach publicznych27. Niewątpliwie bowiem
środkami zagranicznymi były zdefiniowane ustawowo „środki europejskie”28. 26 Zgodnie z art. Współpracy; 4) środki na realizację wspólnej polityki rolnej; 5a) środki przeznaczone na realizację
Inicjatywy na rzecz zatrudnienia ludzi młodych; 5b) środki Europejskiego Funduszu Pomocy Naj-
bardziej Potrzebującym; 5c) środki pochodzące z instrumentu „Łącząc Europę”, o którym mowa
w rozporządzeniu Parlamentu Europejskiego i Rady (UE) nr 1316/2013 z dnia 11 grudnia 2013 r.
ustanawiającym instrument „Łącząc Europę”, zmieniającym rozporządzenie (UE) nr 913/2010
oraz uchylającym rozporządzenia (WE) nr 680/2007 i (WE) nr 67/2010 (Dz. Urz. UE L 348
z 20.12.2013, s. 129 ze zm.). Definicja beneficjenta zawarta w ustawie o Narodowym Planie Rozwoju
oraz w ustawie o zasadach prowadzenia polityki rozwoju 2 pkt 11 ustawy o NPR wprowadzono definicję legalną uzupełniająca, że publiczne
środki wspólnotowe to „środki finansowe pochodzące z budżetu Wspólnot Europejskich,
a w szczególności z funduszy strukturalnych, o których mowa w przepisach Unii Europejskiej
w sprawie Europejskiego Funduszu Rozwoju Regionalnego, w sprawie Europejskiego Funduszu
Społecznego, w sprawie wsparcia rozwoju obszarów wiejskich z Europejskiego Funduszu Orienta-
cji i Gwarancji Rolnej (EFOiGR) oraz zmieniających i uchylających niektóre rozporządzenia, roz-
porządzeniu w sprawie Instrumentu Finansowego Wspierania Rybołówstwa oraz rozporządzeniu
ustanawiającym Fundusz Spójności, ujęte w załączniku do ustawy budżetowej, służące realizacji
Narodowego Planu Rozwoju”. 27 Ustawa z dnia 30 czerwca 2005 r. o finansach publicznych (Dz.U. z 2005 r., nr 249, poz. 2104 ze
zm.); ustawa z dnia 27 sierpnia 2009 r. o finansach publicznych (Dz.U. z 2009 r., nr 157, poz. 1240
ze zm.). 27 Ustawa z dnia 30 czerwca 2005 r. o finansach publicznych (Dz.U. z 2005 r., nr 249, poz. 2104 ze
zm.); ustawa z dnia 27 sierpnia 2009 r. o finansach publicznych (Dz.U. z 2009 r., nr 157, poz. 1240
ze zm.). 28 Zgodnie z art. 5 pkt 5 u.f.p. ilekroć w ustawie jest mowa o środkach europejskich – rozumie się
przez to środki, o których mowa w art. 5 ust. 3 pkt 1, 2, 4 i 5a–5c. W istocie oznacza to: 1) środki
pochodzące z funduszy strukturalnych, Funduszu Spójności, Europejskiego Funduszu Rybac-
kiego oraz Europejskiego Funduszu Morskiego i Rybackiego, z wyłączeniem środków, o których
mowa w pkt 5 lit. a i b; 2) niepodlegające zwrotowi środki z pomocy udzielanej przez państwa
członkowskie Europejskiego Porozumienia o Wolnym Handlu (EFTA), z wyłączeniem środków,
o których mowa w pkt 5 lit. c i d: a) Norweskiego Mechanizmu Finansowego, b) Mechanizmu
Finansowego Europejskiego Obszaru Gospodarczego, c) Szwajcarsko-Polskiego Programu 28 Zgodnie z art. 5 pkt 5 u.f.p. ilekroć w ustawie jest mowa o środkach europejskich – rozumie się
przez to środki, o których mowa w art. 5 ust. 3 pkt 1, 2, 4 i 5a–5c. W istocie oznacza to: 1) środki
pochodzące z funduszy strukturalnych, Funduszu Spójności, Europejskiego Funduszu Rybac-
kiego oraz Europejskiego Funduszu Morskiego i Rybackiego, z wyłączeniem środków, o których
mowa w pkt 5 lit. a i b; 2) niepodlegające zwrotowi środki z pomocy udzielanej przez państwa
członkowskie Europejskiego Porozumienia o Wolnym Handlu (EFTA), z wyłączeniem środków,
o których mowa w pkt 5 lit. 36 Ustawa z dnia 24 czerwca 1994 r. o własności lokali (Dz.U. z 2020 r., poz.1910), art. 6. 29 H.H. Fredriksen, EEA Main Agreement and Secondary EU Law Incorporated into the Annexes
and Protocols, w: C. Baudenbacher (red.), The Handbook of EEA Law, Heidelberg–New York–
Dordrecht–London 2015, s. 95 i n. Definicja beneficjenta zawarta w ustawie o Narodowym Planie Rozwoju
oraz w ustawie o zasadach prowadzenia polityki rozwoju c i d: a) Norweskiego Mechanizmu Finansowego, b) Mechanizmu
Finansowego Europejskiego Obszaru Gospodarczego, c) Szwajcarsko-Polskiego Programu 132 Robert Talaga W istocie tak określone środki obejmowały nie tylko środki unijne, lecz także
środki pochodzące z Europejskiego Obszaru Gospodarczego. W efekcie środki,
z jakich można było korzystać, pochodziły również z państw nieczłonkowskich,
które uruchomiły specjalne fundusze dla słabiej rozwiniętych krajów członkow-
skich, w zamian za możliwość korzystania z dobrodziejstw wspólnego rynku29. Do
takich środków miały jednak zastosowanie odrębne regulacje, na podstawie których
były one wydatkowane. Konieczne było również uwzględnienie, że ustawodawca
posłużył się, definiując beneficjenta, pojęciem osoby „realizującej projekt”. W tym
względzie konieczne było wzięcie pod uwagę również definicji „projektu”30. Status
beneficjenta mógł mieć przede wszystkim podmiot prawa krajowego posiadający
zdolność prawną w rozumieniu przepisów prawa cywilnego, czyli każda osoba
fizyczna31, każda osoba prawna32, a także jednostka organizacyjna, której ustawa
przyznaje zdolność prawną33 – w szczególności spółki osobowe: spółka jawna,
spółka partnerska, spółka komandytowa oraz spółka komandytowo-akcyjna34,
spółki kapitałowe w organizacji35 oraz wspólnoty mieszkaniowe36. Dopiero w ostat-
niej kolejności, po uwzględnieniu wszystkich wskazówek systemowych określo-
nych ustawowo oraz pozaustawowo, dopuszczalne pozostawało uwzględnienie 29 H.H. Fredriksen, EEA Main Agreement and Secondary EU Law Incorporated into the Annexes
and Protocols, w: C. Baudenbacher (red.), The Handbook of EEA Law, Heidelberg–New York–
Dordrecht–London 2015, s. 95 i n. 29 H.H. Fredriksen, EEA Main Agreement and Secondary EU Law Incorporated into the Annexes
and Protocols, w: C. Baudenbacher (red.), The Handbook of EEA Law, Heidelberg–New York–
Dordrecht–London 2015, s. 95 i n. 30 Zgodnie z art. 5 pkt 9 u.z.p.p.r. projekt to „przedsięwzięcie realizowane w ramach programu ope-
racyjnego na podstawie decyzji lub umowy o dofinansowanie, zawieranej między beneficjentem
a instytucją zarządzającą, instytucją pośredniczącą lub instytucją wdrażającą”. 30 Zgodnie z art. 5 pkt 9 u.z.p.p.r. projekt to „przedsięwzięcie realizowane w ramach programu ope-
racyjnego na podstawie decyzji lub umowy o dofinansowanie, zawieranej między beneficjentem
a instytucją zarządzającą, instytucją pośredniczącą lub instytucją wdrażającą”. 31 Ustawa z dnia 23 kwietnia 1964 r. – Kodeks cywilny (Dz.U. z 2020 r., poz. 1740), art. 8, dalej jako
k.c. 31 Ustawa z dnia 23 kwietnia 1964 r. – Kodeks cywilny (Dz.U. z 2020 r., poz. 1740), art. 8, dalej jako
k.c. 32 Art. 33 k.c. 32 Art. 33 k.c. 33 Art. 331 § 1 k.c. 33 Art. 331 § 1 k.c. 34 Ustawa z dnia 15 września 2000 r. – Kodeks spółek handlowych (Dz.U. z 2020 r., poz. 1526, 2320),
art. 34 Ustawa z dnia 15 września 2000 r. – Kodeks spółek handlowych (Dz.U. z 2020 r., poz. 1526, 2320),
art. 8 § 1, dalej jako k.s.h. 30 Zgodnie z art. 5 pkt 9 u.z.p.p.r. projekt to „przedsięwzięcie realizowane w ramach programu ope-
racyjnego na podstawie decyzji lub umowy o dofinansowanie, zawieranej między beneficjentem
a instytucją zarządzającą, instytucją pośredniczącą lub instytucją wdrażającą”. 35 Art. 11 § 1 k.s.h. Definicja beneficjenta zawarta w ustawie o Narodowym Planie Rozwoju
oraz w ustawie o zasadach prowadzenia polityki rozwoju 8 § 1, dalej jako k.s.h. 35 Art. 11 § 1 k.s.h. 36 Ustawa z dnia 24 czerwca 1994 r. o własności lokali (Dz.U. z 2020 r., poz.1910), art. 6. Definicja legalna beneficjenta środków publicznych… 133 w procesie wykładni pojęcia beneficjenta znaczeń pojęć składowych definicji legal-
nej ustalonych w oparciu o reguły pozajęzykowe37. 38 Na podstawie art. 30a ust. 1 pkt 1 u.z.p.p.r. pierwotnie ukształtowany został pogląd, zgodnie z któ-
rym zakwalifikowanie do dofinansowania może nastąpić tylko odnośnie do projektu, który wcze-
śniej przeszedł pozytywnie wszystkie etapy oceny – por. wyrok NSA z dnia 20 lipca 2011 r., II GSK
1380/11, LEX nr 1083290, wyrok NSA z dnia 20 lipca 2011 r., II GSK 1381/11, LEX nr 1083291,
wyrok NSA z dnia 13 czerwca 2012 r., II GSK 786/12, LEX nr 1216708. 37 Zob. M. Zieliński, op. cit., s. 205; J. Supernat, Odesłania do zasad prakseologicznych w prawie admi-
nistracyjnym, „Acta Universitatis Wratislaviensis” 1984, nr 763, Prawo CXXVII, s. 9–137. 37 Zob. M. Zieliński, op. cit., s. 205; J. Supernat, Odesłania do zasad prakseologicznych w prawie admi-
nistracyjnym, „Acta Universitatis Wratislaviensis” 1984, nr 763, Prawo CXXVII, s. 9–137.
38 Na podstawie art. 30a ust. 1 pkt 1 u.z.p.p.r. pierwotnie ukształtowany został pogląd, zgodnie z któ-
rym zakwalifikowanie do dofinansowania może nastąpić tylko odnośnie do projektu, który wcze-
śniej przeszedł pozytywnie wszystkie etapy oceny – por. wyrok NSA z dnia 20 lipca 2011 r., II GSK
1380/11, LEX nr 1083290, wyrok NSA z dnia 20 lipca 2011 r., II GSK 1381/11, LEX nr 1083291,
wyrok NSA z dnia 13 czerwca 2012 r , II GSK 786/12, LEX nr 1216708 37 Zob. M. Zieliński, op. cit., s. 205; J. Supernat, Odesłania do zasad prakseologicznych w prawie admi-
nistracyjnym, „Acta Universitatis Wratislaviensis” 1984, nr 763, Prawo CXXVII, s. 9–137.
38 Na podstawie art. 30a ust. 1 pkt 1 u.z.p.p.r. pierwotnie ukształtowany został pogląd, zgodnie z któ- Przedmiotowe ograniczenia nabycia statusu beneficjenta Ograniczenia przedmiotowe dotyczące możliwości uzyskania statusu beneficjenta
można rozpatrywać z punktu widzenia zachowania zasady równego traktowania
wobec prawa wszystkich podmiotów ubiegających się o dofinansowanie ich przed-
sięwzięć z funduszy unijnych przeznaczonych na realizację polityki spójności. W tym względzie wymogi stawiane wobec podmiotów aplikujących o takie środki
publiczne mogły być zróżnicowane z uwagi na potrzeby poszczególnych kon-
kursów o dofinansowanie. Za każdym razem musiały być one jednak takie same
wobec wszystkich podmiotów ubiegających się o środki przeznaczone na realizację
danego celu strategicznego określonego w programie operacyjnym. W przypadku
możliwości uzyskania statusu beneficjenta istotny był pogląd rozdzielający odrębne
i nietożsame pojęcia dotyczące wymogu pozytywnego przejścia wszystkich etapów
oceny projektu oraz wymogu zakwalifikowania projektu do dofinansowania38. W efekcie instytucja zarządzająca programem operacyjnym (odpowiedzialna za jego
przygotowanie i realizację) była zobowiązana do weryfikacji dokonanej uprzednio
pozytywnej oceny projektu w aspekcie jej zgodności z kryteriami wyboru projek-
tów. W konsekwencji po pozytywnym przejściu wszystkich etapów postępowania
konkursowego, a przed podpisaniem umowy o dofinansowanie, przeprowadzana
była tzw. wizyta monitorująca w celu potwierdzenia założeń zgłoszonego wniosku
o dofinansowanie. W praktyce mogło być wiele przyczyn niedochowania warun-
ków regulaminu konkursu, które rzutowały na brak możliwości zawarcia umowy
o dofinansowanie. Przykładowo jedną z takich przyczyn mogło być ustalenie
podczas weryfikacji wniosku, że projekt, pomimo pozytywnego przejścia wszyst-
kich etapów jego oceny, nie spełniał kryteriów mających zastosowanie dla danego
programu (działania). Nie miało w tym względzie znaczenia, czy wynikająca stąd
przeszkoda do zawarcia umowy istniała w momencie złożenia projektu, czy też
powstała tuż przed zawarciem tej umowy o dofinansowanie. W takim przypadku 134 Robert Talaga wnioskodawca, którego projekt pozytywnie przeszedł wszystkie etapy oceny, ale
mimo to nie spełniał kryteriów mających zastosowanie dla danego programu
(działania), nie nabywał prawa do dofinansowania, rozumianego jako prawo do
pomocy, o którą się ubiegał39. Brak spełnienia warunków określonych w projek-
cie, dla których wstępnie przewidziane zostało dofinansowanie, w konsekwencji
mogło skutkować ponowną weryfikacją i uniemożliwieniem podpisania umowy
o dofinansowanie oraz usunięciem z listy rankingowej40. W świetle prawa nie było
podstaw do związania instytucji zarządzającej wadliwie dokonaną oceną projektu,
jeżeli zgłoszony projekt nie spełniał któregoś z kryteriów wyboru z przyczyn, które
pojawiły się po sporządzeniu list rankingowych zgłoszonych projektów, a zostały
ujawnione przed zawarciem umowy o dofinansowanie z wnioskodawcą, który tym
samym nie mógł nabyć statusu beneficjenta. 40 WSA stwierdził, że przez „kompletność i zdatność do użytku” należy rozumieć stan, w którym
rzecz posiada nie tylko wymagane elementy konstrukcyjne pozwalające na funkcjonowanie
zgodnie z przeznaczeniem, lecz także spełnienie wymogów prawnych, potwierdzone stosownym
zezwoleniem, pozwoleniem, atestem, homologacją lub podobnymi aktami pozwalającymi na
legalne użytkowanie. Samo posiadanie znaku CE nie pozwala uznać, że określone przedmioty są
kompletne, a więc gotowe do celów określonych w projekcie przewidzianym do dofinansowania,
jeżeli nie okazano dokumentacji wydanej przez organ właściwej jednostki dozoru technicznego –
por. wyrok WSA w Poznaniu z dnia 25 listopada 2015 r., III SA/Po 1038/15, LEX nr 1947037. 41 Taki obowiązek wynikał natomiast z „Krajowych wytycznych dotyczących kwalifikowania
wydatków w ramach funduszy strukturalnych i Funduszu Spójności w okresie programowania
2007–2013” oraz z „Wytycznych Instytucji Zarządzającej WRPO na lata 2007–2013 w sprawie
kwalifikowalności kosztów w ramach Działania 1.1, 1.2, 1.4 i 1.6”. 39 Wyrok WSA w Poznaniu z dnia 20 maja 2015 r., III SA/Po 378/15, LEX nr 1807739; wyrok WSA
w Poznaniu z dnia 1 lipca 2015 r., III SA/Po 492/15, LEX nr 1807771; wyrok WSA w Poznaniu
z dnia 9 maja 2017 r., III SA/Po 218/17, LEX nr 2286958. 39 Wyrok WSA w Poznaniu z dnia 20 maja 2015 r., III SA/Po 378/15, LEX nr 1807739; wyrok WSA
w Poznaniu z dnia 1 lipca 2015 r., III SA/Po 492/15, LEX nr 1807771; wyrok WSA w Poznaniu
z dnia 9 maja 2017 r., III SA/Po 218/17, LEX nr 2286958.
40 WSA stwierdził, że przez „kompletność i zdatność do użytku” należy rozumieć stan, w którym
rzecz posiada nie tylko wymagane elementy konstrukcyjne pozwalające na funkcjonowanie
zgodnie z przeznaczeniem, lecz także spełnienie wymogów prawnych, potwierdzone stosownym
zezwoleniem, pozwoleniem, atestem, homologacją lub podobnymi aktami pozwalającymi na
legalne użytkowanie. Samo posiadanie znaku CE nie pozwala uznać, że określone przedmioty są
kompletne, a więc gotowe do celów określonych w projekcie przewidzianym do dofinansowania,
jeżeli nie okazano dokumentacji wydanej przez organ właściwej jednostki dozoru technicznego –
por. wyrok WSA w Poznaniu z dnia 25 listopada 2015 r., III SA/Po 1038/15, LEX nr 1947037.
41 Taki obowiązek wynikał natomiast z „Krajowych wytycznych dotyczących kwalifikowania
wydatków w ramach funduszy strukturalnych i Funduszu Spójności w okresie programowania
2007–2013” oraz z „Wytycznych Instytucji Zarządzającej WRPO na lata 2007–2013 w sprawie
kwalifikowalności kosztów w ramach Działania 1.1, 1.2, 1.4 i 1.6”. 42 Wyrok WSA w Poznaniu z dnia 23 września 2015 r., III SA/Po 713/15, LEX nr 1934876.
43 Wyrok WSA w Poznaniu z dnia 10 listopada 2015 r., III SA/Po 952/15, LEX nr 1977299; postano-
wienie WSA w Poznaniu z dnia 10 kwietnia 2015 r., III SA/Po 243/15, LEX nr 1761107.
44 Wyrok WSA w Poznaniu z dnia 16 lipca 2015 r., III SA/Po 582/15, LEX nr 1807795. Przedmiotowe ograniczenia nabycia statusu beneficjenta Przykładowo wizyta monitorująca
mogła mieć na celu potwierdzenie prawidłowego udzielenia dofinansowania pod
kątem zgodności z wnioskiem środków trwałych lub wartości niematerialnych
i prawnych przedstawionych do refundacji bądź potwierdzenie prawa własności
środków lub wartości niematerialnych i prawnych przedstawionych do refunda-
cji, albo weryfikację wydatków przedstawionych do refundacji. W tym względzie
zakaz podwójnego finansowania przyjęty w ustawodawstwie unijnym ma charakter
bezterminowy i musi być spełniony w każdym momencie, a więc także w dacie
podpisania umowy o dofinansowanie i wypłacenia dotacji. Ten sam wydatek dwu-
krotnie przedstawiony do współfinansowania ze środków funduszy strukturalnych
Unii Europejskiej uniemożliwiał zatem zawarcie umowy z wnioskodawcą z uwagi
na zakaz podwójnego finansowania tych samych wydatków41. Przyczyną uniemoż-
liwiającą nabycie statusu beneficjenta mogło być więc naruszenie obowiązku złoże-
nia przez wnioskodawcę informacji lub oświadczenia o braku pomocy publicznej Definicja legalna beneficjenta środków publicznych… 135 w odniesieniu do tych samych kosztów kwalifikowanych wskazanych w projekcie
zgodnie z zasadą zakazu podwójnego finansowania mającej swe źródło w unijnych
zasadach oszczędności, wydajności i skuteczności zarządzania finansami budżetu
UE. Taka sytuacja mogła wystąpić w przypadku uprzedniego zakupu środków
trwałych i oprogramowania dokonanego przez wnioskodawcę sfinansowanego
z kredytu inwestycyjnego MSP, którego zabezpieczeniem było udzielone poręczenie. Nie było w takim przypadku możliwości rezygnacji z przedmiotowego poręczenia
udzielonego na mocy wcześniejszej umowy poręczenia, a w konsekwencji umowa
o dofinansowanie nie mogła zostać zawarta, ponieważ wniosek o dofinansowanie
był wyłączony z możliwości podpisania umowy i usunięty z listy rankingowej42. W przypadku uprzedniego uzyskania wsparcia z funduszy strukturalnych Unii
Europejskiej (np. w postaci pożyczki w ramach inicjatywy JEREMIE) przeznaczo-
nych na koszty przedsięwzięcia, które zostały następnie ujęte w złożonym wniosku
o dofinansowanie, nie mogła być również podpisana umowa o dofinansowanie pro-
jektu43. Jej zawarcie naruszałoby bowiem zakaz podwójnego finansowania, albo-
wiem ten sam wydatek dwukrotnie został przedstawiony do współfinansowania ze
środków funduszy strukturalnych Unii Europejskiej najpierw w formie pożyczki,
a następnie w formie dotacji. Szerzej rzecz ujmując, wizyta monitorująca, która
potwierdziła, że miało miejsce sprzeczne z ustawą o rachunkowości korygowanie
wsteczne dokumentów księgowych (uznane za nieprawidłowość związaną z księ-
gowaniem wydatków uniemożliwiających uznanie ich za kwalifikowane), była też
podstawą do negatywnej oceny zgłoszonego wniosku projektowego i jego wyłą-
czenia z listy przewidzianej do dofinansowania44. W takiej sytuacji wnioskodawca
ubiegający się o dofinansowanie nie mógł podpisać umowy o dofinansowanie pro-
jektu z przyczyn przedmiotowych dotyczących zgłoszonego projektu, a zatem nie
mógł stać się beneficjentem środków publicznych przeznaczonych na realizację
polityki spójności. 45 Wyrok TK z dnia 12 grudnia 2011 r., P 1/11, OTK-ZU 2011, nr 10A, poz. 115.
46 Zob. R. Lewicka, Znaczenie tzw. „nieformalnych” źródeł prawa w działaniach administracji publicz-
nej, w: P. Chmielnicki, A. Dybała (red.), Nowe kierunki działań administracji publicznej w Polsce
i Unii Europejskiej, Warszawa 2009, s. 95–108; K. Wlaźlak, O swoistych źródłach prawa administra-
cyjnego – wybrane problemy badawcze, w: P. Chmielnicki, A. Dybała (red.), Nowe kierunki działań
administracji publicznej w Polsce i Unii Europejskiej, Warszawa 2009, s. 114–117. 47 Dz. Urz. UE L 187 z 26.6.2014 r., s. 1–78. Podmiotowe ograniczenia uzyskania statusu beneficjenta Ograniczenia podmiotowe wprowadzonej do systemu prawnego definicji legal-
nej beneficjenta można oceniać z perspektywy zachowania zasady przejrzystości
prawa. W tym względzie należało zwrócić uwagę na uwarunkowania wynikające 136 Robert Talaga z przepisów o charakterze ustawowym, które odnosiły się do zakresu zastoso-
wania pojęcia beneficjenta. W praktyce pewne ograniczenia wynikały zarówno
z przepisów o charakterze uniwersalnym dotyczącym całego systemu prawnego,
jak i z przepisów o ograniczonym zakresie oddziaływania, np. dotyczących finan-
sów publicznych, kończąc na regulacjach dotyczących systemów realizacji polityki
spójności. W tym względzie w dalszej kolejności możliwe było dookreślenie wpro-
wadzonej definicji beneficjenta na potrzeby realizacji polityki spójności poprzez
uwzględnienie przepisów o charakterze podustawowym. Co więcej, konieczne
było również uwzględnienie dokumentów prawnych niższego rzędu, w tym także
dokumentów programowych (np. uzupełnień programów operacyjnych będących
częścią systemów realizacji programów operacyjnych). W istocie nie stanowiły one
prawa powszechnie obowiązującego, a w efekcie ich moc wiążąca była ograniczona45. W praktyce były one jednak stosowane jako regulacje stanowiące rodzaj „swoistych
źródeł prawa”46. Oznaczało to traktowanie ich jako merytoryczne rozwinięcie
regulacji o charakterze powszechnie obowiązującym. Zakres ich dopuszczalnego
zastosowania mógł mieć jednak zastosowanie jedynie pomocnicze i drugorzędne. Niejednokrotnie mogło ono być kluczowe dla ustalenia właściwego zakresu pojęcia
wnioskodawcy i beneficjenta w określonej sprawie. Przykładowo w świetle uregulo-
wań u.z.p.p.r. na potrzeby uznania za beneficjenta jednostki organizacyjnej niepo-
siadającej osobowości prawnej, wymóg posiadania zdolności prawnej lub zdolności
sądowej nie był tożsamy z definicją przedsiębiorstwa określoną w art. 1 załącznika
I do rozporządzenia Komisji (UE) nr 651/2014 z dnia 17 czerwca 2014 roku uznają-
cego niektóre rodzaje pomocy za zgodne z rynkiem wewnętrznym w zastosowaniu
art. 107 i 108 Traktatu (tekst mający znaczenie dla EOG)47. Regulamin określo-
nego konkursu o udzielenie dofinansowania mógł jednak umożliwiać ubieganie
się o wsparcie tylko pewnym grupom przedsiębiorców (mikroprzedsiębiorcom,
małym przedsiębiorcom lub średnim przedsiębiorcom), prowadzącym działalność
gospodarczą potwierdzoną wpisem do odpowiedniego rejestru prowadzonego na
terytorium Rzeczypospolitej Polskiej. Zgodnie z regulaminem określonego kon-
kursu udzielane dofinansowanie mogło być jednak uznane za regionalną pomoc
inwestycyjną oraz pomoc na usługi doradcze, udzielaną zgodnie z przepisami roz- Definicja legalna beneficjenta środków publicznych… 137 porządzenie KE nr 651/201448. W rezultacie wnioskodawcą, który po pozytywnej
ocenie zgłoszonego projektu mógł nabyć status beneficjenta pomocy publicznej,
była każda taka forma organizacyjna, która zgodnie z unijnymi przepisami ode-
słania posiadała przymiot mikoprzedsiębiorstwa, małego przedsiębiorstwa bądź
średniego przedsiębiorstwa49. 48 Regulamin konkursu dostępny na stronie: www.bgk.pl/przedsiebiorstwa/kredyt-na-innowacje-
-technologiczne/skorzystaj-z-programu-poddzialanie-322-kredyt-na-innowacje-technologiczne-
po-ir. 52 Taki pogląd został zaprezentowany w odniesieniu do pojęcia producenta rolnego na potrzeby
transferowanych płatności rolnośrodowiskowych – por. uchwała NSA z dnia 30 maja 2012 r.,
II GPS 2/12, ONSAiWSA 2012, nr 4, poz. 63. 49 Zob. K. Brysiewicz, Kiedy przedsiębiorstwo jest powiązane – ubieganie się o środki europejskie przez
mikro-, małe i średnie przedsiębiorstwa, „Zeszyty Naukowe Sądownictwa Administracyjnego”
2014, nr 1, s. 78–99. 53 Spółka cywilna to umowa kreująca trwały stosunek prawny rozłożony w czasie, zobowiązujący
wspólników do dążenia w oznaczony sposób (oznaczone działanie) do osiągnięcia wspólnego celu
gospodarczego obiektywnie osiągalnego i prawnie dopuszczalnego. Spółka cywilna, choć nie jest
odrębnym podmiotem prawa, oznacza zobowiązanie się do osiągnięcia wspólnego celu gospo-
darczego, a więc do wspólnego działania, które należy traktować jako jedno działanie dwóch lub
więcej wspólników, nie zaś jako sumę ich działań indywidualnych. 50 Wyrok WSA w Poznaniu z dnia 1 lipca 2015 r., III SA/Po 492/15, LEX nr 1807771.
51 Art. 860 k.c. Podmiotowe ograniczenia uzyskania statusu beneficjenta W regulaminie konkursu o dofinansowanie mogły
być wreszcie określone względem spółek kapitałowych wymogi dotyczące zacho-
wania statusu wnioskodawcy w trakcie procedury aplikowania o środki publiczne,
mogące uzyskać status beneficjenta. Jeżeli przed podpisaniem umowy o dofinan-
sowanie ustalono, że w wyniku połączenia spółki kapitałowej wnioskującej o dofi-
nansowanie z przejmowaną spółką doszło do niedozwolonej „zmiany statusu”
wnioskodawcy (w rozumieniu regulaminu konkursu o dofinansowanie), to taki
podmiot nie mógł uzyskać statusu beneficjenta50. Natomiast złożony pierwotnie
wniosek o dofinansowanie był wyłączony z możliwości zawarcia umowy o dofi-
nansowanie i usunięty z listy rankingowej mimo pozytywnego przejścia procedury
konkursowej, będącej częścią etapu administracyjnego. i Szczególnym przypadkiem spełnienia przesłanek do uzyskania statusu benefi-
cjenta była spółka cywilna51. Zasadniczo mogła ona nabywać taki status jako pod-
miot uprawniony do otrzymywania środków pomocowych52. Spółka cywilna nie
stanowi odrębnego podmiotu prawa, tylko w istocie pozostaje stosunkiem zobo-
wiązaniowym łączącym wspólników53. W praktyce wykonują oni we wspólnym
imieniu i na wspólny rachunek działalność gospodarczą, mimo że w rejestrze przed-
siębiorców lub w ewidencji działalności gospodarczej wpisani są pod własnymi
imionami i nazwiskami (firmami) i opatrzeni wzmianką o prowadzeniu wspólnej 48 Regulamin konkursu dostępny na stronie: www.bgk.pl/przedsiebiorstwa/kredyt-na-innowacje-
-technologiczne/skorzystaj-z-programu-poddzialanie-322-kredyt-na-innowacje-technologiczne-
po-ir. 49 Zob. K. Brysiewicz, Kiedy przedsiębiorstwo jest powiązane – ubieganie się o środki europejskie przez
mikro-, małe i średnie przedsiębiorstwa, „Zeszyty Naukowe Sądownictwa Administracyjnego”
2014, nr 1, s. 78–99. 49 Zob. K. Brysiewicz, Kiedy przedsiębiorstwo jest powiązane – ubieganie się o środki europejskie przez
mikro-, małe i średnie przedsiębiorstwa, „Zeszyty Naukowe Sądownictwa Administracyjnego”
2014, nr 1, s. 78–99. 50 Wyrok WSA w Poznaniu z dnia 1 lipca 2015 r., III SA/Po 492/15, LEX nr 1807771. 51 Art. 860 k.c. 52 Taki pogląd został zaprezentowany w odniesieniu do pojęcia producenta rolnego na potrzeby
transferowanych płatności rolnośrodowiskowych – por. uchwała NSA z dnia 30 maja 2012 r.,
II GPS 2/12, ONSAiWSA 2012, nr 4, poz. 63. 53 Spółka cywilna to umowa kreująca trwały stosunek prawny rozłożony w czasie, zobowiązujący
wspólników do dążenia w oznaczony sposób (oznaczone działanie) do osiągnięcia wspólnego celu
gospodarczego obiektywnie osiągalnego i prawnie dopuszczalnego. Spółka cywilna, choć nie jest
odrębnym podmiotem prawa, oznacza zobowiązanie się do osiągnięcia wspólnego celu gospo-
darczego, a więc do wspólnego działania, które należy traktować jako jedno działanie dwóch lub
więcej wspólników, nie zaś jako sumę ich działań indywidualnych. 54 Spółka cywilna nie jest osobnym podmiotem prawa i co do zasady nie nabywa sama żadnych praw
ani obowiązków, które przynależą łącznie wspólnikom w trakcie trwania spółki (art. 863 K.c.).
Podejmowane przez uczestników spółki czynności prawne we wspólnym imieniu, na wspólny
rachunek wspólników są skuteczne wobec wszystkich wspólników, a nie w indywidualnej sfe-
rze prawnej danego wspólnika (art. 863 k.c.). Poszczególni wspólnicy nie dysponują udziałem
w majątku objętym przez spółkę, tylko pozostają współuprawnieni z tytułu nabywanych praw pod-
miotowych jako uczestnicy wielostronnego, obligacyjno-organizacyjnego stosunku spółki. Wspól-
nicy mogą występować w obrocie łącznie (art. 866 k.c.), ale pozostają uzależnieni od bytu prawnego
spółki, a zatem nie są niezależni od zmian w składzie osobowym. Zmiany składu osobowego nie
rzutują natomiast na trwałość stosunków prawnych nawiązanych z osobami trzecimi (art. 871 k.c.).
Pewna odrębność majątkowa spółki cywilnej (art. 871 k.c.) nie ogranicza możliwości podziału
majątku pomiędzy wspólników. Nie ogranicza ona również możliwości skierowania żądań mająt-
kowych wierzycieli osobistych poszczególnych wspólników do majątku spółki cywilnej. Podmiotowe ograniczenia uzyskania statusu beneficjenta 53 Spółka cywilna to umowa kreująca trwały stosunek prawny rozłożony w czasie, zobowiązujący
wspólników do dążenia w oznaczony sposób (oznaczone działanie) do osiągnięcia wspólnego celu
gospodarczego obiektywnie osiągalnego i prawnie dopuszczalnego. Spółka cywilna, choć nie jest
odrębnym podmiotem prawa, oznacza zobowiązanie się do osiągnięcia wspólnego celu gospo-
darczego, a więc do wspólnego działania, które należy traktować jako jedno działanie dwóch lub
więcej wspólników, nie zaś jako sumę ich działań indywidualnych. 53 Spółka cywilna to umowa kreująca trwały stosunek prawny rozłożony w czasie, zobowiązujący
wspólników do dążenia w oznaczony sposób (oznaczone działanie) do osiągnięcia wspólnego celu
gospodarczego obiektywnie osiągalnego i prawnie dopuszczalnego. Spółka cywilna, choć nie jest
odrębnym podmiotem prawa, oznacza zobowiązanie się do osiągnięcia wspólnego celu gospo-
darczego, a więc do wspólnego działania, które należy traktować jako jedno działanie dwóch lub
więcej wspólników, nie zaś jako sumę ich działań indywidualnych. 138 Robert Talaga działalności w formie spółki cywilnej54. To nie spółka cywilna zatem posiadała sta-
tus przedsiębiorcy jako odrębny podmiot, tylko jej wspólnicy55. Za przedsiębior-
ców (będących beneficjentami) mogli być więc uznani wyłącznie wspólnicy spółki
cywilnej z uwagi na wymóg posiadania zdolności prawnej przez każdy podmiot
prawa krajowego w rozumieniu przepisów prawa cywilnego56. Określone organi-
zacyjnie wspólne przedsięwzięcie (projekt) zainicjowane przez wspólników spółki
cywilnej nie mogło być samodzielnie uznane za beneficjenta. Wystąpienie ze spółki
choćby jednego podmiotu automatycznie oznaczało, że przerywał on realizację
celu operacji (przedsięwzięcia) będącej przedmiotem dofinansowania. W efekcie
były wspólnik spółki cywilnej ubiegającej się o dofinansowanie poza nią nie miał
już statusu beneficjenta57. Niewystarczające było samo stwierdzenie, że spółka
cywilna miała określoną odrębność w stosunku do tworzących ją wspólników58
oraz dopuszczalne było „wstąpienie” do niej nowego wspólnika przejmującego
za zgodą pozostałych dotychczasowe zobowiązania oraz uprawnienia podmio-
tów ustępujących59. W przypadku „wystąpienia” jednego ze wspólników ze spółki
cywilnej tracił on prawa i obowiązki przynależące łącznie podmiotom tworzącym 54 Spółka cywilna nie jest osobnym podmiotem prawa i co do zasady nie nabywa sama żadnych praw
ani obowiązków, które przynależą łącznie wspólnikom w trakcie trwania spółki (art. 863 K.c.). Podejmowane przez uczestników spółki czynności prawne we wspólnym imieniu, na wspólny
rachunek wspólników są skuteczne wobec wszystkich wspólników, a nie w indywidualnej sfe-
rze prawnej danego wspólnika (art. 863 k.c.). Poszczególni wspólnicy nie dysponują udziałem
w majątku objętym przez spółkę, tylko pozostają współuprawnieni z tytułu nabywanych praw pod-
miotowych jako uczestnicy wielostronnego, obligacyjno-organizacyjnego stosunku spółki. Wspól-
nicy mogą występować w obrocie łącznie (art. Podmiotowe ograniczenia uzyskania statusu beneficjenta 866 k.c.), ale pozostają uzależnieni od bytu prawnego
spółki, a zatem nie są niezależni od zmian w składzie osobowym. Zmiany składu osobowego nie
rzutują natomiast na trwałość stosunków prawnych nawiązanych z osobami trzecimi (art. 871 k.c.). Pewna odrębność majątkowa spółki cywilnej (art. 871 k.c.) nie ogranicza możliwości podziału
majątku pomiędzy wspólników. Nie ogranicza ona również możliwości skierowania żądań mająt-
kowych wierzycieli osobistych poszczególnych wspólników do majątku spółki cywilnej. 55 Ustawa z dnia 2 lipca 2004 r. o swobodzie działalności gospodarczej (Dz.U. z 2017 r., poz. 2168
ze zm.), art. 4 ust. 2. Ustawa obowiązywała w okresie stosowania przepisów ustawy o NPR oraz
u.z.p.p.r., ale utraciła swoją moc na podstawie ustawy z dnia 6 marca 2018 r. – Przepisy wprowa-
dzające ustawę – Prawo przedsiębiorców oraz inne ustawy dotyczące działalności gospodarczej
(Dz.U. z 2018 r., poz. 650), art. 192. 56 Wyrok NSA z dnia 12 marca 2014 r., II GSK 2024/12, LEX nr 1488083. 57 W tym względzie raczej odosobnione były poglądy uznające spółkę cywilną za beneficjenta – por. wyrok WSA w Gorzowie Wlkp. z dnia 13 grudnia 2012 r., II SA/Go 529/12, LEX nr 1224013. 58 Uchwała NSA z dnia 15 października 2008 r., II GPS 5/08, ONSAiWSA 2009, nr 1, poz. 3; uchwała
NSA z dnia 30 maja 2012 r., II GPS 2/12, ONSAiWSA 2012, nr 4, poz. 63; wyrok NSA z dnia
8 grudnia 2011 r., II GSK 1134/11, LEX nr 1108399. 59 Uchwała SN z dnia 21 listopada 1995 r., III CZP 160/95, OSNC 1996, nr 3, poz. 33. Definicja legalna beneficjenta środków publicznych… 139 spółkę w trakcie jej trwania, a zatem tracił możliwość zachowania statusu wniosko-
dawcy, a w konsekwencji tracił również możliwość uzyskania statusu beneficjenta
transferowanych środków publicznych. Zmiana wspólnika spółki cywilnej two-
rzyła jednak nowy byt prawny innych podmiotów, który nie zachowywał ciągłości,
ani nie gwarantował dalszego funkcjonowania przedsięwzięcia objętego wcześniej-
szym wnioskiem o dofinansowanie. W przypadku „wstąpienia” nowego wspólnika
uzyskiwał on nowe prawa i obowiązki wynikające z zawiązania umowy spółki. Wnioski W zaprezentowanym opracowaniu podjęto próbę analizy zastosowanej przez usta-
wodawcę konstrukcji legislacyjnej oraz jej konsekwencji, jakie mogą się pojawić
podczas stosowania wprowadzonej definicji beneficjenta na potrzeby realizowanej
polityki spójności. W tym względzie należało przyjąć, że podlegała ona wykładni
i w jej ramach konieczne było uwzględnienie regulacji zawartych nie tylko w prze-
pisach ustawowych, na podstawie których została ona wprowadzona, lecz także
w przepisach szerzej odnoszących się do finansów publicznych, również w przepi-
sach innych ustaw związanych z realizacją polityki spójności. Wreszcie w pewnym
ogólnym zakresie miały do tego rodzaju definicji zastosowanie przepisy ustawowe
o charakterze uniwersalnym dla całego systemu prawnego. Niewątpliwie na kształt
definicji beneficjenta w określonej sprawie mogły mieć też wpływ regulacje zawarte
w określonym systemie realizacji określonego programu operacyjnego, a niekiedy
wręcz w regulaminie określonego konkursu o udzielenie dofinansowania. Jedno-
cześnie tworzyły one możliwość odesłania do przepisów unijnych, których bez-
pośrednie zastosowanie w sprawie mogło nadawać określony zakres rozumienia
i zastosowania pojęcia beneficjenta w określonych typach spraw. Wprowadzona
przez ustawodawcę konstrukcja umożliwiająca określenie zakresu pojęciowego
beneficjenta w praktyce miała jednak dość ograniczoną przejrzystość. Ograniczona
była również jednolitość stosowania wprowadzonej definicji legalnej. Na potrzeby
każdego konkursu o udzielenie dofinansowania jego zakres pojęciowy mógł być
bowiem modyfikowany zarówno przez uregulowania o charakterze zbliżonym do
prawa wewnętrznie obowiązującego, jak i odesłania do bezpośrednio obowiązują-
cych przepisów prawa unijnego. Pewnego rodzaju ograniczenia w zakresie nabycia
statusu beneficjenta mogły również wyniknąć z przyczyn dotyczących realizacji
samego wniosku projektowego, jeżeli nie zostały zachowane wszystkie kryteria
wymagane do uzyskania dofinansowania. Takie sytuacje dotyczące utraty pewnych
cech wnioskodawcy mogły zostać stwierdzone już po przeprowadzeniu postępowa-
nia w przedmiocie udzielenia dofinansowania i po sporządzeniu list rankingowych 140 Robert Talaga projektów konkursowych. W efekcie takie następcze niespełnianie kryteriów udzie-
lenia dofinansowania stwierdzone na etapie wizyty monitorującej uniemożliwiało
zawarcie umowy o dofinansowanie i nabycie tym samym przez wnioskodawcę
statusu beneficjenta. Tego rodzaju przedmiotowe ograniczenia systemowe były
konsekwencją konieczności przestrzegania zasady równego traktowania wobec
prawa wszystkich podmiotów ubiegających się o dofinansowanie. Niejednokrotnie
było to związane z uwzględnieniem ograniczeń wynikających z zasad dotyczących
zarządzania unijnymi funduszami. Podsumowując, warto dostrzec, że poczynione rozważania są pewnego rodzaju
połączeniem poglądów funkcjonujących zarówno w literaturze prawniczej, jak
i w orzecznictwie na potrzeby wydatkowania środków unijnych w ramach poli-
tyki spójności. Z metodologicznego punktu widzenia takie połączenie prezentuje
pewną „koncepcję wykładni prawa”60. Można rozważać, czy jest ona z kolei elemen-
tem „sprawiedliwej wykładni prawa”61. Wnioski W istocie jednak wystarczające wydaje się
rozpatrywanie przeprowadzonej analizy pod kątem „prawidłowej wykładni prawa”
zarówno w aspekcie materialnym, jak i proceduralnym. W tym sensie przeprowa-
dzona analiza może być również w przyszłości traktowana jako punkt odniesie-
nia przy wprowadzaniu i stosowaniu kolejnych konstrukcji prawnych związanych
z określaniem podmiotów korzystających ze środków publicznych. 61 O. Bogucki, Sprawiedliwość wykładni prawa „Acta Iuris Stetinensis” 2019, nr 2, s. 7–22. 60 M. Zirk-Sadowski, T. Bekrycht, Filozofia prawa a teorie wykładni prawa (wybrane zagadnienia),
„Acta Universitatis Lodziensis. Folia Iuridica” 2017, t. 78, s. 6. Bibliografia Baran M., Przekształcenia w systemie prawnych form działania administracji publicznej,
w: R. Hauser, Z. Niewiadomski, A. Wróbel (red.), System Prawa Administracyjnego. Tom 3. Europeizacja prawa administracyjnego, Warszawa 2014. Baran M., Przekształcenia w systemie prawnych form działania administracji publicznej,
w: R. Hauser, Z. Niewiadomski, A. Wróbel (red.), System Prawa Administracyjnego. Tom 3. Europeizacja prawa administracyjnego, Warszawa 2014. Błaś A., Formy aktów administracyjnych w polskim ustawodawstwie, w: J. Boć (red.), Pra-
wo administracyjne, Wrocław 2001. Bogucki O., Sprawiedliwość wykładni prawa, „Acta Iuris Stetinensis” 2019, nr 2. Borowicz K., Zasady prowadzenia polityki rozwoju, Warszawa 2008. Brysiewicz K., System wyboru i oceny projektów współfinansowanych ze środków euro-
pejskich w perspektywie finansowej 2014–2020, „Przegląd Prawa Publicznego” 2014,
nr 10. Definicja legalna beneficjenta środków publicznych… 141 Brysiewicz K., Kiedy przedsiębiorstwo jest powiązane – ubieganie się o środki europejskie
przez mikro-, małe i średnie przedsiębiorstwa, „Zeszyty Naukowe Sądownictwa Ad-
ministracyjnego” 2014, nr 1. Chauvin T., Stawecki T., Winczorek P., Wstęp do prawoznawstwa, Warszawa 2017. Fredriksen H.H., EEA Main Agreement and Secondary EU Law Incorporated into the An-
nexes and Protocols, w: C. Baudenbacher (red.), The Handbook of EEA Law, Heidel-
berg–New York–Dordrecht–London 2015. Gajda A., Spójność gospodarcza i społeczna, w: J. Barcz (red.), Prawo Unii Europejskiej,
Tom II Prawo materialne i polityki, Warszawa 2006. Gregorowicz J., Definicje w prawie i nauce prawa, Łódź 1962. Grzybowski S., Wypowiedź normatywna oraz jej struktura formalna, „Zeszyty Naukowe
Uniwersytetu Jagiellońskiego” 1961, nr 39. Jakimowicz W., Wykładnia w prawie administracyjnym, Kraków–Warszawa 2006. Kaźmierczyk S., Definicja legalna, w: A. Bator (red.), Wprowadzenie do nauk prawnych. Leksykon tematyczny, Warszawa 2012. Lande J., Nauka o normie prawnej, Lublin 1956. Lewicka R., Znaczenie tzw. „nieformalnych” źródeł prawa w działaniach administracji
publicznej, w: P. Chmielnicki, A. Dybała (red.), Nowe kierunki działań administracji
publicznej w Polsce i Unii Europejskiej, Warszawa 2009. Lindhl L., Deduction and Justification in the Law. The Role of Legal Terms and Concepts,
„Ratio Iuris” 2004, t. 17, nr 2. Malinowski A., Polski język prawny. Wybrane zagadnienia, Warszawa 2006. Nowak L., Spór o definicje legalne a pojmowanie „prawodawcy”, „Państwo i Prawo” 1963,
nr 3. Pieńkoś J., Podstawy juryslingwistyki. Język w prawie – prawo w języku, Warszawa 199 Poździk R., Zasady wdrażania funduszy unijnych w latach 2014–2020, „Europejski Prze-
gląd Sądowy” 2014, nr 12. Prutis S., Dobór instrumentów prawnych służących wsparciu rozwoju obszarów wiejskich,
„Studia Iuridica Agraria” 2009, t. VII. Bibliografia Prutis S., Mechanizmy prawne wsparcia rozwoju obszarów wiejskich ze środków Unii
Europejskiej (dobór metody regulacji prawnej), w: A. Doliwa, S. Prutis (red.), Tom
III – Wypieranie prawa administracyjnego przez prawo cywilne, w: D.R. Kijowski,
P.J. Suwaj (red. serii), Kryzys prawa administracyjnego?, Warszawa 2012. Seidler L., Teoria państwa i prawa (część analityczna), Kraków 1951. Stahl M., Prawne formy działania administracji publicznej – ugody, porozumienia admi-
nistracyjne, umowy publicznoprawne, publiczne, administracyjne, umowy cywilne, w:
M. Stahl (red.), Prawo administracyjne. Pojęcia, instytucje, zasady w teorii i orzecz-
nictwie, Warszawa 2013. 142 Robert Talaga Stankiewicz W., Budżet Unii Europejskiej – kierunki ewolucji systemu finansowania
w aspekcie nowej perspektywy finansowej 2007–2013, „Rocznik Integracji Europej-
skiej” 2007, nr 1. Supernat J., Odesłania do zasad prakseologicznych w prawie administracyjnym, „Acta
Universitatis Wratislaviensis” 1984, nr 763, Prawo CXXVII. Świstak M., Tkaczyński J.W., Polityki publiczne – zagadnienia teoretyczne, w: M. Świ-
stak, J.W. Tkaczyński (red.), Wybrane polityki publiczne Unii Europejskiej. Stan praw-
ny i perspektywy, Kraków 2015. Świstak M., Polityka regionalna Unii Europejskiej jako polityka publiczna, Kraków 20 Świstak M., Polityka regionalna Unii Europejskiej jako polityka publiczna, Kraków 2018. śk
k
kł d
l
d l
d
k
l Waśkowski E., Teoria wykładni prawa cywilnego, metodologia dogmaty tyki cywilistycz-
nej w zarysie, Warszawa 1936. Wlaźlak K., O swoistych źródłach prawa administracyjnego – wybrane problemy badaw-
cze, w: P. Chmielnicki, A. Dybała (red.), Nowe kierunki działań administracji publicz-
nej w Polsce i Unii Europejskiej, Warszawa 2009. Wróblewski B., Język prawny i prawniczy, Kraków 1948. Wróblewski J., Zagadnienia wykładni prawa ludowego, Warszawa 1959. Zieliński M., Wykładnia prawa. Zasady. Reguły. Wskazówki, Warszawa 2002. Zirk-Sadowski M., Bekrycht T., Filozofia prawa a teorie wykładni prawa (wybrane zagad-
nienia), „Acta Universitatis Lodziensis. Folia Iuridica” 2017, t. 78. Ziembiński Z., O zwrotach definicyjnych w ustawodawstwie PRL, „Zeszyty Naukowe
Uniwersytetu im. Adama Mickiewicza w Poznaniu. Zeszyt Specjalny”, Poznań 1956. Ziembiński Z., O zwrotach definicyjnych w ustawodawstwie PRL, „Zeszyty Naukowe
Uniwersytetu im. Adama Mickiewicza w Poznaniu. Zeszyt Specjalny”, Poznań 1956. Legal definition of a “beneficiary” of public funds transferred
on the basis of a project financing agreement under the cohesion policy Abstract Defining a given concept by the legislator may result in consequences exceeding the scope of
application of a single legal act. This is undoubtedly the case with the definition of a benefi-
ciary, which was introduced by national legal provisions. On the one hand, it required taking
into account concepts that were universal for the entire legal system. On the other hand, it
was limited by the regulations that were applicable to the implementation of specific strategic
goals set by individual operational programs. The indicated conditions should be taken into
account in the process of interpreting the concept of an entity entitled to benefit from EU
funds. At the same time, the legislative structure applied required taking into account claims
presented by legal scholars and commentators and judicial decisions. The presented views on
the definition of the concept of “beneficiary” used in the framework of the cohesion policy Definicja legalna beneficjenta środków publicznych… 143 have primarily a descriptive aspect, but they are part of a broader scope of the normative
concept of legal interpretation. Keywords: cohesion policy, project financing agreement, beneficiary, legal definition,
interpretation of law Keywords: cohesion policy, project financing agreement, beneficiary, legal definition,
interpretation of law Talaga R., Definicja legalna „beneficjenta” środków publicznych wydatkowanych w ramach
polityki spójności, „Acta Iuris Stetinensis” 2022, nr 1 (vol. 37), 125–143,
DOI: 10.18276/ais.2022.37-07. CYTOWANIE Talaga R., Definicja legalna „beneficjenta” środków publicznych wydatkowanych w ramach
polityki spójności, „Acta Iuris Stetinensis” 2022, nr 1 (vol. 37), 125–143,
DOI: 10.18276/ais.2022.37-07.
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PENGEMBANGAN MODUL TERMOKIMIA DENGAN PENDEKATAN INKUIRI TERPADU PENDIDIKAN KARAKTER UNTUK MENINGKATKAN LOGIKA SISWA
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Jurnal Pendidikan Agama Islam Jurnal Pendidikan Agama Islam Abstrak Beredarnya bahan ajar kimia yang kurang mampu memberikan makna dalam
proses pembelajaran, mengakibatkan siswa semakin jenuh dan bosan mempelajari
materi yang ada di dalam bahan ajar. Sementara itu pembelajaran sekarang
menitikberatkan pada kegiatan siswa, sedangkan guru diposisikan sebagai
fasilitator. Pembelajaran ini diharapkan dapat meningkatkan keikutsertaan dan
menimbulkan rasa keingintahuan dalam belajar, memperbaiki pengertian dan pola
pikir, serta membantu siswa untuk mengembangkan logika. Penelitian ini
merupakan penelitian pengembangan yang bertujuan untuk menghasilkan modul
kimia yang baik pada materi termokimia. Hasil penelitian pengembangan modul ini
menunjukkan bahwa (1) Terdapat perbedaan logika siswa pada kelas eksperimen
dan kelas kontrol, hal ini ditunjukkan dengan rata-rata nilai evaluasi termokimia
kelas eksperimen sebesar 73,08 dan kelas kontrol sebesar 64,56 dan dengan
signifikansi 0,000 (< 0,05). (2) Modul kimia berpendekatan Inkuiri yang
dikembangkan efektif untuk membantu penerapan nilai pendidikan karakter. Hal
ini ditunjukkan dengan adanya peningkatan sikap siswa di setiap pertemuannya
pada kelas eksperimen. Kata kunci: Modul termokimia, inkuiri terpadu, karakter, logika Email: teguhw13@gmail.com Email: teguhw13@gmail.com PENGEMBANGAN MODUL TERMOKIMIA DENGAN PENDEKATAN
INKUIRI TERPADU PENDIDIKAN KARAKTER UNTUK MENINGKATKAN
LOGIKA SISWA Teguh Wibowo
BPOM Semarang 3 Rustaman, N.Y., Pendidikan dan Penelitian Sains dalam Mengembangkan Keterampilan
Berpikir Tingkat Tinggi untuk Membangun Karakter, Bandung: Universitas Pendidikan
Indonesia, 2012, h.15. 2 Suharyadi, et.al.,. “Pengembangan Buku Ajar Berbasis Kontekstual Pada Pokok Bahasan
Asam dan Basa”. Jurnal Riset dan Praktik Pendidikan Kimia, 2013, ed. 1, h. 60. 1 Abdurrahman, Meaningful Learning Re-invensi Kebermaknaan Pembelajaran, Yogyakarta:
Pustaka Pelajar, 2007, H. 15. 131
PROGRES - Volume 3 Nomor 3 Oktober 2015
1 Abdurrahman, Meaningful Learning Re-invensi Kebermaknaan Pembelajaran, Yogyakarta:
Pustaka Pelajar, 2007, H. 15.
2 Suharyadi, et.al.,. “Pengembangan Buku Ajar Berbasis Kontekstual Pada Pokok Bahasan
Asam dan Basa”. Jurnal Riset dan Praktik Pendidikan Kimia, 2013, ed. 1, h. 60.
3 Rustaman, N.Y., Pendidikan dan Penelitian Sains dalam Mengembangkan Keterampilan
Berpikir Tingkat Tinggi untuk Membangun Karakter, Bandung: Universitas Pendidikan
Indonesia, 2012, h.15. Pustaka Pelajar, 2007, H. 15.
2 Suharyadi, et.al.,. “Pengembangan Buku Ajar Berbasis Kontekstual Pada Pokok Bahasan
Asam dan Basa”. Jurnal Riset dan Praktik Pendidikan Kimia, 2013, ed. 1, h. 60.
3 R
t
N Y
P
didik
d
P
liti
S i
d l
M
b
k
K t
il Abstract Circulation of chemistry teaching materials that are less able to provide meaning in
the learning process, resulting in more saturated and bored students learn the
material that is in teaching materials. While the current study focused on the
activities of students, while the teacher is positioned as a facilitator. Learning is
expected to increase participation and generate curiosity in learning, improve
understanding and mindset, as well as helping students to develop logic. This research
is a development that aims to produce a good chemistry module on thermochemical
material .. The results show that the development of this module (1) There are
differences in the logic students in the experimental class and control class, as shown
by the average value of the evaluation of thermochemical class experiment at 73.08 130 PROGRES - Volume 3 Nomor 3 Oktober 2015 Jurnal Pendidikan Agama Islam and 64.56 for the control class and the significance of 0.000 (<0.05). (2) Module
Inquiry berpendekatan chemistry developed to help implement effective character
education values. This is indicated by an increase in students' attitudes at every
meeting in the experimental class. Keywords: Thermochemical modules, integrated inquiry, character, logic Prodjosantoso, A.K. , Pembelajaran Kimia Secara Menarik dan Menyenangkan Pendekatan
elevansi, Semarang: FMIPA UNNES, 2012. p
y
g
g
Brist, A.H., The Effect Of A Contextual Approach To Chemistry Instruction On Students’
ttitudes, Confidence, And Achievement In Science, Montana University, 2012, h. 1. ,
g
,
Op.Cit, Suharyadi, et.al.,. Pengembangan Buku …..h. 66. 132
PROGRES - Volume 3 Nomor 3 Oktober 2015 132
PROGRES - Volume 3 Nomor 3 Oktober 2015
4 Prodjosantoso, A.K. , Pembelajaran Kimia Secara Menarik dan Menyenangkan Pendekatan
Relevansi, Semarang: FMIPA UNNES, 2012.
5 Op.Cit, Suharyadi, et.al.,. Pengembangan Buku …..h. 66.
6 Brist, A.H., The Effect Of A Contextual Approach To Chemistry Instruction On Students’
Attitudes, Confidence, And Achievement In Science, Montana University, 2012, h. 1. A. PENDAHULUAN Proses pembelajaran yang dilakukan oleh guru selama ini masih
kurang melibatkan siswa untuk aktif. Pembelajaran hanya terfokus pada
penyampaian materi yang berujung pada nilai ujian nasional. Masalah
serius yang sedang dihadapi bangsa Indonesia adalah sistem pendidikan
yang berjalan saat ini selalu berorientasi pada pengembangan kognitif (otak
kiri) dan masih kurang memperhatikan pengembangan afektif, empati, dan
rasa (otak kanan)1. Mata pelajaran yang berhubungan dengan pendidikan
karakter pun (seperti kewarganegaraan dan agama) ternyata pada
praktiknya juga lebih menekankan pada aspek hafalan atau hanya sekedar
tahu2. Padahal pembentukan karakter harus dilakukan secara sistematis
dan berkesinambungan yang melibatkan aspek knowledge, feeling, loving,
dan acting. Pembentukan karakter dapat diibaratkan sebagai pembentukan
seseorang menjadi binaragawan yang memerlukan “latihan otot-otot
akhlak” secara terus-menerus agar menjadi kokoh dan kuat3. Pada pembelajaran kimia yang harus diperhatikan adalah
bagaimana siswa mendapatkan pengetahuan (learning to know) konsep
melalui pengalaman praktis dengan cara melakukan observasi atau
eksperimen (learning to do) secara langsung sehingga dirinya berperan 131 Jurnal Pendidikan Agama Islam sebagai ilmuan, mengaplikasikan ilmu kimia untuk menciptakan hal yang
benar demi kepentingan global (learning to live together), dan mempunyai
kemantapan emosional dan intelektual (learning to be). Pelajaran kimia di
tingkat menengah merupakan pelajaran yang dianggap sulit untuk
dipahami, kurang menarik, dan tidak relevan bagi siswa4. Salah satu
penyebabnya adalah kurang minat dan motivasi untuk mempelajari kimia
dengan senang hati, merasa terpaksa atau suatu kewajiban. Selain itu penggunaan metode pembelajaran yang cenderung
monoton dan kurangnya keterlibatan siswa dalam menemukan suatu
konsep dalam proses pembelajaran juga menjadi penyebab kimia kurang
menarik bagi siswa5. Kondisi seperti ini juga dialami di SMAN 1 Lasem. Ketika guru memberikan pembelajaran yang berkenaan dengan konsep,
maka terkadang siswa masih terkesan canggung dan kebinggungan dengan
konsep materi pada pembelajaran. Pembelajaran lebih terkesan bersifat
teacher-centered, guru hanya menyampaikan materi sebagai produk dan
siswa menghafal informasi faktual. Hal ini sejalan dengan yang dinyatakan
oleh Brist bahwa siswa yang mempelajari kimia cenderung dibombardir
dengan fakta terisolasi dan rumus-rumus kimia yang tidak ada hubungan
dengan kehidupan mereka, sehingga mereka cenderung untuk menghafal,
kemudian siswa dengan mudah membuangnya tanpa bekas6. Belajar melibatkan pembentukan makna oleh seseorang dari apa
yang mereka lakukan, lihat, dan dengar. Pada proses belajar seperti ini,
maka kemungkinan besar akan terjadi pembelajaran bermakna. Terdapat
beberapa model pembelajaran berpusat konstruktivisme yang digunakan 132 Jurnal Pendidikan Agama Islam untuk melatih siswa melatih logika, misalnya adalah dengan menggunakan
bahan pembelajaran, salah satunya modul. p y
p
8 Horsley,M., et.al., “The Role Of Textbooks And Other Teaching And Learning Resources In
Higher Education In Australia:Change And Continuity In Supporting Learning”, International
Assosiation for Research of Textbook and Educational Media (IARTEM e-journal). 2010, ed. 3, h.
45. A. PENDAHULUAN Bahan pembelajaran yang
berupa buku teks mampu membantu siswa untuk meningkatkan
pemahaman secara ilmiah7. 7 Niaz, M. , “How to Facilitate Students Conceptual Understanding of Chemistry?-a History
and Philosophy of Science Perspective”, Chemical Education International, 2005, ed. 6, h. 1. 9 Indriyanti, N. & Susilowati, E,. Pengembangan Modul, Surakarta: Universitas Sebelas Mare
2010, h. 3. 9 Indriyanti, N. & Susilowati, E,. Pengembangan Modul, Surakarta: Universitas Sebelas Maret.
2010 h 3 133
PROGRES - Volume 3 Nomor 3 Oktober 2015
Niaz, M. , How to Facilitate Students Conceptual Understanding of Chemistry? a History
and Philosophy of Science Perspective”, Chemical Education International, 2005, ed. 6, h. 1.
8 Horsley,M., et.al., “The Role Of Textbooks And Other Teaching And Learning Resources In
Higher Education In Australia:Change And Continuity In Supporting Learning”, International
Assosiation for Research of Textbook and Educational Media (IARTEM e-journal). 2010, ed. 3, h.
45.
9 Indriyanti, N. & Susilowati, E,. Pengembangan Modul, Surakarta: Universitas Sebelas Maret.
2010, h. 3. 133
ROGRES - Volume 3 Nomor 3 Oktober 2015 10 Sudjana, Teknologi Pengajaran. Bandung: Sinar Baru Algensindo, 2004, h. 132. PROGRES - Volume 3 Nomor 3 Oktober 2015 PROGRES - Volume 3 Nomor 3 Oktober 2015
10 Sudjana, Teknologi Pengajaran. Bandung: Sinar Baru Algensindo, 2004, h. 132. PROGRES - Volume 3 Nomor 3 Oktober 2015 B. KAJIAN PUSTAKA Modul merupakan suatu cara pengorganisasian materi pelajaran
yang memperhatikan fungsi pendidikan. Siswa lebih suka dengan bahan
ajar tertulis (contohnya modul), meskipun siswa menikmati fleksibilitas
yang ditawarkan bahan ajar berbasis ICT8. Strategi pengorganisasian materi
pelajaran mengacu pada upaya penyajian materi pelajaran, dan synthesizing
yang mengacu pada upaya untuk menunjukkan kepada siswa keterikaitan
antara fakta, konsep, prosedur dan prinsip yang terkandung dalam materi
pelajaran9. Berdasarkan hasil data di lapangan bahwa dari sekian materi
pembelajaran kimia, materi termokimia termasuk materi yang sulit. Hal ini
terlihat dari hasil statistika Ujian Nasional di SMAN 1 Lasem yang tercatat
tingkat kesalahan terbanyak berada pada materi tersebut, yang berarti
siswa kurang memahami konsep materi tersebut. Berdasarkan hasil
observasi melalui wawancara kepada 4 guru kimia di Kabupaten Rembang,
menyatakan bahwa siswa mudah mengerjakan soal-soal hitungan, akan
tetapi kesulitan untuk memahami konsep materi tersebut, umumnya siswa
hanya menghafalkan rumus-rumus yang sudah diajarkan. Oleh karena itu 133 Jurnal Pendidikan Agama Islam perlu adanya tindakan perbaikan untuk mempermudah siswa dalam
memahami konsep-konsep tersebut dengan cara melatih siswa untuk
memiliki logika untuk memahami konsep-konsep dalam kimia. Salah satu sumber belajar dan media pembelajaran yang dapat
membantu siswa maupun guru dalam proses pembelajaran adalah modul. Bahan ajar ini termasuk media cetak hasil pengembangan teknologi cetak
yang berupa buku. Modul juga dapat menjadi buku pegangan guru di
samping buku lainnya. Modul merupakan sumber belajar yang dapat
dikembangkan oleh guru sebagai fasilitator dalam kegiatan pembelajaran. Sudjana mendefinisikan modul merupakan satu unit program
belajar mengajar terkecil secara rinci menggariskan: (1) Tujuan
instruksional yang akan dicapai, (2) Topik yang akan dijadikan dasar proses
belajar-mengajar, (3) Pokok-pokok materi yang dipelajari, (4) Kedudukan
dan fungsi modul dalam kesatuan program yang lebih luas, (5) Peranan
guru dalam proses belajar-mengajar, (6) Alat-alat dan sumber yang akan
dipergunakan, (7) Kegiatan-kegiatan belajar yang harus dilakukan dan
dihayati siswa secara berurutan, (8) Lembaran kerja yang harus diisi oleh
siswa, dan (9) Program evaluasi yang akan dilaksanakan10. Berdasarkan beberapa pengertian di atas, modul didefinisikan
sebagai alat yang berisi materi dengan tujuan agar siswa dapat belajar
secara mandiri atau dengan bimbingan guru dalam kegiatan belajar
mengajar dan untuk mengevaluasi yang dirancang secara sistematis dan
menarik untuk mencapai kompetensi yang diharapkan pada tujuan
pembelajaran. Secara prinsip tujuan pembelajaran adalah agar siswa
berhasil menguasai bahan pelajaran sesuai dengan indikator yang telah
ditetapkan. 134 Jurnal Pendidikan Agama Islam Inkuiri berasal dari bahasa inggris “inquiry”, yang dapat diartikan
sebagai pertanyaan, pemeriksaan, atau penyelidikan. y
,
,
g
j
,
gy
,
12 Gulo, W. , Strategi Belajar Mengajar, Jakarta: PT Grasindo, 2002, h. 84. ,
,
g p
j
f
p
, J
,
,
14 Hamalik, O., Proses Belajar Mengajar, Jakarta: PT Bumi Aksara, 2009, h.219. 11 Suyanti, R. Dwi, Strategi Pembelajaran Kimia, Yogyakarta: Graha Ilmu, 2010, h. 43) g
j
g j
J
13 Wena, M., Strategi pembelajaran Inovatif Kontemporer, Jakarta: Bumi Aksara, 2009, h. 76. ,
,
j
g j
, J
,
,
Ryan, K & Bohlin, K. E., Building Character in Schools: Practical Ways to Bring Moral
struction to Life. San Francisco: Jossey Bass, 1999, h. 5. 135
PROGRES - Volume 3 Nomor 3 Oktober 2015 PROGRES - Volume 3 Nomor 3 Oktober 2015 p Q
y,
Q
J
,
,
Solso. Robert L., Psikologi Kognitif, Jakarta: Erlangga, 2010, h. 405. Baldis & Sean R., “Character Education in the Classroom; A Personal Approach”. English
eeadership Quarterly, ProQuest Education Journals. 2002, ed. 4, h. 26. 16 Fajri, Z & Aprilia, R., Kamus Lengkap Bahasa Indonesia, Jakarta: Dhifa Publisher, 2003, h.
422. ,
g
g
f, J
gg ,
,
Jacob, C., Kemampuan Penalaran Logis Siswa. Surabaya: SPS IKIP, 2007, h. 29. 1
PROGRES - Volume 3 Nomor 3 Oktober 2015 Leeadership Quarterly, ProQuest Education Journals. 2002, ed. 4, h. 26.
18 Solso. Robert L., Psikologi Kognitif, Jakarta: Erlangga, 2010, h. 405. 136
PROGRES - Volume 3 Nomor 3 Oktober 2015
19 Jacob, C., Kemampuan Penalaran Logis Siswa. Surabaya: SPS IKIP, 2007, h. 29. p Q
y,
Q
J
,
,
18 Solso. Robert L., Psikologi Kognitif, Jakarta: Erlangga, 2010, h. 405. PROGRES - Volume 3 Nomor 3 Oktober 2015 B. KAJIAN PUSTAKA Dapat pula diartikan
sebagai proses bertanya dan mencari tahu jawaban terhadap pertanyaan
ilmiah yang diajukan. Dengan kata lain, inkuiri adalah suatu proses untuk
memperoleh dan mendapatkan informasi dengan melakukan observasi atau
eksperimen untuk mencari jawaban atau memecahkan masalah dengan
bertanya dan mencari tahu11. Gulo menyatakan bahwa strategi inkuiri adalah suatu rangkaian
kegiatan belajar yang melibatkan secara maksimal seluruh kemampuan
siswa untuk mencari dan menyelidiki secara sistematis, kritis, logis, analitis,
sehingga siswa dapat merumuskan sendiri penemuannya dengan penuh
percaya diri12. Strategi inkuiri dikembangkan untuk mengajar para siswa
memahami proses meneliti dan menerangkan suatu kejadian. Menurut
Schuman kesadaran siswa terhadap proses inkuiri dapat ditingkatkan
sehinga mereka dapat diajarkan prosedur pemecahan masalah secara
ilmiah13. Oleh karena itu, siswa harus disadarkan bahwa pendapat orang
lain dapat memperkaya pengetahuan yang dimiliki. Pengajaran inkuiri dibentuk atas dasar diskoveri, sebab seorang
siswa harus menggunakan kemampuannya berdiskoveri dan kemampuan
lainnya. Dalam inkuiri, seseorang bertindak sebagai seorang ilmuwan
(scientist), melakukan eksperimen, dan mampu malakukan proses mental
berinkuiri14. Secara istilah, kata karakter berasal dari bahasa Yunani, yaitu
charassein yang berarti “to engrave”15. Dalam kamus besar bahasa 135 PROGRES - Volume 3 Nomor 3 Oktober 2015 Jurnal Pendidikan Agama Islam Indonesia, karakter diartikan sebagai sifat-sifat kejiwaan, akhlak atau budi
pekerti yang menjadi ciri khas seseorang16. Orang berkarakter berarti orang
yang berkepribadian, berperilaku, bersifat, bertabiat, atau berwatak. Baldis dan Sean dalam tulisannya menjelaskan bahwa pendidikan
karakter merupakan usaha yang dengan sengaja untuk melatih nilai-nilai
kebaikan17.. Sengaja yang dimaksud di sini adalah merencanakan,
merumuskan dan memprogramkan kegiatan-kegiatan sekolah dalam upaya
penanaman nilai pendidikan karakter. Ini menjadi semangat tersendiri bagi
pelaku pendidikan agar implementasi pendidikan karakter dapat berjalan
sesuai dengan target yang diharapkan bersama. Berdasarkan berbagai pendapat di atas, maka dapat disimpulkan
bahwa pendidikan karakter merupakan usaha sadar dan terencana dalam
upaya untuk meningkatkan pengetahuan, keterampilan maupun sikap yang
dibutuhkan agar seseorang berperilaku sesuai dengan nilai-nilai luhur,
norma, etika, maupun aturan yang berlaku. Sehingga pada akhirnya nilai-
nilai karakter itu dapat dipahami, dihayati, dan diaplikasikan dalam
kehidupan sehari-hari oleh siswa. Logika merupakan ilmu berpikir, sedangkan berpikir adalah proses
umum untuk menentukan sebuah isu dalam pikiran18. Logika merupakan
salah satu jenis penalaran yang lebih khusus. Menurut Glass dan Holyoak
bahwa penalaran meliputi berbagai simpulan pengetahuan mutahir dan
keyakinan19. Penalaran, pengambilan keputusan dan pemecahan masalah
merupakan proses kognitif yang saling berhubungan. Pengambilan
keputusan meliputi usaha untuk mencapai setiap variasi dari berbagai tipe 136 Jurnal Pendidikan Agama Islam tujuan. B. KAJIAN PUSTAKA Logika adalah bentuk khusus dari penalaran dalam upaya
pengambilan inferensi dan konklusi yang digambarkan oleh premis20. Kattsoff
mengemukakan
bahwa
logika
merupakan
ilmu
pengetahuan mengenai penyimpulan yang lurus terhadap suatu objek21. Ilmu pengetahuan ini menguraikan tentang aturan-aturan serta cara-cara
untuk mencapai kesimpulan, setelah didahului oleh suatu perangkat
premis. Dapat disimpulkan logika merupakan kekuatan memahami dan
menarik suatu kesimpulan berdasarkan data-data (premis) yang ada. Logika juga merupakan pembentuk (cara berpikir) bukan sebagai bentukan
(hasil
pemikiran),
sehingga
dominasinya
terletak
pada
kekuatan
pengetahuan, teori dan sejumlah pengetahuan lain. Logika merupakan salah
satu kemampuan penting dan keterampilan yang perlu dimiliki dan
merupakan fitrah dari manusia. Dengan logika ini, manusia berpikir dan
membedakan mana yang benar dan salah, mampu berpikir untuk terus
mempertahankan kelangsungan hidupnya dan keturunannya, mampu
berkreasi
dan
menciptakan
teknologi
yang
dapat mempermudah
kehidupannya, serta manusia mampu terus berkembang dan meningkatkan
kemampuannya dalam beradaptasi dengan lingkungan yang dinamis dan
berubah secara berkelanjutan. g,
J ,
g
y
gy
,
,
21 Kattsoff, L., Pengantar Filsafat, Yogyakarta: Tiara Wacana Yogya, 2004, h. 24. 20 Sternberg, Robert J. , Cognitive Psychology. USA: Thomson Wadworth, 2006, h. 425. PROGRES - Volume 3 Nomor 3 Oktober 2015 22 Borg & Gall,. Educational Research, An Introduction. New York: Longman Inc, 2003, h. 590.
23 Creswell, J., Research Desaign; Qualitative, Quantitative, and Mix Methods Approaches.
California: SAGA, 2009. H. 161. C. METODE PENELITIAN Desain penelitian yang digunakan dalam penelitian ini adalah
Research
and
Development
(R
dan
D). Penelitian
ini
bersifat 137 PROGRES - Volume 3 Nomor 3 Oktober 2015 Jurnal Pendidikan Agama Islam mengembangkan sebuah produk yaitu modul kimia berpendekatan inkuiri
terpadu nilai pendidikan karakter. Keberadaan ini menjadi penting untuk
menjadi kajian dalam proses pengembangan sebuah produk dalam upaya
mendukung proses pembelajaran. Pada penelitian ini sesuai dengan teori dari Borg dan Gall yang
menyatakan bahwa ada beberapa tahapan dalam penelitian model Research
and
Development,
yaitu
(1)
research
and
information
collecting,
pengumpulan informasi dan penelitian awal, (2) planning, perencanaan, (3)
develop preliminary form of product, pengembangan format atau model, (4)
preliminary field testing, persiapan uji coba tes di lapangan, (5) main
product revision, revisi terhadap produk yang akan diujicobakan di
lapangan, (6) main field testing, tes di lapangan, (7) operational product
revisions, revisi setelah mendapatkan masukan dari tes lapangan, (8)
operational field testing, pelaksanaan tes uji coba model atau tes
pembelajaran, (9) final product revision, revisi terakhir produk, (10)
dominition and implementation22. Implementasi dilakukan kelas XI SMAN Lasem. Desain implementasi
digunakan Posttest Only Control-Group Design23. Langkah-langkah yang
ditempuh dalam proses implementasi yang lebih luas sesuai dengan desain
adalah: (1) menentukan sekelompok subjek penelitian; (2) mencobakan
produk modul (X); (3) mengadakan posttest (Y) setelah proses belajar
mengajar dengan produk modul hasil pengembangan dilakukan; dan (4)
membandingkan hasil Y1 dan Y2 untuk mengkaji keefektivitasan dari
produk modul PROGRES - Volume 3 Nomor 3 Oktober 2015 138
PROGRES - Volume 3 Nomor 3 Oktober 2015 D. HASIL PENELITIAN DAN PEMBAHASAN
Hasil Penelitian 138 Jurnal Pendidikan Agama Islam 1. Hasil Tes Evaluasi Termokimia 2.
Hasil Observasi Penanaman Pendidikan Karakter 2. Hasil Observasi Penanaman Pendidikan Karakter Selain menguji perbedaan logika siswa pada kelas eksperimen dan
kelas kontrol, juga dilakukan observasi terhadap penanaman nilai
pendidikan karakter pada kelas eksperimen setiap pertemuannya. Data
observasi tersebut digunakan untuk melihat sejauh mana modul yang
dikembangkan mampu membantu penerapan nilai pendidikan karakter
pada siswa. Observasi terhadap sikap siswa dilakukan menggunakan
metode peer assesment, dimana 1 siswa dinilai oleh 2 siswa yang lain. Hal
ini bertujuan untuk memudahkan penulis mengetahui sikapnya dan
menjaga objektivitas penilaian selama pembelajaran. Berdasarkan hasil
penelitian terlihat adanya peningkatan sikap siswa berdasarkan nilai-nilai
karakter yang diteliti menggunakan metode peer assesment meskipun
belum terlalu besar peningkatannya. 1.
Hasil Tes Evaluasi Termokimia 1. Hasil Tes Evaluasi Termokimia 1. Hasil Tes Evaluasi Termokimia Pada tahap implementasi modul dilaksanakan selama 5 pertemuan
proses pembelajaran menggunakan modul dan 1 pertemuan untuk tes
evaluasi materi termokimia. Implementasi modul yang dikembangkan
menggunakan metode eksperimen, dimana digunakan 2 kelas sebagai kelas
eksperimen yang menggunakan modul pada proses pembelajaran dan 2
kelas kontrol yang pada proses pembelajarannya tidak menggunakan
modul. Pemilihan kelas eksperimen dan kontrol dilakukan secara acak,
sesuai dengan hasil asumsi awal uji homogenitas. Kelas XI MIA 1 dan XI MIA
2 digunakan sebagai kelas eksperimen, sedangkan kelas XI MIA 3 dan XI
MIA 4 digunakan sebagai kelas kontrol. Pada akhir pertemuan dilakukan tes evaluasi termokimia,
digunakan untuk mengetahui keefektifan penggunaan modul yang
dikembangkan dengan cara membandingkan nilai pada kelas eksperimen
dan kelas kontrol. Berdasarkan data penelitian terlihat ada perbedaan antara kelas
eksperimen dan kelas kontrol. Hal itu ditunjukkan dengan adanya
perbedaan pada jumlah siswa yang tuntas (memenuhi KKM = 75) dan rata-
rata nilai. Setelah mengetahui adanya pengaruh yang lebih baik terhadap
logika siswa antara kelas eksperimen dan kelas kotrol, maka selanjutnya
dilihat apakah perbedaan tersebut signifikan atau tidak menggunakan uji
paired sampel t-test. 139
PROGRES - Volume 3 Nomor 3 Oktober 2015
Pada kolom equal variance assumed ternyata sig (2-tailed) kurang
dari 0,05 maka H0 ditolak, artinya terdapat perbedaan yang signifikan
antara kelas eksperimen yaitu kelas dengan pembelajaran menggunakan
modul termokimia yang berpendekatan inkuiri terpadu pendidikan
karakter dengan kelas kontrol yaitu kelas dengan pembelajaran tanpa
menggunakan modul termokimia yang berpendekatan inkuiri terpadu 139 Jurnal Pendidikan Agama Islam pendidikan karakter. Untuk mengetahui kelas mana yang mempunyai nilai
rataan yang lebih tinggi, kita gunakan analisis Group Statistik yang hasilnya
dapat dilihat bahwa rata-rata kelas eksperimen = 73,08 lebih tinggi dari
rata-rata kelas kontrol = 64,56. Ini berarti bahwa tingkat logika siswa kelas
eksperimen lebih baik dari logika siswa kelas kontrol. 26 Sudarmin & Septianu, E.,. “Pengembangan Modul IPA Terpadu Tema Perubahan Zat
Berbasis Discovery Untuk Meningkatkan Keterampilan Generik dan Hasil Belajar Siswa”.
Unnes Science Education Journal. 2014, ed. , h.3. 24 Bauer, K., “Textbooks and Teaching Resources: A Case Study from the Early Childhood
Classroom”. International Assosiation for Research of Textbook and Educational Media
(IARTEM e-journal). 2010, ed. 3, h. 86. (
j
)
,
,
25 Conolly, M., Using Modules to Teach General Chemistry. USA: University of St.Thomas, 2012,
h. 24. 24 Bauer, K., “Textbooks and Teaching Resources: A Case Study from the Early Childhoo
Classroom”
International Assosiation for Research of Textbook and Educational Med Bauer, K., “Textbooks and Teaching Resources: A Case Study from the Early Childhood ,
,
g
y
y
Classroom”. International Assosiation for Research of Textbook and Educational Med
(IARTEM e-journal). 2010, ed. 3, h. 86. 27 Saptorini. “Peningkatan Keterampilan Generik Sains bagi Mahasiswa Melalui Perkuliahan
Praktikum Kimia Analisis Intrumen Berbasis Inkuiri”. Jurnal Inovasi Pendidikan Kimia. 2008,
ed. 2, h.190. Saptorini. “Peningkatan Keterampilan Generik Sains bagi Mahasiswa Melalui Perkuliahan 142
PROGRES - Volume 3 Nomor 3 Oktober 2015 p
g
p
g
Praktikum Kimia Analisis Intrumen Berbasis Inkuiri”. Jurnal Inovasi Pendidikan Kimia. 200
ed. 2, h.190. 1.
Karakteristik Modul Termokimia 140
PROGRES - Volume 3 Nomor 3 Oktober 2015
Penelitian dan pengembangan modul dengan pendekatan inkuiri
terpadu pendidikan karakter pada materi termokimia dimulai dengan studi
pendahuluan untuk menganalisis kondisi siswa, kebutuhan guru terhadap
modul, dilanjutkan dengan studi literasi yang berkaitan dengan modul yang
dikembangkan dan dilakukan uji validasi ahli materi dan media. Terkait
kondisi siswa yang terjadi di SMAN 1 Lasem, terlihat bahwa ketersediaan
bahan ajar masih belum memadai. Apalagi SMAN 1 Lasem merupakan salah 140 Jurnal Pendidikan Agama Islam satu sekolah yang menengah atas yang menjadi percontohan di Kabupaten
Rembang. Oleh karena itu perlu adanya bahan ajar yang memadai untuk
mendukung proses pembelajaran siswa. Hal ini juga sesuai dengan yang
disampaikan Bauer bahwa bahan ajar yang memadai merupakan salah satu
penentu kualitas dan keefektifan pembelajaran24. Untuk itu bahan ajar yang
dikembangkan oleh penulis pada penelitian ini adalah berupa modul. Melalui modul penulis dapat untuk mengatur penyajian sesuai dengan
kebutuhan siswa dan siswa mampu memanfaatkan modul tersebut secara
mandiri. Hal tersebut juga didasari oleh Conolly yang berpendapat bahwa
melalui sebuah modul dapat merubah pembelajaran sesuai dengan
karakteristik yang diinginkan25. 2. Keefektifan Termokimia Terhadap Logika Siswa Penelitian ini, bermaksud untuk mengembangkan modul yang tidak
hanya sekedar memberikan penyajian materi yang banyak, namun juga
memberikan ruang kepada siswa untuk menggunakan logikanya dalam
menyelesaikan suatu permasalahan. Sudarmin
dan
Septianu
juga
menyatakan bahawa modul mampu meningkatkan inferensi logika siswa26. Meskipun hasil penelitian Saptorini menyatakan bahwa inferensi logika
dikategorikan sebagai keterampilan generik yang sulit dikembangkan,
karena inferensi logika merupakan kemampuan berfikir kompleks untuk
menyusun dan merumuskan kesimpulan tergolong belum dapat dikatakan 141 PROGRES - Volume 3 Nomor 3 Oktober 2015 Jurnal Pendidikan Agama Islam berkembang pada tingkatan SMA27. Modul termokimia dikatakan efektif
terhadap logika siswa apabila nilai evaluasi terkarena mokimia siswa yang
menggunakan modul pada proses pembelajarannya lebih tinggi daripada
nilai evaluasi termokimia siswa yang tidak menggunakan modul pada
proses pembelajarannya. Untuk melihat keefektifannya, menerapkan
metode eksperimen dengan menggunakan kelas ekperimen (kelas yang
menggunakan modul pada proses pembelajarannya) dan kelas kontrol
(kelas yang tidak menggunakan modul pada proses pembelajarannya). Pada
prosedur ini digunakan nilai post-test, dengan membandingkan nilai kedua
kelas tersebut. PROGRES - Volume 3 Nomor 3 Oktober 2015 3.
Peran Modul Terhadap Penanaman Nilai Pendidikan Karakter 3. Peran Modul Terhadap Penanaman Nilai Pendidikan Karakter Modul yang dikembangkan, selain mengajarkan logika siswa dalam
menyelesaikan suatu masalah, namun juga memuat nilai-nilai pendidikan
karakter. Harapannya adalah modul yang dikembangkan ini mampu
membantu penerapan pendidikan karakter pada proses pembelajaran. Sehingga siswa sudah dibiasakan dengan sikap-sikap yang memang
dibutuhkan. Nilai karakter siswa dapat dilihat pada sikap siswa selama
pembelajaran. Nilai karakter yang diharapkan muncul antara lain jujur,
disiplin, kreatif, mandiri, rasa ingin tahu, gemar membaca, peduli
lingkungan dan tanggung jawab. Terkait penyajian materi pada modul
harus memuat nilai kebaikan dan ketauladanan. Komposisi pengetahuan
yang disampaikan seimbang dengan nilai karakter yang dibutuhkan. Penerapan modul ini selama pembelajaran, juga menilai sikap siswa dengan
cara peer assesment. Melalui metode ini diharapkan mendapatkan hasil
yang objektif, dimana 1 siswa hanya mengamati 2 siswa. Sehingga fokus 142 Jurnal Pendidikan Agama Islam objek yang diamati tidak terlalu banyak28. Namun pada awal pembelajaran
siswa sudah diberikan petunjuk tentang penilaian pada hal ini. Berdasarkan
observasi yang sudah dilakukan, terlihat adanya peningkatan sikap siswa di
setiap pertemuannya. Berdasarkan hasil observasi yang diperoleh, siswa masih kurang
dalam
mencari
informasi
di
luar
secara
mandiri. Siswa
hanya
mengandalkan informasi yang disampaikan oleh guru di sekolah. Bahkan
ketika ada tugas di sekolah, siswa hanya mengandalkan mengerjakan tugas
di tempat les, tanpa mengetahui secara rinci dan jelas dari penyelesaian
tugas tersebut. Siswa belum percaya diri pada kemampuan yang dimiliki,
sehingga siswa perlu ditanamkan dan dipupuk oleh guru agar siswa dapat
lebih bangga terhadap potensi yang dimilikinya. Hal ini sejalan dengan
definisi pendidikan karakter yang disampaikan oleh Thomas Lickona yang
menyatakan bahwa pendidikan karakter merupakan suatu usaha yang
disengaja untuk membantu seseorang sehingga dapat memahami,
memperhatikan, dan melakukan nilai-nilai etika yang inti29. 28 White, Eddy, “Student Perspectives of Peer Assessment for Learning in a Public Speaking
Course”. Asian EFL Journal, 2009, h. 9. 143
PROGRES - Volume 3 Nomor 3 Oktober 2015
J
29 Sudrajat, A., “Mengapa Pendidikan Karakter”. Jurnal Pendidikan Karakter, 2011, h. 49. Keterbatasan Penelitian Penelitian dan pengembangan modul dengan pendekatan inkuiri
terpadu pendidikan karakter mempunyai beberapa keterbatasan dalam
pengembangan dan implementasi. Keterbatasan penelitian tersebut antara
lain: (1) modul hanya menyajikan satu materi, belum bisa dibuat untuk satu
semester atau satu tahun; (2) penelitian ini belum kebermaknaan modul ini
untuk kompetesi ranah psikomotorik; (3) peneliti hanya menggunakan data
nilai evaluasi akhir, sehingga belum mampu mengamati proses logika yang
dilakukan oleh siswa. 143 Jurnal Pendidikan Agama Islam PROGRES - Volume 3 Nomor 3 Oktober 2015 E. SIMPULAN Berdasarkan hasil penelitian dan pembahasan, diperoleh simpulan
tentang produk modul kimia dengan pendekatan inkuiri terpadu
pendidikan karakter yang dikembangkan sebagai bahan ajar untuk
meningkatkan logika siswa. Secara rinci, simpulan dari penelitian ini adalah
sebagai berikut: 1. Modul kimia berpendekatan inkuri yang dikembangkan mampu
meningkatkan logika siswa. Hal ini ditunjukkan dengan rata-rata nilai
evaluasi termokimia kelas eksperimen sebesar 73,08 dan kelas kontrol
sebesar 64,56 dan dengan signifikansi kurang dari 0,05. 2. Modul kimia berpendekatan Inkuiri yang dikembangkan efektif untuk
membantu penerapan nilai pendidikan karakter. Hal ini ditunjukkan
dengan adanya peningkatan sikap siswa di setiap pertemuannya pada
kelas eksperimen. 144 PROGRES - Volume 3 Nomor 3 Oktober 2015 Jurnal Pendidikan Agama Islam DAFTAR PUSTAKA Abdurrahman. 2007. Meaningful Learning Re-invensi Kebermaknaan
Pembelajaran. Yogyakarta: Pustaka Pelajar. Baldis & Sean R. 2004. “Character Education in the Classroom; A Personal
Approach”. English Leeadership Quarterly, ProQuest Education
Journals. 26 (4). Bauer, K. 2010. Textbooks and Teaching Resources: A Case Study from the
Early Childhood Classroom. International Assosiation for Research of
Textbook and Educational Media (IARTEM e-journal). 3(2): 81-96. Borg & Gall. 2003. Educational Research, An Introduction. New York:
Longman Inc. Brist, A.H. 2012. The Effect Of A Contextual Approach To Chemistry
Instruction On Students’ Attitudes, Confidence, And Achievement In
Science. Proposal for Science Master. Master Programme di Montana
University Conolly, M. 2012. Using Modules to Teach General Chemistry. University of
St.Thomas Creswell, J. 2009. Research Desaign; Qualitative, Quantitative, and Mix
Methods Approaches. California: SAGA Fajri, Z & Aprilia, R. 2003. Kamus Lengkap Bahasa Indonesia. Jakarta: Dhifa
Publisher. Gulo, W. 2008. Strategi Belajar Mengajar. Jakarta: PT Grasindo. Hamalik, O. 2009. Proses Belajar Mengajar. Jakarta: PT Bumi Aksara. Horsley,M., Knight, B., & Huntly, H. 2010. “The Role Of Textbooks And Other
Teaching And Learning Resources In Higher Education In
Australia:Change
And
Continuity
In
Supporting
Learning”. 145 PROGRES - Volume 3 Nomor 3 Oktober 2015 Jurnal Pendidikan Agama Islam International Assosiation for Research of Textbook and Educational International Assosiation for Research of Textbook and Educational Media (IARTEM e-journal). 3(2):43-61. Indriyanti, N. Yunita & Susilowati, E. 2010. Pengembangan Modul. Surakarta: Universitas Sebelas Maret.. Jacob, C. 1997. Kemampuan Penalaran Logis. Surabaya: SPS IKIP. Kattsoff, L. 2004. Pengantar Filsafat. Yogyakarta: Tiara Wacana Yogya. Niaz, M. 2005. “How to Facilitate Students Conceptual Understanding of
Chemistry?-a History and Philosophy of Science Perspective”. Chemical Education International. 6 (1). Prodjosantoso, A.K. 2008.Pembelajaran Kimia Secara Menarik dan
Menyenangkan Pendekatan Relevansi. Semarang: FMIPA UNNES. Rustaman,
N.Y. 2012. Pendidikan
dan
Penelitian
Sains
dalam
Mengembangkan Keterampilan Berpikir Tingkat Tinggi untuk
Membangun Karakter, Bandung: Universitas Pendidikan Indonesia.. Ryan, K & Bohlin, K. E. 1999. Building Character in Schools: Practical Ways to
Bring Moral Instruction to Life San Francisco: Jossey Bass Rustaman,
N.Y. 2012. Pendidikan
dan
Penelitian
Sains
dalam
Mengembangkan Keterampilan Berpikir Tingkat Tinggi untuk
Membangun Karakter, Bandung: Universitas Pendidikan Indonesia.. Ryan, K & Bohlin, K. E. 1999. Building Character in Schools: Practical Ways to
Bring Moral Instruction to Life. San Francisco: Jossey Bass. Saptorini. 2008. “Peningkatan Keterampilan Generik Sains bagi Mahasiswa
Melalui Perkuliahan Praktikum Kimia Analisis Intrumen Berbasis
Inkuiri”. Jurnal Inovasi Pendidikan Kimia. 2(1): 190-198. Solso. Robert L., Maclin Otto H. & Maclin Kimberly. PROGRES - Volume 3 Nomor 3 Oktober 2015 DAFTAR PUSTAKA 2009. Psikologi Kognitif. Jakarta: Erlangga. Sternberg, Robert J. 2006. Cognitive Psychology. USA: Thomson Wadworth Sternberg, Robert J. 2006. Cognitive Psychology. USA: Thomson Wadworth
Sudarmin & Septianu, E. 2014. “Pengembangan Modul IPA Terpadu Tema
Perubahan
Zat
Berbasis
Discovery
Untuk
Meningkatkan
Keterampilan Generik dan Hasil Belajar Siswa”. Unnes Science
Education Journal. 3 (3). Sudjana. 2004. Teknologi Pengajaran. Bandung: Sinar Baru Algensindo. 146 PROGRES - Volume 3 Nomor 3 Oktober 2015 Jurnal Pendidikan Agama Islam Sugiyono. 2010. Metode Penelitian Pendidikan; Pendekatan Kuantitaif,
Kualitatif dan R & D. Bandung: Alfabeta
Suharyadi, Permanasari,A., dan Hernani. 2013. “Pengembangan Buku Ajar
Berbasis Kontekstual Pada Pokok Bahasan Asam dan Basa”. Jurnal
Riset dan Praktik Pendidikan Kimia.1(1): 60-68. Suyanti, R. Dwi. 2010. Strategi Pembelajaran Kimia. Yogyakarta: Graha Ilmu. Wena, M. 2009. Strategi pembelajaran Inovatif Kontemporer. Jakarta: Bumi
Aksara. White, Eddy. 2009. “Student Perspectives of Peer Assessment for Learning
in a Public Speaking Course”. Asian EFL Journal. 1 (1). Sugiyono. 2010. Metode Penelitian Pendidikan; Pendekatan Kuantitaif,
Kualitatif dan R & D. Bandung: Alfabeta
Suharyadi, Permanasari,A., dan Hernani. 2013. “Pengembangan Buku Ajar
Berbasis Kontekstual Pada Pokok Bahasan Asam dan Basa”. Jurnal
Riset dan Praktik Pendidikan Kimia.1(1): 60-68. Suyanti, R. Dwi. 2010. Strategi Pembelajaran Kimia. Yogyakarta: Graha Ilmu. Wena, M. 2009. Strategi pembelajaran Inovatif Kontemporer. Jakarta: Bumi
Aksara. White, Eddy. 2009. “Student Perspectives of Peer Assessment for Learning
in a Public Speaking Course”. Asian EFL Journal. 1 (1). Sugiyono. 2010. Metode Penelitian Pendidikan; Pendekatan Kuantitaif,
Kualitatif dan R & D. Bandung: Alfabeta Kualitatif dan R & D. Bandung: Alfabeta
Suharyadi, Permanasari,A., dan Hernani. 2013. “Pengembangan Buku Ajar
Berbasis Kontekstual Pada Pokok Bahasan Asam dan Basa”. Jurnal
Riset dan Praktik Pendidikan Kimia.1(1): 60-68. Suyanti, R. Dwi. 2010. Strategi Pembelajaran Kimia. Yogyakarta: Graha Ilmu Wena, M. 2009. Strategi pembelajaran Inovatif Kontemporer. Jakarta: Bumi
Aksara. White, Eddy. 2009. “Student Perspectives of Peer Assessment for Learning
in a Public Speaking Course”. Asian EFL Journal. 1 (1). 147 PROGRES - Volume 3 Nomor 3 Oktober 2015
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https://openalex.org/W3123899587
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https://europepmc.org/articles/pmc8429363?pdf=render
|
English
| null |
Epidemiology of knife carrying among young British men
|
Social psychiatry and psychiatric epidemiology
| 2,021
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cc-by
| 6,247
|
Abstract Purpose Knife carrying has caused considerable public concern in the UK. But little is known of the epidemiology and
characteristics of men who carry knives. We investigated associations with socioeconomic deprivation, area-level factors,
and psychiatric morbidity. Methods Cross-sectional surveys of 5005 British men, 18–34 years, oversampling Black and Minority Ethnic (BME) men,
lower social grades, and in London Borough of Hackney and Glasgow East. Participants completed questionnaires cover-
ing violent behaviour and psychiatric morbidity using standardised self-report instruments. Socioeconomic deprivation
measured at small area level. Results Prevalence of knife carrying was 5.5% (4.8–6.9) and similar among white and BME subgroups. However, prevalence
was twice the national rate in Glasgow East, and four times higher among Black men in Hackney, both areas with high levels
of background violence and gang activity. Knife carrying was associated with multiple social problems, attitudes encour-
aging violence, and psychiatric morbidity, including antisocial personality disorder (AOR 9.94 95% CI 7.28–13.56), drug
dependence (AOR 2.96 95% CI 1.90–4.66), and paranoid ideation (AOR 6.05 95% CI 4.47–8.19). There was no evidence
of a linear relationship with socioeconomic deprivation. Conclusion Men who carry knives represent an important public health problem with high levels of health service use. It
is not solely a criminal justice issue. Rates are increased in areas where street gangs are active. Contact with the criminal
justice system provides opportunity for targeted violence prevention interventions involving engagement with integrated
psychiatric, substance misuse, and criminal justice agencies. Keywords Knife-carrying · Violence · Psychiatric morbidity · Ecological model · Socioeconomic deprivation Jeremy Coid1,2 · Yingzhe Zhang1,3 · Yamin Zhang1 · Junmei Hu4 · Lindsay Thomson5 · Paul Bebbington6 ·
K
ld
Bh i2 Received: 28 May 2020 / Accepted: 6 January 2021 / Published online: 27 January 2021
© The Author(s) 2021 Social Psychiatry and Psychiatric Epidemiology (2021) 56:1555–1563
https://doi.org/10.1007/s00127-021-02031-x Social Psychiatry and Psychiatric Epidemiology (2021) 56:1555–1563
https://doi.org/10.1007/s00127-021-02031-x Social Psychiatry and Psychiatric Epidemiology (2021) 56:1555–1563
https://doi.org/10.1007/s00127-021-02031-x Social Psychiatry and Psychiatric Epidemiology (2021) 56:1555–1563
https://doi.org/10.1007/s00127-021-02031-x ORIGINAL PAPER Introduction Yingzhe Zhang: co-first author. Firearms account for the majority of intentional deaths in
young men worldwide, mainly due to their accessibility. In the UK, however, knife crime accounts for more deaths
than firearms, because firearm accessibility has been strictly
and successfully controlled. Nevertheless, use and carry-
ing of knives by young people is perceived as a growing
problem in the UK. The British Medical Association has
called for knife crime to be tackled as a public health con-
cern [1] and politicians have proposed that healthcare profes-
sionals be legally responsible for identifying and reporting
perpetrators of knife crime [2]. However, disproportionate
media reporting of a “violence epidemic” may have over-
estimated the increase in violence [3]. Furthermore, serious
violence, including murder, declined internationally over the
past 2 decades [4]. In UK, rates of violence-related injury * Jeremy Coid
j.w.coid@qmul.ac.uk 1
Brain Research Center and Mental Health Center, West
China Hospital of Sichuan University, 28th Dianxin
South West Street, Wuhou District, Chengdu 610041,
People’s Republic of China 2
Wolfson Institute of Preventive Medicine, Queen Mary
University of London, London, UK 3
West China School of Public Health, Sichuan University,
Chengdu, Sichuan, China 4
West China School of Basic Medical Sciences and Forensic
Medicine, China, Forensic Psychiatry, Sichuan University,
Chengdu, China 5
Forensic Psychiatry, University of Edinburgh, Edinburgh,
UK 6
University College London, London, UK 6
University College London, London, UK (0123
1 3456789)
3 1556 Social Psychiatry and Psychiatric Epidemiology (2021) 56:1555–1563 remained lower during 2019 than the mid-2000s. Changes
in police recording of offences may have also over-inflated
these perceived changes. However, a small increase in deaths
of men involving knives or sharp instruments occurred in
England and Wales during 2018 [5], and persisted into 2019,
prompting continuing media and public concern. London Borough of Hackney and Glasgow East, Scotland,
output areas characterised by high levels of violence and
gang activity. The same sampling principles applied to each
survey type. The self-administered questionnaire piloted in a previous
survey was adapted and informed consent obtained from
respondents. Respondents completed pencil and paper ques-
tionnaires in privacy and were paid £5 for participation. Little is known of the personal characteristics or epi-
demiology of men who carry knives in the UK. If these
recent, modest increases in national rates are due to iso-
lated pockets of violence in one region or city, this would be
of concern. Method i
Reasons for violence They were asked whether violence
was instrumental (to obtain money, drugs or sex), they
had deliberately looked for a fight, often ruminated about
violence, found violence exciting, easily lost their temper,
became violent if disrespected, would typically obtain a
weapon and look for someone who had threatened them. Violence, violent attitudes, and child maltreatment The aims of this paper were to investigate (1) prevalence
of young British men who carry knives and associations of
knife carrying with demographic factors, including ethnicity,
socioeconomic status, area-level effects, and specific geo-
graphical locations known to be associated with high levels
of violence and gang activity, (2) associations between car-
rying knives and attitudes towards violence, (3) associations
with psychiatric morbidity. All participants were questioned about violent behaviour
using questions from previous UK surveys [16, 17]. Characteristics of violence They were asked ‘‘Have you
been in a physical fight, assaulted, or deliberately hit anyone
in the past 5 years’ and if they had carried a knife. They
were asked about outcome of violence, victims, number
of incidents, whether violence occurred at sporting events,
involved gang fights, whether they were gang members, and
if they had ever used a weapon in a fight, Survey measures The Psychosis Screening Questionnaire [10] described five
symptoms and screened participants for psychosis when ≥ 2
criteria were met. The Hospital Anxiety and Depression
Scale [11] was used to define Anxiety and Depression based
on scores of > 11 in the past week. Scores > 20 on the Alco-
hol Use Disorders Identification Test [12], and scores > 25
on the Drug Use Identification Test [13] were used to iden-
tify alcohol or drug dependence, respectively. Questions from the Structured Clinical Interview for
DSM-IV Personality Disorders Screening Questionnaire
[14] identified Antisocial Personality disorder (ASPD) when
3 or more of 7 items for adult antisocial personality were
present, and Conduct disorder when 3 or more of 15 items
before age 15 years. More information is needed on men who carry knives to
identify whether knife carrying is a significant public health
problem that warrants public concern. Furthermore, whether
there are associations between ethnicity, unemployment, and
area level effects. There are no previous studies of knife car-
rying to indicate whether psychiatric morbidity is associated. At household-level, we included quintiles of area-level
scores of Index of Multiple Deprivation (IMD) which meas-
ures levels of deprivation in small areas called ‘lower layer
super output areas’ [15]. Introduction It would suggest a more targeted public health
approach is needed in certain localities rather than nation-
wide campaigns or criminal justice actions [3]. A contro-
versial response to knife carrying has been police stop-and-
search policy, with black people in the UK nine times more
likely to be stopped and searched than white people [6]. In
London, however, where stop and searches are highest, more
victims and perpetrators of homicides involving knives are
of black and minority ethnic (BME) background. It has been
suggested this may be related to an upsurge of gang-related
violence in these communities [7]. However, London con-
trasts with observations of a marked decline over the past
decade of violence by teenage males, together with homicide
rates and gang violence observed in Scotland, previously the
highest in Europe, particularly in Glasgow [8]. Data collection This study has been previously described [9]. The survey
was carried out in 2011 based on random location sampling. Individual sampling units (census areas of 150 households)
were randomly selected within British regions in propor-
tion to their population to derive a representative sample
of young men (18–34 years) from England, Scotland, and
Wales. There were four additional, boost surveys, including
young BME men, and those from lower social grades and, Victimization They were asked if they had been a victim
of violence, feared violent assault, experienced domestic
violence, sexual assault. Criminality They were asked about previous criminal
convictions for violence and robbery, whether imprisoned, 1 3 Social Psychiatry and Psychiatric Epidemiology (2021) 56:1555–1563 1557 could easily obtain a firearm, and whether friends encour-
aged them in committing crimes. psychiatric morbidity, which were adjusted for demographic
influences. l
The study was approved by Queen Mary University of
London ethics committee. All analyses were performed
using SPSS 25.0. Adverse childhood experiences They were asked if they
had witnessed violence in their home, physical, sexual
abuse, or neglect, been in care, or experienced a serious
injury before age 16. Life events and daily living We asked about contempo-
rary factors such as whether they had a close relationship,
had moved home in the preceding year, had a separation or
divorce; been fired from their job, had serious money prob-
lems, no educational qualifications, were not in education
employment or training, had experienced life-threatening
injury or homelessness since the age of 16. Demography and sampling The weighted sample included 5005 men, 18–34 years of
age: 1915 (38.3%) main survey; 1017 (20.3%) BME sample;
596 (11.9%) lower social classes; 712 (14.2%) Hackney; and
765 (15.3%) Glasgow East. Of the total sample, 381 (7.6%)
reported carrying a knife in the past 5 years. Table 1 shows
men who carried knives were younger, UK-born, single,
unemployed, and Black, but fewer of south Asian origin. More were in the boost surveys of lower social classes,
Hackney, and Glasgow East, but not BME men. Knife car-
rying showed highest prevalence in Hackney and Glasgow
East. In the main sample, the overall population rate for
young men 18–34 years based on the main representative
survey of England, Scotland and Wales was 5.5% (4.8–6.9),
with the rate for white men 5.8% (4.5–7.2). The repre-
sentative BME boost indicated there were no significant Statistical analysis We compared demographic characteristics of men carrying
knives compared with the rest. We describe the distribu-
tions knife-carrying across the booster and main surveys. We
undertook logistic regression modelling to test for associa-
tions. We tested small area level effects of socioeconomic
deprivation and social status (unemployed), separately in the
main and combined booster surveys, using quintiles of the
Index of Multiple Deprivation (IMD) scores [15]. We then
tested associations with risk factors for violence, childhood
and adulthood victimization and trauma, and live events, and Table 1 Demographic characteristics of men who have carried knife and others (n = 5005)
*p < 0.05
**p < 0.01
***p < 0.001
Characteristic
Other men n = 4624
Carried knife n = 381
OR
95% CI
n (%)
n (%)
Non-UK born
641 (14.2)
33 (8.7)
0.59**
0.41–0.85
Single
2833 (61.9)
268 (70.7)
1.48***
1.18–1.86
Unemployed
1679 (37.1)
206 (57.2)
2.27***
1.83–2.82
Ethnicity
White (reference)
2949 (63.9)
243 (63.8)
Ref
Black
635 (13.8)
80 (21.0)
1.51**
1.15–1.97
South asian
964 (20.9)
51 (13.4)
0.63**
0.46–0.86
Other
69 (1.5)
7 (1.8)
1.15
0.51–2.60
Survey type
Main (reference)
1,810 (39.1)
105 (27.7)
Ref
Ethnic minorities
959 (20.7)
58 (15.2)
1.04
0.75–1.44
Lower social classes
544 (11.8)
52 (13.6)
1.63**
1.15–2.30
London, Hackney
630 (13.6)
82 (21.5)
2.22***
1.64–3.00
Glasgow East
681 (14.7)
84 (22.0)
2.10***
1.56–2.84
Mean (SD)
Mean (SD)
Age (years)
26.27 ± 4.97
25.27 ± 5.28
0.96***
0.94–0.98 Table 1 Demographic characteristics of men who have carried knife and others (n = 5005) 1558 Social Psychiatry and Psychiatric Epidemiology (2021) 56:1555–1563 Table 4 shows independent associations between psychi-
atric morbidity and carrying a knife. Following adjustments,
there was no association with depression, but all other forms
of psychopathology were significantly higher, particularly
ASPD and conduct disorder. Men carrying knives were also
significantly more likely than other men to report all forms
of psychiatric service use. We investigated whether there
were age trends in associations with psychiatric morbidity
observed in Table 4. We found that two conditions showed
a significant trend for increasing prevalence with age. First,
anxiety disorder: 18–21 years, (n = 34, 28.6%); 22–25 years,
(n = 23, 30.3%); 26–29 years, (n = 26, 39.4%); 30–34 years,
(n = 60, 54.5%); (Chi-square trend = 19.11, p < 0.001). Statistical analysis Sec-
ond, drug abuse: 18–21 years, (n = 20, 17.7%); 22–25 years,
(n = 15, 19.2%); 26–29 years, (n = 22, 32.4%); 30–34 years,
(n = 34, 32.7%), (Chi-square trend = 9.82, p < 0.05). We
investigated whether this corresponded to prevalence of
gang membership in each age band, but there was no sig-
nificant age trend for the latter (Chi-square trend = 1.26, NS). differences between White, Black (5.9%, 3.6–8.2), or South
Asian (5.1%, 2.9–7.3) men at the population level.f differences between White, Black (5.9%, 3.6–8.2), or South
Asian (5.1%, 2.9–7.3) men at the population level. Table 2 shows effects of unemployment and socioeco-
nomic deprivation measured at small area level for (1) main
survey (2) total combined sample. Main survey participants
were stratified into five levels with equal numbers according
to IMD scores where they lived. The same quintile ranges
previously created were then applied to the combined sam-
ple, including boost surveys. Findings for the main survey
indicated that, adjusted for age, quintiles 3–5 showed similar
prevalence. All were significantly higher than reference cat-
egory of men from least deprived areas, but not quintile 2. Following adjustment for unemployment, all three quintiles
with higher IMD scores still showed significantly higher
odds of association for knife carrying compared to refer-
ence quintile 1. There was no gradient in odds of association
observed between quintiles 2–5 before or after adjustment in
either the main or all surveys combined. Table 3 shows independent associations between knife
carrying and risk factors for future violence, including previ-
ous violence, criminality, and attitudes towards and charac-
teristics of violence. All characteristics of previous violence
showed strong positive associations with knife carrying. Model 2 is adjusted for age and socioeconomic status (unemployment) Discussion Given strong associations with physical harm and psychiat-
ric morbidity, carrying knives should be a cause of concern
for public health services. We found that 1 in 18 men age
18–34 years in Britain reported having carried a knife with
personal characteristics indicating risks for future violence. We did not find higher rates of knife carrying among any
specific ethnic group using representative samples of the Table 3 also shows that men who reported carrying knives
had experienced multiple childhood victimization, maltreat-
ment and trauma, and adverse life events in adulthood. They
were unlikely to be in close relationships. They were more
likely to have moved house in the past year, no qualifica-
tions, were not in employment, education or training, and
part of a criminal peer group. Adjusted for non-UK birth, being single, unemployment, ethnicity, age, and survey type Discussion Table 2 Effects of small area level socioeconomic deprivation and social status (unemployment) on knife carrying
Model 1 is adjusted for age
Model 2 is adjusted for age and socioeconomic status (unemployment)
Socioeconomic deprivation
(main survey)
n = 1916
Other men n = 1810
Carried knife n = 106
Model 1
Carried knife n = 106
Model 2
n (%)
n (%)
OR
95% CI
AOR
95% CI
1 Least deprived, n = 384
377 (20.8)
7 (6.6)
Ref
–
Ref
–
2, n = 385
368 (20.3)
17 (16.0)
2.44
0.99–6.06
1.81
0.72–4.58
3, n = 384
351 (19.4)
33 (31.1)
5.24***
2.25–12.18
4.23***
1.80–9.95
4, n = 383
361 (19.9)
22 (20.8)
3.42**
1.42–8.22
2.91*
1.20–7.03
5 Most deprived, n = 380
353 (19.5)
27 (25.5)
4.27***
1.81–10.06
3.29**
1.37–7.85
Socioeconomic deprivation (all
surveys)
n = 5005
Other men n = 4624
Carried knife n = 381
Model 1
Carried knife n = 381
Model 2
n (%)
n (%)
OR
95% CI
AOR
95% CI
1 Least deprived, n = 459
446 (9.6)
14 (3.7)
Ref
–
Ref
–
2, n = 596
565 (12.2)
30 (7.9)
1.67
0.87–3.19
1.44
0.72–2.86
3, n = 758
708 (15.3)
50 (13.1)
2.25**
1.22–4.14
2.09*
1.10–3.96
4, n = 1249
1134 (24.5)
116 (30.4)
3.31***
1.87–5.86
3.32***
1.83–6.03
5 most deprived, n = 1943
1772 (38.3)
172 (45.0)
3.17***
1.81–5.55
2.87***
1.59–5.16 Table 2 Effects of small area level socioeconomic deprivation and social status (unemployment) on knife carrying 1 3 Social Psychiatry and Psychiatric Epidemiology (2021) 56:1555–1563 1559 Table 3 Independent Associations between knife carrying and individual and interpersonal factors (n = 5005)
Adjusted for non-UK birth, being single, unemployment, ethnicity, age, and survey type
*p < 0.05
**p < 0.01
Other men n = 4,624
Carried knife n = 381
AOR
95% CI
n (%)
n (%)
Characteristics of violence
Any violence in past 5 year
1264 (27.6)
344 (90.5)
23.82***
16.63–34.12
≥ 3 violent incidents
785 (17.4)
220 (65.1)
8.47***
6.56–10.93
Gang fight
99 (2.2)
163 (43.5)
30.63***
22.57–41.57
Intimate partner violence
111 (2.4)
83 (22.3)
11.79***
8.46–16.45
Instrumental violence
106 (2.3)
184 (48.8)
38.54***
28.65–51.85
Violence at sports events
199 (4.3)
155 (41.3)
14.01***
10.73–18.30
Used weapon in a fight
227 (5.0)
180 (49.6)
18.88***
14.46–24.66
Used weapon if threatened
277 (6.0)
198 (51.8)
24.19***
18.24–32.09
Outcome of violence
Perpetrator injured
429 (9.4)
153 (41.1)
6.38***
4.99–8.15
Other person injured
523 (11.4)
173 (46.6)
6.68***
5.24–8.52
Police involved
280 (6.1)
115 (31.0)
6.80***
5.19–8.92
Victimization
Fear violent victimization
752 (17.2)
145 (40.2)
3.07***
2.42–3.90
Assaulted and injured
641 (13.9)
125 (32.8)
2.79***
2.17–3.58
Attitudes to violence
Excited by violence
191 (4.2)
187 (51.2)
22.76***
17.45–29.70
Violent ruminations
284 (6.5)
185 (52.6)
16.20***
12.54–20.93
Violent if disrespect
742 (18.2)
262 (74.2)
12.90***
9.85–16.90
Easily lose temper
470 (10.9)
213 (61.7)
13.05***
10.13–16.80
Looked for fight
139 (3.1)
173 (46.4)
24.48***
18.52–32.36
Associated criminality
Previous violence conviction
237 (5.1)
115 (30.2)
7.50***
5.68–9.92
Previous robbery conviction
49 (1.1)
38 (10.0)
9.56***
6.03–15.15
Ever in prison
136 (2.9)
99 (26.1)
11.58***
8.42–15.91
Gang member
43 (1.0)
69 (19.2)
23.07***
14.89–35.75
Could obtain firearm
323 (7.4)
205 (62.5)
22.72***
17.30–29.83
Childhood victimization/trauma
Bullying
1181 (25.5)
146 (38.3)
1.76***
1.40–2.22
Witnessed violence in home
408 (8.8)
160 (42.0)
7.53***
5.88–9.63
Sexual abuse
105 (2.3)
26 (6.8)
3.68***
2.33–5.82
Physical abuse
255 (5.5)
74 (19.4)
4.30***
3.18–5.80
Neglect
188 (4.1)
75 (19.7)
5.79***
4.26–7.87
Adult victimization/life events
Domestic violence
110 (2.4)
36 (9.5)
4.59***
3.03–6.96
Sexual assault
43 (0.9)
18 (4.7)
5.02***
2.78–9.04
Life threatening injury
103 (2.2)
44 (11.5)
5.88***
3.95–8.75
Separation/divorce
340 (7.4)
49 (12.9)
1.96***
1.39–2.77
Fired from job
734 (15.9)
91 (23.9)
1.69***
1.29–2.21
Homelessness
258 (5.6)
116 (30.5)
7.26***
5.50–9.57
Serious money problems
683 (14.8)
184 (48.3)
5.92***
4.66–7.51
Daily living
Close relationship
2490 (58.0)
139 (29.5)
0.47***
0.35–0.62
Moved home past year
1138 (25.1)
122 (33.1)
1.70***
1.33–2.16
No educational qualifications
529 (11.4)
105 (27.6)
2.46***
1.89–3.21
Not in education employment training
856 (19.2)
148 (40.9)
2.24***
1.62–3.10
Encouraged by friends into crime
294 (6.7)
155 (46.7)
10.62***
8.16–13.82 dent Associations between knife carrying and individual and interpersonal factors (n = 5005) 3 Social Psychiatry and Psychiatric Epidemiology (2021) 56:1555–1563 1560 Table 4 Independent Associations between knife carrying and psychiatric morbidity (n = 5005)
a Adjusted for other psychiatric morbidity outcomes, non-UK birth, being single, unemployment, ethnicity, age, and survey type
b Adjusted for non-UK birth, being single, unemployment, ethnicity, age, and survey type
c Adjusted for other psychosis outcomes, non-UK birth, being single, unemployment, ethnicity, age, and survey type
*p < 0.05
**p < 0.01
***p < 0.001
Measure
Other men n = 4624
Carried knife n = 381
AOR
95% CI
n (%)
n (%)
Psychiatric morbidity
Anxietya
597 (13.1)
143 (38.4)
1.10
0.75–1.61
Depressiona
426 (9.4)
71 (19.3)
1.47
0.96–2.25
Alcohol dependencea
370 (8.2)
138 (39.3)
2.35***
1.65–3.33
Drug dependencea
80 (1.8)
91 (25.1)
2.96***
1.90–4.66
Antisocial personality disordera
434 (9.8)
220 (62.1)
9.94***
7.28–13.56
Suicide attemptb
226 (5.0)
78 (21.5)
4.58***
3.38–6.21
Conduct disorderb
879 (19.4)
260 (69.3)
10.53***
8.18–13.55
Psychosis (PSQ ≥ 2)a
221 (4.9)
96 (26.2)
1.80**
1.19–2.74
PSQ items
Hypomania c
161 (3.5)
47 (12.3)
1.87**
1.20–2.92
Thought insertionc
112 (2.4)
40 (10.5)
1.22
0.73–2.05
Paranoia delusionc
301 (6.5)
143 (37.5)
6.05***
4.47–8.19
Delusional mood/perceptionc
261 (5.6)
83 (21.8)
1.26
0.84–1.90
Hallucinations
142 (3.1)
51 (13.4)
1.26
0.79–2.02
Psychiatric service useb
Consulted medical practitioner
374 (8.1)
76 (20.2)
3.10***
2.32–4.13
Consulted psychiatrist or psychologist
82 (1.8)
36 (9.5)
5.00****
3.23–7.75
Psychiatric admission
145 (3.2)
45 (12.2)
3.50***
2.40–5.10
Psychotropic medication
174 (3.9)
51 (13.9)
3.31***
2.30–4.77 Table 4 Independent Associations between knife carrying and psychiatric morbidity (n = 5005) r other psychiatric morbidity outcomes, non-UK birth, being single, unemployment, ethnicity, age, and survey type Adjusted for other psychiatric morbidity outcomes, non-UK birth, being single, unemployment, ethnicity, age
Adjusted for non-UK birth, being single, unemployment, ethnicity, age, and survey type general population. Discussion However, there may be specific area-
level effects in Hackney for BME men and Glasgow east
(where all participants were white) which require further
investigation in similar, atypical locations with high rates. Men who carry knives were of lower social class. However,
when we specifically investigated associations with socio-
economic deprivation (SED) at national level, there was no
simple linear association. Knife carrying was less common
in the 40% of areas characterised by lowest levels of SED,
but showed similar rates across the remaining 60% and
did not correspond to increasing levels of SED. They were
also somewhat younger in the sample and there was a trend
of declining prevalence with age. However, a sub-group
showed persistence in the 30–34 years age group. misuse, psychiatric morbidity and service use. These
associated factors can be considered within an ecological
model of violence [18] (see Fig. 1) in which a complex
series of adverse, inter-related factors have impacted on
these men over the life-course. Key individual biological
and personal history factors include persistence of conduct
disorder into adulthood as ASPD, the most prevalent psy-
chiatric condition. Our study must therefore be compared
to studies of younger persons where prevalence is much
higher and the peak age of carrying knives is 14 years,
but where the majority desist leaving a “hard core” with
multiple interpersonal and individual level problems [19]. The latter correspond to our sample, with multiple antiso-
cial behaviours and associated psychopathology including
anxiety, suicidal behaviour and substance misuse [20–22]. Anxiety and drug misuse may have contributed to these
men persisting in carrying knives into later age than would
have been expected. Violence tends to show a progressive
decline in prevalence at the population level from mid-
teenage years onwards. However, our findings for carrying
knives did not follow this trend, suggesting a marker for a her psychiatric morbidity outcomes, non-UK birth, being single, unemployment, ethnicity, age, and survey type Ecological model Men who self-reported that they had carried knives
showed multiple problems of violence, criminality,
adverse childhood experiences, educational and occu-
pational disadvantage, traumatic life events, substance 3 Social Psychiatry and Psychiatric Epidemiology (2021) 56:1555–1563 1561 Fig. 1 Ecological model of knife carrying Fig. 1 Ecological model of knife carrying relationship shown with poverty. However, low social capi-
tal is frequently associated with SED and may have influ-
enced knife carrying in Glasgow where communities have
not shown similar resilience to the effects of poverty and
deprivation as other cities in the UK [29]. more severe and persistent form of antisocial behaviour. The association with psychosis corresponds to previous
findings for both violence [16] and gang membership [9]
and was largely explained by symptoms of paranoid idea-
tion. Meta-analysis has shown paranoid ideation is most
strongly associated with violence at the population level
[23]. Our cross-sectional method meant we could not conclude
whether there had been epidemic spread of carrying knives
and whether this had become a societal and cultural norm. Knife carrying appeared evenly distributed through 60% of
areas in Britain compared to the 40% showing lowest levels
of SED. SED was not associated with carrying knives in a
Scottish survey of younger people [19]. Nevertheless, two
urban locations, surveyed because of high levels of violence
and gang activity, did show exceptional levels. This is of
major concern and supports the notion that a small number
of urban areas with high concentrations of young men with
multiple problems could be vulnerable to epidemic spread,
leading to high levels of knife crime and requiring targeted
interventions in these areas [3]. Our cross-sectional method
also meant we could not demonstrate epidemics of knife
crime related to gang activity. Nevertheless, US gang-related
murders are confirmed as having an epidemic-like process
of social contagion, similar to infectious disease [30], and
which could be applied to future study of knife-related crime
in Britain. The interpersonal experience of witnessing violence in
the home in the ecological model (Fig. 1) was the most
commonly reported childhood experience [24]. Childhood
physical abuse is the most consistent predictor of violence,
particularly when compounded by additional forms of mal-
treatment such as sexual abuse and neglect [25]. Bullying
by peers corresponds to weapon carrying, including firearms
[26]. Ecological model Nevertheless, other individual factors should be con-
sidered in both childhood and adulthood, including intention
to intimidate others, facilitate robbery, deliberately injure,
or simply for perceived power and status that carrying a
weapon provides [27]. Inability to maintain close relationships meant these men
did not experience protective factors of a supportive intimate
partner. Many were part of a criminal peer group. Although
most men who carried knives were not gang members, this
factor showed one of the strongest associations with knife
carrying, corresponding to dramatic increase in violent and
criminal behaviour observed after joining a gang [28]. Societal and cultural norms which create an environment
that accepts or condones knife carrying and violence were
not specifically measured in this study. Societal factors have
implications for population-level preventive interventions,
are usually broad factors that reduce inhibitions against vio-
lence, and in previous studies have included poverty, eco-
nomic, social and gender inequalities, poor social security, Associations between socioeconomic deprivation and
knife carrying were complex and did not show a simple
linear relationship, questioning whether societal norms for
this behaviour were largely independent of these factors. The observed trends suggest that men who carry knives are
considerably fewer in more affluent areas, with no simple 1 3 3 Social Psychiatry and Psychiatric Epidemiology (2021) 56:1555–1563 1562 low social capital, social and cultural norms, masculinity
linked to violence, weak legal and criminal justice sanctions,
weapon availability, and population displacement [18]. Our
study did not support a key role for all these factors, includ-
ing poverty. However, we have shown syndemic effects
between psychosis, substance dependence, high risk sexual
behaviour and crime and violence, with knife carrying as
a component of the latter, in Hackney, east London. This
disproportionally affected young Black men [31]. However,
despite black men in Hackney showing a prevalence of knife
carrying four times the national average, black men across
Britain showed a slightly lower prevalence than white men
suggesting that they had overcome disadvantages, with no
overall association between knife carrying and ethnicity. strategies in schools have been recommended based on
observations that young persons are more likely to desist,
although many young persons who carry knives will leave
school at the earliest opportunity [19]. It is unclear whether decline in knife-related crime in
Scotland was related to any specific interventions. Implications Funding The Maurice and Jacqueline Bennett Charitable Trust and
National Institute of Health Research Program Grant (NIHR RP-PG-
6407-10500), Programme Grants for Applied Research. Knife carrying represents an extensive, closely related
series of Public Health problems. Population-level inter-
ventions may be appropriate for juveniles but are not
strongly supported for men 18–34 years on the basis of
these findings. It is important that the fall in convictions
for violent crime 2017–2018 in Scotland occurred predom-
inantly among those age 13–19 years, with little change
among those 25 years and above [8]. Early prevention Ecological model The
Violence Reduction Unit of Police Scotland was estab-
lished in 2005 at a time of rising homicide rates and is
considered the most likely explanation [32]. The unit
adopted a Public Health approach [18] but did not involve
Public Health Agencies and was based on a successful
programme implemented by police and social services in
the USA [33]. Its aims were to reduce violence by working
with health, education and social work agencies to achieve
societal and individual attitudinal change by focusing on
enforcement and contain and manage individuals who
carry weapons and are involved in violent behaviour. Emphasis on enforcement was balanced by a rehabilitative
approach in which desistance was rewarded with support
to find employment, education, and healthcare, including
treatment for substance abuse. Limitations Our study has several limitations. Because knife carrying
was measured at any time over the past 5 years, some partici-
pants may have ceased carrying knives by the time of survey. The cross-sectional method prevented firm conclusions on
direction of association. The possibility that knife carrying
in association with violent behaviour leads to psychiatric
morbidity must therefore be considered, together with bi-
directionality of many associations we have described. Fur-
thermore, we were unable to investigate cohort effects of age
on knife carrying. High levels of psychiatric morbidity indicate need for
additional involvement of mental health in an integrated
approach with criminal justice agencies. Although few men-
tal healthcare professionals are currently trained to contrib-
ute to public mental health prevention programmes, young
men who carry knives are already accessing mental health
services according to our findings. Our findings also suggest
that interventions should be targeted in specific geographi-
cal locations, particularly inner-London, where continuing
trends of more knife crime suggest that stop-and-search pol-
icy has had little impact. Support for police from local com-
munities was essential in development of the Public Health
model in Scotland. However, factors of ethnicity may make
this more difficult in English cities. Findings that Black men
are no more likely to carry knives nationally indicate the
need to target men primarily on the basis of their criminal
activities, with histories of violence and gang membership. Further research is needed into why certain inner urban areas
generate clusters of multiple risks, including knife carrying,
high levels of violence, and gang activity, and factors which
increase or impede the transmission of these public health
problems between communities. Self-report may have underestimated true prevalence of
knife carrying, because socially undesirable behaviours tend
to be less frequently reported. Our definition of gang mem-
bership did not correspond to an accepted definition and
was deliberately broad to avoid eliminating cases. However,
self-reported gang membership is generally accepted as a
key component. Random location sampling does not provide detailed
information on number of young men who declined to
participate. However, because the method is based on the
National Census, participants were identified and included
according to representative strata and actual frequency in
the population. This method has considerable advantages
for investigating health-related behaviours such as violence
and criminality. Compliance with ethical standards Conflict of interest On behalf of all authors, the corresponding author
states that there is no conflict of interest. 1 3 1563 Social Psychiatry and Psychiatric Epidemiology (2021) 56:1555–1563 Ethical approval All human and animal studies have been approved by
the appropriate ethics committee and have, therefore, been performed
in accordance with the ethical standards laid down in the 1964 Declara-
tion of Helsinki and its later amendments. Ethical approval All human and animal studies have been approved by
the appropriate ethics committee and have, therefore, been performed
in accordance with the ethical standards laid down in the 1964 Declara-
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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
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(2021) 9:63
Linek et al. Movement Ecology
https://doi.org/10.1186/s40462-021-00298-y
Open Access
RESEARCH
A partial migrant relies upon a range‑wide
cue set but uses population‑specific weighting
for migratory timing
Nils Linek1,2* , Paweł Brzęk3, Phillip Gienapp4, M. Teague O’Mara1,5, Ivan Pokrovsky1,6,7, Andreas Schmidt1,
J. Ryan Shipley1, Jan R. E. Taylor3, Juha Tiainen8,9, Tamara Volkmer1,2, Martin Wikelski1,2,10 and Jesko Partecke1,2*
Abstract
Background: Many birds species range over vast geographic regions and migrate seasonally between their breeding
and overwintering sites. Deciding when to depart for migration is one of the most consequential life-history decisions
an individual may make. However, it is still not fully understood which environmental cues are used to time the onset
of migration and to what extent their relative importance differs across a range of migratory strategies. We focus on
departure decisions of a songbird, the Eurasian blackbird Turdus merula, in which selected Russian and Polish populations are full migrants which travel relatively long-distances, whereas Finnish and German populations exhibit partial
migration with shorter migration distances.
Methods: We used telemetry data from the four populations (610 individuals) to determine which environmental
cues individuals from each population use to initiate their autumn migration.
Results: When departing, individuals in all populations selected nights with high atmospheric pressure and minimal
cloud cover. Fully migratory populations departed earlier in autumn, at longer day length, at higher ambient temperatures, and during nights with higher relative atmospheric pressure and more supportive winds than partial migrants;
however, they did not depart in higher synchrony. Thus, while all studied populations used the same environmental
cues, they used population-specific and locally tuned thresholds to determine the day of departure.
Conclusions: Our data support the idea that migratory timing is controlled by general, species-wide mechanisms,
but fine-tuned thresholds in response to local conditions.
Keywords: Songbird migration, Departure decision, Control mechanisms, Environmental cues
Background
For migratory species, the timing of annual migration is
an integral component of their life history [1]. Although
optimal departure for autumn and spring migration is
consequential for fitness and survival throughout the
year [2, 3], the timing and duration of migration often
varies widely among individuals, populations and species
*Correspondence: nlinek@ab.mpg.de; partecke@ab.mpg.de
1
Max Planck Institute of Animal Behavior, Radolfzell, Germany
Full list of author information is available at the end of the article
[4, 5]. The critical decision when to initiate migration is
thought to be based in significant parts on information
gathered from the local environment [6–8]. Understanding the mechanisms that underly variation in migration
timing and the consequences to individuals survival and
fitness is paramount to our understanding of migration
as a whole [9, 10].
Migration is a complex life-history stage, and its underlying spatiotemporal organisation is assumed to be part
of an endogenous, genetically inherited migration program encoding when, where, and how far to migrate
© 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://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.
Linek et al. Movement Ecology
(2021) 9:63
[11–13]. The complexity can be visualised in a continuum
between rigid obligate migration and flexible facultative
migration [14] (Fig. 1a). Obligate migration is typically
characterised by its regularity, consistency, and predictability and is often associated with fully migratory
species (such as Common Cuckoos Curculus canorus,
Red-backed Shrikes Lanius collurio, or Thrush Nightingales Luscinia luscinia [15]; Fig. 1b). On the other
extreme, facultative migrants such as Pine Siskins Spinus
pinus supposedly base migratory decisions mainly on
Page 2 of 14
environmental conditions that, e.g. predict food abundance [16]. This irruptive migration (Fig. 1b) can flexibly
respond to changing local conditions. Thus, it is thought
to be less regulated by genetic control mechanisms that
would be potentially more rigid in their expression.
Independent of where species or populations lie along
the migration continuum, individuals rely on environmental cues to assess local proximate information to
determine the timing of migration [2]. Environmental
cues which contain information about present and future
Fig. 1 a Proximate control mechanisms for migration: Two extremes along a continuous gradient. On one side facultative (environmentally
induced) and on the other end, obligate migration (intrinsic control mechanisms). b Different migration types (ranging from irruptive to full
migration) which are linked to specific proximate control mechanisms (see (a)). c Locations of study sites for Eurasian blackbirds (Turdus merula).
While the Spanish population of blackbirds is fully resident, departures from partial (Germany and Finland) and fully (Poland and Russia) migratory
populations are used in this study
Linek et al. Movement Ecology
(2021) 9:63
conditions are likely selected based on their historical relationship to fitness [17], but can also vary in their
precision and reliability regarding such conditions. Day
length, for instance, provides information on coarse longterm trends ranging from weeks to months (e.g., seasonal
climate trends) and are used by obligate migrants to time
their migration [18, 19]. Migration is initiated either by
triggering internal migration programs directly to specific day length thresholds or by calibrating internal clock
mechanisms of individuals [18]. On a finer temporal scale
in the order of days to weeks, cues such as ambient temperature can provide information about the near future,
e.g. upcoming challenges in thermoregulation [20] or
information on resource abundance [21], because of the
strong influence of temperature on availability of prey
insects [22] and worms [23]. Finally, cues, which offer
information about the immediate future, such as rapidlychanging atmospheric conditions related to weather (e.g.,
wind characteristic [24], overcast conditions [25], and
atmospheric pressure [26]), likely influence departure
decisions in many species and populations. This influence is because of prevailing weather conditions can
directly affect immediate energetic costs and survival
during migration [25, 27] or in the case of cloud cover,
opportunities to use celestial navigation [28, 29].
Although all migratory species likely integrate information from some environmental cues operating at different
time scales to determine the optimal timing of departure [30], the exact set of cues used and the magnitude
of their effects likely vary between species depending on
their migration strategies and even within individuals
along their migration routes [31]. A comparison of longand medium-distance migratory species, for instance,
showed that long-distance migrants were least selective
at a stopover site for environmental cues such as temperature, atmospheric pressure, and wind conditions compared to medium-distance migratory species [32].
Similar differences in cue selection and responsiveness
may also occur between different populations of the same
species, which vary in their migration strategies. For
example, propensity to migrate often varies across longitudinal, latitudinal, and altitudinal gradients, and wideranging species such as the Eurasian blackbird Turdus
merula often have non-migratory (in southern Europe,
i.e. Spain), partially migratory (central Europe and coastal
areas), and fully migratory populations in continental
northeast Europe (Fig. 1b, c). In general, populations in
breeding areas with harsh environmental conditions during the non-breeding season in the northern or eastern
parts of the species range exhibit a higher propensity to
migrate or full migration. This is accompanied by potential longer migration distances than populations in milder
regions with a lower proportion of migrants and shorter
Page 3 of 14
migration distances (Fig. 1) [33–36]. In an obligate partial migrant, the blackcap (Sylvia atricapilla), propensity
to migrate and migration distance (measured as nocturnal activity) are genetically correlated [37]. Due to earlier and potentially faster deterioration of environmental
conditions during autumn and longer migration distance,
fully migratory populations in the north or east likely
face stronger time constraints to initiate their migratory
journey. In addition, for obligate migrants, there is likely
a stronger selection for reliable accuracy of migration
onset regarding favourable flight conditions en route and
on stopover sites [38], because of the potential fitness and
survival consequences of mistiming in these populations.
In contrast, for partially migratory populations, the
selection for accurate timing of migration onset may be
relaxed because those breed in areas with milder environmental conditions in the autumn and have potentially
shorter migration distances [34]. This potential lack of
environmental pressure during winter might also lead
to certain risk assessment scenarios where birds decide
to overwinter at their breeding site, but if winter conditions become unbearable, they suffer severe fitness consequences or need to perform some form of emergency
winter migration [39–41]. Accordingly, partially and fully
migratory populations of the same species may feature
different decision criteria in evaluating the optimal timing of departure for migration, i.e., they may differ in the
use of specific environmental cues for their departure
decisions (Fig. 1a, b).
To understand how migratory strategy determines
which environmental cues are used to initiate migration
departure, we used radio-telemetry to track Eurasian
blackbirds from populations exhibiting either partial or
full migration. By combining environmental data with
individual-level tracking data, we compared migration
departures of individuals from four distinct populations along a gradient of migratory strategies (Fig. 1c).
We expected migrants from fully migratory populations
in Russia and Poland to depart earlier in the season followed by migrants of the partial migrant population in
Finland and lastly migrants of the partial migrant population in Germany.
We tested the hypothesis that birds from fully migratory populations which breed in areas characterised by
harsh environmental winter conditions will base their
departure decisions on cues that are more predictable
across years and reliable for far-away overwintering sites,
such as day length, and less upon more flexible environmental cues such as ambient temperature. We predicted
this would result in a more synchronised and rigid timing of migration among individuals and years. In contrast, individuals from partially migratory populations
that likely migrate shorter distances and where some part
Linek et al. Movement Ecology
(2021) 9:63
of the population remains resident year-round may use
cues containing more immediate information about local
environmental conditions such as immediate changes
in the ambient temperature. Individual flexibility, which
leads to a greater population-level variation in departure
dates, should result in less synchrony of departures.
As a consequence of differences in departure timing
of migrants originating from fully and partially migratory populations, we examined the hypothesis of whether
migrating individuals of different populations use a similar, species-wide set of cues with either identical or population-specific threshold values.
We further predicted that favourable weather conditions should affect both fully and partially migratory populations similarly because the decision to migrate should
follow the same general principles of minimised survival
risk and energetic costs during flight independent of the
migration strategy. However, due to differences in time
constraints and consequences of delayed onset of migration, we predicted that departure decisions of individuals of fully migratory populations should be less affected
by ephemeral weather conditions than those of partially
migratory populations.
Methods
Study sites and species
We monitored 610 Eurasian blackbirds (Turdus merula)
at four geographically distant study sites (Fig. 1): The sites
with partially migratory populations included southern Germany (47.7801° N, 9.0203° E, 651 a.m.s.l.) where
we monitored 527 blackbirds (291 males, 207 females
and 29 birds of unknown sex during nine field seasons
from 2009 to 2017) and southern Finland (61.0975° N,
25.1579° E, 154 a.m.s.l.) where we monitored 34 individuals in three field seasons from 2014 to 2016 (21 males, 13
females). Both regions consist of mixed deciduous and
coniferous forests and are characterised by large bodies of water, which moderate the climate towards milder
temperatures. However, during winter (2009–2017 during December and January), the Finnish study site has,
on average colder temperatures (− 6.1 °C) compared to
the German site (− 3.8 °C). Fully migratory populations
were monitored in Russia (55.4582° N, 37.1791° E, 182
a.m.s.l.) where we radio-tracked 30 blackbirds (23 males,
seven females during three field seasons: 2014–2016)
and in north-east Poland (53.3483° N, 22.5927° E, 106
a.m.s.l.) where 19 individuals were studied (15 males and
four females during two field seasons: 2015–2016). Those
regions can be defined as continental with cold winters
(average temperature for December and January in Russia: − 9 °C, Poland: − 4.9 °C), and they consist primarily of coniferous forests. At all study sites, we captured
birds from May until August using mist nets. Based on
Page 4 of 14
previous ring recoveries during winter, we expected
migratory birds from all sites to leave in a southwestern direction with the two fully migratory populations
(Russia and Poland) covering longer migration distances
[33–36]. Both adults and immatures were included. The
age and sex of captured birds were determined based on
differences in the colouration of plumage and beak [42]
(Additional file 1: Table S1).
Capture and tracking methods
After capture, each bird was fitted with an aluminium
band with a unique ID and a backpack with a radio transmitter (≤ 2.6 g produced either by 1. Sparrow Systems,
Fisher, IL, USA (2009–2012, 2014–2015), 2. the Swiss
Ornithological Institute, Sempach, Switzerland (2014),
or 3. Holohil Systems Ltd., Canada (2013)) via a leg-loop
harness. All radio transmitters have sent a radio impulse
every 1.5 to 3 s from deployment until at least next
spring/summer. The mean weight of the captured blackbirds was 88 g resulting in an additional load of 3% with
the radio transmitter, which has a battery life of at least
nine months. To determine the status (presence/absence
and alive/dead), non-breeding strategy (migrant versus
resident) and the timing of departures of individuals,
we deployed one to five automated receiver units (ARU,
Sparrow Systems, Fisher, IL, USA) at haphazard locations on each study site [43]. Each ARU searched for up
to 60 selected frequencies within a maximum timeframe
of 240 s. ARUs were connected to H-antennas (ATS,
Isanti, MN, USA), mounted at the height of 3 to 12 m.
24-h ARU monitoring enabled us to precisely determine
departure events via a rapid decline of signal strength of
the radio transmitters. We used ARU data sightings and
manual tracking to ensure the absence of an individual
within a 2.5 km radius. Manual tracking was done via a
combination of handheld H antenna (Andreas Wagener
Telemetry Systems, Köln, DE) and Yaesu VR 500 receiver
(Vertex Standard USA, Cypress, CA, USA). For the German population, we also used car-mounted Yagi-antenna
(AF Antronics, Inc., Urbana, IL, USA) and an airplane
equipped with two H-antennas and two Biotrack receivers (Lotek, Newmarket, ON, Can) to ensure departure of
an individual within a 20 km radius of the study site and
to validate the 2.5 km radius, which we used to define
departures in the other study areas. For seven German
birds (one in 2009, 2010 and five in 2015), the departure could not be identified on an ARU recordings, and
the departure date was calculated subsequently as the
mean between the last time the bird was tracked and the
first date missing. All post-breeding departures between
the 2nd of September and the 24th of November were
included in our analysis. Later departures occurred only
in the German population and have been phenotypically
Linek et al. Movement Ecology
(2021) 9:63
different based on previous studies [39]. Hence they were
classified as winter migration or irruptive migration
events. This study also included published data from our
German study site collected before 2016 [39, 44].
Environmental variables
We selected day length as a possible cue since photoperiod has known biological significance in controlling
timing for migration [45]. Day length was calculated in
hours per day via the maptools package using R statistical software V. 3.3.2 [46, 47]. We included local ambient
temperature at 2 m above ground in our model as the
ambient temperature indicates future energetic costs due
to thermoregulation [20]. The temperature may also provide information about the availability of food resources
in the near future [48]. Because ambient temperature
is highly correlated with day length and will be colder
as the season progresses during autumn, we calculated
the residuals of temperature each night out of a linear
regression of date and temperature, defined as residual
temperature. This calculation allowed us to measure the
deviation from the expected temperature that would historically occur at this point of time within a year and is
perceived as colder or warmer on average. Since cloud
cover strongly impacts navigation at night [28], we also
included the percentage of cloud cover in our model. To
represent essential characteristics of flight conditions, we
used atmospheric pressure (at surface level), wind direction and speed (at surface level) and used this to calculate
likely tailwind assistance and crosswind in meters per
second. These cues likely provide immediate information
about upcoming weather conditions in the subsequent
hours and days [26, 49].
The ambient temperature, atmospheric pressure, cloud
cover and wind conditions used in the analyses were
interpolated from the nearest weather stations in each
location by the National Centers for Environmental Prediction [50]. The data were derived for the four breeding
locations in a 6-h resolution, using the RNCEP package
[51], and a mean for each night between 6 and 12 pm was
calculated. Due to the fact that blackbirds leave the ARU
signal range relatively quickly with their departure, unfortunately, no statements can be made about the behaviour
and environmental conditions after initial departure.
Therefore we estimated the same mean south-west
migration direction for all four populations [52] based
on ring recoveries of blackbirds. Tailwind assistance was
calculated for a species mean departure direction of 225°
by tailwind assistance = windspeed ∗ cos(wind direction − mean departure direction). Crosswinds perpendicular to the assumed mean departure direction have been
calculated by crosswinds = windspeed ∗ sin(wind direction − mean departure direction [53].
Page 5 of 14
Data analysis
Statistical analyses were performed using R statistical
software V. 3.3.2 [46].
To account for the unbalanced sample sizes between
the sites, we also performed the following analysis with
a balanced sample size in all populations, by reducing the
German data set to the same years as the other sampled
locations. This analysis provided similar results with the
same order of importance in the predictors.
In order to compare timing and departure conditions
between populations and migration types, the equality
of variances between different sites and their parameters
were ensured using Levene’s test [54] before running the
corresponding linear models with site and migration
type as explanatory variables in separate models (package stats). Least-squared means post-hoc tests were
performed when sites or migration types differed in the
conditions during their departure nights. P-values of
multiple comparisons were adjusted via the Benjamini–
Hochberg method (package emmeans) [55]. Since the
normalised mean atmospheric pressure and mean wind
result in a measured value of zero, a one-sample t-test
was used to test for a general preference of these environmental conditions at departure days across all populations. Variances in departure timing between populations
were compared using Bartlett’s test of homogeneity of
variances (package stats) [56] while we adopted a 5% significance threshold for all models.
After testing for occurring differences in departure conditions between populations, we investigated
the potential influence of these environmental variables on individual departure decisions. Therefore, we
used a time-dependent Cox proportional hazard model
implemented in the survival package [57]. The Cox proportional hazard model describes the probability of
departure over time as a function of a baseline probability which can be modified by fixed variables like population or time-varying explanatory variables like weather
conditions and day length [58]. All weather parameters
were scaled by subtracting the mean and dividing it by
the standard deviation for each population and year to
make their effect sizes comparable within and between
each population within the model. This was also done to
account for general differences between the microhabitats and annual variation as study site-specific differences
in meteorological conditions are corrected for in scaled
variables which allows to only focus on perceived changes
in each population. However, we scaled day length only
within each site but across all years, as day length over
time does not vary between years. We included the sex,
age and unique ID of the birds in the model, but it had no
significant influence on the outcome and was therefore
omitted.
Linek et al. Movement Ecology
(2021) 9:63
We restricted the analysis to a timeframe of 47 days
before the first migratory departure in each year and
site, as 47 days is the mean migration window across all
four populations based on our radio-telemetry measures
(Additional file 1: Table S2). We used an information-theoretic approach for model selection from a global starting
model to calculate all possible models via the “dredge”
function (package MuMin) [59] (i.e., all combinations of
including or excluding each variable and its interaction
with populations). To evaluate the likelihood of a model
being ‘best’ among all other candidate models and estimate their relative predictor importance [60], we ranked
them based on their Akaike weights derived from differences of their Akaike information criterion (AIC) corrected for a small sample size, AICc [61]. Out of all model
combinations, we selected those within a ΔAICc < 2 [62]
from the top model, which included those with strong to
moderate support (Additional file 1: Table S3).
The estimated parameters for all predictors included in
these top models were then calculated by model averaging [63] with the “model.avg” function (package MuMin)
[59]. Predictors of the global model but with no meaningful influence were not included in the top model subset.
Least-squared means post-hoc tests on the most complex
of the candidate models were performed when the averaged model results showed a significant interaction of
environmental cues and populations. P-values of multiple
comparisons were adjusted via the Benjamini–Hochberg
method (package emmeans) [55].
Finally, for the Cox proportional hazard model, we ran
a Schoenfeld test (cox.zph function, package survival)
[57] to ensure the model assumption are met and further
double-checked for correlations between all used variables within the timeframe of analysis (Additional file 1:
Table S4).
Results
Proportions of migrants
In total, we observed 212 departures during autumn
across all four populations (Additional file 1: Table S1).
In Germany, 136 birds of the population migrated, while
391 remained at the breeding site. As expected, the German population can be defined as a partially migratory
population, with 26% of birds migrating on average. Our
radio telemetry data also verified that the Finnish blackbird population is partially migratory. Out of the 34 monitored birds, we observed 27 migrants, resulting in 74%
migrants at the Finnish study site. In Russia (30 migrants)
and Poland (19 migrants), no resident individuals were
observed, and thus these populations are defined as fully
migratory.
Page 6 of 14
Departure timing during autumn migration
Our long-term monitoring revealed that all departures
for autumn migration happened after sunset during the
night (mean: 21:40, range: 18:30 to 02:00 UTC).
On a night-to-night basis, blackbirds from the Russian
and Polish populations departed the earliest in the year,
followed by the Finnish and later the German populations [F3,215 = 26.88, p < 0.01] (Fig. 2). By pooling both the
two fully migratory (Russian and Polish) and the two partially migratory (Finnish and German) populations, the
two migratory strategies (fully versus partially migratory)
differed in the timing of the night-to-night departure
decision [F1,217 = 64.75, p < 0.01]. Fully migratory populations left their breeding site 13 ± 2 days earlier than the
partially migratory populations (Fig. 2). However, the
length of the window when migrants departed (= variance in departure dates) did not differ among the four
populations [df = 3, k = 4.18, p > 0.24] or the two migration types [df = 1, k = 0.75, p > 0.38] (Additional file 1:
Table S2).
Environmental conditions during departures
While no general differences in environmental conditions
were found between study sites throughout the overall
migration window (i.e. similar temperature ranges, wind
conditions etc.) (Additional file 1: Figure S1a–S1f ), comparisons of environmental conditions during nights with
migratory departures between populations showed significant differences (Fig. 3a–f ) and pronounced variance
(Additional file 1: Table S5).
Day length
Day length at departure differed among the four populations [F3,215 = 16.68, p < 0.01]. Post-hoc tests indicated
that these differences were most pronounced between
the two migration types (partial vs. full migration)
[F1,217 = 45.70, p < 0.01], with 0.7 ± 0.1 h longer daylength
at departure dates for full than partial migrants (Fig. 3a).
Ambient temperature
Populations also departed at different ambient temperatures [F3,215 = 4.23, p < 0.006]. Blackbirds of the
Polish study site departed at warmer temperatures
(mean ± standard error: 7 ± 0.9 °C) than conspecifics
at the Finnish (2.6 ± 0.8 °C) [β = 4.4, SE = 1.2, t = 3.6,
p < 0.01] and German study sites (4.3 ± 0.3 °C) [β = 2.7,
SE = 1.0, t = 2.7, p = 0.02]. However, the temperature
(4.4 ± 0.7 °C) during departures of blackbirds at the Russian study site could not be found to be different compared to the other populations. When comparing the
two migration types, blackbirds from fully migratory
populations departed when it was 1.4 ± 0.7 °C warmer
Linek et al. Movement Ecology
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Page 7 of 14
Fig. 2 Timing of autumn migration events across all years for each population. The solid black line marks the population median date for departure,
while the dashed lines left and right mark the corresponding first and third quantiles. Day 1 = equals 1st. January
than blackbirds from partially migratory populations
[F1,217 = 4.31, p = 0.04] (Fig. 3b).
Russian study sites (41.2 ± 2.9%) [β = − 17.4, SE = 4.3,
t = − 4.1, p < 0.01] (Fig. 3d).
Atmospheric pressure
Tailwind assistance
The four populations also differed in the relative
atmospheric pressure during the nights of departures
[F3,215 = 6.33, p < 0.01]. Relative atmospheric pressure
was higher at departures of blackbirds from the Polish
study site compared to conspecifics of the German site
study site [β = 1.2, SE = 0.3, t = 4.0, p < 0.01]. Fully migratory populations departed on nights with higher relative
atmospheric pressure than partially migratory populations [β = 0.66, SE = 0.2, t = 3.3, p < 0.01]. Across all populations, the atmospheric pressure was higher on nights
when birds departed than on nights when birds did not
depart [M = 0.56 ± 0.17, t(218) = 6.47, p < 0.01] (Fig. 3c).
Cloud cover
All populations favoured nights with a cloud cover of
less than 50% for their departures [M = 33.36 ± 2.21,
t(218) = − 14.84, p < 0.01]. However, populations also
differed in actual cloud cover during departure nights
[F3,215 = 5.57, p < 0.01]. Departures at the Finnish study
site occurred during nights with significant lower cloud
cover (23.8 ± 3.1%) compared to those at the German
(33.5 ± 1.4%) [β = − 9.8, SE = 3.4, t = − 2.9, p = 0.01] and
In general, tailwind assistance on departure nights differed among populations
[F3,215 = 19.47, p < 0.01].
Tailwind on departure nights at the German study
site (− 0.3 ± 0.2 m/s) was lower than at Russian
(3.0 ± 0.5 m/s) [β = − 3.3, SE = 0.5, t = − 6.6, p < 0.01],
Polish (2.2 ± 0.6 m/s) [β = − 2.5, SE = 0.6, t = − 4.1,
p < 0.01] and Finnish (1.6 ± 0.5 m/s) [β = − 1.9, SE = 5.3,
t = − 3.5, p < 0.01] sites. Departures from the Russian site occurred during the strongest tailwinds (up to
7 m/s) across all populations, even stronger than during
departures at the Finnish site [β = 1.4, SE = 0.7, t = 2.1,
p < 0.05]. Blackbirds from partially migratory populations
left with lower tailwind assistance than those from fully
migratory populations
[F1,217 = 42.16, p < 0.01]. However, they did not differ from zero [M = − 0.004 ± 0.39,
t(169) = − 0.02, p = 0.98]. Fully migratory populations,
in contrast, preferred nights where tailwind assistance
in their approximated direction of migration was clearly
present [M = 2.71 ± 0.77, t(48) = 7.11, p < 0.01] (Fig. 3e).
Crosswinds
Departures from all four populations differed in their
crosswind conditions [F3,215 = 18.03, p < 0.01] (Fig. 3f ).
During departures at the German study site crosswinds
Linek et al. Movement Ecology
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Page 8 of 14
Fig. 3 Comparison of day length (a), ambient temperature (b), scaled atmospheric pressure (c), cloud cover (d), tailwind assistance (e) and
crosswind (f) at departure nights during autumn migration between blackbird populations. Black solid lines represent the median, boxes represent
first and third quartiles, and whiskers describe the 95% confidence intervals. The dashed red line in (c) represents mean atmospheric pressure
during the entire departure window, while it marks the absence of tail and crosswinds in (e) and (f). Populations that were identified as different via
post hoc tests are joined by brackets noted with the corresponding significant levels (* ≤ 0.05, ** ≤ 0.01, ***≤ 0.001)
are mainly towards north-west [M = 0.99 ± 0.25,
t(142) = 7.76, p < 0.01] and the strongest in this direction
compared to all other populations (Finland: [β = 1.06,
SE = 0.38, t = 2.79, p < 0.01], Russia: [β = 1.7, SE = 0.36,
t = 4.69, p < 0.01] and Poland: [β = 2.7, SE = 0.44, t = 6.1,
p < 0.01]). However, while being different between sites,
crosswinds could not be found to be different from
zero at the study sites in Finland [M = − 0.07 ± 0.85,
t(26) = − 0.16, p = 0.87] and Russia [M = − 0.71 ± 1.02,
t(29) = − 1.43, p = 0.16]. In contrast, blackbirds at the
Polish study site selected nights for departure with crosswinds primarily towards south-east [M = − 1.71 ± 0.69,
t(18) = − 5.19, p < 0.01] and also differed significantly
from crosswind conditions during departure nights at
the Finnish study site [β = − 1.64, SE = 0.54, t = − 3.02,
p < 0.01].
Used cues for individual timing of migratory departures
The best averaged Cox proportional hazard model
showed that all investigated environmental cues, day
length [sum of model weight = 1, p < 0.01], residual
ambient temperature [sum of model weight = 0.73,
p = 0.05], atmospheric pressure [sum of model
weight = 1, p = 0.01] and cloud cover [sum of model
weight = 1, p < 0.01] as well as tail [sum of model
weight = 1, p = 0.02] and crosswind affected [sum of
model weight = 1, p < 0.01] the decision of Russian, Polish, Finnish and German populations to depart during
autumn migration (Table 1). Across sites, day length
was the most influential predictor, with shorter day
lengths increasing departure probability [β = − 3.65,
SE = 1.17], followed by the various cues describing
flight conditions. In those high atmospheric pressure [β = 0.30, SE = 0.12], increased tailwind assistance [β = 0.29, SE = 0.13] and decreased crosswinds
[β = 0.59, SE = 0.11] had a positive effect on the departure probability. Cloud cover instead had a negative
effect with obstructed skies reducing the departure
probability of blackbirds [β = − 0.42, SE = 0.11]. The
overall residual temperature was the predictor with the
smallest but still significant effect. Colder than normal
temperatures increased departure probabilities across
all populations [β = − 0.17, SE = 0.09].
Linek et al. Movement Ecology
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Page 9 of 14
Table 1 Averaged model for the effects of weather variables and population on departure probability of individual blackbirds
Parameter
Day length
Residual ambient temperature
Atmospheric pressure
Cloud cover
Tailwind assistance
Crosswind
Population (Finland) × Day length
Population (Russia) × Day length
Population (Poland) × Day length
Estimate (β) ± SE
− 3.65 ± 1.17
− 0.17 ± 0.09
0.30 ± 0.12
− 0.42 ± 0.11
0.29 ± 0.13
0.59 ± 0.11
0.73 ± 0.54
1.95 ± 0.81
1.82 ± 0.64
95% CI
− 5.95 ± − 1.35
− 0.34 ± 0.01
0.06 ± 0.53
p-value
< 0.01
0.05
0.01
− 0.64 ± − 0.21
< 0.01
0.38 ± 0.81
< 0.01
0.36 ± 3.54
0.02
0.04 ± 0.53
− 0.33 ± 1.79
0.56 ± 3.08
0.02
0.18
< 0.01
Population (Finland) × Atmospheric pressure
− 0.27 ± 0.37
− 0.99 ± 0.45
0.46
Population (Poland) × Atmospheric pressure
1.00 ± 0.40
0.21 ± 1.79
0.01
− 1.33 ± − 0.14
0.02
0.37 ± 0.28
− 0.17 ± 0.92
0.18
0.11 ± 0.27
− 0.42 ± 0.64
Population (Russia) × Atmospheric pressure
Population (Finland) × Cloud cover
Population (Russia) × Cloud cover
Population (Poland) × Cloud cover
Population (Finland) × Tailwind assistance
Population (Russia) × Tailwind assistance
Population (Poland) × Tailwind assistance
− 0.36 ± 0.37
0.38 ± 0.44
− 0.74 ± 0.31
− 0.29 ± 0.46
1.31 ± 0.44
Population (Finland) × Crosswind
− 0.63 ± 0.25
Population(Poland) × Crosswind
− 1.28 ± 0.30
Population (Russia) × Crosswind
− 0.29 ± 0.39
− 1.08 ± 0.36
− 0.48 ± 1.24
0.33
0.39
− 1.19 ± 0.60
0.44 ± 2.17
0.52
< 0.01
0.68
− 1.12 ± − 0.14
0.01
− 1.86 ± − 0.69
< 0.01
− 1.05 ± 0.47
0.45
Adjusted Akaike’s Information Criterion (AICc) has been used to determine the final candidate models (Additional file 1: Table S1)
Average model estimates, adjusted standard errors (SE), 95% confidence intervals (CIs) and associated p-values of parameters included in the candidate models.
p-values ≤ 0.05 are given in bold font. The reference category for species is the German population
The best averaged cox proportional hazard model also
revealed significant two-way interactions between populations and timing-relevant cues (Table 1). This suggests population-specific differences in the magnitude of
influence on departure decision probabilities. Post-hoc
tests on the interaction between populations and day
length indicated that departure probability of blackbirds
from both fully migratory (Russia and Poland) populations was less strongly affected by decreasing day length
than that of migrants of the German and Finnish partially migratory study populations [β = 0.69, SE = 0.33,
t = 2.09, p = 0.04]. The significant two-way interaction between populations and atmospheric pressure
indicated that blackbirds of the Polish study site had a
higher departure probability with higher relative atmospheric pressure compared to migrants of the German
study site [β = 1, p = 0.01] (Table 1), and post-hoc tests
revealed that the influence of atmospheric pressure was
also greater compared to the Russian migrants [p = 0.02]
(Additional file 1: Table S6). In Russian migrants, the significant two-way interaction between species and cloud
cover indicated that they had been more influenced in
their departure probability by cloud cover compared to
migrants of the German partially migratory population
[β = − 0.74, p = 0.02] (Table 1). Also, the significant
two-way interaction between the Russian population
and tailwind assistance together with post-hoc tests
indicated that Russian migrants had a higher departure
probability [β = 1.36, SE = 0.40, p < 0.01] when tailwind
assistance was stronger compared to all other migrants
(Table 1, Additional file 1: Table S6). A significant interaction between the Finnish and Polish populations with
crosswind suggested that German migrants have been
influenced to a greater extent by winds from the southeast than migrants from the Finnish [β = − 0.63, p = 0.01]
and Polish [β = − 1.28, p < 0.01] populations (Table 1).
Observed Polish migrants, on the other hand, had a
higher departure probability with stronger crosswinds
from the northwest compared to migrants from Germany [β = 1.29, p < 0.01] and migrants from the Russia
site [β = 1.15, p = 0.02] (Additional file 1: Table S6).
Discussion
By combining environmental data and radio tracking
of Eurasian blackbirds from four populations with different migration strategies, we identified a common
species-wide cue set used for the decision of migratory
departures, but also confirmed variation in migratory
Linek et al. Movement Ecology
(2021) 9:63
timing itself and revealed effects of cues that differed in
their magnitude between populations exhibiting different migration strategies.
Fully migratory populations from Russia and Poland
departed earlier in the year than their partial migrant
conspecifics in Finland and Germany. This result is
consistent with previous studies [40], and we hypothesise that several factors like earlier deteriorating environmental conditions at the breeding site, relatively
longer migration distances and thus longer migration
drive earlier departures in Russia and Poland [52]. Considering the potentially greater consequences of mistiming in migrants, which migrate longer distances,
we predicted higher synchrony in departure dates as
they might be under stronger time constraints during
migration. However, our results did not support this
idea, as we found no significant difference in the variation of departures between partial (relatively shorter
migration distances) and full migrants (relatively longer
migration distances). While time constraints have been
shown for spring migration to secure high-quality
breeding territories [9, 64] or reproductive partners
[65, 66], our results about initial departure conditions
during autumn migration are not consistent with previous assumptions [67]. We suggest that also during the
typically slower and extended autumn migration [68],
fully migratory populations of blackbirds may not be
under stronger selection for optimal departure timing
to leave their breeding sites than their partial migrant
conspecifics. However, as we only recorded the initial
departure, we cannot draw any conclusions about time
constraints en-route, especially since stopover locations and durations remain unknown.
The environmental conditions studied had been similar across study sites during the migration window and
spanned the same ranges (Fig. 3; Additional file 1: Figure S1a-S1f ). However, they differed during nights with
departures. This would suggest that local differences in
the investigated environmental factors did not cause the
timing differences between populations. Hence there did
not appear to be a uniform species-wide threshold for
the cues blackbirds use for departure decisions across
their breeding range (Fig. 3). Similar to obligate and
potentially long-distance migrants, the fully migratory
blackbird populations left earlier in the year at longer
day lengths (Fig. 3a) and higher ambient temperatures
(Fig. 3b). Therefore, ambient conditions were significantly milder than conditions preceding departures in
partially migratory populations. This finding, in combination with the finding that all sites had similar environmental conditions throughout the migration window
(Additional file 1: Figure S1a–S1f ), supports the idea that
anticipated resources at the wintering destination [18] or
Page 10 of 14
longer flight distances with optimal flight conditions may
inform departure decisions of obligate full migrants [69].
Migrants of all populations appeared to depart during
periods of high relative atmospheric pressure, which is a
signal for favourable flight conditions [26] already found
as an important cue for migratory timing [49]. In particular, the departures of blackbirds at the Polish study site
occurred during nights with particularly high atmospheric pressures (Fig. 3c), pointing towards a higher
selectivity for such nights. Cloud cover during departures
was 33 ± 2% across all populations and showed a general
preference for departures at nights with little cloud cover.
Clear skies likely facilitate celestial navigation [28, 29]
which is thought to be of critical importance for migration in many species [70, 71].
Tailwind assistance and crosswinds at departure nights
showed distinct population-specific differences along a
geographic pattern. While the investigated north-eastern
Russian and Polish migrants departed with more tailwind
assistance than the migrants from Germany and Finland,
the departures in the German population did not occur
during nights with either clear head or tailwind. However, especially the blackbirds from the Russian study site
migrated on nights when high tailwind assistance was
likely to reduce the energetic cost of migration [72]. The
contrasts in crosswind conditions on departure nights
between populations are particularly evident in the
most south-westerly population of German birds, which
departed more often with winds coming from the southeast, and the Polish birds, which started their migration
mainly on days with predominantly wind coming from
the northwest.
Based on Cox proportional hazard models, all variables, i.e. day length, residual temperature, atmospheric
pressure, cloud cover, and wind conditions, significantly
influenced the night-to-night probability of departure
decision in all blackbirds regardless of their migratory
propensity (Table 1). Decreasing day length was the
most important factor for night-to-night departure decisions of all studied populations, followed by cloud cover,
atmospheric pressure, tailwind, crosswind and residual
temperature.
As in long-distance migratory species, decreasing day
length seems to be also an important cue for departure decisions in the partial migrant blackbird, a short
to medium distance migratory species, during autumn
migration. For example, (1) peak autumn migration
season across Europe in Blackbirds is during October
[52] and individuals do not migrate before their specific migration window even when they experience suitable flight conditions (e.g. high atmospheric pressure and
adequate wind conditions) and (2) captive blackbirds in
a temperature-controlled common garden experiment
Linek et al. Movement Ecology
(2021) 9:63
exhibited nocturnal restlessness during October as well
[73].
However, the likely fine-tuning of the timing of
departure on a night-to-night basis appears to be more
modulated via imminent flight conditions like wind characteristics and atmospheric pressure, predicting potentially unfavourable flight conditions [27], while also
preferring clear skies, which might be necessary for navigation [29]. As such, flight conditions were the second
most important factors across all populations (Table 1).
These conditions likewise stood out in the first part of
our analysis, where all populations showed a strong preference for days with below-average cloud cover, high
atmospheric pressure, and population-specific wind conditions compared to residual temperature (Fig. 3). Those
conditions are generally characterised by no rain and
favourable flight conditions.
Of the environmental factors we studied, temperature
was the factor that had the least influence on departure
probability. However, in our full and partial migrant
populations (residual) temperatures lower than average
increased the probability for departure decisions as they
likely contain local environmental information about
diminishing food resources and increasing thermoregulatory costs [74] (Table 1).
However, the magnitude of the effects of all cues,
except residual temperature, differed between full and
partial migrant populations. While day length is still the
strongest predictor for departure timing for full migrants
in Russia and Poland, it was less influential in departure
decisions than for partial migrants from Finland and
Germany. This appears to contradict our general understanding of how day length influences the seasonal organisation of migration [32, 75] and our expectation that
obligate full migrants rely more strongly on predictive
cues like day length than facultative partial migrants. One
possible explanation for this finding could be that the
effects of daylength are masked by other more relevant
factors such as appropriate weather conditions essential
for every migratory flight. Migrant blackbirds from Russia and Poland, which likely travel longer distances, in
particular, may be naturally subject to significantly more
energetic constraints and higher fitness consequences
[76, 77] compared to migrants from Finland and Germany. They may be more sensitive to the costs of flight
energetics [78, 79] and hence are more prone to select
nights with optimal flight conditions, which are characterised by high atmospheric pressure and best possible
wind support to a greater extent for departure and migration (Table 1, Fig. 3).
In contrast, there is likely a smaller need for optimal
flight conditions in short-distance migrants, such as the
blackbirds from the German population, as they only
Page 11 of 14
travel about 1–3 days to their wintering areas (Linek
et al., 2022, in prep). This hypothesis is supported by
two-way interactions between populations and several
weather variables (Table 1). Full migrants from Poland
had a higher probability for departure at nights with
higher atmospheric pressure and with a stronger crosswind from the northwest compared to partial migrants in
Germany. Similarly, full migrants from the Russian study
side had a higher probability of departure at nights with
clearer skies and stronger tailwind assistance than partial migrants. Additional data are necessary to verify this
hypothesis. The greater preference of full migrants for
particularly good flight conditions and the fact that we
could not observe a hard-wired threshold for specific day
lengths [80] across populations is consistent with the fact
that we did not find greater synchrony in departure dates
and highlights the population-specific weighting of environmental conditions for migratory departures.
However, the overall importance of all six environmental variables across all populations suggests that partial migrants from our German and Finnish study sites
behave similarly to the other two fully migratory populations by selecting suitable flight conditions for their
departures (Fig. 1c, Table 1). Previous research supports
our finding that crosswinds and tailwind assistance seem
generally important [32, 67]. The combination of lateral
drift en route and increased energy expenditure imposed
by crosswinds [81] seems crucial for all populations and
may outweigh the potential time constraints [82] faced
by relatively early-departing populations in Russia and
Poland.
Conclusion
In summary, despite discovering that migrants of all populations appeared to depart during favourable weather
conditions, suggesting that they are sensitive to the costs
of flight energetics [83] and available visual cues [84],
we found large differences not only in departure timing
between populations and migration types (full and partial migrants) but also in the environmental conditions
at their respective departures. These population-specific
differences in day length, ambient temperature, atmospheric pressure, and wind characteristics likely indicate
local adjustments to their specific environment. In combination, the investigated cues allow initiation, fine-tuning and adjustments to short term weather conditions
within a broader migration window, but blackbirds from
different populations appear to use population-specific
local cue thresholds and weightings when integrating
environmental information to initiate migration decisions (also see [85]).
All the cues tested in our study are also correlated
to some extent to other environmental conditions and
Linek et al. Movement Ecology
(2021) 9:63
thus may serve as a proxy for other still unknown cues
which birds use from their environment to time their
migration optimally [2]. In this respect, factors such as
food availability, which so far could not be adequately
measured for our populations, but likely varies between
study sites and influences overall body conditions [86],
could be of particular importance [87–89]. In addition,
other events in earlier life-history stages, e.g., a delay
during breeding, are also shown to influence individual
departure decisions [49]. Nevertheless, it is necessary
to precisely understand what control mechanisms birds
use for optimal migration timing to identify the numerous challenges migratory birds have and will have to
face [90] including habitat fragmentation [91], shorter
breeding times [92], changes in temperature [93] and
habitat alteration or loss [94]. Because of the life-history consequences of poorly timed migratory decisions,
it is of critical importance to understand at a population level what mechanism animals use to initiate
migration and predict how they will respond to rapid
environmental changes in the near future [95].
Recent studies [96, 97] advanced the field of migration
on a community level and studied individual flexibility
in cues usage [98, 99], yet they lack the combination
of individual-level resolution, necessary to determine
cue-response relationships, and simultaneous sampling
among different species. Studies like the present, using
individual tracking data from different populations of
the same species, may reveal common control mechanisms and population-specific adaptations, predicting
the magnitude of vulnerability to climate change among
populations. Future studies should implement common
garden experiments or reciprocal translocations to illuminate differences in intrinsic, potentially genetic, cue
evaluation or population-specific cue thresholds to
expand our knowledge about the control of migration
in a period of unprecedented global change.
Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s40462-021-00298-y.
Additional file 1: Tables and figures for additional information on the collected data, environmental conditions and statistical analysis.
Acknowledgements
We thank Marion Soresina, Nicole Blaser and the numerous field assistants
who conducted fieldwork at all four study sites. We also thank Paul Schaeffer,
Cornelia Wingfield Twining, Kamran Safi, and Matthias Loretto for statistical
advice and the International Max Planck Research School for Organismal Biology. The work outside of Germany was made possible by the support of John
Loehr and Sirkka Rantala at the Lammi Biological Station, University of Helsinki,
Finland and the support from the field station of the University of Białystok,
in Gugny, which was used as a base for the work in Poland and the Malinki
Biological Station of the A.N. Severtsov Institute of Ecology and Evolution RAS
in Russia.
Page 12 of 14
Authors’ contributions
NL and JP designed the research and wrote the manuscript. NL analysed
the data. JRS, MW and JP revised the manuscript. MTO and PG helped to
design the analysis. NL, AS, JT, PB, JT, IP, TV, MW and JP conducted fieldwork
and helped with manuscript revisions. All authors (NL, TV, PG, JRS, MTO, AS,
JT, PB, JT, IP, MW and JP) gave final approval for publication, agree to be held
accountable for the work performed therein and have no competing interests.
All authors read and approved the final manuscript.
Funding
Open Access funding enabled and organized by Projekt DEAL. Funding was
provided by the Max-Planck-Gesellschaft and by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s ExcellenceStrategy – EXC 2117 – 422037984.
Availability of data materials
The dataset supporting the conclusions of this article is available from the
corresponding authors on reasonable request.
Declarations
Ethics approval and consent to participate
This research was approved by the responsible ethic commissions and
ministries in each country. Germany: Regierungspräsidium Freiburg,
35-9185.81/G-16/115, 35-9185.81/G-13/29, 35-9185.81/G-09/08; Finland:
Regional State Administrative Agency (Southern Finland, team ELLA),
Permit Number: ESAVI/3165/04. 10. 07/2014; Russia: We followed §44 and
§6 of the Federal Law of the Russian Federation No. 52 from 24.04.1995
(last update 18.02.2020) “On Wildlife; Poland: Local Ethical Committee for
Animal Experimentation in Białystok; Poland (permits 12/2014, 46/2014) and
Ministry of Environment, Poland, decisions DLP-III-4102-119/9205/15/MD and
DLP-III-4102-119/9206/15/M.
Consent for publication
Not applicable.
Competing interests
We declare we have no competing interests.
Author details
1
Max Planck Institute of Animal Behavior, Radolfzell, Germany. 2 Department
of Biology, University of Konstanz, Konstanz, Germany. 3 Faculty of Biology,
University of Białystok, Białystok, Poland. 4 Michael-Otto-Institut im NABU,
Bergeshusen, Germany. 5 Department of Biological Sciences, Southeastern
Louisiana University, Hammond, USA. 6 Institute of Plant and Animal Ecology,
UB RAS, Ekaterinburg, Russia. 7 Institute of Biological Problems of the North,
FEB RAS, Magadan, Russia. 8 Natural Resources Institute Finland, Helsinki,
Finland. 9 Lammi Biological Station, University of Helsinki, Lammi, Finland.
10
Centre for the Advanced Study of Collective Behaviour, University of Konstanz, Konstanz, Germany.
Received: 8 October 2021 Accepted: 5 December 2021
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Postponed or immediate drainage of infected necrotizing pancreatitis (POINTER trial): study protocol for a randomized controlled trial
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Postponed or immediate drainage of infected necrotizing pancreatitis (POINTER trial): study
protocol for a randomized controlled trial
Grinsven, Janneke van; Dijk, Sven M. van; Dijkgraaf, Marcel G.; Boermeester, Marja A.; Bollen,
Thomas L.; Bruno, Marco J.; Goor, H. van; Santvoort, H.C. van; Besselink, Marc G. 2019, Article / Letter to editor (Trials, 20, (2019), article 239)
Doi link to publisher: https://doi.org/10.1186/s13063-019-3315-6 Version of the following full text: Publisher’s version
Downloaded from: http://hdl.handle.net/2066/203594
Download date: 2024-10-24 Postponed or immediate drainage of
infected necrotizing pancreatitis (POINTER
trial): study protocol for a randomized
controlled trial Janneke van Grinsven1,2*
, Sven M. van Dijk1,2, Marcel G. Dijkgraaf3, Marja A. Boermeester1, Thomas L. Bollen4,
Marco J. Bruno5, Sandra van Brunschot6,7, Cornelis H. Dejong8,9, Casper H. van Eijck10, Krijn P. van Lienden11,
Djamila Boerma2, Peter van Duijvendijk12, Muhammed Hadithi13, Jan Willem Haveman14, René W. van der Hulst15,
Jeroen M. Jansen16, Daan J. Lips17, Eric R. Manusama18, I. Quintus Molenaar7, Donald L. van der Peet19,
Alexander C. Poen20, Rutger Quispel21, Alexander F. Schaapherder22, Erik J. Schoon23, Matthijs P. Schwartz24,
Tom C. Seerden25, B. W. Marcel Spanier26, Jan Willem Straathof27, Niels G. Venneman28, Wim van de Vrie29,
Ben J. Witteman30, Harry van Goor31, Paul Fockens6, Hjalmar C. van Santvoort2,7†, Marc G. Besselink1*† and for the
Dutch Pancreatitis Study Group STUDY PROTOCOL Postponed or immediate drainage of
infected necrotizing pancreatitis (POINTER
trial): study protocol for a randomized
controlled trial Abstract * Correspondence: j.vangrinsven@pancreatitis.nl; m.g.besselink@amc.uva.nl 1Department of Surgery, Amsterdam UMC, University of Amsterdam, G4.196,
PO Box 26000, 1105 AZ Amsterdam, Netherlands
Full list of author information is available at the end of the article 1Department of Surgery, Amsterdam UMC, University of Amsterdam, G4.196,
PO Box 26000, 1105 AZ Amsterdam, Netherlands
Full list of author information is available at the end of the article Abstract Background: Infected necrosis complicates 10% of all acute pancreatitis episodes and is associated with 15–20%
mortality. The current standard treatment for infected necrotizing pancreatitis is the step-up approach (catheter drainage,
followed, if necessary, by minimally invasive necrosectomy). Catheter drainage is preferably postponed until the stage of
walled-off necrosis, which usually takes 4 weeks. This delay stems from the time when open necrosectomy was the
standard. It is unclear whether such delay is needed for catheter drainage or whether earlier intervention could actually
be beneficial in the current step-up approach. The POINTER trial investigates if immediate catheter drainage in patients
with infected necrotizing pancreatitis is superior to the current practice of postponed intervention. Methods: POINTER is a randomized controlled multicenter superiority trial. All patients with necrotizing pancreatitis are
screened for eligibility. In total, 104 adult patients with (suspected) infected necrotizing pancreatitis will be randomized
to immediate (within 24 h) catheter drainage or current standard care involving postponed catheter drainage. Necrosectomy, if necessary, is preferably postponed until the stage of walled-off necrosis, in both treatment arms. The
primary outcome is the Comprehensive Complication Index (CCI), which covers all complications between randomization
and 6-month follow up. Secondary outcomes include mortality, complications, number of (repeat) interventions, hospital
and intensive care unit (ICU) lengths of stay, quality-adjusted life years (QALYs) and direct and indirect costs. Standard
follow-up is at 3 and 6 months after randomization. Methods: POINTER is a randomized controlled multicenter superiority trial. All patients with necrotizing pancreatitis are
screened for eligibility. In total, 104 adult patients with (suspected) infected necrotizing pancreatitis will be randomized
to immediate (within 24 h) catheter drainage or current standard care involving postponed catheter drainage. Necrosectomy, if necessary, is preferably postponed until the stage of walled-off necrosis, in both treatment arms. The
primary outcome is the Comprehensive Complication Index (CCI), which covers all complications between randomization
and 6-month follow up. Secondary outcomes include mortality, complications, number of (repeat) interventions, hospital
and intensive care unit (ICU) lengths of stay, quality-adjusted life years (QALYs) and direct and indirect costs. Standard
follow-up is at 3 and 6 months after randomization. (Continued on next page) * Correspondence: j.vangrinsven@pancreatitis.nl; m.g.besselink@amc.uva.nl
Hjalmar C van Santvoort and Marc G Besselink are shared senior author
†Hjalmar C. van Santvoort and Marc G. Besselink contributed equally to this
work. Note: Note:
To cite this publication please use the final published version (if applicable). To cite this publication please use the final published version (if applicable). Grinsven et al. Trials (2019) 20:239
https://doi.org/10.1186/s13063-019-3315-6 Open Access (Continued from previous page) Discussion: The POINTER trial investigates if immediate catheter drainage in infected necrotizing pancreatitis reduces
the composite endpoint of complications, as compared with the current standard treatment strategy involving delay of
intervention until the stage of walled-off necrosis. Trial registration: ISRCTN, 33682933. Registered on 6 August 2015. Retrospectively registered. Keywords: Necrotizing pancreatitis, Infection, Step-up approach, Timing, Drainage, Complication, Randomized
controlled trial Methods Acute pancreatitis is one of the most common gastrointes-
tinal conditions requiring acute hospital admission [1]. Around 20–30% of these patients develop necrotizing pan-
creatitis [2]. Infected necrotizing pancreatitis occurs in a
third of these patients and is associated with 15–20%
mortality [3, 4], despite radiological, endoscopic or surgical
interventions [3–6]. The current, 2013 international treat-
ment guidelines [7, 8] recommend a step-up approach,
based on the results of the Dutch PANTER trial [3]. The
first step of this step-up approach is catheter drainage,
preferably once the (extra) pancreatic collection has orga-
nized and has become fully encapsulated (walled-off necro-
sis). This process is usually complete 4 weeks after the
onset of disease. During this time, intravenous antibiotic
treatment is used which may obviate the need for any
intervention in a small subset of patients [4]. If catheter
drainage does not resolve the clinical signs of infection and
sepsis, surgical [3] or endoscopic [9] necrosectomy is per-
formed as the next step. The trial protocol is written in accordance with the Stand-
ard protocol items: recommendation for interventional
trials (SPIRIT) guidelines (see Fig. 1 and the Additional
file 1: SPIRIT checklist) [18]. Study aim The POINTER trial aims to determine whether immediate
catheter drainage in patients with (suspected) infected
necrotizing pancreatitis is superior to the current standard
of postponed catheter drainage with regard to clinical out-
come and cost-effectiveness. The hypothesis is that
pro-active diagnosis of infected necrosis and immediate
catheter drainage prevents further clinical deterioration in
these patients, reducing complications and possibly death,
and reduces length of hospital stay and costs, as compared
to postponing catheter drainage using antibiotics, prefera-
bly until the stage of walled-off necrosis. © The Author(s). 2019 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. Grinsven et al. Trials (2019) 20:239 Page 2 of 10 (Continued from previous page) Study design and setting Postponing all interventions for infected necrosis until
the stage of walled-off necrosis has been standard practice
for many years. The rationale for this delay lies in the pre-
vention of the “extra hit” (i.e. a pro-inflammatory reaction)
of open surgery in these already critically ill patients, and
in the relationship between early open necrosectomy and
mortality [10]. In line with this practice, catheter drainage
in the current step-up approach has also been postponed
until the stage of walled-off necrosis. Meanwhile, intraven-
ous antibiotics are administered to reduce systemic illness
from the infected necrosis, which may lead to increased in-
cidence of Candida infections and antibiotic resistance
[10]. Notably, several observational studies have suggested
that encapsulation is not mandatory for safe and successful
catheter drainage [3, 11–15]. In other conditions, such as
pancreatic fistula after pancreatic resection, early (percu-
taneous) catheter drainage has also proven to be safe and
successful [16]. Furthermore, an international survey
among expert pancreatologists demonstrated “equipoise”
between immediate and postponed catheter drainage of in-
fected necrotizing pancreatitis [17]. The aim of immediate
catheter
drainage
is
to
prevent
further
clinical
deterioration. POINTER is a randomized controlled multicenter super-
iority trial, including hospitalized adult patients with
proven or suspected infected necrotizing pancreatitis. In
total, 25 centers are participating in the trial, including all
8
Dutch
university
medical
centers. Endpoints
are
assessed by an adjudication committee, blinded to the
assigned treatment arm, based on clinical case descrip-
tions and endpoint definitions. The Data and Safety Moni-
toring Committee (DSMC) monitors patient safety. Inclusion criteria (see Additional file 2: Table S1 and
Additional file 3: Table S2)
The inclusion criteria are: Inclusion criteria (see Additional file 2: Table S1 and
Additional file 3: Table S2) The inclusion criteria are: – Proven infected necrotizing pancreatitis (0–35 days
after the onset of disease) or clinical suspected
infected necrosis (15–35 days after the onset of
disease) – Catheter drainage of the necrotic collection is
technically feasible, as deemed by the expert panel
and/or treating physician (i.e. enough encapsulation
and liquefaction) q
– Age ≥18 years Grinsven et al. Trials (2019) 20:239 Page 3 of 10 Fig. 1 SPIRIT schedule of enrollment, interventions, and assessments. *In the case of no clinical improvement. Study design and setting βBaseline variables: age, sex, center,
body mass index, etiology of pancreatitis, American Society of Anesthesiologist’s classification, co-morbidity, disease severity, suspected or proven
infected necrosis, time from admission to randomization, time from admission to tertiary referral. ^Primary outcome: Comprehensive Complications
Index #Secondary outcomes: mortality complications number of (repeat) interventions hospital and intensive care unit lengths of stay quality- Exclusion criteria Exclusion criteria
The exclusion criteria are: The exclusion criteria are: – Onset of acute pancreatitis > 35 days ago – Indication for emergency laparotomy because of an
abdominal catastrophe (e.g. bleeding, bowel
perforation or abdominal compartment syndrome) Treatment groups – Previous retroperitoneal intervention for necrotizing
pancreatitis (ascites drainage is permitted and All patients with signs of infected necrotizing pancrea-
titis are pro-actively assessed for the presence of infected Grinsven et al. Trials (2019) 20:239 Grinsven et al. Trials (2019) 20:239 Page 4 of 10 Page 4 of 10 necrosis, on imaging (contrast enhanced computed tom-
ography (CECT) or magnetic resonance imaging (MRI)). In the case of unclear signs of infection, percutaneous
FNA is performed in patients with necrotizing pancrea-
titis within 14 days after onset of disease who have had
clinical signs of infection (i.e. new (multiple) organ fail-
ure or 2/3 parameters raised: temperature, CRP or
leukocyte count) for 2 days consecutively . After the first
14 days, clinical signs of infection suffice for diagnosing
infected necrosis (having no other focus for infection,
e.g. pneumonia), based on our experiences in previous
trials [3, 9]. Obviously, in the case of clinical doubt, FNA
is still allowed after the first 14 days. The presence of gas
in the (extra) pancreatic necrosis on CECT is considered
proven infected necrosis in all patients, regardless of the
disease stage (i.e. before or after 14 days). necrosis, either by imaging (gas configurations), fine-
needle aspiration (FNA), or clinical signs of infection,
which may include persistent organ failure. See Add-
itional file 4: Figure S1 for the inclusion flowchart. FNA
is only used on indication and not as a screening tool. Patients who fulfill the eligibility criteria are randomly
assigned to group A or B. Since diagnosing infected ne-
crosis and making decisions on whether to perform in-
vasive intervention in these patients are challenging, the
Dutch pancreatitis expert panel [19] is 24 hours per day,
7 days per week, to assess indications for intervention
and eligibility for randomization as was done in the
PANTER [3] and TENSION [9] trials. Step-up approach
h Both a percutaneous surgical and an endoscopic step-up
approach are permitted in the POINTER trial, depend-
ing on the location of the necrotic collection(s), the ex-
tent of encapsulation and the preference of the treating
physician. Transluminal (transgastric or transduodenal),
endoscopic or percutaneous catheter drainage via the
retroperitoneal route may be performed. In the case of
no clinical improvement within the first 72 h after initial
catheter drainage, an additional or upsizing drainage
procedure is performed (Additional file 4: Figure S1). Necrosectomy is performed when there is no clinical im-
provement after this second drainage procedure and no
further possibilities for optimized or additional drainage. Control group (B) Control group (B)
Group B will have postponed catheter drainage and re-
ceive antibiotics, preferably until the (extra) pancreatic
necrotic collection has reached the stage of walled-off ne-
crosis. In case of no clinical improvement within 72 h after
drainage, the possibility of additional drainage is evaluated,
including drain revision or drain upsizing. In case of no
clinical improvement thereafter and no possibilities for
additional catheter drainage, minimally invasive necrosect-
omy is performed. No clinical improvement is defined as
new organ failure or 2/3 parameters that do not decrease
(temperature, CRP and leukocyte count). See flowchart in
Additional file 4: Figure S1. Intervention group (A) Group A will receive immediate catheter drainage within
24 h after randomization, while starting (or continuing)
antibiotic treatment. In the case of no clinical improve-
ment within 72 h thereafter, the possibility of additional
drainage is evaluated, including drain revision or drain up-
sizing. In the case of no clinical improvement thereafter,
and no possibilities for additional catheter drainage, min-
imally invasive necrosectomy is performed once the (extra)
pancreatic collection has developed into walled-off necro-
sis. No clinical improvement is defined as new organ fail-
ure or 2/3 parameters that do not decrease (temperature,
C-reactive protein (CRP) and leukocyte count). See flow-
chart in Additional file 4: Figure S1. Supportive treatment pp
Current standard management of acute (necrotizing)
pancreatitis is extensively described in the International
Association of Pancreatology (IAP)/American Pancreatic
Association (APA) guidelines of 2013 [7]. These inter-
national guidelines have been adopted by the Dutch
Pancreatitis Study Group (DPSG) and the national pro-
fessional organizations involved. In accordance with
these guidelines, patients receive fluid resuscitation, pain
management and enteral feeding (or oral, if enteral feed-
ing is not tolerated). Antibiotic prophylaxis is not given. The source of infection will be investigated in patients
with clinical signs of infection and deterioration, by
means of analysis of blood, urine, sputum, and ascites
and by diagnostic imaging (e.g. chest x-ray and abdom-
inal CECT). Once the focus of infection is identified, tar-
geted antibiotics are given or, in when there infected
necrosis is suspected and there is persistent deterior-
ation, broad spectrum antibiotics with optimal tissue
penetration are started empirically. The latter usually
consists of meropenem or imipenem, based on the local
antibiotics protocol. Randomization, blinding and treatment allocation Randomization, blinding and treatment allocation Randomization, blinding and treatment allocation
Patients are randomized using a centrally operated com-
puter (ALEA system) with variable block randomization
for allocation concealment between group A (immediate
catheter drainage) and group B (postponed catheter
drainage). Stratification at randomization is applied for the
following factors: presence of organ failure (yes versus no),
disease duration (day 0–20 versus day 21–35) and center
(expected high number of included patients versus other
centers). For randomization of a patient, physicians can
contact the study coordinator via telephone (www.pancrea-
titis.nl) 24 h per day, 7 days per week in order to check the
eligibility criteria and to verify whether informed consent
has been obtained. Blinding of patients and physicians to
treatment strategy is not feasible, since both treatments are
highly different timing-wise. Patients are coded by a
numeric randomization code (anonymized). Primary endpoint The primary endpoint is the Comprehensive Complica-
tions Index (CCI) [20, 21], including all complications
other than pre-existent complications (e.g. treatment for
infected (extra) pancreatic necrosis) occurring after
randomization until 6 months after randomization, and
graded according to the Clavien-Dindo classification. These complications are assessed by an adjudication
committee, blinded for assigned treatment arm, based
on clinical case descriptions using definitions. Secondary endpoints Secondary endpoints are mortality, new-onset (multiple)
organ failure, bleeding requiring intervention, perfor-
ation of a visceral organ requiring intervention, entero-
cutaneous
and
pancreatic
fistula,
incisional
hernia
(including burst abdomen), wound infections, endocrine
and exocrine pancreas insufficiency, number of patients
with severe complications (Clavien-Dindo III or higher),
number of patients per Clavien-Dindo classification,
number of surgical, endoscopic and radiological (repeat)
interventions, length of hospital stay, length of ICU ad-
mission, quality-adjusted life years (QALYs) and total
direct and indirect costs (see Additional file 7 for rele-
vant definitions). Also, for mutual comparison, the pri-
mary endpoint of the previous PANTER trial will be a
secondary endpoint of the POINTER trial (i.e. a com-
posite of major complications: new-onset multiple organ Diagnosing infected necrosis All patients with signs of infected necrotizing pancrea-
titis are screened for eligibility (Additional file 4: Figure
S1). We differentiate between diagnosis of infected ne-
crosis within the first 14 days after onset of disease and
diagnosis thereafter. In the first 14 days a proven infec-
tion is mandatory, as in this early course of the disease it
is impossible to distinguish systemic inflammatory re-
sponse syndrome (SIRS) from sepsis. This proof requires
either a positive gram stain or culture from FNA or gas
configurations in the (peri) pancreatic collection with Grinsven et al. Trials (2019) 20:239 Grinsven et al. Trials (2019) 20:239 Page 5 of 10 After percutaneous catheter drainage, additional necro-
sectomy should be performed surgically (e.g. videoscopic
assisted retroperitoneal debridement (VARD)) and after
endoscopic transluminal drainage, endoscopic translumi-
nal necrosectomy is performed. Additional catheter
drainage or necrosectomy may be performed using the
“other” approach only when (additional) necrotic collec-
tions are not technically approachable using the stand-
ard second step. Both step-up approaches are performed
to conform with the PANTER and TENSION trial pro-
tocols [3, 9]; see also Additional file 5: Figure S2 and
Additional file 6: Figure S3. Randomized studies can be
influenced by underestimation of learning curves, there-
fore in the POINTER trial the participating centers must
have documented expertise, defined as having performed
at least 10 independent VARD procedures, 10 independ-
ent endoscopic transluminal drainage procedures and 10
independent endoscopic transluminal necrosectomies. This number has been chosen to achieve a balance be-
tween volume and feasibility, and from experience with
other trials of our study group including necrotizing
pancreatitis patients [3, 9]. In the case of there being in-
sufficient local experience available (e.g. during week-
ends), the patient is transferred to a tertiary referral
center with sufficient experience. failure, enterocutaneous fistula or perforation of a vis-
ceral
organ
requiring
intervention,
intra-abdominal
bleeding requiring intervention or death during admis-
sion or during the 3 months after discharge) [3]. Sample size The sample size was calculated based on the primary
endpoint, the CCI. A mean CCI score of 40 (with stand-
ard deviation of 27) for postponed catheter drainage is
based on the number of complications identified in the
step-up arm of the PANTER trial [3] and TENSION trial
[9]. Analysis by Student’s t test will have 80% power to
detect a clinically relevant reduction of 15 to a CCI score
of 25 [21] at a significance level of 0.05; for a sample size
that equals 2 × 51, this will result in 102 evaluable pa-
tients. Assuming a dropout rate of about 2%, then 104
patients need to be included. A drop-out rate of 2% is
relatively low, but was chosen from experience with
other trials in our study group including patients with
necrotizing pancreatitis, in which no there was no loss
to follow-up or drop-outs [3, 9]. Monitoring and quality assurance Clinical trial monitoring is performed by an independent
monitor. The trial monitor checks and verifies documents
and reports in the trial patients’ electronic or paper records
at every site. The frequency may be changed based on the
total enrollment period and enrollment rate. The monitor
checks the site files according to the Medical Research In-
volving Human Subjects Act (WMO)/Good Clinical Prac-
tice (GCP) standards, as to whether and how essential
documents are collected/administered and verifies all re-
ported severe adverse events (SAEs). Also, the monitor
verifies protocol compliance. A monitoring report is com-
piled after each monitoring visit at each specific site. Statistical analysis All randomized patients are evaluated for primary and
secondary endpoints at 6 months after randomization. Using primary source data, an adjudication committee
will assess the occurrence of the primary and secondary
outcomes blinded to treatment allocation, after the last
patient has completed the predefined follow up 6
months after randomization. The primary analysis is based on intention-to-treat prin-
ciples. For exploratory reasons per-protocol analysis will
also performed. A tabular listing of all patients excluded
from the intention-to-treat populations will be provided
together
with
the
reasons
for
exclusion. For
the
intention-to-treat population the protocol deviations in
each randomization arm are listed. Predefined subgroup
analyses will be performed in patients with and without
(multiple) organ failure and disease duration (cutoff 20
days) at the time of randomization and per center (high
expected number of included patients and other centers). Baseline values Baseline criteria (all < 24 h prior to randomization) are
age, sex, center, body mass index, etiology of pancreatitis,
American Society of Anesthesiologist’s (ASA) classifica-
tion, co-morbidity, disease severity (SIRS, ICU admission,
single or multiple organ failure, Acute Physiology and
Chronic Health Evaluation (APACHE) II score, Multiple
Organ Dysfunction Score (MODS) [26], Sequential Organ
Failure Assessment (SOFA) score [27], CRP), CT severity
index (CTSI), suspected or proven infected necrosis, time
from admission to randomization (days) and time from
admission to tertiary referral (days). Follow up
h f ll The follow-up duration is 6 months from randomization. Outpatient follow-up visits take place at the discretion
of the responsible physician, but in any case, follow up
at 3 and 6 months after randomization can be consid-
ered as standard care. All patients undergo imaging
(preferably
CECT)
at
3
and
6 months
post
randomization. Furthermore, exocrine and endocrine
pancreatic function is measured at these points in time,
with blood glucose measurements and fecal elastase
tests, respectively. No blood or fecal samples are stored
at the DPSG data center. The treating physician is re-
sponsible for the application, interpretation and treat-
ment when needed. Also, every patient receives a Grinsven et al. Trials (2019) 20:239 Page 6 of 10 Page 6 of 10 combined
questionnaire
at
home
(Short
Form-36
(SF-36) [22], Euroqol 5 dimensions (EQ-5D) [23], iMedi-
cal Consumption Questionnaire (iMCQ) [24] and iPro-
ductivity Cost Questionnaire (iPCQ) [25]) at 3 and 6
months. Data from patient records are collected until 6
months post randomization. will be compared using Student’s t test, Wilcoxon’s rank
sum test, the chi square (χ2) test or Fischer;s exact test,
as appropriate. In the event of imbalance between
groups at baseline, regression analysis will be used to
correct for the effect of the covariates. The economic evaluation will address the question of
whether or not immediate catheter drainage in patients
with infected necrosis is cost-effective compared to the
current management of postponed catheter drainage. A
cost-effectiveness analysis and a cost-utility analysis will
be performed, both from a societal perspective. The pri-
mary outcome parameters in the cost-effectiveness and
cost-utility analyses, respectively, are the costs per unit
of the CCI score and the costs per QALY. Safety Physicians who are involved in the trial will be asked to re-
port all adverse events to the coordinating investigator. The independent Data Safety Monitoring Committee
(DSMC) will evaluate safety parameters at regular inter-
vals. The DSMC consists of five members: two surgeons, a
gastroenterologist, a radiologist and an epidemiologist. Evaluations are planned after patient number 25, 50 and
75 have completed their 6-month follow up. Deceased pa-
tients and every SAE that is possibly trial-related will be
discussed unblinded by the DSMC, 25 patients at a time
in 4 sessions. During the inclusion period of the study the
DSMC performs interim analyses only on safety. Only for
safety reasons as
assessed
by the
DSMC
will
the
POINTER trial be prematurely terminated. There will be
no interim analysis of treatment effect. Adverse events are
reported using the online module (https://www.toetsin-
gonline.nl) of the Dutch Central Committee on Research
involving human subjects. All analyses will be performed in SPSS for Windows
or SAS System for Windows. All data handling and ana-
lyses will be saved in a syntax/program file. Results will
be presented with all centers combined. A two-tailed p
value <0.05 is considered statistically significant. No cor-
rections for multiple tests will be applied. The primary outcome is a sum of all complications
that are weighted for their severity (multiplication of the
median reference values from patients and physicians),
the CCI [20, 21]. Comparison of the primary endpoint
will be expressed in terms of the absolute difference in
mean CCI score and standard deviation (SD). Subse-
quent analyses will be directed at the secondary end-
points. Data will be presented as mean ± SD and in the
case of skewed distributions as median and range. Values Discussion Early
necrosectomy is now recognized as having a major im-
pact in the critically ill patient, whereas postponed
necrosectomy allows the immune system to recover
from the pro-inflammatory response due to pancreatitis. Since the step-up approach is now considered stand-
ard of care, the issue of the optimal timing of catheter
drainage has become highly relevant. Current literature
reports that 35–64% of patients with infected necrotiz-
ing pancreatitis can be treated with catheter drainage
alone, without the need for invasive necrosectomy [3, 5]. Therefore, several expert pancreatologists have stated
that they already practice immediate catheter drainage
in patients with infected necrosis. In a recent inter-
national survey performed in preparation for this study,
55% of expert pancreatologists stated that they typically
postpone catheter drainage by using antibiotics, whereas
the other 45% proclaimed to drain immediately after
diagnosing infected necrosis [17]. Thus, in practice im-
mediate catheter drainage is already being performed in
individual patients, regardless of the effect of antibiotics
alone or the degree of encapsulation: (percutaneous)
catheter drainage in infected necrosis has been described
retrospectively in several cohorts [32] at a median of 2
weeks (9–15 days) after the onset of disease instead of at
4 weeks (28 days). After the first 14 days, clinical signs of infected necro-
sis are much more reliable. In the PANTER trial [3] it
was possible to attain 91% accuracy in the identification
of infected necrosis based on clinical criteria. Patients
can be randomized in the POINTER trial after the first
14 days, based on the clinical diagnosis of infected ne-
crosis, as was done in the PANTER trial [3]. Patient outcomes were significantly better in the
step-up-approach arm of the PANTER trial compared to
the control group (primary open necrosectomy), but
mortality (19%) did not differ between the two groups
[3]. The POINTER trial assesses whether early detection
of infected necrosis and immediate catheter drainage im-
prove outcomes, to further reduce mortality and mor-
bidity. Earlier intervention may prevent patients from
further deterioration while often waiting several weeks
before undergoing invasive intervention, and thereby re-
duce complications and length of hospital stay and im-
prove patient quality of life. In the control group, the
effect of antibiotics is awaited while letting the (extra) Patients are randomized to undergo either immediate
(< 24 h) or postponed (in walled-off necrosis) catheter
drainage of infected collections. Discussion According to current evidence-based international
guidelines [7, 8], suspected or proven infected necrosis
in patients with clinical signs of infection is an indication
for invasive intervention. There should be a strong re-
luctance towards intervening in sterile collections [7,
28]. Currently, intervention is advised when the infected
necrosis has become walled-off, which occurs typically 4
weeks after the onset of disease [29]. This practice of
postponing interventions until the stage of walled-off ne-
crosis is based on literature published when open necro-
sectomy was the standard intervention [10, 30, 31]. Early
necrosectomy is now recognized as having a major im-
pact in the critically ill patient, whereas postponed
necrosectomy allows the immune system to recover
from the pro-inflammatory response due to pancreatitis. Since the step-up approach is now considered stand-
ard of care, the issue of the optimal timing of catheter
drainage has become highly relevant. Current literature
reports that 35–64% of patients with infected necrotiz-
ing pancreatitis can be treated with catheter drainage
alone, without the need for invasive necrosectomy [3, 5]. Therefore, several expert pancreatologists have stated
that they already practice immediate catheter drainage
in patients with infected necrosis. In a recent inter-
national survey performed in preparation for this study,
55% of expert pancreatologists stated that they typically
postpone catheter drainage by using antibiotics, whereas
the other 45% proclaimed to drain immediately after
diagnosing infected necrosis [17]. Thus, in practice im-
mediate catheter drainage is already being performed in
individual patients, regardless of the effect of antibiotics
alone or the degree of encapsulation: (percutaneous)
catheter drainage in infected necrosis has been described
retrospectively in several cohorts [32] at a median of 2
weeks (9–15 days) after the onset of disease instead of at
4 weeks (28 days). Patient outcomes were significantly better in the According to current evidence-based international
guidelines [7, 8], suspected or proven infected necrosis
in patients with clinical signs of infection is an indication
for invasive intervention. There should be a strong re-
luctance towards intervening in sterile collections [7,
28]. Currently, intervention is advised when the infected
necrosis has become walled-off, which occurs typically 4
weeks after the onset of disease [29]. This practice of
postponing interventions until the stage of walled-off ne-
crosis is based on literature published when open necro-
sectomy was the standard intervention [10, 30, 31]. Discussion Infected necrosis is a potentially lethal complication of
acute pancreatitis, typically requiring invasive interven-
tion. The treatment of infected necrotizing pancreatitis
is associated with lengthy hospital stay and high costs. Grinsven et al. Trials (2019) 20:239 Page 7 of 10 Page 7 of 10 Page 7 of 10 Grinsven et al. Trials (2019) 20:239 The POINTER trial is the first randomized controlled
trial designed to determine the optimal timing of cath-
eter drainage in infected necrotizing pancreatitis: i.e. immediate or postponed, once walled-off necrosis has
occurred. pancreatic necrotic collections become walled-off. Anti-
biotic treatment alone may suffice in a small minority of
patients [4], which is another possible benefit for the
control group. g
p
Infected necrosis can be diagnosed on imaging (e.g. CECT) by the presence of gas in the (extra) pancreatic
necrotic collection, irrespective of the source of the gas
(i.e. through gas-forming bacteria or loss of integrity of
the gastrointestinal tract). Collections with gas are seen
in up to 42% of patients with infected necrosis [33] and
can occur in every phase of the disease [34, 35]. Infected
necrosis can also be confirmed by a positive gram stain
or culture gathered with FNA. In a recent study, infected
necrosis was confirmed by FNA in 86% (of 28 patients),
which was similar to diagnosis based on clinical symp-
toms (80% of 92 patients) or gas identified on imaging
(94% of 88 patients) [33]. Until recently, FNA was not
routinely used in the Netherlands for diagnosing in-
fected necrosis [17], as its outcome did not influence
treatment because invasive intervention was postponed
until the stage of walled-off necrosis, even in the case of
a positive culture. In the POINTER trial, however, it is
pivotal to detect infected necrosis as early as possible so
as to perform immediate drainage (group A). Differenti-
ating SIRS from sepsis is, however, very difficult in the
first 14 days of the disease. Therefore, in the absence of
gas on imaging but with clinical signs of infection in the
first 14 days, a positive gram stain or culture after FNA
is obligatory prior to randomization. Since the false
negative rate of FNA is relatively high [33], a second
FNA is advised in patients with persistent deterioration
and a primary negative FNA. Clinical signs alone are suf-
ficient to diagnose (suspected) infected necrotizing pan-
creatitis after the first 14 days [3]. Discussion Both the surgical and
the endoscopic step-up approach are allowed, depending
on the location of the necrotic collection(s), the extent
of encapsulation and the preference of the treating phys-
ician. It is known that both approaches are effective and
safe and that not all (peri) pancreatic collections are ap-
proachable using a single technique [6, 9]. The CCI [20, 21] is the primary endpoint of the trial. Patients with infected necrotizing pancreatitis often have
a long disease course with multiple complications, and
therefore the CCI score is considered a representative Grinsven et al. Trials (2019) 20:239 Grinsven et al. Trials (2019) 20:239 Page 8 of 10 Page 8 of 10 tool to take into account all these complications. The in-
dividual complications (e.g. organ failure and mortality),
number of (repeat) interventions, hospital and ICU
lengths of stay, QALYs and direct and indirect costs are
also analyzed as secondary end points. MG Besselink, MD PhD, dept. of surgery, Amsterdam UMC, University of
Amsterdam (chair); HC van Santvoort, MD PhD, dept. of surgery, St. Antonius
Hospital Nieuwegein and University Medical Center Utrecht; J van Grinsven,
MD PhD, dept. of surgery, Amsterdam UMC, University of Amsterdam and St. Antonius Hospital Nieuwegein; SM van Dijk, MD, dept. of surgery, Amsterdam
UMC, University of Amsterdam and St. Antonius Hospital Nieuwegein; MA
Boermeester, MD PhD, dept. surgery, Amsterdam UMC, University of Amsterdam;
TL Bollen, MD PhD, dept. of radiology, St. Antonius Hospital Nieuwegein; MJ
Bruno, MD PhD, dept. of gastroenterology and hepatology, Erasmus MC
University Medical Center Rotterdam; MG Dijkgraaf, PhD, clinical research unit,
Amsterdam UMC, University of Amsterdam; KP van Lienden, MD PhD, dept. of
radiology, Amsterdam UMC, University of Amsterdam; P Fockens, MD, PhD, dept. of gastroenterology and hepatology, Amsterdam UMC, University of Amsterdam. Data safety monitoring committee Patients are stratified based on organ failure at baseline,
since it is known that patients with organ failure have
poorer outcomes, as compared to patients with no organ
failure. Patients are also stratified on disease duration (cut-
off 20 days) since this is obviously related to the onset of
walled-off necrosis. Finally, patients are stratified on ex-
pected high versus low volumes of patient inclusion. JPH Drenth, MD PhD, dept. of gastroenterology and hepatology, Radboud
University Medical Center Nijmegen (chair); DJ Gouma, MD PhD, dept. of surgery,
Amsterdam UMC, University of Amsterdam; LPS Stassen, MD PhD, dept. Trial status The
trial
was
registered
on
6
August
2015
as
ISRCTN33682933 (http://www.isrctn.com/ISRCTN33682933). The first patient was randomized on 4 August 2015. To date, 88 of the 104 patients have been randomized
and the inclusion of patients is on schedule. The
trial
was
registered
on
6
August
2015
as
ISRCTN33682933 (http://www.isrctn.com/ISRCTN33682933). Discussion of
surgery, Maastricht University Medical Center; J Stoker, MD PhD, dept. of radiology,
Amsterdam UMC, University of Amsterdam; S van Dieren, MD PhD,
epidemiologist, Amsterdam UMC, University of Amsterdam. In conclusion, the POINTER trial is a multicenter ran-
domized controlled trial that investigates whether imme-
diate catheter drainage reduces the CCI in patients with
infected necrotizing pancreatitis, as compared to post-
poned catheter drainage. Acute pancreatitis expert panel MA Boermeester, MD PhD, dept. of surgery, Amsterdam UMC, University of
Amsterdam; TL Bollen, MD PhD, dept. of radiology, St Antonius Hospital
Nieuwegein; MJ Bruno, MD PhD, dept. of gastroenterology and hepatology,
Erasmus MC University Medical Center Rotterdam; VC Cappendijk, MD PhD, dept. of radiology, Jeroen Bosch Hospital ‘s-Hertogenbosch; CH Dejong, MD PhD, dept. of surgery, Maastricht University Medical Center; CH van Eijck, MD PhD, dept. of
surgery, Erasmus MC University Medical Center Rotterdam; P Fockens, MD PhD,
dept. of gastroenterology and hepatology, Amsterdam UMC, University of
Amsterdam; H van Goor, MD PhD, dept. of surgery, Radboud University Medical
Center Nijmegen; MG Besselink, MD PhD, dept. of surgery, Amsterdam UMC,
University of Amsterdam; JW Haveman, MD PhD, dept. of surgery, University
Medical Center Groningen, University of Groningen; HS Hofker, MD PhD, dept. of
surgery, University Medical Center Groningen, University of Groningen; JS Laméris,
MD PhD, dept. of radiology, Amsterdam UMC, University of Amsterdam; KP van
Lienden, MD PhD, dept. of radiology, Amsterdam UMC, University of Amsterdam;
VB Nieuwenhuijs, MD PhD, dept. of surgery, Isala Clinics Zwolle; JW Poley, MD
PhD, dept. of gastroenterology and hepatology, Erasmus MC University Medical
Center Rotterdam. Additional files Antonius Hospital Nieuwegein and University Medical Center Utrecht (co-
principal investigator); J van Grinsven, MD PhD, dept. of surgery, Amsterdam UMC,
University of Amsterdam and St. Antonius Hospital Nieuwegein (study
coordinator); SM van Dijk, MD, dept. of surgery, Amsterdam UMC, University of
Amsterdam and St. Antonius Hospital Nieuwegein (study coordinator). Clinical centers and principal investigators (all in the Netherlands)
1. Amsterdam UMC, University of Amsterdam, PO 22660, 1100 DD
Amsterdam; MG Besselink, dept. of surgery. 2. Albert Schweitzer Hospital, PO 444, 3300 AK Dordrecht; W van de Vrie,
dept. of gastroenterology and hepatology. 3. Amphia Hospital Breda, PO 90158, 4800 RK Breda; T Seerden, dept. of
gastroenterology and hepatology. 4. Catharina Hospital, PO 1350, 5623 EJ Eindhoven; EJ Schoon, dept. of
gastroenterology and hepatology. 5. Erasmus MC University Medical Center, PO 2040, 3000 CA Rotterdam; MJ
Bruno, dept. of gastroenterology and hepatology. 6. Gelre Hospital, PO 9014, 7300 DS Apeldoorn; P van Duijvendijk, dept. of surgery. 7. Hospital Gelderse Vallei, PO 9025, 6710 HN Ede; BJ Witteman, dept. of
gastroenterology and hepatology. 8. Isala Clinics, PO 10400, 8000 GK Zwolle; AC Poen, dept. of Additional file 7: Relevant definitions. (DOCX 17 kb) Abbreviations APA: American Pancreatic Association; APACHE: Acute Physiology and
Chronic Health Evaluation; ASA: American Society of Anesthesiologist’s
classification; CCI: Comprehensive Complication Index; CECT: Contrast
enhanced computed tomography; CRP: C-reactive protein; CTSI: Computer
Tomography Severity Index; DPSG: Dutch Pancreatitis Study Group;
DSMC: Data Safety Monitoring Committee; FNA: Fine needle aspiration;
GCP: Good Clinical Practice; IAP: International Association of Pancreatology;
ICU: Intensive care unit; iMCQ: iMedical Consumption Questionnaire;
iPCQ: iProductivity Cost Questionnaire; MODS: Multiple Organ Dysfunction
Score; MRI: Magnetic resonance imaging; QALY: Quality-adjusted life year;
SAE: Serious adverse event; SD: Standard deviation; SIRS: Systemic
inflammatory response syndrome; SOFA: Sequential Organ Failure
Assessment; SSI: Surgical site infection; VARD: Videoscopic assisted
retroperitoneal debridement; WMO: Medical research involving human
subjects act (in Dutch, Wet Medisch-wetenschappelijk Onderzoek met mensen) Acknowledgements
Steering/adjudication committee Additional files Additional file 1: SPIRIT checklist. (DOCX 46 kb)
Additional file 2: Table S1. Inclusion and exclusion criteria. (DOCX 14 kb)
Additional file 3: Table S2. Criteria for infected necrotizing pancreatitis. (DOCX 14 kb)
Additional file 4: Figure S1. Inclusion and randomization flowchart. (DOCX 33 kb)
Additional file 5: Figure S2. Surgical step-up approach [32, 37, 38]. (DOCX 253 kb)
Additional file 6: Figure S3. Endoscopic step-up approach [32, 37, 38]. (DOCX 169 kb)
Additional file 7: Relevant definitions. (DOCX 17 kb) AF Schaapherder, MD PhD, dept. of surgery, Leids University Medical Center;
R Timmer, MD PhD, dept. of gastroenterology and hepatology, St Antonius
Hospital Nieuwegein; HC van Santvoort, MD PhD, dept. of surgery, St
Antonius Hospital Nieuwegein and University Medical Center Utrecht. Key staff at coordinating centers y
g
MG Besselink, MD PhD, dept. of surgery, Amsterdam UMC, University of
Amsterdam (principal investigator); HC van Santvoort, MD PhD, dept. of surgery,
St. Antonius Hospital Nieuwegein and University Medical Center Utrecht (co-
principal investigator); J van Grinsven, MD PhD, dept. of surgery, Amsterdam UMC,
University of Amsterdam and St. Antonius Hospital Nieuwegein (study
coordinator); SM van Dijk, MD, dept. of surgery, Amsterdam UMC, University of
Amsterdam and St. Antonius Hospital Nieuwegein (study coordinator). Clinical centers and principal investigators (all in the Netherlands)
1. Amsterdam UMC, University of Amsterdam, PO 22660, 1100 DD
Amsterdam; MG Besselink, dept. of surgery. 2. Albert Schweitzer Hospital, PO 444, 3300 AK Dordrecht; W van de Vrie,
dept. of gastroenterology and hepatology. 3. Amphia Hospital Breda, PO 90158, 4800 RK Breda; T Seerden, dept. of
gastroenterology and hepatology. 4. Catharina Hospital, PO 1350, 5623 EJ Eindhoven; EJ Schoon, dept. of
gastroenterology and hepatology. 5. Erasmus MC University Medical Center, PO 2040, 3000 CA Rotterdam; MJ
Bruno, dept. of gastroenterology and hepatology. 6. Gelre Hospital, PO 9014, 7300 DS Apeldoorn; P van Duijvendijk, dept. of surgery. 7. Hospital Gelderse Vallei, PO 9025, 6710 HN Ede; BJ Witteman, dept. of
gastroenterology and hepatology. 8. Isala Clinics, PO 10400, 8000 GK Zwolle; AC Poen, dept. of
gastroenterology and hepatology. 9. Jeroen Bosch Hospital, PO 90153, 5200 ME 's-Hertogenbosch; DJ Lips,
dept. of surgery. 10. Spaarne Gasthuis, PO 417, 2000 AK Haarlem; RW van der Hulst, dept. of
gastroenterology and hepatology MG Besselink, MD PhD, dept. of surgery, Amsterdam UMC, University of
Amsterdam (principal investigator); HC van Santvoort, MD PhD, dept. of surgery,
St. Steering/adjudication committee Competing interests 15. Meander Medical Center, PO 1502, 3800 BM, Amersfoort; MP Schwartz, dept. of gastroenterology and hepatology. The authors declare that they have no competing interests. 16. Medical Center Leeuwarden, PO 888, 8901 BR Leeuwarden; E Manusama, dept. of surgery. Funding
Th POINT The POINTER trial is an investigator-initiated trial. Financial support is provided by
Fonds NutsOhra (grant number 1404–044), the Netherlands and the Amsterdam
UMC, University of Amsterdam, the Netherlands. The Sponsors have no influence
on the design of the study, data collection, results or publication. of Surgery, Jeroen Bosch Hospital, s Hertogenbosch, Netherlands. 18Department of Surgery, Medical Center Leeuwarden, Leeuwarden,
Netherlands. 19Department of Surgery, Amsterdam UMC, Vrije Universiteit
Amsterdam, Amsterdam, Netherlands. 20Department of Gastroenterology and
Hepatology, Isala Clinics Zwolle, Zwolle, Netherlands. 21Department of
Gastroenterology and Hepatology, Reinier de Graaf Gasthuis Delft, Delft,
Netherlands. 22Department of Surgery, Leids University Medical Center
Leiden, Leiden, Netherlands. 23Department of Gastroenterology and
Hepatology, Catharina Hospital Eindhoven, Eindhoven, Netherlands. 24Department of Gastroenterology and Hepatology, Meander Medical Center
Amersfoort, Amersfoort, Netherlands. 25Department of Gastroenterology and
Hepatology, Amphia Hospital Breda, Breda, Netherlands. 26Department of
Gastroenterology and Hepatology, Rijnstate Hospital Arnhem, Arnhem,
Netherlands. 27Department of Gastroenterology and Hepatology, Maxima
Medical Center Veldhoven, Veldhoven, Netherlands. 28Department of
Gastroenterology and Hepatology, Medisch Spectrum Twente Enschede,
Enschede, Netherlands. 29Department of Gastroenterology and Hepatology,
Albert Schweitzer Hospital Dordrecht, Dordrecht, Netherlands. 30Department
of Gastroenterology and Hepatology, Hospital Gelderse Vallei Ede, Ede,
Netherlands. 31Department of Surgery, Radboud University Medical Center
Nijmegen, Nijmegen, Netherlands. Publisher’s Note
S
i
N
i 17. Medisch Spectrum Twente, PO 50000, 7500 KA Enschede; NG Venneman, MD PhD, dept. of gastroenterology and hepatology Springer Nature remains neutral with regard to jurisdictional claims in published
maps and institutional affiliations. 18. OLVG Amsterdam, PO 95500, 1090 HM Amsterda gastroenterology and hepatology. 19. Radboud University Nijmegen Medical Cen . Radboud University Nijmegen Medical Center, PO 9101, 6500 HB Authors’ contributions JvG drafted the manuscript. SMvD, HCvS and MGB co-authored the writing
of the manuscript. JvG, HCvS, MAB, TLB, MJB, SvB, CHD, MGD, CHvE, KPvL, PF
and MGB participated in the design of the study during several meetings of
the Dutch Pancreatitis Study Group. JvG, MGB and MGD calculated the sam-
ple size. All authors critically assessed the study design, edited the manu-
script and read and approved the final manuscript. Availability of data and materials Patients are coded by a numeric randomization code (anonymized) and the
principal investigators are the only ones to have access to this code. The
source data are kept by the project leader for 15 years at the datacenter of
the DPSG. All data generated or analyzed during this study will be included
in the published results. Consent for publication 13. Maastricht University Medical Center, PO 5800, 6202 AZ Maastricht; CH
Dejong, dept. of surgery. Not applicable, the study results will not contain any data from any
individual person. Not applicable, the study results will not contain any data from any
individual person. Dejong, dept. of surgery. 14. Maxima Medical Center, PO 7777, 5500 MB Veldhoven; JW Straathof, dept. of gastroenterology and hepatology. of gastroenterology and hepatology. 1.
Peery AF, Dellon ES, Lund J, Crockett SD, McGowan CE, Bulsiewicz WJ, et al.
Burden of gastrointestinal diseases in the United States: 2012 update.
Gastroenterology. 2012;143:1179–87. Author details
1 6Department
of Gastroenterology and Hepatology, Amsterdam UMC, University of
Amsterdam, Amsterdam, Netherlands. 7Department of Surgery, University
Medical Center Utrecht, Cancer Center, Utrecht, Netherlands. 8Department of
Surgery, Maastricht University Medical Center+, Maastricht, Netherlands. 9NUTRIM School for Nutrition and Translational Research in Metabolism,
Maastricht, Netherlands. 10Department of Surgery, Erasmus MC University
Medical Center Rotterdam, Rotterdam, Netherlands. 11Department of
Radiology, Amsterdam UMC, University of Amsterdam, Amsterdam,
Netherlands. 12Department of Surgery, Gelre Hospital Apeldoorn, Apeldoorn,
Netherlands. 13Department of Gastroenterology and Hepatology, Maasstad
Hospital Rotterdam, Rotterdam, Netherlands. 14Department of Surgery,
University Medical Center Groningen, University of Groningen, Groningen,
Netherlands. 15Department of Gastroenterology and Hepatology, Spaarne
Gasthuis Haarlem, Haarlem, Netherlands. 16Department of Gastroenterology
and Hepatology, OLVG Amsterdam, Amsterdam, Netherlands. 17Department
of Surgery, Jeroen Bosch Hospital, ’s-Hertogenbosch, Netherlands. 18D
f S
M di
l C
L
d
L
d y
20. Reinier de Graaf Gasthuis, PO 5011, 2600 GA Delft; R Quispel, dept. of 20. Reinier de Graaf Gasthuis, PO 5011, 26 gastroenterology and hepatology. g
gy
gy
21. Rijnstate Hospital, PO 9555, 6800 TA Arnhem; BMW Spanier, dept. of 22. St Antonius Hospital, PO 2500, 3430 EM Nieuwegein; D Boerma, dept. of
surgery. 23. University Medical Center Groningen, PO 30001, 9700 RB Groningen; JW
Haveman, dept. of surgery. g y
24. University Medical Center Utrecht, PO 85500, 3508 GA Utrecht; IQ 24. University Medical Center Utrecht, PO 85500, 3508 GA Utrecht; IQ
Molenaar, dept. of surgery. 25. Amsterdam UMC, Vrije Universiteit van Amsterdam, PO 7057, 1007 MB
Amsterdam; DL van der Peet, dept. of surgery. Independent physician PJ Tanis, MD PhD, dept. of surgery, Amsterdam UMC, University of
A
t d PJ Tanis, MD PhD, dept. of surgery, Amsterdam UMC, University of
A
t d PJ Tanis, MD PhD, dept. of surgery, Amsterdam UMC, University Author details
1 Author details
1Department of Surgery, Amsterdam UMC, University of Amsterdam, G4.196,
PO Box 26000, 1105 AZ Amsterdam, Netherlands. 2Department of Surgery, St. Antonius Hospital Nieuwegein, Nieuwegein, Netherlands. 3Clinical Research
Unit, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands. 4Department of Radiology, St. Antonius Hospital Nieuwegein, Nieuwegein,
Netherlands. 5Department of Gastroenterology and Hepatology, Erasmus MC
University Medical Center Rotterdam, Rottedam, Netherlands. 6Department
of Gastroenterology and Hepatology, Amsterdam UMC, University of
Amsterdam, Amsterdam, Netherlands. 7Department of Surgery, University
Medical Center Utrecht, Cancer Center, Utrecht, Netherlands. 8Department of
Surgery, Maastricht University Medical Center+, Maastricht, Netherlands. 9NUTRIM School for Nutrition and Translational Research in Metabolism,
Maastricht, Netherlands. 10Department of Surgery, Erasmus MC University
Medical Center Rotterdam, Rotterdam, Netherlands. 11Department of
Radiology, Amsterdam UMC, University of Amsterdam, Amsterdam,
Netherlands. 12Department of Surgery, Gelre Hospital Apeldoorn, Apeldoorn,
Netherlands. 13Department of Gastroenterology and Hepatology, Maasstad
Hospital Rotterdam, Rotterdam, Netherlands. 14Department of Surgery,
University Medical Center Groningen, University of Groningen, Groningen,
Netherlands. 15Department of Gastroenterology and Hepatology, Spaarne
Gasthuis Haarlem, Haarlem, Netherlands. 16Department of Gastroenterology
and Hepatology, OLVG Amsterdam, Amsterdam, Netherlands. 17Department
of Surgery, Jeroen Bosch Hospital, ’s-Hertogenbosch, Netherlands. 18Department of Surgery, Medical Center Leeuwarden, Leeuwarden,
Netherlands. 19Department of Surgery, Amsterdam UMC, Vrije Universiteit
Amsterdam, Amsterdam, Netherlands. 20Department of Gastroenterology and
Hepatology, Isala Clinics Zwolle, Zwolle, Netherlands. 21Department of
Gastroenterology and Hepatology, Reinier de Graaf Gasthuis Delft, Delft,
Netherlands. 22Department of Surgery, Leids University Medical Center
Leiden, Leiden, Netherlands. 23Department of Gastroenterology and
Hepatology, Catharina Hospital Eindhoven, Eindhoven, Netherlands. 24Department of Gastroenterology and Hepatology, Meander Medical Center
Amersfoort, Amersfoort, Netherlands. 25Department of Gastroenterology and
Hepatology, Amphia Hospital Breda, Breda, Netherlands. 26Department of
Gastroenterology and Hepatology, Rijnstate Hospital Arnhem, Arnhem,
Netherlands. 27Department of Gastroenterology and Hepatology, Maxima
Medical Center Veldhoven, Veldhoven, Netherlands. 28Department of
Gastroenterology and Hepatology, Medisch Spectrum Twente Enschede,
Enschede, Netherlands. 29Department of Gastroenterology and Hepatology,
Albert Schweitzer Hospital Dordrecht, Dordrecht, Netherlands. 30Department
of Gastroenterology and Hepatology, Hospital Gelderse Vallei Ede, Ede,
Netherlands. 31Department of Surgery, Radboud University Medical Center
Nijmegen, Nijmegen, Netherlands. Nijmegen; H van Goor, dept. of surgery. 1Department of Surgery, Amsterdam UMC, University of Amsterdam, G4.196,
PO Box 26000, 1105 AZ Amsterdam, Netherlands. 2Department of Surgery, St. Antonius Hospital Nieuwegein, Nieuwegein, Netherlands. 3Clinical Research
Unit, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands. 4Department of Radiology, St. Antonius Hospital Nieuwegein, Nieuwegein,
Netherlands. 5Department of Gastroenterology and Hepatology, Erasmus MC
University Medical Center Rotterdam, Rottedam, Netherlands. Acknowledgements
S
i
/ dj di
i 10. Spaarne Gasthuis, PO 417, 2000 AK Haarlem; RW van der Hulst, dept. of
gastroenterology and hepatology. Steering/adjudication committee Page 9 of 10 Page 9 of 10 Grinsven et al. Trials (2019) 20:239 Grinsven et al. Trials (2019) 20:239 11. Leids University Medical Center, PO 9600, 2300 RC Leiden; AF
Schaapherder, dept. of surgery. 12. Maasstad Hospital Rotterdam, PO 9100, 3007 AC Rotterdam; M Hadithi,
dept. of gastroenterology and hepatology. 13. Maastricht University Medical Center, PO 5800, 6202 AZ Maastricht; CH
Dejong, dept. of surgery. 14. Maxima Medical Center, PO 7777, 5500 MB Veldhoven; JW Straathof, dept. of gastroenterology and hepatology. 15. Meander Medical Center, PO 1502, 3800 BM, Amersfoort; MP Schwartz,
dept. of gastroenterology and hepatology. 16. Medical Center Leeuwarden, PO 888, 8901 BR Leeuwarden; ER
Manusama, dept. of surgery. 17. Medisch Spectrum Twente, PO 50000, 7500 KA Enschede; NG Venneman,
MD PhD, dept. of gastroenterology and hepatology. 18. OLVG Amsterdam, PO 95500, 1090 HM Amsterdam; JM Jansen, dept. of
gastroenterology and hepatology. 19. Radboud University Nijmegen Medical Center, PO 9101, 6500 HB
Nijmegen; H van Goor, dept. of surgery. 20. Reinier de Graaf Gasthuis, PO 5011, 2600 GA Delft; R Quispel, dept. of
gastroenterology and hepatology. 21. Rijnstate Hospital, PO 9555, 6800 TA Arnhem; BMW Spanier, dept. of
gastroenterology and hepatology. 22. St Antonius Hospital, PO 2500, 3430 EM Nieuwegein; D Boerma, dept. of
surgery. 23. University Medical Center Groningen, PO 30001, 9700 RB Groningen; JW
Haveman, dept. of surgery. 24. University Medical Center Utrecht, PO 85500, 3508 GA Utrecht; IQ
Molenaar, dept. of surgery. 25. Amsterdam UMC, Vrije Universiteit van Amsterdam, PO 7057, 1007 MB
Amsterdam; DL van der Peet, dept. of surgery. Independent physician
PJ Tanis, MD PhD, dept. of surgery, Amsterdam UMC, University of
Amsterdam. representatives. This applies only to patients who are temporarily
incapacitated because of the severity of pancreatitis. representatives. This applies only to patients who are temporarily
incapacitated because of the severity of pancreatitis. 11. Leids University Medical Center, PO 9600, 2300 RC Leiden; A Schaapherder, dept. of surgery. 12. Maasstad Hospital Rotterdam, PO 9100, 3007 AC Rotterdam; M Hadithi,
dept. of gastroenterology and hepatology. dept. of gastroenterology and hepatology. Ethics approval and consent to participate The study is conducted according to the principles of the Declaration of Helsinki
(59th version, October 2008) and in accordance with Dutch Law on research
with humans (Medical Research Involving Human Subjects Act (WMO)). Central
ethical approval has been confirmed from the medical research ethics
committee of the Academic Medical Center Amsterdam on 19 June 2015 (METC
2015_033, NL52361.018.15) and we will not begin recruiting at other centers in
the trial until local ethical approval has been obtained. Important protocol
modifications will be communicated to the central and local ethics committees. Patients are preferably recruited by the principal investigators. If this is not
possible for practical reasons, he/she is replaced by a designated substitute
(the study coordinator, local treating physicians or a study nurse) who is fully
informed and aware of the study procedures and requirements. Written
informed consent is obtained from each participant. In the case of
incapacitated patients, informed consent is obtained from the patients’ legal Received: 13 November 2018 Accepted: 21 March 2019 2.
Banks PA, Freeman ML. Practice Parameters Committee of the American
College of Gastroenterology. Practice guidelines in acute pancreatitis. Am J
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drainage size matter? World J Gastroenterol. 2008;14:725–30. 33. Van Baal MC, Bollen TL, Bakker OJ, van Goor H, Boermeester MA, Dejong
CH, et al. The role of routine fine-needle aspiration in the diagnosis of
infected necrotizing pancreatitis. Surgery. 2014;155:442–8. 12. Freeny PC, Hauptmann E, Althaus SJ, Traverso LW, Sinanan M. Percutaneous
CT-guided catheter drainage of infected acute necrotizing pancreatitis:
techniques and results. AJR Am J Roentgenol. 1998;170:969–75. 34. Besselink MG, van Santvoort HC, Boermeester MA, Nieuwenhuijs VB, van
Goor H, Dejong CH, et al. Timing and impact of infections in acute
pancreatitis. Br J Surg. 2009;96:267–73. 13. Lee JK, Kwak KK, Park JK, Yoon WJ, Lee SH, Ryu JK, et al. The efficacy of
nonsurgical treatment of infected pancreatic necrosis. Pancreas. 2007;34:
399–404. 35. Van Grinsven J, van Brunschot S, van Baal MC, Besselink MG, Fockens P, van
Goor H, et al. Natural history of gas configurations and encapsulation in
necrotic collections during necrotizing pancreatitis. J Gastrointest Surg. 2018;22:1557–64. 14. Mortelé KJ, Girshman J, Szejnfeld D, Ashley SW, Erturk SM, Banks PA, et
al. CT-guided percutaneous catheter drainage of acute necrotizing
pancreatitis: clinical experience and observations in patients with sterile
and infected necrosis. AJR Am J Roentgenol. 2009;192:110–6. 36. Schneider A, Löhr JM, Singer MV. The M-ANNHEIM classification of
chronic pancreatitis: introduction of a unifying classification system
based on a review of previous classifications of the disease. J
Gastroenterol. 2007;42:101–19. 15. References Zerem E, Imamović G, Sušić A, Haračić B. Step-up approach to infected
necrotising pancreatitis: a 20-year experience of percutaneous drainage in a
single centre. Dig Liver Dis. 2011;43:478–83. 37. Van Brunschot S, Bakker OJ, Besselink MG, Bollen TL, Fockens P, Gooszen
HG, et al. Treatment of necrotizing pancreatitis. Clin Gastroenterol Hepatol. 2012;10:1190–201. 16. Poon RT, Fan ST, Lo CM, Ng KK, Yuen WK, Yeung C, et al. External drainage
of pancreatic duct with a stent to reduce leakage rate of
pancreaticojejunostomy after pancreaticoduodenectomy a prospective
randomized trial. Ann Surg. 2007;246:425–35. 38. Da Costa DW, Boerma D, van Santvoort HC, Horvath KD, Werner J, Carter
CR, et al. Staged multidisciplinary step-up management for necrotizing
pancreatitis. Br J Surg. 2014;101:e65–79. 17. Van Grinsven J, van Brunschot S, Bakker OJ, Bollen TL, Boermeester MA,
Bruno MJ, et al. Diagnostic strategy and timing of intervention in
infected necrotizing pancreatitis: an international expert survey and
case vignette study. HPB (Oxford). 2016;18:49–56. 18. Chan AW, Tetzlaff JM, Gøtzsche PC, Altman DG, Mann H, Berlin JA, et al. SPIRIT 2013 explanation and elaboration: guidance for protocols of clinical
trials. BMJ. 2013;346:e7586. 19. Van Grinsven J, van Brunschot S, van Santvoort HC, Dutch Pancreatitis Study
Group. The value of a 24/7 online nationwide multidisciplinary expert panel
for acute necrotizing pancreatitis. Gastroenterology. 2017;152:685–8 e6. 20. Slankamenac K, Graf R, Barkun J, Puhan MA, Clavien PA. The comprehensive
complication index: a novel continuous scale to measure surgical morbidity. Ann Surg. 2013;258:1–7. 21. Slankamenac K, Nederlof N, Pessaux P, de Jonge J, Wijnhoven BP, Breitenstein S,
et al. The comprehensive complication index: a novel and more sensitive
endpoint for assessing outcome and reducing sample size in randomized
controlled trials. Ann Surg. 2014;260:757–62. 22. Ware JE Jr, Sherbourne CD. The MOS 36-item short-form health survey (SF-
36). I. Conceptual framework and item selection. Med Care. 1992;30:473–83. 23. Brooks R. EuroQol: the current state of play. Health Policy. 1996;37(1):53–72. 23. Brooks R. EuroQol: the current state of play. Health Policy. 1996;37(1):53–72. 24. Bouwmans C, Hakkaart- van Roijen L, Koopmanschap M, Krol M, Severens H,
Brouwer W. Handleiding iMTA Medical Consumption Questionnaire (iMCQ). Rotterdam: iMTA, Erasmus Universiteit; 2013. Q
p y
y
;
( )
24. Bouwmans C, Hakkaart- van Roijen L, Koopmanschap M, Krol M, Severens H,
Brouwer W. Handleiding iMTA Medical Consumption Questionnaire (iMCQ). Rotterdam: iMTA, Erasmus Universiteit; 2013.
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https://europepmc.org/articles/pmc2971811?pdf=render
|
Latin
| null |
Ethyl 2-(3,3-dibutylthioureido)-4,5,6,7-tetrahydrobenzo[<i>b</i>]thiophene-3-carboxylate
|
Acta crystallographica. Section E
| 2,009
|
cc-by
| 4,756
|
organic compounds = 0.26 mm1
T = 292 K
0.36 0.30 0.25 mm Acta Crystallographica Section E
Structure Reports
Online
ISSN 1600-5368 Acta Crystallographica Section E
Structure Reports
Online
ISSN 1600-5368 = 107.711 (12)
V = 2189.8 (4) A˚ 3
Z = 4
Mo K radiation
Data collection
Bruker SMART CCD area-detector
diffractometer
Absorption correction: multi-scan
(SADABS; Sheldrick, 1996)
Tmin = 0.903, Tmax = 0.938 Data collection
Bruker SMART CCD area-detector
diffractometer
Absorption correction: multi-scan
(SADABS; Sheldrick, 1996)
Tmin = 0.903, Tmax = 0.938 12194 measured reflections
4036 independent reflections
3460 reflections with I > 2(I)
Rint = 0.027 12194 measured reflections
4036 independent reflections
3460 reflections with I > 2(I)
Rint = 0.027 Table 1
Hydrogen-bond geometry (A˚ , ). Key indicators: single-crystal X-ray study; T = 292 K; mean (C–C) = 0.006 A˚;
disorder in main residue; R factor = 0.073; wR factor = 0.153; data-to-parameter
ratio = 14.4. D—H A
D—H
H A
D A
D—H A
N1—H1A O2
0.86 (3)
1.89 (2)
2.643 (4)
145 (3) In the title compound, C20H32N2O2S2, the cyclohexene ring is
disordered over two half-boat conformations with occupancy
factors of 0.71:0.29. One n-butyl chain is also disordered over
two positions with occupancy factors of 0.83:0.17. The
molecular conformation is stabilized by an intramolecular
N—H O hydrogen bond. Data collection: SMART (Bruker, 2001); cell refinement: SAINT
(Bruker, 2001); data reduction: SAINT; program(s) used to solve
structure: SHELXS97 (Sheldrick, 2008); program(s) used to refine
structure:
SHELXL97
(Sheldrick,
2008);
molecular
graphics:
PLATON (Spek, 2009); software used to prepare material for
publication: SHELXTL97 (Sheldrick, 2008). We gratefully acknowledge financial support of this work by
the
National
Basic
Research
Program
of
China
(2003CB114400), the National Natural Science Foundation of
China (20372023, 20102001), the Educational Commission of
Hubei Province of China (grant No. D20092406, B20092412),
the Shiyan Municipal Science and Technology Bureau (grant
No. 20061835), and Yunyang Medical College (grant Nos. 2007QDJ14, 2007QDJ15, 2007ZQB19). Related literature For the synthesis and biological activity of thienopyrimidin-
4(3H)-one derivatives, see: De Laszlo et al. (1992a,b); Taguchi
et al. (1993a,b); Walter (1999a,b); Ding et al. (2004); Santagati
et al. (2003); Abbott GmbH Co KG (2004a, 2004b); Walter &
Zeun (2004); Ford et al. (2004a,b); Duval et al. (2005);
Waehaelae et al. (2004a,b). For a description of the Cambridge
Structural Database, see: Allen (2002). For related structures,
see: Xu et al. (2005); Zeng et al. (2005, 2006, 2007, 2008, 2009);
Wang et al. (2007, 2008); Zheng et al. (2007); Xie et al. (2008). Supplementary data and figures for this paper are available from the
IUCr electronic archives (Reference: RZ2377). Experimental
Crystal data
C20H32N2O2S2
Mr = 396.60
Monoclinic, P21=c
a = 10.9311 (11) A˚
b = 21.715 (3) A˚
c = 9.6841 (3) A˚
o3094
Wang et al. doi:10.1107/S1600 Ethyl 2-(3,3-dibutylthioureido)-4,5,6,7-
tetrahydrobenzo[b]thiophene-3-
carboxylate Refinement
R[F 2 > 2(F 2)] = 0.073
wR(F 2) = 0.153
S = 1.21
4036 reflections
280 parameters
13 restraints Supplementary data and figures for this paper are available from the
IUCr electronic archives (Reference: RZ2377). Hong-Mei Wang, Jing Xu, Xiao-Hua Zeng* and Jia-Hua
Tian Hong-Mei Wang, Jing Xu, Xiao-Hua Zeng* and Jia-Hua
Tian H atoms treated by a mixture of
independent and constrained
refinement
max = 0.30 e A˚ 3
min = 0.23 e A˚ 3 Institute of Medicinal Chemistry, Yunyang Medical College, Shiyan 442000, People’s
Republic of China
Correspondence e-mail: zengken@126.com
Received 23 October 2009; accepted 9 November 2009 Institute of Medicinal Chemistry, Yunyang Medical College, Shiyan 442000, People’s
Republic of China
d
l
k Received 23 October 2009; accepted 9 November 2009 Received 23 October 2009; accepted 9 November 2009 Table 1
Hydrogen-bond geometry (A˚ , ). Sheldrick, G. M. (1996). SADABS. University of Go¨ttingen, Germany.
Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122.
Spek, A. L. (2009). Acta Cryst. D65, 148–155.
Taguchi, M., Ota, T. & Hatayama, K. (1993a). Chem. Abstr. 119, 160309m.
Taguchi, M., Ota, T. & Hatayama, K. (1993b). World Patent 9303040.
Waehaelae, K., Lilienkampf, A., Alho, S., Huhtinen, K., Johansson, N.,
Koskimies, P. & Vihko, K. (2004a). Chem. Abstr. 142, 74590.
Waehaelae, K., Lilienkampf, A., Alho, S., Huhtinen, K., Johansson, N.,
Koskimies, P. & Vihko, K. (2004b). World Patent 2004110459.
Walter, H. (1999a). Chem. Abstr. 130, 237580e.
Walter, H. (1999b). World Patent 9911631.
Walter, H. & Zeun, R. (2004). Chimia, 57, 692–696.
Wang, H.-M., Chen, L.-L., Hu, T. & Zeng, X.-H. (2008). Acta Cryst. E64,
o2404. References Abbott GmbH & Co. KG (2004a). Chem. Abstr. 141, 89095. (
)
Abbott GmbH & Co. KG (2004b). German Patent 10259382. Allen, F. H. (2002). Acta Cryst. B58, 380–388. Bruker (2001). SMART and SAINT. Bruker AXS Inc., Madison, Wisconsin,
USA. De Laszlo, S. E., Patchett, A. A., Allen, E. E. & Greenlee, W. J. (1992a). Chem. Abstr. 118, 22249v. De Laszlo, S. E., Patchett, A. A., Allen, E. E. & Greenlee, W. J. (1992b). Eur. Patent 502725. Ding, M. W., Xu, S. Z. & Zhao, J. F. (2004). J. Org. Chem. 69, 8366–8371. Duval, E., Case, A., Stein, R. L. & Cuny, G. D. (2005). Bioorg. Med. Chem. Lett. 15, 1885–1889. Experimental Ford, J., Palmer, N. J., Atherall, J. F., Madge, D. J., Sherborne, B., Bushfield, M. & Stevens, E. B. (2004a). Chem. Abstr. 142, 74599. Crystal data Ford, J., Palmer, N. J., Atherall, J. F., Madge, D. J., Sherborne, B., Bushfield, M. & Stevens, E. B. (2004b). World Patent 2004111057. & Stevens, E. B. (2004b). World Patent 2004111057. Santagati, A., Marrazzo, A. & Granata, G. (2003). J. Heterocycl. Chem. 40,
869–873. o3094
Wang et al. Acta Cryst. (2009). E65, o3094–o3095 Acta Cryst. (2009). E65, o3094–o3095 doi:10.1107/S1600536809047230 Acta Cryst. (2009). E65, o3094–o3095 Zheng, A.-H., Long, J.-Y., Zeng, X.-H. & Wang, H.-M. (2007). Acta Cryst. E63,
o1142–o1144. (
)
Walter, H. & Zeun, R. (2004). Chimia, 57, 692–696. Wang, H.-M., Chen, L.-L., Hu, T. & Zeng, X.-H. (2008). Acta Cryst. E64,
o2404. ,
(
)
y
,
Spek, A. L. (2009). Acta Cryst. D65, 148–155. organic compounds Wang, H.-M., Zeng, X.-H., Zheng, A.-H., Tian, J.-H. & He, T.-Y. (2007). Acta
Cryst. E63, o4365. Xie, H., Meng, S.-M., Fan, Y.-Q. & Guo, Y. (2008). Acta Cryst. E64, o2434. Xu, S.-Z., Cao, M.-H., Hu, Y.-G., Ding, M.-W. & Xiao, W.-J. (2005). Acta Cryst. E61, o2789–o2790. Zeng, X.-H., Deng, S.-H., Qu, Y.-N. & Wang, H.-M. (2009). Acta Cryst. E65,
o1142–o1143. Zeng, X.-H., Ding, M.-W. & He, H.-W. (2006). Acta Cryst. E62, o731–o732. g
g
(
)
y
Zeng, G., Li, Q. & Hu, Y. (2008). Acta Cryst. E64, o535. Zeng, G., Li, Q. & Hu, Y. (2008). Acta Cryst. E64, o535. Zeng, X.-H., Wang, H.-M., Luo, Z.-G., Ding, M.-W. & He, H.-W. (2005). Acta
Cryst. E61, o4160–o4161. ,
(
)
Walter, H. & Zeun, R. (2004). Chimia, 57, 692–696. Zeng, X.-H., Zhao, L.-H., Luo, H. & Long, J.-Y. (2007). Acta Cryst. E63, o3004. Zheng, A.-H., Long, J.-Y., Zeng, X.-H. & Wang, H.-M. (2007). Acta Cryst. E63,
o1142–o1144. Wang et al. C20H32N2O2S2
o3095 Acta Cryst. (2009). E65, o3094–o3095 Comment The derivatives of heterocycles containing the thienopyrimidine system, which are well known bioisosteres of quinazolines,
are of great importance because of their remarkable biological properties, including antimicrobial or antifungal activities (De
Laszlo et al., 1992a,b; Walter, 1999a,b; Ding et al., 2004; Walter & Zeun, 2004), significant 5-HT1A and 5-HT1B receptor
activities (Taguchi et al., 1993a,b; Abbott GmbH & Co. KG, 2004a,b), potential selective COX-2 enzyme inhibitor activity
(Santagati, et al., 2004), 17beta-hydroxysteroid dehydrogenase inhibitor activity (Waehaelae et al., 2004a,b), potassium
channel inhibitor activity (Ford et al., 2004a,b), and tissue transglutaminase inhibitor activity (Duval et al., 2005). Recently,
our group has been engaged in the preparation of derivatives of thienopyrimidin-4(3H)-one via aza-Wittig reaction of beta-
ethoxycarbonyl iminophosphorane with CS2. As a continuation of our research for new biologically active heterocycles, the
title compound was obtained as an intermediate product from beta-ethoxycarbonyl iminophosphorane in CS2 and structur-
ally characterized in order to elucidate the cyclization mechanism involved in the reaction. In the title compound (Fig. 1), bond lengths within the benzothiophene ring system are in good agreement with those
observed for closely related structures (Xu et al., 2005; Zeng et al., 2005, 2006, 2007, 2008, 2009; Wang et al., 2007, 2008;
Zheng et al., 2007; Xie et al., 2008), and in the ranges of values observed in previously reported structures in the Cambridge
Structural Database (Version 5.26; Allen, 2002). The theiophene ring is planar, with a maximum displacement of 0.008 (3)Å
for atom C8. The attached cyclohexene ring is disordered over two half-chair conformations with site occupancy factors of
0.71:0.29. A n-butyl chain is also disordered over two positions with site occupancy factors of 0.83:0.17. The molecular
conformation is stabilized by an intramolecular N—H···O hydrogen bond (Table 1). The crystal packing is enforced only
by van der Waals interactions. Ethyl 2-(3,3-dibutylthioureido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate H.-M. Wang, J. Xu, X.-H. Zeng and J.-H. Tian H.-M. Wang, J. Xu, X.-H. Zeng and J.-H. Tian H.-M. Wang, J. Xu, X.-H. Zeng and J.-H. Tian Experimental To a solution of ethyl 2-thiocyanato-4,5,6,7-tetrahydrobenzo[b]thiophene -3-carboxylate (3 mmol) prepared according to
Zeng et al. (2005) in CH3CN (15 ml) was added dibutylamine (3 mmol) at room temperature, and the reaction mixture was
stirred for 6 h. The solvent was removed under reduced pressure and the residue was recrystallized from EtOH to give the
title compound in yield of 81% (m.p. 442 K). Elemental analysis:calculated for C20H32N2O2S2: C, 60.57; H, 8.13; N, 7.06. Found: C, 59.34; H, 8.55; N, 6.67%. Crystals suitable for X-ray diffraction analysis were obtained by slow evaporation of
a hexane/dichloromethane (1:3 v/v) solution at room temperature. Acta Cryst. (2009). E65, o3094-o3095 [ doi:10.1107/S1600536809047230 ] Acta Cryst. (2009). E65, o3094-o3095 [ doi:10.1107/S1600536809047230 ] supplementary materials supplementary materials Refinement The C4, C5 carbon atoms of the cyclohexene ring and the C15, C16 carbon atoms of one n-butyl chain are disordered over
two positions with site occupancy factors of 0.71:0.29 and 0.83:0.17, respectively. During the refinement, the C—C bond
lengths involving the disordered carbon atoms have been constrained to be 1.54 (1) Å. The H atom attached to atom N1
was located in a difference Fourier map and allowed to ride with a restraint of N—H = 0.86 Å. Other H atoms were placed sup-1 supplementary materials at calculated positions and treated as riding atoms, with C—H = 0.96–0.97 Å, and Uiso(H) = 1.2 Ueq(C) or 1.5 Ueq(C) for
methyl H atoms. at calculated positions and treated as riding atoms, with C—H = 0.96–0.97 Å, and Uiso(H) = 1.2 Ueq(C) or 1.5 Ueq(C) for
methyl H atoms. Figures
F
p
a Fig. 1. The molecular structure of the title compound showing the atom-labeling scheme. Dis-
placement ellipsoids are drawn at 50% probability level. H atoms are represented by circles of
arbitrary size. Only the major components of the disordered groups are shown. Fig. 1. The molecular structure of the title compound showing the atom-labeling scheme. Dis-
placement ellipsoids are drawn at 50% probability level. H atoms are represented by circles of
arbitrary size. Only the major components of the disordered groups are shown. Ethyl 2-(3,3-dibutylthioureido)-4,5,6,7-tetrahydrobenzo[b]thiophene- 3-carboxylate Crystal data
C20H32N2O2S2
F000 = 856
Mr = 396.60
Dx = 1.203 Mg m−3
Monoclinic, P21/c
Mo Kα radiation, λ = 0.71073 Å
Hall symbol: -P 2ybc
Cell parameters from 9055 reflections
a = 10.9311 (11) Å
θ = 2.2–27.6º
b = 21.715 (3) Å
µ = 0.26 mm−1
c = 9.6841 (3) Å
T = 292 K
β = 107.711 (12)º
Block, colourless
V = 2189.8 (4) Å3
0.36 × 0.30 × 0.25 mm
Z = 4 F000 = 856
Dx = 1.203 Mg m−3
Mo Kα radiation, λ = 0.71073 Å
Cell parameters from 9055 reflections
θ = 2.2–27.6º
µ = 0.26 mm−1
T = 292 K
Block, colourless
0.36 × 0.30 × 0.25 mm Data collection
Bruker SMART CCD area-detector
diffractometer
4036 independent reflection
Radiation source: fine-focus sealed tube
3460 reflections with I > 2σ
Monochromator: graphite
Rint = 0.027
T = 292 K
θmax = 25.5º
φ and ω scans
θmin = 2.1º
Absorption correction: multi-scan
(SADABS; Sheldrick, 1996)
h = −13→13
Tmin = 0.903, Tmax = 0.938
k = −21→26
12194 measured reflections
l = −11→11 Absorption correction: multi-scan
(SADABS; Sheldrick, 1996)
Tmin = 0.903, Tmax = 0.938
12194 measured reflections supplementary materials where P = (Fo
2 + 2Fc
2)/3
S = 1.21
(Δ/σ)max < 0.001
4036 reflections
Δρmax = 0.30 e Å−3
280 parameters
Δρmin = −0.23 e Å−3
13 restraints
Extinction correction: none
Primary atom site location: structure-invariant direct
methods where P = (Fo
2 + 2Fc
2)/3
(Δ/σ)max < 0.001
Δρmax = 0.30 e Å−3
Δρmin = −0.23 e Å−3
Extinction correction: none Refinement Secondary atom site location: difference Fourier map
Hydrogen site location: inferred from neighbouring
sites H atoms treated by a mixture of
independent and constrained refinement H atoms treated by a mixture of
independent and constrained refinement w = 1/[σ2(Fo
2) + (0.0275P)2 + 2.0031P] w = 1/[σ2(Fo
2) + (0.0275P)2 + 2.0031P] sup-2 supplementary materials Special details Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance mat-
rix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations
between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of
cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F2, convention-
al R-factors R are based on F, with F set to zero for negative F2. The threshold expression of F2 > σ(F2) is used only for calculating R-
factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F2 are statistically about twice as large
as those based on F, and R- factors based on ALL data will be even larger. actional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x
y
z
Uiso*/Ueq
Occ. (<1)
C1
1.0984 (4)
−0.00230 (16)
0.7833 (4)
0.0631 (9)
C2
1.1175 (5)
−0.0488 (2)
0.9023 (5)
0.0856 (12)
H2A
1.0446
−0.0765
0.8804
0.103*
0.71
H2B
1.1237
−0.0280
0.9928
0.103*
0.71
H2C
1.0714
−0.0361
0.9687
0.103*
0.29
H2D
1.0832
−0.0882
0.8616
0.103*
0.29
C3
1.2396 (7)
−0.0852 (4)
0.9174 (11)
0.097 (2)
0.71
H3A
1.2660
−0.1048
1.0119
0.117*
0.71
H3B
1.2207
−0.1175
0.8448
0.117*
0.71
C4
1.3461 (8)
−0.0488 (4)
0.9018 (8)
0.093 (2)
0.71
H4A
1.4202
−0.0754
0.9147
0.112*
0.71
H4B
1.3686
−0.0177
0.9771
0.112*
0.71
C4'
1.315 (2)
−0.0763 (7)
0.8441 (17)
0.077 (5)
0.29
H4'1
1.4018
−0.0918
0.8832
0.092*
0.29
H4'2
1.2627
−0.1081
0.7835
0.092*
0.29
C3'
1.2569 (14)
−0.0586 (11)
0.9758 (17)
0.113 (9)
0.29
H3'1
1.2705
−0.0918
1.0458
0.135*
0.29
H3'2
1.2968
−0.0215
1.0251
0.135*
0.29
C5
1.3142 (4)
−0.01737 (18)
0.7532 (4)
0.0751 (10)
H5A
1.3787
0.0136
0.7554
0.090*
0.71
H5B
1.3163
−0.0476
0.6803
0.090*
0.71
H5C
1.3789
0.0116
0.8067
0.090*
0.29
H5D
1.3339
−0.0286
0.6653
0.090*
0.29
C6
1.1838 (3)
0.01239 (15)
0.7127 (3)
0.0577 (8)
C7
1.1347 (3)
0.05951 (15)
0.6064 (3)
0.0551 (8) sup-3 supplementary materials supplementary materials sup-4
C8
1.0133 (3)
0.07897 (14)
0.6033 (3)
0.0542 (7)
C9
1.1975 (3)
0.08581 (17)
0.5067 (4)
0.0634 (9)
C10
1.3820 (4)
0.0835 (2)
0.4231 (5)
0.0951 (14)
H10A
1.3581
0.1259
0.3967
0.114*
H10B
1.4735
0.0824
0.4730
0.114*
C11
1.3540 (5)
0.0462 (2)
0.2931 (5)
0.1069 (16)
H11A
1.3741
0.0040
0.3193
0.160*
H11B
1.4048
0.0602
0.2340
0.160*
H11C
1.2646
0.0498
0.2398
0.160*
C12
0.8369 (3)
0.15459 (17)
0.5066 (4)
0.0659 (9)
C13
0.6951 (5)
0.2433 (2)
0.4072 (6)
0.1029 (15)
H13A
0.6756
0.2414
0.4983
0.123*
H13B
0.7147
0.2858
0.3911
0.123*
C14
0.5827 (5)
0.2238 (3)
0.2904 (6)
0.1242 (19)
H14A
0.5686
0.1800
0.2980
0.149*
0.83
H14B
0.5957
0.2317
0.1973
0.149*
0.83
H14C
0.5373
0.1905
0.3203
0.149*
0.17
H14D
0.6066
0.2106
0.2064
0.149*
0.17
C15
0.4615 (9)
0.2616 (5)
0.3037 (12)
0.181 (5)
0.83
H15A
0.3847
0.2396
0.2496
0.217*
0.83
H15B
0.4641
0.2612
0.4047
0.217*
0.83
C16
0.4496 (12)
0.3174 (5)
0.2607 (16)
0.232 (7)
0.83
H16A
0.5305
0.3381
0.2977
0.348*
0.83
H16B
0.3864
0.3376
0.2953
0.348*
0.83
H16C
0.4228
0.3184
0.1567
0.348*
0.83
C15'
0.4923 (18)
0.2749 (12)
0.213 (3)
0.090 (8)
0.17
H15C
0.5365
0.3140
0.2372
0.108*
0.17
H15D
0.4727
0.2690
0.1094
0.108*
0.17
C16'
0.372 (2)
0.2782 (14)
0.248 (5)
0.125 (13)
0.17
H16D
0.3180
0.3090
0.1892
0.188*
0.17
H16E
0.3897
0.2888
0.3487
0.188*
0.17
H16F
0.3298
0.2390
0.2303
0.188*
0.17
C17
0.9008 (4)
0.23002 (19)
0.3483 (5)
0.0814 (11)
H17A
0.8536
0.2535
0.2637
0.098*
H17B
0.9449
0.1968
0.3162
0.098*
C18
0.9998 (4)
0.2719 (2)
0.4523 (5)
0.0897 (13)
H18A
0.9553
0.3058
0.4813
0.108*
H18B
1.0437
0.2487
0.5388
0.108*
C19
1.0961 (5)
0.2970 (2)
0.3882 (5)
0.1017 (15)
H19A
1.0523
0.3158
0.2955
0.122*
H19B
1.1485
0.2635
0.3709
0.122*
C20
1.1828 (5)
0.3447 (2)
0.4862 (6)
0.126 (2)
H20A
1.1321
0.3792
0.4981
0.190*
H20B
1.2465
0.3582
0.4432
0.190*
H20C
1.2244
0.3266
0.5791
0.190*
N1
0.9493 (3)
0.12572 (14)
0.5138 (3)
0.0637 (7)
H1A
0.997 (3)
0.1387 (16)
0.464 (3)
0.076*
N2
0.8108 (3)
0.20414 (16)
0.4177 (4)
0.0801 (9)
O1
1.3122 (2)
0.06116 (13)
0.5196 (3)
0.0769 (7) sup-4 supplementary materials pp
y
sup-
O2
1.1519 (3)
0.12628 (13)
0.4189 (3)
0.0869 (8)
S1
0.95706 (9)
0.03954 (4)
0.72506 (10)
0.0662 (3)
S2
0.74212 (10)
0.13005 (5)
0.60210 (13)
0.0823 (3)
Atomic displacement parameters (Å2)
U11
U22
U33
U12
U13
U23
C1
0.080 (2)
0.060 (2)
0.0549 (19)
−0.0045 (17)
0.0284 (17)
−0.0010 (16)
C2
0.106 (3)
0.085 (3)
0.075 (3)
0.006 (2)
0.041 (2)
0.018 (2)
C3
0.109 (6)
0.097 (6)
0.093 (6)
0.026 (4)
0.040 (5)
0.035 (5)
C4
0.094 (5)
0.099 (6)
0.083 (6)
0.014 (5)
0.021 (5)
0.019 (4)
C4'
0.083 (12)
0.078 (11)
0.068 (10)
0.020 (9)
0.021 (9)
−0.004 (8)
C3'
0.18 (2)
0.106 (17)
0.053 (10)
0.055 (16)
0.040 (12)
0.009 (9)
C5
0.079 (2)
0.074 (2)
0.077 (3)
0.010 (2)
0.030 (2)
0.006 (2)
C6
0.0649 (19)
0.0570 (19)
0.0541 (18)
−0.0018 (16)
0.0225 (15)
−0.0035 (15)
C7
0.0634 (19)
0.0523 (17)
0.0542 (18)
−0.0054 (15)
0.0248 (15)
−0.0023 (14)
C8
0.0610 (18)
0.0520 (18)
0.0511 (17)
−0.0049 (15)
0.0194 (15)
−0.0051 (14)
C9
0.068 (2)
0.062 (2)
0.067 (2)
−0.0079 (17)
0.0303 (18)
0.0015 (18)
C10
0.081 (3)
0.113 (4)
0.108 (4)
−0.012 (3)
0.053 (3)
0.013 (3)
C11
0.111 (4)
0.130 (4)
0.102 (4)
−0.005 (3)
0.065 (3)
0.004 (3)
C12
0.062 (2)
0.065 (2)
0.068 (2)
0.0013 (17)
0.0149 (17)
−0.0078 (18)
C13
0.098 (3)
0.094 (3)
0.116 (4)
0.013 (3)
0.031 (3)
0.008 (3)
C14
0.090 (4)
0.154 (5)
0.118 (4)
0.010 (4)
0.015 (3)
0.014 (4)
C15
0.111 (7)
0.256 (15)
0.161 (9)
0.068 (8)
0.021 (6)
0.083 (9)
C16
0.180 (11)
0.235 (14)
0.296 (17)
0.081 (11)
0.096 (11)
0.137 (14)
C15'
0.09 (2)
0.087 (19)
0.09 (2)
0.015 (16)
0.029 (17)
0.017 (16)
C16'
0.10 (3)
0.10 (2)
0.18 (4)
−0.02 (2)
0.05 (3)
0.03 (2)
C17
0.084 (3)
0.076 (3)
0.079 (3)
0.008 (2)
0.017 (2)
0.016 (2)
C18
0.092 (3)
0.086 (3)
0.087 (3)
−0.001 (2)
0.020 (2)
0.009 (2)
C19
0.109 (4)
0.089 (3)
0.104 (4)
−0.005 (3)
0.027 (3)
0.015 (3)
C20
0.119 (4)
0.102 (4)
0.137 (5)
−0.025 (3)
0.007 (4)
0.003 (4)
N1
0.0641 (17)
0.0633 (18)
0.0680 (19)
0.0034 (14)
0.0262 (14)
0.0037 (15)
N2
0.073 (2)
0.076 (2)
0.090 (2)
0.0137 (17)
0.0216 (18)
0.0066 (19)
O1
0.0683 (15)
0.0907 (19)
0.0836 (18)
−0.0001 (14)
0.0410 (14)
0.0126 (14)
O2
0.094 (2)
0.0862 (19)
0.095 (2)
0.0115 (16)
0.0501 (17)
0.0313 (16)
S1
0.0714 (6)
0.0700 (6)
0.0664 (6)
−0.0047 (4)
0.0345 (4)
0.0008 (4)
S2
0.0682 (6)
0.0924 (8)
0.0923 (8)
−0.0011 (5)
0.0333 (5)
−0.0054 (6)
Geometric parameters (Å, °)
C1—C6
1.352 (4)
C12—N2
1.353 (5)
C1—C2
1.498 (5)
C12—N1
1.362 (4)
C1—S1
1.732 (4)
C12—S2
1.672 (4)
C2—C3'
1.488 (15)
C13—C14
1.457 (6)
C2—C3
1.520 (8)
C13—N2
1.502 (5)
C2—H2A
0.9700
C13—H13A
0.9700
C2—H2B
0.9700
C13—H13B
0.9700
C2—H2C
0.9700
C14—C15'
1.522 (16)
C2—H2D
0.9700
C14—C15
1.597 (9) Atomic displacement parameters (Å2) Atomic displacement parameters (Å2) sup-5 supplementary materials supplementary materials sup-6
C3—C4
1.452 (11)
C14—H14A
0.9700
C3—H3A
0.9700
C14—H14B
0.9700
C3—H3B
0.9700
C14—H14C
0.9700
C4—C5
1.534 (7)
C14—H14D
0.9700
C4—H4A
0.9700
C15—C16
1.274 (11)
C4—H4B
0.9700
C15—H15A
0.9700
C4'—C5
1.552 (14)
C15—H15B
0.9700
C4'—C3'
1.63 (2)
C16—H16A
0.9600
C4'—H4'1
0.9700
C16—H16B
0.9600
C4'—H4'2
0.9700
C16—H16C
0.9600
C3'—H3'1
0.9700
C15'—C16'
1.454 (19)
C3'—H3'2
0.9700
C15'—H15C
0.9700
C5—C6
1.505 (5)
C15'—H15D
0.9700
C5—H5A
0.9700
C16'—H16D
0.9600
C5—H5B
0.9700
C16'—H16E
0.9600
C5—H5C
0.9700
C16'—H16F
0.9600
C5—H5D
0.9700
C17—N2
1.461 (5)
C6—C7
1.435 (4)
C17—C18
1.532 (6)
C7—C8
1.385 (4)
C17—H17A
0.9700
C7—C9
1.460 (4)
C17—H17B
0.9700
C8—N1
1.380 (4)
C18—C19
1.480 (6)
C8—S1
1.715 (3)
C18—H18A
0.9700
C9—O2
1.219 (4)
C18—H18B
0.9700
C9—O1
1.333 (4)
C19—C20
1.524 (6)
C10—C11
1.449 (6)
C19—H19A
0.9700
C10—O1
1.458 (4)
C19—H19B
0.9700
C10—H10A
0.9700
C20—H20A
0.9600
C10—H10B
0.9700
C20—H20B
0.9600
C11—H11A
0.9600
C20—H20C
0.9600
C11—H11B
0.9600
N1—H1A
0.86 (3)
C11—H11C
0.9600
C6—C1—C2
126.3 (4)
C10—C11—H11B
109.5
C6—C1—S1
113.3 (3)
H11A—C11—H11B
109.5
C2—C1—S1
120.4 (3)
C10—C11—H11C
109.5
C3'—C2—C1
110.3 (8)
H11A—C11—H11C
109.5
C1—C2—C3
109.7 (4)
H11B—C11—H11C
109.5
C3'—C2—H2A
132.0
N2—C12—N1
114.3 (3)
C1—C2—H2A
109.7
N2—C12—S2
124.0 (3)
C3—C2—H2A
109.7
N1—C12—S2
121.7 (3)
C3'—C2—H2B
81.6
C14—C13—N2
112.5 (4)
C1—C2—H2B
109.7
C14—C13—H13A
109.1
C3—C2—H2B
109.7
N2—C13—H13A
109.1
H2A—C2—H2B
108.2
C14—C13—H13B
109.1
C3'—C2—H2C
112.7
N2—C13—H13B
109.1
C1—C2—H2C
109.8
H13A—C13—H13B
107.8
C3—C2—H2C
134.6
C13—C14—C15'
116.0 (11)
H2A—C2—H2C
76.3
C13—C14—C15
108.2 (6)
C3'—C2—H2D
106.3
C13—C14—H14A
110.1
C1—C2—H2D
109.6
C15'—C14—H14A
131.1 1.452 (11)
C14—H14A
0.9700
0.9700
C14—H14B
0.9700
0.9700
C14—H14C
0.9700
1.534 (7)
C14—H14D
0.9700
0.9700
C15—C16
1.274 (11)
0.9700
C15—H15A
0.9700
1.552 (14)
C15—H15B
0.9700
1.63 (2)
C16—H16A
0.9600
0.9700
C16—H16B
0.9600
0.9700
C16—H16C
0.9600
0.9700
C15'—C16'
1.454 (19)
0.9700
C15'—H15C
0.9700
1.505 (5)
C15'—H15D
0.9700
0.9700
C16'—H16D
0.9600
0.9700
C16'—H16E
0.9600
0.9700
C16'—H16F
0.9600
0.9700
C17—N2
1.461 (5)
1.435 (4)
C17—C18
1.532 (6)
1.385 (4)
C17—H17A
0.9700
1.460 (4)
C17—H17B
0.9700
1.380 (4)
C18—C19
1.480 (6)
1.715 (3)
C18—H18A
0.9700
1.219 (4)
C18—H18B
0.9700
1.333 (4)
C19—C20
1.524 (6)
1.449 (6)
C19—H19A
0.9700
1.458 (4)
C19—H19B
0.9700
0.9700
C20—H20A
0.9600
0.9700
C20—H20B
0.9600
0.9600
C20—H20C
0.9600
0.9600
N1—H1A
0.86 (3)
0.9600
126.3 (4)
C10—C11—H11B
109.5
113.3 (3)
H11A—C11—H11B
109.5
120.4 (3)
C10—C11—H11C
109.5
110.3 (8)
H11A—C11—H11C
109.5
109.7 (4)
H11B—C11—H11C
109.5
132.0
N2—C12—N1
114.3 (3)
109.7
N2—C12—S2
124.0 (3)
109.7
N1—C12—S2
121.7 (3)
81.6
C14—C13—N2
112.5 (4)
109.7
C14—C13—H13A
109.1
109.7
N2—C13—H13A
109.1
108.2
C14—C13—H13B
109.1
112.7
N2—C13—H13B
109.1
109.8
H13A—C13—H13B
107.8
134.6
C13—C14—C15'
116.0 (11)
76.3
C13—C14—C15
108.2 (6)
106.3
C13—C14—H14A
110.1
109.6
C15'—C14—H14A
131.1 sup-6 supplementary materials su
C3—C2—H2D
78.4
C15—C14—H14A
110.1
H2B—C2—H2D
133.7
C13—C14—H14B
110.1
H2C—C2—H2D
107.8
C15—C14—H14B
110.1
C4—C3—C2
114.6 (6)
H14A—C14—H14B
108.4
C4—C3—H3A
108.6
C13—C14—H14C
111.9
C2—C3—H3A
108.6
C15'—C14—H14C
112.0
C4—C3—H3B
108.6
H14B—C14—H14C
130.0
C2—C3—H3B
108.6
C13—C14—H14D
111.0
H3A—C3—H3B
107.6
C15'—C14—H14D
95.9
C3—C4—C5
112.3 (7)
C15—C14—H14D
131.3
C3—C4—H4A
109.2
H14C—C14—H14D
108.9
C5—C4—H4A
109.2
C16—C15—C14
117.9 (11)
C3—C4—H4B
109.2
C16—C15—H15A
107.8
C5—C4—H4B
109.2
C14—C15—H15A
107.8
H4A—C4—H4B
107.9
C16—C15—H15B
107.8
C5—C4'—C3'
108.2 (12)
C14—C15—H15B
107.8
C5—C4'—H4'1
110.1
H15A—C15—H15B
107.2
C3'—C4'—H4'1
110.1
C16'—C15'—C14
114.7 (19)
C5—C4'—H4'2
110.1
C16'—C15'—H15C
108.6
C3'—C4'—H4'2
110.1
C14—C15'—H15C
108.6
H4'1—C4'—H4'2
108.4
C16'—C15'—H15D
108.6
C2—C3'—C4'
104.1 (12)
C14—C15'—H15D
108.6
C2—C3'—H3'1
110.9
H15C—C15'—H15D
107.6
C4'—C3'—H3'1
110.9
C15'—C16'—H16D
109.5
C2—C3'—H3'2
110.9
C15'—C16'—H16E
109.5
C4'—C3'—H3'2
110.9
H16D—C16'—H16E
109.5
H3'1—C3'—H3'2
109.0
C15'—C16'—H16F
109.5
C6—C5—C4
111.3 (4)
H16D—C16'—H16F
109.5
C6—C5—C4'
110.0 (8)
H16E—C16'—H16F
109.5
C6—C5—H5A
109.4
N2—C17—C18
111.4 (4)
C4—C5—H5A
109.4
N2—C17—H17A
109.3
C4'—C5—H5A
133.0
C18—C17—H17A
109.3
C6—C5—H5B
109.4
N2—C17—H17B
109.3
C4—C5—H5B
109.4
C18—C17—H17B
109.3
H5A—C5—H5B
108.0
H17A—C17—H17B
108.0
C6—C5—H5C
109.9
C19—C18—C17
113.1 (4)
C4'—C5—H5C
110.8
C19—C18—H18A
108.9
H5B—C5—H5C
130.8
C17—C18—H18A
108.9
C6—C5—H5D
108.9
C19—C18—H18B
108.9
C4—C5—H5D
131.5
C17—C18—H18B
109.0
C4'—C5—H5D
109.0
H18A—C18—H18B
107.8
H5C—C5—H5D
108.1
C18—C19—C20
112.1 (4)
C1—C6—C7
111.5 (3)
C18—C19—H19A
109.2
C1—C6—C5
120.8 (3)
C20—C19—H19A
109.2
C7—C6—C5
127.6 (3)
C18—C19—H19B
109.2
C8—C7—C6
112.3 (3)
C20—C19—H19B
109.2
C8—C7—C9
120.4 (3)
H19A—C19—H19B
107.9
C6—C7—C9
127.2 (3)
C19—C20—H20A
109.5
N1—C8—C7
122.5 (3)
C19—C20—H20B
109.5 78.4
C15—C14—H14A
133.7
C13—C14—H14B
107.8
C15—C14—H14B
114.6 (6)
H14A—C14—H14B
108.6
C13—C14—H14C
108.6
C15'—C14—H14C
108.6
H14B—C14—H14C
108.6
C13—C14—H14D
107.6
C15'—C14—H14D
112.3 (7)
C15—C14—H14D
109.2
H14C—C14—H14D
109.2
C16—C15—C14
109.2
C16—C15—H15A
109.2
C14—C15—H15A
107.9
C16—C15—H15B
108.2 (12)
C14—C15—H15B
110.1
H15A—C15—H15B
110.1
C16'—C15'—C14
110.1
C16'—C15'—H15C
110.1
C14—C15'—H15C
108.4
C16'—C15'—H15D
104.1 (12)
C14—C15'—H15D
110.9
H15C—C15'—H15D
110.9
C15'—C16'—H16D
110.9
C15'—C16'—H16E
110.9
H16D—C16'—H16E
109.0
C15'—C16'—H16F
111.3 (4)
H16D—C16'—H16F
110.0 (8)
H16E—C16'—H16F
109.4
N2—C17—C18
109.4
N2—C17—H17A
133.0
C18—C17—H17A
109.4
N2—C17—H17B
109.4
C18—C17—H17B
108.0
H17A—C17—H17B
109.9
C19—C18—C17
110.8
C19—C18—H18A
130.8
C17—C18—H18A
108.9
C19—C18—H18B
131.5
C17—C18—H18B
109.0
H18A—C18—H18B
108.1
C18—C19—C20
111.5 (3)
C18—C19—H19A
120.8 (3)
C20—C19—H19A
127.6 (3)
C18—C19—H19B
112.3 (3)
C20—C19—H19B
120.4 (3)
H19A—C19—H19B
127.2 (3)
C19—C20—H20A
122.5 (3)
C19—C20—H20B sup-7
78.4
C15—C14—H14A
110.1
133.7
C13—C14—H14B
110.1
107.8
C15—C14—H14B
110.1
114.6 (6)
H14A—C14—H14B
108.4
108.6
C13—C14—H14C
111.9
108.6
C15'—C14—H14C
112.0
108.6
H14B—C14—H14C
130.0
108.6
C13—C14—H14D
111.0
107.6
C15'—C14—H14D
95.9
112.3 (7)
C15—C14—H14D
131.3
109.2
H14C—C14—H14D
108.9
109.2
C16—C15—C14
117.9 (11)
109.2
C16—C15—H15A
107.8
109.2
C14—C15—H15A
107.8
107.9
C16—C15—H15B
107.8
108.2 (12)
C14—C15—H15B
107.8
110.1
H15A—C15—H15B
107.2
110.1
C16'—C15'—C14
114.7 (19)
110.1
C16'—C15'—H15C
108.6
110.1
C14—C15'—H15C
108.6
108.4
C16'—C15'—H15D
108.6
104.1 (12)
C14—C15'—H15D
108.6
110.9
H15C—C15'—H15D
107.6
110.9
C15'—C16'—H16D
109.5
110.9
C15'—C16'—H16E
109.5
110.9
H16D—C16'—H16E
109.5
109.0
C15'—C16'—H16F
109.5
111.3 (4)
H16D—C16'—H16F
109.5
110.0 (8)
H16E—C16'—H16F
109.5
109.4
N2—C17—C18
111.4 (4)
109.4
N2—C17—H17A
109.3
133.0
C18—C17—H17A
109.3
109.4
N2—C17—H17B
109.3
109.4
C18—C17—H17B
109.3
108.0
H17A—C17—H17B
108.0
109.9
C19—C18—C17
113.1 (4)
110.8
C19—C18—H18A
108.9
130.8
C17—C18—H18A
108.9
108.9
C19—C18—H18B
108.9
131.5
C17—C18—H18B
109.0
109.0
H18A—C18—H18B
107.8
108.1
C18—C19—C20
112.1 (4)
111.5 (3)
C18—C19—H19A
109.2
120.8 (3)
C20—C19—H19A
109.2
127.6 (3)
C18—C19—H19B
109.2
112.3 (3)
C20—C19—H19B
109.2
120.4 (3)
H19A—C19—H19B
107.9
127.2 (3)
C19—C20—H20A
109.5
122.5 (3)
C19—C20—H20B
109.5 sup-7 supplementary materials supplementary materials 8
C8—S1
125.5 (2)
H20A—C20—H20B
109.5
C8—S1
112.0 (2)
C19—C20—H20C
109.5
C9—O1
121.9 (3)
H20A—C20—H20C
109.5
C9—C7
124.6 (3)
H20B—C20—H20C
109.5
C9—C7
113.5 (3)
C12—N1—C8
130.4 (3)
—C10—O1
111.0 (4)
C12—N1—H1A
121 (3)
—C10—H10A
109.4
C8—N1—H1A
109 (3)
C10—H10A
109.4
C12—N2—C17
123.9 (3)
—C10—H10B
109.4
C12—N2—C13
120.2 (4)
C10—H10B
109.4
C17—N2—C13
114.9 (3)
A—C10—H10B
108.0
C9—O1—C10
117.8 (3)
—C11—H11A
109.5
C8—S1—C1
90.86 (16)
C1—C2—C3'
23.4 (10)
C6—C7—C9—O2
−179.4 (4)
C1—C2—C3'
−155.7 (8)
C8—C7—C9—O1
−178.1 (3)
C1—C2—C3
−9.1 (7)
C6—C7—C9—O1
0.8 (5)
C1—C2—C3
171.9 (5)
N2—C13—C14—C15'
−145.7 (11)
—C2—C3—C4
−57.1 (15)
N2—C13—C14—C15
171.8 (5)
C2—C3—C4
39.8 (10)
C13—C14—C15—C16
76.9 (14)
C3—C4—C5
−59.9 (11)
C15'—C14—C15—C16
−32.1 (19)
C2—C3'—C4'
−54.3 (15)
C13—C14—C15'—C16'
−106 (3)
C2—C3'—C4'
40.2 (10)
C15—C14—C15'—C16'
−19 (2)
C4'—C3'—C2
71.5 (17)
N2—C17—C18—C19
177.6 (4)
C4—C5—C6
45.6 (9)
C17—C18—C19—C20
172.7 (4)
C4—C5—C4'
−48.1 (16)
N2—C12—N1—C8
171.8 (3)
—C4'—C5—C6
−50.6 (14)
S2—C12—N1—C8
−7.6 (5)
—C4'—C5—C4
47.6 (13)
C7—C8—N1—C12
−172.7 (3)
C1—C6—C7
−179.1 (4)
S1—C8—N1—C12
5.7 (5)
C1—C6—C7
0.0 (4)
N1—C12—N2—C17
−7.3 (5)
C1—C6—C5
−1.7 (6)
S2—C12—N2—C17
172.2 (3)
C1—C6—C5
177.4 (3)
N1—C12—N2—C13
−175.5 (3)
C5—C6—C1
−15.9 (6)
S2—C12—N2—C13
3.9 (5)
—C5—C6—C1
17.0 (8)
C18—C17—N2—C12
−81.1 (5)
C5—C6—C7
161.0 (5)
C18—C17—N2—C13
87.7 (4)
—C5—C6—C7
−166.1 (7)
C14—C13—N2—C12
−92.5 (5)
C6—C7—C8
0.8 (4)
C14—C13—N2—C17
98.3 (5)
C6—C7—C8
−176.3 (3)
O2—C9—O1—C10
−0.9 (6)
C6—C7—C9
−178.1 (3)
C7—C9—O1—C10
178.9 (3)
C6—C7—C9
4.8 (6)
C11—C10—O1—C9
−92.2 (5)
C7—C8—N1
177.3 (3)
N1—C8—S1—C1
−177.4 (3)
C7—C8—N1
−3.7 (5)
C7—C8—S1—C1
1.1 (3)
C7—C8—S1
−1.3 (4)
C6—C1—S1—C8
−0.7 (3)
C7—C8—S1
177.7 (3)
C2—C1—S1—C8
178.5 (3)
C7—C9—O2
1.8 (6)
ogen-bond geometry (Å, °)
H···A
D—H
H···A
D···A
D—H··
H1A···O2
0.86 (3)
1.89 (2)
2.643 (4)
145 (3) 125.5 (2)
H20A—C20—H20B
109.5
112.0 (2)
C19—C20—H20C
109.5
121.9 (3)
H20A—C20—H20C
109.5
124.6 (3)
H20B—C20—H20C
109.5
113.5 (3)
C12—N1—C8
130.4 (3)
111.0 (4)
C12—N1—H1A
121 (3)
109.4
C8—N1—H1A
109 (3)
109.4
C12—N2—C17
123.9 (3)
109.4
C12—N2—C13
120.2 (4)
109.4
C17—N2—C13
114.9 (3)
108.0
C9—O1—C10
117.8 (3)
109.5
C8—S1—C1
90.86 (16)
23.4 (10)
C6—C7—C9—O2
−179.4 (4)
−155.7 (8)
C8—C7—C9—O1
−178.1 (3)
−9.1 (7)
C6—C7—C9—O1
0.8 (5)
171.9 (5)
N2—C13—C14—C15'
−145.7 (11)
−57.1 (15)
N2—C13—C14—C15
171.8 (5)
39.8 (10)
C13—C14—C15—C16
76.9 (14)
−59.9 (11)
C15'—C14—C15—C16
−32.1 (19)
−54.3 (15)
C13—C14—C15'—C16'
−106 (3)
40.2 (10)
C15—C14—C15'—C16'
−19 (2)
71.5 (17)
N2—C17—C18—C19
177.6 (4)
45.6 (9)
C17—C18—C19—C20
172.7 (4)
−48.1 (16)
N2—C12—N1—C8
171.8 (3)
−50.6 (14)
S2—C12—N1—C8
−7.6 (5)
47.6 (13)
C7—C8—N1—C12
−172.7 (3)
−179.1 (4)
S1—C8—N1—C12
5.7 (5)
0.0 (4)
N1—C12—N2—C17
−7.3 (5)
−1.7 (6)
S2—C12—N2—C17
172.2 (3)
177.4 (3)
N1—C12—N2—C13
−175.5 (3)
−15.9 (6)
S2—C12—N2—C13
3.9 (5)
17.0 (8)
C18—C17—N2—C12
−81.1 (5)
161.0 (5)
C18—C17—N2—C13
87.7 (4)
−166.1 (7)
C14—C13—N2—C12
−92.5 (5)
0.8 (4)
C14—C13—N2—C17
98.3 (5)
−176.3 (3)
O2—C9—O1—C10
−0.9 (6)
−178.1 (3)
C7—C9—O1—C10
178.9 (3)
4.8 (6)
C11—C10—O1—C9
−92.2 (5)
177.3 (3)
N1—C8—S1—C1
−177.4 (3)
−3.7 (5)
C7—C8—S1—C1
1.1 (3)
−1.3 (4)
C6—C1—S1—C8
−0.7 (3)
177.7 (3)
C2—C1—S1—C8
178.5 (3)
1.8 (6)
D—H
H···A
D···A
D—H···A
0.86 (3)
1.89 (2)
2.643 (4)
145 (3) sup-8 supplementary materials g. supplementary materials 1 Fig. 1 sup-9
|
https://openalex.org/W4377046988
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https://www.nature.com/articles/s41418-023-01173-6.pdf
|
English
| null |
USP28 controls SREBP2 and the mevalonate pathway to drive tumour growth in squamous cancer
|
Cell death and differentiation
| 2,023
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cc-by
| 14,607
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1German Cancer Research Center, Division of Tumor Metabolism and Microenvironment, Im Neuenheimer Feld 581, 69120 Heidelberg, Germany. 2Protein Stability and Cancer
Group, Department of Biochemistry and Molecular Biology, Theodor-Boveri-Institute, Biocenter, Am Hubland, 97074 Würzburg, Germany. 3Institute of Biochemistry II, Goethe
University Frankfurt, Theodor-Stern-Kai 7, Haus 75, 60590 Frankfurt am Main, Germany. 4Institute of Pathology, Julius Maximilians University and Comprehensive Cancer Center
(CCC) Mainfranken, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany. 5Theodor Boveri Institute, Department of Biochemistry and Molecular Biology, Biocenter, University of
Würzburg, 97074 Würzburg, Germany. 6Internal Medicine VIII-Clinical Tumor Biology, University of Tübingen, Otfried-Müller-Straße 14, 72076 Tübingen, Germany. 7Instituto
Maimónides de Investigación Biomédica de Córdoba (IMIBIC), Córdoba, Spain. 8Departamento de Biología Celular, Fisiología e Inmunología, Universidad de Córdoba, Córdoba,
Spain. 9Hospital Universitario Reina Sofía, Córdoba, Spain. 10Present address: Institute of Biochemistry II, Goethe University Frankfurt, Theodor-Stern-Kai 7, Haus 75, 60590
Frankfurt am Main, Germany. 11These authors contributed equally: Carina R. Maier, Oliver Hartmann. ✉email: markus.diefenbacher@uni-wuerzburg.de;
almut.schulze@dkfz-heidelberg.de ARTICLE
OPEN
USP28 controls SREBP2 and the mevalonate pathway to drive
tumour growth in squamous cancer Carina R. Maier1,2,11, Oliver Hartmann
2,11, Cristian Prieto-Garcia
2,10, Kamal M. Al-Shami1, Lisa Schlicker1, Felix C. E. Vogel1,
Silke Haid1, Kevin Klann3, Viktoria Buck4, Christian Münch
3, Werner Schmitz5, Elias Einig
6, Bastian Krenz
5,
Marco A. Calzado
7,8,9, Martin Eilers
5, Nikita Popov6, Mathias T. Rosenfeldt
4, Markus E. Diefenbacher
2✉and
Almut Schulze
1✉ Carina R. Maier1,2,11, Oliver Hartmann
2,11, Cristian Prieto-Garcia
2,10, Kamal M. Al-Shami1, Lisa Schlicker1, Felix C. E. Vogel1,
Silke Haid1, Kevin Klann3, Viktoria Buck4, Christian Münch
3, Werner Schmitz5, Elias Einig
6, Bastian Krenz
5,
Marco A. Calzado
7,8,9, Martin Eilers
5, Nikita Popov6, Mathias T. Rosenfeldt
4, Markus E. Diefenbacher
2✉and
Almut Schulze
1✉ © The Author(s) 2023 © The Author(s) 2023 © The Author(s) 2023 SREBP2 is a master regulator of the mevalonate pathway (MVP), a biosynthetic process that drives the synthesis of dolichol, heme A,
ubiquinone and cholesterol and also provides substrates for protein prenylation. Here, we identify SREBP2 as a novel substrate for
USP28, a deubiquitinating enzyme that is frequently upregulated in squamous cancers. Our results show that silencing of USP28
reduces expression of MVP enzymes and lowers metabolic flux into this pathway. We also show that USP28 binds to mature
SREBP2, leading to its deubiquitination and stabilisation. USP28 depletion rendered cancer cells highly sensitive to MVP inhibition
by statins, which was rescued by the addition of geranyl-geranyl pyrophosphate. Analysis of human tissue microarrays revealed
elevated expression of USP28, SREBP2 and MVP enzymes in lung squamous cell carcinoma (LSCC) compared to lung
adenocarcinoma (LADC). Moreover, CRISPR/Cas-mediated deletion of SREBP2 selectively attenuated tumour growth in a KRas/p53/
LKB1 mutant mouse model of lung cancer. Finally, we demonstrate that statins synergise with a dual USP28/25 inhibitor to reduce
viability of SCC cells. Our findings suggest that combinatorial targeting of MVP and USP28 could be a therapeutic strategy for the
treatment of squamous cell carcinomas. Cell Death & Differentiation (2023) 30:1710–1725; https://doi.org/10.1038/s41418-023-01173-6 Received: 20 December 2022 Revised: 16 April 2023 Accepted: 25 April 2023
Published online: 18 May 2023 www.nature.com/cdd INTRODUCTION
h
b DNA damage response (DDR) in cancer cells by deubiquitinating
and stabilising the checkpoint kinase 2 (CHK2), the TP53-binding
protein 1 (TP53BP1) and Claspin (CLSPN) [6, 7], thereby preventing
apoptosis but establishing cell cycle arrest to facilitate DNA repair
[8]. However, the exact function of USP28 in regulating DNA
double-strand repair is still under investigation [9, 10]. The ubiquitin-proteasome system controls a wide range of cellular
processes. Conjugation of the 76 amino acid polypeptide ubiquitin
to target proteins is achieved through the sequential action of E1,
E2 and E3 enzymes that first activate and then conjugate ubiquitin
to the target protein. The complexity of the system is achieved by
the large number of E3 enzymes (up to 1000) that differ in their
molecular structure and show substantial substrate specificity
[1, 2]. The ubiquitination process is counteracted by deubiquiti-
nating enzymes (DUB), a family of over 100 isopeptidases, that
hydrolyse the peptide bond between the C-terminus of ubiquitin
and a substrate (e.g. another ubiquitin molecule) [3]. Ubiquitin-
specific proteases (USP) make up almost half of the DUBs encoded
by the human genome. Interestingly, USP28 counteracts the activity of the F-box and
WD repeat domain containing protein 7 (FBXW7), a tumour
suppressor protein that is part of the SCF (Skp1, Cullin-1, F-box)
protein complex and facilitates the ubiquitination and destabilisa-
tion of MYC among other proteins. USP28 antagonises FBXW7,
resulting in MYC stabilisation [11–13]. However, in some cell types,
USP28 can also deubiquitinate and stabilise FBXW7 itself [14],
leading to the downregulation of FBXW7 targets [15]. y
g
USP28 was shown to control the stability of a number of cancer-
relevant proteins, most notably MYC [4]. However, USP28 also
promotes tumour suppressive pathways, for example by deubi-
quitinating TP53, a function that is counteracted by nuclear
caspase 8 during tumour relapse [5]. USP28 also modulates the Recently, USP28 was shown to be upregulated in squamous cell
carcinoma (SCC) where it leads to the deubiquitination and
stabilisation of ΔNP63 [16]. Genetic deletion of USP28 via CRISPR/
Cas9 blocked tumour formation in a mouse model of lung squamous
cell carcinoma (LSCC) driven by mutant KrasG12D in combination with Received: 20 December 2022 Revised: 16 April 2023 Accepted: 25 April 2023
Published online: 18 May 2023 Official journal of CDDpress C.R. Maier et al. 1711 deletion of Trp53 and Lkb1 [16]. shUSP28 #1
TGCTGTTGACAGTGAGCGCACAAGAGATTAGAAATATAAATAGTGAAGCCA
CAGATGTATTTATATTTCTAATCTCTTGTATGCCTACTGCCTCGGA
shUSP28 #2
TGCTGTTGACAGTGAGCGATACAAGAGATTAGAAATATAATAGTGAAGCCA
CAGATGTATTATATTTCTAATCTCTTGTAGTGCCTACTGCCTCGGA
shSREBF2 #1
TGCTGTTGACAGTGAGCGCTCTGTATATATTTAAACCTAATAGTGAAGCCA
CAGATGTATTAGGTTTAAATATATACAGATTGCCTACTGCCTCGGA
shSREBF2 #2
TGCTGTTGACAGTGAGCGAAAGGCCATTGATTACATCAAATAGTGAAGCCA
CAGATGTATTTGATGTAATCAATGGCCTTCTGCCTACTGCCTCGGA
CRISPR guide RNA sequences
sgCtrl fwd humanCACCGCGAGGTATTCGGCTCCGCG
sgUSP28_1 fwd humanCACCG GAGTTGATGGTTGGCCAGTT
sgUSP28_2 fwd humanCACCG ACCCCAATCCCAATGACTGG
sgSREBF2_#1 fwd mouseCACCGCTTCAGCGTGGTCAACACAA
sgSREBF2_#2 fwd mouseCACCGAGCGACCGTCTGTACCGTGG shUSP28 #1
TGCTGTTGACAGTGAGCGCACAAGAGATTAGAAATATAAATAGTGAAGCCA
CAGATGTATTTATATTTCTAATCTCTTGTATGCCTACTGCCTCGGA
shUSP28 #2
TGCTGTTGACAGTGAGCGATACAAGAGATTAGAAATATAATAGTGAAGCCA
CAGATGTATTATATTTCTAATCTCTTGTAGTGCCTACTGCCTCGGA
shSREBF2 #1
TGCTGTTGACAGTGAGCGCTCTGTATATATTTAAACCTAATAGTGAAGCCA
CAGATGTATTAGGTTTAAATATATACAGATTGCCTACTGCCTCGGA
shSREBF2 #2
TGCTGTTGACAGTGAGCGAAAGGCCATTGATTACATCAAATAGTGAAGCCA
CAGATGTATTTGATGTAATCAATGGCCTTCTGCCTACTGCCTCGGA
CRISPR guide RNA sequences
sgCtrl fwd humanCACCGCGAGGTATTCGGCTCCGCG
sgUSP28_1 fwd humanCACCG GAGTTGATGGTTGGCCAGTT
sgUSP28_2 fwd humanCACCG ACCCCAATCCCAATGACTGG
sgSREBF2_#1 fwd mouseCACCGCTTCAGCGTGGTCAACACAA
sgSREBF2_#2 fwd mouseCACCGAGCGACCGTCTGTACCGTGG g
g
The sterol regulatory element binding proteins (SREBP1a,
SREBP1c and SREBP2, encoded by the SREBF1 and SREBF2 genes,
respectively) are basic helix-loop-helix leucine zipper transcription
factors that control the expression of genes involved in fatty acid
and cholesterol biosynthesis [19]. While SREBP1a and SREBP1c
show selectivity towards regulation of fatty acid biosynthesis,
SREBP2 controls the expression of enzymes of the mevalonate
pathway (MVP), which has multiple metabolic outputs that connect
to biosynthetic and signalling processes in cancer, including
providing substrates for the prenylation of small G proteins [20]. Key steps in the MVP are catalysed by the enzymes 3-Hydroxy-3-
Methylglutaryl-CoA Synthase 1 (HMGCS1), 3-Hydroxy-3-Methylglu-
taryl-CoA Reductase (HMGCR) and Farnesyl-Diphosphate Farnesyl-
transferase 1 (FDFT1). SREBPs are expressed as inactive precursors
that reside in the membrane of the endoplasmic reticulum (ER). While
the
precise
mechanism
of
activation
of
SREBP1
is
incompletely understood, both SREBP1 and SREBP2 are activated
by proteolytic cleavage in a sterol-dependent manner [19]. Once
nuclear, the N-terminal portion of the protein comprising the DNA
binding and transactivation domains is subject to further regula-
tion, including FBWX7-dependent ubiquitination [21, 22]. Interest-
ingly, binding of FBWX7 to SREBP depends on phosphorylation of
the conserved CDK2-phosphodegron (CPD) motif by glycogen
synthase kinase-3β (GSK3B) [21]. Given the antagonism between
FBWX7 and USP28 outlined above, it therefore seems possible that
SREBPs could also be substrates for USP28-dependent deubiqui-
tination and stabilisation. RNAseq and data analysis q
y
RNA was extracted using RNeasy columns (Qiagen). mRNA was isolated
using NEBNext® Poly(A) mRNA Magnetic Isolation Module and library
preparation was performed with NEBNext® Ultra™RNA Library Prep Kit for
Illumina following the manufacturer’s instructions. Libraries were size-
selected using Agencourt AMPure XP Beads (Beckman Coulter) followed by
amplification
with
12
PCR
cycles. Library
quantification
and
size
determination was performed with an Experion system (Bio-Rad) and
libraries were sequenced with NextSeq500 (Illumina) at DKFZ Genomics
Core facility. Reads were aligned to the human genome (hg19) with
TopHat2, Bowtie v0.12.8 using default parameters. Mapped reads per gene
(EnsEMBL GRCh37, release 74) were counted using the “summarizeOver-
laps” function in the GenomicAlignments R package, non-expressed genes
were removed (mean read count per gene over all samples >1) and TMM
normalized with EdgeR. Gene set enrichment analyses were performed
with the C2 and Hallmark collection from the MSigDB v5.2 with default
parameters and 1000 permutations. Principle component analysis (PCA)
was performed with the prcomp function from R after centering
sequencing depth-normalized expression values of all expressed genes
(n = 20,434). RNAseq data are available at GEO (GSE204703). Here, we report the identification of SREBP2 as a novel substrate
of USP28 in squamous cancer cells. Depletion of USP28 resulted in
the destabilisation of mature SREBP2, reduced expression of MVP
enzymes and rendered cancer cells highly sensitive to MVP
inhibition by statins, which was rescued by precursors of protein
prenylation. We also found that USP28 and SREBP2 proteins are
highly expressed in tissues from human LSCC patients compared
to adenocarcinoma and that deletion of Srebp2 reduced tumour
formation in a KRas/p53/LKB1 mutant mouse model of lung
cancer. Moreover, statins synergised with a dual USP28/25
inhibitor in reducing viability of human LSCC cells. Our findings
suggest that USP28 targets SREBP2 to drive LSCC tumour growth. Proteomics analysis Whole cell proteome analysis of A431 cells expressing shRNAs targeting
USP28 or non-targeting controls and treated with 1 µg/mL doxycycline for
72 h has been described previously [10, 18]. Pathway analysis of USP28
regulated proteins (FDR p-value ≤0.05) was performed with the PANTHER
tool using the statistical overrepresentation test and PANTHER pathways
with default settings [19]. INTRODUCTION
h
b Moreover, acute deletion of Usp28
using a dual recombinase model resulted in regression of tumours
formed by induction of mutant KrasG12D in combination with Fbw7
deletion [17]. Furthermore, pharmacologic inhibition of USP28
negatively affected LSCC tumour growth and was well tolerated
in vivo [16, 17]. In addition, USP28 supports oncogenic transforma-
tion of respiratory cells and its inhibition synergises with therapeutic
strategies targeting the EGFR/RAS/ERK pathway in transformed lung
epithelial
cells
[18]. Therefore,
understanding
the
molecular
mechanisms by which USP28 supports tumour formation is vital
for the rational targeting of LSCC. and cloned into LT3-GEPIR vector (Addgene #111177). Viral particles were
produced in HEK293T cells and stable populations were selected using
puromycin (2 µg/mL). For CRISPR/Cas9 mediated gene silencing, sgRNA
sequences were designed using CHOPCHOP (https://chopchop.cbu.uib.no)
and cloned into pX458 (Addgene #48138). U2OS cells were transfected and
selected with puromycin and single clones were picked. Silencing and
knockout efficiency was confirmed using qPCR and western blotting. shUSP28 #1
TGCTGTTGACAGTGAGCGCACAAGAGATTAGAAATATAAATAGTGAAGCCA
CAGATGTATTTATATTTCTAATCTCTTGTATGCCTACTGCCTCGGA
shUSP28 #2
TGCTGTTGACAGTGAGCGATACAAGAGATTAGAAATATAATAGTGAAGCCA
CAGATGTATTATATTTCTAATCTCTTGTAGTGCCTACTGCCTCGGA
shSREBF2 #1
TGCTGTTGACAGTGAGCGCTCTGTATATATTTAAACCTAATAGTGAAGCCA
CAGATGTATTAGGTTTAAATATATACAGATTGCCTACTGCCTCGGA
shSREBF2 #2
TGCTGTTGACAGTGAGCGAAAGGCCATTGATTACATCAAATAGTGAAGCCA
CAGATGTATTTGATGTAATCAATGGCCTTCTGCCTACTGCCTCGGA
CRISPR guide RNA sequences
sgCtrl fwd humanCACCGCGAGGTATTCGGCTCCGCG
sgUSP28_1 fwd humanCACCG GAGTTGATGGTTGGCCAGTT
sgUSP28_2 fwd humanCACCG ACCCCAATCCCAATGACTGG
sgSREBF2_#1 fwd mouseCACCGCTTCAGCGTGGTCAACACAA
sgSREBF2_#2 fwd mouseCACCGAGCGACCGTCTGTACCGTGG Tissue culture and reagents Detection of cholesterol and CoQ10 by LC MS
Cells were incubated in DMEM medium without glucose (Sigma, D5030)
supplemented with 25 mM [U13C]-glucose (Cambridge Isotope Labora-
tories, Inc.). After 48 h, cells were washed with 0.5 ml cold ammonium
acetate (154 mM), snap-frozen in liquid nitrogen and scraped off in 0.5 mL
ice-cold MeOH/H2O (80/20 v/v). The suspension was transferred to a glass
tube and another 0.5 ml ice-cold MeOH/H2O (80/20 v/v) were added. Internal standards 7-dehydrocholesterol-d7 (Sigma-Aldrich) and coenzyme
Q9 (Sigma-Aldrich) were used in the final concentrations of 2 and
0.24 µmol/l, respectively. After addition of 120 µl 0.2 M HCl, 360 µl
chloroform, 400 µl chloroform and 400 µl of water with vigorous mixing
between the pipetting steps, samples were centrifuged at 3,000 g for
10 min. 700 µl of the lower phase were collected and taken to dryness
under a stream of nitrogen gas at 40 °C. The resulting residues were
dissolved in 50 µl isopropanol and taken for LC-MS analysis. Tissue culture and reagents Tissue culture and reagents g
Cell lines were obtained from ATCC (American Type Culture Collection,
Chicago, IL, USA) and used at low passage. Cells were cultured in DMEM
(A431, U2OS, HEK293T) or RPMI 1640 (NCI-H520, LUDLU1, CALU-1, NCI-
H522, NCI-H1299, A549) supplemented with 10% foetal calf serum (FCS),
2 mmol/L glutamine and 100 U/mL penicillin/streptomycin (all Sigma). Delipidated FCS was prepared as previously described [23]. Cells were
grown at 37 °C in a humidified incubator at 5% CO2 and regularly tested for
absence of mycoplasma. Cell-permeable cholesterol, Co-enzyme Q10,
Geranylgeranyl pyrophosphate ammonium salt, (R)-Mevalonic acid-lithium
salt, MG-132 and Simvastatin were from Sigma. Nucleosides (100×,
EmbryoMax) were from Merck. AZ1 was from Selleckchem. Ni-NTA pull-down assay
HEK293T
ll
f HEK293T cells were transfected with plasmids encoding 6His-tagged K48-
only ubiquitin, HA-tagged mSREBP2 and USP28-WT or USP28-CA using the
Polyjet reagent (Signagen). 48 h after transfection, cells were lysed in Urea
buffer (8 M Urea, 1%TX-100, 300 mM NaCl, 25 mM Imidazole) in PBS and
briefly sonicated. Cleared lysates were incubated with Ni-NTA beads
overnight to capture ubiquitinated proteins. Beads were washed three
times with lysis buffer and precipitated proteins were denatured in sample
buffer for separation on SDS-PAGE. Proximity ligation assay (PLA) Paraffin-embedded sections of human and murine samples were cut into
4 µm sections with a microtome (Leica). Slides were de-paraffinized and
rehydrated using the following protocol: 3 × 5 min in Xylene, 2 × 2 min in
ethanol (100%), 2 × 2 min in ethanol (95%), 2 × 2 min in ethanol (70%), 2 min
in ethanol (50%) and 5 min in H2O. Antigen retrieval was performed with
10 mM sodium citrate buffer (pH 6.0) in a microwave oven at 800 W, 650 W
and 360 W for 5 min each. Samples were permeabilised with TBS 0.1 % Triton
X-100 for 10 min and blocked with TBS containing 3 % H2O2 for 10 min. Human samples were stained with anti-SREBF2 (R&D, MAB7119), anti-USP28
(Sigma, HPA006778), anti-SREBF1 (PTG, 14088-1-AP) and anti-HMGCS1 y
g
y
Cells were grown on chambered coverslips (Ibidi), fixed, permeabilised and
PLA was carried out using the Duolink kit (Sigma) according to
manufacturer’s instructions. Slides were mounted with in situ Mounting
Medium (Sigma) containing DAPI. Signals were detected using a confocal
SP8 microscope (Leica). Analysis of Cancer Genome Atlas (TCGA) data and survival
analysis y
Raw gene expression data of lung adenocarcinoma (LADC) and lung
squamous carcinoma (LSCC) were extracted from TCGA datasets (firehose
legacy) using cBioPortal (http://cbioportal.org). mRNA expression data
(RNAseq V2 RSEM) were log2 transformed and compared using a non-
parametric two-tailed Mann-Whitney test. Correlation analyses were
performed using GEPIA2 [24]. Kaplan–Meier plots and survival analyses
were performed using the KM plotter tool [25]. Cell lysis, cell fractionation and western blotting Cell lysis, cell fractionation and western blotting
Cells were lysed in RIPA buffer (150 mM NaCl, 50 mM Tris pH 8.0, 1% (v/v)
NP-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS) with protease and
phosphatase inhibitors for 30 min and cleared by centrifugation. Proteins
were quantified using BCA (Thermo Scientific). Cell fractionation as
performed using NE-PER™Nuclear and Cytoplasmic Extraction kit (Thermo
Scientific). Proteins were separated on SDS-PAGE and blotted onto PVDF
membrane (Immobilon), treated with blocking solution (Thermo Scientific)
and incubated with primary and secondary antibodies. Signals were
detected on an Odyssey scanner
(LI-COR)
or
ChemiDoc
(BioRad). Antibodies used were: anti-SREBP2 (R&D, AF7119), anti-USP28 (Sigma,
HPA006778), anti-USP25 (Sigma, HPA018297), anti-HMGCS1 (Abcam,
ab155787), anti-calreticulin (Stressgen, SPA-600), anti-Lamin A/C (Protein-
tech, 10298-1), anti-ubiquitinated proteins (EMD Millipore, FK2 04-263),
anti-actin
(Sigma,
A3854),
anti-vinculin
(Sigma,
V9131). Fluorescent
secondary antibodies were from LI-COR and BioRad. HR-coupled anti-
bodies were from GE Healthcare. Immunofluorescence g
For colony formation experiments, cells were seeded in 6 cm plates at very
low density and treated with doxycycline or solvent as indicated. For cell
viability assay, cells were seeded in 96 well plates and treated as indicated. After incubation, cells were washed with PBS and fixed for 10 min in 3.7%
paraformaldehyde (PFA). Cells were washed again and stained with 0.1%
crystal violet solution for 1 h. Plates were rinsed in water, dried and
extracted using 10% acetic acid. Absorbance was measured at 550 nm. Cells were grown on glass slides (Ibidi), washed with PBS and fixed with 3.7
% PFA for 10 min. Cells were permeabilized with 0.2 % Triton X-100 for
10 min, washed with PBS and blocked with 3 % BSA in PBS for 30 min. Primary antibodies were diluted in 1 % BSA in PBS and incubated overnight
at 4 °C. Cells were washed twice with PBS and incubated with fluorescently
labelled secondary antibodies (Alexa488 and Alexa633, Invitrogen) for 1 h in
the dark. Finally, cells were washed three times with PBS and mounted with
mounting medium containing DAPI (Duolink®, Sigma). Concentration of
primary antibodies was experimentally determined (1:100–1:50), secondary
antibodies were used in a concentration of 1:200. Gene silencing and CRISPR/Cas9 knock-out For direct IP, cells were lysed
in RIPA buffer. Immunoprecipitation was performed using 0.75 mg of
Dynabeads™Protein A/G (Invitrogen), 5 μg of anti-SREBP2 (R&D, AF7119)
or 5 µg of anti-USP28 (Sigma, HPA006778) and 400 μg (U2OS) or 1 mg (A431)
of protein lysate. Goat or rabbit IgG was used as a control. for 30 min. Pellets were lysed in fractionation buffer (20 mM HEPES pH 7.4,
10 mM KCl, 2 mM MgCl2, 1 mM EDTA, 1 mM EGTA) supplemented with
protease inhibitors for 30 min, passed through a 27-gauge needle, incubated
for 20 min and cleared by centrifugation. Pellets were washed and extracted
in PBS with 0.1% SDS. Genomic DNA was sheared by passing through a 22-
gauge needle. SREBP2-enriched fraction was cleared by centrifugation,
protein concentration was determined by BCA. For direct IP, cells were lysed
in RIPA buffer. Immunoprecipitation was performed using 0.75 mg of
Dynabeads™Protein A/G (Invitrogen), 5 μg of anti-SREBP2 (R&D, AF7119)
or 5 µg of anti-USP28 (Sigma, HPA006778) and 400 μg (U2OS) or 1 mg (A431)
of protein lysate. Goat or rabbit IgG was used as a control. Human lung cancer tissue microarrays (TMA) Human lung cancer tissue microarrays (TMA)
Human lung cancer samples were obtained from the Pathology Depart-
ment at the University Hospital Würzburg (Germany) with informed
consent from all patients. Experiments were in agreement with the
principles set out in the WMA Declaration of Helsinki and the Department
of Health and Human Services Belmont Report. Samples were approved
under Ethics Approval 17/01/2006 (University Hospital Würzburg). Addi-
tional human samples were obtained from the Instituto Maimónides de
Investigación Biomédica de Córdoba (IMIBIC), Córdoba, Spain after
informed consent was given approved under ethical approval licences
Decret 439/2010 (Hospital Universitario Reina Sofia). TMAs were prepared
as previously described [26]. In brief, paraffin moulds were cast using an
Arraymold Kit (IHC World, Kit D, IW-115, core diameter 2 mm, 36 cores). Human samples were cut and stained using haematoxylin and eosin and
digitalized using a 3D Histech slide scanner (panoramic FLASH). Tumour
and non-transformed tissues were identified, manually ‘punched’ and
transferred. Upon completion, 3-µm thick sections were cut using a
microtome and processed. Transient transfection Cells were transfected using Polyethylenimine (PEI, branched) (Sigma). Plasmid-DNA and PEI were separately prepared in Optimem in DNA:PEI
ratio of 1:2. Both solutions were incubated for 5 min and afterwards mixed
and incubated for 20 min at RT. Cells were washed once with PBS and
medium was changed to transfection medium (DMEM, 2% FCS). DNA-PEI
solution was added dropwise to the cells and incubated up to 24 h. Gene silencing and CRISPR/Cas9 knock-out For LC-MS analysis of cholesterol and ubiquinone, 3 µl of sample was
applied to a C8 column (Accucore C8, 2.6 µm particles, 50 × 2.1 mm, For inducible gene silencing, shRNA sequences targeting human USP28
and SREBF2 were designed using splashRNA (http://splashrna.mskcc.org) Cell Death & Differentiation (2023) 30:1710 – 1725 C.R. Maier et al. C.R. Maier et al. 1712 Thermo Scientific). Mobile phase buffer A consisted of 0.1% formic acid in
CH3CN/H2O (10/90, v/v) and mobile phase buffer B consisted of 0.1%
formic acid in CH3CN/iPrOH/H2O (45/45/10, v/v/v). After sample application
at 40 °C, the LC gradient program was 20% solvent B for 2 min, followed by
a linear increase to 99.5% solvent B within 5 min, then maintaining 99.5%
solvent B for 28 min, then returning to 20% B within 1 min. The column
was equilibrated at 20% B for 5 min prior every injection. The flow rate was
maintained at 350 µl/min. The eluent was directed to the ESI source of the
MS instrument from 4.0 min to 35.0 min after sample injection. MS analysis
was performed on a Q-Exactive plus mass spectrometer (Thermo
Scientific). For the detection of cholesterol, the following scan and HESI
source parameters were applied in positive mode: 350–400 m/z; resolution:
70,000; AGC-Target 3E6; maximum injection time: 200 ms; sheath gas: 30;
auxiliary gas: 5; aux gas heater temperature: 350 °C; spray voltage: 3.2 kV;
capillary temperature: 320 °C, S-lens RF level: 55.0. For the detection of
coenzyme Q10, the following scan and HESI source parameters were
applied in polarity switching mode: 200–1600 m/z; resolution: 70,000; AGC-
Target 1E6; maximum injection time: 50 ms; sheath gas: 30; auxiliary gas: 5;
aux gas heater temperature: 350 °C; spray voltage: 3.2 kV; capillary
temperature: 320 °C, S-lens RF level: 55.0. Signal determination and
quantitation was performed using El-Maven Software Version 0.12.0
(https://elucidata.io/el-maven/) and natural abundance correction was
performed using FluxFix (http://fluxfix.science/). for 30 min. Pellets were lysed in fractionation buffer (20 mM HEPES pH 7.4,
10 mM KCl, 2 mM MgCl2, 1 mM EDTA, 1 mM EGTA) supplemented with
protease inhibitors for 30 min, passed through a 27-gauge needle, incubated
for 20 min and cleared by centrifugation. Pellets were washed and extracted
in PBS with 0.1% SDS. Genomic DNA was sheared by passing through a 22-
gauge needle. SREBP2-enriched fraction was cleared by centrifugation,
protein concentration was determined by BCA. Immunoprecipitation (IP) Cells were harvested by scraping, dithiobis[succinimidylpropionate] (DSP,
Sigma) was added in a final concentration of 0.8 mM in PBS and incubated Cell Death & Differentiation (2023) 30:1710 – 1725 C.R. Maier et al. 1713 structural homology to USP28 [30, 31], was unaffected (Fig. 1a). Protein extracts of doxycycline-treated cells were submitted to
quantitative LC-MS analysis [10] to identify global changes in
cellular proteome. Pathway analysis (www.pantherdb.org) of
proteins
downregulated
after
USP28
depletion
(FDR ≤0.05)
revealed strong regulation of pathways linked to the ubiquitin-
proteasome system, as well as DNA replication and cell cycle
control (Fig. 1b). Interestingly, regulation of cholesterol biosynth-
esis was also strongly associated with USP28 silencing (Fig. 1b). Detailed analysis revealed significant downregulation of proteins
mapping to the upper part of the cholesterol biosynthesis
pathway, also known as the mevalonate pathway (MVP), which
facilitates the synthesis of isoprenoids that can be used as
substrates for protein prenylation [20]. Proteins of the lower part
of cholesterol synthesis pathway downstream of SQLE were less
strongly downregulated or even induced (Figs. 1c and S1a). (Abcam, ab155787). Murine samples were stained with anti-USP28 (Sigma,
HPA006778), Nkx2-1 (TTF1; Santa Cruz sc-13040) anti-HMGCS1 (Abcam,
ab155787), anti-SREBF2 (R&D, MAB7119), anti-Δnp63 (4A4) (Ventana,
05867061001) and anti-Cytokeratin 5 (Bimake, A5439). Slides were devel-
oped with DAB staining solution (SignalStainR DAB Substrate Kit, Cell
Signaling 8059 S) and counterstained with haematoxylin (Sigma H3136). Slides were scanned at 40x resolution using a Pannoramic DESK II slide
scanner (3D Histech) and analysed using QuPath (version 0.3.2). (Abcam, ab155787). Murine samples were stained with anti-USP28 (Sigma,
HPA006778), Nkx2-1 (TTF1; Santa Cruz sc-13040) anti-HMGCS1 (Abcam,
ab155787), anti-SREBF2 (R&D, MAB7119), anti-Δnp63 (4A4) (Ventana,
05867061001) and anti-Cytokeratin 5 (Bimake, A5439). Slides were devel-
oped with DAB staining solution (SignalStainR DAB Substrate Kit, Cell
Signaling 8059 S) and counterstained with haematoxylin (Sigma H3136). Slides were scanned at 40x resolution using a Pannoramic DESK II slide
scanner (3D Histech) and analysed using QuPath (version 0.3.2). Animal experiments p
All in vivo experiments were approved by the Regierung Unterfranken
and the ethics committee under the licence numbers 2532-2-362, 2532-2-
367
and
2532-2-374. The
mouse
strain
used
was
B6(C)-Gt(ROSA)
26Sorem1.1(CAG-cas9*,-EGFP)Rsky/J (Jackson Laboratories, stock #028555). All
animals were housed in standard cages in a pathogen-free facility on a
12 h light/dark cycle with ad libitum access to food and water. Animal
health monitoring via sentinel animal screening is carried out in
accordance with FELASA 2014 guidelines and conducted every 3 months. Adult mice (7–8 weeks old) were anaesthetized with isoflurane and
intratracheally intubated with 60 µl AAV (1 × 10^11 PFU) diluted in PBS. Viruses were quantified using Coomassie staining protocol [29] and via
qPCR
(https://www.addgene.org/protocols/aav-titration-qpcr-using-sybr-
green-technology/). At the indicated time point, animals were sacrificed
by cervical dislocation and lungs were dissected and fixed using 4 %
neutral buffered formalin (Sigma). Tumour burden was determined by
calculating the percent tumour area relative to total lung area for each
animal using QuPath 0.3.2 (https://qupath.github.io/). No animals were
excluded from the analysis and tissues were analysed in a blinded manner. g
g
p
y
pp g
We next applied stable isotope labelling using U-13C-Glucose
to follow MVP metabolite flux in A431 cells. Surprisingly, the
cholesterol pool remained largely unlabelled, suggesting that these
cells obtain cholesterol mostly through uptake rather than de novo
synthesis (Fig. 1h). In contrast, a large proportion of ubiquinone
(CoQ10), a metabolite containing ten isoprenyl subunits in its side-
chain,
showed
a
high
degree
of
labelling. Depletion
of
USP28 significantly reduced the proportion of labelled CoQ10,
indicating downregulation of the MVP, while cholesterol labelling
was only mildly affected (Fig. 1i). MVP regulation by USP28 was also
confirmed in U2OS osteosarcoma cells, where knockout of USP28
reduced expression of HMGCS1 and resulted in a small but
significant decrease in cholesterol levels (Fig. S1h, i). AAV production More-
over, we observed a substantial overlap between genes regulated
by USP28 or SREBP2, with several of the overlapping genes also
found among the significantly regulated proteins (Fig. 1g). Together, these data indicate that USP28 alters the expression
of SREBP2 target genes, particularly those mapping to the MVP. 3 SREBP2 associates and colocalises with USP28 SREBP2 associates and colocalises with USP28
As USP28 silencing resulted in downregulation of SREBP2 target
genes without affecting expression of SREBF2 mRNA (Fig. S1d), we
next investigated whether USP28 regulates SREBP2 protein level. Immunoblot analysis of USP28-silenced U2OS cells (Fig. 2a)
revealed a marked reduction in expression of HMGCS1 as well
as a loss of the mature form of SREBP2, which forms multiple
bands of approximately 65–70 kDa due to post-translational
modifications [32]. In contrast, the 125 kDa full-length SREBP2
protein was not affected. Immunofluorescence staining showed
mostly nuclear localisation of USP28, while SREBP2 is found both
in the nucleus and in a perinuclear region (Fig. 2b), likely
representing the ER-membrane. Moreover, individual cells dis-
playing low USP28 staining (marked by arrowheads) were mostly Reporting summary
Further information on research design is available in the Nature Portfolio
Reporting Summary linked to this article. AAV production We next applied RNA sequencing (RNAseq) to establish the
transcriptional response to USP28 silencing in A431 cells. Gene set
enrichment analysis revealed strong downregulation of direct
ΔNP63 target genes in response to silencing of USP28 (Fig. 1d),
confirming our previous findings [16]. Furthermore, genes involved
in cholesterol biosynthesis also showed significant downregulation
(Fig. 1d, e), suggesting that the reduction in MVP pathway proteins
is the consequence of altered transcriptional activity. Efficient
downregulation of USP28 and HMGCS1 mRNA and protein was
also
achieved
when
cells
were
treated
with
0.5 µg/ml
of
doxycycline, confirming specificity of the effect (Figs. 1f and S1b). Transcription of MVP genes is primarily controlled by SREBP2, a
basic helix-loop-helix transcription factor that is activated upon
low
intracellular
sterol
levels
via
regulated
intramembrane
processing [20]. We, therefore, investigated the transcriptional
response to SREBF2 silencing in A431 cells. Silencing of SREBF2
resulted in clear downregulation of cholesterol biosynthesis
enzymes and also affected ΔNP63 target genes (Fig. S1c–g),
suggesting that USP28 and SREBP2 functionally overlap. More-
over, we observed a substantial overlap between genes regulated
by USP28 or SREBP2, with several of the overlapping genes also
found among the significantly regulated proteins (Fig. 1g). Together, these data indicate that USP28 alters the expression
of SREBP2 target genes, particularly those mapping to the MVP. We next applied stable isotope labelling using U-13C-Glucose
to follow MVP metabolite flux in A431 cells. Surprisingly, the
cholesterol pool remained largely unlabelled, suggesting that these
cells obtain cholesterol mostly through uptake rather than de novo
synthesis (Fig. 1h). In contrast, a large proportion of ubiquinone
(CoQ10), a metabolite containing ten isoprenyl subunits in its side-
chain,
showed
a
high
degree
of
labelling. Depletion
of
USP28 significantly reduced the proportion of labelled CoQ10,
indicating downregulation of the MVP, while cholesterol labelling
was only mildly affected (Fig. 1i). MVP regulation by USP28 was also
confirmed in U2OS osteosarcoma cells, where knockout of USP28
reduced expression of HMGCS1 and resulted in a small but
significant decrease in cholesterol levels (Fig. S1h, i). Transcription of MVP genes is primarily controlled by SREBP2, a
basic helix-loop-helix transcription factor that is activated upon
low
intracellular
sterol
levels
via
regulated
intramembrane
processing [20]. We, therefore, investigated the transcriptional
response to SREBF2 silencing in A431 cells. Silencing of SREBF2
resulted in clear downregulation of cholesterol biosynthesis
enzymes and also affected ΔNP63 target genes (Fig. S1c–g),
suggesting that USP28 and SREBP2 functionally overlap. Statistical analysis Statistical analysis was performed using R 3.10 (http://www.r-project.org) or
Graphpad Prism 9 (Graphpad Software Inc.). Drug synergy was calculated
using the synergyfinder tool (https://synergyfinder.fimm.fi) with ZIP model. Cell Death & Differentiation (2023) 30:1710 – 1725 AAV production To produce AAV particles, 5 million HHEK293T cells per dish were seeded
in 15 cm plates and cultivated until a confluence of ~60–70 %. Cells were
transfected with the pRepCap (pRC, Cell Biolabs Inc.), the cis-plasmid
(pAAV) and the pAdDeltaF6 (Addgene #112867) in a 1:1:2 molar ratio by
mixing the DNA in 2 ml DMEM (w/o FCS) and adding polyethylenimine
(PEI, Polysciences (DNA:PEI ratio of 1:2). The mixture was incubated 15 min
at room temperature and added dropwise to the plates. To harvest AAV
particles, cells and supernatants were collected after 96 h and transferred
into a 50 ml conical tube. At first, NaCl was added (final concentration
0.5 M) and slowly mixed for 1 h at 4 °C. Next, Chloroform was added (final
concentration 10 %) and slowly mixed for 30 min at 4 °C. The suspension
was centrifuged at 2000 × g for 30 min at 4 °C. Water phase was transferred
into a new conical tube and PEG 8000 was added (final concentration
10 %) and mixed well. AAV was precipitated overnight at 4 °C. After the
centrifugation at 2000 × g for 20 min at 4 °C the pellet was dissolved in AAV
resuspension buffer (PBS + 0.001% pluronic F68 + 200 mM NaCl) (~100 µl/
15 cm dish used) and protease inhibitor and DNase/RNase were added. Reaction was incubated for 2 h at 37 °C, chloroform in a ratio of 1:1
was added and samples were centrifuge at 12,000 × g for 5 min at 4 °C. The
chloroform step was repeated and the water phase was collected. Virus
titre was determined by quantitative PCR using primers against the ITR of
the AAV as previously reported [27]. This procedure was previously
described [28]. We next applied RNA sequencing (RNAseq) to establish the
transcriptional response to USP28 silencing in A431 cells. Gene set
enrichment analysis revealed strong downregulation of direct
ΔNP63 target genes in response to silencing of USP28 (Fig. 1d),
confirming our previous findings [16]. Furthermore, genes involved
in cholesterol biosynthesis also showed significant downregulation
(Fig. 1d, e), suggesting that the reduction in MVP pathway proteins
is the consequence of altered transcriptional activity. Efficient
downregulation of USP28 and HMGCS1 mRNA and protein was
also
achieved
when
cells
were
treated
with
0.5 µg/ml
of
doxycycline, confirming specificity of the effect (Figs. 1f and S1b). USP28 regulates the mevalonate pathway To study the consequences of USP28 depletion in squamous cell
carcinoma, we generated A431 cells expressing doxycycline-
inducible shRNA sequences targeting USP28 (#1 and #2) or non-
targeting control (shRenilla). Doxycycline treatment for 96 h
induced almost complete depletion of USP28 protein, while the
related deubiquitinating enzyme USP25, which shows significant Cell Death & Differentiation (2023) 30:1710 – 1725 C.R. Maier et al. 1714 evoid of nuclear SREBP2 (Fig. 2b), with nuclear staining
tensities of USP28 and SREBP2 showing positive correlation
g. S2a). Cell fractionation in A431 cells revealed that USP28 and
ll length SREBP2 were found in the cytoplasm and cytoplasmic
membranes (Fig. 2c), as indicated by the presence of th
membrane protein calreticulin (CALR) and cytoplasmic G
USP28 also colocalised with mature SREBP2 in the nuclear fra
identified by the presence of MYC and lamin A/C (Fig. 2c). Cell Death & Differentiation (2023) 30:1710 nuclear staining
ositive correlation
d that USP28 and
and cytoplasmic
membranes (Fig. 2c), as indicated by the presence of the ER
membrane protein calreticulin (CALR) and cytoplasmic GAPDH
USP28 also colocalised with mature SREBP2 in the nuclear fraction
identified by the presence of MYC and lamin A/C (Fig. 2c). devoid of nuclear SREBP2 (Fig. 2b), with nuclear staining
intensities of USP28 and SREBP2 showing positive correlation
(Fig. S2a). Cell fractionation in A431 cells revealed that USP28 and
full length SREBP2 were found in the cytoplasm and cytoplasmic devoid of nuclear SREBP2 (Fig. 2b), with nuclear staining
intensities of USP28 and SREBP2 showing positive correlation
(Fig. S2a). Cell fractionation in A431 cells revealed that USP28 and
full length SREBP2 were found in the cytoplasm and cytoplasmic membranes (Fig. 2c), as indicated by the presence of the ER-
membrane protein calreticulin (CALR) and cytoplasmic GAPDH. USP28 also colocalised with mature SREBP2 in the nuclear fraction,
identified by the presence of MYC and lamin A/C (Fig. 2c). Cell Death & Differentiation (2023) 30:1710 – 1725 USP28 deubiquitinates and stabilises SREBP2 We next determined whether USP28 regulates SREBP2 stability. First,
we treated USP28 wild-type or KO U2OS cells with cycloheximide
(CHX) to block translation in a time-course experiment. Deletion of
USP28 accelerated the loss of both full-length and mature SREBP2
compared to wild-type cells (Fig. 3a, b). Similar results were also
obtained after USP28 silencing (Fig. S3a, b). Furthermore, silencing of
USP28 in A431 prevented the stabilisation of mature SREBP2
following proteasome inhibition by MG-132 (Fig. 3c). and shUSP28#2, respectively). The MVP has multiple metabolic outputs that participate in
numerous different cellular processes, including protein prenyla-
tion and the mitochondrial electron transfer chain (ETC) (Fig. 4c)
[20]. We have shown previously that the MVP supplies ubiquinone
(CoQ10) to support enhanced pyrimidine nucleotide biosynthesis
in TP53 deficient colon cancer cells [34]. To determine which MVP
output is crucial for cell survival, we treated USP28-silenced A431
cells with simvastatin in combination with either mevalonate, cell-
permeable cholesterol, CoQ10, nucleosides or geranyl-geranyl-
pyrophosphate (GG-PP), the substrate for the prenylation of small
GTPases [35, 36]. Mevalonate restored cell viability of simvastatin
treated cells, confirming the specificity of the drug (Fig. 4d, e). In
contrast to the other treatments, only GG-PP restored cell viability
at least partially, indicating that the provision of substrates for
protein prenylation is an essential output of the MVP in these cells. In order to explore potential mechanisms downstream of
SREBP2 in SCC cells, we further analysed the RNAseq data from
A431 cells after SREBF2 silencing. Interestingly, SREBF2 depletion
caused a marked induction of gene expression signatures
connected to interferon and T cell receptor signalling (Fig. 4f). Among the strongest regulated genes within these gene sets were
those coding for major histocompatibility complex class I and II
(MHC-I, MHC-II) proteins (Fig. S4c), suggesting that depletion of
SREBF2 could trigger enhanced antigen presentation. This could
be reminiscent of a mechanism recently described for pancreatic
cancer where deletion of oncogenic Kras blocked immune evasion
via the induction of MHC gene expression [37]. To address the mode of regulation of SREBP2 by USP28, we
used U2OS cells to co-express mature SREBP2 together with either
wild-type USP28 (WT) or a mutant variant, in which the catalytic
cysteine (Cys 171) within the USP domain was replaced by alanine
(CA). This showed that wild-type USP28 increases the levels of
mature SREBP2, which was not observed with the CA mutant
(Fig. 3d). C.R. Maier et al. 1715 Fig. 1
USP28 regulates the mevalonate pathway (MVP). a A431 cells expressing inducible shRNA sequences targeting USP28 (shUSP28#1
and shUSP28#2) or non-targeting control (shRen) were treated with 1 µg/ml of doxycycline (DOX) for 96 h. Protein lysates were analysed for
expression of USP28 and USP25 by immunoblotting. Vinculin is shown as loading control. b Proteins differentially regulated by USP28
(shUSP28#1) silencing for 72 h in A431 cells [10] (FDR ≤0.05 compared to shRen, total of 2200 proteins) were subjected to pathway analysis
using the PANTHER tool. c Differential expression of MVP proteins as determined by proteomics analysis. Pathway map is coloured by log2FC
(*q-value ≤0.05, n = 3). d A431 cells expressing an inducible shRNA sequence targeting USP28 (shUSP28#2) or non-targeting control (shRen)
were treated with 1 µg/ml of doxycycline (DOX) for 120 h. Changes in gene expression were analysed and enrichment plots for gene sets
mapping to direct ΔNP63 targets and cholesterol biosynthesis are shown. e Validation of downregulation of mevalonate pathway genes
following USP28 silencing using 1 µg/ml of doxycycline (DOX) for 96 h. Data are presented as mean ± SD of three independent experiments
(*p < 0.05, ****p < 0.0001, unpaired two-tailed t-test with FDR). f A431 cells expressing an inducible shRNA sequence targeting USP28
(shUSP28#2) or non-targeting control (shRen) were treated with 0.5 µg/ml of doxycycline (DOX) for 96 h. Expression of USP28 and HMGCS1
were analysed by immunoblotting. Actin is shown as loading control. g Overlap between genes downregulated by silencing of USP28 and
SREBP2 in A431 cells. Among the 252 overlapping genes are 8 mevalonate pathway enzymes (boxed) that also showed downregulation in the
proteomics dataset. h A431 cells were incubated with medium containing 25 mM [U13C]-glucose for 48 h. Metabolites were extracted and
analysed by LC-MS. Fractions of labelled and unlabelled ubiquinone (CoQ10) and cholesterol are shown. Data are presented as mean ± SD of
three independent experiments. i A431 cells expressing inducible shRNA sequences targeting USP28 (shUSP28#1 and shUSP28#2) or non-
targeting control (shRen) were treated with 1 µg/ml of doxycycline (DOX) for 120 h. During the last 48 h, cells were incubated with medium
containing 25 mM [U13C]-glucose. Metabolites were extracted and analysed by LC-MS. Fractions of labelled and unlabelled ubiquinone
(CoQ10) and cholesterol are shown. Data are presented as mean ± SD of three independent experiments (n.s. Depletion of USP28 renders cancer cells highly sensitive to
MVP inhibition Depletion of USP28 renders cancer cells highly sensitive to
MVP inhibition We next employed proximity ligation assays to investigate
association of the two proteins in situ. Indeed, a strong enhance-
ment of nuclear and cytoplasmic PLA signals was observed when
the probes for USP28 and SREBP2 were combined (Fig. 2d). USP28
was also detected in SREBP2 immunoprecipitates from lysates of
A431 and U2OS cells (Fig. 2e) and both proteins could be
coprecipitated from U2OS cells (Fig. 2f). Combined, these results
indicate that USP28 and SREBP2 co-localise and interact, suggesting
that USP28 could function as a deubiquitinating enzyme for SREBP2. However, it is possible that the interaction between SREBP2 and
USP28 is indirect and involves additional, unknown proteins. Having identified USP28 as a regulator of SREBP2, we next
investigated whether targeting the USP28/SREBP2 axis would
affect viability of squamous cancer cells. Consistent with previous
findings [10], we observed that silencing of USP28 reduced colony
formation in A431 cells (Fig. S4a, b). Interestingly, USP28 silencing
rendered A431 cells highly sensitive to MVP inhibition by the
HMGCR-inhibitor simvastatin (Fig. 4a, b), suggesting cooperation
between the two pathways. While USP28 silencing or simvastatin
treatment alone resulted in an approximately 50% reduction,
combining these two treatments resulted in an 80–90% reduction
in cell number (combination index 0.49 and 0.33 for shUSP28#1
and shUSP28#2, respectively). non-significant, *p < 0.05,
****p < 0.0001, one-way ANOVA with post-hoc Dunnett’s test). Cell Death & Differentiation (2023) 30:1710 – 1725 USP28 deubiquitinates and stabilises SREBP2 USP28 WT but not CA mutant also enhanced levels of
c-Myc and mature SREBP1a (Fig. S3c, d), indicating a similar mode
of regulation. Furthermore, USP28 WT but not CA mutant
decreased levels of mature SREBP2 in a pull-down assay using
6His-tagged K48-only ubiquitin (Fig. 3e), while silencing of USP28
increased the amount of SREBP2 ubiquitination detected by
immunoprecipitation in A431 cells (Fig. 3f). FBXW7
has
been
implicated
in
the
ubiquitination
and
destabilisation of both SREBP1 and SREBP2 [21] and several
established targets of USP28 are also substrates for FBXW7 [33]. Interestingly, deletion of the CPD motif in SREBP2, which
abolished the destabilisation of SREBP2 by FBXW7, only had a
partial effect on the ability of USP28 to stabilise SREBP2 (compare
3.9- to 1.7-fold increase in mSREBP2 protein levels, Fig. 3g). This
was in contrast to similar experiments performed with SREBP1,
where mutation of the CPD completely blocked regulation by
both FBXW7 and USP28 (Fig. S3e). This suggests that USP28 might
also target ubiquitin residues in SREBP2 introduced by other
ubiquitin ligases, potentially explaining the strong stabilising effect
on this transcription factor. USP28 and SREBP2 are overexpressed in SCC
USP28 has been identified as a major oncogenic regulator in
squamous cell carcinoma [33] and its genetic deletion or inhibition
was shown to block tumour growth in mouse models of LSCC
[16, 17]. Analysis of TCGA data revealed that a set of cholesterol USP28 and SREBP2 are overexpressed in SCC Cell Death & Differentiation (2023) 30:1710 – 1725 C.R. Maier et al. biosynthesis genes showed positive correlation with USP28
i
i
i
l
ll
i
d
d
i
l
Furthermore, expression of USP28, SREBF2, HMGCS1 and
i
ifi
tl
hi h
i
LSCC
d t
LADC (Fi
Fig. 2
USP28 regulates SREBP2 via close interaction. a U2OS cells expressing inducible shRNA sequences targeting USP28 (shUSP2
shUSP28#2) or non-targeting control (shRen) were treated with 1 µg/ml of doxycycline (DOX) for 72 or 96 h. Protein lysates were anal
expression of USP28, full length (flSREBP2) and mature SREBP2 (mSREBP2) and HMGCS1 by immunoblotting. Actin is shown as
control. b U2OS cells were stained for USP28 and SREBP2 by immunofluorescence. DAPI was used to mark nuclei. Cells with low
expression of USP28 and SREBP2 are marked with arrowheads. c A431 cells were subjected to cell fractionation into cytoplasm and
Fractions were analysed for expression of USP28 as well as full length (flSREBP2) and mature SREBP2 (mSREBP2). Calreticulin served as
for ER-membranes, c-Myc and Lamin A/C as markers for nuclei and GAPDH as marker for cytoplasm. Unspecific bands for flSREBP2 are
by asterisks. Two independent replicate fractionations are shown. d U2OS cells were analysed by proximity ligation assay using an
specific for USP28 and SREBP2 either alone or in combination. Number of PLA signals per cell were analysed in >42 cells from at lea
biologically independent samples. Data are presented as mean ± SD (*p < 0.05, **p < 0.01, one-way ANOVA with post-hoc Dunnet
e A431 and U2OS cells were subjected to cross-linking with DSP. Cells were fractionated and lysates were subjected to immunopreci
using antibodies detecting SREBP2 or matched immunoglobulin controls (IgG). Input and precipitates were analysed for presenc
length and mature SREBP2 as well as USP28. IgG bands are marked by asterisks. f Lysates of U2OS cells were subje
immunoprecipitation using antibodies detecting SREBP2 (left) or USP28 (right). Precipitates were analysed for presence of full-len
mature SREBP2 as well as USP28. C.R. Maier et al. 1716 Fig. 2
USP28 regulates SREBP2 via close interaction. a U2OS cells expressing inducible shRNA sequences targeting USP28 (shUSP28#1 and
shUSP28#2) or non-targeting control (shRen) were treated with 1 µg/ml of doxycycline (DOX) for 72 or 96 h. Protein lysates were analysed for
expression of USP28, full length (flSREBP2) and mature SREBP2 (mSREBP2) and HMGCS1 by immunoblotting. Actin is shown as loading
control. USP28 and SREBP2 are overexpressed in SCC b U2OS cells were stained for USP28 and SREBP2 by immunofluorescence. DAPI was used to mark nuclei. Cells with low nuclear
expression of USP28 and SREBP2 are marked with arrowheads. c A431 cells were subjected to cell fractionation into cytoplasm and nuclei. Fractions were analysed for expression of USP28 as well as full length (flSREBP2) and mature SREBP2 (mSREBP2). Calreticulin served as marker
for ER-membranes, c-Myc and Lamin A/C as markers for nuclei and GAPDH as marker for cytoplasm. Unspecific bands for flSREBP2 are marked
by asterisks. Two independent replicate fractionations are shown. d U2OS cells were analysed by proximity ligation assay using antibodies
specific for USP28 and SREBP2 either alone or in combination. Number of PLA signals per cell were analysed in >42 cells from at least three
biologically independent samples. Data are presented as mean ± SD (*p < 0.05, **p < 0.01, one-way ANOVA with post-hoc Dunnett’s test). e A431 and U2OS cells were subjected to cross-linking with DSP. Cells were fractionated and lysates were subjected to immunoprecipitation
using antibodies detecting SREBP2 or matched immunoglobulin controls (IgG). Input and precipitates were analysed for presence of full
length and mature SREBP2 as well as USP28. IgG bands are marked by asterisks. f Lysates of U2OS cells were subjected to
immunoprecipitation using antibodies detecting SREBP2 (left) or USP28 (right). Precipitates were analysed for presence of full-length and
mature SREBP2 as well as USP28. Fig. 2
USP28 regulates SREBP2 via close interaction. a U2OS cells expressing inducible shRNA sequences targeting USP28 (shUSP28#1 and
shUSP28#2) or non-targeting control (shRen) were treated with 1 µg/ml of doxycycline (DOX) for 72 or 96 h. Protein lysates were analysed for
expression of USP28, full length (flSREBP2) and mature SREBP2 (mSREBP2) and HMGCS1 by immunoblotting. Actin is shown as loading
control. b U2OS cells were stained for USP28 and SREBP2 by immunofluorescence. DAPI was used to mark nuclei. Cells with low nuclear
expression of USP28 and SREBP2 are marked with arrowheads. c A431 cells were subjected to cell fractionation into cytoplasm and nuclei. Fractions were analysed for expression of USP28 as well as full length (flSREBP2) and mature SREBP2 (mSREBP2). Calreticulin served as marker
for ER-membranes, c-Myc and Lamin A/C as markers for nuclei and GAPDH as marker for cytoplasm. Unspecific bands for flSREBP2 are marked
by asterisks. Two independent replicate fractionations are shown. USP28 and SREBP2 are overexpressed in SCC Levels of USP28 and mature SREBP2 were determined by immunoblotting. Actin is shown as loading control. e HEK293 cells were transfected with expression vectors coding for HA-tagged USP28 or HA-tagged mSREBP2 together with His6-Ubiquitin
(K48-only). Ubiquitinated proteins were purified using Ni-NTA and analysed by immunoblotting (Ub). Presence of USP28 and SREBP2 was
confirmed in the input. f A431 cells expressing inducible shRNA sequences targeting USP28 (shUSP28#2) were treated with 1 µg/mL
doxycycline (DOX) or solvent (ethanol) for 96 h with 20 µM MG132 being added during the last 6 h. Cells were lysed and subjected to
immunoprecipitation using antibodies detecting SREBP2. Precipitates were analysed for the presence of ubiquitinated proteins (Ub). Efficient
silencing of USP28 and presence of full length and mature SREBP2 was confirmed in the input. Actin is shown as a loading control. IgG bands
are marked by asterisks. g U2OS cells were transfected with either wild type or CPD mutant (Ser 432 and 436 to Ala) HA-tagged mature
SREBP2 together with HA-tagged USP28 or Flag-FBXW7. Levels of USP28, FBXW7 and mature SREBP2 were determined by immunoblotting. Actin is shown as loading control. Fig. 3
USP28 controls the stability of mature SREBP2. a U2OS cells were transfected with gRNAs targeting USP28 (KO USP28) and parental
controls were treated with 50 µg/ml cycloheximide for the indicated times. Levels of USP28 as well as full length (flSREBP2) and mature
SREBP2 (mSREBP2) were determined by immunoblotting. Actin is shown as loading control. b Quantification of full-length and mature
SREBP2 signals relative to actin from (a). c A431 cells expressing inducible shRNA sequences targeting USP28 (shUSP28#2) were treated with
0.5 µg/ml of doxycycline (DOX) or solvent (ethanol, EtOH) for 72 h. During the last 6 h, 20 µM MG-132 was added. Cells were lysed and
analysed for expression of full-length (flSREBP2) and mature SREBP2 (mSREBP2). Actin is shown as loading control. d U2OS cells were
transfected with expression vectors coding for HA-tagged mature SREBP2 (aa 1-484, HA-mSREBP2) together with HA-tagged wild type (WT) or
catalytically inactive (CA) USP28. Levels of USP28 and mature SREBP2 were determined by immunoblotting. Actin is shown as loading control. e HEK293 cells were transfected with expression vectors coding for HA-tagged USP28 or HA-tagged mSREBP2 together with His6-Ubiquitin
(K48-only). Ubiquitinated proteins were purified using Ni-NTA and analysed by immunoblotting (Ub). Presence of USP28 and SREBP2 was
confirmed in the input. USP28 and SREBP2 are overexpressed in SCC d U2OS cells were analysed by proximity ligation assay using antibodies
specific for USP28 and SREBP2 either alone or in combination. Number of PLA signals per cell were analysed in >42 cells from at least three
biologically independent samples. Data are presented as mean ± SD (*p < 0.05, **p < 0.01, one-way ANOVA with post-hoc Dunnett’s test). e A431 and U2OS cells were subjected to cross-linking with DSP. Cells were fractionated and lysates were subjected to immunoprecipitation
using antibodies detecting SREBP2 or matched immunoglobulin controls (IgG). Input and precipitates were analysed for presence of full
length and mature SREBP2 as well as USP28. IgG bands are marked by asterisks. f Lysates of U2OS cells were subjected to
immunoprecipitation using antibodies detecting SREBP2 (left) or USP28 (right). Precipitates were analysed for presence of full-length and
mature SREBP2 as well as USP28. Furthermore, expression of USP28, SREBF2, HMGCS1 and FDFT1
was significantly higher in LSCC compared to LADC (Fig. 5a). It
should be noted that SREBP2 can also induce transcription from
the SREBF2 gene via a sterol response element (SRE) located in its Furthermore, expression of USP28, SREBF2, HMGCS1 and FDFT1
was significantly higher in LSCC compared to LADC (Fig. 5a). It
should be noted that SREBP2 can also induce transcription from
the SREBF2 gene via a sterol response element (SRE) located in its biosynthesis genes showed positive correlation with USP28
expression in cervical squamous cell carcinoma and endocervical
adenocarcinoma (CESC), head and neck squamous cell carcinoma
(HNSC) as well as in LSCC, but not in normal lung tissue (Fig. S5a). biosynthesis genes showed positive correlation with USP28
expression in cervical squamous cell carcinoma and endocervical
adenocarcinoma (CESC), head and neck squamous cell carcinoma
(HNSC) as well as in LSCC, but not in normal lung tissue (Fig. S5a). Cell Death & Differentiation (2023) 30:1710 – 1725 3
USP28 controls the stability of mature SREBP2. a U2OS cells were transfected with gRNAs targeting USP28 (KO USP28) and paren
ntrols were treated with 50 µg/ml cycloheximide for the indicated times. Levels of USP28 as well as full length (flSREBP2) and mat
EBP2 (mSREBP2) were determined by immunoblotting. Actin is shown as loading control. b Quantification of full-length and mat
EBP2 signals relative to actin from (a). c A431 cells expressing inducible shRNA sequences targeting USP28 (shUSP28#2) were treated w
µg/ml of doxycycline (DOX) or solvent (ethanol, EtOH) for 72 h. USP28 and SREBP2 are overexpressed in SCC During the last 6 h, 20 µM MG-132 was added. Cells were lysed a
alysed for expression of full-length (flSREBP2) and mature SREBP2 (mSREBP2). Actin is shown as loading control. d U2OS cells w
nsfected with expression vectors coding for HA-tagged mature SREBP2 (aa 1-484, HA-mSREBP2) together with HA-tagged wild type (WT)
alytically inactive (CA) USP28. Levels of USP28 and mature SREBP2 were determined by immunoblotting. Actin is shown as loading cont
HEK293 cells were transfected with expression vectors coding for HA-tagged USP28 or HA-tagged mSREBP2 together with His6-Ubiqu
48-only). Ubiquitinated proteins were purified using Ni-NTA and analysed by immunoblotting (Ub). Presence of USP28 and SREBP2 w
nfirmed in the input. f A431 cells expressing inducible shRNA sequences targeting USP28 (shUSP28#2) were treated with 1 µg/
xycycline (DOX) or solvent (ethanol) for 96 h with 20 µM MG132 being added during the last 6 h. Cells were lysed and subjected
munoprecipitation using antibodies detecting SREBP2. Precipitates were analysed for the presence of ubiquitinated proteins (Ub). Efficie
encing of USP28 and presence of full length and mature SREBP2 was confirmed in the input. Actin is shown as a loading control. IgG ban
marked by asterisks. g U2OS cells were transfected with either wild type or CPD mutant (Ser 432 and 436 to Ala) HA-tagged mat
EBP2 together with HA-tagged USP28 or Flag-FBXW7. Levels of USP28, FBXW7 and mature SREBP2 were determined by immunoblotti
tin is shown as loading control. C.R. Maier et al. C.R. Maier et al. 1717 Fig. 3
USP28 controls the stability of mature SREBP2. a U2OS cells were transfected with gRNAs targeting USP28 (KO USP28) and parental
controls were treated with 50 µg/ml cycloheximide for the indicated times. Levels of USP28 as well as full length (flSREBP2) and mature
SREBP2 (mSREBP2) were determined by immunoblotting. Actin is shown as loading control. b Quantification of full-length and mature
SREBP2 signals relative to actin from (a). c A431 cells expressing inducible shRNA sequences targeting USP28 (shUSP28#2) were treated with
0.5 µg/ml of doxycycline (DOX) or solvent (ethanol, EtOH) for 72 h. During the last 6 h, 20 µM MG-132 was added. Cells were lysed and
analysed for expression of full-length (flSREBP2) and mature SREBP2 (mSREBP2). Actin is shown as loading control. d U2OS cells were
transfected with expression vectors coding for HA-tagged mature SREBP2 (aa 1-484, HA-mSREBP2) together with HA-tagged wild type (WT) or
catalytically inactive (CA) USP28. USP28 and SREBP2 are overexpressed in SCC Individual analysis showed
that LUAD patients with high SREBF2 expression show poor overall survival, which is similar to that of LSCC patients
(Fig. S5d). Together, these data indicate that both USP28 and
SREBP2 are upregulated in human squamous cell carcinoma,
particularly the LSCC subtype. Finally, Kaplan–Meier analysis of human lung cancer patient data
revealed that both USP28 and SREBF2 were indicative of poor
survival in a mixed cohort (Fig. 5e). Individual analysis showed
that LUAD patients with high SREBF2 expression show poor Cell Death & Differentiation (2023) 30:1710 – 1725 USP28 and SREBP2 are overexpressed in SCC f A431 cells expressing inducible shRNA sequences targeting USP28 (shUSP28#2) were treated with 1 µg/mL
doxycycline (DOX) or solvent (ethanol) for 96 h with 20 µM MG132 being added during the last 6 h. Cells were lysed and subjected to
immunoprecipitation using antibodies detecting SREBP2. Precipitates were analysed for the presence of ubiquitinated proteins (Ub). Efficient
silencing of USP28 and presence of full length and mature SREBP2 was confirmed in the input. Actin is shown as a loading control. IgG bands
are marked by asterisks. g U2OS cells were transfected with either wild type or CPD mutant (Ser 432 and 436 to Ala) HA-tagged mature
SREBP2 together with HA-tagged USP28 or Flag-FBXW7. Levels of USP28, FBXW7 and mature SREBP2 were determined by immunoblotting. Actin is shown as loading control. This revealed that USP28, SREBP2 and HMGCS1, but not SREBP1,
were significantly higher expressed in LSCC tumours (n = 33)
compared to LADC tumours (n = 75) (Figs. 5b, c, S5b). Furthermore,
nuclear staining for USP28 showed positive correlation with SREBP2
and HMGCS2 expression in LSCC but not in LADC (Figs. 5d and S5c). promoter [38]. Thus, stabilisation of mature SREBP2 by USP28 can
also enhance SREBF2 mRNA expression. This revealed that USP28, SREBP2 and HMGCS1, but not SREBP1,
were significantly higher expressed in LSCC tumours (n = 33)
compared to LADC tumours (n = 75) (Figs. 5b, c, S5b). Furthermore,
nuclear staining for USP28 showed positive correlation with SREBP2
and HMGCS2 expression in LSCC but not in LADC (Figs. 5d and S5c). We next analysed a tissue microarray comprising over 100
tissue samples of human non-small lung cancer by immunohis-
tochemistry combined with quantitative image analysis (QuPath). Cell Death & Differentiation (2023) 30:1710 – 1725 C.R. Maier et al. C.R. Maier et al. 1718 Finally, Kaplan–Meier analysis of human lung cancer patient data
revealed that both USP28 and SREBF2 were indicative of poor
survival in a mixed cohort (Fig. 5e). Individual analysis showed
that LUAD patients with high SREBF2 expression show poor
overall survival, which is similar to that of LSCC patients
(Fig. S5d). Together, these data indicate that both USP28 and
SREBP2 are upregulated in human squamous cell carcinoma
particularly the LSCC subtype. Cell Death & Differentiation (2023) 30:1710 – 1725 Finally, Kaplan–Meier analysis of human lung cancer patient data
revealed that both USP28 and SREBF2 were indicative of poor
survival in a mixed cohort (Fig. 5e). DISCUSSION
SREBP t SREBP transcription factors integrate multiple cellular signals to
drive the expression of genes controlling fatty acid and cholesterol
biosynthesis [19, 20]. Here we have investigated the regulation of
SREBP2 by the deubiquitinating enzyme USP28. We found that
depletion of USP28 reduces the expression of multiple MVP
proteins, most prominently enzymes that catalyse the sequential
condensation of acetoacetyl-CoA to form isoprenoid chains that
are used for protein prenylation and for the synthesis important
cellular metabolites, including CoQ10, dolichol and cholesterol. Using 13C-glucose tracing, we found that A431 cells display very
low levels of de novo cholesterol biosynthesis but divert glucose-
derived carbons into the synthesis of isoprenoids. We have
recently shown that squalene epoxidase (SQLE) is upregulated in
aggressive prostate cancer through a micro-RNA dependent
mechanism to drive de novo cholesterol biosynthesis [40]. Our
current findings suggest that different cancer entities dis-
tinctly modulate critical nodes of the MVP to fulfil specific
metabolic demands. We, therefore, used the same AAV-based CRISPR/Cas9 strategy
to target Srebf1 or Srebf2 in KPL tumours (KPLS1 and KPLS2). The
efficiency of the gRNAs used to delete Srebf1 or Srebf2 was first
established using T7E1 analysis and immunoblotting (Fig. S5e, f). While deletion of Srebf1 did not alter tumour load (Fig. S6i),
deletion of Srebf2 resulted in a significant reduction in tumour
burden in age matched KPLS2 compared to KPL mice (Fig. 6d, e). We also observed a significant reduction in the percentage of
Srebp2 positive nuclei and Hmgcs1 positive cells (Fig. 6f),
confirming efficient target deletion. Consistent with our observa-
tion that SREBP2 regulates expression of TP63 targets in human
SCC cells (see Fig. S1e), we also observed a reduction in ΔNp63
positive nuclei in KPLS2 tumours (Fig. 6g, h). Together, these
results strongly implicate SREBP2 in lung tumour growth and
suggest that SREBP2 contributes to SCC transcriptional pro-
grammes via regulating ΔNP63. Our results also demonstrate that USP28 regulates the MVP by
increasing the stability of SREBP2, the transcription factor responsible
for the expression of most MVP enzymes. While both proteins
localise to and interact in the nucleus, we did observe some level of
interaction of SREBP2 and USP28 also in the cytoplasm. So far, most
studies have described USP28 as a primarily nuclear protein. One
potential explanation for cytoplasmic localisation could be alter-
native splicing, leading to broader subcellular distribution of USP28. Indeed, alternatively spliced products of USP28 have been described
[41]. DISCUSSION
SREBP t Nevertheless, USP28-dependent SREBP2 stabilisation most likely
targets the mature form, as it was detected in our experiments when
only the isolated N-terminal part of the protein was expressed. Interestingly, mutation of the CPD, which mediates GSK3β-
dependent phosphorylation and subsequent recognition by the
SCF/FBXW7 ubiquitin ligase, did not fully abolish the regulation of
SREBP2 by USP28. This suggests that mature SREBP2 can also be a
substrate for other ubiquitinating enzymes. An early study described
degradation of nuclear SREBPs by the ubiquitin-proteasome system
without identifying the ubiquitin ligases involved [42]. The only other C.R. Maier et al. 1719 Fig. 4
Inhibition of USP28 and MVP synergise in reducing cell viability. a A431 cells expressing inducible shRNAs targeting USP28
(shUSP28#1 or shUSP28#2) or control cells were treated with 1 µg/ml doxycycline (Dox) or solvent (ethanol, EtOH) for 72 h together with
10 µM simvastatin (Sim) or solvent (DMSO). Cell viability was determined by crystal violet staining. Data are presented as mean± SD of three
independent experiments (n.s. non-significant, ****p < 0.00001, one-way ANOVA with post-hoc Tukey test). b Growth curves of shUSP28#1 or
control (shRen) treated with 1 µg/ml doxycycline (Dox) or solvent (ethanol, EtOH) together with 10 µM simvastatin (Sim) or solvent (DMSO). Data are presented as mean± SD of three independent experiments (n.s. non-significant, *p < 0.05, ****p < 0.00001, one-way ANOVA with post-
hoc Tukey test). c Schematic of the different metabolic outputs of the MVP. GG-PP = geranyl-geranyl-pyrophosphate, CoQ10 = ubiquinone. d Cells as in (a) were treated with 1 µg/ml doxycycline or solvent (ethanol, EtOH) for 72 h together with 10 µM simvastatin (Sim) or solvent
(DMSO) plus the indicated compounds: 100 µM mevalonate, 1 µM cell-permeable cholesterol, 5 µM ubiquinone (CoQ10), nucleosides (30 µM C,
G, A, U each; 10 µM T) or 5 µM GG-PP. Cell viability was determined by crystal violet staining. Representative images of three independent
replicates are shown. e Quantitation of data shown in (d). Data are presented as mean± SD of three independent replicates (n.s. non-
significant, **p < 0.01, ****p < 0.0001, unpaired two-tailed Student’s t-test between EtOH vs EtOH plus simvastatin; #p < 0.05, ####p < 0.0001, one-
way ANOVA with post-hoc Dunnett’s test compared to DOX plus simvastatin). f GSEA enrichment plots for gene sets mapping to interferon
and TCR signalling comparing cells depleted for SREBF2 (SREBF2#2) and control (shRen). Deletion of SREBF2 reduces tumour formation in a LSCC
mouse model whether LADC and LSCC cell lines respond different to inhibition of
the MVP by statins. While LADC cells (H1299 and EKVX) were highly
sensitive to simvastatin treatment, two LSCC cell lines (LUDLU and
H21170) showed a remarkable resistance to this drug (Fig. 7a). We,
therefore, treated A431 and the two LSCC cell lines with a
combination of simvastatin and the dual USP28/25 inhibitor AZ1
[39]. We also restricted exogenous lipid availability by culturing cells
in 1% FCS. Interestingly, drug synergy between simvastatin and AZ1
was only observed when cells were exposed to lipid deplete
conditions (Figs. 7b and S7a). These results indicate that combined
targeting of USP28 and MVP could be efficient in eliminating SCC
cells. To study the role of SREBP2 in LSCC, we employed a CRISPR/Cas9
mouse model in which lung tumours are induced by intratracheal
delivery of adeno-associated virus (AAV) particles into Rosa26:Cas9
transgenic mice (Fig. S6a). This strategy was used to introduce the
oncogenic mutation G12D into the Kras locus via a repair template
in combination with inactivation of Tp53 either alone (KP) or
together with a Stk11/Lkb1 deletion (KPL). We have previously
shown that KPL mice develop LSCC at high frequency, while only a
minority of lesions present with a LADC phenotype [16]. In this
model, deletion of Usp28 prevented LSCC formation, resulting in a
reduced tumour load and enhanced survival [16]. We first investigated lung tissue sections from KP and KPL mice
by immunohistochemistry staining. This revealed that in both KP
and KPL mice, tumour lesions showed upregulation of the Srebp2
target Hmgcs1 (Fig. S6b–e). Tumours from KPL mice also displayed
a high percentage of nuclei that were positive for Usp28
(Fig. S6d, e). We next compared tumours from the SCC and ADC
subtypes, as determined by keratin 5 (Krt5), thyroid transcription
factor 1 (Ttf-1/Nkx2-1) and Usp28 staining, in KPL mice. Interest-
ingly, while both tumour subtypes showed increased Hmgcs1
levels compared to normal tissue, Hmgcs1 expression was
significantly higher in SCC compared to ADC (Fig. S6f, g). We also
analysed lung tumour tissues after CRISPR/Cas9 deletion of Usp28
in KPL tumours (KPLU). These tumours showed a marked drop in
the percentage of nuclei that are positive for SREBP2 (Fig. 6a, b). Furthermore, expression of HMGCS1 and FDFT1 was also reduced
in lysates from KPLU compared to KPL tumours (Fig. S6h),
providing additional evidence that Usp28 regulates Srebp2 in lung
cancer. Statins synergise with a dual USP28/25 inhibitor to reduce
viability in SCC cells Haematoxylin and eosin (H&E) staining is als
b Boxplot showing quantification of Srebp2 staining in KPL and KPLU tumours. Percent positive cells are shown. (KPL: n = 9; KPL
***p < 0.001, Mann–Whitney test). c Tissue sections from KPL and KPLS2 tumours were stained for the adenocarcinoma marker thyroid tran
factor 1/NK2-homeobox 1 (Ttf-1/Nkx2-1), the squamous marker keratin 5 (Krt5), Srebp2 and Hmgcs1 by immunohistochemistry. H&E staini
shown. d H&E staining of representative lung tissue sections from KPL and KPLS2 mice. e Ratio of tumour area relative to total lung area in
KPLS2 mice. Data are displayed as mean ± SD (KPL: n = 11; KPLS2: n = 13; Mann-Whitney test). f Boxplot showing quantification of Sre
Hmgcs1 staining in KPL and KPLS2 tumours. Percent positive nuclei or positive cells are shown. (KPL: n = 7; KPLS2: n = 9; ***p < 0.00
Whitney test). g Tissue sections from KPL and KPLS2 tumours were stained for ΔNp63. h Boxplot showing quantification of ΔNp63 staini
and KPLS2 tumours. Percent positive nuclei are shown. (KPL: n = 6; KPLS2: n = 6; ***p < 0.05, Mann-Whitney test). Fig. 6
Deletion of Srebf2 attenuates tumour formation in a mouse model of lung squamous cell carcinoma. a Tissue sections from KPL and
KPLU lung tumours were stained for Usp28, Srebp2 and Hmgcs1 by immunohistochemistry. Haematoxylin and eosin (H&E) staining is also shown. b Boxplot showing quantification of Srebp2 staining in KPL and KPLU tumours. Percent positive cells are shown. (KPL: n = 9; KPLU: n = 7;
***p < 0.001, Mann–Whitney test). c Tissue sections from KPL and KPLS2 tumours were stained for the adenocarcinoma marker thyroid transcription
factor 1/NK2-homeobox 1 (Ttf-1/Nkx2-1), the squamous marker keratin 5 (Krt5), Srebp2 and Hmgcs1 by immunohistochemistry. H&E staining is also
shown. d H&E staining of representative lung tissue sections from KPL and KPLS2 mice. e Ratio of tumour area relative to total lung area in KPL and
KPLS2 mice. Data are displayed as mean ± SD (KPL: n = 11; KPLS2: n = 13; Mann-Whitney test). f Boxplot showing quantification of Srebp2 and
Hmgcs1 staining in KPL and KPLS2 tumours. Percent positive nuclei or positive cells are shown. (KPL: n = 7; KPLS2: n = 9; ***p < 0.001, Mann-
Whitney test). g Tissue sections from KPL and KPLS2 tumours were stained for ΔNp63. h Boxplot showing quantification of ΔNp63 staining in KPL
and KPLS2 tumours. Statins synergise with a dual USP28/25 inhibitor to reduce
viability in SCC cells In order to investigate the translational significance of targeting the
USP28/SREBP2 axis in squamous cancer, we first investigated Cell Death & Differentiation (2023) 30:1710 – 1725 C.R. Maier et al. C.R. Maier et al. 1720 BP2 is upregulated in squamous cell carcinoma. a Violin plots showing expression of USP28, SREBF2, HMGCS1 and FD
ocarcinoma (ADC) and lung squamous carcinoma (SCC). Data are from TCGA (Firehose legacy). Significance was ca
tney test. ****p < 0.0001. b Tissue sections from a human NSCLC TMA were stained for USP28, SREBP2 HMGCS1 a
stochemistry. Haematoxilin and eosin (H&E) staining is also shown. Representative images for SCC are shown. c Box
ion of USP28, SREBP2, HMGCS and SREBP1 staining in SCC and ADC tumours. Percent positive cells are shown. (SCC
< 0.001, Mann-Whitney test). d Pearson correlation between expression of USP28, SREBP2 and HMGCS in SCC tumour
coefficient, p = two tailed t-test). e Kaplan–Meier plots of overall survival of lung cancer patients (N = 1925) dichotom
based on median USP28 or SREBF2 expression score. Survival differences were calculated with the log-rank test. Fig. 5
SREBP2 is upregulated in squamous cell carcinoma. a Violin plots showing expression of USP28, SREBF2, HMGCS1 and FDFT1 in human
Lung adenocarcinoma (ADC) and lung squamous carcinoma (SCC). Data are from TCGA (Firehose legacy). Significance was calculated using
Mann–Whitney test. ****p < 0.0001. b Tissue sections from a human NSCLC TMA were stained for USP28, SREBP2 HMGCS1 and SREBP1 by
immunohistochemistry. Haematoxilin and eosin (H&E) staining is also shown. Representative images for SCC are shown. c Boxplots showing
quantification of USP28, SREBP2, HMGCS and SREBP1 staining in SCC and ADC tumours. Percent positive cells are shown. (SCC: n = 33; ADC:
n = 75; ***p < 0.001, Mann-Whitney test). d Pearson correlation between expression of USP28, SREBP2 and HMGCS in SCC tumours (R = Pearson
correlation coefficient, p = two tailed t-test). e Kaplan–Meier plots of overall survival of lung cancer patients (N = 1925) dichotomised into ‘high’
and ‘low’ based on median USP28 or SREBF2 expression score. Survival differences were calculated with the log-rank test. Cell Death & Differentiation (2023) 30:1710 – 1725 C.R. Maier et al. 1721 Fig. 6
Deletion of Srebf2 attenuates tumour formation in a mouse model of lung squamous cell carcinoma. a Tissue sections from
KPLU lung tumours were stained for Usp28, Srebp2 and Hmgcs1 by immunohistochemistry. Statins synergise with a dual USP28/25 inhibitor to reduce
viability in SCC cells Percent positive nuclei are shown. (KPL: n = 6; KPLS2: n = 6; ***p < 0.05, Mann-Whitney test). Cell Death & Differentiation (2023) 30:1710 – 1725 C.R. Maier et al. 1722 Fig. 7
Simvastatin synergises with a dual USP28/25 inhibitor to reduce viability of SCC cells. a Human ADC (H1299 and EKXV) and SCC
(LUDLU and H2170) cells were treated with different concentrations of simvastatin for 72 h. Cell viability was determined using crystal violet
staining. b A431, H2170 and LUDLU cells were treated with indicated concentrations of simvastatin (Sim) or AZ1 in medium containing 1% FCS
for 72 h. Drug synergy was calculated using ZIP model. c Working model for the regulation of SREBP2 by USP28. Created with BioRender.com. Fig. 7
Simvastatin synergises with a dual USP28/25 inhibitor to reduce viability of SCC cells. a Human ADC (H1299 and EKXV) and SC
(LUDLU and H2170) cells were treated with different concentrations of simvastatin for 72 h. Cell viability was determined using crystal viole
staining. b A431, H2170 and LUDLU cells were treated with indicated concentrations of simvastatin (Sim) or AZ1 in medium containing 1% FC
for 72 h. Drug synergy was calculated using ZIP model. c Working model for the regulation of SREBP2 by USP28. Created with BioRender.com Fig. 7
Simvastatin synergises with a dual USP28/25 inhibitor to reduce viability of SCC cells. a Human ADC (H1299 and EKXV) and SCC
(LUDLU and H2170) cells were treated with different concentrations of simvastatin for 72 h. Cell viability was determined using crystal violet
staining. b A431, H2170 and LUDLU cells were treated with indicated concentrations of simvastatin (Sim) or AZ1 in medium containing 1% FCS
for 72 h. Drug synergy was calculated using ZIP model. c Working model for the regulation of SREBP2 by USP28. Created with BioRender.com. Cell Death & Differentiation (2023) 30:1710 – 1725 Cell Death & Differentiation (2023) 30:1710 – 1725 Cell Death & Differentiation (2023) 30:1710 – 1725 C.R. Maier et al. 1723 ubiquitin ligases, in addition to FBXW7, described to regulate
SREBP2 so far are TRC8 (also known as ring finger protein 139,
RNF139) and ITCH (Itchy E3 Ubiquitin Protein Ligase). TRC8
ubiquitinates the full-length protein to prevent SREBP2 processing
[43, 44], while ITCH potentially targets nuclear SREBP2 [45]. While
further investigations are needed to identify additional ubiquitin
ligases for SREBP2, the possibility that USP28 might also target
ubiquitin chains assembled by other ubiquitin ligases on SREBP2
could explain the strong effect of USP28 on SREBP2 stability. Our
working model for the regulation of SREBP2 by USP28 is displayed in
Fig. 7c. Notably, it is unclear whether the interaction between SREBP2
and USP28 is direct or whether it involves additional proteins. ER-membrane of macrophages [54]. It is also possible that
impaired prenylation of RAS or RHO proteins leads to the
activation of IFN signalling, for example by triggering ER-stress
as reported previously [55]. It will also be interesting to investigate
whether combinatorial treatment using the more selective USP28
inhibitors that are currently under pre-clinical evaluation [17]
together with some of the established therapeutics targeting MVP
activity [56] could exacerbate the induction of inflammation and
stress signalling, resulting in improved anti-tumour effects. ER-membrane of macrophages [54]. It is also possible that
impaired prenylation of RAS or RHO proteins leads to the
activation of IFN signalling, for example by triggering ER-stress
as reported previously [55]. It will also be interesting to investigate
whether combinatorial treatment using the more selective USP28
inhibitors that are currently under pre-clinical evaluation [17]
together with some of the established therapeutics targeting MVP
activity [56] could exacerbate the induction of inflammation and
stress signalling, resulting in improved anti-tumour effects. While further experiments are required to reveal the full
complexity of cancer-relevant pathways controlled by USP28
and SREBP2, the results provided by our study add an additional
layer to the regulation of SREBP2 activity and open novel
translational avenues for the treatment of squamous tumours. USP28 is highly upregulated in LSCC, where it stabilises the
transcription factor ΔNP63 that is essential for the maintenance of
the squamous lineage [16]. We observed that USP28 depletion
reduced the viability of A431 SCC cells and rendered them highly
sensitive to MVP inhibition by simvastatin and that this effect could
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Investigation, CRM, OH, KMA-S, SH, KK, VB; Data Analysis, ECEV, CM, EE, Material, BK, MAC,
ME, MTR; Writing – Original Draft, AS and CRM; Writing – Review & Editing, AS, CRM, MED;
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Chem. 1996;271:26461–4. ACKNOWLEDGEMENTS We would like to thank the Light Microscopy and Genomics Core Units at DKFZ and
Celine Reifenbach for technical assistance. This work was funded by the German
Research Foundation (DFG, GRK 2243) and the German Cancer Aid (DKH 70114554). KMA-S acknowledges funding by the German Academic Exchange Service (DAAD). 34. Kaymak I, Maier CR, Schmitz W, Campbell AD, Dankworth B, Ade CP, et al. Mevalonate Pathway Provides Ubiquinone to Maintain Pyrimidine Synthesis and
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and facilitate the progression from G1 to S phase in growth-stimulated rat FRTL-5
cells. J Biol Chem. 1997;272:13–6. FUNDING Open Access funding enabled and organized by Projekt DEAL. 39. Wrigley JD, Gavory G, Simpson I, Preston M, Plant H, Bradley J, et al. Identification
and Characterization of Dual Inhibitors of the USP25/28 Deubiquitinating Enzyme
Subfamily. ACS Chem Biol. 2017;12:3113–25. COMPETING INTERESTS 40. Kalogirou C, Linxweiler J, Schmucker P, Snaebjornsson MT, Schmitz W, Wach S,
et al. MiR-205-driven downregulation of cholesterol biosynthesis through SQLE-
inhibition identifies therapeutic vulnerability in aggressive prostate cancer. Nat
Commun. 2021;12:5066. Cell Death & Differentiation (2023) 30:1710 – 1725 Reprints and permission information is available at http://www.nature.com/
reprints 44. Lee JP, Brauweiler A, Rudolph M, Hooper JE, Drabkin HA, Gemmill RM. The TRC8
ubiquitin ligase is sterol regulated and interacts with lipid and protein biosyn-
thetic pathways. Mol Cancer Res. 2010;8:93–106. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims
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To cite this version: Mahdi Ftouh, Nesrine Kalboussi, Nabil Abid, Souad Sfar, Nathalie Mignet, et al.. Contribution of
Nanotechnologies to Vaccine Development and Drug Delivery against Respiratory Viruses. PPAR
Research, 2021, 2021, pp.1-28. 10.1155/2021/6741290. hal-03423555 Contribution of Nanotechnologies to Vaccine
Development and Drug Delivery against Respiratory
Viruses
Mahdi Ftouh, Nesrine Kalboussi, Nabil Abid, Souad Sfar, Nathalie Mignet,
Badr Bahloul To cite this version:
Mahdi Ftouh, Nesrine Kalboussi, Nabil Abid, Souad Sfar, Nathalie Mignet, et al.. Contribution of
Nanotechnologies to Vaccine Development and Drug Delivery against Respiratory Viruses. PPAR
Research, 2021, 2021, pp.1-28. 10.1155/2021/6741290. hal-03423555 HAL Id: hal-03423555
https://cnrs.hal.science/hal-03423555v1
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Volume 2021, Article ID 6741290, 28 pages
https://doi.org/10.1155/2021/6741290 Hindawi
PPAR Research
Volume 2021, Article ID 6741290, 28 pages
https://doi.org/10.1155/2021/6741290 Mahdi Ftouh
,1 Nesrine Kalboussi
,1,2 Nabil Abid
,3,4 Souad Sfar
,1
Nathalie Mignet
,5 and Badr Bahloul
1 1Drug Development Laboratory LR12ES09, Faculty of Pharmacy, University of Monastir, Tunisia
2Sahloul University Hospital, Pharmacy Department, Sousse, Tunisia 3Department of Biotechnology, High Institute of Biotechnology of Sidi Thabet, University of Manouba, BP-66, 2020 Ariana,
Tunis, Tunisia Laboratory of Transmissible Diseases and Biological Active Substances LR99ES27, Faculty of Pharmacy, Universit
Rue Ibn Sina, 5000 Monastir, Tunisia 5University of Paris, INSERM, CNRS, UTCBS, Faculté de Pharmacie, 4 avenue de l’Observatoire, 75006 Paris, France University of Paris, INSERM, CNRS, UTCBS, Faculté de Pharmacie, 4 avenue de l’Observatoire, 75006 Paris, Fra Correspondence should be addressed to Nesrine Kalboussi; kalboussi.nessrine@gmail.com Received 6 August 2021; Accepted 8 October 2021; Published 27 October 2021 Received 6 August 2021; Accepted 8 October 2021; Published 27 October 2021 Academic Editor: Marcelo H. Napimoga Academic Editor: Marcelo H. Napimoga Copyright © 2021 Mahdi Ftouh et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. According to the Center for Disease Control and Prevention (CDC), the coronavirus disease 2019, a respiratory viral illness linked
to significant morbidity, mortality, production loss, and severe economic depression, was the third-largest cause of death in 2020. Respiratory viruses such as influenza, respiratory syncytial virus, SARS-CoV-2, and adenovirus, are among the most common
causes of respiratory illness in humans, spreading as pandemics or epidemics throughout all continents. Nanotechnologies are
particles in the nanometer range made from various compositions. They can be lipid-based, polymer-based, protein-based, or
inorganic in nature, but they are all bioinspired and virus-like. In this review, we aimed to present a short review of the
different nanoparticles currently studied, in particular those which led to publications in the field of respiratory viruses. We
evaluated those which could be beneficial for respiratory disease-based viruses; those which already have contributed, such as
lipid nanoparticles in the context of COVID-19; and those which will contribute in the future either as vaccines or antiviral
drug delivery systems. We present a short assessment based on a critical selection of evidence indicating nanotechnology’s
promise in the prevention and treatment of respiratory infections. 1. Introduction that, it spread to 29 nations or regions [2–4]; (ii) SARS-
CoV-2, genetically linked to that of 2002 and animal CoVs,
has been detected since December 2019 [5]. Besides the high
mortality rate, all of these viruses share several common fea-
tures; they are RNA viruses and exhibit a remarkable rate of
recombination and/or reassortment, with less extent for
CoVs, which hampers the development of effective antivirals
and specific vaccines. The high mutation rate and recombi-
nation/reassortment of several RNA viruses advocate the
continued emergence of novel viruses that pose a continued
threat to global health and economic systems. Yet, it results
in uncontrolled pandemics, such as SARS-CoV-2. Therapeu-
tic antiviral medicines can be utilized as a treatment during Several viruses were identified in the 20th and 21st centuries. We limit the number of viruses in this review to those linked
to major pandemics caused by respiratory viruses (Rvs) that
marked these two centuries. In chronological order, five
pandemics were caused by influenza viruses: Spanish influ-
enza (1918-1920), Asian flu (1957), Hong Kong flu (1968),
Russian flu (H1N1, 1977), and avian influenza H5N1
(1997, 2003 and 2018) [1]. Additionally, two pandemics
were caused by two coronaviruses (CoVs): (i) the Severe
Acute Respiratory Syndrome CoV (SARS-CoV) originally
sprang out around South China in the fall of 2002. After 2 PPAR Research the early stages of a disease epidemic when conventional
vaccinations are unavailable. Antiviral medicines, on the
other hand, should not be cytotoxic and must overcome
many obstacles to be effective at a specific location. Accord-
ing to its mechanism of action, its target might be extracellu-
lar to block virus/cell receptor interaction or intracellular to
hamper the virus replication steps [6]. Additionally, antiviral
drugs need to escape from the immune system components
and avoid their degradation. Therefore, nanodelivery vehi-
cles could enhance the activity of antiviral drugs and their
pharmacokinetic profile while reducing their systemic toxic-
ity. Furthermore, nanotechnology is considered helpful not
only to improve the antiviral molecule delivery but also to
supply the viral component(s) to the immune system before
infection, enhancing the immune response to respond once
the infection occurs. 1. Introduction Eight human respiratory viruses circulate commonly in
all age groups: adenovirus (ADV), human bocavirus (HboV;
parvovirus), human coronavirus (HcoV) and coronavirus
related to the SARS-CoV (severe acute respiratory syn-
drome), human influenza virus (HIV), human metapneu-
movirus (HMPV; paramyxovirus), human parainfluenza
virus (HPIV), human respiratory syncytial virus (HRSV),
and human rhinovirus (HRV). Despite the fact that these
respiratory viruses cause a large number of illnesses, there
are now just a few preventative or therapeutic measures
available. In the next section, we highlight features of a number of
viruses inducing common respiratory diseases. 2.1. Human Respiratory Syncytial Virus (HRSV). The virus
was originally identified as a cause of coryza in chimpanzees
in 1956. It was soon discovered in children suffering from
bronchiolitis and pneumonia. Because it produces multinu-
clear giant cell syncytia in tissue culture, it was named HRSV
in 1957 [8, 9]. In 2016, HRSV was reclassified by the Inter-
national Committee on Taxonomy of Viruses (ICTV), and
since then the virus has been named human orthopneumo-
virus; however, we keep using the old name in the present
review. HRSV is a ubiquitous virus and can infect very early
in life. It is the most common viral pathogen causing severe
lower respiratory tract infection (LRTI) and a primary cause
of hospitalizations in young children, putting a significant
strain on health care resources. Children less than 6 months
account for nearly half of all LRTI hospital admissions and
in-hospital fatalities caused by HRSV [10]. Presently, most
people acquire HRSV infection before the second or third
year of life, and reinfections are common throughout life. However, reinfection in older adults can occur causing
upper respiratory tract infection and causing LRTI in 90%
of the cases [11]. As a result, HRSV is a major cause of hos-
pitalization, especially during the epidemic with both adults’
and children’s health being compromised. HRSV-associated
hospitalization rates in adults approached those associated
with influenza in 2015, with 3.4 million hospitalizations
and 95,000–150,000 fatalities worldwide, and over 175,000
hospitalizations in children under the age of five in the
United States per year [10, 12]. In this review, we will look at how nanotechnology
might help in the treatment of respiratory viral infections. We begin with a background in virology, elucidating general
characteristics of viruses, emphasizing common features of a
specific group of viruses inducing respiratory diseases. 2. Respiratory Viruses Viruses are obligatory intracellular entities that are incapable
of self-replication. It can guide the infected cell machinery to
create additional virus particles. The genetic material of
most viruses is either RNA or DNA, but not both at the
same time. The nucleic acid molecules (DNA or RNA)
may be single- or double-stranded (ssNA). The nucleic acid
of an infectious virus particle (virion) is encased inside the
capsid, made up of many replicates of a single or several dis-
tinct proteins. Some viruses, known as enveloped viruses,
have a lipid envelope that is generated from the host cellular
membrane during budding. Those viruses lacking this lipid
membrane are called nonenveloped viruses. The range of
hosts that a virus may infect is commonly used to categorize
it. A virus that solely infects bacteria, for example, is known
as a bacteriophage, or simply a phage. Several events high-
light the lytic cycle of viral replication: adsorption, penetra-
tion, replication, and release of new virus particles. The
outcome of these events is the death of the infected cell [7]. From a taxonomic point of view, HRSV is classified as a
member of the Paramyxoviridae family [13], order of Mono-
negavirales, with a pleomorphic (80–350 nm in diameter)
enveloped virus that is a member of the subfamily Orthopar-
amyxovirinae and is the type-species member of the Orthop-
neumovirus genus [14]. HRSV is an enclosed virus with a
single-strand negative-sense RNA genome ((-) ssRNA) that
is approximately 15 kb in size. The nucleocapsid protein
(N), nucleocapsid-associated proteins (M2-1, P, and L),
one M2-2 protein (the second ORF of the M2 gene), one
matrix protein (M), three transmembrane proteins (F, G,
and SH), and two nonstructural proteins (NS1 and NS2)
are all encoded by the genome [15, 16]. Only a small number of viral infections have clinical
characteristics that may be used to determine the infection’s
aetiology. More often than not, generalized viral infections
make the clinical picture less distinct, and as a result, a vari-
ety of viruses can cause illnesses with identical clinical symp-
toms, or syndromes. The wide range of virus infections may include gastroen-
teritis and respiratory infections. Therefore, the diagnosis of
virus infections depends on the infected cell types and
organs showing the most virus concentration. Major con-
cern was given to respiratory diseases, as they constitute a
real health threat in the absence of effective antiviral drugs
and vaccines. 1. Introduction The
use of nanotechnology to combat viral infections, with an
emphasis on self-assembled nanoparticles, will next be
addressed in depth. Finally, the use of nanotherapeutics in
the recent coronavirus epidemic will be presented. 2. Respiratory Viruses The latter two proteins are expressed for IFN and apo-
ptosis inhibition [16, 17] (Figure 1). The virus spreads
between hosts via respiratory droplets or contaminated
objects or surfaces. It infects the apical ciliated epithelial cells 3 PPAR Research Fusion protein F
Attachment
glycoprotein G
RNA
Small hydrophobic
protein
Matrix protein M
Figure 1: Schematic representation of HRSV. Fusion protein F
Attachment
glycoprotein G
RNA
Small hydrophobic
protein
Matrix protein M
Figure 1: Schematic representation of HRSV. enza disease are often missing from low- and middle-income
countries. A, B, C, and D are the four types of influenza
viruses known thus far. Seasonal outbreaks of illness are
caused by human influenza A and B viruses every winter
across the world, with type C viruses causing sporadic mild
upper respiratory symptoms [32, 33]. Influenza D viruses
are predominantly found in cattle and are not known to
infect humans. The influenza A virus (IAV), on the other hand, causes
the most serious clinical illness and is the most prevalent
cause of seasonal epidemics and pandemics [34]. IAV strains
are classified into subtypes based on two proteins found on
the virus’s surface: haemagglutinin (HA) and neuraminidase
(NA). Both of these have a significant role in the develop-
ment of disease. [35]. Influenza viruses are enveloped viruses
of the family Orthomyxoviridae with a segmented ((-)
ssRNA)) genome. The genome consists of eight segments
[36, 37]. The termini of viral RNA connect with the viral
RNA-dependent RNA polymerase (RdRp), which is made
up of three protein subunits (PB1, PB2, and PA), whereas
the rest of the viral RNA is bound by an oligomeric nucleo-
protein (NP) [38–40]. At least 12 viral proteins are encoded
by the genome, the majority of which are required for effec-
tive virus replication and virion production. The influenza
virus genome is transcribed and replicated in the nucleus,
unlike other ((-) ssRNA)) viruses. The M1 protein, which
is located outside of the ribonucleoprotein complex and
forms a layer underneath the lipid cell-derived envelope, is
a significant structural component of the virus particles
[41, 42] (Figure 2). The envelope of lipid cells is made up
of three membrane proteins: HA, NA, and ion channel pro-
tein (M2). Fever, myalgia, headaches, malaise, sore throat,
dry cough, and nasal congestion are all symptoms of influ-
enza [43–45]. Gastrointestinal symptoms such as nausea,
vomiting, and diarrhea are also frequent [46]. 2. Respiratory Viruses The incuba-
tion period for influenza is 1 to 4 days (from infection to
onset of symptoms) [35]. Most people recover without med-
ical intervention within a week after infection, although
infection can sometimes lead to hospitalization and death,
especially in those with underlying medical problems,
infants/young children, and the elderly. Although the mech-
anism(s) underlying the RNA virus’s evolution is (are) still
not fully understood, the prevalence of new variants of
IAV is driven by antigenic shift and drift phenomena [47]. Figure 1: Schematic representation of HRSV. of the upper respiratory tract (URT) after 2 to 8 days of
incubation in the nasopharyngeal or conjunctival mucosa
[18]. Following cell attachment with the host cell membrane
via G glycoprotein, the prefusion form of the HRSV-F glyco-
protein links to nucleolin on the cell surface, causing mem-
brane fusion and virus particle entry [19, 20]. The
nucleocapsid is released shortly after that, including the viral
genome and the N, P, M2-1, and L proteins [21–24]. The lat-
ter protein initiates the transcription and replication of the
genome [25]. Finally, virus assembly takes place at the plasma mem-
brane, where nucleocapsids bind to membrane viral glyco-
proteins that are found on the cell membrane [16]. The
virus release occurs after clustering new mature virions at
the apical surface [26]. p
Altogether, the virus replication in host cells may induce
several complications, such as airway and alveolar obstruc-
tions, oedema, and pneumonia due to necrosis of respiratory
epithelial cells, hypersecretion of mucus, and the accumula-
tion of cellular debris [16, 18]. The immune response devel-
oped against HRSV is somehow impaired and does not
induce long-term protection. The fact that most HRSV
infections in healthy people are mild suggests that prior
infection does produce significant immune protection [27]. The F and G proteins are the main antigenic targets of neu-
tralizing antibodies around which vaccine and drug research
have been putting the spotlight on. Palivizumab is a recom-
binant humanized monoclonal antibody and the only
licensed prophylaxis treatment against HRSV, targeting the
HRSV-F protein. However, HRSV resistance to palivizumab
has been reported [28]. No vaccine has been approved yet. The hurdles in HRSV vaccine development include princi-
pally the immature immune system of neonates and the
induction of low-affinity neutralizing antibodies [29]. 2. Respiratory Viruses The antigenic drift refers to the evolution of a virus as a
result of changes in its genes that occur over time when the
virus replicates. These mutations cause changes in the HA
and NA surface proteins of the influenza virus, which are
recognized by the immune system and capable of inducing
an immunological response, including the development of
antibodies that can prevent infection. 2.2. Human Influenza Virus. The WHO estimates that more
than 650,000 people die each year from flu-related respira-
tory illnesses throughout the world [30]. Since 2010, the
CDC estimates that influenza has caused 9 million to 45 mil-
lion illnesses, 140,000 to 810,000 hospitalizations, and
12,000 to 61,000 mortalities in the United States [31]. These
numbers, often neglected by the public, expose influenza’s
weight on our population and our health care providers. The impact of these mutations on the immune system’s
ability to identify novel variations is determined by the num-
ber of mutations acquired as well as their locations within
surface proteins. The immune system may not be able to
detect and prevent the newest influenza strains. As a result,
a person is once again vulnerable to flu infection. To keep
up with evolving influenza viruses, flu vaccine formulation
must be examined and modified each year. In the spring of Country disease estimations play a crucial role in
informing decisions about national influenza prevention
and control programmes. Although reliable data about influ- 4 PPAR Research Neuraminidase NA
Hemagglutinin HA
Lipid bilayer
Matrix protein M1
RNA
Ion channel M2
Figure 2: Schematic representation of an influenza virus. nomic trade. With 185 million confirmed COVID-19 cases,
including 4 million deaths, reported by July 2021, the illness
soon became a pandemic, posing a global danger to human
health and global economic commerce [63]. The replicase
gene, which encodes nonstructural proteins (NSPs), takes
up two-thirds of the genome (approximately 20 kb), whereas
structural and accessory proteins take up only around 10 kb. The spike (S), membrane (M), envelope (E), and nucleocap-
sid (N) proteins are coded by the 3′ end of the viral genome
(Figure 3). The angiotensin-converting enzyme 2 (ACE 2)
receptor binds to the spike protein, which allows SARS-
CoV and SARS-CoV-2 to infect cells [64, 65]. Figure 2: Schematic representation of an influenza virus. 2. Respiratory Viruses The ACE 2 receptor is not only present in epithelia of the
lung but also, with different expression rates, in oral and
nasal mucosa, nasopharynx, stomach, small intestine, colon,
skin, lymph nodes, arterial and venous endothelial cells, thy-
mus, bone marrow, spleen, liver, kidney, and brain which
could explain the pathogenesis encountered following viral
infection [66]. CoVs are widely spread through respiratory
droplets. Infection can occur following direct exposure and
inhalation of droplets or indirect contact with nasal, con-
junctival, or oral mucosa [64]. Sia and collaborators (2020)
described the pathogenesis and transmission of SARS-
CoV-2 in golden hamsters, a perfect animal model with
ACE 2 receptors able to support SARS-CoV and SARS-
CoV-2 virus replication [67, 68]. They proved the presence
of SARS-CoV-2 viral antigens in nasal mucosa, and bron-
chial epithelial 2 to 5 days after inoculation. The study
showed replication of the virus in the upper respiratory tract
pursued by lower infection within the lungs with a strong
innate immune response which could explain the pulmonary
symptoms like those of SARS and MERS, i.e., fever, dry
cough, pharyngitis, shortness of breath, joint pain, and tired-
ness in infected patients [67–69]. IgA, IgM, and IgG anti-
bodies
were
detected
after
the
symptomatic
onset,
indicating a B-cell-mediated humoral immune response
against the nucleocapsid protein N and the spike protein S. Viral clearance is dependent on the T-cell immune response
to suppress infected cells and stop viral replication [69]. Pro-
tective immunity duration post-SARS-CoV-2 infection is an
uncertain point and subject of debates. However, studies
suggest that immunological memory could last 3–8 months
[70, 71]. All treatments used are not specific for SARS-
CoV-2, but they tend to lower the complication risk,
enhance the patient’s overall comfort, and decrease his hos-
pital stay. Fortunately, more than 50 vaccine candidates are
currently in trials [72] with more than 10 approved world-
wide,
including
BioNTech/Pfizer
BNT162b2,
Moderna
mRNA-1273, Gamaleya Sputnik V, Oxford/AstraZeneca
AZD1222, Sinopharm BBIBP-CorV, Sinovac CoronaVac,
Sinopharm Inactivated, FBRI EpiVacCorona, and CanSinoc
Ad5-nCoV [34, 73]. Among these, both Moderna and BioN-
Tech mRNA encoding for the spike protein are encapsulated
with lipid nanoparticles to protect the mRNA from degrada-
tion by nucleases and provide its cell internalization [74, 75],
while Gamaleya Sputnik V, Oxford/AstraZeneca, and CanSi-
noc vaccines are based on adenoviruses as vectors with E1
and E3 deletions encoding for the full-length S protein
[74]. 3. Classical Antivirals and Vaccines 3.1. Anti-RSV. RSV treatment is mainly supportive. Main-
taining hydration and oxygenation within the physiological
norms is the basis of RSV management. Nevertheless, bron-
chodilators, corticotherapy, decongestants, and antiviral
agents have been tried in multiple studies with no significant
impact on the course of the illness [81]. Ribavirin (a nucleo-
side analogue) is approved for the treatment of patients with
severe RSV infection only [82]. Its efficacy has not been
proven, and there is no sufficient evidence to confidently
state whether or not it is clinically effective in mild to mod-
erate RSV infection. Therefore, its routine use is not recom-
mended in RSV patients, and only severe RSV for LRTI
should be treated with ribavirin [81, 83]. Prevention could
be established with palivizumab, a monoclonal antibody
(mAb) directed against the RSV fusion (F) protein [81]. However, a double-blind randomized clinical trial on over
400 infants did not provide significant effectiveness [84]. The high cost of both antiviral treatment ($1,192 to $2,085
a month) [85] and antibody prevention ($1,866 per vial in
the US) [86] raised ongoing cost-effectiveness controversies. Failing to meet sufficient effectiveness with these com-
pounds, researchers are more oriented to vaccine develop-
ment,
even
though
several
antiviral
agents
such
as
enzaplatovir, presatovir, and caplacizumab are currently
under investigation [87]. Vaccination against RSV remains
on hold as there is no licensed vaccine available so far. In
addition, RSV vaccine development is very complicated
due to antigenic diversity, the immunization of young
infants who may respond inadequately to vaccination, and
the history of the formalin-inactivated RSV vaccine. The lat-
ter was used in the early 1960s; it not only failed to protect,
but also generated an exaggerated clinical response in infants
[88]. Despite the immense effort by researchers putting up
many types of vaccines in clinical trials [89], no conven-
tional vaccine was potent enough to get licensed. Respiratory disease, gastroenteritis, and keratoconjuncti-
vitis are the clinical diseases expressed by adenoviruses
depending on their cell tropism. Among the HAdV-
associated respiratory diseases, serotypes 1–7, 11, 14, 16,
21, and 50, are considered to be the main pathogens that
cause respiratory tract infection [79]. Infections are more
frequent in young children because of the lack of humoral
immunity. Epidemics can occur in healthy adults, particu-
larly military recruits, or children causing an URT infection
(mild colds) in most cases, while severe cases appear mostly
in immunocompromised patients with LRTI. 2. Respiratory Viruses The disease quickly became a pandemic and consti-
tuted a global threat for both human health and world eco- PPAR Research 5 Spike protein
Lipid bilayer
membrane
Membrane
protein M
Envelope
protein E
RNA
Figure 3: Schematic representation of CoVs. Figure 3: Schematic representation of CoVs. fied version of a chimpanzee adenovirus, known
as
ChAdOx1. It can be introduced into cells, but it cannot rep-
licate inside them [79]. The Sputnik V vaccine is based on a
heterologous recombinant adenovirus approach using ade-
novirus 26 (Ad26) and adenovirus 5 (Ad5) as vectors for
SARS-CoV-2 spike glycoprotein expression [80]. SARS-CoV-2. The world’s leading vaccine approved in 55
countries is BioNTech/Pfizer’s LNP vaccine being the first
ever RNA-based vaccine, licensed for human use with more
than 90% efficacy [76]. This approval brings to light the
open future of nanotechnology in the field of vaccination. 2.4. Human Adenoviruses (HAdVs). Human adenovirus
(HAdV) infections represent 5 to 10% of pediatric and 1 to
7% of adult respiratory tract infections (RTI) [77]. HAdVs
are nonenveloped double-stranded linear DNA viruses
coated with a 70–150 nm sized icosahedral nucleocapsid. This latter is made of 3 different capsomeres:hexons, penton
bases, and penton fibers through which the virus binds to
the host cell’s Coxsackie B and adenovirus receptor (CAR). The viral entry is a receptor-mediated endocytosis assisted
by cell surface integrins. Acidity within the endocyte cleaves
the fibers and exposes pentons that promote endosomal
membrane lysis and capsid release in the cytoplasm [78]
(Figure 4). Thereafter, the viral capsid reaches the nucleus
and attaches to the nuclear pore complex (NPC) via an
interaction with the hexon. The viral genome is then injected
into the nucleus to proceed to early and late replication
phases [78]. Fifty-one human adenovirus serotypes have
been identified and distributed in six species, A–F. 2. Respiratory Viruses Sinopharm and Sinovac vaccines are inactivated 2009, an H1N1 virus including genes from North American
swine, Eurasian swine, humans, and birds emerged and
swiftly spread, resulting in a pandemic [48, 49]. The WHO
developed a Global Influenza Surveillance Network to track
antigenic changes and provide yearly influenza vaccine com-
position recommendations [50]. Many vaccines are available
in the market, but protection is down to 45% against 2019–
2020 seasonal influenza A and B viruses according to the
CDC [51]. Antiviral drugs and vaccines developed against
the flu lack efficiency and broad spectrum coverage. 2.3. Human Coronaviruses (HCoVs). Coronaviridae is a fam-
ily that belongs to the Nidovirales order and the Coronavir-
ineae suborder. The Coronaviridae family (CoVs) are
subdivided into 4 genera: alpha, beta, gamma, and delta. Unlike alpha and beta CoVs, which only infect mammalian
species, gamma and delta CoVs infect a wider range of ani-
mals, including birds. CoV infections in humans and ani-
mals mostly cause respiratory and gastrointestinal illnesses
[52, 53]. They are enveloped positive-sense single-stranded RNA
viruses ((+)ssRNA), with club-like spikes protruding from
their surface, a huge RNA genome, and a unique replication
mechanism. They may induce a wide range of illnesses in
animals, including enteritis in cows and pigs, upper respira-
tory disease in chickens, and fatal respiratory infections in
human. For a long time, CoVs were not thought to be partic-
ularly harmful to humans, as demonstrated by HcoV-229E,
HcoV-OC43, HcoV-NL63, and HcoV-HKU1. They have
been recognized for a long time to cause seasonal, generally
minor respiratory tract infections. However, the severe acute
respiratory syndrome (SARS) outbreaks in Guangdong
Province, China, in 2002 and 2003 [54–57] followed by the
Middle East respiratory syndrome CoV (MERS-CoV) in
Middle Eastern countries [58], have demonstrated that CoVs
may cause more serious respiratory infections in humans. SARS-CoV and MERS-CoV were reported to be directly
transmitted to humans by market civets and dromedary
camels, respectively. Both viruses, however, are believed to
have originated in bats [59, 60]. The emergence of a new
SARS, caused by a newly emerged human CoV strain in
December 2019 in China [61, 62], named SARS-CoV-2,
has further shown the unexpected severe character of these
CoVs. 3. Classical Antivirals and Vaccines More than
80% of laboratory-confirmed influenza cases were prevented
with oral oseltamivir prophylaxis treatment in households
[92]. Following its oral administration, the dose is widely
distributed in the body and excreted as oseltamivir carboxyl-
ate (the active form) mainly through the kidneys with a
short 1–3 hours half-life [93]. Zanamivir Relenza® (NAI)
commercialized as an oral inhaled powder is also an alterna-
tive for influenza A and B treatment or prophylaxis. Monto
et al. showed through a randomized clinical trial that zana-
mivir
was
67%
efficacious
in
preventing
laboratory-
confirmed clinical influenza cases [94]. Both antiviral com-
pounds are most effective when treatment is initiated within
48 hours of the onset of symptoms. Other antiviral agents
such as intravenous peramivir or oral baloxavir may be used
for treatment [95]. Besides oseltamivir and zanamivir,
amantadine and its analogue rimantadine represent the first
antivirals licensed against influenza. They act as replication
blockers through their interaction with the viral M2 protein. Antiviral therapy with neuraminidase inhibitors is asso-
ciated with several side effects, such as bronchospasm and
reduction in airflow related to zanamivir and nausea, vomit-
ing, and abdominal pain related to oseltamivir in 10% of
treated patients [97]. Flu vaccination is available with differ-
ent types of vaccines in the market. The CDC recommends
the use of any licensed vaccine including inactivated influ-
enza vaccine (Fluzone®), recombinant influenza vaccine
(Flublok®), or live attenuated influenza vaccine (FluMist®)
[98]. All the abovementioned vaccines offer protection
against the selected strains by WHO. In fact, every two years,
WHO analyses surveillance data and laboratory and clinical
studies to determine the circulating flu viruses that should be
included in the vaccine [99]. The conventional vaccine
approach comes with many limits, such as virus reactivation
regarding live attenuated vaccine, lack of immune response
to recombinant vaccine needing the addition of a potent
adjuvant, and the administration of multiple dosages. More-
over, the short length of protection is the setback of a killed
vaccine. 3.3. Anti-SARS-CoV. There is no specific anti-SARS treat-
ment so far, but many potential antiviral molecules have
been investigated in vitro and in patients. Following the
SARS-CoV 2003 outbreak, ribavirin (RBV) has been used
at therapeutic doses as a standard anti-SARS agent in Hong
Kong, Canada, and other countries. However, in vitro and
in vivo studies indicated that the virus was modestly sensi-
tive to ribavirin at high doses [77]. 3. Classical Antivirals and Vaccines Untreated
HAdV can lead to viral dissemination and high mortality
[77, 78]. Treatment is symptomatic and antiviral therapy
using ribavirin trifluridine and cidofovir for severe AdV
infections has been reported. Live oral vaccines against
AdV types 4 and 7 are very effective in preventing respira-
tory infection and are routinely used by United States sol-
diers, but not yet available to civilians [77]. Since AdVs
readily infect humans, they have been used as gene therapy
vectors and vaccine delivery systems. In the COVID-19 pan-
demic, the Oxford/AstraZeneca team worked with a modi- 6 6 PPAR Research Penton
Penton base
Hexon
Double stranded
DNA
Fiber
Figure 4: Schematic representation of adenovirus. Figure 4: Schematic representation of adenovirus. Thus, they inhibit viral uncoating and entry into the cell. Both of these agents are only effective against influenza A
infections with high rates of resistance exceeding 30% and
up to 80% [81], which makes these drugs useless in the treat-
ment of influenza infection [96]. 3.2. Anti-Influenza. Although influenza viruses cause mild
illness with quick recovery in healthy adults, elderly and
immunocompromised patients are often exposed to clinical
complications needing medical care. The main part of treat-
ment in influenza patients is supportive therapy with fever
and hypoxemia management, although the Guidelines by
the Infectious Diseases Society of America (IDSA) recom-
mend antiviral treatment for any patient with suspected or
confirmed influenza who is hospitalized; has severe, compli-
cated, or progressive illness; or is at a higher risk for influ-
enza complications [90]. Several antiviral medications are
recommended for treatment and chemoprophylaxis of influ-
enza that depends on the evolution of symptoms and age of
patients: oral oseltamivir (influenza A and B), inhaled zana-
mivir (influenza A and B), intravenous peramivir (influenza
A and B), and oral baloxavir (influenza A and B). There are
some restrictions in their use, and they will be discussed
below. Oral oseltamivir Tamiflu® (neuraminidase inhibitor
(NAI)) is the gold standard antiviral in these three circum-
stances. With proven efficacy, it is prescribed for both treat-
ment and prophylaxis of influenza in adults and children
older than one year old. In 2000, Treanor and collaborators
(2000). demonstrated a reduced illness duration and severity
by more than 30% and 35%, respectively [91]. In addition,
the highly protective efficacy of oseltamivir was shown when
used as postexposure prophylaxis in households. 4. Nanoparticles as a Vaccine/Drug
Delivery System Introduced in 1959 by the physicist Richard Feynman, nano-
technology is now an integral part of science. Its use in med-
icine came with an outstanding breakthrough in delivery
systems. With the possibility to control size, shape, and com-
pounds, nanoparticles (NPs) offer an advanced ground for
the diagnosis and treatment of diseases. Furthermore, NPs
can carry drugs either by encapsulation or by conjugation
with the possibility of active or passive targeting. With such
rigorous
control,
promising
pharmacokinetics
can
be
achieved, such as prolonged drug half-life, enhanced drug
efficacy, and decreased toxicity [113]. Many nanobased
drugs and nanodelivery systems are FDA and EMA
approved for human use, and more similar drugs are being
investigated in clinical trials [114]. NPs can be classified into
several types, according to size, morphology, and physical
and chemical properties. They are generally classified into
organic (dendrimers, micelles, liposomes, ferritin, etc.) and
inorganic particles (iron oxide nanoparticles, silver or gold
nanoparticles, carbon dots, etc.) [115]. 4.1. Liposomes. Liposomes were first described by Alec Bang-
ham, in 1961. Since then, they have been investigated as
plausible drug delivery systems [116]. They are spherical
nanovesicles, consisting of one or more lipid bilayers. These
layers can either be composed of natural or synthetic phos-
pholipids, usually phosphatidylcholine [117] (Figure 5). The liposome size ranges between 30 and 1000 nm, depend-
ing on the preparation method. This unique structure allows
loading hydrophilic drugs in the aqueous core, as well as
hydrophobic therapies within the acyl hydrocarbon chains
of the lipid bilayers [118]. On the basis of their size and
number of bilayers, liposomes can be classified into multila-
mellar vesicles (MLV, with several lamellar phase lipid bilay-
ers), small unilamellar liposome vesicles (SUV, with one
lipid bilayer), and large unilamellar vesicles (LUV) [117]. Their production is a simple procedure, consisting of three
main steps: lipid drying, lipid hydration, and purification. p
y
Vaccination against SARS-CoV-2 is one of the most out-
standing achievements in human medical history. One year
after the virus discovery, the FDA issued the first emergency
use authorization for the Pfizer/BioNTech COVID-19 vac-
cine [73]. This was the first mRNA and nanoparticle vaccine
to be approved for human use. Soon after, many others got
licensed, such as the Moderna mRNA-1273 vaccine, the
Oxford/AstraZeneca AZD1222, the Janssen (Johnson &
Johnson) Ad26.COV2.S, and the Sinovac CoronaVac [73]. 3. Classical Antivirals and Vaccines Furthermore, ribavirin
showed side effects including haemolytic anaemia in 33%
to 73%, hypocalcaemia in 58%, and hypomagnesaemia in
46% of patients [100]. Besides, clinical studies evaluating
the efficacy of ribavirin against SARS-CoV were inconclu-
sive. Confronting the new SARS pandemic, a recent retro-
spective
cohort
study
including
2,037
patients
with
COVID-19 concluded that RBV/IFN-α therapy did not
show any benefit in improving patient outcomes [101]. Many other antiviral compounds, such as interferons, were
used in the hope of efficiently managing the 2003 and 2019 PPAR Research 7 adenovirus-based vaccines against SARS-CoV-2 such as
Sputnik V, Janssen Ad26.COV2.S, Oxford/AstraZeneca
AZD1222, and CanSino Ad5-nCoV [73]. adenovirus-based vaccines against SARS-CoV-2 such as
Sputnik V, Janssen Ad26.COV2.S, Oxford/AstraZeneca
AZD1222, and CanSino Ad5-nCoV [73]. outbreaks. Protease inhibitors have been used in Hong Kong
with promising result. Ritonavir and lopinavir association
(Kaletra), used in SARS-CoV patients, was associated with
lower death rates and intubation rates and a significantly
lower incidence of ARDS [100]. Otherwise, ritonavir and
lopinavir (LPVr) used in clinical trials with COVID-19
patients reported possible effectiveness with reduced mortal-
ity at 28 days and shortened ICU admissions and time to
discharge. The conducted studies are limited methodologi-
cally and quantitatively, as they lack a respectful sample size. More sophisticated and larger studies need to be further
conducted to determine the efficacy and safety of LPVr for
COVID-19 [102]. On the other hand, remdesivir is the only
antiviral agent approved by the FDA for COVID-19 patients
that was recommended by the Infectious Diseases Society of
America (IDSA) to be used for hospitalized patients with
severe COVID-19, defined as patients with SpO2 ≤94%, or
patients who require supplemental oxygen, mechanical ven-
tilation, or extracorporeal mechanical oxygenation [103]. Due to the huge urge to control 2019s devastating pandemic,
the biomedical community conducted numerous clinical tri-
als on previously known antiviral agents for the treatment of
COVID-19, i.e., remdesivir. Some studies proclaim that
remdesivir was potent against SARS-CoV-2, exhibiting a
shorter time to recovery, better clinical status, and less mor-
tality [104, 105]. However, WHO concluded that it had little
or no effect on patients [106, 107]. The drug continues to be
endorsed by the FDA and EMA in critical conditions, but
further studies need to be undertaken to prove its efficacy. 4. Nanoparticles as a Vaccine/Drug
Delivery System Many others are
still under clinical investigation, such as Arikayce, an inhaled
liposomal formulation of amikacin for the treatment of seri-
ous chronic lung infections [120]. levels in malignancies when compared to free doxorubicin
[121]. Since its approval, Doxil has been widely used [122,
123] and liposomes are now the leading nanostructure, as
an approved delivery systems. Further liposome nanoformu-
lations were licensed, such as AmBIsome and Abelcet for
amphotericin with reduced nephrotoxicity and Vyxeos for
daunorubicin and cytarabine codelivery. Many others are
still under clinical investigation, such as Arikayce, an inhaled
liposomal formulation of amikacin for the treatment of seri-
ous chronic lung infections [120]. Many immunoliposomes are also under promising clin-
ical investigations. These liposomes are endowed with sur-
face antibodies or ligands for targeted site drug delivery,
reducing side effects and toxicity [124]. Liposomes are also
an interesting antigen carrier for vaccines as they can carry
viral proteins, DNA, and RNA. Moreover, they are capable
of inducing cellular or humoral immune response depend-
ing on their charge, size, and lipid composition [125]. For
instance, unsaturated lipids were shown to induce Th2
responses, whereas saturated lipids promote Th1 immune
response [126]. While small-sized liposomes have been
shown to be uptaken by DCs, larger ones are phagocytosed
by macrophages. Moreover, small liposomes (100 nm)
would mostly induce Th2-dependent responses, while larger
liposomes
(400–1000 nm)
would
stimulate
Th1-based
responses [126]. Since their first application as a vaccine
delivery system in 1974, two liposome-based vaccines have
been approved for human use: Inflexal V for influenza and
Epaxal for hepatitis A [127]. For DNA- and mRNA-based vaccines, the lipid compo-
sition should be adjusted to provide tight interactions with
the negative charges of nucleic acids. As the number of pos-
itively charged phospholipids is scarce, synthetic lipids are
usually designed and auto-assembled similarly to liposomes,
leading to the so-called lipid nanoparticles (LNP). Cationic
liposomes or LNPs not only protect DNA and RNA from
degradation but are often associated with a greater immune
response due to favorable electrostatic interactions with
anionic cell membranes. Nevertheless, positively charged
carriers also suffer from rapid elimination from the blood-
stream upon systemic injection, which is why ionisable lipids
have been proposed [128]. These lipids, positively charged at
low pH and neutral at physiological pH, provide both
nucleic acid interaction and long-circulation time. 4. Nanoparticles as a Vaccine/Drug
Delivery System The concomitant dis-
covery of the role of siRNA or mRNA and lipidic autoas-
sembled systems led to the amazing rapidity to conceive a
molecule encoding for the spike of SARS-CoV-2. The simul-
taneous progress made in the last thirty years on RNAs and
liposomes dedicated to nucleic acid delivery finally led to an
efficient delivery system for the first siRNA approved drug,
which reached the market in 2018 [129]. This overall success
led to the rapid design of the actual mRNA delivery systems
in the context of SARS-CoV-2. Thanks to these advances,
RNA liposomal vaccines against SARS-CoV-2 have been
approved by the FDA and EMA less than a year after the
worldwide diffusion of the virus. The Pfizer and BioNTech
vaccine made with mRNA and lipidic nanoparticles showed
90% efficacy on 3000 patients, and the Moderna LNP RNA
vaccine reached a 94.1% effectiveness (CDC). Little is known
concerning the mRNA-lipid structure, even though some
information is now reported [130]. Nevertheless, this effi-
cacy and the easy design of mRNA opens the way for future
lipid-based drug delivery systems [131]. Many other lipid-
based vaccines against SARS-CoV-2 and influenza are still
under investigation (Table 1). This sophisticated nanocarrier
offers broad flexibility in drug or vaccine delivery with better
pharmacokinetics such as higher stability concerning fragile
molecules or proteins as discussed earlier, sustained release
of cargo, lower therapeutic doses, and reduced toxicity of
the encapsulated agent. Moreover, liposomes can play a
key role in vaccination as some lipid compounds have adju-
vant properties. Although the DDS is known to be moder-
ately
immunogenic
or
nonimmunogenic,
it
can
be
rendered immunogenic by modifying its surface [132]. The
main liposome drawbacks concern cationic lipids, which
exhibit toxicity and low intracellular release of their payload. However, ionisable lipids partially improve these side effects. From charged to uncharged, the downside is their high cost
of production, which limited their ordinary usage till the
COVID-19 crisis. Now that the production process and site
are up and running, it offers a broader perspective on lipid-
based carriers. levels in malignancies when compared to free doxorubicin
[121]. Since its approval, Doxil has been widely used [122,
123] and liposomes are now the leading nanostructure, as
an approved delivery systems. Further liposome nanoformu-
lations were licensed, such as AmBIsome and Abelcet for
amphotericin with reduced nephrotoxicity and Vyxeos for
daunorubicin and cytarabine codelivery. 4. Nanoparticles as a Vaccine/Drug
Delivery System However, China led the way to the first approved and effec-
tive vaccine in June 2020, but only for limited use in the Chi-
nese military. It is a DNA vaccine coding for the spike
protein incorporated in a nonreplicating adenovirus as a
delivery system to human cells [73]. The second in the race
was Russia with approval of its Sputnik V vaccine for wide-
spread emergency use in August 2020 [73]. Nanoformulations of existing drugs with low bioavail-
ability or high toxicity have benefitted from the stability
and improved biodistribution that liposomes provide. In this
context, PEGylated liposomes (with a hydrophilic polymer
chain) were developed to overcome short half-life and insta-
bility due to hydrophobic interactions with opsonins and
rapid uptake via the mononuclear phagocytic system [119]. The main example of this achievement is Doxil, a PEGylated
liposomal formulation of doxorubicin that was indicated for
the treatment of patients with ovarian cancer, for whom dis-
ease has progressed or recurred after platinum-based che-
motherapy. Liposomes were the first nanostructures to get
Investigational New Drug (IND) status by the FDA [120]. Soon after, Doxil was approved in 1995 for better cardiac
safety, fewer side effects, and a 16-fold enhancement of drug 3.4. Antiadenovirus. There is no clinically approved antiviral
agent against AdV infections; however, multiple drugs have
been tested, such as ribavirin [108, 109] and cidofovir [110]. In 2011, the adenovirus vaccine was used by the US mil-
itary to reduce acute respiratory disease. The vaccine is an
oral live virus type 4 and type 7 produced by Teva Pharma-
ceutical Industries Ltd. under contract to the U.S. Army. There is currently no adenovirus vaccine available to the
general public [111]. However, adenovirus is a common vac-
cine vector under promising clinical trials for other patho-
gens such as tuberculosis, Ebola, Zika, HIV, and SARS-
CoV-2 [73, 112]. The 2020s pandemic represented the right
set of circumstances to give the green light to several 8 PPAR Research 8 Hydrophilic
head
Phospholipid
bilayer
Hydrophobic
tail
Aqueous core
Figure 5: Schematic representation of liposomal drug delivery systems. Figure 5: Schematic representation of liposomal drug delivery systems. densed state of innovation. In parallel, amazing discoveries
have been made in the field of RNAs. 4. Nanoparticles as a Vaccine/Drug
Delivery System Associ-
ated with exchangeable lipid-PEG to provide nucleic acid
release after cell internalization, these liposomes are a con- 4.2. Polymeric
Nanoparticles. Polymeric
nanoparticles
(PNPs) are one of the most investigated NPs. Endowed with
biocompatibility and meticulous control over shape, size,
and components, they offer a magnificent drug/vaccine
delivery system. PNPs are made from natural polymers PPAR Research 9 Table 1: Nanoparticle-based vaccines against respiratory viruses. Material
Size
(nm)
Virus
Antigen/epitope
Adjuvant
Vaccine
trial
phase
Administration
route
Benefits
References
(year)
Liposomes
DLPC
liposomes
30–
100
Influenza
(H1N1)
M2, HA, and NP
highly conserved
peptides
MPL and
trehalose 6,6′
-dimycolate
In vivo
Mice
Intranasal
(i) Protection against diverse influenza strains
(ii) Highly specific T-cell responses that sharply limit viral
replication following infection
[143] (2010)
Lipid
nanoparticles
(LPN)
43–54
Influenza
(H1N1)
(H3N2)
Hemagglutinin (split
vaccine)
CpG-ODN
(TLR 9
agonist)
In vivo
Mice
Subcutaneous
(i) Improve adjuvant effects in vivo with greater production
of cytokines and costimulatory molecules CD80 and CD86
on DCs
(ii) Improved T-cell responses and protection over
heterologous and heterosubtypic strain
[144] (2019)
—
SARS-CoV-2
mRNA encoding
full-length, prefusion
stabilized spike
protein
—
Licensed
Intramuscular
(deltoid)
Safety, efficacy
[73, 145]
(2021)
Moderna
—
SARS-CoV-2
Full spike mRNA
—
Licensed
Intramuscular
(deltoid)
Safety, efficacy
[73, 145]
(2021)
BioNTech SE
and Pfizer
—
SARS-CoV-2
Spike mRNA
—
Phase 2b/
3
Intramuscular
Safety, efficacy
[73, 145]
(2021)
CureVac
Polymeric nanoparticles
PLGA
225.4
Bovine
parainfluenza
3 virus
(BPI3V)
BPI3V proteins
—
In vivo
Calves
Intranasal
Greater mucosal IgA responses
[146] (2015)
200–
300
Swine
influenza
virus (H1N2)
Inactivated virus
H1N2 antigen
—
In vivo
Pigs
Intranasal
Reduce the clinical disease and induce cross-protective cell-
mediated immune response in a pig model
[147] (2017)
γ-PGAa
100–
200
Influenza
(H1N1)
Hemagglutinin
—
In vivo
Mice
Subcutaneous/
intranasal
Sufficient cross-protective immune responses against
influenza virus infection in mice
Effective cross-protection with minimal side effects
[148] (2009)
PGA-PLL
150–
600
RSV
G protein
CX3C motif
—
In vivo
Mice
Subcutaneous
Induce blocking antibodies that prevent the interaction of
the RSV G protein with the fractalkine receptor (CX3CR1)
and protect mice against RSV replication and disease
pathogenesis
[149] (2015)
Chitosan
140
Influenza
(H1N1)
H1N1 antigen
—
In vivo
Mice
Intranasal
(i) Stimulate macrophages to produce IL-1β and IL-6
(ii) Stimulate spleen lymphocytes to produce IL-2 and IFN-
γ
[150] (2015)
HA2 and NP
In vitro
—
[151] (2020) Table 1: Nanoparticle-based vaccines against respiratory viruses. 4. Nanoparticles as a Vaccine/Drug
Delivery System 10 PPAR Research Table 1: Continued. Material
Size
(nm)
Virus
Antigen/epitope
Adjuvant
Vaccine
trial
phase
Administration
route
Benefits
References
(year)
220–
500
All influenza
viruses
Trimethyl
chitosan TMC
np
(i) Systemic adaptive immunity
(ii) Conserved proteins delivered in an adjuvanted
nanoparticle system
300–
350
Influenza
(H1N1)
HA-split
—
In vivo
Mice
Intranasal
(i) Vaccine innocuousness
(ii) Effective and safe delivery vehicle/adjuvant for the
influenza vaccine
[152] (2014)
571.7
Swine
influenza
virus (H1N2)
Killed swine
influenza antigen
—
In vivo
Pigs
Intranasal
(i) In CNP-KAg vaccinated pigs challenged with
heterologous virus, reduced severity of macroscopic and
microscopic influenza-associated pulmonary lesions were
observed
(ii) Chitosan SwIAV nanovaccine delivered by IN route
elicited strong cross-reactive mucosal IgA and cellular
immune responses in the respiratory tract that resulted in a
reduced nasal viral shedding and lung virus titers in pigs
[153] (2018)
200–
250
Influenza
(H1N1)
M2e
Heat shock
protein 70c
In vivo
Mice
Intranasal
(i) Induce a long lasting M2e-specific humoral and cellular
immune responses
(ii) Provide full protection against a 90% lethal dose (LD90)
of the influenza virus A/PR/8/34 (H1N1). [154] (2018)
HPMA/
NIPAM
12–25
RSV
F protein
TLR-7/8
agonist
In vivo
Mice
Subcutaneous
The improved pharmacokinetic profile by particulate
polymer-TLR-7/8a was also associated with reduced
morbidity and enhanced vaccine immunogenicity for
inducing antibodies and T-cell immunity
(i) Optimizing adjuvant design to elicit broad-based
antibody and T-cell responses with protein antigens
(ii) Protective immunity
[155, 156]
(2018, 2015)
Polyanhydride 200–
800
RSV
F and G
glycoproteins
—
In vivo
Calves
Intranasal
Enhanced interactions with antigen-presenting cells that are
necessary in the initiation of efficacious immune responses
BRSV-F/G nanovaccine is highly immunogenic, and with
optimization, has the potential to significantly reduce the
disease burden associated with RSV infection in both
humans and animals
[157, 158]
Self-assembling proteins and peptide-based nanoparticles
N
nucleocapsid
protein of
RSV
15
RSV
RSV phosphoprotein
R192G
detoxified E. coli
enterotoxin
LT
In vivo
Mice
Intranasal
Efficient and safe intranasal vaccine against RSV
[159] (2008)
15
RSV
FsII
Montanide™
Gel 01
In vivo
Mice
Intranasal
N-specific cellular immunity and F-specific antibodies for
protection
[160] (2016) PPAR Research 11 Table 1: Continued. 4. Nanoparticles as a Vaccine/Drug
Delivery System Material
Size
(nm)
Virus
Antigen/epitope
Adjuvant
Vaccine
trial
phase
Administration
route
Benefits
References
(year)
15
Influenza
(H1N1)
M2e
Montanide™
Gel 01
In vivo
Mice
Intranasal
Nucleoprotein nanoring is a potent carrier for mucosal
delivery of vaccine antigens
[161] (2013)
Ferritin
12.5
Influenza
(H1N1)
M2e
—
In vivo
Mice
Intranasal
High immunogenicity, cross-protection, and convenient
administration, as well as being economical and suitable for
large-scale production
[162] (2018)
Q11
—
Influenza
(H1N1)
Acid polymerase
—
In vivo
Mice
Intranasal
Immunogenic, noninflammatory, and promote more lung-
resident memory CD8+ T cells compared to subcutaneous
immunization
[163] (2018)
Self-
assembling
proteins
55
RSV
F protein (prefusion) Squalene-oil-
in-water
In vivo
Mice
Subcutaneous
In mice and nonhuman primates, the full-valency
nanoparticle immunogen displaying 20 DS-Cav1 trimers
induced neutralizing antibody responses ∼10-fold higher
than trimeric DS-Cav1
[164] (2019)
Protein
recombinant
nanoparticle
ResVax
—
RSV
F protein
Aluminum
phosphate
Phase III
Humans
First RSV vaccine to show Phase 3 efficacy
Favorable safety and tolerability data
[165] (2020)-
Failed to hit
its primary
endpoint
Self-
assembling
proteins
Influenza
(H1N1,
H3N2)
HA, M2e
—
In vivo
Mice
Intramuscular
Controlled release
Protective potency
[166] (2018)
Inorganic nanoparticles
Gold
12
Influenza
M2e
CpG
In vivo
Mice
Intranasal
Generate robust anti-M2e serum IgG antibodies in mice
[167] (2017)
Gold
nanorods
21 × 57 RSV
F protein
—
In vitro
—
A potent method for immunizing against viruses such as
RSV with surface glycoproteins that are targets for the
human immune response
[168] (2013)-
Ferritin
—
RSV
F protein
AF03
In vivo
Mice
Intramuscular
This pre-F vaccine increased the generation of NAbs
targeting the desired pre-F conformation, an attribute that
facilitates the development of an effective RSV vaccine
[169] (2020)-
Others
VLP
—
SARS-CoV-2
Full-length,
prefusion spike
protein
Saponin-based
Matrix-M™
Phase 3
Intramuscular
Safety
[73, 145,
170]
(2021)
Novavax
80–
120
Influenza
(H1N1)
Hemagglutinin
—
In vivo
Mice
Intranasal
Intranasal immunization with VLPs containing HA
induced high serum and mucosal antibody titers and
neutralizing activity against PR8 and A/WSN/33 (H1N1)
viruses
A promising strategy for the development of a safe and
effective vaccine
[171] (2007) 12 12 PPAR Research Table 1: Continued. 4. Nanoparticles as a Vaccine/Drug
Delivery System (chitosan, albumin, alginate, and heparin) or synthetic poly-
mers ((N-(2-hydroxypropyl) methacrylamide, copolymer,
polyethylene glycol (PEG), and poly(lactic acid)/poly(lac-
tic-co-glycolic acid)), as their name suggests. They are uti-
lized in a number of biological applications [130]. There
are two forms of polymeric NPs: nanospheres and nanocap-
sules. The nanospheres have drugs adsorbed on their surface
or incorporated in the matrix, whereas nanocapsules have
drugs enclosed in their core (Figure 6). Polymer NPs may
be made in a variety of shapes and sizes, ranging from
10 nm to 1000 nm [120]. Some polymeric NPs can help with
drug release for weeks without accumulating in the body;
they can also help with regulated release, improved cellular
uptake, drug molecule protection from degradation, site-
specific delivery, minimal toxicity, and theranostic proper-
ties [118]. Therefore, polymeric NPs are being explored as
potential carriers for a variety of medicines, including cancer
therapies, cardiovascular disease treatments, diabetes treat-
ments, and vaccines [118]. The predominance of polymers
among authorized and currently studied nanodrugs demon-
strates their value in enhancing traditional diagnostic and
therapeutic medicine (indicated in Table 1). stantial biological barrier to NP distribution and bioavail-
ability. To address these limitations, several modified
surface PNPs have been studied. Much like liposomes,
PEGylation of polymeric NPs is a key to bypass the RES. Grafting hydrophilic groups, such as polyethylene glycol
(PEG), hide the hydrophobic surface and limit recognition
by the immune system. Moreover, they inhibit hydrophobic
and electrostatic interactions with plasma opsonins, giving
longer drug half-life and better distribution [134]. Using this
technology, interferon gamma beta-1a is now administered
once every two to four weeks, instead of daily administration
for the treatment of relapsing forms of multiple sclerosis
thanks to a licensed PEGylated formula named Plegridy®
[120]. In addition, ligands and antibodies can be grafted on
the polymeric surface, giving the system further affinity to
targets. PLGA-based NPs can protect antigens from degra-
dation for four weeks, which is very helpful when it comes
to mucosal vaccination [133]. Moreover, PLGA NPs pro-
mote antigen internalization by APCs and facilitate antigen
processing and presentation to naïve lymphocytes [134]. The most common technique used for the preparation of
PLGA nanoparticles is the emulsification-solvent evapora-
tion technique that allows the encapsulation of hydrophobic
drugs. Other techniques, such as the double emulsion
W/O/W, were used to encapsulate hydrophilic drugs, like
peptides, proteins, and nucleic acids. 4. Nanoparticles as a Vaccine/Drug
Delivery System aterial
Size
(nm)
Virus
Antigen/epitope
Adjuvant
Vaccine
trial
phase
Administration
route
Benefits
References
(year)
80–
120
Influenza
(H1N1,
H3N2, and
H5N1)
M2e
—
In vivo
Mice
Intranasal
Cross-protection by inducing humoral and cellular immune
responses
[172] (2018)
—
Influenza
(H3N2)
HA
Matrix-M1
Phase 1/
2a
In vivo
ferret
Broadly protective immunity and improved vaccine efficacy [173] (2020)
80–
120
RSV
F protein and G
glycoprotein of RSV
and M1 protein of
Influenza
—
In vivo
Mice
Intranasal
Enhanced protection against live RSV challenges
Significant decreases in lung viral replication and obvious
attenuation of histopathological changes associated with
viral infections
[174] (2017)
—
RSV
RSV-F
Aluminum
phosphate
Phase 3
Humans
(i) Tolerated without dose-related increases in adverse
events
(ii) A 7- to 19-fold increase in the anti-F IgG and a 7- to 24-
fold increase in the antigenic site II binding and
palivizumab competitive antibodies
[175] 2013
Not effective
in a large
phase 3 trial
80–
120
MERS-CoV
MERS spike protein
(S)
Alum
In vivo
Mice
Intramuscular
High-titer antibodies in mice
[176] (2014)
80–
120
MERS-CoV
Recombinant MERS-
CoV S
Matrix-M1
In vivo
Mice
Intramuscular
High-titer anti-S neutralizing antibody and protected mice
from MERS-CoV infection in vivo
[177] (2017)
80–
120
MERS-CoV
MERS-CoV VLPs
With protein S, E,
and M
Alum
Rhesus
macaques Intramuscular
Excellent immunogenicity in rhesus macaques
[178] (2016)
COMb
40
Influenza
(H1N1)
Hemagglutinin
ISCOMATRIX
In vivo
Sheep/
mice
Intranasal
(i) Induce serum haemagglutination inhibition (HAI) titres
in mice far superior to those obtained with nonadjuvanted
vaccine delivered subcutaneously
(ii) Induce mucosal IgA responses in the lung, nasal
passages, and large intestine, together with high levels of
serum IgA
(iii) Improved protection
[179, 180]
(2008, 2003)
irosomes
150
Influenza A
Virus envelope
proteins
Virosomes
Licensed
Intramuscular
Good immunogenicity in both healthy and
immunocompromised elderly, adults, and children. [181] (2009)
breviations: DLPC—dilauroylphosphatidylcholine; MPL—monophosphoryl lipid A; CpG-ODN—unmethylated cytosine-guanine dinucleotide oligodeoxynucleotides; PGA—gamma-polyglutamic acid; PGA-
L—poly(γ-glutamic acid)-poly(L-lysine); HA—hemagglutinin; NA—neuraminidase; IL—interleukin; HPMA—N-(2-hydroxypropyl)methacrylamide; NIPAM—poly(N-isopropylacrylamide); TLR—Toll-like
eptor; ISCOM—immune stimulating complexes; SwIAV—swine influenza A virus; TMC np—trimethyl chitosan nanoparticle. 13 PPAR Research 13 Polymeric membrane
Aqueous core
Drug
Polymeric core and matrix
Drug
Figure 6: The two main types of polymeric nanoparticles known as the nanosphere (matrix system) and the nanocapsule (reservoir system)
with different drug-loading modalities. Polymeric core and matrix Drug Figure 6: The two main types of polymeric nanoparticles known as the nanosphere (matrix system) and the nanocapsule (reservoir system)
with different drug-loading modalities. 4. Nanoparticles as a Vaccine/Drug
Delivery System Figure 8: Schematic representations of micelle. going from hours to several months. The two major mecha-
nisms associated with drug release from PLGA-based DDSs
are diffusion and degradation/erosion [135]. Astonishing
therapy outcomes associated with PLGA nanoparticles were
achieved, with greater cargo stability, longer circulation
time, longer half-life, and extended release. The polymeric
nanoparticle, Capoxone® (glatiramer acetate), approved by
the FDA in 1996, is indicated for the treatment of multiple
sclerosis symptoms, acting as an immunomodulator with
greater circulation and stability [120]. Multiple other poly-
meric nanoparticles were licensed, such as Neulasta® (Pegfil-
grastim),
for
the
treatment
of
chemotherapy-induced
neutropenia, and Adynovate (antihemophilic factor) and
Rebinyn (coagulation factor IX), for the treatment of acute
bleeding in hemophilia A and B, respectively [120]. As far
as we know, no antiviral drug with polymeric formulation
has been approved; however, nanoformulations of Efavirenz
and Lopinavir have demonstrated effective suppression of
HIV in preclinical studies [136]. Considered as a pivotal
DDS, PLGA nanoparticles have a recognized position in
drug and vaccine delivery. This position stands behind dif-
ferent and numerous clinical trials and studies cited in
Table 1. Influenza, parainfluenza, and respiratory syncytial
viruses were the center of attention in PLGA nanoparticle-
based vaccines. going from hours to several months. The two major mecha-
nisms associated with drug release from PLGA-based DDSs
are diffusion and degradation/erosion [135]. Astonishing
therapy outcomes associated with PLGA nanoparticles were
achieved, with greater cargo stability, longer circulation
time, longer half-life, and extended release. The polymeric
nanoparticle, Capoxone® (glatiramer acetate), approved by
the FDA in 1996, is indicated for the treatment of multiple
sclerosis symptoms, acting as an immunomodulator with
greater circulation and stability [120]. Multiple other poly-
meric nanoparticles were licensed, such as Neulasta® (Pegfil-
grastim),
for
the
treatment
of
chemotherapy-induced
neutropenia, and Adynovate (antihemophilic factor) and
Rebinyn (coagulation factor IX), for the treatment of acute
bleeding in hemophilia A and B, respectively [120]. As far
as we know, no antiviral drug with polymeric formulation
has been approved; however, nanoformulations of Efavirenz
and Lopinavir have demonstrated effective suppression of
HIV in preclinical studies [136]. Considered as a pivotal
DDS, PLGA nanoparticles have a recognized position in
drug and vaccine delivery. This position stands behind dif-
ferent and numerous clinical trials and studies cited in
Table 1. Influenza, parainfluenza, and respiratory syncytial
viruses were the center of attention in PLGA nanoparticle-
based vaccines. 4. Nanoparticles as a Vaccine/Drug
Delivery System PLGA nanoparticles
can be developed by the nanoprecipitation method, also
called the interfacial deposition method [134]. Other tech-
niques exist, such as the spray-drying method [134]. Drug
loading into NPs is achieved by two methods: whether dur-
ing the NP production or the adsorption of the drug on NPs
after their production. This type of drug delivery system
(DDS) comes with controlled release of loaded substances, 4.2.1. PLGA Nanoparticles. Poly(lactic-co-glycolic acid)
(PLGA) NPs are the most widely used PNPs, since their
FDA and EMA approval. PLGA is a synthetic polymer, con-
sisting of linked monomers of lactic acid and glycolic acid. Both of these elements are biodegradable, biocompatible,
and efficiently processed by the body via the Krebs cycle
after hydrolysis of the polymer [133]. Before any processing,
NPs, circulating within the bloodstream encounter opsonins. Opsonized NPs get internalized via the RES, which leads
them to the liver or the spleen. This system exhibits a sub- 14 PPAR Research PPAR Research Branching units
Core
Surface groups
Internal voids
Figure 7: Schematic representations of dendrimers. Figure 7: Schematic representations of dendrimers. Hydrophilic head
Hydrophobic tail
Hydrophobic core
Figure 8: Schematic representations of micelle. have low drug loading even with their high encapsulation
efficiency. Compared with liposomes and their sustained
release, these polymeric NPs usually offer high burst release
of cargo. This latter cannot be sensitive to low pH (such as
RNA or DNA) due to acid formation during PLGA break-
down. Furthermore, the scale-up and high cost of these sys-
tems can be roadblocks for their development [137] 4.2.2. Chitosan Nanoparticles. Chitosans (CS), biopolymeric
nanoparticles, are made of the second most abundant natu-
ral polysaccharide, chitin. This sugar is made up of a mixture
of
-(1-4)-linked-D-glucosamine
and
N-acetyl-D-glucos-
amine, as well as N-(2-hydroxypropyl) methacrylamide/N-
isopropylacrylamide, which may be found in crustacean
shells like shrimp or crab shells and fungus cell walls. First,
it was discovered by French Professor Henri Braconnot in
1811. Since then, continuous research and application trials
have been conducted within the pharmaceutical field due
to its attractive features, to finally reach approval by the
US FDA for tissue engineering and drug delivery [138]. It
is a cationic, highly basic and biocompatible polymer with
low toxicity and immunogenicity. CS has been reported to
enhance drug bioavailability for both oral and nasal routes
due to its mucoadhesive property and the positive surface
charge,
allowing
interaction
with
the
cell’s
negative
membrane. 4. Nanoparticles as a Vaccine/Drug
Delivery System PAMAM, PLL, and carbosi-
lane dendrimers have been studied as antiviral NPs against
influenza via sialic acid functionalization. Sialic acid linked
to PAMAM dendrimers completely protected against infec-
tion in a murine influenza pneumonitis model [184]. A
recent study, using PAMAM dendrimers as an influenza
H5-DNA vaccine delivery system, evokes successful protec-
tion against the H5N1 virus [185]. Having such a low molec-
ular weight,
dendrimers are weakly immunogenic to
nonimmunogenic when it comes to vaccine delivery. Protein
conjugation, which implies higher molecular weight and
multifunctional immunogens, could overcome this problem
[186]. In this regard, multiple antigenic peptide (MAP) den-
drimers were introduced as VDS with the ability to present
multiple copies of an antigen or multiple antigens to the
immune system, simultaneously [186]. Dendrimers are
expensive DDS with high costs for their synthesis. Moreover,
their surface-group nature/generation-dependent cytotoxic-
ity, which can be significant with cationic dendrimers, con-
stitutes a hurdle for their clinical application. Their tunable
chemical and physical properties, cargo solubility, increase
of lipophilic drugs, and possible targeted delivery or multi-
antigenic vaccine presentation, on the other hand, impose
them back as a significant DDS and encourage the pursuit
of new studies [187]. Figure 9: Schematic representation of a gold nanoparticle with
different cargo possibilities. nanoformulations have poor stability, making it a challenge
to produce shelf-stable NPs [140]. CS derivatives have been
developed in order to improve permeability and mucoadhe-
sive property. Also, hydrophobic drugs for chemotherapy
can be incorporated with these modified amphiphilic CS
[141]. This form of NPs could be a suitable way to deliver
antiviral drugs, characterized by their high hydrophilicity
and thus very low per os bioavailability. Chitosan vaccines containing influenza, diphtheria, and
pertussis antigens for nasal delivery were prepared by Illum
et al. In systemic vaccine delivery, chitosan acts as an adju-
vant. Activation of macrophages occurs after the uptake of
chitosan. Chitosan has been widely used for DNA mucosal
vaccines. The same authors have developed a chitosan-
based DNA flu vaccine along with other chitosan antigen-
based vaccines still in clinical trials [142], as summarized
in Table 1. As discussed earlier, chitosan proposes low toxicity with
mucoadhesive properties, making it appropriate for mucosal
routes. However, this DDS lacks sufficient stability, making
it a hurdle for industrial high-scale production. It is only
appropriate for the encapsulation of some hydrophilic drugs
such as proteins and RNA, but its low immunogenicity may
be unsolicited [140]. 4. Nanoparticles as a Vaccine/Drug
Delivery System Emulsion cross-linking, emulsion-droplet coalescence,
ionic gelation, reverse micellar technique, and a chemically
modified chitosan method are five ways for its manufacture. Ionic gelation, on the other hand, is the most frequent tech-
nique for making chitosan NPs. Sodium tripolyphosphate
(TPP) is used to cross-link CS precursors, resulting in
large-sized (100–300 nm) polydisperse particles [139]. A pitfall associated with CS is its restrained spectrum of
drug loading. Indeed, CS NPs are only able to load hydro-
philic drugs like various antiviral molecules, proteins, and
RNAs. The incorporation of drugs may be performed either
during nanoformation or by incubating preformed nanopar-
ticles in the drug solution [140]. Following administration,
CS tend to release their cargo by three main mechanisms:
desorption, diffusion, and polymer erosion release. [140]. Polymeric delivery systems come with great assets such
as EMA and FDA approval for human use. Their biodegrad-
ability and biocompatibility, therefore, have low toxicity. Besides, with PLGA NPs, we can secure sustained release
and targeted delivery of cargo to specific organs or cells
[137]. Finally, the production methods and formulations
are well described in the literature. However, PLGA NPs Although CS has been studied for various administration
routes, the mucosal delivery is considered the suitable one,
due to the mucoadhesive signature. On the other hand, CS PPAR Research 15 Drug
Antibody
PEG
DNA
RNA
Grafting group
Grafting group
Grafting group
S
S
Au
S
Figure 9: Schematic representation of a gold nanoparticle with
different cargo possibilities. Drug
Antibody
PEG
DNA
RNA
Grafting group
Grafting group
Grafting group
S
S
Au
S trolled size, weight, and drug release, low toxicity, high load-
ing capacity, targeted delivery, and excellent cell uptake [118,
183]. Drug Dendrimers can be attained whether through a divergent
method or a convergent approach. In these two techniques,
monomers are added sequentially by click chemistry or lego
chemistry to the initiator [118, 183]. Meticulous control of
the dendrimer’s pharmacological and physicochemical char-
acteristics is conducted by using different polymers, mono-
mers, and functional groups [118, 182, 183]. Several kinds
of dendrimers have been reported, such as polyamidoamine
(PAMAM) dendrimers, polypropylene imine (PPI) dendri-
mers, liquid crystalline dendrimers, peptide dendrimers, gly-
codendrimers, and poly-L-lysine dendrimers (PLL). The
latter, functionalized with naphthalene disulfonate groups,
is the only approved dendrimer for the prevention of bacte-
rial vaginosis, known as VivaGel® [118]. The approval con-
cerns
the
EU
region
and
Australia,
while
FDA
authorization is still pending. 4. Nanoparticles as a Vaccine/Drug
Delivery System More are under clinical
investigation, and most of them are cancer therapeutics such
as cisplatin and paclitaxel [120]. In immunology, micelles
have been studied as an adjuvant loading system or vaccine
delivery system with promising results. 4.4.1. Gold NPs. Gold has been exploited for its putative
medical properties throughout the history of civilisation. It
was used in the early 20th century to help alleviate rheuma-
toid arthritis [191]. Recently, gold nanoparticles (GPN) have
gained great interest as a transporter for pharmaceutical
compounds or vaccines due to their plasmonic property,
which could offer novel means of drug release. Surface plas-
mon resonance (SPR) is a resonance phenomenon caused by
the interaction of metal NP conduction electrons with inci-
dent photons [190, 192]. Gold NPs are hybrids, displaying an inorganic core typ-
ically surrounded by an organic shell. The core governs the
physical properties, while the chemical nature of the mono-
layer dictates the solubility and the reactivity of the particles
[182, 183]. Subsequently, their size, pharmacokinetics, and
physicochemical qualities can be meticulously tailored
according to the need. AuNPs with a 10–150 nm size range
are produced via reduction of gold salts with the presence
of a stabilizing agent to prevent aggregation. More stable,
monodisperse, and smaller NPs (6–8 nm) are achieved using
recent advances and technology. Therefore, they are called
gold nanoclusters [193]. Through interaction, gold may be
directly coupled utilizing thiolated (-SH) molecules to pro-
duce stable monolayer-protected particles [194]. This thiol-
mediated ligand-gold bond is highly stable in the extracellu-
lar environment. On the other hand, the monolayer is rap-
idly cleaved within the cell due to high glutathione levels
providing a mechanism for internal release [194]. Targeted
delivery is feasible with size control and surface functionali-
zation using cell penetrating peptides and surface cell recep-
tor ligands [194] as shown schematically in Figure 9. Understanding its pharmacokinetics is a fundamental step
to develop a potent drug and vaccine delivery system. Au
is masked by a hydrophobic monolayer immediately recog-
nized by the RES and internalized by macrophages and other
phagocytic cells, following administration. This could be
very interesting in vaccine delivery, as the phagocytes navi-
gate to lymph nodes and activate the immune system. Yet, Micelles are much like dendrimers with only lipophilic
substance encapsulation possible, low entity-loading capac-
ity, and mean in vivo stability. Nonetheless, PMs can offer
increased cargo solubility, improving its bioavailability. 4. Nanoparticles as a Vaccine/Drug
Delivery System All these pitfalls limit researchers’ ori-
entation to chitosans as a DDS. 4.3. Micelles. The concept of “micelles” was introduced to
describe self-assembling structures with particle diameters
from 5 to 100 nm range [118]. The amphiphilic molecules
aggregate at a certain temperature (critical micelle tempera-
ture (CMT)) and a well-determined concentration (critical
micelle concentration (CMC)) [118]. The core of the
micelles is formed by the hydrophobic fragments, carrying
poorly water-soluble drugs (Figure 8), while the hydrophilic
surface can be conjugated to the hydrophilic active compo-
nent and then allow higher drug loading and minimal pre-
mature release [188]. 4.2.3. Dendrimers. Dendrimers are polymeric nanosized par-
ticles, made of a central polymer or a molecule from which
emanates symmetrical branches forming 3D hyperbranched
monodisperse structures (Figure 7). These well-defined
homogeneous structures are made of three units: a central
core, an inner shell, and an outer shell containing numerous
functionalities [182, 183]. Their unique architecture makes
them excellent candidates as drug or vaccine delivery agents. In fact, substances can be either entrapped in the inner shell
voids or carried on their surface via electrostatic interactions
or chemical conjugation [118]. Moreover, these tree-like
structures offer a broad spectrum of assets, such as con- Additionally, targeted drug delivery is a promising
approach with ligand or antibody surface grafting. Various
multifunctional
micellar
formulations
were
elegantly
designed and are under active investigations [189]. Endowed 16 PPAR Research Polymer layer
Fe304 or Fe203 core
Attachment group
RNA
Drug
Antigen
PEG
Antibody
DNA
Figure 10: Schematic representation of an iron oxide. Antibody Figure 10: Schematic representation of an iron oxide. by greater stability in vivo, polymeric micelles (PMs) hold
enhanced qualities compared to conventional micelles. PMs are made of amphiphilic polymers and, most likely,
the FDA-approved PEG. Other polymers have been utilized,
such
as
poly(N-isopropylacrylamide),
poly(methacrylic
acid), and biodegradable poly(esters), which includes poly(-
glycolic acid), poly(D-lactic acid), poly(D,L-lactic acid),
copolymers of lactide/glycolide, and poly(ε-caprolactone)
[190]. Various techniques like solvent extraction, dialysis,
and solution casting methods are used for formulating poly-
meric micelles, and with the appropriate components, tai-
lored structures and characteristics of micellar NPs can be
achieved, offering optimum drug loading and release. Estra-
sorb, a promising DDS with a FDA-approved formulation
(oestradiol hemihydrate, Novavax, Inc.), is indicated for
moderate-to-severe vasomotor symptoms associated with
menopause. This micellar formulation led to stable levels
of oestradiol for eight to 14 days. 4. Nanoparticles as a Vaccine/Drug
Delivery System Nonetheless, AuNPs offer
unprecedented characteristics as a delivery system, with high
yield control of size, functionalization, drug release, and
pharmacokinetics. One of AuNps’ disadvantages is the ten-
dency to accumulate in the liver and spleen, which may lead
to toxicity. Coating with biocompatible materials can
decrease their build-up and facilitate their elimination [133]. of AuNPs, conjugated with the M2e protein of influenza A,
demonstrated protective immune response in mice against
the H1N1, H3N2, and H5N1 influenza A viruses [167]. Unfortunately, no gold inorganic NP-based vaccines have
been approved so far by the FDA. Nonetheless, AuNPs offer
unprecedented characteristics as a delivery system, with high
yield control of size, functionalization, drug release, and
pharmacokinetics. One of AuNps’ disadvantages is the ten-
dency to accumulate in the liver and spleen, which may lead
to toxicity. Coating with biocompatible materials can
decrease their build-up and facilitate their elimination [133]. 4.4.3. Quantum Dots. Quantum dots (QDs) are tiny nano-
crystals with a size range of 2–10 nm and are named for their
shape. An inorganic core comprised of semiconducting ele-
ments like silicon, cadmium selenide, cadmium sulfide, or
indium arsenide forms the architecture of these devices. This
core has unique properties such as minimal tissue penetra-
tion, restricted light scattering, narrow emission bands, easy
synthesis with a high surface-to-volume ratio, and low light
scattering [205]. Consisting of hundreds to a few thousand
atoms, the core of a QD is a layered structure with a metallic
shell (i.e., ZnS) [206]. With their exceptional optical proper-
ties, QDs are able to reveal other NP delivery systems’ bio-
logical reactions and pharmacokinetics. In fact, QDs can be
loaded in other DDS with no effects on their characteristics
or behaviours [206]. This system has been extensively
explored as a theranostic for sensing, imaging, and therapy. As a drug carrier, they display enhanced bioavailability, con-
trolled release, greater stability, and targeted delivery [118]. As an antiviral carrier, their antiviral activity was demon-
strated against several viruses, such as SARS-CoV-2, pseu-
dorabies virus, and respiratory syndrome virus [207, 208]. 4.4.2. Iron Oxide. Iron oxide (IO) is a chemical compound
made up of iron and oxygen that is involved in a variety of
geological and biological processes. Magnetite (Fe3O4),
maghemite (g-Fe2O3), and hematite (-Fe2O3) are the three
most prevalent forms of IO found in nature [197]. IO NPs
have a similar structure design to AuNPs. 4. Nanoparticles as a Vaccine/Drug
Delivery System Fur-
thermore, they can come with great control over drug release
along with extended in vivo circulation time [187]. 4.4. Inorganic Nanoparticles. Inorganic substances can be
extremely toxic to our human body, although insignificant
amounts can be tolerated. In fact, these elements are making
their way up as promising career platforms for therapeutics
as well as vaccines. Inorganic nanoparticles (INPs) come
with rigorous control over structure and physicochemical
properties for rigorous and precise drug delivery. INPs dis-
play different varieties of NPs. In the present review, we
put together the most propitious in three categories. 17 17 PPAR Research Surface antigen
presentation
Phospholipid
membrane
Matrix protein
Enveloped VLP
Single protein
Triple protein
Double protein
Capsid
Naked VLP
Figure 11: Schematic representation of virus-like particles. Surface antigen
presentation
Phospholipid
membrane
Matrix protein
Enveloped VLP
Single protein
Triple protein
Double protein
Capsid Naked VLP Figure 11: Schematic representation of virus-like particles. 14
12
10
8
6
4
2
0
1968
1978
1988
1998
2008
2018
year
Publication's count
Liposome
Polymeric nanoparticles
Self-assembling proteins
Peptide-based nanoparticles
Gold nanoparticles
Iron oxide
Quantum dots
Figure 12: Number of publications related to RSVs according to the type of nanoparticles over a period of 50 years Publication's count Figure 12: Number of publications related to RSVs according to the type of nanoparticles over a period of 50 years threshold for the induction of potent cellular responses
and T-cell polyfunctionality by GNPs lies between 10 nm
and 22nm [195]. AuNPs, conjugated with recombinant
influenza haemagglutinin trimers and flagellin, showed
enhanced and protective mucosal cellular immunity against
the influenza virus [196]. Another intranasal formulation this interaction displays a hard pitfall for drug delivery
because of rapid elimination. In this manner, PEG functio-
nalization is the key to skip this reticuloendothelial system
uptake. p
According to a study of size effect on GNP lymph node
delivery and cytotoxic T-lymphocyte responses, the size 18 PPAR Research Pharmaceuticals) and as an MRI contrast agent (Gastro-
MARK™; umirem®; AMAG Pharmaceuticals) [204]. This
demonstrates the safety of IO, opening an avenue for
another drug delivery system that needs to be well debriefed. of AuNPs, conjugated with the M2e protein of influenza A,
demonstrated protective immune response in mice against
the H1N1, H3N2, and H5N1 influenza A viruses [167]. Unfortunately, no gold inorganic NP-based vaccines have
been approved so far by the FDA. 4. Nanoparticles as a Vaccine/Drug
Delivery System In fact, their core,
usually made of maghemite or magnetite, is layered by a bio-
compatible polymer, or less commonly, an inorganic coating
[194, 198]. [
,
]
The organic molecule surface modification serves many
purposes: it stabilizes NPs in a biological solution, provides
functional groups at the surface for subsequent derivatiza-
tion, and prevents rapid absorption by the reticuloendothe-
lial
system
(RES)
[199,
200]. Formulated
into
nanostructures, they offer great biocompatibility, high sur-
face volume ratio, low toxicity, and simple separation meth-
odology. Furthermore, their superparamagnetic features
display a simple targeted pharmaceutical delivery, via an
external magnetic field applied near a specific organ or tis-
sue. Therefore, they were intensely investigated as a drug
delivery system and contrast agents for MRI [120]. SPIONs
are created via a variety of physical, chemical, and biological
methods. These techniques result in varied NP forms and, as
a result, diverse characteristics [197]. Cargo can either be
conjugated to surface moieties or encapsulated inside the
particle outer layer or within coembedded mesoporous par-
ticles (Figure 10). For a synergistic combination with the
iron
oxide’s
hyperthermic
characteristics,
stimuli-
responsive components for encapsulation can enable trig-
gered cargo release [194]. In summary, IO NPs can be mag-
netically directed to a disease site, tracked via contrast
imaging, heated at the affected sites, and provide triggered
drug release [194]. With such rigorous control of drug deliv-
ery, IO NPs have been studied in vitro and in vivo for a wide
range of applications in medicine, including as an MRI con-
trast agent and as a form of IV and intranasal [198] drug
delivery, and for cancer treatment [201]. Within this frame-
work, IO NPs’ antiviral activity has been demonstrated
in vitro [202], reporting their potential activity against influ-
enza and SARS-CoV-2. A study by Abo-zeid et al. revealed
that both Fe2O3 and Fe3O4 NPs interacted efficiently with
the receptor-binding domain (RBD) of the SARS-CoV-2 S
glycoprotein, leading to viral inactivation [203]. However,
no SPION has been approved as a vaccine or antiviral drug
delivery system; however, these nanostructures have been
approved for intravenous iron replacement therapy in
chronic kidney disease (Feraheme™/ferumoxytol; AMAG 4.5. Self-Assembling Protein Nanoparticles and VLPs 4.5. Self-Assembling Protein Nanoparticles and VLPs 4.5.1. Self-Assembling Protein Nanoparticles (SAPNs). Self-
assembling protein nanoparticles (SAPNs) are structures
with an expected size range from 20 to 100 nm. These pro-
teins are constituted by coiled-coil domains composed of
α-helical highly versatile amino acid sequences. The latter
are characterized by seven-residue repeats, called heptad
repeats (Hrs), in which polar residues and charged ones sta-
bilize this unique formation with intramolecular interac-
tions. Such interactions force the coiled-coil protein to self-
assemble into nanoparticles [209]. Using a computational
protein design, scientists were able to control the nanoparti-
cle’s size, shape, and characteristics and develop highly
immunogenic epitopes that self-assemble into roughly
spherical NPs [210]. As a consequence, we are able to deliver
a cargo or display a repeated sequence of one or numerous
antigens giving place to a strong immune response. Since
1981, a SAPN vaccine has been approved against the hepati-
tis B virus. The formulation was based on the surface antigen
of the virus (HbsAg) that self-assembled into spherical par-
ticles with an average diameter of ~22nm. Since then,
numerous studies have been conducted to develop efficient
SAPN-based vaccines against malaria, toxoplasmosis, HIV,
SARS, and influenza [211]. The Malaria Protein 014
(FMP014) vaccine combined with a saponin molecule
(ALF with QS-21) derived from the bark of the Quillaja spe-
cies (ALFQ) reached phase 1 in clinical trials [212]. It was
reported that immunization with a SAPN B-cell epitope of
SARS-CoV-1 enhanced SARS neutralization in vitro and
increased antibody titer production in mice [213]. Unfortu-
nately, no further clinical trials have been conducted due to 19 PPAR Research virus regression. Furthermore, a universal influenza vaccine
was developed by Karch et al. with complete protection
against lethal influenza A challenge in mice [214]. nanoparticles had a 5.8-fold increase in the absorption than
the marketed formulation [220]. As a matter of fact, nano-
particles are able to protect drugs from stomach acidity
and they can facilitate uptake via transcytosis by M cells, epi-
thelial cells, and Peyer’s patches which results in higher bio-
availability
[221–223]. In
regard
to
biodistribution,
nanoparticles have achieved unprecedented control over
substance release. Nanoformulations of acyclovir [219],
zidovudine [224], efavirenz [225], and ribavirin [226] were
associated with sustained release, while controlled and mod-
ulated release was attained with both nanoencapsulated rito-
navir and lamivudine, under specific stimuli [227, 228]. Additionally, half-life is an important factor in drug efficacy
and compliance. 4.5. Self-Assembling Protein Nanoparticles and VLPs Usually, antiviral agents have a limited
half-life which require
daily
multiple
administrations. Nanoparticle-based drugs have shown greater and longer
half-lives due to cargo protection against the chemical reac-
tions of metabolism (ex. aebynin (coagulation factor IX),
adynovate (antihemophilic factor), and arikayce (amikacin
liposome inhalation suspension)) [229]. Safety is the first
drug attribute to be evaluated in a phase I clinical trial. When side effects are frequent and significant, this turning
point can limit the administration dose, and hence, treat-
ment outcome. Safety is particularly improved via nanopar-
ticle drug delivery systems. As an illustration, polymeric
nanoparticle ribavirin has a lower possibility of accumula-
tion in red blood cells compared to conventional ribavirin,
and therefore, less haemolytic anaemia occurrence [230]. Use of nanoformulations was correlated with reduced toxic-
ity and increased safety compared with conventional medi-
cines. For instance, Curosurf (poractant alfa in a LNP),
Doxil (doxorubicin HCl liposome), Abelcet, and AmBIsome
(liposomal amphotericin B), DepoCyt (liposomal cytara-
bine), and Marqibo (liposomal vincristine) are all nanobased
drugs that were granted approval by the FDA based on lower
toxicity compared with conventional formulation counter-
parts [229]. All these examples show that nanoparticles can
improve the benefit-to-risk ratio and could be useful for
antiviral drug delivery in the context of RSVs. 4.5.2. Virus-Like
Particles
(VLPs). Virus-like-particles
(VLPs) are self-assembling polyprotein NPs, imitating the
structure, organization, and the conformation of viruses
with no genetic material as schematized in Figure 11. There-
fore, these VLPs are noninfectious but efficiently internal-
ized by antigen-presenting cells (APCs), and antigens can
be presented with both major histocompatibility complex
MHC classes 1 and 2, eliciting both humoral and cellular
immune responses [215]. The structure can be made of a single or multiple capsid
proteins covered by a cell membrane such as influenza VLPs
or uncovered like papillomavirus VLPs. The production of
such structures passes through injecting of recombinant
plasmid with genes coding for the necessary structural pro-
tein in an expression system such as bacterial E. coli, yeast
cell, baculovirus/insect cell, mammalian, and recently,
plants [215]. VLPs range from 20 to 200 nm in size and are able to
mimic the virus structure with the possibility of incorporat-
ing various types of antigens, giving broad spectrum cover-
age over highly mutational viruses, such as influenza. The
possible addition of adjuvants confers an enhanced immune
response. 4.5. Self-Assembling Protein Nanoparticles and VLPs Vaccines against Human Papilloma Virus (HPV),
such as Cervarix®, Gardasil®, and Gardasil9®, and vaccines
against Hepatitis B Virus (HBV), such as the 3rd generation
Sci-B-Vac™, are commercially available VLP-based vaccines
[216]. A number of developed VLP vaccines are under clin-
ical investigation (Table 1), and two of the most promising
ones are Influenza and Norwalk. Chimeric VLPs [215] and
Novavax’s SARS-CoV-2 vaccine adjuvanted with their
Matrix-M™are currently in phase III clinical trials [73,
145, 170]. This proves the safety and efficiency of such nano-
vaccines for human use and gives researchers a promising
alternative for vaccine formulation. The VLP delivery system represents a cheaper and safer
system for vaccination. A strong B-cell and T-cell response
with no adjuvant is one of its assets in immunotherapy. As
a result, VLPs trigger a protective response at low doses. However, their production can be tricky as separation is a
complicated procedure. Another disadvantage of these
DDS is the high cost of production and the sophisticated
equipment required [217, 218]. g
y
Apart from small drug delivery, the interest of NPs has
also been raised for the delivery of biotherapeutics. From
the demonstration of antigen protection to vaccine applica-
tion, there is only one step. Nanoparticles have therefore
been proposed as vaccine drug delivery systems. The metic-
ulous control over the structure, size, and chemical features
of
nanoparticles
mediates
their
cell
interactions
and
entrance. Uptake by dendritic cells might be facilitated with
small NP size even though no difference related to the size
was obtained regarding the immunological response in non-
human primates [231]. In terms of composition, positively
charged nanoparticles enhance the uptake of mRNA leading
to a significant improvement of gene expression in mice
[232]. The most recent example is the novel Pfizer/BioN-
Tech and Moderna mRNA COVID-19 vaccines composed
of lipid nanoparticles. These nanoobjects carry the mRNA,
protect it from nuclease degradation, and allow its cell
uptake. The role of the lipid carrier as adjuvant is another
issue which should be carried out, as the charge alone might
not be responsible for the adjuvanticity [233]. Nanoparticles Abbreviations Although NPs are of great interest, many challenges are
confronted in their development. Characterization of these
systems is a complicated process. Size, charge, architecture,
and composition need to be well known and characterized. Multiple nanoplatforms with interindividual differences in
formulations, in response to drug/vaccine delivery system
requirements, make it hard to develop a standard nanoparti-
cle. As a result, the safety and toxicity profiles of each novel
nanoparticle developed need to be characterized to avoid
unpredictable side effects. In addition, nanoparticle pharma-
cokinetics, structure-activity relation, and cargo loading and
release are specific parameters for every DDS which needs to
be well controlled and identified. The FDA through its
Nanotechnology Characterization Lab (US-NCL) has pro-
vided protocols to standardize the characterization and eval-
uation
methods
to
study
nanoparticles
dedicated
to
cancer [234]. CDC:
The Centers for Disease Control and Preventio
CoVs:
Corona viruses
RVs:
Respiratory viruses
RNA:
Ribonucleic acid
mRNA:
Messenger ribonucleic acid
DNA:
Deoxyribonucleic acid
ssRNA:
Single-stranded ribonucleic acid
ADV:
Adenovirus
HAdV:
Human adenovirus
HMPV:
Human metapneumovirus
HboV:
Human bocavirus
SARS:
Severe acute respiratory syndrome
HIV:
Human influenza virus
HPIV:
Human parainfluenza virus
HRSV:
Human respiratory syncytial virus
HRV:
Human rhinovirus
ICTV:
International Committee on Taxonomy of
Viruses
LRTI:
Lower respiratory tract infection
URTI:
Upper respiratory tract infection
URT:
Upper respiratory tract
IFN:
Interferon
WHO:
World Health Organization
IAV:
Influenza A virus
NP:
Nucleoprotein
MERS:
Middle East respiratory syndrome
LNP:
Lipid nanoparticle
RTI:
Respiratory tract infections
NPC:
Nuclear pore complex
ARTI :
Acute respiratory tract infection
IDSA:
Infectious Diseases Society of America
NAI:
Neuraminidase inhibitor
RBV:
Ribavirin
ICU:
Intensive care unit
FDA:
Food and Drug Administration The European counterpart, the EU-NCL, extended to
health applications. These networks importantly contributed
to the harmonization with issued guidance documents to
translate more efficiently nanodrugs towards commercialisa-
tion. Related publications also showed the importance using
several techniques to analyse more finally the nanoparticle
shape and size distribution [235], highlighting also separa-
tion techniques which should be implemented prior to mea-
suring the size distribution [236]. Industrial production of
nanoparticles has also hampered their commercialisation. COVID-19 showed the difficulty of producing these vaccines
at a large scale in a relatively short period of time. Of course,
the management of this crisis is a strong future guidance for
the next nanoparticles to be produced [231, 237] . 5. Conclusions and Perspectives Year after year, nanotechnology applied to drug/vaccine
delivery has been proving itself. Nanoparticles are versatile
systems, and researchers tend to carve their features depend-
ing on the unmet clinical need. As a drug delivery system, nanoparticles were able to
enhance cargo pharmacokinetics, safety, and efficacy. Phar-
macokinetic-wise, the absorption of hydrophilic drugs such
as many antiviral agents (efavirenz, aciclovir [219], and lopi-
navir) is greater when formulated in a nanostructure. For
instance, oral administration of efavirenz loaded in PLGA 20 PPAR Research few years. We can also comment on the fact that the amount
of publications related to respiratory diseases still remains
rather low as regard to the application of nanotechnologies
for cancer. From the commercialization and production
point of view, lipid-based nanoparticles are still ahead; how-
ever, as reported in Table 1 with polymers and the summa-
rized expected results from phase 3 clinical trials with VLPs,
we should also give worth consideration to these nanoparti-
cles in the future. Therefore, the advances in both nanopar-
ticle design, characterization, and scale-up, together with a
better knowledge of the structure and mechanisms of virus
entry, offer strong opportunities in the near future, to
develop nanoparticles with additional features for RSVs. mRNA can easily be tuned to address various variants of
the same virus. The design of lipids, polymers, and novel
nanoparticulate structures will possibly allow tuning into
the immune response level. As a prospect, the design of
the next generation of nanoparticles in the field of vaccines
and response to RSVs should be a combination of delivery
and adjuvant properties. This is the current challenge of
researchers in the field. can be functionalised with specific DC surface receptor
(CD11c, CD40, and DEC205) ligands to improve their spe-
cific uptake. Moreover, their surface charge and composition
can be tuned to increase their interactions and cell uptake,
including mucosa/gut-associated lymphoid tissues. This is
an interesting quality for mucosal vaccines (oral or nasal)
against respiratory viruses. As a matter of fact, mucosal
administration of vaccines comes with strong local immu-
nity and production of specific IgA antibodies difficult to
achieve with other vaccinal routes (IM and SC). Local IgA
antibodies are able to opsonize the virus prior to cell entry;
the first step of viral infection. Intranasal vaccination is an
attractive route against respiratory viruses as it combines
strong local immunity within the site of infection and sys-
temic immunity. 5. Conclusions and Perspectives Table 1 contains multiple intranasal nano-
particle vaccines against RVs offering many key advantages. Nanoparticles as vaccine delivery systems are promising
innovations as they can simulate viral pathogens in structure
and in a number of physical/chemical properties. All respira-
tory viruses have different architectures, antigens, and
pathology pathways. Therefore, researchers are obligated to
tailor nanoparticle features depending on cargo interactions
and release needed. Abbreviations From our survey and the reported data on Figure 12, we
could see that liposomes were the earliest nanotechnologies
reported in the literature followed by polymers; they are
reported earlier than protein-based and inorganic-based
nanoparticles and that all of them are increasing in the last 21 PPAR Research EMA:
European Medicines Agency
NP:
Nanoparticle
SUV:
Small unilamellar vesicles
LUV:
Large unilamellar vesicles
IND:
Investigational new drug
LNP:
Lipid nanoparticle
DC:
Dendritic cell
PEG:
Polyethylene glycol
siRNA:
Small interfering RNA
PNP:
Polymeric nanoparticle
PLGA:
Poly(lactic-co-glycolic acid)
RES:
Reticuloendothelial system
DDS:
Drug delivery system
CS:
Chitosan
TPP:
Tripolyphosphate
PAMAM: Polyamidoamine
PPI:
Polypropylene imine
VDS:
Vaccine delivery system
CMT:
Critical micelle temperature
CMC:
Critical micelle concentration
INP:
Inorganic nanoparticles
GNP:
Gold nanoparticle
SPR:
Surface plasmon resonance
AuNP:
Gold nanoparticle
MRI:
Magnetic resonance imaging
SPION:
Superparamagnetic iron oxide nanoparticles
IV:
Intravenous
IM:
Intramuscular
SC:
Subcutaneous
RBD:
Receptor-binding domain
QDs:
Quantum dots
VLP:
Virus-like particle
SAPN:
Self-assembling protein nanoparticle
MHC:
Major histocompatibility complex
HPV:
Human papillomavirus
HBV:
Hepatitis B virus
Ig:
Immunoglobulin
NCL:
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Published on: Apr 18, 2021 License: Creative Commons Attribution 4.0 International License (CC-BY 4.0) California San Antonio Review 2 California San Antonio Review San Antonio Review
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Effects of intraoperative PEEP optimization on postoperative pulmonary complications and the inflammatory response: study protocol for a randomized controlled trial
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Ruszkai et al. Trials (2017) 18:375
DOI 10.1186/s13063-017-2116-z Ruszkai et al. Trials (2017) 18:375
DOI 10.1186/s13063-017-2116-z Open Access * Correspondence: molnar.lajos.zsolt@med.u-szeged.hu
2Department of Anaesthesiology and Intensive Therapy, University of Szeged,
Semmelweis u. 6, Szeged 6725, Hungary
Full list of author information is available at the end of the article Effects of intraoperative PEEP optimization
on postoperative pulmonary complications
and the inflammatory response: study
protocol for a randomized controlled trial Zoltán Ruszkai1, Erika Kiss2, Ildikó László2, Fanni Gyura1, Erika Surány1, Péter Töhötöm Bartha1,
Gergely Péter Bokrétás1, Edit Rácz1, István Buzogány3, Zoltán Bajory4, Erzsébet Hajdú4 and Zsolt Molnár2* © The Author(s). 2017 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. * Correspondence: molnar.lajos.zsolt@med.u-szeged.hu
2Department of Anaesthesiology and Intensive Therapy, University of Szeged,
Semmelweis u. 6, Szeged 6725, Hungary
Full list of author information is available at the end of the article
© The Author(s). 2017 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. Abstract Background: Patients undergoing general anesthesia and mechanical ventilation during major abdominal surgery
commonly develop pulmonary atelectasis and/or hyperdistention of the lungs. Recent studies show benefits of
lung-protective mechanical ventilation with the use of low tidal volumes, a moderate level of positive end-expiratory
pressure (PEEP) and regular alveolar recruitment maneuvers during general anesthesia, even in patients with healthy
lungs. The purpose of this clinical trial is to evaluate the effects of intraoperative lung-protective mechanical ventilation,
using individualized PEEP values, on postoperative pulmonary complications and the inflammatory response. Methods/design: A total number of 40 patients with bladder cancer undergoing open radical cystectomy and urinary
diversion (ileal conduit or orthotopic bladder substitute) will be enrolled and randomized into a study (SG) and a control
group (CG). Standard lung-protective ventilation with a PEEP of 6 cmH2O will be applied in the CG and an optimal PEEP
value determined during a static pulmonary compliance (Cstat)-directed PEEP titration procedure will be used in the SG. Low tidal volumes (6 mL/Kg ideal bodyweight) and a fraction of inspired oxygen of 0.5 will be applied in both groups. After surgery both groups will receive standard postoperative management. Primary endpoints are postoperative
pulmonary complications and serum procalcitonin kinetics during and after surgery until the third postoperative day. Secondary and tertiary endpoints will be: organ dysfunction as monitored by the Sequential Organ Failure Assessment
Score, in-hospital stay, 28-day and in-hospital mortality. Discussion: This trial will assess the possible benefits or disadvantages of an individualized lung-protective mechanical
ventilation strategy during open radical cystectomy and urinary diversion regarding postoperative pulmonary
complications and the inflammatory response. Trial registration: ClinicalTrials.gov, ID: NCT02931409. Registered on 5 October 2016. Keywords: Positive end-expiratory pressure, Static pulmonary compliance, Lung-protective ventilation, Radical
cystectomy, Postoperative pulmonary complications, Procalcitonin Keywords: Positive end-expiratory pressure, Static pulmonary compliance, Lung-protective ventilation, Radical
cystectomy, Postoperative pulmonary complications, Procalcitonin Ruszkai et al. Trials (2017) 18:375 Page 2 of 9 Page 2 of 9 g
Objectives of the study Theoretically, in patients with healthy lungs, during
general anesthesia and mechanical ventilation, inad-
equate PEEP values may lead to decreased pulmonary
compliance and gas exchange disorders due to pulmon-
ary atelectasis and/or hyperinflation of the lungs. In our
clinical trial, optimal PEEP values will be determined
during a static pulmonary compliance-directed PEEP ti-
tration procedure to protect from hyperdistention, and
regular ARMs will be performed using the sustained air-
way pressure by the continuous positive airway pressure
(CPAP) method, applying 30 cmH2O of PEEP for 30 s,
to prevent atelectasis [5, 13, 14]. The main objectives of this trial are to compare the effects
of a standard LPV applying 6 cmH2O of PEEP to a LPV
using an individually titrated optimal PEEP on: (1) oxy-
genation and PPC, (2) the degree of inflammatory
response evaluated by early PCT kinetics (0, 2, 6, 12, 24,
48 and 72 h after surgical incision) and (3) to evaluate the
relationship between the degree of inflammation and post-
operative pulmonary and extrapulmonary complications. Background reflects the host response. Therefore, there is some ra-
tionale in monitoring PCT kinetics as an indicator of the
host inflammatory response. Patients undergoing general anesthesia and mechanical
ventilation during major abdominal surgery commonly
develop pulmonary atelectasis and/or hyperinflation of
the lungs leading to complications either intraoperatively
or in the postoperative period, resulting in ventilator-
induced lung injury (VILI) [1, 2]. The aim of this investigator-initiated, double-center,
single-blinded (subject), prospective, randomized con-
trolled trial is to evaluate the effects of intraoperative
LPV, applying an individually titrated optimal PEEP, on
postoperative pulmonary complications (PPC) and the
inflammatory response in patients undergoing radical
cystectomy and urinary diversion (ileal conduit or ortho-
topic bladder substitute). We hypothesized that optimiz-
ing intraoperative mechanical ventilation (incorporating
LPV, ideal PEEP and ARM) can attenuate the inflamma-
tory response as compared to conventional modes of
mechanical ventilation, and hence may result in im-
proved postoperative oxygenation, prevent the occur-
rence of VILI, and reduce the incidence of organ
dysfunction. These anticipated advantages may also im-
prove postoperative recovery and survival rates, shorten
in-hospital stay and reduce health care-related costs. Lung-protective mechanical ventilation (LPV), by ap-
plying “low” tidal volumes (TV = 6 mL/Kg of ideal body-
weight, IBW), optimal positive end-expiratory pressure
(PEEP) and regular alveolar recruitment maneuvers
(ARM) in case of acute respiratory distress syndrome
(ARDS) have been shown to be advantageous in critically
ill patients. Recent studies have also shown positive re-
sults of LPV and regular ARM during general anesthesia
in patients with healthy lungs [3, 4]. The main advan-
tages of this strategy are improved gas exchange and
prevention of either pulmonary atelectasis or VILI [5–7]. However, the effects of applying an optimal level of
PEEP have not entirely been evaluated. There are several types of PEEP titration methods such
as dead space fraction (Vds/Vt)-guided or static pulmon-
ary compliance (Cstat)-directed techniques [8–12]. Study endpoints The primary outcome variables are PPC and PCT kinet-
ics. PPC are defined as new infiltrates or atelectasis on a
chest X-ray, abnormal breathing sounds on auscultation,
respiratory failure defined as PaO2/FiO2 < 300 or the
need for noninvasive or invasive ventilatory support
within the first three postoperative days. PCT kinetics
will be evaluated during and after surgery. Blood sam-
ples will be taken at 0, 2, 6, 12, 24, 48 and 72 h after sur-
gical incision. According to recent data it is expected
that PCT values will peak at approximately 24 h after
surgery and that they should decline by approximately
50% daily in the case of an uneventful postoperative
course. Therefore, in addition to the absolute values
the change between T0–T24–T48 will also be evalu-
ated [16, 22]. On the one hand major abdominal surgery induces an
inflammatory response that is necessary for postopera-
tive recovery (e.g., wound healing), but on the other
hand an overwhelming inflammatory response may also
lead to adverse events (AEs) such as organ dysfunction
[15–19]. Radical cystectomy is considered major surgery;
hence, there is an increased risk of postoperative compli-
cations. Inappropriate mechanical ventilation during
general anesthesia can also lead to an amplified inflam-
matory response, which theoretically may worsen the
postoperative outcome via several mechanisms. How-
ever, the relationship between LPV and the postoperative
inflammatory response after radical cystectomy has not
been investigated yet. Secondary
outcome
variables
are
extrapulmonary
complications: incidence of circulatory failure, gastro-
intestinal and renal dysfunction, hematologic and coagu-
lation disorders and infection (Table 1). There is strong correlation between the degree of in-
flammatory response and serum procalcitonin (PCT)
concentrations [20, 21]; hence, there is some rationale in
the belief that monitoring the inflammatory response by
regular PCT measurements in the postoperative period Tertiary endpoints are intensive care unit (ICU) days,
in-hospital stay, in-hospital and 28-day mortality. Ruszkai et al. Preoperative assessment and admission During standard institutional preoperative assessment,
the patient’s eligibility for radical cystectomy and urinary
diversion will be evaluated. Medical history, laboratory
and chest X-ray or computed tomography (CT) scan,
12-lead electrocardiogram (ECG), ASA physical status,
BMI, Respiratory Failure Risk Index (RFRI), nutritional
risk screening (NRS 2002 tool) and, if required (in case
of history of smoking or coronary artery disease), results
of spirometry, echocardiography and ergometry will be
recorded. Participants fulfilling the inclusion criteria will
be asked for their signed informed consent. Study endpoints Trials (2017) 18:375 Page 3 of 9 Table 1 Secondary endpoints
Endpoint
Time frame Detailed description
Circulatory failure
28 days
Hypotension – MAP < 65 mmHg
Severe cardiac arrhythmia – 40/min
< HR > 150/min
ScvO2 < 70%
dCO2 > 7 mmHg
Serum lactate > 2 mmol/L
Severe metabolic acidosis
(actual bicarbonate < 18 mmol/L)
Acute coronary syndrome
Acute left ventricular failure
Pulmonary embolism
Cardiac arrest
Gastrointestinal
dysfunction
28 days
Constipation
Ileus
Anastomotic leakage
Reoperation
Disorders of liver function
Renal dysfunction
28 days
RIFLE criteria
Hematologic and
coagulation disorders
28 days
Severe bleeding
Coagulopathy – INR > 1.5
Infection
28 days
Any infection except from
pneumonia
MAP mean arterial pressure, HR heart rate, ScvO2 central venous oxygen
saturation, dCO2 central venous-to-arterial carbon dioxide gap, INR
International Normalized Ratio Participants will be randomized to the SG or CG in a ra-
tio of 1:1. Randomization will be carried out by a
computer-generated blocked randomization list with 10
blocks of four patients per block. Allocation will be stored
in sealed, opaque and numbered envelopes. Participants
will be included and allocated in numerical order. All original records (CRF and relevant correspond-
ence) will be archived and secured for 15 years, and then
destroyed according to the hospital standards concern-
ing destruction of confidential information. Selection of the participants Patients with bladder cancer scheduled for open radical
cystectomy and urinary diversion will be screened and
recruited during routine perioperative assessment. Par-
ticipants fulfilling the inclusion criteria will be asked for
their signed informed consent. Withdrawal of consent
may be initiated by the participant at any time during
the trial. Inclusion criteria are age over 18 years, patients with
bladder cancer undergoing radical cystectomy and urin-
ary diversion (ileal conduit or orthotopic bladder substi-
tute) and provision of signed informed consent. Exclusion criteria are age below 18 years, American
Society of Anesthesiologists (ASA) physical status IV,
history of severe chronic obstructive pulmonary disease
(COPD, GOLD grades III or IV), history of severe or un-
controlled bronchial asthma, history of severe restrictive
pulmonary disease, pulmonary metastases, history of any
thoracic surgery, need for thoracic drainage before sur-
gery, renal replacement therapy prior to surgery, con-
gestive heart failure (NYHA grades III or IV), extreme
obesity (Body Mass Index, BMI > 35 Kg/m2) and lack of
patient’s consent. MAP mean arterial pressure, HR heart rate, ScvO2 central venous oxygen
saturation, dCO2 central venous-to-arterial carbon dioxide gap, INR
International Normalized Ratio MAP mean arterial pressure, HR heart rate, ScvO2 central venous oxygen
saturation, dCO2 central venous-to-arterial carbon dioxide gap, INR
International Normalized Ratio Study design This is an investigator-initiated, double-center, parallel-
group, single-blinded, interventional, prospective, ran-
domized controlled trial conducted at the Department of
Anesthesiology and Intensive Care of Péterfy Sándor Hos-
pital Budapest and at the Department of Anesthesiology
and Intensive Therapy of University of Szeged. The first
patient will be randomized in October 2016. This
protocol conforms to the Consolidated Standards of
Reporting
Trials
(CONSORT)
guidelines. Figure
1
shows the Standard Protocol Items: Recommendation
for Interventional Trials (SPIRIT) schedule of enroll-
ment,
interventions
and
assessments. The
SPIRIT
2013 Checklist is given in Additional file 1. Blinding, data collection, randomization and record-
keeping This is a single-blinded (participant) study. Patient data,
intraoperative and postoperative measurements, fluid
balance, respiratory parameters, laboratory results and
clinical status (Sequential Organ Failure Assessment
(SOFA) score) will be collected onto Case Report Forms
(CRF). CRF and the patient evaluation chart will not be
assessed in front of the patient. After admission to the Department of Urology (on
the day before surgery) a central venous catheter will
be placed, a blood sample will be taken from included
patients for baseline levels of PCT (T0), a chest X-ray
will be performed and, if there are no exclusion cri-
teria, patients will be randomized into one of the Ruszkai et al. Trials (2017) 18:375 Page 4 of 9 Fig. 1 Standard Protocol Items: Recommendation for Interventional Trials (SPIRIT) schedule of enrollment, interventions and assessments. DOS day
of surgery, POD postoperative day, SOFA Sequential Organ Failure Assessment, ICU intensive care unit ig. 1 Standard Protocol Items: Recommendation for Interventional Trials (SPIRIT) schedule of enrollment, interventions and a
f surgery, POD postoperative day, SOFA Sequential Organ Failure Assessment, ICU intensive care unit tems: Recommendation for Interventional Trials (SPIRIT) schedule of enrollment, interventions and assessments. DOS day
tive day, SOFA Sequential Organ Failure Assessment, ICU intensive care unit Fig. 1 Standard Protocol Items: Recommendation for Interventional Trials (SPIRIT) schedule of enrollment, interventions and assessments. DOS day
of surgery, POD postoperative day, SOFA Sequential Organ Failure Assessment, ICU intensive care unit Intraoperative care study groups. Patients will be given oral carbohydrate
loading (maltodextrin) 12, 8 and 2 h before surgery,
1000 mL of crystalloid solution will be given and
antimicrobial prophylaxis will be introduced using
ciprofloxacin and metronidazole 30 min before surgi-
cal incision. Antimicrobial prophylaxis will be contin-
ued for 72 h (2 × 400 mg ciprofloxacin and 3 ×
500 mg metronidazole per day). Deep vein thrombosis
prophylaxis will be carried out using low-molecular-
weight heparin (LMWH). Before induction of anesthesia an epidural catheter and
an arterial cannula will be inserted for invasive arterial
blood pressure monitoring and blood gas sampling. Immediately after induction of anesthesia and orotra-
cheal intubation, once a steady state has been reached
(Table 2), all patients will be submitted to an ARM using
the sustained airway pressure by the CPAP method, ap-
plying 30 cmH2O PEEP for 30 s. After ARM, PEEP will
be set to 6 cmH2O in the CG (“standard PEEP”) and Ruszkai et al. Trials (2017) 18:375 Page 5 of 9 Table 2 Steady state after induction of anesthesia
Parameter
Value
Hemodynamics
Mean arterial pressure
65 mmHg < MAP < 90 mmHg
Heart rate
50/min < HR < 100/min
Ventilation
SpO2
≥96%
EtCO2
35–40 mmHg
Anesthetics
EtSevo
1.0 MAC
MAP mean arterial pressure, HR heart rate, SpO2 peripheral capillary oxygen
saturation, EtCO2 end-tidal carbon dioxide partial pressure, EtSevo end-tidal
sevoflurane concentration, MAC minimal alveolar concentration Table 2 Steady state after induction of anesthesia will be performed to eliminate bias caused by the eleva-
tion of IAP. Patients’ clinical progress and secondary endpoints will
be monitored by daily SOFA scores, laboratory and
physical examinations. Postoperative hydration and vasopressor therapy will
be directed by MAP, ScvO2, dCO2 and arterial lactate
levels. PRBC units will be transfused if decreased
hemoglobin (Hb) levels result in tissue oxygenation
disorders or become symptomatic (hypotension, dizzi-
ness or weakness develop). Fresh frozen plasma will be
given if the prothrombin International Normalized Ratio
(INR) > 1.5. Platelet suspension units will be given ac-
cording to the Transfusion Guidelines of the Hungarian
National Blood Transfusion Service. Study arms and assigned intraoperative interventions Study arms and assigned intraoperative interventions
A total number of 40 patients with bladder cancer sub-
mitted to general anesthesia and open radical cystec-
tomy and urinary diversion will be enrolled in this study. An equal number of patients will be randomized into
the two groups. Patients randomized into the SG group undergo an al-
veolar recruitment maneuver using the sustained airway
pressure by the CPAP method, applying 30 cmH2O
PEEP for 30 s followed by a decremental PEEP titration
procedure directed by Cstat. During the PEEP titration
procedure, PEEP will be decreased from 14 cmH2O by 2
cmH2O every 4 min, until a final PEEP of 6 cmH2O is
reached. On each level of PEEP, ABGs will be collected
and evaluated. Optimal PEEP is considered as the PEEP
value resulting the highest possible Cstat measured by
the ventilator. After the PEEP titration procedure, lung-
protective mechanical ventilation will be performed
using optimal PEEP and low tidal volumes and ARM will
be performed every 60 min. From postoperative day 4 (POD 4 to POD 28, follow-up) From postoperative day 4 (POD 4 to POD 28, follow-up)
During the follow-up period, secondary endpoints, in-
hospital stay, 28-day and in-hospital mortality will also
be evaluated. During surgery, in cases of hypotension, intravenous
norepinephrine will be started to maintain mean arterial
pressure above 65 mmHg. For intraoperative fluid man-
agement patients will receive 3 mL/Kg/h of balanced
crystalloid solution until end of surgery. In cases of
bleeding, a 200-mL colloid (hydroxyethyl starch, HES)
solution bolus and crystalloid substitution will be given. Packed red blood cell (PRBC) transfusion will be given
whenever the attending anesthetist feels it necessary. Figure 2 shows the CONSORT flowchart of the trial. Intraoperative care MAP mean arterial pressure, HR heart rate, SpO2 peripheral capillary oxygen
saturation, EtCO2 end-tidal carbon dioxide partial pressure, EtSevo end-tidal
sevoflurane concentration, MAC minimal alveolar concentration MAP mean arterial pressure, HR heart rate, SpO2 peripheral capillary oxygen
saturation, EtCO2 end-tidal carbon dioxide partial pressure, EtSevo end-tidal
sevoflurane concentration, MAC minimal alveolar concentration LPV (TV = 6 mL/Kg IBW, FiO2 = 0.5) will be performed. In the SG (“optimal PEEP”) PEEP will be determined
during a Cstat-directed decremental PEEP titration pro-
cedure. During surgery ARM will be repeated and arterial
and central venous blood gas samples (ABGs, CVBGs) will
be evaluated every 60 min. In case of decreased oxygen
saturation (SpO2 < 94%) rescue ARM will be performed
using a FiO2 of 1.0. PCT levels will be measured 2, 6, 12,
24, 48 and 72 h after surgical incision. In both groups, patients will be allowed to drink clear
fluids immediately after surgery and the use of chewing
gum will be encouraged. Prokinetics and an oral liquid
diet using a drinking formula will be started on POD 1
and patients will begin active mobilization. The nasogas-
tric tube will be removed on the morning of POD 1. Arterial blood pressure, heart rate (HR) and end-tidal
carbon dioxide tension (EtCO2) will be monitored con-
tinuously. Cstat, airway resistance (Raw), Vds/Vt, core
temperature and train-of-four relaxometry data will be
recorded every 15 min. Postoperative care After extubation, patients will be admitted to the ICU. ABGs and CVBGs will be collected and evaluated (pH,
base
excess
(BE),
standard
bicarbonate
(stHCO3−),
ScvO2), PaO2/FiO2 and central venous-to-arterial carbon
dioxide gap (dCO2) will be calculated every 6 h until
72 h after surgery. On the first postoperative day (POD),
a chest X-ray will be performed and repeated on the fol-
lowing days if the development of pulmonary complica-
tions are suspected. The chest X-ray will be evaluated by
an independent, trained radiologist who will not be in-
volved in the study. Continuous epidural analgesia and
intermittent intravenously administered analgesia (para-
cetamol or metamizol) will be introduced, and evaluated
effective if a Numeric Pain Rating Scale (NPRS) score is
lower than 3 points. Patients randomized into the CG group will undergo
an alveolar recruitment maneuver using the sustained
airway pressure by the CPAP method, applying 30
cmH2O PEEP for 30 s followed by low-tidal-volume
LPV using a PEEP value of 6 cmH2O and ARM will be
repeated every 60 min. During postoperative care, continuous intraabdominal
pressure (IAP) monitoring via a direct intraperitoneal
catheter, placed before closure of the abdominal wall, Page 6 of 9 Ruszkai et al. Trials (2017) 18:375 Fig. 2 Consolidated Standards of Reporting Trials (CONSORT) flowchart. PEEP positive end-expiratory pressure, PCT procalcitonin, ABGs arterial blood
gas sample, CVBGs central venous blood gas sample, Cstat static pulmonary compliance, Vds/Vt dead space fraction, Raw airway resistance, MAP mean
arterial pressure, ARM alveolar recruitment maneuver, PRBC packed red blood cell, FFP fresh frozen plasma, IAP intraabdominal pressure Fig. 2 Consolidated Standards of Reporting Trials (CONSORT) flowchart. PEEP positive end-expiratory pressure, PCT procalcitonin, ABGs arterial blood
gas sample, CVBGs central venous blood gas sample, Cstat static pulmonary compliance, Vds/Vt dead space fraction, Raw airway resistance, MAP mean
arterial pressure, ARM alveolar recruitment maneuver, PRBC packed red blood cell, FFP fresh frozen plasma, IAP intraabdominal pressure Adverse events and interruption of the trial Every patient included in the trial will receive daily visits
from an intensive care therapist and urologist in charge
from POD 1 until leaving the hospital. During ICU stay,
and if necessary on the intermediate care unit, all pa-
tients will be continuously monitored. The study nurse
will be responsible for collecting blood samples and will
record relevant required data onto CRF. During the out-
of-hospital follow-up period (until POD 28) patients’
progress, particularly deterioration will be checked by
daily phone-call visits. Radical cystectomy and urinary diversion is considered
major surgery with an operating time lasting for several
hours. This gives the potential for inappropriate intraop-
erative ventilatory management causing further harm by
exacerbating
the
surgery-induced
inflammatory
re-
sponse, hence causing more postoperative complica-
tions. Titrating PEEP and performing regular ARMs
during the anesthesia of these patients certainly has a
strong pathophysiological rationale with potential bene-
fits as indicated by recent clinical trials [4–7, 14], but
this strategy is also cumbersome, time consuming and,
due to the numerous blood gas samplings required, may
be costly. Therefore, testing our hypothesis in a clinical
study is necessary to answer these questions. The investigators will monitor the patients for any ad-
verse events (AEs), which are defined as severe or pro-
longed hypotension (systolic blood pressure < 90 mmHg)
and significant cardiac arrhythmias associated with the
PEEP titration procedure. AEs will be documented on
the CRF and the principal investigator will be informed. Serious adverse events (SAEs) are defined as severe baro-
trauma leading to pneumothorax, significant prolongation
of hospitalization, persistent or significant disability or in-
capacity, and severe deterioration (life-threatening state or
even death) associated with the PEEP titration procedure. All treatment-related SAEs will be recorded and reported
to the Hungarian Scientific and Medical Research Council
Ethics Committee and the Local Ethics Committees. If any
SAEs occur, the trial will be interrupted and an investiga-
tion will be performed. The investigators will monitor the patients for any ad-
verse events (AEs), which are defined as severe or pro-
longed hypotension (systolic blood pressure < 90 mmHg)
and significant cardiac arrhythmias associated with the
PEEP titration procedure. AEs will be documented on
the CRF and the principal investigator will be informed. Duration of the trial The annual number of open radical cystectomy and
urinary diversion is around 100 in the two study centers. Recruitment of the participants is expected within
18 months. The final data collection and estimated com-
pletion date of the trial is March 2018. Data distribution will be tested by the Kolmogorov-
Smirnov analysis. Normally distributed data will be pre-
sented as mean and standard deviation (SD) and skewed
data as median (interquartile range). Comparing related
samples, the paired and unpaired t test will be used for
normally distributed data and the Wilcoxon signed rank
test and Mann-Whitney U test for skewed data. Differ-
ences in proportions will be evaluated using the Fisher’s
exact test, and risk ratio with associated 95% confidence
interval (CI). Analysis of the primary endpoint (PPC)
will be carried out by the unpaired Student’s t test (95%
CI). A two-way, repeated-measures analysis of variance
(two-way RM ANOVA) will be used to compare the
groups’ serum PCT levels. The relationship between
PCT levels and organ dysfunctions will be evaluated
using the Pearson correlation. Statistical analysis of SOFA
scores, ICU days, in-hospital stay, in-hospital and 28-day
mortality data of groups will be implemented by the chi-
square test. A P value < 0.05 will be considered significant. Data monitoring Data will be analyzed by the research team in collabor-
ation with a medically versed biostatistician after com-
pletion of the trial. There will be no interim analysis. Statistical analysis will be conducted on an intention-to-
treat basis. IBM SPSS 20.0 statistical software will be
used for analysis. Data monitoring will be performed centrally for quality
control purposes by an external, independent physician,
who will not be involved in the study. Monitoring will
evaluate the progress of the study and verify the accur-
acy and completeness of the data recording (CRF, source
data, Informed Consent Forms and outcome variables). Ruszkai et al. Trials (2017) 18:375 Ruszkai et al. Trials (2017) 18:375 Page 7 of 9 Page 7 of 9 It is expected that the majority of source data will be
recorded onto CRF; nonetheless, before starting the data
analysis, the mechanism and pattern of missing data will
be evaluated and these findings will be used to deter-
mine whether they have had an impact on the statistical
analysis and results and how they can be managed. Discussion This investigator-initiated, pragmatic, interventional, pro-
spective, randomized controlled trial will assess the
possible benefits and disadvantages of an individualized
lung-protective mechanical ventilation strategy during
open radical cystectomy and urinary diversion as indicated
mainly by PPC and the inflammatory response. PPC can develop after major abdominal surgery. Im-
paired gas exchange may lead to secondary disorders
(delayed return of gastrointestinal function, renal dys-
function, cardiac disorders, etc.) resulting in prolonged
hospitalization time and increased cost of hospital care
[15–17]. The impact of an inappropriate intraoperative
mechanical ventilation-caused inflammatory response –
both systemic and intrapulmonary –, on these complica-
tions is still uncertain. Surgery induces an inflammatory response that is neces-
sary for postoperative recovery [18–21]. Inappropriate
mechanical ventilation can also cause an inflammatory re-
sponse, which can lead to AEs such as pulmonary compli-
cations and distant organ dysfunction. Applying an
individualized lung-protective ventilatory strategy during
general anesthesia may reduce the degree of inflammation
and decrease the incidence of pulmonary and extrapul-
monary complications in the postoperative period, thereby
contributing to shorter hospitalization time and reduced
cost of hospital care [3–5]. Competing interests
Th
h
d
l
h Additional file 1: SPIRIT 2013 Checklist: recommended items to
address in a clinical trial protocol and related documents. (DOCX 52 kb) Abbreviations Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. ABGs: Arterial blood gas samples; ARDS: Acute respiratory distress syndrome;
ARM: Alveolar recruitment maneuver; ASA: American Society of
Anesthesiologists; BE: Base excess; BMI: Body Mass Index; CG: Control group;
CI: Confidence interval; COPD: Chronic obstructive pulmonary disease;
CPAP: Continuous positive airway pressure; CRF: Case Report Form;
Cstat: Static pulmonary compliance; CT: Computer tomography;
CVBGs: Central venous blood gas samples; dCO2: Central venous-to-arterial
carbon dioxide gap; ECG: Electrocardiogram; EtCO2: End-tidal carbon dioxide
tension; FiO2: Fraction of inspired oxygen; GOLD: Global Initiative for Chronic
Obstructive Lung Disease; HR: Heart rate; IAP: Intraabdominal pressure;
IBW: Ideal bodyweight; ICU: Intensive care unit; INR: International Normalized
Ratio; LMWH: Low-molecular-weight heparin; LPV: Lung-protective
ventilation; MAP: Mean arterial pressure; NPRS: Numeric Pain Rating Scale;
NRS 2002: Nutritional risk screening; NYHA: New York Heart Association;
PaO2: Partial pressure of arterial oxygen; PCT: Procalcitonin; PEEP: Positive
end-expiratory pressure; POD: Postoperative day; PPC: Postoperative
pulmonary complications; PRBC: Packed red blood cells; Raw: Airway
resistance; RFRI: Respiratory Failure Risk Index; ScvO2: Central venous oxygen
saturation; SD: Standard deviation; SG: Study group; SOFA: Sequential Organ
Failure Assessment; SPIRIT: Standard Protocol Items: Recommendation for
Interventional Trials; SpO2: Oxygen saturation; stHCO3−: Standard bicarbonate;
TV: Tidal volume; Vds/Vt: Dead space fraction; VILI: Ventilator-induced lung
injury Availability of data and materials 7. Whalen FX, Gajic O, Thompson GB, et al. The effects of the alveolar
recruitment maneuver and positive end-expiratory pressure on arterial
oxygenation during laparoscopic bariatric surgery. Anesth Analg. 2006;
102:298–305. 7. Whalen FX, Gajic O, Thompson GB, et al. The effects of the alveolar
recruitment maneuver and positive end-expiratory pressure on arterial
oxygenation during laparoscopic bariatric surgery. Anesth Analg. 2006;
102:298–305. Not applicable. Received: 19 January 2017 Accepted: 21 July 2017 Received: 19 January 2017 Accepted: 21 July 2017 References 1. Slutsky AS, Ranieri VM. Ventilator-induced lung injury. N Engl J Med. 2013;
369:2126–36. 1. Slutsky AS, Ranieri VM. Ventilator-induced lung injury. N Engl J Med. 2013;
369:2126–36. 2. Ricard J-D, Dreyfuss D, Saumon G. Ventilator-induced lung injury. Eur Respir
J. 2003;22 Suppl 42:2–9. 2. Ricard J-D, Dreyfuss D, Saumon G. Ventilator-induced lung injury. Eur Respir
J. 2003;22 Suppl 42:2–9. 3. Futier E, Constantin J-M, Paugam-Burtz C, et al. A trial of intraoperative
low-tidal-volume ventilation in abdominal surgery. N Engl J Med. 2013;
369:428–37. 3. Futier E, Constantin J-M, Paugam-Burtz C, et al. A trial of intraoperative
low-tidal-volume ventilation in abdominal surgery. N Engl J Med. 2013;
369:428–37. 4. Hemmes SN, Gama De Abreu M, Pelosi P, et al. High versus low positive
end-expiratory pressure during general anaesthesia for open abdominal
surgery (PROVHILO trial): a multicentre randomised controlled trial. Lancet. 2014;384:495–503. 4. Hemmes SN, Gama De Abreu M, Pelosi P, et al. High versus low positive
end-expiratory pressure during general anaesthesia for open abdominal
surgery (PROVHILO trial): a multicentre randomised controlled trial. Lancet. 2014;384:495–503. Acknowledgements We thank all the contributors and collaborators of our trial for their support. 5. Sutherasan Y, Vargas M, Pelosi P. Protective mechanical ventilation in the
non-injured lung: review and meta-analysis. Crit Care. 2014;18:211. Additional file Competing interests
The authors declare that they have no competing interests. Funding 6. Futier E, Constantin J-M, Pelosi P, et al. Intraoperative recruitment maneuver
reverses detrimental pneumoperitoneum-induced respiratory effects in
healthy weight and obese patients undergoing laparoscopy. Anesthesiology. 2010;113:1310–9. g
This study is supported by internal departmental funding from Péterfy
Sándor Hospital and the University of Szeged. This study is supported by internal departmental funding from Péterfy
Sándor Hospital and the University of Szeged. Anesthesiology. 2010;113:1310–9. Adverse events and interruption of the trial Serious adverse events (SAEs) are defined as severe baro-
trauma leading to pneumothorax, significant prolongation
of hospitalization, persistent or significant disability or in-
capacity, and severe deterioration (life-threatening state or
even death) associated with the PEEP titration procedure. All treatment-related SAEs will be recorded and reported
to the Hungarian Scientific and Medical Research Council
Ethics Committee and the Local Ethics Committees. If any
SAEs occur, the trial will be interrupted and an investiga-
tion will be performed. The potential implications of our results can further
improve our knowledge on the effects of optimal intra-
operative ventilatory strategies and, in the case of posi-
tive results, these may not only be applicable to patients
with bladder cancer undergoing radical cystectomy and Page 8 of 9 Page 8 of 9 Ruszkai et al. Trials (2017) 18:375 Ruszkai et al. Trials (2017) 18:375 urinary diversion, but presumably to all patients under-
going similar types of major abdominal surgery. urinary diversion, but presumably to all patients under-
going similar types of major abdominal surgery. with the Declaration of Helsinki and was prospectively registered on 5 October
2016 at https://clinicaltrials.gov with the trial identification number NCT02931409. Participants fulfilling the inclusion criteria will sign an Informed Consent
Form during their perioperative assessment. Withdrawal of consent may be
initiated by the participant at any time during the trial. Trial status
The trial is ongoing. Authors’ contributions ZR and ZM wrote the manuscript together with IL, EK and PTB. The study
protocol was designed by ZR and ZM in close collaboration with IB and ZB. IL was involved in designing the statistical methods of the study. FG, ES,
GPB, ER and EH will collaborate in patient recruitment and collection of data. ZM is the study director and ZR is the principal investigator of the trial. All
authors read and approved the final manuscript. 8. Mols G, Priebe H-J, Guttmann J. Alveolar recruitment in acute lung injury. Br
J Anaesth. 2006;96:156–66. 8. Mols G, Priebe H-J, Guttmann J. Alveolar recruitment in acute lung injury. Br
J Anaesth. 2006;96:156–66. 9. Talley HC, Bentz N, Georgievski J, et al. Anesthesia providers’ knowledge and
use of alveolar recruitment maneuvers. J Anesth Clin Res. 2012;3:325. 9. Talley HC, Bentz N, Georgievski J, et al. Anesthesia providers’ knowledge and
use of alveolar recruitment maneuvers. J Anesth Clin Res. 2012;3:325. 10. Chacko J, Rani U. Alveolar recruitment maneuvers in acute lung injury/acute
respiratory distress syndrome. Indian J Crit Care Med. 2009;13:1–6. 10. Chacko J, Rani U. Alveolar recruitment maneuvers in acute lung injury/acute
respiratory distress syndrome. Indian J Crit Care Med. 2009;13:1–6. 11. Siobal MS, Ong H, Valdes J, et al. Calculation of physiologic dead space:
comparison of ventilator volumetric capnography to measurements by
metabolic analyzer and volumetric CO2 monitor. Respir Care. 2013;58:
1143–51. Author details
1 1Department of Anaesthesiology and Intensive Care, Péterfy Sándor Hospital,
Péterfy Sándor u. 8-20, Budapest 1076, Hungary. 2Department of
Anaesthesiology and Intensive Therapy, University of Szeged, Semmelweis u. 6, Szeged 6725, Hungary. 3Department of Urology, Péterfy Sándor Hospital,
Péterfy Sándor u. 8-20, Budapest 1076, Hungary. 4Department of Urology,
University of Szeged, Kálvária sgt.57, Szeged 6725, Hungary. 16.
Lindberg M, Hole A, Johnsen H, et al. Reference intervals for procalcitonin
and C-reactive protein after major abdominal surgery. Scand J Clin Lab
Invest. 2002;62:189–94.
17.
Sido B, Teklote JR, Hartel M, et al. Inflammatory response after abdominal
surgery. Best Pract Res Clin Anaesthesiol. 2004;18:439–54.
18.
Sarbinowski R, Arvidsson S, Tylman M, et al. Plasma concentration of
procalcitonin and systemic inflammatory response syndrome after
colorectal surgery. Acta Anaesthesiol Scand. 2005;49:191–6.
19.
Mokart D, Merlin M, Sannini A, et al. Procalcitonin, interleukin 6 and
Systemic Inflammatory Response Syndrome (SIRS): early markers of
postoperative sepsis after major surgery. Br J Anaesth. 2005;94:767–73.
20.
Bogár L, Molnár Z, Tarsoly P, et al. Serum procalcitonin level and leukocyte
antisedimentation rate as early predictors of respiratory dysfunction after
oesophageal tumour resection. Crit Care. 2006;10:R110.
21.
Minami E, Ito S, Sugiura T, et al. Markedly elevated procalcitonin in early
postoperative period in pediatric open heart surgery: a prospective cohort
study. J Intensive Care. 2014;2:38.
22.
Trasy D, Tánczos K, Németh M, et al. Early procalcitonin kinetics and
appropriateness of empirical antimicrobial therapy in critically ill patients. A
prospective observational study. J Crit Care. 2016;34:50–5. Ethics approval and consent to participate
Th
d
d b
h H
S 12. El-Baradey GF, El-Shamaa NS. Compliance versus dead space for optimum
positive end expiratory pressure determination in acute respiratory distress
syndrome. Indian J Crit Care Med. 2014;18:508–12. The study was approved by the Hungarian Scientific and Medical Research
Council Ethics Committee (Egészségügyi Tudományos Tanács Tudományos
Kutatás Etikai Bizottság ETT-TUKEB; chairperson: Professor Dr. Zsuzsanna
Schaff; registration number 21586-4/2016/EKU) on 17 June 2016 and the
Local Ethics Committee of Péterfy Sándor Hospital Budapest (Péterfy Sándor utcai
Kórház Intézeti Kutatásetikai Bizottság IKEB; chairperson: Dr. Mária Vas; registration
number CO-338-045) on 12 September 2016 and the Regional Ethics Committee
of the University of Szeged (Regionális Humán Orvosbiológiai Tudományos és
Kutatásetikai Bizottság RKEB; chairperson: Dr. Tibor Wittmann; registration number
149/2016-SZTE) on 19 September 2016. This study is conducted in accordance 13. Pelosi P, Gama De Abreu M, Rocco PRM. New and conventional strategies
for lung recruitment in acute respiratory distress syndrome. Crit Care. 2010;
14:210. 14. Vargas M, Sutherasan Y, Gregoretti C, et al. PEEP role in ICU and operating
room: from pathophysiology to clinical practice. Sci World J. 2014. doi:10. 1155/2014/852356. 14. Vargas M, Sutherasan Y, Gregoretti C, et al. PEEP role in ICU and operating
room: from pathophysiology to clinical practice. Sci World J. 2014. doi:10. 1155/2014/852356. 15. Desborough JP. The stress response to trauma and surgery. Br J Anaesth. 2000;85:109–17. 15. Desborough JP. The stress response to trauma and surgery. Br J Anaesth. 2000;85:109–17. Page 9 of 9 22.
Trasy D, Tánczos K, Németh M, et al. Early procalcitonin kinetics and
appropriateness of empirical antimicrobial therapy in critically ill patients. A
prospective observational study. J Crit Care. 2016;34:50–5. 21.
Minami E, Ito S, Sugiura T, et al. Markedly elevated procalcitonin in early
postoperative period in pediatric open heart surgery: a prospective cohort
study. J Intensive Care. 2014;2:38. 16.
Lindberg M, Hole A, Johnsen H, et al. Reference intervals for procalcitonin
and C-reactive protein after major abdominal surgery. Scand J Clin Lab
Invest. 2002;62:189–94. Ruszkai et al. Trials (2017) 18:375 Ruszkai et al. Trials (2017) 18:375 16. Lindberg M, Hole A, Johnsen H, et al. Reference intervals for procalcitonin
and C-reactive protein after major abdominal surgery. Scand J Clin Lab
Invest. 2002;62:189–94. 16. Lindberg M, Hole A, Johnsen H, et al. Reference intervals for procalcitonin
and C-reactive protein after major abdominal surgery. Scand J Clin Lab
Invest. 2002;62:189–94. 16. Lindberg M, Hole A, Johnsen H, et al. Reference intervals for procalcitonin
and C-reactive protein after major abdominal surgery. Scand J Clin Lab
Invest. 2002;62:189–94. 17. Sido B, Teklote JR, Hartel M, et al. Inflammatory response after abdominal
surgery. Best Pract Res Clin Anaesthesiol. 2004;18:439–54. 18. Sarbinowski R, Arvidsson S, Tylman M, et al. Plasma concentration of
procalcitonin and systemic inflammatory response syndrome after
colorectal surgery. Acta Anaesthesiol Scand. 2005;49:191–6. 19. Mokart D, Merlin M, Sannini A, et al. Procalcitonin, interleukin 6 and
Systemic Inflammatory Response Syndrome (SIRS): early markers of
postoperative sepsis after major surgery. Br J Anaesth. 2005;94:767–73. 20. Bogár L, Molnár Z, Tarsoly P, et al. Serum procalcitonin level and leukocyte
antisedimentation rate as early predictors of respiratory dysfunction after
oesophageal tumour resection. Crit Care. 2006;10:R110. 21. Minami E, Ito S, Sugiura T, et al. Markedly elevated procalcitonin in early
postoperative period in pediatric open heart surgery: a prospective cohort
study. J Intensive Care. 2014;2:38. 22. Trasy D, Tánczos K, Németh M, et al. Early procalcitonin kinetics and
appropriateness of empirical antimicrobial therapy in critically ill patients. A
prospective observational study. J Crit Care. 2016;34:50–5. 17. Sido B, Teklote JR, Hartel M, et al. Inflammatory response after abdominal
surgery. Best Pract Res Clin Anaesthesiol. 2004;18:439–54. 18. Sarbinowski R, Arvidsson S, Tylman M, et al. Plasma concentration of
procalcitonin and systemic inflammatory response syndrome after
colorectal surgery. Acta Anaesthesiol Scand. 2005;49:191–6. Ruszkai et al. Trials (2017) 18:375 • We accept pre-submission inquiries
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Anesthetic effects and body weight changes associated with ketamine-xylazine-lidocaine administered to CD-1 mice
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RESEARCH ARTICLE a1111111111
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a1111111111 Anesthetic effects and body weight changes
associated with ketamine-xylazine-lidocaine
administered to CD-1 mice Urshulaa Dholakia1☯, Stuart C. Clark-Price1☯*, Stephanie C. J. Keating1☯, Adam W. Stern2 Urshulaa Dholakia1☯, Stuart C. Clark-Price1☯*, Stephanie C. J. Keating1☯, Adam W. Stern2
1 Department of Veterinary Clinical Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois,
United States of America, 2 Department of Pathobiology, University of Illinois at Urbana-Champaign, Urbana,
Illinois, United States of America Urshulaa Dholakia1☯, Stuart C. Clark-Price1☯*, Stephanie C. J. Keating1☯, Adam W. Stern2
1 Department of Veterinary Clinical Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois,
United States of America, 2 Department of Pathobiology, University of Illinois at Urbana-Champaign, Urbana,
Illinois, United States of America Urshulaa Dholakia1☯, Stuart C. Clark-Price1☯*, Stephanie C. J. Keating1☯, Adam W. Stern2
1 Department of Veterinary Clinical Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois,
United States of America, 2 Department of Pathobiology, University of Illinois at Urbana-Champaign, Urbana,
Illinois, United States of America 1 Department of Veterinary Clinical Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois,
United States of America, 2 Department of Pathobiology, University of Illinois at Urbana-Champaign, Urbana,
Illinois, United States of America ☯These authors contributed equally to this work. * sccp@illinois.edu a1111111111
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a1111111111 Abstract Anesthesia for mice is commonly performed through the injection of parenteral agents via
the intraperitoneal (IP) route. Variability in anesthetic sensitivities has been noted in mice
resulting in inconsistencies in anesthetic depth and/or mortality. Anesthetic protocols that
improve consistency and safety are needed. The objectives of this study were to assess the
effects of intraperitoneal (IP) ketamine (95 mg/kg) and xylazine (7 mg/kg) alone or combined
with lidocaine at 4, 8, or 16 mg/kg on time to loss (LRR) and return (RRR) of righting reflex,
duration of immobilization and loss of pedal withdrawal response (PWR), body weight and
histopathology in CD-1 mice. In a prospective, randomized trial, 36 male CD-1 mice, 4–6
weeks of age were randomly assigned to 5 groups: saline (SA, n = 4); ketamine-xylazine
(KX, n = 8); ketamine-xylazine-lidocaine 4 mg/kg (KXL4, n = 8); ketamine-xylazine-lidocaine
8 mg/kg (KXL8, n = 8); ketamine-xylazine-lidocaine 16 mg/kg (KXL16, n = 8). Two mice in
each group were euthanized at day 2 post-injection and the remaining mice were euthanized
at day 11 post-injection. After IP injection, LRR and RRR, duration of immobilization and
loss of PWR, body weight and histopathology were evaluated. LRR occurred sooner in mice
receiving KXL16 compared with KX, with median (range) times of 78 (62–104) and 107 (91–
298) seconds, respectively. Loss of PWR occurred in 1, 5, 4, 6 mice for groups KX, KXL4,
KXL8, and KXL16 respectively. Median (range) duration of absent PWR was longer in mice
receiving KXL16 at 13 (0–30) minutes, compared to KX at 0 (0–9) minutes. Duration of
immobilization and RRR were not different between groups. Weight loss occurred 2 days
following anesthesia but was not different between groups. Weight gain was significantly
greater in all lidocaine groups 11 days post-injection compared to KX. No mortality or histo-
pathologic abnormalities were observed in any group. Lidocaine administered with ketamine
and xylazine shortens the onset of anesthesia in mice and improves anesthetic depth with-
out prolonging recovery time. Introduction Anesthesia for small rodents is commonly performed through the injection of one, or a mix-
ture of parenteral agents [1–3]. The intraperitoneal (IP) route of administration is often uti-
lized for laboratory mice when there is an inability to use inhalant anesthesia for research-
specific requirements, lack of inhalational anesthesia equipment, or due to technical limita-
tions of intravenous or intramuscular injections in small species [4]. Ketamine is frequently
combined with xylazine to induce and maintain anesthesia for a variety of procedures in labo-
ratory mice [5]. However, a great deal of variability in anesthetic sensitivities has been noted
based on strain, sex, and age, such that inconsistencies in anesthetic depth and/or mortality
among individual rodents remains a concern [6–8]. Using balanced anesthesia to reduce total
doses of each individual agent in order to minimize adverse effects, while providing sufficient
analgesia, is especially important for small rodents [4]. Numerous studies have shown advan-
tages of the addition of sedative and/or analgesic medications to improve the quality or dura-
tion of anesthesia in rodents [1,4,6,9]. Systemically administered lidocaine has been used as an adjunct to balanced anesthesia pro-
tocols in several species, including rodents, rabbits, pigs, and humans [10–14]. Lidocaine acts
primarily upon voltage-gated sodium channels to inhibit generation and propagation of action
potentials in electrically active tissues such as nerves, muscle, and cardiac muscle. Other postu-
lated mechanisms of action include the induction of acetylcholine release in the CSF, inhibi-
tion of glycine receptors, and release of endogenous opioids in human and veterinary patients
[10,15]. Lidocaine reduces isoflurane requirements for surgery in dogs and horses [16,17],
contributes to multimodal analgesia in combination with other agents for the treatment of
postoperative pain in dogs, cats, goats, horses, calves, rats, and humans [15], and has anti-
inflammatory in humans, horses, and dogs [10,15,18–20]. Local anesthetics such as lidocaine
have demonstrated in vitro antimicrobial effects on bacterial isolates from humans and horses
[21,22]. In addition, lidocaine is readily available as a sterile, pharmaceutical-grade product,
inexpensive, and not subject to state or DEA controlled substance regulation. Intravenous
infusion of lidocaine to C57BL/6 mice has been shown to reduce ketamine and medetomidine
maintenance anesthetic drug requirements [13]. In rats, the inclusion of lidocaine to intraperi-
toneally-administered pentobarbital has been shown to reduce pain behavior in comparison to
injection of pentobarbital alone, and reduce c-fos expression, an indicator of stress, in spinal
cord neurons [23]. OPEN ACCESS Citation: Dholakia U, Clark-Price SC, Keating SCJ,
Stern AW (2017) Anesthetic effects and body
weight changes associated with ketamine-xylazine-
lidocaine administered to CD-1 mice. PLoS ONE 12
(9): e0184911. https://doi.org/10.1371/journal. pone.0184911 Editor: Jyotshna Kanungo, National Center for
Toxicological Research, UNITED STATES
Received: July 12, 2017
Accepted: September 1, 2017
Published: September 14, 2017
Copyright: © 2017 Dholakia 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: Jyotshna Kanungo, National Center for
Toxicological Research, UNITED STATES
Received: July 12, 2017
Accepted: September 1, 2017
Published: September 14, 2017 Copyright: © 2017 Dholakia 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. Competing interests: The authors have declared
that no competing interests exist. 1 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0184911
September 14, 2017 Ketamine-xylazine-lidocaine in CD-1 mice Introduction Despite studies demonstrating the benefits of systemically administered
lidocaine, there is limited literature evaluating clinical use of lidocaine for general anesthetic
protocols in mice. The current study was performed to determine how the inclusion of lido-
caine in ketamine/xylazine-anesthetized mice would affect the duration of anesthesia, response
to noxious stimulation, changes in post-anesthetic body weight and histopathologic findings
following intraperitoneal injection in CD-1 mice. The hypothesis tested was that the addition
of lidocaine to an IP injection of ketamine and xylazine in mice would result in a more rapid
induction and a deeper plane of anesthesia. Experimental groups Mice were randomly assigned by use of a random number generator (random.org) to one of 5
groups: saline control (SA, n = 4); ketamine-xylazine (KX, n = 8); ketamine-xylazine-lidocaine
4mg/kg (KXL4, n = 8); ketamine-xylazine-lidocaine 8mg/kg (KXL8, n = 8); ketamine-xyla-
zine-lidocaine 16mg/kg (KXL16, n = 8). Two mice in each group were euthanized at day 2
post-injection and the remaining mice were euthanized at day 11 post-injection. Animals were
euthanized by carbon dioxide inhalation according to American Veterinary Medical Associa-
tion Guidelines for the Euthanasia of Animals, 2013. Cervical dislocation was subsequently per-
formed to ensure death, after respiratory arrest. All mice were submitted for gross necropsy
and histopathology of thoracic and abdominal organs. A minimum sample size of 7 mice per
experimental group was determined to be necessary to detect a difference between groups of a
60 second decrease in time to LRR with a sigma of 60, and alpha of 0.05, and a power set to
0.80. The sigma value was obtained from a previous study evaluating xylazine-ketamine anes-
thesia in mice [24]. To ensure statistical relevancy and to account for the potential for error
during the study, the number of mice was increase to 8 per experimental group. Anesthetic dilution and administration All anesthetic agents were USP grade, commercially available formulations. Drugs were
diluted using sterile 0.9% sodium chloride for injection (Hospira), and stored separately in
sterile glass vials at room temperature for no more than 7 days. Ketamine hydrochloride (Zeta-
mine, VetOne) was diluted to a concentration of 10mg/mL; xylazine hydrochloride (Anased,
Akorn Inc) was diluted to a concentration of 2mg/mL; and lidocaine hydrochloride 2% (Hos-
pira) was diluted to 2mg/mL (for groups KXL4 and KXL8) and to 4mg/mL for administration
to group KXL16. The maximum dose of 16 mg/kg of lidocaine was selected as it remains
below toxic doses in mice [25,26]. Control animals were administered 0.5 mL of sterile 0.9%
sodium chloride. Ketamine (95 mg/kg) and xylazine (7 mg/kg) were administered to all experi-
mental groups. Ketamine and xylazine doses were determined based on the authors’ previous
experience, and fall within published reference ranges recommended for mice. Lidocaine was
administered according to experimental group. Anesthetics were combined into a single
syringe for intraperitoneal administration just prior to dosing. Mice were manually restrained
and injected with similar technique, using a 1mL tuberculin syringe with 25g needle into the
right lower quadrant of the abdomen. Total volumes for IP injection were no greater than
20μL/g. Ketamine-xylazine-lidocaine in CD-1 mice and then daily throughout the study period. This study was approved by the University of Illi-
nois Urbana-Champaign Institutional Animal Care and Use Committee (protocol #15195),
and mice were housed within the university’s AAALAC-accredited animal facility. Husbandry,
health monitoring, and experimental procedures were in compliance with the recommenda-
tions of the Guide for the Care and Use of Laboratory Animals (NRC, 2011). Animals Thirty six, male CD-1(ICR) mice (age 4–6 wk) were acquired from Charles River Laboratories
(Wilmington, MA USA). Mice were housed in compatible groups within individually venti-
lated microisolator cages (Microvent, Allentown Caging Equipment), provided with free
choice standard rodent diet (Teklad Rodent Diet 8604, Envigo), bottled tap water, autoclaved
corncob bedding, and cotton nesting material. All mice were weighed prior to the experiment, PLOS ONE | https://doi.org/10.1371/journal.pone.0184911
September 14, 2017 2 / 11 Ketamine-xylazine-lidocaine in CD-1 mice hind toes. The same investigator (UD) performed all PWR to maintain consistency. Digital
pressure was chosen over mechanical forceps in order to avoid traumatic injury to the toes,
with purposeful withdrawal of the limb considered a positive response. The time to return of
righting reflex (RRR) was recorded for each mouse. Once alert and ambulatory, mice were
returned to their home cage. All procedures took place between 0900 and 1200, to control for
circadian variation. Samples At the designated experimental time points, mice were euthanized and immediately submitted
to the Veterinary Diagnostic Laboratory, University of Illinois at Urbana-Champaign for
blinded necropsy examination and histopathology. Tissue specimens collected were preserved
in 10% buffered formalin with fixation times of approximately 24–72 hours prior to process-
ing. Samples were embedded in paraffin and sections cut at 3 μm, and stained with hematoxy-
lin and eosin (HE). Statistics Data were analyzed for normality with a Kolmogorov-Smirnov test. Times to loss and return
of righting reflex, and duration of loss of PWR were analyzed with a Kruskall-Wallis test and a
post hoc Dunn’s multiple comparisons test when significant. A Cochran-Armitage test for
trend was used to analyze the loss of PWR. Percent change in body weights 11 days post-injec-
tion was calculated in all surviving mice as [(weight on day 11—weight on day zero)/weight on
day zero] X 100 and were analyzed with ANOVA and a post hoc Tukey-Kramer multiple com-
parisons test and reported as mean ± SD. A commercial statistical program was utilized for all
analysis (InStat1, GraphPad Software, Inc. La Jolla, CA, USA). A P value of <0.05 was consid-
ered significant. KX, ketamine (95 mg/kg)-xylazine (7 mg/kg), KXL4, ketamine-xylazine-lidocaine (4 mg/kg), KXL8, ketamine-
xylazine-lidocaine (8 mg/kg), KXL16, ketamine-xylazine-lidocaine (16 mg/kg), LRR, loss of righting reflex,
RRR, return of righting reflex, TDI, total duration of immobilization, PWR, pedal withdrawal response.
a Indicates significant difference (P < 0.05) from KX. https://doi.org/10.1371/journal.pone.0184911.t001 Anesthesia and data collection Mice were weighed individually immediately prior to dosing. Following IP injection, mice
were placed into a clean, bedded cage. Simultaneously, a stopwatch was initiated to record the
time to loss of righting reflex (LRR), and all subsequent time measures. Upon LRR, mice were
removed from the cage and placed in dorsal recumbency on a disposable absorbent pad, on
top of a thermostatically-controlled pad (Small Animal Heated Pad, H&K) for thermal sup-
port. Eyes were lubricated using artificial tears ointment (Rugby Laboratories). Pedal with-
drawal response (PWR) was checked at one minute intervals, using digital pressure on the 3 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0184911
September 14, 2017 PLOS ONE | https://doi.org/10.1371/journal.pone.0184911
September 14, 2017 Ketamine-xylazine-lidocaine in CD-1 mice Table 2. Mean ± SD body weight change after intraperitioneal injection with ketamine/xylazine ± lidocaine in mice. KX
KXL4
KXL8
KXL16
Baseline body weight, g
34.9 ± 2.2
34.5 ± 4.3
32.4 ± 1.5
32.0 ± 2.0
Weight (%) change, 2 days post
-1.6 ± 1.6
-0.5 ± 1.7
-1.3 ± 1.3
-0.8 ± 2.1
Weight (%) change, 11 days post
2.2 ± 2.9
7.4 ± 4.1a
7.3 ± 2.0a
11.9 ± 2.0b
KX, ketamine (95 mg/kg)-xylazine (7 mg/kg), KXL4, ketamine-xylazine-lidocaine (4 mg/kg), KXL8, ketamine-xylazine-lidocaine (8 mg/kg), KXL16, ketamine-
xylazine-lidocaine (16 mg/kg). a Indicates significant difference (P < 0.05) from group KX. Table 2. Mean ± SD body weight change after intraperitioneal injection with ketamine/xylazine ± lidocaine in mice. KX
KXL4
KXL8
KXL16
Baseline body weight, g
34.9 ± 2.2
34.5 ± 4.3
32.4 ± 1.5
32.0 ± 2.0
Weight (%) change, 2 days post
-1.6 ± 1.6
-0.5 ± 1.7
-1.3 ± 1.3
-0.8 ± 2.1
Weight (%) change, 11 days post
2.2 ± 2.9
7.4 ± 4.1a
7.3 ± 2.0a
11.9 ± 2.0b Table 2. Mean ± SD body weight change after intraperitioneal injection with ketamine/xylazine ± lidocain D body weight change after intraperitioneal injection with ketamine/xylazine ± lidocaine in mice. ange after intraperitioneal injection with ketamine/xylazine ± lidocaine in mice. Weight (%) change, 11 days post
2.2 ± 2.9
7.4 ± 4.1a
7.3 ± 2.0a
11.9 ± 2.0b
KX, ketamine (95 mg/kg)-xylazine (7 mg/kg), KXL4, ketamine-xylazine-lidocaine (4 mg/kg), KXL8, ketamine-xylazine-lidocaine (8 mg/kg), KXL16, ketamine-
xylazine-lidocaine (16 mg/kg). KX, ketamine (95 mg/kg)-xylazine (7 mg/kg), KXL4, ketamine-xylazine-lidocaine (4 mg/kg), KXL8, ketamine-xylazine-lidocaine (8 mg/kg), KXL16, ketamine-
xylazine-lidocaine (16 mg/kg). kg), KXL4, ketamine-xylazine-lidocaine (4 mg/kg), KXL8, ketamine-xylazine-lidocaine (8 mg/kg), KXL16, ketamine- https://doi.org/10.1371/journal.pone.0184911.t002 group (12.5%); in 5 of 8 mice in the KXL4 group (62.5%); in 4 of 8 mice in the KXL8 group
(50%); and in 6 of 8 mice in the KXL16 group (75%). There was a significant linear trend
toward loss of PWR (P = 0.027) as the dose of lidocaine was increased. The total duration of
loss of PWR in group KXL16 was significantly greater than the KX group (P = 0.027). There
was no statistical difference in duration of loss of PWR between any other groups. Addition-
ally, only mice in group KXL16 maintained a consistent loss of PWR over consecutive minutes,
whereas mice in other groups demonstrated intermittent loss and return of PWR. Body weight data are summarized in Table 2. Pre-injection (baseline) body weight was not
significantly different between control or anesthetic groups, and all anesthetic groups demon-
strated a loss of body weight during the first 2 days post-injection. There was no significant dif-
ference in the percent of weight loss experienced in the first 2 days between anesthetic groups. There were 6 mice remaining for each anesthetic group on day 11 that were included in the
day 11 body weight analysis. However, only 2 mice remained alive in the control group at that
time point. Due to the low number of animals, the control group was not included in the body
weight statistical analysis. Body weight 11 days post-injection increased 2.2 ± 2.9% in group
KX, 7.4 ± 4.1% in group KXL4, 7.3 ± 2.0% in group KXL8, and 11.9 ± 2.0% in group KXL16
from baseline. Mice in group KXL4 (p<0.05), KXL8 (p<0.05), and KXL16 (p<0.001) had a
significantly greater increase in post-anesthetic body weight compared to mice in group KX. Histopathology The lung, heart, liver, kidney, spleen, small intestine and mesentery, and abdominal body wall
were examined from all mice. All mice had a minimal to mild vacuolar hepatopathy (consis-
tent with glycogen) and was considered an incidental finding. Two mice in the KXL16 group
(one 2-day mouse and one 11-day mouse) had small numbers of cytoplasmic vacuoles within
the cardiac interstitium and was not considered to be a clinically relevant finding. No other
cardiac changes including inflammation, fibrosis or necrosis were observed in any sections of
heart examined. Mesothelial cells lining the abdominal body wall were non-reactive. No
abnormalities were found on any other tissue sample from all mice. Tissue from the central
nervous system of mice was not examined. Necropsy All mice were in good overall health and body condition. No gross abnormalities were
observed in any of the day 2 post-injection mice or the day 11 post-injection mice. Results All mice recovered from anesthetic trials in all groups, with no mortality or other adverse
events observed. Times for LRR, RRR and PWR for each group are summarized in Table 1. Mice in all anesthetic groups lost righting reflex following IP injection. Time to LRR for group
KXL16 was significantly shorter than the KX group (P = 0.002). There was no statistical differ-
ence between times to LRR between any other groups. All mice in all anesthetic groups
regained righting reflex by two hours after IP injection. There was no statistical difference
between times to RRR between groups. Loss of PWR occurred in only 1 of 8 mice in the KX Table 1. Median (range) summary data of mice after intraperitioneal injection with ketamine/
xylazine ± lidocaine. KX
KXL4
KXL8
KXL16
LRR (seconds)
107 (91–298)
101 (85–162)
102 (88–141)
78 (62–104)a
RRR (minutes)
47 (36–67)
53 (31–68)
41 (32–54)
42 (37–49)
TDI (minutes)
39 (30–53)
41 (26–47)
27 (21–45)
32 (28–38)
Loss of PWR (minutes)
9 (9)
6 (1–13)
11(1–17)
16.50(11–30)a
Number of mice that lost PWR in group (%)
1/8 (12.5)
5/8 (62.5)
4/8 (50)
6/8 (75)
KX, ketamine (95 mg/kg)-xylazine (7 mg/kg), KXL4, ketamine-xylazine-lidocaine (4 mg/kg), KXL8, ketamine-
xylazine-lidocaine (8 mg/kg), KXL16, ketamine-xylazine-lidocaine (16 mg/kg), LRR, loss of righting reflex,
RRR, return of righting reflex, TDI, total duration of immobilization, PWR, pedal withdrawal response. a Indicates significant difference (P < 0.05) from KX. Median (range) summary data of mice after intraperitioneal injection with ketamine/
± lidocaine Table 1. Median (range) summary data of mice after intraperitioneal injection with ketamine/
xylazine ± lidocaine. KX
KXL4
KXL8
KXL16
LRR (seconds)
107 (91–298)
101 (85–162)
102 (88–141)
78 (62–104)a
RRR (minutes)
47 (36–67)
53 (31–68)
41 (32–54)
42 (37–49)
TDI (minutes)
39 (30–53)
41 (26–47)
27 (21–45)
32 (28–38)
Loss of PWR (minutes)
9 (9)
6 (1–13)
11(1–17)
16.50(11–30)a
Number of mice that lost PWR in group (%)
1/8 (12.5)
5/8 (62.5)
4/8 (50)
6/8 (75) Table 1. Median (range) summary data of mice after intraperitioneal injection with ketamine/
xylazine ± lidocaine. KX, ketamine (95 mg/kg)-xylazine (7 mg/kg), KXL4, ketamine-xylazine-lidocaine (4 mg/kg), KXL8, ketamine-
xylazine-lidocaine (8 mg/kg), KXL16, ketamine-xylazine-lidocaine (16 mg/kg), LRR, loss of righting reflex,
RRR, return of righting reflex, TDI, total duration of immobilization, PWR, pedal withdrawal response. a Indicates significant difference (P < 0.05) from KX. 4 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0184911
September 14, 2017 Ketamine-xylazine-lidocaine in CD-1 mice studies demonstrating beneficial effects of systemically administered lidocaine as an adjunct to
general anesthesia. Lidocaine has previously been demonstrated to reduce the minimum alveo-
lar concentration of inhalant anesthetics in rabbits [12], dogs [16,27], horses [17], and to
reduce opioid requirements in humans [28]. Lidocaine is also used as a co-induction agent in
some species. Intravenous administration of 2 mg/kg lidocaine prior to induction with propo-
fol has been shown to decrease consciousness [29], and to minimize and/or eliminate pain
associated with propofol injection [30]. Inclusion of lidocaine with pentobarbital administered
intraperitoneally to male Wistar Hanover GALAN rats reduced Fos-like immunoreactivity of
spinal neurons by one-third in comparison to pentobarbital alone [23]. That study concluded
that the addition of lidocaine to a moderately painful, alkaline solution (pentobarbital) reduced
nociceptive inputs from injection, thereby serving as a refinement for animal welfare. In consideration of these previous studies, we hypothesized that the addition of lidocaine to
other injectable anesthetic solutions used in mice, such as ketamine/xylazine, would demon-
strate similar beneficial clinical characteristics. All mice administered lidocaine-supplemented
anesthesia experienced a rapid and smooth induction following IP injection, with a zero failure
rate, as all mice lost righting reflex and became immobilized. Additionally, LRR was signifi-
cantly faster in mice administered the KXL16 combination than KX alone. The admixture of
lidocaine with ketamine hydrochloride, an acidic solution supplied at pH of 3.5–5.5, into the
same syringe may have alleviated pain associated with IP injection in our mice, resulting in
less excitement and/or distress. Lidocaine is known to have a rapid onset of action, less than
two minutes [15]. Alternatively, lidocaine may have had a more direct, centrally-acting effect
to enhance the actions of ketamine and xylazine for more rapid induction of unconsciousness
[10]. The higher lidocaine dose required to elicit this effect approaches a similar range to that
used in a prior study evaluating paw-licking responses to formalin in male Swiss albino mice
[31]. In this experiment, IP lidocaine doses of 20–30 mg/kg were required to produce signifi-
cant antinociceptive effects. The dose ranges used in the current study were initially based
upon those used in another study in which lidocaine was given as a continuous intravenous
infusion at 2, 4, and 8 mg/kg/hr, with no hemodynamic issues noted to occur in the mice [13]. PLOS ONE | https://doi.org/10.1371/journal.pone.0184911
September 14, 2017 Discussion To the authors’ knowledge, this study represents the first evaluation of intraperitoneal lido-
caine to supplement ketamine-xylazine anesthesia in mice. The results are supportive of earlier 5 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0184911
September 14, 2017 However, larger groups,
cross-over designed studies, different strains of mice, and other combinations and doses of
lidocaine should be studied to further define doses and techniques that provide more consis-
tent results. Despite the observed variability, the loss of PWR was expected based on the previously
reported properties of lidocaine. Specifically, intravenous lidocaine has been noted to decrease
laryngeal reflexes at intubation in dogs [41,42], and children [43]. In addition, lidocaine con-
tinuous-rate infusion confers an anesthetic-sparing effect in mice [13], and allows for dose
reduction of inhalants and opioids through perioperative analgesic effects [11,27]. p
g p
p
g
[
]
In rodents, post anesthetic weight loss has been noted to persist for up to 48 hours, and is
nonspecific with regard to use of inhalant or parenteral agents [44–47]. Within the present
study, all mice other than the control group lost weight during the first 2 days following anes-
thesia. Mice did not demonstrate other clinical signs of distress, and were otherwise active,
hydrated, and apparently healthy. Similarly, in a study of Long-Evans and Sprague-Dawley
rats, animals were noted to lose 2.5% ± 0.56 of their body weight 24 hours following anesthesia
alone using ketamine/xylazine/acepromazine [45]. Acute loss of body weight, and/or body
condition, is often used as an indicator of pain or distress, and as an objective measure for
humane endpoints for laboratory animals. Few studies clearly differentiate weight loss effects
between anesthetic protocols and other contributing factors [48]. Therefore, the mice in this
study were monitored over the course of 11 days following recovery to assess for influences of
the anesthetic protocol on longer-term body weight and overall health. The inclusion of lido-
caine into the anesthetic protocol did not affect weight loss in the first 2 days following anes-
thesia at any of the doses administered. However, mice in all anesthetic groups eventually
regained weight by day 11 post-injection, and most interestingly, all lidocaine-supplemented
groups showed significantly higher weight gains at 11 days. Mice in group KXL16 had the
greatest weight gain at day 11 post-injection and thus the greatest difference compared to mice
in group KX. The physiologic mechanism for greater weight gain is not clear, as a single-bolus
of lidocaine administered at the time of anesthesia would be unlikely to remain clinically rele-
vant in terms of serum levels for 11 days. The maximum dose of 16 mg/kg of lidocaine was designed to remain below other published
values for CNS toxicity [25], and cardiotoxicity in mice [26]. In contrast to studies investigating other anesthetic adjunctive agents, the duration of anes-
thesia for male CD-1 mice in the current study was not dose-dependent with lidocaine; even at
the highest dose used, the duration of immobilization and time to RRR remained comparable
to that of ketamine/xylazine used alone. Previous adjunctive agents attempted in mice include
acepromazine [32,33], midazolam [34], and numerous permutations of other alpha-2 agonists,
dissociatives, and opioids [2,6,35]. In most cases, the addition of long-acting drugs resulted in
increased duration of immobilization, which potentially delays recovery from hypothermia, a
significant factor contributing to rodent mortality. In particular, use of acepromazine as an
adjunct to ketamine/medetomidine in female BALB/c mice was shown to significantly prolong
the time to recovery [36]. The relatively short duration of action of lidocaine, with a half-life of
0.5–2 hours following intravenous administration [15], may have contributed to its rapid elim-
ination and facilitated a more rapid recovery compared to other anesthetic adjuncts. Similar to prior studies and reviews of injectable anesthesia protocols for mice, great vari-
ability in the response to external stimuli of individual mice to ketamine-xylazine and lido-
caine supplemented groups was observed [6–8]. This may be related to methods of evaluation,
as limb movements do not necessarily correspond to conscious perception [37–39]; however,
many institutions utilize loss of PWR as a sign of adequate surgical depth [8,36]. Lidocaine has
previously been evaluated for stability as a diluted solution for up to 14 days, and no incompat-
ibilities have been noted [40]. Therefore, it is unlikely that mixture of the drug with saline, PLOS ONE | https://doi.org/10.1371/journal.pone.0184911
September 14, 2017 6 / 11 Ketamine-xylazine-lidocaine in CD-1 mice ketamine, or xylazine contributed to the variability as a result of issues with stability or
potency. Instead, it may be attributed to differing rates of IP absorption, or the nature of indi-
vidual animals. Despite the observed variability, mice in group KXL16 showed a consistent
and statistically significant increase in duration of loss of PWR compared to group KX. Addi-
tionally, there was a dose-dependent effect of lidocaine on loss of PWR. It appears that as the
dose of lidocaine is increased, mice are more likely to lose PWR. PLOS ONE | https://doi.org/10.1371/journal.pone.0184911
September 14, 2017 Conclusions The administration of lidocaine concurrently with ketamine and xylazine resulted in a faster
onset of general anesthesia, improves anesthetic depth, and does not extend the duration of
time to recovery of CD-1 mice. Additionally, a significant increase in time of reduced PWR for
the KXL16 mice was noted. Optimum effective doses for other strains of mice, older animals
or those with co-existing conditions may vary from the present study, and should be deter-
mined separately based on initial pilot studies. Further studies investigating the use of intraper-
itoneal lidocaine in mice is warranted. Supporting information S1 File. Raw data. Raw data of mice after intraperitioneal injection with ketamine/
xylazine ± lidocaine. Author Contributions Conceptualization: Urshulaa Dholakia, Stuart C. Clark-Price, Stephanie C. J. Keating. Conceptualization: Urshulaa Dholakia, Stuart C. Clark-Price, Stephanie C. J. Keating. Data curation: Stuart C. Clark-Price. Data curation: Stuart C. Clark-Price. Formal analysis: Stuart C. Clark-Price. Formal analysis: Stuart C. Clark-Price. Funding acquisition: Stuart C. Clark-Price Funding acquisition: Stuart C. Clark-Price. Investigation: Urshulaa Dholakia, Stuart C. Clark-Price, Stephanie C. J. Keating, Adam W. Stern. Methodology: Urshulaa Dholakia, Stuart C. Clark-Price, Stephanie C. J. Keating, Adam W. Stern. Project administration: Stuart C. Clark-Price. Writing – original draft: Urshulaa Dholakia. Writing – review & editing: Urshulaa Dholakia, Stuart C. Clark-Price, Stephanie C. J. Keating,
Adam W. Stern. Ketamine-xylazine-lidocaine in CD-1 mice A weakness of this study was the lack of blinding of the investigator assessing PWR. This
could have led to unintentional bias in the assessment of PWR. However, the other parameters
assessed (LRR, RRR, and body weight) are objective measures and likely not affected by lack of
blinding. Additionally, because of randomization of treatments, on any given day of assess-
ment, the order of mice receiving any of the four treatments was random and therefore it
would not have been possible to assess for any trend in response until the study was completed
and statistical analysis was performed minimizing bias. With that said, future studies will uti-
lize blinding during assessment. Gross and histopathology was performed without knowledge
of treatment groups. It is possible that lidocaine promoted a faster return
to normal feeding behaviors and/or normal gastrointestinal function after recovery from
immobilization. All groups of mice were fed a similar diet, ad libitum. However, feed and
water consumption of each group was not quantified, therefore it is unknown if the lidocaine
groups returned to feeding behaviors sooner. Reduction of inflammation or irritation of the
gastrointestinal tract from the administration of ketamine and/or xylazine by the inclusion of
lidocaine was considered as a possible cause of the increased weight gain in the lidocaine
groups. However, necropsy of 2-day post and 11-day post-injection mice did not reveal any
gross or histologic evidence of gastrointestinal abnormalities in any mice of all groups. Large,
retrospective studies in the human literature are equivocal with regard to the benefits of sys-
temic lidocaine for promoting faster return to normal gastrointestinal function and reducing
hospital stay [49]. Other reviews suggest greatly improved patient comfort, and reduced time
to hospital discharge [11,49]. Post-operative ileus is one complication following general anes-
thesia in horses, and can often be effectively managed using intravenous lidocaine prophylacti-
cally [50]. Further investigation in regard to the effects of lidocaine on food and water intake
of mice following anesthesia is warranted. 7 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0184911
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https://projecteuclid.org/journals/abstract-and-applied-analysis/volume-2014/issue-none/On-Nonnegative-Solutions-of-Fractional-q-Linear-Time-Varying-Dynamic/10.1155/2014/247375.pdf
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On Nonnegative Solutions of Fractional<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M1"><mml:mrow><mml:mi>q</mml:mi></mml:mrow></mml:math>-Linear Time-Varying Dynamic Systems with Delayed Dynamics
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1. Introduction or with the nonnegative solutions of dynamic systems in a
fractional context. See, for instance, [32–34]. On the other
hand, see [13–22, 32–39] for a background on quantum and
fractional calculus and some related applications to dynamic
systems. There are abundant background results concerning the
exact and approximate solutions of fractional differential
equations [1–4], fractional derivatives involving products
of polynomials [5, 6], fractional derivatives and fractional
powers of operators [7–9], and boundary value problems
concerning fractional calculus in a theoretical context and
also concerning a wide range of applications like, for instance,
control theory, robotics, signal processing, heat transfer,
lossless transmission lines, and so forth [1–22]. In particular,
some generalized operators of fractional integration have
been recently applied to the product of generalized Bessel
functions of the first class in [6] leading to multivariable
generalized Lauricella-type functions. Furthermore, related
generalized fractional integrals are also discussed in that
paper. On the other hand, new unified integral formulas
involving products of Srivastava-type polynomials and the
𝐻-function as well as fractional integration for Appell’s
functions are discussed in [23, 24].h The Caputo 𝑞-difference scheme has been proposed in
[40] and then the problems of initial values are investigated in
[41, 42]. In particular, a Caputo-type 𝑞-fractional initial value
problem is solved in [41] with the solution being formulated
in terms of a new 𝑞-Mittag-Leffler function. On the other
hand, the related investigation in [42] is focused on analytical
aspects of 𝑞-fractional calculus while the variational iteration
method is extended “ad hoc” to 𝑞-fractional calculus in order
to solve the Caputo 𝑞-fractional initial value problem. There
is also a recent monograph [43] available on the fractional
𝑞-difference methodology which is of potential interest for
readers interested in quantum fractional calculus. Also, it has
to be pointed out that an increasing research interest is being
devoted to the use of fractional calculus in the analysis of
mathematical models based on partial differential equations. In particular, the fractal heat conduction problem is solved
by proposing a local fractional variation iteration method
in [44]. On the other hand, the solutions of the Helmholtz
equation involving local fractional derivative operators are
investigated in [45] combined with series expansion and
variational iteration methods. This paper is concerned with the investigation of nonneg-
ative solutions of fractional 𝑞-differential dynamic systems
with point delays and some related asymptotic properties
formulated by the Caputo fractional derivative. Hindawi Publishing Corporation
Abstract and Applied Analysis
Volume 2014, Article ID 247375, 19 pages
http://dx.doi.org/10.1155/2014/247375 Hindawi Publishing Corporation
Abstract and Applied Analysis
Volume 2014, Article ID 247375, 19 pages
http://dx.doi.org/10.1155/2014/247375 Hindawi Publishing Corporation
Abstract and Applied Analysis
Volume 2014, Article ID 247375, 19 pages
http://dx.doi.org/10.1155/2014/247375 M. De la Sen Institute for Research and Development of Processes, Faculty of Science and Technology, University of Basque Country,
Campus of Leioa, Barrio Sarriena, P.O. Box 48940, Leioa, Spain Institute for Research and Development of Processes, Faculty of Science and Technology, University of Basque Cou
Campus of Leioa, Barrio Sarriena, P.O. Box 48940, Leioa, Spain Correspondence should be addressed to M. De la Sen; manuel.delasen@ehu.es Correspondence should be addressed to M. De la Sen; manuel.delasen@ehu.es Received 8 January 2014; Accepted 8 February 2014; Published 8 May 2014 Academic Editor: Dumitru Baleanu Copyright © 2014 M. De la Sen. This is an open access article distributed under the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This paper is devoted to the investigation of nonnegative solutions and the stability and asymptotic properties of the solutions
of fractional differential dynamic linear time-varying systems involving delayed dynamics with delays. The dynamic systems are
described based on 𝑞-calculus and Caputo fractional derivatives on any order. Abstract and Applied Analysis 2 𝑀
=
(𝑀𝑖𝑗)
∈
R𝑛×𝑛is a Metzler matrix if 𝑀𝑖𝑗
≥
0; for all (𝑖, 𝑗̸= 𝑖) ∈𝑛×𝑛. 𝑀R𝑛×𝑛is the set of Metzler matrices
of order 𝑛.h It might be pointed out that positive dynamic models are
an essential tool to describe some real world applications as,
for instance, medical, biological, or epidemic models. It has
to be pointed out that a major advantage of the use of 𝑞-
calculus is that it does not need the existence of limits or
restrictive regularity conditions on the functions dealt with
in order to establish the formulation. In particular, derivatives
and higher-order derivatives of a wide class of functions exist
almost everywhere under the 𝑞-calculus framework [37]. In this context, the 𝑞-calculus formalism on differ-integral
systems is close to the classical one on difference systems
with the additional advantage that the parameter running the
samples can be chosen to be real so that it links the selection
of the sampling points in a multiplicative fashion. Such
sampling points are backward-in-time dependent on each
time instant for which the 𝑞-fractional solution is evaluated
while asymptotically vanishing to zero as the number of used
samples increases to infinity for each given time instant where
the quantum fractional solution is being computed. The maximum real eigenvalue, if any, of a real matrix 𝑀,
is denoted by 𝜆max(𝑀). The maximum real eigenvalue, if any, of a real matrix 𝑀,
is denoted by 𝜆max(𝑀). 𝑥≥𝑦, 𝑥> 𝑦, and 𝑥≫𝑦mean, respectively, 𝑥−𝑦≥0,
𝑥−𝑦> 0, and 𝑥−𝑦≫0 for 𝑥, 𝑦being any real scalars,
vectors, or matrices of compatible dimensions or orders.h 𝑥≥𝑦, 𝑥> 𝑦, and 𝑥≫𝑦mean, respectively, 𝑥−𝑦≥0,
𝑥−𝑦> 0, and 𝑥−𝑦≫0 for 𝑥, 𝑦being any real scalars,
vectors, or matrices of compatible dimensions or orders.h p
The following fundamental result of [32] is concerned
with the unique left-sided solution on R0+ of the differential
fractional system (36). Theorem 1. Consider the Caputo fractional differential system
of order 𝛼with 𝑝≥0 (potentially repeated) delays and 0 ≤𝑞≤
𝑝distinct delays: ( 𝐶𝐷
𝛼
𝑎+𝑥) (𝑡) :=
1
Γ (𝑘−𝛼) ∫
𝑡
𝑎
(𝑡−𝜏)𝑘−𝛼−1 (𝑥(𝑘) (𝜏)) 𝑑𝜏
=
𝑝
∑
𝑖=0
𝐴𝑖(𝑡−ℎ𝑖) + 𝐵𝑢(𝑡) ;
𝑡∈[𝑎, 𝑏]
(2) (2) 1.1. Notation. Abstract and Applied Analysis Z, R, and C are the sets of integer, real, and
complex numbers, Z+ and R+ are the positive integer and real
numbers, and N ≡Z0+ := Z+ ∪{0} ;
R0+ := R+ ∪{0} ;
C+ := {𝑧∈C : Re 𝑧> 0} ;
C0+ := {𝑧∈C : Re 𝑧≥0} ,
𝑛:= {1, 2, . . . , 𝑛} . (1) on R0+, for any 𝛼∈C0+ with 𝑘= [Re 𝛼] + 1 if 𝛼∉Z+
and 𝑘= 𝛼if 𝛼∈Z+; 𝑘−1, 𝑘∈Z0+, 0 = ℎ0 < ℎ1 <
ℎ2 < ⋅⋅⋅< ℎ𝑝= ℎ< ∞are distinct constant delays,
𝐴0, 𝐴𝑖∈R𝑛×𝑛(𝑖∈𝑝:= {1, 2, . . . , 𝑝}), are the matrices of
dynamics for each delay ℎ𝑖, 𝑖∈𝑝∪{0}, 𝐵∈R𝑛×𝑚is the control
matrix and with initial condition of the state being given by 𝑘𝑛-
real vector functions 𝜑𝑗: [−ℎ, 0] →R𝑛, with 𝑗∈𝑘−1 ∪{0},
which are absolutely continuous except eventually in a set of
zero measure of [−ℎ, 0] ⊂R of bounded discontinuities with
𝜑𝑗(0) = 𝑥𝑗(0) = 𝑥(𝑗) (0) = 𝑥𝑗0 𝑗∈𝑘−1 ∪{0} and 𝑢: R0+ →
R𝑚is a bounded piecewise continuous control function. Then,
the unique left-sided solution of (2) is given by (1) The following notation is used to characterize different levels
of positivity of matrices. p
y
R𝑛×𝑚
0+
:= {𝑀= (𝑚𝑖𝑗) ∈R𝑛×𝑚: 𝑚𝑖𝑗≥0; for all (𝑖, 𝑗) ∈
𝑛× 𝑚} is the set of all 𝑛× 𝑚real matrices of nonnegative
entries. If 𝑀∈R𝑛×𝑚then 𝑀≥0 is used as a simpler notation
for 𝑀∈R𝑛×𝑚
0+ . p
y
R𝑛×𝑚
0+
:= {𝑀= (𝑚𝑖𝑗) ∈R𝑛×𝑚: 𝑚𝑖𝑗≥0; for all (𝑖, 𝑗) ∈
𝑛× 𝑚} is the set of all 𝑛× 𝑚real matrices of nonnegative
entries. If 𝑀∈R𝑛×𝑚then 𝑀≥0 is used as a simpler notation
for 𝑀∈R𝑛×𝑚
0+ . 0+
R𝑛×𝑚
+
:= {0 ̸= 𝑀= (𝑚𝑖𝑗) ∈R𝑛×𝑚: 𝑚𝑖𝑗≥0; for all (𝑖, 𝑗) ∈
𝑛× 𝑚} is the set of all nonzero 𝑛× 𝑚real matrices of
nonnegative entries (i.e., those with at least one of their
entries being positive). If 𝑀∈R𝑛×𝑚then 𝑀> 0 is used as a
simpler notation for 𝑀∈R𝑛×𝑚
+
. 0+
R𝑛×𝑚
+
:= {0 ̸= 𝑀= (𝑚𝑖𝑗) ∈R𝑛×𝑚: 𝑚𝑖𝑗≥0; for all (𝑖, 𝑗) ∈
𝑛× 𝑚} is the set of all nonzero 𝑛× 𝑚real matrices of
nonnegative entries (i.e., those with at least one of their
entries being positive). If 𝑀∈R𝑛×𝑚then 𝑀> 0 is used as a
simpler notation for 𝑀∈R𝑛×𝑚
+
. Abstract and Applied Analysis 𝑥(𝑡) =
𝑘−1
∑
𝑗=0
(Φ𝛼𝑗0 (𝜏) 𝑥𝑗0 p
+
R𝑛×𝑚
++
:= {𝑀= (𝑀𝑖𝑗) ∈R𝑛×𝑚: 𝑀𝑖𝑗> 0; for all (𝑖, 𝑗) ∈
𝑛× 𝑚} is the set of all 𝑛× 𝑚real matrices of positive entries. If 𝑀∈R𝑛×𝑚then 𝑀≫0 is used as a simpler notation for
𝑀∈R𝑛×𝑚
++ . The superscript 𝑇denotes the transpose and 𝑀𝑇
𝑖
and 𝑀𝑗are, respectively, the 𝑖th row and the jth column of the
matrix 𝑀. p
+
R𝑛×𝑚
++
:= {𝑀= (𝑀𝑖𝑗) ∈R𝑛×𝑚: 𝑀𝑖𝑗> 0; for all (𝑖, 𝑗) ∈
𝑛× 𝑚} is the set of all 𝑛× 𝑚real matrices of positive entries. If 𝑀∈R𝑛×𝑚then 𝑀≫0 is used as a simpler notation for
𝑀∈R𝑛×𝑚
++ . The superscript 𝑇denotes the transpose and 𝑀𝑇
𝑖
and 𝑀𝑗are, respectively, the 𝑖th row and the jth column of the
matrix 𝑀. +
𝑝
∑
𝑖=1
∫
ℎ𝑖
0
Φ𝛼(𝑡−𝜏) 𝐴𝑖𝜑𝑗(𝜏−ℎ𝑖) 𝑑𝜏)
+
𝑝
∑
𝑖=1
∫
𝑡
ℎ𝑖
Φ𝛼(𝑡−𝜏) 𝐴𝑖𝑥(𝜏−ℎ𝑖) 𝑑𝜏
+ ∫
𝑡
0
Φ𝛼(𝑡−𝜏) 𝐵𝑢(𝜏) 𝑑𝜏;
𝑡∈R0+
(3) (3) ∫
𝑡
ℎ
Φ𝛼(𝑡−𝜏) 𝐴𝑖𝑥(𝜏−ℎ𝑖) 𝑑𝜏 A close notation to characterize the positivity of vectors
is the following. + ∫
𝑡
0
Φ𝛼(𝑡−𝜏) 𝐵𝑢(𝜏) 𝑑𝜏;
𝑡∈R0+ R𝑛
0+ := {V = (V1, V2, . . . , V𝑛)𝑇∈R𝑛: V𝑖≥0; for all 𝑖∈𝑛}
is the set of all 𝑛real vectors of nonnegative components. If
V ∈R𝑛then V ≥0 is used as a simpler notation for V ∈R𝑛
0+. with 𝑘= [Re 𝛼] + 1 if 𝛼∉Z+ and 𝑘= 𝛼if 𝛼∈Z+, where with 𝑘= [Re 𝛼] + 1 if 𝛼∉Z+ and 𝑘= 𝛼if 𝛼∈Z+, where with 𝑘= [Re 𝛼] + 1 if 𝛼∉Z+ and 𝑘= 𝛼if 𝛼∈Z+, where p
0+
R𝑛
+ := {0 ̸= V = (V1, V2, . . . , V𝑛)𝑇∈R𝑛: V𝑖≥0; for all 𝑖∈
𝑛} is the set of all 𝑛real nonzero vectors of nonnegative
components (i.e., at least one component is positive). If V ∈R𝑛
then V > 0 is used as a simpler notation for V ∈R𝑛
+. p
0+
R𝑛
+ := {0 ̸= V = (V1, V2, . . . , V𝑛)𝑇∈R𝑛: V𝑖≥0; for all 𝑖∈
𝑛} is the set of all 𝑛real nonzero vectors of nonnegative
components (i.e., at least one component is positive). If V ∈R𝑛
then V > 0 is used as a simpler notation for V ∈R𝑛
+. 1. Introduction See, for
instance, [25–31] for formulations of functional dynamic
systems under delays. Some of these papers are concerned
with fundamental properties of positive dynamic systems Abstract and Applied Analysis Abstract and Applied Analysis 𝑥(𝑡) ≡𝑥(0) (𝑡) =
𝑘
∑
𝑖=1
𝑡𝛼−𝑖𝑥(𝛼−𝑖) (0) (a) If 𝑎> 0 then it is positive if 𝑥(0) ≥0 and 0 ≤
𝑡< 1/[(1 −𝑞)𝑎] but any 𝑞-solution is unbounded if
𝑥(0) ̸= 0 and |𝑥(𝑡)/𝑥(𝑞𝑡)| > 1 if 0 ≤𝑡< 1/[(1 −𝑞)𝑎]
and |𝑥(𝑡)/𝑥(𝑞𝑡)| = +∞if 𝑡= 1/[(1 −𝑞)𝑎]. (a) If 𝑎> 0 then it is positive if 𝑥(0) ≥0 and 0 ≤
𝑡< 1/[(1 −𝑞)𝑎] but any 𝑞-solution is unbounded if
𝑥(0) ̸= 0 and |𝑥(𝑡)/𝑥(𝑞𝑡)| > 1 if 0 ≤𝑡< 1/[(1 −𝑞)𝑎]
and |𝑥(𝑡)/𝑥(𝑞𝑡)| = +∞if 𝑡= 1/[(1 −𝑞)𝑎]. (a) If 𝑎> 0 then it is positive if 𝑥(0) ≥0 and 0 ≤
𝑡< 1/[(1 −𝑞)𝑎] but any 𝑞-solution is unbounded if
𝑥(0) ̸= 0 and |𝑥(𝑡)/𝑥(𝑞𝑡)| > 1 if 0 ≤𝑡< 1/[(1 −𝑞)𝑎]
and |𝑥(𝑡)/𝑥(𝑞𝑡)| = +∞if 𝑡= 1/[(1 −𝑞)𝑎]. (b) If 𝑎= 0 then 𝑥(𝑡) = 𝑥(0) so that the 𝑞-solution is
constant, globally stable, and nonnegative if 𝑥(0) ≥0. (b) If 𝑎= 0 then 𝑥(𝑡) = 𝑥(0) so that the 𝑞-solution is
constant, globally stable, and nonnegative if 𝑥(0) ≥0. (c) If 𝑎< 0 then 𝑥(𝑡) = 𝑥(𝑞𝑡)/(1 + (1 −𝑞)|𝑎|𝑡) ≤𝑥(𝑞𝑡) ≤
𝑥(0); 𝑡≥0 with the first inequality being obvious
and the second one being proved by contradiction. Assume that the second inequality is false. Then, there
is 𝑡> 0, so that there is 𝑞𝑡> 0, such that for any 𝑛∈N,
the subsequent contradiction is got: Note that the solution is positive for any order 𝛼= 𝑘if 𝑎≥0
and 𝑥(𝑖)(0) ≥0 for 𝑖= 1, 2, . . . , 𝑘and 𝑥(0)(0) = 𝑥(0) = 𝑥0. Assume that, for 𝑎< 0, there is some 𝑡> 0 such that 𝑥(𝑡) = 0
and 𝑥𝑘−1(𝜏) < 0 for 𝜏∈(𝑡, 𝑡+ 𝑇) and some 𝑇> 0 since
it follows from continuity arguments that if it is negative at a
point it is also negative on some interval containing the point. Then, the following contradiction arises: 𝑥(0) < 𝑥(𝑞𝑡)
= (1 + (1 −𝑞) |𝑎| 𝑡) 𝑥(𝑡) ≤𝑥(𝑞2𝑡)
= (1 + (1 −𝑞) |𝑎| 𝑞𝑡) 𝑥(𝑞𝑡)
≤⋅⋅⋅≤(1 + (1 −𝑞) |𝑎| 𝑞𝑛𝑡) 𝑥(𝑞𝑛+1𝑡)
≤lim sup
𝑛→+∞(1 + (1 −𝑞) |𝑎| 𝑞𝑛𝑡) 𝑥(𝑞𝑛+1𝑡)
=
lim
𝑛→+∞(1 + (1 −𝑞) |𝑎| 𝑞𝑛𝑡) 𝑥(𝑞𝑛+1𝑡) = 𝑥(0) . Abstract and Applied Analysis Φ𝛼𝑗0 (𝑡) := 𝑡𝑗𝐸𝛼,𝑗+1 (𝐴0𝑡𝛼) ;
Φ𝛼(𝑡) := 𝑡𝛼−1𝐸𝛼,𝛼(𝐴0𝑡𝛼) ,
𝐸𝛼,𝑗(𝐴0𝑡𝛼) :=
∞
∑
ℓ=0
(𝐴0𝑡𝛼)
ℓ
Γ (𝛼ℓ+ 𝑗);
𝑗∈𝑘−1 ∪{0, 𝛼}
(4) Φ𝛼𝑗0 (𝑡) := 𝑡𝑗𝐸𝛼,𝑗+1 (𝐴0𝑡𝛼) ;
Φ𝛼(𝑡) := 𝑡𝛼−1𝐸𝛼,𝛼(𝐴0𝑡𝛼) , Φ𝛼𝑗0 (𝑡) := 𝑡𝑗𝐸𝛼,𝑗+1 (𝐴0𝑡𝛼) ; Φ𝛼(𝑡) := 𝑡𝛼−1𝐸𝛼,𝛼(𝐴0𝑡𝛼) , (4) +
R𝑛
++ := {V = (V1, V2, . . . , V𝑛)𝑇∈R𝑛: V𝑖> 0; for all 𝑖∈𝑛} is
the set of all 𝑛real vectors of positive components. If V ∈R𝑛
then V ≫0 is used as a simpler notation for V ∈R𝑛
++. 𝐸𝛼,𝑗(𝐴0𝑡𝛼) :=
∞
∑
ℓ=0
(𝐴0𝑡𝛼)
ℓ
Γ (𝛼ℓ+ 𝑗);
𝑗∈𝑘−1 ∪{0, 𝛼} 3 Abstract and Applied Analysis for 𝑡≥0 and Φ𝛼0(𝑡) = Φ𝛼(𝑡) = 0 for 𝑡< 0, where 𝐸𝛼,𝑗(𝐴0𝑡𝛼)
are the Mittag-Leffler functions. The formulation within the 𝑞-calculus framework leads to If 𝑞-calculus is used then the integral formulas of
Theorem 1 are not necessary to calculate the fractional
Caputo-type solutions of any order as shown in the subse-
quent example. 𝑥(𝛼−1) (𝑡) = 𝑥(𝛼−1) (0) + ∫
𝑡
0
𝑎𝑥(𝜏1) 𝑑𝜏1,
𝑥(𝛼−2) (𝑡) = 𝑥(𝛼−2) (0) + 𝑡𝑥(𝛼−1) (0)
+ ∫
𝑡
0
∫
𝜏1
0
𝑎𝑥(𝜏2) 𝑑𝜏2 𝑑𝜏1, 𝑥(𝛼−1) (𝑡) = 𝑥(𝛼−1) (0) + ∫
𝑡
0
𝑎𝑥(𝜏1) 𝑑𝜏1,
𝑥(𝛼−2) (𝑡) = 𝑥(𝛼−2) (0) + 𝑡𝑥(𝛼−1) (0) Example 2. Consider the differential equation
̇𝑥(𝑡) = 𝑎𝑥(𝑡),
𝑥(0) = 𝑥0. The standard solution for any real 𝑞∈(0, 1)
and 𝑡≥0 is 𝑥(𝑡) = 𝑒𝑎(1−𝑞)𝑡𝑥(𝑞𝑡). It is globally stable (resp.,
globally asymptotically stable) for 𝑎≤0 (resp., for 𝑎< 0) and
nonnegative for any 𝑎∈R if 𝑥(0) ≥0.h + ∫
𝑡
0
∫
𝜏1
0
𝑎𝑥(𝜏2) 𝑑𝜏2 𝑑𝜏1, g
y
The use of 𝑞-calculus for 𝐷𝑞𝑥(𝑡) := (𝑥(𝑡)−𝑥(𝑞𝑡))/(1−𝑞)𝑡=
𝑎𝑥(𝑡) yields 𝑥(𝛼−𝑗) (𝑡) =
𝑗
∑
𝑖=1
𝑡𝑗−𝑖𝑥(𝑗−𝑖) (0)
(9) 𝑥(𝑡) = 𝑥(𝑞𝑡) + (1 −𝑞) 𝑎𝑡𝑥(𝑡) ;
(5) (9) (5) + 𝑎∫
𝑡
0
∫
𝜏𝑗−1
0
⋅⋅⋅∫
𝜏1
0
𝑥(𝜏𝑗) 𝑑𝜏𝑑𝜏2 𝑑𝜏1, + 𝑎∫
𝑡
0
∫
𝜏𝑗−1
0
⋅⋅⋅∫
𝜏1
0
𝑥(𝜏𝑗) 𝑑𝜏𝑑𝜏2 𝑑𝜏1, then, the solution becomes then, the solution becomes 𝑥(𝑡) =
𝑥(𝑞𝑡)
1 −(1 −𝑞) 𝑎𝑡. (6) (6) 𝑥(𝑡) ≡𝑥(0) (𝑡) =
𝑘
∑
𝑖=1
𝑡𝛼−𝑖𝑥(𝛼−𝑖) (0)
+ 𝑎∫
𝑡
0
∫
𝜏𝑘−1
0
⋅⋅⋅∫
𝜏1
0
𝑥(𝜏𝑘) 𝑑𝜏𝑑𝜏2 𝑑𝜏1. Abstract and Applied Analysis (15) (𝑎−𝑏)(0) = 1,
(𝑎−𝑏)(𝑧) = 𝑎𝑧
∞
∏
𝑘=0
[ 𝑎−𝑏𝑞𝑘
𝑎−𝑏𝑞𝑧+𝑘] ,
(15) 𝑥(𝑡)
(𝑎−𝑏)(0) = 1,
(𝑎−𝑏)(𝑧) = 𝑎𝑧
∞
∏
𝑘= 0 ≤𝑥(𝑡)
(𝑎−𝑏)(0) = 1,
(𝑎−𝑏)(𝑧) = 𝑎𝑧
∞
∏
𝑘=0
[ 𝑎−𝑏𝑞𝑘
𝑎−𝑏𝑞𝑧+𝑘] ,
( 0 ≤𝑥(𝑡) 0 ≤𝑥(𝑡) (15) =
𝑘
∑
𝑖=1
𝑡𝑘−𝑖𝑥(𝑘−𝑖) (0)
−|𝑎| ∫
𝑡
∫
𝜏𝑘−1
∫
𝜏1
𝑥(𝜏𝑘) 𝑑𝜏𝑑𝜏2 𝑑𝜏1
(11)
𝑧(∈R)
̸= 0;
𝑎, 𝑏∈R. If 𝑧∈Z+then the 𝑞-power function is
𝑧−1 =
𝑘
∑
𝑖=1
𝑡𝑘−𝑖𝑥(𝑘−𝑖) (0)
−|𝑎| ∫
𝑡
0
∫
𝜏𝑘−1
0
. . . ∫
𝜏1
0
𝑥(𝜏𝑘) 𝑑𝜏𝑑𝜏2 𝑑𝜏1
= 𝑥(0) −|𝑎| ∫
𝑡
0
∫
𝜏𝑘−1
0
. . . ∫
𝜏1
0
𝑥(𝜏𝑘) 𝑑𝜏𝑑𝜏2 𝑑𝜏1
⇒lim
𝑡→∞𝑥(𝑡) = 0
(11) =
𝑘
∑
𝑖=1
𝑡𝑘−𝑖𝑥(𝑘−𝑖) (0)
−|𝑎| ∫
𝑡
0
∫
𝜏𝑘−1
0
. . . ∫
𝜏1
0
𝑥(𝜏𝑘) 𝑑𝜏𝑑𝜏2 𝑑𝜏1
= 𝑥(0) −|𝑎| ∫
𝑡
0
∫
𝜏𝑘−1
0
. . . ∫
𝜏1
0
𝑥(𝜏𝑘) 𝑑𝜏𝑑𝜏2 𝑑𝜏1
⇒lim
𝑡→∞𝑥(𝑡) = 0
(11)
𝑧(∈R)
̸= 0;
𝑎, 𝑏
If 𝑧∈Z+then the 𝑞-power function is
(𝑎−𝑏)(0) = 1,
(𝑎−𝑏)(𝑧) =
𝑧−1
∏
𝑘=0
[𝑎−𝑏
leading, in particular, to (𝑎−𝑏)(1) = ∏0
𝑘=0[𝑎−
𝑏. Formula (16) is the 𝑞-analog of the Pochham
(𝑞-shifted factorial) [36]. The 𝑞-derivative of a fu =
𝑘
∑
𝑖=1
𝑡𝑘−𝑖𝑥(𝑘−𝑖) (0) 𝑧(∈R)
̸= 0;
𝑎, 𝑏∈R. If 𝑧∈Z+then the 𝑞-power function is If 𝑧∈Z+then the 𝑞-power function is −|𝑎| ∫
𝑡
0
∫
𝜏𝑘−1
0
. . . ∫
𝜏1
0
𝑥(𝜏𝑘) 𝑑𝜏𝑑𝜏2 𝑑𝜏1
= 𝑥(0) −|𝑎| ∫
𝑡
0
∫
𝜏𝑘−1
0
. . . ∫
𝜏1
0
𝑥(𝜏𝑘) 𝑑𝜏𝑑𝜏2 𝑑𝜏1
⇒lim
𝑡→∞𝑥(𝑡) = 0
(11)
If 𝑧∈Z+then the 𝑞-
(𝑎−𝑏)(0) =
leading, in particul
𝑏. Formula (16) is t
(𝑞-shifted factorial) (11) (𝑎−𝑏)(0) = 1,
(𝑎−𝑏)(𝑧) =
𝑧−1
∏
𝑘=0
[𝑎−𝑏𝑞𝑘]
(16) (16) leading, in particular, to (𝑎−𝑏)(1) = ∏0
𝑘=0[𝑎−𝑏𝑞𝑘] = 𝑎−
𝑏. Formula (16) is the 𝑞-analog of the Pochhammer symbol
(𝑞-shifted factorial) [36]. The 𝑞-derivative of a function 𝑓is
defined by and then global (resp., global asymptotic) stability holds for
𝑎≤0 (resp., for 𝑎< 0) while the solutions are nonnegative
for any nonnegative initial conditions and any real 𝑎. Lemma 3. The following properties hold. Lemma 3. The following properties hold. and 𝑡𝑒
=
𝑡𝑒(𝑥0) is large enough, since 𝑟(𝑡)
=
∫
𝑡
0 ∫
𝜏𝑘−1
0
. . . ∫
𝜏1
0 |𝑥(𝜏𝑘)|𝑑𝜏𝑑𝜏2 𝑑𝜏1 is strictly increasing since it
possesses a nonnegative integrand, so that there is 𝑡∗∈[0, 𝑡𝑒)
such that 𝑥(𝑡∗) = 0 and then 𝑥(𝑡) = 0; for all 𝑡≥𝑡∗, that
is, the solution reaches the zero-equilibrium in finite time
and global asymptotic stability is guaranteed. To evaluate the
error between the 𝑞-calculus solution and the standard one,
denote them, respectively, as 𝑥(𝑡) = 𝑥∗(𝑡) + 𝑒(𝑡) and 𝑥∗(𝑡)
and then define the extended vector V(𝑡) = (𝑥∗(𝑡), 𝑒(𝑡))𝑇. The
substitution of both solutions yields (i) The
commutation
property
𝐷𝑗
𝑞(𝐷𝑘
𝑞𝑓)(𝑧)
=
𝐷𝑘
𝑞(𝐷𝑗
𝑞𝑓)(𝑧); 𝑓𝑜𝑟𝑎𝑙𝑙𝑗, 𝑘∈Z0+, 𝑓𝑜𝑟𝑎𝑙𝑙𝑞∈(0, 1)
holds for any real 𝑧̸= 0. (ii) The
commutation
property
𝐷𝑗
𝑞(𝐷𝑘
𝑞𝑓)(𝑧)
=
𝐷𝑘
𝑞(𝐷𝑗
𝑞𝑓)(𝑧); 𝑓𝑜𝑟𝑎𝑙𝑙𝑗, 𝑘∈Z0+, 𝑓𝑜𝑟𝑎𝑙𝑙𝑞∈(0, 1)
holds for 𝑧
=
0 for any 𝑗,𝑘, 𝑛∈Z0+, such that
𝑛= 𝑗+ 𝑘, provided that (𝐷𝑖
𝑞𝑓)(0) exists for any
𝑖∈𝑛= {1, 2, . . . , 𝑛}. V (𝑡) = [[
[
𝑒𝑎(1−𝑞)𝑡
0
(1 −𝑞) 𝑎𝑡
1 −(1 −𝑞) 𝑎𝑡
1
1 −(1 −𝑞) 𝑎𝑡
]]
]
V (𝑞𝑡) ;
∀𝑡∈R0+. (13) (iii) (𝐷𝑛
𝑞(𝑓+ 𝑔))(𝑧) = (𝐷𝑛
𝑞𝑓)(𝑧) + (𝐷𝑛
𝑞𝑔)(𝑧) or any real
𝑧̸= 0, and also for 𝑧= 0 if (𝐷𝑖
𝑞𝑓)(0) exists for any
𝑖∈𝑛= {1, 2, . . . , 𝑛}. (iv) (13) (13) (𝐷𝑛
𝑞𝑓) (𝑧) =
1
(1 −𝑞)
𝑛𝑧𝑛
𝑛
∑
𝑗=0
(−1)𝑗(𝑛
𝑗) 𝑓(𝑞𝑗𝑧) ;
(19) Note that for 𝑎̸= 0, the first eigenvalue of the matrix of
dynamics tends to +∞if 𝑎> 0 and to zero if 𝑎< 0 while the
second one converges to zero as 𝑡→∞in both cases. Thus,
the error between both solutions converges asymptotically to
zero if 𝑎̸= 0. If 𝑎= 0 then V(𝑡) = V(𝑞𝑡), for all 𝑡∈R0+. (19) for all 𝑛∈Z0+, 𝑓𝑜𝑟𝑎𝑙𝑙𝑞∈(0, 1) holds for any real
𝑧̸= 0 and also for 𝑧= 0 if for all 𝑛∈Z0+, 𝑓𝑜𝑟𝑎𝑙𝑙𝑞∈(0, 1) holds for any real
𝑧̸= 0 and also for 𝑧= 0 if (𝐷𝑛
𝑞𝑓) (0) =
1
(1 −𝑞)𝑛lim
𝑧→0 (
𝑛
∑
𝑗=0
(−1)𝑗(𝑘
𝑗)
𝑓(𝑞𝑗𝑧)
𝑧𝑛
)
(20) Abstract and Applied Analysis These
stability properties are independent of the nonnegativity of
the solutions since if 𝑥(0) < 0 then by continuity of the
solutions 𝑥(𝑡) < 0 in some interval [0, 𝑡𝑒) so that (𝐷𝑞𝑓) (𝑧) = 𝑓(𝑞𝑧) −𝑓(𝑧)
(𝑞−1) 𝑧
,
(𝐷𝑞𝑓) (0) = lim
𝑧→0 (𝐷𝑞𝑓) (𝑧)
(17) (17) [35, 36] and the 𝑞-derivative of high 𝑛-order of a function 𝑓
is defined by (𝐷0
𝑞𝑓)(𝑧) = 𝑓(𝑧) and 𝑥(𝑡) = −|𝑥(0)| + |𝑎| ∫
𝑡
0
∫
𝜏𝑘−1
0
. . . ∫
𝜏1
0
𝑥(𝜏𝑘)𝑑𝜏𝑑𝜏2 𝑑𝜏1;
𝑡∈[0, 𝑡𝑒)
(12) (𝐷𝑛
𝑞𝑓) (𝑧) = 𝐷𝑞(𝐷𝑛−1
𝑞
𝑓) (𝑧) ,
𝑛∈Z0+. (18) 𝑡∈[0, 𝑡𝑒) (18) Lemma 3. The following properties hold. Abstract and Applied Analysis (7) 𝑥(0) < 𝑥(𝑞𝑡) 𝑥(0) < 𝑥(𝑞𝑡)
= (1 + (1 −𝑞) |𝑎| 𝑡) 𝑥(𝑡) ≤𝑥(𝑞2𝑡)
= (1 + (1 −𝑞) |𝑎| 𝑞𝑡) 𝑥(𝑞𝑡)
≤⋅⋅⋅≤(1 + (1 −𝑞) |𝑎| 𝑞𝑛𝑡) 𝑥(𝑞𝑛+1𝑡)
≤lim sup
𝑛→+∞(1 + (1 −𝑞) |𝑎| 𝑞𝑛𝑡) 𝑥(𝑞𝑛+1𝑡)
=
lim
𝑛→+∞(1 + (1 −𝑞) |𝑎| 𝑞𝑛𝑡) 𝑥(𝑞𝑛+1𝑡) = 𝑥(0) . (7) 𝑥(0) < 𝑥(𝑞𝑡) 𝑥(0) < 𝑥(𝑞𝑡)
= (1 + (1 −𝑞) |𝑎| 𝑡) 𝑥(𝑡) ≤𝑥(𝑞2𝑡)
= (1 + (1 −𝑞) |𝑎| 𝑞𝑡) 𝑥(𝑞𝑡)
≤⋅⋅⋅≤(1 + (1 −𝑞) |𝑎| 𝑞𝑛𝑡) 𝑥(𝑞𝑛+1𝑡)
≤lim sup
𝑛→+∞(1 + (1 −𝑞) |𝑎| 𝑞𝑛𝑡) 𝑥(𝑞𝑛+1𝑡)
=
lim
𝑛→+∞(1 + (1 −𝑞) |𝑎| 𝑞𝑛𝑡) 𝑥(𝑞𝑛+1𝑡) = 𝑥(0) . (7)
point it is also negative on som
Then, the following contradic
0 > 𝑥(𝑡+ 𝑇)
=
𝛼
∑
𝑖=1
𝑇𝑘−𝑖𝑥(𝑖) (𝑡)
+ |𝑎| ∫
𝑇
0
∫
𝜏𝑘−1
0
⋅⋅⋅∫
𝜏1
0 (7) (10) + |𝑎| ∫
𝑇
0
∫
𝜏𝑘−1
0
⋅⋅⋅∫
𝜏1
0
𝑥(𝜏𝑘+ 𝑡)𝑑𝜏𝑑𝜏2 𝑑𝜏1
≥|𝑎| ∫
𝑇
0
∫
𝜏𝛼−1
0
. . . ∫
𝜏1
0
𝑥(𝜏𝑘+ 𝑡)𝑑𝜏𝑑𝜏2 𝑑𝜏1 > 0
(10) Since 𝑥(𝑡) is bounded for 𝑡≥0 for any given 𝑥(0) then
lim𝑡→∞𝑥(𝑡) = 0. Therefore, for 𝑎< 0 the 𝑞-differential
equation has a nonnegative 𝑞-solution on R0+ for any 𝑥(0) ≥0
and it is globally asymptotically stable. On the other hand,
the Caputo fractional solution of real order 𝛼of the associate
fractional differential equation is so that any Caputo fractional solution of any real order 𝛼
is nonnegative for 𝑡≥0 and any given initial conditions
𝑥(𝑖)(0) ≥0 for 𝑖= 0, 1, . . . , 𝑘−1. Since any solution is
nonnegative for any nonnegative initial conditions, then if 𝑥(𝛼) (𝑡) = 𝑑𝑥(𝛼) (𝑡)
𝑑𝑡𝛼
= 𝑎𝑥(𝑡) ,
𝑥(0) = 𝑥0, 𝑡≥0. (8) (8) 4 Abstract and Applied Analysis 𝑎< 0, one gets for the case of interest 𝑥(𝑖)(0) = 0 for
𝑖= 1, 2, . . . , 𝑘
The 𝑞-power function is
∞
[
𝑏𝑘
] 𝑎< 0, one gets for the case of interest 𝑥(𝑖)(0) = 0 for
𝑖= 1, 2, . . . , 𝑘
The 𝑞-power function is (𝑎−𝑏)(0) = 1,
(𝑎−𝑏)(𝑧) = 𝑎𝑧
∞
∏
𝑘=0
[ 𝑎−𝑏𝑞𝑘
𝑎−𝑏𝑞𝑧+𝑘] ,
𝑧(∈R)
̸= 0;
𝑎, 𝑏∈R. 2. Preliminaries on Fractional
𝑞-Differential Systems (20) Fundamental definitions of 𝑞-calculus are [35–38] Proof. First, note that for 𝑧̸= 0 the identity (𝐷𝑛
𝑞𝑓)(𝑧)
=
𝐷𝑞(𝐷𝑛−1
𝑞
𝑓)(𝑧) always holds for 𝑛∈Z0+, with (𝐷0
𝑞𝑓)(𝑧) = [𝑎]𝑞= 1 −𝑞𝑛
1 −𝑞,
𝑎∈R, 𝑞∈(0, 1) . (14)
Proof. First, note that for 𝑧̸= 0 the identity (𝐷𝑛
𝑞𝑓)(𝑧)
=
𝐷𝑞(𝐷𝑛−1
𝑞
𝑓)(𝑧) always holds for 𝑛∈Z0+, with (𝐷0
𝑞𝑓)(𝑧) = [𝑎]𝑞= 1 −𝑞𝑛
1 −𝑞,
𝑎∈R, 𝑞∈(0, 1) . (14) (14) 5 Abstract and Applied Analysis 𝑓(𝑧), which ensures the existence of (𝐷𝑛
𝑞𝑓)(𝑧) for 𝑛∈Z0+ and
𝑧̸= 0. Proceed now by complete induction and assume that,
for some given 𝑘∈Z0+, the commutation property below
holds for the existing (𝐷𝑛
𝑞𝑓)(𝑧) for any real 𝑧̸= 0: 𝑓(𝑧), which ensures the existence of (𝐷𝑛
𝑞𝑓)(𝑧) for 𝑛∈Z0+ and
𝑧̸= 0. Proceed now by complete induction and assume that,
for some given 𝑘∈Z0+, the commutation property below
holds for the existing (𝐷𝑛
𝑞𝑓)(𝑧) for any real 𝑧̸= 0: for 𝑛= 0, 1, . . . , 𝑘from (19) (first identity) and the definition
of the operator z𝑞(second identity). Then, one gets from the
definition of the 𝑞-derivative in (17) the use of the first and
second identities of (25) for (𝐷𝑛
𝑞𝑓)(𝑧), with 𝑛= 𝑘, the identity
(z𝑞𝑓)(𝑧) = 𝑓(𝑞𝑧), and, finally, the second identity in (25) for
𝑛= 𝑘+ 1: (𝐷𝑛
𝑞𝑓) (𝑧) = 𝐷𝑛−1
𝑞
(𝐷𝑞𝑓) (𝑧) = 𝐷𝑞(𝐷𝑛−1
𝑞
𝑓) (𝑧) . (21) (21) (𝐷𝑘+1
𝑞
𝑓) (𝑧) = (𝐷𝑞(𝐷𝑘+1
𝑞
𝑓)) (𝑧)
=
((1 −z𝑞)
𝑘𝑓) (𝑧) −((1 −z𝑞)
𝑘𝑓) (𝑞𝑧)
(1 −𝑞)
𝑘𝑧𝑘(1 −𝑞) 𝑧
=
((1 −z𝑞)
𝑘𝑓) (𝑧) −((1 −z𝑞)
𝑘z𝑞𝑓) (𝑧)
(1 −𝑞)𝑘+1𝑧𝑘+1
=
((1 −z𝑞)
𝑘+1𝑓) (𝑧)
(1 −𝑞)𝑘+1𝑧𝑘+1
=
1
(1 −𝑞)
𝑘+1𝑧𝑘+1
𝑘+1
∑
𝑗=0
(−1)𝑗(𝑘+ 1
𝑗) 𝑓(𝑞𝑗𝑧)
(26 (𝐷𝑘+1
𝑞
𝑓) (𝑧) = (𝐷𝑞(𝐷𝑘+1
𝑞
𝑓)) (𝑧) Then, by using the commutation property of the operator
(𝐷𝑞𝐷𝑛−1
𝑞
) on 𝐷𝑞𝑓for any real 𝑧̸= 0 h
y
g
p
(𝐷𝑞𝐷𝑛−1
𝑞
) on 𝐷𝑞𝑓for any real 𝑧̸= 0 (𝐷𝑞𝐷𝑛−1
𝑞
) (𝐷𝑞𝑓) (𝑧) = (𝐷𝑛−1
𝑞
𝐷𝑞) (𝐷𝑞𝑓) (𝑧)
= (𝐷𝑞𝐷𝑛
𝑞𝑓) (𝑧) = (𝐷𝑛
𝑞𝐷𝑞𝑓) (𝑧)
= (𝐷𝑛+1
𝑞
𝑓) (𝑧) . (22) The commutation of the operator composition 𝐷𝑛
𝑞𝐷𝑞for any
nonzero 𝑧∈Z+ has been proven. Now, for any integer 𝑧≥2
we can find integers 𝑗, 𝑘∈Z+ (being nonunique for 𝑧≥3)
such that 𝑗+ 𝑘= 𝑛. 2. Preliminaries on Fractional
𝑞-Differential Systems Hence, (𝐷𝑗
𝑞𝐷𝑘
𝑞𝑓)(𝑧) = (𝐷𝑘
𝑞𝐷𝑗
𝑞𝑓)(𝑧) =
(𝐷𝑗+𝑘
𝑞𝑓)(𝑧) and the result follows by taking an arbitrary 𝑛and
nonzero 𝑧∈Z+. Property (i) has been proved. Property (ii)
can be proved in the same way for 𝑗+ 𝑘= 𝑛and any 𝑛∈Z0+
such that (𝐷𝑖
𝑞𝑓)(0) exists for 𝑖∈𝑛= {1, 2, . . . , 𝑛}. Properties
(i)–(iii) have been proved. (26) (26) so that if (19) holds for any given real 𝑧̸= 0 and 𝑛= 0, 1, . . . , 𝑘
for any given 𝑘∈Z+ then it also holds for 𝑛= 0, 1, . . . ., 𝑘+ 1
and such a nonzero real 𝑧. Then, by complete induction, (19)
is true for any nonnegative integer 𝑛. If (𝐷𝑛
𝑞𝑓)(0) exists for
any 𝑛∈Z0+ the result also applies for 𝑧= 0. On the other hand, the 𝑞-derivative operator (17) is a
linear operator [37], so that (𝐷𝑞(𝑓+ 𝑔)) (𝑧) = (𝐷𝑞𝑓) (𝑧) + (𝐷𝑞𝑔) (𝑧) . (23) (23) Property (i) and (23) yield for 𝑧̸= 0, and also for 𝑧= 0 if
(𝐷𝑖
𝑞𝑓)(0) exists for any 𝑖∈𝑛= {1, 2, . . . , 𝑛}, the following
relationships: Assume that 𝑓: [𝑎, 𝑏]
→
C𝑛for some real interval
[𝑎, 𝑏] ⊂R. Then, the Riemann-Liouville left-sided fractional
𝑞-derivative RL𝐷𝛼
𝑞𝑎+𝑓of order 𝛼∈C0+of the vector function
𝑓in [𝑎, 𝑏] is point-wise defined as (𝐷𝑛
𝑞(𝑓+ 𝑔)) (𝑧) = (𝐷𝑞(𝐷𝑛−1
𝑞
(𝑓+ 𝑔))) (𝑧)
= (𝐷𝑛−1
𝑞
(𝐷𝑞(𝑓+ 𝑔))) (𝑧)
= (𝐷𝑛−1
𝑞
((𝐷𝑞𝑓) (𝑧) + (𝐷𝑞𝑔))) (𝑧)
= 𝐷𝑞((𝐷𝑛−1
𝑞
𝑓) (𝑧) + (𝐷𝑛−1
𝑞
𝑔))) (𝑧)
= (𝐷𝑛
𝑞𝑓) (𝑧) + (𝐷𝑛
𝑞𝑔) (𝑧) . (24) ( RL𝐷𝛼
𝑞𝑎+ 𝑓) (𝑡)
:=
1
Γ𝑞(𝑘−𝛼) (𝐷𝑘
𝑞(∫
𝑡
𝑎
𝑓(𝜏)
(𝑡−𝑞𝜏)𝛼+1−𝑘𝑑𝜏𝑞)) ;
(27) ( RL𝐷𝛼
𝑞𝑎+ 𝑓) (𝑡) (27) 𝑡∈[𝑎, 𝑏] , 𝑡∈[𝑎, 𝑏] , where 𝑘= [Re 𝛼] + 1 and Γ : C \ Z0−
→
C, where
Z0−:= {𝑛∈Z : 𝑛≤0}, is the 𝑞-gamma function defined
as Γ𝑞(𝑧) := (1 −𝑞)(𝑧−1)/(1 −𝑞)𝑧−1 = (1/(1 −𝑞)𝑧−1)∏∞
𝑗=0[(1 −
𝑞𝑗)/(1 −𝑞𝑧+𝑗−1)]; 𝑧∈C \ Z0−which satisfies the following
relations [35, 36]: Hence, Property (iii) follows.i Hence, Property (iii) follows.i To prove Property (iv), define the time 𝑞-delay operator
z𝑞on 𝑓as (z𝑞𝑓)(𝑧) = 𝑓(𝑞𝑧) so that (z𝑗
𝑞𝑓)(𝑧) = 𝑓(𝑞𝑖𝑧),
for all 𝑗∈Z0+ with (z0
𝑞𝑓)(𝑧) = 𝑓(𝑧). Now, for any nonzero
real 𝑧, assume that the property is true for 𝑛= 0, 1, . . . 2. Preliminaries on Fractional
𝑞-Differential Systems , 𝑘and
some given 𝑘∈Z+. Thus, Γ𝑞(𝑧+ 1) = (1 −𝑞)
(𝑧)(1 −𝑞)
−𝑧= [𝑧]𝑞Γ𝑞(𝑧) ,
(28) (28) (𝐷𝑛
𝑞𝑓) (𝑧) =
1
(1 −𝑞)𝑛𝑧𝑛
𝑛
∑
𝑗=0
(−1)𝑗(𝑛
𝑗) 𝑓(𝑞𝑗𝑧) (𝐷𝑛
𝑞𝑓) (𝑧) =
1
(1 −𝑞)𝑛𝑧𝑛
𝑛
∑
𝑗=0
(−1)𝑗(𝑛
𝑗) 𝑓(𝑞𝑗𝑧)
=
((1 −z𝑞)
𝑛𝑓) (𝑧)
(1 −𝑞)𝑛𝑧𝑛
(25) where [𝑧]𝑞
=
(1 −𝑞𝑧)/(1 −𝑞) for 𝑧
∈
R is a 𝑞-real
number. Remember that the usual gamma function is defined
by Γ(𝑧)
:=
∫
∞
0 𝜏𝑧−1𝑒−𝜏𝑑𝜏; 𝑧
∈
C \ Z0−. Now, we can
replace the standard simple and higher-order derivatives
under the integral symbol by their 𝑞-derivative versions to (25) Abstract and Applied Analysis 6 build the Caputo fractional 𝑞-derivative 𝐶𝐷𝛼
𝑞𝑎+ 𝑓by using the
identities It turns out that, under the given assumptions, 𝑥: R0+ →
R𝑛
0+ and then the solution is nonnegative; for all 𝑡∈R0+ since
𝑒𝐴0𝑡∈R𝑛×𝑛
0+ ; for all 𝑡∈R0+ since 𝐴0 ∈𝑀R𝑛×𝑛. On the other
hand, (𝐷𝑘
𝑞𝑓) (𝜏) = 𝑓(𝑘)
𝑞
(𝜏) =
𝑑𝑘
𝑞𝑓(𝜏)
𝑑𝜏𝑘
𝑞
= 𝐷𝑞(𝐷𝑘−1
𝑞
𝑓) (𝜏) = 𝐷𝑘−1
𝑞
(𝐷𝑞𝑓) (𝜏)
(29) (𝐷𝑘
𝑞𝑓) (𝜏) = 𝑓(𝑘)
𝑞
(𝜏) =
𝑑𝑘
𝑞𝑓(𝜏)
𝑑𝜏𝑘
𝑞
(29) (29) 𝑥𝑖(𝑡) = 𝑒𝑇
𝑖𝑒𝐴0𝑡𝑥0 𝑥𝑖(𝑡) = 𝑒𝑇
𝑖𝑒𝐴0𝑡𝑥0
+
𝑛
∑
𝑗=1
(
𝑝
∑
𝑖=1
∫
ℎ𝑖
0
𝑒𝑇
𝑖𝑒𝐴0(𝑡−𝜏)
× (𝐴𝑖𝑗𝜑𝑗(𝜏−ℎ𝑖) 𝑑𝜏
+
𝑝
∑
𝑖=1
∫
𝑡
ℎ𝑖
𝑒−𝐴0𝜏𝐴𝑖𝑗𝑥𝑗(𝜏−ℎ𝑖) 𝑑𝜏))
+
𝑚
∑
𝑗=1
𝑝
∑
𝑖=1
∫
𝑡
ℎ𝑖
𝑒𝑇
𝑖𝑒𝐴0(𝑡−𝜏)𝐵𝑖𝑗𝑢𝑗(𝜏−ℎ𝑖) 𝑑𝜏,
∀𝑡∈R0+,
𝑖∈𝑛. (33) = 𝐷𝑞(𝐷𝑘−1
𝑞
𝑓) (𝜏) = 𝐷𝑘−1
𝑞
(𝐷𝑞𝑓) (𝜏) +
𝑛
∑
𝑗=1
(
𝑝
∑
𝑖=1
∫
ℎ𝑖
0
𝑒𝑇
𝑖𝑒𝐴0(𝑡−𝜏) in [𝑎, 𝑏]; then the Caputo left-sided fractional 𝑞-derivative
𝐶𝐷𝛼
𝑞𝑎+ 𝑓of order 𝛼∈C0+ of the vector function 𝑓in [𝑎, 𝑏] ⊂
R+ is point-wise defined as ( 𝐶𝐷𝛼
𝑞𝑎+ 𝑓) (𝑡)
=
1
Γ𝑞(𝑘−𝛼) ∫
𝑡
𝑎
(𝑡−𝑞𝜏)
𝑘−𝛼−1 (𝐷𝑘
𝑞𝑓(𝜏)) 𝑑𝑞𝜏;
(30) +
𝑝
∑
𝑖=1
∫
𝑡
ℎ𝑖
𝑒−𝐴0𝜏𝐴𝑖𝑗𝑥𝑗(𝜏−ℎ𝑖) 𝑑𝜏)) (30) +
𝑚
∑
𝑗=1
𝑝
∑
𝑖=1
∫
𝑡
ℎ𝑖
𝑒𝑇
𝑖𝑒𝐴0(𝑡−𝜏)𝐵𝑖𝑗𝑢𝑗(𝜏−ℎ𝑖) 𝑑𝜏, 𝑡∈[𝑎, 𝑏] , where 𝑘= [Re 𝛼] + 1 if 𝛼∉Z0+ and 𝑘= 𝛼if 𝛼∈Z0+. Note that the existence of 𝑓∈𝐶𝑘−1((𝑎, 𝑏), R𝑛) in (30) is not
required, as it is required in the standard fractional calculus
for the existence of Caputo derivatives since the existence
of the standard and higher-order fractional 𝑞-derivatives is
ensured. ∀𝑡∈R0+,
𝑖∈𝑛. 2. Preliminaries on Fractional
𝑞-Differential Systems (33) It is easy to see that it is always possible to get 𝑥𝑖(𝑡) < 0 by
construction if any of the conditions 𝜑: [−ℎ, 0] →R𝑛
0+,
𝐴𝑖∈R𝑛×𝑛
0+ ; for all 𝑖∈𝑝, 𝑢: R0+
→
R𝑚
0+, or 𝐴0 ∈
𝑀R𝑛×𝑛⇔𝑒𝐴0𝑡∈R𝑛
+; for all 𝑡∈R0+ fails for some 𝜑:
[−ℎ, 0]
→
R𝑛
0+ and 𝑢: R0+
→
R𝑚
0+ by taking some
large component in either the initial condition function or
the control function corresponding to a negative entry of the
matrix whose positivity condition fails. It is easy to see that it is always possible to get 𝑥𝑖(𝑡) < 0 by
construction if any of the conditions 𝜑: [−ℎ, 0] →R𝑛
0+,
𝐴𝑖∈R𝑛×𝑛
0+ ; for all 𝑖∈𝑝, 𝑢: R0+
→
R𝑚
0+, or 𝐴0 ∈
𝑀R𝑛×𝑛⇔𝑒𝐴0𝑡∈R𝑛
+; for all 𝑡∈R0+ fails for some 𝜑:
[−ℎ, 0]
→
R𝑛
0+ and 𝑢: R0+
→
R𝑚
0+ by taking some
large component in either the initial condition function or
the control function corresponding to a negative entry of the
matrix whose positivity condition fails. Example 4. Consider the differential dynamic system Example 4. Consider the differential dynamic system ̇𝑥(𝑡) =
𝑝
∑
𝑖=0
𝐴𝑖(𝑡−ℎ𝑖) + 𝐵𝑢(𝑡) ;
𝑡∈[0, ∞) ,
(31) where 0 = ℎ0 < ℎ1 < ℎ2 < ⋅⋅⋅< ℎ𝑝= ℎ< ∞are
distinct constant delays, 𝐴0, 𝐴𝑖∈R𝑛×𝑛; for all 𝑖∈𝑝are the
matrices of dynamics for each delay ℎ𝑖, 𝑖∈𝑝∪{0}, 𝐵∈R𝑛×𝑚
is the control matrix and with initial condition of the state
being given the real vector function 𝜑: [−ℎ, 0]
→
R𝑛,
which is absolutely continuous except eventually in a set of
zero measure of [−ℎ, 0] ⊂R of bounded discontinuities with
𝜑(0) = 𝑥(0) = 𝑥0 𝑗∈𝑝, and 𝑢: R0+ →R𝑚is a bounded
piecewise-continuous control function. Some results about
the nonnegativity of the solutions of (31) by using standard,
fractional and 𝑞-calculus follow below. where 0 = ℎ0 < ℎ1 < ℎ2 < ⋅⋅⋅< ℎ𝑝= ℎ< ∞are
distinct constant delays, 𝐴0, 𝐴𝑖∈R𝑛×𝑛; for all 𝑖∈𝑝are the
matrices of dynamics for each delay ℎ𝑖, 𝑖∈𝑝∪{0}, 𝐵∈R𝑛×𝑚
is the control matrix and with initial condition of the state
being given the real vector function 𝜑: [−ℎ, 0]
→
R𝑛,
which is absolutely continuous except eventually in a set of
zero measure of [−ℎ, 0] ⊂R of bounded discontinuities with
𝜑(0) = 𝑥(0) = 𝑥0 𝑗∈𝑝, and 𝑢: R0+ →R𝑚is a bounded
piecewise-continuous control function. The following extension of Proposition 7 is obvious. The following extension of Proposition 7 is obvious. Proposition 8. Proposition 7 still holds if 𝐴𝑖: R0+ →R𝑛×𝑛;
𝑖∈𝑛∪{0}. ∫
𝑡
𝑎
(𝑡−𝑞𝜏)𝑘−𝛼−1 (𝐷𝑘
𝑞𝑥) (𝜏) 𝑑𝑞𝜏
= ∫
𝑡
0
(𝑡−𝑞𝜏)
𝑘−𝛼−1 (𝐷𝑘
𝑞𝑥) (𝜏) 𝑑𝑞𝜏
−∫
𝑎
0
(𝑡−𝑞𝜏)𝑘−𝛼−1 (𝐷𝑘
𝑞𝑥) (𝜏) 𝑑𝑞𝜏
−(1 −𝑞) 𝑎
∞
∑
𝑛=0
(1 −𝑞𝑛+1)
𝑘−𝛼−1𝑎𝑘−𝛼−1𝑞𝑛(𝐷𝑘
𝑞𝑥) (𝑞𝑛𝑎)
(40) 2. Preliminaries on Fractional
𝑞-Differential Systems (ii) 𝑥(𝑡) ≥0;𝑓𝑜𝑟𝑎𝑙𝑙𝑡∈R0+ if (−𝐴0) ∈𝑀R𝑛×𝑛
+
with 𝐴0𝑖𝑖=
−1; 𝑓𝑜𝑟𝑎𝑙𝑙𝑖∈𝑛and 𝐴0𝑖𝑗≤0 with 𝐴0𝑖𝑗< 0 for only
one 𝑗∈𝑛for each 𝑖∈𝑛. 1
Γ𝑞(0) =
1 −𝑞
(1 −𝑞)
(1) = 1. (38) (38) Proof. The solution of (34) is Proof. The solution of (34) is 𝑥(𝑡) = (𝐼−𝐴0)−1 (𝑥(𝑞𝑡) + (1 −𝑞) 𝑡
𝑝
∑
𝑖=1
𝐴𝑖𝑥(𝑡−ℎ𝑖)) ; Now consider, from (2) and (24), the linear time-varying dif-
ferential functional left-sided Caputo fractional 𝑞-differential
system of order 𝛼 ∀𝑡∈R0+ ∀𝑡∈R0+ 0+
(35) ( 𝐶𝐷𝛼
𝑞0+ 𝑥) (𝑡) = (1 −𝑞)
𝑘−𝛼−1
∞
∏
𝑗=0
[1 −𝑞𝑘+𝑗−𝛼
1 −𝑞𝑗+1 ]
× ∫
𝑡
𝑎
(𝑡−𝑞𝜏)
𝑘−𝛼−1 (𝐷𝑘
𝑞𝑥) (𝜏) 𝑑𝑞𝜏
=
𝑝
∑
𝑖=0
𝐴𝑖(𝑡) 𝑥(𝑡−ℎ𝑖) + 𝐵(𝑡) 𝑢(𝑡)
(39) provided that (𝐼−𝐴0)−1 exists. Since 𝜑:
[−ℎ, 0] →R𝑛
0+,
𝑢: R0+ →R𝑚
0+, 𝐴𝑖∈R𝑛×𝑛
0+ , for nonnegativity of the solutions
the existence of (𝐼−𝐴0)−1 ∈R𝑛×𝑛
+
is also required. Note that
if (𝐼−𝐴0) ∈R𝑛×𝑛
+
and nonsingular then (𝐼−𝐴0)−1 ∈R𝑛×𝑛
+
if and only if (𝐼−𝐴0) ∈R𝑛×𝑛
+
is monomial (or generalized
permutation matrix, i.e., it has only a nonzero entry per
row and then only a nonzero entry per column). Due to
its structure, this condition can be fulfilled with 𝐴0 being
diagonal, that is, 𝐴0𝑖𝑗= 0 for 𝑖, 𝑗( ̸= 𝑖) ∈𝑛in order that
(𝐼−𝐴0) is monomial and (𝐼−𝐴0) ∈R𝑛×𝑛
+ . Then, (𝐼−𝐴0)−1 ∈
R𝑛×𝑛
0+
if and only if for all 𝑖∈𝑛either 1 > 𝐴0𝑖𝑖> 0 or
𝐴0𝑖𝑖≤0. Hence, Property (i) follows directly. Property (ii)
follows since, under the given constraints, (−𝐴0) ∈𝑀R𝑛×𝑛,
with 𝐴0𝑖𝑖= −1for all 𝑖∈𝑛, (𝐼−𝐴0) ∈R𝑛×𝑛
+
and Metzler,
monomial (then nonsingular) so that (𝐼−𝐴0)−1 ∈R𝑛×𝑛
+ . Hence, Property (ii) follows. provided that (𝐼−𝐴0)−1 exists. Since 𝜑:
[−ℎ, 0] →R𝑛
0+,
𝑢: R0+ →R𝑚
0+, 𝐴𝑖∈R𝑛×𝑛
0+ , for nonnegativity of the solutions
the existence of (𝐼−𝐴0)−1 ∈R𝑛×𝑛
+
is also required. Note that
if (𝐼−𝐴0) ∈R𝑛×𝑛
+
and nonsingular then (𝐼−𝐴0)−1 ∈R𝑛×𝑛
+
if and only if (𝐼−𝐴0) ∈R𝑛×𝑛
+
is monomial (or generalized
permutation matrix, i.e., it has only a nonzero entry per
row and then only a nonzero entry per column). Due to
its structure, this condition can be fulfilled with 𝐴0 being
diagonal, that is, 𝐴0𝑖𝑗= 0 for 𝑖, 𝑗( ̸= 𝑖) ∈𝑛in order that
(𝐼−𝐴0) is monomial and (𝐼−𝐴0) ∈R𝑛×𝑛
+ . 2. Preliminaries on Fractional
𝑞-Differential Systems Some results about
the nonnegativity of the solutions of (31) by using standard,
fractional and 𝑞-calculus follow below. Proposition 6 (Theorem 4.1(iii) of [32]). Any solution (37)
to any Caputo fractional differential system of fractional order
𝛼∈C0+ is nonnegative independent of the delays; that is,
𝑥(𝑡) ∈R𝑛
0+𝑓𝑜𝑟𝑎𝑙𝑙𝑡∈[−ℎ, 𝑡) ∩R0+ for some 𝑡∈R0+, for any
set of delays satisfying 0 = ℎ0 < ℎ1 < ℎ2 < ⋅⋅⋅< ℎ𝑝≤ℎ< ∞
and any absolutely continuous functions of initial conditions
𝜑𝑗: [−ℎ, 0]
→
R𝑛
0+, 𝑓𝑜𝑟𝑎𝑙𝑙𝑗∈𝑘−1 ∪{0} and any
piecewise continuous control 𝑢: R0+ →R𝑚
0+, if and only if
𝐴0 ∈𝑀R𝑛×𝑛for 𝑡∈R0+ being sufficiently small. Furthermore,
𝑥(𝑡) ∈R𝑛
0+𝑓𝑜𝑟𝑎𝑙𝑙𝑡∈[−ℎ, 0) ∪R0+ either if, in addition,
𝐴0 ≥0 or if 𝐴0 is nilpotent or if 0 < 𝛼≤𝑘= 1, 𝐴𝑖∈R𝑛×𝑛
0+
(𝑓𝑜𝑟𝑎𝑙𝑙𝑖∈𝑝) and 𝐵∈R𝑛×𝑚
0+ . Proposition 5. The solution of (31) is nonnegative on [0, ∞)
for any 𝜑: [−ℎ, 0] →R𝑛
0+ and 𝑢: R0+
→R𝑚
0+ if and only if,
𝐴𝑖∈R𝑛×𝑛
0+ 𝑓𝑜𝑟𝑎𝑙𝑙𝑖∈𝑝and 𝐴0 ∈𝑀R𝑛×𝑛. Proposition 7. Consider the 𝑞-calculus version of (31) under
similar initial conditions: Proof. The sufficiency part of the proof is direct since the
unique left-sided mild solution is given by (𝐷𝑞𝑥) (𝑡) := 𝑥(𝑡) −𝑥(𝑞𝑡)
(1 −𝑞) 𝑡
=
𝑝
∑
𝑖=0
𝐴𝑖(𝑡−ℎ𝑖) + 𝐵𝑢(𝑡) ;
𝑡∈[0, ∞) . (34) (𝐷𝑞𝑥) (𝑡) := 𝑥(𝑡) −𝑥(𝑞𝑡)
(1 −𝑞) 𝑡
=
𝑝
∑
𝑖=0
𝐴𝑖(𝑡−ℎ𝑖) + 𝐵𝑢(𝑡) ;
(34) 𝑥(𝑡) = 𝑒𝐴0𝑡(𝑥0 +
𝑝
∑
𝑖=1
∫
ℎ𝑖
0
𝑒−𝐴0𝜏𝐴𝑖𝜑(𝜏−ℎ𝑖) 𝑑𝜏
+
𝑝
∑
𝑖=1
∫
𝑡
ℎ𝑖
𝑒−𝐴0𝜏𝐴𝑖𝑥(𝜏−ℎ𝑖) 𝑑𝜏
+ ∫
𝑡
0
𝑒−𝐴0𝜏𝐵𝑢(𝜏) 𝑑𝜏) ;
∀𝑡∈R0+. (34) 𝑡∈[0, ∞) . 𝑡∈[0, ∞) . Assume that 𝜑: [−ℎ, 0] →R𝑛
0+, 𝑢: R0+ →R𝑚
0+, 𝐴𝑖∈
R𝑛×𝑛
0+ 𝑓𝑜𝑟𝑎𝑙𝑙𝑖∈𝑝. Then, Assume that 𝜑: [−ℎ, 0] →R𝑛
0+, 𝑢: R0+ →R𝑚
0+, 𝐴𝑖∈
R𝑛×𝑛
0+ 𝑓𝑜𝑟𝑎𝑙𝑙𝑖∈𝑝. Then, (32) (i) 𝑥(𝑡) ≥0; 𝑓𝑜𝑟𝑎𝑙𝑙𝑡∈R0+ if either 1 > 𝐴0𝑖𝑖> 0 or
𝐴0𝑖𝑖≤0 and 𝐴0𝑖𝑗= 0 for all 𝑖, 𝑗( ̸= i)∈𝑛. (i) 𝑥(𝑡) ≥0; 𝑓𝑜𝑟𝑎𝑙𝑙𝑡∈R0+ if either 1 > 𝐴0𝑖𝑖> 0 or
𝐴0𝑖𝑖≤0 and 𝐴0𝑖𝑗= 0 for all 𝑖, 𝑗( ̸= i)∈𝑛. Abstract and Applied Analysis Abstract and Applied Analysis 7 7 (b) If 𝛼∈Z0+ then 𝑘= 𝛼, and (b) If 𝛼∈Z0+ then 𝑘= 𝛼, and (ii) 𝑥(𝑡) ≥0;𝑓𝑜𝑟𝑎𝑙𝑙𝑡∈R0+ if (−𝐴0) ∈𝑀R𝑛×𝑛
+
with 𝐴0𝑖𝑖=
−1; 𝑓𝑜𝑟𝑎𝑙𝑙𝑖∈𝑛and 𝐴0𝑖𝑗≤0 with 𝐴0𝑖𝑗< 0 for only
one 𝑗∈𝑛for each 𝑖∈𝑛. 2. Preliminaries on Fractional
𝑞-Differential Systems Then, (𝐼−𝐴0)−1 ∈
R𝑛×𝑛
0+
if and only if for all 𝑖∈𝑛either 1 > 𝐴0𝑖𝑖> 0 or
𝐴0𝑖𝑖≤0. Hence, Property (i) follows directly. Property (ii)
follows since, under the given constraints, (−𝐴0) ∈𝑀R𝑛×𝑛,
with 𝐴0𝑖𝑖= −1for all 𝑖∈𝑛, (𝐼−𝐴0) ∈R𝑛×𝑛
+
and Metzler,
monomial (then nonsingular) so that (𝐼−𝐴0)−1 ∈R𝑛×𝑛
+ . Hence, Property (ii) follows. (39) with 𝑘−1 < 𝛼(∈R+) ≤𝑘; 𝑘−1, 𝑘∈Z0+, 0 = ℎ0 < ℎ1 <
ℎ2 < ⋅⋅⋅< ℎ𝑝= ℎ< ∞being distinct constant delays; 𝐴0 :
R0+ →R𝑛, 𝐴𝑖: R0+ →R𝑛(𝑖∈𝑝:= {1, 2, . . . , 𝑝}), are the
bounded matrix functions of dynamics for each delay ℎ𝑖, 𝑖∈
𝑝∪{0}, and 𝐵: R0+ →R𝑛×𝑚is the control matrix function. The initial conditions are given by 𝑘𝑛-real vector functions
𝜑𝑗: [−ℎ, 0] →R𝑛, with 𝑗∈𝑘−1 ∪{0} and with 𝜑𝑗(0) =
𝑥𝑗(0) = 𝑥(𝑗)(0) = 𝑥𝑗0, and 𝑢: R0+ →R𝑚is a bounded
piecewise continuous control function. The Jackson integral
in the integral term in (39) becomes with 𝑘−1 < 𝛼(∈R+) ≤𝑘; 𝑘−1, 𝑘∈Z0+, 0 = ℎ0 < ℎ1 <
ℎ2 < ⋅⋅⋅< ℎ𝑝= ℎ< ∞being distinct constant delays; 𝐴0 :
R0+ →R𝑛, 𝐴𝑖: R0+ →R𝑛(𝑖∈𝑝:= {1, 2, . . . , 𝑝}), are the
bounded matrix functions of dynamics for each delay ℎ𝑖, 𝑖∈
𝑝∪{0}, and 𝐵: R0+ →R𝑛×𝑚is the control matrix function. The initial conditions are given by 𝑘𝑛-real vector functions
𝜑𝑗: [−ℎ, 0] →R𝑛, with 𝑗∈𝑘−1 ∪{0} and with 𝜑𝑗(0) =
𝑥𝑗(0) = 𝑥(𝑗)(0) = 𝑥𝑗0, and 𝑢: R0+ →R𝑚is a bounded
piecewise continuous control function. The Jackson integral
in the integral term in (39) becomes 3. Fractional 𝑞-Differential Dynamic Systems
of Order 𝛼with Internal Point Delays The fractional Caputo 𝑞-differential dynamic systems of order
𝛼can become modified from (2). The functions Γ𝑞(𝑘−𝛼),
𝐷𝑘
𝑞(∫
𝑡
𝑎(𝑡−𝑞𝜏)𝑘−𝛼−1𝑓(𝜏)𝑑𝜏𝑞),
and
∫
𝑡
𝑎(𝑡−𝑞𝜏)𝑘−𝛼−1(𝐷𝑘
𝑞𝑓)
(𝜏)𝑑𝑞𝜏are now displayed to be used later in order to solve
such a differential system. The combination of (2) with (24)
with the replacement of 𝑓(𝑡) with 𝑥(𝑡) leads to The fractional Caputo 𝑞-differential dynamic systems of order
𝛼can become modified from (2). The functions Γ𝑞(𝑘−𝛼), 𝑞∫𝑎
𝑞
∫𝑎
𝑞
(𝜏)𝑑𝑞𝜏are now displayed to be used later in order to solve
such a differential system. The combination of (2) with (24)
with the replacement of 𝑓(𝑡) with 𝑥(𝑡) leads to (40) 1
Γ𝑞(𝑘−𝛼) ∫
𝑡
𝑎
(𝑡−𝑞𝜏)𝑘−𝛼−1 (𝐷𝑘
𝑞𝑥) (𝜏) 𝑑𝑞𝜏
=
𝑝
∑
𝑖=0
𝐴𝑖𝑥(𝑡−ℎ𝑖) + 𝐵𝑢(𝑡) ;
𝑡∈[𝑎, 𝑏] . for 𝑡≥𝑎. By replacing the dummy argument 𝜏under the two
integral symbols in (40) by 𝑞𝑛𝑡and 𝑞𝑛𝑎, respectively, since
(see [37]): for 𝑡≥𝑎. By replacing the dummy argument 𝜏under the two
integral symbols in (40) by 𝑞𝑛𝑡and 𝑞𝑛𝑎, respectively, since
(see [37]): (36) (36) ∫
𝑡
0
𝑓(𝜏) 𝑑𝑞𝜏= (1 −𝑞) 𝑡
∞
∑
𝑛=0
𝑓(𝑞𝑛𝑡) 𝑞𝑛. (41) (41) (a) If 𝛼∈R+ and 𝛼∉Z0+ then 𝑘< 𝛼+ 1, and 1
Γ𝑞(𝑘−𝛼) = (1 −𝑞)𝑘−𝛼−1
(1 −𝑞)
(𝑘−𝛼−1)
= (1 −𝑞)𝑘−𝛼−1
∞
∏
𝑗=0
[1 −𝑞𝑘+𝑗−𝛼
1 −𝑞𝑗+1 ] . (37 1
Γ𝑞(𝑘−𝛼) = (1 −𝑞)𝑘−𝛼−1
(1 −𝑞)
(𝑘−𝛼−1) The series in (41) is convergent for all 𝑡∈R0+ since the
function 𝑓satisfies |𝑓(𝑡)| < 𝐵𝑡𝐶in a right neighborhood
of 𝑡= 0 for some real constants 𝐵> 0 and 𝐶> −1. So,
the Jackson integral in (40) is convergent since ‖(𝐷𝑘
𝑞𝑥)(𝑡)‖ <
𝐵𝑡𝐶+𝛼+1−𝑘in some right neighbourhood of 𝑡= 0 for real The series in (41) is convergent for all 𝑡∈R0+ since the
function 𝑓satisfies |𝑓(𝑡)| < 𝐵𝑡𝐶in a right neighborhood
of 𝑡= 0 for some real constants 𝐵> 0 and 𝐶> −1. So,
the Jackson integral in (40) is convergent since ‖(𝐷𝑘
𝑞𝑥)(𝑡)‖ <
𝐵𝑡𝐶+𝛼+1−𝑘in some right neighbourhood of 𝑡= 0 for real (37) (37) = (1 −𝑞)𝑘−𝛼−1
∞
∏
𝑗=0
[1 −𝑞𝑘+𝑗−𝛼
1 −𝑞𝑗+1 ] . Abstract and Applied Analysis 8 Lemma 10. Assume that ‖(𝐷𝑘
𝑞𝑥)(𝑡)‖𝑡𝑘+𝛾−𝛼−1 is bounded in a
right neighborhood [0, 𝑡0) of 𝑡= 0 for some 𝑡0 ∈R+ and some
real constant 1 > 𝛾≥0. Define Lemma 10. 3. Fractional 𝑞-Differential Dynamic Systems
of Order 𝛼with Internal Point Delays Assume that ‖(𝐷𝑘
𝑞𝑥)(𝑡)‖𝑡𝑘+𝛾−𝛼−1 is bounded in a
right neighborhood [0, 𝑡0) of 𝑡= 0 for some 𝑡0 ∈R+ and some
real constant 1 > 𝛾≥0. Define constants 𝐵> 0 and 𝐶> −1 [37, 39]. Equations (40) and
(39), together with (18), Lemma 3 (see (19)), and (17), yield: constants 𝐵> 0 and 𝐶> −1 [37, 39]. Equations (40) and
(39), together with (18), Lemma 3 (see (19)), and (17), yield: ( 𝐶𝐷
𝛼
𝑞𝑎+𝑥) (𝑡)
= (1 −𝑞)𝑘−𝛼−1
∞
∏
𝑗=0
[1 −𝑞𝑘+𝑗−𝛼
1 −𝑞𝑗+1 ]
× (1 −𝑞) [𝑡
∞
∑
𝑛=0
(1 −𝑞𝑛+1)
𝑘−𝛼−1𝑡𝑘−𝛼−1𝑞𝑛
× 𝐷((𝐷𝑘−1
𝑞
𝑥)) (𝑞𝑛𝑡) −𝑎
∞
∑
𝑛=0
(1 −𝑞𝑛+1)
𝑘−𝛼−1
× 𝑎𝑘−𝛼−1𝑞𝑛𝐷((𝐷𝑘−1
𝑞
𝑥)) (𝑞𝑛𝑎) ]
𝑝
∑
(
) 𝑆(𝑡, 𝑛) :=
𝑛
∑
𝑖=0
(1 −𝑞𝑖+1)
𝑘−𝛼−1𝑡𝑘−𝛼𝑞𝑖(𝐷𝑘
𝑞𝑥) (𝑞𝑖𝑡) ;
∀𝑡∈R0+,
∀𝑛∈Z0+. (43) (43) ∀𝑡∈R0+,
∀𝑛∈Z0+. Then, the following identities hold: Then, the following identities hold: 𝑛
∑
𝑖=0
(1 −𝑞𝑖+1)
𝑘−𝛼−1𝑡𝑘−𝛼𝑞𝑖[(𝐷𝑘
𝑞𝑥) (𝑞𝑖+1𝑡) −(𝐷𝑘
𝑞𝑥) (𝑞𝑖𝑡)]
= 𝑞𝛼−𝑘(𝑆(𝑞𝑡, 𝑛) −𝑆(𝑡, 𝑛)) ;
∀𝑡∈R0+, ∀𝑛∈Z0+,
(44) 𝑛
∑
𝑖=0
(1 −𝑞𝑖+1)
𝑘−𝛼−1𝑡𝑘−𝛼𝑞𝑖[(𝐷𝑘
𝑞𝑥) (𝑞𝑖+1𝑡) −(𝐷𝑘
𝑞𝑥) (𝑞𝑖𝑡)] =
𝑝
∑
𝑖0
𝐴𝑖(𝑡) 𝑥(𝑡−ℎ𝑖) + 𝐵(𝑡) 𝑢(𝑡) . 𝑖=0
(42) = 𝑞𝛼−𝑘(𝑆(𝑞𝑡, 𝑛) −𝑆(𝑡, 𝑛)) ;
∀𝑡∈R0+, ∀𝑛∈Z0+,
(44)
∞
∑
𝑖=0
(1 −𝑞𝑖+1)
𝑘−𝛼−1𝑡𝑘−𝛼𝑞𝑖[(𝐷𝑘
𝑞𝑥) (𝑞𝑖+1𝑡) −(𝐷𝑘
𝑞𝑥) (𝑞𝑖𝑡)]
= 𝑞𝛼−𝑘(𝑆∗(𝑞𝑡) −𝑆∗(𝑡)) ;
∀𝑡∈[0, 𝑡0) ,
(45)
𝑆∗(𝑡) =
1
(1 −𝑞)𝛼+1𝑡𝛼𝑥(𝑡) + ̂𝑆𝑁𝑡(̂𝑞𝑁𝑡) + ̃𝑆𝑁𝑡(̃𝑞𝑁𝑡) ,
(46)
̂𝑆𝑁𝑡(̂𝑞𝑁𝑡) =
1
(1 −𝑞)𝛼+1𝑡𝛼
𝑘
∑
𝑗=1
(−1)𝑗(𝑘
𝑗) 𝑥(𝑞𝑗𝑡)
+
𝑁
∑
𝑖=1
𝑘
∑
𝑗=0
(−1)𝑗
𝑞𝑖
(1 −𝑞𝑖+1) (1 −𝑞)
𝑘𝑡𝛼
× (𝑘
𝑗) 𝑥(𝑞𝑗+𝑖𝑡) ,
(47)
̃𝑆𝑁𝑡(̃𝑞𝑁𝑡) =
∞
∑
𝑖=𝑁+1
𝑘
∑
𝑗=0
(−1)𝑗
𝑞𝑖
(1 −𝑞𝑖+1)(1 −𝑞)
𝑘𝑡𝛼(𝑘
𝑗) 𝑥(𝑞𝑗+𝑖𝑡)
(48) (42) 0+,
(44) The
following
auxiliary
results
hold
concerning
the
product ∏𝑛
𝑗=0[(1 −𝑞𝑘+𝑗−𝛼)/(1 −𝑞𝑗+1)] and the series
∑∞
𝑛=0 (1 −𝑞𝑛+1)
𝑘−𝛼−1𝑡𝑘−𝛼−1𝑞𝑛𝐷((𝐷𝑘−1
𝑞
𝑥))(𝑞𝑛𝑡). ∞
∑
𝑖=0
(1 −𝑞𝑖+1)
𝑘−𝛼−1𝑡𝑘−𝛼𝑞𝑖[(𝐷𝑘
𝑞𝑥) (𝑞𝑖+1𝑡) −(𝐷𝑘
𝑞𝑥) (𝑞𝑖𝑡)] ∞
∑
𝑖=0
(1 −𝑞𝑖+1)
𝑘−𝛼−1𝑡𝑘−𝛼𝑞𝑖[(𝐷𝑘
𝑞𝑥) (𝑞𝑖+1𝑡) −(𝐷𝑘
𝑞𝑥) (𝑞𝑖𝑡)]
= 𝑞𝛼−𝑘(𝑆∗(𝑞𝑡) −𝑆∗(𝑡)) ;
∀𝑡∈[0, 𝑡0) ,
(45) = 𝑞𝛼−𝑘(𝑆∗(𝑞𝑡) −𝑆∗(𝑡)) ;
∀𝑡∈[0, 𝑡0) , Lemma 9. Define 𝑃𝑛
:=
(∏𝑛
𝑗=0[(1 −𝑞𝑘+𝑗−𝛼)/(1 −𝑞𝑗+1)]). 3. Fractional 𝑞-Differential Dynamic Systems
of Order 𝛼with Internal Point Delays Then, 𝑃𝑛+1 = 𝑔𝑛𝑃𝑛for all 𝑛∈Z0+, where (45) (45) (45)
𝑆∗(𝑡) =
1
(1 −𝑞)𝛼+1𝑡𝛼𝑥(𝑡) + ̂𝑆𝑁𝑡(̂𝑞𝑁𝑡) + ̃𝑆𝑁𝑡(̃𝑞𝑁𝑡) ,
(46)
̂𝑆𝑁𝑡(̂𝑞𝑁𝑡) =
1
(1 −𝑞)𝛼+1𝑡𝛼
𝑘
∑
𝑗=1
(−1)𝑗(𝑘
𝑗) 𝑥(𝑞𝑗𝑡)
+
𝑁
∑
𝑖=1
𝑘
∑
𝑗=0
(−1)𝑗
𝑞𝑖
(1 −𝑞𝑖+1) (1 −𝑞)
𝑘𝑡𝛼
× (𝑘
𝑗) 𝑥(𝑞𝑗+𝑖𝑡) ,
(47)
̃𝑆𝑁𝑡(̃𝑞𝑁𝑡) =
∞
∑
𝑖=𝑁+1
𝑘
∑
𝑗=0
(−1)𝑗
𝑞𝑖
(1 −𝑞𝑖+1)(1 −𝑞)
𝑘𝑡𝛼(𝑘
𝑗) 𝑥(𝑞𝑗+𝑖𝑡)
(48) (a) 𝑃0 = 0 and 𝑔𝑛= 1, for all 𝑛∈Z0+, so that 𝑃𝑛≡0, if
𝛼∈Z0+; (b) 𝑃0
= (1 −𝑞𝑘−𝛼)/(1 −𝑞) = (𝑘−𝛼)𝑞and 𝑔𝑛
:=
(𝑘+ 𝑛+ 1 −𝛼)𝑞/ ∑𝑛
𝑖=0 𝑞𝑖< 1, for all 𝑛∈Z0+, if 𝛼∉
Z0+; (b) 𝑃0
= (1 −𝑞𝑘−𝛼)/(1 −𝑞) = (𝑘−𝛼)𝑞and 𝑔𝑛
:=
(𝑘+ 𝑛+ 1 −𝛼)𝑞/ ∑𝑛
𝑖=0 𝑞𝑖< 1, for all 𝑛∈Z0+, if 𝛼∉
Z0+;
(c) ∃lim𝑛→∞𝑃𝑛= 𝑃∗. (47) (c) ∃lim𝑛→∞𝑃𝑛= 𝑃∗. (c) ∃lim𝑛→∞𝑃𝑛= 𝑃∗. Proof. Note that Equation (49) follows by defining
𝛾𝑖:= (1 −𝑞𝑖+1)
(𝑘−𝛼−1)/𝑖𝑞< 1 with 𝛾𝑖
𝑖≤𝛾𝑖< 1 for some
1 > 𝛾> 𝑞; for all i (≥N)∈Z0+ and some 𝑁∈Z0+, or
equivalently 1 > 𝛾> 𝑞max (1, max
𝑖≥𝑁+1
1
(1 −𝑞𝑖+1)(1+𝛼−𝑘)/𝑖)
(50) (50) (1 −𝑞𝑖+1)
1+𝑎−𝑘≥(𝑞
𝛾)
𝑖
⇐⇒1 ≥𝑞𝑖+1 + (𝑞
𝛾)
𝑖/(1+𝑎−𝑘)
. (54) for any given 𝑁∈Z0+. (54) f
y g
0+
Equations (46)-(47) also hold for 𝑘
=
𝛼(∈
Z0+) by
replacing 𝑘→𝛼everywhere 𝑘appears. Consider f
y g
0+
Equations (46)-(47) also hold for 𝑘
=
𝛼(∈
Z0+) by
replacing 𝑘→𝛼everywhere 𝑘appears. Consider 𝑆(𝑡, 𝑛+ 1) = 𝑆(𝑡, 𝑛) + 𝑑(𝑡, 𝑛) (𝐷𝑘
𝑞𝑥) (𝑞𝑛+1𝑡) ;
∀𝑡∈R0+,
∀𝑛∈Z0+,
∃lim
𝑛→∞[(1 −𝑞𝑛+2)
𝑘−𝛼−1𝑡𝑘−𝛼𝑞𝑛+1 (𝐷𝑘
𝑞𝑥) (𝑞𝑛+1𝑡)]=0;
(51) 𝑆(𝑡, 𝑛+ 1) = 𝑆(𝑡, 𝑛) + 𝑑(𝑡, 𝑛) (𝐷𝑘
𝑞𝑥) (𝑞𝑛+1𝑡) ;
∀𝑡∈R0+,
∀𝑛∈Z0+, Hence, (46)–(48) follow from (55) for some strictly decreas-
ing real sequence {𝜀𝑁} ∈R+ such that |𝑥(𝑞𝑗+𝑖𝑡)| ≤| 𝑥(0)|+𝜀𝑁;
for all 𝑖(≥N)∈Z0+ for 𝑗= 0, 1, . . . , 𝑘. Hence, (46)–(48) follow from (55) for some strictly decreas-
ing real sequence {𝜀𝑁} ∈R+ such that |𝑥(𝑞𝑗+𝑖𝑡)| ≤| 𝑥(0)|+𝜀𝑁;
for all 𝑖(≥N)∈Z0+ for 𝑗= 0, 1, . . . , 𝑘. (51) ∃lim
𝑛→∞[(1 −𝑞𝑛+2)
𝑘−𝛼−1𝑡𝑘−𝛼𝑞𝑛+1 (𝐷𝑘
𝑞𝑥) (𝑞𝑛+1𝑡)]=0; ∀𝑡∈R0+, The quantum Caputo fractional solutions have explicit
expressions as formulated in the subsequent result. where 𝑑(𝑡, 𝑛) = (1 −𝑞𝑛+2)
𝑘−𝛼−1𝑡𝑘−𝛼𝑞𝑛+1. Proof. Since ‖(𝐷𝑘
𝑞𝑥)(𝑡)‖𝑡𝑘+𝛾−𝛼−1 is bounded in a right neigh-
bourhood [0, 𝑡0) of 𝑡= 0 for some real constant 1 > 𝛾≥0
then the Jackson integral Theorem 11. Proof. Note that Proof. Note that Proof. Note that (1) 𝑃𝑛+1/𝑃𝑛= ((1−𝑞𝑘+𝑛+1−𝛼)/(1−𝑞𝑛+1))/((1−𝑞𝑘+𝑛−𝛼)/(1−
𝑞𝑛)) →1 as 𝑛→∞;
(2) if 𝑘= 𝛼(∈Z0+), 𝑃0 = 0,
𝑃1 = 0, 𝑃𝑛+1/𝑃𝑛= 1 𝑦𝑃=
𝑃𝑛= 𝑃0 = (𝑘−𝛼)𝑞= [𝑘−𝛼]𝑞= [0]𝑞= 0; (1) 𝑃𝑛+1/𝑃𝑛= ((1−𝑞𝑘+𝑛+1−𝛼)/(1−𝑞𝑛+1))/((1−𝑞𝑘+𝑛−𝛼)/(1−
𝑞𝑛)) →1 as 𝑛→∞; (1) 𝑃𝑛+1/𝑃𝑛= ((1−𝑞𝑘+𝑛+1−𝛼)/(1−𝑞𝑛+1))/((1−𝑞𝑘+𝑛−𝛼)/(1−
𝑞𝑛)) →1 as 𝑛→∞; ̃𝑆𝑁𝑡(̃𝑞𝑁𝑡) =
∞
∑
𝑖=𝑁+1
𝑘
∑
𝑗=0
(−1)𝑗
𝑞𝑖
(1 −𝑞𝑖+1)(1 −𝑞)
𝑘𝑡𝛼(𝑘
𝑗) 𝑥(𝑞𝑗+𝑖𝑡)
(48) (2) if 𝑘= 𝛼(∈Z0+), 𝑃0 = 0,
𝑃1 = 0, 𝑃𝑛+1/𝑃𝑛= 1 𝑦𝑃=
𝑃𝑛= 𝑃0 = (𝑘−𝛼)𝑞= [𝑘−𝛼]𝑞= [0]𝑞= 0; (48) (3) if 𝑘̸= 𝛼(∈R+), with 𝛼∉Z0+, then 𝑃0
= (1 −
𝑞𝑘−𝛼)/(1−𝑞) = (𝑘−𝛼)𝑞, 𝑃1 = ((1−𝑞𝑘−𝛼)/(1−𝑞))((1−
𝑞𝑘+1−𝛼)/(1 −𝑞2)) = (1/(1 + 𝑞))(𝑘−𝛼)𝑞(𝑘+ 1 −𝛼)𝑞=
((𝑘+ 1 −𝛼)𝑞/(1 + 𝑞))𝑃0, 𝑃2
= ((1 −𝑞𝑘+2−𝛼)/(1 −
𝑞3))𝑃1 = (1/(1 + 𝑞+ 𝑞2))((1 −𝑞𝑘+2−𝛼)/(1 −𝑞))𝑃1 =
(1/(1 + 𝑞+ 𝑞2))(𝑘+ 2 −𝛼)𝑞𝑃1, 𝑃𝑛+1 = 𝑔𝑛𝑃𝑛with 𝑔𝑛=
(𝑘+ 𝑛+ 1 −𝛼)𝑞/ ∑𝑛
𝑖=0 𝑞𝑖and 𝑔𝑛< 1. Assume this is
false so that 𝑔𝑛≥1. Then, 1−𝑞𝑛−𝑞𝑘+𝑛+1−𝛼+𝑞𝑘+2𝑛+1−𝛼≥
1 −𝑞𝑛+1 −𝑞𝑘+𝑛−𝛼+ 𝑞𝑘+2𝑛+1−𝛼and, equivalently, (−𝑞𝑛−
𝑞𝑘+𝑛+1−𝛼) ≥(−𝑞𝑛+1 −𝑞𝑘+𝑛−𝛼), or 𝑞𝑛(1−𝑞) ≤𝑞𝑘+𝑛−𝛼(1−
𝑞) ⇒1 ≤𝑞𝑘−𝛼< 1 if 𝛼< 𝑘< 𝛼+ 1 (a contradiction). (3) if 𝑘̸= 𝛼(∈R+), with 𝛼∉Z0+, then 𝑃0
= (1 −
𝑞𝑘−𝛼)/(1−𝑞) = (𝑘−𝛼)𝑞, 𝑃1 = ((1−𝑞𝑘−𝛼)/(1−𝑞))((1−
𝑞𝑘+1−𝛼)/(1 −𝑞2)) = (1/(1 + 𝑞))(𝑘−𝛼)𝑞(𝑘+ 1 −𝛼)𝑞=
((𝑘+ 1 −𝛼)𝑞/(1 + 𝑞))𝑃0, 𝑃2
= ((1 −𝑞𝑘+2−𝛼)/(1 −
𝑞3))𝑃1 = (1/(1 + 𝑞+ 𝑞2))((1 −𝑞𝑘+2−𝛼)/(1 −𝑞))𝑃1 =
(1/(1 + 𝑞+ 𝑞2))(𝑘+ 2 −𝛼)𝑞𝑃1, 𝑃𝑛+1 = 𝑔𝑛𝑃𝑛with 𝑔𝑛=
(𝑘+ 𝑛+ 1 −𝛼)𝑞/ ∑𝑛
𝑖=0 𝑞𝑖and 𝑔𝑛< 1. Assume this is
false so that 𝑔𝑛≥1. Then, 1−𝑞𝑛−𝑞𝑘+𝑛+1−𝛼+𝑞𝑘+2𝑛+1−𝛼≥
1 −𝑞𝑛+1 −𝑞𝑘+𝑛−𝛼+ 𝑞𝑘+2𝑛+1−𝛼and, equivalently, (−𝑞𝑛−
𝑞𝑘+𝑛+1−𝛼) ≥(−𝑞𝑛+1 −𝑞𝑘+𝑛−𝛼), or 𝑞𝑛(1−𝑞) ≤𝑞𝑘+𝑛−𝛼(1−
𝑞) ⇒1 ≤𝑞𝑘−𝛼< 1 if 𝛼< 𝑘< 𝛼+ 1 (a contradiction). for any given 𝑁∈Z0+ if 𝛼∉Z0+, where 𝑘= [Re 𝛼] + 1,
̂𝑞𝑁= (𝑞, 𝑞2, . . . , 𝑞𝑁+𝑘), ̃𝑞𝑁= (𝑞𝑁+1, 𝑞𝑁+2, 𝑞𝑁+3, . . .), 𝑆∗(𝑡) =
lim𝑛→∞𝑆(𝑡, 𝑛); 𝑓𝑜𝑟𝑎𝑙𝑙𝑡∈R0+, since such a limit function
exists; 𝑓𝑜𝑟𝑎𝑙𝑙𝑡∈R0+, and ̃𝑆𝑁𝑡(𝑡)
→
0 as 𝑁
→
∞;
𝑓𝑜𝑟𝑎𝑙𝑙𝑡∈R0+ ̃𝑆𝑁𝑡(̃𝑞𝑁𝑡)≤( 𝛾
𝑞𝑘)
𝑁+1 (𝑘+ 1) (‖𝑥(0)‖ + 𝜀𝑁)
(1 −𝛾) (1 −𝑞)𝑘𝑡𝛼
×
𝑘
∑
𝑗=0
(−1)𝑗(𝑘
𝑗)
,
(49) (49) Abstract and Applied Analysis 9 where {𝜀𝑁} is a nonnegative real sequence which converges to
0; 𝛾∈R+ is subject to where {𝜀𝑁} is a nonnegative real sequence which converges to
0; 𝛾∈R+ is subject to since 𝑞𝑛→0 as 𝑛→∞. Proof. Note that Consider the left-sided 𝑞-fractional Caputo solu-
tion of or fractional order 𝛼of the functional differential system ( 𝐶𝐷
𝛼
𝑞0+𝑥) (𝑡) =
𝑝
∑
𝑖=0
𝐴𝑖(𝑡) 𝑥(𝑡−ℎ𝑖) + 𝐵(𝑡) 𝑢(𝑡) ;
𝑡∈[0, ∞)
(55) ( 𝐶𝐷
𝛼
𝑞0+𝑥) (𝑡) =
𝑝
∑
𝑖=0
𝐴𝑖(𝑡) 𝑥(𝑡−ℎ𝑖) + 𝐵(𝑡) 𝑢(𝑡) ;
𝑡∈[0, ∞) ∫
𝑡
0
(𝑡−𝑞𝜏)𝑘−𝛼−1 (𝐷𝑘
𝑞𝑥) (𝜏) 𝑑𝑞𝜏
= (1 −𝑞)
∞
∑
𝑛=0
(1 −𝑞𝑛+1)
𝑘−𝛼−1𝑡𝑘−𝛼𝑞𝑛(𝐷𝑘
𝑞𝑥) (𝑞𝑛𝑡)
(52) (52) (55) with initial condition of the state 𝑥: [−ℎ, ∞) →R𝑛being
defined by 𝑘𝑛-real vector functions 𝜑𝑗: [−ℎ, 0] →R𝑛, with
𝑗∈𝑘−1 ∪{0} and 𝜑𝑗(0) = 𝑥𝑗(0) = 𝑥(𝑗)(0) = 𝑥𝑗0 𝑗∈𝑘−1 ∪
{0}, and 𝑢: R0+ →R𝑚is a bounded piecewise continuous
control function and ℎ= ℎ𝑝= max1≤𝑖≤𝑝ℎ𝑖.ht converges to a function 𝐹(𝑡) on [0, 𝑡0) which is a 𝑞-
antiderivative of (𝑡−𝑞𝜏)𝑘−𝛼−1(𝐷𝑘
𝑞𝑥)(𝜏) and which is con-
tinuous at 𝑡
=
0 with 𝐹(0)
=
0 and is a unique
(𝑡−𝑞𝜏)𝑘−𝛼−1(𝐷𝑘
𝑞𝑥)(𝜏) in this class of functions [39]. Note that
𝑆(𝑡, 𝑛) ≤𝑆∗(𝑡) = lim𝑛→∞𝑆(𝑡, 𝑛); for all 𝑛∈Z0+; 𝑡∈[0, 𝑡0)
since the series 𝑆(𝑡, 𝑛) is convergent, since the Jackson integral
(52) is convergent for each fix 𝑡∈[0, 𝑡0), and non-decreasing
with 𝑛since it is consists of nonnegative terms. It follows from
(43), with the replacement 𝑡→𝑞𝑡, that 𝑝
𝑝
Thus, the unique left-sided solution of (55) is calculated
almost everywhere via analytical expressions obtained from
Lemma 10 as follows. (i) If 𝛼∉Z0+ so that 𝑘= [Re 𝛼] + 1 then one gets for any
given 𝑁∈Z+ that (i) If 𝛼∉Z0+ so that 𝑘= [Re 𝛼] + 1 then one gets for any
given 𝑁∈Z+ that 𝑆(𝑞𝑡, 𝑛) = 𝑞𝑘−𝛼[
𝑛
∑
𝑖=0
(1 −𝑞𝑖+1)
𝑘−𝛼−1𝑡𝑘−𝛼𝑞𝑖(𝐷𝑘
𝑞𝑥) (𝑞𝑖+1𝑡)]
= 𝑞𝑘−𝛼(𝑆(𝑡, 𝑛)
+
𝑛
∑
𝑖=0
(1 −𝑞𝑖+1)
𝑘−𝛼−1𝑡𝑘−𝛼𝑞𝑖
× [(𝐷𝑘
𝑞𝑥) (𝑞𝑖+1𝑡) −(𝐷𝑘
𝑞𝑥) (𝑞𝑖𝑡)] ) ;
(53) 𝑥(𝑡) = (
(𝑃𝑁+ 𝜌𝑁)
(1 −𝑞)
2𝛼−𝑘+1𝑡𝛼𝐼−𝐴0 (𝑡))
−1
× (
𝑝
∑
𝑗=1
𝐴𝑗(𝑡) 𝑥(𝑡−ℎ𝑗) −(𝑃𝑁+ 𝜌𝑁) (1 −𝑞)
𝑘−𝛼
× [̂𝑆𝑁𝑡(̂𝑞𝑁𝑡) + ̃𝑆𝑁𝑡(̃𝑞𝑁𝑡)] + 𝐵𝑢(𝑡) ) ; × [(𝐷𝑘
𝑞𝑥) (𝑞𝑖+1𝑡) −(𝐷𝑘
𝑞𝑥) (𝑞𝑖𝑡)] ) ; (53) )
(56) (56) for all 𝑡∈[0, 𝑡0) and hence (44) follows. By taking limits
as 𝑛
→
∞in (44), one gets (46)–(48) since the limit
𝑆∗(𝑡)
=
lim𝑛→∞𝑆(𝑡, 𝑛) exists for all 𝑡
∈
R0+ by using
Lemma 3(iv) for the expansion of the 𝑞-derivative of order 𝑘 𝑓𝑜𝑟𝑎𝑙𝑙𝑡∈𝑇𝑅𝑁𝐾, where 𝑇𝑅𝑁𝑘:= {𝑡∈R+ : (((𝑃𝑁+
𝜌𝑁)/(1 −𝑞)2𝛼−𝑘+1𝑡𝛼)𝐼−𝐴0(𝑡)) 𝑖𝑠𝑛𝑜𝑛𝑠𝑖𝑛𝑔𝑢𝑙𝑎𝑟}. Proof. Note that 𝑓𝑜𝑟𝑎𝑙𝑙𝑡∈𝑇𝑅𝑁𝐾, where 𝑇𝑅𝑁𝑘:= {𝑡∈R+ : (((𝑃𝑁+
𝜌𝑁)/(1 −𝑞)2𝛼−𝑘+1𝑡𝛼)𝐼−𝐴0(𝑡)) 𝑖𝑠𝑛𝑜𝑛𝑠𝑖𝑛𝑔𝑢𝑙𝑎𝑟}. 10
Abstract and Applied Analysis Abstract and Applied Analysis Abstract and Applied Analysis 10 (ii) If 𝑘= (𝛼∈Z0+) then one gets for any given 𝑁∈Z+
that (ii) If 𝑘= (𝛼∈Z0+) then one gets for any given 𝑁∈Z+
that 𝜌𝑁= 1 from (37)-(38) so that (58) (with 𝑘= 𝛼) becomes
simplified as follows: 𝑥(𝑡) = (
1
(1 −𝑞)
𝛼+1𝑡𝛼𝐼−𝐴0 (𝑡))
−1
× (
𝑝
∑
𝑗=1
𝐴𝑗(𝑡) 𝑥(𝑡−ℎ𝑗) −̂𝑆𝑁𝑡(̂𝑞𝑁𝑡)
−̃𝑆𝑁𝑡(̃𝑞𝑁𝑡) + 𝐵(𝑡) 𝑢(𝑡) )
= (
1
(1 −𝑞)𝛼+1𝑡𝛼𝐼−𝐴0 (𝑡))
−1
× (
𝑝
∑
𝑗=1
𝐴𝑗(𝑡) 𝑥(𝑡−ℎ𝑗)
+
1
(1 −𝑞)
𝛼+1𝑡𝛼𝑥(𝑡) −𝑆∗(𝑡) + 𝐵(𝑡) 𝑢(𝑡)) ;
(57)
𝑓𝑜𝑟𝑎𝑙𝑙𝑡
∈
𝑇𝑅𝛼, where 𝑇𝑅𝛼
:=
{𝑡
∈
R+
:
((1/(1
𝑞)𝛼+1𝑡𝛼)𝐼
𝐴(𝑡)) 𝑖𝑠𝑛𝑜𝑛𝑠𝑖𝑛𝑔𝑢𝑙𝑎𝑟} = [
1
(1 −𝑞)𝛼+1𝑡𝛼𝑥(𝑡) + ̂𝑆𝑁𝑡(̂𝑞𝑁𝑡) + ̃𝑆𝑁𝑡(̃𝑞𝑁𝑡)]
=
𝑝
∑
𝑗=0
𝐴𝑗(𝑡) 𝑥(𝑡−ℎ𝑗) + 𝐵(𝑔) 𝑢(𝑡)
if 𝑘= (𝛼∈Z0+
( (60) Thus, the (unique) left-sided solution of (55) follows directly
from (59) and (60). A variant of Theorem 11 might be obtained in a simple way
by selecting the initial point 𝑎= 𝑎(𝑡) = 𝑡−𝜁so that the real
interval [𝑡−𝑎(𝑡), 𝑡] has a constant measure for each 𝑡(≥h)
∈R+ independent of 𝑡. For that purpose, one implements
the quantum fractional left-sided Caputo derivative with the
operator ( 𝐶𝐷
𝛼
𝑞(𝑡−𝜁)+𝑥)(𝑡) replacing ( 𝐶𝐷
𝛼
𝑞0+𝑥)(𝑡) while fixing
𝑎(𝑡) = 𝑡−𝜁in (42) to reformulate (55) with the appropriate
“ad hoc” modifications of (56) and (57). +
1
(1 −𝑞)
𝛼+1𝑡𝛼𝑥(𝑡) −𝑆∗(𝑡) + 𝐵(𝑡) 𝑢(𝑡)) ;
(57) Theorem 12. Consider the left-sided 𝑞-fractional Caputo solu-
tion of or fractional order 𝛼∈R0+ of the functional differential
system )
(57) )
(57) 𝑓𝑜𝑟𝑎𝑙𝑙𝑡
∈
𝑇𝑅𝛼, where 𝑇𝑅𝛼
:=
{𝑡
∈
R+
:
((1/(1 −𝑞)𝛼+1𝑡𝛼)𝐼−𝐴0(𝑡)) 𝑖𝑠𝑛𝑜𝑛𝑠𝑖𝑛𝑔𝑢𝑙𝑎𝑟}. ( 𝐶𝐷𝛼
𝑞(𝑡−𝜁)+ 𝑥) (𝑡)
=
𝑝
∑
𝑖=0
𝐴𝑖(𝑡) (𝑡−ℎ𝑖) + 𝐵𝑢(𝑡) ;
𝑡∈[0, ∞)
(61) ( 𝐶𝐷𝛼
𝑞(𝑡−𝜁)+ 𝑥) (𝑡) (61) Proof. Proof. Note that One can evaluate the approximation of (42) for 𝑎= 0
and 𝛼∉Z0+ by using (46) as for some prefixed 𝜁∈R+, with initial condition of the state
[ ℎ
)
R𝑛b i
d fi
d b 𝑘
l
t
f
ti for some prefixed 𝜁∈R+, with initial condition of the state
𝑥: [−ℎ, ∞) →R𝑛being defined by 𝑘𝑛-real vector functions
𝜑𝑗: [−ℎ, 0] →R𝑛
0+, with 𝑗∈𝑘−1 ∪{0}, which are bounded
with 𝜑𝑗(0) = 𝑥𝑗(0) = 𝑥(𝑗)(0) = 𝑥𝑗0 𝑗∈𝑘−1 ∪{0}, and
𝑢: R0+ →R𝑚is a bounded piecewise continuous control
function and ℎ= ℎ𝑝= max1≤𝑖≤𝑝ℎ𝑖.ht ( 𝐶𝐷
𝛼
𝑞0+𝑥) (𝑡) = (𝑃𝑁+ 𝜌𝑁) (1 −𝑞)
𝑘−𝛼
× [
1
(1 −𝑞)
𝛼+1𝑡𝛼𝑥(𝑡) + ̂𝑆𝑁𝑡(̂𝑞𝑁𝑡)
+̃𝑆𝑁𝑡(̃𝑞𝑁𝑡) ]
=
𝑝
∑
𝑗=0
𝐴𝑗(𝑡) 𝑥(𝑡−ℎ𝑗) + 𝐵(𝑡) 𝑢(𝑡)
if 𝛼∉Z0+ f
𝑝
≤𝑖≤𝑝
𝑖
Thus, the unique left-sided solution of (55) is calculated
almost everywhere via analytical expressions as follows. (i) If 𝛼∉Z0+ so that 𝑘= [Re 𝛼] + 1 then one gets for any
given 𝑁∈Z+ that (i) If 𝛼∉Z0+ so that 𝑘= [Re 𝛼] + 1 then one gets for any
given 𝑁∈Z+ that 𝑥(𝑡) = (
(𝑃𝑁+ 𝜌𝑁)
(1 −𝑞)2𝛼−𝑘+1 𝑡𝛼𝐼−𝐴0 (𝑡))
−1
× (
𝑝
∑
𝑗=1
𝐴𝑗(𝑡) 𝑥(𝑡−ℎ𝑗) + 𝐵(𝑡) 𝑢(𝑡)
+ (𝑃𝑁+ 𝜌𝑁) (1 −𝑞)
𝑘−𝛼
× (̂𝑆𝑁(𝑡−𝜁) (̂𝑞𝑁(𝑡−𝜁)) (58) where 𝜌𝑁:= 𝑃∗−𝑃𝑁→0,
̃𝑆𝑁𝑡(̃𝑞𝑁𝑡) →0
as 𝑁→∞;
∀𝑡∈R0+
(59) (59) with 𝜌𝑁→0, 𝜎𝑁(𝑡) →0 as 𝑁→∞, for all 𝑡∈R0+. On the other hand, for 𝑎= 0 and 𝑘= 𝛼(∈Z0+), note that
1/Γ(0) = (1 −𝑞)𝑘−𝛼(∏∞
𝑗=0[(1 −𝑞𝑘+𝑗−𝛼)/(1 −𝑞𝑗+1)]) = 𝑃𝑁+ with 𝜌𝑁→0, 𝜎𝑁(𝑡) →0 as 𝑁→∞, for all 𝑡∈R0+. On the other hand, for 𝑎= 0 and 𝑘= 𝛼(∈Z0+), note that
1/Γ(0) = (1 −𝑞)𝑘−𝛼(∏∞
𝑗=0[(1 −𝑞𝑘+𝑗−𝛼)/(1 −𝑞𝑗+1)]) = 𝑃𝑁+ )
(62) Abstract and Applied Analysis 11 11 Proof. One gets from Lemma 10, (48) that 𝑓𝑜𝑟𝑎𝑙𝑙𝑡∈𝑇𝑅𝑁𝐾, where 𝑇𝑅𝑁𝑘:= {𝑡∈R+ : (((𝑃𝑁+
𝜌𝑁)/(1 −𝑞)2𝛼−𝑘+1𝑡𝛼)𝐼−𝐴0(𝑡)) 𝑖𝑠𝑛𝑜𝑛𝑠𝑖𝑛𝑔𝑢𝑙𝑎𝑟}, where
̂𝑆𝑁(𝑡−𝜁)(̂𝑞𝑁𝑡) and ̃𝑆𝑁(𝑡−𝜁)(̃𝑞𝑁𝑡) are got by replacing 𝑡→
(𝑡−𝜁) in their counterparts ̂𝑆𝑁𝑡(̂𝑞𝑁𝑡) and ̃𝑆𝑁𝑡(̃𝑞𝑁𝑡)
defined in (47)-(48). Proof. Proof. Note that One gets from Lemma 10, (48) that ̃𝑆𝑁(𝑡−𝜁) (̃𝑞𝑁(𝑡−𝜁)) −̃𝑆𝑁𝑡(̃𝑞𝑁𝑡)
≤
∞
∑
𝑖=𝑁+1
𝑘
∑
𝑗=0
(−1)𝑗
𝑞𝑖
(1 −𝑞𝑖+1) (1 −𝑞)𝑘(𝑘
𝑗)
× (
𝑥(𝑞𝑗+𝑖(𝑡−𝜁))
(𝑡−𝜁)𝛼
−
𝑥(𝑞𝑗+𝑖𝑡)
𝑡𝛼
)
(ii) If 𝑘= (𝛼∈Z0+) then one gets for any given 𝑁∈Z+that 𝑥(𝑡) = (
1
(1 −𝑞)
𝛼+1 𝑡𝛼𝐼−𝐴0 (𝑡))
−1
× (
𝑝
∑
𝑗=1
𝐴𝑗(𝑡) 𝑥(𝑡−ℎ𝑗) + 𝐵(𝑡) 𝑢(𝑡)
+ ̂𝑆𝑁(𝑡−𝜁) (̂𝑞𝑁(𝑡−𝜁)) + ̃𝑆𝑁(𝑡−𝜁)
× (̃𝑞𝑁(𝑡−𝜁)) −̂𝑆𝑁𝑡(̂𝑞𝑁𝑡) −̃𝑆𝑁𝑡(̃𝑞𝑁𝑡) ) ;
(63) ≤2
𝑞𝑁+1
(1 −𝑞)𝑘+1(1 −𝑞𝑁+1)
× sup
𝑖≥𝑁
[
1
(𝑡−𝜁)𝛼(𝑥(𝑞𝑗+𝑖(𝑡−𝜁)), 𝑥(𝑞𝑗+𝑖𝑡))] ; ∀𝑡(≥𝜁) ∈R0+ ∀𝑡(≥𝜁) ∈R0+ +
(67) 0+
(67) )
(63) since 𝑓𝑜𝑟𝑎𝑙𝑙𝑡
∈
𝑇𝑅𝛼, where 𝑇𝑅𝛼
:=
{𝑡
∈
R+
:
((1/(1 −𝑞)𝛼+1𝑡𝛼)𝐼−𝐴0(𝑡)) 𝑖𝑠𝑛𝑜𝑛𝑠𝑖𝑛𝑔𝑢𝑙𝑎𝑟}, where
̂𝑆𝑁(𝑡−𝜁)(̂𝑞𝑁𝑡) and ̃𝑆𝑁(𝑡−𝜁)(̃𝑞𝑁𝑡) are got by replacing 𝑡→
(𝑡−𝜁) in their counterparts ̂𝑆𝑁𝑡(̂𝑞𝑁𝑡) and ̃𝑆𝑁𝑡(̃𝑞𝑁𝑡)
defined in (47)-(48). 𝑓𝑜𝑟𝑎𝑙𝑙𝑡
∈
𝑇𝑅𝛼, where 𝑇𝑅𝛼
:=
{𝑡
∈
R+
:
((1/(1 −𝑞)𝛼+1𝑡𝛼)𝐼−𝐴0(𝑡)) 𝑖𝑠𝑛𝑜𝑛𝑠𝑖𝑛𝑔𝑢𝑙𝑎𝑟}, where
̂𝑆𝑁(𝑡−𝜁)(̂𝑞𝑁𝑡) and ̃𝑆𝑁(𝑡−𝜁)(̃𝑞𝑁𝑡) are got by replacing 𝑡→
(𝑡−𝜁) in their counterparts ̂𝑆𝑁𝑡(̂𝑞𝑁𝑡) and ̃𝑆𝑁𝑡(̃𝑞𝑁𝑡)
defined in (47)-(48). 𝑓𝑜𝑟𝑎𝑙𝑙𝑡
∈
𝑇𝑅𝛼, where 𝑇𝑅𝛼
:=
{𝑡
∈
R+
:
((1/(1 −𝑞)𝛼+1𝑡𝛼)𝐼−𝐴0(𝑡)) 𝑖𝑠𝑛𝑜𝑛𝑠𝑖𝑛𝑔𝑢𝑙𝑎𝑟}, where
̂𝑆𝑁(𝑡−𝜁)(̂𝑞𝑁𝑡) and ̃𝑆𝑁(𝑡−𝜁)(̃𝑞𝑁𝑡) are got by replacing 𝑡→
(𝑡−𝜁) in their counterparts ̂𝑆𝑁𝑡(̂𝑞𝑁𝑡) and ̃𝑆𝑁𝑡(̃𝑞𝑁𝑡)
defined in (47)-(48). ∞
∑
𝑖=𝑁+1
𝑞𝑖
1 −𝑞𝑖+1 ≤
∞
∑
𝑖=𝑁+1
𝑞𝑖
1 −𝑞𝑖
≤
∞
∑
𝑖=𝑁+1
𝑞𝑖
1 −𝑞𝑁+1 ≤
𝑞𝑁+1
(1 −𝑞) (1 −𝑞𝑁+1). (68) (68) Proof. It is omitted since it follows directly as that of
Theorem 11 by using (42) with 𝑎= 𝑎(𝑡) = 𝑡−𝜁, for all 𝑡∈
R0+. In a similar way, one can obtain The following auxiliary result is useful to then formulate
the solution calculation under a finite truncation of the
infinite series associated with the Jackson integral. ̃𝑆𝑁𝑡(̃𝑞𝑁𝑡)≤
𝑞𝑁+1
(1 −𝑞)𝑘+1(1 −𝑞𝑁+1)
× sup
𝑖≥𝑁
[ 1
𝑡𝛼(𝑥(𝑞𝑗+𝑖𝑡))] ;
∀𝑡∈R0+
(69) (69) Lemma 13. The following properties hold. Lemma 13. The following properties hold. Lemma 13. The following properties hold. (i) For any 𝜀∈R+ and any finite real 𝑇1 > 𝜀and 𝑇2 > 𝜁+𝜀,
the limits below exist: and hence Property (i) follows since one has from (67) and
(69) for some finite positive real constants 𝑀1 = 𝑀(𝜀, 𝑇1)
and 𝑀2 = 𝑀(𝜁+ 𝜀, 𝑇2) that and hence Property (i) follows since one has from (67) and
(69) for some finite positive real constants 𝑀1 = 𝑀(𝜀, 𝑇1)
and 𝑀2 = 𝑀(𝜁+ 𝜀, 𝑇2) that lim
𝑁→∞
̃𝑆𝑁𝑡(̃𝑞𝑁𝑡) = 0;
𝑡∈[𝜀, 𝑇1) ,
(64a)
lim
𝑁→∞[̃𝑆𝑁𝑡(̃𝑞𝑁𝑡) −̃𝑆𝑁(𝑡−𝜁) (̃𝑞𝑁(𝑡−𝜁))] = 0;
𝑡∈[𝜁+ 𝜀, 𝑇2) . (64b) (64a) (64a) ̃𝑆𝑁𝑡(̃𝑞𝑁𝑡)≤
𝑞𝑁+1
(1 −𝑞)
𝑘+1(1 −𝑞𝑁+1) (64b) × sup
𝜀≤𝑡≤𝑇1
sup
𝑖≥𝑁
[ 1
𝜀𝛼(𝑥(𝑞𝑗+𝑖(𝑡)))] (ii) Assume, furthermore, that the solution 𝑥: R0+ →R𝑛
of (61) has the following property: ≤
𝑞𝑁+1𝑀1
(1 −𝑞)
𝑘+1(1 −𝑞𝑁+1)
;
∀𝑡∈[𝜀, 𝑇1) ,
̃𝑆𝑁𝑡(̃𝑞𝑁𝑡) −̃𝑆𝑁(𝑡−𝜁) (̃𝑞𝑁(𝑡−𝜁))
≤
2𝑞𝑁+1𝑀2
(1 −𝑞)𝑘+1(1 −𝑞𝑁+1)
;
∀𝑡∈[𝜁+ 𝜀, 𝑇2
( lim sup
𝑡→∞
(sup
𝑖≥𝑁
𝑥(𝑞𝑖𝑡)
𝑡𝛼
−
𝑥(𝑞𝑖(𝑡−𝜁))
(𝑡−𝜁)𝛼
−𝑂(𝑡−𝜁)𝛼) = 0. (65) (65) Then, the limit below exists: lim
𝑡→∞lim
𝑁→∞[̃𝑆𝑁𝑡(̃𝑞𝑁𝑡) −̃𝑆𝑁(𝑡−𝜁) (̃𝑞𝑁(𝑡−𝜁))] = 0. (66) lim
𝑡→∞lim
𝑁→∞[̃𝑆𝑁𝑡(̃𝑞𝑁𝑡) −̃𝑆𝑁(𝑡−𝜁) (̃𝑞𝑁(𝑡−𝜁))] = 0. (66) (66) (70) Abstract and Applied Analysis Abstract and Applied Analysis 12 Note that if the solution of (61) satisfies (65) then
for any real 𝜀∈R+ and 𝜁+ 𝜀≤𝑇< ∞, where Note that if the solution of (61) satisfies (65) then
for any real 𝜀∈R+ and 𝜁+ for any real 𝜀∈R+ and 𝜁+ 𝜀≤𝑇< ∞, where for any real 𝜀∈R+ and 𝜁+ 𝜀≤𝑇< ∞, where for any real 𝜀∈R+ and 𝜁+ 𝜀≤𝑇< ∞, where Note that if the solution of (61) satisfies (65) then ̃𝑆𝑁(𝑡−𝜁) (̃𝑞𝑁(𝑡−𝜁)) −̃𝑆𝑁𝑡(̃𝑞𝑁𝑡)
≤
∞
∑
𝑖=𝑁+1
𝑘
∑
𝑗=0
(−1)𝑗
𝑞𝑖
(1 −𝑞𝑖+1) (1 −𝑞)
𝑘
× (𝑘
𝑗) (
𝑥(𝑞𝑗+𝑖(𝑡−𝜁))
(𝑡−𝜁)𝛼
−
𝑥(𝑞𝑗+𝑖𝑡)
𝑡𝛼
)
≤𝐾𝑆𝑞𝑁+1
1 −𝑞sup
𝑖≥𝑁
(
𝑥(𝑞𝑖(𝑡−𝜁))
(𝑡−𝜁)𝛼
−
𝑥(𝑞𝑖𝑡)
𝑡𝛼
)
. (71)
Then,
li
̃𝑆
(̃ (𝑡
𝜁))
̃𝑆
(̃ 𝑡)
𝑇𝑅𝑘𝛼:= {𝑡∈[𝜁+ 𝜀, 𝑇) :
(
𝑃∗
(1 −𝑞)2𝛼−𝑘+1 𝑡𝛼𝐼−𝐴0 (𝑡)) 𝑖𝑠𝑛𝑜𝑛𝑠𝑖𝑛𝑔𝑢𝑙𝑎𝑟} . Lemma 13. The following properties hold. (75)
If,
in
addition,
𝐴0
is
nonsingular
and
lim sup𝑡→∞(sup𝑖≥𝑁‖𝑥(𝑞𝑖𝑡)/𝑡𝛼
−
𝑥(𝑞𝑖(𝑡
−
𝜁))/(𝑡−𝜁)𝛼‖ −𝑂(𝑡−𝜁)𝛼) = 0 then
lim
𝑡→∞lim
𝑁→∞
𝑥(𝑡) + 𝐴−1
0 (𝑡) ̃𝑆𝑁(𝑡−𝜁) (̃𝑞𝑁(𝑡−𝜁)) −̃𝑆𝑁𝑡(̃𝑞𝑁𝑡)
≤
∞
∑
𝑖=𝑁+1
𝑘
∑
𝑗=0
(−1)𝑗
𝑞𝑖
(1 −𝑞𝑖+1) (1 −𝑞)
𝑘
× (𝑘
𝑗) (
𝑥(𝑞𝑗+𝑖(𝑡−𝜁))
(𝑡−𝜁)𝛼
−
𝑥(𝑞𝑗+𝑖𝑡)
𝑡𝛼
)
𝑇𝑅𝑘𝛼:= {𝑡∈[𝜁+ 𝜀, 𝑇) :
(
𝑃∗
(1 −𝑞)2𝛼−𝑘+1 𝑡𝛼𝐼−𝐴0 (𝑡)) 𝑖𝑠𝑛𝑜𝑛𝑠𝑖𝑛𝑔𝑢𝑙𝑎𝑟} . (75) ̃𝑆𝑁(𝑡−𝜁) (̃𝑞𝑁(𝑡−𝜁)) −̃𝑆𝑁𝑡(̃𝑞𝑁𝑡) 𝑇𝑅𝑘𝛼:= {𝑡∈[𝜁+ 𝜀, 𝑇) :
(
𝑃∗
(1 −𝑞)2𝛼−𝑘+1 𝑡𝛼𝐼−𝐴0 (𝑡)) 𝑖𝑠𝑛𝑜𝑛𝑠𝑖𝑛𝑔𝑢𝑙𝑎𝑟} . (75) (75)
≤𝐾𝑆𝑞𝑁+1
1 −𝑞sup
𝑖≥𝑁
(
𝑥(𝑞𝑖(𝑡−𝜁))
(𝑡−𝜁)𝛼
−
𝑥(𝑞𝑖𝑡)
𝑡𝛼
)
. (71)
Then,
lim sup
𝑡→∞
̃𝑆𝑁(𝑡−𝜁) (̃𝑞𝑁(𝑡−𝜁)) −̃𝑆𝑁𝑡(̃𝑞𝑁𝑡)
≤𝐾𝑆𝑞𝑁+1
1 −𝑞lim sup
𝑡→∞
(sup
𝑖≥𝑁
(
𝑥(𝑞𝑖(𝑡−𝜁))
(𝑡−𝜁)𝛼
−
𝑥(𝑞𝑖𝑡)
𝑡𝛼
)
)
(72)
so that
If,
in
addition,
𝐴0
is
nonsingular
and
lim sup𝑡→∞(sup𝑖≥𝑁‖𝑥(𝑞𝑖𝑡)/𝑡𝛼
−
𝑥(𝑞𝑖(𝑡
−
𝜁))/(𝑡−𝜁)𝛼‖ −𝑂(𝑡−𝜁)𝛼) = 0 then
lim
𝑡→∞lim
𝑁→∞
𝑥(𝑡) + 𝐴−1
0 (𝑡)
× (
𝑝
∑
𝑗=1
𝐴𝑗(𝑡) 𝑥(𝑡−ℎ𝑗) + 𝐵(𝑡) 𝑢(𝑡)
+ 𝑃∗(1 −𝑞)𝑘−𝛼(̂𝑆𝑁(𝑡−𝜁) (̂𝑞𝑁(𝑡−𝜁))
)
≤𝐾𝑆𝑞𝑁+1
1 −𝑞sup
𝑖≥𝑁
(
𝑥(𝑞𝑖(𝑡−𝜁))
(𝑡−𝜁)𝛼
−
𝑥(𝑞𝑖𝑡)
𝑡𝛼
)
. (71)
Then,
lim sup
𝑡→∞
̃𝑆𝑁(𝑡−𝜁) (̃𝑞𝑁(𝑡−𝜁)) −̃𝑆𝑁𝑡(̃𝑞𝑁𝑡)
If,
in
addition,
𝐴0
is
nonsingular
and
lim sup𝑡→∞(sup𝑖≥𝑁‖𝑥(𝑞𝑖𝑡)/𝑡𝛼
−
𝑥(𝑞𝑖(𝑡
−
𝜁))/(𝑡−𝜁)𝛼‖ −𝑂(𝑡−𝜁)𝛼) = 0 then
lim
𝑡→∞lim
𝑁→∞
𝑥(𝑡) + 𝐴−1
0 (𝑡) ≤𝐾𝑆𝑞𝑁+1
1 −𝑞sup
𝑖≥𝑁
(
𝑥(𝑞𝑖(𝑡−𝜁))
(𝑡−𝜁)𝛼
−
𝑥(𝑞𝑖𝑡)
𝑡𝛼
)
. (71)
Then,
If,
in
addition,
𝐴0
is
nonsingular
and
lim sup𝑡→∞(sup𝑖≥𝑁‖𝑥(𝑞𝑖𝑡)/𝑡𝛼
−
𝑥(𝑞𝑖(𝑡
−
𝜁))/(𝑡−𝜁)𝛼‖ −𝑂(𝑡−𝜁)𝛼) = 0 then
If,
in
addition,
𝐴0
is
nonsingular
and
lim sup𝑡→∞(sup𝑖≥𝑁‖𝑥(𝑞𝑖𝑡)/𝑡𝛼
−
𝑥(𝑞𝑖(𝑡
−
𝜁))/(𝑡−𝜁)𝛼‖ −𝑂(𝑡−𝜁)𝛼) = 0 then If,
in
addition,
𝐴0
is
nonsingular
and
lim sup𝑡→∞(sup𝑖≥𝑁‖𝑥(𝑞𝑖𝑡)/𝑡𝛼
−
𝑥(𝑞𝑖(𝑡
−
𝜁))/(𝑡−𝜁)𝛼‖ −𝑂(𝑡−𝜁)𝛼) = 0 then (71) Then, lim
𝑡→∞lim
𝑁→∞
𝑥(𝑡) + 𝐴−1
0 (𝑡) Then,
lim sup
𝑡→∞
̃𝑆𝑁(𝑡−𝜁) (̃𝑞𝑁(𝑡−𝜁)) −̃𝑆𝑁𝑡(̃𝑞𝑁𝑡)
lim
𝑡→∞lim
𝑁→∞
𝑥(𝑡) + 𝐴−1
0 (𝑡) lim sup
𝑡→∞
̃𝑆𝑁(𝑡−𝜁) (̃𝑞𝑁(𝑡−𝜁)) −̃𝑆𝑁𝑡(̃𝑞𝑁𝑡) lim sup
𝑡→∞
̃𝑆𝑁(𝑡−𝜁) (̃𝑞𝑁(𝑡−𝜁)) −̃𝑆𝑁𝑡(̃𝑞𝑁𝑡) lim sup
𝑡→∞
𝑆𝑁(𝑡−𝜁) (𝑞𝑁(𝑡−𝜁)) −𝑆𝑁𝑡(𝑞𝑁𝑡)
≤𝐾𝑆𝑞𝑁+1
1 −𝑞lim sup
𝑡→∞
(sup
𝑖≥𝑁
(
𝑥(𝑞𝑖(𝑡−𝜁))
(𝑡−𝜁)𝛼
−
𝑥(𝑞𝑖𝑡)
𝑡𝛼
)
)
(72)
o that
0 ≤lim sup
𝑡→∞
lim
𝑁→∞
̃𝑆𝑁(𝑡−𝜁) (̃𝑞𝑁(𝑡−𝜁)) −̃𝑆𝑁𝑡(̃𝑞𝑁𝑡)
(
𝑁1
)
(73)
× (
𝑝
∑
𝑗=1
𝐴𝑗(𝑡) 𝑥(𝑡−ℎ𝑗) + 𝐵(𝑡) 𝑢(𝑡)
+ 𝑃∗(1 −𝑞)𝑘−𝛼(̂𝑆𝑁(𝑡−𝜁) (̂𝑞𝑁(𝑡−𝜁))
−̂𝑆𝑁𝑡(̂𝑞𝑁𝑡)) )
= 0. Lemma 13. The following properties hold. (78) (78) 13 Abstract and Applied Analysis Abstract and Applied Analysis Abstract and Applied Analysis If, in addition, 𝐴0 is nonsingular and lim sup𝑡→∞
(sup𝑖≥𝑁‖𝑥(𝑞𝑖𝑡)/𝑡𝛼−𝑥(𝑞𝑖(𝑡−𝜁))/(𝑡−𝜁)𝛼‖−𝑂(𝑡−𝜁)𝛼) =
0 then 0𝑖𝑖( )
p𝑡→∞
0𝑖𝑖( )
𝑓𝑜𝑟𝑎𝑙𝑙𝑖, 𝑗( ̸= 𝑖) ∈𝑛, 𝑓𝑜𝑟𝑎𝑙𝑙𝑡∈R0+ or 𝑓
𝑗
𝑓
0+
(C2) (−𝐴0(𝑡))
∈
𝑀R𝑛×𝑛
+ , 𝐴0𝑖𝑖 (C2) (−𝐴0(𝑡))
∈
𝑀R𝑛×𝑛
+ , 𝐴0𝑖𝑖(𝑡)
=
−(𝑃𝑁+
𝜌𝑁)/(1 −𝑞)2𝛼−𝑘+1𝑡𝛼, 𝑓𝑜𝑟𝑎𝑙𝑙𝑖∈𝑛, 𝐴0𝑖𝑗(𝑡) ≤0 with 𝐴0𝑖𝑗(𝑡) < 0
for only one 𝑗∈𝑛for each 𝑖∈𝑛, 𝑓𝑜𝑟𝑎𝑙𝑙𝑡∈R0+. lim
𝑡→∞lim
𝑁→∞
𝑥(𝑡) + 𝐴−1
0 (𝑡) Proof. Since 𝛼∉Z0+then 𝑘= [Re 𝛼] + 1 and one gets from
(55)–(58), (49), and (80) that 𝑥(𝑡) ≥(
𝑃𝑁+ 𝜌𝑁
(1 −𝑞)2𝛼−𝑘+1 𝑡𝛼𝐼−𝐴0 (𝑡))
−1
× (
𝑝
∑
𝑗=1
𝐴𝑗(𝑡) 𝑥(𝑡−ℎ𝑗) −(𝑃𝑁+ 𝜌𝑁) (1 −𝑞)𝑘−𝛼
× [
[
̂𝑆𝑁𝑡(̂𝑞𝑁𝑡) + ( 𝛾
𝑞𝑘)
𝑁+1 (𝑘+ 1) (‖𝑥(0)‖ + 𝜀𝑁)
(1 −𝛾) (1 −𝑞)𝑘𝑡𝛼
×
𝑘
∑
𝑗=0
(−1)𝑗(𝑘
𝑗)
̂𝑒]
]
+𝐵(𝑡) 𝑢(𝑡) ) ≥0;
∀𝑡∈R0+
(81) (79) 4. Nonnegativity of the 𝑞-Solutions of Caputo
Fractional Order 𝛼 Assume also that 𝛼∉Z0+, 𝐵(𝑡) =
(𝑏𝑖𝑗(𝑡)) ∈R𝑛×𝑛
+
is a monomial diagonal matrix (i.e., 𝑏𝑖𝑖>
0 and 𝑏𝑖𝑗= 0; 𝑓𝑜𝑟𝑎𝑙𝑙𝑖, 𝑗( ̸= 𝑖) ∈𝑛) and that the control
components satisfy the constraints for some 𝑁∈Z+ and some
real nonnegative strictly decreasing sequence {𝜀𝑛}: (81) (81) since
(((𝑃𝑁+ 𝜌𝑁)/(1 −𝑞)2𝛼−𝑘+1𝑡𝛼)𝐼−𝐴0(𝑡))
−1
∈
R𝑛×𝑛
+
for all 𝑡
∈
R0+ under any of conditions (C1) or
(C2) where ̂𝑒∈R𝑛is a real vector with all its components
being one, since since
(((𝑃𝑁+ 𝜌𝑁)/(1 −𝑞)2𝛼−𝑘+1𝑡𝛼)𝐼−𝐴0(𝑡))
−1
∈
R𝑛×𝑛
+
for all 𝑡
∈
R0+ under any of conditions (C1) or
(C2) where ̂𝑒∈R𝑛is a real vector with all its components
being one, since since
(((𝑃𝑁+ 𝜌𝑁)/(1 −𝑞)2𝛼−𝑘+1𝑡𝛼)𝐼−𝐴0(𝑡))
−1
∈
R𝑛×𝑛
+
for all 𝑡
∈
R0+ under any of conditions (C1) or
(C2) where ̂𝑒∈R𝑛is a real vector with all its components
being one, since 𝑢𝑖(𝑡) ≥max (0 , 𝑏−1
𝑖𝑖(𝑡)
× ( (𝑃𝑁+ 𝜌𝑁) (1 −𝑞)𝑘−𝛼
× [
[
𝑒𝑇
𝑖̂𝑆𝑁𝑡(̂𝑞𝑁𝑡) −( 𝛾
𝑞𝑘)
𝑁+1
× (𝑘+ 1) (‖𝑥(0)‖ + 𝜀𝑁)
(1 −𝛾) (1 −𝑞)𝑘𝑡𝛼
𝑘
∑
𝑗=0
(−1)𝑗(𝑘
𝑗)
]
]
−
𝑝
∑
𝑗=1
𝑒𝑇
𝑖𝐴𝑗𝑥(𝑡−ℎ𝑗))) ;
∀𝑖∈𝑛, ∀𝑡∈R0+,
(80) (
𝑝
∑
𝑗=1
𝐴𝑗(𝑡) 𝑥(𝑡−ℎ𝑗) −̂𝑆𝑁𝑡(̂𝑞𝑁𝑡) + ( 𝛾
𝑞𝑘)
𝑁+1
× (𝑘+ 1) (‖𝑥(0)‖ + 𝜀𝑁)
(1 −𝛾) (1 −𝑞)𝑘𝑡𝛼
𝑘
∑
𝑗=0
(−1)𝑗(𝑘
𝑗)
̂𝑒
+𝐵(𝑡) 𝑢(𝑡) ) ≥0;
∀𝑡∈R0+
(82) (
𝑝
∑
𝑗=1
𝐴𝑗(𝑡) 𝑥(𝑡−ℎ𝑗) −̂𝑆𝑁𝑡(̂𝑞𝑁𝑡) + ( 𝛾
𝑞𝑘)
𝑁+1 × [
[
𝑒𝑇
𝑖̂𝑆𝑁𝑡(̂𝑞𝑁𝑡) −( 𝛾
𝑞𝑘)
𝑁+1 × (𝑘+ 1) (‖𝑥(0)‖ + 𝜀𝑁)
(1 −𝛾) (1 −𝑞)𝑘𝑡𝛼
𝑘
∑
𝑗=0
(−1)𝑗(𝑘
𝑗)
̂𝑒
+𝐵(𝑡) 𝑢(𝑡) ) ≥0;
∀𝑡∈R0+
(82) (82) × (𝑘+ 1) (‖𝑥(0)‖ + 𝜀𝑁)
(1 −𝛾) (1 −𝑞)𝑘𝑡𝛼
𝑘
∑
𝑗=0
(−1)𝑗(𝑘
𝑗)
]
] from (80) and any of (C1)-(C2) implies the existence of
(((𝑃𝑁+ 𝜌𝑁)/(1 −𝑞)2𝛼−𝑘+1𝑡𝛼)𝐼−𝐴0(𝑡))
−1 ∈R𝑛×𝑛
+ ; for all 𝑡∈
R0+. from (80) and any of (C1)-(C2) implies the existence of
(((𝑃𝑁+ 𝜌𝑁)/(1 −𝑞)2𝛼−𝑘+1𝑡𝛼)𝐼−𝐴0(𝑡))
−1 ∈R𝑛×𝑛
+ ; for all 𝑡∈
R0+. (80) (80) where 𝑒𝑖is the unit Euclidean vector of R𝑛with its 𝑖th
component being one. Then, the solution of (55) is nonnegative,
that is, 𝑥: [−ℎ, 0] ∪R0+ →R𝑛
0+ under a nonnegative real
control 𝑢: R0+ →R𝑛
0+ if one of the following conditions holds. where 𝑒𝑖is the unit Euclidean vector of R𝑛with its 𝑖th
component being one. Lemma 13. The following properties hold. (76) 𝑡→∞
≤𝐾𝑆𝑞𝑁+1
1 −𝑞lim sup
𝑡→∞
(sup
𝑖≥𝑁
(
𝑥(𝑞𝑖(𝑡−𝜁))
(𝑡−𝜁)𝛼
−
𝑥(𝑞𝑖𝑡)
𝑡𝛼
)
)
(72)
× (
𝑝
∑
𝑗=1
𝐴𝑗(𝑡) 𝑥(𝑡−ℎ𝑗) + 𝐵(𝑡) 𝑢(𝑡)
+ 𝑃∗(1 −𝑞)𝑘−𝛼(̂𝑆𝑁(𝑡−𝜁) (̂𝑞𝑁(𝑡− ≤𝐾𝑆𝑞𝑁+1
1 −𝑞lim sup
𝑡→∞
(sup
𝑖≥𝑁
(
𝑥(𝑞𝑖(𝑡−𝜁))
(𝑡−𝜁)𝛼
−
𝑥(𝑞𝑖𝑡)
𝑡𝛼
)
)
(72) so that 0 ≤lim sup
𝑡→∞
lim
𝑁→∞
̃𝑆𝑁(𝑡−𝜁) (̃𝑞𝑁(𝑡−𝜁)) −̃𝑆𝑁𝑡(̃𝑞𝑁𝑡) 0 ≤lim sup
𝑡→∞
lim
𝑁→∞
̃𝑆𝑁(𝑡−𝜁) (̃𝑞𝑁(𝑡−𝜁)) −̃𝑆𝑁𝑡(̃𝑞𝑁𝑡) (76) (73) (73) ≤( lim
𝑁→∞
𝐾𝑆𝑞𝑁+1
1 −𝑞) lim sup
𝑡→∞
(0(𝑡−𝜁)𝛼) = 0
(73)
(ii) If 𝑘= (𝛼∈Z
) then one gets ≤( lim
𝑁→∞
𝐾𝑆𝑞𝑁+1
1 −𝑞) lim sup
𝑡→∞
(0(𝑡−𝜁)𝛼) = 0 (ii) If 𝑘= (𝛼∈Z0+) then one gets and then ∃lim𝑡→∞lim𝑁→∞‖̃𝑆𝑁(𝑡−𝜁)(̃𝑞𝑁(𝑡−𝜁))−̃𝑆𝑁𝑡(̃𝑞𝑁𝑡)‖ =
0. Hence, Property (ii) follows. and then ∃lim𝑡→∞lim𝑁→∞‖̃𝑆𝑁(𝑡−𝜁)(̃𝑞𝑁(𝑡−𝜁))−̃𝑆𝑁𝑡(̃𝑞𝑁𝑡)‖ =
0. Hence, Property (ii) follows. lim
𝑁→∞
𝑥(𝑡) −(
1
(1 −𝑞)
𝛼+1 𝑡𝛼𝐼−𝐴0 (𝑡))
−1
× (
𝑝
∑
𝑗=1
𝐴𝑗(𝑡) 𝑥(𝑡−ℎ𝑗) + 𝐵𝑢(𝑡) 0. Hence, Property (ii) follows. The subsequent result follows directly from Theorem 12
and Lemma 13. Theorem 14. The following properties hold for 𝛼∈R0+. lim
𝑁→∞
𝑥(𝑡) −(
1
(1 −𝑞)
𝛼+1 𝑡𝛼𝐼−𝐴0 (𝑡))
−1
× (
𝑝
∑
𝑗=1
𝐴𝑗(𝑡) 𝑥(𝑡−ℎ𝑗) + 𝐵𝑢(𝑡) The subsequent result follows directly from Theorem 12
and Lemma 13. Theorem 14. The following properties hold for 𝛼∈R0+. (i) If 𝛼∉Z0+ so that 𝑘= [Re 𝛼] + 1 then one gets Theorem 14. The following properties hold for 𝛼∈R0+. + ̂𝑆𝑁(𝑡−𝜁) (̂𝑞𝑁(𝑡−𝜁)) −̂𝑆𝑁𝑡(̂𝑞𝑁𝑡) )
= 0;
𝑡∈𝑇𝑅𝛼
(77) (i) If 𝛼∉Z0+ so that 𝑘= [Re 𝛼] + 1 then one gets lim
𝑁→∞
𝑥(𝑡) −(
𝑃∗
(1 −𝑞)2𝛼−𝑘+1 𝑡𝛼𝐼−𝐴0 (𝑡))
−1
× (
𝑝
∑
𝑗=1
𝐴𝑗𝑥(𝑡−ℎ𝑗) + 𝐵(𝑡) 𝑢(𝑡) for any real 𝜀∈R+ and 𝜁+ 𝜀≤𝑇< ∞, where for any real 𝜀∈R+ and 𝜁+ 𝜀≤𝑇< ∞, where + 𝑃∗(1 −𝑞)𝑘−𝛼(̂𝑆𝑁(𝑡−𝜁) (̂𝑞𝑁(𝑡−𝜁))
−̂𝑆𝑁𝑡(̂𝑞𝑁𝑡)) )
= 0;
𝑡∈𝑇𝑅𝑘𝛼
(74)
𝑇𝑅𝛼: = {𝑡∈[𝜁+ 𝜀, 𝑇) :
(
1
(1 −𝑞)𝛼+1 𝑡𝛼𝐼−𝐴0 (𝑡)) 𝑖𝑠𝑛𝑜𝑛𝑠𝑖𝑛𝑔𝑢𝑙𝑎𝑟} . (78) + 𝑃∗(1 −𝑞)𝑘−𝛼(̂𝑆𝑁(𝑡−𝜁) (̂𝑞𝑁(𝑡−𝜁))
−̂𝑆𝑁𝑡(̂𝑞𝑁𝑡)) )
= 0;
𝑡∈𝑇𝑅𝑘𝛼
(74) 𝑇𝑅𝛼: = {𝑡∈[𝜁+ 𝜀, 𝑇) : 𝑇𝑅𝛼: = {𝑡∈[𝜁+ 𝜀, 𝑇) : (
1
(1 −𝑞)𝛼+1 𝑡𝛼𝐼−𝐴0 (𝑡)) 𝑖𝑠𝑛𝑜𝑛𝑠𝑖𝑛𝑔𝑢𝑙𝑎𝑟} . (78) (
1
(1 −𝑞)𝛼+1 𝑡𝛼𝐼−𝐴0 (𝑡)) 𝑖𝑠𝑛𝑜𝑛𝑠𝑖𝑛𝑔𝑢𝑙𝑎𝑟} . 4. Nonnegativity of the 𝑞-Solutions of Caputo
Fractional Order 𝛼 The following two results hold for the nonnegativity for
all time of the solutions of (55) under certain nonnegative
controls. × [
[
̂𝑆𝑁𝑡(̂𝑞𝑁𝑡) + ( 𝛾
𝑞𝑘)
𝑁+1 (𝑘+ 1) (‖𝑥(0)‖ + 𝜀𝑁)
(1 −𝛾) (1 −𝑞)𝑘𝑡𝛼
×
𝑘
∑
𝑗=0
(−1)𝑗(𝑘
𝑗)
̂𝑒]
]
+𝐵(𝑡) 𝑢(𝑡) ) ≥0; Theorem 15. Consider the Caputo fractional 𝑞-differential
system of order 𝛼∈R0+ (55) under a piecewise-continuous
control vector function 𝑢: R0+
→
R𝑛
0+ and any set of
vector functions of initial conditions 𝜑𝑗: [−ℎ, 0] →R𝑛
0+,
𝑓𝑜𝑟𝑎𝑙𝑙𝑗∈𝑘−1 ∪{0}. Assume also that 𝛼∉Z0+, 𝐵(𝑡) =
(𝑏𝑖𝑗(𝑡)) ∈R𝑛×𝑛
+
is a monomial diagonal matrix (i.e., 𝑏𝑖𝑖>
0 and 𝑏𝑖𝑗= 0; 𝑓𝑜𝑟𝑎𝑙𝑙𝑖, 𝑗( ̸= 𝑖) ∈𝑛) and that the control
components satisfy the constraints for some 𝑁∈Z+ and some
real nonnegative strictly decreasing sequence {𝜀𝑛}: Theorem 15. Consider the Caputo fractional 𝑞-differential
system of order 𝛼∈R0+ (55) under a piecewise-continuous
control vector function 𝑢: R0+
→
R𝑛
0+ and any set of
vector functions of initial conditions 𝜑𝑗: [−ℎ, 0] →R𝑛
0+, ∀𝑡∈R0+ 𝑓𝑜𝑟𝑎𝑙𝑙𝑗∈𝑘−1 ∪{0}. 4. Nonnegativity of the 𝑞-Solutions of Caputo
Fractional Order 𝛼 Then, the solution of (55) is nonnegative,
that is, 𝑥: [−ℎ, 0] ∪R0+ →R𝑛
0+ under a nonnegative real
control 𝑢: R0+ →R𝑛
0+ if one of the following conditions holds. Theorem 16. Under similar assumptions for the control, the
matrix 𝐵and the initial conditions as in Theorem 15, assume Abstract and Applied Analysis Abstract and Applied Analysis 14 since there is ((1/(1 −𝑞)𝛼+1𝑡𝛼)𝐼−𝐴0(𝑡))
−1 ∈R𝑛×𝑛
+ , for all 𝑡∈
R0+under any of conditions (C3) or (C4). Then, that 𝛼= 𝑘∈Z0+, 𝐵(𝑡) = (𝑏𝑖𝑗(𝑡)) ∈R𝑛×𝑛
+
and that the control
components satisfy the constraints 𝑥(𝑡) ≥(
1
(1 −𝑞)
𝛼+1 𝑡𝛼𝐼−𝐴0 (𝑡))
−1
× (
𝑝
∑
𝑗=1
𝐴𝑗(𝑡) 𝑥(𝑡−ℎ𝑗) −̂𝑆𝑁𝑡(̂𝑞𝑁𝑡)
+ 𝐵(𝑡) 𝑢(𝑡) −( 𝛾
𝑞𝑘)
𝑁+1 (𝛼+ 1) (‖𝑥(0)‖ + 𝜀𝑁)
(1 −𝛾) (1 −𝑞)𝑘𝑡𝛼
×
𝛼
∑
𝑗=0
(−1)𝑗(𝛼
𝑗)
̂𝑒) ≥0;
∀𝑡∈R0+
(85) 𝑢𝑖(𝑡) ≥max (0, 𝑏−1
𝑖𝑖(𝑡) 𝑢𝑖(𝑡) ≥max (0, 𝑏−1
𝑖𝑖(𝑡) × (𝑒𝑇
𝑖̂𝑆𝑁𝑡(̂𝑞𝑁𝑡) −( 𝛾
𝑞𝑘)
𝑁+1 × (𝑘+ 1) (‖𝑥(0)‖ + 𝜀𝑁)
(1 −𝛾) (1 −𝑞)𝑘𝑡𝛼
𝛼
∑
𝑗=0
(−1)𝑗(𝛼
𝑗)
−
𝑝
∑
𝑗=1
𝑒𝑇
𝑖𝐴𝑗(𝑡) 𝑥(𝑡−ℎ𝑗))) ; ×
𝛼
∑
𝑗=0
(−1)𝑗(𝛼
𝑗)
̂𝑒) ≥0;
∀𝑡∈R0+ (85)
)
(85) ∀𝑖∈𝑛,
∀𝑡∈R0+ ∀𝑖∈𝑛,
∀𝑡∈R0+ from (83) and any of conditions (C3) or (C4) imply from from (83) and any of conditions (C3) or (C4) imply from
Proposition 7 the existence of ((1/(1 −𝑞)𝛼+1𝑡𝛼)𝐼−𝐴0(𝑡))
−1 ∈
R𝑛×𝑛
+ , for all 𝑡∈R0+. from (83) and any of conditions (C3) or (C4) imply from
Proposition 7 the existence of ((1/(1 −𝑞)𝛼+1𝑡𝛼)𝐼−𝐴0(𝑡))
−1 ∈
R𝑛×𝑛
+ , for all 𝑡∈R0+. (83) (83) Remark 17. If the 𝑞-Caputo fractional differential system (61)
is considered instead of (55), Theorems 15 and 16 apply with
the replacements for some 𝑁∈Z+ and some real nonnegative strictly decreasing
sequence {𝜀𝑛}. Then, 𝑥: [−ℎ, 0] ∪R0+
→
R𝑛
0+ under a
nonnegative real control 𝑢: R0+ →R𝑛
0+ if one of the following
conditions holds. 𝑘 for some 𝑁∈Z+ and some real nonnegative strictly decreasing
sequence {𝜀𝑛}. Then, 𝑥: [−ℎ, 0] ∪R0+
→
R𝑛
0+ under a
nonnegative real control 𝑢: R0+ →R𝑛
0+ if one of the following
conditions holds. 4. Nonnegativity of the 𝑞-Solutions of Caputo
Fractional Order 𝛼 ̂𝑆𝑁𝑡(̃𝑞𝑁𝑡)→̂𝑆𝑁(𝑡−𝜁) (̃𝑞𝑁(𝑡−𝜁)) −̂𝑆𝑁𝑡(̃𝑞𝑁𝑡);
̃𝑆𝑁𝑡(̃𝑞𝑁𝑡)→̃𝑆𝑁(𝑡−𝜁) (̃𝑞𝑁(𝑡−𝜁)) −̃𝑆𝑁𝑡(̃𝑞𝑁𝑡)
(86) (C3) Either (1 −𝑞)𝑘−𝛼/(1 −𝑞)𝛼+1𝑡𝛼> 𝐴0𝑖𝑖(𝑡) > 0 or
𝐴0𝑖𝑖(𝑡) ≤0 with lim sup𝑡→∞𝐴0𝑖𝑖(𝑡) < 0 and 𝐴0𝑖𝑗(𝑡) =
0for all 𝑖, 𝑗( ̸= i) ∈𝑛; for each 𝑡∈R0+, or (86) f
𝑗
f
0
(C4) (−𝐴0(𝑡)) ∈𝑀R𝑛×𝑛
+
with 𝐴0𝑖𝑖(𝑡) = −(1 −𝑞)𝑘−𝛼/(1 −
𝑞)𝛼+1𝑡𝛼; 𝑓𝑜𝑟𝑎𝑙𝑙𝑖∈𝑛and 𝐴0𝑖𝑗(𝑡) ≤0 with 𝐴0𝑖𝑗(𝑡) < 0 for only
one 𝑗∈𝑛for each 𝑖∈𝑛; for each 𝑡∈R0+. for any given 𝜁∈R+ and for all 𝑡(≥𝜁)∈R+ with the second
replacement leading to the replacement below in (80)–(85)
from (67) so as to keep the validity of both theorems under
the modified nonnegative controls (80) and (83), respectively: Proof. Since 𝛼(=k) ∈Z0+, one gets from (55)–(58), (49), and
(83) that ( 𝛾
𝑞𝑘)
𝑁+1 (𝑘+ 1) (‖𝑥(0)‖ + 𝜀𝑁)
(1 −𝛾) (1 −𝑞)𝑘𝑡𝛼
→2
𝑞𝑁+1
(1 −𝑞)𝑘+1(1 −𝑞𝑁+1)
× sup
𝑖≥𝑁
[
1
(𝑡−𝜁)𝛼(𝑥(𝑞𝑗+𝑖(𝑡−𝜁)), 𝑥(𝑞𝑗+𝑖𝑡))] ;
∀𝑡(≥𝜁) ∈R0+. (87) 𝑥(𝑡) ≥(
1
(1 −𝑞)
𝛼+1 𝑡𝛼𝐼−𝐴0 (𝑡))
−1
× (
𝑝
∑
𝑗=1
𝐴𝑗(𝑡) 𝑥(𝑡−ℎ𝑗) −(𝑃𝑁+ 𝜌𝑁) (1 −𝑞)
𝑘−𝛼
× [
[
̂𝑆𝑁𝑡(̂𝑞𝑁𝑡) + ( 𝛾
𝑞𝑘)
𝑁+1
× (𝑘+ 1) (‖𝑥(0)‖ + 𝜀𝑁)
(1 −𝛾) (1 −𝑞)𝑘𝑡𝛼
×
𝛼
∑
𝑗=0
(−1)𝑗(𝛼
𝑗)
̂𝑒]
]
+𝐵(𝑡) 𝑢(𝑡) ) ≥0
∀𝑡∈R (87) Theorem 15 is extendable for a nondiagonal square or a
rectangular control matrix as follows. × (𝑘+ 1) (‖𝑥(0)‖ + 𝜀𝑁)
(1 −𝛾) (1 −𝑞)𝑘𝑡𝛼
×
𝛼
∑
𝑗=0
(−1)𝑗(𝛼
𝑗)
̂𝑒]
]
+𝐵(𝑡) 𝑢(𝑡) ) ≥0; Theorem 18. Consider (55) with 𝛼∉Z0+ under a piecewise-
continuous control vector function 𝑢
:
R0+
→
R𝑚
0+,
with 𝑚
≤
𝑛, and any set of vector functions of initial
conditions 𝜑𝑗: [−ℎ, 0] →R𝑛
0+, 𝑓𝑜𝑟𝑎𝑙𝑙𝑗∈𝑘−1 ∪{0}. Assume also that 𝐵(𝑡) = (𝑏𝑖𝑗(𝑡)) ∈R𝑛×𝑚
+
has positive rows
𝑏𝑇
𝑖(𝑡)
=
(𝑏𝑖1(𝑡), 𝑏𝑖2(𝑡), . . . , 𝑏𝑖𝑚(𝑡))
∈
R𝑛
+, that the control
components of 𝑢(𝑡) are generated as 𝑢𝑗(𝑡) = 𝜆𝑗(𝑡)𝑚(𝑡), where ∀𝑡∈R0+ ∀𝑡∈R0+ Abstract and Applied Analysis Abstract and Applied Analysis 15 Abstract and Applied Analysis 𝜆𝑗: R0+
→
R0+ and the solution nonnegativity lower-
bounding function 𝑚: R0+
→R+ satisfy the constraints
inf𝑡∈R0+min𝑖∈𝑛(∑𝑚
𝑗=1 𝑏𝑖𝑗(𝑡)𝜆𝑗(𝑡)) > 0, and (i) The solution of (55) is nonnegative and bounded if 𝛼∉
Z0+ and either condition (C1) or condition (C2) of Theorem 15
holds. 4. Nonnegativity of the 𝑞-Solutions of Caputo
Fractional Order 𝛼 If, in addition, 𝑢0(𝑡) →0 as 𝑡→∞then the solution
𝑥: R0+ →R𝑚
0+ of (55) converges asymptotically to 𝑥𝑒= 0
so that (55) is globally asymptotically stable for any control law
and any set of vector functions of initial conditions satisfying
the given constraints. (i) The solution of (55) is nonnegative and bounded if 𝛼∉
Z0+ and either condition (C1) or condition (C2) of Theorem 15
holds. If, in addition, 𝑢0(𝑡) →0 as 𝑡→∞then the solution
𝑥: R0+ →R𝑚
0+ of (55) converges asymptotically to 𝑥𝑒= 0
so that (55) is globally asymptotically stable for any control law
and any set of vector functions of initial conditions satisfying
the given constraints. 𝑚(𝑡) ≥max
𝑖∈𝑛
{
{
{
(
𝑚
∑
𝑗=1
𝑏𝑖𝑗(𝑡) 𝜆𝑗(𝑡))
−1
× ( (𝑃𝑁+ 𝜌𝑁) (1 −𝑞)𝑘−𝛼
× [
[
( 𝛾
𝑞𝑘)
𝑁+1 (𝑘+ 1) (‖𝑥(0)‖ + 𝜀𝑁)
(1 −𝛾) (1 −𝑞)
𝑘𝑡𝛼
×
𝑘
∑
𝑗=0
(−1)𝑗(𝑘
𝑗)
]
]
−
𝑝
∑
𝑗=1
𝑒𝑇
𝑖𝐴𝑗(𝑡) 𝑥(𝑡−ℎ𝑗) −𝑒𝑇
𝑖̂𝑆𝑁𝑡(̂𝑞𝑁𝑡))
}
}
}
∀𝑡∈R0+
(88 g
(ii) Property (i) also holds if 𝛼= 𝑘∈Z0+ and either
condition (C3) or condition (C4) of Theorem 16 holds. g
(ii) Property (i) also holds if 𝛼= 𝑘∈Z0+ and either
condition (C3) or condition (C4) of Theorem 16 holds. Proof. Since rank 𝐵(𝑡) = 𝑛for all 𝑡∈R0+ and, since 𝑚≥𝑛,
the right generalized inverse of 𝐵(𝑡), 𝐵−1
right(𝑡) = (𝐵(𝑡)𝐵𝑇(𝑡))−1
exists for all 𝑡∈R0+ and then (89) is well-posed. One gets
from Theorem 15, (84), after substituting (89) into (81) 𝑥(𝑡) ≥(
𝑃𝑁+ 𝜌𝑁
(1 −𝑞)2𝛼−𝑘+1 𝑡𝛼𝐼−𝐴0 (𝑡))
−1
𝑢0 (𝑡) ≥0;
∀𝑡∈R0+
(91) (91) ∀𝑡∈R0+
(88) ∀𝑡∈R0+
(88) ∀𝑡∈R0+ for some 𝑁∈Z+ and some nonnegative strictly decreasing
sequence {𝜀𝑛}. Then, the solution of (55) is nonnegative under
a nonnegative real control if either (C1) or (C2) of Theorem 15
holds. since 𝑢0 : R0+ →R𝑚
0+ and either condition (C1) or condition
(C2) of Theorem 15 holds. Then, 𝑥: R0+ →R𝑛
0+ and it is
bounded since 𝑢0 : R0+ →R𝑚
0+ is bounded. Note also that
a similar upper-bounding expression for 𝑥(𝑡) can be got by
rearranging signs in (81) resulting to be since 𝑢0 : R0+ →R𝑚
0+ and either condition (C1) or condition
(C2) of Theorem 15 holds. Then, 𝑥: R0+ →R𝑛
0+ and it is
bounded since 𝑢0 : R0+ →R𝑚
0+ is bounded. 4. Nonnegativity of the 𝑞-Solutions of Caputo
Fractional Order 𝛼 Note also that
a similar upper-bounding expression for 𝑥(𝑡) can be got by
rearranging signs in (81) resulting to be The proof of Theorem 18 is direct and then omitted. Close
extensions to Theorem 18 for Theorem 16 (𝛼= 𝑘∈Z0+) and
for Remark 17 (the Caputo fractional derivative operator is
defined on finite sliding intervals) can be directly made for a
nondiagonal square or a rectangular control matrix. Theorem 19. Consider (55) under any set of bounded vector
functions of initial conditions 𝜑𝑗: [−ℎ, 0] →R𝑛
0+, 𝑓𝑜𝑟𝑎𝑙𝑙𝑗∈
𝑘−1 ∪{0}. Assume also that 𝐵(𝑡) ∈R𝑛×𝑚
+
, with 𝑚≥𝑛and
rank 𝐵(𝑡) = 𝑛, 𝑓𝑜𝑟𝑎𝑙𝑙𝑡∈R0+, and that the following control
law is used for some given bounded vector function 𝑢0 : R0+ →
R𝑚
0+: 𝑥(𝑡) ≤(
𝑃𝑁+ 𝜌𝑁
(1 −𝑞)
2𝛼−𝑘+1 𝑡𝛼𝐼−𝐴0 (𝑡))
−1
× (
𝑝
∑
𝑗=1
𝐴𝑗(𝑡) 𝑥(𝑡−ℎ𝑗) −(𝑃𝑁+ 𝜌𝑁) (1 −𝑞)
𝑘−𝛼
× [
[
̂𝑆𝑁𝑡(̂𝑞𝑁𝑡) −( 𝛾
𝑞𝑘)
𝑁+1 𝑢(𝑡) = 𝐵𝑇(𝑡) (𝐵(𝑡) 𝐵𝑇(𝑡))
−1 𝑢1 (𝑡) ,
(89)
𝑢1 (𝑡) = 𝑢0 (𝑡) −
𝑝
∑
𝑗=1
𝐴𝑗(𝑡) 𝑥(𝑡−ℎ𝑗)
+ (𝑃𝑁+ 𝜌𝑁) (1 −𝑞)𝑘−𝛼
× [
[
̂𝑆𝑁𝑡(̂𝑞𝑁𝑡) + ( 𝛾
𝑞𝑘)
𝑁+1 (𝑘+ 1) (‖𝑥(0)‖ + 𝜀𝑁)
(1 −𝛾) (1 −𝑞)𝑘𝑡𝛼
×
𝑘
∑
𝑗=0
(−1)𝑗(𝑘
𝑗)
̂𝑒]
]
. (90) (89) + (𝑃𝑁+ 𝜌𝑁) (1 −𝑞)𝑘−𝛼
× [
[
̂𝑆𝑁𝑡(̂𝑞𝑁𝑡) + ( 𝛾
𝑞𝑘)
𝑁+1 (𝑘+ 1) (‖𝑥(0)‖ + 𝜀𝑁)
(1 −𝛾) (1 −𝑞)𝑘𝑡𝛼
×
𝑘
∑
𝑗=0
(−1)𝑗(𝑘
𝑗)
̂𝑒]
]
. (90) × (𝑘+ 1) (‖𝑥(0)‖ + 𝜀𝑁)
(1 −𝛾) (1 −𝑞)𝑘𝑡𝛼
×
𝑘
∑
𝑗=0
(−1)𝑗(𝑘
𝑗)
̂𝑒]
]
+ 𝐵(𝑡) 𝑢(𝑡) ) ≥0; × (𝑘+ 1) (‖𝑥(0)‖ + 𝜀𝑁)
(1 −𝛾) (1 −𝑞)𝑘𝑡𝛼 ×
𝑘
∑
𝑗=0
(−1)𝑗(𝑘
𝑗)
̂𝑒]
]
+ 𝐵(𝑡) 𝑢(𝑡) ) ≥0; (90) ∀𝑡∈R0+ Then, the following properties hold. Then, the following properties hold. Then, the following properties hold. (92) Abstract and Applied Analysis 16 𝑘−1 ∪{0}. Assume also that 𝐵(𝑡) ∈R𝑛×𝑚
+
, with 𝑚≥𝑛and
rank 𝐵(𝑡) = 𝑛, 𝑓𝑜𝑟𝑎𝑙𝑙𝑡∈R0+.Then, the following properties
hold. 𝑘−1 ∪{0}. Assume also that 𝐵(𝑡) ∈R𝑛×𝑚
+
, with 𝑚≥𝑛and
rank 𝐵(𝑡) = 𝑛, 𝑓𝑜𝑟𝑎𝑙𝑙𝑡∈R0+.Then, the following properties
hold. 4. Nonnegativity of the 𝑞-Solutions of Caputo
Fractional Order 𝛼 so that 0 ≤(
1
(1 −𝑞)𝛼+1 𝑡𝛼𝐼−𝐴0 (𝑡))
−1
𝑢0 (𝑡) ≤𝑥(𝑡)
≤(
1
(1 −𝑞)
𝛼+1 𝑡𝛼𝐼−𝐴0 (𝑡))
−1
× (𝑢0 (𝑡) + ( 𝛾
𝑞𝑘)
𝑁+1 (𝑘+ 1) (‖𝑥(0)‖ + 𝜀𝑁)
(1 −𝛾) (1 −𝑞)𝑘𝑡𝛼
×
𝑘
∑
𝑗=0
(−1)𝑗(𝑘
𝑗)
̂𝑒) . (i) If 𝛼∉Z0+ and the control law (89)-(90) is replaced
with (89) and (96) then under either condition (C1) or
condition (C2) of Theorem 15 𝑥(𝑡) ≥(
(𝑃𝑁+ 𝜌𝑁)
(1 −𝑞)2𝛼−𝑘+1 𝑡𝛼𝐼−𝐴0 (𝑡))
−1
× (𝑢0 (𝑡) + ( 𝛾
𝑞𝑘)
𝑁+1 (𝑘+ 1) (‖𝑥(0)‖ + 𝜀𝑁)
(1 −𝛾) (1 −𝑞)
𝑘
×
𝑘
∑
𝑗=0
(−1)𝑗(𝑘
𝑗)
( 1
𝑡𝛼
0
−1
𝑡𝛼) ̂𝑒) ≥0;
(97) (93) (93) (97) Thus, if 𝑢0(𝑡) →0 as 𝑡→∞then 𝑥(𝑡) →0 as 𝑡→∞
since (𝛾/𝑞𝑘)
𝑁+1(𝑘+ 1)(‖𝑥(0)‖ + 𝜀𝑁)/(1 −𝛾)(1 −𝑞)𝑘𝑡𝛼→0
as 𝑡→∞and Property (i) has been proved. Property (ii) is
similar to the solution satisfying ∀𝑡∈R0+, lim inf
𝑡→∞(𝑥(𝑡) −𝐴−1
0 (𝑡) (𝑢0 (𝑡) + 𝜀̂𝑒)) ≥0
(98) lim inf
𝑡→∞(𝑥(𝑡) −𝐴−1
0 (𝑡) (𝑢0 (𝑡) + 𝜀̂𝑒)) ≥0
(98) (98) 𝑥(𝑡) ≥(
1
(1 −𝑞)𝛼+1 𝑡𝛼𝐼−𝐴0 (𝑡))
−1
𝑢0 (𝑡) ≥0;
∀𝑡∈R0+. (94) for any given 𝜀∈R+ and 𝑁∈Z+ and some 𝑡0 =
𝑡0(𝑁, 𝜀) ∈R+. As a result, lim inf𝑡→∞(𝑥(𝑡)−𝐴−1
0 𝜀̂𝑒) ≥
0 if 𝑢0(𝑡) →0 as 𝑡→∞. (94) (94) (ii) If 𝛼= 𝑘∈Z0+ and (90) is replaced with (96) for 𝑘= 𝛼
then under either condition (C3) or condition (C4) of
Theorem 16 Note that (𝛾/𝑞𝑘)
𝑁+1(𝑘+ 1)(‖𝑥(0)‖ + 𝜀𝑁)/(1 −𝛾)(1 −𝑞)𝑘𝑡𝛼
converges to zero as 𝑁→∞and as 𝑡→∞. Thus, for any
given 𝜀∈R+ and 𝑁∈Z+ there is 𝑡0 = 𝑡0(𝑁, 𝜀) ∈R+ such that 𝑥(𝑡) ≥(
1
(1 −𝑞)𝛼+1 𝑡𝛼𝐼−𝐴0 (𝑡))
−1
× (𝑢0 (𝑡) + ( 𝛾
𝑞𝑘)
𝑁+1 (𝑘+ 1) (‖𝑥(0)‖ + 𝜀𝑁)
(1 −𝛾) (1 −𝑞)
𝑘
×
𝑘
∑
𝑗=0
(−1)𝑗(𝑘
𝑗)
( 1
𝑡𝛼
0
−1
𝑡𝛼) ̂𝑒) ≥0;
∀𝑡∈R0+
(99) ( 𝛾
𝑞𝑘)
𝑁+1 (𝑘+ 1) (‖𝑥(0)‖ + 𝜀𝑁)
(1 −𝛾) (1 −𝑞)𝑘𝑡𝛼
𝑘
∑
𝑗=0
(−1)𝑗(𝑘
𝑗)
≤𝜀;
∀𝑡(≥𝑡0) ∈R+. (95) (95) (99) ∀𝑡(≥𝑡0) ∈R+. As a result, if 𝑢1(𝑡) in (90) is replaced with 𝑢1 (𝑡) = 𝑢0 (𝑡) −
𝑝
∑
𝑗=1
𝐴𝑗(𝑡) 𝑥(𝑡−ℎ𝑗) + (𝑃𝑁+ 𝜌𝑁) (1 −𝑞)
𝑘−𝛼 ∀𝑡∈R0+ and (98) holds. × [
[
̂𝑆𝑁𝑡(̂𝑞𝑁𝑡) + ( 𝛾
𝑞𝑘)
𝑁+1
× (𝑘+ 1) (‖𝑥(0)‖ + 𝜀𝑁)
(1 −𝛾) (1 −𝑞)𝑘𝑡𝛼
0
×
𝑘
∑
𝑗=0
(−1)𝑗(𝑘
𝑗)
̂𝑒]
]
. (96) × [
[
̂𝑆𝑁𝑡(̂𝑞𝑁𝑡) + ( 𝛾
𝑞𝑘)
𝑁+1 Example 21. From Theorem 11, (57), one concludes that (55),
under any set of nonnegative set of real vector functions of
initial conditions on [−ℎ, 0] and any admissible nonnegative
control vector function, has a limiting nonnegative 𝑞-solution
of (Caputo) fractional order 𝛼∈Z+ satisfying lim inf
𝑡→∞(𝑥(𝑡) −(
1
(1 −𝑞)𝛼+1 𝑡𝛼𝐼−𝐴0 (𝑡))
−1
× (
𝑝
∑
𝑖=1
𝐴𝑖(𝑡) 𝑥(𝑡−ℎ𝑖) + 𝐵(𝑡) 𝑢(𝑡))) ≥0
(100) ×
𝑘
∑
𝑗=0
(−1)𝑗(𝑘
𝑗)
̂𝑒]
]
. (100) (96) The following result is a consequence of Theorem 19 and
Theorems 15-16 under a control law given by (89) and (96). The following result is a consequence of Theorem 19 and
Theorems 15-16 under a control law given by (89) and (96). if ∃((1/(1 −𝑞)𝛼+1𝑡𝛼)𝐼−𝐴0(𝑡))
−1for all 𝑡∈R0+in order that
the 𝑞-solution of (Caputo) fractional order 𝛼∈Z+ of (55)
exist for 𝑡∈R0+ some of the two sets of constraints below
holds: Corollary 20. Consider (55) under any set of bounded vector
functions of initial conditions 𝜑𝑗: [−ℎ, 0] →R𝑛
0+, 𝑓𝑜𝑟𝑎𝑙𝑙𝑗∈ Abstract and Applied Analysis 17 (1) One has lim inf𝑡→∞(𝐴0(𝑡) −(1/(1 −𝑞)𝛼+1𝑡𝛼)𝐼)
−1 ≤
0, lim inf𝑡→∞𝐴𝑖(𝑡)
≥
0; for all 𝑖
∈
𝑝,
lim inf𝑡→∞𝐵(𝑡) ≥0. The
first
above
condition
implies
∃lim𝑡→∞(−𝐴−1
0 (𝑡)) = −𝐴−1
0∞≥0 so that (−𝐴0∞) is a
monomial matrix which implies that if 𝐴0∞∈𝑀R𝑛×𝑛
then it is diagonal nonsingular with negative diagonal
entries. (1) One has lim inf𝑡→∞(𝐴0(𝑡) −(1/(1 −𝑞)𝛼+1𝑡𝛼)𝐼)
−1 ≤
0, lim inf𝑡→∞𝐴𝑖(𝑡)
≥
0; for all 𝑖
∈
𝑝,
lim inf𝑡→∞𝐵(𝑡) ≥0. The
first
above
condition
implies
∃lim𝑡→∞(−𝐴−1
0 (𝑡)) = −𝐴−1
0∞≥0 so that (−𝐴0∞) is a
monomial matrix which implies that if 𝐴0∞∈𝑀R𝑛×𝑛
then it is diagonal nonsingular with negative diagonal
entries. (𝑡1, 𝑡2, 𝑡3, . . .) such that 𝑡𝑖+1 = 𝑞−𝑚𝑖(𝑡𝑖)𝑡𝑖for all 𝑖∈Z+ and some
given sequence {𝑚𝑖(𝑡𝑖)} of positive integers. (𝑡1, 𝑡2, 𝑡3, . . .) such that 𝑡𝑖+1 = 𝑞−𝑚𝑖(𝑡𝑖)𝑡𝑖for all 𝑖∈Z+ and some
given sequence {𝑚𝑖(𝑡𝑖)} of positive integers. Step 3 (sequence of interval lengths to construct the discrete
solution recursively). Fix a string of interval (nonzero) mea-
sures (𝜁1(𝑡1), 𝜁2(𝑡2), 𝜁3(𝑡3), . . and (98) holds. .), which are the measures of the
computation intervals [𝑡𝑖−𝜁𝑖(𝑡𝑖), 𝑡𝑖], for all 𝑖∈Z+ in (61),
such that 𝜁𝑖+1(𝑡𝑖+1) = 𝑞−𝑗𝑖(𝑡𝑖+1)𝑡𝑖, for all 𝑖∈Z+ for some
sequence {𝑗𝑖(𝑡𝑖+1)}, of nonnegative integers, with 𝑗𝑖(𝑡𝑖+1) ≤
𝑚𝑖(𝑡𝑖), satisfying 1 ≤𝑞−𝑚𝑖(𝑡𝑖) −𝑞−𝑗𝑖(𝑡𝑖+1), for all 𝑖∈Z+ so as
to guarantee the algorithm running constraints: (2) One
has
∃lim𝑡→∞(−𝐴−1
0 (𝑡))
=
−𝐴−1
0∞,
−𝐴−1
0∞(lim inf𝑡→∞𝐴𝑖(𝑡))
≥
0; for all 𝑖
∈
𝑝,
−𝐴−1
0∞(lim inf𝑡→∞𝐵(𝑡)) ≥0. (2) One
has
∃lim𝑡→∞(−𝐴−1
0 (𝑡))
=
−𝐴−1
0∞,
−𝐴−1
0∞(lim inf𝑡→∞𝐴𝑖(𝑡))
≥
0; for all 𝑖
∈
𝑝,
−𝐴−1
0∞(lim inf𝑡→∞𝐵(𝑡)) ≥0. 𝑡𝑖≤(𝑞−𝑚𝑖(𝑡𝑖) −𝑞−𝑗𝑖(𝑡𝑖+1)) 𝑡𝑖< 𝑡𝑖+1 = 𝑞−𝑚𝑖(𝑡𝑖)𝑡𝑖;
∀𝑖∈Z+. (101) (101) Note that it has been used that for 𝛼> 0, ̂𝑆𝑁(̂𝑞𝑁𝑡) →0,
̃𝑆𝑁(̂𝑞𝑁𝑡) →0 as 𝑡→∞, for all 𝑁∈Z0+ from (47)-(48). If 𝛼= 0 then the existence of 𝐴−1
0∞is not required
and the first constraint of each of the above sets is changed
to the existence of lim inf𝑡→∞(𝐴0(𝑡) −(1 −𝑞)−1𝐼)−1
≤
0, that is, lim sup𝑡→∞𝐴0(𝑡)
≤
(1 −𝑞)−1𝐼provided that
lim sup𝑡→∞‖𝐴0(𝑡)‖2 < (1 −𝑞)−1 to guarantee the nonsingu-
larity of (𝐴0(𝑡) −(1 −𝑞)−1𝐼). Acknowledgments y
0
𝑞
If 𝛼∉Z0+ and 𝑘= [𝛼] + 1 then the above constraints
have to be amended via (56) with the replacement ((1/(1 −
𝑞)𝛼+1𝑡𝛼)𝐼) →((𝑃∗/(1 −𝑞)2𝛼−k+1𝑡𝛼)𝐼). The author is very grateful to the Spanish Government for
its support through Grant DPI2012-30651 and to the Basque
Government for its support through Grants IT378-10 and
SAIOTEK S-PE13UN039. He is also grateful to the University
of Basque Country for its support through Grant UFI 2011/07
and to the referees for their useful comments on the first
version of the paper. The author is very grateful to the Spanish Government for
its support through Grant DPI2012-30651 and to the Basque Government for its support through Grants IT378-10 and
SAIOTEK S-PE13UN039. He is also grateful to the University
of Basque Country for its support through Grant UFI 2011/07
and to the referees for their useful comments on the first
version of the paper. On the other hand, note that the various above results and
properties given in the above remarks within this section lead
directly to sufficiency-type conditions for the nonnegativity
of the quantum fractional solutions and their limit values
under the proposed control laws. Remark 22. If the 𝑞-Caputo fractional differential system
(61) is considered instead of (55), then Theorem 19 and
Corollary 20 hold under modified equations (90), (92),
(93), (95), and (96) being subject to replacements (87) and
̂𝑆𝑁𝑡(̃𝑞𝑁𝑡) →(̂𝑆𝑁(𝑡−𝜁)(̃𝑞𝑁(𝑡−𝜁)) −̂𝑆𝑁𝑡(̃𝑞𝑁𝑡)) for any given
𝜁∈R+ and for all 𝑡(≥𝜁) ∈R+(see Remark 17). On the
other hand, modified equations (97) and (99) remain valid
under the above replacements together with the additional
replacements 1/𝑡𝛼
0
→
(1/𝑡𝛼
0 −1/(𝑡0 −𝜁)𝛼) and 1/𝑡𝛼
→
(1/𝑡𝛼−1/(𝑡−𝜁)𝛼). A variant of the disposal can be made with
the replacement of 𝜁with a function 𝜁: R0+ →R+ with
𝜁(𝑡) ≤𝑡, for all 𝑡∈R0+. Conflict of Interests The author declares that there is no conflict of interests
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https://openalex.org/W2071646533
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https://hal.science/hal-00912399/document
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English
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Caterpillars and Fungal Pathogens: Two Co-Occurring Parasites of an Ant-Plant Mutualism
|
PloS one
| 2,011
|
cc-by
| 8,254
|
To cite this version: Olivier Roux, Régis Céréghino, Pascal J. Solano, Alain Dejean. Caterpillars and fungal pathogens: two
co-occurring parasites of an ant-plant mutualism. PLoS ONE, 2011, vol. 6 (5), pp. 1-8. 10.1371/jour-
nal.pone.0020538. hal-00912399 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-00912399
https://hal.science/hal-00912399v1
Submitted on 2 Dec 2013 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés. HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
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. Distributed under a Creative Commons Attribution 4.0 International License Abstrad ln mutualisms, each interacting species obtains resources from its partner that it would obtain less efficiently if alone, and so
derives a net fitness benefit. ln exchange for shelter (domatia) and food, mutualistic plant-ants protect their host
myrmecophytes from herbivores, encroaching vines and fungal pathogens. Although selective filters enable
myrmecophytes to host those a nt species most favorable to their fitness, sorne insects can by-pass these filters, exploiting
the rewards supplied whilst providing nothing in return. This is the case in French Guiana for Cecropia obtusa (Cecropiaceae)
as Pseudocabima guianalis caterpillars (Lepidoptera, Pyralidae) can colonize saplings before the installation of their
mutualistic Azteca ants. The caterpillars shelter in the domatia and feed on food bodies (FBs) whose production increases as
a result. They delay colonization by ants by weaving a silk shield above the youngest trichilium, where the FBs are produced,
blocking access to them. This probable temporal priority effect also allows female moths to lay new eggs on trees that
already shelter caterpillars, and so to occupy the niche longer and exploit Cecropia resources before colonization by ants. However, once incipient a nt colonies are able to develop, they prevent further colonization by the caterpillars. Although no
higher herbivory rates were noted, these caterpillars are ineffective in protecting their host trees from a pathogenic fungus,
Fusarium moniliforme (Deuteromycetes), that develops on the trichilium in the absence of mutualistic ants. Therefore, the
Cecropia treelets can be parasitized by two often overlooked species: the caterpillars that shelter in the domatia and feed on
FBs, delaying colonization by mutualistic ants, and the fungal pathogen that develops on old trichilia. The cost of greater FB
production plus the presence of the pathogenic fungus likely affect tree growth. • E-mail: olivier.roux@ird.fr driven by chemical compounds [13--16]; however ta enter into the
domatia of certain myrmecophyte species, founding ant queens
must be the right size or be able ta recognize and ta gnaw an
entrance hale into the prostomata or thinner area, generally devoid
ofvessels [10],[17-19]. Caterpillars and Fungal Pathogens: Two Co-Occurring
Parasites of an Ant-Piant Mutualism Olivier Roux1*, Régis Céréghino2'3, Pascal J. Solano4 , Alain Dejean1'3 1 CNRS, Écologie des Forêts de Guyane (UMR-CNRS 8172), Campus Agronomique, Kourou, France, 2 CNRS, UMR 5245, Ecolab (Laboratoire d'E
Toulouse, France, 3 Université de Toulouse, UPS, Toulouse, France, 4Aniane, France Open Archive TOULOUSE Archive Ouverte (OATAO)
OATAO is an open access repository that collects the work of Toulouse researchers and
makes it freely available over the web where possible.
This is an author-deposited version published in : http://oatao.univ-toulouse.fr/
Eprints ID : 10156
To link to this article : DOI:10.1371/journal.pone.0020538
URL : http://dx.doi.org/10.1371/journal.pone.0020538
o cite this version : Moreau, Corrie S. and Roux, Olivier and Céréghino, Régis and Solano, Pascal
and Dejean, Alain. Caterpillars and fungal pathogens: two co-occurring parasites of an ant-plant
mutualism. (2011) PLoS ONE, vol. 6 (n° 5). pp. 1-8. ISSN 1932-6203 Open Archive TOULOUSE Archive Ouverte (OATAO)
OATAO is an open access repository that collects the work of Toulouse researchers and
makes it freely available over the web where possible. To link to this article : DOI:10.1371/journal.pone.0020538
URL : http://dx.doi.org/10.1371/journal.pone.0020538 To cite this version : Moreau, Corrie S. and Roux, Olivier and Céréghino, Régis and Solano, Pascal
J. and Dejean, Alain. Caterpillars and fungal pathogens: two co-occurring parasites of an ant-plant
mutualism. (2011) PLoS ONE, vol. 6 (n° 5). pp. 1-8. ISSN 1932-6203 Any correspondance concerning this service should be sent to the repository
administrator: staff-oatao@listes-diff.inp-toulouse.fr Introduction Mutualisms are interspecific interactions involving two or more
species where each partner obtains resources that it would obtain
less efficiently if alone, and so derives a net fitness benefit [1-3]. These mutualistic partnerships are transmitted from one genera-
tion to the next in one of two ways. ln vertical transmission, hosts
transmit symbiont offspring directly to their own offspring [4],[5],
while in horizontal transmission the partners need ta renew their
association after each reproductive event [3], [6]. Biotic pollina-
tion, seed dispersal by animais, ant-plant associations and
interactions between rhizobia or mycorrhiza and plant roots are
transmitted horizontally and can be mutualistic [4],[6--9]. Nevertheless, these mechanisms do not keep the mutualism
between myrmecophytes and plant-ants free from conflict,
competition and/or exploitation by other ants or by non-ant
species. ln this context of competition for resources, the abilities of
species are generally unequal, leading ta hierarchically-organized
systems with dominant and subordinate species. Ta survive, weaker
competitors must develop colonization strategies, be resistant ta
perturbations, manage with fewer resources or have good longevity
with the aim of conserving their access ta the "niche" [20--22]. One
alternative way ta obtain an advantage over a better competitor is ta
be the frrst ta obtain access ta resources and ta monopolize them. Often this advantage allows poor competitors ta persist longer in
habitats than they would otherwise [23],[24]. This phenomenon is
known as "temporal priority" and has been documented in many
taxa such as mycorrhizal fungi [25-27], plants [28],[29], amphib-
ians [24], [30] and insects [23], [31]. Myrmecophytes (orant-plants) are involved in mutualisms with a
limited number of so-called plant-ants that they shelter in domatia
(i.e. hollow branches or thorns and leaf pouches) and usually provide
with food through extra-floral nectaries (EFNs) and/or food bodies
(FBs). ln retum, plant-ants protect their host plant from herbivores,
competitors, encroaching vines and fungal pathogens [1 0]. Because
the transmission of ant-plant mutualisms is horizontal, myrmeco-
phytes have evolved severa! types of selective fllters enabling them ta
host those ant species most favorable ta their fitness [11],[12]. Host-
plant selection by founding ant queens, for example, seems ta be It is well known that mutualistic plant-ant species compete for
their host-plant [14],[32-34]. Moreover, mutualistic ants are not the only ones competing for this resource. lndeed, parasites of
ant-myrmecophyte mutualisms-mostly ant species-are able to
colonize the myrmecophytes, but do not provide them with
protection [8], [9]. The caterpillars' diet To assess if the caterpillars feed exclusively on the FBs, in 2001
and 2002, we selected 83 saplings on which we had found at least Figure 1. Trlchlllum of Cecropia. A- Azteca affari worlœr.; removing
food bodies (arrow) from a Cecropia obtusa trichilium. 8- Pseudocabima
guianalis caterpillars occupying the upper part of a C obtusa. They
gnawed an entrance hole into the prostomata (p) in order to enter into
the hollow internodes. Secreted strands of silk caver the trichilium and
keep the witherecl stipules (DS) from falling. C- Trichilium of C obtusa
infested by Fusarium moniliforme. D- Penetration by a filament of
F. moniliforme (arrows) inside a food body (FB). E-F. moniliforme
spreading throughout a food body (FB and black arrows). The cells of
the trichilium at the base of the FB seem to react to the presence of
fungus (white arrow). The myrmecophyte CemJjlia obtura (Cecropiacae), the focal
species of this study, is mutualistically associated with several A,eteca
species (Dolichoderinae) whose founding queens and workers
recognize the zone where the prostomata is situated and so
establish colonies in the internodal domatia [13],[40]. In addition
to shelter, the plant provides the ,Azteea colonies with food in the
form of g1ycogen-rich Müllerian bodies produced by the trichilia,
pads of dense trichomes situated at the base of the leaf petiole, and
lipid-rich pearl bodies produced beneath young leaves. Mutualistic
A,eteca workers generally protect their host trees from defoliating
insects, encroaching vines and fungal pathogens [17], [41-43], but
this is not the case for saplings [44],[45]. Because the transmission of the A;;.teca--Cecropia mutualism is
horizontal, the size of the internodes plus the production rate of
the food bodies do not permit A,eteca colonies to develop before the
saplings reach ca. 1 rn in height [44],[46]. Before Ctcropio. saplings
reach this minimwn size, herbivores and parasites may use this
absence of mutualistic plant-ants to opportunistically tak.e over the
Cecropia's resources; whereas, after this period of rime, potential
invaders must overcome the biotic defenses conferred by plant-
ants [44]. We noted that recently-perturbed areas are rapidly occupied by
thousands of C. obtusa saplings, permitting sorne caterpillars to live
in the domatia and feed on the FBs, and that caterpillar presence
was associated with the development of a fungus on the old
trichilia. Introduction This was fust shawn for Pseudmnyrmex
nigropiiosur that colonizes myrmecophytic Aca&ia and consumes
their EF'Ns and FBs, but exhibits no defensive behavior [35]. Sorne non-ant insects are also able to colonize and parasitize
myrmecophytes, benefiting from the shelter and food provided by
the plant in different ways; for example, the larvae of the clerid
beetle Phyllobamu.s sp. parasitize myrmecophytic Pip" trees,
feeding on bath the FBs and on mutualistic ants. The fitness of
the host trees is reduced due ta a greater investment in FB
production and a decrease in biotic protection by the guest ants,
with a subsequent increase in herbivory [36]. Also, females of the
chrysomelid beetle CrMlomera sp. open an entrance hole in the
prostomata of Curopia trees in the same manner as mutualistic
A,eteca ants. They then lay eggs in the domatia and the larva.e feed
on young leaves [3 7]. Ethics Statement This study was conducted according to relevant national and
international guidelines. Study sites We conducted this study between 2000 and 2009 in French
Guiana near the Petit Saut dam (5°03'39n N-53°02'36"W) and near
the Montagne rks singes (5°04'19.6"N-52°41'42.5"W). We selected
and tagged C. obtura that were ca. 1.15 rn to 1.30 rn tall (N = 61 0)
between 2000 and 2006 in a cleared 1.5 ha zone situated near the
dam, and on plants growing along a straight, recently-opened dirt
raad near the Montagne rks singes (N = 64) between 2006 and 2009. In these areas, C. obtura is mostly associated with A;;.teca afjari and
A. o'IJO.ticeps whose colonies exploit Müllerian bodies (Fig. lA), tend
hemipterans in the host tree domatia and prey on insects landing
on the leaves [42]. These insects, often less numerous and less aggressive than
ants, must find weaknesses in the mutualism to be able to colonize
their host tree and exploit its resources. Using chernical mirnicry
or camouflage to counter ant aggressiveness is one solution for
getting past ant defenses [38],[39]; however, being the fll'St to
arrive to colonize new treelets might also represent a serious
advantage because the biotic defense provided by mutualistic
plant-ants is not yet in place and nearly leaves the plant without
indirect deferues. Fungal infestation of the trichilia one caterpillar. Preliminary studies conducted during a 24-hour
period permitted us to leam that FBs are produced during the
afternoon, between 15:00 and dusk (see also [47-50]), and that
caterpillars are active during FB production. During a more
comprehensive survey, we observed their behavior for 5 minutes
per sapling in the morning between 8:00 and 11 :00 as weil as
during FB production in the aftemoon between 15:00 and 18:00. Observations were also made at night between 21:00 and 5:00 to
ensure that the caterpillars were not active noctumally. Between 2003 and 2005, we recorded the number of trees
whose trichilia had a fungal infestation out of 610 C. obtusa saplings
sheltering an Azteca colony (N = 349), caterpillars (N = 83), or not
occupied (N = 178). We scored the presence versus absence of
fungal infestation on the trees as "!" and "0", respectively, and
compared the results using the Kruskal-Wallis test and Dunn's
post-hoc test. To analyze how ant or caterpillar presence affected fungal
development, we took samples of 20 trichilia with and 20 without
developed mycelium from C. obtusa in ali cases (i.e. trichilia taken
from trees sheltering ants, caterpillars or unoccupied) and
cultivated the mycelium in aseptic conditions in Sabouraud's
nutritive substrate (N = 120 trichilia). We analyzed these samples
under a microscope to verifY how the mycelium develops on the
trichilia. We frrst fixed the sampled trichilia with FAA (formalin,
acetic acid, alcohol), and then embedded them in paraffin. We
stained tissue sections with basic fuchsin light -green or toluidine
blue contrasted with sodium molybdate. Food body production, herbivory, ant and caterpillar
presence and tree growth To quantify FB production, in 2003 we selected 30 saplings,
eacb bearing at !east five leaves: 10 sheltered A. a!fari, 10 sheltered
caterpillars and the remaining 10 were unoccupied. Because FB
production increases when they are removed by ants or
experimentally [1 7], we conducted experiments where bath ants
and caterpillars were prevented access to the upper zone of the
trees where most of the FB production occurs. We did this by
placing a ring of Tanglefoot® around the trunk under the
lowermost leaf to isola te the top of each sapling, and th us prevent
the ants and caterpillars from having access to the leaves and to
the FBs. We then plugged the entrance hales situated in this upper,
isolated section with a spot of Tanglefoot®. The ants and
caterpillars could, nevertheless, move freely in and out of the
domatia using the lower entrance hales giving them access to the
lower part of the trunk and to aider leaves with inactive trichilia. We placed aluminum foi! shelters around the isolated trichilia to
protect them from flying insects and to gather the FBs that
dropped off [48],[51]. For each sapling and during 20 days, at ca. 19:00, we removed the FBs produced that day and that had fallen
from the !east mature trichilia onto the aluminum foi!, and
counted them. We compared the results using a repeated measures
ANOVA followed by a Newman-Keuls' post-hoc test for multiple
comparisons (GraphPad Prism 4.03 software). Voucher specimens of the adult maths obtained after the
metamorphosis of the caterpillars were identified as Pseudocabima
guianalis (Lepidoptera, Pyralidae, Phyticinae) and were deposited at
the Systematic Entomology Laboratory of the United States
Department of Agriculture (USDA), Beltsville, Maryland. Fungal
samples were identified as Fusarium moniliforme (Deuteromycetes)
and were deposited at the Laboratoire de biologie et taxonomie des
microchampignons, Muséum National d'Histoire Naturelle, Paris, France. The caterpillars Observations made on the 83 saplings sheltering caterpillars
permitted us to note that the frrst instar caterpillars lived under a
silk shelter that they built between the stipules developing around
the terminal bud, the trunk and the youngest leaf. They only left
this shelter between 15:00 and 18:00 to feed on the FBs produced
daily during that rime period by the youngest trichilia, or
sometimes by the other tricbilia. As the shoot grows, the stipules,
which normally drop off, are trapped by the silk (Fig. lB; Fig. S 1 a). From their third instar, ca. 1.5-cm-long caterpillars, like ants,
gnawed the prostomata in arder to shelter in the last internode. They wove a silk shield above the upper part of the tree trunk and
the youngest trichilium (Fig. lB, Fig. Si b-d), and left the domatia
only to feed on the FBs under the shelter of strands of silk. Pupation occurred inside the domatia. Larval and pupal
development took about 30 days. Just-emerging maths leave the
trunk by flying out through the stomata that are widened by the
caterpillars when they are in their last larval stage. We also tested, in 2003-2004, if the presence of ants and
caterpillars affected the presence of defoliating insects by scoring
the amount of herbivory on the oldest leaf on 90 saplings
(30 sheltered A. a!fari, 30 sheltered caterpillars, and the 30 others
were unoccupied), each bearing at !east five leaves. We chose the
oldest leaves because they provide an idea of the history of the
defoliation over the preceding ca. 18 months which corresponds to
the lifespan of C. obtusa leaves [42]. We evaluated the percentage of
foliar surface eaten by insects (FSE) using the following scale:
(!) leafintact; (2) slightly attacked: O%<FSE:=;25%; (3) somewhat
attacked: 25%<FSEo=;50%; (4) very attacked: 50%<FSEo=;75%;
and (5) extremely attacked: FSE>75%. We compared the results
using the Kruskal-Wallis test. The caterpillars' diet To broaden our understanding of the biological
interactions and coexistence of these caterpillars within the
A,eteca-CemJjlia mutualism, we conducted a correlation study where
we posed the following questions. (1) Do caterpillars mainly or
even exclusively feed on the FBs and, if so, does this activity
increase FB production as is known for mutualistic plant-ants [36]? (2) Is caterpillar presence associated with the greater herbivory of
Cecropia saplings? (3) Does this presence favor fungal development
on the trichilia with deleterious consequences for the plant? (4)
Can A;;.teca ants prevent colonization by caterpillars, or, inversely
can caterpillars delay or even prevent colonization by ants thus
allowing them to exploit Cecropia saplings longer? Figure 1. Trlchlllum of Cecropia. A- Azteca affari worlœr.; removing
food bodies (arrow) from a Cecropia obtusa trichilium. 8- Pseudocabima
guianalis caterpillars occupying the upper part of a C obtusa. They
gnawed an entrance hole into the prostomata (p) in order to enter into
the hollow internodes. Secreted strands of silk caver the trichilium and
keep the witherecl stipules (DS) from falling. C- Trichilium of C obtusa
infested by Fusarium moniliforme. D- Penetration by a filament of
F. moniliforme (arrows) inside a food body (FB). E-F. moniliforme
spreading throughout a food body (FB and black arrows). The cells of
the trichilium at the base of the FB seem to react to the presence of
fungus (white arrow). Figure 1. Trlchlllum of Cecropia. A- Azteca affari worlœr.; removing
food bodies (arrow) from a Cecropia obtusa trichilium. 8- Pseudocabima
guianalis caterpillars occupying the upper part of a C obtusa. They
gnawed an entrance hole into the prostomata (p) in order to enter into
the hollow internodes. Secreted strands of silk caver the trichilium and
keep the witherecl stipules (DS) from falling. C- Trichilium of C obtusa
infested by Fusarium moniliforme. D- Penetration by a filament of
F. moniliforme (arrows) inside a food body (FB). E-F. moniliforme
spreading throughout a food body (FB and black arrows). The cells of
the trichilium at the base of the FB seem to react to the presence of
fungus (white arrow). Fungal infestation of the trichilia Fungal presence To verifY how caterpillars can delay or even prevent ant
colonization and vice versa, every 8 months betweenjune 2006 and
June 2008, we noted which ant species or if caterpillars sheltered in
the domatia and fed on the FBs on the 64 C. obtusa situated near
the Montagne des singes. An additional survey was conducted injuly
2009. lnjuly 2008, we measured the height of the trees that had
sheltered (!) Azteca colonies during the experimental period
(A. ovaticeps: N = 1 0; A. a!fari: N = 22), (2) neither ants nor
caterpillars (N = 1 0), or (3) caterpillars during the en tire experi-
mental period or that had been replaced by an Azteca colony only
during the last part of the experimental period (N = 14). Ali of
these trees are the same age as they developed just after the dirt
raad was built near the Montagne des singes and have a similar
exposure to the sun and to rain. We compared the results using an
ANOVA and Newman-Keuls' post-hoc test. Fusarium moniliforme was present on 323 of the 610 C. obtusa
saplings (53.0%), sometimes completely covering the trichilia
(Fig. 1 C). The percentage of infested individuals was significantly
lower among saplings sheltering an Azteca colony than those
sheltering P. guianalis caterpillars or not occupied, while the
difference between the latter two cases was not significant (Fig. 2). Normally, FBs are sub-spherical and homogeneous with reserve
cells bordered by a cell wall and a thin cuticle. The penetration of
the F. moniliforme mycelium into an FB occurs once it is already
formed, but not necessarily completely developed. ln Figure ID, a
filament of F. moniliforme can be seen entering into an FB. The
bordering cells are in necrosis, as are the frrst reserve cells. The
trichomes around the FB react strongly to the presence of the
fungus, saturating their cell walls with lignin (Fig. lE); whereas the :ll! 100
b
b
E
0
90
E Vl
80
Q) :::J
Vl Cl
0 c:
70
.s:::
:::J
;:->-
60
Vl.O
l? -c
= Q)
50
a. -"'
co u
Vlj!! 40
oro
83
178
Q)
Q)
30
ｲｮｾ＠co
Q)
ë ;: 20
Q)
ｾ＠
10
Q)
Il.. 0
a
1
349
1
Azteca
Caterpillars
Unoccupied
Figure 2. Trlchlllum lnfested by Fusarium monilifomle. Fungal presence Percentages of Cecropia obtusa saplings whose trichilia were attacked by Fusarium
moniliforme in three situations: saplings sheltering an Azteca colony, saplings sheltering Pseudocabima guianalis caterpillars, and unoccupied saplings
(N = number of saplings in each case). Statistical comparisons; Kruskai-Wallis test; H3
610 = 338.1; P<0.001; Dunn's multiple comparison test, different
letters indicate significant differences at P<0.001. :ll! 100
b
b
E
0
90
E Vl
80
Q) :::J
Vl Cl
0 c:
70
.s:::
:::J
;:->-
60
Vl.O
l? -c
= Q)
50
a. -"'
co u
Vlj!! 40
oro
83
178
Q)
Q)
30
ｲｮｾ＠co
Q)
ë ;: 20
Q)
ｾ＠
10
Q)
Il.. 0
a
1
349
1
Azteca
Caterpillars
Unoccupied
Figure 2. Trlchlllum lnfested by Fusarium monilifomle. Percentages of Cecropia obtusa saplings whose trichilia were attacked by Fusarium
moniliforme in three situations: saplings sheltering an Azteca colony, saplings sheltering Pseudocabima guianalis caterpillars, and unoccupied saplings
(N = number of saplings in each case). Statistical comparisons; Kruskai-Wallis test; H3
610 = 338.1; P<0.001; Dunn's multiple comparison test, different
letters indicate significant differences at P<0.001. Figure 2. Trlchlllum lnfested by Fusarium monilifomle. Percentages of Cecropia obtusa saplings whose trichilia were attacked by Fusarium
moniliforme in three situations: saplings sheltering an Azteca colony, saplings sheltering Pseudocabima guianalis caterpillars, and unoccupied saplings
(N = number of saplings in each case). Statistical comparisons; Kruskai-Wallis test; H3
610 = 338.1; P<0.001; Dunn's multiple comparison test, different
letters indicate significant differences at P<0.001. FB cells show no reaction either in the cell wall or in the
cytoplasm. Once inside the FB, the mycelium progressively
invades ail of the cells, down to the base of the FB (Fig. lE). The first cells of the inner triclûlium react to the presence of the
mycelium when the FB is highly invaded. They seem to contain
more tannin and are more elongated. of the experiment-so 3 years after the beginning of the survey-six
trees still sheltered caterpillars. During this en tire lapse of time, the
trees occupied by A. a!fari or A. ovatictps were never colonized by
caterpillars. In J uly 2009, four CecropiJJ trees still sheltered
caterpillars. Food body production, herbivory and tree growth We noted a proliferation of the mycelium in in vitro cultures in
Petri dishes with bath healthy and infected trichilia, betraying the
presence of spores in ail cases (i.e. trichilia taken from trees
sheltering ants, caterpillars or unoccupied). Ant and caterpillar occupancy of Cecropia obtusa trees Ant and caterpillar occupancy of Cecropia obtusa trees
Out of the 610 C. obtusa saplings studied near the Petit ｳｾｴ＠
dam,
only 349 (57 .2%) sheltered A. alfori or A. ｯｶ｡ｴｩ｣ｾｳ＠
colonies. Among
the others, 178 (29.2%) were totally unoccupied, while the
remaining 83 (13.6%) sheltered three to six P. guimullis caterpillars
at different larval stages (Fig. Sic). For the 64 C. obtusa surveyed
during 3 years near the MonltJgne lhs singes, at the start of the survey
the perœntage of C. obtusa sheltering P. gu.ianoJis caterpillars was by
far superior (39.1% or 25 trees out of 64; Fig. 3), illustrating that
there are variations between areas. When present, caterpillars
were also more numerous with some trees sheltering up to 12
caterpillars. Saplings were also associated with the two Curopio-ant
species typical of the area, A. a!fari and A. ovatictps, as weil as,
unexpectedly, the tire ant Solenopsis saevis.rima (tree N"7). Also, six
trees were unoccupied at the start of the survey, and three of the
25 saplings bearing caterpillars were also occupied by A. aljari
(trees N" 24, 43 and 52). This dual hosting was also observed later
in the swvey for two additional trees (trees N" 14 and 41 ), but after
a few months, ail five trees were occupied only by kteca colorùes. Note that, in the end, tree N° 52 was colonized by A. ｯｶ｡ｴｩｴＺｾｳＮ＠
Over the course of the different surveys, unoccupied trees were
colonized by caterpillars (four cases) or directly by Azteca ants (trees
N° 19 and 25). Although S. saevissima workers exploited the FBs
and were aggressive towards flying insects landing on their host
tree foliage, tree N"7 was colonized in the end by caterpillars
(Fig. 3). While the two Az/tca species occupied more and more trees
over time, the number of trees sheltering P. guianalis caterpillars
lirst increased and then decreased. They were replaced by A. aljari
or A. ovaJi.ceps colonies on 15 and 10 trees, respectively. At the end Fungal presence FB production was significantly higher for saplings sheltering
kteca ants than for those sheltering caterpillars and for the latter
compared to unoccupied saplings (Fig. 4). We did not note significant differences in the perœntage of
foliar surface eaten by defoliating insects between the C. obtusa
sheltering an Af;teca colony, caterpillars, or not occupied by either
ants or caterpillars (Kruskal-Wallis test, H 2
90= 1.813; P>0.05). Nevertheless, caterpillar presence a.ffected trec growth as those
sheltering kteca colonies during this experimental period were
significandy taller at the end of the survey than those sheltering
caterpillars or those that were unoccupied (Fig. 5). The differences
were not significant between trees sheltering colonies of the two
kteca species, or between trees sheltering caterpillars or that were
unoccupied. grouped to ensure the leglblllty of the figure. and so do not correspond
to their geographie distribution.
doi:1 0.1371/jou rnal.pone.0020538.g003 Tree
number
1
2
20
54
56
57
58
61
63
64
19
5
18
31
30
33
3
52
59
60
32
15
Surveys
2007
2008
47
Caterpillars
48
1
S. saevissima
49
34
6
14
25
Unoccupied
40
13
42
41
43
24
10
11
12
35
36
26
4
8
9
16
17
21
22
23
27
28
29
37
38
39
44
45
46
50
51
53
55
62
2009
Figure 3. Host successional patterns for Ceaopm treelets. Tree
numbe
1
2
20
54
56
57
58
61
63
64
19
5
18
31
30
33
3
52
59
60
32
15
47
48
1
49
34
6
14
25
40
13
42
41
43 This type of increase in FB production is usually stimulated
when mutualistic ants remove the FBs because the space thus
made available reduces the pressure on the trichilia and favors the
production of the next group of FBs [17], [54]. In the absence of
mutualistic ants, FB production remains law, but is Iùgh enough to
be attractive to founding ant queens [17],[54). Because our
experimental design did not allow Az,teca ants, caterpillars or other
insects access to the trichilia, FB production should have been
reduced to the same level as on unoccupied trees. Y et, this was not
the case, suggesting tbat a factor other than FB removal plays a
role, such as the plant obtaining nutrients from its ant or
caterpillar inhabitants [55]. When parasitic ants are present, the establishment of mutualistic
species is durably prevented, and plant fitness is lessened due to
increased herbivory [8]. The presence of non-ant parasites does
not imply the exclusion of mutualistic ants, but host tree leaves can
suffer herbivory if these insects feed on the plant [3 7] or on plant-
ants that are therefore unable to protect their host trees [36]. However, here, the presence of P. guianalis caterpillars was not
associated with greater herbivory compared to trees sheltering
Azteca colonies or unoccupied trees because saplings rely on
secondary anti-defoliator compounds and structures for their
protection [37],[44], [ 45]. Caterpillars Even though the saplings did not suffer greater herbivory, the
protective mutualism is very disrupted as, when present, Az,teca
colonies significandy limit the development of the Fusarium
mycelium. Indeed, we show that in the absence of mutualistic
ants, Fusarium developed on the trichilia of both unoccupied trees
and trees sheltering caterpillars. Discussion Al! in ail, these results constitute a new step in understanding the
nature of the parasitism of the Azteca-CuropitJ mutualism by non-
ant insects. Like Coelomera chrysomelid beetles [3 7] and mutualistic
ants, P. guianalis caterpillars at their third larval stage recognize the
prostomata and gnaw an entrance hale to shelter in the host tree
domatia. The difference with the damage caused by Coelomera is
that it is direct as they feed on young leaves [3 7], whereas
P. guimullis caterpillars, like mutualistic ants, feed on the FBs
produced by the plant. They are indirectly detrimental ta their
host tree because they allow Fusarium to develop on the triclûlium. Moreover, although it is possible that female maths select the most
productive trees for their offspring [52], the caterpillars seems to
induce an ｩｮ｣ｲ･｡ｾ･＠
in FB production, as do kteca workers and
clerid beede (Phyllobaenus sp.) larvae on Piper [36]. However, unlike
sorne mutualistic ants [53), P. guimullis caterpillars do not provide
any services in return for this increase in FB production. grouped to ensure the leglblllty of the figure. and so do not correspond
to their geographie distribution. doi:1 0.1371/jou rnal.pone.0020538.g003 grouped to ensure the leglblllty of the figure. and so do not correspond
to their geographie distribution. doi:1 0.1371/jou rnal.pone.0020538.g003 grouped to ensure the leglblllty of the figure. and so do not correspond
to their geographie distribution.
doi:1 0.1371/jou rnal.pone.0020538.g003 Moreover, this fungus is known to
produce growth-inhibiting mycotoxines that are also responsible
for necrosis in plants [56],[57] and insects [58],[59]. Consequent-
ly, likely due to the presence of this pathogen plus the cost of
producing FBs, the growth rate of the trees that sheltered
caterpillars during the survey conducted at the Montagne des singes
was affected if compared to those that sheltered Azt«a colonies
during the same period (Fig. 5). Therefore, mutualistic Azt,eca likely control the extent of the
fungal infection in the same way that, by defending myrmeco-
phytic Piper from stem-boring insects, PJWJQk ants reduce fungal
infections [60]. On the other band, when deprived of their
mutualistic Crematogaster ants, myrmecophytic Macaranga suffer
from both shoot borers and pathogenic fungi [61]. lndeed, ants'
antifungal activity is well known [37],[60],[62] and can be due to
chernicals produced by the venom, the metapleural or the
mandibular glands [63--66] or results from the activity of symbiotic
bacteria [6 7]. On the plant side, it bas been noted that sorne
myrmecophyte species have lost their intrinsic physiological
defenses against fungal infection [60],[68]. The spores of Fusarium
can be disseminated by bath wind and insects, particularly
Lepidoptera larvae that are resistant [58], explaining why the
P. guianalis caterpillars were not infected by Fusarium, while the host
plant trichilia were. Because we did not note a difference in the
amount of herbivory between ant-inhabited and ant-free CtcropitJ,
one can hypothesize that Fusarium might be the main selective
driving force in the present situation. In that case, the earlier the
C. obtusa treelets shelter mutualistic Azteca colonies, the more they
will grow due to the antifugal activity of the ants (particularly by
suppressing spore germination [69]). Later, as the trees grow and
their ability to synthesize secondary antiherbivore compounds
lessens, Azteca workers, that belong to larger and larger colonies, Figure 3. Host successional patterns for Ceaopm treelets. Host
successional patterns for each Cecropia sapling monitored during the 3-
year survey on the dirt road near the Montagne des singes. Dashes
correspond to trees shelterlng both caterplllars and A. a/far/. Trees were 140
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Figure 4. Food body production. grouped to ensure the leglblllty of the figure. and so do not correspond
to their geographie distribution.
doi:1 0.1371/jou rnal.pone.0020538.g003 Comparison of the mean food body production per leaf and per day (±SE) by the youngest tricl1ilia on Cecropia
obtusa saplings during 20 successive days in three situations: saplings sheltering an Azteco alfori colony, saplings sheltering Pseudocabima guionolis
caterpillars, and unoccupied saplings (10 individuals in each case). Statistical comparisons; repeated measures ANOVA: F2
30=64.81; P<0.001;
Newman-Keuls' post-hoc test: different letters indicate significant differences at P<0.001. Figure 4. Food body production. Comparison of the mean food body production per leaf and per day (±SE) by the youngest tricl1ilia on Cecropia
obtusa saplings during 20 successive days in three situations: saplings sheltering an Azteco alfori colony, saplings sheltering Pseudocabima guionolis
caterpillars, and unoccupied saplings (10 individuals in each case). Statistical comparisons; repeated measures ANOVA: F2
30=64.81; P<0.001;
Newman-Keuls' post-hoc test: different letters indicate significant differences at P<0.001. When caterpillars do successfully colonize a tree, severa! overlapping generations can be observed-the youngest sheltering
under the stipules developing around the terrrùnal bud, the trunk
and the youngest leaf, and the oldest in the host tree domatia. During the hours of FB production, both young and old
caterpillars share the FBs on the trichilia. Smaller caterpillars
likely benefit from the silk woven above the trichilia by larger
mates as protection from competing ants, predators and/ or
parasitoids which seem repelled, and so do not walk on it {pers. obs.). The overlap between different generations of caterpillars
plus the fact that certain trees can be occupied during severa! years
imply that female P. guian4lis moths lay eggs on trees already
sheltering caterpillars. will take over and provide their host trees with biotic protection
from herbivorous insects [11],[17],[42]. We cannot exclude that the occupancy by caterpillars could be
favored by the plant's characteristics or rrùcro-environmental
conditions rather than by the competitive and colonizing abilities
of the insects. Nevertheless, because C. obtusa is a pioneer species
that develops in large numbers in recently cleared areas, sorne
insect species with a high rate of dispersal can be the flrst to reach
the resources provided by the trees (see [51] for Reduvidae feeding
on C. obtura FBs before the installation of AQ.!ca colonies). It is thus
likely that a temporal priority enabled P. guian4lis caterpillars to
install themselves on certain trees prior to the arrivai of the plant's
mutualistic Azteca ants with which they are involved in competitive
exclusion (see also [23],[24] for a temporal priority concerning
ant-myrmecophyte mutualisms). References 19. Brouat C, Garcia N, Andary C, McKey D (2001) Plant Jock and ant key:
pairwise coevolution of an exclusion fùter in an ant-plant mutualism. Proc R Soc
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2098-2107. g
8. Yu DW (2001) Parasites of mutualisms. Biol] Linn Soc 72: 529-546. 9. Clement LW, Koppen SCW, Brand WA, Heil M (2008) Strategies of a parasite
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interactions among plants, arbuscular mycorrhizal fungi, and fungal endophytes. Oikos 117: 310-320. of the ant-Acacia mutualism. Behav Eco! Sociobiol 62: 953-962. 10. Rico-Gray V, Oliveira P, eds (2007) The ecology and evolution of ant-plant
interactions. Chicago: The University of Chicago press. 331 p. 10. Rico-Gray V, Oliveira P, eds (2007) The ecology and evolutio interactions. Chicago: The University of Chicago press. 331 p. 11
D
id
DW M K
D (1993) Th
l i
l
f 28. Bertness MD, Shumway SW (1993) Competition and facilitation in marsh
plants. Amer Nat 142: 718-724. 11. Davidson DW, McKey D (1993) The evolutionary ecology of symbiotic ani-
plant relationships. J Hymenopt Res 2: 13-83. 29. Ladd B, FacelliJM (2008) Priority effects produced by plant litter result in non-
additive competitive effects. Oecologia 157: 687-ii96. 12. Ward PS (1993) Systematic studies on Pseudomyrmex acacia-ants (Hymenoptera:
Forrnicidae: Pseudomyrmecinae).J Hymenopt Res 2: 117-168. 30. Lawler SP, Morin PJ (1993) Temporal overlap, competition, and priority effects
in larval anurans. Ecology 74: 174--182. 13. Longino JT (1989) Geographie variation and community structure in an ant-
plant mutualism: Al;teca and Cecropia in Costa Rica. Biotropica 21: 126-132. Longino JT (1989) Geographie variation and community structure in an a 31. Hodge S, Arthur W, Mitchell P (1996) Effects of temporal priority on
interspecific interactions and community development. Oikos 76: 350-358. 14. Vasconcelos HL (1993) Ant colonization of Maieta guianensis seedlings, an
Amazon ant-plant. Oecologia 95: 439-443. 32. Davidson DW, Snelling RR, LonginoJT (1989) Competition among ants for
myrmecophytes and the significance of plant trichomes. Biotropica 21: 64--73. 15. Edwards DP, Hassall M, Sutherland ｾＬ＠
Yu DW (2006) Assembling a
mutualism: ant symbionts locale their host plants by detecting volatile chemicals. Insect Soc 53: 172-176. 33. Yu DW, Davidson DW (1997) Experimental studies of species-specificity in
Cecropia-ant relationships. Eco! Mon 67: 273-294. 16. Jurgens A, Feldhaar H, Feldmeyer B, Fiala B (2006) Chemical composition of
leaf volatiles in Macaranga species (Euphorbiaceae) and their potential role as
olfactory eues in host-localization of foundress queens of specifie ant partners. Biochem Syst Eco! 34: 97-113. 34. Fonseca CR, Benson WW (2003) Ontogenetic succession in Amazonian ant
trees. Oikos 102: 407-412. 35. Janzen DH (1975) Pseudomyrmex nigropilosa: a parasite of a mutualism. Science
188: 936-937. 17. Davidson DW (2005) Cecropia and its biotic defenses. In: Berg CC, Ros 17. Acknowledgments We are grateful to the Laboratoire Environnement de Petit Saut for its
logistical help, A. Yockey-Dejean for proof-reading the manuscript, M. A. Solis (USDA, Beltsville, Maryland, USA) and E. Bury (Muséwn National
d'Histoire Naturelle, Paris, France) for identifYing the P. guianalis and F. moniliforme, respectively. We would also like to thank anonyrnous referees
for their helpful comments. Supporting Information Figure Sl Caterpillars on Cecropia treelets. a Upper part
of a young Cecropia obtusa sheltering Pseudocabina guianalis caterpil-
lars. Strands of silk produced by the caterpillars keep the stipules of
two leaves against the trunk (yellow arrow). An entrance hale
gnawed by a caterpillar is visible (white arrow). Note that the
leaves were not attacked by defoliating insects. b A forth instar
caterpillar eating food bodies on the youngest trichilia on a tree,
sorne strands of silk are visible. c Three larval stages eating food
bodies on the same trichilia. d A forth instar caterpillar eating food
bodies on a trichilia that began to be infected by Fusarium
moniliforme. e Severa! caterpillars at different stages on a trichilia,
sorne strands of silk are visible. ln conclusion, P. guianalis caterpillars are able to "break the
code" [36] of the Azteca-Cecropia mutualism by recognizing the
prostomata and exploiting the resources Cecropia normally supplies
to mutualistic Azteca; they even induce greater FB production. Although no higher herbivory rates were noted, these caterpillars
are ineffective in keeping a fungus from developing on the trichilia
of their host trees, something that mutualistic Azteca ants can do. By denying mutualistic ants access to FBs and young leaves,
P. guianalis caterpillars become a more formidable competitor of
mutualistic ants and so are parasites of both Cecropia saplings and
the Azteca-Cecropia mutualism. Author Contributions Conceived and designed the experiments: AD PJS. Performed the
experiments: AD PJS. Analyzed the data: OR RC AD. Contributed
reagents/materials/analysis tools: AD. Wrote the paper: OR AD. Conceived and designed the experiments: AD PJS. Performed the
experiments: AD PJS. Analyzed the data: OR RC AD. Contributed
reagents/materials/analysis tools: AD. Wrote the paper: OR AD. grouped to ensure the leglblllty of the figure. and so do not correspond
to their geographie distribution.
doi:1 0.1371/jou rnal.pone.0020538.g003 When an incipient Azttca colony successfully colonizes their host
tree, large caterpillars seem to deny the first workers access to the 250 -
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0
A. ovaticeps
A. alfari
Caterpillars
b
T
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Unoccupied
Figure 5. Helght of CemJpiatreelets. Mean height of trees (±SE) that sheltered colonies of one of the two Azteca species, Pseudocobima guionolis
caterpillars, or that sheltered neither Azteca nor caterpillars during the experimental period. Statistical comparisons (normality and equal variance
tests passed); ANOVA: F2
56,8.56; P<0.0001. Newman-Keuls' post-hoc test: different letters lndlcate slgnlflcant differences at P< O.OS. Figure 5. Helght of CemJpiatreelets. Mean height of trees (±SE) that sheltered colonies of one of the two Azteca species, Pseudocobima guionolis
caterpillars, or that sheltered neither Azteca nor caterpillars during the experimental period. Statistical comparisons (normality and equal variance
tests passed); ANOVA: F2
56,8.56; P<0.0001. Newman-Keuls' post-hoc test: different letters lndlcate slgnlflcant differences at P< O.OS. most productive trichilia by weaving silk above them. They, thus,
indirecdy slow dawn colony growth as these foraging workers only
have access to the lower, less-productive trichilia often already
covered by F. monili:forme. Consequendy, the development of the
colonies depends mosdy on the ants attending hemipterans in the
internodes of the host trees (if any). So, although caterpillars can
delay Azteca colonization, the Cecropia trees are fmally exclusively
occupied by Azteca ants. Furthermore, once Azteca colonizes a
Cecropia tree, the workers exploit the FBs on the upper, most-
productive trichilia and patrol the foliage, rendering the situation
irreversible by preventing colonization by caterpillars. lndeed, the
Azteca workers, that are able to capture insects the size of a female
P. guianalis math [42], probably destroy any insect eggs that have
been successfully laid on their host plant's foliage (see [70] and
references therein). References ｄｾ･｡ｮ＠
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Long-term Administration of CU06-1004 Ameliorates Cerebrovascular Aging and BBB Injury in Aging Mouse Model: A Randomized Control Trial
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Research Article Keywords: CU06-1004, Blood-brain barrier, Aging, Brain microvascular endothelial cell (BMEC), Reactive
oxygen species (ROS), Cerebrovasculature, Inflammation, Neurodegenerative disorders Posted Date: December 12th, 2022 Abstract A series of behavioral tests revealed
improved motor and cognitive function in aged mice that received CU06-1004. Conclusion: These findings suggest CU06-1004 has promise as a therapeutic for delaying age-related
cerebrovascular impairment and improving cognitive function in old age. DOI: https://doi.org/10.21203/rs.3.rs-1845446/v2 License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Additional Declarations: No competing interests reported. Version of Record: A version of this preprint was published at Fluids and Barriers of the CNS on February
1st, 2023. See the published version at https://doi.org/10.1186/s12987-023-00410-x. Page 1/28 Abstract Background: Age-related changes in the cerebrovasculature, including blood-brain barrier (BBB) disruption
and vascular dementia are emerging as potential risks for many neurodegenerative diseases. Therefore,
endothelial cells that constitute the cerebrovasculature play a key role in preventing brain injury. Our
previous study showed that CU06-1004, endothelial cell dysfunction blocker, prevented vascular leakage
and enhanced vascular integrity in ischemic reperfusion injury and normalization of tumor vasculature. Here, we evaluate the effects of CU06-1004 on age-related decline in cerebrovascular function of aged
mice brain. Results: In this study, we investigated the protective effects of CU06-1004 on reducing oxidative stress-
induced damage in human brain microvascular endothelial cells (HBMECs). HBMECs were treated with
hydrogen peroxide (H2O2) to establish an oxidative stress-induced cellular injury model. Pretreatment with
CU06-1004 considerably reduced oxidative stress-induced cytotoxicity, ROS generation, senescence-
associated β-galactosidase activity, and senescence markers in HBMECs. Additionally, pretreatment with
CU06-1004 decreased the expression levels of inflammatory proteins, compared to H2O2 treatment alone. Results: In this study, we investigated the protective effects of CU06-1004 on reducing oxidative stress-
induced damage in human brain microvascular endothelial cells (HBMECs). HBMECs were treated with
hydrogen peroxide (H2O2) to establish an oxidative stress-induced cellular injury model. Pretreatment with
CU06-1004 considerably reduced oxidative stress-induced cytotoxicity, ROS generation, senescence-
associated β-galactosidase activity, and senescence markers in HBMECs. Additionally, pretreatment with
CU06-1004 decreased the expression levels of inflammatory proteins, compared to H2O2 treatment alone. Based on the cytoprotective effect of CU06-1004 in HBMECs, we further examined the vascular protective
effects of CU06-1004 on cerebrovascular aging in aged mice. Long-term administration of CU06-1004
alleviated age-associated cerebral microvascular rarefaction and cerebrovascular senescence in the aged
mouse brain. CU06-1004 supplementation also reduced extravasation of plasma IgG by improving BBB
integrity in the aged mouse brain. This improvement in BBB integrity was associated with reduced
neuronal injury and cognition memory dysfunction in aged mice. A series of behavioral tests revealed
improved motor and cognitive function in aged mice that received CU06-1004. Based on the cytoprotective effect of CU06-1004 in HBMECs, we further examined the vascular protective
effects of CU06-1004 on cerebrovascular aging in aged mice. Long-term administration of CU06-1004
alleviated age-associated cerebral microvascular rarefaction and cerebrovascular senescence in the aged
mouse brain. CU06-1004 supplementation also reduced extravasation of plasma IgG by improving BBB
integrity in the aged mouse brain. This improvement in BBB integrity was associated with reduced
neuronal injury and cognition memory dysfunction in aged mice. Background The blood-brain barrier (BBB) is a physical barrier composed of brain microvascular endothelial cells
(BMECs) that limits the movement of substances from circulating blood into the brain, maintaining brain
homeostasis. The BMECs play an important role in BBB integrity by forming tight junctions in the cerebral
blood vessels (1, 2). However, as aging progresses, the BMECs are damaged by various stimuli and environmental factors,
losing the ability to proliferate, migrate, and repair damage (3, 4). The damaged BMECs then become
senescent, an irreversible growth arrest. As senescent cells accumulate in organs, the release of high
levels of inflammatory cytokines, matrix metalloproteinases, and immune regulators is induced. This
results in the surrounding microenvironment changing to a senescence-associated secretory phenotype
(SASP). Development of the SASP is thought to be the main cause of aged-related diseases, and this
phenotype can develop if only 2 ~ 3% of the endothelial cells become senescent. Therefore, Senescence Page 2/28 Page 2/28 Page 2/28 of BMECs in the cerebrovascular system could have major implications for cerebrovascular diseases and
neurodegenerative disorders, including BBB disruption, mild cognitive impairment, and vascular dementia
(5–7). Accumulating evidence has demonstrated oxidative stress and inflammation are the primary
factors driving cellular senescence (8, 9). Inflammatory cytokines secreted by senescent cells trigger
further inflammation and senescence in surrounding tissue. Additionally, increased presence of
inflammatory cytokines reduces levels of endogenous antioxidative enzymes and induces accumulation
of reactive oxygen species (ROS) in tissue. Accumulation of ROS in the cerebrovascular system, which is
particularly sensitive to oxidative stress, leads to BBB disruption mediated by oxidative stress (10–12). For these reasons, many studies have shown the structure and function of the BBB deteriorates during
aging process, leading to increased permeability of BBB (13–15). Disruption of the BBB is associated
with the loss of motor neurons, neuroinflammation, and cognitive impairment (16). However, few
pharmaceutical interventions have been identified as therapeutic candidates for preserving BBB
functionality and preventing cerebrovascular aging (17–19). CU06-1004 is a small molecule known to activate the cAMP/Rac/cortactin pathway, strengthening the
tight junction barrier in endothelial cells and blocking hyperpermeability (20, 21). Acute CU06-1004
treatment for ischemia/reperfusion-induced BBB injury lowered cerebral edema and astrocyte end-foot
disruption by stabilizing endothelial cell junctions (22). Based on these previous findings, we conducted this study to investigate the effects of CU06-1004 on
age-related decline in cerebrovascular function of aged mice brain. Background To investigate the role and potential
molecular mechanisms of CU06-1004 in the aged brain, we used both an oxidative stress injury cell
model, induced by H2O2, and a natural aging mouse model. Our results showed CU06-1004 inhibited
oxidative stress-induced HBMEC senescence and inflammation through NF-kB signaling suppression. Furthermore, we observed the novel finding that long-term oral administration of CU06-1004 improved
age-associated cerebral microvascular rarefaction in aged mice. Notably, treatment with CU06-1004
increased the expression of tight junction, which was important for BBB maintenance in cerebral
microvasculature. Consequently, CU06-1004 treatment attenuated neuropathological changes in the aged
brain. We also found CU06-1004 treatment rescued impaired cognition deficits and enhanced muscle
function in 23-month-old-mice. In summary, our results demonstrated CU06-1004 can effectively
ameliorate age-associated cerebrovascular aging and brain injury, suggesting CU06-1004 has potential
as an effective drug to protect against age-related cerebrovascular diseases. Drug treatment CU06-1004 was synthesized as described previously (20). Briefly, CU06-1004 was synthesized via
tetrahydropyran deprotection and subsequent glycosidation with 4,6-di-O-acetyl-2,3-didieoxyhex-2-
enopyran in the presence of an acid. A working solution of CU06-1004 (10 µg/µl) was prepared in
dimethyl sulfoxide (DMSO, Sigma, # D2650) for in vitro experiments. The 72-week-old mice were divided Page 3/28 into two groups, old-vehicle (n = 15) and CU06-1004 (10 mg/kg) (n = 15). CU06-1004 was dissolved in
olive oil (Sigma, # O1514) for oral administration. Both treatments were orally administered 6 days a
week using a Zonde needle (100 µl, Jeung Do Bio & Plant Co, # JD-S124) for 6 months, from 18 to 24
months of age. Primary Cultures Of Human Brain Microvascular Endothelial Cells
(Hbmecs) Human brain microvascular endothelial cells (HBMECs) were purchased from ScienCell (Cat. No. 1000)
and cultured in EGM-2 media (Lonza, CC-3156). The media was supplemented with the EGM-2
SingleQuots™ kit (Lonza, CC-4176), 20% FBS, and 1% penicillin/streptomycin (P/S, Cat. No. 0503). Cells
were routinely passaged at 80−90% confluency, and cells between passages 3 and 6 were used for
experiments. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2. Experimental Animals Male C57BL/6J mice (72-week-old) were purchased from Charles River Laboratories Japan (Yokohama,
Kanagawa, Japan). Additionally, 6-week-old male C57BL/6J mice (DBL, Korea) were used as young mice
and 24-month-old male C57BL/6J mice were used as aged mice. All mice were housed under controlled
conditions (24°C ± 1°C, 12 h light/dark cycles, 55% humidity, and specific-pathogen-free) and provided
with free access to food and water. All animal facilities and experiments were performed in accordance
with the Korean Food and Drug Administration guidelines. All procedures were approved by the
Institutional Animal Care and Use Committee at Yonsei University (permit number: IACUC-A-202010-1154-
01). Measurement Of Intracellular Reactive Oxygen Species (Ros) The formation of ROS was measured using a ROS-sensitive dye, 2’,7’-dichlorodihydrofluorescein diacetate
(H2-DCFDA, Invitrogen, #D399), as an indicator. HBMECs were seeded at 1 ⋅ 104 cells/well in a black, clear
bottom 96-well plate containing 100 µl of culture medium and then incubated at 5% CO2 and 37°C
overnight. The following day, HBMECs were starved of media for 2 h and then pretreated with CU06-1004
(10 µg/ml) for 1 h. The media were removed, and the cells were washed twice with PBS. This was
followed by stimulation of ROS development via incubation with 100 µM H2O2 for 2 h. The cells were
then incubated with 10 µM H2-DCFDA for 30 min at 37°C. The fluorescent product formation was
quantified with a spectrofluorometer at 485/520 nm. The fluorescent cells were then washed twice with
PBS and observed using a fluorescence microscope (Microscope, Olympus DX51; Camera, Olympus
DP72). Rna Isolation And Reverse Transcription Polymerase Chain Reaction
(Rt-pcr) RNA isolation and reverse transcription polymerase chain reaction (RT-PCR)
Total RNA was extracted from HBMECs using easy-BLUE™ (iNtRON, #17061). Total RNA was reverse
transcribed into cDNA using M-MLV Reverse Transcriptase (Promega Corporation, #M1701) in the
presence of oligo(dT) primers and dNTP. The following temperature protocol was used for reverse
transcription: Denaturation at 70°C for 5 min, annealing at 25°C for 10 min, and extension at 42°C for 50
min. The following primers were used for PCR: p21: 5’-GCTTCATGCCAGCTACTTCC-3’(forward), 5’-
CCCTTCAAAGTGCCATCTGT-3’ (reverse), p16: 5’-CCTCGTGCTGATGCTACTGA-3’(forward), 5’-
CATCATCATGACCTGGTCTTCT-3’ (reverse), GAPDH: 5′-CCACCCATGGCAAATTCC-3′(forward), 5′-
TCGCTCCTGGAAGATGGTG-3′(reverse). All results were normalized to GAPDH expression levels. Measurement Of Cell Viability Colorimetric 3-(4,5-dimetylthialzol-2-yl)-2,5-diphenyltertrazolium bromide (MTT, Thermo Fisher Scientific,
#M6494) assay was used to measure cell viability. MTT is reduced to formazan by mitochondrial
dehydrogenases, and the absorbance (570 nm) is directly proportional to viable cell count. HBMECs were
seeded into a gelatin-coated 24-well plate at 1 ⋅ 105 cells/well and incubated at 37°C in EGM-2 medium
overnight. The following day the cells were treated with either CU06-1004 or H2O2. The cells were then
washed with 1x PBS and incubated for 4 h at 37°C with MTT solution (0.1 mg/ml) for evaluating cell
viability. After the 4h incubation, the MTT solution was removed and a 50:50 solution of dimethyl
sulfoxide and ethanol was added (200 µl/well) to solubilize formazan crystals. Absorbance was detected
at a wavelength of 540 nm and cell viability was calculated as a percentage of absorbance detected from
the control cells. Page 4/28 Senescence-associated-β-galactosidase (SA-β-gal) staining. The samples were fixed with 3.7% formaldehyde for 10 min and washed with cold 1x PBS for 15 min at
room temperature. Samples were washed twice more with PBS and then incubated at 37°C without CO2
for 24h with senescence-associated β-gal staining solution, containing 1mg/mL 5-bromo-4-chloro-3-
indolyl-β-D-galactopyranoside (X-gal, MERCK, #B4252), 5mM potassium ferrocyanide, 5mM potassium
ferricyanide, 150Mm NaCl, 2mM MgCl2. 0.01% Nonidet-P-40. After the 24h incubation, samples were
washed with PBS and then observed for aging by degree of blue color development (23). Staining and
imaging were observed under a phase-contrast microscope (Nikon, Japan). Quantitative immunofluorescent microscopy of cerebral immunoglobulin G extravasation. The integrity of the BBB was determined by detection of cerebral perivascular extravasation of the
plasma protein immunoglobulin G (IgG), a widely used and established method (24). Brain tissues were
immersion-fixed in 4% paraformaldehyde for 24 h and immersed in 15% and 30% sucrose each day. The Page 5/28 Page 5/28 tissues were then frozen in OCT compound and stored at -80 °C. Brain cryosections of 25 µm were placed
on Polysine™-coated microscope slides (Leica, #3800050CL). The sections were prefixed in acetone for
30 min at -70°C. Unspecific binding was blocked with 10% goat serum in PBS for 30 min. Goat anti-
mouse IgG conjugated with Alexa 488 (Invitrogen, #A28175) at a concentration of 1:50 antibody diluent
solution was applied to the sections and sections were incubated at 4°C for 20 h. After washing the
sections with 0.2% TBST and 1 x PBS, the sections were mounted with mounting solution (DAKO,
#S3023). The immunofluorescent images were taken using a Confocal 980 (LSM 980 META; Carl-Zeiss). For each cortex and hippocampus region 5–6 images were randomly taken from each brain section, and
all images were used for subsequent quantitative analysis. Quantitation Of Il-6 And Tnf-α By Elisa Blood samples through cardiac puncture were obtained in SST tube (Becton Dickinson, # BD365967) and
incubated for 30min at RT. Then, the murine serum was collected following sample centrifugation for 10
min at 1500 rpm. The serum concentrations of IL-6 and TNF-α were determined using Quantikine ELISA
Kit (R&D systems, #M6000B, #MTA00B) according to the manufacturer protocol. Histology And Immunohistochemical Analysis Following 6 months of drug administration, the 24-month-old mice were anesthetized using avertin (2, 2,
2-Tribromoethanol, Sigma Aldrich, #T48402), 250 mg/kg of body weight, and perfused with 0.9% saline
solution into the apex of the left ventricle. The brain tissue was removed and fixed in 4% Following 6 months of drug administration, the 24-month-old mice were anesthetized using avertin (2, 2,
2-Tribromoethanol, Sigma Aldrich, #T48402), 250 mg/kg of body weight, and perfused with 0.9% saline
solution into the apex of the left ventricle. The brain tissue was removed and fixed in 4%
paraformaldehyde in PBS (pH 7.4) overnight at 4°C. Following overnight fixing, brain tissue was
incubated in 15% sucrose overnight at 4°C and then transferred to 30% sucrose at 4°C until the tissue
sank. Fixed tissue was encapsulated with Tissue-Tek OCT embedding medium for 30 min at room
temperature, transferred to an embedding mold filled with OCT, frozen on dry ice, and stored at -70°C. Frozen sections (25-µm-thick) were cut at -20°C, and slides were stored at -80°C until stained for
immunofluorescence. The sections were prefixed in acetone for 30 min at -70°C and air dried. The OCT
was washed off with running tap water. The sections were incubated in blocking solution for 1 hour at
room temperature and then incubated overnight at 4°C in CD31 primary antibody (1:200; Abcam,
#ab24590), GFAP (1:200; Millipore, #MAB360), claudin-5 (1:200; Invitrogen, #35-2500), and occludin
(1:200; Invitrogen, #711500). After incubation, sections were washed 3 times with 0.2% Triton X-100 in
PBS (10 min/wash), and further incubated separately in 488-conjugated secondary antibody (1:400;
Invitrogen, #A28175), 594-conjugated secondary antibody (1:400; Invitrogen, #A21297), and 4′,6-
diamidino-2-phenylindole (DAPI; 1:1000, Duolink, #D9542). The sections were analyzed using a confocal
microscope (LSM 880 META; Carl-Zeiss). Wire hang test This test evaluated the forelimb strength of mice. The apparatus consisted of a stainless-steel wire (90
cm length, 2 mm in diameter), secured horizontally between two vertical stands, 30 cm above a soft
padded surface. The wire hang test was conducted at 23 months of age. The mouse was forced to grasp
the central position of the wire with its forepaws, and the time it took to fall from the wire to the pad was
measured. When the time was over 150 s the mouse was released from the wire, and the time was
recorded as 150 s. The trial was conducted three times for each mouse and the averaged value was used
for evaluation. The resting time between consecutive attempts was 3 min. Western Blot Analysis Western Blot Analysis Page 6/28 Western blotting was performed as previously described (25). Briefly, HBMECs were lysed using RIPA
buffer (100 mM Tris-Cl, 5 mM EDTA, 50 mM NaCl, 50 mM β-Glycero-phosphate, 50 mM NaF, 0.1 mM
Na3VO4, 0.5% NP-40, 1% Triton X-100, and 0.5% Sodium deoxycholate) at 4°C. Sample protein
concentration was quantified using the SMART™ BCA Protein Assay Kit (iNtRON, #21071). Next, 25 µg of
cell lysates were separated by sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
and transferred to nitrocellulose membranes. The membranes were blocked with 3% bovine serum
albumin in 0.1% TBST and probed with primary antibodies. The membranes were then incubated with
horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Life Science)
secondary antibodies. β-actin was used as the loading control. The primary antibodies were obtained
from Cell Signaling Technology and were used at a 1:1000 dilution: phospho-IκB-α (#9242), IκB-α
(#2859). The other primary antibodies used were ICAM-1 (1:1000, Santa Cruz Biotechnology, #SC-8439),
VCAM-1 (1:1000, Santa Cruz Biotechnology, #SC-13160), COX-2 (1:1000, Santa Cruze Biotechnology,
#SC-376861) and β-actin (1:2000, Thermo Fisher Scientific, #MA5-15739). Rotarod Test This test assessed motor coordination by placing animals on a Rotarod device (Four Lane Rotarod; Ugo
Basile, Italy, #MSW-007) that consists of an accelerating rod that the mouse must balance on. If a mouse
loses its balance and falls the rod automatically stops and records the time to fall as well as the speed at
fall. Prior to the first test, mice were habituated to the testing system until they were able to stay on the
rod at a constant speed of 2 rpm for approximately 1 min. During testing, each animal was exposed to
the apparatus three times for 300 s per trial. The initial speed of the Rotarod was set to 4 rpm and
increased to 50 rpm over 300 s. When the mouse fell the session was over, and the Ugo Basile program
stopped the timer (26). T-maze Alteration T-maze Alteration Page 7/28 Spatial working memory was assessed using a simple T-maze test (27). Each trial consisted of a sample
run and a choice run. In the sample run, one of the goal arms was blocked, forcing the mouse to enter the
other goal arm (e.g., the left arm). Prior to beginning the choice run, a 30 s delay was introduced between
trials. The blocks were removed, and the mouse was given a free choice of either arm in the choice run. Even without a reward, driven by curiosity, mice usually selected the previously unvisited arm (e.g., the
right arm). The animal was considered to have made the correct choice (+) if it visited the previously
unsampled arm but incorrect (-) if it visited the previously sampled arm. A total of 10 free choices made
by the mice were measured and the percentage of correct arm choices during the choice trials was
calculated. Each arm of the T-maze was cleaned between sessions using ethanol to remove any olfactory
cues, which may have affected the behavior of the next mouse tested. GraphPad Prism 8 software (GraphPad Software, La Jolla, CA) was used for statistical analyses. GraphPad Prism 8 software (GraphPad Software, La Jolla, CA) was used for statistical analyses. Statistical significance was determined by using as mean ± standard deviation (SD) or mean ± standard
error of the mean (SEM) and P values less than 0.05 were considered statistically significant. All
experiments were performed at least three times, and representative results are shown. CU06-1004 reduces H 2 O 2 -induced inhibition of HBMEC growth. CU06-1004 reduces H 2 O 2 -induced inhibition of HBMEC growth Prior to investigating the protective properties of CU06-1004 (Fig. 1A) against H2O2 treatment, the
cytotoxic potential of CU06-1004 was examined on HBMEC. The MTT assay indicated CU06-1004 did not
show any cytotoxic effects at concentrations ranging from 1 to 20 µg/ml (1, 2, 5, 10, and 20 µg/ml). Conversely, HBMEC growth was significantly increased in a dose-dependent manner (Fig. 1B). Further
MTT analyses revealed that while 50 µM H2O2 did not inhibit cell growth, 100 µM H2O2 significantly
inhibited the growth of HBMECs (Fig. 1C). Notably however, CU06-1004 pretreatment effectively reversed
H2O2-induced inhibition of cell growth (Fig. 1D). These results demonstrated CU06-1004’s ability to
prevent H2O2-induced inhibition of HBMEC growth. CU06-1004 reverses H 2 O 2 -induced senescence in HBMECs. To investigate the effect of CU06-1004 on HBMECs with senescent phenotype, cells were treated with 100
µM H2O2 to induce cellular senescence (Fig. 3A). At 3 days post-H2O2 exposure the presence of senescent
HBMECs was confirmed by enlarged shape and cytoplasmic granularity of exposed cells. After 5 days,
The SA-β-Gal staining assay was used to detect the level of HBMEC senescence induced by H2O2. A 3-
fold increase in percentage of SA-β-Gal+ cells was observed in H2O2-treated group compared to control
cells. However, pretreatment with CU06-1004 significantly inhibited this effect and reduced the percentage
of SA-β-Gal+ cells to 2-fold compared to control cells (Fig. 3B). Senescence is a state of permanent
cellular arrest that is established and maintained by the expression of cyclin-dependent kinase inhibitors
(CKIs). As p16INK4a and p21 are known to mediate permanent cell cycle arrest (31, 32), we determined
mRNA levels associated with these genes by RT-PCR. The expression of p16 INK4a and p21 was
downregulated in the CU06-1004 treatment group, confirming the anti-senescence effect of CU06-1004 in
HBMECs (Fig. 3C-E). CU06-1004 inhibits H 2 O 2 -induced ROS generation and alleviates the inflammatory response of
HBMECs. CU06-1004 inhibits H 2 O 2 -induced ROS generation and alleviates the inflammatory response of
HBMECs. To elucidate the possible mechanisms by which CU06-1004 prevented inhibition of HBMEC growth we
used H2-DCFDA. H2-DCFDA is a cell-permeable fluorescent dye which fluoresces upon oxidation by ROS,
and it was used to determine the effect of CU06-1004 on H2O2-induced oxidative stress in HBMECs. Exposure of HBMECs to H2O2 for 2 h significantly enhanced ROS generation compared to untreated
HBMECs. However, pretreatment with CU06-1004 for 1 h significantly inhibited the H2O2-induced increase
in ROS generation in a dose-dependent manner (Fig. 2A-B). Page 8/28 Page 8/28 Many studies have determined oxidative stress induces an inflammatory response either directly or
indirectly, and oxidative stress and inflammation are primary mechanisms related to onset of age-related
vascular endothelial dysfunction (28–30). Therefore, to investigate whether the inflammatory response
induced by oxidative stress was alleviated by CU06-1004, we determined the expression of inflammatory
proteins in H2O2‑treated HBMECs. As shown in Fig. 2C-G, CU06-1004 treatment effectively inhibited the by
H2O2-induced expression of ICAM-1 and VCAM-1, as well as the expression of COX-2, an inflammation
mediating enzyme. Furthermore, NF-κB, as a transcription factor, is considered a major mediator of the
inflammatory response. Notably, the level of phosphorylated IκBα, an indicator of NF-κB activity, was
significantly reduced in HBMECs treated with CU06-1004. Together, these results suggest CU06-1004 has
a protective effect from ROS-induced damage by inhibiting the inflammatory response in HBMECs. The disruption of BBB integrity during aging could be prevented by CU06-1004. We then investigated whether age-induced cerebral microvascular rarefaction affects the BBB integrity. The integrity of the BBB was determined by detection of cerebral extravasation of the plasma protein
immunoglobulin G (IgG). As shown in Fig. 5A-B, IgG was almost absent from the parenchyma of the
cortex and hippocampus of young mice but was highly abundant in aged mice parenchyma. Notably, this
abundance was significantly decreased in the old-1004 group. As BBB permeability is highly dependent
on cerebrovascular endothelial tight junctions, we next examined the integrity of these junctions in the
brains of young and aged mice. Compared with young mice, aged mice (old-vehicle) cerebral vessels
expressed less claudin-5 and occludin proteins. Notably, claudin-5 was upregulated in aged mice that
received CU06-1004 treatment (old-1004). However, there was no difference in expression levels of
occludin between the aged mice groups (Fig. 5C-D). We used electron microscopy to further examine the
minor changes of tight junction complexes responsible for cerebrovascular leakage. In the young mice,
ultrastructural analysis showed seamless tight junctions within a smooth endothelial layer surrounded by
astrocyte endfeet. Although it was confirmed that the capillary wall became thicker and the astocytic
endfeet was considerably swollen in the old mice, the tight junctional complex was less damaged in old-
1004 group than old-vehicle (Figure S1). These results indicate aging accelerates the onset of BBB
dysfunction and long-term administration of CU06-1004 could prevent damage to BBB integrity
associated with aging. CU06-1004 alleviates age-associated cerebral microvascular rarefaction and vascular aging in aged mice. Based on results showing the anti-inflammatory and anti-senescence effects of CU06-1004 on HBMECs
we further examined whether long-term administration of CU06-1004 (10 mg/kg via oral gavage) to 18-
month-old mice (late middle-age) had a protective effect on cerebrovascular aging. First, we measured
the maximal cortical diameter and the ratio of brain to body weight at the postmortem examination. The
mean cortical diameter was decreased by 8% in 24-month-old mice (late age) compared to 6-week-old
mice (young age). However, no differences were detected between the old-vehicle and old-1004 group. (Fig. 4A-B). Additionally, the brain to body weight ratio was significantly reduced in 24-month-old mice
(late age) but was less reduced in old-1004 group (Fig. 4C). Next, to examine brain microvasculature
changes in aging, the brain vasculature patterning in the cortex and hippocampus of aged mice brain
was analyzed through immunofluorescent staining of endothelial markers. There was significant
reduction in capillary vessel density in old mice microvasculature compared to young mice
microvasculature. However, the old-1004 group, old mice treated with CU06-1004, showed greater Page 9/28 Page 9/28 capillary vessel density and higher numbers of branch points, compared to old mice receiving only the
vehicle, old-vehicle. This indicates CU06-1004 improves vessel maintenance and inhibits cerebral
microvascular rarefaction in aged mice (Fig. 4D-E). Additionally, SA-β-Gal+ cells were clearly observed in
vessels located in the brain cortex of aged mice, but cell prevalence decreased in the old-1004 group
(Fig. 4F). These results suggest long-term administration of CU06-1004 protects against aging-induced
microvascular rarefaction and vascular aging. CU06-1004 treatment in late middle-age improved motor function and recognition memory dysfunction. Next, we quantified the neuronal nuclear protein A60-positive (NeuN+) cells in the brains of aged mice. The number of NeuN+ cells was significantly reduced and incompact in the cortex and the CA1 region of
the hippocampus in aged mouse brain compared to young mouse brain. However, NeuN+ cell numbers
and compactness were restored following CU06-1004 treatment (old-1004), demonstrating the efficacy of
CU06-1004 in protecting against neuronal damage in the aged brain (Fig. 7A-B). We then examined
whether CU06-1004 treated aged mice showed behavioral and cognitive recovery (Fig. 7C). Motor
function and working memory activities were performed with old-vehicle and old-1004 mice at 23
months. The Rotarod test and wire hang test are classic methods for evaluating motor coordination of
the limbs and balance in aged animals. As shown in Fig. 7D, both old-vehicle and old-1004 groups
exhibited shorter average time to fall off the accelerating rotating rod compared to the young group, but
no significant difference was detected between the old-vehicle and old-1004 group. In the wire hang test,
the old-1004 group showed a marked increase in hanging time (about by 3-fold) compared to the average
hanging time of the old-vehicle group, indicating CU06-1004 enhances motor coordination and forelimb
muscle strength (Fig. 7E). The T-maze test was used as a spontaneous alternation task for assessing
spatial working memory. Aged mice demonstrated a significantly lower percentage of correct
spontaneous alternation choices, indicating an impairment in working memory. However, the increase in
percentage of correct spontaneous alternation choices between old-vehicle and old-1004 groups
indicated a significant effect of CU06-1004 treatment (Fig. 7F). These results suggest long-term
administration of CU06-1004 reduces neuromuscular strength impairment caused by aging and damage
to spatial working memory caused by neuronal cell damage. CU06-1004 attenuates neuropathological changes in the aged brain. Studies have shown BBB dysfunction amplifies neuroinflammation and may act as a key process in the
development of neuroinflammation (33). Therefore, we determined astrocyte activation in brain tissues of
young and aged mice, as this is a widely accepted hallmark of neuroinflammation in aged mice brain. Histopathological alterations were evaluated using immunohistochemistry and immunofluorescence
staining. As shown in Fig. 6A-B, cytoplasmic staining showing GFAP-positive (activated) astrocytes in
brain sections of the hippocampus was significantly increased in aged mice compared to young mice. Double immunofluorescence staining showed increased GFAP activation in the hippocampus of aged
mice (old-vehicle), suggesting that aging causes upregulation of activated astrocytes. Notably, we found
long-term administration of CU06-1004 reduced systemic TNF-α and IL-6 levels, which appeared to
increase with aging (Fig. 6C). Additionally, we analyzed expression levels of inflammatory proteins from
brain tissue extracts. In brain tissue, the expression levels of proteins such as ICAM-1, VCAM-1, and COX-2
were lower in aged mice that received CU06-1004 (old-1004) than in aged mice that did not (old-vehicle) Page 10/28 Page 10/28 (Fig. 6D-G). Collectively, these results indicate long-term administration of CU06-1004 exerts anti-
inflammatory and neuroprotective effects in aged mice. Discussion Aging is a biological process in which the structure and function of all organs progressively deteriorate
over time (34). Aging is also a major risk factors for developing various vascular diseases, including,
cardiovascular diseases, strokes, eye diseases, and neurodegenerative diseases. Similarly, the vascular
system, which supplies oxygen and nutrients throughout the body, is affected by the aging process and
becomes more susceptible to diseases in the aged population. Therefore, it is very important to develop
novel therapies that can slow the aging process and more effectively treat aging-related diseases (35). CU06-1004, an endothelial cell dysfunction blocker, has been shown to prevent vascular leakage and
enhance vascular integrity in ischemic reperfusion injury and normalization of tumor vasculature. However, the mechanisms underlying the role of CU06-1004 in oxidative stress-induced HBMEC
senescence, inflammation, and age-related cerebrovascular dysfunctions remain unknown. In this study,
the brains of aged mice showed higher SA-β-galactosidase activity than young mice brains. Capillaries of
the young mice brains were interconnected in tubular structures while the capillaries of the aged mice Page 11/28 Page 11/28 Page 11/28 brain were fragmented and disconnected in both the cortex and hippocampal regions. This implied that
BMECs that had become senescent, a state of irreversible cell growth inhibition, contributes to the
decrease in cerebral capillary density during aging (Fig. 4). Mice that are 18−24 months of age are used
to represent humans that are 56−69 years of age. Additionally, humans aged 55−85 years have shown a
significant decrease in microvasculature density in brain tissue, similar to that observed in Alzheimer’s
disease patients. In normal aging, cerebrovascular loss causes chronic hypoperfusion to the brain,
eventually leading to cognitive impairment and vascular dementia. Therefore, maintaining
cerebrovascular homeostasis is important for preventing cerebrovascular aging and brain pathology. Moreover, we observed cerebral microvascular rarefaction in aged brain tissue causes incomplete BBB
integrity, which in turn leads to exceedingly high trans-endothelial permeability and increased passive
extravasation of plasma IgG. Here we show long-term administration of CU06-1004 in aged mice alleviates age-associated cerebral
microvascular rarefaction and inhibits the leakage of plasma IgG into the brain parenchyma by
suppressing cellular senescence and upregulating stability of claudin-5, the most enriched tight junction
protein in the aged mouse brain (Fig. 5). It is also known that BBB integrity is strongly affected by oxidative stress. Increased ROS production
contributes to cerebral endothelium dysfunction and increased permeability of the BBB (36). Discussion Long-term administration of
CU06-1004 in aged mice alleviates symptoms associated with motor and cognitive deficits, including,
cerebral microvascular rarefaction, neuronal losses, and chronic neuroinflammation. Collectively, these
results suggest CU06-1004 could be a useful therapeutic for preventing cerebrovascular aging and age-
associated brain injury. In conclusion, this study showed cerebrovascular aging may contribute to age-related cerebrovascular
damage and neuroinflammation in the aged brain. Additionally, it was confirmed that CU06-1004 protects
the endothelial cells of cerebral blood vessels, HBMECs, against oxidative stress-induced senescence and
inflammation through ROS scavenging, leading to reduced cytotoxicity. Long-term administration of
CU06-1004 in aged mice alleviates symptoms associated with motor and cognitive deficits, including,
cerebral microvascular rarefaction, neuronal losses, and chronic neuroinflammation. Collectively, these
results suggest CU06-1004 could be a useful therapeutic for preventing cerebrovascular aging and age-
associated brain injury. Discussion Additionally,
cerebral endothelial cells have high concentrations of mitochondria, increasing the risk of oxidative
damage in cells (37). The oxidation-inflammatory theory of aging also proposes that age-associated
oxidative stress is a driving factor of cellular senescence (9). Consistent with previous studies, we
observed H2O2-induced generation of excessive free radicals activated HBMEC senescence and led to
cells exhibiting classical SASPs characteristics such as an enlarged cell shape, cytoplasmic granularity,
and increased SA-β-galactosidase activity. Furthermore, it was observed that H2O2 exposure activated cell
cycle inhibition pathways, p16INK4a/p21, and strongly suppressed cell proliferative capacity. Alternatively,
HBMECs supplemented with CU06-1004 were characterized by attenuated SA-β-galactosidase activity
and marked downregulation of inflammatory proteins associated with SASP, potentially due to CU06-
1004-mediated NF-κB inhibition. Additionally, CU06-1004 treatment appeared to prevent senescence-
associated cell cycle arrest by inhibiting cell cycle suppressors p16INK4a and p21. This was then
confirmed, as HBMECs treated with CU06-1004 showed improved proliferative capacity following H2O2
exposure compared to control cells (Fig. 3). Overall, these results indicated CU06-1004 inhibited the
development of oxidative stress-induced senescence-associated features and the inflammatory response
in HBMECs. As chronic systemic inflammation increases with aging, cerebrovasculature becomes damaged due to
proinflammatory cytokines and proinflammatory molecules (38–41). Chronic systemic inflammation is
characterized by low-grade and persistent inflammation, leading to tissue degeneration. Additionally,
chronic low-grade inflammation contributes to various age-related pathologies in aging tissue, including
tissues of the nervous and musculoskeletal systems (28, 42, 43). Notably, we confirmed that long-term Page 12/28 Page 12/28 Page 12/28 administration of CU06-1004 reduced the degree of systemic inflammation caused by plasma
concentrations of proinflammatory cytokines, including TNF-α and IL-6. These results suggest prevention
of vascular damage by CU06-1004 may inhibit inflammation in the brain as well as other tissues (Fig. 6). We also showed that aged mice treated with CU06-1004 had improved muscle strength and recognition
memory in behavior tests (Fig. 7). These findings emphasize the importance of the BBB in maintaining
normal function of the central nervous system, thereby resisting neuronal injury, and improving cognitive
function. In conclusion, this study showed cerebrovascular aging may contribute to age-related cerebrovascular
damage and neuroinflammation in the aged brain. Additionally, it was confirmed that CU06-1004 protects
the endothelial cells of cerebral blood vessels, HBMECs, against oxidative stress-induced senescence and
inflammation through ROS scavenging, leading to reduced cytotoxicity. Conclusions Collectively, our data showed that CU06-1004, a known endothelial dysfunction blocker, acts as a
neuroprotective against age-related cerebrovascular impairment by exerting anti-senescence and anti-
inflammatory effects in HBMECs. And long-term administration of CU06-1004 alleviated age-associated
cerebral microvascular rarefaction and cerebrovascular aging thereby improving BBB integrity, and BBB
integrity was associated with reduced neuronal injury, reduced cognition memory dysfunction, and
improved motor and cognitive function in aged mice. These findings suggest supplementation of CU06-
1004 has great promise as a therapeutic for delaying age-related cerebrovascular impairment and
improving cognitive function in old age. BBB BBB
Blood-brain barrier
HBMEC Human brain microvascular endothelial ce
IL-6
Interleukin-6
TNF-α
Tumor necrosis factor alpha
ICAM-1
Intercellular adhesion
VCAM-1
Vascular adhesion molecule-1 Page 13/28 Page 13/28 NF-ĸB
Nuclear factor-kappa B
IkBα
nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha)
COX-2
Cyclooxygenase-2
ROS
Reactive oxygen species
IgG
Immunoglobulin G
SASP
Senescence-associated secretory phenotype
GFAP
Glial fibrillary acidic protein
CD31
Cluster of differentiation 31
NeuN
Neuronal nuclear protein
Declarations NF-ĸB
Nuclear factor-kappa B
IkBα
nuclear factor of kappa light polypeptide gene
COX-2
Cyclooxygenase-2
ROS
Reactive oxygen species
IgG
Immunoglobulin G
SASP
Senescence-associated secretory phenotype
GFAP
Glial fibrillary acidic protein
CD31
Cluster of differentiation 31
NeuN
Neuronal nuclear protein NF-ĸB
Nuclear factor-kappa B
IkBα
nuclear factor of kappa light polypeptide gene en
COX-2
Cyclooxygenase-2
ROS
Reactive oxygen species
IgG
Immunoglobulin G
SASP
Senescence-associated secretory phenotype
GFAP
Glial fibrillary acidic protein
CD31
Cluster of differentiation 31
NeuN
Neuronal nuclear protein NF-ĸB Availability of data and materials Not applicable. Ethics approval and consent to participate This study was approved by the Institutional Animal Care and Use Committee of Yonsei University
(approval number; IACUC-A-202010-1154-01). Competing interests The authors declare that they have no competing interests. Mice behavior test graphic was created with
BioRender (http://biorender.com) Consent for publication Not applicable. Funding Not applicable. Authors’ contributions Acknowledgements Not applicable. Authors’ contributions Page 14/28 Page 14/28 Page 14/28 Page 14/28 We thank CURACLE Co., Ltd. For providing us with the CU06-1004. HJ designed the project and planned
the experiments. HJ performed all experiments and quantifications. HJ, MY and SY performed behavior
tests of mice. HJ and YGK discussed the results and wrote the manuscript. HJ, MY, HZ and YM
contributed to proofreading the manuscript. YGK supervised and corrected manuscript. All authors read
and approved the final manuscript. References 1. Stamatovic SM, Keep RF, Andjelkovic AV. Brain endothelial cell-cell junctions: how to "open" the blood
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associated diseases. J Gerontol A Biol Sci Med Sci. 2014;69(Suppl 1):4–9. 42. Franceschi C, Campisi J. Chronic inflammation (inflammaging) and its potential contribution to age-
associated diseases. J Gerontol A Biol Sci Med Sci. 2014;69(Suppl 1):4–9. 43. Qu C, Song H, Shen J, Xu L, Li Y, Qu C, et al. Mfsd2a Reverses Spatial Learning and Memory
Impairment Caused by Chronic Cerebral Hypoperfusion via Protection of the Blood-Brain Barrier. Front Neurosci. 2020;14:461. 43. Qu C, Song H, Shen J, Xu L, Li Y, Qu C, et al. Mfsd2a Reverses Spatial Learning and Memory
Impairment Caused by Chronic Cerebral Hypoperfusion via Protection of the Blood-Brain Barrier. Front Neurosci. 2020;14:461. Page 17/28 Page 17/28 Figures Figures Figure 1 Figure 1 CU06-1004 reduces H2O2 -induced inhibition of HBMEC growth. CU06-1004 reduces H2O2 -induced inhibition of HBMEC growth. Page 18/28 Page 18/28 Page 18/28 (A) Chemical structure of CU06-1004. (B) HBMECs were incubated with increasing concentrations of
CU06-1004 (1, 2, 5, 10, 20 μg/ml) for 48 h. Cell viability was determined using 3-(4, 5-dimethylthiazol-2-
yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. (C) Cell viability of HBMECs treated with increasing
concentrations of H2O2 for 24 h. Cell viability was determined using MTT assay. (D) HBMECs were
pretreated with CU06-1004 (5-20 μg/ml) for 1 h before 100 μM H2O2 exposure. After 24 h, cell viability
was determined using MTT assay. All data are presented as the mean ± SEM, ***P < 0.001, n.s. not
significant. (A) Chemical structure of CU06-1004. (B) HBMECs were incubated with increasing concentrations of
CU06-1004 (1, 2, 5, 10, 20 μg/ml) for 48 h. Cell viability was determined using 3-(4, 5-dimethylthiazol-2-
yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. (C) Cell viability of HBMECs treated with increasing
concentrations of H2O2 for 24 h. Cell viability was determined using MTT assay. (D) HBMECs were
pretreated with CU06-1004 (5-20 μg/ml) for 1 h before 100 μM H2O2 exposure. After 24 h, cell viability
was determined using MTT assay. All data are presented as the mean ± SEM, ***P < 0.001, n.s. not
significant. (A) Chemical structure of CU06-1004. (B) HBMECs were incubated with increasing concentrations of
CU06-1004 (1, 2, 5, 10, 20 μg/ml) for 48 h. Cell viability was determined using 3-(4, 5-dimethylthiazol-2-
yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. (C) Cell viability of HBMECs treated with increasing
concentrations of H2O2 for 24 h. Cell viability was determined using MTT assay. (D) HBMECs were
pretreated with CU06-1004 (5-20 μg/ml) for 1 h before 100 μM H2O2 exposure. After 24 h, cell viability
was determined using MTT assay. All data are presented as the mean ± SEM, ***P < 0.001, n.s. not
significant. Page 19/28 CU06-1004 inhibits H2O2-triggered reactive oxygen species (ROS) generation and alleviates the
inflammatory response in HBMECs. (A) Representative fluorescent images indicating ROS production in HBMECs. HBMECs were pretreated
with 5 and 10 μg/ml CU06-1004 for 1 h, followed by incubation with 100 μM H2O2 for 2 h. Cells were then Page 20/28 Page 20/28 labeled with 2’, 7’-dichlorodihydrofluorescein diacetate (H2-DCFDA) to measure ROS production. (B) The
levels of ROS were detected by fluorescence microscopy with DCF-DA as the fluorescent probe. Quantitative analysis was performed by measuring the fluorescence intensity relative to the control cells. Each value represents the mean ± SEM of three independent experiments (n=3 experiments, *P <0.05). (C)
CU06-1004 suppresses ROS-mediated nuclear factor kappa-B (NF-κB) activation in HBMECs. HBMECs
were pretreated with CU06-1004 for 1 h and treated with H2O2 for 6 h. (D-G) Quantitative analysis of
ICAM-1/β-actin, VCAM-1/β-actin, p-IκBα/IκBα and COX-2/β-actin ratios. All ratios were analyzed using
western blotting. β-actin served as the internal control (n = 3 per group). All data are presented as the
mean ± SEM, *P <0.05, **P < 0.01, ***P < 0.001. labeled with 2’, 7’-dichlorodihydrofluorescein diacetate (H2-DCFDA) to measure ROS production. (B) The
levels of ROS were detected by fluorescence microscopy with DCF-DA as the fluorescent probe. Quantitative analysis was performed by measuring the fluorescence intensity relative to the control cells. Each value represents the mean ± SEM of three independent experiments (n=3 experiments, *P <0.05). (C)
CU06-1004 suppresses ROS-mediated nuclear factor kappa-B (NF-κB) activation in HBMECs. HBMECs
were pretreated with CU06-1004 for 1 h and treated with H2O2 for 6 h. (D-G) Quantitative analysis of
ICAM-1/β-actin, VCAM-1/β-actin, p-IκBα/IκBα and COX-2/β-actin ratios. All ratios were analyzed using
western blotting. β-actin served as the internal control (n = 3 per group). All data are presented as the
mean ± SEM, *P <0.05, **P < 0.01, ***P < 0.001. Page 21/28 CU06-1004 reverses H2O2-induced senescence in HBMECs. (A) Senescence-associated β-galactosidase staining in HBMECs treated with 100 mM H2O2 for 1 to 2
days, with or without CU06-1004, and then maintained in fresh media for 5 days. Representative
microscopic images were captured with phase-contrast microscopy. (B) Quantification of SA-β-gal- Page 22/28 positive cells shown in Figure 3A. (C-E) The relative mRNA levels of p16 and p21 in senescent HBMECs
following exposure to H2O2 or CU06-1004 were quantified by RT-PCR. All data are presented as the mean
± SEM, **P < 0.01, ***P < 0.001. positive cells shown in Figure 3A. (C-E) The relative mRNA levels of p16 and p21 in senescent HBMECs
following exposure to H2O2 or CU06-1004 were quantified by RT-PCR. All data are presented as the mean
± SEM, **P < 0.01, ***P < 0.001. The effect of long-term administration of CU06-1004 on cerebrovascular aging in aged mice. Page 23/28 (A) Representative images of brain morphology of 6-week-old (young), 24-month-old vehicle (old-veh),
and 24-month-old mice that received CU06-1004 (old-1004). (B-C) Quantification of maximal brain
diameter and brain/body weight ratio (brain index) in young, old-vehicle, and old-1004 groups. (D-E)
Representative images and quantification of capillary vessel density in the cortex and hippocampus of
young, old-vehicle, and old-1004 group. Scale bar = 50 µm. (F) SA-β-gal expression in brain cortex of
young, old-vehicle and old-1004 groups (n = 6~10 per group). All data are presented as the mean ± SD, *P
< 0.05, ***P < 0.001, n.s. not significant. (A) Representative images of brain morphology of 6-week-old (young), 24-month-old vehicle (old-veh),
and 24-month-old mice that received CU06-1004 (old-1004). (B-C) Quantification of maximal brain
diameter and brain/body weight ratio (brain index) in young, old-vehicle, and old-1004 groups. (D-E)
Representative images and quantification of capillary vessel density in the cortex and hippocampus of
young, old-vehicle, and old-1004 group. Scale bar = 50 µm. (F) SA-β-gal expression in brain cortex of
young, old-vehicle and old-1004 groups (n = 6~10 per group). All data are presented as the mean ± SD, *P
< 0.05, ***P < 0.001, n.s. not significant. (A) Representative images of brain morphology of 6-week-old (young), 24-month-old vehicle (old-veh),
and 24-month-old mice that received CU06-1004 (old-1004). (B-C) Quantification of maximal brain
diameter and brain/body weight ratio (brain index) in young, old-vehicle, and old-1004 groups. (D-E)
Representative images and quantification of capillary vessel density in the cortex and hippocampus of
young, old-vehicle, and old-1004 group. Scale bar = 50 µm. (F) SA-β-gal expression in brain cortex of
young, old-vehicle and old-1004 groups (n = 6~10 per group). All data are presented as the mean ± SD, *P
< 0.05, ***P < 0.001, n.s. not significant. Page 24/28 Page 24/28 Figure 5 Figure 5 Impaired blood-brain barrier (BBB) integrity and reduced tight junction protein coverage in aged mice were
rescued following CU06-1004 administration. Impaired blood-brain barrier (BBB) integrity and reduced tight junction protein coverage in aged mice were
rescued following CU06-1004 administration. (A) Confocal microscopic images of plasma IgG extravasation as a marker of blood-brain barrier
disruption. Scale bar = 50 µm. (B) Quantitative analyses were assessed on values of IgG mean intensity in
cerebral cortex and hippocampus in young, old-vehicle, and old-1004 groups. (C) Double immunostaining
of claudin-5/CD31 (Left) and occludin/CD31 (Right). The effect of long-term administration of CU06-1004 on cerebrovascular aging in aged mice. Representative images show the cerebral
abundance of claudin-5 (green) and occludin (green) in cerebral vessels of the young, old-vehicle, and old-
1004 groups. Scale bar = 20 µm. (D) Quantitative analysis of claudin-5/CD31 and occludin/CD31
coverage ratio groups (n = 6~10 per group). All data are presented as the mean ± SD, *P < 0.05, **P < 0.01,
***P < 0.001, n.s. not significant. Page 25/28 CU06-1004 reduces neuronal loss and cognition deficit in aged mice. (A) Neuronal nuclei were visualized using immunofluorescence staining in brain tissues of young, old-
vehicle, and old-1004 group. Scale bar = 50 µm. (B) The number of NeuN-positive cells per mm2 was
calculated in the cortex and CA1 regions in hippocampus of young, old-vehicle, and old-1004 group. All
data are presented as the mean ± SD, *P <0.05, ***P < 0.001. (C-F) Behavior tests were conducted using
aged mice at 23 months of age. (D) Rotarod test; (E) Wire hang test; (F) T-maze test. Mice were given oral-
injection of olive oil (vehicle) or CU06-1004 (10mg/kg) for 6 months and behavioral tests were performed
at 23 months separately. (n=10-12 mice per group). The results are presented as the mean ± SEM, *P
<0.05, ***P < 0.001, n.s. not significant. Effects of CU06-1004 on neuropathological changes in aging brain. Aging affects induction of astrocytic activation. (A) Histopathological analysis of astrocyte activation in
the brains of young, old-vehicle, and old-1004 groups. Histopathological alterations were evaluated using
immunohistochemistry (DAB) and immunofluorescence staining. The brown cytoplasmic staining of Page 26/28 Page 26/28 GFAP-positive astrocytes in the hippocampus was observed in each group. Double immunofluorescence
showed increased GFAP activation in the hippocampus of aged mice compared to young mice. Scale bar
= 50 µm. (B) Astrocyte activation was quantified using fluorescent intensity. (C) Serum levels of TNF-α
and IL-6 in young, old-vehicle, and old-1004 mice. (D-G) Expression of inflammatory proteins in brain
tissue extracts. β-actin was the internal control(n = 5~7 per group). All data are presented as the mean ±
SD, *P <0.05, **P < 0.01, ***P < 0.001. GFAP-positive astrocytes in the hippocampus was observed in each group. Double immunofluorescence
showed increased GFAP activation in the hippocampus of aged mice compared to young mice. Scale bar
= 50 µm. (B) Astrocyte activation was quantified using fluorescent intensity. (C) Serum levels of TNF-α
and IL-6 in young, old-vehicle, and old-1004 mice. (D-G) Expression of inflammatory proteins in brain
tissue extracts. β-actin was the internal control(n = 5~7 per group). All data are presented as the mean ±
SD, *P <0.05, **P < 0.01, ***P < 0.001. Page 27/28 FigureS1.tif FigureS1.tif
FigureS2.tif Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. FigureS1.tif FigureS2.tif Page 28/28
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English
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Explaining the global spatial distribution of organic crop producers
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Agricultural systems
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cc-by
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A R T I C L E I N F O Keywords:
Organic farming
Sustainable agriculture
Market access
Spatial analysis
Global farm distribution Organic farming has been proposed as a feasible way to reduce the environmental impacts of agriculture,
provide better products to consumers, and improve farmers' income. How organic farmers are distributed
worldwide, however, remains unknown. Using publicly accessible registries of organic crop farmers we mapped
their distribution globally and related it to local socio-economic, climatic, and soil characteristics. We show that
organic crop farmers are mostly present in areas with favorable socio-economic and climatic conditions, both
globally but also within countries. Within developed countries, the locations of organic crop farmers often do not
differ significantly from the locations of conventional crop farmers. In developing countries, there are, however,
larger differences and organic crop farmers concentrate in the more accessible and developed regions. Our
results suggest that crop farmers in poor areas may not have sufficient access to certification and markets. To
promote the spread of organic farming, certification and other incentives could target farmers in areas with
lower market access and higher levels of poverty which could improve value chains for organic products in these
areas. warn that upscaling organic farming could lead to disruptions in food
security and more widespread environmental degradation through
displacement effects (Connor, 2008; Leifeld et al., 2013; Muller et al.,
2017). In some cases, organic farming has been reported to cause in-
creased poverty and inequality between rich and poor farmers
(Beuchelt and Zeller, 2011; Getz and Shreck, 2006; Gómez Tovar et al.,
2005). Žiga Maleka,⁎, Koen F. Tieskensb, Peter H. Verburga,c a Institute for Environmental Studies (IVM), Vrije Universiteit Amsterdam, De Boelelaan 1087, 1081 HV Amsterdam, the Netherlands
b Department of Environmental Health, Boston University, School of Public Health, 715 Albany Street, Boston, MA 02118, USA
c Swiss Federal Institute for Forest Snow and Landscape Research, WSL Zürcherstrasse 111, CH-8903 Birmensdorf, Switzerland https://doi.org/10.1016/j.agsy.2019.102680
Received 26 June 2019; Received in revised form 23 August 2019; Accepted 26 August 2019
⁎ Corresponding author.
E-mail addresses: z.malek@vu.nl (Ž. Malek), tieskens@bu.edu (K.F. Tieskens), p.h.verburg@vu.nl (P.H. Verburg).
0308-521X/ ©
2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license
(http://creativecom
m
ons.org/licenses/BY/4.0/). Agricultural System
s 176 (2019) 102680 Agricultural System
s 176 (2019) 102680 Explaining the global spatial distribution of organic crop producers Žiga Maleka,⁎, Koen F. Tieskensb, Peter H. Verburga,c https://doi.org/10.1016/j.agsy.2019.102680
Received 26 June 2019; Received in revised form 23 August 2019; Accepted 26 August 2019
0308-521X/ ©
2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license
(http://creativecom
m
ons.org/licenses/BY/4.0/). 2.1. Data collection 2.1. Data collection We collected data on certified organic crop farmers from publicly
available datasets (full details on data collection in the Supplementary
material). The locations were collected from national repositories on
organic producers, or webpages and recent reports from major certifiers
and official institutions (Tables S1 and S2). The IFOAM – Organics
International definition of organic farming was followed when col-
lecting producer's data to ensure consistency. Organic farming is de-
fined as agricultural activities, with limited or forbidden use of syn-
thetic inputs such as fertilizers and pesticides (IFOAM, 2017; Seufert
et al., 2017). Some countries have different terms describing organic
agriculture, such as biological (Italy, Spain, France), ecological (Central
European countries), or both (Germany, Canada). Although these are
different terms, the practices of production are falling under the IFOAM
definition (Seufert et al., 2017). Only data on currently certified field
operations and crop farmers with a physical address were included. Certificates that were surrendered, withdrawn, or under conversion
were disregarded. Some registries provide information on the exact
type of crops produced, while most only provide information on the
type of organization (producer, handler, processer), or type of agri-
cultural activity (e.g. plant production). We, therefore, did not differ-
entiate between different crops produced by organic farmers. We ex-
cluded records on handlers and processers of organic products. Addresses of livestock farms often did not equal the location where land
was managed under organic principles. Organic feed that was used in
such farms was produced elsewhere. To avoid such inconsistencies
between certificate location and operation address, livestock farms
were excluded from our analysis (unless they also produced crops or
animal feed themselves). Inclusion and exclusion criteria are discussed
in more detail in the Supplementary Methods. y
Besides analyzing the spatial distributions within countries or re-
gions (Supplementary material), we also performed global analyses. A
logistic regression was conducted using data from all countries with
representative data. To study potential variation in the spatial dis-
tribution of organic crop farmers in countries with different shares of
organic farming, separate regressions were performed for groups of
countries where organic farming is nationally significant, countries that
are significant global producers, and countries where organic agri-
culture remains a niche activity. We hypothesized that within countries
in different development stages and with different shares of organic
farming the spatial determinants of organic agricultural may be struc-
turally different. 1. Introduction Advancements in agriculture and cropland intensification have
contributed to increases in food production crucial for ensuring global
food security (Meyfroidt, 2018; Muller et al., 2017). At the same time,
intensification through increased use of synthetic inputs has led to
negative environmental and social consequences (Verburg et al., 2013). Different strategies have emerged to reduce the environmental impacts
of agriculture, while at the same time supporting food security; organic
agriculture is among the most promising (Reganold and Wachter, 2016;
Seufert and Ramankutty, 2017). Both the proponents and opponents of organic farming often fail to
consider the spatial context in which organic farming is occurring
(Seufert et al., 2012). Knowledge on the spatial distribution and the
location characteristics influencing organic certification is limited to
local- or country-scale examples. Proximity to markets in larger urban
centers has been identified as an important spatial driver in several
European examples (Frederiksen and Langer, 2004; Ilbery and Maye,
2010), as well as in Kenya (Ayuya et al., 2015), Korea (Choi, 2016) and
the United States (Kniss et al., 2016; Kuo and Peters, 2017). In England,
organic farmers tend to be located on good soils (Gabriel et al., 2009),
whereas in Germany, they were preferentially located in areas with
poorer soils and a higher share of natural areas (Auerswald et al., 2003;
Schmidtner et al., 2012). Organic farmers in the United States are more
likely found in areas with a more temperate climate and varied terrain
(Kuo and Peters, 2017). However, depending on crop type, these pat-
terns may deviate, as has been demonstrated for organic grape Evidence suggests numerous environmental benefits of organic
farming: reduced soil loss and increased organic matter, lower carbon
emissions, and higher biodiversity values (Bengtsson et al., 2005;
Blackman and Naranjo, 2012; Gomiero et al., 2011). Certified organic
farming can lead to higher profits (Crowder and Reganold, 2015), im-
proving the income of smallholder farmers (Ayuya et al., 2015; Barrett
et al., 2001; Bolwig et al., 2009; DeFries et al., 2017). For certain crop
types, organic farming can under the right conditions maintain yields or
even
improve
the
production
efficiency
(Seufert
et
al.,
2012;
Tzouvelekas et al., 2001). However, such benefits are contested (Hole
et al., 2005), particularly when it comes to looking at environmental
benefits per crop unit (Seufert and Ramankutty, 2017). Some studies Ž. Malek, et al. 1. Introduction Agricultural System
s 176 (2019) 102680 automatic geocoding, some farmers had to be identified manually. Some addresses were descriptive, or the place of the field operation was
not immediately recognized by Google. Addresses in regions that do not
use the Latin alphabet were also mostly not identified automatically. This was the case for several parts of the world – Middle East and North
Africa, Former Soviet Union, and South-east Asia. Details on geocoding
and manual identification of addresses, are provided in Supplementary
Methods. production in California (Kniss et al., 2016). Moreover, organic farmers
are found to concentrate spatially due to strong influence of other
farmers in their direct vicinity (Ilbery and Maye, 2010; Schmidtner
et al., 2012; Wollni and Andersson, 2014). Overarching trends in these
observations, or which factors affect the distribution of organic farmers
on a global level, have not been investigated. Understanding the global distribution and the local spatial context
of organic farmers is important for numerous reasons. First, identifying
the areas where organic farming is present today is crucial to support
expansion of certified cropland in the future (Tayleur et al., 2018). Second, to improve access to organic certification, an understanding of
its spatial distribution is needed (Tayleur et al., 2017). Moreover, to be
fully aware of potential benefits and consequences of organic farming,
we need to understand its intersections with local environments and
cultures (Getz and Shreck, 2006). To support such analysis, our objec-
tive was to systematically map the locations of organic crop farmers
worldwide based on publicly available data from national authorities
and certifiers. We present and analyze, for the first time, the global
spatial distribution of organic crop farming. The only similar mapping
attempt so far focused on certified commodity crops in tropical coun-
tries and did not cover organic certification (Tayleur et al., 2018). Of-
ficial statistics already give an overview of the distribution of organic
farmers globally: the majority of organic crop farmers are smallholders
in developing countries, but a considerable portion of organic cropland
is located in developed countries (Europe, North America and Australia
together represent 50% of organic areas although they only have 6.3%
of all farmers) (Willer and Lernoud, 2018). We went beyond such ag-
gregate statistics by studying the spatial distribution within countries
and the influence of local socio-economic, climate, and soil and terrain
characteristics on the spatial distribution of organic crop farmers. 1. Introduction Geocoded addresses were allocated on a 1 km spatial grid, using an
equal area projection in a geographic information system (ESRI, 2015). This way, the uncertainty related to the potential distance between the
physical address of the crop farmer and the corresponding field op-
eration was reduced. We excluded duplicate records where it was clear
that the certificate presents the same farmer, which can hold multiple
certificates for different crops. Such certificates were identified by the
same farmer name, and same address. We included only one record
from such duplicates. 2.2. Data analysis We studied the potential effect of numerous socio-economic, cli-
matic, soil and terrain variables on the spatial distribution of organic
crop farmers. Depending on the characteristics of the available data,
two different types of analysis were performed. In countries with re-
presentative data (i.e. all organic farmers were included in the dataset)
binomial logistic regressions were performed using locations of organic
crop farmers as presence and a similar sized (random) sample of all
other arable lands as absence data. Logistic regression is a common
approach when studying the spatial determinants of land use and land
management (Neumann et al., 2011; van Asselen and Verburg, 2012;
Van Dessel et al., 2011). We performed logistic regression using SPSS
24 (IBM Corp., 2016). In countries with unrepresentative data (i.e., the
set of organic producers identified was not complete), we applied
maximum entropy modeling that is appropriate to such data structures
(Phillips et al., 2006; Wisz and Guisan, 2009). Data was considered as
representative in countries where the numbers overlapped with re-
ported statistics on organic producers (Willer and Lernoud, 2018). Also,
data from countries where lower numbers of organic farmers were re-
corded but where the data was provided by official governmental in-
stitutions (e.g. government agencies responsible for organic farming,
Table S1) were considered as representative – such countries were
France and Italy. 2.1. Data collection This may in-
dicate that results are indeed sensitive to location inaccuracies in some
contexts, and implies potential uncertainties of our results. At the same
time, this can also describe the heterogeneity of the regional spatial
distribution of organic crop farmers. We hypothesized that bio-physical variables that limit or encourage
agricultural activities and crop choices might influence the choice for
engaging in organic farming. Temperature, precipitation, potential
evapotranspiration (PET), and the aridity index (Hijmans et al., 2005)
were selected as climatic variables as they determine potential vege-
tation growth and represent different aspects of cultivation suitability
for different crop types (Licker et al., 2010; Panagos et al., 2015). To
study the influence of terrain, we focused on altitude and slope that can
pose constraints to agricultural activities (Havlík et al., 2011). We
considered seven soil variables that describe different physical and
chemical soil characteristics: shares of clay and sand, cation exchange
capacity (CEC), soil pH, drainage and depth, and organic content
(ISRIC, 2018; Stoorvogel et al., 2016). Soil variables define the suit-
ability for crop production by characterizing soil structure, fertility, and
ability to store water, while the different factors have different influ-
ences on different crop types (Gardner et al., 1999). All spatial data
were projected to an equal area projection and converted to a 1 km
spatial resolution. All explanatory factors are described in more detail
in the supplementary material (Table S3). In countries and regions where our points presented only a portion
of total producers (Table S1), we performed maximum entropy mod-
eling using MaxEnt (Phillips et al., 2017, 2006). MaxEnt is suitable to
study the factors influencing the spatial distribution in case of limited
presence-only data, with high uncertainty on the exact location of ab-
sence-data (Elith et al., 2011; Fithian and Hastie, 2013). MaxEnt has a
good predictive power particularly with small datasets (Wisz and
Guisan, 2009), useful for countries and regions where we had limited
data on organic farmers. Although mostly used in ecological studies to
study species distribution, it has also been applied in studying agri-
cultural management or cropland extent. Among others, it has been
used to study the distribution of specific crops, potential shifts in their
spatial extent due to climate change, and carbon sequestration potential
of specific land management types (Duan and Zhou, 2013; Liu et al.,
2015; Luedeling and Neufeldt, 2012; Machovina and Feeley, 2013). 2.1. Data collection The global poverty map (Elvidge et al., 2009), and gridded GDP
data (Nordhaus, 2006) were used to represent the influence of the level
of development at a location (Kummu et al., 2018). organic crop farmers were mapped outside the cropland mask (due to
the uncertainties related to the cropland map). We added the areas
where we mapped organic crop farmers to the cropland mask, to not
exclude them from our analysis. To evaluate the predictive ability of
our regression models, we calculated the Receiver Operating Char-
acteristics (ROC) and the Area Under Curve (AUC) values (Supple-
mentary material). The AUC was also interpreted as an indicator of the
extent to which organic farmers differ from conventional farmers based
on spatial location characteristics. Low AUC values indicate that dif-
ferences in the spatial distribution of organic farmers, compared to
conventional farmers, cannot be explained by the included spatial
variables, indicating a probable low difference in the spatial location of
conventional and organic farming. To study the robustness of our
models, additional tests were performed. First, to check if our models
performed better than a random model, we also calculated models
using a randomly allocated presence data set (within the cropland
area). Without exception, all test regressions had AUC values of around
0.5 while no model had an AUC higher than 0.53. Secondly, the re-
gressions were performed using different random samples of absence
points. Our regression results did not change significantly for different
samples for most countries (the regressions consisted of the same
variables with the overall relationships and their strengths remaining
similar). In five countries, regressions with a different random sample
had slightly different results (Supplementary material). Additionally,
three countries had to be excluded (Armenia, Kazakhstan and Uruguay)
as the regressions differed significantly with each different random
absence sample. These countries all have only a few dozen organic
farmer observations within relatively large total farming areas, ex-
plaining this sensitivity. Finally, we performed additional logistic re-
gressions using spatial data that was aggregated to 10 km resolution for
a number of larger countries (Argentina, Australia, Brazil, Canada and
USA) to test sensitivity to location accuracy and data resolution (Sup-
plementary material). We observed that the regressions differed parti-
cularly in terms of local soil characteristics (Argentina, Australia and
Brazil), but also rural population (Argentina, Brazil and USA), market
accessibility and irrigation (Australia, Brazil and USA). 2.1. Data collection Countries were defined as nationally significant in
case the national share of organic cropland covered > 2% of total
cropland. Significant global producers were countries with > 1% of
total global organic cropland. Other countries were defined as niche
countries. The extent of our analyses and the defined global regions are
described in Supplementary material (Figs. S1 and S2). For all criteria,
the latest data from the IFOAM was used (Willer and Lernoud, 2018). We additionally studied the spatial distribution of cooperatives of or-
ganic producers. We performed the logistic regression for Peru (a
country where such data was accessible), using locations of co-
operatives (Fig. S3). The addresses were geocoded using Google Maps (Google Maps,
2017) and mapcarta (Mapcarta, 2017). While most addresses allowed It was hypothesized that access to markets (both by exporting pro-
ducts
and
access
to
more
wealthy
consumers),
and
access
to 2 Ž. Malek, et al. Agricultural System
s 176 (2019) 102680 information and mechanisms related to organic certification were
processes that influence the spatial allocation of organic crop farms. We
therefore used data representing the distribution of population, agri-
cultural activities, distance to markets and level of economic develop-
ment. Population density and the density of rural population were ex-
pected to affect the distribution of organic crop farmers by representing
potential consumers and agricultural activities at a location (Neumann
et al., 2015). The market access index was used to study the accessi-
bility to national and international markets, the effect of available ca-
pital to farmers, and role of road and transport infrastructure (Verburg
et al., 2011). The market access index goes beyond only representing
the distribution of population and cities as it accounts for the distance
to major markets, but also the type of infrastructure, and terrain
characteristics influencing the travel time (comparison with the dis-
tribution of population in the Supplementary Material Table S3 with
detailed documentation in Verburg et al., 2011). This way it helps
identifying areas where it is, among others, easier to access financial
mechanisms, receive information on organic certification, buy agri-
cultural inputs, and finally, making it less costly to transport products to
markets. We used data on areas equipped with irrigation (Siebert et al.,
2013; Siebert et al., 2005) as a proxy to study the effect of the level of
agricultural mechanization and infrastructure (Kuemmerle et al.,
2013). 3.1. First global map of organic crop farmers Globally, organic crop farmers are located in areas with more ben-
eficial socio-economic, climatic and soil conditions (Table 1, Table S5). They can be found in more densely populated areas with better access
to markets, and areas with lower poverty levels. Moreover, it is more
likely that we find them in areas equipped with irrigation. They are
often located in areas with more rainfall, higher temperatures and
lower evapotranspiration. The influence of soil characteristics on the
spatial distribution of organic crop farmers is less pronounced. Overall,
they tend to be more frequently situated in areas with shallow, less
drained soils, a higher pH, and higher organic matter and sand content
(Table 1, Table S5). While they are more likely found at lower eleva-
tions, also a positive association was found with steeper slopes. We mapped 112,724 certified crop farmers in 150 countries around
the world (Figs. 1 and 2). There is a higher density of organic crop
farmers in high-income countries, particularly closer to larger cities
(e.g. Fig. 2a, c–e). In high income countries, organic crop farmers are
often found equally spread in high densities across the total cropland
area within the individual countries (e.g. Fig. 2b). Two main exceptions
to this pattern are found in Argentina and Australia, where, together,
1739 farmers are spread over > 9200 km2 organic cropland (Willer and
Lernoud, 2018). For comparison, over 3000 organic crop farmers in
Slovenia cultivate an area that equals 0.8% of the organic cropland of
Argentina and Australia combined (Willer and Lernoud, 2018). Despite the large number of crop farmers mapped, our collection of
certified farm locations includes only 5% of all global certified organic
crop farmers. However, together the farmers included in our data cul-
tivate 38% of the total global organic cropland. Cropland represents
36% of the total global organic area (Willer and Lernoud, 2018). We
likely recorded considerably more crop farmers in countries where one
certificate presents a cooperative or a similar collective association - a
predominant way of organic production in numerous countries of Latin
America, Africa and the former Soviet Union (Bravo-Monroy et al.,
2016; Jena et al., 2012; Willer and Lernoud, 2018)ypo. Although
membership data on individual cooperative certificates is not available,
certifiers report that 711 cooperative certificates from Latin America
correspond to 63,000 producers (whereas we treated them as 711
certificates) (BIOLATINA, 2018). 2.1. Data collection The map presents the total number of all collected data aggregated to 100 × 100 km
spatial units (Eckert IV equal area projection). Countries with representative/unrepresentative data are defined in the Table S1. Cropland (Fritz et al., 2015) is
marked with gray. 2.1. Data collection Similar to the logistic regression analysis, we limited the MaxEnt ana-
lysis to cropland areas only. Details of our MaxEnt application are de-
scribed in the Supplementary material. Correlations between variables were calculated to be able to reduce
multicollinearity for each country or region. Highly correlated variables
(Pearson correlation coefficient > 0.8) were excluded. For the logistic
regression, the data was standardized at the level of analysis (country,
region, global). Forward conditional regressions were conducted in countries with
representative data using a balanced sample of presence (organic crop
farmers) and absence points for each country or region. For absence
points, we randomly selected a spatially balanced sample distributed on
a cropland mask to reduce potential bias due to spatial autocorrelation. Such an approach has been demonstrated as optimal when sampling
absence data (Hirzel and Guisan, 2002), particularly to reduce the
uncertainty related to biased sampling, and sampling of locations,
where organic crop farmers are unlikely to occur. Before sampling the
absence data, we excluded the locations of organic crop farmers, to
remove pseudo-absence data (Wisz and Guisan, 2009). We used the
IIASA-IFPRI hybrid cropland map (Fritz et al., 2015) as a cropland mask
for all countries and regions outside Europe. In Europe, the CORINE
land cover map was used (EEA, 2015). In Belgium, Denmark and Slo-
venia, the data did not allow for differentiation between crop and li-
vestock farmers. In these three countries we also considered pastures,
together with the cropland mask. In Australia and Argentina, a few Farmers' decisions to adopt organic farming are heavily influenced
by other farmers in their surroundings who converted to organic
(Allaire et al., 2015a; Gabriel et al., 2009; Schmidtner et al., 2012;
Wollni and Andersson, 2014). We therefore also looked at neighbor-
hood effects by counting the number of occurrences of finding multiple
organic crop farmers located within the same 1 km cell. 3 Ž. Malek, et al. Agricultural System
s 176 (2019) 102680 Fig. 1. Global spatial distribution of collected organic crop farmers' certificates. The map presents the total number of all collected data aggregated to 100 × 100 km
spatial units (Eckert IV equal area projection). Countries with representative/unrepresentative data are defined in the Table S1. Cropland (Fritz et al., 2015) is
marked with gray. Fig. 1. Global spatial distribution of collected organic crop farmers' certificates. 3. Results 3.2. Organic crop farmers are concentrated in areas with favorable socio-
economic, climate and soil conditions 3.1. First global map of organic crop farmers Data on organic crop farmer dis-
tribution is representative for 42 countries, mostly in North America,
Oceania, and considerable parts of Europe and South America (Tables
S1, S4). However, data on organic crop farmers in important organic
producing countries such as China, Germany, Spain, and Poland are
missing. In Africa and Asia (together with the former USSR), we only
cover 6 countries with representative data, and mapped 0.1 and 1% of
all farmers respectively (Table S4). Small differences are found between countries with different shares
of organic agriculture (Table 1, Table S5). In countries where organic
farming has a high share, rural population and irrigation are not sig-
nificant, and organic crop farmers are more likely to occur on soils with
a lower organic content. In niche countries, where the share of organic
farming is low, the occurrence of organic crop farmers is negatively
correlated to rural population. When looking at individual countries and regions, again, the posi-
tive effect of favorable socio-economic, climatic and soil characteristics
on the distribution of organic crop farmers is observed (Tables 1 and 2,
and Tables S6, S7 and S8). Particularly poverty has a negative effect on
the likelihood that an organic crop farmer is present at a location,
which we identified in 34 of the 42 countries and regions analyzed
(Tables S6 and S8). However, there are also clear differences between
countries. The negative influence of poverty is not limited to lower
income countries – organic crop farmers are also negatively related to
poverty in upper-middle- and high-income countries such as Argentina,
Canada, Chile, China, and several members of the European Union. In
Australia, Egypt, and the United States, organic crop farmers are even
more likely to be found in areas with higher degrees of poverty/lower
wealth (Table 1). In most European countries, rural population density 4 Agricultural System
s 176 (2019) 102680 Ž. Malek, et al. Detailed distribution of organic crop farmers in a) Japan, b) Italy, c) South Africa, d) Eastern Australia, and e) United States of America. The map p
mber of all collected data within a 25 × 25 km spatial unit (Eckert IV equal area projection). Data for all these countries is representative. The lege
gures. Major cities are settlements with over 200,000 inhabitants (Natural Earth, 2018). Fig. 2. 3.1. First global map of organic crop farmers Detailed distribution of organic crop farmers in a) Japan, b) Italy, c) South Africa, d) Eastern Australia, and e) United States of America. The map presents the
total number of all collected data within a 25 × 25 km spatial unit (Eckert IV equal area projection). Data for all these countries is representative. The legend is valid
for all figures. Major cities are settlements with over 200,000 inhabitants (Natural Earth, 2018). 5 Agricultural System
s 176 (2019) 102680 Ž. Malek, et al. ized maximum entropy results for organic crop farmers in selected regions and countries with unrepresentative data. Values present the contribution of a variable to the spatial distribution of organic crop farmers
d the direction of the influence (+ −means that a variable is positive to a certain value and then negative). Empty cells present variables with a contribution below 1%. Variables with * contribute > 1% in
al runs without poverty. AUC (area under curve) is a measure of the goodness of fit of the maximum entropy model. Complete maximum entropy results for all studied countries are provided in Table S8. Sample size
Population density
Rural population
Market access
Poverty
Irrigation
Soil drainage
Organic content
Soil pH
Slope
Altitude
Temperature
Precipitation
AUC
East
185
44.5
1
1.2
−20.8
4
−2.5
−2.1
± 1.8
15.1
0.96
West
93
8.2
4.2
31.8
−6.8
−2.2
−1.8
9.3
0.96
216
4.8
6
−13.5
1.9
−4.9
3.3
4.8
−4.6
0.98
America
343
7.4
2.2
8.8
−25.8
−4.1
−2.9
1.8
± 1.2
14.8
7.3
6.9
2.6
0.92
269
1.7*
−1.6*
3.1*
−74.1
−2.5
1.4
−1.8
± 1.7
−5
2.2
0.9
r
392
3.3*
−1.8*
1.7
−72.4
3.1
2.7
−0.6*
3.2
3.7*
−2.8
0.96
1692
2.9
−41.6
−7.4
3.7
−1.1
−3.7
−2.7
5
7.4
0.86
East and North Africa
224
2.5
−1.7
5.7
−28.6
−7.6
−2.3
1.3
−39.6
1.4
−1
0.95
1698
1.1
1*
−78.3
4.3*
2.4
−1.7
1.6
± 2.9
−0.2*
1.1
0.9
766
−13.3
−5.6
7
−35.7
−4.3
± 2.4
−3.1
−3.7
20.1
−2.5
0.92
ast Asia - mainland
178
14.3
4.8
−22.2
± 6.1
3.1
−5.2
−5.9
17.9
−8.5
0.9
ast Asia - maritime
263
−5.2
3.4
20.3
−29
−2.8
−3.5
± 1.3
−3.1
17.5
2.5
−2.8
0.95
418
± 3.4
−45
−2.7
−6.5
2.3
−4.1
20.2
0.92 Table 1
Summarized regression results for organic crop farmers globally and in selected countries with representative data. 3.1. First global map of organic crop farmers All regression coefficients are reported only for variables that have p < .05 significance (forward
conditional regression), variables marked with * have p < .01 significance. Empty cells mean a variable is not significant for that country. AUC (area under curve) is a measure for the goodness of fit of the regression
model. Complete regression results for all studied countries are provided in Tables S5, S6 and S7. Sample size
Population density
Rural population
Market access
Poverty
Irrigation
Soil drainage
Soil organic content
Soil pH
Slope
Altitude
Temperature
Precipitation
AUC
Argentina
556
0.66*
−1.79*
0.50*
−0.92*
0.44*
0.84*
−0.37*
0.97
Australia
1183
1.78*
0.11*
0.34*
−0.08*
−0.20*
0.34*
0.46*
0.30*
0.91
Austria
18,551
−0.13*
−0.03
−0.07*
−0.10*
0.18*
−0.12*
0.23*
0.63
Belgium
2015
−0.21*
−0.17*
−0.12*
−0.11
0.64*
0.74
Brazil
9004
0.92*
0.75*
0.17*
−0.33*
−0.09*
0.16*
0.12*
−0.23*
0.11*
0.94
Canada
2568
0.79*
0.25*
0.12
−0.35*
0.17*
−6.50*
0.71*
0.89
Egypt
289
0.07
1.12*
0.49
−1.16*
−0.85*
−1.50*
−10.48*
0.89
France
11,624
0.06*
−0.05*
0.04*
−0.27*
−0.05*
−0.07*
0.03
0.11*
0.11*
0.23*
0.07*
0.63
Italy
18,985
0.06*
−0.08*
−0.34*
−1.41*
−0.41*
0.04*
−0.44*
0.06*
0.28*
0.35*
−0.04*
0.80
Japan
1485
0.47*
0.35*
−0.82*
−0.21
0.49*
0.82
New Zealand
523
3.52*
0.25*
−0.22
0.21*
0.22
−1.43*
−0.67*
1.05*
0.87
India - Odisha
4275
0.24*
−0.38*
−0.32*
−0.21*
0.24*
−1.19*
−0.37*
0.13*
0.71*
0.68*
0.84
Peru - cooperatives
311
−0.26*
0.24*
0.15
0.71
Slovenia
3516
−0.13*
0.17*
0.07*
−0.13*
1.01*
0.53*
0.70
South Africa
119
3.0
−1.08*
0.28
0.52
−0.35
0.92
Ukraine
294
0.29*
−0.59*
0.24
−0.57*
0.80*
0.80
USA
11,457
0.23*
0.36*
0.00*
0.26*
−0.17*
−0.22*
−0.51*
−0.82*
0.78
Global - all
98,752
0.58*
0.09*
0.37*
−0.43*
0.18*
−0.04*
0.03*
0.04*
0.33*
−0.09*
1.00*
0.33*
0.88
Global - Nationally significant
71,692
0.24*
0.16*
−0.53*
−0.07*
0.33*
0.06*
0.38*
0.25*
0.85
Globally significant
22,945
0.57*
0.14*
0.29*
0.21*
−0.12*
0.03*
0.07*
0.22*
−0.29*
−0.9*
0.59*
0.83
Global - Niche
4115
0.23*
−0.07*
0.20*
−0.61*
0.18*
0.09*
−0.25*
1.13*
0.65*
0.90 Table 1
Summarized regression results for organic crop farmers globally and in selected countries with representative data. All regression coefficients are reported only for variables that have p < .05 significance (forward
conditional regression), variables marked with * have p < .01 significance. Empty cells mean a variable is not significant for that country. AUC (area under curve) is a measure for the goodness of fit of the regression
model. 3.1. First global map of organic crop farmers Complete regression results for all studied countries are provided in Tables S5, S6 and S7. Sample size
Population density
Rural population
Market access
Poverty
Irrigation
Soil drainage
Soil organic content
Soil pH
Slope
Altitude
Temperature
Precipitation
AUC
Argentina
556
0.66*
−1.79*
0.50*
−0.92*
0.44*
0.84*
−0.37*
0.97
Australia
1183
1.78*
0.11*
0.34*
−0.08*
−0.20*
0.34*
0.46*
0.30*
0.91
Austria
18,551
−0.13*
−0.03
−0.07*
−0.10*
0.18*
−0.12*
0.23*
0.63
Belgium
2015
−0.21*
−0.17*
−0.12*
−0.11
0.64*
0.74
Brazil
9004
0.92*
0.75*
0.17*
−0.33*
−0.09*
0.16*
0.12*
−0.23*
0.11*
0.94
Canada
2568
0.79*
0.25*
0.12
−0.35*
0.17*
−6.50*
0.71*
0.89
Egypt
289
0.07
1.12*
0.49
−1.16*
−0.85*
−1.50*
−10.48*
0.89
France
11,624
0.06*
−0.05*
0.04*
−0.27*
−0.05*
−0.07*
0.03
0.11*
0.11*
0.23*
0.07*
0.63
Italy
18,985
0.06*
−0.08*
−0.34*
−1.41*
−0.41*
0.04*
−0.44*
0.06*
0.28*
0.35*
−0.04*
0.80
Japan
1485
0.47*
0.35*
−0.82*
−0.21
0.49*
0.82
New Zealand
523
3.52*
0.25*
−0.22
0.21*
0.22
−1.43*
−0.67*
1.05*
0.87
India - Odisha
4275
0.24*
−0.38*
−0.32*
−0.21*
0.24*
−1.19*
−0.37*
0.13*
0.71*
0.68*
0.84
Peru - cooperatives
311
−0.26*
0.24*
0.15
0.71
Slovenia
3516
−0.13*
0.17*
0.07*
−0.13*
1.01*
0.53*
0.70
South Africa
119
3.0
−1.08*
0.28
0.52
−0.35
0.92
Ukraine
294
0.29*
−0.59*
0.24
−0.57*
0.80*
0.80
USA
11,457
0.23*
0.36*
0.00*
0.26*
−0.17*
−0.22*
−0.51*
−0.82*
0.78
Global - all
98,752
0.58*
0.09*
0.37*
−0.43*
0.18*
−0.04*
0.03*
0.04*
0.33*
−0.09*
1.00*
0.33*
0.88
Global - Nationally significant
71,692
0.24*
0.16*
−0.53*
−0.07*
0.33*
0.06*
0.38*
0.25*
0.85
Globally significant
22,945
0.57*
0.14*
0.29*
0.21*
−0.12*
0.03*
0.07*
0.22*
−0.29*
−0.9*
0.59*
0.83
Global - Niche
4115
0.23*
−0.07*
0.20*
−0.61*
0.18*
0.09*
−0.25*
1.13*
0.65*
0.90
Table 2
Summarized maximum entropy results for organic crop farmers in selected regions and countries with unrepresentative data. Values present the contribution of a variable to the spatial distribution of organic crop farmer
in %, and the direction of the influence (+ −means that a variable is positive to a certain value and then negative). Empty cells present variables with a contribution below 1%. Variables with * contribute > 1% in
additional runs without poverty. AUC (area under curve) is a measure of the goodness of fit of the maximum entropy model. Complete maximum entropy results for all studied countries are provided in Table S8. 3.1. First global map of organic crop farmers In developing countries, organic farmers mostly produce com-
modity crops for export to more developed markets with only a minor
portion being produced for domestic consumption (Willer and Lernoud,
2018). Being close to established supply chains of commodity crops
(Ton, 2013) can, therefore, explain the identified positive effect of
market accessibility. Furthermore, crop farmers who are better con-
nected to markets are also more likely to receive more information on
the benefits of organic farming, whereas farmers in remote areas are
mostly influenced informally (Wollni and Andersson, 2014). In nearly all analyzed countries and regions, organic crop farmers
are positively related to precipitation and temperature (Tables 1, 2, and
Tables S5, S6, S8). In a few countries, however, they tend to occur in
drier areas. These are countries in north and south Africa, south-east
Asia, Ecuador, Italy, Peru, and Brazil (Table 1, Tables S6 and S8). In
countries spanning over different climatic regions such as Argentina,
Brazil, China, Mexico and the United States, organic crop farmers are
more likely found in more temperate areas with lower temperatures
(Tables 1, 2). There are fewer similarities among countries in terms of the effect of
soil and terrain conditions, demonstrating that their role depends on
the regional/local context (and potentially depends on different crop
types specific for some regions). The effect of soils is more similar in
South American countries, looking at the influence of clay, pH and soil
depth (Tables 1, 2, Tables S4, S6). In Australia, Croatia, Italy, Turkey,
the United States, the Indian state of Odisha, and countries in West
Africa combinations of soil conditions indicate that organic farmers
cultivate land less suitable for cropland activities (Tables 1,2 and Tables
S6 and S8). In South and West Africa, mainland and maritime South-
East Asia, France, Italy and Slovenia, and numerous South American
countries, organic crop farmers are more likely found at higher alti-
tudes. In Mexico and the rest of Central America, East Africa and China,
they tend to occur in mid-altitudes (Table 2, Table S8). Organic crop
farmers on steeper slopes are more likely found in developed countries,
probably indicating a high share of permanent crops (e.g. vineyards and
orchards) (Willer and Lernoud, 2018). 3.1. First global map of organic crop farmers Sample size
Population density
Rural population
Market access
Poverty
Irrigation
Soil drainage
Organic content
Soil pH
Slope
Altitude
Temperature
Precipitation
AUC
Africa - East
185
44.5
1
1.2
−20.8
4
−2.5
−2.1
± 1.8
15.1
0.96
Africa - West
93
8.2
4.2
31.8
−6.8
−2.2
−1.8
9.3
0.96
Bolivia
216
4.8
6
−13.5
1.9
−4.9
3.3
4.8
−4.6
0.98
Central America
343
7.4
2.2
8.8
−25.8
−4.1
−2.9
1.8
± 1.2
14.8
7.3
6.9
2.6
0.92
China
269
1.7*
−1.6*
3.1*
−74.1
−2.5
1.4
−1.8
± 1.7
−5
2.2
0.9
Ecuador
392
3.3*
−1.8*
1.7
−72.4
3.1
2.7
−0.6*
3.2
3.7*
−2.8
0.96
India
1692
2.9
−41.6
−7.4
3.7
−1.1
−3.7
−2.7
5
7.4
0.86
Middle East and North Africa
224
2.5
−1.7
5.7
−28.6
−7.6
−2.3
1.3
−39.6
1.4
−1
0.95
Mexico
1698
1.1
1*
−78.3
4.3*
2.4
−1.7
1.6
± 2.9
−0.2*
1.1
0.9
Peru
766
−13.3
−5.6
7
−35.7
−4.3
± 2.4
−3.1
−3.7
20.1
−2.5
0.92
Southeast Asia - mainland
178
14.3
4.8
−22.2
± 6.1
3.1
−5.2
−5.9
17.9
−8.5
0.9
Southeast Asia - maritime
263
−5.2
3.4
20.3
−29
−2.8
−3.5
± 1.3
−3.1
17.5
2.5
−2.8
0.95
Turkey
418
± 3.4
−45
−2.7
−6.5
2.3
−4.1
20.2
0.92
6 6 Ž. Malek, et al. Agricultural System
s 176 (2019) 102680 does not play a significant role. Organic crop farmers are in Austria,
Ireland, and Italy more likely found in areas less connected to markets
(Table 1). In several of the studied countries and regions (19) organic
crop farmers are negatively related to the extent of irrigation (Tables S6
and S8). This shows, that irrigation is not necessarily relevant to the
occurrence of organic agriculture on a local scale, depending on re-
gional agricultural contexts. GDP, and irrigation also suggest that organic crop farmers are more
likely to be found in areas with better socio-economic conditions. This
is supported by local studies, where particularly distance to markets
and urban population have been identified to play a role in the con-
version to organic farming, both for reaching affluent domestic markets
as well as for better access to international markets (Allaire et al.,
2015b; Giovannucci and Ponte, 2005; Karki et al., 2011; Koesling et al.,
2008). 3.1. First global map of organic crop farmers Higher likelihood for organic
crop farmers in areas with higher altitudes, steeper slopes and poorer
soils can, however, also be explained by economic and agronomic ob-
stacles in converting to organic farming on more suitable areas (e.g. profitability of conventional agriculture on fertile plains can present an
obstacle to conversion). Additionally, our findings on how locations of organic crop farmers
in developed organic markets are more similar to conventional farmers
can be explained by specific regional and historic contexts. First, these
highly developed organic markets are mostly smaller (European)
countries, where consumers and export markets are close to producers –
making it possible to sell products locally (e.g. Ilbery and Maye, 2010). These are countries with high levels of overall socio-economic devel-
opment, with a relatively large consumer base (more wealthier con-
sumers). Such countries also have a well-developed certification sector,
good transport and institutional infrastructure. Finally, organic certifi-
cation has been present for a while in developed organic markets such
as the European Union, where regulation on organic production of
agriculture already exists since 1991 (EC, 1991; Padel et al., 2009). In
less developed organic markets (“niche” countries), organic certifica-
tion is at a much earlier stage and may still be developing. Organic farming affects the whole farming system and is not an
adoption of a single technique – in developed countries, converting can
mean a significant change to established farming practices, and can
result in failure due to risks in the transition period (Kerselaers et al.,
2007). Farmers in poor areas who cannot afford such experimentation
could, therefore, be less likely to convert. Evidence from developing
countries, however, also suggests that conversion to organic farming
might be a smaller obstacle, as agriculture can be organic by default,
but not yet certified (Ayuya et al., 2015; Bolwig et al., 2009). Conver-
sion to organic farming in such conditions can be merely a continuation
of existing cropland management, fertilization and pest control prac-
tices (Bolwig et al., 2009). Nevertheless, optimization of farm opera-
tions, implementation of biological plant protection and fertilization,
with potential changes to crop types would be necessary to achieve a
successful conversion to organic. This is especially the case when there
is poor cropland management due to lower accessibility (or lack of fi-
nances) to agricultural inputs. In developed organic markets, we identified less evident influences
of spatial characteristics on the spatial distribution of organic crop
produces. 3.1. First global map of organic crop farmers This is demonstrated by their spatial pattern, weak regression
coefficients and low AUCs (area under curve of the Receiver Operating
Characteristic) (Table 1, Table S5). We observed this mostly in Eur-
opean countries: Austria, Czech Republic, France, Ireland, and parti-
cularly Denmark and the Netherlands (Table S6). Countries with the
smallest differences in location between conventional and organic
farming are also countries with the highest shares of organic farmers, as
indicated by the negative relationship between the AUC and the share
of organic farmers (Fig. S4). In this study, we focused on a set of socio-economic variables that
can explain a considerable part of the spatial distribution of organic
crop farmers. However, explaining the socio-economic processes that
limit or drive conversion is complex. Organic farmers are a hetero-
geneous group with a variety of attitudes towards the choice of farming
method, including changes to lifestyle and environmental values
(Darnhofer et al., 2005; Malek et al., 2019; Tzouramani et al., 2014). Moreover, there are large differences in the spatial determinants for
certification of different crop types (Tayleur et al., 2018). The data
collected for this study did not allow distinguishing producers of dif-
ferent crop types. Other potential obstacles for certification we were
unable to address are related to bureaucracy and the required financial
resources, lower education and insufficient information, and organiza-
tional support (Barrett et al., 2001; Beltrán-Esteve et al., 2012;
Boncinelli et al., 2017; Salazar, 2014; Veldstra et al., 2014). In some countries we observed strong clustering of organic produ-
cers in specific locations, demonstrated by the ratio between the total
number of organic crop farmers and the number of unique locations
(Table S9). For example, around 9000 organic crop farmers in Brazil are
located on 1250 unique locations. Countries with a considerable share
of organic farmers that are characterized by a high share of unique
locations are mostly European countries like Belgium, Czech Republic,
Denmark, France and the Netherlands. 5. Conclusion Organic farming is promoted as a way to provide food in a more
sustainable way, reducing the environmental impacts of agriculture. Our results indicate, that, particularly in countries where organic
agriculture is less developed, organic crop farmers are present in areas
with relatively favorable socio-economic conditions. To sustain trends
of increases in organic production in these countries (Willer and
Lernoud, 2018), efforts are needed to support access to certification for
farmers in poorer regions and by providing better market access. In
developing countries, organic certification often implies a continuation
of existing cropland management (Ayuya et al., 2015; Bolwig et al.,
2009). Therefore, it is mainly the certification and access to value
chains to reach consumers that is hampering a further expansion of
certified organic production. Targeting such areas with less-favorable
socio-economic conditions can indeed pose a higher risk for estab-
lishing a steady and successful supply of organic products due to a
potentially higher rate of certification failures and problems in estab-
lishing value chains. Nevertheless, this way organic farming can be-
come a tool for improving famers' livelihoods while at the same time
limiting the input of artificial fertilizers and pesticides. The process can,
therefore, be seen as a component of sustainable intensification stra-
tegies. Ayuya, O.I., Gido, E.O., Bett, H.K., Lagat, J.K., Kahi, A.K., Bauer, S., 2015. Effect of
certified organic production systems on poverty among smallholder farmers: em-
pirical evidence from Kenya. World Dev. 67, 27–37. https://doi.org/10.1016/j. worlddev.2014.10.005. Barrett, H.R., Browne, A.W., Harris, P.J.C., Cadoret, K., 2001. Smallholder farmers and
organic certification: accessing the EU market from the developing world. Biol. Agric. Hortic. 19, 183–199. https://doi.org/10.1080/01448765.2001.9754920. Beltrán-Esteve, M., Picazo-Tadeo, A.J., Reig-Martínez, E., 2012. What makes a citrus
farmer go organic? Empirical evidence from Spanish citrus farming. Span. J. Agric. Res. 10, 901. https://doi.org/10.5424/sjar/2012104-2957. Bengtsson, J., Ahnström, J., Weibull, A.-C., 2005. The effects of organic agriculture on
biodiversity and abundance: a meta-analysis: organic agriculture, biodiversity and
abundance. J. Appl. Ecol. 42, 261–269. https://doi.org/10.1111/j.1365-2664.2005. 01005.x. Beuchelt, T.D., Zeller, M., 2011. Profits and poverty: Certification's troubled link for
Nicaragua's organic and fairtrade coffee producers. Ecol. Econ. 70, 1316–1324. https://doi.org/10.1016/j.ecolecon.2011.01.005. https://doi.org/10.1016/j.ecolecon.2011.01.005. BIOLATINA, 2018. Listas de Operadores Certificados. List of Certified Operators, Certified
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on location choices is similar to what has been observed for certified
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s 176 (2019) 102680 4.2. The role of data and collective associations The datasets analyzed in this study are publicly available from the
sources listed in the article or the Supplementary material. All data
prepared in this paper on the approximate locations of organic farmers
are available freely on https://dataverse.nl/dataverse/BETA (upon
publication of the paper). Our inventory shows that significant efforts for improving the ac-
cessibility of data on organic farmers are necessary. Only then will it be
possible to identify location characteristics that drive and limit certified
organic farming in developing countries in more detail as well. To
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considerably more organic crop farmers than the number of mapped
certificates suggests. In some regions (e.g. India and Latin America) one
certificate often presents a group association (e.g. cooperative). Exact
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available for only 29 of these cooperative certificates, but these certi-
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locations suggest. When assuming that our records cover all certificates
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ditional crop farmers (Willer and Lernoud, 2018) and 12 more countries
would have representative data. Acknowledgments The authors would like to thank Helga Willer from the Research
Institute of Organic Agriculture (FiBL) Switzerland, Tegan Renner from
the Atlantic Canada Organic Regional Network (ACORN), Annemie
Lambert from BioForum Vlaanderen, and Lianne Gouma and Nynke
Schulp from Vrije Universiteit Amsterdam for their help in data col-
lection. The research in this paper was supported by the European
Research Council ERC grant GLOLAND (no. 311819). This paper con-
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Spatial Distribution of Droughts in the Titicaca Lake Basin
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Resumo A presente pesquisa avaliou a distribuição espacial do risco de seca na bacia hidrográfica do lago Titicaca localizada em
território peruano para uma escala de distrito, com base em perigo e vulnerabilidade. O perigo foi quantificado com o
déficit de precipitação usando Índice de Precipitação Padronizado (SPI, por suas siglas em inglês), para uma escala de
tempo de três meses; e a vulnerabilidade foi obtida de acordo com os indicadores socioeconômicas e físicas da bacia. Os
resultados mostram que a seca é fortemente modulada por condições anômalas de TSM dos oceanos circundantes, prin-
cipalmente pela região do Pacífico. Na Bacia do Lago Titicaca, foi identificado que aproximadamente 50% dos distritos
apresentam alto a muito alto risco de seca, localizados principalmente no Noroeste, centro-leste, e centro-sul da bacia. Esses distritos apresentam um maior déficit de precipitação e apresentam indicadores mais vulneráveis ao perigo de seca
devido ao facto de depender economicamente de uma agricultura de sequeiro precária e de um sistema de pecuária
extensiva e mista. Nesta área estão as províncias mais importantes da região alto-andino, como Puno, San Roman,
Azángaro, Melgar e Carabaya, e destacam-se distritos como Puno e Juliaca considerado o capital econômico do depar-
tamento de Puno. Palavras-chave: Bacia hidrográfica do lago Titicaca, seca, risco, perigo, vulnerabilidade. Autor de correspondência: Wendy A. Alonso, waramayo@lamolina.edu.pe. Revista Brasileira de Meteorologia, v. 37, n. 3, 289 304, 2022
DOI: http://dx.doi.org/10.1590/0102-77863730054 Revista Brasileira de Meteorologia, v. 37, n. 3, 289 304, 2022
DOI: http://dx.doi.org/10.1590/0102-77863730054 rbmet.org.br Article 1Departamento de Ordenamiento Territorial y Construcción, Universidad Nacional Agraria La
Molina, Lima, Perú.
2Servicio Nacional de Meteorología e Hidrología del Perú, Lima, Perú.
3Departamento de Recursos Hídricos, Universidad Nacional Agraria La Molina, Lima, Perú. Received: 7 February 2021 - Accepted: 8 February 2022 Abstract The present research has assessed the spatial distribution of drought risk in the Titicaca Lake Basin located in Peruvian
territory for a district scale, based on hazard and vulnerability. Drought hazard has been quantified with the deficit of
precipitation using the Standardized Precipitation Index (SPI) for a time scale of 3-months, and the vulnerability has
been obtained according to the socio-economic and physical indicators of the Basin. The results show that the drought is
strongly modulated by anomalous SST conditions of the surrounding Oceans, mainly by the Pacific region. Over the
Titicaca Lake Basin was identified that about 50% of districts present a high to very high risk of drought mainly, in the
northwestern, central-east, and central-south of the Basin. These districts have a larger deficit of precipitation and
showed indicators that are more vulnerable to the drought hazard due to that economically depends on precarious
rainfed agriculture and an extensive and mixed livestock system. Within this area are the most important provinces of
the high-Andean region, such as Puno, San Roman, Azángaro, Melgar, and Carabaya, and outstanding districts as Puno
and Juliaca, considered the economic capital of the department of Puno. Keywords: Titicaca Lake basin, drought, risk, hazard, vulnerability. Wendy A. Alonso1
, Waldo Lavado-Casimiro2
, Raúl Espinoza-Villar1
,
Eduardo Chávarri-Velarde3 Wendy A. Alonso1
, Waldo Lavado-Casimiro2
, Raúl Espinoza-Villar1
,
Eduardo Chávarri-Velarde3 1Departamento de Ordenamiento Territorial y Construcción, Universidad Nacional Agraria La
Molina, Lima, Perú. 2Servicio Nacional de Meteorología e Hidrología del Perú, Lima, Perú. 3Departamento de Recursos Hídricos, Universidad Nacional Agraria La Molina, Lima, Perú. 1Departamento de Ordenamiento Territorial y Construcción, Universidad Nacional Agraria La
Molina, Lima, Perú. 2Servicio Nacional de Meteorología e Hidrología del Perú, Lima, Perú. 3Departamento de Recursos Hídricos, Universidad Nacional Agraria La Molina, Lima, Perú. 1. Introduction planet (Ortega, 2013). It is therefore considered by many
to be the most complex but least understood of all-natural
hazards (WMO, 2016). It differs from other hazards such
as floods, tropical cyclones, and earthquakes because it
builds up slowly over a considerable period and can per- Drought is one of the most damaging natural hazards
related to climate (Sönmez et al., 2005), and is considered
to affect more people than any other natural disaster on the Spatial distribution of droughts in the Titicaca Lake Basin 290 sist for years after the event has ended, making it difficult
to determine the beginning and end (Tannehill, 1947).i 2019). During the austral winter (in the absence of the
SAMS influence), the moist eastern flux is replaced by
westerly winds, which provide dry air related to the atmo-
spheric stability over the Pacific Ocean. This strong sea-
sonality explains that almost all precipitation occurs in 1-3
month (Lavado et al., 2012). g
g
(
)
Usually, droughts are classified according to type, in
meteorological, agricultural, and hydrological, and they
are differentiated by their intensity, duration, and spatial
extent (OMM, 2012). Meteorological drought is defined as
a threshold of precipitation deficit that is reached during a
period previously determined (OMM, 2006). The majority
of available drought-related indices focus on the assess-
ment of meteorological drought, for example, Standard
Precipitation Index (SPI), Palmer Index (PI), Simple Pre-
cipitation Intensity Index (SPII) (Rojas, 2021). The index
most used is the Standardized Precipitation Index (SPI),
which is recommended by the World Meteorological
Organization (WMO) as the standard drought index (Ser-
afini et al., 2019) given its simplicity and flexibility for the
study of precipitation on various time scales (Endara et al.,
2019). Mckee et al. (1993) originally developed the SPI to
quantify the precipitation deficit, for different time scales
of 3, 6, 12, 24, and 48 months. Short-term scales such as
months may be important for agricultural interests, while
long-term scales such as years may be transcendental for
interests in water supply management (Guttman, 1998). The present research assesses the spatial distribution
of drought risk in the Titicaca Lake Basin located in Per-
uvian territory for a district territorial division, based on
hazard and vulnerability. 1.1. Geographical and climatic characteristics of the
Titicaca Lake basin The hydrographic Basin of Lake Titicaca is located
in South America, south of Peru and northwest of Bolivia,
between 14°05' and 16°50' north longitude and 68°10' and
71°05' west longitude, on the Peruvian territory covers an
area of 48,910 km2 including the part corresponding to the
Lake, with an altitude varying between 3,812 and
5,500 m.a.s.l. In addition, has a cold and dry climate, with
a maximum temperature of 18 °C, a minimum of -2 °C,
and an average of 10 °C. The spatial distribution of annual
mean precipitation has a decreasing pattern from north to
south. In general, it varies from 200 to 1,400 mm (INEI,
2013). In Peru, the droughts are quite often since last dec-
ades, according to Lovón (1985). Between 2000 and 2010,
163 drought events with different frequencies were repor-
ted at the national level, being higher on the Basin of Paci-
fic, followed by the Titicaca Lake Basin (ANA, 2013). The Titicaca Lake Basin is located in the southern region
of Peru, an area where several drought events have occur-
red (Vega, 2016). According to the National Institute of
Civil Defense (INDECI, by acronym in Spanish), histori-
cally the area of the southern highlands of Peru is the area
most prone to the occurrence of droughts, this area
includes the departments of Puno, Cusco, Tacna, Moque-
gua, Arequipa and Apurimac, with 70% of the population
economically active in rainfed agriculture and livestock. One of the most critical occurred at the end of 1982 and
the beginning of 1983, it was an exceptional drought that
fundamentally affected the highland the Puno and Cusco
departments (Lovón, 1985). In Peruvian territory, the Titicaca Lake Basin is
located within the high-Andean region of the department
of Puno, occupying approximately 63% of its surface. This is composed of 13 provinces and 90 districts, as can
be seen in Fig. 1 and listed in Table 1. The district with the
largest area is Santa Rosa district with 2,472 km2, belong-
ing to the province of El Collao, followed by Nuñoa dis-
trict with 2,209 km2, located in the province of Melgar. Likewise, the Titicaca Lake Basin is made up of 16 hydro-
graphic units, as can be seen in Fig. 2. The hydrographic
unit of Azángaro has the largest area with 8,754 km2, fol-
lowed by the Pucará with 5,541 km2. 1. Introduction The drought hazard is estimated
with the precipitation deficit monthly rainfall obtained
from the latest version of product PISCO - Precipitation
v1.2 (Peruvian Interpolation of the SENAMHIs Climato-
logical and Hydrological Stations) for the period 1981 to
2016 (36 years), and then is quantified with the Standar-
dized Precipitation Index (SPI) for a time scale of 3-
months. The vulnerability is obtained according to socio-
economic and physical indicators of the hydrographic
Basin of Lake Titicaca. 1.1. Geographical and climatic characteristics of the
Titicaca Lake basin The Lake Titicaca Basin region features several
characteristics that influence the spatial-temporal rainfall
distribution, and many studies have shown that regions
with lakes (e.g., Lake Titicaca Basin) are affected by a
combination of orographic and convective precipitation
(Chuchón and Pereira, 2022). The rainy season over the
Altiplano occurs during the austral summer linked to the
active phase of the South America Monsoon System-
SAMS (Zhou and Lau 1998; Vera et al., 2006). The peak
of precipitation occurs in January, and is characterized by
intense convective
activity
combined
with moisture
advection from the Amazon Basin (Aceituno and Mon-
tecinos 1993; Chaffaut et al., 1998; Vuille et al., 1998;
Garreaud et al., 2003; Vizy and Cook, 2007; Mayta et al., 2.1.1. Precipitation data Monthly rainfall is obtained from product PISCO-
Precipitation v1.2 (Peruvian Interpolation of the SENAM-
HIs Climatological and Hydrological Stations) for the per- 291 Alonso et al. Figure 1 - The Titicaca Lake Basin located in Peruvian Territory at the district scale. Figure 1 - The Titicaca Lake Basin located in Peruvian Territory at the district scale. Figure 1 - The Titicaca Lake Basin located in Peruvian Territory at the district scale. computed as the anomaly of the average of the monthly
SST over 55° W-15° W, 5° N-25° N region and is avail-
able from NOAA Climate Indices. iod 1981 to 2016 (36 years). PISCO - precipitation is a spa-
tial precipitation database produced and validated by the
National Meteorological and Hydrological Service of Peru
(SENAMHI, by acronym in Spanish) at a monthly time
step and a grid resolution of 0.05°. The series starts in Jan-
uary 1981 until 2016 (Aybar et al., 2017) and can be freely
downloaded through the SENAMHI website. The valida-
tion of PISCO-precipitation in the hydrographic regions of
Peru is detailed by Endara (2017) and Porto (2021). 2.1.3. Socioeconomic and physical indicators The indicators were quantified using the latest
National Censuses 2017: Population XII, Housing VII,
and Indigenous Communities III; National Agricultural
Census IV - 2012. Additionally, the Census population,
statistical information of Economically Active Population,
and Human Development Index are obtained from web-
sites and can be freely downloaded. 2.1.2. Oceanic indices To assess the impacts of El Niño-Southern Oscilla-
tion (ENSO) in precipitation, we used the eastern (E) and
central (C) index which represent anomalous surface
warming in the east and center Pacific, as can be seen in
Fig. 3 documented in detail by Takahashi et al. (2011) and
is available at the Climate Monitoring Indices of the Geo-
physical Institute of Peru. In addition, to measure the
impacts of the anomalous condition over Tropical North
Atlantic (TNA), we used the TNA index. This index is 2.2.1. Precipitation analysis The rainy months are identified for drought monitor-
ing in the Titicaca Lake Basin due to that the main eco-
nomic activities in the Basin depend on rainfall for their
development. The fluctuation of the rainy months and their
slope-index are calculated to know if increase or decrease Spatial distribution of droughts in the Titicaca Lake Basin 292 the statistical test non-parametric Mann-Kendall at a 95%
of confidence level. the precipitation trends, and detect if they present statis-
tical significance. The trend analysis was carried out with tical significance. The trend analysis was carried out with
of confidence level. Table 1 - Provinces and Districts on the Titicaca Lake Basin located in Peruvian Territory. 2.2.1. Precipitation analysis Azángaro Province composed of the following
districts:
Huancané Province composed of the
following districts:
Puno Province composed of the following districts:
A01 Achaya
127.15 km2
A31
Cojata
885.50 km2
A62 Acora
1,925.01 km2
A02 Arapa
338.04 km2
A32
Huancané
389.32 km2
A63 Atuncolla
131.29 km2
A03 Asillo
405.52 km2
A33
Huatasani
106.89 km2
A64 Capachica
104.03 km2
A04 Azángaro
724.22 km2
A34
Inchupalla
298.75 km2
A65 Chucuito
115.84 km2
A05 Caminaca
146.67 km2
A35
Pusi
147.28 km2
A66 Coata
99.88 km2
A06 Chupa
152.92 km2
A36
Rosaspata
307.61 km2
A67 Huata
129.17 km2
A07 José Domingo Choquehuanca 67.25 km2
A37
Taraco
197.09 km2
A68 Mañazo
280.54 km2
A08 Muñani
788.72 km2
A38
Vilque Chico 511.83 km2
A69 Paucarcolla
174.39 km2
A09 Potoni
625.62 km2
Lampa Province composed of the following
districts:
A70 Pichacani
1,644.94 km2
A10 Saman
204.79 km2
A39
Cabanilla
386.78 km2
A71 Plateria
243.94 km2
A11 San Antón
518.23 km2
A40
Calapuja
143.05 km2
A72 Puno
462.69 km2
A12 San José
399.34 km2
A41
Lampa
663.87 km2
A73 San Antonio
42.69 km2
A13 San Juan de Salinas
105.18 km2
A42
Nicasio
133.93 km2
A74 Tiquillaca
345.09 km2
A14 Santiago de Pupuja
320.47 km2
A43
Ocuviri
869.62 km2
A75 Vilque
195.83 km2
A15 Tirapata
201.65 km2
A44
Palca
497.14 km2
San Antonio de Putina Province composed of the
following districts:
Carabaya Province composed of the following
districts:
A45
Paratia
750.66 km2
A76 Ananea
970.92 km2
A16 Ajoyani
385.54 km2
A46
Pucara
527.98 km2
A77 Pedro Vilca Apaza 143.65 km2
A17 Crucero
858.88 km2
Lampa Province composed of the following
districts:
A78 Putina
1,043.07 km2
A18 Usicayos
14.71 km2
A47
Santa Lucia
1,381.93 km2
A79 Quilcapuncu
527.70 km2
Chucuito Province composed of the following
districts:
A48
Vilavila
161.80 km2
San Román Province composed of the following
districts:
A19 Desaguadero
162.26 km2
Melgar Province composed of the following
districts:
A80 Cabana
193.94 km2
A20 Huacullani
632.24 km2
A49
Antauta
658.23 km2
A81 Cabanillas
552.15 km2
A21 Juli
778.17 km2
A50
Ayaviri
1,021.73 km2
A82 Caracoto
282.31 km2
A22 Kelluyo
486.70 km2
A51
Cupi
217.50 km2
A83 Juliaca
533.99 km2
A23 Pisacoma
958.76 km2
A52
Llalli
221.64 km2
Sandia Province composed of the following
districts:
A24 Pomata
404.24 km2
A53
Macari
693.31 km2
A84 Cuyocuyo
190.03 km2
A25 Zepita
527.55 km2
A54
Nuñoa
2,208.76 km2
Yunguyo Province composed of the following
districts:
El Collao Province composed of the following
districts:
A55
Orurillo
399.26 km2
A85 Copani
59.25 km2
A26 Capazo
1,048.26 km2
A56
Santa Rosa
806.45 km2
A86 Cuturapi
23.92 km2
A27 Conduriri
850.62 km2
A57
Umachiri
333.11 km2
A87 Ollaraya
26.73 km2
A28 Ilave
895.13 km2
Moho Province composed of the following
districts:
A88 Tinicachi
3.62 km2
A29 Pilcuyo
153.22 km2
A58
Conima
67.70 km2
A89 Unicachi
5.92 km2
A30 Santa Rosa
2,472.45 km2
A59
Huayrapata
405.49 km2
A90 Yunguyo
175.97 km2
A60
Moho 507.83 km2
A61
Tilali
51.30 km2 Table 1 - Provinces and Districts on the Titicaca Lake Basin located in Peruvian Territory. 2.2.1. Precipitation analysis 293 Alonso et al. 2.2.2. Drought hazard calculation 2.2.2. Drought hazard calculation SPI value
Drought category
Probability of occurrence (%)
0.00 a - 0.99
Slight drought
~ 24
-1.00 a -1.49
Moderate drought
9.2
-1.50 a -1.99
Severe drought
4.4
≤-2.00
Extreme drought
2.3
~ 40
Source: McKee et al. (1993). Figure 2 - Hydrographic Units of the Titicaca Lake Basin located in Pe-
ruvian Territory. y
For the calculation of drought hazard, each district is
assigned a weight and a rating using the analytical hier-
archy process (AHP), according to the drought category
(moderate, severe, or extreme) that depends on the values
obtained from the SPI and the percentage of drought
occurrence. AHP is a decision-making method that sys-
tematically evaluates alternatives to derive priorities and
importance when evaluation criteria are numerous and
complex (Young-Sik et al., 2020). The percentage of
drought occurrence is calculated by dividing the number
of specific drought events (moderate, severe, or extreme)
by the total events of the analyzed period. Table 3 shows
the weights and qualifications assigned according to
drought category and percentage of drought occurrence. The drought occurrence and hazard maps are devel-
oped for a time scale of 3-months at the district scale. The
maps are made using the kriging interpolation method
which is widely recognized as a standard approach for
interpolating surfaces based on scalar measurements of with unit values of the E-index, and the C-index. Source: Takahashi et al. (2011). Table 2 - Category of droughts defined by SPI values. SPI value
Drought category
Probability of occurrence (%)
0.00 a - 0.99
Slight drought
~ 24
-1.00 a -1.49
Moderate drought
9.2
-1.50 a -1.99
Severe drought
4.4
≤-2.00
Extreme drought
2.3
~ 40
Source: McKee et al. (1993). Table 2 - Category of droughts defined by SPI values. SPI value
Drought category
Probability of occurrence (%)
0.00 a - 0.99
Slight drought
~ 24
-1.00 a -1.49
Moderate drought
9.2
-1.50 a -1.99
Severe drought
4.4
≤-2.00
Extreme drought
2.3
~ 40
Source: McKee et al. (1993). Table 2 - Category of droughts defined by SPI values. Figure 2 - Hydrographic Units of the Titicaca Lake Basin located in Pe-
ruvian Territory. Source: McKee et al. (1993). Figure 3 - The patterns of sea surface temperature anomalies associated with unit values of the E-index, and the C-index. Source: Takahashi et al. (2011). 2.2.2. Drought hazard calculation source of economic income in the region and it depends on
the precipitation for its development. Table 2 shows the
category of droughts defined by SPI values. There is evi-
dence that the high-Andean region of Puno presents a
strong
signal
associated
with
anomalous
conditions
observed in the Pacific region (i.e., El Niño). Therefore,
the correlation between the Peruvian precipitation (from
PISCO for January to March season and the 1980-2016
period) against the TNA index, E index, and C index is
analyzed. The precipitation deficit of meteorological drought
in the Titicaca Lake Basin is quantified using the Standar-
dized Precipitation Index (SPI) for a temporary scale of 3-
months. The short time scales (1, 2, or 3 months), can pro-
vide an early warning of drought and help assess drought
severity (OMM, 2012). In the Titicaca Lake Basin, it can
be essential because the agricultural activity is the primary period) against the TNA index, E index, and C index is
analyzed. For the calculation of drought hazard, each district is
assigned a weight and a rating using the analytical hier-
archy process (AHP), according to the drought category
(moderate, severe, or extreme) that depends on the values
obtained from the SPI and the percentage of drought
occurrence. AHP is a decision-making method that sys-
tematically evaluates alternatives to derive priorities and
importance when evaluation criteria are numerous and
complex (Young-Sik et al., 2020). The percentage of
drought occurrence is calculated by dividing the number
of specific drought events (moderate, severe, or extreme)
by the total events of the analyzed period. Table 3 shows
the weights and qualifications assigned according to
drought category and percentage of drought occurrence. The drought occurrence and hazard maps are devel-
oped for a time scale of 3-months at the district scale. The
maps are made using the kriging interpolation method
which is widely recognized as a standard approach for
interpolating surfaces based on scalar measurements of
Figure 2 - Hydrographic Units of the Titicaca Lake Basin located in Pe-
ruvian Territory. Figure 3 - The patterns of sea surface temperature anomalies associated with unit values of the E-index, and the C-index. Source: Takahashi et al. (2011). Table 2 - Category of droughts defined by SPI values. 2.2.2. Drought hazard calculation Figure 3 - The patterns of sea surface temperature anomalies associated with unit values of the E-index, and the C-index. Source: Takahashi et al. (2011). Spatial distribution of droughts in the Titicaca Lake Basin 294 Table 3 - Weights and qualifications are assigned according to drought
category and PO. Drought
category
Weights
Percentage of drought
occurrence (PO)
Qualification
Moderate
1.0
≤9.0
1.0
9.1 - 10.0
2.0
10.1 - 11.0
3.0
≥11.1
4.0
Severe
2.0
≤3.5
1.0
3.6 - 4.5
2.0
4.6 - 5.5
3.0
≥5.6
4.0
Very severe
3.0
≤1.5
1.0
1.6 - 2.0
2.0
2.1 - 2.5
3.0
≥2.6
4.0
Source: Shahid and Behrawan (2008). ing to the most relevant activities and characteristics of the
region which are negatively affected due to the impact of
drought events. Table 4 shows the socioeconomic and
physical indicators detailed. Table 3 - Weights and qualifications are assigned according to drought
category and PO. The socioeconomic and physical indicators are divi-
ded into 4 classes: low, moderate, high, and very high,
using the natural breaks method (Jenks). The natural break
method is used to derive the classes. This method creates a
range according to an algorithm that uses the average of
each range to distribute the data more evenly across the
ranges (Shahid and Behrawan, 2008). This method
ensures that the ranges are well represented by their avera-
ges and that the data values within each range are fairly
close together (Smith, 1986). GIS software ArcView 9.0 is
used to identify the natural breakpoints in the data. The vulnerability to drought is obtained with the
index that is the average of the scores assigned to each
indicator as shown in the Eq. (2) according to Johnson
et al. (2016). The vulnerability index is classified into
four ranges with the Natural Break method (Jenks),
according to their 25%, 50% and 75% percentiles, then
scores of 1.0, 2.0, 3.0 and 4.0 are placed to distinguish
their levels of vulnerability (low, moderate, high and very
high). Finally, the drought vulnerability map is prepared
at the district level differentiated the vulnerability levels
with colors. Source: Shahid and Behrawan (2008). different points (Shahid and Behrawan, 2008). The
Drought Hazard Index (DHI) was derived using Eq. (1)
according to Hungsoo et al. (2015). 2.2.2. Drought hazard calculation DHI = (MDw × MDr) þ (SDw × SDr)
þ (VSDw × VSDr)
ð1Þ ð1Þ where MDw is the weight assigned to moderate drought;
MDr is rating assigned to the moderate drought percentage
of drought occurrence; SDw is the weight assigned to
severe drought; SDr is rating assigned to the severe drought
percentage of drought occurrence; VSDw is the weight
assigned to extreme drought; VSDr is rating assigned to the
extreme drought percentage of drought occurrence. DVI = Dp þ Id þ Ae þ Rhm þ Pg þ Da
ð
Þ
Number of indicators
ð2Þ ð2Þ where Dp is the score assigned to population density; Id is
the score assigned to the Human Development Index; Ae
is the score assigned to Active Economic Activity in agri-
culture, livestock, hunting, and forestry; Rhm is the score
assigned to the female/male relation; Pg is the score
assigned to the livestock population; Sa is the score
assigned to the rainfed agricultural area. 2.2.3. Vulnerability to drought calculation The vulnerability to drought is obtained by Eq. (2),
which is based on 4-socioeconomic and 2-physical indica-
tors of the Titicaca Lake Basin. The Titicaca Lake Basin is
located within the high-Andean region of the department
of Puno, which is one of the departments with more pov-
erty at the national level, with main economic activities
that depend on the rains for their development such as
rainfed agriculture and livestock. For this reason, the
socioeconomic and physical indicators are chosen accord- Table 4 - Socioeconomic and physical indicators.
Socioeconomic
indicator
Population Density: The number of people per unit area and is usually expressed in inhabitants per km2.Human Development
Index (IDH): It is calculated by the United Nations Development Programme (PNUD). Its levels range to go from 0 to 1. The
factors taken into account to measure it are the average annual income per person, access to education and health services, and
life expectancy.Economically Active Population (PEA) in Agriculture, Livestock, Hunting and Forestry: Represents the percen-
tage of people who depend mainly on agriculture and livestock, as well as hunting and forestry.Female / Male relation: Repre-
sents the relation of female to male residents in a region. According to the Intergovernmental Panel on Climate Change (IPCC),
women are more affected by the climate crisis than men.
Physical indicator
Livestock Production: The amount of livestock production in a region: alpacas, sheep, cattle, pigs, and other species.Rainfed
Agricultural: Represents the agricultural area in hectares under rainfed irrigation. This indicator is highly vulnerable to drought
events, due to the crops only receive the water provided by rainfall. 2.2.4. Drought risk calculation Drought risk is obtained through the Drought Risk
Index (DRI) which is a function of hazard and vulner-
ability, as shown by Eq. (3) according to Shahid and Beh-
rawan (2008). DRI = DHI × DVI
ð3Þ ð3Þ DRI = DHI × DVI
ð3Þ Table 4 - Socioeconomic and physical indicators. Socioeconomic
indicator
Population Density: The number of people per unit area and is usually expressed in inhabitants per km2.Human Development
Index (IDH): It is calculated by the United Nations Development Programme (PNUD). Its levels range to go from 0 to 1. The
factors taken into account to measure it are the average annual income per person, access to education and health services, and
life expectancy.Economically Active Population (PEA) in Agriculture, Livestock, Hunting and Forestry: Represents the percen-
tage of people who depend mainly on agriculture and livestock, as well as hunting and forestry.Female / Male relation: Repre-
sents the relation of female to male residents in a region. According to the Intergovernmental Panel on Climate Change (IPCC),
women are more affected by the climate crisis than men. Physical indicator
Livestock Production: The amount of livestock production in a region: alpacas, sheep, cattle, pigs, and other species.Rainfed
Agricultural: Represents the agricultural area in hectares under rainfed irrigation. This indicator is highly vulnerable to drought
events, due to the crops only receive the water provided by rainfall. Table 4 - Socioeconomic and physical indicators. Socioeconomic
indicator
Population Density: The number of people per unit area and is usually expressed in inhabitants per km2.Human Development
Index (IDH): It is calculated by the United Nations Development Programme (PNUD). Its levels range to go from 0 to 1. The
factors taken into account to measure it are the average annual income per person, access to education and health services, and
life expectancy.Economically Active Population (PEA) in Agriculture, Livestock, Hunting and Forestry: Represents the percen-
tage of people who depend mainly on agriculture and livestock, as well as hunting and forestry.Female / Male relation: Repre-
sents the relation of female to male residents in a region. According to the Intergovernmental Panel on Climate Change (IPCC),
women are more affected by the climate crisis than men. Physical indicator
Livestock Production: The amount of livestock production in a region: alpacas, sheep, cattle, pigs, and other species.Rainfed
Agricultural: Represents the agricultural area in hectares under rainfed irrigation. 2.2.4. Drought risk calculation This indicator is highly vulnerable to drought
events, due to the crops only receive the water provided by rainfall. Table 4 - Socioeconomic and physical indicators. sical indicator
Livestock Production: The amount of livestock production in a region: alpacas, sheep, cattle, pigs, and other species.Rainfed
Agricultural: Represents the agricultural area in hectares under rainfed irrigation. This indicator is highly vulnerable to drought
events, due to the crops only receive the water provided by rainfall. 295 Alonso et al. 0171, 0155, 0175, and Ramis. Ramis shows the lowest
slope-index with -0.052, followed by the Huancané with
-0.033. In contrast, the hydrographic units located in the
north and west show fluctuation and slope-index positives,
which indicates an increase in the precipitation concerning
the average. The trend analysis from the Mann-Kendall
test on the rainfall with a 95% of confidence level not
show statistical significance in any period of the time ser-
ies analyzed. where DRI is the drought risk index; DHI is the drought
hazard index; DVI is the drought vulnerability index. Figure 4 shows the schematic depiction of the methodo-
logy used in this study to calculate the drought risk. 3.1. Precipitation analysis Figure 5 shows the spatial distribution of the annual
mean precipitation (1981-2016) in the hydrographic units
of the Titicaca Lake Basin and the rainfall for the January
to March period. The precipitation over the Titicaca pla-
teau is important to study because the development of the
main industries in this region depends on it (Romero,
2013). The hydrographic units with the highest rainfall are
located in the west, center, and east of the Titicaca Lake
Basin, while those in the north and south show the lowest
rainfall. The rainy months are January, February, and
March (hereafter, JFM) due to during this period the pre-
cipitation is higher compared to the rest of the year, repre-
senting more than 50% of the annual accumulated
precipitation. July is the driest month, while January is the
wettest month (Andrade, 2017). During the JFM season,
the hydrographic units located in the southern and eastern
of the Basin show fluctuation and slope-index negative,
which indicates decrease precipitation concerning the
average. These Basins are Huancané, Ilave, Suches, Call-
accame, Mauri Chico, and the Inter-Basins of 0157, 0173, 3.2. Drought hazard Figure 6 shows the districts with the highest fluc-
tuation of the Standardized Precipitation Index (SPI) for a
time scale of 3-months in the period 1981 to 2016. In the
Fig. 6.a the 3-months SPI for rainy season (JFM) is ana-
lyzed and not the winter period because a significant dry
period during the winter season may have few impacts
(Wilhite, 2000). During JFM is not detected significant
changes in the SPI fluctuation, so not show statistical sig-
nificance in any period of the time series analyzed. In
1983,
1987,
1990
and
1992,
occurred
extreme
(SPI ≤-2.00), severe (-1.50 ≤SPI ≤-1.99) and moderate
(-1.0 ≤SPI ≤-1.49) drought events with categories and
intensities different. The longest extreme drought occur-
ring in 1983 with a period of 60 days, while the severe
droughts in 1990 and 1992 lasted 37 and 40 days. The
SPI reached values of -3.76 in 1983 in Vilavila district;
-2.82 in 1990 in Antauta district; -2.69 in 1987 in Orurillo Figure 4 - Schematic depiction of the methodology used in this study. Figure 4 - Schematic depiction of the methodology used in this study. 296 Spatial distribution of droughts in the Titicaca Lake Basin 296 Figure 5 - Spatial distribution of the annual mean precipitation (1981-2016) in the hydrographic units of the Titicaca Lake Basin and the rains of January,
February, and March (JFM). Figure 5 - Spatial distribution of the annual mean precipitation (1981-2016) in the hydrographic units of the Titicaca Lake Basin and the rains of January,
February, and March (JFM). district and -2.62 in 1992 in San Antonio district. Severe
and moderate droughts were detected in 1982, 1989,
1998, 2005, 2006, 2009 and 2016 in districts of the high-
Andean region of Puno, and only moderate drought were
observed in 1988, 1991, 1993, 1995, 1996, 2000, 2003,
2008, 2010, 2011, 2013, 2014 and 2015. According to
ANA (2013), the drought event in 2011 affected various
departments, such as Arequipa, Cajamarca, Lambayeque,
Piura, La Libertad, Lima, Moquegua, Tacna, Amazonas,
Huánuco, San Martín, Junín and Puno. The drought
recorded in 1982-83 resulted from a rainfall deficit during
El Niño conditions (analyzed in next section) when
upper-level west wind anomalies inhibited the influx of
moisture from the east (Sulca et al., 2018), causing losses
in the Puno region in agriculture that were much larger
than in livestock (Claverías, 2016). 3.2. Drought hazard on Index (SPI) for a time scale of 3-months in the 1981 to 2016 peri Figure 6 - Districts with the highest fluctuation of the Standardized Precipitation Index (SPI) for a time scale of 3-months in the 1981 to 2016 period. he highest fluctuation of the Standardized Precipitation Index (SPI) for a time scale of 3-months in the 1981 to 2016 period. Figure 6 - Districts with the highest fluctuation of the Standardized Precipitation Index (SPI) for a time scale of 3 present a significant rainfall deficit during the El Niño
phenomenon (Lagos et al., 2008; Lavado and Espinoza
2014). closeness of the years of their occurrence is not just a
coincidence. The climate of South America (SA) has long
held an intimate connection with El Niño (Cai et al.,
2020). The main climate anomalies that characterize the
El Niño in South America are related to a modification of
the Walker circulation and to a southern shift of the Inter-
tropical Convergence Zone, they include manifestation of
the significant rainfall deficits in the Altiplano of south-
eastern Peru and Bolivia, the Brazilian northeastern,
northtern of South America and southern of Central
America (Ortlieb and Macharé, 1993). In Peru, El Niño
phenomenon mainly generates climatic alteration that is
manifested in intense rains in the north and severe
droughts in the altiplano region of the south (Fig. 7)
where is the hydrographic drainages of Lake Titicaca that closeness of the years of their occurrence is not just a
coincidence. The climate of South America (SA) has long
held an intimate connection with El Niño (Cai et al.,
2020). The main climate anomalies that characterize the
El Niño in South America are related to a modification of
the Walker circulation and to a southern shift of the Inter-
tropical Convergence Zone, they include manifestation of
the significant rainfall deficits in the Altiplano of south-
eastern Peru and Bolivia, the Brazilian northeastern,
northtern of South America and southern of Central
America (Ortlieb and Macharé, 1993). In Peru, El Niño
phenomenon mainly generates climatic alteration that is
manifested in intense rains in the north and severe
droughts in the altiplano region of the south (Fig. 7)
where is the hydrographic drainages of Lake Titicaca that Figure 7 shows the correlation between PISCO-pre-
cipitation for the JFM season and the C, E, and TNA
index. 3.2. Drought hazard Likewise, in 1992
drought coincidentally occurred with El Niño year and
was the most severe and affected 16 departments in Peru:
Amazonas, Cajamarca, La Libertad, Ancash, Junín, Huá- district and -2.62 in 1992 in San Antonio district. Severe
and moderate droughts were detected in 1982, 1989,
1998, 2005, 2006, 2009 and 2016 in districts of the high-
Andean region of Puno, and only moderate drought were
observed in 1988, 1991, 1993, 1995, 1996, 2000, 2003,
2008, 2010, 2011, 2013, 2014 and 2015. According to
ANA (2013), the drought event in 2011 affected various
departments, such as Arequipa, Cajamarca, Lambayeque,
Piura, La Libertad, Lima, Moquegua, Tacna, Amazonas,
Huánuco, San Martín, Junín and Puno. The drought
recorded in 1982-83 resulted from a rainfall deficit during
El Niño conditions (analyzed in next section) when
upper-level west wind anomalies inhibited the influx of
moisture from the east (Sulca et al., 2018), causing losses
in the Puno region in agriculture that were much larger
than in livestock (Claverías, 2016). Likewise, in 1992
drought coincidentally occurred with El Niño year and
was the most severe and affected 16 departments in Peru:
Amazonas, Cajamarca, La Libertad, Ancash, Junín, Huá- nuco, Huancavelica, Pasco, Lima, Cusco, Apurímac,
Ayacucho, Arequipa, Tacna, Moquegua and Puno (Endara
et al., 2019). On the other hand, it should be noted that in
1983 and 1992 droughts followed a volcanic eruption in
the Tropic, El Chichón in April 1982 in Mexico, and Mt. Pinatubo in June 1991 in the Philippines (Imfeld et al.,
2019).i Figure 6.b shows the precipitation deficit analyzed
with the 3-months SPI in every month of the year, finding
59 districts out of 90 (59/90) present negative fluctuation
and 31/90 districts present positive fluctuation for the per-
iod 1981 to 2016. In the analysis of the SPI, it has been
registered 19,317 drought events during the period from
1981 to 2016, of which: 68% (13,074) is slight drought;
18% (3,535) is moderate drought; 9% (1,788) is severe
drought, and 5% (920) is extreme drought. According to Caviedes (1973), ENSO and drought
are indeed the consequence of the same large-scale varia-
tion in the tropical and equatorial circulation, and that the 297 Alonso et al. Figure 6 - Districts with the highest fluctuation of the Standardized Precipitation Index (SPI) for a time scale of 3-months in the 1981 to 2016 period. 3.2. Drought hazard Precipitation extremes in the southern Andean re-
gion are highly impacted by SST anomalous conditions in
the central and eastern Pacific, showing negative correla-
tions (precipitation deficit) (Figs. 7.b and 7.c). On the
other hand, impacts of anomalous conditions in the tropi-
cal Atlantic, during the rainy season, is barely detected
(Fig. 7.a). These results are in agreement with findings
observed in Fig. 6, where SPI peaks (positive and nega-
tive) are strongly associated with El Niño and La Niña
events recorded in recent decades. Table 5 shows ENSO Spatial distribution of droughts in the Titicaca Lake Basin 298 Table 5 - ENSO influence on hydrographic units of the Titicaca Lake
Basin through the E (eastern El Niño) and C (central El Niño) index
based on Fig. 7. Hydrografic units
C/E indices
Azángaro
C
Callaccame
C
Coata
C
Huancané
E
Ilave
C
Ilpa
C
Mauri
C
Mauri Chico
C
Pucará
C
Suches
-
Interc. 0155
C
Interc. 0157
C
Interc. 0171
-
Interc. 0173
C
Interc. 0175
C
Interc. Ramis
- influence on hydrographic units of the Titicaca Lake Basin
through the E (eastern El Niño) and C (central El Niño)
index based on Fig. 7. Table 1 shows the districts that are
within the hydrographic units. The Basins of Azángaro,
Callaccame, Coata, Ilave, Ilpa, Mauri, Mauri Chico,
Pucará and Inter-Basins of 0155, 0157, 0173, 0175, pre-
sent a very strong correlation with the C index which indi-
cates correlation with cold, moderately-warm, and neutral
events in the central Pacific, and the Basin of Huancané
presented more correlation with the E index, which indi-
cates warm events in the equatorial Pacific. Table 5 - ENSO influence on hydrographic units of the Titicaca Lake
Basin through the E (eastern El Niño) and C (central El Niño) index
based on Fig. 7. Figure 8 shows the probability of drought occur-
rence (PO) in drought categories: moderate, severe, and
extreme, in the districts of the Titicaca Lake Basin. The
PO levels are low, moderate, high, and very high, and
according to each category of drought, the following has
been found: Moderate drought: In this category are 23 districts out
of 90 (23/90) that present very-high PO; 26/90 have
high PO; 23/90 maintain a moderate PO and 18/90 have
a low level of PO. 3.2. Drought hazard The districts with high to very-high
PO are located in the northeast, central, and southeast of
the Titicaca Lake Basin, covering 54% of the Basin. e Titicaca Lake Basin, covering 54% of the Basin. Severe drought: In this case, show 23 districts out of 90
(23/90) that present very-high PO; 25/90 have high PO;
22/90 maintain a moderate PO and 20/90 have a low
level of PO. The districts with high to very-high PO are
located in the north, west, and south-east, and represent
49% of the Basin. months SPI (in every month), obtained using Table 3. The results suggest that 37 districts out of 90 (37/90)
show very-high drought hazard, 9/90 districts present
high hazard, and 24/90 show moderate hazard. The dis-
tricts with a high degree of exposure to drought events
(high to very-high hazard) predominate in the northeast
and center-south of the Titicaca Lake Basin, covering
51% of the area. In addition, the districts located in the
part of the center-north and south of the Basin show low
to moderate risk except for Capazo district. The drought
hazard is caused by the precipitation deficit that can be
associated with the occurrence of special climatic condi-
tions. Extreme drought: In this analysis, it has been found that
27 districts out of 90 (23/90) present very-high PO; 26/
90 have high PO; 17/90 maintain a moderate PO and
20/90 have a low level of PO. The districts with high to
very-high PO are located in the northwest, northeast,
and center-south, and covers 68% of the Basin. Figure 9 shows the drought hazard of the Titicaca
Lake Basin, at the district scale for a time scale of 3- Figure 7 - Correlation between the Peruvian precipitation against the (a) TNA index, (b) E index, and (c) C index. Precipitation data comes from PISCO
for January to March season and the 1980-2016 period. Blue (red) shadings indicate positive (negative) correlation, where statistically significant values
(95% of confidence level) are plotted in hatching. Figure 7 - Correlation between the Peruvian precipitation against the (a) TNA index, (b) E index, and (c) C index. Precipitation data comes from PISCO
for January to March season and the 1980-2016 period. Blue (red) shadings indicate positive (negative) correlation, where statistically significant values
(95% of confidence level) are plotted in hatching. 299 Alonso et al. 3.2. Drought hazard Figure 8 - The Probability of Drought Occurrence in the Titicaca Lake Basin at the district scale for a scale time of 3-months for different drought cate-
gories. Figure 8 - The Probability of Drought Occurrence in the Titicaca Lake Basin at the district scale for a scale time of 3-months for different drought cate-
gories. Figure 8 - The Probability of Drought Occurrence in the Titicaca Lake Basin at the district scale for a scale time of 3-mo
gories. 3.3. Vulnerability to drought Figure 10.b shows the Economically Active Population
(PEA) in the agriculture, livestock, hunting, and for-
estry sectors. The districts with high to very-high vul-
nerability are mainly located in the north, center-east,
and southeast, covering 60% of the Titicaca Lake Basin. In this area are the larger territorial surface of districts
such as Santa Rosa, Nuñoa and Acora. Figure 10 shows the vulnerability to drought of so-
cioeconomic and physical indicators at the district scale of
the Titicaca Lake Basin. The results are discussed below: Figure 10.a shows the population density at the district
scale. The districts located in the center-east and south-
east (except Pisacoma district) present high to very-
high population density and cover 29% of the Basin. In
this area are the districts of Juliaca and Puno that show
a high population density. Figure 10.c shows the Human Development Index
(IDH). The districts located in the northeast and south
of the Basin have high to very-high vulnerability and
occupy 43% of the area. In this zone is the Carabaya
province, which according to the National Institute of
Statistics and Informatics (INEI) presents a higher per-
centage of poverty, as the districts of Crucero and Usi-
cayos. Figure 9 - Drought Hazard in the Titicaca Lake Basin at the district scale
for a 3-month SPI. Figure 10.d shows the relationship between women and
men. The districts located in the center-north have a lar-
ger population of women than men, occupying 38% of
the Titicaca Lake Basin. According to the Intergovern-
mental Panel on Climate Change (IPCC), the climate
crisis affects women more than men. The provinces
with the largest number of districts within this zone are
Azángaro, Carabaya, Moho, and San Román. Figure 10.e shows livestock production. Most of the
districts of the Basin of Lake Titicaca present high to
very-high livestock production, covering 80% of the
Basin. According to Ardiles (2015), the Puno depart-
ment is the most important in the country in terms of
livestock production and the main domestic supplier of
meat. The districts with the highest risk are: Acora,
Ilave, Nuñoa, Santa Lucia, Santa Rosa, Juli, Pichacani,
Lampa and Azángaro. In addition, the districts with the
least number of livestock units are located in the eye- Figure 9 - Drought Hazard in the Titicaca Lake Basin at the district scale
for a 3-month SPI. 3.4. Drought risk Figure 12 shows the drought risk for a time scale
of 3-months in the Titicaca Lake Basin at the district
scale. The results show that 25 districts out of 90 (25/90)
present very-high risk, 20/90 districts with high risk, and
24/90 districts with moderate risk. The districts with
high and very high drought risk are localized in the
northwestern, center-east, and center-south of the Titi-
caca Lake Basin, occupying 53% of the Basin. The dis-
tricts with low to moderate drought risk are located in
the zones of southern and center-western of the Basin. The provinces main with a larger number of districts in
the zones with high and very-high drought risk are:
Azángaro province that present one of the higher popu-
lation densities and has a larger development in livestock
(Romero, 2013), and inside are the districts: Achaya,
Chupa, Saman, Arapa, Asillo, and San Anton. Another
province is Melgar, where are the districts: Orurillo,
Macari, Ayaviri, Nuñoa, and Santa Rosa. The Nuñoa
district, present the second-largest district area within the
Titicaca Lake Basin with high livestock production and
agricultural area under rainfed. Additionally, the Cara-
baya province presents a higher percentage of poverty
and contains the districts of Usicayos and Crucero. The
Usicayos district is the second poorest district at the
national level with a 96.9% incidence of poverty (INEI,
2013). The provinces that stand out the most for their
economy are Puno and San Román. The San Román
province has the districts: Cabana, Caracoto and Juliaca. The Puno province contain the districts: Acora, Picha-
cani, Puno, Mañazo, Plateria, Paucarcolla, Atuncolla,
Huata, Chucuito, Capachica and Coata. The Acora dis-
trict present the third-largest district area within the Titi-
caca Lake Basin with high livestock production and
agricultural area under rainfed. The cities of Juliaca and
Puno have a high population density that is 4 and
3 times higher than the departmental average, and con-
centrate most of the industry, commerce, as well as
infrastructure, and state services of the Puno department
(Ardiles, 2015). The city of Juliaca is considered as the
economic capital of the department of Puno; there local,
regional, national and cosmopolitan entrepreneurs com-
pete (Apaza, 2016). Figure 11 - Vulnerability to drought on the Titicaca Lake Basin at the
district scale. 3.4. Drought risk important districts such as Puno and the city of Juliaca
which is considered a gigantic permanent market and is
the economic capital of the department of Puno (Apaza,
2016). Likewise, the districts of Ayaviri, Muñani, Juli
and Azángaro, have a larger surface extension and pre-
sent more women than men. The districts with moderate
to low vulnerability are located in the west, southwest,
and northeast. There are areas with a low vulnerability
that border areas with high or very high vulnerability,
due to the socio-economic and physical indicators are
independent in each district. Figure 12 - Drought risk on the Titicaca Lake Basin at the district scale
for a 3-month SPI. Spatial distribution of droughts in the Titicaca Lake Basin Spatial distribution of droughts in the Titicaca Lake Basin 300 Figure 10 - Vulnerability to drought of socioeconomic and physical indicators at the district scale of the Titicaca Lake Basin. Figure 10 - Vulnerability to drought of socioeconomic and physical indicators at the district scale of the Titicaca Lake Basin. igure 10 - Vulnerability to drought of socioeconomic and physical indicators at the district scale of the Titicaca Lake Basin. ability, and 22/90 with moderate vulnerability. The dis-
tricts with high and very-high vulnerability are located in
the center-north and southeast of the Basin, occupying
47% of the surface. The Puno region is considered the
main producer of livestock in the departments of the
high-Andean zone of Peru, however, still maintains
levels of monetary poverty higher than the national aver-
age (Paredes and Escobar, 2018). The districts within the
Titicaca Lake Basin that present high levels of poverty at
the national level, higher livestock production, and
depend on the agricultural sector in special rainfed agri-
cultural are Macari, Zepita, Vilque chico, Asillo, Orurillo
and Saman. In addition, in the zone of greatest vulne-
rability are the provinces with the highest population
density as Puno, Huancané, Azángaro, San Román, and brow of the jungle, and in the southern such as Cutur-
api, Unicachi and Tinicachi.i Figure 10.f shows the rainfed agricultural area, finding
that the districts located in the center-north and south-
east (except the districts of Capazo, Pisacoma, Hua-
cullani) of the Titicaca Lake Basin, have high to very-
high vulnerability to drought and occupy 45% of the
area. The main provinces are Puno, Azángaro, Melgar,
Chucuito, Huancané, Lampa, El Collao, and San
Román. Figure 11 shows vulnerability to drought at the dis-
trict scale in the Titicaca Lake Basin. The map is divided
into districts with low, moderate, high, and very-high
vulnerability obtained 33 districts out of 90 (33/90) with
very-high vulnerability, 13/90 districts with high vulner- 301 Alonso et al. Figure 11 - Vulnerability to drought on the Titicaca Lake Basin at the
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(OMM-Nº
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Commons Attribution License (type CC-BY), which permits unrestricted use, distribution and
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en el área rural de Puno. Journal of High Andean
Research, v. 20, n. 1, p. 39-60, 2018. WMO. Handbook of Drought Indicators and Indices. Inte-
grated Drought Management Programme (IDMP),
Integrated Drought Management Tools and Guidelines
Series 2. Geneva: WMO, p. 52, 2016. PORTO, A. Validación del Producto Grillado Pisco de Preci-
pitación v2.1 con la Data Observada de las Estaciones Internet Resources Census population, statistical information of Economically
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cursivo/publicaciones_digitales/Est/Lib1627/. Census population, statistical information of Economically
Active Population, https://www.inei.gob.pe/media/MenuRe
cursivo/publicaciones_digitales/Est/Lib1627/. SENAMHI, http://www.senamhi.gob.pe. Climate Monitoring Indices of the Geophysical Institute of Peru,
http://met.igp.gob.pe/variabclim/indices.html. Human Development Index, https://www.pe.undp.org/content/
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Calculating the power to examine treatment‐covariate interactions when planning an individual participant data meta‐analysis of randomized trials with a binary outcome
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R E S E A R C H A R T I C L E R E S E A R C H A R T I C L E This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the
original work is properly cited.
© 2022 The Authors. Statistics in Medicine published by John Wiley & Sons Ltd.
4822
wileyonlinelibrary com/journal/sim
Statistics in Medicine 2022;41:4822–4837 Calculating the power to examine treatment-covariate
interactions when planning an individual participant data
meta-analysis of randomized trials with a binary outcome 2Centre for Statistics in Medicine, Nuffield
Department of Orthopaedics,
Rheumatology and Musculoskeletal
Sciences, University of Oxford, Oxford, UK
3NIHR Oxford Biomedical Research
Centre, Oxford University Hospitals NHS
Foundation Trust, Oxford, UK This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the
original work is properly cited.
© 2022 The Authors Statistics in Medicine published by John Wiley & Sons Ltd 10970258, 2022, 24, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/sim.9538 by Test, Wiley Online Library on [06/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are Received: 17 March 2022
Revised: 30 June 2022
Accepted: 12 July 2022 Received: 17 March 2022
Revised: 30 June 2022
Accepted: 12 July 2022
DOI: 10.1002/sim.9538 Revised: 30 June 2022
Accepted: 12 July 2022 DOI: 10.1002/sim.9538 Calculating the power to examine treatment-covariate
interactions when planning an individual participant data
meta-analysis of randomized trials with a binary outcome Richard D. Riley1
Miriam Hattle1
Gary S. Collins2,3
Rebecca Whittle1
Joie Ensor1
002/sim.9538 by 1Centre for Prognosis Research, School of
Medicine, Keele University, Keele,
Staffordshire, UK
2Centre for Statistics in Medicine, Nuffield
Department of Orthopaedics,
Rheumatology and Musculoskeletal
Sciences, University of Oxford, Oxford, UK
3NIHR Oxford Biomedical Research
Centre, Oxford University Hospitals NHS
Foundation Trust, Oxford, UK
Correspondence
Richard D. Riley, Centre for Prognosis
Research, School of Medicine, Keele
University, Keele, Staffordshire ST5 5BG,
UK. Email: r.riley@keele.ac.uk
Funding information
Cancer Research UK, Grant/Award
Number: C49297/A27294; Medical
Research Council, Grant/Award Number:
MR/V038168/1; NIHR Biomedical
Research Centre, Oxford 1Centre for Prognosis Research, School of
Medicine, Keele University, Keele,
Staffordshire, UK 1Centre for Prognosis Research, School of
Medicine, Keele University, Keele,
Staffordshire, UK Before embarking on an individual participant data meta-analysis (IPDMA)
project, researchers and funders need assurance it is worth their time and cost. This should include consideration of how many studies are promising their IPD
and, given the characteristics of these studies, the power of an IPDMA includ-
ing them. Here, we show how to estimate the power of a planned IPDMA of
randomized trials to examine treatment-covariate interactions at the participant
level (ie, treatment effect modifiers). We focus on a binary outcome with binary
or continuous covariates, and propose a three-step approach, which assumes
the true interaction size is common to all trials. In step one, the user must spec-
ify a minimally important interaction size and, for each trial separately (eg, as
obtained from trial publications), the following aggregate data: the number of
participants and events in control and treatment groups, the mean and SD for
each continuous covariate, and the proportion of participants in each category
for each binary covariate. This allows the variance of the interaction estimate
to be calculated for each trial, using an analytic solution for Fisher’s informa-
tion matrix from a logistic regression model. Step 2 calculates the variance of the
summary interaction estimate from the planned IPDMA (equal to the inverse of
the sum of the inverse trial variances from step 1), and step 3 calculates the cor-
responding power based on a two-sided Wald test. Stata and R code are provided,
and two examples given for illustration. Extension to allow for between-study
heterogeneity is also considered. Statistics in Medicine. 2022;41:4822–4837. 1
INTRODUCTION Individual participant data (IPD) meta-analysis projects herald much promise,1 but are potentially time-consuming, often
taking upwards of 2 years to engage with trial investigators; to obtain, clean, harmonize and meta-analyze the IPD; and to
publish and disseminate results. Therefore, before embarking on an IPD meta-analysis project, researchers and funders
may want to be reassured that the time and resources required are worth their investment. This should include considera-
tion of how many trials are likely to provide their IPD and, based on this, estimation of the potential power of the planned
IPD meta-analysis.2 In our experience, power calculations and sample size justifications are rarely considered in protocols or publica-
tions of IPD meta-analysis projects. It might be argued that obtaining IPD is almost always worth the investment, in
order to best appraise, synthesize, and summarize the existing evidence. However, if researchers knew before IPD col-
lection that their planned IPD meta-analysis may have, say, only 30% power to detect a clinically important effect, then
they might reconsider whether the project should be initiated. Conversely, if a planned IPD meta-analysis has a poten-
tial power of over 80%, then researchers and funders would be more reassured that the required resources are worth
investment. In this article, we propose how to estimate the power of a planned IPD meta-analysis project, in advance of IPD
collection, where the primary objective is to synthesize IPD from randomized trials to examine treatment-covariate inter-
actions at the participant level (also known as effect modifiers or moderators). The drive to identify treatment-covariate
interactions stems from the idea of stratified (personalized or precision) medicine, where treatment decisions are tailored
to each individual conditional on their covariate values. The availability of IPD from existing trials improves the ability
and power to examine treatment-covariate interactions, compared to single trials or a traditional meta-analysis of pub-
lished aggregate data. Hence, the primary objective of many IPD meta-analysis projects is to examine treatment-covariate
interactions, especially those synthesizing IPD from randomized trials. Previous work in this area has focused mainly on continuous outcomes,2-4 or simulation-based approaches,5,6 but here
we focus on analytic solutions for binary outcomes. In Section 2 we introduce a two-stage IPD meta-analysis framework
for the synthesis of interaction estimates from randomized trials. Correspondence Correspondence
Richard D. Riley, Centre for Prognosis
Research, School of Medicine, Keele
University, Keele, Staffordshire ST5 5BG,
UK. Email: r.riley@keele.ac.uk Funding information
Cancer Research UK, Grant/Award
Number: C49297/A27294; Medical
Research Council, Grant/Award Number:
MR/V038168/1; NIHR Biomedical
Research Centre, Oxford K E Y W O R D S
individual participant data (IPD), meta-analysis, power, treatment effect modifier,
treatment-covariate interaction K E Y W O R D S
individual participant data (IPD), meta-analysis, power, treatment effect modifier,
treatment-covariate interaction This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the
original work is properly cited. © 2022 The Authors. Statistics in Medicine published by John Wiley & Sons Ltd. 4822
wileyonlinelibrary.com/journal/sim
Statistics in Medicine. 2022;41:4822–4837. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the
original work is properly cited. g
p
p
y
© 2022 The Authors. Statistics in Medicine published by John Wiley & Sons Ltd. Statistics in Medicine. 2022;41:4822–4837. RILEY et al. 4823 1
INTRODUCTION However, before IPD are obtained, we assume estimates
of the desired interactions are not available from the trials to be included in the IPD meta-analysis project (if they were,
the need for IPD may be unjustified). Rather, we assume users can extract basic aggregate data from each trial, such as the
number of participants and events, the proportion in each category (eg, proportion male) for binary covariates, and
the mean and SD of continuous covariates. In Section 3 we show how this aggregate data can be used to approximate the
variance of each trial’s interaction estimate from a logistic regression model, adapting analytic (closed-form) solutions
proposed by Demidenko.7 Section 4 explains how to subsequently use these trial variances to calculate the corresponding
power of the planned IPD meta-analysis project. Section 5 illustrates the proposal with two examples, and our results are
compared to an approximate approach proposed by Kovalchick and Cumberland.8 Section 6 considers extension to allow
for between-trial heterogeneity in the true interaction size, and how to focus on precision rather than power. Finally,
Section 7 concludes with discussion. Stata code is provided in the Supplementary material, and R code is available at
www.github.com/gscollins1973. 2.1
First-stage Consider IPD from a parallel group trial, comparing a treatment (xij = 1) to a control (xij = 0). Let zij be a participant-level
covariate (eg, the age of participant j in trial i), observed for all participants in each trial, and consider that a binary
outcome is of interest (ie, yij = 0 or 1, where 0 denotes no event and 1 denotes an event occurred), such as whether
pre-eclampsia occurred during pregnancy. To estimate the treatment-covariate interaction in each trial separately, a
logistic regression could be fitted as follows, yij ∼Bernoulli
(
pij
)
ln
(
pij
1 −pij
)
= 𝛼i + 𝛽ixij + 𝛾izij + 𝜆ixijzij
(1) (1) where pij is the probability of the outcome event for participant j in trial i. The trial subscript i is not strictly
required, but we include it to emphasise that the logistic regression is applied to each of the i = 1 to S trials sepa-
rately, leading to S estimates of each parameter (one for each trial). Equation (1) is usually estimated using maximum
likelihood estimation, and this is our focus here, although adaptations are potentially important if sparse data are a
concern.13,14 The treatment-covariate interaction term, 𝜆i, indicates the expected change in treatment effect (log odds ratio) for
a one-unit increase in zij for trial i, and is adjusted for the prognostic effect (𝛾i) of the covariate of interest (zij) and the
reference treatment effect (𝛽i). Other prognostic factors could also be adjusted for, but we do not consider this here. For a
continuous covariate, Equation (1) assumes the effect of the interaction is linear (although extension to non-linear trends
is important in practice4,15). implement two-stage and one-stage approaches, and broad discussion of modeling interactions in IPD meta-analysis is
given elsewhere.1,4 implement two-stage and one-stage approaches, and broad discussion of modeling interactions in IPD meta-analysis is
given elsewhere.1,4 We now provide details on the two-stage approach for estimating a treatment-covariate interaction from an IPD
meta-analysis of S trials with a binary outcome. This provides the foundation for our power calculations which follow in
subsequent sections. 2
A TWO-STAGE APPROACH TO ESTIMATING A
TREATMENT-COVARIATE INTERACTION IN AN IPD META-ANALYSIS
WITH A BINARY OUTCOME In this article we focus on a two-stage IPD meta-analysis for summarizing a treatment-covariate interaction. In the
first stage, the treatment-covariate interactions are estimated using the IPD in each trial separately; in the second
stage, these interaction estimates are pooled using a chosen meta-analysis model.3 This two-stage approach can be
implemented using ipdmetan in Stata.9 By only pooling interaction estimates derived from within-trial information (ie,
based at the participant level), this approach automatically avoids trial-level confounding and aggregation bias that
may occur in meta-regression based on across-trial information,10,11 in a multivariate meta-analysis that jointly synthe-
sizes interactions and reference treatment effects,4 or in one-stage IPD meta-analysis models that do not separate out
within-trial and across-trial relationships.4,12 Our website (www.ipdma.co.uk) provides examples of statistical code to 4824 RILEY et al. 3
CALCULATING THE VARIANCE OF A TREATMENT-COVARIATE
INTERACTION ESTIMATE FOR A BINARY OUTCOME IN A SINGLE TRIAL this section we describe analytical (closed-form) solutions for var
(
̂𝜆i
)
in a single randomized Information matrix. Such solutions are challenging to obtain as, unlike for continuous outco ( )
lated with the value of ̂𝜆i itself. The reason is that for generalized linear models such as the logistic regression model in
Equation (1), each participant-level variance is a function of the participant’s predicted outcome values from the fitted
model. In other words, rather than considering one variance term per trial for a continuous outcome (ie, a single resid-
ual variance, 𝜎2
i , or one residual variance term per treatment group), for binary outcomes a separate variance term (𝜎2
ij)
is required for each participant, conditional on their covariate values. Specifically, for binary outcomes, a participant’s
response variance is pij
(1 −pij
) and thus depends on their expected outcome probability (pij), which is conditional on
the baseline risk in the trial and the prognostic effect of any covariates, including the interaction and treatment terms. This makes closed-form solutions problematic, and so Kovalchik and Cumberland suggested approximating these vari-
ances to enable a closed-form solution (based on matrix algebra, and implemented within the package ipdmeta in R).8
Their approach requires strong assumptions, such as replacing each participant’s pij
(1 −pij
) with pi (1 −pi), where pi is
the overall outcome risk in the participant’s corresponding group within trial i. Such approximations may not be reliable;
for example, the error in the approximate closed-form power estimates of Kovalchik and Cumberland is often over 10%.8
Their approach also assumes a one-stage model that amalgamates within-trial and across-trial information,8 and so will
generally over-estimate the actual power. ( ) To address these concerns, we propose to rather use and extend the analytic solutions for var
(
̂𝜆i
)
derived by Demi-
denko et al,7 which are asymptotically exact based on Fisher’s information matrix. We derive this for a binary covariate
(Section 3.1) and then for a continuous covariate (Section 3.2). Section 4 utilizes the solutions for the power calculation
for IPD meta-analysis projects. 2.2
Second stage where ̂𝜏2 is the (REML) estimated between-trial variance in interaction. where ̂𝜏2 is the (REML) estimated between-trial variance in interaction. In order to consider the potential power of an IPD meta-analysis project, we need to ascertain the potential value of
var(̂𝜆) in advance. Fundamentally this depends on the trial variances (var
(
̂𝜆i
)
), and so the next section proposes how
these may be ascertained in advance of IPD collection. 2.2
Second stage The first stage produces S estimates of the treatment-covariate interaction (̂𝜆i) and its variance (var
(
̂𝜆i
)
), one pair for
each trial in the IPD meta-analysis. In the second stage, the ̂𝜆i values are synthesised using either a common-effect model
(ie, the true interaction is assumed the same in all trials, denoted by 𝜆), ̂𝜆i ∼N
(
𝜆, var
(
̂𝜆i
))
(2) (2) or a random-effects model (ie, the true interactions in the trials are assumed randomly drawn from a normal
distribution, with a mean of 𝜆and between-study variance of 𝜏2): or a random-effects model (ie, the true interactions in the trials are assumed randomly drawn from a normal
distribution, with a mean of 𝜆and between-study variance of 𝜏2): ̂𝜆i ∼N
(
𝜆i, var
(
̂𝜆i
))
𝜆i ∼N (𝜆, 𝜏2)
(3) (3) Maximum likelihood estimation can be used to fit Equation (2), whereas restricted maximum likelihood (REML) is
recommended to fit model Equation (3).16 The summary estimate of 𝜆will be a weighted average, and it summarises
the difference in the expected treatment effect for two participants who differ in zij by one unit. That is, 𝜆represents
the summary difference in log odds ratios (ie, exp(𝜆) gives a ratio of odds ratios) for two individuals that differ in zij by
one-unit. The smaller the var
(
̂𝜆i
)
for a trial, the more weight it has in the meta-analysis. 4825 RILEY et al. 4825 For the common-effect model, the variance of the summary interaction estimate is For the common-effect model, the variance of the summary interaction estimate is var(̂𝜆) =
1
∑S
i=1
(
var
(
̂𝜆i
))−1 ,
(4) var(̂𝜆) =
1
∑S
i=1
(
var
(
̂𝜆i
))−1 ,
(4) (4) where S is the total number of trials in the IPD meta-analysis. where S is the total number of trials in the IPD meta-analysis. where S is the total number of trials in the IPD meta-analysis. ffects model, the variance of the summary interaction estimate is, For the random-effects model, the variance of the summary interaction estimate is, For the random-effects model, the variance of the summary interaction estimate is, var(̂𝜆) =
1
∑S
i=1
(
var
(
̂𝜆i
)
+ ̂𝜏2
)−1 ,
(5) (5) where ̂𝜏2 is the (REML) estimated between-trial variance in interaction. where ̂𝜏2 is the (REML) estimated between-trial variance in interaction. 3.1
Binary covariate unit information matrix can be expanded into a closed-form solution of7: Demidenko shows the unit information matrix can be expanded into a closed-form solution Demidenko shows the unit information matrix can be expanded into a closed-form solution of7: Demidenko shows the unit information matrix can be expanded into a closed-form solution of7: Ii =
exp (𝛼i)
(1 + exp (𝛼i))2 M1 Pr (xij = 0, zij = 0)
+
exp (𝛼i + 𝛽i)
(1 + exp (𝛼i + 𝛽i))2 M2 Pr (xij = 1, zij = 0)
+
exp (𝛼i + 𝛾i)
(1 + exp (𝛼i + 𝛾i))2 M3 Pr (xij = 0, zij = 1)
(8) Ii =
exp (𝛼i)
(1 + exp (𝛼i))2 M1 Pr (xij = 0, zij = 0)
+
exp (𝛼i + 𝛽i)
(1 + exp (𝛼i + 𝛽i))2 M2 Pr (xij = 1, zij = 0)
+
exp (𝛼i + 𝛾i)
(1 + exp (𝛼i + 𝛾i))2 M3 Pr (xij = 0, zij = 1)
+
exp (𝛼i + 𝛽i + 𝛾i + 𝜆i)
(1
(
𝛽
𝜆))2 M4 Pr (xij = 1, zij = 1) ,
(8) +
exp (𝛼i + 𝛾i)
(1 + exp (𝛼i + 𝛾i))2 M3 Pr (xij = 0, zij = 1)
(8) (8) p
+
exp (𝛼i + 𝛽i + 𝛾i + 𝜆i)
(1 + exp (𝛼i + 𝛽i + 𝛾i + 𝜆i))2 M4 Pr (xij = 1, zij = 1) , +
exp (𝛼i + 𝛽i + 𝛾i + 𝜆i)
(1 + exp (𝛼i + 𝛽i + 𝛾i + 𝜆i))2 M4 Pr (xij = 1, zij = 1) , +
exp (𝛼i + 𝛽i + 𝛾i + 𝜆i)
(1 + exp (𝛼i + 𝛽i + 𝛾i + 𝜆i))2 M4 Pr (xij = 1, zij = 1) , where where M1 =
⎡
⎢
⎢
⎢
⎢
⎢⎣
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
⎤
⎥
⎥
⎥
⎥
⎥⎦
M2 =
⎡
⎢
⎢
⎢
⎢
⎢⎣
1
1
0
0
1
1
0
0
0
0
0
0
0
0
0
0
⎤
⎥
⎥
⎥
⎥
⎥⎦
M3 =
⎡
⎢
⎢
⎢
⎢
⎢⎣
1
0
1
0
0
0
0
0
1
0
1
0
0
0
0
0
⎤
⎥
⎥
⎥
⎥
⎥⎦
M4 =
⎡
⎢
⎢
⎢
⎢
⎢⎣
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
⎤
⎥
⎥
⎥
⎥
⎥⎦
. 3.1
Binary covariate Therefore, to derive the unit information matrix after fitting the logistic regression of Equation (1) to a partic-
ular trial, we need to specify the estimated values of parameters 𝛼, 𝛽, 𝛾, and 𝜆alongside the four joint probabilities
Pr
(
xij = a, zij = b
)
, estimated as the proportion of participants in the trial classified as xij = 0, zij = 0; proportion classified
as xij = 1, zij = 0; proportion classified as xij = 0, zij = 1; and proportion classified as xij = 1, zij = 1. We can then derive the
asymptotic variance of the interaction estimate, using Equation (6) (ie, var(̂𝜆) = I−1
i (4, 4)∕ni). y
p
g
q
i
∕
We will adapt this approach (specifically Equation (8) followed by Equation (6)) when extending to the IPD
meta-analysis setting in Section 4, to estimate the potential var
(
̂𝜆i
)
of a binary covariate for each trial planned to be in
the IPD meta-analysis project. i
We will adapt this approach (specifically Equation (8) followed by Equation (6)) when extending to the IPD
meta-analysis setting in Section 4, to estimate the potential var
(
̂𝜆i
)
of a binary covariate for each trial planned to be in
the IPD meta-analysis project. To demonstrate the robustness of this approach, we used IPD from a randomized trial that assessed the effects of
an internet-accessed sexually transmitted infection (e-STI) testing and results service (SH:24) on uptake of STI testing,
compared to usual care. The IPD for the trial are freely available in the supplementary material of Wilson et al,17 for which
we kindly thank the authors, and a primary outcome of interest was self-reported STI testing at 6 weeks. We examined the
variance of the treatment-age interaction estimated from (i) fitting a logistic regression model (via maximum likelihood
estimation) or (ii) our approach (ie, Equation (8) followed by Equation (6)) assuming the same estimates of 𝛼i, 𝛽i, 𝛾i,
and 𝜆i as the logistic regression estimates. We did this for the full dataset (1739 participants: 921 intervention group, 818
usual care group) and then for sequentially lower sample sizes (selected at random) down to 200 participants. 3.1
Binary covariate Let zij be a binary covariate, such as zij = 1 for males and zij = 0 for females. Then, Demidenko shows that the asymptotic
variance of ̂𝜆i after fitting the logistic regression model in Equation (1) is,7 var
(
̂𝜆i
)
= I−1
i (4, 4)∕ni,
(6) (6) where ni is the total sample size of trial i, and I−1
i (4, 4) denotes the 4,4 element of the inverse matrix of Fisher’s unit
information matrix (I) (we use the word “unit” as it is independent of sample size). 4826 RILEY et al. Given the design matrix X = (1, xij, zij, xijzij
)′, the 4 by 4 unit information matrix for the ith trial can be expressed as, Ii = E(x,z)
(
exp (𝛼i + 𝛽ixij + 𝛾izij + 𝜆ixijzij
)
(1 + exp (𝛼i + 𝛽ixij + 𝛾izij + 𝜆ixijzij
))2 XX′
)
= E(x,z)
⎛
⎜
⎜
⎜
⎜
⎜⎝
exp (𝛼i + 𝛽ixij + 𝛾izij + 𝜆ixijzij
)
(1 + exp (𝛼i + 𝛽ixij + 𝛾izij + 𝜆ixijzij
))2
⎡
⎢
⎢
⎢
⎢
⎢⎣
1
xij
zij
xijzij
xij
xij
xijzij
xijzij
zij
xijzij
zij
xijzij
xijzij
xijzij
xijzij
xijzij
⎤
⎥
⎥
⎥
⎥
⎥⎦
⎞
⎟
⎟
⎟
⎟
⎟⎠
,
(7) (7) where E(x,z)(B) denotes the expected value of B over the joint distribution of x and z. Note that the expected value of a
matrix B is formed by the matrix of expected values of the elements of B. 3.1
Binary covariate (B) Continuous covariate—age, assuming it was normally distributed based on the observed mean and SD
for each trial F I G U R E 1
Comparison of the variance of a treatment-covariate interaction from the randomized trial of Wilson et al,17 as estimated
from (i) fitting a logistic regression model to the IPD (via maximum likelihood estimation using the logit command in Stata) or (ii) our
approach (ie, for a binary covariate Equation (8) followed by Equation (6); for a continuous covariate, Equation (9) followed by Equation (6))
applied to the aggregate data assuming the same estimates of 𝛼i, 𝛽i, 𝛾i, and 𝜆i as the logistic regression estimates. Stata code is available in the
Supplementary material. Each point corresponds to a different sample size, ranging from 1739 participants (far left) to 200 participants (far
right). (A) Binary covariate—sex. (B) Continuous covariate—age, assuming it was normally distributed based on the observed mean and SD
for each trial 3.1
Binary covariate The Stata
simulation code is provided in the Supplementary material, and the findings are shown in Figure 1A, which confirm that
the proposed approach gives variances of interaction estimates identical to those obtained from fitting a logistic regression
model when examining a binary covariate. 10970258, 2022, 24, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/sim.9538 by Test, Wiley Online Library on [06/03/2 RILEY et al. 4827 F I G U R E 1
Comparison of the variance of a treatment-covariate interaction from the randomized trial of Wilson et al,17 as estimated
from (i) fitting a logistic regression model to the IPD (via maximum likelihood estimation using the logit command in Stata) or (ii) our
approach (ie, for a binary covariate Equation (8) followed by Equation (6); for a continuous covariate, Equation (9) followed by Equation (6))
applied to the aggregate data assuming the same estimates of 𝛼i, 𝛽i, 𝛾i, and 𝜆i as the logistic regression estimates. Stata code is available in the
Supplementary material. Each point corresponds to a different sample size, ranging from 1739 participants (far left) to 200 participants (far
right). (A) Binary covariate—sex. (B) Continuous covariate—age, assuming it was normally distributed based on the observed mean and SD
for each trial F I G U R E 1
Comparison of the variance of a treatment-covariate interaction from the randomized trial of Wilson et al,17 as estimated
from (i) fitting a logistic regression model to the IPD (via maximum likelihood estimation using the logit command in Stata) or (ii) our
approach (ie, for a binary covariate Equation (8) followed by Equation (6); for a continuous covariate, Equation (9) followed by Equation (6))
applied to the aggregate data assuming the same estimates of 𝛼i, 𝛽i, 𝛾i, and 𝜆i as the logistic regression estimates. Stata code is available in the
Supplementary material. Each point corresponds to a different sample size, ranging from 1739 participants (far left) to 200 participants (far
right). (A) Binary covariate—sex. RILEY et al. 4828 We will adapt this approach (specifically Equation (9) followed by Equation (6)) when extending to the IPD
meta-analysis setting in the next section, to estimate the potential var
(
̂𝜆i
)
for each trial planned to be in the IPD
meta-analysis project, based on aggregate data available for each trial in terms of the number of participants, events and
an assumed covariate distribution. To demonstrate the robustness of this approach, we again used the randomized trial
of Wilson et al,17 now considering the estimated variance of a treatment-age interaction from (i) fitting a logistic regres-
sion model with (ii) our approach (ie, Equation (9) followed by Equation (6)) assuming the same estimates of 𝛼i, 𝛽i, 𝛾i,
and 𝜆i as the logistic regression estimates, but with the age covariate simulated based on an assumed normal distribu-
tion (with means and SDs matching those observed in the IPD for each group). The results are shown in Figure 1B, for
trial sample sizes from 200 to 1739 as before. Unlike in the binary covariate situation, our proposal is no longer exact,
due to the assumed distribution of the covariate being an approximation. However, it still works well in this example, as
the variance estimates for the treatment-age interaction from our proposal are very similar to those from fitting the logis-
tic regression model to the IPD directly. This is despite the observed distribution of age not being particularly close to a
normal distribution (Supplementary Material). 4
CALCULATING THE POWER OF A POTENTIAL IPD META-ANALYSIS
PROJECT TO ESTIMATE A TREATMENT-COVARIATE INTERACTION WITH
A BINARY OUTCOME We return to the main focus of our article: how to calculate the power of an IPD meta-analysis project aiming to estimate
a treatment-covariate interaction for a binary outcome. We reiterate that our aim is to do this in advance of IPD collection
and we assume that trials from which IPD are desired have not reported interaction estimates and their variances previ-
ously. The overall power of the IPD meta-analysis is not simply the sum of the power of each individual trial, but rather is
a function of the var
(
̂𝜆i
)
for the trials (the estimated variances of the trial-specific interaction estimates), which defines
the variance of the summary interaction estimate from the meta-analysis. ( ) We outline a three-step process. Step 1 describes how to derive an estimate of var
(
̂𝜆i
)
for each trial using routinely
reported aggregate data from trial publications, alongside assumptions about effect sizes (in particular, the assumed
magnitude of the treatment-covariate interaction). Step 2 takes these variances and uses them to derive an estimate of
the meta-analysis summary result for the treatment-covariate interaction. Then, third, the power of the planned IPD
meta-analysis is derived based on these previous values. Stata code to implement the approach is provided in the Supple-
mentary material, and takes just a few seconds to run for a binary covariate, and less than a few minutes for a continuous
covariate in the examples provided. R code is available at www.github.com/gscollins1973. 3.2
Continuous covariate We now extend the work of Demidenko to a continuous covariate (zij),7 again utilizing the solution of Equation (6) that
var
(
̂𝜆i
)
= I−1
i (4, 4)∕ni. For a continuous covariate, the Fisher’s unit information matrix for the logistic regression model
in Equation (1) can be written as: Ii = E(x,z)
(
exp (𝛼i + 𝛽ixij + 𝛾izij + 𝜆ixijzij
)
(1 + exp (𝛼i + 𝛽ixij + 𝛾izij + 𝜆ixijzij
))2 XX′
)
= E(x,z)
⎛
⎜
⎜
⎜
⎜
⎜⎝
exp(𝛼i+𝛽ixij+𝛾izij+𝜆ixijzij)
(1+exp(𝛼i+𝛽ixij+𝛾izij+𝜆ixijzij))
2
⎡
⎢
⎢
⎢
⎢
⎢⎣
1
xij
zij
xijzij
xij
x2
ij
xijzij
x2
ijzij
zij
xijzij
z2
ij
xijz2
ij
xijzij
x2
ijzij
xijz2
ij
x2
ijz2
ij
⎤
⎥
⎥
⎥
⎥
⎥⎦
⎞
⎟
⎟
⎟
⎟
⎟⎠
= E(x,z)(B),
(9) (9) where B is a 4 by 4 matrix. where B is a 4 by 4 matrix. where B is a 4 by 4 matrix. The expected value (E(x,z)(B)) depends on the joint distribution of the continuous covariate and the treatment/control
group allocation (ie, joint distribution of x and z), and also the values of the logistic regression parameters (𝛼i, 𝛽i, 𝛾i, 𝜆i)
themselves. For this reason, and unlike for a binary covariate, it is not possible to modify Equation (9) into a closed-form
solution for Ii. However, one way to derive E(x,z)(B) post estimation, is to calculate each of the 16 components of B for each
participant in the trial using the estimated logistic regression parameters, and then their means provide their expected
values and thus form Ii. We can then derive the asymptotic variance of the interaction estimate, using Equation (6) (ie,
var
(
̂𝜆i
)
= I−1
i (4, 4)∕ni). 4.1.1
Binary covariate Based on the assumed values of 𝛼i, 𝛽i, 𝛾i, and 𝜆i, and the aggregate data extracted, we can apply Equation (8) followed
by Equation (6) to obtain an estimate of var
(
̂𝜆i
)
. 10970258, 2022, 24, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/sim.9538 by Test, Wiley Online Library on [06/03/20 RILEY et al. 4829 TA B L E 1
Aggregate data required from each trial to implement the proposed power calculation for the interaction between
treatment effect and a binary covariate
Total participants in the trial (ni)
Total participants in control group (nCi)
Total participants in treatment group (nTi)
Number of outcome events in the control group (eCi)
Number of outcome events in the treatment group (eTi)
Proportion of patients in the trial with xij = 0 and zij = 0 (Pr (xij = 0, zij = 0))
Proportion of patients in the trial with xij = 0 and zij = 1 (Pr (xij = 0, zij = 1))
Proportion of patients in the trial with xij = 1 and zij = 0 (Pr (xij = 1, zij = 0))
Proportion of patients in the trial with zij = 1 and xij = 1 (Pr (xij = 1, zij = 1)) TA B L E 1
Aggregate data required from each trial to implement the proposed power calculation for the interaction between
treatment effect and a binary covariate TA B L E 2
Aggregate data required from each trial to implement the proposed power calculation for the interaction between
treatment effect and a continuous covariate TA B L E 2
Aggregate data required from each trial to implement the proposed power calculation for the interaction between
treatment effect and a continuous covariate TA B L E 2
Aggregate data required from each trial to implement the proposed power calculation for the interaction between
treatment effect and a continuous covariate
Total participants in the trial (ni)
Total participants in control group (nCi)
Total participants in treatment group (nTi)
Number of outcome events in the control group (eCi)
Number of outcome events in the treatment group (eTi)
Characteristics to define the continuous covariate’s assumed distribution (eg, mean and SD in each group) Characteristics to define the continuous covariate’s assumed distribution (eg, mean and SD in each group) outcome in trial i in the control group for a participant with the mean value of zij. 4.1.1
Binary covariate This can be approximated by the overall
log-odds of the outcome event in the control group of trial i, which can be derived from log(eCi∕(nCi −eCi)). The treatment
effect (𝛽i) for a participant with the mean value of zij can be approximated by the overall treatment effect (log odds ratio)
in the trial. If not reported directly, this log odds ratio can be calculated manually from nCi, nTi, eCi, and eTi. p
y
g
y
In terms of the prognostic effect (𝛾i) of the covariate, we suggest assuming this is zero for simplicity, or considering a
range of possible values (see examples later). For the key parameter (𝜆i), we suggest identifying a minimally important
value via discussion with clinical experts within the IPD meta-analysis project team. It is simplest to assume 𝜆i is common
for all trials (ie, 𝜆i = 𝜆), although we discuss extensions to allow for between-study heterogeneity in Section 6. g
y
g
y
Based on the assumed values of 𝛼i, 𝛽i, 𝛾i, and 𝜆i, and the aggregate data extracted, we can apply Equation (8) followed
by Equation (6) to obtain an estimate of var
(
̂𝜆i
)
. 4.1.1
Binary covariate For each trial promising their IPD (or for which IPD are sought), the first step is to apply Equation (8) followed by
Equation (6) to obtain an estimate of var
(
̂𝜆i
)
. ( )
To implement the approach, we need to obtain the aggregate data from each trial listed in Table 1. Some of the items
listed can be derived from other aggregate data listed, but all items are included for completeness. This set of aggregate data
are usually available from trial publications, especially for commonly reported baseline covariates like sex (eg, in a table
summarizing baseline characteristics per group, often referred to as “Table 1”), but if not, the original trial investigators
could be contacted to provide this information. If only the overall proportion with zij = 1 (or = 0) is available, then, as
these are randomized trials, it could be assumed that the same proportion occurs in both treatment and control groups. We also need to make assumptions about the values of the logistic regression parameters in each of the trials
(𝛼i, 𝛽i, 𝛾i, 𝜆i). Without loss of generalisability, let us assume zij will be centered by its trial-specific mean when fitting the
logistic regression of Equation (1). This does not change the interpretation of the interaction term (𝜆i) or the prognostic
effect of the covariate (𝛾i), but it helps to interpret the two other parameters. That is, 𝛼i now becomes the log-odds of the 10970258, 2022, 24, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/sim.9538 by Test, Wiley Online Library on [06/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Cre RILEY et al. 4.1.1
Binary covariate 4829
TA B L E 1
Aggregate data required from each trial to implement the proposed power calculation for the interaction between
treatment effect and a binary covariate
Total participants in the trial (ni)
Total participants in control group (nCi)
Total participants in treatment group (nTi)
Number of outcome events in the control group (eCi)
Number of outcome events in the treatment group (eTi)
Proportion of patients in the trial with xij = 0 and zij = 0 (Pr (xij = 0, zij = 0))
Proportion of patients in the trial with xij = 0 and zij = 1 (Pr (xij = 0, zij = 1))
Proportion of patients in the trial with xij = 1 and zij = 0 (Pr (xij = 1, zij = 0))
Proportion of patients in the trial with zij = 1 and xij = 1 (Pr (xij = 1, zij = 1))
TA B L E 2
Aggregate data required from each trial to implement the proposed power calculation for the interaction between
treatment effect and a continuous covariate
Total participants in the trial (ni)
Total participants in control group (nCi)
Total participants in treatment group (nTi)
Number of outcome events in the control group (eCi)
Number of outcome events in the treatment group (eTi)
Characteristics to define the continuous covariate’s assumed distribution (eg, mean and SD in each group)
outcome in trial i in the control group for a participant with the mean value of zij. This can be approximated by the overall
log-odds of the outcome event in the control group of trial i, which can be derived from log(eCi∕(nCi −eCi)). The treatment
effect (𝛽i) for a participant with the mean value of zij can be approximated by the overall treatment effect (log odds ratio)
in the trial. If not reported directly, this log odds ratio can be calculated manually from nCi, nTi, eCi, and eTi. In terms of the prognostic effect (𝛾i) of the covariate, we suggest assuming this is zero for simplicity, or considering a
range of possible values (see examples later). For the key parameter (𝜆i), we suggest identifying a minimally important
value via discussion with clinical experts within the IPD meta-analysis project team. It is simplest to assume 𝜆i is common
for all trials (ie, 𝜆i = 𝜆), although we discuss extensions to allow for between-study heterogeneity in Section 6. 4830 Based on the assumed values of 𝛼i, 𝛽i, 𝛾i, and 𝜆i, and the aggregate data extracted, we can obtain an estimate of var
(
̂𝜆i
)
for each trial by calculating Fisher’s unit information matrix as described in Section 3.2. This is implemented in our Stata
and R code. Essentially, we mimic what occurs in software behind the scenes, post-estimation of a logistic regression
model. That is, for each trial separately, our code: i. generates a large dataset (eg, 1 million participants) that matches the trial aggregate data provided in terms of the
proportion of participants in the treatment and control groups, the proportion with an outcome event in each group,
and the distribution of z in each group (eg, a normal distribution with a specified mean and SD); i. generates a large dataset (eg, 1 million participants) that matches the trial aggregate i. generates a large dataset (eg, 1 million participants) that matches the trial aggregate data provided in terms of the
proportion of participants in the treatment and control groups, the proportion with an outcome event in each group,
and the distribution of z in each group (eg, a normal distribution with a specified mean and SD); tribution of z in each group (eg, a normal distribution with a specified mean and SD); ii. calculates Ii = E(x,z)(B) conditional on the values of 𝛼i, 𝛽i, 𝛾i, and 𝜆i specified for that trial (Equation 9);
iii. uses Equation (6) to calculate var
(
̂𝜆i
)
= I−1
i (4, 4)∕ni. ii. calculates Ii = E(x,z)(B) conditional on the values of 𝛼i, 𝛽i, 𝛾i, and 𝜆i specified for that trial (Equation 9);
iii. uses Equation (6) to calculate var
(
̂𝜆i
)
= I−1
i (4, 4)∕ni. 5
APPLIED EXAMPLES We now apply our proposed method to two examples. 4.2
Step 2: Estimate the variance of the summary treatment-covariate from
the planned IPD meta-analysis Step 1 produces S estimates of var
(
̂𝜆i
)
, one for each trial. The variance of the summary interaction estimate from an IPD
meta-analysis of these trials can then be estimated, depending on whether step 1 assumed 𝜆i was common or random
across trials. In particular, when assuming 𝜆i is common (𝜆i = 𝜆), we can use Equation (5) to calculate the anticipated
estimate of var(̂𝜆) for the IPD meta-analysis project: var(̂𝜆) =
1
∑S
i=1
(
var
(
̂𝜆i
))−1 . 4.3
Step 3: Calculate the power of the planned IPD meta-analysis The final step is to calculate the power of the planned IPD meta-analysis project to detect 𝜆. Assuming a common
interaction for all trials, and based on a Wald-test and a 5% statistical significance level, the power is approximately: Power = Prob
⎛
⎜
⎜
⎜⎝
̂𝜆
√
var(̂𝜆)
> 1.96
⎞
⎟
⎟
⎟⎠
+ Prob
⎛
⎜
⎜
⎜⎝
̂𝜆
√
var(̂𝜆)
< −1.96
⎞
⎟
⎟
⎟⎠
= Φ
⎛
⎜
⎜
⎜⎝
−1.96 +
̂𝜆
√
var(̂𝜆)
⎞
⎟
⎟
⎟⎠
+ Φ
⎛
⎜
⎜
⎜⎝
−1.96 −
̂𝜆
√
var(̂𝜆)
⎞
⎟
⎟
⎟⎠
. (10) (10) Here, Φ(x) is the probability of sampling a value < x from a standard normal distribution, var(̂𝜆) is the estimated
variance of the summary interaction estimate (as obtained in step 2), and ̂𝜆can be replaced with the assumed true 𝜆(as
defined in step 1). The power estimate is usually multiplied by 100 and reported as a %. 4.1.2
Continuous covariate The approach to estimate var
(
̂𝜆i
)
for a continuous covariate is similar to that described for a binary covariate, and the
aggregate data required from each trial publication are shown in Table 2. There is the added complexity of having to
specify the assumed distribution of the continuous covariate. For simplicity, this might be assumed to be a normal dis-
tribution (with a different mean and SD for each trial), especially as the mean and SD of key continuous covariates are
usually reported in a trial publication (in the table of trial participant characteristics). However, depending on the covari-
ate and information available, other distributions (eg, uniform) might be sensible to consider, and indeed a different type
of distribution could be used in each trial if necessary. Similar to the binary covariate setting, assumptions are needed about the values of parameters 𝛼i, 𝛽i, 𝛾i, and 𝜆i. Again,
without loss of generalisability, centering zij by its mean allows 𝛼i to be approximated by the overall log-odds of the
outcome event in the control group in trial i, and 𝛽i can be approximated by the overall treatment effect (log odds ratio)
in trial i. As before, we suggest to let 𝛾i = 0 (or consider a range of values), and choose 𝜆i after discussion with clinicians
on what constitutes a minimally relevant interaction effect size. RILEY et al. 10970258, 2022, 24, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/sim.9538 by Test, Wiley Online Library on [06/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA arti Note: Assuming 𝛼i is the observed log-odds of the outcome event in the control group in trial i, 𝛽i is the observed overall treatment effect (log odds ratio) in trial
i, there is no prognostic effect of the covariate (𝛾i = 0) and 𝜆is log(1.3) for sex (males compared to females) and log(1.3) for a 10-year increase in age. Note: Assuming 𝛼i is the observed log-odds of the outcome event in the control group in trial i, 𝛽i is the observed overall treatment effect (log odds ratio) in trial
i, there is no prognostic effect of the covariate (𝛾i = 0) and 𝜆is log(1.3) for sex (males compared to females) and log(1.3) for a 10-year increase in age. prevention of gastrointestinal bleeding for patients with cirrhosis and esophageal varices. IPD were obtained from four
randomized trials involving a total of 286 patients randomized to active treatment and 383 to a control (placebo), and
aggregate data from the trials are shown in Table 3. We now ask the question: assuming this aggregate data could be obtained in advance (eg, from trial publications or
investigators), what is the estimated power of a planned IPD meta-analysis to examine treatment-covariate interactions? We focus on the covariates sex and age, and use the three-step process described in Section 4 to undertake our power
calculations. As in previous considerations of this meta-analysis,8 we assume 𝜆= log(1.3) for the treatment-sex interaction
and 𝜆= log(1.027) for the treatment-age interaction. These correspond to an odds ratio that is 30% higher for males
compared to females (ie, the treatment is less effective for males compared to females), and an odds ratio that is 30%
higher for every 10-year increase in age. We also assume that the interaction is common (the same) for all trials. Age is
also assumed normally distributed in each trial, with a mean and SD as given in the table. The results of the power calculation are shown in Table 4, assuming that the prognostic effect of the covariates is zero
(𝛾i = 0). The power is low for both covariates. There is a power of 8.8% to detect the assumed treatment-sex interaction,
and a power of 26% to detect the assumed treatment-age interaction. The power estimates barely change when allowing
for a moderate prognostic effect of each covariate. 10970258, 2022, 24, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/sim.9538 by Test, Wiley Online Library on [06/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA arti 4831 RILEY et al. 10970258, 2022, 24, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/sim.9538 by Test, Wiley Online Library on [06/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA arti 4831 TA B L E 3
Aggregate data from 4 randomized trials included in the IPD meta-analysis project of Poynard et al,18 as reported by
Kovalchik and Cumberland8 TA B L E 3
Aggregate data from 4 randomized trials included in the IPD meta-analysis project of Poynard et al,18 as reported by
Kovalchik and Cumberland8
Trial
Total participants
(events) control
Total participants
(events) treatment
Age in years:
mean (SD)
control
Age in years:
mean (SD) treatment
Male,
% control
Male,
% treatment
1
112 (30)
118 (19)
54 (11)
54 (9)
71
71
2
89 (28)
85 (16)
53 (11)
55 (11)
73
67
3
49 (11)
30 (1)
55 (9)
53 (7)
71
73
4
53 (13)
53 (13)
57 (12)
55 (11)
76
74 egate data from 4 randomized trials included in the IPD meta-analysis project of Poynard et al,18 as reported b
berland8 from 4 randomized trials included in the IPD meta-analysis project of Poynard et al,18 as reported by TA B L E 3
Aggregate data from 4 randomized trials included in the IPD meta-analysis project of Poynard et al,18 as reported by
Kovalchik and Cumberland8 1
112 (30)
118 (19)
54 (11)
54 (9)
71
71
2
89 (28)
85 (16)
53 (11)
55 (11)
73
67
3
49 (11)
30 (1)
55 (9)
53 (7)
71
73
4
53 (13)
53 (13)
57 (12)
55 (11)
76
74
TA B L E 4
Results of our power calculation for the Poynard example, using the three-step process described in Section 3 based on the
aggregate data shown in Table 3
Variance estimate
of each trial’s interaction
estimate
(
var
(
̂𝝀i
) )
Power (%) based
on each trial
separately
Weight (%)
in the planned
IPD meta-analysis
Study
Sex
Age
Sex
Age
Sex
Age
Trial 1
0.512
0.00115
6.55
12.06
41.17
35.05
Trial 2
0.596
0.00107
6.33
12.62
35.36
37.74
Trial 3
5.513
0.0229
5.14
5.34
3.82
1.76
Trial 4
1.073
0.00159
5.74
10.09
19.65
25.45
Variance estimate of
summary interaction
estimate (var(̂𝝀))
Power (%) of
planned IPD
meta-analysis
Planned IPD meta-analysis
Sex
Age
Sex
Age
All 4 trials
var(̂𝜆) = 0.211
var(̂𝜆) = 0.0004
8.82
25.66 TA B L E 4
Results of our power calculation for the Poynard example, using the three-step process described in Section 3 based on the
aggregate data shown in Table 3 r power calculation for the Poynard example, using the three-step process described in Section 3 based on the
l 3 TA B L E 4
Results of our power calculation for the Poynard example, using the three-step process described in Section 3 based on the
aggregate data shown in Table 3
Variance estimate
of each trial’s interaction
estimate
(
var
(
̂𝝀i
) )
Power (%) based
on each trial
separately
Weight (%)
in the planned
IPD meta-analysis
Study
Sex
Age
Sex
Age
Sex
Age
Trial 1
0.512
0.00115
6.55
12.06
41.17
35.05
Trial 2
0.596
0.00107
6.33
12.62
35.36
37.74
Trial 3
5.513
0.0229
5.14
5.34
3.82
1.76
Trial 4
1.073
0.00159
5.74
10.09
19.65
25.45
Variance estimate of
summary interaction
estimate (var(̂𝝀))
Power (%) of
planned IPD
meta-analysis
Planned IPD meta-analysis
Sex
Age
Sex
Age
All 4 trials
var(̂𝜆) = 0.211
var(̂𝜆) = 0.0004
8.82
25.66
Note: Assuming 𝛼i is the observed log-odds of the outcome event in the control group in trial i, 𝛽i is the observed overall treatment effect (log odds ratio) in trial
i, there is no prognostic effect of the covariate (𝛾i = 0) and 𝜆is log(1.3) for sex (males compared to females) and log(1.3) for a 10-year increase in age. 5.1
Example 1: Efficacy of beta-adrenergic-antagonist drugs in the prevention
of gastrointestinal bleeding Our first example considers the power of an IPD meta-analysis conducted by Poynard et al,18 and is an example considered
in previous methodology papers.3,8 The project aimed to examine the efficacy of beta-adrenergic-antagonist drugs in the 5.2
Example 2: Examining effect modifiers for exercise interventions to improve pain
in osteoarthritis In the Subgrouping and TargetEd Exercise pRogrammes for OsteoArthritis (STEER OA) project, IPD was obtained from
existing randomized trials to examine potential participant-level characteristics that interact with the effect of exercise
interventions among people with knee and/or hip OA.19 Although pain and function outcomes were mostly analyzed
on a continuous scale, one binary outcome of interest was whether the patient had a reduction of pain (compared to
baseline pain) by 3-6 months. Ultimately, 31 trials provided their IPD, and here we retrospectively consider the potential
power of this IPD meta-analysis for examining whether age or sex modify the effect of exercise for this binary outcome. The aggregate data for each trial are provided in Table 5, in terms of the total participants and outcome events for each
treatment (exercise program) and control group, and the summary characteristics of age (mean, SD) and sex (percentage
male). For simplicity, we assume the same interaction sizes are of interest as in the previous example (𝜆= log(1.3) for the
treatment-sex interaction and 𝜆= log(1.027) for the treatment-age interaction), and that the true interaction is the same
in each trial. We also assume the prognostic effect of age and sex is zero, and that age is normally distributed. Applying
the three-step process described in Section 4 to undertake our power calculations, we find the planned IPD meta-analysis
project has a power of 90.4% for age and 41.9% for sex (Table 6). Hence, the power is reassuringly high for age, but
only moderate for sex. The power for sex is partly hampered by sex not being recorded for two trials (trials 18 and 19),
and only females being recruited in another (trial 20), which means these three trials provide no information about the
treatment-sex interaction. ley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License The anticipated percentage weight for each trial is also revealing (Table 6) as it showcases which trials are most impor-
tant for obtaining their IPD. For example, in terms of the age covariate, trials 1, 15, and 28 are each expected to provide
over 10% of the weight toward the summary meta-analysis result. Indeed, repeating our power calculation just using these
three trials gives a power of 48% for age, which is over half the power of the full IPD meta-analysis of all 31 trials. 10970258, 2022, 24, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/sim.9538 by Test, Wiley Online Library on [06/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA arti For example, when assuming the prognostic effect of sex is an odds
ratio of 1.5 (𝛾i = ln(1.5)), the power is 9.0%, compared to 8.8% when assuming there is no prognostic effect. Given the low
power, it raises considerable doubts as to the value of doing this IPD meta-analysis project in terms of reliably estimating
the interactions. In particular, if known in advance of IPD collection, the low power might persuade researchers and
funders that it is not worth their investment; or perhaps it may provide incentive to pursue IPD from additional trials,
if they exist. 4832 RILEY et al. The anticipated contribution of each of the trials is similar for trials 1, 2, and 4, as revealed by the power estimates
when taking each trial in isolation, and the anticipated percentage weight in the IPD meta-analysis (Table 4). Trial 3 has
the fewest number of participants and events, and the lowest SD of age, and so contributes relatively little; indeed, the
percentage weight toward the summary interaction is just 3.82% and 1.76% for sex and age, respectively. It is clear that trial
3 basically provides no information towards the power calculation, which is unsurprising given the small sample sizes
and sparse events, and so obtaining IPD from this trial is of low priority if the focus is solely on the treatment interaction
with age or sex (but there may be other objectives that still warrant prioritizing the IPD for this trial). We also compared our power estimates to those calculated using the approach of Kovalchik and Cumberland.8 Recall,
their method uses a more approximate approach that (in addition to within-trial information) also allows across-trial
information to contribute toward the power calculation, which is not recommended in practice due to potential for aggre-
gation bias.4 However, in this example, the power estimates using Kovalchik and Cumberland’s approach are 8.7% for
sex and 26% for age, almost identical to our estimates of 8.8% and 26% based solely on within-trial information. The simi-
larity is because the across-trial information is negligible in this example, as the mean covariate values vary only slightly
across trials, as shown in Table 3. The percentage male varies only from 71% to 76%, and the mean age varies only from
53 to 57 years. 5.2
Example 2: Examining effect modifiers for exercise interventions to improve pain
in osteoarthritis Hence,
collecting the IPD from trials 1, 15, and 28 should be prioritized, in terms of examining age. Also note that, as emphasized in Section 4, the power contribution of each trial depends on the covariate distribution
and variance, not just the total number of participants and events. For example, although trial 4 has 199 participants and
104 events, and is thus one of the larger trials, it has a relatively low SD for age, and this leads to an anticipated percentage
weight of only 2.4% toward the IPD meta-analysis examining the treatment-age interaction. In contrast to the previous example, our power estimates are much lower than the approximate estimates obtained
using the approach of Kovalchick and Cumberland,8 for both sex (41.9% vs 46.9%) and age (90.4% vs 95.3%). Recall that the
approach of Kovalchick and Cumberland also allows across-trial information to contribute toward the power calculation,
which here is influential as the mean covariate value varies considerable across trials (unlike in the first example), as seen
in Table 5. For example, the percentage male ranges from about 9% to 59%, and the mean age ranges from about 57 to
75 years. 10970258, 2022, 24, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/sim.9538 by Test, Wiley Online Library on [0 RILEY et al. 4834 RILEY et al. TA B L E 6
Results of our power calculation for the STEER-OA example, using the three-step process described in Section 3 based on
the aggregate data shown in Table 5 TA B L E 6
Results of our power calculation for the STEER-OA example, using the three-step process described in Section 3 based on
the aggregate data shown in Table 5 TA B L E 6
Results of our power calculation for the STEER-OA example, using the three-step process described in Section 3 based on
the aggregate data shown in Table 5 p
p ,
g
p p
the aggregate data shown in Table 5
Variance estimate of
each trial’s interaction
estimate (var
(
̂𝝀i
)
)
Power (%) based on
each trial separately
Weight (%) in the
planned IPD
meta-analysis
Study
Sex
Age
Sex
Age
Sex
Age
Trial 1
0.50
0.0006
6.60
17.86
4.49
10.02
Trial 2
1.88
0.0067
5.42
6.19
1.19
0.97
Trial 3
0.80
0.0029
5.99
7.75
2.79
2.21
Trial 4
0.39
0.0027
7.05
7.96
5.74
2.38
Trial 5
0.37
0.0015
7.16
10.38
6.04
4.28
Trial 6
0.24
0.0013
8.41
11.12
9.49
4.86
Trial 7
0.78
0.0018
6.02
9.52
2.87
3.62
Trial 8
0.95
0.0039
5.84
7.07
2.36
1.67
Trial 9
0.94
0.0021
5.84
8.82
2.37
3.06
Trial 10
2.43
0.0158
5.33
5.50
0.92
0.41
Trial 11
0.36
0.0012
7.22
11.92
6.21
5.48
Trial 12
2.64
0.0061
5.30
6.31
0.85
1.06
Trial 13
1.46
0.0040
5.54
6.98
1.53
1.60
Trial 14
1.24
0.0052
5.64
6.52
1.81
1.23
Trial 15
0.22
0.0006
8.61
18.06
10.05
10.18
Trial 16
0.41
0.0009
6.96
13.97
5.48
7.06
Trial 17
0.70
0.0024
6.13
8.34
3.20
2.68
Trial 18
-
0.0026
-
8.11
-
2.50
Trial 19
-
0.0126
-
5.63
-
0.51
Trial 20
-
0.0371
-
5.21
-
0.17
Trial 21
3.06
0.0161
5.26
5.49
0.73
0.40
Trial 22
6.88
0.0196
5.11
5.40
0.33
0.33
Trial 23
0.67
0.0017
6.19
9.87
3.35
3.89
Trial 24
0.71
0.0031
6.11
7.61
3.13
2.11
Trial 25
7.32
0.0106
5.11
5.75
0.31
0.61
Trial 26
2.85
0.0171
5.28
5.46
0.78
0.38
Trial 27
0.37
0.0011
7.18
12.62
6.12
6.02
Trial 28
0.19
0.0005
9.35
22.63
12.05
13.65
Trial 29
1.84
0.0059
5.43
6.34
1.22
1.09
Trial 30
0.64
0.0013
6.25
11.07
3.51
4.82
Trial 31
2.05
0.0091
5.39
5.87
1.09
0.71
Planned IPD
meta-analysis
Variance estimate
of summary interaction
estimate (var(̂𝝀))
Power (%) of
planned IPD
meta-analysis
Sex
Age
Sex
Age
All trials
0.022
0.000064
41.88
90.42
Note: Assuming 𝛼i is the observed log-odds of the outcome event in the control group in trial i, 𝛽i is the observed overall treatment effect (log odds ratio) in
trial i, there is no prognostic effect of the covariate (𝛾i = 0) and 𝜆is log(1.3) for sex (males compared to females) and log(1.3) for a 10-year increase in age. 5.2
Example 2: Examining effect modifiers for exercise interventions to improve pain
in osteoarthritis 4833 TA B L E 5
Aggregate data from 31 randomized trials included in the IPD meta-analysis project of STEER-OA,19 where events
correspond to patients reporting to being in pain TA B L E 5
Aggregate data from 31 randomized trials included in the IPD meta-analysis project of STEER-OA,19 where events
correspond to patients reporting to being in pain
Trial
Total participants
(events) control
Total participants
(events) treatment
Age in years:
mean (SD) control
Age in years:
mean (SD)
treatment
Male,
% control
Male,
% treatment
1
68 (39)
142 (88)
64.25 (12.21)
65.29 (11.46)
22.06
30.99
2
24 (9)
24 (15)
67.17 (8.13)
65.17 (6.72)
20.83
37.50
3
44 (18)
45 (30)
64.59 (7.55)
64.52 (9.05)
54.55
48.89
4
99 (40)
100 (64)
62.54 (5.36)
60.96 (5.92)
30.30
40.00
5
74 (40)
148 (75)
62.28 (6.77)
63.93 (9.39)
36.49
28.38
6
159 (55)
153 (56)
69.63 (6.26)
69.86 (6.82)
37.74
36.60
7
63 (22)
63 (40)
64.94 (9.43)
63.21 (8.38)
26.98
22.22
8
41 (19)
111 (83)
69.61 (6.10)
70.39 (6.28)
17.07
29.73
9
43 (16)
45 (28)
60.37 (9.91)
61.76 (9.49)
46.51
24.44
10
16 (9)
23 (8)
75.19 (4.58)
73.57 (7.3)
25.00
21.74
11
108 (60)
109 (73)
68.23 (7.98)
67.94 (8.54)
35.19
34.86
12
23 (10)
25 (7)
61.30 (7.06)
65.03 (8.91)
26.09
12.00
13
35 (13)
36 (29)
61.53 (7.80)
63.34 (9.55)
31.43
33.33
14
49 (20)
56 (32)
65.20 (5.73)
65.43 (5.27)
12.24
21.43
15
140 (71)
278 (193)
66.76 (8.72)
66.52 (8.25)
31.43
28.78
16
147 (78)
71 (50)
59.11 (9.99)
57.79 (10.42)
59.18
59.15
17
54 (16)
53 (33)
63.56 (8.73)
65.62 (8.21)
46.30
43.40
18
78 (44)
80 (40)
68.61 (6.13)
69.02 (6.55)
NA
NA
19
40 (17)
40 (26)
58.20 (4.26)
57.25 (3.98)
NA
NA
20
44 (29)
43 (30)
63.91 (2.36)
63.81 (2.41)
0
0
21
9 (6)
19 (13)
70.44 (7.83)
66.37 (5.55)
44.44
47.37
22
11 (5)
21 (16)
71.18 (5.25)
72.43 (6.52)
9.09
14.29
23
54 (27)
55 (38)
57.21 (9.82)
58.4 (10.00)
48.15
43.64
24
54 (21)
55 (23)
68.60 (6.87)
66.85 (7.41)
27.78
34.55
25
20 (10)
20 (15)
67.10 (5.36)
66.14 (8.74)
35.00
5.00
26
17 (11)
17 (10)
70.76 (4.71)
69.59 (6.74)
23.53
23.53
27
102 (66)
101 (64)
66.59 (9.56)
64.18 (8.52)
45.10
37.62
28
156 (78)
235 (110)
61.89 (9.59)
61.54 (9.58)
35.90
36.17
29
27 (16)
28 (20)
78.93 (8.30)
78.89 (6.91)
33.33
21.43
30
102 (55)
98 (77)
67.78 (9.22)
68.29 (8.45)
20.59
22.45
31
23 (4)
23 (7)
66.78 (7.27)
67.57 (7.86)
52.17
60.87 ey.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Note: Assuming 𝛼i is the observed log-odds of the outcome event in the control group in trial i, 𝛽i is the observed overall treatment effect (log odds ratio) in
trial i, there is no prognostic effect of the covariate (𝛾i = 0) and 𝜆is log(1.3) for sex (males compared to females) and log(1.3) for a 10-year increase in age.
Bold values are the key results. 6.2
Focusing on precision rather than power In addition to (or even instead of) calculating power, researchers may also want to check the potential precision of
their planned IPD meta-analysis, in terms of whether confidence intervals will be sufficiently narrow to inform clinical
decision-making. For example, assuming no between-trial heterogeneity in the treatment-covariate interaction, an antici-
pated 95% confidence interval for the interaction from an IPD meta-analysis can be calculated using ̂𝜆± (1.96 ×
√
var(̂𝜆) ),
where ̂𝜆is the assumed interaction and var(̂𝜆) is taken from Step 2 of our approach. ey.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 6.1
Allowing for heterogeneity To allow for between-trial heterogeneity in a treatment-covariate interaction, we can extend the power calculation to, heterogeneity in a treatment-covariate interaction, we can extend the power calculation to, allow for between-trial heterogeneity in a treatment-covariate interaction, we can extend the Power = T
⎛
⎜
⎜
⎜⎝
−tS−1,0.975 +
̂𝜆
√
var(̂𝜆)
⎞
⎟
⎟
⎟⎠
+ T
⎛
⎜
⎜
⎜⎝
−tS−1,0.975 −
̂𝜆
√
var(̂𝜆)
⎞
⎟
⎟
⎟⎠
,
(11) (11) where T(x) is the probability of sampling a value < x from a t-distribution with a mean of zero and S −1 degrees of free-
dom, and S is the number of trials assumed to provide their IPD. Crucially var(̂𝜆) must be derived from the random-effects
solution of Equation (5), and so an assumed value of ̂𝜏(the between-trial SD of interactions) must also be given, which
is not straightforward to ascertain. A t-distribution is used (rather than a normal distribution) to help reflect extra uncer-
tainty due to ̂𝜏being estimated in practice (akin to the use of the t-distribution in the Hartung-Knapp-Sidik-Jonkman
[HKSJ] approach for deriving 95% confidence intervals after fitting a random-effects meta-analysis20,21). For example, returning to the STEER-OA example from Section 5, and considering the power to detect a treatment-age
interaction of 𝜆= log(1.027), let us apply Equation (11) (rather than Equation (10)) to allow for between-study hetero-
geneity, which we assume is 𝜏= 0.015, as this would reflect quite large variability relative to the summary interaction of
1.027, such that the interaction may be negligible in some settings. The power is estimated to be 81.9%, which is somewhat
lower than when assuming a common-effect (90.4%), as expected, but still reassuringly high. Equation (11) is only an approximation, and this is of most concern when there are a small number of trials and the
true 𝜏is close to zero, as then 𝜏is particularly poorly estimated in practice (often with upward bias). A simulation-based
approach would be a better reflection of the uncertainty in that situation.5 Bold values are the key results. iley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License RILEY et al. 4835 7
DISCUSSION Since IPD meta-analysis projects with large power are more likely to detect important effects of interest, they should
(other things being equal) be a higher priority for funding. Therefore, careful assessments of power and sample size are an
important part of planning and commissioning IPD meta-analysis projects that aim to estimate a particular effect. In this
article, we have proposed a new approach for calculating the power when designing an IPD meta-analysis project aiming
to pool randomized trials to estimate a treatment-covariate interaction for a binary outcome. Our three-step approach
uses an asymptotic solution for calculating variances of interaction estimates, which is more exact compared to previous
suggestions. Further, we showed how routinely reported aggregate data from trial publications can be used to inform the
power calculation, to ensure the power is conditional on the actual characteristics (eg, sample size in each group, number
of events, covariate distributions) of each trial for which IPD is promised or desired. This requires the distribution of the
covariate to be assumed, with is potentially difficult for continuous covariates, for which we recommended using either
a normal distribution or uniform distribution as a pragmatic approach. We also showed how power calculations reveal which trials contribute most to the power, which could be used along-
side other information (eg, relevance of trial population, outcome definitions, follow-up length, and so forth), to inform
decisions regarding which trials are prioritized for obtaining their IPD. This last point might be viewed as contentious, as
some may argue that IPD should always be sought from all trials. The counterargument is that IPD meta-analysis projects 4836 RILEY et al. are time-consuming (and thus costly), and sometimes limited resources are available or there is an urgent need for quick
answers (eg, in a pandemic); then, it may be justifiable to focus efforts on obtaining IPD from the subset of trials that pro-
vide the most information to answer the research question reliably and in the shortest time-frame for the population of
interest. are time-consuming (and thus costly), and sometimes limited resources are available or there is an urgent need for quick
answers (eg, in a pandemic); then, it may be justifiable to focus efforts on obtaining IPD from the subset of trials that pro-
vide the most information to answer the research question reliably and in the shortest time-frame for the population of
interest. ORCID Richard D. Riley
https://orcid.org/0000-0001-8699-0735
Miriam Hattle
https://orcid.org/0000-0003-1542-6277
Gary S. Collins
https://orcid.org/0000-0002-2772-2316
Rebecca Whittle
https://orcid.org/0000-0003-1793-0135 Joie Ensor
https://orcid.org/0000-0001-7481-0282 7
DISCUSSION As with any sample size calculation, our approach is a pragmatic one to help gauge potential power under plausible
assumptions. In practice, the actual power (and confidence interval width of results) of the IPD meta-analysis project will
change if the modeling assumptions are incorrect (eg, in terms of the amount of IPD actually received from existing trials,
the prognostic effect of the covariate, the assumed distribution of the continuous covariate, etc.). Thus, funding appli-
cations for IPD meta-analysis projects might display a range of power calculations (or expected precision of estimates)
conditional on a range of assumptions. For example, they might estimate power (or expected precision) when optimisti-
cally assuming IPD will be obtained from all trials, but also when cautiously assuming IPD will only be provided by trials
that already provided verbal or written agreement. In summary, we have provided a new approach for estimating the potential power or precision of an IPD meta-analysis
project aiming to estimate a treatment-covariate interaction with a binary outcome from randomized trials. We hope the
approach, and our associated Stata and R code, improves the uptake of power calculations in future IPD meta-analysis
projects, to help researchers and funders gauge the likelihood of success. In the near future we will also launch a dedicated
package called ipdmapower, to implement this and other power calculations for IPD meta-analysis projects. FUNDING INFORMATION Richard D. Riley, Miriam Hattle, Rebecca Whittle, Gary S. Collins, and Joie Ensor were supported by funding from the
MRC Better Methods Better Research panel (Grant reference: MR/V038168/1). Gary S. Collins was supported by the NIHR
Biomedical Research Centre, Oxford, and Cancer Research UK (program Grant: C49297/A27294). ACKNOWLEDGEMENT We thank the associate editor and two reviewers for their comments that helped impro We thank the associate editor and two reviewers for their comments that helped improve the article upon revision. DATA AVAILABILITY STATEMENT The work presented involves applying equations using aggregated or simulated data, and therefore no actual
individual-level data is available for sharing. Simulation and example code is provided in the Supplementary Material,
and aggregate data for examples are shown in the paper itself. Poynard T, Cales P, Pasta L, et al. Beta-adrenergic-antagonist drugs in the prevention of gastrointestinal bleeding in patients with cirrhosis
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a systematic review update and individual participant data meta-analysis protocol. BMJ Open. 2017;7(12):e018971. 20. Hartung J, Knapp G. A refined method for the meta-analysis of controlled clinical trials with binary outcome. Stat Med. 2001;20(24):3875-3889. 20. Hartung J, Knapp G. A refined method for the meta-analysis of controlled clinical trials with binary outcome. Stat Med. 2001;20(24):3875-3889. 21. Sidik K, Jonkman JN. A simple confidence interval for meta-analysis. Stat Med. 2002;21(21):3153-3159. 21. Sidik K, Jonkman JN. A simple confidence interval for meta-analysis. Stat Med. 2002;21(21):3153-3159. 21. Sidik K, Jonkman JN. A simple confidence interval for meta-analysis. Stat Med. 2002;21(21):3153-315 10970258, 2022, 24, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/sim.9538 by Test, Wiley Online Library on [06/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the ap RILEY et al. 4837 wo-stage individual participant data meta-analysis and generalized forest plots. Stata J. 2015;15(2):369-396. 10. Fisher DJ, Copas AJ, Tierney JF, Parmar MKB. A critical review of methods for the assessment of patient-level interactions in individual
participant data meta-analysis of randomized trials, and guidance for practitioners. J Clin Epidemiol. 2011;64(9):949-967. 10. Fisher DJ, Copas AJ, Tierney JF, Parmar MKB. A critical review of methods for the assessment of patient-level interactions in individual
participant data meta-analysis of randomized trials, and guidance for practitioners. J Clin Epidemiol. 2011;64(9):949-967. g
11. Thompson SG, Kaptoge S, White I, et al. Statistical methods for the time-to-event analysis of individual participant data from multiple
epidemiological studies. Int J Epidemiol. 2010;39(5):1345-1359. 11. Thompson SG, Kaptoge S, White I, et al. Statistical methods for the time-to-event analysis of individual participant data from multiple
epidemiological studies. Int J Epidemiol. 2010;39(5):1345-1359. p
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12. Hua H, Burke DL, Crowther MJ, Ensor J, Tudur Smith C, Riley RD. One-stage individual participant data meta-analysis models: estima-
tion of treatment-covariate interactions must avoid ecological bias by separating out within-trial and across-trial information. Stat Med. 2017;36(5):772-789. 12. Hua H, Burke DL, Crowther MJ, Ensor J, Tudur Smith C, Riley RD. One-stage individual participant data meta-analysis models: estima-
tion of treatment-covariate interactions must avoid ecological bias by separating out within-trial and across-trial information. Stat Med. 2017;36(5):772-789. 12. Hua H, Burke DL, Crowther MJ, Ensor J, Tudur Smith C, Riley RD. One-stage individual participant data meta-analysis models: estima-
tion of treatment-covariate interactions must avoid ecological bias by separating out within-trial and across-trial information. Stat Med. 2017;36(5):772-789. 13. Firth D. Bias reduction of maximum likelihood estimates. Biometrika. 1993;80(1):27-38. 13. Firth D. Bias reduction of maximum likelihood estimates. Biometrika. 1993;80(1):27-38. 15. Royston P, Sauerbrei W. A new approach to modelling interactions between treatment and continuous covariates in clinical trials by using
fractional polynomials. Stat Med. 2004;23(16):2509-2525. 16. Langan D, Higgins JPT, Jackson D, et al. A comparison of heterogeneity variance estimators in simulated random-effects meta-analyses. Res Synth Methods. 2019;10(1):83-98. 17. Wilson E, Free C, Morris TP, et al. Internet-accessed sexually transmitted infection (e-STI) testing and results service: a randomised,
single-blind, controlled trial. PLoS Med. 2017;14(12):e1002479. 18. REFERENCES 1. Riley RD, Tierney JF, Stewart LA. Individual Participant Data Meta-Analysis: A Handbook for Healthcare Research. Chichester, West
Sussex: Wiley; 2021. 1. Riley RD, Tierney JF, Stewart LA. Individual Participant Data Meta-Analysis: A Handbook for Healthcare Research. Chichester, West
Sussex: Wiley; 2021. 2. Riley R. D, Ensor J. Power calculations for planning an IPD meta-analysis. In: Riley RD Tierney J, Stewart LA, eds. Individual Participant
Data Meta-Analysis: A Handbook for Healthcare Research. Chichester, West Sussex: Wiley 2021. 3. Simmonds MC, Higgins JP. Covariate heterogeneity in meta-analysis: criteria for deciding between meta-regression and individual patient
data. Stat Med. 2007;26(15):2982-2999. 4. Riley RD, Debray TPA, Fisher D, et al. Individual participant data meta-analysis to examine interactions between treatment effect and
participant-level covariates: statistical recommendations for conduct and planning. Stat Med. 2020;39(15):2115-2137. 5. Ensor J, Burke DL, Snell KIE, Hemming K, Riley RD. Simulation-based power calculations for planning a two-stage individual participant
data meta-analysis. BMC Med Res Methodol. 2018;18(1):41. Kontopantelis E, Springate DA, Parisi R, Reeves D. Simulation-based power calculations for mixed effects m
J Stat Softw. 2016;74(12):25. 6. Kontopantelis E, Springate DA, Parisi R, Reeves D. Simulation-based power calculations for mixed effects modeling: ipdpower in stata. J Stat Softw. 2016;74(12):25. p
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J Stat Softw. 2016;74(12):25. 7
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ith bi
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St t M d 2008 27(1) 36 46 f
(
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7. Demidenko E. Sample size and optimal design for logistic regression with binary interaction. Stat Med. 2 8. Kovalchik SA, Cumberland WG. Using aggregate data to estimate the standard error of a treatment-covariate interaction in an individual
patient data meta-analysis. Biom J. 2012;54(3):370-384. SUPPORTING INFORMATION SUPPORTING INFORMATION How to cite this article: Riley RD, Hattle M, Collins GS, Whittle R, Ensor J. Calculating the power to examine
treatment-covariate interactions when planning an individual participant data meta-analysis of randomized trials
with a binary outcome. Statistics in Medicine. 2022;41(24):4822-4837. doi: 10.1002/sim.9538 iley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
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XCIX.—A new sulphide of nitrogen
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Journal of the Chemical Society. Transactions
| 1,910
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public-domain
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BURT: A NEW STJLPHIDE OF NITROQEN. 1171 1171 Downloaded by Freie Universitaet Berlin on 14/05/2015 18:24:58. View Article Online / Journal Homepage / Table of Contents for this issue View Article Online / Journal Homepage / Table of Contents for this issue XC1X.-A
New Xulphide of Nitrogen.
By FRANK
PLAYFAIR
BURT. By FRANK
PLAYFAIR
BURT WHEN the yellow sulphide of nitrogen, N,S,, is heated in a vacuum. it sublimes without decomposition. At looo the vapour pressure is
appreciable, and a sublimate soon. makes its appearance when one
end of an exhausted glass tube containing a little of the sulphide
is heated in a steam-jacket. Published on 01 January 1910. Downloaded by Freie Universitaet Berlin o j
I n the course of some attempts to effect quantitative analysis of
the vapour by means of silver gauze, it was observed that, whilst
at temperatures above 200° decomposition was complete, very little
action took place when the silver was kept at temperatures of
100--120°. Advantage was taken of this fact to remove the last
traces of free sulphur from nitrogen sulphide. Into a vertical glass tube about 1 cm. in diameter, 1-2
grams
of powdered nitrogen sulphide were dropped, and ,a roll of silver
gauze was pushed in above so as just to touched the sulphide. The
tube was then drawn out 3-4
cm. beyond the top of the gauze,
and attached by quill tubing to a mercury pump. After exhaustion
in the cold, the vertical tube was heated in a steam-jacket up to
the level of the top of the silver. The sulphide slowly volatilised,
and the bulk of it deposited as a crystalline crust immediately
above the silver. During the experiment the gauze was gradually
covered with a film of silver sulphide, and a very small quantity
of nitrogen was continuously evolved. After a few hours’ heating, when there was a conspicuous
subIimate of the yellow sulphide, a faint blue film was observed
lining the quill tubing leading to the pump. This film gradually
increased in quantity and in extent in both directions. If the
experiment was pushed sufficiently far, the blue deposit crept down
the tube until it overlapped the crystals of yellow sulphide, forming
an intermediate green zone, while it spread a foot or more along
the quill tubing in the direction of the pump. The colour of thc
deposit increased to a fine, deep blue when viewed by transmitted
light, until finally those portions of the tube which were most thickly
coated became opaque. By reflected light, the colour was bronze,
with a metallic lustre. It should be noted here that the formation of a blue substance
was observed by 0. C. M. Davis when heating the compound
N,S,,SbCl, in a vacuum, but the quantity obtained was too small
t o investigate. When all the yellow sulphicle had been sublimed, the tube wa 4 H 2 View Article Online BURT: A NEW SULPHIDE OF NITROGEN. 1172 cooled, air was let in, and the portion containing the blue sub-
stance was broken off for examination. Published on 01 January 1910. Downloaded by Freie Universitaet Berlin o The film was extremely
thin, and yielded an unweighable quantity of bronze-coloured fluff
when scraped out with a knife. 01 January 1910. Downloaded by Freie Universitaet Berlin on 14/05/2015 18:24:58. p
The substance was conveniently examined by treating with
various reagents small pieces of quill tubing which were coated with
the film. It was found to be quite insoluble when boiled with
several organic liquids which dissolve the yellow sulphide, such
as ether, chloroform, benzene, or carbon disulphide. On shaking
or warming a piece of the tubing in water, the film rapidly scaled
off, and was slowly decomposed. On adding a few drops of bromine
water, solution was accelerated, and the resulting liquid gave a
precipitate of barium sulphate when mixed with a solution of a
barium salt. When another portion was warmed with a solution of sodium
hydroxide, ammonia was evolved. When heated in air, the substance was decomposed with the
evolution of vapours having an odour somewhat like that of iodine,
and recalling that of a foul gun-barrel. This may have been due
t o the presence of the liquid sulphide of nitrogen, N,S,, described
by Muthmann and Clever (Zeitsch. anorg. Chem., 1897, 13, ZOO). When heated in a vacuum, the substance showed no signs of
volatilising at 100°, and it could be freed in this manner from any
accompanying yellow sulphide. When a small quantity was heated in a melting-point tube,
decomposition occurred without liquefaction, the substance turning
yellow at about the melting point of the yellow sulphide (188O). A small tube containing a few specks of the compound was
exhausted and sealed up, and then rapidly heated to a temperature
a few degrees below the melting point of the yellow sulphide in
the vapour of boiling aniline. A blue ring was observed to creep
up the tube in the wake of the rising aniline vapour, and when
the tube was completely submerged, it was seen to be filled with a
yellow vapour, which afterwards deposited as crystals on the cooling
walls. ‘On examining these crystals with a lens, two distinct types
were seen to be present, one consisting of the small, highly refract-
ing crystals characteristic of a sublimate of yellow sulphide of
nitrogen, and the other of brown, feathery needles. There were
also some milky patches that were probably sulphur. A little
nitrogen was found in the tube when the end was broken under
mercury. Published on 01 January 1910. Downloaded by Freie Universitaet Berlin o Many unsuccessful attempts were made to obtain a quantity of
the substance sufficient for analysis. The temperature of the silver
was varied between looo and 130° with apparently little effect. View Article Online 1173 BURT: A NEW SULPHIDE OF NITROGEN. When a very long column of silver gauze-about
100 cm.--was
used, no blue compound at all was formed. Downloaded by Freie Universitaet Berlin on 14/05/2015 18:24:58. ,
p
Nitrogeii
sulphide was being sublimed in a vacuum over quartz wool, heated
to about 300O; analysis was by no means complete, and among a
variety of products were some large, bronze-coloured crystals, which
deposited in the connecting tubing leading to the pump. On
examiliation these crystals proved to be identical with the blue
substance already described. They broke with a bronze-coloured
fracture, were insoluble in organic solvents, and gave the samc
odour on warming. A single large crystal, weighing about four
milligrams, was selected for analysis. It was weighed in a small
quartz bucket on an assay balance, and then introduced into a
glass tube closed at one end; a small piece of silver gauze was
pushed in above it, and the tube was drawn out and connected with
a mercury pump by means of a sealed glass junction. After
exhaustion, the silver gauze was heated with a naked flame, and
the crystal was “sublimed” over it. The lower end of the gauze
was coated with sulphide, and the nitrogen evolved was pumped
off and transferred to a small constant-volume point gas-burette,
where it was measured. The volume reduced
to N.T.P. was
1.034 C.C. Taking the weight of a normal litre of nitrogen as
1.2514 grams, the
A weighable quantity was finally obtained by accident. Weight of gas obtained
= 0’00129 gram
Weight of the crystal
= 0.00433 ,,
Weight of sulphur (by difference) = 0’00304 ,, Then, dividing by the respective atomic weights : showing a deviation of only 3 per cent. from the values required by
a compound of the composition (NS),, a satisfactory agresment. in
view of the very small quantity of material employed. The new sulphide therefore contains the same relative proportions
of nitrogen and sulphur as the yellow sulphide, and affords an
iiiteresting example of inorganic polymerism. The mode of formation of this blue sulphide of nitrogen by the
action of silver on the sulphide, N4S4, is not easy to explain. Published on 01 January 1910. Downloaded by Freie Universitaet Berlin o Although its appearance is always accompanied by evolution of
nitrogen and the formation of silver sulphide, any quantitative
relationship between these phenomena is difficult to detect, because
of the very small extent to which the reaction occurs. The fact
that the blue sulphide is deposited beyond the yellow on a cooler
part of the tube in spite of the demonstrably higher vapour pressure View Article Online BURT: A NEW SULPHIDE OF NITROGEN. 1174 of the latter suggests that the new sulphide is formed in situ from
a volatile and unstable intermediate compound. wnloaded by Freie Universitaet Berlin on 14/05/2015 18:24:58. p
This hypothesis is supportcd by the following observations. Whenever the blue compound was being prepared, the phosphoric
oxide between the reaction tube and the mercury pump acquired a
red colour. This occurred even when the reaction
tube was
separated from the pump by many feet of connecting tubing,
indicating the presence of a gas or vapour, which combined with
or was decomposed by the phosphoric oxide. The red colour
changed to green in the presence of air. The mercury in the pump
chamber was also attacked. A U-tube cooled in liquid air and a
mercury gauge were then introduced into the system. Besides the
usua.1 blue film, small quantities of a brown and a white substance
were condensed in the U-tube in different zones. The white sub-
stance volatilised as soon as the liquid air was removed, and con-
densed again when the air was replaced, its vapour pressure being
clearly indicated by the fall and rise df mercury in the gauge. When communication was established between the U tube and the
pump, with the liquid-air vessel removed, the phosphoric oxide was
at once discoloured. I n another experiment the U-tube and its contents, while still
cooled in liquid air, were sealed off after every trace of nitrogen
had been pumped out. The blue film in the U-tube increased on
keeping, and a little nitrogen was apparently formed. On the
other hand, there was still something present which was condensible
in liquid air after the tube had been kept for several days. It is
possible that this was sulphur dioxide derived from the traces of
oxygen which silver always contains. I n conclusion, I desire to thank Mr. F. L. Usher for assistance in
the experiments and for many helpful suggestions. Published on 01 January 1910. Downloaded by Freie Universitaet Berlin o The results of these experiments suggest that the immediate
precursor of the blue sulphide of nitrogen is a gas or very volatile
liquid at the ordinary temperature; that it contains relatively less
sulphur, being formed from the yellow sulphide by partial reduc-
tion ; and that it decomposes on keeping into the blue sulphide and
nitrogen. These suggestions axe put forward tentatively, because
the amount of substance dealt with was so small that quantitative
treatment was impossible. It is at least probable that the sulphides of nitrogen may prove
as numerous as their oxygen analogues. I n conclusion, I desire to thank Mr. F. L. Usher for assistance in
the experiments and for many helpful suggestions. UNIVERSITY
COLLEGE
LONDON,
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English
| null |
The Implicitome: A Resource for Rationalizing Gene-Disease Associations
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PloS one
| 2,016
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cc-by
| 12,280
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RESEARCH ARTICLE OPEN ACCESS Citation: Hettne KM, Thompson M, van Haagen
HHHBM, van der Horst E, Kaliyaperumal R, Mina E,
et al. (2016) The Implicitome: A Resource for
Rationalizing Gene-Disease Associations. PLoS ONE
11(2): e0149621. doi:10.1371/journal.pone.0149621 The Implicitome: A Resource for Rationalizing
Gene-Disease Associations Kristina M. Hettne1☯*, Mark Thompson1☯, Herman H. H. B. M. van Haagen1☯, Eelke van der
Horst1, Rajaram Kaliyaperumal1, Eleni Mina1, Zuotian Tatum1, Jeroen F. J. Laros1, Erik
M. van Mulligen1,2, Martijn Schuemie2, Emmelien Aten1, Tong Shu Li5,
Richard Bruskiewich6, Benjamin M. Good5, Andrew I. Su5, Jan A. Kors2, Johan den
Dunnen1, Gert-Jan B. van Ommen1, Marco Roos1, Peter A.C. ‘t Hoen1, Barend Mons1,3,
Erik A. Schultes1,4 Kristina M. Hettne1☯*, Mark Thompson1☯, Herman H. H. B. M. van Haagen1☯, Eelke van der
Horst1, Rajaram Kaliyaperumal1, Eleni Mina1, Zuotian Tatum1, Jeroen F. J. Laros1, Erik
M. van Mulligen1,2, Martijn Schuemie2, Emmelien Aten1, Tong Shu Li5, g
,
j
,
,
g
,
Richard Bruskiewich6, Benjamin M. Good5, Andrew I. Su5, Jan A. Kors2, Johan den
Dunnen1, Gert-Jan B. van Ommen1, Marco Roos1, Peter A.C. ‘t Hoen1, Barend Mons1,3,
Erik A. Schultes1,4 1 Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands,
2 Department of Medical Informatics, Erasmus University Medical Center Rotterdam, Rotterdam, The
Netherlands, 3 Dutch Techcentre for Life Sciences, Utrecht, The Netherlands, 4 Leiden Institute for
Advanced Computer Science, Leiden, The Netherlands, 5 Department of Molecular and Experimental
Medicine, The Scripps Research Institute, La Jolla, CA, United States of America, 6 STAR Informatics /
Delphinai Corporation, Port Moody, BC, Canada ☯These authors contributed equally to this work. * k.m.hettne@lumc.nl ☯These authors contributed equally to this work. Abstract High-throughput experimental methods such as medical sequencing and genome-wide
association studies (GWAS) identify increasingly large numbers of potential relations
between genetic variants and diseases. Both biological complexity (millions of potential
gene-disease associations) and the accelerating rate of data production necessitate
computational approaches to prioritize and rationalize potential gene-disease relations. Here, we use concept profile technology to expose from the biomedical literature both
explicitly stated gene-disease relations (the explicitome) and a much larger set of implied
gene-disease associations (the implicitome). Implicit relations are largely unknown to, or
are even unintended by the original authors, but they vastly extend the reach of existing bio-
medical knowledge for identification and interpretation of gene-disease associations. The
implicitome can be used in conjunction with experimental data resources to rationalize both
known and novel associations. We demonstrate the usefulness of the implicitome by ratio-
nalizing known and novel gene-disease associations, including those from GWAS. To facili-
tate the re-use of implicit gene-disease associations, we publish our data in compliance
with FAIR Data Publishing recommendations [https://www.force11.org/group/fairgroup]
using nanopublications. An online tool (http://knowledge.bio) is available to explore estab-
lished and potential gene-disease associations in the context of other biomedical relations. Editor: Tudor Groza, Garvan Institute of Medical
Research, AUSTRALIA
Received: September 22, 2015
Accepted: February 3, 2016
Published: February 26, 2016
Copyright: © 2016 Hettne 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: Tudor Groza, Garvan Institute of Medical
Research, AUSTRALIA
Received: September 22, 2015
Accepted: February 3, 2016
Published: February 26, 2016
Copyright: © 2016 Hettne 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 data files relevant to
this publication have been submitted to the
DataDryad repository with DOI: doi:10.5061/dryad. gn219. Funding: This project was sponsored by the Dutch
Center of Medical Systems Biology (CMSB),
ODEX4all (NWO 650.002.002), Melton Foundation,
and a grant from the John Templeton Foundation. Funding from the European Commission (FP-7
project RD-Connect, grant agreement No. 305444),
and the Innovative Medicines Initiative Joint
Undertaking under grant agreement No. ☯These authors contributed equally to this work.
* k.m.hettne@lumc.nl Introduction of financial contribution from the European Union’s
Seventh Framework Programme (FP7/2007-2013)
and in-kind contribution of EFPIA companies are
gratefully acknowledged. This work was also
supported by the National Institutes of Health
(U54GM114833 and GM089820). Delphinai
Corporation has an open ended "payasyougo"
subcontract with the Scripps Institute to develop the
Knowledge.bio system, which is MITopensourced,
with joint copyright attribution. Scripps Institute has
USA NIH approval to fund the collaboration with
Delphinai Corporation. The funders had no role in
study design, data collection and analysis, decision to
publish, or preparation of the manuscript. of financial contribution from the European Union’s
Seventh Framework Programme (FP7/2007-2013)
and in-kind contribution of EFPIA companies are
gratefully acknowledged. This work was also
supported by the National Institutes of Health
(U54GM114833 and GM089820). Delphinai
Corporation has an open ended "payasyougo"
subcontract with the Scripps Institute to develop the
Knowledge.bio system, which is MITopensourced,
with joint copyright attribution. Scripps Institute has
USA NIH approval to fund the collaboration with
Delphinai Corporation. The funders had no role in
study design, data collection and analysis, decision to
publish, or preparation of the manuscript. Organizing the knowledge space around disease pathophysiology and phenotypes is crucial for
data interpretation [1,2] and translational medicine [3]. Presently however, most geneticists
attempt to rationalize large and complex datasets using keyword-based searches to interrogate
existing knowledge resources. Although keywords can effectively retrieve individual docu-
ments, they fail to scale with either data complexity or data volume. For keyword searches to
work comprehensively, ambiguities in terminology and meaningful relations between key-
words must be factored in by the human operator. Without constructing sophisticated queries,
large troves of relevant information typically remain elusive to the clinician and researcher. In contrast to keyword approaches, we use concept profiles to expose the associative infor-
mation contained in MEDLINE [http://www.ncbi.nlm.nih.gov/pubmed] abstracts as a docu-
ment-independent, weighted semantic network of disambiguated biomedical concepts. From
this network we then expose all gene-disease associations forming a “Literature Wide Associa-
tion Study” or LWAS. The vast majority of gene-disease associations are implicit, that is, they
are associated by their mutual association to intermediate concepts. We call the network of
implicit relations the gene-disease implicitome. Competing Interests: Richard Bruskiewich is the
Founder/CEO of Delphinai Corporation, a Canadian
federally incorporated startup company performing
scientific information systems development for the life
sciences. "STAR Informatics" is a trade name of this
company. Kristina M. The Implicitome: A Resource for Rationalizing Gene-Disease Associations Introduction Hettne has performed paid
consultancy since November 1, 2015, for Euretos b.v,
a startup founded in 2012 that develops knowledge
management and discovery services for the life
sciences, with the Euretos Knowledge Platform as a
marketed product. The paid consultancy did not
specifically fund this study. This does not alter the
authors' adherence to PLOS ONE policies on sharing
data and materials. Concept profile technology behind LWAS has its roots in Swanson’s hidden relationship
model [4], where it is assumed that one set of literature contains direct relationships between
concept X and concept Y, while a separate disjoint set of literature contains direct relationships
between concept Y and concept Z. Combining these literature sets permits the inference of a
relationship between X and Z via the linking concept Y [5][6]. This approach has been previ-
ously applied in the functional annotation of proteins [7], interpretation of gene expression
data [8–11], chemical toxicological classification [12], and protein-protein interaction predic-
tion [13]. In the present study, we exploit the linking Y concepts to inform and rationalize the
putative gene-disease relations. More specifically, in our case, the gene would be the X concept
and the disease the Z concept. The Y concepts can belong to any biomedical concept category,
such as another gene or disease, or a biological process, phenotype, or drug. Here, we use LWAS to show how information in the concept profiles can be used to priori-
tize and interpret new gene candidates implicated in disease. For example, this information can
be used to build cases for causality in pathogenicity or cases for genetic modifiers. We first vali-
date our LWAS by manually analyzing 105 top-ranking potentially novel gene-disease associa-
tions for relevant information that implicates genes in the etiology of disease phenotypes. We
also report on a detailed case study involving causative genes for the rare genetic disorder
Seckel Syndrome. Then in a retrospective analysis, we show how to appropriately integrate
LWAS with Genome Wide Association Study (GWAS) data (14) so that empirically derived
associations can be rationalized using the totality of the biomedical literature. Together, these
analyses demonstrate that LWAS complements other data sources when interpreting the bio-
medical significance of newly reported gene candidates causing disease or contributing to
pathophysiology. Abstract 115191
(Open PHACTS), resources of which are composed 1 / 21 PLOS ONE | DOI:10.1371/journal.pone.0149621
February 26, 2016 LWAS: Concept profile analysis exposes implicit gene-disease
associations A detailed description of concept profile technology can be found in Materials and Methods. Briefly, there are three steps to the analysis: (1) We index the biomedical terminology in a large
corpus of MEDLINE abstracts (dating from January 1980 to July 2014) using an extensive the-
saurus, mapping and disambiguating terms to over 600,000 biomedical concepts; (2) For each PLOS ONE | DOI:10.1371/journal.pone.0149621
February 26, 2016 2 / 21 The Implicitome: A Resource for Rationalizing Gene-Disease Associations Fig 1. Gene-Disease LWAS using concept profiles and networks of implicit information. a) Concepts X and Z share an association in a hypothetical
concept network via an explicit link (co-occurrence) and multiple implicit links (indirect connections via an intermediate concept, Y1, Y2, and Y3). The concept
profile for concept X is depicted where the weights (w) between concepts reflect the co-occurrence frequencies of each concept in the data source. b)
Concept profiles for concepts X and Z have explicit links to concepts Y1, Y2, and Y3 but no explicit link between themselves, as reflected in their
corresponding concept profiles. c) The intermediate shared concepts between concept profiles X and Z constitute implicit information, indirectly linking X and
Z (red dotted line). The strength of the implicit link (match score) is computed as the inner product of the weights of matching concepts in the concept profiles. d & e) The distribution of concept profile size for gene (median 1142, maximum 56,028) and disease (median 995, maximum 81,562) concepts. f) The
distribution of number of overlapping concepts between gene and disease concept profiles (median 180, maximum overlap 40,725). Only 23 concept pairs
had no overlapping concepts. g) Concept profiles for the human gene CWH43 (left) and the disease “Hyperphosphatesia with Mental Retardation” (right)
which share no explicit co-occurrence. The 37 overlapping concepts are shown clustered in between. Both the number and weights of these overlapping
links contribute to the strength of the implicit association. h) The distribution of match scores (higher numbers indicating stronger associations) for the 204
million LWAS-derived gene-disease pairs for both the explicit (black) and implicit (red) associations. p
g 1. Gene-Disease LWAS using concept profiles and networks of implicit information. a) Concepts X and Z sha Fig 1. Gene-Disease LWAS using concept profiles and networks of implicit information. doi:10.1371/journal.pone.0149621.g001 LWAS: Concept profile analysis exposes implicit gene-disease
associations a) Concepts X and Z share an association in a hypothetical
concept network via an explicit link (co-occurrence) and multiple implicit links (indirect connections via an intermediate concept, Y1, Y2, and Y3). The concept
profile for concept X is depicted where the weights (w) between concepts reflect the co-occurrence frequencies of each concept in the data source. b)
Concept profiles for concepts X and Z have explicit links to concepts Y1, Y2, and Y3 but no explicit link between themselves, as reflected in their
corresponding concept profiles. c) The intermediate shared concepts between concept profiles X and Z constitute implicit information, indirectly linking X and
Z (red dotted line). The strength of the implicit link (match score) is computed as the inner product of the weights of matching concepts in the concept profiles. d & e) The distribution of concept profile size for gene (median 1142, maximum 56,028) and disease (median 995, maximum 81,562) concepts. f) The
distribution of number of overlapping concepts between gene and disease concept profiles (median 180, maximum overlap 40,725). Only 23 concept pairs
had no overlapping concepts. g) Concept profiles for the human gene CWH43 (left) and the disease “Hyperphosphatesia with Mental Retardation” (right)
which share no explicit co-occurrence. The 37 overlapping concepts are shown clustered in between. Both the number and weights of these overlapping
links contribute to the strength of the implicit association. h) The distribution of match scores (higher numbers indicating stronger associations) for the 204
million LWAS-derived gene-disease pairs for both the explicit (black) and implicit (red) associations. doi:10.1371/journal.pone.0149621.g001 recognized gene and disease concept in MEDLINE a weighted list (profile) of all other concepts
is constructed from the observed co-occurrence frequency in each abstract (Fig 1); (3) The
strength of the association between a pair of concepts, i.e., the match score, is determined by
both the number and the weights of the overlapping concepts between their profiles. Of the 417,561,711 possible gene-disease pairs (19,113 genes x 21,847 diseases in our thesau-
rus) more than half (213,489,335) lacked sufficient literature representation to build a concept PLOS ONE | DOI:10.1371/journal.pone.0149621
February 26, 2016 3 / 21 The Implicitome: A Resource for Rationalizing Gene-Disease Associations profile for either one or both of the concepts in the pair (we address the heterogeneity in litera-
ture abundance below). Match scores were computed for the remaining 204,072,376 pairs and
normalized to a percentile. LWAS: Concept profile analysis exposes implicit gene-disease
associations Typically, gene-disease pairs exposed by concept profiles share a
few hundred overlapping concepts, but in some cases we found overlaps that extended to tens
of thousands (Fig 1f and 1g). Importantly, concept profiles indicate the individual contribution
of each concept to the overall match score. In the detailed examples of gene-disease associa-
tions below, we show how this information can be very useful when rationalizing the gene-dis-
ease associations. Explicit associations (a gene and disease that co-occur in the same abstract) account for
only 0.73% (1,479,895) of the total number of associations. The match scores of the explicit
associations are among the strongest, with the distribution peaking in the 99.4 percentile (Fig
1h, black line). The vast majority of the associations, however, are implicit (Fig 1h, red line). Implicit gene-disease associations generally have lower match scores than explicit associations,
but there is nonetheless substantial overlap between the two distributions. Hence, there are
numerous gene-disease associations that have not been made explicit in the literature but for
which there is strong evidence of a meaningful relation. In the range of the nearly 1.5 million
explicit gene-disease match scores (extending to the 86th percentile), there are more than 25
million implicit associations (S1 Table). Some genes and diseases are more extensively studied than others and this is reflected in the
biomedical literature (S2 and S3 Tables). Fig 2a and 2b depict the MEDLINE abundance of
gene and of disease concepts as rank-ordered distributions of the number of abstracts in which
they are mentioned. The biomedical literature is biased toward publications mentioning a rela-
tively small number of commonly studied genes and diseases (steep part of curve on the left). A
relatively small number of genes (fewer than 10%) and diseases (fewer than 5%) account for
80% of all MEDLINE publications. Although match score tends to decrease with publication
abundance there are millions of gene-disease pairs having low publication abundance yet
match scores in the same range as gene-disease pairs that are highly published (Fig 2c and 2d). Hence, concept profiles can in many cases compensate for strong biases in literature
abundance. We have made individual gene-disease associations and their match scores accessible as a
public data set and via an online tool (see Materials and Methods). PLOS ONE | DOI:10.1371/journal.pone.0149621
February 26, 2016 Validation of the top LWAS associations The 105 highest ranking implicit gene-disease associations and their top connecting concepts
are listed in S4 Table. By applying our manual assessment procedure described in the Materials
and Methods section, we found that the associations could be grouped into four types (Fig 3). Type I: gene-disease associations where a family member of the gene is causing the disease (71
cases; for example the gene-disease pair TTBK1-SCA11, where TTBK1 and TTBK2 are iso-
forms of the TTBK gene, and a mutation in the TTBK2 form is causing the disease [14] or
where a disease has a very broad phenotypic overlap with other disorders, e.g. the gene-disease
pair WDR62-MCPH4, where MCPH4 is a subtype of autosomal recessive primary microceph-
aly (MCPH) for which WDR62 is already found to be the causing gene for subtype MCPH2
[15], and the second most mutated gene in a large family study of MCPH [16]). These types of
candidates are not necessarily false positives but can frequently be labeled as obvious. Type II:
false positive gene-disease associations resulting from co-occurrences reflecting negative find-
ings reported in the literature (4 cases). Type III: false positive gene-disease associations result-
ing from concept mapping errors, such as homonyms (11 cases). Type IV: Gene-disease
associations that are novel and show promise for follow up investigations (19 cases). PLOS ONE | DOI:10.1371/journal.pone.0149621
February 26, 2016 4 / 21 PLOS ONE | DOI:10.1371/journal.pone.0149621
February 26, 2016 Distribution of the top 105 highest-ranking implicit gene-disease pairs determined by manual
inspection: Type I Gene family member (n = 71) represents gene-disease associations where a family member of the gene is causing the disease or a
disease with very large phenotypic overlap; Type II Negation (n = 4) and Type III Homonym (n = 11) represent different classes of LWAS false positives
composing 14% of the cases. Type IV Novel association (n = 19) indicates gene-disease associations of promise for follow up investigations. Fig 3. The relative distribution of LWAS association types. Distribution of the top 105 highest-ranking implicit gene-disease pairs determined by manual
inspection: Type I Gene family member (n = 71) represents gene-disease associations where a family member of the gene is causing the disease or a
disease with very large phenotypic overlap; Type II Negation (n = 4) and Type III Homonym (n = 11) represent different classes of LWAS false positives
composing 14% of the cases. Type IV Novel association (n = 19) indicates gene-disease associations of promise for follow up investigations. doi:10.1371/journal.pone.0149621.g003 vitamin D3 levels [21]. Thus, LWAS implicates CYP2R1 in SLO syndrome since defects may
potentially lead to 7-dihydrocholesterol accumulation. In the second Type IV example, CWH43 is linked to the disorder hyperphosphatasia with
mental retardation. The top ranking connecting concept is PGAP2. PGAP2 defects cause Hyper-
phosphatasia with mental retardation syndrome 3 and are thought to be related to the function
of PGAP2 in the maturation of glycosylphosphatidylinositol (GPI) modifications to proteins
that anchor them in the plasma membrane [22,23]. CWH43 is a gene that has not yet been con-
nected to disease but its protein product has sequence homology with PGAP2 and has been
demonstrated to be involved in remodeling of the lipid moiety of the GPI anchor in yeast [24]. The Implicitome: A Resource for Rationalizing Gene-Disease Associations Fig 2. Correction of literature bias in the match score. a,b) Distribution of genes and diseases recognized by LWAS when sorted by publication
abundance (log number of MEDLINE abstracts). Red lines indicate the 5-abstract cut-off, below which concept profiles are not constructed. c,d) Distribution
of gene and disease rank orders, binned in 10 percentile intervals (x-axis). Higher numbers indicating stronger associations (y-axis). Fig 2. Correction of literature bias in the match score. a,b) Distribution of genes and diseases recognized by LWAS when sorted by publication
abundance (log number of MEDLINE abstracts). Red lines indicate the 5-abstract cut-off, below which concept profiles are not constructed. c,d) Distribution
of gene and disease rank orders, binned in 10 percentile intervals (x-axis). Higher numbers indicating stronger associations (y-axis). doi:10.1371/journal.pone.0149621.g002 Although the false positive results among the high scoring gene-disease associations (14%)
confirm that improvements can still be made in the automatic mapping of biomedical concepts
[17], Type IV results demonstrate that LWAS is nevertheless exposing potentially millions of
biomedically important yet still unknown gene-disease associations and the relevant informa-
tion for rationalizing them. To illustrate, we mention here two examples of Type IV associa-
tions: First, the gene CYP2R1 had a strong association with Smith-Lemli-Opitz (SLO)
syndrome. The top-ranking connecting concepts are 7-dihydrocholesterol reductase (DHCR7;
defects in this gene cause SLO syndrome [18,19] and cholesta-5,7-dien-3beta-ol (= 7-dihydro-
cholesterol). A pathological hallmark of SLO is increased levels of 7-dihydrocholesterol [20]. 7-dihydrocholesterol is a precursor of vitamin D3 and defects in CYP2R1 are known to affect PLOS ONE | DOI:10.1371/journal.pone.0149621
February 26, 2016 5 / 21 The Implicitome: A Resource for Rationalizing Gene-Disease Associations Fig 3. The relative distribution of LWAS association types. Distribution of the top 105 highest-ranking implicit gene-disease pairs determined by manual
inspection: Type I Gene family member (n = 71) represents gene-disease associations where a family member of the gene is causing the disease or a
disease with very large phenotypic overlap; Type II Negation (n = 4) and Type III Homonym (n = 11) represent different classes of LWAS false positives
composing 14% of the cases. Type IV Novel association (n = 19) indicates gene-disease associations of promise for follow up investigations. Fig 3. The relative distribution of LWAS association types. The Implicitome: A Resource for Rationalizing Gene-Disease Associations Table 1. Ranked list of genes having high match scores to Seckel Syndrome based on overlapping concepts in their concept profiles generated
until July 2009. NUP85 is ambiguous, with PCNT as the synonym causing a homonym problem with the PCNT gene, and a large overlap of articles. ANTXR1 was previously labeled as ATR, causing the same sort of problem as for NUP85, with an overlap of articles with ATR. Only ATR had been identified
as a causative gene for Seckel Syndrome by July 2009. Bold formatting indicates gene-disease associations derived by implicit information only (i.e., having
no co-occurrences in the literature up to July 2009). Rank
Gene
name
Co-
occurrences
in MEDLINE
abstracts
until July
2009
Top-ranking concept
explaining link
between gene and
Seckel syndrome
Causative
gene for
Seckel
syndrome
PMIDs and publication date of publication (italics) describing
causative mutations in that gene
1
ATR
7
ATR
Yes
19504344,16015581,15616588,15496423,15309689,14571270,12640452;
April 2003
2/3
PCNT
3
Seckel syndrome
19546241,18157127,16278902
2/3
NUP85
2
Seckel syndrome
19546241,18157127
4
ANTXR1
2
ANTXR1
19504344,12640452
5
MCPH1
3
MCPH1
19546241,17102619,16217032
6
NBN
2
NBN
18664457,15616588
7
MCPH2
0
Primary microcephaly
Implicit link: no co-occurrence in Medline up to July 2009
8
FANCD2
2
FANCD2
15616588,15314022
9
ATRIP
0
ATR
Implicit link: no co-occurrence in Medline up to July 2009
10
DNMT1
1
DNMT1
17015478
11
MDC1
1
MDC1
18664457
12
FANCC
1
Fanconi's Anemia
10232749
13
PALB2
6
Fanconi's Anemia
17224058,15314022,10232749,7686032,6465473,3115102
14
CENPJ
0
Microcephaly
Yes
Implicit link: no co-occurrence in Medline up to July 2009. 20522431; June
2010
15
ASPM
0
Microcephaly
Implicit link: no co-occurrence in Medline up to July 2009
16
CHEK1
5
CHEK1
19504344,18077418,17015478,16217032,15616588
17
PROP1
0
dwarfism
Implicit link: no co-occurrence in Medline up to July 2009
18
MMAB
2
MMAB
19504344,15314022
19
TOPBP1
0
ATR
Implicit link: no co-occurrence in Medline up to July 2009
20
FOXL2
1
FOXL2
16015581
doi:10.1371/journal.pone.0149621.t001 retrospective analysis using a Medline corpus of abstracts published before July 2009. By look-
ing backwards in time, we demonstrate how overlapping concepts between gene and disease
concept profiles can help expert researchers to identify and functionally rationalize the role of
candidate genes in disease pathophysiology. Seckel Syndrome is a rare autosomal recessive disorder characterized by growth retardation,
mental retardation, microcephaly, and a characteristic “bird-headed” facial appearance (receding
forehead and micrognathia with a prominent nose). LWAS assisted rationalization of causative genes for Seckel Syndrome LWAS can expose information that implicates genes in the etiology of disease phenotypes. Focusing on the genetic disorder Seckel Syndrome [MIM 210600], we perform a 5-year PLOS ONE | DOI:10.1371/journal.pone.0149621
February 26, 2016 6 / 21 PLOS ONE | DOI:10.1371/journal.pone.0149621
February 26, 2016 Unfortunately, in 2009 we could not have detected the relation between CEP152 and Seckel
Table 2. Top ranking overlapping concepts between Seckel Syndrome & CENPJ. The contribution of each concept to the overall match score is given
as a percentage. Rank
Overlapping concept
Identifier
Contribution (%)
1
Microcephaly
UMLS:C0025958
17.77
2
Primary microcephaly
UMLS:0431350
17.31
3
MCPH1
OMIM:251200
11.86
4
Mcph1
EG:244329
11.44
5
MCPH1
EG:36272
11.44
6
MCPH1
EG:100125976
11.41
7
MCPH1
EG:79648
7.54
8
PCNT
EG:5116
4.38
9
osteodysplastic primordial dwarfism
UMLS:C0432244
1.94
10
NUP85
EG:79902
1.11
11
MOPD II
OMIM:210720
0.93
12
pericentrin
UMLS:C0252534
0.77
13
Dwarfism
UMLS:C0013336
0.72
14
Centrosome
GO:0005813
0.55
15
Genes, Recessive
UMLS:C0017361
0.32
doi:10.1371/journal.pone.0149621.t002
The Implicitome: A Resource for Rationalizing Gene-Disease Associations Table 2. Top ranking overlapping concepts between Seckel Syndrome & CENPJ. The contribution of each concept to the overall match score is given
as a percentage. Rank
Overlapping concept
Identifier
Contribution (%)
1
Microcephaly
UMLS:C0025958
17.77
2
Primary microcephaly
UMLS:0431350
17.31
3
MCPH1
OMIM:251200
11.86
4
Mcph1
EG:244329
11.44
5
MCPH1
EG:36272
11.44
6
MCPH1
EG:100125976
11.41
7
MCPH1
EG:79648
7.54
8
PCNT
EG:5116
4.38
9
osteodysplastic primordial dwarfism
UMLS:C0432244
1.94
10
NUP85
EG:79902
1.11
11
MOPD II
OMIM:210720
0.93
12
pericentrin
UMLS:C0252534
0.77
13
Dwarfism
UMLS:C0013336
0.72
14
Centrosome
GO:0005813
0.55
15
Genes, Recessive
UMLS:C0017361
0.32
doi:10.1371/journal.pone.0149621.t002 epts between Seckel Syndrome & CENPJ. The contribution of each concept to the overall match score is given The second highest-ranking LWAS gene after ATR in Table 1 is pericentrin (PCNT). In
2009 there were three articles where PCNT and Seckel syndrome were mentioned in the same
abstract. One of them actually claims that mutations in PCNT can cause Seckel Syndrome [26]. It is now believed that mutations in PCNT cause the phenotypically very similar disease Pri-
mary microcephaly and microcephalic osteodysplastic primordial dwarfism type II (MOPD
II). LWAS postulates many other genes associated with primary microcephaly, such as
MCPH1 (rank 4), MCPH2 (rank 7), ASPM (rank 15), and must therefore also be considered as
candidates causing or impacting the development of Seckel syndrome. This is to be expected
since microcephaly is an important pathological hallmark of Seckel syndrome. Additional
genes associated with Seckel syndrome were suggested by LWAS through function in the cell
cycle check point. Seckel syndrome shows phenotypic overlap
with type II microcephalic osteodysplastic primordial dwarfism (MOPD2, MIM:210720). In 2009, the OMIM database indicated the following loci for Seckel Syndrome: chromo-
somes 18p11-q11 (SCKL2, MIM:606744), 14q (SCKL3, MIM:608664) and 13q12 (SCKL4,
MIM:613676), but causative mutations were only found in a single gene: ATR (ataxia-telangi-
ectasia and RAD3-related protein, SCKL1) [25]. As expected, LWAS identified ATR as the
highest-ranking gene associated with Seckel Syndrome, mainly based on explicit links (direct
co-occurrences in seven Medline abstracts, Table 1). Table 1 ranks genes having high match
scores to Seckel Syndrome based on overlapping concepts in the concept profiles generated
until July 2009. Implicit links (no co-occurrences) are in bold font. PLOS ONE | DOI:10.1371/journal.pone.0149621
February 26, 2016 7 / 21 The Implicitome: A Resource for Rationalizing Gene-Disease Associations The second highest-ranking LWAS gene after ATR in Table 1 is pericentrin (PCNT). In
2009 there were three articles where PCNT and Seckel syndrome were mentioned in the same
abstract. One of them actually claims that mutations in PCNT can cause Seckel Syndrome [26]. It is now believed that mutations in PCNT cause the phenotypically very similar disease Pri-
mary microcephaly and microcephalic osteodysplastic primordial dwarfism type II (MOPD
II). LWAS postulates many other genes associated with primary microcephaly, such as
MCPH1 (rank 4), MCPH2 (rank 7), ASPM (rank 15), and must therefore also be considered as
candidates causing or impacting the development of Seckel syndrome. This is to be expected
since microcephaly is an important pathological hallmark of Seckel syndrome. Additional
genes associated with Seckel syndrome were suggested by LWAS through function in the cell
cycle check point. This is also in line with the current knowledge about the molecular patho-
physiology of Seckel syndrome, as all causative genes known so far indeed have a function in
the control mechanisms that ensure the fidelity of cell division. Additional gene mutations causing Seckel syndrome were published in the period after July
2009. These gene mutations were highly ranked in the LWAS using literature before that date
(Table 1). For example, in June 2010 CENPJ was published as a causative gene for Seckel syn-
drome. Concept profiles showed CENPJ ranked 14th out of 12,391 genes having concept pro-
files, and as the third gene without direct co-occurrences (Table 1). The concept microcephaly
contributed the most to the CENPJ-Seckel Syndrome match score (Table 2). Microcephaly is
an important pathological feature of Seckel syndrome and CENPJ already had been associated
with microcephaly in April 2005 [27]. In the terms of Swanson’s relationships model, Micro-
cephaly would be the key linking Y-concept. More recently (January 2011-February 2014), mutations in for example RBBP8, CEP152
and DNA2 were found to cause Seckel syndrome: SCKL2, October 2011 [28]; SCKL5, January
2011 [29]; SCKL8, February 2014 [30], respectively. In 2009, RBBP8 ranked 209/12,391. The
highest ranking concepts for the implicit association of RBBP8 and Seckel Syndrome were ATR
and cell cycle checkpoint. DNA2 ranked 296/12,391 with ATR, MEC1, and replication fork as
linking concepts, implicating genes with a function in cell cycle control (similar to ATR). PLOS ONE | DOI:10.1371/journal.pone.0149621
February 26, 2016 This is also in line with the current knowledge about the molecular patho-
physiology of Seckel syndrome, as all causative genes known so far indeed have a function in
the control mechanisms that ensure the fidelity of cell division. Additional gene mutations causing Seckel syndrome were published in the period after July
2009. These gene mutations were highly ranked in the LWAS using literature before that date
(Table 1). For example, in June 2010 CENPJ was published as a causative gene for Seckel syn-
drome. Concept profiles showed CENPJ ranked 14th out of 12,391 genes having concept pro-
files, and as the third gene without direct co-occurrences (Table 1). The concept microcephaly
contributed the most to the CENPJ-Seckel Syndrome match score (Table 2). Microcephaly is
an important pathological feature of Seckel syndrome and CENPJ already had been associated
with microcephaly in April 2005 [27]. In the terms of Swanson’s relationships model, Micro-
cephaly would be the key linking Y-concept. More recently (January 2011-February 2014), mutations in for example RBBP8, CEP152
and DNA2 were found to cause Seckel syndrome: SCKL2, October 2011 [28]; SCKL5, January
2011 [29]; SCKL8, February 2014 [30], respectively. In 2009, RBBP8 ranked 209/12,391. The
highest ranking concepts for the implicit association of RBBP8 and Seckel Syndrome were ATR
and cell cycle checkpoint. DNA2 ranked 296/12,391 with ATR, MEC1, and replication fork as
linking concepts, implicating genes with a function in cell cycle control (similar to ATR). Unfortunately, in 2009 we could not have detected the relation between CEP152 and Seckel 8 / 21 PLOS ONE | DOI:10.1371/journal.pone.0149621
February 26, 2016 The Implicitome: A Resource for Rationalizing Gene-Disease Associations Syndrome, since we had no concept profile for CEP152 due to its low literature abundance at
the time (having fewer than 5 abstracts). CEP152 is thus an example of how an overall scarcity
in the existing genetic knowledge space effectively delimits a deductive approach in identifying
causative genes for Seckel Syndrome. The contribution of an ATRIP variant to Seckel syndrome has been reported, but not con-
firmed [31]. ATRIP was the second highest ranking gene in 2009 with no co-occurrence with
Seckel syndrome (Table 1). ATR was the top connecting concept, which is logical since the
gene product of ATRIP is the ATR-interacting protein ATRIP. LWAS-assisted interpretation of GWAS hits To illustrate how implicit information can be used to complement gene-disease associations
obtained using other independent approaches, we analyzed the overlap between LWAS and
GWAS. To do this, we focused on GWAS hits from the NHGRI GWAS Catalog spanning the
interval between January 2013 and August 2014 (according to the PubMed publication date
[http://www.ncbi.nlm.nih.gov/pubmed]), and compared this to LWAS from a literature corpus
spanning 1980 until July 2012 to ensure that our literature associations were not influenced by
the reporting of the GWAS hits in the literature. We applied multiple filters to the GWAS Cata-
log in order to achieve a comparable set of GWAS-LWAS gene-disease associations. A detailed
description of these filtering steps is given in the Materials and Methods section. p
g
p
g
The GWAS Catalog filtering steps resulted in 238 GWAS records representing 35 diseases, all
published in 2013 or later (S5 Table). Of these gene-disease associations, 194 had concept profiles
and could be exposed from LWAS. 45 of these 194 gene-disease pairs (23%) pass the 95-percentile
cutoff (4.6-fold enrichment; 5% would be random). At the 99th percentile, there were 12 (6%)
gene-disease pairs (6-fold enrichment; 1% would be random). The overlap between these inde-
pendent datasets can be quantitatively defined by partitioning the data based on the stringency of
the associations. For GWAS, this includes the commonly used p-value cutoffs of 10−5 and 10−8. For LWAS this includes the 99th-percentile cutoff and 95th-percentile cutoff. Fig 4 depicts the cor-
respondence of LWAS and GWAS rankings, for the overlapping gene-disease associations. These
LWAS hits can be used to automatically focus relevant knowledge in the scientific literature to
assist the expert in the interpretation of novel GWAS associations. For example, the gene-disease
association with the most stringent p-value and highest percentile score from our study is ERAP1
and Behçet’s disease. The overlapping concepts with the highest contribution score (HLA-B, 53%
and Ankylosing spondylitis, 25%) are explicitly mentioned as evidence in the eventual landmark
GWAS paper published after 2012 [32]: HLA-B51 (a HLA-B serotype) as interacting with
ERAP1, and ERAP1 as one of the three risk loci shared with Ankylosing spondylitis and psoriasis,
which are thought to involve pathogenic pathways similar to Behçet’s disease. Importantly, of the 45 overlapping LWAS-GWAS hits above the 95th percentile, there were
16 gene concepts appearing in fewer than 100 abstracts. LWAS-assisted interpretation of GWAS hits This result demonstrates the power of
LWAS in providing relevant information even for rarely published genes (S5 Table). PLOS ONE | DOI:10.1371/journal.pone.0149621
February 26, 2016 Discussion We have shown that LWAS, a method that is based solely on the information available in the
literature, can be used in conjunction with experimental data resources to interpret known
associations, to anticipate novel associations, and to provide context and rationalization for
both known and novel associations. The relative proportion of LWAS gene-disease associations
that are novel and show promise for follow-up investigations is likely to decrease with lower
match scores. However, as the vast majority of gene-disease associations have yet to be explic-
itly stated, the implicitome provides the broadest possible knowledge base in which gene- 9 / 21 PLOS ONE | DOI:10.1371/journal.pone.0149621
February 26, 2016 Keeping such a dictionary up to date with a minimal amount of identifier mapping
errors is cumbersome and time-consuming. Therefore we are looking into other, open
resources as basis for thesaurus creation and maintenance such as the BioPortal repository of
biomedical ontologies [33] and WikiData [http://www.wikidata.org], which are offering other
integrated knowledge sources in addition to the Unified Medical Language System (UMLS). g
g
g
g
y
Secondly, while the bioinformatics community has made great progress in the normaliza-
tion of gene mentions (mainly thanks to the BioCreative challenges [http://www.biocreative. org/]), disease mentions remain notoriously difficult to normalize [34]. Although our concept
recognizer Peregrine has been shown to outperform MetaMap [35], a standard tool developed
by the NLM for mapping UMLS metathesaurus concepts from text [36] our list of gene-disease
associations nonetheless contains both false positives arising from errors in concept mapping
as well as false negatives arising from errors due to incomplete thesaurus coverage and concept
mapping. In our analysis of the top 105 gene-disease associations, we found 11 errors related to
concept mapping. This number is expected to vary depending on the disease and gene, since
some disease and gene names are more ambiguous than others. Recent research suggests that
generation of word-concept statistical models from a knowledge base can aid in the task of
word sense disambiguation [37], but the scaling of these models is not well studied and integra-
tion of these types of models into concept mapping systems awaits future investigation. As a
way to indicate concept ambiguity in our thesaurus, we currently report on the number of
within-thesaurus case-insensitive homonyms for each concept (see the field “homonymFre-
quency” in for example http://rdf.biosemantics.org/emco/v1.5/concepts/C1836621). More
advanced methods can be envisioned [38] but in any case LWAS is vastly more efficient than
digging up that same information by manual keyword searches of the literature. Thirdly, the information in the literature is biased towards a relatively small number of
commonly studied genes and diseases. Consequently, more than half of the possible gene-dis-
ease pairs from our thesaurus lacked sufficient literature representation to build a concept pro-
file for either one or both of the concepts in the pair. Still, our resource is more than 400 times
larger than a very recent gene-disease association database generated by the kernel-based
machine learning method BeFree [39]. The Implicitome: A Resource for Rationalizing Gene-Disease Associations Fig 4. Overlapping implicit gene-disease associations between LWAS and GWAS. Green area: GWAS p-value cutoff of 10−5, yellow area: GWAS p-
value cutoffs of 10−8, red horizontal area: LWAS 99th-percentile cutoff, blue horizontal area: LWAS 95th-percentile cutoff. Fig 4. Overlapping implicit gene-disease associations between LWAS and GWAS. Green area: GWAS p-value cutoff of 105, yellow area: GWAS p-
value cutoffs of 10−8, red horizontal area: LWAS 99th-percentile cutoff, blue horizontal area: LWAS 95th-percentile cutoff. Fig 4. Overlapping implicit gene-disease associations between LWAS and GWAS. Green area: GWAS p-value cutoff of 105, yellow area: GWAS p-
value cutoffs of 10−8, red horizontal area: LWAS 99th-percentile cutoff, blue horizontal area: LWAS 95th-percentile cutoff. doi:10.1371/journal.pone.0149621.g004 disease pairs can be interpreted. Investigating how properties such as network statistics and
graph visualization techniques can aid in the automatic reproduction of the different types of
LWAS categories currently determined by hand (type I-IV, described in the results section) is a
topic for future work. disease pairs can be interpreted. Investigating how properties such as network statistics and
graph visualization techniques can aid in the automatic reproduction of the different types of
LWAS categories currently determined by hand (type I-IV, described in the results section) is a
topic for future work. GWAS hits are the outcome of high-throughput screens having no dependence on prior bio-
medical knowledge. Thus, the correspondence of implicit LWAS associations to subsequently
discovered GWAS associations indicates that the corpus of biomedical knowledge is tracking,
and in some cases may even be anticipating future biomedical discovery. LWAS, therefore, PLOS ONE | DOI:10.1371/journal.pone.0149621
February 26, 2016 10 / 21 The Implicitome: A Resource for Rationalizing Gene-Disease Associations complements GWAS: LWAS can detect associations for GWAS hits that are not yet sufficiently
powered, and can provide information helping to rationalize mechanisms of pathogenicity;
GWAS, on the other hand, has the potential of finding completely novel associations for which
there are few or no antecedents in the literature. When integrated, LWAS and GWAS become a
powerful tool allowing experts to intelligently prioritize gene-disease pairs for further study. Thus, tapping the implicitome to provide context and supporting evidence is of vital importance
when new gene-disease associations are uncovered by high-throughput approaches. Our application of concept profiles has clear limitations. Firstly, the underlying thesaurus
used for mapping the gene and disease concepts in text is based on numerous integrated dictio-
naries. PLOS ONE | DOI:10.1371/journal.pone.0149621
February 26, 2016 This difference might partly be explained by the sen-
tence-based scope of the previously mentioned method, while our method relies on co-occur-
rence statistics on the abstract level. Furthermore, in LWAS, the focus lies not on prediction
but rather on exposing all the information knowledge space as it currently exists. Integrating
results from LWAS with machine learning approaches [39] and semantic interpreters [40] is a
topic for future research. As a first step in this direction our Knowledge.Bio application [http://
www.knowledge.bio/] integrates the top one percent of our gene-disease associations from the
Implicitome with SemMedDB [41]. Starting with a gene-disease association hypothesis,
Knowledge.Bio can be used to rationalize the association as being causative, or part of the path-
ogenic pathway that can possibly be influenced by drugs. Instead of reading all the literature on
SLOS (788 citations in PubMed as of November 6, 2015) and CYP2R1 (190 citations in
PubMed as of November 6, 2015) separately, the Y concepts gives a starting point for PLOS ONE | DOI:10.1371/journal.pone.0149621
February 26, 2016 11 / 21 The Implicitome: A Resource for Rationalizing Gene-Disease Associations exploration using directed statements from SemMedDB. The filtering options in knowledge. bio allows for steering the exploration towards for example either more evidence for casuality
or possible treatment options (for an example on SLOS, see [http://www.slideshare.net/goodb/
poster-knowledgebio-an-interactive-tool-for-literaturebased-discovery]). Biases in literature coverage reflect powerful historical trends in research and funding prior-
ities in the life sciences. Yet, the uncertainty coefficient used to compute weights in concept
profiles, as well as the large numbers of implicit links composing the semantic network (num-
ber of overlapping concepts between concept profiles) mitigate the effects of literature abun-
dance biases with respect to the information content carried by the network. Hence, even
rarely studied gene or disease concepts can have high match scores if they have enough over-
lapping concepts, as demonstrated in the GWAS study. Adding information from the numer-
ous structured databases now available might fill some of the knowledge gaps surrounding
rarely studied diseases and/or genes. Towards this end, we have adopted the nanopublication
model [42,43] to facilitate the dissemination and integration of LWAS data with other data
resources. A nanopublication links a description of the gene-disease association, its percentile-
rank in this LWAS, and provenance metadata as a stand-alone semantically enabled publica-
tion. Materials and Methods We start by briefly describing the algorithms behind concept profiles (for an extensive descrip-
tion see [9]), followed by the manual assessment steps for validating the top 105 associations,
the filtering steps used in the GWAS study, and the method and design choices behind expos-
ing the gene-disease associations as nanopublications. We end with an overall description of
the software and pipeline. Conclusions We have demonstrated the application of LWAS in rationalizing the implicit links between
genes and diseases. The implicitome provides a rich theoretical, practical and historical context
for interpreting data from essentially any other biomedical resource. Nanopublication of
LWAS results are increasingly essential when confronting inherently complex biological sys-
tems where both the numbers of biomedical concepts and the resulting combinatorial explo-
sion of possible associations lie beyond human knowledge and reasoning capacity. LWAS nanopublications can be queried and cited, and are machine-readable, thus intero-
perating with other semantic data resources. Although we publish here LWAS-derived gene-disease associations as nanopublications, it
is possible to expose from MEDLINE associations for any other concept pair (for example pro-
tein-protein [13], gene-chemical [12], or drug-drug [44]). Hence, LWAS has fundamental and
universal application in any biomedical data-integration effort. S6 Table lists examples of
resources directly compatible with the nanopublication format. Prior knowledge, whether it is abundant or sparse (and even in cases where gene or disease
concept profiles could not be constructed) is always an important factor when triaging millions
of theoretically possible gene-disease associations arising in high-throughput empirical studies.,
By defining the boundaries of knowledge space (some 400 million possible gene-disease associa-
tions) and mapping those subject areas that are insufficiently studied, LWAS can provide
research priority recommendations that could optimize discovery returns on investment [45]. Concept profile generation Exposing the network of implicit information from text was a three-step procedure involving
concept indexing, concept profile creation and concept profile matching. First, we applied an PLOS ONE | DOI:10.1371/journal.pone.0149621
February 26, 2016 12 / 21 The Implicitome: A Resource for Rationalizing Gene-Disease Associations Open Source concept recognition software called Peregrine employing simple contextual heu-
ristics to resolve homonyms and map ambiguous protein, gene and disease names and spellings
to unique biomedical concepts [46]. Peregrine has been shown to have F-measures (harmonic
mean of precision and recall) of 0.765 for gene mentions [46] and 0.569 for disease mentions
[35]. Other than the recognition of terms, we employed no other natural language processing
procedures, such as noun or verb recognition. Coupled to Peregrine we used an extensive
curated in-house thesaurus of biomedical concepts based on the 2010AB UMLS [47] aug-
mented with concepts from Entrez Gene [48], OMIM, UniProt [49], the Human Gene Nomen-
clature Database [50], and JoChem [51]. The gene thesaurus was expanded so as to improve
recall by taking into account synonyms due to common spelling variations [52]. The UMLS
thesaurus was adapted for efficient natural language processing by avoiding terms that were
overly ambiguous, and complicated clinical terms not likely to be found in text [53]. We also
excluded UMLS semantic categories considered irrelevant for biological information about
genes [10]. The thesaurus contained 687,718 biomedical concepts in total, of which 19,113 are
genes from different species and 21,847 are diseases. Using Peregrine, over 17 million MED-
LINE documents (titles, Medical Subject Headings, and abstract text) dating from January
1980 up to the stated time points were then indexed. Stop words were removed and words
were stemmed to their uninflected form by the lexical variant generator normalizer [54]. In the second step, concept profiles were constructed for all concepts. A concept profile is
an M-dimensional vector wx = (wx1,wx2,. . .,wxM) where x is a particular concept and M is the
total number of concepts in the thesaurus. The weight wxy indicates the strength of the associa-
tion of concept x to concept y and is computed from pairwise concept-concept co-occurrence
frequencies within individual abstracts. Co-occurrence is characterized by four contingencies:
both concepts occur, neither concepts occurs, or one concept occurs without the other. Based
on these contingencies, association strength between x and y is computed using the symmetric
uncertainty coefficient [5,55]. PLOS ONE | DOI:10.1371/journal.pone.0149621
February 26, 2016 Concept profile generation This information-theoretical measure takes the a priori probabil-
ity of direct relations into account, and is a way to normalize co-occurrence statistics for litera-
ture and concept usage biases. In particular, it gives extra weight to those concepts that have
very specific associations. As an example, consider the concept DMD (the gene) and the corre-
sponding disease Duchenne Muscular Dystrophy. In the vast majority of MEDLINE abstracts,
both concepts will be absent. However, there will still be many abstracts where these concepts
appear together. Relatively few abstracts will mention one concept but not the other. This
results in a high value for the uncertainty coefficient reflecting the strong association between
DMD and Duchenne. In contrast, the concepts “human” and DMD will yield low association
score. In this case, “human” is a generic concept and there will be many abstracts where human
and DMD occur together, but also many other abstracts where human occurs regardless of any
other concept. We know from previous [13] and related [56] work that co-occurrence within
the same abstract is, with some margin of error, an effective indicator of associations made
explicit by the author (consider the earlier example of the mutated DMD gene causing the dis-
ease Duchenne Muscular Dystrophy). In the third and last step, we use the previously established explicit associations between
concept pairs to expose implicit associations from the text. To do this we compute an associa-
tion score between concepts based on the similarity or match between their concept profiles. This match score is based on the connecting concepts, i.e., those concepts that appear in the
concept profiles for both X and Y (even if X and Y themselves never co-occur explicitly in liter-
ature). The match score is implemented as the inner product of two concept vectors, so a
higher match score indicates a stronger association (reflecting the number of shared concepts
between X and Y and the weight (wxy) of those concepts). We then calculate the relative associ-
ation as a percentile rank over the ensemble of associations. 13 / 21 PLOS ONE | DOI:10.1371/journal.pone.0149621
February 26, 2016 The Implicitome: A Resource for Rationalizing Gene-Disease Associations Manual assessment steps The 105 highest ranking implicit gene-disease associations and their top connecting concepts
were manually assessed by searching the Online Mendelian Inheritance in Man (OMIM) (14)
and PubMed databases with an expanded synonym search for information about the gene and
the disease separately, in conjunction with the top connecting concept (see S4 Table). GWAS catalogue filtering steps Gene-disease associations were removed if they 1) were already reported in earlier studies as a
GWAS hit in the GWAS catalogue, 2) mentioned “response (to)” as part of the disease/trait
description or 3) could not be mapped unambiguously to a gene or disease concept from our
thesaurus using the gene symbol as reported by the authors of the study. Mapping of gene sym-
bols and disease descriptions to thesaurus concepts was achieved by querying the Peregrine
web service [http://implicitome.cloud.tilaa.nl/conceptlinker/service/index/indexText]. This
service recognizes concepts in free text and uses the same indexing engine as used for generat-
ing the concept profiles. Queries to the service return matching concepts, their semantic types
and a number of external database identifiers. These identifiers reference major biomedical
data sources, such as GO and UMLS. Concept suggestions with the correct semantic type, i.e. “gene” (T028) or “disease” (T047), were mapped back to the corresponding concept in our the-
saurus using the “mapDatabaseIDListToConceptIDs” method of the Concept Profile Analysis
(CPA) web service [57]. Case sensitive matches between GWAS gene/disease description and
concept label was preferred when more than one concept was suggested. For all GWAS entries
that were successfully mapped to concept pairs, we removed those that had an explicit co-
occurrence in literature and those that did not have a concept profile. This was determined by
utilizing the “findCoOccurringDocuments” and the “filterConceptsWithProfile” CPA methods
respectively. For the remaining entries, match scores were retrieved by invoking the “getSimi-
larConceptProfiles” method, and mapped to a percentile rank by projection onto the match
scores of 1500 randomly selected gene-disease associations that were retrieved from the same
service. For duplicate GWAS gene-disease associations, only the p-value of the most significant
observation was considered. GWAS hits with p-values smaller than 10−8 were classified as
“high significance”, hits with p-values between 10−8 and 10−5 as “intermediate significance”. PLOS ONE | DOI:10.1371/journal.pone.0149621
February 26, 2016 Exposing LWAS as nanopublications In order to promote reuse and machine-readability, we exposed gene-disease associations from
our LWAS as nanopublications [43] following the guidelines of the Open PHACTS consortium
[58] (available at http://nanopub.org/guidelines). Each of these nanopublications has an identi-
cal format describing the LWAS-derived association for a gene-disease pair. At its highest level
of description, a nanopublication has three essential components: an Assertion, Provenance,
and PublicationInfo. The Assertion contains statements composing a single scientific claim. The Provenance contains statements about how the Assertion “came to be” including attribu-
tion and the methodological context from which the Assertion was derived helping others to
estimate the trustworthiness of the claim. PublicationInfo documents attribution to the crea-
tors (people and/or institutions) of the nanopublication itself. PublicationInfo also includes
citation and reuse information such as date and time-stamp, licensing and versioning
information. At the level of implementation, a nanopublication is a schema built on top of existing
semantic technology that uses statements in the form of subject-predicate-object triples to
encode associative knowledge. The statements are serialized using the Resource Description
Framework (RDF) such that all the entities are identified using resolvable Uniform resource PLOS ONE | DOI:10.1371/journal.pone.0149621
February 26, 2016 14 / 21 The Implicitome: A Resource for Rationalizing Gene-Disease Associations identifiers (URIs). URIs ensure machine readability and data interoperability, and permit auto-
mated search and reasoning. The nanopublications herein are composed of a total of 20 triples
employing URIs from seven existing public ontologies. The Assertion in our gene-disease nanopublication is a claim that a particular gene has an
association with a particular disease with a relative strength denoted by a percentile rank. The
SemanticScience Integrated Ontology (SIO) provided the key ontological terms when modeling
this association. The gene and the disease in the association are identified using URIs from an
RDF version of the Peregrine thesaurus that was used to index MEDLINE in the first step of
our concept profile pipeline. Using these thesaurus URIs it is possible to retrieve for example
concept definitions, labels and identifiers under which the concept is known in its original data
sources: e.g. MeSH, OMIM or ChEBI. Note that the latter feature promotes interoperability of
the data contained in the nanopublications: the associations that are exposed by the nanopubli-
cations can relatively easily be integrated with any other dataset using those external
identifiers. Exposing LWAS as nanopublications The provenance model of the gene-disease nanopublication links to an RDF resource identi-
fier that denotes the process from which the assertion was derived. In this case it refers to the
concept profile-based method that determined the association with the specified relative
strength as indicated by the percentile score. We note that more detailed provenance could be
attached to the nanopublication provenance. For example, an extended provenance model
could link to the source code repository and the specific code version, thus enabling a full prov-
enance trace for the nanopublication assertion. The whole implicitome contains 204,072,376 nanopublications, of which 202,592,481 have
no co-occurrence in the literature. We provide an additional set of nanopublications represent-
ing the explicitome: gene disease pairs that Peregrine indexing identified to be in the same
abstract. This set contains 7,858,449 nanopublications, each of which asserts a single gene-dis-
ease co-occurrence and the PubMed ID of the publication in which they co-occur. The RDF for both the implicitome and explicitome nanopublication datasets is available
through the data repository (doi:10.5061/dryad.gn219/5 and doi:10.5061/dryad.gn219/4). In
addition, the associations are provided as a CSV file (doi:10.5061/dryad.gn219/6). Example
nanopublications can be viewed using the nanopublication viewer that is available at http://rdf. biosemantics.org/ under the tab “Nanopublications”. Clicking on the URI will display the
nanopublication in the viewer at the bottom of the page. Two more visualization options are
available on the right side of the URI link, one using a graph viewer and another that will sim-
ply render the content in the browser. Acknowledgments This project was sponsored by the Dutch Center of Medical Systems Biology (CMSB), ODEX4-
all (NWO 650.002.002), Melton Foundation, and a grant from the John Templeton Founda-
tion. The opinions expressed in this publication are those of the authors and do not necessarily
reflect the views of the John Templeton Foundation. Funding from the European Commission
(FP-7 project RD-Connect, grant agreement No. 305444), and the Innovative Medicines Initia-
tive Joint Undertaking under grant agreement No. 115191 (Open PHACTS), resources of
which are composed of financial contribution from the European Union’s Seventh Framework
Programme (FP7/2007-2013) and in-kind contribution of EFPIA companies are gratefully
acknowledged. This work was also supported by the National Institutes of Health
(U54GM114833 and GM089820). This work was carried out on the Dutch national e-infra-
structure with the support of SURF Foundation. We would like to thank Tobias Messemaker
and Yu Sun for their expert advice on the Seckel Syndrome case study, and Jaap van den Herik
of the Leiden Center for Data Science for carefully reading the manuscript. S6 Table. Nanopublication-compatible resources.
(XLSX) S6 Table. Nanopublication-compatible resources. (XLSX) Software It contains both concept ID to PMID mappings (“in which abstract does the concept
occur?”) and PMID to concept ID mappings (“which concepts occur in a given abstract?”). For
easier recreation of the concept profiles, match scores and subsequent analyses, we make this
database available as a download (doi:10.5061/dryad.gn219/2), so that the time-consuming
MEDLINE indexing step may be skipped. Berkeley DB key-value store was used to achieve efficient storage and retrieval of the indexed
concepts. It contains both concept ID to PMID mappings (“in which abstract does the concept
occur?”) and PMID to concept ID mappings (“which concepts occur in a given abstract?”). For
easier recreation of the concept profiles, match scores and subsequent analyses, we make this
database available as a download (doi:10.5061/dryad.gn219/2), so that the time-consuming
MEDLINE indexing step may be skipped. Software The datasets and graphs in this paper were created using a software pipeline consisting of a
variety of programming languages, tools and data sources. In this section we provide a sum-
mary description to facilitate reproduction and extension of this work. Fig 5 shows an overview
of the software pipeline, which starts at the top with the MEDLINE data: subsequent steps
going down and sideways. A version of this figure with clickable links to respective code and
data repositories is available (doi:10.5061/dryad.gn219/3). Data resources and intermediate
data processing products are shown as rectangles, while the ovals show different software com-
ponents (colored by programming language). Detailed documentation can be found in the
source code for each component at the public Github code repository at https://github.com/
BiosemanticsDotOrg/GeneDiseasePaper. All software components and datasets are available under an open source license, with the
exception of the MEDLINE corpus and the (UMLS-based) thesaurus. For these, a license is PLOS ONE | DOI:10.1371/journal.pone.0149621
February 26, 2016 15 / 21 The Implicitome: A Resource for Rationalizing Gene-Disease Associations required that may be obtained from the NIH U.S. National Library of Medicine. Information
about individual concepts from our thesaurus can be found at http://rdf.biosemantics.org/fct. Our code contains a modified version of the Peregrine indexer, which is available in the Github
repository The standard Peregrine version is available from https://trac nbic nl/data-mining A
Fig 5. Overview of LWAS workflow (concept profile creation and analysis). doi:10.1371/journal.pone.0149621.g005 Fig 5. Overview of LWAS workflow (concept profile creation and analysis). Fig 5. Overview of LWAS workflow (concept profile creation and analysis). doi:10.1371/journal.pone.0149621.g005 doi:10.1371/journal.pone.0149621.g005 required that may be obtained from the NIH U.S. National Library of Medicine. Information
about individual concepts from our thesaurus can be found at http://rdf.biosemantics.org/fct. Our code contains a modified version of the Peregrine indexer, which is available in the Github
repository. The standard Peregrine version is available from https://trac.nbic.nl/data-mining. A required that may be obtained from the NIH U.S. National Library of Medicine. Information
about individual concepts from our thesaurus can be found at http://rdf.biosemantics.org/fct. Our code contains a modified version of the Peregrine indexer, which is available in the Github
repository. The standard Peregrine version is available from https://trac.nbic.nl/data-mining. A 16 / 21 PLOS ONE | DOI:10.1371/journal.pone.0149621
February 26, 2016 The Implicitome: A Resource for Rationalizing Gene-Disease Associations Berkeley DB key-value store was used to achieve efficient storage and retrieval of the indexed
concepts. S4 Table. Details for 105 highest ranking implicit associations. Gene and disease by label;
connecting concepts (contribution to match score percentage); classification.
(XLSX) S4 Table. Details for 105 highest ranking implicit associations. Gene and disease by label;
connecting concepts (contribution to match score percentage); classification. (XLSX) S4 Table. Details for 105 highest ranking implicit associations. Gene and disease by label;
connecting concepts (contribution to match score percentage); classification. (XLSX) S4 Table. Details for 105 highest ranking implicit associations. Gene and disease by label;
connecting concepts (contribution to match score percentage); classification. (XLSX) Supporting Information S1 Table. Distribution of match score values. Histogram of the complete range of match
scores for both implicit and explicit associations. (XLSX) S1 Table. Distribution of match score values. Histogram of the complete range of match
scores for both implicit and explicit associations. (XLSX)
S2 Table. Literature abundance for genes. Comma separated: concept ID, label and number
of occurrences. (CSV)
S3 Table. Literature abundance for diseases. Comma separated: concept ID, label and num-
ber of occurrences. (CSV)
S4 Table. Details for 105 highest ranking implicit associations. Gene and disease by label;
connecting concepts (contribution to match score percentage); classification. (XLSX)
S5 Table. GWAS records after filtering. (XLSX)
S6 Table. Nanopublication-compatible resources. (XLSX) S2 Table. Literature abundance for genes. Comma separated: concept ID, label and number
of occurrences.
(CSV) S2 Table. Literature abundance for genes. Comma separated: concept ID, label and number
of occurrences. (CSV) S3 Table. Literature abundance for diseases. Comma separated: concept ID, label and num-
ber of occurrences. (CSV) S3 Table. Literature abundance for diseases. Comma separated: concept ID, label and num-
ber of occurrences. (CSV) S3 Table. Literature abundance for diseases. Comma separated: concept ID, label and num-
ber of occurrences. (CSV) Author Contributions Conceived and designed the experiments: KMH MT HHHBMH EH JD GJBO PACH BM EAS
MS AS. Performed the experiments: KMH MT HHHBMH EH RK PACH EAS. Analyzed the
data: KMH MT HHHBMH EH RK EA PACH EAS BMG TSL. Contributed reagents/ 17 / 21 17 / 21 PLOS ONE | DOI:10.1371/journal.pone.0149621
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Marketing Perspective in Social Enterprise Performance: A Bibliometric Analysis
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ABSTRACT Social enterprise performance is a subject that is being intensively researched
in recent years because the effects are claimed to be accelerated a country›s economic
development. The purpose of this study is to analyze the correlation of marketing
regarding social enterprise performance so that it can present theoretical frameworks
and guides for new researchers. The Scopus, Crossref, and google scholar databases
were used in this research. This research uses qualitative methods from the literature
review. The most cited paper is “Social Marketing: An Approach to Planned Social
Change”. The most prolific years were 2015 to 2019. This research resulted in three
major topics that are currently being researched, namely social marketing, enterprise,
and performance. Furthermore, the results of this study also indicate future research
opportunities. Keywords: marketing, social enterprise performance, bibliometric analysis,
journal of social entrepreneurship Keywords: marketing, social enterprise performance, bibliometric analysis,
journal of social entrepreneurship Social Enterprise Performance Social enterprise performance (SEP) is the performance of social
business actors who apply a practical, innovative, and sustainable
approach to have an impact on the lower economic class community
and overcome the surrounding social problems. SEP consists of two
measurement dimensions, namely social performance and economic
performance (Park & Shin, 2021). In other words, the performance
of social entrepreneurship is judged to be good based on how many
social benefits the entrepreneur provides, and the economic impact on
the company. Often social enterprises cannot integrate and balance the
economic interests/profitability of the company and the social impact
on society. Meanwhile, conventional entrepreneurship measurement
models have also begun to be adopted in social entrepreneurship
activities. According to (Somers, 2005), the balanced scorecard can be
used to measure social enterprise performance in terms of economic
profit, social profit, and environmental profit. Furthermore, social and
environmental profits flow to the community. Otherwise, economic
profits flow back into the social enterprise and to ethical investors. Introduction Social enterprise performance is material that is at an early
stage to be researched. Various researchers measure social enterprise
performance through various dimensions such as personal issues
and business elements (Liang et al., 2015), social performance
(Liu et al., 2015; Miles, 2014; Shin & Park, 2019), and financial
performance (Miles, 2014; Park & Shin, 2021). However, very few
have researched social enterprise performance based on a marketing
perspective. There is a missing link between marketing and social
entrepreneurship (Ghods, 2019). This research is expected to provide
a brief highlight to researchers and social entrepreneurship actors
to be able to improve their performance from the marketing side. The purpose of this study is to analyze the correlation between
marketing in social enterprise performance so that can present a
theoretical framework and guidelines for the new researchers. This Vol. 1 No. 1, Oktober 2022 12 Siti Alhamra Salqaura, Wan Rizca Amelia, Alfifto, Haryaji Catur Putera H study discusses the Top 5 most cited publications, year of publication,
and visualization topic areas in terms of marketing in social enterprise
performance. Literature Review (JOCRISE) Journal of Critical Realism in Socio-Economics Method There are two steps applied in this research. First, the authors
collect the data using publish or perish software. At an advanced level,
these data are frameworked using Vos Viewer software. This study
uses Scopus, Crossref, and Google Scholar databases source. These
data sources are limited to journal articles only. The language selected
is English only. The period for the data source is not determined in
this study. The authors included the keywords and title “marketing
social enterprise performance”. Next, sample articles are downloaded
in .ris format, Afterward, the authors create a term co-occurrence map
based on the text data. The title and abstract fields are extracted and
choose “5” as the minimum number of occurrences of a term. As the
result, from 6070 terms, a total of 351 meet the threshold. Moreover,
for each of the 351 terms, a relevance score will be calculated. Based Marketing Perspektive In Social Enterprise Performance: A Bibliometric Analysis 13 on this score, the most relevant terms will be selected. And the default
setting choice is to select the 60% most relevant terms. So that, amount
211 terms are selected. Result & Discussion The result of this research is divided into discussions namely
Top 5 most cited publications, year of publication, and visualization
topic area in terms of marketing in social enterprise performance. Top 5 most cited publications Building upon data analysis, the authors determined the Top
5 most cited publications based on Crossref, Google Scholar, and
Scopus databases respectively as Table 1 below. Table 1 Crossreff Database Table 1 Crossreff Database
Cites
Authors
Title
Year
569
Kotler & Zaltman
Social Marketing: An
Approach to Planned Social
Change
1971
483
Luo et al., 2009
The Debate over Doing
Good: Corporate Social
Performance, Strategic
Marketing Levers, and
Firm-Idiosyncratic Risk
2009
292
Defourny &
Nyssens
Social enterprise in
Europe: recent trends and
developments
2008
259
Korschun et al.,
Corporate Social
Responsibility, Customer
Orientation, and the Job
Performance of Frontline
Employees
2014 Vol. 1 No. 1, Oktober 2022 14 Siti Alhamra Salqaura, Wan Rizca Amelia, Alfifto, Haryaji Catur Putera H 244
Kang, Frank
Germann
Rajdeep Grewal
et al.,
Washing Away Your
Sins? Corporate Social
Responsibility, Corporate
Social Irresponsibility, and
Firm Performance
2016 244
Kang, Frank
Germann
Rajdeep Grewal
et al.,
Washing Away Your
Sins? Corporate Social
Responsibility, Corporate
Social Irresponsibility, and
Firm Performance
2016 244 Table 1 shows the most cited paper in the crossref database was
“Social Marketing: An Approach to Planned Social Change” which
have published in 1971 with 569 citations. This paper conducted an
analysis and examined four social campaigns to ascertain whether
elements or traits were responsible for social marketing success or
failure. This study discovered eminently successful regard to five
factors i.e. the force, the direction, the mechanism, the adequacy and
compatibility, and the distance. The result concludes that the more
a social campaign resembled those of a product campaign, the more
successful the social campaign. Moreover, followed by “The Debate
over Doing Good: Corporate Social Performance, Strategic Marketing
Levers, and Firm-Idiosyncratic Risk” which has been cited 483 times. This paper proposed a theoretical framework based on marketers
and investors perspectives. This research found that higher corporate
social performance lowers undesirable firm-idiosyncratic risk. pi
y
Furthermore, the third most-cited literature is entitled “Social
enterprise in Europe: recent trends and developments” which has
been cited 292 times. This paper assessed comparatively different
institutions such as legal frameworks, public policies, supporting
structures, and so forth in European Union (EU) countries. This
study found that even though the concept of social enterprise has
not gained the same recognition all over EU countries, even poorly
understood, the fact is social enterprise is growing over there and
experienced in the majority of EU countries. (JOCRISE) Journal of Critical Realism in Socio-Economics (JOCRISE) Journal of Critical Realism in Socio-Economics Top 5 most cited publications The fourth most-cited
paper is “Corporate Social Responsibility, Customer Orientation, and
the Job Performance of Frontline Employees” by cited 259 times. This
paper explored the frontline employee’s opinion in terms of corporate
social responsibility. This study discovered that frontline employees
identify with the organization (i.e., organizational identification) and
with customers (i.e., employee-customer identification) in proportion
to how much management and customers (respectively) perceive the
company›s CSR activities to be supported. And the last fifth most-cited
article is “Washing Away Your Sins? Corporate Social Responsibility,
Corporate Social Irresponsibility, and Firm Performance” and this has (JOCRISE) Journal of Critical Realism in Socio-Economics Marketing Perspektive In Social Enterprise Performance: A Bibliometric Analysis 15 been cited 244 times. This paper investigated the correlation between
corporate social responsibility and firm performance. Based on this
research, showed that corporate social responsibility are likely benefit
financially to the firm. Otherwise, corporate social responsibility
could not erase the corporate social irresponsibility as redemption. Table 2 Google Scholar Database
Cites Authors
Title
Year
168
Liu G, TY Eng, S
Takeda
An investigation of
marketing capabilities
and social enterprise
performance in the UK
and Japan
2015
59
Kwilinski A,
K Pajak, O
Halachenko, S
Vasylchak
Marketing tools for
improving enterprise
performance in the
context of social and
economic security of
the state: innovative
approaches to assessment
2019 Table 2 Google Scholar Database Table 2 presents the most cited paper in the Google Scholar
database “An investigation of marketing capabilities and social
enterprise performance in the UK and Japan” which was launched
in 2015 with 168 times citations. This literature investigated the
effect of marketing capabilities on social enterprise performance. Disparate from the commercial entrepreneurship theory, the finding
showed that not all marketing capabilities are associated with social
enterprise performance. The second most-cited is “Marketing tools
for improving enterprise performance in the context of social and
economic security of the state: innovative approaches to assessment”
which has been cited 59 times. This paper assessed the activity
of enterprises. This research results in the measures, methods,
and marketing tools based on innovative technologies which can
significantly increase the efficiency performance of enterprises in
sanatorium and resort areas. The rest of the research literature in the
google scholar database was not cited yet (zero “0” cited) in terms of
marketing is social enterprise performance. Vol. 1 No. Top 5 most cited publications 1, Oktober 2022 16 Siti Alhamra Salqaura, Wan Rizca Amelia, Alfifto, Haryaji Catur Putera H Siti Alhamra Salqaura, Wan Rizca Amelia, Alfifto, Haryaji Catur Putera H Table 3 Scopus Database
Cites Authors
Title
Year
88
Liu G, , TY Eng,
S Takeda
An Investigation of Marketing
Capabilities and Social
Enterprise Performance in the
UK and Japan
2015
37
M. P. Miles,
Social Enterprises and the
Performance Advantages
of a Vincentian Marketing
Orientation
2014
16
H. Musa
Social media marketing and
online small and medium
enterprises performance:
Perspective of Malaysian
small and medium enterprises
2016
12
W. Basri
Examining the impact
of artificial intelligence
(Ai)-assisted social media
marketing on the performance
of small and medium
enterprises: Toward effective
business management in the
saudi arabian context
2020
8
Lee & Chandra
Dynamic and Marketing
Capabilities as Predictors
of Social Enterprises’
Performance
2020 Table 3 Scopus Database
Cites Authors
Title
Year
88
Liu G, , TY Eng,
S Takeda
An Investigation of Marketing
Capabilities and Social
Enterprise Performance in the
UK and Japan
2015
37
M. P. Miles,
Social Enterprises and the
Performance Advantages
of a Vincentian Marketing
Orientation
2014
16
H. Musa
Social media marketing and
online small and medium
enterprises performance:
Perspective of Malaysian
small and medium enterprises
2016
12
W. Basri
Examining the impact
of artificial intelligence
(Ai)-assisted social media
marketing on the performance
of small and medium
enterprises: Toward effective
business management in the
saudi arabian context
2020
8
Lee & Chandra
Dynamic and Marketing
Capabilities as Predictors
of Social Enterprises’
Performance
2020 Table 3 Scopus Database Table 3 shows the most-cited paper in the Scopus database. Its
result is equivalent to the most-cited in the google scholar database
i.e. “An Investigation of Marketing Capabilities and Social Enterprise
Performance in the UK and Japan” with 88 times cited in the Scopus
database. Subsequently, the second most-cited literature is “Social
Enterprises and the Performance Advantages of a Vincentian
Marketing Orientation” and this has been cited 37 times. This paper
explored social entrepreneurship in Australia. Should those social
enterprises (SEs) become more marketing oriented or not? Vincentian- (JOCRISE) Journal of Critical Realism in Socio-Economics Marketing Perspektive In Social Enterprise Performance: A Bibliometric Analysis 17 based values have been used as a measurement of a Vincentian
marketing orientation (VMO) to leverage business in service to
the poor. The result found that VMO has a strong correlation with
social, economic, and environmental performance. Top 5 most cited publications These findings
suggest that the SEs can benefit by leveraging marketing and
capabilities to better serve their benefits and stakeholder. The third
most-cited article is “Social media marketing and online small and
medium enterprises performance: Perspective of Malaysian small
and medium enterprises” by cited 16 times. This study investigated
the effectiveness of social media marketing implementation on
organizational focuses on SMEs in Malaysia. Moreover, the result
showed that brand reputation and image (BRI), customer brand
reputation (CBA), and customer engagement (CE) have a medium
correlation attachment with organizational performance amount
69,2%. The fourth most-cited literature is “Examining the impact
of artificial intelligence (Ai)-assisted social media marketing on the
performance of small and medium enterprises: Toward effective
business management in the Saudi Arabian context” which has
been cited 12 times. This research investigated the implementation
of artificial intelligence-assisted social media marketing (AISMM)
in terms of enhancing SME›s performance (SMEP). Lastly, The
fifth most-cited script is “Dynamic and Marketing Capabilities as
Predictors of Social Enterprises’ Performance” by cited 8 times. This
article explored the core element of an organization’s absorptive
capacity. Furthermore, absorptive capacity is downgraded to
marketing capabilities and measured by financial performance and
social performance. An SE’s absorptive capacity does not directly
translate into the financial and social performance but requires
the leveraging mechanism of marketing capabilities to realize this
performance. Year of Publications The research related to social enterprise performance
has increased from year to year. The growth of scientific article
publications related to social enterprise performance can be seen in
the following graph (Figure 1). Vol. 1 No. 1, Oktober 2022 18 Siti Alhamra Salqaura, Wan Rizca Amelia, Alfifto, Haryaji Catur Putera H Figure 1. Year of Publication
Based on statistic analysis in Figure 1, the result shows that
the trend of publication in terms of social enterprise performance
increased significantly since 2006 and the culmination in 2015-2019. This study is in accordance with the research by (Hira et al., 2021;
Rey-Martí et al., 2016). However, the next range of years from 2019-
2024 is still progressing. Figure 1. Year of Publication Figure 1. Year of Publication Figure 1. Year of Publication Figure 1. Year of Publication Based on statistic analysis in Figure 1, the result shows that
the trend of publication in terms of social enterprise performance
increased significantly since 2006 and the culmination in 2015-2019. This study is in accordance with the research by (Hira et al., 2021;
Rey-Martí et al., 2016). However, the next range of years from 2019-
2024 is still progressing. (JOCRISE) Journal of Critical Realism in Socio-Economics Visualization Topic Area Based on VosViewer analysis, there were 5 clusters formed i.e. blue, green, red, yellow, and purple. The network between those dots
showed the relationship between one topic and another. Vosviewer
software can display bibliometric mapping comprehensively by
revealing three different visualizations; network visualization (Figure
2), overlay visualization (Figure 3), and density visualization (Figure
4). The same color network showed the keywords connectivity in
some topics. The size of the dot or circle indicates the appearance of
keywords in the titles and abstracts. Therefore, The more keywords
emanate, the bigger size of the circle/ dot. (JOCRISE) Journal of Critical Realism in Socio-Economics 19 Marketing Perspektive In Social Enterprise Performance: A Bibliometric Analysis Figure 2. Visualization Topic Using Network Visualization Figure 2. Visualization Topic Using Network Visualization Figure 2 shows clusters in each of the topic areas studied. Based
on the bibliometric analysis, presents that there were few keywords of
the topic developed previously namely enterprise, social marketing,
performance, and management. So the keywords “performance” and
“management” are the close topic in social enterprise performance
based on a marketing perspective. The smaller circle around the
figure shows that many topics need to be explored. For instance,
social enterprise performance, financial performance, and social
performance are infinitesimal. It could be a clue for any future study. Figure 3. Visualization Topic Area Using Overlay Visualization Figure 3. Visualization Topic Area Using Overlay Visualization Vol. 1 No. 1, Oktober 2022 Vol. 1 No. 1, Oktober 2022 Vol. 1 No. 1, Oktober 2022 20 Siti Alhamra Salqaura, Wan Rizca Amelia, Alfifto, Haryaji Catur Putera H Figure 3 presents the trend over the years related to marketing
in social enterprise performance research. It is worth noting that social
enterprise performance research based on a marketing perspective
starts in 2008. The blue circle showed the topic studied at the
beginning of this research namely social marketing. The brighter the
color, the newer the research. The keywords enterprise has green
color indicates the topic explored in the middle of the development
around 2010-2016. The yellow color showed the recent topics research
explored starting from 2018 such as performance, social enterprise
performance, and marketing strategy. The yellow color could be the
gap in order to enrich this topic and also for future research directions. Figure 4. Visualization topic area using density visualization Figure 4. Visualization topic area using density visualization Figure 4. Visualization topic area using density visualization
Figure 4 displays the depth of the research. Bright colors
indicate a lot of research that has been done. The darker the density
area indicates that there is still little research on the topic. Therefore,
need more exploration exclusively in terms of social enterprise
performance and enterprise marketing. Figure 4 displays the depth of the research. Bright colors
indicate a lot of research that has been done. The darker the density
area indicates that there is still little research on the topic. Therefore,
need more exploration exclusively in terms of social enterprise
performance and enterprise marketing. (JOCRISE) Journal of Critical Realism in Socio-Economics Conclusion Social enterprise performance is believed to be able to accelerate
the growth of a country to make a country with a prosperous income
level of the majority of the people. This has been proven by several
developed countries, especially in the United Kingdom (Defourny
& Nyssens, 2010; Liu et al., 2015), United States (Cui & Jernigan,
n.d.; Janelle, 2006; Stern & McKoy, 2001; Terjesen, 2017; Young,
2001) and Japan (Kawamoto, 2020; Liu et al., 2015; Nakao & Nishide,
2020). However, empirical research related to social enterprise (JOCRISE) Journal of Critical Realism in Socio-Economics Marketing Perspektive In Social Enterprise Performance: A Bibliometric Analysis 21 performance itself is still very little done. As evidence, this study
has demonstrated the existence of a super tiny circle exist for social
enterprise performance solely. This article attempts to visualize and
configure systematic literature sharing and then analyze it through
a bibliometric approach. This bibliometric review was used to
recognize the key themes in marketing research perspective or scope
respectively that has been carried out so far and so beneficial for
determining novelty in order to conduct further research. Social enterprise performance is believed to be able to accelerate
the growth of a country to make a country with a prosperous income
level for the majority of the people. This has been proven in several
developed countries, especially in the United Kingdom (Defourny &
Nyssens, 2010; Liu et al., 2015), United States (Cui & Jernigan, n.d.;
Janelle, 2006; Stern & McKoy, 2001; Terjesen, 2017; Young, 2001) and
Japan (Kawamoto, 2020; Liu et al., 2015; Nakao & Nishide, 2020). However, empirical research related to social enterprise performance
itself is still very little done. As evidence, this study has demonstrated
the existence of a super tiny circle for social enterprise performance
solely. This article attempts to visualize and configure systematic
literature sharing and then analyze it through a bibliometric
approach. This bibliometric review was used to recognize the key
themes in marketing research perspective or scope respectively that
have been carried out so far and so beneficial for determining novelty
in order to conduct further research. Limitations and recommendation for further research The limitations of this bibliometric research are the databases
used only from crossref, google scholar, and Scopus. For further
research, need to conduct exploration for other databases such as a
web of science, dimension, semantic scholars, and so on. Otherwise,
exploration of another keyword should be consider. (JOCRISE) Journal of Critical Realism in Socio-Economics References Basri, W. (2020). Examining the impact of artificial intelligence
(Ai)-assisted social media marketing on the performance of
small and medium enterprises: Toward effective business
management in the saudi arabian context. International Journal
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Social Responsibility, Customer Orientation, and the Job
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& Kuzior, P. (2019). Marketing Tools for Improving Enterprise
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scopus_id/84991200300 Vol. 1 No. 1, Oktober 2022 24 Siti Alhamra Salqaura, Wan Rizca Amelia, Alfifto, Haryaji Catur Putera H Nakao, K., & Nishide, Y. (2020). The development of social
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Synthesis and structure study of new manganese and indium based phosphate: (Ca,Ba)<sub>1.222</sub>Mn<sub>0.923</sub>In<sub>1.570</sub>(PO<sub>4</sub>)<sub>3</sub>
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Synthesis and structure study of new manganese and indium based
phosphate: (Ca,Ba)1.222Mn0.923In1.570(PO4)3 Elhassan Benhsina*, Abderrazzak Assani, Mohamed Saadi and Lahcen El Ammari
Laboratoire de Chimie Appliquée des Matériaux, Centre des Sciences des Matériaux, Faculty of Sciences, Mohammed V
University in Rabat, Avenue Ibn Battouta, BP 1014, Rabat, Morocco. *Correspondence e-mail: h.benhsina@gmail.com Abstract. Phosphates are an important class of materials which are well-known due to their
structural diversity and their important potential application in many fields of sciences. A member
of this family, represented by transition metal phosphates, is in the forefront of important
researchers. A new manganese and indium based phosphate is successfully isolated by means of
solid state reaction. Its corresponding crystal structure is isotypic to the alluaudite and crystallizes in
the monoclinic system with the space group C2/c and cell parameters a=12.4697(5)Å ;
b=12.9365(5)Å ; c=6.5625(3)Å and β=115.603(1)°. The framework of this phosphate is built up on
the basis of MnO6, InO6 and PO4 polyhedra. The InO6 octahedra are linked together via common
edge, leading to the formation of In2O10 dimers. The In2O10 dimers share edge with MnO6
octahedra, generating chains along the [101] direction. The PO4 tetrahedra connect two chains
giving arise to a three-dimensional framework with hexagonal tunnels where the barium and
calcium are located. En déployant plusieurs méthodes de synthèse :
synthèse
par
diffusion
à
l’état
solide,
synthèse
hydrothermale ou synthèse par chimie douce, et en
utilisant la diffraction sur monocristal ou sur poudre,
nous avons pu isoler et caractériser plusieurs phosphates
de
type
alluaudite. A
titre
d’exemple,
NaMg3(HPO4)2(PO4)
[3],
AgMg3(HPO4)2(PO4)
[4],
Na2Co2Fe(PO4)3[5] et Na1,67Zn1,67Fe1,33(PO4)3 [6]. MATEC Web of Conferences 149, 01085 (2018)
CMSS-2017 MATEC Web of Conferences 149, 01085 (2018)
CMSS-2017 https://doi.org/10.1051/matecconf/201814901085 © 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/). 2 Expérimental Le nouveau phosphate (Ca,Ba)1,222Mn0,923In1,570(PO4)3
a été obtenu par la réaction de diffusion à l’état solide. Les réactifs (nitrates de calcium (Riedel-de haën, 98%),
nitrate de baryum (Riedel-de haën, 98.5%), carbonate de
manganèse (Riedel-de haën, 99%), iodure d’indium
(Ventron, 98.5) et l’acide phosphorique (Riedel-de haën,
85%)) sont dissouts dans l’eau distillée avec quelque
goutte d’acide nitrique. Le mélange réactionnel ainsi
obtenu est maintenu sous agitation pendant 24 heures
avant d’être porté à sec. La poudre obtenue est ensuite
broyée dans un mortier en agate et est portée
progressivement à la fusion à une température de
1080°C, dans un creuset en platine. Après un
refroidissement lent avec une vitesse de 5°C/heure
jusqu’à la température ambiante, nous obtenons des
cristaux de taille suffisante pour la diffraction des rayons
X sur monocristal. 1 Introduction Les
matériaux
phosphatés
connaissent
un
développement considérable en raison de leur diversité
structurale et de leurs importantes et potentielles
applications dans de nombreux domaines de la
technologie moderne. Nous pouvons citer, à titre
d’exemple, le phosphate LiFePO4 [1] de structure type
olivine, dont l’importance est amplement justifiée par son
application dans les batteries au lithium commercialisés à
l’échelle mondiale. De même, les propriétés électriques
de LiMnPO4 [2], de structure alluaudite, avoisinent celles
de LiFePO4 et représente l’un des matériaux les plus
prometteurs dans le stockage d’énergie en tant que
cathode pour les batteries. Par conséquent, l’un des
principaux axes de recherche développés dans notre
laboratoire est axé sur la synthèse et la caractérisation
structurale
de
nouveaux
matériaux
phosphatés,
apparentés essentiellement à la très convoitée famille
alluaudite. Ce choix est justifié par les propriétés
physico-chimiques de ces matériaux. En effet, la structure du premier phosphate de cette
famille alluaudite est déterminée par P.B. Moore [7]. Ces
phosphates cristallisent dans un système monoclinique
avec un groupe d’espace C2/c. Moore a montré que cette
famille peut être représentée par la formule générale
A(2)A(1)M(1)M(2)2(PO4)3 où A représente des sites
cationiques pouvant être occupés par des cations de
moyen ou grande taille et de faible charge (généralement
monovalent ou divalent) alors que les sites cationiques de
type M représentent un environnement octaédrique
généralement occupé par des éléments de transitions. Toutefois, le site de type M(1) est très distordu alors que
celui de type M(2) est presque régulier. https://doi.org/10.1051/matecconf/201814901085 MATEC Web of Conferences 149, 01085 (2018)
CMSS-2017 Temperature (K)
296
a (Å)
12,4697 (5)
b(Å)
12,9365 (5)
c(Å)
6,5625 (3)
β (°)
115,603 (1)
V (Å3)
954,68 (7)
Z
4
Type de radiation
Mo Kα
(mm-1)
16,79
Collection des données
Nombre de réflexions mesurées
9990
Nombre réflexions
indépendantes
1822
Nombre de réflexions avec
[I>2(I)]
1703
Rint
0,029
(sin θ/λ)max (Å-1)
0,769
Affinement
R[F2> 2(F2)]
0,022
wR(F2)
0,056
S
1,13
Nombre de paramètres
102 Dans ce travail, nous présentons la synthèse et la
caractérisation structurale du nouveau phosphate à base
d’indium
et
de
manganèse
(Ca,Ba)1,222Mn0,923In1,570(PO4)3. 3.1 Collection des données et résolution
structurale La résolution structurale a été réalisée en utilisant
plusieurs réflexions collectées par le diffractomètre à
quatre cercle de type Brüker X8 APEX[8] des Unités
d'Appui Techniques à la Recherche Scientifique
(UATRS) relevant du Centre National pour la recherche
Scientifique et Technique CNRST (Maroc). En effet,
seules les intensités répondant au critère statistique
I>2(I) ((I) étant l’écart type sur l’intensité) ont été
utilisées pour localiser les positions des atomes. Ces
données ont été corrigées des effets de Lorentz
polarisation et des effets d’absorption. Le tableau 1
montre les conditions d’enregistrement et les données
cristallographiques de (Ca,Ba)1,222Mn0,923In1,570(PO4)3. La
résolution et l’affinement de la structure ont été réalisés
par la méthode directe couplée aux synthèses de Fourier
différences successives, à l’aide des programmes
SHELXT2014 [9] et SHELXL2014 [10]. Les
conditions
d’extinction
observées
pour
(Ca,Ba)1,222Mn0,923In1,570(PO4)3 sont compatibles avec les
groupes d’espace C2/c et Cc. La structure de ce
phosphate a été résolue dans le groupe centro-symétrique
(C2/c). Précisons que la méthode des moindres carrés,
appliquée sur le carré des modules des facteurs de
structure F2, a été utilisée pour affiner les différents
paramètres structuraux (positions atomiques (x, y, z),
paramètres de déplacement isotropes (Uiso) ou anisotropes
(Uaniso) et occupation des sites cristallographiques). Les
facteurs d’accord, obtenus à la fin du dernier cycle
d’affinement, R(F) = 2,20%, wR(F2) = 5,60% et GooF=
S= 1,13. Ces valeurs attestent de la bonne qualité de
l’affinement. Le tableau 2 regroupe les positions atomiques, les
déplacements atomiques isotropes équivalents ainsi que
les taux d’occupations des sites cristallographiques. Le
tableau 3 rassemble les facteurs de déplacements
atomiques
anisotropes
dans Tableau 1: Données cristallographiques et conditions Tableau 1: Données cristallographiques et conditions
d’enregistrement de (Ca,Ba)1,222Mn0,923In1,570(PO4)3. ab eau :
o
ées c sta og ap ques et co d t o s
d’enregistrement de (Ca,Ba)1,222Mn0,923In1,570(PO4)3. Donnée du cristal
Formule chimique
(Ca,Ba)1,222Mn0,923In1,570(PO4)3
Système cristallin
Monoclinique
Groupe d’espace
C2/c d’enregistrement de (Ca,Ba)1,222Mn0,923In1,570(PO4)3. Donnée du cristal
Formule chimique
(Ca,Ba)1,222Mn0,923In1,570(PO4)3
Système cristallin
Monoclinique
Groupe d’espace
C2/c (Ca,Ba)1,222Mn0,923In1,570(PO4)3. 2 2 MATEC Web of Conferences 149, 01085 (2018)
CMSS-2017 https://doi.org/10.1051/matecconf/201814901085 Tableau 2: Positions atomiques, déplacements atomiques isotropes équivalents et taux d’occupations des sites cristallographiques dans
(Ca,Ba)1,222Mn0,923In1,570(PO4)3. Tableau 2: Positions atomiques, déplacements atomiques isotropes équivalents et taux d’occupations des sites cristallographiques dans
(Ca,Ba)1,222Mn0,923In1,570(PO4)3. Tableau 2: Positions atomiques, déplacements atomiques isotropes équivalents et taux d’occupations des sites cristallographiques d
(Ca,Ba)1,222Mn0,923In1,570(PO4)3. Wyck. x
y
z
Ueq
Occ. 3.1 Collection des données et résolution
structurale In1
8f
0,721 (2)
0,3477 (1)
0,6327 (3)
0,009(1)
0,785(1)
Mn1
4e
1,00
0,7375 (1)
0,750
0,010 (1)
0,923 (2)
Ca1
4e
0,500
0,4866 (1)
0,250
0,033 (1)
0,922 (1)
Ba1
8f
0,522 (1)
-0,0006 (1)
0,466 (1)
0,026 (1)
0,151 (1)
P1
8f
0,7397 (1)
0,6104 (1)
0,6341 (1)
0,009 (1)
1
P2
4e
0,5000
0,2114 (1)
0,2500
0,008 (1)
1
O1
8f
0,7263 (2)
0,6795 (1)
0,8136 (2)
0,012 (1)
1
O2
8f
0,6681(2)
0,6628 (1)
0,4025 (3)
0,014 (1)
1
O3
8f
0,5461 (2)
0,2796 (1)
0,4621 (2)
0,014 (1)
1
O4
8f
0,4069(2)
0,1369 (1)
0,2639 (4)
0,023 (1)
1
O5
8f
0,6867 (2)
0,5042 (1)
0,6345 (3)
0,019 (1)
1
O6
8f
0,872 (2)
0,6031 (1)
0,6862 (4)
0,024 (1)
1 Tableau 3 : Facteurs de déplacements atomiques anisotropes dans le phosphate (Ca,Ba)1,222Mn0,923In1,570(PO4)3. U11
U22
U33
U12
U13
U23
In1
0,01137 (9)
0,00810 (9)
0,00950 (9)
-0,00101 (5)
0,00593 (6)
-0,00118 (5)
Mn1
0,0112 (2)
0,0114 (2)
0,0107 (2)
0,000
0,00719 (2)
0,000
Ca1
0,0148 (4)
0,0279 (5)
0,0400 (6)
0,000
-0,0036 (3)
0,000
Ba1
0,0258 (6)
0,0121 (4)
0,0267 (6)
0,0006 (4)
-0,0013 (4)
0,0012 (4)
P1
0,0135 (2)
0,0064 (2)
0,0081 (2)
0,00066 (2)
0,00576 (2)
0,00012 (2)
P2
0,0078 (3)
0,0095 (3)
0,0074 (3)
0,000
0,0031 (2)
0,000
O1
0,0175 (7)
0,0109 (6)
0,0083 (6)
-0,0002 (5)
0,0070 (5)
-0,0016 (5)
O2
0,0252 (8)
0,0104 (6)
0,0080 (6)
0,0017 (5)
0,0081 (6)
0,0019 (5)
O3
0,0120 (6)
0,0211 (8)
0,0066 (6)
0,0015 (5)
0,0027 (5)
-0,0030 (5)
O4
0,0104 (7)
0,0180 (8)
0,0394 (2)
0,0009 (6)
0,0085 (7)
0,0145 (7)
O5
0,0289 (9)
0,0078 (7)
0,0242 (8)
-0,0004 (6)
0,0150 (7)
0,0024 (6)
O6
0,0170 (7)
0,0175 (8)
0,0412 (2)
0,0021 (6)
0,0164 (8)
-0,0042 (7) Tableau 3 : Facteurs de déplacements atomiques anisotropes dans le phosphate (Ca,Ba)1,222Mn0,923In1,570(PO4)3. 3 3 3 MATEC Web of Conferences 149, 01085 (2018)
CMSS-2017 https://doi.org/10.1051/matecconf/201814901085 Les atomes d’indium et de baryum occupent
partiellement les sites 8f avec des taux d’occupation de
0,785(1) et 0,151 (1) respectivement. Quant aux atomes
de manganèse et de calcium, ils occupent partiellement
les sites 4e avec des taux d’occupation de 0,993(2) et
0,922(1) respectivement. Les autres atomes (phosphore et
oxygène) occupent totalement les sites 8f à l’exception de
P2 qui est localisé sur le site 4e. Ceci conduit à la
formulation suivante : Ca0,922Ba0,300Mn0,923In1,570(PO4)3. Ce
type
de
non-
stœchiométrie est souvent rencontré dans les structures de
type
alluaudite :
Na1,72Mn3,28(AsO4)3[11],
Ag1,49Mn2,90(AsO4)3[12],
Na1,67Mn2,17(MoO4)3[13],
Na2,44Mn1,79(SO4)3[14]. Ca0,922Ba0,300Mn0,923In1,570(PO4)3. Ce
type
de
non-
stœchiométrie est souvent rencontré dans les structures de
type
alluaudite :
Na1,72Mn3,28(AsO4)3[11],
Ag1,49Mn2,90(AsO4)3[12],
Na1,67Mn2,17(MoO4)3[13],
Na2,44Mn1,79(SO4)3[14]. 4 Description de la structure Les distances et les angles significatifs dans le phosphate
(Ca,Ba)1,222Mn0,923In1,570(PO4)3 sont regroupés dans le
tableau 4. Tableau 4: Distances (Ǻ) et angles interatomiques (°) dans la structure de(Ca,Ba)1,222Mn0,923In1,570(PO4)3. Distances P(1) - O (Ǻ)
Distances P(2) - O (Ǻ)
P1—O5
1,5252 (2)
P2—O3xii
1,5344 (2)
P1—O6
1,5355 (2)
P2—O3
1,5345 (2)
P1—O2
1,5472 (2)
P2—O4
1,5410 (2)
P1—O1
1,5479 (2)
P2—O4xii
1,5411 (2)
Distance moyenne
1,5389 Å
Distance moyenne
1,5376 Å
Angles O - P(1) - O (°)
Angles O - P(2) - O (°)
O5—P1—O6
111,68 (11)
O3xii—P2—O3
154,72 (7)
O5—P1—O2
109,24 (10)
O3xii—P2—O4
90,36 (6)
O6—P1—O2
110,74 (11)
O3—P2—O4
109,79 (13)
O5—P1—O1
109,08 (10)
O3xii—P2—O4xii
114,63 (9)
O6—P1—O1
109,27 (10)
O3—P2—O4xii
107,58 (11)
O2—P1—O1
106,69 (9)
O4—P2—O4xii
102,65 (15)
Distances In - O (Ǻ)
Distances Mn - O (Ǻ)
In1—O5
2,0694 (2)
Mn1—O3vi
2,2660 (2)
In1—O4i
2,1073 (2)
Mn1—O3viii
2,2660 (2)
In1—O2ii
2,1424 (2)
Mn1—O6ix
2,2734 (2)
In1—O1iii
2,1501 (2)
Mn1—O6
2,2735 (2)
In1—O3
2,1605 (2)
Mn1—O2x
2,2905 (2)
In1—O1iv
2,2591 (2)
Mn1—O2xi
2,2766 (2)
Distance moyenne
2,1481Å
Distance moyenne
2,3434 Å
Angles O – In– O (°)
Angles O – Mn– O (°)
O5—In1—O4i
95,63 (7)
O3vi—Mn1—O3viii
152,22 (9)
O5—In1—O2ii
85,41 (7)
O3vi—Mn1—O6ix
116,17 (7)
O4i—In1—O2ii
110,19 (8)
O3viii—Mn1—O6ix
85,77 (7)
O5—In1—O1iii
104,96 (7)
O3vi—Mn1—O6
85,77 (7)
O4i—In1—O1iii
85,63 (7)
O3viii—Mn1—O6
116,17 (7)
O2ii—In1—O1iii
160,45 (6)
O6ix—Mn1—O6
80,23 (9)
O5—In1—O3
103,80 (7)
O3vi—Mn1—O2x
90,83 (6)
O4i—In1—O3
159,40 (6)
O3viii—Mn1—O2x
73,45 (6) leau 4: Distances (Ǻ) et angles interatomiques (°) dans la structure de(Ca,Ba)1,222Mn0,923In1,570(PO4)3. 4 Description de la structure Codes de symétrie : (i) x+1/2, -y+1/2, z+1/2; (ii) x, -y+1, z+1/2; (iii) x, -y+1, z-1/2; (iv) -x+3/2, y-1/2, -z+3/2; (v) -x+3/2, -y+1/2, -
z+1; (vi) -x+3/2, y+1/2, -z+3/2; (vii) –x+1, -y+1, -z+1; (viii) x+1/2, y+1/2, z; (ix) -x+2, y, -z+3/2; (x) x+1/2, -y+3/2, z+1/2; (xi) -
x+3/2, -y+3/2, -z+1; (xii) -x+1, y, -z+1/2; (xiii) -x+1, -y+1, -z; (xiv) -x+1, -y, -z+1; (xv) x-1/2, -y+1/2, z-1/2; (xvi) x-1/2, y-1/2, z;
(xvii) x, -y, z-1/2; (xviii) x, -y, z+1/2. Ces chaînes sont connectées via les sommets des
tétraèdres P1O4 et P2O4, pour former une charpente
tridimensionnelle délimitant des tunnels dans lesquels
les atomes de baryum et de calcium se logent (Figure
2). La structure de ce phosphate est formée de
tétraèdres PO4, d’octaèdres InO6 et MnO6. Deux octaèdres d’indium mettent des arêtes en
commun pour former un dimer In2O10. Ces derniers sont
liés alternativement par les octaèdres de manganèse
MnO6 pour construire des chaînes parallèle à la
direction [101] (Figure 1). Figure
2
:
Projection
de
la
structure
(Ca,Ba)1,222Mn0,923In1,570(PO4)3 selon l'axe c. Figure 1: Chaînes formées par les octaèdres MnO6 et InO6
parallèle à [101]. Figure
2
:
Projection
de
la
structure
(Ca,Ba)1,222Mn0,923In1,570(PO4)3 selon l'axe c. 4 Description de la structure 4 MATEC Web of Conferences 149, 01085 (2018)
CMSS-2017 https://doi.org/10.1051/matecconf/201814901085 O2ii—In1—O3
78,58 (6)
O6ix—Mn1—O2x
85,01 (6)
O1iii—In1—O3
82,82 (6)
O6—Mn1—O2x
161,49 (7)
O5—In1—O1iv
171,49 (7)
O3vi—Mn1—O2xi
73,45 (6)
O4i—In1—O1iv
80,08 (6)
O3viii—Mn1—O2xi
90,83 (6)
O2ii—In1—O1iv
89,18 (6)
O6ix—Mn1—O2xi
161,49 (7)
O1iii—In1—O1iv
82,15 (6)
O6—Mn1—O2xi
85,01 (6)
O3—In1—O1iv
81,49 (6)
O2x—Mn1—O2xi
111,45 (8)
Distances Ca - O (Ǻ)
Distances Ba- O (Ǻ)
Ca1—O5
2,6005 (2)
Ba1—O4
2,308 (2)
Ca1—O5xii
2,6006 (2)
Ba1—O6xv
2,377 (2)
Ca1—O5iii
2,7446 (2)
Ba1—O6v
2,381 (2)
Ca1—O5vii
2,7446 (2)
Ba1—O4xiv
2,384 (2)
Ca1—O3
2,9577 (2)
Ba1—O6iv
2,480 (2)
Ca1—O3xii
2,958 (2)
Ba—Oiiv
2,7204
Ca1—O2
2,9623 (2)
Distance moyenne
2,4417 Å
Ca1—O2xii
2,9623 (2)
Distance moyenne
2,8163 Å
Codes de symétrie : (i) x+1/2, -y+1/2, z+1/2; (ii) x, -y+1, z+1/2; (iii) x, -y+1, z-1/2; (iv) -x+3/2, y-1/2, -z+3/2; (v) -x+3/2, -y+1/2, -
z+1; (vi) -x+3/2, y+1/2, -z+3/2; (vii) –x+1, -y+1, -z+1; (viii) x+1/2, y+1/2, z; (ix) -x+2, y, -z+3/2; (x) x+1/2, -y+3/2, z+1/2; (xi) -
x+3/2, -y+3/2, -z+1; (xii) -x+1, y, -z+1/2; (xiii) -x+1, -y+1, -z; (xiv) -x+1, -y, -z+1; (xv) x-1/2, -y+1/2, z-1/2; (xvi) x-1/2, y-1/2, z;
(xvii) x, -y, z-1/2; (xviii) x, -y, z+1/2. O2ii—In1—O3
78,58 (6)
O6ix—Mn1—O2x
85,01 (6)
O1iii—In1—O3
82,82 (6)
O6—Mn1—O2x
161,49 (7)
O5—In1—O1iv
171,49 (7)
O3vi—Mn1—O2xi
73,45 (6)
O4i—In1—O1iv
80,08 (6)
O3viii—Mn1—O2xi
90,83 (6)
O2ii—In1—O1iv
89,18 (6)
O6ix—Mn1—O2xi
161,49 (7)
O1iii—In1—O1iv
82,15 (6)
O6—Mn1—O2xi
85,01 (6)
O3—In1—O1iv
81,49 (6)
O2x—Mn1—O2xi
111,45 (8)
Distances Ca - O (Ǻ)
Distances Ba- O (Ǻ)
Ca1—O5
2,6005 (2)
Ba1—O4
2,308 (2)
Ca1—O5xii
2,6006 (2)
Ba1—O6xv
2,377 (2)
Ca1—O5iii
2,7446 (2)
Ba1—O6v
2,381 (2)
Ca1—O5vii
2,7446 (2)
Ba1—O4xiv
2,384 (2)
Ca1—O3
2,9577 (2)
Ba1—O6iv
2,480 (2)
Ca1—O3xii
2,958 (2)
Ba—Oiiv
2,7204
Ca1—O2
2,9623 (2)
Distance moyenne
2,4417 Å
Ca1—O2xii
2,9623 (2)
Distance moyenne
2,8163 Å
étrie : (i) x+1/2
y+1/2 z+1/2; (ii) x
y+1 z+1/2; (iii) x
y+1 z 1/2; (iv) x+3/2 y 1/2
z+3/2; (v) x+3/2 Codes de symétrie : (i) x+1/2, -y+1/2, z+1/2; (ii) x, -y+1, z+1/2; (iii) x, -y+1, z-1/2; (iv) -x+3/2, y-1/2, -z+3/2; (v) -x+3/2, -y+1/2, -
z+1; (vi) -x+3/2, y+1/2, -z+3/2; (vii) –x+1, -y+1, -z+1; (viii) x+1/2, y+1/2, z; (ix) -x+2, y, -z+3/2; (x) x+1/2, -y+3/2, z+1/2; (xi) -
x+3/2, -y+3/2, -z+1; (xii) -x+1, y, -z+1/2; (xiii) -x+1, -y+1, -z; (xiv) -x+1, -y, -z+1; (xv) x-1/2, -y+1/2, z-1/2; (xvi) x-1/2, y-1/2, z;
(xvii) x, -y, z-1/2; (xviii) x, -y, z+1/2. Remerciements : Les
auteurs
remercient
les
Unités
d'Appui
Techniques à la Recherche Scientifique (UATRS),
relevant du Centre National pour la recherche
Scientifique et Technique (CNRST) (Maroc) pour la
collecte des données de la diffraction de rayons X. Figure 1: Chaînes formées par les octaèdres MnO6 et InO6
parallèle à [101]. 5 MATEC Web of Conferences 149, 01085 (2018)
CMSS-2017 https://doi.org/10.1051/matecconf/201814901085 Références 1. L.-X. Yuan et al., Energy Environ. Sci., vol. 4, no. 2,
pp. 269–284, (2011).. 2. J. Kim, H. Kim, K. Park, Y. Park, and S. Lee, vol. 4,
pp. 8632–8636, (2014). 3. A. Assani, M. Saadi, M. Zriouil, . & L. El Ammari,
Acta Cryst. E67, i5 (2011a). 4. A. Ould Saleck, A. Assani, M. Saadi, C. Mercier, C. Follet, and L. El Ammari, Acta Crystallogr. Sect. E
Crystallogr. Commun., vol. 71, no. 7, pp. 813–815,
(2015). 5. A. Bouraima, A. Assani, M. Saadi, T. Makani, and 5. A. Bouraima, A. Assani, M. Saadi, T. Makani, and
L. El Ammari, Acta Crystallogr. Sect. E Crystallogr. Commun., vol. 71, no. Ii, pp. 558–560, (2015). L. El Ammari, Acta Crystallogr. Sect. E Crystallogr. Commun., vol. 71, no. Ii, pp. 558–560, (2015). 6. J. Khmiyas, A. Assani, M. Saadi, and L. El Ammari,
Acta Crystallogr. Sect. E Crystallogr. Commun., vol. 71, no. Iii, pp. 690–692, (2015). pp
(
)
7. P. B. Moore, Am. Mineral., vol. 56, p. 1955, (1971). 8. Bruker APEX2 and SAINT. Bruker AXS Inc.,
Madison, Wisconsin, USA. (2005). 9. G. M. Sheldrick, Acta Cryst.A71, 3–8 (2015a). 10. G. M. Sheldrick, Acta Cryst.C71, 3-8 (2015b). 11. B. Ayed, M. Krifa & A. Haddad Acta Cryst. C58,
i58, i98-100 (2002). 12. B. Ayed & A. Haddad , Acta Cryst E59, i77-i79
(2003). 13. C. Bouzidi, M. Faouzi Zid, A. Driss & W. Frigui. Acta Cryst. E70, i18–i19 (2014). 14. Debasmita Dwibedi, Rafael B. Araujo, Sudip
Chakraborty, Pradeep P. Shanbogh, alini G. Sundaram,
Rajeev Ahuja, & Prabeer Barpanda J. Mater. Chem. A,
(2015). 14. Debasmita Dwibedi, Rafael B. Araujo, Sudip
Chakraborty, Pradeep P. Shanbogh, alini G. Sundaram, Rajeev Ahuja, & Prabeer Barpanda J. Mater. Chem. A,
(2015). 6 6
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https://openalex.org/W2587765579
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https://research-information.bris.ac.uk/ws/files/108426425/srep42352.pdf
|
English
| null |
Dynamics of epidemic diseases on a growing adaptive network
|
Scientific reports
| 2,017
|
cc-by
| 13,682
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Demirel , G., Barter, E., & Gross, T. (2017). Dynamics of epidemic
diseases on a growing adaptive network. Scientific Reports, 7, Article
42352. https://doi.org/10.1038/srep42352 Demirel , G., Barter, E., & Gross, T. (2017). Dynamics of epidemic
diseases on a growing adaptive network. Scientific Reports, 7, Article
42352. https://doi.org/10.1038/srep42352 Demirel , G., Barter, E., & Gross, T. (2017). Dynamics of epidemic
diseases on a growing adaptive network. Scientific Reports, 7, Article
42352. https://doi.org/10.1038/srep42352 Publisher's PDF, also known as Version of record
License (if available):
CC BY
Link to published version (if available):
10.1038/srep42352 Dynamics of epidemic diseases on a
growing adaptive network Güven Demirel1, Edmund Barter2 & Thilo Gross2 The study of epidemics on static networks has revealed important effects on disease prevalence of
network topological features such as the variance of the degree distribution, i.e. the distribution of
the number of neighbors of nodes, and the maximum degree. Here, we analyze an adaptive network
where the degree distribution is not independent of epidemics but is shaped through disease-induced
dynamics and mortality in a complex interplay. We study the dynamics of a network that grows
according to a preferential attachment rule, while nodes are simultaneously removed from the network
due to disease-induced mortality. We investigate the prevalence of the disease using individual-based
simulations and a heterogeneous node approximation. Our results suggest that in this system in the
thermodynamic limit no epidemic thresholds exist, while the interplay between network growth and
epidemic spreading leads to exponential networks for any finite rate of infectiousness when the disease
persists. received: 12 October 2016
accepted: 08 January 2017
Published: 10 February 2017 received: 12 October 2016
accepted: 08 January 2017
Published: 10 February 2017 Throughout human history epidemic diseases have been a constant threat. The plague of Athens killed 25–35
percent of the city’s population as early as 430 BC1. A bubonic plague epidemic killed between 75000 and 100000
inhabitants of London between April 1665 and January 1666, when its population was about 4600001. Smallpox
became a major threat to Europe throughout the eighteenth century, where growing cities like London were espe-
cially vulnerable to the disease on account of continuously providing immigrants that are susceptible to the virus1. After a brief respite during the 20th century epidemics are on the rise again. For instance, around 655000 people
died of malaria in 20102 and more than 100000 confirmed cases of Zika infection have occurred in the Americas
in the past year3. Combating such epidemic diseases efficiently in the mega-cities of the future is likely to require
a heightened understanding of the dynamics of the diseases and the social networks on which they spread. In the past two decades, epidemic spreading has been extensively studied on different complex net-
works to understand the influence of the social contact network structure on the disease prevalence4–7. Two commonly studied models of infectious diseases are Susceptible-Infected-Susceptible (SIS) and
Susceptible-Infected-Removed (SIR) models. University of Bristol – Bristol Research Portal
General rights University of Bristol – Bristol Research Portal
General rights This document is made available in accordance with publisher policies. Please cite only the
published version using the reference above. Full terms of use are available:
http://www.bristol.ac.uk/red/research-policy/pure/user-guides/brp-terms/ www.nature.com/scientificreports www.nature.com/scientificreports 1Management Science & Entrepreneurship, Essex Business School, University of Essex, Southend-on-Sea, UK.
2Department of Engineering Mathematics, University of Bristol, Bristol, UK. Correspondence and requests for
materials should be addressed to E.B. (email: edmund.barter@bristol.ac.uk) Scientific Reports | 7:42352 | DOI: 10.1038/srep42352 www.nature.com/scientificreports/ A relatively recent addition is to consider also the feedback of the epidemic on the social network structure,
which can be indirect, e.g. by triggering behavioral changes of agents6,32, or direct by removing agents due to
hospitalization, quarantine or death. A relatively recent addition is to consider also the feedback of the epidemic on the social network structure,
which can be indirect, e.g. by triggering behavioral changes of agents6,32, or direct by removing agents due to
hospitalization, quarantine or death. p
q
Modeling the network response to an ongoing epidemic leads to an adaptive network, a system with interplay
between the dynamics of the network and the dynamics on the network takes place33,34. Epidemics on adaptive
networks can exhibit complex emergent dynamics32,35–38 (e.g. sustained oscillations, bistability, and hysteresis)
and emergent topological properties32,35,39,40 (e.g. heterogeneous degree distributions and assortative degree cor-
relations). Furthermore, the study of the social response to epidemics is interesting from an applied point of view
because it could enable enhanced vaccine control41 and effective quarantine strategies42. f
q
g
In comparison to social responses to epidemics32,35–39,43–48, network growth and direct topological feedback
via the removal of nodes have received less attention. Previous works considered the case where network growth
and death processes are balanced and thus the population stays in equilibrium and fluctuates around a fixed sys-
tem size49,50. The case of continuous growth was studied in Ref. 51, where new nodes attach preferentially to high
degree non-infected nodes. It was observed that a transition from a scale-free topology to an exponential one
takes place as the infectiousness decreases. Reference 52 study the effects of network growth and demographics
on the dynamics of an SIS disease simultaneously spreading on the network, showing that the epidemic threshold
vanishes in the thermodynamic limit. In another study, network growth and node removals have also been incor-
porated in a single model53. However, the authors focused on epidemic oscillations and did not consider topo-
logical effects in detail. Another related work focused on the interplay between network growth and dynamical
behavior in the context of evolutionary game theory54, where new players preferentially attach to those receiving
higher payoffs. g
p yf
Here, we consider the growth of a network by preferential attachment from which nodes are simultaneously
removed due to an SIR epidemic. www.nature.com/scientificreports/ The appeal of this model lies in its paradoxical nature, in absence of the disease,
preferential attachment leads to the formation of scale-free topologies in which the epidemic threshold vanishes,
such that the disease can invade. However, an established SIR disease will quickly infect and remove nodes of high
degree such that the variance of the degree distribution is decreased and epidemic thresholds reappear, potentially
leading to the extinction of the disease. g
Now, consider the following line of reasoning: Observing a scale-free topology implies that the epidemic is
extinct. But an extinct epidemic implies scale-free structure and hence vanishing epidemic threshold precluding
extinction. Logically, the only possible solution is that the epidemic persists (unconditionally) in a network that
is not scale-free. In other words, one would expect that the coevolution of epidemic state and network structure
should lead to a vanishing epidemic threshold in an exponential network.h g p
p
The argument presented above is admittedly hand-wavy. One objection against this line of reasoning that
comes to mind immediately is that the paradox can also be resolved temporally, such that the epidemic goes
extinct while the network is exponential. A disease-free scale-free network can then develop later since the dis-
ease cannot be reintroduced since no infected agents are left. However, this temporal resolution is only feasible in
finite networks. In the thermodynamic limit it can be easily shown that a finite number of infected survive even
below the epidemic threshold, which precludes complete extinction. p
p
p
In the remainder of this paper we present a detailed dynamical analysis of the epidemic model using a hetero-
geneous node approximation along with detailed numerical computations. We show that disease-induced mortal-
ity reduces the variance of the degree distribution to a finite value, but not sufficiently far to cause the extinction
of the epidemic. Thus, a balance is reached where the finite variance of the degree distribution is matched to the
infectiousness of the pathogen. Therefore, the epidemic threshold vanishes in the thermodynamic limit, con-
firming the hand-waving argumentation above. We also identify a parameter region, where, after all, a temporal
resolution of the paradox is observed. Scientific Reports | 7:42352 | DOI: 10.1038/srep42352 Dynamics of epidemic diseases on a
growing adaptive network For SIS diseases on complex networks, the crucial determinant of epidemic spreading is the average maximum
degree or the degree cutoff of the contact network where the hub node with the maximum degree and its neigh-
bors act as an active incessant source for the endemic state8,9. Therefore, the epidemic threshold vanishes in the
thermodynamic limit in networks where the maximum degree increases with the system size, which is true for
most random networks unless there is a natural restriction on degree, for instance, due to cognitive limitations7.f For SIR diseases on complex networks, differently from SIS diseases, the fate of the disease is determined
by the degree distribution, i.e. the probability distribution of finding an individual with a given number of con-
tacts8,10,11. If the variance of this distribution is finite, then there is generally a threshold of infectiousness, which
the disease has to exceed to reach a finite fraction of the population. By contrast, if the variance of the degree
distribution is infinite, such as in certain scale-free networks, then the epidemic threshold vanishes and the SIR
disease percolates on the network for all non-zero infectiousness levels via hierarchical spreading from hub nodes
to lower degree nodes12. Thus, scale-free networks allow unlikely diseases with low infectiousness to spread and
become endemic. Today, the structure of social networks is recognized as a key factor with direct implications for epidemic
dynamics and potential counter measures13–17. This insight has motivated the integration of real world network
data into epidemic models18–23. Furthermore, theoretical models have been extended by including several prop-
erties of real-world networks such as degree constraints24, degree correlations25, clustering26–28, information fil-
tering29, social hierarchy30, and nonuniform transmission probabilities31. 1Management Science & Entrepreneurship, Essex Business School, University of Essex, Southend-on-Sea, UK. 2Department of Engineering Mathematics, University of Bristol, Bristol, UK. Correspondence and requests for
materials should be addressed to E.B. (email: edmund.barter@bristol.ac.uk) Scientific Reports | 7:42352 | DOI: 10.1038/srep42352 1 www.nature.com/scientificreports/ www.nature.com/scientificreports/ The heterogeneous node approximation consists of a set of ordinary differential
equations for the densities [Ak], the abundance of nodes in the class Ak, which is the set of nodes of state A ∈ {S, I}
and degree k, normalized by the total number of nodes N. The total density of S-nodes is denoted as [S], [S] = ∑k[Sk]. The density [I] is defined analogously such that [S] + [I] = 1.ii hi
Before deriving the full equations for the specific system under investigation, we first illustrate the general
structure of the heterogeneous moment expansion ≡
+ ∆
−
∆
. ∆→
A
A
t
t
A
t
t
d
dt
[
]
lim [
(
)]
[
( )]
k
t
k
k
0 Here, two types of terms contribute to d[Ak]/dt: a) changes in the abundance of nodes in the class Ak, and b)
changes in the normalization factor N. ll
l
d
h
h
f
d
d
d
l
h Here, two types of terms contribute to d[Ak]/dt: a) changes in the abundance of nodes in the class Ak, and b)
changes in the normalization factor N. For illustration, let us consider the process that removes infected individuals at a per capita rate r. For the
moment we assume that these removals do not change N, as we treat the change in N separately below. When an
infected node of degree k is removed, the abundance of nodes of type Ik, N(I,k), decreases by 1 so that the density
[Ik] is reduced by 1/N. Considering that (Δt)rN(I,k)(t) such removal events take place within a time step Δt, we
obtain the rate of change for the density [Ik] due to process a): = −
= −
. I
r N
t
N t
r I
d
dt
[ ]
( )
( )
[ ]
k
I k
k
( , ) We now express the effect of the modification of the normalization factor N due to such removal events. When an infected node of arbitrary degree k′ is removed, the densities of all degree classes Ak are affected due
to the modified normalization factor. In total, (Δt)rNI removal events take place within a time step Δt resulting
in [Ak(t + Δt)] = [Ak(t)]N(t)/(N(t) − 1), when isolated from the other changes of type a). www.nature.com/scientificreports/ mechanism that is used here can also be considered as an approximate description for the hospitalization of
infected individuals, which effectively removes the links. Furthermore, diseases in which dead hosts continue
transmitting the disease can be captured in the same framework by a reduced removal rate r, taking into account
the finite time between the death and the actual removal of the body. mechanism that is used here can also be considered as an approximate description for the hospitalization of
infected individuals, which effectively removes the links. Furthermore, diseases in which dead hosts continue
transmitting the disease can be captured in the same framework by a reduced removal rate r, taking into account
the finite time between the death and the actual removal of the body. i
y
Unless mentioned otherwise, the following set of parameter values is used throughout the paper: m0 = 6,
m = 5, q = 0.01. In agent-based simulations the network is simulated until N reaches 107 or the time reaches 104. dynamics on and of complex network
called coarse-graining or moment-c
us node approximation, also called h
where the network evolution is captu Analytical treatment. The dynamics on and of complex networks can be captured by a set of coupled ordi-
nary differential equations, in so-called coarse-graining or moment-closure approximations7,39,57–64. In the next
section we develop a heterogeneous node approximation, also called heterogeneous mean-field or degree-based
mean-field approximation7,11,65,66, where the network evolution is captured in a set of equations for the node den-
sities in different degree-classes.f f
g
Since the heterogeneous node approximation is in the form of a high-dimensional system of ordinary differ-
ential equations, we follow two directions for reducing the dimensionality of analysis. First we apply the mathe-
matical triple jump approach67 to transform the infinite dimensional ordinary differential equation system in the
thermodynamic limit to a two-dimensional partial differential equation system. Second we develop an alternative
approach that reduces the heterogeneous node approximation to a low-dimensional ordinary differential equa-
tion system assuming random graph properties, which is later in the paper shown to be capable of estimating the
network dynamics to high accuracy when away from the epidemic threshold. Heterogeneous approximation. Scientific Reports | 7:42352 | DOI: 10.1038/srep42352 Methods
I
hi
i In this section, we first introduce the model and then develop our theoretical approach that describes the evolu-
tion of the network dynamics based on coarse-graining approximations. Model. We study the spreading of a susceptible-infected-removed (SIR) disease55 on an evolving network. In
this network, a given node is either susceptible (state S) or already infected with the disease (state I). We start with
a fully connected network of m0 nodes and consider three dynamical processes: a) the arrival of nodes, b) disease
transmission, and c) the removal of nodes.h In the following we measure all rates per capita, including the arrival rate. This implies that larger populations
have a proportionally larger influx of individuals, which appears plausible e.g. for growing cities, where the attrac-
tivity of the city increases with size. It is analogous to the use of per capita birth rates in models of population
dynamics. We note that this assumption is necessary to keep the model well-defined in the thermodynamic limit. d
h
l
d
l
d
f
d
h h d
h New nodes arrive in the population at a constant per capita rate q and are already infected with the disease with
probability w. Arriving nodes immediately establish links with m of the nodes selected according to the preferential
attachment rule56: A new node establishes a link with a particular node i of degree ki with a probability proportional
to ki/∑jkj. Therefore, an incoming node’s links will attach to a node of degree k with probability kpk/〈k〉,
where pk denotes the degree distribution (the probability that a randomly picked node has degree k) and 〈k〉 =
∑ kpk is the mean degree.h pk
g
Disease transmission occurs at rate p on every link connecting a susceptible and an infected node. Therefore,
nodes with higher degree are proportionally more likely to catch and spread the disease. Removal of infected nodes takes place at rate r. Because we describe a fatal disease from which recovery
is not possible, removed nodes and their links are entirely deleted and do not re-appear at a later stage. The
removal of links along with the nodes depicts the rapid removal of corpses in the human population. The removal Scientific Reports | 7:42352 | DOI: 10.1038/srep42352 2 www.nature.com/scientificreports/ Writing the complete set of changes caused by infection, node arrival, and node removal processes, we derive
he moment expansion for the densities [Sk] and [Ik] www.nature.com/scientificreports/ Therefore, the rate of
change of type b) due to removal events is =
−
−
=
−
. ∆→
∆→
A
rN
t
A
t
N t
N t
A
t
r I t
A
t
N t
N t
d
dt
[
]
lim
( ) [
( )]
( )
( )
1
[
( )]
lim
[ ( )][
( )]
( )
( )
1
k
t
I
k
k
t
k
0
0 Noting that every node is updated on average once in a unit time, i.e. Δt = 1/N, and taking the thermodynamic
limit, we obtain the renormalization rate =
. A
r I
A
d
dt
[
]
[ ][
]
k
k Writing the complete set of changes caused by infection, node arrival, and node removal processes, we derive
the moment expansion for the densities [Sk] and [Ik] Scientific Reports | 7:42352 | DOI: 10.1038/srep42352 3 www.nature.com/scientificreports/ ∑
∑
∑
∑
∑
∑
δ
δ
=
−
+
−
+
−
−
−
+
−
+
=
+
−
+
−
−
+
+
−
+
−
<
<
−
′
′
′
+
′
′
′
−
′
′
′
+
′
′
′
t
S
q
w
m
k
k S
k
S
S
p
S I
r
S
I
S I
I
S
I
q w
m
k
k I
k
I
I
p
S I
r
I
I
I I
I
I
I
k
k
d
d
[
]
((1
)
(
[
]
(
1)[
])
[
])
[
]
(
[
]
[
]
[ ][
]),
d
dt
[ ]
(
(
[ ]
(
1)[
])
[ ])
[
]
(
[
]
[
]
[ ][ ]
[ ]), 0
(1
k
k m
k
k
k
k
k k
k
k
k
k
k k
k
k
k m
k
k
k
k
k k
k
k
k
k
k k
k
k
,
1
1
,
1
1
M (1) here, the quantity [AkBk′] denotes the density of links between nodes of type Ak and Bk′, where A, B ∈ {S, I}, and k
and k′ are the respective degrees. www.nature.com/scientificreports/ For understanding the equation governing the evolution of the density [Sk], consider that new nodes with
degree m arrive at rate q and have state S with probability 1−w. Thus, the density [Sm] increases at rate q(1−w). A newly arriving node builds a link to a node in the Sk class with probability k[Sk]/〈k〉 and causes it to pass into
the Sk+1 class. Because m such links are established by each newly arriving node, the density [Sk] decreases by
qmk[Sk]/〈k〉. Similarly, nodes in the Sk−1 class pass into the Sk class at rate qm(k−1)[Sk−1]/〈k〉. Additionally, as
explained above, we need to renormalize the density [Sk] when a node arrives. This corresponds to a loss of the
[Sk] density of q[Sk]. y
q
At rate p, nodes within the Sk class become infected through their links with infected nodes of arbitrary degree k′,
causing them to pass into the Ik class. The total density of such links is ∑k′[SkIk′]. h
Finally, nodes within the Sk class pass into the Sk−1 class due to the removal of their infected neighbors of arbi-
trary degree k′. Given the density of such links, the density of [Sk] decreases by r∑k′[SkIk′]. Similarly, nodes in the
Sk+1 class pass into the Sk class corresponding to a gain of r∑k′[Sk+1Ik′]. As infected nodes are removed, the density
of all degree classes increases due to the renormalization leading to a gain of r[I][Sk] for the density [Sk]. The rate
equation for the density [Ik] is constructed analogously, with the addition of a term for the removal of infected
nodes with degree k at the rate r, r[Ik]. g
As the equations for node densities [Ak] depend on link densities [AkBk′], Eq. (1) is not closed. In order to close
the system, the moment expansion should be truncated by the moment-closure approximation, in which the den-
sities of larger subgraphs are estimated in terms of the densities of smaller ones. Here, we use the heterogeneous
node approximation to close the system at the node level ≈
′
. ′
′
A B
kk A
B
k
[
]
[
][
]
(2)
k
k
k
k (2) We have assumed that the nodes with the same degree can be considered identical and state and degree corre-
lations between neighboring nodes are negligible. www.nature.com/scientificreports/ The mixing assumption generally requires a mixing or anneal-
ing process that makes it possible to replace the adjacency matrix structure with the degree distribution8, which
is provided here by the constant removal and addition of nodes and links. p
y
Using the node approximation of Eq. (2), we reach δ
δ
=
−
+
−
+
−
−
−
+
+
−
+
=
+
−
+
−
−
+
+
+
−
+
−
<
<
−
+
−
+
t
S
q
w
m
k
k S
k
S
S
pz I k S
r z I
k
S
k S
I
S
I
q w
m
k
k I
k
I
I
pz I k S
r z I
k
I
k I
I
I
I
k
k
d
d
[
]
((1
)
(
[
]
(
1)[
])
[
])
[ ] [
]
(
[ ]((
1)[
]
[
])
[ ][
]),
d
dt
[ ]
(
(
[ ]
(
1)[
])
[ ])
[ ] [
]
(
[ ]((
1)[
]
[ ])
[ ][ ]
[ ]), 0
(3)
k
k m
k
k
k
I
k
I
k
k
k
k
k m
k
k
k
I
k
I
k
k
k
k
,
1
1
,
1
1
M (3) where zI = 〈kI〉/〈k〉 and 〈kI〉 is the mean degree of infected nodes. In the following we refer to Eq. (3) as the heterogeneous approximation. The main drawback of such hetero-
geneous approximations is the high dimensionality of the system of equations, which complicates the analytical
solution and thus typically necessitates extensive numerical studies except for the analysis of special conditions. Furthermore, since it is not possible to numerically integrate an infinite dimensional system of differential equa-
tions, we need to introduce a degree cut-off kM by assuming ∑
∑
=
+
∞
=
p
p
k
k
k
k
k
k
1
0
M
M
. The higher the degree
cut-off, kM, the more precise the heterogeneous approximation becomes. Mathematical triple jump approach. The mathematical triple jump approach of ref. 67 consists of three steps. First, a high-dimensional ordinary differential equation system is developed to capture the dynamics under the
types of heterogeneity that are identified to be of utmost importance. www.nature.com/scientificreports/ S
S
k
k
S
I
k
S
k S
d
dt
[ ]
d
dt
[
],
d
dt
d
dt
([
]
[ ]),
d
dt
[ ]
d
dt
[
]
(6)
k
k
k
k
k
S
k
k (6) Using Eqs (3) and (6), we obtain Using Eqs (3) and (6), we obtain sing Eqs (3) and (6), we obtain =
−
−
−
〉
〉
〈〉
+
〈〉=
−〈〉+
〉
−〈
+
〈
〉=
−
−〈
+
〉
〉
−
〈
〉
〈〉
〈
〉−〈
〉
−〈
〉〈
〉
〈〉
. S
q
w
S
p k
k
k
S I
r S I
k
q
m
k
r
k
S
k
S
k
q
w
m
k
S
m k
k
p k
I
k
k
k
r k
k
k
I
d
dt
[ ]
(1
[ ])
[ ][ ]
[ ][ ],
d
dt
(2
)
(2
[ ]
(1
[ ])),
d
dt
(1
)(
)
[ ]
[ ] (
)
[ ]
S
I
S
S
S
S
I
S
S
S
I
2
2 (7) In the following, we refer to Eq. (7) as the coarse-grained heterogeneous approximation, which does not
involve any further approximations other than those discussed above. 2 Because we have not derived an equation for the second moment of the susceptible degree distribution kS
2 ,
Eq. (7) does not constitute a closed dynamical system. We address this problem by replacing kS
2 by 〈kS〉2 + 〈kS〉
in an additional approximation. We note that this approximation is valid exactly when the network has a Poisson
degree distribution. It can therefore be thought of as a ‘random-graph approximation’. This approximation will
certainly fail in the case of scale-free networks because of the degree distribution’s diverging variance, i.e. →∞
kS
2
, in which case we will resort to the heterogeneous approximation and its PDE description in the ther-
modynamic limit for the analysis of the model. However, as will become apparent below, the system obtained by
the random-graph approximation still performs well for distributions with large finite variance. www.nature.com/scientificreports/ R t
I
S
Q
t
k
S
R
t
k
I
( , 1)
[ ]
1
[ ],
( , 1)
[ ],
( , 1)
[ ]
x
S
x
I Using these quantities, we obtain the partial differential equations γ
α
β
β
β
=
−
+
−
+
+
−
+
+
+
Q t x
q
w x
r
q Q t x
qmx
qm
p
r
x
r
Q
t x
( ,
)
(1
)
(
)
( ,
)
1
[
(
(
) )
]
( ,
)
t
m
x
2 γ
α
β
β
β
=
−
+
−
+
+
−
+
+
+
Q t x
q
w x
r
q Q t x
qmx
qm
p
r
x
r
Q
t x
( ,
)
(1
)
(
)
( ,
)
1
[
(
(
) )
]
( ,
)
(4)
t
m
x
2
γ
α
β
β
β
α
β
=
−
+
−
+
+
−
−
+
+
. R t x
qwx
q
r
R t x
x
r
qmx R
t x
p x Q
t x
( ,
)
[
(1
)] ( ,
)
1
(1
)(
)
( ,
)
( ,
)
(5)
t
m
x
x (4) γ
α
β
β
β
α
β
=
−
+
−
+
+
−
−
+
+
. R t x
qwx
q
r
R t x
x
r
qmx R
t x
p x Q
t x
( ,
)
[
(1
)] ( ,
)
1
(1
)(
)
( ,
)
( ,
)
(5
t
m
x
x (5) Homogeneous approximation. As a second alternative approach we develop a low-dimesional approximation
by summing over the degree classes in Eq. (3). We consider the susceptible proportion of the population [S], the
mean degree 〈k〉, and the mean degree of susceptibles 〈kS〉 which evolve according to Homogeneous approximation. As a second alternative approach we develop a low-dimesional approximation
by summing over the degree classes in Eq. (3). We consider the susceptible proportion of the population [S], the
mean degree 〈k〉, and the mean degree of susceptibles 〈kS〉 which evolve according to ∑
∑
∑
=
〈〉=
+
〈
〉
=
. www.nature.com/scientificreports/ Second, the obtained system of ordinary
differential equations is transformed to a low-dimensional partial differential equation system in the thermody-
namic limit using moment generating functions. Finally, the partial differential equation system is analyzed using
the tools of dynamical systems theory.hiih y
y
y
The first step has already been completed to find the heterogeneous approximation in Eq. (3). The second step
is done below in this section, while the last step is carried out in the next section.i p
We first introduce the quantities Q(t, x) = ∑k[Sk(t)]xk and R(t, x) = ∑k[Ik(t)]xk, which are the generating func-
tions of the degree distributions of susceptible and infected populations. The time derivatives of Q(t, x) and R(t,
x) are given by Scientific Reports | 7:42352 | DOI: 10.1038/srep42352 4 www.nature.com/scientificreports/ ∑
∑
≡
≡
Q t x
S t
t
x R t x
I
t
t
x
( ,
)
d[
( )]
d
( ,
)
d[
( )]
d
t
k
k
k
t
k
k
k The partial derivatives with respect to x are defined analogously by The partial derivatives with respect to x are defined analogously by ∑
∑
≡
≡
−
−
Q
t x
S t
kx
R
t x
I
t
kx
( ,
)
[
( )]
( ,
)
[
( )]
x
k
k
k
x
k
k
k
1
1 The functions Q(t, x) and R(t, x) are particularly useful because they are related to the moments as given
below: The functions Q(t, x) and R(t, x) are particularly useful because they are related to the moments as given
below: γ
α
β
≡
=
=
−
≡
=
≡
=
. Scientific Reports | 7:42352 | DOI: 10.1038/srep42352 Results y
g
g
In order to quantify the topological transition from the scale-free to the exponential degree distribution, we
plot the variance σ2 of the degree distribution as a function of infectiousness p and fraction of infected arrivals
w in Fig. 2. As either parameter increases, the disease prevalence in the steady-state, [I]*, increases and removal
occurs at a high rate. As a result the degree distribution becomes narrower and the degree variance decreases. It is
apparent that very high values of the variance are only found for low infectiousness p, whereas higher infectious-
ness quickly leads to narrow distributions. Above we computed the variance σ2 of the degree distribution of networks. One concern in any computation
of this kind is finite-size effects. In the heterogeneous approximation these effects appear directly in form of the
maximal degree that is considered in the approximation. In agent-based simulation a similar cut-off exists as the
maximal degree in a network of finite size is bounded by the number of nodes. Hence all moments of the degree
distribution, including the variance, must be finite regardless of the shape of the degree distribution. However, if
the finite networks are drawn from an ensemble that becomes scale-free in the thermodynamic limit the variance
σ2 is often found to increase logarithmically with the imposed cut-off 24.if t
g
y
pf
We now rule out that low values of the degree variance σ2, observed above, were due to finite-size effects
by considering the variance σ2 as a function of the degree cut-off kc (Fig. 2 inset). For the case of p = 0, where
we observed scale-free behavior, we find that the observed variance increases logarithmically as expected. Conversely, for the finite values of infectiousness p the observed σ2 is insensitive to a sufficiently large cut-off. In summary these results show that fatal diseases should relatively quickly destroy scale-free structure of social
networks at all but the smallest removal rate and/or infectiousness.f Let us now investigate the effect of the emergent network topology on the prevalence of the disease. Plots of
the disease prevalence as a function of the infectiousness p and the fraction of infected arrivals w are shown in
Figs 3 and 4. Figure 3 shows that the heterogeneous approximation (dashed lines) is in very good agreement with
the agent-based model for the whole range of parameters p and w. Results In the previous section, we first present the analysis of heterogeneous and homogeneous approximations and
confirm the results by comparison with individual-based simulations of the network. Later we present a detailed
analysis of the epidemic threshold. We finally discuss the emergence of dynamics that involve epidemic cycles. General properties of the network and disease prevalence. Before we launch into a detailed discus-
sion of the model, let us consider the limiting case of network evolution in the absence of the epidemic. In this
case the model is identical to the Barabási-Albert model of network growth56, which is known to lead to scale-free
topologies, where the degree distribution follows a power law pk ∝ k−γ with exponent γ = 3 and thus the degree
variance σ2 diverges in the disease-free state. Because the density of infected vanishes in the absence of the epi-
demic, it is also evident that the degree distribution must be scale-free independent of the parameters p and r. g
p
p
p
In the present model the emergence of scale-free topologies is thus expected in the limit where the disease
goes extinct or remains limited to a finite number of infected nodes N. When the epidemic is present, high
degree nodes are disproportionately likely to become infected and subsequently removed, which can be expected
to prevent the formation of scale-free topologies.i p
p
g
We confirm this intuition by plotting degree distributions for various parameter sets in Fig. 1. We show a
comparison of the heterogeneous approximation with individual-based simulations. The figure shows a good
agreement between the modeling approaches and confirms basic intuition. When all arriving nodes are sus-
ceptible (w = 0), a scale-free degree distribution with the expected exponent γ = 3 is formed for p = 0. At finite
infectiousness p, the topology changes from scale-free to exponential. The same behavior is observed at higher
rates of infected arrivals, 0 < w < 1. When all arriving nodes are already infected (w = 1), the distribution has a bimodal form for p = 0 with high
degree contribution coming from the initial susceptibles which never get infected. At positive infectiousness p,
these individuals eventually die and the mode at high degrees disappears. www.nature.com/scientificreports/ U i
th
d
h
i
ti
bt i =
−
−
−
〈
〉〈
〉
〈〉
+
〈〉=
−〈〉+
〈
−〈〉
+
〈
〉=
−
−〈
〉
+
〈
〉
〈〉
−
〈
〉〈
〉
〈〉
−
〈
〉〈
〉
〈〉
S
q
w
S
p k
k
k
S I
r S I
k
q
m
k
r
k
S
k
S
k
q
w
m
k
S
m k
k
p k
k
I
k
r k
k
k
I
d
dt
[ ]
(1
[ ])
[ ][ ]
[ ][ ],
d
dt
(2
)
(2
[ ]
(1
[ ])),
d
dt
(1
)(
)
[ ]
[ ]
[ ],
(8)
S
I
S
S
S
S
S
I
S
I 〈〉
〈〉
〈
〈〉
〈
〉=
−
−〈
〉
+
〈
〉
〈〉
−
〈
〉〈
〉
〈〉
−
〈
〉〈
〉
〈〉
q
k
q
w
m
k
S
m k
k
p k
k
I
k
r k
k
k
I
dt
d
dt
(1
)(
)
[ ]
[ ]
[ ],
(8)
S
S
S
S
S
I
S
I (8) Scientific Reports | 7:42352 | DOI: 10.1038/srep42352 5 www.nature.com/scientificreports/ where [I] and 〈kI〉 are given by the equations [S] + [I] = 1 and 〈kS〉[S] + 〈kI〉[I] = 〈k〉, such that the system consti-
tutes a closed model. In the following we refer to this model as the homogeneous approximation. where [I] and 〈kI〉 are given by the equations [S] + [I] = 1 and 〈kS〉[S] + 〈kI〉[I] = 〈k〉, such that the system consti-
tutes a closed model. In the following we refer to this model as the homogeneous approximation. Results The precision of the homogeneous approxima-
tion is also high for a large range of parameter values. As illustrated in Fig. 4, the absolute error in estimation of
the disease prevalence of the approximation is maximal for intermediate values of infectiousness p, but still less
than 0.05. The only qualitative discrepancy between the approximation and the agent-based model emerges at
low infectiousness p for zero infected arrivals (w = 0). Here, the homogeneous model predicts the existence of an
epidemic threshold, whereas in the agent-based simulation and the heterogeneous approximation the disease is
found to persist even for very low levels of infectiousness p. p
y
p
Summarizing the results shown so far, we can say that ongoing epidemic dynamics quickly leads to the for-
mation of networks with finite variance. Generally,one would expect that such networks should exhibit a finite
epidemic threshold. Nevertheless, the heterogeneous approximation and simulations indicate that the epidemic
can persist in these networks for any finite positive value of the infectiousness. Analysis of the epidemic threshold. Here, we investigate in greater detail the apparent absence of the
threshold, which is implied by the general analysis above. Throughout the argument we will only consider the case
where all arriving agents are susceptible, w = 0.hi The epidemic threshold is commonly defined as the minimal value of the infectiousness, below which a ran-
domly picked node is susceptible with probability 1. In any finite network this implies that below the epidemic
threshold each individual node is susceptible. By contrast in the thermodynamic limit there can still be a finite
number of infected nodes as long as the density of such nodes in the network is zero, i.e. [I] = 0. In the following Scientific Reports | 7:42352 | DOI: 10.1038/srep42352 6 www.nature.com/scientificreports/ d
h
b l
h
id
i
h
h ld
h di
f
b
i
h
h
b
ill
10
−8
10
−4
10
0
Pk
10
−8
10
−4
10
0
Pk
10
0
10
1
10
2
10
3
10
−8
10
−4
10
0
k
Pk
10
−8
10
−4
10
0
p=0
p=0.0004
p=0.001
p=0.01
p=0.1
p=1.0
10
−8
10
−4
10
0
10
0
10
1
10
2
10
3
10
−8
10
−4
10
0
k
w=0.50
w=1.00
Pk ~ k−3
Pk ~ k−3
Pk ~ k−3
w=0.00
Pk ~ k−3
Pk ~ k−3
Pk ~ k−3
Figure 1. Results Degree distribution for varying infectiousness p and infected arrival fraction w. Left: individual-
based simulations. Right: heterogeneous approximation. Scale-free degree distributions are observed when
disease infectiousness vanishes (p = 0). For increasing infectiousness the degree distribution quickly becomes
exponential. Parameters: r = q = 0.01, m0 = 6, m = 5. Shown are averages over 1000 simulation runs. 10
−8
10
−4
10
0
Pk ~ k−3 10
−8
10
−4
10
0
Pk ~ k−3 0
0 10
0 10
0
10
1
10
2
10
3
10
−8
10
−4
10
0
k
Pk
p=0
p=0.0004
p=0.001
p=0.01
p=0.1
p=1.0
10
0
10
1
10
2
10
3
10
−8
10
−4
10
0
k
w=1.00
Pk ~ k−3
Pk ~ k−3
Figure 1. Degree distribution for varying infectiousness p and infected arrival fraction w. Left: individual-
based simulations. Right: heterogeneous approximation. Scale-free degree distributions are observed when
disease infectiousness vanishes (p = 0). For increasing infectiousness the degree distribution quickly becomes Figure 1. Degree distribution for varying infectiousness p and infected arrival fraction w. Left: individual-
based simulations. Right: heterogeneous approximation. Scale-free degree distributions are observed when
disease infectiousness vanishes (p = 0). For increasing infectiousness the degree distribution quickly becomes
exponential. Parameters: r = q = 0.01, m0 = 6, m = 5. Shown are averages over 1000 simulation runs. we denote the state below the epidemic threshold as the disease-free state, but recognize that there may be still a
nite number of infected individuals. we denote the state below the epidemic threshold as the disease-free state, but recognize that there may be still a
finite number of infected individuals. We now calculate the epidemic threshold from Eq. (3) by looking at the stability of the disease-free state repre-
sented by [I] = 0 and hence [Ik] = 0 for all k. The disease-free state is stable if all eigenvalues of the corresponding
Jacobian matrix have negative real parts. The epidemic threshold is characterized by a bifurcation point where the
leading eigenvalue of the Jacobian is zero. The disease-free state becomes unstable while a stationary state with
di
l
b
t bl b
d th
id
i th
h ld We now calculate the epidemic threshold from Eq. (3) by looking at the stability of the disease-free state repre-
sented by [I] = 0 and hence [Ik] = 0 for all k. Results The disease-free state is stable if all eigenvalues of the corresponding
Jacobian matrix have negative real parts. The epidemic threshold is characterized by a bifurcation point where the
leading eigenvalue of the Jacobian is zero. The disease-free state becomes unstable while a stationary state with
non-zero disease prevalence becomes stable beyond the epidemic threshold. We now calculate the epidemic threshold from Eq. (3) by looking at the stability of the disease free state repre
sented by [I] = 0 and hence [Ik] = 0 for all k. The disease-free state is stable if all eigenvalues of the corresponding
Jacobian matrix have negative real parts. The epidemic threshold is characterized by a bifurcation point where the
leading eigenvalue of the Jacobian is zero. The disease-free state becomes unstable while a stationary state with
non-zero disease prevalence becomes stable beyond the epidemic threshold. Scientific Reports | 7:42352 | DOI: 10.1038/srep42352 7 www.nature.com/scientificreports/ 0
0.0025
0.005
0.0075
0.01
p
0
0.25
0.5
0.75
1
w
10
50
100
200
300
102
103
104
kc
0
200
400
2
Figure 2. Variance σ2 of the degree distribution. Degree variance σ2 is plotted as a function of the
infectiousness p and the infected arrival fraction w from agent-based simulations. The degree variance σ2
decreases with increasing infectiousness p and fraction of infected arrivals w. Inset: Degree variance σ2 as a
function of the degree cut-off kc. A transition from a cut-off dependent (p = 0, scale-free) to an independent
(p = 0.0004, p = 0.001, and p = 0.005, exponential) regime is observed as the infectiousness p is increased. In network simulations (circles), nodes were restricted to at most kc neighbors. In the heterogeneous
approximation (solid lines) the cut-off kc is directly imposed as kM. Parameters: r = q = 0.01, m0 = 6, m = 5,
kc = 5 × 103. 0
0.0025
0.005
0.0075
0.01
p
0
0.25
0.5
0.75
1
w
10
50
100
200
300
102
103
104
kc
0
200
400
2 Figure 2. Variance σ2 of the degree distribution. Degree variance σ2 is plotted as a function of the
infectiousness p and the infected arrival fraction w from agent-based simulations. The degree variance σ2
decreases with increasing infectiousness p and fraction of infected arrivals w. Inset: Degree variance σ2 as a
function of the degree cut-off kc. Results A transition from a cut-off dependent (p = 0, scale-free) to an independent
(p = 0.0004, p = 0.001, and p = 0.005, exponential) regime is observed as the infectiousness p is increased. In network simulations (circles), nodes were restricted to at most kc neighbors. In the heterogeneous
approximation (solid lines) the cut-off kc is directly imposed as kM. Parameters: r = q = 0.01, m0 = 6, m = 5,
kc = 5 × 103. Figure 2. Variance σ2 of the degree distribution. Degree variance σ2 is plotted as a function of the
infectiousness p and the infected arrival fraction w from agent-based simulations. The degree variance σ2
decreases with increasing infectiousness p and fraction of infected arrivals w. Inset: Degree variance σ2 as a
function of the degree cut-off kc. A transition from a cut-off dependent (p = 0, scale-free) to an independent
(p = 0.0004, p = 0.001, and p = 0.005, exponential) regime is observed as the infectiousness p is increased. In network simulations (circles), nodes were restricted to at most kc neighbors. In the heterogeneous
approximation (solid lines) the cut-off kc is directly imposed as kM. Parameters: r = q = 0.01, m0 = 6, m = 5,
kc = 5 × 103. 10
−4
10
−3
10
−2
10
−1
10
0
0
0.25
0.5
0.75
1
p
[I]
w = 0.00
w = 0.25
w = 0.50
w = 0.75
w =1.00
0
0.25
0.5
0.75
1
0
0.25
0.5
0.75
1
w
[I]
p=0
p=0.001
p=0.01
p=0.1
Figure 3. Dependence of the disease prevalence [I]* on infectiousness p (top) and fraction of infected
arrivals w (bottom). The disease prevalence increases monotonically with infectiousness p and fraction of
infected arrivals w. In agent-based simulations (circles), [I]* is calculated over the surviving runs among 103
total realizations. Homogeneous approximation (solid lines) is the analytical solution of Eq. (8). Heterogeneous
approximation (dashed lines) is the stationary value of the numerical integration of Eq. (3). Parameters:
r = q = 0.01, m0 = 6, m = 5. 10
−4
10
−3
10
−2
10
−1
10
0
0
0.25
0.5
0.75
1
p
[I]
w = 0.00
w = 0.25
w = 0.50
w = 0.75
w =1.00 0
0.25
0.5
0.75
1
0
0.25
0.5
0.75
1
w
[I]
p=0
p=0.001
p=0.01
p=0.1 Figure 3. Results Dependence of the disease prevalence [I]* on infectiousness p (top) and fraction of infected
arrivals w (bottom). The disease prevalence increases monotonically with infectiousness p and fraction of
infected arrivals w. In agent-based simulations (circles), [I]* is calculated over the surviving runs among 103
total realizations. Homogeneous approximation (solid lines) is the analytical solution of Eq. (8). Heterogeneous
approximation (dashed lines) is the stationary value of the numerical integration of Eq. (3). Parameters:
r = q = 0.01, m0 = 6, m = 5. Scientific Reports | 7:42352 | DOI: 10.1038/srep42352 8 www.nature.com/scientificreports/ www.nature.com/scientificreports/ p
w
0
0.005
0.01
0
0.25
0.5
0.75
1
p
w
0
0.0025
0.005
0.0075
0.01
0
0.25
0.5
0.75
1
0
0.01
0.02
0.03
0.04
0.05
0.06
p
0
0.005
0.01
0
0.2
0.4
0.6
0.8
1
individual−based
simulation
homogeneous
approximation
difference
Figure 4. Performance of the homogeneous approximation in estimating the disease prevalence [I]* in
the (p, w) parameter space. Top-left: individual-based simulations. Top-right: homogeneous approximation. Bottom: absolute difference between individual-based simulations and the homogeneous approximation. Homogeneous approximation performs well for a large range of infectiousness p and infected arrival fractions w. Parameters: r = q = 0.01, m0 = 6,m = 5. p
w
0
0.005
0.01
0
0.25
0.5
0.75
1
p
0
0.005
0.01
0
0.2
0.4
0.6
0.8
1
individual−based
simulation
homogeneous
approximation p
p
w
0
0.0025
0.005
0.0075
0.01
0
0.25
0.5
0.75
1
0
0.01
0.02
0.03
0.04
0.05
0.06
p
difference p Figure 4. Performance of the homogeneous approximation in estimating the disease prevalence [I]* in
the (p, w) parameter space. Top-left: individual-based simulations. Top-right: homogeneous approximation. Bottom: absolute difference between individual-based simulations and the homogeneous approximation. Homogeneous approximation performs well for a large range of infectiousness p and infected arrival fractions w. Parameters: r = q = 0.01, m0 = 6,m = 5. The Jacobian matrix is obtained by the linearization of Eq. (3) around the steady state. We will shortly refer
to the Jacobian matrix evaluated at the disease-free steady state as the Jacobian and denote it J = [Jij], where i, j =
0, 1, 2, …, kM. Results The Jacobian has a block matrix structure Θ
Θ
=
J
0
C
(9)
S
I (9) where ΘS ≡ ∂SdS/dt, ΘI ≡ ∂IdI/dt, and C ≡ ∂IdS/dt, all evaluated at the disease-free state and with the definitions
≡
…
(
)
S
S
S
S
S
[
], [ ], [
],
, [
]
k
0
1
2
M
and ≡
…
I
I
I
I
I
([ ], [ ], [ ],
, [
])
k
0
1
2
M .h where ΘS ≡ ∂SdS/dt, ΘI ≡ ∂IdI/dt, and C ≡ ∂IdS/dt, all evaluated at the disease-free state and with the definitions
≡
…
(
)
S
S
S
S
S
[
], [ ], [
],
, [
]
k
0
1
2
M
and ≡
…
I
I
I
I
I
([ ], [ ], [ ],
, [
])
k
0
1
2
M .h (
)
M
M
The epidemic threshold is characterized by zero eigenvalue of the Jacobian, which is given by the solution of
=
detJ 0. From Eq. (9), the determinant of the Jacobian can be obtained from the equality of
Θ
Θ
=
detJ
det
det
S
I. Taking the partial derivatives of Eq. Results (3), we obtain Scientific Reports | 7:42352 | DOI: 10.1038/srep42352 9 www.nature.com/scientificreports/
Θ =
+
u v
A ,
(10)
S
S
S
S
Θ =
+
u v
A ,
S
S
S
S
Θ =
+
u v
A ,
S
S
S
S (10) where uS = [uS(k)], vS = [vS(k)], and AS = [AS(j, k)], for j, k = 1, 2, …, kM + 1, with matrix entries where uS = [uS(k)], vS = [vS(k)], and AS = [AS(j, k)], for j, k = 1, 2, …, kM + 1, with matrix entries =
=
−
−
−
≤
≤
−
−
=
+
=
−
−
−
−
u
k
k
q
m
k
S
k
S
k
k
q
m
k
S
k
k
v
k
k
( )
0,
1
4
((
1)[
]
(
2)[
]),
2
4
(
2)[
,
1
( )
1
S
k
k
k
S
1
2
M
2
M =
+
k
k
1
M =
−
+
=
−
=
+
A
j k
q j
j
k
q j
j
k
( ,
)
(
1)
2
,
(
1)
2
,
1
0,
otherwise
S otherwise From the matrix determinant lemma,
Θ =
+
−
det
(1
v A
u )detA
S
S
S
1
S
S
. From the matrix determinant lemma,
Θ =
+
−
det
(1
v A
u )detA
S
S
S
1
S
S
. From the matrix determinant lemma,
Θ =
+
−
det
(1
v A
u )detA
S
S
S
1
S
S
. From the matrix determinant lemma,
Θ =
+
−
det
(1
v A
u )detA
S
S
S
1
S
S
. From the matrix determinant lemma,
Θ =
+
−
det
(1
v A
u )detA
S
S
S
1
S
S
. Scientific Reports | 7:42352 | DOI: 10.1038/srep42352 Results +
=
−
(1
v A u )
0,
(11)
S
S
S
1 (11) which can be computed exactly given that, for
≡
−
Q
A
S
q
2
S
1 and QS = [QS(j, k)], the entries of QS are given by the
recursion equation which can be computed exactly given that, for
≡
−
Q
A
S
q
2
S
1 and QS = [QS(j, k)], the entries of QS are given by the
recursion equation =
>
−
+
=
<
+
−
=
=
+
−
+
+
<
Q
j k
k
j
k
k
j
j
k
k
j
k
k
k
Q
j k
k
j
( ,
)
0,
1
1
,
and
1
1
2
,
1
1
1
( ,
1),
(12)
S
S
M
M
−
+
+
<
k
k
Q
j k
k
j
1
1
( ,
1),
(12)
S (12) We note that the matrix QS provides also the densities [Sk] in the disease-free steady state for finite degree
cut-off kM. The vector of steady state susceptible densities, S, can be solved from We note that the matrix QS provides also the densities [Sk] in the disease-free steady state for finite degree
cut-off kM. The vector of steady state susceptible densities, S, can be solved from = −
S
2Q b,
(13)
S = −
S
2Q b,
S (13) where =
=
{
b
1,
i
m
0,
otherwise
(14)
i (14) We have now established an explicit solution for
Θ =
det
0
S
for any kM, which is obtained by inserting Eqs (12)
and (13) into Eq. (11). We have now established an explicit solution for
Θ =
det
0
S
for any kM, which is obtained by inserting Eqs (12)
and (13) into Eq. (11). We now follow the same procedure to solve
Θ =
det
0
I
, which constitutes the other possible solution of
d J 0 Thi
l
i
i
i
b q
We now follow the same procedure to solve
Θ =
det
0
I
, which constitutes the other possible solution o
=
etJ 0. Results This solution is given by +
=
−
(1
v A u )
0,
(15)
I
I
1
I
+
=
−
(1
v A u )
0,
I
I
1
I
(15) where =
−
−
u k
p
m
k
S
( )
2
(
1)
I
k
1
=
−
v k
k
( )
1
I
=
−−
+
=
−
=
+
A j k
r
q j
j
k
q j
j
k
( ,
)
(
1)
2
,
(
1)
2
,
1
0,
otherwise
I The matrix inverse
≡
−
Q
A
I
I
1 is solved from the recursive equation The matrix inverse
≡
−
Q
A
I
I
1 is solved from the recursive equation Scientific Reports | 7:42352 | DOI: 10.1038/srep42352 10 www.nature.com/scientificreports/ Figure 5. The epidemic threshold P* calculated from Eq. (15) as a function of the degree cut-off kM. The
epidemic threshold vanishes in the thermodynamic limit. Parameters: r = q = 0.01, w = 0, m0 = 6, m = 5. Figure 5. The epidemic threshold P* calculated from Eq. (15) as a function of the degree cut-off kM. The
epidemic threshold vanishes in the thermodynamic limit. Parameters: r = q = 0.01, w = 0, m0 = 6, m = 5. =
>
−
+
+
=
−
+
+
+
<
Q j k
k
j
r
q k
k
j
q k
r
q k
Q j k
k
j
( ,
)
0,
1
(
1)/2
,
(
1)
2
(
1)
( ,
1),
(16)
I
I (16) The epidemic threshold needs to satisfy one of Eqs (11) or (15) for any finite degree cut-off kM. Our investiga-
tions show that Eq. (11) is ruled out and the epidemic threshold is given by Eq. (15). Figure 5 shows the epidemic
threshold calculated from Eq. (15) for increasing values of degree cut-off kM, which approaches zero in the ther-
modynamic limit. This result is contraintuitive, as the epidemic threshold vanishes even in a system where the
degree variance is finite.i gi
For verification, we now take a complementary approach and calculate the epidemic threshold from Eqs (4)
and (5). Scientific Reports | 7:42352 | DOI: 10.1038/srep42352 Results Parameters: r = q = 0.01, w = 0,
m0 = 6, m = 5. 101
102
103
104
M0
10-2
10-1
100
fraction of surviving runs
p=0.0003
p=0.0004
p=0.0005
p=0.0010 Figure 6. The fraction of surviving runs for low infectiousness p as a function of the initial network size M0 . The fraction of surviving runs increases as the initial network size M0 increases. In order to ensure the same
initial average degree m, the Barabási-Albert growth model with m = 5 and m0 = 6 is iterated until the network
reaches size M0, then the full model simulation with disease dynamics starts. Then nodes are assigned states and
the infection, removal, and network growth processes take place simultaneously. Parameters: r = q = 0.01, w = 0,
m0 = 6, m = 5. We note that for large networks the probability of a node being infected scales as kpk. Therefore in the case of
the scale-free degree distribution, pk ~ k−3, which is bound to form at the threshold, if one existed, the mean degree
of infected nodes, 〈kI〉, is undefined. Hence in the limit of large networks pc → 0 and the threshold vanishes. We note that for large networks the probability of a node being infected scales as kpk. Therefore in the case of
the scale-free degree distribution, pk ~ k−3, which is bound to form at the threshold, if one existed, the mean degree
of infected nodes, 〈kI〉, is undefined. Hence in the limit of large networks pc → 0 and the threshold vanishes. Further, in the thermodynamic limit the network cannot reach a disease-free steady state. In the thermody-
namic limit, when γ = 0 a finite number of nodes will be infected. In this situation preferential attachment leads
to a degree distribution with infinite variance, as node removal is so small. At the moment the variance of the
degree distribution becomes infinite, we anticipate 〈kI〉 to also become infinite and the limit pc to go to zero. With
the epidemic limit at zero the disease will be abel to spread in the network.i p
p
We note that in finite networks the temporal dynamics can lead to a disease-free scale-free state. In this case
the network goes through an initial exponential phase where an epidemic threshold exists that leads to the extinc-
tion of the epidemic. Results Setting time derivatives to 0 and x = 1 in both Eqs (4) or (5), we obtain γ
γ
βα
α
β
−
+
+
+
=
. r
q
r
p
(
)
0
2 γ
γ
βα
α
β
−
+
+
+
=
. r
q
r
p
(
)
0
2 for the case w = 0. Using the definitions in Eq. (4) we find for the case w = 0. Using the definitions in Eq. (4) we find γ
γ
−
+
−
+
=
. r
p k
k
k
p k
k
k
q
r
(
)
0
I
S
I
S
2 As expected there are two solutions for the steady state fraction of nodes infected, γ. Factoring out the unin-
fected state γ = 0 gives the non-trivial root γ =
+
−
−
. q
r
p
r
p
(17)
k
k
k
k
k
k
I
S
I
S (17) For this root to give a value of 0 ≤ γ ≤ 1 requires
+
<
q
r
p
(
)
c
k
k
k
I
S . k
k One can now ask if
+
=
q
r
p
(
)
c
k
k
k
I
S constitutes a finite threshold below which the endemic state becomes
unphysical. At such a threshold the number of infected nodes would become zero, leading to a double root at
γ = 0. At this point 〈k〉 ≈ 〈ks〉 and therefore ≈
+
. p
q
r
k
(18)
c
I (18) Scientific Reports | 7:42352 | DOI: 10.1038/srep42352 11 www.nature.com/scientificreports/ 101
102
103
104
M0
10-2
10-1
100
fraction of surviving runs
p=0.0003
p=0.0004
p=0.0005
p=0.0010
Figure 6. The fraction of surviving runs for low infectiousness p as a function of the initial network size M0 . The fraction of surviving runs increases as the initial network size M0 increases. In order to ensure the same
initial average degree m, the Barabási-Albert growth model with m = 5 and m0 = 6 is iterated until the network
reaches size M0, then the full model simulation with disease dynamics starts. Then nodes are assigned states and
the infection, removal, and network growth processes take place simultaneously. Results Subsequently, a scale-free topology develops, in which the epidemic threshold vanishes. However the epidemic cannot reappear as no infected are left which could reignite the epidemic. We emphasize
that this mechanism is a finite-size effect. The finite-size extinction of the epidemic is demonstrated explicitly in
Fig. 6, which shows that the fraction of individual-based simulation runs in which the epidemic persists increases
with the initial network size M0. For populations that start with more than a few individuals the effect can be
neglected and epidemics persist indefinitely. Further dynamics. Precluding the finite-size effect described above, simulation runs in the parameter range
considered so far approach a finite prevalence for any positive value of infectiousness. The paradox outlined above
is thus resolved by alternative a) mentioned in the introduction, that is the model does not have an epidemic
threshold although the second moment of the degree distribution remains finite. As a final step in our exploration
present some evidence that the temporal solution of the paradox, alternative b), is also possible.fi Up to now we have considered relatively small removal rates r. At sufficiently high removal rate, the evolution
of the disease and the topology exhibits dynamics different from the convergence to a stationary state mainly
due to the dominance of finite size effects. Figure 7 shows a representative evolution at non-zero fraction of
infected arrivals w. At the combination of high infectiousness p and high removal rate r, the dynamics resembles
a homoclinic trajectory in the 〈k〉−[I] plane. The disease spreads quickly over the network and covers the whole
population immediately. Then disease-induced removals dominate and the population becomes extinct until
new healthy individuals arrive and the disease spreading restarts, which leads to cycles of population growth and
collapse. p
When all arrivals are susceptible, i.e. w = 0, the epidemics in the finite population disappears entirely and the
collapse-and-growth cycle cannot be completed. A disease-free scale-free network then emerges. Based on the
arguments above one can suspect that the same behavior cannot occur in the thermodynamic limit. Instead it is
likely that the observed dynamics forms part of a homoclinic cycle, where long phases of very low disease preva-
lence are disrupted by sharp outbreaks.h y
The observed dynamics at high removal rates would correspond to diseases with very high mortality where
infected individuals die almost immediately. Scientific Reports | 7:42352 | DOI: 10.1038/srep42352 Results This is reminiscent of the massive pandemics in history, where
humanity was exposed to new pathogens with very high virulence. While such diseases could lead to the deaths
of large fractions of populations, continuous supply of healthy individuals through immigration provided new
hosts to the disease and introduced new bursts of disease spreading which caused repeated epidemic cycles. Scientific Reports | 7:42352 | DOI: 10.1038/srep42352 12 www.nature.com/scientificreports/ 0
10
<k>
p=0.05 0
10
<k>
0
1
0
10
[I]
<k>
0
1
[I]
p=1000
p=0.05
p=0.40
p=1
Figure 7. The dynamical evolution in the [I]−〈k〉 plane for high removal rate r. At high infectiousness p,
the observed behavior resembles a homoclinic trajectory. The network grows from a healthy initial state until
an infected individual eventually arrives and the disease quickly spreads over the network and the infected
individuals subsequently die. Homogeneous approximation (red circles) fails to capture this behavior and
predicts a stable equilibrium. Parameters: q = 0.01, r = 0.10, m0 = 6, m = 5, w = 0.1. p=0.40 0
1
0
10
[I]
<k>
0
p=1 0
1
0
10
[I]
<k>
0
1
[I]
p=1000
p=1h 1 1 0 [I] [I] Figure 7. The dynamical evolution in the [I]−〈k〉 plane for high removal rate r. At high infectiousness p,
the observed behavior resembles a homoclinic trajectory. The network grows from a healthy initial state until
an infected individual eventually arrives and the disease quickly spreads over the network and the infected
individuals subsequently die. Homogeneous approximation (red circles) fails to capture this behavior and
predicts a stable equilibrium. Parameters: q = 0.01, r = 0.10, m0 = 6, m = 5, w = 0.1. Discussion
h Discussion
In the present paper we have investigated the dynamics of a fatal SIR disease in a growing population. Our main
finding is that no epidemic threshold exists in this model. Although the variance of the degree distribution
remains finite in the evolved topologies topologies “unlikely” diseases with very low infectiousness can persist
indefinitely. In the present paper we have investigated the dynamics of a fatal SIR disease in a growing population. Our main
finding is that no epidemic threshold exists in this model. Although the variance of the degree distribution
remains finite in the evolved topologies topologies “unlikely” diseases with very low infectiousness can persist
indefinitely. i
y
We presented a detailed analytical exploration for the case of low removal rate, where the prevalence of the
disease reaches a stationary level. In the growing population the ongoing epidemic dynamics eliminates the nodes
of high degree and thus leads to the formation of topologies for which the variance of the degree distribution is
finite. However, this mechanism only reduces the width of the degree distribution so far that the epidemic can
still persist. Therefore, the disease itself can not reduce the degree variance so much to cause its own extinction. For any finite value of the infectiousness the network adapts its topology such that the variance of the degree
distribution is lowered to point where the epidemic can still be sustained, which explains the observed absence
of the epidemic threshold.fi p
When the removal rate is sufficiently high, network simulations indicate that the disease can no longer stabi-
lize at a stationary level and instead growth-and-collapse cycles are observed. Such dynamics are reminiscent of
homoclinic orbits, which have for instance been observed in an adaptive-network model of social cooperation68.h homoclinic orbits, which have for instance been observed in an adaptive-network model of social cooperation68. The dynamical feedback between the population structure and the epidemic disease has been so far studied
in a number of articles in the past couple of years32,35–39,43,44,46,47. However, models captured mostly social interac-
tions in non-fatal diseases. A significant obstacle to progress in this line of work if that the network evolution in
most models is driven by behavioral changes of individuals (whom to meet, how often to wash hands). Scientific Reports | 7:42352 | DOI: 10.1038/srep42352 www.nature.com/scientificreports/ cannot easily be compared with real world data. By contrast, in the model proposed here, the social network
evolves due to demographic processes such as migration and death on which data may be easier to obtain.h g p
p
g
y
The model introduced here can be extended in several directions. For instance, dead hosts may not be imme-
diately removed from the system and continue to spread the disease for a finite time until eventually being
removed, the disease may not certainly lead to death such that infected individuals can recover, newly arriving
individuals may establish links with only susceptible individuals, but not infected individuals, and susceptible
individuals may rewire their links with infected neighbors to other susceptible individuals as a precaution against
the epidemic. Independently of the specific mechanism, the disease-mortality combined with the network growth
process can be expected to hold in all systems in which the dynamics of the population, in absence of the epi-
demic, leads to topologies with diverging variance of the degree distribution. p
g
g
g
g
We believe that the proposed model is relevant for epidemics in rapidly growing cities, especially in the devel-
oping countries. In this context, connecting the model to real world data will be feasible in the future. In these
situations we anticipate there is no epidemic threshold as the relevant populations often comprise many millions
and are hence sufficiently large that the thermodynamic limit is a reasonable approximation, and the arrival of
infected immigrants reintroduces diseases to the population. One can easily imagine extensions of the present
model that incorporate policy measures such as vaccination, quarantine, or regulation of migration. We hope that
this will in the future lead to the formulation of more efficient policies for combating epidemic diseases. References Efficient immunization strategies for computer networks and populations. Phys. Rev. Lett. 91, 247901 (2003).fi 16. Holme, P. Efficient local strategies for vaccination and network attack. Europhys. Lett. 68, 908–914 (2004). fi
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despite the ever increasing availability of data, for behavior often no records exist such that model predictions Scientific Reports | 7:42352 | DOI: 10.1038/srep42352 13 www.nature.com/scientificreports/ Author Contributions All authors contributed to and reviewed the manuscript. G.D prepared the figures. All authors contributed to and reviewed the manuscript. G.D prepared the figures. Acknowledgementsh Acknowledgements
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J. Phys. 12, 093015 (2010). Additional Informationi Competing financial interests: The authors declare no competing financial interests. How to cite this article: Demirel, G. et al. Dynamics of epidemic diseases on a growing adaptive network. Sc
Rep. 7, 42352; doi: 10.1038/srep42352 (2017). Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps an
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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 | 7:42352 | DOI: 10.1038/srep42352 15
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English
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Analysis of Interest in Shopping for Goods Online at Mitha Mum Shop Palu
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International Journal of Health, Economics, and Social Sciences (IJHESS)
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cc-by-sa
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International Journal of Health, Economics, and Social Sciences International Journal of Health, Economics, and Social Sciences Vol-2, Issue-1, 2020
e-ISSN: 2685-6689 (IJHESS) Analysis of Interest in Shopping for Goods Online at
Mitha Mum Shop Palu Haris Abd. Kadir(1), Sarifuddin(2), Hasmitha Ma’rifatul(3*),
1,2,3Faculty of Economics Universitas Muhammadiyah Palu
*Corresponding Author, Email: hasmithamarifatul@gmail.com Haris Abd. Kadir(1), Sarifuddin(2), Hasmitha Ma’rifatul(3*),
1,2,3Faculty of Economics Universitas Muhammadiyah Palu
*Corresponding Author, Email: hasmithamarifatul@gmail.com ABSTRACT This study aims to determine the interest in shopping for goods online at Mitha Mum Shop Palu. The results of the research on the distribution of respondents on the statement of wanting to buy
products at Mitha Mum Shop Palu because of the variety of answers to agree as many as 15 people
or 16.3% strongly, Agree as many as 59 people or 64.1% and neutral as many as 18 people or 19.6
%. The results of the distribution of respondents to statements referencing Mitha Mum Shop Palu
products to others answered strongly agree as many as 22 people or 23.9%, Agree as many as 49
people or 53.3%, neutral as many as 20 people or 21.7% and disagree as many as one people or
1.1%. The results of the distribution of respondents to the question feel happy to buy products at
Mitha Mum Shop Palu that respondents who answered strongly agree as many as 18 people or
19.6%, agree as much as 51 or 55.4%, and neutral as many as 23 people or 25%. The results of the
distribution of respondents to questions looking for information on the latest products at Mitha
Mum Shop Palu that respondents who answered strongly agree were 23 people or 25%, Agree as
many as 55 or 59.8% and neutral as many as 14 people or 15.2%. Keywords - Flower Shopping, Goods Online, Shop Palu Published by : Universitas Muhammadiyah
P l INTRODUCTION Mitha Mum is an online shop site where individuals or groups can buy and sell
products and services online. This online shop is a place to sell or offer a wide variety of
products or services such as fashion, property, sports equipment, household appliances
and so on. The online shop is now a marketing centre for buying and selling online, which is
spread in various parts of the world. Including what is being done by Mitha Mum Shop,
which is an online shop to offer the goods and services it provides. Online business is increasing without limited time and place. Buying and selling with
the internet as a medium of contact and the website as a marketing catalogue is more
practical and efficient because it does not require direct meetings between sellers and
buyers. Even online businesses have many advantages, namely in terms of service,
effectiveness, security, and also popularity. There are several reasons why online shops are
increasingly in demand, namely: 1. Lower prices compared to prices in stores. 2. Quiet
atmosphere when shopping. 3. Save on transportation costs. 4. No need to jostle around the 134 Published by : Universitas Muhammadiyah
P l International Journal of Health, Economics, and Social Sciences Vol-2, Issue-1, 2020
e-ISSN: 2685-6689 Vol-2, Issue-1, 2020
e-ISSN: 2685-6689 (IJHESS) store. 5. Can shop at any time; consumers do not have to take the time to go to the store. These factors encourage business people to sell goods online shop either independently or
in groups (organizations). store. 5. Can shop at any time; consumers do not have to take the time to go to the store. These factors encourage business people to sell goods online shop either independently or
in groups (organizations). METHODOLOGY Sugiyono (2016 :), said that data analysis is the process of systematically searching
and compiling data obtained from observations, interviews, questionnaires and
documentation. After the data and all the required materials have been collected, the
authors separate and report the data according to its type and presented in the form of
frequency tables and percentage values. Arikunto (2003) presents the presentation
formula as follows: P = Percentage F = Data Frequency N = Number of Respondents To find out the interest in shopping for goods online at Mitha Mum Shop Palu, the authors
make a measure as follows: 1) Strongly Agree, 2) Agree, 3) Neutral, 4) Disagree and 5)
Strongly Disagree To find out the interest in shopping for goods online at Mitha Mum Shop Palu, the authors
make a measure as follows: 1) Strongly Agree, 2) Agree, 3) Neutral, 4) Disagree and 5) )
g y
g
,
)
g
,
)
,
)
g
)
Strongly Disagree Strongly Disagree Published by : Universitas Muhammadiyah
l RESULT & DISCUSSION The statement of interest in shopping on the indicator I want to buy products at
Mitha Mum Shop Palu because of its variety
Table 1. Want to Buy Products at Mitha Mum Shop Palu Because of its Variety
Answer Category
Frekuensi
Persentase (%)
Netral
18
19.6
Agree
59
64.1
Strongly Agree
15
16.3
Total
92
100 From the results of the distribution of table 1, respondents in the statement that they
want to buy products at Mitha Mum Shop Palu because of the variety of answers to strongly
agree as many as 15 people or 16.3%, Agree as many as 59 people or 64.1% and neutral as
many as 18 people or 19, 6%. as shown in Table 5.3. based on this statement, the
respondent stated that the desire to buy a product, because Mitha Mum Shop Palu provides
a variety of products so that consumers are easy to choose what product they like. Published by : Universitas Muhammadiyah 135 International Journal of Health, Economics, and Social Sciences
(IJHESS) International Journal of Health, Economics, and Social Sciences Vol-2, Issue-1, 2020
(IJHESS) e-ISSN: 2685-6689 Vol-2, Issue-1, 2020
e-ISSN: 2685-6689 (IJHESS) Statement of Shopping Interest on Indicators Referencing Mitha Mum Shop Palu
Products to Others
Table 2. Referencing Mitha Mum Shop Palu Products to Others
Answer Category
Frekuensi
Persentase (%)
Not Agree
1
1.1
Netral
20
21.7
Agree
49
53.3
Strongly Agree
22
23.9
Total
92
100 hopping Interest on Indicators Referencing Mitha Mum Shop Palu
ers From the results of the distribution table 2, respondents to answer strongly agree as
many as 22 people or 23.9%, agree as many as 49 people or 53.3%, neutral as many as 20
people or 21.7% and do not agree as many as 1 person or 1.1%. as shown in Table 5.4. based on this statement, the respondents stated that after buying products at Mitha Mum
Shop Palu, they felt comfortable and satisfied. So that these consumers recommend or refer
family and friends to buy products at Mitha Mum Shop Palu. Statement of Shopping Interest on Indicators of Feeling Happy Buying Products at
Mitha Mum Shop Palu
Table 3. DISCUSSION The results of the analysis of this study indicate that shopping interest affects Mitha
Mum Shop Palu Services, it can be explained that shopping interest is the desire of
consumers to have a product from Mitha Mum Shop Palu. Consumer buying interest in
Mitha Mum Shop Palu is an attitude of consumer behaviour. The importance of buying part
is to fulfil the daily needs of consumers to carry on their life. The quality of service provided by Mitha Mum Shop Palu is a critical strategic variable
for consumers in buying back existing products or goods at Mitha Mum Shop Palu; this is
done to achieve long-term satisfaction with customers and also to attract new customers. To accomplish this goal, Mitha Mum Shop Palu pays attention to workers' actions and
equips their work with sufficient knowledge of right service strategies, especially in the
online sales system. Mitha Mum Shop Palu has a wide variety of products for sale, namely clothes, flowers,
pots, wall hangings, and various other items. The facilities offered by Mitha Mum Shop Palu
are complete so that consumers are satisfied to choose the product they like to have. One
example of the Mitha Mum Shop Palu product, if we want to buy live flowers, they are
offered pots, soil or a frame for flower beds. Items sold at Mitha Mum Shop Palu online are by the condition of the goods. This is
proof that the service promised by Mitha Mum Shop Palu to consumers is very accurate
and reliable. The polite attitude or appearance displayed by Mitha Mum Shop Palu in
serving consumers also fosters a sense of trust in consumers in buying products at Mitha
Mum Shop Palu. The services provided by Mitha Mum Shop Palu to consumers in the interest to buy
existing products are supported by Howard's theory cited in Durianto and Liana (2004),
purchase part is something related to consumer plans to purchase certain products and
how many units of the work are needed. At a specific period. As for the research support mentioned above, based on the research results of Endi
and Lilis (2014) it shows that the trust variable has a significant effect on purchase
intention, while the perceived convenience and perceived benefits do not have a
substantial impact on purchase intention. RESULT & DISCUSSION Feeling Happy Buying Products at Mitha Mum Shop Palu
Answer Category
Frekuensi
Persentase (%)
Netral
23
25.0
Agree
51
55.4
Strongly Agree
18
19.6
Total
92
100 From the results of the distribution of respondents to the question, they feel happy
buying products at Mitha Mum Shop Palu, that respondents who answered strongly agree
as many as 18 people or 19.6%, agree as much as 51 or 55.4%, and neutral as many as 23
people or 25%. as shown in Table 3. From the results of the distribution of respondents, it illustrates that consumers who
purchase products at Mitha Mum Shop Palu feel happy, because the desire to buy products
is in accordance with the conditions of the product being purchased and the price offered
by Mitha Mum Shop Palu is quite cheap. 136
Published by : Universitas Muhammadiyah
Statement of Shopping Interest on Indicators of Finding Information on the Latest
Products at Mitha Mum Shop Palu
Table 4. Finding Information on the Latest Products at Mitha Mum Shop Palu
Answer Category
Frekuensi
Persentase (%)
Netral
14
15.2
Agree
55
59.8
Strongly Agree
23
25.0 136 Published by : Universitas Muhammadiyah
P l International Journal of Health, Economics, and Social Sciences
(IJHESS) International Journal of Health, Economics, and Social Sciences
(IJHESS) Vol-2, Issue-1, 2020
e-ISSN: 2685-6689 From the results of the distribution of respondents to questions looking for
information on the latest products at Mitha Mum Shop Palu, 23 people or 25% of
respondents who answered strongly agree, Agree as many as 55 or 59.8% and neutral as
many as 14 people or 15.2%. as shown in Table 4. y
p
p
From the results of the distribution of respondents to the questions in table 5.6,
consumers are always looking for information on the latest products from Mitha Mum Shop
Palu, via Facebook, Instagram or other social media. The existence of Mitha Mum Shop Palu
by selling online, makes it easier for consumers to see products only from home or at work. Published by : Universitas Muhammadiyah
Palu CONCLUSIONS Based on the results of the analysis and discussion of the research results, it can be
concluded that: Based on the results of the analysis and discussion of the research results, it can be
concluded that: 1. From the results of the distribution of respondents on the statement of wanting to buy
products at Mitha Mum Shop Palu because of the variety of answers to strongly agree
as many as 15 people or 16.3%, Agree as many as 59 people or 64.1% and neutral as
many as 18 people or 19.6 %. 2. From the results of the distribution of respondents to statements reflecting Mitha Mum
Shop Palu products to others, 22 people answered strongly or 23.9%, Agree as many as
49 people or 53.3%, neutral as many as 20 people or 21.7% and disagree as many as 1
person or 1 , 1%. 3. From the results of the distribution of respondents to the question, they feel happy
buying products at Mitha Mum Shop Palu, that respondents who answered strongly
agree as many as 18 people or 19.6%, agree as much as 51 or 55.4%, and neutral as
many as 23 people or 25%. 4. From the results of the distribution of respondents to questions looking for the latest
product information at Mitha Mum Shop Palu, 23 people or 25% of respondents who
answered strongly agree, Agree as many as 55 or 59.8% and neutral as many as 14
people or 15.2%. DISCUSSION This means that in online trading, trust is the
most critical factor for Tokobagus.com online sales business people to pay attention to. 137 Published by : Universitas Muhammadiyah
Pal International Journal of Health, Economics, and Social Sciences Vol-2, Issue-1, 2020
e-ISSN: 2685-6689 Vol-2, Issue-1, 2020
e-ISSN: 2685-6689 Whereas the results of research by Nanda and Wahyono (2016) show that the variable
reputation has a negative and insignificant effect on purchase intention, but standing has a
positive and significant impact on purchase intention through the variable trust as a
mediating variable; Website quality variables have a negative and insignificant effect on
purchase intention, but website quality has a positive and significant impact on purchase
intention through trust as a mediating variable. The trust variable has a positive and
significant effect on purchase intention. 8. Nanda Bella Fidanty Shahnaz, Wahyono. 2016. Faktor Yang Mempengaruhi Minat Beli
Konsumen Di Toko Online. Management Analysis Journal. ISSN. 2252-6552. Hal. 389-
399 International Journal of Health, Economics, and Social Sciences Vol-2, Issue-1, 2020
(IJHESS) e-ISSN: 2685-6689 Published by : Universitas Muhammadiyah
P l REFERENCES 1. Arikunto, S. 2006. Metode Penelitian Kualitatif. Bumi Aksara. Jakarta. 1. Arikunto, S. 2006. Metode Penelitian Kualitatif. Bumi Aksara. Jakarta. 2. Nugroho J. Setiadi. 2013. Perilaku Konsumen. Edisi Revisi. PT. Kharisma Putra Utama. Jakarta. 4. Sugiyono. 2016. Metode Penelitian Kuantitatif, Kualitatif dan R&D, Cetakan ke-24. Alfabeta. Bandung. 5. Suparyanto & Rosad. 2015. Manajemen Pemasaran. IN MEDIA. Bogor. 5. Suparyanto & Rosad. 2015. Manajemen Pemasaran. IN MEDIA. Bogor. 6. Durianto dan Liana S, 2004. Strategi Menaklukkan Pasar; Melalui Riset Ekuitas dan
Perilaku Merek, PT. Gramedia Pustaka Utama, Jakarta. 7. Endi Rekarti dan Lilis Hertina. 2014. Beberapa Faktor Yang Berpengaruh Terhadap
Minat Beli Online Pada Situs Jual Beli Tokobagus.com. Jurnal Ilmu Ekonomi Dan
Sosial. No.3 Hal. 311-318. Published by : Universitas Muhammadiyah
P l 138 International Journal of Health, Economics, and Social Sciences Vol-2, Issue-1, 2020
(IJHESS) e-ISSN: 2685-6689
8. Nanda Bella Fidanty Shahnaz, Wahyono. 2016. Faktor Yang Mempengaruhi Minat Beli
Konsumen Di Toko Online. Management Analysis Journal. ISSN. 2252-6552. Hal. 389-
399 Vol-2, Issue-1, 2020
e-ISSN: 2685-6689 (IJHESS) 8. Nanda Bella Fidanty Shahnaz, Wahyono. 2016. Faktor Yang Mempengaruhi Minat Beli
Konsumen Di Toko Online. Management Analysis Journal. ISSN. 2252-6552. Hal. 389-
399 Published by : Universitas Muhammadiyah
P l 139
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https://www.nature.com/articles/s41598-023-44670-x.pdf
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English
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Lemon peel essential oil and its nano-formulation to control Agrotis ipsilon (Lepidoptera: Noctuidae)
|
Scientific reports
| 2,023
|
cc-by
| 8,664
|
OPEN Therefore, to control pests, many nations are switch-
ing from chemical-based agriculture to “green” agriculture, which makes extensive use of biopesticides7–9. Novel
strategies have included the innovation of new types of insecticides, and reevaluation and use of conventional
pest control agents. Previous studies proved that pest communities have positive and negative interactions with
a wide range of plants. Negative interaction with pests, which causes harm to the plants, has led to the develop-
ment of various chemical defense mechanisms which considered good sources of new toxic compounds for
pests10–12. Due to plant insect infection a broad range of secondary metabolites are produced to get adapted, either
as part of their normal program of growth and development or in response to biotic stress13. Those secondary
metabolites have toxic, repellent, and/or anti-nutritional effects on the pests14. Several organic compounds have
been extracted from plants as volatile oils and other plant ingredients which show potential toxic effects against
pest species15. The agro-industrial wastes such as Pomegranate Peels Extract and Orange Waste were used in
pest control7,16. In this context, essential oil extracted from 4 citrus species displayed potent toxicity against Cal‑
losobruchus maculatus as a natural biopesticide17,18. In recent years, new technological approaches to overcome
existing challenges in agriculture such as, lack in natural resources, and emergence of the new pests and climatic
1Plant Biochemistry Department, National Research Centre, Dokki, Giza 12622, Egypt. 2Basic Applied Science
Institute, Egypt-Japan University of Science and Technology (E-JUST), P.O. Box 179, New Borg El‑Arab City,
Alexandria 21934, Egypt. 3Biochemistry Department, Faculty of Agriculture, Cairo University, Giza 12613,
Egypt. 4Pests and Plant Protection Department, Agricultural and Biological Research Institute, National Research
Centre, Dokki, Giza 12622, Egypt. *email: amotagly@cu.edu.eg Increasing food production is one of the main challenges to meet the global needs of the growing world
population1. Unfortunately, agriculture crop loss caused by insect pests is considered one of the main causes for
less intensive production2. Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae), black cutworm, is one of the
extreme destructive pests that attack various crops such as corn, cotton, wheat, and many vegetables in many
regions over the world. An extreme scale of economic loss through the destruction of plants foliar and roots. A. ipsilon moths deposit many hundred eggs on vegetation. once the eggs hatch, the early larval stages of A. ipsilon feed on the foliage of the crop. www.nature.com/scientificreports www.nature.com/scientificreports OPEN Habiba A. Ahmed 1, Amr A. Nassrallah 2,3*, M. A. Abdel‑Raheem 4 & Huda H. Elbehery 4 Due to excessive use of synthetic pesticides the pest resistance developed along with pesticide
residues accumulation in crops. Therefore, many nations are switching from chemical-based
agriculture to “green” agriculture for pest control. The destructive pest black cutworm, Agrotis ipsilon
(Hufnagel) (Lepidoptera: Noctuidae), is a polyphagous species that economically lead to extensive
loss of a broad range of crops including corn, cotton, wheat, and many vegetables through the
damage of foliar and roots. In this study, lemon peel essential oil (LPEO) was subjected to nano-
formulation using polyethylene glycol as nanocarrier. The lethal activity of LPEO and its nano-form
(LPEO-NPs) were tested against A. ipsilon second larval instar using feeding bioassay at different
concentrations. Growth and developmental parameters, including larval and pupal duration, larval
and pupal mortality, malformations % and adult emergence were evaluated. Results showed that
LPEO exhibited insecticidal activity and causes different levels of effects on the development of A. ipsilon according to its concentration and formulation. In addition, at 75 mg/ml LPEO and LPEO-
NPs significantly increased the larval mortality to 80.00% and 90.00%, respectively. The overall data
revealed that insecticidal toxicity of LPEO was increased by nano-formulation. 1
Vol :(01234
Scientific Reports | (2023) 13:17922
| https://doi.org/10.1038/s41598-023-44670-x
Increasing food production is one of the main challenges to meet the global needs of the growing world
population1. Unfortunately, agriculture crop loss caused by insect pests is considered one of the main causes for
less intensive production2. Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae), black cutworm, is one of the
extreme destructive pests that attack various crops such as corn, cotton, wheat, and many vegetables in many
regions over the world. An extreme scale of economic loss through the destruction of plants foliar and roots. A. ipsilon moths deposit many hundred eggs on vegetation. once the eggs hatch, the early larval stages of A. ipsilon feed on the foliage of the crop. Therefore, the Black cutworm invasion caused significant yield reductions,
especially at the seedling stage3. The current strategy for A. ipsilon control relies heavily on the use of several
pesticides, which harm the environment and poses a threat to public health due to food accumulation and ground
water or occasional exposure4,5. The need for efficient biodegradable and selective insecticides has increased due
to the issues produced by these pesticides and their residues6. Gas chromatography‑mass spectrometryh g
The GC–MS analysis of lemon essential oil was separated and identified using a Hewlett-Packard 5890 gas
chromatograph, coupled to VG Analytical 70-250S mass spectrometer. The GC was equipped with a fused silica
capillary CP-Sil 5 CB column (25 m × 0.25 mm i.d., film thickness 0.40 μm, from Chromback, Varian). Helium
was used as carrier gas at a flow rate of 1 mL/min. The oven program started with an initial temperature of 80 °C
held for 2 min and then the oven temperature was heated at 10 °C/min to 270 °C and finally held isothermally
for 20 min. For GC–MS detection, an election ionization system, with ionization energy of 70 eV was used. A
scan rate of 0.6 s (cycle time: 0.2 s) was applied, covering a mass range from 35 to 600 amu. Nano‑lemon essential oil preparation Polyethylene glycol 4000 (PEG) was used in this study as nano carrier. In brief, 50 g of PEG 4000 was melted at
60 °C using hotplate stirrer, a mixture of 10 g of lemon essential oil dissolved into 2 ml of tween 80 was added
dropwise to the melted PEG while stirring for 2 h at 15,000 rpm. The mixture was cooled at 4 °C for 24 h and
ground in a refrigerated mortar. Finally, the product was sieved using a stainless-steel sieve (230 mesh), stored
at room temperature in an airtight container25,26. Material and methods All chemicals used in this study were analytically grade purchased from Sigma Aldrich (USA). Fourier‑transform infrared spectroscopy (FTIR)h Fourier‑transform infrared spectroscopy (FTIR)h The association with the nanocarrier and the functional groups were evaluated using Fourier-transform infrared
pectroscopy (Bruker Optics Tensor 27, Bruker Corporation, Billerica, MA, USA). Zeta Potentialh The surface charges of the nanoparticles in water suspension at room temperature were determined by zeta
potential Malvern Zeta Sizer (NanoZS, Malvern, UK). Essential oil extraction Essential oil was extracted using cold pressing technique from dried pesticide-free certified Lemon peel obtained
from El Marwa company, 6th October-Egypt. www.nature.com/scientificreports/ changes have been applied. Currently, researchers are developing traditional formulations to treat insects rely
on high efficiency, through enhances solubility of insoluble active ingredients, ability for slow release, improved
stability to avoid prematurely degradation, develop mobility and higher pesticidal activity. Nano-formulation is
a new alternative strategy, utilizing natural products used as potential anti-insecticides due to physicochemical
alteration, including size, shape, and charge19. Nanotechnology has become one of the most powerful technolo-
gies in the current years for managing many pests. This technology will lead to drastic changes in the agriculture
field including future pest management. Over the past 20 years, various pesticide molecules offer a solution to
problems like pesticide resistance and side effects have been approved of which 50% are NPs20. Nano formula-
tions can solve specific agricultural issues due to their small size, high surface-area-to-volume ratio, and capac-
ity to give unique crop protection approaches21. In addition, agricultural nanomaterials can play a high role in
integrated pest management (IPM) programs through maintaining the number of pests below levels that may
lead to economic losses with minimal impact on the environment22. In this regard, citrus essential oils as emul-
sions and included in polyethylene glycol (PEG) nanoparticles (EO-NPs exhibited a lethal against the invasive
tomato Tuta absoluta23. Nano emulsions are used because they have greater stability compared to traditional
emulsions. The advantages of the nano emulsions are prominent due to the small droplet size at the interface
is highly significant. Due to its special physicochemical characteristics, nanotechnology is developing rapidly
in many sectors, especially cosmetics, pharmaceutics, agriculture, and food industries24. Therefore, this study
purposed to evaluate the insecticidal efficiency of lemon peel essential oil (LPEO) and Lemon peel Essential oil
nanoparticles (LPEO-NPs) against A. ipsilon larvae. p
Transmission electron microscopy (TEM)h p
Transmission electron microscopy (TEM)h py
The structure of the prepared nanoparticles was visualized using transmission electron microscopy (TEM) with
a FEI TECNAI G2 F20 S-TWIN (Thermo Fisher Scientific, Waltham, MA, USA). OPEN Therefore, the Black cutworm invasion caused significant yield reductions,
especially at the seedling stage3. The current strategy for A. ipsilon control relies heavily on the use of several
pesticides, which harm the environment and poses a threat to public health due to food accumulation and ground
water or occasional exposure4,5. The need for efficient biodegradable and selective insecticides has increased due
to the issues produced by these pesticides and their residues6. Therefore, to control pests, many nations are switch-
ing from chemical-based agriculture to “green” agriculture, which makes extensive use of biopesticides7–9. Novel
strategies have included the innovation of new types of insecticides, and reevaluation and use of conventional
pest control agents. Previous studies proved that pest communities have positive and negative interactions with
a wide range of plants. Negative interaction with pests, which causes harm to the plants, has led to the develop-
ment of various chemical defense mechanisms which considered good sources of new toxic compounds for
pests10–12. Due to plant insect infection a broad range of secondary metabolites are produced to get adapted, either
as part of their normal program of growth and development or in response to biotic stress13. Those secondary
metabolites have toxic, repellent, and/or anti-nutritional effects on the pests14. Several organic compounds have
been extracted from plants as volatile oils and other plant ingredients which show potential toxic effects against
pest species15. The agro-industrial wastes such as Pomegranate Peels Extract and Orange Waste were used in
pest control7,16. In this context, essential oil extracted from 4 citrus species displayed potent toxicity against Cal‑
losobruchus maculatus as a natural biopesticide17,18. In recent years, new technological approaches to overcome
existing challenges in agriculture such as, lack in natural resources, and emergence of the new pests and climatic 1Plant Biochemistry Department, National Research Centre, Dokki, Giza 12622, Egypt. 2Basic Applied Science
Institute, Egypt-Japan University of Science and Technology (E-JUST), P.O. Box 179, New Borg El‑Arab City,
Alexandria 21934, Egypt. 3Biochemistry Department, Faculty of Agriculture, Cairo University, Giza 12613,
Egypt. 4Pests and Plant Protection Department, Agricultural and Biological Research Institute, National Research
Centre, Dokki, Giza 12622, Egypt. *email: amotagly@cu.edu.eg | https://doi.org/10.1038/s41598-023-44670-x Scientific Reports | (2023) 13:17922 www.nature.com/scientificreports/ Insect and plant rearing p
g
Black Cutworm A. ipsilon larvae obtained from organic greenhouses of Faculty of Agriculture, Cairo University,
Egypt. A. ipsilon were reared laboratory on castor plant leaves for many generations in the absence of pesticides
inside polyester net cages (50 × 60 × 80 cm) at 25 ± 2 °C and 70–75% RH, with a photoperiod of 16:8 (L: D). Strips
of black net were used as site of egg deposition. The newly deposited eggs were collected and placed in 1000 ml
glass vials covered with muslin fixed tightly with a rubber band. The newly hatched larvae supplied daily with
fresh castor leaves till the pupation. The pupae were incubated in glass jars until adult emergence and provided
with pieces of cotton wool soaked in 10% honey solution as food supplement. The 2nd A. ipsilon larval instars
of S. frugiperda were obtained from the colony for experiments. https://doi.org/10.1038/s41598-023-44670-x Scientific Reports | (2023) 13:17922 | www.nature.com/scientificreports/ Statistical analysish y
The data were statistically analyzed using one-way ANOVA in SPSS computer program; means were compared
using Duncan’s Multiple Range Test at the 0.05 level of probability27. Nano‑characterization Lemon peel essential oil loaded into polyethylene glycol (PEG) was subjected to physical characterization. Vari-
ous techniques covering the main techniques used in nanoparticles characterization were used including TEM,
FTIR and zeta potential. The results of TEM indicated that the generated LPEO-NPs were in nano-size when
the size of the particles falls within the sub-micrometer range compared to respective controls Fig. 1. Moreover,
the TEM observations confirmed that the size LPEO-NPs consisting of clusters of spherical properties approxi-
mately 75 nm of size. Toxicological bioassayh The examination toxicity of lemon peel essential oil (LPEO) and its nano-form (LPEO-NPs) on the second larval
instar of A. ipsilon were executed using the leaf dipping method; where clean castor leaves were dipped for 20 s
into LPEO or LPEO-NPs at different concentrations selected based on preliminary experiment (6.25, 11.63,
21.65, 40.30 and 75) dissolved in TWEEN 80 as emulsifier or deionized water, respectively. PEG and PEG-NPs
particles alone at 75 mg/ml did not show any toxicity effects as compared to negative control (water and TWEEN
80+). All tested concentrations for toxicity study were performed in 3 replicates; each one had ten larvae of 2nd
larval instar of A. ipsilon. The larvae were left to feed for one day on the treated leaf, and then, the larvae were
provided daily with new untreated castor leaves. The experiments were examined daily to record the develop-
ment and growth, including larval and pupal duration, larval and pupal mortality, percentage of malformations
and adult emergence. FTIR characterization FTIR spectrum showed slight changes in surface-modified nanocarrier and nano-formulations (Fig. 2). In LPEO-
NPs spectrum showed a clearly two peaks were shifted at 405.75 and 2164.45 cm−1, a new peak at 611.69 cm−1,
which strongly confirmed the surface modification compared with PEG-NPs control. These peaks could have
corresponded to the presence of loaded of LPEO. Ethics approval and consent to participateh pp
p
p
This experiment does not involve human experiments and animal experiments. The use of black cutworm, Agrotis
ipsilon (Hufnagel) (Lepidoptera: Noctuidae) was supervised by prof. Abdel-Raheem M. A. All methods were
performed in accordance with the relevant guidelines and regulations. Scientific Reports | (2023) 13:17922 | Lemon essential oil composition
Th
f h
l
l p
The composition of the essential oil extracted from lemon peel was determined using GC–MS analysis. As
shown in Table 2, GC–MS analysis identified 13 different compounds in the essential oil extracted from lemon
peel representing 100% of total oil. D-Limonene was the most abundant compound with 57.65%, followed by
γ-Terpinene with 14.45% and β-Pinene with 11.43% of total compounds. Anyway, several papers have reported
that essential oil of lemon peel composition showed high variation between cultivar and growing place28. Zeta Potential Measurementh The changes in zeta potential values for LPEO-NPs compared to PEG-NPs were shown in Fig. 3 and Table 1. The results indicated that both PEG-NPs and LPEO-NPs showed negative surface charge in deionized water
of − 10.1 ± 0.5 mV and − 11.5 ± 0.9 mV, respectively. As well as the poly dispersed index PDI value was 0.447
and 0.355, respectively indicated the homogeneity of the formulations. These results indicate that our nano-
formulation lemon peel essential oil was nano size. Figure 1 . TEM images for nanoformulations of PEGNPs control (A) TEM image: LPEO-NPs (B). Figure 1 . TEM images for nanoformulations of PEGNPs control (A) TEM image: LPEO-NPs (B). https://doi.org/10.1038/s41598-023-44670-x Scientific Reports | (2023) 13:17922 | https://doi.org/10.1038/s41598-023-44670-x Scientific Reports | (2023) 13:17922 | m/scientificreports/
Figure 2 . FTIR spectra of prepared nanoparticles. LPEO in red, LPEO-NPs in blue and PEG-NPs in black. Figure 3 . Particale size measurement of PEG-NPs (A) and LPEO-NPs (B) suspended in deionized water. www.nature.com/scientificreports/ cientificreports/
Figure 2 . FTIR spectra of prepared nanoparticles. LPEO in red, LPEO-NPs in blue and PEG-NPs in black. Lemon essential oil composition
Th
iti
f th
ti l
il
t
t d f
l
l
d t
i
d
i
GC MS
l
i
A
Figure 2 . FTIR spectra of prepared nanoparticles. LPEO in red, LPEO-NPs in blue and PEG-NPs in black. Figure 3 . Particale size measurement of PEG-NPs (A) and LPEO-NPs (B) suspended in deionized water. Table 1. Zeta potential and PDI of produced nano-essential oil. Parameter
PEG-NPs control
Lemon EO-NPs
Zeta potential
− 10.1
− 11.5
PDI
0.447
0.355 Figure 2 . FTIR spectra of prepared nanoparticles. LPEO in red, LPEO-NPs in blue and PEG-NPs in black. Figure 2 . FTIR spectra of prepared nanoparticles. LPEO in red, LPEO-NPs in blue and PEG-NPs in black. Figure 3 . Particale size measurement of PEG-NPs (A) and LPEO-NPs (B) suspended in deionized water. Figure 3 . Particale size measurement of PEG-NPs (A) and LPEO-NPs (B) suspended in deionized water. Figure 3 . Particale size measurement of PEG-NPs (A) and LPEO-NPs (B) suspended in deionized water. Table 1. Zeta potential and PDI of produced nano-essential oil. Parameter
PEG-NPs control
Lemon EO-NPs
Zeta potential
− 10.1
− 11.5
PDI
0.447
0.355 Table 1. Zeta potential and PDI of produced nano-essential oil. Table 1. Zeta potential and PDI of produced nano-essential oil. Toxicological bioassayh Accordingly, the LPEO and LPEO-NPs led to a delayed
effect on the larvae formation via reducing the number of emerged larvae that reached adulthood by treatment
with the highest concentration of LPEO-NPs 75 mg/ml compared to control, therefore confirming their dis-
ruptive effect on insect growth Table 3. Adult Emergence % declined to (13.33 ± 8.2% and 6.67 ± 3.33%) at high
concentration (75 mg/ml) of LPEO and LPEO-NPs respectively. All adult emerged from LPEO-NPs treatment
were malformed and died. The surviving larvae fed on castor leaves treated with 21.63 mg/ml of LPEO developed
into pupae. However, among the 63.33% emerged adults, 16.67% exhibited deformations properties.h concentrations (75 mg/mL) of LPEO and LPEO-NPs compared to negative control with the duration period of
19.50 ± 0.29, 22.30 ± 0.37 and 10.63 days, respectively. Accordingly, the LPEO and LPEO-NPs led to a delayed
effect on the larvae formation via reducing the number of emerged larvae that reached adulthood by treatment
with the highest concentration of LPEO-NPs 75 mg/ml compared to control, therefore confirming their dis-
ruptive effect on insect growth Table 3. Adult Emergence % declined to (13.33 ± 8.2% and 6.67 ± 3.33%) at high
concentration (75 mg/ml) of LPEO and LPEO-NPs respectively. All adult emerged from LPEO-NPs treatment
were malformed and died. The surviving larvae fed on castor leaves treated with 21.63 mg/ml of LPEO developed
into pupae. However, among the 63.33% emerged adults, 16.67% exhibited deformations properties.h p p
,
g
g
,
p
p
The mortality of the larval and pupae stage A. ipsilon was shown in Table 4. In this context, nano-formulations
of LPEO significantly increased the larval mortality to 90.00%, while LPEO treatment displayed 80.00% at
75 mg/ml. However, no significant larval mortality was observed in negative control. Although we observed that
larvae mortality % were concentration dependent at higher concentrations 21.63, 40.3 and75 mg/ml, the pupal
mortality was 10.0, 20.0 and 6.67% respectively in LPEO treatment and 40, 30 and 3.33% in the case of LPEO-
NPs. Surprisingly, lower concentration at 6.25 and 11.63 mg/ml showed higher pupal mortality compared to
higher concentration with pupal mortality of 26.67 and 33.33% in response to LPEO while LPEO-NPs exhibited
30 and 43.33%, respectively Table 4. These results can be explained by the accumulative resistance due to high
dose treatment. Toxicological bioassayh The results indicated that high concentrations of essential oil and nano essential oil (75 mg/mL) caused
86.67 + 8.82% mortality, while the LPEO-NPs exhibited 93.33 + 3.33% Fig. 4. The toxicity effect of LPEO and
LPEO-NPs against the 2nd instars larvae of A. ipsilon were shown in Table 3. As a result, an acute toxic effect at
larvae and adult stages of A. ipsilon was observed due to LPEO and LPEO-NPs treatment. In addition, significant
toxic effects were obtained in response to higher concentrations and significantly prolonged the duration of
larvae compared to the control. Furthermore, both LPEO and LPEO-NPs treatment significantly increased the
larval duration at 75 mg/ml with a duration period of 21.25 ± 0.48 and 23.6 ± 0.51 days, respectively. However,
the larval duration at 6.25 mg/ml was 17.81 ± 0.26 and 18.40 ± 0.52 days compared to 16.63 ± 0.38 days for con-
trol. Additionally, pupal duration was extremely longer when larvae were fed on castor leaves treated with high https://doi.org/10.1038/s41598-023-44670-x Scientific Reports | (2023) 13:17922 | www.nature.com/scientificreports/ Table 2. Lemon peel essential oil composition. Compound
RT
Area Sum %
β-Thujene
6.189
0.91
α-Pinene
6.366
3.49
Sabinene
7.333
3.01
β-Pinene
7.43
11.43
β-Myrcene
7.745
2.66
D-Limonene
8.844
57.65
γ-Terpinene
9.605
14.45
Terpinolene
10.417
0.94
β-Citral
14.674
1
α-Citral
15.47
1.57
Neryl acetate
17.942
0.67
cis-α-Bergamotene
19.83
0.94
β-Bisabolene
21.627
1.27 Table 2. Lemon peel essential oil composition. Table 2. Lemon peel essential oil composition. 0
10
20
30
40
50
60
70
80
90
100
6.25
11.63
21.65
40.3
75
Mortality %
Concentrations (mg/ml)
LPEO
LPEO-NPs
Figure 4. Percentage Accumulated mortality of A. ipsilon treated with different concentrations of LPEO and 0
10
20
30
40
50
60
70
80
90
100
6.25
11.63
21.65
40.3
75
Mortality %
Concentrations (mg/ml)
LPEO
LPEO-NPs
Figure 4. Percentage Accumulated mortality of A. ipsilon treated with different concentrations of LPEO and
LPEO-NPs. Figure 4. Percentage Accumulated mortality of A. ipsilon treated with different concentrations of LPEO and
LPEO-NPs. concentrations (75 mg/mL) of LPEO and LPEO-NPs compared to negative control with the duration period of
19.50 ± 0.29, 22.30 ± 0.37 and 10.63 days, respectively. Toxicological bioassayh **Highly
significant. LPEO-NPs at 6.25 mg/ml does not allow larval to completely molting as shown in Fig. 5B,C compared to
untreated larval Fig. 5A,D or formed Larval-pupal intermediates as shown in Fig. 5N,O in LPEO and Fig. 5P–R
in LPEO-NPs compared to untreated larval Fig. 5A,D, respectively. Although, some of the larvae succeed to the
pupae stage but it failed to get emerging. The symptoms of pupa failure were observed as showed in Fig. 5F,E
and G,H failed to get rid of their larval or pupal molted skin at 2.5 mg/ml LPEO and 11.63 mg/ml LPEO-NPs,
respectively. Some of the emerged adults were had some deformations, there were deformed (folded) wings from
the adults that emerged from the cutworm treated with 12.5 mg/ml LPEO and 6.25 mg/ml LPEO-NPs Fig. 5K,L
while other adult failed to get rid pupal skin Fig. 5M. Fig. 5J,K and L,M at 11.63 mg/ml LPEO and 6.25 mg/ml
LPEO-NPs, respectively compared to normal adult Fig. 5I. As shown in Table 5 showed, the highest percentage
of adult and total malformation was obtained at 6.25 mg/ml of LPEO-NP with 23.33 ± 6.67% and 40 + 11.55%,
respectively. LPEO-NPs at 6.25 mg/ml does not allow larval to completely molting as shown in Fig. 5B,C compared to
untreated larval Fig. 5A,D or formed Larval-pupal intermediates as shown in Fig. 5N,O in LPEO and Fig. 5P–R
in LPEO-NPs compared to untreated larval Fig. 5A,D, respectively. Although, some of the larvae succeed to the
pupae stage but it failed to get emerging. The symptoms of pupa failure were observed as showed in Fig. 5F,E
and G,H failed to get rid of their larval or pupal molted skin at 2.5 mg/ml LPEO and 11.63 mg/ml LPEO-NPs,
respectively. Some of the emerged adults were had some deformations, there were deformed (folded) wings from
the adults that emerged from the cutworm treated with 12.5 mg/ml LPEO and 6.25 mg/ml LPEO-NPs Fig. 5K,L
while other adult failed to get rid pupal skin Fig. 5M. Fig. 5J,K and L,M at 11.63 mg/ml LPEO and 6.25 mg/ml
LPEO-NPs, respectively compared to normal adult Fig. 5I. As shown in Table 5 showed, the highest percentage
of adult and total malformation was obtained at 6.25 mg/ml of LPEO-NP with 23.33 ± 6.67% and 40 + 11.55%,
respectively. Toxicological bioassayh In this regard, we cannot exclude alternative cytotoxic mechanisms and targeted key elements
including penetration level and defense mechanism. Based on the findings, the highest concentration of the tested
oil caused a high level of larval mortality, and we did not record any larval malformations at 75 mg/ml Table 5.hi The finding exhibited that the treatment with essential oil showed insect growth regulatory activity against
A. ipsilon larvae and produced several abnormal larvae, pupae, and adults Fig. 5. Whereas both LPEO and Scientific Reports | (2023) 13:17922 | https://doi.org/10.1038/s41598-023-44670-x www.nature.com/scientificreports/ Table 3. Effect of LPEO and LPEO-NPs on some development parameters of Agrotis ipsilon. Means in a
Column followed with the same letter(s) are not significantly different at 5% level of probability. **Highly
significant. Treatment
Concentration (mg/ml)
Larval duration
Pupal duration
Adult emergence %
Mean ± S.E
LPEO
75
21.25 ± 0.48b
19.50 ± 0.29c
13.33 ± 8.82ef
40.3
20.89 ± 0.31b
18.89 ± 0.26cd
23.33 ± 8.82def
21.65
19.67 ± 0.37cd
18.67 ± 0.37cd
63.33 ± 14.53b
11.63
18.73 ± 0.27def
18.09 ± 0.28d
46.67 ± 3.33bcd
6.25
17.82 ± 0.26f. 18.18 ± 0.30d
56.67 ± 8.82bc
LPEO-NP
75
23.60 ± 0.51a
22.80 ± 0.37a
6.67 ± 3.33f. 40.3
21.38 ± 0.46b
21.50 ± 0.42b
13.33 ± 3.33ef
21.65
20.38 ± 0.38bc
19.63 ± 0.46c
30.00 ± 10.00cdef
11.63
19.09 ± 0.28de
19.18 ± 0.18cd
40.00 ± 20.82bcde
6.25
18.40 ± 0.52ef
18.90 ± 0.31cd
53.33 ± 3.33bcd
Control
0.00
16.63 ± 0.38g
10.63 ± 0.38e
100.00 ± 0.00a
F
21.721**
71.58**
8.13**
Sig
0.00
0.00
0.00 Treatment
Concentration (mg/ml)
Larval duration
Pupal duration
Adult emergence %
Mean ± S.E
LPEO
75
21.25 ± 0.48b
19.50 ± 0.29c
13.33 ± 8.82ef
40.3
20.89 ± 0.31b
18.89 ± 0.26cd
23.33 ± 8.82def
21.65
19.67 ± 0.37cd
18.67 ± 0.37cd
63.33 ± 14.53b
11.63
18.73 ± 0.27def
18.09 ± 0.28d
46.67 ± 3.33bcd
6.25
17.82 ± 0.26f. 18.18 ± 0.30d
56.67 ± 8.82bc
LPEO-NP
75
23.60 ± 0.51a
22.80 ± 0.37a
6.67 ± 3.33f. 40.3
21.38 ± 0.46b
21.50 ± 0.42b
13.33 ± 3.33ef
21.65
20.38 ± 0.38bc
19.63 ± 0.46c
30.00 ± 10.00cdef
11.63
19.09 ± 0.28de
19.18 ± 0.18cd
40.00 ± 20.82bcde
6.25
18.40 ± 0.52ef
18.90 ± 0.31cd
53.33 ± 3.33bcd
Control
0.00
16.63 ± 0.38g
10.63 ± 0.38e
100.00 ± 0.00a
F
21.721**
71.58**
8.13**
Sig
0.00
0.00
0.00 Table 3. Toxicological bioassayh Effect of LPEO and LPEO-NPs on some development parameters of Agrotis ipsilon. Means in a
Column followed with the same letter(s) are not significantly different at 5% level of probability. **Highly
significant. Table 3. Effect of LPEO and LPEO-NPs on some development parameters of Agrotis ipsilon. Means in a
Column followed with the same letter(s) are not significantly different at 5% level of probability. **Highly
significant. Table 4. Percentage Larval and Pupal mortality of Agrotis ipsilon treated with LPEO and LPEO-NP. Means
in a Column followed with the same letter(s) are not significantly different at 5% level of probability. **Highly
significant. Treatment
Concentration (mg/ml)
Larval mortality %
Pupal mortality %
Mean ± SE
LPEO
75
80.00 ± 15.28a
6.67 ± 6.67cd
40.3
56.67 ± 17.64ab
20.00 ± 10.00abcd
21.65
26.67 ± 8.82bc
10.00 ± 5.77bcd
11.63
20.00 ± 10.00c
33.33 ± 12.02ab
6.25
16.67 ± 8.82c
26.67 ± 8.82abc
LPEO-NP
75
90.00 ± 0.00a
3.33 ± 3.33d
40.3
56.67 ± 8.82ab
30.00 ± 10.00abc
21.65
30.00 ± 10.00bc
40.00 ± 0.00a
11.63
30.00 ± 17.32bc
30.00 ± 5.77abc
6.25
3.33 ± 3.33c
43.33 ± 3.33a
Control
0.00
0.00 ± 0.00c
0.00 ± 0.00d
F
7.54**
4.50**
Sig
0.00
0.00 Table 4. Percentage Larval and Pupal mortality of Agrotis ipsilon treated with LPEO and LPEO-NP. Means
in a Column followed with the same letter(s) are not significantly different at 5% level of probability. **Highly
significant. Treatment
Concentration (mg/ml)
Larval mortality %
Pupal mortality %
Mean ± SE
LPEO
75
80.00 ± 15.28a
6.67 ± 6.67cd
40.3
56.67 ± 17.64ab
20.00 ± 10.00abcd
21.65
26.67 ± 8.82bc
10.00 ± 5.77bcd
11.63
20.00 ± 10.00c
33.33 ± 12.02ab
6.25
16.67 ± 8.82c
26.67 ± 8.82abc
LPEO-NP
75
90.00 ± 0.00a
3.33 ± 3.33d
40.3
56.67 ± 8.82ab
30.00 ± 10.00abc
21.65
30.00 ± 10.00bc
40.00 ± 0.00a
11.63
30.00 ± 17.32bc
30.00 ± 5.77abc
6.25
3.33 ± 3.33c
43.33 ± 3.33a
Control
0.00
0.00 ± 0.00c
0.00 ± 0.00d
F
7.54**
4.50**
Sig
0.00
0.00 Table 4. Percentage Larval and Pupal mortality of Agrotis ipsilon treated with LPEO and LPEO-NP. Means
in a Column followed with the same letter(s) are not significantly different at 5% level of probability. **High
significant. Table 4. Percentage Larval and Pupal mortality of Agrotis ipsilon treated with LPEO and LPEO-NP. Means
in a Column followed with the same letter(s) are not significantly different at 5% level of probability. Discussion Larval death was the first indicator of the insecticidal efficacy of
(LPEO) and (LPEO-NPs). The results showed that, maximum percentage mortality was recorded at the high
concentrations of LPEO and LPEO-NPs. The nano-emulsion of Purslane Oil exhibited 85.00% larval mortality
of A. ipsilon30.hit p
The tested concentrations significantly prolonged the duration of larvae after treated by (LPEO) and (LPEO-
NPs) when compared to the control. These results agreed with the previously reported results obtained by31 who
reported that the dehydrated f Piper auritum’s ethanolic extract affected the survival of Spodoptera frugiperda
by extending the larval and pupal stages and shortening the duration of the adult stage, with the strength of
the effect depending on the extract’s concentration. A slight significance in larval duration elongation treated
with LC15 and LC50 of Lemongrass essential oil compared with control was reported by32. Similarly, bulk, nano-
emulsion and loaded nano-emulsion of neem oil led to larval duration elongation to 22.71, 24.40 and 24.28 days,
respectively compared to control 18.82 days33. Similar findings confirmed that the larval duration of A. ipsilon
was 22.61 and 24.30 days for purslane oil bulk and its nano-emulsion, respectively compared to 18.72 days in
control30. In addition, the pupal duration of all tested concentrations was longer than control. Similar data
expressed by30 indicated that pupal duration of A. ipsilon was prolonged 10.00 days compared to control (). All
tested oil caused a reduction in the percentage adult emergence. These results were also agreed with previous
obtained data reported by34 who found that Nano-formulated Jojoba oil (0.1%) caused 15.30% reduction for
adult emergence. Number of emerged malformed individuals was added to the number of normal ones which
explains the high percentage of adult emergence (63.33%) at concentration 25 mg/ml of LPEO. Whereas higher
concentration resulted in high mortality. Despite all surviving larvae in high concentrations pupated, the percentage of pupal mortality was lower than
in the lower concentrations. In agreement with work reported by31, the pupal mortality of Spodoptera frugiperda
was higher in all treatments of the larvae exposed to the LC35 of ethanolic extract of Piper auritum than LC50
and LC56. Additionally, nano-formulation using PEG of citrus peel essential oil extracted from different species
including orange peel and lemon exhibited lethal activity against tomato T. absoluta have been reported by21.hii The overall obtained data confirms that pupal and adult malformation% were significantly increased due
to larva LPEO treatment. Discussion Agro-waste-derived material converted into valuable nano-product recruiting researchers as feasible alterna-
tive fertilizers, plant protection and remove the hazard pesticides in the field of plant biochemistry. Citrus peel
and tomato pomaces materials are nano-management turning into valuable pharmaceutical applications by our
group26,29. Herein, Lemon peel Essential oil was reformulated into nanoform as potential insecticide against
black cut warm. The nano-characterizations using TEM, FTIR and zeta potential indicate that our product was Scientific Reports | (2023) 13:17922 | https://doi.org/10.1038/s41598-023-44670-x www.nature.com/scientificreports/ bl
lf
f
l
l
d
d l
f
l
d
d b
h
Treatment
Concentration (mg/
ml)
Larval Malformation
%
Pupal malformation
%
Adult malformation
%
Total malformation %
Mean ± SE
LPEO
75
0.00 ± 0.00a
0.00 ± 0.00c
0.00 ± 0.00c
0.00 ± 0.00d
40.3
0.00 ± 0.00a
0.00 ± 0.00c
0.00 ± 0.00c
0.00 ± 0.00d
21.65
3.33 ± 3.33a
6.67 ± 3.33abc
16.67 ± 12.02ab
26.67 ± 6.67abc
11.63
0.00 ± 0.00a
6.67 ± 3.33abc
3.33 ± 3.33bc
10.00 ± 0.00cd
6.25
3.33 ± 3.33a
6.67 ± 3.33abc
0.00 ± 0.00c
10.00 ± 0.00cd
LPEO-NP
75
0.00 ± 0.00a
0.00 ± 0.00c
6.67 ± 3.33bc
6.67 ± 3.33d
40.3
3.33 ± 3.33a
16.67 ± 3.33a
10.00 ± 0.00abc
30.00 ± 5.77ab
21.65
3.33 ± 3.33a
3.33 ± 3.33bc
0.00 ± 0.00c
6.67 ± 3.33d
11.63
0.00 ± 0.00a
13.33 ± 8.82ab
3.33 ± 3.33bc
16.67 ± 12.02bcd
6.25
3.33 ± 3.33a
13.33 ± 3.33ab
23.33 ± 6.67a
40.00 ± 11.55a
Control
0.00
0.00 ± 0.00a
0.00 ± 0.00c
0.00 ± 0.00c
0.00 ± 0.00d
F
0.60ns
2.85*
3.09*
5.31**
Sig
0.80
0.02
0.01
0.00 Table 5. Malformations % of Larval, pupal, and Adult of Agrotis ipsilon produced by treatment with
different concentrations of LPEO and LPEO-NP. Means in a Column followed with the same letter(s) are not
significantly different at 5% level of probability. **Highly significant, *significant, ns = non-significant. in nanoform. The LPEO-NPs displayed nano characteristics, when the size was less than 50 nm, and the charge
was − 11.5 ± 0.9 mV. As well as the electron microscope images TEM showed the new shape of the LPEO-NPs,
these nano-properties have enhanced the efficiency of the LPEO.hiff p
pfi
y
The finding showed that LPEO caused different levels of effects on biological parameters of A. ipsilon accord-
ing to its concentration and formulation. Table 5. Malformations % of Larval, pupal, and Adult of Agrotis ipsilon produced by treatment with
d ff
f
d
l
f ll
d
h h
l
( ) different concentrations of LPEO and LPEO-NP. Means in a Column followed with the same letter(s) are n
significantly different at 5% level of probability. **Highly significant, *significant, ns = non-significant. Table 5. Malformations % of Larval, pupal, and Adult of Agrotis ipsilon produced by treatment with
different concentrations of LPEO and LPEO-NP Means in a Column followed with the same letter(s) are n Table 5. Malformations % of Larval, pupal, and Adult of Agrotis ipsilon produced by treatment with
different concentrations of LPEO and LPEO-NP. Means in a Column followed with the same letter(s) are not
i
ifi
tl diff
t t 5% l
l f
b bilit
**Hi hl
i
ifi
t * i
ifi
t
i
ifi
t Discussion However, LPEO-NP slightly enhances pupal and adult malformation% compared to
control. In comparing to respective controls, the results confirm that, the tested EOLP had a significant effect
on the percentage of pupal and adult malformation where when larva treated with nano oil lead to raise the
malformation. In general, morphological malformations occurred in response to a lack in chitin synthesis there-
fore inhibiting the process of ecdysis. Our findings point to an inhibition role of LPEO and LPEO-NP in chitin
synthesis. In this context, Similar explanation was reported by33,35 who reported that the combination garlic and mint oil has a particular mode of action which interferes with
the rate of deposition chitin. The newly formed cuticle become less hard, so it cannot stand the internal pres-
sure during the molting and thus resulting in an inability to get rid of the exuviae and finally in death. Also,
Liburd et al., 2000 confirmed that, the inhibition of chitinase activity can be explained by the properties of the
mint oil as a growth regulator which leads to death, mostly because the insects cannot get rid of the exuviae. In
this context, Amin et al. 2019 reported that, the effects of the oils on pests may attribute to their effect on pest
neuro endocrine system and juvenile hormone leading to hormonal imbalance causing deformation. Basedow Scientific Reports | (2023) 13:17922 | https://doi.org/10.1038/s41598-023-44670-x www.nature.com/scientificreports/ Figure 5 . Normal stages (A, D, I) and Various stages malformations of Agrotis ipsilon treated with different
concentrations of LPEO and LPEO-NP at different concentrations. Figure 5 . Normal stages (A, D, I) and Various stages malformations of Agrotis ipsilon treated with different
concentrations of LPEO and LPEO-NP at different concentrations. et al.,2012 mentioned that the treatment of beeswax with Neem Azal-T/S resulted in abnormal development and
death of larval and pupal stages of greater wax moth.f Also, it has been reported that the 2nd larval instar of A. ipsilon was treated with different formulations of
purslane oil caused prolongation in pupal duration, increasing the pupal mortality% as well as pupae malfor-
mation% by30. Citrus species are enriched in their contents of secondary metabolites, including polyphenolics,
flavonoids, alkaloids and terpenoids36. The existence of terpenoids and flavonoids as a common phytochemical
component in citrus extracts explainig their potential role in the toxicity against mosquitoes37. www.nature.com/scientificreports/ nanoencapsulation system using β-cyclodextrin/Gum Arabic (βCD/GA) inclusion. accordingly, the protection
activity of Citronella Essential Oil (CEO) against Spodoptera littoralis was enhanced after its incorporation into
the different chitosan nano-systems40. larvae of Rhynchophorus ferrugineus was more affected with the nano oils
than the bulk40,41. The overall obtained results of this study concluded that, plant-derived essential oils can be
used as a promising alternative pest management. Lemon Peel waste is hopeful as a natural biopesticide against
the cutworm, A. ipsilon larvae. Furthermore, the application of Lemon Peel Essential Oil in nano form led to
enhance its insecticidal toxicity. nanoencapsulation system using β-cyclodextrin/Gum Arabic (βCD/GA) inclusion. accordingly, the protection
activity of Citronella Essential Oil (CEO) against Spodoptera littoralis was enhanced after its incorporation into
the different chitosan nano-systems40. larvae of Rhynchophorus ferrugineus was more affected with the nano oils
than the bulk40,41. The overall obtained results of this study concluded that, plant-derived essential oils can be
used as a promising alternative pest management. Lemon Peel waste is hopeful as a natural biopesticide against
the cutworm, A. ipsilon larvae. Furthermore, the application of Lemon Peel Essential Oil in nano form led to
enhance its insecticidal toxicity. Received: 10 May 2023; Accepted: 11 October 2023 Received: 10 May 2023; Accepted: 11 October 2023 References 6. Mesbah, A. H., Tayeb, H. E., Kordy, M. A., El-Shershaby, M. A. & El-Wakil, H. N. Latent effect of two formulated botanical fine
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Prod Res. 68, 25–32 (2016). 18. Amin, L. R., Abo El-Enin, S. A., Abdel-Ghany, A. M., El-Badawy, S. S. & Amin, T. R. Physicochemical and phytochemical analysis
of some Citrus peel extracts, and their activity against the cotton leafworm, Spodopteralittoralis (Boisd) Lepidoptera: Noctuidae. Egypt. J. Agric. Res. 95(3), 1083–1096 (2017). gyp
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5. Salem, S. A., Abd El-Salam, A. M. E., Abdel-Raheem, M. A., Farage, N. A. & El-Hawary, F. M. Field studies to assess the efficiency
of bio-extracts against the scourge of onion crops, Thrips tabaci Lindeman in Egypt. Der Pharma Chemica 8(20), 74–77 (2016). 6 Mesbah A H Tayeb H E Kordy M A El Shershaby M A & El Wakil H N Latent effect of two formulated botanical fine ili
5. Salem, S. A., Abd El-Salam, A. M. E., Abdel-Raheem, M. A., Farage, N. A. & El-Hawary, F. M. Field studies to assess the efficiency
of bio-extracts against the scourge of onion crops, Thrips tabaci Lindeman in Egypt. Der Pharma Chemica 8(20), 74–77 (2016). 6. Mesbah, A. H., Tayeb, H. E., Kordy, M. A., El-Shershaby, M. A. & El-Wakil, H. N. Latent effect of two formulated botanical fine
dusts on Agrotis ipsilon (Hufn ) generation Alex Sci Exchange J 37 221 230 (2016) 5. Salem, S. A., Abd El Salam, A. M. E., Abdel Raheem, M. A., Farage, N. A. & El Hawary, F. M. Field studies to assess the efficiency
of bio-extracts against the scourge of onion crops, Thrips tabaci Lindeman in Egypt. Der Pharma Chemica 8(20), 74–77 (2016). Discussion The insecti-
cidal activity of Cymbopogon citratus (Lemongrass) is attributed to the action of major constituents citral and
limonene38,39. The previous results showed that LPEO and LPEO-NP have potential toxic effect on different
stages of the cutworm. Moreover, lemon peel essential oil nano-formulation enhances insecticidal toxicity. Another, results obtained by40 exhibited that insecticidal toxicity of cinnamon essential oil was increased by Scientific Reports | (2023) 13:17922 | https://doi.org/10.1038/s41598-023-44670-x www.nature.com/scientificreports/ Competing interests h p
g
The authors declare no competing interests. Author contributions H.A.A., H.H.E., A.A.N. and E.M.A. designed the experiments. H.A.A., H.H.E. and A.A.N. performed the experi-
ments and data analysis, and drafted the manuscript. All authors read and prepare the final manuscript. H.A.A., H.H.E., A.A.N. and E.M.A. designed the experiments. H.A.A., H.H.E. and A.A.N. performed the experi-
ments and data analysis, and drafted the manuscript. All authors read and prepare the final manuscript. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 33. Amin, A. R. H., Bayoumi, A. E. D., Dimetry, N. Z. & Youssef, D. A. Efficiency of Nano-formulations of neem and peppermint
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its histological effect on albino rats. Middle East J. Appl. Sci 8(4), 1404–1412 (2018). gf
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strains of Anopheles stephensi Liston and Aedes aegypti L. Parasitol Res. 111, 173–178 (2012).l 8. Oliveira, E. R. et al. Toxicity of Cymbopogon flexuosus essential oil and citral for Spodoptera frugiperda. Ciência e Agrotecnologia
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0. Elbehery, H. H. & Ibrahim, S. S. Cinnamon essential oil loaded β-cyclodextrin/gum Arabic nanoparticles affecting life table
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1. Ibrahim, S. S. Polyethylene glycol nanocapsules containing syzygium aromaticum essential oil for the management of lesser grain
borer. Rhyzopertha dominica. Food Biophys. 17(4), 523–534 (2022). Author contributions Funding g
Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in coopera-
tion with The Egyptian Knowledge Bank (EKB). g
Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in coopera-
tion with The Egyptian Knowledge Bank (EKB). References 15(3)
413–419 (2017). 5. González, J. O., Gutiérrez, M. M., Ferrero, A. A. & Band, B. F. Essential oils nanoformulations for stored-product pest control–
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27. Duncan, D. B. Multiple range and multiple F tests. Biometrics 11(1), 1–42 (1955). 27. Duncan, D. B. Multiple range and multiple F tests. Biometrics 11(1), 1–42 (1955). 28. Njoku, V. I. & Evbuomwan, B. O. Analysis and comparative study of essential oil extracted from Nigerian orange, lemon and lime
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0. Siam, A. N. Nano formulation of essential oils against agrotis ipsilon (Hufn.) Larvae (Lepidoptera: Noctuidae). Acad. J. Entomol
14(2), 30–38 (2021). 31. Jiménez-Durán, A. et al. Biological activity of phytochemicals from agricultural wastes and weeds on Spodoptera frugiperda (je
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2. Moustafa, M. A. et al. Insecticidal activity of lemongrass essential oil as an eco-friendly agent against the black cutworm Agrotis
ipsilon (Lepidoptera: Noctuidae). Insects 12(08), 737 (2021). https://doi.org/10.1038/s41598-023-44670-x Scientific Reports | (2023) 13:17922 | Additional information Correspondence and requests for materials should be addressed to A.A.N. Correspondence and requests for materials should be addressed to A.A.N. Correspondence and requests for materials should be addressed to A.A.N. © The Author(s) 2023 Reprints and permissions information is available at www.nature.com/reprints. 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
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Adjudicação de direitos e escolhas políticas na assistência social: o STF e o critério de renda do BPC
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[*] Este artigo recebeu o 1º lugar no
Concurso de Artigos Jurídicos sobre
Direito à Assistência Social (Tema 3:
Assistência Social como direito uni-
versal e seu papel na efetivação dos
direitos humanos), promovido pelo
Ministério do Desenvolvimento So-
cial e Combate à Fome, pela Funda-
ção Oswaldo Cruz (prodisa/fio-
cruz) e pelo Centro de Pesquisa em
Direito Sanitário da Universidade de
São Paulo. RESUMO Este trabalho discute as implicações da decisão do stf que
amplia o número de beneficiários do Benefício de Prestação Continuada ao flexibilizar o limite de renda para seu rece-
bimento. A Corte realizou uma escolha distributiva e política com custos de oportunidade significativos, o que fica claro
quando os efeitos dessa decisão são comparados ao de políticas como o Bolsa Família e o Brasil Carinhoso. PALAVRAS-CHAVE: Benefício de Prestação Continuada; Supremo
Tribunal Federal; judicialização; direito social; Bolsa Família;
assistência social. Rights Adjudication and Policy Choices in Social
Assistance: the Brazilian Supreme Federal Court
and the Maximum Income Threshold for the
Benefício de Prestação Continuada (BPC)
ABSTRACT O stf e o critério de renda do bpc* http://dx . doi.org / 10.25091/
S01013300201500030007 Daniel Wei L. Wang
Natália Pires de Vasconcelos Rights Adjudication and Policy Choices in Social
Assistance: the Brazilian Supreme Federal Court
and the Maximum Income Threshold for the
Benefício de Prestação Continuada (BPC)
ABSTRACT This paper analyzes the implications of the Brazilian Federal
Supreme Court judgment that expanded the number of people eligible for a social benefit called Benefício de Prestação
Continuada. This judgment has policy implications and opportunity costs that become clear when the expected impact of
this decision is compared to the cost and effectiveness of other social policies such as Bolsa Família and Brasil Carinhoso. KEYWORDS: Benefício de Prestação Constinuada; Brazilian Federal
Supreme Court; judicialization of politics; social rights; Bolsa Família;
social assistance. Supreme Court; judicialization of politics; social rights; Bolsa Família;
social assistance. A Constituição Federal é inequívoca com relação ao
compromisso que assume com o combate à pobreza e à exclusão so-
cial. A cidadania e a dignidade da pessoa humana são fundamentos do
Estado brasileiro (art. 1º), que tem, dentre outros objetivos, construir
uma sociedade livre, justa e solidária; erradicar a pobreza e a margi-
nalização e reduzir as desigualdades sociais e regionais; além de pro-
mover o bem de todos, sem preconceitos de origem, raça, sexo, cor,
idade ou qualquer outra forma de discriminação (art. 3º). A Constitui-
ção, quando trata dos “direitos e garantias fundamentais” (título II),
também prevê um amplo rol de direitos sociais (art. 6º): os direitos à NOVOS ESTUDOS 103 ❙❙ novembro 2015 135 educação, à saúde, à alimentação, ao trabalho, à moradia, ao lazer, à se-
gurança, à previdência social, à proteção da maternidade e da infância
e à assistência aos desamparados. Esses direitos ganham densidade ao longo do texto constitucional. No caso da assistência aos desamparados, o art. 203 da Constituição
trata dos objetivos da assistência social e, em seu inciso v, estabelece
“a garantia de um salário mínimo de benefício mensal à pessoa porta-
dora de deficiência e ao idoso que comprovem não possuir meios de
prover à própria manutenção ou de tê-la provida por sua família, con-
forme dispuser a lei”. É sobre esse benefício, o Benefício de Prestação
Continuada (bpc), que trata este artigo. Embora seja uma prestação assistencial prevista constitucio-
nalmente desde 1988, ele foi apenas regulamentado em 1993 pela
Lei Orgânica da Assistência Social (Loas), com alterações promovi-
das pelas Leis n. 12.435 e n. 12.470, ambas de 2011, e pelos Decretos
n. 6.214, de 2007, e n. 6.564, de 2008. [2] Hoffmann; Bentes, 2010; Piove-
san, 2008; Wang, 2008; Ferraz, 2011. [1] Coutinho, 2013. ÃO DE DIREITOS E ESCOLHAS POLÍTICAS NA ASSISTÊNCIA SOCIAL ❙❙ Daniel Wei L. Wang, Natália Pires de Vasconcelos Rights Adjudication and Policy Choices in Social
Assistance: the Brazilian Supreme Federal Court
and the Maximum Income Threshold for the
Benefício de Prestação Continuada (BPC)
ABSTRACT O bpc é um dos elementos do
Sistema Único de Assistência Social, uma complexa rede de políticas
públicas não contributivas que visa à prestação do direito constitu-
cional à assistência social,1 e também um direito social reconhecido
constitucionalmente. Uma das consequências da constitucionalização de direitos so-
ciais, sobretudo quando possuem esse status de direitos humanos
fundamentais, é a sua judicialização. Os exemplos mais conhecidos
no Brasil de judicialização de direitos sociais são as ações judiciais
contra o poder público com pedidos de medicamento (direito à saúde)
e de matrículas em creches (direito à educação).2 O mesmo fenômeno
tem ocorrido com o bpc, um caso muito menos estudado, porém com
impacto orçamentário e sobre o desenho de sua própria política pú-
blica potencialmente maior que o da judicialização de direitos como
saúde e educação. O art. 20, §3o, da Lei Orgânica de Assistência Social (Loas), ao
regulamentar o art. 203, inciso v, da Constituição Federal, estabelece
que, para fins de recebimento de bpc, “considera-se incapaz de prover
a manutenção da pessoa com deficiência ou idosa a família cuja renda
mensal per capita seja inferior a 1/4 (um quarto) do salário mínimo”. Pessoas cujas famílias estão acima desse critério de renda, contudo,
têm levado ao Judiciário demandas questionando a sua constitucio-
nalidade. Argumentam, de maneira geral, que tal critério de renda res-
tringiria seu direito constitucional de assistência social porque, ainda
que tenham uma renda familiar per capita acima do critério estabe-
lecido pela Loas, vivem em famílias que são incapazes de prover seu
sustento. Essa questão foi analisada em diversas decisões no Supre-
mo Tribunal Federal (stf) e, recentemente, teve sua repercussão geral
reconhecida e julgada no Recurso Extraordinário (re) 567.985. 136 ADJUDICAÇÃO DE DIREITOS E ESCOLHAS POLÍTICAS NA ASSISTÊNCIA SOCIAL ❙❙ Daniel Wei L. Wang, Natália Pires de Vasconcelos Assim como acontece com outros direitos sociais, a constitucio-
nalização e judicialização do bpc coloca ao Judiciário o desafio de de-
cidir, de um lado, sem “usurpar” as funções usualmente atribuídas ao
Executivo e ao Legislativo por razões de legitimidade democrática e
capacidade institucional e, de outro, sem “abdicar” de sua função de
controlador desses outros poderes na concretização de direitos.3 Este artigo, primeiramente, descreve como o stf tem lidado com
esse dilema entre “abdicação” e “usurpação” quando decide sobre o
critério de renda do bpc. [3] Michelman, 2003, p.16; Ferraz,
2013. [4] Fuller, 1978. [4] Fuller, 1978. Jurisprudência do stf anterior ao re 567.985 Jurisprudência do stf anterior ao re 567.985 O benefício de prestação continuada é questionado perante o stf
pelo menos desde 1993.5 Nesse ano, pouco antes da aprovação da Lei
Orgânica da Assistência Social (Loas), um grupo de pessoas porta-
doras de deficiência impetrou o Mandado de Injunção 448/rs, sob
relatoria do ministro Marco Aurélio, demandando que o stf decla-
rasse a mora do Congresso Nacional em regulamentar o benefício e
determinasse um prazo de 45 dias para aprovação da nova lei ou a con-
cessão direta de um salário mínimo aos impetrantes. O stf só julgou
a ação em 5 de setembro de 1994, acolhendo em parte a pretensão dos
demandantes para apenas declarar o Legislativo em mora, algo que
somente seria completamente sanado em 1996 com a vigência do De-
creto 1.744/1995.6 Apesar de regulamentado, a concessão do benefício
continuou sendo questionada judicialmente, sobretudo quanto à exi-
gência de renda per capita familiar máxima de 1/4 de salário mínimo. [5] Considera-se aqui, obviamente,
a judicialização apenas do benefício
de prestação continuada da Assis-
tência Social, previsto pelo art. 203,
inciso v da Constituição de 1988 e
regulamentado pela Loas. Isso por-
que existem também os benefícios de
prestação continuada oferecidos pela
Previdência Social, como aposenta-
dorias, pensões por morte, auxílios,
rendas mensais vitalícias, abonos de
permanência em serviço, os salários-
-família e maternidade (conferir em
http://www.previdencia.gov.br/esta-
tisticas/menu-de-apoio-estatisticas-
-anuario-estatistico-da-previden-
cia-social-2007-beneficios/, acesso
em: 12 nov. 2013). Esses benefícios,
sua correção e critérios, já eram am-
plamente judicializados mesmo an-
tes da Constituição de 1988, como
se pode conferir pela jurisprudência
do stf (por exemplo: re 74.311/gb,
rel. min. Djaci Falcão, j. 3 out. 1972; re
87.690/ce, rel. min. Xavier de Albu-
querque, j. 19 ago. 1977; e re 147.684/
df, rel. min. Sepúlveda Pertence, j. 26
jun. 1992). Assim, em 1998 o procurador-geral da República ajuizou a Ação
Direta de Inconstitucionalidade (adi) 1.232, requerendo que o stf
reconhecesse a inconstitucionalidade do critério de 1/4 de salário mí-
nimo estabelecido na Loas. Segundo os argumentos do demandante,
a Constituição determinou que “[a] assistência social será prestada
a quem dela necessitar” (art. 203) e, portanto, o art. Rights Adjudication and Policy Choices in Social
Assistance: the Brazilian Supreme Federal Court
and the Maximum Income Threshold for the
Benefício de Prestação Continuada (BPC)
ABSTRACT Como demonstraremos, a jurisprudência
do stf sobre o tema sempre oscilou entre ora respeitar a decisão
legislativa, que limita a concessão do benefício apenas para pessoas
de renda familiar per capita abaixo de 1/4 de salário mínimo, ora
determinar sua inconstitucionalidade por desconsiderar as neces-
sidades de pessoas não compreendidas por esse critério. Essa osci-
lação refletiu-se nos debates do pleno da Corte, bem como em suas
decisões monocráticas, marcados pela convivência de diferentes
interpretações sobre a questão. Para uniformizar sua jurisprudência e sanar essa incerteza e du-
alidade, o stf declarou a repercussão geral no re 567.985, caso que
assumiria então a função de recurso paradigma ao problema. Na de-
cisão desse recurso, ao tentar escapar da crítica de “abdicar” das suas
funções de protetor de direitos constitucionais, decidiu pela incons-
titucionalidade do critério de renda do bpc, o que a princípio permi-
te que pessoas com renda acima de 1/4 de salário mínimo per capita
tornem-se elegíveis para receber o benefício. Ao mesmo tempo, para
também esquivar-se da crítica de ter “usurpado” funções legislativas
e executivas, decidiu pela não nulidade do art. 20, §3o da Loas, sem
estabelecer novos parâmetros à concessão do benefício e deixando a
decisão para os juízes em cada caso. p
j
Em seguida à análise da jurisprudência do stf, este artigo argu-
menta que a solução dada pelo Tribunal para essa questão não foi
plenamente adequada, uma vez que o direito à assistência social foi
interpretado e aplicado sem suficiente consideração da política de
assistência social que o concretiza. Na medida em que a efetivação
do direito à assistência social não é analisada como uma questão
de justiça distributiva e policêntrica,4 a qual, portanto, afeta muitos
outros não contemplados no processo judicial e de forma quase im-
previsível, ela foi reduzida à solução de uma disputa bilateral entre
um indivíduo versus o Estado. Como se espera discutir neste artigo,
essa simplificação do problema leva os ministros do stf a perderem
de vista que a possível ampliação de direitos da assistência social via
adjudicação é inexoravelmente uma escolha distributiva e política
entre diferentes necessidades, valores e demandas sociais que com-
petem por recursos escassos. NOVOS ESTUDOS 103 ❙❙ novembro 2015 137 137 [6] Penalva et al., 2010. O CRITÉRIO DE RENDA DO BENEFÍCIO DE PRESTAÇÃO CONTINUADA NO STF Jurisprudência do stf anterior ao re 567.985 ÃO DE DIREITOS E ESCOLHAS POLÍTICAS NA ASSISTÊNCIA SOCIAL ❙❙ Daniel Wei L. Wang, Natália Pires de Vasconcelos Jurisprudência do stf anterior ao re 567.985 20, §3o, da Loas,
para ser constitucional, não poderia ser interpretado como o único
critério para determinar a falta de meios para uma família prover o
sustento para o idoso ou pessoa com deficiência, mas apenas como
uma forma dentre várias outras para se comprovar tal situação. A decisão da maioria dos ministros nesse caso julgou pela consti-
tucionalidade do dispositivo. O voto do ministro Nelson Jobim, que
encabeçou a posição majoritária, julgou que a Constituição foi clara ao
determinar que “compete à lei dispor a forma de comprovação”, de tal
modo que a inclusão de outros critérios dependeria de mudança legis-
lativa e não de interpretação pelo stf. O ministro Sepúlveda Pertence
acrescentou ainda que provavelmente a lei seria omissa na previsão
de outras hipóteses necessárias à comprovação da miserabilidade. No
entanto, a ação direta de inconstitucionalidade não era, para ele, o re-
médio adequado para sanar essa omissão. A posição dissidente do relator ministro Ilmar Galvão e do mi-
nistro Néri da Silveira, ao contrário, acompanhou o parecer da Pro-
curadoria-Geral da República, o qual recomendava que o texto fosse
declarado inconstitucional sem redução de texto, por meio da técnica
de interpretação conforme a Constituição. Para esses dois ministros
vencidos, a única interpretação constitucional possível do critério que 138 ADJUDICAÇÃO DE DIREITOS E ESCOLHAS POLÍTICAS NA ASSISTÊNCIA SOCIAL ❙❙ Daniel Wei L. Wang, Natália Pires de Vasconcelos 138 ADJUDICAÇÃO DE DIREITOS E ESCOLHAS POLÍTICAS NA ASSISTÊNCIA SOCIAL ❙❙ Daniel Wei L. Wang, Natália Pires de Vas não “afastaria grande parte dos destinatários do benefício” deveria
permitir compreendê-lo apenas como uma presunção juris et juris, ou
seja, exigência suficiente para comprovação de miserabilidade, mas
não necessária, já que outros critérios também poderiam servir para
determinar a real situação de incapacidade econômica da família do
idoso ou portador de deficiência. Apesar de dissidente e minoritária, essa interpretação do critério
de 1/4 de salário mínimo per capita foi assumida por muitas decisões
em tribunais federais inferiores, o que ensejou novas manifestações
do stf, primeiro monocraticamente7 e depois em plenário no julga-
mento do Agravo Regimental da Reclamação 2.303, em maio de 2004. Nessa decisão, o stf, liderado pelo voto da relatora ministra Ellen
Gracie, reiterou o precedente e julgou que a decisão reclamada (que
concedera o benefício de prestação continuada sem o cumprimento
de 1/4 de salário mínimo) estava em desacordo com o julgamento do
stf na adi 1.232. Também na decisão desse agravo formou-se outra dissidência
minoritária, sobretudo com o voto do ministro Carlos Ayres Britto. Este recuperou a posição vencida na adi 1.232 para então votar pela
procedência do recurso e, portanto, pela interpretação que compreen-
de a exigência de 1/4 de salário mínimo com um critério objetivo que
não impede “que o particular pretendente ao benefício mensal possa
eventualmente comprovar, mesmo ultrapassando esse parâmetro de
ganhos per capita, a necessidade de assistência material-financeira”. Mesmo depois do julgamento do referido agravo, a posição mi-
noritária do stf continuou a ser adotada em muitos tribunais infe-
riores8 que concediam o benefício de prestação continuada a idosos
e portadores de deficiência com renda familiar per capita superior a
1/4 de salário mínimo. Isso levou a novos recursos e reclamações in-
terpostos pelo Instituto Nacional do Seguro Social (inss) perante o
stf, alegando novamente o descumprimento do precedente da Corte
firmado na adi 1.232. [8] Penalva et al., 2010. Os próprios ministros, no entanto, começariam a se posicionar
monocraticamente de forma favorável à concessão dos benefícios
mesmo quando não cumprido esse critério de renda. [7] Conferir: ai 471.005 agr, rel.
min. Gilmar Mendes, j. 17 dez. 2003;
ai 475.496, rel. min. Marco Aurélio,
j. 19 mar. 2004; e Rcl 2303, rel. min.
Ellen Gracie, j. 1 abr. 2004. [9] Conferir: Rcl 3.891, rel. min. Car-
los Velloso, j. 1 dez. 2005; Rcl 3.805,
rel min. Cármen Lúcia, j. 9 out. 2006;
Rcl 3.975 mc, rel. min. Sepúlveda
Pertence, j. 23 fev. 2006; Rcl 4.156,
rel. min. Eros Grau, j. 13 mar. 2006;
Rcl 4.137, rel. min. Eros Grau, j. 13 mar.
2006; Rcl 4.270, rel. min. Eros Grau,
j. 17 abr. 2006; Rcl. 4.280, rel. min.
Sepúlveda Pertence, j. 20 jun. 2006;
Rcl 4.145, rel. min. Marco Aurélio, j. 30
abr. 2006; Rcl 4.374, rel. min. Gilmar
Mendes, j. 1 fev. 2007. [8] Penalva et al., 2010. [8] Penalva et al., 2010. 138 ADJUDICAÇÃO DE DIREITOS E ESCOLHAS POLÍTICAS NA ASSISTÊNCIA SOCIAL ❙❙ Daniel Wei L. Wang, Natália Pires de Vasconcelos Nas decisões da
Corte entre maio de 2004 e fevereiro de 20089 (quando declarada a
repercussão geral do re 567.985 sobre o tema), os ministros indivi-
dualmente passaram a vincular-se cada vez mais à posição até então
dissidente, não apenas argumentando que a adi 1.232 não teria deter-
minado o critério de 1/4 como o único e suficiente para aferir pobreza,
mas também que a interpretação conjunta das regras constitucionais,
da previdência e assistência social, permitiria compreender renda e
pobreza de forma mais abrangente. Nas palavras de Carlos Velloso ao
recuperar e ratificar a decisão recorrida, por exemplo: NOVOS ESTUDOS 103 ❙❙ novembro 2015 139 Ora, em decorrência da Emenda Constitucional n. 31/2000, que ins-
tituiu o Fundo de Combate e Erradicação da Pobreza, e foi regulamenta-
do pela Lei Complementar n. 111/2001, que no §2o do art. 3o remeteu ao
Executivo a definição do conceito de linha de pobreza, forçoso é reconhecer
que o valor da linha de pobreza definido pelo inciso i do art. 4o do Decreto
n. 3.997/2001 também se aplica para fins de obtenção do benefício assisten-
cial de prestação continuada de que tratam o art. 203, inciso v, da Constitui-
ção Federal de 1988 e o art. 20 da Lei n. 8.742/93, sob pena de afronta ao
princípio da dignidade da pessoa humana (art. 1º, inciso iii, cf/88), já que
não se pode admitir que a uma pessoa na linha de pobreza seja negado um
benefício de Assistência Social.10 Apesar de individualmente os ministros se posicionarem contrá-
rios a uma interpretação estrita e suficiente do critério de 1/4, o plená-
rio do stf permaneceu reafirmando a posição estabelecida na adi,11
dando provimento a recursos e reclamações do inss que alegassem
descumprimento desse precedente. Essa situação levou à convivência
de duas posições diferentes da Corte: o critério de 1/4 ora era visto
como insuficiente e até superável para a constatação da condição de
miserabilidade e concessão do benefício, ora era confirmado invocan-
do-se a posição majoritária da adi 1.232. [11] Como informa o ministro Teori
Zavascki em seu voto no re 567.985:
“O Plenário, todavia, continuava
mantendo o que foi decidido na adi
1.232/df (v. g.: Rcl-mc-AgR 4.427/
RS, Pleno, rel. min. Cezar Peluso, j. 6
jun. 2007, dj 29 jun. 2007, p. 23; Rcl
2.323/pr, Pleno, rel. min. Eros Grau,
j. 7 abr. 2005, dj 20 maio 2005, p. 8;
Rcl-agr 2.303/rs, Pleno, rel. min. [10] Rcl 3.891, j. 1 dez. 2005. 138 ADJUDICAÇÃO DE DIREITOS E ESCOLHAS POLÍTICAS NA ASSISTÊNCIA SOCIAL ❙❙ Daniel Wei L. Wang, Natália Pires de Vasconcelos El-
len Gracie, j. 13 maio 2004, dj 1 abr. 2005, p. 5)”. Recursos semelhantes, normalmente ajuizados pelo inss quando
perdia nas instâncias federais a quo poderiam, então, obter destinos
bastante diversos caso fossem decididos monocraticamente ou leva-
dos a plenário. Em razão da convivência desses dois possíveis resulta-
dos em meio às decisões do stf e na tentativa de, de uma vez por todas,
vincular todos os demais tribunais à sua posição majoritária, em feve-
reiro de 2008 o stf decide pela repercussão geral do re 567.985, de
relatoria do ministro Marco Aurélio. O re 567.985 O recurso extraordinário 567.985 foi julgado em 10 de abril de
2013. Interposto pelo inss contra decisão da Turma Recursal da Se-
ção Judiciária do Estado do Mato Grosso, questionava a concessão
judicial do benefício de prestação continuada a indivíduo que não
preenchia o critério de 1/4 de salário mínimo per capita como renda
familiar, alegando descumprimento do quanto determinado pelo pre-
cedente da adi 1.232. A decisão recorrida não só assumia que esse critério não seria
necessário para a concessão do benefício como teria se modificado
para meio salário mínimo com o advento das Leis n. 9.533/1997 (lei
que autoriza a União a conceder apoio financeiro aos municípios
que instituírem programas de renda mínima) e n. 10.689/2003 140 ADJUDICAÇÃO DE DIREITOS E ESCOLHAS POLÍTICAS NA ASSISTÊNCIA SOCIAL ❙❙ Daniel Wei L. Wang, Natália Pires de Vasconcelos (Lei do Programa Nacional de Acesso à Educação). Outros argu-
mentos a favor dessa posição são trazidos pelo defensor público-
-geral da União e pelo Instituto de Bioética, Direitos Humanos e
Gênero (Anis), que figuravam como interessados no caso. Dentre
esses argumentos estão: 1/4 de salário mínimo não se mostraria
mais um padrão adequado para aferir a miserabilidade individual
e familiar; outras leis teriam modificado esse critério (tais como as
citadas pela decisão recorrida bem como o Decreto n. 3.887/2001,
que instituiu o Fundo de Combate e Erradicação da Pobreza); e um
valor mínimo maior serviria como melhor parâmetro a concessão
do benefício, pois evitaria os erros de focalização que já estariam
presentes na política, além de aumentar a população legalmente
beneficiária com um custo adicional de “apenas” 3% sobre o orça-
mento previdenciário total. Duas visões sobre o problema concorreram na decisão desse caso. A primeira, que fundamentou a posição vencedora, é capitaneada pe-
los ministros Marco Aurélio e Gilmar Mendes. Para ambos, o critério
de 1/4 de salário mínimo sofreria de um vício de omissão parcial, já
que não seria suficiente para aferir a real condição de miserabilidade
do beneficiário, que, em muitos casos, poderia ter renda per capita fa-
miliar superior, mas ainda assim ser considerado pobre e necessitado
do benefício. Para os ministros, a determinação constitucional desse
benefício exigiria apenas que o indivíduo comprovasse sua necessi-
dade. Isso porque o direito ao benefício decorreria da própria Cons-
tituição, e não da política social criada com a Loas, que apenas o teria
operacionalizado. O re 567.985 Mais ainda, assumir que o critério de 1/4 seria uma
forma suficiente de o Estado identificar os mais necessitados permi-
tiria desprezar todos aqueles que, não cumprindo o critério, teriam
ainda assim seu direito à dignidade humana ameaçado por viverem
em situação de pobreza. Esse critério, inclusive, teria passado por processo de “inconsti-
tucionalização” paulatina causada por mudanças fáticas e jurídicas
derivadas, sobretudo, da criação de outros programas de transferência
de renda, como o Bolsa Família, e da adoção de outras políticas sociais
que utilizam o valor de meio salário mínimo para aferição da pobre-
za. Esse processo estaria conduzindo a um esvaziamento da decisão
tomada na adi 1.232, reiterado pelas mudanças na própria jurispru-
dência recente do stf. Nenhum dos dois ministros, contudo, argu-
mentou pela superação desse precedente, ou seja, a simples declaração
de inconstitucionalidade do critério de 1/4 de salário mínimo, já que
reconhecer a nulidade nesse caso levaria a um “vácuo” jurídico que
poderia prejudicar a concessão de novos benefícios. É sobre a solução
desse problema — o que fazer com o critério de 1/4 — que os dois
ministros divergem, como se verá a seguir. 141 NOVOS ESTUDOS 103 ❙❙ novembro 2015 141 A segunda visão do problema, que fundamenta os votos dissi-
dentes dos ministros Teori Zavascki e Ricardo Lewandowski, ar-
gumenta que a discussão em questão se apresentaria exatamente
nos mesmos termos daquela da adi 1.232. Portanto, seria possível
rever essa decisão em recurso extraordinário apenas se o critério
de renda da Loas tivesse sofrido uma inconstitucionalidade su-
perveniente, o que para os ministros vencidos não ocorrera. Como
assinala em seu voto o ministro Teori Zavascki, ao entender ser
impossível comparar o bpc com outras políticas com objetivos e
contornos próprios: O benefício decorrente do art. 203, v, da Constituição, de natureza
individual, disciplinado no art. 20, §3º da Lei 8.742/1993, tem confi-
guração e pressupostos normativos próprios, insuscetíveis de equipara-
ção com outros benefícios sociais, de natureza familiar, como o da Lei
10.689/2003, que cria o Programa Nacional de Acesso à Alimentação,
ou o da Lei 9.533/1997, que incentiva a criação de programas de garan-
tia de renda mínima associados a ações socioeducativas. 142 ADJUDICAÇÃO DE DIREITOS E ESCOLHAS POLÍTICAS NA ASSISTÊNCIA SOCIAL ❙❙ Daniel Wei L. Wang, Natália Pires de Vasconcelos O re 567.985 Por isso mesmo,
não se pode supor que a mudança na legislação em relação aos requisitos
ou pressupostos para a concessão de um desses benefícios autorize a con-
clusão de que os mesmos pressupostos ou requisitos devam ser aplicados
aos demais. A partir da posição majoritária, o stf negou provimento ao re-
curso, mas optou por não declarar a nulidade do texto. O ministro
Marco Aurélio, relator do caso, entendeu que o critério de 1/4 de-
veria ser considerado constitucional, desde que sua interpretação
e a necessidade de cumpri-lo ou eventualmente ultrapassá-lo cou-
besse aos juízes a quo pela análise caso a caso. O ministro Gilmar
Mendes, por outro lado, também temeroso de que a declaração de
inconstitucionalidade levasse à nulidade do critério, sugere outra
solução ao problema, esta adotada pela Corte: a declaração parcial de
inconstitucionalidade sem redução do texto. Apesar de propor que a
Corte determinasse um prazo de pouco mais de dois anos (até 31 de
dezembro de 2015) para que o Congresso editasse uma nova legis-
lação sobre a matéria, tal proposta do ministro não obteve a maioria
de 2/3 necessária à modulação de efeitos da decisão. Ao final, a Corte decidiu por negar provimento ao recurso, de-
clarando incidentalmente a inconstitucionalidade parcial do §3º do
art. 20 da Lei n. 8.742/1993, sem redução de seu texto. Desse modo,
o critério de 1/4 continuaria vigente e aplicável, mas sem excluir ou-
tros critérios ou fontes de prova que pudessem aferir a condição de
miséria no caso concreto, ao menos enquanto se espera nova regula-
mentação do Congresso Nacional, sem prazo para ocorrer. O STF E SUA ESCOLHA DE POLÍTICA PÚBLICA A importância do bpc é inegável no combate à pobreza e à desi-
gualdade, assim como na proteção social que uma renda estável ofere-
ce a idosos e pessoas portadoras de deficiência.12 O bpc tem ampliado
seu alcance desde sua criação. A idade mínima para recebimento do
benefício, inicialmente de setenta anos, foi reduzida para 68 e atual-
mente é de 65 anos. Também o conceito de deficiência foi ampliado
para incluir não apenas a dimensão médica, mas também o contexto
social e ambiental em que o indivíduo vive.13 Além disso, o aumento
significativo do salário mínimo na última década elevou o patamar
real de elegibilidade para se tornar beneficiário do programa, bem
como os gastos com benefícios. Observa-se, assim, que o bpc tem se
expandido, o que se reflete tanto na ampliação do número de benefi-
ciários como no gasto com o bpc nas últimas décadas (ver Gráfico 1). O critério de renda de 1/4 do salário mínimo per capita, porém, não
foi objeto de alteração legislativa, apesar dos 53 projetos de lei que
entre 1994 e 2010 propuseram seu aumento, mas que não obtiveram
apoio suficiente no Executivo e Legislativo para aprovação.14 Esse cri-
tério absoluto de renda é criticado por alguns especialistas. Medeiros,
Diniz e Squinca, por exemplo, consideram que “uma linha de pobreza
única que não considere particularidades das famílias é inadequada
nesse caso. Esse tipo de linha tende a tratar do mesmo modo famílias
que têm necessidades bastante diferentes”.15 Com base nessa premis-
sa, a ampliação judicial da cobertura do bpc por meio da flexibilização
do critério de renda é vista por alguns como um avanço na promoção
do direito à assistência social.16 [14] Miranda, 2012, p. 33. Sensibilizar-se com a situação desses demandantes é humano, e
a possibilidade de ajudá-los por meio da adjudicação e da linguagem
dos direitos é bastante atrativa. O stf, sobretudo no re 567.985,
entendeu cumprir sua função de controlar os outros poderes para
a realização de direitos fundamentais e de objetivos estabelecidos
na Constituição (solidariedade, dignidade, erradicação da pobreza,
assistência aos desemparados). A opinião da maioria dos ministros
pela flexibilização do critério de 1/4 de salário mínimo baseou-se
nas necessidades daqueles que, embora considerados pelos juízes
em situações de vulnerabilidade social, ficariam excluídos do bpc
por estarem acima do limiar de renda estabelecido em lei. [12] Jaccoud; Hadjab; Cheibub, 2010,
p. 16; Medeiros; Sawaya Neto; Gran-
ja, 2009; Medeiros; Diniz; Squinca,
2006; Medeiros; Britto; Soares,
2007; Datamétrica, 2010; Soares et
al., 2006. [16] Ivo; Silva, 2011; Penalva; Diniz;
Medeiros, 2010; Pereira, 2013. [15] Medeiros; Diniz; Squinca, 2006,
p. 12. [13] Jaccoud; Hadjab; Cheibub, 2010,
p. 6-7. 142 ADJUDICAÇÃO DE DIREITOS E ESCOLHAS POLÍTICAS NA ASSISTÊNCIA SOCIAL ❙❙ Daniel Wei L. Wang, Natália Pires de Vasconcelos 142 ADJUDICAÇÃO DE DIREITOS E ESCOLHAS POLÍTICAS NA ASSISTÊNCIA SOCIAL ❙❙ Daniel Wei L. Wang, Natália Pires de Vasconcelos [14] Miranda, 2012, p. 33. NOVOS ESTUDOS 103 ❙❙ novembro 2015 O STF E SUA ESCOLHA DE POLÍTICA PÚBLICA A decisão
expandiu por via judicial a abrangência do benefício para que, assim,
protegesse os direitos desses cidadãos a receber esse benefício da
assistência social. A avaliação quanto à extensão do alcance do bpc, no entanto,
assim como a de qualquer política pública fundada nos princípios
de solidariedade social, não pode ser feita com base apenas em quem NOVOS ESTUDOS 103 ❙❙ novembro 2015 143 se beneficia dessa expansão. Em um cenário de recursos públicos
escassos e inúmeras demandas sociais, qualquer escolha por uma
determinada política para um determinado grupo implica que ou-
tras políticas e outros grupos deixarão de ser atendidos. Conforme
afirmou Wang, “a escassez de recursos exige que o Estado faça esco-
lhas, o que pressupõe preferências e que, por sua vez, pressupõem
preteridos”.17 Portanto, qualquer política deve ser avaliada também
com base no seu custo de oportunidade, aquilo que poderia ser ga-
nho e aqueles que poderiam ser beneficiados com o uso alternativo
dos mesmos recursos. É preciso ter claro que por trás do discurso jurídico na decisão
do stf no re 567.985 está uma escolha de política pública. Valer-se
da linguagem do direito e dos direitos para defender certos grupos,
em casos como esse, é fazer escolhas distributivas e políticas entre
diferentes necessidades e demandas sociais que competem por re-
cursos escassos.18 Uma boa política de assistência social deve considerar a necessi-
dade de um dado grupo em comparação com a necessidade de outros
igualmente ou mais vulneráveis. Ela deve, portanto, levar em consi-
deração quem são aqueles na sociedade cujas necessidades são mais
prementes, definindo prioridades em um contexto de escassez de re-
cursos de modo a maximizar os benefícios e obter um equilíbrio entre
a abrangência da política (a quantidade de pessoas que ela atinge) e
sua intensidade (o tamanho do benefício a ser oferecido). Essas são
questões de escolha política, de eficiência econômica, justiça distribu-
tiva e valores sociais que tendem a ser negligenciadas quando se anali-
sa a questão apenas sob a ótica das necessidades de alguns indivíduos,
por mais relevantes que elas sejam, deslocadas de um contexto social
maior. Esses elementos estão praticamente ausentes (com exceção da
breve colocação do ministro Teori Zavascki, acima exposta) na decisão
do re 567.985 pelo stf. O stf considerou que o limiar de renda estabelecido pela Loas
é parcialmente inconstitucional por determinar um limite absoluto
de renda familiar per capita para recebimento do benefício. [18] Kennedy, 2004, p. xxiii; Wal-
dron, 2010; Griffith, 1979, p. 15;
Koskenniemi, 2010; Shapiro; Sweet,
2002, p. 180; Wesson, 2012. [17] Wang, 2008, p. 540. ÃO DE DIREITOS E ESCOLHAS POLÍTICAS NA ASSISTÊNCIA SOCIAL ❙❙ Daniel Wei L. Wang, Natália Pires de Vasconcelos [17] Wang, 2008, p. 540. O STF E SUA ESCOLHA DE POLÍTICA PÚBLICA Ao não
estabelecer outro limite de renda para a concessão do benefício ou
formas de interpretar o critério de 1/4, contudo, a Corte deixa a crité-
rio dos demais tribunais e juízes avaliar a necessidade de cada indi-
víduo no caso concreto. Uma solução como tal impõe aos gestores a
difícil tarefa de decidir sobre a concessão de benefícios sem critérios
claros, sujeitando suas decisões à constante e incerta revisão, sem
que se saiba minimamente o que esperar de cada decisão judicial. Do ponto de vista dos demandantes que recorrem ao Judiciário para
ver reconhecido seu direito ao benefício, a situação é de igual impre-
visibilidade e de provável iniquidade. A possibilidade de variadas 144 ADJUDICAÇÃO DE DIREITOS E ESCOLHAS POLÍTICAS NA ASSISTÊNCIA SOCIAL ❙❙ Daniel Wei L. Wang, Natália Pires de Vasconcelos interpretações por parte de diferentes juízes sobre os critérios para
concessão do bpc transforma o Judiciário em uma loteria que pode
atribuir resultados diferentes a pessoas em situação semelhante. Ademais, essa situação de incerteza deve permanecer mesmo que a
Loas venha a ser modificada legislativamente para incluir um novo
patamar de renda, já que, segundo a decisão no re 567.985, é a ne-
cessidade do indivíduo, a ser aferida no caso concreto, que determi-
na seu direito de receber o bpc. Por outro lado, embora a decisão não tenha sido clara quanto a
isso, a comparação do bpc com outras políticas de assistência social
sugere que o stf pode aceitar um critério de renda mais alto para a
concessão do benefício, a ser estabelecido por lei. Essa possibilidade,
porém, ignora o já mencionado trade-off entre a extensão de uma
política e o tamanho de seu benefício, ideia que será retomada nos
parágrafos a seguir. Embora o real impacto orçamentário da decisão do stf seja incer-
to, estimou-se em 2010 que, na hipótese de o critério de renda familiar
ser elevado para meio salário mínimo per capita em vez de 1/4 (uma
das propostas que foi discutida pelo stf), e todas as outras variáveis
permanecendo constantes, haveria um aumento de 48% no número
de pessoas elegíveis para recebimento do benefício, o que representa-
ria, à época, um impacto orçamentário de 8,9 bilhões de reais.19 Esse
mesmo dado foi mencionado, inclusive, pelo ministro Marco Aurélio
no relatório do re 567.985. [19] Penalva; Diniz; Medeiros, 2010,
p. 63. [20] Penalva; Diniz; Medeiros, 2010. NOVOS ESTUDOS 103 ❙❙ novembro 2015 O STF E SUA ESCOLHA DE POLÍTICA PÚBLICA Penalva, Diniz e Medeiros20 argumentam
que esse seria um custo absorvível pela capacidade orçamentária da
União, uma vez que representaria “apenas” 2,4% do orçamento da
seguridade social. [20] Penalva; Diniz; Medeiros, 2010. Os números, por si só, já impressionam. Contudo, o gasto com
a flexibilização do critério de renda do bpc é mais bem dimensiona-
do se compararmos o bpc com outras políticas de assistência social,
como o Programa Bolsa Família (pbf) e o Programa Brasil Carinhoso
(pbc). Isso torna mais claro como a decisão da maioria dos ministros
no re 567.985 representa de fato uma escolha política com altos cus-
tos de oportunidade. Como já mencionado, o fato de que os recursos são escassos faz
com que as políticas de assistência social precisem lidar com o trade-off
entre abrangência e tamanho do benefício. O bpc oferece o benefí-
cio mais alto de todos os programas de assistência social (um salário
mínimo) e, de acordo com dados extraídos da Matriz de Informação
Social do Ministério de Desenvolvimento Social, atendeu cerca de 3,9
milhões de indivíduos em 2013 a um custo de aproximadamente 31,5
bilhões de reais. O pbf, por sua vez, é uma política que optou pela abrangência,
atingindo um grande número de pessoas, ainda que oferecendo um NOVOS ESTUDOS 103 ❙❙ novembro 2015 145 benefício menor. O pbf é voltado a famílias em situação de pobreza
(renda per capita inferior a 140 reais) e pobreza extrema (com renda
per capita inferior a setenta reais). Divide-se esse benefício em duas
partes, uma composta pelo benefício básico e outra formada por um
adicional pago por criança e jovem na família. Os valores pagos são
relativamente baixos se comparados ao bpc, já que variam entre 32 e
306 reais, a depender do nível de pobreza da família e do número de
crianças e adolescentes que fazem parte dela. Em 2013, de acordo os
dados da Matriz de Informação Social do Ministério de Desenvolvi-
mento Social, o pbf beneficiou 14 milhões de famílias a um custo de
pouco mais de 24 bilhões de reais. O STF E SUA ESCOLHA DE POLÍTICA PÚBLICA Além do foco nos mais pobres e miseráveis, o pbf tem também foco
em beneficiar crianças e adolescentes, a faixa etária onde está a maior
proporção de miseráveis no Brasil.21 Isso pode ser observado no fato de
que, dentro do pbf, o benefício que teve maior valorização real (descon-
tada a inflação) foi o adicional por criança, sendo este o responsável pelo
aumento da transferência média por beneficiário do pbf.22 O foco nesse
grupo foi ainda mais reforçado com a criação do pbc, uma política com-
plementar ao pbf, pela Medida Provisória n. 570/2012 (transformada
na Lei Ordinária n. 12.722/2012). Essa política é voltada para famílias
com crianças de zero a quinze anos que, mesmo após receberem o pbf,
ainda não conseguem ultrapassar o limiar de extrema pobreza (renda
per capita de setenta reais). O orçamento do pbc estimado para esse
programa em 2013 foi de 1,74 bilhão de reais e existe previsão de que ele
reduza o nível de miséria no Brasil para menos de 1% da população.23 O resultado dessas escolhas de abrangência e focalização do pbf
reflete-se no sucesso da política internacionalmente reconhecida e ad-
mirada por oferecer marcante impacto em diversos indicadores sociais,
como pobreza, miséria e mortalidade infantil,24 sendo ela, dentre todas
as políticas de assistência social, a que mais contribuiu para a redução
da desigualdade social.25 E tudo isso a um custo relativamente baixo.26 [24] Campello; Neri, 2013; Rasella et
al., 2013. [25] Soares et al., 2006, p. 36; Hoff-
mann, 2013. [26] Coutinho, 2013. [27] Penalva; Diniz; Medeiros, 2010. Se aplicarmos a metodologia utilizada por Penalva, Diniz e Me-
deiros27 para calcular o impacto orçamentário da flexibilização do
critério de renda da Loas para recebimento do bpc no período de
2005-2013, chegamos à conclusão de que, caso o limiar de renda
para recebimento do bpc tivesse sido elevado para meio salário mí-
nimo per capita em todos os anos, o gasto extra com o bpc repre-
sentaria mais do que 60% daquilo que foi gasto com o pbf e para
beneficiar um número muito menor de pessoas (ver Gráfico 1). Esse
gasto adicional (corrigido pela inflação com base no ano de 2005)
em 2013 seria também o equivalente a 8,6 vezes o valor nominal do
orçamento do Programa Brasil Carinhoso. [23] Osório; Souza, 2012. [24] Campello; Neri, 2013; Rasella et
al., 2013. [22] Osorio; Souza, 2012, p. 4. [21] Ipea, 2013, p. 49. [25] Soares et al., 2006, p. 36; Hoff-
mann, 2013. [26] Coutinho, 2013. ÃO DE DIREITOS E ESCOLHAS POLÍTICAS NA ASSISTÊNCIA SOCIAL ❙❙ Daniel Wei L. Wang, Natália Pires de Vasconcelos [22] Osorio; Souza, 2012, p. 4. [27] Penalva; Diniz; Medeiros, 2010. [24] Campello; Neri, 2013; Rasella et
al., 2013.
[25] Soares et al., 2006, p. 36; Hoff-
mann, 2013.
[26] Coutinho, 2013.
[27] Penalva; Diniz; Medeiros, 2010. [27] Penalva; Diniz; Medeiros, 2010. O STF E SUA ESCOLHA DE POLÍTICA PÚBLICA Importa ressaltar que essa é apenas uma estimativa do impacto que
a decisão do stf pode ter, uma vez que ele não definiu um novo limiar Gráfico 1 – Comparação entre custo e benefícios do bpc e pbf 16
14
12
10
8
6
4
2
0
25
20
15
10
5
0
2005
2006
2007
2008
2009
2010
2011
2012
2013
Gasto em bilhões de Reais de 2005
Indivíduos ou famílias em milhões
Benefícios bpc (indivíduo)
Benefícios bpc (indivíduo) extra estimado com
elevação do critério de renda (1/2 salário mínimo)
Benefícios bpf (família)
Gasto bpc
Gasto bpc extra estimado com elevação
do critério de renda (1/2 salário mínimo)
Gasto bpf
Fonte: mds — Matriz de Informação Social. Elaboração própria. Inflação corrigida pelo ipca com ano-base de 2005. Fonte: ibge 16
14
12
10
8
6
4
2
0
25
20
15
10
5
0
2005
2006
2007
2008
2009
2010
2011
2012
2013
Gasto em bilhões de Reais de 2005
Indivíduos ou famílias em milhões
(
)
G
bpc 16
14
12
10
8
6
4
2
0
25
20
15
10
5
0
2005
2006
2007
2008
2009
2010
2011
2012
2013
Gasto em bilhões de Reais de 2005
Indivíduos ou famílias em milhões de renda. O real impacto, portanto, vai depender de como a questão será
tratada nos tribunais inferiores, no Legislativo e em futuras decisões do
próprio stf. Porém, essa estimativa e a comparação com o pbf e o pbc
têm por intuito mostrar que a decisão da Corte, ao flexibilizar ou alte-
rar o critério de 1/4 de salário mínimo per capita, assume possivelmente
um custo de oportunidade altíssimo. A depender de como o impacto
orçamentário será absorvido pelo governo, isso pode impedir, dificultar
ou atrasar a expansão de outras políticas assistenciais (tanto em maior
cobertura quanto em aumento do valor dos benefícios pagos). Como se observou no Gráfico 1, os gastos com o bpc têm crescido
nos últimos anos, de tal modo que controlar os custos dessa política
por meio da manutenção do critério de 1/4 de salário mínimo per ca-
pita não significa necessariamente descaso com os mais necessitados,
insensibilidade social ou, como afirmaram alguns ministros do stf,
uma omissão estatal em atualizar uma regra ultrapassada. [28] Soares et al., 2006. NOVOS ESTUDOS 103 ❙❙ novembro 2015 [28] Soares et al., 2006. O STF E SUA ESCOLHA DE POLÍTICA PÚBLICA Tal critério
pode ser visto também como uma forma de garantir a focalização do
próprio bcp nos mais pobres, um elemento importante para o suces-
so da política no combate à desigualdade,28 além de permitir que re-
cursos da assistência social estejam disponíveis para viabilizar outras
políticas ainda mais focalizadas nos mais necessitados. A decisão do stf, assim, não só assumiu uma escolha política de po-
tencial grande impacto como também contrariou os caminhos da própria
política de assistência social instituída no Brasil até então. Observa-se
que a decisão forçaria a ampliação de uma política de relativo alto custo NOVOS ESTUDOS 103 ❙❙ novembro 2015 147 (alto benefício), se comparada com outras políticas de assistência social,
a um público de renda familiar per capita um pouco mais elevada (acima
de 181 reais, de acordo com o salário mínimo de 2014), indo de encontro à
posição assumida até o momento pelo governo federal que oferece bene-
fícios menores para grandes grupos, com maior foco nos mais miseráveis
(com renda familiar per capita menor do que setenta reais). Ao decidir sobre o critério de renda do bpc, faltou à decisão do re
567.985 uma análise contextualizada do benefício dentro de toda a po-
lítica de assistência social e dos custos de oportunidade da ampliação
desse benefício, o que talvez permitisse à maioria do stf compreender a
necessidade de focalização e controle dos custos dessa e de outras políti-
cas. O ponto é que, para a maioria da Corte, a dimensão de política social
do bpc é ocultada pela sua dimensão de direito garantido pelo art. 203, v,
da Constituição e, sobretudo, pelo princípio da dignidade humana, em
um caminho argumentativo que privilegia uma relação direta entre o
beneficiário individual e seu direito constitucional, ignorando a política
pública e as escolhas políticas que se colocam em meio aos dois. Conclusão O argumento central deste artigo propõe que a proteção judicial do
direito à assistência social não pode estar dissociada da política de assis-
tência social. Considerando que essa é uma política de caráter não con-
tributivo calcada nos princípios da solidariedade social e universalidade
— financiada por toda a sociedade para todos que dela precisam —, ela
deve ser informada por valores, demandas e necessidades sociais, pela
sua eficiência econômica e por preocupações com justiça distributiva. O argumento central deste artigo propõe que a proteção judicial do
direito à assistência social não pode estar dissociada da política de assis-
tência social. Considerando que essa é uma política de caráter não con-
tributivo calcada nos princípios da solidariedade social e universalidade — financiada por toda a sociedade para todos que dela precisam —, ela
deve ser informada por valores, demandas e necessidades sociais, pela
sua eficiência econômica e por preocupações com justiça distributiva. Um dos riscos da adjudicação de direitos sociais, contudo, é trans-
formar questões policêntricas como essa em uma disputa bilateral entre
o litigante e o governo. Quando isso ocorre, a política social e todas as
difíceis escolhas que estão necessariamente por trás dela perdem seu
espaço central na lide judicial, que passa a focar-se quase exclusivamente
na necessidade dos litigantes. Essa foi fundamentalmente a forma como
a maioria do stf analisou a constitucionalidade do critério de 1/4 de
salário mínimo per capita para recebimento do bpc no re 567.985. Pelo teor dessa decisão, que abre a possibilidade de concessão do
benefício a pessoas com renda familiar per capita acima do critério es-
tabelecido na Loas, a palavra final sobre a condição de miserabilidade
de cada demandante acima desse patamar de renda tornou-se respon-
sabilidade e critério dos juízes caso a caso. Além da incerteza e imprevi-
sibilidade deixada aos tribunais inferiores, gestores e futuros benefici-
ários sobre como, respectivamente, julgar, gerir e o que esperar do bpc,
torna-se provável que a necessidade de cada indivíduo seja novamente
avaliada fora do contexto de uma política de assistência social. 148 ADJUDICAÇÃO DE DIREITOS E ESCOLHAS POLÍTICAS NA ASSISTÊNCIA SOCIAL ❙❙ Daniel Wei L. Wang, Natália Pires de Vasconcelos 148 ADJUDICAÇÃO DE DIREITOS E ESCOLHAS POLÍTICAS NA ASSISTÊNCIA SOCIAL ❙❙ Daniel Wei L. Wang, Natália Pires de Vasconcelos Com essa decisão, o stf opta por ampliar uma política que oferece
um benefício relativamente alto (o maior de todos da assistência so-
cial) para um grupo de pessoas com renda per capita acima do limiar
proposto por lei. Essa opção tem custos significativos, especialmente
se comparada a outras políticas como o Bolsa Família e o Brasil Cari-
nhoso, que oferecem pequenos benefícios, mas com amplo alcance en-
tre os mais miseráveis. Nesses custos não se contabiliza apenas quan-
to se espera gastar como consequência da decisão, mas, sobretudo, o
quanto deixou de ser ganho com o uso alternativo desses recursos
para, por exemplo, ampliar a cobertura ou o valor dos benefícios de
outras políticas de assistência social. A comparação entre o bpc e outras políticas de assistência social
não tem por objetivo argumentar que um aumento do critério de ren-
da para recebimento do benefício seria uma escolha política equivoca-
da. Uma avaliação sobre a oportunidade e conveniência dessa medida
exigiria uma análise muito mais completa do que a que seria possível
desenvolver neste trabalho. Este artigo pretende apenas apontar que,
diante desse tipo de questão — a qual envolve decisões políticas e
grande alocação de recursos orçamentários, possui efeitos policêntri-
cos e cuja solução depende de informações técnicas e conhecimento
dos valores, necessidades e preferências sociais —, existem boas ra-
zões para uma postura mais deferente do Judiciário com relação às
escolhas do Executivo e do Legislativo, sobretudo quando se trata
de uma política que apresentou diversos avanços, como é o caso da
assistência social no Brasil. Essa deferência não se dá por um apego
formal à doutrina da separação de poderes, mas por razões democráti-
cas e epistêmicas, ou seja, o reconhecimento pelo Judiciário de que os
outros poderes, em regra, possuem maior legitimidade e capacidade
institucional para decidir questões dessa natureza.29 Em suma, a escolha política de limitar a ampliação de um benefí-
cio de assistência social não significa necessariamente uma violação a
um direito causada por indiferença ou omissão estatal. Essa limitação
pode ser, como no caso do bpc e da assistência social, uma opção go-
vernamental que define prioridades e focalização. [29] King, 2012; Vermeule, 2006;
Sunstein; Vermeule, 2002; Kavanagh,
2008, p. 192. Daniel Wei L. Wang é professor de direito da Queen Mary University of London.
Natália Pires de Vasconcelos é doutoranda em direito constitucional na Universidade de
São Paulo. 148 ADJUDICAÇÃO DE DIREITOS E ESCOLHAS POLÍTICAS NA ASSISTÊNCIA SOCIAL ❙❙ Daniel Wei L. Wang, Natália Pires de Vasconcelos Por esse raciocínio,
uma decisão judicial que respeita o critério adotado pela política pú-
blica nessas circunstâncias não significa que o Judiciário abdica de sua
função na defesa de direitos, mas demonstra o entendimento de que
o direito à assistência social não é apenas daqueles que conseguem
chegar aos tribunais, mas de toda a população que precisa ser assistida
por essa política. Recebido para publicação
em 26 de março de 2014. NOVOS ESTUDOS
CEBRAP
103, novembro 2015
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national Journal of Human Rights, Humanitarianism, and Development, v. 1, n. 1, 2010. national Journal of Human Rights, Humanitarianism, and Development, v. 1, n. 1, 2010. Medeiros, Marcelo; Britto, Tatiana; Soares, Fábio. “Transferência de renda no Brasil”. Novos Estudos — Cebrap, n. 79, nov. 2007. iros, Marcelo; Britto, Tatiana; Soares, Fábio. “Transferência de renda no B 79, nov. 2007. 150 ADJUDICAÇÃO DE DIREITOS E ESCOLHAS POLÍTICAS NA ASSISTÊNCIA SOCIAL ❙❙ Daniel Wei L. Wang, Natália Pires de Vasconcelos Medeiros, Marcelo; Diniz, Debora; Squinca, Flávia. Transferências de renda para a população com deficiência no Brasil:
uma análise do Benefício de Prestação Continuada. Brasília: Ipea, Texto para Discussão n. 1.184, 2006. Disponí-
vel em: http://www.ipea.gov.br/portal/index.php?option=com_content&view=article&id=4782. Acesso
em: 10 nov. 2013. Medeiros, Marcelo; Sawaya Neto, Melchior; Granja, Fábio H. A distribuição das transferências, público-alvo e cobertura
do Benefício de Prestação Continuada (BPC). Brasília: Ipea, Texto para Discussão n. 1.416, 2009. Disponível em:
http://www.ipea.gov.br/portal/index.php?option=com_content&view=article&id=4948. Acesso em: 10
nov. 2013. Michelman, Frank. “The Constitution, Social Rights, and Liberal Political Justification”. International Journal of
Constitutional Law, v. 1, n. 1, p. 13-34, 2003. Miranda, Geralda Luiza de. Preferências, instituições e política na formulação e implementação do Benefício de Prestação Con-
tinuada. Brasília: Ipea, Texto para Discussão n. 1734, 2012. Disponível em: http://www.ipea.gov.br/portal/ tinuada. Brasília: Ipea, Texto para Discussão n. 1734, 2012. Disponível em: http://www.ipea.gov.br/portal/
index.php?option=com_content&view=article&id=15082. Acesso em: 10 nov. 2013. p
p
p
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index.php?option=com_content&view=article&id=15082. Acesso em: 10 nov. 2013. Osório, Rafael G.; Souza, Pedro Herculano Guimarães F. de. O Bolsa Família depois do Brasil Carinhoso: uma análise do p
potencial de redução da pobreza extrema. Brasília: Ipea, Nota Técnica n. 14, 2012. potencial de redução da pobreza extrema. Brasília: Ipea, Nota Técnica n. 14, 2012. Penalva, Janaína; Diniz, Debora; Medeiros, Marcelo. “O Benefício de Prestação Continuada no Supremo Tribunal
Federal”. Sociedade e Estado, v. 25, n. 1, abr. 2010. Federal”. Sociedade e Estado, v. 25, n. 1, abr. 2010. Pereira, Luciano Meneguetti. “Políticas públicas, direitos fundamentais e Poder Judiciário: uma análise crítica Pereira, Luciano Meneguetti. “Políticas públicas, direitos fundamentais e Poder Judiciário: uma análise crítica
do Benefício de Prestação Continuada (BPC)”. Revista Brasileira de Políticas Públicas, v. 3, n. 1, p. 25-50, jan./ Pereira, Luciano Meneguetti. “Políticas públicas, direitos fundamentais e Poder Judiciário: uma análise crítica
do Benefício de Prestação Continuada (BPC)”. Revista Brasileira de Políticas Públicas, v. 3, n. 1, p. REFERÊNCIAS BIBLIOGRÁFICAS 25-50, jan./
jun. 2013. Piovesan, Flavia. “Brazil: Impact and Challenges of Social Rights in the Courts”. In: Langford, Malcolm (Org.). Social Rights Jurisprudence: Emerging Trends in International and Comparative Law. Cambridge, UK: Cambridge
University Press, 2008. Rasella, Davide; Aquino, Rosana; Santos, Carlos A. T.; Paes-Sousa, Rômulo; Barreto, Maurício L. “Effect of a Con-
ditional Cash Transfer Programme on Childhood Mortality: A Nationwide Analysis of Brazilian Municipa-
lities”. The Lancet, v. 382, n. 9.886, p. 57-64, jul. 2013. p
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Shapiro, Martin; Sweet, Alec Stone. On Law, Politics and Judicialization. Oxford: Oxford University Press, 2002. Soares, Fábio Veras et al. Programas de transferência de renda no Brasil: impactos sobre a desigualdade. Brasília: Ipea, Texto Soares, Fábio Veras et al. Programas de transferência de renda no Brasil: impactos sobre a desigualdade. Brasília: Ipea, Texto
para Discussão n. 1228, 2006. Disponível em: http://www.anpec.org.br/encontro2006/artigos/A06A156 ,
g
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para Discussão n. 1228, 2006. Disponível em: http://www.anpec.org.br/encontro2006/artigos/A06A156. pdf. Acesso em: 10 nov. 2013. Sunstein, Cass R., Vermeule, Adrian. “Interpretation and Institutions”. U Chicago Law & Economics, Olin Working
Paper n. 156; U Chicago Public Law Research Paper n. 28, jul. 2002. Vermeule, Adrian. Judging Under Uncertainty: An Institutional Theory of Legal Interpretation. Cambridge; Londres: Har-
vard University Press, 2006. Waldron, Jeremy. “Socioeconomic Rights and Theories of Justice”. NYU School of Law, Public Law Research Paper
n. 10-79, 29 out. 2010. Wang, Daniel Wei Liang. “Escassez de recursos, custos dos direitos e reserva do possível na jurisprudência do
stf”. Revista Direito GV, v. 4, n. 2, dez. 2008. Wesson, Murray. “Disagreement and the Constitutionalization of Social Rights”. Human Rights Law Review, v. 12,
n. 2, 2012. NOVOS ESTUDOS 103 ❙❙ novembro 2015 151
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NOTICIAS MÉDICAS
Nuevas publicaciones nacionales
e internacionales
Hospital Metropolitano
New national and international publications
Metropolitan Hospital
Recibido: 08-06-2023
Publicado: 29-09-2023
DOI: https://doi.org/10.47464/MetroCiencia/vol31/3/2023/104-109
Revista MetroCiencia
Volumen 31, Número 3, 2023
ISSNp: 1390-2989 ISSNe: 2737-6303
Editorial Hospital Metropolitano
revistametrociencia.com.ec
Nuevas publicaciones nacionales e internacionales
Hospital Metropolitano 2023
Academic Publications
Metropolitan Hospital 2023
P
resentamos nuevas publicaciones
nacionales e internacionales de gran
importancia para el personal de salud. Las publicaciones que llevan el logo del
Hospital Metropolitano tienen, como autores, médicos de nuestra institución. Las personas que quieran aportar en esta sección,
lo pueden hacer enviando sus comentarios
a
educacion@hmetro.med.ec,referencia
Noticias Metro Ciencia.
Historia de la cirugía en Quito
Ha sido un privilegio especial participar en
la edición de este libro que cumple con el
deber ineludible de toda sociedad de reconocer la labor inconmensurable de los
forjadores de una de las actividades más
nobles y humanísticas del conglomerado
humano como es preservar la salud de los
ciudadanos. En el tratamiento de las dolencias y diversas patologías de los pacientes,
la cirugía ha sido a través de las diferentes
épocas, uno de los principales recursos
que han desarrollado los médicos para aliviarlas y conseguir, además, una curación
definitiva.
Si bien la cirugía es un arte manual, una intervención requiere un conocimiento profundo
e integral de la enfermedad y se constituye,
por otro lado, en un acto de gran humanismo al introducirse en la compleja anatomía
de un ser humano para entregarle salud y
bienestar físico y espiritual. Esta interacción
entre el cirujano y el paciente se reviste de
ética y honestidad, con enorme respeto a
la dignidad humana, conforme a lo que se
denomina cirugía centrada en la persona.
Al ser el depositario de la confianza del paciente y su familia, el cirujano tiene la grave
responsabilidad de realizar sus procedimientos con la mayor destreza y delicadeza
pues todo acto quirúrgico conlleva diversos
grados de riesgos de morbilidad y aún de
mortalidad. Una comunicación estrecha entre el cirujano, el paciente y la familia, para
que estos últimos sean conscientes de los
riesgos, es sumamente importante para evitar una ulterior mala interpretación entre una
complicación científicamente prevista y una
injusta denominación de mala práctica.
Usted es libre de:
Compartir — copiar y redistribuir el material en cualquier medio o formato.
La práctica de la cirugía ha tenido una tremenda evolución desde los procedimientos
prehistóricos, los de las antiguas civilizaciones y aquellos del barbero medieval, hasta
la presente utilización de recursos técnicos
Adaptar — remezclar, transformar y
construir a partir del material para cualquier propósito, incluso comercialmente.
*Correspondencia: drsantiagocampos@gmail.com
Campos-Miño S
complejos que incluyen intervenciones endoscópicas, laparoscópicas y robóticas,
programas computarizados complejos e
instrumentos y equipos sofisticados de gran
precisión. La actividad actual de un cirujano
requiere, además de acumular experiencia,
una educación continua permanente y una
intensa actividad académica y de enseñanza, complementada con la investigación y
la producción de literatura científica a nivel
nacional e internacional.
La obra que ahora presentamos, titulada
“Historia de la Cirugía en Quito”, relata el
desarrollo de la cirugía a través de la historia, pero sobre todo lo ocurrido en Quito, en
los siglos XX y actual, a partir de la fundación de la primera clínica Pasteur en 1926 y
el Hospital Eugenio Espejo en 1933. Consta
de 21 artículos, escritos por 32 autores, casi
todos cirujanos, algunos maestros de la especialidad y otros cirujanos en actividad, los
cuales, con enorme afán y dedicación, han
descrito del desenvolvimiento y desarrollo
de la cirugía desde los pioneros hasta los
especialistas actuales que utilizan los más
modernos recursos tecnológicos y científicos. En los diferentes capítulos se ha hecho
una amena y documentada descripción de
la evolución de la cirugía en el mundo, en
nuestro país, en nuestra ciudad y en cada
uno de las principales instituciones hospitalarias públicas y privadas de Quito. Las
fotografías y las imágenes de las personas
y lugares en donde se desarrolló la cirugía
en Quito, estamos seguros de que serán del
completo agrado de los lectores e impactarán emocionalmente a quienes recorran las
páginas de este libro.
106
106
Figura 1. Pacheco-Ojeda LA (editor), Mendoza-Orquera AG (coeditora), Alarcón-Benítez A (coeditor)
Historia de la cirugía en Quito, Sociedad Ecuatoriana
de Cirugía, Capítulo Pichincha, Quito, 2023. ISBN:
9789942884886
Dr. Luis Antonio Pacheco Ojeda
Cirujano General y Oncólogo
Hospital Metropolitano
Quito, Ecuador
Torre Médica 2, of. 213,
calle San Gabriel y Arteta y Calisto.
https://orcid.org/0000-0002-8192-5763
MetroCiencia Vol. 31 Nº 3 (2023)
NOTICIAS MEDICAS: Nuevas publicaciones nacionales e internacionales
Hospital Metropolitano 2023
Diagnóstico y manejo del asma severo
de tratamiento difícil en adolescentes y
adultos. Agosto 2023
Ultrasonido cardiopulmonar.
Springer. Agosto 2023
Editorial
Prevalencia de malnutrición y soporte
nutricional en UCIP Latinoamericanas.
Pediatric Critical Care Medicine. Agosto
2023
Neurocirugía para pacientes. Dr. Juan
Francisco Lasso. Agosto 2023
FEATURE ARTICLE
Downloaded from http://journals.lww.com/pccmjournal by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hC
ywCX1AWnYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC4/OAVpDDa8KKGKV0Ymy+78= on 10/23/2023
Malnutrition and Nutrition Support in Latin
American PICUs: The Nutrition in PICU
(NutriPIC) Study
OBJECTIVES: To characterize the nutritional status of children admitted to Latin
American (LA) PICUs and to describe the adequacy of nutrition support in reference to contemporary international recommendations.
DESIGN: The Nutrition in PICU (NutriPIC) study was a combined point-prevalence study of malnutrition carried out on 1 day in 2021 (Monday 8 November)
and a retrospective cohort study of adequacy of nutritional support in the week
preceding.
SETTING: Four-one PICUs in 13 LA countries.
PATIENTS: Patients already admitted to the PICU of 1 month to 18 years old on
the study day were included in the point-prevalence study. For the retrospective
arm, we included patients receiving nutritional support on the study day and with
a PICU length of stay (LOS) greater than or equal to 72 hours. Exclusion criteria
were being a neonate, conditions that precluded accurate anthropometric measurements, and PICU LOS greater than 14 days.
Santiago Campos-Miño, MD, MSc1
Artur Figueiredo-Delgado, PhD2
Patricia Zárate, MD3
Patricia Zamberlan, PhD2
Eliana Muñoz-Benavides, MD4
Jorge A. Coss-Bu, MD5
on behalf of the Nutrition
Committee, Latin American
Society of Pediatric Intensive
Care (SLACIP)
INTERVENTIONS: None.
MEASUREMENTS AND MAIN RESULTS: Of 316 patients screened, 5 did
not meet age criteria. There were 191 of 311 patients who were included in the
point-prevalence study and underwent anthropometric evaluation. Underweight
and length for age less than –2 z scores were present in 42 of 88 children (47.7%)
and 41 of 88 children (46.6%) less than 24 months old, and 14 of 103 (13.6%) and
(23/103) 22.3% of 103 children greater than or equal to 24 months, respectively. Evidence of obesity (body mass index > 2 z score) was present in 7 of 88
children (5.7%) less than 24 months old and 13 of 103 children (12.6%) greater
than or equal to 24 months. In the 115 of 311 patients meeting criteria for the
retrospective arm, a total of 98 patients reported complete nutritional data. The
7-day median (interquartile range) adequacy for delivered versus recommended
enteral energy and protein requirement was 114% (75, 154) and 99% (60, 133),
respectively.
CONCLUSIONS: The NutriPIC study found that in 2021 malnutrition was
highly prevalent especially in PICU admissions of less than 24 months old.
Retrospectively, the 7-day median nutritional support appears to meet both energy and protein requirements.
KEY WORDS: critically ill; malnutrition; nutrition adequacy; nutrition support;
obesity; pediatrics
T
he historical pediatric critical care research data on nutrition, largely
over 10 years old, indicate that during critical illness nutritional deficiencies and malnutrition are associated with greater odds of morbidity
and mortality (1). These studies report a prevalence of malnutrition ranging
from 15% to 50%, with a particular problem in Latin America (2, 3). Obesity
Pediatric Critical Care Medicine
Copyright © 2023 by the Society of
Critical Care Medicine and the World
Federation of Pediatric Intensive and
Critical Care Societies
DOI: 10.1097/PCC.0000000000003337
www.pccmjournal.org
1
Copyright © 2023 by the Society of Critical Care Medicine and Wolters Kluwer Health, Inc. All Rights Reserved.
MetroCiencia Vol. 31 Nº 3 (2023)
107
107
Campos-Miño S
PUNTAJE DE VExUS FETAL. Revista Chilena de Anestesia 2023
Caso Clínico
Ética en medicina intensiva. Editorial
Springer. Julio 2023
revistachilenadeanestesia.cl
DOI: 10.25237/revchilanestv52n5-19
Puntuación de VExUS fetal
Score de VExUS fetal
Edgardo Banille1,*, Alfredo Rodríguez-Portelles2, Javier Ponce3, Betina Vega1, Ricardo Ledesma1,a, Carlos Maure1,b, Mario Espósito1,b,
Marco Emmanuel Jiménez Texcalpa4
1
Servicio de Cardiología Pediátrica de Clínica del Sol. Córdoba. Argentina.
2
Unidad de Cuidados intensivos pediátricos Hospital Padre Carollo. Quito, Ecuador.
3
Servicio de Terapia Intensiva Pediátrica. Hospital Rawson. San Juan, Argentina.
4
Servicio de Terapia Intensiva. Centro Médico de Occidente. Guadalajara, México.
a
Servicio de Imágenes.
b
Servicio de Neonatología de Clínica Universitaria Reyna Fabiola. Córdoba, Argentina.
Ninguno de los autores presenta conflicto de intereses.
Fecha de recepción: 17 de abri de 2023 / Fecha de aceptación: 23 de mayo de 2023
ABSTRACT
We present a male patient of 1 day of life, preterm newborn (PTNB): 36 weeks of gestation and large for gestational age (LGA): Weight: 3,500
grams. Prenatal diagnosis of hypertrophic cardiomyopathy was made. She was born by cesarean section due to maternal history of type I diabetes
and hypothyroidism for 2 years of age, treated with NPH insulin 40 IU in the morning and corrections according to glycemia and levothyroxine 125
mg/day. The primary objective is to analyze the mechanisms involved in fetal heart failure and the physiopathogenesis of water overload by means
of a scoring system (VExUS score). The score is obtained with Doppler profiles of abdominal vessels (inferior vena cava, suprahepatic veins, portal
vein and renal veins). We tried to find a correlation with the fetal heart failure score described by Huhta and Paul in 1996, which combines cardiac
size and function with Doppler flow parameters of fetal abdominal vessels. Both scores reflected severe congestion. The secondary objective is to
observe the evolution of heart failure and venous congestion with the treatment implemented.
Key words: Nenatate, miocardiopaty, VExUS.
RESUMEN
Se presenta un paciente masculino de 1 día de vida, recién nacido pretérmino (RNPT): 36 semanas de gestación y grande para edad gestacional
(GEG): Peso: 3.500 gramos. Se realiza diagnóstico prenatal de miocardiopatía hipertrófica. Nace por operación cesárea por antecedentes
maternos con diabetes tipo I e hipotiroidismo desde los 2 años de edad, tratada actualmente con insulina NPH 40 UI por la mañana y correcciones
según glucemia y levotiroxina 125 mg/día. El objetivo primario es analizar los mecanismos involucrados en la insuficiencia cardíaca fetal y la
fisiopatogenia de la sobrecarga hídrica por intermedio de un sistema de puntaje (score VExUS). El mismo se obtiene con los perfiles Doppler de los
vasos abdominales (vena cava inferior, venas suprahepáticas, vena porta y venas renales) (Figuras 1). Tratamos de buscar una correlación con el
score de insuficiencia cardíaca fetal descripto por Huhta y Paul en el año 1996, este score combina el tamaño y la función cardíaca con parámetros
de flujo Doppler de vasos abdominales fetales (Figura 2). Ambos scores reflejaron congestión severa. El objetivo secundario, es observar la
evolución de la insuficiencia cardíaca y de la congestión venosa con el tratamiento implementado.
Palabras clave: Neonato, miocardiopatía, VexUS.
ebanille@hotmail.com
*ORCID: https://orcid.org/0000-0002-7433-3153
558
Rev. Chil. Anest. 2023; 52 (5): 558-564
Santiago Campos-Miño
Editor en Jefe, MetroCiencia
Hospital Metropolitano
Quito, Ecuador
https://orcid.org/0000-0003-4686-7358
108
108
MetroCiencia Vol. 31 Nº 3 (2023)
Primera Jornada de Investigación
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Human germline heterozygous gain-of-function<i>STAT6</i>variants cause severe allergic disease
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The Journal of experimental medicine/The journal of experimental medicine
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van der; Yang, J.; Chan, K.W.;
Rosa Duque, J.S.D.; Lee, P.P.W.; Ho, M.H.K.; Chung, B.H.; Le, H.T.M.; Yang, W; Rohani, P.;
Fouladvand, A.; Rokni-Zadeh, H.; Changi-Ashtiani, M.; Miryounesi, M.; Puel, A.; Shahrooei, M.;
Finocchi, A.; Rossi, P.; Rivalta, B.; Cifaldi, C.; Novelli, A.; Passarelli, C.; Arasi, S.; Bullens, D.; Sauer,
K.; Claeys, T.; Biggs, C.M.; Morris, E.C.; Rosenzweig, S.D.; O'Shea, J.J.; Wasserman, W.W.;
Bedford, H.M.; Karnebeek, C.D.M. van; Palma, P.; Burns, S.O.; Meyts, I.; Casanova, J.L.; Lyons, J.J.;
Parvaneh, N.; Nguyen, A.T.; Cancrini, C.; Heimall, J.; Ahmed, H.; McKinnon, M.L.; Lau, Y.L.; Béziat,
V.; Turvey, S.E. 2023, Article / Letter to editor (Journal of Experimental Medicine, 220, 5, (2023), pp. e20221755,
article e20221755) 2023, Article / Letter to editor (Journal of Experimental Medicine, 220, 5, (2023), pp. e20221755,
article e20221755) Doi link to publisher: https://doi.org/10.1084/jem.20221755 Doi link to publisher: https://doi.org/10.1084/jem.20221755 Human germline heterozygous gain-of-function STAT6 variants cause severe allergic disease. Sharma, Mehul; Leung, D.; Momenilandi, M.; Jones, L.C.W.; Pacillo, L.; James, A.E.; Murrell, J.R.;
Delafontaine, S.; Maimaris, J.; Vaseghi-Shanjani, M.; Bel, K.L. Del; Lu, H.Y.; Chua, G.T.; Cesare, S. Di; Fornes, O.; Liu, Zhongyi; Matteo, G. Di; Fu, M.P.; Amodio, D.; Tam, I.Y.S.; Chan, G.S.W.;
Sharma, A.A.; Dalmann, J.; Lee, R. van der; Blanchard-Rohner, G.; Lin, S.; Philippot, Q.; Richmond,
P.A.; Lee, J.J.; Matthews, A.; Seear, M.; Turvey, A.K.; Philips, R.L.; Brown-Whitehorn, T.F.; Gray,
C.J.; Izumi, K.; Treat, J.R.; Wood, K.H.; Lack, J.; Khleborodova, A.; Niemela, J.E.; Yang, Xingtian;
Liang, R.; Kui, L.; Wong, C.S.; Poon, G.W.K.; Hoischen, A.; Made, C.I. van der; Yang, J.; Chan, K.W.;
Rosa Duque, J.S.D.; Lee, P.P.W.; Ho, M.H.K.; Chung, B.H.; Le, H.T.M.; Yang, W; Rohani, P.;
Fouladvand, A.; Rokni-Zadeh, H.; Changi-Ashtiani, M.; Miryounesi, M.; Puel, A.; Shahrooei, M.;
Finocchi, A.; Rossi, P.; Rivalta, B.; Cifaldi, C.; Novelli, A.; Passarelli, C.; Arasi, S.; Bullens, D.; Sauer,
K.; Claeys, T.; Biggs, C.M.; Morris, E.C.; Rosenzweig, S.D.; O'Shea, J.J.; Wasserman, W.W.;
Bedford, H.M.; Karnebeek, C.D.M. van; Palma, P.; Burns, S.O.; Meyts, I.; Casanova, J.L.; Lyons, J.J.;
Parvaneh, N.; Nguyen, A.T.; Cancrini, C.; Heimall, J.; Ahmed, H.; McKinnon, M.L.; Lau, Y.L.; Béziat,
V.; Turvey, S.E. 2023, Article / Letter to editor (Journal of Experimental Medicine, 220, 5, (2023), pp. e20221755,
article e20221755) Human germline heterozygous gain-of-function STAT6 variants cause severe allergic disease. Sharma, Mehul; Leung, D.; Momenilandi, M.; Jones, L.C.W.; Pacillo, L.; James, A.E.; Murrell, J.R.;
Delafontaine, S.; Maimaris, J.; Vaseghi-Shanjani, M.; Bel, K.L. Del; Lu, H.Y.; Chua, G.T.; Cesare, S. Di; Fornes, O.; Liu, Zhongyi; Matteo, G. Di; Fu, M.P.; Amodio, D.; Tam, I.Y.S.; Chan, G.S.W.;
Sharma, A.A.; Dalmann, J.; Lee, R. van der; Blanchard-Rohner, G.; Lin, S.; Philippot, Q.; Richmond,
P.A.; Lee, J.J.; Matthews, A.; Seear, M.; Turvey, A.K.; Philips, R.L.; Brown-Whitehorn, T.F.; Gray,
C.J.; Izumi, K.; Treat, J.R.; Wood, K.H.; Lack, J.; Khleborodova, A.; Niemela, J.E.; Yang, Xingtian;
Liang, R.; Kui, L.; Wong, C.S.; Poon, G.W.K.; Hoischen, A.; Made, C.I. Human germline heterozygous gain-of-function
STAT6 variants cause severe allergic disease Mehul Sharma1*, Daniel Leung2*, Mana Momenilandi3,4*, Lauren C.W. Jones1**, Lucia Pacillo5,6,7**, Alyssa E. James8**,
Jill R. Murrell9**, Selket Delafontaine10,11**, Jesmeen Maimaris12,13**, Maryam Vaseghi-Shanjani1**, Kate L. Del Bel1**,
Henry Y. Lu14,15,16**, Gilbert T. Chua2,17, Silvia Di Cesare5,7, Oriol Fornes18,19, Zhongyi Liu2, Gigliola Di Matteo6,7, Maggie P. Fu20,21,
Donato Amodio6, Issan Yee San Tam2, Gavin Shueng Wai Chan22, Ashish A. Sharma23, Joshua Dalmann1, Robin van der Lee18,19,
G´eraldine Blanchard-Rohner1,24, Susan Lin1, Quentin Philippot3,4, Phillip A. Richmond1,18, Jessica J. Lee18,25, Allison Matthews18,26,
Michael Seear1, Alexandra K. Turvey1, Rachael L. Philips27, Terri F. Brown-Whitehorn28, Christopher J. Gray29, Kosuke Izumi29,
James R. Treat30, Kathleen H. Wood9, Justin Lack31, Asya Khleborodova31, Julie E. Niemela32, Xingtian Yang2, Rui Liang2, Lin Kui2,33,
Christina Sze Man Wong34, Grace Wing Kit Poon35, Alexander Hoischen36, Caspar I. van der Made36, Jing Yang2, Koon Wing Chan2,
Jaime Sou Da Rosa Duque2, Pamela Pui Wah Lee2, Marco Hok Kung Ho2,37, Brian Hon Yin Chung2, Huong Thi Minh Le38, Wanling Yang2,
Pejman Rohani39, Ali Fouladvand40, Hassan Rokni-Zadeh41, Majid Changi-Ashtiani42, Mohammad Miryounesi43, Anne Puel3,4,60,
Mohammad Shahrooei44, Andrea Finocchi5,7, Paolo Rossi5,45, Beatrice Rivalta5,6,7, Cristina Cifaldi7, Antonio Novelli46, Chiara Passarelli46,
Stefania Arasi47, Dominique Bullens48,49, Kate Sauer50,51, Tania Claeys52, Catherine M. Biggs1, Emma C. Morris12,13,
Sergio D. Rosenzweig32, John J. O’Shea27, Wyeth W. Wasserman18, H. Melanie Bedford26,53, Clara D.M. van Karnebeek18,54, Paolo Palma5,6,
Siobhan O. Burns12,13***, Isabelle Meyts10,11***, Jean-Laurent Casanova3,4,55,56,60***, Jonathan J. Lyons8***, Nima Parvaneh57***,
Anh Thi Van Nguyen58***, Caterina Cancrini5,7***, Jennifer Heimall28***, Hanan Ahmed59***, Margaret L. McKinnon19***,
Yu Lung Lau2****, Vivien B´eziat3,4,60****, and Stuart E. Turvey1**** STAT6 (signal transducer and activator of transcription 6) is a transcription factor that plays a central role in the
pathophysiology of allergic inflammation. We have identified 16 patients from 10 families spanning three continents with a
profound phenotype of early-life onset allergic immune dysregulation, widespread treatment-resistant atopic dermatitis,
hypereosinophilia with esosinophilic gastrointestinal disease, asthma, elevated serum IgE, IgE-mediated food allergies, and
anaphylaxis. The cases were either sporadic (seven kindreds) or followed an autosomal dominant inheritance pattern (three
kindreds). All patients carried monoallelic rare variants in STAT6 and functional studies established their gain-of-function
(GOF) phenotype with sustained STAT6 phosphorylation, increased STAT6 target gene expression, and TH2 skewing. Precision
treatment with the anti–IL-4Rα antibody, dupilumab, was highly effective improving both clinical manifestations and
immunological biomarkers. This study identifies heterozygous GOF variants in STAT6 as a novel autosomal dominant allergic
disorder. Human germline heterozygous gain-of-function
STAT6 variants cause severe allergic disease We anticipate that our discovery of multiple kindreds with germline STAT6 GOF variants will facilitate the
recognition of more affected individuals and the full definition of this new primary atopic disorder. ARTICLE .............................................................................................................................................................................
*M. Sharma, D. Leung, and M. Momenilandi contributed equally to this paper; **L.C.W. Jones, L. Pacillo, A.E. James, J.R. Murrell, S. Delafontaine, J. Maimaris, M. Vaseghi-
Shanjani, K.L. Del Bel, and H.Y. Lu contributed equally to this paper; ***S.O. Burns, I. Meyts, J.L. Casanova, J.J. Lyons, N, Parvaneh, A.T.V. Nguyen, C. Cancrini, J. Heimall,
H. Ahmed, and M.L McKinnon contributed equally to this paper;
****Y.L. Lau, V. Beziat, and S.E. Turvey contributed equally to this paper. Correspondence to Stuart
E. Turvey: sturvey@cw.bc.ca; Mehul Sharma: msharma@bcchr.ca. *M. Sharma, D. Leung, and M. Momenilandi contributed equally to this paper; **L.C.W. Jones, L. Pacillo, A.E. James, J.R. Murrell, S. Delafontaine, J. Maimaris, M. Vaseghi-
Shanjani, K.L. Del Bel, and H.Y. Lu contributed equally to this paper; ***S.O. Burns, I. Meyts, J.L. Casanova, J.J. Lyons, N, Parvaneh, A.T.V. Nguyen, C. Cancrini, J. Heimall,
H. Ahmed, and M.L McKinnon contributed equally to this paper;
****Y.L. Lau, V. Beziat, and S.E. Turvey contributed equally to this paper. Correspondence to Stuart
E. Turvey: sturvey@cw.bc.ca; Mehul Sharma: msharma@bcchr.ca.
© 2023 Sharma et al. This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/). © 2023 Sharma et al. This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/). Note: To cite this publication please use the final published version (if applicable). ARTICLE
Human germline heterozygous gain-of-function
STAT6 variants cause severe allergic disease
Mehul Sharma1*, Daniel Leung2*, Mana Momenilandi3,4*, Lauren C.W. Jones1**, Lucia Pacillo5,6,7**, Alyssa E. James8**,
Jill R. Murrell9**, Selket Delafontaine10,11**, Jesmeen Maimaris12,13**, Maryam Vaseghi-Shanjani1**, Kate L. Del Bel1**,
Henry Y. Lu14,15,16**, Gilbert T. Chua2,17, Silvia Di Cesare5,7, Oriol Fornes18,19, Zhongyi Liu2, Gigliola Di Matteo6,7, Maggie P. Fu20,21,
Donato Amodio6, Issan Yee San Tam2, Gavin Shueng Wai Chan22, Ashish A. Sharma23, Joshua Dalmann1, Robin van der Lee18,19,
G´eraldine Blanchard-Rohner1,24, Susan Lin1, Quentin Philippot3,4, Phillip A. Richmond1,18, Jessica J. Lee18,25, Allison Matthews18,26,
Michael Seear1, Alexandra K. Turvey1, Rachael L. Philips27, Terri F. Brown-Whitehorn28, Christopher J. Gray29, Kosuke Izumi29,
James R. Treat30, Kathleen H. Wood9, Justin Lack31, Asya Khleborodova31, Julie E. Niemela32, Xingtian Yang2, Rui Liang2, Lin Kui2,33,
Christina Sze Man Wong34, Grace Wing Kit Poon35, Alexander Hoischen36, Caspar I. van der Made36, Jing Yang2, Koon Wing Chan2,
Jaime Sou Da Rosa Duque2, Pamela Pui Wah Lee2, Marco Hok Kung Ho2,37, Brian Hon Yin Chung2, Huong Thi Minh Le38, Wanling Yang2,
Pejman Rohani39, Ali Fouladvand40, Hassan Rokni-Zadeh41, Majid Changi-Ashtiani42, Mohammad Miryounesi43, Anne Puel3,4,60,
Mohammad Shahrooei44, Andrea Finocchi5,7, Paolo Rossi5,45, Beatrice Rivalta5,6,7, Cristina Cifaldi7, Antonio Novelli46, Chiara Passarelli46,
Stefania Arasi47, Dominique Bullens48,49, Kate Sauer50,51, Tania Claeys52, Catherine M. Biggs1, Emma C. Morris12,13,
Sergio D. Rosenzweig32, John J. O’Shea27, Wyeth W. Wasserman18, H. Melanie Bedford26,53, Clara D.M. van Karnebeek18,54, Paolo Palma5,6,
Siobhan O. Burns12,13***, Isabelle Meyts10,11***, Jean-Laurent Casanova3,4,55,56,60***, Jonathan J. Lyons8***, Nima Parvaneh57***,
Anh Thi Van Nguyen58***, Caterina Cancrini5,7***, Jennifer Heimall28***, Hanan Ahmed59***, Margaret L. McKinnon19***,
Yu Lung Lau2****, Vivien B´eziat3,4,60****, and Stuart E. Turvey1**** J. Exp. Med. 2023 Vol. 220 No. 5
e20221755 E. Turvey: sturvey@cw.bc.ca; Mehul Sharma: msharma@bcchr.ca.
© 2023 Sharma et al. This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/). Identification of 16 patients from 10 families with severe
early-onset allergic disease heterozygous for rare damaging
STAT6 variants We studied 16 patients from 10 kindreds with severe early-onset
allergic disease spanning three continents. Patients were iden-
tified by their expert clinicians as candidates for genetic as-
sessment based on their extreme phenotype and, in some cases,
their family history (see clinical narratives in Data S1; Tables S4 2 of 18 Journal of Experimental Medicine
https://doi.org/10.1084/jem.20221755 Journal of Experimental Medicine
https://doi.org/10.1084/jem.20221755 and S5; and Fig. 1 A). The patients were from diverse ethnicities,
specifically European (Kindred D, F, and J), Middle Eastern
(Kindred A and C), Hispanic (Kindred B), South Asian (Kindred
H), East Asian (Kindred E), and Southeast Asian (Kindred Y). The cases were either sporadic (seven kindreds) or affected
multiple individuals of either sex over different generations
consistent with autosomal dominant (AD) inheritance (Kindreds
C, F, and J). All patients carried monoallelic rare variants in
STAT6 (NM_001178079.2). The consensus negative selection
score of STAT6 reveals a negative selection score that overlaps
with known AD IEIs (Rapaport et al., 2021), consistent with the
AD inheritance pattern observed in Kindreds C, F, and J (Fig. 1 B). In addition, and also consistent with an AD disorder, by se-
quencing both healthy parents (when available) we established
that the STAT6 mutation was de novo in Patient 2 (P2), P5, P10,
and P12 (from Kindreds B, D, G, and I, respectively; Fig. 1 A). The
disease was fully penetrant in the families studied, as all STAT6
variant carriers were affected. None of the variants have
previously been reported in population databases (Fig. 1 C;
i.e., gnomAD). All the variants were private to the studied
kindreds, except the p.D419G variant which was common in
Kindreds A and E. Pathogenicity prediction models identify all
of these variants to be pathogenic, evidenced by high patho-
genic CADD (Rentzsch et al., 2021), SIFT (Sim et al., 2012), and
Polyphen-2 (Adzhubei et al., 2010) scores (Tables S4 and S5). (GOF) variants in the gene STAT6 found in 16 individuals from 10
unrelated families spanning three continents. Signal transducer
and activator of transcription 6 (STAT6) is the main tran-
scription factor that mediates the biological effects of IL-4,
a key cytokine necessary for type 2 differentiation of
T cells, B cell survival, proliferation, and class switching to
IgE (Goenka and Kaplan, 2011; Takeda et al., 1996; Villarino
et al., 2020; Villarino et al., 2017), as well as that of IL-13, a
cytokine linked to anaphylaxis (Gowthaman et al., 2019). Affected
individuals
experienced
early-onset
severe,
sometimes fatal, multisystem allergic disease which was
refractory to conventional treatments. Notably, precision
therapeutics aimed at targeting exaggerated STAT6 sig-
naling were beneficial in those who received them. .............................................................................................................................................................................
1Dept. of Pediatrics, BC Children’s Hospital, University of British Columbia, Vancouver, Canada;
2Dept. of Paediatrics and Adolescent Medicine, School of Clinical Medicine,
The University of Hong Kong, Hong Kong, China;
3Laboratory of Human Genetics of Infectious Diseases, Necker Branch, INSERM, Necker Hospital for Sick Children, Paris,
France;
4Imagine Institute, University of Paris-Cit´e, Paris, France;
5Dept. of System Medicine, Pediatric Chair, University of Tor Vergata, Rome, Italy;
6Academic Dept. of
Pediatrics (DPUO), Unit of Clinical Immunology and Vaccinology, IRCCS Bambin Ges`u Children Hospital, Rome, Italy;
7Research Unit of Primary Immunodeficiency, IRCCS
Bambin Ges`u Children Hospital, Rome, Italy;
8Translational Allergic Immunopathology Unit, Laboratory of Allergic Diseases, National Institute of Allergy and Infectious
Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD, USA;
9Pathology and Laboratory Medicine, Division of Genomic Diagnostics, Children’s Hospital of
Philadelphia, Philadelphia, PA, USA;
10Dept. of Microbiology, Immunology and Transplantation, Laboratory for Inborn Errors of Immunity, KU Leuven, Leuven, Belgium;
11Dept. of Pediatrics, Pediatric Immunodeficiencies Division, University Hospitals Leuven, Leuven, Belgium;
12Institute of Immunity and Transplantation, Division of
Infection and Immunity, University College London, London, UK;
13Dept. of Immunology, Royal Free London NHS Foundation Trust, London, UK;
14Division of
Hematology/Oncology, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA;
15Dept. of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical
School, Boston, MA, USA;
16Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA;
17Allergy Centre, Union Hospital, Hong Kong,
China;
18Centre for Molecular Medicine and Therapeutics, BC Children’s Hospital Research Institute, Vancouver, Canada;
19Dept. of Medical Genetics, University of British
Columbia, Vancouver, Canada;
20Dept. of Medical Genetics, The University of British Columbia, Vancouver, Canada;
21Genome Science and Technology Program, Faculty
of Science, The University of British Columbia, Vancouver, Canada;
22Dept. of Pathology, Queen Mary Hospital, Hong Kong, China;
23Dept. of Pathology, Emory University,
Atlanta, GA, USA;
24Unit of Immunology and Vaccinology, Division of General Pediatrics, Dept. of Woman, Child, and Adolescent Medicine, Geneva University Hospitals and
Faculty of Medicine, University of Geneva, Geneva, Switzerland;
25Genome Science and Technology Graduate Program, University of British Columbia, Vancouver,
Canada;
26Dept. of Paediatrics, University of Toronto, Toronto, Canada;
27Molecular Immunology and Inflammation Branch, National Institute of Arthritis, Musculoskeletal
and Skin Diseases, NIH, Bethesda, MD, USA;
28Dept. of Pediatrics, Division of Allergy and Immunology, Children’s Hospital of Philadelphia, Philadelphia, PA, USA;
29Pediatrics, Division of Human Genetics, Children’s Hospital of Philadelphia, Philadelphia, PA, USA;
30Pediatrics, Division of Pediatric Dermatology, Perelman School of
Medicine, University of Pennsylvania, Philadelphia, PA, USA;
31NIAID Collaborative Bioinformatics Resource, NIAID, NIH, Bethesda, MD, USA;
32Immunology Service,
Clinical Center, NIH, Bethesda, MD, USA;
33Dept. of Pediatrics, University of California San Diego, La Jolla, CA, USA;
34Dept. of Medicine, Divison of Dermatology, The
University of Hong Kong, Hong Kong, China;
35Dept. of Paediatrics and Adolescent Medicine, Queen Mary Hospital, Hong Kong, China;
36Dept. of Human Genetics,
Radboud University Medical Center, Nijmegen, Netherlands;
37Virtus Medical, Hong Kong, China;
38Pediatric Center, Vinmec Times City International General Hospital,
Hanoi, Vietnam;
39Pediatrics, Pediatric Gastroenterology and Hepatology Research Center, Pediatrics Centre of Excellence, Children’s Medical Center, University of Medical
Sciences, Tehran, Iran;
40Pediatrics, Allergy and Clinical Immunology, Lorestan University of Medical Sciences, Khoramabad, Iran;
41Dept. of Medical Biotechnology,
School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran;
42School of Mathematics, Institute for Research in Fundamental Sciences, Tehran, Iran;
43Dept. of
Medical Genetics, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran;
44Microbiology and Immunology, Laboratory of Clinical Bacteriology
and Mycology, KU Leuven, Leuven, Belgium;
45DPUO, Research Unit of Infectivology and Pediatrics Drugs Development, Bambino Ges`u Children Hospital IRCCS, Rome,
Italy;
46Laboratory of Medical Genetics, Translational Cytogenomics Research Unit, Bambino Ges`u Children Hospital IRCCS, Rome, Italy;
47Allergy Unit, Area of
Translational Research in Pediatric Specialities, Bambino Ges`u Children’s Hospital, IRCCS, Rome, Italy;
48Dept. of Microbiology, Immunology and Transplantation, Allergy
and Clinical Immunology Research Group, KU Leuven, Leuven, Belgium;
49Dept. of Pediatrics, Pediatric Allergy Division, University Hospitals Leuven, Leuven, Belgium;
50Dept. of Pediatrics, Pediatric Pulmonology Division, AZ Sint-Jan Brugge, Brugge, Belgium;
51Dept. of Pediatrics, Pediatric Pulmonology Division, University Hospitals
Leuven, Leuven, Belgium;
52Dept. of Pediatrics, Pediatric Gastroenterology Division, AZ Sint-Jan Brugge, Brugge, Belgium;
53Genetics Program, North York General
Hospital, Toronto, Canada;
54Depts. of Pediatrics and Clinical Genetics, Amsterdam University Medical Centers, Amsterdam, Netherlands;
55Howard Hughes Medical
Institute, The Rockefeller University, New York, NY, USA;
56Department of Pediatrics, Necker Hospital for Sick Children, AP-HP, France;
57Department of Pediatrics,
Children’s Medical Center, Tehran University of Medical Sciences, Tehran, Iran;
58Dept. of Immunology, Allergy and Rheumatology, Division of Primary Immunodeficiency,
Vietnam National Children’s Hospital, Hanoi, Vietnam;
59Faculty of Health Sciences, McMaster University, Hamilton, Canada;
60St. Giles Laboratory of Human Genetics of
Infectious Diseases, Rockefeller Branch, The Rockefeller University, New York, NY, USA. Introduction Vaseghi-Shanjani et al., 2021). Identifying new PADs accel-
erates the diagnosis and treatment of affected individuals and
can uncover new molecular targets for preventing and treating
common allergic disease. Asthma and related atopic diseases, including atopic der-
matitis, food allergy, allergic rhinitis, and eosinophilic gas-
trointestinal diseases, are estimated to affect ∼20% of the
global population imposing immense health and economic
burdens (Dierick et al., 2020). Identifying human single-
gene defects that lead to severe allergic disease—so-called
primary atopic disorders (PADs)—is a powerful strategy to
define the cellular and molecular mechanisms that drive
human allergic inflammation (Lyons and Milner, 2018; Currently there are only a few known inborn errors of im-
munity (IEIs) underlying severe allergic disease (Milner, 2020). Indeed, most cases are of unknown etiology, particularly those
that are isolated or sporadic. In this study, we describe a novel
human PAD caused by germline heterozygous gain-of-function © 2023 Sharma et al. This article is available under a Creative Commons License (Attribution 4.0 International, as described at https 1 of 18 Rockefeller University Press
J. Exp. Med. 2023 Vol. 220 No. 5
e20221755 https://doi.org/10.1084/jem.20221755 Rockefeller University Press
J. Exp. Med. 2023 Vol. 220 No. 5
e20221755 https://doi.org/10.1084/jem.20221755 Human germline STAT6 gain-of-function variants Sharma et al.
Human germline STAT6 gain-of-function variants Sharma et al. Human germline STAT6 gain-of-function variants Figure 1. 16 patients with severe allergic disease and STAT6 variants in different protein domains. (A) Family pedigree of the 16 patients from 10
different families. Filled symbols = affected individual; unfilled symbols = unaffected individual. (B) Consensus negative selection (CoNeS) score for STAT6 in
Sharma et al. Journal of Experimental Medicine
3 of 18
Human germline STAT6 gain-of-function variants
https://doi.org/10.1084/jem.20221755
Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud Univ ress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 pdf by Radboud University user on 13 June 2023 Figure 1. 16 patients with severe allergic disease and STAT6 variants in different protein domains. (A) Family pedigree of the 16 patients from 10
different families. Filled symbols = affected individual; unfilled symbols = unaffected individual. (B) Consensus negative selection (CoNeS) score for STAT6 in Sharma et al. Journal of Experimental Medicine
Human germline STAT6 gain-of-function variants
https://doi.org/10.1084/jem.20221755 Journal of Experimental Medicine
3 of 18
https://doi.org/10.1084/jem.20221755 Remarkably, nine patients from six kindreds carried a variant
affecting amino acid D419. Importantly, variants leading to
amino acid changes at p.D419, p.D519, and p.P643 can be found
in the Catalogue of Somatic Mutations in Cancer (COSMIC)
database as recurrent somatic variants in lymphoma with some
experimental evidence for a GOF phenotype for variants at
p.D419 (Yildiz et al., 2015; Zam`o et al., 2018; Fig. S1 A). The
reported variants lie in different protein domains of STAT6,
including the DNA-binding domain (p.E382 and p.D419), the
linker domain (p.D519), and the SH2 domain (p.K595), while
p.P643 lies in close proximity to the p.Y641 phosphorylation
site (Fig. 1, D–E). Although the variants were located within
different domains of the STAT6 protein, modeling of STAT6
interacting with DNA reveals that all the identified variants
(with the exception of p.P643) lie near the protein–DNA in-
terface and result in amino acids changes leading to increased
electro-positivity at physiological pH (Fig. 1 E). Notably, E382
and D419 are located in regions responsible for protein–DNA
recognition (Li et al., 2016). Changes in these variants decrease
the electro-negativity of the protein near the DNA-binding
interface and are predicted to enhance STAT6 binding to
DNA (Fig. 1 E and Fig. S1 B). In aggregate, these data suggest that
the STAT6 germline monoallelic variants identified in the pa-
tients underlie severe allergic disease by a GOF mechanism. Functional analysis of the STAT6 variants confirms their
GOF activity To assess the functional impact of the STAT6 variants, we se-
lected HEK293 cells as our model system, as these cells lack
endogenous STAT6 but express other components of the IL-4R
pathway (Fig. 3 A; Mikita et al., 1996). HEK293 cells were
transfected with each of the 10 patient STAT6 variants along
with three different controls: WT STAT6, a predicted damaging
STAT6 population variant found in the gnomAD healthy popu-
lation database (p.A321V), and a biochemically inactive STAT6
variant (p.Y641F; Wick and Berton, 2000). To investigate STAT6
transcription factor activity, we conducted luciferase assays
with three different reporter plasmids containing STAT6 bind-
ing sites (Li et al., 2016). While there were some difference re-
lated to the specific combinations of reporter plasmids and
patient variants, there was evidence of GOF activity with all
STAT6 patient variants resulting in higher promoter activity at
baseline and after stimulation compared to the controls (Fig. 3 B
and Fig. S3, A and B). The phosphorylation status of STAT6
variants was also quantified by flow cytometry and was con-
firmed to be higher at baseline and after stimulation compared
to WT (Fig. 3, C and D; and Fig. S3 C). STAT6 phosphorylation
was not detectable by flow cytometry for the p.P643R variant;
however, increased baseline phosphorylation was confirmed by
immunoblotting (Fig. 3 E and Fig. S3, D and E). This may point to
a conformational change in tertiary structure of STAT6 near the
phosphorylation site p.Y641 for this variant that rendered it
inaccessible to the flow cytometry antibody. ess.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 Unifying clinical features of the 16 patients with severe
allergic disease g
The patients in the cohort were aged from 3 to 60 yr. Full clinical
narratives are provided in Data S1, and their clinical features are
summarized in Fig. 2 A. All had severe allergic disease which
began in their first year of life. Severe, treatment-resistant
atopic dermatitis (15/16) and food allergies (15/16) were the
most common clinical manifestations, followed by asthma (11/16)
and eosinophilic gastrointestinal disease (10/16) and severe epi-
sodes of anaphylaxis (9/16). Clinical laboratory testing was notable
for eosinophilia and markedly elevated serum IgE levels (Fig. 2, B
and C). Other aspects of the clinical laboratory and immunological
work up were largely unremarkable, although clinical hallmarks
of chronic systemic inflammation were present in some patients
(i.e., elevations in white blood cell counts, platelets, and serum
immunoglobulin levels). T, B, and natural killer (NK) cell numbers
were all typically in the normal range (Fig. S2). Clinical biopsies
confirmed the presence of eosinophils in the skin and gastroin-
testinal tract (Fig. 2, E–G). Endoscopic visualization of the
esophagus revealed trachealization and furrowing consistent
with eosinophilic esophagitis (Bolton et al., 2018; Fig. 2, H and I). We next evaluated if the increased promoter activity leads to
global transcriptomic changes. As transcriptomic studies on
HEK293 cells after IL-4 stimulation have been challenging to
interpret (Yildiz et al., 2015), we stably expressed p.E382Q and
p.D419G STAT6 by lentiviral transduction in Jurkat T cells,
which express endogenous STAT6 and serve as a model of het-
erozygosity (Kim et al., 2012). Here, we observed that WT-,
p.E382Q-, and p.D419G-transduced cells clustered separately
from each other both at baseline and after stimulation with IL-4
(Fig. 3 F). Differential gene expression analysis revealed signif-
icantly increased transcript abundance of known STAT6 target
genes, including IL4R (Goenka and Kaplan, 2011), CISH (Yildiz
et al., 2015), and XBP1 (Bettigole et al., 2015) in p.E382Q and
p.D419G transduced cells when compared to WT transduced
control (Fig. 3 G). Interestingly, p.E382Q and p.D419G had 67 and In addition to atopic disease, half of the patients presented
with recurrent skin, respiratory, and viral infections, although
there were no deep-seated or fatal infections. Short stature (less
than third percentile for age) was a common feature (7/16), and 4 of 18 Journal of Experimental Medicine
https://doi.org/10.1084/jem.20221755 Human germline STAT6 gain-of-function variants relation to the score for known IEI genes reported with inheritance pattern of either AD, AR, or both (AD + AR). (C) Frequency and CADD score for missense
(black) and predicted LOF (pLOF, blue) STAT6 variants reported in a public database and STAT6 variants reported in our patient cohort (red). The dotted line
corresponds to the mutation significance cutoff (MSC). (D) Schematic illustrating the protein domains of STAT6. Amino acid location of the variants shown are
highlighted, with the length of the bar corresponding to the number of patients reported with variants at that site. (E) Structural model of the DNA-STAT6
homodimer complex showing location of the different STAT6 variants in relation to the DNA-binding interface. Remarkably, nine patients from six kindreds carried a variant
affecting amino acid D419. Importantly, variants leading to
amino acid changes at p.D419, p.D519, and p.P643 can be found
in the Catalogue of Somatic Mutations in Cancer (COSMIC)
database as recurrent somatic variants in lymphoma with some
experimental evidence for a GOF phenotype for variants at
p.D419 (Yildiz et al., 2015; Zam`o et al., 2018; Fig. S1 A). The
reported variants lie in different protein domains of STAT6,
including the DNA-binding domain (p.E382 and p.D419), the
linker domain (p.D519), and the SH2 domain (p.K595), while
p.P643 lies in close proximity to the p.Y641 phosphorylation
site (Fig. 1, D–E). Although the variants were located within
different domains of the STAT6 protein, modeling of STAT6
interacting with DNA reveals that all the identified variants
(with the exception of p.P643) lie near the protein–DNA in-
terface and result in amino acids changes leading to increased
electro-positivity at physiological pH (Fig. 1 E). Notably, E382
and D419 are located in regions responsible for protein–DNA
recognition (Li et al., 2016). Changes in these variants decrease
the electro-negativity of the protein near the DNA-binding
interface and are predicted to enhance STAT6 binding to
DNA (Fig. 1 E and Fig. S1 B). In aggregate, these data suggest that
the STAT6 germline monoallelic variants identified in the pa-
tients underlie severe allergic disease by a GOF mechanism. 5/16 had other skeletal issues such as pathologic fractures and
generalized hypermobility. Finally, two of the patients died from
their disease; one from anaphylaxis at the age of 20 yr and an-
other from a cerebral aneurysm at age 35 yr. These clinical
presentations highlight the severity of the multi-system disease
found in this patient cohort. Sharma et al.
Human germline STAT6 gain-of-function variants Sharma et al. Human germline STAT6 gain-of-function variants Human germline STAT6 gain-of-function variants relation to the score for known IEI genes reported with inheritance pattern of either AD, AR, or both (AD + AR). (C) Frequency and CADD score for missense
(black) and predicted LOF (pLOF, blue) STAT6 variants reported in a public database and STAT6 variants reported in our patient cohort (red). The dotted line
corresponds to the mutation significance cutoff (MSC). (D) Schematic illustrating the protein domains of STAT6. Amino acid location of the variants shown are
highlighted, with the length of the bar corresponding to the number of patients reported with variants at that site. (E) Structural model of the DNA-STAT6
homodimer complex showing location of the different STAT6 variants in relation to the DNA-binding interface. Journal of Experimental Medicine
https://doi.org/10.1084/jem.20221755 Sharma et al. Human germline STAT6 gain-of-function variants Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud Uni Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 Figure 2. Major clinical features of the 16 patients. (A) Tabulation and comparison of the clinical phenotype for 16 patients. Please note blood eosinophil
and IgE values were only available for 15 patients. (B) IgE concentration in whole blood for 15 out of the 16 patients. Shaded area represents IgE < 240 µg/liter, pdf by Radboud University user on 13 June 2023 Figure 2. Major clinical features of the 16 patients. (A) Tabulation and comparison of the clinical phenotype for 16 patients. Please note blood eosinophil
and IgE values were only available for 15 patients. (B) IgE concentration in whole blood for 15 out of the 16 patients. Shaded area represents IgE < 240 µg/liter, Journal of Experimental Medicine
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https://doi.org/10.1084/jem.20221755 Journal of Experimental Medicine
5 of 18
https://doi.org/10.1084/jem.20221755 Human germline STAT6 gain-of-function variants Patients with GOF STAT6 variants have slower STAT6
dephosphorylation kinetics after IL-4 stimulation and an
enhanced TH2 signature To further investigate the role of STAT6 GOF variants in pri-
mary cells, STAT6 phosphorylation was quantified in patient
samples. Unexpectedly, we found no differences in the percentage
of phospho-STAT6 positive cells between patients and healthy
controls after IL-4 stimulation over a 60 min time course nor after
stimulation with different doses of IL-4 (Fig. S4, A and B). How-
ever, differences emerged when we monitored the kinetics of
STAT6 dephosphorylation after stimulation (Fig. 4, A and B; and
Fig. S4 C). Specifically, washing out of IL-4 led to slower de-
phosphorylation kinetics of STAT6 in most patient cells compared
to healthy controls (Fig. 4, A and B; and Fig. S4 D), supporting a
GOF mechanism in patient lymphocytes. We did note that one of
our kindreds did not display delayed dephosphorylation (Fig. S4 D), suggesting that this might not be the only GOF mecha-
nism. Indeed, increased STAT6 activity without phosphorylation
has previously been reported in follicular lymphoma research
studying the p.D419 mutational hotspot (Yildiz et al., 2015). JAK inhibitors and IL-4Rα monoclonal antibody can be used as
potential therapeutics for patients with STAT6 GOF variants
Due to the severity of the multi-system allergic disease experi-
enced by the patients in our cohort, various treatment
approaches were used over the years. Corticosteroids,
administered topically and systemically, were the mainstay of
treatment for most patients. Unfortunately, even high doses of
corticosteroids were unable to control the allergic inflammation
and they were responsible for many side-effects including cat-
aracts and osteoporosis. Other treatments used in this cohort
that proved ineffective included topical tacrolimus, oral metho-
trexate, and mepolizumab (an anti–IL-5 monoclonal antibody). Given that the STAT6 axis is critical for the differentiation of
TH2 cells (Kaplan et al., 1996), a subset of CD4+ helper T cells that
is a major contributor to the pathogenesis of allergic disease, we
next investigated TH2 signatures in these patients. Patients
showed skewing towards TH2 pathway activity compared to
healthy controls based on higher levels of the TH2 cytokines IL-5,
IL-13, and IL-4 as measured by flow cytometry (Fig. 4, C and D), or
through transcriptomic signature by single-cell RNA sequencing
(scRNAseq; Fig. 4 E). High throughput proteomics also validated
the increased IL-4 expression, but not high IL-13 expression (Fig. S4 E). Differences in proportions of other subsets of helper T cells
were restricted to higher IL-21+ cells in patient memory CD4+
T cells (Fig. which is the typical upper limit of normal. (C) Eosinophil count in whole blood for 15 out of the 16 patients. Shaded area represents counts <0.5 × 109/liter,
which is the typical upper limit of normal. The horizontal broken line denotes an eosinophil count of 1.5 × 109/liter, since hypereosinophilic syndrome is
traditionally defined as peripheral blood eosinophilia >1.5 × 109/liter persisting ≥6 mo. (D) Photograph of widespread and severe atopic disease. (E) Pho-
tomicrograph of the skin biopsy showing marked pseudoepitheliomatous hyperplasia with acanthosis, hyperkeratosis, and focal parakeratosis, suggestive of
lichen simplex chronicus (H&E stain, original magnification 2×). Moderate chronic inflammation within the papillary dermis in which scattered eosinophils
(white arrows) are conspicuous (inset, H&E stain; original magnification, 40×). (F) Photomicrograph of duodenal biopsy showing abundant eosinophils (white
arrows) amongst lymphocytes (H&E stain; original magnification, 40×). (G) Photomicrograph of gastric antral biopsy showing abundant infiltrate of eosinophils
(arrows) amongst lymphocytes and plasma cells (H&E stain; original magnification, 40×). (H and I) Endoscopic images showing (H) furrowing and (I) tra-
chealization in the middle esophagus, suggestive of eosinophilic esophagitis. Sulczewski et al., 2021), and CD4+ T cells express high GATA3. scRNAseq further demonstrated that IL4R, a gene encoding a
key receptor that mediates STAT6 activation, was upregulated
in all B and T cell subsets (Fig. 4 F and Fig. S4 G). 80 uniquely increased hits, which did not overlap with WT nor
with each other (Fig. 3 H, Fig. S3 F, and Table S6). This suggests
that the altered activity of both p.E382Q and p.D419G is not re-
stricted to enhanced activity of known STAT6 targets alone. Further investigation through gene set enrichment analyses
(GSEA) showed increased enrichment in IL-4-STAT6 target genes
at baseline (Fig. 3 I and Table S7), increased enrichment in T
helper 2 (TH2) drivers after stimulation (Elo et al., 2010; Fig. 3 J
and Table S7), and increased enrichment in proliferation path-
ways for p.D419G consistent with its known oncogenic activity
(Ritz et al., 2009; Tate et al., 2019; Yildiz et al., 2015; Fig. S3 G). Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud Un Using flow cytometry, we confirmed that IL-4Rα expression
was significantly higher on both naive and memory CD4+ T cells
of seven patients from three different kindreds compared to
nine healthy controls (Fig. 5 A). Patients with GOF STAT6 variants have slower STAT6
dephosphorylation kinetics after IL-4 stimulation and an
enhanced TH2 signature S4 F). scRNAseq showed that patient B cells expressed
high IGHE and low IGHG3 (Fig. 4 F), reflecting patterns opposite
of those seen in STAT6 knockout mice (Shimoda et al., 1996; Having demonstrated that heterozygous STAT6 variants lead
to higher STAT6 activity and TH2 immunological skewing, we
tested in vitro whether targeting the STAT6 pathway could be
clinically beneficial. Based on our findings of higher phospho-
rylation of STAT6, and higher expression of IL-4Rα, we selected
the JAK inhibitors, ruxolitinib and tofacitinib, and the anti–IL-4Rα
antibody, dupilumab, as drugs to test in vitro as they are all used
clinically for treatment of allergic disease (Bacharier et al., 2021;
Beck et al., 2014; Bissonnette et al., 2016; Kim et al., 2020). We
demonstrated that both ruxolitinib and tofacitinib effectively de-
creased the patient-specific enhanced STAT6 phosphorylation/
activation in HEK293 cells at baseline and after IL-4 stimulation,
whereas dupilumab inhibited IL-4 mediated increase in
STAT6 activity (Fig. 6 A and Fig. S5 A). We further confirmed in 6 of 18 Journal of Experimental Medicine
https://doi.org/10.1084/jem.20221755 IL-4Rα expression was also
found to be higher in non-class switched memory and class
switched memory B cells of unstimulated patient primary cells
(Fig. 5 B). These findings suggest higher baseline activity of
STAT6 in patient cells driving IL-4Rα expression similar to that
seen in our Jurkat model (Fig. 3, G–J; and Tables S6 and S7). Finally, we measured the expression of CD23 (the low-affinity
IgE receptor, FcεRII) which is known to be upregulated by
STAT6 (Fig. S3 B; Kneitz et al., 2000) and we found higher ex-
pression of CD23 on all subsets of patient B cells at baseline
(Fig. 5 B) and following stimulation with IL-4 (Fig. S4 H). Taken
together, these experiments conducted using primary patient
cells further confirm the STAT6 GOF phenotype. ress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 Human germline STAT6 gain-of-function variants Sharma et al. Human germline STAT6 gain-of-function variants which is the typical upper limit of normal. (C) Eosinophil count in whole blood for 15 out of the 16 patients. Shaded area represents counts <0.5 × 109/liter,
which is the typical upper limit of normal. The horizontal broken line denotes an eosinophil count of 1.5 × 109/liter, since hypereosinophilic syndrome is
traditionally defined as peripheral blood eosinophilia >1.5 × 109/liter persisting ≥6 mo. (D) Photograph of widespread and severe atopic disease. (E) Pho-
tomicrograph of the skin biopsy showing marked pseudoepitheliomatous hyperplasia with acanthosis, hyperkeratosis, and focal parakeratosis, suggestive of
lichen simplex chronicus (H&E stain, original magnification 2×). Moderate chronic inflammation within the papillary dermis in which scattered eosinophils
(white arrows) are conspicuous (inset, H&E stain; original magnification, 40×). (F) Photomicrograph of duodenal biopsy showing abundant eosinophils (white
arrows) amongst lymphocytes (H&E stain; original magnification, 40×). (G) Photomicrograph of gastric antral biopsy showing abundant infiltrate of eosinophils
(arrows) amongst lymphocytes and plasma cells (H&E stain; original magnification, 40×). (H and I) Endoscopic images showing (H) furrowing and (I) tra-
chealization in the middle esophagus, suggestive of eosinophilic esophagitis. Journal of Experimental Medicine
https://doi.org/10.1084/jem.20221755 Sharma et al. Human germline STAT6 gain-of-function variants Figure 3. STAT6 variants lead to increased STAT6 activity in HEK293 cells and Jurkat T cells. (A) Schematic illustrating classical IL-4–mediated STAT6
activation, dimerization, and phosphorylation. (B) Luciferase assay of STAT6 activity on a plasmid containing a 4× STAT6 binding site (TTCCCAAGAA; the
Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud Un ess.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 221755.pdf by Radboud University user on 13 June 2023 pdf by Radboud University user on 13 June 2023 Figure 3. STAT6 variants lead to increased STAT6 activity in HEK293 cells and Jurkat T cells. (A) Schematic illustrating classical IL-4–mediated STAT6
activation, dimerization, and phosphorylation. (B) Luciferase assay of STAT6 activity on a plasmid containing a 4× STAT6 binding site (TTCCCAAGAA; the Journal of Experimental Medicine
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https://doi.org/10.1084/jem.20221755 S5 B), repeated
whole blood RNAseq showed global transcriptomic changes that
were suggestive of mildly increased eosinophilic and allergic
gene signatures after 38 d, followed by a shift of the tran-
scriptome towards healthy controls after 123 and 492 d, re-
spectively (Fig. 6 C and Fig. S5, C–E). scRNAseq confirmed a
decrease in TH2 gene signatures 2 yr following initiation of
dupilumab, accompanied by a decrease in the expression of IL-
4R on both naive CD4+ and CD8+ T cells (Fig. 6, D–E). Clinically,
these changes were accompanied by dramatic increase in
growth velocity, improved skin condition as quantified by
SCORAD (SCORing atopic dermatitis) and EASI (eczema area
and severity index) scores, and the ability to wean from oral
corticosteroids (Fig. 6, F–H). Similarly, P1 experienced re-
markable clinical benefits with dupilumab with improved
skin inflammation (Fig. 6 D), resolution of pruritus, and the
ability to discontinue oral daily high dose corticosteroids. In
addition to resolution of skin inflammation with dupilumab, P2
was able to discontinue swallowed budesonide without a flare
in the severity of her eosinophilic esophagitis. Our preclinical
data also suggested that JAK inhibitors may be beneficial (Fig. 6,
A and B), and P4 had received tofacitinib (5 mg/d) for 2 mo at
the time this manuscript was finalized. His initial response
to tofacitinib was encouraging with less dysphagia, less
esophageal food impaction, improved endoscopic appearance
of the esophagus, and a marked reduction in the number of
intraepithelial eosinophils. ss.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 STAT6 is intimately linked to the biology of allergic inflam-
mation. The central and most studied role of STAT6 is in me-
diating the biological effects of IL-4, a cytokine necessary for TH2
differentiation, B cell survival, proliferation, and class switching
to IgE (Elo et al., 2010; Takeda et al., 1996), as well as in driving
M2 macrophage polarization (Ginhoux et al., 2016). In T cells,
STAT6 activation induces the expression of GATA3, the master
regulator of TH2 differentiation, which in turn enhances ex-
pression of IL-4, IL-5, and IL-13, cytokines necessary for
promoting allergic responses by activating mast cells and
eosinophils (Sloka et al., 2011). The presence of greater TH2 cell
populations, or TH2 cells producing copious amounts of IL-4/IL-
5/IL-13, could be a driver of the observed allergic phenotype
presented in our patients. (F) Principal component analysis (PCA) comparing unstimulated and stimulated
(100 ng/ml IL-4 for 4 h) WT (green), p.E382Q (blue), and p.D419G (purple) STAT6-transduced Jurkat T cells. Individual symbols represent technical replicates of
one transduced pool for each genotype. PC1 and PC2 contribution is shown in brackets. (G) Normalized counts comparing stimulated WT (green) vs. p.E382Q
(blue) or p.D419G (purple), for IL4R, CISH, and XBP1. (H) Heatmap representation of normalized counts of a transcription set defined as IL-4 targets in
transduced Jurkat T cells. (I and J) Asterisk indicates adjusted P value <0.05. GSEA plots for (I) curated STAT6 target genes comparing WT vs. either p.E382Q
(blue) or p.D419G (purple) at baseline, or (J) IL-4-TH2 targets genes comparing WT vs. either p.E382Q (blue) or p.D419G (purple) after stimulation with IL-4. Normalized enrichment score and adjusted P value are shown. Source data are available for this figure: SourceData F3. targeted therapeutic option, improving both clinical manifes-
tations of disease and immunological biomarkers. patient primary cells that tofacitinib accelerated STAT6 de-
phosphorylation following IL-4 stimulation (Fig. 6 B). These
data suggest the potential clinical benefit of directly targeting
the IL-4/STAT6 pathway in patients with STAT6 GOF variants. Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud U Although the full phenotype(s) of individuals with GOF
STAT6 variants will only be uncovered through the identifica-
tion of additional affected individuals, we propose to classify
human germline AD GOF STAT6 syndrome as a PAD (Lyons and
Milner, 2018; Milner, 2020; Vaseghi-Shanjani et al., 2021). Based
on our study, possible clinical “red flags” for this new disorder
include: (i) early life onset; (ii) peripheral blood eosinophilia;
(iii) elevated serum IgE; (iii) widespread, treatment-resistant
atopic dermatitis; (iv) multiple food and drug allergies; (v) se-
vere (and even fatal) anaphylaxis; (vi) recurrent skin and res-
piratory infections; (vii) eosinophilic gastrointestinal disorder,
including eosinophilic esophagitis; (viii) asthma; (ix) allergic
rhinoconjunctivitis; (x) short stature; and possibly (xi) vascular
malformations of the brain. p
y
p
Once their STAT6 GOF variant was identified, three of the
patients were started on dupilumab and all showed significant
clinical improvement. P6, who has been on treatment with du-
pilumab for over 2 yr, serves as an illustrative example. Mir-
roring peripheral blood eosinophil counts (Fig. Discussion We present a combination of clinical, genetic, molecular, and
transcriptional evidence of a new human disorder caused by
germline AD GOF STAT6 variants in 16 patients with life-long
severe allergic disease. These variants led to sustained STAT6
phosphorylation, increased STAT6 target gene expression, and
TH2 skewed transcriptional profile. Importantly, we demonstrate
in three patients that dupilumab treatment is a highly effective 8 of 18 Journal of Experimental Medicine
https://doi.org/10.1084/jem.20221755 Journal of Experimental Medicine
https://doi.org/10.1084/jem.20221755 Elevated IgE in partnership with mast
cells is important for both acute and chronic manifestations of
allergic disorders and can be an additional driver of the allergic
diathesis (Galli and Tsai, 2012). STAT6 hyperactivation in air-
way epithelial cells and resident dendritic cells can further
create an environment favoring asthma and chronic lung dis-
ease, as this would induce production of chemokines that pro-
mote TH2 cells and eosinophil recruitment (Matsukura et al.,
2001; Medoff et al., 2009). Population genetics provide further
support for the central role that STAT6 plays in the development
of human allergic disease. Multiple independent genome-wide
association studies (GWAS) have found that polymorphisms in
STAT6 associate with many allergic conditions (Table 1). Our
study expands this appreciation of the role of STAT6 in human
disease by establishing that heterozygous GOF variants cause a
monogenic form of severe allergic disease. underlined bases represent two half-sites for STAT6-specific binding) for WT-, different STAT6 variant–transfected HEK293 cells before and after stimulation
with IL-4 (0.02 ng/ml for 4 h); n = 3. (C) Phospho-STAT6 (Y641) expression in WT- and STAT6 variant–transfected HEK293 cells before and after treatment with
IL-4 (10 ng/ml for 30 min). Gating strategy for pSTAT6+ cells can be found in Fig. S3 C. (D) Quantification of C; n = 4. (E) Immunoblot in HEK293 cells
transfected with WT-, inactive- (p.Y641F), p.P643R-, and p.D419G-STAT6 variants for pSTAT6, and Myc-tag before and after treatment with IL-4 (10 ng/ml for
30 min); n = 3. Full-length immunoblot for this can be found in Fig. S3, D and E. (F) Principal component analysis (PCA) comparing unstimulated and stimulated
(100 ng/ml IL-4 for 4 h) WT (green), p.E382Q (blue), and p.D419G (purple) STAT6-transduced Jurkat T cells. Individual symbols represent technical replicates of
one transduced pool for each genotype. PC1 and PC2 contribution is shown in brackets. (G) Normalized counts comparing stimulated WT (green) vs. p.E382Q
(blue) or p.D419G (purple), for IL4R, CISH, and XBP1. (H) Heatmap representation of normalized counts of a transcription set defined as IL-4 targets in
transduced Jurkat T cells. (I and J) Asterisk indicates adjusted P value <0.05. GSEA plots for (I) curated STAT6 target genes comparing WT vs. either p.E382Q
(blue) or p.D419G (purple) at baseline, or (J) IL-4-TH2 targets genes comparing WT vs. either p.E382Q (blue) or p.D419G (purple) after stimulation with IL-4. Normalized enrichment score and adjusted P value are shown. Source data are available for this figure: SourceData F3. underlined bases represent two half-sites for STAT6-specific binding) for WT-, different STAT6 variant–transfected HEK293 cells before and after stimulation
with IL-4 (0.02 ng/ml for 4 h); n = 3. (C) Phospho-STAT6 (Y641) expression in WT- and STAT6 variant–transfected HEK293 cells before and after treatment with
IL-4 (10 ng/ml for 30 min). Gating strategy for pSTAT6+ cells can be found in Fig. S3 C. (D) Quantification of C; n = 4. (E) Immunoblot in HEK293 cells
transfected with WT-, inactive- (p.Y641F), p.P643R-, and p.D419G-STAT6 variants for pSTAT6, and Myc-tag before and after treatment with IL-4 (10 ng/ml for
30 min); n = 3. Full-length immunoblot for this can be found in Fig. S3, D and E. Human germline STAT6 gain-of-function variants Sharma et al. Human germline STAT6 gain-of-function variants Human germline STAT6 gain-of-function variants underlined bases represent two half-sites for STAT6-specific binding) for WT-, different STAT6 variant–transfected HEK293 cells before and after stimulation
with IL-4 (0.02 ng/ml for 4 h); n = 3. (C) Phospho-STAT6 (Y641) expression in WT- and STAT6 variant–transfected HEK293 cells before and after treatment with
IL-4 (10 ng/ml for 30 min). Gating strategy for pSTAT6+ cells can be found in Fig. S3 C. (D) Quantification of C; n = 4. (E) Immunoblot in HEK293 cells
transfected with WT-, inactive- (p.Y641F), p.P643R-, and p.D419G-STAT6 variants for pSTAT6, and Myc-tag before and after treatment with IL-4 (10 ng/ml for
30 min); n = 3. Full-length immunoblot for this can be found in Fig. S3, D and E. (F) Principal component analysis (PCA) comparing unstimulated and stimulated
(100 ng/ml IL-4 for 4 h) WT (green), p.E382Q (blue), and p.D419G (purple) STAT6-transduced Jurkat T cells. Individual symbols represent technical replicates of
one transduced pool for each genotype. PC1 and PC2 contribution is shown in brackets. (G) Normalized counts comparing stimulated WT (green) vs. p.E382Q
(blue) or p.D419G (purple), for IL4R, CISH, and XBP1. (H) Heatmap representation of normalized counts of a transcription set defined as IL-4 targets in
transduced Jurkat T cells. (I and J) Asterisk indicates adjusted P value <0.05. GSEA plots for (I) curated STAT6 target genes comparing WT vs. either p.E382Q
(blue) or p.D419G (purple) at baseline, or (J) IL-4-TH2 targets genes comparing WT vs. either p.E382Q (blue) or p.D419G (purple) after stimulation with IL-4. Normalized enrichment score and adjusted P value are shown. Source data are available for this figure: SourceData F3. Sharma et al.
Human germline STAT6 gain-of-function variants Sharma et al. Human germline STAT6 gain-of-function variants Figure 4. Primary lymphocytes of STAT6 GOF patients display higher STAT6 activity and TH2 skewing. (A) Histograms showing phosphorylation of
STAT6 in healthy control (blue) and patient (red) LCLs that were stimulated with IL-4 (10 ng/ml) for 15 min, washed with PBS and subsequently incubated in IL-
4–free media for 15, 30, and 60 min. Gating strategy for pSTAT6+ cells can be found in Fig. S4 C. (B) Quantification of pSTAT6+ cells from three separate
experiments done in A, multiple unpaired t test corrected for multiple comparison using the Benjamini–Hochberg method. ***, P < 0.001. (C) Frequency of IL5+,
Sharma et al. Journal of Experimental Medicine
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Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 p
p
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y Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud Un aded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_ press.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 Figure 4. Primary lymphocytes of STAT6 GOF patients display higher STAT6 activity and TH2 skewing. (A) Histograms showing phosphorylation of
STAT6 in healthy control (blue) and patient (red) LCLs that were stimulated with IL-4 (10 ng/ml) for 15 min, washed with PBS and subsequently incubated in IL-
4–free media for 15, 30, and 60 min. Gating strategy for pSTAT6+ cells can be found in Fig. S4 C. (B) Quantification of pSTAT6+ cells from three separate
experiments done in A, multiple unpaired t test corrected for multiple comparison using the Benjamini–Hochberg method. ***, P < 0.001. (C) Frequency of IL5+, Journal of Experimental Medicine
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https://doi.org/10.1084/jem.20221755 Sharma et al. Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud Un Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 ress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 Beyond defining the phenotype of STAT6 GOF, we also pre-
sent laboratory and clinical evidence supporting the effective-
ness of dupilumab and tofacitinib treatment in these patients. It
was notable that the three patients (P1, P2, and P6) who received
dupilumab have experienced dramatic improved atopic derma-
titis and could be weaned off systemic corticosteroids. P6, who
had short stature and delayed bone age before starting the bio-
logic agent, experienced rapid height and weight gain following
initiation of dupilumab. In addition to the documented clinical
benefits of dupilumab therapy in patients with STAT6 GOF, we
also present early data suggesting that the JAK inhibitors,
ruxolitinib or tocafitinib, may be effective in this patient
population. It is noteworthy that the oldest patient in this cohort, P7
(p.D419H), experienced recurrent B cell lymphoma—follicular
lymphoma at 49 yr and diffuse large B cell lymphoma at age 60
yr. Activating somatic mutations in STAT6 are well documented
in B cell lymphoma with amino acid D419 being a particular
mutational hotspot (Ritz et al., 2009; Tate et al., 2019; Yildiz
et al., 2015). The patient’s p.D419H variant has been reported
multiple times as a somatic mutation in COSMIC, as have other
variants found in our patient cohort (i.e., p.D419, p.D519, and
p.P643). More patients will need to be identified and followed to
fully define the phenotype caused by germline STAT6 GOF
variants, but it is biologically plausible that these patients may
be at higher risk of developing B cell malignancies warranting
enhanced clinical vigilance. While this study has many strengths, notably the extreme
allergic phenotype of the 16 patients combined with in-depth
functional assessment of their STAT6 variants; because of the
global nature of our cohort, the study does have limitations. First, patients were identified by their local expert clinicians as
candidates for genetic assessment based on their extreme al-
lergic phenotype and, in some cases, their family history. As a
result, we do not have prospectively defined inclusion criteria. Second, the global nature of the cohort and variation in local
access to medications such as dupilumab limited our ability to
run the same assays on pre-treatment primary cells from all
patients. Sharma et al. Human germline STAT6 gain-of-function variants IL13+, and IL4+ cells in memory CD4+ T cells of one representative patient, along with one representative healthy control. (D) Quantification of C showing IL5+,
IL13+, and IL4+ cells in patients along with 15 age-matched healthy controls. **, P < 0.01; ***, P < 0.001. (E) Uniform manifold approximation and projection
(UMAP) visualization of scRNAseq done on enriched T cells comparing one patient with one age-matched healthy control. (F) Dot plot showing expression of
selected differentially expressed genes (adjusted P value < 0.05) observed in scRNAseq between patient and healthy control and associated with T cells, B cells,
monocytes, or dendritic cells. The fatal cerebral aneurysm in P10 (p.E382Q) was not clini-
cally anticipated, but it is possible that the STAT6 GOF variant
also increased the risk of developing cerebral aneurysms. In-
deed, P1 (p.D419G) also had multiple rare anatomical variants in
the arteries of the Circle of Willis. Intracranial aneurysms have
been reported in patients with both STAT3 loss-of-function
(LOF) and STAT1 GOF (Chandesris et al., 2012; Dadak et al., 2017;
Toubiana et al., 2016). Increased activation of other STAT family
members, including STAT2, STAT3, and STAT5 have also been
observed in human abdominal aortic aneurysms (STAT6 was not
evaluated), although it is not clear whether enhanced STAT
phosphorylation contributes to aneurysms or is the result of
inflammation caused by aneurysms (Liao et al., 2012). As more
individuals with STAT6 GOF variants are identified, the possi-
bility of cerebral vascular anomalies warrants investigation. recurrent skin and lung infections, and elevated serum IgE
(Freeman and Milner, 2020; Vaseghi-Shanjani et al., 2021;
Zhang et al., 2018). AD hyper-IgE syndrome caused by dominant
negative variants in STAT3 (i.e., Job’s syndrome or STAT3 LOF) is
the best characterized of these conditions, but this list of dis-
orders also includes other AD (IL6ST; Beziat et al., 2020) and
autosomal recessive (AR; ZNF341 [B´eziat et al., 2018], IL6ST
[Shahin et al., 2019]) disorders (Bergerson and Freeman, 2019;
Minegishi, 2021). Notably, the patients we identified with STAT6
GOF variants did exhibit some of the extra-immunological
features typical of STAT3 LOF, specifically hyperextensible
joints, pathologic fractures, and vascular anomalies (Bergerson
and Freeman, 2019). Sharma et al. Despite these limitations, our study does identify GOF
variants in STAT6 as a novel monogenic allergic disorder. We
also present clinical and single cell evidence of the effectiveness
of dupilumab in STAT6 GOF patients. We anticipate that this
discovery will facilitate the recognition and targeted treatment
of more affected individuals and, with time, a full definition of
the human genotype-phenotype relationship caused by germline
human STAT6 GOF variants will emerge, including under-
standing the risk of lymphoma. A GOF STAT6 model (designated STAT6VT) has previously
been described in vitro (Daniel et al., 2000) and has been used
to study chronic atopic dermatitis in mouse models (Bruns
et al., 2003; DaSilva-Arnold et al., 2018). STAT6VT has the
substitution of two amino acid residues, at positions 547 and
548, in the SH2 domain resulting in a STAT6 mutant that is
constitutively active in an IL-4–independent manner and is
unresponsive to IL-4 stimulation (Daniel et al., 2000). The
humans we identified with STAT6 GOF variants and STAT6VT
mice share a number of key features of the allergic diathesis,
including elevated serum IgE and chronic atopic dermatitis. Very recently, a report was published describing a father and
his two sons with severe allergic disease who were all heter-
ozygous for the GOF STAT6 variant p.E377K (Suratannon et al.,
2022). This new family shares many of the features we report
in our cohort of 10 families (Fig. 2 A), further emphasizing
that patients with early onset severe allergic disease should be
assessed for underlying monogenic gene defects, including
STAT6. Based on our findings reported in this study, we suggest that
heterozygous GOF variants in STAT6 be added to the list of AD
causes of the hyper-IgE phenotype. While each of the conditions
known to cause a hyper-IgE phenotype has some specific clinical There is now a growing list of human single gene defects
that cause the classic hyper-IgE phenotypic triad of eczema, 10 of 18 Journal of Experimental Medicine
https://doi.org/10.1084/jem.20221755 Journal of Experimental Medicine
https://doi.org/10.1084/jem.20221755 Human germline STAT6 gain-of-function variants Sharma et al.
Human germline STAT6 gain-of-function variants Sharma et al. Human germline STAT6 gain-of-function variants Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 Figure 5. Primary lymphocytes of STAT6 GOF patients display high expression of STAT6 target genes. (A) Expression of IL4Rα in naive and memory
CD4+ cells is quantified as % positive cells in primary patient cells (n = 7, red) and healthy controls (n = 9, blue). Gating strategy for naive and memory and CD4+
Sharma et al. Journal of Experimental Medicine
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Human germline STAT6 gain-of-function variants
https://doi.org/10.1084/jem.20221755
Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud Uni Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 m/article-pdf/220/5/e20221755/1448977/jem_20221755 /article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 221755.pdf by Radboud University user on 13 June 2023 Figure 5. Primary lymphocytes of STAT6 GOF patients display high expression of STAT6 target genes. (A) Expression of IL4Rα in naive and memory
CD4+ cells is quantified as % positive cells in primary patient cells (n = 7, red) and healthy controls (n = 9, blue). Gating strategy for naive and memory and CD4+ Journal of Experimental Medicine
11 of 18
https://doi.org/10.1084/jem.20221755 Sharma et al. Human germline STAT6 gain-of-function variants Human germline STAT6 gain-of-function variants is presented along with a dot plot for a fluoresence minus one (FMO) IL-4Rα sample to display IL-4Rα+ gating, as well as a representative dot plot for a patient
and healthy control. (B) Expression of CD23 and IL4Ra in naive, non-class switched memory and memory B cells is quantified as % positive cells in primary
patient cells (n = 7, red) and healthy controls (n = 9, blue). Gating strategy for B cell subsets is presented along with a dot plot for an FMO IL-4Rα sample to
display IL-4Rα+ gating, as well as a representative dot plot for a patient and healthy control. Unpaired t test. **, P < 0.01; ***, P < 0.001. is presented along with a dot plot for a fluoresence minus one (FMO) IL-4Rα sample to display IL-4Rα+ gating, as well as a representative dot plot for a patient
and healthy control. (B) Expression of CD23 and IL4Ra in naive, non-class switched memory and memory B cells is quantified as % positive cells in primary
patient cells (n = 7, red) and healthy controls (n = 9, blue). Gating strategy for B cell subsets is presented along with a dot plot for an FMO IL-4Rα sample to
display IL-4Rα+ gating, as well as a representative dot plot for a patient and healthy control. Unpaired t test. **, P < 0.01; ***, P < 0.001. is presented along with a dot plot for a fluoresence minus one (FMO) IL-4Rα sample to display IL-4Rα+ gating, as well as a representative dot plot for a patient
and healthy control. (B) Expression of CD23 and IL4Ra in naive, non-class switched memory and memory B cells is quantified as % positive cells in primary
patient cells (n = 7, red) and healthy controls (n = 9, blue). Gating strategy for B cell subsets is presented along with a dot plot for an FMO IL-4Rα sample to
display IL-4Rα+ gating, as well as a representative dot plot for a patient and healthy control. Unpaired t test. **, P < 0.01; ***, P < 0.001. features (e.g., viral skin infections are a defining feature of
DOCK8 deficiency; Biggs et al., 2017; Chu et al., 2012), there is
considerable clinical overlap and clinically approved testing of
these pathways is rarely available. Sharma et al. Therefore, we recommend
genetic testing as the most efficient initial diagnostic approach
to patients who experience severe allergic disease beginning
very early in life. Finally, we demonstrate that dupilumab and
JAK inhibition may be an effective targeted treatment options
for patients with GOF STAT6 variants. features (e.g., viral skin infections are a defining feature of
DOCK8 deficiency; Biggs et al., 2017; Chu et al., 2012), there is
considerable clinical overlap and clinically approved testing of
these pathways is rarely available. Therefore, we recommend
genetic testing as the most efficient initial diagnostic approach
to patients who experience severe allergic disease beginning
very early in life. Finally, we demonstrate that dupilumab and
JAK inhibition may be an effective targeted treatment options
for patients with GOF STAT6 variants. Identification of STAT6 variant via next-generation sequencing
Based on local availability, research or clinical next-generation
sequencing of the genomic DNA was performed using either
whole exome or a targeted panel approach (as described previ-
ously; B´eziat et al., 2021; Campbell et al., 2022; Chovanec et al.,
2022; Hebert et al., 2022; Murrell et al., 2022; Tarailo-Graovac
et al., 2016). After bioinformatic analysis, de novo and inherited
STAT6 variants that were predicted to be damaging and that
segregated with disease were identified in each family (Tables
S4 and S5). Luciferase reporter assays Luciferase reporter plasmids encoding a 4× STAT6 binding site
(TTCCCAAGAA; the underlined bases represent the two half-
sites for STAT6-specific binding), encoding the promoter site
for CCL26, and encoding the promoter site for FCER2 were used
to assess WT and variant STAT6 promoter activity (Li et al.,
2016; Yildiz et al., 2015). See supplemental methods at the end
of the PDF for details. Table 1. Number of published GWAS studies linking polymorphisms (SNPs) in STAT6 to common allergic diseases in the population
Phenotype
Number of published
associations
References
Asthmaa
14
Daya et al., 2019; Demenais et al., 2018; Ferreira et al., 2019; Han et al., 2020; Johansson et al.,
2019; Olafsdottir et al., 2020; Pickrell et al., 2016; Pividori et al., 2019; Sakaue et al., 2021; Shrine
et al., 2019; Valette et al., 2021; Zhu et al., 2020; Zhu et al., 2018; Zhu et al., 2019
Eosinophil count
7
Astle et al., 2016; Chen et al., 2020; H¨oglund et al., 2022; Kachuri et al., 2021; Kichaev et al., 2019;
Sakaue et al., 2021; Vuckovic et al., 2020
Allergic disease
3
Ferreira et al., 2017; Ferreira et al., 2020; Zhu et al., 2018
Atopic dermatitis/eczema
3
Johansson et al., 2019; Kichaev et al., 2019; Tanaka et al., 2021
Serum IgE level
2
Daya et al., 2021; Granada et al., 2012
Allergic sensitization
2
Bønnelykke et al., 2013; Waage et al., 2018
Allergic rhinitis
1
Johansson et al., 2019
Eosinophilic gastrointestinal
disorders
1
Sleiman et al., 2014
Significant genome-wide associations (P < 5 × 10−8) between STAT6 SNPs and all relevant allergic traits were obtained through the National Human Genome
Research Institute–European Bioinformatics Institute GWAS Catalog (https://www.ebi.ac.uk/gwas/). aIncludes asthma, childhood-onset asthma, adult-onset asthma, and atopic asthma. Table 1. Number of published GWAS studies linking polymorphisms (SNPs) in STAT6 to common allergic disease Significant genome-wide associations (P < 5 × 10−8) between STAT6 SNPs and all relevant allergic traits were obtained through the National Human Genome
Research Institute–European Bioinformatics Institute GWAS Catalog (https://www.ebi.ac.uk/gwas/). aIncludes asthma, childhood-onset asthma, adult-onset asthma, and atopic asthma. Significant genome-wide associations (P < 5 × 10−8) between STAT6 SNPs and all relevant allergic traits were obtained through the National Human Genome
Research Institute–European Bioinformatics Institute GWAS Catalog (https://www.ebi.ac.uk/gwas/). aIncludes asthma, childhood-onset asthma, adult-onset asthma, and atopic asthma. Journal of Experimental Medicine
12 of 18
https://doi.org/10.1084/jem.20221755 Journal of Experimental Medicine
12 of 18 Generation and expression of STAT6 variant plasmids STAT6 variants described in this study were generated through
site-directed mutagenesis (SDM) for transfection purposes. Ex-
pression of WT STAT6 or STAT6 variants were induced tran-
siently in HEK293 cells using lipofectamine, or stably in Jurkat
T cells using a lenti-viral approach. See supplemental methods at
the end of the PDF for details. All procedures performed in the study were in accordance with
the ethical standards of the institutional research committee
and with the 1964 Helsinki declaration and its later amend-
ments or comparable ethical standards. All study participants
and/or their parents/guardians provided written informed
consent. Research study protocols were approved by local
institutions, specifically: The University of British Columbia
Clinical Research Ethics Board (H09-01228, H15-00641),
University College London Research Ethics Committee (04/
Q0501/119, 06/Q0508/16), University of Hong Kong Insti-
tutional Review Board (UW 08-301), National Institutes of
Health Institutional Review Board (NCT01164241), Child-
ren’s Hospital of Philadelphia Institutional Review Board
(19-016617), Children’s Hospital Bambino Gesù Ethical
Committee (1702_OPBG_2018). Sharma et al. Human germline STAT6 gain-of-function variants Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 Figure 6. JAK inhibitors and IL-4Rα antibody can be used as potential therapeutics for patients with GOF STAT6 variants. (A) Quantification of
phospho-STAT6 expression in transfected HEK293 cells left untreated (black) pre-treated with ruxolitinib (10 μM, 1 h; pink), tofacitinib (10 μM, 1 h; green), or
Sharma et al. Journal of Experimental Medicine
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Human germline STAT6 gain-of-function variants
https://doi.org/10.1084/jem.20221755
Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 /jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 Figure 6. JAK inhibitors and IL-4Rα antibody can be used as potential therapeutics for patients with GOF STAT6 variants. (A) Quantification of
phospho-STAT6 expression in transfected HEK293 cells left untreated (black) pre-treated with ruxolitinib (10 μM, 1 h; pink), tofacitinib (10 μM, 1 h; green), or Figure 6. JAK inhibitors and IL-4Rα antibody can be used as potential therapeutics for patients with GOF STAT6 variants. (A) Quantification of
phospho-STAT6 expression in transfected HEK293 cells left untreated (black) pre-treated with ruxolitinib (10 μM, 1 h; pink), tofacitinib (10 μM, 1 h; green), or Journal of Experimental Medicine
13 of 18
https://doi.org/10.1084/jem.20221755 Sharma et al.
Human germline STAT6 gain-of-function variants Acknowledgments (a) Jurkat cells: To model transcriptomics changes caused by
STAT6 variants, Jurkat T cells were transduced with either
c.1144G>C, p.E382Q, c.1256A>G, p.D419G, or WT STAT6. The
cells were either left unstimulated or stimulated with 100 ng/ml
of IL-4 for 4 h and subsequently used for RNA extraction and
sequencing. (b) Whole blood: Bulk RNAseq was done on 10
samples: one patient sample before dupilumab treatment initi-
ation, four patient samples after dupilumab treatment initiation,
and five healthy controls. (c) scRNAseq: Performed on PBMCs
and enriched T cells from the patient sample before and 2 yr
after dupilumab treatment, along with one age-matched healthy
control. See supplemental methods at the end of the PDF for
details. g
We thank the National Institute for Health and Care Research
(NIHR) BioResource volunteers for their participation, and
gratefully acknowledge NIHR BioResource centers, National
Health Service Trusts, and staff for their contribution. A list of
NIHR BioResource Rare Diseases Consortium members is
available at the end of the PDF. We would also like to ac-
knowledge the Biomedical Research Centre Sequencing Core for
their assistance with RNAseq and processing. We acknowledge
the extended clinical care team at the National Institute of Al-
lergy and Infectious Diseases who helped in the care and eval-
uation of Patient 11. We also thank the extended clinical team at
the Department of Paediatrics and Adolescent Medicine, Queen
Mary Hospital, Hong Kong, China for providing expert care and
support. We thank the Genomics Core of the Centre for Pan-
orOmic Sciences of the University of Hong Kong for their pro-
fessional performance of bulk and scRNAseq. Finally, we thank
the patients and their families for their trust and support. Flow cytometry
( ) (a) Phospho-flow cytometry: STAT6 phosphorylation was de-
termined via phospho-flow cytometry for STAT6-transfected
HEK293 cells, peripheral blood mononuclear cells (PBMCs),
T cell blasts, and EBV-transformed lymphoblastoid B cell lines
(LCLs). (b) Intracellular cytokine staining: Intracellular cytokine
staining was conducted on nine patient PBMCs, alongside 15
healthy controls, to study CD4+ T helper subsets as previously
described (Sharma et al., 2022). (c) CD23 and IL-4Rα expression
was studied on seven patient PBMCs and nine healthy control
PBMCs. See supplemental methods at the end of the PDF for
details. Structural modeling This work was supported by grants from the Canadian In-
stitutes of Health Research (PJT 178054; S.E. Turvey), Genome
British Columbia (SIP007; S.E. Turvey), and BC Children’s
Hospital Foundation. S.E. Turvey holds a Tier 1 Canada Research
Chair in Pediatric Precision Health and the Aubrey J. Tingle
Professor of Pediatric Immunology. M. Sharma was supported The effects of the STAT6 variants on the protein function
and structure were analyzed using three-dimensional models. SWISS-MODEL (Waterhouse et al., 2018) was used to model the
variants based on a template structure of the human STAT6
transcription factor solved as a homodimer and in complex with 14 of 18 Journal of Experimental Medicine
https://doi.org/10.1084/jem.20221755 Journal of Experimental Medicine
https://doi.org/10.1084/jem.20221755 Online supplemental material Clinical narratives for each patient are presented as Data S1. Fig. S1 is a detailed structural model showing the DNA and STAT6
variant interface. Fig. S2 shows complete blood count and the
immunological workup of all the patients. Fig. S3 shows addi-
tional in vitro data confirming the GOF nature of the STAT6
variants. Further workup of the primary patient cells is shown
in Fig. S4. Additional IL4Rα antibody and JAK inhibitor treat-
ment data of cells and patients is presented in Fig. S5. Table S1
lists primers used for site-directed mutagenesis. Table S2 lists
antibodies used for phospho-flow on different immune subsets. Table S3 lists antibodies used for TH phenotyping in patient
PBMCs. Pathogenicity prediction of the variants are presented in
Tables S4 and S5. Tables S6 and S7 are gene lists from GSEA
analysis shown in Fig. 3. Supplemental methods are listed at the
end of the PDF. dupilumab (10 nM, 1 h; blue), before and after stimulation with IL-4 (10 ng/ml, 30 min). Individual points represent separate transfections for each genotype
(n = 4). Gating strategy for pSTAT6+ cells can be found in Fig. S3 C. One-way ANOVA and Tukey’s post-hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (B) Quantification of pSTAT6+ cells in patient (red) and healthy control (blue) LCLs stimulated with IL-4 (10 ng/ml for 15 min), washed and incubated in
tofacitinib (10 μM) for 15, 30, and 60 min. Dotted translucent lines are indicative of no tofacitinib treatment (Fig. 4 B); n = 1. Gating strategy for pSTAT6+ cells
can be found in Fig. S4 C. (C) Cell type proportion gene signature as determined by the software Cibersort, in a patient undergoing dupilumab treatment for 2 yr
and five healthy controls. Cell labels are listed on the right. (D) Donut plot showing frequencies of CD4+ T helper subsets in one patient, an age-matched
healthy control (Fig. 4 E), and a 2-yr post-dupilumab treatment patient sample as measured by scRNAseq on enriched T cells. Frequency of TH2 cells is
quantified in the donut plots of the different samples. (E) Violin plots showing expression of IL4R in the patient (red), healthy control (blue), and a 2-yr post-
dupilumab sample (green). (F) Eczema scoring, EASI and SCORAD, for two patients after treatment with multiple doses of dupilumab. (G and H) Photographs
of hands showing (G) the severity of atopic dermatitis before and (H) the improvement after dupilumab treatment. DNA (PDB: 4Y5W, resolution: 3.1 ˚A, chains A, C, M, and N; Li
et al., 2016). Structures were visualized with UCSF Chimera
(Pettersen et al., 2004). Immunoblotting Immunoblotting was conducted as previously described (Sharma
et al., 2022) to assess the phosphorylation status of p.P643R
STAT6 variant, as phosphorylation of this variant could not be
detected via flow cytometry, using an antibody against the ty-
rosine 641 phosphorylation site. See supplemental methods at
the end of the PDF for details. Histology
l
f Formalin-fixed, paraffin-embedded gastric, duodenal, and skin
tissue were sectioned and subjected to H&E staining. Sharma et al. Human germline STAT6 gain-of-function variants The Laboratory of Human
Genetics of Infectious Diseases is supported by the Howard
Hughes Medical Institute, the Rockefeller University, the St. Giles
Foundation, the French National Research Agency (ANR) under
the “Investments for the Future” program (ANR-10-IAHU-01),
ANR CARMIL2 (ANR-21-CE15-0034), Instituts Th´ematiques Mul-
tiorganismes (ITMO) Cancer of Aviesan, and Institut National du
Cancer (INCa) within the framework of the 2021–2030 Cancer
Control Strategy (on funds administered by the Institut National
de la Sant´e et de la Recherche M´edicale), the Integrative Biology of
Emerging Infectious Diseases Laboratory of Excellence (ANR-10-
LABX-62-IBEID), the French Foundation for Medical Research
(EQU201903007798), the Square Foundation, Institut National de
la Sant´e et de la Recherche M´edicale, and Paris University Cit´e. This work was also supported by Children’s Hospital Bambino
Ges`u, where L. Pacillo and B. Rivalta were supported by 4-yr
doctoral scholarships. C. Cifaldi and C. Cancrini were supported by
the Italian Ministry of Health; C. Cifaldi was supported with
a 5x1000 Children’s Hospital post-doctoral scholarship, and
C. Cancrini holds a Development of Innovative Diagnostic and
Therapeutic Approaches for PID grant (Programma di rete, NET-
2011-02350069) and Ricerca Corrente. Additionally, the optimi-
zation of the Olink platform was supported by the PENTA
Foundation, funded through an independent grant by ViiV
Healthcare UK, named EPIICAL. Y.L. Lau is supported by the So-
ciety for the Relief of Disabled Children, Jeffrey Modell Founda-
tion, Doris Zimmern Endowed Professorship in Community Child
Health, and Chung Ko Lee and Cheung Yuen Kan Education and
Research Fund. D. Leung is supported by the Croucher Founda-
tion. J.S.D. Rosa Duque is supported by a donation in memory of
Dr. Ton Lung Quong and Reverend Marion Quong. Z. Liu, R. Liang,
and X. Yang are supported by the Edward and Yolanda Wong
Fund. The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript. Author contributions: Conceptualization, methodology, for-
mal analysis, investigation, visualization, writing—original Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud Un press.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 Disclosures: S. Delafontaine is supported by the Personal
Research Foundation Flanders grant 11F4421N. T.F. Brown-
Whitehorn reported grants from DBV Technology, “other” from
DBV Technology, and grants from Regeneron outside the sub-
mitted work. J.R. Treat reported personal fees from Sanofi/Re-
generon outside the submitted work. D. Bullens reported grants
from Research Foundation Flanders and Sanofi Genzyme
outside the submitted work. E.C. Morris reported personal fees
from Quell Therapeutics Limited outside the submitted work. J.J. O’Shea reported a patent for Janus kinase inhibitors with
royalties paid (National Institutes of Health). P. Palma reported
grants from the ViiV Foundation and Chiesi Foundation outside
the submitted work. S.O. Burns has received grant support from
CSL Behring and personal fees or travel expenses from CSL
Behring, Baxalta US Inc, Glaxo Smith Kline, and Biotest. I. Meyts
reported grants from CSLBehring (paid to institution) and
“other” from Boehringer-Ingelheim SAB (paid to institution)
outside the submitted work. J. Heimall reported grants from
Regeneron, CSL Behring, Enzyvant, and ADMA; and personal
fees from CSL Behring, UpToDate, Enzyvant, ADMA, and CIRM
outside the submitted work. No other disclosures were reported. Submitted: 14 October 2022
Revised: 12 December 2022
Accepted: 9 February 2023 Submitted: 14 October 2022
Revised: 12 December 2022
Accepted: 9 February 2023 References Adzhubei, I.A., S. Schmidt, L. Peshkin, V.E. Ramensky, A. Gerasimova, P. Bork, A.S. Kondrashov, and S.R. Sunyaev. 2010. A method and server
for predicting damaging missense mutations. Nat. Methods. 7:248–249. https://doi.org/10.1038/nmeth0410-248 Author contributions: Conceptualization, methodology, for-
mal analysis, investigation, visualization, writing—original
draft preparation, writing—review and editing: M. Sharma, D. Leung, M. Momenilandi, L.C.W. Jones, L. Pacillo, A.E. James,
J.R. Murrell, S. Delafontaine, J. Maimaris, M. Vaseghi-Shanjani,
K.L. Del Bel, and H.Y. Lu. Investigation, resources, writing—
review and editing: G.T. Chua, S. Di Cesare, O. Fornes, Z. Liu, G. Di Matteo, M.P. Fu, D. Amodio, I.Y.S. Tam, G.S.W. Chan, A.A. Aran, D., A.P. Looney, L. Liu, E. Wu, V. Fong, A. Hsu, S. Chak, R.P. Naikawadi, P.J. Wolters, A.R. Abate, et al. 2019. Reference-based
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Astle, W.J., H. Elding, T. Jiang, D. Allen, D. Ruklisa, A.L. Mann, D. Mead, H. Bouman, F. Riveros-Mckay, M.A. Kostadima, et al. 2016. The allelic Journal of Experimental Medicine
https://doi.org/10.1084/jem.20221755 Journal of Experimental Medicine
https://doi.org/10.1084/jem.20221755 15 of 18 by a CIHR Frederick Banting and Charles Best Canada Graduate
Scholarships Doctoral Award and University of British Columbia
Four Year Doctoral Fellowship (4YF). H.Y. Lu is supported by a
Canada Graduate Scholarship, 4YF, Killam Doctoral Scholarship,
Friedman Award for Scholars in Health, and a BC Children’s
Hospital Research Institute Graduate Studentship. M. Vaseghi-
Shanjani is funded by the Vanier Canada Graduate Scholarship
and 4YF. The work by J. Heimall was supported by the Elizabeth
Paige Lavin Endowed Chair fund. This project has also been
funded in part with federal funds from the Division of Intra-
mural Research of the National Institute of Allergy and Infec-
tious Diseases, National Institutes of Health. The content of this
publication does not necessarily reflect the views or policies of
the Department of Health and Human Services, nor does mention
of trade names, commercial products, or organizations imply en-
dorsement by the US Government. The Laboratory of Human
Genetics of Infectious Diseases is supported by the Howard
Hughes Medical Institute, the Rockefeller University, the St. Giles
Foundation, the French National Research Agency (ANR) under
the “Investments for the Future” program (ANR-10-IAHU-01),
ANR CARMIL2 (ANR-21-CE15-0034), Instituts Th´ematiques Mul-
tiorganismes (ITMO) Cancer of Aviesan, and Institut National du
Cancer (INCa) within the framework of the 2021–2030 Cancer
Control Strategy (on funds administered by the Institut National
de la Sant´e et de la Recherche M´edicale), the Integrative Biology of
Emerging Infectious Diseases Laboratory of Excellence (ANR-10-
LABX-62-IBEID), the French Foundation for Medical Research
(EQU201903007798), the Square Foundation, Institut National de
la Sant´e et de la Recherche M´edicale, and Paris University Cit´e. This work was also supported by Children’s Hospital Bambino
Ges`u, where L. Pacillo and B. Rivalta were supported by 4-yr
doctoral scholarships. C. Cifaldi and C. Cancrini were supported by
the Italian Ministry of Health; C. Cifaldi was supported with
a 5x1000 Children’s Hospital post-doctoral scholarship, and
C. Cancrini holds a Development of Innovative Diagnostic and
Therapeutic Approaches for PID grant (Programma di rete, NET-
2011-02350069) and Ricerca Corrente. Additionally, the optimi-
zation of the Olink platform was supported by the PENTA
Foundation, funded through an independent grant by ViiV
Healthcare UK, named EPIICAL. Y.L. Lau is supported by the So-
ciety for the Relief of Disabled Children, Jeffrey Modell Founda-
tion, Doris Zimmern Endowed Professorship in Community Child
Health, and Chung Ko Lee and Cheung Yuen Kan Education and
Research Fund. D. Leung is supported by the Croucher Founda-
tion. J.S.D. Rosa Duque is supported by a donation in memory of
Dr. Ton Lung Quong and Reverend Marion Quong. Z. Liu, R. Liang,
and X. Yang are supported by the Edward and Yolanda Wong
Fund. The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript. A th
t ib ti
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t
li
ti
th d l
f Sharma, J. Dalmann, R. van der Lee, G. Blanchard-Rohner, S. Lin,
Q. Philippot, P.A. Richmond, J.J. Lee, A. Matthews, M. Seear, A.K. Turvey, R.L. Philips, T.F. Brown-Whitehorn, C.J. Gray, K. Izumi,
J.R. Treat, K.H. Wood, J. Lack, A. Khleborodova, J.E. Niemela, X. Yang, R. Liang, L. Kui, C.S.M. Wong, G.W.K. Poon, A. Hoischen,
C.I. van der Made, J. Yang, K.W. Chan, J.S.D. Rosa Duque, P.P.W. Lee, M.H.K. Ho, B.H.Y. Chung, H.T.M. Le, W. Yang, P. Rohani, A. Fouladvand, H. Rokni-Zadeh, M. Changi-Ashtiani, M. Mir-
younesi, A. Puel, M. Shahrooei, A. Finocchi, P. Rossi, B. Rivalta,
C. Cifaldi, A. Novelli, C. Passarelli, S. Arasi, D. Bullens, K. Sauer, T. Claeys, C.M. Biggs, E.C. Morris, S.D. Rosenzweig,
J.J. O’Shea, W.W. Wasserman, H.M. Bedford, C.D.M. van Karne-
beek, and P. Palma. Conceptualization, methodology, investiga-
tion, validation, visualization, resources, funding acquisition,
project administration, supervision, writing—original draft
preparation, writing—review and editing: S.O. Burns, I. Meyts, J.-
L. Casanova, J.J. Lyons, N. Parvaneh, A.T.V. Nguyen, C. Cancrini,
J. Heimall, H. Ahmed, M.L. McKinnon, Y.L. Lau, V. Beziat, and S.E. Turvey. by a CIHR Frederick Banting and Charles Best Canada Graduate
Scholarships Doctoral Award and University of British Columbia
Four Year Doctoral Fellowship (4YF). H.Y. Lu is supported by a
Canada Graduate Scholarship, 4YF, Killam Doctoral Scholarship,
Friedman Award for Scholars in Health, and a BC Children’s
Hospital Research Institute Graduate Studentship. M. Vaseghi-
Shanjani is funded by the Vanier Canada Graduate Scholarship
and 4YF. The work by J. Heimall was supported by the Elizabeth
Paige Lavin Endowed Chair fund. This project has also been
funded in part with federal funds from the Division of Intra-
mural Research of the National Institute of Allergy and Infec-
tious Diseases, National Institutes of Health. The content of this
publication does not necessarily reflect the views or policies of
the Department of Health and Human Services, nor does mention
of trade names, commercial products, or organizations imply en-
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sequencing and the management of neurometabolic disorders. N. Engl. J. Med. 374:2246–2255. https://doi.org/10.1056/NEJMoa1515792 Sharma et al. Human germline STAT6 gain-of-function variants Journal of Experimental Medicine
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https://doi.org/10.1084/jem.20221755 Sharma et al. Sharma et al. Human germline STAT6 gain-of-function variants Human germline STAT6 gain-of-function variants Supplemental material Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud Un Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 press.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 Journal of Experimental Medicine
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https://doi.org/10.1084/jem.20221755 https://doi.org/10.1084/jem.20221755 Sharma et al. https://doi.org/10.1084/jem.20221755 Human germline STAT6 gain-of-function variants gure S1. Pathogenic STAT6 germline variants lie in different protein domains and are frequently identified as somatic variants. (A) Somatic mu-
tion counts for different amino acid changed as reported by COSMIC for STAT6. Red highlighted changes are those germline variants also identified in our
hort that cause severe allergic disease. (B) Structural model of the DNA-STAT6 homodimer complex showing location of the different STAT6 variants in
lation to the DNA-binding interface Specifically zoom-ins for variants at each location are shown in relation to previously described variants known to affect
Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud Un Figure S1. Pathogenic STAT6 germline variants lie in different protein domains and are frequently identified as somatic variants. (A) Somatic mu-
tation counts for different amino acid changed as reported by COSMIC for STAT6. Red highlighted changes are those germline variants also identified in our
cohort that cause severe allergic disease. (B) Structural model of the DNA-STAT6 homodimer complex showing location of the different STAT6 variants in
relation to the DNA-binding interface. Specifically, zoom-ins for variants at each location are shown in relation to previously described variants known to affect
STAT6 function. re S1. Pathogenic STAT6 germline variants lie in different protein domains and are frequently identified as somatic variants. (A) Somatic mu-
on counts for different amino acid changed as reported by COSMIC for STAT6. Red highlighted changes are those germline variants also identified in our press.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 pdf by Radboud University user on 13 June 2023 Figure S1. Pathogenic STAT6 germline variants lie in different protein domains and are frequently identified as somatic variants. (A) Somatic mu-
tation counts for different amino acid changed as reported by COSMIC for STAT6. Red highlighted changes are those germline variants also identified in our
cohort that cause severe allergic disease. (B) Structural model of the DNA-STAT6 homodimer complex showing location of the different STAT6 variants in
relation to the DNA-binding interface. Specifically, zoom-ins for variants at each location are shown in relation to previously described variants known to affect
STAT6 function. Journal of Experimental Medicine
https://doi.org/10.1084/jem.20221755 Journal of Experimental Medicine
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https://doi.org/10.1084/jem.20221755 Journal of Experimental Medicine
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https://doi.org/10.1084/jem.20221755 Sharma et al. Human germline STAT6 gain-of-function variants Figure S2. Complete blood counts and immunological workup of patients with pathogenic STAT6 variants. (A–G) Complete blood count for 15 out of
the 16 patients and age-based references (orange-shaded area) for the following populations: (A) hemoglobin, (B) platelets, (C) white blood cells, (D) lym-
phocytes, (E) neutrophils, (F) basophils, and (G) monocytes. (H–L) Immunological workup for 15 out of the 16 patients showing age-based references (orange-
shaded area) and populations quantification for: (H) CD3+ T cells, (I) CD4+ CD3+ T cells, (J) CD8+ CD3+ T cells, (K) NK cells, and (L) CD19+ B cells. (M–O)
Immunoglobulin concentrations for 15 out of the 16 patients showing age-based references (orange-shaded area): (M) IgA, (N) IgM, and (O) IgA. Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 20 Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud Un Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 ress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 Figure S2. Complete blood counts and immunological workup of patients with pathogenic STAT6 variants. (A–G)
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l t l Figure S2. Complete blood counts and immunological workup of patients with pathogenic STAT6 variants. (A–G) Complete blood count for 15 out of
the 16 patients and age-based references (orange-shaded area) for the following populations: (A) hemoglobin, (B) platelets, (C) white blood cells, (D) lym-
phocytes, (E) neutrophils, (F) basophils, and (G) monocytes. (H–L) Immunological workup for 15 out of the 16 patients showing age-based references (orange-
shaded area) and populations quantification for: (H) CD3+ T cells, (I) CD4+ CD3+ T cells, (J) CD8+ CD3+ T cells, (K) NK cells, and (L) CD19+ B cells. (M–O)
Immunoglobulin concentrations for 15 out of the 16 patients showing age-based references (orange-shaded area): (M) IgA, (N) IgM, and (O) IgA. Journal of Experimental Medicine https://doi.org/10.1084/jem.20221755 Human germline STAT6 gain-of-function variants Human germline STAT6 gain-of-function variants ure S3. In vitro assays demonstrate that STAT6 variants lead to increased STAT6 activity. (A and B) Luciferase assay of STAT6 activity on a plasmid
taining (A) CCL26 promoter and (B) FcεR2 promoter for WT-, different STAT6-variant transfected HEK293 cells before and after stimulation with IL-4
Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 ure S3. In vitro assays demonstrate that STAT6 variants lead to increased STAT6 activity. (A and B) Luciferase assay of STAT6 activity on a plasmid
taining (A) CCL26 promoter and (B) FcεR2 promoter for WT-, different STAT6-variant transfected HEK293 cells before and after stimulation with IL-4
0 ng/ml for 40 h), n = 3. (C) Gating strategy for determining % positive HEK293 pSTAT6 cells: dot plot for fluoresence minus one (FMO) is presented and was
d for establishing pSTAT6+ cells. (D and E) Full-length immunoblots of the cropped immunoblots shown in Fig. 3 E, showing HEK293 cells transfected with
-, inactive- (p.Y641F), p.P643R-, and p.D419G- STAT6 variants for (D) Myc-tag and β-actin, as well as (E) pSTAT6 before and after treatment with IL-4
ng/ml for 30 min). (F) Significantly upregulated (i) and downregulated (ii) genes upon IL-4 treatment in WT (green), p.E382Q (blue), and p.D419G (purple) in
kat cells as shown through Venn diagram. (G) Sample level enrichment analyses of significantly enriched immune pathways from MSigDB Hallmark in
timulated and IL-4–stimulated samples, comparing WT vs. either p.E382Q or p.D419G. Heatmap is normalized across the rows and shown as relative
ression. rma et al. Journal of Experimental Medicine
S4
Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud Un Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 press.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 Figure S3. In vitro assays demonstrate that STAT6 variants lead to increased STAT6 activity. (A and B) Luciferase assay of STAT6 activity on a plasmid
containing (A) CCL26 promoter and (B) FcεR2 promoter for WT-, different STAT6-variant transfected HEK293 cells before and after stimulation with IL-4
(100 ng/ml for 40 h), n = 3. (C) Gating strategy for determining % positive HEK293 pSTAT6 cells: dot plot for fluoresence minus one (FMO) is presented and was
used for establishing pSTAT6+ cells. (D and E) Full-length immunoblots of the cropped immunoblots shown in Fig. Journal of Experimental Medicine 3 E, showing HEK293 cells transfected with
WT-, inactive- (p.Y641F), p.P643R-, and p.D419G- STAT6 variants for (D) Myc-tag and β-actin, as well as (E) pSTAT6 before and after treatment with IL-4
(10 ng/ml for 30 min). (F) Significantly upregulated (i) and downregulated (ii) genes upon IL-4 treatment in WT (green), p.E382Q (blue), and p.D419G (purple) in
Jurkat cells as shown through Venn diagram. (G) Sample level enrichment analyses of significantly enriched immune pathways from MSigDB Hallmark in
unstimulated and IL-4–stimulated samples, comparing WT vs. either p.E382Q or p.D419G. Heatmap is normalized across the rows and shown as relative
expression. Journal of Experimental Medicine
https://doi.org/10.1084/jem.20221755 Sharma et al. Human germline STAT6 gain-of-function variants Measure of STAT6 activity in patient primary lymphocytes. (A) 1-h time course to measure phosphorylation of STAT6 in different populations
es from five patients (red) and one healthy control (blue) after stimulation with IL-4 (10 ng/ml). (B) Dose response in LCLs of patient one (red) vs. control (blue) after stimulation of cells with various doses of IL-4 15 min. (C) Gating strategy to determine % pSTAT6 positive cells in LCLs: dot plot
resented and was used for establishing pSTAT6+ cells. (D) Histograms showing phosphorylation of STAT6 in healthy control (blue) and patients
e p.D419Y (red, n = 2), p.D519H (purple, n = 4), p.D419N (pink, n = 1), and healthy controls (blue, n = 5) in T cell blasts that were stimulated with IL-
for 15 min, washed with PBS, and subsequently incubated in IL-4–free media for 60 min. Quantification of pSTAT6+ cells is presented and
o max stimulation (noted at 15 min). Two-way ANOVA followed by ˇS´ıd´ak’s multiple comparisons was conducted. **, P < 0.01. (E) Readout of 92
or P5 using throughput Olink proteomics. Eight healthy control distribution are shown as a violin plot in blue. The patient is shown as a red circle. s, IL-4 and IL-13, are highlighted in yellow. (F) T helper cell distribution for nine patients (red) and 15 age-matched healthy controls (blue) each. tomic comparison of naive CD4+ and naive CD8+ T cells between P6 and one healthy control measured through scRNAseq. Red genes are enriched
ue genes are enriched in healthy control. The two dotted lines are the P value and adjusted P value respectively. (H) Quantification of % CD23
Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud Un ress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 Figure S4. Measure of STAT6 activity in patient primary lymphocytes. (A) 1-h time course to measure phosphorylation of STAT6 in different populations
of lymphocytes from five patients (red) and one healthy control (blue) after stimulation with IL-4 (10 ng/ml). (B) Dose response in LCLs of patient one (red) vs. one healthy control (blue) after stimulation of cells with various doses of IL-4 15 min. (C) Gating strategy to determine % pSTAT6 positive cells in LCLs: dot plot
for FMO is presented and was used for establishing pSTAT6+ cells. Sharma et al. (D) Histograms showing phosphorylation of STAT6 in healthy control (blue) and patients
with genotype p.D419Y (red, n = 2), p.D519H (purple, n = 4), p.D419N (pink, n = 1), and healthy controls (blue, n = 5) in T cell blasts that were stimulated with IL-
4 (10 ng/ml) for 15 min, washed with PBS, and subsequently incubated in IL-4–free media for 60 min. Quantification of pSTAT6+ cells is presented and
normalized to max stimulation (noted at 15 min). Two-way ANOVA followed by ˇS´ıd´ak’s multiple comparisons was conducted. **, P < 0.01. (E) Readout of 92
biomarkers for P5 using throughput Olink proteomics. Eight healthy control distribution are shown as a violin plot in blue. The patient is shown as a red circle. Key cytokines, IL-4 and IL-13, are highlighted in yellow. (F) T helper cell distribution for nine patients (red) and 15 age-matched healthy controls (blue) each. (G) Transcriptomic comparison of naive CD4+ and naive CD8+ T cells between P6 and one healthy control measured through scRNAseq. Red genes are enriched
in patient; blue genes are enriched in healthy control. The two dotted lines are the P value and adjusted P value respectively. (H) Quantification of % CD23
positive cells in naive, non-class switched memory, and class-switched memory B cells between patients (red, n = 7) and healthy controls (blue, n = 9) after
stimulation with IL-4 (10 ng/ml) for 20 h. Unpaired t test. *, P < 0.05; **, P < 0.01. S5 Journal of Experimental Medicine
https://doi.org/10.1084/jem.20221755 Sharma et al. Journal of Experimental Medicine
https://doi.org/10.1084/jem.20221755 S6 Sharma et al. Human germline STAT6 gain-of-function variants Figure S5. STAT6 activity can be therapeutically targeted and can resolve clinical disease severity. (A) Quantification of luciferase assay in HEK293
transfected cells pre-treated with ruxolitinib (10 μM, 1 h), tofacitinib (10 μM, 1 h), or dupilumab (10 nM, 1 h), before and after stimulation with IL-4 (0.02 ng/ml,
4 h). n = 4. One-way ANOVA and Tukey’s post-hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (B) Eosinophil counts before and following initiation of
treatment with dupilumab are presented. Dots in red corresponds to transcriptomic data from this patient presented in Fig. 6 C. (C) PCA comparing whole
blood bulk RNAseq of P6 before treatment with dupilumab and four time points after treatment, alongside five healthy controls. (D) Heatmap signatures of
differentially expressed genes comparing pre-treatment patient samples against five healthy controls. Genes are row normalized. (E) Key genes, previously
described to be biomarkers for allergic disease (Lemonnier et al., 2020) in whole blood RNA are presented for the patient samples. Gray shaded area is the
range for the expression of these genes in five healthy controls. Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud Un press.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 Figure S5. STAT6 activity can be therapeutically targeted and can resolve clinical disease severity. (A) Quantification of luciferase assay in HEK293
transfected cells pre-treated with ruxolitinib (10 μM, 1 h), tofacitinib (10 μM, 1 h), or dupilumab (10 nM, 1 h), before and after stimulation with IL-4 (0.02 ng/ml,
4 h). n = 4. One-way ANOVA and Tukey’s post-hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (B) Eosinophil counts before and following initiation of
treatment with dupilumab are presented. Dots in red corresponds to transcriptomic data from this patient presented in Fig. 6 C. (C) PCA comparing whole
blood bulk RNAseq of P6 before treatment with dupilumab and four time points after treatment, alongside five healthy controls. (D) Heatmap signatures of
differentially expressed genes comparing pre-treatment patient samples against five healthy controls. Genes are row normalized. (E) Key genes, previously
described to be biomarkers for allergic disease (Lemonnier et al., 2020) in whole blood RNA are presented for the patient samples. Gray shaded area is the
range for the expression of these genes in five healthy controls. Phospho-STAT6 detection in PBMCs and T cell blasts p
Freshly isolated PBMCs were rested for 1 h in RPMI medium (without FCS) at a final concentration of 0.5 × 106 cells/ml in a 96-well
V-bottom plate. Cells were stained by Aqua Live/Dead Cell Stain Kit (Thermo Fisher Scientific; DCM; 10 min in 37°C). After removing
the DCM by washing with RPMI, cells were left unstimulated or were stimulated with IL4 (100 ng/μl) at different time points (0 min,
5 min, 15 min, 30 min, 60 min in 37°C). After the desired time point stimulation, the cells were fixed by adding 100 μl of prewarmed
BD Phosflow Fix Buffer I (BDB557870) directly to the wells (10 min in 37°C). Then cells were washed with 200 μl FACS buffer (PBS +
2% DMSO + 2 mM EDTA) and permeabilized in 100 μl of cold BD Phosflow Perm Buffer III (Cat# 558050) for 20 min at 4°C. The cells
were washed two times with 200 μl FACS buffer and stained using a cocktail of antibodies (Table S2) for 60 min at room tem-
perature and darkness. Samples were acquired on a FACS Gallios machine. p
p
q
For the generation of T cells blasts, T cells were first isolated from PBMCs using EasySep Human T Cell Isolation Kit (Cat# 17951;
Stemcell). Subsequently, the cells were treated with exogenous IL-2 (100 U/ml) and ImmunoCult Human CD3/CD28 T cell Activator
(Cat# 10971; Stemcell) for 12 d and maintained at a concentration of 1 million cells/ml every 2 d. Phosphorylation and dephos-
phorylation of STAT6 was measured as described above at time points: 0, 15 min of IL-4 stimulation (10 ng/ml), and 60 min after
wash out of IL-4. The following antibodies were used: STAT6 PE-CF594 (Cat# 564148; BD Biosciences), p-STAT6 AF647 (Cat# 612601;
BD Biosciences), and Fixable Viability Stain 780 (Cat# 565388; BD Biosciences). Samples were acquired on the BD FACSymphony. Intracellular cytokine staining on patient PBMCs To carry out intracellular cytokine detection/TH phenotyping, PBMCs from the patient and five age-matched controls were stim-
ulated with PMA/ionomycin for 4 h at 37°C in the presence of GolgiStop (Cat# 554724; BD Biosciences). Cells were stained with an
antibody panel (Table S3) using the eBioscience Foxp3 Transcription Factor Staining Buffer Set (Cat# 00-5523-00; Invitrogen,
Thermo Fisher Scientific). Samples were acquired on the BD FACSymphony flow cytometer (BD Biosciences). Data were analyzed
using FlowJo (BD Biosciences). Journal of Experimental Medicine
https://doi.org/10.1084/jem.20221755 Journal of Experimental Medicine
https://doi.org/10.1084/jem.20221755 S7 Transient and stable expression of STAT6 variants Transient expression of STAT6 variants in HEK293 cells were accomplished using a Lipofectamine 3000 kit (Thermo Fisher Sci-
entific) according to the manufacturer’s recommendations. Briefly, HEK293 cells were seeded at 2.0 × 105 cells/well in a 24-well plate
in 0.5 ml of DMEM with 10% FBS (Gibco, Life Technologies) and incubated for 24 h at 37°C. Cells were transfected with 250 ng of
plasmid DNA using the P3000 and Lipofectamine 3000 reagents and harvested after 24 h. Stable expression of STAT6 in Jurkat T cells was accomplished using the lentivirus approach as previously described (Kutner
et al., 2009; Fung et al., 2021). Briefly, Jurkat T cells were infected with lentiviral particles in the presence of 5 μg/ml polybrene
(Sigma-Aldrich) and spinoculated at 800 g for 30 min at 32°C, cultured, and expanded in complete RPMI-1640 (GE Healthcare)
supplemented with 10% FBS. Expanded cells were sorted on GFP expression using a BD FACS Aria (BD Biosciences) cell sorter. Generation of STAT6 variant plasmids f
p
Plasmids used for transfection studies contained full-length STAT6 in a pCMV6 entry vector with a C-terminal GFP tag (Cat#
RG210065; OriGene Technologies). To generate variants listed in Table S3, a Q5 site-directed mutagenesis kit (Cat# E0554S; New
England Biolabs) was used according to the manufacturer’s recommendations, with primer pairs noted in Table S1. To generate lentivirus vectors, WT, p.E382Q, and p.D419G STAT6 from the above plasmids were cloned into a GFP-tagged Lenti
vector (Cat# PS100071; OriGene Technologies) using EcoRI-HF (Cat# R3101) and NotI-HF (Cat# R3189) both from New England
Biolabs. The three STAT6 plasmids were packaged using third-generation packaging plasmids and transfected into HEK293T cells. Culture media was collected, centrifuged, filtered, concentrated, and stored at −80°C before use. All new variant plasmids were confirmed by Sanger sequencing and purified from 10-beta competent E. coli using a QIAprep Spin
Miniprep Kit (Qiagen). Supplemental methods Phospho-STAT6 detection in HEK293 cells Phospho-STAT6 activity was quantified by flow cytometry in transfected HEK293 cells stimulated with 10 ng/ml of IL-4 (Cat# 204-
IL-020; R&D Systems) for 15 min or transfected cells pre-treated with either 10 μM ruxolitinib (Cat# tlrl-rux; Invivogen), 10 μM
tofacitinib (Cat# S50001; Selleckchem), or 10 nM dupilumab for 2 h before stimulation as previously described (Lu et al., 2021). Briefly, transfected and stimulated cells were fixed using BD Cytofix (Cat# 554655; BD Biosciences) for 20 min at 4°C and per-
meabilized using Perm III for 30 min on ice (Cat# 558050; BD Biosciences). The cells were then stained with STAT6 PE-CF594 (Cat#
564148; BD Biosciences) and p-STAT6 AF647 (Cat# 612601; BD Biosciences). pSTAT6 expression was measured in GFP+STAT6+ cells
from WT and STAT6 variants samples on an LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software (BD
Biosciences). Sharma et al. Human germline STAT6 gain-of-function variants Olink high-throughput proteomics Olink high-throughput proteomics Olink high-throughput proteomics Serum proteins for P5 were analyzed using a multiplex technology based on proximity-extension assays (Lundberg et al., 2011). We
measured specific proteins using the Olink panel of inflammation. Briefly, the kit consisted of a microtiter plate for measuring 92
protein biomarkers in 88 samples, and each well contained 96 pairs of DNA-labeled antibody probes. To minimize inter- and in-
tra-run variation, the data were normalized using both an internal control (extension control) and an inter-plate control, and then
transformed using a pre-determined correction factor. The pre-processed data were reported in arbitrary units as normalized
protein expression (NPX) that enables individual protein analysis across a sample set analyzed in log2 scale, wherein a higher NPX
correlates with higher protein expression. The data were pre-processed using the NPX Manager Software and OlinkAnalyze R
package (v1.3.0). The data for two key TH2 cytokines, IL-4 and IL-13, are highlighted in Fig. S4 E. Luciferase reporter assay with CCL26 and FcεR2 promoter f
p
y
p
Luciferase reporter plasmids with CCL26 and FcεR2 promoter were also used to study STAT6 activity and were constructed from
pMCS-Gaussia Dura Luc vector (#16190; ThermoFisher Scientific). 293FT cells (#51-0035; Invitrogen) were seeded in 24-well plates
overnight at a density of 200,000 cells and transfected with 245 ng of pCCL26/pFceR2V2-Gaussia Dura Luc, 245 ng of a plasmid
encoding WT and variant STAT6, and 10 ng pCMV-red firefly Luc vector (#16156; ThermoFisher Scientific) using TransIT-2020
transfection reagent (#MIR5400; Mirus). Transfected cells were subsequently stimulated with 100 ng/ml of IL-4 (#200-04; Pe-
protech) or left untreated. Cell lysates were prepared and processed using Pierce Gaussia-Firefly Luciferase Dual Assay Kit (#16182;
Thermo Fisher Scientific) according to the manufacturer’s recommendations. Luciferase activity was measured on the luminometer
(Fusion alpha-FP, PerkinElmer Packard), and signals of Gaussia luciferase were normalized to those of red firefly luciferase. Whole blood RNAseq Human peripheral blood was collected in EDTA tubes. Erythrocytes were removed with RBC Lysis Solution (#158904; Qiagen) and
the resultant white cell pellet was washed once with PBS. RNA was extracted with TRIzoL (#15596026; Ambion). Library preparation
and Illumina sequencing (pair-end sequencing of 151 bp) were done at the Genomics Core of the Centre for PanorOmic Sciences in
LKS Faculty of Medicine of The University of Hong Kong. Complementary DNA (cDNA) libraries were prepared by KAPA mRNA
HyperPrep Kit KR1352-v3.16 (#08098123702; Roche). One microgram of total RNA was used as starting material. The manufacturer’s
protocol was followed. In brief, poly-A containing mRNA was collected using poly-T oligo-attached magnetic beads. The purified
mRNA was fragmented to 200–300 bp by incubating at 94°C for 6 min in the presence of magnesium ions. The fragmented mRNA
was then applied as template to synthesize the first-strand cDNA using random hexamer-primer and reverse transcriptase. In the
second strand cDNA synthesis, the mRNA template was removed and a replacement strand was generated to form the blunt-end
double-stranded (ds) cDNA. The ds-cDNA underwent 39 adenylation and ligation with xGen Dual Index UMI Adapters (Integrated
DNA Technologies). The adaptor-ligated libraries were enriched by 10 cycles of PCR. The libraries were denatured and diluted to
optimal concentration. Illumina NovaSeq 6000 was used for pair-end 151 bp sequencing. Journal of Experimental Medicine
https://doi.org/10.1084/jem.20221755 Journal of Experimental Medicine
https://doi.org/10.1084/jem.20221755 S8 Luciferase reporter assays with 4× STAT6 binding sites A luciferase reporter plasmid encoding a 4× STAT6 binding site (TTCCCAAGAA) was used to assess WT and variant STAT6 promoter
activity (Li et al., 2016). The p4xSTAT6-Luc2P plasmid (Addgene plasmid #35554; Addgene; http://n2t.net/addgene:35554; RRID:
Addgene_35554) was a gift from Axel Nohturfft (St. George’s University of London, London, UK). Briefly, HEK293 cells were seeded
in 24-well plates overnight at a density of 200,000 cells and transfected with 250 ng of p4xSTAT6-Luc2P, 250 ng of a plasmid
encoding a GFP-tagged WT or variant STAT6, and 10 ng Renilla luciferase (R-Luc) using Lipofectamine 3000. Transfected cells were
subsequently stimulated with 0.2 ng/ml of IL-4 (Cat# 6507-IL-010; R&D Systems) or left unstimulated. Additionally, for inhibitor
assays, transfected cells were incubated with either 10 μM ruxolitinib (Cat# tlrl-rux, Invivogen), 10 μM tofacitinib (Cat# S50001;
Selleckchem), or 10 nM dupilumab (Cat# HY-P9926; MedChemExpress) for 1 h before stimulation. Cell lysates were prepared and
processed using a Dual-Glo Luciferase Assay Kit (Cat# E2920; Promega) according to the manufacturer’s recommendations. Lu-
ciferase activity was measured on the Infinite M200 plate reader (Tecan). CD23 and IL-4Rα staining on lymphocytes g
y p
y
Expression of IL-4Rα and CD23 was studied on primary patient B cells (within PBMCs). Briefly, primary patient cells were incubated
in RPMI with or without IL-4 (10 ng/ml). After 20 h, the cells were washed and stained with the following antibodies: BV510 mouse
anti-human CD3 (Cat# 563109; BD; clone: UCHT1), APC-R700 mouse anti-human CD4 (Cat# 565995; BD; clone: SK3), BUV395 mouse
anti-human CD8 (Cat# 740303; BD; clone: HIT8a), BUV737 mouse anti-human CD19 (Cat# 741829; BD; clone: HIB19), BV786 mouse
anti-human CD27 (Cat# 563328; BD; clone: L128), PerCP-Cy5.5 mouse anti-human CD45RA (Cat# 563429; BD; clone: HI100), BV605
mouse anti-human IgD (Cat# 563313; BD; clone: IA6-2), BB515 mouse anti-human IgM (Cat# 564622; BD; clone: G20-127), BV421
mouse anti-human CD23 (Cat# 562707; BD, clone: M-L233), PE mouse anti-human CD124 (IL-4Rα; Cat# 355003; BioLegend; clone:
G077F6), and Fixable Viability Stain 780 (Cat# 565388; BD). Cells were acquired on the BD FACSymphony. Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud Universi Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 Human germline STAT6 gain-of-function variants Sharma et al. Human germline STAT6 gain-of-function variants Journal of Experimental Medicine
https://doi.org/10.1084/jem.20221755 Jurkat RNAseq RNA was extracted in triplicate as previously described (Lu et al., 2021) using a RNeasy Mini Plus Kit (Qiagen) according to the
manufacturer’s recommendations. RNA was prepared following the standard protocol for the NEBNext Ultra II Stranded mRNA
(New England Biolabs) and sequenced on the Illumina NextSeq 500 with Paired End 42 × 42 bp reads. De-multiplexed read se-
quences were aligned to a reference sequence using RNA-Seq Alignment app (v1.1.1) on Illumina Basespace, using Spliced Tran-
scripts Alignment to a Reference (STAR) aligner and Cufflinks 2 for assembly and estimation of gene expression. Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud Un Expression data were normalized to reads between samples using the edgeR package in R (R Foundation). Normalized counts
were filtered to remove low counts using the filterByExpr function in edgeR (Chen et al., 2016). PCA was done on log2(normalized
counts+0.25) in R using the PCA function. Differential expression between unstimulated and stimulated samples for all three STAT6-
transduced Jurkat T cells was accomplished using Limma (Ritchie et al., 2015). Differentially expressed genes were defined as those
with fold change (FC) >1.25 and adjusted P value <0.05. Pathway analysis was done by first performing GSEA with 1,000 permutations using the Molecular Signatures Database Hall-
mark module. Signal-to-noise ratio was used for gene ranking and the obtainednormalized enrichment scores and P values were
further adjusted using the Benjamini–Hochberg method. Pathways with an adjusted P value <0.05 were considered significant. Leading-edge genes from significant pathways between STAT6 WT and STAT6 c.1256A>G Jurkat were identified (no significantly
upregulated pathways were identified between STAT6 WT and STAT6 c.1144G>C). Expression levels of these genes were then de-
termined in each of the three groups (WT, c.1144G>C, c.1256A>G) under both stimulated and unstimulated conditions. Sample level
enrichment analyses scores were computed as previously described (Kulpa et al., 2019). Briefly, z-scores were computed for gene
sets of interest for each sample. The mean expression levels of significant genes were compared to the expression of 1,000 random
gene sets of the same size. The difference between observed and expected mean expression was then calculated and represented on
heatmaps. GSEA was also done on two other gene sets: (i) STAT6 targets and (ii) IL-4 TH2 targets. STAT6 targets were defined as the set of
genes that were significantly upregulated upon IL-4 stimulation in WT-STAT6–transduced Jurkat T cells. Jurkat RNAseq Enrichment of this gene set
was determined at both baseline and after IL-4 stimulation between WT and p.E382Q STAT6, and WT and p.D419G STAT6 Jurkat
T cells. IL-4-STAT6 TH2 skewing targets were previously reported (Elo et al., 2010) STAT6 target genes that are significantly up-
regulated in response to IL-4 treatment and that lead to TH2 skewing. Raw data are deposited in the Gene Expression Omnibus with
accession number GSE222646. scRNAseq scRNAseq was done on a patient sample before and after dupilumab treatment along with one age-matched healthy control. Human
peripheral blood was collected in lithium heparin tubes and PBMCs were isolated using Lymphoprep (AS #04-03-9391/02; Alere
Technologies). Single-cell suspension was obtained by passing through a 30 μm Pre-Separation Filter (#130-041-407; Miltenyi Biotec
GmbH). Dead cells were removed by Dead Cell Removal Kit (#130-090-101; Miltenyi Biotec GmbH). T cell isolation was performed
using Pan T cell Isolation Kit (#130-096-535; Miltenyi Biotec GmbH). Single-cell encapsulation and library preparation are done at
Genomics Core of the Centre for PanorOmic Sciences in LKS Faculty of Medicine of The University of Hong Kong. Single cell en-
capsulation and cDNA libraries were prepared by Chromium Single Cell 59 Reagent Kit v1 and Chromium Single Cell A Chip Kit, or by
Chromium Next GEM (gel beads-in-emulsion) Single Cell 59 Reagent Kit v2 (Dual Index) and Chromium Next GEM Chip K Single Cell Journal of Experimental Medicine
https://doi.org/10.1084/jem.20221755 S9 Journal of Experimental Medicine
https://doi.org/10.1084/jem.20221755 Sharma et al. Human germline STAT6 gain-of-function variants De-multiplexed read sequences were aligned to a reference sequence and transcript expression were estimated using the
DRAGEN RNA pipeline on Illumina Basespace. Expression data was normalized to reads between samples using the edgeR package in
R (R Foundation). Normalized counts were filtered to remove low counts using the filterByExpr function in edgeR (Chen et al., 2016). PCA was done on log2(normalized counts+0.25) in R using the PCA function. Differential expression was conducted using Limma
(Ritchie et al., 2015), on patient pre-treatment sample against 5 healthy controls. Differentially expressed genes (adjusted P value
<0.05) were visualized using pheatmap in R. Cell type abundance was estimated using the LM22 signature matrix file in Cibersort
(Newman et al., 2015). Cell types were reduced to nine immunologically relevant cell types: neutrophils, eosinophils, mast cells,
dendritic cells, monocytes, NK cells, CD4 T cells, CD8 T cells, memory B cells, and naive B cells. Raw data are deposited in the Gene
Expression Omnibus with accession number GSE215390. Western blot Cell lysates were prepared by lysis of HEK293 cells in a modified radio immunoprecipitation assay buffer with the Halt protease and
phosphatase inhibitors cocktail (Thermo Fisher Scientific). Cell lysates were separated by 10% SDS-PAGE and transferred onto
polyvinylidene difluoride membranes (Immobilon-FL; MilliporeSigma). Membranes were blocked with 5% BSA in Tris-buffered
saline with Tween-20, incubated with primary antibodies for 18 h at 4°C, incubated with secondary antibodies for 1 h at room
temperature, and imaged using a LI-COR Odyssey infrared scanner (LI-COR Biosciences). The primary antibodies used were the
following: STAT6 (Cat# 9362), pSTAT6 (Cat# 9361), Myc-tag (Cat# 2276), and β-actin (Cat# 3700), all from Cell Signaling Tech-
nologies. The secondary antibodies used were the following: goat anti-rabbit IgG DyLight 800 conjugated (611-145-002-0.5; Rockland
Immunochemicals) and goat anti-mouse IgG IRDye 680RD (926-6870; LI-COR). Sharma et al. Human germline STAT6 gain-of-function variants Kit. Cells in suspension were counted and loaded into individual wells of 10X Chromium Single Cell chip (10X Genomics). Single cells
were then encapsulated into GEM by 10X Chromium Single Cell Controller. Single Cell 59 Reagent Kit was used to perform
downstream steps: reverse transcription is performed on GEMs, followed by cDNA clean-up and amplification. The ds-cDNA went
through enzymatic fragmentation and adapter ligation. Index PCR and SPRIselect size selection were then done as per the manu-
facturer’s protocol. Library size and concentration are determined by Qubit, quantitative PCR, and Bioanalyzer assays. The libraries
were denatured and diluted to optimal concentration. Illumina NovaSeq 6000 was used for pair-end 151 bp sequencing with No-
vaSeq 6000 S4 Reagent Kit v1.0 and v1.5 (#20012866 and #20028312; Illumina). The raw FASTQ data was obtained, and the cellranger count function was then applied to generate single cell feature counts for
every single library (Cell Ranger v5.0.1). The output of filtered feature-barcode matrix containing only detected cell-associated
barcodes was used for further downstream analysis. The default parameters and options were applied for cellranger count after
providing the input FASTQ data and the relevant prebuilt references of the Human dataset (GRCh38) downloaded from the 10X
Genomic website (https://support.10xgenomics.com/single-cell-gene-expression/software/downloads/latest/). The filtered expression matrix generated by Cell Ranger for each sample was analyzed and processed with the R package Seurat
(v3.2.0). Quality control involved first filtering for quality based on the following criteria: mapping to over 200 unique genes, the
fraction of mitochondrial transcripts was <10%, and the fraction of unique hemoglobin transcripts was <1%. Scaling and clustering
was performed on PBMCs and enriched T cells separately. Dimensionality reduction using PCA was done on the 2,000 most variable
genes and UMAP was based on the first 20 PCs. Cell identities were first annotated with SingleR using fine labeling from the
Novershtern hematopoietic dataset (Novershtern et al., 2011; Aran et al., 2019) and Monaco Immune dataset (Monaco et al., 2019) for
PBMCs and enriched T cells respectively. The annotation was refined manually based on the UMAP clustering patterns, grouping the
SingleR labels for PBMCs into 10 main populations: naive B cells, memory B cells, naive CD4+, memory CD4+, naive CD8+, memory
CD8+, monocytes, dendritic cells, NK cells, and others. Journal of Experimental Medicine
https://doi.org/10.1084/jem.20221755 Journal of Experimental Medicine
https://doi.org/10.1084/jem.20221755 S10 Sharma et al. For differential gene expression analyses, we utilized the Seurat im-
plementation of negative binomial test, assuming an underlying negative binomial distribution in RNAseq data (Robinson and
Smyth, 2007; Hafemeister and Satija, 2019). Downloaded from http://rupress.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 ss.org/jem/article-pdf/220/5/e20221755/1448977/jem_20221755.pdf by Radboud University user on 13 June 2023 All the computational resources were obtained from the cluster of High Performance Computing Facility of the Centre for
PanorOmic Sciences, University of Hong Kong. Raw data are deposited in the Gene Expression Omnibus with accession number
GSE215405. NIHR BioResource Rare Diseases PID Consortium members Zoe Adhya, Hana Alachkar, Ariharan Anantharachagan, Richard Antrobus, Gururaj Arumugakani, Chiara Bacchelli, Helen Bax-
endale, Claire Bethune, Shahnaz Bibi, Barbara Boardman, Claire Booth, Michael Browning, Mary Brownlie, Siobhan Burns, Anita
Chandra, Hayley Clifford, Nichola Cooper, Sophie Davies, John Dempster, Lisa Devlin, Rainer Doffinger, Elizabeth Drewe, David
Edgar, William Egner, Tariq El-Shanawany, Bobby Gaspar, Rohit Ghurye, Kimberley Gilmour, Sarah Goddard, Pavel Gordins, Sofia
Grigoriadou, Scott Hackett, Rosie Hague, Lorraine Harper, Grant Hayman, Archana Herwadkar, Stephen Hughes, Aarnoud Huis-
soon, Stephen Jolles, Julie Jones, Peter Kelleher, Nigel Klein, Taco Kuijpers (principal investigator), Dinakantha Kumararatne, James
Laffan, Hana Lango Allen, Sara Lear, Hilary Longhurst, Lorena Lorenzo, Jesmeen Maimaris, Ania Manson, Elizabeth McDermott,
Hazel Millar, Anoop Mistry, Valerie Morrisson, Sai Murng, Iman Nasir, Sergey Nejentsev, Sadia Noorani, Eric Oksenhendler, Mark
Ponsford, Waseem Qasim, Ellen Quinn, Isabella Quinti, Alex Richter, Crina Samarghitean, Ravishankar Sargur, Sinisa Savic, Sur-
anjith Seneviratne, Carrock Sewall, Fiona Shackley, Ilenia Simeoni, Kenneth G.C. Smith (principal investigator), Emily Staples, Hans
Stauss, Cathal Steele, James Thaventhiran, Moira Thomas, Adrian Thrasher (principal investigator), Steve Welch, Lisa Willcocks,
Sarita Workman, Austen Worth, Nigel Yeatman, and Patrick Yong. Provided online are Table S1, Table S2, Table S3, Table S4, Table S5, Table S6, Table S7, and Data S1. Table S1 lists primers used for
site-directed mutagenesis. Table S2 lists antibodies used for phospho-flow on different immune subsets. Table S3 lists antibodies
used for TH phenotyping in patient PBMCs. Table S4 shows variant annotation and pathogenicity prediction of the variants
reported within the DNA-binding domain of STAT6 for 10 patients. Table S5 shows variant annotation and pathogenicity
prediction of the variants reported outside of the DNA-binding domain of STAT6 for six patients. Table S6 lists genes upregulated
(gray) and downregulated (red) in transduced WT-, p.E382Q-, and p.D419G-transduced Jurkats that meet the cutoff of fold-change
(FC) > 1.25 and adjusted P value < 0.05. Table S7 lists genes in the leading edge driving the enrichment of two pathways (IL-4/
STAT6 pathway and TH2 pathway) between transduced WT- vs. p.E382Q- and WT- vs. p.D419G- Jurkats. Data S1 describes clinical
narratives for each patient. Sharma et al. Journal of Experimental Medicine https://doi.org/10.1084/jem.20221755
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English
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Left Axis Deviation in Brugada Syndrome: Vectorcardiographic Evaluation during Ajmaline Provocation Testing Reveals Additional Depolarization Abnormalities
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International journal of molecular sciences
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cc-by
| 6,502
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Left Axis Deviation in Brugada Syndrome: Vectorcardiographic
Evaluation during Ajmaline Provocation Testing Reveals
Additional Depolarization Abnormalities an der Ree 1,†
, Jeroen Vendrik 1,†, Jan A. Kors 2, Ahmad S. Amin 1, Arthur A. M. Wilde 1
,
1 3
1 an der Ree 1,†
, Jeroen Vendrik 1,†, Jan A. Kors 2, Ahmad S. Amin 1, Arthur A. M. Wilde 1
,
n 1,3 and Pieter G. Postema 1,* Martijn H. van der Ree 1,†
, Jeroen Vendrik
Hanno L. Tan 1,3 and Pieter G. Postema 1,* 1
Heart Center, Department of Clinical and Experimental Cardiology, Amsterdam UMC, University of
Amsterdam, Cardiovascular Sciences, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands;
mailto:m.h.vanderree@amsterdamumc.nl (M.H.v.d.R.); j.vendrik@amsterdamumc.nl (J.V.);
a.s.amin@amsterdamumc.nl (A.S.A.); a.a.wilde@amsterdamumc.nl (A.A.M.W.);
h.l.tan@amsterdamumc.nl (H.L.T.) 1
Heart Center, Department of Clinical and Experimental Cardiology, Amsterdam UMC, University of
Amsterdam, Cardiovascular Sciences, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands;
mailto:m.h.vanderree@amsterdamumc.nl (M.H.v.d.R.); j.vendrik@amsterdamumc.nl (J.V.);
a.s.amin@amsterdamumc.nl (A.S.A.); a.a.wilde@amsterdamumc.nl (A.A.M.W.);
h.l.tan@amsterdamumc.nl (H.L.T.) 2
Department of Medical Informatics, Erasmus MC, University Medical Center Rotterdam, Doctor
Molewaterplein 40, 3015 GD Rotterdam, The Netherlands; j.kors@erasmusmc.nl
3
Netherlands Heart Institute Moreelsepark 1 3511 EP Utrecht The Netherlands 2
Department of Medical Informatics, Erasmus MC, University Medical Center Rotterdam, Doctor
Molewaterplein 40, 3015 GD Rotterdam, The Netherlands; j.kors@erasmusmc.nl p
j
3
Netherlands Heart Institute, Moreelsepark 1, 3511 EP Utrecht, The Netherlands *
Correspondence: p.g.postema@amsterdamumc.nl *
Correspondence: p.g.postema@amsterdamumc.nl †
These authors contributed equally to this work. Abstract: Patients with Brugada syndrome (BrS) can show a leftward deviation of the frontal QRS-
axis upon provocation with sodium channel blockers. The cause of this axis change is unclear. In
this study, we aimed to determine (1) the prevalence of this left axis deviation and (2) to evaluate its
cause, using the insights that could be derived from vectorcardiograms. Hence, from a large cohort
of patients who underwent ajmaline provocation testing (n = 1430), we selected patients in whom a
type-1 BrS-ECG was evoked (n = 345). Depolarization and repolarization parameters were analyzed
for reconstructed vectorcardiograms and were compared between patients with and without a >30◦
leftward axis shift.
Citation: van der Ree, M.H.; Vendrik,
J.; Kors, J.A.; Amin, A.S.; Wilde,
A.A.M.; Tan, H.L.; Postema, P.G. Left
Axis Deviation in Brugada Syndrome:
Vectorcardiographic Evaluation
During Ajmaline Provocation Testing
Reveals Additional Depolarization
Abnormalities. Int. J. Mol. Sci. 2021,
22, 484. https://doi.org/10.3390/
ijms22020484 Received: 31 August 2020
Accepted: 28 December 2020
Published: 6 January 2021 Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional clai-
ms in published maps and institutio-
nal affiliations. Keywords: Brugada syndrome; vectorcardiogram; left axis deviation; ventricular arrhythmias International Journal of
Molecular Sciences International Journal of
Molecular Sciences International Journal of
Molecular Sciences International Journal of
Molecular Sciences Left Axis Deviation in Brugada Syndrome: Vectorcardiographic
Evaluation during Ajmaline Provocation Testing Reveals
Additional Depolarization Abnormalities We found (1) that the prevalence of a left axis deviation during provocation testing
was 18% and (2) that this left axis deviation was not explained by terminal conduction slowing
in the right ventricular outflow tract (4th QRS-loop quartile: +17 ± 14 ms versus +13 ± 15 ms,
nonsignificant) but was associated with a more proximal conduction slowing (1st QRS-loop quartile:
+12[8;18] ms versus +8[4;12] ms, p < 0.001 and 3rd QRS-loop quartile: +12 ± 10 ms versus +5 ± 7 ms,
p < 0.001). There was no important heterogeneity of the action potential morphology (no difference
in the ventricular gradient), but a left axis deviation did result in a discordant repolarization (spatial
QRS-T angle: 122[59;147]◦versus 44[25;91]◦, p < 0.001). Thus, although the development of the
type-1 BrS-ECG is characterized by a terminal conduction delay in the right ventricle, BrS-patients
with a left axis deviation upon sodium channel blocker provocation have an additional proximal
conduction slowing, which is associated with a subsequent discordant repolarization. Whether this
has implications for risk stratification is still undetermined. 1. Introduction In patients suspected of Brugada syndrome (BrS), documenting the spontaneous
type-1 BrS-ECG or, after, provocation testing with a cardiac sodium channel blocker is
a required criterion for the BrS-diagnosis [1]. During such provocation testing, the QRS
axis may deviate towards the left [2,3]. As the right ventricular outflow tract (RVOT) is a
critical area in the development of the type-1 BrS-ECG and its associated malignant arrhyth-
mias [1,4], this left axis deviation may be caused, among others, by exaggerated conduction
slowing in the RVOT exceeding the conduction slowing that is already associated with
the development of the type-1 ECG [1,4,5]. This could result in a diminished rightward Copyright: © 2021 by the authors. Li-
censee MDPI, Basel,
Switzerland. This article is an open access article
distributed under the terms and con-
ditions of the Creative Commons At-
tribution (CC BY)
license (https://
creativecommons.org/licenses/by/
4.0/). https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 484. https://doi.org/10.3390/ijms22020484 Int. J. Mol. Sci. 2021, 22, 484 2 of 9 vector and consequently a more pronounced and leftward vector, resulting in a leftward
axis deviation [1]. Alternatively, this leftward deviation may be due to more proximal
conduction abnormalities. Currently, the prevalence of a left axis deviation in drug-induced
BrS and its origin are unknown. Importantly, when the underlying pathophysiological
mechanisms underlying the ECG variations in BrS are unraveled, this could contribute to
risk stratification. vector and consequently a more pronounced and leftward vector, resulting in a leftward
axis deviation [1]. Alternatively, this leftward deviation may be due to more proximal
conduction abnormalities. Currently, the prevalence of a left axis deviation in drug-induced
BrS and its origin are unknown. Importantly, when the underlying pathophysiological
mechanisms underlying the ECG variations in BrS are unraveled, this could contribute to
risk stratification. While the 12-lead ECG is unsuitable for determining the origin of a leftward axis
deviation, vectorcardiography provides more three-dimensional electrophysiological data
and consequently more spatiotemporal information [4,6,7]. For this reason, the vectorcar-
diogram is potentially able to distinguish between conduction slowing in the RVOT and
more proximal conduction abnormalities, thereby providing the opportunity to discover
the origin of the left axis deviation. Furthermore, the vectorcardiogram is also able to pro-
vide additional information on repolarization characteristics [4,8,9], which could provide
additional insights on the electrophysiological effects of axis deviations. 1. Introduction In this study, we evaluated vectorcardiograms of a large cohort of patients suspected
of BrS who underwent provocation testing in order to (1) determine the prevalence of a left
axis deviation in patients with a positive ajmaline test result and (2) to evaluate the cause
of this left axis deviation. 2.1. Baseline Characteristics Figure 1 shows the flowchart of the selection of patients. In total, 345 patients in whom
ajmaline testing elicited a type-1 BrS-ECG (positive test) were identified from a total cohort
of 1430 patients who underwent provocation testing (24.1%). In total, 320 of these 345
(92.8%) patients were included for this study, and 25 (7.2%) patients were excluded due
to incomplete data or an indeterminable axis. The prevalence of a left axis deviation in
patients with a positive test and determinable QRS-axis was 17.5% (n = 56/320). Patients
with a left axis deviation at baseline were more often female and were significantly shorter
and lighter; the BMI, however, did not significantly differ (Table 1). The age, histories of
(possible) arrhythmias, the indication for testing and the administered ajmaline dose, as
well as the percentage of the ajmaline maximal target dose, did not differ (Table 1). The
presence of an SCN5A mutation was also not significantly different between the groups. Please note that genetic testing was not performed in all patients, in particular when genetic
testing in a family member had already revealed the absence of a potentially causative
mutation. The baseline ECG parameters are presented in Table 1. R REVIEW
3
f 10 Figure 1. Flowchart of the patient selection. Table 1. Baseline characteristics. Figure 1. Flowchart of the patient selection. Figure 1. Flowchart of the patient selection. Figure 1. Flowchart of the patient selection. Int. J. Mol. Sci. 2021, 22, 484 3 of 9 Table 1. Baseline characteristics. Table 1. Baseline characteristics. 2.1. Baseline Characteristics Characteristic
Left Axis Deviation (n = 56)
No Left Axis Deviation (n = 264)
Age, years
46 ± 13
45 ± 13
Male sex
20 (35.7) *
143 (54.2)
Length, cm
171 ± 10 *
175 ± 11
Weight, kg
71 ± 16 *
78 ± 14
BMI, kg/m2
24 ± 4
26 ± 4
History of SVT, n (%)
0 (0.0)
6 (2.6)
History of VT/VF, n (%)
1 (2.2)
11 (4.8)
History of Syncope, n (%)
13 (28.9)
45 (20.4)
Family history of SCD/ SUD, n (%)
25 (47.2)
134 (52.3)
Family history of BrS, n (%)
31 (55.4)
135 (51.1)
Genetic testing, n (%)
35 (62.5)
154 (58.3)
Likely pathogenic or pathogenic SCN5A variant, n (%)
9 (25.7 ‡)
18 (11.7 ‡)
Indication for Test
ECG suspicious for BrS, n (%)
7 (12.5)
56 (21.2)
Symptoms (Syncope, VT/VF or AF), n (%)
6 (10.7)
18 (6.8)
Family screening BrS, n (%)
32 (57.1)
134 (50.8)
Family screening SCD/ SUD, n (%)
11 (19.6)
56 (21.2)
Ajmaline administered (mg)
72 ± 19
74 ± 24
Percentage of maximum ajmaline target dose
administered (%)
100 ± 22
95 ± 28
ECG Parameters
Heart rate (bpm)
66 ± 10
68 ± 1
PR-interval
168 ± 30
168 ± 29
QRS-duration (ms)
100 ± 14 *
105 ± 15
QTc-interval (ms)
425 ± 22
424 ± 28
Normal QRS-axis, n (%)
53 (94.6)
224 (84.8)
Left QRS-axis, n (%)
3 (5.4)
23 (8.7)
Data are presented as the mean±SD or n (%).* = p < 0.05. ‡: of patients who underwent genetic testing, nonsignificant (p = 0.057). BMI:
body mass index, BrS: Brugada syndrome, SCD: sudden cardiac death, SVT: supraventricular tachycardia, SUD: sudden unexplained death,
VT: ventricular tachycardia, VF: ventricular fibrillation. Data are presented as the mean±SD or n (%).* = p < 0.05. ‡: of patients who underwent genetic testing, nonsignificant (p = 0.057). BMI:
body mass index, BrS: Brugada syndrome, SCD: sudden cardiac death, SVT: supraventricular tachycardia, SUD: sudden unexplained death,
VT: ventricular tachycardia, VF: ventricular fibrillation. Data are presented as the mean±SD or n (%).* = p < 0.05. ‡: of patients who underwent genetic testing, nonsignificant (p = 0.057). BMI:
body mass index, BrS: Brugada syndrome, SCD: sudden cardiac death, SVT: supraventricular tachycardia, SUD: sudden unexplained death,
VT: ventricular tachycardia, VF: ventricular fibrillation. 2.2. Baseline Vectorcardiogram Parameters Patients with a positive test and a left axis deviation upon provocation testing had, at
baseline, shorter QRS-durations when compared to patients without a left axis deviation
upon provocation testing (100 ± 14 ms versus 105 ± 15 ms, p < 0.05). In the 3rd quartile
of the QRS-loop, conduction was slightly faster in patients with a left axis deviation
upon provocation (10[8;12] ms versus 12[10;14] ms, p < 0.05). There was no difference in
the depolarization–repolarization interaction in both groups (Table 2, QRS-T angle and
ventricular gradient). 4 of 9 Int. J. Mol. Sci. 2021, 22, 484 Table 2. Vectorcardiographic parameters in patients with a left axis deviation of >30◦at baseline, at ajmaline peak, and the
change between the baseline and ajmaline peak. Characteristic
Baseline
Peak
Change in Vectorcardiographic
Parameters between the
Baseline and Ajmaline Peak
Left Axis
Deviation
(n = 56)
No Left Axis
Deviation
(n = 264)
Left Axis
Deviation
(n = 56)
No Left Axis
Deviation
(n = 264)
Left Axis
Deviation
(n = 56)
No Left Axis
Deviation
(n = 264)
QRS duration, ms
100 ± 14 *
105 ± 15
143 ± 21 *
136 ± 20
+43 ± 16 †
+31 ± 21
Duration of q1, ms
33 ± 4
33 ± 5
45 ± 8 *
42 ± 8
+12[8;18] †
+8[4;12]
Duration of q2, ms
15 ± 3
15 ± 3
18 ± 6 *
20 ± 5
+2[–2;6] †
+6[2;8]
Duration of q3, ms
10[8;12] *
12[10;14]
22[14;30] †
14[10;20]
+12 ± 10 †
+5 ± 7
Duration of q4, ms
42 ± 10
44 ± 11
58 ± 16
57 ± 14
+17 ± 14
+13 ± 15
Frontal axis
QRS-loop, ◦
29 ± 24
29 ± 36
–74[–107;3] †
29[0;40]
–98[–128;–24] †
–6[–27;–1]
Spatial QRS T angle,
◦
67 ± 27
62 ± 34
122[59;147] †
44[25;91]
39 ± 46 †
–1 ± 43
Ventricular gradient,
mV.ms
37 ± 16
41 ± 17
35 ± 13
38 ± 14
–1 ± 7
–3 ± 7
Max. J-amplitude in
V1-V2ic3 (µV)
98.5[59;129]
89[52;142]
311[220;397]
342[245;440]
236 ± 185
246 ± 165
Data are presented as the mean±SD. median [IQR]. Max.: maximum; ms: milliseconds; mV: Millivolts; µV: Microvolts; q1: first quartile of
the QRS-loop; q2: second quartile of the QRS-loop; q3: third quartile of the QRS-loop; q4: fourth quartile of the QRS-loop. 2.2. Baseline Vectorcardiogram Parameters * = p < 0.05 and
† = p ≤0.001 for patients with a left axis deviation compared to patients with no left axis deviation. Table 2. Vectorcardiographic parameters in patients with a left axis deviation of >30◦at baseline, at ajmaline peak, and the
change between the baseline and ajmaline peak. Data are presented as the mean±SD. median [IQR]. Max.: maximum; ms: milliseconds; mV: Millivolts; µV: Microvolts; q1: first quartile of
the QRS-loop; q2: second quartile of the QRS-loop; q3: third quartile of the QRS-loop; q4: fourth quartile of the QRS-loop. * = p < 0.05 and
† = p ≤0.001 for patients with a left axis deviation compared to patients with no left axis deviation. 2.3.1. Depolarization Abnormalities At the peak ajmaline dose, progressive conduction slowing had occurred in all four
quartiles in both groups (Table 2). Patients with a left axis deviation showed more of a
conduction delay when compared to patients without a left axis deviation (QRS duration:
+43 ± 17 ms versus +30 ± 21 ms, p < 0.001). This conduction delay in patients with a left
axis deviation occurred primarily in the first (+12[8;18] ms versus +8[4;12] ms, p < 0.001)
and third (+12 ± 10 ms versus + 5 ± 7 ms, p < 0.001) quartiles. In the second quartile,
conduction slowing was less pronounced in patients with a left axis deviation (+2[−2;6] ms
versus +6[2;8] ms, p <0.001). Noticeably, the conduction delay in the fourth quartile was
similar in both groups (Table 2). In addition, there were no differences in the amount of
maximal right-precordial ST-elevation (measured at the J-point) between the two groups. In
Figure 2, the QRS-T loops of two representative patients at baseline and ajmaline peak are
shown; from this figure, the conduction slowing at the peak ajmaline dose when compared
to baseline (especially in the patient with left axis deviation) can be appreciated. Figure 3
shows the proportion of patients with a left axis deviation and an increase of the conduction
intervals above the median value of the cohort. For the first (63.3% versus 43.7%, p < 0.05)
and third quartiles (72.7% versus 37.6%, p < 0.001) this proportion was significantly higher
compared to patients without a left axis deviation (Figure 3). For the second quartile, in
contrast, the proportion of patients with conduction slowing was larger in the patients
without a left axis deviation (21.8% versus 35.4%, p = 0.059). The proportion of patients
with conduction slowing above the median of the entire cohort in the fourth quartile was
similar in both groups (52.7% versus 46.4%, p = 0.458) (Figure 3). 5 of 9 Int. J. Mol. Sci. 2021, 22, 484
Int. J. Mol. Sci. 2021, 21, (a) (b)
Figure 2. The QRS-T loop in the transverse plane of two representative patients at baseline and ajmaline peak. (a) Patient
without a left axis deviation. The QRS-axis in the frontal plane changed 3° to the left. 2.3.1. Depolarization Abnormalities At baseline, the QRS-duration was
94 ms and increased to 106 ms at ajmaline peak; the changes in the duration of the quartiles of the QRS-loops were: q1 +0
ms, q2 +2 ms, q3 +2 ms and q4 +8 ms. The QRS-T angle changed with –19° from 27° at baseline to 8° at ajmaline peak; (b)
Patient with a left axis deviation. The QRS-axis in the frontal plane changed 60° to the left. At baseline, the QRS-duration
was 92 ms and increased to 138 ms at ajmaline peak; the changes in the duration of the quartiles of the QRS-loops were:
q1 +14 ms, q2 -2 ms, q3 +18 ms and q4 +16 ms. The QRS-T angle changed with +60° from 40° at baseline to 100° at ajmaline
peak. Dashes of the QRS-T loop: 2-ms intervals. The black arrow indicates the QRS-loop direction. Figure 2. The QRS-T loop in the transverse plane of two representative patients at baseline and ajmaline peak. (a) Patient
without a left axis deviation. The QRS-axis in the frontal plane changed 3◦to the left. At baseline, the QRS-duration was 94
ms and increased to 106 ms at ajmaline peak; the changes in the duration of the quartiles of the QRS-loops were: q1 +0
ms, q2 +2 ms, q3 +2 ms and q4 +8 ms. The QRS-T angle changed with –19◦from 27◦at baseline to 8◦at ajmaline peak; (b)
Patient with a left axis deviation. The QRS-axis in the frontal plane changed 60◦to the left. At baseline, the QRS-duration
was 92 ms and increased to 138 ms at ajmaline peak; the changes in the duration of the quartiles of the QRS-loops were: q1
+14 ms, q2 -2 ms, q3 +18 ms and q4 +16 ms. The QRS-T angle changed with +60◦from 40◦at baseline to 100◦at ajmaline
peak. Dashes of the QRS-T loop: 2-ms intervals. The black arrow indicates the QRS-loop direction. I t J Mol S i 2021 21
FOR PEER REVIEW
6
f 10 (b) (a) (a) (b) (a) Figure 2. The QRS-T loop in the transverse plane of two representative patients at baseline and ajmaline peak. (a) Patient
without a left axis deviation. The QRS-axis in the frontal plane changed 3° to the left. 2.3.1. Depolarization Abnormalities At baseline, the QRS-duration was
94 ms and increased to 106 ms at ajmaline peak; the changes in the duration of the quartiles of the QRS-loops were: q1 +0
ms, q2 +2 ms, q3 +2 ms and q4 +8 ms. The QRS-T angle changed with –19° from 27° at baseline to 8° at ajmaline peak; (b)
Patient with a left axis deviation. The QRS-axis in the frontal plane changed 60° to the left. At baseline, the QRS-duration
was 92 ms and increased to 138 ms at ajmaline peak; the changes in the duration of the quartiles of the QRS-loops were:
q1 +14 ms, q2 -2 ms, q3 +18 ms and q4 +16 ms. The QRS-T angle changed with +60° from 40° at baseline to 100° at ajmaline
peak. Dashes of the QRS-T loop: 2-ms intervals. The black arrow indicates the QRS-loop direction. Figure 2. The QRS-T loop in the transverse plane of two representative patients at baseline and ajmaline peak. (a) Patient
without a left axis deviation. The QRS-axis in the frontal plane changed 3◦to the left. At baseline, the QRS-duration was 94
ms and increased to 106 ms at ajmaline peak; the changes in the duration of the quartiles of the QRS-loops were: q1 +0
ms, q2 +2 ms, q3 +2 ms and q4 +8 ms. The QRS-T angle changed with –19◦from 27◦at baseline to 8◦at ajmaline peak; (b)
Patient with a left axis deviation. The QRS-axis in the frontal plane changed 60◦to the left. At baseline, the QRS-duration
was 92 ms and increased to 138 ms at ajmaline peak; the changes in the duration of the quartiles of the QRS-loops were: q1
+14 ms, q2 -2 ms, q3 +18 ms and q4 +16 ms. The QRS-T angle changed with +60◦from 40◦at baseline to 100◦at ajmaline
peak. Dashes of the QRS-T loop: 2-ms intervals. The black arrow indicates the QRS-loop direction. Figure 2. The QRS-T loop in the transverse plane of two representative patients at baseline and ajmaline peak. (a) Patient
without a left axis deviation. The QRS-axis in the frontal plane changed 3° to the left. At baseline, the QRS-duration was
94 ms and increased to 106 ms at ajmaline peak; the changes in the duration of the quartiles of the QRS-loops were: q1 +0
ms, q2 +2 ms, q3 +2 ms and q4 +8 ms. 3.1. Main Findings In this study, we show that (1) the prevalence of a left axis deviation during ajmaline
provocation testing in patients with a positive ajmaline test is 17.5% and (2) that this left
axis deviation is not caused by conduction slowing in the RVOT but is due to additional
conduction slowing in the more proximal conduction system. g
p
y
The observed left axis deviation could possibly occur due to excessive conduction
slowing in the RVOT, as this is the most commonly affected region in BrS patients [1,4]. Our
results, however, show that the RVOT conduction, as mirrored by the conduction slowing
in the 4th quartile of the QRS-loop, slows in equal amounts between patients with a left
axis deviation and patients without a left axis deviation while they develop the type-1 ECG. This demonstrates that the RVOT region is equally affected in both groups and that the
cause of the left axis deviation must originate more proximally in the conduction system. Conduction slowing in BrS patients who developed a left axis deviation was indeed most
prominent in the 1st and 3rd quartiles of the QRS-loop, most likely representing additional
septal and free wall depolarization abnormalities, respectively [7,10–12]. Apparently,
in these patients with a left axis deviation, conduction seems to be more extensively
affected. This could potentially explain some part of the varying arrhythmogenic risk in
BrS-patients [1]. These conductional differences cannot be explained from our results by the
currently known (likely) pathogenic SCN5A mutation, as the mutation status did not differ
significantly between groups. Still, loss-of-function SCN5A mutations will often result in a
more general conduction disease. Surprisingly, at baseline, conduction in the 3rd quartile
is slightly faster in patients with a left axis deviation, whilst out of the four quarters, this
quartile showed the most conduction slowing at the peak ajmaline. If conduction in the
free wall actually is the most extensively affected region, this faster conduction at baseline
is remarkable and against our expectations. The underlying mechanism and the clinical
implications of this finding are unclear and could be the focus of future research. In addition,
global repolarization appeared to be more discordant from depolarization in patients with
a left axis deviation, as indicated by increased, borderline abnormal (>105–135◦), QRST-
angles. In the general population, abnormal (>135◦) and borderline abnormal (105–135◦)
QRS-T angles are strong predictors for cardiac death. 3.1. Main Findings One could therefore hypothesize that
patients with a positive test with a concomitant left axis deviation might have a higher
arrhythmogenic risk. y
g
As to the origin of the characteristic ST-elevation (or J-point elevation) that is associated
with the development of a type-1 BrS-ECG, previous studies suggested that excitation
failure at the RVOT was elementary in this process [13,14]. In the current study, the
amount of ST-elevation did not differ between those ajmaline-positive patients with or
without a left axis deviation upon ajmaline provocation. Whether vectorcardiography,
which is dependent on spatial vectors, would be able to add insights to the occurrence
and localization of an excitation failure in the development of the type-1 ECG during
the simultaneous slowing of conduction during type-1 ECG development is currently
uncertain. 2.3.1. Depolarization Abnormalities The QRS-T angle changed with –19° from 27° at baseline to 8° at ajmaline peak; (b)
Patient with a left axis deviation. The QRS-axis in the frontal plane changed 60° to the left. At baseline, the QRS-duration
was 92 ms and increased to 138 ms at ajmaline peak; the changes in the duration of the quartiles of the QRS-loops were:
q1 +14 ms, q2 -2 ms, q3 +18 ms and q4 +16 ms. The QRS-T angle changed with +60° from 40° at baseline to 100° at ajmaline
peak. Dashes of the QRS-T loop: 2-ms intervals. The black arrow indicates the QRS-loop direction. Figure 2. The QRS-T loop in the transverse plane of two representative patients at baseline and ajmaline peak. (a) Patient
without a left axis deviation. The QRS-axis in the frontal plane changed 3◦to the left. At baseline, the QRS-duration was 94
ms and increased to 106 ms at ajmaline peak; the changes in the duration of the quartiles of the QRS-loops were: q1 +0
ms, q2 +2 ms, q3 +2 ms and q4 +8 ms. The QRS-T angle changed with –19◦from 27◦at baseline to 8◦at ajmaline peak; (b)
Patient with a left axis deviation. The QRS-axis in the frontal plane changed 60◦to the left. At baseline, the QRS-duration
was 92 ms and increased to 138 ms at ajmaline peak; the changes in the duration of the quartiles of the QRS-loops were: q1
+14 ms, q2 -2 ms, q3 +18 ms and q4 +16 ms. The QRS-T angle changed with +60◦from 40◦at baseline to 100◦at ajmaline
peak. Dashes of the QRS-T loop: 2-ms intervals. The black arrow indicates the QRS-loop direction. Figure 3. Proportion of patients with conduction slowing above the median value of the entire
cohort. Figure 3. Proportion of patients with conduction slowing above the median value of the entire cohort. Figure 3. Proportion of patients with conduction slowing above the median value of the entire
cohort. Figure 3. Proportion of patients with conduction slowing above the median value of the entire cohort. Int. J. Mol. Sci. 2021, 22, 484 6 of 9 2.3.2. Repolarization Abnormalities 2.3.2. Repolarization Abnormalities The spatial QRS-T angle significantly differed at the peak ajmaline dose (Table 2): in
patients with a left axis deviation, the spatial QRS-T angle rose to borderline abnormal
values (>105–135◦) whilst remaining normal in the patients without a left axis deviation
(122[59;147]◦versus 44[25;91]◦, p < 0.001). The heterogeneity of the action potential mor-
phology, as expressed by the ventricular gradient, did not significantly change or differ
between the two groups at the peak ajmaline dose (Table 2). 3.2. Future Perspective Future studies may investigate whether the occurrence of a left axis deviation during
ajmaline testing in BrS-patients partly determines their arrhythmogenic risk. Clearly, a Int. J. Mol. Sci. 2021, 22, 484 7 of 9 greater ability to stratify the arrhythmogenic risk in BrS patients would make it possible to
optimize treatments accordingly. greater ability to stratify the arrhythmogenic risk in BrS patients would make it possible to
optimize treatments accordingly. 4.2.1. Electrocardiographic Recordings 4.2.1. Electrocardiographic Recordings Modified 12-lead ECGs—with V3 and V5 placed cranially to V1 and V2 over the third
intercostal space—were recorded at baseline and at one minute after each ajmaline bolus. 3.3. Limitations Vectorcardiograms were not recorded using additional orthogonal Frank XYZ leads
but were reconstructed from 12-lead ECGs. Despite the fact that the reconstruction process
using matrix multiplication has been validated, we cannot exclude some degree of vari-
ability from Frank leads [15]. However, we used the same lead setup in every patient and
compared within-patient changes. In addition, by using the reconstructed vectorcardio-
grams, we enabled a future comparison of our data with other cohorts for whom digital
12-lead ECG data is available. In addition, as this was a retrospective exploratory study, we were not informed
about the follow-up data of these patients so as to determine potential differences in
arrhythmogenic risk or differences in the progression of the clinical phenotype as indicated
by the presence or absence of a (ajmaline-induced) left axis deviation. 4.1. Patients and Sodium Channel Provocation Testing 4.1. Patients and Sodium Channel Provocation Testing In this study, out of a cohort of 1430 patients who underwent provocation testing
during the period from 2009 to 2015, a selection of patients was made based on the
provocation test result and the cardiac axis. All patients for whom the provocation test was
positive (see below) and whose electrocardiogram were available for a vectorcardiogram
reconstruction were selected for this study; those with an abnormal or negative test were
excluded. Tests were defined as (1) positive if a type-1 BrS-ECG occurred [1], (2) abnormal
if arrhythmias or an excessive QRS-widening of ≥40% occurred, and (3) negative if the
target dose was reached and none of the criteria above were met. Ajmaline was used as
the sodium channel blocker and was infused intravenously in boluses of 10 mg/min until
the maximum dose (1 mg/kg) was reached or until a positive or abnormal test result was
obtained. None of the patients had exhibited a spontaneous type-1 BrS ECG before the
provocation test. These patients underwent provocation testing because of symptoms (e.g.,
unexplained syncope or documented ventricular arrhythmias), a baseline ECG that raised
the suspicion of BrS, family screening for BrS or family screening in the context of a sudden
cardiac death or sudden unexplained death. 4.2. Electrocardiographic Recordings, Analysis and Definitions 4.2.2. Electrocardiographic Analysis ECGs were analyzed with the electrocardiographic analysis system MEANS, and
its markers settings (e.g., P-wave onset, P-wave offset, QRS onset, etc.) were manually
inspected and adjusted if deemed necessary [16]. Vectorcardiograms were reconstructed
from the 12-lead ECG using the matrix multiplication as previously described [15]. The
vectorcardiographic analysis consisted of dividing the spatial QRS-loop in four quartiles
of equal length. Subsequently, the durations (ms) in the transverse plane were measured
out of these four quartiles. Furthermore, to study the depolarization and repolarization
interaction, the spatial QRS-T angle (◦) and the vector magnitude (mV·ms) (i.e., ventric-
ular gradient) of the spatial QRS-T integral were determined. An abnormally increased
QRS-T angle indicates global discordant repolarization, values of 105–135◦(borderline
abnormal) and >135 (abnormal) are associated with fatal cardiac arrhythmias in the general
population [8]. The ventricular gradient is considered a three-dimensional measure of the
heterogeneity of the action potential morphology [9]. In order to calculate the change in
the conduction intervals, the vectorcardiographic parameters at baseline were deducted Int. J. Mol. Sci. 2021, 22, 484 8 of 9 from the parameters at the peak ajmaline dose. To further evaluate the four quartiles of
the QRS-loop between patients with a left axis deviation and patients without a left axis
deviation, the proportion of patients with an increase above the median value of the entire
cohort was compared between the two groups. from the parameters at the peak ajmaline dose. To further evaluate the four quartiles of
the QRS-loop between patients with a left axis deviation and patients without a left axis
deviation, the proportion of patients with an increase above the median value of the entire
cohort was compared between the two groups. 4.3. Statistical Analysis The statistical analysis was performed with SPSS Statistics (version 25.0, IBM Corpo-
ration, Armonk, New York, USA). Categorical variables are presented as frequencies and
group percentages. To compare such variables, the Fisher-exact test was used. Continuous
variables are expressed as the mean ± standard deviation in the case of a normal distribu-
tion or the median [interquartile range] in the case of a skewed distribution. Histograms
and Q–Q plots were used to evaluate the distribution of variables with continuous data. The unpaired two-tailed t-test was used to compare normally distributed variables; in the
case of a skewed distribution, the Mann–Whitney U test was used. A p-value of <0.05 was
accepted as the level of statistical significance. 4.2.3. Electrocardiographic Definitions A left axis deviation was defined in this study as a leftward shift of the frontal QRS
axis of >30◦at the peak ajmaline dose as compared to the baseline. We defined the fourth
quartile of the QRS-loop—in patients with a positive ajmaline test—as representative of
RVOT conduction [4]. 5. Conclusions In BrS-patients, a left axis deviation during ajmaline testing occurs in a significant
number of patients. This leftward shift in axis is not caused by an additional conduction
slowing of the RVOT but appears to occur as a consequence of an additional, more proximal
conduction slowing. Whether a left axis deviation can also be used for the stratification of
arrhythmia risk is currently undetermined. Author Contributions: Conceptualization, P.G.P. and A.A.M.W. and M.H.v.d.R.; methodology, P.G.P. and A.A.M.W. and M.H.v.d.R.; software, J.A.K.; validation, J.A.K.; formal analysis, M.H.v.d.R. and
J.V.; investigation, P.G.P., A.A.M.W., A.S.A. and H.L.T.; resources P.G.P., A.A.M.W. and H.L.T.;
data curation, M.H.v.d.R., J.V. and J.A.K.; writing—original draft preparation, M.H.v.d.R., J.V. and
P.G.P.; writing—review and editing, A.A.M.W., A.S.A. and H.L.T.; visualization, M.H.v.d.R., P.G.P.;
supervision, P.G.P.; project administration, M.H.v.d.R. and J.V.; funding acquisition, A.A.M.W. and
H.L.T. All authors have read and agreed to the published version of the manuscript. Funding: This work has received funding from the European Union’s Horizon 2020 research and
innovation programme under acronym ESCAPE-NET, registered under grant agreement No. 733381,
and the COST Action PARQ (grant agreement No CA19137) supported by COST (European Coopera-
tion in Science and Technology). Institutional Review Board Statement: Ethical review and approval were waived for this study, the
sodium channel provocation tests were performed as part of routine clinical care. Informed Consent Statement: Patient consent was waived, the sodium channel provocation tests
were performed as part of routine clinical care. The data was subsequently collected and anonymized
by the treating physician. Informed Consent Statement: Patient consent was waived, the sodium channel provocation tests
were performed as part of routine clinical care. The data was subsequently collected and anonymized
by the treating physician. Data Availability Statement: The data presented in this study are available on request from the
corresponding author. The data are not publicly available due to privacy reasons. Data Availability Statement: The data presented in this study are available on request from the
corresponding author. The data are not publicly available due to privacy reasons. Acknowledgments: We acknowledge the support from the Netherlands CardioVascular Research
Initiative, the Dutch Heart Foundation, Dutch Federation of University Medical Centres, the Nether-
lands Organisation for Health Research and Development and the Royal Netherlands Academy of
Sciences (Predict2). References 1. Antzelevitch, C.; Yan, G.-X.; Ackerman, M.J.; Borggrefe, M.; Corrado, D.; Guo, J.; Gussak, I.; Hasdemir, C.; Horie, M.; Huikuri,
H.; et al. J-Wave syndromes expert consensus conference report: Emerging concepts and gaps in knowledge. Europace 2016, 13,
665–694. [CrossRef] 1. Antzelevitch, C.; Yan, G.-X.; Ackerman, M.J.; Borggrefe, M.; Corrado, D.; Guo, J.; Gussak, I.; Hasdemir, C.; Horie, M.; Huikuri,
H.; et al. J-Wave syndromes expert consensus conference report: Emerging concepts and gaps in knowledge. Europace 2016, 13,
665–694. [CrossRef] 2. Morita, H.; Morita, S.T.; Nagase, S.; Banba, K.; Nishii, N.; Tani, Y.; Watanabe, A.; Nakamura, K.; Kusano, K.F.; Emori, T.; et al. Ventricular Arrhythmia Induced by Sodium Channel Blocker in Patients with Brugada Syndrome. J. Am. Coll. Cardiol. 2003, 42,
1624–1631. [CrossRef] [PubMed] 3. Wilde, A.A.M.; Antzelevitch, C.; Borggrefe, M.; Brugada, J.; Brugada, R.; Brugada, P.; Corrado, D.; Hauer, R.N.W.; Kass, R.S.;
Nademanee, K.; et al. Proposed diagnostic criteria for the Brugada syndrome: Consensus report. Circulation 2002, 106, 2514–2519. [CrossRef] [PubMed] 4. Postema, P.G.; van Dessel, P.F.H.M.; Kors, J.A.; Linnenbank, A.C.; van Herpen, G.; Ritsema van Eck, H.J.; van Geloven, N.;
de Bakker, J.M.T.; Wilde, A.A.M.; Tan, H.L. Local Depolarization Abnormalities Are the Dominant Pathophysiologic Mechanism
for Type 1 Electrocardiogram in Brugada Syndrome. A Study of Electrocardiograms, Vectorcardiograms, and Body Surface
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g
j
5. Pappone, C.; Mecarocci, V.; Manguso, F.; Ciconte, G.; Vicedomini, G.; Sturla, F.; Votta, E.; Mazza, B.; Pozzi, P.; Borrelli, V.; et al. New electromechanical substrate abnormalities in high-risk patients with Brugada syndrome. Heart Rhythm 2020, 17, 637–645. [CrossRef] [PubMed] [CrossRef] [PubMed] 6. Pérez Riera, A.R.; Uchida, A.H.; Filho, C.F.; Meneghini, A.; Ferreira, C.; Schapacknik, E.; Dubner, S.; Moffa, P. Significance of
Vectorcardiogram in the Cardiological Diagnosis of the 21st Century. Clin. Cardiol. 2007, 30, 319–323. [CrossRef] [PubMed] g
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7. Pérez-Riera, A.R.; Ferreira Filho, C.; de Abreu, L.C.; Ferreira, C.; Yanowitz, F.G.; Femenia, F.; Brugada, P.; Baranchuk, A. Do patients with electrocardiographic Brugada type 1 pattern have associated right bundle branch block? A comparative 7. Pérez-Riera, A.R.; Ferreira Filho, C.; de Abreu, L.C.; Ferreira, C.; Yanowitz, F.G.; Femenia, F.; Brugada, P.; Baranchuk, A. Do patients with electrocardiographic Brugada type 1 pattern have associated right bundle branch block? A comparative
vectorcardiographic study. Europace 2012, 14, 889–897. [CrossRef] [PubMed] g
p
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p
[
] [
]
8. 5. Conclusions Acknowledgments: We acknowledge the support from the Netherlands CardioVascular Research
Initiative, the Dutch Heart Foundation, Dutch Federation of University Medical Centres, the Nether-
lands Organisation for Health Research and Development and the Royal Netherlands Academy of
Sciences (Predict2). Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. 9 of 9 Int. J. Mol. Sci. 2021, 22, 484 References Kardys, I.; Kors, J.A.; Van der Meer, I.M.; Hofman, A.; Van der Kuip, D.A.M.; Witteman, J.C.M. Spatial QRS-T angle predicts
cardiac death in a general population. Eur. Heart J. 2003, 24, 1357–1364. [CrossRef] 9. Draisma, H.H.M.; Schalij, M.J.; van der Wall, E.E.; Swenne, C.A. Elucidation of the spatial ventricular gradient and its link with
dispersion of repolarization. Heart Rhythm 2006, 3, 1092–1099. [CrossRef] [PubMed] 10. Durrer, D.; van Dam, R.T.; Freud, G.E.; Janse, M.J.; Meijler, F.L.; Arzbaecher, R.C. Total excitation of the isolated human heart. Circulation 1970, 41, 899–912. [CrossRef] 11. Lambiase, P.D.; Rinaldi, A.; Hauck, J.; Mobb, M.; Elliott, D.; Mohammad, S.; Gill, J.S.; Bucknall, C.A. Non-contact left ventricular
endocardial mapping in cardiac resynchronisation therapy. Heart 2004, 90, 44–51. [CrossRef] [PubMed] 12. Postema, P.G.; van Dessel, P.F.H.M.; de Bakker, J.M.T.; Dekker, L.R.C.; Linnenbank, A.C.; Hoogendijk, M.G.; Coronel, R.; Tijssen,
J.G.P.; Wilde, A.A.M.; Tan, H.L. Slow and discontinuous conduction conspire in Brugada syndrome: A right ventricular mapping
and stimulation study. Circ. Arrhythm. Electrophysiol. 2008, 1, 379–386. [CrossRef] [PubMed] 13. Hoogendijk, M.G.; Potse, M.; Vinet, A.; de Bakker, J.M.T.; Coronel, R. ST segment elevation by current-to-load mismatch: An
experimental and computational study. Heart Rhythm 2011, 8, 111–118. [CrossRef] [PubMed] 14. Hoogendijk, M.G.; Opthof, T.; Postema, P.G.; Wilde, A.A.M.; De Bakker, J.M.T.; Coronel, R. The Brugada ECG pattern a marker of
channelopathy, structural heart disease, or neither? Toward a unifying mechanism of the Brugada syndrome. Circ. Arrhythmia
Electrophysiol. 2010, 3, 283–290. [CrossRef] [PubMed] p y
15. Kors, J.A.; Van Herpen, G.; Sittig, A.C.; Van Bemmel, J.H. Reconstruction of the frank vectorcardiogram from standard electrocar-
diographic leads: Diagnostic comparison of different methods. Eur. Heart J. 1990, 11, 1083–1092. [CrossRef] [PubMed] p y
15. Kors, J.A.; Van Herpen, G.; Sittig, A.C.; Van Bemmel, J.H. Reconstruction of the frank vectorcardiogram from standard electrocar-
diographic leads: Diagnostic comparison of different methods. Eur. Heart J. 1990, 11, 1083–1092. [CrossRef] [PubMed]
16. Van Bemmel, J.H.; Kors, J.A.; van Herpen, G. Methodology of the modular ECG analysis system MEANS. Methods Inf. Med. 1990, 15. Kors, J.A.; Van Herpen, G.; Sittig, A.C.; Van Bemmel, J.H. Reconstruction of the frank vectorcardiogram from standard electrocar-
diographic leads: Diagnostic comparison of different methods. Eur. Heart J. 1990, 11, 1083–1092. [CrossRef] [PubMed]
16. Van Bemmel, J.H.; Kors, J.A.; van Herpen, G. Methodology of the modular ECG analysis system MEANS. Methods Inf. Med. 1990,
29, 346–353. [PubMed] 16. Brugada Syndrome
Electrocardiogram Brugada Syndrome
Electrocardiogram References Van Bemmel, J.H.; Kors, J.A.; van Herpen, G. Methodology of the modular ECG analysis system MEANS. Methods Inf. Med. 1990,
29, 346–353. [PubMed]
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Postnatal Morphological and Histochemical Type Differentiation of the Porcine Muscle Fibres
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Acta veterinaria Brno
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ACTA VET. BRNO 1995, 64:211·224 ACTA VET. BRNO 1995, 64:211·224 * Address for correspondence:
I.Kaman
Sumavska25
602 00 Bmo, Czech Republic POSTNATAL MORPHOLOGICAL AND HISTOCHEMICAL TYPE
DIFFERENTIATION OF THE PORCINE MUSCLE FIBRES I.KAMAN* Department of Pathological Morphology, Faculty of Veterinary Medicine, University of Veterinary
and Phannaceutical Sciences, 612 42 Bmo Abstract Pig, Duroc breed, muscle, fibre differentiation, type transformation, histochemistry Pig, Duroc breed, muscle, fibre differentiation, type transformation, histochemistry Abstract Kaman 1.: Postnatal Morphological and Histochemical Type Differentiation of the Porcine
Muscle Fibres. Acta vet. Bmo 1995,64:211·224. Kaman 1.: Postnatal Morphological and Histochemical Type Differentiation of the Porcine
Muscle Fibres. Acta vet. Bmo 1995,64:211·224. Histological and histochemical detection of myosin ATPase, SDH and glycogen activity were
used to study seven topographically and functionally different muscles of 23 Duroc pigs aged 2-4
hours, 7 days, and I, 3 and 6 months. The aim of the study was to find out whether type
differentiation of muscle fibres (MFs) is detennined genetically or whether it is variable, and if so,
in what ways, to what extent, and what is its relationship with "load myopathies", and to help
answer the question of the "giant fibre", MF splitting, and of suitability of individual types of
differentiation histochemical methods. Our findings indicated that muscle fibres of neonates were fully morphologically aJ)d adequately
histochemically differentiated, with MFII predominating. In addition to the basic MF I and II types,
the myosin ATPase reaction helped us differentiate also the intermediary IIC type with its MS, SS
and SM subtypes converging to type I; in some muscles we were able to demonstrate the IIA and
lIB types differentiated to a different extent. In the first month of life, muscles were differentiated
in: I) muscles with a predominance of MF II, anaerobic (m. longissimus dorsi, m. triceps brachii,
m. gracilis and m. semimembranosus), metabolically equipped for "load myopathies"; 2) muscles
with a high to dominant proportion (45·70%) of oxidative MF I (m. tibialis cranialis, m. trapezius,
m. stemomastoideus). No marked further conversion was found at the age of 3 months, and at the
age of 6 months only a minimum conversion occurred. Deep zones of muscles belonging to group
1 muscles showed a higher relative ratio of MF I than the surface ones. Change in the proportion
of MF I:MF II took place by gradual conversion of intermediary MFs IIC of their subtypes
MS ~ SS ~ SM in type I. Detection of SDH activity did not allow an objective type differentiation of MFs in piglets
younger than I month. The glycogen content was not always in correlation with an adequate MF
type. In glycolytic muscles, completely isolated MF lIB, of the "giant fibre" type, affected by
dystrophic changes were identified. No longitudinally split MFs were demonstrated. Pig, Duroc breed, muscle, fibre differentiation, type transformation, histochemistry Breeds of pigs raised for meat that are intensively selected for extreme muscle growth in
certain parts of the body, marked reduction of the proportion of fatty tissue and long trunks
are, according to Haas (1972), characterised by insufficient adaptability, which is
manifested by a number metabolic disorders. Bickhardt et al. (1972) characterised them
as a specific syndrome and called them "load myopathies". Although different breeds
exhibited considerable differences in their sensitivity to the syndrome (H 0 p e 1984; U h r in
et al. 1986), load myopathies have remained a topical issue because breeding animals with
a genetic disposition to those disorders are still being used, causing considerable economic
and breeding losses. Intensive research of load myopathies looking into many different
aspects has been developed all around the world. It became obvious that functional and * Address for correspondence:
I.Kaman
Sumavska25
602 00 Bmo, Czech Republic 212 morphological aspects of the muscle fibre present a host of questions related, in particular,
to the type differentiation and MF distribution in the muscle bundle (MB), which determine
the qualitative and quantitative character of the muscle. q
q
Ashmore et al. (1972), Bader (1981) and Davies (1972) reported that it was
genetically determined. Schlegel (1982) confirmed it both in the domestic and the wild
pig. On the other hand, Binkhorst (1969), Beermann and Cassens (1977),
Horak (1979), Suzuki and Cassens (1980), Salomon et al. (1983), Fazarinc et
al.( 1991) point out that the type distribution of MF in the muscle varies in relation to the age,
breed, physical activity, diet, etc. Its relation to meat quality has been stressed by
Ashmore etal. (1973), Kawaida etal. (1978) and KulBkova and Uhrin (1986). (
),
(
)
(
)
Research into porcine MFs has been carried out mainly on the m. longissimus dorsi
(Todorov and Petro v 1969; Swatland; Cassens 1973; K10sowska et al. 1976;
Szentkuti et al. 1981; Salomon et al. 1983; Rotkiewicz 1981; Uhrin et al. 1986) or a few other, structurally analogous, muscles (Petrov 1970, Hende De Van et
al. 1972; Bader 1983), which lead to a concept of their specifically identical structure and
to a term "porcine muscle". p
Differences are encountered in information on the incidence and importance of the so-
called "giant fibres" in striated muscles (Todorov and Petrov 1969; Klosowska et al. 1976; Hraste et al. 1980; Szentkuti et al. 1981; Bader 1983; Finger et al. Pig, Duroc breed, muscle, fibre differentiation, type transformation, histochemistry 1986;
Fazarinc etal.1991) and also on the longitudinal splitting of muscle fibres (Todorov and
Petro v 1969; Petro v 1970; Schmalbruch 1976; N0stvold 1981; Uhrin and
Kuli~kova 1985). Our aim was to study the development of morphological and histochemical type
differentiation of muscle fibres of Duroc pigs from their birth to the age of six months, and
to find out if MF type distribution in muscles was stable, genetically determined, or if it was
variable. If variable, we intended to identify the principle and process of type transformation
and muscle fibres conversion and relate the results to the load myopathies syndrome, "giant
fibres" and longitudinal muscle fibre splitting. We also wanted to assess histochemical
methods used from the point of view of reliability, repeatability and, most importantly,
objective results. Results Results Neonatal Period M. longissimus dorsi -both cryostat and paraffin HE-stained histological sections showed
full morphological differentiation with muscle fibres completely filled with myofibrils and
peripherally located cell nuclei. Fibres were mostly tightly packed together and were
becoming polygonal in cross-section (CS). The difference in the thickness between thicker
primary fibres, now corresponding to type I fibres and thinner secondary fibres, now
corresponding to MF n, was obliterated to various degrees. The muscle seemed to be
relatively compact, and consequently the primary, and sometimes even secondary, muscle
bundles (MBs) were not clearly delineated. y
In spite of this predominant characteristic feature, the superficial zone of the muscle was
less compact with clearly recognisable bundles, which did not lie close to each other, and
neither did muscle fibres, which were circular in cross-section. Type I and n fibres can be
easily differentiated without any special staining or histochemical reactions because the
former may be up to 3 times thicker than the latter; MFs are fully lined with myofibrils. In
thickness and topography, the intermediary MF nc fall in between the MF I and MF n (Fig. 3a, b). The remains of the myotubular lumen may sometimes be found in MF I which were
situated in the centre of primary MBs. Fully differentiated neuromuscular spindles (NMS)
were found regularly. They contained 1-3 markedly thick MFs and 3-8 (sometimes even
more) 2-5 times thinner fibres. Cell nuclei were usually situated subsarcolemmally, but
intrafibrallocation was also frequent. Strikingly well-developed muscle fibres and bundles were found regularly around blood
vessels. When taking specimens, we saw time and again that the m. longissimus dorsi in
Duroc pigs was much better supplied with blood than in any other breeds raised for meat. Myosin ATPase detection - ATPase reaction following alkaline preincubation at pH 9.2
(Fig. 3b) differentiated 1, less frequently 2, weakly reacting, frequently markedly thicker,
alkali labile primary MF I in the centre of the primary muscle bundle. Dominant in the MB
were 8-25 strongly reacting alkaline stable secondary MF II. Near the MF I, 1-3 (most
frequently 2) muscle fibres could be distinguished which fell, by thickness and reaction
intensity, between MF I and MF n. They were intermediary MF of type nc, comprised of
MS, SS and SM subtypes, differring from one another by the intensity of reaction when
transformed to MF I. Materials and Methods Twenty-three Duroc pigs of both sexes from a small breeding herd were used. They were divided into five age
groups, L e. 2 - 24 hours, 7 days, and 1,3 and 6 months of age, with a mean body mass of 1.4, 2, 4.75, 28.5 and
105 kg, respectively. g
p
y
Muscle tissue was excised bioptically under general ether narcosis or Hypnodil Lp. with Stresnil Lm. premedication. In the case of the slaughter group, excisions were made immediately after slaughter from the m. longissimus dorsi, m. trapezius, caput longum m. tricipitis brachii, m. gracilis, m. semimembranosus, m. tibialis
cranialis and m. sternomastoideus. Excisions were divided in two parts. The smaller one, a cube 0.5 cm in size, was
covered in talc and frozen to about -190°C. The blocks were then stored at -60 °C. Later, at -20°C, they were cut
into 10 J.IID cryostat sections exactly perpendicular to muscle fibres and studied by histochemical (D u bow i tz and
Brooke 1973; Lojda and Papou§ek 1978; Suzuki, Cassens 1980a) and, after hematoxylin-eosin staining
(HE), histological methods. Type classification of muscle fibres in series sections was based on the detection of myosin ATPase activity after
alkaline (pH 9.2; 9.4) and acid (PH 4.3; 4.6) preincubation, and a comparison of the two reactions. The other method
used was based on the detection of the oxidative enzyme succinate dehydrogenase (SDH). The glycogen
distribution in MF was ascertained by the periodic acid Shiff (PAS) reaction. The rest of muscle excisions were fixed in a neutral 10% formaldehyde, HE stained and cut into 8 - 10 J.IID paraffm
histological sections. The thickness of MF in the sections and its variations were ascertained by means of an
eyepiece and a stage micrometer. y p
g
Of the maze of classification systems, we chose the Brooke and Kaiser (1970) nomenclature for the MF
type differentiation and the Suzuki and Cassens (1980 a,b) nomenclature for the differentiation of
transformation subtypes of IIC intermediary muscle fibres. 213 Results Their reliable differentiation and identification was only possible by
a comparison between ATPase reactions after alkaline and acid preincubations. p
p
Myosin ATPase reaction after acid preincubation at pH 4.3 (Fig. 3a) presented an inverse
image to the ATPase reaction after alkaline preincubation. The acid labile MF n, reacting
strongly after alkaline preincubation, were now reacting weakly, while acid stable MF I,
reacting weakly after alkaline preincubation, were now reacting strongly. Besides MF I,
sections of different intensity were also recorded in a group of 2-3, rarely up to 5, MFs of
the intermediary type nc grouped around the MF I, which we considered conversion
subtypes MS, SS and SM. The MS subtype reacted weakly after acid preincubation and
strongly after alkaline preincubation, the SS subtype reacts strongly after both types of
preincubation, and the SM subtype reacts strongly after acid preincubation and moderately
after alkaline preincubation. Other MFs of the secondary developmental population of the
primary MB belonged to MF II, which in neonates may already be differentiated in some
places to a varying extent into MF llA and llB (Fig. 3a,b). While the MF llA will maintain
negative or almost imperceptibly positive ATPase reaction after acid preincubation, the MF
llB exhibited moderate to medium strong reaction. After alkaline preincubation, both types
reacted strongly, although the IIA type almost imperceptibly less strongly than the llB type. SDH - the reaction was highly positive and constant In transversal sections groups of 214 most frequently three MFs in the centre of primary bundles were found. They were
conspicuous by a higher content of the reaction product, delineating the fibre contours not
only intermyofibrilarly but frequently also subsarcolemmally. They were oxidative MF
I and intermediary MF IIC, differing from the surrounding fibres of type II, which were only
slightly delineated or completely undelineated and reacted with less intensity. For the age
group studied, the SDH reaction cannot be used for an accurate MF type differentiation. g
p
yp
PAS reaction to glycogen is positive, to a considerable extent confluent and clearly
differentiated in places. The non-reacting muscle fibres in the centre of the primary bundle
correspond to the MF I type. The 1-3 adjoining fibres, with a very weak reaction, correspond
to the intermediary MF IIC type. They differ from intensively reacting muscle fibres of the
glycolytic type II on the MB periphery. Results PAS reaction was not, however, limited to muscle
fibres only, and reactions in interfibral and interfascicular areas were fairly common. y,
y
M. trice.ps brachii. caput lon&um - mostly compact muscle, with a majority of primary
muscle bundles fused together. Extrafusal fibres were completely filled with myofibrils
whose cell nuclei were located peripherally, the thickness of both MF populations is
approximately the same, with half of them still circular in cross-section. Neuromuscular
spindles were also fully differentiated. p
y
Routine myosin ATPase reaction identified an absolute predominance of type II fibres
with 1 (sometimes 2) Type I fibres in the centre of the primary muscle bundle. SDH reaction was clearly positive. The type differentiation of muscle fibres was still
incomplete in places, elsewhere 1-3 Illore intensively reacting MFs can be found in the
centre of primary MBs. p
y
PAS reaction was highly positive (Fig. 2). The glycogen distribution was already more or
less clearly differentiated in individual places. Muscle fibres of type I reacted negatively
and 1-3 adjoining intermediary muscle fibres showed a weak reaction. j
g
y
M. ~cilis - morphologically fully differentiated (Fig. 4). Muscle fibres of both
populations were still circular and relatively small in cross-section, and similar in thickness. Primary muscle bundles were clearly defined, muscle fibres in them were more or less
separated with interstitium and circular, rather than polygonal, in cross-section. ATPase reaction after alkaline preincubation showed that the muscle had an absolute
predominance ofMF II, with only one negatively reacting MF I in the centre of the primary
MB. Two to four types of MF can usually be differentiated. Other types can be differentiated
only at a comparison with acid ATPase. A reaction after preincubation at pH 4.3
differentiated conversion subtypes MS, SS and SM, and even, although rarely, MF lIA and
lIB (Fig. 4). SDH reaction was positive. It, however, only allowed for differentiation between MF I and
MF II. In some places, the PAS reaction identified glycogen only in fibres, elsewhere also
between fibres, in some places uniformly in all fibres, in some places muscle fibres were
differentiated into MF I and MF II, on a few occasions even lIe. M. semimembranosus exhibited adequate features of full differentiation in two
modifications. The deeper zone of the muscle seemed more compact, its closely adjoining
muscle fibres of mostly polygonal CS mainly of the same thickness. Results In the remaining,
superficial and less compact zone, muscle fibres and even muscle bundles communicated
more loosely. Delineated to a varying extent by interstitium, they were of a corresponding
circular CS and exhibited marked variability in thickness. Intrafibrilarly located cell nuclei
and myotubule rudiments were very rare. Neuromuscular spindles were fully differentiated. muscle fibres of mostly polygonal CS mainly of the same thickness. In the remaining,
superficial and less compact zone, muscle fibres and even muscle bundles communicated
more loosely. Delineated to a varying extent by interstitium, they were of a corresponding
circular CS and exhibited marked variability in thickness. Intrafibrilarly located cell nuclei
and myotubule rudiments were very rare. Neuromuscular spindles were fully differentiated. The detection of ATPase activity yielded predictable results. First of all, histochemical
differentiation between alkali lability and acid stability of MFs on the one hand and alkali
stability and acid lability on the other may not be very clearly developed yet. In parts of the The detection of ATPase activity yielded predictable results. First of all, histochemical
differentiation between alkali lability and acid stability of MFs on the one hand and alkali
stability and acid lability on the other may not be very clearly developed yet. In parts of the 215 section, or sometimes even on entire sections, reactions cannot be evaluated. Assessable
areas and normally reacting histological sections exhibited a unified type structure of the
primary bundle, which most frequently had a single centrally located MF I in it. Other
histological sections showed two or three different zones in them. In the zones
corresponding to the superficial and deep layers of the muscle, almost all primary bundles
had 1 and 5-6 type I fibres respectively, with different levels of intensive reactions after acid
preincubation and weak reactions after alkaline preincubation. Between the two, there was
an intermediary level with 2-4 MF I. This documents histochemical as well as metabolic and
functional differences in the structure of m. semimembranosus, whose identification will
depend on the type and site of sampling. SDH reaction was, to a certain extent, analogous to the complexity of ATPase reactions. The results, generally speaking, were less conclusive than those in previous muscles. The
PAS reaction, mostly limited to muscle fibres only, was monotonous and differentiated in
places only. M. tibialis cranialis - histological sections transversed the entire thickness of the muscle. Results They were characterised by an intensive HE affinity and basic attributes of fully
differentiated MFs of mainly polygonal es, which was due to a high compactness of the
muscle. In spite of the predominantly uniform thickness of muscle fibres, thicker type
I fibres may sometimes be identified and, exceptionally, also myotubes. Muscle fibres
several times thinner than the rest were not exceptional. At a junction of several fibres, or
where they were pushed against another fibre, they looked very much like a split or splitting
fibres. Histochemically, they may resemble the nearest fibre but may be completely
different. Myosin ATPase reaction at pH 9.4 preincubation will commonly differentiate three types
of fibres, the dark-staining type II fibres, the light-staining or non-reacting type I fibres, and
the intermediary type lIe fibres. The differentiated inversion at pH 4.3 preincubation (Fig. 5) can differentiate between four types of muscle fibres, i. e. MF I, II and lIe, containing all
three transformation subtypes MS, SS and SM. Most frequently, primary MBs contained 3
to 5, rarely 9 and - in aggregated bundles - up to 13 type I and lIe muscle fibres with
a differentiated intensity of their reaction product. The higher number of acid stable fibres
in the MB clearly differentiated the m. tibialis cranialis from the previous muscles. What
makes it particularly interesting is the fact that this muscle's primary MBs in neonates
contained only one and exceptionally two type I muscle fibres. Markedly increased numbers
of intermediary types of muscle fibres were found. Rather than concentrated in the centre of
muscle bundles only, they were also disseminated in it in various patterns. y
y
p
Type II muscle fibres, numerically predominating in neonates, may, in places, be
histochemically differentiated into IIA and lIB types (Fig. 5). Although it delineated fibres of primary bundles to various degrees, the SDH reaction
made it only occasionally possible to identify the more intensively reacting fibres there. The
PAS reaction was usually considerably different, and to a large extent confiuent, in various
parts of histological sections. In spite of that, more intensively reacting glycolytic fibres may
- to a certain extent and in some places only - be differentiated from less intensively reacting
oxidative fibres. The m. trapezius exhibited all the characteristics of an adequately morphologically
differentiated muscle: muscle fibres were of the same thickness, they were fIlled with
myofibrils, and the CS was polygonal. Results In spite of that, primary MFs sometimes contained
1-4 circular fibres of a slightly larger es. Occasionally, this muscle also contained extremely
thin fibres. HE staining was exceptionally intensive in histological sections in general and
in oxidative and type lIe fibres in particular, which also partly differed in structure. It is, 216 however, unsuitable for an objective identification of even the basic types of mu ,
j
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The myosin ATPase reaction after acid and pH 9.4 preincubations can commonly
differentiate 4 and 3 degrees of intensity, respectively. They corresponded to
a differentiation of fibres into type I, II and IIC which, however, cannot be unambiguously
identified without a comparison between acid and alkali ATPase. The main difference lies
in the higher number of intermediary type IIC fibres or, more accurately, of their subtypes. Primary bundles most frequently contained 3-5, sometimes up to 8, intensively reacting type
I and IIC muscle fibres, of which 1-3 were type I fibres, the remaining being type IIC
subtypes, i. e. MS, SS and SM. These acid stable fibres were not as centrally located as they
were in the first four muscles, and sometimes complicated MBs were found with 14 or more
acid stable muscle fibres. The SOH reaction was usually quite strong over the entire cross-section. Muscle fibres of
higher and lower enzymatic activity corresponding to type I and IIC fibres and type II fibres
respectively may be differentiated from each other. A reliable type differentiation in this age
group and muscle, however, is not possible on the basis of the SOH reaction. The PAS
reaction was frequently only local in character. Occasionally, weakly reacting light-
coloured type IIC, practically non-reacting type I, and more strongly reacting type II fibres
may be differentiated. These reactions do not allow an objective type differentiation. M. sternomastoideus - complete morphological differentiation was evident from a large
number of HE mounts. In spite of that, thinner secondary fibres, or only very slightly thicker
primary fibres - some with a hint of a tubular lumen - in the primary bundle, were still in
evidence in places. In extrafusal fibres, cell nuclei were located clearly subsarcolemmally. In intrafusal fibres, on the other hand, they were located both intrafibrilarly and
sarcolemmally. In this muscle, it is not at all difficult to find up to seven NMS in
a histological section. The detection of the myosin ATPase yielded a characteristic pattern. Results With alkaline
preincubation, 2-3 and sometimes even 4 degrees of reaction can commonly be
differentiated. In the primary bundle, 2-10 (most frequently 3-5) alkali labile fibres with
a uniformly or non-uniformly weak reactions were located. After acid preincubation, the
density of the reaction product allowed the differentiation of 4-5 types of fibres. Primary
MBs contained 2-16 (the more complicated one up to 21) fibres of various acid stability. In
many cases, they represented a half of the total number of fibres in the primary bundle, and
sometimes even more. Such muscle fibres did not, however, form a central agglomerate but
were as a rule irregularly distributed inside and also along the edges of primary bundles. The SOH reaction - majority of muscle fibres was clearly delineated by subsarcolemmally
located reaction product but a more intensive reaction, or a hint of it, corresponding to the
localisation of type I or IIC fibres, occurred in places only. The PAS reaction was uniform
in some places and differentiated in others. The most intensive reaction came from type II
fibres, less intensive from type IIC, and the least intensive - or even negative - reaction from
type I fibres. Glycogen was sometimes identified outside muscle fibres in primary and
secondary bundles. Examinations of muscles in the first age group pointed out to marked structural similarities
between them. For further investigation in other age groups, we divided the muscles into two
groups. Group 1 included the m.longissimus dorsi, m. triceps brachii, m. semimembranosus
and m. gracilis, and Group 2 m. tibialis cranialis, m. trapezius and m. sternomastoideus. Piglets aged seven days Both groups of muscles were characterised by a more polygonal CS of their fibres. The
muscles were also more compact, with a tendency to level off the differences in thickness 217 between primary and secondary populations of fibres. Centrally located cell nuclei and
remains of tubular lumina were a rare exception. In the glycolytic muscle type (m. longissimus dorsi), intensively HE-staining type I and DC muscle fibres in the centre of
primary muscle bundles was found frequently. This phenomenon, however, was not
constant and did not suffice for type identification. In a 7-day-old piglet, we found
a tendency towards "giant fibres" in an anaerobic type of muscles (m. triceps brachii). y
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ATPase reaction showed no fundamental differences from that in the neonates. Results It did,
however, allow, generally speaking, a more unambiguous type identification. Reactions of
myosin ATPase after alkaline, and particularly after acid, preincubations showed a more
balanced thickness of both basic types of MFs. SOH - type differentiation of muscle fibres
by means of SOH detection has clearly advanced and allows now a relatively clear
identification of muscle fibres of type I as well as type D and DC. In 7-day-old piglets, the
SOH reaction allowed an assessment of the ratio between oxidative and anaerobic muscle
fibres. PAS reaction was constantly positive, medium to strong in intensity, bound to muscle
fibres only. In relation to MF, it was either monotonous or differentiated. If monotonous, the
intensity of its reaction was usually lower. The most intensive reaction was observed in type
II, i.e. glycolytic, fibres, and the least intensive reaction, or none at all, in the type I, i.e. oxidative, fibres. The intensity of their reaction placed the intermediary DC MFs in between
the two. Piglets aged one month one 218 month), we found 1-6 (most frequently 3) type I fibres, surrounded by 5-7 type IIC fibres
(Fig. 8). The number of IIC MFs in the rest of the bundle was reduced by the fibres that have
converted to type I. And while in neonates, converted fibres in muscles of this groups were
mostly of the MS subtype, the now dominant is the SS sUbtype. The MF I precursors, the
last of conversion subtype MS, and MS forms were also found there. yp
The fact that, at birth, Group 2 muscles (m. trapezius, m. tibialis cranialis, m. sternomastoideus) contained a higher number of intermediary fibres that Group 1 muscles
can only be demonstrated by comparing results of the myosin ATPase after alkaline and acid
preincubation. Although the differences may be considerable, they are not as big as the
differences that take place during the postnatal MF conversion, leading to fundamental
changes in the MF I: MF II ratio. In one-month-old pigs, type I fibres represented about 40-
70% of all MFs in Group 2 muscles (Fig. 10). p
(
g
)
The most extensive conversion of type II into type I fibres was found in the m. sternomastoideus, where type I MFs make up about 70% (Fig. 9a,b), i.e. it is a clearly tonic,
red muscle although an absolute majority of its fibres at birth belonged to type II. Conversion
was also responsible for the approximately 40% representation of type I fibres in the m. tibialis cranialis although, at birth, the primary muscle bundle contained only 1-2 fibres of
that type, and for their approx. 50% representation in the m. trapezius. yp
pp
p
p
Type II muscle fibres, i.e. also the IIA and lIB types in addition of the IIC type, are now
also fully histochemically differentiated. Compared to the total number of type II fibres, the
number of type IIA fibres is relatively low. yp
y
SDH reactions in individual muscles were quite different. The MF I and IIC reacted
usually quite well. Type II muscle fibres were not clearly, if at all, delineated to be reliably
differentiated from their neighbouring type II fibres, and their quantification on the basis of
the SDH reaction was generally impossible. While the histochemical reaction product in
Group 1 muscles was mostly or exclusively located·interfibrilarly, in Group 2, and in the m. Piglets aged one month Piglets aged one month HE - the most important feature, besides a further increase in the compactness of muscle
tissue due to the growing thickness of muscle fibres, is the thickness ratio inversion between
types of muscle fibres. While in the early postnatal period generallYr and in surface layers
of anaerobic muscles particularly, muscle fibres of type I were thicker than those of type D,
this disproportion disappeared within a week and at the age of one month, the type D fibres
were thicker, particularly in the peripheries of primary MBs, i. e. in the region of type D
fibres. In contrast with this development, extremely thin MFs w~re found in the region of
thick MFs. These extra thin fibres, which may be several times thinner than the rest of fibres,
usually adjoined the thick ones very closely and were strongly suggestive of "daughter"
MFs. In transversal sections, particularly the frozen ones, groups of ysually 3-5 MFs with
a "perforated" structure were clearly identifiable in primary bundles. The were more
intensively HE-staining lipoid-rich type I and DC, i.e. intermediary, fibres. In animals of this age, myosin ATPase with alkaline anp particularly with acid
preincubation is able to differentiate not only between basic fibre types I and D, but also
between all others, i.e. llA, lIB and the conversion DC type with its transformational
subtypes MS, SS and SM. As soon as in the 2nd week after birth, characteristic patterns of MFs in primary bundles
may be discerned which were considered permanent and called "a typical porcine muscle"
by many authors. They were represented by a cluster of type I and DC fibres in the centre of
the primary bundle and surrounded by type D (llA, lIB) fibres, which made up an absolute
majority of MFs in the primary bundle. We have, however, determined that this pattern
exists only in muscles with a predominance of type II fibres, i. e. glycolytic ones, but not in
muscles with a type I fibres predominance, and in completely oxidative muscles. yp
p
p
y
While in neonates, centres of such primary bundles typically contained a single type I fibre
surrounded by 1-3, less frequently by up to 5, type DC fibres depending on the size of the
bundle, and the rest of the bundle contained 8-25 type D fibres, in this age group (i. e. Piglets aged one month The minimum quantity of type
I fibres in the superficial zone suggests a fast muscle, while the clearly larger number of
fibres of this type in the deeper zone suggest a tendency to a tonic muscle. Figs 12a, b also
demonstrate the presence of fibres from type IIA and lIB. At this age, myosin ATPase can reliably differentiated muscle fibres of type II into MFs
IIA (reacting negatively after acid preincubation) and MFs lIB (reacting weakly or
moderately after the same preincubation). While the reaction after alkaline preincubation of
both types ofMFs is strong, that of type lIB fibres is somewhat stronger. The number of type
IIA fibres is generally low compared to the number of type lIB fibres. At this age, the SDH reaction completely delineated individual MFs of type II, but did not
differentiate between types lIA and lIB. Oxidative fibres of type I and those ofintermediary
type IIC were characterised by a highly positive reaction. Type IIC fibres can be identified
as a whole but individual transformation subtypes are unidentifiable. In order to accurately
differentiate between type I and IIC, this method should be combined with inverse ATPase
reaction after alkaline preincubation. The PAS reaction was subdued to weak, and differed both in individual animals and
muscles. Localised predominantly in type II fibres, glycogen was also occasionally
demonstrated, although with difficulties, in type IIC fibres. Pigs aged six months HE -histological sections were characterised by marked polygonal character of the muscle
fibres' CS and compactness of tissue. With the exception of the m. tibialis cranialis and
partly also the m. sternomastoideus, MFs inside muscle bundles appeared thinner than along
their peripheries. Thin "daughter" fibres occurred only occasionally. Strikingly circular
MFs, often thicker than the surrounding ones and suggestive of "giant fibres" were not
difficult to find. These MFs, however, HE-stained more intensively than other MFs, with
cell nuclei mostly subsarcolemmally. In these MFs, ATPase detection pointed to type II
fibres. ATPase - changes in the development of type differentiation were, compared to the
situation at three months, conspicuous. Phasic muscles with an absolutely dominant
proportion of type II fibres have retained the typical chessboard pattern of types I and II
distribution. Piglets aged one month tibialis anterior particularly, it was also distributed subsarcolemroally to a large extent,
which helped to clearly delineate both intermediary and, even more so, oxidative muscle
fibres. The PAS reaction - it was clearly defined but subdued in this age group. It allowed
a reliable identification of non-reacting type I fibres in the middle of primary muscle bundles
and strongly-reacting type II fibres as a whole. It was, however, difficult, if not downright
impossible, to differentiate between the IIA, lIB and the intermediary IIC fibres. Pigs aged three months HE - overall histological characteristics were analogous with those in the previous age
group. We occasionally found MFs of a perfectly round transversal CS, which contrasted
with surrounding fibres (Figs 12b, 15). They were also usually thicker, with enhanced or
reduced susceptibility to HE-staining, and dystrophic changes of varying extent. They were
most frequently identified on the periphery of muscle bundles. Appearing mostly in muscles
with a predominance of Type II fibres, they resembled fibres described in literature as "giant
fibres". In paraffin histological sections, we recorded several cases of a suspected longitudinal
split of MFs. The findings were not, however, convincing enough, or the problem
sufficiently investigated, for us to give an unambiguous answer to that question. Myosin ATPase - using a comparison between acid and alkaline preincubation reactions,
we were able to demonstrate continuing developments in the muscle fibre type
differentiation. In the group of muscles comprising the m. longissimus dorsi, m. triceps
brachii, m. gracilis and m. semimembranosus, type II fibres have retained their highly 219 dominant position. Although transformations have about quadrupled the number of type
I fibres, they still represent only about 12% of total MFs in those muscles. Among
transformation types, the SS and SM subtypes are dominant, while the MS subtype is
receding. While Group 2 muscles (m. tibialis cranialis, m. trapezius and m. sternomastoideus) characterised by an exceptionally intensive and extensive conversion of
type II into type I fibres which fundamentally changes the character of muscles have retained
their pro-conversion character, it is much less intensive and has no major impact on the type
structure of muscle fibres in the bundle. In the m. semimembranosus of the neonates, we demonstrated differences in the type
distribution of MFs in its superficial and deep layers. In the caput longum of the m. tricipitis
brachii of 3-months-old pigs, we made analogous fmdings. Piglets aged one month And although the number of type I fibres had increased 3-4 times, there were
only few exceptions of more than 2-7 type I fibres in the primary bundle because their
original number in neonates was so very small (1-2). Not all type I fibres were always in
a cluster. Some may be quite isolated (Fig. 18). In muscles with predominantly type II fibres
(e.g. m.longissimus dorsi), type I fibres made up about 12%. The thickness of MFs had changed considerably. Type II fibres were generally thicker
than type I fibres. But there were MFs of type II which were clearly thinner than type I fibres. The thickest type II fibre in muscles with a predominance of muscles of this type was found 220 in the m. semimembranosus (125 J.1IIl), while the thickest type I fibre in the same muscle was
only 115 J.1IIl. Average thickness of its type II and I fibres was 96 J.1IIl and 82 J.1IIl respectively;
in the m. gracilis 85 J.1IIl and 72 J.1IIl. g
In spite of the fact that tonic muscles contained a higher percentage of intermediary musc-
le fibres as opposed to phasic muscles at birth, this difference was by far not as great as the
one that occurred later due to postnatal increase of MF I type and decrease of MF II type. Although there was some conversion in these muscles observed between months 3 to 6, the
changes were not conspicuous . .In m. tibialis cranialis the conversion of muscle fibres from
type II into type I reached approximately 45 % (Fig. 14), in m. trapezius (Fig. 13ab) was
this ratio ca 50:50 %, and about 60-70 % in m. sternomastoideus. ,
It is interesting to note that the thickness of the two basic fibre types, i.e. type I and II, was
very similar and also that they were relatively thinner than those in phasic muscles. In m. trapezius, the maximum thickness was 91 J.1IIl, with an average of 81 J.1IIl and 73 J.1IIl (type
I and II, respectively). Higher average values of type I fibres than type II fibres were also
recorded in m. tibialis cranialis. In the m. triceps, type II fibres were thicker than type I fibres. p , yp
yp
The SDH reaction was highly reliable in all cases. It provides a sharp differentiation
between oxidative MFs on the one hand and anaerobic MFs on the other. Piglets aged one month Compared to the
myosin ATPase method, it allowed for about a 30% increase in the number of identified
oxidative MFs. A more accurate identification of intermediary MF types and
a differentiation of type I fibres was, however, rather questionable if it is not combined with
another (conversion) method. (
)
The PAS reaction in animals 6 months old was mild and selectively limited to type II
fibres. In some cases, intermediary MFs also partly reacted. Discussion In newborn Duroc piglets, muscle fibres of skeletal muscles were fully morphologically
and adequately histochemically differentiated. Among objective differentiation criteria, we
included myofibrils embedded in MFs, subsarcolemmally located cell nuclei, a shift towards
a polygonal CS, a tendency towards equalising thickness of the two populations of MFs
(corresponding to Type I and II fibres) and histochemical (particularly myosin ATPase)
differentiation if MFs. The centre of primary bundles of the neonatal m. longissimus dorsi, m. triceps brachii, m. gracilis and m. semimembranosus contain 1 and exceptionally 2 type I muscle fibres. Attached to them are 1-3 (rarely 4) intermediary muscle fibres of type IIC. The m. trapezitls
contains 1-4 MFs of type I and up to 8 MFs of type IIC in their MS, SS and SM subtypes;
the m. sternomastoideus contains 2-4 MFs of type I and 2-10 usually differently reacting
MFs of type IIC. yp
Davies (1972), Bader (1981) and Schlegel (1982) reported that type distribution of
MFs in the m. longissimus dorsi and m. semimembranosus is genetically fixed and
practically not subject to any changes. The list may be extended to include the m. gracilis
and caput longum m. tricipitis brachii, i.e. muscles with an absolute predominance of type
II fibres. In the other muscle group (m. tibialis cranialis, m. trapezius and m. sternomastoideus), however, we recorded an intensive conversion of type IIC fibres into
type I fibres at the age of one month which led to a fundamental, even dominant, change in
the muscle structure. The most extensive conversion (about 70%) of MFs of type I in the
muscle bundle was identified in the m. sternomastoideus. At the age of three months, the
progress of conversion was no longer very significant, and at 6 months it was completely
insignificant. At the same time, the thickness ofMFs peaked. The relative thickness ofMFs 221 changed compared the situation among neonates, but type n fibres were not always the
thickest. These fmdings correlate significantly with results reported by Suzuki and
Cassens (1980b) and, to a certain extent, with those reported by Fazarinc et al. (1991). Although significantly different with respect to early postnatal stages, our findings are also
analogous with Horak's (1979) results from adolescent periods of his study involving
miniature pigs. Discussion Although he used myosin ATPase detection after acid preincubation for
histochemical differentiation, he failed to compare the results with an inverse reaction after
alkaline preincubation and mistook intermediary type nc and its MS, SS and SM subtypes
(characterised by a strong, although differentiated, reaction after acid preincubation
similarly to type I fibres) for type I fibres. This was mainly reflected in a significant
overestimation of the number, and percentage representation, of type I fibres in the muscle
bundle, in the characteristic of the dynamics of their development in the postnatal ontogeny
and their interrelations with other muscle fibre types. yp
Objective description of the process of transformation of MF nc subtypes can only be
accomplished by comparing the myosin ATPase activity after alkaline preincubation with
its inverse reaction after acid preincubation; the acid labile and alkali stable MS subtypes are
transformed into acid and alkali stable SS subtypes, which, in their turn, through acid stable
and alkali labile SM SUbtypes in muscle fibres of type I. yp
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B eermann et al. (1978) recorded different histochemical patterns in superficial and deep
zones of m. semitendinosus. Using myosin ATPase reaction, we were able to differentiate
three different zones in the neonatal m. semimembranosus, with 1,2-4 and 5-6 type I fibres
in the superficial, medial or transitory and deep zones, respectively. A similar situation was
found in the caput longum of the m. triceps brachii in 3-month-old pigs. These findings point
to histochemical type differences in the structure of individual muscles which may not be
identified morphologically. If not taken into account when samples are taken from muscles
and evaluated, it may lead to controversial results. In basic characteristics, the superficial
and deep zones correspond to the phasic and tonic types of muscles, respectively. p
p
p
yp
,
p
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We believe that methods used, rather than any real differences, are the true cause of
discrepancies in type differentiation and muscle fibres distribution. Using black Sudan,
Todorov and Petrov (1969), for instance, concluded that the differentiation between
"dark" and "light" muscle fibres takes place in the first month. Discussion (1986) also in the wild pig, although S zen tku ti et al. (1980) failed to confmn
the finding in the wild pig. We found an enormously thick muscle fibre of type II, i.e. a suspect giant fibre, at the
periphery of the primary muscle bundle of a glycolytic muscle (m. triceps brachii) of a seven-
day old Duroc piglet. Other characteristics of the GF (circular cross-section, intrafibral
nucleus and possibly also some dystrophic changes) were only observed in older animals
(Figs 12b, 15). They were histochemically identified as type lIB fibres (Fig. 12b). Structural
dystrophic changes are already visible. They are the thickest fibres, insufficiently supplied
with blood, glycolytic, with a demanding anaerobic metabolism, very vulnerable. On the
other hand, they are characterised by the most intensive growth, guaranteeing an enormously
fast increase in muscling, which is what these breeds of pigs are exclusively selected for. g
p g
y
In our study, fibres of the GF type were found only occasionally, and dystrophic changes
only very rarely. Similar results were reported by T 0 d 0 r 0 v and Pet r 0 v (1969) in pigs
1-7 months old whose breed was, however, not mentioned. They considered them as light
MFs transformed from dark MFs, as fibres that differentiated first and therefore to the
highest degree that are, in a sense, worn-out, and the dystrophy as a physiological process. The minimum incidence of GFs in our material reflects positively on the husbandry used in
the breeding herd which supplied us with animals, and a degree of resist~ce of the Duroc
breed against "load myopathies". The process in the fibres might be considered
physiological to the extent of degenerative changes described by Martinov (1937) in
human muscles which he called physiological degeneration and which was confirmed by
other authors in various vertebrates. We did not find any lengthways splitting MFs as they were very vividly described and
documented by Todorov and Petrov (1969) in 2-3 year old pigs of contemporary breeds
raised for meat. They arrived at a definitive conclusion that muscles grow postnatally not only
by hypertrophy but also hyperplasia of muscle fibres. The general idea of splitting fibres is also
supported by N 0S t v 0 I d (1981 ). Discussion On the basis of myosin ATPase
reaction after acid preincubation and NHD-TR reaction, H 0 r a k (1979) reported 5-10% ratio
of type I fibres already in neonatal muscles which show a predominance of type n fibres and
in muscles of a mixed type in adults, and 20% ratio in muscles with type I dominance. Using
SDH activity detection, KulHkova and Uhrin (1986)foundthatthehighestnumberofred
MFs in glycolytic muscles is at birth and may reach up to 49.6%. This is in contradiction not
only with the results of MF type differentiation using myosin ATPase activity detection as
reported by Suzuki and Cassens (1980b), but also with our results. Our findings showed
that SDH reaction cannot reliably differentiate MF types in muscles of newborn piglets and,
apparently, intermediary type nc fibres were misinterpreted as type I fibres Most authors report higher levels of glycogen in glycolytic type n fibres. Our results
support this only partially. Similar conclusion was also made by Uhrin and
KulHkova (1985). Reactions were frequently confluent, on other occasions MFs had no
glycogen. In animals of more advanced age where muscle excisions were not made
intravitally, PAS reactions were mild. Glycogen seems to be quickly depleted post mortem,
and glycolytic process may be more intensive in type n fibres. The stress before and during
muscle excisions plays undoubtedly a certain role. p y
y
Giant fibres (GF) are described as a symptom of poorer quality of meat and load
myopathies in slaughter pigs (Cassens et al. 1969; Klosowska and Klosowski 1976; 222 Klosowska et al. 1976; Schmitt and Dumont 1980). The authors believe that the
frequency with which they are found depends on the breed (higher incidence in stress-prone
animals: Belgian Landrace and Pietrain), muscle type, diet and husbandry. Their
conspicuously large size, circular transversal cross-section and possibly also morphological
changes are linked to their enormous energy reserves of glycogen and function, intensive
anaerobic glycolysis and impaired acid-base balance leading to hydropic degeneration
(B ader 1982). Fazarinc et al. (1991) demonstrated that usually one muscle fibre of this
kind at various stages of development can be found in a majority of primary muscle bundles
in the m. longissimus dorsi of a 6-month-old stressed pig. g
p g
Hraste et al. (1969), on the other hand, described GFs in pigs of primitive breeds,
Fi n g e r et al. PostnataIni morfologicka a histochemick8 typova diferenciace svaloveho vIakna prasete Prozkoumali jsme 7 topograficky a funkcne rozdHnych svalli 23 prasat plemene Duroc
ve veku 2-4 h, 7 d, 1, 3 a 6 mesicli histologicky a histochemicky detekci aktivity myozino-
ve ATPazy, SDH a glykogenu. CHern bylo zjistit zdaje typova distribuce svalovych vla-
ken (SV) ve svalech geneticky determinovana anebo promenna. Je-li promenna, pakjakjm
zplisobem, v jakem stupni,a jaky je jeji vztah k zatezovym myopatiim. CHern bylo take pfi-
spet k objasneni "giant fibres" stepeni SV a vhodnosti typu diferenciacni histochemicke
metody. SV novorozencli byla pine morfologicky a adekvatne histochemicky diferencovana; domi-
novala SV II. Reakci myozinove A TPazy jsme rozliSili krome zakladnich typli SV I a II tez
intermediami typ IIC ajeho podtypy MS,SS a SM konvergujici v typ I; v nekterych svalech
j sme prokazali i v rliznem stupni diferencovane typy IIA lIB. Behem 1. mesice po narozeni
se svaly rozlisily ve: 1. svaly s pl'etrvavajici pl'evahou SVII, anaerobnich (m. longissimus
dorsi, m. triceps brachii, m. gracilis a m. semimembranosus), metabolicky disponovane pro
zatezove myopatie; 2. svaly s vysokym aZ dominantnim (45-70%) podHem oxidativnich SV
I (m. tibialis cranialis, m trapezius, m. stemomastoideus). Ve veku 3 mesicli se jiz dalsi kon-
verze podstatne neprojevila, a zcela minimalne v 6 mesicich. Hluboke partie svalli 1. svalo-
ve skupiny vykazovaly vyssi relativni poml SV Inez povrchove. Detekce aktivity SDH neumoznila objektivni typovou identifikaci SV u selat mladsich 1
mesice. Obsah glykogenu nekoreloval vZdy s adekvatnim typem SV. V glykolytickjch sva-
lech jsme identifikovali zcela ojedinela, dystrofickymi zmenarni postizena SV lIB, typu
"giant fibres". Neprokazalijsme podeIne stepena SV. Discussion We are aware of the limited significance of our observations,
but Uhrin and KuliSkova (1985) also failed to demonstrate any muscle fibre splitting in
Duroc pigs. The incidence of fibres several times thinner than the rest of them in the primary
muscle bundle in animals 1-6 months old might support the concept of new muscle fibres. New
MFs are more likely to grow from satellite cells, rather than by their longitudinal splitting, as
documented by S c h m al bru c h (1976) in his ultramicroscopy studies, and which, viewed by
a light microscope, appear as fragments of longitudinal splitting of muscle cells. g
p
pp
g
g
p
g
Many authors consider a structural sample of a muscle bundle with 1-7 muscle fibres of
type I in its centre and type II (IIA, lIB) fibres embedded in its periphery as a constant and
typical model or unit of the "porcine muscle. It is a fiction, whose origin might be traced
down to an almost exclusive study of the glycolytic m. longissimus dorsi (or its structural
analogue). This model does not apply to muscles with a higher proportion of oxidative
muscle fibres, and even less so for typically oxidative, slow muscles. 223 PostnataIni morfologicka a histochemick8 typova diferenciace svaloveho vIakna prasete References 67: 311-
319 319
RENDE VAN DEN, C., MUYLLE, E., OY AERT, W., ROOSE DE, P. 1972: Changes in muscle characteristics in
growing pigs, histochemical and electron microscopic stUdy. Zbl. Vet. Med. A, 19: 102-110 g
g p g
p
y
HORAK, V. 1979: Heterogenita a ontogeneticicY vyvoj typli svalovych vlaken u miniprasat a drlibek(In Czech),
PhD. thesis, UUFG,Li~hov,174p. HRASTE, A., ZOBUND2UA, M., BEGO, U. 1980: Prilog pojevi divoskih vlakna u m. longissimus dorsi svinja
primitivnich pasmina. Veter. Arch. 50: 51-59 p
p
KAMAN, J. 1995: Morphological and histochemical type differentiation of muscle fibers in newborn pigs: its
relationship to topical myopathies. Acta vet. Bmo 64:35-47 p
p
y p
KAWAIDA,H.,HIRATA,I.,MIYAUCHI, Y., KANMATSUSE,N., KAKO, Y.,KOJIMA,M.1978: Studies on
meat quality and its estimation technics of Kagoshima Berkshire. I. Correlation between muscle fibre diameter
and some characteristics of longissimus dorsi muscle. Jap. J. Swine Husb. Res. 15: 22-28 g
p
KLOSOWSKA, D., KLOSOWSKI, B. 1976: Wstepne obserwacje nad wplywem sezonu na niektore cechy
histologicznej budowy miesnia najdluszego grzbietu swill rasy wielkej bialej polskiej. Zesz. probl. Post. Nank. rol.l80: 447-451 KLOSOWSKA, D., KLOSOWSKI, B., GRAJEWSKA, S. 1976: Histologiczny obraz miema najdluzszego
grzbietu swill mieszencov rasy Norweskiej landrace i Wielkej bialej polskej zywionych paszami
o zroznicowanym poziomie bialka. Zesz. probl. Post. Nank. rol. 180: 441-446 y
p
p
KUUSKOV A, L., UHRIN, V. 1986: Postnat4lny rast jednotlivych typov svalovych vlaken niektorych svalov
prasiat. 2ivoc. vjr. 31: 143-152
v~lavmf p
j
LOJDA, Z., PAPOUSEK, F. 1978: Zaklady histochemickeho pn1kazu enzymu. O'stav dal§fho v~lavmf zdi-avot. kadrIi, Bmo, 119 p. p
N0STVOLD, S. O. 1981: Anatomy of skeletal muscle tissue. Proc. Symp. ,,Porcine stress and solution to the meat
quality". Jeloy, Norway, 1980. Agricult. Food Res. Soc., Norway PETROV, J. 1970: Porodni i polovi razli~ija v diametyra na muskulnite vlakna i krechkostta na mesoto u prasta,
otgle~dani pri razli~en re~. Sp. 2ivotnovydni nauki.7: 83-98 PETROV, J. 1970: Porodni i polovi razli~ija v diametyra na muskulnite vlakna i krechkostta na mesoto u prasta,
otgle~dani pri razli~en re~. Sp. 2ivotnovydni nauki.7: 83-98
PETROV, J. 1976: liber die postnatale Entwick}ungiderSkeletmuskelzellenbei Schwein. Zbl. Vet. Med. C 5: 224- p
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PETROV, J. 1976: liber die postnatale Entwick}ungider Skeletmuskelzellen bei Schwein. Zbl. Vet. Med
243 ROTKIEWICZ, T. 1980: Badanie morfologiczne i histochemiczne miesnia nejdluszego grzbietu (musculus
longissimus dorsi) u roznych ras swill. Zesz., nank. Wyz. Szk. roIn. Olszt. 12: 49-93 g
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y
SALOMON, F., MICHEL, G., GRUSCHWITZ, F. References ASHMORE, C. R., ADDIS, P. B., DOERR, L. 1973: Development of muscle fibers in the fetal pig. J. Anim. Sci. 36:1088-1093 ASHMORE, C. R., TOMPKINS, G., DOERR, L. 1972: Postnatal development of muscle fiber types i
animals. J. Anim. Sci. 34:37-41 BADER, R. 1982: Histologische Befunde aus licht- und elektronmikroskopischen Untersuchungen an der
Skelettrnuskulatur von gesunden ausgemasteten Schweinen der Deutschen Landrasse. Zbl. Vet. Med. A,
29:458-476 BADER, R. 1983: Vergleichende Untersuchungen an der Skelettrnuskulatur von Wild- und Hausschweinen. Berl. MUnch. Tierarztl. Wschr. 96:89-97 BADER, R. 1981: Enzymhistochemische, histometrische, histologische und elektronmikroskopische
Untersuchungen an der Skelettrnuskulatur gesunder ausgemasteter Schweine der Deutschen Landrasse in
unterschiedlichen Haltungssystemen. Berl. Freie Univ., Diss. Med. Vet. BEERMANN, D. H., CASSENS, R. G. 1977: Indirect fluorescence of primary and secondary myofibers in
developing porcine muscle. J. Histochem. Cytochem. 25: 439-442 NN, D. H. 1978: A second look at fiber type differentiation in porcine skeletal muscle. J. Anim. Sci. 46
2 BICKHARDT, K., CHEVALIER, H. J.,GIESE, W., REINHARD, H. 1. 1972: Akute Riickenmuskelnekrose und
Belastungsmyopathie beim Schwein. Fortschritte der Vet. Med., BerlinIHamburg, S.lll g
y p
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BINKHORST, R. A. 1969: The effects of training on some isometric contraction characteristics of a fast muscle. PfIiigers Arch. ges. Physiol. Berlin, Gottingen, 309: 193-202 BROOKE, M. H., KAISER, K. K. 1970: Muscle fiber types: How many and what kind? Arch. Neurol. 23: 369-379
CASSENS, R. G., COOPER, C. C., BRISKEY, J. 1969: The occurrence and histochemical characterization of giant
fibers in the muscle of growing and adult animals. Acta Neuropathol. 12: 300-308 g
g
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DAVIES, A. S. 1972: Postnatal changes in the histochemical fibre types of porcine skeletal muscle. 1. Anat. 113:
213-240 DUBOWITZ, V., BROOKE, M.H. 1972: Muscle biopsy: A modern approach. Saunders Company Ltd., London,
474p. FAZARINC, G., BAVDEK, S., LORGER, J. 1991: Postnatale histokemicne in morfometricne sprembe
v skeletnem mi§icju domacega pra§ica. Zb. Vet. fak. Ljubljana, 28: 151-165 224 FINGER, W., DZAPO, v., WASMUTH, R. 1986: Morphometrische Untersuchung am m.longissimus dorsi von
Schweinerassen unterschiedlicher Konstitution. Zschr. Ziicht. Zuchtungsbiol. 103: 59-68 g
72: Untersuchungen zur Erfassung des Adaptationsvermligens beim Hausschwein. Schriftreihe des
k-Inst f Tierzucht u Tierernlihrung Marienseell'renthorst 98 p g
HAASE, S. 1972: Untersuchungen zur Erfassung des Adaptationsvermligens beim Hausschwein. Schri
Max-Planck-Inst. f. Tierzucht u. Tierernlihrung, Marienseell'renthorst, 98 p. g
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HANZLtKOV A, V. 1980: Histochemical patterns in normal and splaylegged muscle fibers. Histochem. References 1983: On the development of fiber types composition and
diameter in porcine longissimus (Sus scrofa domesticus). Anat. Anz. 154: 69-79 SCHLEGEL, O. 1982: Untersuchungen iiber die Fasertypenverteilung und Fasertypenquerschnittsfllichen in m. longissimus dorsi und m. semitendinosus vo~ ~erten Haus- und immobil gehaltenen Wildschweinen.lnaug. Diss., Tierllrztl. Hochschule, Hannover.'
I t SCHMALBRUCH, H. 1976: Muscle fiber splitting Iuxt regeneration in disea.se<i human muscle. Neuropathol. and
Applied Neurobiol. 2: 3-19
' pp
SUZUKI, A., CASSENS, R. G. 1980a: pH sensitivity of myosin adcmosine triphosphatase and subtypes of
myofibres in porcine muscle. Histochem. J. 12:687-693 y
p
SUZUKI, A., CASSENS, R. G. 1980b: A hist(jchemical study of my6fiber types in muscle of the growing pig. J. Anim. Sci. 51:1449-1461 SWATLAND, H. J., CASSENS, R. G. 1973: Prenatal development, histochemistry and innervation of porcine
muscles. J. Anim. Sci. 36:343-354
" SZENTKUTI, L. -NIEMEYER, B., SCHLEGEL, 0.: 1981: Vergleichende Untersuchung von Muskelfasertypen
mit der Myosin-ATPase-Reaktion im M. LOngiSsimus dorsi von Haus- und Wildschweinen. Dtsch. Tierllrztl. Wschr.88:393-448 TODOROV, A., PETROV, J. 1969: Entwick}ung, und Verllnderung der Skelettmuskelfaser (Differenzierung,
Hyperp1asie und physiologische Degeneration) heim Schwein nach der Geburt. Anat. Anz. 125: 88-108 TODOROV, A., PETROV, J. 1969: Entwick}ung, und Verllnderung der Skelettmuskelfaser (Differenzierung,
Hyperp1asie und physiologische Degeneration) heim Schwein nach der Geburt. Anat. Anz. 125: 88-108
UHRIN, V., KUL1SKOV A, L. 1985: Porovnanie typov svalovych vlaken roznych plemien a kr!uncov o§fpanYch. yp p
p y
g
g
UHRIN, V., KUL1SKOV A, L. 1985: Porovnanie typov svalovych vlaken roznych plemien a kr!uncov
Proc. XlIth Genetic Days, Nitra, 234-236
' UHRIN, V., KUL1SKOvA, L., POLTARSKY J., BULLA, J.1986: Mikroskopicka stavba niektorych svalov
o§fpanych zaradenych do hybridi~neho prograinu. 2ivoc. vjroba 31:839-847 Plate I. Kaman J.: Postnatalni sval. vhikna prasete ... pp. 211-224. Fig.l.: Neonatal piglet, transversal histological cross-section (CS), m. semimembranosus, markedly
compact muscle tissue. Completed morphological differentiation of extra- and intrafusal muscle
fibres (MFs) , characterised by MFs completely filled with myofibrils, with subsarcolemmally
located cell nuclei, polygonal CS, obliteration of differences in thickness between the first and
second developmental population of MFs. NMS = neuromuscular spindle; currently occurring
intrafibral nuclei, disproportion in the thickness of intrafusal MFs. HE, x 100. Fig.l.: Neonatal piglet, transversal histological cross-section (CS), m. semimembranosus, markedly
compact muscle tissue. References Completed morphological differentiation of extra- and intrafusal muscle
fibres (MFs) , characterised by MFs completely filled with myofibrils, with subsarcolemmally
located cell nuclei, polygonal CS, obliteration of differences in thickness between the first and
second developmental population of MFs. NMS = neuromuscular spindle; currently occurring
intrafibral nuclei, disproportion in the thickness of intrafusal MFs. HE, x 100. Fig.l.: Neonatal piglet, transversal histological cross-section (CS), m. semimembranosus, markedly
compact muscle tissue. Completed morphological differentiation of extra- and intrafusal muscle
fibres (MFs) , characterised by MFs completely filled with myofibrils, with subsarcolemmally
located cell nuclei, polygonal CS, obliteration of differences in thickness between the first and
second developmental population of MFs. NMS = neuromuscular spindle; currently occurring
intrafibral nuclei, disproportion in the thickness of intrafusal MFs. HE, x 100. Fig.2.: Neonatal piglets, m .. triceps brachii, c. longum. PAS reaction. Glycogen not only
interfibrilarly but also interfascicularly. Intrafibral reaction graded in places, strong in MFs II,
weaker in MFs IIC, negative in MFs I (see explanatory notes). x 100. Fig.2.: Neonatal piglets, m .. triceps brachii, c. longum. PAS reaction. Glycogen not only
interfibrilarly but also interfascicularly. Intrafibral reaction graded in places, strong in MFs II,
weaker in MFs IIC, negative in MFs I (see explanatory notes). x 100. Plate II. Fig.3a.: Neonatal piglet. CS of a rather superficial, less compact, zone of the m. longissimus dorsi. Primary and secondary muscle bundles (MBs) patterned rather loosely, clearly delineated; muscle
fibres of mainly circular CS. Myosin ATPase activity after pH 4.3 preincubation differentiated all
basic MF types, including the MS, SS and SM subtypes of the conversion IIC type. Difference in
thickness between primary (type I) and secondary type II) muscle fibres still noticeable. MFs
adequately filled with myofibrils; x 160. Plate II. Plate II. Fig.3a.: Neonatal piglet. CS of a rather superficial, less compact, zone of the m. longissimus dorsi. Primary and secondary muscle bundles (MBs) patterned rather loosely, clearly delineated; muscle
fibres of mainly circular CS. Myosin ATPase activity after pH 4.3 preincubation differentiated all
basic MF types, including the MS, SS and SM subtypes of the conversion IIC type. Difference in
thickness between primary (type I) and secondary type II) muscle fibres still noticeable. MFs
adequately filled with myofibrils; x 160. Fig.3a.: Neonatal piglet. CS of a rather superficial, less compact, zone of the m. longissimus dorsi. References Primary and secondary muscle bundles (MBs) patterned rather loosely, clearly delineated; muscle
fibres of mainly circular CS. Myosin ATPase activity after pH 4.3 preincubation differentiated all
basic MF types, including the MS, SS and SM subtypes of the conversion IIC type. Difference in
thickness between primary (type I) and secondary type II) muscle fibres still noticeable. MFs
adequately filled with myofibrils; x 160. Fig.3b.: Inversion image to 3a: myosin ATPase reaction after alkaline preincubation at pH 9.2. Legend to MF types in both images is identical. x 160. Fig.3b.: Inversion image to 3a: myosin ATPase reaction after alkaline preincubation at pH 9.2. Legend to MF types in both images is identical. x 160. Fig.3b.: Inversion image to 3a: myosin ATPase reaction after alkaline preincubation at pH 9.2. Legend to MF types in both images is identical. x 160. Plate III. Plate III. FigA.: Neonatal piglet. Myosin ATPase detection after preincubation at pH 4.3 demonstrated a high
degree of histochemical and morphological differentiation. The most intensive reaction was
observed in the completely acid stable MF I, intensive reaction in conversion MFs with their SS,
SM subtypes, and moderate in MS subtype. The reaction also differentiated type II fibres into IIA
and lIB. M. gracilis, typical glycolytic muscle with I MF of type I in the primary MB and with
absolute predominance of type II fibres; . x 160. FigA.: Neonatal piglet. Myosin ATPase detection after preincubation at pH 4.3 demonstrated a high
degree of histochemical and morphological differentiation. The most intensive reaction was
observed in the completely acid stable MF I, intensive reaction in conversion MFs with their SS,
SM subtypes, and moderate in MS subtype. The reaction also differentiated type II fibres into IIA
and lIB. M. gracilis, typical glycolytic muscle with I MF of type I in the primary MB and with
absolute predominance of type II fibres; . x 160. FigA.: Neonatal piglet. Myosin ATPase detection after preincubation at pH 4.3 demonstrated a high
degree of histochemical and morphological differentiation. The most intensive reaction was
observed in the completely acid stable MF I, intensive reaction in conversion MFs with their SS,
SM subtypes, and moderate in MS subtype. The reaction also differentiated type II fibres into IIA
and lIB. M. gracilis, typical glycolytic muscle with I MF of type I in the primary MB and with
absolute predominance of type II fibres; . x 160. Fig.5.: Neonatal piglet. References Series CS from m. tibialis cranialis, as in m. gracilis, ATPase reaction at pH
4.3 demonstrated I (exceptionally 2) acid stable type I fibres. Compared to m. gracilis, however, its
primary MBs contain a large number (up to 12) conversion MFs, with a predominance of SM
subtype, which reacts almost analogically as type I fibres. Polygonal muscle fibres are equally thick
and fully filled with myofibrils; x 100. Fig.5.: Neonatal piglet. Series CS from m. tibialis cranialis, as in m. gracilis, ATPase reaction at pH
4.3 demonstrated I (exceptionally 2) acid stable type I fibres. Compared to m. gracilis, however, its
primary MBs contain a large number (up to 12) conversion MFs, with a predominance of SM
subtype, which reacts almost analogically as type I fibres. Polygonal muscle fibres are equally thick
and fully filled with myofibrils; x 100. Fig.5.: Neonatal piglet. Series CS from m. tibialis cranialis, as in m. gracilis, ATPase reaction at pH
4.3 demonstrated I (exceptionally 2) acid stable type I fibres. Compared to m. gracilis, however, its
primary MBs contain a large number (up to 12) conversion MFs, with a predominance of SM
subtype, which reacts almost analogically as type I fibres. Polygonal muscle fibres are equally thick
and fully filled with myofibrils; x 100. Plate IV. Fig. 6a.: Neonatal piglet. CS from m. stemomastoideus. ATPase at pH 4.3. Mostly one and
exceptionally 3 thick, primary acid stable type I fibres in primary MB, very intensive reaction. Conspicuously large number of conversion subtypes (the most advanced SM subtype already
converging to type I fibres). In the centre, primary MB with an almost completed conversion of type
II fibres into transformation subtypes, with a predominance ofMS subtype. x 160. Plate IV. Fig. 6a.: Neonatal piglet. CS from m. stemomastoideus. ATPase at pH 4.3. Mostly one and
exceptionally 3 thick, primary acid stable type I fibres in primary MB, very intensive reaction. Conspicuously large number of conversion subtypes (the most advanced SM subtype already
converging to type I fibres). In the centre, primary MB with an almost completed conversion of type
II fibres into transformation subtypes, with a predominance ofMS subtype. x 160. Fig. 6b.: Inversion image of6a. Myosin ATPase reaction at pH 9.4. x 160. Fig. 6b.: Inversion image of6a. Myosin ATPase reaction at pH 9.4. x 160. PlateV. Fig. 7.: Seven-day-old piglet. Muscle fibre type differentiation by SDH reaction in m.longissimus
dorsi. References Reaction markedly differentiated than in a newborn. Oxidative fibres react with a higher
intensity than the rest. Their clear delineation, particularly between individual type II fibres, is still
impossible in places; x 100. Fig. 7.: Seven-day-old piglet. Muscle fibre type differentiation by SDH reaction in m.longissimus
dorsi. Reaction markedly differentiated than in a newborn. Oxidative fibres react with a higher
intensity than the rest. Their clear delineation, particularly between individual type II fibres, is still
impossible in places; x 100. Fig. 7.: Seven-day-old piglet. Muscle fibre type differentiation by SDH reaction in m.longissimus
dorsi. Reaction markedly differentiated than in a newborn. Oxidative fibres react with a higher
intensity than the rest. Their clear delineation, particularly between individual type II fibres, is still
impossible in places; x 100. Fig. 8.: One-month-old piglet. CS from m. triceps brachii. ATPase at pH 4.5. Although the number
of type I fibres has increased from 1 up to 4, type II fibres have retained absolute majority. Numerous
transformation subtypes (MS, SS, SM) differentiated by intensity of their reaction, and
differentiated type IIA and lIB fibres; x 63. Fig. 8.: One-month-old piglet. CS from m. triceps brachii. ATPase at pH 4.5. Although the number
of type I fibres has increased from 1 up to 4, type II fibres have retained absolute majority. Numerous
transformation subtypes (MS, SS, SM) differentiated by intensity of their reaction, and
differentiated type IIA and lIB fibres; x 63. Plate VI. Fig. 9a.: One-month-old piglet Myosin ATPase activity at pH 9.4 in the cross-section from the m. stemomastoideus depicts the opposite situation from that in Fig. 8.: Although primary MBs in
neonates contained only 1-3 fibres of type I (light), they now make up an complete majority of fibres
in the muscle. Individual transformation MFs and type IIA and IIB fibres can still be found. x 100. Fig. 9a.: One-month-old piglet Myosin ATPase activity at pH 9.4 in the cross-section from the m. stemomastoideus depicts the opposite situation from that in Fig. 8.: Although primary MBs in
neonates contained only 1-3 fibres of type I (light), they now make up an complete majority of fibres
in the muscle Individual transformation MFs and type IIA and IIB fibres can still be found x 100 Fig. 9a.: One-month-old piglet Myosin ATPase activity at pH 9.4 in the cross-section from the m. stemomastoideus depicts the opposite situation from that in Fig. References 8.: Although primary MBs in
neonates contained only 1-3 fibres of type I (light), they now make up an complete majority of fibres
in the muscle. Individual transformation MFs and type IIA and IIB fibres can still be found. x 100. Fig. 9b.: Inversion image of 9a; ATPase at pH 4.35. Acid stable MF I, black. Infrequent MF IIA,
IIB and conversion MFs are shown in graded shades grey. x 100. Fig. 9b.: Inversion image of 9a; ATPase at pH 4.35. Acid stable MF I, black. Infrequent MF IIA,
IIB and conversion MFs are shown in graded shades grey. x 100. Plate VII. Plate VII. Fig. 10.: One-month-old piglet. M. trapezius. ATPase reaction after alkaline preincubation
documents the conversion of a muscle with complete predominance of type II fibres into a mixed-
type muscle with over 50% of Type I fibres (the lightest); x 63. Fig. 10.: One-month-old piglet. M. trapezius. ATPase reaction after alkaline preincubation
documents the conversion of a muscle with complete predominance of type II fibres into a mixed-
type muscle with over 50% of Type I fibres (the lightest); x 63. Fig. 10.: One-month-old piglet. M. trapezius. ATPase reaction after alkaline preincubation
documents the conversion of a muscle with complete predominance of type II fibres into a mixed-
type muscle with over 50% of Type I fibres (the lightest); x 63. Fig.ll.: Three-months-old pig. M. triceps brachii. CS, PAS reaction to glycogen is moderate. Glycogen distribution is not entirely identical with type differentiation of muscle fibres. MF I - the
lightest, MF II - the darkest, MF IIC - intermediary; x 63. Fig.ll.: Three-months-old pig. M. triceps brachii. CS, PAS reaction to glycogen is moderate. Glycogen distribution is not entirely identical with type differentiation of muscle fibres. MF I - the
lightest, MF II - the darkest, MF IIC - intermediary; x 63. Plate VIII. Fig.12a.: Three-month-old pig. Muscle fibre type distribution in m. triceps brachii. The number of
MF I (black) is only slightly elevated, dominance of MF IT remains highly significant. Lighter MF
ITA, darker MF 1m and isolated MF I are clearly identifiable at the periphery of primary bundles. ATPase at pH 4.3. x 100. Plate VIII. Fig.12a.: Three-month-old pig. Muscle fibre type distribution in m. triceps brachii. The number of
MF I (black) is only slightly elevated, dominance of MF IT remains highly significant. References Lighter MF
ITA, darker MF 1m and isolated MF I are clearly identifiable at the periphery of primary bundles. ATPase at pH 4.3. x 100. Fig. 12b.: Inversion image of Fig. 12a. ATPase at pH 9.4. In the bottom right part of the picture
a "giant fibre" with dystrophic changes can be seen. x 100. Fig. 12b.: Inversion image of Fig. 12a. ATPase at pH 9.4. In the bottom right part of the picture
a "giant fibre" with dystrophic changes can be seen. x 100. Plate IX. Plate IX. Fig.13a.: Six-month old pig. M. trapezius. ATPase at pH 9.4. Conversion no longer makes any
significant progress. Transformation MFs, particularly their more advanced stages, are still clearly
identifiable; x 63. Fig.13a.: Six-month old pig. M. trapezius. ATPase at pH 9.4. Conversion no longer makes any
significant progress. Transformation MFs, particularly their more advanced stages, are still clearly
identifiable; x 63. Fig.l3b.: Inversion image of Fig. 13a. ATPase at pH 4.3; x 63. Fig.l3b.: Inversion image of Fig. 13a. ATPase at pH 4.3; x 63. PlateX. PlateX. Fig.l4.: Six-month-old pig. MF type distribution in m. tibialis cranialis. Routine ATPase. Conversion of type II fibres into type I (the lightest) has not reached 50%. MF II are clearly
differentiated into types IIA and IIB; x 63. . Fig.l4.: Six-month-old pig. MF type distribution in m. tibialis cranialis. Routine ATPase. Conversion of type II fibres into type I (the lightest) has not reached 50%. MF II are clearly
differentiated into types IIA and IIB; x 63. . Fig.IS.: Six-month-old pig. M. semimembranosus. ATPase at pH 9.4 demonstrates a continued
absolute predominance of glycolytic MFs of type II differentiated into MF IIA (grey) and IIB (dark
grey). Type I muscle fibres (the lightest) are either in clusters or individually located in primary
bundles. A giant fibre can be seen in the bottom right part of the image. Fig.IS.: Six-month-old pig. M. semimembranosus. ATPase at pH 9.4 demonstrates a continued
absolute predominance of glycolytic MFs of type II differentiated into MF IIA (grey) and IIB (dark
grey). Type I muscle fibres (the lightest) are either in clusters or individually located in primary
bundles. A giant fibre can be seen in the bottom right part of the image.
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Case Report: Neotendon regeneration and repair of gluteus tendon tear at 1-year follow-up after ultrasound guided platelet rich plasma tenotomy
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Case Report: Neotendon regeneration and repair of gluteus
tendon tear at 1-year follow-up after ultrasound guided
platelet rich plasma tenotomy [version 1; peer review: 1
approved with reservations, 1 not approved] Case Report: Neotendon regeneration and repair of gluteus
tendon tear at 1-year follow-up after ultrasound guided
platelet rich plasma tenotomy [version 1; peer review: 1
approved with reservations, 1 not approved] tendon tear at 1 year follow up after ultrasound guided
platelet rich plasma tenotomy [version 1; peer review: 1
approved with reservations, 1 not approved]
Arockia Doss
Image Guided Therapy Clinic, Nedlands, Western Australia, 6009, Australia
Corresponding author: Arockia Doss (rocki.doss@imageguidedtherapyclinic.com)
Competing interests: No competing interests were disclosed. Grant information: The author(s) declared that no grants were involved in supporting this work. Copyright: © 2014 Doss A. 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. Data
associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public
domain dedication). How to cite this article: Doss A. Case Report: Neotendon regeneration and repair of gluteus tendon tear at 1-year follow-up after
ultrasound guided platelet rich plasma tenotomy [version 1; peer review: 1 approved with reservations, 1 not approved]
F1000Research 2014, 3:284 https://doi.org/10.12688/f1000research.5719.1
First published: 19 Nov 2014, 3:284 https://doi.org/10.12688/f1000research.5719.1
First published: 19 Nov 2014, 3:284
https://doi.org/10.12688/f1000research.5719.1
Latest published: 19 Nov 2014, 3:284
https://doi.org/10.12688/f1000research.5719.1
v1
Abstract
Greater trochanteric pain syndrome (GTPS) is a common condition
resulting in posterolateral hip pain typically in perimenopausal
women. Gluteal tendinopathy is the underlying pathology and
contributes to health care cost burden as a poorly managed tendon
disorder. There is no established effective treatment for gluteal
tendon pathology in GTPS. This article describes clinical, imaging and
life style improvements after percutaneous tendon repair using
autologous platelet rich plasma tenotomy under ultrasound imaging
guidance in a case of GTPS. The improvements observed in this
patient add to the call for urgent medical and economical need for
more research on percutaneous tendon repair. Keywords
GTPS, gluteal tendinopathy, tendon repair, tenotomy, ultrasound
imaging
Open Peer Review
Approval Status
1
2
version 1
19 Nov 2014
view
view
Nikolaos Gougoulias, Frimley Park Hospital,
Frimley, UK
1. Jason K.F. Wong, University of Manchester,
Manchester, UK
2. Any reports and responses or comments on the
article can be found at the end of the article. F1000Research 2014, 3:284 Last updated: 28 MAR 20 Arockia Doss Image Guided Therapy Clinic, Nedlands, Western Australia, 6009, Australia Image Guided Therapy Clinic, Nedlands, Western Australia, 6009, Australia First published: 19 Nov 2014, 3:284
https://doi.org/10.12688/f1000research.5719.1
Latest published: 19 Nov 2014, 3:284
https://doi.org/10.12688/f1000research.5719.1
v1 Abstract Greater trochanteric pain syndrome (GTPS) is a common condition
resulting in posterolateral hip pain typically in perimenopausal
women. Gluteal tendinopathy is the underlying pathology and
contributes to health care cost burden as a poorly managed tendon
disorder. There is no established effective treatment for gluteal
tendon pathology in GTPS. This article describes clinical, imaging and
life style improvements after percutaneous tendon repair using
autologous platelet rich plasma tenotomy under ultrasound imaging
guidance in a case of GTPS. The improvements observed in this
patient add to the call for urgent medical and economical need for
more research on percutaneous tendon repair. Keywords
GTPS, gluteal tendinopathy, tendon repair, tenotomy, ultrasound
imaging Corresponding author: Arockia Doss (rocki.doss@imageguidedtherapyclinic.com)
Competing interests: No competing interests were disclosed. Grant information: The author(s) declared that no grants were involved in supporting this work. Copyright: © 2014 Doss A. 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. Data
associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public
domain dedication). How to cite this article: Doss A. Case Report: Neotendon regeneration and repair of gluteus tendon tear at 1-year follow-up after
ultrasound guided platelet rich plasma tenotomy [version 1; peer review: 1 approved with reservations, 1 not approved]
F1000Research 2014, 3:284 https://doi.org/10.12688/f1000research.5719.1
First published: 19 Nov 2014, 3:284 https://doi.org/10.12688/f1000research.5719.1
imaging Introduction with partial split tears (image not shown). Under ultrasound imaging
(GE Logic 9, 9MHz probe) control, 1% lignocaine 5 cc was infiltrated
through a 22 G needle into and around the minimus tendon. Percu-
taneous tenotomy was performed into the foot print and the adjacent
gluteal cuff under real time imaging guidance. 4–5 cc of autologous
PRP (REGEN Switzerland, Adistem Hong Kong) was infiltrated. Repeated percutaneous tenotomy with PRP of the medius tendon
was performed in a similar manner 12 days later. Routine rehabilita-
tion with range of motion exercises, graded activity and a strength-
ening program was commenced. She took no time off work. Her
symptoms improved within 4 weeks. At 6 months post treatment,
she enjoyed a walking holiday in the hilly terrains of the Kimberly,
Western Australia without limitation. Clinical and ultrasound exam
follow-up at 12 months revealed a weight reduction of 6 kg with a
near normal BMI of 25.1. Her daily pain had resolved, with moder-
ate pain only on ascending stairs. There was minimal greater tro-
chanteric tenderness and no limitation of hip abduction. Ultrasound
of the gluteal tendons revealed that neotendon tissue had replaced
the degenerative tendinotic tissue with obliteration of the previously
known tear defect (Figure 2). Greater trochanteric pain syndrome (GTPS) is a common cause of
posterolateral hip pain typically seen in peri and post menopausal
women. Coexistent obesity, ilio-tibial band (ITB) syndrome, low
back pain, osteoarthritis are added risk factors1. Evaluation using
imaging and histology techniques revealed degenerative tendinosis
with tears of the gluteal tendons2–4. The primary underlying pathol-
ogy of GTPS is a gluteal tendinopathy with or without tears of the
tendon5. Although GTPS is widely referred to as greater trochanteric
‘bursitis’, there is no inflammation of the greater trochanteric bursa
on histological evaluation of surgical specimens6. The natural history of GTPS is favourable in most cases and responds
to physical therapy, weight loss, non-steroidal anti-inflammatory
drugs and behaviour modification1. However in some patients the
condition causes significant disability and necessitates interven-
tion. Corticosteroid injection into the bursa and surgical repair of
any torn gluteal tendons are the current common treatment options. Corticosteroid injections are controversial in a degenerating tendi-
nopathy7. Corticosteroid injections carry the risk of a dampening
effect and progressive worsening of tendon pathology8. Case report For those patients who do not respond to conservative measures,
the current options for treating tendinopathy by corticosteroid injec-
tions are ineffective7. Corticosteroid injections may result in pro-
gression of a catabolic tendon environment with worsening of
tendon degeneration, dampening of protective pain sensory feed-
back resulting in over-activity and tendon rupture. Surgical repair is
reserved as the very last option and data on efficacy is limited to ret-
rospective case series10–12. Data from 2008 suggest that direct surgi-
cal costs and indirect costs from bone and joint disorders amount to
USD 915 billion in the United States13. This is likely to escalate in
the next few decades13. Therefore there is a medical and economic
need to treat tendon disorders with innovative methods that will
address these issues. The ideal procedure should aim to repair the
tendon with minimum direct and indirect expenditure, be repeat-
able in a single course of treatment, reproducible across health care
systems and widely available. A 56 year old female Caucasian patient presented with 6 months of
progressive left sided greater trochanteric pain syndrome. She com-
plained of pain every day for many months, was unable to climb
stairs and experienced moderate stiffness of the outer hip during
early morning awakening. Pre-procedural clinical examination
revealed an overweight individual with a BMI of 27, valgus knees,
gynacoid pelvis, localised tenderness of the gluteus minimus and
medius insertions into the facets of the greater trochanter, painful
limitation of passive and active hip abduction and provocation to
resisted abduction due to gluteal tendon dysfunction. She wished to
be very active, commence a walking holiday and reduce her body
weight. She refused both corticosteroid injection therapy and sur-
gery. Full written informed consent to treat and publish the data was
obtained from the patient. Ultrasound scan showed a 10 mm × 12 mm
high grade full thickness tear of the degenerating gluteus medius
tendon insertion (Figure 1) and gluteus minimus tendinopathy Figure 2. Ultrasound of the gluteal tendons showing that
neotendon tissue had replaced the degenerative tendinotic
tissue with obliteration of the previously known tear defect. Figure 1. Ultrasound showing full thickness tear of the
degenerating gluteus medius tendon insertion (10 mm × 12 mm
high grade). Figure 2. Ultrasound of the gluteal tendons showing that
neotendon tissue had replaced the degenerative tendinotic
tissue with obliteration of the previously known tear defect. Figure 1. Discussion
d
h l Tendon pathologies in an ageing population contribute to a signifi-
cant bulk of musculoskeletal pain syndromes resulting in a major
health burden worldwide. They represent the third highest health
care expenditure, costing AUD$517 million per annum in Australia9. To date there are no effective treatment strategies that uniformly
address the pathology of degeneration in tendinopathy7. Introduction A pioneer-
ing percutaneous treatment for all tendoligamentous and cartilage
tears using autologous platelet rich plasma (PRP) tenotomy under
high resolution imaging control was routine clinical management
in the author’s practice. The same treatment was performed in a
patient with GTPS. This report is the 1-year follow up on the clini-
cal outcome and imaging appearance of this patient. Competing interests: No competing interests were disclosed. Competing interests: No competing interests were disclosed. Grant information: The author(s) declared that no grants were involved in supporting this work. le: Doss A. Case Report: Neotendon regeneration and repair of gluteus tendon tear at 1-year follow-up aft
atelet rich plasma tenotomy [version 1; peer review: 1 approved with reservations, 1 not approved]
3:284 https://doi.org/10.12688/f1000research.5719.1 ,
p
g
shed: 19 Nov 2014, 3:284 https://doi.org/10.12688/f1000research.5719.1 Page 1 of 10 F1000Research 2014, 3:284 Last updated: 28 MAR 2022 Case report In this patient, there was a combination of clinical improvement
together with imaging follow-up demonstrating neotissue tendon
regeneration within the gluteus medius tendon tear. The reduction
in body weight and the improved BMI are important markers for
improved outcome in lower limb tendon disorders. This implies
improved mobility and overall health due to life style modifications
subsequent to the treatment. The second step is the understanding that the macrostructure of the
musculotendinous junction, the full extent of the tendon and the
enthesis at the tendon bone fibrocartilage interface are implicated
in chronic tendinopathy. Therefore any treatment should be aimed
at correction of the entire extent of this anatomy where possible. Delivery of therapeutic material into the affected tendon should
fulfill the requirement of being able to penetrate 1) through splits
and tears within the substance of the tendon, 2) surrounding para-
tendon and the 3) fibrocartilage footprint of the insertional portion
of the tendon, ensuring that as much affected tendon is treated as
possible. The author has previously published data on neotissue regeneration
in a full thickness rotator cuff tear with complete abolition of pain
that lasted nearly two years post percutaneous repair in an elderly
patient19. As far as the author is aware this is the second report on
neotissue tendon regeneration following ultrasound guided percu-
taneous liquid PRP tenotomy and the first report of percutaneous
repair of a gluteus medius tendon tear. In addition, there is regional attrition of more than one tendon in
any given region and any treatment should address augmentation of
these regional tendons to ensure additional ‘scaffolding’ of a pro-
gressively weakened attritional environment. Placement of therapy
through the neighbouring attritional tissues may contribute to addi-
tional scaffolding via ‘neotissue’ regeneration around a weakened
tendon environment. For example, in gluteal minimus/medius tears,
the iliotibial band, gluteus maximus interface should be augmented. As far as the author is aware, this concept of percutaneous tendon
augmentation via biologicals has not been previously described in
the literature. This single report cannot form the basis of routine clinical applica-
tion in other dissimilar service delivery models. Clinical use of this
technique should be subject to regular clinical follow-up and out-
come evaluation. Further research is needed prior to routine clinical
use outside these parameters. In conclusion, the treatment of degenerative tendon tears has entered
a new paradigm. Case report Ultrasound showing full thickness tear of the
degenerating gluteus medius tendon insertion (10 mm × 12 mm
high grade). Page 2 of 10 F1000Research 2014, 3:284 Last updated: 28 MAR 2022 F1000Research 2014, 3:284 Last updated: 28 MAR 2022 F1000Research 2014, 3:284 Last updated: 28 MAR 2022 improvement, failure of intervention and reflect the various prepa-
rations of therapeutic material and the mode of delivery of PRP usu-
ally as an injection17,18. The role of high resolution imaging control
in the delivery of therapeutic material is either absent or unclear in
these prior reports. The search for new options should begin with an understanding of
the basic anatomy, physiology and pathophysiology of tendon dis-
orders. Tendons do not possess adequate vascular or nerve supply14. The ability of the human body to repair tendons is therefore inher-
ently limited. The paratendon structures surrounding tendon fibrils
contain neurovascular tissue that enables neuronal mediation and
reparative tendon homeostasis via immunomodulatory and inflam-
matory molecular pathways. Dysfunctional neuronal mediation and
an inadequate tendon homeostasis are the causes of chronic tendon
disorders. An understanding of this inadequacy is the key to treating
chronic painful dysfunction in tendon disorders14. This case report is unique in the simultaneous correlation of clini-
cal and imaging findings as part of routine clinical practice. This
ensured an accurate diagnosis of the afflicted tendons and was fol-
lowed by delivery of the percutaneous treatment precisely into the
culprit tendon and the augmentation of surrounding tissues using
high resolution imaging control. This procedure represents a unique
delivery of care model where clinical evaluation, imaging confir-
mation and percutaneous treatment were all performed by a single
treating specialist dedicated to image-guided orthopaedic inter-
vention. On this basis, the results here shown are incomparable to
other existing published studies that use a different service delivery
model. It is intuitive to suggest that the initial step should aim for the cor-
rection of limitation of neurovascular supply of tendons. Such a
correction may facilitate neuronal mediation and improve immu-
nomodulatory and inflammatory molecular pathways. This may
pave the way for an adequate, albeit prolonged normal healing
response. On this basis, biologicals that possess an anti-inflammatory/
immunomodulatory effect that promote neo-tissue regeneration and
neo-angiogenesis may offer a solution. The optimal biological mate-
rial should facilitate neuronal mediation and tendon homeostasis. Competing interests p
g
No competing interests were disclosed. Case report In this patient, percutaneous tendon repair under
imaging guidance with autologous PRP tenotomy resulted in neo-
tissue tendon regeneration of a gluteal tendon tear. This correlated
with improvement in the clinical syndrome, implementation of life
style modification, reduced BMI with very minimal loss of revenue
due to time off work. This suggests that percutaneous repair may
efficiently address the issues of the pathology and help in minimiz-
ing a sedentary life style in an increasingly overweight population
with very minimal or no loss of time from work. This in turn should
reduce healthcare costs to a stretched health system, loss of revenue
to the patient and the indirect costs to the community. There is an
economical and medical need for more research on this new para-
digm shift in tendon repair. Autologous PRP is a supra physiologic concentration of platelets
containing various growth factors secreted by the alpha granules in
platelets. PRP has gained increasing and controversial popularity
in the past few years. Application of PRP in treating degenerative
rotator cuff lesions is made on the basis of its role in the regula-
tion of matrix gene expression and cell proliferation15. The applica-
tion of PRP into the enthesis is based on the regenerative effects
on meniscal cells that share similar fibrocartilage histology16. Pre-
vious reports of autologous PRP are mixed, with some showing Page 3 of 10 Page 3 of 10 Page 3 of 10 F1000Research 2014, 3:284 Last updated: 28 MAR 2022 Grant information
The author(s) declared that no grants were involved in supporting
this study. ;
( )
PubMed Abstract� | Publisher Full Text | Free Full Text 15. Jo CH, Kim JE, Yoon KS, et al.: Platelet-rich plasma stimulates cell proliferation
and enhances matrix gene expression and synthesis in tenocytes from human
rotator cuff tendons with degenerative tears. Am J Sports Med. 2012; 40(5):
1035–1045. Acknowledgements Acknowledgements
The author is grateful to his referring health professionals and
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gluteus medius tendon insertion. Arthroscopy. 2008; 24(2): 130–136. PubMed Abstract� | Publisher Full Text
5. Alvarez-Nemegyei J, Canoso JJ: Evidence-based soft tissue rheumatology: III:
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8. Hart L: Corticosteroid and other injections in the management of
tendinopathies: a review. Clin J Sport Med. 2011; 21(6): 540–1. PubMed Abstract� | Publisher Full Text
9. http://www.aihw.gov.au/WorkArea/DownloadAsset.aspx?id=6442457279. 10. Davies JF, Stiehl JB, Davies JA, et al.: Surgical treatment of hip abductor tendon
tears. J Bone Joint Surg Am. 2013; 95(15): 1420–5. PubMed Abstract� | Publisher Full Text
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follow-up. Arthroscopy. 2013; 29(12): 1941–7. PubMed Abstract� | Publisher Full Text 12. Domb BG, Botser I, Giordano BD: Outcomes of endoscopic gluteus medius
repair with minimum 2-year follow-up. Am J Sports Med. 2013; 41(5): 988–97. PubMed Abstract� | Publisher Full Text 13. The Burden Of Musculoskeletal Diseases. Reference Source 13. The Burden Of Musculoskeletal Diseases. Reference Source 14. Ackermann PW: Neuronal regulation of tendon homoeostasis. Int J Exp Pathol. 2013; 94(4): 271–286. PubMed Abstract� | Publisher Full Text | Free Full Text 035
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PubMed Abstract� | Publisher Full Text 16. Ishida K, Kuroda R, Miwa M, et al.: The regenerative effects of platelet-rich
plasma on meniscal cells in vitro and its in vivo application with biodegradable
gelatin hydrogel. Tissue Eng. 2007; 13(5): 1103–12. PubMed Abstract� | Publisher Full Text 17. Zhang J, Wang JH: PRP treatment effects on degenerative tendinopathy - an
in vitro model study. Muscles Ligaments Tendons J. 2014; 4(1): 10–7. eCollection
2014. PubMed Abstract | Publisher Full Text | Free Full Text Consent Written informed consent for medical treatment and publication of
this anonymized report was obtained from the patient. PubMed Abstract | Publisher Full Text | Free Full Text 18. Wang JH: Can PRP effectively treat injured tendons? Muscles Ligaments
Tendons J. 2014; 4(1): 35–7. eCollection 2014. PubMed Abstract� | Publisher Full Text | Free Full Text 19. Doss A: Neotendon infilling of a full thickness rotator cuff foot print tear
following ultrasound guided liquid platelet rich plasma injection and
percutaneous tenotomy: favourable outcome up to one year [v1; ref status:
indexed, http://f1000r.es/xz]. F1000Res. 2013; 2: 23. eCollection 2013. PubMed Abstract� | Publisher Full Text | Free Full Text 10. Davies JF, Stiehl JB, Davies JA, et al.: Surgical treatment of hip abductor tendon
tears. J Bone Joint Surg Am. 2013; 95(15): 1420–5. PubMed Abstract� | Publisher Full Text 11. McCormick F, Alpaugh K, Nwachukwu BU, et al.: Endoscopic repair of full-thickness Page 4 of 10 Page 4 of 10 Jason K.F. Wong Institute of Inflammation and Repair, University of Manchester, Manchester, UK I think this is a controversial area whereby it is not possible to evaluate the effectiveness of PRP
specifically on the patients improvement in symptoms. We all have patients who respond in some
way to a change in treatment, behaviour or management which do not work again when trialed. There are too many confounding factors here that make this observation uncertain, for example
the patient could have got better without treatment, the needling could have caused the site to
bleed and the clotting could have healed the defect, fluid injected itself could have caused it to
heal, the US scan is not confirmation of neotendon as organised scar can have similar
appearances and a biopsy would have to be performed to confirm. Many different possibilities
exists that without proper study and experimental design means that this is an interesting
observation which may be biased and incorrect. Based on the literature published in this field to
date, it cannot be supported to change clinical practice without a proper randomised control study
based on significantly higher patient numbers. I know that this practice goes on in many sports
clinics at much financial gain to independent practicing sports clinicians. Competing Interests: No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of
expertise to state that I do not consider it to be of an acceptable scientific standard, for
reasons outlined above. Author Response 03 Jan 2015 Author Response 03 Jan 2015 F1000Research 2014, 3:284 Last updated: 28 MAR 2022 Open Peer Review Version 1 © 2014 Wong J. This is an open access peer review report 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. Purpose of Case Reports Case reports such as this article, serve the purpose of describing novel, innovative or new
treatment options by describing the outcome in a single or series of cases. Case reports do
not offer ‘data’ on efficacy. Case reports offer qualitative descriptions that reflect day to day
clinical practice. There are many variables leading to bias in a case report. Controlling bias
and proving efficacy of such a new treatment is the purpose of controlled studies and trials. Case reports are meant to be the subject of future rigorous studies and are not meant to
change routine clinical practice. This is made very clear in my article. The weight of
establishing efficacy rests on future controlled studies. Until such time, ultrasound guided
platelet rich plasma (PRP) tenotomy in gluteal tendon tears should not be used in routine
clinical practice without audit and follow up arrangements. This is also made very clear in
my paper. Therefore, to ‘Not Approve’ this case report on the basis of ‘lack of efficacy’ suggests that the
reviewer has not taken into account the very nature and purpose of case reports and has
not provided due credit to the points that were acknowledged in my article anyway. One should not forget that case reports described the first heart transplant for
cardiomyopathy 2 and the self experimentation by Barry Marshall with Helicobacter pylori
to prove the pathogenesis of peptic ulcers 3. Arockia Doss, Image Guided Therapy Clinic, Nedlands, Western Australia, Australia Arockia Doss, Image Guided Therapy Clinic, Nedlands, Western Australia, Austra I wish to thank the reviewer for his time to write a review for this case report on the repair
of a gluteal tendon tear following ultrasound image guided Platelet Rich Plasma (PRP)
tenotomy. It is regrettable that this reviewer has not approved this case report. Approval
and indexation in Pubmed is crucial for furthering study in a new area of controversy and Page 5 of 10 F1000Research 2014, 3:284 Last updated: 28 MAR 2022 interest. A similar case report on tendon repair in rotator cuff tear published in
F1000Research was subsequently approved and indexed in PubMed1. interest. A similar case report on tendon repair in rotator cuff tear published in
F1000Research was subsequently approved and indexed in PubMed1. The reviewer states that there is not enough evidence of efficacy data from a single patient
related outcome and there is lack of evidence that the treatment actually worked. He also
states that PRP procedures are performed for financial gain and his approval of this paper
may result in widespread routine clinical use of PRP. I wish to make the following points in response to this review. Why is this case report important in gluteal tendon tears? The concept of being able to repair a tendon with a relatively inexpensive out of hospital
procedure is innovative. This innovative approach has not been given due credit by the
reviewer. Tendon repair is currently perceived as being a surgical procedure in a hospital. Surgical tendon repair procedures are performed in private hospitals for those with
private health fund insurance schemes in the Western world. This means that
patients without private health cover do not have access to a hospital based surgical
procedure and are left to live in pain for a long time. This leads to a sedentary life
style and associated co-morbidities in a large proportion of patients. 1. Irrespective of currently available surgical or non surgical options, tendon disorders
are a major cause of morbidity in the western world and any innovation should be
welcome. 2. Irrespective of currently available surgical or non surgical options, tendon disorders
are a major cause of morbidity in the western world and any innovation should be
welcome. 2. There is a move towards keeping patients out of hospital due to escalating costs and
risks of infection from antibiotic resistant microorganisms in a hospital environment. This case report is timely and reflects the current trend of out of hospital procedures
that may potentially offer an alternative to in hospital surgical repair. 3. There is a move towards keeping patients out of hospital due to escalating costs and
risks of infection from antibiotic resistant microorganisms in a hospital environment. This case report is timely and reflects the current trend of out of hospital procedures
that may potentially offer an alternative to in hospital surgical repair. 3. Reports such as this offer proof of concept of regenerative percutaneous tendon
repair procedures. 4. Reports such as this offer proof of concept of regenerative percutaneous tendon
repair procedures. 4. Is there a need for a tendon biopsy ? The reviewer suggests the need for biopsy to prove healing of the treated tendon tear. When patients improve after treatment of a tendon, a biopsy to prove regeneration of a
healed tendon is impractical, unnecessary and may be harmful. The British Medical Journal
published a case report on tendon regeneration in a case of rotator cuff tendon tear
following percutaneous treatment 4. Good clinical outcome coupled with restoration of
normal tendon appearances on magnetic resonance imaging formed the basis of the
conclusion of ‘tendon regeneration’ by the authors. Regeneration of tendon was concluded
without tendon biopsy in that case report. Page 6 of 10 F1000Research 2014, 3:284 Last updated: 28 MAR 2022 Peer review Bias Open access publishing with open peer review offers a platform for unbiased dissemination
of articles irrespective of the source of the article - whether the article is from a small
private practice or from an academic department. Richard Smith (Former Editor, British
Medical Journal) describes the bias of accepting and publishing articles from big name
institutions as the Mathew effect: `To those who have, shall be given; to those who have not
shall be taken away even the little that they have’ 5. The comment ‘sports physicians performing PRP procedures with much financial gain’
suggests such a bias of the reviewer against private practices. The reviewers’ comment
assumes that private practices perform such procedures for the revenue from such
procedures. This is a regrettable and largely incorrect view. Such new procedures are
offered by private practitioners or demanded by patients due to the lack of other options in
an area of unmet medical need, as patients have tried existing options with no benefit. The
comment implies that good medical write ups are not to be expected from private non
academic practices. Purpose of this review The comment ‘based on the literature published in this field to date, it (PRP) cannot be
supported to change clinical practice without a proper randomised control study’ suggests Page 7 of 10 F1000Research 2014, 3:284 Last updated: 28 MAR 2022 that the reviewer rejects this case report on the basis of other studies and the possibility
that PRP may be used widely in clinical practice. I find that such a conclusion does not serve
the purpose of this review. that the reviewer rejects this case report on the basis of other studies and the possibility
that PRP may be used widely in clinical practice. I find that such a conclusion does not serve
the purpose of this review. Conclusion The weight of careful responsible reviews rests on all of us who have the ability to change
medical practice on the basis of good evidence. Where there is no such evidence in an area
of unmet medical need as seen with tendon disorders that result in a huge health economic
burden, should we not provide practice based evidence? When such practice based
evidence is written up, we need a review without any prejudice to the source of the article. Unfortunately this review does not help us take a forward step to reduce the health burden
from musculoskeletal disorders. Competing Interests: I am the author of this article. Competing Interests: I am the author of this article. Reviewer Report 24 November 2014 https://doi.org/10.5256/f1000research.6114.r6747 000 esea cg.
0
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ultrasound guided liquid platelet rich plasma injection and percutaneous tenotomy:
favourable outcome up to one year [v1; ref status: indexed, http://f1000r.es/xz]. F1000Researcg. 2013; 2 (23). PubMed Abstract | Free Full Text | Publisher Full Text |
Reference Source F1000Research 2014, 3:284 Last updated: 28 MAR 2022 https://doi.org/10.5256/f1000research.6114.r6747 © 2014 Gougoulias N. This is an open access peer review report 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. Page 8 of 10 Nikolaos Gougoulias Trauma and Orthopaedics Department, Frimley Park Hospital, Frimley, UK The author presented the outcome of one case of tendon healing, followed for one year, after PRP
injection. First I would like to comment on the terminology. "Tenotomy" may be a misleading
term. Tenontomy means "cutting a tendon". Is this what the author did? Or just an injection. This
needs clarification. Furthermore, one cannot prove that it was the PRP that aided tendon healing, or the injection
itself. Thee are various good quality randomized studies that showed no difference in the
outcomes of PRP versus placebo injections. These should be cited in the discussion. A single case
is not even indicative of the effectiveness of PRP. In theory PRPs may have anti-inflammatory and
reparative properties, may have shown "promising" results in the lab, but their clinical
effectiveness is questioned and controversial. Taking into consideration that their use is financially
beneficial to doctors and the industry, their use cannot be widespread, unless good quality
evidence is available. Competing Interests: No competing interests were disclosed. Competing Interests: No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of
expertise to confirm that it is of an acceptable scientific standard, however I have
significant reservations, as outlined above. Author Response 24 Nov 2014
Arockia Doss, Image Guided Therapy Clinic, Nedlands, Western Australia, Australia
First of all, thank you for your time with valuable comments and review. Tenotomy can be performed by a percutaneous technique with multiple points of
fenestrations through a tendon (Chiavaras MM, Jacobson JA. Ultrasound-guided tendon
fenestration. Semin Musculoskelet Radiol. 2013 Feb;17(1):85-90. doi: 10.1055/s-0033-
1333942. Epub 2013 Mar 13. Review. PubMed PMID: 23487340), (Housner JA, Jacobson JA,
Misko R. Sonographically guided percutaneous needle tenotomy for the treatment of
chronic tendinosis. J Ultrasound Med. 2009 Sep;28(9):1187-92. PubMed PMID: 19710216). With a tenotomy the tendon is fenestrated subcutaneously in an attempt to change a
chronic tendinopathy to an acute condition, thus allowing the opportunity to heal. This is a
distinctly different procedure to an 'injection' where the needle is not moved within the
tendon. It is made clear in the paper that this single patient case report does not constitute evidence
for routine application. The purpose of this paper is to document tendon tear repair seen on ultrasound in
conjunction with clinical improvement. Nikolaos Gougoulias This should form the basis of future rigorous studies
on efficacy of a combined technique of PRP tenotomy, PRP alone or tenotomy alone, versus
Page 9 of 10 I confirm that I have read this submission and believe that I have an appropriate level of
expertise to confirm that it is of an acceptable scientific standard, however I have
significant reservations, as outlined above. Author Response 24 Nov 2014 Arockia Doss, Image Guided Therapy Clinic, Nedlands, Western Australia, Australia Arockia Doss, Image Guided Therapy Clinic, Nedlands, Western Australia, Australia
First of all, thank you for your time with valuable comments and review. First of all, thank you for your time with valuable comments and review. First of all, thank you for your time with valuable comments and review. Tenotomy can be performed by a percutaneous technique with multiple points of
fenestrations through a tendon (Chiavaras MM, Jacobson JA. Ultrasound-guided tendon
fenestration. Semin Musculoskelet Radiol. 2013 Feb;17(1):85-90. doi: 10.1055/s-0033-
1333942. Epub 2013 Mar 13. Review. PubMed PMID: 23487340), (Housner JA, Jacobson JA,
Misko R. Sonographically guided percutaneous needle tenotomy for the treatment of
chronic tendinosis. J Ultrasound Med. 2009 Sep;28(9):1187-92. PubMed PMID: 19710216). With a tenotomy the tendon is fenestrated subcutaneously in an attempt to change a
chronic tendinopathy to an acute condition, thus allowing the opportunity to heal. This is a
distinctly different procedure to an 'injection' where the needle is not moved within the
tendon. It is made clear in the paper that this single patient case report does not constitute evidence
for routine application. The purpose of this paper is to document tendon tear repair seen on ultrasound in
conjunction with clinical improvement. This should form the basis of future rigorous studies
on efficacy of a combined technique of PRP tenotomy, PRP alone or tenotomy alone, versus Page 9 of 10 F1000Research 2014, 3:284 Last updated: 28 MAR 2022 Such case reports offer practice based evidence rather than evidence based practice. Nikolaos Gougoulias Competing Interests: I am the author of this article The benefits of publishing with F1000Research:
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1 Introduction In 2022, we can celebrate the 100th anniversary of the formation of the Deutsche Geophysikalische Gesellschaft (DGG,
German Geophysical Society). The DGG was founded as Deutsche Seismologische Gesellschaft (DSG, German Seismological
Society), which changed its name to Deutsche Geophysikalische Gesellschaft two years later, in 1924. The 100th anniversary
of the DGG foundation opened for the opportunity to investigate its wider background. To understand this, one has to look
15
back in the development of the international cooperation in seismology during the decades before World War I (WWI). The
following text is the slightly altered, translated version of an article (Schweitzer, 2022) originally published in Germany by
the DGG as part of a set of articles under the common title Geophysik im Wandel (Geophysics in Change). Foundation of the Deutsche Geophysikalische Gesellschaft (DGG) in
its international context Johannes Schweitzer 1
1NORSAR, Kjeller, 2027, Norway Correspondence to: Johannes Schweitzer (Johannes.schweitzer@norsar.no)
5 Abstract. The article describes the international seismological cooperation at the beginning of the last century and how this
cooperation changed due to World War I. These changes were the direct reasons leading to the foundation of the Deutsche
Seismologische Gesellschaft in 1922, which changed its name to Deutsche Geophysikalische Gesellschaft two years later. The
second part of the article shortly describes the further development of the relationship between German geophysicists and their
international colleagues until the beginning of World War II. 10 History of
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Geo- and Space
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Discussions
Open Access https://doi.org/10.5194/hgss-2022-12
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c⃝Author(s) 2022. CC BY 4.0 License. 2 The Early Roots of International Cooperation in Seismology Conferences and diplomatic negations before Belgium, Bulgaria, Chile, Congo, Germany, Greece, Hungary, Italy, Japan,
30
Mexico, The Netherlands, Norway, Portugal, Romania, Russia, Spain, Switzerland and the United States of America signed
an interstate convention for the following 12 years to form the Internationale Seismologische Assoziation (ISA, International
Seismological Association). After some changes of the statutes, also Canada, France, Great Britain and Serbia became ISA
members in 1907 and Argentina joined in 1913. Following Rebeur-Paschwitz’s idea, ISA established under the directorship of Gerland a Zentralbureau (Central Office) in
35
Strasbourg, at Gerland’s university institute, to organize the international seismological data exchange and to publish world-
wide bulletins. With this, the global centre for the international cooperation in seismology was located in Strasbourg, Alsace. In 1910, after Gerland’s retirement, Oskar August Ernst Hecker (1864-1938, see e.g., Kohlschütter, 1938, Jacobs & Börngen,
2019) became the second director of ISA’s Central Office and of the Imperial Main Station. More details about the
establishment of ISA, its activities and the work of the Zentralbureau including many additional references can be found in
40
Schweitzer (2003) and Schweitzer and Lay (2019) 2 The Early Roots of International Cooperation in Seismology In 1895, shortly before his death, Ernst von Rebeur-Paschwitz (1861-1895, see e.g., Davison, 1895; Kertz, 2002) published
20
two path-breaking articles (Rebeur-Paschwitz, 1895a; b), in which he proposed the installation of a worldwide network of
seismic stations with common instrumentation, to monitor the seismicity of the Earth and to investigate the Earth’s interior. He also proposed the foundation of an international institute (bureau) that should be responsible for collecting and publishing
all reports about felt and/or instrumentally observed earthquakes as completely as possible. Rebeur-Paschwitz’s ideas were taken up by Georg Cornelius Karl Gerland (1833-1919, see e.g., Sapper, 1940; Kertz, 2002),
25
who established the Kaiserliche Haupstation für Erdbebenforschung (Imperial Main Station for Earthquake Research) at the
University of Strasbourg and could finally, in 1901, invite to the 1. Internationale Seismologische Konferenz (1st International Rebeur-Paschwitz’s ideas were taken up by Georg Cornelius Karl Gerland (1833-1919, see e.g., Sapper, 1940; Kertz, 2002),
25
who established the Kaiserliche Haupstation für Erdbebenforschung (Imperial Main Station for Earthquake Research) at the
University of Strasbourg and could finally, in 1901, invite to the 1. Internationale Seismologische Konferenz (1st International 1 History of
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c⃝Author(s) 2022. CC BY 4.0 License. Seismological Conference) in Strasbourg, Alsace, at that time Germany. The main topic of this conference was to establish an
institutionalized framework for the international cooperation in seismology. It took two additional International Seismological
Conferences and diplomatic negations before Belgium, Bulgaria, Chile, Congo, Germany, Greece, Hungary, Italy, Japan,
30
Mexico, The Netherlands, Norway, Portugal, Romania, Russia, Spain, Switzerland and the United States of America signed
an interstate convention for the following 12 years to form the Internationale Seismologische Assoziation (ISA, International Seismological Conference) in Strasbourg, Alsace, at that time Germany. The main topic of this conference was to establish an
institutionalized framework for the international cooperation in seismology. It took two additional International Seismological Conferences and diplomatic negations before Belgium, Bulgaria, Chile, Congo, Germany, Greece, Hungary, Italy, Japan,
30
Mexico, The Netherlands, Norway, Portugal, Romania, Russia, Spain, Switzerland and the United States of America signed
an interstate convention for the following 12 years to form the Internationale Seismologische Assoziation (ISA, International
Seismological Association). After some changes of the statutes, also Canada, France, Great Britain and Serbia became ISA
members in 1907 and Argentina joined in 1913. 3 ISA Becomes a Victim of WWI The political and social “earthquakes” in Europe because of WWI changed everything. Strasbourg, Alsace became again part
of France and with this the ISA Central Bureau became part of the post-war problems between France and Germany. During of France and with this the ISA Central Bureau became part of the post-war problems between France and Germany. During
WWI and until the end of 1918, the Central Bureau and the Imperial Main Station in Strasbourg continued working as usual,
45
to the extent possible, but as documented in its published Newsletters (ISA, 1913-1917) and the last activity report of Hecker
(see Kövesligethy, 1922) with reduced staff. On 23 December 1918, all German employees of the Central Office in Strasbourg
had to leave the Central Office, and were deported out of France on 6 January 1919, without any possibility of carrying their
personal belongings still located in the Bureau with them (Hecker, 1924; Sapper, 1940). The Central Office and the Imperial WWI and until the end of 1918, the Central Bureau and the Imperial Main Station in Strasbourg continued working as usual,
45
to the extent possible, but as documented in its published Newsletters (ISA, 1913-1917) and the last activity report of Hecker
(see Kövesligethy, 1922) with reduced staff. On 23 December 1918, all German employees of the Central Office in Strasbourg
had to leave the Central Office, and were deported out of France on 6 January 1919, without any possibility of carrying their
personal belongings still located in the Bureau with them (Hecker, 1924; Sapper, 1940). The Central Office and the Imperial Main Station, most of the instrumentation, the scientific material and the libraries of both the Central Office and the Imperial
50
Main Station became inaccessible for German seismologists; unfortunately, much of this material got lost for ever. Already shortly before the end of WWI, in October 1918, the Royal Society in London had invited to an ‘Inter-Allied
Conference on International Scientific Organizations’ with participants from eight countries, who excluded in a quite emotional
resolution any future cooperation in international research organisations with colleagues from the Central Powers (Germany, Main Station, most of the instrumentation, the scientific material and the libraries of both the Central Office and the Imperial
50
Main Station became inaccessible for German seismologists; unfortunately, much of this material got lost for ever. 3 ISA Becomes a Victim of WWI Already shortly before the end of WWI, in October 1918, the Royal Society in London had invited to an ‘Inter-Allied
Conference on International Scientific Organizations’ with participants from eight countries, who excluded in a quite emotional
resolution any future cooperation in international research organisations with colleagues from the Central Powers (Germany, Austria-Hungary, Bulgaria and the Ottoman Empire) (Nature, 1918). This statement was the result of strong national feelings
55
after four years of war in Europe with never-before-seen cruelties and loss of human lives. However, the statement had far-
reaching consequences, because this exclusion of the Central Powers became part of the statutes of the Conseil International
des Recherches (International Research Council, IRC), when it was newly founded together with its Unions (as e.g., the herein
relevant International Union of Geodesy and Geophysics (IUGG)) in Brussels, Belgium, in July 1919. Even states, which had Austria-Hungary, Bulgaria and the Ottoman Empire) (Nature, 1918). This statement was the result of strong national feelings
55
after four years of war in Europe with never-before-seen cruelties and loss of human lives. However, the statement had far-
reaching consequences, because this exclusion of the Central Powers became part of the statutes of the Conseil International
des Recherches (International Research Council, IRC), when it was newly founded together with its Unions (as e.g., the herein
relevant International Union of Geodesy and Geophysics (IUGG)) in Brussels, Belgium, in July 1919. Even states, which had 2 History of
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c⃝Author(s) 2022. CC BY 4.0 License. been neutral during WWI, could only join the Council with support by a 2/3 majority of the members. With this construction,
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the IRC was, as Cock (1983) wrote, ‘quite openly part of the general post-war policy’. been neutral during WWI, could only join the Council with support by a 2/3 majority of the members. With this construction,
60
the IRC was, as Cock (1983) wrote, ‘quite openly part of the general post-war policy’. been neutral during WWI, could only join the Council with support by a 2/3 majority of the members. With this construction,
60
the IRC was, as Cock (1983) wrote, ‘quite openly part of the general post-war policy’. 4 The Foundation of the Deutsche Seismologische Gesellschaft (DSG) in 1922 The first General Assembly of IUGG was planned in Rome for May 2022. But there was still an unsolved problem with ISA. The original convention from 1904 had been signed for 12 years. The last ISA meeting before WWI had been in Manchester, The first General Assembly of IUGG was planned in Rome for May 2022. But there was still an unsolved problem with ISA. The original convention from 1904 had been signed for 12 years. The last ISA meeting before WWI had been in Manchester,
UK, in 1911. The next meeting was planned for St. Petersburg, Russia, in 1914, but this meeting had to be cancelled because
65
of the outbreak of the war. In 1916, when the contract period of the ISA convention officially ended, ISA member states were
fighting on both sides of the WWI fronts and a new convention or a renewal of the expiring one was not possible. Also, because
of the London 1918 resolution, there was no general interest to revitalize the ISA after WWI. To formalize the end of the
interstate convention, a joint 3rd General Assembly and 5th Conference of the Permanent Commission of ISA was organized
in Strasbourg, France, on 24-25 April 1922. At the end of this final meeting, the ISA was dissolved (Kövesligethy, 1922). 70
Then, the delegates journeyed on to meet again for the 1st General Assembly of the IUGG in Rome, one week later, on 2-10 UK, in 1911. The next meeting was planned for St. Petersburg, Russia, in 1914, but this meeting had to be cancelled because
65
of the outbreak of the war. In 1916, when the contract period of the ISA convention officially ended, ISA member states were
fighting on both sides of the WWI fronts and a new convention or a renewal of the expiring one was not possible. Also, because
of the London 1918 resolution, there was no general interest to revitalize the ISA after WWI. To formalize the end of the
interstate convention, a joint 3rd General Assembly and 5th Conference of the Permanent Commission of ISA was organized UK, in 1911. The next meeting was planned for St. Petersburg, Russia, in 1914, but this meeting had to be cancelled because
65
of the outbreak of the war. 4 The Foundation of the Deutsche Seismologische Gesellschaft (DSG) in 1922 In 1916, when the contract period of the ISA convention officially ended, ISA member states were
fighting on both sides of the WWI fronts and a new convention or a renewal of the expiring one was not possible. Also, because
of the London 1918 resolution, there was no general interest to revitalize the ISA after WWI. To formalize the end of the
interstate convention, a joint 3rd General Assembly and 5th Conference of the Permanent Commission of ISA was organized in Strasbourg, France, on 24-25 April 1922. At the end of this final meeting, the ISA was dissolved (Kövesligethy, 1922). 70
Then, the delegates journeyed on to meet again for the 1st General Assembly of the IUGG in Rome, one week later, on 2-10
May 1922. There, during this Assembly, the new Seismology Section of IUGG was established (Rothé, 1922). However, based
on the statutes of the IRC, IUGG membership was based on state memberships and the Central Powers were explicitly excluded
from joining IRC as members and no German seismologists were allowed to participate in the Assembly. in Strasbourg, France, on 24-25 April 1922. At the end of this final meeting, the ISA was dissolved (Kövesligethy, 1922). 70
Then, the delegates journeyed on to meet again for the 1st General Assembly of the IUGG in Rome, one week later, on 2-10
May 1922. There, during this Assembly, the new Seismology Section of IUGG was established (Rothé, 1922). However, based
on the statutes of the IRC, IUGG membership was based on state memberships and the Central Powers were explicitly excluded
from joining IRC as members and no German seismologists were allowed to participate in the Assembly. In summer 1922, the German seismologists were internationally isolated and needed a forum for meetings and discussions. 75
Therefore, Emil Wiechert (1861-1928, see e.g., Mulligan, 2001; Kertz, 2002; Schweitzer & Ritter, 2012; Jacobs & Neunhöfer
(2013); Jacobs & Börngen, 2019) and Oskar Hecker invited German seismologists to a special meeting for the afternoon of 19
September 1922, during the yearly conference of the Deutsche Naturforscher und Ärzte (German natural scientists and
physicians), in Leipzig (Wiener & Bauschinger, 1923; Kohlschütter, 1938). During this meeting, Hecker proposed the In summer 1922, the German seismologists were internationally isolated and needed a forum for meetings and discussions. 4 The Foundation of the Deutsche Seismologische Gesellschaft (DSG) in 1922 75
Therefore, Emil Wiechert (1861-1928, see e.g., Mulligan, 2001; Kertz, 2002; Schweitzer & Ritter, 2012; Jacobs & Neunhöfer
(2013); Jacobs & Börngen, 2019) and Oskar Hecker invited German seismologists to a special meeting for the afternoon of 19
September 1922, during the yearly conference of the Deutsche Naturforscher und Ärzte (German natural scientists and
physicians), in Leipzig (Wiener & Bauschinger, 1923; Kohlschütter, 1938). During this meeting, Hecker proposed the In summer 1922, the German seismologists were internationally isolated and needed a forum for meetings and discussions. 75
Therefore, Emil Wiechert (1861-1928, see e.g., Mulligan, 2001; Kertz, 2002; Schweitzer & Ritter, 2012; Jacobs & Neunhöfer
(2013); Jacobs & Börngen, 2019) and Oskar Hecker invited German seismologists to a special meeting for the afternoon of 19
September 1922, during the yearly conference of the Deutsche Naturforscher und Ärzte (German natural scientists and
physicians), in Leipzig (Wiener & Bauschinger, 1923; Kohlschütter, 1938). During this meeting, Hecker proposed the foundation of the Deutsche Seismologische Gesellschaft (DSG) as new home for the German seismologists and in parallel a
80
counterweight to the German hostile institutions founded by the IUGG (Hecker, 1922: ‘zur gleichen Zeit ein Gegengewicht
gegen die von der deutschfeindlichen Union géodesique et géophysique begründeten Landesinstitute‘) and presented some
drafted statutes. The 24 seismologists present spontaneously endorsed the idea and on 21 September they founded this society
by adopting its statutes (Jacobs & Börngen, 2019). foundation of the Deutsche Seismologische Gesellschaft (DSG) as new home for the German seismologists and in parallel a
80
counterweight to the German hostile institutions founded by the IUGG (Hecker, 1922: ‘zur gleichen Zeit ein Gegengewicht
gegen die von der deutschfeindlichen Union géodesique et géophysique begründeten Landesinstitute‘) and presented some
drafted statutes. The 24 seismologists present spontaneously endorsed the idea and on 21 September they founded this society
by adopting its statutes (Jacobs & Börngen, 2019). There were of course many more people in Germany interested in seismology than the 24 founding members present in Leipzig. 85
Hecker, just elected Geschäftsführende Vorsitzende (acting Chair = Secretary) of the DSG, informed by letter colleagues not
present in Leipzig about the new society and asked for their support by becoming DSG members (Hecker, 1922). Hecker’s
letters also contained a copy of § 2 of the DSG statutes with the objectives of the society (here translated from German; see
also Fig. 5 The Development During the Following years
100 5 The Development During the Following years
100
For 4-5 October 1923, Hecker had invited to the first meeting of the new society in Jena, where he had managed the foundation
of the new Reichsanstalt für Erdbebenforschung (Imperial Institute for Earthquake Research) as successor to the former
Imperial Main Station in Strasbourg (Hecker, 1924) and which had just officially started its work (1 October 1923). The
exclusion of the German colleagues from all IUGG activities was of course also true for the other geophysical disciplines and For 4-5 October 1923, Hecker had invited to the first meeting of the new society in Jena, where he had managed the foundation
of the new Reichsanstalt für Erdbebenforschung (Imperial Institute for Earthquake Research) as successor to the former
Imperial Main Station in Strasbourg (Hecker, 1924) and which had just officially started its work (1 October 1923). The
exclusion of the German colleagues from all IUGG activities was of course also true for the other geophysical disciplines and following a proposal by Carl Mainka (1873-1943; Anonymous, 1944; Kertz, 2002) the DSG widened its focus by changing
105
the statutes and renaming the society into Deutsche Geophysikalische Gesellschaft (DGG) during the 2nd DSG assembly in
Innsbruck, Austria (Wiechert, 1925; Jacobs & Neunhöfer (2013). During the same meeting, the DSG also decided to start the
publication of an own scientific journal for its members, the Zeitschrift für Geophysik (ZfG, Journal of Geophysics). Both
changes resulted in a jump of DGG memberships after 1924 (Table 1). following a proposal by Carl Mainka (1873-1943; Anonymous, 1944; Kertz, 2002) the DSG widened its focus by changing
105
the statutes and renaming the society into Deutsche Geophysikalische Gesellschaft (DGG) during the 2nd DSG assembly in
Innsbruck, Austria (Wiechert, 1925; Jacobs & Neunhöfer (2013). During the same meeting, the DSG also decided to start the
publication of an own scientific journal for its members, the Zeitschrift für Geophysik (ZfG, Journal of Geophysics). Both
changes resulted in a jump of DGG memberships after 1924 (Table 1). following a proposal by Carl Mainka (1873-1943; Anonymous, 1944; Kertz, 2002) the DSG widened its focus by changing
105
the statutes and renaming the society into Deutsche Geophysikalische Gesellschaft (DGG) during the 2nd DSG assembly in
Innsbruck, Austria (Wiechert, 1925; Jacobs & Neunhöfer (2013). 5 The Development During the Following years
100 During the same meeting, the DSG also decided to start the
publication of an own scientific journal for its members, the Zeitschrift für Geophysik (ZfG, Journal of Geophysics). Both
changes resulted in a jump of DGG memberships after 1924 (Table 1). In parallel with the negations about a German membership in the League of Nations in Geneva, the general scientific isolation
110
of Germany, Austria, Hungary and Bulgaria became more and more questioned by former neutral states, but also by many
colleagues from the Allied Nations. Common scientific interests between single German scientists became more important
than ‘old political’ problems and German researchers were addressed with invitations for memberships in commissions of the
different Unions and it looks like that in particular the IUGG was not belonging to the strong supporters of a German exclusion. In parallel with the negations about a German membership in the League of Nations in Geneva, the general scientific isolation
110
of Germany, Austria, Hungary and Bulgaria became more and more questioned by former neutral states, but also by many
colleagues from the Allied Nations. Common scientific interests between single German scientists became more important
than ‘old political’ problems and German researchers were addressed with invitations for memberships in commissions of the
different Unions and it looks like that in particular the IUGG was not belonging to the strong supporters of a German exclusion. In parallel with the negations about a German membership in the League of Nations in Geneva, the general scientific isolation
110
of Germany, Austria, Hungary and Bulgaria became more and more questioned by former neutral states, but also by many
colleagues from the Allied Nations. Common scientific interests between single German scientists became more important
than ‘old political’ problems and German researchers were addressed with invitations for memberships in commissions of the
different Unions and it looks like that in particular the IUGG was not belonging to the strong supporters of a German exclusion. This impression transpires from a 19 pages long report by Ernst Kohlschütter (1870-1942; Maurer, 1942) for the German
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Reichsministerium des Inneren (Imperial Ministry of the Interior), in March 1927, about his international contacts and
experiences from participating in international conferences (Kohlschütter, 1927). Also, the increasing number of DGG
members with foreign addresses show an improvement in international relations (Table 1). 4 The Foundation of the Deutsche Seismologische Gesellschaft (DSG) in 1922 History of
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Open Access History of
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Open Access 95 Promoting all other seismological research and education related interests.” The news about the DSG foundation must have been distributed quite fast and a list of DSG members from 15 December 1922
contained already 46 members (Table 1) of which 7 were registered with addresses outside of Germany (Austria, Canada,
Italy, Spain, Switzerland). During the following years, the membership list continued to slowly grow (Table 1). 4 The Foundation of the Deutsche Seismologische Gesellschaft (DSG) in 1922 1): There were of course many more people in Germany interested in seismology than the 24 founding members present in Leipzig. 85
Hecker, just elected Geschäftsführende Vorsitzende (acting Chair = Secretary) of the DSG, informed by letter colleagues not
present in Leipzig about the new society and asked for their support by becoming DSG members (Hecker, 1922). Hecker’s
letters also contained a copy of § 2 of the DSG statutes with the objectives of the society (here translated from German; see
also Fig. 1): There were of course many more people in Germany interested in seismology than the 24 founding members present in Leipzig. 85
Hecker, just elected Geschäftsführende Vorsitzende (acting Chair = Secretary) of the DSG, informed by letter colleagues not
present in Leipzig about the new society and asked for their support by becoming DSG members (Hecker, 1922). Hecker’s
letters also contained a copy of § 2 of the DSG statutes with the objectives of the society (here translated from German; see
also Fig. 1): “The German Seismological Society intends fostering theoretical and applied seismology and related topics. It tries to achieve
90
this by “The German Seismological Society intends fostering theoretical and applied seismology and related topics. It tries to achieve
90
this by “The German Seismological Society intends fostering theoretical and applied seismology and related topics. It tries to achieve
90
this by 3 3 https://doi.org/10.5194/hgss-2022-12
History of
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c⃝Author(s) 2022. CC BY 4.0 License. 1. Supporting the scientific exchange of ideas between its members,
2. Supporting the scientific relations to related corporations,
3. If applicable, editing scientific publications or participating in already exiting publication projects,
4. Organizing scientific meetings,
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5. Promoting all other seismological research and education related interests.”
The news about the DSG foundation must have been distributed quite fast and a list of DSG members from 15 December 1922
contained already 46 members (Table 1) of which 7 were registered with addresses outside of Germany (Austria, Canada,
Italy, Spain, Switzerland). During the following years, the membership list continued to slowly grow (Table 1). https://doi.org/10.5194/hgss-2022-12
History of
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c⃝Author(s) 2022. CC BY 4.0 License. 5 The Development During the Following years
100 At the end of the 1920s, the DGG
had become a quite international society, with more than 30 % of its membership residing outside of Germany (Table 1), This impression transpires from a 19 pages long report by Ernst Kohlschütter (1870-1942; Maurer, 1942) for the German
115
Reichsministerium des Inneren (Imperial Ministry of the Interior), in March 1927, about his international contacts and
experiences from participating in international conferences (Kohlschütter, 1927). Also, the increasing number of DGG
members with foreign addresses show an improvement in international relations (Table 1). At the end of the 1920s, the DGG
had become a quite international society, with more than 30 % of its membership residing outside of Germany (Table 1), contrary to the year 2022, when only 145 (12 %) of the 1202 members had an address outside of Germany. Even foreign
120
colleagues are documented to have participated the yearly DGG assemblies as e.g., by the entries in the guest book of the
Taunus Observatory near Frankfurt am Main (Fig. 2). 4 History of
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Open Access https://doi.org/10.5194/hgss-2022-12
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c⃝Author(s) 2022. CC BY 4.0 License. However, due to the construction of the statutes, German scientists could officially participate in Unions of the IRC only after
Germany becomes an IRC member. Even an individual participation in IUGG bodies as guests was not supported by the
German government and the DGG, because the German government was focusing on solving the general problem of a German
25
IRC membership for all adherent Unions together to conditions, which were politically acceptable for Germany. Such
conditions were formulated by the DGG and adopted in a resolution during its 3rd assembly in Göttingen, in 1925. This
resolution was then not only published in the assembly minutes (DGG, 1926), but also sent in March 1926 by Hecker, the However, due to the construction of the statutes, German scientists could officially participate in Unions of the IRC only after
Germany becomes an IRC member. Even an individual participation in IUGG bodies as guests was not supported by the German government and the DGG, because the German government was focusing on solving the general problem of a German
125
IRC membership for all adherent Unions together to conditions, which were politically acceptable for Germany. 5 The Development During the Following years
100 But Gutenberg wrote also that he was willing to enter in private correspondence with the
Commission members and that he would like to be informed about the work of the Commission since, somewhen, Germany
would anyhow become a Union member (“… bin ich gerne zu persönlicher Korrespondenz mit deren Mitgliedern bereit und initiated a longer correspondence between Gutenberg and the DGG Vorstand (Executive Board) with the result that Gutenberg
135
followed the wish of the DGG Executive Board and kindly refused the invitation to become a Commission member in a letter
to Rothé from 23 April 1929. But Gutenberg wrote also that he was willing to enter in private correspondence with the
Commission members and that he would like to be informed about the work of the Commission since, somewhen, Germany
would anyhow become a Union member (“… bin ich gerne zu persönlicher Korrespondenz mit deren Mitgliedern bereit und würde mich freuen, wenn ich über die Arbeit der Kommission auf dem Laufenden gehalten würde, da doch einmal Deutschland
140
in die Union eintreten wird, …”). From the same year (1929), the Gutenberg collection also contains a longer correspondence
between Gutenberg, Kohlschütter, Walter Davis Lambert (1879-1968; Whitten, 1973), who was influential in the Geodesy
Section of the IUGG and the Geodesy Section of the American Geophysical Union, and the Secretary General of IUGG and
of the IRC Henry George Lyons (1864-1944; Dale, 1944) with discussions on how to solve the persisting problem of a German
membership. 145 würde mich freuen, wenn ich über die Arbeit der Kommission auf dem Laufenden gehalten würde, da doch einmal Deutschland
140
in die Union eintreten wird, …”). From the same year (1929), the Gutenberg collection also contains a longer correspondence
between Gutenberg, Kohlschütter, Walter Davis Lambert (1879-1968; Whitten, 1973), who was influential in the Geodesy
Section of the IUGG and the Geodesy Section of the American Geophysical Union, and the Secretary General of IUGG and
of the IRC Henry George Lyons (1864-1944; Dale, 1944) with discussions on how to solve the persisting problem of a German
membership. 145 5 The Development During the Following years
100 Such
conditions were formulated by the DGG and adopted in a resolution during its 3rd assembly in Göttingen, in 1925. This
resolution was then not only published in the assembly minutes (DGG, 1926), but also sent in March 1926 by Hecker, the
DGG Vorsitzender (Chair), to (at least) the German Reichskanzler (Imperial Chancellor) and the Imperial Ministry of the German government and the DGG, because the German government was focusing on solving the general problem of a German
125
IRC membership for all adherent Unions together to conditions, which were politically acceptable for Germany. Such
conditions were formulated by the DGG and adopted in a resolution during its 3rd assembly in Göttingen, in 1925. This
resolution was then not only published in the assembly minutes (DGG, 1926), but also sent in March 1926 by Hecker, the
DGG Vorsitzender (Chair), to (at least) the German Reichskanzler (Imperial Chancellor) and the Imperial Ministry of the Interior (Hecker, 1926). 130
From the correspondence of Beno Gutenberg (1889-1960; Schweitzer, 1989; Knopoff, 1998; 1999; Kertz, 2002; Jacobs &
Börngen, 2019), as still stored in the Gutenberg Collection of the Caltech Archive, it is known that this problem was still
unsolved in 1929. At the beginning of 1929, Gutenberg had been invited by Edmond Rothé (1873-1942; Perrier, 1942;
Anonymous 1948) to become a member in one of the Commissions of the Seismological Section of the IUGG. This invitation Interior (Hecker, 1926). 130
From the correspondence of Beno Gutenberg (1889-1960; Schweitzer, 1989; Knopoff, 1998; 1999; Kertz, 2002; Jacobs &
Börngen, 2019), as still stored in the Gutenberg Collection of the Caltech Archive, it is known that this problem was still
unsolved in 1929. At the beginning of 1929, Gutenberg had been invited by Edmond Rothé (1873-1942; Perrier, 1942;
Anonymous 1948) to become a member in one of the Commissions of the Seismological Section of the IUGG. This invitation initiated a longer correspondence between Gutenberg and the DGG Vorstand (Executive Board) with the result that Gutenberg
135
followed the wish of the DGG Executive Board and kindly refused the invitation to become a Commission member in a letter
to Rothé from 23 April 1929. 6 Germany becomes IUGG Member This blocked situation continued until 1930, when for the first time German researchers participated in the IUGG General
Assembly in Stockholm, Sweden, as guests and the DGG Executive Board decided to prepare together with other disciplines
a German IUGG membership. Also, the IRC had moved and already asked the Central Power states to become IRC members. In 1931, after long discussions within the IRC (Cock, 1983), the IRC adopted a major change of its statutes, which resulted in
0
a larger independence of the Union with own rights to manage their memberships and a name change to International Council
of Scientific Unions (ICSU), which existed in this form until 2018. This blocked situation continued until 1930, when for the first time German researchers participated in the IUGG General
Assembly in Stockholm, Sweden, as guests and the DGG Executive Board decided to prepare together with other disciplines
a German IUGG membership. Also, the IRC had moved and already asked the Central Power states to become IRC members. In 1931, after long discussions within the IRC (Cock, 1983), the IRC adopted a major change of its statutes, which resulted in
0
a larger independence of the Union with own rights to manage their memberships and a name change to International Council
of Scientific Unions (ICSU), which existed in this form until 2018. 150 Reports about the ongoing negotiations regarding the German IUGG membership were on the agenda of the DGG membership
meetings during the following years. The major issue to be solved was an internal one: the German government had to find a Reports about the ongoing negotiations regarding the German IUGG membership were on the agenda of the DGG membership
meetings during the following years. The major issue to be solved was an internal one: the German government had to find a 5 History of
Geo- and Space
Sciences
Discussions
Open Access https://doi.org/10.5194/hgss-2022-12
Preprint. Discussion started: 18 November 2022
c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/hgss-2022-12
Preprint. Discussion started: 18 November 2022
c⃝Author(s) 2022. CC BY 4.0 License. way to finance the yearly IUGG membership fee and the travel costs of the delegates to the IUGG General Assemblies. 155
Therefore, in the following IUGG Assemblies in Lisbon, Portugal (1933) and Edinburgh, UK (1936), German scientists still
participated as guests but became more and more involved in IUGG activities. 6 Germany becomes IUGG Member A visible sign of this is e.g., the
165
foundation of the Federation of European Seismologists in 1950, which as European Seismological Commission (ESC) was
in 1951 incorporated into the now named International Association of Seismology and Physics of the Earth's Interior (IASPEI)
and subsequently of the IUGG: the first ESC President (1951-1956) became the German Wilhelm Hiller (1899-1980;
Schneider, 1980). 7 Outlook
170 However, what became already visible in the 1920s, was confirmed during the further development of geophysics: a self-
chosen or forced long-term isolation of individual states from the international discourse leads to worse science for everyone
involved New scientific findings in geophysics depend on the (free) exchange of global observations and data That is why it
180 However, what became already visible in the 1920s, was confirmed during the further development of geophysics: a self-
chosen or forced long-term isolation of individual states from the international discourse leads to worse science for everyone However, what became already visible in the 1920s, was confirmed during the further development of geophysics: a self-
chosen or forced long-term isolation of individual states from the international discourse leads to worse science for everyone
involved. New scientific findings in geophysics depend on the (free) exchange of global observations and data. That is why it
180
is important to maintain cross border contacts and not to let them break off even when there are political differences between However, what became already visible in the 1920s, was confirmed during the further development of geophysics: a self-
chosen or forced long-term isolation of individual states from the international discourse leads to worse science for everyone
involved. New scientific findings in geophysics depend on the (free) exchange of global observations and data. That is why it
180
is important to maintain cross-border contacts and not to let them break off even when there are political differences between
the states. The bridge-building function of scientific exchange in conflict situations has often been documented. The IUGG
with its associations and the DGG have also repeatedly contributed to this over the past 100 years. involved. New scientific findings in geophysics depend on the (free) exchange of global observations and data. That is why it
180
is important to maintain cross-border contacts and not to let them break off even when there are political differences between
the states. The bridge-building function of scientific exchange in conflict situations has often been documented. The IUGG
with its associations and the DGG have also repeatedly contributed to this over the past 100 years. 180 involved. New scientific findings in geophysics depend on the (free) exchange of global observations and data. 6 Germany becomes IUGG Member The long-standing DGG Chair (1927-1929,
1930-1936) Kohlschütter was directly involved in the discussions and was installed by the German government as Chair of
the Deutsche Vereinigung für Geodäsie und Geophysik (German Union for Geodesy and Geophysics). Finally, on 6 February way to finance the yearly IUGG membership fee and the travel costs of the delegates to the IUGG General Assemblies. 155
Therefore, in the following IUGG Assemblies in Lisbon, Portugal (1933) and Edinburgh, UK (1936), German scientists still
participated as guests but became more and more involved in IUGG activities. The long-standing DGG Chair (1927-1929,
1930-1936) Kohlschütter was directly involved in the discussions and was installed by the German government as Chair of
the Deutsche Vereinigung für Geodäsie und Geophysik (German Union for Geodesy and Geophysics). Finally, on 6 February 1937, Kohlschütter could inform the IUGG that Germany is joining the Union (Kohlschütter, 1937). Then, during the following
160
IUGG General Assembly in Washington D.C., in September 1939, the few German scientists, who made it to the conference
despite the start of a new war in Europe, could be specially welcomed as members and no longer guests of the International
Association of Seismology (Naumann & Rothé, 1940, p. 226). The reorganization of the international scientific landscape after World War II (WWII) was then no longer characterized by 1937, Kohlschütter could inform the IUGG that Germany is joining the Union (Kohlschütter, 1937). Then, during the following
160
IUGG General Assembly in Washington D.C., in September 1939, the few German scientists, who made it to the conference
despite the start of a new war in Europe, could be specially welcomed as members and no longer guests of the International
Association of Seismology (Naumann & Rothé, 1940, p. 226). The reorganization of the international scientific landscape after World War II (WWII) was then no longer characterized by new exclusion rules for individual countries, but by the will to cooperate and communicate. A visible sign of this is e.g., the
165
foundation of the Federation of European Seismologists in 1950, which as European Seismological Commission (ESC) was
in 1951 incorporated into the now named International Association of Seismology and Physics of the Earth's Interior (IASPEI)
and subsequently of the IUGG: the first ESC President (1951-1956) became the German Wilhelm Hiller (1899-1980;
Schneider, 1980). new exclusion rules for individual countries, but by the will to cooperate and communicate. 7 Outlook
170 Founded in the scientific isolation of Germany in the post-war years of WWI, the DGG has developed into a quite agile
centenarian with around 1200 members. I am sure that the 24 founding fathers did not dream of such a successful development
when the society was founded in 1922. The initial reasons for a separate national society described here have disappeared over
time. What has remained, however, is the desire to have a formal framework for the coordination of joint scientific activities Founded in the scientific isolation of Germany in the post-war years of WWI, the DGG has developed into a quite agile
centenarian with around 1200 members. I am sure that the 24 founding fathers did not dream of such a successful development
when the society was founded in 1922. The initial reasons for a separate national society described here have disappeared over
time. What has remained, however, is the desire to have a formal framework for the coordination of joint scientific activities Founded in the scientific isolation of Germany in the post-war years of WWI, the DGG has developed into a quite agile
centenarian with around 1200 members. I am sure that the 24 founding fathers did not dream of such a successful development
when the society was founded in 1922. The initial reasons for a separate national society described here have disappeared over
time. What has remained, however, is the desire to have a formal framework for the coordination of joint scientific activities and for the exchange of scientific results. For many German early career scientists, a first oral or poster presentation during
175
one of the yearly DGG assemblies was often the beginning of a long-lasting career in geophysics and, last but not least, the
DGG offers the possibility of regular meetings between colleagues and friends. However, what became already visible in the 1920s, was confirmed during the further development of geophysics: a self- and for the exchange of scientific results. For many German early career scientists, a first oral or poster presentation during
175
one of the yearly DGG assemblies was often the beginning of a long-lasting career in geophysics and, last but not least, the
DGG offers the possibility of regular meetings between colleagues and friends. Acknowledgements I have collected material for this article over the last few decades. Many friends and colleagues have helped me with this, and
185
I thank them all. Special thanks go to Jo Wassermann for the copies of letters from C. W. Lutz's files, to Alexander Rudloff,
Ellen Gottschämmer, Franz Jacobs and Michael Lindenfeld for their help in finding old DGG membership lists, to Kasper
Fischer for the most recent DGG membership numbers and to Myrto Pirli for a critical review of the text. I have collected material for this article over the last few decades. Many friends and colleagues have helped me with this, and
185
I thank them all. Special thanks go to Jo Wassermann for the copies of letters from C. W. Lutz's files, to Alexander Rudloff,
Ellen Gottschämmer, Franz Jacobs and Michael Lindenfeld for their help in finding old DGG membership lists, to Kasper
Fischer for the most recent DGG membership numbers and to Myrto Pirli for a critical review of the text. 7 Outlook
170 That is why it
180
is important to maintain cross-border contacts and not to let them break off even when there are political differences between
the states. The bridge-building function of scientific exchange in conflict situations has often been documented. The IUGG
with its associations and the DGG have also repeatedly contributed to this over the past 100 years. 6 History of
Geo- and Space
Sciences
Discussions
Open Access https://doi.org/10.5194/hgss-2022-12
Preprint. Discussion started: 18 November 2022
c⃝Author(s) 2022. CC BY 4.0 License. References Kohlschütter, E. (1927). Stimmungsbericht hinsichtlich des Beitritts Deutschlands zum Internationalen Forschungsrate
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Academy Press, Washington D.C. 1998, 35 pp. Knopoff, L. (1999). Beno Gutenberg-June 4, 1889-January 25, 1960. DGG Mitteilungen, 4/1999, Knopoff, L. (1999). Beno Gutenberg-June 4, 1889-January 25, 1960. DGG Mitteilungen, 4/1999, 2-15. References 240 Nature, editorial office (1918). Inter-allied conference on international scientific organisations. Nature, 102, 133. 240
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Comptes Rendue de Séances de la Septième Conférence, Réunie a Washington D. C., du 4 au 15 Septembre 1939, rédigés par
le Secrétaire de la Conférence F. Neumann, Publiés dans les deux langues par le Secrétaire Général E. Rothé. Librairie Édouard
Privat, Toulouse, 359 pp., see also http://download.iaspei.org/meetings/1930-1939/IUGG1939.pdf . Perrier, G. (1942). Edmond Rothé. Bull. Géodésique, 69, 64-66, doi: 10.1007/BF03031303. 245
Rebeur-Paschwitz, E. v. (1895a). Horizontalpendel-Beobachtungen auf der Kaiserlichen Universitäts-Sternwarte zu Strassburg
1892-1894. Beiträge zur Geophysik, 2, 211-536. Rebeur-Paschwitz, E. v. (1895a). Horizontalpendel-Beobachtungen auf der Kaiserlichen Universitäts-Sternwarte zu Strassburg
1892-1894. Beiträge zur Geophysik, 2, 211-536. 8 History of
Geo- and Space
Sciences
Discussions
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c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/hgss-2022-12
Preprint. Discussion started: 18 November 2022
c⃝Author(s) 2022. CC BY 4.0 License. Rebeur-Paschwitz, E. v. (1895b). Vorschläge zur Errichtung eines internationalen Systems von Erdbeben-Stationen. Beiträge
zur Geophysik, 2, 773-782. Rebeur-Paschwitz, E. v. (1895b). Vorschläge zur Errichtung eines internationalen Systems von Erdbeben-Stationen. Beiträge
zur Geophysik, 2, 773-782. Rothé, E. (1922). Union Géodésique et Géophysique Internationale, Section de Sismologie, Comptes Rendue de Séances de
250
la Première Conférence, Réunie a Rome, du 2 au 10 mai 1922, rédigés par le Secrétaire E. Rothé. Librairie Édouard Privat,
Toulouse, 94 pp., siehe auch http://download.iaspei.org/meetings/1922-1929/IUGG1922.pdf. Sapper, K. (1940). Georg Gerland (1833-1919) / Professor der Geographie. Lebensbilder aus Hessen und Waldeck, 2, 150-
162. Toulouse, 94 pp., siehe auch http://download.iaspei.org/meetings/1922-1929/IUGG1922.pdf. Sapper, K. (1940). Georg Gerland (1833-1919) / Professor der Geographie. Lebensbilder aus Hessen und Waldeck, 2, 150-
162. Sapper, K. (1940). Georg Gerland (1833-1919) / Professor der Geographie. Lebensbilder aus Hessen und Waldeck, 2, 150-
162. Schmidt, A. (1927). Tagung und Hauptversammlung der Deutschen Geophysikalischen Gesellschaft. Zeitschrift für
255
Geophysik, 3, 316. Schmidt, A. (1927). Tagung und Hauptversammlung der Deutschen Geophysikalischen Gesellschaft. Zeitschrift für
255
Geophysik, 3, 316. Schneider, G. (1980). Wilhelm Hiller. Jahresheft der Gesellschaft für Naturkunde in Württemberg, 135, 279-284. Schweitzer, J. & J. R. R. Ritter (2012). Emil Wiechert (1861-1928). DGG Mitteilungen, 1/2012, 27-31, 2012. Schweitzer, J. & T. Lay (2019). IASPEI: its origins and the promotion of global seismology. History of Geo- and Space Schneider, G. (1980). Wilhelm Hiller. Jahresheft der Gesellschaft für Naturkunde in Württemberg, 135, 279-284. Schweitzer, J. & J. R. Wiener, O. & J. Bauschinger (1923). Geophysik. Verhandlungen der Gesellschaft deutscher Naturforscher und Ärzte, 87.
Versammlung zu Leipzig vom 17.-24. September 1922, 351. References R. Ritter (2012). Emil Wiechert (1861-1928). DGG Mitteilungen, 1/2012, 2 Schweitzer, J. & T. Lay (2019). IASPEI: its origins and the promotion of global seismology. Schweitzer, J. & T. Lay (2019). IASPEI: its origins and the promotion of global seismology. History of Geo- and Space
Sciences, Spec. Volume 10, 173-180, doi: 10.5194/hgss-10-173-2019. 260 Sciences, Spec. Volume 10, 173-180, doi: 10.5194/hgss-10-173-2019. 260
Schweitzer, J. (1989). Beno Gutenberg (1889-1960). DGG Mitteilungen 3/1989, 7-10. Schweitzer, J. (2003). Early German contributions to modern seismology, Chapter 79.24 Germany, Part A, in: Lee, W. H. K.,
H. Kanamori, P. C. Jennings, and C. Kisslinger (eds.) (2003). IASPEI International Handbook of Earthquake and Engineering
Seismology, Part B, Academic Press, ISBN 0-12-440658-0, Handbook CD #2 (CD ISBN 0-12-440654-8) file J. (1989). Beno Gutenberg (1889-1960). DGG Mitteilungen 3/1989, 7-10. /79_24germany/ch79_24parta.pdf, 58 pp. 265
Schweitzer J. (2022). Die Gründung der Deutschen Geophysikalischen Gesellschaft im Rahmen der internationalen
Beziehungen zu Beginn des zwanzigsten Jahrhunderts. In: DGG (ed.) Geophysik im Wandel, 2022, https://dgg-
online.de/WordPress_01/wp-content/uploads/2022/08/DGG100_Schweitzer_v4.pdf
Whitten, C. A. (1973). Walter Davis Lambert 1879-1968. Biographical Memoirs, National Academy of Sciences. National Academy Press, Washington D.C. 1973, 146-162. 270
Wiechert, E. (1925). Mitteilungen. Zeitschrift für Geophysik, 1, 217. Wiener, O. & J. Bauschinger (1923). Geophysik. Verhandlungen der Gesellschaft deutscher Naturforscher und Ärzte, 87. Versammlung zu Leipzig vom 17.-24. September 1922, 351. 9 9 Figure 1: Facsimile of the attachment to Hecker’s letter (Hecker, 1922) containing § 2 of the DSG statutes, written in old German
5
handwriting, as adopted on 22 September 1922. https://doi.org/10.5194/hgss-2022-12
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c⃝Author(s) 2022. CC BY 4.0 License. History of
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Open Access History of
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Open Access Figure 1: Facsimile of the attachment to Hecker’s letter (Hecker, 1922) containing § 2 of the DSG statutes, written in old German
275
handwriting, as adopted on 22 September 1922. Figure 1: Facsimile of the attachment to Hecker’s letter (Hecker, 1922) containing § 2 of the DSG statutes, written in old German
275
handwriting, as adopted on 22 September 1922. 10 https://doi.org/10.5194/hgss-2022-12
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c⃝Author(s) 2022. CC BY 4.0 License. Figure 2: As an example of the international contacts of the DGG, a page from the guest book of the Taunus Observatory near
Frankfurt am Main from 1927 (courtesy Seismology Group, Goethe University Frankfurt). This page not only documents the visit
of the Danish seismologist Inge Lehmann (1888-1993; e.g., Emmerich & Schweitzer, 1988; Kertz, 2002) on September 22, 1927, but
280
also some of the participants at the 5th DGG annual meeting in Frankfurt/Main, who visited the observatory as part of the conference
program on the afternoon of September 28, 1927 (Schmidt, 1927). From this entry we know that at least five of the conference
participants came from abroad (whether it was solely for the conference or another reason for staying in Germany is an open
question): Victor Conrad (1876-1962; e.g. Hader, 1962; Kertz, 2002) (Vienna, Austria), Walter Davis Lambert (1879-1968)
(Washington D.C., USA) here together with his sister Mary B. Lambert (New York City, USA), Pavel Mikhailovich Nikiforov (1884-
285
1944) (Leningrad, Soviet Union), Leo Wenzel Pollak (1888-1964; Kistermann, 1999) (Prague, Czechoslovakia) and Adalbert Prey
(1873-1949; Ferrari d'Occhieppo, 1981) (Prague, Czechoslovakia) 280 12 https://doi.org/10.5194/hgss-2022-12
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Open Access Table 1: DGG Memberships during the first 10 years, as regularly published as a separate list or as published in the
DGG journal Zeitschrift für Geophysik (ZfG). * copy existing in the archive of the author. https://doi.org/10.5194/hgss-2022-12
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c⃝Author(s) 2022. CC BY 4.0 License. Date
Source
Members
(personal &
institutional)
of which with an address
outside of Germany
19 / 21 September 1922
(foundation)
See e.g., DSG membership list
October 1923
24
1 (4 %)
15 December 1922
DSG membership list *
46
8 (17 %)
October 1923
DSG membership list *
62
13 (21 %)
1 March 1925
DGG membership list, ZfG 1,
173-176
118
29 (25 %)
End of 1925
ZfG 1, 222
131
29 (22 %)
End of 1926
ZfG 2, 48; ZfG 2, 160; ZfG 2,
304
154
38 (25 %)
1 March 1927
DGG membership list,
attachment to ZfG 3
145
37 (26 %)
End of 1927
ZfG 3, 200; ZfG 3, 380
165
44 (27 %)
Spring 1928
ZfG 4, 160
177
52 (29 %)
1 June 1928
DGG membership list,
attachment to ZfG 4
170
52 (31 %)
1 July 1929
DGG membership list,
attachment to ZfG 5
187
58 (31 %)
1 October 1930
DGG membership list,
attachment to ZfG 6
216
74 (34 %)
1 October 1931
DGG membership list,
attachment to ZfG 7
212
70 (33 %)
1 November 1932
DGG membership list,
attachment to ZfG 8
197
64 (32 %) rships during the first 10 years, as regularly published as a separate list or as published in th
rnal Zeitschrift für Geophysik (ZfG). * copy existing in the archive of the author. 290 13 13
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Initial Validation of Surface Ocean Properties in MITgcm Arctic Regional Model
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Initial Validation of Surface Ocean Properties in MITgcm Arctic
Initial Validation of Surface Ocean Properties in MITgcm Arctic
Regional Model
Regional Model Arash Bigdeli
University of Rhode Island, arash_bigdeli@uri.edu Follow this and additional works at: https://digitalcommons.uri.edu/theses
Terms of Use
All rights reserved under copyright. University of Rhode Island
University of Rhode Island
DigitalCommons@URI
DigitalCommons@URI
Open Access Master's Theses
2015
Initial Validation of Surface Ocean Properties in MITgcm Arctic
Initial Validation of Surface Ocean Properties in MITgcm Arctic
Regional Model
Regional Model
Arash Bigdeli
University of Rhode Island, arash_bigdeli@uri.edu Open Access Master's Theses Recommended Citation
Recommended Citation Recommended Citation
Recommended Citation
Bigdeli, Arash, "Initial Validation of Surface Ocean Properties in MITgcm Arctic Regional Model" (2015). Open Access Master's Theses. Paper 819. https://digitalcommons.uri.edu/theses/819 This Thesis is brought to you by the University of Rhode Island. It has been accepted for inclusion in Open Access
Master's Theses by an authorized administrator of DigitalCommons@URI. For more information, please contact
digitalcommons-group@uri.edu. For permission to reuse copyrighted content, contact the author directly. INITIAL VALIDATION OF SURFACE OCEAN
PROPERTIES IN MITGCM ARCTIC REGIONAL MODEL BY
ARASH BIGDELI MASTER OF SIENCE IN OCEANOGRAPHY THESIS MASTER OF SIENCE IN OCEANOGRAPHY THESIS OF
ARASH BIGDELI ABSTRACT In ice-covered regions it can be challenging to determine air-sea exchange – for
heat and momentum, but also for gases like carbon dioxide and methane. The harsh
environment and relative data scarcity make it difficult to characterize even the
physical properties. Here, we seek a mechanistic interpretation for the rate of air-sea
gas exchange (k) derived from radon-deficits. These require an estimate of the water
column history extending 30 days prior to sampling. We used coarse resolution
(36km) regional configuration of the MITgcm with fine near surface vertical spacing
(2m) to evaluate the capability of the model to reproduce conditions prior to sampling. The model is used to estimates sea-ice velocity, concentration and mixed-layer depth
experienced by the water column .We then compared the model results to existing
field data including satellite, moorings and Ice-tethered profilers. We found that sea-
ice coverage have 88 to 98% accuracy, sea-ice velocities have 78% correlation which
resulted in 2 km/day error in 30 day trajectory of sea-ice. Model showed the capacity
to capture mixed layer evolution trends although with a bias and water velocities
showed only 29% correlation with actual data. Using the capacity of the model to
produce same order of magnitude of water speed we calculated an average radius of
possible origins of water parcel equal to 10.3 km. ACKNOWLEDGMENTS I would like to thank my advisor Dr. Brice Loose. He gave me the chance to
explore and patiently stayed by me while I find my own way through the endless
opportunities world of oceanography provides. This work could not been done without
his support both as an advisor and a friend. I also would like to thank my committee members for their knowledge and
advice, Dr. Tetsu Hara and specially Dr. Stephan Grilli, without their help I would
have not been able to set foot on my current path. I am appreciative for NSF funding that supported this research. Fatemeh Faghihzadeh my dear wife who offered me unconditional love and
support through every step of this journey Lastly I want to say eternal thanks to my late father. iii PREFACE The following thesis is presented in manuscript form and is being prepared for
publication in Ocean Science journal. iv TABLE OF CONTENTS v
ABSTRACT .................................................................................................................. ii
ACKNOWLEDGMENTS .......................................................................................... iii
PREFACE .................................................................................................................... iv
TABLE OF CONTENTS ............................................................................................. v
LIST OF TABLES ..................................................................................................... vii
LIST OF FIGURES .................................................................................................. viii
MANUSCRIPT 1 ......................................................................................................... 10
CHAPTER 1 ............................................................................................................... 11
INTRODUCTION ....................................................................................................... 11
CHAPTER 2 ............................................................................................................... 17
METHOD ..................................................................................................................... 17
CHAPTER 3 ............................................................................................................... 19
RESULTS .................................................................................................................... 19
3.1 Sea ice concentration ..................................................................................................... 19
3.2 Sea ice velocity and trajectory ........................................................................................ 21
3.1 Temperature and salinity, and Geopotential height ...................................................... 23
3.3.1 Geopotential height ................................................................................................. 23
3.3.1 Vertical Salinity Temperature profiles ..................................................................... 24
3.4 Density profiles ............................................................................................................... 26
3.5 Mixed layer depth ........................................................................................................... 27
3.6 Circulation ....................................................................................................................... 29
3.7 Velocities in the water column beneath drifting sea ice ................................................ 30
3.7.1 Sources of error in water column current velocities ............................................... 33
CHAPTER 4 ............................................................................................................... 36
SUMMARY ................................................................................................................. 36 v BIBLIGRAPHY ......................................................................................................... 39
FIGURES .................................................................................................................... 47
TABLES ...................................................................................................................... 67 vi vi LIST OF TABLES TABLE
PAGE TABLE Table 1. Summary of comparison between the simulation, estimation and data. ....... 67 vii vii FIGURE FIGURE
PAGE
FIG 1 A graphic illustration of two possible back trajectory of a single sampling
station ........................................................................................................................... 47
FIG 2Arctic Sea-ice extent from 1992-2012 ................................................................ 47
FIG 3Averaged satellite sea ice cover from 2006-2013, Solid red line marking 80%
cover and orange line marking 60%, Blue dots show the analysis grid, stars show the
location of the three points Cyan P1, Green P2, Red P3 where time series data is
graphed in Fi ................................................................................................................ 48
FIG 4Time history of Sea-Ice fraction from top P1, P2 and P3, Satellite data compared
with model results A0 and A1 ...................................................................................... 48
FIG 5 Root mean square error averaged over time from 2006 to 2013,black mask
covers the grid points on the ground ............................................................................ 49
FIG 6 Monthly averaged Annual Root Mean Square Error ......................................... 49
FIG 7 Velocity components from A0 and A1 compared with drift velocity of ITP-53
..................................................................................................................................... .50
FIG 8 30 day trajectory of ITP 53, starting August 2011 to August 2012 ................... 50
FIG 9 Simulated and observed trajectory of ITP53, Aug 06 2011 to Aug 13 2012 .... 51
FIG 10 MITgcm geopotential height referenced to 400(m), 2008-2011 ..................... 51
FIG 11 Obtained from ITP 1 on 13-Dec-2006 at 74.80°N and 131.44°W .................. 52
FIG 12 Obtained from ITP 1 on 28-Aug-2006 at 76.96°N and 133.32°W .................. 52
FIG 13 Obtained from ITP-43 on 27-Nov-2010 at 75.41°N and 143.09°W ............... 53
FIG 14 Obtained from ITP-13 on 30-Jul-2008 at 75.00°N and 132.78°W .................. 53
FIG 15 ITP-62 distribution of sampling stations and the circle centered at 75N 147 W
and radius of 330 (km) ................................................................................................. 54 PAGE viii FIG 16 Observed upper ocean density, ITP 62 Sep 04 2012 to 12 Aug 2013 ............. 54
FIG 17 Simulated upper ocean density following ITP-62 ........................................... 54
FIG 18 Sea Ice fraction and Modeled Ice thickness interpolated into ITP-62 drift ..... 55
FIG 19 Different methods applied to simulated density profiles from a)ITP 1 winter
b)ITP 43 winter c)ITP 1 summer d) ITP13 summer .................................................... 56
FIG 20 Mixed layer depth time series based on M1, simulation and data from ITP-62
...................................................................................................................................... 57
FIG 21 Mixed layer depth time series based on M2, simulation and data from ITP-62
...................................................................................................................................... 57
FIG 22 a) Mixed layer evolution during ITP 62 drift b) Sea-Ice fraction c) Wind Speed
10m above the surface d) Air temperature ................................................................... FIGURE 59
FIG 23 Cross Correlation of Mixed layer depth with wind speed and air temperature
...................................................................................................................................... 59
FIG 24 Model velocities averaged at 180-250(m) , 2006-2012 ................................... 60
FIG 25: Model velocities averaged from 1-15 (m), 2006-2012 ................................... 61
FIG 26 ITP-V Speed simulated and observed ............................................................. 62
FIG 27 ITP-V Eastward velocity observed and simulated........................................... 62
FIG 28 ITP-V Northward velocity observed and simulated ........................................ 63
FIG 29 ITP-V correlation versus depth ........................................................................ 63
FIG 30 Mooring-D Observed and simulated Speed ..................................................... 64
FIG 31 Mooring-D eastward velocity observed, simulated and estimated .................. 65
FIG 32 Mooring-D northward velocity observed, simulated and Estimated ............... 66 ix MANUSCRIPT 1 Submitted to Ocean Science journal ARCTIC REGIONAL MODEL 10 INTRODUCTION The ocean surface is a dynamic region where momentum, heat and salt, as well as
biogeochemical compounds are exchanged with the atmosphere and with the deep
ocean. These fluxes drive vertical mixing that generates the mixed layer. On the top of
the mixed layer, at the sea-air interface, gases of biogenic origin and geochemical
significance are exchanged with the atmosphere. Theory indicates that the aqueous
viscous sublayer, which has a length scale of 20 to 200 μm, (Jähne and Haubecker
1998) is the primary bottleneck for air-water exchange. Limitations in measurement at
this critical scale have led to approximations of sea-air gas exchange based on indirect
measurements. Four approaches involving data are typically used (Bender et al. 2011),
1) Parametrization of the turbulent kinetic energy (TKE) at the base of the viscous
sublayer 2) Tracing purposefully injected gases (Ho et al. 2006; Nightingale et al. 2000) 3) Micro Meteorological methods (H. J. Zemmelink et al. 2006, 2008;
Blomquist et al. 2010; Salter et al. 2011), and 4) Radon-deficit Method. Here, we
examine the radon-deficit method (4), together with a parameterization of the TKE
forcing (1) that theoretically leads to the observed deficit in mixed-layer radon. 1) Parametrization of the turbulent kinetic energy (TKE) at the base of the viscous
sublayer 2) Tracing purposefully injected gases (Ho et al. 2006; Nightingale et al. 2000) 3) Micro Meteorological methods (H. J. Zemmelink et al. 2006, 2008;
Blomquist et al. 2010; Salter et al. 2011), and 4) Radon-deficit Method. Here, we
examine the radon-deficit method (4), together with a parameterization of the TKE
forcing (1) that theoretically leads to the observed deficit in mixed-layer radon. TKE is mostly dominated by wind speed on open ocean interface (Wanninkhof 1992;
Ho et al. 2006; Wanninkhof and McGillis 1999; Nightingale et al. 2000; Sweeney et
al. 2007; Takahashi et al. 2009). In the polar oceans wind energy and atmospheric
forcing are transferred in a more complex manner as a result of sea ice cover (Loose et 11 11 al. 2009, 2014; Legge et al. 2015). Sea ice drift due to Ekman flow (McPhee and
Martinson 1992), freezing and melting of ice leads on the surface ocean (Morison et
al. 1992) and short period waves (Wadhams et al. 1986; Kohout and Meylan 2008) all
constitute important sources of momentum transfer. Considering the scarcity of data
on marginally covered sea-ice zones (Johnson et al. INTRODUCTION 2007; Gerdes and KöBerle 2007),
especially during arctic winter time, comparing and validating these models are quite
challenging. The radon deficit method involves sampling 222Rn and 226Ra in the mixed layer to
examine any difference in the concentration or (radio) activity of the two species. Radon is a gas, radium is a cation; in absence of gas exchange 222Rn and 226Ra enter
secular equilibrium meaning the amount of 222Rn produced is equal to decay rate of
226Ra. Any missing 222Rn in the mixed layer is attributed to exchange with
atmosphere (Peng et al. 1979). Since the 222Rn concentration in air is very low, less than 5% (Smethie et al. 1985)
and considering that concentration is proportional to activity/decay rate A, we can use
Eq. (1) to determine gas exchange. Where k gas transfer velocity in (m d-1), AE is the
activity or decay rate of 222Rn which in secular equilibrium is equal to 226Ra
activity, AM is 222Rn measured decay rate in mixed layer, λ is decay constant of
222Rn (0.181 d-1) and h is the mixed layer depth ,
(1) ,
(1) (1) Gas transfer velocities from equation (1) reflect the memory of 222Rn for a period of
two to four weeks (Bender et al. 2011), which is four to eight times the half-life of 12 12 222Rn (3.8 days).The mixed layer depth, h, is calculated from the measurements
performed at the hydrographic stations during 222Rn sampling process. While a valuable tool this method is based on two premises, a) an invariant mixed
layer and b) invariant TKE forcing during the entire period of “memory” that gas in
the mixed-layer has experienced. To further illustrate the discrepancy caused by
utilizing the invariant assumptions, described above, consider a mixed layer that
rapidly changes by a factor of 2 just prior to sampling for radon. If the mixed-layer
becomes shallower (stratification) h – defined by the density profile - will be smaller
by factor of 0.5 while AE/AM in the mixed layer remains the same. Based on equation
1, this causes k to be half of its true value. That is, prior to stratification TKE forcing
was sufficient to ventilate the ocean to a depth greater than the apparent h (Bender et
al. 2011). 222Rn (3.8 days).The mixed layer depth, h, is calculated from the measurements
performed at the hydrographic stations during 222Rn sampling process. INTRODUCTION 222Rn (3.8 days).The mixed layer depth, h, is calculated from the measurements
performed at the hydrographic stations during 222Rn sampling process. While a valuable tool this method is based on two premises, a) an invariant mixed
layer and b) invariant TKE forcing during the entire period of “memory” that gas in
the mixed-layer has experienced. To further illustrate the discrepancy caused by
utilizing the invariant assumptions, described above, consider a mixed layer that
rapidly changes by a factor of 2 just prior to sampling for radon. If the mixed-layer
becomes shallower (stratification) h – defined by the density profile - will be smaller
by factor of 0.5 while AE/AM in the mixed layer remains the same. Based on equation
1, this causes k to be half of its true value. That is, prior to stratification TKE forcing
was sufficient to ventilate the ocean to a depth greater than the apparent h (Bender et
al. 2011). Conversely, if the mixed layer deepens due to mixing, h increases and a new parcel of
water with AE/AM= 1 is added to mixed layer, causing the activity ratio to come
closer to unity. These two influences on equation 1 (increasing H and AE/AM
approaching unity) work against each other, but the net effect is to cause k to appear
larger. The change of factor of 2 in mixed layer depth in less than two weeks has been
observed during several studies (Acreman and Jeffery 2007; Ohno et al. 2008; Kara
2003). The “memory” of gas exchange forcing that radon experiences is further complicated
by the presence of sea ice. Consider two alternate water parcel drift paths that lead to
the 222Rn sampling station in sea-ice zone (Fig. 1). Path B demonstrates a history
which water column spends most its back trajectory under sea-ice. Path A shows a 13 water column which experiences stratification and shallowing of mixed layer depth
equal to δh when drifting through water that is completely uncovered by ice. During
most of Path B gas transfer happens in form of diffusion through sea-ice and it will
have a very low k (Crabeck et al. 2014; Loose et al. 2011). In contrast Path A will
have a greater radon deficit, but a smaller h because of stratification. INTRODUCTION In either case, it
is critical to take into account the time history of gas exchange forcing, including
changes in the mixed-layer and ice cover, which has led to the apparent radon deficit
at the time of measurement. water column which experiences stratification and shallowing of mixed layer depth
equal to δh when drifting through water that is completely uncovered by ice. During
most of Path B gas transfer happens in form of diffusion through sea-ice and it will
have a very low k (Crabeck et al. 2014; Loose et al. 2011). In contrast Path A will
have a greater radon deficit, but a smaller h because of stratification. In either case, it
is critical to take into account the time history of gas exchange forcing, including
changes in the mixed-layer and ice cover, which has led to the apparent radon deficit
at the time of measurement. This observation about drift paths in the sea ice zone strongly implies that we must
consider both time and space in estimating the forcing conditions that are recorded in
the radon deficit. In other words, we require a Lagrangian back trajectory of water
parcels to track the evolution of mixed layer and its relative velocity 4 weeks prior to
sampling. Although satellite data, Ice tethered drifters (Krishfield et al. 2008) and moorings
(Krishfield et al. 2014; Proshutinsky et al. 2009) have provided valuable seasonal and
spatial information about the sea ice zone, they do not track individual water parcels
and tend to convolve space and time variations. The spatial limitation of these data
pose a challenge to producing a a back trajectory of the water parcel hence we used a
3D numerical model to simulate these data. This is the principle goal of our study – we want to know if a coarse resolution model
that can be run on a desktop multi-core processor can provide us with spatial and
temporal information to better model the back trajectory of a radon-labelled water
parcel. The null hypothesis might therefore be, that simple assumptions such as an 14 14 invariant mixed-layer, Ekman ice drift and area-averaging of sea ice cover are as good
or better at reproducing the radon-based estimates of k. INTRODUCTION We set up a numerical simulation that captures near surface phenomena as a means to
access the history of a water parcel sampled for radon, and the forcing conditions that
led to the observed radon deficit. For this study we are focusing on near surface
phenomena in the Arctic using the Arctic Regional configuration of the MITgcm with
the domain of ECCO2 (Marshall et al. 1997; Menemenlis et al. 2008; Losch et al. 2010; Heimbach et al. 2010; Nguyen et al. 2011). A number of Arctic ocean-ice
models which have been compared as part of Arctic Ocean Models Intercomparison
Project (AOMIP) (Proshutinsky et al. 2001; Lindsay and Rothrock 1995; Proshutinsky
et al. 2008) in regard to their capability to represent main ice-ocean dynamics. This
model has shown to have better correlation with remotely sensed sea ice data (Johnson
et al. 2012) and high near surface vertical resolution. It is worth noting that model studies using higher resolution evaluate model skill are
already in print. For example, Holloway et al., (2011) use a 4 (km) version of the
MITgcm regional model has a high skill in producing near surface velocities and
capturing eddies. So, the choice to use the 36 (km) resolution model may seem
anachronistic. However, we approach this problem as geochemists who are searching
for a data interpretation tool. We seek higher resolution in the top 100 m of the water
column, as compared to these prior studies. Furthermore, the 36 (km) model can be
run on a desktop multi-core processor, and the simulation is completed in less than a
week. 15 The remainder of the article is organized as follows: In section 2 we introduce the
changes we made to increase the near surface resolution. Section 3.1 and 3.2 contain
the comparison of the new results and original model outputs including sea-ice
concentration, velocity and trajectory to observed data from satellite and Ice tethered
profiler. Sections 3.3, 3.4 and 3.5 are used to study the model output salinity and
temperature structure resulting upper ocean density structure and mixed layer. Sections 3.6 and 3.7 evaluate the qualitative model circulation and correlation in near
surface water velocity. Lastly, section 4 shows the summary of our results. In each
section, we attempt to compare model output to direct observations, and to simple
assumptions – such as those that are commonly made in geochemical studies of the
upper ocean. INTRODUCTION These comparisons help define the relative value of the model output to
geochemical data interpretation. 16 METHOD The model has a horizontal resolution of 36km, and 50 vertical layers employing the
z* coordinate system (Adcroft and Campin 2004) and the maximum model depth of
6150m. The surface forcing is sourced from ERA40 and ECMWF numerical weather
forecast, and the boundary conditions are taken from output of the ECCO2 global
model (Menemenlis et al. 2008) and 25 year Japanese Reanalysis Project
(JRA25)(Onogi et al. 2007). Initial conditions are from World Ocean Atlas 2005
(Antonov et al.; Locarnini et al.) and initial sea ice conditions are from (Zhang and
Rothrock 2003), model is allowed to spin up from 1979 to 1992. The vertical mixing
uses K profile parameterization (KPP) developed by Large et al. (1994) and salt plume
parameterization of Nguyen et al. (2009). We introduced a new vertical grid spacing (hereafter referred to as A1), 2 meter steps
near surface which continue to the depth of 50 meter which then gradually increase to
a maximum interval of 650 meters in deep ocean contrasting to the optimized model
(called A0) which had 10 meter intervals near surface, progressively increasing to 450
meter at maximum depth. All the boundary conditions from ECCO2 have been
interpolated to match the new grid system. Due to stability condition of the model we
also reduced the time steps of the model from 2400 seconds to 600 seconds. The
model simulates the time period from January 1992 to September 2013. This new grid
system allows us to capture near surface small details which cannot be represented
with the coarser grid system. 17 Satellite estimation of sea-ice cover at 25km horizontal resolution (Comiso 2000) is
being interpolated on a grid system and then compared with A0 and A1. Then sea-ice
drift gathered by 28 Ice Tethered Profilers (ITP) (Krishfield et al. 2008) which has
more than 2 month of data in Beaufort Sea between 2006 and 2013 has been used to
do the ice velocity and trajectory comparison. We selected only ITPs with 2 or more
months of drift data in the Beaufort Sea. We compared near surface water velocity data from ITP-V (Williams et al. 2010) and
upward looking Acoustic Doppler Current Profiler installed on McLane Moored
Profiler (MMP) (Proshutinsky et al. 2009; Krishfield et al. 2014) to A1 in order to
compute the accuracy and feasibility of calculating back trajectory of parcels located
at the mixed layer. METHOD Using salinity and temperature profiles from ITPs (Krishfield et al. 2008) we
calculated mixed layer depth and the average geopotential height between 2006 and
2013 and compared it to 2m vertical resolution model output(A1). 18 3.1 Sea ice concentration Both A0 and A1 model scenarios are used to study sea ice extent (area of cells with
more than 15% ice cover) from 1992-2013. At the Arctic Basin scale, the model
depicts a decrease in the September minimum sea ice extent of 0.82 million square
kilometres per decade or 10% of the starting value at 1992 compared to satellite data
which display a 1.2 million square kilometres per decade, the model show an over
estimation of sea-ice extent (Fig. 2). For further analysis we introduced a grid system
covering the Beaufort Gyre and interpolated the data from satellite (Comiso 2008) and
A0 and A1 on to the grid. The analysis grid extends from 70° to 80° north and 130° to 170° west, covering most
of Beaufort Gyre (Fig. 3). Grid points can be divided into two main geographic zones
that are marked out based on sea ice cover. The first zone contains grid points where
the annual average sea ice cover is greater than 80%. These sets of points are fully
covered by sea-ice most of the year. The second zone can be described as “marginally
ice covered” wherein the ocean surface is free of ice for some fraction of the year. We
chose 3 points within this sea ice geography to compare the seasonal and interannual
behavior of the model with satellite ice cover. The points are located at 80°,131.82°
(P1), 70.82° 169.82° (P2), and 74.76° north and 163.51° west (P3). The ice cover at P1, P2 and P3 (Fig. 4) can be divided into 3 ice phases: (a) Fully
covered in ice, (b) Open water and (c) a transition between (a) and (b). P3, which is 19 the furthest south, has all three phases. In contrast P1 ice cover only dips below 60%
for two brief periods during the 7 year time series depicted in Fig 4 - once in 2008 and
again in 2012. These three points illustrate where and when the model has the greatest
challenge reproducing the actual sea ice cover. At the extremities of the ice pack,
where the water is predominantly covered by 100% or 0% ice (P1 and P3), the model
captures the seasonal advance and retreat and the percent ice cover itself is accurate. 3.1 Sea ice concentration Overall, the periods of transition (melt and freeze) coincide with the greatest RMSE. The 2m revised grid (A1), with smaller vertical intervals near the surface has produced a greater RMSE
than the optimized model (A0). We are still exploring why this took place. It is possible that the
convective parameterization in the ice model is somehow negatively affected by short i.e. 2 m vertical
layers. To reproduce changes in the mixed layer will require the greater resolution reflected in the A1
model run. 3.1 Sea ice concentration However, in the transition regions that are characterized by marginal ice for much of
the year (P2), the model has more difficulty reproducing accurate sea ice cover as well
as the timing of the advance and retreat. These results are the same for both A0 and
A1 experiments, and this behavior is consistent with the description that has been
explained by Johnson et al. (2007), that models have a higher accuracy predicting sea
ice concentration in central arctic and less accuracy near periphery and lower latitudes. The spatial sensitivity of the model can be observed using root mean square (RMS)
error Eq. (2), calculated over the 1992-2013 period (Fig. 5). The area with most error
coincides with area between the 80% and 60% contour lines (Fig. 3) and is
concentrated primarily in the Western Beaufort. The RMSE error of 0.2 is the
maximum value away from land, this same level of error can also be found near land
which is caused by fast-ice generation. Fast Ice in the model is replaced with pack of
drifting sea ice; this error is common between numerical models and has been studied
during AOMIP (Johnson et al. 2012, p. 20). (2) The spatial sensitivity of the model can be observed using root mean square (RMS)
error Eq. (2), calculated over the 1992-2013 period (Fig. 5). The area with most error
coincides with area between the 80% and 60% contour lines (Fig. 3) and is
concentrated primarily in the Western Beaufort. The RMSE error of 0.2 is the
maximum value away from land, this same level of error can also be found near land
which is caused by fast-ice generation. Fast Ice in the model is replaced with pack of
drifting sea ice; this error is common between numerical models and has been studied
during AOMIP (Johnson et al. 2012, p. 20). (2) (2) (2) If we compare the monthly climatology for sea ice cover over the 1992-2013 period, the RMS error
between model and satellite data is least during the early winter months (e.g. Jan-Mar) when sea ice is 20 close to its maximum extent. Comparing A0 and A1 Fig. 5 depicts an increase in RMSE during July,
August, September and October and a minor decrease in May and November. The RMSE appears to be
greater during the summer months of ice retreat, and slightly less during the autumn months of ice
advance. 3.2 Sea ice velocity and trajectory Ekman turning causes ice and water to move at divergent angles with respect to each
other. Ice moves the fastest, with mean values of 0.09 m s-1 (Cole et al. 2014), and the
water column progressively winds down in velocity, along the Ekman spiral. Stratification in the Arctic leads to a confinement of the shear stress closer to the air-
sea interface and also produces greater divergent flow vectors between ice and water
(McPhee 2012). In the marginal ice zone or in regions where ice is converging or
diverging, these motions, relative to the motion of the water column can produce
significant changes in the water column momentum budget as well as air-sea fluxes. Thankfully, the ITPs can provide us with a measure of the real ice drift. For purposes of comparison with the model and ITP drifts estimated a simple sea-ice
velocity vector field Eq. (3) using reanalysis wind data (Onogi et al. 2007), with “a”
and θ as empirical coefficients equal to 0.019 and 28 degree (Cole et al. 2014), This
simple estimation neglects feedback from Ekman spiral under the ice which has been
implemented in more sophisticated analytical solutions (Park and Stewart 2015). 21 (3) To generate a more quantitative comparison between the results we utilized the same
method used by Timmermans et al. (2011), comparing ice velocity components (u-v)
of A0, A1 and estimated velocity from Eq. (3) to ITP velocity and finding the
correlation coefficient of each experiment versus the daily averaged actual drift
velocity of the ITPs (Fig. 6) By averaging over all the ITPs operating in Beaufort Gyre during 2006 to 2013, A0
had 0.78, A1 0.77 and simple estimation had 0.65 correlation with actual velocity
components. As stated in the introduction, one of the principle objectives of this study is to evaluate
the capability of the MITgcm to produce useful forcing field on the time-scale of
mixed layer memory for biological and geochemical compounds, particularly the
volatile ones. For a trace gas such as oxygen or radon, we only require adequate
representation of the time-history of hydrographic conditions that act upon on a tracer
for ~30 days, because this is the approximate renewal time of the mixed layer, based
on a representative value of air-sea exchange. Consequently, we need the ice that
covers our Lagrangian water parcel, so we calculated 30 day trajectories of sea-ice. 3.2 Sea ice velocity and trajectory We generated the ice floe trajectories by step-wise integration of the daily-average ice
model output of velocity and direction in order to follow the individual ice floe. To
illustrate, we have selected one of the 28 ITPs - ITP53 during its operation in Beaufort
Sea from August 6, 2011 to August 13, 2012. To generate a statistically large sample size, this calculation is preformed such that
every day is treated as a starting point for simulation and the trajectory of that parcel is 22 22 traced for a 30 day period. Figure 7 shows the result of this calculation for ITP 53. Taking the separation between the actual ITP trajectory after 30 days and the model
trajectory, we determine the average drift error. Using 28 ITPs and 11744 operation
days, for a total of N = 352320 data points. The average separation is 59.3 (km) for A0
and 62.5 (km) for A1 that is equal to 1.97 and 2.08 (km/day) error respectively. This
amounts to a 5% increase in the error from A0 to A1. Same calculation for trajectories
based on Ekman drift sea-ice velocities computed from Eq. (3) yield 4.3 km/day error. In other words, the coarse resolution model error is approximately half as large as if
we assumed a simple Ekman drift for the ice trajectory. The path of the ice, seen through its trajectory, appears to show a dependence of the
choice of resolution in the vertical grid cell, but we cannot say for certain if it
improves or deteriorates the results. ITP-52 has correlation coefficient of 0.87 on
northward direction and 0.82 on eastward direction but due to accumulation of error
both A0 and A1 fail to represent the actual trajectory of ITP-52 using just the start
point and letting error accumulate through the entire ITP path that is 374 results in a
pronounced difference in end points (Fig. 10). 3.3.1 Geopotential height McPhee (2013) showed that the accumulation of freshwater in the Beaufort Gyre led
to an increase in geopotential height and a strong geostrophic flow that may have
feedbacks for the advection of ice out of the Canadian Arctic. Here, we have used the
model to calculate geopotential height (Fig. 12), referenced to the 400-m isobath and 23 averaged from 2008-2011, with velocity vectors calculated based on the thermal wind
equation. We can compare our result to hydrographic reports generated from ITPs and
CTD cast during the same time period which has been reported in previous literature
(McPhee 2013). Hydrographic data show a doming of about 45+ (cm) relative to geoid in Beaufort
Gyre with the maximum centered at 74.5° N and 150° W (Kwok and Morison 2011, p. 20). Our model results show the same dome and correct sign, producing the correct
geostrophic flow direction with the magnitude of 100+ (cm). The model shows the
center of the dome to be around 80° N and 140W. Discrepancy between the
magnitudes is in line with the results from sea-ice concentration section, with model
over predicting the sea-ice extent hence generating excess fresh water. In a big picture
this shows the capability of the model to predict the freshening of top 400 meter. 3.3.1 Vertical Salinity Temperature profiles We chose 4 profiles in Beaufort sea to represent the simulated vertical salinity and
temperature. The first two sets of profiles are from ITP-1 winter (Fig. 11) and summer
2006 (Fig. 11). The third set is from ITP-43 during winter 2010 (Fig. 12) and the
fourth is from ITP-13 during summer 2008 (Fig. 13). During winter time, a well mixed layer reaches below 15 meters in the model T and S
profiles (Fig. 11), followed by a very large gradient. The mixed-layer temperature is
close to the local freezing point in a condition called “ice bath” (Shaw et al. 2009). The ITP profiles are similar; however the ITP mixed layer depth is deeper by nearly
10 meters, indicating more ice formation and convective heat loss over this water
column, as compared to the model water column. In summer (Fig. 11) the model 24 mixed layer shoals to approximately 5 meters depth following two local temperature
extrema, the bigger maximum is at ~35 meters generated by intrusion of Pacific
Summer Water (PSW) which is a dominant feature in Canada basin. The second
smaller maximum happens around 10 meters called Summer Mixed Layer (Shimada et
al. 2001) or Near-Surface Temperature Maximum (NSTM) (Jackson et al. 2010)
which is a seasonal feature generated by shortwave solar heat diffusion (Donald K. Perovich 1990). These two well-defined phenomena are broadly descriptive of the
summer surface layer in the Beaufort Gyre. They are; however, absent from the ITP
data at this location, indicating a different ice and heat budget time history. The
mismatch in temperature is one of the challenges that numerical models face when
reproducing the cold halocline. Recent studies show that eddies with diameter of
approximately 30 km (Nguyen et al. 2012) have a major contribution in transport of
PW from shelf break into Canadian basin, which requires a model with finer
horizontal resolution. Data and model profiles in Fig. 12 show better agreement in the shape and the
absolute value of the T and S profiles. Both model and ITP data have a 20 meter deep
mixed layer during 2010 winter. The model in this case does not show as much change
in vertical temperature structure compared to actual data. In the profile from ITP-13
(Fig. 3.3.1 Vertical Salinity Temperature profiles 13) the model again over estimates the temperature beneath the mixed layer,
although certain features including the NSTM can still be found near 10 meters, yet
not as pronounced since it is very close to PSW. Bearing in mind that density in the
Arctic is dominated by changes in salinity, we move forward to density profiles from
this point on. 25 25 3.4 Density profiles We compare the MITgcm density to the time series of density profiles from ITP-62
(Fig. 15-16) during the course of nearly a year starting in Sep 2012. A black mask
indicates locations where there is no data from ITP-62 - particularly in the upper 7
meters of the water column. ITP-62 transited through Canadian basin, density profiles
have both temporal and spatial changes in them (Fig. 14). We are able to discern some broad similarities in the model and ITP density profiles. From September to mid-November, the density profile above 50 (m) tended to
increase, consistent with the period of cooling and ice formation. From December
through March, both ITP and model density profiles remain relatively constant. Between March and April, ITP-62 appears to drift through a unique water parcel, with
lower density above 70 (m). The same feature can be observed in the MITgcm density. However, on a smaller scale, there is significantly more variation in the ITP data than
what the model represents. For exploring the reason behind the density signals we are going to use the simulated
fraction of sea ice cover and ice thickness (Fig. 17). The dominating effect appears to
result from sea ice fraction and when there is continuously covered area. The changes
from sea ice thickness can be observed in volume of fresh water in the water column. For exploring the reason behind the density signals we are going to use the simulated
fraction of sea ice cover and ice thickness (Fig. 17). The dominating effect appears to
result from sea ice fraction and when there is continuously covered area. The changes
from sea ice thickness can be observed in volume of fresh water in the water column. A peak in near surface density can be seen late in March when a decrease in ice
fraction from 100% to 90% exposed the surface water to cold atmosphere, which
generated newly formed sea ice and inserted brine into the water column. This signal
will be further discussed on mixed-layer section. 26 3.5 Mixed layer depth There are many different methods in the literature for calculating mixed layer depth
(Brainerd and Gregg 1995; Wijesekera and Gregg 1996; Thomson and Fine 2003; de
Boyer Montégut et al. 2004; Lorbacher et al. 2006; Shaw et al. 2009). The methods
can be divided into two main types (Dong et al. 2008): The first type of algorithm
looks for the depth (zMLD) at which there has been a density increase of δρ between the
ocean surface and zMLD. A typical range of values for δρ are 0.005 (kg m-3) to 0.125
(kg m-3) (Brainerd and Gregg 1995; de Boyer Montégut et al. 2004). The second type
uses slightly different criteria, where the base of the mixed layer is determined as the
depth where the gradient of density (∂ρ/∂z) equals or exceeds a threshold; typical
numbers for (∂ρ/∂z) are 0.005 (kg m-4) to 0.05 (kg m-4) (Brainerd and Gregg 1995;
Lorbacher et al. 2006). A more sophisticated approach to type 1 of this criteria is to
utilize a differential between (ρ100m - ρsurface) as the cut of point (instead of using a
fixed δρ) to account for the effects of surface ρ changes during winter and summer
(Shaw et al. 2009). Here, we have implemented two of these methods M1 and M2,
with M1 using δρ equal to 0.2 of (ρ100m–ρsurface) (Shaw et al. 2009) and M2 with a
gradient (∂ρ/∂z) cut off point equal to 0.02 (kg m-4) which matches innate model
parametrization of MLD (Nguyen et al. 2009). We compare these 2 methods by applying them to the profiles from Fig. (10-11-12-13)
which result in (Fig. 18). In case (a) and (b) M1 produces a mixed layer depth that is 8
to 12 meters deeper, compared to the other method. A visual examination of profiles
appears to indicate that the M1 criteria may be too flexible of a criteria. The results
from M2 appear to be intermittently “realistic”, whereas M1 can be difficult to 27 implement on high resolution data with greater small-scale variability. In practice, we
find M2 is the most straight-forward to implement. 3.5 Mixed layer depth The wind appears to have led to a divergence in ice cover, which
in-turn exposed the ocean to the cold atmosphere, leading to a loss of buoyancy and an
increase in the mixed-layer depth. In this regard, the wind appears to play a facilitating
role, leading to sea ice divergence, but there is no evidence to support that a strong
wind event caused the mixed layer to deepen. For further exploring the forcing that drives the mixed layer, we use the Japanese
reanalysis (JRA-25) (Onogi et al. 2007) wind speed, air temperature data and sea-ice
fraction from the model and interpolated them on the path of ITP-62. The prominent
feature during March to April can be explained by the reduction in ice cover to 95% ,
which resulted in water column exposure to low or moderate wind from 5 (ms-1) to 10
(ms-1) and low air temperatures, which in turn increased mixing and deepening of ML
by 100% (Fig. 17). The wind appears to have led to a divergence in ice cover, which
in-turn exposed the ocean to the cold atmosphere, leading to a loss of buoyancy and an
increase in the mixed-layer depth. In this regard, the wind appears to play a facilitating
role, leading to sea ice divergence, but there is no evidence to support that a strong
wind event caused the mixed layer to deepen. Further exploring the dependency of ML evolution to wind and temperature we
calculated cross correlation between ML-wind and ML-temperature (Fig. 22). This
analysis shows responses with respect to air temperature happen with no significant
lag and wind with 10 days of delay which would result in a maximum 0.36 correlation
for wind and 0.39 correlation for temperature. Without accounting for the effect of sea
ice cover, which can somewhat modulate momentum inputs by the wind and strongly
modulate heat and moisture fluxes, it appears that neither temperature nor wind has a
dominant effect on MLD in the Arctic, but rather both play a role. 3.5 Mixed layer depth It should be mentioned that it is difficult to consistently compare performance of the
M1(δρ) and M2 methods on ITP and model data, because the model data extends to
the free surface, but the ITP data stops at 7 (m) depth and it has been shown that
summer mixed layer in the Canada basin can be less than 12 meters (Toole et al. 2010). To account for this effect, we apply an additional restriction wherein any
profile whose mixed-layer depth is less than 2 (m) below the shallowest ITP
measurement is discarded. This restriction effectively removes any ML depths
shallower than 10 meters due to ITP sampler not resolving the upper 8 meters of water
column. In some cases, a remnant mixed-layer from the previous winter may exist in
the water column. In this case, the methods incorrectly identify the remnant ML as
actual ML depth. To compare the methods over a longer time period, we calculated the mixed-layer
depth from model data and ITP-62 data along the ITP-62 drift track. We used both M1
and M2 to determine the ML depth for the model data and for ITP-62 data (Fig. 19-
20). M1 and M2 show almost similar results. The model shows a shallower ML
compared to the ITP data; the most prominent feature in early March corresponds to a
sudden change in density found in (Fig. 15). During the months of June and July, the
model predicts zero mixed layers with stratification almost to the surface. The ITP
data, beginning at 8 m cannot reflect this stratification, but we know this model result
to be plausible based upon our comparisons with shipboard CTD profiles (data not
shown here) and what is known about ice melting and stratification. 28 For further exploring the forcing that drives the mixed layer, we use the Japanese
reanalysis (JRA-25) (Onogi et al. 2007) wind speed, air temperature data and sea-ice
fraction from the model and interpolated them on the path of ITP-62. The prominent
feature during March to April can be explained by the reduction in ice cover to 95% ,
which resulted in water column exposure to low or moderate wind from 5 (ms-1) to 10
(ms-1) and low air temperatures, which in turn increased mixing and deepening of ML
by 100% (Fig. 17). 3.6 Circulation The time-averaged upper ocean circulation in the Arctic has been described by
oceanographers based upon the origin of the water masses that enter and exit the
Arctic Ocean. These are primarily the Atlantic water (AW) and Pacific Water (PW). 29 After entering the Arctic ocean AW and PW loose heat and sink, AW to between 200
(m) and 800 (m) (Golubeva and Platov 2007) and PW to between 50 and 150m (Steele
et al. 2004). During the AOMIP intercomparison study it was observed that Arctic
models can produce two AW circulation in opposing directions, with observations
suggesting a cyclonic circulation in Arctic basins (Lindsay and Rothrock 1995). Potential vorticity influx from sub-Arctic oceans (Yang 2005) and unresolved eddy
parametrization (Holloway et al. 2007) have been shown to diminish this discrepancy
and create consistent cyclonic circulation among all models (Proshutinsky et al. 2011). We evaluated the capability of the model to reproduce the general circulation of Arctic
by averaging output ocean velocities from A1 experiment during a time span of seven
years from 2006 to 2012. We focused on two depth intervals 1-15 (m) depth to
represent Ekman layer (Cole et al. 2014) (Fig. 24) and the second set is 180-250 m to
model AW circulation (Fig. 23). The flow field (Fig. 23) representing AW displays a counter-clockwise topographic
boundary current in the Beaufort and Chukchi seas matching AW cyclonic circulation
(Lindsay and Rothrock 1995; Proshutinsky et al. 2011). In contrast there is a
clockwise boundary current in the southern Canadian basin (Fig. 24). These
circulation patterns qualitatively match the hydrographic description of the time-mean
circulation in the Arctic. 3.7 Velocities in the water column beneath drifting sea ice We have very little information from direct observations that permit us to track a
water parcel especially beneath sea ice. This is one area where model output could be
critical as there are not obvious alternatives. To judge the consistency of the model 30 water current field, we compared 2D model water velocity to data gathered from two
sources: (1) from ADCPs mounted on moorings that were deployed starting in 2008 in
Beaufort Gyre (Proshutinsky et al. 2009) and (2) the ITP-V sensor equipped with
MAVS (Modular Acoustic Velocity Sensors) (Williams et al. 2010), which was the
only operating ITP before 2013 which had an acoustic sensor mounted on it. water current field, we compared 2D model water velocity to data gathered from two
sources: (1) from ADCPs mounted on moorings that were deployed starting in 2008 in
Beaufort Gyre (Proshutinsky et al. 2009) and (2) the ITP-V sensor equipped with
MAVS (Modular Acoustic Velocity Sensors) (Williams et al. 2010), which was the
only operating ITP before 2013 which had an acoustic sensor mounted on it. We compared the magnitude of velocity without accounting for flow direction, i.e. removing the effects of Ekman turning (Cole et al. 2014) in order to find a 2D-
correlation over the duration of ITP-V working days which is from Oct 9, 2009 to Mar
31, 2010 (Fig. 25). The ITP data has been daily averaged so it matches the time
interval of the model structure. Due to ITPs mechanical limitation on reporting
velocities shallower than ~6 meters, the comparison starts from 7 meters. The first result that emerges is that ITP velocity shows a lot more structure, changing
speed at a much higher time frequency most probably caused by eddies which has
been shown to be a characteristic feature in the top layers of Beaufort Sea (Zhao et al. 2014). Figure 26-27 depicts the simulated and observed velocity components for ITP-
V, to better compare the results the data and simulation have been filtered by 4 days
low pass filter corresponding to 36 (km) in distance which the ITP travels effectively
low pass filtering the results. The absolute 2D-correlation between the simulated and observed velocities for
eastward direction is 20%, northward direction 22% and for the speed is 20%, these
numbers represent r and not r2. We also compared the correlation versus depth and
correlation of the velocity components and current speed (Fig. 28). 3.7 Velocities in the water column beneath drifting sea ice If averaged over
depth the northward velocities will have about 28% correlation with depth averaged 31 31 data and also the correlation increases with depth after 40 meters. One possible reason
for this lack of correlation near surface is effects of stratification in the model due to
its shallower mixed layer. Another source of ocean current data from the Arctic are ADCPs mounted on the
moorings which has been deployed as part of Beaufort Gyre Observation System
(BGOS), these profilers are located on the top float of subsurface moorings, measuring
water velocities within the upper 30 meters of water column (Krishfield et al. 2014). Mooring D was the first mooring outfitted with an ADCP in 2005. Figure 29 shows
the current speed measured by this mooring from October 2010, covering 1032 days
of data located at 73.99° N and 139.98° W. Simulation results follow certain features of the actual data (Fig. 29), high velocities
around Jan-2011, Oct-2011 and Oct-2012 which is caused by exposure of water
column surface to winds. In general, higher velocity magnitudes are observed which
are not represented in the simulation. For further exploring these high velocity features in the mooring data, we used wind
speed from Japanese Reanalysis data (Onogi et al. 2007), sea-ice cover from satellite,
constant sea-ice draft of 2m, sea-ice velocity estimation based on Sect. 3.2, Eq. (3) to
construct a simple Ekman spiral based on Eq. (4) and Eq. (5). Our assumptions include
a neutrally buoyant layer, constant dimensionless eddy viscosity (K* =0.2) (McPhee
and Martinson 1992; McPhee 2012) and that our target depth is outside of the
boundary (log) layer sea ice (Cole et al. 2014). , ,
(4) (4) 32 32 32 ,
(5)
,
(6) (5) (6) With z the distance from the water interface and τ(z) representing the shear vector, f
Coriolis parameter and Cice is the fraction of sea-ice cover. The interface friction
velocity vector (u*0) is calculated based on the type of interface. In case of water-ice
interface u*0 is based on law of the wall (McPhee 2008). Total Ekman velocity is Cice
weighted summation of Ekman vectors based on ice and air (Eq. 6), we then compared
the velocity components based on this method, simulation and data (Fig. 30-31). 3.7.1 Sources of error in water column current velocities There is 29% correlation between the simulation and mooring data. This is nearly
identical to the poor correlation observed between the model and the ITP velocities. These discrepancies between model and data can be caused by several reasons. The
first is the model resolution is too coarse to resolve mesoscale and submesoscale
eddies, which have characteristic lengths of 10 km or smaller (Boccaletti et al. 2007;
Timmermans et al. 2008). Because these eddies would all hypothetically fit within one
of our 36-km grid boxes, this model configuration lacks the ability to represent them. Eddy resolving models have better agreement with velocity data (Holloway et al. 2011). Secondly the parameterization of sea-ice in the current model utilizes
“levitating” sea-ice (Losch et al. 2010), meaning the sea-ice always stays on top of the
free surface. This assumption neglects the forcing caused by sea-ice advection
whenever surface velocity is different than the sea-ice velocity (Campin et al. 2008). Neglecting the actual draft of sea-ice also introduces an error in calculating the correct 33 distance from boundary which in turn makes the direction of velocity in Ekman spiral
unreliable. Omission of other physics such as tidal waves, which is a common trait
between arctic models, may as well have an effect on the velocity fields. Having established that the correlation between the model output and mooring or ITP
data is generally poor (i.e. 29% as reported above), we can further ask whether, in the
absence of data, we would be just as well served by assuming that the current speeds
are purely Ekman in nature. Using Eq. 4-6, as described above, we generated the
Ekman spiral under partial ice cover to measure the current speed using only
reanalysis winds. As with the model data, we compute the vertically-averaged current
speed form 0 to 20 m depth. Using this assumption of Ekman drift and reanalysis
winds, it is possible to reproduce the mooring velocity magnitude with 18%
correlation. Whereas the 29% correlation between model and mooring data is
unacceptable in most cases, it is still possible to argue that this is an improvement over
the Ekman drift assumption. This is easily explained by the fact that the model
captures more of the processes driving currents, including inertial oscillations. 3.7.1 Sources of error in water column current velocities In spite of the poor correlation between model and data, it captures many of the
processes that drive net velocity, including Ekman, geostrophic, internal wave, inertial
and subinertial processes. Consequently, we can use the model to determine the net
drift and compare it to a typical geochemical assumption for Lagrangian drift. Here we
refer to the approach employed by Rutgers Van Der Loeff et al. (2014). While
evaluating radon deficits in the water column beneath variable ice cover, those authors
assumed 0.5 cm/s bulk of mixed-layer speed and an elapsed time of 48 days to achieve
a total drift of ~ 20 km. The 20 km becomes the radius over which ice cover is 34 34 averaged to account for variations in surface area for exchange. We computed the 48
day back trajectory of water parcels originating from Mooring D for two years using
simulated velocities and found out that the average distance between the start and end
points is 10.3 Km, this number is 49% less that what was mentioned on previous
literature (Rutgers Van Der Loeff et al. 2014). This difference is due to water parcel
trajectory deviating from a straight line and having a circular motion between the start
and end point. averaged to account for variations in surface area for exchange. We computed the 48
day back trajectory of water parcels originating from Mooring D for two years using
simulated velocities and found out that the average distance between the start and end
points is 10.3 Km, this number is 49% less that what was mentioned on previous
literature (Rutgers Van Der Loeff et al. 2014). This difference is due to water parcel
trajectory deviating from a straight line and having a circular motion between the start
and end point. 35 35 SUMMARY We have used a 36-km version of the MITgcm to evaluate whether coarse-resolution
model output can be used to compensate for lack of data in the Arctic. The goal was to
understand if/how to use model output to interpret geochemical tracer fields. This
systematic comparison of upper ocean processes has revealed the following. With regard to configuration, which was necessary to capture vertical profiles in near
surface ocean, our experiments showed that changing the vertical resolution of model
grid cells reduced the accuracy of the sea-ice trajectory and concentration. This is
likely due to the fact that the model output was tuned with a different vertical grid
spacing (Nguyen et al. 2011) and the tuning coefficients are no longer optimal, once
we change that spacing. It has been shown by (Holloway et al. 2011) that optimized
parametrization of the model is sensitive to horizontal resolution; vertical resolution
may as well have the same effect on the model. We observed good correlation (78%) between model ice velocity and ITP drift. The
rapid accumulation of small errors in the drift speed and direction leads to significant
differences in the predicted and actual drift trajectory. We found errors in drift
trajectory of 2km/day. This relatively strong correlation is perhaps not surprising,
considering that more than half of the ice drift variability can be explained by Ekman
drift alone, and the model receives wind as an input parameter. It is also worth noting
that by doubling the model resolution, error decreases by more than half, i.e. 0.8
(km/day) accuracy by using a 18-km model (Nguyen et al. 2011). 36 The estimation of mixed layer depth is challenging: No algorithm performs well in all
situations; CTD profiles from drifting buoys often do not include the top 7-10 m of the
surface ocean where stratification can be important, and because the density structure
of the ocean is affected by vertical fluxes and by geostrophy. In these model-data
comparisons we found model MLD to be biased consistently shallower, but this in part
depends on a surface value, which is not recorded in moored drifters such as ITPs. The
evolution of the mixed layer showed that MLD correlates equally well (or poorly if
constrained by sea-ice) with wind and temperature trends (36% and 39% respectively). SUMMARY Despite the potential for bias in the model MLD, the time series of ITP and model
MLD reveal that the model captures variability at a similar frequency. It appears 10-
50% changes in MLD happen regularly over the course of days and major changes
(i.e. 100-200 %) appear to be event-driven as opposed to gradual seasonal evolution. The estimation of mixed layer depth is challenging: No algorithm performs well in all
situations; CTD profiles from drifting buoys often do not include the top 7-10 m of the
surface ocean where stratification can be important, and because the density structure
of the ocean is affected by vertical fluxes and by geostrophy. In these model-data
comparisons we found model MLD to be biased consistently shallower, but this in part
depends on a surface value, which is not recorded in moored drifters such as ITPs. The
evolution of the mixed layer showed that MLD correlates equally well (or poorly if
constrained by sea-ice) with wind and temperature trends (36% and 39% respectively). Despite the potential for bias in the model MLD, the time series of ITP and model
MLD reveal that the model captures variability at a similar frequency. It appears 10-
50% changes in MLD happen regularly over the course of days and major changes
(i.e. 100-200 %) appear to be event-driven as opposed to gradual seasonal evolution. The potential for such event-driven changes in the MLD in the time prior to sampling
are clearly important to consider when evaluating surface ocean geochemical tracer
fields, although it may be sufficient to look for large excursions in the wind, air
temperature and ice cover from reanalysis data. The A1 experiment showed a good visual representation of Arctic general circulation
but on finer scales, individual flows did not match the data from ITPs or Mooring. This discrepancy may be the result of two causes - the lack of eddy resolving
resolution and the lack of realistic ice physics. Holloway et al. (2011) have shown that
by increasing the resolution to less than 9 (km) a good agreement can be reached for
10 m vertical resolution. Our current lack of representation of velocity fields will lead 37 37 to outcomes that are broadly similar, but locally incorrect. We further saw this effect
on temperature profiles modeled on ITP path. to outcomes that are broadly similar, but locally incorrect. SUMMARY We further saw this effect
on temperature profiles modeled on ITP path. We compare each of the variables affecting gas-exchange and summarize our
comparison on Table-1. Overall, we find that a coarse-resolution model can yield as
much as 50% improvement over simple assumptions that are loosely constrained by
data (e.g. assuming Ekman drift or using a radius to average ice properties over a
given time period). However, the coarse resolution model is not up to the task of
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2014: Characterizing the eddy field in the Arctic Ocean halocline. J. Geophys. Res. BIBLIGRAPHY Oceans, 119, 8800–8817, doi:10.1002/2014JC010488. 46 FIG 1 A graphic illustration of two possible back trajectory of a single sampling station
FIG 2Arctic Sea-ice extent from 1992-2012 using A0 FIG 1 A graphic illustration of two possible back trajectory of a single sampling station FIG 1 A graphic illustration of two possible back trajectory of a single sampling station llustration of two possible back trajectory of a single sampling station FIG 2Arctic Sea-ice extent from 1992-2012 using A0 FIG 2Arctic Sea-ice extent from 1992-2012 using A0 47 FIG 3 Averaged satellite sea ice cover from 2006-2013, Solid red line marking 80% cover and
orange line marking 60%, Blue dots show the analysis grid, stars show the location of the three
points Cyan P1, Green P2, Red P3 where time series data is graphed in Fi FIG 3 Averaged satellite sea ice cover from 2006-2013, Solid red line marking 80% cover and
orange line marking 60%, Blue dots show the analysis grid, stars show the location of the three
points Cyan P1, Green P2, Red P3 where time series data is graphed in Fi FIG 4Time history of Sea-Ice fraction from top P1, P2 and P3, Satellite data compared with
model results A0 and A1 FIG 4Time history of Sea-Ice fraction from top P1, P2 and P3, Satellite data compared with
model results A0 and A1 48 Fig 5. Root mean square error averaged of A1 over time from 2006 to 2013; black mask covers
the grid points on the ground
Fig 6. Monthly averaged Annual Root Mean Square Error Fig 5. Root mean square error averaged of A1 over time from 2006 to 2013; black mask covers
the grid points on the ground Fig 5. Root mean square error averaged of A1 over time from 2006 to 2013; black mask covers
the grid points on the ground 5. Root mean square error averaged of A1 over time from 2006 to 2013; black mask c
the grid points on the ground
Fig 6. Monthly averaged Annual Root Mean Square Error Fig 6. Monthly averaged Annual Root Mean Square Error 49 Fig 7. Velocity components from A0 and A1 compared with drift velocity of ITP-53
Fig 8. 30 day trajectory of ITP 53, starting August 2011 to August 2012 Fig 7. BIBLIGRAPHY Velocity components from A0 and A1 compared with drift velocity of ITP-53 Fi 8 30 d
t
j
t
f ITP 53
t
ti
A
t 2011 t A
t 2012 Fig 8. 30 day trajectory of ITP 53, starting August 2011 to August 2012 50 50 Fig 9. Simulated and observed trajectory of ITP53, Aug 06 2011 to Aug 13 2012
Fig 10. MITgcm geopotential height referenced to 400(m), 2008-2011 Fig 9. Simulated and observed trajectory of ITP53, Aug 06 2011 to Aug 13 2012 ig 9. Simulated and observed trajectory of ITP53, Aug 06 2011 to Aug 13 2012
Fig 10. MITgcm geopotential height referenced to 400(m), 2008-2011 Fig 10. MITgcm geopotential height referenced to 400(m), 2008-2011 Fig 10. MITgcm geopotential height referenced to 400(m), 2008-2011 51 Fig 11. Obtained from ITP 1 on 13-Dec-2006 at 74.80°N and 131.44°W Fig 11. Obtained from ITP 1 on 13-Dec-2006 at 74.80°N and 131.44°W Fig 12: Obtained from ITP 1 on 28-Aug-2006 at 76.96°N and 133.32°W Fig 12: Obtained from ITP 1 on 28-Aug-2006 at 76.96°N and 133.32°W 52 Fig 13. Obtained from ITP-43 on 27-Nov-2010 at 75.41°N and 143.09°W Fig 13. Obtained from ITP-43 on 27-Nov-2010 at 75.41°N and 143.09°W Fig 14: Obtained from ITP-13 on 30-Jul-2008 at 75.00°N and 132.78°W Fig 14: Obtained from ITP-13 on 30-Jul-2008 at 75.00°N and 132.78°W 53 Fig 15. ITP-62 distribution of sampling stations and the circle centered at 75N 147 W and radius
of 330 (km) Fig 15. ITP-62 distribution of sampling stations and the circle centered at 75N 147 W and radius
of 330 (km)
Fig 16. Observed upper ocean density, ITP 62 Sep 04 2012 to 12 Aug 2013
Fig 17. Simulated upper ocean density following ITP-62 15. ITP-62 distribution of sampling stations and the circle centered at 75N 147 W and radius
of 330 (km) Fig 15. ITP-62 distribution of sampling stations and the circle centered at 75N 147 W and radius
of 330 (km) Fig 16. Observed upper ocean density, ITP 62 Sep 04 2012 to 12 Aug 2013 Fig 16. Observed upper ocean density, ITP 62 Sep 04 2012 to 12 Aug 2013 Fig 16. Observed upper ocean density, ITP 62 Sep 04 2012 to 12 Aug 2013 Fig 17. Simulated upper ocean density following ITP-62 Fig 17. Simulated upper ocean density following ITP-62 54 54 Fig. BIBLIGRAPHY 18: Sea Ice fraction and Modeled Ice thickness interpolated into ITP-62 drift Fig. 18: Sea Ice fraction and Modeled Ice thickness interpolated into ITP-62 drift Fig. 18: Sea Ice fraction and Modeled Ice thickness interpolated into ITP-62 drift 55 55 Fig 19: Different methods applied to simulated density profiles from a)ITP 1 winter b)ITP 43
winter c)ITP 1 summer d) ITP13 summer Fig 19: Different methods applied to simulated density profiles from a)ITP 1 winter b)ITP 43
winter c)ITP 1 summer d) ITP13 summer 56 Fig 20: Mixed layer depth time series based on M1, simulation and data from ITP-62 Fig 20: Mixed layer depth time series based on M1, simulation and data from ITP-62 Fig 20: Mixed layer depth time series based on M1, simulation and data from ITP-62 Fig 21: Mixed layer depth time series based on M2, simulation and data from ITP-62 Fig 21: Mixed layer depth time series based on M2, simulation and data from ITP-62 Fig 21: Mixed layer depth time series based on M2, simulation and data from ITP-62 57 57 Fig 22: a) Mixed layer evolution during ITP 62 drift b) Sea-Ice fraction c) Wind Speed 10m above
the surface d) Air temperature Fig 22: a) Mixed layer evolution during ITP 62 drift b) Sea-Ice fraction c) Wind Speed 10m above Fig 22: a) Mixed layer evolution during ITP 62 drift b) Sea-Ice fraction c) Wind Speed 10m above
the surface d) Air temperature 58 58 Fig 23: Cross Correlation of Mixed layer depth with wind speed and air temperature Fig 23: Cross Correlation of Mixed layer depth with wind speed and air temperature 59 59 Fig 24. Model velocities averaged at 180-250(m) , 2006-2012 Fig 24. Model velocities averaged at 180-250(m) , 2006-2012 60 60 Fig 25. Model velocities averaged from 1-15 (m), 2006-2012 Fig 25. Model velocities averaged from 1-15 (m), 2006-2012 61 61 Fig 26. ITP-V Speed simulated and observed
Fig 27. ITP-V Eastward velocity observed and simulated Fig 26. ITP-V Speed simulated and observed Fig 26. ITP-V Speed simulated and observed
Fig 27. ITP-V Eastward velocity observed and simulated Fig 26. ITP-V Speed simulated and observed Fig 26. ITP-V Speed simulated and observed Fig 27. ITP-V Eastward velocity observed and simulated 62 Fig 28. ITP-V Northward velocity observed and simulated
Fig 29. ITP-V correlation versus depth Fig 28. ITP-V Northward velocity observed and simulated Fig 29. BIBLIGRAPHY ITP-V correlation versus depth 63 Fig 30. Mooring-D Observed and simulated Speed Fig 30. Mooring-D Observed and simulated Speed 64 64 Fig 31. Mooring-D eastward velocity observed, simulated and estimated Fig 31. Mooring-D eastward velocity observed, simulated and estimated 65 65 Fig 32. Mooring-D northward velocity observed, simulated and Estimated Fig 32. Mooring-D northward velocity observed, simulated and Estimated 66 TABLES TABLES Sea ice
fraction
Sea ice
drift
Sea ice
velocity
Mixed layer
depth
Water current
velocity for
back trajectory
calculation
Coarse
resolution
3D
simulation
Between
98% to 88%
correlation
2 Km d -1
error
78%
correlation
Model
follows
mixed layer
evolution
trend
29% correlation
between model
velocity and
mooring data
Estimation
from direct
observation
Satellite data
readily
available
Remote
sensing
estimates
available. Coverage is
spotty. Remote sensing
estimates
available. Coverage is
spotty. Mooring or
ITP, but not
available
inside
Lagrangian
water parcel
Mooring or ITP,
but not available
inside lagrangian
water parcel
Estimation
from simple
assumptions
No need for
estimation
4.2 Km d -1
error
65%
correlation
Not
quantified
18% correlation
between Ekman
flow and
mooring data. Take home
message
Use satellite
data
Use satellite
data where
available. Model
trajectories
may have
value as
ensemble
averages
Sea ice velocity
from model has
a good
correlation and
is valuable
Absolute
value of
MLD can be
biased, but
model can
reveal the
potential for
changes
Ekman
assumption is
easier to
implement,
albeit less
accurate by 11%. Simulations
indicate a radius
(10.3 km) for
averaging
properties. 67
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The coefficient of technical readiness as an indicator of the effectiveness of the strategy of technical operation of lifting and transport equipment of terminals
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The coefficient of technical readiness as an
indicator of the effectiveness of the strategy of
technical operation of lifting and transport
equipment of terminals Igor Zub1,*, Yuri Yezhov1, and Nikolai Stenin1
1Admiral Makarov State University of Maritime and Inland Shipping, Russia 1Admiral Makarov State University of Maritime and Inland Shipping, Russia Abstract. Technical operation of lifting and transport equipment is one of
the key problems of ports and terminals. The functions of technical operation
include maintenance and repair of lifting and transport equipment. The
imperfection of the maintenance and repair system leads to downtime of
lifting and transport equipment, which affects the cost of loading and
unloading operations. The main requirements for technical operation are to
ensure the quality and speed of loading and unloading operations with
minimal operating costs. One of the indicators for assessing the quality of
technical operation is the coefficient of technical readiness, which shows
that the probability that the lifting and transport equipment will be in a
working state at any time, except for periods in which operation is not
provided. These periods include the time when the lifting and transport
equipment is being maintained and repaired. Technical operation strategy is
selected based on the technical readiness coefficient. © 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/). * Corresponding author: zubiv@gumrf.ru E3S Web of Conferences 244, 08009 (2021)
EMMFT-2020 E3S Web of Conferences 244, 08009 (2021)
EMMFT-2020 https://doi.org/10.1051/e3sconf/202124408009 E3S Web of Conferences 244, 08009 (2021)
EMMFT-2020 E3S Web of Conferences 244, 08009 (2021)
EMMFT-2020 TO is a complex organizational and technical system, which is a subsystem of a
stevedoring company (StC), and has an important direction in the strategic development plan. When creating a strategic plan for the development of the StC, a technical policy strategy is
worked out that ensures competitiveness. Currently, there may be several StCs located on the
territory of the port, which are separate enterprises and compete with each other. The main
areas of the technical policy of the StC include: areas of the technical policy of the StC include:
- selection of LTE (manufacturer model range new or from the secondary market); p
y
- selection of LTE (manufacturer, model range, new or from the secondary market); - service life of the LTE (operation of the LTE up to: a certain operating time of engine
hours; maximum technical condition; a certain calendar operating time); - service life of the LTE (operation of the LTE up to: a certain operating time of engine
hours; maximum technical condition; a certain calendar operating time); - strategy of TO (carrying out maintenance (M): by the schedule, in the certain operating
time, the number of processed goods; carrying out repair: (R) according to the schedule, to
technical condition, in the case of failure); - strategy of TO (carrying out maintenance (M): by the schedule, in the certain operating
time, the number of processed goods; carrying out repair: (R) according to the schedule, to
technical condition, in the case of failure); )
- selection of repair personnel (full-time or signing a contract with an outsourcing
organization). - selection of repair personnel (full-time or signing a contract with an outsourcing
organization). 2 Research methods On the basis of the analysis and synthesis of scientific research, the analysis of the possibility
of using the technical readiness coefficient (KTR) as an indicator of the effectiveness of the
chosen TO strategy is carried out. Based on the generalization of the results obtained, it was
concluded that the KTR of LTE can serve not only as an indicator of the effectiveness of the
TO strategy, but also influence its choice. 1 Introduction Technical operation (TO) of lifting and transport equipment (LTE) is one of the key
problems of ports and terminals. The functions of technical operation include maintenance
and repair (MR) of the LTE. The imperfection of the MR system leads to downtime of the
LTE, which affects the cost of loading and unloading operations (LUO). The main
requirements for technical operation are to ensure the quality and speed of loading and
unloading operations with minimal operating costs. Currently, there is no single approach to
the technical operation of port lifting and transport equipment. This situation can be explained
by the following reasons: 1) outdated rules of technical operation of lifting and transport equipment (RD 31.01.02-
04) [1, 2]; 1) outdated rules of technical operation of lifting and transport equipment (RD 31.01.02-
04) [1, 2];
2) th
i t
f th
d
i
t
d 2) the variety of the used equipment used; 3) the lack of modern scientific studies of the processes of technical operation of the port
LTE. https://doi.org/10.1051/e3sconf/202124408009 E3S Web of Conferences 244, 08009 (2021)
EMMFT-2020 E3S Web of Conferences 244, 08009 (2021)
EMMFT-2020 https://doi.org/10.1051/e3sconf/202124408009 When deciding whether to reduce the LUO time by changing the technology, it is
necessary to address the question of economic feasibility. In this case, events can develop in
two ways: y
1) The change of the LUO technology does not entail a change in the LTE fleet; 2) The change of the LUO technology is only possible with a complete or partial
replacement of LTE fleet. Both in the first and in the second variants, the main indicators are: cargo traffic, the
productivity of the LTE fleet, the development strategy of the StC. The “efficiency-cost” indicator can serve as an express analysis of the feasibility of
introducing new equipment [4]: Кef.-cost = PLTE /С, (1) (1) where PLTE - is the performance of the new LTE, C - is the cost of the new LTE and operating
costs. Next, the unit cost of a unit of productivity (TEU/rub., m3/rub., ton/rub., etc.) of the LTE
is determined: kunit cost = 1/Кef.-cost, (2) (2) kunit cost = 1/Кef.-cost, whence it follows that: kunit cost = С/ PLTE. (3) kunit cost = С/ PLTE. (3) When comparing indicators К ef.-cost.1 – for the new LTE and К ef.-cost.2 – for the operated
LTE, it is advisable to replace the LTE if the inequality is met: Кef.-cost.2 – К ef.-cost.1 > 0, (4) (4) Кef.-cost.2 – К ef.-cost.1 > 0, or kunit cost.2 – kunit cost.1 < 0. (5) (5) If equation (5) is less than zero for any values kunit cost.1 and kunit cost.2, then it is economically
feasible to introduce a new LTE. The operation of the LTE is associated with a reduction in its resource. The reduction of
the resource is due to the aging processes of the metal, which results in the formation of
cracks in the metal structures, which lead to changes in the operating parameters, as a result
of which a failure or accident may occur. Corrosion and friction processes occurring in the
friction pairs lead to wear and destruction of parts of aggregates and mechanisms. To ensure
the operational state of the LTE, regardless of the financial state of the StC, a TO system is
being developed. 3 Results The TO strategy should be optimal in terms of the indicator that characterizes the quality of
operation of the LTE. When choosing the optimal TO strategy, the possibility of adjusting
the rules of technical operation (RTO) is taken into account. The indicators of the quality of
operation of the LTE include: operation of the LTE include: - coefficient of technical readiness of KTR; - the probability of completing the task (operational readiness coefficient) Kop; - coefficient of technical use of KTU; - average profit per unit of calendar time S; - average costs per unit of operating time C. To select the optimal strategy, it is proposed to use the minimax method, which consists
in first finding the worst (in terms of quality) among the distribution functions that
characterize the operation of a technical object, and then determining the optimal control
under these conditions [3]. The TO strategy of LTE is formed on the basis of: - available operational experience (failure statistics, operating time to failure); - the coefficient of technical readiness of KTR; - the coefficient of technical use of KTU. The trend in the development of modern StC terminals involves reducing the downtime
of vehicles (V) on the LUO. This indicator can be achieved by maintaining and ensuring the
operability of the LTE. The processing speed of the vehicle is affected not only by a working
LTE, but also by the LUO technology, the availability of a reserve and the use of modern
high-performance LTE. Not all ports or StCs located on their territory can have a reserve
LTE or perform technical re-equipment of the terminal due to the high cost of modern LTE. 2 E3S Web of Conferences 244, 08009 (2021)
EMMFT-2020 The main task of the TO of LTE is to maintain and / or restore a working, and in some
cases, serviceable technical condition, as well as to monitor compliance with the current
norms and rules during the operation of the LTE. The efficiency of the LTE is ensured both
by the prevention and timely elimination of failures, and by the organization of diagnostics
of nodes and mechanisms. Based on the analysis of the obtained diagnostic results, the
reliability of LTE is predicted. The availability of statistical data and diagnostic results allows
calculating the average operating time for failure of individual units and mechanisms. To
prevent an emergency stop of the LTE, preventive technical measures are carried out, spare
parts are ordered, which reduces the time spent by LTE under repair. Based on statistical data
and diagnostic results, routine maintenance and repair works (MR) are planned, which allows
forming a staff of repair personnel and effectively manage them, regardless of their
organizational form (full-time or outsourced organization). The implementation of MR schedules does not always give the desired result – the
absence of failures. The search for optimal management of TO has identified various 3 E3S Web of Conferences 244, 08009 (2021)
EMMFT-2020 https://doi.org/10.1051/e3sconf/202124408009 strategies, which are based on different principles for restoring and sustaining the efficiency
of LTE of organizational and technical measures. The recovery time (trecovery) of LTE depends
on the development and implementation of organizational and technical measures, which is
one of the criteria for evaluating the TO system and is subject to optimization. strategies, which are based on different principles for restoring and sustaining the efficiency
of LTE of organizational and technical measures. The recovery time (trecovery) of LTE depends
on the development and implementation of organizational and technical measures, which is
one of the criteria for evaluating the TO system and is subject to optimization. The most common system of organizational and technical measures to maintain and
restore the working condition of technical facilities is the system of planned preventive
repairs (PPR), within which a system of MR is being developed. In the PPR system, each
technical object undergoes a repair cycle during operation through certain periods of time,
which can be expressed in calendar terms of inter-repair periods or depending on the
operating time of the engine hours. E3S Web of Conferences 244, 08009 (2021)
EMMFT-2020 The PPR system implies a rational staffing table and
distribution of employees of the repair service, the order of spare parts and consumables,
tools, auxiliary equipment used for the repair of LTE components and mechanisms. For each
planned repair, a technological card of the repair work is developed, and the start and end
time of the repair is planned. p
p
At the same time, the PPR system has a number of disadvantages: p
p
At the same time, the PPR system has a number of disadvantages: - equipment repairs are carried out without the actual need; - equipment repairs are carried out without the actual need; - the need to take the equipment out of operation, followed by full or partial disassembly
to carry out defect detection of parts; - the need to take the equipment out of operation, followed by full or partial disassembly
to carry out defect detection of parts; the need to take the equipment out of operation, followed by full or partial disassembly
to carry out defect detection of parts; to carry out defect detection of parts; - replacement of components and parts with a large residual resource with new ones. Analyzing the shortcomings of the PPR, a number of authors [5, 6, 7, 8, 9, 10, 11, 12, 13]
consider that with the transition to a market economy, the PPR system has lost its relevance,
and the system of repairs for technical condition is more effective. Depending on the
operating conditions, the criteria for carrying out repairs on the technical condition are
selected. These criteria include: control of parameters, when the equipment is taken out for
repair when the parameters reach the critical zone, and reliability, when the criteria is the
reliability of the equipment. When controlling the parameters, the frequency of repairs is set
depending on the technical condition of the parts, the process of failure of which is gradual,
relative to the limits of their tolerances. When monitoring the reliability of equipment, the
volume and frequency of repairs are set depending on the level of reliability of the equipment
[4, 14, 15]. E3S Web of Conferences 244, 08009 (2021)
EMMFT-2020 E3S Web of Conferences 244, 08009 (2021)
EMMFT-2020 Equation (7) represents the utilization coefficient (Ku) of the LTE in the condition j, if
this is the objective condition of operation. In this case (μj/lij) - is the objective function of
the technical operation process and this function can be considered as the Ku, and the
maximum of the function as the criteria for the optimality of technical operation. At the same
time, the Ku shows the ratio of the time when the LTE is in working or operable condition
for a given period of operation, to the time of working or operable condition and all downtime
for maintenance and repairs for the considered period of operation. In the absence of cargo
traffic, the LTE can be taken out for preventive works, but this will immediately reduce the
Ku, therefore, the Ku at full load of the terminal can serve as an additional indicator of the
efficiency of technical operation. Two TO strategies are considered in work [16]: the Ca strategy - only emergency repairs
are carried out, and the C0 strategy - periodic repairs are carried out. When the LTE is
operating without failure for the specified repair period (tмрп) then preventive repair is carried out. The criteria for selecting the strategy were adopted: criteria for selecting the strategy were adopte g
gy
- minimum cost of repair work for a certain operational period R(t) → min; - statistics of failures of LTE units and mechanisms; - statistics of failures of LTE units and mechanisms; - coefficient of technical readiness of LTE KTR → max. - coefficient of technical readiness of LTE KTR → max. When performing mathematical calculations, the authors came to the conclusion that the
repair costs during the time between failures are less than in the production of preventive
repairs, therefore, according to the authors, it is not economically profitable to carry out
preventive repairs. When performing mathematical calculations, the authors came to the conclusion that the
repair costs during the time between failures are less than in the production of preventive
repairs, therefore, according to the authors, it is not economically profitable to carry out
preventive repairs. E3S Web of Conferences 244, 08009 (2021)
EMMFT-2020 Analyzing the shortcomings of the PPR, a number of authors [5, 6, 7, 8, 9, 10, 11, 12, 13]
consider that with the transition to a market economy, the PPR system has lost its relevance, In addition to these two repair strategies, there is a strategy for repairing equipment af
its failure [14, 15, 16, 17]. Any strategy must be effective and have a feasibility study. One of the parameters that
can be used to evaluate the effectiveness of the LTE is the coefficient of technical readiness
(KTR) [3]. LTE has different technical conditions, but it can only be in one condition at the same
time i = 1, 2, …, n out of the many possible ones (i ∈ E). At the initial moment of time t = 0,
the LTE is in a working or operable condition. During operation t, the LTE changes from
condition i to j. In the condition j, the LTE stays for a random time before moving to the next
condition. The probability of such a transition will have the form [5, 18]: Pij(t) = PijFij(t), (6) Pij(t) = PijFij(t), (6) ere Fij(t) – function of distribution of transition time from the condition i to j. where Fij(t) – function of distribution of transition time from the condition i to j. es with communicating conditions, we obtain the following equation: For finite processes with communicating conditions, we obtain For finite processes with communicating conditions, we obtain the following equation: lij
t
P
j
ij
=
)
(
lim
, (7) (7) where lij – average time to the first hit of the LTE from the condition i to j, μj – average time
of staying in the condition j. where lij – average time to the first hit of the LTE from the condition i to j, μj – average time
of staying in the condition j. 4 https://doi.org/10.1051/e3sconf/202124408009 E3S Web of Conferences 244, 08009 (2021)
EMMFT-2020 In real operating conditions, it is characteristic that the time-to-failure distribution
function Fa(t) and the preventive repair distribution function Fp(t) do not coincide: Fa(t) ≠ Fp(t) (8) (8) Fa(t) ≠ Fp(t) The inequality (8) allows choosing the optimal strategy from the strategies C0 and Ca. Continuing the research on the choice of the TO strategy, the R(t) and KTR functions were
presented in the form of [19]: ( )
( )
( )
+
+
=
t
t
p
a
a
p
a
p
p
a
dt
t
F
t
F
dt
t
F
t
F
t
F
с
t
F
c
t
R
0
0
)
(
)
(
)
(
)
(
, (9)
KТR(t) =
1
R1(t)+1, (10) ( )
( )
( )
+
+
=
t
t
p
a
a
p
a
p
p
a
dt
t
F
t
F
dt
t
F
t
F
t
F
с
t
F
c
t
R
0
0
)
(
)
(
)
(
)
(
, (9) (9) (10) KТR(t) =
1
R1(t)+1, (10) where cа and cр – average costs for emergency and preventive repair, respectively; R1(t) –
cost function, when replaced in the function R(t) cа and cр to Ta and Tp – the average time
spent on emergency and preventive repair, respectively. It follows from (10) that the
maximum KTR is reached at the minimum point of the function R1(t). When distributing the operating time after emergency and preventive repairs according
to exponential laws: Fa(t) = 1 – e-at, Fp(t) = 1 – ept, a, p > 0, (11) (11) and when the following inequality is satisfied: (12) k < 1/(1 + п), с = ср/са, k = р/а, (12) k < 1/(1 + п), с = ср/са, k = р/а, in accordance with economic indicators, the С0 strategy is optimal compared to the Са
strategy. But in case when p = a, Fa(t) = Fp(t), carrying out preventive repairs will be
impractical, and the optimal strategy in this case will be Са. in accordance with economic indicators, the С0 strategy is optimal compared to the Са
strategy. But in case when p = a, Fa(t) = Fp(t), carrying out preventive repairs will be
impractical, and the optimal strategy in this case will be Са. E3S Web of Conferences 244, 08009 (2021)
EMMFT-2020 5 E3S Web of Conferences 244, 08009 (2021)
EMMFT-2020 https://doi.org/10.1051/e3sconf/202124408009 We consider in the work [20] the problem of finding the lower confidence bound of the
KTR for systems with recoverable elements by a method based on the use of confidence
bounds for the reliability parameters of individual elements with the same confidence
coefficient. The essence of this method is that this coefficient for a single element passes to
the entire system and a change in the technical condition of one part can lead to changes in
the technical condition of the unit or mechanism. The organization of preventive works for MR in the TO system allows maintaining the
KTR at the optimal level, which can be set: KTR → opt., the evaluation of the efficiency of the
operation of the StC terminal’s of LTE fleet can be carried out using the KTR, which is a
comprehensive indicator of the reliability of a technical object and is determined by the
equation: КТR =
Тр
Тр+ТВР, (13) (13) where Tр and TВР – accordingly, the execution time of LUO of LTE and restoration of the
technical object's efficiency, hours: where Tр and TВР – accordingly, the execution time of LUO of LTE and restoration of the
technical object's efficiency, hours: ТВР = ТM + Тsc.m + Тem.r, (14) (14) where ТM – time spent on maintenance, Тsc.m – the time spent on scheduled maintenance,
Тem.r – time spent on emergency repairs. where ТM – time spent on maintenance, Тsc.m – the time spent on scheduled maintenance,
Тem.r – time spent on emergency repairs. The concept of interval KTR is introduced to assess the reliability of LTE at the time
interval required for performing LUO [17]. In this case, the KTR is considered as an indicator
of the performance of the LTE of its functional purpose with a high probability. When
determining the KTR, the equation (13) is presented as: ТВР
Тр =
1−КТR
КТR . (15) (15) From the equation (15), it is possible to determine the ratio of the duration of TВР and Tр,
which is assigned at the KTR required to perform the technological operation. If we consider LTE as a system with independent recovery of elements, then the lower
confidence bound for KTR is constructed by the method of confidence bounds for the
reliability parameters of individual elements of the LTE with the same confidence coefficient. 4 Discussion The considered TO strategies throughout the life cycle of the LTE depend on the adopted
technical policy of the StC. The effectiveness of the functioning and management of the TO
system of LTE requires further research. The existing separate studies of TO strategies are
not combined into a single methodological complex, and were developed under certain
operating conditions of technical objects. The effectiveness of the chosen TO strategy can be estimated by the value of the KTR,
since this coefficient takes into account only the downtime of the LTE during emergency
repairs. Planned MR works are not taken into account when calculating the KTR, since at this
moment the LTE is in working or operable condition. When calculating the Ku, all LTE downtime is taken into account, even when routine
maintenance is performed in “windows”, so the Ku cannot be used as an indicator of the
effectiveness of the selected TO strategy. of LTE. This approach to determining the definition of KTR and ТВР is possible only with one-time
work. At StC terminals, this approach can only be applied when handling single shipments
or cargo (for example, non-dimensional cargo). In the equation (13), the ТВР indicator, which
is subject to optimization, in particular, when reducing the downtime for emergency repairs,
entails an increase in the value of KTR. The equation (10) given in work [19] expresses the
economic dependence of the KTR on the quantity and quality of the repair and restoration
work carried out. The use of KTR shows the effectiveness of the TO strategy and takes into
account the reliability of the LTE in the process of its operation. E3S Web of Conferences 244, 08009 (2021)
EMMFT-2020 The confidence factor for LTE is maintained when transition from individual elements to the
LTE as a whole. Since the system availability factor KTR (u) = exp[f(u)], we can also calculate the lower
γ- confidence bound for the coefficient of system readiness [19]: КТR
¯
= КТR(u
¯ ) = ∏
Кi(ui)
m
i=1
, (16) (16) where ui = ui(γ) –lower confidence bounds for the parameters of individual elements with the
same confidence coefficient; γ, т – is the number of LTE subsystems. Equation (14) shows that the lower confidence bound for the KTR is preserved during the
transition from individual elements to the LTE as a whole. Failure prevention leads to an
increase of KTR and minimizes operating costs. The impact of KTR on the cost of LUO can be estimated through a measure of
efficiency, which can be represented as the MEF coefficient: МEF =
ΔКТR
Δ(ТВР
Тр ), (17) (17) where ΔКТR= Кi+1 – Ki –the increment of the technical readiness coefficient of the LTE during
the transition from one technical condition i to another (i + 1), where ΔКТR= Кi+1 – Ki –the increment of the technical readiness coefficient of the LTE during
the transition from one technical condition i to another (i + 1), 6 https://doi.org/10.1051/e3sconf/202124408009 E3S Web of Conferences 244, 08009 (2021)
EMMFT-2020 1
+
−
=
i
р
ВР
i
р
ВР
р
ВР
Т
Т
Т
Т
Т
Т
– increment of time ТВР and Тр when changing reliability
f LTE References 1. RD 31.1.02-04. Rules for the technical operation of lifting and transport equipment in
commercial seaports (CJSC “Central Research and Design Institute of the Navy”, St. Petersburg, 2004) 2. I.V. Zub, Y.E. Ezhov, V.A. Sidorenko, Bulletin of the Admiral Makarov State University
of Maritime and Inland Shipping 10(6), 1152-1161 (2018) DOI: 10.21821/2309-5180-
2018-10-6-1152-1161. 3. D.V. Varnakov, V.V. Varnakov, M.E. Dezhatkin, Bulletin of the Ulyanovsk State
Agricultural Academy 2(38), 168 – 173 (2017) DOI 10.18286/1816-4501-2017-2-168-
173 4. S.I. Makarenko, Control, communication, and security systems 1, 278-287 (2016) 5. N.N. Smirnov, A.A. Itskovich, Maintenance and repair of aircraft equipment according
to the condition (Transport, M., 1987) 6. G.D. Kokorev, I.A. Uspensky, I.N. Nikolotov, Agro engineering 3, 72 – 75 (2009) 7. V.S. Kvaginidze, N.A. Koretskaya, N.N. Chupeikina, V.V. Akimenko, A.V. Akhremenkov, Mining Information and Analytical Bulletin (scientific and technical
journal) S3, 310 – 314 (2011) 8. E.M. Bashirova, U.F. Yumaguzin, R.T. Yulberdin, Transportation and storage of
petroleum products and hydrocarbons 1, 18-22 (2014) 7 7 https://doi.org/10.1051/e3sconf/202124408009 E3S Web of Conferences 244, 08009 (2021)
EMMFT-2020 9. A.T. Martirosyan,
Internet-journal
“NAUKOVEDENIE”
9(6)
(2017)
https://naukovedenie.ru/PDF/164EVN617 10. A.L. Manakov, A.Y. Kirpichnikov, T.K. Tyunyukova, Bulletin of the Irkutsk State
Technical University 5(100), 127-131 (2015) 11. O.N. Osnovina, L.M. Boeva, Mining Information and Analytical Bulletin (scientific and
technical journal) 3, 251-255 (2015) 12. E.E. Bueshev, A.N. Potapov, Scientific Bulletin of the military-industrial complex of
Russia 4, 80 – 86 (2016) 13. G. Medvedeva, A. Museridze, Y. Tikhonova, A. Kryukov, A. Zaydullin, SAPR
(computer-aided design tools) and Graphics 1, 84-89 (2013) 14. A.I. Vinnik, N.G. Makarenko, A.M. Smirnov, A.A. Shargaev, Bulletin of the Samara
Scientific Center of the Russian Academy of Sciences 18.1(2), 161 – 165 (2016) 15. V.A. Ivanov, A.A. Feshchenko, Bulletin of the Perm National Research Polytechnic
University. Mechanical engineering, materials science 20(3), 82-89 (2018) DOI:
10.15593/2224-9877/2018.3.10 16. I.I. Vainshtein, G.E. Mikhalchenko, Y.V. Vainshtein, K.V. Safonov, Bulletin of
Reshetnev Siberian State University of Science and Technology 2(54), 20-25 (2014) 17. A.T. Lebedev, A.A. Seregin, A.G. Arzhenovsky, Improving the efficiency of the
functioning of machines and equipment of the agro-industrial complex by managing the
reliability of their systems 2(46), 4-11 (2019) 18. A.N. Dyakov, Proceedings of the Military Space Academy named after A. F. Mozhaisky
660, 180 – 189 (2018) 19. I.I. Vainshtein, G.E. Mikhalchenko, Y.V. Vainshtein, K.V. Safonov, Vestnik Reshetnev
Siberian State University of Science and Technology 16(3), 645-650 (2015) 20. I.V. Pavlov, S.V. References Razgulyaev, Bulletin of the Bauman Moscow State Technical
University. Natural sciences 4, 15 – 22 (2015) 8 8
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Development of the Uterine Cervix and Its Implications for the Pathogenesis of Cervical Cancer
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Springer eBooks
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Essentials of Diagnostic Gynecological
Pathology Series Editors
Naveena Singh
London, UK Series Editors
Naveena Singh
London, UK
W. Glenn McCluggage
Lebanon, UK W. Glenn McCluggage
Lebanon, UK The Essentials of Diagnostic Gynecological Pathology series is sponsored by
the British Association of Gynecological Pathologists. These lavishly illus-
trated books cover the pathology of the wide but sometimes rare range of
conditions involving this area. The volumes in this series are written to be
useful diagnostically to general as well as specialist gynecological histopa-
thologists and pathologists in training. Gynecologists, oncologists, derma-
tologists, genitourinary physicians and cancer nurse specialists will find
expert insights here that will help in the treatment and counselling of their
patients. More information about this series at http://www.springer.com/series/10115 C. Simon Herrington
Editor Editor
C. Simon Herrington
University of Edinburgh
Edinburgh Cancer Research Centre
Institute of Genetics and Molecular Medicine
Western General Hospital
Edinburgh
UK Editor
C. Simon Herrington
University of Edinburgh
Edinburgh Cancer Research Centre
Institute of Genetics and Molecular Medicine
Western General Hospital
Edinburgh
UK Essentials of Diagnostic Gynecological Pathology
ISBN 978-3-319-51255-6 ISBN 978-3-319-51257-0 (eBook)
DOI 10.1007/978-3-319-51257-0 Library of Congress Control Number: 2017941256 © Springer International Publishing AG 2017
This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or
part of the material is concerned, specifically the rights of translation, reprinting, reuse of
illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way,
and transmission or information storage and retrieval, electronic adaptation, computer software,
or by similar or dissimilar methodology now known or hereafter developed. y
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The use of general descriptive names, registered names, trademarks, service marks, etc. in this
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The registered company is Springer International Publishing AG p
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The registered company is Springer International Publishing AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland Preface It is a great privilege to edit this volume on cervical pathology as part of the
series of books on gynecological pathology. The structure of the book is simi-
lar to other volumes in the series, beginning with a discussion of normal
structure, in this case focusing particularly on the cervical transformation
zone and the putative cells of origin of neoplastic cervical lesions. Cervical
disease is dominated by the effects of human papillomavirus (HPV) infec-
tion, and the content of the book reflects this, with both a specific chapter on
HPV and discussion of HPV and its relationship to cervical lesions in several
of the other chapters. Chapters on cervical screening and the management of
cervical cancer are followed by sequential treatment of benign, preinvasive,
and invasive squamous and glandular lesions of the cervix, both HPV- and
non-HPV-related. Essentials of Diagnostic Gynecological Pathology
ISBN 978-3-319-51255-6 ISBN 978-3-319-51257-0 (eBook)
DOI 10.1007/978-3-319-51257-0 The final chapters focus on mesenchymal and mixed
tumors, and other neoplasms including neuroendocrine tumors. Appendices
outlining specimen handling, tumor staging, and the use of frozen section
diagnosis are also included. I am extremely grateful to the chapter contributors for their excellent con-
tributions. Thanks go also to the publishers, who have seen the project through
to completion. Finally, I would like to thank the series editors for asking me
to edit this volume, which I hope will be of value to those who read it. Edinburgh, UK C. Simon Herrington C. Simon Herrington Preface to the Series Over the last few decades, the study of cervical cancer has elucidated not only
mechanisms of oncogenesis but also the fantastic potential of an effective
cancer prevention strategy. Given this knowledge, it is lamentable that this
preventable cancer continues to afflict more than half a million women world-
wide annually. More than three-quarters of all cases occur in low-resource
nations, and in wealthier nations, there are striking differences between privi-
leged and underprivileged areas. The differences in incidence are mirrored in
mortality statistics, with far higher proportions of fatal cases in women who
have never been screened. It is important therefore to continuously strive towards improving out-
comes, starting with diagnostic accuracy. While the human papillomavirus
(HPV) accounts for the vast majority of cervical cancers, recent years have also
thrown light on the biology of the less frequent but generally more aggressive
HPV-independent cancers which are mainly of glandular type. Advances in
diagnostic adjuncts including biomarkers and efforts to standardize terminol-
ogy and classification have the potential to improve diagnostic reproducibility. Disease outcomes have improved with newer treatment strategies designed to
reduce the adverse effects of treatment and to promote fertility-conserving
options, while clinical trials continue to address unanswered questions. The British Association of Gynaecological Pathologists (BAGP) was
formed with the objectives to promote the health of women by the study
of the pathology of gynecological diseases, to advance the knowledge and
practice of gynecological pathology, and to improve the accuracy of patho-
logical diagnosis. The BAGP fulfills these objectives through educational
meetings, courses, collaborative projects, surveys, and posting of edu-
cational material on its website (www.thebagp.org). Endorsement of this
textbook series is part of its goal to promote accuracy and precision in diag-
nostic gynecological pathology. It is with pleasure and pride that we present this third volume of the series. Essentials of Diagnostic Gynecological Pathology
ISBN 978-3-319-51255-6 ISBN 978-3-319-51257-0 (eBook)
DOI 10.1007/978-3-319-51257-0 Gynecological pathology forms a major part of the workload of most histopa-
thology laboratories. The female genital tract is complex, and the main inten-
tion behind producing this series is to provide detailed information on specific
areas in a compact and affordable format. We hope that this timely update
will be of interest to trainee and consultant pathologists worldwide. Naveena Singh
W. Glenn McCluggage London, UK
Belfast, UK London, UK
Belfast, UK vii Contents 1 Development of the Uterine Cervix and Its Implications
for the Pathogenesis of Cervical Cancer�������������������������������������� 1
Anton H.N. Hopman and Frans C.S. Ramaekers
2 Human Papillomaviruses (HPVs)������������������������������������������������ 21
Kate Cuschieri and Ramya Bhatia
3 Cervical Screening: History, Current Algorithms,
and Future Directions�������������������������������������������������������������������� 45
John H.F. Smith
4 Surgical and Nonsurgical Management of Cervical
Cancer: Current Practice and Future Directions ���������������������� 67
Melanie E. Powell and Tim Mould
5 Benign Lesions of the Cervix�������������������������������������������������������� 79
C. Simon Herrington
6 Cervical Squamous Intraepithelial Lesions�������������������������������� 91
Anne M. Mills and Mark H. Stoler
7 Squamous Cell Carcinoma of the Cervix������������������������������������ 115
Naveena Singh and Lars-Christian Horn
8 Endocervical Adenocarcinoma In Situ/Cervical
Glandular Intraepithelial Neoplasia and Adenocarcinoma
of the Usual Type���������������������������������������������������������������������������� 149
Rosemary H. Tambouret and David C. Wilbur
9 Non-Human-Papillomavirus (HPV)-Related
Adenocarcinomas and Their Precursors������������������������������������� 185
Yoshiki Mikami
10 Mesenchymal and Mixed Epithelial-Mesenchymal
Neoplasms of the Cervix���������������������������������������������������������������� 201
W. Glenn McCluggage
11 Other Cervical Neoplasms������������������������������������������������������������ 213
Martin C. Chang and Terence J. Colgan ix Contents x Appendix 1: Surgical Cut Up of Cervical Specimens������������������������ 237
Appendix 2: Dataset for Reporting Cervical Neoplasia �������������������� 247
Appendix 3: TNM and FIGO Staging of Cervical
Carcinoma (ICD-O C53) ���������������������������������������������������������������������� 253
Appendix 4: Frozen Section Analysis in Cervical Carcinoma���������� 257
Index�������������������������������������������������������������������������������������������������������� 261 Contributors Ramya Bhatia, PhD, MRes, BSc University of Edinburgh, HPV Research
Group, Division of Pathology, Queens Medical Research Institute,
Edinburgh, UK Martin C. Chang, MD, PhD Department of Laboratory Medicine and
Pathobiology, University of Toronto, Mount Sinai Hospital, Pathology and
Laboratory Medicine, Toronto, ON, Canada Terence J. Colgan, MD Laboratory Medicine and Pathobiology, University
of Toronto, Mount Sinai Hospital, Department of Pathology and Laboratory
Medicine, Toronto, ON, Canada Kate Cuschieri, BSc, PhD, FRCPath Royal Infirmary of Edinburgh, NHS
Lothian, Scottish HPV Reference Laboratory, Edinburgh, UK C. Simon Herrington, MA, DPhil, FRCP, FRCPE, FRCPath University
of Edinburgh, Edinburgh Cancer Research Centre, Institute of Genetics and
Molecular Medicine, Western General Hospital, Edinburgh, UK Anton H.N. Essentials of Diagnostic Gynecological Pathology
ISBN 978-3-319-51255-6 ISBN 978-3-319-51257-0 (eBook)
DOI 10.1007/978-3-319-51257-0 Hopman, PhD Department of Molecular Cell Biology,
GROW-School for Oncology and Developmental Biology, Maastricht
University Medical Center, Maastricht, The Netherlands Anton H.N. Hopman, PhD Department of Molecular Cell Biology,
GROW-School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands Lars-Christian Horn, MD, PhD University Hospital of Leipzig, Institute
of Pathology, Division of Breast, Gynecologic and Perinatal Pathology,
Leipzig, Germany W. Glenn McCluggage, FRCPath Belfast Health and Social Care Trust,
Department of Pathology, Belfast, UK Yoshiki Mikami, PhD, MD Department of Diagnostic Pathology,
Kumamoto University Hospital, Chuo-ku, Kumamoto, Japan Anne M. Mills, MD University of Virginia Health System,
Department of Pathology, Charlottesville, VA, USA Tim Mould, MBBS, MA, DM, FRCOG Gynaecological Cancer Centre,
University College, London Hospitals, London, UK Melanie E. Powell, MD, FRCR, FRCP Clinical Oncology, St. Bartholomew’s Hospital, Barts Health NHS Trust, London, UK Bartholomew’s Hospital, Barts Health NHS Trust, London, UK xi xi Contributors xii Frans C.S. Ramaekers, PhD Department of Molecular Cell Biology,
GROW-School for Oncology and Developmental Biology, Maastricht
University Medical Center, Maastricht, MD, The Netherlands Naveena Singh, MD, FRCPath Barts Health NHS Trust, Department of
Cellular Pathology, London, UK John H.F. Smith, BSC, MB, BS, FRCPath Department of Histopathology
and Cytology, Royal Hallamshire Hospital, Sheffield, UK Mark H. Stoler, MD University of Virginia Health System, Department of
Pathology, Charlottesville, VA, USA Rosemary
H. Tambouret,
MD Massachusetts
General
Hospital,
Department of Pathology, and Harvard Medical School, Boston, MA, USA David C. Wilbur, MD Massachusetts General Hospital and Harvard Medical
School, Department of Pathology, Boston, MA, USA
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Nutritional Interactions in the Cnidarian-Dinoflagellate Symbiosis and their Role in Symbiosis Establishment
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Nutritional Interactions in the Cnidarian-Dinoflagellate
Symbiosis and their Role in Symbiosis Establishment Ashley E. Sproles Victoria University of Wellington
2017 2 2 Abstract Mass bleaching events induced by climate change are threatening coral reef ecosystems
worldwide. Elevated seawater temperatures cause breakdown of the coral’s endosymbiotic
relationship with dinoflagellate algae from the genus Symbiodinium. Corals rely on these
symbionts for the majority of their metabolic carbon (provided by photosynthetic products)
and for efficient nitrogen cycling in the reef ecosystem. Researchers have predicted that corals
may potentially adapt to higher ocean temperatures according to the Adaptive Bleaching
Hypothesis (ABH), which states that bleaching can facilitate a change in symbiont
communities, allowing more thermally tolerant symbionts to become dominant. Hosting
thermally tolerant symbionts thus grants the coral a higher resistance to bleaching. However,
confidence in this adaptation method has wavered due to the nutritional impairments of hosts
harbouring thermally tolerant symbionts under normal environmental conditions. It is also
unknown if coral species that do not currently associate with thermally tolerant symbionts
would be able to successfully switch their symbiont communities. Here, I explore the
mechanisms driving nutritional exchange in the symbiosis and determine the effect of
establishing a non-native (heterologous) association with a thermally tolerant symbiont. A
combined approach of bioinformatic analysis with proteomic and isotopic labelling
experiments is used to uncover a link between host-symbiont cellular integration, the potential
for nutrient exchange, and the success of establishing a symbiosis. In Chapter 2, I characterized membrane protein sequences discovered in publicly available
cnidarian and Symbiodinium transcriptomes and genomes to identify potential transporters of
sugars into cnidarian cells and nitrogen products into Symbiodinium cells. I examined the
facilitated glucose transporters (GLUT), sodium/glucose cotransporters (SGLT), and
aquaporin (AQP) channels in the cnidarian host as mechanisms for sugar uptake, and the
ammonium and high-affinity nitrate transporters (AMT and NRT2, respectively) in the algal
symbionts as mechanisms for nitrogen uptake. Homologous protein sequences were used for
phylogenetic analysis and tertiary structure deductions. In cnidarians, I identified putative
glucose transporters of the GLUT family and glycerol transporting AQP proteins, as well as
sodium monocarboxylate transporters and sodium myo-inositol cotransporters homologous to
SGLT proteins. I predict that cnidarians use GLUT proteins as the primary mechanism for
glucose uptake, while glycerol moves into cells by passive diffusion. I also identified putative 3 3 AMT proteins in several Symbiodinium clades and putative NRT2 proteins only in a single
clade. I further observed a high expression of putative AMT proteins in Symbiodinium, which
may have resulted from adaptations to conditions experienced inside the host cell. Abstract This study
is the first to identify transporter sequences from a diversity of cnidarian species and
Symbiodinium clades. The phylogenetic patterns seen in chapter 2 led to the hypothesis that symbiont types may have
different influences on host-symbiont cellular integration. In chapter 3, I explored this notion
using the model cnidarian Aiptasia. A population of anemones was rendered aposymbiotic
using a menthol-bleaching method developed by my colleagues and I, and anemones then
experimentally infected with either the native (homologous) symbiont (Symbiodinium
minutum, clade B1) or a thermally tolerant heterologous symbiont (Symbiodinium trenchii,
clade D1a). The response of the host proteome to these associations was examined by analysing
the extracted host proteins with liquid chromatography-nano-electrospray-tandem mass
spectrometry (LC-nano-ESI-MS/MS), and identifying resulting peptides against a cnidarian
database. Proteins were compared between B1-colonised anemones, D1a-colonised anemones,
and aposymbiotic anemones to determine which proteins were affected by the different
treatments. Overall, I found that the response of D1a-colonised anemones mimicked that of
aposymbiotic anemones to some degree, and showed signs of less efficient carbon and nitrogen
pathways. Additionally, I discovered that the symbiosome protein NPC2 was upregulated only
in B1-colonised anemones and could therefore be an important determinant of symbiont
success. I further investigated the apparent inadequacy of carbon and nitrogen pathways in hosts
harbouring Symbiodinium D in chapter 4, using isotope labelling and sub-cellular imaging. Carbon and nitrogen fluxes between experimentally infected Aiptasia with both homologous
(Symbiodinium B1) and heterologous (Symbiodinium D1a) symbionts were compared over
multiple time points during symbiosis establishment. This was accomplished by incubating
anemones in 13C- and 15N-labelled seawater to measure metabolite transfer to the host, and
anemones were also fed 13C- and 15N-labelled Artemia to measure reverse translocation to the
symbiont. Carbon and nitrogen enrichment in host tissues and symbiont cells were imaged
using nanoscale secondary ion mass spectrometry (nanoSIMS). In both the earlier stages of 4 4 symbiosis establishment (2 days post-inoculation) and later stages of symbiont proliferation
(14 days post-inoculation), Symbiodinium B1 reached significantly higher population densities
(> 300%) than Symbiodinium D1a. Differences in symbiont nitrogen uptake from host tissues
were detectable, however they were likely due to nitrogen limitation imposed by the
comparatively high densities of the homologous symbiont. Acknowledgements I owe much gratitude to the many brilliant scientists I have met during my life that have inspired
me to continue pursuing academic research. I have incredible appreciation for my
undergraduate professors from Florida Institute of Technology, Dr. Ralph Turingan, Dr. Richard Turner, and Dr. Rob van Woesik, who provided me with exceptional instruction during
my undergraduate studies and motivated me to continue exploring the marine environment. Additionally, I thank Dr. Joshua Voss from Harbor Branch Oceanographic Institute for being
a mentor during my first experience in coral reef research, and the entire Voss lab for their
valued knowledge and friendship. I am also grateful to Dr. Clay Cook for sharing with me his
infinite wisdom on the cnidarian-dinoflagellate symbiosis, and for encouraging me to
participate in this project. Of course, this thesis would not have been possible without the help
of my supervisor, Dr. Simon Davy, whom I thank for believing in me and providing me with
much helpful advice and opportunities for academic collaboration. I would also like to thank
my secondary supervisor, Dr. Clint Oakley, for his assistance with my proteomics work and
perseverance with working out any technical difficulties we faced. A big thank you also goes
out to the rest of the Davy lab members for providing a positive and intellectually stimulating
work environment. This research was possible thanks to the financial support of the Royal Society of New
Zealand’s Marsden Fund, and many thanks are also due to the VUW Faculty of Science for
awarding me a Faculty Strategic Research Grant that allowed me to present my research at the
2016 International Coral Reef Symposium. I was lucky to be afforded collaborative research
trips to Hawaii, Oregon, and Switzerland throughout my study, where those at the hosting
institutions helped in making my research goals possible. Thank you to Nathan Kirk and Sheila
Kitchen (Oregon State University) for teaching me bioinformatics and phylogenetics, Thomas
Krueger and Anders Miebom (École Polytechnique Fédérale de Lausanne) for hosting me in
their lab and allowing me to use the nanoSIMS, also thanks to Ruth Gates, Hollie Putnam, and
the rest of the Gates lab (Hawaii Institute of Marine Biology) for their assistance with my field
work in Hawaii, to Tina Carvalho (University of Hawaii at Manoa) for allowing me to book
out the only bench space in the microscopy lab for an entire two weeks, and to St. Abstract While Symbiodinium B1 cells
maintained a high degree of carbon fixation throughout symbiosis establishment, less than 50%
of Symbiodinium D1a cells were found to be fixing carbon in the later stages of establishment,
despite still receiving substantial amounts of host-derived nitrogen. These findings support my
previous prediction that Symbiodinium D1a affects the host’s carbon and nitrogen pathways,
acting as a less mutually beneficial symbiont. Overall, my results provide new insights into the underlying factors determining the success of
symbiotic interactions. The results indicate that cnidarian hosts and their dinoflagellate
symbionts have adapted to the symbiotic life-style by cellular integration, thereby making
compatibility of certain biological processes a key factor in determining symbiosis success. Furthermore, the findings presented here show how the potential for nutritional exchange may
be linked to host-symbiont compatibility and hence specificity. This information is relevant
when making predictions about which corals may successfully adapt to climate change via the
ABH. 5 5 6 6 Acknowledgements John
Wakefield and Jane Anderson (University of Otago) for allowing me the use of their facilities
to process an excessive amount of samples in a very short time frame. Special 7 7 acknowledgements are also due for Arthur Grossman (The Carnegie Institute), for critiquing
my work to the highest standards and providing excellent suggestions to my experimental
designs, and for Virginia Weis (OSU), for acting almost as an additional supervisor by
investing a considerable amount of time and effort into helping design, execute, and edit my
work. Throughout my time at Victoria University I have been lucky to acquire many friends that have
provided valuable moral support. I would like to especially thank my fellow Davy lab member,
Jennifer Matthews, for being an incredible friend and constant source of advice and support
throughout the past four years. I would also like to thank Grace Paske, Emily McGrath, Megan
Schaffer, Becky Focht, Steph Price, Kirsty Yule, Amanda Taylor, and Katie Hillyer for keeping
me sane with many fun-filled evenings and adventures away from the lab. I sincerely thank my parents, Karen and Bud, for teaching me to be ambitious in all my life
endeavours and supporting me unconditionally throughout my academic journey. I also thank
my brother, Dan, for graciously letting me attempt to claim the Dr. Sproles title before him. Lastly, many thanks to Josh for his willingness to move 8,000 miles away from our home
country to support me during my studies, and for having much patience and sense of adventure
along the way. 8 8 List of Abbreviations 13C
Isotopically-labelled carbon
15N
Isotopically-labelled nitrogen
3D
Three dimensional
AA
Amino acids
AB
Accumulation body
ABC
ATP-binding cassette
ABH
Adaptive bleaching hypothesis
AMT
Ammonium transporter
ANOVA
Analysis of Variance
APC
Acid-polyamine-organocation
ATP
Adenosine triphosphate
AQP
Aquaporin
BHMT
Betaine-Homocysteine S-Methyltransferase
BLAST
Basic local alignment search tool
BSA
Bovine serum albumin
C
Carbon
CA
Carbonic anhydrase
CCM
Carbon concentrating mechanism
Chl
Chloroplast
CHT
Choline transporter
CO2
Carbon dioxide
COACH
Consensus approach
CS
Circumsporozoite
CZAR
Carbon contribution of algae to animal respiration
DCMU
3-(3,4-dichlorophenyl)-1,1-dimethylurea
df
Degrees of freedom
DIC
Dissolved inorganic carbon
DIN
Dissolved inorganic nitrogen
DMSO
Dimethyl sulfoxide
DNA
Deoxyribonucleic acid
EM
Electron microscopy 13C
15N
3D
AA
AB
ABC
ABH
AMT
ANOVA
APC
ATP
AQP
BHMT
BLAST
BSA
C
CA
CCM
Chl
CHT
CO2
COACH
CS
CZAR
DCMU
df
DIC
DIN
DMSO
DNA 13C
Isotopically-labelled carbon
15N
Isotopically-labelled nitrogen
3D
Three dimensional
AA
Amino acids
AB
Accumulation body
ABC
ATP-binding cassette
ABH
Adaptive bleaching hypothesis
AMT
Ammonium transporter
ANOVA
Analysis of Variance
APC
Acid-polyamine-organocation
ATP
Adenosine triphosphate
AQP
Aquaporin
BHMT
Betaine-Homocysteine S-Methyltransferase
BLAST
Basic local alignment search tool
BSA
Bovine serum albumin
C
Carbon
CA
Carbonic anhydrase
CCM
Carbon concentrating mechanism
Chl
Chloroplast
CHT
Choline transporter
CO2
Carbon dioxide
COACH
Consensus approach
CS
Circumsporozoite
CZAR
Carbon contribution of algae to animal respiratio
DCMU
3-(3,4-dichlorophenyl)-1,1-dimethylurea
df
Degrees of freedom
DIC
Dissolved inorganic carbon
DIN
Dissolved inorganic nitrogen
DMSO
Dimethyl sulfoxide
DNA
Deoxyribonucleic acid
EM
Electron microscopy 13C
15N
3D
AA
AB
ABC
ABH
AMT
ANOVA
APC
ATP
AQP
BHMT
BLAST
BSA
C
CA
CCM
Chl
CHT
CO2
COACH
CS
CZAR
DCMU
df
DIC
DIN
DMSO
DNA
EM 9 9 ER
Endoplasmic reticulum
FDR
False discovery rate
FSW
Filtered seawater
GLM
Generalized linear model
GLUT
GO
Facilitated glucose transporter
Gene ontology
G-protein
Guanine nucleotide binding protein
h
Hour(s)
H2O
Water
HCl
Hydrochloric acid
HCO3-
Bicarbonate ion
HMIT
Proton/myo-inositol cotransporter
HPLC
High performance liquid chromatography
HRF
Host release factors
I-TASSER
Iterative threading assembly refinement
IRMS
Isotope-ratio mass spectrometry
ITS2
Internal transcriber spacer 2
K15NO3
Potassium nitrate
LC-nano-ESI-MS/MS
Liquid chromatography-nano electrospray-tandem mass spectrometry
LD
Lipid droplets
MAFFT
Multiple alignment using fast fourier transform
Mep
Methylammonium permease
MFI
Mean fluorescence index
MFS
Major facilitator superfamily
min
Minute(s)
MS
Mass spectrometry
MS/MS
Tandem mass spectrometry
N
Nitrogen
NaHCO3
Sodium bicarbonate
NaNO3
Sodium nitrate
NanoSIMS
Nanoscale secondary ion mass spectrometry
NCBI
National Center for Biotechnology Information
NIP
Nodulin-26-like proteins 10 NMDS
Non-metric multidimensional scaling
NPC2
Niemann-Pick type C
NRT2
High-affinity nitrate transporter 2
OCT
Organic cation transporters
OsO4
Osmium tetroxide
P:R
Ratio of maximum gross photosynthesis to respiration
PCR
Polymerase chain reaction
Pgross
Maximum gross photosynthesis
PI
Precursor intensity
PPM
Parts per million
Pyr
Pyrenoid
qPCR
Quantitative real-time PCR
RNA
Ribonucleic acid
ROI
Region of interest
ROS
Reactive oxygen species
SGLT
Sodium/glucose cotransporter
SLC
Solute carrier
SMCT
Sodium-coupled monocarboxylate transporters
SMIT
Sodium/myo-inositol cotransporter
Symb
Symbiosome
TM
Transmembrane
TP1
Time point 1
TP2
Time point 2
UA
Uric acid crystals
UniProtKB
UniProt knowledgebase NMDS
Non-metric multidimensional scaling
NPC2
Niemann-Pick type C
NRT2
High-affinity nitrate transporter 2
OCT
Organic cation transporters
OsO4
Osmium tetroxide
P:R
Ratio of maximum gross photosynthesis to respiration
PCR
Polymerase chain reaction
Pgross
Maximum gross photosynthesis
PI
Precursor intensity
PPM
Parts per million
Pyr
Pyrenoid
qPCR
Quantitative real-time PCR
RNA
Ribonucleic acid
ROI
Region of interest
ROS
Reactive oxygen species
SGLT
Sodium/glucose cotransporter
SLC
Solute carrier
SMCT
Sodium-coupled monocarboxylate transporters
SMIT
Sodium/myo-inositol cotransporter
Symb
Symbiosome
TM
Transmembrane
TP1
Time point 1
TP2
Time point 2
UA
Uric acid crystals
UniProtKB
UniProt knowledgebase 11 List of Figures List of Figures Figure 1.1
Transmission electron micrographs of symbiotic Aiptasia tissue. 13C
15N
3D
AA
AB
ABC
ABH
AMT
ANOVA
APC
ATP
AQP
BHMT
BLAST
BSA
C
CA
CCM
Chl
CHT
CO2
COACH
CS
CZAR
DCMU
df
DIC
DIN
DMSO
DNA 21
Figure 1.2
The six phases of symbiosis establishment. 23
Figure 1.3
The cnidarian-dinoflagellate symbiosis supports coral reefs. 25
Figure 1.4
Two methods of symbiont community change under the ABH. 29
Figure 1.5
Intercellular metabolite exchange between host and symbiont. 32
Figure 1.6
Mechanisms of secondary active transport and channel proteins. 33
Figure 2.1
Maximum likelihood phylogenetic inference of GLUT proteins. 48
Figure 2.2
Protein structural analysis of selected sequences from each
family. 51
Figure 2.3
Maximum likelihood phylogenetic inference of SGLT sequences. 53
Figure 2.4
Maximum likelihood phylogenetic inference of AQP sequences. 55
Figure 2.5
Maximum likelihood phylogenetic inference of AMT sequences. 58
Figure 2.6
Maximum likelihood phylogenetic inference of NRT2 sequences. 61
Figure 3.1
NMDS plot showing grouping of Aiptasia protein samples. 86
Figure 3.2
Venn diagram comparison of annotated proteins exclusive to one
or two treatment groups. 88
Figure 3.3
Biological processes and molecular function GO terms for
successfully mapped proteins significantly different between B1-
and D1a-colonised anemones. 89
Figure 3.4
Functional implications of upregulated proteins in Aiptasia, along
a continuum of symbiotic success. 102
Figure 4.1
Infection success of Symbiodinium types B1 and D1a in Aiptasia. 116
Figure 4.2
Nitrogen and carbon enrichment of host and symbiont tissues in
heterotrophy and autotrophy experiments. 118
Figure 4.3
NanoSIMS images of nitrogen and carbon enrichment within
Aiptasia tissue across time points. 120
Figure 5.1
Conceptual diagram of the cellular processes during varying
degrees of symbiont compatibility. 134
Figure A1
Motifs used for manual editing of the GLUT alignment. 177
Figure A2
Motifs used for manual editing of the SGLT alignment. 177
Figure A3
Motifs used for manual editing of the AQP alignment. 178 12 12 Figure A4
Motifs used for manual editing of the AMT alignment. 178
Figure A5
Motifs used for manual editing of the AQP alignment. 178
Figure C1
Predicted symbiosome membranes. 187
Figure C2
Density gradient symbiosome isolation steps. 188 Figure A4
Motifs used for manual editing of the AMT alignment.
178
Figure A5
Motifs used for manual editing of the AQP alignment.
178
Figure C1
Predicted symbiosome membranes.
187
Figure C2
Density gradient symbiosome isolation steps.
188 13 List of Tables List of Tables Table 3.1
Proteins significantly different between Symbiodinium B1 and
aposymbiotic treatments. 77
Table 3.2
Proteins significantly different between Symbiodinium D1a and
aposymbiotic treatments. 81
Table 3.3
Proteins significantly different between Symbiodinium B1 and
D1a treatments. 85
Table 4.1
Range of N and C enrichment values between the time points
and symbiont types. 119
Datasheet A1
Characterization data for newly identified cnidarian GLUT
sequences. 179
Disc
Datasheet A2
Characterization data for newly identified cnidarian SGLT
sequences. 179
Disc
Datasheet A3
Characterization data for newly identified cnidarian AQP
sequences. 179
Disc
Datasheet A4
Characterization data for newly identified Symbiodinium AMT
sequences. 179
Disc
Datasheet A5
Characterization data for newly identified Symbiodinium NRT2
sequences. 179
Disc
Table A1
List of resources used in custom cnidarian and Symbiodinium
BLAST databases. 180
Table A2
Reference proteins used as queries for homology searches of
each protein group. 183
Table B1
Carbon enrichment of host tissue and symbiont cells in the
heterotrophy experiment. 185
Table B2
Nitrogen enrichment of host tissue and symbiont cells in the
autotrophy experiment. 186 14 Table of Contents
Abstract ...................................................................................................................................... 3
Acknowledgements .................................................................................................................... 7
List of Abbreviations ................................................................................................................. 9
List of Figures .......................................................................................................................... 12
List of Tables ........................................................................................................................... 14
Chapter 1: General Introduction .............................................................................................. 19
1.1 Endosymbiosis as a Basis for Entire Ecosystems ....................................................... 19
1.2 Cnidarian-Dinoflagellate Symbiosis ........................................................................... 20
1.2.1 Structure of the cnidarian-dinoflagellate symbiosis ............................................ 20
1.2.2 Uptake, recognition, and regulation of symbionts ............................................... 22
1.3 Significance of Coral Reefs ........................................................................................ 25
1.3.1 Ecological importance ......................................................................................... 25
1.4 Symbiont Diversity and Specificity ............................................................................ 26
1.4.1 Genetic diversity within Symbiodinium............................................................... 26
1.4.2 Host-symbiont specificity ..................................................................................... 27
1.4.3 Thermally tolerant symbionts as a mechanism of adaptation ............................. 29
1.5 Metabolite Exchange between Partners ...................................................................... 30
1.5.1 Translocation of photosynthetic products ............................................................ 30
1.5.2 Reverse translocation........................................................................................... 31
1.5.3 Mechanisms of inter-partner transport ................................................................ 33
1.6 “Omics” Investigations of Nutritional Transport ....................................................... 34
1.6.1 Transcriptomics and genomics ............................................................................ 34
1.6.2 Identification of symbiosis-associated proteins ................................................... 35
1.6.3 Aiptasia as a model organism .............................................................................. 36
1.7 Aims and Objectives ................................................................................................... 37
Chapter 2: Phylogenetic characterization of transporter proteins in the cnidarian-
dinoflagellate symbiosis........................................................................................................... 41
2.1. Introduction ............................................................................................................... 41
2.2. Methods ..................................................................................................................... 43
2.2.1 Identification of cnidarian and Symbiodinium transporter proteins .................. 43
2.2.2 Retrieval of protein homologs .............................................................................. 44
2.2.3 Sequence alignment and phylogenetic analysis ................................................... 44
2.2.4 Structural predictions and characterization of proteins ...................................... 45
2.3. Figure A4
Motifs used for manual editing of the AMT alignment.
178
Figure A5
Motifs used for manual editing of the AQP alignment.
178
Figure C1
Predicted symbiosome membranes.
187
Figure C2
Density gradient symbiosome isolation steps.
188 Results and Discussion .............................................................................................. 46 Table of Contents 15 2.3.1 Cnidarian GLUT proteins .................................................................................... 46
2.3.2 Cnidarian SGLT proteins ..................................................................................... 50
2.3.3 Metazoan AQP proteins ....................................................................................... 54
2.3.4 Symbiodinium AMT proteins ............................................................................... 56
2.3.5 Symbiodinium NRT2 proteins ............................................................................. 59
2.4. Conclusions ............................................................................................................... 62
Chapter 3: Proteomic changes show that thermally-tolerant Symbiodinium may impair host
cellular processes ..................................................................................................................... 65
3.1 Introduction ................................................................................................................ 65
3.2 Methods ...................................................................................................................... 68
3.2.1 Experimental organisms ...................................................................................... 68
3.2.2 Infection of Aiptasia with Symbiodinium ............................................................ 69
3.2.3 Analysis of infection success ................................................................................ 70
3.2.4 Symbiont verification ........................................................................................... 71
3.2.5 Protein extraction ................................................................................................ 72
3.2.6 Identification of proteins using LC-MS/MS ......................................................... 72
3.2.7 Statistical analysis ............................................................................................... 74
3.3 Results ........................................................................................................................ 75
3.3.1 Infection success .................................................................................................. 75
3.3.2 Proteins unique to treatment groups.................................................................... 76
3.4 Discussion ................................................................................................................... 89
3.4.1 Impact of symbiont density................................................................................... 90
3.4.2 Proteins associated with symbiotic and aposymbiotic states .............................. 90
3.4.3 Proteins upregulated in symbiotic versus aposymbiotic anemones ..................... 92
3.4.3.1 Previously identified proteins in symbiotic Aiptasia ....................................... 92
3.4.3.2 Redox and breakdown processes ...................................................................... 94
3.4.3.3 Cell signalling ................................................................................................... 94
3.4.3.4 Tissue structure and muscle functioning ........................................................ 95
3.4.3.5 Cellular and metabolic processes ..................................................................... 95
3.4.4 Homologous (B1) versus heterologous (D1a) symbiont effects on host proteome
....................................................................................................................................... 96
3.4.4.1 Metabolic processes .......................................................................................... 97
3.4.4.2 Proteolysis and gene processing ...................................................................... 97
3.4.4.3 Immune responses ............................................................................................ 98
3.4.4.4 Multifunctional cellular processes................................................................... 99 16 3.4.4.5 Protein assembly ............................................................................................. 100
3.4.5 Conclusions ........................................................................................................ 101
Chapter 4: Symbiodinium type effects nutrient exchange and symbiont success in Aiptasia 105
4.1 Introduction .............................................................................................................. 105
4.2 Methods .................................................................................................................... 107
4.2.1 Experimental organisms .................................................................................... 107
4.2.2 Infection of Aiptasia with Symbiodinium .......................................................... 107
4.2.3 Analysis of colonisation success ........................................................................ 109
4.2.4 Confirmation of symbiont genotype ................................................................... 110
4.2.5 Isotopic labelling of carbon and nitrogen compounds ...................................... 111
4.2.5.1 Preparation of isotope solutions .................................................................... 111
4.2.5.2 Heterotrophic nitrogen translocation ............................................................ 112
4.2.5.3 Autotrophic C and N fixation and translocation........................................... 112
4.2.6 NanoSIMS sample preparation and enrichment measurements ........................ 113
4.2.7 NanoSIMS image analysis ................................................................................. 114
4.2.8 Statistical analysis ............................................................................................. 114
4.3 Results ...................................................................................................................... 115
4.3.1 Infection success ................................................................................................ 115
4.3.2 Heterotrophy experiment ................................................................................... 116
4.3.3 Autotrophy Experiment ...................................................................................... 119
4.4 Discussion ................................................................................................................. 121
4.4.1 Infection dynamics of Symbiodinium B1 versus Symbiodinium D1a ............... 121
4.4.2 The effect of symbiont type on heterotrophic metabolic translocation .............. 122
4.4.3 The effect of symbiont type on autotrophic metabolite translocation ................ 123
4.4.4 Conclusions ........................................................................................................ Figure A4
Motifs used for manual editing of the AMT alignment.
178
Figure A5
Motifs used for manual editing of the AQP alignment.
178
Figure C1
Predicted symbiosome membranes.
187
Figure C2
Density gradient symbiosome isolation steps.
188 124
Chapter 5: General Discussion............................................................................................... 127
5.1 Summary ................................................................................................................... 127
5.2 Host-symbiont integration determines functionality of symbiosis ........................... 128
5.2.1 How has the integration of cellular processes allowed for adaptation to a
symbiotic life-style? .................................................................................................... 128
5.2.2 Does the degree of cellular compatibility determine where host-symbiont pairs
fall on the mutualism-parasitism continuum? ............................................................ 132
5.3 Implications for coral adaptability via thermally tolerant symbionts ....................... 136
5.4 Limitations and future work ..................................................................................... 137
5.5 Conclusions .............................................................................................................. 138 3.4.4.5 Protein assembly .. 17 References .............................................................................................................................. 141
Appendix A: Protein sequences and genetic resources.......................................................... 177
Appendix B: Carbon and nitrogen enrichment values ........................................................... 185
Appendix C: Additional Symbiosome Work ......................................................................... 187
Publications ............................................................................................................................ 189 18 1.1 Endosymbiosis as a Basis for Entire Ecosystems Symbiosis is defined as the close interaction between different species living together
over a long period of time (Freedman 2014). The term as we know it today was first coined in
1877 by German botanist Heinrich Anton de Bary, who observed that lichens were two
organisms, a fungus and an alga, living essentially as one (cited in Sapp 2004). The remainder
of the nineteenth century was a pinnacle of exploration into the complexities of symbiotic
associations. In 1881, Karl Andreas Heinrich Brandt concluded from his observations, that
photosynthetic pigments found in animals were due to symbiotic plant invaders within the
animal cells (Krueger 2016). Nearly a century thereafter, ‘The Endosymbiont Theory’ was first
formulated to describe the origin of all plant and animal cells. The theory postulated that
chloroplasts and mitochondria originated from symbiotic prokaryotes which evolved into the
fully integrated organelles of eukaryotes that we see today (Margulis 1971; Werner 1992). Symbiotic interactions can take a variety of forms, which are described by the following three
categories: (1) parasitism, where one partner benefits at a cost to the other; (2) commensalism,
where neither partner benefits nor is harmed; and (3) mutualism, where both partners benefit
from the relationship (Roossinck 2005). To date, numerous microorganisms have been
described as endosymbionts that form complex mutualisms with plants and animals. These
symbioses involve cellular integration of the symbiont into the host’s cells, and can become
the basis of entire ecosystems by facilitating nutrient cycling. For instance, legume plants
harbour populations of nitrogen-fixing soil bacteria (rhizobia) within their root nodules that
facilitate nitrogen-carbon exchange with the host (Werner 1992; Udvardi & Day 1997). Many
plants outside the legume family also utilize nitrogen-fixing symbionts in various plant
organelles (Santi et al. 2013). This mechanism is essential to many terrestrial ecosystems, since
nitrogen is a limiting factor for plant growth and nitrogen gas cannot be fixed by plants alone
(Santi et al. 2013). A range of marine invertebrates also forms ecologically significant
endosymbioses. In particular, the relationship between sulphur-oxidizing chemoautotrophic
bacteria and deep-sea marine invertebrates involves an exchange of nutrients that is so efficient
the animals completely lack a mouth and gut (Cavanaugh 1994). Symbiotic associations with
photosynthetic algae and bacteria are common among poriferans and cnidarians, primarily
because the relationships allows these animals to adopt features which help them survive in 19 otherwise uninhabitable locations, and in particular nutrient-poor seas (Venn et al. 2008). 1.1 Endosymbiosis as a Basis for Entire Ecosystems Studies of the symbiosis between the freshwater Hydra and green alga Chlorella have
discovered an intimate integration of host and symbiont metabolism that likely occurred as a
result of co-evolution between the two partners (Muscatine 1965; Muscatine & Lenhoff 1965;
Cook 1972; Kovačević et al. 2010). With functional similarities to the Hydra-Chlorella
association is the most well-known and ecologically important marine symbiosis of all, is that
between cnidarians and dinoflagellate algae of the genus Symbiodinium, which will be the
focus of the research described here. 1.2.1 Structure of the cnidarian-dinoflagellate symbiosis The algal symbionts reside within the cnidarian host’s gastrodermal cells, which can contain
up to six cells each (Muscatine et al. 1998). Surrounding each algal cell is a multilayer series
of membranes called the symbiosome membrane complex (Fig. 1.1; Wakefield et al. 2000;
Wakefield and Kempf 2001). The complex consists of an outer, host-derived membrane that is
formed by a phagosome undergoing early arrest (Palincsar et al. 1988; Rands et al. 1993;
Mohamed et al. 2016), and multiple symbiont-derived inner layers (Taylor 1971; Trench &
Winsor 1987). The inner membranes are thought to be sloughed-off layers of symbiont theca
(Taylor 1968; Trench & Blank 1987; Schwarz et al. 1999; Wakefield et al. 2000; Wakefield &
Kempf 2001). The symbiosome serves as the interface for all molecular exchanges between
host and symbiont and is also key to avoiding host digestion of the algal cells (Fitt & Trench
1983; Barott et al. 2015). It is still unknown exactly how and when during symbiosis
establishment the symbiosome is formed; however, a crucial component in symbiosome
biogenesis might be the retention of certain membrane proteins which regulate membrane
fusion with host endocytic vesicles, such as ApRab4 in the symbiont (Hong et al. 2009) and
sym32 in the host (Reynolds et al. 2000; Schwarz & Weis 2003). 20 Figure 1.1 Transmission electron micrographs of Symbiodinium cells within Aiptasia gastrodermal tissue. (A)
Symbiodinium cell housed inside a multi-layered membrane complex (Symb, symbiosome), containing
photosynthetic organelles (Chl, chloroplasts), a nucleus with chromosomes (C), lipid droplets (LD), uric acid
crystals (UA), and an accumulation body (AB). (B) Detail of the Symbiodinium pyrenoid (Pyr), emerging from
the chloroplast. Micrographs by A. Sproles. Figure 1.1 Transmission electron micrographs of Symbiodinium cells within Aiptasia gastrodermal tissue. (A)
Symbiodinium cell housed inside a multi-layered membrane complex (Symb, symbiosome), containing
photosynthetic organelles (Chl, chloroplasts), a nucleus with chromosomes (C), lipid droplets (LD), uric acid
crystals (UA), and an accumulation body (AB). (B) Detail of the Symbiodinium pyrenoid (Pyr), emerging from
the chloroplast. Micrographs by A. Sproles. Symbiodinium cells contain uric acid crystals (Fig. 1.1), which are produced as nitrogen stores
that can be readily utilized when environmental conditions become nitrogen-depleted (Clode
et al. 2009; Kopp et al. 2013). Cells also may contain a single-stalked pyrenoid capped by a
starch granule emerging from the chloroplast, as well as lipid droplets and accumulation bodies
(Fig. 1.1; Wakefield et al. 2000). 1.2.2 Uptake, recognition, and regulation of symbionts 1.2.2 Uptake, recognition, and regulation of symbionts Cnidarian hosts can obtain endosymbiotic Symbiodinium by either horizontal or vertical
transmission. Horizontal transmission involves acquisition of symbionts from the surrounding
environment, while vertical transmission involves the host parent passing symbionts to its
offspring (Stat et al. 2006)). Species that use horizontal transmission have the benefit of
potentially being able to form associations with a wide variety of symbiont types, while vertical
transmission guarantees the host a symbiont that has already been selected for as successful in
their species (Douglas 1998; Loh et al. 2001; Barneah et al. 2004). Most scleractinian coral
species produce aposymbiotic larvae, requiring them to acquire symbionts via horizontal
transmission (Babcock & Heyward 1986; Richmond & Hunter 1990; Abrego et al. 2009). Cnidarian larvae are only able to acquire symbionts from their environment once they have
formed a mouth, and usually do so while feeding (Schwarz et al. 1999; Bucher et al. 2016). Symbiodinium cells are attracted to the cnidarians by chemical cues, such as ammonia, that are
given off as they are feeding (Fitt 1984). In order to avoid digestion by the animal, a host-
symbiont recognition phase must occur during initial contact of algal and host cells (Fig 1.2). Symbiodinium cells release species-specific glycoconjugates that act as a form of inter-partner
signalling to the host (Markell et al. 1992; Markell & Trench 1993; Markell & Wood-Charlson
2010). Additionally, the algal cell walls are lined with an assemblage of glycoproteins that can
serve as recognition factors during symbiosis establishment, by binding to lectins on the animal
cells surface (Lin et al. 2000; Wood-Charlson et al. 2006; Wood-Charlson & Weis 2009; Logan
et al. 2010; Neubauer et al. 2016). These glycan/lectin interactions are part of an innate immune
response that, in vertebrates, is used to recognize and destroy pathogenic invaders (Mcguinness
et al. 2003). The signalling pathways of this response may play a role in both recognition and
maintenance of accepted symbionts (Poole et al. 2016; Bay et al. 2011). Certain genes have
also been hypothesized to play a role in symbiont recognition, such as scavenger receptors,
Sym32, carbonic anhydrase, and Niemann-Pick type C (NPC2) genes (Rodriguez-Lanetty et
al. 2006; Ganot et al. 2011; Dani et al. 2014). As algal cells make contact with the host, they are phagocytosed (Smith 1979; Fitt & Trench
1983). 1.2.1 Structure of the cnidarian-dinoflagellate symbiosis Pyrenoids act as starch reserves in most algae, and may
facilitate the distribution of photosynthetic products from the chloroplast to other organelles
(Griffiths 1970). Lipid droplet formation in Symbiodinium is enhanced by environmental
stressors such as elevated temperatures and nitrogen starvation (Jiang et al. 2014; Pasaribu et
al. 2016), and is thought to play a key role in the exchange of compounds between partners
(Kellogg & Patton 1983; Patton & Burris 1983; Pasaribu et al. 2014). Accumulation bodies are
poorly understood, but most likely act similarly to lysosomes, in being a ‘trash dump’ for the
cell (Taylor 1968; Wakefield et al. 2000). Interestingly, they are also enriched in the symbiosis-
related protein Sym32, a cell adhesion protein (Schwarz & Weis 2003). Symbiodinium cells contain uric acid crystals (Fig. 1.1), which are produced as nitrogen stores
that can be readily utilized when environmental conditions become nitrogen-depleted (Clode
et al. 2009; Kopp et al. 2013). Cells also may contain a single-stalked pyrenoid capped by a
starch granule emerging from the chloroplast, as well as lipid droplets and accumulation bodies
(Fig. 1.1; Wakefield et al. 2000). Pyrenoids act as starch reserves in most algae, and may
facilitate the distribution of photosynthetic products from the chloroplast to other organelles
(Griffiths 1970). Lipid droplet formation in Symbiodinium is enhanced by environmental
stressors such as elevated temperatures and nitrogen starvation (Jiang et al. 2014; Pasaribu et
al. 2016), and is thought to play a key role in the exchange of compounds between partners
(Kellogg & Patton 1983; Patton & Burris 1983; Pasaribu et al. 2014). Accumulation bodies are
poorly understood, but most likely act similarly to lysosomes, in being a ‘trash dump’ for the
cell (Taylor 1968; Wakefield et al. 2000). Interestingly, they are also enriched in the symbiosis-
related protein Sym32, a cell adhesion protein (Schwarz & Weis 2003). 21 1.2.2 Uptake, recognition, and regulation of symbionts After the recognition and phagocytosis phases, sorting occurs as the host decides
whether to reject or accept each newly acquired symbiont (Fig. 1.2). Although initial symbiont
uptake in new coral recruits may be fairly unspecific, as larvae mature they begin to exhibit 22 more specificity for certain symbiont types (Weis et al. 2001; Little et al. 2004; Thornhill et
al. 2006). During this sorting phase, rejected cells are destroyed via host cell apoptosis
(programmed cell death; Dunn and Weis 2009), while the accepted cells proliferate within the
host via cell division (Wilkerson et al. 1983, 1988). Figure 1.2 The six phases of symbiosis establishment (according to description in Davy et al. 2012). 1) Initial
contact between Symbiodinium and host cells, where recognition is facilitated by cell surface molecules from each
partner. 2) Phagocytosis of algal cell by the host cell. 3) Sorting of algal cells into accepted symbionts (green
arrow) to be housed in symbiosome membranes, and rejected cells (red arrow) which are removed via apoptosis,
autophagy, or exocytosis. 4) Proliferation of symbiont population by cell division. 5) Dynamic stability of
symbiont population. 6) Dysfunction of symbiont cells due to natural degradation or stress. Image credit: A. Sproles. Figure 1.2 The six phases of symbiosis establishment (according to description in Davy et al. 2012). 1) Initial
contact between Symbiodinium and host cells, where recognition is facilitated by cell surface molecules from each
partner. 2) Phagocytosis of algal cell by the host cell. 3) Sorting of algal cells into accepted symbionts (green
arrow) to be housed in symbiosome membranes, and rejected cells (red arrow) which are removed via apoptosis,
autophagy, or exocytosis. 4) Proliferation of symbiont population by cell division. 5) Dynamic stability of
symbiont population. 6) Dysfunction of symbiont cells due to natural degradation or stress. Image credit: A. Sproles. The accepted cells then transition into a haploid vegetative state and are stored in the host’s
endodermal cells (Davy & Turner 2003; Santos & Coffroth 2003). Regulatory mechanisms are
enforced to keep symbiont density at a relatively constant level, achieving dynamic stability by
balancing population numbers through nutrient limitation and removal of excess algae
(Muscatine & Pool 1979; Jones & Yellowlees 1997; Koike et al. 2004). 1.3.1 Ecological importance The cnidarian-dinoflagellate symbiosis is widespread throughout the world’s oceans, acting as
a foundation for the construction of vast coral reef ecosystems (Fig. 1.3). Coral reefs serve as
biodiversity hot spots in waters with very low nutrient availability and plankton abundance
(Muscatine & Porter 1977). Additionally, they support a high diversity of marine fauna while
providing
a
home
for The cnidarian-dinoflagellate symbiosis is widespread throughout the world’s oceans, acting as
a foundation for the construction of vast coral reef ecosystems (Fig. 1.3). Coral reefs serve as
biodiversity hot spots in waters with very low nutrient availability and plankton abundance
(Muscatine & Porter 1977). Additionally, they support a high diversity of marine fauna while
providing
a
home
for
millions of endemic species
and act as a nursery for
many juvenile fish (Reaka-
Kudla
1997;
Knowlton
2001). The endosymbiotic
algae are critical to the
existence
of
the
reef
system, since they provide
the
animal
with
the
majority of its required
metabolic carbon (Lasker
1981; Muscatine et al. 1981; Richmond 1993). The
algae
are
also
proficient
at
nitrogen
cycling, which allows reef
systems to have such high
productivity
within
oligotrophic waters (Suzuki et al. 1995). Nitrogen recycling occurs when nitrogenous products
Figure 1.3 The cnidarian-dinoflagellate symbiosis supports coral reef
ecosystems. (A) Histological section of a cnidarian tentacle showing
Symbiodinium cells within host gastrodermal tissue (20x mag). (B) Detail of
tentacles on the mushroom coral Fungia scutaria. (C) Individual coral
colonies of different morphologies. (D) Coral reef with high coral diversity
in Bali, Indonesia. Micrographs and photographs by A. Sproles. the world’s oceans, acting as
Fig. 1.3). Coral reefs serve as
lity and plankton abundance
versity of marine fauna while
providing
a
home
for
millions of endemic species
and act as a nursery for
many juvenile fish (Reaka-
Kudla
1997;
Knowlton
2001). The endosymbiotic
algae are critical to the
existence
of
the
reef
system, since they provide
the
animal
with
the
majority of its required
metabolic carbon (Lasker
1981; Muscatine et al. 1981; Richmond 1993). The
algae
are
also
proficient
at
nitrogen
cycling, which allows reef
systems to have such high
productivity
within
rs when nitrogenous products
host excretions or dissolved
mino acids, some of which are
t al. 2006; O’Neil & Capone
or the algae’s o n cell lar Figure 1.3 The cnidarian-dinoflagellate symbiosis supports coral reef
ecosystems. (A) Histological section of a cnidarian tentacle showing
Symbiodinium cells within host gastrodermal tissue (20x mag). 1.2.2 Uptake, recognition, and regulation of symbionts Symbiont proliferation
is slowed while in hospite by controlling the amount of nutrient flux from the host (Miller &
Yellowlees 1989; Leletkin 2000), or by reducing rates of cell division (reviewed in Davy et al. 2012). Surplus algae are removed through apoptosis, autophagy (digestion of cells), or
exocytosis (expulsion of symbiont into environment) (Titlyanov et al. 1996; Dunn et al. 2004,
2007b; Ainsworth et al. 2007; Weis 2008; Paxton et al. 2013; Hawkins et al. 2013, 2014). 23 Inter-partner signalling pathways are also important to the regulation of symbiont populations
(Rodriguez-Lanetty et al. 2006; Detournay & Weis 2011). In particular, the sphingosine
rheostat pathway acts using a complement of two molecular signals: one that promotes
apoptosis of the symbiotic cells and one that inhibits it, allowing control over symbiont
population numbers (Detournay & Weis 2011). Another, called the transforming growth factor
beta (TGFβ) pathway, also functions as part of the immune response to allow tolerance of the
symbiont (Detournay et al. 2012). The last phase of symbiosis, dysfunction, is the breakdown
of the association due to senescence (natural cell degradation) or stress (Sawyer & Muscatine
2001; Hanes & Kempf 2013; Tolleter et al. 2013; Dani et al. 2016), and involves removal of
algal cells through one of the previously mentioned mechanisms. 24 1.3.1 Ecological importance (B) Detail of
tentacles on the mushroom coral Fungia scutaria. (C) Individual coral
colonies of different morphologies. (D) Coral reef with high coral diversity
in Bali, Indonesia. Micrographs and photographs by A. Sproles. Figure 1.3 The cnidarian-dinoflagellate symbiosis supports coral reef
ecosystems. (A) Histological section of a cnidarian tentacle showing
Symbiodinium cells within host gastrodermal tissue (20x mag). (B) Detail of
tentacles on the mushroom coral Fungia scutaria. (C) Individual coral
colonies of different morphologies. (D) Coral reef with high coral diversity
in Bali, Indonesia. Micrographs and photographs by A. Sproles. oligotrophic waters (Suzuki et al. 1995). Nitrogen recycling occurs when nitrogenous products
(ammonium and nitrate) are obtained by the algae from either host excretions or dissolved
inorganic material in the seawater, and then used to assimilate amino acids, some of which are
translocated back to the host (Wang & Douglas 1999; Tanaka et al. 2006; O’Neil & Capone
2008). However, the assimilated amino acids are also used for the algae’s own cellular
processes with sometimes only small amounts of nitrogen products being returned to the host 25 (Wang & Douglas 1998; Lipschultz & Cook 2002). Photosynthetic products transferred to the
host also enhance coral calcification rates (Colombo-Pallotta et al. 2010; Holcomb et al. 2014;
Bertucci et al. 2015), which aids in building the reef. The complex reef structure is thereby
able to act as a safe haven against turbulent waters and predation for millions of marine fauna
(Pearse & Muscatine 1971). Unfortunately, coral reefs worldwide are threatened by climate change. Incidents of bleaching
have been increasing dramatically since the 1980s, and are causing degradation of many of the
world’s coral reefs ecosystems (Hoegh-Guldberg 1999). In most cases, bleaching (i.e. the loss
of the symbiotic algae and/or their photosynthetic pigments) causes mortality of the coral
unless it is able to quickly re-establish its symbiont population (Jokiel 2004). Current
predictions show that reefs across the globe are amidst the longest bleaching event on record
(2014 – present) with extended summer-like temperatures each season that are expected to
continue (Heron et al. 2016). 1.4.1 Genetic diversity within Symbiodinium The species Symbiodinium microadriaticum was first described in 1962 to describe the
symbionts of the jellyfish Cassiopeia sp. (Freudenthal 1962). This was thought to be the only
member of this genus until Trench and Blank (1987) introduced three new species: S. goreauii
from a sea anemone, S. kawagutii from a coral, and S. pilosum from a zoanthid. Later
researchers began to use molecular techniques to examine the DNA of the small ribosomal
subunit of Symbiodinium within reef corals. This revealed large genetic diversity within the
species, and further physiological differences between genotypes led to suggestions that many
more species might be present (Rowan & Powers 1991). Six additional Symbiodinium species
were then identified from a bivalve, corals, and sea anemones (McNally 1994; Trench 1997). As diversity studies continued to use ribosomal subunit DNA, genetically distinct clades of
Symbiodinium were designated as A, B, C, and D (Carlos et al. 1999). Further developments
came with the discovery that the ribosomal internal transcribed spacer (ITS) regions from
Symbiodinium could be used as a marker since it had a better molecular resolution than previous
techniques (LaJeunesse 2001; Rodriguez-Lanetty 2003). This, coupled with other methods
such as denaturing gradient gel electrophoresis (DGGE), allowed further classification into 26 sub-clades or ‘types’ (e.g. A3, B1, D1a) to better reflect the physiological and ecological
differences within each clade (van Oppen et al. 2001; LaJeunesse & T. 2002; LaJeunesse et al. 2004; Sampayo et al. 2009; Finney et al. 2010). Currently, around 70 different types within
nine divergent clades (A-I) and 22 species of Symbiodinium have been described, although
about half of these clades (F-I) associate primarily with foraminiferans rather than metazoans,
or are free-living (LaJeunesse 2001; Stat et al. 2006; Pochon & Gates 2010; LaJeunesse et al. 2012; Jeong et al. 2014; Hume et al. 2015; LaJeunesse et al. 2015; Lee et al. 2015). Physiology and tolerance to environmental stress vary widely between different types of
Symbiodinium. Some, such as those from clade A and clade D, are more tolerant to thermal
stress and therefore relay a higher resistance of bleaching to the host (Thornhill et al. 2006;
Loram et al. 2007; LaJeunesse et al. 2009; Stat & Gates 2011; Ladner et al. 2012; Barshis et
al. 2014; Lee et al. 2015). 1.4.1 Genetic diversity within Symbiodinium Some Symbiodinium types have evolved tolerances of, are oxidative
stress in some members of clade C (Hawkins & Davy 2012), cold stress in some clade B types
(Thornhill et al. 2008), and high light stress in some clade A and D types (Reynolds et al. 2008;
Hennige et al. 2008; Yuyama et al. 2016). Attributes like respiration rate, photosynthetic
efficiency and production, and nitrogen assimilation can also differ between symbiont types
(Baker et al. 2013; Starzak et al. 2014; Hawkins et al. 2016; McIlroy et al. 2016). The large
genetic and functional diversity of cnidarian symbionts is now clearer, thanks to the constant
development of new identification techniques. Further study of functional differences within
Symbiodinium will allow more accurate predictions of how coral reefs might respond to the
stressors of climate change. 1.4.2 Host-symbiont specificity Host-symbiont specificity is defined as a clearly non-random pattern of association where the
ratio of observed host-symbiont combinations compared with the range of possible
combinations is very small (Baker 2003). An early hypothesis as to why specificity occurs was
posed by Trench (1997), who concluded that specificity was likely due to the co-adaptation of
the host and symbiont to environmental conditions. Early experiments on infectivity of
aposymbiotic (i.e. lacking symbionts) hosts with different algal strains observed these non-
random patterns of symbiont uptake in gorgonians and sea anemones (Kinzie 1974; Kinzie &
Chee 1979), followed by the finding of different algal cell surface antigens (Kinzie & Chee 27 1982). It is now known that metazoans possess lectins with a glycan-binding function (as
explained in section 1.2.2), which is known to be used in cell-cell adhesion and recognition
(Wood-Charlson & Weis 2009; Logan et al. 2010). Experiments show that some cnidarian
lectins have a specificity for binding mannose (Vidal-Dupiol et al. 2009) and galactose side-
chains (Koike et al. 2004), the latter of which were also found to be present on the cell surface
of the symbionts (Fig 1.3). This suggests that glycan-lectin binding may be a major determinant
of host-symbiont specificity (Bay et al. 2011). Molecular approaches have revealed patterns of host-symbiont specificity in nature, where
hosts exhibit strong selectivity for some symbiont types over others (van Oppen et al. 2001;
Santos et al. 2003; LaJeunesse et al. 2004; Stat et al. 2009; Mieog et al. 2009; Abrego et al. 2009; Xiang et al. 2013). Usually, this entails a symbiont assemblage of one dominant type
existing with one or more ‘background’ symbiont type(s) that exist in only low population
densities (Héloïse et al. 2017; Ziegler et al. 2017). Experimental methods have found
heterologous (i.e. non-native) algae to infect hosts less successfully than homologous (i.e. native) algal types (Weis et al. 2001; Belda-Baillie et al. 2002; Dunn & Weis 2009; Starzak et
al. 2014), illustrating the specificity for some symbionts over others. However, over longer
periods of time, heterologous algal types may be able to establish long-term associations with
host corals, as demonstrated by the invasion of clade D Symbiodinium in Caribbean corals
(Pettay et al. 2015), and a newly discovered association of clade B Symbiodinium in corals
from French Polynesia (Héloïse et al. 2017). 1.4.2 Host-symbiont specificity Specificity may also be caused by symbiont
transmission method, since horizontal transmission allows more interactions with generalist
algae (Fabina et al. 2012), or host depth (Frade et al. 2008) due to the fact that some types may
be more or less sensitive to light. Quality of metabolic exchange and energetic costs for the
host could also be an underlying factor that contributes to specificity (Cooper et al. 2011;
Starzak et al. 2014), but this has been rarely studied at the cellular level. 28 1.4.3 Thermally tolerant symbionts as a mechanism of adaptation 1.4.3 Thermally tolerant symbionts as a mechanism of adaptation Although many cnidarians have high specificity for one symbiont type (Santos et al. 2004;
Thornhill et al. 2009), many have the advantage of being able to harbour multiple algal types
(Baker 2003; Silverstein et al. 2012). This feature can prove essential during times of stress
when changing symbiont population composition allows the more tolerant algae to become
dominant (Jones et al. 2008; Abrego & Ulstrup 2008; LaJeunesse et al. 2009, 2010; Thomas et
al. 2014). This phenomenon has been defined as the Adaptive Bleaching Hypothesis (Fig. 1.4),
which proposes that bleaching
provides the opportunity for the
host to be repopulated with a
different symbiont assemblage
(Buddemeier & Fautin 1993;
Fautin & Buddemeier 2004). Baker (2003) has described the
two methods of symbiont
community change that may
arise after such an event as:
‘symbiont
shuffling’,
a
quantitative change in the
relative abundance of existing
symbiont communities within
colonies
(Fig. 1.4A);
and
‘symbiont switching’, a qualitative change by recombination with symbionts acquired from the
environment (Fig. 1.4B). Symbiont shuffling occurs in many instances after bleaching events,
often with symbionts from clade D becoming dominant (LaJeunesse et al. 2009; Kemp et al. 2014). Algae from Symbiodinium clade D (primarily Symbiodinium trenchii, type D1a) act as
opportunists by existing as background symbionts until the dominant population is removed
by thermally-induced bleaching, at which point clade D symbionts proliferate to high densities
that increase coral survival (Stat & Gates 2011; Kemp et al. 2014; Bay et al. 2016). The ability
to successfully function under thermal stress, and its identity as a host-generalist, make D1a an
ideal candidate for coral adaptation through symbiont shuffling (Berkelmans & van Oppen
2006; LaJeunesse et al. 2014). It has been questioned whether symbiont switching is possible,
considering that observations of this phenomenon in the field are rare. Nevertheless, switching
Figure 1.4 Depiction of the two methods of symbiont community
change (as described by Baker et al. (2003) under the adaptive
bleaching hypothesis. (A) Symbiont shuffling, a quantitative change in
existing symbiont communities. (B) Symbiont switching, a qualitative
change by recombination with symbionts acquired from the
environment. Image credit: A. Sproles. Although many cnidarians have high specificity for one symbiont type (Santos et al. 2004;
Thornhill et al. 2009), many have the advantage of being able to harbour multiple algal types
(Baker 2003; Silverstein et al. 2012). 1.4.3 Thermally tolerant symbionts as a mechanism of adaptation This feature can prove essential during times of stress
when changing symbiont population composition allows the more tolerant algae to become
dominant (Jones et al. 2008; Abrego & Ulstrup 2008; LaJeunesse et al. 2009, 2010; Thomas et
al. 2014). This phenomenon has been defined as the Adaptive Bleaching Hypothesis (Fig. 1.4), Figure 1.4 Depiction of the two methods of symbiont community
change (as described by Baker et al. (2003) under the adaptive
bleaching hypothesis. (A) Symbiont shuffling, a quantitative change in
existing symbiont communities. (B) Symbiont switching, a qualitative
change by recombination with symbionts acquired from the
environment. Image credit: A. Sproles. Figure 1.4 Depiction of the two methods of symbiont community
change (as described by Baker et al. (2003) under the adaptive
bleaching hypothesis. (A) Symbiont shuffling, a quantitative change in
existing symbiont communities. (B) Symbiont switching, a qualitative
change by recombination with symbionts acquired from the
environment. Image credit: A. Sproles. ‘symbiont switching’, a qualitative change by recombination with symbionts acquired from the
environment (Fig. 1.4B). Symbiont shuffling occurs in many instances after bleaching events,
often with symbionts from clade D becoming dominant (LaJeunesse et al. 2009; Kemp et al. 2014). Algae from Symbiodinium clade D (primarily Symbiodinium trenchii, type D1a) act as
opportunists by existing as background symbionts until the dominant population is removed
by thermally-induced bleaching, at which point clade D symbionts proliferate to high densities
that increase coral survival (Stat & Gates 2011; Kemp et al. 2014; Bay et al. 2016). The ability
to successfully function under thermal stress, and its identity as a host-generalist, make D1a an
ideal candidate for coral adaptation through symbiont shuffling (Berkelmans & van Oppen
2006; LaJeunesse et al. 2014). It has been questioned whether symbiont switching is possible,
considering that observations of this phenomenon in the field are rare. Nevertheless, switching 29 to a clade D symbiont has been demonstrated experimentally (Boulotte et al. 2016), illustrating
that it is fundamentally possible. However, the overall potential for clade D symbionts is
hindered by their sub-optimal functioning under normal environmental conditions (Little et al. 2004; Jones & Berkelmans 2011; Baker et al. 2013; Pernice et al. 2014), and the potential
combined effects of other environmental factors induced by climate change (Hoadley et al. 2016). 1.5.1 Translocation of photosynthetic products One of the most widely studied aspects of cnidarian-dinoflagellate symbiosis is the nutrient
exchange between symbiont and host. The algal endosymbionts capture light to produce fixed
carbon via photosynthesis, of which up to 99% is translocated to the host in the form of essential
sugars, lipids, amino acids, and glycoproteins (Muscatine & Cernichiari 1969; Davies 1984;
Muscatine 1984; Davy et al. 2012). The photosynthate can satisfy from 50-100% of the host’s
daily carbon requirements for respiration, growth, and maintenance (Muscatine et al. 1981;
Edmunds and Davies 1986; Davies 1991), depending on the light regime and species
concerned. (Falkowski et al. 1984; Davies 1991). Early studies on photosynthate transport to
the host focused on glycerol, as it was thought to be incorporated into cnidarian lipid pools or
used as metabolic fuel (Muscatine 1967; Schmitz & Kremer 1977; Hofmann & Kremer 1981;
Battey & Patton 1984; Colombo-Pallotta et al. 2010). It is now known that glycerol is exuded
as a stress response from Symbiodinium cells, and also may be translocated to the host as a
minor metabolite (Lewis & Smith 1971; Whitehead & Douglas 2003; Burriesci et al. 2012;
Suescun-Bolivar et al. 2016). Fixed carbon for animal metabolism is now primarily thought to
be translocated to the host in the form of glucose (Muscatine et al. 1967; Ishikura et al. 1999;
Burriesci et al. 2012; Hillyer et al. 2016). Lipids are also translocated from the symbiont to
host, as storage molecules like wax esters and triacylglycerol, and as sterols and fatty acids
such as palmitic, stearic, and oleic acid (von Holt & von Holt 1968a; Yamashiro et al. 1999;
Hillyer et al. 2016). Fatty acids and phospholipids may also have a role in the recognition,
proliferation, and overall success of symbiotic associations, since inhibition of these
compounds impairs symbiont infection rates (Wang et al. 2013b). Additional products released
by the symbiont are organic acids (von Holt & von Holt 1968a; b, Trench 1971a; b; Whitehead 30 & Douglas 2003), glycolic acid (Cernichiari et al. 1969), and amino acids (von Holt & von
Holt 1968a; b; Lewis & Smith 1971). Overall, translocation of fixed carbon is the central
feature of the symbiosis, as it is incorporated into all host tissues (Tremblay et al. 2012; Kopp
et al. 2015; Freeman et al. 2016). 1.5.1 Translocation of photosynthetic products The potential for carbon translocation could be an underlying
cause of symbiont selectivity, since some Symbiodinium types do not translocate as much
carbon to hosts as others (Starzak et al. 2014; Pernice et al. 2014). 1.5.2 Reverse translocation The reverse translocation of inorganic and organic compounds from within the host’s
gastrodermal cells provides essential nutrients to the symbionts. The symbionts utilize CO2
derived from host respiration as well as dissolved inorganic carbon (DIC) from the seawater,
transported in the form of bicarbonate ions through the host epithelial cells (Allemand et al. 1998). Inorganic carbon is necessary for both algal photosynthesis and host calcification,
therefore carbonic anhydrase proteins are essential as they work in the carbon-concentrating
mechanism of the symbiosis to catalyse the absorption of carbon for these processes (Furla et
al. 2000; Bertucci et al. 2013; Barott et al. 2015). Two of the other major nutrients that the
algae require are phosphorus and nitrogen, both of which are known to be essential
macronutrients for all organisms (Cook et al. 1988). Phosphate uptake for symbiont nutrition
has been demonstrated both in the dark (Cates & McLaughlin 1979; Deane & O’Brien 1981)
and in the light, where it is said to be enhanced by photosynthesis (Jackson & Yellowlees
1990). Nitrogen may be obtained by the algal symbionts from several sources: nitrogen fixed
by bacterial colonies within the holobiont (Lesser et al. 2007; Freeman et al. 2016), dissolved
inorganic nitrogen from the seawater (O’Neil & Capone 2008; Kopp et al. 2013, 2015), or
nitrogen excreted by the host as waste from heterotrophic feeding on zooplankton (Porter 1974;
Steen 1986; Anthony & Fabricius 2000; Houlbrèque et al. 2004). Nitrogenous compounds from
these sources are taken up by the symbiont in the forms of both ammonium (Muller-Parker et
al. 1994; Pernice et al. 2012) and nitrate (D’Elia et al. 1983; Miller & Yellowlees 1989; Grover
et al. 2003). The symbiont uses these compounds for amino acid assimilation, some of which
are used for building algal proteins, and others are recycled back to the host (Lewis & Smith
1971; Tanaka et al. 2006). Additionally, host-derived fatty acids, lipids, and the amino acid
glycine are translocated to the symbionts (von Holt 1968; Wang & Douglas 1999; Imbs et al. 2014) 31 Figure 1.5 Summary of intercellular metabolite exchange within cnidarian-dinoflagellate associations. Phosphate,
bicarbonate, and nitrate ions are acquired from the seawater and passed through the host cells, then through the
symbiosome membrane for utilization by Symbiodinium cells. Host-derived carbon dioxide, ammonium, lipids,
and glycine are also translocated to the symbiont. The symbiont uses the carbon and nitrogen compounds to
photosynthesize and assimilate amino acids, respectively. 1.5.2 Reverse translocation Photosynthetic products and amino acids are then
translocated to the host. Image credit: A. Sproles. Figure 1.5 Summary of intercellular metabolite exchange within cnidarian-dinoflagellate associations. Phosphate,
bicarbonate, and nitrate ions are acquired from the seawater and passed through the host cells, then through the
symbiosome membrane for utilization by Symbiodinium cells. Host-derived carbon dioxide, ammonium, lipids,
and glycine are also translocated to the symbiont. The symbiont uses the carbon and nitrogen compounds to
photosynthesize and assimilate amino acids, respectively. Photosynthetic products and amino acids are then
translocated to the host. Image credit: A. Sproles. Modern technology may hold the key to deeper understanding of nutritional transport between
cnidarians and Symbiodinium. Nanoscale secondary ion mass spectrometry (nanoSIMS) has
been successfully used to quantify and localize carbon and nitrogen pools within coral and
symbiont cells using 13C and 15N isotope tracers (Clode et al. 2007; Pernice et al. 2012; Kopp
et al. 2013, 2015). This technique has been applied to measure carbon and nitrogen exchange
between a coral that can harbour two different symbiont types, finding that those from clade C
were able to fix and pass more carbon to the host than those from clade D (Pernice et al. 2014). So far the studies have mainly focused on nitrogen assimilation and are only just starting to
include carbon fixation and the differences in translocation between symbiont types (Clode et
al. 2007; Pernice et al. 2012, 2014, Kopp et al. 2013, 2014). However, characterization of 32 nutritional fluxes between different host-symbiont pairs during symbiosis establishment could
uncover more details about the mechanisms that underlie host-symbiont specificity. Figure 1.6 Functional diagrams of secondary active transport
proteins (A-C) and channel proteins (D). A) Uniporter, a
solute is transported along its concentration gradient. B)
Symporter, a solute is transported against its concentration
gradient with an ion along its concentration gradient. C)
Antiporter, a solute and an ion are transported along their
concentration gradients together. D) Channel, solutes flow
passively along their concentration gradient. Image credit: A. Sproles 1.5.3 Mechanisms of inter-partner
transport The exact mechanisms of transport
between host and symbiont are still not
entirely
known,
however
exchanged
molecules would certainly need to traverse
the symbiosome membrane (Fig. 1.5). While little is known about the identity of
symbiosome transporters, predictions can
be made based on information from related
systems and overall cell biology. Passive
diffusion is only able to account for a small
percentage of membrane transport, with
polar molecules such as amino acids and
carbohydrates unable to pass through the
cell membrane without facilitation by
active transporter proteins (Vander Heiden
et al. 2009). For example, only about 15%
of
DIC
necessary
for
symbiont
photosynthesis can be obtained by cellular
diffusion, meaning that carbon is likely
transported actively by either bicarbonate
and CO2 pumps, or symport and antiport
mechanisms (Allemand et al. 1998). Almost all of the metabolites described in
section 1.5.1-1.5.2 can be transported
across the plasma membrane by either
secondary active transporters in the major
facilitator superfamily (MFS) or passively The exact mechanisms of transport
between host and symbiont are still not
entirely
known,
however
exchanged
molecules would certainly need to traverse
the symbiosome membrane (Fig. 1.5). While little is known about the identity of
symbiosome transporters, predictions can
be made based on information from related
systems and overall cell biology. Passive
diffusion is only able to account for a small
percentage of membrane transport, with
polar molecules such as amino acids and
carbohydrates unable to pass through the
cell membrane without facilitation by
active transporter proteins (Vander Heiden
et al. 2009). For example, only about 15%
of
DIC
necessary
for
symbiont
photosynthesis can be obtained by cellular
diffusion, meaning that carbon is likely
transported actively by either bicarbonate
and CO2 pumps, or symport and antiport
mechanisms (Allemand et al. 1998). Almost all of the metabolites described in
section 1.5.1-1.5.2 can be transported
across the plasma membrane by either
secondary active transporters in the major
facilitator superfamily (MFS) or passively Figure 1.6 Functional diagrams of secondary active transport
proteins (A-C) and channel proteins (D). A) Uniporter, a
solute is transported along its concentration gradient. B)
Symporter, a solute is transported against its concentration
gradient with an ion along its concentration gradient. C)
Antiporter, a solute and an ion are transported along their
concentration gradients together. D) Channel, solutes flow
passively along their concentration gradient. Image credit: A. Sproles 33 through channel proteins (Fig 1.6). 1.5.3 Mechanisms of inter-partner
transport The MFS are secondary carriers that occur ubiquitously
throughout life and transport small molecules using energy from a chemiosmotic ion gradient
(Pao et al. 1998); it is the largest superfamily of secondary active transporters, encompassing
29 protein families that transport sugars, drugs, organophosphates, Krebs cycle intermediates,
inorganic anions (such as nitrate/nitrite, phosphate, and cyanate), nucleosides, and
monocarboxylic acids (Saier et al. 1999). Each protein family in the MFS has individually
defined motifs, but they all share one highly conserved five-residue motif and thirteen-residue
motif characteristic for MFS transporters (Pao et al. 1998). The structure of MFS proteins is
the same regardless of whether they are a symporter, uniporter, or antiporter: 12 transmembrane
α-helices (TMs) joined by hydrophilic loops with both the N and C terminus inside the
cytoplasm (Law et al. 2008). Other transporter families that could possibly be involved in
nutrient transport include: outer membrane porins (channels that non-selectively allow passage
of solutes; Fig. 1.6), ABC transporters, ligand-gated ion channels, amino acid-polyamine-
choline transporters, and the ATPases P type, F type, V type, and A type; however, transporters
from these families are much less diverse and abundant than those from the MFS superfamilies
(Saier 1994). Nutrient transporters are highly conserved macromolecules throughout all
domains of life, so the known proteins within these families could very likely have orthologous
counterparts within the cnidarian-dinoflagellate symbiosis. 1.6.1 Transcriptomics and genomics With publicly available transcriptome and genome sequences for both partners, the potential
for bioinformatics as an effective tool for elucidating proteins involved in these symbiotic
associations has never been greater. There are currently 29 cnidarian transcriptomes and four
genomes available, including those of Aiptasia (Lehnert et al. 2012; Baumgarten et al. 2015). In addition, there are at least nine transcriptomes and three genomes available for different
Symbiodinium types (Appendix A, Table A1). Reviewed transporter protein sequences are also
freely available through NCBI, UniProtKB, and various other databases, providing
opportunities for investigative searches of interesting proteins within these genetic resources. Identification of C-type lectins within the cnidarian Nematostella was accomplished using the
genomic database, which greatly influenced information on cell-surface recognition 34 mechanisms (Wood-Charlson & Weis 2009). Additionally, sequences from symbiotic vs. aposymbiotic samples of the same cnidarian species have been compared in order to
successfully detect changes due to symbiosis. Interestingly, while few transcriptional changes
happen in larvae newly engaged in symbiosis (Yuyama et al. 2005; Deboer et al. 2007;
Voolstra et al. 2009), many genes are up-regulated – including several that encode for
transporters of glucose, fatty acids, sterols, amino acids, ammonium, carbonic-anhydrase,
phosphate, sulphate, and zinc – in adult symbiotic anemones compared with aposymbiotic
counterparts (Rodriguez-Lanetty et al. 2006; Lehnert et al. 2014). This method has also
allowed identification of genes involved in light-enhanced calcification (Bertucci et al. 2015)
and symbiont selectivity (Wolfowicz et al. 2016). Transcriptomic studies have also been used
to identify evolving proteins (Voolstra et al. 2011; Kitchen et al. 2015; Bellis et al. 2016), such
as those that are linked to thermal sensitivity in corals and their symbionts (Barshis et al. 2014;
Howells et al. 2016; Levin et al. 2016). Additionally, these resources have helped to detect
conserved genes and metabolic pathways in Symbiodinium, and describe genetically unique
areas between the clades (Rosic et al. 2014), as well as identify symbiont proteins involved in
nutrient uptake (Leggat et al. 2007; Aranda et al. 2016). Bioinformatics has the potential to
detect membrane transport proteins that exchange metabolites between different host-symbiont
pairs, yet a comprehensive analysis of this kind has not yet been done. 1.6.2 Identification of symbiosis-associated proteins Although identification of genes involved in the symbiosis provides useful information, it is
important to use this alongside proteomic analysis in the laboratory to yield the most conclusive
results. Previous methods to assess symbiosis-associated proteins have used 2D gel
electrophoresis (Weis & Levine 1996), but recent techniques allow for more specific protein
identification. Liquid chromatography tandem mass spectrometry (LC-MS/MS) is able to
analyse cnidarian peptides that are identified using databases generated from cnidarian and
Symbiodinium genetic resources (Peng et al. 2010; Oakley et al. 2016). Immunocytochemistry
can then be used to localize proteins to tissues within the symbiotic association (Yuyama &
Watanabe 2008; Dani et al. 2014; Barott et al. 2015). These techniques have led to discovery
of a few likely symbiosome membrane proteins (Peng et al. 2010; Dani et al. 2014; Barott et
al. 2015), proteins associated with coral disease (Garcia et al. 2016), and a full characterization
of the proteomic changes in Aiptasia that take place in response to establishment of symbiosis 35 (Oakley et al. 2016). Finding these proteins has allowed better predictions of the cellular
processes at work during these interactions. For instance, these results have revealed the sterol-
transporting NPC2 protein to be of potential significance in the cnidarian-dinoflagellate
symbiosis, as it is upregulated in symbiotic anemones and is localized to symbiosome
membranes (Dani et al. 2014; Oakley et al. 2016). Another finding shows that an ion pump
(vacuolar H+-ATPase) is located in the symbiosome and helps to acidify the algal environment
(Barott et al. 2015). Many other functional implications can be drawn from the few studies that
have successfully executed proteomic identifications in the cnidarian-dinoflagellate symbiosis,
and continuation of this type of work will hopefully be able to uncover some of the mysteries
that still persist in the field. 1.6.3 Aiptasia as a model organism The sea anemone Aiptasia sp. ( = ExAiptasia) has become a globally-adopted, tractable model
for studies of the cnidarian-Symbiodinium symbiosis, as it is easily kept under laboratory
conditions and can be experimentally bleached and re-infected with different strains of
Symbiodinium (Belda-Baillie et al. 2002; Xiang et al. 2013; Starzak et al. 2014; Hambleton et
al. 2014; Matthews et al. 2016). Access to this model cnidarian overcomes some of the
difficulties of working with live corals, and allows more in-depth investigations of the
processes behind symbiosis establishment and persistence. Like many corals, Aiptasia
produces non-symbiotic larvae that acquire symbionts from their environment, and undergo
the same phases of symbiosis establishment described above (Bucher et al. 2016). In fact, the
sea anemone has the same symbiont selection patterns during the larval stage as two different
Acropora coral species (Wolfowicz et al. 2016). The genome of Aiptasia has been assembled
along with the transcriptomes of the anemone in symbiotic and aposymbiotic states (Lehnert
et al. 2014; Baumgarten et al. 2015), leaving great potential for bioinformatics-based studies. So far, the sea anemone has provided an ideal model system for examining gene expression
(Kuo et al. 2004; Dunn et al. 2007a; Lehnert et al. 2014), bleaching mechanisms (Sawyer &
Muscatine 2001; Hanes & Kempf 2013; Fransolet et al. 2013; Paxton et al. 2013; Bieri et al. 2016), carbon and nitrogen metabolism (Swanson et al. 1998; Smith et al. 2004; Clode et al. 2009; Dunn et al. 2012; Hillyer et al. 2016), protein expression (Oakley et al. 2016), symbiont
selectivity (Hambleton et al. 2014; Bellis et al. 2016; Wolfowicz et al. 2016), ocean
acidification (Gibbin & Davy 2014), bacterial associations (Palincsar et al. 1989; Röthig et al. 36 2016), and immune pathways (Hawkins et al. 2013; Poole et al. 2016) in the cnidarian-
dinoflagellate symbiosis, although this list is not exhaustive. The usage of Aiptasia allows the
biological systems within coral reefs to be examined without having to compromise any corals
themselves, making it a valuable research tool in the field of cnidarian-dinoflagellate
symbiosis. The combination of “omics” research with other isotopic labelling experiments
could be powerful in studying this model cnidarian system. 1.7 Aims and Objectives The primary aim of this research was to identify whether carbon and nitrogen exchange in the
cnidarian-dinoflagellate symbiosis is impacted by symbiont type, and infer how any difference
may influence the potential of corals to adapt to warming oceans under climate change. This
was addressed using a combination of bioinformatic analysis and experimental investigations
using proteomics and nanoSIMS techniques. The mechanisms of nutritional exchange across
the symbiotic interface are largely unknown, so these were first explored to characterize
evolutionary patterns leading to the integration of host-symbiont metabolite exchange; next,
the functional implications of cnidarians associating with a thermally-tolerant, opportunistic
Symbiodinium type were assessed. My specific objectives were therefore to: 1. Identify and characterize the membrane transporter sequences of the major exchanged
metabolites (glucose, glycerol, ammonium, and nitrate) in the cnidarian-dinoflagellate
symbiosis, across a range of cnidarian species and Symbiodinium clades. Hypothesis: Cnidarians will possess sequences homologous to human GLUT, SGLT,
and AQP proteins for the uptake of glucose and glycerol from symbionts, while
Symbiodinium will possess sequences homologous to plant AMT and NRT2 proteins
for the uptake of ammonium and nitrate from the host. 2. Construct phylogenies of the characterized transporters, to determine how conserved nutrient
transport is within the symbiotic associations and if diversification due to symbiosis has
occurred during protein evolution. Hypothesis: Transporter proteins will be highly conserved from humans to cnidarians,
and from Arabidopsis to Symbiodinium, since they provide an essential cellular 37 function. Furthermore, diversification of protein sequences is likely to have occurred
during the evolution of symbiosis. 3. Use the model system of Aiptasia, to identify the response of a host proteome to a
heterologous, thermally-tolerant symbiont type (Symbiodinium trenchii, type D1a) compared
to a homologous symbiont type under normal environmental conditions. Hypothesis: The heterologous symbiont will elicit proteins indicative of a less
successful carbon and nitrogen metabolism then those of a host with homologous
symbionts. Moreover, proteins involved in other important symbiotic processes will be
down-regulated in heterologous symbionts. 4. Compare intercellular carbon and nitrogen translocation from symbiont to host, as well as
carbon and nitrogen ‘reverse translocation’ between a heterologous, thermally-tolerant
symbiont type (Symbiodinium trenchii, type D1a) with that of a homologous symbiont type
(Symbiodinium minutum, type B1) in Aiptasia hosts under normal environmental conditions. 4. 1.7 Aims and Objectives Compare intercellular carbon and nitrogen translocation from symbiont to host, as well as
carbon and nitrogen ‘reverse translocation’ between a heterologous, thermally-tolerant
symbiont type (Symbiodinium trenchii, type D1a) with that of a homologous symbiont type
(Symbiodinium minutum, type B1) in Aiptasia hosts under normal environmental conditions. Hypothesis: The bi-directional translocation of both carbon and nitrogen will be lower
when the host associates with Symbiodinium D1a than B1 at non-stressful temperatures,
due to poorer integration of the novel symbiosis. Hypothesis: The bi-directional translocation of both carbon and nitrogen will be lower
when the host associates with Symbiodinium D1a than B1 at non-stressful temperatures,
due to poorer integration of the novel symbiosis. Hypothesis: The bi-directional translocation of both carbon and nitrogen will be lower
when the host associates with Symbiodinium D1a than B1 at non-stressful temperatures,
due to poorer integration of the novel symbiosis. 5. Determine whether nutrient (C and N) exchange changes with time during the colonization
of the host by different symbiont types (homologous and heterologous). 5. Determine whether nutrient (C and N) exchange changes with time during the colonization
of the host by different symbiont types (homologous and heterologous). 5. Determine whether nutrient (C and N) exchange changes with time during the colonization
of the host by different symbiont types (homologous and heterologous). Hypothesis: Nutritional exchange will increase with time, from the early to latter stages
of symbiosis establishment, but to a greater extent when infected by a homologous
rather than heterologous symbiont type. Hypothesis: Nutritional exchange will increase with time, from the early to latter stages
of symbiosis establishment, but to a greater extent when infected by a homologous
rather than heterologous symbiont type. 38 The research comprising this thesis has been written as a series of stand-alone manuscripts,
therefore some degree of overlap in the introduction of each chapter may be present. All
laboratory and fieldwork, statistical analyses, and writing were completed by the author, with
further assistance from others as follows: The research comprising this thesis has been written as a series of stand-alone manuscripts,
therefore some degree of overlap in the introduction of each chapter may be present. 1.7 Aims and Objectives All
laboratory and fieldwork, statistical analyses, and writing were completed by the author, with
further assistance from others as follows: Chapter 2: This chapter is formatted as an individual manuscript with combined results and
discussion, as this improves the clarity and interpretation of the very complex data sets
included. It has been submitted for publication in the journal Molecular Phylogenetics and
Evolution and is currently ‘under review’: Sproles, A. E., Kirk, N. L., Kitchen, S. A., Oakley,
C. A., Grossman, A. R., Weis, V. M., Davy, S. K. Phylogenetic characterization of transporter
proteins in the cnidarian-dinoflagellate symbiosis. N. L. Kirk and S. A. Kitchen assisted with
study design, data analysis and writing. C. A. Oakley, A. R. Grossman, V. M. Weis, and S. K. Davy assisted with study design and writing. Chapter 3: This chapter is formatted as an individual manuscript in preparation for publication:
Sproles, A. E., Matthews, J. M., Oakley, C. A., Grossman, A. R., Weis, V. M., Davy, S. K. Proteomic changes show that thermally-tolerant Symbiodinium may impair host cellular
processes. Experimental infection of Aiptasia and infection success measurements were
conducted equally with J. M. Matthews. C. A. Oakley assisted with protein preparations,
experimental design, and writing. V. M. Weis and A. R. Grossman assisted with experimental
design. S. K. Davy assisted with experimental design and writing. Chapter 4: This chapter is formatted as an individual manuscript in preparation for publication:
Sproles, A. E., Krueger, T., Oakley, C. A., Grossman, A. R., Weis, V. M., Miebom, A., Davy,
S. K. Symbiodinium type effects nutrient exchange and symbiont success in Aiptasia. T. Krueger assisted with experimental design and nanoSIMS preparation and analysis. C. A. Oakley, A. R. Grossman, V. M Weis and A. Miebom assisted with experimental design, and
S. K. Davy assisted with experimental design and writing. 39 40 2.1. Introduction The cnidarian-dinoflagellate symbiosis is an endosymbiotic relationship involving intercellular
exchange of many compounds. Dinoflagellates of the genus Symbiodinium reside within
cnidarian endodermal cells and provide the host with the majority of its fixed carbon as
photosynthetic products, with sugars being the major trafficked metabolites (Gordon & Leggat
2010; Davy et al. 2012; Kopp et al. 2015). In exchange, the cnidarian provides nitrogenous
compounds, generated as by-products from heterotrophic feeding, to the algal symbionts
(Allemand et al. 1998; Houlbrèque et al. 2004; Davy et al. 2012). Successful metabolite
exchange between the host and symbiont is key to the health and survival of reef-building
corals, as it allows the partners to obtain necessary nutrients that are often limiting in
oligotrophic oceans (Muscatine et al. 1981; O’Neil & Capone 2008). Characterization of transported metabolites has long been a focus of cnidarian-dinoflagellate
research. Glucose is now considered to be the primary carbon product translocated to the host
from the symbiont (Burriesci et al. 2012; Hillyer et al. 2016), although evidence from early
studies pointed to glycerol (Muscatine 1967; Lewis & Smith 1971; Schmitz & Kremer 1977). It is now thought that glycerol is primarily released by Symbiodinium cells in isolation (e.g. in
culture), while glucose is the major photosynthetic product translocated from the symbiont to
the host in hospite (Ishikura et al. 1999; Whitehead & Douglas 2003; Burriesci et al. 2012;
Hillyer et al. 2016). High levels of glycerol are also released as an osmolyte in response to
stress (Suescún-Bolívar et al. 2012). In the opposite direction, the major nitrogen metabolite
transported to the symbiont by the host is thought to be ammonium, while nitrate is an
important nutrient obtained from seawater (Muller-Parker et al. 1994; Pernice et al. 2012; Kopp
et al. 2013). Nitrogenous compounds are used for the synthesis of amino acids that are then
incorporated into symbiont proteins (Wang & Douglas 1998) or translocated back to the animal
(Wang & Douglas 1999; Tanaka et al. 2006). 41 Although these carbon and nitrogen metabolites are an important source of energy and nutrition
in the symbiosis, the transport mechanisms are still poorly understood. All molecules entering
or leaving a cell must traverse the plasma membrane, most either by active transport or passive
diffusion through transporter or channel proteins (Lodish et al. 2000). 2.1. Introduction In the cnidarian-
dinoflagellate symbiosis, Symbiodinium cells are housed within a host-derived vacuole called
the symbiosome, which acts as the interface between host and symbiont (Fitt & Trench 1983;
Wakefield et al. 2000; Wakefield & Kempf 2001). Therefore, the exchange of compounds must
be facilitated by proteins in the cellular membranes of each of the partners as well as in the
symbiosome interface (Fig. 1.5). Insights on nutrient transport can be gained from studies of other systems. Glucose primarily
enters eukaryotic cells through transporters in two families of compounds: facilitated glucose
transporters (GLUT) and sodium-coupled glucose transporters (SGLT) (Wood & Trayhurn
2003; Scheepers et al. 2015). Members of both families have been extensively characterized in
humans and other animals (Escher & Rasmuson-Lestander 1999; Doege et al. 2000, 2001;
Joost et al. 2002; Wright 2013). While few invertebrate GLUT and SGLT homologs have been
described (Escher & Rasmuson-Lestander 1999; Feng et al. 2013), proteins from both of these
families are encoded by genes of the cnidarian Aiptasia pallida (also known as ExAiptasia
pallida) (Baumgarten et al. 2015). Previous studies have used proteomics (Peng et al. 2010;
Oakley et al. 2016) and transcriptomics (Lehnert et al. 2014) to identify proteins involved in
the symbiosis; however, transporters specific to the symbiosome membrane have not been
firmly established. Glycerol can be trafficked by aquaporin (AQP) channels, which are intrinsic
water channels that transport small molecules (Hara-Chikuma & Verkman 2006; Rojek et al. 2008). Since most eukaryotes already use AQP to facilitate the diffusion of water, I predict that
cnidarians likely have AQP members capable of glycerol transport as well. Currently, three genes encoding ammonium transporters (AMT proteins) and two encoding
high-affinity nitrate transporters (NRT proteins) have been identified in Symbiodinium
transcriptomes (Mayfield et al. 2010; Brown et al. 2011; Van Den Heuvel 2012). The recent
genome assembly of Symbiodinium kawagutii (Clade F) has also revealed genes encoding
AMT and NRT family members (Lin et al. 2015). However, these studies have only focused
on a single Symbiodinium type and did not consider the large genetic diversity within the genus. 42 Currently, the Symbiodinium genus is classified into nine divergent clades (A–I), with each
(except clade E) further divided into sub-clades or “types” (e.g., A3, B1, D1a) and some
described species (Coffroth & Santos 2005; Pochon & Gates 2010; LaJeunesse et al. 2012). 2.1. Introduction Genetic distances between these clades were found to be greater than the distances separating
different species of free-living dinoflagellates (Santos et al. 2002; Takabayashi et al. 2004;
Sampayo et al. 2009; Krueger et al. 2015). Therefore, analysis of these proteins from a diversity
of Symbiodinium types may reveal differences in their conservation. The goal of this study was to identify and characterize potential membrane transport
mechanisms employed by both partners of the cnidarian-dinoflagellate symbiosis by
performing informatics/phylogenetic investigations of well-described sugar and nitrogen
transport proteins that are highly conserved among eukaryotes. I investigated five protein
families: three families of secondary active transporters, GLUT, SGLT, and NRT2, and two
families of channel proteins, AQP and AMT. I used a phylogenetic approach to identify
cnidarian homologs to GLUT, SGLT, and AQP, and Symbiodinium homologs to AMT and
NRT2. I then performed protein structural predictions, conservation analyses, and structural
domain identifications to help establish whether or not these proteins function in metabolite
exchange within the cnidarian-dinoflagellate symbiosis. Insights gained from these analyses
will help inform further functional investigations of the symbiosome membrane and enhance
our understanding of nutrient exchange in the cnidarian-dinoflagellate symbiosis. 2.2.2 Retrieval of protein homologs Sequences from the cnidarian and Symbiodinium databases with high sequence similarity to
the reference proteins were retrieved and used for phylogenetic analyses. Those protein
sequences were also used to search the NCBI non-redundant (nr) database using BLASTp,
retrieving sequences with an E value cut-off of ≤ 1 × 10-5 and excluding hypothetical proteins
(Appendix A, Datasheets A1–A5). The top five annotated BLAST hits for each sequence were
used as additional homologs in the phylogenetic analyses (Appendix A, Datasheets A1–A5). 2.2.1 Identification of cnidarian and Symbiodinium transporter proteins Separate analyses were completed to categorize the major transporters of the cnidarian host
and Symbiodinium symbiont. Cnidarian and Symbiodinium transcriptomes and genomes were
downloaded from publically available sources (Appendix A, Table A1), and each was
formatted into a searchable database. Reference sequences for each protein family were
obtained from UniProt (Appendix A, Table A2); these included Homo sapiens GLUT, SGLT,
and AQP, and Arabidopsis thaliana AMT and NRT2 (Appendix A, Table A2). Putative
orthologues to the reference sequences were identified in the cnidarian and Symbiodinium 43 databases by reciprocal BLAST (Telford 2007; Voolstra et al. 2011) with a bit-score cut-off of
80 for any given alignment. To identify cnidarian sugar transporters, H. sapiens reference
proteins (Appendix A, Table A2) were used as BLAST input queries to search cnidarian
databases and identify Symbiodinium nitrate and ammonium transporters; A. thaliana reference
proteins (Appendix A, Table A2) were similarly used as BLAST input queries for searching
the Symbiodinium databases. The best scoring matches were then converted to predicted
proteins using custom Perl scripts (obtained from Eli Meyer, https://github.com/Eli-Meyer). 2.2.3 Sequence alignment and phylogenetic analysis Protein family sequences were aligned using MAFFT v. 7.017 (default parameters) (Katoh &
Standley 2013). Alignments were manually edited in Aliview (Larsson 2014) to eliminate false
positive matches (Bucka-Lassen et al. 1999) by removing protein fragments (sequences of <
150 amino acids) and sequences that did not align with any of the defined motifs. Motifs used
were: the GLUT GR, GRR, and PETK motifs (Appendix A, Fig. A1), all of which are
considered necessary for sugar transport function (Joost & Thorens 2001); the SGLT Y, GG,
and D motifs (Appendix A, Fig. A2), which are the most conserved (≥77%) between all pro-
and eukaryotic taxa (Wright & Turk 2004); the two NPA motifs and one D residue of AQP
(Appendix A, Fig. A3), since NPA motifs form the aqueous pore and represent the AQP
signature while the D residue is present in aquaglyceroporins and widens the pore opening
allowing the transfer of larger solutes (Agre et al. 2002); the AMT D, AG, VH, and G motifs
(Appendix A, Fig. A4), which are responsible for ammonium recognition and transport 44 function (Marini et al. 2006); the NRT2 conserved F residue along with the proposed NNP
signature A/G-G-W-G/A-N/D-M/L-G and the structurally important GG and GAGG glycine
clusters (Appendix A, Fig. A5) (Trueman et al. 1996; Forde 2000). function (Marini et al. 2006); the NRT2 conserved F residue along with the proposed NNP
signature A/G-G-W-G/A-N/D-M/L-G and the structurally important GG and GAGG glycine
clusters (Appendix A, Fig. A5) (Trueman et al. 1996; Forde 2000). Phylogenetic analysis of each protein family was conducted using maximum-likelihood
inference in RAxML v8.0.26 (Stamatakis 2014). The PROTGAMMAAUTO function was
used to empirically determine the best substitution model for each dataset (highest likelihood
score of starting parsimony tree); all datasets in this study were best suited to using the AUTO
model. The topology with the highest likelihood score for each protein tree was selected from
100 replicate trees, and then branch support was calculated using 100 bootstraps. Trees were
rooted to the most ancestral protein recovered by BLAST homology searches, where ancestry
was determined in previous studies. Phylogenetic inference was then used to draw conclusions
on the protein identities of novel cnidarian and Symbiodinium sequences (Eisen et al. 1997;
Eisen 1998; Zmasek & Eddy 2001; Sjölander 2004). 2.2.4 Structural predictions and characterization of proteins Additional sequence analyses were conducted to further characterize the identified cnidarian
and Symbiodinium proteins. The number of transmembrane (TM) helices and subcellular
localization for each protein were predicted using the MemPype webserver, which outperforms
other localization methods with an accuracy rate of 70% (Pierleoni et al. 2011), and protein
domains were determined using the InterProScan (Quevillon et al. 2005) in Geneious v. 7.1.9. One protein sequence per gene family was also selected for protein structural analysis. Sequences from the cnidarian Nematostella vectensis with high similarity to the reference
sequences were used for analysis of the cnidarian proteins because they were closest in amino
acid length and had the highest similarity to the reference proteins. Furthermore, the N. vectensis genome had the most complete annotations. A sequence from Symbiodinium clade C
was used for AMT structural analysis to further examine the sequences within the observed
protein expansion, and a sequence from Symbiodinium clade A was chosen for NRT2 structural
analysis since it was the only clade with sequences retrieved for this protein family. The three-
dimensional structure and protein function were predicted using I-TASSER (Zhang 2008; Roy
et al. 2010). I-TASSER aligns predictions with the most similar protein structures from the 45 Protein Data Bank (http://www.rcsb.org/pdb) and evaluates the structural similarity using a
TM-score ranging from 0 – 1, where 1 indicates a perfect match. Ligands and ligand-binding
sites are also predicted as part of I-TASSER using the COACH algorithm (Yang et al. 2013),
which evaluates the confidence of the prediction using a C-score ranging from 0 – 1, where 1
indicates 100% confidence in the prediction. To determine the degree of structural conservation
within each cnidarian and Symbiodinium protein family, all newly identified sequences were
aligned within their family using MAFFT v. 7.017 and submitted to the ConSurf server
(Ashkenazy et al. 2010) along with the corresponding predicted 3D structure. Conservation
scores for each residue were calculated using the Bayesian method with the best evolutionary
substitution model determined empirically as WAG (Mayrose et al., 2004), and scores were
subsequently depicted onto the protein structures using Polyview 3D (Porollo & Meller 2007). 2.3.1 Cnidarian GLUT proteins A total of 23 cnidarian sequences were identified as GLUT homologs, representing putative
glucose transporters (Appendix A, Table A1). Sequence lengths were highly variable, with
some sequences containing fewer than the 12 TM domains that are typical of proteins within
the major facilitator superfamily (MFS), which includes GLUT proteins (Reddy et al. 2012). These sequences may represent incomplete proteins that were not excluded from our analysis
because they contain the conserved protein motifs (Appendix A, Table A1). Only cnidarian
sequences from GLUT classes 1 and 2 were identified. All sequences include domains for MFS
sugar transporters, while some also contain domains associated with specific GLUT members. Our phylogenetic analysis of the GLUT proteins resolved the sequences into four main clades
of solute transporters (Fig. 2.1): (1) organic cation transporters (OCT); (2) class 1 and 2 GLUT
proteins; (3) GLUT6 and GLUT8; and (4) GLUT10, GLUT12, and HMIT (proton/myo-inositol
transporter). One divergent protein from Porites australiensis (Fig. 2.1) branches from near the
root of the tree rather than clustering with any of the four main clades. This protein shares the
highest sequence similarity with the yeast glucose transporter RGT2 (Appendix A, Table A1). 46 Since the P. australiensis transcriptome was generated from RNA derived from the entire
holobiont rather than just host material, it is likely that this deduced protein represents RNA
contamination from the algal symbiont or bacteria (Shinzato et al. 2014). Robust support is
only observed for the OCT clade, with the other major clades being less well resolved. OCT is
another MFS protein family that is closely related to the sugar porter family (Saier et al. 1999)
(Fig. 2.1, clade 1). These proteins facilitate diffusion of organic cations such as
neurotransmitters, amino and fatty acids, and nucleotides in vertebrates, but they have not been
well studied in invertebrate systems (Eraly et al. 2004; Ciarimboli 2008). The clustering of the GLUT sequences resulted in a branching order that follows previously
constructed metazoan GLUT phylogenies (Wilson-O’Brien et al. 2010). Class 3 GLUT
proteins are ancestral to the other GLUTs, and they have diverged into three well supported
sub-clades (GLUT6 and GLUT8; GLUT10 and GLUT12; and HMIT), while class 1 and 2
proteins form a well-supported clade as sister groups that evolved more recently (Wilson-
O’Brien et al. 2010; Scheepers et al. 2015). 2.3.1 Cnidarian GLUT proteins Overall, I identified cnidarian homologs to all class
1 and class 3 GLUT members, except for GLUT6 (Appendix A, Table A1). 47 Figure 2.1 Maximum likelihood phylogenetic inference of GLUT sequences. The clustering of the GLU
sequences resulted in a branching order that follows previously constructed metazoan GLUT phylogen
(Wilson-O’Brien et al., 2010). Class 3 GLUT proteins are ancestral to the other GLUTs, and they have diverg
into three well supported sub-clades (GLUT6 and GLUT8; GLUT10 and GLUT12; and HMIT), while class 1 a
2 proteins form a well-supported clade as sister groups that evolved more recently (Scheepers et al., 2015; Wilso
O’Brien et al., 2010). Overall, I identified cnidarian homologs to all class 1 and class 3 GLUT members, exce
for GLUT6 (Appendix A, Table A1). Figure 2.1 Maximum likelihood phylogenetic inference of GLUT sequences. The clustering of the GLUT
sequences resulted in a branching order that follows previously constructed metazoan GLUT phylogenies
(Wilson-O’Brien et al., 2010). Class 3 GLUT proteins are ancestral to the other GLUTs, and they have diverged
into three well supported sub-clades (GLUT6 and GLUT8; GLUT10 and GLUT12; and HMIT), while class 1 and
2 proteins form a well-supported clade as sister groups that evolved more recently (Scheepers et al., 2015; Wilson-
O’Brien et al., 2010). Overall, I identified cnidarian homologs to all class 1 and class 3 GLUT members, except
for GLUT6 (Appendix A, Table A1). 48 Ten cnidarian putative GLUT8 proteins were identified (Fig. 2.1). GLUT8 is of particular
interest in the cnidarian-dinoflagellate symbiosis because it has a high affinity for glucose
(Uldry & Thorens 2004) and exhibits increased mRNA levels in symbiotic A. pallida relative
to those in aposymbiotic anemones of the same species (Lehnert et al. 2014). As it is localized
to endosomal compartments in mice, GLUT8 is predicted to function as a hexose transporter
in intracellular membranes (Augustin et al. 2005). Four of the identified cnidarian GLUT8
proteins in this study were predicted to be localized to internal membranes, while six were
predicted to exist in the plasma membrane (Appendix A, Table A1). Internal GLUT proteins
may reside in the symbiosome membrane, which is an internal vacuolar membrane of host
origin that is derived from an early endosome (Fitt & Trench 1983; Rands et al. 1993;
Wakefield & Kempf 2001). Three GLUT8 proteins were expressed in N. vectensis and two in
P. australiensis. All proteins in the non-symbiotic cnidarian N. 2.3.1 Cnidarian GLUT proteins vectensis were predicted to be
localized to the plasma membrane. The two GLUT8 proteins in the symbiotic cnidarian, P. australiensis, had different predicted localizations based on the MemLoci algorithm; one is
likely in the plasma membrane while the other appears to reside in internal membranes. These
analyses raise the possibility that symbiotic cnidarians have a distinct GLUT8 that is associated
with the symbiosome membrane where it facilitates movement of glucose from the symbiont
to the host. The deduced 3D structure of the cnidarian GLUT8 protein closely resembles the crystal
structure of human GLUT1 (TM score = 0.89, Fig. 2.2). All ten structural hits from the Protein
Data Bank were GLUT proteins from other species ranging from bacteria to vertebrates
(Appendix A, Datasheet A1, Tab C). Among the cnidarian GLUT sequences, conservation was
highest for residues on the inner surface of the translocation pore, where the active binding
sites are presumably located (Fig. 2.2). The predicted ligand was maltose (Fig. 2.2), a
disaccharide composed of two glucose molecules (Berg et al. 2002). However, confidence of
this prediction was low (C-score = 0.11), so I cannot make definitive conclusions about the
solutes transported by the cnidarian GLUT protein. Overall, the results of the structural analysis
support the hypothesis that the identified cnidarian proteins shared a common ancestor with
human GLUT proteins. 49 2.3.2 Cnidarian SGLT proteins 2.3.2 Cnidarian SGLT proteins A total of 23 cnidarian predicted sequences are homologs to SGLT proteins. While most
sequences contain the 12 TM helices indicative of the amino acid-polyamine- organocation
superfamily (APC), to which the SGLTs belong (Saier 2000), three sequences appear to be
protein fragments (Appendix A, Datasheet A2). Only one cnidarian sequence was identified as
likely to be a true SGLT (N. vectensis SLGT4). This sequence clustered with two other
cnidarian SGLT sequences that were previously identified in A. pallida. Similarly, one
cnidarian sequence was identified as a choline transporter (CHT) with high similarity to the A. pallida CHT protein. The majority of cnidarian sequences were identified as putative sodium
myo-inositol transporters (SMIT) or sodium-coupled monocarboxylate transporters (SMCT)
(Fig. 2.3). All of the identified SGLT homologs were predicted to be localized to either the
plasma membrane or internal membranes and contained the sodium solute symporter domain
(Appendix A, Datasheet A2). All proteins in the three main clades (CHT, SMCT, and SGLT) are part of the SLC5 gene
family and function as sodium ion symporters. Recovered cnidarian proteins did not share high
sequence similarity to the SGLT1-5 reference proteins, but they were closely related to SMCT
or SMIT proteins (Fig. 2.3). The SMCT1 protein (encoded by SLC5A8) can transport fatty
acids, carboxylic acids, and vitamins; it also functions as a tumour suppressor factor in humans
by allowing the uptake of the fatty acid butyrate, which induces apoptosis (Gupta et al. 2006;
Ganapathy et al. 2008). Cnidarian SMCT proteins were primarily predicted to be localized to internal membranes, with
fewer in the plasma membrane. Additionally, multiple SMCT sequences were found in two of
the symbiotic coral species, Fungia scutaria and P. australiensis (Appendix A, Datasheet A2,
Tab D). Symbiotic cnidarians are thought to regulate internal algal populations through
apoptosis of symbiont-containing gastrodermal cells (Muscatine & Pool 1979; Dunn et al. 2007b; Dunn & Weis 2009). If this is the case, the SMCT proteins could be located in the
symbiosome membrane to illicit this regulatory response by inducing host cell degradation. 50 Figure 2.2 Protein structural
analysis of selected sequences
from each gene family. Predicted
3D structures (left), conservation
analysis (middle), and predicted
ligands
(right)
of
cnidarian
proteins (A-C) and Symbiodinium
proteins (D-E): (A) GLUT, as
deduced from the amino acid
sequence
of
N. 2.3.2 Cnidarian SGLT proteins vectensis_4
GLUT8 and aligned with the
human GLUT1 protein (PDB ID
= 4pyp), (B) SMIT, as deduced
from the N. vectensis_5 SMIT
sequence and aligned with the
K294A mutant of VSGLT (PDB
ID = 2xq2), (C) AQP, as deduced
from the N. vectensis_4 AQP3
sequence and aligned with the E. coli glycerol facilitator GLPF
(PDB ID = 1fx8). Figure 2.2 Protein structural
analysis of selected sequences
from each gene family. Predicted
3D structures (left), conservation
analysis (middle), and predicted
ligands
(right)
of
cnidarian
proteins (A-C) and Symbiodinium
proteins (D-E): (A) GLUT, as
deduced from the amino acid
sequence
of
N. vectensis_4
GLUT8 and aligned with the
human GLUT1 protein (PDB ID
= 4pyp), (B) SMIT, as deduced
from the N. vectensis_5 SMIT
sequence and aligned with the
K294A mutant of VSGLT (PDB
ID = 2xq2), (C) AQP, as deduced
from the N. vectensis_4 AQP3
sequence and aligned with the E. coli glycerol facilitator GLPF
(PDB ID = 1fx8). (D) AMT, as deduced from
Symbiodinium
C1_25
AMT
sequence and aligned with the S. cerevisiae
methylammonium
permease Mep2 (PDB ID =
5aexA), and (E) NRT2, as
deduced
from
the
SymbiodiniumA_24
NRT2
sequence and aligned with the E. coli
glycerol-3-phosphate
transporter GlpT (PDB ID =
1pw4). Predicted structures of
each protein are coloured by
structure (alpha helix = pink, coil
= white, with yellow arrows
ribbons
showing
sequence
direction) and aligned with the
top matching structure from the
Protein Data Bank, depicted by a
cyan mesh ribbon. Conservation
scores are rendered onto the
protein models using a colour
gradient,
from
blue
(low
conservation, score = 1) to red
(high conservation, score = 9),
with yellow regions indicating
insufficient data for analysis. Ligand predictions show the
ligand at the predicted binding
sites with transparent protein
models coloured by secondary
structure (helices = red, coils =
blue). 51 The SGLT protein clade has high bootstrap support, and the tree shows SGLT and SMIT
sequences clustering together within vertebrate and invertebrate sub-clades, with one sub-clade
consisting entirely of cnidarian proteins (Fig. 2.3). Nine cnidarian proteins in this clade were
identified as SMITs, which are closely related to SGLTs of the SLC5 gene family. These
proteins function similarly to proton myo-inositol transporters (HMIT), primarily transporting
myo-inositol (Bourgeois et al. 2005). 2.3.2 Cnidarian SGLT proteins SMIT1 can also transport a variety of sugars, including
glucose, although it does so with low affinity (Wright 2013), while SMIT2 can transport D-
chiro-inositol (Coady et al. 2002). Myo-inositol is important for the growth of mammalian
cells and is a key component in phospholipid biosynthesis and eukaryotic signalling pathways
(Holub 1986). The identification of both HMIT and SMIT transporters in cnidarians suggests
an uncharacterized role for myo-inositol in cnidarian metabolism or cell structure. A SMIT protein sequence from N. vectensis (N. vectensis_5 SMIT) was used for 3D structural
analysis (Fig. 2.2). The predicted tertiary structure of the protein most closely resembles that
of the K294A mutant of the VSGLT protein from Escherichia coli (TM score = 0.78). Overall
residue conservation was low, with the most conserved residues lining the protein pore (Fig. 2.2). The ligands were predicted to be sodium (C-score = 0.21) and galactose (C-score = 0.19),
which only weakly supports the idea that this protein functions as a sodium-coupled sugar
transporter (Fig. 2.2). Proteins in the SGLT family are known to transport galactose, some with
a high affinity (Zhao & Keating 2007); however, experimental analysis is needed to confirm
this function in cnidarians. 52 52 Figure 2.3 Maximum likelihood phylogenetic inference of SGLT sequences. The tree was rooted by the bacterial
sodium solute symporter (SSS) homolog from Acanthamoeba castellanii. The number of amino acid substitutions
is indicated by the scale bar, and bootstrap support for nodes >50% is represented by shaded circles. Three main
clades were resolved: (1) choline transporters (CHT), (2) sodium-coupled monocarboxylate transporters (SMCT),
and (3) sodium/glucose cotransporters (SGLT and SMIT). The cluster of vertebrate SMIT proteins is collapsed to
enhance tree clarity. The sequence used for structural analysis is marked by a yellow star (N. vectensis_5 SMIT). Figure 2.3 Maximum likelihood phylogenetic inference of SGLT sequences. The tree was rooted by the bacterial
sodium solute symporter (SSS) homolog from Acanthamoeba castellanii. The number of amino acid substitutions
is indicated by the scale bar, and bootstrap support for nodes >50% is represented by shaded circles. Three main
clades were resolved: (1) choline transporters (CHT), (2) sodium-coupled monocarboxylate transporters (SMCT),
and (3) sodium/glucose cotransporters (SGLT and SMIT). The cluster of vertebrate SMIT proteins is collapsed to
enhance tree clarity. The sequence used for structural analysis is marked by a yellow star (N. vectensis_5 SMIT). 53 2.3.3 Metazoan AQP proteins 2.3.3 Metazoan AQP proteins A total of 16 cnidarian sequences were identified as AQP homologs, all with at least five TM
domains typical of AQPs (Appendix A, Datasheet A3). In cnidarians, the majority of the AQP
homologs were predicted to be localized to internal membranes (Appendix A, Datasheet A3). All sequences contained domains present in members of the major intrinsic protein family and
in AQP transporters, while some also had domains associated with specific AQPs (Appendix
A, Datasheet A3). Our phylogenetic analysis resulted in the emergence of two sister clades: (1)
aquaglyceroporins; and (2) classical aquaporins (Fig. 2.4). All sequences identified as
aquaglyceroporins contained the conserved aspartate (D) residue that signifies a larger protein
pore (Agre et al. 2002). Aquaglyceroporin proteins all share a common ancestor, but these proteins were shown to have
diverged into vertebrate and invertebrate lineages before the emergence of the different protein
members. The cnidarian aquaglyceroporin sequences were identified as either AQP3 or AQP9. These sub-clades of exclusively cnidarian sequences are highly supported and include two
previously identified AQP3 sequences from A. pallida, suggesting that the other cnidarian
sequences are also AQP3 proteins. In mammals, aquaglyceroporins can transport a diversity of
solutes other than glycerol, including urea and water (Rojek et al. 2008). AQP3 primarily
transports water and glycerol, while AQP9 can also transport larger molecules, such as lactate,
purines, and pyrimidines (Ishibashi et al. 2011). Experimental evidence is needed to confirm
which of these small solutes the cnidarian AQP proteins transport. 54 Figure 2.4 Maximum likelihood phylogenetic inference of AQP sequences. The tree was rooted by the bacterial
homolog from Desulfuromonas acetoxidans. The number of amino acid substitutions is indicated by the scale bar,
and bootstrap support for nodes >50% is represented by shaded circles. The proteins resolved into two main
clades: (1) aquaglyceroporins and (2) classical aquaporins. The sequence used for structural analysis is marked
by a yellow star (N. vectensis_4 AQP3). Figure 2.4 Maximum likelihood phylogenetic inference of AQP sequences. The tree was rooted by the bacterial
homolog from Desulfuromonas acetoxidans. The number of amino acid substitutions is indicated by the scale bar,
and bootstrap support for nodes >50% is represented by shaded circles. The proteins resolved into two main
clades: (1) aquaglyceroporins and (2) classical aquaporins. The sequence used for structural analysis is marked
by a yellow star (N. vectensis_4 AQP3). 2.3.3 Metazoan AQP proteins The clade of classical aquaporins diverges into a sub-clade of nodulin 26-like proteins (NIP)
and a sub-clade of metazoan proteins. The NIP clade is highly supported and contains mostly
proteins from vascular plants, along with one cnidarian NIP (Fig. 2.4). The identification of a
cnidarian NIP is surprising since these proteins are known as plant-specific water channels that
function similarly to aquaglyceroporins (Ishibashi et al. 2011). Nodulin 26 is an integral
symbiosome membrane protein in nitrogen-fixing soybean nodules that acts as both an ion
channel and a water channel (Fortin et al. 1986; Weaver et al. 1994); however, the cnidarian
NIP in this study was present in a non-symbiotic species and may be representative of
contamination from the cnidarian’s environment. 55 The sub-clade of metazoan classical aquaporins contains cnidarian sequences sharing a
common ancestor with vertebrate and invertebrate AQP1, as well as vertebrate AQP4 and
AQP0 (Fig. 2.4). While AQP1 and AQP4 are water-only channels, AQP0 has a variety of
functions in other organisms (Agre et al. 2002). AQP0 is the lens major intrinsic protein,
originally found in lens fibre cells of vertebrate eyes and later discovered to function in
increasing the water permeability of animal cells (Gorin et al. 1984; Mulders et al. 1995). Its
suspected structural role in cell adhesion making AQP0 unique in the aquaporin family
(Fotiadis et al. 2000). Cell adhesion proteins are important in the cnidarian-dinoflagellate
symbiosis for cell-cell interactions, such as signalling between partners (Reynolds et al. 2000);
cnidarian AQP0 may function in this capacity. One AQP9 sequence from N. vectensis (N. vectensis_4 AQP3) was used for structural analysis
of the cnidarian AQP protein (Fig. 2.2). The deduced structure was very similar to that of the
E. coli glycerol facilitator, GLPF (Fig. 2.2, TM score = 0.90). Additional hits from the Protein
Data Bank were protein structures of aquaglyceroporins from other species (Appendix A,
Datasheet A3, Tab C). Furthermore, the predicted ligand (Fig. 2.2) was glycerol (C-score =
0.46), and the most highly conserved residues among all cnidarian AQPs were those lining the
protein pore (Fig. 2.2). Overall, the structural analysis supports the prediction that these
proteins function as glycerol channels. 2.3.4 Symbiodinium AMT proteins A total of 40 Symbiodinium sequences were putatively identified as AMT proteins. Most of
these sequences contained 10–12 TM domains, which is typical of AMT proteins (Thomas &
Mullins 2000; von Wiren et al. 2000), while five sequences were shorter, containing only seven
to nine TM helices. The sequences were primarily identified as general AMT proteins rather
than specific members or isoforms and were predicted to be localized to either the cell
membrane or internal membranes (Appendix A, Datasheet A4, Tab D). All sequences
contained the domain for ammonium transport (Appendix A, Datasheet A4, Tab D). 56 Phylogenetic analysis showed the divergence of five main AMT clades: (1) bacterial cyclases;
(2) metazoan-like AMT proteins; (3) diaphoretickes AMT proteins; (4) alveolate AMT
proteins; and (5) bacterial-like AMT proteins (Fig. 2.5). The clade of bacterial cyclases is not
well supported and includes the enzymes adenylate cyclase and guanylate cyclase exclusively
from bacterial species. Several clades of AMT proteins were identified in the phylogenetic analysis. The first clade
shows AMT proteins diverging into three sub-clades, although not all are well resolved (Fig. 2.5). Clade 2 has robust support and shows metazoan AMT proteins emerging alongside a
cluster of exclusively Symbiodinium proteins. This clade includes four A. pallida sequences in
the metazoan cluster and five of the Symbiodinium sequences that contain the domain
characteristic of AMT3. The finding of Symbiodinium AMTs sharing a common ancestor and
sequence similarities with AMTs of the cnidarian host A. pallida raises the possibility of
horizontal gene transfer between symbiotic partners, which was also found to be the case in the
Aiptasia genome assembled by Baumgarten et al. (2015). Twenty-nine Aiptasia gene products
within the genome were found to best align with Symbiodinium proteins, and formed a distinct
clade together after phylogenetic analysis, further supporting the hypothesis of horizontal gene
transfer between Symbiodinium and cnidarians (Baumgarten et al. 2015). Clade 3 is only weakly supported overall and includes proteins from organisms within the
expansive diaphoretickes super-group (Adl et al. 2012). Nevertheless, a well-supported sub-
clade shows a Symbiodinium AMT sequence (SymbiodiniumC1_12) grouping with vascular
plant and red algal proteins. This Symbiodinium sequence also had high sequence similarity to
the AMT1-3, which in plants has a high affinity for the uptake of both methylammonium and
ammonium (von Wiren et al. 2000). 2.3.4 Symbiodinium AMT proteins Consequently, the upregulation of AMT proteins in this Symbiodinium type may have evolved
as an adaptation to survive the varying nitrogen conditions experienced in different host cell
environments. The second lineage including Symbiodinium AMT proteins is a well-supported
clade showing divergence from bacterial proteins into alveolate and red algal proteins (Fig. 2.5,
clade 5). Overall, our analysis of the Symbiodinium AMT proteins and their homologs has
revealed a large diversity of these transporters within this dinoflagellate species, which may
have arisen through multiple evolutionary events caused by horizontal gene transfer,
duplication, or classical evolution. For structural analysis of the Symbiodinium AMT proteins, one sequence from Symbiodinium
clade C (SymbiodiniumC1_25) that falls within the alveolate cluster was used. The predicted
3D structure most closely resembles the structure of the Saccharomyces cerevisiae
methylammonium permease Mep2 (TM score = 0.84, Fig. 2.2). Residues lining the inside of
the protein channel are highly conserved (Fig. 2.2), suggesting their importance in transport
function. Ligand predictions (Fig. 2.2) had very low confidence scores but favoured
methylamine (C-score = 0.1) and ammonia (C-score = 0.09). The information gained from this
structural analysis provides preliminary evidence that these highly conserved proteins serve as
ammonium channels. 2.3.4 Symbiodinium AMT proteins While clade 4 is also lacking adequate bootstrap support,
it contains a highly supported sub-clade that includes a large amount of expressed
Symbiodinium C1 AMT proteins in relation to other alveolates (Fig. 2.5). While expression of
multiple AMT proteins is observed for almost all alveolate species in the analysis,
Symbiodinium C1 was found to express a total of 28 proteins, while other alveolate species
have only one to four expressed AMT proteins. 57 8
Figure 2.5 Maximum likelihood phylogenetic inference of AMT sequences. The tree was rooted by the bacterial
homolog from a Spirochaeta sp. The number of amino acid substitutions is indicated by the scale bar, and
bootstrap support for nodes >50% is represented by shaded circles. The proteins resolved into five main clades:
(1) bacterial cyclases, (2) metazoan-like ammonium transporters, (3) plant-like ammonium transporters, (4)
chromalveolate ammonium transporters, and (5) bacterial-like ammonium transporters. The sequence used for
structural analysis is marked by a yellow star (Symbiodinium C1_25 AMT). Figure 2.5 Maximum likelihood phylogenetic inference of AMT sequences. The tree was rooted by the bacterial
homolog from a Spirochaeta sp. The number of amino acid substitutions is indicated by the scale bar, and
bootstrap support for nodes >50% is represented by shaded circles. The proteins resolved into five main clades:
(1) bacterial cyclases, (2) metazoan-like ammonium transporters, (3) plant-like ammonium transporters, (4)
chromalveolate ammonium transporters, and (5) bacterial-like ammonium transporters. The sequence used for
structural analysis is marked by a yellow star (Symbiodinium C1_25 AMT). 58 Symbiodinium C1 is known as a generalist symbiont that occurs worldwide, forming
associations with a variety of invertebrate hosts (LaJeunesse 2005; Stat et al. 2009). Consequently, the upregulation of AMT proteins in this Symbiodinium type may have evolved
as an adaptation to survive the varying nitrogen conditions experienced in different host cell
environments. The second lineage including Symbiodinium AMT proteins is a well-supported
clade showing divergence from bacterial proteins into alveolate and red algal proteins (Fig. 2.5,
clade 5). Overall, our analysis of the Symbiodinium AMT proteins and their homologs has
revealed a large diversity of these transporters within this dinoflagellate species, which may
have arisen through multiple evolutionary events caused by horizontal gene transfer,
duplication, or classical evolution. Symbiodinium C1 is known as a generalist symbiont that occurs worldwide, forming
associations with a variety of invertebrate hosts (LaJeunesse 2005; Stat et al. 2009). 2.3.5 Symbiodinium NRT2 proteins Only four sequences in total were identified as Symbiodinium NRT2 homologs, all of which
were from Symbiodinium clade A, although a sequence from clade C and an unspecified
Symbiodinium sequence within the NCBI database were also related to NRT2. The four
sequences had high similarity with one another (74–79%) and ranged in length from 549–613
amino acids with 11–12 TM helices, typical of MFS transporters (Appendix A, Datasheet A5). Half of these NRT2 homologs were predicted to be localized to the plasma membrane and the 59 other half to internal membranes, while all sequences contained the domains for MFS and were
related to high affinity nitrate transporter 2.3 (Appendix A, Datasheet A5, Tab D). Phylogenetic analysis of the NRT2 proteins showed divergence of: (1) eukaryotic; and (2)
bacterial nitrate transporters (Fig. 2.6). Surprisingly, very few NRT sequences were recovered
from the Symbiodinium databases, and no other alveolate NRT2 sequences were identified (Fig. 2.6). Expression of NRT2 proteins are highly regulated by environmental nitrate
concentrations. As part of the high-affinity uptake system, the proteins are upregulated during
nitrogen starvation (< 0.5 mM) and repressed when nitrate levels are high (> 0.5 mM)
(Okamoto et al. 2003; Lezhneva et al. 2014). If the Symbiodinium NRT2 protein level is
regulated in the same way, then the gene would be repressed when the cells are grown in the
commonly used f/2 culture medium, since it contains 0.882 mM nitrate (Guillard 1975). However, as copies of the NRT2 genes were not discovered in the Symbiodinium genomes
surveyed, identification of the protein may indicate that this gene has either been lost in some
derived species of this genus but retained by the more ancestral Symbiodinium clade A, or that
the proteins discovered were from contaminants in the clade A transcriptome. The expression of nitrate transporters in only Symbiodinium clade A could be due to the free-
living nature of some dinoflagellate types within this clade (Hirose et al. 2008; Reimer et al. 2010), allowing the cells to more readily utilize nitrate from seawater. Ammonium is excreted
by the host, while nitrate is not, and ammonium has been predicted to be the primary nitrogen
source for symbiotic Symbiodinium cells (Kopp et al. 2013; Pernice et al. 2014). 2.3.5 Symbiodinium NRT2 proteins Many
Symbiodinium types that have evolved to be strictly symbiotic may have lost the need for high-
affinity nitrate transport, while those with a partially or wholly free-living lifestyle may have
retained it. While I had initially hypothesized that the identified Symbiodinium NRT2 homologs are nitrate
transporters, the structural analysis indicates otherwise. The predicted 3D structure of the
putative Symbiodinium NRT2 sequence (Symbiodinium A-24 NRT) was most similar to that of
the glycerol-3-phosphate transporter from E. coli (TM score = 0.73, Fig. 2.2), rather than any
nitrate transporters. Overall conservation was low, and one of the TM helices was only found 60 in a single sequence (Fig. 2.2). The ligand was predicted as monoacylglycerol (Fig. 2.2) but
with low confidence (C-score = 0.06). While the identified potential NRT2 homologs resemble
integral membrane transport proteins, they are very likely MFS transporters with a different
function. Figure 2.6 Maximum likelihood phylogenetic inference of NRT2 sequences. The tree was rooted by the clade of
bacterial sequences. The number of amino acid substitutions is indicated by the scale bar, and bootstrap support
for nodes >50% is represented by shaded circles. Proteins resolved into two clades: (1) eukaryotic and (2) bacterial
nitrate transporters. The sequence used for protein structural analysis is marked with a yellow star (Symbiodinium
A-24 NRT). Figure 2.6 Maximum likelihood phylogenetic inference of NRT2 sequences. The tree was rooted by the clade of
bacterial sequences. The number of amino acid substitutions is indicated by the scale bar, and bootstrap support
for nodes >50% is represented by shaded circles. Proteins resolved into two clades: (1) eukaryotic and (2) bacterial
nitrate transporters. The sequence used for protein structural analysis is marked with a yellow star (Symbiodinium
A-24 NRT). 61 2.4. Conclusions This study identified sugar transport proteins from cnidarians and nitrogen transport proteins
from their dinoflagellate symbionts. Homology searches identified previously undescribed
cnidarian GLUT, SGLT, and AQP homologs and Symbiodinium AMT and NRT2 homologs. Phylogenetic and structural analyses demonstrated high conservation of most of the
investigated protein families, suggesting that the characterized transport functions of the
reference sequences are likely retained in the identified cnidarian and Symbiodinium sequences. Considerably more cnidarian sequences were identified in the GLUT family than in the SGLT
family, signifying that GLUT proteins could be involved in the transport of glucose between
the symbiont and cnidarian host. Genes encoding for glucose transporters are present in the
Symbiodinium genome, indicating the ability of the sugar to be exported to the symbiosome
space via this mechanism (Lin et al. 2015), where cnidarian GLUT proteins could facilitate
uptake into the animal cells. Furthermore, potentially related transporters of the SLC5 gene
family known to transport other compounds, such as monocarboxylates and myo-inositol, were
identified in the cnidarians. Protein sequences for cnidarian aquaglyceroporins were also
discovered, suggesting a potential mechanism for glycerol translocation into cnidarian cells. Many of the sugar transporters discussed here have predicted localizations to internal
membranes, which are defined in the MemPype pipeline as “the endoplasmic reticulum, the
nuclear membranes, the Golgi apparatus, the vesicles, the vacuoles, the lysosomes, the
peroxisome, the microsomes, and the endosome” (Pierleoni et al. 2011). Since the symbiosome
is derived from an early endosome (Fitt & Trench 1983; Wakefield & Kempf 2001), I presume
that symbiosome-localized proteins would fall into the “internal membrane” category. Transporters predicted as localized to only internal membranes were OCT and AQP9
(Appendix A, Datasheets A2–A3). Investigations of these proteins might identify them as being
associated exclusively with the symbiosome, which would be helpful in the establishment of
specific marker proteins for identification of symbiosome membranes. 62 Additionally, more Symbiodinium AMT sequences were identified than NRT sequences, which
is likely due to ammonium being the primary nitrogen source for symbiotic dinoflagellates
(D’Elia et al. 1983; Grover et al. 2002). Based on our findings, I suggest the use of AMT
transporters to investigate the nutritional conditions experienced by symbionts in hospite. Since
expression of these proteins is known to be regulated by environmental ammonium
concentrations, AMT transporter expression patterns by Symbiodinium cells in hospite could
reveal information about the nutrient conditions experienced within symbiotic host cells. 2.4. Conclusions It has
previously been hypothesized that the host can regulate intracellular symbiont populations by
limiting their growth and nutrient supply (Muscatine & Pool 1979; Falkowski et al. 1993; Jones
& Yellowlees 1997; Wooldridge 2010). In plants and various algal species, AMT isoforms are
transcriptionally regulated by environmental conditions, in a manner similar to that for genes
encoding NRT2 proteins, where high-affinity isoforms are expressed during nitrogen starvation
and low-affinity isoforms are expressed under ample nutrient availability (Gazzarrini et al. 1999; González-Ballester et al. 2004; Lezhneva et al. 2014). Therefore, verification of
regulated expression in Symbiodinium and analysis of AMT proteins during colonization
experiments may help to establish whether or not the host imposes nitrogen limitations on the
symbiont population. The resources used to compile cnidarian and Symbiodinium databases in this study included
mostly transcriptomic data. Molecular phylogenies conducted on proteins rather than DNA are
more accurate at establishing relationships between lineages, since proteins are the molecules
on which natural selection acts (Salemi & Vandamme 2003). While transcriptomes contain
highly useful information about the proteins being transcribed during the times of sampling,
they represent only a snapshot of an organism’s genome and not a comprehensive view of its
total coding capacity. The results of this study can be interpreted as putative identification of
symbiont and host transport proteins, while the absence of any proteins in our datasets does not
necessarily indicate their absence from the organism. Absence of proteins indicates that they
were not expressed at the time of sampling for the transcriptome, yet the organism may still
have the genes for these proteins; only an analysis of the organism’s genome would be able to
confirm this information. All new protein identifications described here are inferred from
homology and require verification through experimental analyses. While the results cannot be
used to deduce the full range of cnidarian and Symbiodinium proteins from each of these five
transporter families, they do give us a previously unseen glimpse of a subset of transporter 63 proteins that are likely used in the cnidarian-dinoflagellate symbiosis. Using currently available
genetic resources, I have taken the first steps towards uncovering mechanisms used in the
exchange of carbon and nitrogen between the partner organisms in the cnidarian-Symbiodinium
symbiosis. With the emergence of new genomic resources and proteomic-based experiments,
we are likely to develop a more complete picture of transporter functions and mechanisms in
symbiotic associations. 64 3.1 Introduction The cnidarian-dinoflagellate symbiosis allows for the sustainability of biodiverse coral reef
ecosystems within nutrient-limited oceans (Muscatine & Porter 1977). A range of cnidarian
species can act as the host – such as corals, sea anemones, and jellyfish – while the symbionts
are dinoflagellates from the genus Symbiodinium (Schoenberg & Trench 1980). Genetic
diversity within the genus is extensive; the genus is classified into nine divergent clades, which
are further subdivided into sub-clades or ‘types’, along with a handful of described species
(Trench and Blank 1987; Coffroth and Santos 2005; Pochon and Gates 2010; LaJeunesse et al. 2012; LaJeunesse et al. 2015; Lee et al. 2015). Physiology and host specificity vary between
Symbiodinium types, with a range of thermal tolerances (Rowan et al. 1997; Berkelmans & van
Oppen 2006) and abilities to form associations with either multiple or just a single host species
(van Oppen et al. 2001; Baker 2003; Santos et al. 2004; Xiang et al. 2013). The underlying
cause of host-symbiont specificity is still not entirely clear, although it is linked to cellular
interactions between partners (Lin et al. 2000; Bay et al. 2011; Davy et al. 2012). Coral reefs worldwide are threatened by climate change, which is warming the oceans and
inducing breakdown of the symbiosis, a process also known as coral bleaching (Hoegh-
Guldberg et al. 2007). Coral bleaching often leads to host mortality, unless a symbiont
population can quickly be re-established (Jokiel 2004). Some Symbiodinium types are more
tolerant to higher temperatures (such as many members of clades A and D), therefore providing
the host with more resistance to bleaching (Tchernov et al. 2004; Hennige et al. 2011;
LaJeunesse et al. 2014; Hume et al. 2016). These thermally-tolerant algae can act as
opportunists, where they replace the dominant symbiont type once its population numbers
decline (Baker & Kelly 2004; Jones et al. 2008; LaJeunesse et al. 2009). Symbiodinium from
clade D are the most common opportunists during times of thermal stress (Rowan 2004;
LaJeunesse et al. 2009; Stat & Gates 2011). More specifically, Symbiodinium type D1a
(Symbiodinium trenchii) is the most globally distributed symbiont type that commonly
populates heat-stressed corals (LaJeunesse et al. 2014; Hoadley et al. 2016). 3.1 Introduction The presence of
opportunistic, thermally-tolerant algae has led to a theory that corals may adapt to climate 65 change by ‘shuffling’ or ‘switching’ their symbiont assemblages to accommodate clade D
Symbiodinium (or other thermally tolerant symbiont types) as the dominant partner, thereby
conferring a higher thermal tolerance on the entire holobiont (Baker et al. 2003; Berkelmans
& van Oppen 2006; Baskett et al. 2009). Symbiont shuffling is described as a change in the
relative abundance of existing symbiont types within a mixed assemblage of homologous (i.e. naturally occurring) symbionts, while symbiont switching is described as a complete change
from the dominant homologous symbiont type to a heterologous (i.e. not naturally occurring)
type acquired from the environment (Baker 2003). S. trenchii is thought to be a Pacific Ocean
dinoflagellate that was introduced to the Atlantic and has since become invasive, as it is the
only clade D symbiont found in the Caribbean (Pettay et al. 2015). In various Caribbean corals,
S. trenchii exists as a background symbiont that becomes dominant when the host is under
environmental stress (Correa et al. 2009; Grottoli et al. 2014; Kennedy et al. 2015). This
demonstrates an interesting phenomenon that lies somewhere between the previously defined
symbiont shuffling and switching, where a non-native symbiont colonizes heterologous hosts
over time and can out-compete the homologous type under stress, but never completely
replaces the homologous symbiont and returns to only minimal background populations once
environmental conditions stabilize (Thornhill et al. 2006). This gives us evidence that
heterologous symbioses can occur in nature, but it is unknown if they are capable of completely
replacing the homologous type in the long-term. Symbiotic interactions between cnidarians and Symbiodinium are thought to exist on a
continuum from mutualism to parasitism (Lesser et al. 2013). Clades with thermally tolerant
symbiont types can exhibit more parasitic qualities by impairing host metabolism and
reproduction (Little et al. 2004; Stat et al. 2008; Jones & Berkelmans 2011; Pernice et al. 2014). For instance, both Symbiodinium clades A and D translocate less fixed carbon to their
hosts than do other symbiont types (Stat et al. 2008; Pernice et al. 2014). Additionally, corals
at the Keppel Islands infected with clade A were found to have the lowest survival and growth
rates compared to corals infected with two other symbiont types (Mieog et al. 2009). 3.1 Introduction Clade D
also has an impact on growth rate, as seen by the two- to three-fold slower growth of juvenile
corals infected with clade D compared to juveniles infected with Clade C (Little et al. 2004). Likewise, corals harbouring clade D contain less stored lipid, with smaller and less numerous
eggs produced during spawning compared to counterparts harbouring clade C (Jones &
Berkelmans 2011). However, while these previous studies provide evidence that clade D is a 66 less mutually beneficial symbiont, conferring reduced fitness even to its regular host species,
the molecular effects of hosting this symbiont have never been investigated in a novel
symbiosis. This therefore hinders our ability to understand the implications of hosting a
heterologous, and potentially less beneficial, symbiont type. Indeed, cnidarians can be
experimentally infected with a range of heterologous Symbiodinium types that can form long-
term associations with the new host, though these are typically less successful than their
homologous counterparts (Weis et al. 2001; Belda-Baillie et al. 2002; Dunn & Weis 2009). Examining the response of symbiotic cnidarians to infection by different symbiont types at the
molecular, cellular and physiological levels, therefore provides a means of understanding the
diverse, complex processes that undoubtedly underlie the formation of a successful cnidarian-
dinoflagellate symbiosis, and which may dictate the capacity for partner switching and
holobiont adaptation in the face of climate change. Protein expression can be used to determine the effect of environmental conditions on an
organism’s cellular processes (Bradley et al. 2002). Therefore, proteomic analysis can be a
powerful tool for elucidating the impacts of different symbiotic partners on a host cnidarian. Early research in this field reported differences in protein profiles between symbiotic and
aposymbiotic host anemones (Anthopleura elegantissima) using Western blots, providing
evidence that dinoflagellate symbionts directly affect host molecular biology (Weis & Levine
1996). Later, the cnidarian genes encoding for two of the symbiosis-upregulated proteins were
identified as carbonic anhydrase (Weis & Reynolds 1999) and sym32, a cell adhesion protein
(Reynolds et al. 2000). Investigations aimed at identifying additional host proteins involved in
symbiosis have since begun using advanced quantitative methods such as liquid
chromatography-nano electrospray-tandem mass spectrometry (LC-nano-ESI-MS/MS). Peng
and co-workers (2010) conducted a proteomic analysis of isolated symbiosome membranes
(i.e. host gastrodermal vesicles housing symbiont cells), identifying a small fraction of proteins
derived from both host and symbiont (Peng et al. 2010). 3.1 Introduction More recently, Oakley and co-workers
(2016) identified host proteins of the model cnidarian Aiptasia that are associated with the
symbiotic state, finding that host metabolism shifts to accommodate symbiotic nutrient
exchange (Oakley et al. 2016). Furthermore, Oakley et al. (submitted) used the same methods
to identify Aiptasia proteins associated with thermal stress, finding that heat shock induced a
number of stress-related proteins in the host, while slower acclimation to a higher temperature
had virtually no effect on the host proteome. While progress has been made on the 67 characterization of proteins involved in cnidarian-dinoflagellate cellular processes, there have
yet to be any investigations on the impact of different symbiont types on the host proteome. In this study, I compared the effect of a heterologous clade D Symbiodinium versus a
homologous clade B Symbiodinium on the symbiotic cnidarian Aiptasia at the proteomic level. Aiptasia is an important, globally-adopted model system for the study of the cnidarian-
dinoflagellate symbiosis (Weis et al. 2008). This approach provided a platform to elucidate the
molecular processes and biological functions that determine the successful establishment and
persistence of the cnidarian-dinoflagellate symbiosis, and the implications of harbouring a
novel, thermally-tolerant but potentially less mutually beneficial symbiont type. Ultimately,
this approach sheds light on the adaptive potential of the symbiosis by means of changing the
dominant symbiont type, and informs subsequent, more specific examination of the coral-
dinoflagellate symbiosis. In this study, I compared the effect of a heterologous clade D Symbiodinium versus a
homologous clade B Symbiodinium on the symbiotic cnidarian Aiptasia at the proteomic level. Aiptasia is an important, globally-adopted model system for the study of the cnidarian-
dinoflagellate symbiosis (Weis et al. 2008). This approach provided a platform to elucidate the
molecular processes and biological functions that determine the successful establishment and
persistence of the cnidarian-dinoflagellate symbiosis, and the implications of harbouring a
novel, thermally-tolerant but potentially less mutually beneficial symbiont type. Ultimately,
this approach sheds light on the adaptive potential of the symbiosis by means of changing the
dominant symbiont type, and informs subsequent, more specific examination of the coral-
dinoflagellate symbiosis. 3.2.1 Experimental organisms Individuals of the symbiotic sea anemone Aiptasia sp. (= ExAiptasia sp.) (n ~ 750) were
collected from a laboratory stock population, originating from an unknown Pacific location,
and divided evenly into three 1.5 L beakers. Beakers were filled with 0.22 µm-filtered seawater
(FSW) and aerated with Hailea® air pumps, and then placed in separate water baths maintained
at 25oC with WEIPRO® MX-1019 temperature controllers. Light was provided by AQUA-
GLO T8 fluorescent lightbulbs at ~95 µmol photons m-2 s-1 on a 12 h:12 h light: dark cycle. These temperature and light conditions were maintained throughout the experiment. Anemones were allowed to settle within beakers for 72 h, and then rendered aposymbiotic
using the menthol treatment described in Matthews et al. (2016). At the end of treatment, a
subset (n = 20) of anemones was checked for residual symbionts with a confocal microscope
(Olympus Provis AX70, 40x magnification). Each anemone was placed into a glass-bottom
FluoroDish™ (World Precision Instruments, FL, USA) and examined at 40x magnification 68 under a 543 nm laser with a 600-700 nm emission filter, exciting the chlorophyll
autofluorescence from any remaining symbionts. Aposymbiotic individuals were divided
amongst 15 containers with screw-top lids and placed into water baths designated for the three
treatment groups: Symbiodinium B1 (S. minutum); Symbiodinium D1a (S. trenchii); and
aposymbiotic. Symbiodinium B1 and D1a were sub-cultured from laboratory stocks and grown in f/2 medium
(Bigelow Laboratory, USA) at 25oC under an irradiance of 100 µmol photons m-2 s-1 (OSRAM
DULUX L 36W 4000K white fluorescent bulbs) on a 12 h:12 h light: dark cycle for six weeks
prior to the infection experiment. A 1-mL aliquot from each culture was centrifuged for 1 min
at 16,000 x g to pellet algae. The supernatant was then discarded and the pellet stored in 500
µL DMSO buffer [20% dimethyl sulfoxide and 0.5 M ethylene diaminetetraacetic acid (EDTA)
in NaCl-saturated water] at 4oC prior to genotyping (see below). 3.2.2 Infection of Aiptasia with Symbiodinium Prior to infection, anemones were starved for approximately one week to encourage the uptake
of symbionts as a feeding response during the infection experiment. A subset of anemones from
each treatment group (n ~ 20) were also examined with confocal microscopy (see above for
details) one day prior to infection to ensure that they had maintained an aposymbiotic status. Symbiodinium cells were harvested from the culture flasks and pipetted into 50 mL Falcon™
tubes. The tubes were centrifuged for 5 min at 1,000 x g to pellet algae, and then the supernatant
was discarded. Algal cells were then re-suspended in 20 mL FSW, and these steps were
repeated twice to wash the cells. After the final resuspension, cell density was quantified using
an Improved Neubauer haemocytometer (six counts per sample). Cell densities of each
Symbiodinium type were brought to a similar concentration (~ 3 x 106 mL-1) by dilution of the
denser suspension with FSW. Anemones were infected during the midpoint of the light cycle. A dilute suspension of freshly-
hatched Artemia nauplii to induce phagocytosis was added to the Symbiodinium cultures and 69 gently mixed. Aerators were removed from the anemone containers 15 min before infection to
encourage extension of tentacles, and a glass pipette then used to distribute the appropriate
Symbiodinium suspension evenly onto the oral discs of all anemones in the corresponding
treatment groups. Anemones in the aposymbiotic group were not infected with any
Symbiodinium culture. After infection, all aerators were replaced and the FSW refreshed after
two days. This infection process was repeated weekly for three weeks, and the anemones
sampled at the fourth week. 3.2.3 Analysis of infection success Symbiont populations were assessed at the end of the experiment (four weeks post-infection). Individual anemones from the B1 and D1a treatment groups (n = 5 per treatment) were
homogenized in 1 mL FSW, using Eppendorf micropestles followed by an IKA T-10 electric
saw-tooth homogenizer to further break up animal tissue. The homogenate was separated into
host and symbiont fractions by centrifugation for 5 min at 16,000 x g; the supernatant (host
fraction) was removed and stored at -20oC for protein analysis, and the pellet (symbiont
fraction) re-suspended in 100 µL FSW with 1% formalin and stored at 4oC for cell counts. Algal cells were counted with an Improved Neubaur haemocytometer at 100x magnification
with a light microscope (six counts per sample). Cell counts were then normalized to host
protein content as measured by the Bradford assay (Bradford, 1976). Anemones from the
aposymbiotic treatment were observed under confocal microscopy as described previously, to
determine if any background symbiont re-establishment may have affected experimental
anemones. At the end of the experiment, three anemones each from the B1 and D1a treatments
were processed to separate host and algal fractions as before. The algal fraction was stored in
DMSO at 4oC to use for Symbiodinium genotyping (see below). 70 3.2.4 Symbiont verification DNA was extracted from the initial Symbiodinium cultures (n = 2) and the isolated symbiont
fractions (n = 3 per symbiotic treatment) using the CTAB/phenol-chloroform protocol of Baker
et al. (2004). Samples stored in DMSO were centrifuged at 16,000 x g for 5 min to pellet algal
cells, and the DMSO then removed. Algal pellets were added to 1% SDS in DNA buffer (0.4
M NaCl, 50 mM EDTA, ddH2O, pH = 8), vortexed, and then incubated for 60 min at 65oC. After incubation, a 100 µL aliquot of the sample was added to 5 µL of Proteinase K and
incubated overnight at 37oC. Room temperature CTAB mix was added to twice the volume
(210 µL), and then incubated for 30-60 min at 65oC. After allowing samples to cool, 315 µL
chloroform were added and left on a rotating mixer for 2-3 h. Samples were centrifuged at
10,000 x g for 10 min, and the supernatant (~250 µL) then transferred to a new tube. Twice the
volume (500 µL) of chilled 100% ethanol was mixed into samples, which were then kept at -
20oC for 30 min to precipitate DNA. 3.2.5 Protein extraction Three weeks post-infection, anemones (n = 6 per treatment) were harvested for proteomic
analysis. Individuals were homogenized with an IKA T-10 electric saw-tooth homogenizer,
and then separated into host and symbiont fractions by centrifugation as described in section
3.2.3. EppendorfTM LoBind microcentrifuge tubes for protein research were used in all of the
following extraction steps. For each sample, 15 µg protein were combined with a lysis buffer
consisting of 5% sodium deoxycholate, 100 mM dithiothreitol and 50 mM triethylammonium
bicarbonate. The samples were then incubated for 15 min at 90oC, followed by 15 min at 60oC. Next, samples were centrifuged at 16,000 x g for 5 min to pellet cell debris and the supernatant
transferred to a new tube. Iodoacetamide was added to a final concentration of 50 mM and
samples then incubated in the dark, at room temperature, for 30 min. Samples were
subsequently diluted ten-fold with HPLC-grade water (Sigma-Aldrich, Auckland, NZ) and
vortexed briefly to mix. Next, 1 µg trypsin was added to each tube followed by incubation at
37oC for 18 h to allow enzyme digestion. After incubation, trifluoroacetic acid was added to a
final concentration of 0.5% and vortexed to mix. Samples were then centrifuged for 10 min at
16,000 x g to pellet precipitate and the supernatant transferred to a new tube. Sample volumes
were then concentrated to < 500 µL using an Eppendorf Concentrator 5301 set to 45oC, for
approximately 1 h. The resulting peptides were desalted with Millipore® Ziptips C18 (Omix
Bond Elut, Agilent Technologies) before resuspension in 0.1% formic acid. All materials were
acquired from Sigma Aldrich (Auckland, New Zealand). 3.2.3 Analysis of infection success Next, samples were centrifuged for 10 min at 10,000 x g
and the supernatant discarded. The samples were dried in an Eppendorf Concentrator 5301 for
30 min at 45oC, after which 100 µL 0.3 M NaOAc were added to dissolve the pellet. Then, 200
µL 100% ethanol were added, and the sample mixed and stored at -20oC for 2 h. After
defrosting, samples were centrifuged for 10 min at 10,000 x g and the supernatant discarded. The pellets were dried thoroughly as before, and then re-suspended in 50 µL TE buffer and
analysed with PCR. Extracted DNA was used for PCR with the outer primers ITSintfor2 (forward; 5'-
GAATTGCAGAACTCCGTG-3')
and
ITS2Rev2
(reverse;
5'-
CCTCCGCTTACTTATATGCTT-3'). The thermal cycling regime had an initial denaturation
step of 3 min at 95ºC, followed by 40 cycles of 15 s at 95ºC, 15 s at 56ºC, and 10 s at 72ºC
(performed with an Applied Biosystems Veriti thermo-cycler). Each reaction contained 1 µL
DNA template, 12.5 µL 1x MyTaq PCR reaction mix (Bioline, Randolph, MA, USA), 1.5 µL
each primer, 2 µL 10 mg/mL bovine serum albumin (Sigma Alrich, Auckland, New Zealand),
and 6.5 µL deionised sterile water to a final volume of 25 µL. PCR products were cleaned with
ExoSAP-IT (USB Corporation, Cleveland, OH, USA) and sequenced by the Macrogen
Sequencing Service (Macrogen Inc., Seoul, South Korea). Sequences were aligned with
Geneious v. 7.0 (Biomatters Ltd., Auckland, New Zealand) and a BLAST search was carried 71 out against Symbiodinium ITS2 sequences in GenBank. Sequences of the initial infection
cultures were compared with those extracted from anemones at the end of the experiment to
confirm symbiont identities. 3.2.6 Identification of proteins using LC-MS/MS Peptides were analysed by liquid chromatography-tandem mass-spectrometry in a LTQ
Orbitrap XL (Thermo Scientific) using settings based on those described in Oakley et al. (2016). Peptide separation was achieved using liquid chromatography (Ultimate 3000, Dionex) 72 with an Acclaim PepMap C18 column (#160321, Thermo Scientific), while the column oven
was set to 35oC. Chromeleon Xpress software (v2.11.0.2914, Dionex) was used to conduct a
300-min nonlinear gradient from 96% buffer A (0.1% formic acid) to 50% buffer B (80%
acetonitrile, 0.1% formic acid) at a flow rate of 0.3 µL min-1. GOAT software (version 1.0.1)
(Trudgian et al. 2014) was used to optimize the gradient, based on the initial run, to yield
maximal protein identifications. Peptides were ionized with an electrospray needle source at 1.9 kV spray voltage and injected
into an LTQ Orbitrap XL (Thermo Scientific), operated using Thermo Xcalibur (v2.1, Thermo
Scientific) software. The Orbitrap analysed MS spectra at 30,000 resolution before collision
fractionation, and the ion trap then analysed MS/MS spectra by taking the top eight MS peaks
while rejecting +1 charge states. A dynamic exclusion of 180 s was used to minimize
repetitious peptide analysis. Other instrument settings were based on those of Kalli and Hess
(2012). The resulting spectra were searched against a custom Aiptasia database of 320,798 total
sequences, and were processed by MScDb software (Marx et al. 2013) to reduce peptide-level
redundancy. The database was constructed from the Aiptasia genome (Baumgarten et al. 2015),
all cnidarian sequences from UniProt (28/4/14 release, Swiss-Prot and TrEMBL), all open
reading frames from the Aiptasia CC7 transcriptome (Lehnert et al. 2012), and a contaminant
database (CRAPome v1.0) (Mellacheruvu et al. 2013). MS peaks were extracted by Proteome
Discoverer (v2.1, Thermo Scientific) and analysed using Sequest (Thermo Fisher Scientific)
and X! Tandem (The GPM, v CYCLONE (2010.12.01.1)). Both Sequest and X! Tandem
searches assumed trypsin as the digestion enzyme, with a fragment ion mass tolerance of 0.60
Da and a parent ion tolerance of 10 PPM. Both searches also included carbamidomethyl of
cysteine as a fixed modification. In Sequest, oxidation of methionine and carbamylation of the
n-terminus were specified as variable modifications. In X! Tandem, Glu->pyro-Glu of the n-
terminus, ammonia-loss of the n-terminus, gln->pyro-Glu of the n-terminus, oxidation of
methionine, and carbamylation of the n-terminus were specified as variable modifications. 73 Scaffold (v4.4.8, Proteome Software, Inc.) was used to validate MS/MS based peptide and
protein identifications. 3.2.6 Identification of proteins using LC-MS/MS Peptide identifications were accepted if they were established at > 99%
probability by the Scaffold Local FDR algorithm. Protein identifications were accepted if they
were established at > 99% probability and contained a minimum of two identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm (Nesvizhskii et al. 2003). Proteins that contained similar peptides and could not be differentiated based on MS/MS
analysis alone were grouped together to satisfy the principles of parsimony. Proteins sharing
significant peptide evidence were grouped into clusters. 3.2.7 Statistical analysis Symbiont cell density and P:R values were tested for significant differences between B1 and
D1a groups using a t-test in SPSS statistical software (v. 20, IBM Corporation). Symbiont cell density and P:R values were tested for significant differences between B1 and
D1a groups using a t-test in SPSS statistical software (v. 20, IBM Corporation). Proteins were quantified by Precursor Intensity (PI) in Scaffold and statistical analyses were
conducted in R v. 3.3.1 (www.r-project.org). To identify protein clusters with significant
differences between treatments, a custom R script (developed by S.P. Wilkinson, Victoria
University of Wellington) for multivariate analysis with post hoc identifications was used,
according to the parameters described in Oakley et al. (submitted). First, a scaling factor was
applied to correct for any differences in protein loading between samples. This was done by
calculating a pairwise matrix of the scaling factors as each sample (x) against each other sample
(y), as the median of the vector: 𝑥𝑖
𝑦𝑖
𝑖= 1, 2, 3, … , 𝑚 Where m is the total number of proteins that were detected in the total number (18) of samples. The sample with the lowest protein loading depth from the reference treatment, cell type B1,
was identified as the matrix column with the lowest average scaling factor. All other samples
were then normalized using the reference sample column. To fit the model to parametric
assumptions, PI values were assigned for low-abundance protein clusters where concentrations
were below the detection limit of the instrument (‘non-detects’). Protein clusters consisting of
> 75% non-detects were removed from the dataset, and any non-detects in the remaining
clusters were imputed by robust regression on order statistics (assuming a log-normal
distribution) using the R package ‘NADA’. Next, protein clusters that were significantly 74 different between treatments were identified. For multivariate analysis of treatment effects, the
dataset was log transformed and Bray-Curtis dissimilarities calculated for each pairwise sample
comparison. Analysis of variance by permutation of dissimilarities was carried out using the
‘adonis’ function in the ‘vegan’ R package (v2.2-1). To test for differences in individual protein
clusters between treatments, a generalized linear model (GLM) with treatment effects was
fitted and compared against an intercept-only null model using an analysis of deviance X2 test,
where α = 0.05. 3.2.7 Statistical analysis Protein clusters with significantly different concentrations between treatments
were identified with the false discovery rate (FDR) correction method of Benjamini &
Hochberg (1995), at a q-threshold of 0.05. The procedure was repeated 100 times, with
randomization of imputed values to prevent the regression on order statistics imputation from
skewing the results. Proteins that were significantly different between treatments were retrieved from the database
and BLAST searched against the UniProtKB database using Geneious v.10.0.2. The top
reviewed hit for each protein with an E value ≤ 1 x 10-5 was used to assign an annotation, and
any without a match under this threshold were designated as hypothetical proteins. Blast2GO
Basic v 4.0.7 (Conesa et al. 2005) was used to map gene ontology (GO) terms to significantly
different proteins. 3.3.1 Infection success Examination of anemones in the aposymbiotic control treatment at the end of the experiment
revealed no re-establishment of background symbionts. Additionally, Symbiodinium
genotyping confirmed symbionts at the end of the experiments matched the Symbiodinium B1
and Symbiodinium D1a cultures originally used for infection. Symbiont cell densities in
anemones infected with Symbiodinium B1 (average of 1.12 x 10+7 ± 1.16 x 10+6 std. error) were
1.5-fold higher (d.f. = 1, t = 2.36, p = 0.04) than those in anemones infected with Symbiodinium
D1a (average of 7.29 x 10+6 ± 1.19 x 10+6). 75 3.3.2 Proteins unique to treatment groups 3.3.2 Proteins unique to treatment groups A total of 1,608 protein clusters (from here on referred to solely as “proteins”) were identified
between all treatments, while 31 decoy sequences were discarded and 276 sequences were not
analysed further due to having more than 75% non-detects. From the remaining 1,301 proteins,
966 had at least one value imputed. Across all 100 iterations, multivariate p-values for the
differentially-expressed proteins in the GLM analysis were < 0.05. Overall, 142 identified
proteins were significantly different between the three treatments (i.e. aposymbiotic;
Symbiodinium B1; Symbiodinium D1a). After accounting for hypothetical proteins, 128
sequences were annotated using sequence similarities from the UniProtKB database. Between
the B1 and aposymbiotic treatments, 105 significantly different proteins were found (Table
3.1); between the D1a and aposymbiotic treatments, 108 significantly different proteins were
found (Table 3.2); and between the B1 and D1a treatments, 22 significantly different proteins
were found (Table 3.3). While 124 of these proteins were found in all treatment groups, 18
were exclusive to only one or two of the treatments (Fig. 3.1). 76 Table 3.1. Proteins significantly different between Symbiodinium B1 and aposymbiotic treatments, in order of
fold-change from B1 to Apo. Colour scale in first column indicates degree of protein upregulation in B1 (green)
to aposymbiotic (red) anemones. Green and pink shaded rows are those proteins previously found to be
significantly different between symbiotic and aposymbiotic anemones, respectively, in Oakley et al. (2016). 3.3.2 Proteins unique to treatment groups g
y
y
p
y
,
p
y,
y
(
)
B1-Apo
Fold
Change
Sequence ID
Proteins
in
Cluster
Best Annotation
Match
Accession
Matched
Species
E value
%
Pairwise
Identity
1175.53
AIPGENE22527
1
Protein NPC2 homolog
Q9VQ62
Drosophila
melanogaster
2.98E-26
37.9%
383.67
AIPGENE22473
1
Niemann Pick type C2
protein homolog
P79345
Bos taurus
1.80E-19
29.9%
62.28
AIPGENE3015
1
DELTA-alicitoxin-
Pse2a
P58911
Phyllodiscus
semoni
4.56E-151
63.7%
49.27
AIPGENE1278
1
Unconventional
myosin-XVIIIa
Q92614
Homo sapiens
0
33.1%
30.12
Q6S3M2
1
Glutamine synthetase
P09606
Rattus
norvegicus
0
68.4%
28.59
AIPGENE6975
2
Fibronectin type III
domain-containing
protein
B8VIW9
Acropora
millepora
5.65E-165
33.3%
28.15
BAB83090.1
1
Carbonic anhydrase 2
Q8UWA5
Tribolodon
hakonensis
4.18E-41
44.7%
21.90
AIPGENE2901
1
Carbonic anhydrase 2
Q8UWA5
Tribolodon
hakonensis
1.90E-56
38.0%
21.87
AIPGENE13275
1
Circumsporozoite
protein
P06915
Plasmodium
berghei
4.20E-17
37.0%
21.73
AIPGENE9787
2
Tropomyosin-1
P39921
Hydra vulgaris
3.50E-17
32.1%
21.70
AIPGENE1237
1
Hypothetical protein
KXJ28962.1
ExAiptasia
pallida
1.20E-15
100%
21.19
AIPGENE27968
1
Hypothetical protein
KXJ18477.1
ExAiptasia
pallida
3.18E-77
100%
20.27
AIPGENE21668
1
EGF and laminin G
domain-containing
protein
B8UU78
Acropora
millepora
0
36.5%
18.96
BAB89358.1
2
Calcyphosin-like
protein
Q6P8Y1
Mus musculus
6.70E-69
47.8%
16.41
AIPGENE11024
2
Hemicentin-2
A2AJ76
Mus musculus
9.61E-17
24.5%
16.33
AIPGENE22517
4
Isoform 4 of Golgin
subfamily B member 1
Q14789-4
Homo sapiens
2.60E-87
20.9%
15.94
EDO34868.1
1
40S ribosomal protein
S19
Q8T5Z4
Branchiostoma
belcheri
4.38E-70
69.1%
13.31
AIPGENE9034
1
Serine/arginine-rich
splicing factor 4
Q8VE97
Mus musculus
2.31E-64
56.7%
12.90
EDO49557.1
1
Muscle M-line
assembly protein unc-
89
KXJ27993.1
ExAiptasia
pallida
1.25E-35
68%
12.73
EDO46723.1
1
Calponin-1
P26932
Gallus
1.24E-18
32.9%
12.71
EDO35342.1
1
Hypothetical protein
KXJ25875.1
ExAiptasia
pallida
8E-93
100%
12.50
A7S6S5
1
Glutamine synthetase
P15103
Bos taurus
0
67.6%
12.38
EDO43201.1
1
Actin-binding LIM
protein 1
O14639
Homo sapiens
1.58E-87
41.4%
11.68
Q09Y99
1
Caspase-7
P55214
Mesocricetus
auratus
1.34E-67
45.6%
10.55
A7RP49
2
Solute carrier family 8
member 1
P48765
Bos taurus
0
54.0%
9.93
EDO40093.1
2
von Willebrand factor
D and EGF domain-
containing protein
Q8N2E2
Homo sapiens
2.24E-25
32.6%
9.76
EDO39668.1
1
Tropomyosin
Q8WR63
Trichinella
pseudospiralis
7.34E-16
30.6% 77 77 Table 3.1 continued Table 3.1 continued Table 3.1 continued
9.30
AIPGENE26879
1
NADH dehydrogenase
iron-sulfur protein 6,
mitochondrial
P52503
Mus musculus
3.50E-39
56.1%
9.16
EDO41848.1
1
Tropomodulin-1
A0JNC0
Bos taurus
2.22E-74
40.4%
8.28
EDO29941.1
1
Multifunctional protein
ADE2
P38024
Gallus gallus
1.15E-180
65.6%
8.06
AIPGENE7641
1
Flotillin-2
O61492
Drosophila
melanogaster
0
65.0%
7.87
AIPGENE435
2
Succinate--CoA
ligasesubunit alpha,
mitochondrial
P13086
Rattus
norvegicus
4.58E-154
72.1%
7.83
AIPGENE6089
2
Ras-like protein RAS2
P38976
Hydra vulgaris
1.01E-89
74.1%
7.78
A7SGL1
1
Protein disulfide-
isomerase A6
Q922R8
Mus musculus
0
60.0%
7.50
AIPGENE21644
1
Muscle M-line
assembly protein unc-
89
O01761
Caenorhabditis
elegans
0.00E+00
26.5%
7.34
EDO43005.1
1
Aprataxin and PNK-
like factor
Q8IW19
Homo sapiens
4.33E-21
32.0%
7.28
AIPGENE3143
1
DELTA-thalatoxin-
Avl2a
Q76DT2
Actineria
villosa
0.00E+00
59.7%
6.67
KXJ26680.1
1
Rootletin
Q5TZA2
Homo sapiens
0
40.6%
6.57
A7RZ15
6
Erythrocyte band 7
integral membrane
protein
P27105
Homo sapiens
2.40E-129
72.3%
6.44
AIPGENE20022
3
Hypothetical protein
KXJ13090.1
ExAiptasia
pallida
0
99%
6.10
AIPGENE6448
2
cGMP-dependent
protein kinase 1
O77676
Oryctolagus
cuniculus
0
63.6%
5.89
EDO43151.1
1
Hypothetical protein
KXJ24863.1
ExAiptasia
pallida
3.48E-23
100%
5.71
AIPGENE7839
1
Hypothetical protein
KXJ11047.1
ExAiptasia
pallida
0
99%
5.48
AIPGENE13174
1
Hypothetical protein
KXJ30118.1
Terebra
subulata
4.05E-91
99%
4.92
AIPGENE19216
1
Acid ceramidase
A5A6P2
Pan troglodytes
1.18E-102
41.6%
4.61
AIPGENE2351
1
Peroxiredoxin-6
Q9TSX9
Sus scrofa
1.01E-69
66.9%
4.41
AAR24460.1
2
Tropomyosin
Q95VA8
Trichinella
spiralis
2.53E-53
46.8%
4.05
A7STP5
1
GTPase Kras
P79800
Meleagris
gallopavo
4.80E-105
86.7%
3.66
EDO48626.1
2
Heat shock protein HSP
90-beta
O57521
Danio rerio
0
78.5%
3.63
AIPGENE28926
1
Myotrophin
Q91955
Gallus gallus
4.07E-19
38.6%
3.49
AIPGENE29213
3
Gelsolin-like protein 1
Q7JQD3
Lumbricus
terrestris
1.56E-122
51.2%
3.00
AIPGENE13249
1
Microtubule-associated
protein RP/EB family
member 1
Q6V291
Coturnix
coturnix
4.74E-94
51.7%
2.85
AIPGENE3056
1
Calpain-9
O35920
Rattus
norvegicus
0
46.0%
2.69
AIPGENE700
1
Tubulin
polymerization-
promoting protein
family member 2
Q3T077
Bos taurus
2.42E-42
50.6%
2.62
AIPGENE23774
4
Myosin-like antigen
P21249
Onchocerca
volvulus
4.91E-07
50.6%
2.55
AIPGENE16749
1
Talin-2
Q9Y4G6
Homo sapiens
0
49.2%
2.38
EEN66183.1
1
Protein NPC2 homolog
Q9VQ62
Drosophila
melanogaster
6.57E-24
49.4% 78 Table 3.1 continued Table 3.1 continued Table 3.1 continued
2.01
AIPGENE27021
1
U-actitoxin-Avd8a
P0DMZ3
Anemonia
viridis
5.43E-19
45.8%
1.96
AIPGENE19130
1
Myophilin
Q24799
Echinococcus
granulosus
6.57E-36
41.4%
1.85
EDO46722.1
2
Myophilin
Q24799
Echinococcus
granulosus
2.96E-31
37.9%
1.84
EDO36481.1
2
ZP domain-containing
protein
G8HTB6
Acropora
millepora
4.01E-75
39.3%
1.75
EDO45013.1
37
Actin
P12716
Pisaster
ochraceus
3.25E-113
89.4%
1.69
AAH90759.1
2
14-3-3 protein epsilon;
Short=14-3-3E
P62258
Homo sapiens
8.35E-109
65.6%
1.63
AIPGENE28714
3
Hypothetical protein
KXJ10810.1
ExAiptasia
pallida
0
99%
1.50
KXJ18065.1
3
Catalase
P04040
Homo sapiens
0
71.9%
0.75
A7SHH5
1
Protein disulfide-
isomerase
P05307
Bos taurus
0
59.5%
0.56
EDO37116.1
1
Putative
aminopeptidase
W07G4.4
Q27245
Caenorhabditis
elegans
5.84E-26
44.7%
0.56
EDO42526.1
2
Hydroxysteroid
dehydrogenase-like
protein 2
A4FUZ6
Bos taurus
0
62.3%
0.55
EDO49845.1
1
Beta-hexosaminidase
subunit alpha
P06865
Homo sapiens
2.85E-158
47.6%
0.52
AIPGENE12299
4
Glyceraldehyde-3-
phosphate
dehydrogenase
O57479
Columba livia
0.00E+00
73.2%
0.50
EDO44966.1
6
Abscisic-aldehyde
oxidase
Q7G9P4
Arabidopsis
thaliana
0
35.6%
0.50
KXJ18065.1
3
Catalase
P04040
Homo sapiens
0
71.9%
0.50
AIPGENE26156
2
Cathepsin L
Q26636
Sarcophaga
peregrina
1.54E-125
55.3%
0.46
AIPGENE26035
1
Lysine-specific
demethylase 8
B2GUS6
Xenopus
tropicalis
8.26E-18
27.3%
0.42
AIPGENE4246
4
Cholinesterase
P21927
Oryctolagus
cuniculus
1.20E-107
35.7%
0.41
AIPGENE13554
2
Creatinase
P38487
Bacillus sp. 3.3.2 Proteins unique to treatment groups (strain B-0618)
7.61E-134
66.6%
0.41
AIPGENE27013
1
Hypothetical protein
KXJ29487.1
ExAiptasia
pallida
1.27E-154
99%
0.40
EDO48107.1
2
Dipeptidyl peptidase 2
Q9EPB1
Rattus
norvegicus
1.99E-112
51.9%
0.37
EEN57628.1
2
Alpha-aminoadipic
semialdehyde
dehydrogenase
Q2KJC9
Bos taurus
0
67.2%
0.35
AIPGENE2301
2
Carbonyl reductase
[NADPH] 1
P48758
Mus musculus
5.66E-84
50.8%
0.30
AIPGENE473
3
Cathepsin Z
P05689
Bos taurus
3.04E-129
64.3%
0.29
EDO47653.1
2
Aldehyde
dehydrogenase,
mitochondrial
P11884
Rattus
norvegicus
0
72.7%
0.28
AIPGENE22562
1
Inosine-uridine
preferring nucleoside
hydrolase
P83851
Leishmania
major
2.64E-19
31.3%
0.27
AIPGENE7506
2
Pirin-like protein
Q9SEE4
Solanum
lycopersicum
1.10E-74
43.6%
0.27
AIPGENE6148
1
Lysosomal alpha-
mannosidase
Q60HE9
Macaca
fascicularis
0
49.1%
0.24
EDO36900.1
1
Nematocyst expressed
protein 6
K7Z9Q9
Nematostella
vectensis
2.55E-74
58.8% 79 0.24
EDO40873.1
3
Creatinase
P19213
Flavobacterium
sp. (strain U-
188)
0
61.8%
0.20
EDO44805.1
1
Universal stress protein
A-like protein
Q8LGG8
Arabidopsis
thaliana
1.10E-18
28.3%
0.16
EDO46736.1
1
Homogentisate 1,2-
dioxygenase
O09173
Mus musculus
0
67.6%
0.15
EDO31703.1
4
Transmembrane
protease serine 3
Q8K1T0
Mus musculus
7.08E-53
40.9%
0.14
AIPGENE13027
2
Alpha-N-
acetylgalactosaminidase
Q90744
Gallus gallus
8.01E-158
55.0%
0.13
AIPGENE10977
1
Betaine--homocysteine
S-methyltransferase 1
(BHMT1)
Q5M8Z0
Xenopus
tropicalis
0
61.9%
0.13
AIPGENE5163
1
Urocanate hydratase
Q96N76
Homo sapiens
0
65.8%
0.11
KXJ15780.1
1
Mesencephalic
astrocyte-derived
neurotrophic factor
homolog
B4QX46
Drosophila
simulans
2.01E-60
57.8%
0.10
EDO45852.1
1
Cathepsin L
Q95029
Drosophila
melanogaster
9.41E-132
59.7%
0.08
A7RLS5
1
Aldehyde
dehydrogenase,
mitochondrial
P05091
Rattus
norvegicus
0
71.8%
0.08
A7RIN6
1
S-methylmethionine--
homocysteine S-
methyltransferase
(BHMT2)
Q5RF32
Pongo abelii
7.54E-137
57.6%
0.07
EDO39870.1
1
Beta-galactosidase
Q58D55
Bos taurus
1.22E-147
45.8%
0.06
52902
1
Heat shock 70 kDa
protein A
P09446
Caenorhabditis
elegans
5.94E-44
88.5%
0.06
AIPGENE28006
2
Alpha-N-
acetylglucosaminidase
P54802
Homo sapiens
0
47.7%
0.04
AIPGENE12842
1
Hypothetical protein
KXJ12770.1
ExAiptasia
pallida
0
99%
0.04
AIPGENE25444
1
Oxidoreductase
HTATIP2
Q9Z2G9
Mus musculus
1.28E-30
37.5%
0.03
12437
1
Calmodulin-like protein
1
Q9ZQE6
Arabidopsis
thaliana
2.45E-04
33.8%
0.00
AIPGENE8259
1
Persulfide dioxygenase
ETHE1, mitochondrial
O95571
Homo sapiens
5.54E-90
56.0%
0.00
EDO47743.1
3
Betaine--homocysteine
S-methyltransferase 1
(BHMT1)
Q95332
Sus scrofa
6.96E-120
72.4% 80 Table 3.2 Proteins significantly different between Symbiodinium D1a and aposymbiotic treatments, in order of
fold-change from D1a to Apo. Colour scale in first column indicates degree of protein upregulation in D1a
(yellow) to aposymbiotic (red) anemones. Green and pink shaded rows were also found to be significantly
different proteins between symbiotic and aposymbiotic anemones, respectively, in Oakley et al. (2016). 3.3.2 Proteins unique to treatment groups different proteins between symbiotic and aposymbiotic anemones, respectively, in Oakley et al. (2016). 3.3.2 Proteins unique to treatment groups D1a-
Apo
Fold
Change
Sequence ID
Proteins
in
Cluster
Best Annotation
Match
Accession
Matched
Species
E value
%
Pairwise
Identity
199.95
EDO34868.1
1
40S ribosomal protein
S19
Q8T5Z4
Branchiostoma
belcheri
4.38E-70
69.1%
184.64
AIPGENE3015
1
DELTA-alicitoxin-
Pse2a
P58911
Phyllodiscus
semoni
4.56E-
151
63.7%
89.17
AIPGENE6975
2
Fibronectin type III
domain-containing
protein
B8VIW9
Acropora
millepora
5.65E-
165
33.3%
50.82
AIPGENE9787
2
Tropomyosin-1
P39921
Hydra vulgaris
3.50E-17
32.1%
47.21
AIPGENE27968
1
Hypothetical protein
KXJ18477.1
ExAiptasia
pallida
3.18E-77
100%
45.25
AIPGENE21668
1
EGF and laminin G
domain-containing
protein
B8UU78
Acropora
millepora
0
36.5%
40.13
BAB89358.1
2
Calcyphosin-like
protein
Q6P8Y1
Mus musculus
6.70E-69
47.8%
39.74
EDO42416.1
2
Cell surface
glycoprotein 1
Q06852
Clostridium
thermocellum
1.26E-10
45.2%
35.61
A7RIX7
2
Proteasome subunit
alpha type-6
Q9QUM9
Mus musculus
7.65E-
160
84.1%
35.42
EDO37221.1
1
40S ribosomal protein
S6
P62755
Rattus
norvegicus
6.46E-
123
84.8%
32.46
AIPGENE1237
1
Hypothetical protein
KXJ28962.1
ExAiptasia
pallida
1.20E-15
100%
23.85
EDO39668.1
1
Tropomyosin
Q8WR63
Xenopus laevis
7.34E-16
30.6%
22.19
AIPGENE16941
1
60S acidic ribosomal
protein P0
Q9DG68
Rana sylvatica
2.59E-
153
70.5%
21.86
EDO43151.1
1
Hypothetical protein
KXJ24863.1
ExAiptasia
pallida
3.48E-23
100%
21.85
EDO36394.1
1
Predicted protein
XP_001628457.1
Nematostella
vectensis
1.14E-
105
63.0%
21.26
EDO43201.1
1
Actin-binding LIM
protein 1
O14639
Homo sapiens
1.58E-87
41.4%
19.68
AIPGENE22517
4
Isoform 4 of Golgin
subfamily B member 1
Q14789-4
Homo sapiens
2.60E-87
20.9%
19.14
AIPGENE3128
1
DELTA-alicitoxin-
Pse2a
P58911
Phyllodiscus
semoni
0.00E+00
59.6%
17.09
BAB83090.1
1
Carbonic anhydrase 2
Q8UWA5
Tribolodon
hakonensis
4.18E-41
44.7%
16.71
EDO45498.1
2
Beta-hexosaminidase
Q04786
Vibrio
vulnificus
1.04E-
171
37.6%
16.38
AIPGENE2901
1
Carbonic anhydrase 2
Q8UWA5
Tribolodon
hakonensis
1.90E-56
38.0%
16.08
EDO41848.1
1
Tropomodulin-1
A0JNC0
Bos taurus
2.22E-74
40.4%
15.30
AIPGENE1278
1
Unconventional
myosin-XVIIIa
Q92614
Homo sapiens
0
33.1%
14.32
EDO29941.1
1
Multifunctional protein
ADE2
P38024
Gallus gallus
1.15E-
180
65.6%
13.93
A7SGL1
1
Protein disulfide-
isomerase A6
Q922R8
Mus musculus
0
60.0%
13.37
EDO39721.1
1
Guanine nucleotide-
binding protein subunit
beta-2-like 1
O42248
Danio rerio
0
80.8%
13.26
AIPGENE7641
1
Flotillin-2
O61492
Drosophila
melanogaster
0
65.0% 81 Table 3.2 continued Table 3.2 continued Table 3.2 continued
13.24
AIPGENE3067
2
DELTA-thalatoxin-
Avl2a
Q76DT2
Actineria
villosa
0
62.5%
13.15
47580
2
Rootletin
Q5TZA2
Homo sapiens
0
40.6%
12.86
EDO26161.1
1
Hypothetical protein
KXJ12230.1
ExAiptasia
pallida
7.80E-
147
100%
12.32
AIPGENE9796
1
SH3 domain-binding
glutamic acid-rich-like
protein 3
Q91VW3
Mus musculus
6.15E-20
43.3%
10.89
AIPGENE20022
3
Hypothetical protein
KXJ13090.1
ExAiptasia
pallida
0
99%
10.65
A7RZ15
6
Erythrocyte band 7
integral membrane
protein
P27105
Homo sapiens
2.40E-
129
72.3%
10.38
AAR24460.1
2
Tropomyosin
Q95VA8
Trichinella
spiralis
2.53E-53
46.8%
9.43
AIPGENE435
2
Succinate--CoA ligase
[ADP/GDP-forming]
subunit alpha,
mitochondrial
P13086
Rattus
norvegicus
4.58E-
154
72.1%
9.37
AIPGENE16099
1
Collagen alpha-3(VI)
chain
P15989
Gallus gallus
5.61E-15
26.1%
9.33
AIPGENE26879
1
NADH dehydrogenase
[ubiquinone] iron-sulfur
protein 6, mitochondrial
P52503
Mus musculus
3.50E-39
56.1%
9.28
AIPGENE3143
1
DELTA-thalatoxin-
Avl2a
Q76DT2
Actineria
villosa
0.00E+00
59.7%
9.00
EDO46723.1
1
Calponin-1
P26932
Gallus gallus
1.24E-18
32.9%
8.73
AIPGENE19216
1
Acid ceramidase
A5A6P2
Pan troglodytes
1.18E-
102
41.6%
8.72
AIPGENE21644
1
Muscle M-line
assembly protein unc-
89
O01761
Caenorhabditis
elegans
0.00E+00
26.5%
8.58
AIPGENE23527
1
Universal stress protein
Slr1101
P72745
Synechocystis
sp. 3.3.2 Proteins unique to treatment groups (strain PCC
6803)
2.67E-10
36.6%
8.58
AIPGENE17132
1
Spindle assembly
abnormal protein 6
homolog
Q5ZMV2
Gallus gallus
1.55E-
137
41.4%
8.37
EDO37155.1
2
Protein disulfide-
isomerase A6
Q63081
Rattus
norvegicus
4.49E-
178
56.5%
8.13
Q09Y99
1
Caspase-7
P55214
Mesocricetus
auratus
1.34E-67
45.6%
8.09
EDO40200.1
1
Pleckstrin homology
domain-containing
family B member 2
Q5F3S2
Gallus gallus
2.95E-18
33.7%
7.74
AIPGENE6448
2
cGMP-dependent
protein kinase 1
O77676
Oryctolagus
cuniculus
0
63.6%
7.41
AIPGENE20759
1
Amiloride-sensitive
amine oxidase
P36633
Rattus
norvegicus
1.97E-
171
36.7%
7.10
AIPGENE13174
1
Hypothetical protein
KXJ30118.1
ExAiptasia
pallida
4.05E-91
99%
7.01
EDO40093.1
2
von Willebrand factor
D and EGF domain-
containing protein
Q8N2E2
Homo sapiens
2.24E-25
32.6%
6.94
AIPGENE6089
2
Ras-like protein RAS2
P38976
Hydra vulgaris
1.01E-89
74.1%
6.93
AIPGENE4457
1
Matrilin-3
O42401
Gallus gallus
1.17E-20
35.4%
6.45
EDO38232.1
1
Delta(3,5)-Delta(2,4)-
dienoyl-CoA isomerase,
mitochondrial
O35459
Mus musculus
1.04E-
127
58.5%
5.44
EDO39405.1
1
Actin-related protein
2/3 complex subunit 3
Q9JM76
Mus musculus
1.44E-84
67.6% 82 Table 3.2 continued Table 3.2 continued
5.05
AIPGENE29213
3
Gelsolin-like protein 1
Q7JQD3
Lumbricus
terrestris
1.56E-
122
51.2%
5.00
9270
1
Vitelline membrane
outer layer protein 1
homolog
Q7Z5L0
Homo sapiens
1.10E-25
34.4%
4.52
AIPGENE3056
1
Calpain-9
O35920
Rattus
norvegicus
0
46.0%
4.24
AIPGENE1342
1
Aflatoxin B1 aldehyde
reductase member 2
Q8CG45
Rattus
norvegicus
6.08E-
119
53.3%
4.20
EDO48626.1
2
Heat shock protein HSP
90-beta
O57521
Danio rerio
0
78.5%
3.63
AIPGENE7839
1
Hypothetical protein
KXJ11047.1
ExAiptasia
pallida
0
100%
3.61
AIPGENE2351
1
Peroxiredoxin-6
Q9TSX9
Sus scrofa
1.01E-69
66.9%
3.57
AIPGENE28926
1
Myotrophin
Q91955
Gallus gallus
4.07E-19
38.6%
3.42
EDO42442.1
1
Death-associated
protein kinase 3
O43293
Homo sapiens
7.62E-51
33.3%
3.33
AIPGENE29242
2
Gelsolin-like protein 2
Q8MPM1
Lumbricus
terrestris
7.76E-
123
53.3%
3.32
AIPGENE23774
4
Isoform 4 of Golgin
subfamily B member 1
Q14789-4
Homo sapiens
1.50E-
104
20.1%
3.13
AIPGENE12489
1
Actin-depolymerizing
factor 1
Q39250
Arabidopsis
thaliana
1.33E-15
31.0%
2.93
AIPGENE13249
1
Microtubule-associated
protein RP/EB family
member 1
Q6V291
Coturnix
coturnix
4.74E-94
51.7%
2.68
EDO45013.1
37
Actin
P12716
Pisaster
ochraceus
3.25E-
113
89.4%
2.58
AIPGENE27021
1
U-actitoxin-Avd8a
P0DMZ3
Anemonia
viridis
5.43E-19
45.8%
2.21
AIPGENE700
1
Tubulin
polymerization-
promoting protein
family member 2
Q3T077
Bos taurus
2.42E-42
50.6%
1.82
AIPGENE19130
1
Myophilin
Q24799
Echinococcus
granulosus
6.57E-36
41.4%
1.66
AAH90759.1
2
14-3-3 protein epsilon
P62258
Homo sapiens
8.35E-
109
65.6%
1.63
AIPGENE28714
3
Hypothetical protein
KXJ10810.1
ExAiptasia
pallida
0
99%
1.43
EDO46722.1
2
Myophilin
Q24799
Echinococcus
granulosus
2.96E-31
37.9%
0.61
AIPGENE13749
2
Selenium-binding
protein 1
Q569D5
Xenopus
tropicalis
0
65.7%
0.58
EDO42526.1
2
Hydroxysteroid
dehydrogenase-like
protein 2
A4FUZ6
Bos taurus
0
62.3%
0.57
EDO37116.1
1
Putative
aminopeptidase
W07G4.4
Q27245
Caenorhabditis
elegans
5.84E-26
44.7%
0.53
$47926
3
Catalase
P04040
Homo sapiens
0
71.9%
0.51
EDO44966.1
6
Abscisic-aldehyde
oxidase
Q7G9P4
Arabidopsis
thaliana
0
35.6%
0.51
EDO48107.1
2
Dipeptidyl peptidase 2
Q9EPB1
Rattus
norvegicus
1.99E-
112
51.9%
0.50
AIPGENE27853
2
Fumarylacetoacetate
hydrolase domain-
containing protein 2
Q6GLT8
Xenopus laevis
2.77E-
135
62.8%
0.47
AIPGENE4246
4
Cholinesterase
P21927
Oryctolagus
cuniculus
1.20E-
107
35.7%
0.43
EDO49845.1
1
Beta-hexosaminidase
subunit alpha
P06865
Homo sapiens
2.85E-
158
47.6% 83 0.43
AIPGENE2301
2
Carbonyl reductase
[NADPH] 1
P48758
Mus musculus
5.66E-84
50.8%
0.42
AIPGENE473
3
Cathepsin Z
P05689
Bos taurus
3.04E-
129
64.3%
0.39
EDO49709.1
2
N(4)-(Beta-N-
acetylglucosaminyl)-L-
asparaginase
O02467
Spodoptera
frugiperda
2.24E-
132
57.6%
0.38
AIPGENE26156
2
Cathepsin L
Q26636
Sarcophaga
peregrina
1.54E-
125
55.3%
0.34
EEN57628.1
2
Alpha-aminoadipic
semialdehyde
dehydrogenase
Q2KJC9
Bos taurus
0
67.2%
0.33
AIPGENE7184
1
Transmembrane
protease serine 3
Q8K1T0
Mus musculus
1.59E-57
41.7%
0.32
EDO40873.1
3
Creatinase
P19213
Flavobacterium
sp. 3.3.2 Proteins unique to treatment groups (strain U-
188)
0
61.8%
0.29
AIPGENE8932
1
Heterogeneous nuclear
ribonucleoprotein U-
like protein 1
Q8VDM6
Mus musculus
1.16E-
108
35.4%
0.27
AIPGENE27013
1
Hypothetical protein
KXJ29487.1
ExAiptasia
pallida
1.27E-
154
99%
0.26
EDO47653.1
2
Aldehyde
dehydrogenase,
mitochondrial
P11884
Rattus
norvegicus
0
72.7%
0.25
EDO37584.1
1
Lysosomal alpha-
mannosidase
O00754
Homo sapiens
0
51.2%
0.23
AIPGENE6148
1
Lysosomal alpha-
mannosidase
Q60HE9
Macaca
fascicularis
0
49.1%
0.18
AIPGENE13027
2
Alpha-N-
acetylgalactosaminidase
Q90744
Gallus gallus
8.01E-
158
55.0%
0.17
EDO46736.1
1
Homogentisate 1,2-
dioxygenase
O09173
Mus musculus
0
67.6%
0.16
AIPGENE7223
1
Transmembrane
protease serine 6
Q9DBI0
Mus musculus
1.80E-56
41.8%
0.14
AIPGENE1156
1
Guanylate-binding
protein 4
Q96PP9
Homo sapiens
1.89E-19
36.8%
0.11
AIPGENE7506
2
Pirin-like protein
Q9SEE4
Solanum
lycopersicum
1.10E-74
43.6%
0.10
AIPGENE12842
1
Hypothetical protein
KXJ12770.1
ExAiptasia
pallida
0
99%
0.08
AIPGENE25444
1
Oxidoreductase
HTATIP2
Q9Z2G9
Mus musculus
1.28E-30
37.5%
0.08
AIPGENE27740
1
Insoluble matrix shell
protein 1
P86982
Ruditapes
philippinarum
1.44E-14
37.8%
0.06
AIPGENE28006
2
Alpha-N-
acetylglucosaminidase
P54802
Homo sapiens
0
47.7%
0.04
EDO45852.1
1
Cathepsin L
Q95029
Drosophila
melanogaster
9.41E-
132
59.7%
0.03
52902
1
Heat shock 70 kDa
protein A (hsp-1)
P09446
Caenorhabditis
elegans
5.94E-44
88.5%
0.03
AIPGENE12842
1
Hypothetical protein
KXJ12770.1
ExAiptasia
pallida
1.32E-39
57%
0.01
AIPGENE8259
1
Persulfide dioxygenase
ETHE1, mitochondrial
O95571
Homo sapiens
5.54E-90
56.0% 84 Table 3.3 Proteins significantly different between Symbiodinium B1 and D1a treatments, in order of fold-change
from B1 to D1a. Colour scale in first column indicates degree of protein upregulation in B1 (green) to D1a
(yellow) anemones. Green and pink highlighted rows were also found to be significantly different proteins
between symbiotic and aposymbiotic anemones, respectively, in Oakley et al. (2016). between symbiotic and aposymbiotic anemones, respectively, in Oakley et al. (2016). 3.3.2 Proteins unique to treatment groups B1-D1a
Fold
Change
Sequence ID
Proteins
in
Cluster
Best Annotation
Match
Accession
Matched Species
E value
%
Pairwise
Identity
4384.86
AIPGENE22527
1
Protein NPC2
homolog
Q9VQ62
Drosophila
melanogaster
2.98E-26
37.9%
526.93
AIPGENE22473
1
Epididymal secretory
protein E1 (NPC2)
P79345
Bos taurus
1.80E-19
29.9%
26.04
Q6S3M2
1
Glutamine synthetase
P09606
Rattus norvegicus
0
67.2%
25.61
AIPGENE23106
2
Legumain
Q95M12
Bos taurus
8.61E-54
41.4%
23.25
A7S6S5
1
Glutamine synthetase
P15103
Bos taurus
0
67.6%
15.27
AIPGENE9034
1
Serine/arginine-rich
splicing factor 4
Q8VE97
Mus musculus
2.31E-64
56.7%
15.21
AIPGENE13275
1
Circumsporozoite
protein
P06915
Plasmodium
berghei
4.20E-17
37.0%
10.29
EDO43005.1
1
Aprataxin and PNK-
like factor
Q8IW19
Homo sapiens
4.33E-21
32.0%
8.24
AIPGENE14763
1
Neurolysin
Q5R9V6
Pongo abelii
8.60E-66
55.9%
2.49
AIPGENE7506
2
Pirin-like protein
Q9SEE4
Solanum
lycopersicum
1.10E-74
43.6%
2.47
AIPGENE8932
1
Heterogeneous
nuclear
ribonucleoprotein U-
like protein 1
Q8VDM6
Mus musculus
1.16E-
108
35.4%
1.61
AIPGENE13749
2
Selenium-binding
protein 1
Q569D5
Xenopus tropicalis
0
68.2%
0.76
AIPGENE12496
1
78 kDa glucose-
regulated protein
(GRP-78, Hsp70, BiP)
P07823
Mesocricetus
auratus
0
79.2%
0.66
EDO45013.1
37
Actin
P12716
Pisaster ochraceus
3.25E-
113
89.4%
0.31
AIPGENE1342
1
Aflatoxin B1 aldehyde
reductase member 2
(rAFAR2)
Q8CG45
Rattus norvegicus
6.08E-
119
53.3%
0.26
AIPGENE22562
1
Inosine-uridine
preferring nucleoside
hydrolase (NSNH)
P83851
Leishmania major
2.64E-19
31.3%
0.22
AIPGENE10977
1
Betaine--
homocysteine S-
methyltransferase 1
(BHMT1)
Q5M8Z0
Xenopus tropicalis
0
61.9%
0.14
A7RIN6
1
Betaine--
homocysteine S-
methyltransferase 2
(BHMT2)
Q5RF32
Pongo abelii
7.54E-
137
57.6%
0.10
ARMET
1
Mesencephalic
astrocyte-derived
neurotrophic factor
homolog
B4QX46
Drosophila
simulans
2.01E-60
57.8%
0.09
EDO26161.1
1
Hypothetical
KXJ12230.1
ExAiptasia pallida
7.80E-
147
100%
0.08
EDO34868.1
1
40S ribosomal protein
S19
Q8T5Z4
Branchiostoma
belcheri
4.38E-70
69.1%
0.01
EDO47743.1
3
Betaine--
homocysteine S-
methyltransferase 1
(BHMT1)
Q95332
Sus scrofa
6.96E-
120
72.4% 85 Non-metric multidimensional scaling (NMDS) of the proteins resulted in an excellent
representation of protein patterns reduced to two dimensions (stress = 0.051). Grouping
patterns can be seen between the three treatment groups, with most differences occurring along
the NMDS2 axis (Fig. 3.1). The aposymbiotic group is skewed negatively away from the two
symbiotic groups on NMDS2, while the D1a group has a smaller range along NMDS2 than the
B1 group. Non-metric multidimensional scaling (NMDS) of the proteins resulted in an excellent
representation of protein patterns reduced to two dimensions (stress = 0.051). Grouping
patterns can be seen between the three treatment groups, with most differences occurring along
the NMDS2 axis (Fig. 3.1). 3.3.2 Proteins unique to treatment groups The aposymbiotic group is skewed negatively away from the two
symbiotic groups on NMDS2, while the D1a group has a smaller range along NMDS2 than the
B1 group. Figure 3.1 NMDS plot showing grouping of Aiptasia protein samples (based on precursor intensity). Black
crosses represent individual proteins, while coloured dots represent the protein profile of each replicate anemone
in a particular treatment. Grouping patterns are encompassed by coloured ellipses according to treatment. Figure 3.1 NMDS plot showing grouping of Aiptasia protein samples (based on precursor intensity). Black
crosses represent individual proteins, while coloured dots represent the protein profile of each replicate anemone
in a particular treatment. Grouping patterns are encompassed by coloured ellipses according to treatment. Eight annotated proteins were found in both the B1 and D1a treatments but not in aposymbiotic
anemones, while the D1a and aposymbiotic treatments shared three proteins that were absent
from the B1 treatment, and the B1 and aposymbiotic treatments shared just one protein that
was absent from the D1a treatment (Fig. 3.2). The proteins unique to the two symbiotic
treatments included two that are necessary for basic cell functioning: 40S ribosomal protein
S19, which is required for ribosome biosynthesis (Danilova et al. 2008), and the muscle M-
line assembly protein unc-89, which is necessary for proper anchoring of myosin filaments via 86 muscle M lines (Benian et al. 1996). Also included in the symbiotic-only proteins are two cell-
signalling proteins: acid ceramidase and a guanine nucleotide-binding protein (GNBP aka G
protein) (Vetter and Whitinghofler 2001; Bernardo et al. 1995). Additional proteins specific to
the symbiotic state are involved in lipid and nitrogen metabolism, mRNA splicing, and cell
adhesion. The three proteins shared exclusively by the D1a and aposymbiotic groups are all
enzymes involved in the breakdown of ATP, amino acids, and aldehydes, while the protein
shared exclusively by the B1 and aposymbiotic groups, cathepsin L, is an enzyme involved in
the degradation of lysosomal proteins (Homma et al. 1994). Two Neimann pick C2 protein (NPC2) homologs and a serine/arginine-rich splicing factor
were discovered only in anemones infected with Symbiodinium B1, while a third NPC2
homolog was found in all treatments but was only significantly different between the B1 and
aposymbiotic groups, with upregulation (2.38-fold) in the B1 treatment (Table 3.1). A
persulfide dioxygenase ETHE1 protein was found only in aposymbiotic anemones, while no
proteins were found uniquely in anemones infected with Symbiodinium D1a. 3.3.2 Proteins unique to treatment groups 87 Figure 3.2 Venn diagram comparison of annotated proteins exclusive to only one or two treatment groups. Figure 3.2 Venn diagram comparison of annotated proteins exclusive to only one or two treatment group Figure 3.2 Venn diagram comparison of annotated proteins exclusive to only one or two treatment groups. Of the 22 proteins that were significantly different between B1- and D1a-colonised anemones,
18 were successfully mapped to at least one GO term (Fig. 3.3). The majority of these proteins
are involved in cellular and metabolic processes, and mostly function in binding and catalytic
activity (Fig. 3.3). While the majority of GO terms are mapped to a similar proportion of
upregulated proteins from each symbiotic treatment, only proteins upregulated in D1a-
colonised anemones were linked to multi-cellular organismal processes, immune system
process, locomotion, and structural molecule activity (Fig. 3.3). 88 Figure 3.3 Biological process (BP) and molecular function (MF) GO terms for successfully mapped proteins that
were significantly different between B1- and D1a-colonised anemones, illustrating the proportions of each that
were upregulated in either treatment. Figure 3.3 Biological process (BP) and molecular function (MF) GO terms for successfully mapped proteins that
were significantly different between B1- and D1a-colonised anemones, illustrating the proportions of each that
were upregulated in either treatment. 3.4.1 Impact of symbiont density 3.4.1 Impact of symbiont density The density of Symbiodinium B1 was 1.5-fold higher than that of D1a, and it is unclear whether
this may have affected differences in protein expression between treatments. Three proteins in
particular exhibited fold-changes that approximately corresponded to the difference in
symbiont density. These were the selenium-binding protein, 78kDa glucose-regulated protein,
and actin. While it is noted here that the observed differences of the former two proteins may
potentially be driven by cell density, there is also a possibility that the expression of these
proteins is actually affected by symbiont type, and this is discussed in more detail below. As
for actin, the fold-change from B1-D1a almost exactly matched the symbiont density
difference, so it is either a unique coincidence or the two parameters are related. Actin is found
ubiquitously throughout life and is most commonly known for its role as the thick filament in
eukaryote muscle fibres (Cooper 2000; Gunning et al. 2015). In Aiptasia, it is an important
component for maintaining structural integrity of the tissues, and inhibition of the protein can
lead to animal mortality (Dunn et al. 2007a). Ultimately, the probable link between actin
expression levels and symbiont density would need further investigation to confirm this. 3.4 Discussion This study aimed to determine the molecular impact of a heterologous, thermally-tolerant
opportunist symbiont type on a model cnidarian host, under normal environmental conditions. The results describe here expand on previous findings that reported a number of proteins to be
unique to the symbiotic and aposymbiotic states, by identifying proteins that are enhanced by
a native versus non-native symbiotic association. My findings confirmed the importance of
some proteins to the symbiotic state, such as glutamine synthetase and G-proteins, while
differentiating others as indicative of either an optimal or sub-optimal association. Most
notably, I found that NPC2-like proteins are integral components of a successful homologous
symbiosis. Additionally, I found that BHMT, which was previously only detected in
aposymbiotic anemones, is expressed at high levels in the heterologous symbiosis with
Symbiodinium D1a and therefore may indicate incompatibilities in the association. Overall, my
findings suggest that Symbiodinium D1a is indeed a less mutually beneficial symbiont to
Aiptasia hosts under normal environmental conditions. 89 3.4.2 Proteins associated with symbiotic and aposymbiotic states While six annotated proteins were found solely in the two symbiotic treatments, only one
protein was found to be unique to aposymbiotic anemones. The two symbiotic proteins known
to be involved in cell signalling are the G-protein and acid ceramidase. G-proteins act as a
molecular switch to regulate processes such as cell growth, differentiation, and transport
(Vetter & Wittinghofer 2001). Of particular relevance, in the cnidarian Renilla koellikeri, G-
proteins were found to stimulate the enzyme adenylate cyclase, suggesting that a receptor-
dependent G-protein signal transduction system operates in cnidarians (Awad & Anctil 1993). Furthermore, a proteomics study by Peng et al. (2010) discovered a G-protein-coupled receptor
in symbiosome fractions of Aiptasia, indicating that G-protein signal transduction likely plays
a role in symbiont recognition and/or regulation at the symbiotic interface. The other signalling
protein found in symbiotic anemones, acid ceramidase, is a lysosomal enzyme that catalyses
the hydrolysis of ceramide to free fatty acids and sphingosine (Bernardo et al. 1995). 90 Symbiodinium produces a unique ceramide, symbioramide-C16 (Nakamura et al. 1998), which
could be translocated to the host as a substrate for this reaction. While free fatty acids are a
major component of each partner’s metabolism and are predicted to play a critical role in the
success of the symbiosis (Dunn et al. 2012; Wang et al. 2013a), sphingosine acts in cell
signalling and apoptosis (Cuvillier 2002). The sphingosine rheostat pathway has been
identified as the homeostatic pathway triggered by the immune response for inter-partner
communication of regulatory signals (Rodriguez-Lanetty et al. 2006; Detournay & Weis 2011). This pathway includes two complementary molecular signals: one which promotes apoptosis
of the symbiotic cells and one that inhibits it; these pathways act in concert as a mechanism for
the host to control proliferation of the symbiont population (Detournay & Weis 2011). The
presence of these two proteins only in symbiotic Aiptasia confirms their importance in
communication between the partners of the symbiosis. Three of the proteins found only in symbiotic anemones function primarily in maintaining cell
structure. Hemicentin-2 acts as an extracellular adhesive, holding cells together to preserve the
integrity of tissues (Vogel et al. 2006; Xu et al. 2013). Hemicentins have previously been found
in cnidarians and may also have a secondary role in immunity or cell signalling (Schwarz et al. 2008; Barshis et al. 2013; Brekhman et al. 2015; Oakley et al. 2016). 3.4.2 Proteins associated with symbiotic and aposymbiotic states Protein unc-89 is required
for proper anchoring of myosin filaments via muscle M lines (Benian et al. 1996), while the
40S ribosomal protein S19 is necessary for ribosome biosynthesis (Danilova et al. 2008). The
downregulation of these proteins in aposymbiotic anemones could lead to developmental
defects of tissues, but more research on this would be necessary. One protein specific to the
symbiotic treatments, glutamine synthetase, is involved in nitrogen metabolism and has
previously been reported to be characteristic of symbiotic Aiptasia (Oakley et al. 2016). Ammonium can be assimilated by both the host and symbiont separately, through the glutamine
synthetase/glutamine oxoglutarate aminotransferase cycle (Pernice et al. 2012). The expression
of this enzyme only in symbiotic anemones indicates that aposymbiotic anemones assimilate
less nitrogen when they no longer need to control symbiont population numbers through
nitrogen limitation (Miller and Yellowlees 1989; Wang & Douglas 1998; Yellowlees et al. 2008). 91 The only protein expressed solely in aposymbiotic anemones was sulphur deoxygenase ETHE1
(aka persulphide deoxygenase). This protein is involved in the mitochondrial oxidation
pathway, acting as a catalyst for the oxidation of a persulphide molecule to sulphite
(Hildebrandt & Grieshaber 2008). Sulphite is an important intermediate in sulphur metabolism
pathways, however sulphite on its own is toxic to both plant and animal cells (Hänsch &
Mendel 2005; Niknahad & O’Brien 2008). When sulphite is not readily metabolized into
sulphate or other products, it can inhibit certain enzymes and attack various substrates (Heber
& Hueve 1997). Furthermore, it can lead to an increase in oxygen consumption and production
of reactive oxygen species (ROS) (Niknahad & O’Brien 2008); the accumulation of ROS in
symbiotic cnidarians is known to induce host cell apoptosis and is linked to symbiotic
breakdown (Lesser 2006; Weis 2008). The upregulation of sulphur deoxygenase could
therefore cause an accumulation of toxic sulphite in aposymbiotic Aiptasia cells, especially if
any substrates used for sulphite metabolism are usually obtained from the symbionts. This
finding suggests that the aposymbiotic state could be physiologically detrimental to Aiptasia
and could possibly explain, in part, why they do not occur in an aposymbiotic state in nature. 3.4.3.1 Previously identified proteins in symbiotic Aiptasia Only one previous study has identified differences in the host proteome between symbiotic and
aposymbiotic Aiptasia (Oakley et al. 2016). The present study confirmed many of the findings
of this previous work, including the elevated expression of NPC2 proteins, glutamine
synthetase and flotillin-2 in symbiotic anemones. All three of these proteins enhance host
metabolism by acting in lipid or nitrogen processing, indicating the metabolic benefit of the
symbiosis to the host. Additionally, the current study showed the upregulation of catalase,
creatinase, transmembrane protease serine, betaine-homocysteine S-methyltransferase
(BHMT), and fumarylacetoacetase in aposymbiotic anemones relative to symbiotic ones. Many
of these proteins aid in the breakdown of proteins and other products, and are thought to be
more highly expressed in aposymbiotic anemones due to their higher reliance on heterotrophy
(Oakley et al. 2016). Interestingly, however, when comparing the host proteome of anemones
infected with Symbiodinium D1a to that of aposymbiotic anemones, some results differ from 92 the previous study of Oakley et al. (2016), where the anemones containing homologous
Symbiodinium B1 were compared with the aposymbiotic state. In particular, these authors
reported selenium-binding proteins to be upregulated 10-fold in symbiotic vs. aposymbiotic
anemones, while here the same proteins were downregulated 0.6-fold in D1a-colonised
anemones. Additionally, amine oxidase, which Oakley et al. (2016) found to be downregulated
in B1-colonised anemones, was upregulated 7.41-fold in D1a-colonised anemones. These two
proteins are involved in intra-Golgi protein transport and histamine breakdown, respectively
(Porat et al. 2000; Mcgrath et al. 2009). This means that D1a symbionts may induce inhibition
of proper protein synthesis by the Golgi, while also breaking down histamine at an increased
rate. Histamine is an important molecule in the immune response of vertebrates, mainly being
associated with the deleterious effects of allergic reactions, and also having other potentially
beneficial roles (Schneider et al. 2002). While research on invertebrate histamine is scarce, it
is known to act as a major neurotransmitter, as in vertebrates (Roeder et al. 2003). The
enhanced breakdown of histamine in D1a-colonised anemones could be a way for the
symbionts to evade a host immune response that may attack the foreign symbionts. This
evidence illustrates how Symbiodinium D1a affects the host proteome differently than
Symbiodinium B1, possibly impairing cellular function in Aiptasia more than the aposymbiotic
state, while simultaneously protecting the foreign symbiont. 3.4.3.2 Redox and breakdown processes Many previously unreported proteins were likewise found to be upregulated in both symbiotic
treatments, and may also be indicative of processes that are important during the earlier stages
of symbiosis establishment. Only two proteins involved in oxidative stress were found:
mitochondrial complex I (NADH: Ubiquinone Oxidoreductase), which is the major site of ROS
generation within mitochondria (Galkin & Brandt 2005); and peroxiredoxin 6 which acts in
antioxidant defence by repairing damaged cell membranes (Manevich & Fisher 2005). This
might indicate the expression of normally functioning cellular redox systems, where ROS are
produced at low levels and can act in cell signalling (Circu & Aw 2010), however it is difficult
to make this assumption since these proteins only make up a small part of the overall respiratory
electron transport system. A number of proteins involved in cytolytic and proteolytic activity
were also found. These include two novel anemone toxins, PsTX-60A and AvTX-60A, that are
nematocyst components serving to attack and kill foreign cells (Nagai et al. 2002; Oshiro et al. 2004). The presence of these toxins indicates the development of an effective defence system
and method of food capture, via nematocysts, in symbiotic anemones. The caspase-7 protease,
which can induce cell apoptosis (Marcelli et al. 1998), and the calpain-9 protease, which is
activated second to caspase during apoptosis (Wood & Newcomb 1999), were both up-
regulated in symbiotic anemones. Cnidarians can control their symbiont populations by
inducing either apoptosis or autophagy of their symbiont-colonised gastrodermal cells, with
caspase as a participant in the former (Dunn et al. 2007b; Dunn & Weis 2009). As these two
mechanisms are known to work in tandem, our findings may implicate calpain as an additional
component of the autophagy mechanism in symbiotic cnidarians. 3.4.3.1 Previously identified proteins in symbiotic Aiptasia Two carbonic anhydrase proteins were found to be highly upregulated (16-29 fold) in both
symbiotic treatments when compared to the aposymbiotic state. Carbonic anhydrase is an
integral part of the carbon concentrating mechanism used by symbiotic cnidarians to transport
DIC from seawater to the symbiont’s chloroplast (Weis 1991; Bertucci et al. 2013). Oakley et
al. (2016) found upregulation of an H+-ATPase in symbiotic anemones, which is a protein that
commonly works in conjunction with carbonic anhydrase to transport protons across the
symbiosome and allow the conversion of HCO3 to CO2; the CO2 can then be utilized by the
symbiont (Furla et al. 2000; Barott et al. 2015). Anemones in the present study were sampled
just four weeks after establishing a symbiont population, whereas Oakley et al. (2016) used
anemones involved in a lifelong symbiosis, so it is clear that cellular processes that are critical
to the long-term success of the symbiosis, such as those involved with DIC delivery, operate
even during the early stages of symbiont colonisation of the host. 93 3.4.3.4 Tissue structure and muscle functioning Structural changes to host gastrodermal cells occur during symbiosis establishment, by the
formation of specialized vesicles called symbiosomes that house symbiont cells (Fitt & Trench
1983; Wakefield & Kempf 2001). The structural proteins upregulated in symbiotic anemones
could reflect proteins which aid in the creation of symbiosomes from late endosomes and their
subsequent maintenance. These include fibronectin type III proteins, which are cell-surface
glycoproteins that act in cell adhesion and have been previously reported as a component of
cnidarian mesoglea (Zhang et al. 1994; Koide et al. 1998). Also found were the cytoskeletal
rootletin protein (Yang et al. 2002) and the microtubule-binding protein EB1 that aids in cell
division and migration (Juwana et al. 1999). A number of muscle proteins were also
upregulated, including calponin-1 and myophilin, which are involved in actin- and protein-
binding (Gimona et al. 1990; Martin et al. 1995), tropomyosin and tropomodulin, which play
critical roles in muscle contraction and filament organization (Weber et al. 1994), a gelsolin-
like protein that promotes filament assembly (Sun et al. 1999), and myotrophin, which
promotes muscle cell-growth and -assembly (Sen et al. 1990). These findings suggest that both
cell structural integrity and muscle development are enhanced by the symbiotic state. 3.4.3.3 Cell signalling Signalling molecules are necessary for cnidarian hosts to communicate with their dinoflagellate
symbionts. Here, a number of proteins associated with signalling pathways were upregulated
in the symbiotic treatments. This included two signalling proteins from the Ras superfamily,
RAS2, which acts in plasma membrane signalling and responses to nutrient limitation (Tatchell
et al. 1985), and GTPase KRas, which acts in regulation of cell proliferation (Yang et al. 2012;
Zimmermann et al. 2013). Also upregulated in the symbiotic state were a protein kinase central 94 to the nitric oxide/cGMP signalling pathway (Hofmann et al. 2000), the 14-3-3 epsilon protein
responsible for regulation of signalling pathways (Fu et al. 2000), and a calcyphosin-like
protein that acts in calcium-dependent signalling cascades (Dong et al. 2008). Future
investigations on host-symbiont signalling should include these pathways for further study, as
they could be key to successful symbiosis establishment. 3.4.3.5 Cellular and metabolic processes The majority of upregulated proteins in the symbiotic anemones are involved in cellular and
metabolic processes. A number of these were involved in transport systems, including a golgin
protein responsible for vesicle transport to the Golgi apparatus (Malsam 2005), a
sodium/calcium antiporter protein in the plasma membrane (Komuro et al. 1992), and the
transmembrane stomatin protein, which regulates ion channel transport (Price et al. 2004). 95 Additionally, a succinyl-coA synthetase involved in the TCA cycle (Fraser et al. 1999) was
also upregulated in symbiotic anemones, illustrating the role of symbionts in host carbohydrate
metabolism. Inter-partner transport mechanisms are especially important when considering the
high percentage of photosynthetic carbon products that need to be transported to the host for
use in its metabolism. Previously, a host-derived proton pump was localized to the symbiosome
membrane in two coral species, and was predicted to act in host regulation of symbiont
physiology by acidifying the internal environment (Barott et al. 2015). It is therefore possible
that the transporter proteins identified in this study may also be components of the symbiosome
membrane, acting similarly on symbiont physiology by regulating the symbiont’s access to
other molecules. Additionally, a succinyl-coA synthetase involved in the TCA cycle (Fraser et al. 1999) was
also upregulated in symbiotic anemones, illustrating the role of symbionts in host carbohydrate
metabolism. Inter-partner transport mechanisms are especially important when considering the
high percentage of photosynthetic carbon products that need to be transported to the host for
use in its metabolism. Previously, a host-derived proton pump was localized to the symbiosome
membrane in two coral species, and was predicted to act in host regulation of symbiont
physiology by acidifying the internal environment (Barott et al. 2015). It is therefore possible
that the transporter proteins identified in this study may also be components of the symbiosome
membrane, acting similarly on symbiont physiology by regulating the symbiont’s access to
other molecules. 3.4.4.1 Metabolic processes Other proteins that were upregulated in B1- versus D1a-colonised anemones include two
isoforms of glutamine synthetase (26- and 23-fold upregulation). Glutamine synthetase is the
enzyme used by microalgae to catalyse the conversion of ammonia and glutamate into the
amino acid glutamine (Syrett & Peplinska 1988). It is also thought to be the primary enzyme
responsible for inorganic nitrogen incorporation and host protein synthesis in algal-invertebrate
symbioses (Pernice et al. 2012), due to its presence in host tissues of symbiotic corals, sea
anemones, giant clams, and freshwater hydra (Miller & Yellowlees 1989; Rees et al. 1994;
Wang & Douglas 1998; Smith et al. 2004). Interestingly, previous studies have consistently
found glutamine synthetase to be upregulated in symbiotic host tissues compared to
aposymbiotic ones (Lipschultz & Cook 2002), so the upregulation of this enzyme in the
homologous infection only, suggests that hosting the heterologous D1a may result in slower
nitrogen incorporation into host tissues. Alternatively, it may indicate that D1a symbionts in
the experiment have not yet triggered the host population control mechanism of nitrogen
limitation (Miller & Yellowlees 1989; Wang & Douglas 1998; Yellowlees et al. 2008). 3.4.4 Homologous (B1) versus heterologous (D1a) symbiont effects on host proteome By comparing the host proteome of anemones infected with two different symbiont types that
differ in infection success, one homologous and one heterologous, we gain new insights into
the processes involved in the establishment of a successful symbiosis. Our results suggest that
the protein most indicative of a successful symbiosis is NPC2, which was found to be highly
upregulated in B1-colonised anemones when compared to D1a-colonised anemones (4384.86-
and 526.93-fold upregulation, respectively). The present study therefore confirms the presence
of NPC2 in cnidarians and its close association with the symbiotic state (Kuo et al. 2004; Ganot
et al. 2011; Dani et al. 2014; Lehnert et al. 2014; Oakley et al. 2016), while also providing
evidence that its activity is linked to the ultimate colonisation success of the invading symbiont
type. Here, we found that two of three identified NPC2 homologs are exclusive to anemones
infected with homologous symbionts, while the third NPC2 homolog was upregulated only in
the homologous association compared to the aposymbiotic treatment. NPC2 works in
conjunction with the integral membrane protein NPC1 in the binding and export of cholesterol
from lysosomes and late endosomes (Xu et al. 2008). Four NPC2 genes have been found in the
cnidarian, Anemonia viridis, with the protein product of one gene being localized to the host
gastrodermal cells (Dani et al. 2014). Because of its general role and localization, it has been
suggested that it may be associated with the symbiosome membrane, where it could play a role
in sterol transport from symbiont to host (Dani et al. 2014; Oakley et al. 2016). Additionally,
it has been speculated that the cnidarian NPC2 protein might act in symbiont recognition and 96 persistence due to its role in immune signalling pathways of other invertebrates (Shi et al. 2012;
Dani et al. 2014). If this is the case, our results might indicate the inefficiency of heterologous
symbionts when transporting essential lipids to the host, or the inefficiency of heterologous
symbionts when initiating the adequate signalling pathway for host-symbiont compatibility. 3.4.4.2 Proteolysis and gene processing The proteases neurolysin and legumain were also only upregulated in B1-infected anemones. These proteins act in the regulation of cell proliferation pathways by metabolizing bioactive
compounds (Paschoalin et al. 2007; Dall & Brandstetter 2013). Neurolysin is an enzyme
secreted by melanoma tumours that regulates tumour proliferation by triggering an angiogenic
switch (Paschoalin et al. 2007). Legumain, which mainly acts in lysosomal compartments, is
overexpressed in the majority of human cancer tumours and is linked to enhancing tissue
invasion and promoting tumour growth (reviewed in Dall and Brandstetter 2016). The presence 97 of these enzymes could indicate that B1-colonised anemones may have more control over the
growth and proliferation of their symbiont population than when colonised by D1a. B1-
colonised anemones may also have more efficient RNA and DNA processing mechanisms, as
illustrated by the upregulation of a heterogeneous ribonucleoprotein and a serine/arginine-rich
splicing factor (Kohtz et al. 1994; Kzhyshkowska et al. 2003), and the PALF nuclease
responsible for DNA repair (Kanno et al. 2007), all in only B1-colonised anemones. This could
mean that D1a-colonised anemones are more susceptible to mutations and transcription errors. Overall, these results point to better integration of host-symbiont metabolism and gene
processing in the B1- versus D1a-colonised anemones. 3.4.4.3 Immune responses Proteins involved in immune responses were also upregulated in B1- versus D1a-colonised
anemones. One highly upregulated protein (15-fold increase) was identified as the
circumsporozoite (CS) protein. The CS protein is the main surface antigen produced by the
mature sporozoite of the malaria parasite, Plasmodium berghei, and is highly upregulated in
the infective stage of the parasite compared to non-infective stages (Aikawa et al. 1981;
Yoshida et al. 1981; Nardin et al. 1982). The CS protein is important to the parasite as it is
thought to play a critical role in invasion of host cells by binding to regions of the host cell’s
plasma membrane that are exposed to circulating blood (Aikawa et al. 1981; Cerami et al. 1992). The Aiptasia CS protein homolog identified here could similarly function to promote
infectivity of host cells, and its upregulation in B1-colonised hosts might help explain why
Symbiodinium B1 infects Aiptasia more rapidly than Symbiodinium D1a. The cnidarian CS
protein should be studied further for its potentially pivotal role as a Symbiodinium surface
ligand that binds to host cells to aid in infectivity. Also upregulated in B1- versus D1a-colonised anemones was a pirin-like protein. The human
pirin protein functions in cell migration and is upregulated during cell death (Gelbman et al. 2007; Miyazaki et al. 2010). Pirin proteins are highly conserved between all kingdoms of life
(Dunwell et al. 2001), however their functions differ between organisms, and range from roles
in programmed cell death to seed germination (Orzaez et al. 2001; Lapik & Kaufman 2003;
Adams & Jia 2005). Because of the versatility of this protein, a specific role in symbiotic 98 cnidarians is difficult to determine, though it could very likely be involved apoptosis pathways
given the number of other apoptosis-associated proteins that were found here. cnidarians is difficult to determine, though it could very likely be involved apoptosis pathways
given the number of other apoptosis-associated proteins that were found here. 3.4.4.4 Multifunctional cellular processes A host methionine pathway would explain the presence
of BHMT in all treatments in the present study, however the upregulation of this protein in
D1a-colonised and aposymbiotic anemones requires further explanation. Of particular note in
this regard, a transcriptomic study of a coral holobiont (Porites australiensis) found that half
of the enzymes for the methionine synthesis pathway were expressed by the host while the
other half were expressed by the symbiont, suggesting that the methionine pathway in the
cnidarian-dinoflagellate symbiosis involves intimate cooperation between the partners
(Shinzato et al. 2014). Additionally, Symbiodinium cells are known to synthesize their own two
betaine compounds, zooxanthellabetaine-A and –B (Nakamura et al. 1998), and these
compounds are potentially substrates in the host methionine reaction. Given this, an inability
to obtain these compounds from less compatible symbionts might result in an accumulation of
methionine-production enzymes, such as BHMT, in the host. 3.4.4.4 Multifunctional cellular processes Proteins upregulated in D1a-colonised anemones are involved in a number of important cell
processes. These include aflatoxin B1 aldehyde reductase (AKR7A2), a nucleoside hydrolase,
and betaine-homocysteine S-methyltransferases 1 and 2 (BHMT1 and BHMT2). AKR7A2 in
humans is involved in the production of neurotransmitters and may play a role in detoxification,
tumour resistance, and regulation of osmolarity in the kidneys (O’Connor et al. 1999; Lyon et
al. 2007). Nucleoside hydrolase is an enzyme used by protozoan parasites for purine salvaging
and A. thaliana for pyrimidine breakdown (Parkin et al. 1991; Shi et al. 1999; Jung et al. 2009). The function in Aiptasia is most likely similar to that of A. thaliana since neither have a
parasitic role, potentially showing an upregulation of pyrimidine breakdown in D1a-colonised
anemones. Upregulation of this breakdown could explain the downregulation in gene
processing proteins described above, and together may indicate a reduced ability for RNA and
DNA synthesis. Three BHMT proteins were highly upregulated (up to 100-fold) in D1a-colonised anemones
when compared to the B1-colonised anemones, which is interesting considering that they are
typically characteristic of the aposymbiotic state (Rodriguez-Lanetty et al. 2006; Oakley et al. 2016). Indeed, BHMT levels were the same in D1a-colonised and aposymbiotic anemones. BHMT converts homocysteine to methionine via a methyl donor from betaine, therefore acting
in both the regulation of homocysteine levels and in methionine metabolism (Finkelstein &
Martin 1984). Other than being a critical component in methionine metabolism, protein
‘moonlighting’ is possible for BHMT; though other functions have only been speculated upon,
and range from associations with tubulin to lysosomal membranes (Pajares & Pérez-Sala 2006). In mammals, feeding restrictions and folate-deficient diets were linked with increased BHMT
activity (Park & Garrow 1999; Halsted et al. 2002). It is possible that the increased BHMT in
aposymbiotic and D1a-colonised Aiptasia is linked to a lack of carbon energy, since they would
usually obtain this from the symbionts. On the other hand, the upregulation of BHMT could
signify that compatible symbionts are required for methionine metabolism in the symbiosis. 99 Consistent with this, it was previously found that bleached Aiptasia could only synthesize two
of the essential amino acids - threonine and methionine - leading to the conclusion that these
two amino acids were synthesized by the host while the others were translocated from the
symbionts (Wang & Douglas 1998). 3.4.4.5 Protein assembly Two upregulated proteins in D1a-colonised anemones may indicate problems with protein
assembly. A neurotropic factor protein called Armet was upregulated 10-fold in these
anemones. Armet is a protein that is secreted in response to an accumulation of misfolded
proteins in the endoplasmic reticulum (ER), to protect against ER stress-induced cell death
(Apostolou et al. 2008; Tadimalla et al. 2008; Hellman et al. 2011). Armet has been localized
to both the ER and Golgi apparatus, and found to protect against apoptosis while also inhibiting
cell proliferation (Apostolou et al. 2008). Concurrently, a 78 kDa glucose-regulating protein
(aka BiP) that also plays a role in the ER protein folding stress-response (Bertolotti et al. 2000;
Oka et al. 2013) and is upregulated in heat-shocked anemones (Oakley et al. unpublished data)
was slightly more abundant in D1a-colonised anemones. The upregulation of these proteins
suggests that Aiptasia host cells are stressed when infected with Symbiodinium D1a, and this
may, in part, be due to defects in protein folding in the ER. Additionally, the presence of Armet
as an immune response to Symbiodinium D1a may be another explanation for the lesser success
of this heterologous symbiont type. Two upregulated proteins in D1a-colonised anemones may indicate problems with protein
assembly. A neurotropic factor protein called Armet was upregulated 10-fold in these
anemones. Armet is a protein that is secreted in response to an accumulation of misfolded
proteins in the endoplasmic reticulum (ER), to protect against ER stress-induced cell death
(Apostolou et al. 2008; Tadimalla et al. 2008; Hellman et al. 2011). Armet has been localized
to both the ER and Golgi apparatus, and found to protect against apoptosis while also inhibiting
cell proliferation (Apostolou et al. 2008). Concurrently, a 78 kDa glucose-regulating protein
(aka BiP) that also plays a role in the ER protein folding stress-response (Bertolotti et al. 2000;
Oka et al. 2013) and is upregulated in heat-shocked anemones (Oakley et al. unpublished data)
was slightly more abundant in D1a-colonised anemones. The upregulation of these proteins
suggests that Aiptasia host cells are stressed when infected with Symbiodinium D1a, and this
may, in part, be due to defects in protein folding in the ER. Additionally, the presence of Armet
as an immune response to Symbiodinium D1a may be another explanation for the lesser success
of this heterologous symbiont type. 3.4.4.5 Protein assembly 100 Finally, the 40S ribosomal protein S19 (Rps19), which is necessary for the maturation of the
40S ribosomal subunit (Flygare et al. 2007), was also upregulated in D1a-colonised anemones. Rps19 is essential for ribosome construction and was present in anemones within all treatment
groups, however it may have been upregulated in the presence of D1a due to its secondary role
in immunity. In particular, Rps19 dimers are able to activate a phagocytosis pathway that
eliminates invasive organisms (Revollo et al. 2005; Mollnes et al. 2013), once again
highlighting a lack of compatibility between Symbiodinium D1a and Aiptasia. 3.4.5 Conclusions The results of this study improve our understanding of how different symbiont types affect the
host cnidarian proteome, with major implications for symbiosis function and the potential for
the establishment of novel host-symbiont pairings. Furthermore, the identification of host
proteins that exhibit changed levels only four weeks after infection provide insight into the
cellular processes important in the early stages of symbiosis establishment and symbiont
proliferation (Fig. 3.4). My findings confirm NPC2 and glutamate synthetase as integral
components of a successful symbiosis, and emphasize the importance of cell signalling
pathways and structural proteins to symbiosis success. Significant differences were found
between B1- and D1a-colonised anemones, leading me to conclude that heterologous infections
with Symbiodinium D1a may cause reduced fitness of the host. Indeed, D1a-colonised anemones upregulated proteins indicative of stress or aposymbiosis. Conversely, when colonised by the homologous Symbiodinium B1, the host anemone exhibits
better function of carbon, nitrogen and lipid metabolism, as well as more active pathways for
the regulation of symbiont cell proliferation (Fig. 3.4). 101 Figure 3.4 Functional implications of upregulated proteins in Aiptasia, along a continuum
of symbiotic success. The continuum ranges from aposymbiotic anemones (no symbiont
population; red) to B1-colonised anemones (homologous symbiont population; green),
with D1a-colonised anemones (heterologous symbiont population; yellow) falling in
between the two. Functional descriptions are coloured according to their placement along
the scale. Image credit: A. Sproles. Figure 3.4 Functional implications of upregulated proteins in Aiptasia, along a continuum
of symbiotic success. The continuum ranges from aposymbiotic anemones (no symbiont
population; red) to B1-colonised anemones (homologous symbiont population; green),
with D1a-colonised anemones (heterologous symbiont population; yellow) falling in
between the two. Functional descriptions are coloured according to their placement along
the scale. Image credit: A. Sproles. Furthermore, only those anemones colonised by Symbiodinium B1 possessed elevated levels
of NPC2 proteins, for which there is increasing evidence of a major role in the establishment
of a successful cnidarian-dinoflagellate association. Given these numerous patterns, relating
both to the structural and functional integrity of the symbiosis, it seems likely that a
heterologous symbiosis between Symbiodinium D1a and Aiptasia would not be optimal in the
long-term under normal environmental conditions. 3.4.5 Conclusions This finding is especially significant in light
of the widespread interest in the biogeographical spread and thermal resilience of this symbiont
type, as if similar effects are induced in corals, then this would help to explain why this
Symbiodinium type rarely dominates in the field. Even in its regular, homologous associations,
Symbiodinium D1a is known to trade functional efficiency for thermal tolerance (Stat & Gates
2011), and the discoveries presented here suggest that it affects proper functioning of host
cellular processes in heterologous associations. This raises substantial doubts about whether
this symbiont type could be a ‘nugget of hope’ for coral reefs, as previously suggested Furthermore, only those anemones colonised by Symbiodinium B1 possessed elevated levels
of NPC2 proteins, for which there is increasing evidence of a major role in the establishment
of a successful cnidarian-dinoflagellate association. Given these numerous patterns, relating
both to the structural and functional integrity of the symbiosis, it seems likely that a
heterologous symbiosis between Symbiodinium D1a and Aiptasia would not be optimal in the
long-term under normal environmental conditions. This finding is especially significant in light
of the widespread interest in the biogeographical spread and thermal resilience of this symbiont
type, as if similar effects are induced in corals, then this would help to explain why this
Symbiodinium type rarely dominates in the field. Even in its regular, homologous associations,
Symbiodinium D1a is known to trade functional efficiency for thermal tolerance (Stat & Gates
2011), and the discoveries presented here suggest that it affects proper functioning of host
cellular processes in heterologous associations. This raises substantial doubts about whether
this symbiont type could be a ‘nugget of hope’ for coral reefs, as previously suggested 102 (Berkelmans & van Oppen 2006), though whether these same traits persist over longer periods
of symbiosis establishment warrants study, so that we can better determine the adaptive
capacity of reef corals in the face of climate change. (Berkelmans & van Oppen 2006), though whether these same traits persist over longer periods
of symbiosis establishment warrants study, so that we can better determine the adaptive
capacity of reef corals in the face of climate change. (Berkelmans & van Oppen 2006), though whether these same traits persist over longer periods
of symbiosis establishment warrants study, so that we can better determine the adaptive
capacity of reef corals in the face of climate change. 103 104 4.1 Introduction The exchange of carbon and nitrogen are essential aspects of the mutualistic relationship
between cnidarians and dinoflagellate algae of the genus Symbiodinium (Muscatine 1990;
Lipschultz & Cook 2002). Photosynthetic products from the algal symbionts can meet up to
100% of the cnidarian’s metabolic energy demand, thereby supporting important processes
such as respiration, growth, reproduction and coral calcification (Vandermeulen et al. 1972;
Falkowski et al. 1984; Muscatine 1984). Fixed carbon from photosynthesis is translocated
mostly as the sugar glucose (Burriesci et al. 2012; Hillyer et al. 2016). Glucose is a necessary
metabolite as it is utilized in glycolysis to produce TCA cycle intermediates for the ultimate
production of ATP energy (Fernie et al. 2004). The translocation of adequate fixed carbon is
essential for host survival, as it is incorporated into all cnidarian tissue layers (Tremblay et al. 2012; Kopp et al. 2015; Freeman et al. 2016). The symbionts act as important primary
producers in coral reef ecosystems where waters are typically nutrient-limited (Mustatine &
Porter 1977; Hatcher 1988). This high productivity is due to efficient nitrogen cycling, and
symbionts have the ability to take up dissolved nitrate, ammonium, and urea from the seawater
along with excretory ammonium translocated from the host (Rahav et al. 1989; Grover et al. 2002, 2003, 2006, 2008). The symbionts use these nitrogenous compounds to synthesise amino
acids that are used in the algae’s cellular processes or provided back to the host (Lewis & Smith
1971; Tanaka et al. 2006). The genus Symbiodinium contains high genetic diversity, being separated into nine distinct
clades (A-I) which are further divided into ‘types’ or species (Trench & Blank 1987; Coffroth
& Santos 2005; Pochon & Gates 2010; Lajeunesse et al. 2012; LaJeunesse et al. 2015; Lee et
al. 2015). Functional characteristics vary greatly between the different algal clades (and types)
in a variety of respects, including thermal sensitivity, host infectivity and metabolite production
(Rowan et al. 1997; van Oppen et al. 2001; Santos et al. 2004; Starzak et al. 2014). Some of
these symbionts act as generalists, being able to form associations with a wide range of different
host species across multiple oceans, while others are specific to only a small handful of hosts
(LaJeunesse 2005; Frade et al. 2008; Fabina et al. 2012, 2013). Additionally, hosts can display 105 this type of selectivity for symbionts (Baker 2003). 4.1 Introduction This phenomenon is called host-symbiont
specificity, and the mechanisms underpinning its existence are not yet entirely clear
(Hambleton et al. 2014). It is of particular interest in the realm of coral adaptability to climate
change, because some Symbiodinium types are more thermally tolerant, and therefore more
resistant to symbiotic breakdown via bleaching (Tchernov et al. 2004; Jones & Berkelmans
2011; Hume et al. 2016). However, these thermally tolerant symbiont types do not naturally
associate with all coral species, and usually only exist in low background populations under
normal environmental conditions. Of particular note is Symbiodinium from clade D,
particularly type D1a (Symbiodinium trenchii), which can overrun a host’s dominant symbiont
population when seawater temperatures become thermally stressful (Jones et al. 2008;
LaJeunesse et al. 2009). A major benefit of hosting D1a is the enhanced thermal tolerance of
the entire holobiont, and its ability to remain metabolically active under very high seawater
temperatures (Baker et al. 2013). However, under normal environmental conditions, D1a
presents major drawbacks, as the host may suffer decreased growth, reproduction and
metabolite exchange (Jones & Berkelmans 2010, 2011; Baker et al. 2013; Starzak et al. 2014;
Pernice et al. 2014). This makes Symbiodinium D1a an especially interesting candidate for
studying the implications of a change in the dominant member of the symbiont population, and
ultimately the functional attributes that lead, in part, to host-symbiont specificity. Symbiodinium types vary in the amount of fixed carbon they contribute to the host (Starzak et
al. 2014; Pernice et al. 2014), and nitrogen assimilation rates differ between symbiont types
(Baker et al. 2013, Pernice et al. 2014). However, few have investigated whether similar
patterns exist with respect to the amount of host-derived nitrogen translocated to the symbionts. Symbiodinium types vary in the amount of fixed carbon they contribute to the host (Starzak et
al. 2014; Pernice et al. 2014), and nitrogen assimilation rates differ between symbiont types
(Baker et al. 2013, Pernice et al. 2014). However, few have investigated whether similar
patterns exist with respect to the amount of host-derived nitrogen translocated to the symbionts. Carbon and nitrogen assimilation may be important during the initial establishment of a
symbiosis, as illustrated by the green Hydra – Chlorella symbiosis, where excreted symbiont
metabolites signal nutritional potential to the host (Hohman et al. 1982; McAuley & Smith
1982). 4.2.1 Experimental organisms Individuals of the sea anemone Aiptasia sp. (n = 300), originating from an unknown Pacific
location, were divided evenly into three 1.5-L beakers filled with 0.22 µm-filtered seawater
(FSW). Beakers were placed in three separate water baths maintained at 25oC with WEIPRO®
MX-1019 temperature controllers and the water was aerated with Hailea® air pumps. Illumination by AQUA-GLO T8 fluorescent lightbulbs at ~95 µmol photons m-2 s-1 was
provided on a 12 h:12 h light: dark cycle. Anemones were rendered aposymbiotic using
menthol incubations over a period of four weeks (Matthews et al. 2016), and then kept in the
described experimental set-up for the duration of the experiment. Symbiodinium minutum (type
B1) and Symbiodinium trenchii (D1a) cultures were prepared according to the methods in
section 3.2.1. 4.1 Introduction This system of signalling may be linked to symbiont specificity in cnidarian-
dinoflagellate associations, however symbiont specificities often change at some point between
initial symbiont uptake and cell proliferation (Starzak et al. 2014; Little et al. 2004; Bay et al. 2011; Weis et al. 2001). 106 In this study, I investigated the exchange of carbon and nitrogen metabolites between different
host-symbiont pairs to uncover the functional dynamics triggered by different symbiont types. Using nanoscale secondary ion mass-spectrometry (nanoSIMS), I was able to image symbiont
cells within host tissues directly, and provide precise quantifications of the carbon and nitrogen
enrichment derived from the assimilation and inter-partner translocation of isotope tracers. To
address the possible link to host-symbiont specificity, I examined these interactions both in the
presence of a homologous (i.e. native) and heterologous (i.e. non-native) symbiont type during
the earlier phases of symbiosis establishment, with particular focus on the opportunistic but
thermally tolerant Symbiodinium D1a. I then considered the patterns observed in light of the
potential long-term persistence of these host-symbiont pairings. 4.2.2 Infection of Aiptasia with Symbiodinium All seawater used in the experiments was 0.22-µm filtered. Anemones were starved for one
week following recovery from menthol treatment to increase the eventual uptake of introduced
symbionts. Symbiodinium f/2 culture medium (NCMA Bigelow Laboratory, USA) was 107 refreshed two days before infection, and Artemia sp. nauplii were hatched one day before
infection. All anemones were examined under the confocal microscope (Olympus Provis
AX70, 40x magnification) to check for background symbionts. This was achieved by placing
anemones into a glass-bottom FluoroDish™ (World Precision Instruments, FL, USA) and
examining them under a 543 nm laser with a 600-700 nm emission filter, exciting the
chlorophyll autofluorescence from symbionts. Any anemones found to be harbouring
symbionts were removed from the experimental population. Because replicate anemones needed to be kept separate from their source populations during
sampling (isotopic labelling), a system using six-well plates was adopted prior to infection. Anemones intended for sampling at the first time point (2 days) were removed from their stock
populations and allowed to settle individually in plate wells prior to inoculation with
Symbiodinium, to avoid the risk of mechanical detachment affecting symbiosis establishment
at this early stage in the colonisation process. In total, four six-well plates containing a subset
of anemones (n = 5) were prepared 24 h before infection, and labelled as follows:
Symbiodinium B1 autotrophy; Symbiodinium D1a autotrophy; Symbiodinium B1 heterotrophy;
and Symbiodinium D1a heterotrophy. Inoculation with Symbiodinium was carried out in the
plates, along with the rest of the anemone stock populations from each treatment, as described
below. Symbiodinium cultures were prepared for inoculation according to the methods outlined in
section 3.2.2. Aerators were removed to encourage tentacle extension. A glass pipette was used
to drop the corresponding Symbiodinium culture suspension onto the oral disc of each anemone
in the re-infected treatments, while no culture suspension was added to the aposymbiotic
treatment. FSW in all treatments was replaced after 24 h, and samples for the first time point
(TP1) were collected after 2 days. Anemones remained in the experimental setup for two weeks
following inoculation, at which point another set of anemones was transferred to the six-well
plates for sampling at the second time point (TP2). The first time-point was chosen as 2 days
after infection to observe the nutritional interactions occurring during the earlier phases (i.e. recognition, engulfment, and sorting) of symbiont establishment. 4.2.2 Infection of Aiptasia with Symbiodinium The second time-point was
chosen as two-weeks after infection, to observe interactions likely to be in the ‘proliferation’
phase of symbiosis establishment. 108 4.2.3 Analysis of colonisation success 4.2.3 Analysis of colonisation success Colonisation success was evaluated at 2 days after inoculation (TP1) and 14 days after
inoculation (TP2) by quantifying symbiont populations using haemocytometer counts and
mean fluorescence index (MFI). For cell counts, anemones (n = 5 per treatment) were
homogenized in 1 mL FSW with an IKA T-10 electric saw-tooth homogenizer and separated
into host and symbiont fractions, by centrifuging for 5 min at 16,000 x g. The host protein in
the supernatant was quantified using the Bradford Assay (Bradford 1976), and the algal pellet
was re-suspended in 100 µL FSW with 1% formalin. Symbiont cells were counted with an
Improved Neubaur haemocytometer at 100x magnification under a light microscope (six counts
per sample) and normalised to host protein. To measure infection success using MFI, whole
anemones from each treatment were examined by confocal microscopy (Olympus Provis
AX70, at 100× magnification) to detect the chlorophyll autofluorescence of any
dinoflagellates. Anemones (n = 5) were selected at random from each treatment, and placed in
a “relaxation solution” [50% FSW, 50% 0.37 M magnesium chloride (MgCl2)] for 15 min
before being moved to a FluoroDishTM glass-bottom confocal dish (World Precision
Instruments, Florida USA). Anemone tissues were viewed under TD1 (transmitted detector 1)
set to the 473 nm laser, and symbiont chlorophyll autofluorescence was detected using 543 nm
excitation and a 600–700nm emission filter. Ten images were acquired in the z-plane for three
tentacles of each replicate anemone (n = 5/treatment) and cell fluorescence was quantified as
a proxy for symbiont density by measuring the mean 600-700 nm fluorescence intensity of
tentacle gastrodermis using ImageJ software (National Institutes of Health, Bethesda, MD,
USA). Chlorophyll autofluorescence intensity for each pixel was measured against a threshold
value for the background, determined by measuring the MFI at 600 nm of an aposymbiotic
gastrodermal section. Colonisation success was evaluated at 2 days after inoculation (TP1) and 14 days after
inoculation (TP2) by quantifying symbiont populations using haemocytometer counts and
mean fluorescence index (MFI). For cell counts, anemones (n = 5 per treatment) were
homogenized in 1 mL FSW with an IKA T-10 electric saw-tooth homogenizer and separated
into host and symbiont fractions, by centrifuging for 5 min at 16,000 x g. The host protein in
the supernatant was quantified using the Bradford Assay (Bradford 1976), and the algal pellet
was re-suspended in 100 µL FSW with 1% formalin. 4.2.3 Analysis of colonisation success The respiration rate (mL O2 h-
1) was measured for 30 min in the dark or until a constant rate was attained, after which time
the rate of net photosynthesis was measured for 30 min or until constant, at a saturating
irradiance of 400 µmol photons m-2 s-1. The water in the chamber was refreshed with each
change of anemone. A FSW-only control was also measured for 30 min in the dark and light
in order to account for oxygen fluctuations associated with microbial contamination. Gross
photosynthesis was calculated by the addition of net photosynthesis to dark respiration
(Muscatine et al. 1981). The average ratio of hourly gross photosynthesis to respiration (P:R)
was calculated to determine the potential productivity of each anemone. 4.2.3 Analysis of colonisation success Symbiont cells were counted with an
Improved Neubaur haemocytometer at 100x magnification under a light microscope (six counts
per sample) and normalised to host protein. To measure infection success using MFI, whole
anemones from each treatment were examined by confocal microscopy (Olympus Provis
AX70, at 100× magnification) to detect the chlorophyll autofluorescence of any
dinoflagellates. Anemones (n = 5) were selected at random from each treatment, and placed in
a “relaxation solution” [50% FSW, 50% 0.37 M magnesium chloride (MgCl2)] for 15 min
before being moved to a FluoroDishTM glass-bottom confocal dish (World Precision
Instruments, Florida USA). Anemone tissues were viewed under TD1 (transmitted detector 1)
set to the 473 nm laser, and symbiont chlorophyll autofluorescence was detected using 543 nm
excitation and a 600–700nm emission filter. Ten images were acquired in the z-plane for three
tentacles of each replicate anemone (n = 5/treatment) and cell fluorescence was quantified as
a proxy for symbiont density by measuring the mean 600-700 nm fluorescence intensity of
tentacle gastrodermis using ImageJ software (National Institutes of Health, Bethesda, MD,
USA). Chlorophyll autofluorescence intensity for each pixel was measured against a threshold
value for the background, determined by measuring the MFI at 600 nm of an aposymbiotic
gastrodermal section. Maximum photosynthetic and dark respiratory O2 fluxes were also measured at the end of the
experiment (TP2), to assess photosynthetic function of each symbiotic treatment. A single
anemone (n = 3 per symbiotic treatment) was placed on top of a piece of nylon mesh inside a
10-mL glass chamber filled with FSW, and left to settle on the mesh for 1 – 2 h. A magnetic
stir bar was placed underneath the mesh floor to circulate the water inside the chamber, and
was set to the lowest setting to avoid disturbing the anemone. The chamber was then sealed
with a glass lid and rubber O-ring. Oxygen and temperature probes (FIBOX 3; PreSens GmbH, 109 Germany) were inserted into the lid to measure oxygen % air saturation in the chamber. The
chamber was placed inside a 500 mL beaker filled with FSW heated to 25oC within the
experimental water bath to regulate the chamber’s temperature. 4.2.4 Confirmation of symbiont genotype DNA was extracted from Symbiodinium cultures and algae isolated from anemones TP2, using
a method adapted from Logan et al. (2010). Samples in DMSO were centrifuged at 16,000 x g
for 5 min to pellet algae, and the DMSO then removed. Algal pellets were re-suspended in 300
µL guanidinium buffer (50% w/v guanidinium isothiocyanate; 50 mM Tris pH 7.6; 10 µM
EDTA; 4.2% w/v sarkosyl; 2.1% v/v β-mercaptoethanol), before being incubated for 7 d at 4oC
to gently break open algal cells. Following incubation, samples were heated for 10 min at 72oC,
and then centrifuged at 16,000 x g for 5 min. The supernatant was combined with an equal
volume of cold isopropanol in a new tube, and samples then chilled at -20oC overnight to
precipitate DNA. Following DNA precipitation, samples were centrifuged at 16,000 x g for 10
min at 4oC and the supernatant discarded. The pellet was washed by adding 200 µL 70%
ethanol, vortexing, and centrifuging at 16,000 x g for 10 min at 4oC. Samples were then dried
in a laminar flow hood for 1 h before re-suspension in 50 µL 10 mM TE buffer by milling
without beads at 30 Hz for 1 min. DNA extractions were then used for qPCR. To provide immediate verification of symbiont identities, qPCR with clade-specific primers
was used (Yamashita et al. 2011). The clade D primers SymD28S-1F (forward; 5’-AAT GCT
TGT GAG CCC TGG TC-3’) and SymD28S-1R (reverse; 5’-AAG GCA ATC CTC ATG CGT
ATG-3’), and the clade B primers SymB28S-1F (forward; 5’-CAC ATG TCG TGC TGA GAT
TGC-3’) and SymbB28S-1R (reverse; 5’-CTC GCA TGC TGA GAA ACA CTG-3’) were
used. The thermal cycling regime had an initial denaturation step of 10 min at 95oC followed 110 by 40 cycles of 15 s at 95oC and 1 min at 60oC (performed with Applied Biosystems StepOne
system). Each reaction contained 2 µL DNA template, 5 µL SYBR Green Mastermix (Life
Technologies), 0.5 µL bovine serum albumin (Sigma Alrich, Auckland, New Zealand), 1.7 µL
PCR grade H2O, 0.4 µL forward primer, and 0.4 µL reverse primer. Samples where the clade-
specific primer matched the clade of Symbiodinium present showed amplification, and were
used to compare the identity of the inoculation cultures with the TP2 symbionts. 4.2.5 Isotopic labelling of carbon and nitrogen compounds At each sampling time point, the anemones in six-well plates (see section 4.2.2) were labelled
with 13C and 15N. The anemones in the “heterotrophy” plates were each fed 13C- and 15N-
labelled Artemia, in order to track these nutrients as they were incorporated into host tissues
and translocated to symbiont cells. The anemones in the “autotrophy” plates were incubated in
13C-and 15N-labelled seawater to track the nutrients as they were assimilated by the symbionts
and translocated to host tissues. All incubations were conducted under the conditions described
in section 4.2.1, with light provided by AQUA-GLO T8 fluorescent lightbulbs at ~95 µmol
photons m-2 s-1, and lids were placed loosely over the plates to prevent evaporation while
maintaining oxygen exchange. 4.2.5.1 Heterotrophic nitrogen translocation Cultures of the green alga Tetraselmis were grown in 15N- and 13C- labelled f/2 medium for 5
months, with sub-culturing every 2 weeks, to yield highly-enriched algae. Tetraselmis in
unlabelled f/2 medium was also cultured for the same time-period, to use as reference samples. Artemia nauplii were hatched and divided into two groups, with one group being fed labelled
Tetraselmis and the other fed unlabelled Tetraselmis for reference samples, for 10 days before
harvesting. At TP1 and TP2, anemones from the B1 and D1a treatments were fed three labelled
shrimp each (determined from preliminary trials of host labelling intensity). Six hours post-
feeding, anemones were placed in an EM fixative (2% paraformaldehyde, 2.5% glutaraldehyde,
0.1 M sodium cacodylate) and stored at 4oC for 24 h. Anemone tentacles were excised and
returned to the same fixative solution. The unlabelled Tetraselmis and Artemia were each
collected in a 50 mL Falcon tube and centrifuged for 10 min at 4,000 x g to pellet. The
supernatant was discarded and the pellets were frozen at -80oC and freeze-dried for later
enrichment analysis with the nanoSIMS. 4.2.5.1 Preparation of isotope solutions 13C-labelled FSW was obtained by adding 5 mL 1 M HCl to 2 L autoclaved 0.22 µm FSW, to
convert the existing 12C in the water to CO2, and then aerating the water under a fume hood for
3 h to remove the CO2. The carbon in the medium was then replaced by adding 2 mM
NaH13CO3 and the pH was balanced to ~ 7.8 using 1 M NaOH. 15N- and 13C-labelled f/2 culture
medium was then prepared by adding the components of an f/2 medium kit (Bigelow
Laboratory, MA) to the 13C-labelled FSW, while replacing the NaNO3 component with
Na15NO3. An unlabelled f/2 medium was also prepared for reference samples in the same
manner, except using NaHCO3 and the NaNO3 included with the f/2 medium kit. Isotopically-
labelled FSW for the autotrophy experiment was prepared by adding K15NO3 to a final
concentration of 30 µM into the 13C-labelled FSW. K15NO3 was used in lieu of K15NH4, since
the goal was to observe only symbiont assimilated nitrogen, and the host is also able to 111 assimilate ammonium (Kopp et al. 2013). Unlabelled FSW for the reference sample containing
2 mM NaHCO3 and 30 µM KNO3 was also prepared. All reagents were obtained from Sigma
Alrich (Auckland, New Zealand). assimilate ammonium (Kopp et al. 2013). Unlabelled FSW for the reference sample containing
2 mM NaHCO3 and 30 µM KNO3 was also prepared. All reagents were obtained from Sigma
Alrich (Auckland, New Zealand). 4.2.5.1 Autotrophic C and N fixation and translocation The FSW in each well (15 mL) containing an anemone (n = 5) was replaced with 13C- and 15N-
labelled FSW, and incubated for 6 h (determined from preliminary trials of symbiont labelling
intensity). One anemone from the B1 treatment was also incubated in unlabelled FSW as a
control. After incubation, anemones were placed into EM fixative as above and stored at 4oC
for 24 h. Anemone tentacles were then excised as before and returned to the fixative. 112 4.2.6 NanoSIMS sample preparation and enrichment measurements Fixative was removed from anemone tentacles and they were post-fixed for 1 h in 1% OsO4. Next, the samples were dehydrated through an ethanol series (15%, 30%, 50%, 70%, 90%, 3 x
100%), followed by 10 min in propylene oxide (10 min in each solution). Samples were
incubated in a 1:1 propylene oxide: resin mixture for ~ 18 h before embedding in Procure 812
resin (ProSciTech, Australia). Semi-thin sections (500 nm) of each resin block were cut with a
Diatome Ultra 45o Diamond Knife. Sections were stretched using a Max Wax heated pen and
transferred to 10 mm round glass coverslips using a Perfect Loop (Electron Microscopy
Sciences, USA). Both Tetraselmis and Artemia lyophilized samples were smeared onto a 10
mm round glass cover slip. Regions of interest (ROIs) on each cover slip were identified under a light microscope (40x
magnification) as those containing Symbiodinium cells within host tissue and were
photographed to use as image maps in subsequent analysis. Cover slips were then coated with
7 nm gold and ROIs were imaged with a NanoSIMS ion probe (conducted at École
Polytechnique Fédérale de Lausanne, Switzerland). About 10 Symbiodinium cells were imaged
per anemone at TP1, while around 20-40 Symbiodinium cells were imaged per anemone at
TP2, reflecting the increase in symbiont density and hence ease of locating cells over time. In
the autotrophy experiment, a reduced number of replicates (n = 4 anemones; n ≥ 12 symbionts
total/treatment) was obtained at TP1 due to the inability of the instrument to detect much carbon
translocation from symbiont to host at such cell densities. Samples were bombarded with a 16
keV primary ion beam of (1-2 pA) Cs+ focused to a spot size between 100 – 150 nm on the
sample surface. 4.2.5.1 Autotrophic C and N fixation and translocation Secondary molecular ions 12C14N-, 12C15N-, 12C12C- and 12C13C- were
simultaneously collected in electron multipliers at a mass resolution (M/∆M) of ~ 9000, enough
to resolve the 12C15N- and 13C14N- ions from possibly problematic interferences, while charge
compensation was not necessary. Images of 40 x 40 µm with 256 x 256 pixels were obtained
for 12C14N-, 12C15N-, 12C12C- and 12C13C- by rastering the primary ion beam across the sample
with a dwell-time of 5 ms. A drift correction was applied, and the 15N/14N and 13C/14C maps
obtained by taking the ratio between 12C15N- and 12C14N- images or 12C13C- and 12C12C- images. 15N and 13C enrichments were expressed in the delta notation (δ15N and δ13C in ‰) as calculated by: 113 𝛿15𝑁= (
Nmes
Nnat −1) x 103 𝛿15𝑁= (
Nmes
Nnat −1) x 103 Where Nmes is the measured 15N/14N ratio and Nnat is the natural 15N/14N ratio measured in
unlabelled control anemone samples. Where Nmes is the measured 15N/14N ratio and Nnat is the natural 15N/14N ratio measured in
unlabelled control anemone samples. 𝛿13𝐶= (
Cmes
Cnat −1) x 103 𝛿13𝐶= (
Cmes
Cnat −1) x 103 Where Cmes is the measured 13C/12C ratio and Cnat is the natural 13C/12C ratio measured in
unlabelled control anemone samples from the autotrophy experiment. Both Nnat and Cnat were
measured on sections of unlabelled anemone tissue each day of NanoSIMS analysis to correct
for any potential instrument drift. Measured enrichments from NanoSIMS images were
considered significant when above the natural fluctuations of the 15N/14N and 13C/12C ratios
from different images of unlabelled tissue, defined as the average of δ15N or δ13C ± 3σ. 4.2.7 NanoSIMS image analysis L’IMAGE® software was used to analyse NanoSIMS images. ROIs were defined within the
images by first tracing the outline of dinoflagellate cells, and then separately tracing the
gastrodermal tissue (avoiding intercellular space and mesoglea) and subtracting the
dinoflagellate ROIs, i.e. to gather enrichment data for the symbiont and host, respectively. ROI
enrichment values were averaged between replicates for each sample. For Tetraselmis and
Artemia samples, the entire area containing lyophilized sample was selected as an ROI. 4.2.8 Statistical analysis Symbiont cell density values could not be transformed to fit normality assumptions, so the non-
parametric Kruskal-Wallis test was used to test for differences (quantified using MFI) between
symbiont types at the two time-points. The hourly ratios of gross photosynthesis to dark
respiration (P:R) at TP2 were tested for differences between symbiont types using a one-way
ANOVA. 114 Nitrogen enrichment from the heterotrophy experiment and carbon enrichment from the
autotrophy experiment were compared between anemones infected with Symbiodinium type
B1 versus D1a. Any negative enrichment values were changed to zero and included in mean
calculations. Average host and symbiont enrichment values for each anemone were used, since
raw data could not be normalized through transformations and did not have equal sample sizes. Nitrogen enrichment values (in δ15N ‰) were log10 transformed to meet normality
assumptions. For the heterotrophy experiment, a two-way ANOVA was conducted to test
whether nitrogen enrichment was significantly influenced by symbiont type or time point. Separate tests were conducted for enrichment values of the host tissues and symbiont cells. For
the autotrophy experiment, a two-way ANOVA was similarly used to determine if carbon
enrichment in the host tissues and symbiont cells was influenced by either symbiont type or
time point. Nitrogen enrichment from the heterotrophy experiment and carbon enrichment from the
autotrophy experiment were compared between anemones infected with Symbiodinium type
B1 versus D1a. Any negative enrichment values were changed to zero and included in mean
calculations. Average host and symbiont enrichment values for each anemone were used, since
raw data could not be normalized through transformations and did not have equal sample sizes. Nitrogen enrichment values (in δ15N ‰) were log10 transformed to meet normality
assumptions. For the heterotrophy experiment, a two-way ANOVA was conducted to test
whether nitrogen enrichment was significantly influenced by symbiont type or time point. Separate tests were conducted for enrichment values of the host tissues and symbiont cells. For
the autotrophy experiment, a two-way ANOVA was similarly used to determine if carbon
enrichment in the host tissues and symbiont cells was influenced by either symbiont type or
time point. 4.3.1 Infection success Symbiodinium genotyping confirmed successful colonisation by Symbiodinium B1 and D1a in
their respective experimental treatments. A significant difference in cell density between B1
and D1a was seen at both time points (Fig. 4.1), where B1 was approximately 13x more dense
than D1a at TP1 (d.f. = 1, χ2 = 6.82, p < 0.01), and approximately 3x more dense at TP2 (d.f. =
1, χ2 = 6.82, p < 0.01). Furthermore, at 2 days post-infection, B1 symbionts had successfully
colonised all anemone tentacles, while D1a symbionts had left many tentacles un-colonised. There were no differences between symbiont types with respect to gross photosynthesis (One-
way ANOVA; F (1, 4) = 0.533, p = 0.51), photosynthesis per symbiont cell (One-way
ANOVA; F (1, 4) = 1.38, p = 0.31) or P:R ratio (One-way ANOVA; F (1, 4) = 4.98, p = 0.09). The average hourly P:R ratio of B1-colonised anemones was 2.6 and D1a-colonised anemones
was 1.8. 115 Figure 4.1 Infection success of Symbiodinium types B1 and D1a in the cnidarian host Aiptasia. A) Symbiont
population density within anemone tentacles as assessed by mean fluorescence index (MFI) at 2 days [TP1] and
14 days [TP2] post infection, shown with +/- standard error of the mean. Significant differences between symbiont
types are indicated by a red asterisk (*p < 0.05). B) Confocal images of anemone tentacles infected with both
symbiont types at the two time points, showing red autofluorescence of symbiont cells. Scale bars are 100 µm. Figure 4.1 Infection success of Symbiodinium types B1 and D1a in the cnidarian host Aiptasia. A) Symbiont
population density within anemone tentacles as assessed by mean fluorescence index (MFI) at 2 days [TP1] and
14 days [TP2] post infection, shown with +/- standard error of the mean. Significant differences between symbiont
types are indicated by a red asterisk (*p < 0.05). B) Confocal images of anemone tentacles infected with both
symbiont types at the two time points, showing red autofluorescence of symbiont cells. Scale bars are 100 µm. 4.3.2 Heterotrophy experiment Tetraselmis cells successfully incorporated 15N-nitrate and 13C-bicarbonate (δ13C: 4885 ‰ and
δ15N: 1,170,772 ‰), while Artemia successfully incorporated the labelled algae (δ13C: 4266 ‰
and δ15N: 113,031 ‰). Carbon enrichment values (δ13C ‰) for both the host tissues and
symbiont cells were mostly negative or lower than the window of error, so were excluded from
further analysis (Appendix B, Table B1). Nitrogen enrichment (δ15N ‰) in host tissues of B1-
colonised anemones was 74% lower, while in D1a-colonised anemones it was 60% lower, at
14 days post-inoculation than at 2 days (two-way ANOVA; F (3, 19) = 12.81, p = 0.003). Between the same time points, B1 symbiont cell enrichment was 90% lower and D1 symbiont 116 cell enrichment was 28% lower (two-way ANOVA; F (3, 19) = 44.67, p < 0.0001) (Fig. 4.2;
Table 4.1). At 2 days post inoculation, no differences in 15N enrichment were observed between symbiont
types (Fig. 4.3A, B). However, at 14 days post-inoculation, Symbiodinium D1a cells appeared
more enriched with 15N than Symbiodinium B1 cells (Fig.4.3C, D). While host nitrogen
enrichment was not affected by symbiont type during this time (two-way ANOVA; F (3, 19) =
0.391, p = 0.541), nitrogen enrichment of the dinoflagellate cells was significant (two-way
ANOVA; F (1, 19) = 6.88, p = 0.018). Ultimately, B1 cells were only an average of 20% as
enriched with nitrogen as D1a cells. Particles highly enriched in nitrogen (up to δ15N 215,000
‰; Table 1) were also noted within the host gastrodermal tissues, which were likely
phagocytosed Artemia particles (Fig. 4.3A-D). 117 gure 4.2 Nitrogen and carbon enrichment of host tissues and symbiont cells. Arrows indicate flow of labelle
mpounds in the system. A) In the heterotrophy experiment, labelled Artemia was fed to symbiotic Aiptasia an
nitrogen was incorporated into host tissues, then translocated to the symbionts. B) In the autotroph
periment, symbiotic Aiptasia were incubated in labelled seawater and the carbon was fixed by the symbiont
d then translocated to the host tissues. Bars represent the mean delta values across all replicate anemones, whi
otted points over each bar show the mean delta value for each replicate anemone (n = 5). Bars are shown wi
standard error of the mean. Significant differences between symbiont types are indicated by a red asterisk (*
0.05; **p < 0.01). Figure 4.2 Nitrogen and carbon enrichment of host tissues and symbiont cells. 4.3.2 Heterotrophy experiment Arrows indicate flow of labelled
compounds in the system. A) In the heterotrophy experiment, labelled Artemia was fed to symbiotic Aiptasia and
the nitrogen was incorporated into host tissues, then translocated to the symbionts. B) In the autotrophy
experiment, symbiotic Aiptasia were incubated in labelled seawater and the carbon was fixed by the symbionts,
and then translocated to the host tissues. Bars represent the mean delta values across all replicate anemones, while
plotted points over each bar show the mean delta value for each replicate anemone (n = 5). Bars are shown with
+/- standard error of the mean. Significant differences between symbiont types are indicated by a red asterisk (*p
< 0.05; **p < 0.01). 118 4.3.3 Autotrophy Experiment 4.3.3 Autotrophy Experiment Symbiodinium cells successfully utilized dissolved 13C-bicarbonate in the seawater for carbon
fixation. While some symbiont incorporation of dissolved 15N-nitrate was detectable, δ15N
enrichment values were mostly below the window-of-error and were therefore excluded from
further analysis (Appendix B, Table B2). Carbon enrichment (δ13C) of dinoflagellate cells was
significantly affected by symbiont type during both time points (two-way ANOVA; F (3, 17)
= 9.934, p < 0.01). At 2 days post-inoculation (Fig. 4.3E, F), Symbiodinium D1a cells were an
average of 41% less enriched in carbon than Symbiodinium B1 cells (Fig. 4.2). At 14 days post-
inoculation (Fig. 4.3G, H), Symbiodinium B1 cells were clearly more enriched with carbon than
Symbiodinium D1a cells, where D1a symbionts were an average of 75% less enriched than B1
symbionts (Fig. 4.2). Values for carbon enrichment in host tissues were low and there was no
effect of symbiont type (two-way ANOVA; F (3, 17) = 3.75, p = 0.073). Negative or zero δ13C values were interpreted as symbionts that were not fixing carbon (Fig. 4.3H; Table 4.1). At TP1, 100% of type B1 symbionts analysed (n = 12) were fixing carbon,
while 85% of type D1a symbionts analysed (n = 13) were fixing carbon. At TP2, 89% of type
B1 symbionts (n = 205) surveyed were fixing carbon, while nearly 50% of D1a symbionts
surveyed (n = 132) were fixing carbon. Table 4.1 Range of nitrogen and carbon enrichment values between the time points and symbiont types, as
measured using nanoSIMS image analyses. TP1
B1
D1a
Heterotrophy
Autotrophy
Heterotrophy
Autotrophy
Host
δ15N 1,845 - 1,6077
δ13C 0 - 184
δ15N 497 - 44,131
δ13C 0 - 132
Symbiont
δ15N 132 - 11,371
δ13C 382 - 1,660
δ15N 71 - 7,821
δ13C 0 - 2,728
Phagocytosed
food
δ15N 14,395 - 215,286
N/A
δ15N 1,787 - 194,186
N/A
TP2
B1
D1a
Heterotrophy
Autotrophy
Heterotrophy
Autotrophy
Host
δ15N 616 - 2,575
δ13C 0 - 659
δ15N 783 - 6,585
δ13C 0 - 182
Symbiont
δ15N 27 - 1,915
δ13C 0 - 3,579
δ15N 264 - 5,279
δ13C 0 - 1,510
Phagocytosed
food
δ15N 5,221 - 98,634
N/A
δ15N 13,568 - 137,195
N/A Table 4.1 Range of nitrogen and carbon enrichment values between the time points and symbiont types, as
measured using nanoSIMS image analyses. 4.3.3 Autotrophy Experiment 119 120
Figure 4.3 Imaging of 15N and 13C carbon enrichment within symbiotic Aiptasia tissue between 2 days post-inoculation [TP1] and 14 days post-inoculation [TP2] with
Symbiodinium B1 and Symbiodinium D1a. Colour scale indicates values in delta notation, where black signifies no enrichment and white signifies enrichment higher than δ
20,000. Symbiont cells are outlined in cyan, with remaining enriched areas being anemone gastrodermal tissue. (A-D) 15N enrichment of Symbiodinium B1-colonised Aiptasia
(A, C) and Symbiodinium D1a-colonised Aiptasia (B, D) at TP1 (A-B) and TP2 (C-D). (E-H) 13C enrichment of Symbiodinium B1-colonised Aiptasia (E, G) and Symbiodinium
D1a-colonised Aiptasia (F, G) at TP1 (E, F) and TP2 (G, H). White arrows point to symbiont cells that are not fixing any carbon. Figure 4.3 Imaging of 15N and 13C carbon enrichment within symbiotic Aiptasia tissue between 2 days post-inoculation [TP1] and 14 days post-inoculation [TP2] with
Symbiodinium B1 and Symbiodinium D1a. Colour scale indicates values in delta notation, where black signifies no enrichment and white signifies enrichment higher than δ
20,000. Symbiont cells are outlined in cyan, with remaining enriched areas being anemone gastrodermal tissue. (A-D) 15N enrichment of Symbiodinium B1-colonised Aiptasia
(A, C) and Symbiodinium D1a-colonised Aiptasia (B, D) at TP1 (A-B) and TP2 (C-D). (E-H) 13C enrichment of Symbiodinium B1-colonised Aiptasia (E, G) and Symbiodinium
D1a-colonised Aiptasia (F, G) at TP1 (E, F) and TP2 (G, H). White arrows point to symbiont cells that are not fixing any carbon. 120 4.4 Discussion This study assessed the dynamics of carbon and nitrogen metabolism during two phases of
symbiosis establishment, while comparing the differences between a homologous
(Symbiodinium B1) and heterologous symbiont type (Symbiodinium D1a). The reduced
translocation of photosynthetic carbon to the host by D1a is consistent with its inability to
colonise hosts as readily as the homologous symbionts. In fact, the lack of carbon fixation by
a little over half of D1a cells in the longer-term raises questions about the likelihood of its
ultimate success in providing energy to the host; however, D1a appears to adequately receive
host-derived nitrogen throughout symbiosis establishment, indicating that the heterologous
symbiosis is a less mutually beneficial symbiont. 4.4.2 The effect of symbiont type on heterotrophic metabolic translocation Anemones in the two treatments incorporated similar levels of 15N into host tissues. However,
a difference in the translocation of labelled nitrogen to symbionts during TP2 was observed,
where D1a symbionts were enriched about four times as much as B1 symbionts (Fig. 4.3). Differences in symbiont density between the two Symbiodinium types may have contributed to
this outcome, since the population density of B1 was three-fold more than that of D1a at TP2,
and hence D1a may have had less competition from neighbouring cells if N was a limited
resource (Cook & D’Elia 1987; Falkowski et al. 1993). However, despite the population of B1
being 13-fold more dense than that of D1a at TP1, no difference in symbiont 15N enrichment
was seen at this earlier time point. This could simply be because N was not limiting at this
earlier stage, though an alternative explanation would be that B1 is more efficient at taking up
nitrogen from the host in the initial stages of colonisation, so countering any increased inter-
cellular competition for resources. Furthermore, it is also possible that D1a is less
‘metabolically adept’ during these early stages. Consistent with this, previous studies have
reported that members of Symbiodinium clade D assimilate less DIN than do their clade C
counterparts under normal seawater temperatures (Baker et al. 2013; Pernice et al. 2014),
though this trend reverses once temperatures become thermally stressful (30oC) (Baker et al. 2013). The possible reasons for the differential N-uptake are therefore unclear, as both type-
specific differences and density-dependent effects could be involved, and the contributions of
these towards the patterns seen could shift during host colonisation. Further experiments when
the anemones contain equal densities of different symbiont types would help to shed some light Anemones in the two treatments incorporated similar levels of 15N into host tissues. However,
a difference in the translocation of labelled nitrogen to symbionts during TP2 was observed,
where D1a symbionts were enriched about four times as much as B1 symbionts (Fig. 4.3). Differences in symbiont density between the two Symbiodinium types may have contributed to
this outcome, since the population density of B1 was three-fold more than that of D1a at TP2,
and hence D1a may have had less competition from neighbouring cells if N was a limited
resource (Cook & D’Elia 1987; Falkowski et al. 1993). 4.4.1 Infection dynamics of Symbiodinium B1 versus Symbiodinium D1a Quantification of symbiont population density at two time points during the infection process
allowed for a comparison of the rate of symbiosis establishment between the algal types. Not
surprisingly, the homologous type (B1) was able to fully colonise the anemones at a greater
rate and reached higher symbiont densities than the heterologous type (D1a). MFI was used to
assess infection success throughout the experiment because this method provides visual
corroboration of symbiont population densities within Aiptasia tentacles, which were also the
site of nanoSIMS measurements. Symbiodinium B1 was able to quickly establish symbiont
populations in all host tentacles by TP1, while Symbiodinium D1a was slower to establish
symbiont populations in host tentacles, leaving many un-colonised at TP1. Oxygen fluxes after
two weeks of infection, determined by P:R ratios, indicated that both symbiont types were
producing enough carbon to sustain animal respiratory needs in the light (P:R > 1). Previous
studies are unanimous in finding a similar result with respect to colonisation by homologous
versus heterologous symbionts, where both can successfully invade the host, but with the
heterologous symbiont doing so more slowly and often achieving lower cell densities (Weis et
al. 2001; Belda-Baillie et al. 2002; Dunn & Weis 2009; Starzak et al. 2014). The reasons for
this pattern are uncertain, but could be related to complementary glycans on the cell surface of
the homologous symbiont (Koike et al. 2004; Logan et al. 2010; Bay et al. 2011), important
inter-partner signalling processes (Schwarz & Weis 2003; Wood-Charlson et al. 2006; 121 Detournay et al. 2012) or up- or down-regulation of the host’s immune defences (Davy et al. 2012). Although densities of B1 were more than 10-fold higher than those of D1a at TP1, this
gap had narrowed to ~3-fold by TP2. This may be due to B1 populations reaching maximum
capacity while D1a populations were still in the proliferation phase. The results presented here
suggest that most D1a cells are rejected during the recognition and sorting phases, but those
few that do become successfully established as symbionts are able to proliferate to some
degree. The colonisation patterns seen here are therefore representative of the establishment
process for an optimal symbiosis (Symbiodinium B1) and a sub-optimal symbiosis
(Symbiodinium D1a). 4.4.2 The effect of symbiont type on heterotrophic metabolic translocation However, despite the population of B1
being 13-fold more dense than that of D1a at TP1, no difference in symbiont 15N enrichment
was seen at this earlier time point. This could simply be because N was not limiting at this
earlier stage, though an alternative explanation would be that B1 is more efficient at taking up
nitrogen from the host in the initial stages of colonisation, so countering any increased inter-
cellular competition for resources. Furthermore, it is also possible that D1a is less
‘metabolically adept’ during these early stages. Consistent with this, previous studies have
reported that members of Symbiodinium clade D assimilate less DIN than do their clade C
counterparts under normal seawater temperatures (Baker et al. 2013; Pernice et al. 2014),
though this trend reverses once temperatures become thermally stressful (30oC) (Baker et al. 2013). The possible reasons for the differential N-uptake are therefore unclear, as both type-
specific differences and density-dependent effects could be involved, and the contributions of
these towards the patterns seen could shift during host colonisation. Further experiments when
the anemones contain equal densities of different symbiont types would help to shed some light 122 on this issue. Nevertheless, these findings contribute further to our understanding of
heterotrophic N metabolism in the cnidarian-dinoflagellate symbiosis and its role in symbiont
population regulation. While Artemia were highly enriched with 13C (δ13C: 4885 ‰) when fed to anemones, the
enrichment values for both host tissues and symbiont cells were very low or undetectable,
which may reflect the sensitivity limits of the instrument. Cnidarians use carbon derived from
heterotrophy for building proteins and membranes throughout their body, where it tends to
reside for a long time (Bachar et al. 2007). This heterotrophy-derived carbon is also a potential
factor for enhancing coral bleaching resistance by prohibiting intercellular CO2 limitation
(Wooldridge 2014). Therefore, the quantification of heterotrophic carbon between symbiont
treatment types does present an interesting topic for further investigations, but studies should
focus on using more sensitive techniques such as bulk isotope ratio-mass spectrometry. 4.4.3 The effect of symbiont type on autotrophic metabolite translocation Symbiodinium B1 fixed 41-75% more carbon than Symbiodinium D1a while in symbiosis with
Aiptasia throughout symbiosis establishment. However, no differences in translocation to the
host (indicated by levels of host tissue 13C-enrichment) were found. Enrichment in host tissues
was very low, and at times undetectable in anemone tissues hosting both symbiont types. It is
unknown if this is again attributable to the sensitivity threshold of the instrument, or if neither
symbiont was releasing much carbon during the experiment. Furthermore, 11% of D1a cells
showed no evidence of carbon fixation at the first time point, with this proportion increasing
to more than 50% of the population by the later time point; this compared to 100 and 89% of
functional B1 cells at the first and later time points, respectively. The small decline in the
photosynthetic performance of the homologous symbionts can potentially be explained by
regular senescence and degradation processes that occur in a steady-state population (Trench
1974; Titlyanov et al. 1996; Jones & Yellowlees 1997). However, the high portion of D1a cells
that ceased to fix carbon, and the sharp decline over time, suggests symbiont dysfunction;
further studies on cellular viability will help to clarify this issue. The lower capacity for clade D Symbiodinium to assimilate carbon and nitrogen in comparison
to the more dominant clade C symbiont has been demonstrated previously (Baker et al. 2013; 123 Pernice et al. 2014). This ultimately is a disadvantage to the host, which would have to rely on
other carbon sources to meet its daily energy demands. Translocation of photosynthetic
products from symbiont to host can be regulated by putative host release factors (HRFs) that
may vary widely between host species and exhibit different host-symbiont specificities (Sutton
& Hoegh-Guldberg 1990; Gates et al. 1995, 1999; Cook & Davy 2001), while cell signalling
molecules can also regulate photosynthetic performance and even completely inhibit it (Wang
& Douglas 1997; Grant et al. 2001). It is therefore possible that, in addition to enhanced cellular
dysfunction, the reduced photosynthesis of D1a could be due to incompatibilities with
unknown Aiptasia signalling molecules that usually enhance photosynthesis and translocation
by its symbionts. Nitrate from the seawater can only be utilized by the symbionts, since the coral host lacks the
reductase enzymes necessary for assimilation (Tanaka et al. 2006). 4.4.3 The effect of symbiont type on autotrophic metabolite translocation Symbionts convert nitrate
to ammonium using nitrate reductase, which is the assimilated into amino acids for use in
building both host and symbiont proteins (Crossland & Barnes 1977; Swanson et al. 1998). Here, enrichment resulting from nitrate uptake from the seawater, in both symbiont cells and
host tissues, was limited. Other studies have similarly found that dissolved nitrate is taken up
at a much slower rate (up to 23-times) than is DIC by the symbionts (Tanaka et al. 2006). If
ammonium had been used in place of nitrate, higher uptake values would have likely been
obtained, since ammonium is the primary nitrogen source for symbionts (Kopp et al. 2013). 4.4.4 Conclusions NanoSIMS is a powerful analytical tool for investigating C and N metabolism in the cnidarian-
dinoflagellate symbiosis. Recently, studies have successfully used the technique to visualize
intracellular sites of C and N assimilation within symbiotic tissues using incubations in 13C and
15N isotope solutions (Clode et al. 2007; Pernice et al. 2012, 2014, Kopp et al. 2013, 2015). Chemical fixation techniques are suitable for investigations involving elements bound to
macromolecules (Grovenor et al. 2006), such as photosynthetically fixed carbon and
assimilated nitrogen. However, fixation chemicals and resin may introduce C and N with
natural isotope ratios that may slightly dilute the 13C and 15N tracers (Stadermann et al. 2005,
Musat et al. 2014), which could have contributed to the almost undetectable enrichment values 124 obtained for 15N in the autotrophy experiment and 13C in the heterotrophy experiment. Freeze-
substitution methods could be used in future investigations to avoid dilution of the tracers by
fixation chemicals, and to track any diffusible ions in the symbiosis since this method prevents
the redistribution of such elements within the cell (Grovenor et al. 2006, Feng et al. 2015). The results presented here are the first to use nanoSIMS to investigate metabolite fluxes during
symbiosis establishment, as well as the influence of symbiont genotype on these fluxes. Additionally, this study is one of the first to assess the translocation of heterotrophically-
derived compounds from host to symbiont. The results demonstrate how Symbiodinium D1a
acts as a less mutually beneficial symbiont for Aiptasia when compared with the homologous
Symbiodinium B1 under normal temperatures, during the early and late stages of symbiosis
establishment. Overall, Symbiodinium D1a was less effective at fixing and hence translocating
carbon (at the population level) to the host during both early and late stages of symbiosis
establishment. These interactions occur alongside a less successful colonisation of the host,
raising the possibility that infection success and metabolic exchange are linked in determining
symbiont colonisation success. However, more information would be needed to determine if
the nutritional patterns seen are cause or consequence of host-symbiont compatibility. Clade D symbionts can cause reduced coral holobiont fitness by impairing growth rates and
reproduction (Little et al. 2004; Jones & Berkelmans 2010, 2011; Lesser et al. 2013). The
results from my experiment support the previous findings of clade D symbionts being less
efficient at certain cellular processes. 5.1 Summary The exchange of carbon and nitrogen is a central feature driving the persistence of the
cnidarian-dinoflagellate symbiosis (Muscatine & Porter 1977; Tremblay et al. 2012; Pernice et
al. 2014). Therefore, the aim of this thesis was to characterize the cellular mechanisms essential
for inter-partner nutritional exchange in this symbiosis, and to determine the effects of hosting
a thermally-tolerant, but putatively less mutually beneficial symbiont type. This information is
necessary to assess the possibility of symbiont switching as a means of adapting to climate
change, since the successful establishment of a symbiosis may be linked to the nutritional
potential between host-symbiont pairs. First, bioinformatics was used to elucidate, for the first
time, protein sequences of membrane transporters likely to act in the exchange of metabolites
between host and symbiont (Chapter 2). These sequences consisted primarily of glucose,
glycerol, nitrogen, and ammonium transporters, and were found to be conserved among a
diversity of species, while patterns between different Symbiodinium clades were also evident. Next, proteomic and isotopic-labelling experiments were conducted to examine the effects of
hosting the thermally tolerant symbiont type, Symbiodinium D1a, on cellular processes within
a symbiotic association (Chapter 3 and 4). My findings corroborated the importance of carbon
and nitrogen translocation to the successful establishment and persistence of the symbiosis, and
showed that Symbiodinium D1a was a less successful partner than the normal symbiont type
(Symbiodinium B1). The results presented here demonstrate that, under non-stressful
environmental conditions, Symbiodinium D1a provides less benefits to the host when engaged
in a heterologous association with the model cnidarian Aiptasia than that of a homologous
association. These findings raise the following important questions: How has the integration of
cellular processes allowed for adaptation to a symbiotic life-style? Does the degree of
compatibility between host and symbiont cell processes determine where partnerships fall on
the mutualism – parasitism continuum? And, what are the implications of thermally-tolerant
heterologous symbionts for the adaptability of coral reefs to climate change? These questions
will be considered further here, with reference to Fig. 5.1, summarising the important cellular
interactions and how they are affected by the different symbiotic associations. The exchange of carbon and nitrogen is a central feature driving the persistence of the
cnidarian-dinoflagellate symbiosis (Muscatine & Porter 1977; Tremblay et al. 2012; Pernice et
al. 2014). 4.4.4 Conclusions While over half of the D1a symbionts neglected to
provide any fixed carbon to the host in the latter stages of host colonisation, all were receiving
nitrogen from the host. Parasitism is defined by one partner living inside another, obtaining
beneficial nutrients while the host gains no benefit from the relationship (Park & Allaby 2013). The D1a symbiosis does continue to provide carbon to the host, albeit at lesser quantities, and
therefore is merely a sub-optimal symbiont rather than truly parasitic. These findings reinforce
the conclusions from Chapter 3, that a symbiosis between Aiptasia and Symbiodinium D1a may
not be sustainable long-term under normal environmental conditions. Aiptasia represents a
model organism for the cnidarian-dinoflagellate system (Weis 2008; Lehnert et al. 2012;
Wolfowicz et al. 2016), and interactions between other cnidarian species and Symbiodinium
D1a could well experience similar inadequacy in metabolic exchange. Clade D symbionts can cause reduced coral holobiont fitness by impairing growth rates and
reproduction (Little et al. 2004; Jones & Berkelmans 2010, 2011; Lesser et al. 2013). The
results from my experiment support the previous findings of clade D symbionts being less
efficient at certain cellular processes. While over half of the D1a symbionts neglected to
provide any fixed carbon to the host in the latter stages of host colonisation, all were receiving
nitrogen from the host. Parasitism is defined by one partner living inside another, obtaining
beneficial nutrients while the host gains no benefit from the relationship (Park & Allaby 2013). The D1a symbiosis does continue to provide carbon to the host, albeit at lesser quantities, and
therefore is merely a sub-optimal symbiont rather than truly parasitic. These findings reinforce
the conclusions from Chapter 3, that a symbiosis between Aiptasia and Symbiodinium D1a may
not be sustainable long-term under normal environmental conditions. Aiptasia represents a
model organism for the cnidarian-dinoflagellate system (Weis 2008; Lehnert et al. 2012;
Wolfowicz et al. 2016), and interactions between other cnidarian species and Symbiodinium
D1a could well experience similar inadequacy in metabolic exchange. 125 126 5.1 Summary Therefore, the aim of this thesis was to characterize the cellular mechanisms essential
for inter-partner nutritional exchange in this symbiosis, and to determine the effects of hosting
a thermally-tolerant, but putatively less mutually beneficial symbiont type. This information is
necessary to assess the possibility of symbiont switching as a means of adapting to climate
change, since the successful establishment of a symbiosis may be linked to the nutritional
potential between host-symbiont pairs. First, bioinformatics was used to elucidate, for the first
time, protein sequences of membrane transporters likely to act in the exchange of metabolites
between host and symbiont (Chapter 2). These sequences consisted primarily of glucose,
glycerol, nitrogen, and ammonium transporters, and were found to be conserved among a
diversity of species, while patterns between different Symbiodinium clades were also evident. Next, proteomic and isotopic-labelling experiments were conducted to examine the effects of
hosting the thermally tolerant symbiont type, Symbiodinium D1a, on cellular processes within
a symbiotic association (Chapter 3 and 4). My findings corroborated the importance of carbon
and nitrogen translocation to the successful establishment and persistence of the symbiosis, and
showed that Symbiodinium D1a was a less successful partner than the normal symbiont type
(Symbiodinium B1). The results presented here demonstrate that, under non-stressful
environmental conditions, Symbiodinium D1a provides less benefits to the host when engaged
in a heterologous association with the model cnidarian Aiptasia than that of a homologous
association. These findings raise the following important questions: How has the integration of
cellular processes allowed for adaptation to a symbiotic life-style? Does the degree of
compatibility between host and symbiont cell processes determine where partnerships fall on
the mutualism – parasitism continuum? And, what are the implications of thermally-tolerant
heterologous symbionts for the adaptability of coral reefs to climate change? These questions
will be considered further here, with reference to Fig. 5.1, summarising the important cellular
interactions and how they are affected by the different symbiotic associations. 127 5.2.1 How has the integration of cellular processes allowed for adaptation to a symbiotic
life-style? 5.2.1 How has the integration of cellular processes allowed for adaptation to a symbiotic
life-style? The establishment of symbiotic associations can cause co-evolution of the partners as they
adapt to conditions experienced while in symbiosis (Kawaida et al. 2013; Guerrero et al. 2013). Symbiotic corals first evolved within the mid-to-late Triassic period (225-199 mya; Stanley Jr.,
1981), allowing for a long history of natural selection on the partners as a cohesive unit. Thus,
selection pressures likely drove the integration of host and symbiont cellular processes seen
within modern cnidarian-dinoflagellate relationships. Most remarkable is the integration of
carbon metabolism (Fig. 5.1). Uptake of DIC is achieved through a carbon concentrating
mechanism (CCM) involving host carbonic anhydrase (CA), that converts seawater carbon to
a form that can be utilized in host calcification and symbiont photosynthesis (Furla et al. 2000). This integrated mechanism likely evolved to satiate both partners’ carbon demands using just
one process, but it is unable to function efficiently when the partners are separated (Chapter
3). The downregulation of important CCM proteins in aposymbiotic individuals is due to their
location in the symbiosome (Barott et al. 2015; Oakley et al. 2016). The membrane protein
VHA (vacuolar H+ -ATPase) pumps H+ into the symbiosome where it reacts with carbonic
anhydrase in the conversion of HCO3- to CO2, that is then used for symbiont photosynthesis
and translocation of the resulting sugars to the host (Barott et al. 2015). The primary sugar
translocated to the host is glucose, while glycerol is released by the dinoflagellates largely as a
stress response (Burriesci et al. 2012; Suescun-Bolivar et al. 2016). The mechanism of glucose
transport to the host is likely through GLUT transporters in the symbiosome membrane
(Chapter 2), with GLUT8 suggested as a likely candidate for the marking of isolated
symbiosome vesicles (Ling et al. 2016). Overall, the integration of carbon metabolism between
host and symbiont enhances the nutritional potential of each partner and provides benefits that
neither would achieve on their own. Inositol has not been highly studied in the symbiosis, but its roles in host-symbiont pathways
are now becoming clearer. The phosphatidylinositol signalling system (PtdIns) and the inositol
phosphate metabolism pathway (InsP) are common to at least four different Symbiodinium 128 clades, and may to be critical to the foundation of the symbiosis (Rosic et al. 2014). 5.2.1 How has the integration of cellular processes allowed for adaptation to a symbiotic
life-style? Myo-
inositol is a central reactant to both of these pathways, which produce metabolites used in signal
transduction, such as vesicular trafficking, secretion, actin assembly, and nuclear messaging
(Anderson et al. 1999; Abel et al. 2001). Cnidarians likely transport myo-inositol across the
symbiosome membrane (Chapter 2), so that symbionts can use it as a critical building block
in these pathways. In a similarly integrated association, the legume-rhizobia symbiosis,
signalling between host and symbiont is mediated via phosphatidylinositide-regulated
endocytosis (Peleg-Grossman et al. 2007). Therefore, inositol in the cnidarian-dinoflagellate
symbiosis may can be considered a putative means of communication between host and
symbiont. Along these lines are potential glycosylphosphatidylinositol (GPI) anchors on the
symbiont cell surface that may be important for recognition by cnidarian hosts (Davy et al. 2012). More research should be conducted on the role of inositol in host-symbiont interactions,
as it could be a multifaceted molecule important to the establishment and persistence of
compatible associations. The exchange of ions across the symbiotic interface is another important feature of host-
symbiont integration. Ion transport is important to the symbiosis as it regulates pH of the
symbiosome space (Gibbin et al. 2014; Barott et al. 2015) and drives the transport of myo-
inositol, as well as other molecules, through membrane co-transporters (Pao et al. 1998). The
symbiotic state is necessary for adequate expression of sodium/calcium antiporters and other
transmembrane ion regulators, and possibly upregulates the production of free fatty acids
(Chapter 3). Fatty acids are other important products coupled with ion transport from host to
symbiont (Imbs et al. 2014), likely though either SMCT or OCTN proteins in the symbiosome
membrane (Chapter 2). Integrated host-symbiont nitrogen metabolism is a further major adaptation to a symbiotic life-
style, as symbionts are provided with host-derived nitrogen from heterotrophic feeding
(Chapter 4). Ammonium is the primary nitrogen source for symbiotic corals, as it can be
assimilated by both the host and symbiont (Pernice et al. 2012; Kopp et al. 2013). Nitrate and
urea dissolved in the seawater are also nitrogen products that can be important nutrients for
symbiotic corals, with urea being preferred over nitrate (Grover et al. 2003, 2006). Other than
water and glycerol, aquaporin channel proteins can also likely transport urea, purines, and 129 pyrimidines through the symbiosome (Chapter 2). Aquaporins are protein channels in both
cnidarian and Symbiodinium cell membranes (Fig. 5.2.1 How has the integration of cellular processes allowed for adaptation to a symbiotic
life-style? 5.1), so these solutes would be able to flow
freely between the partners (Lin et al. 2015). When nitrogen availability is high, symbionts
store the nutrient as uric acid crystals to use as reserves for when conditions are nitrogen limited
(Clode et al. 2007; Kopp et al. 2013). This may account for increased translocation of host
nitrogen to symbionts during early stages of symbiosis when nitrogen is freely available, so
that they can rely on these nitrogen stores in later phases of symbiosis when high symbiont
densities cause nitrogen limitation (Chapter 4). Formation of these uric acid crystals is also
important to the host, since the crystals contain purine rings that may be used for host purine
salvaging, facilitated by host nucleoside hydrolase (NH) and aquaporin channels in the
symbiosome (Chapter 2 & 3). One apparent adaptation to a shared nitrogen metabolism is the upregulation of AMT proteins
seen in Symbiodinium. A previous genomic study found an expansion of ammonium transporter
genes in the dinoflagellates that showed species-specific patterns between Symbiodinium
minutum, Symbiodinium kawagutii, and Symbiodinium microadriaticum genomes (Aranda et
al. 2016). However, examination of Symbiodinium transcripts was better able to show how
adaptation to integrated nitrogen metabolism may differ between clades, as more
transcriptomes are publicly available (Chapter 2). The most ancestral clade, Symbiodinium A
may have retained some of its reliance on seawater nitrate over host ammonium, while the
more derived clades have lost the need to transcribe a nitrate transporter repertoire due to an
increasing reliance on host-derived ammonium (Chapter 2). Interestingly, the more typically
free-living Symbiodinium clade F (Symbiodinium kawagutii) transcribed few ammonium and
nitrate transporting proteins, despite having the genes for them (Lin et al. 2015). Symbiodinium
C expressed the greatest diversity of ammonium uptake proteins, which is likely an adaptation
to its qualities as a host-generalist and ability to thrive in a diversity of environments
(LaJeunesse 2005; Lin et al. 2015). Mechanisms of ammonium transport in the symbiosis further illustrate the congruent
evolutionary paths leading to host-symbiont integration. Both Symbiodinium and cnidarians
possess ammonium channel proteins (Chapter 2), and their expression in the host is
downregulated when not engaged in symbiosis (Lehnert et al. 2014; Oakley et al. 2016). 130 Therefore, these channels likely work in tandem to allow the flow of ammonium molecules
between partners. 5.2.1 How has the integration of cellular processes allowed for adaptation to a symbiotic
life-style? Co-evolution of the host and symbiont ammonium channels is evident by the
amino acid conservation seen between Aiptasia AMT sequences and Symbiodinium AMT
sequences from clades A, B, C that cluster together in phylogenetic analysis (Chapter 2). Interestingly, Aiptasia is able to form associations with symbionts from all of these clades
(Chen et al. 2016; Wolfowicz et al. 2016), demonstrating that evolutionary adaptations may be
key to successful associations. Additionally, the protein sequence for Symbiodinium adenylate
cyclase is closely related to a metazoan-like clade of Symbiodinium ammonium transporters. Although this result initially seemed inexplicable, it may also be related to the co-evolution of
cnidarian and Symbiodinium proteins, since G-proteins induced by the symbiosis are known to
stimulate adenylate cyclase (Chapter 3). Therefore, these cnidarian G-proteins could be
stimulating Symbiodinium adenylate cyclase as part of a signalling pathway. The integration of cell-signalling pathways is responsible for successful recognition and
regulation of symbiont populations. Apoptosis and autophagy pathways are one of the primary
ways that hosts can regulate symbiont proliferation and remove damaged or unwanted
symbionts (Dunn et al. 2007b; Dunn & Weis 2009). Host proteins expressed at the symbiotic
interface are likely to help facilitate this pathway by transporting apoptosis-inducing products
into the symbiosome space (Chapter 2). The presence of a properly integrated symbiosome is
critical to the host’s abilities to remove unwanted symbiont cells, as apoptosis pathways are
highly downregulated without the presence of a symbiotic relationship (Chapter 3). Animal cell adhesion proteins evolved new roles under the selection pressures of symbiosis,
by gaining the ability to mediate cell-to-cell interactions in the symbiosome membrane. The
Sym32 protein is usually expressed on the apical edges of cnidarian gastrodermal cells, but
becomes part of the symbiosome upon establishment of symbiosis (Reynolds et al. 2000;
Schwarz & Weis 2003). Additional cell adhesion proteins that may act similarly are the
cnidarian AQP0 protein (Chapter 2), and the glycoproteins hemicentin and fibronectin
(Chapter 3). Glycoproteins on the Symbiodinium cell surface are known to act in symbiont
recognition by the host (Lin et al. 2000; Bay et al. 2011). This recognition process is executed
by the glycoproteins binding to lectins on the foreign symbiont cell’s outer surface,
consequently activating phagocytosis of the cell (McGuinness et al. 2003). Glycan-lectin 131 signalling ultimately determines whether cells are accepted as symbionts or rejected (Koike et
al. 2004; Wood-Charlson et al. 2006; Logan et al. 2010; Bay et al. 2011). 5.2.1 How has the integration of cellular processes allowed for adaptation to a symbiotic
life-style? The presence of
these symbiosis-enhanced host proteins could indicate that Symbiodinium cell surface
glycoproteins are incorporated into the symbiosome membrane after phagocytosis. The cnidarian animal seems to have evolved a dependence on Symbiodinium metabolites to
maintain its overall health and physiology. The symbionts trigger more efficient production of
nematocyst toxins, which allow the animal to more efficiently defend itself against predators
and capture food (Chapter 3). Additionally, the symbionts promote muscle fibre assembly
(Chapter 3), while the absence of symbionts can cause toxic reactants to build up in host
tissues when the solutes needed to complete the reaction pathway are usually provided by the
symbiont (Chapter 3). Corals must adapt to elevated seawater temperatures in order to survive the imminent effects
of climate change. Many think that this will happen according to the Adaptive Bleaching
Hypothesis (ABH), with more thermally-tolerant symbionts becoming dominant in corals
(Buddemeier & Fautin 1993; Fautin & Buddemeier 2004; Berkelmans & van Oppen 2006). However, the functional diversity among Symbiodinium types poses the question of whether
the more tolerant symbionts will be wholly compatible with all host species on a cellular level. While integration of host and symbiont cells must occur for a relationship to be established,
my results suggest that there are varying degrees of cellular compatibility and that these are
linked to where the association falls on a scale from mutualism – parasitism – complete
dysfunction (Fig. 5.1). A previously described continuum for symbiotic corals categorized Symbiodinium from clades
A and D as being more towards the parasitism end of the scale, due to the reduced fitness they
impart on hosts under normal environmental conditions (Stat et al. 2008; Lesser et al. 2013). This is problematic since these two clades are also the most tolerant to environmental stress 132 (Loram et al. 2007; Yuyama et al. 2016; Silverstein et al. 2017). Symbiodinium from clade A
is likely to be the most parasitic since it is the most ancient, and therefore it may be less
physiologically integrated with the host than are more derived clades of Symbiodinium
(Chapter 2). This lower compatibility has been highlighted by background populations of
Symbiodinium A, that exhibited lower rates of carbon fixation and translocation than did the
dominant symbionts from clade C Symbiodinium (Stat et al. 2008). 5.2.1 How has the integration of cellular processes allowed for adaptation to a symbiotic
life-style? In homologous infections,
clade D Symbiodinium has also been observed as less efficient at nutrient assimilation and
translocation than clade C counterparts (Baker et al. 2013; Pernice et al. 2014). Consistent with
these previous observations, in the current study more than half of the symbiont cells from
Symbiodinium clade D did not photosynthesize at all in Aiptasia, but were still able to receive
host-derived nitrogen (Chapter 4). This demonstrates how less-than-optimal host-symbiont
nitrogen and carbon pathways are characteristic of symbioses involving clade D Symbiodinium. This could be related to incompatible cellular aspects such as host release factors (HRFs). Symbiodinium types incubated with HRFs from the same host species fix and release different
quantities of photosynthetic carbon (Stat et al. 2008). HRFs can consist of different compounds
such as amino acids (Gates et al. 1995, 1999; Wang & Douglas 1997; Stat et al. 2008), but they
differ greatly in their symbiont-specificity (Sutton & Hoegh-Guldberg 1990). However, it
should be recognised that HRFs are now widely regarded as artefact of experiments with
isolated symbionts and homogenized host tissues (Davy et al. 2012). The reduced ability of
Symbiodinium D cells to fix carbon could also be related to incompatibilities within the carbon
concentrating mechanism, which would ultimately provide less carbon for photosynthesis (Fig. 5.1). 133 134 134 Figure 5.1 Conceptual diagram of the cellular processes during varying degrees of symbiont compatibility. Purple =
proteins; pink = charged molecules; blue = transporters; orange = organic compounds. Molecules and processes
downregulated in comparison to the homologous association are filled in red; upregulated molecules are outlined in
bold. In a homologous association, H+ ions are pumped through the symbiosomes by vATPase (1), where they react
with carbonic anhydrase (CA) to convert bicarbonate ions to CO2 to be used in symbiont photosynthesis. Photosynthesis
(Photo) produces sugars, mainly glucose, that are translocated to the host through GLUT proteins (2). Glycerol is
released by the symbiont and potentially translocated to the host through AQP proteins (3). Myo-inositol from the host
is transported to the symbiont through HMIT and SMIT proteins (4) where it acts in the symbiont’s phosphatidylinositol
signalling system (PtdIns) and inositol phosphate metabolism (InsP). Nitrogen is taken up by the symbiont from the host
in the form of ammonium by AMT transporters (5) in the symbiosome and symbiont cell membrane, and is also taken
up from the seawater in the form of nitrate, through NRT2 transporters (6), and urea, through AQP proteins (3). The
nitrogen compounds are used for amino acid assimilation or stored as uric acid crystals (UA). Symbiont betaine (Bet)
and homocysteine (Hcy) are translocated to the host to be used in the methionine pathway (Meth), catalysed by BHMT. Selenium binding proteins (SBP) act in protein transport (Prot) within the Golgi apparatus. GPI-anchors and
glycoproteins (Glyco) on the symbiont cell surface, as well as NPC2, the circumsporozoite protein (CS), and the cell
adhesion protein Sym32 all act in recognition processes. In a heterologous association, CA is downregulated, which further impairs photosynthesis and reduces translocation of
sugars to the host, and NPC2 and CS are downregulated. BHMT is upregulated, indicating dysfunction of methionine
metabolism, and selenium binding proteins are downregulated indicating dysfunction in protein transport of the Golgi. In aposymbiotic anemones, ETHE1 is upregulated indicating dysfunction in the mitochondrial (Mit) oxidation pathway,
and succinyl coA is downregulated indicating dysfunction in the TCA cycle. NPC2 and CS are downregulated, as are
vesicle transport to the Golgi and inter-Golgi protein transport. In aposymbiotic anemones, ETHE1 is upregulated indicating dysfunction in the mitochondrial (Mit) oxidation pathway,
and succinyl coA is downregulated indicating dysfunction in the TCA cycle. NPC2 and CS are downregulated, as are
vesicle transport to the Golgi and inter-Golgi protein transport. Regulation of symbiont cell proliferation is a prime feature of mutualistic associations, used to
ensure that nutritional resources do not become unevenly allocated to the symbionts (Muscatine
& Pool 1979; Detournay & Weis 2011). However, heterologous clade D Symbiodinium can
suppress many of the constituents of these pathways in the host, meaning that cell proliferation
is not able to be adequately regulated (Chapter 3). This could provide an opportunity for the
symbionts to have uncontrolled growth that may deprive the host of nutrients. This was
potentially the case in another infection experiment, where heterologous symbionts were able
to reach population densities much higher than the homologous symbionts (Starzak et al. 2014). Clade D Symbiodinium also induces the breakdown of molecules involved in the animal’s
immune response, possibly making it more susceptible to diseases (Chapter 3). This may have
been the case for Symbiodinium A, which was found to have a significant association with
diseased or abnormal corals over healthy corals (Stat et al. 2008). Additionally, Symbiodinium
D impairs protein and DNA synthesis, which could be a side-effect of elevated pyrimidine
breakdown (Chapter 3). Overall, clade D Symbiodinium has many negative effects on host
cellular processes despite its ability to form an association. In Aiptasia - Symbiodinium D associations, central enzymes of the nitrogen assimilation
pathway in the host were downregulated (Chapter 3), yet nitrogen from heterotrophy was still 135 easily incorporated and provided to the symbiont (Chapter 4). This indicates that the
symbionts may impair the host’s ability to assimilate dissolved ammonium in the seawater, but
not their ability to utilize nutrients from feeding. The most notable incompatibility is within
the methionine assimilation pathway, which relies on solutes from each partner for proper
functioning (Nakamura et al. 1998; Wang & Douglas 1998). Dysfunction of the pathway is
apparent in both aposymbiotic anemones and in Symbiodinium D-colonised anemones by
accumulation of the intermediate, BHMT, which is generally consumed in the functioning
reaction (Chapter 3). The decreased nutritional potential for Symbiodinium D associations may
also be due to missing putative recognition molecules from the onset of symbiosis, such as the
NPC2 and CS proteins (Fig. 5.1), which could be important for the development of successful
mutualistic relationships (Chapter 3). Overall, the degree of cellular compatibility between
host and symbiont can reveal the type of symbiotic relationship being observed, which
ultimately determines symbiont success. 5.3 Implications for coral adaptability via thermally tolerant symbionts Opportunistic, thermally tolerant symbiont types are less beneficial, and sometimes
harmful, to cnidarian hosts at non-stressful temperatures (Little et al. 2004; Jones &
Berkelmans 2011; Pernice et al. 2014), especially when the host is not well-adapted to their
presence (Chapters 3 & 4). However, when in symbiosis with their native host species, these
symbiont types are able to retain high nutritional potential under the conditions provided by a
warming climate (Jones & Berkelmans 2010; Baker et al. 2013; Silverstein et al. 2017). Therefore, symbiont shuffling to thermally tolerant Symbiodinium types is a viable option for
corals already harbouring one of these types in background populations. The caveat to this
adaptation process would be reduced growth of the corals as temperatures fluctuate between
stressful and tolerable across seasons (Brown et al. 1999; Warner et al. 2002). However, as
ocean temperatures warm, to be consistently near the current tolerance limits of corals,
associations with homologous Symbiodinium A and D will become more globally dominant to
improve coral survival (Howells et al. 2016; Sampayo et al. 2016). Unfortunately, coral species
that have not previously adapted to these symbiont types are unlikely to survive due to the
negative effects that will be induced by the symbionts under fluctuating temperatures (Chapter
3 & 4). Seawater temperatures are rising at an alarming rate, much faster than the 136 environmental pressures that have shaped host-symbiont specificities (Hoegh-Guldberg et al. 2007; Heron et al. 2016). Therefore, novel associations with heterologous symbiont types will
likely not have enough time to evolve compatible cellular mechanisms (Chapter 2). This scenario would cause an overall decrease in genetic diversity on coral reefs, as only coral
species with previously existing populations of thermally-tolerant symbionts would survive. Moreover, there would be a shift in dominance to mostly Symbiodinium A and D. Genetic
diversity in plant and animal populations allows for population growth and resistance to disease
(Williams 2001; Tarpy 2002; Hughes & Boomsa 2004). A lesser resistance to disease in coral
populations may be further amplified by the ability of some thermally-tolerant symbiont types
to suppress host immune processes (Chapter 3). Coral diseases such as black band disease and
white syndrome are accelerated under thermal stress, further complicating the situation (Jones
et al. 2004; Bruno et al. 2007). 5.3 Implications for coral adaptability via thermally tolerant symbionts Ultimately, if the rate of ocean warming was reduced, then
corals might have a better chance to adapt to changing conditions, via the ABH or by
developing compatible symbioses with novel symbiont types, and so avoid a reduction in
genetic diversity and coral cover. 5.4 Limitations and future work However, methods for analysis of
the symbiont fraction have yet to be developed, and could be vital to future projects aimed at
understanding the cellular changes undergone by symbionts integrating into host cells. Many
of the proteins I discovered seemed to be symbiosome membrane proteins (Chapter 2 & 3). Immunolabelling would be useful to further investigate the location of these proteins and
confirm whether they are components of the symbiotic interface, as this technique has
previously been successful in localizing NPC2 and vATPase to the symbiosome (Dani et al. 2014; Barott et al. 2015). While nanoSIMS has provided novel imaging of the nutrient
exchange between cnidarians and their symbiotic dinoflagellates, the instrument was not able
to obtain many values for carbon enrichment in host tissues. I was unable to tell if these low
values indicate the absence of translocation or if the levels were beyond the sensitivity
threshold of the instrument. Future studies should couple nanoSIMS translocation experiments
with bulk isotope-ratio mass spectrometry (IRMS) measurements to offset the potential
problems of instrument sensitivity (Tanaka et al. 2006; Popa et al. 2007; Grover et al. 2008). Establishing a baseline is important before attempting to observe the impacts of temperature
manipulations on a system. Due to this, and the complexity of experimental analyses, all of my
experiments were conducted under normal seawater conditions (Chapters 3 & 4). However,
the functioning of thermally-tolerant symbiont types changes with temperature (Baker et al. 2013; Silverstein et al. 2017). Therefore, further information on the functionality of
heterologous Symbiodinium D1a as a suboptimal symbiont would benefit from repeating these
experiments at various elevated temperatures. 5.4 Limitations and future work While data mining is an extremely useful tool for bioinformatic analyses of the symbiosis,
investigations are limited to those of cnidarian species and Symbiodinium types with
transcriptomes and genomes that have already been reliably sequenced (Chapter 2). While
new resources are being released frequently, bioinformatic analysis will also have to take into
consideration that the organisms were sampled under different conditions. Therefore, future
work examining protein sequences would benefit from a comparative transcriptomics approach
of hosts infected with different symbiont types under controlled conditions. This method has
been used to show genetic differences between symbiotic and aposymbiotic individuals, and
has identified some transmembrane sequences (Lehnert et al. 2014; Aranda et al. 2016). This
method could help elucidate whether integration of transporter proteins in the symbiosome is
successful between different symbiotic associations, and whether this led to some of the
patterns observed in my research. 137 Proteomic analysis using LC-MS/MS has allowed the discovery of host proteins important to
the symbiotic state (Peng et al. 2010; Oakley et al. 2016). However, methods for analysis of
the symbiont fraction have yet to be developed, and could be vital to future projects aimed at
understanding the cellular changes undergone by symbionts integrating into host cells. Many
of the proteins I discovered seemed to be symbiosome membrane proteins (Chapter 2 & 3). Immunolabelling would be useful to further investigate the location of these proteins and
confirm whether they are components of the symbiotic interface, as this technique has
previously been successful in localizing NPC2 and vATPase to the symbiosome (Dani et al. 2014; Barott et al. 2015). While nanoSIMS has provided novel imaging of the nutrient
exchange between cnidarians and their symbiotic dinoflagellates, the instrument was not able
to obtain many values for carbon enrichment in host tissues. I was unable to tell if these low
values indicate the absence of translocation or if the levels were beyond the sensitivity
threshold of the instrument. Future studies should couple nanoSIMS translocation experiments
with bulk isotope-ratio mass spectrometry (IRMS) measurements to offset the potential
problems of instrument sensitivity (Tanaka et al. 2006; Popa et al. 2007; Grover et al. 2008). Proteomic analysis using LC-MS/MS has allowed the discovery of host proteins important to
the symbiotic state (Peng et al. 2010; Oakley et al. 2016). 5.5 Conclusions Overall, my work has shown that the exchange of carbon and nitrogen products is a process
shaped by the compatibility of host and symbiont, and that this partly determines the success
of a symbiotic relationship. My findings further the work of Oakley et al. (2016), who
identified proteins that were upregulated in the symbiotic versus aposymbiotic state, by 138 differentiating proteins upregulated by mutualistic versus parasitic, or less beneficial,
symbiosis. My findings have also furthered the work of Pernice et al. (2014), by observing the
fluxes of carbon and nitrogen in symbiotic tissues during the earlier phases of symbiosis
establishment, and observing the effect of heterologous clade D Symbiodinium on these fluxes
compared to the homologous Symbiodinium type. An understanding of the mechanisms driving
host-symbiont specificity are important for determining how corals may adapt to climate
change (LaJeunesse et al. 2010; Stat & Gates 2011; Davy et al. 2012; Silverstein et al. 2012),
especially if the only hope for coral survival is symbiont community change to a thermally
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Figure A1 Motifs used for manual editing of the GLUT alignment. Partial alignment of the Homo sapiens
GLUT sequences, showing conserved domains with functional motifs used to screen identified protein
sequences, outlined in red shaded boxes. Included are residues 170–180 with the GR motif, residues 420–430
with the GRR motif, and residues 640–650 with the PETK motif. Appendix A: Protein sequences and genetic resources Appendix A: Protein sequences and genetic resources Figure A1 Motifs used for manual editing of the GLUT alignment. Partial alignment of the Homo sapiens
GLUT sequences, showing conserved domains with functional motifs used to screen identified protein
sequences, outlined in red shaded boxes. Included are residues 170–180 with the GR motif, residues 420–430
with the GRR motif, and residues 640–650 with the PETK motif. Figure A2 Motifs used for manual editing of the SGLT alignment. Partial alignment of the Homo sapiens SGLT
sequences, showing structural motifs used to screen identified protein sequences, outlined in red shaded boxes. Included are residues 129–170 with the Y, GG, and D motifs. Figure A2 Motifs used for manual editing of the SGLT alignment. Partial alignment of the Homo sapiens SGLT
sequences, showing structural motifs used to screen identified protein sequences, outlined in red shaded boxes. Included are residues 129–170 with the Y, GG, and D motifs. 177 Figure A3 Motifs used for manual editing of the AQP alignment. Partial alignment of the Homo sapiens
aquaglyceroporin sequences, showing functional and structural motifs used to screen identified protein
sequences, outlined in red shaded boxes. Included are residues 90–100 with the first NPA motif, and resides
530–540 with the second NPA motif and the D residue. Figure A4 Motifs used for manual editing of the AMT alignment. Partial alignment of the Arabidopsis thaliana
AMT sequences, showing functional and structural motifs used to screen identified protein sequences, outlined
in red shaded boxes. Included are residues 210–238 with the D, AG, VH, and G motifs responsible for
ammonium recognition and transport function. Figure A5 Motifs used for manual editing of the NRT alignment. Partial alignment of the Arabidopsis thaliana
NRT2 sequences, showing functional and structural motifs used to screen identified protein sequences, outlined
in red shaded boxes. Included are residues 100–110 with a conserved F residue, 333–342 with a proposed NNP
signature A/G-G-W-G/A-N/D-M/L-G, and 680–690 and 949–960 with structurally important conserved glycine
residues GG and GAGG, respectively. Figure A3 Motifs used for manual editing of the AQP alignment. Partial alignment of the Homo sapiens
aquaglyceroporin sequences, showing functional and structural motifs used to screen identified protein
sequences, outlined in red shaded boxes. Included are residues 90–100 with the first NPA motif, and resides
530–540 with the second NPA motif and the D residue. Figure A3 Motifs used for manual editing of the AQP alignment. Appendix A: Protein sequences and genetic resources Partial alignment of the Homo sapiens
aquaglyceroporin sequences, showing functional and structural motifs used to screen identified protein
sequences, outlined in red shaded boxes. Included are residues 90–100 with the first NPA motif, and resides
530–540 with the second NPA motif and the D residue. Figure A3 Motifs used for manual editing of the AQP alignment. Partial alignment of the Homo sapiens
aquaglyceroporin sequences, showing functional and structural motifs used to screen identified protein
sequences, outlined in red shaded boxes. Included are residues 90–100 with the first NPA motif, and resides
530–540 with the second NPA motif and the D residue. Figure A4 Motifs used for manual editing of the AMT alignment. Partial alignment of the Arabidopsis thaliana
AMT sequences, showing functional and structural motifs used to screen identified protein sequences, outlined
in red shaded boxes. Included are residues 210–238 with the D, AG, VH, and G motifs responsible for
ammonium recognition and transport function. Figure A4 Motifs used for manual editing of the AMT alignment. Partial alignment of the Arabidopsis thaliana
AMT sequences, showing functional and structural motifs used to screen identified protein sequences, outlined
in red shaded boxes. Included are residues 210–238 with the D, AG, VH, and G motifs responsible for
ammonium recognition and transport function. Figure A5 Motifs used for manual editing of the NRT alignment. Partial alignment of the Arabidopsis thaliana
NRT2 sequences, showing functional and structural motifs used to screen identified protein sequences, outlined
in red shaded boxes. Included are residues 100–110 with a conserved F residue, 333–342 with a proposed NNP
signature A/G-G-W-G/A-N/D-M/L-G, and 680–690 and 949–960 with structurally important conserved glycine
residues GG and GAGG, respectively. Figure A4 Motifs used for manual editing of the AMT alignment. Partial alignment of the Arabidopsis thaliana
AMT sequences, showing functional and structural motifs used to screen identified protein sequences, outlined
in red shaded boxes. Included are residues 210–238 with the D, AG, VH, and G motifs responsible for
ammonium recognition and transport function. Figure A5 Motifs used for manual editing of the NRT alignment. Partial alignment of the Arabidopsis thaliana
NRT2 sequences, showing functional and structural motifs used to screen identified protein sequences, outlined
in red shaded boxes. Included are residues 100–110 with a conserved F residue, 333–342 with a proposed NNP
signature A/G-G-W-G/A-N/D-M/L-G, and 680–690 and 949–960 with structurally important conserved glycine
residues GG and GAGG, respectively. Appendix A: Protein sequences and genetic resources Figure A5 Motifs used for manual editing of the NRT alignment. Partial alignment of the Arabidopsis thaliana
NRT2 sequences, showing functional and structural motifs used to screen identified protein sequences, outlined
in red shaded boxes. Included are residues 100–110 with a conserved F residue, 333–342 with a proposed NNP
signature A/G-G-W-G/A-N/D-M/L-G, and 680–690 and 949–960 with structurally important conserved glycine
residues GG and GAGG, respectively. Figure A5 Motifs used for manual editing of the NRT alignment. Partial alignment of the Arabidopsis thaliana
NRT2 sequences, showing functional and structural motifs used to screen identified protein sequences, outlined
in red shaded boxes. Included are residues 100–110 with a conserved F residue, 333–342 with a proposed NNP
signature A/G-G-W-G/A-N/D-M/L-G, and 680–690 and 949–960 with structurally important conserved glycine
residues GG and GAGG, respectively. Figure A5 Motifs used for manual editing of the NRT alignment. Partial alignment of the Arabidopsis thaliana
NRT2 sequences, showing functional and structural motifs used to screen identified protein sequences, outlined
in red shaded boxes. Included are residues 100–110 with a conserved F residue, 333–342 with a proposed NNP
signature A/G-G-W-G/A-N/D-M/L-G, and 680–690 and 949–960 with structurally important conserved glycine
residues GG and GAGG, respectively. 178 Datasheet A1 (on disc) Characterization data for newly identified cnidarian GLUT sequences. (Tab A)
Summary of characteristics for each cnidarian GLUT sequence used in the phylogenetic analysis, with the
sequence submitted to I-TASSER highlighted. (Tab B) Accession numbers for each homolog sequence from
other species used in the GLUT phylogenetic tree. (Tab C) I-TASSER output for structural predictions of the N. vectensis GLUT8 sequence. (Tab D) MemPype results for the newly identified GLUT sequences. Datasheet A2 (on disc) Characterization data for newly identified cnidarian SGLT sequences. (Tab A)
Summary of characteristics for each cnidarian SGLT sequence used in the phylogenetic analysis, with the
sequence submitted to I-TASSER highlighted. (Tab B) Accession numbers for each homolog sequence from
other species used in the SGLT phylogenetic tree. (Tab C) I-TASSER output for structural predictions of the N. vectensis SMIT sequence. (Tab D) MemPype results for the newly identified SGLT sequences. Datasheet A3 (on disc) Characterization data for newly identified cnidarian AQP sequences. (Tab A) Summary
of characteristics for each cnidarian AQP sequence used in the phylogenetic analysis, with the sequence
submitted to I-TASSER highlighted. (Tab B) Accession numbers for each homolog sequence from other species
used in the AQP phylogenetic tree. Appendix A: Protein sequences and genetic resources (Tab C) I-TASSER output for structural predictions of the N. vectensis_4
AQP3 sequence. (Tab D) MemPype results for the newly identified AQP sequences. Datasheet A4 (on disc) Characterization data for newly identified Symbiodinium AMT sequences. (Tab A)
Summary of characteristics for each Symbiodinium AMT sequence used in the phylogenetic analysis, with the
sequence submitted to I-TASSER highlighted. (Tab B) Accession numbers for each homolog sequence from
other species used in the AMT phylogenetic tree. (Tab C) I-TASSER output for structural predictions of the
SymbiodiniumC1_25 sequence. (Tab D) MemPype results for the newly identified AMT sequences. Datasheet A5 (on disc) Characterization data for newly identified Symbiodinium NRT2 sequences. (Tab A)
Summary of characteristics for each Symbiodinium NRT2 sequence used in the phylogenetic analysis, with the
sequence submitted to I-TASSER highlighted. (Tab B) Accession numbers for each homolog sequence from
other species used in the AMT phylogenetic tree. (Tab C) I-TASSER output for structural predictions of the
Symbiodinium A-24 sequence. (Tab D) MemPype results for the newly identified NRT2 sequences. 179 Table A1 List of resources used in custom cnidarian and Symbiodinium BLAST databases, their origin, and publication if available. Species with an asterisk (*) after the
name indicate those that were in symbiosis at the time of sampling. Table A1 List of resources used in custom cnidarian and Symbiodinium BLAST databases, their origin, and publication if available. Species with an asterisk (*) after the
name indicate those that were in symbiosis at the time of sampling. Table A1 List of resources used in custom cnidarian and Symbiodinium BLAST databases, their origin, and publication if available. Species with an asterisk (*) after th
name indicate those that were in symbiosis at the time of sampling. Cnidaria Resources
Species
Common name
Symbiotic? Genome (G) or
Transcriptome
(T)
Source
Publication
Acropora digitifera
Staghorn coral
Yes
G
OIST Marine Genomics Unit
Shinzato et al. 2011
Anthopleura elegantissima
Aggregating
anemone
Yes
T
Weis and Meyer lab
Kitchen et al. 2015
Acropora millepora
Branching stony
coral
Yes
T
Compagen
Brower et al. 1997
Acropora palmata*
Elkhorn coral
Yes
T
Compagen
Polato et al. 2011
Aiptasia pallida
Pale anemone
Yes
T
personal comm. Lehnert et al. 2012
Anemonia viridis*
Snakelocks
anemone
Yes
T
Compagen
Sabourault et al. 2009
Aurelia aurita
Moon jellyfish
No
T
Compagen
Fuchs et al. 2014
Clytia hemisphaerica
Hydrozoan
No
T
Compagen
Lapebie et al. Appendix A: Protein sequences and genetic resources 2014
Edwardsiella lineata
Lined sea
anemone
No
T
EdwardsiellaBase
Stefanik et al. 2014
Fungia scutaria
Mushroom coral
Yes
T
Weis and Meyer lab
Kitchen et al. 2015
Hydra magnapapillata
Hydra
Yes
G
Compagen
Chapman et al. 2010
Hydra vulgaris*
Hydra
Yes
T
Compagen
Pan et al. 2014
Hydractinia
symbiolongicarpus
Hermit crab
hydroid
No
T
Dryad Digital Repository
Plachetzki et al. 2014 180 Madracis auretenra*
Yellow pencil
coral
Yes
T
Meyer lab
http://people.oregonstate.edu/~meyere/d
ata.html
Montastraea cavernosa*
Great star coral
Yes
T
Weis and Meyer lab
Kitchen et al. 2015
Metridium senile
Plumose
anemone
No
G
Compagen
Beagley et al. 1998
Montastrea (Orbicella)
faveolata*
Mountain star
coral
Yes
T
Compagen
Schwarz et al. 2008
Nematostella vectensis
Starlet sea
anemone
No
G
DOE Joint Genome Institute
Putnam et al. 2007
Pocillopora damicornis* Cauliflower coral
Yes
T
PocillaporaBase
Traylor-Knowles et al. 2011
Pseudodiploria strigosa
Symmetrical
brain coral
Yes
T
Meyer lab
http://people.oregonstate.edu/~meyere/d
ata.html
Platygyra carnosus*
Brain coral
Yes
T
PcarnBase
Sun et al. 2013
Platygyra daedalea
Brain coral
Yes
T
Meyer lab
http://people.oregonstate.edu/~meyere/d
ata.html
Tubastraea coccinea *
Orange-cup coral
Yes
T
Meyer lab
Personal comm. Tubastraea micrantha*
Black sun coral
Yes
T
Meyer lab
Personal comm. Polypodium hydriforme
Parasitic hydroid
No
T
European Nucleotide Archive
Shpirer et al. 2014
Porites compressa*
Finger coral
Yes
T
Meyer lab
Personal comm. Porities astreoides*
Mustard hill
coral
Yes
T
Matz lab
Kenkel et al. 2013
Porites evermanni*
Finger coral
Yes
T
Meyer lab
Personal comm. Porites australiensis*
Finger coral
Yes
T
OIST Marine Genomics Unit
Shinzato et al. 2014
Porites lobata*
Lobe coral
Yes
T
Meyer lab
personal comm. 181 Seriatopora
hystrix*
Thin birdsnest
coral
Yes
T
Weis and Meyer lab
Kitchen et al. 2015
Stylophora pistillata*
Smooth
cauliflower coral
Yes
T
Centre Scientifique de Monaco
Karako-Lampert et al. 2014
Symbiodinium Resources
Species or Type
Symbiotic? Resource
Source
Publication
Symbiodinium minutum
(B1)
Yes
G
OIST Marine Genomics Unit
Shoguchi et al. 2013
Symbiodinium CassKB8
(A1)
Yes
T
iPlant Datastore
Bayer et al. 2012
Symbiodinium kawagutii
CCMP2468 (F)
No
T
iPlant Datastore
Zhang et al. 2013
Symbiodinium (C1)
Yes
T
iPlant Datastore
Keeling et al. 2014
Symbiodinium (C3)*
Yes
T
Palumbi Lab Website
Ladner et al. 2012
Symbiodinium (D2)*
Yes
T
Palumbi Lab Website
Ladner et al. 2013 182 Table A2 Reference proteins used as queries for homology searches of each protein group. Table A2 Reference proteins used as queries for homology searches of each protein group. Appendix A: Protein sequences and genetic resources Facilitated Glucose Transporters
UniProt ID
Protein
Tree ID
Species
P11166
Solute carrier family 2, facilitated glucose
transporter 1
GLUT1
Homo sapiens
P11168
Solute carrier family 2, facilitated glucose
transporter 2
GLUT2
Homo sapiens
P11169
Solute carrier family 2, facilitated glucose
transporter 3
GLUT3
Homo sapiens
P14672
Solute carrier family 2, facilitated glucose
transporter 4
GLUT4
Homo sapiens
P22732
Solute carrier family 2, facilitated glucose
transporter 5
GLUT5
Homo sapiens
Q9UGQ3
Solute carrier family 2, facilitated glucose
transporter 6
GLUT6
Homo sapiens
Q6PXP3
Solute carrier family 2, facilitated glucose
transporter 7
GLUT7
Homo sapiens
Q9NY64
Solute carrier family 2, facilitated glucose
transporter 8
GLUT8
Homo sapiens
Q9NRM0
Solute carrier family 2, facilitated glucose
transporter 9
GLUT9
Homo sapiens
O95528
Solute carrier family 2, facilitated glucose
transporter 10
GLUT10
Homo sapiens
Q9BYW1
Solute carrier family 2, facilitated glucose
transporter 11
GLUT11
Homo sapiens
Q8TD20
Solute carrier family 2, facilitated glucose
transporter 12
GLUT12
Homo sapiens
Q96QE2
Proton myo-inositol cotransporter
HMIT
Homo sapiens
Sodium/Glucose Transporters
UniProt ID
Protein
Tree ID
Species
P13866
Sodium/glucose cotransporter 1
SGLT1
Homo sapiens
P31639
Sodium/glucose cotransporter 2
SGLT2
Homo sapiens
C7EWH7
Sodium/glucose cotransporter 3
SGLT3
Homo sapiens
Q2M3M2
Sodium/glucose cotransporter 4
SGLT4
Homo sapiens
A0PJK1
Sodium/glucose cotransporter 5
SGLT5
Homo sapiens
Aquaglyceroporins
UniProt ID
Protein
Tree ID
Species 183 Q92482
Aquaporin-3
AQP3
Homo sapiens
O14520
Aquaporin-7
AQP7
Homo sapiens
O43315
Aquaporin-9
AQP9
Homo sapiens
Q96PS8
Aquaporin-10
AQP10
Homo sapiens
Ammonium Transporters
UniProt ID
Protein
Tree ID
Species
P54144
Ammonium transporter 1 member 1
AMT1-1
Arabidopsis thaliana
Q9ZPJ8
Ammonium transporter 1 member 2
AMT1-2
Arabidopsis thaliana
Q9SQH9
Ammonium transporter 1 member 3
AMT1-3
Arabidopsis thaliana
Q9SVT8
Ammonium transporter 1 member 4
AMT1-4
Arabidopsis thaliana
Q9LK16
Ammonium transporter 1 member 5
AMT1-5
Arabidopsis thaliana
Nitrate Transporters
UniProt ID
Protein
Tree ID
Species
O82811
High affinity nitrate transporter 2.1
NRT2.1
Arabidopsis thaliana
Q9LMZ9
High affinity nitrate transporter 2.2
NRT2.2
Arabidopsis thaliana
Q9FJH7
High affinity nitrate transporter 2.3
NRT2.3
Arabidopsis thaliana
Q9FJH8
High affinity nitrate transporter 2.4
NRT2.4
Arabidopsis thaliana
Q9LPV5
High affinity nitrate transporter 2.5
NRT2.5
Arabidopsis thaliana
Q9LXH0
High affinity nitrate transporter 2.6
NRT2.6
Arabidopsis thaliana 184 Appendix B: Carbon and nitrogen enrichment values Table B1 Carbon enrichment of host tissue and symbiont cells in the heterotrophy experiment. Time
point
Tissue
type
Symbiont
type
Replicate
anemone
Ave. C Enrichment
(δ13C)
δ13C std. Appendix A: Protein sequences and genetic resources error
1 Host
B1
1
38.64
5.30
1 Host
B1
2
197.68
40.7
1 Host
B1
3
41.59
15.03
1 Host
B1
4
13.27
2.90
1 Host
B1
5
11.63
1.91
1 Host
D1a
1
20.32
3.10
1 Host
D1a
2
4.91
3.23
1 Host
D1a
3
13.75
5.73
1 Host
D1a
4
38.12
11.06
1 Host
D1a
5
1.102
0.91
2 Host
B1
1
9.76
0.37
2 Host
B1
2
0
0
2 Host
B1
3
7.49
5.30
2 Host
B1
4
0
0
2 Host
B1
5
0
0
2 Host
D1a
1
0.91
0.91
2 Host
D1a
2
3.14
2.48
2 Host
D1a
3
8.11
6.06
2 Host
D1a
4
5.19
5.19
2 Host
D1a
5
0
0
1 Symbiont B1
1
15.46
1.93
1 Symbiont B1
2
0
0
1 Symbiont B1
3
18.88
2.39
1 Symbiont B1
4
12.73
2.97
1 Symbiont B1
5
11.00
3.56
1 Symbiont D1a
1
3.34
1.52
1 Symbiont D1a
2
0
0
1 Symbiont D1a
3
2.565
1.44
1 Symbiont D1a
4
5.69
3.27
1 Symbiont D1a
5
6.59
2.18
2 Symbiont B1
1
11.68
1.74
2 Symbiont B1
2
5.41
1.28
2 Symbiont B1
3
9.72
3.15
2 Symbiont B1
4
0.63
0.35
2 Symbiont B1
5
0.98
0.51
2 Symbiont D1a
1
2.44
1.04
2 Symbiont D1a
2
6.03
1.58
2 Symbiont D1a
3
7.35
1.76
2 Symbiont D1a
4
7.18
1.48
2 Symbiont D1a
5
5.11
0.82 185 Table B2 Nitrogen enrichment of host tissue and symbiont cells in the autotrophy experiment. Table B2 Nitrogen enrichment of host tissue and symbiont cells in the autotrophy experiment. Time
point
Tissue
type
Cell
type
Replicate
anemone
Ave. N Enrichment
(δ15N)
δ15N std. Appendix C: Additional Symbiosome Work I also attempted the
method using Percoll® gradients and Ficoll® gradients, but again found that the gradients did
not successfully isolate
the
symbiosome
membranes from the rest
of the host homogenate
and intact Symbiodinium
cells. I proceeded to test
the FM-143 stain on
Symbiodinium
cultures,
finding that it was able to
stain the dinoflagellate
cell
membrane,
rather
than being specific to the
symbiosome. I therefore
concluded that FM-143 dye was not an accurate marker for the symbiosome, as was previously
published. An appropriate marker for the symbiosome membrane is needed before isolation of
the membranes is possible, which can be successfully accomplished using immunolabelling
using an antibody for a known symbiosome protein (Dani et al. 2014; Barott et al. 2015). While
other research groups have begun using immunocytochemistry techniques for this purpose, I
Figure C1 Predicted symbiosome membranes, stained with FM-143, viewed
under 40x magnification and the FITC filter. Scale bar is 10 µm. Optimization of the symbiosome isolation protocol from Kazandjian et al. (2008) and Starzak
(2012) was attempted over a two-year period. I was able to identify membranes predicted to be
symbiosomes, however they could not be isolated in concentrations high enough for accurate
analysis. Isolation was first attempted using sucrose density gradients, as per the original
protocol (Kazandjian et al. 2008), using 20 anemones. The predicted symbiosomes were
stained with FM-143 and observed under the FITC filter of a fluorescence microscope (Figure
C1). However, the top “golden layer” from where the resulting symbiosomes were harvested,
was consistently found to contain many contaminants as well (Figure C2). I also attempted the
method using Percoll® gradients and Ficoll® gradients, but again found that the gradients did Figure C1 Predicted symbiosome membranes, stained with FM-143, viewed
under 40x magnification and the FITC filter. Scale bar is 10 µm. Figure C1 Predicted symbiosome membranes, stained with FM-143, viewed
under 40x magnification and the FITC filter. Scale bar is 10 µm. concluded that FM-143 dye was not an accurate marker for the symbiosome, as was previously
published. An appropriate marker for the symbiosome membrane is needed before isolation of
the membranes is possible, which can be successfully accomplished using immunolabelling
using an antibody for a known symbiosome protein (Dani et al. 2014; Barott et al. 2015). Appendix A: Protein sequences and genetic resources error
1 Host
B1
1
8.73
1.54
1 Host
B1
2
0.73
0.73
1 Host
B1
3
0
0
1 Host
B1
4
7.48
2.25
1 Host
D1a
1
0
0
1 Host
D1a
2
0
0
1 Host
D1a
3
0
0
1 Host
D1a
4
0
0
2 Host
B1
1
3.27
1.42
2 Host
B1
2
5.44
0
2 Host
B1
3
10.42
1.85
2 Host
B1
4
10.51
1.86
2 Host
B1
5
10.20
1.66
2 Host
D1a
1
2.21
0.60
2 Host
D1a
2
6.23
0.49
2 Host
D1a
3
6.12
3.12
2 Host
D1a
4
7.55
4.02
2 Host
D1a
5
9.17
2.31
1 Symbiont
B1
1
202.77
12.40
1 Symbiont
B1
2
130.29
51.14
1 Symbiont
B1
3
13.01
13.01
1 Symbiont
B1
4
115.01
20.35
1 Symbiont
D1a
1
0
0
1 Symbiont
D1a
2
0
0
1 Symbiont
D1a
3
3.86
1.97
1 Symbiont
D1a
4
12.22
10.33
2 Symbiont
B1
1
16.67
1.70
2 Symbiont
B1
2
55.03
3.74
2 Symbiont
B1
3
30.81
2.74
2 Symbiont
B1
4
61.55
3.98
2 Symbiont
B1
5
71.59
5.37
2 Symbiont
D1a
1
14.29
2.08
2 Symbiont
D1a
2
24.37
1.79
2 Symbiont
D1a
3
36.34
3.02
2 Symbiont
D1a
4
17.58
3.39
2 Symbiont
D1a
5
42.86
7.03 186 Appendix C: Additional Symbiosome Work Optimization of the symbiosome isolation protocol from Kazandjian et al. (2008) and Starzak
(2012) was attempted over a two-year period. I was able to identify membranes predicted to be
symbiosomes, however they could not be isolated in concentrations high enough for accurate
analysis. Isolation was first attempted using sucrose density gradients, as per the original
protocol (Kazandjian et al. 2008), using 20 anemones. The predicted symbiosomes were
stained with FM-143 and observed under the FITC filter of a fluorescence microscope (Figure
C1). However, the top “golden layer” from where the resulting symbiosomes were harvested,
was consistently found to contain many contaminants as well (Figure C2). I also attempted the
method using Percoll® gradients and Ficoll® gradients, but again found that the gradients did
not successfully isolate
the
symbiosome
membranes from the rest
of the host homogenate
and intact Symbiodinium
cells. I proceeded to test
the FM-143 stain on
Symbiodinium
cultures,
finding that it was able to
stain the dinoflagellate
cell
membrane,
rather
than being specific to the
symbiosome. I therefore
concluded that FM-143 dye was not an accurate marker for the symbiosome, as was previously
published. An appropriate marker for the symbiosome membrane is needed before isolation of
the membranes is possible, which can be successfully accomplished using immunolabelling
using an antibody for a known symbiosome protein (Dani et al. 2014; Barott et al. 2015). While
other research groups have begun using immunocytochemistry techniques for this purpose, I
carried out bioinformatic analysis of nutrient transporters in various cnidarian-dinoflagellate
symbiosis to provide information on more protein candidates for the symbiosome. Figure C1 Predicted symbiosome membranes, stained with FM-143, viewed
under 40x magnification and the FITC filter. Scale bar is 10 µm. Optimization of the symbiosome isolation protocol from Kazandjian et al. (2008) and Starzak
(2012) was attempted over a two-year period. I was able to identify membranes predicted to be
symbiosomes, however they could not be isolated in concentrations high enough for accurate
analysis. Isolation was first attempted using sucrose density gradients, as per the original
protocol (Kazandjian et al. 2008), using 20 anemones. The predicted symbiosomes were
stained with FM-143 and observed under the FITC filter of a fluorescence microscope (Figure
C1). However, the top “golden layer” from where the resulting symbiosomes were harvested,
was consistently found to contain many contaminants as well (Figure C2). Appendix C: Additional Symbiosome Work While
other research groups have begun using immunocytochemistry techniques for this purpose, I
carried out bioinformatic analysis of nutrient transporters in various cnidarian-dinoflagellate
symbiosis to provide information on more protein candidates for the symbiosome. concluded that FM-143 dye was not an accurate marker for the symbiosome, as was previously
published. An appropriate marker for the symbiosome membrane is needed before isolation of
the membranes is possible, which can be successfully accomplished using immunolabelling
using an antibody for a known symbiosome protein (Dani et al. 2014; Barott et al. 2015). While
other research groups have begun using immunocytochemistry techniques for this purpose, I
carried out bioinformatic analysis of nutrient transporters in various cnidarian-dinoflagellate
symbiosis to provide information on more protein candidates for the symbiosome. 187 F
s
A
t
a
f
h
o
(
a
2
c
l
m
1
m
r
l
(
d
l
p
1
t
s
s
t Figure C2 Density gradient
symbiosome isolation steps. Anemones were homogenised,
then passed through a syringe
and needle to strip membranes
from intact algal cells. The
homogenate was then loaded
onto
a
density
gradient
(sucrose, Percoll®, or Ficoll®)
and centrifuged at 4000 x g for
2
hours. Images
1A-1D
correspond to the resolved
layers, viewed under 20x
magnification, scale bars are
100µm. The
symbiosome
membranes are meant to be
resolved into the top “golden
layer” in the loading zone
(1A). However, other cellular
debris was also found in this
layer. Symbiosomes were also
potentially visible in layers
1B-1D. Therefore, I concluded
that this method was not
successful
in
isolating
symbiosomes from the rest of
the anemone homogenate. 188 Publications
Matthews JL, Sproles AE, Oakley CA, Grossman AR, Weis VM, Davy SK (2016) Menthol-
induced bleaching rapidly and effectively provides experimental aposymbiotic sea
anemones (Aiptasia sp.) for symbiosis investigations. Journal of Experimental
Biology, 219, 306–310. ABSTRACT of symbionts, maintained in an aposymbiotic state for extended
periods, and successfully re-infected with a variety of Symbiodinium
types (Belda-Baillie et al., 2002; Schoenberg and Trench, 1980;
Starzak et al., 2014). The ease of experimental manipulation,
combined with the rapidly increasing resolution of genomic,
proteomic and metabolic databases for Aiptasia sp., makes this
symbiotic anthozoan an ideal candidate for biochemical and genetic
experiments that will further our understanding of the cellular
processes
underlying
the
cnidarian–dinoflagellate
symbiosis
(Lehnert et al., 2012; Peng et al., 2010; Sunagawa et al., 2009;
Weis et al., 2008). Experimental manipulation of the symbiosis between cnidarians and
photosynthetic dinoflagellates (Symbiodinium spp.) is crucial to
advancing the understanding of the cellular mechanisms involved
in host–symbiont interactions, and overall coral reef ecology. The
anemone Aiptasia sp. is a model for cnidarian–dinoflagellate
symbiosis, and notably it can be rendered aposymbiotic (i.e. dinoflagellate-free) and re-infected with a range of Symbiodinium
types. Various methods exist for generating aposymbiotic hosts;
however, they can be hugely time consuming and not wholly
effective. Here, we optimise a method using menthol for production
of
aposymbiotic
Aiptasia. The
menthol
treatment
produced
aposymbiotic hosts within just 4 weeks (97–100% symbiont loss),
and the condition was maintained long after treatment when
anemones were held under a standard light:dark cycle. The ability
of Aiptasia to form a stable symbiosis appeared to be unaffected by
menthol exposure, as demonstrated by successful re-establishment
of the symbiosis when anemones were experimentally re-infected. Furthermore, there was no significant impact on photosynthetic or
respiratory performance of re-infected anemones. Currently, the most commonly used method to render anemones
(including Aiptasia sp.) aposymbiotic combines temperature stress
(heat or cold shock) followed by dark treatment and/or chemical
inhibition
of
photosynthesis
with
3-(3,4-dichlorophenyl)-
1,1-dimethylurea
(DCMU)
(e.g. Belda-Baillie
et
al.,
2002;
Lehnert et al., 2014; Xiang et al., 2013). This is a slow, laborious
process that often requires months of preparation (Starzak et al.,
2014; Xiang et al., 2013), and may not result in the full eradication
of in hospite endosymbionts (Belda-Baillie et al., 2002; Schoenberg
and Trench, 1980; Wang and Douglas, 1998). ,
;
g
g
,
)
Recently, Wang et al. (2012) described a method of menthol-
induced bleaching in the corals Isopora palifera and Stylophora
pistillata. Menthol is known to cause local anaesthetic effects in
neuronal and skeletal muscles via blocking voltage-operated
sodium channels (Haeseler et al., 2002). Publications Matthews JL, Sproles AE, Oakley CA, Grossman AR, Weis VM, Davy SK (2016) Menthol-
induced bleaching rapidly and effectively provides experimental aposymbiotic sea
anemones (Aiptasia sp.) for symbiosis investigations. Journal of Experimental
Biology, 219, 306–310. Matthews JL, Sproles AE, Oakley CA, Grossman AR, Weis VM, Davy SK (2016) Menthol-
induced bleaching rapidly and effectively provides experimental aposymbiotic sea
anemones (Aiptasia sp.) for symbiosis investigations. Journal of Experimental
Biology, 219, 306–310. The following manuscript outlines the optimisation of a method for producing aposymbiotic
individuals of the model cnidarian, Aiptasia. This work was carried out by myself and a
fellow lab member, and was integral to chapters 2 and 3, which used this method to produce
experimental animals for the infection studies. Since publication, this method has been
utilized by other research groups investigating infection dynamics of the cnidarian-
dinoflagellate symbiosis using Aiptasia as a model system, and will continue to allow these
types of experiments to be more easily conducted. 189 Published by The Company of Biologists Ltd | Journal of Experimental Biology (2016) 219, 306-310 doi:10.1242/jeb.128934 METHODS & TECHNIQUES 1School of Biological Sciences, Victoria University of Wellington, Wellington 6140,
New Zealand. 2Department of Plant Biology, The Carnegie Institution, Stanford,
CA 94305, USA. 3Oregon State University, Department of Integrative Biology, 3029
Cordley Hall, Corvallis, OR 97331, USA. Menthol-induced bleaching rapidly and effectively provides
experimental aposymbiotic sea anemones (Aiptasia sp.) for
symbiosis investigations Jennifer L. Matthews1, Ashley E. Sproles1, Clinton A. Oakley1, Arthur R. Grossman2, Virginia M. Weis3 and
Simon K. Davy1,* Jennifer L. Matthews1, Ashley E. Sproles1, Clinton A. Oakley1, Arthur R. Grossman2, V
Simon K. Davy1,* ABSTRACT This cellular response
has led to its use as a marine anaesthetic (Alexander, 1964; Lauretta
et al., 2014). Menthol is known to act on a variety of different
membrane receptors, including transient receptor potential (TRP)
M8, which results in an increase in intracellular Ca2+ concentrations
and causes a cold sensation in vertebrates (Hans et al., 2012;
McKemy et al., 2002; Okazawa et al., 2000; Peier et al., 2002). As
proposed by Wang et al. (2012), the mechanism of menthol-induced
bleaching might be attributable to Ca2+-stimulated exocytosis (Pang
and Südhof, 2010). Preliminary experiments conducted by Wang
et al. (2012) also suggest that menthol inhibition of Symbiodinium
photosystem II activity may play a role in the expulsion of the algal
cells or the digestion of the Symbiodinium cells by the host. Although the exact mechanism by which menthol induces symbiont
expulsion is not yet clear, the findings of Wang and co-authors
provide a platform for developing the use of menthol to generate
aposymbiotic Aiptasia sp. KEY WORDS: Cnidarian–dinoflagellate symbiosis, Symbiodinium,
Coral reefs Experimental organisms Symbiotic individuals of Aiptasia sp. (n=300) were harvested from
a long-term laboratory stock maintained in 1 µm filtered seawater
(FSW) at 25°C, with light provided by AQUA-GLO T8 fluorescent
bulbs at ∼95 µmol photons m−2 s−1 (12 h:12 h light:dark cycle). Anemones were maintained in 1 µm FSW aerated with a Hailea®
aquarium air pump at 2 l min−1 and at 25°C using the WEIPRO®
Temperature Controller MX-1019. These lighting and aquarium
conditions were maintained throughout the 19-week experiment. The anemones were evenly divided among the treatments (menthol,
cold shock and untreated) and allowed to settle for 72 h before
beginning a 4-week treatment period. Menthol (20% w/v in ethanol; Sigma-Aldrich, Auckland, NZ) was
added to 1 µm FSW at a final concentration of 0.19 mmol l−1. This
concentration resulted in successful bleaching without causing
mortality. A higher concentration of 0.38 mmol l−1 as suggested by
Wang et al. (2012) was trialled prior to this experiment, but caused
mortality of over 50% of the anemones (J.L.M., unpublished data). The anemones were incubated in the menthol/FSW solution for 8 h,
after which the menthol/FSW was removed and the anemones were
incubated in 1 µm FSW for 16 h. DCMU (100 mmol l−1 dissolved
in EtOH, Sigma-Aldrich) was added to a final concentration of
5 µmol l−1 to prevent nuisance algal blooms and to limit the re-
establishment of any residual symbiont cells by inhibiting
photosynthesis. This 24-h cycle was repeated for four consecutive
days. The 8-h menthol/FSW incubation occurred during the 12-h
light period. The 4-day treatment was repeated after a 3-day break,
during which anemones were maintained under the lighting and
temperature conditions stated above, with biweekly feeding with
Artemia sp. nauplii. The Symbiodinium culture (ID: FLAp2) was grown at 25°C and
an irradiance of 100 μmol photons m−2 s−1 on a 12 h:12 h light:dark
cycle. Symbiodinium cells were sub-cultured from a laboratory stock
and grown in silica-free f/2 medium (AusAqua Pty, SA, Australia)
for 6 weeks before use in the infection study. To genotype the
Symbiodinium cells, DNA was extracted as described by Hill et al. (2014) from an algal pellet derived from a 1 ml aliquot of the
Symbiodinium suspension. Samples were immediately used for
PCR, using the thermal cycling regime and reaction mixture of
Hill et al. (2014), and the outer primers ITSintfor2 and ITS2Rev2. Experimental organisms PCR products were cleaned with ExoSAP-IT (USB Corporation,
OH, USA) and sequenced by the Macrogen Sequencing Service For cold shock, the water was replaced with 4°C 1 µm FSW, and
placed in a refrigerator at 4°C for 4 h. Afterwards, the water was
replaced with 25°C 1 µm FSW containing a final concentration of
50 µmol l−1 DCMU (100 mmol l−1 dissolved in EtOH, Sigma-
Aldrich) and held under the lighting and aquarium conditions Fig. 1. Effects of menthol and cold shock on
algal cell density in Aiptasia sp. (A) Density of
Symbiodinium cells in menthol-treated (closed
circles) and cold-shocked (open circles)
anemones over a 19-week period. The anemones
were treated for the first 4 weeks, then left to
recover in regular filtered seawater for the
remaining 15 weeks. Outliers (defined as
exceeding 1.5× interquartile range) were detected
in the menthol-treated anemones at weeks 4 and
11, and are indicated by a red cross. Values are
mean±s.e.m.; n=5. (B) Confocal microscopy
images of anemones during menthol treatment
and cold shock (weeks 1–4). Yellow arrows
indicate Symbiodinium cells in host tissues. (C,D) Confocal fluorescence images of menthol-
treated and cold-shocked anemones, respectively,
15 weeks post-treatment (i.e. week 19). Scale bars
are 100 µm. 0
5
10
Week
20
15
Symbiont density (cells mg–1 host protein)
A
0
1e+6
2e+6
3e+6
4e+6
5e+6
6e+6
Menthol
Cold shock
Menthol outliers 0
5
10
Week
20
15
Symbiont density (cells mg–1 host protein)
D
C
A
0
1e+6
2e+6
3e+6
4e+6
5e+6
6e+6
Menthol
Cold shock
Menthol outliers
Fig. 1. Effects of menthol a
algal cell density in Aiptas
Symbiodinium cells in menth
circles) and cold-shocked (o
anemones over a 19-week p
were treated for the first 4 we
recover in regular filtered sea
remaining 15 weeks. Outliers
exceeding 1.5× interquartile
in the menthol-treated anem
11, and are indicated by a re
mean±s.e.m.; n=5. (B) Confo
images of anemones during
and cold shock (weeks 1–4)
indicate Symbiodinium cells
(C,D) Confocal fluorescence
treated and cold-shocked ane
15 weeks post-treatment (i.e
are 100 µm. 0
1
2
3
4
Cold shock
Menthol
0
5
10
Week
20
15
Symbiont density (cells mg–1 host protein)
D
C
B
A
0
1e+6
2e+6
3e+6
4e+6
5e+6
6e+6
Menthol
Cold shock
Menthol outliers
Fig. 1. Effects of menthol and cold shock on
algal cell density in Aiptasia sp. METHODS & TECHNIQUES METHODS & TECHNIQUES (Macrogen, Seoul, South Korea). Sequences were aligned with
Geneious v. 7.0 (Biomatters, Auckland, NZ) and a BLAST search
was carried out against Symbiodinium ITS2 sequences in GenBank. Using this approach, the Symbiodinium genotype was confirmed as
B1. those of untreated and healthy symbiotic anemones. Ultimately, we
describe a method to assist the many researchers around the world
who use the Aiptasia model system to further our understanding of
the coral–dinoflagellate symbiosis. INTRODUCTION The symbiosis between corals and dinoflagellate algae of the genus
Symbiodinium promotes the success of coral reefs in nutrient-poor
tropical seas. However, there are still large gaps in our knowledge
about how this symbiosis is established and maintained (Davy et al.,
2012; Weis and Allemand, 2009; Weis et al., 2008). Moreover, we
do not fully understand how and why this symbiosis breaks down
under stress (e.g. bleaching) (Weis, 2008). These knowledge
gaps hinder our capacity to understand the function of coral reefs,
and to predict how coral reefs might respond to our changing
environment. Journal of Experimental Biology To elucidate the cellular basis of the coral–dinoflagellate
relationship, the symbiotic sea anemone Aiptasia sp. has been
widely adopted as a model system because of its robust nature
and the ability to produce large populations easily in the laboratory
(Weis et al., 2008). Furthermore, Aiptasia sp. can be cleared We therefore tested the applicability of this approach to Aiptasia
sp., in terms of its effectiveness and impact on the capacity for
symbiosis re-establishment. In particular, we determined whether
menthol
can
rapidly
and
effectively
produce
aposymbiotic
anemones, and whether these anemones can be experimentally re-
infected with symbiotic dinoflagellates at cell densities similar to *Author for correspondence (Simon.Davy@vuw.ac.nz) Received 25 July 2015; Accepted 5 November 2015 306 Journal of Experimental Biology (2016) 219, 306-310 doi:10.1242/jeb.128934 Experimental organisms (A) Density of
Symbiodinium cells in menthol-treated (closed
circles) and cold-shocked (open circles)
anemones over a 19-week period. The anemones
were treated for the first 4 weeks, then left to
recover in regular filtered seawater for the
remaining 15 weeks. Outliers (defined as
exceeding 1.5× interquartile range) were detected
in the menthol-treated anemones at weeks 4 and
11, and are indicated by a red cross. Values are
mean±s.e.m.; n=5. (B) Confocal microscopy
images of anemones during menthol treatment
and cold shock (weeks 1–4). Yellow arrows
indicate Symbiodinium cells in host tissues. (C,D) Confocal fluorescence images of menthol-
treated and cold-shocked anemones, respectively,
15 weeks post-treatment (i.e. week 19). Scale bars
are 100 µm. 307
Journal of Experimental Biology A C C Journal of Experimental Biology D D Journal of Experimental Biology 0
1
2
3
4
Cold shock
Menthol
Week
B B 0 3 4 1 2 307 Journal of Experimental Biology (2016) 219, 306-310 doi:10.1242/jeb.128934 METHODS & TECHNIQUES described above. The anemones were incubated under these
conditions for 3 days, and the treatment was then repeated. The
anemones were unfed for the duration of the cold shock, as per the
protocol described by Steen and Muscatine (1987). Anemones were
cold shocked a total of eight times (twice per week) during the
treatment period. at an irradiance of ∼100 µmol photons m−2 s−1. Net photosynthesis
and respiration were corrected for background levels using a FSW-
only control, and gross photosynthesis was calculated by the
addition of net photosynthesis to dark respiration (Muscatine et al.,
1981). The average ratio of gross photosynthesis to respiration (P:R)
for 24 h was calculated for each anemone, by assuming a 12 h:12 h
light:dark cycle and that respiration was constant in the light and
dark, and the average ratio for each treatment was calculated. After the 4-week treatment period, all anemones were maintained
in 1 µm FSW at 25°C under the irradiance regime described above,
with biweekly feeding, for an additional 15 weeks. Their symbiotic
status was assessed via confocal fluorescence microscopy and cell
density estimates (described below) at weeks 1–5, 7, 9, 11 and 19. After the 4-week treatment period, all anemones were maintained
in 1 µm FSW at 25°C under the irradiance regime described above,
with biweekly feeding, for an additional 15 weeks. Statistical tests The non-parametric Mann–Whitney U-test was used to test for
differences between treatments with respect to symbiont density
(SPSS statistical software, v. 20, IBM Corporation). Separate
analyses were run for the bleaching and post-bleaching periods. The untreated group of anemones was kept under the same
thermal and light regimes as the menthol-treated and cold-shocked
anemones, with biweekly feeding throughout the entire 19-week
experiment. They were assessed for symbiont density at the same
time points as the experimental anemones, to obtain an average
symbiont density of untreated, healthy anemones. A one-way ANOVA was performed on the photosynthetic rate,
respiration rate and log-transformed P:R valuesto determinewhether
significant differences were present between the re-infected
menthol-treated and untreated symbiotic anemones at the end of
the 19-week experimental period. Experimental organisms Their symbiotic
status was assessed via confocal fluorescence microscopy and cell
density estimates (described below) at weeks 1–5, 7, 9, 11 and 19. The untreated group of anemones was kept under the same
thermal and light regimes as the menthol-treated and cold-shocked
anemones, with biweekly feeding throughout the entire 19-week
experiment. They were assessed for symbiont density at the same
time points as the experimental anemones, to obtain an average
symbiont density of untreated, healthy anemones. Re-infection of aposymbiotic anemones The capacity to re-infect menthol-treated anemones with algal
symbionts was tested; this was not possible with cold-shocked
anemones because they were not fully aposymbiotic. Anemones
were infected with cultured homologous Symbiodinium type B1, i.e. symbionts originally isolated from Aiptasia sp. A drop of
concentrated Symbiodinium suspension (3×106 cells ml−1) was
pipetted onto the oral disc of each anemone, followed by a drop of
Artemia sp. nauplii to invoke a feeding response and encourage
Symbiodinium uptake (Davy et al., 1997). This process was repeated
4 h later, and the water was replaced with 1 µm FSW 8 h after the
second infection. Confocal microscopy Whole anemones from each treatment were examined at the same
time points as cell density assessments by confocal microscopy
(Olympus Provis AX70, at 100× magnification) to detect the
chlorophyll autofluorescence of any dinoflagellates. Anemones
were selected at random from each treatment, and placed in a
‘relaxation solution’ [50% FSW, 50% 0.37 mol l−1 magnesium
chloride
(MgCl2)]
for
15 min
before
being
moved
to
a
FluoroDish™
glass-bottom
confocal
dish
(World
Precision
Instruments, FL, USA). The autofluorescence emission of the
Symbiodinium cells was excited using a 559 nm laser and captured
at 647±10 nm. This was performed to check the rates of bleaching
and re-infection, as well as to detect any background Symbiodinium
populations in the post-treatment anemones. B C C RESULTS AND DISCUSSION Menthol treatment induced
97–100% symbiont loss within
14–28 days, with 60% of anemones losing all symbionts. In
comparison, cold shock never induced 100% loss within the same A
B
C
0
2
4
6
8
10
12
14
Week
Symbiont density (cells mg–1 host protein)
0
1e+6
2e+6
3e+6
4e+6
5e+6
Aposymbiotic, menthol treated
Re-infected, menthol treated
Symbiotic, untreated
Fig. 2. Repopulation of menthol-treated Aiptasia sp. with algal symbionts. (A) Symbiont cell density over a 12-week period after anemones were
experimentally re-infected with the homologous Symbiodinium type B1 (closed
triangles). Also shown are symbiont densities in symbiotic, untreated
anemones (closed square) and menthol-treated anemones that were not
experimentally re-infected (open circles). Values are means±s.e.m.; n=5. (B,C) Confocal images showing, respectively, the re-established symbiont
population 12 weeks after reinfection versus tissues in a non-infected
aposymbiotic anemone. Scale bars are 100 µm. A
0
2
4
6
8
10
12
14
Week
Symbiont density (cells mg–1 host protein)
0
1e+6
2e+6
3e+6
4e+6
5e+6
Aposymbiotic, menthol treated
Re-infected, menthol treated
Symbiotic, untreated Symbiont density and protein content Symbiodinium populations were quantified with a haemocytometer
and light microscope (100× magnification; n=8 replicate counts per
sample) using whole-anemone homogenate (n=5). Cell density was
normalised to soluble protein content, measured via the Bradford
assay (Bradford, 1976). Cell density was measured at weeks 1–5, 7,
9, 11 and 19. References Senses 37, 463-469. Hill, R., Fernance, C., Wilkinson, S. P., Davy, S. K. and Scott, A. (2014). Symbiont
shuffling during thermal bleaching and recovery in the sea anemone Entacmaea
quadricolor. Mar. Biol. 161, 2931-2937. Lauretta, D., Hä ussermann, V., Brugler, M. R. and Rodrı́guez, E. (2014). Isoparactis fionae sp. nov. (Cnidaria: Anthozoa: Actiniaria) from Southern
Patagonia with a discussion of the family Isanthidae. Organ. Divers. Evol. 14,
31-42. Lehnert, E. M., Burriesci, M. S. and Pringle, J. R. (2012). Developing the anemone
Aiptasia as a tractable model for cnidarian–dinoflagellate symbiosis: the
transcriptome of aposymbiotic A. pallida. BMC Genomics 13, 271. Lehnert, E. M., Mouchka, M. E., Burriesci, M. S., Gallo, N. D., Schwarz, J. A. and
Pringle, J. R. (2014). Extensive differences in gene expression between
symbiotic and aposymbiotic cnidarians. G3 4, 277-295. McKemy, D. D., Neuhausser, W. M. and Julius, D. (2002). Identification of a cold
receptor reveals a general role for TRP channels in thermosensation. Nature 416,
52-58. As expected, given its anaesthetic properties (Alexander, 1964;
Lauretta et al., 2014), menthol induced tentacle relaxation and
unresponsiveness in anemones for the duration of the treatment. Once the seawater was replaced with regular FSW, the anemones
were able to regain full movement and feeding capabilities within
∼15 min. This is in stark contrast to the residual effects of cold
shock on anemones, where long-lasting tentacle retraction and
occasional mortality were observed. Muscatine, L., McCloskey, L. and Marian, R. (1981). Estimating the daily contribution
of carbon from zooxanthellae to coral animal respiration. Oceanography 26,
601-611. Okazawa, M., Terauchi, T., Shiraki, T., Matsumura, K. and Kobayashi, S. (2000). l-Menthol-induced [Ca2+]i increase and impulses in cultured sensory neurons. Neuroreport 11, 2151-2155. 2 Pang, Z. P. and Sü dhof, T. C. (2010). Cell biology of Ca2+-triggered exocytosis. Curr. Opin. Cell Biol. 22, 496-505. Peier, A. M., Moqrich, A., Hergarden, A. C., Reeve, A. J., Andersson, D. A.,
Story, G. M., Earley, T. J., Dragoni, I., McIntyre, P. and Bevan, S. (2002). ATRP
channel that senses cold stimuli and menthol. Cell 108, 705-715. In summary, we provide a rapid and effective method for the
generation of aposymbiotic Aiptasia sp. that will facilitate studies of
symbiosis
establishment
and
maintenance,
host–symbiont
recognition, and symbiosis function with this model system. In
particular, menthol treatment has the potential to rapidly create large
stocks of aposymbiotic anemones without compromising their
infectivity. References Alexander, R. M. (1964). Visco-elastic properties of the mesogloea of jellyfish. J. Exp. Biol. 41, 363-369. Belda-Baillie, C. A., Baillie, B. K. and Maruyama, T. (2002). Specificity of a model
cnidarian–dinoflagellate symbiosis. Biol. Bull. 202, 74-85. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye binding. Anal. biochem. 72, 248-254. Davy,
S.,
Turner,
J. and
Lucas,
I. (1997). The
nature
of
temperate
anthozoandinoflagellate symbioses. In Proc. 7th Int. Coral Reef Symp., vol. 2,
pp. 1307-1312. One key use foraposymbiotic Aiptasia sp. is forexperimental work
that involves re-infection with symbiotic algae of different types and
from different host species. It was therefore important to test the
infectivity of the menthol-treated anemones, and establish whether
there were any significant long-term impacts of menthol treatment
versus no treatment on physiological performance (photosynthesis
and respiration) of the re-infected anemones. When infected with
Symbiodinium cells (ITS2 type B1), symbiont density increased
slowly during the initial 2 weeks, followed by a rapid increase during
weeks 2–12 (Fig. 2A). Confocal microscopy confirmed that menthol-
treated anemones were fully repopulated after 12 weeks (Fig. 2B). The infection pattern and maximum cell density were similar to those
of other re-infection studies using heat- and cold-shock bleached
Aiptasia and type B1 Symbiodinium, where a steep increase in cell
density was observed in the first 4 weeks, followed by stabilisation
(Belda-Baillie et al., 2002; Schoenberg and Trench, 1980; Starzak
et al., 2014). Additionally, the photosynthetic rate (P=0.432; n=4),
respiration rate (P=0.347; n=4) and ratio of photosynthesis to
respiration (P=0.368; n=4) in these anemones were similar to those of
untreated symbiotic anemones. This indicates that their capacity to re-
form a functional symbiosis was not compromised, further
highlighting the value of this method. Davy,
S.,
Turner,
J. and
Lucas,
I. (1997). The
nature
of
temperate
anthozoandinoflagellate symbioses. In Proc. 7th Int. Coral Reef Symp., vol. 2,
pp. 1307-1312. Davy, S. K., Allemand, D. and Weis, V. M. (2012). Cell biology of cnidarian-
dinoflagellate symbiosis. Microbiol. Mol. Biol. Rev. 76, 229-261. Haeseler, G., Maue, D., Grosskreutz, J., Bufler, J., Nentwig, B., Piepenbrock, S., Dengler, R. and Leuwer, M. (2002). Voltage-dependent block of neuronal and
skeletal muscle sodium channels by thymol and menthol. Eur. J. Anaesthesiol. 19, 571-579. Hans, M., Wilhelm, M. and Swandulla, D. (2012). Menthol suppresses nicotinic
acetylcholine receptor functioning in sensory neurons via allosteric modulation. Chem. Competing interests
The authors declare no competing or financial interests time frame (Fig. 1A,B), and any residual symbionts in the menthol-
treated anemones did not re-establish a full symbiosis once returned
to normal seawater conditions over the course of our experiment
(Fig. 1A,C). Competing interests
The authors declare no competing or financial interests. The authors declare no competing or financial interests. Author contributions
J.L.M., A.E.S., C.A.O., A.R.G., V.M.W. and S.K.D. conceived and designed the
experiments. J.L.M. and A.E.S. carried out the experiments and subsequent data
analysis. J.L.M., A.E.S., C.A.O., A.R.G., V.M.W. and S.K.D. wrote the manuscript. (
g
)
Although statistically, the patterns of symbiont loss during weeks
1–4 were not different between the menthol and cold-shock
treatments
(Mann–Whitney
U=113,
d.f.=4,
P=0.423,
n=5;
Fig. 1A),
only
the
menthol-treated
anemones
successfully
maintained aposymbiotic status in the longer term. In both
treatments, symbiont density remained near zero during the first
week of recovery (weeks 4–5; Fig. 1A); however, subsequently there
was a significant difference between the cell densities of menthol- and
cold-shock-treated
anemones
(Mann–Whitney
U=35,
d.f.=4,
P<0.0005; n=5), with the menthol-treated anemones remaining
mostly symbiont-free (0–0.05% of pre-bleaching density) and the
cold-shocked anemones becoming fully re-populated (weeks 5–9;
Fig. 1A). Confocal microscopy confirmed these measurements
(Fig. 1C). Menthol treatment therefore has considerable potential
for the rapid and effective generation of aposymbiotic Aiptasia sp. References Ultimately, this method will help to accelerate the rate of
discovery as we try to better understand the function of coral reefs,
and their response and adaptive capacity in the face of climate
change. Peng, S. E., Wang, Y. B., Wang, L. H., Chen, W. N. U., Lu, C. Y., Fang, L. S. and
Chen, C. S. (2010). Proteomic analysis of symbiosome membranes in Cnidaria–
dinoflagellate endosymbiosis. Proteomics 10, 1002-1016. Schoenberg, D. A. and Trench, R. K. (1980). Genetic variation in Symbiodinium (=
Gymnodinium) microadriaticum Freudenthal, and specificity in its symbiosis with
marine
invertebrates. III. Specificity
and
infectivity
of
Symbiodinium
microadriaticum. Proc. R. Soc. B Biol. Sci. 207, 445-460. Starzak, D. E., Quinnell, R. G., Nitschke, M. R. and Davy, S. K. (2014). The
influence of symbiont type on photosynthetic carbon flux in a model cnidarian–
dinoflagellate symbiosis. Mar. Biol. 161, 711-724. g
y
Steen, R. G. and Muscatine, L. (1987). Low temperature evokes rapid exocytosis of
symbiotic algae by a sea anemone. Biol. Bull. 172, 246-263. Sunagawa, S., Wilson, E. C., Thaler, M., Smith, M. L., Caruso, C., Pringle,
J. R., Weis, V. M., Medina, M. and Schwarz, J. A. (2009). Generation and
analysis of transcriptomic resources for a model system on the rise: the sea Acknowledgements Funding This work was supported by a grant from the Royal Society of New Zealand Marsden
Fund (grant no. 1202 awarded to S.K.D., A.R.G. and V.M.W.). This work was supported by a grant from the Royal Society of New Zealand Marsden
Fund (grant no. 1202 awarded to S.K.D., A.R.G. and V.M.W.). O2 flux Fig. 2. Repopulation of menthol-treated Aiptasia sp. with algal symbionts. (A) Symbiont cell density over a 12-week period after anemones were
experimentally re-infected with the homologous Symbiodinium type B1 (closed
triangles). Also shown are symbiont densities in symbiotic, untreated
anemones (closed square) and menthol-treated anemones that were not
experimentally re-infected (open circles). Values are means±s.e.m.; n=5. (B,C) Confocal images showing, respectively, the re-established symbiont
population 12 weeks after reinfection versus tissues in a non-infected
aposymbiotic anemone. Scale bars are 100 µm. Fifteen weeks after reinfection, maximum photosynthetic and dark
respiratory O2 fluxes were measured to compare re-infected
menthol-treated anemones against untreated anemones (n=4). A
FIBOX 3 fibre optic oxygen transmitter and oxygen probe (PreSens,
Germany) were used as described by Starzak et al. (2014). The
respiration rate (ml O2 h−1) was measured for 30 min in the dark,
after which the rate of net photosynthesis was measured for 30 min 308 Journal of Experimental Biology (2016) 219, 306-310 doi:10.1242/jeb.128934 METHODS & TECHNIQUES Acknowledgements
We are grateful to Malindi Gammon and Josh Brian for laboratory assistance. We are grateful to Malindi Gammon and Josh Brian for laboratory assistance. Wang, J. and Douglas, A. (1998). Nitrogen recycling or nitrogen conservation in an
alga-invertebrate symbiosis? J. Exp. Biol. 201, 2445-2453. anemone Aiptasia pallida and its dinoflagellate endosymbiont. BMC Genomics
10, 258. Weis, V. M. (2008). Cellular mechanisms of Cnidarian bleaching: stress causes the
collapse of symbiosis. J. Exp. Biol. 211, 3059-3066. g
y
p
Wang, J. T., Chen, Y. Y., Tew, K. S., Meng, P. J. and Chen, C. A. (2012).
Physiological and biochemical performances of menthol-induced aposymbiotic
corals. PLoS One 7, e46406. Weis, V. M., Davy, S. K., Hoegh-Guldberg, O., Rodriguez-Lanetty, M. and
Pringle, J. R. (2008). Cell biology in model systems as the key to understanding
corals. Trends Ecol. Evol. 23, 369-376. Journal of Experimental Biology (2016) 219, 306-310 doi:10.1242/jeb.128934 Xiang, T., Hambleton, E. A., DeNofrio, J. C., Pringle, J. R., Grossman, A. R. and
Lin,
S.
(2013).
Isolation
of
clonal
axenic
strains
of
the
symbiotic
dinoflagellate Symbiodinium and their growth and host specificity. J. Phycol.
49, 447-458. anemone Aiptasia pallida and its dinoflagellate endosymbiont. BMC Genomics
10, 258.
Wang, J. and Douglas, A. (1998). Nitrogen recycling or nitrogen conservation in an
alga-invertebrate symbiosis? J. Exp. Biol. 201, 2445-2453.
Wang, J. T., Chen, Y. Y., Tew, K. S., Meng, P. J. and Chen, C. A. (2012).
Physiological and biochemical performances of menthol-induced aposymbiotic
corals. PLoS One 7, e46406.
Weis, V. M. (2008). Cellular mechanisms of Cnidarian bleaching: stress causes the
collapse of symbiosis. J. Exp. Biol. 211, 3059-3066. Weis, V. M. and Allemand, D. (2009). Physiology. What determines coral health?
Science 324, 1153-1155.
Weis, V. M., Davy, S. K., Hoegh-Guldberg, O., Rodriguez-Lanetty, M. and
Pringle, J. R. (2008). Cell biology in model systems as the key to understanding
corals. Trends Ecol. Evol. 23, 369-376.
Xiang, T., Hambleton, E. A., DeNofrio, J. C., Pringle, J. R., Grossman, A. R. and
Lin,
S.
(2013).
Isolation
of
clonal
axenic
strains
of
the
symbiotic
dinoflagellate Symbiodinium and their growth and host specificity. J. Phycol.
49, 447-458. Weis, V. M. and Allemand, D. (2009). Physiology. What determines coral health?
Science 324, 1153-1155. Weis, V. M. and Allemand, D. (2009). Physiology. What determines coral health?
Science 324, 1153-1155. Acknowledgements We are grateful to Malindi Gammon and Josh Brian for laboratory assistance. 309 Journal of Experimental Biology (2016) 219, 306-310 doi:10.1242/jeb.128934 METHODS & TECHNIQUES METHODS & TECHNIQUES Xiang, T., Hambleton, E. A., DeNofrio, J. C., Pringle, J. R., Grossman, A. R. and
Lin,
S. (2013). Isolation
of
clonal
axenic
strains
of
the
symbiotic
dinoflagellate Symbiodinium and their growth and host specificity. J. Phycol. 49, 447-458. Weis, V. M. (2008). Cellular mechanisms of Cnidarian bleaching: stress causes the
collapse of symbiosis. J. Exp. Biol. 211, 3059-3066. Journal of Experimental Biology 310
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The professional and educational ecosystem as a driver of development collaboration between engineering education and production
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© 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/). https://doi.org/10.1051/matecconf/202031102003 https://doi.org/10.1051/matecconf/202031102003 IX Czarnowski Readings
MATEC Web of Conferences 311, 02003 (2020) UDC 334.7 (045); JEL Classification: D83, E24 The professional and educational ecosystem as
a driver of development collaboration between
engineering education and production IX Czarnowski Readings
MATEC Web of Conferences 311, 02003 (2020) https://doi.org/10.1051/matecconf/202031102003 The professional and educational ecosystem as
a driver of development collaboration between
engineering education and production Mikhail Flek1, Ekaterina Ugnich2
1Professor, D-r of Technical Sciences, Deputy Managing Director, Rostvertol PLC, «Aircraft
Engineering» Department Chair, Rostov-on-Don, Russia; D., associate professor Don State Technical University, Rostov-on-Don, Russia Abstract. The formation and development of human capital as to the
current and future needs of production is a serious challenge for modern
enterprises. Human capital, which is based on knowledge and skills, is the
most important factor of production that determines the competitiveness and
efficiency of the enterprise. One of the ways to solve this problem is the
formation of the enterprise vocational education ecosystem, by combining
with various educational organizations. The authors formulate the concept
of a professional and educational ecosystem as a type of socio-economic
ecosystem. The importance of interaction between ecosystem subjects as the
basis for the emergence of synergistic effects is emphasized. The theoretical
conclusions are supported by empirical material on the example of the
professional and educational ecosystem of a large helicopter enterprise. The
characteristic of the enterprise's professional and educational ecosystem is
given. Emphasis is placed on the structural and specific description of the
professional and educational ecosystem. The reasons for the formation of
the professional and educational ecosystem of the enterprise and the way of
its development are analyzed. The analysis of the effectiveness of the
professional and educational ecosystem can be based on the stakeholder
theory, based on the assessment of the results of the ecosystem by its main
participants. In order to analyze the effectiveness of the professional and
educational ecosystem of the enterprise, a survey of its participants was
conducted on the importance of knowledge and skills acquired as a result of
training, and satisfaction with them. On the basis of the poll results, a
perceptual map was built by the analysis of Needs&Gaps. She demonstrated
the most important attributes, describing the results of the professional and
educational ecosystem from the perceptual of importance and satisfaction of
participants in this ecosystem. The construction of the perceptual map made
it possible to draw a conclusion about the high assessment of the participants
of the ecosystem of practical skills acquired in the learning process. The
results of the analysis of the professional and educational ecosystem of the
enterprise confirm its effectiveness in solving the problem of human capital
formation. Keywords: human capital, enterprise, professional and educational
ecosystem, production system, production, engineering education,
collaboration. Introduction This goal is achieved by solving
the following tasks:
reveal the essence of the professional and educational ecosystem of the enterprise
and its role in the formation of the human capital of the enterprise,
reveal the essence of the professional and educational ecosystem of the enterprise
and its role in the formation of the human capital of the enterprise,
analyze the sources of formation and development of the professional and
educational ecosystem on the example of a helicopter-building enterprise,
describe the structure of the professional and educational ecosystem enterprise,
evaluate the results of the functioning of the professional and educational ecosystem
enterprise. Introduction In the context of the establishment of a post-industrial society and the increase in the
intellectualization of the economy, a significant problem for many domestic enterprises is the
gaS beWZeeQ Whe UeTXiUed ³TXaliW\´ Rf hXPaQ UeVRXUceV aQd Whe SURSRVed education system. According to a study by the Boston Consulting Group, Russia ranks 89th in terms of the
availability of qualified personnel. At the same time, 35% of graduates of domestic colleges
and 25% of graduates of universities [6] cannot find a job in the specialty acquired, mainly
due to the lack of vacancies. Moreover, for enterprises, especially high-tech, the problem of
RbVRleVceQce Rf kQRZledge iV UeleYaQW, ViQce WheiU ³half-life´ iQ VcieQce-based industries is
less than 2.5 years. The solution to these and other problems is impossible without improving
the mechanisms of the education system as a source of formation and development of the
human capital of the enterprise - the most important production resource in which the
knowledge, skills, and experience of workers are concentrated [8]. g ,
,
p
[ ]
Large enterprises in different ways try to solve the problem of personnel gaps in the
necessary qualifications. One such way is the creation of corporate universities. In Russia
today there are more than 30 of them. Corporate universities, as a rule, implement programs
of further vocational education, that is, they form only professional competencies. The
"foundation" of the human capital is laid from the school bench - it is formed by the education
system and family. This problem is partially eliminated by another way to improve the
"quality" of the formation of the human capital - the combination of enterprise efforts with
various educational organizations. Within the framework of such an association, basic
departments are often created, the principle of which is based on dual practice-oriented
training, which has proved its effectiveness both in Russia and abroad [19]. There is also a
more complex version of such an association - the formation of the professional and
educational ecosystems, which are based on the ongoing acquisition of the necessary
knowledge, skills, and experience on the principle of lifelong learning. The aim of this work is to study the features of the formation and development of the
professional and educational ecosystem of the enterprise as a driver for the development of
the collaboration of engineering education and production. Methods The inability of the existing provisions of the neoclassical economy to adequately study
the dynamics of economic development, to explain the change in technological structures
and scientific and production cycles [18] makes us reconsider many approaches and
directions of economic theory [7]. Modern conditions for the development of the economy
require new methodological approaches that can assess the feasibility of the collaboration
between entities, as well as the potential of the communities of various economic agents
formed on their basis. It is the systematic approach that underlies this work that allows one
to create a single research space for the whole complex of socio-economic phenomena. The
systemic paradigm is based on the concept of ecosystems, which originally arose in biology 2 2 IX Czarnowski Readings
MATEC Web of Conferences 311, 02003 (2020) https://doi.org/10.1051/matecconf/202031102003 and has interdisciplinary potential. The concept of ecosystems allows one to consider it from
the point of view of collaborations and interrelations between constituent components [14]. The concept of ecosystems is increasingly used to study open systems with a different number
of heterogeneous participants having diverse relationships [17]. In this regard, we consider it
appropriate to apply it to the study of the collaboration of the enterprise with educational
organizations. The professional and educational ecosystem is a special socio-economic
ecosystem. Its basis is the collaboration of participants, a network of collaborating and
competing organizations that offer related products and services [1]. If in the biological
ecosystem biomass is the product (result), then in the professional and educational ecosystem
it is human capital. and has interdisciplinary potential. The concept of ecosystems allows one to consider it from
the point of view of collaborations and interrelations between constituent components [14]. The concept of ecosystems is increasingly used to study open systems with a different number
of heterogeneous participants having diverse relationships [17]. In this regard, we consider it
appropriate to apply it to the study of the collaboration of the enterprise with educational
organizations. The professional and educational ecosystem is a special socio-economic
ecosystem. Its basis is the collaboration of participants, a network of collaborating and
competing organizations that offer related products and services [1]. If in the biological
ecosystem biomass is the product (result), then in the professional and educational ecosystem
it is human capital. Methods p
To assess the effectiveness of the professional and educational ecosystem in this study,
we used the theory of stakeholders [13] and a direct subjective methodology [5], based on
Whe aVVeVVPeQW Rf Whe ecRV\VWeP¶V UeVXlWV b\ iWV PaiQ SaUWiciSaQWV, aV Zell aV a ViWXaWiRQal
analysis method [20], which allows studying current phenomena in real conditions. The
empirical basis for evaluating the effectiveness of the professional and educational ecosystem
of Rostvertol PJSC is the results of a survey conducted at the end of 2018 of four different
groups of its various participants regarding the importance and satisfaction of the knowledge
and skills that are forming in this ecosystem, which form the basis of the human capital of
the enterprise. Based on the results of the questionnaire, a perception map was constructed
using the Needs & Gaps analysis method [15], which was able to clearly demonstrate the
advantages and disadvantages of the functioning of the professional and educational
ecosystem, as well as its development reserves. Results In determining the content of socio-economic ecosystems, which include professional and
educational, it is necessary to emphasize the simultaneous implementation of cooperation
and competition. Since there is no common understanding of the socio-economic ecosystem,
specifying the most capacious definition of G.B. Kleiner [2], we define professional and
educational ecosystem as a complex dynamic system, geographically localized, consisting of
the collaboration entities that create a specific product (result) and implement processes of
cooperation and competition simultaneously with the formation of synergistic effects. Based
on this definition, a professional and educational ecosystem is not just a combination of
educational institutions with a partner enterprise but their complex dynamic formations,
where synergistic effects are achieved - effects when the effectiveness of the system increases
dXe WR Whe cRllabRUaWiRQ Rf iWV YaUiRXV elePeQWV iQ QaWXUe. The SUefi[ ³ecR´ WR Whe ZRUd
³V\VWeP´ iQ WhiV caVe ePShaVi]eV Whe Qeed fRU a ceUWaiQ cRllabRUaWiRQ eQYiURQPeQW fRU
participants based on their horizontal connections, where synergistic effects arise. The socio-economic ecosystem, according to G.B. Kleiner [3], includes four components:
organizational, infrastructure, communication and logistics, and innovation (Fig. 1). Thus,
the organizational component is a combination of organizations and independent individuals
functioning as part of an ecosystem. This component can be described as a cluster. The
infrastructural component is the mechanisms, rules, and regulations that shape the
environment of its participants. The communication and logistics component is characterized
by integration and communication mechanisms that ensure the process of the collaboration
between participants. The innovative component represents innovative impulses, that is,
measures aimed at adapting the ecosystem to changes in the external environment. From this
perspective, one can characterize the professional and educational ecosystem (Fig. 1). 3 IX Czarnowski Readings
MATEC Web of Conferences 311, 02003 (2020) https://doi.org/10.1051/matecconf/202031102003 Fig. 1. The components of the professional and educational ecosystem. Fig. 1. The components of the professional and educational ecosystem. Thus, the successful functioning of the professional and educational ecosystem is ensured
by the collaboration of various components and their reproduction. The main direction of all
components of the professional and educational ecosystem is the joint creation of value -
human capital of the enterprise [11]. An illustration of an example of the professional and educational ecosystem of PJSC
Rostvertol, the largest helicopter-building enterprise included in the Russian Helicopters
holding, allows one to demonstrate the specifics of this mechanism in the formation of the
human capital of the enterprise. Results The search for ways to solve the problem of matching the
³TXaliW\´ of the human capital of the enterprise to its current and future production tasks led
to the fact that in 2002, enterprise initiated the creation of an educational space, which was
then transformed, in 2015, into a professional and educational cluster, which at the moment
is already a professional and educational ecosystem. The main reasons for creating enterprise educational space in 2002 are: the need for continuous training of workers in view of the special nature of the
enterprise ² the manufacturer of aviation equipment ² due to the continuous
updating of the range and modification of manufactured products, the development of
new technologies, materials, and high-tech equipment; the need for confidentiality in the transfer of knowledge and the formation of qualified
personnel, dictated by the specifics of the military-industrial complex; natural aging and the departure of qualified engineers from production amid
worsening labor mobility throughout the country. Specialized colleges, schools, and, founded in the same year 2002, together with Don
State Technical University (DSTU) the basic department of Aircraft Engineering, became
participants of the educational space of PJSC Rostvertol. Specialized colleges, schools, and, founded in the same year 2002, together with Don
State Technical University (DSTU) the basic department of Aircraft Engineering, became
participants of the educational space of PJSC Rostvertol. 4 IX Czarnowski Readings
MATEC Web of Conferences 311, 02003 (2020) https://doi.org/10.1051/matecconf/202031102003 In the future, as a result of strengthening the relationships between the participants of the
educational space, it was transformed into a professional and educational cluster (Fig. 2). The
fundamental difference between the educational space and the professional and educational
cluster is that in the first there is no concentration of efforts in a certain area, there are no
strong relationships between the participants, the organizational structure is not structured. Fig. 2. Formation of a professional and educational ecosystem (space - cluster - ecosystem)
educational space
Professional and educational
ecosystem
Professional and educational
cluster Fig. 2. Formation of a professional and educational ecosystem (space - cluster - ecosystem) The goal of the professional and educational cluster of PJSC Rostvertol is to develop an
integrated training system for workers and specialists for the aviation industry along the chain
"Secondary general education - Secondary vocational education - Higher education - Further
professional education". Results Thus, the formation of a future specialist occurs starting from
school. Participants of the professional and educational cluster [9] are presented in table 1. Table 1. Professional and educational cluster members
Organization level
Representative
organizations
Main functionality
Basic educational organizations
Secondary general
education
sponsored schools of Rostov-
on-Don
Lay the "foundation" of the human
capital,
provide
entrants
with
subsequent cluster links
Secondary
vocational
education
Don Industrial and Technical
College. BN Slusar; Aviation
college
Training aviation industry workers
and specialists for enterprise
Higher education
DSTU: basic department of
Aircraft Engineering
Training of qualified specialists with
higher education in the aviation
industry for enterprise
Further professional
education
DSTU: Institute of Aviation
Industry Management and
Innovation
Additional professional training and
retraining of enterprise workers,
advanced training, the formation of
new competencies of workers in
target areas
Structural units of the base enterprise
Further professional
education
Personnel Training Center,
Aviation Training Center
Additional professional training for
enterprise
workers,
advanced
training
Non-formal
education in the
course of work
Workshops and departments
of the enterprise
Practice and exchange of experience
in the workplace 5 5 IX Czarnowski Readings
MATEC Web of Conferences 311, 02003 (2020) IX Czarnowski Readings
MATEC Web of Conferences 311, 02003 (2020) https://doi.org/10.1051/matecconf/202031102003 Thus, the participants of the professional and educational cluster (Table 1) are represented
not only by educational institutions from schools to universities but also by structural units
of enterprise [10]. The further development of the organizational structure of the professional and
educational cluster in the ecosystem is due to the turbulence of the external environment
under the influence of the strengthening of integration processes and the challenges of digital
production. The need for ecosystems is due to the fact that in modern conditions, to create a
product that meets all the necessary requirements, it is necessary to combine the resources
and competencies of several organizations. The ecosystem provides a new higher
organization level for production [4]. So, if the product of the educational space is educational
services, the product of the professional and educational ecosystem is human capital. If the educational space of PJSC Rostvertol included several educational organizations
that are not interconnected, then the professional and educational cluster represents an
already defined organizational structure of participants based on their collaboration. Results The
professional and educational ecosystem in addition to the totality of the participants and their
collaboration, includes the environment, certain integration mechanisms that develop the
collaboration of participants and the adaptation mechanism, which serves as the basis for the
sustainability and self-development of the ecosystem. The success of the professional and educational ecosystem is characterized by the
effectiveness of the formation of the human capital of the enterprise. A number of researchers
suggest evaluating the functioning of socio-economic ecosystems based on the development
Rf Whe WheRU\ Rf ³VWakehRldeUV´ [13]. FRU WhiV, iW iV QeceVVaU\ WR ideQWif\ SaUWieV (SaUWiciSaQWV)
who are interested in the effective functioning of the ecosystem, to determine their value
relationships and results of activities. Mixing up the subjectivity of stakeholders, a definite
assessment system is formed that characterizes the effectiveness of the functioning of the
socio-economic ecosystem. The effectiveness of a professional and educational ecosystem
should be evaluated, first of all, on the basis of availability and satisfaction with knowledge
and skills that make up the human capital of the enterprise [12]. Among the ³VWakehRldeUV´
of the professional and educational ecosystem four main groups can be distinguished: of the professional and educational ecosystem, four main groups can be distinguish - executives of the enterprise and its structural divisions; - professors of educational organizations of the enterprise; - enterprise workers (graduates, previously trained in educational organizations of the
professional and educational ecosystem); p
y
)
- students currently studying in the educational organizations of this ecosystem. In order to analyze the effectiveness of the system of education and training of specialists
in the professional and educational ecosystem of PJSC Rostvertol, in November 2018, a
survey of representatives of these four groups of participants was conducted. On a five-point
scale, respondents rated the importance and satisfaction of the results of training in a
professional and educational ecosystem: In order to analyze the effectiveness of the system of education and training of specialists
in the professional and educational ecosystem of PJSC Rostvertol, in November 2018, a
survey of representatives of these four groups of participants was conducted. On a five-point
scale, respondents rated the importance and satisfaction of the results of training in a
professional and educational ecosystem: - general professional knowledge necessary for an engineer, - special professional knowledge directly related to the aircraft industry, - general cultural knowledge, - practical skills. Results The survey results of four groups of respondents are presented in table 2. The survey results of four groups of respondents are presented in table 2. In general, all groups gave a satisfactory rating (at least 3 points) of both importance and
satisfaction with knowledge and skills. The high importance of specialized professional
knowledge was noted by enterprise executives (4.92) with a satisfaction value of 4.31. The
least satisfaction with knowledge, both general professional and special, was noted by
graduates - enterprise workers (values 3.39 and 3.56, respectively). They are also the least
satisfied with the practical skills acquired in the learning process (3.74). This can be
explained by the rapid obsolescence of specialized knowledge. 6 6 IX Czarnowski Readings
MATEC Web of Conferences 311, 02003 (2020) https://doi.org/10.1051/matecconf/202031102003 IX Czarnowski Readings Table 2. Survey results on the importance and satisfaction of knowledge and skills gained as a result
of training
Indicators
("attributes")
importance
satisfaction
executives
professors
graduates
students
executives
professors
graduates
students
General
professional
knowledge (1)
4,65
4,26
4,02
4,01
4,58
3,99
3,39
3,63
Special
professional
knowledge (2)
4,92
4,48
4,27
4,49
4,31
4,24
3,56
4,42
General cultural
knowledge (3)
4,59
4,40
3,71
3,67
4,19
4,00
4,04
4,01
Practical skills (4)
4,72
4,55
4,34
4,53
4,63
4,11
3,74
4,03
Note. The table shows the average values according to the respondents' assessment of the importance
and satisfaction with knowledge and skills on a five-point scale (5 - very important/absolutely satisfied;
1 - does not matter/absolutely not satisfied). At the same time, executives noted the importance and
satisfaction of this knowledge for enterprise workers; professors - for students (future workers);
department graduates (enterprise workers) and students - own knowledge. Note. The table shows the average values according to the respondents' assessment of the importance
and satisfaction with knowledge and skills on a five-point scale (5 - very important/absolutely satisfied;
1 - does not matter/absolutely not satisfied). At the same time, executives noted the importance and
satisfaction of this knowledge for enterprise workers; professors - for students (future workers);
department graduates (enterprise workers) and students - own knowledge. FRU a PRUe deWailed deVcUiSWiRQ Rf Whe ³TXaliW\´ aVVeVVPeQW of the human capital of the
professional and educational ecosystem, we will construct a perception map by the Needs &
Gaps analysis method [15]. Perception maps of four groups of respondents are presented in Figure 3. The numbering
Rf ³aWWUibXWeV´ (1-4) is presented in Table 2. 7 7 Results The Needs & Gaps method is based on the results of respondents'
assessment of importaQce aQd VaWiVfacWiRQ ZiWh ceUWaiQ ³aWWUibXWeV´ RQ a 5-point scale. To
construct a perception map, the survey results presented in Table 2 were used. To construct a perception map, a coordinate plane was constructed for each of the four
groups of respondents. Origin points (average values for importance and satisfaction for the
eQWiUe VeW Rf ³aWWUibXWeV´) aUe SUeVeQWed iQ Wable 3. Table 3. Origin points for the perception map of each group of "participants" of the professional and
educational ecosystem
Indicator
Executives
Professors
Graduates
Students
Importance
4,71
4,23
4,09
4,18
Satisfaction
4,43
4,09
3,68
4,02 3. Origin points for the perception map of each group of "participants" of the professional and
educational ecosystem Perception maps of four groups of respondents are presented in Figure 3. The numbering
Rf ³aWWUibXWeV´ (1-4) is presented in Table 2. Conclusions The professional and educational ecosystem is a driver for the development of integration
of educational organizations and enterprises, the main task of which is to provide enterprises
with hXPaQ caSiWal ZiWh Whe UeTXiUed "TXaliW\". The UeTXiUePeQWV fRU a ceUWaiQ ³TXaliW\´ of
the human capital of the enterprise are set by the features of modern production, due to a
change in technological structure, the challenges of global competition and the rapid
obsolescence of knowledge in science-based industries. In other words, the professional and
educational ecosystem is aimed at achieving unity in the development of productive forces
and production relations. The development process of the professional and educational
ecosystem itself is based on the complexity of its structure and increased adaptation to
changing external conditions based on constant updates. The effectiveness of a professional and educational ecosystem can be judged by
evaluating the results of its functioning. One of the options for assessing the socio-economic
ecRV\VWeP caQ be Whe aSSlicaWiRQ Rf Whe WheRU\ Rf ³VWakehRldeUV´ aQd a diUecW VXbjecWiYe
methodology based on a survey of various groups of ecosystem participants. The assessment
of the professional and educational ecosystem of the helicopter-building enterprise as a whole
indicates its effectiveness. Participants of the professional and educational ecosystem noted
that its basic advantage is the acquisition of practical skills by carriers of the human capital
of the enterprise. Prospects for the development of the professional and educational
ecosystem are seen in strengthening the collaboration of its participants and expanding their
composition. 7 IX Czarnowski Readings
MATEC Web of Conferences 311, 02003 (2020) https://doi.org/10.1051/matecconf/202031102003 IX Czarnowski Readings Fig. 3. Perception maps of participants of the professional and educational ecosystem Fig. 3. Perception maps of participants of the professional and educational ecosystem The upper right quadrant (I) is characterized by high importance and high satisfaction of
Whe ³aWWUibXWe´. The lRZeU UighW TXadUaQW (II) iV Whe TXadUaQW Rf VecRQdaU\ beQefiWV RU gaSV. Attributes located in the second quadrant need support and development. The upper left
quadrant (III) characterizes the underlying flaw. The lower left quadrant (IV) is characterized
by low importance with low satisfaction. Improving the attributes located there requires more
substantial efforts but is necessary for the qualitative formation of the human capital of the
enterprise. It is noteworthy that all groups of respondents noted that practical skills
(³aWWUibXWe´ 4) aUe Whe baVic adYaQWage Rf Whe SURfeVViRQal aQd edXcaWiRQal ecRV\VWeP (fRU all
respondents, it is in quadrant I). Analyzing the perception map, it can be noted that according to all participants in the
ecosystem, its basic advantage is the acquisition of practical skills by the carriers of the
human capital of the enterprise (quadrant I). Regarding the secondary benefits of the
professional and educational ecosystem (quadrant II), the opinions of the respondents were
divided. So, enterprise executives and its structural divisions attributed secondary
professional knowledge to secondary advantages, and graduates of ecosystems (enterprise
workers) general cultural knowledge. Two other groups of participants (professors and 8 8 IX Czarnowski Readings
MATEC Web of Conferences 311, 02003 (2020) IX Czarnowski Readings
MATEC Web of Conferences 311, 02003 (2020) https://doi.org/10.1051/matecconf/202031102003 VWXdeQWV) did QRW ideQWif\ aQ\ ³aWWUibXWe´. RegaUdiQg Whe baVic lack of the professional and
educational ecosystem (quadrant III), the opinions of the respondents were again divided. Enterprise executives, as well as graduates (enterprise workers), marked the basic lack of
obtaining special professional knowledge, and professors - obtaining general professional
and general cultural knowledge. In general, the professional and educational ecosystem has
confirmed its importance for enterprise in terms of the formation of its human capital,
however, the mechanisms for transferring professional knowledge in the ecosystem require
further improvement and development. 4. Kuzmichev A.D. Economic policy and lean production // World of the new economy. -
2016. - No. 3. p.46-53 References 1. Karpinskaya V.A. Ecosystem as a unit of economic analysis. Systemic problems of
domestic mesoeconomics, microeconomics, enterprise economics: materials of the
Second Conference of the Department for modeling production facilities and complexes
of the Central Economic Mathematical Institute of the Russian Academy of Sciences
(Moscow, January 12, 2018). M .: Central Economic Mathematical Institute, 2018 2. Kleiner G.B. The enterprise ecosystem in the light of systemic economic theory/Strategic
planning and enterprise development: proceedings of the Nineteenth All-Russian
Symposium. Moscow, April 10-11, 2018 M.: Central Economic Mathematical Institute
of the Russian Academy of Sciences, 2018. Section 1. 2. Kleiner G.B. The enterprise ecosystem in the light of systemic economic theory/Strategic
planning and enterprise development: proceedings of the Nineteenth All-Russian
Symposium. Moscow, April 10-11, 2018 M.: Central Economic Mathematical Institute
of the Russian Academy of Sciences, 2018. Section 1. y
3. Kleiner G.B. Socio-economic ecosystems in the light of the system paradigm. System
Analysis in Economics - 2018: Proceedings of the V International Scientific and Practical
Conference - Biennale (November 21±23, 2018)/Ed. by G.B. Kleiner, S.E. Schepetova. M.: Prometheus, 2018. p. 5-14 3. Kleiner G.B. Socio-economic ecosystems in the light of the system paradigm. System
Analysis in Economics - 2018: Proceedings of the V International Scientific and Practical
Conference - Biennale (November 21±23, 2018)/Ed. by G.B. Kleiner, S.E. Schepetova. M.: Prometheus, 2018. p. 5-14 4. Kuzmichev A.D. Economic policy and lean production // World of the new economy. -
2016. - No. 3. p.46-53 9 9 9 IX Czarnowski Readings
MATEC Web of Conferences 311, 02003 (2020) https://doi.org/10.1051/matecconf/202031102003 5. Maltseva V. A. The skill mismatch concept and the problem of assessing the mismatch
of cognitive skills // Educational Issues. ± 2019. - ʋ3. p.43-76 g
p
6. Monitoring the employment of graduates. Ministry of Science and Education of the
Russian Federation, M., 2017 7. Popov E.V., Simonova V.L., Tikhonova A.D. A factor model for the development of
innovative ecosystems // Innovations. - 2019. - No. 10 (252). p. 88-100 8. Falko S.G., Ivanova N.U., Ankudinova M.L. Features of the formation of the human
capital in innovative organizations/Innovation Management - 2018: Materials of an
international scientific and practical conference/Ed. by R.M. Nizhegorodskiy, N.P. Goridko± Novocherkassk: Platov South-Russian State Polytechnic University (NPI),
2018. p.65-71 9. Flek M.B., Ugnich E.A. Professional and educational cluster as an ecosystem: 146-159
development in a digital transformation // Journal of Economic Regulation. 2018. Vol. 9,
No. 4. p. g
20. Yin R.K. Case Study Research Design and Methods. Thousand Oaks, CA: Sage, 200 References 146-159 10. Flek M.B., Ugnich E.A. The role of the basic department in the formation of the human
capital of the enterprise // Public Administration. Electronic bulletin. 2018. No. 67. p.293-
313 11. Human capital: theory and practice of management in socio-economic systems:
monograph/ed. by R. M. Nizhegorodtseva and S. D. Reznik. - M .; Penza, 2008 .-- 394 p. 12. Flek M.B., Ugnich E.A. Human Capital Formation of the Enterprise: the Role and
Experience of the Specialized Department of the Pillar University // Advances in
Economics, Business and Management Research. ± 2019. - Vol.79. P. 289-291 DOI:
https://doi.org/10.2991/iscfec-19.2019.81 13. Freeman R. E. Strategic Management: A Stakeholder Approach. - Boston, 1984 14. Moore J.F. Predators and prey: A new ecology of competition. Harvard Business Review,
May-June, 1993 15. Galport, N., Azzam, T. Evaluator Training Needs and Competencies: A Gap Analysis //
American Journal of Evalution. 2017. Vol.38. No1. P.80-100 16. Jarvi, K, Almpanopoulou, A., Ritala, P. Organization of knowledge ecosystem:
Prefigurative and partial forms // Research Policy. 2018. 47(8). P.1523-1537 17. Pravdiuk N., Pokynchereda V., Pravdiuk M. The human capital of an enterprise: theory
and assessment methodology // Baltic Journal of Economic Studies. ± 2019. ʋ2. VRl.5. P.176-183 DOI: https://doi.org/10.30525/2256-0742/2019-5-2-176-183 18. Schwab K. The Fourth Industrial Revolution. Crown Business, New York, 2017 19. Remington T. F. Public-Private Partnerships in VET: Translating the German Model of
Dual Education/National Research University Higher School of Economics, Institute of
Education. Moscow: HSE Publishing House, 2017 20. Yin R.K. Case Study Research Design and Methods. Thousand Oaks, CA: Sage, 2003 10 10
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https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0007214&type=printable
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English
| null |
Early Events Associated with Infection of Epstein-Barr Virus Infection of Primary B-Cells
|
PloS one
| 2,009
|
cc-by
| 14,922
|
Early Events Associated with Infection of Epstein-Barr
Virus Infection of Primary B-Cells Sabyasachi Halder, Masanao Murakami, Subhash C. Verma, Pankaj Kumar, Fuming Yi, Erle S. Robertson*
Department of Microbiology and Abramson Comprehensive Cancer Center, Tumor virology Program, University of Pennsylvania School of Medicine, Philadelphia,
Pennsylvania United States of America Department of Microbiology and Abramson Comprehensive Cancer Center, Tumor virology Program, University of Pennsylvania School of Medicine, Philadelphia,
Pennsylvania, United States of America Abstract 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: 1R01CA137894-01 and 5R01CA108461-05 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: erle@mail.med.upenn.edu by Burkitt lymphoma (BL) cells, viral gene expression is restricted to
the two EBER genes, the BART transcripts, and EBNA1 (EBV
nuclear antigen 1) [9]. In latency II additional expression of three
latent-membrane proteins, LMP-1, LMP-2A and LMP-2B is
observed and is most frequently seen in Hodgkin’s lymphoma. Latency III is seen in lymphoproliferative diseases developed in
immunocompromised individuals and EBV-transformed lympho-
blastoid cell lines [7]. In this group all six EBNAs, all three LMPs
and the two EBERs are expressed [9,10]. Type IV latency is less
strictly defined and pertains to infectious mononucleosis patients
and patients with a post-transplant lymphoproliferative disease [9]. Some individuals also presents with the so called putative latency
program (latency 0), in which no detectable latent gene expression is
detected [10]. The principal mediators of EBV-induced growth and
cellular transformation of B lymphocytes in vitro include EBNA2,
EBNA3A, 3C and LMP1 proteins [11]. The EBNA genes are
important for transformation of primary B lymphocytes, leading to
transactivation and regulation of other cellular and viral genes
[4,12]. These proteins are involved in augmentation of the
expression of genes coding for CD21, CD23, LMP1 and LMP2
proteins in B lymphocytes [4,11,13]. Abstract Epstein Barr virus (EBV) is closely associated with the development of a vast number of human cancers. To develop a system
for monitoring early cellular and viral events associated with EBV infection a self-recombining BAC containing 172-kb of the
Epstein Barr virus genome BAC-EBV designated as MD1 BAC (Chen et al., 2005, J.Virology) was used to introduce an
expression cassette of green fluorescent protein (GFP) by homologous recombination, and the resultant BAC clone, BAC-
GFP-EBV was transfected into the HEK 293T epithelial cell line. The resulting recombinant GFP EBV was induced to produce
progeny virus by chemical inducer from the stable HEK 293T BAC GFP EBV cell line and the virus was used to immortalize
human primary B-cell as monitored by green fluorescence and outgrowth of the primary B cells. The infection, B-cell
activation and cell proliferation due to GFP EBV was monitored by the expression of the B-cell surface antigens CD5, CD10,
CD19, CD23, CD39, CD40 , CD44 and the intercellular proliferation marker Ki-67 using Flow cytometry. The results show a
dramatic increase in Ki-67 which continues to increase by 6–7 days post-infection. Likewise, CD40 signals showed a gradual
increase, whereas CD23 signals were increased by 6–12 hours, maximally by 3 days and then decreased. Monitoring the viral
gene expression pattern showed an early burst of lytic gene expression. This up-regulation of lytic gene expression prior to
latent genes during early infection strongly suggests that EBV infects primary B-cell with an initial burst of lytic gene
expression and the resulting progeny virus is competent for infecting new primary B-cells. This process may be critical for
establishment of latency prior to cellular transformation. The newly infected primary B-cells can be further analyzed for
investigating B cell activation due to EBV infection. Citation: Halder S, Murakami M, Verma SC, Kumar P, Yi F, et al. (2009) Early Events Associated with Infection of Epstein-Barr Virus Infection of Primary B-Cells. PLoS
ONE 4(9): e7214. doi:10.1371/journal.pone.0007214 Citation: Halder S, Murakami M, Verma SC, Kumar P, Yi F, et al. (2009) Early Events Associated with Infection of Epstein-Barr Virus Infection of Primary B-Cells. PLoS
ONE 4(9): e7214. doi:10.1371/journal.pone.0007214
Editor: Linqi Zhang, Comprehensive AIDS Reseach Center, China Received May 29, 2009; Accepted August 27, 2009; Published September 28, 2009 Received May 29, 2009; Accepted August 27, 2009; Published September 28, 2009 Copyright: 2009 Halder et al. PLoS ONE | www.plosone.org September 2009 | Volume 4 | Issue 9 | e7214 Introduction Ki-67 is a monoclonal antibody that
recognizes
a
proliferation
related
human
nuclear
antigen
expressed during G1, S, and G2/M phase of the cell cycle but
not in resting (G0 phase) cells [43,44]. BZLF1(also referred to as Zta or ZEBRA) serves as a checkpoint
for initiation of the replicative cycle [15]. BZLF1 is a DNA-
binding protein, and its expression precedes the switch from latent
to lytic infection [4]. BZLF1 is a viral transactivator protein known
to be directly involved in triggering expression of the lytic genes
and downregulation of latent genes, culminating in cell death and
release of infectious virions [15]. This protein up-regulates
expression of other immediate early genes as well as its own
expression [16]. This immediate early expression in turn up-
regulates the expression of early genes such as viral DNA
polymerase (BALF5) and thymidine kinase [4,17]. The major
proteins of the lytic phase are the EBV DNA polymerase, BALF5
[18] and the late lytic cascade, major capsid protein, BcLF1[14]. Two small RNAs (EBER-1 and EBER-2) represent the most
abundant EBV RNA expressed during latent infection and
undergo continuous expression in EBV-positive tumors, indepen-
dently of the latency type [19,20]. Conventionally, herpesvirus mutants are generated by homol-
ogous recombination in infected cells with DNA fragments or
plasmids carrying the mutant allele as described almost 30 years
ago [21,22,23]. As a consequence, recombination between the
herpesvirus genome and the mutant allele gives rise to a mixed
population that consists of the wild-type and mutant virus, such
that their separation is necessary and important for evaluation of
the phenotype. This approach has been proven to be quite tedious
with gammaherpesviruses, (i.e. EBV), because so far no host cell
type has been shown to fully support the lytic, productive phase of
these viruses. In the case of EBV, to study latent genes it is first
essential to obtain an immortalized cell line latently infected with
the mutant virus, which takes place often in combination with
wild-type virus if the gene is essential. To separate these viruses in
a second step, the latently infected cell needs to support the lytic
phase to produce infectious virions important for establishment of
another latently infected, immortalized B cell line exclusively
carrying the viral mutant or can be passed into an already
immortalized cell line like Ramos or BL41[13]. Introduction Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that
establishes latent infection in B lymphocytes. EBV is associated
with various lymphoid and epithelial malignancies, such as
Burkitt’s lymphoma, nasopharyngeal carcinoma, lymphoprolifer-
ative diseases in immunosuppressed patients, and gastric carcino-
ma [1] . The principal target cells for EBV infection are human
primary B lymphocytes, but the virus can also infect other
lymphocytes
and
epithelial
cells [2,3,4,5,6]. EBV
has two
alternative lifestyles: latent (non-productive) infection, and lytic
(productive) replication [7]. Following primary infection, EBV
persists within memory B lymphocytes in a latent state for the life
of the host. A low level of reactivation during the lytic cycle allows
viral shedding into the saliva and transmission of the virus in vivo
[7]. EBV binds to B-lymphocytes through interaction of the
glycoprotein gp350/220 with the complement receptor CD21[8]. In vitro, EBV transforms peripheral human B lymphocytes into
indefinitely proliferating lymphoblastoid cell lines (LCLs) that
allows for genetic manipulation of the virus [7]. Latently infected B cells maintain EBV genomes as 184-kb
circular plasmids, referred to as episomes, and express only a limited
number of viral gene products [7]. At present, four patterns of EBV
latency are recognized [9,10]. In type I latency, represented mainly The lytic cascade of Epstein-Barr virus infection is divided into
three phases of regulated gene expression, immediate early, early
and late [14]. Synthesis of the viral encoded transactivator, PLoS ONE | www.plosone.org September 2009 | Volume 4 | Issue 9 | e7214 1 Early Events in EBV Infection to be important for the apoptosis of antigen-receptor induced
lymphocytes and for the maintenance of tolerance by B cells [36]. Additionally, previous work reported that CD5 expression is
down-regulated by EBV during transformation of CD19 positive
B-cells [37]. The B-cell activation marker CD23 has been shown
to be upregulated by EBV infection and induced at high level in
EBV-transformed lymphocytes [38,39]. EBNA-2 and LMP-1
cooperatively induces CD23 [13] as well as the human CD40
antigen which is a 50-kD glycosylated phosphoprotein [40]. LMP-
1 is a transmembrane protein with a structure reminiscent of G
protein coupled receptors, but its signaling activities are similar to
that of CD40 which belongs to the TNF receptor family [41]. In
addition, depending on the cellular context, LMP1 can induce
specific B-cell activation antigens such as CD39, CD23, CD21,
CD40 and CD44 [13,42]. Introduction Because B cell
immortalization is a prerequisite to establishment of a mutant
EBV LCL, this approach excludes the genetic analysis of genes
that are essential for B cell immortalization in vitro [24,25,26]. Gp350 is the glycoprotein found most abundantly on the surface
of EB virions as well as on the surface of EBV-infected cells in
which EBV is lytically replicating [45]. It is the glycoprotein of the
virion that binds to the EBV receptor CD21 (or complement
receptor type II, CR2) and initiates infection ([8,46].The another
glycoprotein, gp110 encoded by EBV BALF4 ORF is expressed
during the lytic phase of EBV not only at nuclear membrane but
also at the cellular membrane and has been shown genetically to
be essential for virus maturation [47,48,49]. Acyclovir [ACV; 9-(2-hydroxyethoxy methyl) guanine] is one of
a class of antiviral compounds effective in curbing a variety of
herpesvirus infection in vitro and in animal models [50]. In the
EBV system ACV is an effective inhibitor of viral DNA replication
in productively infected cells but is essentially devoid of any effect
on the replication of viral DNA in latently infected cells, where
cellular control mechanisms regulate EBV DNA synthesis[51,52]. In this report, we generated a recombinant EBV containing a
GFP cassette cloned in the BAC vector backbone designated as
BAC GFP-EBV in an effort to study the molecular changes during
early infection of primary B-cell induced by the virus. [53]. Previous studies by other groups which generated fluorescence
tagged EBV proved difficult in our hands [54,55]. However, the
MD1 system generated by Wang and collegues [29] proved to be
manipulatable in our hands and so we decided to introduce a
fluorescence marker for monitoring infection in this system. The
green fluorescent protein is a suitable marker because it fluoresces
strongly and stably in mammalian cells and can be monitored by
noninvasively strategies in living cells [56,57]. In this study, a GFP,
puromycin and ampicillin cassette was introduced into the EBV-
BAC [29] to establish the BAC based recombinant EBV GFP
designated as GFP-EBV by homologous recombination, and the
recombinant virus DNA was shuttled from E.coli to mammalian
cells. The induced GFP-EBV virus was used for the infection of
Peripheral Blood Mononuclear cells (PBMC) and establishment of
lymphoblastoid cell lines (LCLs). Using the GFP-EBV infected
PBMCs, we monitored a range of immunophenotypic changes. PLoS ONE | www.plosone.org Introduction Several B-cell surface antigen markers such as CD5, CD10, CD19,
CD23, CD39, CD40 and CD44 [40], as well as the intercellular
proliferation protein Ki-67 were used during initial infection of
EBV. We also analyzed the latent and lytic the protein profiles
during early infection of primary B-cell by recombinant EBV. Our
results suggested that EBV infection to B-cells involves an initial
burst of lytic replication which may be critical for the many
signaling events involving anticrine and paracrine factors which
eventually leads to B-cell transformation and establishment of
latency after 2–4 weeks in culture. [
,
,
]
The introduction of the bacterial artificial chromosome (BAC)
system into the genetics of herpesviruses brought a new dimension
to the field [27]. In the BAC system, the entire viral genome can
be propagated in Escherichia coli, and mutations can be rapidly and
precisely introduced into viral genes [27]. To facilitate the
generation of recombinant viruses, the EBV genome was first
cloned as a bacterial artificial chromosome (BAC). F-plasmid
sequences for prokaryotic replication [28], kanamycin resistance
marker for prokaryotic selection, and a cytomegalovirus promoter-
driven puromycin resistance cassette for eukaryotic selection were
inserted into the BamHI site of a plasmid containing EBV BamW
DNA and then transfected into B95-8 cells [29]. Following the
successful cloning of other herpesviruses, the B95-8 strain of EBV
was cloned in a BAC vector [29]. The system employed an
epithelial cell background as the virus producing cell and virus
production was induced by transfecting an expression vector
encoding a viral immediate protein BZLF1 [30,31] . In vitro EBV infection results in human B-lymphocytes
activation and perpetual proliferation [32,33]. EBV infected cells
grow in tight clumps and express a number of B-cell activation
molecules including CD5, CD23, CD39, CD40 and CD44 and
proliferation surface antigen marker CD10 as well as intracellular
proliferation marker Ki-67 [34]. CD5 expression on B-cell can be
up-regulated by various activating agents, which indicate that
CD5 is a B-cell activation antigen [35]. CD5 has also been shown September 2009 | Volume 4 | Issue 9 | e7214 September 2009 | Volume 4 | Issue 9 | e7214 PLoS ONE | www.plosone.org 2 Early Events in EBV Infection 0.65% agarose gel by running for 16 h–20 h at 40V and visualized
by ethidium bromide staining and UV exposure. The digestion
pattern was analyzed and compared with wild type MD1BAC. Cells and virus cultures BJAB was used as EBV negative cell line and LCL1 & LCL2
were used as EBV positive cell lines [58]. BAC-EBV was
propagated in EL350 [29] and GFP-Amp cassette was incorpo-
rated into BAC-EBV by homologous recombination. BAC GFP-
EBV was transferred into HEK 293T cells and the BAC GFP-
EBV infected Lymphoblastoid cell lines (LCLs) were established
from primary B-cell (Immunology core of UPENN). PBMCs were
obtained from UPENN immunology core from de-identified
different donors for multiple infection studies. All B-cells were
grown in RPMI1640 with 10% fetal bovine serum, and adherent
cells in Delbecco’s Modified Essential Medium (DMEM) supple-
mented
with
10%
fetal
bovine
serum
(FBS),
50
mg/ml
streptomycin,
and
50U
penicillin
(medium)
(Bio-Whittaker,
Walkersville, MD). 1 mg/ml final concentration of Puromycin
was used for selection of cells transfected or infected with the BAC
GFP-EBV. Introduction The resolved gel was transferred onto the Genescreen tranfer
membrane (PerkinElmer, Waltham, MA, USA) and the cassette
was visualized by hybridizing with the EBV BamHI I fragment as
a probe. Transformation of recombinant BAC GFP-EBV into
bacteria Oligonucleotide primers corresponding to the different region of
EBV genome were synthesized 59 to 39. The sequence of
oligonucleotides and the product length are shown in table 1. The genomic DNA was prepared from approximately 56104
cells. Following centrifugation to remove medium, cells were
resuspended in 0.26phosphate buffered saline, boiled for 10 min,
and mixed with 0.1 volume of 10-mg/ml proteinase K (Sigma,
Marlborough, MA), and the mixture was incubated for 30 min at
55uC. Proteinase K was inactivated by incubation at 95uC for
20 min. PCR analysis was performed by using Perkin-Elmer
thermal cycler with 5 ml of DNA in a 50 ml reaction. PCR-
amplified DNA was analyzed by electrophoresis using 2% agarose
gels and visualized by staining with ethidium bromide and UV
exposure. doi:10.1371/journal.pone.0007214.t001 The construction of BAC GFP-EBV genome For incorporation of GFP in the BAC EBV genome, we
considered
the
region
of
EBV
genome
from
149,116
to
154,747 bp. The GFP DNA was introduced into the site of the
B95.B deletion at 152,008 and the GFP cassette contained a
BamHI site and was flanked by 50 bp downstream and upstream
sequence from 152,008 bp [59]. 3646 bp of the GFP cassette
(GFP-Puro-Amp) with one BamHI site was electroporated into the
bacterial cells EL350 with BAC EBV (MD1BAC) and the cassette
was incorporated by homologous recombination where positive
clones were screened for ampicillin resistance. The amplicon was
transfected into electrocompetent bacterial cells EL350 MD1BAC
by electroporation at 1.75 kV. BAC GFP-EBV DNA was
extracted from the positive clones and subjected to BamHI
digestion overnight. The digested products were resolved on Infection of primary human B cells with GFP-EBV p
y
Lymphoblastoid cell lines were generated by infections of 16106
primary B-cells incubating with virus suspension in 1 ml of RPMI
1640 (10% Fatal Bovine Serum) medium in the presence of
cyclosporin A (Sigma, Marlborough, MA) and incubated for 4 hrs
in 37uC. Cells were centrifuged for 5 min at 1500 rpm, the
supernatant discarded, pelleted cells were resuspended in fresh
RPMI 1640 (10% FBS) medium in 96 well plates. The infection was
checked by the visualization of GFP expression using fluourescence
microscopy. The transfected green cells were enriched by selection
with puromycin. The infected cells were then transferred to 48 well
plates and expanded to larger well plates until the cultures
continued to grow continuously in complete media. The number
of clones generated for BAC GFP-EBV LCL was also shown in
table 2. To monitor the early stage of infection after 4 hrs
incubation with EBV and PBMC, the cells were then washed two
times with fresh RPMI 1640 (10% FBS) medium to remove excess
virus and fresh medium was added. This infection step is designated
as Infection I. The supernatant from primary infection (Infection I)
of 26107 PBMC by GFP-EBV were used infection of fresh PBMC
(16106) in a similar manner and infection further monitored by
GFP fluorescence. This step of infection was designated as Infection
II. Infection I was also monitored by adding 25 mM acyclovir and
the supernatant from infection I were also used for infection II to
determine if virus produced was due to replication during lytic
replication during lytic infection in Infection I and not due to virus
passed on from the initial infection . Real time PCR Total RNA were prepared from 56106 GFP EBV infected
PBMCs after different times post-infection (6h, 12h, 24 h, 48h, 72h,
96h, 120h and 168 hours) using Trizol reagent (Invitrogen, Inc.,
Carlsbad, CA) according to manufacturer’s instructions. cDNA was
synthesized using a Superscript II RT kit (Invitrogen, Inc.,
Carlsbad, CA) according to the manufacturer’s instructions. The
specific primers used for the amplification of latent genes (EBNA-1,
EBNA-2, LMP-1), lytic genes (BZLF1, BcLF1 and BALF5) and
internal control GAPDH are shown in Table 3. The lytic gene,
BALF5, was also amplified in the presence of 25 mM ACV. Table 3. Primers used in q-Real time PCR[59]. Primer Name
DNA Sequence (59-39)
Product length
EBNA-1
59-CATTGAGTCGTCTCCCCTTTGGAAT-39
150 bp
59-TCATAACAAGGTCCTTAATCGCATC-39
EBNA-2
59-GAGACCAGAGCCAAACACCTCCAGT-39
150 bp
59-TTAGGGGTTGCCGTGTGTGAATTTC-39
LMP-1
59-CCCGCACCCTCAACAAGCTACCGAT-39
150 bp
59-TTGTCAGGACCACCTCCAGGTGCGC-39
BZLF1
59-AACCGCTCCGACTGGGTCGTGGTTT-39
150 bp
59-CCAGGTTGAGGTGCTTCTCCCCCGG-39
BcLF1
59-CCTCTTGGAATGCAGCTGGGGCCAG-39
150 bp
59-CCAATTATGACCTGCTGCGGCTGGA-39
BALF5
59-GCTGGCCTTGAGGGCGCTGAGGACT-39
259 bp
59-CACCCACGGAAGCCCTCTGGACTTC-39
doi:10.1371/journal.pone.0007214.t003 Flow cytometry analysis of EBV infected peripheral blood
mononuclear cells Flow cytometry analysis of EBV infected peripheral blood
mononuclear cells Table 2. No. of clones generated in HEK293T and LCLs of BAC
GFP-EBV. Peripheral Blood Mononuclear Cells (PBMCs) (procured from
Immuology core, University of Pennsylvania Medical School,
Philadelphia, PA) were infected with BAC GFP- EBV and the
infected cells were fixed with 0.5% paraformaldehyde at 6 h, 12 h,
24 h, 48 h, 72 h, 96 h, 120 h and 168 h post-infection for 1 hour
at 4uC and washed three times with buffer W (1X PBS with 0.1%
BSA and 0.001% NaN3). A broad panel of fluorochrome-
conjugated monoclonal antibodies was used for detection of the
following cell surface markers: CD3, CD5, CD10, CD19, CD23,
CD39, CD40, CD44 and Ki-67, respectively. The surface antigen
markers contained different conjugates such as Phycoerythrin (PE),
Phycoerythrin-Cy7 (PE-Cy7), Per CP, PercpCy 5.5 and Allophy-
cocyanin (APC). doi:10.1371/journal.pone.0007214.t002 Induction of virus from 293T cells containing GFP EBV
BACmid Flow cytometry was carried out on an 8-color flow cytometry
instrument CYANADP (Wistar Institute, Philadelphia, PA) with
Cell-Quest software (Becton-Dickinson, San Jose, CA) used in
accordance with the manufacturer’s instructions. Instrument
settings were adjusted so that fluorescence of cells from uninfected
controls, in the case of GFP readings, or negative controls (i.e.,
with antibody omitted in antibody labeling) fell within the first
decade of a four decade logarithmic scale on which emission is
displayed. Flow cytometry plots showed at least 20000 events. The
data were analyzed by FlowJo software (Becton-Dickinson, San
Jose, CA). The expression levels of different surface antigen
markers as well as an intracellular proliferating marker were
analyzed from GFP positive EBV infected cells. The extent of
infected B-cell and T-cell population from total cell populations
were analyzed from CD19 and CD3 positive cells. Cells harboring BAC GFP-EBV were induced to release virus
by culturing for 5 days in complete RPMI 1640 medium
containing phorbol ester TPA (12-O-tetradecanoylphorbol-13-
acetate, 20 ng/ml) and butyric acid (BA, 3 mM; both from
Sigma). Cell suspensions were centrifuged at 1,800 rpm for 10 min
and the supernatant was filtered through a 0.45 micron cellulose
acetate filter. The viral particle were concentrated by ultracentri-
fugation at 27K rpm at 4uC and stored at 280uC. Tranfection of GFP BAC-EBV into 293T cells HEK293T cell were transfected with 5 to 10 mg of BAC GFP-
EBV DNA by lipofectamine 2000 (Invitrogen, Inc., Carlsbad, CA)
according
to
manufacturer
instruction. After
2
days
post
transfection, cells were trypsinised and plated at 104 cells per well
in 96-well tissue culture plates in DMEM medium and the
medium was replaced with fresh 1 mg/ml puromycin-containing
DMEM medium every three days. Puromycin-resistant clones
(shown in Table 2) were screened for the presence of episomally
maintained BAC GFP-EBV by visualization of GFP protein. Table 1. Primers used in PCR analysis[59]. Table 1. Primers used in PCR analysis[59]. Table 1. Primers used in PCR analysis[59]. Primers
Product size
Oligonucleotide sequence
EBV genome
BamHIT sense
350 bp
59-CCCCCTTTTCCGCATCAG-39
140112-140461 bp
BamHIT antisense
59-AGTCCGGATTGGGCACCA-39
BamHIK sense
341 bp
59-GCTGCTTTCCTCGGATGCC-39
112231-112571 bp
BamHIK antisense
59-CTGGGATGGGGAGCGGAG-39
BamHI E sense
1128 bp
59-TACTGCCACCAGTACCACAACA-39
97001-98128 bp
BamHI E antisense
59-GGCCGACATTCTCCAAGATAA-39
BamHI H sense
194 bp
59-CTCTGCCACCTGCAACACTA-39
49117-49310 bp
BamHI H antisense
59-ATTTGGGGTGCTTTGATGAG-39
Fragment C sense:
761 bp
59-GCAGGGCTCGCAAAGTATAG-39
11095-11855 bp
Fragment C antisense
59-TGCGGAAGTGACACCAAATA-39
Puro sense
220 bp
59-CGTGCAGTGCTTCAGCCGCTACCCC-39
152856-153075 bp
Puro antisense
59-CTTGTGCCCCAGGATGTTGCCGTCC-39
EGFP Sense
552 bp
59-GACGTAAACGGCCACAAGTT-39
152716-153267 bp
EGFP antisense
59-CTGGGTGCTCAGGTAGTGGT-39
E3Ct1t2 sense
152 bp
59-AGAAGGGGAGCGTGTGTTGT-39
99939-100091 bp
E3Ct1t2 antisense
59-GGCTCGTTTTTGACGTCGGC-39
EBVGFP sense
820 bp
59-GGGCTCGTTTAAACAAAGTCTCATC-39
151921-152740 bp
EBVGFP antisense
59-CGCTGAACTTGTGGCCGTTTACGTC-39
doi:10 1371/journal pone 0007214 t001 September 2009 | Volume 4 | Issue 9 | e7214 PLoS ONE | www.plosone.org 3 Early Events in EBV Infection Table 2. No. of clones generated in HEK293T and LCLs of BAC
GFP-EBV. Cell
No. of clones
293T
4,10,11,12,15
BAC GFP-EBV
LCL
10,11,14,15
BAC GFP-EBV
doi:10.1371/journal.pone.0007214.t002 Table 2. No. of clones generated in HEK293T and LCLs of BAC
GFP-EBV. Cell
No. of clones
293T
4,10,11,12,15
BAC GFP-EBV
LCL
10,11,14,15
BAC GFP-EBV
doi:10.1371/journal.pone.0007214.t002 Western Blotting Cell lysates were electrophoresed on SDS-PAGE gels and
transferred to 0.45 micron nitrocellulose membranes. Blots were
then probed using specific primary antibodies (S12) for LMP-1
[60] and human serum (KJ) for EBNA-1 and A10 for EBNA-3C
[61] with required dilutions. This was followed by incubation with
fluorescence labeled secondary antibodies, Alexa Fluor 680 and
Alexa Fluor 800 (Molecular Probes Inc., Carlsbad, CA; and
Rockland Inc., Gilbertsville, PA, respectively) diluted at 1:20,000. Blots were visualized and analyzed using LICOR Odyssey
imaging system and Odyssey software (Li-Cor, Lincoln, NE). PLoS ONE | www.plosone.org September 2009 | Volume 4 | Issue 9 | e7214 4 Early Events in EBV Infection The target gene was amplified from cDNA using SYBR green
real-time master mix (MJ Research Inc.,Waltham,MA), 1 mM
each primer and 5 ml of the cDNA product in a total volume of
20 ml. Thirty-five cycles of 1 min at 94uC, 1 min at 55uC and
1 min at 72uC, followed by 10 min at 72uC, were performed in an
MJ Research Opticon II thermocycler (MJ Research Inc.,
Waltham, MA). Each cycle was followed by two plate reads, with
the first at 72uC and the second at 85uC. A melting curve analysis
was performed to verify the specificity of the amplified products,
and the values for the relative quantitation were calculated by the
DDCt method [62]. All experiments were performed in triplicate. of two bands after cassette introduction was confirmed by
Southern blot analysis using the BamHI I fragment as a probe
(Figure 1B, right panel). Inclusion of the GFP cassette at this site
was also confirmed by amplifying the junction region of EBV gene
and GFP cassette. The amplification of an 820 bp size PCR
product, which is shown in Figure 2, is identical with the predicted
size shown in schematic
diagram. The
GFP incorporated
MD1BAC was then designated as BAC GFP-EBV. To test whether the BAC GFP-EBV clone was competent for
viral replication and B-cell immortalization, the BAC GFP-EBV
was induced for replication in 293T cells [29,54]. Highly pure
BAC GFP-EBV DNA was prepared and transfected into HEK
293T cell. Two days after transfection, 10–20% of the cells
typically showed GFP expression. Transfected cells were selected
by puromycin in 96 well plates. After Four to six weeks of
puromycin selection, 5 stable HEK 293T clones of BAC GFP-
EBV expressing GFP were selected for further analysis. Immunofluorescence GFP infected PBMC cells after different time intervals of
postinfection were dried onto slides and fixed using a 1:1 mixture
of acetone and methanol. After fixation cells were extensively
washed in PBS and incubated in blocking buffer [PBS supplement-
ed with 0.1% Triton-X 100, 0.2% fish skin gelatin (Sigma)] at room
temperature for 30 min. Endogenous expression of gp350 and
gp110 were detected using mouse monoclonal antibody (1:500
dilution), and rabbit (1:250 dilution) respectively. Primary antibod-
ies were diluted in blocking buffer and incubated with fixed cells for
1 h at RT. Slides were washed three times (5 min each) with PBS
and incubated with appropriate secondary antibody (1:2000) for 1 h
at RT followed by three times washes (5 min each) with PBS. The last wash contained 49, 69-diamidino-2-phenylindole (DAPI;
Promega Inc., Madison, WI) for nuclear staining. Goat anti-mouse
antibody Alexa Fluor 594 and goat anti-rabbit antibody Alexa Fluor
594 were purchased from Molecular Probes Inc.(Carlsbad, CA). Slides were then washed in PBS and mounted using Prolong anti-
fade (Molecular Probes Inc, Carlsbad, CA). Fluorescence was
viewed by confocal microscopy and analyzed with Fluoview 300
software from Olympus Inc. (Melville, NY). Incorporation of GFP into BAC EBV (MD1BAC) and
selection in mammalian epithelial cells To incorporate the GFP cassette into the BAC vector sequences
containing the entire EBV genome, designated as MD1BAC [29],
we transfected a GFP cassette which contained GFP, puromycin
and ampicillin resistance genes under the control of the CMV
promoter into EL350 by homologous recombination between the
viral genome (Fig. 1A). Ampicillin was introduced along with the
GFP cassette into EBV genome with the BAC vector backbone for
selection in E.coli [29]. Viral genome position 152,008 was chosen
for insertion of the cassette as there is no identifiable open reading
frame from 151,959 to 152,058 i.e. 100 bp is unique sequence in
B95-B EBV genome [59]. The GFP cassette was amplified from GFP-pBSpuro cassette
[63] with 50 nucleotides of EBV genome (151,959-152,008 and
152,008-152,058) at either terminal of the cassette (Figure 1A); the
amplified cassette was then transfected in EL350 with MD1BAC
for homologous recombination and the clones were screened on
amp-kan plate (kanamycin was already inserted into BAC EBV in
MD1BAC [29]). The DNA was extracted from amp-kan resistant
EL350 colonies and digested with BamHI. The digestion pattern
of BAC GFP-EBV is shown in Figure 1B (left panel). The GFP
cassette had a BamHI site, therefore the BamHI fragment
containing the 152,008 site split into two fragments of 4,981 bp
and 4,297 bp after BamHI digestion (Figure 1B, arrow heads). The digestion profile compared with MD1BAC clearly indicated
that there were full EBV genomes with the GFP cassette
incorporated at the desired site (Figure 1B, left panel). Generation Western Blotting The GFP
signals for two such clones are shown in Figure 3B. Persistence of
the full length EBV genome was determined by PCR analysis of
hirt extracted DNA from these stable cell lines using 5 primer sets
across the entire genome (Figure 3A). The PCR results suggested
the presence of full length intact EBV containing GFP and
Puromycin markers within the BAC GFP-EBV. Amplification of
the different regions across the EBV genome confirmed that these
stable cells were able to maintain the intact BAC GFP-EBV. Infection of primary B-cell with BAC GFP-EBV and
establishment of LCLs BAC GFP-EBV virus from the stable 293T cells were induced
by treating with chemical inducers, TPA and butyric acid for 4
days [29]. The supernatant containing GFP EBV was used to
infect PBMC after concentrating by ultracentrifugation. Human
primary B-cells were infected by using different volumes of the
concentrated virus. The infected cells were incubated at 37uC and
grown overnight. The cells having high GFP expression and B-cell
clumping were then diluted and plated into 96 well plates and
incubated for 3–4 weeks. The infection was monitored by GFP
expression. Approximately, 20–30% of the cells were positive for
green flourescence after 36 hours of infection. Four weeks later,
these green cells were clearly transformed to LCLs. The GFP
content in LCL was enhanced by puromycin screening, and stable
LCLs with GFP expression were obtained from the above pool of
cells as shown in Figure 4B. To monitor whether or not the GFP-
EBV positive LCLs were intact, the genome was analyzed by PCR
amplification across different regions of the EBV genome
(Figure 4A). PCR analysis of the different region of the EBV
genome in LCLs suggested that the LCL maintained an intact
EBV genome along with GFP cassette (Figure 4A). Analysis of EBV latent proteins expression in GFP-EBV
positive LCLs In latency type III, six nuclear antigens (EBNA1, EBNA2,
EBNA3A, EBNA3B, EBNA3C, and EBNA-LP), three latent
membrane proteins (LMP1, LMP2A, and LMP2B), two small
nonpolyadenylated RNAs (EBER-1 and EBER-2), and transcripts
from the BamHI-A region (BARTs) are expressed [64]. To further
investigate the establishment of latent infection due to GFP-EBV,
the latent protein expression profile of the critical latent antigens in
these GFP-EBV transformed LCLs were analysed. The protein
expression in HEK 293T containig BAC GFP-EBV and GFP-
EBV infected LCLs were analysed by immunoblotting along with
BJAB as negative control and previously created LCLs as a
positive control (Figure 5). Antibodies detecting EBNA3C and PLoS ONE | www.plosone.org September 2009 | Volume 4 | Issue 9 | e7214 5 Early Events in EBV Infection Figure 1. Strategy for insertion of the GFP cassette into BAC EBV (MD1BAC). (A) BamHI region of EBV genome (149116 bp -154747) [59]
was targeted for homologous recombination. A DNA fragment was generated by PCR amplification of a GFP/AMP cassette with a BamHI site using
primers incorporating 50 nucleotides of the EBV genome upstream of the 152008 nucleotide and downstream of the 152009 nucleotide at the 59
termini. The fragment was electroporated into E.coli EL350 carrying EBV bacmid (MD1BAC) and expressing recombinase to allow homologous
recombination. After recombination the BamHI site splits to two fragments compared to BACMD1. (B) The BamHI digestion pattern of BAC GFP-EBV
and MD1BAC was visualised in 0.65% agarose (left panel). The fragments which split in to two fragments are indicated by white arrows. BamHI I
fragment was used as a probe for southern blot analysis (left panel). The southern blot analysis showed that the 5.6 bp fragment from MD1BAC
(white arrow) and two BAC GFP-EBV fragments ,5 kb and ,4.3 kb. doi:10.1371/journal.pone.0007214.g001 Figure 1. Strategy for insertion of the GFP cassette into BAC EBV (MD1BAC). (A) BamHI region of EBV genome (149116 bp -154747) [59]
was targeted for homologous recombination. A DNA fragment was generated by PCR amplification of a GFP/AMP cassette with a BamHI site using
primers incorporating 50 nucleotides of the EBV genome upstream of the 152008 nucleotide and downstream of the 152009 nucleotide at the 59
termini. The fragment was electroporated into E.coli EL350 carrying EBV bacmid (MD1BAC) and expressing recombinase to allow homologous
recombination. After recombination the BamHI site splits to two fragments compared to BACMD1. Analysis of EBV latent proteins expression in GFP-EBV
positive LCLs (B) The BamHI digestion pattern of BAC GFP-EBV
and MD1BAC was visualised in 0.65% agarose (left panel). The fragments which split in to two fragments are indicated by white arrows. BamHI I
fragment was used as a probe for southern blot analysis (left panel). The southern blot analysis showed that the 5.6 bp fragment from MD1BAC
(white arrow) and two BAC GFP-EBV fragments ,5 kb and ,4.3 kb. doi:10.1371/journal.pone.0007214.g001 markers (CD5, CD10, CD23, CD39, CD40, and CD44) as well as
the intracellular proliferation marker (Ki-67) at different times
post-infection. EBNA1 and the oncoprotein LMP-1 were used to detect the
specific antigens. The expression patterns of these proteins are
shown in Figure 5. Detection of EBNA-1, LMP-1 and EBNA-3
proteins clearly showed that the recombinant GFP-EBV was
capable of transforming primary B-cells into LCLs and was also
able to maintain a type III latency program. Since EBV also infects T-cells along with B-cell, we investigated
EBV infected T-cell population by analyzing the expression of
CD3 antigen at different times postinfection. The expression of
CD3 in GFP positive cells showed that 20% of the CD3 positive T
cells were positive by 6 hours infection increasing to 38% by
24 hours. However, this signal was dramically decreased by
48 hours and went down further to 8% after 168 hrs (Figure 6A,
left panel). Of significance, the extent of B-cell infectivity measured
by GFP positive CD19 expression was very high within 6 hours EBV infection induces B-cell activation and cell
proliferation To investigate the immunophenotypic effects of EBV infection
on B-cell during early infection, we analyzed the expression profile
of several B-cell activation and proliferation surface antigen PLoS ONE | www.plosone.org September 2009 | Volume 4 | Issue 9 | e7214 6 Early Events in EBV Infection Figure 2. Verification of recombinant BAC GFP-EBV. (A) The DNA from bacterial colonies screened by kanamycin-ampicillin together and
sequence analysis of the inserted GFP/amp into MD1BAC genome by PCR amplification, with one primer of EBV genome and another primer from
GFP. Amplification of the 820 bp fragment was shown from DNA observed from 4 independent isolates (2–5 lanes) with MD1BAC as negative control. (B) Schematic of EBV showing position of the markers and GFP. doi:10.1371/journal.pone.0007214.g002 Figure 2. Verification of recombinant BAC GFP-EBV. (A) The DNA from bacterial colonies screened by kanamycin-ampicillin togeth
sequence analysis of the inserted GFP/amp into MD1BAC genome by PCR amplification, with one primer of EBV genome and another prime
GFP. Amplification of the 820 bp fragment was shown from DNA observed from 4 independent isolates (2–5 lanes) with MD1BAC as negative c
(B) Schematic of EBV showing position of the markers and GFP. doi:10.1371/journal.pone.0007214.g002 Figure 2. Verification of recombinant BAC GFP-EBV. (A) The DNA from bacterial colonies screened by kanamycin-ampicillin together and
sequence analysis of the inserted GFP/amp into MD1BAC genome by PCR amplification, with one primer of EBV genome and another primer from
GFP. Amplification of the 820 bp fragment was shown from DNA observed from 4 independent isolates (2–5 lanes) with MD1BAC as negative control. (B) Schematic of EBV showing position of the markers and GFP. doi:10.1371/journal.pone.0007214.g002 about 30% by 6 hours postinfection increasing to about 50% by 72
hours post-infection and then showed a rapid decline immediately
after which continued to 7 days later (Figure 6C, left panel). Importantly, a well-known B-cell activation and proliferation
marker CD40 was detected in about 15% of the GFP-positive cells
and gradually increased to over 50% by 7 days postinfection
(Figure 6C, midle panel). The expression of CD44 was unchanged
throughout the events of early infection (Figure 6C, right panel). However, the percentage of EBV positive cells as determined by
GFP expressing CD39 was significant, approximately 90%, but
gradually decreased to 42% by 7 days postinfection (data not
shown). postinfection increasing to above 90% after 7 days postinfection
(Figure 6A, right panel). PLoS ONE | www.plosone.org September 2009 | Volume 4 | Issue 9 | e7214 EBV infection induces B-cell activation and cell
proliferation PBMCs were infected by GFP-EBV and the infected cells were
subjected to flow cytometry analysis at different times post-
infection. The expression of the indicated surface antigen markers
were determined up to 7 days for GFP positive EBV infected cells. The expression of the CD5 cell surface marker did show a
gradual change during the course of early infection. By 6 hours
post-infection only 28% of the GFP positive cells were expressing
CD5 (Figure 6B). This was increased to 54% by 5 days later. Interestingly the percent of GFP positive cells that were CD5
positive were dramatically less by 6–7 days suggesting that this
initial increase was not sustained after 5 days with development of
LCLs (Figure 6A, left panel). However, the CD10 activation
marker showed that about 30% of the GFP-positive cells expressed
CD10 by 6 hours postinfection and this was relatively unchanged
by 7 days postinfection (Figure 6A, middle panel). The expression
pattern of CD23 during early infection with GFP-EBV was
interesting with an increase in the percentage of GFP positive cells To study proliferation due to infection of primary B-cell by
GFP-EBV on B-cell, we monitored expression of the intracellular
proliferation marker Ki-67 [34] by Flow Cytometry. Ki-67 was
expressed at 48 hrs of post-infection of GFP EBV and its signal was
consistently increased up to 7 days post-infection (Figure 6B, right
panel). Importantly, analysis of LCLs showed extremely high levels
of Ki-67 suggesting a requirement for Ki-67, or that it has a PLoS ONE | www.plosone.org 7 September 2009 | Volume 4 | Issue 9 | e7214 Early Events in EBV Infection Figure 3. Transfection of Full length BAC GFP-EBV into 293T cells. (A) BAC GFP-EBV DNA was transfected to 293T and stable cell line
generated as screened by puromycin. Different genes encoding by EBV (BamHI T, BamHI K, BamHI E, BamHI H and BamHI C) as well as puromycin and
GFP were analyzed by PCR amplification taking 5 different transfected clones (4, 10, 11, 12 and 15). BJAB and LCL were taken as negative and positive
control, respectively. (B) Phase contrast (left panel) and Flourescence images (right panel) showed two indifferent (number 4 and 12) containing BAC
GFP-EBV transfected into 293T stable cell line screened by puromycin. doi:10.1371/journal.pone.0007214.g003 Figure 3. Transfection of Full length BAC GFP-EBV into 293T cells. EBV infection induces B-cell activation and cell
proliferation (A) BAC GFP-EBV DNA was transfected to 293T and stable cell line
generated as screened by puromycin. Different genes encoding by EBV (BamHI T, BamHI K, BamHI E, BamHI H and BamHI C) as well as puromycin and
GFP were analyzed by PCR amplification taking 5 different transfected clones (4, 10, 11, 12 and 15). BJAB and LCL were taken as negative and positive
control, respectively. (B) Phase contrast (left panel) and Flourescence images (right panel) showed two indifferent (number 4 and 12) containing BAC
GFP-EBV transfected into 293T stable cell line screened by puromycin. doi:10 1371/journal pone 0007214 g003 Figure 4. LCLs established by infection with GFP-EBV. (A) PBMC cells were infected by GFP- EBV and made GFP positive LCLs selected with
puromycin. The proliferating cells are clustered and GFP positive. BAC GFP-EBV transfected 293T cell clones and LCL established EBV encoded
different region of EBV (E3CT1T2, BamHI H, BamHI K, BamHI T and BamHI C) as well as puromycin and GFP were checked by PCR amplification taking
2 different infected LCL clones (11 and 14). BJAB was used as negative control and LCL1 & LCL2 were used as positive controls, respectively. (B) Phase-
contrast (left) and fluorescence (right) images of 2 different established (11 and 14) GFP-EBV LCLs. doi:10.1371/journal.pone.0007214.g004 Figure 4. LCLs established by infection with GFP-EBV. (A) PBMC cells were infected by GFP- EBV and made GFP positive LCLs selected with
puromycin. The proliferating cells are clustered and GFP positive. BAC GFP-EBV transfected 293T cell clones and LCL established EBV encoded
different region of EBV (E3CT1T2, BamHI H, BamHI K, BamHI T and BamHI C) as well as puromycin and GFP were checked by PCR amplification taking
2 different infected LCL clones (11 and 14). BJAB was used as negative control and LCL1 & LCL2 were used as positive controls, respectively. (B) Phase-
contrast (left) and fluorescence (right) images of 2 different established (11 and 14) GFP-EBV LCLs. doi:10.1371/journal.pone.0007214.g004 September 2009 | Volume 4 | Issue 9 | e7214 PLoS ONE | www.plosone.org 8 Early Events in EBV Infection Figure 5. Latent gene expression. Latent proteins expressions were
analyzed in cells infected with in BAC GFP-EBV stably transfected 293T
and LCLs made from BAC GFP-EBV infection. Expression of EBV EBNA-1,
EBNA-3C and LMP-1 were determined by Western blotting. doi:10.1371/journal.pone.0007214.g005 during the initial stages of infection i.e. The progeny GFP-EBV produced during early infection is
infectious To investigate further our hypothesis that early lytic infection
produces viral progeny capable of infecting new cells, we used
supernatant collected during the early time points to infect new
cells. PBMCs were infected with supernatant collected at the
different times and GFP-EBV was monitored by GFP expression
using a fluorescence microscope as well as FACS to 7 days
(Figure 8A and B, left panel). After 24 hours post-infection, GFP
signals and clumping of the primary B-cells were visualized. FACS
analysis also showed that 5% of the cells were GFP positive by 12
hours and increased to 20% after 72 hrs post infection (Figure 8A
and 8B). Importantly, we showed that the lytic genes were
expressed at a higher level when compared to latent genes
suggesting a burst of lytic infection and particle release. To
examine whether the lytic replication cycle produces virion
particles released into the supernatant at each time point post
infection, the supernatant was collected and used to infect fresh
PBMCs. The infection of fresh PBMCs was monitored for GFP
expression. The result showed that the supernatant from 72 hours
post infection was capable of infecting fresh PBMCs followed by
GFP expression and cell clumping (Figure 8B, right panel). The
percentage of GFP expressing cells as determined by FACS was 1–
2% from 72 h to 168 h. This result strongly suggested a burst of
lytic replication and release of virion particles during the initial
stages of EBV infection. However, the number of infected cells was
significantly higher when the supernatant from 7 days of post-
infection was used to infect new cells. Figure 5. Latent gene expression. Latent proteins expressions were
analyzed in cells infected with in BAC GFP-EBV stably transfected 293T
and LCLs made from BAC GFP-EBV infection. Expression of EBV EBNA-1,
EBNA-3C and LMP-1 were determined by Western blotting. doi:10.1371/journal.pone.0007214.g005 critical role in initiating and maintaining the transformed state
induced by the virus. For further investigation of the immunophenotypic effects of
EBV infection on B-cell during early infection, we also analyzed
the expression profiles of several B-cell activation and proliferation
surface antigen markers (CD5, CD10, CD23, CD39, CD40, and
CD44) as well as the intracellular proliferation marker (Ki-67) at
different times post-infection in non-infected B-cells i.e. GFP
negative cells. The progeny GFP-EBV produced during early infection is
infectious Flowcytometry analysis from GFP-negative cells
showed that there was no significant change in surface antigen
markers
(CD5,
CD10,
CD23,
CD40
and
CD44)
or
the
intercellular marker Ki-67 during early infection as shown in
figure 6D and figure 6E. Rapid induction of EBV lytic replication during early
infection of human primary B-cells EBV establishes different types of latency characterized by
differential expression of a group of latency proteins [14]. To
evaluate the gene expression pattern during early events of
infection, GFP-EBV was used to infect PBMCs and latent and lytic
gene expression were analyzed. Latent expression of EBNA-1,
EBNA-2 and LMP-1 and the lytic genes immediate early
transactivator (BZLF1), major capsid protein (BcLF-1) and DNA
polymerase (BALF5) were monitored. The transcript levels of
these genes were determined from the total RNA extracted from
GFP-EBV infected PBMCs at different time points post-infection
up to 7 days (Figure 7). EBV infection induces B-cell activation and cell
proliferation it came on at 6 hrs post-
infection and peaked at 24 hrs (Figure 7B, left panel). Interestingly,
the BZLF1 transcripts decreased after 24 hrs but began to increase
again by 96–120 hours post-infection. Another lytic gene BcLF-1
was detected at 6 hrs post-infection and continued to increase
throughout the course of study. The expression of DNA
polymerase (BALF5) during early infection peaked by 12 hours
post-infection followed by a gradual reduction to 96 hrs. However,
the level of BALF5 was also increased again by 7 days (Figure 7B). Interestingly, the levels of lytic transcripts as determined by Real
time PCR was greater than that compared to the latent genes
during early stage of B-cell infection by GFP-EBV. PLoS ONE | www.plosone.org Production of progeny GFP-EBV particles during early
infection is inhibited by acyclovir To investigate lytic gene expression as well as the release of
virion particles during early stages of infection, we monitored
GFP-EBV infection to PBMCs in the presence of 25 mM ACV. PBMCs were infected with GFP-EBV with and without 25 mM
ACV at the different times post-infection and infection was
monitored by GFP expression using a fluorescence microscope as
well as FACS analysis up to 7 days (Figure 9A, left panel and 9C). After 24 hours post-infection, GFP signals and clumping of the
primary B-cells were clearly observed. FACS analysis also showed
that 5% of the cells were GFP positive by 24 hours and increased
to 20% after 72 hrs post infection, whereas in the presence of
25 mM ACV, the extent of GFP expression decreased (Figure 9A,
right panel and 9B). It was observed that GFP expression was 13–
15% in presence of ACV, whereas in absence of ACV it was
,20%. The proliferation marker Ki-67 was also measured by flow
cytometry in presence of ACV (Figure 9, right panel). The results
showed that in the presence of 25 mM ACV, the expression of Ki-
67 decreased by 1.5 fold. To test the inhibition effect of ACV on
viral DNA polymerase synthesis, we checked the level of viral The copies of latent gene transcripts at different time points
post-infection of GFP-EBV was determined by semi-quantitative
real time PCR as shown in Figure 7A. Expression of EBNA-1 was
barely detectable at 24 hrs, but was clearly detected at 48 hrs and
peaked by 120 hrs. EBNA2 signals were evident by 6 hrs and
plateaued by 24 hrs post-infection (Figure 7A, middle panel). EBNA-2 transcript was consistently detected through 7 days of
infection at similar levels up to 7 days throughout the course of the
study. Similarly, LMP-1 transcript levels was detectable at 6 h and
reached at maximum level by 24 hours of post-infection
(Figure 7A, right panel). Interestingly, the level of LMP-1
transcripts remained consistent up to 7 days post infection. The mRNA levels of lytic genes BZLF1, BcLF1 and BALF5
were also determined using real time qPCR at different time points
post infection. We found that the BZLF-1 gene was expressed September 2009 | Volume 4 | Issue 9 | e7214 PLoS ONE | www.plosone.org 9 Early Events in EBV Infection Figure 6. Expression of B-cell proliferation and activation markers due to GFP-EBV early infection. Production of progeny GFP-EBV particles during early
infection is inhibited by acyclovir (A) Th
designated time of GFP-EBV postinfection (6h, 12h, 24h, 48h, 72h, 96h, 120h and 168h) of early infection, the expression of latent genes EBNA-1
EBNA-2 and LMP-1 mRNAs were examined by qReal Time PCR. (B) The lytic genes mRNAs BZLF1 which is the immediate early transcriptional and
replication protein, major capsid protein BcLF1 and DNA polymerase BALF5 mRNA were also examined by qReal Time PCR after GFP-EBV infection a
similiar intervals stated above. To determine quality of the RNA, GAPDH mRNA was also amplified by RT-PCR. The fold change was calculated by th
DDCt method Each data point shown is the average of three identical experiments 6 SD was shown in error bar Figure 7. Latent and lytic gene expression during GFP-EBV early infection. 56106 of PBMC cells were infected with GFP-EBV. (A) The
designated time of GFP-EBV postinfection (6h, 12h, 24h, 48h, 72h, 96h, 120h and 168h) of early infection, the expression of latent genes EBNA-1,
EBNA-2 and LMP-1 mRNAs were examined by qReal Time PCR. (B) The lytic genes mRNAs BZLF1 which is the immediate early transcriptional and
replication protein, major capsid protein BcLF1 and DNA polymerase BALF5 mRNA were also examined by qReal Time PCR after GFP-EBV infection at
similiar intervals stated above. To determine quality of the RNA, GAPDH mRNA was also amplified by RT-PCR. The fold change was calculated by the
DDCt method. Each data point shown is the average of three identical experiments. 6 SD was shown in error bar. doi:10.1371/journal.pone.0007214.g007 DNA polymerase (BALF5) gene expression by Real Time PCR in
the presence of ACV shown in Figure 9D. The results clearly
showed that in the presence of 25 mM ACV, the expression of
BALF5 mRNA decreased by 4–5 fold decreased compared to the
absence of ACV. These results suggest that in the presence of
ACV, the rate of lytic replication is inhibited which results in lower
infection of GFP-EBV and B-cell proliferation. expression was dramatically decreased. At 168h post-infection, the
level of gp110 expression was higher in the absence of ACV as
compared to the levels in the presence of ACV. The same result
was also seen for gp350 expression (Figure 10B). Production of progeny GFP-EBV particles during early
infection is inhibited by acyclovir Thus, the late
protein expression profiles in ACV suggested that the productive
cycle was inhibited by acyclovir and further supportive our
conclusions that the bursts of replication and virion particle
production during the early stages of infection is likely to be crucial
for establishment of latency and transformation of the infected
primary B-cells. To examine the effect of ACV on the lytic replication cycle as
well as the production of virion particles released into the
supernatant at each time point postinfection, the supernatants
from infection-I were collected and used to infect fresh PBMCs
(infection-II). Infection-II was monitored by visualization GFP
using fluorescence microscopy (Figure 9B). The GFP results
showed that the release of virion particle was inhibited in presence
of ACV at Infection-I. This data strongly supports the conclusion
that virion particles produced due to burst of lytic replication
during early stages of infection. PLoS ONE | www.plosone.org Production of progeny GFP-EBV particles during early
infection is inhibited by acyclovir 26106 PBMC were
EBV and (A) describe CD3 and CD19 GFP population. (B) the expression of proliferation markers (CD5, CD10 and Ki-67) and (C) a
(CD23, CD40 and CD44) were measured by Flow cytometry after different time post-infection (6h, 12h, 24h, 48h, 96h, 120h and 16
infected cells (i.e. GFP-positive cells). (D) The expression of proliferation markers (CD5, CD10 and Ki-67) and (E) activation markers
CD23) were measured by Flow cytometry after different time post-infection (6 h, 12 h, 24 h, 48 h, 96 h, 120 h and 168 h) with GFP-EB
cells (i.e. GFP-negative B-cells). doi:10.1371/journal.pone.0007214.g006 Figure 6. Expression of B-cell proliferation and activation markers due to GFP-EBV early infection. 26106 PBMC were infected by GFP-
EBV and (A) describe CD3 and CD19 GFP population. (B) the expression of proliferation markers (CD5, CD10 and Ki-67) and (C) activation markers
(CD23, CD40 and CD44) were measured by Flow cytometry after different time post-infection (6h, 12h, 24h, 48h, 96h, 120h and 168h) with GFP-EBV
infected cells (i.e. GFP-positive cells). (D) The expression of proliferation markers (CD5, CD10 and Ki-67) and (E) activation markers (CD40, CD44 and
CD23) were measured by Flow cytometry after different time post-infection (6 h, 12 h, 24 h, 48 h, 96 h, 120 h and 168 h) with GFP-EBV non-infected B-
cells (i.e. GFP-negative B-cells). doi:10.1371/journal.pone.0007214.g006 September 2009 | Volume 4 | Issue 9 | e7214 PLoS ONE | www.plosone.org 10 Early Events in EBV Infection Figure 7. Latent and lytic gene expression during GFP-EBV early infection. 56106 of PBMC cells were infected with GFP-EBV. (A) The
designated time of GFP-EBV postinfection (6h, 12h, 24h, 48h, 72h, 96h, 120h and 168h) of early infection, the expression of latent genes EBNA-1,
EBNA-2 and LMP-1 mRNAs were examined by qReal Time PCR. (B) The lytic genes mRNAs BZLF1 which is the immediate early transcriptional and
replication protein, major capsid protein BcLF1 and DNA polymerase BALF5 mRNA were also examined by qReal Time PCR after GFP-EBV infection at
similiar intervals stated above. To determine quality of the RNA, GAPDH mRNA was also amplified by RT-PCR. The fold change was calculated by the
DDCt method. Each data point shown is the average of three identical experiments. 6 SD was shown in error bar. doi:10.1371/journal.pone.0007214.g007 Figure 7. Latent and lytic gene expression during GFP-EBV early infection. 56106 of PBMC cells were infected with GFP-EBV. Discussion Using the BAC system, the viral genome can be propagated in
Escherichia coli, and mutations can be rapidly and precisely
introduced into any of the viral genes. To facilitate the generation
of recombinant viruses, the EBV genome was first cloned into the
bacterial artificial chromosome (BAC) [28,29]. The resultant BAC
based EBV genome was able to make virus and was capable of B-
cell immortalization [29]. To monitor infection of primary B-cell
by EBV, we introduced the GFP ORF into the EBV BAC by
homologous recombination and the resultant construct was
designated as BAC GFP-EBV [29]. The construction of BAC
GFP-EBV was confirmed by exhaustive restriction enzyme
digestion pattern, PCR analysis of the junctions, selected regions
of EBV as well as southern blot analysis. In addition, the new BAC To support the above results that lytic replication occurred in
EBV infected cells , we checked the expression of late genes
glycoproteins gp110 and gp350 by detection of the protein sing
fluorescence microscopy. PBMCs were infected with GFP-EBV. The
expressions
of
glycoprotein
(gp110
and
gp350)
were
monitored by immunoflourescence analysis in the presence and
absence of ACV at different times post-infection (Figure 10A and
10B). The results showed that in absence of ACV gp110 was
expressed at 96 h whereas in presence of ACV, the gp110 PLoS ONE | www.plosone.org September 2009 | Volume 4 | Issue 9 | e7214 11 Early Events in EBV Infection Figure 8. Lytic burst of EBV virus during early infection of GFP-EBV. PBMC cells were infected with GFP-EBV and at specific times
postinfection the supernanat was collected and used infect fresh PBMC cells. (A) Phase-contrast (left) and fluorescence (right) images of GFP-EBV
infected PBMC cells are shown after specific times postinfection (24 hrs, 72 hrs, 120 hrs and 168 hours) (left panel). Phase-contrast (left) and
fluorescence (right) images of PBMC cells infected with supernatant from the above mentioned times post-infection (from 24hrs, 72hrs, 120hrs and
168h) (right panel). B. Flow cytometry analysis of GFP expression at post-infection of different time intervals (6h, 12h, 24h, 48h, 72h, 96h, 120h and
168h) (left panel), and after infection of fresh PBMC from supernatant of infected cells (right panel). doi:10.1371/journal.pone.0007214.g008 Figure 8. Lytic burst of EBV virus during early infection of GFP-EBV. PBMC cells were infected with GFP-EBV and at specific times
postinfection the supernanat was collected and used infect fresh PBMC cells. Discussion (A) Phase-contrast (left) and fluorescence (right) images of GFP-EBV
infected PBMC cells are shown after specific times postinfection (24 hrs, 72 hrs, 120 hrs and 168 hours) (left panel). Phase-contrast (left) and
fluorescence (right) images of PBMC cells infected with supernatant from the above mentioned times post-infection (from 24hrs, 72hrs, 120hrs and
168h) (right panel). B. Flow cytometry analysis of GFP expression at post-infection of different time intervals (6h, 12h, 24h, 48h, 72h, 96h, 120h and
168h) (left panel), and after infection of fresh PBMC from supernatant of infected cells (right panel). doi:10.1371/journal.pone.0007214.g008 GFP-EBV clone was also competent for virus replication and B-
cell immortalization. The induction of 293T cells stably main-
taining the BAC GFP-EBV by chemical inducers produced
progeny recombinant virus which was used to infect primary B-
cells leading to the generation of LCLs expressing GFP. These
studies strongly suggested that the recombinant GFP-EBV had
similar infectious properties when compared to the wild type EBV. The advantage of this GFP expressing BAC-EBV system is that
EBV infection and propagation in mammalian cells can now be
monitored from the early stages post-infection to transformation of
B-cells and generation of LCLs. the early stages post-infection. We used a panel of surface antigen
markers CD5, CD10, CD19, CD23, CD39, CD40 & CD44 and
the intracellular marker Ki-67 to correlate B-cell activation with
proliferation during the early stages of infection up to 7 days. GFP
positive cells were evaluated for expression of the surface proteins
indicated. CD19, a specific surface antigen marker for B-cells
[66,67], was detected in greater than 90% of the GFP positive
cells. This strongly suggested that the GFP-EBV infected cells were
predominantly B-cells among the mixed population of PBMCs. The cell surface protein CD5 expression on B cell can be up-
regulated by a number of agents which results in B-cell activation
[35]. CD5 has been shown to be important for apoptosis of
antigen-receptor induced B lymphocytes [68]. The expression of
CD5 was also shown to be regulated by EBV [37]. CD5
expression was suppressed in EBV transformed cells suggesting
that that the virus may down-regulate its expression to prevent
apoptosis of the transformed cell. Our data during the early stages The infection of human B lymphocytes by EBV in vitro results in
immortalization of the infected cells and augmentation of
numerous B-cell surface antigens[65]. PLoS ONE | www.plosone.org September 2009 | Volume 4 | Issue 9 | e7214 Discussion We wanted to obtain a
more detailed picture of the early events after EBV infection and
to monitor changes in expression of cell surface markers as a result
of infection. We used infection of PBMCs by GFP-EBV to monitor September 2009 | Volume 4 | Issue 9 | e7214 PLoS ONE | www.plosone.org 12 Early Events in EBV Infection Figure 9. The progeny virus produced in the primary infection is inhibited by acyclovir. PBMCs were infected with GFP-EBV in
presence and absence of 25 mM of ACV (infection-I) and at specific times postinfection the supernatant was collected and used
infect fresh PBMCs cells (infection II). (A) Phase-contrast (left) and fluorescence (right) images of GFP-EBV infected PBMC cells are shown after
specific times postinfection (24 hrs, 72hrs, 120 hrs and 168 hours) in absence of ACV (left panel) and in presence of 25 mM ACV (right panel). (B) Phase-
contrast (left) and fluorescence (right) images of PBMC cells infected with supernatant from the above mentioned times post-infection from 24hrs,
72hrs, 120hrs and 168h (infection-II) in absence (left panel) and presence of 25 mM ACV (right panel). (C) Flow cytometry analysis of GFP (left panel)
and Ki-67(right panel) expression at post-infection of different time intervals (6h, 12h, 24h, 48h, 72h, 96h, 120h and 168h) in absence and presence of
25 mM ACV. D. DNA polymerase BALF5 mRNA was also examined by qReal Time PCR after GFP-EBV infection at similiar intervals stated above in
absence and presence of 25 mM ACV. To determine quality of the RNA, GAPDH mRNA was also amplified by RT-PCR. The fold change was calculated
by the DDCt method. Each data point shown is the average of three identical experiments. 6 SD was shown in error bar. doi:10.1371/journal.pone.0007214.g009 Figure 9. The progeny virus produced in the primary infection is inhibited by acyclovir. PBMCs were infected with GFP-EBV in
presence and absence of 25 mM of ACV (infection-I) and at specific times postinfection the supernatant was collected and used
infect fresh PBMCs cells (infection II). (A) Phase-contrast (left) and fluorescence (right) images of GFP-EBV infected PBMC cells are shown after
specific times postinfection (24 hrs, 72hrs, 120 hrs and 168 hours) in absence of ACV (left panel) and in presence of 25 mM ACV (right panel). Discussion (B) Phase-
contrast (left) and fluorescence (right) images of PBMC cells infected with supernatant from the above mentioned times post-infection from 24hrs,
72hrs, 120hrs and 168h (infection-II) in absence (left panel) and presence of 25 mM ACV (right panel). (C) Flow cytometry analysis of GFP (left panel)
and Ki-67(right panel) expression at post-infection of different time intervals (6h, 12h, 24h, 48h, 72h, 96h, 120h and 168h) in absence and presence of
25 mM ACV. D. DNA polymerase BALF5 mRNA was also examined by qReal Time PCR after GFP-EBV infection at similiar intervals stated above in
absence and presence of 25 mM ACV. To determine quality of the RNA, GAPDH mRNA was also amplified by RT-PCR. The fold change was calculated
by the DDCt method. Each data point shown is the average of three identical experiments. 6 SD was shown in error bar. doi:10.1371/journal.pone.0007214.g009 was maintained after 6 hrs followed by activation and cell
proliferation. of EBV infection showed that CD5 levels increased from 6 hrs to 5
days but was depressed after 5 days to relatively low levels seen in
LCLs. Our data supports previous studies monitoring CD5 levels
in transformed B-cell [37]. However, during the initial days of
EBV infection (up to 5 days), the increased expression of CD5 is
likely to be due to signaling as a result of virus-host interaction and
resulting B-cells activation. Also, the infection of primary B-cell by
EBV leads to killing of a large percentage of cells due to lytic
replication during the early stage of infection. This resulting cell
death may also be a reason for up-regulation of CD5 expression
during the early stages of infection. CD10, another cell surface
protein, preferentially expressed in germinal center is an activation
marker for B-cells as the germinal center is the site for activation
and proliferation of B-cells [69]. The expression of CD10 was
unchanged after 6 hrs post-infection during the course of early
infection of EBV. This suggests that the initial interaction between
EBV and B-cells led to a rapid change in CD10 expression, which of EBV infection showed that CD5 levels increased from 6 hrs to 5
days but was depressed after 5 days to relatively low levels seen in
LCLs. Our data supports previous studies monitoring CD5 levels
in transformed B-cell [37]. September 2009 | Volume 4 | Issue 9 | e7214 Discussion However, during the initial days of
EBV infection (up to 5 days), the increased expression of CD5 is
likely to be due to signaling as a result of virus-host interaction and
resulting B-cells activation. Also, the infection of primary B-cell by
EBV leads to killing of a large percentage of cells due to lytic
replication during the early stage of infection. This resulting cell
death may also be a reason for up-regulation of CD5 expression
during the early stages of infection. CD10, another cell surface
protein, preferentially expressed in germinal center is an activation
marker for B-cells as the germinal center is the site for activation
and proliferation of B-cells [69]. The expression of CD10 was
unchanged after 6 hrs post-infection during the course of early
infection of EBV. This suggests that the initial interaction between
EBV and B-cells led to a rapid change in CD10 expression, which The B-cell activation markers CD23, CD40 and CD44 have
been shown to be associated with EBV infection [13]. Addition-
ally, the viral proteins EBNA-2 and LMP-1 cooperatively induce
the cell surface protein, CD23 [13]. CD23 expression induced by
infection of B-cell with GFP-EBV showed an interesting trend in
that CD23 expression increased and reached maximum levels by
72 hrs but then dramatically decreased by 7 days. However,
EBNA-2 was expressed at the initial stage of early infection and
reached maximum within 24 hours. These results suggest that the
expression of EBNA-2 at the initial stage activates the CD23
expression which continued to increase up to 72 hrs. In addition,
LMP-1 expression was detected after 48 hours which is also known
to up-regulate CD23 expression [70]. Moreover, the increase in
lytic replication by EBV during the early stages post infection is
also expected to lead to cell death. This provides a possible PLoS ON PLoS ONE | www.plosone.org September 2009 | Volume 4 | Issue 9 | e7214 13 Early Events in EBV Infection Figure 10. Glycoprotein expression during early stage of
infection in presence of acyclovir. Endogenous expression of (A)
gp110 and (B) gp350 were detected using mouse monoclonal antibody
(1:200 dilution), and rabbit respectively (1:250 dilution). Primary
antibodies were diluted in blocking buffer and incubated with fixed
cells for 1 h at RT. Discussion Slides were washed three times (5 min each) with PBS
and incubated with appropriate secondary antibody (1:2000) for 1 h at
RT followed by three times washes (5 min each) with PBS. The last wash
contained 49, 69-diamidino-2-phenylindole (DAPI; Promega Inc., Madi-
son, WI) for nuclear staining. Goat anti-mouse antibody Alexa Fluor 594
and goat anti-rabbit antibody Alexa Fluor 594 were purchased from
Molecular Probes Inc. (Carlsbad, CA). Slides were then washed in PBS
and mounted using Prolong anti-fade (Molecular Probes Inc, Carlsbad,
CA). Fluorescence was viewed by confocal microscopy and analyzed
with Fluoview 300 software from Olympus Inc. (Melville, NY). The
images were sequentially captured using an Olympus confocal
microscope. All panels are representative pictures from similar repeat
experiments. d i
/j
l infection also activates the CD40 signaling pathway which
suppresses apoptosis and promotes proliferation of infected cells
[73]. CD44 is a cell adhesion molecule which exists in multiple
isoforms associated with tumorigenesis and metastasis [74]. The
level of CD44 was detected by 6 hrs post-infection and was
maintained
throughout
our
study
suggesting
an
important
contribution to the proliferation and transformation process. Ki-67 is a nuclear antigen that is expressed in proliferating cells
during the different phases of the cell cycle and its expression is used
as a marker for cell proliferation [34]. It is reported that Ki-67 is
expressed in CD19 positive cells in B-CLL [42]. It was also observed
that Ki-67 positive cells infected with EBV express EBNA2 [42]. We
showed that expression of Ki-67 post-infection of B-cell by GFP-EBV
was detected by 48 hrs and consistently increased after 7 days as well
as in EBV transformed cells. Therefore, B-cell proliferation is most
likely initiated a few hours after EBV infection eventually leading to
B-cell transformation and LCLs. The delayed expression of Ki-67 (at
48 hours) is likely to be due to lytic replication of infected cells at the
early stages of infection where the infected cells may not survive but
produces progeny capable of infecting new cells that eventually
persists and establishes latency. This result strongly supports the
hypothesis that during the early stages of EBV infection primary B-
cells undergo lytic replication important for persistence of the virus,
latency and transformation to LCLs. It is also possible that the lytic
genes may contribute and is important to the upregulation of cellular
genes important for driving cell proliferation and transformation. Discussion In an effort to understand the latent and lytic gene expression
profile during early stage of EBV infection we used semi-
quantitative real time PCR to determine the levels of transcript
for the latent genes EBNA-1, EBNA-2 and LMP-1 [7] as well as
the immediate early, early and late lytic genes BZLF1, BALF5 and
BcLF1 [14]. Data from the Real time PCR showed that the latent
genes EBNA-1, EBNA-2 and LMP-1 were expressed along with
the lytic genes BZLF1, BALF5 and BcLF1. Figure 10. Glycoprotein expression during early stage of
infection in presence of acyclovir. Endogenous expression of (A)
gp110 and (B) gp350 were detected using mouse monoclonal antibody
(1:200 dilution), and rabbit respectively (1:250 dilution). Primary
antibodies were diluted in blocking buffer and incubated with fixed
cells for 1 h at RT. Slides were washed three times (5 min each) with PBS
and incubated with appropriate secondary antibody (1:2000) for 1 h at
RT followed by three times washes (5 min each) with PBS. The last wash
contained 49, 69-diamidino-2-phenylindole (DAPI; Promega Inc., Madi-
son, WI) for nuclear staining. Goat anti-mouse antibody Alexa Fluor 594
and goat anti-rabbit antibody Alexa Fluor 594 were purchased from
Molecular Probes Inc. (Carlsbad, CA). Slides were then washed in PBS
and mounted using Prolong anti-fade (Molecular Probes Inc, Carlsbad,
CA). Fluorescence was viewed by confocal microscopy and analyzed
with Fluoview 300 software from Olympus Inc. (Melville, NY). The
images were sequentially captured using an Olympus confocal
microscope. All panels are representative pictures from similar repeat
experiments. doi:10 1371/journal pone 0007214 g010 Alfieri et al.[75] prevoiusly showed latent gene expression by
immunostainig during initial stages of infection (up to 3 days) of
PBMCs by EBV. They showed that EBNA-1 expressed at 20–32 hrs
post-infection and reached levels seen in LCLs at 46–70 hrs post-
infection, whereas EBNA-2 expressed at 16 hrs post-infection and
LMP-1 expressed in 48 h post-infection. Expression of EBNA-1 and
EBNA-2 was similiar in our studies. However, we observed that
LMP-1 was expressed at an earlier time post-infection (maximum at
24 hours) when compared to the previous report (maximum at 48
hours) [75]. However, Yuan et al. [76] reported that induction of
lytic infection by IgG crosslinking, EBNA and LMP mRNA were
expressed which supports our result. September 2009 | Volume 4 | Issue 9 | e7214 Discussion Since the gene expression
profile of our results suggests that after infection with GFP-EBV,
PBMCs establishes latent infection as well as lytic replication at
during the early stages of post-infection. As infected cells are
undergoing latent and lytic replication at the same time and are in
the overall cell population, it would be difficult to measure the
precise number of cells in the lytic or latent phase of infection and the
cells expressing LMP-1 during the early stages of infection. Further,
studies are ongoing to determine whether or not the expression
patterns we see are directly related to latent or lytic replication. doi:10.1371/journal.pone.0007214.g010 explanation for the observed down-regulation of CD23 after 72
hrs. However, other possibilities exist in that additional latent
antigens may also contribute to CD23 regulation and that the
early increase in CD23 levels are important for persistence or
signaling events that eventually leads to cell proliferation and
transformation. The
Cell
surface
protein
CD40
is
mainly
expressed in antigen presenting cells, and plays a critical role in
B-cell activation by providing cell survival signals via interaction
with the CD40 ligand (CD40L) expressed on the surface of
activated T-cells [71]. It is reported that signaling CD40 and its
ligand CD40L contributes and is likely to be critical for the
antiapoptotic function of EBV and B cell transformation in the
presence of LMP1 after EBV infection [72]. Our results showed
that the expression level of CD40 from GFP positive cells (infected
cells) increased from 6 hrs post-infection and continued to increase
up to 7 days, and are maximally expressed in transformed EBV
positive LCLs. The increased expression of CD40 due to EBV Since BZLF1 was strongly expressed during the early phase of
infection the lytic genes are then induced resulting in progeny and
infection of new cells. Expression of the DNA polymerase (BALF5)
and major capsid protein BcLF1, also indicate that virion particles
were produced which was confirmed by infection of fresh PBMCs
using supernatant from primary infection. The infection of fresh
PBMCs strongly showed that virion particles were produced
during the early stages of infection (Figure 8). To determine if the
production of virion particles occurred during the early stages of September 2009 | Volume 4 | Issue 9 | e7214 PLoS ONE | www.plosone.org 14 Early Events in EBV Infection Figure 11. Schematic representation of events during early
infection of PBMCs by GFP-EBV leading to B cell transformation. Discussion doi:10.1371/journal.pone.0007214.g011 infection, we used acyclovir in the course of infection. The
inhibition of viral DNA polymerase expression as well as the late
lytic protein (gp110 and gp350) expression due to addition of ACV
strongly supported our conclusion that virion particles produced
were due lytic replication and not from virus produced on from the
initial infection (see Figure 9 and Figure 10). Additionally, the
insertion of the cassette in the B95-8 deletion site was the least
deleterious in affecting changes in lytic replication as BZLF1 is
located at the distant position in the genome from the insertion site
and that induction of the other late genes are dependent on
BZLF1 expression. Thus, we were confident that the early lytic
replication is important to contributing to establishment of latency
and transformation of primary infected B-cells. Additionally, since
latent genes were also produced during the early infection, there is
most likely a finely tuned mechanism for early induction of lytic
genes and viral progeny production important for triggering cell
proliferation and a switch from a lytic type infection to a latent
infection in the newly infected cells. LCL were initially believed to arise from direct outgrowth of in
vivo-infected EBV–carying B-cells (One step mechanism)[77]. However, Rickinson et al. [78,79] showed that cell lines can also
be generated in two steps: release of virus from infected cells
during the initial period of in vitro cultivation, followed by the
secondary immortalization of normal B-cells in vitro. Lewin et
al.[80] showed experimentally that the 2-step mechanism is more
common. Our results also suggest that the 2-step mechanism (the
virus particles, due to productive cycle at the early stage of
infection, competent for infection of uninfected B-cells and
immortalization of B-cells) is the most probable for infected
primary B-cells to be driven to transformation by EBV infection in
vitro (see Figure 11). However, studies are ongoing to carefully
address these questions and will provide a more detailed molecular
mechanism of this process. Additionlly, the induction of lytic genes
may also be committed to a small population of the infected cells
that eventually
dies but are critical for induction of the
proliferative genes and switching to a latent type infection. It
would certainly be important to determine whether the infected
cells can undergo same level of lytic replication, survive and is
eventually switch to latent infection and transformation or whether Figure 11. References Fields virology 4th ed. Philadelphia: Lippincott Williams & Wilkins, Philadel-
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experiments: SH FY. Analyzed the data: SH. Contributed reagents/
materials/analysis tools: SH MM SCV PK ESR. Wrote the paper: SH
ESR. Conceived and designed the experiments: SH ESR. Performed the
experiments: SH FY. Analyzed the data: SH. Contributed reagents/
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DMK PM, Griffin DE, Lamb RA, Martin MA, Roizman B, Straus SE, eds. PLoS ONE | www.plosone.org September 2009 | Volume 4 | Issue 9 | e7214 September 2009 | Volume 4 | Issue 9 | e7214 15 Early Events in EBV Infection Early Events in EBV Infection Early Events in EBV Infection Early Events in EBV Infection Neuhierl B, Feederle R, Hammerschmidt W, Delecluse HJ (2002) Glycoprotein
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34: 560–568. PLoS ONE | www.plosone.org September 2009 | Volume 4 | Issue 9 | e7214 September 2009 | Volume 4 | Issue 9 | e7214 16
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NA ROTA DOS CABOCLOS
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1 Como citar: RABELO, Miriam; FLAKSMAN, Clara. Na rota dos caboclos. Debates do
NER, Porto Alegre, v. 2, n. 38, p. 145 - 180, 2020. 2 Doutora em Ciências Sociaia/Antropologia pela University of Liverpool, LIVERPOOL,
Inglaterra. Atualmente é professora titular do Departamento de Sociologia e do Programa
de Pós-Graduação em Ciências Sociais da Universidade Federal da Bahia, Brasil. E-mail:
mcmrabelo@uol.com.br. 3 Doutora em Antropologia Social pela Universidade Federal do Rio de Janeiro, Brasil.
Atualmente realiza pós-doutorado (Faperj) no Programa de Pós-Graduação em Antropo
logia Social do Museu Nacional/Universidade Federal do Rio de Janeiro, Brasil. E-mail:
claramflaksman@gmail.com. DOI: https://doi.org/10.22456/1982-8136.106250 DOI: https://doi.org/10.22456/1982-8136.106250 Palavras-chave: Caboclos; Religiões Afroindígenas; Movimento; Trajetórias. Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 THE PATHWAYS OF THE CABOCLOS Abstract: Present in many different Afroindigenous religions of Brazil, caboclos
are plastic entities that escape precise definitions and stand out for their move
ment. Taking seriously the caboclos’s inclination toward movement, this text
avoids questions regarding their origin or nature, in favor of an approach that
accompanies their journeys, seeking to identify the effects of their passage through
the lives of the people and houses (terreiros) where they act. It argues that in the
terreiros where they play a prominent role, caboclos perform a mobile topology
that brings together in variable ways different times, landscapes, and practices. In
their movement, the caboclos help to compose the terreiro as a space where new
connections can always be produced. If the result of this composition is a mixture,
it is important to emphasize that the sense of mixing here is social: it refers to the
possibility of different beings mingling and living together. Keywords: Caboclos; Afroindigenous religions; Movement; Trajectories. Keywords: Caboclos; Afroindigenous religions; Movement; Trajectories. NA ROTA DOS CABOCLOS1 Miriam C. M. Rabelo2
Clara Flaksman3 Miriam C. M. Rabelo2 Resumo: Presentes em muitas religiões de matriz afroindígena, caboclos são
entidades plásticas, que escapam a definições precisas e se destacam pelo seu
movimento. Levando a sério essa propensão dos caboclos ao movimento, este texto
evita perguntas relativas à origem ou natureza dessas entidades em favor de uma
abordagem que acompanha seus trajetos, buscando identificar os efeitos de suas
passagens pelas vidas das pessoas e terreiros baianos onde atuam. Conforme mostra,
nas casas em que têm papel de destaque, os caboclos performam uma topologia
móvel que aproxima e junta de maneiras variáveis tempos, paisagens e práticas
diversas. No seu movimento, os caboclos ajudam a compor o terreiro como espaço
em que novas conexões podem se produzir. Se o resultado dessa composição pode
ser chamado de mistura, é importante ressaltar que o sentido de mistura aqui é
social: remete à possibilidade de convivência entre seres diferentes. 2 Doutora em Ciências Sociaia/Antropologia pela University of Liverpool, LIVERPOOL,
Inglaterra. Atualmente é professora titular do Departamento de Sociologia e do Programa
de Pós-Graduação em Ciências Sociais da Universidade Federal da Bahia, Brasil. E-mail:
mcmrabelo@uol.com.br. 3 Doutora em Antropologia Social pela Universidade Federal do Rio de Janeiro, Brasil. Atualmente realiza pós-doutorado (Faperj) no Programa de Pós-Graduação em Antropo
logia Social do Museu Nacional/Universidade Federal do Rio de Janeiro, Brasil. E-mail:
claramflaksman@gmail.com. Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 146 Miriam C. M. Rabelo, Clara Flaksman Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 4 Não deixando de levar em conta, em primeiro lugar, a polissemia do termo: “caboclo”
tem uma ampla variedade de significados. Inicialmente, podemos traçar a distinção mais
evidente: refere-se tanto a pessoas quanto a entidades espirituais. Dentro dessa primeira
variação, há outras diferenciações, referentes aos tipos de seres (tanto pessoas quanto
entidades) que são denominados “caboclos”. O caboclo ao qual nos referimos aqui é
a denominação utilizada para uma ampla gama de entidades espirituais, cultuadas em
diversas nações do candomblé da Bahia. Encontram-se, sob essa nomenclatura, tanto
espíritos de índios quanto de boiadeiros, marinheiros e assim por diante, como veremos
ao longo deste trabalho. CHEGANDO Caboclos são entidades em movimento, que habitam paragens distantes,
de onde chegam ou irrompem em meio a galopes, saltos, cambaleios. Ao
se fazerem presentes, arrastam consigo pedaços de seus mundos, atraindo
espectadores que não tardam a ser puxados para dentro, para sambar com
eles e, quem sabe, dar lugar a mais um deles. Quando se juntam para dançar
(ou “vadiar”), mar, sertão e mato muitas vezes se justapõem, quase se tocando. Vêm de suas aldeias para trabalhar, mas também beber, fumar e prosear. O
vínculo com eles pode durar o tempo de uma vida, mas também pode ser
subitamente desfeito. E quando partem de vez, tendo cumprido sua missão,
deixam órfãos não só seus filhos ou médiuns, mas também clientes, amigos,
confidentes e companheiros de farra. São entidades plásticas – parecem
integrar-se ou adaptar-se a qualquer situação. Estão presentes no candomblé,
na umbanda e no espiritismo, em locais públicos e espaços domésticos, NA ROTA DOS CABOCLOS Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 5 Nos referimos aqui aos projetos associados “Na rota dos caboclos”, que contou com o
apoio do PPGCS-UFBA e “Caminhos e Moradas dos Caboclos na Bahia” desenvolvido
com apoio do CNPq. NA ROTA DOS CABOCLOS 147 sejam eles espaços religiosos ou não. Convivem com os orixás e com espí
ritos desencarnados e, da mesma forma, como alguns preferem cantigas e
rezas, outros gostam mesmo é de sambar, beber, fumar charutos, e há ainda
aqueles que apreciam as duas coisas. Sua mobilidade e plasticidade fazem
deles mediadores por excelência, conectando tempos e paisagens, práticas
e entidades diferentes. Neste texto, ao invés de propor uma definição dos caboclos – juntando
alguns de seus atributos mais salientes, em uma perspectiva essencialista -
queremos tratar de algumas dimensões do seu movimento. Na verdade,
tendo percebido a dificuldade de responder à pergunta “o que ou quem são
mesmo os caboclos4?”, decidimos nos aproximar dessas entidades acompa
nhando seus trajetos, encontrando os efeitos de suas passagens pelas vidas
das pessoas e terreiros onde atuam. Mais do que desvendar a “origem” ou
a “natureza” do caboclo, pretendemos traçar os seus caminhos (termo que
no candomblé significa tanto trajetória percorrida quanto destino) e lançar
luz sobre sua multiplicidade. Nosso material vem de fontes diversas: de nosso próprio trabalho
etnográfico, de trabalhos de outros pesquisadores das religiões de matriz
africana e de depoimentos públicos de lideranças. Com poucas exceções
tratamos aqui de terreiros baianos em que o culto aos caboclos ocupa lugar
de destaque, a maioria deles na região do Recôncavo Baiano onde iniciamos Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 148 Miriam C. M. Rabelo, Clara Flaksman pesquisa5, mas também um terreiro na Chapada Diamantina, no interior do
estado, pesquisado por uma de nós em finais dos anos 1980 (Rabelo, 1990). pesquisa5, mas também um terreiro na Chapada Diamantina, no interior do
estado, pesquisado por uma de nós em finais dos anos 1980 (Rabelo, 1990). Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 6 Federação Nacional do Culto Afro-Brasileiro, entidade responsável por expedir alvarás
de funcionamento para casas filiadas e manter o registro das suas atividades religiosas. Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 CAMINHOS Em geral, os caboclos chegam na vida (e no corpo) das pessoas para
trabalhar – curar, aconselhar, resolver problemas de toda sorte. Muitos se
referem a seu trabalho como uma missão - o caminho (destino/percurso)
de um caboclo é sua missão - e é o tempo da missão que determina sua
permanência. Uma vez que baixam, tendem a acompanhar seus filhos por
toda vida. Mas assim como chegam, podem bem partir. E se seus filhos
porventura morrem antes que tenham completado sua missão, podem bem
procurar outros (corpos) humanos para trabalhar. Vejamos alguns desses
percursos nas casas e nos corpos de algumas pessoas. Roquinha é mãe de santo de uma casa de candomblé de Salvador. Passou
muitos anos na umbanda, antes de se aproximar do candomblé. No centro
de umbanda que frequentava recebia o caboclo Boiadeiro que trabalhava na
mesa branca e dava consulta regularmente. Uma doença grave a conduziu
para o candomblé. Ela foi feita (iniciada) no candomblé e depois de um
longo percurso recebeu o cargo de mãe de santo. Boiadeiro é hoje presença
constante no seu terreiro. A vinda do caboclo para o candomblé foi nego
ciada: para que ficasse no terreiro, seria preciso que lhe permitissem atuar
na mesa branca. Trabalhar por caridade, ajudando aos necessitados, era a
missão que tinha a cumprir na terra. Boiadeiro não queria (e não quer) parte
com as coisas de candomblé. Atualmente preside as sessões de mesa branca,
realizadas quinzenalmente no terreiro. Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 NA ROTA DOS CABOCLOS NA ROTA DOS CABOCLOS 149 Nas sessões, uma grande mesa é transportada para o centro do barracão. Forrada com uma toalha branca, a mesa abriga uma profusão de objetos:
pipocas espalhadas por toda sua extensão, velas, vasos com flores, charutos,
bebidas para os caboclos, imagens de santos: Cosme e Damião, Santa Bárbara,
São Jorge e a Virgem Maria. Quando começa a sessão a mãe de santo e os
filhos da casa sentam-se em volta da mesa. Atrás deles, de pé, distribuem-se
os demais participantes, em sua maioria clientes. A sessão começa com reza. Um Pai Nosso é entoado para cada um dos clientes que antecipadamente
deixou seu nome com a equede responsável pela organização do evento. Quando termina a longa lista de clientes, os caboclos começam a chegar,
embalados pelas rezas, que ainda não cessaram. Alguns vêm de forma violenta
derrubando seus médiuns dos banquinhos. Mas a ordem não demora a ser
restabelecida para mais uma rodada de orações: Ave-Marias e Salve Rainhas. Ao final, todos levantam e dão-se as mãos, formando um grande círculo
– membros da assistência incluídos. Cantigas de caboclo, então, sucedem
as rezas. Os caboclos se desprendem do círculo e logo recebem folhas para
dar passe. Entre eles está Boiadeiro – o mais procurado pelos presentes,
que fazem fila para serem atendidos. Muitos conversam com o caboclo,
contando-lhe seus problemas e pedindo conselhos; quando é necessário,
uma equede toma nota das receitas que ele prescreve. No bairro, Boiadeiro
tem fama de curador caridoso e eficiente. Paulo vive no município de Sapeaçu, em zona de sítios, mais recuada
da cidade. No seu sítio, muitas árvores fazem sombra na área central de
terra batida onde estão sua residência e o terreiro – classificada como "giro
de caboclo", conforme documento da Fenacab6 emoldurado na parede do
barracão. Sentamo-nos para conversar debaixo de uma mangueira, de frente
para um barranco que margeia o terreno de Paulo. 6 Federação Nacional do Culto Afro-Brasileiro, entidade responsável por expedir alvarás
de funcionamento para casas filiadas e manter o registro das suas atividades religiosas. Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 150 Miriam C. M. Rabelo, Clara Flaksman Paulo conta que tinha 17 anos quando um caboclo lhe pegou pela
primeira vez. Era Gentio. Paulo conta que tinha 17 anos quando um caboclo lhe pegou pela
primeira vez. Era Gentio. Ele me pegou e desceu essa mata aqui. Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 NA ROTA DOS CABOCLOS 151 não. Você está aqui porque Eru que lhe trouxe". Eru era o caboclo dela. E aí
na hora que ela falou que foi Eru que me trouxe, ele bafou ela. Bafou ela e me
abraçou, e aí caiu a lágrima e caiu a lágrima e aí ele me disse: "É, meu filho,
só você que vai continuar o que eu quero". "Sobre?" "É, a velha Maria está
indo embora". Aí chora ele, chora eu, chora ele, chora eu. E ele disse: "Olhe,
ninguém tem o que você tem, quem anda aqui na minha casa. Continue o
que eu lhe ensinei que ninguém vai poder com você. Lembre de mim aonde
você for". Aí aquilo ficou na minha cabeça. Mas mesmo com o recado de Eru, Paulo não assumiu o lugar de D. Maria depois de sua morte - nem nenhum de seus filhos. O terreiro perma
neceu sem liderança, e depois de algum tempo Eru veio para expressar seu
descontentamento - no corpo da filha de Paulo. Ele [Eru] pegou a minha filha dormindo, e botou aí no meio da sala e largou
a língua no que ele queria. [Falou] dos filhos de dona Maria que abandonou. Ela tem casa em Cabuçu, abandonada, o terreiro lá dela de Irará os filhos
abandonaram. Então o caboclo está numa revolta muito grande, só que eu
não tenho como resolver, tu tá entendendo? Eu não tenho como resolver. O
que eu posso resolver é alimentar ele. E ele, espiritualmente, ele resolva da
maneira deles, porque eu não posso ir lá dizer aos filhos de dona Maria que
isso não está bom, que isso está errado. Eu não posso fazer isso, porque eles
tinham que ter a noção que o que ela deixou tinha que continuar, né? Na sua filha, Paulo vê a continuidade de seu trabalho e, embora não
pareça muito disposto a falar sobre isso, também a volta do caboclo Eru de
dona Maria. Se o corpinho franzino da menina de doze anos desponta agora
como lugar escolhido por Eru para seguir trabalhando, Paulo bem sabe que,
assim que a notícia se espalhar, esse lugar será contestado pelos outros filhos
da mãe-de-santo vereadora. Mas também sabe que os caboclos escolhem e
fazem seus caminhos. Foi assim com ele: jogado na mata por Gentio, foi
levado a experimentar no corpo não só a energia do caboclo, mas também
seu elemento. NA ROTA DOS CABOCLOS Aqui é o lugar chamado "represa". Ali
embaixo, depois daquela mata ali embaixo é água. Dá 2km de água aí. Ele
me pegou, atravessou esta água aí, caiu na mata aí, levou três dias dentro pelo
mato. Sem roupa, sem lençol, sem nada. Três dias perdido no mato. Aí minha
mãe foi na casa de um rezador e o rezador disse que eu estava na mata, que
o caboclo estava comigo. Aí foi, o homem fez o trabalho, chamou, ele veio. Todo lascado... aí teve que cuidar. [Ele disse] que minha sina era trabalhar,
ajudar o povo. Eu que resolvesse. Se eu não quisesse tinha outra solução pra
mim, mas eu como não queria perder minha vida, né? Depois de alguns anos, o rezador que havia sido capaz de controlar o
caboclo de Paulo, e que cuidara dele, veio a falecer. Desprotegido, Paulo
experimentou novo período de sofrimento. Resolveu, então, procurar outra
pessoa que pudesse tomar conta do seu caboclo. Foi ao encontro de um
amigo rezador que trabalhava com o caboclo Sultão das Matas. Ao vê-lo,
Sultão disse que não poderia cuidar dele. "Mas não se incomode não, que
eu tenho um lugar para lhe levar". Conduziu-o então a casa de dona Maria. Maria Bacelar, que veio a ser a mãe de santo de Paulo por dezenove anos,
era uma personagem famosa da vida religiosa e política de Irará, município
situado a uns 140 km de Sapeaçu, em região conhecida como porta do sertão
da Bahia. Além de mãe de santo era vereadora influente e diretora de uma
escola municipal que funcionava em sua fazenda, junto com o terreiro de
umbanda. A festa do caboclo Eru, seu guia espiritual, era evento importante
da cidade. Quando D. Maria estava perto de morrer, Paulo sonhou com
ela lhe chamando para dar um presente a Oxum. Foi encontrá-la em Irará: Eu saí tão cedo, quando eu cheguei lá ela estava rezando o terço na frente da
porta. Ela me olhou assim, mandou eu aguardar. Ela terminou de fazer as
orações dela. Depois ela me chamou. Eu disse: "Ó, veia, eu tive um sonho
assim, assim, assim". Ela disse: "Eu sei, meu filho, você não está aqui à toa Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 NA ROTA DOS CABOCLOS Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 ra 1- Pôster de Mata Virgem no terreiro de Toinho (foto de Antônia Oliveira, 2018) NA ROTA DOS CABOCLOS Quando precisou de alguém para cuidar dele e de seus guias,
foi Sultão das Matas – o caboclo de um amigo rezador – que o conduziu 152 Miriam C. M. Rabelo, Clara Flaksman àcasa de dona Maria. E foi Eru que o reconduziu a Irará, quando sua mãe
de santo estava prestes a morrer. Toinho foi criado por Dona Nilza, mãe de santo de uma casa de umbanda
em um bairro da periferia de Santo Amaro, cidade próxima de Sapeaçu. Já
muito idosa e doente, ela não dá mais sessão e as famosas festas de caboclo
que animavam sua casa são agora lembranças do passado. Foi uma filha de dona Nilza que nos encaminhou para a casa de Toinho
– “ele vai saber explicar tudo pra vocês”. É uma casa modesta com varanda
na frente em uma rua de casas geminadas, muito diferente da paisagem rural
do terreiro de Paulo. Entramos direto no que parece ser o barracão, nossa
impressão confirmada pelas prateleiras com imagens de santos e quadros
religiosos nas paredes: Mãe d’Água, Preto Velho, Caboclo Mata Virgem. Figura 1- Pôster de Mata Virgem no terreiro de Toinho (foto de Antônia Oliveira, 2018) Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 NA ROTA DOS CABOCLOS 153 Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 Toinho então nos contou a história de dona Nilza: Hoje mãe está com 85 anos, ela é de Oxalá com Oxum, ela trabalha com o
caboclo Rei das Estrelas que foi batizado por Rei de Congo e Rei José. As
histórias de caboclo lá são muito profundas. Porque mãe quando começava
rezar a cabana, ela começava a rezar as treze noites para Santo Antônio, quando
chegava no dia 14 ela começava a vestir os índios. Quando era o dia primeiro
de julho, ela armava a cabana. Era a cabana maior que existia em Santo Amaro. Começava em julho e terminava no último sábado de setembro. Todo dia se
referenciava ali, ali todo dia se cantava, ali todo dia se louvava. Quando as
frutas iam ficando ruins, eram retiradas e colocadas nas águas, no mato, até
completar a cabana. A cabana era de Campo Verde, caboclo Campo Verde. Sendo que o caboclo da casa, o patrono, o nosso rei, era o Rei das Estrelas. Só que ela também tinha outro espírito que trabalhava de dia (sexta-feira)
que chama Doutor Antônio Manuel da Luz, é um médico. Esse fez muita
caridade, fez muitas ações, tem provas vivas ainda, graças a Deus. E aí, veio
eu. Eu ali no meio, eu ali no meio. Quando eu acordei, mesmo, eu já estava
dentro do negócio. Os caboclos demoraram a chegar na vida de Toinho, apesar de seu
envolvimento na sessão de D. Nilza. A entidade que veio primeiro foi exu,
o “escravo”, e Toinho passou um tempo dando consulta com ele. Mas certa
vez, quando um cliente veio em busca de exu, Mata Virgem passou na frente,
como nos contou Toinho: Ele veio e tomou o espaço. E hoje é ele quem manda e desmanda aqui
dentro. É Mata Virgem. A palavra primeira e a última é dele. E depois disso
eu trabalhava no mercado, vendia café na época. Ele mandou que eu viesse
pra casa, descansar porque eu estava muito cansado. Isso tem 17 anos, eu
estou descansando até hoje. Não voltei mais no mercado, ele quem mandou. Abaixo de Deus, é o vento que eu respiro. Então quando o Mata Virgem
apareceu, eu trabalhava com o escravo, só fazia minha consulta. E quando
o Mata Virgem apareceu, ele determinou que vinha pra trabalhar, ele queria
candomblé, ele queria atabaque e ele queria a casa aberta. Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 154 Miriam C. NA ROTA DOS CABOCLOS humanos com quem trabalharam, o tempo de sua missão como caboclos
e o tempo de suas vidas como índios, marujos, boiadeiros, aparecem, cada
um, no horizonte do outro. Conectam lugares; quando chegam seus nomes
já trazem paisagens móveis - Treme-Terra, Sultão das Matas, Mata Virgem,
Rei das Estrelas, Gentileiro – que muitas vezes se encontram em um ritmo
acelerado. E no seu movimento podem mesmo redesenhar o espaço do terreiro,
abrindo nichos, passagens, zonas de encontro e convivência, como fez o
caboclo de Roquinha ao demandar que a sessão de mesa branca fosse trazida
para dentro do terreiro de candomblé, garantindo que no terreiro houvesse
espaço também para quem não quer manter relações com o candomblé. Os caboclos são mestres da composição. Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 Toinho então nos contou a história de dona Nilza: M. Rabelo, Clara Flaksman Quando Mata Virgem chegou Toinho refez o caminho de Dona Nilza. Levou o caboclo para batizar com Dona Estela, mãe de santo do município
de Terra Nova, não muito distante de Santo Amaro. O caboclo Rei do
Congo, com quem Dona Estela trabalha, havia batizado Rei das Estrelas,
caboclo de D. Nilza. Nada mais certo que batizasse também Mata Virgem. “Então a gente tem Rei das Estrelas como pai e Rei de Congo como avô”. À história das relações de parentesco entre os caboclos, sobrepõe-se outra,
de parentesco entre os humanos em cujos corpos Mata Virgem trabalhou e
ainda a história da vida de Mata Virgem, índio Tupinambá: Mata Virgem, ele conta que ele era de uma tia avó minha. Ele trabalhou. Essa
tia avó tinha 35 anos de morta e ele voltou e retornou a terra para trabalhar. Ele conta a história que ele morreu, ele era um homem trabalhador, era um
índio que morreu numa tribo Tupinambá. E ele voltou ao terreiro para traba
lhar. É o caboclo da festa, é o caboclo que trabalha, é o caboclo do conselho. É o caboclo que quando chega uma pessoa no desespero, na aflição, atende
as pessoas. É um caboclo querido pela comunidade, graças a Deus. Mata Virgem ocupou o espaço do exu que baixava em Toinho. Quando
chegou, mudou a rotina de Toinho: queria casa aberta, ciclo de festas e
obrigações. Selou o vínculo de Toinho com o mundo de sua mãe adotiva,
e o conectou ao passado mais remoto de sua família. Mas impôs suas prefe
rências: enquanto na casa de D. Nilza prevalecia a mesa branca, o terreiro
de Toinho é de giro. *** Os percursos dos caboclos deixam em seu rastro muitos tipos de conexão. Produzem laços de parentesco e amizade: caboclos são batizados por outros
caboclos, cuidam dos seus parceiros humanos, ligam-se a outros humanos
como curadores, conselheiros ou companheiros de copo, ligam humanos a
seus territórios, ativam laços esquecidos e criam outros inesperados. Multi
plicam e conectam diferentes planos temporais – os diferentes tempos dos Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 155 7 Mãe de santo de um dos terreiros mais antigos de Salvador, o Ilé Iyá Omi Asé Yamasé
(mais conhecido como Gantois), onde Landes fez grande parte da pesquisa de campo
que deu origem ao livro "Cidade das Mulheres". COMPOSIÇÕES No livro "Cidade das Mulheres", Ruth Landes relata seu encontro com
Sabina, uma “mãe cabocla”. Landes fora acompanhada por Edison Carneiro
ao terreiro de Sabina, localizado no bairro da Barra. Quando Sabina teve
oportunidade de explicar a Landes o seu candomblé, falou dos caboclos: Esse templo é protegido por Jesus e Oxalá e pertence ao Bom Jesus da Lapa. É uma casa dos espíritos caboclos, os antigos índios brasileiros, e não vem
dos africanos iorubá ou do Congo. Os antigos índios da mata mandam os
espíritos deles nos guiar, e alguns espíritos de índios mortos há centenas
de anos. Salvamos primeiro os deuses iorubá nas nossas festas porque não
podemos deixá-los de lado; mas depois salvamos os caboclos porque foram
os primeiros donos da terra em que vivemos. Foram os donos e, portanto,
agora são os nossos guias, vagando no ar e na terra. Eles nos protegem (Landes,
2002, p. 232). 156 Miriam C. M. Rabelo, Clara Flaksman Já é sabido que Sabina não era bem vista nos meios mais ortodoxos do
candomblé de Salvador; Landes se refere ao desprezo que Mãe Menininha7
nutria não só por ela, mas pelo candomblé de caboclo em geral. Depoimentos
colhidos por Donald Pierson, durante sua estadia na Bahia na década de
1930, mostram que esse desprezo era compartilhado entre os adeptos das
casas jeje-nagôs, ciosos da sua herança africana. Falava-se então dos “imita
dores” caboclos. Um dos interlocutores de Pierson, pai de santo de uma
casa nagô assim se referiu a um conhecido pai de santo de terreiro caboclo: Seus avós, que é que eles sabiam? Foram educados na seita? Será que deixaram o
cargo para ele? Não! Ele veio do sertão e quer fundar um candomblé. Aprendeu
um pouco de gêge, um pouco de nagô, um pouco de congo, umpouco dessas
coisas de índio e assim por diante. Que mistura desgraçada!” (Pierson, 1971,
p.305). “Se seja mistura, é bobage”, disse-lhe outro. Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 8 Caboclos de pena são a classe de caboclos conhecidos como "índios", associados a
elementos de origem ameríndia ou afroindígena. Alguns de seus nomes são: Sete Flechas,
Sultão das Matas, Tomba Morro, Treme-Terra, Juremeiro, Pena Branca. “Se seja mistura, é bobage”, disse-lhe outro. A definição que Toinho nos oferece de seu terreiro pode ser tomada
como um contraponto interessante aos comentários que Pierson colheu
sobre o candomblé de caboclo: Existe a linha da umbanda, existe o giro de caboclo, eu já sou giro de caboclo. Porque eu trabalho com caboclo, trabalho com candomblé e trabalho com
a umbanda. Então se chama giro de caboclo porque vem uma coleção, uma
mistura, um conjunto de várias mãos seguras. Para Sabina, nas casas de caboclo é preciso louvar os orixás primeiro,
por uma questão de cortesia, de educação: não se pode deixá-los de lado. Coleção e mistura são os termos usados por Toinho para descrever o giro de
caboclo, modalidade de prática que define tanto seu terreiro quanto o de 7 Mãe de santo de um dos terreiros mais antigos de Salvador, o Ilé Iyá Omi Asé Yamasé
(mais conhecido como Gantois), onde Landes fez grande parte da pesquisa de campo
que deu origem ao livro "Cidade das Mulheres". Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 157 NA ROTA DOS CABOCLOS Paulo. Se coleção ressalta a pluralidade que esses terreiros abrigam, mistura
não pode significar indistinção. Seu sentido aqui remete mais à convivência
(pessoas que se misturam) do que à produção (fruto de mistura): o modelo é
social e não químico. Por que estariam os caboclos associados à mistura? Se
lembrarmos que os caboclos são entidades em movimento, e que em casas
como a de Toinho e Paulo estão à frente das atividades do terreiro, então
talvez possamos concluir disso que cabe também a eles parte importante do
trabalho de composição do terreiro como espaço em que novas conexões
podem se produzir. *** No terreiro de Paulo, em meio às muitas árvores e plantas, uma bandeira
branca erguida por uma haste comprida revela aos desavisados que ali se
trabalha com forças invisíveis. Convida aos interessados em se beneficiar
dessas forças a buscar a ajuda de quem sabe mobilizá-las. Passamos por ela,
enquanto Paulo percorre conosco o espaço, mostrando plantas e lugares,
contando a história do terreiro e de suas entidades. - Aqui, venha ver aqui uma coisa. Você vai se apaixonar por esse cabra,
venha ver ele aqui… Ó ele aí, ó o Boiadeiro aí! 158 Miriam C. M. Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 9 Dois de julho é a data em que se comemora a independência da Bahia. Os caboclos
(indígenas) são tidos como heróis da independência, não só celebrados nos festejos
oficiais da data, como também nas casas de candomblé de Salvador e Recôncavo Baiano
(muitas das quais fazem festa de caboclo no dia). “Se seja mistura, é bobage”, disse-lhe outro. Rabelo, Clara Flaksman otografia 2 - Casa de Boiadeiro, terreiro de Paulo (foto de Antônia Oliveira, 2018) Fotografia 2 - Casa de Boiadeiro, terreiro de Paulo (foto de Antônia Oliveira, 2018) Uma casinha de telha sem a parede da frente abriga uma estátua de
Boiadeiro, o corpo negro, calça, gibão e chapéu de couro. Ao seu lado
imagens bem menores de caboclos de pena8 e outra menor ainda de Jesus
Cristo. À frente uma quartinha vermelha com folhas de espada d’Ogum e
uma vela de sete dias acesa. Ao lado um cacho de licuri. - Meu caboclo é Gentio. Aí Boiadeiro já é outra entidade. Vêm vários,
mas quem manda é Gentio. Ele é que domina tudo. Ele é que é o dono
daqui, tá entendendo? O caboclo que manda aqui nessa casa... Vêm todos,
mas a ordem é dele. O que ele diz que é isso, é isso. - Você trabalha mais na linha de caboclo? - Você trabalha mais na linha de caboclo? - Caboclo, caboclo. - Caboclo, caboclo. 8 Caboclos de pena são a classe de caboclos conhecidos como "índios", associados a
elementos de origem ameríndia ou afroindígena. Alguns de seus nomes são: Sete Flechas,
Sultão das Matas, Tomba Morro, Treme-Terra, Juremeiro, Pena Branca. 159 NA ROTA DOS CABOCLOS - Na linha de orixá também? - Na linha de orixá também? - Tudo. Aqui no orixá é Ogum. Eu sou de Ogum com Iemanjá, entendeu? - Tudo. Aqui no orixá é Ogum. Eu sou de Ogum com Iemanjá, entendeu? Paulo abre a porta de uma casa pequena, de um só cômodo, e explica
que é ali que a comida das festas é dividida. Amontoados em um canto,
vários inhames enramados. Ele nos explica que aquele é o inhame de Ogum,
a ser oferecido para ele, com vistas a obter prosperidade e "coisas boas
dentro de casa". Um pouco mais adiante, mostra-nos a gruta do caboclo. Segundo ele, a cada Dois de Julho9 oferece frutas e mel para Gentio - sem
bebidas alcoólicas. Chegamos então à árvore de Boiadeiro, uma jaqueira. Paulo então explica: - Esse pé de jaqueira é de Boiadeiro, de jaca dura. É o pé de Boiadeiro. No churrasco dele, na festa dele, ele não samba dentro de casa, samba do
lado de fora. Boiadeiro manda cercar tudo de palha de dendê e o couro
come aí. É carne, é cerveja. Que Boiadeiro gosta, né? Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 “Se seja mistura, é bobage”, disse-lhe outro. De cervejada. A festa
de Iemanjá eu fiz [na praia de] Cabuçu, eu dou presente de Iemanjá com
Marujo. Recebo ele também, quando me pega eu levo dois dias doente. Embora Paulo faça festas para Boiadeiro e para Marujo, a sessão semanal
que acontece em seu terreiro é comandada por Gentio. “Aí ele é que vai
dando ordem pros outros, pros médiuns. Cada médium, ele solta uma
entidade, aí se torna um grupo ali de caboclo com ele junto. É uma força
muito grande, é uma força muito grande”. Na sessão os caboclos dançam
no barracão, mas não bebem, só água e mel. Dona Maria instruiu Paulo a
não os acostumar a beber. - Porque já pensou? Tem dias que eu atendo das seis da manhã às seis
da tarde. E [o caboclo] não sai não. Nem pra eu ir no banheiro, não sai não. E não sei, rapaz. Eles me pegam em festa aqui, dez da noite é a saída dele, Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 160 Miriam C. M. Rabelo, Clara Flaksman dez da noite, e vai até seis da manhã. Couro comendo aí e sem largar pra
nada. Mas porque vem com as forças deles mesmo. Agora meu Boiadeiro
bebe, meu Marujo bebe, meu Marujo fuma. Já é outras coisas. Mas o da
mesa mesmo, o dono da mesa… Uma vez por ano, Paulo também dá comida para os exus, mas sem
toque, porque a linha da sua casa não permite. No terreiro de Paulo tudo
é feito na linha branca: -Na linha do dendê eu não mexo, entendeu? Nada não vai azeite (de dendê)
aqui. Nada, nada. Obrigação de santo nenhum. Minha linha não permite
dendê não, não aceita dendê não. E nem cortar. Raspar sim, mas cortar10
não. (...) Ela (dona Maria) também não mexia não. (...) Nós [também] não
mexemos em nada de morto. Cemitério nós não vamos. (...) Mata eu adoro,
adoro mata, adoro água. Qualquer mata se me chamar eu entro, não tenho
medo. Mas cemitério, eu tenho pavor de quem morre. E aí é a vida. -Na linha do dendê eu não mexo, entendeu? Nada não vai azeite (de dendê)
aqui. Nada, nada. Obrigação de santo nenhum. Minha linha não permite
dendê não, não aceita dendê não. E nem cortar. Raspar sim, mas cortar10
não. (...) Ela (dona Maria) também não mexia não. Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 "Cortar" é uma forma de se referir ao abate cerimonial nas religiões de matriz african “Se seja mistura, é bobage”, disse-lhe outro. (...) Nós [também] não
mexemos em nada de morto. Cemitério nós não vamos. (...) Mata eu adoro,
adoro mata, adoro água. Qualquer mata se me chamar eu entro, não tenho
medo. Mas cemitério, eu tenho pavor de quem morre. E aí é a vida. A casa de Paulo, como a de dona Maria, é de linha branca - não se corta
para as entidades, não se toca para exu, oferendas só com mel e azeite doce. Também é lugar de confluência de muitas linhas: de caboclo, orixá, exu,
preto-velho. E é espaço recortado, definido mesmo, pelas relações entre os
caboclos, tendo à frente Gentio, o dono de tudo, segundo o próprio Paulo. Em cada uma dessas formulações, temos um modo próprio de desenhar o
espaço do terreiro e distribuir suas diferenças. Em duas delas linha é pala
vra-chave – na primeira, define a tradição da casa, o modo de atuar que
Paulo aprendeu com sua mãe de santo e que ele buscou imprimir na conduta
dos seus caboclos; na segunda o modo de lidar com cada tipo de entidade
(orixás, exus, caboclos, etc.) que em geral define a casa como candomblé,
mesa branca, umbanda ou giro. Mas ao ser definido pelos caboclos, o
espaço do terreiro ganha novos contornos: as diferenças entre linhas como
tradições ou entre linhas como modos de trabalhar para diferentes tipos Cortar" é uma forma de se referir ao abate cerimonial nas religiões de matriz africana. 161 NA ROTA DOS CABOCLOS Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 NA ROTA DOS CABOCLOS de entidades (que se traduzem em diferenças entre candomblé e umbanda
ou giro e mesa branca, por exemplo) se convertem em diferenças entre os
temperamentos dos caboclos da casa. Cada caboclo projeta uma zona própria
de afeto e influência capaz de atrair outros caboclos e humanos, fazendo-os
gravitar em seu entorno. As linhas, neste caso, orientam-se e convergem para
o foco (temporário) de atração. Gentio comanda a sessão, chama e junta
os caboclos da casa para trabalhar, mas só com água e mel, conforme foi
acostumado. Qualquer caboclo pode chegar, mas a ordem é sua – nada de
bebida. Quando a festa é de Boiadeiro, a cerveja corre solta para humanos
e caboclos. E quando se trata de festejar Marujo, marinheiro beberrão, sua
afinidade com o mar, permite que sua festa seja feita junto com Iemanjá,
divindade iorubana das águas salgadas. *** Não muito distante do terreiro de Toinho, está o Centro de Caboclo
Jaguaraci de Mãe Zenaide. Fomos procurá-la por indicação de Toinho que,
orgulhoso, disse tratar-se de uma filha de santo bem mais velha do que
ele e que já tinha casa aberta. A casa tem uma varanda fechada na frente
que dá entrada a uma sala de estar; ao final de um corredor, chega-se ao
barracão. Dona Zenaide não se encontra e é Nayran, sua filha (biológica)
quem nos recebe. Toda terça feira, mainha dá sessão aqui, onde tem os clientes, amigos, irmãos
de santo que vêm, aí tem a sessão, orações, pede pelas vidas das pessoas que
estão em hospitais, doentes em cima da cama, proteção às pessoas, aí depois
de tudo o caboclo responde, o caboclo decide, os que estão sentados na mesa
também respondem, aí depois tem um passe, como se fosse uma limpeza de
corpo, que faz é o guguru, que no caso é a pipoca, que passa nas pessoas, e
aí depois tem o encerramento com muita cantiga, alguma coisa pro pessoal
que veio comer e aí o pessoal vai embora, tod a terça feira de 15 em 15 é
realizada a sessão. Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 162 Miriam C. M. Rabelo, Clara Flaksman D. Zenaide tem dois caboclos, Gentileiro e Jaguaraci, ambos caboclos
de pena, embora Gentileiro tenha mais de uma forma: “o caboclo de Mainha
é o caboclo que na verdade o povo diz que ele é um caboclo de couro... Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 NA ROTA DOS CABOCLOS tem
gente que conhece ele como um caboclo índio e tem gente que conhece
ele como um caboclo de couro, porque ele pode vir trajado das duas coisas,
tanto do couro como da pena”. Gentileiro é o caboclo da sessão; Jaguaraci
“é mais pro candomblé”. Ele foi responsável por uma famosa adição à casa
de dona Zenaide: uma jiboia, guardada em um armário transformado em
viveiro, no corredor que dá para o barracão. Uma foto pregada na parede
do barracão exibe a jiboia enrolada no pescoço de Jaguaraci, pela porta de
vidro do armário a vemos enroscada em um pedaço de pau. Nayran conta
que Jaguaraci vivia pedindo que lhe trouxessem uma cobra e que até o dia
em que ganhou a jiboia de uma mãe de santo que lhe tinha muito apreço,
a casa de dona Zenaide era constantemente visitada por cobras. 163 NA ROTA DOS CABOCLOS Fotografia 3- Foto do caboclo Jaguaraci na parede do barracão de dona Zenaide (foto de
Antônia Oliveira, 2018) Fotografia 3- Foto do caboclo Jaguaraci na parede do barracão de dona Zenaide (foto de
Antônia Oliveira, 2018) Como Jaguaraci, o caboclo de Nayran - Juremeiro – não tem afi
m a sessão - “é só de festa”. “Às vezes até me apanha na sessão, m Como Jaguaraci, o caboclo de Nayran - Juremeiro – não tem afinidade
com a sessão - “é só de festa”. “Às vezes até me apanha na sessão, mas não Como Jaguaraci, o caboclo de Nayran - Juremeiro – não tem afinidade
com a sessão - “é só de festa”. “Às vezes até me apanha na sessão, mas não Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 164 Miriam C. M. Rabelo, Clara Flaksman fica não, por causa do respeito”. Interessante notar que é o respeito que
mantém o caboclo da gira distante da sessão: a questão central aqui é menos
a preservação de uma tradição contra misturas, do que a consideração pelo
espaço do outro – a vinda de um caboclo festeiro na sessão ameaça a boa
convivência, no terreiro, entre caboclos de sessão e de giro. Nayran é de candomblé. No decorrer da conversa nos damos conta
que também ela é mãe de santo, embora não fique claro se já tem filhos
iniciados. 11 O Bembé do Mercado é uma celebração religiosa afro-brasileira realizada na cidade de
Santo Amaro, no Recôncavo Baiano. Teve início em 1889, em comemoração à abolição
da escravatura, e fruto do empenho das comunidades de terreiro de Santo Amaro.
Comporta hoje três dias de festa de rua em honra aos orixás. Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 12 No candomblé, tomar rum é dançar ao som dos tambores, ou ser provocado pelos
tambores a dançar. NA ROTA DOS CABOCLOS 165 de suas festas. Assim como vimos nos arranjos espaciais das casas de Paulo
e Nayran, aproximações e distanciamentos no tempo são orientados tanto
pelas inclinações dos distintos caboclos, quanto por uma preocupação com
a boa convivência entre eles. Na casa de Toinho, toda quarta feira tem sessão de caboclo. Durante
a quaresma, essa se resume à mesa branca, mas no resto do ano é sessão de
giro. Toca-se também para os orixás no giro de caboclo, ele explica, mas
quando é festa de orixá, tudo é feito no ketu. No calendário da casa, além das sessões, tem-se uma sequência de festas:
Ogum, orixá de frente de Toinho, em janeiro; Mata Virgem, em julho;
Obaluaê, em agosto; caruru de Cosme e Damião em setembro e o presente
das águas em dezembro. As duas primeiras são as festas mais importantes,
vários dias de preparativos e vários dias de celebração. Perguntamos se os
caboclos vêm na festa de Ogum. Sempre quando depois que a gente faz o xirê, depois que Ogum toma rum12,
a gente serve a feijoada para a comunidade, tem aquela uma hora de descanso
e depois a gente vira pra caboclo. O caboclo aqui em casa não fica fora de
nada, de nenhuma situação, nem eles mesmos não se permitem. Ontem eu
tentei até dar uma escapulidinha, [mas] ele vem. E hoje estou lutando para que eu deixe também um legado, porque casa de
caboclo aqui em Santo Amaro tá bem pouco... Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 NA ROTA DOS CABOCLOS Emoldurados na parede do barracão estão seu registro de sacerdo
tisa de culto afro e o alvará de seu terreiro de candomblé ketu, o Ilê Ase Oju
Oba Layó, ambos expedidos pela Fenacab. Também na parede do barracão
vê-se a moção de reconhecimento concedida à d. Zenaide pelo Bembé do
Mercado de Santo Amaro11. Entre Nayran e dona Zenaide estabeleceu-se
uma espécie de acordo de cooperação, ou de “divisão de trabalhos”: “Ela
cuida das coisas dela e... tem filhos de santo dela... que ela cuida também... uma comida, faz as coisas tudo direitinho. Aí quando aparece alguém que
seja pra poder raspar, ela já passa pra mim”. Nayran explica que no barracão são celebradas as festas de seu orixá,
Xangô, e do caboclo Juremeiro – “agora sempre separadamente, quando é
festa de Ogum (orixá de D. Zenaide) é de Ogum, quando é festa de Xangô... agora geralmente quando é a festa do caboclo, aí a gente faz junto”. O fato de serem mãe e filha certamente contribuiu para que esse arranjo
tomasse forma. Mas também contribuíram as inclinações de seus caboclos. *** As diferenças entre caboclos (e entre eles e os orixás) distribuem-se
não só no espaço dos terreiros, como também no tempo – no calendário Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 NA ROTA DOS CABOCLOS CASA DE CABOCLO Abaixo temos três excertos, de diferentes fontes, que tratam do caboclo
e seu culto em religiões de matriz africana: 12 No candomblé, tomar rum é dançar ao som dos tambores, ou ser provocado pelos
tambores a dançar. Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 166 Miriam C. M. Rabelo, Clara Flaksman Já em São Luís fala-se mais de uma ‘quase invasão’ de entidades espirituais
caboclas nos terreiros de Mina. Adianta-se que, uma vez acolhidas pelos pais de
santo, estas entidades ensinaram novos cânticos e danças e aqueles passaram a
realizar para elas ritos especiais impregnados de elementos culturais indígenas
("dança batendo os pés ou imitando guerra, uso de arco e flecha, maracá e de
adornos de penas", etc.) (M. Ferreti, 1994, p.23). Têm-se falado aqui, às vezes muitos oradores disseram, que o candomblé,
chamado assim ou assado, foi de um mais velho do que outro. Mas o caboclo
é mais velho, porque os outros vieram de lá pra cá, e ele já estava aqui. Ninguém foi buscar “ele” não. Ele já estava aqui. Ele é o dono da terra. E
por que, agora, se expurga o dono de suas casas? Se acha que caboclo não
tem prestígio, se acha que caboclo não é “feito”, se acha que ele não tem pai,
nem mãe, que nasceu num oco-de-pau? Caboclo não nasceu assim, não. Ele
tem pai, mãe, tem tudo. Caboclo é uma “nação”, tem bandeira, tanto quanto
outra qualquer, mas não é bandeira de Tempo como botam. Ele tem bandeira,
porque a bandeira dele é a Nacional. É a Bandeira Brasileira, verde, amarela,
“Ordem e Progresso”. O Brasil não é uma nação? O caboclo não é brasileiro? (Ferreira, 1984, p. 65). Eu acho que a porta do candomblé é o caboclo, caboclo que traz, caboclo
que leva. Exu é o mensageiro, mas quem traz é o caboclo. É caboclo que tem
a liberdade, é caboclo que tem a ousadia, é o caboclo que entra no mato na
hora que ele quer, sai a hora que ele quer, então eu acho que o candomblé
tem muito a ver e a dever aos caboclos sim. Caboclo é a entrada e saída de
tudo... (Pai-de-santo de Salvador em entrevista a Ana Sheldon, 2019). Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 CASA DE CABOCLO O primeiro trecho apresentado é parte da narrativa que pais e mães
de santo ofereceram a Mundicarmo Ferreti sobre a entrada do culto aos
caboclos no tradicional Tambor de Mina: “uma quase invasão”. O segundo
foi extraído de palestra sobre o candomblé de caboclo proferida por Almiro
Miguel Ferreira, em 1983, durante o Encontro de Nações de Candomblé, Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 NA ROTA DOS CABOCLOS 167 teria uma finalidade política: servia como categoria usada pelas casas que se definiam
pela fidelidade à tradição africana, para marcar sua distinção com respeito a terreiros
vistos como sincréticos ou misturados. NA ROTA DOS CABOCLOS realizado em Salvador. Enquanto os informantes de Ferreti apontavam para
a entrada dos caboclos nas práticas religiosas africanas do Tambor de Mina,
Ferreira enfatizava a anterioridade dos caboclos – não precisou ninguém os
buscar, estavam já aqui muito antes dos orixás terem sido trazidos. Até por
volta de 1944, conta Ferreira, o candomblé de caboclo tinha presença forte
em Salvador; com o prestígio crescente do candomblé africano, sacerdotes
do culto aos caboclos teriam migrado e refeito seus cultos nos moldes do
candomblé keto, jeje ou angola. Como resultado, por volta de 1944 o
candomblé de caboclo teria se extinguido na Bahia (Ferreira, 1984), vencido
pelo modelo do culto aos orixás. Apesar das diferenças - o discurso da anterioridade contrasta claramente
com o discurso da intrusão (às vezes formulado como uma história de
mistura ou corrupção) - esses dois trechos levantam questões que durante
certo tempo13 ocuparam a atenção de pesquisadores das religiões de matriz 13 As referências a uma modalidade de candomblé, conhecida como candomblé de caboclo,
são bastante antigas e praticamente todas giram em torno da determinação de sua origem. Em 1906 Nina Rodrigues (2010) a descreveu como resultante de um processo de adição
de fragmentos de crenças ameríndias ao que era essencialmente o candomblé africano,
tal como praticado entre os povos de origem banto (cuja pobreza ritual, segundo ele, os
fazia mais abertos a incorporações estrangeiras). Poucos anos depois, em 1919, Manuel
Querino (1938) propôs tratar-se de uma modalidade de culto surgida da convivência
íntima entre indígenas (catequizados) e africanos. Mas foi a posição de Rodrigues que
dominou o debate na primeira metade do século XX. Tanto Arthur Ramos (1940)
quanto Edison Carneiro (1948) viram o candomblé de caboclo como essencialmente
um candomblé banto a que foram acrescentados elementos do espiritismo e da mítica
ameríndia (apesar de concordar com Ramos nesse ponto, Carneiro não falhou em
observar que os encantados caboclos também se faziam presentes em casas nagôs). A
associação direta entre candomblé banto/angola e culto aos caboclos (presente, nas
análises de Rodrigues, Ramos e Carneiro), levou Santos (1995) a questionar se a categoria
candomblé de caboclo de fato demarcaria um campo empírico próprio. Para este autor,
ao invés de circunscrever uma modalidade distinta de candomblé, o termo serviria para
indicar os terreiros em que o culto aos caboclos tinha proeminência (muitos dos quais
se definiam como nação caboclo). Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 NA ROTA DOS CABOCLOS No mundo dos candomblés baianos, seu emprego 168 Miriam C. M. Rabelo, Clara Flaksman africana. Como e quando os caboclos ganharam espaço nessas religiões? Teria realmente existido um culto dedicado exclusivamente aos caboclos
que se extinguiu nos anos 1940, conforme frisa Ferreira (1984), ou seria o
candomblé de caboclo apenas um nome dado aos candomblés que primeiro,
e de modo mais conspícuo, abriram suas portas aos caboclos, sacrificando
assim a tradição africana (quase uma categoria de acusação, conforme
aponta Santos, 1995)? É possível dizer que essas questões, embora difíceis de responder (até
porque tratam de uma história marcada por muitas especificidades e variações
regionais), valem a pena serem perseguidas – afinal podem esclarecer aspectos
importantes relativos à formação do campo religioso afroindígena na Bahia
e país. Mas o modelo cronológico linear pressuposto na sua formulação – a
ideia de que podemos traçar uma linha de desenvolvimento/transformação
a partir de um ponto localizado de origem – pode ter como efeito tornar
invisíveis os muitos e complexos trajetos através dos quais caboclos se cruzam,
entram nas vidas das pessoas (humanas) e vêm a conviver com orixás (vimos
alguns desses caminhos nas histórias de Toinho, Paulo e Nayran). Os relatos de Ferreti e Ferreira também têm algo a dizer sobre esse
último ponto. Os caboclos chegaram na Mina sem ser convidados, nego
ciaram entrada, ensinaram seus modos e cantigas (e provavelmente também
aprenderam). Conforme Ferreira, em Salvador, perderam com o tempo
seu culto próprio, mas adaptaram-se às regras das casas de candomblé de
tradição africana. Passaram, nos dois casos, a dividir espaço com voduns,
orixás ou inquices. Ambas narrativas testemunham a persistência dos cabo
clos. E enfatizam seu movimento: o percurso de quem chega, se acomoda
ou reacomoda segundo as possibilidades da situação. O depoimento do pai de santo de Salvador traz um elemento novo: não
só fala da presença dos caboclos em cultos de matriz africana como permite teria uma finalidade política: servia como categoria usada pelas casas que se definiam
pela fidelidade à tradição africana, para marcar sua distinção com respeito a terreiros
vistos como sincréticos ou misturados. Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 169 NA ROTA DOS CABOCLOS Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 NA ROTA DOS CABOCLOS articular uma perspectiva bem diferente para abordar a relação entre essas
entidades e os orixás. Aqui já não se trata nem de afirmar a anterioridade
dos caboclos, nem de relatar sua entrada nos cultos de origem africana: a
pertença dos caboclos ao candomblé é tomada como fato. O importante
é definir seu lugar, ou melhor, sua função – definir os caboclos pelo que
eles fazem: levar e trazer. Interessante a diferença sutil entre caboclo e Exu:
este é o mensageiro, mas quem leva e traz (transporta) é o caboclo. Talvez
essa diferença possa ser traduzida em outros termos ainda: se Exu domina
a arte da tradução que permite a comunicação entre humanos e deuses, o
caboclo domina a arte do transporte - porque é “da terra”, conhece bem seus
territórios e tem a ousadia necessária para percorrê-los livremente. As cantigas de caboclo ilustram bem esse ponto: nelas os caboclos
chegam trazendo para dentro do terreiro territórios de mata, sertão e água. No jarê, candomblé de caboclo da Chapada Diamantina, região central do
estado da Bahia, é assim também que chegam os orixás: como caboclos. Enquanto nos terreiros do Recôncavo que se identificam como giro de
caboclo ou umbanda, os orixás são cultuados com os caboclos (segundo
diferentes formas de distanciamento e aproximação), no jarê, são cultuados
como caboclos ou simplesmente são caboclos (Senna, 1984; Rabelo, 1990,
1993; Bannagia, 2015), em um processo que Senna (1984) definiu como
“caboclarização”. Para encerrar nosso percurso pelo mundo dos caboclos, vamos ao terreiro
de seu Agenor, curador de jarê que uma de nós (Miriam) conheceu em 1987. *** Agenor tinha um pequeno lote no Corujão, comunidade rural loca
lizada no município de Nova Redenção, área de agricultura camponesa
na Chapada Diamantina. Vivia, como a maioria dos seus vizinhos, do
cultivo da terra, mas também do seu trabalho como curador de jarê. Antes
de trabalhar no couro (como o jarê era também conhecido, em referência
aos tambores que batiam nas festas para louvar os caboclos), foi curador
de sessão (seus caboclos ou guias chegavam para rezar e dar assistência aos Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 170 Miriam C. M. Rabelo, Clara Flaksman clientes que o procuravam). Quando depois de uma vida de andanças – ao
sabor da disponibilidade de terra para plantar – se estabeleceu no Corujão,
aproximou-se do jarê. NA ROTA DOS CABOCLOS Teve como mestre no jarê um famoso curador do
município vizinho de Ibiquera. Nas festas de jarê, que passaram a ocorrer
todo sábado em seu modesto terreiro, começava sempre com um circuito
de rezas em latim que, uma vez findado, dava lugar à dança dos caboclos. Estes “vadiavam” ao som dos tambores até altas horas da noite, quando
Agenor suspendia a brincadeira das entidades para dar início às atividades
de cura. Trabalhava com seu caboclo Antônio Silvino, um Preto-Velho,
também referido como Baluaê. Fotografia 4 - Caboclo Antônio Silvino, jarê de Agenor (foto de Miriam Rabelo, 1987)
Era também Antônio Silvino quem abria a festa dos caboclos, chegando
com o corpo curvado pelo peso dos anos. O caboclo falava com uma voz
cansada, mas suas palavras tinham o tom de desafio. “Eu não tenho papa
na língua”, costumava dizer. Os cantos entoados por Antônio Silvino e
pelos outros caboclos que chegavam no terreiro descreviam os territórios de
onde vinham e por onde passavam, desenhando um quadro do movimento Fotografia 4 - Caboclo Antônio Silvino, jarê de Agenor (foto de Miriam Rabelo, 1987) ografia 4 - Caboclo Antônio Silvino, jarê de Agenor (foto de Miriam Rabelo, 1987) Era também Antônio Silvino quem abria a festa dos caboclos, chegando
com o corpo curvado pelo peso dos anos. O caboclo falava com uma voz
cansada, mas suas palavras tinham o tom de desafio. “Eu não tenho papa
na língua”, costumava dizer. Os cantos entoados por Antônio Silvino e
pelos outros caboclos que chegavam no terreiro descreviam os territórios de
onde vinham e por onde passavam, desenhando um quadro do movimento Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 171 NA ROTA DOS CABOCLOS a que se refere o pai de santo entrevistado por Sheldon. Seguem algumas
cenas retiradas do relato de uma festa realizada na casa de Agenor em 1987. Depois de uma baforada no charuto, Antônio Silvino clamou: “Viva Cristo
Rei! Viva Nossa Senhora! Viva Rei Ogum! Viva Tomba Morro! Viva Tranca
Rua! Viva o velho Antônio Silvino!” A cada salva, os presentes respondiam:
“Viva!” Dona Rosa, mulher de Agenor, entregou um cajado ao caboclo do
marido. Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 NA ROTA DOS CABOCLOS Antônio Silvino puxou uma cantiga, dançando com o corpo apoiado
no cajado: Ói o véio, ói o véio
que lá vem beirando o mar
Ói o véio, de tão véio
já num guenta mais andar Ói o véio, ói o véio
que lá vem beirando o mar
Ói o véio, de tão véio
já num guenta mais andar
Baluaê, nego véio
O meio do mundo é aqui
vem visitar meu peji Baluaê, nego véio
O meio do mundo é aqui
vem visitar meu peji O povo fez coro animado: “O meio do mundo é aqui”. O caboclo entregou
o cajado e cantou, remando com os braços erguidos: Canoeiro bate o remo
pra canoa não virar
Canoeiro bate o remo
que eu quero atravessar À certa altura, parou e fez um sinal da cruz. O gesto sinalizou a chegada de
outro caboclo. Era o Ogum: Ogum, Ogum é curador
Ogum é dos primeiros
que nesta casa chegou Depois de entoar alguns cantos e distribuir bênçãos aos presentes, Ogum
se retirou do corpo de Agenor. Em retribuição à cachaça que o curador Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 172 Miriam C. M. Rabelo, Clara Flaksman mandou servir aos tocadores, um deles puxou outra cantiga pra Ogum. O
ritmo dos tambores se intensificou. Vã, ajudante de Agenor, e duas mulheres
da vizinhança foram tomados por Ogum. O Ogum de Vã foi o primeiro a
puxar cantiga em meio a passadas largas e seguras, mas logo se foi deixando o
rapaz visivelmente abalado num canto do barracão. Com Ogum, as mulheres
seguiram dançando fazendo girar com velocidade as longas saias floridas. Boa noite rei
rei de tremer terra
Chegou rei Ogum
que tava vencendo guerra Boa noite rei
rei de tremer terra
Chegou rei Ogum
que tava vencendo guerra Areia, areiá
vamos jogar areia no mar
Eu não vim escondido
Eu vim vadiar A dança dos caboclos seguiu ainda por muito tempo. “Isso aí, caboclo!” –
gritaram os tocadores, contentes com o samba. Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 NA ROTA DOS CABOCLOS Também satisfeito, o curador
cantou: A casa pode, papai
O terreiro aguenta seus filhos A casa pode, papai A casa pode, papai O terreiro aguenta seus filhos O terreiro aguenta seus filhos Comprovando a abertura e força da casa para suportar a presença de seus
filhos, o jarê de Agenor viu ainda chegarem para “vadiar” as Titias Nagôs e
Iansã, Sultão das Matas, Sete Serras, Gentio, Tomba Morro, Oxóssi, Xangô
e Gentileiro. 173 NA ROTA DOS CABOCLOS Fotografia 5 - Caboclo Sultão, jarê de Agenor, 1987 (foto de Miriam Rabelo,
1987) ografia 5 - Caboclo Sultão, jarê de Agenor, 1987 (foto de Miriam Rab
1987) ESPAÇOS E ENCONTROS Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 ESPAÇOS E ENCONTROS Em uma conferência de 1973, intitulada “O encontro entre deuses
africanos e espíritos indígenas” (publicada em português em 2006), Bastide
propõe uma tipologia das religiões afro-brasileiras a partir das relações, aí
encontradas, entre orixás e espíritos ameríndios. O primeiro tipo corresponde Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 174 Miriam C. M. Rabelo, Clara Flaksman à separação nítida entre duas modalidades religiosas que coexistem sem
se interpenetrar – o catimbó e o candomblé ou, mais especificamente, o
candomblé iorubá que não aceita a entrada dos caboclos. No segundo tipo,
exemplificado pelos candomblés bantos (ou de nação angola), observa-se a
incorporação do culto dos caboclos ao espaço do terreiro. Essa incorporação,
entretanto, se dá de um modo que preserva a autonomia entre caboclo e orixá
– cultuados em festas separadas e segundo princípios diferentes. O terceiro
tipo corresponde à macumba carioca. Orixás e caboclos são aí honrados
em uma mesma cerimônia, mas segundo uma ordem (os deuses africanos
primeiro, os caboclos quando estes já se foram) que evita a mistura. Compa
rada aos candomblés bantos, explica Bastide, a macumba teria produzido
uma contração do tempo que mantém separadas as duas entidades. Assim,
conclui o autor, apesar das diferenças, candomblé iorubá, candomblé banto
e macumba são reveladores de uma mentalidade – africana - que rejeita a
confusão dos gêneros, substituindo a mistura ou interpenetração por diversas
formas de “compartimentação das entidades espirituais” (Ibid, 2006, p. 224). à separação nítida entre duas modalidades religiosas que coexistem sem
se interpenetrar – o catimbó e o candomblé ou, mais especificamente, o
candomblé iorubá que não aceita a entrada dos caboclos. No segundo tipo,
exemplificado pelos candomblés bantos (ou de nação angola), observa-se a
incorporação do culto dos caboclos ao espaço do terreiro. Essa incorporação,
entretanto, se dá de um modo que preserva a autonomia entre caboclo e orixá ,
q
p
– cultuados em festas separadas e segundo princípios diferentes. O terceiro
tipo corresponde à macumba carioca. Orixás e caboclos são aí honrados
em uma mesma cerimônia, mas segundo uma ordem (os deuses africanos
primeiro, os caboclos quando estes já se foram) que evita a mistura. Compa
rada aos candomblés bantos, explica Bastide, a macumba teria produzido
uma contração do tempo que mantém separadas as duas entidades. ESPAÇOS E ENCONTROS Assim,
conclui o autor, apesar das diferenças, candomblé iorubá, candomblé banto
e macumba são reveladores de uma mentalidade – africana - que rejeita a
confusão dos gêneros, substituindo a mistura ou interpenetração por diversas
formas de “compartimentação das entidades espirituais” (Ibid, 2006, p. 224). Finalmente, observa Bastide, no espiritismo de umbanda teríamos
uma efetiva integração entre os dois cultos. Os orixás deixam de incorporar
em uma religião centrada no culto aos espíritos desencarnados (entre os
quais se incluem os caboclos), mas são alçados à condição de chefes das
falanges em que se distribuem esses espíritos. Pode-se notar que, mesmo
aqui, divindades africanas e caboclos seguem separados, ainda que por uma
fronteira bem mais tênue. A chave da tipologia é uma noção de distância espacial (medida pelo
grau de aproximação ou separação entre os locais de culto aos orixás e aos
caboclos) e temporal (medida pela aproximação/separação entre os períodos
rituais dedicados a uma ou outra dessas entidades). Em “Os Problemas do
Sincretismo Religioso” (volume 2 de “As Religiões Africanas do Brasil”,
1985), Bastide já havia desenvolvido tipologia semelhante para tratar do
sincretismo entre o catolicismo e a religiosidade de matriz africana. Embora
aí o foco de sua análise não seja o encontro entre deuses africanos e espíritos NA ROTA DOS CABOCLOS Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 NA ROTA DOS CABOCLOS 175 indígenas, vale a pena nos determos na classificação que emprega, pois
esclarece aspectos do texto de 1973. Bastide propõe tratar do sincretismo a partir de três planos de análise:
o plano ecológico, o plano dos ritos e estruturas cerimoniais e o plano das
representações coletivas. Aos dois primeiros correspondem, grosso modo,
os vetores de análise que utiliza ao discutir o sincretismo afro-indígena e
por isso trataremos especificamente deles aqui. O arranjo de objetos em que se inscreve a presença de orixás e santos
católicos e sua distribuição pelo terreiro (formando zonas ou regiões) constitui
fonte para o entendimento do sincretismo naquilo que o autor denomina
de sua dimensão ecológica. Uma vez que o espaço ecológico é regido pela
justaposição – objetos materiais podem apenas estar ao lado ou próximos
uns dos outros, mas nunca se fundir – “graus de sincretismo aqui se mani
festarão pelo maior ou menor grau de aproximação ou afastamento entre
o que se poderia chamar de regiões católicas e de regiões africanas num
mesmo espaço sagrado” (Bastide, 1985, p.376). Assim tem-se uma variação
que vai da existência de dois altares em um mesmo terreiro, situados em
lugares diferentes - o altar católico no salão de danças, o africano no peji - à
adição de decorações católicas nos altares africanos ou de quadros de santos
católicos nas paredes do peji - “os dois espaços teoricamente separados,
tendem, pois a se interpenetrar” (Ibid, p. 377) -, até a abolição mesma
do peji (zona africana) e a coexistência em um mesmo altar de elementos
materiais católicos e africanos. O segundo plano tem o tempo como uma de suas dimensões e traz a
questão de como se relacionam ritos católicos e africanos no calendário de
festas dos terreiros ou no transcurso de uma mesma celebração. Conforme
Bastide, o plano temporal “nos põe em presença dos mesmos problemas
e das mesmas soluções que o plano espacial” (p. 378). Também aqui são
identificados tipos que variam conforme o grau de separação ou interpe
netração entre as sequências rituais. É possível usar a tipologia de Bastide para caracterizar as casas que
descrevemos. Em linhas gerais parecem se aproximar do “tipo” macumba ou Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 176 Miriam C. M. Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 NA ROTA DOS CABOCLOS Rabelo, Clara Flaksman se situar entre o candomblé angola e a macumba – manifestações religiosas
em que os caboclos ocupam compartimentos próximos, mas separados,
daqueles reservados aos deuses africanos e suas formas de culto. Várias configurações aqui descritas parecem se conformar a esse tipo. Caminhando pela área externa do terreiro de Paulo, encontramos lugares
bem demarcados – grutas, árvores, pedras, pequenas construções – lugares
de caboclo e lugares de orixá. Na área interna, o altar católico situado em
um canto do barracão e o peji em um quarto separado marcam uma zona
católica e uma zona africana e sugerem sequências rituais bem diferentes. No
terreiro de Toinho, o calendário de celebrações mantém a separação entre
festa de orixá (realizada no keto) e festa de caboclo (embora, os caboclos
“não fiquem fora de nada) e no espaço compartilhado de dona Zenaide e
Nayran, sessão de caboclo e candomblé não se confundem. Em todos esses
lugares, a “mistura” parece ser evitada pela existência de compartimentos
(tanto espaciais quanto temporais). Mas se a ideia de um espaço/tempo mensurável por distâncias mais ou
menos fixas e passível de divisão em compartimentos parece acenar com um
caminho para classificação e comparação entre terreiros de acordo com o
modo como caboclos e orixás são aí postos em relação (e para uma teoria
do sincretismo afro-indígena-católico, como pretendia Bastide), a intensa
movimentação dos caboclos que registramos aqui sugere que esse modelo
só funciona às custas de uma simplificação excessiva. Uma das fraquezas do modelo de Bastide é que nele tanto o espaço,
quanto o tempo são reduzidos à extensão. Enquanto o espaço é a extensão
em que estão distribuídos objetos, o tempo é a extensão em que se distribuem
sequências que podem estar mais ou menos separadas e que nos tipos mais
sincréticos tendem a se interpenetrar ou mesmo se fundir. Nesse modelo, o
movimento só pode ser descrito como deslocamento de um ponto a outro
ou de um compartimento a outro em uma série tempo-espacial. O quadro muda completamente quando espaço e tempo são pensados
a partir do movimento – ou a partir de uma abordagem que ressalta sua
produção prática. Se antes a única questão que o movimento dos caboclos NA ROTA DOS CABOCLOS 177 NA ROTA DOS CABOCLOS permitia articular dizia respeito à preservação ou não de uma distância
com relação aos orixás, agora a atenção se volta para as diferentes formas
espaciais que o movimento dessas entidades contribui para produzir e às
possibilidades de convivência implicadas em cada uma delas. Ao seguirmos o movimento dos caboclos a partir dessa mudança de foco,
nos deparamos com uma topologia móvel. Na casa de Paulo, os caboclos
Gentio e Boiadeiro recortam, com seus diferentes temperamentos, as outras
divisões do terreiro, criando centros de força que puxam entidades, objetos
e lugares ora em uma, ora em outra direção. No espaço compartilhado de
dona Zenaide e Nayran, afinidades entre Jaguaraci e Juremeiro instauram
uma zona de interseção entre o candomblé de uma e a umbanda da outra:
na casa de Zenaide, caboclos mais chegados ao candomblé tendem a se
manter afastados da mesa branca (“por questão de respeito”), mas podem
bem se juntar aos caboclos do candomblé de Nayran. E na casa de Toinho,
a ubiquidade dos caboclos – que não podem ficar de fora de nada – sugere
que “virar (a festa) pra caboclo” é menos operar um deslocamento temporal
(de um compartimento de tempo a outro), do que fazer girar o espaço
para deixar ver o que estava temporariamente oculto (mas presente) - ou
talvez trazer para frente o que, na festa dos orixás, funcionava como pano
de fundo. A depender de como são “provocados” ou “ativados” por afetos
e afinidades que funcionam como verdadeiras forças de atração, espaços (e
objetos) podem assumir formas diferentes. Esses processos de espacialização falam de uma ecologia – ponto que
Bastide não falhou em perceber. Mas a questão ecológica interessante que
levantam não diz respeito às relações de justaposição entre objetos rígidos,
senão às possibilidades de convivência entre seres – caboclos, orixás e os
humanos que veem seus corpos transformados pela companhia dessas
entidades. É justamente no sentido de convivência que Toinho emprega a palavra
mistura: em referência às possibilidades de compor um coletivo a partir de
uma coleção e à força que advém de “um conjunto de várias mãos seguras”. Essa ideia está presente também na festa de Agenor, quando o terreiro é 178 Miriam C. M. Rabelo, Clara Flaksman performado justamente como o espaço – “o centro do mundo” – capaz de
atrair e aguentar a movimentação intensa de entidades e territórios – o mar,
o mato, o sertão. NA ROTA DOS CABOCLOS No esquema de Bastide, o jarê corresponderia justamente à mistura
como interpenetração e confusão de gêneros, degradação da memória africana
sob efeito da pobreza e dispersão a que foram expostas as populações de
afrodescendentes nas zonas rurais. Mas se pensamos a ecologia nos termos
de Toinho, talvez possamos dizer que enquanto na umbanda e na gira, cabo
clos e orixás experimentam aproximações e convivem em arranjos que são
produzidos, em grande medida, sob efeito da movimentação dos primeiros,
no jarê é justamente porque vêm como caboclos, portando territórios de
água, mata e sertão, que os orixás podem conviver no terreiro. Ou ainda:
no jarê, caboclo é justamente a maneira de existir junto de caboclos e orixás. Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 O QUE DIZER, ENTÃO, DOS CABOCLOS? Neste texto seguimos alguns rastros deixados pelos percursos dos cabo
clos nos terreiros e nos corpos de pais e mães de santo baianos que mantêm
com eles estreita relação. Entidades que escapam a definições precisas, os
caboclos se destacam pelo movimento: chegam trazendo mata, mar e sertão,
invadem, persistem, se juntam com orixás e espíritos desencarnados, vivem
no leva e traz, desenhando territórios afetivos e arrastando consigo humanos,
outras entidades, objetos. Definem seus próprios caminhos e ao fazê-lo
multiplicam conexões não só entre tempos e lugares diferentes, mas entre
práticas e formas de existir diferentes. Para traçarmos as rotas dos caboclos e aos poucos desenharmos as
cartografias de suas trajetórias, buscamos aqui acompanhar seu movimento e
dos caboclos cria zonas de atração e performa uma topologia móvel, a partir
da qual se abrem diferentes possibilidades de convivência. Essas caracterís
ticas irão inevitavelmente nos escapar enquanto estivermos presos a uma
concepção de movimento como mero deslocamento no espaço. NA ROTA DOS CABOCLOS 179 É bem verdade que orixás também se conectam a humanos e a outros
orixás por afinidades e afetos, produzindo aproximações e separações na
dinâmica interna dos seus terreiros. Além disso, movimento e traçado de
conexões caracterizam também os modos de algumas entidades do panteão
africano, em especial Exu. A questão aqui não é se apenas o caboclo detém
essa propensão ao movimento e aos afetos. O que nos interessa ressaltar é
que no movimento de quem chega, persiste, se junta e compõe, caboclos
maximizam e estendem possibilidades de conexão e convivência, contri
buindo para produzir arranjos variados nos terreiros e na vida das pessoas
que são tocadas por eles. Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 Debates do NER, Porto Alegre, ano 20, n. 38, p. 145-180, ago./dez. 2020 REFERÊNCIAS BANNAGIA, Gabriel. As Forças do Jarê: religiao de matriz afriacana na
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das Letras. 2006 [1973]. CARNEIRO, Edison. Candomblés da Bahia. Rio de Janeiro: Ed. de Ouro,
1969 (1948). FERREIRA, Almiro Miguel. Candomblé de Caboclo. In: Encontro das Nações
de Candomblé. Salvador: Inamá e Universidade Federal da Bahia, 1984. FERRETI, Mundicarmo. Terra de Caboclos. São Luís: SECMA, 1994. LANDES, Ruth. A Cidade das Mulheres. Rio de Janeiro: Editora UFRJ,
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Paulo, Ed. Nacional, 1971 [1942]. QUERINO, Manuel. Costumes Africanos no Brasil. Rio de Janeiro: Civili
zação Brasileira, 1938 (1919). RABELO, Miriam C.M. Play and Struggle: dimensions of the religious expe
rience of peasants from Nova Redenção, Northeast Brazil. Tese de doutorado,
Universidade de Liverpool, Reino Unido, 1990. RABELO, Miriam C.M. Jarê Cult Centres and Eccesiastical Base Commu
nities: the creation of identity among peasants in Nova Redenção, Northeast
Brazil. In: ROSTAS, Susana; DROOGERS, André (orgs.). The Popular Use
of Popular Religion in Brazil. Amsterdam: CEDLA, 1993 (p. 29-51). RAMOS, Arthur. O Negro Brasileiro. Recife: Fundação Joaquim Nabuco,
1940 (1934). RODRIGUES, Nina. Os Africanos no Brasil. Centro Edelstein de Pesquisas
Sociais, Biblioteca Virtual de Ciências Humanas, 2010 (1938). SANTOS, Jocélio Teles dos. O Dono da Terra: o caboclo nos candomblés da
Bahia. Salvador: Editora Sarah Letras, 1995. SENNA, Ronaldo. Jarê: manifestação religiosa na Chapada Diamantina. Tese
de doutorado, Universidade de São Paulo, 1984. Recebido em: 07/08/2020
Aprovado em: 03/09/2020
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Impact of economic risks on the financial stability of agricultural enterprises
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Impact of economic risks on the financial stability of agricultural
enterprises
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1 Elena Dadayan1,*, Anna Storozheva1, and Ekaterina Letyagina1 1Krasnoyarsk State Agricultural University, 660049, Russia, Krasnoyarsk, Mira 90 1Krasnoyarsk State Agricultural University, 660049, Russia, Krasnoyarsk, Mira 90 Abstract. This article features a topical research, which is determined by the state of the economic sphere
of life of modern Russian society. It focuses on the agricultural economic field of activity represented by
agricultural enterprises. In the process of researching issues raised in the article, we have used general and
specific scientific research methods, such as observation, analysis and synthesis, deduction and induction. They are what made it possible to identify economic risks that affect the financial stability of economic
entities. We have focused on the financial risks of agricultural enterprises. Activities in agriculture are risky,
and the growth of agricultural production, financial stability, etc., depends on how subjects overcome
economic risks of various types. It is no secret that the growth of agricultural production is largely shaped
by export economic operations. Thus, this leads to a drop in domestic consumer demand. Economic risks
can lead to financial insolvency of agricultural enterprises. The article focuses on the specifics of the
bankruptcy procedure of an agricultural organization. We provide quantitative indicators on declaring a
debtor bankrupt by sectors of the economy for 2017 and 2018, including ones for bankruptcies of
agricultural enterprises in the Krasnoyarsk region. Using systematic approach, we come to the conclusion
about the main problems associated with the financial insolvency of agricultural enterprises. * Corresponding author: dadaelena@yandex.ru 1 Introduction nature of the harvest, which ultimately determines the
profitability of the agricultural enterprise, does not
always allow complying with the requirement of
systematic pay and financial incentives for workers. In
agriculture, production and financial and economic
results vary immeasurably more than in other industries,
depending on the prevailing weather and climatic
conditions. It is quite natural that activities in agriculture are very
risky. Not only the growth of agricultural production, but
also the continued viability and financial sustainability
of the agricultural producer depends on the ways and
means its actors will use to overcome or mitigate
economic risks. We believe that when determining the financial
condition of the agricultural producer, it is necessary to
consider not only the seasonal nature and weather and
climate conditions, but also the complexity of
technological processes, remoteness from the market for
products, low profitability, etc. Agriculture, crop production, animal husbandry and
other agricultural sectors continue to play a significant
role in the life support of society. At the same time, the
Russian Government seeks to actively develop these
areas. Russian citizens can now receive land resources
for the implementation of agricultural and other
activities on preferential terms. Thus, cases of financial insolvency of agricultural
enterprises, which then determine the beginning of the
bankruptcy procedure, become quite common. However, we should note that the growth of
agricultural production is largely driven by export
economic operations. At the same time, this growth is
constrained by the constantly falling domestic consumer
demand. For example, consumption of milk and dairy
products per capita in 2018 compared to 2013 decreased
by 20 kg. Every second citizen consumes less than
rational norms of fish. The same situation is with meat:
according to statistics, from July 2018 to June 2019,
Russians purchased 4.3% less meat products than during
the same period of the previous year. Analysts recorded
a decrease in sales in all categories of meat products,
from poultry and pork to sausages. © 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/). ICEPP-2020
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e3s DP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0
ses/by/4.0/). 2 Methods Moreover, the
duration of the cycle (period) in normal conditions,
depending on the specifics of the agricultural industry
(animal husbandry, crop production, and their specific
types), can range from three to eighteen months and
practically cannot be reduced. • external, more typical for agricultural organizations,
which are independent of the enterprise and include,
among other things, natural factors. Another equally important feature mentioned earlier
is the natural and seasonal nature of agricultural
production. The maximum value of loads is determined
by a unique set of natural factors and significant
production actions (in crop production, for example, they
are weather, climate, precipitation, and soil quality
during planting and harvesting periods). This specificity is primarily related to the use of
agricultural land as the main means of production. In
addition, the activities of agricultural organizations
depend in no small part on natural and climatic
conditions, are seasonal in nature, which, in one way or
another, is reflected by the legislator in its regulation [1]. Mukhin I.N. notes that one of the most objective
methods for assessing the degree of insolvency risk is
the Davydova-Belikov model, developed at the Irkutsk
State Economic Academy. The model includes four
factors serving as the base for the regression equation,
using which we can determine the final indicator of
financial insolvency risk [2]. These two traits define such feature as low fund
return, capital turnover, and high level of overhead. Indeed, during certain periods, agricultural producers are
in high need to use a large amount of mechanical
equipment at the same time, which in general might
never be used again during the year, but its maintenance
or repairs burden on the cost of production. Additionally,
we need to take into account that a part of agriculture
produce needs to be laid aside for the internal needs of
agricultural producer for future reproduction (seeds,
feed, etc.), which reduces the commerciality of
production. In view of the specifics of their activities, the
bankruptcy procedure of agricultural organizations has
several features and nuances. According to Ruschitskaya
O.A., this is due to the fact that "as a rule, in the
procedures provided by the Federal Law, the financial
recovery procedures and the external management of
agricultural organizations do not restore the solvency of
the organizations, and as a result, they go under the
hammer. At best, the owner changes. At worst, no one
needs the property of agricultural bankrupts. 2 Methods In the process of researching the issues raised in the
article, we have used general and specific scientific
research methods to consider the influence of economic
risks on agricultural enterprises. We can observe the
process of the influence of economic risks on the
procedure for declaring a debtor bankrupt in various
sectors of the economy in Russia as a whole and in the
Krasnoyarsk region in particular. We demonstrate the
tendency of a gradual increase in the number of
applications for declaring a debtor bankrupt in relation to In addition, agricultural production remains special in
terms of return on investment and profit. The seasonal ICEPP-2020
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e3s legal entities and peasant farms throughout the country. However, the growth of such cases in the Krasnoyarsk
region is decreasing. Considering the above, it is quite
clear that it is necessary to investigate the issues of
regulation of the specifics of bankruptcy of agricultural
organizations and to determine the main factors guiding
the fate of agricultural producers at the stage of
bankruptcy. legal entities and peasant farms throughout the country. However, the growth of such cases in the Krasnoyarsk
region is decreasing. Considering the above, it is quite
clear that it is necessary to investigate the issues of
regulation of the specifics of bankruptcy of agricultural
organizations and to determine the main factors guiding
the fate of agricultural producers at the stage of
bankruptcy. This raises the need to properly and effectively
investigate and determine the financial condition of the
company, "including the use of digital technologies that
contribute to the development of the digital economy as
a whole" [4]. At the same time, when assessing the financial
condition of the agricultural producer, it is necessary to
consider the specific features of the agricultural industry
that determine this process: Y.V. Zinchenko proposed two main reasons for the
bankruptcy of producers, with which we certainly agree:
• internal, which depend on the investment, operating
and financial activities of the company, low level of staff
qualifications; For example, cyclicality (periodicity) is an important
characteristic of agricultural production. 2 Methods And there
appear more and more empty buildings that look
reproachfully with their eye sockets of broken windows"
[3]. Moreover, another significant difference is the
inability to quickly recover the investment, as in the
agricultural process it is possible to return capital only
when the entire cycle is completed. Meanwhile,
production risks are determined by the significant
influence of natural and seasonal factors and the time it
takes for finding the consumer for the agricultural
products, return of receivables, etc. This can cause the
turnover rate can increase further, and income, in its
turn, decrease. In the context of inflation that is now
typical of all economies of the world this can lead to a
significant depreciation of profits, and as a result to
financial insolvency and even bankruptcy. These circumstances led to the allocation in the
legislation of the Russian state of systematic rules of
legal regulation of the process of recognition of an
agricultural
organization
as
insolvent,
which
is
concentrated in paragraph 3 of Chapter IX of the Federal
Law of 26.10.2002 No. 127-FZ "On insolvency
(bankruptcy)" (further – Law on Bankruptcy). The above traits create conditions in which the use of
a narrow intra-industry specialization exceeds reasonable
economic risks. The optimal and necessary for any
agricultural producer organization is the combination of
various agricultural industries, in particular livestock and
crop production, or the combination of products of the
same agricultural orientation (growing cows, pigs,
horses, small cattle, etc.). This achieves the most optimal
and efficient use of resources when a part of the
produced product of one type of agricultural production
is used in another. It also ensures efficient and effective
employment of agricultural workers. Section 177 of the Law on Bankruptcy establishes a
legal definition of an agricultural organization, naming
as such legal entities whose main activities are: Section 177 of the Law on Bankruptcy establishes a
legal definition of an agricultural organization, naming
as such legal entities whose main activities are:
• agricultural production, which generates at least fifty
per cent of total revenue;
d
i
d
i
f
i
l
l
d
h • agricultural production, which generates at least fifty
per cent of total revenue; • agricultural production, which generates at least fifty
per cent of total revenue; • production and processing of agricultural products, the
proceeds of which account for at least fifty per cent of
total revenue. 2 Methods Thus, in order to recognize a legal entity as an
agricultural organization, the court needs to assess the
nature of its activities and analyze the financial part of
the organization. A very important specific feature of agricultural
production, which should undoubtedly be taken into 2 ICEPP-2020
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e3s account, is formed by the main resource used in such
production – land. Its considerable length and territorial
spread can frequently result in large distances from the
markets of manufactured goods and, consequently, in an
increase in transportation costs [5]. Lvov O.A. and Peganova O.M. note that knowledge
of economic prerequisites and analysis of factors and
causes of bankruptcy processes are necessary to manage
the crisis of socio-economic systems, allowing us to
predict the onset of a crisis at enterprises and take timely
preventive measures [5]. We should also note that the specificity of
agricultural products (its biological origin) determines
the presence of a significant factor of randomness, that
cannot always be controlled by agricultural producers,
i.e., all other things being equal, product reproduction
can be different, not always fitting forecasts of product
performance. It is worth agreeing with the position of these
authors. Indeed, the stability of any producer depends on
the correct calculations and assessment of economic
risks. In addition, analysis of the financial condition of
the enterprise allows us to identify the company at risk
and prevent bankruptcy in most cases. Agricultural enterprises, moreover, are forced to
strictly follow the principle of continuity of the process. At the same time, not all the parts of the process can be
automated and it is not always possible to track the
quality of each operation that makes up the process. Once again, given the specifics of agricultural
enterprises, the arbitrator can properly and more fully
assess the financial condition of the agricultural
enterprise, as well as draw the right conclusions about
the future of agricultural enterprises. Looking at the distribution of figures in the statistics
for 2017 and 2018 on the industry structure of
companies declared bankrupt, we should point out that
the number of reports of the debtor's bankruptcy and the
opening of competitive proceedings for legal entities
operating in agriculture, forestry, hunting and fishing are
at 569 and 558 respectively, and it ranks seventh out of
twenty possible. 2 Methods Thus, the specific features of agricultural production
listed by us should be considered when determining the
state of financial and economic activity in the
bankruptcy procedure. This will allow to take into
account all the peculiarities of the economic entity and
correctly establish the procedure of bankruptcy, as well
as contribute to the successful implementation of this
procedure in relation to a particular legal entity. In addition, there is no significant downward trend in
this indicator, and there has been no growth either. We should note that to date, the period of financial
and economic detail of the debtor which should be
subject to analysis by the arbitration court or the
competitive manager when categorizing the organization
as agricultural has not been established by law. Thus, the bankruptcy procedure, including that of
agricultural producers, often ends with the recognition of
the debtor as bankrupt and the opening of competitive
proceedings (Table 1). Considering foreign experience, we note that the
problem of economic risk was studied in the works of
such authors as G.A. Karolyi, T.M. Ruefli, K. Cuthbertson, P. Gripaios, M. Morishima, etc. 3 Results and analysis The industry structure of companies declared
bankrupt. the bankruptcy case) have been filed in the Arbitration
Court of Krasnoyarsk Region over the past five years. 4 Results and conclusions Number of reports of bankruptcy and opening of
competitive proceedings against legal entities and peasant
farms in Krasnoyarsk region for 2015-2018. 4 Results and conclusions We believe that it is possible to draw the following
conclusions: 1. The financial sustainability of any producer,
including
agricultural,
depends
on
the
correct
calculations and assessment of economic risks. 2. To maintain the viability of the agricultural
enterprise at risk to be declared bankrupt, an analysis of
the financial condition of an enterprise needs to be
carried out. 2. To maintain the viability of the agricultural
enterprise at risk to be declared bankrupt, an analysis of
the financial condition of an enterprise needs to be
carried out. 3. Analysis of the debtor's financial condition shows
solvency, financial stability, his business activity. All the
main indicators of the debtor's activity reflected in the
financial and economic activity report have a clear
tendency either to decline or increase. They indicate the
deterioration or improvement of the financial condition
of the company. 4. At the same time, we believe that in determining
the financial condition of the agricultural producer it is
necessary to understand and take into account the basic
characteristics of agricultural production, namely: 4. At the same time, we believe that in determining
the financial condition of the agricultural producer it is
necessary to understand and take into account the basic
characteristics of agricultural production, namely: • the period of operating production in agriculture is
determined by the calendar year; • agricultural production is associated with highly labor-
intensive processes; • often the agricultural producer is far away from the
direct markets of manufactured products; • agriculture tends to have low profitability; • agriculture tends to have low profitability; • agricultural production and its success are determined
by seasonal natural conditions; • financial stability of agricultural producers directly
depends on government support, subsidies, and financial
aid. Looking at the regions of Russia, the Krasnoyarsk
region in particular, we should note that the trend of four
years indicates a gradual increase in the number of
reports of the debtor being declared bankrupt and the
opening of competitive proceedings against legal entities
and peasant farms (Table 2). 5. We should note that public agricultural production
policies (protectorate and support for agricultural
producers) are reflected in the low numbers of
agricultural enterprises declared bankrupt. 5. We should note that public agricultural production
policies (protectorate and support for agricultural
producers) are reflected in the low numbers of
agricultural enterprises declared bankrupt. Table 2. 3 Results and analysis In this regard, court practice shows that most often the
share of revenue for agricultural enterprises is
determined, based on the data of the latest balance sheet
(the ruling of Federal Antimonopoly Service of the
Moscow district of 29.07.2008 No. KG-A40/6402-08,
case No A41-K2-5510/06). In theory of law, there is a position that it would be
more appropriate to analyze revenue data based on the
seasonal nature of the debtor's activities. According to
Tkachev V.N., the Law on Bankruptcy should be
supplemented by a rule on the need to take into account
the financial and economic activities of the debtor not
only during the year preceding the date of the relevant
procedure, but also the average figure for the entire
period of the debtor's existence. Moreover, the choice of
the calculation method should be given to the debtor
himself, which will better protect the interests of the
agricultural debtor [4]. We share this point of view and
believe that when a bankruptcy case is filed, the status of
the debtor and the possibility of applying Special Law on
Bankruptcy
rules
established
for
agricultural
organizations to it should be determined on the basis of
the documents submitted in the case, including financial
and accounting records. 3 ICEPP-2020
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e3s Table 1. The industry structure of companies declared
bankrupt. No
Number of reports of bankruptcy
and opening of competitive
proceedings against legal entities
and peasant farms (by the debtor's
industry)
2017
2018
1 Trade
3832
3701
2 Construction
2767
2670
3 Manufacturing and processing
1488
1435
4 Real estate transactions
1465
1405
5 Professional, scientific and technical
activities
673
732
6 Transportation and storage
629
638
7 Agriculture,
forestry,
hunting,
fishing, and fish farming
569
558
8 Financial and insurance activities
319
352
9 Supplying electric energy, gas, and
steam; air conditioning
292
324
10 Administrative and related additional
services
282
243
11 Hotels and catering companies
208
207
12
Water
supply,
drainage,
waste
collection and disposal, liquidation of
environmental pollution
174
184
13 Information
and
communication
activities
192
160
14 Mining
153
135
15 Other types of services
57
64
16 Activities in culture, sports, leisure,
and entertainment
60
55
17 Health and social services
54
54
18 Public administration and military
security; social security
2
8
19 Education
18
8
20 No data
204
184
Total
13541
13117 Table 1. References [1]
Y.V. Zinchenko, L.L. Orekhova, Causes of
bankruptcy of enterprises in Russia, Young
Researcher of Don, 1, 4, 110-114 (2017). Region
name
2015 2016 2017 2018
Number of
bankrupt
companies
as % of
companies
in the
region, 2018
Increase in
the
number of
bankrupt-
cies,
2018 to
2017, %
Krasnoyarsk
Region
155 164 172 171
0.24%
-0.58% [2]
I.N. Mukhin, Bankruptcy of enterprises and the
system of assessing their solvency, Alley of
Science, 16, 1, 647-650 (2017). [3]
O.A. Ruschitskaya,
Social
aspects
of
the
insolvency of agricultural enterprises, Agrarian
Bulletin of the Urals, 8, 126, 102 (2014). [4]
V.N. Tkachev, Insolvency (bankruptcy) of special
categories of subjects of competition law:
theoretical and practical problems of legal
regulation , Walters Clover, Moscow, 162 (2007). At the same time, the number of cases of bankruptcy
of agricultural enterprises in Krasnoyarsk region is
insignificant compared to the number of bankruptcies of
legal entities related to other types of economic activity. Thus, depending on the organizational and legal form of
debtors, 13 applications (including applications to join [5]
O.A. Lvova, O.M. Peganova, Factors and reasons
for the bankruptcy of companies in the current 4 ICEPP-2020
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e3s economy, State administration, Electronic Bulletin
44, 64-82 (2014). [6] [6]
S.M. Kurbatova, L.Yu. Aisner, V.V. Naumkina,
Some aspects of the essence and legal regulation of
agriculture digitalization as one of the priorities of
the
modern
state
policy
of
agriculture
development, IOP Conference Series: Earth and
Environmental Science Krasnoyarsk Science and
Technology City Hall of the Russian Union of
Scientific and Engineering Associations, 32021
(2019). [7]
L.Yu. Aisner, O.D. Naumov, M.G. Nedelina,
Efficiency of land use in the Russian Federation in
the post-reform period: problems and prospects,
IOP Conference Series: Earth and Environmental
Science, 421, 032014 (2020). [8]
Statistical
bulletin
of
the
United
Federal
Bankruptcy Registry as of 31.12.2018. [Electronic
resource]. Available at: http://www.fedresurs.ru
(Accessed: 15.01.2020). [9]
G.A. Karolyi,
Predicting
risk:
some
new
generalizations, Management science, 38, 1, 57-74
(1992). [10] T.M. Ruefli, Strategic risk: an ordinal approach,
Management science, 38, 12, 1707-1731 (1992). [11] K. Cuthbertson, P. Gripaios, The macroeconomy: a
guide for business, Routledge, London, 152
(1993). [12] M. Morishima, Equilibrium, stability, and growth:
a multi-sectoral analysis, Clarendon Press, Oxford,
(1964). [13] L.N. Tempan, N.D. Eriashvili, Risk management in
a financial crisis, A textbook, Unity, Moscow, 160
(2017). [14] F. References Knight, The concept of risk and uncertainty,
Almanac: Theory and History of Economic and
Social Institutions and Systems, Moscow, 5, 23-24
(1994). [15] A.S. Shapkin, V.A. Shapkin, Economic and
financial risks. Valuation, management, investment
portfolio, 7th ed., Dashkov and K, Moscow, 554
(2008). [16] J.A. Schumpeter,
Theory
of
Economic
Development, Direct Media, Moscow, 400 (2007). [17] E. Holmes, Risk Management, Eksmo, Moscow,
137 (2007). 5
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Understanding HIV Positive Women’s Experiences with Antenatal Care in Rural Maputo Province, Mozambique
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Abstract Introduction: Motherhood among women living with HIV/AIDS is considered perilous in most of the countries of
Sub-Saharan Africa. Objectives: To analyse women’s compliance to biomedical norms recommended to reduce the risk of mother-to-
child transmission of HIV during pregnancy and childbirth in the rural province of Maputo. Methodology: A qualitative study was performed, consisting of in-depth interviews and focus group discussions
with women who had become mothers, in-depth interviews with community health workers and semi-structured
interviews with mother and child health nurses. We used Bourdieu’s theory of practice as a guiding framework to
analyse the data. Results: Our findings showed that participants complied with some recommended biomedical norms to reduce
the risk of mother-to-child transmission of HIV, such as subsequent antenatal visits, adherence to antiretroviral
therapy and childbirth at the health facility. However, they did not comply with the timing of the first antenatal care,
use of modern health care system to treat illness episodes and use of condom during pregnancy. Conclusion: The study results suggest that compliance to the recommended prevention of mother-to-child
transmission is the result of complex interactions in which participants rely on knowledge and resources within both
the family and community and the modern health care system. Awareness among health care workers of pregnant
women’s perspectives as well as an adequate education about the timing of the first antenatal care and the benefits
of antenatal care in both the health facility and community could thus enable to improve prevention of mother-to-
child transmission of HIV. Keywords: Antenatal care; Experience of pregnancy; HIV positive
women; Mozambique HIV infected women also face stigma and discrimination in their
communities, contributing to a limited or lack of access to adequate
health services and stress [7]. Understanding HIV Positive Women’s Experiences with Antenatal Care in Rural
Maputo Province, Mozambique Carlos Eduardo Cuinhane1,2, Gily Coene3, Kristien Roelens4 and Christophe Vanroelen5
1Department of Sociology, Eduardo Mondlane University, Maputo, Mozambique
2Vrije Universiteit Brussel (Brussels University), RHEA, Centre for Research in Gender, Diversity and Intersectionality, Brussels, Belgium
3Department of Philosophy and Ethics, Vrije Universiteit Brussel (Brussels University), RHEA, Centre for Research in Gender and Diversity, Brussels, Belgium
4Department of Obstetrics & Gynaecology, Faculty of Medicine & Health Sciences, Ghent University, Ghent University Hospital, Belgium
5Department of Sociology, Vrije Universiteit Brussel (Brussels University), Brussels, Belgium *Corresponding author: Carlos Eduardo Cuinhane, Vrije Universiteit Brussel (Brussels University), RHEA - Centre for Research in Gender, Diversity and
Intersectionality, Brussels, Belgium, Tel: (+258) 824080890; Fax: (+258) 21485402; E-mail: c.cuinhane@hotmail.com Received date: July 20, 2016; Accepted date: Aug 22, 2016; Published date: Sep 02, 2016 Received date: July 20, 2016; Accepted date: Aug 22, 2016; Published date: Sep 02, 2016 CE, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
d reproduction in any medium, provided the original author and source are credited. Copyright: © 2016 Cuinhane CE, et al. This is an open-access article distributed under the terms of the Creative Commons Att
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: © 2016 Cuinhane CE, 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. Clinics in Mother and Child Health Clinics in Mother and Child Health 13:3 DOI: 10.4172/2090-7214.1000246 Cuinhane et al., Clinics Mother Child Health 2016,
13:3
DOI: 10.4172/2090-7214.1000246 Cuinhane et al., Clinics Mother Child Health 2016,
13:3
DOI: 10.4172/2090-7214.1000246 Clinics Mother Child Health, an open access journal
ISSN:2090-7214 Page 2 of 9 In this regard, Bourdieu distinguishes between
economic, cultural, social and symbolic forms of capital. In Mozambique many women do a HIV test for the first time at
ANC. When they are diagnosed HIV positive, they start ARTs
immediately and are integrated in a “One-stop” model treatment at the
maternity clinic. This model consists of offering both mother and child
health HIV service in the same room during pregnancy and
breastfeeding [3]. Late ANC may result in high risk of MTCT of HIV,
estimated at 10-25% between 14-36 weeks of pregnancy and 25-50%
during the last month of pregnancy [15]. Most pregnant women still do not meet a minimal of 4 ANC visits,
and not all women attending the 4 ANC visits give birth in a health
facility. In Sub-Saharan Africa, in 2014, only 49% of women reported
at least four ANC visits and only 52% of childbirths were attended by
skilled health personnel [16]. In 2011, the Demographic Health Survey
(DHS) at a national level showed that in Mozambique, 91% of women
attended at least one ANC, 51% attended 4 ANC visits, and 55% gave
birth at a health facility [17]. Likewise, the rate of MTCT in Sub-
Saharan Africa was 14% in 2014; while Mozambique reported 9% of
MTCT of HIV, despite providing ARTs to 91% of pregnant women
living with HIV [7]. From Bourdieu’s theory we can consider the health care system and
the family/community as historically relative autonomy developed
social fields. The family/community, particularly in rural areas, is still
largely structured by traditional or cultural values, norms, ways of
knowing and doing whilst the health care system relies on biomedical
norms, scientific standards and procedures. In both fields, there are
different assumptions about what to know, and how to behave. Through attending ANC, pregnant women move between these two
fields, and respond to the – often conflicting – demands or logics of
these fields. Thus, Bourdieu’s framework enabled to explain how
pregnant women accessed and used different resources throughout
pregnancy and childbirth and how such access influenced the use of
biomedical norms. Adherence to ANC has been burdened by structural barriers, such
as lower maternal education and cultural factors like the perception of
a pregnancy as a healthy physical state. Therefore, some women visit
health professionals when they perceive a treat to their well-being. Page 2 of 9 Page 2 of 9 living with HIV, ANC enables access to ARTs to early prevent MTCT
[12,13], counselling about benefits of giving birth at a health facility,
safe practices of breastfeeding and early postnatal care [2]. individual actions, while the latter stresses individual attitudes and
decision-making processes. To overcome this dichotomy, we used the
theory of Pierre Bourdieu, and in particular his Theory of Practice, as
an explanatory framework for this study [27]. This framework enabled
us to understand the inherent complexities of the participant’s
experiences and practices with regard to attending antenatal care and
childbirth at a health facility. Most African Sub-Saharan countries, including Mozambique, have
adopted WHO [1] guidelines that recommend at least 4 visits of ANC
for women whose pregnancy is progressing normally, with the first
visit in the first trimester (before 12 weeks of pregnancy). Similarly,
Mozambique is implementing an “Opt out” HIV testing model and “B
+” approach for pregnant women since 2013 [14]. The Opt-out HIV
testing model consists of counselling and testing all pregnant women
and their male partners, whilst the B+ approach comprises of
providing triple ARTs to all HIV-infected pregnant women, starting as
early as 14 weeks of gestation and continuing into lifelong therapy [2]. Bourdieu’s theory conceptualizes the relationship between social
structures and individual agency and thus helps to understand why
persons act in a certain – predictable although not completely
determined - way. According to Bourdieu, persons belong to one or
more fields or structured social spaces that are governed by historically
developed rules and norms about how to act and interact. Throughout
different socialisation processes, people develop a habitus or an
embodied disposition to perceive, think, feel, speak, evaluate and act in
a certain way [27]. In contrast to structuralist approaches, Bourdieu
does not assume that norms and rules simply impose themselves on
individuals. Rather, they are used and applied strategically to advance
one’s interests or to obtain a better position, taking into account the
requirements of the specific circumstances. Depending on the amount
and use of relevant resources, one can have more or less possibilities to
act. These resources – Bourdieu uses the term capital - are not only
economical, but can also include the language that one speaks, the
educational qualifications that one has, the network of social relations
one can rely on, knowledge and experience learned from different
social contexts [28]. Page 2 of 9 Also
the belief that pregnancy disclosure could lead to spiritual
complications constitutes a barrier [18]. Other structural barriers
comprise of difficulties to reach a health facility, long waiting times at
the health facility [19] and lack of knowledge of the HIV status [20]. A
lack of uptake of HIV tests and ARTs adherence during pregnancy are
associated with the lack of ability to pay for the transport costs from
the place of residence to the health facility, non-disclosure of the HIV
status to the client’s sexual partner [21], lack of education [22],
emotional stress and side effects of ARTs [23]. Study design and study sites This qualitative study was conducted in Namaacha and Manhiça
rural districts of the Maputo province, located in the south of
Mozambique. Namaacha district had a population of 51,257 in 2015
[29] and 10 health facilities. Manhiça district had about 263,736
inhabitants [29] and 14 health facilities and one rural hospital. These
study sites were relevant for this study because they have the highest
prevalence of HIV/AIDS in the country, accounting for 26% of all
pregnant women living with HIV [30]. Although the aforementioned studies highlight important issues on
ANC and PMTCT, there is a paucity of research about practices and
compliance of biomedical norms during pregnancy and childbirth of
women living with HIV. Therefore, the objective of this study was to
analyse women’s compliance to biomedical norms in order to reduce
the risk of MTCT of HIV during pregnancy and childbirth. Understanding how women perceive and practise PMTCT is
important to improve maternal and child health care and to reduce the
risk of mother-to-child transmission of HIV during pregnancy and
childbirth. Citation:
Cuinhane CE, Coene G, Roelens K, Vanroelen C (2016) Understanding HIV Positive Women’s Experiences with Antenatal Care in
Rural Maputo Province, Mozambique. Clinics Mother Child Health 13: 246. doi:10.4172/2090-7214.1000246 Introduction Several studies [8,9] reported that mother-to-child transmission
(MTCT) of HIV occurs during pregnancy, labour and breastfeeding. When HIV pregnant women follow the prescribed PMTCT, the
transmission is almost eliminated [10]. However, lack of coverage and
follow-up of antenatal care (ANC) influence the effectiveness of
PMTCT in developing countries [11]. Moreover, this process requires
early adherence and compliance to several biomedical norms that are
often conflicting with social norms at community level. The journey to motherhood among pregnant women living with
HIV/AIDS is considered perilous in most of the countries of Sub-
Saharan Africa. Carrying a pregnancy implies to follow biomedical
norms recommended by campaigns on prevention of mother-to-child
transmission (PMTCT) of HIV. These recommendations consist of
attending first antenatal care from the first trimester of the pregnancy
[1], as well as adherence and compliance to antiretroviral therapy
(ARTs) to prevent transmission of HIV during the pregnancy, as
recommended by the World Health Organization [2] and the Ministry
of Health of Mozambique [3,4]. Moreover, women are recommended
to give childbirth at a health facility, to adhere to ARTs for the new-
born, to practise exclusive breastfeeding in the first six months of life
[2,5] and to adhere to lifelong ARTs for their own health [6]. However, ANC plays a critical role in the health of pregnant women regardless
of their HIV status. It is a key entry point to receive preventive health
services such as nutritional support, prevention and treatment of
several diseases (malaria, tuberculosis, tetanus, toxic immunization)
[1]. Moreover, women receive counselling about postpartum care for
themselves and their new-born and child spacing. Among women Volume 13 • Issue 3 • 1000246 Clinics Mother Child Health, an open access journal
ISSN:2090-7214 Cuinhane CE, Coene G, Roelens K, Vanroelen C (2016) Understanding HIV Positive Women’s Experiences with Antenatal Care in
Rural Maputo Province, Mozambique. Clinics Mother Child Health 13: 246. doi:10.4172/2090-7214.1000246 Citation:
Cuinhane CE, Coene G, Roelens K, Vanroelen C (2016) Understanding HIV Positive Women’s Experiences with Antenatal Care in
Rural Maputo Province, Mozambique. Clinics Mother Child Health 13: 246. doi:10.4172/2090-7214.1000246 Cuinhane CE, Coene G, Roelens K, Vanroelen C (2016) Understanding HIV Positive Women’s Experiences with Antenatal Care in
Rural Maputo Province, Mozambique. Clinics Mother Child Health 13: 246. doi:10.4172/2090-7214.1000246 Citation: Procedures The study obtained ethical clearance from the Faculty of Medicine
of Eduardo Mondlane University and Maputo Central Hospital
Bioethics committee. Verbal information about the objective of the
study was provided, and a written consent was presented to each
participant to make an informed choice on whether or not to
participate in the study. All participants read and signed the informed
consent forms. Those who could not read and sign, choose someone of
their trust to translate the information into the local language and to
sign on their behalf. Three sociologists participated in the data
collection: one male main researcher (the first author) and two female
assistant researchers, who were trained on the objectives of the study
and data collection instruments. The main researcher coordinated the
field work, interviewed the participants and moderated the FGDs. Results The study applied purposive sampling to assess 59 HIV positive
women who had given birth and were breastfeeding (25 in Namaacha
district and 34 in Manhiça district). These participants were important
to assess the trajectory’s experience of women from early pregnancy to
childbirth. To select the participants, the main researcher approached
all mothers at a postnatal visit in a child at risk clinic (CRC); and those
who agreed to participate were interviewed. A total of 59 in-depth
interviews and 5 focus group discussion (FGDs) (three in Manhiça
district and two in Namaacha district) with HIV positive women, and
6 semi-structured interviews with maternal and child health (MCH)
nurses were performed. Moreover, 6 in-depth interviews with
community health workers (CHWs) were carried out. Each FGD had
between 6-10 participants. Both individual in-depth interviews and
FGDs were conducted in Portuguese – the national language – for
those who could read and write it and in local vernacular language
(Rhonga) for those who could not understand Portuguese. Characteristics of study participants The demographic characteristics of the study participants are
displayed in Table 1. The majority of participants were between 18-34
years, married or living with a partner. Some participants lacked
formal education. Most participants were farmers and economically
dependent of their partners, and had more than one child. Characteristics of participants
Manhiça
(n=34)
Namaacha
(n=25)
Total
Age range
18-24
14
10
24
25-34
17
11
28
35-39
3
4
7
Educational Level
None
10
4
14
Less than primary education (1-6 years)
8
6
14
Primary education completed or more (7-9
years)
15
12
27
Secondary education completed (12 years)
1
3
4
Marital Status
Single
5
7
12
Married/Living with a partner
27
17
44
Divorced/Widow
2
1
3
Number of Children
1-2
20
14
34
3-4
11
7
18
5-6
3
4
7
Occupation
Factor worker
0
1
1
Farmer
24
19
43
Housemaid
2
1
3
Own business/sale
6
1
7
Student
2
1
3
Teacher
0
2
2
Table 1: Demographic participants of the study participants. We used in-depth interviews to collect information about practices
and experiences of pregnancy care and childbirth among HIV positive
women. In addition, the FGD’s assessed different experiences about
pregnancy care. The semi-structured interviews with MCH nurses
gathered information about women’s adherence to ANC as well about
the compliance to biomedical norms among MCH nurses, whose role
is, among other activities, to advise pregnant women about safe
pregnancy care practices and the management of ARTs. The in-depth
interviews with CHWs assessed the mechanisms used to mobilize and
enhance the relationship between pregnant women and health care
providers. Recruitment of participants and data collection The recruitment and interviews of the study participants took place
between January and March 2015 in six health facilities that implement
a PMTCT of HIV program. Three health facilities were selected in each
district, out of which one was located at the head quarter and two in Studies on adherence to ANC have been dominated by structural
approaches [24,25] and behavioural approaches [26]. The former
emphasizes the material, economic and social conditions to explain Volume 13 • Issue 3 • 1000246 Clinics Mother Child Health, an open access journal
ISSN:2090-7214 Citation:
Cuinhane CE, Coene G, Roelens K, Vanroelen C (2016) Understanding HIV Positive Women’s Experiences with Antenatal Care in
Rural Maputo Province, Mozambique. Clinics Mother Child Health 13: 246. doi:10.4172/2090-7214.1000246 Cuinhane CE, Coene G, Roelens K, Vanroelen C (2016) Understanding HIV Positive Women’s Experiences with Antenatal Care in
Rural Maputo Province, Mozambique. Clinics Mother Child Health 13: 246. doi:10.4172/2090-7214.1000246 Citation: Page 3 of 9 (SPSS, version 23) was used to analyse quantitative data emerging from
the data set. (SPSS, version 23) was used to analyse quantitative data emerging from
the data set. the neighbourhood. The district headquarter is relatively urbanized,
whilst the neighbourhood has more rural characteristics. These
discrepancies were taken into account for the purpose of the analysis. Citation:
Cuinhane CE, Coene G, Roelens K, Vanroelen C (2016) Understanding HIV Positive Women’s Experiences with Antenatal Care in
Rural Maputo Province, Mozambique. Clinics Mother Child Health 13: 246. doi:10.4172/2090-7214.1000246 Data analysis A thematic analysis approach was applied to obtain the key themes
emerging from the data. The analysis involved five stages. First,
interviews were transcribed and then translated from Portuguese to
English. Second, each transcription was read more than once, and
initial codes were generated. Third, the codes were used to identify and
search different themes across the data. Fourth, the identified themes
were revised according to the research objectives, and final themes
were defined. Codes and themes emerging from the data set were
discussed and approved by all researchers involved in this study. The
last stage of thematic analysis consisted of producing a report of the
research outcomes. Moreover, a statistic package for Social Sciences Table 1: Demographic participants of the study participants. Table 1: Demographic participants of the study participants. Moreover, nearly all participants were members of Protestant
church, whilst others were members of Catholic and Muslim. Findings
of this study comprise four key themes: 1) knowledge of HIV positive
status, 2) timing of the first ANC visit, 3) pregnancy care after the first Clinics Mother Child Health, an open access journal
ISSN:2090-7214 Volume 13 • Issue 3 • 1000246 Cuinhane CE, Coene G, Roelens K, Vanroelen C (2016) Understanding HIV Positive Women’s Experiences with Antenatal Care in
Rural Maputo Province, Mozambique. Clinics Mother Child Health 13: 246. doi:10.4172/2090-7214.1000246 Citation:
Cuinhane CE, Coene G, Roelens K, Vanroelen C (2016) Understanding HIV Positive Women’s Experiences with Antenatal Care in
Rural Maputo Province, Mozambique. Clinics Mother Child Health 13: 246. doi:10.4172/2090-7214.1000246 on:
Cuinhane CE, Coene G, Roelens K, Vanroelen C (2016) Understanding HIV Positive Women’s Experiences with Antenatal Care in
Rural Maputo Province, Mozambique. Clinics Mother Child Health 13: 246. doi:10.4172/2090-7214.1000246 Citation: Page 4 of 9 However, few participants used pharmaceutical tests and blood tests at
the health facility to confirm the pregnancy. ANC and 4) childbirth practices. In each theme, participants reported
different experiences which enabled to analyse their compliance to
biomedical norms. Reasons for timing the first ANC Most participants attended the first ANC when they perceived
illness episodes, and were seeking care for themselves. These
participants used previous knowledge learned from MCH nurses to
know that pregnant women were only recommended to take
medication from a health facility. This knowledge enabled participants
to seek the first ANC between 8 and 12 weeks of gestational age. Moreover, some participants who knew their HIV positive status prior
to pregnancy also attended the first ANC between 8-12 weeks because
their partners, who were also HIV positive, and MCH nurses advised
them. However, other participants in the same situation attended the
first ANC at 16 weeks. Most of these participants perceived that their
babies were not at risk because they were already taking ARTs before
the pregnancy. They also reported knowing that HIV positive women
had to take ARTs to prevent MTCT of HIV and book ANC at 12
weeks. Figure 1: Distribution of participants regarding knowledge of HIV
status prior to pregnancy. The timing of the first ANC visit This theme comprises four subthemes: the first explores how women
self-diagnosed pregnancy, the second describes social norms that
women followed before the first ANC, the third present’s reasons that
women reported for booking the first ANC and the fourth shows ANC
visits throughout pregnancy. Attendance to the first ANC between 13-24 weeks was also
motivated by a perception of illness episodes, such as feeling unwell,
headache and malaria; the need to confirm if they were pregnant,
advice from their partners, mothers and peers. Some participants
recognized that it was too late to attend the first ANC at 16 weeks, but
they could not attend earlier due to work obligation, lived far from the
health facility and others were living in South Africa. Participants
explained that when they were in South Africa with their partners,
they were afraid to book their first ANC because they were
undocumented migrants. However, some participants reported that
MCH nurses had advised them to attend the first ANC when the
gestational age was before 20 weeks or when the belly was visible, as
illustrated by a narrative of one of the participants: Knowledge of HIV positive status All participants said that a health facility was the only place to get
advice and care for pregnancy after diagnosing the pregnancy. However, few participants sought other complementary sources of
healing such as church pastors and witchdoctors prior to the ANC. Participants who consulted pastors perceived illness episodes and they
sought treatment and protection for pregnancy. Some participants
reported that they did not use such practices in previous pregnancies
because they did not experience illness episodes, while others said that
it was a common practice. Participants sought a witchdoctor because
they wanted to inform their ancestors that they were pregnant, and ask
for their spiritual protection. These participants said they engaged in
the same practice that they usually do during pregnancy, as one of the
participants explained: Figure 1 shows the distribution of participants regarding knowledge
of their HIV positive status prior to the first ANC. The majority of
participants of both study sites knew their HIV positive status at the
first ANC visit. Almost all participants, including those who already
knew their HIV positive status, reported that they were tested for HIV
at the first ANC. Only one participant was not tested because she
disclosed her HIV positive status and was taking ARTs. According to
MCH nurses, HIV test should not be repeated to confirm HIV
positivity; but, some women do not disclose their positive status at the
ANC, and therefore, they are tested as if it were the first time. Figure 1: Distribution of participants regarding knowledge of HIV
status prior to pregnancy. “I went to a witchdoctor to inform the ancestors that I am pregnant. The ancestors protect the baby and me and they also ensure a safe
childbirth. I always proceed the same way whenever I am pregnant. I
did the same in my previous pregnancies”. (37 years, 5 children,
Manhiça). Clinics Mother Child Health, an open access journal
ISSN:2090-7214 Self-diagnosis of pregnancy Participants used different ways to self-diagnose their pregnancy
prior to ANC. Most participants used biological signs of the body, such
as lack of menstruation after one month or two months, tiredness and
weakness of the body. Other signs like changing in dietary preferences
(lack of appetite, and choice of food) and perception of illness
episodes, such as a headache, seasickness and vomiting were also used. These signs were learned from books for those who could read and
from the health facility for those who had experience of motherhood. Some participants also learned to diagnose their pregnancy from
school, whilst others used knowledge learned from parents and peers. The majority of participants suggested that one can recognise a
pregnancy as early as 2 weeks and between one and two months. “I attended the first ANC when my belly was visible because when I
went to health facility for the first time the gestational age was one
month and 2 weeks [6 weeks], and MCH nurses sent me back. I
wanted treatment because I knew I was HIV positive, but they told me
that the belly was not visible. They advised me to attend the ANC when Volume 13 • Issue 3 • 1000246 Clinics Mother Child Health, an open access journal
ISSN:2090-7214 Cuinhane CE, Coene G, Roelens K, Vanroelen C (2016) Understanding HIV Positive Women’s Experiences with Antenatal Care in
Rural Maputo Province, Mozambique. Clinics Mother Child Health 13: 246. doi:10.4172/2090-7214.1000246 Citation: Page 5 of 9 the belly was visible. (…) I waited until the belly was visible, and I went
back to the health facility at four months [16 weeks]”. (20 years, 1 child,
Namaacha). the belly was visible. (…) I waited until the belly was visible, and I went
back to the health facility at four months [16 weeks]”. (20 years, 1 child,
Namaacha). health facility”. Participants in the FGDs reported that MCH nurses
advised to attend the first ANC at 12 weeks of the gestational age. They
also said that MCH nurses only get angry with pregnant women if they
attend ANC after 20 weeks. Indeed, there is a difference in terms of
advises the MCH nurses give to women. Some MCH nurses confirmed
advising women to attend the first ANC at 20 weeks and others at 12
weeks. Nevertheless, MCH nurses reported that some women on ARTs
still continue attending the first ANC at 28 weeks. Citation:
Cuinhane CE, Coene G, Roelens K, Vanroelen C (2016) Understanding HIV Positive Women’s Experiences with Antenatal Care in
Rural Maputo Province, Mozambique. Clinics Mother Child Health 13: 246. doi:10.4172/2090-7214.1000246 Table 2 presents the gestational age of the participants at the first
ANC according to their social characteristics. The earliest gestational
age at the first ANC was 8 weeks and the latest was 28 weeks. Most
participants attended the first ANC between 13-24 weeks, regardless of Knowledge of HIV positive status and gestational age of
pregnancy Figure 2 shows the distribution of gestational age at the first
antenatal care visit among participants in relation to knowledge of
HIV positive status prior to pregnancy. Most participants who knew
their HIV positive status prior to the pregnancy attended their first
ANC between 13 and 24 weeks, while a few attended the first ANC
between 1 and 12 weeks of gestational age. Figure 2: Gestational age at first antenatal visit in relation to
knowledge of HIV positive status prior to pregnancy. Figure 2: Gestational age at first antenatal visit in relation to
knowledge of HIV positive status prior to pregnancy. Table 3: Number of ANC visits throughout pregnancy. Self-diagnosis of pregnancy doi:10.4172/2090-7214.1000246 Citation:
Cuinhane CE, Coene G, Roelens K, Vanroelen C (2016) Understanding HIV Positive Women’s Experiences with Antenatal Care in
Rural Maputo Province, Mozambique. Clinics Mother Child Health 13: 246. doi:10.4172/2090-7214.1000246 Citation: Primary education completed or more
(7-9 years)
3
12
2
10
Secondary education completed (12
years)
0
1
0
3
Marital Status
Single
1
4
4
3
Married/Living with a partner
4
23
3
14
Divorced/Widow
1
1
0
1
Number of Children
1-2
2
18
3
11
3-4
3
8
3
4
5-6
1
2
1
3
Occupation
Factor worker
0
0
0
1
Farmer
4
20
7
12
Housemaid
1
1
0
1
Own business/sale
1
5
0
1
Student
0
2
0
1
Teacher
0
0
0
2
Table 3: Number of ANC visits throughout pregnancy
Page 6 of 9 Page 6 of 9 place of residence, their age, level of education, marital status, number
of children and occupation. Those participants who consulted a church
pastor or witchdoctor attended the first ANC between 16-24 weeks,
and other started at 8 weeks. place of residence, their age, level of education, marital status, number
of children and occupation. Those participants who consulted a church
pastor or witchdoctor attended the first ANC between 16-24 weeks,
and other started at 8 weeks. Number of ANC visits throughout pregnancy Figure 3 presents the number of ANC visits throughout pregnancy
according to knowledge of HIV positive status of the participants. Almost all participants who knew their HIV positive prior to the
pregnancy attended 4 and more ANC visits. Nevertheless, most
participants who knew their HIV positive status at the first ANC also
attended 4 and more ANC visits. All participants said ANC enabled to
check the position of the foetus, their weight, access to tablets and
ARTs for the mother and prevent MTCT and treat illnesses. However,
participants reported participating in almost all ANC visits to avoid
reprimands from MCH nurses during childbirth at the health facility. In their experience and perceptions, women who do not frequently
attend ANC suffer because MCH nurses do not treat them adequately
during childbirth. Table 3 shows the number of ANC visits throughout pregnancy
according to the social characteristics of participants. All participants
attended at least two ANC visits. Most participants attended 4 ANC
and more, regardless their age, level of education, marital status,
number of children and occupation. Characteristics of participants
Number
of
ANC
visits
during
pregnancy
Manhiça (n=34)
Namaacha (n=25)
2-3
4
and
more
2-3
4
and
more
Age range
18-24
3
11
3
7
25-34
3
14
2
9
35-39
0
3
2
2
Educational Level
None
0
10
2
2
Less than primary education (1-6
years)
3
5
3
3 Self-diagnosis of pregnancy Participants explained that late ANC attendance (25-36 weeks) was
due to lack of knowledge about their HIV positive status, lack of
money for transport and lack of information about the recommended
time for the first ANC. Some participants with unknown HIV status
prior to the pregnancy reported that they would have attended the first
ANC earlier if they had known they were HIV positive prior to the
pregnancy. Moreover, other participants delayed the first ANC because
they were “feeling lazy” and “did not want to get tired to go to the ANC visits throughout pregnancy they were “feeling lazy” and “did not want to get tired to go to the
Characteristics of participants
Number of weeks at the first ANC
Manhiça (n=34)
Namaacha (n=25)
1-12
13-24
25-36
1-12
13-24
25-36
Age range
18-24
5
8
1
2
6
2
25-34
3
13
1
5
5
1
35-39
2
1
0
0
4
0
Educational Level
None
3
7
0
1
1
2
Less than primary education (1-6 years)
1
6
1
1
5
0
Primary education completed or more (7-9 years)
6
8
1
4
7
1
Secondary education completed (12 years)
0
1
0
1
2
0
Marital Status
Single
1
3
1
1
4
2
Married/Living with a partner
9
17
1
6
10
1
Divorced/Widow
0
2
0
0
1
0
Number of Children
1-2
8
11
1
5
7
2
3-4
1
10
0
1
5
1
1
1
1
1
3
0
Occupation
Factor worker
0
0
0
0
1
0
Farmer
7
15
2
7
9
3
Housemaid
0
2
0
0
1
0
Own business/sale
2
4
0
0
1
0
Student
1
1
0
0
1
0
Teacher
0
0
0
0
2
0
Table 2: Gestational age of pregnancy at the first ANC visit. Table 2 presents the gestational age of the participants at the first
age at the first ANC was 8 weeks and the latest was 28 weeks. Most
5-6 Table 2 presents the gestational age of the participants at the first
ANC according to their social characteristics. The earliest gestational Volume 13 • Issue 3 • 1000246 Clinics Mother Child Health, an open access journal
ISSN:2090-7214 itation:
Cuinhane CE, Coene G, Roelens K, Vanroelen C (2016) Understanding HIV Positive Women’s Experiences with Antenatal Care in
Rural Maputo Province, Mozambique. Clinics Mother Child Health 13: 246. Practices of pregnancy care This section describes women’s practices regarding disclosure of
HIV status to the partner for women who knew their HIV positive
status for the first time at the ANC, adherence to ARTs, use of condom
and management of illness episodes during pregnancy. Volume 13 • Issue 3 • 1000246 Citation:
Cuinhane CE, Coene G, Roelens K, Vanroelen C (2016) Understanding HIV Positive Women’s Experiences with Antenatal Care in
Rural Maputo Province, Mozambique. Clinics Mother Child Health 13: 246. doi:10.4172/2090-7214.1000246 Citation:
Cuinhane CE, Coene G, Roelens K, Vanroelen C (2016) Understanding HIV Positive Women’s Experiences with Antenatal Care in
Rural Maputo Province, Mozambique. Clinics Mother Child Health 13: 246. doi:10.4172/2090-7214.1000246 Citation: Page 7 of 9 Management of illness during pregnancy A considerable number of participants reported illness episodes
during pregnancy. The most common illnesses were constipation,
fever, cough, headache, feet pain, stomach-ache, body pain, malaria,
dizziness, vomiting, diarrhoea and wounds in the vagina. Participants
associated these illnesses with pregnancy and HIV. Some participants
sought the health care to treat illnesses; few pursued praying with the
church pastors, whilst some used traditional remedies to treat
toothache and vomiting. In addition, some participants (suffering from
feet pain, stomach-ache, headache, pain of the body and nausea) did
not seek any treatment because they perceived these illness episodes as
normal for pregnant women. The majority of participants said that
they decided themselves to treat the illness, while few were advised by
their partners or mothers-in-law. Figure 3: Number of ANC visits according to knowledge of HIV
positive status. Adherence to ART during ANC All participants said that they received ARTs at the maternity clinic
and they reported complying mostly to prevent HIV transmission to
the baby and for their own health. Moreover, participants reported that
it was mandatory to take ARTs to prevent HIV infection to the baby. MCH nurses said that most women complied with ART during
pregnancy because they were afraid their baby would be born HIV-
infected. CHWs also helped women to comply with ARTs and
emphasized the need to protect the baby. Participants who did not
disclose their HIV positive status could take ARTs without problem
because they told their partners that those medicines were prescribed
for the safety of the baby during pregnancy and breastfeeding. Disclosure of HIV status to the partner Few participants disclosed their HIV positive status to their
partners after HIV diagnosis. In most cases their partners already
knew their HIV status prior to the pregnancy. In other cases they
accepted the invitation letter from the health facility and went to do
HIV test together. Of those that did not inform their partners, they
said that they were not yet psychologically prepared to disclose their
HIV positive status to their partners, while other feared stigma and
discrimination, and therefore preferred their husband to find out
themselves. Participants reported giving the letters of invitation from
the health facility to their partners, but most of them said they were
not able to go to the health facility due to work obligations. Some participants reported seeking other sources of healing, such as
church pastors, expecting them to pray for them whenever an illness
episode was perceived, and prior to childbirth. The pastor’s prayers
were believed to help to prevent and treat evil spirits that could cause
problems to pregnant women and during childbirth. Illness episodes were associated with the sex of the baby. Participants perceived a baby boy pregnancy as a risk pregnancy
compared to a baby girl pregnancy. They said that when a woman is
pregnant of a baby boy, she is more vulnerable to get sick throughout
pregnancy than when she is pregnant of a baby girl. Participants
reported seeking for a church pastor to prevent the perceived risk of
pregnancy. MCH nurses reported that participants’ partners rarely went to the
health facility when they were invited because they already knew they
would be HIV-tested. However, they said that they advise the pregnant
women to take ARTs regardless of disclosure of their HIV positive
status to their partners. Community health workers (CHWs) also said
that there was lack of male participation because some already knew
their HIV positive status, but did not yet disclose to their partners,
while others were married and impregnated their neighbours; and
therefore, they do not accept to accompany pregnant women to the
health facility. Use of condom during pregnancy Figure 3: Number of ANC visits according to knowledge of HIV
positive status. Few participants used a condom during pregnancy. Participants
reported that they were not using a condom because their partners
were absent during pregnancy, while others said that it was difficult
because they had not disclosed their HIV status to their partners. Some participants who had disclosed HIV status said their partner
accepted to use a condom during the pregnancy to protect the baby. Practices and experience on childbirth The majority of participants reported to have given birth at the
health facility. Participants said that they gave birth at the health
facility because it was “good” and “secure for the baby” and because
they “trusted” the health providers to protect the baby and themselves. Moreover, participants reported that it was recommended to give birth
at the facility to prevent HIV transmission to the baby, and because the
baby had to take syrup after the childbirth. Few participants, who gave
birth at home, reported failure to give birth at the health facility
because they lived far, and there was no transport to take them when
the labour started. Participants said their mother helped to give birth
at home, and suddenly went to the health facility. Most of the
participants said that they decided themselves upon the place to give
birth, and all said they were advised by MCH nurses to give birth at the
health facility. Few participants said the decision of the birthplace was
given by partners, mothers and grandmothers. Clinics Mother Child Health, an open access journal
ISSN:2090-7214 Conclusion The study showed that the participants accessed and used different
resources that influenced compliance to biomedical norms during
ANC and childbirth. Participants relied on their knowledge and
experience about pregnancy care, advice of the family, MCH nurses
and support of CHWs, and other forms of healing (church pastors and
witchdoctors) acquired in multiple interactions in the family and
community and modern health system. Participants followed some
biomedical norms to prevent MTCT of HIV, such as subsequent ANC
visits, compliance to ARTs and childbirth at the health facility, obeying
the logic of the modern health system. However, most participants did
not comply with the recommended time for the first ANC because of
work obligations, lack of money for transport, migration, erroneous
advices of MCH nurses, lack of knowledge of the HIV positive status
and about the recommended time for the first ANC, and social norms. Moreover, the majority of participants did not use a condom
throughout pregnancy and some participants used other forms of
healing to address illness episodes rather than the modern health
system; following thus the logic of their family and community. Late attendance to the first ANC seems to be influenced by the way
the biomedical norms are transmitted and perceived: some MCH
nurses advised pregnant women to attend the first ANC when the belly
was visible or at 20 weeks of the gestational age, whilst participants
perceived that MCH nurses reprimanded them if they attended the
first ANC visit after this period. Moreover, social norms, such as
consulting a church pastor or witchdoctor for spiritual protection prior
to the ANC, as well as seeking modern health care when an illness
episode was perceive seemed to contribute to the late ANC. Other
factors that influenced late first ANC were: work obligations
hampering an appointment, migration, lack of knowledge of the HIV
positive status, lack of knowledge about the recommended time for the
first ANC, lack of money for transport, and the perception of the ANC
as an exhausting process. Early ANC was associated with availability
and access to resources, such as health facility to treat the perceived
symptoms of illness, advice from the partners who knew their HIV
positive status and advise of MCH nurses on timing of the first ANC. This result is in line with previous studies in Sub-Saharan Africa [18]. Discussion The study suggests that women’s compliance to biomedical norms
during pregnancy and childbirth was influenced by the availability and
access to multiple resources, such as knowledge and experience of
pregnancy care and access to modern health care system and Volume 13 • Issue 3 • 1000246 Clinics Mother Child Health, an open access journal
ISSN:2090-7214 Citation:
Cuinhane CE, Coene G, Roelens K, Vanroelen C (2016) Understanding HIV Positive Women’s Experiences with Antenatal Care in
Rural Maputo Province, Mozambique. Clinics Mother Child Health 13: 246. doi:10.4172/2090-7214.1000246 Citation: Page 8 of 9 document reasons for not attending the ANC or use of the health
facility. document reasons for not attending the ANC or use of the health
facility. traditional health care. Thus, participants’ practices reflected their
knowledge and experiences, and seemed to respond the demand of
both the modern health care system and the family and community
norms. Findings of the study showed that participants used knowledge
acquired from the modern health care system, school and from
socialisation in the family and community to early diagnose their
pregnancy. However, most participants attended the first ANC visit
(between13 and 24 weeks) out of the period recommended by WHO
and the Ministry of Health in Mozambique. Similar findings have been
documented in South Africa [31], Ethiopia [32] and Uganda [33]. traditional health care. Thus, participants’ practices reflected their
knowledge and experiences, and seemed to respond the demand of
both the modern health care system and the family and community
norms. Findings of the study showed that participants used knowledge
acquired from the modern health care system, school and from
socialisation in the family and community to early diagnose their
pregnancy. However, most participants attended the first ANC visit
(between13 and 24 weeks) out of the period recommended by WHO
and the Ministry of Health in Mozambique. Similar findings have been
documented in South Africa [31], Ethiopia [32] and Uganda [33]. References 1. WHO (2002) WHO Antenatal Care Randomized Trial: Manual for the
implementation of the new model. Geneva: WHO. 1. WHO (2002) WHO Antenatal Care Randomized Trial: Manual for the
implementation of the new model. Geneva: WHO. 2. WHO (2013) Consolidated guidelines on the use of antiretroviral drugs
for the treating and preventing HIV infection. Recommendations for a
public health approach. Geneva: WHO. 2. WHO (2013) Consolidated guidelines on the use of antiretroviral drugs
for the treating and preventing HIV infection. Recommendations for a
public health approach. Geneva: WHO. 3. MISAU (2011) Plano Nacional de eliminação da transmissão vertical do
HIV (2012-2015). Maputo: MISAU. 3. MISAU (2011) Plano Nacional de eliminação da transmissão vertical do
HIV (2012-2015). Maputo: MISAU. 4. MISAU (2014) Prevenção da transmissão vertical do HIV. Manual do
bolso. Maputo: MISAU. 4. MISAU (2014) Prevenção da transmissão vertical do HIV. Manual do
bolso. Maputo: MISAU. 5. Kellerman SE, Ahmad S, Feeley-Summer T, Jay J, Kim M, et al. (2013)
Beyond prevention of mother-to-child transmission: keeping HIV-
exposed and HIV- positive child health and alive. AIDS 27: S225-S233. 6. WHO (2015) Guideline on when to start antiretroviral therapy and on
pre-exposure prophylaxis for HIV. Geneva: WHO. 6. WHO (2015) Guideline on when to start antiretroviral therapy and on
pre-exposure prophylaxis for HIV. Geneva: WHO. Conclusion The study suggests that there is a need of an adequate health
education for both women and men about HIV and reproductive
health in the family and community and at the health facilities,
emphasizing the benefits of antenatal care to both the baby and the
couple. In addition, more ethnographic studies are needed to access
the motivations of women who do not use the health facility during
ANC and childbirth. The findings showed that despite late first ANC attendance,
participants complied with the follow-up visits and ART adherence, as
it has been documented across Sub-Saharan Africa [34-36]. The
engagement into these practices might have been influenced by the
need to protect the baby against HIV and the preparedness for
childbirth. Women perceived that when they do not miss ANC, they
are likely less reprimanded at the health facility during childbirth. Furthermore, participants received ART monthly from the MCH clinic
to prevent MTCT and for their our health. The majority of participants
gave birth at the health facility following MCH nurse advices, the need
to prevent MTCT of HIV and a perception of benefits for the mother
and the baby. This result is consistent to others studies that suggested
that the choice to give birth at the facility is related to potential health
benefits for the mother and the baby and the perception of good care
from the providers [37,38]. However, like in most Sub-Saharan Africa
countries [39,40], participants did not use a condom throughout the
pregnancy and did not disclose their HIV positive status to their
partners due to fear of stigma and discrimination. Moreover, some
participants followed social norms, such as consulting a church pastor
or a witchdoctor to treat the illness episodes during pregnancy. Similar
practice was also reported in the central region of Mozambique [41]. These practices seem to be influenced by social norms about how to act
and interact during pregnancy. Acknowledgement This study was funded by a project grant from the Desafio,
Development Program in Reproductive Health; HIV/AIDS and Family
Matters; Eduardo Mondlane University, Mozambique and Flemish
Interuniversity Council (VLIR). Citation:
Cuinhane CE, Coene G, Roelens K, Vanroelen C (2016) Understanding HIV Positive Women’s Experiences with Antenatal Care in
Rural Maputo Province, Mozambique. Clinics Mother Child Health 13: 246. doi:10.4172/2090-7214.1000246 Limitation INS, INE, ICF (2010) Inquérito Nacional de Prevalência, Riscos
comportamentais e Informação sobre o HIV e SIDA em Moçambique
2009. Maputo: MISAU. 13. Mandelbrot L, Tubiana R, Le Chenadec J, Dollfus C, Faye A, et al. (2015)
No
perinatal
HIV-1Transmission
from
women
with
effective
antiretroviral therapy stating before pregnancy. Clin Infec Dis 61:
1715-1725. 31. Clouse K, Pettifor A, Shearer K, Maskew M, Bassett J, et al. (2013) Loss to
follow-up before and after delivery among women testing HIV positive
during pregnancy in Johnnesburg, South Africa. Trop Int Health 18:
451-460. 14. MISAU (2015) Relatório annual das actividades relacionadas ao HIV/
SIDA. Maputo: MISAU. 32. Gebremeskel F, Dibaba Y, Admassu B (2015) Timing of first antenatal care
attendance and associated factors among pregnant women in Arba Minch
Town and Arba Minch District, Gamo Goza Zone, South Ethiopia. Journal of Environmental and Public Health. 15. de Cock KM, Fowler MG, Mercier E, de Vincenzi I, Saba J, et al. (2000)
Prevention of mother-to-child HIV transmission in poor resource
countries. JAMA 283: 1175-1182. 33. Kisuule I, Kaye DK, Najjuka F, Ssematimba SK, Arinda A, et al. (2013)
Timing and reasons for coming late for the first antenatal care visit by
pregnant women at Mulago Hospital, Kampala, Uganda. BMC Pregnancy
Childbirth 13: 121. 16. UN (2014) Relatório de situação de 2014 sobre a saúde maternal e
neonatal e infantile. Ethiopia: União Africana. 17. INE, MISAU (2013) Inquérito Demográfico e de Saúde 2011. Maputo:
MISAU. 34. Gupta S, Yamada G, Mpembeni R, Frumence G, Callaghan-Karou JA, et
al. (2014) Factors associated with four or more antenatal care visits and its
decline among pregnant women in Tanzania between 1999 and 2010. PLoS ONE 9: e101893. 18. Finlayson K, Downe S (2013) Why do women not use antenatal services
in low-and-middle income countries? A meta-synthesis of qualitative
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the health facility and reducing maternal mortality: a systematic review. Ethiop J Health Sci 24: 93-104. 19. Deressa W, Same A, Asefa A, Teshome G, Enqusellassie F (2014)
Utilization of PMTCT and associated factors among pregnant women
attending antenatal clinics in Addis Ababa, Ethiopia. BMC Pregnancy
and Childbirth 14: 328. 36. Ebuy H, Yebyo H, Alemayehu M (2015) Levels of adherence and
predictors of adherence to Option B+ PMTCT Programme in Tigre,
Northern Ethiopia. Int J Infect Dis 33: 123-129. Limitation 7. UNAIDS (2015) 2015 Progress Report on the Global Plan. Geneva: Joint
United Nation Program on HIV/AIDS. 7. UNAIDS (2015) 2015 Progress Report on the Global Plan. Geneva: Joint
United Nation Program on HIV/AIDS. This study is subject to methodological limitations. First, the reports
of ANC were retrospective, and problems of recall may have affected
consistency of women’s narratives in terms of practices engaged during
pregnancy. Second, the study did not access other HIV positive women
who probably did not attend CRC and therefore it was not possible do 8. Bertolli J, St Louis ME, Simonds RJ, Nieburg P, Kamenga M, et al. (1996)
Estimating the time of mother-to-child transmission of human
immunodeficiency virus in a breast-feeding population in Kinshasa,
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ISSN:2090-7214 Citation: Page 9 of 9 Page 9 of 9 the infant through breastfeeding: new developments. AJOG 197:S113-
S122. the infant through breastfeeding: new developments. AJOG 197:S113-
S122. 26. Andersen RM (1995) Revising the behavioural model and access to
medical care: Does it matter? J Health Soc behav 36: 1-10. 10. WHO (2006) Antiretroviral drugs for treatment pregnant women and
preventing HIV infection in Infants: Towards universal access. Recommendations for public health approach. Geneva: WHO.h 27. Bourdieu P (1977) Outline of a theory of practice. USA: Cambridge
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prevention of mother-to-child transmission of HIV cascade analysis tool:
supporting health Managers to improve facility-level service delivery. BMC Res notes 7: 743. 29. INE (2010) Projeções anunais da população total, urbana e rural dos
distritos da província de Maputo 2007-2040. Maputo: Instituto Nacional
de Estatística. 12. de Vincenzi (2011) Triple antiretroviral compared with zidovudine and
single-dose nevirapine prophylaxis during pregnancy and breastfeeding
for prevention of mother-to-child transmission of HIV-1 (Kesho Bora
study): a randomised controlled trial. Lancet Infect Dis 11: 171-180. 30. Citation:
Cuinhane CE, Coene G, Roelens K, Vanroelen C (2016) Understanding HIV Positive Women’s Experiences with Antenatal Care in
Rural Maputo Province, Mozambique. Clinics Mother Child Health 13: 246. doi:10.4172/2090-7214.1000246 Clinics Mother Child Health, an open access journal
ISSN:2090-7214 Limitation 20. Gill MM, Machekano R, Isavwa A, Ahmisbwe A, Oyebanji O, et al. (2015)
The association between HIV status and antenatal care attendance amng
pregnant women in rural hospital in Lesotho. J Acquir Immune Defic
Syndr 68: e33-e38. 37. Bhattacharyya S, Srivastava A, Roy R, Avan BI (2016) Factors influencing
women’s preferences for health facility deliveries in Jharkhand State,
India: a cross sectional analysis. BMC Pregnancy and childbirth 16: 50. 21. Gunn JKL, Asaolu IO, Center KI, Gibson SJ, Whitman P, et al. (2016)
Antenatal care and uptake of HIV test among pregnant women in Sub-
Saharan Africa: a cross-sectional study. J Int AIDS Soc 19: 20605. 38. Schnack A, Rempis E, Decker S, Braun V, Rubaihayo J, et al. (2016)
Prevention of mother-to-child transmission in HIV in Option B+ era:
Uptake and adherence during pregnancy in Western Uganda. AIDS
Patient Care and STDs 30. 22. El-Khatib Z, Ekstrom AM, Coovadia A, Abrams EJ, Petzold M, et al. (2011) Adherence and virologic suppression during the 24 weeks on
antiretroviral therapy among women in Johannesburg, South Africa- a
prospective cohort study. BMC Public Health 11: 88. 39. Kharsany ABM, Karim QA (2016) HIV infection and AIDS in Sub-
Saharan Africa: Current status, challenges and opportunities. The Open
AIDS Journal 10: 34-48. 23. Nachega JB, Uthman OA, Anderson J, Peltzer K, Wampold S, et al. (2012)
Adherence to antiretroviral therapy during and after pregnancy in low-
income, middle-income, and high-income countries: a systematic review
and meta-analysis. AIDS 26: 2030-2052. 40. Egbe TO, Tazinya RA, Halle-Ekane GE, Egbe E, Achidi EA (2016)
Estimating HIV incidence during pregnancy and knowledge of
prevention of mother-to-child transmission with an ad hoc analysis of
potential cofactors. Journal of Pregnancy. 24. Backett EM, Davies AM, Petros-Barvazian A (1984) The risk approach in
health care. With special reference to maternal and child health, including
family planning. Geneva: World Health Organization. 41. Chapman R (2010) Family Secrets. Risking Reproduction in Central
Mozambique. Nashville: Vanderbilt University Press. 25. Gerein N, Mayhew S, Lubben M (2003) A framework for a new approach
to antenatal care. International Journal of Gynecology and Obstetrics 80:
175-182. Volume 13 • Issue 3 • 1000246 Clinics Mother Child Health, an open access journal
ISSN:2090-7214
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Ecohydrologic impacts of rangeland fire on runoff and erosion: A literature synthesis
| null | 2,016
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Ecohydrologic Impacts of Rangeland Fire on Runoff and Erosion:
Ecohydrologic Impacts of Rangeland Fire on Runoff and Erosion:
A Literature Synthesis
A Literature Synthesis Follow this and additional works at: https://digitalcommons.usu.edu/sagestep_reports Part of the Life Sciences Commons Part of the Life Sciences Commons Utah State University
Utah State University
DigitalCommons@USU
DigitalCommons@USU
Reports
Publications
7-2016
Ecohydrologic Impacts of Rangeland Fire on Runoff and Erosion:
Ecohydrologic Impacts of Rangeland Fire on Runoff and Erosion:
A Literature Synthesis
A Literature Synthesis
Frederick B. Pierson
USDA Agricultural Research Service
C. Jason Williams
USDA Agricultural Research Service Publications Recommended Citation
Recommended Citation Pierson, F.B., and C. J. Williams. 2016. Ecohydrologic impacts of rangeland fire on runoff and erosion: A
literature synthesis. US Department of Agriculture, Forest Service, Rocky Mountain Research Station, Fort
Collins, CO. RMRS-GTR-351. 110 p. This Report is brought to you for free and open access by
the Publications at DigitalCommons@USU. It has been
accepted for inclusion in Reports by an authorized
administrator of DigitalCommons@USU. For more
information, please contact digitalcommons@usu.edu. United States Department of Agriculture Authors Frederick B. Pierson is the Research Leader and a Supervisory Research Hydrologist
and C. Jason Williams is a Research Hydrologist with the Northwest Watershed Research
Center, USDA Agricultural Research Service, Boise, Idaho. To contact, email F.B. Pierson at,
fred.pierson@ars.usda.gov. Frederick B. Pierson is the Research Leader and a Supervisory Research Hydrologist
and C. Jason Williams is a Research Hydrologist with the Northwest Watershed Research
Center, USDA Agricultural Research Service, Boise, Idaho. To contact, email F.B. Pierson at,
fred.pierson@ars.usda.gov. Ecohydrologic Impacts of Rangeland
Fire on Runoff and Erosion: A Literature
Synthesis Frederick B. Pierson and C. Jason Williams Rocky Mountain
Research Station Rocky Mountain
Research Station General Technical Report
RMRS-GTR-351 General Technical Report
RMRS-GTR-351 Forest
Service July 2016 Pierson, Frederick B.; Williams, C. Jason. 2016. Ecohydrologic impacts of rangeland
fire on runoff and erosion: A literature synthesis. Gen. Tech. Rep. RMRS-GTR-351. Fort Collins, CO: U.S. Department of Agriculture, Forest Service, Rocky Mountain Research
Station. 110 p. Pierson, Frederick B.; Williams, C. Jason. 2016. Ecohydrologic impacts of rangeland
fire on runoff and erosion: A literature synthesis. Gen. Tech. Rep. RMRS-GTR-351. Fort Collins, CO: U.S. Department of Agriculture, Forest Service, Rocky Mountain Research
Station. 110 p. Abstract Fire can dramatically influence rangeland hydrology and erosion by altering ecohydrologic
relationships. This synthesis presents an ecohydrologic perspective on the effects of fire on
rangeland runoff and erosion through a review of scientific literature spanning many decades. The objectives are: (1) to introduce rangeland hydrology and erosion concepts necessary for
understanding hydrologic impacts of fire; (2) to describe how climate, vegetation, and soils
affect rangeland hydrology and erosion; and (3) to use examples from literature to illustrate
how fire interacts with key ecohydrologic relationships. The synthesis is intended to provide a
useful reference and conceptual framework for understanding and evaluating impacts of fire
on rangeland runoff and erosion. Keywords: ecohydrology, fire effects, infiltration, rangeland hydrology, runoff, soil erosion,
soil water repellency, ecohydrology resilience, hillslope hydrology, hydrologic response,
postfire response, soil response Cover Photo. Prescribed fire at the Reynolds Creek Experimental Watershed, Idaho (photo: USDA
Agricultural Research Service, Northwest Watershed Research Center, Boise, Idaho). Acknowledgments We sincerely thank Thomas Thurow and Bradford Wilcox for their reviews and comments,
which greatly helped to improve this manuscript. We greatly appreciate the manuscript
reviews and recommended revisions provided by the Steering Committee for the Great Basin
Fire Science Exchange, including Mark Brunson, Steve Bunting, Jeanne Chambers, Cheri
Howell, Elizabeth Leger, Mike Pellant, and Brad Schultz. We also thank Eugénie MontBlanc
and Lael Gilbert for extensive editing of the manuscript and for editorial improvements to
the manuscript. This work was supported by the U.S. Department of Agriculture, Agricultural
Research Service, Northwest Watershed Research Center, the Great Basin Fire Science
Exchange (funded by the Joint Fire Science Program), and the Sagebrush Steppe
Treatment Evaluation Project (funded by the Joint Fire Science Program, the Bureau of Land
Management, and the National Interagency Fire Center). This is Contribution Number 93 of
the Sagebrush Steppe Treatment Evaluation Project. All Rocky Mountain Research Station publications are produced by U.S. government employees and are in the
public domain and available at no cost. Even though U.S. Forest Service publications are not copyrighted, they are
formatted according to U.S. Department of Agriculture standards and research findings and formatting cannot be
altered in reprints. Altering content or formatting, including the cover and title page, is strictly prohibited. Acknowledgments CONVERSION FACTORS CONVERSION FACTORS CONVERSION FACTORS
Multiply
By
To obtain
degree Celsius (°C)
1.8, and then
add 32
degree Fahrenheit (°F)
millimeter (mm)
3.94 × 10−2
inch (in)
centimeter (cm)
3.94 × 10−1
inch (in)
meter (m)
3.28
feet (ft)
kilometer (km)
6.21 × 10−1
mile (mi)
gram (g)
3.53 × 10−2
ounce (oz)
kilogram (kg)
2.205
pound (lb)
square meter (m2)
10.76
square foot (ft2)
square meter (m2)
2.47 × 10−4
acre (ac)
hectare (ha)
2.47
acre (ac)
square kilometer (km2)
3.86 × 10−1
square mile (mi2)
square kilometer (km2)
247
acre (ac)
cubic meter (m3)
35.3
cubic foot (ft3)
liter (L)
3.53 × 10−2
cubic foot (ft3)
millimeter per hour (mm h−1)
3.94 × 10−2
inch per hour (in h−1)
liter per second (L s−1)
3.53 × 10−2
cubic foot per second (ft3 s−1)
cubic meter per second (m3 s−1)
35.3
cubic foot per second (ft3 s−1)
gram per square meter (g m−2)
4.46 × 10−3
ton per acre (t ac−1)
kilogram per hectare (kg ha−1)
4.46 × 10−4
ton per acre (t ac−1)
megagram (Mg)
1.102
ton (t) Introduction........................................................................................................................1
Section 1: Hillslope and Watershed Hydrology..............................................................2
The Hydrologic Cycle......................................................................................................2
Precipitation....................................................................................................................3
Interception.....................................................................................................................7
Infiltration.........................................................................................................................8
Soil Water Storage and Ground Water Recharge.........................................................10
Evapotranspiration........................................................................................................11
Surface Runoff and Streamflow....................................................................................12
Water Balance...............................................................................................................14
Section 2: Sediment Detachment, Transport, and Mass Wasting...............................15
Rainsplash and Sheetflow Processes...........................................................................15
Concentrated Flow Processes......................................................................................17
Gully Erosion Processes...............................................................................................18
Mass Movement Processes..........................................................................................19
Spatial and Temporal Variations of Processes..............................................................21
Section 3: Climate-Vegetation-Soil-Hydrology Interactions........................................23
Climate as a Driver of Hydrologic Response................................................................23
Vegetation Influences on Water Availability at the Surface and Near-Surface..............26
Interception Effects on Water Availability............................................................................26
Canopy Influence on Soil Microclimate, Evapotranspiration, and
Antecedent Soil Water.................................................................................................30
Cover Influences on Infiltration, Runoff, and Water Transfer and Storage....................32
Vegetation and Cover Influences on Infiltration and Runoff Generation.............................33
Plant Use of Subsurface Water and Its Influence on Runoff...............................................43
Effects of Soil Water Repellency on Runoff Generation.....................................................46
Cover Influences on Sediment Detachment and Transport..........................................50
Surface Protection from Raindrop Detachment..................................................................50
Cover Effects on Sheetflow Erosion...................................................................................51
Cover Effects on Concentrated Flow Erosion.....................................................................51
Section 4: Impacts of Fire on Rangeland Runoff and Erosion....................................53
Fire Behavior and Regimes on Rangelands.................................................................53
Fire Effects that Dictate Hydrologic Response..............................................................54
Reduced Interception and Surface Protection....................................................................54
Physical, Chemical, and Biological Alteration of Soils........................................................56
Exacerbation, Alteration, and Formation of Soil Water Repellency....................................57
Fire Effects on Infiltration and Runoff Generation.........................................................61
Rainsplash and Sheetflow Processes.................................................................................62
Hillslope- to Watershed-Scale Runoff.................................................................................67
Fire Effects on Soil Erosion by Water............................................................................69
Rainsplash and Sheetflow Processes.................................................................................69
Concentrated Flow Processes............................................................................................71
Hillslope- to Watershed-Scale Erosion................................................................................72
Assessing Postfire Hydrologic Vulnerability and Risk...................................................75
Determining Site Susceptibility and Hydrologic Recovery............................................77
Summary and Conclusions............................................................................................79 ii USDA Forest Service RMRS-GTR-351. 2016. Introduction Fire initiates disturbances that alter soil properties and the condition and structure of
vegetation and ground cover (Miller et al. 2013), potentially resulting in amplified runoff
and soil loss (Shakesby and Doerr 2006; Robichaud et al. 2010; Pierson et al. 2011;
Williams et al. 2014b). (Note that in this synthesis, citations are listed in chronologi-
cal order to reflect the progression of knowledge on a given topic.) The hydrologic
and erosional responses to fire vary with the intensity, severity, and spatial scale of the
disturbance, the stability and resilience of the affected ecosystem, and the prevailing
precipitation regime. Our historical understanding of these responses for rangeland eco-
systems comes mostly from anecdotal reports and from short-term, small (0.25 to 1 m2)
field plot studies of hydrologic behavior on gently sloping, semiarid shrub and grassland
sites (Pierson et al. 2002a). In recent years, researchers have expanded the inference space by studying wildfire
and prescribed burning effects on runoff and erosion rates from steeply sloped shrublands
(Pierson et al. 2001a, 2002b, 2008a,b, 2009), woodlands (Pierson et al. 2013, 2014;
Williams et al. 2014a), and shrub-forest interface sites (Johansen et al. 2001). These
studies, however, represent a minor portion of the diverse plant communities and soil
types that occur on sloping rangelands. Despite these limitations, this review presents
fire impacts on rangeland hydrology by (1) explaining fundamental hydrologic processes
necessary for understanding hillslope and watershed runoff and erosion responses;
(2) describing how climate, vegetation, and landscape properties interact with these
principal processes to influence hydrologic and erosional behavior; and (3) providing
examples from literature to illustrate these relationships with respect to fire and postfire
hydrologic recovery. The hillslope scale is emphasized, given the lack of literature on
watershed-scale fire effects for rangelands. Our geographic focus is the western United
States, although we include some literature from other semiarid regions around the globe. We include literature from dry forests because of similarities in the postfire runoff and
erosion processes across the rangeland-dry forest continuum in the Intermountain West
(Williams et al. 2014b). The goal of this review is to provide the reader a background for
understanding rangeland hydrology processes and a conceptual framework from which
to understand how fire affects rangeland hydrology and erosion across a range of site and
vegetative conditions and across precipitation regimes. 1 Section 1: Hillslope and Watershed Hydrology Precipitation The primary water input to the land-atmosphere interface is precipitation. The primary water input to the land-atmosphere interface is precipitation. Precipitation forms when warm, moist air at the Earth’s surface rises into the atmosphere
and cools by adiabatic expansion (Branson et al. 1981; Dingman 2002). During cooling,
water vapor condenses on small particles of matter, forming water droplets. The droplets
remain suspended until gravity overcomes the upward force of the rising air mass, result-
ing in precipitation. Precipitation falls as snow or ice where the air temperature above the
ground surface is 0 °C; otherwise precipitation falls as rain. Uplifting of warm air occurs
through either frontal, convective, or orographic lifting, and the lifting process dictates
the resulting storm type. Frontal lifting occurs when warm air and cool air masses collide, forcing the warm
air mass upwards and over the cool air mass. Warm fronts advancing toward cool air
masses generate prolonged low-intensity (quantity per unit of time), gentle rainfall over
large land areas, whereas large cold fronts advancing toward large moist warm air masses
facilitate high-intensity rainfall of shorter duration in a narrow advancing band. Occluded
fronts occur when a cold air mass overtakes a warm air mass (and collides with another
cold air mass), resulting in cold air everywhere at the surface and warm air above. The
rising warm air over the cool air at the surface generates precipitation, commonly at an
extremely high intensity (Dingman 2002). Convective lifting is associated with localized heating of surface air. Warm surface
air becomes buoyant, rises, and expands due to lower atmospheric pressure. The air mass
cools as it expands and convective clouds form. As the air mass cools, moisture particles
begin to coalesce therein. Convective lifting is most common during warm moist periods,
and generates intense rainfall and hailstorms over small areas scattered across the land-
scape. However, large-scale convective events may occur and, when over a large enough
area, facilitate flash flood events. Orographic lifting occurs when a warm air mass is forced upward along the wind-
ward side of a topographic barrier (such as a mountain range). Precipitation generated
from orographic lifting occurs on the windward side of the barrier and usually increases
with elevation. Rain shadows form on the leeward side of the barrier as the air parcel
crests and warms, and clouds dissipate. The Hydrologic Cycle The hydrologic cycle (fig. 1) refers to the continuous pathways in which water
moves in different phases through the atmosphere; on, into, through, and across the
land surface; to oceans and storage reservoirs; and upwards back into the atmosphere
(Dunne and Leopold 1978; Branson et al. 1981; Maidment 1993; Hornberger et al. 1998;
Dingman 2002; Brutsaert 2005). Knowledge of the hydrologic cycle provides a useful
framework from which to conceptualize and understand vegetation-soil-climate-
hydrology interactions and to understand how fire and other disturbances influence runoff
and erosion behavior (Dunne and Leopold 1978). For a particular hillslope or watershed,
the cycle consists of water inflow, transit and storage, and outflow. Inflow primarily
occurs as precipitation, overland flow and streamflow from upslope areas, and ground
water returns from springs and into streambeds and lakebeds. Transit and storage compo-
nents of water arriving at the land-atmosphere interface include precipitation interception,
infiltration, and water storage on and underneath the land surface. Outflows include
gaseous phase losses to the atmosphere through evaporation and transpiration, and liquid
water losses to plant use, watershed runoff, and deep drainage to aquifers. The remainder
of this section explains the fundamental components of the hydrologic cycle. Figure 1—Illustration of generalized hydrologic cycle for a hillslope showing directional inflows (rainfall,
snowfall, infiltration) and outflows (evapotranspiration, losses to interception, overland flow, deep drainage,
streamflow, and subsurface flow out). Figure 1—Illustration of generalized hydrologic cycle for a hillslope showing directional inflows (rainfall,
snowfall, infiltration) and outflows (evapotranspiration, losses to interception, overland flow, deep drainage,
streamflow, and subsurface flow out). USDA Forest Service RMRS-GTR-351. 2016. USDA Forest Service RMRS-GTR-351. 2016. Precipitation Orographic events are often associated with
frontal or convective events that encounter a topographic barrier. Precipitation is measured as a depth over some duration or period of time (daily,
monthly, seasonally, or annually) and is reported for individual storms as an intensity. Precipitation data for towns, watersheds, and other locales are available from area-
specific climate stations, precipitation gauges, and snow surveys with varying periods
of record (years of data). The various types of precipitation gauges, surveys (fig. 2), and
methods for extrapolating and applying precipitation data from available sources can be
found in most hydrology textbooks (see Dunne and Leopold 1978; Branson et al. 1981;
Dingman 2002; McCuen 2004; Brutsaert 2005). Here we focus our discussion on the
types of precipitation measurements commonly reported. Rainfall depth is the quantity of accumulated rainfall (expressed as a length
measurement such as mm or cm) at a point on the landscape (such as at a rain gauge). Rainfall duration is the time period over which a specified event occurred. Rainfall or
storm intensity is the rainfall rate expressed as depth of accumulation over a specified 3 3 USDA Forest Service RMRS-GTR-351. 2016. Figure 2—(A) Examples of shielded (right) and unshielded (left) dual precipitation gauge system used by
the USDA Agricultural Research Service, Northwest Watershed Research Center, at the Reynolds Creek
Experimental Watershed, Idaho, and (B) snow water equivalent measurement along a snow course transect
of the USDA Natural Resources Conservation Service (photo A: Agricultural Research Service; photo B: Ron
Nichols, Natural Resources Conservation Service). Figure 2—(A) Examples of shielded (right) and unshielded (left) dual precipitation gauge system used by
the USDA Agricultural Research Service, Northwest Watershed Research Center, at the Reynolds Creek
Experimental Watershed, Idaho, and (B) snow water equivalent measurement along a snow course transect
of the USDA Natural Resources Conservation Service (photo A: Agricultural Research Service; photo B: Ron
Nichols, Natural Resources Conservation Service). interval of time (for example, mm h-1 or cm h-1). Depth and duration variables are often
expressed together to define a storm event in terms of a depth-duration relationship
(48 mm during a 45-min interval) or an intensity-duration relationship (64 mm h-1 for
45 min) and are related to specified recurrence intervals (frequency) or return periods (for
example, a 100-year event). A recurrence interval is an estimate of the interval of time between events of a
certain intensity or size. Precipitation A recurrence interval is not the actual time between events of
the specified intensity or size, but rather represents the probability of that event occur-
ring. For example, the 100-year precipitation event has a 1 in 100 chance, or 1 percent
probability, of occurring each year. Most climate stations report depth-duration and
intensity-duration relationships in graphical or tabulated form for a range of return inter-
val storms (depth-duration-frequency or intensity-duration-frequency; fig. 3). Average
annual rainfall is calculated from gauges with long-term records as the total rainfall catch USDA Forest Service RMRS-GTR-351. 2016. Figure 3—Intensity-duration-frequency graphs for (A) climate station 163x20 at 2,170 m elevation in the
snowfall-dominated Reynolds Creek Experimental Watershed, Idaho, 1963 to 1998 (Hanson and Pierson
2001) and (B) climate station 02-8619 at 1,400 m elevation, Tombstone, Arizona, near the summer monsoon
rainfall-dominated Walnut Gulch Experimental Watershed, 1893 to 2000 (Bonnin et al. 2006). Figure 3—Intensity-duration-frequency graphs for (A) climate station 163x20 at 2,170 m elevation in the
snowfall-dominated Reynolds Creek Experimental Watershed, Idaho, 1963 to 1998 (Hanson and Pierson
2001) and (B) climate station 02-8619 at 1,400 m elevation, Tombstone, Arizona, near the summer monsoon
rainfall-dominated Walnut Gulch Experimental Watershed, 1893 to 2000 (Bonnin et al. 2006). USDA Forest Service RMRS-GTR-351. 2016. for a number of complete years of record divided by the number of years used. Rainfall
for a given year may be reported as a percentage of the average annual rainfall (for
example, 120 percent).i Snowfall is most often measured as a storm-specific depth (accumulation) of newly
fallen snow at a point, snowpack accumulation at the land surface over some duration
(daily, monthly, seasonally, or annually), or the snow water equivalent (depth) of newly
fallen snow or the snowpack. In contrast to rainfall, snowfall accumulation stores water
at the land surface and releases it for other hydrologic processes more gradually than
rainfall. Hydrologists are most interested in the snow water equivalent (SWE) within
accumulated snow, as it represents water availability for the hydrologic cycle (fig. 1) (see
Dingman 2002). Snow water equivalent is the depth of water that would result from the complete
melting of the snowpack of a specified area and is a function of the snowpack density
and depth over the defined area of interest. The quantity and timing of water delivery
released from the snowpack depend on SWE and the net energy input into the snowpack
(Dingman 2002). Precipitation Snowpack melting begins after the snowpack temperature is isothermal
at 0 °C. Melt water is retained within the snowpack until the water holding capacity is
exceeded, initiating delivery of snowmelt. Snowmelt (reported as depth of water) refers
to the amount of liquid water leaving the snowpack during a given time period. Other
commonly used terms for snow processes include snowpack ablation and water input. Ablation (measured as a depth of water) is the total loss of water substance (snowmelt
and evaporation) from the snowpack in a given time period. Water input is the total liquid
water (measured as depth of rain and snowmelt) leaving the snowpack during a given
time period. As with rainfall, SWE and other snow measurements are available from nu-
merous regional and local climate and precipitation stations. Snowstorms are reported in
terms of recurrence intervals, and snow water equivalent or snow depth may be reported
as a depth or percentage of average accumulation for various time steps (such as daily,
monthly, or annually). The timing, type, and quantity of precipitation falling at the land-atmosphere
interface are driven mostly by elevation and geography. Rangelands in the northern and
central United States receive substantially more annual precipitation than rangelands in
the desert Southwest (figs. 4 and 5) (see also Branson et al. 1981). In addition to annual
quantity differences, the timing or seasonality of precipitation inputs varies significantly
across U.S. rangelands (fig. 5). Precipitation inputs on southwestern U.S. rangelands (fig. 5E and 5F) occur mostly during the summer monsoonal season (up to 60 to 70 percent
of annual precipitation can occur during July and August) as intense convective thun-
derstorms (Branson et al. 1981; Osborn 1983a,b; Mendez et al. 2003; Wainwright 2006;
Goodrich et al. 2008). Most of the annual precipitation in the northwest and north-central
United States falls November through May and is snowfall-dominated in mountain
locations and rainfall-dominated in valley locations (figs. 6 and 7) (Branson et al. 1981). Rainfall patterns at these locations usually occur as low-intensity, long-duration events,
in late autumn or winter, and during a 4- to 8-week spring rainy season. Rain-on-snow or
rain-on-frozen-soil events are common at mountainous locations in early winter and dur-
ing transitional snow cover periods in spring (Wilcox et al. 1989; Pierson et al. 2001b). Mountain and valley locations experience high-intensity convective storms during the
dry summer months. Precipitation Precipitation differences between the two sites are primarily driven by the differences in elevation. Figure 6—Average monthly precipitation for two climate stations (A – Site 076x59, low elevation; B – 163x20
high elevation) located in the Reynolds Creek Experimental Watershed, Idaho (Hanson and Pierson 2001
Precipitation differences between the two sites are primarily driven by the differences in elevation. Figure 7—Reynolds Creek Experimental
Watershed near Boise, Idaho, where
precipitation is snow dominated in the
uplands and rainfall dominated in the
lowlands (photo: USDA Agricultural
Research Service). Figure 7—Reynolds Creek Experimental
Watershed near Boise, Idaho, where
precipitation is snow dominated in the
uplands and rainfall dominated in the
lowlands (photo: USDA Agricultural
Research Service). Figure 7—Reynolds Creek Experimental
Watershed near Boise, Idaho, where
precipitation is snow dominated in the
uplands and rainfall dominated in the
lowlands (photo: USDA Agricultural
Research Service). Precipitation Annual precipitation is usually greater at higher elevations than on 5 USDA Forest Service RMRS-GTR-351. 2016. Figure 4—(A) Annual precipitation (PRISM Climate Group 2011) and (B) landcover (U.S. Geological Survey
2011) for the western United States. Figure 4—(A) Annual precipitation (PRISM Climate Group 2011) and (B) landcover (U.S. Geological Survey
2011) for the western United States. valley floors due to adiabatic processes (fig. 6) (see Branson et al. 1981; Dingman 2002). Precipitation trends are bimodal for some south-central U.S. rangelands (see Romme
et al. 2009), with peaks occurring in mid-to-late winter and during summer monsoon
months (during which 30 to 40 percent of annual precipitation occurs; see Bowen 1996). Elevational precipitation trends for the central United States are similar to those occurring
on northwest and north-central U.S. rangelands. USDA Forest Service RMRS-GTR-351. 2016. Figure 5—Average monthly precipitation for western U.S. urban centers immediately adjacent to rangelands
(PRISM Climate Group 2011). Panels A–C are indicative of annual precipitation in the Inland Northwest. Panel D is indicative of annual precipitation trends on central U.S. rangelands. Panels E-F demonstrate the
influence of the monsoonal season (July–August) on rainfall in the desert Southwest. Figure 5—Average monthly precipitation for western U.S. urban centers immediately adjacent to rangelands
(PRISM Climate Group 2011). Panels A–C are indicative of annual precipitation in the Inland Northwest. Panel D is indicative of annual precipitation trends on central U.S. rangelands. Panels E-F demonstrate the
influence of the monsoonal season (July–August) on rainfall in the desert Southwest. USDA Forest Service RMRS-GTR-351. 2016. Figure 6—Average monthly precipitation for two climate stations (A – Site 076x59, low elevation; B – 163x20,
high elevation) located in the Reynolds Creek Experimental Watershed, Idaho (Hanson and Pierson 2001). Precipitation differences between the two sites are primarily driven by the differences in elevation. Figure 7—Reynolds Creek Experimental
Watershed near Boise, Idaho, where
precipitation is snow dominated in the
uplands and rainfall dominated in the
lowlands (photo: USDA Agricultural
Research Service). Figure 6—Average monthly precipitation for two climate stations (A – Site 076x59, low elevation; B – 163x20
high elevation) located in the Reynolds Creek Experimental Watershed, Idaho (Hanson and Pierson 2001)
Precipitation differences between the two sites are primarily driven by the differences in elevation. Figure 6—Average monthly precipitation for two climate stations (A – Site 076x59, low elevation; B – 163x20,
high elevation) located in the Reynolds Creek Experimental Watershed, Idaho (Hanson and Pierson 2001). Interception Precipitation arriving at the land-atmosphere interface either is intercepted by
vegetation, rocks, or litter debris or falls unimpeded to the soil surface. Intercepted
precipitation evaporates into the atmosphere (interception loss) or is transferred as
liquid water to the soil surface as throughfall or stemflow (Thurow et al. 1987; Návar
and Bryan 1990; Martínez-Meza and Whitford 1996; Whitford et al. 1997; Wainwright
et al. 1999; Abrahams et al. 2003; Bhark and Small 2003; Carlyle-Moses 2004; Owens
et al. 2006). Throughfall reaches the ground surface by passing directly through spaces USDA Forest Service RMRS-GTR-351. 2016. within and between canopies, and includes canopy drip. Canopy drip begins once canopy
liquid water interception and surface evaporation capacities are exceeded. Stemflow is
precipitation input that reaches the ground surface by running down stems and trunks of
vegetation. Gross or bulk precipitation is the precipitation measured in the open, or above
the canopy. The net precipitation is the gross precipitation arriving at the land-atmosphere
interface minus total interception loss. Intercepted precipitation retained from throughfall
and stemflow is termed “static canopy storage” (Dunkerley 2000).l Interception is strongly influenced by the precipitation frequency and intensity and
by the type and structure of the vegetative community (Branson et al. 1981; Owens et
al. 2006; Dunkerley 2008). Interception losses from a series of small storm and clearing
events are proportionately greater than those from large, prolonged rainfall events
(Rowe 1948; Hamilton and Rowe 1949; Owens et al. 2006). Prolonged events saturate
plant surfaces and the resulting interception rate becomes equal to the evaporation
rate. Intercepted precipitation is evaporated during clear periods between short events,
reestablishing a portion of the interception capacity. This results in a large proportion
of each small storm’s gross precipitation being applied to interception as compared to a
prolonged or large event. Interception is usually greater from conifer than broad-leaved
tree species and is greater from trees species than from shrubs and grasses. Variations in the canopy density of individual plants and the vegetative community
complicate quantification of interception losses over large scales. Therefore interception
terms are commonly reported as a depth or volume of water or as the percentage of
precipitation falling on an individual plant or at a point during the period of interest. Interception losses over large areas are determined by spatially aggregating interception
losses by plant species or area representations (West and Gifford 1976). Interception Dunkerley
(2000) provides a brief overview of interception terminology, measurement methods, and
estimation approaches. Infiltration Porous or rough surfaces are usually more conductive than uniform surfaces. However, high-intensity rainfall can break apart highly conductive porous structure
or aggregates of surface soils, facilitating infilling of soil pores with fine soil particles
(Thornes 1980; Selby 1993). Infilling of pores creates a hydrologic barrier. Compaction
reduces pore size and can also create a hydrologic barrier and reduce the surface soil infil-
tration capacity. Infiltration capacity refers to the maximum rate that water can enter soil
in a given condition. Hydrologic barriers may also form from freezing of surface soils or
swelling of clay soils. Frozen soils near saturation can form “concrete frost” layers with
very low conductivity (Blackburn and Wood 1990; Blackburn et al. 1990). Swelling properties of clay soils may decrease the conductivity of pore spaces
upon wetting, reducing infiltration with increased soil wetness. The presence of organic
matter can increase or decrease the infiltration capacity. Organic matter is associated
with greater aggregate stability (Cerdà 1998b), low bulk density, and formation of large
pores (macropores) or cracks in the soil surface. Aggregate stability and low bulk density
values facilitate maintenance of large pore voids and macropores that transfer water
rapidly downward into or laterally through the soil profile. In contrast, organic matter
may contribute to water-repellent (hydrophobic) conditions, which impede infiltration
(Meeuwig 1971; DeBano and Rice 1973; Doerr et al. 2000, 2009). The formation of
water-repellent soils and the effects on infiltration are discussed in more detail in Section
3, Effects of Soil Water Repellency on Runoff Generation. Infiltration into wet soils is significantly influenced by the saturated hydraulic
conductivity of the soil profile (Knapp 1978; Branson et al. 1981; Selby 1993; Hillel
1998; Dingman 2002). Once the soil profile becomes wet, any further water input is
partially dependent on the redistribution or downward transmittance (percolation) of
existing soil water. Hydraulic conductivity (measured as length per unit time) refers to the
rate at which water is redistributed through the soil profile and is a function of pore space
connectivity (soil porosity and soil structure) and soil wetness. Hydraulic conductiv-
ity increases with increasing soil wetness due to greater connectivity of wet pores (wet
flowpaths). Under saturated conditions, all pore spaces are filled, and higher conductivity
occurs where large and continuous pores represent most of the pore volume. Infiltration The rate at which water infiltrates into the soil profile is influenced by the amount
and arrival rate of water at the ground surface, the ability of the soil to conduct water into
and through the soil profile, and the slope, roughness, and chemical characteristics of the
soil surface (Dunne and Leopold 1978; Knapp 1978; Branson et al. 1981; Selby 1993;
Hillel 1998; Dingman 2002; Brutsaert 2005). Infiltration is reported on a point scale as
a rate (depth per unit of time) or the cumulative depth of water (for example mm or cm)
that infiltrates into the soil profile over some period of time (such as a storm event). For
rainfall events, water availability is a function of the intensity and duration of the storm
(water input rate), interception losses, and the ability of the surface to detain or pond
water (surface detention). Surface detention is a function of the land surface slope, the roughness or
microtopography of the soil surface, and the quantity and structure of litter and woody
debris present. Gentle slopes with rough surfaces and substantial amounts of litter and
debris generally detain more water on the ground surface than steep slopes that are bare
or devoid of ground cover. Snowmelt contributions to infiltration are also influenced by
the water input rate, interception losses, and surface detention. The snowpack generally 8 USDA Forest Service RMRS-GTR-351. 2016. 8 provides a more gradual release of water to the ground surface than does rainfall. However, rapid releases of water from a snowpack may occur during peak snowmelt or
rain-on-snow events (Dingman 2002).i Water infiltrates soil mainly due to a negative pressure gradient or suction (matrix
suction) into the soil matrix and secondarily due to gravity (Knapp 1978; Branson et al. 1981; Selby 1993; Hillel 1998; Hornberger et al. 1998; Dingman 2002; Brutsaert 2005). Matrix suction results from the physical affinity of water to soil-particle surfaces and
pores, and decreases with increasing soil wetness (Hillel 1998). In general, infiltration
is high in the early stages of water input into dry soil, then decreases as the surface soil
becomes increasingly wet, and approaches a relatively steady state (steady state infiltra-
tion rate) as soil becomes saturated. Decreased infiltration over time following rainfall
results mainly from decreased matrix suction with wetting, but also may occur due to
surface sealing (Assouline 2004) and compaction from raindrop impact or shrink/swell
soil properties. USDA Forest Service RMRS-GTR-351. 2016. Soil Water Storage and Ground Water Recharge Some of the water infiltrating the ground surface is retained in the unsaturated zone
as soil water storage, and some passes through the profile into the saturated zone as deep
drainage or ground water recharge. The unsaturated zone encompasses the soil water
or rooting zone, an intermediate zone, and the capillary fringe immediately overlying
the saturated zone (Hornberger et al. 1998; Dingman 2002). The soil water zone is the
uppermost portion of the soil profile where soil water is extracted and used by plants or
evaporated into the atmosphere. The intermediate zone is often referred to as the zone of
aeration and is the area between the soil water zone and the capillary fringe. Water enters
the intermediate zone by percolation from above and exits by gravity drainage. Pore
spaces in the capillary fringe are saturated or are near saturation and pore water is held
there by capillary forces (Hornberger et al. 1998; Dingman 2002). Generally, less than 1
percent of water entering the soil profile passes through the soil water and intermediate
zones into deep storage below the capillary fringe. In this review we restrict our discus-
sion on soil water movement and storage to the unsaturated zone because most surface
responses are associated with soil water content in the rooting zone of the soil profile. Explanations of the soil physics that dictate soil water movement and retention in the
unsaturated and saturated zones are provided in Kramer and Boyer (1995), Hillel (1998),
and Dingman (2002). The water or moisture content of the soil profile dictates water availability for
plants and biological processes, and is a function of the soil texture and structure (Kramer
and Boyer 1995). Soil water content at any point in time is measured as volumetric or
gravimetric water content (see Dingman 2002). Volumetric soil water content is the ratio
of water volume to soil volume, and gravimetric soil water content is the mass of water
per unit mass of dry soil. Soil water content between precipitation events or periods is
referred to as the “antecedent water/moisture content.” The distribution, connectivity,
and size of the soil voids greatly influence the water content and degree of wetness (the
ratio of water content to porosity). Water in capillaries or micropores is tightly held to
soil particles by matrix potential, whereas large and connected pores drain rapidly due
to gravity (Kramer and Boyer 1995). Infiltration For example,
sandy, coarse-grained soils with extensive large pore connectivity will transmit water
downward more rapidly under saturated conditions than fine-grained clayey soils with USDA Forest Service RMRS-GTR-351. 2016. numerous micropores. Therefore, infiltration rates for wet soil conditions approximate
saturated hydraulic conductivity and are generally greater for coarse-grained or well-
aggregated soils. Saturated hydraulic conductivity commonly decreases with soil depth
due to decreases in porosity in deeper portions of soil profiles.il Finally, redistribution and, hence, infiltration are also influenced by the structure
of the soil profile. The presence of an impeding/restrictive layer (layer of low hydraulic
conductivity) may retard water movement during infiltration. The rate of water movement
through restrictive layers is reduced relative to the remainder of the soil profile. Impeded
flow is often overcome through bypass (macropore or preferential) flow. The processes
and measurement methods for water infiltration and redistribution in unsaturated and
saturated soils are described in detail by Hillel (1998) and Dingman (2002). Soil Water Storage and Ground Water Recharge Excluding evaporative demands, total soil water
storage (product of volumetric water content and thickness of the layer) results from the
resolution of the matrix and gravitation forces, and plant water use. 10 USDA Forest Service RMRS-GTR-351. 2016. Water content available for plants is the difference between the field capacity
content and permanent wilting point water content (Kramer and Boyer 1995; Dingman
2002). Field capacity is the water content held against gravity, and refers to the relatively
stable soil water content at which continued downward drainage is negligible. The perma-
nent wilting point (Kramer and Boyer 1995; Dingman 2002) is the soil water content at
which water availability is too low to support plant transpiration demands and the point at
which plants wilt (see Section 1, Evapotranspiration). Field capacity and water retention
are generally greater for clay soils than for sandy soils and are intermediate for loams. Clay soils are compact and cohesive (with numerous micropores) and drain slowly. In
contrast, sandy soils are noncohesive, have large and connective voids, drain rapidly, and
possess limited water storage capacity. The presence of organic matter in soils usually
increases the water storage capacity. Evapotranspiration Evapotranspiration is the primary loss mechanism for precipitation inputs in most
geographic areas of the United States and may constitute 50 to almost 100 percent
of incoming annual precipitation (see Branson et al. 1981; Kramer and Boyer 1995;
Hornberger et al. 1998; Dingman 2002; Brutsaert 2005). Evapotranspiration losses arise
from two primary processes of liquid water conversion to water vapor: evaporation and
transpiration. Evaporation is the process by which liquid or solid water from ground and
vegetative surfaces, from rivers and lakes, and from ice and snow is converted to water
vapor and transferred back into the atmosphere. Evaporation occurs when atmospheric
vapor pressure is lower than vapor pressure of the evaporative surface. In the most basic
sense, evaporation is equal to a coefficient for barometric pressure and wind velocity
multiplied by the difference in maximum vapor pressure at the surface and the vapor
pressure in the air above the evaporative surface (Dalton’s Law) (see Branson et al. 1981
and Dingman 2002). Transpiration is the direct evaporation of liquid water from within the leaves of
plants. During transpiration, water vapor diffuses to the atmosphere from leaf surfaces,
forming a water deficit within foliage cells. This deficit is transmitted from foliage via
water columns within plant xylem tissue through branches, stems, and large roots to
fine roots, where soil water uptake occurs. Transpiration is a biological evaporative
process that is influenced by plant leaf, stem, and root structures, water use strategies,
soil microclimate, water availability, and plant influences on wind and the aboveground
microclimate (Branson et al. 1981; Kramer and Boyer 1995; Hornberger et al. 1998;
Dingman 2002). In order for evapotranspiration to occur there must be (1) a positive net flow of en-
ergy to the evaporative or transpiring surface, (2) water available for conversion to water
vapor, and (3) a flow of vapor away from the surface (see Hornberger et al. 1998 and
Dingman 2002). The conditions that control the net flow of energy determine the energy
available for vaporization of liquid or solid water. Energy arrives at a respective surface
as incoming solar energy and is either reflected or absorbed, depending on the surface
reflectivity (albedo). Light-colored surfaces like freshly fallen snow have much higher
albedo than do dark-colored surfaces such as black soil, and reflect much of the arriving 11 USDA Forest Service RMRS-GTR-351. 2016. solar energy back into the atmosphere. Evapotranspiration Some of the arriving energy is consumed to heat
(sensible heat) the surface and air around it. The evaporation process consumes additional
energy (latent heat) in the conversion of liquid or solid water to water vapor. Therefore
the net flow of energy required for evapotranspiration must satisfy energy reflected and
energy transferred as sensible and latent heat (Kramer and Boyer 1995; Hornberger et al. 1998; Dingman 2002). During water-limited conditions, incoming energy is consumed more in the heating
of the surface and the air than as latent heat for water vaporization. In contrast, in wet
conditions, evapotranspiration is dictated by the quantity of incoming radiant energy,
the dryness of the air, and the efficiency of wind to transport water vapor away from the
surface. Evapotranspiration may be reported as potential or actual evapotranspiration and
is usually provided as a depth of water at a point per period of time (for example, annual
evapotranspiration). Potential evapotranspiration is water loss that occurs when a surface
is fully wet and no soil water deficiencies exist relative to plant water use. Actual evapo-
transpiration refers to water loss that occurs when water availability is reduced below
that found for a wet surface. Methods to measure and calculate evapotranspiration, along
with more detailed explanations of evapotranspiration processes, are found in Kramer and
Boyer (1995), Hornberger et al. (1998), and Dingman (2002). Surface Runoff and Streamflow Precipitation and streamflow shown
are from a convective thunderstorm event (~22 mm rainfall during 2 hours) occurring July 21, 1971, in the
Reynolds Creek Experimental Watershed as recorded at the Tollgate Weir Site 116x83 (USDA Agricultural
Research Service 2011a). Event and base flow contributions to streamflow are separated by the dashed
gray line. Arrows and callouts indicate rising limb, peak discharge, and falling limb of the hydrograph as
responses to the storm event. Figure 8—Example streamflow hydrograph and precipitation hyetograph. Precipitation and streamflow shown
are from a convective thunderstorm event (~22 mm rainfall during 2 hours) occurring July 21, 1971, in the
Reynolds Creek Experimental Watershed as recorded at the Tollgate Weir Site 116x83 (USDA Agricultural
Research Service 2011a). Event and base flow contributions to streamflow are separated by the dashed
gray line. Arrows and callouts indicate rising limb, peak discharge, and falling limb of the hydrograph as
responses to the storm event. Streamflow is the flow rate or discharge of water (measured as volume per unit
of time) along a defined natural channel and is partitioned as either base or event flow
(Dunne and Leopold 1978; Dingman 2002; McCuen 2004). Base flow refers to the
portion of the streamflow that cannot be attributed to a particular precipitation event and
is generally assumed to be ground water return flow into stream channels. Base flow
of a particular stream or drainage network (pattern of streams within a watershed) is
relatively consistent from year to year and depends mostly on the availability of ground
water returns. Stream baseflow may be spatially variable where exchanges of surface
water and ground water facilitate streamflow gains or losses. Event flow is the portion of
streamflow directly resulting from event effective water input, and may also be referred
to as storm runoff or storm flow. Effective water input is the water input from a particular
precipitation event, usually in the form of direct precipitation into streams and Hortonian
or saturated overland flow. Time variability and space variability in event flow generally
increase with increasing watershed size due to temporal and spatial variations in precipi-
tation water input, overland flow generation, and streamflow routing. Watershed or hillslope response to precipitation events is commonly quantified
graphically and analyzed by using streamflow hydrographs and precipitation hyetographs
(fig. 8) (Hornberger et al. 1998; Dingman 2002; McCuen 2004). Hydrographs depict
stream discharge versus time for a point along a stream channel. Surface Runoff and Streamflow The mechanisms for surface runoff generation include Hortonian and saturated
overland flow generation, direct precipitation into stream channels, and ground water
returns to the land surface or stream channels (Horton 1933; Dunne 1978; Selby 1993;
Hornberger et al. 1998; Dingman 2002). Hortonian overland flow (Horton 1933) is
generated when water input on land exceeds the rate at which water can infiltrate the
soil. Hortonian flow (also called infiltration-excess flow) is most common under intense
rainfall in sloping semiarid to arid regions (water-limited) or where surface conductivities
are low. Saturation overland flow (saturation-excess flow) results from continued water
input at the surface of a saturated soil profile. Ponded and saturated soil surfaces shed
any additional water inputs from the atmosphere. The hillslope or watershed area (source
area) contributing to saturated overland flow varies seasonally or during precipitation
events. Hydrologists commonly refer to this variable zone of saturation overland flow as
the “variable source area” (see Selby 1993; Hornberger et al. 1998; Dingman 2002). Infiltration- and saturation-excess overland flows may create sheetflow (interrill) or
concentrated flow (rills), or a combination thereof, on sloping terrain. Sheetflow refers to
overland flow as a thin, relatively spatially connected film or sheet on the land surface. Concentrated flow is runoff that accumulates or converges into well-defined microchan-
nels or rills. Direct precipitation into stream channels occurs during all precipitation
events and can contribute significantly to peak and total event streamflows. Subsurface
return or event flow constitutes only a small portion of event streamflow. However, areas
of ground water mounding or ridging and hillslopes with extensive macropore networks
may contribute substantially to runoff generation and streamflow response (Wilcox et al. 1997; see Dingman 2002). USDA Forest Service RMRS-GTR-351. 2016. 12 Figure 8—Example streamflow hydrograph and precipitation hyetograph. Precipitation and streamflow shown
are from a convective thunderstorm event (~22 mm rainfall during 2 hours) occurring July 21, 1971, in the
Reynolds Creek Experimental Watershed as recorded at the Tollgate Weir Site 116x83 (USDA Agricultural
Research Service 2011a). Event and base flow contributions to streamflow are separated by the dashed
gray line. Arrows and callouts indicate rising limb, peak discharge, and falling limb of the hydrograph as
responses to the storm event. Figure 8—Example streamflow hydrograph and precipitation hyetograph. Surface Runoff and Streamflow Hyetographs quantify
water input (precipitation or snowmelt) at a point versus time and may be shown on a sec-
ondary x- and y-axis of a streamflow hydrograph to view respective stream responses to
water-input events (fig. 8). The shape of the streamflow hydrograph provides qualitative
and quantitative interpretation of watershed response to varying water input. The event
response is evident on the hydrograph by an increase in discharge (rising limb) greater
than base flow, to a peak (peak discharge), followed by a decrease to baseflow (falling
or recession limb) (fig. 8). Hydrologists are particularly interested in watershed response
time, time to peak runoff, peak discharge, and cumulative runoff. The response time and 13 USDA Forest Service RMRS-GTR-351. 2016. time to peak refer to the time it takes for runoff to occur and then to peak following water
input initiation. Peak discharge is the maximum discharge that occurs during a particular
event. Cumulative runoff is the integration of runoff rates with respect to runoff duration. A short response time, a short time to peak runoff, and a steep rising limb indicate rapid
or flashy watershed response to a water input event. The overall response to a particular event, and the resultant hydrograph shape,
depends on the size of the drainage area, soils and geology, slope, and land use/vegetation
patterns (Dingman 2002; McCuen 2004). Large watersheds commonly exhibit delayed
responses to precipitation events unless the event is near the stream outlet or occurs over
a large, contiguous area. Watersheds or hillslopes with low infiltration rates or steep
slopes, or both, typically have short response times, high peak discharge, and steep rising
limbs. Runoff response is delayed and discharge is usually low where extensive vegeta-
tion and ground cover exist and land use favors water retention. Shorter event and peak
response times, higher peak discharges, and steepened rising limbs with respect to similar
rainfall events postdisturbance indicate degraded surface conditions.i An individual hydrograph does not specifically identify the condition eliciting the
response. However, hydrographs can be compared for similar precipitation events over
a range of watershed conditions to infer cause-and-effect relationships where supportive
watershed/hillslope data are available. Infiltration responses to water-input events may
also be quantified in hydrograph form, as the inverse of the runoff relationships (see
Meeuwig 1971; Dunne 1978; Selby 1993; Hillel 1998; Hornberger et al. 1998; Dingman
2002). Water Balance The components of the water cycle with respect to a specified watershed and time
period represent the area water balance (see Branson et al. 1981; Dingman 2002; Wilcox
et al. 2003b). The generalized water balance is expressed as: P + Gin – Q – ET – Gout = ΔS
(Equation 1) Section 2: Sediment Detachment, Transport, and Mass
Wasting Soil erosion refers to the detachment and transport of soil by raindrop impact,
running water, wind, ice flows, and other mass-movement processes (Selby 1993). Soil
detachment is a function of the erosive energy acting on the soil surface and the resis-
tance of the surface to erosion (Thornes 1980; Selby 1993; Toy et al. 2002). In hydrologic
terms, erosive energy acting on the soil surface (shear stress) results from raindrop impact
or the flow of water, or a combination of both. The resistance of the surface (critical
shear stress) is a function of the soil properties, soil cohesiveness, and other surface
characteristics that define the soil erodibility. Erodibility is defined as the vulnerability of
a soil in its current condition to erosion by rainfall, runoff, and wind. Detachment occurs
when the shear stress acting on a soil exceeds the critical shear stress or resistance of the
soil (Foster and Meyer 1972; Nearing et al. 1999; Kinnell 2005). As defined, the removal of detached sediment requires entrainment by a transport
mechanism. Transport may occur by displacement from raindrop impact or entrainment
into flowing water, or both (Kinnell 1988, 1990, 2005). Transport capacity of flowing wa-
ter is dependent on the volume of water, mass of solids versus mass of water, energy loss
as the flow moves downslope, and efficiency of transport (Thornes 1980; Toy et al. 2002;
Kinnell 2005). In this section we summarize these fundamental sediment detachment and
transport mechanisms. Our emphasis is on hillslope processes given the context of this re-
view. Julien (1998), Knighton (1998), and McCuen (2004) provide detailed explanations
of channel or fluvial sediment entrainment, transport, and routing processes beyond the
scope of this review. Soil erosion through wind, creep, weathering, and other mechanisms
not directly involving rainfall and flowing water are discussed in Selby (1993), Toy et al. (2002), Ravi et al. (2007), Sankey et al. (2009), and Field et al. (2012). (Equation 1) where P is precipitation, Gin is incoming ground water, Q is streamflow or surface
runoff, ET is evapotranspiration, Gout is outgoing ground water, and ΔS is the change in
water storage over the period of interest. Over annual scales, the water balance provides
an accounting of the annual water budget. For rangeland ecosystems, runoff usually
amounts to less than 10 percent of the annual water budget (Wilcox et al. 2003b). Nearly
all of the remainder of precipitation falling on rangelands is lost to evapotranspiration. Evapotranspiration may exceed precipitation during dry years. Deep drainage of soil
water beyond the rooting zone as ground water recharge, Gout, is usually less than a few
millimeters (Wilcox et al. 2003b). The net change in water storage is generally considered
to be zero over long time periods (such as several years). Section 3 provides more detail
on the water budget components with respect to rangeland ecosystems. 14 14 USDA Forest Service RMRS-GTR-351. 2016. Section 2: Sediment Detachment, Transport, and Mass
Wasting USDA Forest Service RMRS-GTR-351. 2016. Rainsplash and Sheetflow Processes The combined effects of rainsplash and sheetflow/sheetwash processes are termed
“rainwash” or “interrill” processes (Selby 1993; Toy et al. 2002). Rainwash processes
often represent the dominant erosion processes where microchannel formation is
substantially limited by soil aggregation, soil cohesion, and surface protection. Here, we
describe the two components of rainwash—rainsplash and sheetflow—separately because
their co-occurrence is dependent on overland flow generation. Rainsplash erosion is
the transfer of sediment resulting from raindrop impact. The effects of raindrop impact
include detachment and displacement of soil (see Kinnell 2005), disaggregation of soil
aggregates, and reduced infiltration due to surface sealing (Moss 1991; Assouline 2004;
Kinnell 2005). Sediment detachment and transport by rainsplash occurs in a “splash-
crown” (fig. 9A) with sediment mass declining exponentially outward from the point of
impact on flat surfaces (Thornes 1980; Toy et al. 2002; Kinnell 2005). On sloping terrain,
downslope transport may exceed three times the mass of upslope transport where slopes
are greater than 10 percent (Thornes 1980). Raindrop detachment rates are commonly highest within several minutes after the
onset of rainfall, followed by an exponential decrease to a steady rate; however, rates 15 USDA Forest Service RMRS-GTR-351. 2016. Figure 9—Soil erosion by (A) rainsplash, (B) sheetflow, and (C) concentrated flow processes (photo A: USDA
Natural Resources Conservation Service; photos B and C: USDA Agricultural Research Service, Northwest
Watershed Research Center). Figure 9—Soil erosion by (A) rainsplash, (B) sheetflow, and (C) concentrated flow processes (photo A: USDA
Natural Resources Conservation Service; photos B and C: USDA Agricultural Research Service, Northwest
Watershed Research Center). may be highest at rainfall onset if the supply of detachable sediment is low (see Parsons
et al. 1994). The main source of energy in this process is the kinetic energy of rainfall as
dictated by the raindrop mass and terminal velocity (Gilley and Finkner 1985; Sharma
et al. 1991; Bryan 2000; Salles and Poesen 2000; Salles et al. 2002). Terminal velocity
increases with increasing drop size, and drop size generally increases with increasing
rainfall intensity (Gunn and Kinzer 1949; Salles and Poesen 2000; Salles et al. 2002; Van
Dijk et al. 2002). Thus, the impact energy of rainfall and sediment detachment generally
increases with increasing rainfall intensity. However, cumulative sediment detachment
may be significant from low-intensity storms over long durations. Rainsplash and Sheetflow Processes The decrease in detachment by raindrops during rain events is often associated with
an increase in the depth of sheetflow (Thornes 1980; Moss and Green 1983; Ferrera and
Singer 1985; Gilley et al. 1985; Toy et al. 2002). Flow depths equal to the diameter of
approximately three drops greatly reduce the impact of rainfall (Moss and Green 1983;
Kinnell 1990, 1991, 1993). The shear stress and additional transport capacity of sheetflow
may, however, offset the decrease in raindrop impact, resulting in a net increase in total
sediment production, or yield (Kinnell 2005). In addition to increasing sediment yield,
drop impact from high-intensity rainfall may break apart soil aggregates and facilitate
particle sorting by size and infilling of surface pores with fine material. This process,
referred to as surface sealing (Bradford et al. 1987; Assouline 2004), may create a surface
crust or compact the surface soil, resulting in reduced infiltration (by a factor of 1 to
10) and increased runoff and sediment transport by sheetflow. Soils with high clay and
organic matter content are generally less influenced by raindrop effects than are sandy or
sandy loam soils, and the net effect may be related to soil bulk density. 16 USDA Forest Service RMRS-GTR-351. 2016. Sheetflow generates shear stress that is minor (about 100-fold less) compared to
raindrop impact, but it serves as an additive detachment and transport mechanism for
interrill sediment yield (Thornes 1980; Selby 1993; Toy et al. 2002; Kinnell 2005). Sheetflow does not usually occur as a broad flow across a hillslope; rather, it occurs in
isolated irregular- flow patches of several millimeters depth separated by flow obstacles
(fig. 9B) (Emmett 1970, 1978). Raindrops falling into shallow flow depths create turbu-
lence and detachment, facilitating sediment entrainment. Soil detachment from sheetflow
results from drag created by differential shear stress on the upslope and downslope faces
of the particle, Bernoulli lift in the horizontal direction, and vertical turbulence (see
Thornes 1980; Kinnell 2005). The amount of sediment and the size of particles entrained
depend on the flow velocity and turbulence, both of which generally increase with
increasing slope and flow depth. The rate of transport generally increases with flow depth
to a maximum of about one to three raindrop diameters (Selby 1993). Entrained particles
remain in suspension until a deposition velocity occurs (less than 0.015 m s-1). Rainsplash and Sheetflow Processes The pres-
ence of ice in surface soils may amplify interrill erosion by slightly raising portions of the
surface soils, making them more susceptible to detachment and entrainment by rainsplash
and sheetflow processes (Blackburn and Wood 1990; Blackburn et al. 1990). Concentrated Flow Processes Sediment yield from concentrated flow (rill) processes is several orders of magni-
tude greater than that of sheetflow and rainsplash (Thornes 1980; Wainwright et al. 2000;
Pierson et al. 2008a). Concentrated flow processes may account for 50 to 90 percent
of total sediment yield on slopes with very little vegetation. Concentrated flowpaths or
rills are microchannels of several to tens of centimeters in width and several to 300 mil-
limeters in depth that are easily obliterated between storm events. These microchannels
form when surface roughness elements (microtopography) concentrate sheetflow into
narrow, deeper flowpaths, increasing the velocity and erosive energy of runoff (fig. 9C)
(Emmett 1970, 1978). Concentrated flow detachment and incision occur when the
incoming interrill sediment load is less than the concentrated flow’s transport capacity,
and the shear stress applied to the soil is greater than soil surface critical shear stress or
erodibility (Nearing et al. 1989, 1999; Al-Hamdan et al. 2012b). The shear stress applied
is a function of the density, depth, and velocity of the flowing water, the friction imposed
by the soil and cover, land surface slope, and acceleration due to gravity (see Toy et al. 2002; Al-Hamdan et al. 2012a,b, 2013).l Deposition occurs when the flow transport capacity is exceeded. Sediment detach-
ment and transport are commonly highest at the initiation of concentrated flow and
decrease gradually as more resistive materials are exposed with flowpath incision or
where sediment supply is limited (Nearing et al. 1997; Wainwright et al. 2000; Pierson et
al. 2008a; Al-Hamdan et al. 2012b). On freshly exposed surfaces, parallel concentrated
flowpaths may cross grade or merge by breaking down the divides between microchan-
nels (micropiracy). This process diverts the flow into the deeper, more dominant
flowpaths and generally increases the spacing of concentrated flowpaths in the downslope
direction. 17 USDA Forest Service RMRS-GTR-351. 2016. USDA Forest Service RMRS-GTR-351. 2016. Mass Movement Processes Mass movement erosion occurs when the shear stress applied to a body of soil
material on a slope exceeds the resistance or critical shear stress (shear strength) of the
material to downslope movement and may result directly or indirectly from a particular
water input event (see Sidle et al. 1985; Selby 1993). Shear stress is increased by removal
of lateral soil support, soil profile shifting, overburdening of soils with rain or snow,
ground vibrations, undercutting of banks or ridges, and increased slope steepness. Shear
strength is reduced by loosening of soils with soil shifting, increased buoyancy and capil-
lary tension associated with pore water changes, alteration of soil structure, decreased
root anchoring and elevated water tables due to vegetation alteration, and the presence of
relict weakness planes (such as faults and joints). Mass soil movement processes include
creep, falls/topples, slides, and flows. Here, we briefly summarize the common types and
causes of soil mass movement. Sidle et al. (1985) and Selby (1993) provide extensive
description and explanation of the types, causes, and occurrences of soil mass movements
and present approaches to slope stability assessment and analysis. Soil creep and fall processes generally constitute minor soil loss relative to other
erosion processes. Creep of soil downslope may occur as individual soil particles (particle
creep) or en masse (slope creep). Particle creep occurs due to gravity, particle expansion
and contraction with heating and cooling, and wetting-drying and freeze-thaw processes. Slope creep refers to the slow downslope creep of large soil masses and is a function of
the creep rate and the depth of material in movement. Creep rates usually range between
0.1 and 15 mm y-1 on well-vegetated slopes, but may be as high as 500 mm y-1 on
exposed slopes and areas with frequent freeze-thaw cycles (Selby 1993). Falls result from
the undercutting of slope faces or toe slopes by flowing water or from cliff-top sloughing
after freeze-thaw or wetting-drying periods. Falls may contribute significantly to the
downslope transport of rocks, but sediment contributions from this process are generally
minimal on annual time cycles. Slides occur on failure planes that are either straight (translational) or curved
(slumps), and are the most common form of landslide (Sidle et al. 1985; Selby 1993). Translational slides are more common than slumps. Translational slides usually occur due
to reduced soil strength with saturation and they form in long, shallow (1 to 4 m) linear
features. Gully Erosion Processes Gullies are recently channelized drainage features that transmit ephemeral flow,
usually have steep sides and a head scarp (leading upslope area of exposed soil and rock),
and are more than 30 cm wide and 60 cm deep (see Selby 1993; Toy et al. 2002). These
erosional features commonly form when a master rill deepens and widens its channel,
especially where changes in slope or vegetation patterns occur on unconsolidated materi-
als (fig. 10) (Neary et al. 2012). Gullies may also form where debris and mud flows exit
unstable drainage basins or where large subsurface drainage features collapse. The most
common cause of gully formation is a loss in surface protection associated with a change
in the overlying vegetation or soil disturbance. Gullies forming from rills often have no head scarp, increase in width and depth
downslope toward a master gully, and end in a deposition zone of coalescing fans at
the base of toe slopes. Gullies with head scarps maintain the scarp where soils are
cohesive, but upslope or upstream headcutting occurs if soils are weak (unconsolidated). Peak discharges from gullies typically far exceed the peak discharge of the previously
unchanneled valleys in which they occur. Erosion from gully processes may be severe
where high-intensity rainfall events occur over poorly vegetated surfaces with weakly
consolidated or unconsolidated sediments. Gully erosion most commonly occurs in
pulses, and sediment supply comes mostly from head scarp erosion and bank failures or
sidewall sloughing (Selby 1993). Figure 10—Gully erosion on an unprotected soil (photo: Lynn Betts,
USDA Natural Resources Conservation Service). Figure 10—Gully erosion on an unprotected soil (photo: Lynn Betts,
USDA Natural Resources Conservation Service). 18 Mass Movement Processes High-intensity rainfall saturates the soil profile, reducing the soil strength along
soil material boundaries of different permeability or density. The soil-bedrock interface
is a common translational failure plane where saturated soils are underlain by shallow
bedrock. Overburdening, slope steepening, and ground vibrations are also causes of
translational slides. The rate of movement for translational slides is commonly several
meters per day. Similar to translational slides, slumps form when overburdening under
wet conditions weakens the shear strength of the soil. Slumps, however, are rotational or
curved failure planes, and may initiate long after water input has ceased. They usually oc-
cur in cohesive soils derived from soft rocks like shales, mudstones, and overconsolidated
clays. Downslope progression usually occurs at a rate of a few millimeters per year to
several meters per day. However, slumps that occur in soils with high water content may
generate more substantial downslope transfer of sediment and often result in an earthflow
event at the toe of the failure. 19 USDA Forest Service RMRS-GTR-351. 2016. Flows are gravity-induced mass movements that are intermediate between sliding
and water flows. They occur as debris, earth, or mud flow resulting from wet or dry lique-
faction of coarse debris, fine-grained soil, or clay soil, respectively (Selby 1993). These
fast-moving events (a few meters per day to tens of meters per second) are promoted by
steep slopes, high soil water content, remolding of soil material following other mass
movement events, presence of soil with low liquid limits (easily liquefied soil), ground
vibrations, and the occurrence of soils with open fabrics that facilitate soil movement. Flows typically occur with abundant wetness, but may also occur as dry rock avalanches
or rock fragment flows. They often occur subsequent to an upslope slide that contributes
substantial debris and sediment downslope at a high velocity.lil Debris flows (fig. 11) are flow events consisting of large quantities of debris and
runoff. Debris flows that contain organic matter in large forms, such as trees and logs,
are referred to as “debris torrents.” Here, we consider the discussion that follows to be
similar for debris torrents and flows, and thus refer to both simply as “debris flows.” The
high bulk density and viscosity of debris flows facilitate flow shear strength substantial
enough to transport large boulders and debris. USDA Forest Service RMRS-GTR-351. 2016. Mass Movement Processes Debris flows progress downslope with a
boulder- and debris-laden front followed by slurry and hyperconcentrated flow of coarse-
and fine-soil materials. Flowpaths can extend for many kilometers and commonly cease
in low-gradient alluvial fans with boulder levees (see Selby 1993). Debris flows may occur in 20 to 100 waves during a single event, with thinner fluid
pulses occurring between waves. These events can occur suddenly and pose significant
risk to life, property, and resources due to the high-impact force (5 to 100 times that of
floods) and velocity and the sediment/debris loading possible. For example, an extreme
rainstorm event in central California in 1982 generated more than 200 mm of rainfall
over 32 hours, resulting in more than 18,000 slides (see Ellen and Wiezorek 1988). l Debris flows may occur in 20 to 100 waves during a single event, with thinner fluid
pulses occurring between waves. These events can occur suddenly and pose significant
risk to life, property, and resources due to the high-impact force (5 to 100 times that of
floods) and velocity and the sediment/debris loading possible. For example, an extreme
rainstorm event in central California in 1982 generated more than 200 mm of rainfall
over 32 hours, resulting in more than 18,000 slides (see Ellen and Wiezorek 1988). Debris flows from the slides damaged at least 100 homes and killed 14 people, of whom
10 were buried in their homes. The total cost of the damage was estimated at more than
$280 million. Selby (1993) provides a brief review of this and other catastrophic debris Debris flows from the slides damaged at least 100 homes and killed 14 people, of whom
10 were buried in their homes. The total cost of the damage was estimated at more than
$280 million. Selby (1993) provides a brief review of this and other catastrophic debris Figure 11—Debris flow following a thunderstorm event occurring on
burned forest land (photo: USDA Forest Service). Figure 11—Debris flow following a thunderstorm event occurring on
burned forest land (photo: USDA Forest Service). 20 USDA Forest Service RMRS-GTR-351. 2016. USDA Forest Service RMRS-GTR-351. 2016. flow events of similar magnitude. Cannon et al. Mass Movement Processes (1998, 2001a,b, 2008) explain runoff-
and infiltration-driven triggers for debris flow initiation on burned landscapes and provide
additional examples of the potential impact of debris flows on values-at-risk.llli Earthflows and mudflows are slow- to rapid-moving viscous flows of fine sand,
clay, and silt particles mixed with water. As with debris flows, they often result from
upslope slides, particularly slumps. Slumps of wet soil bulging forward often take the
form of bulbous toes or tongue-like rolls of earth and mud. The downslope movement is
dependent on the weight of the material, slope steepness, shear strength of the material,
and pore water pressures. The rate of movement for earthflows usually ranges from less
than several meters a day to hundreds of meters per hour. Earthflows may affect areas
from several square meters to hectares, but these impacts may require several years and
are commonly of minor degree. Mudflows are highly mobile (with velocities of several
meters per second) and pose greater threat than earthflows to life, property, and resources. For example, the volcanic eruption of Mt. Saint Helens in Washington State in 1980
generated lahar flows (volcanic mudflows) 120 km down the Toutle River and contributed
more than 50 million m3 of sediment into the Lower Columbia River (Pierson 1986). The
Mt. Saint Helens event illustrates changes in flow behavior and deposition from mass
movement initiation to streamflow delivery (see Scott 1988). USDA Forest Service RMRS-GTR-351. 2016. Spatial and Temporal Variations of Processes Sediment detachment and transport may vary dramatically in space and time
(Thornes 1980; Toy et al. 2002). The spatial scaling of sediment yield is a function of the
arrangement and connectivity of surface susceptibility, driving forces (such as rainfall
distribution), and erosion processes occurring within the area of interest (Pierson et al. 2011; Williams et al. 2016a). Rainsplash and sheetflow processes (fig. 9A and 9B) domi-
nate at the small-plot scale (1 to 2 m2) and erosion highly depends on the susceptibility
of the soil surface to raindrop impact. Over large-plot scales (tens of square meters),
sediment yield is more influenced by the fluid-flow entrainment of raindrop- and flow-
detached sediment in sheetflow and concentrated flow (fig. 9C) and the connectivity of
these processes (Williams et al. 2016a). At the hillslope scale, the landscape often has a heterogeneous arrangement of
susceptible conditions and driving forces, resulting in a poorly connected spatial organi-
zation of processes and erosion (Pierson et al. 1994a,b; Abrahams et al. 1995; Bergkamp
1998; Puigdefábregas et al. 1998; Reid et al. 1999; Wilcox et al. 2003a; Puigdefábregas
2005; Williams et al. 2014b). For example, small perturbations on a hillslope may create
small patches of exposed bare soil highly susceptible to rainsplash erosion. High-intensity
rainfall on these patches may generate substantial erosion from raindrop impact, but the
protected surfaces between the perturbations create a disconnect at the larger hillslope
scale, resulting in minor sediment yield. The same landscape with uniform disturbance may undergo substantially more
soil erosion from a similar storm due to an increase in the spatial connectivity of surface
susceptibility, rainsplash detachment, and formation of well-organized sheetflow or
concentrated flow (Williams et al. 2016a). At watershed scales, the distribution of rainfall
or other driving forces is often highly variable, as is erodibility, facilitating even greater 21 disconnect than observed at hillslope scales. In-channel processes also play a role in sedi-
ment delivery over landscape scales. In general, sediment yield per unit area decreases
with increased spatial area due to the inherent loss in connectivity of processes, suscep-
tibility, and driving forces (Pierson et al. 1994a,b; Wilcox et al. 2003a). The collective
arrangement creates a spatially dynamic environment of sediment detachment, transport,
and deposition that is dependent on the respective magnitude and extent of each of these
components’ influence. USDA Forest Service RMRS-GTR-351. 2016. Section 3: Climate-Vegetation-Soil-Hydrology Interactions Precipitation processing and the resultant runoff and erosion processes at point to
landscape scales are a function of climate, vegetation, and soil interactions (Seyfried and
Wilcox 1995; Puigedfàbregas et al. 1999; Pierson et al. 2002a; Robichaud et al. 2010;
Pierson et al. 2011; Williams et al. 2014b). Climate acts as a driving force for runoff gen-
eration and erosion, whereas vegetation and soil properties act as resistive forces. Climate
influences the timing, quantity, type, and intensity (erosive energy) of precipitation falling
at the land-atmosphere interface. Air temperatures and evaporation rates affect available
water and plant establishment and productivity. Canopy interception controls the amount
and erosive energy of precipitation passing through to the ground surface, and influences
the immediate canopy area, climate, and water use. Ground cover, organic matter,
and soil fauna recruitment are a function of plant productivity and climate, and their
interactions regulate soil stability and retention. Ground cover reduces the erosive energy
of raindrops and surface flow, increases aggregate stability, traps and stores sediment,
mediates infiltration rates, and modifies the soil climate and soil fauna activity. Plant root
tissues and soil fauna activity influence the soil bulk density and infiltration capacity. Organic matter and byproducts of fauna activity may inhibit or facilitate soil wettability. Soil development is strongly related to parent materials, erosion processes, climate, and
the vegetative community. Soil porosity and structure affect infiltration, percolation, and
throughflow, all of which influence soil water storage and antecedent moisture conditions. Soil water storage and climate regulate plant productivity and vegetation recruitment. Collectively, these relationships control the spatial and temporal arrangement of runoff
and erosion processes. In this section, we discuss these relationships in detail with respect
to runoff and erosion in preparation for assessing hydrologic impacts of fire. Spatial and Temporal Variations of Processes Temporal variability comes from event oscillations, climate variations, and changes
in land use or disturbance regimes (see Thornes 1980). Short-term variations in erosion
usually refer to changes occurring during a single storm event or over seasonal to annual
time scales. During a storm, the most readily available sediment is eroded first, usually
resulting in an initial pulse of sediment much greater than sediment delivery near the con-
clusion of the storm (Pierson et al. 2008a; Al-Hamdan et al. 2012b). The availability of
sediment is a complex dynamic of sediment detachment and transport that, as previously
mentioned, is highly variable in space. Oscillations in the magnitude and spatial arrange-
ment of driving forces during individual events may create spatially and temporally
variable sediment sources that further influence the temporal response.l Over seasonal and annual timescales, erosion may be influenced by changes in
vegetation, soil conditions, animal activity, or climatic factors. Semiarid areas, for ex-
ample, commonly have low-intensity, long-duration rainfall in winter and high-intensity,
short-duration rainfall in summer (see Branson et al. 1981). These different climate
regimes elicit different erosional responses. The seasonal responses are further influenced
by co-occurring changes in the vegetation cover and soil wetness that may increase or
decrease site erodibility. For example, denser canopy cover on semiarid rangelands dur-
ing summer months, as compared to winter months, offers greater surface protection from
high-intensity rainfall through increased interception. During winter months, low ground
cover and freeze-thaw soil processes may facilitate reduced infiltration and high erosion
rates during low-intensity rainfall (Blackburn and Wood 1990; Blackburn et al. 1990;
Seyfried and Flerchinger 1994; Wilcox 1994). Over timescales that cover multiple years or longer, erosion is more influenced by
climatic changes and land-use disturbances. Prolonged drought conditions may limit plant
growth and recruitment of surface-protecting litter. A series of wet years may stimulate
canopy and ground cover recruitment. Likewise, effects of land use, disturbance, or
postdisturbance rehabilitation may take many years or even decades to influence sediment
yield and may be linked with climatic influences (Allen and Breshears 1998). Finally, all
temporal responses are strongly linked to the time dependence of spatial links in erosion
processes or cumulative effects. 22 22 USDA Forest Service RMRS-GTR-351. 2016. Climate as a Driver of Hydrologic Response The type, intensity, duration, and timing of a precipitation event greatly influence
rangeland hydrologic response. Rainfall events in excess of soil infiltration capacity may
produce substantial runoff, whereas the same quantity of precipitation falling as snow
may generate little or no event runoff. Snow accumulation on the land surface stores
precipitation and delays runoff, allowing more time for evaporative losses and infiltration
(Dingman 2002; McNamara et al. 2005). For rainfall events, the intensity is an important
measure for predicting rangeland response (Branson et al. 1981). High-intensity storms
often greatly exceed the infiltration and storage capacity of the land surface and facilitate
rapid runoff generation and large quantities of streamflow. Runoff from low-intensity
storms is often minimal as long as the rainfall rate does not significantly exceed the
infiltration capacity of the soil (Wilcox et al. 2003a). The duration of a storm event
also influences runoff generation. High-intensity storms over long durations present the
greatest risk for elevated runoff and erosion, whereas low-intensity events over short
durations pose lower risks for runoff and soil loss. However, long-duration low-intensity
events may also generate substantial runoff, especially under saturated soil conditions
(Castillo et al. 2003; Wilcox et al. 2003a). In general, the risk for high runoff and erosion 23 USDA Forest Service RMRS-GTR-351. 2016. is greater the longer the infiltration and storage capacity of the land surface is exceeded. The response is amplified with increasing rainfall intensity. Seasonal variation in runoff behavior from rangelands is related to the prevailing
precipitation regime. In the mountainous north and central United States, high-elevation
range sites are mostly snow dominated while low- to mid-elevation sites are rainfall
dominated with transient winter snowpacks (Branson et al. 1981; Seyfried and Wilcox
1995). The greatest runoff rates from snow-dominated uplands occur during the spring
snowmelt/runoff period or during winter rain-on-snow events (fig. 12A, table 1) (Branson
et al. 1981; Wilcox et al. 1991; Seyfried and Flerchinger 1994; Marks et al. 2001; Pierson
et al. 2001b). The spring runoff season at these sites begins when air temperatures warm
above freezing and the precipitation trend shifts from snowfall to rainfall. Large quanti-
ties of available water and saturated soil conditions amplify runoff (McNamara et al. USDA Forest Service RMRS-GTR-351. 2016. Climate as a Driver of Hydrologic Response Figure 12—Mean monthly streamflow, precipitation, and air temperatures for (A) a semiarid, snow-dominated
drainage in the Reynolds Creek Experimental Watershed, Idaho (USDA Agricultural Research Service
2011a) and (B) an arid, rainfall-dominated drainage in the Walnut Gulch Experimental Watershed, Arizona
(USDA Agricultural Research Service 2011b) (photos: Agricultural Research Service). Figure 12—Mean monthly streamflow, precipitation, and air temperatures for (A) a semiarid, snow-dominated
drainage in the Reynolds Creek Experimental Watershed, Idaho (USDA Agricultural Research Service
2011a) and (B) an arid, rainfall-dominated drainage in the Walnut Gulch Experimental Watershed, Arizona
(USDA Agricultural Research Service 2011b) (photos: Agricultural Research Service). 24 USDA Forest Service RMRS-GTR-351. 2016. Table 1—Ten largest peak flows of record measured on the snowfall-dominated Reynolds Creek
(Outlet Weir, 1963 to 1996), Reynolds Creek Experimental Watershed , Idaho, and rainfall-
dominated Walnut Gulch (Flume 1, 1953 to 2010), Walnut Gulch Experimental Watershed, Arizona. Reynolds Creek, Idaho
Walnut Gulch, Arizona
(Snowfall dominated, 23,372 ha)
(Rainfall dominated, 14,932 ha)
Peak flowa
Peak flowb
Date
(m3 s-1)
Date
(m3 s-1)
23 Dec 1964
109.03
17 Aug 1957
318.61
31 Jan 1963
66.02
02 Aug 1957
183.54
15 Feb 1982
58.97
12 Aug 1972
171.52
11 Jan 1979
47.09
11 Aug 2000
147.50
11 Jun 1977
31.70
10 Sep 1967
132.54
28 Jan 1965
31.53
22 Jul 1964
121.48
21 Jan 1969
25.48
22 Aug 1961
111.26
11 Apr 1982
24.40
10 Aug 1971
102.38
27 Jan 1970
20.64
16 Aug 1958
95.93
02 Mar 1972
19.19
09 Sep 1964
89.58
a Streamflow summaries provided by the USDA Agricultural Research Service, Northwest Watershed Research
Center (USDA Agricultural Research Service 2011a). b Streamflow summaries provided by the USDA Agricultural Research Service, Southwest Watershed Research
Center (USDA Agricultural Research Service 2011b). a Streamflow summaries provided by the USDA Agricultural Research Service, Northwest Watershed Research
Center (USDA Agricultural Research Service 2011a). b Streamflow summaries provided by the USDA Agricultural Research Service, Southwest Watershed Research
Center (USDA Agricultural Research Service 2011b). 2005; Seyfried et al. 2009). Snowmelt runoff is generated mainly from subsurface return
flow in or near stream channels rather than sheetflow or concentrated flow (Flerchinger et
al. 1992; McNamara et al. 2005; Seyfried et al. 2009; Williams et al. 2009). 2005; Seyfried et al. 2009). Snowmelt runoff is generated mainly from subsurface return
flow in or near stream channels rather than sheetflow or concentrated flow (Flerchinger et
al. 1992; McNamara et al. 2005; Seyfried et al. Climate as a Driver of Hydrologic Response 2009; Williams et al. 2009). Runoff at low-to-mid elevations occurs primarily as overland flow due to rainfall
on shallow snowpacks (less than 30 cm snow) and frozen soils (Johnson and Smith 1978;
Wilcox et al. 1989; Blackburn and Wood 1990; Blackburn et al. 1990; Wilcox et al. 1991;
Seyfried and Flerchinger 1994; Seyfried and Wilcox 1995; Marks et al. 2001; Pierson
et al. 2001b). Runoff during summer months usually results from short-duration, high-
intensity rainfall in excess of infiltration (Branson et al. 1981; Wilcox et al. 1991; Pierson
et al. 2001b). For snow-dominated sites and valley locations, infiltration-excess runoff
from high-intensity storms is often limited to small areas resulting from isolated rainfall
patterns and heterogeneous soils and canopy/ground cover (Blackburn 1975; Blackburn
et al. 1992; Pierson et al. 1994a,b). In the desert southwestern United States, some mountainous locations present
intermediate precipitation-runoff patterns, in contrast with the purely snow-dominated or
rainfall-dominated northwest and central U.S. uplands (Wilcox et al. 2003a). Such sites
exhibit both winter snow-dominated and summer rainfall-dominated precipitation-runoff
regimes, but the largest runoff events are usually related to intense monsoon summer
thunderstorms (Wilcox 1994; Breshears et al. 1998; Wilcox et al. 2003a). Valley range-
lands in the desert Southwest also experience the greatest catchment runoff (90 percent
of annual) during the summer monsoon season (fig. 12B, table 1; Osborn and Lane 1969;
Branson et al. 1981; Osborn and Renard 1988; Wilcox et al. 2003a; Stone et al. 2008). Monsoonal storms occur as variable high-intensity (up to 250 mm h-1), short-duration
(5 to 30 min), and spatially limited (10 to 100 km2) events (Osborn 1964; Branson
et al. 1981; Osborn 1983a,b; Osborn and Renard 1988; Renard 1988; Renard et al. 1993; Goodrich et al. 2008). The more intense (more than 100 mm h-1) storms produce 25 USDA Forest Service RMRS-GTR-351. 2016. significant point and hillslope-scale runoff, yielding watershed-scale runoff equivalent to
several years of normal runoff (Branson et al. 1981; Renard et al. 1993). Sediment generation from rangelands is often related to the amount of runoff. For
snow-dominated uplands, peak erosion occurs during snowmelt runoff (Branson et al. 1981; Pierson et al. 2001b). Streamflow sediment concentrations during snowmelt runoff
are most influenced by streambank sloughing and streambed-sediment entrainment
processes, rather than rainsplash and sheetflow. Low- to mid-elevation mountainous sites
may experience the greatest erosion rates from low-intensity, long-duration rain events
during freeze-thaw periods (Seyfried and Flerchinger 1994). Climate as a Driver of Hydrologic Response Freeze-thaw processes
increase surface erodibility, and, combined with lower vegetation and ground cover
in winter months, facilitate increased surface erosion from rainsplash and sheetflow
(Blackburn and Wood 1990; Blackburn et al. 1990; Seyfried and Flerchinger 1994;
Wilcox 1994). Summer convective, high-intensity events at snow-dominated sites may generate
substantial point and hillslope erosion from rainsplash, sheetflow, and concentrated flow. Sediment eroded at the point and hillslope scales during these events usually remains
onsite, stored locally or in ephemeral stream channels, as runoff is commonly too low for
offsite transport (Slaughter and Pierson 2000). Where summer runoff occurs, sediment
concentration per unit of runoff is usually several magnitudes greater than during the
spring runoff period (Johnson and Smith 1978). As with runoff, sediment yield in the
desert Southwest peaks during the summer monsoon season (Branson et al. 1981; Wilcox
et al. 1996; Reid et al. 1999; Wilcox et al. 2003a; Nearing et al. 2007). High-intensity
rainfall during this period magnifies rainsplash, sheetflow, concentrated flow, and flash
flood erosion, generating most of the annual surface erosion from southwestern U.S. rangelands (Branson et al. 1981; Renard et al. 1993). Interception Effects on Water Availability Canopy and ground cover interception at the land-atmosphere interface primarily in-
fluence rangeland hydrology by limiting the amount of water available for infiltration and
runoff (fig. 13). For low-intensity, short-duration rainfall events, most of the precipitation
is captured by plant canopies, litter, and other ground cover, and results in evaporative
losses (Branson et al. 1981; Owens et al. 2006; Dunkerley 2008). High-intensity rainfall
events usually exceed cover storage capacities, resulting in some precipitation routing by
throughflow and stemflow processes in addition to the evaporative losses (Thurow et al. 1987; Návar and Bryan 1990; Martinez-Meza and Whitford 1996; Whitford et al. 1997;
Wainwright et al. 1999; Dunkerley 2000; Abrahams et al. 2003; Bhark and Small 2003;
Carlyle-Moses 2004; Owens et al. 2006; Dunkerley 2008). The percentage of event gross
rainfall captured by cover elements generally decreases as rainfall intensity increases
(Branson et al. 1981; Carlyle-Moses 2004; Owens et al. 2006). Cumulative interception
losses over multistorm and annual time periods vary with the frequency and magnitude of
precipitation events and meteorological conditions (Dunkerley 2008). USDA Forest Service RMRS-GTR-351. 2016. 26 Figure 13—Ponding and interception of artificial rainfall applied on (A) a sagebrush site invaded by western
juniper and (B) the same site 10 years following juniper removal (photos: USDA Agricultural Research
Service, Northwest Watershed Research Center, adapted from Pierson et al. 2007a). Figure 13—Ponding and interception of artificial rainfall applied on (A) a sagebrush site invaded by western
juniper and (B) the same site 10 years following juniper removal (photos: USDA Agricultural Research
Service, Northwest Watershed Research Center, adapted from Pierson et al. 2007a). Interpreting estimates of rainfall interception loss, throughfall, and stemflow from
literature is confounded by the variability in measurement approaches, reported units,
and spatial and temporal experimental scales (Dunkerley and Booth 1999; Dunkerley
2000; Abrahams et al. 2003; Llorens and Domingo 2007; Dunkerley 2008). Estimates
have been reported from event, seasonal, and annual timescales over varying rainfall
intensities, storm patterns, and cumulative precipitation (Branson et al. 1981; Dunkerley
and Booth 1999; Dunkerley 2000; Carlyle-Moses 2004; Llorens and Domingo 2007;
Dunkerley 2008). Results are commonly expressed as a depth of water or percentage
of gross precipitation falling on individual plants or over an entire plant community. Extrapolation of these results to other like plants and communities is tenuous given
variability in vegetation and climate characteristics from one rangeland site to another
(Dunkerley and Booth 1999; Dunkerley 2000, 2008). Interception Effects on Water Availability Shrub- and woodland-community rainfall interception on the annual scale ranges
between 5 and 25 percent (Rowe 1948; Hamilton and Rowe 1949; Pressland 1973; West
and Gifford 1976; Tromble 1983; Thurow et al. 1987; Tromble 1988; Dunkerley and
Booth 1999; Carlyle-Moses 2004). Fewer data are available for herbaceous vegetation
and litter interception. Clark (1940) measured rainfall interception by native prairie
grasses in Nebraska, USA, at levels of 29 to more than 80 percent under low-intensity
artificial rainfall. Thurow et al. (1987) summarized several studies that found grassland
interception of gross annual rainfall ranges from 13 to 56 percent. Thurow et al. (1987)
estimated that interception of gross annual rainfall at two Texas grassland sites with 56
percent (shortgrass) and 62 percent (midgrass) cover was 11 percent and 18 percent,
respectively. Dunkerley and Booth (1999) reported a 32 percent interception of gross
annual rainfall by grass in Australia. Branson et al. (1981) provides a summary of literature on litter interception with
estimates of 2 to 17 percent of gross annual rainfall. These estimates are quite variable
in part because of the methods used to identify when the litter layer stops and the soil
layer begins. For example, Owens et al. (2006) estimated that 5 percent of gross annual
rainfall on Ashe juniper (Juniperus ashei Buchholz) trees at a Texas woodland site was
intercepted by the coarse litter beneath trees, whereas Thurow et al. (1987) determined
that litter (all dead plant material above the mineral soil) intercepted 20 percent of annual
rainfall in a live oak (Quercus virginiana Mill.) motte of the Edwards Plateau, Texas. Routing of intercepted water as stemflow has been estimated at 5 to 17 percent of gross
rainfall for individual rangeland conifers (Thurow and Hester 1997; Owens et al. 2006;
Taucer et al. 2008) and at 3 to 10 percent of gross rainfall for individual shrubs and shrub
communities (Thurow et al. 1987; Návar and Bryan 1990; Martínez-Meza and Whitford
1996; Wainwright et al. 1999; Abrahams et al. 2003; Bhark and Small 2003; Carlyle-
Moses 2004). Canopy interception of snowfall is generally considered of minor hydrologic impor-
tance because most intercepted snow reaches the ground as meltwater or is shed as large
snow masses (Dingman 2002; Storck et al. 2002). General interception estimates suggest
trees can store about 3.8 mm of water as snow (Bonan 2002). Literature on snowfall
canopy interception specific to rangeland plants is extremely limited. Interception Effects on Water Availability General estimates suggest tree,
shrub, and grass foliage can store about 1.3 mm of water (Bonan 2002). Branson et al. (1981) and others (Rowe 1948; Hamilton and Rowe 1949; Hull and
Klomp 1974; West and Gifford 1976; Tromble 1983; Thurow et al. 1987; Tromble 1988;
Návar and Bryan 1990; Martinez-Meza and Whitford 1996; Wood et al. 1998; Dunkerley
and Booth 1999; Dunkerley 2000; Abrahams et al. 2003; Wilcox et al. 2003b; Carlyle-
Moses 2004; Owens et al. 2006; Llorens and Domingo 2007; Dunkerley 2008; Taucer et
al. 2008) provide estimates of event and annual rainfall interception and stemflow for a
variety of rangeland plants and communities. Here, we summarize these data to provide
the reader an idea of the general magnitude of precipitation that returns to the atmosphere
via interception loss. We summarize ranges in the percentage of gross rainfall intercepted
at the event and longer term (seasonal and annual) timescales by individual cover type
and by plant community (see table 2). Gross rainfall interception by rangeland individual
shrubs (Rowe 1948; Hamilton and Rowe 1949; West and Gifford 1976; Branson et al. 1981; Tromble 1983) and conifer trees (Skau 1964; Slaughter 1997; Owens et al. 2006;
Taucer et al. 2008) averages from 50 to 60 percent for low-intensity storms to 5 to 35
percent for high-intensity or large events. Gross rainfall interception by individual shrubs
over multistorm to annual timescales ranges from 5 to 46 percent (Hull 1972; Hull and
Klomp 1974; Thurow et al. 1987; Tromble 1988; Návar and Bryan 1990; Martinez-Meza 27 USDA Forest Service RMRS-GTR-351. 2016. Table 2—Event and annual interception rates reported in literature for various individual rangeland
plant and community types (see Branson et al. 1981). and Whitford 1996; Domingo et al. 1998; Serrato and Diaz 1998), with most reported
values around 5 to 15 percent. plant and community types (see Branson et al. 1981). Event interception
Annual interception
Cover type
as % of gross rainfall
as % of gross rainfall
Individual conifer or shrub
50–60 for Low-Intensity
5–50,
5–35 for High Intensity
5–15 more common
Litter
2–20
2–20
Shrub or woodland community
5–50
5–25
Herbaceous community
15–80
10–55 and Whitford 1996; Domingo et al. 1998; Serrato and Diaz 1998), with most reported
values around 5 to 15 percent. and Whitford 1996; Domingo et al. 1998; Serrato and Diaz 1998), with most reported
values around 5 to 15 percent. Interception Effects on Water Availability The most com-
monly cited references are Hull (1972) and Hull and Klomp (1974). They found dense
shrub cover (2.2 plants per m2) intercepted 37 percent of snowfall at an Idaho rangeland
site. 28 USDA Forest Service RMRS-GTR-351. 2016. Reports of snow interception measurements of conifer species come from
woodland- and forest-dominated sites. Breshears et al. (1997b) reported that snow ac-
cumulation during each of three winter seasons was much greater in areas between tree
canopies than underneath canopies at a twoneedle pinyon/oneseed juniper (Pinus edulis
Englem./J. monosperma (Engelm.) Sarg.) woodland site in New Mexico. Snow water
equivalent in the first year of the 3-year study was about 80 percent greater in openings
between trees than in areas underneath canopies. Schmidt and Gluns (1991) reported
45 to 50 percent canopy interception of snowfall for three conifer species in a forested
setting when snow water equivalent was 10 mm and snow specific gravity was 0.06. Interception decreased to 10 percent for an equivalent storm with snow specific gravity of
0.13 (Schmidt and Gluns 1991). In another forested study, Storck et al. (2002) measured
approximately 60 percent canopy interception of snowfall by four different conifer spe-
cies, with minimal differences between species. Whole plant interception of windblown snowfall by rangeland vegetation is para-
mount in retaining snow against wind scour (Seyfried and Flerchinger 1994; Pomeroy
and Gray 1995; Seyfried and Wilcox 1995; Flerchinger et al. 1998; Liston and Sturm
1998; Flerchinger and Cooley 2000; Marks and Winstral 2001; Marks et al. 2001; Sturm
et al. 2001; Liston et al. 2002; Marks et al. 2002; Winstral and Marks 2002). Wind and
topography interact to redistribute fallen snow on undulating terrain, while vegetation
reduces wind velocities and facilitates deposition (Marks et al. 2001; Marks and Winstral
2001; Marks et al. 2002; Winstral and Marks 2002). Deeper snow accumulations provide
greater insulation for surface soils and plant productivity and prolong snow-covered
periods (Sturm et al. 2001; Liston et al. 2002). The vegetation snow-holding capacity is a function of the vegetation height, density
of plants, and snowpack conditions (Pomeroy and Gray 1995; Liston and Sturm 1998;
Sturm et al. 2001). Overall, accumulation of windblown snow is maximized at the height
of the canopy. Any deposition in excess of canopy height is readily windblown between
events, potentially transported offsite, or lost to wind-driven sublimation (Sturm et al. 2001; Liston et al. 2002). Interception Effects on Water Availability Hutchinson (1965) found that a shrub stand 50 cm in height
stored 25 mm more water than an adjacent area void of shrubs. Flerchinger et al. (1998)
reported that snow depth at a wind-driven rangeland site in Reynolds Creek Experimental
Watershed, Idaho, typically varied by plant community from less than 60 cm in low
sagebrush/grass (Artemisia arbuscula/Poa secunda J. Presl) to 100 cm in mountain big
sagebrush/snowberry (A. tridentata Nutt. ssp. vaseyana (Rydb.) Beetle/Symphoricarpos
spp.) and 100 to 800 cm in an aspen/willow (Populus tremuloides/Salix spp.) stand. Streamflow over a 10-year period at the site highly depended on vegetation retention of
windblown snow (Flerchinger and Cooley 2000). Numerous other studies have reported
similar results from water-limited, snow-dominated rangelands, indicating the retention
of windblown snow is a dominant influence on the timing and quantity of water available
for infiltration, soil storage, plant use, and streamflow generation (Seyfried and Wilcox
1995; Flerchinger et al. 1998; Luce et al. 1998; Flerchinger and Cooley 2000; Marks et
al. 2001; Winstral and Marks 2002; Seyfried et al. 2009; Williams et al. 2009). The vegetation snow-holding capacity is a function of the vegetation height, density
of plants, and snowpack conditions (Pomeroy and Gray 1995; Liston and Sturm 1998;
Sturm et al. 2001). Overall, accumulation of windblown snow is maximized at the height
of the canopy. Any deposition in excess of canopy height is readily windblown between
events, potentially transported offsite, or lost to wind-driven sublimation (Sturm et al. 2001; Liston et al. 2002). Hutchinson (1965) found that a shrub stand 50 cm in height
stored 25 mm more water than an adjacent area void of shrubs. Flerchinger et al. (1998)
reported that snow depth at a wind-driven rangeland site in Reynolds Creek Experimental
Watershed, Idaho, typically varied by plant community from less than 60 cm in low
sagebrush/grass (Artemisia arbuscula/Poa secunda J. Presl) to 100 cm in mountain big
sagebrush/snowberry (A. tridentata Nutt. ssp. vaseyana (Rydb.) Beetle/Symphoricarpos
spp.) and 100 to 800 cm in an aspen/willow (Populus tremuloides/Salix spp.) stand. l Streamflow over a 10-year period at the site highly depended on vegetation retention of
windblown snow (Flerchinger and Cooley 2000). Numerous other studies have reported
similar results from water-limited, snow-dominated rangelands, indicating the retention
of windblown snow is a dominant influence on the timing and quantity of water available
for infiltration, soil storage, plant use, and streamflow generation (Seyfried and Wilcox
1995; Flerchinger et al. Interception Effects on Water Availability 1998; Luce et al. 1998; Flerchinger and Cooley 2000; Marks et
al. 2001; Winstral and Marks 2002; Seyfried et al. 2009; Williams et al. 2009). USDA Forest Service RMRS-GTR-351. 2016. 29 Canopy Influence on Soil Microclimate, Evapotranspiration, and
Antecedent Soil Water In addition to intercepting water, vegetation and litter may further moderate the soil
microclimate and water availability by shading solar radiation and insulating surface soils
(Belsky et al. 1989; Pierson and Wight 1991; Joffre and Rambal 1993; Breshears et al. 1997b, 1998; Domingo et al. 2000; Lebron et al. 2007). The near-surface microclimate
influences soil evaporation and affects soil moisture regimes (Branson et al. 1981;
Breshears et al. 1998; Hillel 1998). Pierson and Wight (1991) reported interspace loca-
tions (areas between shrub canopies, also called intercanopy) in a sagebrush community
(A. tridentata Nutt.) had higher (by 5.2 °C) maximum and lower (by 1.5 °C) minimum
near-surface (0 to 10 cm depth) soil temperatures than coppice locations (areas under-
neath and immediately adjacent to canopies, also called subcanopy) during the spring
season. Small grass clumps and moss clumps within interspaces had little influence on
near-surface soil temperatures. Pierson and Wight (1991) inferred that shrub cover and
the associated litter mounds (coppices) insulated the soil surface from incoming solar
radiation during daylight hours and from sensible heat loss at night. Breshears et al. (1997b) found that interspaces between tree canopies of a twoneedle pinyon/oneseed
juniper woodland exhibited greater (40 to 50 percent more) near-surface solar radiation
than tree coppices, and that preferential shading on the northern side of tree coppices
significantly reduced near-surface solar radiation. Solar radiation differences between
coppice and interspaces were much greater during the summer solstice. Breshears et al. (1997b) also determined that snow water equivalent was greater
in interspace locations than under tree canopies and that the differential accumulation
resulted in temporal variability in the spatial arrangement of soil water. Soils underneath
tree canopies were wetter than interspace soils in early winter following complete melt
of coppice snowpacks and during the monsoon season immediately after intense runoff-
generating rainfall events. Wetter soil conditions on the edges of coppices compared to
interspaces following intense rainfall were assumed partially related to lateral redistribu-
tion of surface runoff from interspace locations to coppices as runon. Interception Effects on Water Availability Interspace soils
were wetter, by 3 percent volumetric moisture content, than coppice soils later in the
winter and in early spring during the interspace snowmelt period. The differential snow
accumulation and melt patterns, largely related to canopy snow interception, exerted a
greater influence on the spatial distribution of soil water (canopy versus interspace loca-
tions) than did the effects of preferential shading (Breshears et al. 1997b). The primary
effect of solar radiation on soil moisture patterns was observed within interspace patches;
north edges with more solar radiation were wetter than south edges during winter and
spring. Breshears et al. (1997b) attributed within-interspace differences to the canopy drip
effects (melting snow) on the warmer south side of trees (north edge of interspaces). Breshears et al. (1998), working at the same site as Breshears et al. (1997b), found
that maximum air temperature was as much as 10 °C greater on interspaces than tree
coppices during late spring through summer and that the associated differences in spatial
temperature produced differences in soil evaporation. Breshears et al. (1998) determined
that spatial differences in soil temperature influenced soil evaporation only when soils
were thawed and were amplified at lower soil water contents (as expressed by soil water
potential). Joffre and Rambal (1993) reported greater water storage under tree canopies 30 USDA Forest Service RMRS-GTR-351. 2016. (with grass) than in unshaded interspaces on three Mediterranean rangeland sites when
precipitation exceeded mean annual levels. Soil water storage was low in interspace
and coppice locations when precipitation was limited, resulting in the loss of about 60
percent and more than 95 percent of precipitation to evapotranspiration in interspaces and
coppices, respectively. Domingo et al. (2000) found that canopy shading in a semiarid
Mediterranean shrubland created a milder microclimate in shrub areas and that diurnal
temperature fluctuations were greater in interspaces. The above noted studies indicate that canopy effects on the near-surface microcli-
mate may influence soil water availability, but that the overall impact is highly dependent
on the quantity of precipitation and other spatial effects (interception, lateral redistribu-
tion). Furthermore, microclimate effects may have greater implications for biological
processes (seed germination and emergence, nutrient and microbial processes) and spatial
vegetation structure than for direct runoff generation (Pierson and Wight 1991; Ludwig
and Tongway 1995; Scholes and Archer 1997; Breshears et al. 1998; Breshears and
Barnes 1999; Reynolds et al. 1999; Belnap et al. 2005; Huxman et al. USDA Forest Service RMRS-GTR-351. 2016. Interception Effects on Water Availability A review of literature on evapotranspiration
rates by various rangeland plant communities is provided by Branson et al. (1981). The
percentage of evapotranspiration occurring as transpiration varies considerably (7 to 80
percent) between plant communities and depends on the amount and timing of precipita-
tion, available energy, plant growth form, and water availability throughout the rooting
depth of the soil profile (Reynolds et al. 2000; Wilcox et al. 2003b; Huxman et al. 2005;
Scott et al. 2006; Stannard and Weltz 2006; Moran et al. 2009). Evaporation from the soil
surface is dependent on surface soil moisture conditions and available energy, and gener-
ally increases with increasing exposure of bare ground (Breshears et al. 1998; Scott et al. 2006; Moran et al. 2009). Weltz and Blackburn 1995; Flerchinger et al. 1996, 1998; Yoder and Nowak 1999b;
Flerchinger and Cooley 2000; Zhang et al. 2001; Huxman et al. 2005; Wilcox et al. 2006;
Wilcox and Thurow 2006; Wilcox et al. 2008). Estimates from literature indicate actual
evapotranspiration from rangeland herbaceous plants, shrubs, trees, and bare soil ranges
from 60 to 100 percent, 60 to 130 percent, 35 to 120 percent, and 70 to 110 percent of
annual precipitation, respectively (fig. 14). A review of literature on evapotranspiration
rates by various rangeland plant communities is provided by Branson et al. (1981). The
percentage of evapotranspiration occurring as transpiration varies considerably (7 to 80
percent) between plant communities and depends on the amount and timing of precipita-
tion, available energy, plant growth form, and water availability throughout the rooting
depth of the soil profile (Reynolds et al. 2000; Wilcox et al. 2003b; Huxman et al. 2005;
Scott et al. 2006; Stannard and Weltz 2006; Moran et al. 2009). Evaporation from the soil
surface is dependent on surface soil moisture conditions and available energy, and gener-
ally increases with increasing exposure of bare ground (Breshears et al. 1998; Scott et al. 2006; Moran et al. 2009). Weltz and Blackburn 1995; Flerchinger et al. 1996, 1998; Yoder and Nowak 1999b; Interception Effects on Water Availability 2005; Ludwig et al. 2005; D’Odorico et al. 2007). The direct effects of evapotranspiration on rangeland runoff generation vary by
precipitation regime and are usually minor relative to their influence on annual and
seasonal water balances (Branson et al. 1981). Annual runoff from rangeland sites usually
represents 0 to 10 percent (but can be as high as 50 percent) of annual precipitation
depending on the type and structure of the plant community, meteorological patterns, and
soils/geology (Carlson et al. 1990; Wilcox et al. 1991; Joffre and Rambal 1993; Wilcox
1994; Weltz and Blackburn 1995; Carlson and Thurow 1996; Wilcox et al. 1996, 1997;
Flerchinger et al. 1998; Flerchinger and Cooley 2000; Wilcox et al. 2003b, 2006; Nearing
et al. 2007; Wilcox et al. 2008). Runoff generation from rainfall-dominated rangelands
primarily occurs as infiltration-excess overland flow and is minimally influenced by
evapotranspiration demands (Branson et al. 1981; Pierson et al. 2001b; Wilcox et al. 2003a; Stone et al. 2008). Exceptions occur following multistorm events or prolonged
low-intensity storms that wet up the near-surface environment, shifting the runoff process
to saturation excess (Wilcox 1994; Castillo et al. 2003). Saturation excess runoff is a function of available soil water storage and rainfall
intensity. Storage capacity is related to antecedent moisture conditions and soil structure
and depth. Under these conditions, evapotranspiration dictates the storage capacity or
degree of saturation in the near-surface environment, strongly influencing, along with
rainfall intensity, the timing and quantity of runoff. Saturation excess overland flow is
more common at snow-dominated sites, during and immediately after peak snowmelt,
and therefore, evaporation of water from the snowpack or saturated soils in these settings
plays an important role in reducing water availability for surface and subsurface flow
(Seyfried and Flerchinger 1994; Marks et al. 2001; Pierson et al. 2001b; Wilcox et al. 2003a; McNamara et al. 2005; Seyfried et al. 2009; Williams et al. 2009). Evapotranspiration demands at snow-dominated sites strongly influence, along
with seasonal water input, the seasonal duration of ephemeral streamflow (McNamara
et al. 2005; Williams et al. 2009). Annual actual and potential evapotranspiration,
inclusive of interception losses, make up more than 90 percent of annual precipita-
tion from rangeland sites, and are limited primarily by the amount of precipitation and
available soil water (Campbell and Harris 1977; Branson et al. 1981; Carlson et al. 1990; 31 USDA Forest Service RMRS-GTR-351. 2016. Weltz and Blackburn 1995; Flerchinger et al. Interception Effects on Water Availability 1996, 1998; Yoder and Nowak 1999b;
Flerchinger and Cooley 2000; Zhang et al. 2001; Huxman et al. 2005; Wilcox et al. 2006;
Wilcox and Thurow 2006; Wilcox et al. 2008). Estimates from literature indicate actual
evapotranspiration from rangeland herbaceous plants, shrubs, trees, and bare soil ranges
from 60 to 100 percent, 60 to 130 percent, 35 to 120 percent, and 70 to 110 percent of
annual precipitation, respectively (fig. 14). A review of literature on evapotranspiration
rates by various rangeland plant communities is provided by Branson et al. (1981). The
percentage of evapotranspiration occurring as transpiration varies considerably (7 to 80
percent) between plant communities and depends on the amount and timing of precipita-
tion, available energy, plant growth form, and water availability throughout the rooting
depth of the soil profile (Reynolds et al. 2000; Wilcox et al. 2003b; Huxman et al. 2005;
Scott et al. 2006; Stannard and Weltz 2006; Moran et al. 2009). Evaporation from the soil
surface is dependent on surface soil moisture conditions and available energy, and gener-
ally increases with increasing exposure of bare ground (Breshears et al. 1998; Scott et al. 2006; Moran et al. 2009). Figure 14—Estimated averages
and ranges (as maximum
and minimum) of annual
evapotranspiration as
percentage of annual
precipitation for various
rangeland plant types as
reported in literature (Carlson
et al. 1990; Joffre and Rambal
1993; Weltz and Blackburn
1995; Carlson and Thurow
1996; Flerchinger et al. 1998;
Yoder and Nowak 1999b;
Brandes and Wilcox 2000;
Flerchinger and Cooley 2000). Figure 14—Estimated averages
and ranges (as maximum
and minimum) of annual
evapotranspiration as
percentage of annual
precipitation for various
rangeland plant types as
reported in literature (Carlson
et al. 1990; Joffre and Rambal
1993; Weltz and Blackburn
1995; Carlson and Thurow
1996; Flerchinger et al. 1998;
Yoder and Nowak 1999b;
Brandes and Wilcox 2000;
Flerchinger and Cooley 2000). Weltz and Blackburn 1995; Flerchinger et al. 1996, 1998; Yoder and Nowak 1999b;
Flerchinger and Cooley 2000; Zhang et al. 2001; Huxman et al. 2005; Wilcox et al. 2006;
Wilcox and Thurow 2006; Wilcox et al. 2008). Estimates from literature indicate actual
evapotranspiration from rangeland herbaceous plants, shrubs, trees, and bare soil ranges
from 60 to 100 percent, 60 to 130 percent, 35 to 120 percent, and 70 to 110 percent of
annual precipitation, respectively (fig. 14). Cover Influences on Infiltration, Runoff, and Water Transfer
and Storage The heterogeneous vegetative and ground cover structure and soil characteristics
across rangeland communities exhibit a high degree of spatial organization and integra-
tion relative to water and soil resource recruitment (Tongway et al. 1989; Tongway and
Ludwig 1990; Pierson et al. 1994b; Dunkerley and Brown 1995; Ludwig et al. 1997;
Scholes and Archer 1997; Breshears and Barnes 1999; Puigdefábregas et al. 1999;
Reynolds et al. 1999; Belnap et al. 2005; Ludwig et al. 2005; Puigdefábregas 2005;
Rango et al. 2006; D’Odorico et al. 2007; Turnbull et al. 2012). The interaction of
vegetation, ground cover, soil properties, climate, and resultant hydrologic processes
on water-limited sites creates stable patches of water, nutrient, and soil accumulation
and retention. Shrub/tree coppices and herbaceous or litter-covered areas create surface
and subsurface conditions that favor infiltration and soil and nutrient retention, whereas
bare areas exhibit higher rates of runoff and soil loss (figs. 15 and 16; Blackburn 1975;
Abrahams et al. 1988; Schlesinger et al. 1990; Seyfried 1991; Blackburn et al. 1992; 32 USDA Forest Service RMRS-GTR-351. 2016. Pierson et al. 1994a,b; Wilcox 1994; Abrahams et al. 1995; Ludwig and Tongway 1995;
Wilcox and Breshears 1995; Parsons et al. 1996; Schlesinger et al. 1996; Wilcox et al. 1996; Whitford et al. 1997; Bergkamp 1998; Davenport et al. 1998; Reid et al. 1999;
Schlesinger et al. 1999, 2000; Dunkerley 2002; Wainwright et al. 2002; Bhark and Small
2003; Wilcox et al. 2003a,c; Pierson et al. 2010; Turnbull et al. 2010a,b; Pierson et al. 2011, 2013, 2014; Williams et al. 2014a). Canopy and ground cover influence the soil microclimate and the recruitment of
soil microbes and microfauna that aid nutrient recycling and further improve infiltration
and soil water storage (Pierson and Wight 1991; Blackburn et al. 1992; Breshears et al. 1997b, 1998; Imeson et al. 1998; Reynolds et al. 1999; Belnap et al. 2005; Ludwig et al. 2005). Heterogeneous vegetation patterns yield horizontally and vertically differential
water use and soil water storage (Walter 1971; Belsky et al. 1989; Belsky et al. 1993;
Joffre and Rambal 1993; Ryel et al. 1996; Breshears et al. 1997a,b, 1998; Breshears and
Barnes 1999; Reynolds et al. 1999; Ludwig et al. 2005). These surface and subsurface
interactions, in an undisturbed condition, result in an organized plant community that
facilitates a positive feedback of biological productivity and hydrologic processes to con-
serve water, soil, and nutrient resources (Ludwig and Tongway 1995; Ludwig et al. Cover Influences on Infiltration, Runoff, and Water Transfer
and Storage 1997;
Davenport et al. 1998; Cammeraat and Imeson 1999; Puigdefábregas et al. 1999; Reid
et al. 1999; Reynolds et al. 1999; Ludwig and Tongway 2000; Ludwig et al. 2000; Pyke
et al. 2002; Wilcox et al. 2003a; Ludwig et al. 2005; Puigdefábregas 2005; Pierson et al. 2010). The stability of the system is defined by its resistance to reduction of these capaci-
ties and by its resiliency to perturbations (Schlesinger et al. 1990; Ludwig and Tongway
1995; Ludwig et al. 1997; Ludwig and Tongway 2000; Pyke et al. 2002; Ludwig et al. 2005; Williams et al. 2014a). Vegetation and Cover Influences on Infiltration and Runoff Generation Higher infiltration rates on coppice mounds versus interspaces are attributed to
deeper surface soil horizons, greater organic matter accumulation and aggregate stability,
lower bulk density, macropores, canopy interception and stemflow, and surface retention
of throughflow and runon underneath and immediately adjacent to the canopy area. Litter
amassment and decomposition underneath shrub and tree canopies (fig. 15) and differ-
ential rainsplash contribute to soil, organic matter, and nutrient accumulation (Blackburn
1975; Blackburn et al. 1992; Parsons et al. 1992; Ludwig and Tongway 1995; Schlesinger
et al. 1996; Puigdefábregas et al. 1999; Reynolds et al. 1999; Schlesinger et al. 1999;
Belnap et al. 2005; Ludwig et al. 2005). Litter and organic matter promote aggregate
stability, macropore formation, and low bulk densities associated with higher infiltration
rates and retain surface water, prolonging time for infiltration (Meeuwig 1970; Blackburn
and Skau 1974; Tromble et al. 1974; Roundy et al. 1978; Wood et al. 1978; Wood and
Blackburn 1981; Beven and Germann 1982; Devaurs and Gifford 1984; Thurow et al. 1986; Johnson and Gordon 1988; Wilcox et al. 1988; Blackburn et al. 1990; Dunne et al. 1991; Seyfried 1991; Pierson et al. 1994a,b; Abrahams et al. 1995; Parsons et al. 1996;
Seyfried and Wilcox 1995; Cerdà 1998b; Wilcox et al. 2003a; Puigdefábregas 2005;
Pierson et al. 2010, 2013, 2014; Williams et al. 2014a). Soil fauna activity is enhanced
by the microclimate, moisture regimes, and nutrient availability underneath canopies. The associated biological activity further improves soil aggregation, macroporosity, and 33 USDA Forest Service RMRS-GTR-351. 2016. Figure 15—Photographs from a Utah juniper woodland site in Utah, showing (A) general direction for
overland flow over contiguous bare interspace, (B) interspace concentrated flowpath during artificial rainfall
simulation, (C) hydrologically stable shrub and (D) tree coppice microsites, (E) moderately stable vegetated
interspace microsite; and (F) hydrologically unstable bare interspace microsite. High-intensity artificial rainfall
simulations performed at the site by Pierson et al. (2010) produced 38, 46, 133, and 313 cumulative grams
of sediment per m2 area from 0.5 m2 plots on the shrub coppice (C), tree coppice (D), and vegetated (E)
and bare (F) interspaces, respectively (photos: USDA Agricultural Research Service, Northwest Watershed
Research Center). Vegetation and Cover Influences on Infiltration and Runoff Generation Figure 15—Photographs from a Utah juniper woodland site in Utah, showing (A) general direction for
overland flow over contiguous bare interspace, (B) interspace concentrated flowpath during artificial rainfall
simulation, (C) hydrologically stable shrub and (D) tree coppice microsites, (E) moderately stable vegetated
interspace microsite; and (F) hydrologically unstable bare interspace microsite. High-intensity artificial rainfall
simulations performed at the site by Pierson et al. (2010) produced 38, 46, 133, and 313 cumulative grams
of sediment per m2 area from 0.5 m2 plots on the shrub coppice (C), tree coppice (D), and vegetated (E)
and bare (F) interspaces, respectively (photos: USDA Agricultural Research Service, Northwest Watershed
Research Center). infiltration (Cammeraat and Imeson 1998; Imeson et al. 1998; Puigdefábregas et al. 1999;
Dunkerley 2002; Belnap et al. 2005; Ludwig et al. 2005). Stemflow concentrates water
input at plant bases, allowing rapid vertical recharge of the soil profile via preferential
flow along root channels (Thurow et al. 1987; Návar and Bryan 1990; Martinez-Meza
and Whitford 1996; Newman et al. 1997; Whitford et al. 1997; Devitt and Smith 2002;
Abrahams et al. 2003; Bhark and Small 2003; Carlyle-Moses 2004; Owens et al. 2006;
Lebron et al. 2007). USDA Forest Service RMRS-GTR-351. 2016. 34 Plant growth form also influences infiltration processes. Infiltration rates are
generally higher for bunchgrasses than sod-forming grasses (Wood and Blackburn 1981;
Knight et al. 1984; Thurow et al. 1986, 1988; Blackburn et al. 1992; Pierson et al. 2002a). Greater vegetative biomass and organic matter accumulation on bunchgrasses than
sodgrasses result in greater rainfall and runoff interception (Knight et al. 1984; Thurow
et al. 1986, 1988). Additionally, biomass and organic matter accumulations under bunch-
grasses most likely favor infiltration-increasing microbial activity (Blackburn et al. 1992). Infiltration under shrub canopies is usually greater than under grass canopies (Wood and
Blackburn 1981, Schlesinger et al. 1999), but the relationship may be reversed depending
on grass biomass (Wilcox et al. 1988). The overall greater infiltration in canopy patches
on shrublands and grasslands increases water availability beneath canopies, which in turn
stimulates biological activity, plant growth, and organic matter and nutrient recruitment. This creates a continuous positive feedback (Schlesinger et al. 1990; Belnap et al. 2005;
Puigdefábregas 2005; D’Odorico et al. 2007). Interspace areas on rangelands, particularly shrublands, are often associated with
surface and subsurface characteristics that inhibit infiltration and soil water storage, and
promote rapid ponding (fig. 13) and runoff initiation. USDA Forest Service RMRS-GTR-351. 2016. Vegetation and Cover Influences on Infiltration and Runoff Generation Interspaces occur with various
amounts of herbaceous cover, or exist as contiguous bare patches (fig. 15; Blackburn et
al. 1992; Pierson et al. 1994a,b; Abrahams et al. 1995; Seyfried and Wilcox 1995; Wilcox
and Breshears 1995; Parsons et al. 1996; Reid et al. 1999; Wilcox et al. 2003a). Well-
vegetated interspaces may exhibit similar surface characteristics as canopy areas to some
degree, but usually generate more surface runoff (table 3; Reid et al. 1999; Bhark and
Small 2003; Wilcox et al. 2003a). On more water-limited or degraded sites, interspaces
have low plant biomass and organic matter (fig. 15F) and thin surface soil accumulations
(Blackburn 1975; Abrahams and Parsons 1991a; Abrahams et al. 1995; Parsons et al. 1996; Wilcox et al. 1996; Pierson et al. 2010). These characteristics result in poor ag-
gregate stability and soil structure, and high bulk densities relative to coppices. They also
facilitate low infiltration rates (Blackburn and Skau 1974; Blackburn 1975; Roundy et
al. 1978; Wood et al. 1978; Thurow et al. 1986; Johnson and Gordon 1988; Wilcox et al. 1988; Blackburn and Wood 1990; Blackburn et al. 1990; Abrahams and Parsons 1991a;
Seyfried 1991; Blackburn et al. 1992; Pierson et al. 1994a,b; Abrahams et al. 1995;
Seyfried and Wilcox 1995; Wilcox and Breshears 1995; Abrahams et al. 1996; Parsons
et al. 1996; Wilcox et al. 1996; Reid et al. 1999; Wilcox et al. 2003a; Pierson et al. 2010,
2011, 2013, 2014; Williams et al. 2014a). In general, surface characteristics of interspace
areas are consistently different from coppices throughout the year, but the magnitude of
the differences and respective influences on infiltration exhibit some seasonality. The
spatial differences in vegetation cover and surface characteristics exert a greater influence
than do seasonal differences on infiltration and runoff generation from sparsely covered
shrublands, whereas seasonal differences in spatially arranged plant biomass might
be of greater influence on infiltration patterns on well-vegetated grass-dominated sites
(Blackburn et al. 1992).il Infiltration in interspace locations is strongly influenced by the expanse of bare
ground, rock cover, or vesicular crusts (Blackburn 1975; Wood et al. 1978; Johnson and
Gordon 1988; Abrahams and Parsons 1991a; Parsons et al. 1992; Abrahams and Parsons
1994; Pierson et al. 1994a; Parsons et al. 1996; Reid et al. 1999; Pierson et al. 2010). Exposure of bare ground to raindrop impact increases potential for surface sealing or 35 USDA Forest Service RMRS-GTR-351. 2016. Vegetation and Cover Influences on Infiltration and Runoff Generation Table 3—Site characteristics, runoff coefficients, and sediment yield reported for rainfall simulation and natural rainfall experiments in the western United States. Craddock and
Pearse (1938)
Cold desert
(Great Basin,
Idaho)
Bunchgrasses
Unb
20.2
30
-
46/60
Artificial
-
Dry
-
-
~35
-
1
1
Unb
20.2
30
-
92/30
Artificial
-
Dry
-
-
~35
-
1
1
Unb
20.2
40
-
46/60
Artificial
-
Dry
-
-
~35
-
<1
1
Unb
20.2
40
-
92/30
Artificial
-
Dry
-
-
~35
-
<1
1
Craddock and
Pearse (1938)
Cold desert
(Great Basin,
Idaho)
Grassland
(Bromus tectorum)
Unb
20.2
30
-
46/60
Artificial
-
Dry
-
-
~20
-
10
31
Unb
20.2
30
-
92/30
Artificial
-
Dry
-
-
~20
-
5
11
Unb
20.2
40
-
46/60
Artificial
-
Dry
-
-
~20
-
26
49
Unb
20.2
40
-
92/30
Artificial
-
Dry
-
-
~20
-
53
331
Craddock and
Pearse (1938)
Cold desert
(Great Basin,
Idaho)
Grassland
(Lupinus spp./Stipa
lettermanii )
Unb
20.2
30
-
46/60
Artificial
-
Dry
-
-
~25
-
44
274
Unb
20.2
30
-
92/30
Artificial
-
Dry
-
-
~25
-
57
511
Unb
20.2
40
-
46/60
Artificial
-
Dry
-
-
~25
-
44
253
Unb
20.2
40
-
92/30
Artificial
-
Dry
-
-
~25
-
55
468
Johnson and
Gordon (1988)
Cold desert
(Great Basin,
Idaho)
Shrub coppice
(Artemisia tridentata)
Unb
~0.9
6
-
127/30
Artificial
-
Dry
15
41
85
25
15
5
Interspace
Unb
~0.9
6
-
127/30
Artificial
-
Dry
29
4
71
13
48
39
Johnson and
Blackburn
(1989)d
Cold desert
(Great Basin,
Idaho)
Shrub steppe
(A. tridentata)
Unb
32.6
10
-
64/60
Artificial
-
Dry
16
39
84
-
2
5
Unb
32.6
6
-
64/60
Artificial
-
Dry
19
12
81
-
11
11
Unb
32.6
9
-
64/60
Artificial
-
Dry
45
16
55
-
17
15
Pierson et al. (1994b)
Cold desert
(Great Basin,
Idaho)
Shrub coppice
(A. tridentata)
Unb
~0.1
7
-
67/60
Artificial
-
4
0
100
100
-
3
1
Vegetated
interspace
Unb
~0.1
7
-
67/60
Artificial
-
4
4
-
96
-
23
23
Bare interspace
Unb
~0.1
7
-
67/60
Artificial
-
2
94
-
6
-
65
381
Reid et al. Vegetation and Cover Influences on Infiltration and Runoff Generation (1999)
Temperate
steppe mountains
(Pajarito Plateau,
New Mexico)
Tree coppice
(Pinus edulis
or Juniperus
monosperma)
Unb
1.0
6-12
-
Variablee
Natural
-
-
0
-
100
-
8e
39e
Vegetated
interspace
Unb
~2
6-12
-
Variablee
Natural
-
-
34
-
66
-
25e
298e
Bare interspace
Unb
~2
6-12
-
Variablee
Natural
-
-
84
-
16
-
37e
1,007e
Study
Climatic
region
(location)
Microsite/
plant community
Treatment/
burn severity
Plot
size
(m2)
Slope
(%)
Time
postfire
(mth)
Rain rate
(mm h-1)
/duration
(min)
Rain
type
WDPT
(s)a
Soil water
(%)b/
conditions
Bare
soil
(%)
Canopy
cover (%)
Ground
cover
(%)
Surface
roughness
(mm)
Runoff
coef. (%)c
Sed. yield
(g m-2) 36 Table 3. Continued
Study
Climatic
region
(location)
Microsite/
plant community
Treatment/
burn severity
Plot
size
(m2)
Slope
(%)
Time
postfire
(mth)
Rain rate
(mm h-1)
/duration
(min)
Rain
type
WDPT
(s)a
Soil water
(%)b/
conditions
Bare
soil
(%)
Canopy
cover (%)
Ground
cover
(%)
Surface
roughness
(mm)
Runoff
coef. (%)c
Sed. yield
(g m-2)
Benavides-
Solorio and
MacDonald
(2001, 2002)f
Temperate
steppe mountains
(Colorado Front
Range, Colorado)
Dry forest
(Pinus ponderosa)
Low-Unb
1.0
20-25
1-3
79/60
Artificial
~65
2
1
-
99
-
55
80
Mod
1.0
20-35
1-3
79/60
Artificial
~50
2
12
-
88
-
58
179
High
1.0
20-45
1-3
79/60
Artificial
~60
2
77
-
23
-
66
1,280
Johansen et al. (2001)
Temperate
steppe mountains
(Pajarito Plateau,
New Mexico)
Dry forest
(P. ponderosa)
Unb
32.5
5
3
60/60g
Artificial
-
~5
48
52
-
23
36
High
32.5
7
3
60/60g
Artificial
-
~5
74
26
-
45
912
Pierson et al. (2001a, 2008a)
Cold desert
(Great Basin,
Nevada)
Shrub coppice
(A. tridentata)
Unb
0.5
30-40
1
85/60
Artificial
200
7
1
100
99
-
30
12
High
0.5
30-40
1
85/60
Artificial
102
1
99
1
1
-
37
41
Interspace
Unb
0.5
30-40
1
85/60
Artificial
220
5
6
74
94
-
49
24
High
0.5
30-40
1
85/60
Artificial
97
1
99
4
1
-
30
21
Pierson et al. (2002b)h
Cold desert
(Great Basin,
Idaho)
Shrub coppice
(A. Vegetation and Cover Influences on Infiltration and Runoff Generation tridentata)
Unb
0.5
35-60
12
67/60
Artificial
-
~14
7
88
93
18
11
2
Mod
0.5
35-60
12
67/60
Artificial
-
~5
97
11
3
12
34
30
High
0.5
35-60
12
67/60
Artificial
-
~5
98
13
2
12
37
22
Interspace
Unb
0.5
35-60
12
67/60
Artificial
-
~14
89
18
12
18
24
4
Mod
0.5
35-60
12
67/60
Artificial
-
~5
95
16
5
12
26
12
High
0.5
35-60
12
67/60
Artificial
-
~5
99
5
1
10
49
148
O’Dea and
Guertin, (2003)
Hot desert
(Sonoran Desert,
Arizona)
Grassland
Unb
30
1-3
-i
Variable
Natural
-
Dry
-
21
-
-
6
1,700i
Mod
30
1-3
-i
Variable
Natural
-j
Dry
-
11
-
-
6
2,800i
Grassland
Unb
30
1-3
-k
Variable
Natural
-
Dry
-
37
-
-
14
1,800k
Mod
30
1-3
-k
Variable
Natural
-j
Dry
-
29
-
-
14
2,200k
Pierson et al. (2007a)
Cold desert
(Great Basin,
Oregon)
Shrub-interspace
zone
Unb
32.5
19
-
55/60
Artificial
-
-
84
6
16
24
25
118
Shrub-interspace
zone – trees
removedl
Unb
32.5
19
-
55/60
Artificial
-
-
64
23
36
36
2
1
Pierson et al. (2007b)m
Cold desert
(Great Basin,
Idaho)
Grassland
(B. tectorum)
Unb
32.5
2
-
64/60
Artificial
-
4
34
54
66
1
2
1 37 Table 3. Continued
Study
Climatic region
(location)
Microsite/plant
community
Treatment/
burn severity
Plot
size
(m2)
Slope
(%)
Time
postfire
(mth)
Rain rate
(mm h-1) /
duration
(min)
Rain
type
WDPT
(s)a
Soil water
(%)b/
conditions
Bare
soil (%)
Canopy
cover
(%)
Ground
cover
(%)
Surface
roughness
(mm)
Runoff
coef. (%)c
Sed. yield
(g m-2)
Michaelides et
al. (2009)
Hot desert
(Chihuahuan
Desert, New
Mexico)
Shrub coppice
(Larrea tridentata)
Unb
1.5
7
-
125/16-30
Artificial
-
7
79
65
21
-
55
97
Interspace
Unb
1.5
3
-
125/16-30
Artificial
-
8
89
1
11
-
49
122
Grassland
(Bouteloua
eriopoda)
Unb
1.5
2
-
125/16-30
Artificial
-
12
96
58
4
-
49
89
Pierson et al. (2009)
Cold desert
(Great Basin,
Idaho)
Shrub coppice
(A. USDA Forest Service RMRS-GTR-351. 2016. Vegetation and Cover Influences on Infiltration and Runoff Generation tridentata)
Unb
0.5
35-50
1
85/60
Artificial
286
7
2
84
98
34
39
17
Mod-High
0.5
35-50
1
85/60
Artificial
261
3
42
10
58
11
76
183
Interspace
Unb
0.5
35-50
1
85/60
Artificial
110
3
25
31
75
18
63
195
Mod-High
0.5
35-50
1
85/60
Artificial
117
4
84
0
16
11
55
705
Shrub-interspace
zone
Unb
32.5
35-50
1
85/60
Artificial
-
2
24
57
76
21
4
8
Mod-High
32.5
35-50
1
85/60
Artificial
208
4
76
0
24
11
27
988
Pierson et al. (2010)
Cold desert
(Great Basin,
Nevada)
Shrub-interspace
zone (intercanopy)
Unb
13
9
-
64/45
Artificial
-
6
26n
39
74
17
24
45
Tree coppice
(P. monophylla or J. osteosperma)
Unb
13
9
-
64/45
Artificial
-
11
3
27o
97
20
3
18
Shrub-interspace
zone (intercanopy)
Unb
13
9
-
102/45
Artificial
-
wet
26n
39
74
17
47
222
Tree coppice
(P. monophylla or J. osteosperma)
Unb
13
9
-
102/45
Artificial
-
wet
3
27o
97
20
5
36
Pierson et al. (2010)
Cold desert
(Great Basin,
Utah)
Shrub-interspace
zone (intercanopy)
Unb
13
14
-
64/45
Artificial
-
7
29p
19
71
26
10
37
Tree coppice
(J. osteosperma)
Unb
13
15
-
64/45
Artificial
-
9
8
21o
92
31
2
13
Shrub-interspace
zone (intercanopy)
Unb
13
14
-
102/45
Artificial
-
wet
29p
19
71
26
44
296
Tree coppice
(J. osteosperma)
Unb
13
15
-
102/45
Artificial
-
wet
8
21o
92
31
10
66 USDA Forest Service RMRS-GTR-351. 2016. 38 Table 3. Continued
Study
Climatic region
(location)
Microsite/plant
community
Treatment/
burn severity
Plot
size
(m2)
Slope
(%)
Time
postfire
(mth)
Rain rate
(mm h-1)
/duration
(min)
Rain
type
WDPT
(s)a
Soil water
(%)b/
conditions
Bare
soil
(%)
Canopy
cover
(%)
Ground
cover
(%)
Surface
roughness
(mm)
Runoff
coef. (%)c
Sed. yield
(g m-2)
Pierson et
al. (2013),
Williams et al. (2014a)
Cold desert
(Great Basin,
Idaho)
Shrub coppice
(Artemisia spp.)
Unb
0.5
20
-
102/45
Artificial
<5
wet
25
117
75
13
20
6
High
0.5
18
12
102/45
Artificial
11
wet
57
21
43
9
23
143
Interspace
Unb
0.5
14
-
102/45
Artificial
<5
wet
46q
20
54
9
63
36
High
0.5
16
12
102/45
Artificial
<5
wet
49q
21
51
8
51
135
Tree coppice
(J. Vegetation and Cover Influences on Infiltration and Runoff Generation occidentalis)
Unb
0.5
21
-
102/45
Artificial
42
wet
0
170
100
12
23
6
High
0.5
17
12
102/45
Artificial
54
wet
50
50
50
8
58
206
Shrub-interspace
zone
Unb
13
19
-
102/45
Artificial
-
wet
28r
18
72
16
50
272
High
13
16
12
102/45
Artificial
-
wet
39r
32
61
17
50
572
Tree coppice
(J. occidentalis)
Unb
13
16
-
102/45
Artificial
-
wet
7
260
93
23
13
48
High
13
18
12
102/45
Artificial
-
wet
25
150
75
21
58
1,083
Pierson et al. (2014)
Cold desert
(Great Basin,
Nevada)
Shrub coppice
(Artemisia spp.)
Unb
0.5
11
-
102/45
Artificial
<5
wet
12
93
88
14
4
6
High
0.5
13
10
102/45
Artificial
<5
wet
49
53
51
8
10
48
Interspace
Unb
0.5
9
-
102/45
Artificial
<5
wet
44s
33
56
9
41
23
High
0.5
10
10
102/45
Artificial
<5
wet
49s
30
51
8
46
41
Tree coppice
(P. monophylla or
J. osteosperma)
Unb
0.5
12
-
102/45
Artificial
48
wet
0
7t
100
12
0
0
High
0.5
15
10
102/45
Artificial
65
wet
10
4t
91
12
28
46
Pierson et al. (2014)
Cold desert
(Great Basin,
Utah)
Shrub coppice
(Artemisia spp.)
Unb
0.5
17
-
102/45
Artificial
<5
wet
18
69
82
13
8
33
High
0.5
16
10
102/45
Artificial
<5
wet
44
28
56
11
29
220
Interspace
Unb
0.5
19
-
102/45
Artificial
<5
wet
52u
19
48
11
56
233
High
0.5
16
10
102/45
Artificial
<5
wet
38u
7
62
9
64
351
Tree coppice
(J. osteosperma)
Unb
0.5
20
-
102/45
Artificial
88
wet
7
22t
93
12
22
98
High
0.5
21
10
102/45
Artificial
125
wet
9
2t
91
12
18
294
a Water drop penetration time (WDPT) is an indicator of strength of soil water repellency as follows: <5 s wettable, 5 to 60 s slightly repellent, 60 to 600 s strongly repellent (Bisdom et al. 1993). b Measured near the soil surface (<5 cm depth). c Runoff coefficient is equal to cumulative runoff divided by cumulative rainfall applied. Value is multiplied by 100 to obtain percentage. d Data shown are for dry run experiments at Coyote Butte, Nancy, and Summit sites, respectively by row. e Runoff and erosion following natural rainfall events were monitored over a 14-month period. USDA Forest Service RMRS-GTR-351. 2016. Vegetation and Cover Influences on Infiltration and Runoff Generation f Data presented for Bobcat Fire only. g Rainfall applied for 60 min under dry conditions, followed by 24-h hiatus, 30 min of rainfall, 30-min hiatus, and 30 min rainfall. Total rain applied was 120 mm. (footnotes continued on next page) 39 h Data presented from south-facing slopes solely. i Cumulative runoff and sediment yield for period of 1 July 1998 to 1 October 1998 resulting from natural rainfall events (100 mm). Fire was in May 1998. j Informal water drop tests showed no postfire soil water repellency at soil surface (O’Dea and Guertin 2003). k Cumulative runoff and sediment yield for period of 1 July 1999 to 1 October 1999 resulting from natural rainfall events (106 mm). Fire was in May 1998. l J. occidentalis trees removed from site by chainsaw cutting 10 years before rainfall simulation experiments. m This is a multiple year study; means shown for soil, cover, runoff, and sediment yield are average of 3 simulation years. n Bare ground was about 64 percent (38 percent rock cover and 26 percent bare soil). o Trees removed from plot by chainsaw immediately before simulations. p Bare ground was about 79 percent (50 percent rock cover and 29 percent bare soil). q Bare ground was about 90 percent across unburned (42 percent rock cover and 46 percent bare soil) and burned (43 percent rock cover and 49 percent bare soil) plots. r Bare ground was about 88 percent (60 percent rock cover and 28 percent bare soil) for unburned plots and 84 percent (45 percent rock cover and 39 percent bare soil) for burned plots. s Bare ground was about 73 percent (29 percent rock cover and 44 percent bare soil) for unburned and 87 percent for burned (38 percent rock cover and 49 percent bare soil) plots. t Trees removed from plot about 12 months before simulation as part of earlier study (Pierson et al. 2010). u Bare ground ≥90 percent across unburned (38 percent rock cover and 52 percent bare soil) and burned (56 percent rock cover and 38 percent bare soil) plots. h Data presented from south-facing slopes solely. i Cumulative runoff and sediment yield for period of 1 July 1998 to 1 October 1998 resulting from natural rainfall events (100 mm). Fire was in May 1998. j Informal water drop tests showed no postfire soil water repellency at soil surface (O’Dea and Guertin 2003). Vegetation and Cover Influences on Infiltration and Runoff Generation k Cumulative runoff and sediment yield for period of 1 July 1999 to 1 October 1999 resulting from natural rainfall events (106 mm). Fire was in May 1998. l J. occidentalis trees removed from site by chainsaw cutting 10 years before rainfall simulation experiments. m This is a multiple year study; means shown for soil, cover, runoff, and sediment yield are average of 3 simulation years. n Bare ground was about 64 percent (38 percent rock cover and 26 percent bare soil). o Trees removed from plot by chainsaw immediately before simulations. p Bare ground was about 79 percent (50 percent rock cover and 29 percent bare soil). q Bare ground was about 90 percent across unburned (42 percent rock cover and 46 percent bare soil) and burned (43 percent rock cover and 49 percent bare soil) plots. r Bare ground was about 88 percent (60 percent rock cover and 28 percent bare soil) for unburned plots and 84 percent (45 percent rock cover and 39 percent bare soil) for burned plots. s Bare ground was about 73 percent (29 percent rock cover and 44 percent bare soil) for unburned and 87 percent for burned (38 percent rock cover and 49 percent bare soil) plots. t Trees removed from plot about 12 months before simulation as part of earlier study (Pierson et al. 2010). u Bare ground ≥90 percent across unburned (38 percent rock cover and 52 percent bare soil) and burned (56 percent rock cover and 38 percent bare soil) plots. 40 development of infiltration-inhibiting surface crusts (Branson et al. 1981; Puigdefábregas
et al. 1999). Decreasing infiltration and increasing runoff with increasing expanse of
bare or vesicular surfaces are well documented in literature (Branson and Owen 1970;
Blackburn et al. 1992; Pierson et al. 1994a; Abrahams et al. 1995; Parsons et al. 1996;
Wilcox et al. 1996; Schlesinger et al. 1999, 2000; Pierson et al. 2002a, 2007a, 2010,
2014; Williams et al. 2014a,b). The effects of rock cover (more than 2 mm) depend
on the size, amount, and embeddedness of the rocks (Wilcox et al. 1988; Poesen et al. 1990; Poesen and Ingelmo-Sanchez 1992). Infiltration is generally positively correlated
with rocks lying on top of the soil matrix due to increased surface roughness and greater
porosity and aggregation around rocks; surface rock extends time to ponding and runoff,
increasing time for infiltration (Poesen et al. Vegetation and Cover Influences on Infiltration and Runoff Generation 1990; Abrahams and Parsons 1994; Poesen
et al. 1994; Valentin 1994; Cerdà 2001; Martínez-Zavala and Jordán 2008). Infiltration
is negatively correlated with embedded rock cover due to a decrease in nonabsorbing
area. Wilcox et al. (1988), Abrahams and Parsons (1991a), and Pierson et al. (2010,
2013) reported negative correlations between rock cover and infiltration in interspace
areas, but did not explicitly evaluate embeddedness. The studies by Wilcox et al. (1988)
and Abrahams and Parsons (1991a) indicate interspace areas occurred in swales and
were more compacted and crusted than coppice areas. Wilcox et al. (1988), Abrahams
and Parsons (1991a), and Pierson et al. (2010) suggested that the negative correlations
were not exclusively associated with rock cover; instead, the relationship was due to
co-occurring low infiltration rates of the bare interspace areas and extensive rock cover. Wilcox et al. (1988) further indicated infiltration was negatively correlated with smaller
size rock cover (2 to 12 mm) and positively correlated with rock cover of intermedi-
ate sizes (26 to 150 mm). Tromble et al. (1974) also reported a negative relationship in
infiltration and small-size rock cover (less than 10 mm). These studies suggest rock cover
can facilitate infiltration and that negative effects of rock cover on infiltration most likely
occur when smaller rocks dominate and the rock cover is embedded rather than freely
lying atop the soil surface (Brakensiek and Rawls 1994). The vegetation- and soils-driven hydrologic heterogeneity (Puigdefábregas 2005)
of rangeland ecosystems creates a mosaic of runoff source and sink areas at the hillslope
scale (figs. 15 and 16) (Schlesinger et al. 1990; Wilcox and Breshears 1995; Schlesinger
et al. 1996; Bergkamp 1998; Puigdefábregas et al. 1999; Reid et al. 1999; Wilcox et
al. 2003a; Ludwig et al. 2005; Turnbull et al. 2012; Williams et al. 2014a). The timing
and quantity of overland flow generation on rangeland sites is strongly correlated to the
quantity and arrangement of canopy and ground cover and bare interspace (Branson et
al. 1981). Runoff occurs more rapidly after the onset of rainfall in sparsely vegetated
interspaces than in vegetated interspace and coppice locations. These relationships, along
with rainfall distribution, are responsible for spatial variability in runoff generation dur-
ing intermediate storm events, but may be dampened by high-intensity or long-duration
rainfall, creating more uniform runoff (Reid et al. 1999; Puigdefábregas 2005). Vegetation and Cover Influences on Infiltration and Runoff Generation (1995) and Parsons et al. (1996) observed that fine-scale vegeta-
tive heterogeneity of a hydrologically stable grassland facilitated runon processes and
that the coarseness of vegetative structure in a degraded shrubland community amplified
runoff with increasing slope length (up to 35 m). Reid et al. (1999) estimated that runon
from bare interspaces (sources) to vegetated interspace areas (sinks) in a hydrologically
stable twoneedle pinyon/oneseed juniper woodland accounted for 12 percent of precipita-
tion over the course of 77 rainfall events. around topographically elevated coppice mounds, grass clumps, or roughness elements
(fig. 16) (Emmett 1970, 1978; Dunne et al. 1991; Seyfried 1991; Parsons et al. 1992;
Thornes 1994; Abrahams et al. 1995; Wilcox and Breshears 1995; Parsons et al. 1996;
Schlesinger et al. 1996, 1999; Wilcox et al. 2003a). Where concentrated, these flowpaths
transfer large volumes of water laterally at greater overland flow depths and velocities
than occur in sheetflow processes (Abrahams et al. 1995; Parsons et al. 1996; Pierson et
al. 2007a). These effects are amplified on steep slopes (Al-Hamdan et al. 2013), although
infiltration and slope steepness have been shown to have a positive correlation on some
rangelands (Wilcox et al. 1988). The interception of flowpaths (mostly due to ponding
behind coppices or topographic features) and subsequent re-infiltration (runon) in coppice
or vegetated hydrologic sinks are thought to stimulate biological productivity and further
facilitate coppice-interspace structure (Schlesinger et al. 1990; Joffre and Rambal 1993;
Dunkerley and Brown 1995; Ludwig and Tongway 1995; Wilcox and Breshears 1995;
Breshears et al. 1997b; Tongway and Ludwig 1997; Bergkamp 1998; Puigdefábregas
et al. 1999; Reid et al. 1999; Bhark and Small 2003; Wilcox et al. 2003a; Ludwig et al. 2005). Abrahams et al. (1995) and Parsons et al. (1996) observed that fine-scale vegeta-
tive heterogeneity of a hydrologically stable grassland facilitated runon processes and
that the coarseness of vegetative structure in a degraded shrubland community amplified
runoff with increasing slope length (up to 35 m). Reid et al. (1999) estimated that runon
from bare interspaces (sources) to vegetated interspace areas (sinks) in a hydrologically
stable twoneedle pinyon/oneseed juniper woodland accounted for 12 percent of precipita-
tion over the course of 77 rainfall events. These studies illustrate that a coarsely arranged source-sink structure, as observed
on degraded sites, potentially generates and releases more surface runoff than a finely
structured source-sink community (Schlesinger et al. 1990; Abrahams et al. Vegetation and Cover Influences on Infiltration and Runoff Generation The hydrologic connectivity and downslope surface hydraulic conductivity dictate
the progression or decay of surface runoff with increased slope length (Abrahams et
al. 1991; Dunne et al. 1991; Pierson et al. 1994b; Wilcox 1994; Abrahams et al. 1995;
Cerdà 1997; Bergkamp 1998; Davenport et al. 1998; Puigdefábregas et al. 1999; Reid
et al. 1999; Wainwright et al. 2000; Wilcox et al. 2003a; Puigdefábregas 2005). Well-
connected flowpaths develop in consecutive source areas where overland flow is routed 41 USDA Forest Service RMRS-GTR-351. 2016. Figure 16—Concentrated flow formed in interspace areas during a high-intensity
rainfall on a shrub steppe site in the Reynolds Creek Experimental Watershed,
Idaho. The lack of runoff from shrub microsites clearly demonstrates the commonly
observed hydrologic stability observed for areas underneath shrub or tree canopies
on rangeland sites (photo: USDA Agricultural Research Service, Northwest
Watershed Research Center). Figure 16—Concentrated flow formed in interspace areas during a high-intensity
rainfall on a shrub steppe site in the Reynolds Creek Experimental Watershed,
Idaho. The lack of runoff from shrub microsites clearly demonstrates the commonly
observed hydrologic stability observed for areas underneath shrub or tree canopies
on rangeland sites (photo: USDA Agricultural Research Service, Northwest
Watershed Research Center). around topographically elevated coppice mounds, grass clumps, or roughness elements
(fig. 16) (Emmett 1970, 1978; Dunne et al. 1991; Seyfried 1991; Parsons et al. 1992;
Thornes 1994; Abrahams et al. 1995; Wilcox and Breshears 1995; Parsons et al. 1996;
Schlesinger et al. 1996, 1999; Wilcox et al. 2003a). Where concentrated, these flowpaths
transfer large volumes of water laterally at greater overland flow depths and velocities
than occur in sheetflow processes (Abrahams et al. 1995; Parsons et al. 1996; Pierson et
al. 2007a). These effects are amplified on steep slopes (Al-Hamdan et al. 2013), although
infiltration and slope steepness have been shown to have a positive correlation on some
rangelands (Wilcox et al. 1988). The interception of flowpaths (mostly due to ponding
behind coppices or topographic features) and subsequent re-infiltration (runon) in coppice
or vegetated hydrologic sinks are thought to stimulate biological productivity and further
facilitate coppice-interspace structure (Schlesinger et al. 1990; Joffre and Rambal 1993;
Dunkerley and Brown 1995; Ludwig and Tongway 1995; Wilcox and Breshears 1995;
Breshears et al. 1997b; Tongway and Ludwig 1997; Bergkamp 1998; Puigdefábregas
et al. 1999; Reid et al. 1999; Bhark and Small 2003; Wilcox et al. 2003a; Ludwig et al. 2005). Abrahams et al. Plant Use of Subsurface Water and Its Influence on Runoff A positive feedback exists between subsurface water acquisition, plant community
structure, and infiltration, and the maintenance of this arrangement (Richards and
Caldwell 1987; Dawson 1993; Burgess et al. 2001; Ludwig et al. 2003; Ryel et al. 2003,
2004; Muñoz et al. 2008; Scott et al. 2008). Herbaceous and woody plants differ in their
ability to obtain the limited soil water on most rangeland sites (Walter 1971; Breshears
and Barnes 1999; Schenk and Jackson 2002). Rangeland plant community density and
structure therefore reflect the ability of the plants to obtain and efficiently use available
soil water (Caldwell 1985; Dawson 1993; Schenk and Jackson 2002). Walter (1971)
proposed a two-layered model of soil water use in water-limited ecosystems based on
rooting depth partitioning among herbaceous and woody species. The model suggests
that herbaceous plants primarily extract soil water from upper soil layers and that woody
plants have the sole access to deeper soil water. Subsequent research has shown woody plants vary in the depth at which they
extract soil water (Peláez et al. 1994; Montaña et al. 1995; Breshears et al. 1997a; Schenk
and Jackson 2002) and that they are capable of competing laterally with herbaceous
species for shallow soil water in interspace areas (Caldwell et al. 1985; Ansley et al. 1991; Peláez et al. 1994; Montaña et al. 1995; Breshears et al. 1997a; Breshears and
Barnes 1999). Schenk and Jackson (2002) summarized rooting depths for herbaceous and
woody plants over a wide range of precipitation regimes. They observed that differences
in rooting depths between herbaceous and woody species tend to decrease with increasing
precipitation. This suggests that as water becomes limited, a more distinct vertical separa-
tion in water use by woody and herbaceous plants emerges (Schenk and Jackson 2002). Breshears and Barnes (1999) pointed out that horizontal as well as vertical gradients
exist due to woody plant lateral acquisition of soil water from interspace areas and that
decreased near-surface soil water favors woody plant recruitment.i Similar processes for horizontal acquisitions of surface water via preferred infiltra-
tion or runon have been proposed to explain maintenance of vegetated islands (Joffre and
Rambal 1993; Dunkerley and Brown 1995; Wilcox and Breshears 1995; Ludwig et al. 1997; Bergkamp 1998; Puigdefábregas et al. 1999; Bhark and Small 2003; Wilcox et al. 2003a; Ludwig et al. 2005) and desertification of water-limited landscapes (Schlesinger
et al. 1990). Vegetation and Cover Influences on Infiltration and Runoff Generation 1995; Parsons 42 USDA Forest Service RMRS-GTR-351. 2016. et al. 1996; Wilcox et al. 1996; Davenport et al. 1998; Bhark and Small 2003). Studies by
Abrahams et al. (1995), Parsons et al. (1996), Wainwright et al. (2000), Michaelides et al. (2009), and Turnbull et al. (2010b, 2012) provide comparative examples of these relation-
ships for fine (grassland) versus coarsely arranged (shrubland) rangeland communities
in southern Arizona. Pierson et al. (2010, 2013) and Williams et al. (2014a) present
examples of similar relationships following conifer encroachment into Great Basin shrub
steppe. USDA Forest Service RMRS-GTR-351. 2016. Plant Use of Subsurface Water and Its Influence on Runoff Plant community structure may also be influenced by plant-specific belowground
water conservation strategies that vertically redistribute (via hydraulic redistribution) soil
water (Caldwell et al. 1998; Horton and Hart 1998; Jackson et al. 2000; Meinzer et al. 2001). Hydraulic redistribution is the passive transfer of soil water through roots upward
as hydraulic lift (Richards and Caldwell 1987; Dawson 1993; Wan et al. 1993; Emerman
and Dawson 1996; Caldwell et al. 1998; Horton and Hart 1998; Yoder and Nowak 1999a;
Mendel et al. 2002; Ludwig et al. 2003; Zou et al. 2005; Muñoz et al. 2008) or downward
as hydraulic descent (Burgess et al. 1998; Schulze et al. 1998; Smith et al. 1999; Jackson
et al. 2000; Burgess et al. 2001; Leffler et al. 2002; Ryel et al. 2002; Hultine et al. Burgess et al. (1998) demonstrated that the reverse of hydraulic lift also occurs. They found that as soils wet up following the dry season, the roots of silkoak (Grevillea
robusta A. Cunn. ex R. Br.) and river redgum (Eucalyptus camaldulensis Dehnh.)
redistributed soil water from the wetter near-surface to drier soil pockets at depth. They
termed the process hydraulic redistribution. In other studies, Ryel et al. (2003, 2004) dis-
covered that pulse rain events in a stand of big sagebrush (A. tridentata Nutt.) delivered
rain at different depths (downward distribution) simultaneously rather than sequentially
from upper to lower soil layers. This process might be expected with macropore or pref-
erential flow; however, the arrival times at the different depths were simultaneous over
a few days rather than hours as commonly reported for macropore and other preferred
flowpaths. Hydraulic descent has also been reported for other water-limited plants includ-
ing a cheatgrass (Bromus tectorum L.) monoculture in Utah, (Leffler et al. 2005), velvet
mesquite (Prosopis velutina Woot.) in southern Arizona (Hultine et al. 2004, Scott et al. 2008), Utah juniper (J. osteosperma (Torr.) Little) in northern Utah (Leffler et al. 2002),
and Arizona walnut (Juglans major Torr.) in southeastern Arizona (Hultine et al. 2003a). The decreased water stress associated with hydraulic redistribution provides dryland
vegetation numerous ecological benefits thought to increase ecosystem primary pro-
ductivity. These include enhanced water-use efficiency and transpiration (Richards and
Caldwell 1987; Caldwell and Richards 1989; Dawson 1993, 1996; Emerman and Dawson
1996; Caldwell et al. 1998; Brooks et al. 2002; Ryel et al. 2002; Hultine et al. 2004; Ryel
et al. 2004; Muñoz et al. Plant Use of Subsurface Water and Its Influence on Runoff Prolonged periods of dry soil conditions coarsen the vegetative structure
in favor of woody plants and shrub/tree islands whereas wet periods facilitate a more
vertically and horizontally heterogeneous community of herbaceous and woody plants
(Schlesinger et al. 1990). Coarsening of the plant community (such as transitions from
grassland to shrubland and shrubland to woodland) through drought or disturbance com-
monly increases bare ground area, hydrologic connectivity of runoff source areas, and 43 USDA Forest Service RMRS-GTR-351. 2016. surface runoff from point to hillslope scales (Abrahams et al. 1995; Parsons et al. 1996;
Wilcox et al. 1996; Davenport et al. 1998; Pierson et al. 2010; Turnbull et al. 2010a,b;
Pierson et al. 2011; Turnbull et al. 2012; Williams et al. 2014a, 2016a). Plant community structure may also be influenced by plant-specific belowground
water conservation strategies that vertically redistribute (via hydraulic redistribution) soil
water (Caldwell et al. 1998; Horton and Hart 1998; Jackson et al. 2000; Meinzer et al. 2001). Hydraulic redistribution is the passive transfer of soil water through roots upward
as hydraulic lift (Richards and Caldwell 1987; Dawson 1993; Wan et al. 1993; Emerman
and Dawson 1996; Caldwell et al. 1998; Horton and Hart 1998; Yoder and Nowak 1999a;
Mendel et al. 2002; Ludwig et al. 2003; Zou et al. 2005; Muñoz et al. 2008) or downward
as hydraulic descent (Burgess et al. 1998; Schulze et al. 1998; Smith et al. 1999; Jackson
et al. 2000; Burgess et al. 2001; Leffler et al. 2002; Ryel et al. 2002; Hultine et al. 2003a,b; Ryel et al. 2003; Hultine et al. 2004; Ryel et al. 2004; Leffler et al. 2005; Scott
et al. 2008) from wetter to drier soil layers along a gradient in water potential. Lateral
redistribution has also been observed (Brooks et al. 2002; Burgess and Bleby 2006), but
its occurrence is less documented than vertical transfers. Hydraulic lift has been docu-
mented in more than 50 woody taxa and herbaceous species (Jackson et al. 2000), but is
most common in deeper-rooted shrubs and trees. During dry periods, plants undergoing
hydraulic lift absorb soil water from moist deep soil layers during evening hours when
transpiration demands are low. The absorbed water is then transferred upwards and
released via roots to the drier near-surface soil during the night. The released water is
subsequently reabsorbed during the next day to meet daily transpiration demands. Plant Use of Subsurface Water and Its Influence on Runoff Plants neighboring hydraulic-lifting species may benefit from soil water redistribu-
tion as well (Caldwell and Richards 1989; Dawson 1993; Yoder and Nowak 1999a; Smith
et al. 1999, Brooks et al. 2002; Leffler et al. 2005; Zou et al. 2005), but these benefits
are probably limited by plant competition and the dry surface soil moisture conditions
in which hydraulic lifting occurs (Caldwell et al. 1998; Ludwig et al. 2004; Muñoz et al. 2008). Dawson (1993) reported that hydraulic lift by sugar maple (Acer saccharum) in
a mesic forest influenced soil moisture conditions up to 5 m from the tree base. Within
this distance, plants neighboring sugar maple used 3 to 60 percent of the lifted water. Neighboring plants that used a high percentage of lifted water exhibited increased
water-use efficiency and greater aboveground growth (Dawson 1993). Yoder and Nowak
(1999a) suggested that hydraulic lift by Mojave yucca (Yucca schidigera) plants in the
water-limited Mojave Desert (located in parts of California, Nevada, Utah, and Arizona),
provided daytime near-surface soil water for neighboring creosote (Larrea tridentata),
rough joinfir (Ephedra nevadensis), burrobush (Ambrosia dumosa), pale desert-thorn
(Lycium pallidum), and Indian ricegrass (Achnatherum hymenoides) plants. All six spe-
cies exhibited hydraulic lift, but only Mojave yucca (a crassulacean acid metabolism, or
CAM, species) lifted water during daytime hours and transpired at night. Neighboring plants that used a high percentage of lifted water exhibited increased
water-use efficiency and greater aboveground growth (Dawson 1993). Yoder and Nowak
(1999a) suggested that hydraulic lift by Mojave yucca (Yucca schidigera) plants in the
water-limited Mojave Desert (located in parts of California, Nevada, Utah, and Arizona),
provided daytime near-surface soil water for neighboring creosote (Larrea tridentata),
rough joinfir (Ephedra nevadensis), burrobush (Ambrosia dumosa), pale desert-thorn
(Lycium pallidum), and Indian ricegrass (Achnatherum hymenoides) plants. All six spe-
cies exhibited hydraulic lift, but only Mojave yucca (a crassulacean acid metabolism, or
CAM, species) lifted water during daytime hours and transpired at night.i Such benefits are not always observed for neighboring plants (Caldwell et al. 1998;
Brooks et al. 2002; Ludwig et al. 2004; Muñoz et al. 2008). In an east African savanna,
Ludwig et al. (2004) determined that any benefit of hydraulic lift by umbrella thorn
(Acacia tortilis) observed for neighboring plants was overwhelmed by near-surface
competition with the water-lifting trees. Ludwig et al. USDA Forest Service RMRS-GTR-351. 2016. Plant Use of Subsurface Water and Its Influence on Runoff 2008; Scott et al. 2008), greater fine-root longevity (Richards
and Caldwell 1987; Dawson 1993; Meinzer et al. 2004), increased microbial activity and
nutrient acquisition (Caldwell et al. 1998; Dawson 1993, 1996; McCulley et al. 2004),
prolonged symbiotic mychorrizal associations during drought (Richards and Caldwell 44 USDA Forest Service RMRS-GTR-351. 2016. 1987; Caldwell et al. 1998; Horton and Hart 1998; Querejeta et al. 2003, 2007), reduced
carbon consumption (Dawson 1993; Caldwell et al. 1998), extension of the growing
season (Ryel et al. 2004; Muñoz et al. 2008; Scott et al. 2008), and decreased competition
for limited water resources (Smith et al. 1999). The primary benefit is, of course, water
conservation. 1987; Caldwell et al. 1998; Horton and Hart 1998; Querejeta et al. 2003, 2007), reduced
carbon consumption (Dawson 1993; Caldwell et al. 1998), extension of the growing
season (Ryel et al. 2004; Muñoz et al. 2008; Scott et al. 2008), and decreased competition
for limited water resources (Smith et al. 1999). The primary benefit is, of course, water
conservation. Few long-term assessments of the ecosystem benefits associated with hydraulic
redistribution exist (Scott et al. 2008). In a 2-year study, Scott et al. (2008) found that
velvet mesquite on a southern Arizona rangeland redistributed soil water from the
near surface to deep soil locations throughout the year, including the dormant season. Downward redistributed winter-season precipitation allowed trees to transpire more
during the dry pre-monsoon period. Hydraulic descent of monsoonal summer rainfall
extended the growing season and allowed for greater photosynthesis and plant productiv-
ity during the seasonal drought. Similar results were reported in another study of velvet
mesquite in southeastern Arizona (Hultine et al. 2004).i Plants neighboring hydraulic-lifting species may benefit from soil water redistribu-
tion as well (Caldwell and Richards 1989; Dawson 1993; Yoder and Nowak 1999a; Smith
et al. 1999, Brooks et al. 2002; Leffler et al. 2005; Zou et al. 2005), but these benefits
are probably limited by plant competition and the dry surface soil moisture conditions
in which hydraulic lifting occurs (Caldwell et al. 1998; Ludwig et al. 2004; Muñoz et al. 2008). Dawson (1993) reported that hydraulic lift by sugar maple (Acer saccharum) in
a mesic forest influenced soil moisture conditions up to 5 m from the tree base. Within
this distance, plants neighboring sugar maple used 3 to 60 percent of the lifted water. Plant Use of Subsurface Water and Its Influence on Runoff (2004) suggested that the trees
outcompeted the grasses for lifted water and that grasses were then limited by the lack
of additional water availability under the more xeric conditions relative to those in the
Dawson (1993) study. Muñoz et al. (2008) also reported that near-surface water use by
xeric community shrubs hydraulically lifting soil water mitigated potential losses to
neighboring plants. Ishikawa and Bledsoe (2000) reported that hydraulic lifting by blue
oak (Q. douglasii) occurred too late in the growing season for neighboring grasses to
benefit. Plants in dry climates with shallow and fibrous roots generally complete seasonal
physiological processes during periods of high near-surface soil water content. As the
near-surface environment dries out, shallow-rooted species die off and competition for 45 USDA Forest Service RMRS-GTR-351. 2016. near-surface resources is reduced. Deep-rooted species delaying hydraulic lift to the near-
surface can then extend the growing season and, by wetting the near-surface, improve
near-surface nutrient acquisition during periods of the year when competition is low
(Caldwell et al. 1998). Benefits to neighboring plants then appear to vary with precipita-
tion regime, type of plant community, and the timing of hydraulic redistribution. The volume of water redistributed is largely a function of available soil water
and the daily evapotranspiration requirements. In a semiarid climate, Ryel et al. (2003)
estimated that 74 percent of precipitation from a 36 mm event and 100 percent of
precipitation from small (less than 8 mm) rainfall events that infiltrated 30 to 150 cm into
the soil profile resulted from hydraulic redistribution by big sagebrush roots. Richards
and Caldwell (1987) reported mountain big sagebrush (A. tridentata Nutt. ssp. vaseyana
(Rydb.) Beetle) hydraulically lifted one-third of its daily evapotranspiration demand. Caldwell and Richards (1989) found that artificial suppression of hydraulic lift reduced
mountain big sagebrush daily transpiration by 25 to 50 percent. Hultine et al. (2003a)
reported daily hydraulic descent amounted to 10 to 60 percent of daily transpiration for
Arizona walnut. Hultine et al. (2004) estimated that diurnal hydraulic descent rates of
velvet mesquite during the winter dormant season were 70 percent of that during the
growing season following monsoon rainfall. Leffler et al. (2005) found that about 6 percent of soil water at 10 to 20 cm depth
underneath a cheatgrass monoculture was hydraulically lifted by cheatgrass during
flowering and seed set. Plant Use of Subsurface Water and Its Influence on Runoff In the same study, senesced cheatgrass in a greenhouse-stored
pot lifted 17 percent of soil water measured in an upper soil layer. Brooks et al. (2002)
determined 28 to 35 percent of water removed daily from the upper 2 m of the soil profile
in coniferous forests of moist Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco) in
Washington and dry ponderosa pine (Pinus ponderosa Dougl. ex Laws) in Oregon, was
replaced by nocturnal hydraulic redistribution. In a mesic climate, Emerman and Dawson
(1996) found that an individual sugar maple tree was capable of lifting 100 L day-1 and
that hydraulic lift provided 25 percent of tree daily water use. Dawson (1996) reported
sugar maple was capable of lifting 25 percent of daily transpiration demand. The studies
cited above are only a sample from literature to demonstrate that hydraulic redistribution
occurs across a range of plant communities (herbaceous and woody) and climate regimes
and may have a substantial impact on ecosystem water balances (Meinzer et al. 2001). Hydraulic redistribution may account for as much as 70 percent of daily transpiration;
however, most values are in the 20 to 35 percent range depending on plant type, anteced-
ent moisture conditions, time of year, and evapotranspiration demand. Effects of Soil Water Repellency on Runoff Generation Soil water repellency is a naturally occurring soil condition that inhibits infiltration. Its occurrence has been well documented on shrubland, chaparral, woodland, and semi-
arid forest ecosystems (Meeuwig 1971; Scholl 1971, 1975; DeBano 1981, 1991; Doerr
et al. 1996, 2000; Jaramillo et al. 2000; DeBano 2001; Huffman et al. 2001; Hubbert et
al. 2006; Lebron et al. 2007; Verheijen and Cammeraat 2007; Woods et al. 2007; Madsen
et al. 2008; Pierson et al. 2008b; Doerr et al. 2009; Pierson et al. 2009, 2010; Robinson
et al. 2010; Bodí et al. 2013; Pierson et al. 2013, 2014; Williams et al. 2014a). Water-
repellent soils form by the coating of particles with hydrophobic compounds leached 46 USDA Forest Service RMRS-GTR-351. 2016. from organic matter accumulations, microbial by-products, or fungal growth under litter
and duff (Savage et al. 1972; Imeson et al. 1992; Bisdom et al. 1993; Doerr et al. 2000). The strength of soil water repellency and its influence on infiltration are a function of the
quantity and type of overlying vegetation, soil texture, and soil water content (Burcar
et al. 1994; Dekker and Ritsema 1994; Bauters et al. 2000; Doerr and Thomas 2000;
Shakesby et al. 2000; Dekker et al. 2001; Huffman et al. 2001; MacDonald and Huffman
2004; Verheijen and Cammeraat 2007; Madsen et al. 2008; Pierson et al. 2008b, 2009,
2010). The type and quantity of vegetation dictate the amount and type of hydrophobic
compounds potentially available. Coarse-textured soils generally are more susceptible
to soil water repellency than fine-textured soils due to their greater particle surface area
(DeBano 1991, Bisdom et al. 1993, Huffman et al. 2001); however, recent research has
demonstrated that strong soil water repellency can occur in fine-textured soils (Doerr et
al. 2000, 2006, 2009). Doerr et al. (2000, 2009) provide a review of occurrence, causes,
hydrologic and erosional effects, and measurement methods of soil water repellency. The strength and persistence of soil water repellency is highly variable in time
and space (DeBano 1971; Witter et al. 1991; Shakesby et al. 1993; Dekker and Ritsema
1994; Doerr and Thomas 2000; Dekker et al. 2001; Huffman et al. 2001; MacDonald
and Huffman 2004; Leighton-Boyce et al. 2005; Verheijen and Cammeraat 2007; Woods
et al. 2007; Madsen et al. 2008; Pierson et al. 2008b, 2009, 2010). USDA Forest Service RMRS-GTR-351. 2016. Effects of Soil Water Repellency on Runoff Generation Soil water repellency
for a particular soil may be present under dry conditions, decrease with soil wetting, and
reappear with soil drying (Shakesby et al. 1993; Doerr et al. 2000). Dekker et al. (2001)
demonstrated that critical soil-water thresholds demarcate wettable and water-repellent
soil conditions. Doerr et al. (2009) suggest from literature that the critical threshold
ranges from 5 percent for organic dune sands to more than 30 percent for fine-textured
soils. Huffman et al. (2001) reported that water repellency in sandy loam soils at
semiarid-forested sites in Colorado became wettable at soil water contents of 12 to 25
percent. Doerr and Thomas (2000) reported that temporal variability in soil water repel-
lency was associated with seasonal rainfall patterns, biological productivity, and wetting
and drying regimes. Pierson et al. (2008b, 2009) found that soil water repellency and the
magnitude of its influence on infiltration and runoff exhibited significant annual vari-
ability at multiple steeply sloped mountain big sagebrush sites in the Inland Northwest,
but the study did not explicitly track soil moisture patterns (fig. 17). In addition to temporal variance, the strength of soil water repellency may be
spatially variable (horizontally and vertically), owing to its presence mostly under
or immediately adjacent to canopy- and litter-covered areas and spatial soil-moisture
gradients (Imeson et al. 1992; Ritsema and Dekker 1994; Dekker et al. 2001; Verheijen
and Cammeraat 2007; Woods et al. 2007; Madsen et al. 2008; Pierson et al. 2008b, 2009,
2010, 2013, 2014; Williams et al. 2014a). On unburned sites, soil water repellency is
commonly stronger at the soil surface and degrades with depth below the mineral surface
(Huffman et al. 2001; Leighton-Boyce et al. 2007; Pierson et al. 2008b, 2009, 2010). The
effects of fire on the occurrence and hydrologic impacts of soil water repellency are dis-
cussed in Section 4, Exacerbation, Alteration, and Formation of Soil Water Repellency. Soil water repellency facilitates runoff initiation either by inhibiting infiltration at
the surface (infiltration-excess runoff) or causing saturation of a shallow soil layer (sat-
uration-excess runoff) immediately overlying a water-repellent zone (Doerr et al. 2000). In either case, runoff initiation may occur rapidly, but infiltration generally increases as 47 47 USDA Forest Service RMRS-GTR-351. 2016. Effects of Soil Water Repellency on Runoff Generation Figure 17—Temporal variability of soil water repellency (measured by using water drop penetration time,
WDPT) effects on infiltration of artificial rainfall into unburned, coarse-textured soils at two sagebrush sites:
(A) Denio Fire (wildfire), Pine Mountain Range, Nevada, (Pierson et al. 2008a) and (B) Breaks Prescribed
Fire, Reynolds Creek Experimental Watershed, Idaho (Pierson et al. 2009). WDPT is an indicator of strength
of soil water repellency as follows: <5 s wettable, 5 to 60 s slightly repellent, 60 to 600 s strongly repellent
(Bisdom et al. 1993). Figure 17—Temporal variability of soil water repellency (measured by using water drop penetration time,
WDPT) effects on infiltration of artificial rainfall into unburned, coarse-textured soils at two sagebrush sites:
(A) Denio Fire (wildfire), Pine Mountain Range, Nevada, (Pierson et al. 2008a) and (B) Breaks Prescribed
Fire, Reynolds Creek Experimental Watershed, Idaho (Pierson et al. 2009). WDPT is an indicator of strength
of soil water repellency as follows: <5 s wettable, 5 to 60 s slightly repellent, 60 to 600 s strongly repellent
(Bisdom et al. 1993). soils become wet (Letey et al. 1962; Meeuwig 1971; Burch et al. 1989; DeBano 2000;
Robichaud 2000; Pierson et al. 2008b, 2009). Increasing infiltration rates over time
(minutes to hours) occurs due to (1) a gradual decrease in repellency with wetting, or (2)
lateral and vertical water transfer through preferential infiltration and flow via macropores
or breaks in the repellent layer (Meeuwig 1971; DeBano 1981; Burch et al. 1989;
Imeson et al. 1992; Hendrickx et al. 1993; Ritsema et al. 1993; Dekker and Ritsema
1994; Ritsema and Dekker 1994, 1995; Dekker and Ritsema 1995, 1996; Ritsema and
Dekker 1996; Ritsema et al. 1997, 1998a, 1998b; Doerr et al. 2000; Jaramillo et al. 2000;
Leighton-Boyce et al. 2007; Pierson et al. 2008b). Vegetation and ground cover store
intercepted rainfall, as well as slow and retain overland flow, allowing more time for
infiltration and soil wetting (Meeuwig 1971; Leighton-Boyce et al. 2007; Pierson et al. 2008a, 2009, 2010, 2013; Williams et al. 2014a). Vegetation and ground cover also pro-
mote macropore development along root channels, animal burrows, and other soil voids
(Belnap et al. 2005; Ludwig et al. 2005). Preferential flow into macropores bypasses
water-repellent layers to wettable soil within the root zone (Meeuwig 1971; DeBano
1981; Burch et al. 1989; Imeson et al. 1992; Hendrickx et al. 1993; Ritsema et al. Effects of Soil Water Repellency on Runoff Generation 1993;
Dekker and Ritsema 1994; Ritsema and Dekker 1994, 1995; Dekker and Ritsema 1996;
Ritsema et al. 1998a, 1998b; Doerr et al. 2000; Jaramillo et al. 2000; Leighton-Boyce et
al. 2007; Pierson et al. 2008b; Robinson et al. 2010). In some cases, the vertical bypass
of water through repellent zones via macropores leaves a dry layer at the soil surface and
wet conditions in the root zone (Meeuwig 1971; Burch et al. 1989; Imeson et al. 1992). The overall effect of preferential flow depends on the extensiveness of the macropore
network and the strength of soil water repellency.ii soils become wet (Letey et al. 1962; Meeuwig 1971; Burch et al. 1989; DeBano 2000;
Robichaud 2000; Pierson et al. 2008b, 2009). Increasing infiltration rates over time
(minutes to hours) occurs due to (1) a gradual decrease in repellency with wetting, or (2)
lateral and vertical water transfer through preferential infiltration and flow via macropores
or breaks in the repellent layer (Meeuwig 1971; DeBano 1981; Burch et al. 1989;
Imeson et al. 1992; Hendrickx et al. 1993; Ritsema et al. 1993; Dekker and Ritsema
1994; Ritsema and Dekker 1994, 1995; Dekker and Ritsema 1995, 1996; Ritsema and
Dekker 1996; Ritsema et al. 1997, 1998a, 1998b; Doerr et al. 2000; Jaramillo et al. 2000;
Leighton-Boyce et al. 2007; Pierson et al. 2008b). Vegetation and ground cover store
intercepted rainfall, as well as slow and retain overland flow, allowing more time for
infiltration and soil wetting (Meeuwig 1971; Leighton-Boyce et al. 2007; Pierson et al. 2008a, 2009, 2010, 2013; Williams et al. 2014a). Vegetation and ground cover also pro-
mote macropore development along root channels, animal burrows, and other soil voids
(Belnap et al. 2005; Ludwig et al. 2005). Preferential flow into macropores bypasses
water-repellent layers to wettable soil within the root zone (Meeuwig 1971; DeBano
1981; Burch et al. 1989; Imeson et al. 1992; Hendrickx et al. 1993; Ritsema et al. 1993;
Dekker and Ritsema 1994; Ritsema and Dekker 1994, 1995; Dekker and Ritsema 1996;
Ritsema et al. 1998a, 1998b; Doerr et al. 2000; Jaramillo et al. 2000; Leighton-Boyce et
al. 2007; Pierson et al. 2008b; Robinson et al. 2010). In some cases, the vertical bypass
of water through repellent zones via macropores leaves a dry layer at the soil surface and
wet conditions in the root zone (Meeuwig 1971; Burch et al. 1989; Imeson et al. 1992). Effects of Soil Water Repellency on Runoff Generation The overall effect of preferential flow depends on the extensiveness of the macropore
network and the strength of soil water repellency.ii The efficacy of naturally occurring soil water repellency on infiltration can be sig-
nificant at point scales, but quantification of the impacts over larger scales is confounded
by spatial variability in hydrophobicity and cover as well as soil properties (Imeson et al. 1992; Doerr et al. 2000; Shakesby et al. 2000; Doerr and Moody 2004; Leighton-Boyce 48 USDA Forest Service RMRS-GTR-351. 2016. et al. 2007; Pierson et al. 2009). DeBano (1971) found that horizontal infiltration was
25 times faster in a soil under wettable conditions as compared to a similar soil under
hydrophobic conditions. Leighton-Boyce et al. (2007) determined that runoff from small-
plot (0.36 m2) rainfall simulations was 16 times higher under water-repellent conditions
than when the same soils were wettable. Madsen et al. (2008) found that pre-wetting
water-repellent surface soils underneath Utah juniper and twoneedle pinyon litter yielded
hydraulic conductivities (as measured by an infiltrometer) 6 to more than 30 times greater
than under water-repellent conditions. Madsen et al. (2008) observed (without taking
specific measurements) that tree coppices retained surface water and routed it laterally
toward preferential wet spots under the tree canopy.lil Vertical preferential flow along wet spots has been referred to as fingered flow
(Ritsema and Dekker 1994; Dekker and Ritsema 1995; Ritsema et al. 1997). Dekker and
Ritsema (1996) reported that fingered flow into dry, strongly water-repellent conditions
generated significant differences up to nearly 30 percent volumetric moisture content
between closely spaced samples of fine-textured soils. In multiyear rainfall simulation
studies of two steeply sloping mountain big sagebrush sites in Nevada and Idaho, Pierson
et al. (2001a, 2008a,b, 2009) found that minimum- and steady-state infiltration rates
(0.5 m2 rainfall simulation plots) on unburned shrub coppices increased 25 to 65 percent
after a between-years decrease in soil water repellency strength by 55 to 75 percent. Minimum- and steady-state infiltration rates on unburned interspaces in the studies by
Pierson et al. (2001a, 2008a) increased by 65 and 55 percent, respectively, after a 55
percent between-years decrease in soil water repellency strength. Pierson et al. (2009)
reported that threefold stronger soil water repellency on shrub coppice than interspace
plots resulted in 31 mm and 49 mm of runoff from shrub coppices and interspaces,
respectively. Effects of Soil Water Repellency on Runoff Generation The contradiction in runoff rates with strength was attributed to interception,
surface retention, and preferential flow (inferred) associated with greater canopy and
ground cover on coppices. Soil moisture and cover conditions for respective coppice and
interspace areas were similar for the unburned condition throughout the Pierson et al. (2001a, 2008a,b, 2009) studies. Clearly, soil water repellency can significantly reduce
infiltration rates over small scales, but the heterogeneity of soil and cover conditions on
undisturbed sites and preferential flowpaths most likely subdue the effects at hillslope
and catchment scales (Meeuwig 1971; Burch et al. 1989; Imeson et al. 1992; Doerr et
al. 2000; Shakesby et al. 2000; Doerr and Moody 2004; Verheijen and Cammeraat 2007;
Pierson et al. 2009). Soil water repellency may provide water conservation and increased plant produc-
tivity for some woody species and may indirectly mitigate runoff generation (Doerr et
al. 2000; Jaramillo et al. 2000; Lebron et al. 2007; Madsen et al. 2008; Robinson et al. 2010). Imeson et al. (1992) suggested that preferential flow to deep storage beneath the
surface water-repellent layer trapped soil water and prevented it from evaporation and
upward capillary transfer. Lebron et al. (2007) and Madsen et al. (2008) observed (in
field observations) that surface water on water-repellent soils under Utah juniper and
twoneedle pinyon was routed to preferential wet spots. They postulated that these loca-
tions provide fingered flow through the water-repellent layer to deep soil storage. Roundy
et al. (1978) hypothesized similar behavior to explain rapid infiltration of simulated
rainfall into water-repellent soils of Utah juniper. Other researchers have proposed
soil water repellency as a routing mechanism to preferential flowpaths and deep soil 49 USDA Forest Service RMRS-GTR-351. 2016. recharge (see Doerr et al. 2000; Lebron et al. 2007). The recharge of deeper soil layers
through preferential flow indirectly influences runoff behavior through increased plant
productivity (Ryel et al. 2003). Water availability deep in the soil profile favors woody
plant recruitment and facilitates a coppice/interspace structure (Breshears and Barnes
1999). Increased plant productivity through greater water availability and transpiration
rates (Ryel et al. 2003) recruits surface plant and litter biomass associated with higher
infiltration rates (Ludwig et al. 1997; Wilcox et al. 2003a, Huxman et al. 2005; Ludwig et
al. 2005). Effects of Soil Water Repellency on Runoff Generation Therefore, surface flow routing by soil water repellency may function similar
to the lateral surface transfers of overland flow (runon) in maintaining shrub, grass, and
tree islands of higher biological activity and water retention (Schlesinger et al. 1990;
Joffre and Rambal 1993; Pierson et al. 1994a,b; Dunkerley and Brown 1995; Ludwig and
Tongway 1995; Seyfried and Wilcox 1995; Wilcox and Breshears 1995; Breshears et al. 1997b; Tongway and Ludwig 1997; Bergkamp 1998; Puigdefábregas et al. 1999; Reid et
al. 1999; Bhark and Small 2003; Wilcox et al. 2003a; Huxman et al. 2005; Ludwig et al. 2005; Robinson et al. 2010). Cover Effects on Sheetflow Erosion Canopy and ground cover reduce sheetflow erosion by controlling the water avail-
able for sediment transport and by recruiting surface roughness elements that disperse
overland flow (Emmett 1970, 1978; Branson et al. 1981; Thurow et al. 1986; Seyfried
1991; Abrahams et al. 1995; Wainwright et al. 2000; Pierson et al. 2002b, 2010, 2013;
Williams et al. 2014a, 2015). Shallow sheetflow (fig. 9B) has little erosive energy, but
is the primary transport mechanism for soil detached by raindrops or by other pre-event
processes (such as freeze-thaw or weather) (Kinnell 1990, 2005). Progressively deeper
flowpaths dampen the erosive energy of raindrops (Moss and Green 1983; Kinnell 1990,
1991, 1993), but may exert enough shear stress on the soil surface to detach and entrain
soil (Foster and Meyer 1972; Kinnell 2005). Collectively, rainsplash and sheetflow are
primary conduits for hillslope sediment delivery except where concentrated flow (fig. 9C)
or rills occur (Kinnell 2005). Higher infiltration rates and rainfall interception associated
with cover elements reduce water availability for transport of eroded material (Blackburn
1975; Branson et al. 1981; Abrahams et al. 1991; Blackburn et al. 1992; Pierson et al. 1994a,b; Abrahams et al. 1995; Reid et al. 1999; Wainwright et al. 2000; Pierson et al. 2008a, 2009, 2010; Williams et al. 2014a). Vegetation, litter, and rocks promote surface
roughness, which dissipates the velocity and energy of runoff where it does occur
(Emmett 1970, 1978; Seyfried 1991; Abrahams and Parsons 1994; Abrahams et al. 1995;
Parsons et al. 1996; Wainwright et al. 2000; Pierson et al. 2002b, 2007a; Al-Hamdan et al. 2012a, 2013). Reduced flow velocities have lower detachment and allow surface runoff
to disperse and sediment to fall out of suspension. Ponding behind shrub mounds, grass
clumps, and litter dams further dissipates rainsplash and facilitates deposition of sediment
delivered from upslope runoff (Emmett 1970, 1978; Seyfried 1991). Within-event soil
loss from well-vegetated areas is generally 2- to 10-fold less than that from sparsely cov-
ered or bare interspaces (table 3), but differences can exceed three orders of magnitude
(Pierson et al. 1994b). Actual differences vary with cover, soil, rainfall, and topography
characteristics. The net effect of cover on interrill processes can reduce rangeland within-
storm soil loss 8- to 10-fold across the plant (less than 1 m2) to patch (tens of square
meters) scales (Pierson et al. Surface Protection from Raindrop Detachment The primary effects of cover on rainsplash erosion are dissipation of rainfall energy,
direct prevention of rainfall contact with the soil surface, and soil stabilization. Recall
that for sediment detachment to occur the erosive energy (shear stress) applied to the
soil surface must exceed the detachment resistance of soil (critical shear stress or shear
strength) (Foster and Meyer 1972; Sharma et al. 1991; Nearing et al. 1999; Kinnell 2005). Canopy and ground cover dissipate the erosive energy of rainfall via interception, thereby
reducing the shear stress applied to the soil surface (Al-Hamdan et al. 2013). Recent stud-
ies have estimated that rangeland canopy and ground cover can reduce rainfall erosivity
approximately 50 percent (Martinez-Mena et al. 1999; Wainwright et al. 1999). Plants
and organic material also contribute to the soil shear strength by anchoring soils and
promoting aggregate stability (Blackburn 1975; Pierson et al. 1994a,b; Cammeraat and
Imeson 1998; Cerdà 1998b; Puigdefábregas et al. 1999; Pierson et al. 2010, 2013, 2014;
Williams et al. 2014a). The surface protection and soil stabilization by cover elements
(figs. 15C and 15D) are paramount in minimizing erosion given that raindrop impact is
the primary sediment contributor to shallow overland flow (Young and Wiersma 1973;
Wainwright et al. 2000; Kinnell 2005). Cover may significantly reduce soil loss even
where surface runoff is substantial (for example during intense rainfall or water-repellent
soil conditions) (Pierson et al. 2009, 2010). Rainsplash erosion rates are seldom quantified separately from overall interrill
erosion rates in rangeland field studies. Parsons et al. (1992, 1994) found the rainsplash
erosion rate on Arizona rangelands was 0.01 to 0.04 g m-2 min-1 on grassland (73 to
86 mm h-1 intensity) and 0.34 g m-2 min-1 on shrubland (145 mm h-1 intensity) during ar-
tificial rainfall experiments (see Wainwright et al. 2000). Rainsplash during the shrubland
experiments eroded about 1.6 times more sediment from areas between plant canopies
than from areas underneath plant canopies (Parsons et al. 1992). The Parsons et al. (1992,
1994) and Wainwright et al. (1999) studies demonstrate the potential influence of cover 50 USDA Forest Service RMRS-GTR-351. 2016. on rainsplash erosion; however, the net effects vary with soil properties (see Bryan 2000;
Kinnell 2005), cover amount/type (Gabet and Dunne 2003), and rainfall characteristics
(Bryan 2000; Salles and Poesen 2000; Salles et al. 2000). Cover Effects on Sheetflow Erosion 1994b, 2009) and, where cover exceeds 50 to 60 percent,
results in minor hillslope soil loss (Gifford 1985; Pierson et al. 1994b, 2008a, 2009, 2010,
2013; Williams et al. 2014a). Cover Effects on Concentrated Flow Erosion The effects of cover elements on concentrated flow erosion (fig. 9C) are similar to
those in sheetflow erosion. The main effects are reduced runoff discharge, flow velocity,
and sediment detachment (Emmett 1970, 1978; Branson et al. 1981; Thurow et al. 1986;
Seyfried 1991; Abrahams et al. 1995; Wainwright et al. 2000; Pierson et al. 2007a, 2008a,
2009; Al-Hamdan et al. 2012a,b, 2013; Williams et al. 2014a). Vegetation and ground
cover reduce water available for concentrated flow formation and thereby decrease 51 USDA Forest Service RMRS-GTR-351. 2016. concentrated flow discharge (Pierson et al. 2007a, 2008a, 2009). Soil detachment by
concentrated flow is well correlated with flow velocity (Pierson et al. 2008a, 2009) and
discharge (Nearing et al. 1997, 1999; Govers et al. 2007; Al-Hamdan et al. 2012b), and
flow velocity is strongly related to discharge (Govers 1992; Nearing et al. 1997, 1999;
Giménez and Govers 2001; Govers et al. 2007; Al-Hamdan et al. 2012a). Grass clumps,
plant bases, coppice mounds (fig. 16), and litter dams create topographic highs that may
concentrate flow where runoff occurs, but concentrated flowpaths on well vegetated/
covered sites generally flow a short distance and disperse (Emmett 1970, 1978; Seyfried
1991; Parsons et al. 1996; Bryan 2000; Wainwright et al. 2000).l The erosive energy and transport capacity of concentrated flow are greatly
reduced when flow intersects ground cover elements (Al-Hamdan et al. 2012a,b, 2013). Roughness created by ground cover counteracts flow energy by amplifying hydraulic
friction until the flow submerges the ground cover (Emmett 1970, 1978; Abrahams and
Parsons 1991b; Abrahams and Parsons 1994; Abrahams et al. 1994, 1995; Parsons et al. 1996; Nearing et al. 1997; Wainwright et al. 2000). Studies from a western juniper (J. occidentalis Hook.) woodland (Pierson et al. 2007a) and a mountain big sagebrush range-
land (Pierson et al. 2009) reported concentrated flow velocities that were 1.5- (woodland
interspaces) to more than 2-fold (recently burned mountain big sagebrush) greater on
degraded hillslopes with 80 percent bare ground than on adjacent hillslopes with 60
percent and 20 percent bare ground, respectively. Sediment yield from concentrated flow
processes was fourfold (Pierson et al. 2009) to eightfold (Pierson et al. 2007a) greater
from the degraded sagebrush and woodland slopes. Sediment transported by concentrated
flow where it does occur on well-vegetated sites often forms miniature alluvial fans ad-
jacent to vegetative clumps (Emmett 1970, 1978; Seyfried 1991). USDA Forest Service RMRS-GTR-351. 2016. Fire Behavior and Regimes on Rangelands The environmental effects of a particular fire or series of fires are often placed in the
context of fire behavior, intensity, severity, and regime (DeBano et al. 1998; Shakesby
and Doerr 2006; Brooks 2008; Keeley 2009). Terms explaining these relationships are
frequently used inconsistently (Lentile et al. 2006; Keeley 2009), especially among
nonfire-specific disciplines like hydrology. Therefore, we present a brief summary of the
fire terms used here to discuss fire effects on rangeland hydrology. Fires are categorized
as ground, surface, or crown type, and each is associated with a particular behavior
(DeBano et al. 1998; Shakesby and Doerr 2006; Brooks 2008). Fire behavior refers to the
rate of spread, residence time, and flame dimensions, and is related to fuel, weather, and
topographic conditions at the time of burning (DeBano et al. 1998, Brooks 2008). Ground
fires are mostly flame-free and burn slowly through duff or decayed organic matter on
the soil surface. Surface fires burn rapidly and consume litter, woody dead material at
or near the surface, herbaceous fuels, shrubs, and small trees. Crown fires burn rapidly
through canopies of trees and tall shrubs, leaving most of the stem and land surface fuels
unburned. An individual fire may comprise one or more of these three primary fire types
(see DeBano et al. 1998).i The term “fire regime” refers to the pattern of repeated burning within a large spatial
expanse (landscape scale) over long time periods and is defined by a characteristic combi-
nation of fire type, frequency, intensity, severity, size, and seasonality (Brooks 2008;
Baker 2009). Fire frequency is the number of fires that occur over a specified period of
time for a particular area, and may be expressed as a return interval/cycle or the length of
time necessary for the area of interest to burn. Seasonality is the period of the year when
fires are most likely to occur. Fire intensity refers to the amount of heat released. Severity
is the degree of impact to soils or vegetation, or to both (see Keeley 2009). Fire behavior, intensity, and severity are affected by the vertical and horizontal con-
tinuity and density of fuels. Fuels, as with fire types, are commonly divided vertically into
layers termed “ground” (duff, roots, and buried or partially buried woody dead materials),
“surface” (litter, herbaceous plants, and low shrubs), and “canopy” (tall shrubs and trees)
fuels (DeBano et al. 1998). Cover Effects on Concentrated Flow Erosion These features indicate
that concentrated flow does redistribute surface soil from bare areas to vegetated zones
on healthy rangelands, but hillslope soil loss from this process is minor under such condi-
tions (Pierson et al. 2007a, 2009). In contrast, concentrated flow becomes the dominant
erosion mechanism on degraded rangelands where ground cover is sparse (Moffet et al. 2007; Pierson et al. 2008a, 2009, 2011, 2013; Williams et al. 2014a,b). 52 Section 4: Impacts of Fire on Rangeland Runoff and
Erosion Section 4: Impacts of Fire on Rangeland Runoff and
Erosion Fire Behavior and Regimes on Rangelands Extensive horizontal continuity of surface fuels, canopy fuels,
or both, facilitates large fires, especially under dry and windy conditions. Breaks in fuel
continuity retard fire progression. Rangeland fuels usually have low horizontal continuity
of surface and canopy fuels—except on more productive sites or following long fire-free
periods (Brooks 2008; Keane et al. 2008). The spread of fire from surface fuels to the
canopy often requires ladder fuels (horizontally connected heterogeneous vertical con-
tinuity). Fire intensity is largely dictated by the amount or density of fuel, or both. Fuel
density (amount of fuel per unit volume of space) influences combustion and the duration
of burning. Loosely packed fuels provide sufficient air supply for higher combustion and
rapid rates of spread relative to densely packed fuels (DeBano et al. 1998; Baker 2009). Fuel moisture conditions and temperature also influence fire intensity by regulating 53 USDA Forest Service RMRS-GTR-351. 2016. flammability and combustion. Dry, hot fuels exhibit higher flammability, combustion, and
rates of spread than moist, cool fuels (DeBano et al. 1998; Baker 2009). The variable most commonly referenced in assessing fire impacts on hydrology
is severity (Pierson et al. 2001a, 2002b; Lewis et al. 2006; Shakesby and Doerr 2006;
Pierson et al. 2008a, 2009; Parsons et al. 2010; Pierson et al. 2014; Williams et al. 2014a,b), although it is not quantified consistently (Keeley 2009). In general, fire severity
is a function of fire intensity and residence time (DeBano et al. 1998). Numerous severity
classification systems exist, but three classes are common: low, moderate, and high
severity (DeBano et al. 1998; Baker 2009; Parsons et al. 2010). Low-severity fires cause
low soil heating (100 to 250 °C), light charring or minor reduction of litter, and virtually
no consumption (but some charring) of duff. Moderate-severity fires char the ground
surface without visible alteration of the mineral soil surface, leave behind some gray- to
black-colored ash, consume litter and most woody debris (except logs), and have surface
temperatures (up to 1 cm depth) of 300 to 400 °C. High-severity fires produce surface
temperatures in excess of 500 °C, create deep ground charring where duff is completely
consumed, visibly affect the upper mineral soil layer (leaving it reddish in color), and
result in white ash from consumption of grasses, litter, and most shrub stems.i Wells et al. (1979) extended severity classification over large expanses based on
total area burned at low, moderate, or high intensities. Fire Behavior and Regimes on Rangelands They suggested the following
framework: (1) low-severity burn (less than 2 percent severely burned, less than
15 percent moderately burned, remainder is unburned or burned at low severity);
(2) moderate-severity burn (less than 10 percent severely burned, more than 15 percent
burned moderately, remainder unburned or burned at low severity); and (3) high-severity
burn (more than 10 percent with patches of high burn severity, more than 80 percent
moderate to severely burned, remainder burned at low severity). Brown (2000) catego-
rized fire regimes loosely related to recurring uniformity of fire severity over a specified
area of interest. The categories are: (1) stand-replacement regime (lethal burning of 80
percent or more of dominant vegetation), (2) understory fire regime (generally nonlethal
to dominant vegetation), and (3) mixed-severity fire regime (recurring fire produces
variable response in time and space, from nonlethal understory to stand-replacement fire). The uniform fine fuels on most grassland communities produce a stand-replacement fire
regime (DeBano et al. 1998; Rice et al. 2008). These fires may be of high intensity, but
generally cause only minor surface soil heating (100 to 400 °C) (Wright and Bailey 1982)
due to the lack of woody fuels and low residence times (DeBano et al. 1998). In contrast,
shrubland ecosystems may exhibit a mixed-severity or stand-replacement fire regime
(Rice et al. 2008) and yield soil surface temperatures of 260 to 700 °C (Wright and Bailey
1982). Parsons et al. (2010) provide guidance on field mapping of burn severity. Fire Effects that Dictate Hydrologic Response Reduced Interception and Surface Protection USDA Forest Service RMRS-GTR-351. 2016. Reduced Interception and Surface Protection Consumption of canopy and ground cover by fire reduces interception capacity and
surface water retention and thus increases the quantity and intensity of water input at the
soil surface and the flow volume and velocity across it (DeBano et al. 1998; Shakesby
and Doerr 2006). The amount of additional water input made available by burning 54 USDA Forest Service RMRS-GTR-351. 2016. Figure 18—(A) Postfire landscape (3 months after wildfire) at the Denio Fire, Pine Mountain Range, Nevada,
and cover removal by fire for (B) shrub coppice and (C) vegetated interspace experimental plots (0.5 m2)
(photos: USDA Agricultural Research Service, Northwest Watershed Research Center). Figure 18—(A) Postfire landscape (3 months after wildfire) at the Denio Fire, Pine Mountain Range, Nevada,
and cover removal by fire for (B) shrub coppice and (C) vegetated interspace experimental plots (0.5 m2)
(photos: USDA Agricultural Research Service, Northwest Watershed Research Center). is dependent on the interception and storage capacity of the postburn cover. General
estimates suggest that the quantity of interception by unburned rangeland trees, shrubs,
and grasses approximates 1 to 2 mm of rainfall per storm (Bonan 2002) depending on the
cover biomass, rainfall intensity and duration, cover moisture content, and the vertical
and horizontal arrangements of cover elements (see Section 3, Vegetation and Cover
Influences on Infiltration and Runoff Generation).l is dependent on the interception and storage capacity of the postburn cover. General
estimates suggest that the quantity of interception by unburned rangeland trees, shrubs,
and grasses approximates 1 to 2 mm of rainfall per storm (Bonan 2002) depending on the
cover biomass, rainfall intensity and duration, cover moisture content, and the vertical
and horizontal arrangements of cover elements (see Section 3, Vegetation and Cover
Influences on Infiltration and Runoff Generation).l The conversion of interception loss (table 2) and stemflow rates to rainfall arrival at
the soil surface is nearly 100 percent where severe burning uniformly removes all canopy
and ground cover elements (fig. 18). Postfire reductions in raindrop dissipation (increases 55 USDA Forest Service RMRS-GTR-351. 2016. in drop intensity) are as important as the increases in the quantity of water (Shakesby
and Doerr 2006). Raindrop energy dictates splash sediment detachment under disturbed
conditions whereas the quantity of overland flow and the downslope velocity govern flow
detachment and transport capacity (Pierson et al. 2008a, 2009; Al-Hamdan et al. 2012b). Reduced Interception and Surface Protection Greater raindrop impact after canopy and ground cover removal results in increased soil
detachment from rainsplash processes (Shakesby and Doerr 2006; Pierson et al. 2001a,
2002b, 2008a, 2009, 2013, 2014; Williams et al. 2014a, 2016a). Reductions in ground
cover (decreased surface roughness) abate surface retention of overland flow, allowing
flow to concentrate and move downslope with greater velocity, erosive energy, and
transport capacity (Shakesby and Doerr 2006; Pierson et al. 2008a, 2009; Al-Hamdan et
al. 2012a,b, 2013; Pierson et al. 2013; Williams et al. 2014a, 2016a). The potential overall
effect is a decrease in the time to runoff generation and an increase in cumulative runoff
and sediment yield over the duration of a storm event. In snow-dominated environments, removal of vegetation by fire may alter snow ac-
cumulation, the timing of runoff initiation, cessation, and peak flow within the year, and
the amount of snowmelt runoff. Burning may also result in increased surface tempera-
tures and snowmelt rates due to greater incoming solar radiation postburn (Tiedemann et
al. 1979). Any reduction in vegetation, therefore, reduces snow accumulation and water
availability for biological processes and streamflow generation. Reduced snow retention
also potentially alters runoff characteristics from summer thunderstorms on water-limited
sites by inhibiting vegetation production and ground cover recruitment. Where snow does
accumulate, runoff responses to mid-winter rain-on-snow events may be substantial after
burning (see Marks et al. 2001; Pierson et al. 2001b). Physical, Chemical, and Biological Alteration of Soils Hydrologically important soil properties (porosity, soil moisture, stability, structure,
and water repellency) are strongly influenced by vegetation, organic debris, and micro-
organisms, which can be removed at varying degrees by burning (DeBano et al. 1998;
Shakesby and Doerr 2006). The magnitude at which these properties are influenced by
burning depends on the degree of soil heating and the amount of organic matter removed. The impact of burning on soils is at a maximum when the entire canopy and all surface
organics are consumed and the mineral surface is exposed. The primary effect of fire
relative to organic matter is to expedite the mineralization process. Postfire recovery of
consumed organic matter may take as long as 5 years on some rangelands (Wright and
Bailey 1982). Soil organic matter is combusted at temperatures above 200 ˚C and is
completely consumed at 500 ˚C (DeBano et al. 1998). These temperatures are well within
the range of surface temperatures commonly reported for grassland and shrubland fires
(Wright and Bailey 1982; Miller et al. 2013). Subsurface soil temperatures on grasslands
usually are unaffected by burning, but can range from approximately 100 to 250 °C
to depths of 5 cm on shrublands (Wright and Bailey 1982; DeBano et al. 1998). The
combustion of organic matter from surface and subsurface soils can alter soil structure,
increase bulk density, and decrease porosity (Giovannini and Lucchesi 1997; Giovannini
et al. 1988; Pierson et al. 2001a,b; Hubbert et al. 2006; Shakesby and Doerr 2006). Soil stability and aggradation can also be reduced through alteration of soil particles
during burning (DeBano et al. 1998; Neary et al. 1999; Andreu et al. 2001; Giovannini et 56 USDA Forest Service RMRS-GTR-351. 2016. al. 2001; Hubbert et al. 2006; Shakesby and Doerr 2006; Mataix-Solera et al. 2011). Soil
temperatures between 150 and 400 °C can drive off hydroxyl groups in clays and increase
erodibility (Giovannini and Lucchesi 1997; DeBano et al. 1998). Collectively, these soil
alterations inhibit infiltration and promote runoff generation and erosion (Hester et al. 1997; DeBano et al. 1998; Shakesby and Doerr 2006). Ash at the soil surface may clog
surface soil pores and further accentuate the runoff response (Campbell et al. 1977; Wells
et al. 1979; Lavee et al. 1995; Neary et al. 1999), although contrary results have been
reported recently for the immediate postfire period (Cerdà and Doerr 2008; Woods and
Balfour 2008; Larsen et al. Physical, Chemical, and Biological Alteration of Soils 2009; Bodí et al. 2012; Ebel et al. 2012). Burning may reduce the role of invertebrates, microorganisms, and mycorrhizae
fungi in facilitating infiltration (Ahlgren and Ahlgren 1960; Wright and Bailey 1982;
DeBano et al. 1998; Shakesby and Doerr 2006). Mycorrhizae and the by-products of soil
fauna promote soil aggregation. Voids created by fauna movement within the soil profile
increase porosity. Soil temperatures above 40 to 210 °C are fatal for most fungi and soil
organisms; organic matter consumption by fire reduces the primary food source for soil
fauna (Wright and Bailey 1982; DeBano et al. 1998; Mataix-Solera et al. 2009). Recovery
of soil fauna and fungi may be rapid (Neary et al. 1999) depending on postfire soil micro-
climate and available nutrients, but can take as much as 3 to 5 years where resources are
limited (Wright and Bailey 1982). Finally, soil moisture retention, a key component of
plant and soil fauna productivity in water-limited ecosystems, is also adversely affected
by the loss of pore structure and surface insulation (against evaporation) by litter (Wright
and Bailey 1982; DeBano et al. 1998). Exacerbation, Alteration, and Formation of Soil Water Repellency Soil heating during burning may enhance, reduce, or create hydrophobic soil condi-
tions (DeBano and Krammes 1966; DeBano et al. 1970; Savage 1974; DeBano et al. 1976; DeBano 1981, 1991; Shakesby et al. 1993; Doerr et al. 1996; DeBano et al. 1998;
DeBano 2000; Doerr et al. 2000; Robichaud 2000; Shakesby et al. 2000; Benavides-
Solorio and MacDonald 2001; Huffman et al. 2001; Doerr et al. 2004; MacDonald and
Huffman 2004; Doerr et al. 2006; Hubbert et al. 2006; Fox et al. 2007; Pierson et al. 2008b; Arcenegui et al. 2008; Doerr et al. 2009; Shakesby 2011). The key determinants
of whether soil water repellency is enhanced, reduced, or created during burning are the
presence of organic matter and the soil temperature reached during burning (DeBano and
Krammes 1966; DeBano et al. 1970; Doerr et al. 1996; DeBano et al. 1998; Doerr et al. 2004, 2009). Naturally occurring soil water repellency (fig. 19) is typically unaltered by
soil temperatures less than 175 °C (DeBano 1981). Soil temperatures between 175 and
270 °C enhance “background” water repellency or may form hydrophobic soil conditions
(DeBano 1981; Doerr et al. 2000, 2009). Water repellency breaks down or is destroyed
at soil temperatures above 270 to 400 °C (Savage 1974; DeBano et al. 1976; Giovannini
and Lucchesi 1997; Doerr et al. 2004).i During fires, combustion of organic matter at the soil surface radiates heat down-
ward into the soil profile and vaporizes organic substances. Some of these substances
are translocated downward along temperature gradients until they cool and condense,
forming a variable-thickness hydrophobic layer parallel to the soil surface (DeBano 1981;
Doerr et al. 1996; DeBano et al. 1998; DeBano 2000; Doerr et al. 2000, 2004, 2009). The 57 USDA Forest Service RMRS-GTR-351. 2016. Figure 19—Dry soil underneath a 2- to 3-cm thick layer of water repellent soil
observed immediately after application of 85 mm of rainfall (see Pierson et
al. 2008b) over a 1-h period following the Breaks Prescribed Fire, Reynolds
Creek Experimental Watershed, Idaho. Soils were strongly water repellent
at the mineral soil surface and to a depth of 2 to 3 cm immediately before
and after the prescribed fire (photo: USDA Agricultural Research Service,
Northwest Watershed Research Center). Figure 19—Dry soil underneath a 2- to 3-cm thick layer of water repellent soil
observed immediately after application of 85 mm of rainfall (see Pierson et
al. Figure 20—Spatial (by microsite) and
annual variation in soil water repellency
(measured by water drop penetration
time, WDPT) measured on unburned
and burned soils underneath shrubs
and in interspaces areas at the Upper
Sheep Creek Prescribed Burn site
in the Reynolds Creek Experimental
Watershed. Year 1 and year 2
measurements are 1 and 2 years
postfire, respectively. WDPT is an
indicator of strength of soil water
repellency as follows: <5 s wettable,
5 to 60 s slightly repellent, 60 to 600 s
strongly repellent (Bisdom et al. 1993). Exacerbation, Alteration, and Formation of Soil Water Repellency Figure 20—Spatial (by microsite) and
annual variation in soil water repellency
(measured by water drop penetration
time, WDPT) measured on unburned
and burned soils underneath shrubs
and in interspaces areas at the Upper
Sheep Creek Prescribed Burn site
in the Reynolds Creek Experimental
Watershed. Year 1 and year 2
measurements are 1 and 2 years
postfire, respectively. WDPT is an
indicator of strength of soil water
repellency as follows: <5 s wettable,
5 to 60 s slightly repellent, 60 to 600 s
strongly repellent (Bisdom et al. 1993). Figure 21—Soil water repellency measured by using the water drop penetration time (WDPT) method on
unburned and burned areas underneath Utah juniper and pinyon (Marking Corral site) and Utah juniper
(Onaqui site) (see Pierson et al. 2010, 2014). WDPT is an indicator of strength of soil water repellency
as follows: <5 s wettable, 5 to 60 s slightly repellent, 60 to 600 s strongly repellent (Bisdom et al. 1993). WDPTs at both sites indicate translocation of prefire repellency from the surface to deeper soil layers (~ 2
to 4 cm) through the first year postfire. The soil water repellency profile 2 years postfire was similar to that
of prefire conditions with stronger repellency at the mineral soil surface. Figure 20—Spatial (by microsite) and
annual variation in soil water repellency
(measured by water drop penetration
time, WDPT) measured on unburned
and burned soils underneath shrubs
and in interspaces areas at the Upper
Sheep Creek Prescribed Burn site
in the Reynolds Creek Experimental
Watershed. Year 1 and year 2
measurements are 1 and 2 years
postfire, respectively. WDPT is an
indicator of strength of soil water
repellency as follows: <5 s wettable,
5 to 60 s slightly repellent, 60 to 600 s
strongly repellent (Bisdom et al. 1993). Figure 21—Soil water repellency measured by using the water drop penetration time (WDPT) method on
unburned and burned areas underneath Utah juniper and pinyon (Marking Corral site) and Utah juniper
(Onaqui site) (see Pierson et al. 2010, 2014). WDPT is an indicator of strength of soil water repellency
as follows: <5 s wettable, 5 to 60 s slightly repellent, 60 to 600 s strongly repellent (Bisdom et al. 1993). WDPTs at both sites indicate translocation of prefire repellency from the surface to deeper soil layers (~
to 4 cm) through the first year postfire. Exacerbation, Alteration, and Formation of Soil Water Repellency 2008b) over a 1-h period following the Breaks Prescribed Fire, Reynolds
Creek Experimental Watershed, Idaho. Soils were strongly water repellent
at the mineral soil surface and to a depth of 2 to 3 cm immediately before
and after the prescribed fire (photo: USDA Agricultural Research Service,
Northwest Watershed Research Center). depth at which the water-repellent layer occurs is related in part to the degree of heating
or fire severity (Huffman et al. 2001). Generally, higher surface temperatures, up to that at
which repellency is destroyed, increase the depth at which soil water repellency is found
(DeBano et al. 1998). Fire-enhanced or fire-induced soil water repellency is commonly
found at or within the first 5 cm of the soil surface, and rapidly decreases in strength with
increasing soil depth (Benavides-Solorio and MacDonald 2001; Huffman et al. 2001;
MacDonald and Huffman 2004; Benavides-Solorio and MacDonald 2005; Pierson et al. 2008b, 2009). The spatial and temporal persistence (figs. 19 and 20) of postburn soil water repel-
lency are highly variable (Imeson et al. 1992; Doerr et al. 2000; Huffman et al. 2001;
MacDonald and Huffman 2004; Hubbert et al. 2006; Woods et al. 2007; Pierson et al. 2008b, 2009, 2013; Bodí et al. 2013; Williams et al. 2014b). The strength of postburn
soil water repellency varies with the spatial continuity of the respective burn. Repellency
strength is usually positively correlated with burn severity, the amount of organic material
present during combustion, and the steepness of the downward soil temperature gradient
during soil heating (DeBano et al. 1998). Steep temperature gradients enhance downward
translocation (fig. 21). The steepness of the gradient is regulated in part by soil water
content and its influence on heat transfer. The persistence of fire-induced soil water repel-
lency commonly ranges from less than 1 to 6 years (DeBano et al. 1976; Huffman et al. 2001; MacDonald and Huffman 2004). The influence of repellency on infiltration is highly variable over seasonal and
annual time scales (fig. 22) and is related to variations in soil moisture content (Shakesby
et al. 1993; Burcar et al. 1994; Dekker and Ritsema 1994; Ritsema and Dekker 1994;
Dekker and Ritsema 1996; Doerr et al. 2000; Shakesby et al. 2000; Benavides-Solorio
and MacDonald 2001; Dekker et al. 2001; MacDonald and Huffman 2004; Hubbert et 58 USDA Forest Service RMRS-GTR-351. 2016. Exacerbation, Alteration, and Formation of Soil Water Repellency The soil water repellency profile 2 years postfire was similar to th
of prefire conditions with stronger repellency at the mineral soil surface. Figure 20—Spatial (by microsite) and
annual variation in soil water repellency
(measured by water drop penetration
time, WDPT) measured on unburned
and burned soils underneath shrubs
and in interspaces areas at the Upper
Sheep Creek Prescribed Burn site
in the Reynolds Creek Experimental
Watershed. Year 1 and year 2
measurements are 1 and 2 years
postfire, respectively. WDPT is an
indicator of strength of soil water
repellency as follows: <5 s wettable,
5 to 60 s slightly repellent, 60 to 600 s
strongly repellent (Bisdom et al. 1993). Figure 21—Soil water repellency measured by using the water drop penetration time (WDPT) method on
unburned and burned areas underneath Utah juniper and pinyon (Marking Corral site) and Utah juniper
(Onaqui site) (see Pierson et al. 2010, 2014). WDPT is an indicator of strength of soil water repellency
as follows: <5 s wettable, 5 to 60 s slightly repellent, 60 to 600 s strongly repellent (Bisdom et al. 1993). WDPTs at both sites indicate translocation of prefire repellency from the surface to deeper soil layers (~ 2
to 4 cm) through the first year postfire. The soil water repellency profile 2 years postfire was similar to that
of prefire conditions with stronger repellency at the mineral soil surface. Figure 21—Soil water repellency measured by using the water drop penetration time (WDPT) method on
unburned and burned areas underneath Utah juniper and pinyon (Marking Corral site) and Utah juniper
(Onaqui site) (see Pierson et al. 2010, 2014). WDPT is an indicator of strength of soil water repellency
as follows: <5 s wettable, 5 to 60 s slightly repellent, 60 to 600 s strongly repellent (Bisdom et al. 1993). WDPTs at both sites indicate translocation of prefire repellency from the surface to deeper soil layers (~ 2
to 4 cm) through the first year postfire. The soil water repellency profile 2 years postfire was similar to that
of prefire conditions with stronger repellency at the mineral soil surface. 59 USDA Forest Service RMRS-GTR-351. 2016. al. 2006; Pierson et al. 2008b; Bodí et al. 2013) and seasonal wetting and drying trends
(Doerr and Thomas 2000; Dekker et al. 2001). Fire Effects on Infiltration and Runoff Generation The degree to which fire affects infiltration and runoff processes depends on the
magnitude of alterations to soil properties, vegetation, and ground cover (DeBano et
al. 1998; Shakesby and Doerr 2006; Pierson et al. 2011; Williams et al. 2014b, 2016a). Runoff generation further depends on water arrival in excess of aboveground storage,
surface storage, and infiltration. Thus, the rainfall intensity and duration as well as the
conditions of the vegetative community and ground surface are key determinants of the
runoff response (Benavides-Solorio and MacDonald 2005; Spigel and Robichaud 2007). Hydrologic response is further influenced by topographic attributes such as hillslope
steepness (Al-Hamdan et al. 2013). The occurrence and hydrologic value of soil fauna,
organic matter, macropores, and soil structure are closely related to the presence of veg-
etation and ground cover (Belnap et al. 2005), particularly in water-limited environments
like rangelands (Ludwig et al. 1997, 2005). Therefore, many assessments of fire effects
on infiltration and runoff consider alteration of vegetation, ground cover, and surface
properties solely and exclude investigation of fire effects on soil organisms, macropores,
and soil organic matter.il Literature clearly indicates that fire influences soil fauna, macropores, and soil
organic matter (Wright and Bailey 1982; DeBano et al. 1998; Neary et al. 1999; Mataix-
Solera et al. 2009; Certini et al. 2011; Mataix-Solera et al. 2011), but their respective
alteration and direct relation to postfire hydrologic responses are rarely specifically
measured or cited. Alteration of canopy, ground cover, and surface soil properties com-
monly serve as a surrogate for the collective fire impact and exhibit strong correlations
with postfire hydrologic response (Benavides-Solorio and MacDonald 2001; Johansen et
al. 2001; Benavides-Solorio and MacDonald 2005; Shakesby and Doerr 2006; Cerdà and
Doerr 2008; Pierson et al. 2008a, 2009, 2013, 2014; Williams et al. 2014a,b). Vegetation
and ground cover intercept rainfall and overland flow, surface roughness promotes
ponding, and surface soil characteristics influence infiltration rates. Ponding delays runoff
generation and allows water to infiltrate through breaks in hydrophobic soils or through
macropores created by root channels, organic matter, or soil fauna. Organic matter input
through plants and soil fauna promotes aggregate stability and infiltration by enhancing
soil structure. From literature, three primary points emerge relative to these relationships:
(1) canopy cover and surface protection are paramount in reducing water availability
for runoff (Cerdà 1998a; Johansen et al. 2001; Pierson et al. 2001a; Benavides-Solorio
and MacDonald 2005; Pierson et al. Exacerbation, Alteration, and Formation of Soil Water Repellency The overall effect of preexisting or fire-
induced soil water repellency is exacerbated by the removal of vegetation and ground
cover (Benavides-Solorio and MacDonald 2001, 2002; Pierson et al. 2008b, 2009, 2013,
2014; Williams et al. 2014a). The increased availability of water arriving at the soil
surface, lack of surface protection, and presence of hydrophobic soil conditions promote
runoff generation and surface soil erosion by raindrop-induced flow, sheetflow, and
concentrated flow processes (DeBano and Krammes 1966; DeBano et al. 1979; Morris
and Moses 1987; Scott and van Wyk 1990; Terry and Shakesby 1993; Inbar et al. 1997;
DeBano 2000; Shakesby et al. 2000; Benavides-Solorio and MacDonald 2001, 2002,
2005; Shakesby and Doerr 2006; Pierson et al. 2008b; Larsen et al. 2009; Pierson et al. 2009, 2014; Williams et al. 2014a,b). Figure 22—Temporal variability of soil water repellency (measured by using water
drop penetration time, WDPT) effects on infiltration of artificial rainfall into unburned
and burned, coarse-textured soils at two sagebrush sites: (A) Denio Wildfire, Pine
Mountain Range, Nevada (Pierson et al. 2008a) and (B) Breaks Prescribed Fire,
Reynolds Creek Experimental Watershed, Idaho (Pierson et al. 2009). WDPT is
an indicator of strength of soil water repellency as follows: <5 s wettable, 5 to 60 s
slightly repellent, 60 to 600 s strongly repellent (Bisdom et al. 1993). Figure 22—Temporal variability of soil water repellency (measured by using water
drop penetration time, WDPT) effects on infiltration of artificial rainfall into unburned
and burned, coarse-textured soils at two sagebrush sites: (A) Denio Wildfire, Pine
Mountain Range, Nevada (Pierson et al. 2008a) and (B) Breaks Prescribed Fire,
Reynolds Creek Experimental Watershed, Idaho (Pierson et al. 2009). WDPT is
an indicator of strength of soil water repellency as follows: <5 s wettable, 5 to 60 s
slightly repellent, 60 to 600 s strongly repellent (Bisdom et al. 1993). USDA Forest Service RMRS-GTR-351. 2016. 60 Rainsplash and Sheetflow Processes Over the last decade, a number of researchers have measured fire effects on infiltra-
tion and runoff over small-plot scales (0.25 to 1 m2) (table 3) (Cerdà 1998a; Robichaud
2000; Benavides-Solorio and MacDonald 2001; Pierson et al. 2001a; Benavidez-Solorio
and MacDonald 2002; Pierson et al. 2002b; Cerdà and Doerr 2005; Groen and Woods
2008; Pierson et al. 2008a,b; Woods and Balfour 2008; Pierson et al. 2009, 2013, 2014;
Williams et al. 2014a,b, 2016a). Small-plot sizes limit inferences to rainsplash and
sheetflow processes solely (Mutchler et al. 1988). These studies have, however, provided
valuable insight into infiltration and runoff generation for both burned and unburned
conditions, and elaborate on the magnitude of runoff response to burning. A series of
rainfall simulation studies on small (0.5 m2) plots (Pierson et al. 2001a, 2002b, 2008a,
2008b, 2009) (see table 3) on steeply sloped (35 to 60 percent) sagebrush rangelands
demonstrate the effects of cover removal, surface alteration, and soil water repellency on
postfire rangeland infiltration and runoff. Pierson et al. (2002b) (see table 3) investigated the hydrologic effects of moderate-
and high-severity burning of sagebrush rangelands on north- and south-facing hillslopes
1 year after the Eighth Street Fire in 1996 near Boise, Idaho. We restrict our discussion of
the study to the south-facing hillslopes for brevity. Prefire total live and litter masses were
32,519 kg ha-1 and 14,372 kg ha-1, respectively, on shrub coppices and 519 kg ha-1 and
1,721 kg ha-1 in interspaces. Moderate- and high-severity burning reduced shrub coppice
total live and litter mass by nearly 100 percent. Moderate burning of interspaces had no
effect on total live mass, but reduced litter mass by 90 percent. High-severity burning of
interspaces reduced total live and litter masses by 74 percent and 96 percent, respectively. Percent bare ground (see table 3) increased on shrub coppices from 7 percent prefire to
nearly 100 percent for the moderate- and high-severity conditions. Interspace bare ground
was high prefire (89 percent) and increased to nearly 100 percent following the burn. Soil
organic matter (2 to 6 percent) was nearly equal across all plots and was not affected by
burning. Near-surface bulk density (0 to 2 cm) was slightly higher on interspace plots
(1.35 g cm-3) than on coppices (1.21 g cm-3) and was not significantly altered by burning. Gravimetric soil-water content ranged from 5 to 14 percent. Fire Effects on Infiltration and Runoff Generation 2008a, 2009, 2013; Williams et al. 2014b); (2) the
effects of decreased surface protection on water availability are markedly influenced
by soil conditions (such as soil water repellency) that inhibit or promote infiltration
and storage (Pierson et al. 2001a,b; Shakesby and Doerr 2006; Pierson et al. 2008a,b,
2009, 2013, 2014; Williams et al. 2014a); and (3) the effects of decreased canopy cover,
surface protection, or the co-occurrence of these effects, are intensified with increasing
rainfall intensity (Inbar et al. 1998; Benavides-Solorio and MacDonald 2005; Spigel
and Robichaud 2007; Larsen et al. 2009; Williams et al. 2014b) and hillslope angle (Al-
Hamdan et al. 2013). 61 USDA Forest Service RMRS-GTR-351. 2016. Rainsplash and Sheetflow Processes Postfire increases in
runoff were also associated with decreasing surface roughness.i A 3-year investigation by Pierson et al. (2001a, 2008a,b) (see table 3, figs. 22A and
24) measured infiltration and runoff of rainsplash and sheetflow following the Denio Fire
in 1999 in Nevada. The fire burned steeply sloping (30 to 40 percent) mountain big sage-
brush at high severity. A convective-type storm was simulated by applying 85 mm h-1
rainfall intensity for 60 min to plots 0.5 m2 in size. Before the fire, total ground cover was
nearly 100 percent in shrub coppice and interspace areas. Shrub canopy cover dominated
in coppice areas whereas grasses dominated interspaces. Canopy cover was consumed
entirely by the wildfire, and bare ground increased from less than 10 percent to more than
95 percent. Ground cover recovered to about 60 percent (40 percent bare ground) follow-
ing the second growing season, but remained significantly different from the unburned
condition (90 percent). Shrub cover was slow to recover (5 percent after two growing
seasons) and litter after the second growing season amassed 30 percent coverage. Surface bulk densities (0 to 4 cm) were not significantly different between prefire
coppice (0.93 g cm-3) and interspace (0.94 g cm-3) areas in the Pierson et al. (2001,
2008a,b) studies. Burning increased bulk density (to 1.21 g cm-3) on both microsites,
presumably by decreasing organic matter content (Pierson et al. 2001a), although organic
matter was not specifically measured. Soil water repellency was strong on all unburned
plots the year of the fire and was reduced more than 50 percent by high-severity burning
on shrub coppice and interspace microsites (Pierson et al. 2008b). Soil water repellency
across all plots (fig. 22A) decreased by 55 percent from the year of the fire (year 0) to 1
year postfire (year 1) and then increased across all plots approximately 40 to 50 percent
from year 1 to year 2 (2 years postfire). Runoff coefficients (table 3) were slightly
greater, but not significantly different, for burned (37 percent) than unburned (30 percent)
shrub coppices immediately postfire, and were significantly higher for the unburned (49
percent) than burned (30 percent) interspaces. Pierson et al. (2001, 2008a,b) explained that slightly greater runoff and significantly
lower minimum infiltration on burned coppices resulted from the removal by fire of
canopy and ground cover over strongly water-repellent soils. Rainsplash and Sheetflow Processes Surface roughness decreased
by 30 percent after moderate- and high-severity burning of coppice microsites and by 30
and 45 percent after moderate- and high-severity burning, respectively, on interspaces. Runoff in the Pierson et al. (2002b) study was measured during simulation of a
summer season convective storm with an intensity of 67 mm h-1 and duration of 60 min. Runoff coefficients (runoff per unit of applied rainfall) from unburned hillslopes were
11 percent for coppice areas and 24 percent for interspace areas. Runoff coefficients for
coppices increased threefold following moderate- and high-severity burning. Runoff
coefficients for interspaces were nearly equal (24 to 26 percent) for unburned and moder-
ate-severity plots, but increased twofold to nearly 50 percent for high-severity burn plots. Greater infiltration and lower runoff on coppices prefire were attributed to interception
and surface water retention associated with high live canopy and litter biomass. Runoff
generally increased with the burn severity, potentially owing to enhanced background
soil water repellency. Soil water repellency was not directly assessed, but the shape of
the infiltration curves clearly indicates the presence of water repellency (steeply decreas-
ing followed by gradual increase throughout simulation) (fig. 23) (see Meeuwig 1971; 62 USDA Forest Service RMRS-GTR-351. 2016. Imeson et al. 1992; Robichaud 2000; Pierson et al. 2001a, 2008b). Postfire increase
runoff were also associated with decreasing surface roughness
Figure 23—Infiltration of artificial
rainfall (67 mm h-1, 60 min) applied
on burned (coppice – BC, interspace
– BI) and unburned (coppice –
UC, interspace – UI) sagebrush
hillslopes following the Eighth Street
Wildfire in 1996 near Boise, Idaho. Data from Pierson et al. (2002b). Figure 23—Infiltration of artificial
rainfall (67 mm h-1, 60 min) applied
on burned (coppice – BC, interspace
– BI) and unburned (coppice –
UC, interspace – UI) sagebrush
hillslopes following the Eighth Street
Wildfire in 1996 near Boise, Idaho. Data from Pierson et al. (2002b). Figure 23—Infiltration of artificial
rainfall (67 mm h-1, 60 min) applied
on burned (coppice – BC, interspace
– BI) and unburned (coppice –
UC, interspace – UI) sagebrush
hillslopes following the Eighth Street
Wildfire in 1996 near Boise, Idaho. Data from Pierson et al. (2002b). Imeson et al. 1992; Robichaud 2000; Pierson et al. 2001a, 2008b). Postfire increases in
runoff were also associated with decreasing surface roughness. Imeson et al. 1992; Robichaud 2000; Pierson et al. 2001a, 2008b). USDA Forest Service RMRS-GTR-351. 2016. Rainsplash and Sheetflow Processes Canopy and litter cover
on unburned coppices mitigated the effects of strong soil water repellency whereas
the removal of cover accentuated the effects of the persistent, but reduced, soil water
repellency on burned coppices (Pierson et al. 2008b). The decrease in runoff from 63 USDA Forest Service RMRS-GTR-351. 2016. Figure 24—(A) The Denio Fire (wildfire), 1999, Pine Mountain Range, Nevada, (B) rainfall
simulations, and (C) 0.5 m plots used in rainfall simulation studies at the site (Pierson et
al. 2001a, 2008a,b) (photos: USDA Agricultural Research Service, Northwest Watershed
Research Center). Figure 24—(A) The Denio Fire (wildfire), 1999, Pine Mountain Range, Nevada, (B) rainfall
simulations, and (C) 0.5 m plots used in rainfall simulation studies at the site (Pierson et
al. 2001a, 2008a,b) (photos: USDA Agricultural Research Service, Northwest Watershed
Research Center). interspace areas was associated with removal of water-shedding senescent vegetation
and fire-reduced strength of soil water repellency. The influence of soil water repellency
on the overall hydrologic response is shown by nearly equal runoff coefficients (10 to
20 percent) and significantly reduced strength (by 55 percent) of soil water repellency
in year 1 across all plots, regardless of differences in canopy and ground cover (Pierson
et al. 2008a,b). A subsequent significant increase in soil water repellency across all
plots in year 2 coincided with a twofold to threefold increase in runoff coefficients for
burned interspaces and all coppices and the reestablishment of differences in minimum
infiltration between burned and unburned coppices (Pierson et al. 2008a,b). In summary,
canopy and ground cover removal dictated water availability whereas the strength of soil
water repellency exerted greater influence on infiltration processes and runoff generation
(Pierson et al. 2008b). interspace areas was associated with removal of water-shedding senescent vegetation
and fire-reduced strength of soil water repellency. The influence of soil water repellency
on the overall hydrologic response is shown by nearly equal runoff coefficients (10 to
20 percent) and significantly reduced strength (by 55 percent) of soil water repellency
in year 1 across all plots, regardless of differences in canopy and ground cover (Pierson
et al. 2008a,b). A subsequent significant increase in soil water repellency across all
plots in year 2 coincided with a twofold to threefold increase in runoff coefficients for
burned interspaces and all coppices and the reestablishment of differences in minimum
infiltration between burned and unburned coppices (Pierson et al. 2008a,b). Rainsplash and Sheetflow Processes In summary,
canopy and ground cover removal dictated water availability whereas the strength of soil
water repellency exerted greater influence on infiltration processes and runoff generation
(Pierson et al. 2008b). Pierson et al. (2008b, 2009) (table 3, figs. 22B and 25) measured infiltration and
runoff from rainfall simulations on small (0.5 m2) plots on burned and unburned moun-
tain big sagebrush rangeland (35 to 50 percent slopes) the year of, and 1 year following
the Breaks Prescribed Fire in the Reynolds Creek Experimental Watershed near Boise,
Idaho. A convective storm was simulated by applying 85 mm h-1 over a 60-min duration. 64 USDA Forest Service RMRS-GTR-351. 2016. Canopy cover prefire was 84 percent on shrub coppices (almost all as shrub cover) and
31 percent on interspaces (22 percent grass). Ground cover was composed almost entirely
of litter and totaled 75 percent for unburned interspaces and 99 percent for unburned
shrub coppices. The fire reduced canopy cover to 10 percent on coppices and 0 percent
on interspaces. Litter cover was reduced to 36 percent and 14 percent for coppices and
interspaces, respectively. After one growing season (year 1), total canopy cover increased
to 40 percent (29 percent forb, 3 percent grass) on burned coppices and 63 percent
(40 percent forb, 22 percent grass) on burned interspaces. Total ground cover in year 1
was 22 percent (15 percent litter) on burned coppices and 32 percent (26 percent litter) on
burned interspaces. Bulk densities (0 to 2 cm) in the Pierson et al. (2009) study were equal (1.13 g cm-3)
on coppice and interspace microsites and were not significantly changed by burning. Soil water repellency was strong and nearly equal on coppice microsites for burned and
unburned conditions the year of the fire (year 0). The strength of soil water repellency
was 55 to 60 percent less on burned and unburned interspaces than on coppices in year
0, but was strong. Soil water repellency was greatly reduced (moderately strong) and
nearly equal across all plots in year 1 (fig. 22B). Canopy and ground cover removal and
strong soil water repellency on burned coppice plots generated significant decreases in
infiltration and increases in runoff postfire (Pierson et al. 2009). Minimum infiltration
decreased 60 percent and runoff doubled on coppice microsites immediately postfire (run-
off coefficient of 76 percent). USDA Forest Service RMRS-GTR-351. 2016. Rainsplash and Sheetflow Processes Runoff coefficients were greater for unburned interspaces
(63 percent) than for coppices (39 percent) even though soil water repellency was twofold
to threefold greater on coppices. Vegetation and ground cover mitigated soil water repel-
lency on unburned coppices. The low cover on unburned interspaces and the presence of strong soil water
repellency facilitated runoff generation. Burning reduced cover on interspace microsites,
but did not increase runoff generation relative to the unburned condition. A significant de-
crease (by 70 percent) in soil water repellency on burned and unburned coppices between
year 0 and year 1 and the nearly uniform moderate soil water repellency across all plots
resulted in a 75 to 90 percent decrease in cumulative runoff. Runoff coefficients in year 1
were 8 to 10 percent across all plots regardless of cover. Total ground cover in year 1 was
significantly less on burned plots (27 percent) than on unburned plots (73 percent) and
canopy cover was significantly less on burned (40 percent) than unburned (84 percent)
coppices. As with the Denio Fire study (Pierson et al. 2001a, 2008a,b), cover influenced
water availability, but the strength of soil water repellency exerted a greater influence
on infiltration (fig. 22B) and runoff generation (table 3). That is, cover influenced the
quantity of water available for runoff generation and soil water repellency, and along with
other soil factors, governed the rate of infiltration for available surface water. The collective studies by Pierson et al. (2001a, 2002b, 2008a,b, 2009) illustrate the
complexity of rainfall-runoff processes on burned and unburned rangelands and are sup-
ported by other recent woodland and well-cited semiarid forested studies (Cerdà 1998a;
Benavides-Solorio and MacDonald 2001, 2002; Pierson et al. 2013, 2014; Williams et
al. 2014a). Recent small-plot scale investigations at a gently sloping western juniper
woodland in Idaho found that severe burning had minimal effect on runoff from mostly
bare interspaces and decadent shrub coppices, but fire-induced litter removal on tree cop-
pices increased runoff of simulated rainfall by twofold to threefold 1 and 2 years postfire 65 USDA Forest Service RMRS-GTR-351. 2016. (table 3; Pierson et al. 2013; Williams et al. 2014a). The wildfire reduced tree coppice
litter cover by a factor of eight, from 98 to 12 percent, and soils underneath tree canopies
were strongly water repellent prefire and postfire. Soils were wettable in interspaces and
underneath shrubs before and after burning. Pierson et al. Rainsplash and Sheetflow Processes (2014) reported contrasting
fire impacts on runoff responses to simulated high-intensity storms 1 and 2 years after
low- to moderate-severity prescribed burns on degraded singleleaf pinyon (P. monophylla
Torr. & Frém.)/Utah juniper (Marking Corral site, Nevada) and Utah juniper (Onaqui site,
Utah) woodlands (table 3). Burning at both sites had minimal impact on runoff generation
from degraded interspaces (table 3). The prescribed burn at the more degraded woodland,
Onaqui, did not significantly reduce litter depths over the strongly water-repellent
soils on tree coppices and resulted in no significant fire effect on runoff generation. In
contrast, burning at the less degraded site, Marking Corral, reduced tree coppice litter
depth twofold, from 40 to 23 mm, and increased runoff coefficients from 0 to 15 and
28 percent for storms of 64 mm h-1 and 102 mm h-1, respectively (Pierson et al. 2014). Soil water repellency was strong before and after burning on tree coppices at both sites
(Pierson et al. 2014). The contrast in runoff responses to burning across the two sites was
attributed to greater tree litter removal at the Marking Corral site on persistent strongly
water-repellent soils (Pierson et al. 2014). The lack of fire effects on runoff generation
from interspaces and shrub coppices at the sites was attributed to the overall degraded
conditions of interspaces before burning and the minimal impact of the moderate-severity
fire on ground cover and soil properties underneath shrub canopies (Pierson et al. 2014). Benavides-Solorio and MacDonald (2001, 2002) (table 3) measured runoff from
burned and unburned areas of a ponderosa pine forest in the Colorado Front Range,
Colorado. A simulated convective rainstorm applied 80 mm h-1 of rainfall for 60 min
to 1.0 m2 plots with unburned and varying burned severity conditions. Although the
study investigated three fire sites, we focus on the primary site, the Bobcat Fire, because
only two replicates existed for each burn severity at other sites (Benavides-Solorio
and MacDonald 2001, 2002). Percent ground cover by burn severity class was 23
percent high-severity, 88 percent moderate-severity, and 99 percent low-severity and
unburned conditions. Slopes averaged 22 to 31 percent and soil moisture averaged 1.0 to
2.0 percent. Runoff coefficients were 66 percent for the high-severity plots, 58 percent
for moderate-severity plots, and 55 percent for the low-severity and unburned plots. Rainsplash and Sheetflow Processes Soils
were strongly water-repellent near the surface and there was very little variation in the
strength of soil water repellency with burn severity. Correlations in runoff and soil water
repellency were not separated for the multiple fires studied, but when all fires were con-
sidered, runoff from high-severity plots was well correlated (r2 = 0.81) with the strength
of natural or fire-enhanced soil water repellency. Runoff was not well correlated with
percent slope or percent bare ground. Benavides-Solorio and MacDonald (2001, 2002)
concluded soil water repellency and soil moisture, as a controller of repellency strength,
were the primary controls on the amount of runoff generated. Cerdà (1998a) used rainfall simulation to study the hydrologic impacts of burning
on moderately steep (25 percent) slopes within a Mediterranean scrubland plant commu-
nity in southeastern Spain. A simulator applied 55 mm h-1 of rainfall to 0.25 m2 plots 7,
18, 29, and 64 months after a wildfire. The site burned at high intensity, and the fire con-
sumed the entire litter layer. Organic matter, soil porosity, and bulk density were similar
across all plots. Ash cover postfire was 3 to 5 cm deep and continuous, but was removed 66 USDA Forest Service RMRS-GTR-351. 2016. by autumn rains before simulation. Total ground cover was about 31 percent during the
first year of simulations and was well established (74 percent) within 18 months. Time
to ponding gradually increased with vegetative recovery from 2 min 17 sec the year of
the fire to 15 min 35 sec 5.5 years postfire. Time to runoff increased over the same time
period from 3 min 47 sec to 25 min 30 sec. The runoff coefficient was 45 percent the year
of the fire, decreased to 14 percent within the first 3 years, and was 6 percent within 5.5
years postfire. The spatial variability in runoff tracked with spatial variability in vegeta-
tive recovery. Cerdà (1998a) found that the relationship between cover and runoff was
negative and exponential, as reported for erosion by Johansen et al. (2001) and Pierson
et al. (2008a, 2009). Runoff was greatly diminished when plant cover recovered to 50 to
60 percent, the third winter following the burn, and approximated that of other, unburned
Mediterranean scrub 5.5 years postfire. In another forested study, Woods and Balfour (2008) measured runoff from ash- and
nonash-covered plots following high-severity wildfire. Rainsplash and Sheetflow Processes Rainfall was applied to 0.5 m2
plots at 75 mm h-1 intensity for 1 hour. The study found that the ash layer provided
1.5 cm of water storage capacity and protected the soil surface from sealing in the imme-
diate postfire period. Time to ponding was 12 min longer and cumulative infiltration was
2.0 cm greater on ash than on ash-free plots. Nine months after the fire, ash and ash-free
plots exhibited similar runoff behavior. Similar ash cover and runoff relationships have
been reported in studies by Cerdà and Doerr (2008), Larsen et al. (2009), and Ebel et al. (2012). Larsen et al. (2009) and Onda et al. (2008) indicate, however, that the positive ash
and infiltration relationships are likely short lived and that soil sealing following winnow-
ing of ash particles may promote runoff, especially where soil water repellency occurs. USDA Forest Service RMRS-GTR-351. 2016. Hillslope- to Watershed-Scale Runoff Fire effects on runoff generation over large-plot (tens of meters) to hillslope scales
is commonly less than observed at small-plot scales due to spatially variable cover
and surface soil conditions (Shakesby and Doerr 2006; Pierson et al. 2009, 2011). This
relationship is illustrated in the aforementioned Pierson et al. (2009) (table 3) study. In
addition to small plots, the study measured runoff from large plots (32.5 m2) (fig. 25B)
in burned and unburned shrub steppe. Runoff coefficients from a simulated convective
storm (85 mm h-1, 60 min) were 27 percent for burned plots and 4 percent for unburned
plots (table 3). Mean runoff coefficients from the same storm on burned and unburned
small plots (0.5 m2) were 2- and more than 10-fold greater, respectively, than on the
large plots (table 3). The decreased runoff from small- to large-plot scales was attributed
to greater spatial variability in infiltration at the large- versus small-plot scale. Although
runoff declined with increasing scale, burned large plots still generated nearly sevenfold
more runoff than unburned large plots. Greater runoff from the burned than unburned
condition was attributed to the uniform threefold reduction in ground cover, 100 percent
reduction in canopy cover, persistence of strong soil water repellency after burning, and
formation of high-velocity concentrated flowpaths on burned plots. These conditions
created more uniform surface hydrologic connectivity on burned than unburned plots
(fig. 25A). Runoff coefficients from all plots were positively correlated with soil water
repellency strength (r2 = 0.56) and percent bare ground (r2 = 0.32). Concentrated flow
experiments in the same study (fig. 26) found that cumulative runoff from consecutive 67 USDA Forest Service RMRS-GTR-351. 2016. Figure 25—(A) The Breaks Prescribed Burn, Reynolds Creek Experimental Watershed, Idaho and (B) large-plot (32.5 m2) rainfall simulations
conducted at the site by Pierson et al. (2009) (photos: USDA Agricultural Research Service, Northwest Watershed Research Center). Figure 25—(A) The Breaks Prescribed Burn, Reynolds Creek Experimental Watershed, Idaho and (B) large-plot (32.5 m2) rainfall simulations
conducted at the site by Pierson et al. (2009) (photos: USDA Agricultural Research Service, Northwest Watershed Research Center). 12-min releases of 7, 12, 15, and 21 L min-1 of concentrated flow were 406 L on burned
plots and 144 L on unburned plots. Runoff from small, large, and concentrated-flow
burned plots was equivalent to that of unburned plots after ground cover recovered to 30
to 40 percent one growing season postfire. USDA Forest Service RMRS-GTR-351. 2016. Figure 26—Concentrated flow
experiments conducted by Pierson
et al. (2009) immediately following
the Breaks Prescribed Fire,
Reynolds Creek Experimental
Watershed, Idaho. Green dye in
flowpath is a visual tracer used
to show concentrated flowpath
formation and direction. Flows
were released at specified rates
(7, 12, 15, and 21 L min-1) for 12-
min intervals each (photo: USDA
Agricultural Research Service,
Northwest Watershed Research
Center). Hillslope- to Watershed-Scale Runoff The return to prefire runoff rates was at least
partially related to a 70 percent reduction in strength (from strong to moderate) of soil
water repellency. Figure 26—Concentrated flow
experiments conducted by Pierson
et al. (2009) immediately following
the Breaks Prescribed Fire,
Reynolds Creek Experimental
Watershed, Idaho. Green dye in
flowpath is a visual tracer used
to show concentrated flowpath
formation and direction. Flows
were released at specified rates
(7, 12, 15, and 21 L min-1) for 12-
min intervals each (photo: USDA
Agricultural Research Service,
Northwest Watershed Research
Center). Figure 26—Concentrated flow
experiments conducted by Pierson
et al. (2009) immediately following
the Breaks Prescribed Fire,
Reynolds Creek Experimental Figure 26—Concentrated flow
experiments conducted by Pierson
et al. (2009) immediately following
the Breaks Prescribed Fire,
Reynolds Creek Experimental
Watershed, Idaho. Green dye in
flowpath is a visual tracer used
to show concentrated flowpath
formation and direction. Flows
were released at specified rates
(7, 12, 15, and 21 L min-1) for 12-
min intervals each (photo: USDA
Agricultural Research Service,
Northwest Watershed Research
Center). 68 USDA Forest Service RMRS-GTR-351. 2016. In another mountain big sagebrush study, Pierson et al. (2008a) reported a 35-fold
difference in runoff between high-severity burned (178 L) and unburned (5 L) plots from
consecutive 12-min concentrated flow releases of 7, 12, and 15 L min-1. Runoff from
small plots and concentrated flow experiments in that study returned to preburn levels
after ground cover increased to about 40 percent and 60 percent, respectively, and soil
water repellency was reduced to moderate levels. At a western juniper woodland in Idaho,
Pierson et al. (2013) and Williams et al. (2014a) found runoff ratios during simulated
64 mm h-1 and 102 mm h-1 intensity rainstorms (45 min, 13 m2 plots) on tree coppices
increased ninefold and fourfold, respectively, 1 year after a high-severity wildfire
(table 3). The increased runoff from tree coppices following the fire occurred due to a
threefold reduction of litter cover, a reduction of more than 4 to 5 cm in litter depth, and
persistence of strongly water-repellent soils postfire (Pierson et al. 2013; Williams et al. 2014a). Burning had no effect on runoff generation from the degraded intercanopy (more
than 80 percent bare soil and rock) at the site the first year after burning. In a semiarid forest setting in New Mexico, Johansen et al. Hillslope- to Watershed-Scale Runoff (2001) (table 3) reported
that runoff from large-plot (32 m2) rainfall simulations on burned and unburned plots was
positively correlated with percent bare soil (r = 0.76), and that the time to runoff genera-
tion was negatively correlated with percent bare soil (r = -0.67). Application of 120
mm of rainfall over 2 simulation hours generated mean runoff coefficients of 45 and 23
percent for burned and unburned conditions, respectively (table 3). Mean ground cover
was 26 percent for burned plots and 52 percent for unburned plots. Soil water repellency
was highly variable in space and strength and had minimal effect on runoff generation. Historical hillslope-scale studies from chaparral communities (see review by
Shakesby and Doerr 2006) are consistent with the semiarid shrub steppe, woodland, and
forested studies by Pierson et al. (2009, 2013), Williams et al. (2014a), and Johansen et
al. (2001). The effects of fire on runoff from rangelands at the watershed scale are lim-
ited. Overall, at the watershed scale, peak discharge rather than cumulative runoff tends
to be greater after burning, and is most pronounced after short-duration, high-intensity,
convective thunderstorms over large expanses of severely burned landscapes (Shakesby
and Doerr 2006). Rainsplash and Sheetflow Processes Higher erosion rates following fire are commonly attributed to decreased aggregate
stability, increased surface exposure to raindrop impact, reduced energy dissipation of
overland flow, and greater surface runoff and overland flow velocity. Greater surface
vulnerability and maximized raindrop energy amplify sediment detachment by rainsplash
processes. Greater water availability and flow velocity provide more efficient downslope
transport and overland flow detachment. Reduced aggregate stability, where it occurs,
magnifies the overall response. The previously mentioned small-plot studies in Nevada
and Idaho by Pierson et al. (2001a, 2002b, 2008a, 2009, 2013, 2014) and in the Colorado
Front Range by Benavides-Solorio and MacDonald (2001, 2002) provide estimates
of postfire erosion increases due to rainsplash and sheetflow processes. Pierson et al. (2002b) (table 3) reported that the more than 90 percent reduction in litter biomass and 70 69 69 USDA Forest Service RMRS-GTR-351. 2016. percent mean reduction in total live biomass 1 year after moderate and severe burning of
south-facing sagebrush hillslopes increased soil erosion by factors of 7 (21 g m-2) and 28
(85 g m-2), respectively. Soil erosion postfire was most pronounced on severely burned
interspace plots (148 g m-2) due to a more than 40 percent reduction in surface roughness. Erosion from severely burned shrub coppice plots was 22 g m-2. Sediment yield from all
plots in the study was negatively correlated with litter biomass (r = -0.59), total ground
cover (r = -0.59), and total canopy cover (r = -0.56).i Pierson et al. (2001a, 2008a) (table 3; fig. 24) measured a threefold increase in
soil erosion from shrub coppice plots (41 g m-2 burned, 12 g m-2 unburned) after fire
consumed nearly 100 percent of canopy and ground cover. Similar bare ground postfire
on burned interspaces generated half the erosion rate that was measured on burned shrub
coppices and produced similar erosion to prefire conditions. The differing responses
were attributed to a more erodible surface, slightly greater runoff, and persistent soil
water repellency on shrub coppices after burning. Erosion 1 year postfire was greatly
reduced across all plots and was similar for burned and unburned conditions. Two years
postfire, burned shrub coppices generated 3- to 14-fold more erosion than any other plot,
burned or unburned. Soil water repellency, time to peak runoff, and minimum infiltration
were the only other variables that exhibited the same temporal trend, implicating runoff
generation and continued greater erodibility as the causal factors. Pierson et al. Rainsplash and Sheetflow Processes (2009) found that 90 percent and 40 percent reductions in canopy and
ground cover increased sediment yield from 17 g m-2 on unburned shrub coppices (80
to 90 percent cover) to 183 g m-2 postfire. Fire-caused reductions of 100 percent canopy
and 80 percent ground cover on interspaces (31 percent and 75 percent canopy and
ground cover prefire) increased sediment yield from 195 g m-2 to 705 g m-2. Fire-induced
increases in erosion on shrub coppices were attributed to greater runoff postfire whereas
significantly increased erodibility, rather than runoff, explained the postfire increase in
erosion from interspaces. In woodland studies in southwestern Idaho, Pierson et al. (2013) and Williams et
al. (2014a) reported that reductions of more than 80 percent of litter cover and 4 to 5 cm
litter depth from tree coppices amplified erosion from small-plot rainfall simulations
(102 mm h-1, 45 min, 0.5 m2) by 18- to 34-fold the first 2 years after severe wildfire
(table 3). In the same studies, more than 80 percent reductions in canopy and litter cover
on shrub coppices increased erosion from simulated storms more than 20-fold 1 year
postfire. Two years postfire, erosion remained elevated on shrub coppice plots, but the
difference in erosion between burned and unburned treatments was not significant. Slight
fire-induced reductions in interspace grass canopy cover (from 15 to 7 percent) at the
site 1 year postfire resulted in a fourfold increase in small-plot scale erosion from burned
interspaces. As with shrub coppices, erosion from burned and unburned interspaces was
not significantly different 2 years postfire. Pierson et al. (2014) cited site-specific differences in soil erodibility as the primary
driver for contrasting erosion rates from simulated rainfall at two woodland sites 1 and 2
years following prescribed fire (table 3). Simulated high-intensity rainfall (102 mm h-1,
45 min, 0.5 m2) generated amplified soil erosion from tree coppices at the less erodible
site (46 to 75 g m-2), but the elevated postfire erosion rates were minor relative to those
of burned tree coppices at the more highly erodible site (242 to 294 g m-2). Runoff rates
from burned tree coppices were similar for the two sites (table 3). 70 USDA Forest Service RMRS-GTR-351. 2016. Benavides-Solorio (2001, 2002; table 3) reported that sediment yield from moder-
ate- and high-severity burned semiarid forested areas were 2- and 16-fold higher than
from plots burned at low severity or unburned. Rainsplash and Sheetflow Processes Percent bare soil (table 3) explained 79
percent of the variability in erosion from all plots, and soil water repellency explained 43
percent of the variability in erosion from plots burned at high severity.i As with runoff, the postfire erosion response appears closely tied to alteration of
cover. A secondary contributing factor appears to be soil water repellency, owing to its
influence on infiltration and on runoff generation. In each of the studies discussed above,
rainfall intensity was held constant. Runoff and erosion both exhibit some dependence
on rainfall intensity as a driving force, and responses may be amplified or dampened by
respective increases or decreases in the rainfall rate (Pierson et al. 2011; Williams et al. 2014b). Concentrated Flow Processes Figure 27—Erosion reported as
sediment per unit of runoff from
large-plot (32.5 m2) rainfall
simulations conducted on
unburned and burned conditions
at the Breaks Prescribed Fire,
Reynolds Creek Experimental
Watershed, Idaho (Pierson et al. 2009). Figure 28—Cumulative sediment
yield versus runoff from rainfall
simulations (102 mm h-1, 45 min)
on unburned and burned tree
coppice and intercanopy (areas
between tree coppices) plots (13
m2) in a western juniper woodland
(Pierson et al. 2013; Williams et al. 2014a). Figure 27—Erosion reported as
sediment per unit of runoff from
large-plot (32.5 m2) rainfall
simulations conducted on
unburned and burned conditions
at the Breaks Prescribed Fire,
Reynolds Creek Experimental
Watershed, Idaho (Pierson et al. 2009). Figure 27—Erosion reported as
sediment per unit of runoff from
large-plot (32.5 m2) rainfall
simulations conducted on
unburned and burned conditions
at the Breaks Prescribed Fire,
Reynolds Creek Experimental
Watershed, Idaho (Pierson et al. 2009). Figure 28—Cumulative sediment
yield versus runoff from rainfall
simulations (102 mm h-1, 45 min)
on unburned and burned tree
coppice and intercanopy (areas
between tree coppices) plots (13
m2) in a western juniper woodland
(Pierson et al. 2013; Williams et al. 2014a). Williams et al. (2014a) and Pierson et al. (2013) also reported a shift from rains-
plash and sheetflow to concentrated flow as the dominant erosion process occurring on
13 m2 rainfall simulation plots following burning of a western juniper woodland (table 3). Erosion from burned tree coppices increased exponentially with increasing runoff postfire
due to the formation of high-velocity concentrated flow paths (fig. 28). Pierson et al. (2009) used simulated consecutive 12 min concentrated flow releases of 7, 12, 15, and
21 L min-1 to quantify erosion solely from concentrated flow processes in unburned and
burned areas of sagebrush rangeland. The cumulative flow releases generated 14,363 g
of sediment immediately postfire and 2,420 g under unburned conditions prefire. Pierson
et al. (2008a) conducted the same concentrated flow experiments as Pierson et al. (2009)
using flow rates of 7, 12, and 15 L min-1 and measured 17,775 g of sediment immediately
following burning and less than 10 g on unburned plots. In both studies, erosion from the
respective simulated concentrated flow rates on burned plots approached that of unburned
plots once ground cover recovered to 60 percent or greater. The large-plot results from
Pierson et al. (2009, 2013) and Williams et al. Concentrated Flow Processes Concentrated flowpaths rarely occur on undisturbed rangelands, but often become
the dominant conduit for overland flow and sediment transport after burning (Moffet et
al. 2007; Pierson et al. 2009; Williams et al. 2014b, 2016a). Following burning, greater
water availability, decreased infiltration, and reduced surface obstructions facilitate
formation of concentrated flowpaths. These relationships are enhanced on steep slopes
and where overland flow is promoted by soil water repellency (Shakesby and Doerr 2006;
Moffet et al. 2007; Pierson et al. 2009; Williams et al. 2014a,b). Concentrated flowpaths
have greater flow depth, velocity, erosive energy, and transport capacity than sheetflow
(Moffet et al. 2007; Pierson et al. 2008a, 2009). The velocity and erosive energy are
a function of the soil grain and form roughness, the eroded flowpath, ground cover,
hillslope angle, and flow discharge (Moffet et al. 2007; Al-Hamdan et al. 2012a,b, 2013). p
gl
g (
)
Pierson et al. (2008a, 2009) (figs. 24, 25, and 26) measured the velocity of simulated
concentrated flowpaths under burned and unburned conditions on sagebrush rangelands
in Idaho and Nevada. In both studies velocity over a range of flow rates was positively
correlated (r2 = 0.59 to 0.70) with and exponentially related to percent bare ground. Velocity decreased non-linearly with decreasing bare ground as vegetation recovered in
the years after burning. Sharp increases in velocity were observed where bare ground
exceeded 50 to 60 percent. Pierson et al. (2009) (table 3) measured increasing erosion
with increasing plot size after burning of sagebrush hillslopes. They attributed the posi-
tive relationship to a switch in the dominant erosion process from rainsplash-sheetflow
to concentrated flow following burning. In the study by Pierson et al. (2009), erosion for
unburned conditions was greater from small plots (rainsplash-sheetflow) than large plots. Following burning, erosion was greater on the larger plots, and the maximum number of
concentrated flowpaths increased from zero for unburned to five for burned large plots. More uniform bare soil conditions, strongly water-repellent soils, and greater hydrologic
connectivity after fire contributed to rapid (3 min) runoff generation and formation of
concentrated flow. Erosion increased from 8 g m-2 for unburned conditions to 988 g m-2
postburn (table 3). Large-plot sediment yield remained greater for burned conditions
until vegetation recovered to nearly 60 percent two growing seasons postfire (fig. 27). No
concentrated flowpaths were observed in burned plots after the second growing season. 71 USDA Forest Service RMRS-GTR-351. 2016. Hillslope- to Watershed-Scale Erosion Hillslope- to Watershed-Scale Erosion Erosion at hillslope to watershed scales is largely dependent on the spatial ar-
rangement of burn severity, bare soil exposure, water-repellent soil conditions, and
rainfall intensity (Shakesby and Doerr 2006). Very few large-scale erosion studies have
been completed on burned rangelands. Therefore insight into large-scale fire effects on
rangeland hydrology comes from studies of dry or semiarid forested sites and the limited
number of hillslope-scale studies for shrub steppe, woodland, and chaparral communities. Benavides-Solorio and MacDonald (2005) used silt fences to measure (see Robichaud
and Brown 2002) postfire erosion on moderate to steep, semiarid forest slopes (25 to
45 percent) in the Colorado Front Range for multiple fires of varying ages and severi-
ties. Contributing areas for the silt fences ranged from 190 to 6,600 m2 and averaged
1,250 m2. Over the 2-year study, percent bare soil explained about 64 percent of the vari-
ability in soil erosion across all plots (n = 48). Sediment yield decreased exponentially
with time after burning, and was highest where bare soil was equal to or greater than 60
percent. After a high-intensity storm, however, extensive concentrated flow networks and
erosion were observed on slopes with 70 to 85 percent ground cover of tree needles (15
to 30 percent bare). Benavides-Solorio and MacDonald (2005) attributed the result to the
high storm erosivity and ground cover type. Soil water repellency was present at least
at slight strength on most plots and was moderately strong on some of the high-severity
burns. As a result, soil water repellency was weakly correlated with sediment production
from all plots (r2 = 0.30), but was more strongly correlated for the high-severity plots
(r2 = 0.40). About 90 percent of the sediment collected during the study was delivered
by high-intensity convective storms whereas 10 percent resulted from frontal rainfall
or snowmelt. Concentrated flow formation played an important role in postfire erosion
rates, particularly in converging topographic positions. Overall sediment yield was well
correlated (r2 = 0.77) with a five-parameter empirical model of percent bare soil, rainfall
erosivity, fire severity, soil water repellency (1 cm depth), and the 84th percentile sedi-
ment grain size. Percent bare soil and rainfall erosivity collectively explained 62 percent
of sediment production variability. A large-plot (32.5 m2) rainfall simulation study of recently burned and unburned
rangeland in southwestern Idaho by Pierson et al. Concentrated Flow Processes (2014a) illustrate the profound influence
that concentrated flow processes have on postfire erosion (table 3). The simulated concen-
trated flow experiments (Pierson et al. 2008a, 2009) demonstrate the erosive potential of
high-velocity concentrated flowpaths on burned rangelands. 72 USDA Forest Service RMRS-GTR-351. 2016. Hillslope- to Watershed-Scale Erosion (2009) found soil water repellency and
sediment yield were moderately correlated (r2 = 0.46) whereas runoff coefficients (table
3) were more strongly correlated with soil water repellency (r2 = 0.56) and weakly cor-
related with bare ground (r2 = 0.32). Sediment yield and repellency in the Pierson et al. (2009) study was positively correlated with runoff coefficients (r2 = 0.83) and with per-
cent bare ground (r2 = 0.76). These correlations suggest that sediment yield in the study
by Pierson et al. (2009) was dependent on runoff generation and bare soil whereas runoff
was more dependent on water-repellent soil conditions in bare areas. The low correlation
in sediment yield and repellency in the Benavides-Solorio and MacDonald (2005) study
indicates that either the strength of soil water repellency was not significant enough
to influence erosion or simply other factors like rainfall intensity or topography and
extensive bare ground exerted more influence on erosion processes. Soil water repellency
in the contrasting Pierson et al. (2009) research was strong at the soil surface immediately
postfire and deteriorated to slight by the end of the 3-year study. Soil water repellency
was much weaker at the sites studied by Benavides-Solorio and MacDonald (2005) and 73 USDA Forest Service RMRS-GTR-351. 2016. showed a general decreasing trend through the study. In a gently sloping (5 to 7 percent)
semiarid forest setting in New Mexico, Johansen et al. (2001) (table 3) found erosion
from high-severity burned large plots (32.5 m2) was 25-fold greater than measured from
unburned plots of the same size. Sediment yield was strongly correlated with percent bare
soil (r = 0.84), and soil water repellency was considered slight. The studies highlighted
here illustrate the influence of ground cover and bare ground on hillslope-scale postfire
erosion response over a range of settings and slopes and demonstrate how soil water
repellency may magnify those effects. Large-scale alteration of surface roughness, canopy, and ground cover on rangeland
soils can trigger flash flooding and mass erosion events where superimposed on steeply
sloping water-repellent hillslopes. These events have received less attention in literature
for rangelands than for forested settings. One year after the Eighth Street Fire (6,070 ha)
in 1996 near Boise, Idaho, a 5- to 10-year return period convective storm (~67 mm h-1)
of 9-min duration on steeply sloped, severely burned sagebrush caused flash flooding
and mud flows in the city of Boise (Pierson et al. 2002b). Hillslope- to Watershed-Scale Erosion Pierson et al. (2002b) (table 3)
conducted rainfall simulations (67 mm h-1) at the site preceding the storm and determined
that elevated runoff and erosion rates from small (0.5 m2) rainfall plots were associated
with extensive bare ground, reduced surface roughness, and soil water repellency. These
effects were greater for south-facing than north-facing slopes. Runoff occurred within 2
to 4 min of rainfall in the Pierson et al. (2002b) study. Flooding during the ensuing storm
was driven by intense rainfall on bare (90 to 100 percent bare ground), water-repellent
soils with reduced water storage capacity and low surface roughness. Most of the rainfall
falling on the south-facing slopes ran off, forming concentrated flow networks (Pierson et
al. 2002b). Moody and Martin (2001) described a similar response to 100-year rainfall storm
following the 4,690 ha Buffalo Creek Fire in steep, forested watersheds of the Colorado
Front Range. More than 60 percent of the burn was high intensity. Within 2 months
postfire, a high-intensity (90 mm h-1, 1 h) rainstorm caused flash flooding that killed two
people and discharged enough sediment into the Strontia Springs Reservoir to reduce
storage capacity by one-third (Agnew et al. 1997; Moody and Martin 2001). Unburned
hillslopes adjacent to the fire generated very little surface runoff (Elliott and Parker
2001). Hillslope erosion increased 150- to 240-fold postfire. Nearly 1,500,000 m3 of sedi-
ment was generated from interrill, rill, and in-channel processes during the first summer
after the fire, and an estimated 86 percent of erosion from the first two summers postfire
was from rill and channel processes (Moody and Martin 2001). Soil water repellency was
not reported for the Buffalo Creek Fire. A torrential rainstorm 2 months after the 800 ha South Canyon Fire in Colorado
generated multiple runoff-triggered debris-flow events that inundated a 13- to 14-ha area
with about 70,000 m3 of soil and engulfed 30 vehicles on an adjacent highway (Cannon
et al. 1998, 2001a). The fire occurred on steeply sloping (30 to 70 percent) woodland and
shrub-dominated hillslopes. Cannon et al. (1998) estimated that rainsplash, sheetflow,
and concentrated flow processes during the storm removed 15 percent of the mineral soil
surface to a depth of 4 cm. Amplified runoff and erosion from rainsplash and sheetflow
on bare soils led to formation of concentrated flow networks and gullies with high erosive
energy and sediment transport capacity. Hillslope- to Watershed-Scale Erosion Erosion per unit of runoff generally increased
downslope as flow paths incised and widened to 30 to 40 cm (Cannon et al. 2001a). The 74 USDA Forest Service RMRS-GTR-351. 2016. examples presented here demonstrate the potential hydrologic responses to high-intensity
rainfall after large-scale alteration of vegetation and ground surface characteristics. A
more detailed chronicle of hillslope- and watershed-scale responses to fire in forested and
chaparral environments can be found in Shakesby and Doerr (2006). Assessing Postfire Hydrologic Vulnerability and Risk The overall postfire hydrologic vulnerability of a site is a function of the site
susceptibility and prevailing climatic regime (fig. 29). Susceptibility is defined by the
vegetation, soil, and topographic characteristics of the site and is much greater where bare
ground is extensive on steep slopes with hydrophobic surface soil conditions (Pierson et
al. 2011; Williams et al. 2014b). In contrast, susceptibility to postfire runoff and erosion is
low where ground cover exceeds 60 percent (Pierson et al. 2008a, 2009, 2013; Williams
et al. 2014a). Susceptibility for a specified area varies in both time and space and depends
on the prefire conditions and the rate of postfire recovery. Hydrologic vulnerability for a
specified susceptibility can be measured as the hydrologic response (runoff, erosion, or
both) to different storm events or rainfall intensities (Williams et al. 2014b). Hydrologic
vulnerability for a given susceptibility then increases with increasing rainfall intensity,
as shown by the different curves in figure 29. Each curve in the figure represents a hypo-
thetical vulnerability (or hydrologic response) associated with a particular storm intensity
over a range of susceptibilities, with susceptibilities defined by the site conditions. For example, hydrologic vulnerability for a low-intensity storm on highly susceptible Figure 29—Conceptual model of hydrologic vulnerability and risk. Hydrologic vulnerability (measured as runoff
and erosion response, Y-axis) is a function of site susceptibility (X-axis) and the prevailing rainfall intensity
(indicated by colors). The level of vulnerability dictates the resources at risk. Concentrated flow processes
dominate the postfire environment where site susceptibility is high (e.g., high bare ground, water-repellent
soils) or rainfall is of moderate to high intensity. Rainsplash processes prevail where susceptibility is low
(well-aggregated soils, land surface well protected by litter cover). Rainsplash, sheetflow, and concentrated
flow processes all contribute to the runoff/erosion response where site susceptibility is indicative of disturbed
conditions (reduced ground cover and aggregate stability, poor soil structure). The overall hydrologic
response is amplified with increasing slope steepness. Figure modified from Pierson et al. (2011) and
Williams et al. (2014b). Figure 29—Conceptual model of hydrologic vulnerability and risk. Hydrologic vulnerability (measured as runoff
and erosion response, Y-axis) is a function of site susceptibility (X-axis) and the prevailing rainfall intensity
(indicated by colors). The level of vulnerability dictates the resources at risk. Concentrated flow processes
dominate the postfire environment where site susceptibility is high (e.g., high bare ground, water-repellent
soils) or rainfall is of moderate to high intensity. Assessing Postfire Hydrologic Vulnerability and Risk Rainsplash processes prevail where susceptibility is low
(well-aggregated soils, land surface well protected by litter cover). Rainsplash, sheetflow, and concentrated
flow processes all contribute to the runoff/erosion response where site susceptibility is indicative of disturbed
conditions (reduced ground cover and aggregate stability, poor soil structure). The overall hydrologic
response is amplified with increasing slope steepness. Figure modified from Pierson et al. (2011) and
Williams et al. (2014b). Figure 29—Conceptual model of hydrologic vulnerability and risk. Hydrologic vulnerability (measured as runoff
and erosion response, Y-axis) is a function of site susceptibility (X-axis) and the prevailing rainfall intensity
(indicated by colors). The level of vulnerability dictates the resources at risk. Concentrated flow processes
dominate the postfire environment where site susceptibility is high (e.g., high bare ground, water-repellent
soils) or rainfall is of moderate to high intensity. Rainsplash processes prevail where susceptibility is low
(well-aggregated soils, land surface well protected by litter cover). Rainsplash, sheetflow, and concentrated
flow processes all contribute to the runoff/erosion response where site susceptibility is indicative of disturbed
conditions (reduced ground cover and aggregate stability, poor soil structure). The overall hydrologic
response is amplified with increasing slope steepness. Figure modified from Pierson et al. (2011) and
Williams et al. (2014b). 75 75 USDA Forest Service RMRS-GTR-351. 2016. conditions is quite low. But hydrologic vulnerability is high for at least a moderate-inten-
sity storm falling on a highly susceptible site (such as a site with more than 60 percent
bare ground and water-repellent soils). The hydrologic vulnerability is then dictated by
the storm intensity and the conditions in which the storm occurs (susceptibility). Concentrated flow processes dictate the response when storm intensity and site
susceptibility are both high postburn. Rainsplash processes dominate and overall vulner-
ability is minimal where site susceptibilities are low, regardless of the rainfall intensity. A
combination of rainsplash, sheetflow, and concentrated flow processes may prevail where
moderate rainfall intensities fall on a moderately susceptibility condition such as on a
degraded site. The overall hydrologic vulnerability further defines the resource at risk
(fig. 29) (Williams et al. 2014b). High-intensity rainfall on moderate to highly susceptible
conditions may cause loss of life (Moody and Martin 2001) or damage to property or in-
frastructures (Pierson et al. 2002b; Klade 2006; Pierson et al. 2011) due to large flooding
or debris flow events, or a combination thereof (Cannon et al. 1998, 2001a; Meyer et al. 2001). Assessing Postfire Hydrologic Vulnerability and Risk Low-intensity storms on highly susceptible conditions may not generate massive
flooding, but may generate enough soil loss to degrade water quality or negatively affect
aquatic habitat. In the framework presented (fig. 29), hydrologic vulnerability curves
can be populated with quantitative data, but the mitigation of risk is value based. Land
managers must consider what hydrologic vulnerability they are willing to accommodate
given the potential risks with which it is associated. Great advances have been made in postfire risk assessment in recent decades
(Robichaud et al. 2007a,b; Pierson et al. 2011; Williams et al. 2014b; Al-Hamdan et al. 2015; Williams et al. 2016a,b). The Erosion Risk Management Tool (ERMiT) is one
recent model developed with a probabilistic framework like the one shown in figure
29 (Robichaud et al. 2007a,b). Models like ERMiT predict the likelihood of a runoff/
erosion response associated with a given storm as the probability of that storm occurrence
given a static susceptibility. The probability of a given storm can be determined from
return interval or intensity/duration/frequency (fig. 3) information for the area of inter-
est. ERMiT and similar models use climate generators to predict storm frequency and
magnitude from a climate station location identified by the user. This approach allows
the user to evaluate hydrologic vulnerability in a predictive way and to make mitigation
decisions based on what may be at risk given a particular storm under a particular set of
site conditions. For example, simulations for defined postfire conditions may show there
is a 20 percent likelihood of a particular storm and an associated infrastructure-damaging
flood event occurring. Managers must then decide whether they are willing to assume that
type of risk, and if not, what could be used to mitigate the response if the event occurs. This, of course, becomes more complicated if a user is interested in predicting potential
response for varying conditions or hydrologic recovery with time or in landscape space. Predictions of this type require the user to run multiple model scenarios, one for each set
of postfire conditions and the same storm frequencies. The Rangeland Hydrology and Erosion Model (RHEM) has been developed
specifically for modeling runoff and erosion from rangelands (Nearing et al. 2011) and is
applicable for predicting runoff and erosion responses on postfire conditions (Al-Hamdan
et al. 2015; Williams et al. 2016a,b). The RHEM model includes optional probability-
based risk assessment output. Assessing Postfire Hydrologic Vulnerability and Risk RHEM model results for different vegetation and ground
cover conditions (e.g., prefire, immediately postfire, 3 years postfire) can be displayed 76 USDA Forest Service RMRS-GTR-351. 2016. simultaneously to assess hydrologic vulnerability across varied surface susceptibility
(Williams et al. 2016b). In summary, postfire hydrologic vulnerability assessment for
large areas or over periods of time benefit from incorporating conceptual and mathemati-
cal models that consider both the probability of storm occurrences and the likelihood of
respective storm occurrences over a range of postfire site conditions. The need to address hydrologic vulnerability and risk mitigation on western range-
lands is increasing due to the increased role of wildfire (Pierson et al. 2011; Williams et
al. 2014b). Annual weed invasion of shrub steppe (Mack 1981; Whisenant 1990; Knapp
1996) and woodlands (Tausch 1999; Miller et al. 2008; Romme et al. 2009), woodland
encroachment (Miller and Tausch 2001; Romme et al. 2009), and increasing global
temperatures (Westerling et al. 2006; Keane et al. 2008) are expected to amplify the
role of fire on western landscapes (Williams et al. 2014b). Cheatgrass is now the major
plant constituent on 4 to 7 million of the 18 million ha of shrub steppe in the Great Basin
(Mack 1981; Knapp 1996). The continuous horizontal fuel structure of cheatgrass-
invaded shrubland promotes more frequent and larger-scale wildland fires than reported
for historical shrub steppe (Whisenant 1990; Peters and Bunting 1994; D’Antonio 2000;
Brooks and Pyke 2001; Brooks et al. 2004; Keane et al. 2008). Current fire return inter-
vals on many cheatgrass areas are 3 to 15 years (Whisenant 1990; Brooks and Pyke 2001;
Brooks et al. 2004); historical fire return intervals on Great Basin sagebrush communities
range from 20 to 100 or more years depending on the productivity of the site (Wright and
Bailey 1982; Keane et al. 2008). Great Basin rangelands with substantial cheatgrass cov-
erage are 10 to 500 times more likely to burn than pristine shrub-bunchgrass communities
(Hull 1965), and fire risk is near 100 percent where cheatgrass coverage approaches 50
percent (Link et al. 2006). Recent infilling of trees in persistent woodlands and wooded shrublands of the
Great Basin has also increased the risk of occurrence of large, high-severity fires (Tausch
1999; Miller and Tausch 2001; Tausch and Hood 2007; Keane et al. 2008; Romme et al. 2009). Determining Site Susceptibility and Hydrologic Recovery Hydrologic susceptibility assessments should include evaluation of those variables
found to significantly affect rangeland hydrology in the postfire environment (Pierson
et al. 2011; Williams et al. 2014b). Our research (Pierson et al. 2001a, 2002b, 2008a,b,
2009, 2013, 2014; Al-Hamdan et al. 2012a,b, 2013; Williams et al. 2014a,b) as well as
others’ research (Benavides-Solorio and MacDonald 2001; Cannon et al. 1998, 2001a,b;
Johansen et al. 2001; Moody and Martin 2001; Benavides-Solorio and MacDonald 2002,
2005; Larsen et al. 2009) indicate these variables include hillslope angle and topography,
percent bare soil, surface soil erodibility, and soil water repellency. Although the influ-
ence of bare soil, erodibility, and soil water repellency are important across a range of
hillslope gradients, the potential magnitude of the hydrologic response may be much
greater for steep hillslopes (DeBano et al. 1998). Furthermore, convergent topography
tends to accentuate concentrated flow (Benavides-Solorio and MacDonald 2005). Results
from numerous studies indicate both runoff and erosion, more so for the latter, are strong-
ly influenced by the expanse of bare ground (Morris and Moses 1987; Benavides-Solorio
and MacDonald 2001; Johansen et al. 2001; Pierson et al. 2001a; Benavides-Solorio and
MacDonald 2002; Pierson et al. 2002b; Benavides-Solorio and MacDonald 2005; Moffet
et al. 2007; Pierson et al. 2008a,b, 2009; Larsen et al. 2009; Pierson et al. 2013; Williams
et al. 2014a, 2016a). For sloping rangelands, a ground cover of 50 to 60 percent is commonly adequate
to protect the soil surface from amplified runoff and erosion during frequently recur-
ring convective thunderstorm events (Gifford 1985; Pierson et al. 2008a, 2009, 2013;
Williams et al. 2014a). A greater ground cover may be necessary to protect against larger,
less frequent, major events (such as a 100-year storm) capable of generating concentrated
flow (Benavides-Solorio and MacDonald 2005; Pierson et al. 2008a, 2009). The presence
of strong soil water repellency further inhibits infiltration, and, when combined with
less than 60 percent ground cover, greatly increases the vulnerability for runoff genera-
tion, concentrated flow, and elevated erosion rates (Pierson et al. 2008a,b, 2009, 2013;
Williams et al. 2014a). The influence of soil water repellency on postfire runoff is well
documented, but the relationships between spatial and temporal repellency strength and
the respective hydrologic responses remain largely unknown. Finally, site erodibility
should be considered (Pierson et al. 2010, 2014). Sites with higher erodibility may
require greater ground cover protection. Assessing Postfire Hydrologic Vulnerability and Risk Cheatgrass invasion into persistent woodlands and wooded shrublands has further
increased the horizontal fuel structure and risk of large-scale fires on many wooded sites
within the Great Basin (Young and Evans 1978; Billings 1994, Tausch 1999; Miller et
al. 2008). Larger, more frequent, uniform, and intense fires increase the spatial expanse
and temporal exposure of these landscapes to accelerated runoff and erosion. Greater
temporal exposure increases both the potential soil loss from low-return interval storms
(1- to 10-year events) and from more damaging, high-intensity thunderstorm events.i The environmental and ecological implications of altered fire regimes are substan-
tial when one considers the magnitude of cumulative soil loss associated with frequently
occurring storms or infrequent but extreme events. Risks to property and human life are
of particular concern at the wildland-urban interface (Craddock 1946; Cannon et al. 1998,
2001a; Meyer et al. 2001; Moody and Martin 2001; Pierson et al. 2002b; Klade 2006;
Pierson et al. 2011; Williams et al. 2014b). The economic consequences are significant
given current U.S. expenditures on postfire risk mitigation (General Accounting Office
2003). 77 USDA Forest Service RMRS-GTR-351. 2016. Determining Site Susceptibility and Hydrologic Recovery The generalities presented here are based on field
studies, but often one or more variables (such as slope and aspect) were held constant
during investigations. Therefore, the collective interaction remains largely unevaluated. Postfire hydrologic recovery assessments are often hampered by the difficulty in
selecting the appropriate reference condition, temporal fluctuations in climate, spatial
variability in postfire surface and vegetation characteristics, and the fact that conditions
required for hydrologic stability differ for runoff versus erosion and for rainsplash/sheet-
flow versus concentrated flow processes (Pierson et al. 2008a, 2009). Large-scale plant
community transitions (D’Antonio and Vitousek 1992; Knapp 1996; DiTomaso 2000;
Brooks and Pyke 2001; Brooks et al. 2004; Rice et al. 2008; Romme et al. 2009) have
created prefire vegetation and surface characteristics that may differ from those that favor
water and soil conservation. Therefore, determination of the appropriate reference for
comparison of postburn runoff and erosion may be difficult. 78 USDA Forest Service RMRS-GTR-351. 2016. Annual variations in climate influence hydrologically important variables like
canopy and ground cover, soil erodibility, and soil water repellency. In a 3-year study,
for example, Pierson et al. (2008a,b; figs. 22 and 24) reported that temporal controls
on naturally occurring soil water repellency exerted a greater influence on runoff from
burned and unburned sagebrush hillslopes than did direct fire effects. Soil erosion from
burned shrub coppices in the study also exhibited significant temporal variability, but the
study did not determine whether this resulted from temporal variation in infiltration/run-
off or erodibility. The study highlighted the need for annual controls and suggested that
results from single-year studies or those without annual unburned controls may produce
unreliable evaluations of hydrologic vulnerability.l Potential spatial variability of site characteristics that influence hydrology like
slope, soil properties, and vegetation should be considered in recovery assessments. For
example, Pierson et al. (2002b) demonstrated that runoff and erosion were significantly
greater on south-facing than north-facing slopes 1 year following wildfire in sagebrush. These differences greatly influenced hydrologic behavior after an intense, flash flood-
generating storm. Analysis of the north-facing slopes solely would not have captured
the potential response of the more vulnerable south-facing slopes. Other fire-influenced
variables like soil water repellency may vary greatly in space depending on variations in
soil properties and burn characteristics (Woods et al. 2007). Lastly, postfire recovery periods differ for runoff and erosion and for different
hydrologic processes. Pierson et al. Determining Site Susceptibility and Hydrologic Recovery (2008a, 2009, 2013) as well as others (Benavides-
Solorio and MacDonald 2001, 2002) have shown that fire-induced increases in erosion
are greater than increases in runoff (table 3). Pierson et al. (2008a, 2009) further dem-
onstrated that postfire erosion may take longer than postfire runoff to return to unburned
levels and that potential erosion from concentrated flow processes may take longer than
that from rainsplash-sheetflow processes to recover. Therefore, studies that focus on one
aspect of hydrologic vulnerability (such as runoff) or on one process (such as rainsplash)
may not accurately evaluate recovery (Pierson et al. 2011). Studies would benefit from
multiyear assessments with annual controls and evaluation of runoff and erosion over
multiple scales that encompass rainsplash, sheetflow, and concentrated flow. But such
all-encompassing studies are often not possible or practical. Investigations that focus on
a single-scale hydrologic parameter or process should consider the potential error associ-
ated with broad inferences of hydrologic recovery. Summary and Conclusions Fire is a natural disturbance on western rangelands that can facilitate amplified
runoff and erosion rates and place natural resources, property, and lives at risk. However,
the degree to which fire increases runoff and erosion rates and the associated risks is
highly variable and depends on many factors. Runoff and erosion from rangelands are
dictated by interactions among climate, vegetation, soils, and topography. Fire alters the
structure of vegetation and ground cover, and may physically or chemically affect soils. Therefore, the magnitude of fire-induced increases in annual or event runoff and erosion
is fundamentally associated with the degree to which fire reduces vegetation and modifies 79 USDA Forest Service RMRS-GTR-351. 2016. 79 soils, and with the local topography and prevailing climate regime (annual scale) or storm
input (event scale).i In general, fire-induced hydrologic vulnerability and risks are low where burning
minimally alters vegetation and ground cover or when precipitation input is low. High-
intensity rainfall on severely burned and steeply sloping hillslopes presents the highest
risk for fire-induced increases in runoff and erosion. Plot- to hillslope-scale studies
presented in this review demonstrate that burning may increase runoff, erosion, or both
during high-intensity rainfall events by factors of 2 to 40 over small-plot scales and more
than 100-fold over large-plot to hillslope scales. Anecdotal reports of large-scale flooding
and debris-flow events from rangelands, woodlands, and semiarid forests, associated with
high-intensity rainfall following burning, document the potential risk to resources (such
as water quality and aquatic habitat), property and infrastructure, and human life. Such
risks are of particular concern for large urban centers along urban-wildland interfaces. It
is important to note the need for postfire risk assessments to consider risk in a probabi-
listic framework that examines the likelihood of specified storm events and the potential
responses for a given set of site conditions or susceptibility. Evaluations of site suscepti-
bility to increased runoff and erosion should focus on the key indicators described in this
review: expanse of bare ground, degree of soil water repellency, slope/topography, and
site-specific soil stability/erodibility. Assessments of risk over long-term intervals should
also evaluate potential changes in site susceptibility associated with prevailing climate
conditions and plant/cover recruitment.i Lastly, the role of fire is changing on western rangelands. Fires are increasing in
frequency, duration, and size across much of the western United States. Summary and Conclusions These changes
potentially increase the overall hydrologic vulnerability of rangelands by spatially and
temporally increasing surface exposure to runoff and erosion processes. We do not
yet know the ramifications of repeated burning and the respective runoff/erosion from
frequently occurring small events or less frequent large events. The materials presented
in this review do, however, provide a foundation from which hydrologic vulnerability
assessments can be developed and conceptual and mathematical models can be advanced
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a mechanism of facilitation in savanna tree-shrub clusters. Oecologia. 145: 32‒40. 110 In accordance with Federal civil rights law and U.S. References Department of Agriculture (USDA) civil rights regu-
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Computational Insights into the Interaction between Cytoadherence Receptor gC1qR and the DBLβ12 Domain of a Plasmodium falciparum PfEMP1 Ligand
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Citation: Bakri, R.; Rehan, M.;
Shamshad, H.; Hafiz, A. Computational Insights into the
Interaction between Cytoadherence
Receptor gC1qR and the DBLβ12
Domain of a Plasmodium falciparum
PfEMP1 Ligand. Life 2021, 11, 993. https://doi.org/10.3390/life11090993 Keywords: malaria; cytoadherence; gC1qR; Plasmodium falciparum erythrocyte membrane protein 1
(PfEMP1); Duffy binding-like (DBL) domain Received: 19 July 2021
Accepted: 15 September 2021
Published: 21 September 2021 Rowaida Bakri 1, Mohd Rehan 2,3,*, Hina Shamshad 1 and Abdul Hafiz 1,4,* 1
College of Medicine, Umm AL Qura University, Makkah 21955, Saudi Arabia; rabakri@uqu.edu.sa (R.B
henathebest@gmail.com (H.S.) 1
College of Medicine, Umm AL Qura University, Makkah 21955, Saudi Arabia; rabakri@uqu.edu.sa (R.B.);
henathebest@gmail.com (H.S.) 2
King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia
3
Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, King Abdulaziz
University, Jeddah 21589, Saudi Arabia 2
King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia
3
Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, King Abdulaziz
University, Jeddah 21589, Saudi Arabia y J
4
Epidemiology, London School of Hygiene and Tropical Medicine, University of London,
London WC1E 7HT, UK 4
Epidemiology, London School of Hygiene and Tropical Medicine, University of London,
London WC1E 7HT, UK *
Correspondence: mrtahir@kau.edu.sa (M.R.); aaahafiz@uqu.edu.sa (A.H.) *
Correspondence: mrtahir@kau.edu.sa (M.R.); aaahafiz@uqu.edu.sa (A.H.) Abstract: Human receptor gC1qR is a 32 kD protein that mediates the cytoadherence of Plasmod-
ium falciparum-infected erythrocytes (IEs) to human brain microvascular endothelial cells (HBMEC)
and platelets. The cytoadherence of IEs to gC1qR has been associated with severe malaria symptoms. The cytoadherence to gC1qR is mediated by the Duffy binding-like β12 (DBLβ12) domain of Plasmod-
ium falciparum erythrocyte membrane protein 1 (PfEMP1), PFD0020c. Here, we report the structural
insights into the binding of the DBLβ12 domain of PfEMP1 with the human receptor gC1qR using
computational methods. A molecular model of the DBLβ12 domain was generated and used for
protein–protein docking with the host receptor gC1qR. The protein–protein docking revealed that
the DBLβ12 asymmetrically interacts with two subunits of the gC1qR trimer at the solution face
of gC1qR. A total of 21 amino acid residues of DBLβ12 interact with 26 amino acid residues in the
gC1qR trimer through 99 nonbonding interactions and 4 hydrogen bonds. Comparative analysis of
binding sites on the DBL domain fold for the two receptors gC1qR and ICAM1 showed that the two
sites are distinct. This is the first study that provides structural insights into DBLβ12 binding with its
receptor gC1qR and may help in designing novel antisevere malaria interventions. life life 1. Introduction Malaria is one of the most devastating parasitic diseases. Malaria has caused about
409,000 deaths in 2019. An estimated 67% of all malaria deaths are among children under
5 years of age [1]. Most of the malaria related deaths are caused by Plasmodium falci-
parum infections, although 5 species of Plasmodium are known to cause human malaria. The P. falciparum infected erythrocytes (IEs) have the unique ability to cytoadhere to host
cells and completely sequester in the blood vasculature of the human host. The P. falciparum-
infected erythrocytes may form rosettes and clumps when they cytoadhere to uninfected
erythrocytes and platelets, respectively. The P. falciparum-infected erythrocytes can also
bind and adhere to microvascular endothelial cells and sequester in the blood vasculature. Sequestration of P. falciparum-infected erythrocytes may lead to obstruction in blood flow,
elevation in locally released cytokine levels, and local hypoxia. Sequestration in the brain
and placenta has been linked to severe malaria. Cytoadherence is thus considered as an
important virulence mechanism of P. falciparum [2,3]. Blocking or reversing the seques-
tration in the brain and placenta may provide novel interventions against cerebral- and
pregnancy-associated malaria, respectively [4,5]. Therefore, the study of receptor–ligand Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
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iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article
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conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). https://www.mdpi.com/journal/life Life 2021, 11, 993. https://doi.org/10.3390/life11090993 Life 2021, 11, 993 2 of 13 interactions involved in the cytoadherence of P. falciparum is important for the research and
development of novel malaria vaccines and antimalaria drugs. interactions involved in the cytoadherence of P. falciparum is important for the research and
development of novel malaria vaccines and antimalaria drugs. p
g
Cytoadherence is mediated by specific receptor–ligand interactions. More than a
dozen human receptors for cytoadherence have been identified, including CD36 [6], in-
tracellular adhesion molecule-1 (ICAM1/CD54) [7], chondroitin sulphate-A (CSA) [8],
endothelial protein C receptor (EPCR) [9], and gC1qR [10]. The parasite partner proteins
in the receptor–ligand interaction pair mediating cytoadherence are known to be P. falci-
parum erythrocyte membrane protein 1 (PfEMP1) family members. PfEMP1 proteins are
variant surface antigens of P. falciparum encoded by var genes [11]. About 60 var genes
exist in a single P. falciparum genome, encoding just as many PfEMP1 proteins. 1. Introduction Transcrip-
tional switching of var gene expression provides a new cytoadherence phenotype to the
parasite. The switching of var gene expression is a tool that allows the parasite to evade the
host’s immune response through antigenic variation [12]. PfEMP1 family members follow
mutually exclusive expression patterns; a single member is expressed on the surface of a
mature stage P. falciparum-infected erythrocyte [13]. g
f
p
y
y
The PfEMP1 proteins are transmembrane proteins that are expressed on the surface of
an infected erythrocyte. PfEMP1 proteins are encoded by var genes. The var genes have
been classified into 5 groups (A, B, C, AB, and BC groups) based on chromosomal location,
gene orientation, and sequence features. Studies have suggested that group A and group
B/A var gene expression in the IEs isolated from human hosts may be associated with
severe malaria [14]. The large extracellular domain of PfEMP1s have multiple adhesive
domains, including Duffy binding-like (DBL) domains and cystine-rich interdomain region
(CIDR) domains. Based on the organization of these adhesive domains within a PfEMP1
protein, PfEMP1s have been further classified into different domain cassettes [15]. p
Earlier, we identified a novel receptor gC1qR that mediates the cytoadherence of
P. falciparum-infected erythrocytes to human brain microvascular endothelial cells and
platelets [10]. Studies suggest that cytoadherence to gC1qR may play an important role in
severe malaria pathogenesis [16,17]. gC1qR protein exists as a homotrimer protein. It has
neither a transmembrane domain, nor a predicted Glycosylphosphatidylinositol (GPI)
anchor sequence [18]. However, the protein is known to be expressed on the surface of
several human cells, including human brain microvascular endothelial cells, dendritic cells,
and platelets [10]. The gC1qR trimer has two distinct faces: the membrane face and the
solution face [18]. In addition to the surface of human cells, gC1qR is also present in blood
serum in soluble form [19]. Activated and proliferating cells secrete a soluble form of
gC1qR (sgC1qR), which is similar to gC1qR in structure and function [20]. A conserved
motif consisting of 174–180 amino acids of sgC1qR is essential for its binding to endothelial
cells through surface bound fibrinogen. Endothelial cells bound sgC1qR can act in an
autocrine and paracrine manner to facilitate vasodilation [21]. Interestingly, mature-stage
P. falciparum-infected erythrocytes can bind to soluble gC1qR [22]. 1. Introduction f
p
y
y
g
q
The malaria parasite ligand that mediates cytoadherence to gC1qR has been identified
as the PFD0020c protein, a member of the PfEMP1 family belonging to group A var
gene. The PfD0020c protein has 5 DBL domains and 2 CIDR domains in its extracellular
adhesive domains. It is the DBLβ12 domain of PfD0020c that interacts with gC1qR [23]. However, the interface region and interacting residues of DBLβ12 have not been explored
yet. Computational methods including sequence information theoretic methods [24,25]
and molecular docking simulation methods [26,27] are useful in the study of functionally
critical residues and interacting residues in receptor–ligand interactions. Here, we have
investigated the interface region and the key interacting residues that mediate the binding
of gC1qR with DBLβ12 using computational methods. Sequence Alignment Analysis Sequence Alignment Analysis The analysis and final illustration preparation of sequence alignment was performed
using Jalview [34]. Molecular Modeling of the DBLβ12 Domain Molecular Modeling of the DBLβ12 Domain Molecular modeling of the DBLβ12 domain of PfEMP1 was carried out using Mod-
eller [30]. The template used for modeling was an ICAM1 binding DBLβ from the PDB. We have used an uncomplexed structure of DBLβ (6s8t) for our modelling to avoid ICAM1
binding-induced conformational changes. A total of 100 models were prepared using
Modeller. The five best models were chosen based on either the lower value of the Modeller
objective function or a DOPE assessment score with a higher value than the GA341 assess-
ment score. Finally, the single best model was selected by Ramachandran plot analysis of
the five best models using Procheck [31]. Molecular Dynamic Simulation Molecular dynamic simulation was carried out by Gromacs with a charmm36-feb2021
force field. The proteins were solvated in a cubic box with a simple point-charge water
molecule, spc216. The solvation box was generated having proteins at the center with
edge distance from the surface of the protein at 10 Å. The whole solvated systems were
neutralized by addition of counter ions (Na+ and Cl−) followed by energy minimization
using the steepest descent method. The periodic boundary conditions were implemented
to avoid surface effects. The energy-minimized system was subjected to NVT and then
NPT equilibration for 100 ps each. The equilibrated DBLβ12 protein and DBLβ12-gC1qR
complexes were simulated for 100 ns. Analysis of Protein–Protein Complex Analysis of Protein–Protein Complex The visual analysis of the protein–protein complex was performed using Pymol
(DeLano WL: PyMOL. San Carlos, CA. DeLano Scientific, 2002), and illustrations were
prepared. The molecular interactions between the DBLβ12 domain and the human gC1qR
were predicted using the Dimplot option of Ligplot+ [33]. Protein–Protein Docking The molecular docking of the DBLβ12 domain and the human gC1qR was carried out
using Patchdock [31], and the predicted complexes were further refined using Firedock [32]. The Patchdock algorithm uses shape complementary criteria for protein–protein docking. The two protein molecules were divided into patches (concave, convex, and flat patches)
and then the patches of the two proteins were superimposed for possible match. Finally,
the obtained protein–protein complexes were scored and ranked using a geometric shape
complementarity score. Data Retrieval The sequence of the PfEMP1 protein of Plasmodium falciparum (Strain 3D7) was ob-
tained from the PlasmoDB database [28] by translating the transcript PFD0020c (New Tran- Life 2021, 11, 993 3 of 13 3 of 13 script ID, PF3D7_0400400). The X-ray crystal structure of human receptor gC1qR in complex
with Factor XII and kininogen with PDB Id: 6szw was obtained from the PDB database. This receptor structure was exploited for the protein–protein docking experiment. Identification of DBLβ12 Domain in PfEMP1 Protein For scanning Duffy binding domains, the PfEMP1 protein sequence was fed into the
pfam database [29] and searched. The DBLβ12 domain was identified as ranging from
843–1030 amino acids of the PfEMP1 protein. However, we considered a broader range
with additional residues at both ends, i.e., 745–1183 amino acids. This stretch of PfEMP1
was used for modeling of the DBLβ12 domain as this was the largest stretch encompassing
the DBLβ12 domain matching its template in the PDB. The template was identified by the
Blastp option of NCBI Blast. 3.1. Molecular Model of DBLβ12 Domain The initial and final amino acid position in a row for the template and model are dis-
played, respectively. The red triangles mark the amino acid positions of DBLβ12 residues interact-
ing with gC1qR, while the green triangles mark the positions of DBLβ residues interacting with
ICAM1. Figure 1. Sequence alignment of the model DBLβ12 with the template DBLβ (PDB ID: 6s8t). The
identically conserved columns are highlighted in blue, and the conserved residues are shown in
white color. The initial and final amino acid position in a row for the template and model are dis-
played, respectively. The red triangles mark the amino acid positions of DBLβ12 residues interact-
ing with gC1qR, while the green triangles mark the positions of DBLβ residues interacting with
ICAM1. Figure 1. Sequence alignment of the model DBLβ12 with the template DBLβ (PDB ID: 6s8t). The iden-
tically conserved columns are highlighted in blue, and the conserved residues are shown in white
color. The initial and final amino acid position in a row for the template and model are displayed,
respectively. The red triangles mark the amino acid positions of DBLβ12 residues interacting with
gC1qR, while the green triangles mark the positions of DBLβ residues interacting with ICAM1. Figure 1. Sequence alignment of the model DBLβ12 with the template DBLβ (PDB ID: 6s8t). The
identically conserved columns are highlighted in blue, and the conserved residues are shown in
white color. The initial and final amino acid position in a row for the template and model are dis-
played, respectively. The red triangles mark the amino acid positions of DBLβ12 residues interact-
ing with gC1qR, while the green triangles mark the positions of DBLβ residues interacting with
ICAM1. Figure 2. Molecular model of DBLβ12. (A) Ribbon representation of the DBLβ12 model colored in
spectrum rainbow with helices labeled with helix number. Color is in gradient from the N-terminal
Figure 2. Molecular model of DBLβ12. (A) Ribbon representation of the DBLβ12 model colored in
spectrum rainbow with helices labeled with helix number. Color is in gradient from the N-terminal
Figure 2. Molecular model of DBLβ12. (A) Ribbon representation of the DBLβ12 model colored in
spectrum rainbow with helices labeled with helix number. Color is in gradient from the N-terminal to
the C-terminal, starting from dark blue, continuing to green and then yellow and finally C-terminal
in red. (B) Topology diagram of DBLβ12 domain with labeled helices. 3.1. Molecular Model of DBLβ12 Domain The gC1qR-binding DBLβ12 domain had 48% identity and 63% similarity with the
template DBLβ that binds ICAM1 (PDB Id: 6s8t). The alignment of the model and template Life 2021, 11, 993 4 of 13 is shown in Figure 1, with identical residue columns highlighted in blue color. The struc-
tural model of the DBLβ12 domain is shown in ribbon representation colored rainbow
spectrum and as a topology diagram in Figure 2. template DBLβ that binds ICAM1 (PDB Id: 6s8t). The alignment of the model and template
is shown in Figure 1, with identical residue columns highlighted in blue color. The struc-
tural model of the DBLβ12 domain is shown in ribbon representation colored rainbow
spectrum and as a topology diagram in Figure 2. The gC1qR-binding DBLβ12 domain had 48% identity and 63% similarity with the
template DBLβ that binds ICAM1 (PDB Id: 6s8t). The alignment of the model and template
is shown in Figure 1, with identical residue columns highlighted in blue color. The struc-
tural model of the DBLβ12 domain is shown in ribbon representation colored rainbow
spectrum and as a topology diagram in Figure 2 is shown in Figure 1, with identical residue columns highlighted in blue color. The struc-
tural model of the DBLβ12 domain is shown in ribbon representation colored rainbow
spectrum and as a topology diagram in Figure 2. template DBLβ that binds ICAM1 (PDB Id: 6s8t). The alignment of the model and template
is shown in Figure 1, with identical residue columns highlighted in blue color. The struc-
tural model of the DBLβ12 domain is shown in ribbon representation colored rainbow
spectrum and as a topology diagram in Figure 2. The gC1qR-binding DBLβ12 domain had 48% identity and 63% similarity with the
template DBLβ that binds ICAM1 (PDB Id: 6s8t). The alignment of the model and template
is shown in Figure 1, with identical residue columns highlighted in blue color. The struc-
tural model of the DBLβ12 domain is shown in ribbon representation colored rainbow
spectrum and as a topology diagram in Figure 2 Figure 1. Sequence alignment of the model DBLβ12 with the template DBLβ (PDB ID: 6s8t). The
identically conserved columns are highlighted in blue, and the conserved residues are shown in
white color. The initial and final amino acid position in a row for the template and model are dis-
played, respectively. 3.1. Molecular Model of DBLβ12 Domain The red triangles mark the amino acid positions of DBLβ12 residues interact-
ing with gC1qR, while the green triangles mark the positions of DBLβ residues interacting with
ICAM1. Figure 1. Sequence alignment of the model DBLβ12 with the template DBLβ (PDB ID: 6s8t). The iden-
tically conserved columns are highlighted in blue, and the conserved residues are shown in white
color. The initial and final amino acid position in a row for the template and model are displayed,
respectively. The red triangles mark the amino acid positions of DBLβ12 residues interacting with
gC1qR, while the green triangles mark the positions of DBLβ residues interacting with ICAM1. spectrum and as a topology diagram in Figure 2. Figure 1. Sequence alignment of the model DBLβ12 with the template DBLβ (PDB ID: 6s8t). The
identically conserved columns are highlighted in blue, and the conserved residues are shown in
white color. The initial and final amino acid position in a row for the template and model are dis-
played, respectively. The red triangles mark the amino acid positions of DBLβ12 residues interact-
ing with gC1qR, while the green triangles mark the positions of DBLβ residues interacting with
ICAM1. p
p
gy
g
g Figure 1. Sequence alignment of the model DBLβ12 with the template DBLβ (PDB ID: 6s8t). The
identically conserved columns are highlighted in blue, and the conserved residues are shown in
white color. The initial and final amino acid position in a row for the template and model are dis-
played, respectively. The red triangles mark the amino acid positions of DBLβ12 residues interact-
ing with gC1qR, while the green triangles mark the positions of DBLβ residues interacting with
ICAM1. Figure 1. Sequence alignment of the model DBLβ12 with the template DBLβ (PDB ID: 6s8t). The iden-
tically conserved columns are highlighted in blue, and the conserved residues are shown in white
color. The initial and final amino acid position in a row for the template and model are displayed,
respectively. The red triangles mark the amino acid positions of DBLβ12 residues interacting with
gC1qR, while the green triangles mark the positions of DBLβ residues interacting with ICAM1. Figure 1. Sequence alignment of the model DBLβ12 with the template DBLβ (PDB ID: 6s8t). The
identically conserved columns are highlighted in blue, and the conserved residues are shown in
white color. 3.1. Molecular Model of DBLβ12 Domain All the residues
are shown as dots (except Glycine, shown as triangles) in four different regions of the plot: most
favorable (A,B,L) in red color, additional allowed (a,b,l,p) in yellow, generously allowed (~a,~b,~l,~p)
in light yellow, and disallowed regions (remaining) in white colors. Glycine residues shown as
triangles are special in having no side chain (only H-atom), so restrictions for being in different
regions of the plot does not apply on them. Table 1. Ramachandran plot analysis of the model with respect to its template. The percentage of
residues are listed in most favorable, additional allowed, generously allowed, and disallowed re-
gions of the plot. Status of the Residues in Ramachandran Plot
Table 1. Ramachandran plot analysis of the model with respect to its template. The percentage of
residues are listed in most favorable, additional allowed, generously allowed, and disallowed regions
of the plot. Protein
Most Favourable
Additional
Allowed
Generously
Allowed
Disallowed
Template
89.0%
10.3%
0.5%
0.3%
Model
87.3%
10.4%
2.0%
0.3%
3.2. Molecular Recognition of Human Receptor gC1qR by the DBLβ12 Domain
Protein
Status of the Residues in Ramachandran Plot
Most
Favourable
Additional
Allowed
Generously
Allowed
Disallowed
Template
89.0%
10.3%
0.5%
0.3%
Model
87.3%
10.4%
2.0%
0.3% g
f
p
g
q
y
β
Protein–protein docking analysis shows that the two proteins bind we
3.2. Molecular Recognition of Human Receptor gC1qR by the DBLβ12 Domain plementary surfaces, the projections, and the recesses in the surfaces were well inter-
locked. The docking results revealed that the DBLβ12 binds to the ligand accessible solu-
tion face of gC1qR and not to the membrane face. This agrees with what is expected as the
solution face is the only face accessible for binding. The absolute value of the binding en-
ergy was high (−18.69 Kcal/mol), thus the complex formed was quite stable. The list of
Protein–protein docking analysis shows that the two proteins bind well, and the com-
plementary surfaces, the projections, and the recesses in the surfaces were well interlocked. The docking results revealed that the DBLβ12 binds to the ligand accessible solution face
of gC1qR and not to the membrane face. This agrees with what is expected as the solution
face is the only face accessible for binding. The absolute value of the binding energy was
high (−18.69 Kcal/mol), thus the complex formed was quite stable. 3.1. Molecular Model of DBLβ12 Domain residue), fall in disallowed regions, as seen in the template as well [35]. Ramachandran plot
analysis establishes that the quality of the model is as good as the template. comparable to that of the template (89.0%). The low percentage of residues, 0.3% (only
one residue), fall in disallowed regions, as seen in the template as well [35]. Ramachan-
dran plot analysis establishes that the quality of the model is as good as the template. (B) (A)
(B)
Figure 3. Ramachandran plot for (A) the model (DBLβ12) and (B) the template used. All the residues
are shown as dots (except Glycine, shown as triangles) in four different regions of the plot: most
favorable (A,B,L) in red color, additional allowed (a,b,l,p) in yellow, generously allowed
(~a,~b,~l,~p) in light yellow, and disallowed regions (remaining) in white colors. Glycine residues
shown as triangles are special in having no side chain (only H-atom), so restrictions for being in
different regions of the plot does not apply on them. bl
h
d
l
l
f h
d l
h
l
h
f
Figure 3. Ramachandran plot for (A) the model (DBLβ12) and (B) the template used. All the residues
are shown as dots (except Glycine, shown as triangles) in four different regions of the plot: most
favorable (A,B,L) in red color, additional allowed (a,b,l,p) in yellow, generously allowed (~a,~b,~l,~p)
in light yellow, and disallowed regions (remaining) in white colors. Glycine residues shown as
triangles are special in having no side chain (only H-atom), so restrictions for being in different
regions of the plot does not apply on them. (B) (A) Figure 3. Ramachandran plot for (A) the model (DBLβ12) and (B) the template used. All the residues
are shown as dots (except Glycine, shown as triangles) in four different regions of the plot: most
favorable (A,B,L) in red color, additional allowed (a,b,l,p) in yellow, generously allowed
(~a,~b,~l,~p) in light yellow, and disallowed regions (remaining) in white colors. Glycine residues
shown as triangles are special in having no side chain (only H-atom), so restrictions for being in
different regions of the plot does not apply on them. Figure 3. Ramachandran plot for (A) the model (DBLβ12) and (B) the template used. 3.1. Molecular Model of DBLβ12 Domain The list of interacting
residues for both the proteins and number of molecular interactions is provided in the
Table 2. The protein–protein interaction plot for the interacting residues of both the proteins
is shown in Figure 4. 3.1. Molecular Model of DBLβ12 Domain Topology diagram shows
sequential secondary structure elements along the whole length of the protein. N and C enclosed in
yellow squares represent N- and C- terminals of the protein. The red cylindrical structures marked
with initial and final amino acid positions represent alpha helices, pink wide-arrows marked with
initial and final amino acid positions represent beta strands, and blue lines marked with direction
‘N-terminal to C-terminal’ represent random coil or loops. bbon representation of the DBLβ12 model colored in Figure 2 Molecular model of DBLβ12 (A) Ribbo spectrum rainbow with helices labeled with helix number. Color is in gradient from the N-terminal
Figure 2. Molecular model of DBLβ12. (A) Ribbon representation of the DBLβ12 model colored in
spectrum rainbow with helices labeled with helix number. Color is in gradient from the N-terminal
Figure 2. Molecular model of DBLβ12. (A) Ribbon representation of the DBLβ12 model colored in
spectrum rainbow with helices labeled with helix number. Color is in gradient from the N-terminal to
the C-terminal, starting from dark blue, continuing to green and then yellow and finally C-terminal
in red. (B) Topology diagram of DBLβ12 domain with labeled helices. Topology diagram shows
sequential secondary structure elements along the whole length of the protein. N and C enclosed in
yellow squares represent N- and C- terminals of the protein. The red cylindrical structures marked
with initial and final amino acid positions represent alpha helices, pink wide-arrows marked with
initial and final amino acid positions represent beta strands, and blue lines marked with direction
‘N-terminal to C-terminal’ represent random coil or loops. The evaluation of the model using Ramachandran plot analysis is shown in Figure 3. Table 1 shows that a high percentage of residues, 87.3%, fall in the most favorable regions
comparable to that of the template (89.0%). The low percentage of residues, 0.3% (only one Life 2021, 11, 993 5 of 13
gure 3. residue), fall in disallowed regions, as seen in the template as well [35]. Ramachandran plot
analysis establishes that the quality of the model is as good as the template. comparable to that of the template (89.0%). The low percentage of residues, 0.3% (only
one residue), fall in disallowed regions, as seen in the template as well [35]. Ramachan-
dran plot analysis establishes that the quality of the model is as good as the template. 3.3. Interface Area and Interacting Residues of DBLβ12-gC1qR Complex DBLβ12 binds the trimeric gC1qR receptor by making interactions with the two
identical monomers in the gC1qR trimer, as shown Figure 4 and Table 2. The three
monomers of gC1qR in the gC1qR trimer are referred to as gC1qRA, gC1qRB, and gC1qRC,
as per their subunit/monomer chain identifier (ID) in the PDB file. These monomers
were identical, and the analysis was performed with respect to these monomer chain IDs. Life 2021, 11, 993 6 of 13 The monomer chain IDs were used here as references and showed that the DBLβ12 bound
to the trimeric receptor and interacted differently with the two identical monomers in
the trimer. Thus, the predicted DBLβ12 interaction was asymmetric with respect to the
three monomers of human receptor gC1qR. The monomer gC1qRA contributed most to the
binding interactions by forming 62 nonbonded contacts and four hydrogen bonds with
its DBLβ12 ligand. While the gC1qRB was forming only 37 nonbonded contacts with its
DBLβ12 ligand. Table 2. Interacting residues of the PfEMP1 DBLβ12 domain and homotrimeric receptor gC1qR
at the interface area with number of nonbonded and hydrogen-bonding interactions. The gC1qR
residues are marked with the name of monomeric subunit in parentheses (A) and (B). Table 2. Interacting residues of the PfEMP1 DBLβ12 domain and homotrimeric receptor gC1qR
at the interface area with number of nonbonded and hydrogen-bonding interactions. The gC1qR
residues are marked with the name of monomeric subunit in parentheses (A) and (B). residues are marked with the name of monomeric subunit in parentheses (A) and (B). DBLβ12 Residues
gC1qR Residues
Hydrogen
Bonds
Non-Bonded
Contacts
Gly-745
Thr-165 (A)
1
Lys-746
Glu-190 (A)
1
Leu-747
Asp-254 (A)
2
Val-748
Ile-203 (A)
1
Asp-906
His-238 (A)
3
Gln-907
His-238 (A)
6
Gln-907
Asp-241 (A)
2
Asn-908
Asp-237 (A)
3
Asn-908
Trp-233 (A)
3
Asn-908
Ala-234 (A)
5
Lys-1000
Asp-241 (A)
1
1
Met-1002
Asp-241 (A)
1
Gly-1149
Asp-202 (A)
1
Lys-1150
Asp-202 (A)
1
2
Val-1152
Trp-233 (A)
5
Val-1152
Tyr-236 (A)
5
Val-1152
Asp-237 (A)
3
Gly-1153
Trp-233 (A)
7
Asn-1155
Leu-231 (A)
1
2
Lys-1158
Asp-229 (A)
1
4
Lys-1164
Glu-190 (A)
4
Asp-906
Phe-85 (B)
7
Gln-907
Gly-78 (B)
3
Gln-907
Ala-81 (B)
2
Lys-1046
Thr-163 (B)
12
Gly-1047
Glu-258 (B)
2
Lys-1048
Glu-258 (B)
1
Lys-1048
Asp-254 (B)
8
Gln-1049
Glu-258 (B)
1
Glu-1176
Ile-139 (B)
1
A total of 21 DBLβ12 residues were predicted to interact with gC1qR. 3.3. Interface Area and Interacting Residues of DBLβ12-gC1qR Complex These 21 interact-
ing residues were Gly-745, Lys-746, Leu-747, Val-748, Asp-906, Gln-907, Asn-908, Lys-1000,
Met-1002, Lys-1046, Gly-1047, Lys-1048, Gln-1049, Gly-1149, Lys-1150, Val-1152, Gly-1153,
Asn-1155, Lys-1158, Lys-1164, and Glu-1176. These 21 DBLβ12 residues contributed to a
total of 99 nonbonded contacts and four hydrogen bonds and stabilized the protein–protein
complex. Among these 21 interacting residues of DBLβ12, four residues (Lys-1000, Lys-
1150, Asn-1155, and Lys-1158) formed one hydrogen bond each. The maximum number of
nonbonded interactions were 12, formed by Lys-1046. The 21 DBLβ12 residues can be mapped to five continuous peptide stretches to fa-
cilitate the identification of targets to disrupt gC1qR-DBLβ12 interaction. These amino
acid stretches include 745–748 (Peptide-1, 4 residues), 906–908 (Peptide-2, 3 residues),
1000–1002 (Peptide-3, 3 residues), 1046–1049 (Peptide-4, 4 residues), and 1149–1176 (Peptide-
5, 28 residues). The peptide-1, peptide-3, and peptide-5 bind subunit gC1qRA. The Peptide-
4 binds subunit gC1qRB. The peptide-2 binds both the subunits gC1qRA and gC1qRB. residues are marked with the name of monomeric subunit in parentheses (A) and (B). 3.3. Interface Area and Interacting Residues of DBLβ12-gC1qR Complex DBLβ12 Residues
gC1qR Residues
Hydrogen
Bonds
Non-Bonded
Contacts
Gly-745
Thr-165 (A)
1
Lys-746
Glu-190 (A)
1
Leu-747
Asp-254 (A)
2
Val-748
Ile-203 (A)
1
Asp-906
His-238 (A)
3
Gln-907
His-238 (A)
6
Gln-907
Asp-241 (A)
2
Asn-908
Asp-237 (A)
3
Asn-908
Trp-233 (A)
3
Asn-908
Ala-234 (A)
5
Lys-1000
Asp-241 (A)
1
1
Met-1002
Asp-241 (A)
1
Gly-1149
Asp-202 (A)
1
Lys-1150
Asp-202 (A)
1
2
Val-1152
Trp-233 (A)
5
Val-1152
Tyr-236 (A)
5
Val-1152
Asp-237 (A)
3
Gly-1153
Trp-233 (A)
7
Asn-1155
Leu-231 (A)
1
2
Lys-1158
Asp-229 (A)
1
4
Lys-1164
Glu-190 (A)
4
Asp-906
Phe-85 (B)
7
Gln-907
Gly-78 (B)
3
Gln-907
Ala-81 (B)
2
Lys-1046
Thr-163 (B)
12
Gly-1047
Glu-258 (B)
2
Lys-1048
Glu-258 (B)
1
Lys-1048
Asp-254 (B)
8
Gln-1049
Glu-258 (B)
1
Glu-1176
Ile-139 (B)
1 DBLβ12 Residues
gC1qR Residues
Hydrogen
Bonds
Non-Bonded
Contacts
Gly-745
Thr-165 (A)
1
Lys-746
Glu-190 (A)
1
Leu-747
Asp-254 (A)
2
Val-748
Ile-203 (A)
1
Asp-906
His-238 (A)
3
Gln-907
His-238 (A)
6
Gln-907
Asp-241 (A)
2
Asn-908
Asp-237 (A)
3
Asn-908
Trp-233 (A)
3
Asn-908
Ala-234 (A)
5
Lys-1000
Asp-241 (A)
1
1
Met-1002
Asp-241 (A)
1
Gly-1149
Asp-202 (A)
1
Lys-1150
Asp-202 (A)
1
2
Val-1152
Trp-233 (A)
5
Val-1152
Tyr-236 (A)
5
Val-1152
Asp-237 (A)
3
Gly-1153
Trp-233 (A)
7
Asn-1155
Leu-231 (A)
1
2
Lys-1158
Asp-229 (A)
1
4
Lys-1164
Glu-190 (A)
4
Asp-906
Phe-85 (B)
7
Gln-907
Gly-78 (B)
3
Gln-907
Ala-81 (B)
2
Lys-1046
Thr-163 (B)
12
Gly-1047
Glu-258 (B)
2
Lys-1048
Glu-258 (B)
1
Lys-1048
Asp-254 (B)
8
Gln-1049
Glu-258 (B)
1
Glu-1176
Ile-139 (B)
1
A total of 21 DBLβ12 residues were predicted to interact with gC1qR. These 21 interact-
ing residues were Gly-745, Lys-746, Leu-747, Val-748, Asp-906, Gln-907, Asn-908, Lys-1000,
Met-1002, Lys-1046, Gly-1047, Lys-1048, Gln-1049, Gly-1149, Lys-1150, Val-1152, Gly-1153,
Asn-1155, Lys-1158, Lys-1164, and Glu-1176. These 21 DBLβ12 residues contributed to a
total of 99 nonbonded contacts and four hydrogen bonds and stabilized the protein–protein
complex. Among these 21 interacting residues of DBLβ12, four residues (Lys-1000, Lys-
1150, Asn-1155, and Lys-1158) formed one hydrogen bond each. The maximum number of
nonbonded interactions were 12, formed by Lys-1046. The 21 DBLβ12 residues can be mapped to five continuous peptide stretches to fa-
cilitate the identification of targets to disrupt gC1qR-DBLβ12 interaction. 3.3. Interface Area and Interacting Residues of DBLβ12-gC1qR Complex These amino
acid stretches include 745–748 (Peptide-1, 4 residues), 906–908 (Peptide-2, 3 residues),
1000–1002 (Peptide-3, 3 residues), 1046–1049 (Peptide-4, 4 residues), and 1149–1176 (Peptide-
5, 28 residues). The peptide-1, peptide-3, and peptide-5 bind subunit gC1qRA. The Peptide-
4 binds subunit gC1qRB. The peptide-2 binds both the subunits gC1qRA and gC1qRB. A total of 21 DBLβ12 residues were predicted to interact with gC1qR. These 21 interact-
ing residues were Gly-745, Lys-746, Leu-747, Val-748, Asp-906, Gln-907, Asn-908, Lys-1000,
Met-1002, Lys-1046, Gly-1047, Lys-1048, Gln-1049, Gly-1149, Lys-1150, Val-1152, Gly-1153,
Asn-1155, Lys-1158, Lys-1164, and Glu-1176. These 21 DBLβ12 residues contributed to a
total of 99 nonbonded contacts and four hydrogen bonds and stabilized the protein–protein
complex. Among these 21 interacting residues of DBLβ12, four residues (Lys-1000, Lys-
1150, Asn-1155, and Lys-1158) formed one hydrogen bond each. The maximum number of
nonbonded interactions were 12, formed by Lys-1046. The 21 DBLβ12 residues can be mapped to five continuous peptide stretches to fa-
cilitate the identification of targets to disrupt gC1qR-DBLβ12 interaction. These amino
acid stretches include 745–748 (Peptide-1, 4 residues), 906–908 (Peptide-2, 3 residues),
1000–1002 (Peptide-3, 3 residues), 1046–1049 (Peptide-4, 4 residues), and 1149–1176 (Peptide-
5, 28 residues). The peptide-1, peptide-3, and peptide-5 bind subunit gC1qRA. The Peptide-
4 binds subunit gC1qRB. The peptide-2 binds both the subunits gC1qRA and gC1qRB. A total of 21 DBLβ12 residues were predicted to interact with gC1qR. These 21 interact-
ing residues were Gly-745, Lys-746, Leu-747, Val-748, Asp-906, Gln-907, Asn-908, Lys-1000,
Met-1002, Lys-1046, Gly-1047, Lys-1048, Gln-1049, Gly-1149, Lys-1150, Val-1152, Gly-1153,
Asn-1155, Lys-1158, Lys-1164, and Glu-1176. These 21 DBLβ12 residues contributed to a
total of 99 nonbonded contacts and four hydrogen bonds and stabilized the protein–protein
complex. Among these 21 interacting residues of DBLβ12, four residues (Lys-1000, Lys-
1150, Asn-1155, and Lys-1158) formed one hydrogen bond each. The maximum number of
nonbonded interactions were 12, formed by Lys-1046. A total of 21 DBLβ12 residues were predicted to interact with gC1qR. These 21 interact-
ing residues were Gly-745, Lys-746, Leu-747, Val-748, Asp-906, Gln-907, Asn-908, Lys-1000,
Met-1002, Lys-1046, Gly-1047, Lys-1048, Gln-1049, Gly-1149, Lys-1150, Val-1152, Gly-1153,
Asn-1155, Lys-1158, Lys-1164, and Glu-1176. These 21 DBLβ12 residues contributed to a
total of 99 nonbonded contacts and four hydrogen bonds and stabilized the protein–protein
complex. Among these 21 interacting residues of DBLβ12, four residues (Lys-1000, Lys-
1150, Asn-1155, and Lys-1158) formed one hydrogen bond each. The maximum number of
nonbonded interactions were 12, formed by Lys-1046. 3.3. Interface Area and Interacting Residues of DBLβ12-gC1qR Complex Asn-1155, Lys-1158, Lys-1164, and Glu-1176. These 21 DBLβ12 residues contributed to a
total of 99 nonbonded contacts and four hydrogen bonds and stabilized the protein–protein
complex. Among these 21 interacting residues of DBLβ12, four residues (Lys-1000, Lys-
1150, Asn-1155, and Lys-1158) formed one hydrogen bond each. The maximum number of
nonbonded interactions were 12, formed by Lys-1046. y
y
The 21 DBLβ12 residues can be mapped to five continuous peptide stretches to fa-
cilitate the identification of targets to disrupt gC1qR-DBLβ12 interaction. These amino
acid stretches include 745–748 (Peptide-1, 4 residues), 906–908 (Peptide-2, 3 residues),
1000–1002 (Peptide-3, 3 residues), 1046–1049 (Peptide-4, 4 residues), and 1149–1176 (Peptide-
5, 28 residues). The peptide-1, peptide-3, and peptide-5 bind subunit gC1qRA. The Peptide-
4 binds subunit gC1qRB. The peptide-2 binds both the subunits gC1qRA and gC1qRB. 7 of 13 Life 2021, 11, 993 Figure 4. Protein–protein interaction plot of DBLβ12 and human receptor gC1qR. (A). The interact-
ing proteins, DBLβ12 and gC1qR, are marked at the top, and their amino acid residues are shown
in columns. The amino acids are shown in elliptical shapes and colored based on their physicochem-
ical properties. The nonbonded contacts between two amino acids are shown as dashed lines in light
orange color. The thickness of the dashed lines corresponds to the number of the interactions be-
tween the two residues. The hydrogen bonds between two residues are shown as blue lines. (B–E). The hydrogen bonds between DBLβ12 and the A subunit of gC1qR are shown in ball and stick rep-
resentations. The balls represent atoms, and the lines joining two atoms represent bonds. The vari-
ous atom types are distinguished based on color (C-atoms, black; N-atoms, blue; O-atoms, red). The
hydrogen bonds are shown as green, thick lines labeled with bond length (Å). The residues with a
pink backbone shown on the top of the hydrogen bond belong to DBLβ12, and the residues with a
light orange backbone shown below the hydrogen bonds belong to A subunit of gC1qR. igure 4. Protein–protein interaction plot of DBLβ12 and human receptor gC1qR. (A). The interacting proteins, DBLβ12 and
C1qR, are marked at the top, and their amino acid residues are shown in columns. The amino acids are shown in elliptical
hapes and colored based on their physicochemical properties. The nonbonded contacts between two amino acids are
hown as dashed lines in light orange color. 3.3. Interface Area and Interacting Residues of DBLβ12-gC1qR Complex The amino acids are shown in elliptical
shapes and colored based on their physicochemical properties. The nonbonded contacts between two amino acids are
shown as dashed lines in light orange color. The thickness of the dashed lines corresponds to the number of the interactions
between the two residues. The hydrogen bonds between two residues are shown as blue lines. (B–E). The hydrogen bonds
between DBLβ12 and the A subunit of gC1qR are shown in ball and stick representations. The balls represent atoms, and the
lines joining two atoms represent bonds. The various atom types are distinguished based on color (C-atoms, black; N-atoms,
blue; O-atoms, red). The hydrogen bonds are shown as green, thick lines labeled with bond length (Å). The residues with a
pink backbone shown on the top of the hydrogen bond belong to DBLβ12, and the residues with a light orange backbone
shown below the hydrogen bonds belong to A subunit of gC1qR. Figure 4. Protein–protein interaction plot of DBLβ12 and human receptor gC1qR. (A). The interact-
ing proteins, DBLβ12 and gC1qR, are marked at the top, and their amino acid residues are shown
in columns. The amino acids are shown in elliptical shapes and colored based on their physicochem-
ical properties. The nonbonded contacts between two amino acids are shown as dashed lines in light
orange color. The thickness of the dashed lines corresponds to the number of the interactions be-
tween the two residues. The hydrogen bonds between two residues are shown as blue lines. (B–E). The hydrogen bonds between DBLβ12 and the A subunit of gC1qR are shown in ball and stick rep-
resentations. The balls represent atoms, and the lines joining two atoms represent bonds. The vari-
ous atom types are distinguished based on color (C-atoms, black; N-atoms, blue; O-atoms, red). The
hydrogen bonds are shown as green, thick lines labeled with bond length (Å). The residues with a
pink backbone shown on the top of the hydrogen bond belong to DBLβ12, and the residues with a
light orange backbone shown below the hydrogen bonds belong to A subunit of gC1qR
Figure 4. Protein–protein interaction plot of DBLβ12 and human receptor gC1qR. (A). The interacting proteins, DBLβ12 and
gC1qR, are marked at the top, and their amino acid residues are shown in columns. The amino acids are shown in elliptical
shapes and colored based on their physicochemical properties. 3.3. Interface Area and Interacting Residues of DBLβ12-gC1qR Complex The thickness of the dashed lines corresponds to the number of the interactions
etween the two residues. The hydrogen bonds between two residues are shown as blue lines. (B–E). The hydrogen bonds
etween DBLβ12 and the A subunit of gC1qR are shown in ball and stick representations. The balls represent atoms, and the
nes joining two atoms represent bonds. The various atom types are distinguished based on color (C-atoms, black; N-atoms,
lue; O-atoms, red). The hydrogen bonds are shown as green, thick lines labeled with bond length (Å). The residues with a
ink backbone shown on the top of the hydrogen bond belong to DBLβ12, and the residues with a light orange backbone
hown below the hydrogen bonds belong to A subunit of gC1qR. Figure 4. Protein–protein interaction plot of DBLβ12 and human receptor gC1qR. (A). The int
ing proteins DBLβ12 and gC1qR are marked at the top and their amino acid residues are s
Figure 4. Protein–protein interaction plot of DBLβ12 and human receptor gC1qR. (A). The interacting protei Figure 4. Protein–protein interaction plot of DBLβ12 and human receptor gC1qR. (A). The interact-
ing proteins, DBLβ12 and gC1qR, are marked at the top, and their amino acid residues are shown
in columns. The amino acids are shown in elliptical shapes and colored based on their physicochem-
ical properties. The nonbonded contacts between two amino acids are shown as dashed lines in light
orange color. The thickness of the dashed lines corresponds to the number of the interactions be-
tween the two residues. The hydrogen bonds between two residues are shown as blue lines. (B–E). The hydrogen bonds between DBLβ12 and the A subunit of gC1qR are shown in ball and stick rep-
resentations. The balls represent atoms, and the lines joining two atoms represent bonds. The vari-
ous atom types are distinguished based on color (C-atoms, black; N-atoms, blue; O-atoms, red). The
hydrogen bonds are shown as green, thick lines labeled with bond length (Å). The residues with a
pink backbone shown on the top of the hydrogen bond belong to DBLβ12, and the residues with a
light orange backbone shown below the hydrogen bonds belong to A subunit of gC1qR
Figure 4. Protein–protein interaction plot of DBLβ12 and human receptor gC1qR. (A). The interacting proteins, DBLβ12 and
gC1qR, are marked at the top, and their amino acid residues are shown in columns. 3.3. Interface Area and Interacting Residues of DBLβ12-gC1qR Complex The nonbonded contacts between two amino acids are
shown as dashed lines in light orange color. The thickness of the dashed lines corresponds to the number of the interactions
between the two residues. The hydrogen bonds between two residues are shown as blue lines. (B–E). The hydrogen bonds
between DBLβ12 and the A subunit of gC1qR are shown in ball and stick representations. The balls represent atoms, and the
lines joining two atoms represent bonds. The various atom types are distinguished based on color (C-atoms, black; N-atoms,
blue; O-atoms, red). The hydrogen bonds are shown as green, thick lines labeled with bond length (Å). The residues with a
pink backbone shown on the top of the hydrogen bond belong to DBLβ12, and the residues with a light orange backbone
shown below the hydrogen bonds belong to A subunit of gC1qR. ackbone shown below the hydrogen bonds belong to A subunit of gC1qR. 3.4. Comparative Analysis of gC1qR-Binding Site and ICAM1 Binding Sites in DBL Domains ackbone shown below the hydrogen bonds belong to A subunit of gC1qR. 3.4. Comparative Analysis of gC1qR-Binding Site and ICAM1 Binding Sites in DBL Domain c bo e s o
be o
e
y
oge
bo
s be o g o A subu i o gC q
3.4. Comparative Analysis of gC1qR-Binding Site and ICAM1 Binding Sites in DBL Domains The structural superposition of DBLβ bound with ICAM1 and DBLβ12 bound with
gC1qR shows that the ICAM1 and gC1qR-binding site regions fell at two separate locations
of the structural fold of the DBLβ domain, as shown in Figure 5. Thus, the interaction
occurs at two distinct sites in the DBL domain with no overlapping regions. The DBLβ domain that specifically binds ICAM1 and the DBLβ12 domain that binds
gC1qR share 48% sequence identity. However, the sequence identity at the interacting
sites of these two domains with their respective receptors was low, suggesting binding
specificity induced selection pressure at these amino acid locations. Among the 21 DBLβ12
residues that interact gC1qR, only four residues (19%) were conserved in the alignment Life 2021, 11, 993 8 of 13
g
BLβ12
t f of DBLβ12 and DBLβ (red marked in Figure 1). Similarly, among 19 residues that inter-
act ICAM1, only two residues (10%) were conserved in the alignment of DBLβ12 and
DBLβ (green marked in Figure 1). These observations suggest that the receptor binding
specificity is determined by the variations in the interacting residues of the DBLβ and
DBLβ12 domains. ICAM1, only two residues (10%) were conserved in the alignment of DBLβ12 and DBLβ
(green marked in Figure 1). These observations suggest that the receptor binding specific-
ity is determined by the variations in the interacting residues of the DBLβ and DBLβ12
domains. of DBLβ12 and DBLβ (red marked in Figure 1). Similarly, among 19 residues that inter-
act ICAM1, only two residues (10%) were conserved in the alignment of DBLβ12 and
DBLβ (green marked in Figure 1). These observations suggest that the receptor binding
specificity is determined by the variations in the interacting residues of the DBLβ and
DBLβ12 domains. ICAM1, only two residues (10%) were conserved in the alignment of DBLβ12 and DBLβ
(green marked in Figure 1). These observations suggest that the receptor binding specific-
ity is determined by the variations in the interacting residues of the DBLβ and DBLβ12
domains. Figure 5. Structural superposition of DBL domains with bound respective receptors. All the protein
subunits are shown in ribbon representation. The two DBL domains DBLβ12 and DBLβ are super-
imposed and colored green and light orange, respectively. The DBLβ receptor ICAM1 is shown in
purple color, while the DBLβ12 receptor gC1qR is shown in blue color. 3.5. Molecular Dynamic Simulation The molecular dynamic simulation was performed on the DBLβ12 model and the
DBLβ12-gC1qR complex to evaluate the stability of the protein model and the complex,
respectively. The DBLβ12 model was subjected to 100 ns simulation, and the RMSD plot
showed the initial increase in RMSD (till approx. 16 ns), and then the plateau was reached. The low deviation in RMSD values during 100 ns simulation indicate the good stability
of the DBLβ12 model. The complex of the DBLβ12-gC1qR proteins was also subjected to
100 ns simulation, and the RMSD plot showed initial increase till 40ns, and then the plateau
was reached. This observation of low deviations in RMSD values of the complex during
the simulation also suggests that the protein complex is stable. c bo e s o
be o
e
y
oge
bo
s be o g o A subu i o gC q
3.4. Comparative Analysis of gC1qR-Binding Site and ICAM1 Binding Sites in DBL Domains The binding sites for the two
receptors are located at different regions on the surface of the DBL domain. Figure 5. Structural superposition of DBL domains with bound respective receptors. All the protein
subunits are shown in ribbon representation. The two DBL domains DBLβ12 and DBLβ are super-
imposed and colored green and light orange, respectively. The DBLβ receptor ICAM1 is shown in
purple color, while the DBLβ12 receptor gC1qR is shown in blue color. The binding sites for the two
receptors are located at different regions on the surface of the DBL domain. 4. Discussion This study has identified and characterized molecular interaction between gC1qR
and DBLβ12 of PfEMP1 PFD0020c of P. falciparum 3D7 using computational methods. This is the first study to characterize the structural details of gC1qR–DBLβ12 interaction in
malaria. Our study has used several rational approaches that have made our computational Life 2021, 11, 993 9 of 13 9 of 13 characterization more realistic. Firstly, to build the model of DBLβ12, we have used a
template of another PfEMP1 DBLβ crystal structure in an unbound state to its receptor,
and thus we have removed the influence of the bound receptor on the ligand in our model. Secondly, the identity and similarity between our model and the template protein sequence
was 48% and 63%, respectively, giving us a fair amount of confidence in our predicted
structure. Thirdly, the length of the amino acid sequence of the model was chosen to
match the length of the template sequence, meaning we have included adjacent stretches
of protein that may have influence on the folding of the DBLβ12 domain in the template
while retaining all the conserved residues that are hallmarks of DBL domain. Fourthly,
the amino acid residues that interact with gC1qR were not much conserved between
the DBLβ12 and DBLβ despite a 48% sequence identity in the two ligands. The amino
acid residues in the two DBL domains ought to be different since the structure of their
receptors are different. And finally, while modeling the interaction, we have not given any
constrains to the computer program to choose any portion or face of the receptor or ligand. Despite using such criteria, our model showed that the DBLβ12 binds to the solution face
of gC1qR. The solution face is the only face of membrane attached gC1qR that is available
for interaction to ligands. g
Interestingly, we found that the two monomers, gC1qRA and gC1qRB, of the gC1qR
trimer interact with the DBLβ12 domain. This interaction is asymmetric with respect to the
gC1qR trimer (Figure A1A) in Appendix A. This type of asymmetric interaction is expected
when the receptor is multimeric and the ligand is a monomer. In fact, the interaction
between gC1qR with factor XII and kininogen (PDB Id: 6szw) is asymmetric as well,
involving only one subunit of gC1qR [36]. 4. Discussion Protein–protein docking results showed that 21 of the DBβ12 residues interacted
with 26 residues of the gC1qR trimer through 99 nonbonding interactions and 4 hydro-
gen bonds. Of the 21 interacting residues of DBLβ12, five residues, Lys-1000, Lys-1150,
Asn-1155, Lys-1158, and Lys-1046, were proposed as key interacting residues since they
formed the major part of the molecular interactions. We have identified 5 stretches of
peptides in DBLβ12 that can be targeted for the disruption of gC1qR-DBLβ12 interactions. We have named these peptide stretches as peptide-1 to peptide-5 to facilitate identification. While peptide-1 to peptide-4 are three to four amino acid long, the peptide-5 is the longest,
comprising 28 amino acids. Peptide-5 can be divided into two, since synthesis of smaller
peptides is less error prone. These peptide regions can be targeted for disrupting gC1qR-
DBLβ12 interactions. The monomer A of the gC1qR trimer makes most of the contacts
with DBLβ12, including all 4 hydrogen bonds. Therefore, we anticipate that disrupting
the interactions between DBLβ12 and gC1qRA will have stronger disruptive effect on the
receptor–ligand interactions as compared to targeting the interactions between DBLβ12
and gC1qRB. Disruption of the interactions between DBLβ12 and both gC1qRA & gC1qRB
may have synergistic effects. Further studies are needed to test these predictions. The in-
formation of key interacting residues and the proposed amino acid stretches may help
in research on the development of an anti-severe malaria vaccine as well as the rational
designing of novel therapeutics to treat and manage severe malaria. The DBLβ domain that binds ICAM1 and the DBLβ12 domain that binds gC1qR share
sequence homology and belong to same domain class. It is intriguing how P. falciparum
employs the different DBL domains to bind a variety of host receptors [37]. We attempted to
understand the binding specificity of these two DBL domains. The structural superposition
of the DBL domains and amino acid conservation analysis of the binding sites of ICAM1
and gC1qR for DBL domains (DBLβ and DBLβ12) revealed that the ICAM1 and gC1qR
bind at distinct locations on the DBL domain surface, and the binding specificity may be
attributed to variations in the amino acid positions found in the binding site regions for the
two receptors despite them having a similar structure fold of the DBL domain. Multiligand receptors like gC1qR generally employ certain residues more frequently
to interact with their ligands. 4. Discussion For instance, the hepatitis C virus core protein interacts
with gC1qR at residues 188–259 [38], a site that overlaps with the DBLβ12 interacting Life 2021, 11, 993 10 of 13 10 of 13 site identified in this study. Similarly, we have found that Trp-233 is a very prominent
amino acid that makes 17 nonbonded interactions with the DBLβ12. The Trp-233 has a
conspicuous position on the solution face of the gC1qR trimer (Figure A1B), and it has also
been reported to be involved in the interaction of gC1qR with kininogen and the generation
of bradykinin as well [39]. It will be interesting to see if the binding of DBLβ12 to gC1qR
can inhibit kininogen binding and bradykinin production. g
g
y
p
The complement component C1q binds at residues 76–93 of gC1qR [40], a site con-
taining three residues (Gly-78, Ala-81, and Phe-85) which interact with DBLβ12. Further,
C1q and PfEMP1 proteins are very large in size. Therefore, the binding of one of the
proteins to gC1qR is likely to hinder the binding of the other protein. If this happens,
the cytoadherence of P. falciparum-infected erythrocytes to gC1qR may affect the localized
complement activation. To our knowledge, this is the first study that reports the interaction between gC1qR
and its pathogen ligand involving two of its three subunits. This asymmetric interac-
tion between gC1qR and the DBLβ12 domain employs several amino acid residues that
gC1qR uses to interact with its other ligands as well [38,40]. However, Ile-139 and Thr-163
seems to be the gC1qR residues that are unique to its interaction with the malaria ligand
DBLβ12. gC1qR is also secreted from human cells and is also present as a soluble recep-
tor in the blood. Serum levels of soluble gC1qR get upregulated during inflammation
and certain cancers. gC1qr has been implicated in cancer progression as an inducer of
angiogenesis and facilitator of metastasis, as well as through the inhibition of innate im-
mune response against cancerous cells through free C1q depletion [41]. Thus, gC1qR is
considered as a potential therapeutic target against certain cancers [42]. Our previous
works have shown that P. falciparum-infected erythrocytes bind to cell surface gC1qR as
well as soluble gC1qR [10,16,22]. It will be interesting to see if during falciparum infection
P. falciparum-infected erythrocytes bind to soluble gC1qR present in blood and modulate its
local concentration and function. We hypothesize that gC1qR ligands of P. Conflicts of Interest: The authors declare no conflict of interest. 4. Discussion falciparum may
provide valuable intervention targets, not only against severe malaria, but also against
certain cancers. In fact, evidence suggests that another P. falciparum cytoadherence receptor,
VAR2CSA, is useful for targeting human cancers [43]. Further studies are needed to explore
the role of this important cytoadherence receptor in severe malaria and other diseases. Author Contributions: Conceptualization, A.H., M.R. and R.B.; methodology, A.H. and M.R.; soft-
ware, M.R., R.B. and H.S.; resources, A.H. and R.B.; data curation, M.R., R.B. and H.S.; writing—
original draft preparation, A.H. and M.R.; writing—review and editing, R.B.; visualization, M.R.;
supervision, A.H.; project administration, A.H. and R.B.; funding acquisition, A.H. and R.B. All au-
thors have read and agreed to the published version of the manuscript. Funding: The research is funded by grant number 14-MED2474-10 from the National Science,
Technology and Innovation Plan (MAARIFAH), the King Abdul-Aziz City for Science and Technology
(KACST), Kingdom of Saudi Arabia. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the
corresponding author. Acknowledgments: We thank National Science, Technology and Innovation Plan (MAARIFAH),
the King Abdul-Aziz City for Science and Technology (KACST), Kingdom of Saudi Arabia for
funding through grant number 14-MED2474-10 and support. We thank the Science and Technology
Unit at Umm Al-Qura University for their continue logistics support. We thank Faizan Ur Rehman,
Institute of Consulting Research and Studies, Umm Al-Qura University for providing computation
system and support. The molecular dynamic simulations in this work were performed at King
Abdulaziz University’s High Performance Computing Center. 11 of 13
p
King Ab- Life 2021, 11, 993 Appendix A
Appendix A
Figure A1. The complex of DBLβ12 and human receptor gC1qR in surface representation. (A). One
view of the complex showing labeled DBLβ12 in green color and the trimeric receptor with each
subunit colored differently. (B). Another view of the complex with interlocked surfaces between the
DBLβ12 and the subunits of gC1qR. The position of conspicuous residue Trp-233 that forms the
maximum nonbonding interactions with the ligand is shown. Figure A1. The complex of DBLβ12 and human receptor gC1qR in surface representation. (A). One
view of the complex showing labeled DBLβ12 in green color and the trimeric receptor with each
subunit colored differently. (B). 4. Discussion Another view of the complex with interlocked surfaces between
the DBLβ12 and the subunits of gC1qR. The position of conspicuous residue Trp-233 that forms the
maximum nonbonding interactions with the ligand is shown. FOR PEER REVIEW Appendix A
Appendix A Appendix A
Appendix A Figure A1. The complex of DBLβ12 and human receptor gC1qR in surface representation. (A). One
view of the complex showing labeled DBLβ12 in green color and the trimeric receptor with each
subunit colored differently. (B). Another view of the complex with interlocked surfaces between the
DBLβ12 and the subunits of gC1qR. The position of conspicuous residue Trp-233 that forms the
maximum nonbonding interactions with the ligand is shown. Figure A1. The complex of DBLβ12 and human receptor gC1qR in surface representation. (A). One
view of the complex showing labeled DBLβ12 in green color and the trimeric receptor with each
subunit colored differently. (B). Another view of the complex with interlocked surfaces between
the DBLβ12 and the subunits of gC1qR. The position of conspicuous residue Trp-233 that forms the
maximum nonbonding interactions with the ligand is shown. OR PEER REVIEW Figure A1. The complex of DBLβ12 and human receptor gC1qR in surface representation. (A). One
view of the complex showing labeled DBLβ12 in green color and the trimeric receptor with each
subunit colored differently. (B). Another view of the complex with interlocked surfaces between the
DBLβ12 and the subunits of gC1qR. The position of conspicuous residue Trp-233 that forms the
maximum nonbonding interactions with the ligand is shown. Figure A1. The complex of DBLβ12 and human receptor gC1qR in surface representation. (A). One
view of the complex showing labeled DBLβ12 in green color and the trimeric receptor with each
subunit colored differently. (B). Another view of the complex with interlocked surfaces between
the DBLβ12 and the subunits of gC1qR. The position of conspicuous residue Trp-233 that forms the
maximum nonbonding interactions with the ligand is shown. R PEER REVIEW A2. The molecular dynamic simulations of the DBLβ12 model and DBLβ12-gC1qR complex. (A) Root mea
n (RMSD) variation with respect to time for simulation of DBLβ12. (B). RMSD variation with respect to
on of DBLβ12-gC1qR complex. Figure A2. The molecular dynamic simulations of the DBLβ12 model and DBLβ12-gC1qR complex. (A) Root mean square deviation (RMSD) variation with respect to time for simulation of DBLβ12. (B). RMSD variation with respect to time for simulation of DBLβ12-gC1qR complex. . nces
t 2020; World Health Organization: Geneva, Switzerland, 2020. References
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World Malaria Report 2020; World Health Organization: Geneva, Switzerland, 2020. 4. Discussion The molecular dynamic simulations of the DBLβ12 model and DBLβ12-gC1qR complex. (A) Root mea
(RMSD) variation with respect to time for simulation of DBLβ12. (B). RMSD variation with respect to
n of DBLβ12-gC1qR complex. Figure A2. The molecular dynamic simulations of the DBLβ12 model and DBLβ12-gC1qR complex. (A) Root mean square deviation (RMSD) variation with respect to time for simulation of DBLβ12. (B). RMSD variation with respect to time for simulation of DBLβ12-gC1qR complex. 1.
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Abstract The infraorbital foramen (IOF) serves as an important anatomical reference for infraorbital nerve block
during different procedures. Its clinical and surgical relevance, associated with conflicting results in its
anatomy description justifies the present study. Thus, this work aims to describe the IOF, qualitatively and
quantitatively, and propose an accurate measurement to define its location. One hundred and seventy-
four IOFs of dry skulls were analyzed and divided in dentate (DE) and edentulous (ED) groups. Distances
were measured between the IOF and the margin of the alveolar arch of the maxilla (MAAM), the inferior
orbital margin (IOM), the intermaxillary suture (IMS), the vertical plane of IOF. A new methodology was
proposed to optimize the identification of IOF based on the IMS distance. Oval-shaped IOFs (n=81/174)
with inferomedial orientation (n=104/174) and with a crest-shaped upper margin (n=163/174) constituted
the norm for this sample. Distance between IOF and MAAM was 29.97 ± 4.09 mm, to IOM 7.27 ± 1.8 mm,
and to IMS 35.09 ± 3.77 mm. ED individuals had IOF closer to MAAM (p=0.0124). The highest point of this
methodology was the lack of statistical differences in the IMS distance in ED and DE specimens, making this
method applicable for both. Therefore, to minimize iatrogenic injuries, clinicians and surgeons must
consider the total or partial presence of teeth, the methodology established to find the IOF, its
inferomedial orientation, and the prevalent crest on its superior margin. Keywords: Infraorbital foramen. Infraorbital nerve. Maxilla. Morphometry. MORPHOMETRY OF THE INFRAORBITAL FORAMEN IN
DENTATE AND EDENTULOUS ADULT HUMANS AND ITS
CLINICAL RELEVANCE João Gabriel ALEXANDER1
, Josemberg da Silva BAPTISTA1 1 Laboratory of Applied Morphology – LEMA, Universidade Federal do Espírito Santo, Espírito Santo, Brazil. Corresponding author:
Josemberg da Silva Baptista
josemberg.baptista@ufes.br How to cite: ALEXANDER, J.G. and BAPTISTA, J.S. Morphometry of the infraorbital foramen in dentate and edentulous adult humans and its
clinical relevance. Bioscience Journal. 2024, 40, e40007. https://doi.org/10.14393/BJ-v40n0a2024-68149 1. Introduction The infraorbital foramen (IOF) is located in the superior region of the maxilla, inferior to the inferior
margin of the orbit (IMO), and related to the most superior part of the zygomaticomaxillary suture, where
the infraorbital artery, vein and nerve (IN) emerge (Zhang et al. 2019). It serves as an important anatomical
and surgical reference for anesthesia, as well as for periorbital, oromaxillofacial, plastic and dental
surgeries, and for IN radiofrequency neurotomy (Aggarwal et al. 2015; Ercikti et al. 2017; Zdilla et al. 2018). Precise knowledge of its location, morphology, and possible anatomical variations prevents neurovascular
injuries in clinical-surgical procedures involving this region, and also guarantees safety during several
surgeries in the anterior and orbital surfaces of the maxilla such as rhinoplasties, tumor surgeries, or Le
Fort osteotomies type 1 (Gupta 2008; Ercikti et al. 2017; Zdilla et al. 2018). Many studies have tried to promote ways to situate the IOF determining distances between the
foramen and adjacent bone points, soft tissue structures, as well as anatomical planes. However,
important inconsistencies have been observed in some results, especially due to anatomical references. To 1 1 Morphometry of the infraorbital foramen in dentate and edentulous adult humans and its clinical relevance highlight the lack of standardization, upon analyzing the distance between IMO and IOF, Aziz et al. found a
mean distance of 8.5 ± 2.2 mm, Ercikti et al. found 8.78 mm, while Aggarwal et al. described that the same
distance was less than 5 mm (that, in 20.3% of cases) (Aziz et al. 2000; Aggarwal et al. 2015; Ercikti et al. 2017). Identifying the specific location of the IOF and performing a procedure based on certain types of
anatomical landmarks can be a complex task, even for experienced physicians (Aggarwal et al. 2015). Hence, there is an urgent need for a standardized method to find the IOF followed by morphometric
descriptions. Considering only bony landmarks such as the nasospinale (NS – anterior nasal spine) and jugale (J)
points, or the median line of the face disregarding soft tissues of the region such as muscles and the buccal
fat pad, as well as variations in the midline or in the IOF, can lead to imprecision in clinical and surgical
procedures in this region (Ercikti et al. 2017; Shin et al. 2020). Specimens The sample contained 101 dried skulls from the collection of the Department of Morphology of the
Centro de Ciencias da Saude. All procedures were authorized by the Ethics Committee for Research with
Human Beings via Plataforma Brasil (CAAE 32180520.0.0000.5060). Analysis of the specimens was
performed at the Laboratory of Studies in Applied Morphology (LEMA) of the Department of Morphology. The sample included only adult skulls, indistinguishable from age and sexual characteristics. Initially, the
IOF was identified in the maxilla, bilaterally, in relation to other adjacent structures: nasal notch, piriform
aperture, zygomatic process of the maxilla, zygomaticomaxillary suture, IMO, canine fossa, and dental
alveoli. Due to the improper condition and/or malformations, 14 skulls were excluded from the initial
sample and 87 followed other stages of qualitative and quantitative analysis, resulting in the analysis of
174 IOFs. 1. Introduction Among other anatomical references, the
extensive use of the maxillary dental arch as a basis for various techniques to block the IN, especially using
the vestibular route, has also revealed great variation: Aggarwal et al. found that IOF was most commonly
aligned to the second premolar (53.4%); Aziz et al. proposed that the first premolar (64%) was the most
common in the American population (Aziz et al. 2000; Aggarwal et al. 2015). Thus, justified by the clinical
and surgical relevance associated with conflicting results in the anatomy of the IOF, the aims of this study
were to analyze the topography and the morphometry of the IOF, propose an accurate method that allows
to identify its location, and verify differences between dentate (DE) and edentulous (ED) individuals. Formation of the dentate (DE) and the edentulous (ED) groups The sample was divided into DE and ED groups. The DE group was composed of hemicranium (both
left and right) that necessarily contained the presence of the teeth or the dental alveoli corresponding to
the maxillary incisors, canines and premolars. The ED group was composed of hemicranium (both left and
right) that presented, as evidence of an antemortem process, the complete or partial absence (minimum 1
and maximum 3) of teeth or dental alveoli corresponding to the maxillary incisors, canines or premolars. The antemortem teeth loss was characterized by total closure of the dental alveoli. No total absence of
teeth was recorded in the ED group. Qualitative analysis Specimens were described according to (Figure 1): • Shape of the IOF: rounded, oval or fusiform; • Shape of the IOF: rounded, oval or fusiform; • Shape of the IOF: rounded, oval or fusiform;
• Orientation of the IOF: regardless of being right or left, it faced medial (nasal), inferomedial or inferior
(labial); • Orientation of the IOF: regardless of being right or left, it faced medial (nasal), inferomedial or inferior
(labial); 2 2 ALEXANDER, J.G. and BAPTISTA, J.S. • Characteristic of the superior margin of the IOF: either presence or absence of a crest on its superior
margin; • Characteristic of the superior margin of the IOF: either presence or absence of a crest on its superior
margin; • Presence of the accessory foramina. Quantitative analysis For quantitative analysis, a photographic stand with an attached camera (NEX-C3, Sony, Japan) was
used. Samples were photographed in the frontal aspect of the cranium (centered in the internasal suture)
as well as with a 30º-cranial inclination (centered in the intermaxillary suture) with a 10mm scale. Measurements were performed using a computer program (ImageJ, National Institutes of Health, United
States of America) with individual scale calibration. The following distances were measured (Figure 1):
Front aspect of the cranium (standard): • Distance between the margin of the alveolar arch of the maxilla (MAAM) and the IOF; • Distance between the margin of the alveolar arch of the maxilla (MAAM) and the IOF; • Distance between the IMO and the IOF; • Distance between the intermaxillary suture (IMS) and the vertical axis where the IOF is located. This
measurement was performed over the margin of the alveolar arch of the maxilla using a millimeter paper
ruler. In this way, the arcuate shape of this part of the maxilla was completely respected;
Front aspect of the cranium (30◦-cranial inclination):
• IOF area. • Distance between the intermaxillary suture (IMS) and the vertical axis where the IOF is located. This
measurement was performed over the margin of the alveolar arch of the maxilla using a millimeter paper
ruler. In this way, the arcuate shape of this part of the maxilla was completely respected;
Front aspect of the cranium (30◦-cranial inclination):
• IOF area. Analysis of results Descriptive statistics were applied to qualitative data. For quantitative analysis, the normal
distribution was ratified and a parametric statistical test was chosen to increase scientific rigor. The
unpaired Student's T-test was chosen to analyze the comparison between DE and ED groups, regardless of
the antimere, with a significance level of 0.05. Bi
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Figure 1. Demonstration of the parameters applied for qualitative and quantitative analysis. Brown circle:
analysis of the infraorbital foramen shape; black line: distance between the infraorbital foramen and the
inferior margin of the orbit; blue line: distance between infraorbital foramen and the margin of the
alveolar arch of the maxilla; green line: distance between intermaxillary suture (passing above the alveolar Figure 1. Demonstration of the parameters applied for qualitative and quantitative analysis. Brown circle:
analysis of the infraorbital foramen shape; black line: distance between the infraorbital foramen and the
inferior margin of the orbit; blue line: distance between infraorbital foramen and the margin of the
alveolar arch of the maxilla; green line: distance between intermaxillary suture (passing above the alveolar Figure 1. Demonstration of the parameters applied for qualitative and quantitative analysis. Brown circle:
analysis of the infraorbital foramen shape; black line: distance between the infraorbital foramen and the
inferior margin of the orbit; blue line: distance between infraorbital foramen and the margin of the
alveolar arch of the maxilla; green line: distance between intermaxillary suture (passing above the alveolar 3 process) and the vertical axis where the infraorbital foramen is located; yellow arrow: analysis of the
presence of the accessory infraorbital foramen (compare the antimeres); red arrow: analysis of the
presence of the crest in the superior margin of the infraorbital foramen; purple arrows: infraorbital
foramen orientation. Scale bar: 1.5cm. Qualitative analysis Orientation
Left
Right
Total (n=174)
Inferomedial
51
53
104
Inferior
21
19
40
Medial
15
15
30 Fourteen accessory foramina were identified, located superomedially in relation to the IOF, 9
belonging to the left antimere and 5 to the right antimere. Only 3 skulls showed bilateral presence of the
accessory foramina. On both antimeres, approximately 46% of the sample (40 skulls of the 87 included)
presented the same foramina in shape, in orientation and in the presence of crest on the superior margin
of the IOF. Other skulls showed at least one difference between the 3 qualitative variables analyzed (Figure
3). Figure 3. Qualitative analysis of the infraorbital foramina. All possible groupings of characteristics related
to shape, orientation, and the presence of the crest in the infraorbital foramina are demonstrated,
according to antimeres and order of prevalence. Figure 3. Qualitative analysis of the infraorbital foramina. All possible groupings of characteristics related
to shape, orientation, and the presence of the crest in the infraorbital foramina are demonstrated,
according to antimeres and order of prevalence. Figure 3. Qualitative analysis of the infraorbital foramina. All possible groupings of characteristics related
to shape, orientation, and the presence of the crest in the infraorbital foramina are demonstrated,
according to antimeres and order of prevalence. Qualitative analysis Among the IOFs analyzed, 80 foramina belonged to the DE group and 94 belonged to the ED group. In both groups, IOFs were located in the maxilla, including the accessory IOFs, keeping an anatomical
relationship medially with the nasal notch and the piriform aperture, superiorly with the IMO, laterally
with the zygomatic process of the maxilla and zygomaticomaxillary suture, and inferiorly with the canine
fossa and dental alveoli (Figure 2). Oval-shaped IOFs (n=81/174) with inferomedial orientation (n=104/174)
and with a crest-shaped upper margin (n=163/174) constituted the norm for this sample. Tables 1 and 2
detail this data. Figure 2. Representation of the qualitative data: images A and B are specimens of the ED group; C and D
are specimens from the DE group. Purple arrow: accessory IOF. Yellow arrow: fusiform IOF. Pink arrow:
rounded IOF. Blue arrow: oval-shaped IOF with medial (nasal) orientation. Red arrow: IOF with inferior
(labial) orientation. Green arrow: IOF with inferomedial orientation. Black arrow: crest on the superior
margin of the IOF. Scale bar: 3cm. Figure 2. Representation of the qualitative data: images A and B are specimens of the ED group; C and D
are specimens from the DE group. Purple arrow: accessory IOF. Yellow arrow: fusiform IOF. Pink arrow:
rounded IOF. Blue arrow: oval-shaped IOF with medial (nasal) orientation. Red arrow: IOF with inferior
(labial) orientation. Green arrow: IOF with inferomedial orientation. Black arrow: crest on the superior
margin of the IOF. Scale bar: 3cm. Table 1. Infraorbital foramen shape. Shape
Left
Right
Total (n=174)
Oval
46
35
81
Rounded
30
37
67
Fusiform
11
15
26 Bioscience Journal | 2024 | vol. 40, e40007 | https://doi.org/10.14393/BJ-v40n0a2024-68149
4 4 4 ALEXANDER, J.G. and BAPTISTA, J.S. Table 2. Infraorbital foramen orientation. Orientation
Left
Right
Total (n=174)
Inferomedial
51
53
104
Inferior
21
19
40
Medial
15
15
30
Fourteen accessory foramina were identified, located superomedially in relation to the IOF, 9
belonging to the left antimere and 5 to the right antimere. Only 3 skulls showed bilateral presence of the
accessory foramina. On both antimeres, approximately 46% of the sample (40 skulls of the 87 included)
presented the same foramina in shape, in orientation and in the presence of crest on the superior margin
of the IOF. Other skulls showed at least one difference between the 3 qualitative variables analyzed (Figure
3). Table 2. Infraorbital foramen orientation. 4. Discussion In the present study, a qualitative and quantitative description of the IOF in the DE and the ED
individuals was performed in relation to adjacent bone structures. Morphometry allowed us to conclude that
the IOF in ED specimens is closer to the MAAM compared to the DE specimens. Although the periodontal
status and the maxillary bone loss could already have been proposed as factors that interfere in the situation
of the IOF (Aggarwal et al. 2015), it had never been proven in the literature, making this result an
unprecedented contribution. Considering the global prevalence of tooth loss at 2.4%, aggravated in third
world countries (such as Brazil, at 3.9%), these findings become even more relevant in clinical-surgical
practice (Kassebaum et al. 2014). Thus, in vestibular anesthesia in the IN, physicians and dentists should be
aware of such data in order to achieve success in the procedure and avoid iatrogenic events. Other IOF localization techniques are widely found in the literature. Most of them consider the
following structures/regions individually: bone, joint and/or cartilaginous structures, such as ala of nose (AN),
columella, anterior nasal spine/nasospinale point, zygomaticomaxillary suture, jugale point, and the median
line of the face. However, these methodologies do not consider the topography of other facial structures
such as the buccal fat pad, facial muscles, and variations in the structures in the median line of the face
(Agthong et al. 2005; Gupta 2008; Chrcanovic et al. 2011; Singh 2011; Aggarwal et al. 2015; Zdilla et al. 2018a; Zdilla et al. 2018b). The major problem in these techniques is the disregard for their effective
application in clinical practice: neglecting a deviation of the nasal septum (in the use of median line of the
face), or considering zygomaticomaxillary suture or jugale point (points not easily palpable) makes clear how
fragile the application of these methods are during procedures related to the IOF. This can lead to iatrogenic
injuries and could increase the need for anesthetic agents followed by complications related to their use
(Ercikti et al. 2017). For this reason, soft tissue landmarks could be viable options during surgeries, but there
are still few studies with relevant samples using such landmarks (Ercikti et al. 2017; Shin et al. 2020). Quantitative analysis The mean, standard deviation and antimeres regarding the IOF measurement are available in Table
3. The DE group confirmed statistically a greater distance between the MAAM and the IOF compared to
the ED group (Table 4). There were no other statistical inferences in measurements performed. Table 3. General morphometry of the infraorbital foramina. Measure (mm)
Left
Right
Mean
Area (mm2)
5,15 ± 1,95
5,06 ± 2,05
5,11 ± 1,99
Distance MAAM to IOF
29,73 ± 3,82
30,22 ± 4,35
29,97 ± 4,09
Distance IMO to IOF
7,31 ± 1,85
7,23 ± 1,76
7,27 ± 1,8
Distance IMS to vertical axis of the IOF
36,01 ± 3,55
34,17 ± 3,78
35,09 ± 3,77 5 5 Morphometry of the infraorbital foramen in dentate and edentulous adult humans and its clinical relevance Table 4. Comparative morphometry of the infraorbital foramen between the dentate and the edentulous
groups. Measure (mm)
DE
ED
Mean difference
“p” value
Area (mm2)
5,32 ± 2,03
4,93 ± 1,96
0,3850
p = 0.2064
Distance MAAM to IOF
30,81 ± 4,82
29,26 ± 3,20
1,549
p = 0.0124*
Distance IMO to IOF
7,39 ± 1,61
7,17 ± 1,95
0,2175
p = 0.4301
Distance IMS to vertical axis of the IOF
35,38 ± 4,33
34,84 ± 3,23
0,5471
p = 0.3427
4 Discussion 4. Discussion The
predominance of an inferomedial orientation (93.67%) and the nature of the bone walls of the IOF bring
practical implications that justify the superolateral orientation of the needle in vestibular anesthesia
procedures, but without its insertion in the IOF. Thus, an adequate approximation to the IOF in order to
acquire precise analgesia in the IN can be achieved safely, avoiding needle tapping into the IOF and
iatrogenic injury to the IN. Yet, in anesthetic methods with the superior approach in the IOF, the prevalent
crest in its superior margin must be considered as it can interfere in the success of the procedure
(Aggarwal et al. 2015; Lee et al. 2006). region of the infraorbital canal (Aziz et al. 2000; Ilayperuma et al. 2010; Aggarwal et al. 2015). The
predominance of an inferomedial orientation (93.67%) and the nature of the bone walls of the IOF bring
practical implications that justify the superolateral orientation of the needle in vestibular anesthesia
procedures, but without its insertion in the IOF. Thus, an adequate approximation to the IOF in order to
acquire precise analgesia in the IN can be achieved safely, avoiding needle tapping into the IOF and
iatrogenic injury to the IN. Yet, in anesthetic methods with the superior approach in the IOF, the prevalent
crest in its superior margin must be considered as it can interfere in the success of the procedure
(Aggarwal et al. 2015; Lee et al. 2006). Incidence of single accessory IOFs and double accessory IOFs ranges from 2.2% to 18.2% and 0.5%
to 1.28% respectively. Despite not identifying the latter, the sample applied in the present research
corroborated with the former (8.04%). Although the anatomical norm establishes that all IN fibers emerge
from the IOF, it is important to understand that some of those emerge separately from the accessory
foramen, which could lead to inadequate analgesia and risk of injury. Health professionals must be aware
of this possibility and the implications involved (Hindy and Abdel-Raouf 1993; Aziz et al. 2000; Kazkayasi et
al. 2001; Bressan et al. 2004; Aggarwal et al. 2015). This work has several limitations. Although characteristics regarding sex and age were identified in
the sample, they were not sufficiently conclusive to allow subdivision of the sample, and this inhibited a
direct relationship between these variables and the data obtained. 4. Discussion Even though the use of dry skulls allowed
for a reasonable sample, they do not belong to clinical practices, which deal with additional anatomical
structures. The measurement proposed in the present research tries to overcome this limitation using
anatomical references that are accessible through physical examination in an attempt to make it more
applicable to clinical routines. Although, in recent years, complementary methods have been developed to assist in approaching the
IOF, it is not always feasible for being time-consuming and expensive, and also due to a shortage of
equipment and qualified personnel (Aggarwal et al. 2015; Tsui 2009). When these are either not available or
are contraindicated, it is necessary to know in-depth the characteristics and the anatomical relationships of
the IOF. According to the most appropriate procedure to approach the IOF in adults, regardless of sex and
race, the following features must be taken into account: the total or partial presence of the individual's
dentition; its distance to MAAM, to IMO, to IMS; the prevalent crest in the superior margin of the IOF; and its
inferomedial orientation. 4. Discussion The results allow for setting a protocol to situate the IOF using easily palpable bone landmarks in an
attempt to overcome the possibility of interference from soft structures and other variables, aiming to
transfer this knowledge to clinical practice in an accurate way. Therefore, a simple rule (made through a strip
of graph paper) can be used to measure the distance between the IMS (passing above the alveolar process)
and the vertical axis, where the infraorbital foramen is located (Fig. 1, Tab 3). Then, the approach must be
through the vestibule mouth for the IF anesthesia, where the needle can be inserted at a depth of
approximately 35mm accompanied by a supraperiosteal injection (or local infiltration). However, a shorter
distance between MAAM and IOF in ED group must be considered. Contrasting the results of the present
study and the observations from previous studies (Hindy and Abdel-Raouf 1993; Aziz et al. 2000; Apinhasmit
et al. 2006; Gupta 2008; Aggarwal et al. 2015; Ercikti et al. 2017), the use of the IMS distance (based on the
vertical plane of the IOF) brought precision and standardization to find the IOF. The highest point of this
methodology was the lack of statistical differences in the IMS distance in ED and DE groups, regardless of
sex, making this method applicable for both. The morphometry of the IOF in relation to the MAAM and the IMO corroborated with other results
found in the literature without statistical differences between antimeres. These results bring reproducibility
of such measurements to clinical practice (Aziz et al. 2000; Kazkayasi et al. 2001; Brandão et al. 2008;
Aggarwal et al. 2015; Ercikti et al. 2017). However, the IMO might not be such a reliable landmark to locate
the exact position of the IOF given its characteristic (long curved line) prone to variations in the location of
the IOF (Ercikti et al. 2017). Although previous studies have discussed that the IOF has sufficient diameter for needle insertion,
it is the measurement of its area and analysis of its shape that illustrate the real dimension of the opening 6 6 ALEXANDER, J.G. and BAPTISTA, J.S. region of the infraorbital canal (Aziz et al. 2000; Ilayperuma et al. 2010; Aggarwal et al. 2015). 5. Conclusions In the present study, the topography and morphometry of the IOF were determined accompanied by
differences between the DE and ED groups in this regard. Clinicians should be aware that the IOF is
presented oval-shaped, with inferomedial orientation, and with a crest-shaped upper margin. Therefore, a
small strip of graph paper can be applied to measure the distance between the IMS and the vertical axis
where the IOF is located, without difference between ED and DE subjects. However, it has to take into
account the shorter distance between MAAM and IOF in ED. Such data can lead to a more appropriate
procedure to approach the IOF in adults, avoiding iatrogenic injuries to IN. Authors' Contributions: ALEXANDER, J.G.: acquisition of data, analysis and interpretation of data, and drafting the article; BAPTISTA, J.S.:
conception and design, analysis and interpretation of data, drafting the article, and critical review of important intellectual content. All authors
have read and approved the final version of the manuscript. Conflicts of Interest: The authors declare no conflicts of interest. Ethics Approval: This work and all its procedures were authorized by the Ethics Committee for Research with Human Beings via Plataforma
Brasil (CAAE 32180520.0.0000.5060). No consent to participate was needed. Acknowledgments: The authors sincerely thank the donated bodies for science so that anatomical research could be performed. Results from
such research can potentially increase mankind's overall knowledge that can then improve patient care. Therefore, these donors and their
families deserve our highest gratitude. Also, we would like to express their most sincere gratitude to the technicians of the Department of
Morphology (Universidade Federal do Espirito Santo) for their support with the materials, and especially Dr. Willian Grassi Bautz (Ph.D.) from
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midface surgery. Clinical Anatomy. 2020, 33(8), 1159–1163. http://doi.org/10.1002/ca.23556 SINGH, R. Morphometric analysis of infraorbital foramen in Indian dry skulls. Anatomy & Cell Biology. 2011, 44(1), 79–83. http://doi.org/10.5115/acb.2011.44.1.79 ZDILLA, M.J., RUSSELL, M.L., and KOONS, A.W. Infraorbital foramen location in the pediatric population: A guide for infraorbital nerve block. Paediatric Anaesthesia. 2018, 28(8), 697–702. http://doi.org/10.1111/pan.13422 ZDILLA, M.J., KOONS, A.W., RUSSELL, M.L. The Infraorbital Foramen Is Located Midway Between the Nasospinale and Jugale: Considerations for
Infraorbital Nerve Block and Maxillofacial Surgery. The Journal of Craniofacial Surgery. 2018, 29(2), 523–527. http://doi.org/10.1097/SCS.0000000000004186 ZHANGE, K.R., BLANDFORD, A.D., HWANG, C.J., et al. Anatomic Variations of the Infraorbital Foramen in Caucasian Versus African American
Skulls. Ophthalmic Plastic and Reconstructive Surgery. 2019, 35(1), 25–28. http://doi.org/10.1097/IOP.0000000000001126 Received: 31 January 2023 | Accepted: 17 October 2023 | Published: 31 January 2024 Received: 31 January 2023 | Accepted: 17 October 2023 | Published: 31 January 2024 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. Bioscience Journal | 2024 | vol. 40, e40007 | https://doi.org/10.14393/BJ-v40n0a2024-68149 8
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English
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Folic acid prevents<i>C. elegans</i>folate deficiency indirectly via bacterial uptake of a breakdown product: a route that can also increase bacterial toxicity and ageing
|
bioRxiv (Cold Spring Harbor Laboratory)
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bioRxiv preprint Abstract Supplementation with the synthetic oxidised folate, folic acid is used to prevent neural tube defects and
other symptoms of folate deficiency. However, several unanswered questions remain over folic acid
efficacy, safety and interactions with gut microbes. Prevention of a development defect caused by folate
deficiency in the nematode worm Caenorhabditis elegans requires > 10 fold higher concentrations of folic
acid compared to folinic acid, a reduced folate. Here we show that the major route for folic acid to restore
normal development is indirect via the Escherichia coli used to feed C. elegans. This route occurs mainly via
the E. coli transporter AbgT, which takes up the folic acid breakdown product para-aminobenzoate-
glutamate (PABA-glu). We found that folic acid preparations, including a commercial supplement, contain
0.3- 4.0 % of this breakdown product. Previously, we have shown that inhibiting bacterial folate synthesis
increases C. elegans lifespan by removing a life-shortening bacterial activity. Here, we show that folic acid
restores bacterial folate synthesis and reverses this lifespan increase. It is still to be determined whether
this bacterial route increases host folate levels in humans and if there are situations where increased
bacterial folate synthesis has negative health complications. Folic acid prevents C. elegans folate deficiency indirectly via bacterial uptake of a breakdown product: a
route that can also increase bacterial toxicity and ageing Folic acid prevents C. elegans folate deficiency indirectly via bacterial uptake of a breakdown product: a
route that can also increase bacterial toxicity and ageing Claire Maynard1, Ian Cummins1, Jacalyn Green2 and David Weinkove1*
1Department of Biosciences, Durham University, South Road, Durham, UK. DH1 3LE
2Midwestern University, Illinois, Downers Grove, IL 60515, USA Claire Maynard1, Ian Cummins1, Jacalyn Green2 and David Weinkove1*
1Department of Biosciences, Durham University, South Road, Durham, UK. DH1 3LE
2Midwestern University, Illinois, Downers Grove, IL 60515, USA * Corresponding author: david.weinkove@durham.ac.uk, +44 191 3341303 Folic acid; Microbiota; Ageing; C. elegans; bacterial metabolism .
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this version posted December 14, 2017. INTRODUCTION The folate cycle is a set of essential biosynthetic reactions known as one carbon metabolism (Ducker and
Rabinowitz, 2017). Folates are a family of molecules with a central aromatic core derived from para-amino
benzoic acid (PABA), a pterin ring that can be modified and chain of one or more glutamates (Green and
Matthews, 2007). At each step of the folate cycle, an enzyme mediates a modification of the pterin ring of
the bound folate that allows the transfer of a chemical group containing one carbon atom (methyl, formyl
etc.) to or from the compound being synthesised (Ducker and Rabinowitz, 2017). Because of this cofactor
role for folate molecules, there are recycled and only required in very small amounts. Animals cannot
synthesise folates and must acquire them from their diet or gut microbes. A symptom of human folate
deficiency is neural tube defects during embryonic development (Ducker and Rabinowitz, 2017). It has been
found the rate of these defects can be lowered by preconception supplementation with folic acid, an
oxidised form of folate not found in nature. In many countries including the US and Canada, there is
mandatory fortification of flour with folic acid and this intervention has successfully decreased the
incidence of birth defects (Bailey et al., 2015). There are some concerns that folic acid supplementation
may have adverse effects (Kim, 2007; Marean et al., 2011; Pickell et al., 2011; Smith et al., 2008), and there
are many unknowns about the efficacy of uptake and biological utilisation of the supplement (Gregory et
al., 2005). However, recent reviews of the evidence by experts acting for government public health bodies
have concluded that the risks are minimal and have recommended either the supplementation of flour or
other food products as a beneficial intervention (Bailey et al., 2015; Boyles et al., 2016;
Public_Health_England, 2017). These reviews do not mention the potential role of gut microbes in
considering the mechanisms and safety of folic acid supplementation. Research from our group and others has shown that inhibiting folate synthesis in Escherichia coli, either by
treatment with the drug sulfamethoxazole (SMX) or mutation of the PABA synthesis pathway (e. g. a pabA
or pabB mutant), extends the lifespan of the nematode Caenorhabditis elegans that feeds on it (Han et al.,
2017; Virk et al., 2012; Virk et al., 2016). While these interventions decrease the folate levels in both E. coli
and C. KEY WORDS Folic acid; Microbiota; Ageing; C. elegans; bacterial metabolism 1 1 . CC-BY 4.0 International license
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bioRxiv preprint INTRODUCTION elegans, there remains sufficient folate available to support normal growth of both (Virk et al.,
2012). Numerous lines of evidence suggest that lifespan is increased because inhibiting folate synthesis
prevents a life-shortening activity of the bacteria rather than because animal folate levels are decreased
(Virk et al., 2016). A mutant in the C. elegans homologue of the human GCPII enzyme (which cleaves
glutamates from polyglutamated folates (Halsted et al., 1998)) shows delayed development and infertility
on low folate E. coli (Virk et al., 2016). This defect is due to folate deficiency because it can be prevented by
adding 1-10 µM folinic acid, a reduced folate. Prevention with folic acid requires much higher
concentrations (100 µM) (Virk et al., 2016). This result may reflect the fact that the only characterised C. elegans folate transporter is FOLT-1, a reduced folate carrier that shows specificity for folinic acid 2 2 . CC-BY 4.0 International license
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bioRxiv preprint (Balamurugan et al., 2007). However, we also discovered that at high concentrations folic acid can partially
reverse the lifespan increase caused by inhibiting E. coli folate synthesis. This finding suggests that E. coli
can take up folic acid (Virk et al., 2012). E. coli does not possess a folate transporter (Nickerson and Webb,
1956; Webb, 1955) but can take up the folic acid breakdown product PABA-glu through the transporter
AbgT and catabolise it to PABA (Carter et al., 2007). PABA can also diffuse across E. coli membranes. PABA
from either source can be used to synthesise folate. (Balamurugan et al., 2007). However, we also discovered that at high concentrations folic acid can partially
reverse the lifespan increase caused by inhibiting E. coli folate synthesis. This finding suggests that E. coli
can take up folic acid (Virk et al., 2012). E. The major route of folic acid prevention of a C. elegans developmental defect requires the E. coli AbgT
transporter In order to test whether folic acid requires E. coli to prevent developmental defects in our C. elegans folate
deficiency model, we tested whether the E. coli abgT genotype influenced the outcome of
supplementation. We first established that the C. elegans gcp-2.1(ok1004) mutant grown on the E. coli
pabA mutant on defined media (DM) has the same growth defect as these worms grown on wild type E. coli
treated with 128 µg/ml SMX (Figure 1a, Virk et al. 2016). Addition of 2.5 µM folinic acid or higher prevented
this growth defect regardless of the abgT genotype (Figure 1b). In contrast, folic acid was found to increase
gcp-2.1 mutant body length on the pabA mutant in a dose-dependent manner, achieving full growth only at
200 µM folic acid (Figure 1c). On the E. coli abgTpabA double mutant, a much weaker response to folic acid
was observed, whereas overexpression of abgT in the E. coli pabA mutant resulted in normal C. elegans
growth at lower concentrations of folic acid (Figure 1c). Analysing the experiment by two-way ANOVA, we
find that there is a significant interaction effect of abgT genotype (F=102.67, p<0.0001) and folic acid
concentration (F=123.55, p<0.0001) on C. elegans gcp-2.1 body length. Mutation of abgT alone did not
influence growth of the gcp-2.1 mutant (Supplementary Figure 1a). These results are consistent with folinic
acid being taken up directly by the worm (Balamurugan et al., 2007), and that the major route of folic acid
uptake requires E. coli and the E. coli AbgT transporter. INTRODUCTION coli does not possess a folate transporter (Nickerson and Webb,
1956; Webb, 1955) but can take up the folic acid breakdown product PABA-glu through the transporter
AbgT and catabolise it to PABA (Carter et al., 2007). PABA can also diffuse across E. coli membranes. PABA
from either source can be used to synthesise folate. In this study, we show that the prevention of delays in C. elegans development by folic acid depends on the
E. coli AbgT transporter, demonstrating that bacterial uptake is the major route of folic acid
supplementation. Consistent with this finding, we show that folic acid increases E. coli folate levels through
the AbgT transporter and we find PABA-glu present in folic acid preparations. We also show that uptake of
PABA-glu by AbgT can reverse the lifespan increase caused by inhibiting E. coli folate synthesis. Thus, this
pathway can have both positive effects on development and negative consequences for ageing. 3 3 . CC-BY 4.0 International license
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bioRxiv preprint RESULTS Folic acid supports growth of E. coli pabA mutants via abgT-dependent uptake of PABA-glu The dependence on E. coli abgT for folic acid to rescue the gcp-2.1 developmental delay strongly suggests
that PABA-glu is available to E. coli following folic acid supplementation. To test the relative ability of E. coli
to take up PABA-glu and folic acid, we added these compounds to pabA, abgT pabA and pabA (abgT OE) E. coli and assessed growth on DM agar plates in PABA-free conditions under which the pabA mutant showed
slower growth than wild type. We found that growth in the presence of folic acid and PABA-glu depended
on abgT expression; 10 µM folic acid rescued growth of the pabA mutant, whereas 100 µM was needed to
achieve an equivalent rescue in the abgT pabA double mutant (Figure 2a). In the presence of 10 µM folic
acid, growth of the pabA strain over-expressing AbgT was greater than of the pabA mutant alone or the
wild type. Supplementation by PABA-glu had a similar effect to folic acid but at a 10-fold lower
concentration (Figure 2a). PABA, which can diffuse across biological membranes (Tran and Nichols, 1991),
rescued bacterial growth at nanomolar concentrations independently of abgT expression (Figure 2a). Overall, rescue of bacterial growth by folic acid can be largely explained by PABA-glu uptake by AbgT while
low concentrations of PABA present in folic acid preparations may explain the ability of 100 µM folic acid to
rescue growth of the E. coli abgT pabA double mutant. Overall, rescue of bacterial growth by folic acid can be largely explained by PABA-glu uptake by AbgT while
low concentrations of PABA present in folic acid preparations may explain the ability of 100 µM folic acid to
rescue growth of the E. coli abgT pabA double mutant. 4 4 . CC-BY 4.0 International license
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this version posted December 14, 2017. Folic acid supports growth of E. coli pabA mutants via abgT-dependent uptake of PABA-glu ;
https://doi.org/10.1101/230227
doi:
bioRxiv preprint 0.0
0.2
0.4
0.6
0.8
1.0
1.2
Control
pabA
SMX
C elegans body length (mm)
WT
gcp-2.1
pabA
a) a) Figure 1. Folic acid prevents developmental delay of a C. elegans folate deficiency model via an E. coli abgT-
dependent route. a) body length of wild-type and gcp-2.1 mutant C. elegans at L4 stage raised on DM agar plates
seeded with WT E. coli (control), pabA mutant or WT E. coli treated with 128 μg/ml SMX. b) body length of gcp-2.1
mutant C. elegans at L4 stage raised on plates seeded with pabA mutant, abgTpabA double mutant or pabA mutan
over-expressing abgT with increasing concentrations of folinic acid and c) folic acid: by two-way ANOVA analyses, w
find that there is a significant interaction effect of strain type (F=102.67, p<0.0001) and folic acid concentration
(F=123.55, p<0.0001) on C. elegans gcp-2.1 body length. Over-expression is conferred by transformation with a hig
copy number plasmid, pJ128. pabA and abgTpabA strain are transformed with the empty vector, pUC19. Error bars
represent standard deviation of C. elegans body length; n ≥40. 0.0
0.2
0.4
0.6
0.8
1.0
Control
pabA
SMX
C elegans body length (mm)
WT
gcp-2.1
pabA
0.0
0.2
0.4
0.6
0.8
1.0
2
4
8
16
32
64
128
gcp-2.1 body length (mm)
[Folic acid] (µM)
pabA
abgTpabA
pabA (abgT OE)
c)
0.0
0.2
0.4
0.6
0.8
1.0
2
4
8
16
32
64
128
gcp-2.1 body length (mm)
[Folinic acid] (µM)
b) Figure 1. Folic acid prevents developmental delay of a C. elegans folate deficiency model via an E. coli abgT-
dependent route. a) body length of wild-type and gcp-2.1 mutant C. Folic acid supports growth of E. coli pabA mutants via abgT-dependent uptake of PABA-glu CC-BY 4.0 International license
available under a
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 Folic acid supports growth of E. coli pabA mutants via abgT-dependent uptake of PABA-glu elegans at L4 stage raised on DM agar plates
seeded with WT E coli (control) pabA mutant or WT E coli treated with 128 μg/ml SMX b) body length of gcp 2
0.0
0.2
0.4
0.6
0.8
1.0
Control
pabA
SMX
C elegans body length (mm
WT
gcp-2.1
pabA
0.0
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1.0
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32
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128
gcp-2.1 body length (mm)
[Folic acid] (µM)
pabA
abgTpabA
pabA (abgT OE)
c)
0.0
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1.0
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gcp-2.1 body length (mm)
[Folinic acid] (µM)
b) 0.0
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gcp-2.1 body length (mm)
b) b) 0.0
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gcp-2.1 body length (mm)
[Folic acid] (µM)
pabA
abgTpabA
pabA (abgT OE)
c)
2
4
8
16
32
64
128
[Folinic acid] (µM) c) Figure 1. Folic acid prevents developmental delay of a C. elegans folate deficiency model via an E. coli abg Figure 1. Folic acid prevents developmental delay of a C. elegans folate deficiency model via an E. coli abgT-
dependent route. a) body length of wild-type and gcp-2.1 mutant C. elegans at L4 stage raised on DM agar plates
seeded with WT E. coli (control), pabA mutant or WT E. coli treated with 128 μg/ml SMX. b) body length of gcp-2.1
mutant C. elegans at L4 stage raised on plates seeded with pabA mutant, abgTpabA double mutant or pabA mutant
over-expressing abgT with increasing concentrations of folinic acid and c) folic acid: by two-way ANOVA analyses, we
find that there is a significant interaction effect of strain type (F=102.67, p<0.0001) and folic acid concentration
(F=123.55, p<0.0001) on C. elegans gcp-2.1 body length. Over-expression is conferred by transformation with a high
copy number plasmid, pJ128. pabA and abgTpabA strain are transformed with the empty vector, pUC19. Error bars
represent standard deviation of C. elegans body length; n ≥40. 5 . CC-BY 4.0 International license
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bioRxiv preprint . Folic acid increases E. coli folate levels in an AbgT-dependent mechanism It order to verify that E. coli growth following folic acid supplementation is attributable to restored bacterial
folate synthesis, we used LC-MS/MS to detect E. coli folate levels under the conditions used in the above
experiment. Levels of the most detectable and thus likely the most abundant THF species, 5-methyl THF-
glu3, are presented in Figure 2b. In the absence of folic acid, folate levels in the pabA mutant strains were
significantly lower than in WT extracts (Figure 2b, Supplementary Figure 2). Addition of folic acid increased
folate levels, where the scale of increase was dependent on abgT expression; with 100 µM folic acid, folate
species were highest in pabA (abgT OE) followed by WT, pabA, and finally lowest in the abgT pabA double
mutant. Other THF species followed the same trend as 5-methyl THF-glu3 and are presented in
Supplementary Figure 2. In summary, folic acid supplementation increases E. coli folate levels in an abgT-
dependent mechanism. 6 6 . CC-BY 4.0 International license
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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 a) Figure 2. Folic acid breakdown supports E. coli growth and increases folate levels via uptake of PABA-glu by AbgT. a)
growth of E. coli WT, pabA, abgTpabA and pabA over-expressing abgT (abgT OE) on agar plates supplemented with
folic acid PABA-glu and PABA as measured by OD600 after 4 days growth at 25℃ (see methods). Each data point is the
average of 8 plates. Error bars represent standard deviation. Asterisks denote the test statistic from Student’s t test
comparison of means, where * = P<0.05 compared to WT growth on the same condition b) 5-methyl THF-glu3 levels in
extracts of E. coli WT, pabA, abgTpabA, pabA (abgT OE) mutants supplemented with 10 µM and 100 µM folic acid. Folate counts from the LC-MS/MS are normalised by dividing by counts of an internal MTX-glu6 spike. .
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bioRxiv preprint Folic acid increases E. coli folate levels in an AbgT-dependent mechanism Extracts were
made after 4 days of bacterial growth at 25°C on solid agar plates. See Supplementary Figure 3 for full folate analysis. Control
100 µM
0.0
0.1
0.1
0.2
0.2
0.3
5-methyl-THF-glu3: MTX-glu6
100 µM
10 µM
0.1
1
[PABA] (µM)
WT
pabA
abgTpabA
pabA (abgT OE)
1
10
[PABA-glu] (µM)
0.1
0.15
0.2
0.25
0.3
0
10
100
Bacterial growth (OD 600)
[Folic acid] (µM)
*
*
*
*
*
*
a)
pabA (abgT OE)
b) 0.1
1
[PABA] (µM)
WT
pabA
abgTpabA
pabA (abgT OE)
1
10
[PABA-glu] (µM)
0.1
0.15
0.2
0.25
0.3
0
10
100
Bacterial growth (OD 600)
[Folic acid] (µM)
*
*
*
*
*
* [PABA] (µM) b) µ
10 µM Figure 2. Folic acid breakdown supports E. coli growth and increases folate levels via uptake of PABA-glu by AbgT. a)
growth of E. coli WT, pabA, abgTpabA and pabA over-expressing abgT (abgT OE) on agar plates supplemented with
folic acid PABA-glu and PABA as measured by OD600 after 4 days growth at 25℃ (see methods). Each data point is the
average of 8 plates. Error bars represent standard deviation. Asterisks denote the test statistic from Student’s t test
comparison of means, where * = P<0.05 compared to WT growth on the same condition b) 5-methyl THF-glu3 levels in
extracts of E. coli WT, pabA, abgTpabA, pabA (abgT OE) mutants supplemented with 10 µM and 100 µM folic acid. Folate counts from the LC-MS/MS are normalised by dividing by counts of an internal MTX-glu6 spike. Extracts were
made after 4 days of bacterial growth at 25°C on solid agar plates. See Supplementary Figure 3 for full folate analysis. 7 7 . CC-BY 4.0 International license
available under a
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bioRxiv preprint . Folic acid preparations contain PABA-glu and PABA It is made
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bioRxiv preprint Folic acid preparations contain PABA-glu and PABA Together, the data presented here indicate that the main route of C. elegans folic acid supplementation is
indirect via E. coli uptake of PABA-glu and PABA. We used LC-MS/MS to test for the presence of these
breakdown products in folic acid preparations from Schircks (used in all other experiments in this study),
Sigma Aldrich and Boots, a UK retailer. We also tested for further folic acid breakdown under the
experimental conditions used here by analysing extracts from agar media supplemented with Schircks folic
acid and incubated at 25 °C for 4 days. We detected PABA-glu in all three folic acid sources at between 0.3%
(Schircks) and 4% (Boots, Figure 3). Under the conditions used for C. elegans experiments PABA-glu
increased to 1.18%, suggesting further breakdown. PABA was found at between 0.01% (Schircks) and 0.06%
(Boots, Figure 3). Figure 3. Folate preparations contain PABA-glu and PABA. Concentrations of PABA-glu and PABA as determined by
LC-MS/MS in 10 and 100 µM folic acid preparations. Samples were folic acid from Schircks, Sigma, Boots and Schircks
folic acid after addition to the agar media and incubation for 4 days at 25 ˚C. Error bars represent standard deviation
over triplicate independent preparations. 0.0
1.0
2.0
3.0
4.0
5.0
10
100
[PABA-glu] (µM)
[Folic acid] (µM)
Schircks
Sigma
Boots
Schircks in media
0.00
0.02
0.04
0.06
0.08
10
100
[PABA] (µM)
[Folic acid] (µM) 0.00
0.02
0.04
0.06
0.08
10
100
[PABA] (µM)
[Folic acid] (µM) 0.0
1.0
2.0
3.0
4.0
5.0
10
100
[PABA-glu] (µM)
[Folic acid] (µM)
Schircks
Sigma
Boots
Schircks in media Figure 3. Folate preparations contain PABA-glu and PABA. Concentrations of PABA-glu and PABA as determined by
LC-MS/MS in 10 and 100 µM folic acid preparations. Samples were folic acid from Schircks, Sigma, Boots and Schircks
folic acid after addition to the agar media and incubation for 4 days at 25 ˚C. Error bars represent standard deviation
over triplicate independent preparations. 8 8 . CC-BY 4.0 International license
available under a
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 . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Folic acid shortens C. elegans lifespan via AbgT-dependent uptake of PABA-glu during adulthood Inhibiting bacterial folate synthesis, without affecting bacterial growth, is known to increase C. elegans
lifespan (Virk et al., 2012; Virk et al., 2016). It was therefore hypothesized that factors that increase
bacterial folate synthesis, such as folic acid (as shown here), may shorten C. elegans lifespan. Consistent
with our previous findings (Virk et al., 2016), we find that C. elegans maintained on any E. coli pabA mutant
are long-lived compared to C. elegans fed wild type E. coli (Figure 4, Supplementary Table 1), whereas the
abgT mutation alone had no impact on C. elegans lifespan (P=0.4312, Supplementary Figure 1b). 10 µM
folic acid was found to decrease C. elegans lifespan on pabA by 9.4% (P=0.0052), and decrease lifespan on
the pabA mutant over-expressing abgT further (by 16.3%, P<0.0001, Figure 4a). In contrast, 10 µM folic acid
had no effect on lifespan on the abgTpabA double mutant (P=0.1901, Figure 4a). 100 µM folic acid
decreased lifespan on pabA E. coli by 23.9% (P<0.0001), whereas this concentration only shortened lifespan
on the abgT pabA double mutant by 4.7% (P=0.0467, Figure 3a). Lifespans on media supplemented with
PABA-glu showed an abgT-dependent response similar to that observed with folic acid supplementation,
but at a 10-fold lower concentration (Figure 4b). In contrast, PABA supplementation shortened C. elegans
lifespan in all cases independently of abgT expression, consistent with its ability to diffuse across biological
membranes (Figure 4c). Folic acid had no effect on the lifespan of C. elegans maintained on WT E. coli
(Supplementary Figure 3). Together, these results suggest that folic acid shortens C. elegans lifespan on
folate-deficient E. coli via AbgT-dependent uptake of PABA-glu during adulthood. 9 9 . CC-BY 4.0 International license
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this version posted December 14, 2017. DISCUSSION In this study we have found that a major route of folic acid uptake by C. elegans is via E. coli. This route is
dependent on the spontaneous breakdown of folic acid into PABA-glu and salvage of this product by the E. coli PABA-glu transporter, AbgT. We found that this route led to increases in the levels of several folate
species in E. coli following folic acid supplementation. This route can also lead to folic acid shortening C. elegans lifespan when worms are cultured on bacteria with impaired folate synthesis. The increase in folate
synthesis caused by folic acid supplementation leads to a bacterial activity/toxicity that is harmful to the
worm over the long term (Figure 5). Is it possible that this bacterial route for folic acid exists in humans? As far as we know, no other studies
have tested folic acid supplements for the presence of PABA-glu or PABA, but several studies have reported
issues with the stability and dissolution of commercial folic acid supplements (Hoag et al., 1997; Sculthorpe
et al., 2001). In light of these issues, manufacturers have adopted a policy of ‘overages’ in order to ensure
sufficient folic acid is released in the small intestine following ingestion (Andrews et al., 2017). The
presence of PABA-glu and PABA in a commercially available folic acid source (Figure 3) combined the
instability of folic acid at the low pH conditions of the stomach, makes it likely that PABA-glu and PABA will
be available to the gut microbiota following supplementation. PABA has been identified as a human fecal
excretory product after ingestion of folic acid (Denko et al.). The abgT gene is widespread in the human gut
microbiota and studies in rodents (Rong et al., 1991) and piglets (Asrar and O'Connor, 2005) have
demonstrated that infusion of labelled PABA into the cecum results in the incorporation of bacterially
synthesized folate in host tissues. Thus the literature suggests that the components for such route exists in
humans, but whether this accounts for significant amounts of folic acid is yet to be seen. We could only
detect this route in C. elegans because of the poor bioavailability of folic acid in our folate deficiency model. Folic acid shortens C. elegans lifespan via AbgT-dependent uptake of PABA-glu during adulthood CC-BY 4.0 International license
available under a
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 Folic acid shortens C. elegans lifespan via AbgT-dependent uptake of PABA-glu during adulthood ;
https://doi.org/10.1101/230227
doi:
bioRxiv preprint 14
16
18
20
22
0
10
100
Mean lifespan (days)
[Folic acid] (µM)
***
***
**
*
***
14
16
18
20
22
0
1
10
Mean lifespan (days)
[PABA-Glu] (µM)
pabA
abgTpabA
pabA (AbgT OE)
***
***
***
**
pabA (abgT OE)
a)
b) a) Figure 4. Folic acid shortens C. elegans lifespan via an E. coli abgT-dependent route during adulthood. Mean
lifespan of WT C. elegans maintained from day 1 of adulthood on pabA, abgTpabA, or pabA (abgT OE) E. coli with
supplementation of (a) folic acid (b) PABA-glu and (c) PABA. Error bars represent standard error. Asterisks denote
the Log-rank non-parametric statistical test of survival, where: *P<0.05; **P<0.01; ***P<0.005 compared to
lifespan on the non-supplemented condition of the same strain. Full lifespan data in Supplementary Table 1. 14
16
18
20
22
0
0.1
1
Mean lifespan (days)
[PABA] (µM)
14
16
18
20
22
0
10
100
Mean lifespan (days)
[Folic acid] (µM)
***
***
**
*
***
14
16
18
20
22
0
1
10
Mean lifespan (days)
[PABA-Glu] (µM)
pabA
abgTpabA
pabA (AbgT OE)
***
***
***
**
pabA (abgT OE)
a)
b)
c) Figure 4. Folic acid shortens C. elegans lifespan via an E. coli abgT-dependent route during adulthood. Mean
lifespan of WT C. elegans maintained from day 1 of adulthood on pabA, abgTpabA, or pabA (abgT OE) E. coli with
supplementation of (a) folic acid (b) PABA-glu and (c) PABA. Error bars represent standard error. Asterisks denote
the Log-rank non-parametric statistical test of survival, where: *P<0.05; **P<0.01; ***P<0.005 compared to
lifespan on the non-supplemented condition of the same strain. Full lifespan data in Supplementary Table 1. 10 . CC-BY 4.0 International license
available under a
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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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 Folic acid is taken up well by humans and leads to increases in serum folate levels, but folic acid is often
found in the oxidised form, suggesting that it is not always bioavailable (Bailey et al., 2015; Gregory et al.,
2005; Patanwala et al., 2014). Further studies are required in order to determine whether folic acid supplementation affects the folate
status of human gut microbes and whether this in turn impacts host health. Interestingly, there are several
diseases associated with an increased abundance of folate-synthesizing gut bacteria, such as inflammatory
bowel disease (Shin et al., 2015) and small intestinal bacterial overgrowth (Camilo et al., 1996), but a causal
relationship between bacterial folate and disease has not been established. The abgT gene is found in the
genomes of several enteric pathogens, including Enterobacter cloacae, N. gonorrhoeae, Salmonella
enterica, Shigella boydii and Staphylococcus aureus in addition to E. coli. Whilst there is much 11 11 consideration about the consequences of folic acid supplementation (Bailey et al., 2015; Boyles et al., 2016;
Public_Health_England, 2017), our work here indicates that folic acid supplement instability and bacterial
genotype are previously unexplored variables that may impact human health and thus warrant
consideration in future reports and studies. Figure 5. Schematic of the impact of folic acid supplementation on C. elegans via indirect uptake of breakdown
products by E. coli. Folic acid is not taken up well by C. elegans directly. We find that the major uptake of folic acid by
C. elegans is dependent on its breakdown into PABA-glu and uptake by the E. coli AbgT transporter. This route
increases bacterial folate synthesis in both wild-type and pabA mutant E. coli. Under conditions of folate-deficiency
(pabA mutant E. coli), increasing bacterial folate via this route is beneficial for C. elegans development. During C. elegans adulthood, this route has a negative impact on longevity as it promotes a bacterial folate-dependent toxicity. 2
O
H
PABA
Chorismate
+ glutamine
AbgT
PabA
FOLATE
CYCLE
PABA-glu
PABA
AbgA
AbgB
THF
2
PABA- glu
E. COLI
C. ELEGANS
FOLATE
SYNTHESIS
DEVELOPMENT
ü Increased
fitness
FOLIC
ACID
PABA
pteridine
glutamic acid
TOXICITY
ADULTHOOD
× Decreased
longevity ADULTHOOD
× Decreased
longevity DEVELOPMENT
ü Increased
fitness Chorismate
+ glutamine pteridine Figure 5. Schematic of the impact of folic acid supplementation on C. elegans via indirect uptake of breakdown
products by E. coli. Folic acid is not taken up well by C. elegans directly. DISCUSSION We find that the major uptake of folic acid by
C. elegans is dependent on its breakdown into PABA-glu and uptake by the E. coli AbgT transporter. This route
increases bacterial folate synthesis in both wild-type and pabA mutant E. coli. Under conditions of folate-deficiency
(pabA mutant E. coli), increasing bacterial folate via this route is beneficial for C. elegans development. During C. elegans adulthood, this route has a negative impact on longevity as it promotes a bacterial folate-dependent toxicity. 12 Folates and related compounds Folic acid, Folinic acid, PABA-Glu, 5-formylTHF-Glu3 , 5-methylTHF-Glu3, methotrexate-Glu6 were obtained
from Schircks, Switzerland. PABA, Vitamin B12 and folic acid were from Sigma Aldrich and folic acid
supplement from Boots, UK. ACKNOWLEDGEMENTS We thank the C. elegans Genetics Center, the C. elegans Knockout Consortium, and NBRP-E.coli at NIG for
strains and we thank Sushmita Maitra and John Mathers for useful comments on the manuscript. This work
was supported by a BBSRC DTP studentship. 13 Culture conditions Defined media (DM) was prepared as described (Virk et al., 2016), except that 10 nM B12 was added. B12,
folic acid and antibiotics are added post-autoclaving for agar plates. DM for liquid culture is filter-sterilised. 0.1 µM PABA added to the liquid DM media used to seed the plates in order to maintain bacterial growth
(apart the growth experiments in Figure 2). 30 µl of 3 ml fresh overnight LB culture is used to inoculate 5ml
DM (15 ml Falcon tube). 25 µg/ml kanamycin (50 ug/ml ampicillin if necessary) added to both LB and DM
pre-incubation. DM liquid cultures are incubated for 18 hr at 37°C, 220 RPM. 14 All strains derived from the Keio collection (Baba et al., 2006). See table. WT-Kan (Virk et al., 2016). abgTpabA double mutant was made using P1 transduction protocol as described in (Moore, 2011). The
abgT over-expression plasmid (pJ128) (Carter et al., 2007) and empty vector (puc19) (Yanisch-Perron et al.,
1985) were transformed into appropriate strains. C. elegans strains used SS104 glp-4(bn2), UF208 (wild-type), and UF209 gcp-2.1(ok1004) (Virk et al., 2016). E. coli preparation and growth assay
Table of E. coli strains used in this study
Strain
Genotype
Plasmid
Characteristics
Source
BW25113/
pGreen 0029
WT
pGreen 0029
kanr
Virk et al 2016
JW3323-1
ΔpabA
n/a
kanr
Baba et al 2006
JW5822-1
ΔabgT
n/a
kanr
Baba et al 2006
CMabgTpabA
ΔabgT ΔpabA
n/a
kanr
This study
CM1
WT
pUC19‡, pGreen 0029
kanr, ampr
This study
CM2
ΔpabA
pUC19
kanr, ampr
This study
CM3
ΔabgT ΔpabA
pUC19
kanr, ampr
This study
CM4
WT (abgT OE)
pJ128‡,
pGreen 0029
kanr, ampr
This study
CM5
ΔpabA (abgT OE)
pJ128
kanr, ampr
This study
CM6
ΔabgT ΔpabA (abgT
OE)
pJ128
kanr, ampr
This study Table of E. coli strains used in this study 15 15 E. coli was prepared as follows for all E. coli and C. elegans experiments. 30 µl of an overnight LB culture of
E. coli was transferred into 5ml DM and incubated for 18 hr at 37 ◦C, 220 RPM. 100 µl the DM culture was
seeded onto DM agar plates and incubated at 25°C for 4 days. E. coli was removed by pipetting 1 ml M9
onto the plate and a glass spreader to scrape off the bacterial lawn. The bacterial suspension was pipetted
into a 1.5 ml Eppendorf and the volume was recorded (v). Tubes were vortexed vigorously to obtain a
homogenised solution. 100 µl was taken and diluted with 900 µl M9 in a cuvette. A spectrophotometer was
used to read bacterial growth at 600 nm. Bacterial growth was calculated by multiplying OD600 by the
volume of the sample (v). E. coli folate extraction Bacterial lawns were scraped from plates into micro centrifuge tubes using M9 solution and kept on ice. Volume (v), multiplied by the OD600 of the solution (diluted 1:5) gives a measure of the amount of material. Samples were concentrated in chilled microcentrifuge and pellets were snap frozen in liquid nitrogen. Pellets were thawed and resuspended in a volume of ice-cold 90% methanol: 10% folate extraction buffer
(FEB: 50 mM HEPES, 50 mM CHES, 0.5% w/v ascorbic acid, 0.2 M DTT, pH 7.85 with NaOH) in proportion to
bacterial content (37.5 × OD600 × v). FEB is spiked with 10 nM methotrexate-Glu6 as an internal standard. Samples were vortexed vigorously and left on ice for 15 min before centrifugation in a cooled
microcentrifuge for 15 min at full speed. Supernatants were used for analysis. Folate LC-MS/MS analysis Folates were detected by multiple reaction monitoring (MRM) analysis using a SCIEX QTRAP 6500
instrument. MRM conditions for folic acid, PABA, PABA-Glu, 5-Me-H4PteGlu3 (5-methylTHF-Glu3) and 5/10-
CHO-H4PteGlu3 (formyl THF3) were optimised by infusion of standards into the instrument. The optimised
conditions for –Glu3 folates were applied to other higher folates using MRM transitions described by Lu et
al., 2007 (Lu et al., 2007). Further confirmation of identity for folates of interest was achieved by
performing enhanced product ion scans and comparing the fragment spectra with known standards. The QTRAP 6500 was operated in ESI+ mode and was interfaced with a Shimadzu Nexera UHPLC system. Samples were separated using a Thermo PA2 C18 column (2.2 µm, 2.1 x 100 mm) with a gradient of 0.1%
formic acid in water (mobile phase A) and acetonitrile (mobile phase B). Samples were maintained at 4⁰C
and 2 µL aliquots were injected. The column was maintained at 40⁰C with a flow rate of 200 µL/min,
starting at 2% B, held for 2 minutes, with a linear gradient to 100% B at 7 minutes, held for 1 minute, before
a 7-minute re-equilibration step at 2% B that was necessary for consistent retention times. The column 16 eluate flow to the MS was controlled via the QTRAP switching valve, allowing analysis between 4 and 8
minutes to minimise instrument contamination. Folates were quantified with reference to external
standards and matrix effects were assessed by spiking of standards into extracted samples. eluate flow to the MS was controlled via the QTRAP switching valve, allowing analysis between 4 and 8
minutes to minimise instrument contamination. Folates were quantified with reference to external
standards and matrix effects were assessed by spiking of standards into extracted samples. Lifespan analysis Survival analyses were performed as described (Virk et al., 2012). glp-4(bn2) worms were maintained at
15°C and shifted to 25°C at the L3 stage. At the L4/young adult stage, animals were placed on bacteria
under the experimental conditions. All lifespan data is in Table S1. Statistical significance was determined
using Log Rank and Wilcoxon tests of the Kaplan-Meier survival model. 17 References Andrews, K.W., Roseland, J.M., Gusev, P.A., Palachuvattil, J., Dang, P.T., Savarala, S., Han, F., Pehrsson, P.R.,
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http://www ncbi nlm nih gov/pubmed/2985470 20 SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION
Supplementary Figure 1. The E. coli abgT deletion has no effect on C. elegans development and lifespan a) body
length of wild-type and gcp-2.1 mutant C. elegans at L4 stage raised on abgT mutant or wild-type E. coli (error bars
represent standard deviation) b) survival curves of wild-type (glp-4) C. elegans on E. coli wild-type and abgT
mutant. See Supplementary Table 1 for further details. 0
0.2
0.4
0.6
0.8
1
WT
gcp-2.1
C. elegans body length (mm)
C. References elegans genotype
WT
abgT
0.0
0.2
0.4
0.6
0.8
1.0
0
5
10
15
20
25
30
Survival
Day of adulthood
WT
abgT
1a)
b) 0
0.2
0.4
0.6
0.8
1
WT
gcp-2.1
C. elegans body length (mm)
C. elegans genotype
WT
abgT
1a) g
g
yp
0.0
0.2
0.4
0.6
0.8
1.0
0
5
10
15
20
25
30
Survival
Day of adulthood
WT
abgT
b) b) Supplementary Figure 1. The E. coli abgT deletion has no effect on C. elegans development and lifespan a) body
length of wild-type and gcp-2.1 mutant C. elegans at L4 stage raised on abgT mutant or wild-type E. coli (error bars
represent standard deviation) b) survival curves of wild-type (glp-4) C. elegans on E. coli wild-type and abgT
mutant. See Supplementary Table 1 for further details. 21 21 Supplementary Figure 2. LC-MS/MS detects lower levels of THFs in pabA mutants, where abgT determines response
to folic acid. Levels of THFs detected in extracts of WT, pabA, abgTpabA, pabA (abgT OE) mutants displayed as a ratio
with an internal MTX-glu6 spike for normalization. Extracts were made after 4 days of bacterial growth at 25°C. Asterisks denote the test statistic from Student’s t test comparison of means, where * = P<0.05. Error bars represent
standard deviation over four replicates. 0.00
0.10
0.20
0.30
0
10
100
Folate: MTX-glu6
5-methyl-THF
*
*
*
0.00
0.15
0.30
0.45
0
10
100
5/10-formyl-THF
*
*
*
0.00
0.35
0.70
0
10
100
Folate: MTX-glu6
[Folic acid] (µM)
5,10-methenyl-THF
*
*
*
0.00
0.10
0.20
0.30
0
10
100
[Folic acid] (µM)
THF
*
*
*
0.00
0.03
0.05
0
10
100
Folate: MTX-glu6
[Folic acid] (µM)
5,10-methylene-THF
WT
pabA
abgTpabA
pabA (abgT OE)
*
* 0.00
0.10
0.20
0.30
0
10
100
Folate: MTX-glu6
5-methyl-THF
*
*
*
0.00
0.15
0.30
0.45
0
10
100
5/10-formyl-THF
*
*
* 0.00
0.15
0.30
0.45
0
10
100
5/10-formyl-THF
*
*
* 0.00
0.10
0.20
0.30
0
10
100
[Folic acid] (µM)
THF
*
*
* 0.00
0.35
0.70
0
10
100
Folate: MTX-glu6
[Folic acid] (µM)
5,10-methenyl-THF
*
*
*
0.00
0.10
0.20
0.30
0
10
100
[Folic acid] (µM)
THF
*
*
* Supplementary Figure 2. LC-MS/MS detects lower levels of THFs in pabA mutants, where abgT determines response
to folic acid. References WT
102
9
15.96
0.48
Control
n/a
n/a Alkaline control
CM1
p
shown)
WT + 10 µM folic acid
98
12
15.96
0.58
0.03%
0.8268
0.4283 10 µM Schircks folic acid
CM1
p
WT + 100 µM folic acid
102
18
16.01
0.48
0.36%
0.866
0.7792 100 µM Schircks folic acid
CM1
p
pabA
113
10
18.35
0.56
Control
n/a
n/a Alkaline control
CM2
p
pabA + 10 µM folic acid
90
11
17.44
0.64
-4.95%
0.2527
0.2605 10 µM Schircks folic acid
CM2
p
pabA + 100 µM folic acid
122
3
15.86
0.40
-13.57%
<.0001*
0.0019* 100 µM Schircks folic acid
CM2
p
abgTpabA
136
14
18.93
0.50
Control
n/a
n/a Alkaline control
CM3
p
abgTpabA + 10 µM folic acid
115
26
19.22
0.52
1.51%
0.6416
0.5684 10 µM Schircks folic acid
CM3
p
abgTpabA + 100 µM folic acid
93
11
19.26
0.60
1.71%
0.5868
0.4482 100 µM Schircks folic acid
CM3
p
pabA (abgT OE)
101
23
18.42
0.56
Control
n/a
n/a Alkaline control
CM5
p
pabA (abgT OE) + 10 µM folic acid
124
0
15.84
0.47
-14.00%
0.0005*
0.0035* 10 µM Schircks folic acid
CM5
p
pabA (abgT OE) + 100 µM folic acid
105
0
15.77
0.50
-14.36%
0.0003*
0.0041* 100 µM Schircks folic acid
CM5
p
WT (abgT OE)
110
9
16.11
0.50
Control
n/a
n/a Alkaline control
CM4
p
WT (abgT OE) + 10 µM folic acid
99
4
16.05
0.57
-0.37%
0.9479
0.8905 10 µM Schircks folic acid
CM4
p
WT (abgT OE) + 100 µM folic acid
127
4
15.78
0.39
-2.02%
0.3698
0.8168 100 µM Schircks folic acid
CM4
p ementary Table 1: Summary of lifespan data
espans conducted on defined media (DM) agar plates incubated at 25ºC
Group name
n
Censor
Mean lifespan Std. References error % change
p value (Log rank) p value (Wilcoxon) Supplement
Bacterial genotype
WT
108
0
15.79
0.53
n/a
n/a
n/a Alkaline control
CM1
pabA
94
3
20.08
0.52
Control
n/a
n/a Alkaline control
CM2
pabA + 0.1 µM PABA
107
0
19.30
0.46
-3.89%
0.0973
0.3296 0.1 µM PABA
CM2
pabA + 1 µM PABA
118
0
15.91
0.45
-20.78%
<.0001*
<.0001* 1 µM PABA
CM2
pabA + 1 µM PABA-Glu
110
2
18.58
0.40
-7.49%
0.0030*
0.0277* 1 µM PABA-Glu
CM2
pabA + 10 µM PABA-Glu
125
10
16.43
0.46
-18.16%
<.0001*
<.0001* 10 µM PABA-Glu
CM2
pabA + 10 µM folic acid
93
5
18.20
0.55
-9.36%
0.0052*
0.0061* 10 µM Schircks folic acid
CM2
pabA + 100 µM folic acid
112
1
15.28
0.45
-23.93%
<.0001*
<.0001* 100 µM Schircks folic acid
CM2
abgTpabA
91
3
20.10
0.44
Control
n/a
n/a Alkaline control
CM3
abgTpabA + 0.1 µM PABA
91
6
18.72
0.49
-6.88%
0.0631
0.0477* 0.1 µM pABA
CM3
abgTpabA + 1 µM PABA
102
1
16.71
0.45
-16.86%
<.0001*
<.0001* 1 µM pABA
CM3
abgTpabA + 1 PABA-Glu
101
11
18.11
0.52
-9.94%
0.1733
0.0076* 1 µM PABA-Glu
CM3
abgTpabA + 10 µM PABA-Glu
106
4
19.00
0.46
-5.49%
0.1817
0.0512 10 µM PABA-Glu
CM3
abgTpabA + 10 µM folic acid
110
1
19.20
0.43
-4.49%
0.1901
0.114 10 µM Schircks folic acid
CM3
abgTpabA + 100 µM folic acid
100
3
19.16
0.40
-4.67%
0.0476*
0.0743 100 µM Schircks folic acid
CM3
pabA (abgT OE)
62
10
19.90
0.52
Contol
n/a
n/a Alkaline control
CM5
pabA (abgT OE) + 0.1 µM PABA
121
1
18.55
0.45
-6.72%
0.0049*
0.0184* 0.1 µM PABA
CM5
pabA (abgT OE) + 1 µM PABA
104
2
16.07
0.50
-19.19%
<.0001*
<.0001* 1 µM PABA
CM5
pabA (abgT OE) + 1 µM PABA-Glu
104
1
17.01
0.43
-14.47%
<.0001*
<.0001* 1 µM PABA-Glu
CM5
pabA (abgT OE) + 10 µM PABA-Glu
117
0
15.05
0.41
-24.33%
<.0001*
<.0001* 10 µM PABA-Glu
CM5
pabA (abgT OE) + 10 µM folic acid
119
0
16.65
0.42
-16.30%
<.0001*
<.0001* 10 µM Schircks folic acid
CM5
pabA (abgT OE) + 100 µM folic acid
117
0
16.19
0.42
-18.61%
<.0001*
<.0001* 100 µM Schircks folic acid
CM5
WT
91
9
13.85
0.44
Control
n/a
n/a n/a
WT
abgT
114
3
12.68
0.35
-8.48%
0.4312
0.0963 n/a
JW5822-1
WT
108
0
14.58
0.40
Control
n/a
n/a Alkaline control
WT
WT + 10 µM folic acid
115
0
15.00
0.39
2.86%
0.4448
0.4588 10 µM Schircks folic acid
WT
WT + 100 µM folic acid
106
1
14.57
0.48
-0.10%
0.566
0.844 100 µM Schircks folic acid
WT
t expt. References Levels of THFs detected in extracts of WT, pabA, abgTpabA, pabA (abgT OE) mutants displayed as a ratio
with an internal MTX-glu6 spike for normalization. Extracts were made after 4 days of bacterial growth at 25°C. Asterisks denote the test statistic from Student’s t test comparison of means, where * = P<0.05. Error bars represent
standard deviation over four replicates. 0.00
0.03
0.05
0
10
100
Folate: MTX-glu6
[Folic acid] (µM)
5,10-methylene-THF
WT
pabA
abgTpabA
pabA (abgT OE)
*
* 0.00
0.03
0.05
0
10
100
Folate: MTX-glu6
[Folic acid] (µM)
5,10-methylene-THF
WT
pabA
abgTpabA
pabA (abgT OE)
*
* 5,10-methylene-THF Supplementary Figure 2. LC-MS/MS detects lower levels of THFs in pabA mutants, where abgT determines response
to folic acid. Levels of THFs detected in extracts of WT, pabA, abgTpabA, pabA (abgT OE) mutants displayed as a ratio
with an internal MTX-glu6 spike for normalization. Extracts were made after 4 days of bacterial growth at 25°C. Asterisks denote the test statistic from Student’s t test comparison of means, where * = P<0.05. Error bars represent
standard deviation over four replicates. 22 Supplementary Figure 3. The impact of folic acid supplementation on C. elegans lifespan on wild-type E. coli. Survival curves of C. elegans maintained from day 1 of adulthood on plates supplemented with 10 µM and 100 µM
folic acid. 0.0
0.2
0.4
0.6
0.8
1.0
0
5
10
15
20
25
30
Survival
Day of adulthood
WT control
WT + 10 µm folic acid
WT + 100 µm folic acid
WT control
WT + 10 µM folic acid
WT + 100 µM folic acid 0.0
0.2
0.4
0.6
0.8
1.0
0
5
10
15
20
25
30
Survival
Day of adulthood
WT control
WT + 10 µm folic acid
WT + 100 µm folic acid
WT control
WT + 10 µM folic acid
WT + 100 µM folic acid Survival Supplementary Figure 3. The impact of folic acid supplementation on C. elegans lifespan on wild-type E. coli. Survival curves of C. elegans maintained from day 1 of adulthood on plates supplemented with 10 µM and 100 µM
folic acid. References 23 abgTpabA + 1 PABA-Glu
101
11
18.11
0.52
-9.94%
0.1733
0.0076* 1 µM PABA-Glu
CM3
p
abgTpabA + 10 µM PABA-Glu
106
4
19.00
0.46
-5.49%
0.1817
0.0512 10 µM PABA-Glu
CM3
p
abgTpabA + 10 µM folic acid
110
1
19.20
0.43
-4.49%
0.1901
0.114 10 µM Schircks folic acid
CM3
p
abgTpabA + 100 µM folic acid
100
3
19.16
0.40
-4.67%
0.0476*
0.0743 100 µM Schircks folic acid
CM3
p
pabA (abgT OE)
62
10
19.90
0.52
Contol
n/a
n/a Alkaline control
CM5
p
pabA (abgT OE) + 0.1 µM PABA
121
1
18.55
0.45
-6.72%
0.0049*
0.0184* 0.1 µM PABA
CM5
p
pabA (abgT OE) + 1 µM PABA
104
2
16.07
0.50
-19.19%
<.0001*
<.0001* 1 µM PABA
CM5
p
pabA (abgT OE) + 1 µM PABA-Glu
104
1
17.01
0.43
-14.47%
<.0001*
<.0001* 1 µM PABA-Glu
CM5
p
pabA (abgT OE) + 10 µM PABA-Glu
117
0
15.05
0.41
-24.33%
<.0001*
<.0001* 10 µM PABA-Glu
CM5
p
pabA (abgT OE) + 10 µM folic acid
119
0
16.65
0.42
-16.30%
<.0001*
<.0001* 10 µM Schircks folic acid
CM5
p
pabA (abgT OE) + 100 µM folic acid
117
0
16.19
0.42
-18.61%
<.0001*
<.0001* 100 µM Schircks folic acid
CM5
p
1
WT
91
9
13.85
0.44
Control
n/a
n/a n/a
WT
p
abgT
114
3
12.68
0.35
-8.48%
0.4312
0.0963 n/a
JW5822-1
n
2
WT
108
0
14.58
0.40
Control
n/a
n/a Alkaline control
WT
p
WT + 10 µM folic acid
115
0
15.00
0.39
2.86%
0.4448
0.4588 10 µM Schircks folic acid
WT
p
WT + 100 µM folic acid
106
1
14.57
0.48
-0.10%
0.566
0.844 100 µM Schircks folic acid
WT
p
at expt. References WT
102
9
15.96
0.48
Control
n/a
n/a Alkaline control
CM1
hown)
WT + 10 µM folic acid
98
12
15.96
0.58
0.03%
0.8268
0.4283 10 µM Schircks folic acid
CM1
WT + 100 µM folic acid
102
18
16.01
0.48
0.36%
0.866
0.7792 100 µM Schircks folic acid
CM1
pabA
113
10
18.35
0.56
Control
n/a
n/a Alkaline control
CM2
pabA + 10 µM folic acid
90
11
17.44
0.64
-4.95%
0.2527
0.2605 10 µM Schircks folic acid
CM2
pabA + 100 µM folic acid
122
3
15.86
0.40
-13.57%
<.0001*
0.0019* 100 µM Schircks folic acid
CM2
abgTpabA
136
14
18.93
0.50
Control
n/a
n/a Alkaline control
CM3
abgTpabA + 10 µM folic acid
115
26
19.22
0.52
1.51%
0.6416
0.5684 10 µM Schircks folic acid
CM3
abgTpabA + 100 µM folic acid
93
11
19.26
0.60
1.71%
0.5868
0.4482 100 µM Schircks folic acid
CM3
pabA (abgT OE)
101
23
18.42
0.56
Control
n/a
n/a Alkaline control
CM5
pabA (abgT OE) + 10 µM folic acid
124
0
15.84
0.47
-14.00%
0.0005*
0.0035* 10 µM Schircks folic acid
CM5
pabA (abgT OE) + 100 µM folic acid
105
0
15.77
0.50
-14.36%
0.0003*
0.0041* 100 µM Schircks folic acid
CM5
WT (abgT OE)
110
9
16.11
0.50
Control
n/a
n/a Alkaline control
CM4
WT (abgT OE) + 10 µM folic acid
99
4
16.05
0.57
-0.37%
0.9479
0.8905 10 µM Schircks folic acid
CM4
WT (abgT OE) + 100 µM folic acid
127
4
15.78
0.39
-2.02%
0.3698
0.8168 100 µM Schircks folic acid
CM4 t expt. hown) p
hown)
|
https://openalex.org/W4213189975
|
https://periodicos.univali.br/index.php/rdp/article/download/18378/10528
|
Portuguese
| null |
AUTONOMIA E PRERROGATIVAS DO MINISTÉRIO PÚBLICO BRASILEIRO EM PERSPECTIVA COMPARADA: CONSTRUINDO UM ÍNDICE DE INDEPENDÊNCIA/AUTONOMIA
|
Revista eletrônica direito e política
| 2,021
|
cc-by-sa
| 24,269
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1 Professor Titular da Faculdade de Direito da Universidade Federal do Paraná (UFPR). Coordenador do
Programa de Pós-Graduação (Mestrado, Doutorado e Pós-Doutorado) da UFPR. Doutor em Ciência
Política pela Universidade Estadual de Campinas (UNICAMP). E-mail: fab_tom@hotmail.com.
2 Professor Adjunto da Faculdade de Direito da UFPR e do Mestrado e Doutorado do Instituto Brasileiro
de Ensino, Desenvolvimento e Pesquisa (IDP-Brasília). Realizou Pós-Doutorado em Direito Constitucional
na Pontifícia Universidade Católica do Rio Grande do Sul (PUC/RS). Doutor em Direito pela UFPR.
Coordenador de Pesquisa da Academia Brasileira de Direito Constitucional (ABDConst). E-mail:
norbertorobl@gmail.com. Fabrício Ricardo de Limas Tomio1 Ilton Norberto Robl Filho2 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021.
Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. Fabrício Ricardo de Limas Tomio1
Ilton Norberto Robl Filho2 Fabrício Ricardo de Limas Tomio1 ABSTRACT The finding of the high degree of autonomy and independence of the Brazilian Public
Prosecutors (after the Federal Constitution of 1988) has been frequent in the national
and comparative specialized literature. The main objective of this article is to develop
an index to measure the autonomy and independence of the Public Prosecutor, for a
more accurate comparative analysis of the Brazilian Public Prosecutor’s Office with
other similar institutions in the world. Secondarily, we compare the index of the Public
Prosecutor to other constitutional powers (Legislative, Executive and Judiciary) to
assess the relationship between the powers and, eventually, suggest a causal
explanation (constitutional making) for the independence/autonomy attributed,
constitutional and statutorily, to the Public Prosecutor. As a methodology, initially, we
used indexes consolidated in the literature that analyze the constitutional powers of
the State (CCP Indexes). Then, approaching the central object, we describe and make
the index of independence/autonomy of the Public Prosecutor (CEPEJ/DIRPOL) based
on institutional prerogatives (de jure) and on Brazilian legislation and using data
organized by CEPEJ for 47 European countries. Preliminary findings and conclusions:
demonstrate that Brazil has almost all the prerogatives and guarantees attributed to
the Public Prosecutor in full, standing in the highest stratum of the index, and,
comparatively, reinforce the (statistically limited) perception that there is a greater
probability of finding a higher degree of institutional prerogatives and guarantees
attributed to the Public Prosecutor in countries that constitutionalize more powers to
the Legislative or in “recent democracies” (more recent transitions of regimes
authoritarian). KEYWORDS: Public Prosecutor; Autonomy; Prerogative; Index. KEYWORDS: Public Prosecutor; Autonomy; Prerogative; Index. RESUMO A constatação do elevado grau de autonomia e independência do Ministério Público
(MP) brasileiro (após Constituição Federal/1988) tem sido frequente na literatura
nacional e comparada. Este artigo tem por objetivo principal desenvolver um índice
para mensurar a autonomia e a independência do MP, permitindo uma análise
comparada mais precisa do MP brasileiro com outras instituições semelhantes no
mundo. Secundariamente, comparamos o índice do MP aos outros poderes
constitucionais (Legislativo, Executivo e Judiciário) para dimensionar a relação entre
os poderes e, eventualmente, sugerir uma explicação causal (constitutional making)
para a independência/autonomia atribuída constitucional e estatutariamente ao MP. Como metodologia, inicialmente, utilizamos índices consolidados na literatura que
analisam os poderes constitucionais do Estado (CCP Índices). Em seguida, abordando
o objeto central, descrevemos e construímos um índice de independência/autonomia
do MP (CEPEJ/DIRPOL) com base em prerrogativas institucionais (de jure),
compilando a legislação brasileira e utilizando os dados organizados pelo CEPEJ para
47 países europeus. Os achados e conclusões preliminares demonstram que o Brasil
apresenta quase todas as prerrogativas e garantias atribuídas ao MP de forma plena,
situando-se no estrato mais elevado do índice, e, comparativamente, reforçam a
percepção, estatisticamente limitada, que há maior probabilidade de encontrar um
grau mais elevado de prerrogativas e garantias institucionais atribuídas ao MP em
países que constitucionalizam mais poderes ao Legislativo ou em “democracias
recentes” (transições mais recentes de regimes autoritários). 1166 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LAVRAS-CHAVE: Ministério Público; Autonomia; Prerrogativa; Índice. PALAVRAS-CHAVE: Ministério Público; Autonomia; Prerrogativa; Índice INTRODUÇÃO Este artigo tem por objetivo principal desenvolver um índice para mensurar a
autonomia e a independência do Ministério Público, permitindo uma análise
comparada mais precisa do Ministério Público brasileiro com outras instituições
estatais semelhantes no mundo. Os principais indicadores comparados existentes não são diretamente relacionados
ao Ministério Público, mas servirão para dimensionar, dentro de um país, a relação 1167 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. entre os poderes e, eventualmente, sugerir uma explicação causal (constitutional
making) para a independência/autonomia do MP. Além disto, pela abrangência da
comparação dos índices do CCP (Comparative Constitutions Project) foi possível
constatar indireta e comparativamente, como hipótese (pelo proxy de Judicial
Independence), a elevada autonomia institucional e constitucional do Ministério
Público brasileiro, pois a estrutura constitucional do Judiciário brasileiro é semelhante
ao do Ministério Público. Dessa forma, nos itens 2 e 3 deste texto, serão expostos e correlacionados os índices
comparativos sobre o Executivo, o Legislativo e o Judiciário a partir do Comparative
Constitutions Project. De outro lado, a seção 4 versa acerca das características
institucionais do Ministério Público brasileiro, analisando ainda os índices existentes
sobre o Ministério Público. Ainda, nessa seção, será construído um índice para analisar
o Ministério Público brasileiro em diálogo e desde a metodologia estabelecida pela
Comissão Europeia para Eficiência do Judiciário (CEPEJ). O item 5, por sua vez, apresenta correlações entre os poderes do Executivo e
Legislativo e a Independência do Judiciário com a independência e com a autonomia
do Ministério Público. INTRODUÇÃO Por fim, nesse item, é escrutinada a relação entre autonomia
do Ministério Público com o restabelecimento da democracia após governos e Estados
autoritários. Na seção 6, as conclusões são apresentadas. 3 ELKINS, Zachary; GINSBURG, Tom; MELTON, James. The comparative constitutions project: A
cross-national historical dataset of written constitutions. Chicago: Mimeo, 2009a. ELKINS,
Zachary; GINSBURG, Tom; MELTON, James. The endurance of national constitutions. Cambridge-
UK: Cambridge University Press, 2009b. ELKINS, Zachary; GINSBURG, Tom; MELTON, James.
Constitutional constraints on executive lawmaking. Working paper, 2012. Disponível em:
http://citeseerx.ist.psu.edu/viewdoc/summary?doi=10.1.1.434.9346. Acesso em 11.10.2021. FISH, M.
Steven; KROENIG, Matthew. The handbook of national legislatures: A global survey. Cambridge-
UK: Cambridge University Press, 2009. MELTON, James; GINSBURG, Tom. Does de jure judicial
independence really matter? A reevaluation of explanations for judicial independence. Journal of Law
and Courts, v. 2, n. 2, p. 187-217, 2014. 1.
QUESTÕES
PRELIMINARES:
ÍNDICES
COMPARATIVOS
DE
INDEPENDÊNCIA DO JUDICIÁRIO E DAS COMPETÊNCIAS E DOS PODERES DO
EXECUTIVO
E
DO
LEGISLATIVO
A
PARTIR
ESPECIALMENTE
DO
COMPARATIVE CONSTITUTIONS PROJECT – CCP 1. QUESTÕES
PRELIMINARES:
ÍNDICES
COMPARATIVOS
DE
INDEPENDÊNCIA DO JUDICIÁRIO E DAS COMPETÊNCIAS E DOS PODERES DO
EXECUTIVO
E
DO
LEGISLATIVO
A
PARTIR
ESPECIALMENTE
DO
COMPARATIVE CONSTITUTIONS PROJECT – CCP Os índices comparativos sobre instituições estatais, os quais buscam mensurar, por
exemplo, os poderes, a independência, a autonomia, são derivados de aspectos
institucionais como distribuição de competências e prerrogativas atribuídas a esses 1168 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. atores institucionais (poderes constitucionais e demais instituições de Estado), por
meio da constituição e/ou da legislação. Dessa forma, permite-se estimar e mensurar
os efeitos da variação institucional no processo decisório, na relação entre os poderes
estatais e órgãos constitucionais autônomos e na formulação das políticas públicas. Esses índices comparativos estabelecem associações e explicações (causalidade)
sobre as intenções dos constituintes (Poder Constituinte Originário e Poder
Constituinte
Derivado
Reformador)
ao
atribuir
graus
distintos
de
competências/prerrogativas a cada poder, a órgão constitucional autônomo e para as
demais instituições estatais. Dessa forma, para construir um índice comparativo de autonomia/independência do
Ministério Público (Prosecutorial Independence – PI - de jure), estabelecendo o status
do Ministério Público brasileiro, serão observadas as seguintes etapas. Em primeiro
lugar, impõe-se compreender a situação das outras instituições centrais do Estado
(no mesmo país e entre os países comparados). Em segundo lugar, analisar-se-ão os
índices (de jure) mais consolidados na literatura sobre instituições, órgãos e poderes
estatais, buscando posteriormente elaborar um índice próprio sobre o Ministério
Público
para
descrever
e
analisar,
comparativamente,
o
grau
de
autonomia/independência do Ministério Público no Brasil. 4 Além dos três índices (Executive Power, Legislative Power e Judicial Independence), o Comparative
Constitutions Project – CCP (índices) apresenta outros três indicadores comparativos: Scope (mede a
porcentagem de 70 tópicos principais da pesquisa do CCP que estão incluídos em qualquer constituição),
Length in Words (relatório do número total de palavras em cada constituição) e Number of Rights (sobre
direitos humanos, um conjunto de 117 direitos específicos encontrados nas constituições nacionais)
(ELKINS, GINSBURG e MELTON, 2009a). 4 Além dos três índices (Executive Power, Legislative Power e Judicial Independence), o Comparative
Constitutions Project – CCP (índices) apresenta outros três indicadores comparativos: Scope (mede a
porcentagem de 70 tópicos principais da pesquisa do CCP que estão incluídos em qualquer constituição),
Length in Words (relatório do número total de palavras em cada constituição) e Number of Rights (sobre
direitos humanos, um conjunto de 117 direitos específicos encontrados nas constituições nacionais)
(ELKINS, GINSBURG e MELTON, 2009a).
5 “§ 2º Ao Ministério Público é assegurada autonomia funcional e administrativa, podendo, observado o
disposto no art. 169, propor ao Poder Legislativo a criação e extinção de seus cargos e serviços auxiliares,
provendo-os por concurso público de provas ou de provas e títulos, a política remuneratória e os planos
de carreira; a lei disporá sobre sua organização e funcionamento. (Redação dada pela Emenda
Constitucional nº 19, de 1998) § 3º O Ministério Público elaborará sua proposta orçamentária dentro dos
limites estabelecidos na lei de diretrizes orçamentárias. § 4º Se o Ministério Público não encaminhar a
respectiva proposta orçamentária dentro do prazo estabelecido na lei de diretrizes orçamentárias, o
Poder Executivo considerará, para fins de consolidação da proposta orçamentária anual, os valores
aprovados na lei orçamentária vigente, ajustados de acordo com os limites estipulados na forma do §
3º. (Incluído pela Emenda Constitucional nº 45, de 2004) (...) § 5º Leis complementares da União e dos
Estados, cuja iniciativa é facultada aos respectivos Procuradores-Gerais, estabelecerão a organização,
as atribuições e o estatuto de cada Ministério Público, observadas, relativamente a seus membros: I -
as seguintes garantias: a) vitaliciedade, após dois anos de exercício, não podendo perder o cargo senão
por sentença judicial transitada em julgado; b) inamovibilidade, salvo por motivo de interesse público,
mediante decisão do órgão colegiado competente do Ministério Público, pelo voto da maioria absoluta
de seus membros, assegurada ampla defesa; (Redação dada pela Emenda Constitucional nº 45, de
2004) c) irredutibilidade de subsídio, fixado na forma do art. 39, § 4º, e ressalvado o disposto nos arts.
37, X e XI, 150, II, 153, III, 153, § 2º, I; (Redação dada pela Emenda Constitucional nº 19, de 1998)
II - as seguintes vedações: a) receber, a qualquer título e sob qualquer pretexto, honorários,
percentagens ou custas processuais; b) exercer a advocacia; c) participar de sociedade comercial, na
forma da lei; d) exercer, ainda que em disponibilidade, qualquer outra função pública, salvo uma de
magistério; e) exercer atividade político-partidária; (Redação dada pela Emenda Constitucional nº 45,
de 2004); f) receber, a qualquer título ou pretexto, auxílios ou contribuições de pessoas físicas, entidades
públicas ou privadas, ressalvadas as exceções previstas em lei. (Incluída pela Emenda Constitucional nº
45, de 2004) § 6º Aplica-se aos membros do Ministério Público o disposto no art. 95, parágrafo único,
V. (Incluído pela Emend Constitucional nº 45, de 2004)”. 1.
QUESTÕES
PRELIMINARES:
ÍNDICES
COMPARATIVOS
DE
INDEPENDÊNCIA DO JUDICIÁRIO E DAS COMPETÊNCIAS E DOS PODERES DO
EXECUTIVO
E
DO
LEGISLATIVO
A
PARTIR
ESPECIALMENTE
DO
COMPARATIVE CONSTITUTIONS PROJECT – CCP Dentre o conjunto de índices comparados, observa-se o construído especialmente
pelos pesquisadores Zachary Elkins, Tom Ginsburg e James Melton, no Comparative
Constitutions Project–CCP, reunindo indicadores extraídos diretamente do texto
constitucional de 190 países e sendo os índices mais abrangentes3. Os três índices 1169 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. (Executive Power, Legislative Power e Judicial Independence) são extraídos do CCP
Data (descritos na página da web do CCP).4 Para a utilização nesta análise, inclusive
por considerações qualitativas, o ranking e os indicadores contínuos são tratados
como classes e separatrizes, dividindo o intervalo de frequência da amostra em partes
iguais (tercis, metade) e agrupando países pelo valor atribuído pelo índice. No caso brasileiro, em virtude especialmente dos artigos 127, §§ 2º, 3º e 4º e 128,
§§ 5º e 6º, Constituição Federal brasileira de 1988 (CF/88)5, o índice de
Independência Judicial (Judicial Independence, CCP) poderia ser tratado como proxy
(substituto) da Independência/Autonomia do Ministério Público (MP). Em outras
palavras,
as
autonomias
funcional,
administrativa
e
financeira
garantidas
constitucionalmente ao MP do Brasil (art. 127, §§ 2º, 3º e 4º, CF/88) podem ser (ELKINS, GINSBURG e MELTON, 2009a). 5 “§ 2º Ao Ministério Público é assegurada autonomia funcional e administrativa, podendo, observado o
disposto no art. 169, propor ao Poder Legislativo a criação e extinção de seus cargos e serviços auxiliares,
provendo-os por concurso público de provas ou de provas e títulos, a política remuneratória e os planos
de carreira; a lei disporá sobre sua organização e funcionamento. 6 “Art. 99. Ao Poder Judiciário é assegurada autonomia administrativa e financeira. § 1º Os tribunais
elaborarão suas propostas orçamentárias dentro dos limites estipulados conjuntamente com os demais
Poderes na lei de diretrizes orçamentárias. § 2º O encaminhamento da proposta, ouvidos os outros
tribunais interessados, compete: I - no âmbito da União, aos Presidentes do Supremo Tribunal Federal
e dos Tribunais Superiores, com a aprovação dos respectivos tribunais; II - no âmbito dos Estados e no
do Distrito Federal e Territórios, aos Presidentes dos Tribunais de Justiça, com a aprovação dos
respectivos tribunais. § 3º Se os órgãos referidos no § 2º não encaminharem as respectivas propostas
orçamentárias dentro do prazo estabelecido na lei de diretrizes orçamentárias, o Poder Executivo
considerará, para fins de consolidação da proposta orçamentária anual, os valores aprovados na lei
orçamentária vigente, ajustados de acordo com os limites estipulados na forma do § 1º deste
artigo. (Incluído pela Emenda Constitucional nº 45, de 2004) § 4º Se as propostas orçamentárias
de que trata este artigo forem encaminhadas em desacordo com os limites estipulados na forma do §
1º, o Poder Executivo procederá aos ajustes necessários para fins de consolidação da proposta
orçamentária anual. (Incluído pela Emenda Constitucional nº 45, de 2004) § 5º Durante a execução
orçamentária do exercício, não poderá haver a realização de despesas ou a assunção de obrigações que
extrapolem os limites estabelecidos na lei de diretrizes orçamentárias, exceto se previamente
autorizadas, mediante a abertura de créditos suplementares ou especiais. (Incluído pela Emenda
Constitucional nº 45, de 2004)”. 1.
QUESTÕES
PRELIMINARES:
ÍNDICES
COMPARATIVOS
DE
INDEPENDÊNCIA DO JUDICIÁRIO E DAS COMPETÊNCIAS E DOS PODERES DO
EXECUTIVO
E
DO
LEGISLATIVO
A
PARTIR
ESPECIALMENTE
DO
COMPARATIVE CONSTITUTIONS PROJECT – CCP Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. comparadas e dimensionadas à independência judicial garantida no artigo 99, CF/886. Acerca das garantias funcionais, tanto na magistratura como no Ministério Público, há
as garantias da vitaliciedade, inamovibilidade e irredutibilidade, segundo os artigos
95, I, II e III7 e 128, § 5º, I, CF/88. De outro lado, uma diferença constitucional
relevante brasileira consiste na escolha do chefe do Ministério Público da União que é
nomeado pelo Presidente da República, após a sabatina do Senado Federal, e na
escolha dos Chefes dos Ministérios Públicos dos Estados e do Distrito Federal, os quais
escolhidos pelo Governador, a partir de lista tríplice dentre os membros da carreira,
de acordo com o artigo 128, §§ 1º e 3º, CF/888, enquanto compete aos tribunais “a)
eleger seus órgãos diretivos e elaborar seus regimentos internos, com observância
das normas de processo e das garantias processuais das partes, dispondo sobre a comparadas e dimensionadas à independência judicial garantida no artigo 99, CF/886. Acerca das garantias funcionais, tanto na magistratura como no Ministério Público, há
as garantias da vitaliciedade, inamovibilidade e irredutibilidade, segundo os artigos
95, I, II e III7 e 128, § 5º, I, CF/88. De outro lado, uma diferença constitucional
relevante brasileira consiste na escolha do chefe do Ministério Público da União que é
nomeado pelo Presidente da República, após a sabatina do Senado Federal, e na
escolha dos Chefes dos Ministérios Públicos dos Estados e do Distrito Federal, os quais
escolhidos pelo Governador, a partir de lista tríplice dentre os membros da carreira,
de acordo com o artigo 128, §§ 1º e 3º, CF/888, enquanto compete aos tribunais “a)
eleger seus órgãos diretivos e elaborar seus regimentos internos, com observância
das normas de processo e das garantias processuais das partes, dispondo sobre a 7 “Art. 95. 6 “Art. 99. Ao Poder Judiciário é assegurada autonomia administrativa e financeira. § 1º Os tribunais
elaborarão suas propostas orçamentárias dentro dos limites estipulados conjuntamente com os demais
Poderes na lei de diretrizes orçamentárias. § 2º O encaminhamento da proposta, ouvidos os outros
tribunais interessados, compete: I - no âmbito da União, aos Presidentes do Supremo Tribunal Federal
e dos Tribunais Superiores, com a aprovação dos respectivos tribunais; II - no âmbito dos Estados e no
do Distrito Federal e Territórios, aos Presidentes dos Tribunais de Justiça, com a aprovação dos
respectivos tribunais. § 3º Se os órgãos referidos no § 2º não encaminharem as respectivas propostas
orçamentárias dentro do prazo estabelecido na lei de diretrizes orçamentárias, o Poder Executivo
considerará, para fins de consolidação da proposta orçamentária anual, os valores aprovados na lei
orçamentária vigente, ajustados de acordo com os limites estipulados na forma do § 1º deste
artigo. (Incluído pela Emenda Constitucional nº 45, de 2004) § 4º Se as propostas orçamentárias
de que trata este artigo forem encaminhadas em desacordo com os limites estipulados na forma do §
1º, o Poder Executivo procederá aos ajustes necessários para fins de consolidação da proposta
orçamentária anual. (Incluído pela Emenda Constitucional nº 45, de 2004) § 5º Durante a execução
orçamentária do exercício, não poderá haver a realização de despesas ou a assunção de obrigações que
extrapolem os limites estabelecidos na lei de diretrizes orçamentárias, exceto se previamente
autorizadas, mediante a abertura de créditos suplementares ou especiais. (Incluído pela Emenda
Constitucional nº 45, de 2004)”.
7 “Art. 95. Os juízes gozam das seguintes garantias: I - vitaliciedade, que, no primeiro grau, só será
adquirida após dois anos de exercício, dependendo a perda do cargo, nesse período, de deliberação do
tribunal a que o juiz estiver vinculado, e, nos demais casos, de sentença judicial transitada em julgado;
II - inamovibilidade, salvo por motivo de interesse público, na forma do art. 93, VIII; III - irredutibilidade
de subsídio, ressalvado o disposto nos arts. 37, X e XI, 39, § 4º, 150, II, 153, III, e 153, § 2º, I.
(Redação dada pela Emenda Constitucional nº 19, de 1998)”.
8 “§ 1º O Ministério Público da União tem por chefe o Procurador-Geral da República, nomeado pelo
Presidente da República dentre integrantes da carreira, maiores de trinta e cinco anos, após a aprovação
de seu nome pela maioria absoluta dos membros do Senado Federal, para mandato de dois anos,
permitida a recondução. (...) § 3º Os Ministérios Públicos dos Estados e o do Distrito Federal e Territórios
formarão lista tríplice dentre integrantes da carreira, na forma da lei respectiva, para escolha de seu
Procurador-Geral, que será nomeado pelo Chefe do Poder Executivo, para mandato de dois anos,
permitida uma recondução”. 1.
QUESTÕES
PRELIMINARES:
ÍNDICES
COMPARATIVOS
DE
INDEPENDÊNCIA DO JUDICIÁRIO E DAS COMPETÊNCIAS E DOS PODERES DO
EXECUTIVO
E
DO
LEGISLATIVO
A
PARTIR
ESPECIALMENTE
DO
COMPARATIVE CONSTITUTIONS PROJECT – CCP (Redação dada pela Emenda
Constitucional nº 19, de 1998) § 3º O Ministério Público elaborará sua proposta orçamentária dentro dos
limites estabelecidos na lei de diretrizes orçamentárias. § 4º Se o Ministério Público não encaminhar a
respectiva proposta orçamentária dentro do prazo estabelecido na lei de diretrizes orçamentárias, o
Poder Executivo considerará, para fins de consolidação da proposta orçamentária anual, os valores
aprovados na lei orçamentária vigente, ajustados de acordo com os limites estipulados na forma do §
3º. (Incluído pela Emenda Constitucional nº 45, de 2004) (...) § 5º Leis complementares da União e dos
Estados, cuja iniciativa é facultada aos respectivos Procuradores-Gerais, estabelecerão a organização,
as atribuições e o estatuto de cada Ministério Público, observadas, relativamente a seus membros: I -
as seguintes garantias: a) vitaliciedade, após dois anos de exercício, não podendo perder o cargo senão
por sentença judicial transitada em julgado; b) inamovibilidade, salvo por motivo de interesse público,
mediante decisão do órgão colegiado competente do Ministério Público, pelo voto da maioria absoluta
de seus membros, assegurada ampla defesa; (Redação dada pela Emenda Constitucional nº 45, de
2004) c) irredutibilidade de subsídio, fixado na forma do art. 39, § 4º, e ressalvado o disposto nos arts. 37, X e XI, 150, II, 153, III, 153, § 2º, I; (Redação dada pela Emenda Constitucional nº 19, de 1998)
II - as seguintes vedações: a) receber, a qualquer título e sob qualquer pretexto, honorários,
percentagens ou custas processuais; b) exercer a advocacia; c) participar de sociedade comercial, na
forma da lei; d) exercer, ainda que em disponibilidade, qualquer outra função pública, salvo uma de
magistério; e) exercer atividade político-partidária; (Redação dada pela Emenda Constitucional nº 45,
de 2004); f) receber, a qualquer título ou pretexto, auxílios ou contribuições de pessoas físicas, entidades
públicas ou privadas, ressalvadas as exceções previstas em lei. (Incluída pela Emenda Constitucional nº
45, de 2004) § 6º Aplica-se aos membros do Ministério Público o disposto no art. 95, parágrafo único,
V. (Incluído pela Emend Constitucional nº 45, de 2004)”. 1170 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. 6 “Art. 99. Ao Poder Judiciário é assegurada autonomia administrativa e financeira. § 1º Os tribunais
elaborarão suas propostas orçamentárias dentro dos limites estipulados conjuntamente com os demais
Poderes na lei de diretrizes orçamentárias. § 2º O encaminhamento da proposta, ouvidos os outros
tribunais interessados, compete: I - no âmbito da União, aos Presidentes do Supremo Tribunal Federal
e dos Tribunais Superiores, com a aprovação dos respectivos tribunais; II - no âmbito dos Estados e no
do Distrito Federal e Territórios, aos Presidentes dos Tribunais de Justiça, com a aprovação dos
respectivos tribunais. § 3º Se os órgãos referidos no § 2º não encaminharem as respectivas propostas
orçamentárias dentro do prazo estabelecido na lei de diretrizes orçamentárias, o Poder Executivo
considerará, para fins de consolidação da proposta orçamentária anual, os valores aprovados na lei 1.
QUESTÕES
PRELIMINARES:
ÍNDICES
COMPARATIVOS
DE
INDEPENDÊNCIA DO JUDICIÁRIO E DAS COMPETÊNCIAS E DOS PODERES DO
EXECUTIVO
E
DO
LEGISLATIVO
A
PARTIR
ESPECIALMENTE
DO
COMPARATIVE CONSTITUTIONS PROJECT – CCP Os juízes gozam das seguintes garantias: I - vitaliciedade, que, no primeiro grau, só será
adquirida após dois anos de exercício, dependendo a perda do cargo, nesse período, de deliberação do
tribunal a que o juiz estiver vinculado, e, nos demais casos, de sentença judicial transitada em julgado;
II - inamovibilidade, salvo por motivo de interesse público, na forma do art. 93, VIII; III - irredutibilidade
de subsídio, ressalvado o disposto nos arts. 37, X e XI, 39, § 4º, 150, II, 153, III, e 153, § 2º, I. (Redação dada pela Emenda Constitucional nº 19, de 1998)”. 8 “§ 1º O Ministério Público da União tem por chefe o Procurador-Geral da República, nomeado pelo
Presidente da República dentre integrantes da carreira, maiores de trinta e cinco anos, após a aprovação
de seu nome pela maioria absoluta dos membros do Senado Federal, para mandato de dois anos,
permitida a recondução. (...) § 3º Os Ministérios Públicos dos Estados e o do Distrito Federal e Territórios
formarão lista tríplice dentre integrantes da carreira, na forma da lei respectiva, para escolha de seu
Procurador-Geral, que será nomeado pelo Chefe do Poder Executivo, para mandato de dois anos,
permitida uma recondução”. 1171 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. competência
e
o
funcionamento
dos
respectivos
órgãos
jurisdicionais
e
administrativo”, segundo o artigo 96, I, CF/88. Ainda, como a estruturação semelhante do Ministério Público e do Judiciário não é um
arranjo constitucional comum à imensa maioria dos 190 Estados nacionais
comparados pelo CCP, a construção de um índice comparativo próprio de
autonomia/independência do MP (Prosecutorial Independence, PI, de jure) torna-se
uma tarefa necessária na compreensão do grau de autonomia do Ministério Público
brasileiro. As garantias e as prerrogativas do judiciário estão mais presentes na constituição da
maior parte dos países. 1.
QUESTÕES
PRELIMINARES:
ÍNDICES
COMPARATIVOS
DE
INDEPENDÊNCIA DO JUDICIÁRIO E DAS COMPETÊNCIAS E DOS PODERES DO
EXECUTIVO
E
DO
LEGISLATIVO
A
PARTIR
ESPECIALMENTE
DO
COMPARATIVE CONSTITUTIONS PROJECT – CCP A consulta ao texto das constituições na base de dados do
CCP demonstra que: de 190 Estados nacionais, 178 constituições (dados atualizados
para 2020) fazem menção explícita à noção de Judicial Independence e todas as
constituições possuem o termo structure of the courts em seu texto. Por outro lado,
161 constituições relatam a presença do termo Public Prosecutor [prosecutor,
prosecutor general, attorney general ou termo semelhante], sem, necessariamente,
atribuir garantias de independência e autonomia ao mais alto representante do
Ministério Público de cada país e menos ainda ao conjunto dos representantes do
Ministério Público. Dessa forma, até preliminarmente, antes da análise dos dados, é
possível definir, comparativamente, a Independência do Ministério Público brasileiro
como superior à Independência do Judiciário, quando contrastado com as garantidas
constitucionais promovidas por outros países. Em relação aos dados do CCP para comparar os poderes constitucionalizados
(governo, legislaturas e tribunais nacionais), há Poder Atribuído Executivo (Executive
Power, CCP), o qual é composto por sete indicadores de poder fixados ao governo
nacional, isto é, (1) poder de iniciar o processo legislativo; (2) poder de emitir
decretos; (3) poder de propor emendas constitucionais; (4) poder de decretar estado
de emergência; (5) poder de veto; (6) poder de impugnar judicialmente a
constitucionalidade da legislação; e (7) poder de dissolver as legislaturas. O Executivo
brasileiro possui cinco desses atributos na Constituição Federal de 1988. Dessa 1172 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. maneira, o Executivo Nacional/Federal brasileiro é superior à maioria dos Estados
nacionais, já que mais da metade das constituições concede ao Executivo nacional
quatro ou menos poderes relacionados a essas competências (ver Gráfico 1). maneira, o Executivo Nacional/Federal brasileiro é superior à maioria dos Estados
nacionais, já que mais da metade das constituições concede ao Executivo nacional
quatro ou menos poderes relacionados a essas competências (ver Gráfico 1). 1.
QUESTÕES
PRELIMINARES:
ÍNDICES
COMPARATIVOS
DE
INDEPENDÊNCIA DO JUDICIÁRIO E DAS COMPETÊNCIAS E DOS PODERES DO
EXECUTIVO
E
DO
LEGISLATIVO
A
PARTIR
ESPECIALMENTE
DO
COMPARATIVE CONSTITUTIONS PROJECT – CCP Sobre o item (4), no estado de sítio, o Presidente no Brasil deve obter a autorização
do Congresso Nacional para sua decretação, após sólido relato dos motivos
determinantes pelo Chefe de Estado e de Governo, de acordo com o artigo 137,
CF/889. Dessa maneira, considera-se que a Presidência não tem o poder de decretar
estado de emergência. Ainda, o Executivo Nacional/Federal não detém o poder de
dissolver as legislaturas, que é o item (7) do índice. Ou seja, comparativa e
constitucionalmente, a competência do Executivo brasileiro pode ser classificada
como um pouco acima da média dos indicadores encontrados nos Estados nacionais. Gráfico 1 – Executive Power – CCP Índices
Fonte: DIRPOL/PPGD/UFPR com os dados do CCP Índices10
“Art. 137. O Presidente da República pode, ouvidos o Conselho da República e o Conselho de Defesa
i
l
li it
C
N
i
l
t
i
ã
d
t
t d
d
íti
d
I
2
7
17
42
29
38
30
25
0
5
10
15
20
25
30
35
40
45
0
1
2
3
4
5
6
7
Quant. Países (n=190)
Índice Executive Power (CCP) Gráfico 1 – Executive Power – CCP Índices Fonte: DIRPOL/PPGD/UFPR com os dados do CCP Índices10 9 “Art. 137. O Presidente da República pode, ouvidos o Conselho da República e o Conselho de Defesa
Nacional, solicitar ao Congresso Nacional autorização para decretar o estado de sítio nos casos de: I -
comoção grave de repercussão nacional ou ocorrência de fatos que comprovem a ineficácia de medida
tomada durante o estado de defesa; II - declaração de estado de guerra ou resposta a agressão armada
estrangeira. Parágrafo único. O Presidente da República, ao solicitar autorização para decretar o estado
de sítio ou sua prorrogação, relatará os motivos determinantes do pedido, devendo o Congresso Nacional
decidir por maioria absoluta”. 10 Organizado pelos pesquisadores do DIRPOL/PPGD/UFPR com os dados do CCP Índices,
https://comparativeconstitutionsproject.org/ccp-rankings/. No destaque, classe com a posição conferida
pela CF/88. 1173 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. 11 Organizado pelos pesquisadores do DIRPOL/PPGD/UFPR com os dados do CCP Índices,
https://comparativeconstitutionsproject.org/ccp-rankings/. No destaque, classe com a posição conferida
pela CF/88. 1.
QUESTÕES
PRELIMINARES:
ÍNDICES
COMPARATIVOS
DE
INDEPENDÊNCIA DO JUDICIÁRIO E DAS COMPETÊNCIAS E DOS PODERES DO
EXECUTIVO
E
DO
LEGISLATIVO
A
PARTIR
ESPECIALMENTE
DO
COMPARATIVE CONSTITUTIONS PROJECT – CCP Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. Por outro lado, o Legislative Power, no Brasil, coloca o Congresso Nacional dentre os
6% dos países que, comparativamente, mais atribuem poder à legislatura nacional
pela constituição (ver Gráfico 2). Dos 190 países, apenas sete possuem o mesmo
índice brasileiro e somente cinco nações detêm maiores valores do índice em relação
ao Brasil. O índice brasileiro é de 0.48, sendo um índice que pode variar entre 0,0 e
1,0. Essa pontuação resulta da média de 32 indicadores binários, representando os
números maiores um conjunto mais robusto de poderes ao Legislativo. O maior valor
do índice (0.62) é atribuído apenas a um país: o Haiti. O segundo índice mais elevado
de poder/competência do Legislativo (0,52) é encontrado em quatro países: Bulgária,
Camboja, Eslováquia e Suécia. O terceiro índice (0,48), além do Brasil, é
compartilhado com outros seis Estados nacionais: Egito, Eritréia, Etiópia, Finlândia,
Ucrânia e Uruguai. Gráfico 2 – Legislative Power – CCP Índices
Fonte: DIRPOL/PPGD/UFPR com os dados do CCP Índices11
1
3
1
9
19
19
16
26
38
29
17
7
4
1
0
5
10
15
20
25
30
35
40
0
0,05 0,09 0,10 0,14 0,19 0,24 0,29 0,33 0,38 0,43 0,48 0,52 0,62
Quant. Países (n=190)
Legislative Power
Índice Legislative Power (CCP) Gráfico 2 – Legislative Power – CCP Índices Índice Legislative Power (CCP) Fonte: DIRPOL/PPGD/UFPR com os dados do CCP Índices11 11 Organizado pelos pesquisadores do DIRPOL/PPGD/UFPR com os dados do CCP Índices,
https://comparativeconstitutionsproject.org/ccp-rankings/. No destaque, classe com a posição conferida
pela CF/88. 1174 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. 1.
QUESTÕES
PRELIMINARES:
ÍNDICES
COMPARATIVOS
DE
INDEPENDÊNCIA DO JUDICIÁRIO E DAS COMPETÊNCIAS E DOS PODERES DO
EXECUTIVO
E
DO
LEGISLATIVO
A
PARTIR
ESPECIALMENTE
DO
COMPARATIVE CONSTITUTIONS PROJECT – CCP Da mesma forma, a constitucionalização da Judicial Independence, no Brasil,
encontra-se no primeiro decil de atribuição de garantias aos tribunais nacionais
(superiores), ou seja, a independência judicial brasileira possui índice dentre os 10%
de países com índices mais elevados do conjunto de 190 nações comparadas (ver
Gráfico 3). As seis garantias constitucionais do indicador mensurados pelo CCP são
as seguintes: (1) constituição contém uma declaração explícita de independência
judicial, (2) constituição prevê que os juízes tenham nomeações vitalícias; (3)
nomeações para o tribunal mais alto envolvem um conselho judicial ou dois (ou mais)
atores (poderes), (4) a remoção é proibida ou limitada, ou requer uma proposta com
voto de maioria qualificada na legislatura, ou apenas um conselho judicial pode propor
a remoção e outro ator político é obrigado a aprovar tal proposta; (5) a remoção se
limita explicitamente a crimes e outras questões de má conduta, traição ou violações
da constituição e (6) os salários judiciais são protegidos de redução (irredutibilidade). Frisa-se que, segundo nossa compreensão, a equipe do CCP classificou erroneamente
o Brasil, porque as seis garantias constitucionais do indicador estão presentes na
CF/88. As bases de dados desagregados do CCP indicam que o Brasil não teria
previsão constitucional no item (4) - a remoção é proibida ou limitada, ou requer uma
proposta com voto de maioria qualificada na legislatura, ou apenas um conselho
judicial pode propor a remoção e outro ator político é obrigado a aprovar tal proposta. Entretanto, por questão de coerência, o índice atribuído pelo CCP será mantido nas
comparações realizadas no presente artigo. 1175 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. Gráfico 3 – Judicial Independence – CCP Índices
Fonte: DIRPOL/PPGD/UFPR com os dados do CCP Índices12
8
48
40
34
41
15
4
0
10
20
30
40
50
60
0
1
2
3
4
5
6
Quant. 12 Organizado pelos pesquisadores do DIRPOL/PPGD/UFPR com os dados do CCP Índices,
https://comparativeconstitutionsproject.org/ccp-rankings/. No destaque, classe com a posição conferida
pela CF/88. 13 O resultado da correlação linear r (Pearson) e regressão linear (R2) entre o índice de judicial
Independence e os índices de poder legislativo e poder executivo são os seguintes: com legislative power
(r = 0,1274; R2 = 0,0162); executive power (r = -0,1840; R2 = 0,0338). A associação entre judicial
independence e executive/legislative power é muito fraca (R2 próximo a zero). O maior poder legislativo
constitucionalizado em um país está levemente associado a maior independência judicial na constituição.
A relação é inversa (negativa) da independência judicial com a constitucionalização de poderes
legislativos ao poder executivo de um país. 14 Organizado pelos pesquisadores do DIRPOL/PPGD/UFPR com os dados do CCP Índices,
https://comparativeconstitutionsproject.org/ccp-rankings/. 1.
QUESTÕES
PRELIMINARES:
ÍNDICES
COMPARATIVOS
DE
INDEPENDÊNCIA DO JUDICIÁRIO E DAS COMPETÊNCIAS E DOS PODERES DO
EXECUTIVO
E
DO
LEGISLATIVO
A
PARTIR
ESPECIALMENTE
DO
COMPARATIVE CONSTITUTIONS PROJECT – CCP Países (n=190)
Judicial Independence
Índice Judicial Independence (CCP) Gráfico 3 – Judicial Independence – CCP Índices Fonte: DIRPOL/PPGD/UFPR com os dados do CCP Índices12 Na definição dada pelo CCP para independência judicial, das seis características
institucionais (prerrogativas constitucionalizadas) que favorecem a independência e
a autonomia judiciais, a CF/88 estabelece cinco ao Poder Judiciário do Brasil. É o
segundo maior índice, compartilhado pelo Brasil e outros 14 países: Albânia,
Bangladesh, Butão, Quênia, Kosovo, Maldivas, Ilhas Marshall, Peru, Serra Leoa,
Eslováquia, Eslovênia, África do Sul, Essautíni-Suazilândia e Uganda. Segundo o
índice do CCP, somente quatro países possuem as seis características de
independência constitucionalizadas: Argentina, Bulgária, Gambia, Nepal. Em síntese e na perspectiva comparada, a constituição brasileira a) garante maior
independência/autonomia ao Judiciário do que poderes à legislatura nacional, mas b)
a independência e a autonomia judiciais e os poderes/competências do Legislativo
são mais robustos do que os poderes atribuídos ao Executivo. Como foi descrito antes, 1176 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. a maioria esmagadora das garantias do judiciário é estendida constitucionalmente ao
Ministério Público. 2. Relações dos Índices Comparativos de Independência do Judiciário e
das Competências e dos Poderes do Executivo e do Legislativo a Partir
Especialmente do Comparative Constitutions Project – CCP Os dados do CCP não permitem fazer uma correlação (estatística) ou estabelecer uma
causalidade entre a constitucionalização das características das instituições
(poderes). Em outras palavras, no constitutional making, a constitucionalização de
prerrogativas e das competências não é um jogo de soma zero, ainda que a relação
entre poderes, órgãos constitucionais autônomos e instituições estatais decorrentes
possa ser investigada por uma abordagem de teoria dos jogos. Dessa forma, a maior
ou a menor independência judicial constitucionalizada, no Estado nacional (que,
conforme já descrito, no Brasil, é um proxy da independência do Ministério Público)
não está fortemente associada, na perspectiva descritiva sobre esse fenômeno, à
maior ou à menor atribuição de poderes/competências ao Executivo ou à legislatura
nacional de um mesmo país.13 1177 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. 15 Organizado pelos pesquisadores do DIRPOL/PPGD/UFPR com os dados do CCP Índices,
https://comparativeconstitutionsproject.org/ccp-rankings/. 1.
QUESTÕES
PRELIMINARES:
ÍNDICES
COMPARATIVOS
DE
INDEPENDÊNCIA DO JUDICIÁRIO E DAS COMPETÊNCIAS E DOS PODERES DO
EXECUTIVO
E
DO
LEGISLATIVO
A
PARTIR
ESPECIALMENTE
DO
COMPARATIVE CONSTITUTIONS PROJECT – CCP Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. Gráfico 4 – Judicial Independence (média) X Executive/Legislative Power
(classes) – CCP Índices
Fonte: DIRPOL/PPGD/UFPR com os dados do CCP Índices14
0
0,5
1
1,5
2
2,5
3
3,5
4
4,5
0
0,05
0,09
0,14
0,19
0,24
0,29
0,33
0,38
0,43
0,48
0,52
0,58
0,62
Judicial Independece
Legislative Power
Judicial Independence (média)
X Legislative Power
0
0,5
1
1,5
2
2,5
3
3,5
4
0
1
2
3
4
5
6
7
Judicial Independence
Executive Power
Judicial Independence (média)
X Executive Power Gráfico 4 – Judicial Independence (média) X Executive/Legislative Power
(classes) – CCP Índices – Judicial Independence (média) X Executive/Legislative Power
(classes) – CCP Índices 0
0,5
1
1,5
2
2,5
3
3,5
4
0
1
2
3
4
5
6
7
Judicial Independence
Executive Power
Judicial Independence (média)
X Executive Power Fonte: DIRPOL/PPGD/UFPR com os dados do CCP Índices14 Na perspectiva descritiva do fenômeno, apenas se observa uma leve tendência de
constitucionalização maior da Judicial Independence com a constitucionalização de
maior Legislative Power e de menor Executive Power. Ou seja, um conjunto de
poderes e competências maiores ao legislativo, na constituição, é frequentemente
acompanhado de uma maior independência judicial, assim como as competências
mais robustas do poder executivo relacionam-se, frequentemente, com a menor
independência judicial (ver Gráfico 4). Ainda, é possível perceber que: (1) não há uma associação clara entre a
constitucionalização de poderes e a independência judicial e, (2) no Brasil, Judiciário,
Legislativo e Executivo são dotados de índices (medidos pelos indicadores do CCP)
semelhantes na constituição, ao contrário dos demais países (exceção ao México) (ver
Gráfico 5). Por outro lado, Chile, França, Portugal e Espanha constitucionalizam mais 1178 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. 17 MAZZILLI, Hugo Nigro. A independência do Ministério Público. Palestra proferida na Associação do
Ministério Público do Rio de Janeiro em 11.03.1996. transcrita em Juris Síntese Millennium [Cd-rom]:
Síntese, jan 2001). Disponível em: http://www.mazzilli.com.br/pages/artigos/indmp.pdf. Acesso em
11.10.2021. P. 07. 18 CARVALHO, Ernani; LEITAO, Natalia. O Novo desenho institucional do Ministério Publico e o
Processo
de
Judicialização
da
Política
[online].
2010.
Disponível
em:
http://www.scielo.br/pdf/rdgv/v6n2/a03v6n2.pdf. Acesso em 03.11.2021. 19 SADEK, Maria Tereza. A Construção de um novo Ministério Público resolutivo. De jure: Revista Jurídica
do Ministério Público do Estado de Minas Gerais, Belo Horizonte, n. 12, p. 130-139, 2009. 1.
QUESTÕES
PRELIMINARES:
ÍNDICES
COMPARATIVOS
DE
INDEPENDÊNCIA DO JUDICIÁRIO E DAS COMPETÊNCIAS E DOS PODERES DO
EXECUTIVO
E
DO
LEGISLATIVO
A
PARTIR
ESPECIALMENTE
DO
COMPARATIVE CONSTITUTIONS PROJECT – CCP Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. eventualmente da causalidade com as prerrogativas e as competências de outras
instituições, órgãos e poderes estatais. eventualmente da causalidade com as prerrogativas e as competências de outras
instituições, órgãos e poderes estatais. 16 MORAES, Alexandre. Direito Constitucional. 13 ed. São Paulo: Atlas, 2003. p. 402. 1.
QUESTÕES
PRELIMINARES:
ÍNDICES
COMPARATIVOS
DE
INDEPENDÊNCIA DO JUDICIÁRIO E DAS COMPETÊNCIAS E DOS PODERES DO
EXECUTIVO
E
DO
LEGISLATIVO
A
PARTIR
ESPECIALMENTE
DO
COMPARATIVE CONSTITUTIONS PROJECT – CCP Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. poderes ao executivo em comparação com as competências do legislativo e com o
nível e a intensidade da independência do Judiciário. A Argentina fixa substanciais
autonomia e independência ao Judiciário, mas estabelece constitucionalmente um
menor conjunto de competências aos demais poderes estatais. poderes ao executivo em comparação com as competências do legislativo e com o
nível e a intensidade da independência do Judiciário. A Argentina fixa substanciais
autonomia e independência ao Judiciário, mas estabelece constitucionalmente um
menor conjunto de competências aos demais poderes estatais. O Brasil e o México, nas suas constituições nacionais, distribuíram mais
equilibradamente as competências e as garantias mensuradas aos três poderes. Nesses países, os índices do Executivo, Judiciário e Legislativo são mais próximos,
porém em ambos o judiciário é o mais fortemente amparado em prerrogativas e
garantias constitucionais (descritas nos índices produzidos pelo CCP). Gráfico 5 – Judicial Independence X Executive Power X Legislative Power –
amostra CCP
DIRPOL/PPGD/UFPR com os dados do CCP Índices15
0
0,2
0,4
0,6
0,8
1
Argentina
Brazil
Chile
France
Italy
Mexico
Portugal
Spain
Executive Power
Legislative Power
Judicial Independence Gráfico 5 – Judicial Independence X Executive Power X Legislative Power –
amostra CCP
Argentina Argentina Brazil Spain Portugal Chile Mexico France Italy DIRPOL/PPGD/UFPR com os dados do CCP Índices15 Feitas essas considerações sobre os índices e suas relações no Executivo, Legislativo
e Judiciário, a próxima seção ingressa no objeto central do texto, o qual trata do
índice de independência/autonomia do Ministério Público, além da sua associação e 1179 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. 3.
Índice de Independência (Autonomia, de Jure) do Ministério Público Com a promulgação da Constituição Federal de 1988, o Ministério Público passou a
gozar de efetiva autonomia institucional (Título IV - Da Organização dos Poderes -
Capítulo IV – Das Funções Essenciais à Justiça, Seção I – Ministério Público), com
independência funcional e autonomias administrativa e financeira. Alexandre de
Moraes assevera que a instituição é "independente no exercício de suas funções, não
ficando sujeito às ordens de quem quer que seja, somente devendo prestar contas
de seus atos à Constituição, às leis e à sua consciência”16. Por sua vez, Mazzilli17
entende que “não se admite possam Ministério Público e seus agentes fazer
simplesmente o que bem quiserem, sem dar contas à lei e a ninguém”. Carvalho e Leitão18 abordam o novo desenho institucional do Ministério Público,
comentando que, embora essa instituição já tenha adquirido diversas prerrogativas
anteriormente, somente com a atual constituição CF/88 foi estabelecido um papel
relevante e autônomo politicamente no âmbito nacional. Ainda, Sadek19 afirma que o 1180 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. Ministério Público no Brasil, diferentemente dos demais ao redor do mundo, apresenta
um perfil institucional próprio, robusto e autônomo: Gostaria de iniciar o desenvolvimento dessa tese com algumas
comparações. Em primeiro lugar, se considerarmos o panorama
internacional, perceberemos que o Ministério Público brasileiro é
singular. Eu costumo brincar dizendo que, quando as pessoas se
gabam exclamando que a jabuticaba só existe no Brasil, retruco,
observando que o que existe de fato só no Brasil é o nosso
Ministério Público. Podemos encontrar instituições análogas na
América Latina, no mundo Europeu e na América do Norte. Em
nenhum dos países, contudo, vamos nos deparar com um
Ministério
Público
que
apresente
um
perfil
institucional
semelhante ou que ostente igual conjunto de atribuições. 21 SADEK, Maria Tereza. A Construção de um novo Ministério Público resolutivo. De jure: Revista Jurídica
do Ministério Público do Estado de Minas Gerais, Belo Horizonte, n. 12, p. 130-139, 2009. p. 131. 20 SADEK, Maria Tereza. A Construção de um novo Ministério Público resolutivo. De jure: Revista Jurídica
do Ministério Público do Estado de Minas Gerais, Belo Horizonte, n. 12, p. 130-139, 2009. p. 130. SADEK, Maria Tereza. A Construção de um novo Ministério Público resolutivo. De jure: Revista Jurídica
do Ministério Público do Estado de Minas Gerais, Belo Horizonte, n. 12, p. 130-139, 2009. p. 130. ,
ç
j
do Ministério Público do Estado de Minas Gerais, Belo Horizonte, n. 12, p. 130-139, 200 EK, Maria Tereza. A Construção de um novo Ministério Público resolutivo. De jure: Revist 20 SADEK, Maria Tereza. A Construção de u a Tereza. A Construção de um novo Ministério Público resolutivo. De jure: Revista Jurídica
úblico do Estado de Minas Gerais, Belo Horizonte, n. 12, p. 130-139, 2009. p. 130. 3.
Índice de Independência (Autonomia, de Jure) do Ministério Público O
nosso Ministério Público, a partir da Constituição de 1988, passou
a ser uma instituição que tem pouca semelhança com seus
congêneres no exterior e tampouco com o Ministério Público
brasileiro do passado. Eu até diria, ousando uma observação
ainda mais radical, que o nome é o mesmo, mas a instituição
não. Várias características mudaram. Entre essas alterações,
ressalte-se, desde logo, a sua localização institucional e o rol de
suas atribuições (grifos nossos).20 Sadek21 relata que o Ministério Público do Brasil, em relação aos demais países da
América Latina, é muito distinto. Por exemplo, na Costa Rica, Colômbia e Paraguai, o
Ministério Público é dependente do Judiciário e, no México, República Dominicana e
Uruguai, relaciona-se com o Executivo. Em análise comparada, grande parte da literatura sobre a autonomia e independência
do Ministério Público descreve de forma estanque as características do Executivo, do
Legislativo e do Judiciário, assim como do Ministério Público, em seu país, sem
estabelecer indicadores que permitam mensurar diferentes graus de competências,
prerrogativas, independência e autonomia. Dentre a literatura, o estudo comparativo 1181 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. feito por Dias e Azevedo22 analisa algumas diferenças entre o Ministério Público em
Portugal, Espanha, França, Itália, Brasil e Argentina. Na literatura, é possível encontrar tentativas de produzir, independentemente da
mensuração de poderes e garantias de outras instituições e poderes, índices de
independência/autonomia do Ministério Público (Prosecutorial Independence-PI). Um
indicador abrangente (para 78 países) foi construído por Van Aaken, Feld e Voigt23
para mensurar a independência formal (de jure PI) e a independência de facto.24 Para classificar o grau de independência formal do MP (de jure PI) em um indicador,
são sumarizadas 21 variáveis institucionais. Por sua vez, o indicador de independência
factual (de facto PI) é composto por sete variáveis retiradas de surveys sobre a
percepção da independência do MP entre especialistas25. Dessa forma, o objetivo dos
autores é relacionar a Prosecutorial Independence (formal e factual) com o combate
à corrupção (ou a percepção do combate à corrupção obtida por surveys com
especialistas e com a população). 22 DIAS, Paulo J.; AZEVEDO, Rodrigo G. de. O papel do Ministério Público: estudos comparados dos
países latino-americanos. São Paulo: Almedina, 2008. 23 VAN AAKEN, Anne; FELD, Lars P.; VOIGT, Stefan. Do independent prosecutors deter political
corruption? An empirical evaluation across seventy-eight countries. American Law and Economics
Review, v. 12, n. 1, p. 204-244, 2010. 24 Os indicadores de independência formal do MP de Van Aaken, Feld e Voigt (2010) são detalhados e
minuciosamente revistos por Carvalho e Leitão (2019, p. 87 e seg.), com conclusões sobre a importância
de desagregar indicadores internos e externos de independência formal para o estudo do caso brasileiro.
Apesar de interessantes as conclusões de Carvalho e Leitão (2019, p. 108), não é possível incorporar
algumas de suas sugestões de mensuração de independência do MP neste artigo, devido à
indisponibilidade de informações legais e constitucionais dos outros países comparados.
25 3.
Índice de Independência (Autonomia, de Jure) do Ministério Público A conclusão associou positivamente de facto PI e
negativamente de jure PI com a percepção de menores índices de corrupção26, 22 DIAS, Paulo J.; AZEVEDO, Rodrigo G. de. O papel do Ministério Público: estudos comparados dos
países latino-americanos. São Paulo: Almedina, 2008. 24 Os indicadores de independência formal do MP de Van Aaken, Feld e Voigt (2010) são detalhados e
minuciosamente revistos por Carvalho e Leitão (2019, p. 87 e seg.), com conclusões sobre a importância
de desagregar indicadores internos e externos de independência formal para o estudo do caso brasileiro. Apesar de interessantes as conclusões de Carvalho e Leitão (2019, p. 108), não é possível incorporar
algumas de suas sugestões de mensuração de independência do MP neste artigo, devido à
indisponibilidade de informações legais e constitucionais dos outros países comparados. 25 VAN AAKEN, Anne; FELD, Lars P.; VOIGT, Stefan. Do independent prosecutors deter political
corruption? An empirical evaluation across seventy-eight countries. American Law and Economics
Review, v. 12, n. 1, p. 204-244, 2010. p. 213-218. 26 De acordo com Van Aaken, Feld e Voigt (2010, p. 205): “The indicators for de jure and de facto PI are
available for seventy-eight and seventy-six countries, respectively. De facto PI turns out to be highly
and robustly significant for explaining variation in perceived corruption: the more independent the
prosecutors factually are, the lower the expected level of corruption, c.p. De jure PI has an unexpected
sign: more independence is correlated with more corruption. But this variable never reaches conventional
levels of significance”. 1182 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. existindo a percepção social de que a corrupção é menor nos países com alto índice
de PI de facto. Nos países com um robusto índice PI de jure, observa-se uma
percepção mais elevada acerca da corrupção. 28 GUTMANNN, Jerg; VOIGT, Stefan: The Independence of Prosecutors and Government Accountability.
ILE Working Paper Series, No. 8, University of Hamburg, Institute of Law and Economics (ILE),
Hamburg, 2017. p. 4. 29 Isto fica claro observando como os autores descrevem o indicador, que foi obtido a partir de dados de
99 países: “The two measures proposed here make [prosecutorial independence] and PA [prosecutorial 27 VAN AAKEN, Anne; FELD, Lars P.; VOIGT, Stefan. Do independent prosecutors deter political
corruption? An empirical evaluation across seventy-eight countries. American Law and Economics
Review, v. 12, n. 1, p. 204-244, 2010. p. 220. accountability] comparable across countries. Each measure is based on one specific question in the WJP’s
general population survey. The questions aim at identifying to what extent the “lack of independence of
prosecutors” or the “corruption of prosecutors” constitute a problem for the criminal investigation system
in the city in which the respondent lives. To indicate the significance of the problem, the respondent can
choose an integer value between 1 and 10 from a scale. The WJP then aggregates all responses at the
country level, such that 0 indicates the absence of PI or a very high level of PA, whereas 1 indicates a
very high level of PI or the absence of PA. In other words, higher values on WJP-indicators reflect a
better quality of governance. All indicator values for 99 countries are documented in Appendix A”
(GUTMANN e VOIGT, 2017, p. 4).
30 31 GUTMANNN, Jerg; VOIGT, Stefan: The Independence of Prosecutors and Government Accountability.
ILE Working Paper Series, No. 8, University of Hamburg, Institute of Law and Economics (ILE),
Hamburg, 2017. 30 GUTMANNN, Jerg; VOIGT, Stefan: The Independence of Prosecutors and Government Accountability.
ILE Working Paper Series, No. 8, University of Hamburg, Institute of Law and Economics (ILE),
Hamburg, 2017. 3.
Índice de Independência (Autonomia, de Jure) do Ministério Público Apesar de relevante, entendemos que esses índices teriam pouca utilidade para os
objetivos do presente estudo, pois não é possível replicar claramente os indicadores
institucionais para mensurar a independência e a autonomia do MP e, sobretudo,
porque o combate à corrupção como efeito da Prosecutorial Independence (logo, a
seleção dos indicadores de jure e de facto associados a esse efeito) não é o foco de
deste texto e desta investigação. Além disso, a informação de independência do MP
extraída dos surveys (de facto) e direcionada à percepção do combate à corrupção
restringe a possibilidade de mensurar a liberdade de atuação do MP em outras áreas,
inclusive em outras questões da esfera política e na execução das políticas públicas. Por fim, Van Aaken, Feld e Voigt27 verificam que os dois indicadores não possuem
uma correlação positiva (de facto PI e de jure PI): a presença de mais garantias
institucionais (constitucionais e legais) do MP em um país não se relaciona com a
maior percepção de especialistas e da população sobre a independência do MP. Outro índice de Prosecutorial Independence (de facto PI) presente na literatura é o
proposto por Gutmann e Voigt28, sendo construídos indicadores também com base
em surveys como do World Justice Project - annual rule of law index, o qual é
composto por oito categorias como ausência de corrupção e justiça criminal. Da
mesma forma, o índice de facto desses autores tem objetivos que distanciam da
possibilidade de sua utilização neste texto29. 1183 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. 3.
Índice de Independência (Autonomia, de Jure) do Ministério Público Partiremos desses dados descritivos para construir a nossa amostra e o índice de
autonomia/independência do MP que chamaremos de: ÍNDICE de Autonomia MP
(CEPEJ/DIRPOL). Essa escolha metodológica limita, neste momento, a comparação
do Brasil com países europeus, o que pretendemos complementar no futuro pela
compilação de dados institucionais de uma amostra ampliada com países da América
Latina. Com base nos dados descritivos do CEPEJ33 para 47 países europeus, construímos um
índice de autonomia/independência do MP [CEPEJ/DIRPOL] que varia de 0-1
(conforme o descrito no Quadro A1 e Tabela A1, no Anexo), contendo os seguintes
indicadores: (1) independência institucional, jurídica e legal do Ministério Público; (2)
regulamento para evitar instruções específicas para processar ou não, dirigido a um
procurador em um tribunal; (3) vitaliciedade/estabilidade dos membros do Ministério
Público e; (4) o papel do Ministério Público (em 14 itens distintos de sua atuação
funcional). Aos dados dos países europeus, extraídos diretamente dos relatórios e das
bases de dados do CEPEJ, foram acrescidas as características institucionais (previstas
em lei e na constituição) específicas e relativas ao Brasil para realizar a comparação. O CEPEJ verifica, através dos itens (1) e (2) do nosso índice, as autonomias
institucional, administrativa, jurídica e legal do Ministério Público, isto é, se órgão e
seus membros são legalmente independentes e se estão subordinados ao Ministério
da Justiça ou a outra autoridade centralizada. O princípio da independência funcional
é avaliado perante o Executivo, Legislativo e sobre as demais autoridades externas
ao MP. Outra questão envolvida na autonomia do MP é a existência de lei ou
regulamento que impede outras autoridades (executivas ou legislativas) de dirigir
instruções específicas a um membro do MP para processar ou não processar um Com base nos dados descritivos do CEPEJ33 para 47 países europeus, construímos um
índice de autonomia/independência do MP [CEPEJ/DIRPOL] que varia de 0-1
(conforme o descrito no Quadro A1 e Tabela A1, no Anexo), contendo os seguintes
indicadores: (1) independência institucional, jurídica e legal do Ministério Público; (2)
regulamento para evitar instruções específicas para processar ou não, dirigido a um
procurador em um tribunal; (3) vitaliciedade/estabilidade dos membros do Ministério
Público e; (4) o papel do Ministério Público (em 14 itens distintos de sua atuação
funcional). 33 CEPEJ. The European Commission for the Efficiency of Justice. European judicial systems -
Efficiency and quality of justice. CEPEJ Studies No. 23, Edition 2016 (2014 data), 2016. 3.
Índice de Independência (Autonomia, de Jure) do Ministério Público Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. Dos 78 países analisados por Gutmann e Voigt30, apenas 14 possuem indicadores de
independência formal do MP (PI, de jure) maiores do que os do Brasil. Como
descreveremos em seguida, apesar de a mensuração da autonomia e da
independência institucional do MP que desenvolveremos neste artigo ser distinta (no
cálculo, na quantidade de países e na abrangência de indicadores institucionais) do
índice de Prosecutorial Independence (de jure PI) proposto por Gutmann e Voigt31,
há uma relativa convergência na atribuição de independência formal comparada do
MP no Brasil: o texto de Gutmann e Voigt e o nosso demonstram a elevada
independência do Ministério Público no Brasil. Como
já
informado,
o
que
pretendemos
é
desenvolver
um
índice
de
Independência/Autonomia do MP com base em prerrogativas institucionais (de jure),
dado que a percepção de independência do MP (de facto) extraída de surveys com
especialistas ou com a população em geral não seria satisfatório para o nosso
objetivo. A maior dificuldade metodológica é o levantamento e a compilação da
legislação infraconstitucional de cada país. Nesse sentido, a base de dados mais
completa e abrangente, além de ter como fonte uma instituição pública relevante,
sobre garantias e prerrogativas institucionais (constituição e legislação) atribuída ao
Ministério Público (ou congêneres), é organizada pelo CEPEJ (The European 1184 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. Commission for the Efficiency of Justice), órgão oficial da Comunidade Europeia32. 32 CEPEJ. The European Commission for the Efficiency of Justice. European judicial systems -
Efficiency and quality of justice. CEPEJ Studies No. 23, Edition 2016 (2014 data), 2016. 3.
Índice de Independência (Autonomia, de Jure) do Ministério Público Aos dados dos países europeus, extraídos diretamente dos relatórios e das
bases de dados do CEPEJ, foram acrescidas as características institucionais (previstas
em lei e na constituição) específicas e relativas ao Brasil para realizar a comparação. O CEPEJ verifica, através dos itens (1) e (2) do nosso índice, as autonomias
institucional, administrativa, jurídica e legal do Ministério Público, isto é, se órgão e
seus membros são legalmente independentes e se estão subordinados ao Ministério
da Justiça ou a outra autoridade centralizada. O princípio da independência funcional
é avaliado perante o Executivo, Legislativo e sobre as demais autoridades externas
ao MP. Outra questão envolvida na autonomia do MP é a existência de lei ou
regulamento que impede outras autoridades (executivas ou legislativas) de dirigir
instruções específicas a um membro do MP para processar ou não processar um 1185 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. investigado. No relatório do CEPEJ34, existem preocupações teóricas e práticas que
diferenciam a independência do Judiciário da autonomia do MP em um Estado de
Direito (rule of law): investigado. No relatório do CEPEJ34, existem preocupações teóricas e práticas que
diferenciam a independência do Judiciário da autonomia do MP em um Estado de
Direito (rule of law): No Estado de Direito, os juízes são independentes dos poderes
executivo e legislativo. A situação é mais complexa em relação
aos promotores públicos, cujo status difere significativamente
entre os Estados. No entanto, esta afirmação também deve ser
qualificada porque em alguns Estados, a independência do
Ministério Público em relação ao poder político pode ser
confirmada no nível estatutário/legal, mas não corresponde à
realidade à luz da tradição histórica de dependência do Ministério
Público. 35 Tradução livre do seguinte trecho: “In a state governed by the rule of law, judges are independent
from the executive and legislative power. The situation is more complex regarding public prosecutors,
whose status differs significantly across states. However this statement must also be qualified because
in some states, the independence of the public prosecution from the political power may be confirmed
at the statutory level, but does not correspond to reality in the light of the historical tradition of public
prosecutors’ dependency. In other states, on the contrary, independence is not recognized in legal acts,
but the tradition and daily practice demonstrate a real de facto independence” CEPEJ. The European
Commission for the Efficiency of Justice. European judicial systems - Efficiency and quality of
justice. CEPEJ Studies No. 23, Edition 2016 (2014 data), 2016.. 34 CEPEJ. The European Commission for the Efficiency of Justice. European judicial systems -
Efficiency and quality of justice. CEPEJ Studies No. 23, Edition 2016 (2014 data), 2016. 36 Tradução livre de: “The institutional context of the prosecution service and particularly its relations
with the executive vary according to the State or entity. However, the principle of functional
independence of prosecutors is emerging as an essential guarantee which has become a true European
standard. This independence is assessed vis-à-vis the executive, the legislative, but also all other
external authorities (…), as well as in terms of the organization model of the public prosecution service
(internal independence)” CEPEJ. The European Commission for the Efficiency of Justice. European
judicial systems - Efficiency and quality of justice. CEPEJ Studies No. 23, Edition 2016 (2014 data),
2016. 3.
Índice de Independência (Autonomia, de Jure) do Ministério Público Em outros Estados, ao contrário, a independência não é
reconhecida na legislação, mas a tradição e a prática cotidiana
demonstram uma independência real de fato.35 No caso do Brasil, a instituição é independente dos demais poderes e autoridades
externas. Nesse sentido, por exemplo, compete a Conselho Nacional do Ministério
Público o controle “da atuação administrativa e financeira do Ministério Público e do
cumprimento dos deveres funcionais de seus membros”, segundo o artigo 130-A, §
2º, CF/88. De outro lado, trata-se de um órgão administrativo do próprio Ministério
Público, sendo 8 dos seus 14 membros originários dos próprios quadros do Ministério
Público da União e dos Estados e Distrito Federal. A independência do MP vem se tornando um padrão na Europa, com a legislação
nacional sendo alterada. Podemos entender melhor essa questão com o item do 34 CEPEJ. The European Commission for the Efficiency of Justice. European judicial systems -
Efficiency and quality of justice. CEPEJ Studies No. 23, Edition 2016 (2014 data), 2016. 1186 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. relatório do CEPEJ sobre a regulamentação para prevenir instruções específicas para
processar (ou não) dirigida a um procurador de justiça em um tribunal: relatório do CEPEJ sobre a regulamentação para prevenir instruções específicas para
processar (ou não) dirigida a um procurador de justiça em um tribunal: O contexto institucional do Ministério Público e, em particular, as
suas relações com o Executivo variam consoante o Estado ou
entidade. No entanto, o princípio da independência funcional dos
procuradores está a emergir como uma garantia essencial que
se tornou uma verdadeira norma europeia. Essa independência
é avaliada face ao executivo, ao legislativo, mas também a todas
as outras autoridades externas (...), bem como em termos do
modelo de organização do Ministério Público (independência
interna)36. 37 Tradução livre de: “After a probationary period, which is usually aimed at training, European public
prosecutors overwhelmingly perform their functions until the age of retirement, which is an important
guarantee of continuity, enabling functional autonomy and facilitating independence” CEPEJ. The
European Commission for the Efficiency of Justice. European judicial systems - Efficiency and
quality of justice. CEPEJ Studies No. 23, Edition 2016 (2014 data), 2016.. 3.
Índice de Independência (Autonomia, de Jure) do Ministério Público Outro critério presente e enfatizado no relatório do CEPEJ é relativo à
vitaliciedade/estabilidade (nomeação e duração do mandato) dos membros do
Ministério Público [item (3), do nosso índice], restando clara a prescrição de que a
independência depende da garantia da permanência no cargo e na carreira: Após um período probatório, que normalmente é destinado à
formação,
os
procuradores
europeus
desempenham
predominantemente as suas funções até a aposentadoria por
idade, o que é uma importante garantia de continuidade,
permitindo autonomia funcional e facilitando a independência37. Após um período probatório, que normalmente é destinado à
formação,
os
procuradores
europeus
desempenham
predominantemente as suas funções até a aposentadoria por
idade, o que é uma importante garantia de continuidade,
permitindo autonomia funcional e facilitando a independência37. No Brasil, conforme já descrito, existem constitucionalmente as garantias da
vitaliciedade, da inamovibilidade e da irredutibilidade de salários, vencimentos e
subsídios, de acordo com o artigo 128, § 5º, I, CF/88. 1187 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. O CEPEJ também reflete e analisa o papel e as atribuições dos promotores e
procuradores em 14 itens distintos de atuação, competência e independência
funcional [conforme o descrito no item (4) do Quadro A1, no Anexo]. Um desses
papéis é a competência para encerrar ou arquivar um caso, impondo ou negociando
a penalidade ou medida, sem requerer uma decisão judicial. Dos 46 países europeus,
24 fixam tal competência ao promotor38. 38 CEPEJ. The European Commission for the Efficiency of Justice. European judicial systems -
Efficiency and quality of justice. CEPEJ Studies No. 23, Edition 2016 (2014 data), 2016.
39 “Art. 17-B. O Ministério Público poderá, conforme as circunstâncias do caso concreto, celebrar acordo
de não persecução civil, desde que dele advenham, ao menos, os seguintes resultados:(Incluído pela Lei
nº 14.230, de 2021) I - o integral ressarcimento do dano; (Incluído pela Lei nº 14.230, de 2021) II - a
reversão à pessoa jurídica lesada da vantagem indevida obtida, ainda que oriunda de agentes privados.
(Incluído pela Lei nº 14.230, de 2021) § 1º A celebração do acordo a que se refere o caput deste artigo
dependerá, cumulativamente: (Incluído pela Lei nº 14.230, de 2021) I - da oitiva do ente federativo
lesado, em momento anterior ou posterior à propositura da ação; (Incluído pela Lei nº 14.230, de 2021)
II - de aprovação, no prazo de até 60 (sessenta) dias, pelo órgão do Ministério Público competente para
apreciar as promoções de arquivamento de inquéritos civis, se anterior ao ajuizamento da
ação; (Incluído pela Lei nº 14.230, de 2021) III - de homologação judicial, independentemente de o
acordo ocorrer antes ou depois do ajuizamento da ação de improbidade administrativa”.
40 “§ 2º O disposto no caput deste artigo não se aplica nas seguintes hipóteses: I - se for cabível
transação penal de competência dos Juizados Especiais Criminais, nos termos da lei; II - se o investigado
for reincidente ou se houver elementos probatórios que indiquem conduta criminal habitual, reiterada ou profissional, exceto se insignificantes as infrações penais pretéritas; III - ter sido o agente beneficiado
nos 5 (cinco) anos anteriores ao cometimento da infração, em acordo de não persecução penal,
transação penal ou suspensão condicional do processo; e IV - nos crimes praticados no âmbito de
violência doméstica ou familiar, ou praticados contra a mulher por razões da condição de sexo feminino,
em favor do agressor. § 3º O acordo de não persecução penal será formalizado por escrito e será firmado
pelo membro do Ministério Público, pelo investigado e por seu defensor. § 4º Para a homologação do
acordo de não persecução penal, será realizada audiência na qual o juiz deverá verificar a sua
voluntariedade, por meio da oitiva do investigado na presença do seu defensor, e sua legalidade. § 5º
Se o juiz considerar inadequadas, insuficientes ou abusivas as condições dispostas no acordo de não
persecução penal, devolverá os autos ao Ministério Público para que seja reformulada a proposta de
acordo, com concordância do investigado e seu defensor. § 6º Homologado judicialmente o acordo de
não persecução penal, o juiz devolverá os autos ao Ministério Público para que inicie sua execução
perante o juízo de execução penal. § 7º O juiz poderá recusar homologação à proposta que não atender
aos requisitos legais ou quando não for realizada a adequação a que se refere o § 5º deste artigo. § 8º
Recusada a homologação, o juiz devolverá os autos ao Ministério Público para a análise da necessidade
de complementação das investigações ou o oferecimento da denúncia”. 3.
Índice de Independência (Autonomia, de Jure) do Ministério Público No caso brasileiro, por exemplo, compete
ao Ministério Público “promover o inquérito civil e a ação civil pública, para a proteção
do patrimônio público e social, do meio ambiente e de outros interesses difusos e
coletivos”, nos termos do artigo 129, III, CF/88. A Lei nº. 7.347/1985, que disciplina
as ações civis públicas, permite que “órgãos públicos legitimados poderão tomar dos
interessados compromisso de ajustamento de sua conduta às exigências legais,
mediante cominações, que terá eficácia de título executivo extrajudicial”, segundo o
artigo 5º, §6º, desse diploma legal. De outro lado, na transação das ações de
improbidade administrativa, o Ministério Público pode celebrar acordo, porém
necessita de homologação judicial, conforme o artigo 17-B, § 1º, Lei Federal nº. 8.429/1992 com alteração da Lei nº. 14.230/202139. Também no acordo de não
persecução penal, nos termos do artigo do Código de Processo Penal, que foi alterado
pela Lei Federal nº. 13.964/2019, o Ministério Público, o investigado e seu defensor
formalizam o acordo, o qual deve ser homologado judicial, de acordo com o artigo
28-A, §§ 2º a 8º40. 1188 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. 3.
Índice de Independência (Autonomia, de Jure) do Ministério Público O Ministério Público no Brasil possui as seguintes prerrogativas e competências
descritas pelo CEPEJ41 para a independência e a autonomia da instituição, como: a)
poder conduzir ou supervisionar a investigação policial, b) realizar investigações,
quando necessário, c) solicitar medidas de investigação ao juiz, d) acusar e propor
ações judiciais, e) formular pedidos que devem ser julgados por sentença judicial, f)
apelar e recorrer, g) supervisionar e fiscalizar o processo de execução e h) outros
poderes significativos como funções em casos civis, administrativos e de insolvência,
assim como a vítima pode contestar uma decisão do promotor público, conforme se
observa especialmente nas funções institucionais constitucionais previstas no artigo
129, I a IX, CF/8842 e na legislação especial como a Lei Federal nº. 7.210/198443, a O Ministério Público no Brasil possui as seguintes prerrogativas e competências
descritas pelo CEPEJ41 para a independência e a autonomia da instituição, como: a)
poder conduzir ou supervisionar a investigação policial, b) realizar investigações,
quando necessário, c) solicitar medidas de investigação ao juiz, d) acusar e propor
ações judiciais, e) formular pedidos que devem ser julgados por sentença judicial, f)
apelar e recorrer, g) supervisionar e fiscalizar o processo de execução e h) outros
poderes significativos como funções em casos civis, administrativos e de insolvência,
assim como a vítima pode contestar uma decisão do promotor público, conforme se
observa especialmente nas funções institucionais constitucionais previstas no artigo
129, I a IX, CF/8842 e na legislação especial como a Lei Federal nº. 7.210/198443, a ou profissional, exceto se insignificantes as infrações penais pretéritas; III - ter sido o agente beneficiado
nos 5 (cinco) anos anteriores ao cometimento da infração, em acordo de não persecução penal,
transação penal ou suspensão condicional do processo; e IV - nos crimes praticados no âmbito de
violência doméstica ou familiar, ou praticados contra a mulher por razões da condição de sexo feminino,
em favor do agressor. § 3º O acordo de não persecução penal será formalizado por escrito e será firmado
pelo membro do Ministério Público, pelo investigado e por seu defensor. § 4º Para a homologação do
acordo de não persecução penal, será realizada audiência na qual o juiz deverá verificar a sua
voluntariedade, por meio da oitiva do investigado na presença do seu defensor, e sua legalidade. 44 “Art. 5o Têm legitimidade para propor a ação principal e a ação cautelar: (Redação dada pela Lei nº
11.448, de 2007) (Vide Lei nº 13.105, de 2015) (Vigência) I - o Ministério Público”. 44 “Art. 5o Têm legitimidade para propor a ação principal e a ação cautelar: (Redação dada pela Lei nº
11.448, de 2007) (Vide Lei nº 13.105, de 2015) (Vigência) I - o Ministério Público”.
45 “Art. 81. A defesa dos interesses e direitos dos consumidores e das vítimas poderá ser exercida em
juízo individualmente, ou a título coletivo. Parágrafo único. A defesa coletiva será exercida quando se
tratar de: I - interesses ou direitos difusos, assim entendidos, para efeitos deste código, os
transindividuais, de natureza indivisível, de que sejam titulares pessoas indeterminadas e ligadas por
circunstâncias de fato; II - interesses ou direitos coletivos, assim entendidos, para efeitos deste
código, os transindividuais, de natureza indivisível de que seja titular grupo, categoria ou classe de
pessoas ligadas entre si ou com a parte contrária por uma relação jurídica base; III - interesses
ou direitos individuais homogêneos, assim entendidos os decorrentes de origem comum. Art. 82.
Para os fins do art. 81, parágrafo único, são legitimados concorrentemente: (Redação dada pela Lei nº
9.008, de 21.3.1995) I - o Ministério Público”.
46 Por exemplo, a lei de recuperação judicial, de recuperação extrajudicial e falência faz menção 25 vezes
ao Ministério Público.
47 “Ex positis, concedo a medida cautelar requerida para suspender a eficácia do artigo 310, §4°, do
Código de Processo Penal (CPP), na redação introduzida pela Lei n° 13.964/2019. Conclusão Ex positis,
na condição de relator das ADIs 6.298, 6.299, 6.300 e 6305, com as vênias de praxe e pelos motivos
expostos: (a) Revogo a decisão monocrática constante das ADIs 6.298, 6.299, 6.300 e suspendo sine
die a eficácia, ad referendum do Plenário, (a1) da implantação do juiz das garantias e seus consectários
(Artigos 3º-A, 3º-B, 3º-C, 3º-D, 3ª-E, 3º-F, do Código de Processo Penal); e (a2) da alteração do juiz
sentenciante que conheceu de prova declarada inadmissível (157, §5º, do Código de Processo Penal);
(b) Concedo a medida cautelar requerida nos autos da ADI 6305, e suspendo sine die a eficácia, ad
referendum do Plenário, (b1) da alteração do procedimento de arquivamento do inquérito policial (28,
caput, Código de Processo Penal); (b2) Da liberalização da prisão pela não realização da audiência de
custodia no prazo de 24 horas (Artigo 310, §4°, do Código de Processo Penal); Nos termos do artigo 10, 47 “Ex positis, concedo a medida cautelar requerida para suspender a eficácia do artigo 310, §4°, do
Código de Processo Penal (CPP), na redação introduzida pela Lei n° 13.964/2019. Conclusão Ex positis,
na condição de relator das ADIs 6.298, 6.299, 6.300 e 6305, com as vênias de praxe e pelos motivos
expostos: (a) Revogo a decisão monocrática constante das ADIs 6.298, 6.299, 6.300 e suspendo sine
die a eficácia, ad referendum do Plenário, (a1) da implantação do juiz das garantias e seus consectários
(Artigos 3º-A, 3º-B, 3º-C, 3º-D, 3ª-E, 3º-F, do Código de Processo Penal); e (a2) da alteração do juiz
sentenciante que conheceu de prova declarada inadmissível (157, §5º, do Código de Processo Penal);
(b) Concedo a medida cautelar requerida nos autos da ADI 6305, e suspendo sine die a eficácia, ad
referendum do Plenário, (b1) da alteração do procedimento de arquivamento do inquérito policial (28,
caput, Código de Processo Penal); (b2) Da liberalização da prisão pela não realização da audiência de
custodia no prazo de 24 horas (Artigo 310, §4°, do Código de Processo Penal); Nos termos do artigo 10, 3.
Índice de Independência (Autonomia, de Jure) do Ministério Público Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. Lei Federal nº. 7.347/198544, a Lei Federal nº. 8.078/199045 e a Lei 11.101/200546,
além de diversas outras leis especiais que estabelecem funções, competências,
prerrogativas e poderes ao Ministério Público. De outro lado, as prerrogativas de i) descontinuar um caso sem precisar de uma
decisão de um juiz e de j) encerrar o caso impondo ou negociando uma sanção ou
medida sem requerer uma decisão judicial são mitigadas na legislação brasileira, pois,
conforme
demonstrado
acima,
a
legislação
brasileira
prevê
em
diversas
oportunidades a necessidade de homologação judicial de acordos. Acerca da
descontinuação de um caso sem necessidade de decisão judicial, a Constituição
Federal brasileira de 1988, no âmbito da ação penal pública, teve “o condão de
extinguir o procedimento ex officio, que podia ser instaurado e julgado pelo órgão
jurisdicional, bem como com o procedimento judicialiforme, que era deflagrado por
portaria de instauração da autoridade policial” (NASCIMENTO, 2017). Por sua vez, de
acordo com o artigo 28, do Código de Processo Penal, que ainda se encontra vigente
em razão da ADI nº. 630547, “Se o órgão do Ministério Público, ao invés de apresentar ao Ministério Público. 47 “Ex positis, concedo a medida cautelar requerida para suspender a eficácia do artigo 310, §4°, do
Código de Processo Penal (CPP), na redação introduzida pela Lei n° 13.964/2019. ,
)
(
,
)
(
g
)
45 “Art. 81. A defesa dos interesses e direitos dos consumidores e das vítimas poderá ser exercida em
juízo individualmente, ou a título coletivo. Parágrafo único. A defesa coletiva será exercida quando se
tratar de: I - interesses ou direitos difusos, assim entendidos, para efeitos deste código, os
transindividuais, de natureza indivisível, de que sejam titulares pessoas indeterminadas e ligadas por
circunstâncias de fato; II - interesses ou direitos coletivos, assim entendidos, para efeitos deste
código, os transindividuais, de natureza indivisível de que seja titular grupo, categoria ou classe de
pessoas ligadas entre si ou com a parte contrária por uma relação jurídica base; III - interesses
ou direitos individuais homogêneos, assim entendidos os decorrentes de origem comum. Art. 82.
Para os fins do art 81 parágrafo único são legitimados concorrentemente: (Redação dada pela Lei nº 3.
Índice de Independência (Autonomia, de Jure) do Ministério Público § 5º
Se o juiz considerar inadequadas, insuficientes ou abusivas as condições dispostas no acordo de não
persecução penal, devolverá os autos ao Ministério Público para que seja reformulada a proposta de
acordo, com concordância do investigado e seu defensor. § 6º Homologado judicialmente o acordo de
não persecução penal, o juiz devolverá os autos ao Ministério Público para que inicie sua execução
perante o juízo de execução penal. § 7º O juiz poderá recusar homologação à proposta que não atender
aos requisitos legais ou quando não for realizada a adequação a que se refere o § 5º deste artigo. § 8º
Recusada a homologação, o juiz devolverá os autos ao Ministério Público para a análise da necessidade
de complementação das investigações ou o oferecimento da denúncia”. 41 41 CEPEJ. The European Commission for the Efficiency of Justice. European judicial systems
- Efficiency and quality of justice. CEPEJ Studies No. 23, Edition 2016 (2014 data), 2016. p. 124
42 “I - promover, privativamente, a ação penal pública, na forma da lei; II - zelar pelo efetivo respeito
dos Poderes Públicos e dos serviços de relevância pública aos direitos assegurados nesta Constituição,
promovendo as medidas necessárias a sua garantia; III - promover o inquérito civil e a ação civil pública,
para a proteção do patrimônio público e social, do meio ambiente e de outros interesses difusos e
coletivos; IV - promover a ação de inconstitucionalidade ou representação para fins de intervenção da
União e dos Estados, nos casos previstos nesta Constituição; V - defender judicialmente os direitos e
interesses das populações indígenas; VI - expedir notificações nos procedimentos administrativos de sua
competência, requisitando informações e documentos para instruí-los, na forma da lei complementar
respectiva; VII - exercer o controle externo da atividade policial, na forma da lei complementar
mencionada no artigo anterior; VIII - requisitar diligências investigatórias e a instauração de inquérito
policial, indicados os fundamentos jurídicos de suas manifestações processuais; IX - exercer outras
funções que lhe forem conferidas, desde que compatíveis com sua finalidade, sendo-lhe vedada a
representação judicial e a consultoria jurídica de entidades públicas”. 43
é
ú
á 43 “Art. 67. O Ministério Público fiscalizará a execução da pena e da medida de seguranç
processo executivo e nos incidentes da execução”. 1189 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. 3.
Índice de Independência (Autonomia, de Jure) do Ministério Público Conclusão Ex positis,
na condição de relator das ADIs 6.298, 6.299, 6.300 e 6305, com as vênias de praxe e pelos motivos
expostos: (a) Revogo a decisão monocrática constante das ADIs 6.298, 6.299, 6.300 e suspendo sine
die a eficácia, ad referendum do Plenário, (a1) da implantação do juiz das garantias e seus consectários
(Artigos 3º-A, 3º-B, 3º-C, 3º-D, 3ª-E, 3º-F, do Código de Processo Penal); e (a2) da alteração do juiz
sentenciante que conheceu de prova declarada inadmissível (157, §5º, do Código de Processo Penal);
(b) Concedo a medida cautelar requerida nos autos da ADI 6305, e suspendo sine die a eficácia, ad
referendum do Plenário, (b1) da alteração do procedimento de arquivamento do inquérito policial (28,
caput, Código de Processo Penal); (b2) Da liberalização da prisão pela não realização da audiência de
custodia no prazo de 24 horas (Artigo 310, §4°, do Código de Processo Penal); Nos termos do artigo 10, 1190 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. a denúncia, requerer o arquivamento do inquérito policial ou de quaisquer peças de
informação, o juiz, no caso de considerar improcedentes as razões invocadas, fará
remessa do inquérito ou peças de informação ao procurador-geral (...)”. a denúncia, requerer o arquivamento do inquérito policial ou de quaisquer peças de
informação, o juiz, no caso de considerar improcedentes as razões invocadas, fará
remessa do inquérito ou peças de informação ao procurador-geral (...)”. §2º, da Lei n. 9868/95, a concessão desta medida cautelar não interfere nem suspende os inquéritos e
os processos em curso na presente data. Aguardem-se as informações já solicitadas aos requeridos, ao
Advogado-Geral da União e ao Procurador-Geral da República. Após, retornem os autos para a análise
dos pedidos de ingresso na lide dos amici curae e a designação oportuna de audiências públicas.
Publique-se. Intimem-se" (STF, ADI nº. 6305, Medida Cautelar, Ministro Luiz Fux, Decisão de
22/01/2020). 48 A pesquisa que gerou a base de dados, adaptados do CEPEJ, e o índice CEPEJ/DIPOL de
autonomia/independência do MP foi realizada pelos pesquisadores do Núcleo de Pesquisa “DIRPOL-
Direito e Política” (PPGD/UFPR), contando com o financiamento do CNPq (Edital 14/2014) e do
PROEX/CAPES. Uma versão inicial da base de dados e indicadores da pesquisa, atualizado e revista neste
artigo, foi utilizada por outros pesquisadores do DIRPOL/PPGD e fez parte das referências presentes na
tese de doutorado de autoria de Jessika T. K Martins (2019), defendida no PPGCP/UFPR. 3.
Índice de Independência (Autonomia, de Jure) do Ministério Público Dessa maneira, o resultado da mensuração dos 47 países europeus e do Brasil, no
índice de independência/autonomia do MP, está expresso no Gráfico 6 (e Tabela A1,
no Anexo).48 Como pode ser visto, o Brasil apresenta quase todas as prerrogativas e
garantias atribuídas ao MP de forma plena e duas prerrogativas e garantias
observadas parcial e mitigadamente. Ou seja, em comparação com os países
europeus e com Israel, a independência e a autonomia do Ministério Público brasileiro
encontram-se no primeiro terço mais elevado do índice. 1167 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas do ministério público brasileiro
em perspectiva comparada: construindo um índice de independência/autonomia. Revista Eletrônica Direito e Política,
Programa de Pós-Graduação Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas do ministério público brasileiro
em perspectiva comparada: construindo um índice de independência/autonomia. Revista Eletrônica Direito e Política,
Programa de Pós-Graduação Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. Gráfico 6 – Índice de Independência/Autonomia do Ministério Público (CEPEJ/DIRPOL) -
Prosecutorial Independence (de jure) – CEPEJ (2016)
Fonte: Dados para o Brasil definidos pelos pesquisadores do Núcleo de Pesquisa DIRPOL/PPGD/UFPR com
base na constituição e legislação. Dados dos países europeus: (CEPEJ, 2016). 0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1,0
Andorra
Suíça
Escócia (UK)
Israel
Malta
Irlanda do…
Azerbaijão
Dinamarca
Rússia
Turquia
Holanda
Mônaco
Alemanha
Belgica
Chipre
Finlândia
Inglaterra (UK)
Suécia
Macedônia
Geórgia
Sérvia
Austria
Moldova
República…
Polônia
Lituânia
França
Hungria
Luxemburgo
Ucrânia
Grécia
România
Irlanda
Itália
Noruega
Armênia
Eslovênia
Estônia
Montenegro
Espanha
Bósnia-…
Brasil
Croácia
Albânia
Bulgária
Letônia
Portugal
Eslováquia
ÍNDICE de Autonomia MP (CEPEJ/DIRPOL) Gráfico 6 – Índice de Independência/Autonomia do Ministério Público (CEPEJ/DIRPOL) -
Prosecutorial Independence (de jure) – CEPEJ (2016)
ÍNDICE de Autonomia MP (CEPEJ/DIRPOL) Fonte: Dados para o Brasil definidos pelos pesquisadores do Núcleo de Pesquisa DIRPOL/PPGD/UFPR com
base na constituição e legislação. Dados dos países europeus: (CEPEJ, 2016). Fonte: Dados para o Brasil definidos pelos pesquisadores do Núcleo de Pesquisa DIRPOL/PPGD/UFPR com
base na constituição e legislação. Dados dos países europeus: (CEPEJ, 2016). 1166 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. 3.
Índice de Independência (Autonomia, de Jure) do Ministério Público Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. Por fim e na Tabela 1, como uma primeira utilização do índice que construímos,
podemos verificar que o agrupamento do índice de independência/autonomia do MP
(CEPEJ), escalonando em classes, posicionaria o Brasil, em comparação com os países
europeus, como um dos que mais atribui garantias e prerrogativas estatutárias ao
Ministério Público. Tabela 1 – Índice de Independência/Autonomia do Ministério Público
(CEPEJ/DIRPOL) – por classes (quintis) - Prosecutorial Independence (de
jure) – CEPEJ (2016) Tabela 1 – Índice de Independência/Autonomia do Ministério Público
(CEPEJ/DIRPOL) – por classes (quintis) - Prosecutorial Independence (de
jure) – CEPEJ (2016)
Autonomia
do MP
(n)
(%)
Países
0,0 a 0,2
3
6,3%
Andorra, Suíça, Escócia (UK)
0,2 a 0,4
3
6,3%
Israel, Malta, Irlanda do Norte (UK)
0,4 a 0,6
8
16,7%
Azerbaijão, Dinamarca, Rússia, Turquia, Holanda,
Mônaco, Alemanha, Belgica
0,6 a 0,8
15
31,3%
Chipre, Finlândia, Inglaterra (UK), Suécia, Macedônia,
Geórgia, Sérvia, Austria, Moldova, República Tcheca,
Polônia, Lituânia, França, Hungria, Luxemburgo
0,8 a 1,0
19
39,6%
Ucrânia, Grécia, România, Irlanda, Itália, Noruega,
Armênia, Eslovênia, Estônia, Montenegro, Espanha,
Bósnia-Herzegovina, Brasil, Croácia, Albânia,
Bulgária, Letônia, Portugal, Eslováquia
Total
48 100,0%
Fonte: Dados para o Brasil definidos pelos pesquisadores do Núcleo de Pesquisa
DIRPOL/PPGD/UFPR com base na constituição e legislação. Dados dos países
europeus: (CEPEJ; 2016). Tabela 1 – Índice de Independência/Autonomia do Ministério Público
(CEPEJ/DIRPOL) – por classes (quintis) - Prosecutorial Independence (de
jure) – CEPEJ (2016) Após a elaboração de índice de (Autonomia, de Jure) do Ministério Público, este texto
apresentará relações com os índices do Comparative Constitucions Project. 3.
Índice de Independência (Autonomia, de Jure) do Ministério Público Após a elaboração de índice de (Autonomia, de Jure) do Ministério Público, este texto
apresentará relações com os índices do Comparative Constitucions Project. 5. RELAÇÕES ENTRE O ÍNDICE DE INDEPENDÊNCIA (AUTONOMIA, DE
JURE) DO MINISTÉRIO PÚBLICO E OS ÍNDICES DO CCP Nos índices do Comparative Constitucions Project (CCP), as competências atribuídas
ao Executivo brasileiro podem ser classificadas como moderadas, com o terceiro ao Executivo brasileiro podem ser classificadas como moderadas, com o terceiro 1166 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. maior índice. Por outro lado, o Legislative Power coloca o Brasil entre o 6% dos países
que
mais
atribui
poder
constitucionalmente
ao
Legislativo. Por
fim,
a
constitucionalização da Judicial Independence brasileira se encontra entre o 10% mais
elevado do conjunto de 190 países comparados. Existe uma tendência de que a constitucionalização de maior independência judicial
reste associada à constitucionalização de maior poder do Legislativo e menor poder
do Executivo, sendo que, no caso do Brasil, os três poderes (Executivo, Judiciário e
Legislativo) têm seus índices semelhantes. No Brasil, os deputados e senadores
constituintes de 1988 fixaram de forma elevada a maioria das prerrogativas
consideradas pelo CCP, atribuindo substancialmente competências e garantias
constitucionalizadas aos poderes estatais. 49 CEPEJ. The European Commission for the Efficiency of Justice. European judicial systems
- Efficiency and quality of justice. CEPEJ Studies No. 23, Edition 2016 (2014 data), 2016. 49 CEPEJ. The European Commission for the Efficiency of Justice. European judicial systems
- Efficiency and quality of justice CEPEJ Studies No 23 Edition 2016 (2014 data) 2016 E quanto ao Ministério Público? Como o Ministério Público brasileiro possui garantias funcionais, institucionais,
orçamentárias quase idênticas àquelas do Judiciário, é lógico afirmar que esses
elementos posicionariam o Ministério Público no Brasil, comparativamente, em estrato
com elevado grau de independência e autonomia. De fato, nos indicadores de
autonomia construídos a partir do CEPEJ49 e do nosso índice CEPEJ/DIRPOL, a
independência/autonomia do MP do Brasil está dimensionada no estrato mais
elevado, na comparação com 46 países europeus e Israel. Frisa-se que o CEPEJ
verifica as garantias de autonomia do Ministério Público, levando em consideração
tanto a legislação infraconstitucional como a legislação constitucional nos países
avaliados, porém, no caso brasileiro, grande parte das garantias e das competências
reside na própria constituição. Em virtude do alto índice de autonomia do Ministério
Público no Brasil, a CF/88 transformou esse órgão em importante ator institucional
com fortes poderes. 1167 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. Correlacionando os resultados do índice do Comparative Constitutional Project e o
índice de Prosecutorial Independence (PI, de jure) (VAN AAKEN; FELD; VOIGT, 2010)
com o índice de Autonomia do MP que construímos (CEPEJ/DIRPOL), podemos
verificar
se
há
alguma
regularidade,
entre
os
países
comparados,
na
institucionalização das prerrogativas e garantias do Ministério Público. 50 O resultado da correlação linear (Pearson) e regressão linear (R2) entre o nosso índice de
autonomia/independência do MP (com base nos dados do CEPEJ, 2016) e os outros índices, expostos no
Gráfico 7, é o seguinte: com legislative power (r = 0,4094; R2 = 0,1676); executive power (r = 0,2749;
R2 = 0,0756); judicial independence (r = 0,2174; R2 = 0,0473); prosecutor independence (r = 0,5828;
R2 = 0,3397). E quanto ao Ministério Público? Quando se
correlaciona o nosso índice de autonomia do Ministério Público com os índices
apresentados anteriormente para os poderes Executivo, Legislativo e Judiciário (CCP),
torna-se possível compreender a relação entre os poderes estatais e esse órgão
constitucionalmente autônomo (Ministério Público), buscando possíveis causalidades
na institucionalização do grau de autonomia do MP nos países comparados. No Gráfico 7, percebe-se que a correlação mais significativa (no conjunto de 47 países
europeus e o Brasil) entre o índice de autonomia/independência do MP proposto e os
índices do CCP encontra-se na associação entre Legislative Power (CCP) e autonomia
do MP (CEPEJ/DIRPOL). Ou seja, há uma correlação positiva e fraca, mas existente,
entre uma maior constitucionalização de Legislative Power e maior institucionalização
de
prerrogativas
ao
MP
na
amostra
analisada. Os
demais
índices
de
constitucionalização das instituições (Executive Power e Judicial Independence, CCP)
não apresentam uma correlação significativa com a autonomia do MP. 50 1168 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. Gráfico 7 – Índice de Independência/Autonomia MP (CEPEJ)/DIRPOL) X
Judicial Independence, Executive/Legislative Power (CCP Índices) e
Prosecutorial Independence (PI, de jure) (VAN AAKEN; FELD; VOIGT, 2010) Gráfico 7 – Índice de Independência/Autonomia MP (CEPEJ)/DIRPOL) X
Judicial Independence, Executive/Legislative Power (CCP Índices) e
Prosecutorial Independence (PI, de jure) (VAN AAKEN; FELD; VOIGT, 2010) p
,
/
g
(
)
Prosecutorial Independence (PI, de jure) (VAN AAKEN; FELD; VOIGT, 2010)
Fonte: Dados para o Brasil definidos pelos pesquisadores do Núcleo de Pesquisa
DIRPOL/PPGD/UFPR com base na constituição e legislação. Dados dos países
europeus: (CEPEJ; 2016). VAN AAKEN; FELD; VOIGT (2010). E quanto ao Ministério Público? R² = 0,1676
0,0
0,2
0,4
0,6
0,8
1,0
0
0,2
0,4
0,6
Índice Autonomia MP (CEPEJ)
Legislative Power (CCP)
Autonomia MP (CEPEJ) x
Legislative Power (CCP)
R² = 0,0756
0,0
0,2
0,4
0,6
0,8
1,0
0
2
4
6
8
Índice Autonomia MP (CEPEJ)
Executive Power (CCP)
Autonomia MP (CEPEJ) x
Executive Power (CCP)
R² = 0,0473
0,0
0,2
0,4
0,6
0,8
1,0
0
2
4
6
Índice Autonomia MP (CEPEJ)
Judicial Independence (CCP)
Autonomia MP (CEPEJ) x
Judicial Independence (CCP)
R² = 0,3397
0,0
0,2
0,4
0,6
0,8
1,0
0
0,2
0,4
0,6
0,8
1
Índice Autonomia MP (CEPEJ)
Prosecutor Independence (PI)
Autonomia MP (CEPEJ) x
Prosecutor Independence (PI) R² = 0,1676
0,0
0,2
0,4
0,6
0,8
1,0
0
0,2
0,4
0,6
Índice Autonomia MP (CEPEJ)
Legislative Power (CCP)
Autonomia MP (CEPEJ) x
Legislative Power (CCP) R² = 0,0756
0,0
0,2
0,4
0,6
0,8
1,0
0
2
4
6
8
Índice Autonomia MP (CEPEJ)
Executive Power (CCP)
Autonomia MP (CEPEJ) x
Executive Power (CCP) Autonomia MP (CEPEJ) x
Executive Power (CCP) Executive Power (CCP) R² = 0,0473
0,0
0,2
0,4
0,6
0,8
1,0
0
2
4
6
Índice Autonomia MP (CEPEJ)
Judicial Independence (CCP)
Autonomia MP (CEPEJ) x
Judicial Independence (CCP) R² = 0,3397
0,0
0,2
0,4
0,6
0,8
1,0
0
0,2
0,4
0,6
0,8
1
Índice Autonomia MP (CEPEJ)
Prosecutor Independence (PI)
Autonomia MP (CEPEJ) x
Prosecutor Independence (PI) Autonomia MP (CEPEJ) x
Judicial Independence (CCP) Autonomia MP (CEPEJ) x
Prosecutor Independence (PI) Prosecutor Independence (PI) Fonte: Dados para o Brasil definidos pelos pesquisadores do Núcleo de Pesquisa
DIRPOL/PPGD/UFPR com base na constituição e legislação. Dados dos países
europeus: (CEPEJ; 2016). VAN AAKEN; FELD; VOIGT (2010). 1169 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. 51 VAN AAKEN, Anne; FELD, Lars P.; VOIGT, Stefan. Do independent prosecutors deter political
corruption? An empirical evaluation across seventy-eight countries. American Law and Economics
Review, v. 12, n. 1, p. 204-244, 2010. E quanto ao Ministério Público? 1 –Índice de Autonomia MP (CEPEJ/DIRPOL) X Legislative Powe
P Índices) (por tercis: País; Autonomia MP; Legislative Power) Quadro 1 –Índice de Autonomia MP (CEPEJ/DIRPOL) X Legislative Power
(CCP Índices) (por tercis: País; Autonomia MP; Legislative Power)
Legislative Power (CCP Índices)
1º Tercil
2º Tercil
3º Tercil
Autonomia do MP (de jure, CEPEJ/DIRPOL)
3º Tercil
Portugal; 0,982; 0,29
Bósnia-Herzegovina;
0,964; 0,24
Armênia; 0,929; 0,29
Itália; 0,893; 0,24
Eslováquia; 0,982; 0,33
Letônia; 0,982; 0,38
Estônia; 0,946; 0,38
Espanha; 0,946; 0,38
Noruega; 0,911; 0,33
Albânia; 0,982; 0,43
Bulgária; 0,982; 0,52
Croácia; 0,964; 0,43
Brasil; 0,964; 0,48
Montenegro; 0,946;
0,43
Eslovênia; 0,929; 0,52
2º Tercil
Grécia; 0,839; 0,24
França; 0,750; 0,24
Irlanda; 0,875; 0,38
Hungria; 0,750; 0,33
Luxemburgo; 0,750;
0,33
Lituânia; 0,732; 0,38
República Tcheca;
0,714; 0,38
Moldova; 0,696; 0,38
Macedônia; 0,661; 0,38
România; 0,839; 0,43
Ucrânia; 0,804; 0,48
Polônia; 0,714; 0,43
Áustria; 0,696; 0,43
Geórgia; 0,679; 0,43
Sérvia; 0,679; 0,43
Suécia; 0,661; 0,52
1º Tercil
Inglaterra (UK); 0,625;
0,09
Chipre; 0,607; 0,19
Alemanha; 0,571; 0,29
Mônaco; 0,500; 0,14
Holanda; 0,464; 0,29
Rússia; 0,446; 0,24
Azerbaijão; 0,411; 0,29
Malta; 0,357; 0,14
Israel; 0,286; 0,19
Andorra; 0,179; 0,19
Belgica; 0,589; 0,38
Turquia; 0,446; 0,38
Suíça; 0,179; 0,33
Finlândia; 0,625; 0,48
Dinamarca; 0,411;
0,43
Fonte: Dados para o Brasil definidos pelos pesquisadores do Núcleo de Pesquisa
DIRPOL/PPGD/UFPR com base na constituição e legislação. Dados dos países
europeus: (CEPEJ; 2016). Quadro 1 –Índice de Autonomia MP (CEPEJ/DIRPOL) X Legislative Power
(CCP Índices) (por tercis: País; Autonomia MP; Legislative Power) 1170 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. E quanto ao Ministério Público? O índice de prosecutorial independence (PI, de jure), desenvolvido por Van Aaken,
Feld e Voigt51, possui, como esperado, maior correlação com o índice de
autonomia/independência do MP que construímos. Para avançar na análise
qualitativa, que não pode ser medida pela correlação linear, realizamos uma
estratificação por tercis dos índices de autonomia do MP (CEPEJ/DIRPOL) e Legislative
Power (CCP) – (ver Quadro 1). Essa apresentação dos dados reforça a percepção que
há maior probabilidade de encontrar um grau mais elevado de prerrogativas e
garantias institucionais atribuídas ao MP, quando os países constitucionalizam mais
poderes ao Legislativo. A associação é limitada, conforme expresso anteriormente,
com grande parte dos países europeus não se enquadrando claramente nessa
tendência de atribuir graus semelhantes de poderes ao Legislativo e autonomia
institucional ao MP. Porém, da mesma forma, o Brasil se encontra no extremo superior
de constitucionalização/institucionalização de prerrogativas ao poder Legislativo ao
MP. Gráfico 7 – Índice de Autonomia MP (CEPEJ/DIRPOL) - Democracias
Consolidadas e Recentes 1171 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. Fonte: Dados para o Brasil definidos pelos pesquisadores do Núcleo de Pesquisa
DIRPOL/PPGD/UFPR com base na constituição e legislação. Dados dos países
europeus: (CEPEJ; 2016). Fonte: Dados para o Brasil definidos pelos pesquisadores do Núcleo de Pesquisa
DIRPOL/PPGD/UFPR com base na constituição e legislação. Dados dos países
europeus: (CEPEJ; 2016). Por fim, incluímos mais uma variável na análise, a consolidação da democracia em
cada país, entendendo democracias menos consolidadas como aquelas que tiveram
longos regimes autoritários (ditadura militar ou socialista) e estabeleceram regimes
democráticos através de uma ruptura marcada por novas constituições nos anos 80
e 90 do século passado. Desta forma, se considerarmos democracias consolidadas
(1; anteriores a década de 80) e recentes (2; posteriores a década de 80, Brasil e
países do leste europeu com constituições resultantes da queda do muro), é possível
ver que há maior probabilidade de encontrar mais garantias e prerrogativas de
autonomia institucional atribuída ao MP em “democracias recentes”. Isto pode
significar que constituições pós anos 80 e de transições mais recentes de regimes
autoritários tendem a reforçar as prerrogativas de autonomia do MP (ver Gráfico 8 e
Quadro 2). No Quadro 2 é possível ver descritiva e qualitativamente a dispersão
apresentada no Gráfico 8. Mesmo havendo muitas democracias recentes (do leste 1172 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. europeu) com menor grau de institucionalização de autonomia do MP, os índices de
autonomia do MP são superiores às democracias consolidadas do mesmo estrato. Fonte: Dados para o Brasil definidos pelos pesquisadores do Núcleo de Pesquisa
DIRPOL/PPGD/UFPR com base na constituição e legislação. Dados dos países
europeus: (CEPEJ; 2016). Esta conclusão é muito importante para o caso do Brasil, pois confirma os poderes
atribuídos ao MP na CF/88, que garantiram maior autonomia e independência. A maior
quantidade de prerrogativas do MP parece ser um produto que se acentua no período
posterior aos anos noventa e está mais fortemente associada à transição de regimes
autoritários nesse período, confirmando que a Constituição brasileira de 1988
também trouxe maior constitucionalização para promover mais garantias à autonomia
do Ministério Público. Gráfico 7 – Índice de Autonomia MP (CEPEJ/DIRPOL) - Democracias
Consolidadas e Recentes Quadro 2 – Índice de Autonomia MP (CEPEJ/DIRPOL) - Democracia
Consolidadas e Recentes
Democracias
Consolidadas
Democracias Recentes
Autonomia do MP (Institucional, CEPEJ)
2º Metade
MÉDIA; 0,868
Portugal; 0,982
Espanha; 0,946
Noruega; 0,911
Itália; 0,893
Irlanda; 0,875
Grécia; 0,839
França; 0,750
Luxemburgo; 0,750
MÉDIA; 0,913
Albânia; 0,982
Bulgária; 0,982
Letônia; 0,982
Eslováquia; 0,982
Bósnia-Herzegovina; 0,964
Brasil; 0,964
Croácia; 0,964
Estônia; 0,946
Montenegro; 0,946
Armênia; 0,929
Eslovênia; 0,929
România; 0,839
Ucrânia; 0,804
Hungria; 0,750
Lituânia; 0,732
1º Metade
MÉDIA; 0,480
Austria; 0,696
Suécia; 0,661
Finlândia; 0,625
Inglaterra (UK); 0,625
Chipre; 0,607
Belgica; 0,589
Alemanha; 0,571
Mônaco; 0,500
Holanda; 0,464
Turquia; 0,446
Dinamarca; 0,411
Malta; 0,357
Israel; 0,286
Andorra; 0,179
Suíça; 0,179
MÉDIA; 0,625
República Tcheca; 0,714
Polônia; 0,714
Moldova; 0,696
Geórgia; 0,679
Sérvia; 0,679
Macedônia; 0,661
Rússia; 0,446
Azerbaijão; 0,411 Quadro 2 – Índice de Autonomia MP (CEPEJ/DIRPOL) - Democracia
Consolidadas e Recentes
Democracias
Consolidadas
Democracias Recentes
Autonomia do MP (Institucional, CEPEJ)
2º Metade
MÉDIA; 0,868
Portugal; 0,982
Espanha; 0,946
Noruega; 0,911
Itália; 0,893
Irlanda; 0,875
Grécia; 0,839
França; 0,750
Luxemburgo; 0,750
MÉDIA; 0,913
Albânia; 0,982
Bulgária; 0,982
Letônia; 0,982
Eslováquia; 0,982
Bósnia-Herzegovina; 0,964
Brasil; 0,964
Croácia; 0,964
Estônia; 0,946
Montenegro; 0,946
Armênia; 0,929
Eslovênia; 0,929
România; 0,839
Ucrânia; 0,804
Hungria; 0,750
Lituânia; 0,732
1º Metade
MÉDIA; 0,480
Austria; 0,696
Suécia; 0,661
Finlândia; 0,625
Inglaterra (UK); 0,625
Chipre; 0,607
Belgica; 0,589
Alemanha; 0,571
Mônaco; 0,500
Holanda; 0,464
Turquia; 0,446
Dinamarca; 0,411
Malta; 0,357
Israel; 0,286
Andorra; 0,179
Suíça; 0,179
MÉDIA; 0,625
República Tcheca; 0,714
Polônia; 0,714
Moldova; 0,696
Geórgia; 0,679
Sérvia; 0,679
Macedônia; 0,661
Rússia; 0,446
Azerbaijão; 0,411 Quadro 2 – Índice de Autonomia MP (CEPEJ/DIRPOL) - Democracias
Consolidadas e Recentes ro 2 – Índice de Autonomia MP (CEPEJ/DIRPOL) - Democracias
Consolidadas e Recentes Autonomia do MP (Institucional, CEPEJ) 1173 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. CONSIDERAÇÕES FINAIS O artigo tem por objetivo principal desenvolver um índice para mensurar a autonomia
e a independência do Ministério Público, permitindo uma análise comparada do
Ministério Público brasileiro com outras instituições estatais semelhantes no mundo. Para realizar esta comparação, inicialmente, utilizamos índices consolidados na
literatura que analisam os poderes centrais do Estado (no mesmo país e entre os
países comparados): Executivo, Legislativo e Judiciário, que serviram para
dimensionar, dentro de um país, a relação entre os poderes e, eventualmente, sugerir
uma explicação causal para a independência/autonomia do MP de cada país. Pela
abrangência (190 países comparados), utilizamos os índices do CCP (Comparative
Constitutions Project). O exame inicial do índice Judicial Independence (CCP) já
permite constatar, indireta e comparativamente, a elevada autonomia institucional e
constitucional do Ministério Público brasileiro, pois a estrutura constitucional do
Judiciário brasileiro é semelhante ao do Ministério Público, sendo um arranjo
constitucional incomum à imensa maioria dos 190 Estados nacionais comparados pelo
CCP. Os principais achados, em relação ao Brasil, permitem verificar que a
constituição brasileira a) garante maior independência/autonomia ao Judiciário do 1174 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. que poderes à legislatura nacional, mas b) a independência e a autonomia judiciais e
os poderes/competências do Legislativo são mais robustos do que os poderes
atribuídos ao Executivo. E a maioria esmagadora das garantias do Judiciário brasileiro
é estendida constitucionalmente ao Ministério Público. CONSIDERAÇÕES FINAIS Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. institucionalização de prerrogativas ao MP na amostra analisada. Esse achado reforça
a percepção, ainda que estatisticamente limitada, sobre a maior probabilidade de
encontrar um grau mais elevado de prerrogativas e garantias institucionais atribuídas
ao MP, quando os países constitucionalizam mais poderes ao Legislativo. institucionalização de prerrogativas ao MP na amostra analisada. Esse achado reforça
a percepção, ainda que estatisticamente limitada, sobre a maior probabilidade de
encontrar um grau mais elevado de prerrogativas e garantias institucionais atribuídas
ao MP, quando os países constitucionalizam mais poderes ao Legislativo. Por fim, introduzimos mais uma variável na análise para compreender o grau de
autonomia/independência do MP na amostra de países comparados: a consolidação
da democracia. Considerando a classificação de democracias consolidadas e recentes,
foi verificado que há maior probabilidade de existir mais garantias e prerrogativas de
autonomia institucional atribuída ao MP em “democracias recentes”. Uma
interpretação possível desse achado é que os constituintes nas transições mais
recentes de regimes autoritários tenderam a reforçar as prerrogativas de autonomia
do Ministério Público. Mesmo salientando o limite explanatório dessa observação
descritiva das frequências na amostra, esta associação entre democracias recentes e
grau de autonomia do MP é muito importante para o caso do Brasil, porque confirma
os poderes atribuídos ao MP na CF/88, que garantiram maior autonomia e
independência. CONSIDERAÇÕES FINAIS que poderes à legislatura nacional, mas b) a independência e a autonomia judiciais e
os poderes/competências do Legislativo são mais robustos do que os poderes
atribuídos ao Executivo. E a maioria esmagadora das garantias do Judiciário brasileiro
é estendida constitucionalmente ao Ministério Público. Em seguida, abordando o objeto central do texto, descrevemos e construímos um
índice de independência/autonomia do Ministério Público, além de buscar associações
e, eventualmente, causalidades que pudessem sustentar uma explicação para a
ampliação, pela CF/1988, das prerrogativas e competências do Ministério Público
brasileiro, que, comparativamente, faz desse órgão estatal um dos MP com mais
elevado grau de autonomia e independência formal. Para tanto, desenvolvemos um
índice de Independência/Autonomia do MP (CEPEJ/DIRPOL) com base em
prerrogativas institucionais (de jure), compilando a legislação brasileira e utilizando
os dados organizados pelo CEPEJ (The European Commission for the Efficiency of
Justice) para 47 países europeus, órgão oficial da Comunidade Europeia. Essa escolha
metodológica limitou a comparação, presente neste artigo, do Brasil com países
europeus. A ampliação da amostra com países da América Latina é uma das agendas
de pesquisa a ser implementada pelo DIRPOL/PPGD. Os primeiros achados, os quais
são resultantes do índice, demonstram que o Brasil apresenta quase todas as
prerrogativas e garantias atribuídas ao MP de forma plena. Isto é, em comparação
com os países europeus, a independência e a autonomia do Ministério Público
brasileiro encontram-se no estrato mais elevado do índice, sendo um dos países que
mais atribui garantias e prerrogativas constitucionais e estatutárias ao Ministério
Público. Situação semelhante àquela descrita na constitucionalização da Judicial
Independence brasileira, a qual está entre o 10% mais elevado do conjunto de 190
países comparados pelo CCP. Na correlação entre o nosso índice de autonomia/independência do MP e os índices
do CCP, um dos principais achados é a associação entre Legislative Power (CCP) e
autonomia do MP (CEPEJ/DIRPOL). Isto é, há uma correlação positiva e fraca, mas
existente entre uma maior constitucionalização de Legislative Power e maior 1175 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. REFERÊNCIAS BIBLIOGRÁFICAS CARVALHO, Ernani; LEITAO, Natalia. O Novo desenho institucional do Ministério
Publico e o Processo de Judicialização da Política [online]. 2010. Disponível
em: http://www.scielo.br/pdf/rdgv/v6n2/a03v6n2.pdf. Acesso em 03.11.2021. CARVALHO, Ernani; LEITAO, Natalia. O Novo desenho institucional do Ministério
Publico e o Processo de Judicialização da Política [online]. 2010. Disponível
em: http://www.scielo.br/pdf/rdgv/v6n2/a03v6n2.pdf. Acesso em 03.11.2021. CARVALHO, Ernani; LEITAO, Natalia. Operationalizing and Measuring Prosecutorial
Independence: The Brazilian Case. In: INGRAM, Matthew C.; KAPISZEWSKI, Diana. (Org.). Beyond High Courts: The Justice Complex in Latin America. Indiana: Notre
Dame Press, 2019, v. 1, p. 81-112. CEPEJ. The European Commission for the Efficiency of Justice. European judicial
systems - Efficiency and quality of justice. CEPEJ Studies No. 23, Edition 2016
(2014 data), 2016. DIAS, Paulo J.; AZEVEDO, Rodrigo G. de. O papel do Ministério Público: estudos
comparados dos países latino-americanos. São Paulo: Almedina, 2008. DIAS, Paulo J.; AZEVEDO, Rodrigo G. de. O papel do Ministério Público: estudos
comparados dos países latino-americanos. São Paulo: Almedina, 2008. 1176 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. ELKINS,
Zachary;
GINSBURG,
Tom;
MELTON,
James. The
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constitutions. Chicago: Mimeo, 2009a. ELKINS, Zachary; GINSBURG, Tom; MELTON, James. The endurance of national
constitutions. Cambridge-UK: Cambridge University Press, 2009b. ELKINS, Zachary; GINSBURG, Tom; MELTON, James. Constitutional constraints
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global survey. Cambridge-UK: Cambridge University Press, 2009. GUTMANNN, Jerg; VOIGT, Stefan: The Independence of Prosecutors and Government
Accountability. ILE Working Paper Series, No. 8, University of Hamburg, Institute
of Law and Economics (ILE), Hamburg, 2017. MARTINS, Jessika Torres Kaminski. Ministério Público, autonomia e competências:
estudo comparado e o caso do MPT na esfera da discriminação. 2019. Tese
(Doutorado em Ciência Política) - Universidade Federal do Paraná. MAZZILLI, Hugo Nigro. MORAES, Alexandre. Direito Constitucional. 13 ed. São Paulo: Atlas, 2 MORAES, Alexandre. Direito Constitucional. 13 ed. São Paulo: Atlas, 2003. SADEK, Maria Tereza. A Construção de um novo Ministério Público resolutivo. De
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Síntese
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http://www.mazzilli.com.br/pages/artigos/indmp.pdf. Acesso em 11.10.2021. MELTON, James; GINSBURG, Tom. Does de jure judicial independence really matter? A reevaluation of explanations for judicial independence. Journal of Law and
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do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. VOIGT, Stefan; WULF, Alexander J. What makes prosecutors independent? Analysing
the
institutional
determinants
of
prosecutorial
independence. Journal
of
Institutional Economics, p. 1-22, 2017. g
of 1178 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. ANEXOS
Quadro A1 – Indicadores - Índice (de jure) de Independência/Autonomia do
Ministério Público (MP) comparado (adaptado de CEPEJ, 2016/DIRPOL)
Indicadores / Índice de autonomia MP comparada (adaptado de CEPEJ,
2016)
(1) Estatuto dos procuradores públicos / Status of public prosecutors (0 - 1):
1.1Estatutariamente independente / Statutorily independente (1=yes;
0=ø)
1.2Sob o Ministério da Justiça ou outra autoridade central / Under the
Ministry of Justice or another central authority (-0,5=yes)
1.3Outro / Other (-0,5=yes)
(2) Regulamento para evitar instruções específicas para processar ou não,
dirigido a um procurador em um tribunal? / Regulation to prevent specific
instructions to prosecute or not, addressed to a prosecutor in a court? 1177 (1=yes; 0=ø)
(3) Mandato do Ministério Público / Term of office of prosecutors (0 - 1):
3.1Compromisso até a aposentadoria / Appointment until retirement
(1=yes; 0=ø)
3.2Contrato Renovável / Renewable contract (-0,5=yes)
(4) Papel do Ministério Público / Role of public prosecutor (14 itens; x=∑yes1-
14/14)
4.1 Papel do procurador para conduzir ou supervisionar a investigação
policial / Role of public prosecutor - to conduct or supervise police
investigation (1/14=yes; 0=ø)
4.2 Papel do procurador para conduzir investigações / Role of public
prosecutor - to conduct investigations (1/14=yes; 0=ø)
4.3 Papel do ministério público – quando necessário para solicitar
medidas de investigação do juiz / Role of public prosecutor- when
necessary, to request investigation measures from the judge
(1/14=yes; 0=ø)
4.4 Papel do procurador público para acusar / Role of public prosecutor-
to charge (1/14=yes; 0=ø)
4.5 Papel do procurador público – apresentar o caso em tribunal / Role of
public prosecutor- to present the case in court (1/14=yes; 0=ø)
4.6 Papel do procurador público – propor uma sentença ao juiz / Role of
public prosecutor- to propose a sentence to the judge (1/14=yes;
0=ø)
4.7 Papel do procurador público para apelar / Role of public prosecutor-
to appeal (1/14=yes; 0=ø)
4.8 Papel do procurador público para supervisionar o processo de
execução / Role of public prosecutor- to supervise the enforcement
procedure (1/14=yes; 0=ø) ANEXOS
Quadro A1 – Indicadores - Índice (de jure) de Independência/Autonomia do
Ministério Público (MP) comparado (adaptado de CEPEJ, 2016/DIRPOL)
Indicadores / Índice de autonomia MP comparada (adaptado de CEPEJ,
2016)
(1) Estatuto dos procuradores públicos / Status of public prosecutors (0 - 1):
1.1Estatutariamente independente / Statutorily independente (1=yes;
0=ø)
1.2Sob o Ministério da Justiça ou outra autoridade central / Under the
Ministry of Justice or another central authority (-0,5=yes)
1.3Outro / Other (-0,5=yes)
(2) Regulamento para evitar instruções específicas para processar ou não,
dirigido a um procurador em um tribunal? / Regulation to prevent specific
instructions to prosecute or not, addressed to a prosecutor in a court? 1177 4.9 Papel do promotor público – descontinuar um caso sem precisar de
uma decisão de um juiz / Role of public prosecutor- to discontinue a
case without needing a decision by a judge (1/14=yes; 0=ø)
4.10 Papel do promotor público encerra o caso impondo ou negociando
uma sanção ou medida sem exigir uma decisão judicial / Role of public
prosecutor- end the case by imposing or negotiating a penalty or
measure without requiring a judicial decision (1=yes; 0=ø)
4.11 Papel do procurador público – outros poderes significativos / Role
of public prosecutor- other significant powers (1/14=yes; 0=ø)
4.12 Papel em processos civis e adminsitrativos / Role in civil and
administrative cases (1/14=yes; 0=ø)
4.13 Papel do procurador público em casos de insolvência - Role of public
prosecutor- role in insolvency cases (1/14=yes; 0=ø)
4.14 Papel do procurador público – A vítima pode contestar uma decisão
do promotor público / Role of public prosecutor- The victim can
dispute a decision of the public prosecuctor (1/14=yes; 0=ø)
Fonte: Organizado pelos pesquisadores do DIRPOL/PPGD/UFPR com base nos
parâmetros propostos pelo CEPEJ (2016) para avaliar a autonomia do Ministério
Público 4.9 Papel do promotor público – descontinuar um caso sem precisar de
uma decisão de um juiz / Role of public prosecutor- to discontinue a
case without needing a decision by a judge (1/14=yes; 0=ø) 4.9 Papel do promotor público – descontinuar um caso sem precisar de
uma decisão de um juiz / Role of public prosecutor- to discontinue a
case without needing a decision by a judge (1/14=yes; 0=ø) 4.9 Papel do promotor público – descontinuar um caso sem precisar de
uma decisão de um juiz / Role of public prosecutor- to discontinue a
case without needing a decision by a judge (1/14=yes; 0=ø) 4.9 Papel do promotor público – descontinuar um caso sem precisar de
uma decisão de um juiz / Role of public prosecutor- to discontinue a
case without needing a decision by a judge (1/14=yes; 0=ø) 4.10 Papel do promotor público encerra o caso impondo ou negociando
uma sanção ou medida sem exigir uma decisão judicial / Role of public
prosecutor- end the case by imposing or negotiating a penalty or
measure without requiring a judicial decision (1=yes; 0=ø) 4.11 Papel do procurador público – outros poderes significativos / Role
of public prosecutor- other significant powers (1/14=yes; 0=ø) p
p
g
p
(
y
)
4.12 Papel em processos civis e adminsitrativos / Role in civil and
administrative cases (1/14=yes; 0=ø) (
y
;
)
4.13 Papel do procurador público em casos de insolvência - Role of public
prosecutor- role in insolvency cases (1/14=yes; 0=ø) 4.14 Papel do procurador público – A vítima pode contestar uma decisão
do promotor público / Role of public prosecutor- The victim can
dispute a decision of the public prosecuctor (1/14=yes; 0=ø) Fonte: Organizado pelos pesquisadores do DIRPOL/PPGD/UFPR com base nos
parâmetros propostos pelo CEPEJ (2016) para avaliar a autonomia do Ministério
Público. 1177 (1=yes; 0=ø)
(3) Mandato do Ministério Público / Term of office of prosecutors (0 - 1):
3.1Compromisso até a aposentadoria / Appointment until retirement
(1=yes; 0=ø)
3.2Contrato Renovável / Renewable contract (-0,5=yes)
(4) Papel do Ministério Público / Role of public prosecutor (14 itens; x=∑yes1-
14/14) (3) Mandato do Ministério Público / Term of office of prosecutors (0 - 1):
3.1Compromisso até a aposentadoria / Appointment until retirement
(1=yes; 0=ø) (
y
;
)
3.2Contrato Renovável / Renewable contract (-0,5=yes)
(4) Papel do Ministério Público / Role of public prosecutor (14 itens; x=∑yes1-
14/14) 4.1 Papel do procurador para conduzir ou supervisionar a investigação
policial / Role of public prosecutor - to conduct or supervise police
investigation (1/14=yes; 0=ø) 4.2 Papel do procurador para conduzir investigações / Role of public
prosecutor - to conduct investigations (1/14=yes; 0=ø) 4.3 Papel do ministério público – quando necessário para solicitar
medidas de investigação do juiz / Role of public prosecutor- when
necessary, to request investigation measures from the judge
(1/14=yes; 0=ø) 4.4 Papel do procurador público para acusar / Role of public prosecutor-
to charge (1/14=yes; 0=ø) 4.5 Papel do procurador público – apresentar o caso em tribunal / Role of
public prosecutor- to present the case in court (1/14=yes; 0=ø) p
p
p
(
y
)
4.6 Papel do procurador público – propor uma sentença ao juiz / Role of
public prosecutor- to propose a sentence to the judge (1/14=yes;
0=ø) )
4.7 Papel do procurador público para apelar / Role of public prosecutor-
to appeal (1/14=yes; 0=ø) 4.8 Papel do procurador público para supervisionar o processo de
execução / Role of public prosecutor- to supervise the enforcement
procedure (1/14=yes; 0=ø) 1179 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas
do ministério público brasileiro em perspectiva comparada: construindo um índice de
independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação
Stricto Sensu em Ciência Jurídica da UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica - ISSN 1980-7791. 1177 4.9 Papel do promotor público – descontinuar um caso sem precisar de
uma decisão de um juiz / Role of public prosecutor- to discontinue a
case without needing a decision by a judge (1/14=yes; 0=ø)
4.10 Papel do promotor público encerra o caso impondo ou negociando
uma sanção ou medida sem exigir uma decisão judicial / Role of public
prosecutor- end the case by imposing or negotiating a penalty or
measure without requiring a judicial decision (1=yes; 0=ø)
4.11 Papel do procurador público – outros poderes significativos / Role
of public prosecutor- other significant powers (1/14=yes; 0=ø)
4.12 Papel em processos civis e adminsitrativos / Role in civil and
administrative cases (1/14=yes; 0=ø)
4.13 Papel do procurador público em casos de insolvência - Role of public
prosecutor- role in insolvency cases (1/14=yes; 0=ø)
4.14 Papel do procurador público – A vítima pode contestar uma decisão
do promotor público / Role of public prosecutor- The victim can
dispute a decision of the public prosecuctor (1/14=yes; 0=ø)
onte: Organizado pelos pesquisadores do DIRPOL/PPGD/UFPR com base nos
râmetros propostos pelo CEPEJ (2016) para avaliar a autonomia do Ministério
Público. 1177 1180 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas do ministério
público brasileiro em perspectiva comparada: construindo um índice de independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação Stricto Sensu em Ciência Jurídica da
UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica -
ISSN 1980-7791. ISSN 1980 7791. Tabela A1 – Dados e Índice de Independência/Autonomia do MP - Prosecutorial Independence
(de jure) [CEPEJ/DIRPOL]
Países
População
Despesa
Total
MP(
(milhõe
s €) (*)
Despes
a por
hab do
MP
(2015. CNMP)
(€) (*)
Salário
Bruto
Anual
Promotor
Público –
Início de
Carreira
(€) (*)
ÍNDICE de
Independênci
a/
Autonomia do
MP
(CEPEJ/DIRP
OL)
(1
)
(**
)
(2
)
(**
)
(3
)
(**
)
(4)
(**)
Albânia
2.893.005
11,88
4,11
8.988
0,982
1,0 1,0 1,0 0,92
9
Alemanha 80.780.728
523,35
6,48
45.294
0,571
0,5 0,0 1,0 0,78
6
Andorra
76.949
0,67
8,70
73.877
0,179
0,0 0,0 0,0 0,71
4
Armênia
3.010.600
6,87
2,28
--
0,929
1,0 1,0 1,0 0,71
4
Áustria
8.584.926
--
--
53.486
0,696
0,0 1,0 1,0 0,78
6
Azerbaijão
9.477.100
51,88
5,47
6.427
0,411
0,5 0,0 0,5 0,64
3
Belgica
11.209.044
--
--
66.182
0,589
0,5 0,0 1,0 0,85
7
Bósnia-
Herzegovi
na
3.827.343
23,72
6,20
23.884
0,964
1,0 1,0 1,0 0,85
7
Brasil
204.450.6
49
4.825,0
4
23,60
95.560
0,964
1,
0
1,
0
1,
0
0,85
7
Bulgária
7.202.198
93,70
13,01
15.317
0,982
1,0 1,0 1,0 0,92
9
Chipre
858.000
15,80
18,41
34.030
0,607
1,0 0,0 1,0 0,42
9
Croácia
4.225.316
40,82
9,66
22.740
0,964
1,0 1,0 1,0 0,85
7
Dinamarca 5.659.715
97,12
17,16
53.623
0,411
0,0 0,0 1,0 0,64
3
Escócia-
UK
5.347.600
--
--
42.501
0,179
0,0 0,0 0,0 0,71
4
Eslováquia
5.421.349
70,10
12,93
28.060
0,982
1,0 1,0 1,0 0,92
9
Eslovênia
2.061.085
17,56
8,52
31.368
0,929
1,0 1,0 1,0 0,71
4 1181 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas do ministério
público brasileiro em perspectiva comparada: construindo um índice de independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação Stricto Sensu em Ciência Jurídica da
UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica -
ISSN 1980-7791. ISSN 1980-7791. 1177 1182
Espanha
46.439.864
270,48
5,82
47.494
0,946
1,0 1,0 1,0 0,78
6
Estônia
1.313.271
10,63
8,09
22.440
0,946
1,0 1,0 1,0 0,78
6
Finlândia
5.471.753
46,22
8,45
48.619
0,625
0,0 1,0 1,0 0,50
0
França
66.317.994
780,76
11,77
41.552
0,750
0,0 1,0 1,0 1,00
0
Geórgia
3.729.500
13,50
3,62
9.996
0,679
0,0 1,0 1,0 0,71
4
Grécia
10.846.979
--
--
30.159
0,839
0,5 1,0 1,0 0,85
7
Holanda
16.902.146
568,73
33,65
81.162
0,464
0,0 0,0 1,0 0,85
7
Hungria
9.855.571
119,74
12,15
16.217
0,750
1,0 0,0 1,0 1,00
0
Inglaterra
-UK
57.408.654
854,56
14,89
--
0,625
1,0 0,0 1,0 0,50
0
Irlanda
4.625.885
37,81
8,17
30.218
0,875
1,0 1,0 1,0 0,50
0
Irlanda do
Norte-UK
1.840.498
--
--
--
0,375
1,0 0,0 0,0 0,50
0
Israel
8.296.600
--
--
22.924
0,286
0,0 0,0 0,5 0,64
3
Itália
60.795.612 1.460,3
7
24,02
56.263
0,893
1,0 1,0 1,0 0,57
1
Letônia
2.001.468
21,77
10,88
19.369
0,982
1,0 1,0 1,0 0,92
9
Lituânia
2.921.262
28,56
9,78
16.195
0,732
1,0 0,0 1,0 0,92
9
Luxembur
go
563.000
--
--
75.316
0,750
1,0 0,0 1,0 1,00
0
Macedônia
2.069.172
6,50
3,14
17.719
0,661
0,0 1,0 1,0 0,64
3
Malta
429.344
--
--
30.628
0,357
0,0 0,0 1,0 0,42
9
Moldova
3.555.159
8,34
2,35
3.217
0,696
1,0 0,0 1,0 0,78
6
Mônaco
37.800
1,36
35,92
46.226
0,500
0,0 0,0 1,0 1,00
0
Montenegr
o
620.029
5,18
8,35
18.453
0,946
1,0 1,0 1,0 0,78
6
Noruega
5.165.802
20,82
4,03
--
0,911
1,0 1,0 1,0 0,64
3 1182 LIMAS TOMIO, Fabrício Ricardo de; ROBL FILHO, Ilton Norberto. Autonomia e prerrogativas do ministério
público brasileiro em perspectiva comparada: construindo um índice de independência/autonomia. Revista Eletrônica Direito e Política, Programa de Pós-Graduação Stricto Sensu em Ciência Jurídica da
UNIVALI, Itajaí, v.16, n.3, 3º quadrimestre de 2021. Disponível em: www.univali.br/direitoepolitica -
ISSN 1980-7791. 1177 Polônia
38.496.000
437,42
11,36
20.849
0,714
1,0 0,0 1,0 0,85
7
Portugal
10.374.822
88,79
8,56
35.699
0,982
1,0 1,0 1,0 0,92
9
República
Tcheca
10.524.783
85,21
8,10
25.124
0,714
1,0 0,0 1,0 0,85
7
Romênia
22.279.183
238,80
10,72
23.676
0,839
0,5 1,0 1,0 0,85
7
Rússia
146.267.28
8
1.346,5
8
9,21
--
0,446
1,0 0,0 0,0 0,78
6
Sérvia
7.114.393
35,55
5,00
17.728
0,679
0,0 1,0 1,0 0,71
4
Suécia
9.747.355
138,46
14,20
61.480
0,661
0,0 1,0 1,0 0,64
3
Suíça
8.237.666
539,21
65,46
116.230
0,179
0,0 0,0 0,0 0,71
4
Turquia
77.695.904
--
--
21.108
0,446
0,0 0,0 1,0 0,78
6
Ucrânia
42.929.000
--
--
5.094
0,804
1,0 1,0 0,5 0,71
4
(*) Dados para o Brasil de 2015. CNMP (Conselho Nacional do Ministério Público). Demais países
CEPEJ (2016). Conversão R$ para €, cotação média BCB-Banco Central do Brasil, 2015/2017. (**) (1) Status of public prosecutors - Statutorily independente; (2) Regulation to prevent
specific instructions to prosecute or not, addressed to a prosecutor in a court?; (3) Term of
office of prosecutors (Mandate); (4) Role of public prosecutor (CEPEJ, 2016, p. 114, 118 e 124). Fonte: Dados para o Brasil definidos pelos pesquisadores do Núcleo de Pesquisa
DIRPOL/PPGD/UFPR com base na constituição e legislação. Dados dos países europeus: (CEPEJ;
2016). https://rm.coe.int/european-judicial-systems-efficiency-and-quality-of-justice-cepej-
stud/1680786b58. (*) Dados para o Brasil de 2015. CNMP (Conselho Nacional do Ministério Público). Demais países
CEPEJ (2016). Conversão R$ para €, cotação média BCB-Banco Central do Brasil, 2015/2017. (**) (1) Status of public prosecutors - Statutorily independente; (2) Regulation to prevent
specific instructions to prosecute or not, addressed to a prosecutor in a court?; (3) Term of
office of prosecutors (Mandate); (4) Role of public prosecutor (CEPEJ, 2016, p. 114, 118 e 124). Fonte: Dados para o Brasil definidos pelos pesquisadores do Núcleo de Pesquisa
DIRPOL/PPGD/UFPR com base na constituição e legislação. Dados dos países europeus: (CEPEJ;
2016). https://rm.coe.int/european-judicial-systems-efficiency-and-quality-of-justice-cepej-
stud/1680786b58. (*) Dados para o Brasil de 2015. CNMP (Conselho Nacional do Ministério Público). Demais países
CEPEJ (2016). Conversão R$ para €, cotação média BCB-Banco Central do Brasil, 2015/2017. (**) (1) Status of public prosecutors - Statutorily independente; (2) Regulation to prevent
specific instructions to prosecute or not, addressed to a prosecutor in a court?; (3) Term of
office of prosecutors (Mandate); (4) Role of public prosecutor (CEPEJ, 2016, p. 114, 118 e 124). 1177 Fonte: Dados para o Brasil definidos pelos pesquisadores do Núcleo de Pesquisa
DIRPOL/PPGD/UFPR com base na constituição e legislação. Dados dos países europeus: (CEPEJ;
2016). https://rm.coe.int/european-judicial-systems-efficiency-and-quality-of-justice-cepej-
stud/1680786b58. 1183
|
https://openalex.org/W2530289567
|
https://www.biodiversitylibrary.org/partpdf/202789
|
English
| null |
LIST OF MARINE SHELLS COLLECTED IN THE NEIGHBOURHOOD OF ADEN BETWEEN 1892 AND 1901
|
Journal of molluscan studies
| 1,902
|
public-domain
| 5,102
|
1 Commander Shopland’s collection was acquired by Mr. Hormusjee Cowasjee
Dinshau, of Aden, who presented it to the National Collection.—KEp. 171 171 Read 14th March, 1902. Tue
shells
enumerated
in
the
following
list
were,
with
the
seven
exceptions noted, collected within a radius of six miles from Steamer
Point, Aden, chiefly on the shores of the Inner Harbour, by dredging
in from three to eleven fathoms in and south of the harbour, and from
the coral brought in from Little Aden for making lime. All
the
species
marked
with
an
asterisk
(*)
have
been
described
from types now in the British Museum (Natural History).' In
compiling
the
list
it
has
been
considered
advisable
to
follow
a
well-known
work
rather
than
attempt
the
almost
hopeless
task
of
adjusting
the
nomenclature
to
the
latest
standards. This
list
has
therefore
been
drawn
up
in
accordance
with
the
‘
Catalog
der
Conchylien-Sammlung
von
F. Paetel.” By
Commander
E,
R.
Sxoprayp. By
Commander
E,
R. Sxoprayp. Read 14th March, 1902. GASTROPODA. ia
Crosseana,
Souverb. ia
Crosseana,
Souverb. egnea,
Gmel. (
) Tanthina
fragilis, Tanthina
fragilis, Tanthina
fragilis,
L
Murex
anguliferus,
5,
Carbonert,
Jous
»,
elavus,
Kien
5,
eyclostomus,
S
,,
Jenestratus,
Che
»,
haustellum,
»»
pinnatus,
Wo
5,
ramosus,
L
,,
rota,
Sowb. ,,
secundus,
Lk
5,
lternispina,
L
5,
varicosus,
So
Pyrula
paradisiaca egnea,
Gmel. Murex
anguliferus, 9»
wgnea
(sulcate
variety). 5,
Carbonert,
Jou »,
elavus, prcta,
Rve. setula,
Rve. ula,
Rve. 5,
eyclostomus,
S a
contracta,
Rve. ,,
Jenestratus,
Ch marmorata,
Rye. »,
haustellum, rubiginosa,
Rve. »»
pinnatus, undosa,
L. 5,
ramosus, forceps,
Perry. ,,
rota, otoma
albifuniculata,
Rve. ,,
secundus, 5,
lternispina, amnicta,
Smith. Baynhami,
Smith. 5,
varicosus, Pyrula
paradisiaca,
M tena,
Rve. eatena,
Rve. 172 PROCEEDINGS OF
THE MALACOLOGICAL SOCIETY Pleurotoma ceechi, Jouss. 5,
etngulifera,
Lk. ,,
erenularis,
Lk. x
>,
Jousseaumt,
Melv. *
,,
latisinuata,
BE. Smith. 5,
Makimonos,
Jouss. ,,
obtusicostata,
Smith. ,,
otitoma,
Jouss. Ranella
spinosa,
Lk. ”? tuberculata, Brod. Bullia
Mauritiana,
Gray. ”? Tahitensis,
Gmel. Phos roseatus, Hinds. Cyllene
Grayi,
Rve. Nassa albescens, v. fenestrata, Dkr. ? »,
Pouloensis,
Jouss. *
5,
tigrina,
Lk. ee
»,
tuberculata,
Gray. a
5,
unitfasciata,
Smith. sy
,,
variabilis,
Smith. . ,,
vtdua,
Rve. 4
,,
vtolacea,
Hinds. x
Defrancia
carinulata,
Souverb. bs
5,
joraminata,
Rye. i
»,
LHorneana,
E. Smith. -
,,
neva,
Rve. "
5,
LPhilippinensis,
Rve. »
rubicunda,
Gould. glo
Daphnella
Boholensis,
Rye. &
,,
ettharella,
Lk. As
,,
erebriplicata,
Rve. 5,
Cuming,
Powis. 4,
eylindrica,
Rve. »,
fenestrata,
Melv. ,,
ressoides,
Rve. Triton
antiquatus,
Hinds. ? ”
arcularia,
L. celata, A. Ad. concinna, Powis. coronata, Lk. dermestina, Gould. Erythrea,
Issel. fissilabris,
A. Ad. gemmulata, Lk. lentiginosa, A. Ad. Marrati,
Smith. nodifera,
Powis. papillosa,
L. persica,
Mart. polychroma,
Melv. pulla, L. variegata, A. Ad. verrucosa, A. Ad. Zailzensis,
Jouss. Eburna
Borneensis,
Sowb. ”
Valentiana,
Swain. Purpura
echinata,
Blain. 2
»»
aquatilis,
Rve. =
,,
bracteatus,
Hinds. -
,,
eancellatus,
Lk. RA
Ns
»»
Vv. decipiens,
Rve. a
,,
¢landestinus,
Chem. ,,
convolutus,
Brod. 5,
labiosus,
Wood. ,,
maculosus,
Chem. »,
prlearis,
L. ,,
Ranzani,
Biane. :
cotra. »
retusus,
Lk. ,,
rubecula,
L. ,,
trilineatus,
Rve. respaceus, Lk. Pe ae aha adicus, Jouss. 5,
Shoplandi,
Jouss. Ranella anceps, Lk. .,
coneinna,
Dunker. 5,
granifera,
Lk. livida,
Rve. be
Ffasciata,
Diy. hippocastanum, L. mancinella,
Lk. persica, L. Rudolphi,
Lk. sacellum, Chem. Ricinula
chrysostoma,
Desh. ? ? FromuSo=- "4 75;
9
»
-+B)
? GASTROPODA. 9
concatenata, Lk. elata, Blain. Jiscellum,
Chem. indigoferum,
Melv. lobata, Blain. marginatum-altigona,
Jouss. nodulosa, C. B. Ad. ricinus, L. tuberculata,
Blain. undata, Chem. Rapana bulbosa, Sol. * Coralliophila Arabica, Melv. bh)
9
”
costularis,
Lk. squamulosa, Rve. violacea, Kien, Ranella
spinosa,
Lk. ”? tuberculata, Brod. Bullia
Mauritiana,
Gray. ”? Tahitensis,
Gmel. Phos roseatus, Hinds. Cyllene
Grayi,
Rve. Nassa albescens, v. fenestrata, Dkr
? . *
ee
. a
. sy
. 4
x
rb. bs
. i
mith. -
"
e. »
glo
e. &
As
? ”
arcularia,
L. celata, A. Ad. concinna, Powis. coronata, Lk. dermestina, Gould. Erythrea,
Issel. fissilabris,
A. Ad. gemmulata, Lk. lentiginosa, A. Ad. Marrati,
Smith. nodifera,
Powis. papillosa,
L. persica,
Mart. polychroma,
Melv. pulla, L. variegata, A. Ad. verrucosa, A. Ad. Zailzensis,
Jouss. Eburna
Borneensis,
Sowb. ”
Valentiana,
Swain. Purpura
echinata,
Blain. 2
=
. -
RA
Rve. a
Ffasciata,
Diy. hippocastanum, L. mancinella,
Lk. persica, L. Rudolphi,
Lk. sacellum, Chem. Ricinula
chrysostoma,
Desh. ? ? o=- "4 75;
9
»
-+B)
? 9
concatenata, Lk. elata, Blain. Jiscellum,
Chem. indigoferum,
Melv. lobata, Blain. marginatum-altigona,
Jouss
nodulosa, C. B. Ad. ricinus, L. tuberculata,
Blain. undata, Chem. Rapana bulbosa, Sol. * Coralliophila Arabica, Melv. bh)
9
”
costularis,
Lk. squamulosa, Rve. violacea, Kien, Pleurotoma ceechi, Jouss. 5,
etngulifera,
Lk. ,,
erenularis,
Lk. x
>,
Jousseaumt,
Melv. *
,,
latisinuata,
BE. Smith. 5,
Makimonos,
Jouss. ,,
obtusicostata,
Smith. ,,
otitoma,
Jouss. »,
Pouloensis,
Jouss. 5,
tigrina,
Lk. »,
tuberculata,
Gray. 5,
unitfasciata,
Smith. ,,
variabilis,
Smith. ,,
vtdua,
Rve. ,,
vtolacea,
Hinds. Defrancia
carinulata,
Souverb
5,
joraminata,
Rye. »,
LHorneana,
E. Smit
,,
neva,
Rve. 5,
LPhilippinensis,
Rve. rubicunda,
Gould. Daphnella
Boholensis,
Rye. ,,
ettharella,
Lk. ,,
erebriplicata,
Rve. 5,
Cuming,
Powis. 4,
eylindrica,
Rve. »,
fenestrata,
Melv. ,,
ressoides,
Rve. Triton
antiquatus,
Hinds. »»
aquatilis,
Rve. ,,
bracteatus,
Hinds. ,,
eancellatus,
Lk. Ns
»»
Vv. decipiens,
R
,,
¢landestinus,
Chem. ,,
convolutus,
Brod. 5,
labiosus,
Wood. ,,
maculosus,
Chem. »,
prlearis,
L. ,,
Ranzani,
Biane. :
cotra. »
retusus,
Lk. ,,
rubecula,
L. ,,
trilineatus,
Rve. respaceus, Lk. Pe ae aha adicus, Jouss. 5,
Shoplandi,
Jouss. Ranella anceps, Lk. .,
coneinna,
Dunker. 5,
granifera,
Lk. livida,
Rve. be
FromuSo= Ranella
spinosa,
Lk. ”? tuberculata, Brod. B)
? tuberculata,
Blain. 5,
Shoplandi,
Jouss. .,
coneinna,
Dunker. )
9
squamulosa, Rve. l 5,
granifera,
Lk. livida,
Rve. GASTROPODA. be ”
violacea, Kien, SHOPLAND :
LIST OF MARINE SHELLS FROM ADEN, Mitra obeliscus, Rve. Magilus
antiquus,
Lk. Ber-
,,
Pacifica,
Rve. bera. 5,
Mflata,
Lk. Ancillaria
acuminata,
Sowhb. 5,
albisuleata,
Sowb. 5, eastanea, Sowb. 5,
exigua,
Sowb. »
sua,
Swain. 5,
ovalis,
Sowb. striolata,
Sowb. Fasciolaria
trapezium,
L. Latirus
Forskali,
Vapp.-Can. »
Lauluccie,
Tapp.-Can. »
polygonus,
L. 5,
smaragdulus,
L. »,
turritus,
Gmel. Vasum cornigerum, Lk. Mitra affinis, Rye. 5,
amabilrs,
Rve. »
armillata,
Rve. 5,
aurantia,
Gmel. 5,
dureolata,
Swain. », bella, Pse. ,,
Bovei,
Kien. 5,
ealigena,
Rve. »,
carnicolor,
Rye. ,,
erculata,
Kien. ,,
¢elathrata,
Rve. 5,
concentrica,
Kve. »
coriacea,
Rve. »
erenifera,
Lk. 5,
erenulata,
Chem. »,
eucumerina,
Lk. ,,
dermestina,
Lk. »
eprscopalrs,
L. »
erred,
Pse. p
ferrugined,
Lk. »,
fissurata,
Lk. 5,
foveolata,
Dkr. »,
fulvescens,
Swain. 55 mnesi, Jouss. 5, tmsculpta, A. Ad. ,,
interlirata,
Rve. 5,
literata,
Lk. », lubens, Rve. »,
marginata,
Sowd. 5,
militaris,
Rve. ,,
mucronata,
Swain. ,,
nebrias,
Melv. »,
Pharaonis,
A. Ad. 5» pretiosa, Rve. »,
procissa,
Rve. 5,
rotundilirata,
Rve. 5, rufescens, A. "Ad. 5,
Ruppelli,
Rve. 5,
scabriuscula,
Gray. 5,
semifasciata,
Lk. »,
Shoplandi,
Mely. »,
tabanula,
Lk. ,,
turgida,
Rve. ,,
ustulata,
Rve. »
variegata,
Sowb. »
vexillum,
Rve. »,
vulpecula,
L. aerampelina,
Melv. Ma ginella clandestina (near),
Broech. », gtbbosa, Jouss. ,,
Mazagonica,
Mely. 5, obtusa, Sowb. :
bera. »,
seripta,
Hinds. ,,
Lerveriana,
Petit. », Verdensis, Smith. Columbella albina, Kien. Me albinodulosa, Gask. ,,
alveolata,
Kien. 5, aspersa, Sowd. ,,
astricta,
Rve. 5,
concinna,
Rve. », conspersa, A. Ad. »
errbraria,
Lk. », jfabula, Rve. 5,
flava,
Brug. 5, LHanleyt, Desh. 5,
lagula,
Duel. »,
lyrata,
Sowb. »
mendicaria,
L. ,,
mercatoria,
L. 5, miser, Sowb. »
propingua,
Smith. 5,
regulus,
Souverb. ,,
Lerpsichore,
Leathes. », 2ea, Melv. ,,
zonata,
Rve. Harpa
minor,
Rumph. 5,
ventricosa,
Lk. from 5,
albisuleata,
Sowb. 5,
rotundilirata,
Rve
5
f
A
"Ad 5,
exigua,
Sowb. gua,
Sowb. 5,
Ruppelli,
Rve. »
sua,
Swain. 5,
scabriuscula,
Gray. »
sua,
Swain. 5,
ovalis,
Sowb. l
b
5,
semifasciata,
Lk. 5,
ovalis,
Sowb. t i l t
S
b Sowb. S
b
5,
semifasciata,
Lk striolata,
Sowb. F
i l
i
t
i
L
»,
Shoplandi,
Mely. striolata,
Sowb. F
i l
i
t
i
L Fasciolaria
trapezium,
L. Fasciolaria
trapezium,
L. »,
tabanula,
Lk. Latirus
Forskali,
Vapp.-Can. Latirus
Forskali,
Vapp.-Can. ,,
turgida,
Rve. GASTROPODA. »
Lauluccie,
Tapp.-Can. »
Lauluccie,
Tapp.-Can. ,,
ustulata,
Rve. »
variegata,
Sowb. »
polygonus,
L. »
vexillum,
Rve. 5,
smaragdulus,
L. »,
vulpecula,
L. »,
turritus,
Gmel. Vasum cornigerum, Lk. Mitra affinis, Rye. Ma ginella clandestina (near),
Broech. 5,
amabilrs,
Rve. »
armillata,
Rve. ,,
Mazagonica,
Mely. f 5,
aurantia,
Gmel. 5, obtusa, Sowb. :
bera. from Ber- 5,
dureolata,
Swain. », bella, Pse. »,
seripta,
Hinds. ,,
Bovei,
Kien. ,,
Lerveriana,
Petit. 5,
ealigena,
Rve. », Verdensis, Smith. »,
carnicolor,
Rye. Columbella albina, Kien. lbi
d l
G
k ,,
erculata,
Kien. ,,
¢elathrata,
Rve. 5,
concentrica,
Kve. 5, aspersa, Sowd. »
coriacea,
Rve. »
erenifera,
Lk. 5,
erenulata,
Chem. »,
eucumerina,
Lk. »
erred,
Pse. f
i
d
Lk p
ferrugined,
Lk. »,
fissurata,
Lk. 5,
foveolata,
Dkr. »,
fulvescens,
Swain. 55 mnesi, Jouss. 5, tmsculpta, A. Ad. ,,
interlirata,
Rve. 5,
literata,
Lk. », lubens, Rve. ,,
mucronata,
Swain. ,,
nebrias,
Melv. 5, 174 CEEDINGS
OF
THE
MALACOLOGICAL SOCIETY. Cassis exarata, Rve. Fauroti,
Jouss. glauca, L. pila,
Rve. rufa, L
torquata, Rve. vibex, L. Dolium Quemanju, Jouss. Sycotypus ficoides, Lk. 3) jeeus, TL:
Lamellaria
perspicua,
Li. Nautica cernica, Jouss. Chinensis,
Lk. Collier, Recl. didyma, Bolt. Forskali,
Chem. maculosa, Lk. mamilla, L. Marochiensis,
Gmel. melanostoma, Gmel. plicatula,
Nutt. Powisiana,
Recl. pulicaris,
Phil. simia, Chem. teniata,
Menke. ae
tela-aranea,
Melv. Naticina
papilla,
Gmel. Sigaretus
Cuvierianus,
Recl. 5,
planulatus,
Recl. Scalaria
clathrus,
L. ,,
decussata,
Lk. Acrilla
gracilis,
H. Ad. ? ”? ? ”? 9
?? ”
? Zerebra albomarginata, Desh. Babylonia,
Lk. cerulescens, Lk. consobrina, Desh. corrugata,
Lk. Gottoensis, EK. Smith. Lamarcki,
Kien,
ligata,
Hinds. nassoides, Hinds. Pellyi, KE. Smith. Souleyeti, Desh. straminea,
Gray. Rve. »,
tessellata,
Gray. textilis,
Hinds. 9
Pyramidella
mitralis,
A. Ad. Paeteli,
Dhrn. ”)
v. serotina, Ad. &
Pyramidella
propingua,
A. Ad. »,
variegata,
A. Ad. Obeliscus dolabratus, L. sulcatus, A. Ad. ,,
terebelloides,
A. Ad. Ringicula
acuta,
Phil. 5,
propinguans,
Hinds. Elusa
bruneomaculata,
Melv. * 4elis exereta, Melv. Eulima
acuta,
Sowb. brevis, Sowb. Martini,
A. Ad. Shoplandi,
Melv. ,,
solidula,
Ad. &
Rye. * Zeiostraca constellata, Melv. Stylifer
exaratus,
A. Ad. »,
jastigvatus,
A. Ad. ,,
solidulus,
Ad. &
Rve. Solarium
cylindraceum,
Chem. dorsuosum, Hinds. hybridum, L. infundibuliforme,
Gmel. levigatum,
Lk. perspectiviunculum,
Chem. perspectivum, L. regium,
Hanley. 5,
variegatum,
Gmel. Conus acuminatus, Hwass. Adansoni,
Lk. Adenensis,
Smith. arenatus,
Hwass. betulinus, L. bullatus, L.: from Berbera. capitaneus, L. catus, Hwass. Ceylonensis,
Hwass. clytospira,
Melv. GASTROPODA. &
Stand. Cuviert, Crosse. Erythreensis,
Beck. Re
,,
v. adustus,
Sowb. Jlavidus,
Lk. fumigatus
(=
Lagardi,
Jouss.),
Hwass. gemmulatus, Sowb. :
Bulhar, 8 fms. generalis, L. geographus, L. wmscriptus, Rve. ;
5
v. Keatt,
lineatus, Chem. ”? ”
* ”
”
”
”? ”
”
from
Sowb. ? Pyramidella
propingua,
A. Ad. »,
variegata,
A. Ad. Obeliscus dolabratus, L. sulcatus, A. Ad. ,,
terebelloides,
A. Ad. Ringicula
acuta,
Phil. 5,
propinguans,
Hinds. Elusa
bruneomaculata,
Melv. * 4elis exereta, Melv. Eulima
acuta,
Sowb. brevis, Sowb. Martini,
A. Ad. Shoplandi,
Melv. ,,
solidula,
Ad. &
Rye. * Zeiostraca constellata, Melv. Stylifer
exaratus,
A. Ad. »,
jastigvatus,
A. Ad. ,,
solidulus,
Ad. &
Rve. Solarium
cylindraceum,
Chem. dorsuosum, Hinds. hybridum, L. infundibuliforme,
Gmel. levigatum,
Lk. perspectiviunculum,
Chem. perspectivum, L. regium,
Hanley. 5,
variegatum,
Gmel. Conus acuminatus, Hwass. Adansoni,
Lk. Adenensis,
Smith. arenatus,
Hwass. betulinus, L. bullatus, L.: from Berbera. capitaneus, L. catus, Hwass. Ceylonensis,
Hwass. clytospira,
Melv. &
Stand. Cuviert, Crosse. Erythreensis,
Beck. Re
,,
v. adustus,
Sowb. Jlavidus,
Lk. fumigatus
(=
Lagardi,
Jouss.),
Hwass. gemmulatus, Sowb. :
Bulhar, 8 fms. generalis, L. geographus, L. wmscriptus, Rve. ;
5
v. Keatt,
lineatus, Chem. ”? ”
* ”
”
”
”? ”
”
from
Sowb
? Cassis exarata, Rve. Fauroti,
Jouss. glauca, L. pila,
Rve. rufa, L
torquata, Rve. vibex, L. Dolium Quemanju, Jouss. Sycotypus ficoides, Lk. 3) jeeus, TL:
Lamellaria
perspicua,
Li. Nautica cernica, Jouss. Chinensis,
Lk. Collier, Recl. didyma, Bolt. Forskali,
Chem. maculosa, Lk. mamilla, L. Marochiensis,
Gmel. melanostoma, Gmel. plicatula,
Nutt. Powisiana,
Recl. pulicaris,
Phil. simia, Chem. teniata,
Menke. ae
tela-aranea,
Melv. Naticina
papilla,
Gmel. Sigaretus
Cuvierianus,
Recl. 5,
planulatus,
Recl. Scalaria
clathrus,
L. ,,
decussata,
Lk. Acrilla
gracilis,
H. Ad. ? ”? ? ”? 9
?? ”
? Zerebra albomarginata, Desh. Babylonia,
Lk. cerulescens, Lk. consobrina, Desh. corrugata,
Lk. Gottoensis, EK. Smith. Lamarcki,
Kien,
ligata,
Hinds. nassoides, Hinds. Pellyi, KE. Smith. Souleyeti, Desh. straminea,
Gray. Rve. »,
tessellata,
Gray. textilis,
Hinds. 9
Pyramidella
mitralis,
A. Ad. Paeteli,
Dhrn. ”)
v. serotina, Ad rufa, L
”? torquata, Rve. 9 Shoplandi,
Melv. * ” maculosa, Lk. infundibuliforme,
Gmel. ”? Powisiana,
Recl. pulicaris,
Phil. perspectiviunculum,
Chem. simia, Chem. perspectivum, L. teniata,
Menke. regium,
Hanley. ” ae
tela-aranea,
Melv. N
i i
ill
G
l 5,
variegatum,
Gmel. Naticina
papilla,
Gmel. Conus acuminatus, Hwass. Sigaretus
Cuvierianus,
Recl. Adansoni,
Lk. 5,
planulatus,
Recl. Adenensis,
Smith. ,,
decussata,
Lk. arenatus,
Hwass. betulinus, L. Acrilla
gracilis,
H. GASTROPODA. Ad. bullatus, L.: from Berbera. Zerebra albomarginata, Desh. Babylonia,
Lk. capitaneus, L. cerulescens, Lk. consobrina, Desh. corrugata,
Lk. Gottoensis, EK. Smith. Lamarcki,
Kien, ligata,
Hinds. nassoides, Hinds. Pellyi, KE. Smith. Souleyeti, Desh. straminea,
Gray. i textilis,
Hinds. 9 9
Pyramidella
mitralis,
A. Ad. Paeteli,
Dhrn. ”) ”) 175 SHOPLAND:
LIST
OF
MARINE
SHELLS
FROM Conus lividus, Hwass. »,
luotificus,
Rve. > moles, L. 5
minimus,
L. »,
mitratus,
Hwass. », nemocanus, Hwass. »,
nussatella,
L. 5,
pusillus,
Chem. », quadratomaculatus, Sowb. :
from Bulhar, 8 fms. »»
quercinus,
Hwass. »,
splendidulus,
Sowb. »
striatus,
L. ,,
sulphuratus,
Hwass. »,
Sumatrensis,
Hwass. ,,
teniatus,
Hwass. ,,
tessellatus,
Hwass. »,
textile,
L. ,,
Lhomasi,
Sowb. Traversianus,
Smith. Strombus
Belutschionste,
Mely. », Columba, Lk. »,
eylindricus,
Swain. », dentatus, L. »
jfloridus,
Lk. 5,
fusiformis,
Sowb. »
grbberulus,
L. », lineatus, Lk. »,
Mauritianus,
Lk. »,
plicatus,
Lk. 5,
Ruppelli,
Rve. »,
terebellatus,
Sowb. »
tricorms,
Lk. », urceus, L. varvabilis,
Swain. Pterocera
bryona,
Gmel. 5, scorpio, L. Rostellaria
curta,
Sowb. ,,
curvtrostris,
Lk. Cyprea
clandestina,
L. 5, eruenta, v. coloba, Melv. » erosa, L., v. nebutes, Melv. »
rythreensis,
Beck. 5,
exusta,
Sowb. »,
jfabula,
Kien. ,,
felina,
Gmel. ,,
jfimbriata,
Gmel. ‘
»,
Vv. macula,
A. Ad. 5» gangrenosa, Sol. », helvola, L. Cyprea
histrio,
Gmel. 5, Lsabella, L. ,»,
lentiginosa,
Gray. »,
Lienardi,
Jouss. »,
Lustert,
Gray. »
lynn,
L. 5,
Mauritiana,
L. 5,
microdon,
Gray. », moneta, L. », nucleus, L. »
ocellata,
L. 5,
pantherina,
Sol. »,
pulchra,
Swain. 5,
punctata,
L. ,,
quadrimaculata,
Gray. 5, talpa, L. »» tegres, L. »
turdus,
Lk. 5,
Lurnert,
Jouss. ,,
undata,
Lk. ,,
vitellus,
L. zigzac, L. Trivia
staphylea,
L. v. limacina, Lk. Ovula laetem Lk. » ovum, L. Birostra spelta, L. Cancellaria elegans, Sowb. »,
Aystrix,
Rve. 5, melanostoma, Sowb. 5,
scalarina,
Lk. Cerithium
albovaricosum,
Smith. 5, asperum, L. 5,
bifasciatum,
Sowb. », c@eruleum, Sowb. 5,
elypeomorus,
Jouss. ,,
columna,
Sowb. »,
eontractum,
Sowb. 5,
echinatum,
Lk. »
Lrythreense,
Lk. 5,
fasciatum,
Mart. »,
fluviatile,
Pot. Mich. »,
Kocht,
Phil. »,
lacteum,
Kien. »» pingue, A. Ad. 5,
recurvum,
Sowb. ,,
rostratum,
Sowb. 5,
Ruppelli,
Phil. »,
scabridum,
Phil. »,
NShoplandi,
Melv. Cyprea
histrio,
Gmel. Conus lividus, Hwass. Cyprea
histrio,
Gmel. 5, Lsabella, L. ,»,
lentiginosa,
Gray. »,
Lienardi,
Jouss. »,
Lustert,
Gray. »
lynn,
L. 5,
Mauritiana,
L. 5,
microdon,
Gray. », moneta, L. », nucleus, L. GASTROPODA. »
ocellata,
L. 5,
pantherina,
Sol. »,
pulchra,
Swain. 5,
punctata,
L. ,,
quadrimaculata,
Gray. 5, talpa, L. »» tegres, L. »
turdus,
Lk. 5,
Lurnert,
Jouss. ,,
undata,
Lk. ,,
vitellus,
L. zigzac, L. Trivia
staphylea,
L. v. limacina, Lk. Ovula laetem Lk. » ovum, L. Birostra spelta, L. Cancellaria elegans, Sowb. »,
Aystrix,
Rve. 5, melanostoma, Sowb. 5,
scalarina,
Lk. Cerithium
albovaricosum,
Smith. 5, asperum, L. 5,
bifasciatum,
Sowb. », c@eruleum, Sowb. 5,
elypeomorus,
Jouss. ,,
columna,
Sowb. »,
eontractum,
Sowb. 5,
echinatum,
Lk. »
Lrythreense,
Lk. 5,
fasciatum,
Mart. »,
fluviatile,
Pot. Mich. »,
Kocht,
Phil. »,
lacteum,
Kien. »» pingue, A. Ad. 5,
recurvum,
Sowb. ,,
rostratum,
Sowb. 5,
Ruppelli,
Phil. »,
scabridum,
Phil. »,
NShoplandi,
Melv. 5, Lsabella, L. », nemocanus, Hwass. »
lynn,
L. »,
nussatella,
L. 5,
Mauritiana,
L. »,
nussatella,
L. 5,
pusillus,
Chem. 5,
microdon,
Gray. 5,
pusillus,
Chem. pusillus,
Chem. 5,
microd », quadratomaculatus, Sowb. :
», moneta, L. from Bulhar, 8 fms. », nucleus, L. »»
quercinus,
Hwass. »»
quercinus,
Hwass. »
ocellata,
L. »,
splendidulus,
Sowb. »,
splendidulus,
Sowb. 5,
pantherina,
Sol. »
striatus,
L. »
striatus,
L. »,
pulchra,
Swain. ,,
sulphuratus,
Hwass. ,,
sulphuratus,
Hwass. 5,
punctata,
L. »,
Sumatrensis,
Hwass. »,
Sumatrensis,
Hwass. ,,
quadrimaculata,
Gray. ,,
teniatus,
Hwass. ,,
tessellatus,
Hwass. »,
textile,
L. »,
textile,
L. 5,
Lurnert,
Jouss. ,,
Lhomasi,
Sowb. ,,
Lhomasi,
Sowb. Traversianus,
Smith. S
b
B l
hi
M Strombus
Belutschionste,
Mely. zigzac, L. h l Trivia
staphylea,
L. »,
eylindricus,
Swain. », dentatus, L. »
jfloridus,
Lk. 5,
fusiformis,
Sowb. »
grbberulus,
L. », lineatus, Lk. »,
Mauritianus,
Lk. 5, melanostoma, Sowb. »,
plicatus,
Lk. 5,
scalarina,
Lk. 5,
Ruppelli,
Rve. »,
terebellatus,
Sowb. »
tricorms,
Lk. », urceus, L. varvabilis,
Swain. Pterocera
bryona,
Gmel. 5, scorpio, L. 5,
elypeomorus,
Jouss. ,,
columna,
Sowb. » erosa, L., v. nebutes, Melv. »
rythreensis,
Beck. »,
lacteum,
Kien. 5,
exusta,
Sowb. »,
jfabula,
Kien. »» pingue, A. Ad. ,,
felina,
Gmel. 5,
recurvum,
Sowb. ,,
jfimbriata,
Gmel. ‘
l », helvola, L. »,
NShoplandi,
Melv. 176 EEDINGS
OF
THE
MALACOLOGICAL SOCIETY. ALACOLOGICAL SOCIETY. Phasianella
nivosa,
Rve. Turbo
coronatus,
vy. granulata,
Gmel. 5,
elegans,
Phil. 5,
petholatus,
L. 5,
pustulatus,
Rve. 5,
radiatus,
Gmel. Leptothyra
leta,
Montr. 5
plula,
Dkr. te 1» Yemenensis, Melv. * Rotella car neolata, Mely. *Delphinula
diplocostira,
Melv. Cardinalia
virgata,
Gmel. Trochus dentatus, Forsk. :
Berbera. GASTROPODA. »,
infundibulum,
v. Erythreus,
Brocchi. Clanculus
Pharaonis,
L. Monodonta dama, Phil. 5, obscura, Wood. Euchelus
bicinctus,
Phil. »,
Delpretei,
Caramagna. .. proximus,
A. Ad. Thalotia
Torresi,
var.,
E. Smith. Zizyphinus scobinatus, A. Ad. Agagus
Agagus,
Jouss. Gibbula
Doria,
Caramagna. Minolia
Catfassii,
Caramagna. ,,
rotelleformis,
Phil. Margarita
variabilis,
A. Ad. Vitrinella
Menegherwi,Caramagna. Haliotis
multiperforata,
Rve. »
varia,
L. Fissurella
Ruppelli,
Sowb. Parmophorus unguis, L. Patella plumbea, Lk. 5,
radians,
Gmel. Buceinulus
solidulus,
L. Hydatina
physis,
Lk. 5, velum, Chem. Bulla ampulla, L. Atys
cylindracea,
Helb. »,
Jerruginea,
Ad,
», naucum, L. Umbrella
Indica,
Lk. from
PODA. Dentalium
Shoplandi,
Jouss. 176
PROCEEDINGS
OF
THE
Cerithium tuberculatum, L. »,
tuberosum,
Fabr. »»
Yerbury?,
Smith. Vertagus cedo-nulli, Sowb. »,
Jasciatus,
Brug. ,,
obeliscus,
Brug. * Bittium chrysomallum, Melv. Triforis ecingulatus, A, Ad. ,,
eollaris,
Hinds. 5,
corrugatus,
Hinds. Littorina
ahenea,
Rve. 5,
grano-costata,
Rve. »,
Vatalensis,
Krauss. ,,
seabra,
L. Modulus
candidus,
Petit. ,,
tectum,
Gmel. Planaxis
breviculus,
Desh. 5, NSavignyt, Desh. ,,
suleatus,
Lk. Rissoina
Bertheloti,
Aud. ,,
¢lathrata,
A. Ad. 5, concinna, A. Ad. ,,
msculpta,
Melv. &
Stand. 5,
insignis,
Ad. &
Rve. 5
pachystoma,
Melv. 5,
Sequensiana,
Issel. ,,
Stdmondiana,
Issel. 5,
sprrata,
Sowb. ,,
tridentata
(=
curta,
Sowb.),
Mich. Onoba delicata, Monteros. Turritella
columnaris,
Kien. ,,
maculata,
Rve. Calyptrea
cicatricosa,
Kve. x
9»,
Ldgariana,
Melv. 5» equestris, L. Crepidula Walshi (= Plana, Ad. &
Rve.),
Herms. Narica
cancellata,
Chem. 5,
ligata,
Reel. Nerita
chrysostoma,
Recl. 5,
plexa,
Chem. »,
polita,
L. 5,
Rumphi,
Recl. Neritina
feuilletti,
Aud. Phasianella
lineolata,
Wood. MALACOLOGICAL SOCIETY. Phasianella
nivosa,
Rve. Turbo
coronatus,
vy. gr
Gmel. 5,
elegans,
Phil. 5,
petholatus,
L. 5,
pustulatus,
Rve. 5,
radiatus,
Gmel. Leptothyra
leta,
Montr. 5
plula,
Dkr. te 1» Yemenensis, Melv. * Rotella car neolata, Mely. *Delphinula
diplocostira,
M
Cardinalia
virgata,
Gmel. Trochus dentatus, Forsk. Berbera. »,
infundibulum,
v. Er
Brocchi. Clanculus
Pharaonis,
L. Monodonta dama, Phil. 5, obscura, Wood. Euchelus
bicinctus,
Phil. »,
Delpretei,
Caramag
.. proximus,
A. Ad. Thalotia
Torresi,
var.,
E. Zizyphinus scobinatus, A. Agagus
Agagus,
Jouss. Gibbula
Doria,
Caramagn
Minolia
Catfassii,
Caram
,,
rotelleformis,
Phil. Margarita
variabilis,
A. A
Vitrinella
Menegherwi,Car
Haliotis
multiperforata,
R
»
varia,
L. Fissurella
Ruppelli,
Sowb
Parmophorus unguis, L. Patella plumbea, Lk. 5,
radians,
Gmel. Buceinulus
solidulus,
L. Hydatina
physis,
Lk. 5, velum, Chem. Bulla ampulla, L. Atys
cylindracea,
Helb. »,
Jerruginea,
Ad,
», naucum, L. Umbrella
Indica,
Lk. SCAPHOPODA. Dentalium octagonum, Lk. Dentalium
Shoplandi,
Jou Cerithium tuberculatum, L. Cerithium tuberculatum, L. 5,
elegans,
Phil. 5,
petholatus,
L. 5,
radiatus,
Gmel. Leptothyra
leta,
Montr. Monodonta dama, Phil. 5, concinna, A. Ad. ,,
msculpta,
Melv. &
Stand. .. proximus,
A. Ad. Thalotia
Torresi,
var.,
E. Smith. Zizyphinus
scobinatus
A
Ad Thalotia
Torresi,
var.,
E. Smith. 5,
insignis,
Ad. &
Rve. Thalotia
Torresi,
var.,
E. Smith
Zizyphinus scobinatus, A. Ad. Zizyphinus scobinatus, A. Ad. 5
pachystoma,
Melv. Agagus
Agagus,
Jouss. 5,
Sequensiana,
Issel. Gibbula
Doria,
Caramagna. ,,
Stdmondiana,
Issel. Minolia
Catfassii,
Caramagna. 5,
sprrata,
Sowb. ( Minolia
Catfassii,
Caramagna
,,
rotelleformis,
Phil. ,,
rotelleformis,
Phil. ,,
tridentata
(=
curta,
Sowb.),
Mich. Margarita
variabilis,
A. Ad. Onoba delicata, Monteros. Vitrinella
Menegherwi,Caramagna. Turritella
columnaris,
Kien. ,
maculata,
Rve. Calyptrea
cicatricosa,
Kve. x
9»,
Ldgariana,
Melv. Phasianella
lineolata,
Wood. Phasianella
lineolata,
Wood. GASTROPODA. »,
tuberosum,
Fabr. »»
Yerbury?,
Smith. Vertagus cedo-nulli, Sowb. »,
Jasciatus,
Brug. ,,
obeliscus,
Brug. * Bittium chrysomallum, Melv. Triforis ecingulatus, A, Ad. ,,
eollaris,
Hinds. 5,
corrugatus,
Hinds. Littorina
ahenea,
Rve. 5,
grano-costata,
Rve. »,
Vatalensis,
Krauss. ,,
seabra,
L. Modulus
candidus,
Petit. ,,
tectum,
Gmel. Planaxis
breviculus,
Desh. 5, NSavignyt, Desh. ,,
suleatus,
Lk. Rissoina
Bertheloti,
Aud. ,,
¢lathrata,
A. Ad. 5, concinna, A. Ad. ,,
msculpta,
Melv. &
Stand. 5,
insignis,
Ad. &
Rve. 5
pachystoma,
Melv. 5,
Sequensiana,
Issel. ,,
Stdmondiana,
Issel. 5,
sprrata,
Sowb. ,,
tridentata
(=
curta,
Sowb.),
Mich. Onoba delicata, Monteros. Turritella
columnaris,
Kien. ,,
maculata,
Rve. Calyptrea
cicatricosa,
Kve. x
9»,
Ldgariana,
Melv. 5» equestris, L. Crepidula Walshi (= Plana, Ad. &
Rve.),
Herms. Narica
cancellata,
Chem. 5,
ligata,
Reel. Nerita
chrysostoma,
Recl. 5,
plexa,
Chem. »,
polita,
L. 5
Rumphi
Recl SCAPHOPODA. Dentalium
Shoplandi,
Jouss. Dentalium octagonum, Lk. Dentalium octagonum, Lk. Dentalium
Shoplandi,
Jouss. ]
=I SHOPLAND:
LIST
OF
MARINE
SHELLS
FROM PELECYPODA. PELECYPODA. Pholadidea
Fauroti,
Jouss. Parapholas
quadrizonalis,
Spelv. Martesia striata, L. Gastrochena
Ruppelli,
Desh. Aspergillum
vaginiferum,
Lk. Solen brevis, Gray. »
corneus,
Lk. », cultelus, L. »,
dactylus,
Jouss. 5,
Gould,
Conrad. truncatus, Sowb. Machera
Taponica,
Dkr. Novaculina xyreces, Melv. Tugonia nobilis, A. Ad. Corbula
Tahitensis,
Lk. Anatina
hispidula,
Cuy. »,
labiata,
Rve. ,,
subrostrata,
Lk. Thracia
Adenensis,
Melv. ,,
Australica,
Rve. Mactra achatina, Chem,
»
erista,
Jouss. 5, decora, Desh. »
Lauroti,
Jouss. »,
v. alba,
Jouss. Ludroria curta, Desh. », wmtermedia, Desh. Standella
Atgyptiaca,
Chem. 5, ¢capillacea, Desh. », NSolanderi, Gray. Raéta
Abercrombiei,
Mely. Cecella Zebuensis, Desh. Asaphis deflorata, L
Psammobia amianta, Melv. »,
contraria,
Desh. 5, elegans, Desh. 55 marmorea, Desh. », occidens, Chem. 5,
pallida,
Desh. »,
rubicunda,
Desh. Weinkaufi,
Crosse. Soletellina
Adamsi,
Desh. Tellina
Adenensis,
Smith. »» easeus, Sowb. 5,
concentrica,
Gould. sy core, J OUSE. 5,
erucigera,
Lk. 5, Deshayesit, Hanley. VOL V —JULY 1902
Tellina edentula, Spglr. »,
foliacea,
L. ,,
holabana,
Melv. »,
mflata,
Chem. 5, lacunosa, Chem. ety «= manumissa, Melv. ,,
methoria,
Mely. 5,
micans,
Hanley. 5, obliqua, Sowb. ,,
ostracea,
Lk. 5,
perplexa,
Hanley. 5,
Pharaonis,
Hanley. ,,
rastellum,
Hanley. »,
rubella,
Desh. rugosa, Born. scobinata, L. ,,
staurella,
Lk. ,,
subpallida,
Smith. ,,
sulcata,
Wood,
»,
virgata,
L. ,,
Yemenensis,
Melv. incre:
cluthratus,
Rye. x
»
epularis,
Melv. »
rythreus,
Bertrand. «5,
scalpellum,
Gray. Scrobicularia
Vaillanti,
Jouss. Amphidesma Chinense, A. Ad,
,, erenatum, Ad. & Ang. 5,
lamellosum,
Sowb. x 9» Shoplandi, Melv. * Cumingia occatilla, Melv. Paphia glabrata, Desh. Mesodésma obtusum, Cross. & F. Tivela ponderosa, Koch. Meretriz
lusoria,
Chem. Callista costata, Chem. 5, eryoma, L. florida,
Lk. ,,
lilacina,
Lk. ,,
umbonella,
Lk. Caryatis
Hebrea,
Lk. 5, pura, Desh. 5,
varians,
Hanley. Lioconcha lentiginosa, Chem. 4,
tegrina,
Lk. Crista pectinata, L. Circe
Arabica,
Chem. 5,
eallipyga,
Born. 12 Jouss. zonalis,
Spelv. . elli,
Desh. ferum,
Lk. uss. rad. owb. Dkr. s, Melv. Ad. Lk. Cuy. Lk. Melv. Rve. Chem,
. ss. ba,
Jouss. sh. Desh. ca,
Chem. Desh. Gray. i,
Mely. Desh. L
ta, Melv. esh. h. Desh. em. h. Desh. Crosse. Desh. Smith. . Gould. k. Hanley. Tellina edentula, Spglr. »,
foliacea,
L. ,,
holabana,
Melv. »,
mflata,
Chem. 5, lacunosa, Chem. ety «= manumissa, Melv. ,,
methoria,
Mely. 5,
micans,
Hanley. 5, obliqua, Sowb. ,,
ostracea,
Lk. 5,
perplexa,
Hanley. 5,
Pharaonis,
Hanley. ,,
rastellum,
Hanley. »,
rubella,
Desh. rugosa, Born. scobinata, L. PELECYPODA. ,,
staurella,
Lk. ,,
subpallida,
Smith. ,,
sulcata,
Wood,
»,
virgata,
L. ,,
Yemenensis,
Melv. incre:
cluthratus,
Rye. x
»
epularis,
Melv. »
rythreus,
Bertrand. «5,
scalpellum,
Gray. Scrobicularia
Vaillanti,
Jouss. Amphidesma Chinense, A. Ad,
,, erenatum, Ad. & Ang. 5,
lamellosum,
Sowb. x 9» Shoplandi, Melv. * Cumingia occatilla, Melv. Paphia glabrata, Desh. Mesodésma obtusum, Cross. & F. Tivela ponderosa, Koch. Meretriz
lusoria,
Chem. Callista costata, Chem. 5, eryoma, L. florida,
Lk. ,,
lilacina,
Lk. ,,
umbonella,
Lk. Caryatis
Hebrea,
Lk. 5, pura, Desh. 5,
varians,
Hanley. Lioconcha lentiginosa, Chem. 4,
tegrina,
Lk. Crista pectinata, L. Circe
Arabica,
Chem. 5,
eallipyga,
Born. 12 Tellina edentula, Spglr. Pholadidea
Fauroti,
Jouss. Tellina edentula, Spglr. »,
foliacea,
L. ,,
holabana,
Melv. »,
mflata,
Chem. 5, lacunosa, Chem. ety «= manumissa, Melv. ,,
methoria,
Mely. 5,
micans,
Hanley. 5, obliqua, Sowb. ,,
ostracea,
Lk. 5,
perplexa,
Hanley. 5,
Pharaonis,
Hanley. ,,
rastellum,
Hanley. »,
rubella,
Desh. rugosa, Born. scobinata, L. ,,
staurella,
Lk. ,,
subpallida,
Smith. ,,
sulcata,
Wood,
»,
virgata,
L. ,,
Yemenensis,
Melv. incre:
cluthratus,
Rye. x
»
epularis,
Melv. »
rythreus,
Bertrand. «5,
scalpellum,
Gray. Scrobicularia
Vaillanti,
Jouss. Amphidesma Chinense, A. Ad,
,, erenatum, Ad. & Ang. 5,
lamellosum,
Sowb. x 9» Shoplandi, Melv. * Cumingia occatilla, Melv. Paphia glabrata, Desh. Mesodésma obtusum, Cross. & F
Tivela ponderosa, Koch. Meretriz
lusoria,
Chem. Callista costata, Chem. 5, eryoma, L. florida,
Lk. ,,
lilacina,
Lk. ,,
umbonella,
Lk. Caryatis
Hebrea,
Lk. 5, pura, Desh. 5,
varians,
Hanley. Lioconcha lentiginosa, Chem. 4,
tegrina,
Lk. Crista pectinata, L. Circe
Arabica,
Chem. 5,
eallipyga,
Born. »,
foliacea,
L. Parapholas
quadrizonalis,
Spelv. Gastrochena
Ruppelli,
Desh. Aspergillum
vaginiferum,
Lk. Solen brevis, Gray. »
corneus,
Lk. 5,
Gould,
Conrad. truncatus, Sowb. truncatus, Sowb. h
k Machera
Taponica,
Dkr. Machera
Taponica,
Dkr. * Novaculina xyreces, Melv. i
bili
d * Novaculina xyreces, Melv. T
i
bili
A
Ad Tugonia nobilis, A. Ad. Tugonia nobilis, A. Ad. Corbula
Tahitensis,
Lk. Anatina
hispidula,
Cuy. Anatina
hispidula,
Cu
»,
labiata,
Rve. ,,
subrostrata,
Lk. * *Thracia
Adenensis,
Melv. ,,
Australica,
Rve. *Thracia
Adenensis,
Melv. A
li
R Mactra achatina, Chem, »
erista,
Jouss. »
erista,
Jouss. 5, decora, Desh. »
Lauroti,
Jouss. »,
v. alba,
Jouss. L d
i
t
D
h Ludroria curta, Desh. », wmtermedia, Desh. Standella
Atgyptiaca,
Chem. 5, ¢capillacea, Desh. », NSolanderi, Gray. Raéta
Abercrombiei,
Mely. Cecella Zebuensis, Desh. Mesodésma obtusum, Cross. & F. PELECYPODA. Asaphis deflorata, L * Psammobia amianta, Melv. »,
contraria,
Desh. 5, elegans, Desh. 55 marmorea, Desh. », occidens, Chem. florida,
Lk. 5,
pallida,
Desh. ,,
lilacina,
Lk. »,
rubicunda,
Desh. W i k
fi
C Weinkaufi,
Crosse. S l t lli
Ad
i
D
h Soletellina
Adamsi,
Desh. Tellina
Adenensis,
Smith. »» easeus, Sowb. 5,
concentrica,
Gould. sy core, J OUSE. 5,
erucigera,
Lk. 5, Deshayesit, Hanley. VOL. V.—JULY, 1902. Crista pectinata, L. sy core, J OUSE. 5,
erucigera,
Lk. Circe
Arabica,
Chem. Circe
Arabica,
Chem. 5, Deshayesit, Hanley. 5, Deshayesit, Hanley. 5,
eallipyga,
Born. 5,
eallipyga,
Born. 12 12 VOL. V.—JULY, 1902. a"~I«x PROCEEDINGS OF THE MALACOLOGICAL SOCIETY. Lucina concinna, H. Ad. 5,
dentifera,
Jouss. 5,
exasperata,
Rve. ,,
Lischeriana,
Issel. », gemma, Kve. 5,
Semperiana,
Issel. Loripes
clausa,
Philippi. Diplodonta
rotundata,
Mont. Pythina
paula,
A. Ad. Scintilla
faba,
Desh. 5, Obockensis, Jouss. ,,
ovulina,
Desh. 5» ptsum, Sowb. Crassatella
radiata,
Sowb. Cardita
antiquata,
Poli. ,,
semiorbiculata,
L. ,,
suleata,
Lk. 5,
variegata,
Brug. Nytilicardia
gubernaculum,
Rve. Mytilus
ater,
Zeleb. 5
pictus,
Born. Crenella
Cumingiana,
Dkr. Modiola
auriculata,
Krauss. »,
lignea,
Rve. ,,
NSirehensis,
Jouss. Lithodomus
cinnamomina,
Lk. ,,
Lrythreensis,
Jouss. ,,
lthophagus,
var.,
Lk. Septifer
excisus,
Wiegm. Avicula
marmorata,
Rve. Meleagrina
margaritrfera,
L. Malleus albus, Lk. Crenatula picta, Gmel. Pinna alta, Sowb. 5, bicolor, Chem. »,
nigra,
Chem. Arca
navicularis,
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Chem. ,,
tortuosa,
L. »,
xebra,
Swain. Barbatia
Domingensis,
Lk. Helblingi,
Chem. imbricata,
Brug. nivea, Gmel. obliquata, Wood. ? ? bl
9
alocardia
clathrata,
Rve. pharca
Natalensis,
Krauss. llea
concamerata,
Chem. Pectunculus
pectentformis,
Lk. Circe corrugata, Chem. ,,
intermedia,
Rve. »,
sertpta,
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éffossa,
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Sowb. 5,
floridus,
var.,
Lk. 5,
geographicus,
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litteratus,
L. .,
Malabaricus,
Chem. ,,
Obscuratus,
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se
y. guadriradiata,
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pinguis,
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radiatus,
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sulcosus,
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textriz,
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foliacea,
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Jouss. flammea,
Lk. 5,
Lamarck,
Gray. 5,
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Chem. Dosinia
alta,
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pubescens,
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radiata,
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porica,
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macrophylla,
Desh. Coralliophaga
Arabica,
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coralliophaga,
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decussata,
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Hemprichi,
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lyra,
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lapicidum,
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assimile,
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Australe,
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rubicundum,
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imbricata,
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Lk. Vulsella
lingua-felis,
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eucullata,
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luculentus,
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plica,
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sanguinolentus,
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Stngaporinus,
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L. 5, sguamosus, v. lividus, Rve. ,,
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Gmel. This file was generated 29 March 2024 at 06:43 UTC Copyright & Reuse Copyright Status: Public domain. The BHL considers that this work is no longer under
copyright protection. This document was created from content at the Biodiversity Heritage Library, the world's
largest open access digital library for biodiversity literature and archives. Visit BHL at
https://www.biodiversitylibrary.org. This file was generated 29 March 2024 at 06:43 UTC
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DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
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v.4, n.11, 2023 v.4, n.11, 2023 RECIMA21 - Ciências Exatas e da Terra, Sociais, da Saúde, Humanas e Engenharia/Tecnologia
1 Discente do Curso de Direito da Universidade Federal do Rio Grande do Norte - UFRN.
2 Docente Efetivo do Curso de Direito da Universidade Federal do Rio Grande do Norte - UFRN. PUBLICADO: 11/2023
RESUMO O trabalho apresenta uma análise dos direitos das pessoas com Transtorno do Espectro Autista (TEA)
na perspectiva da seguridade social, tendo como finalidade debater a construção histórica dos direitos
destas pessoas, desde a descoberta do autismo, até as garantias elencadas a ele, nos mais diversos
dispositivos do ordenamento jurídico brasileiro, suas funcionalidades e finalidades, especialmente no
âmbito da seguridade social. Para tanto, foi realizada uma abordagem qualitativa, buscando
compreender o direito dos autistas em todos os âmbitos, inclusive no da seguridade social, por meio
de uma investigação doutrinária, legislativa e jurisprudencial. Além disso, quanto aos objetivos, pode-
se mencionar que é uma pesquisa descritiva. No tocante aos procedimentos técnicos de pesquisa, é
possível afirmar que foi realizada uma revisão bibliográfica inicial, com o intuito de compreender o
autismo, partindo do seu conceito até o diagnóstico do TEA. Em momento posterior, é feita uma análise
legislativa, explorando o ordenamento jurídico voltado aos autistas, partindo da Constituição Federal
de 1988 e chegando à Lei Orgânica nº 8.742/1993, que concede aos autistas o Benefício de Prestação
Continuada. Em oportunidade conclusiva, foi utilizado, de modo explicativo, o método dedutivo, partindo
da generalização do tema, para chegar à conclusão, com a finalidade de estabelecer uma relação entre
o direito dos autistas e a seguridade social no Brasil. Assim, concluiu-se que, por diversas vezes, por
falta de conhecimento, ou até mesmo por causa das negativas em processos administrativos, as
pessoas com TEA são excluídas dos quadros da seguridade social brasileira. PALAVRAS-CHAVE: Transtorno do Espectro Autista. Direitos do Autista. Seguridade Social. Benefício
de Prestação Continuada. DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
RIGHTS OF PEOPLE WITH AUTISM: AN ANALYSIS OF THE TOPIC IN THE LIGHT OF THE
FEDERAL CONSTITUTION AND SOCIAL SECURITY
DERECHOS DE LAS PERSONAS CON AUTISMO: UN ANÁLISIS DEL TEMA A LA LUZ DE LA
CONSTITUCIÓN FEDERAL Y LA SEGURIDAD SOCIAL Dhelman Salete Melo de Medeiros1, Carlos Francisco do Nascimento2 e4114500
https://doi.org/10.47820/recima21.v4i11.4500 e4114500
https://doi.org/10.47820/recima21.v4i11.4500 ABSTRACT En una oportunidad concluyente, s
explicativa, a partir generalización del tema, para llegar
objetivo de establecer una relación entre los derechos de la
Brasil. Así, se concluyó que, en varias ocasiones, por desc
procesos administrativos, las personas con TEA son exclu
PALABRAS CLAVE: Desorden del espectro autista. Derec
Pago Continuo. RECIMA21 - REVISTA CIENTÍFICA MULTIDISCIPLINAR
ISSN 2675-6218 DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento RESUMEN Este Trabajo presenta los derechos de las personas con Trastorno del Espectro Autista (TEA) la
perspectiva de la seguridad social, con el objetivo de discutir la construcción histórica de los derechos
de estas personas, el descubrimiento del autismo, hasta las garantías enumeradas para el mismo, en
formas más diversas disposiciones del ordenamiento jurídico brasileño, sus funcionalidades y
finalidades. Para ello, se realizó través de un enfoque cualitativo, buscando comprender los derechos
de las personas autistas en todos los ámbitos, incluida la seguridad social, través de investigaciones
doctrinales, legislativas y jurisprudenciales. Además, en cuanto los objetivos, se puede mencionar que
es investigación descriptiva. En cuanto los procedimientos técnicos de investigación, es posible afirmar
que se realizó una investigación bibliográfica inicial, con el objetivo de comprender qué sería el autismo,
desde su concepto hasta el diagnóstico del TEA. En etapa posterior, se realizó un análisis legislativo,
explorando el ordenamiento jurídico dirigido a las personas autistas, a partir de la Constitución Federal
de 1988 y llegando a Ley Orgánica nº 8.742/1993, que concede a las personas autistas la Prestación
de Pago Continuo. En una oportunidad concluyente, se utilizó el método deductivo, de manera
explicativa, a partir generalización del tema, para llegar a la conclusión antes mencionada, con el
objetivo de establecer una relación entre los derechos de las personas autistas y la seguridad social en
Brasil. Así, se concluyó que, en varias ocasiones, por desconocimiento, o incluso por denegaciones en
procesos administrativos, las personas con TEA son excluidas de la seguridad social brasileña. PALABRAS CLAVE: Desorden del espectro autista. Derechos autistas. Seguridad Social. Beneficio de
Pago Continuo. PALABRAS CLAVE: Desorden del espectro autista. Derechos autistas. Seguridad Social. Beneficio de
Pago Continuo. ABSTRACT This work presents the rights of people with Autism Spectrum Disorder (ASD) from the perspective of
social security, with the aim of debating the historical construction of the rights of these people, from the
discovery of autism to the guarantees listed for them in the most diverse provisions of the Brazilian legal
system, their functionalities and purposes. To this end, it was carried out using a qualitative approach,
seeking to understand the rights of autistic people in all spheres, including social security, through
doctrinal, legislative and jurisprudential research. In addition, in terms of objectives, it can be mentioned
that this is a descriptive study. As for the technical research procedures, it is possible to state that an
initial bibliographical survey was carried out in order to understand what autism is, from its concept to
the diagnosis of ASD. Subsequently, a legislative analysis was carried out, exploring the legal system
for autistic people, starting with the 1988 Federal Constitution and ending with Organic Law nº
8,742/1993, which grants autistic people the Continuous Cash Benefit. In conclusion, the deductive
method was used in an explanatory way, starting from the generalization of the theme, to reach the
conclusion mentioned, with the aim of establishing a relationship between the rights of autistic people
and social security in Brazil. Thus, it was concluded that, several times, due to lack of knowledge, or
even due to denials in administrative processes, people with ASD are excluded from the Brazilian social
security system. KEYWORDS: Autism Spectrum Disorder. Autistic Rights. Social Security. Continuous Payment v.4, n.11, 2023 RECIMA21 - REVISTA CIE
ISSN 26
DIREITO DAS P
RESUMEN
Este Trabajo presenta los derechos de las personas co
perspectiva de la seguridad social, con el objetivo de discu
de estas personas, el descubrimiento del autismo, hasta la
formas más diversas disposiciones del ordenamiento
finalidades. Para ello, se realizó través de un enfoque cua
de las personas autistas en todos los ámbitos, incluida la
doctrinales, legislativas y jurisprudenciales. Además, en cu
es investigación descriptiva. En cuanto los procedimientos
que se realizó una investigación bibliográfica inicial, con el
desde su concepto hasta el diagnóstico del TEA. En etapa
explorando el ordenamiento jurídico dirigido a las personas
de 1988 y llegando a Ley Orgánica nº 8.742/1993, que co
de Pago Continuo. DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento Após esses grandes avanços, nas últimas décadas nota-se um considerável crescimento dos
números de diagnóstico do TEA, principalmente em crianças, fase em que o transtorno mais se
manifesta. O que não significa que não existem adultos autistas, exemplo disso é o famoso jogador de
futebol Lionel Messi, argentino de 36 (trinta e seis) anos de idade, eleito como melhor jogador do mundo
por oito vezes. Diante de todo o contexto mundial acerca do TEA, nasceu no ordenamento jurídico brasileiro a
necessidade de implementar leis e garantias que assegurassem a vida destes indivíduos. Com a luta
incessante, principalmente das famílias dos autistas, foi implementada a Lei nº 12.764, instituída em 27
de dezembro de 2012, conhecida como Lei Berenice Piana, que equiparou o TEA a uma deficiência,
ganhando a abrangência de vários efeitos legais já assegurados pela Constituição Federal e demais
dispositivos legais que versam sobre a deficiência. Mais recentemente, além da Lei Berenice Piana, o ordenamento jurídico brasileiro implementou
outro marco aos autistas, a Lei nº 13.977/2020, conhecida como Lei Romeo Mion, que garantiu aos
autistas, “portadores de uma deficiência invisível”, o atendimento prioritário em diversos
estabelecimentos, bem como instituiu a carteira de identificação dessas pessoas, assegurando tudo
aquilo já previsto nas leis anteriores sobre o tema. Além disso, com a equiparação do autismo a deficiência, estes foram abrangidos pela
Assistência Social Brasileira. Conforme a Lei nº 8.742/1993, Lei Orgânica da Assistência Social,
aquelas pessoas com deficiência que não possuem condições de prover sua subsistência ou de tê-la
provida por sua família têm direito ao recebimento de um salário-mínimo vigente no país. Contudo, essa assistência social, garantia disposta tanto na Constituição Federal de 1988 como
na Lei Orgânica da Assistência Social, no que diz respeito às pessoas com autismo, tem sido, por vezes
negada, sendo notória a grande dificuldade dos autistas e suas famílias para subsistirem, face as suas
demandas cotidianas, tratando-se de uma situação de marginalização social, marcando a inclusão
social dessas pessoas de uma forma desafiadora e, por vezes, incerta. Nesse viés, o presente trabalho fora dividido em quatro capítulos. No primeiro capítulo trata-se
da evolução histórica do conceito do Espectro Autista, níveis de comprometimento, diagnóstico e
tratamento do TEA. INTRODUÇÃO O Transtorno do Espectro Autista – TEA, é um tema que deve e merece ser estudado
multidisciplinarmente. Não há como descrever o autismo ou o comportamento das pessoas com
autismo sem antes fazer uma visita aos diversos ramos da ciência, desde o biológico até o social. O
presente trabalho, desse modo, inicialmente, debruça-se sobre a origem histórica do conceito de
autismo, os fatores desencadeadores do seu surgimento e busca compreender quais os direitos que
essas pessoas adquiriram no ordenamento jurídico brasileiro. O TEA, atualmente, é uma síndrome classificada como Transtorno do Desenvolvimento
Intelectual e mesmo com todos os avanços biológicos e tecnológicos, ainda não se consegue
esclarecer firmemente quais são os fatores desencadeantes para o seu surgimento. Desde o início dos
estudos sobre o assunto, já foram apontadas diversas causas para o surgimento do autismo, entre eles:
a prematuridade, fatores genéticos da família, pais acima dos quarenta anos de idade, baixo peso ao
nascer e até mesmo a relação entre a mãe e o nascituro. O TEA, atualmente, é uma síndrome classificada como Transtorno do Desenvolvimento
Intelectual e mesmo com todos os avanços biológicos e tecnológicos, ainda não se consegue
esclarecer firmemente quais são os fatores desencadeantes para o seu surgimento. Desde o início dos
estudos sobre o assunto, já foram apontadas diversas causas para o surgimento do autismo, entre eles:
a prematuridade, fatores genéticos da família, pais acima dos quarenta anos de idade, baixo peso ao
nascer e até mesmo a relação entre a mãe e o nascituro. Atualmente, o autismo define-se como um transtorno que evidencia o déficit em interação
social, falhas de comunicação com outras pessoas, estereótipos e padrões repetitivos, isolamento,
linguagem e habilidades pré-verbais, distúrbios das funções intelectuais, alterações afetivas, distúrbios
psicossomáticos, dentre alguns outros que contribuem para o diagnóstico. O autismo, na maioria dos casos, é diagnosticado até os três primeiros anos da vida de uma
pessoa, mas não impede que seja diagnosticado durante a adolescência ou fase adulta. No Brasil, este
é dado pela Classificação Internacional de Doenças sob a CID-10 F84.0 da OMS, podendo ser
classificado em 03 níveis: primeiro nível, onde os sintomas são mais leves e a pessoa consegue viver
com certa autonomia, segundo nível, com graus intermediários de dependência, em vários aspectos
da vida, e, terceiro nível, em que os acometidos são bastante dependentes de terceiros. 2 2 v.4, n.11, 2023 RECIMA21 - Ciências Exatas e da Terra, Sociais, da Saúde, Humanas e Engenharia/Tecnologia RECIMA21 - REVISTA CIENTÍFICA MULTIDISCIPLINAR
ISSN 2675-6218 DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento Busca-se evidenciar, com isso, o quão o tema é desafiador e instigante, viajando
por uma multidisciplinariedade para que se possa compreender o conceito e a identificação do autismo,
assim como uma rede de apoio centrada em profissionais multidisciplinares para que seja elaborado
um plano de tratamento eficaz e individual para cada pessoa com autismo, pontos fundamentais para
compreender a dimensão do que se trata e o que realmente é o Transtorno do Espectro Autista. Por conseguinte, no segundo capítulo é feito uma análise da tutela Constitucional no
ordenamento jurídico brasileiro, com o fim da análise das garantias elencadas as pessoas com
deficiência, como elas repercutem e de qual maneira são aplicadas às pessoas com autismo, numa
visão analítica da doutrina e legislação pertinentes. Já no terceiro capítulo tratar-se-á da análise legislativa das leis específicas do ordenamento
jurídico brasileiro relativas as pessoas com Transtorno do Espectro Autista, principalmente a Lei 3 3 v.4, n.11, 2023 RECIMA21 - REVISTA CIENTÍFICA MULTIDISCIPLINAR
ISSN 2675-6218 DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento Berenice Piana e a Lei Romeo Mion, que ampliam os direitos dos autistas e buscam efetivar as
garantias já estabelecidas da Lei Maior. Por fim, no quarto capítulo será tratado o direito do autista que não consegue prover o seu
próprio sustento ou tê-lo provido pela sua família, receber um salário-mínimo, no que se denomina de
Benefício de Prestação Continuada, conforme o que dispõe Lei Orgânica da Assistência Social. Com a análise desenvolvida no presente trabalho é possível verificar a dificuldade de conseguir
de fato ser assistido pela Assistência Social, tanto pela burocracia que envolve o procedimento
administrativo referente ao pedido do recebimento do benefício, quanto a dificuldade de submeter-se
ao judiciário para conseguir a efetivação dos direitos dessas pessoas, que foram negadas na via
administrativa, citando como exemplo, o caso do menor D.R.D.A.D. RECIMA21 - Ciências Exatas e da Terra, Sociais, da Saúde, Humanas e Engenharia/Tecnologia DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento mas ao mesmo tempo, sendo pessoas com uma memória excelente, utilizando o termo transtorno
autístico em associação à esquizofrenia infantil, mas diferenciando-o dela, colocando em evidência “um
distúrbio inato do contato afetivo”. mas ao mesmo tempo, sendo pessoas com uma memória excelente, utilizando o termo transtorno
autístico em associação à esquizofrenia infantil, mas diferenciando-o dela, colocando em evidência “um
distúrbio inato do contato afetivo”. Já aos meados do Século XX, dando continuidade aos estudos psiquiátricos da época, Hans
Asperger, médico pediatra, em 1944, publicou o escrito intitulado como “Psicopatologia Autista da
Infância” indo de encontro com o já estudado por Kanner, versando sobre os mesmos sintomas
apontados à síndrome, destacando que a prevalência do autismo se dá em meninos. Em detrimento do proposto até aquele momento, em 1947, Bender entendeu que para aqueles
sintomas estudados até então, se daria o nome de esquizofrenia infantil, haja vista ele considerar o
autismo como sendo a forma mais inicial da esquizofrenia (Gauderer,1992 apud Camargos 2005). Nos estudos sobre o tema, passada a segunda metade do século em comento, alguns
estudiosos, como o psicólogo Bruno Bettelheim, discordando de todos os supramencionados, tratavam
os sintomas correlatos como sendo uma psicose simbiótica, atribuindo a causa da doença ao
relacionamento entre a mãe e o filho, provocados por negligência materna, propagando a teoria das
“mães-geladeiras”, defendendo com veemência que o autismo surgia da vontade dos pais e não de um
fator neurológico ou genético (Bettleheim, 1967). Em busca de aprofundamentos e em continuidade ao histórico das pesquisas sobre o tema,
Michael Rutter, em 1967, considerou que a falta de interesse social, a incapacidade de elaboração de
linguagem responsiva, presença de conduta motora bizarra em padrões de brinquedo bastante
limitados e início precoce, antes dos 30 meses seriam as características principais do autismo, fazendo
certa análise crítica das evidências empíricas até então encontradas. Corroborando com o disposto por Rutter, o Conselho Consultivo Profissional da Sociedade
Nacional para Crianças e Adultos com Autismo dos Estados Unidos definiu o autismo como uma
síndrome que aparece antes dos 30 meses e que apresenta distúrbios nas taxas e sequências do
desenvolvimento, nas respostas e estímulos sensoriais, na fala, na linguagem e capacidades cognitivas
e na interação social (Ritvo; Freedman, 1978). 1. DO TRANSTORNO DO ESPECTRO AUTISTA - TEA Considerado atualmente como sendo um Transtorno do Desenvolvimento Intelectual,
posicionado na Classificação Internacional de Doenças com o código CID-10 F84, o Transtorno do
Espectro Autista, é uma síndrome que mais a mais vem sendo estudada por especialistas dos mais
diversos ramos, mas é tema de pesquisas desde o início do Século XX. O termo “autista”, de acordo com os estudos do médico pediatra Christian Gauderer (1997), foi
utilizado pioneiramente pelo psiquiatra Plouller, no início do século XX, mais precisamente em 1906,
para descrever um sinal crítico de isolamento aos pacientes, principalmente crianças, portadores de
esquizofrenia, o que fazia com que estes perdessem o contato com a realidade, dificultando a
comunicação social para com o meio externo. Entretanto, segundo Camargos (2005), somente em 1911 o psiquiatra Eugen Bleuler conseguiu
difundir o termo autismo na literatura médica, classificando-o como uma perda de contato com a
realidade, ocasionada pela grande dificuldade na comunicação interpessoal, referindo-se originalmente
como um transtorno básico da esquizofrenia, que consistia na limitação das relações pessoais e com
o mundo externo tendo como consequência o isolamento. Posteriormente, Jean Piaget dispôs o autismo como sendo o primeiro estágio no
desenvolvimento, dentro da normalidade, da inteligência das crianças, marcada pela presença de
fenômenos sensitivo-motores sem direção, acreditando que os autistas não possuíam a percepção dos
objetos, criando um mundo de sonhos (Stelzer, 2010). No ano de 1943, Leo Kanner distinguiu o autismo das demais psicoses que até então este era
relacionado, utilizando o termo definido por Bleuler (autismo), para intitular o transtorno atualmente
conhecido como sendo uma doença clínica, levando em consideração um estudo realizado em 11
crianças norte-americanas (oito meninos e três meninas) que apresentavam solidão e invariância,
levando-o a publicar o trabalho “Distúrbios autísticos do contato afetivo”. (Camargos, 2005) Kanner, um dos grandes expoentes acerca do tema até a atualidade, em seus estudos
empíricos e diante da análise do comportamento dos seus pacientes, percebeu que algumas crianças
já no início da vida não respondiam aos estímulos externos, não desenvolvendo interações sociais, 4 v.4, n.11, 2023 RECIMA21 - REVISTA CIENTÍFICA MULTIDISCIPLINAR
ISSN 2675-6218 DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento Deste modo, a partir das definições evidenciadas por Kanner, Rutter e demais estudiosos que
convergiram para um mesmo ideal, houve um lastreamento para os conceitos do autismo pela
Organização Mundial da Saúde e pela American Psychiatric Association na década de 1980, vejamos: RECIMA21 - Ciências Exatas e da Terra, Sociais, da Saúde, Humanas e Engenharia/Tecnologia
“Formaram a base para os critérios diagnósticos do autismo nas duas principais
classificações de transtornos mentais: a CID-9 (OMS, 1984) e o DSM-III-R (APA,
1980). Ambos tinham definições e critérios diagnósticos similares, mas diferenças na
conceituação: enquanto a CID-9 (OMS, 1984) conceituava o autismo como um subtipo
das psicoses com origem específica na infância, evoluindo para esquizofrenia, o DSM-
III-R (APA, 1980) o considerava um tipo de distúrbio global do desenvolvimento,
apresentando
psicopatologia
severa
com
distúrbios
evolutivos
precoces,
caracterizados por atrasos e distorções no desenvolvimento de habilidades sociais,
cognitivas e de comunicação. Com a evolução dos conceitos, o autismo, na CID-10
(OMS, 1993), passou a ser considerado um distúrbio do desenvolvimento, e, no DSM-
IV (APA, 1995), os transtornos globais do desenvolvimento (TGP) foram retirados do
eixo II (prognóstico pobre) e entraram no eixo I (distúrbios mais episódicos e
transitórios), com a possível implicação de que o autismo passou a ser considerado
como distúrbio que responde melhor às intervenções terapêuticas, e seus sintomas 5 v.4, n.11, 2023 “As características essenciais do transtorno do espectro autista são prejuízo
persistente na comunicação social recíproca e na interação social (Critério A) e
padrões restritos e repetitivos de comportamento, interesses ou atividades (Critério
B). Esses sintomas estão presentes desde o início da infância e limitam ou prejudicam
o funcionamento diário (Critérios C e D). O estágio em que o prejuízo funcional fica
evidente irá variar de acordo com características do indivíduo e seu ambiente.
Características
diagnósticas
nucleares
estão
evidentes
no
período
do
desenvolvimento, mas intervenções, compensações e apoio atual podem mascarar
as dificuldades, pelo menos em alguns contextos. Manifestações do transtorno
também variam muito dependendo da gravidade da condição autista, do nível de
desenvolvimento e da idade cronológica; daí o uso do termo espectro. O transtorno
do espectro autista engloba transtornos antes chamados de autismo infantil precoce,
autismo infantil, autismo de Kanner, autismo de alto funcionamento, autismo atípico,
transtorno global do desenvolvimento sem outra especificação, transtorno
desintegrativo da infância e transtorno de Asperger.” (APA, 2014). DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento podem sofrer maiores variações. Os sintomas necessários para determinar um quadro
de autismo foram reduzidos de dezesseis (DSM-III-R – APA 1980) para doze (DSM-
IV – APA, 1995), dentre quatro critérios diagnósticos principais” (Camargos, 2005). Assim, percebe-se que durante o século passado, vários pesquisadores debruçaram-se sobre
o tema, surgindo vários pressupostos teóricos, resultando em diversas nomenclaturas e hipóteses de
causa e diagnósticos para nomear o transtorno autista. Assim, percebe-se que durante o século passado, vários pesquisadores debruçaram-se sobre
o tema, surgindo vários pressupostos teóricos, resultando em diversas nomenclaturas e hipóteses de
causa e diagnósticos para nomear o transtorno autista. De início, pensou-se que o autismo era relacionado à esquizofrenia infantil, logo em seguida,
foi definido como um transtorno básico e limitante da esquizofrenia, chegando à definição de um
problema sensitivo-motor, para poder ganhar status de uma doença clínica de distúrbio afetivo, sendo
visto, por último, como um problema cognitivo. Atualmente, o Transtorno do Espectro Autista é considerado uma síndrome, podendo ser
definido como um distúrbio de desenvolvimento neurológico, apresentando sintomas como: isolamento
desde os primeiros dias de vida, condutas motoras repetitivas e estereotipias, linguagem e habilidades
pré-verbais, distúrbios das funções intelectuais, alterações afetivas, distúrbios psicossomáticos, dentre
alguns outros. De acordo do com o Manual de Diagnóstico e Estatístico de Transtornos Mentais, pode-se
caracterizar o autismo como: “As características essenciais do transtorno do espectro autista são prejuízo
persistente na comunicação social recíproca e na interação social (Critério A) e
padrões restritos e repetitivos de comportamento, interesses ou atividades (Critério
B). Esses sintomas estão presentes desde o início da infância e limitam ou prejudicam
o funcionamento diário (Critérios C e D). O estágio em que o prejuízo funcional fica
evidente irá variar de acordo com características do indivíduo e seu ambiente. Características
diagnósticas
nucleares
estão
evidentes
no
período
do
desenvolvimento, mas intervenções, compensações e apoio atual podem mascarar
as dificuldades, pelo menos em alguns contextos. Manifestações do transtorno
também variam muito dependendo da gravidade da condição autista, do nível de
desenvolvimento e da idade cronológica; daí o uso do termo espectro. RECIMA21 - REVISTA CIENTÍFICA MULTIDISCIPLINAR
ISSN 2675-6218 DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento O transtorno
do espectro autista engloba transtornos antes chamados de autismo infantil precoce,
autismo infantil, autismo de Kanner, autismo de alto funcionamento, autismo atípico,
transtorno global do desenvolvimento sem outra especificação, transtorno
desintegrativo da infância e transtorno de Asperger.” (APA, 2014). “As características essenciais do transtorno do espectro autista são prejuízo
persistente na comunicação social recíproca e na interação social (Critério A) e
padrões restritos e repetitivos de comportamento, interesses ou atividades (Critério
B). Esses sintomas estão presentes desde o início da infância e limitam ou prejudicam
o funcionamento diário (Critérios C e D). O estágio em que o prejuízo funcional fica
evidente irá variar de acordo com características do indivíduo e seu ambiente. Características
diagnósticas
nucleares
estão
evidentes
no
período
do
desenvolvimento, mas intervenções, compensações e apoio atual podem mascarar
as dificuldades, pelo menos em alguns contextos. Manifestações do transtorno
também variam muito dependendo da gravidade da condição autista, do nível de
desenvolvimento e da idade cronológica; daí o uso do termo espectro. O transtorno
do espectro autista engloba transtornos antes chamados de autismo infantil precoce,
autismo infantil, autismo de Kanner, autismo de alto funcionamento, autismo atípico,
transtorno global do desenvolvimento sem outra especificação, transtorno
desintegrativo da infância e transtorno de Asperger.” (APA, 2014). No Brasil, conforme já supramencionado, o diagnóstico do autismo é dado pela Classificação
Internacional de Doenças sob a CID-10 F84.0 da OMS, adotada desde 1996 e pelo DSM-V da American
Psychiatric Association, como Transtorno do Desenvolvimento Intelectual e contido na classificação do
Transtorno do Espectro Autista - TEA, antes chamado de Transtorno do Desenvolvimento Global, que
além do transtorno autista engloba também o transtorno de Asperger, o transtorno desintegrativo da
infância, o transtorno de Rett e o transtorno global do desenvolvimento sem outra especificação do
DSM-IV (APA, 2014). A National Autistic Society (2008) estimou que as causas do autismo poderiam ser muitos
fatores, inclusive vacinas, alergias alimentícias, intoxicação por materiais pesados, exposição precoce
à televisão, idade avançada dos pais, tecnologia sem fio, entre outros fatores (Orrú, 2009ª; Goldstein,
2006). No entanto, até hoje, nunca conseguiu comprovar-se efetivamente a ligação entre tais fatores e
a síndrome. RECIMA21 - Ciências Exatas e da Terra, Sociais, da Saúde, Humanas e Engenharia/Tecnologia 6 6 v.4, n.11, 2023 DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento Diante dos graus de comprometimento do Espectro, nota-se que cada vez mais vem
aumentando o diagnóstico de portadores da doença, que acomete cerca 20 a cada 10 mil nascidos,
com prevalência maior entre as crianças do sexo masculino, mas com gravidade maior quando atinge
as crianças do sexo feminino. Dessarte: Quanto à distribuição por sexo, o autismo é muito mais comum em meninos do que
em meninas, sendo tipicamente relatadas razões de 4:1 ou 5:1. No entanto, quando
as meninas são afetadas, isto ocorre com maior gravidade. As comparações de várias
amostras sugerem que a maioria dos indivíduos autistas funciona dentro da faixa de
retardo mental (APA, 1995; Kaplan e Sadock, 1993; Lewis, Melvin, 1995; OMS, 1993). Concordando, Garfinkel e colaboradores (1992) relatam que os meninos superam as
meninas em todos os estudos de autismo, mas a proporção tem variado de 1,5 a 4,8
meninos para cada menina. Há alguma evidência de que a super-representação de
meninos é mais marcada na presença de QI mais elevado e de sintomas autísticos
mais clássicos (Camargos, 2005). retardo mental (APA, 1995; Kaplan e Sadock, 1993; Lewis, Melvin, 1995; OMS, 1993). Concordando, Garfinkel e colaboradores (1992) relatam que os meninos superam as
meninas em todos os estudos de autismo, mas a proporção tem variado de 1,5 a 4,8
meninos para cada menina. Há alguma evidência de que a super-representação de
meninos é mais marcada na presença de QI mais elevado e de sintomas autísticos
mais clássicos (Camargos, 2005). Se faz importante ressaltar que no Brasil, até o ano de 2022, não era feito nenhum estudo
oficial censitário pelo Instituto Brasileiro de Geografia e Estatística (IBGE), para que se fosse auferido
o exato número de autistas no país, dados esses que passarão a ser essenciais para a realização do
planejamento de políticas públicas acerca do diagnóstico precoce. No diagnóstico do TEA, em prática clínica, alguns especialistas relatam não haver tamanha
distinção entre as espécies acima referenciadas, mas sim, na gravidade do distúrbio, podendo ser
classificado em Grau Leve (Nível 1), Grau moderado (Nível 2), Grau severo (Nível 3), que são
revestidos se limitações e dependências distintas, variado de um indivíduo para outro. RECIMA21 - REVISTA CIENTÍFICA MULTIDISCIPLINAR
ISSN 2675-6218 DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento O Grau leve ou Nível 1, conforme os ensinamentos de Gaiato (2016) é aquele em que os
portadores, quando crianças tem um bom funcionamento, vão bem na escola, não necessitam de ajuda
para o desempenho de tarefas básicas de casa e mesmo possuindo características e sintomas do
espectro, precisam de um tratamento mínimo para o desenvolvimento e funcionalidade futuras. O Grau leve ou Nível 1, conforme os ensinamentos de Gaiato (2016) é aquele em que os
portadores, quando crianças tem um bom funcionamento, vão bem na escola, não necessitam de ajuda
para o desempenho de tarefas básicas de casa e mesmo possuindo características e sintomas do
espectro, precisam de um tratamento mínimo para o desenvolvimento e funcionalidade futuras. De acordo com Gaiato (2016), o Grau moderado ou Nível 2, seria aquele em que os portadores
possuem um nível intermediário do espectro, haja vista serem mais comprometidas, necessitando de
ajuda intensiva na escola e em casa, sendo preciso um tratamento em clínica especializada para que
tenham um “funcionamento mediano” na vida em geral. O Grau severo ou Nível 3 é definido como aquele em que os indivíduos possuem um
diagnostico grave, sendo pessoas bem comprometidas, vez que necessitam do tratamento de
especialistas e acompanhamento constante tanto na escola quanto em casa, sendo pouco funcionais
e dependentes (Gaiato, 2016). O Grau severo ou Nível 3 é definido como aquele em que os indivíduos possuem um
diagnostico grave, sendo pessoas bem comprometidas, vez que necessitam do tratamento de
especialistas e acompanhamento constante tanto na escola quanto em casa, sendo pouco funcionais
e dependentes (Gaiato, 2016). Assim, o autismo se manifesta de diferentes formas nos seus portadores, por isso se chama,
atualmente, como espectro autista, devido à alta dificuldade do diagnóstico, sendo necessária a análise
por uma equipe multiprofissional, com o intuito de afastar outras síndromes ou doenças, não havendo
nenhum fator biológico capaz de determiná-lo, diferentemente da Síndrome de Down, por exemplo, que
apresenta sua constatação até mesmo pela aparência pessoal. RECIMA21 - Ciências Exatas e da Terra, Sociais, da Saúde, Humanas e Engenharia/Tecnologia
Nesse sentido, alguns institutos médicos especializados no diagnóstico do TEA exigem que a
equipe médica multiprofissional apresente familiaridade com pessoas portadoras do TEA, de várias 7 7 v.4, n.11, 2023 RECIMA21 - REVISTA CIENTÍFICA MULTIDISCIPLINAR
ISSN 2675-6218 DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento idades e graus, capacitada para avaliar os múltiplos aspectos do desenvolvimento infantil e aberta à
interdisciplinaridade (Santana, 2013). Após confirmado o diagnóstico e o grau, o autista deve iniciar o
tratamento, na maior brevidade possível, de maneira individualizada e especializada, sendo assistido
de acordo com suas necessidades. Embora a maioria das crianças precise consultar um especialista, como um pediatra
do desenvolvimento-comportamental ou do neurodesenvolvimento, psicólogo,
neurologista ou psiquiatra, para uma avaliação diagnóstica, pediatras gerais e
psicólogos infantis confortáveis com a aplicação dos critérios do DSM-5 podem fazer
um diagnóstico clínico inicial. O diagnóstico clínico pode facilitar o início dos serviços. Neste momento, não existem testes laboratoriais que possam ser usados para fazer
um diagnóstico de TEA, portanto, é necessária uma revisão cuidadosa do histórico
comportamental da criança e observação direta dos sintomas. Para preencher os
critérios diagnósticos, os sintomas devem prejudicar a função. A avaliação formal da
linguagem, das habilidades cognitivas, adaptativas e do estado sensorial é um
componente importante do processo diagnóstico (Susan et al., 2020). Consoante a isso, identifica-se que o autismo não possui cura, até o momento, mas sim
tratamento, que deve ter a finalidade de possibilitar uma vida mais próxima à normalidade aos
portadores, minimizando todas as dificuldades que essas pessoas enfrentam. Além disso, com o mais
avançado conhecimento sobre a síndrome, deve haver cada vez mais melhoria aos tratamentos, para
que os autistas estejam mais próximos da vida daqueles que não padecem dessa excepcionalidade. (Melicio; Vendrametto,2021) Face as singularidades que cada portador do autismo possuí, existem algumas maneiras de
tratamento diferenciadas de acordo com a idade do indivíduo. Na infância, a prioridade deve ser voltada
para a interação social, terapia da fala, amparo da família e descoberta do novo. Na adolescência,
devem ser apresentadas intervenções terapêuticas em grupos sociais em terapias ocupacionais. Na
fase adulta, devem ser observadas as limitações apresentadas no decorrer da vida daquele indivíduo,
incluindo temas como a independência, por exemplo. Devido à heterogeneidade da sintomatologia e à gravidade do TEA, o diagnóstico
pode ser feito em crianças de faixas etárias distintas. RECIMA21 - Ciências Exatas e da Terra, Sociais, da Saúde, Humanas e Engenharia/Tecnologia 2. DA TUTELA CONSTITUCIONAL No Brasil, desde 2012, os portadores da síndrome autista são considerados pessoas com
deficiência. Inicialmente, as pessoas com deficiência, tanto física quando intelectual, eram mantidas à
margem da sociedade e mesmo com a Lei de Inclusão, tais pessoas eram caracterizadas como
pessoas de condutas atípicas, que seriam manifestações de comportamentos típicos de portadores de
síndromes e quadros psicológicos, neurológico ou psiquiátricos que ocasionam atrasos no
desenvolvimento e prejuízos no relacionamento social, em grau que requeira atendimento educacional
especializado (Neves, 2023). Com o passar dos anos, diante dos mais variados movimentos sociais, começou a luta para a
efetivação da garantia do espaço social, da implementação de políticas públicas e direitos sociais dos
autistas e um grande marco dessa batalha foi a instituição, em 2012, da Política Nacional de Proteção
dos Direitos da Pessoa com Transtorno do Espectro Autista que considerou os portadores da síndrome
autista como pessoas com deficiência, para todos os efeitos legais (Brasil, 2012). Nessa perspectiva, vê-se, para além das tutelas específicas, a Constituição da República
Federativa do Brasil de 1988 e na Declaração Universal dos Direitos Humanos, garantias básicas e
fundamentais que devem ser aplicadas a todos os indivíduos, de maneira a efetivar a garantia da
dignidade humana, por meio de direitos básicos elencados na lei maior, como: saúde, alimentação,
educação, trabalho, segurança, lazer, e assistência, de forma a concretizar uma vida digna a todas as
pessoas, pincipalmente aqueles que mais necessitam. Outrossim, nesse sentido dispõe Ingo Sarlet (2011): Temos por dignidade da pessoa humana a qualidade intrínseca e distintiva
reconhecida em cada ser humano que o faz merecedor do mesmo respeito e
consideração por parte do Estado e da comunidade, implicando, neste sentido, um
complexo de direitos e deveres fundamentais que assegurem a pessoa tanto contra
todo e qualquer ato de cunho degradante e desumano, como venham a lhe garantir
as condições existenciais mínimas para uma vida saudável, além de propiciar e
promover sua participação ativa e corresponsável nos destinos da própria existência
e da vida em comunhão com os demais seres humanos, mediante o devido respeito
aos demais seres que integram a rede da vida. Sob essa perspectiva, a Constituição Federal de 1988 elenca, em diversos dispositivos, vários
direitos e garantias especificamente às pessoas portadoras de deficiência, tais quais os Artigos: 6º, 23,
24, 203, 208, 227, dentre outros. De forma geral, o Art. RECIMA21 - REVISTA CIENTÍFICA MULTIDISCIPLINAR
ISSN 2675-6218 O que se observa é a idade em
que os sintomas foram reconhecidos, e não a idade em que eles realmente
começaram. Outrossim, percebe-se que a prevalência é mais abordada do que a
incidência (Susan et al., 2020). Portanto, evidencia-se que mesmo com pouco mais de um século de estudos acerca do tema,
se revelam grandes avanços desde a definição do que seria autismo até ao diagnóstico e tratamento,
que mesmo assim, ainda são dados e feitos com certa dificuldade, vez que em alguns casos, tanto a
omissão do diagnóstico quanto a constatação tardia, podem dificultar o tratamento daquele portador,
sendo melhor para ele a constatação precoce, de preferência até os 30 meses de vida, para que seu
desenvolvimento intelectual se dê o mais próximo da normalidade possível. 8 v.4, n.11, 2023 RECIMA21 - REVISTA CIENTÍFICA MULTIDISCIPLINAR
ISSN 2675-6218 DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento assistência social das pessoas portadoras de deficiência (Brasil, 1988). Conforme a regulamentação
transcrita cumulada com a Lei Orgânica da Assistência Social - Lei 8.742/93, extrai-se que a assistência
social deve resguardar o indivíduo e sua família no enfrentamento de suas dificuldades, com a função
de política pública, o que deve ser protegido pelo Estado. assistência social das pessoas portadoras de deficiência (Brasil, 1988). Conforme a regulamentação
transcrita cumulada com a Lei Orgânica da Assistência Social - Lei 8.742/93, extrai-se que a assistência
social deve resguardar o indivíduo e sua família no enfrentamento de suas dificuldades, com a função
de política pública, o que deve ser protegido pelo Estado. A Assistência Social, conforme Artigo 203 da CRFB/88, será prestada a quem dela necessitar,
independentemente de contribuição à seguridade social e tem por objetivos a habilitação e reabilitação
das pessoas portadoras de deficiência e a promoção de sua integração à vida comunitária, como
também a garantia de um salário-mínimo de benefício mensal à pessoa portadora de deficiência que
comprove não possuir meios de prover sua própria manutenção ou tê-la provida por sua família (Brasil,
1988). Por outro lado, no que se trata da saúde pública, as pessoas com autismo encontram amparo
tanto na Constituição Federal de 1988, em seu Artigo 196, quanto na Lei Federal 7.853/1989 que
dispõe sobre o apoio às pessoas portadoras de deficiência. Enquanto tutela acerca do tratamento
adequado em estabelecimentos de saúde públicos e privados específicos com profissionais
multidisciplinares, aquela prevê que a saúde é um direito de todos e dever do Estado, garantido por
meio de políticas sociais e econômicas, visando acesso universal e igualitário as ações e serviços. Concomitantemente, conforme Art. 277, §1º, II, da CRFB/88, o Poder Público deve promover
programas de prevenção e atendimento especializado. Em paralelo a legislação pátria, no campo da saúde, na Convenção sobre os Direitos da Pessoa
com Deficiência (UNESCO, 2006), os Estados endossaram o entendimento que as pessoas com
deficiência têm o direito de gozar de um elevado estado de saúde e principalmente o direito de não
serem discriminados em razão da sua deficiência, em nenhum âmbito. 2. DA TUTELA CONSTITUCIONAL 6º da CRFB/88 elenca que são direitos sociais a educação, a saúde, o
trabalho, o lazer, a segurança, a Previdência Social, a proteção à maternidade e à infância, a
assistência aos desamparados, na forma da Constituição (Brasil, 1988). Nesse viés, se pode extrair
que tal dispositivo está elencado de modo a assegurar o mínimo para se ter uma vida digna,
estabelecendo a todos os cidadãos, em especial, os portadores de deficiência e hipossuficientes, uma
melhor condição de vida, partindo da igualdade social. Em acréscimo, no tocante à assistência social e a saúde, o Art. 23, II, da CRFB/88 dispõe que
é competência comum da União, dos Estados, municípios e Distrito Federal cuidar da saúde e RECIMA21 - Ciências Exatas e da Terra, Sociais, da Saúde, Humanas e Engenharia/Tecnologia 9 v.4, n.11, 2023 RECIMA21 - REVISTA CIENTÍFICA MULTIDISCIPLINAR
ISSN 2675-6218 DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento RECIMA21 - Ciências Exatas e da Terra, Sociais, da Saúde, Humanas e Engenharia/Tecnologia DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento currículo apropriado, arranjos organizacionais, estratégias de ensino, uso de recurso
e parceria com as comunidades. (Declaração de Salamanca, 1994) Em conseguinte, a Lei 8.069/90 que institui o Estatuto da Criança e do Adolescente (ECA),
garantiu em seu artigo 54, III, o atendimento educacional especializado às crianças e adolescentes com
deficiência, preferencialmente na rede regular de ensino (Brasil, 1990). E em 1996, surgiu a Lei de
Diretrizes e Bases da Educação (LDB), que outorgou em seu capítulo V, a Educação Especial. Além disso, a UNICEF (2006), na Convenção sobre os Direitos da Pessoa com Deficiência,
exige aos Estados a garantia de que as pessoas especiais não estejam excluídas do sistema
educacional somente por possuírem alguma deficiência, e que devem ser criadas políticas públicas que
permitam a sua inclusão, com o desenvolvimento da personalidade, estimulando a criatividade e suas
habilidades intelectuais e físicas. No tocante ao direito ao trabalho, a Constituição Federal, dispõe em seu Art. 7º, XXI, cumulado
com a Lei nº 13.146/2015 – Lei brasileira de Inclusão a proibição de qualquer discriminação no tocante
a salário e critérios de admissão do trabalhador portador de deficiência. Atrelado a isso, a Lei 8.212/1991, que dispõe sobre os Planos de Benefícios da Previdência
Social e dá outras providências, em seu Art. 93 estabelece, in verbis, que: Art. 93. A empresa com 100 (cem) ou mais empregados está obrigada a preencher
de 2% (dois por cento) a 5% (cinco por cento) dos seus cargos com beneficiários
reabilitados ou pessoas portadoras de deficiência, habilitadas, na seguinte
proporção:
I - até 200 empregados.............................................................................................2%;
II - de 201 a 500.......................................................................................................3%;
III - de 501 a 1.000...................................................................................................4%;
IV - de 1.001 em diante. ..........................................................................................5%. V - (VETADO). (grifos nossos) Desta maneira, não só o constituinte como os demais legisladores, admitem o compromisso
em defender, no ambiente do trabalho, as pessoas com deficiência, assegurando que as eventuais
limitações que estes possuam sejam compatíveis com suas atividades profissionais, promovendo a
estes, desenvolvimento intelectual, relações interpessoais, bem como a igualdade entre os cidadãos,
prevista na Constituição de 1988. Além disso, da análise da Lei 13.370/2016, observamos que o servidor público federal que
tenha cônjuge, filho ou dependente com deficiência de qualquer natureza tem direito ao horário
especial, com redução de até 50% da carga horária de trabalho, sem prejuízo dos vencimentos (Brasil,
2016). DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento No ano de 2011, o Governo Federal brasileiro lançou o Plano Nacional dos Direitos da Pessoa
Com deficiência – Viver sem limite, por meio do Decreto 7.612, ressaltando o compromisso do Estado
com as prerrogativas da Convenção supracitada, ratificando-a com equivalência de Emenda
Constitucional, apresentando programas de acesso à educação, inclusão, acessibilidade e saúde
(Brasil, 2011). Quanto a educação, da leitura do Art. 208, III, da Constituição Federal de 1988, vê-se a garantia
do atendimento educacional especializado aos portadores de deficiência, principalmente no ensino
regular (Brasil, 1988). A Lei Federal 7.853/1989 fortaleceu o texto constitucional ao estabelecer “normas
gerais para assegurar o pleno exercício dos direitos pessoais e sociais e sua efetiva integração social
pelas pessoas com deficiência (Brasil, 1989)” Diante disso, em 1994 surge o conceito educação inclusiva, com a Declaração de Salamanca,
ordenada na Conferência Mundial da Educação Especial: Princípio fundamental da escola inclusiva é o de que todas as crianças devem
aprender juntas, sempre que possível, independentemente de quaisquer dificuldades
ou diferenças que elas possam ter. Escolas inclusivas devem reconhecer e responder
às necessidades diversas de seus alunos, acomodando ambos os estilos e ritmos de
aprendizagem e assegurando uma educação de qualidade a todos através de um RECIMA21 - Ciências Exatas e da Terra, Sociais, da Saúde, Humanas e Engenharia/Tecnologia 10 v.4, n.11, 2023 RECIMA21 - REVISTA CIENTÍFICA MULTIDISCIPLINAR
ISSN 2675-6218 DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento Por analogia, a lei deve se estender também a qualquer funcionário público de qualquer esfera,
inclusive os militares, desde que se comprove que não há ninguém que possa acompanhar a pessoa
com deficiência, o que lhe causa prejuízo no desenvolvimento. Consoante ao direito previdenciário, a pessoa com Espectro Autista possui, conforme já fora
citado no tocante a Assistência Social, alguns benefícios previdenciários, sendo assegurado a proteção
previdenciária pelo Benefício de Prestação Continuada instituído pela Lei Orgânica da Assistência
Social, desde que não tenha condições de prover o seu sustento, ou tê-lo provido pela própria família. RECIMA21 - Ciências Exatas e da Terra, Sociais, da Saúde, Humanas e Engenharia/Tecnologia 11 11 v.4, n.11, 2023 DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento Logo, o BPC, é adquirido quando não se cumula qualquer outro benefício de seguridade social
e desde que os requisitos de aprovação estejam preenchidos. Assim, apresenta importante função
social, que é assegurar a proteção e o cuidado para com as pessoas com deficiência. Será dedicado
adiante um tópico específico acerca do BPC/LOAS. Assim, notamos que o ordenamento jurídico brasileiro, precipuamente a Constituição Federal
de 1988, dedicou-se a elencar diversos direitos sociais a pessoa com deficiência a serem ofertados
pelo Estado e pela sociedade. Embora, efetivamente, algumas vezes só sejam conquistados pela luta
dos portadores do Espectro Autista e suas famílias, o ordenamento segue num avanço crescente
relativo ao tema. RECIMA21 - Ciências Exatas e da Terra, Sociais, da Saúde, Humanas e Engenharia/Tecnologia RECIMA21 - REVISTA CIENTÍFICA MULTIDISCIPLINAR
ISSN 2675-6218 DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento RECIMA21 - REVISTA CIENTÍFICA MULTIDISCIPLINAR
ISSN 2675-6218 DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento um importante instrumento no combate à desigualdade social que essas pessoas enfrentavam até
então, agregando aos mesmos uma série de benefícios. um importante instrumento no combate à desigualdade social que essas pessoas enfrentavam até
então, agregando aos mesmos uma série de benefícios. Além disso, a Lei também prevê a criação de diversas políticas públicas para a promoção,
proteção e garantias dos direitos de pessoas com autismo, como a criação de sistemas de informação
sobre autismo, a formação de profissionais para o atendimento dessas pessoas, campanhas de
conscientização, apoio às famílias e algumas outras. O artigo 2º e seus incisos demonstram a relevância que a sociedade se encarrega na inclusão
e no desenvolvimento dos autistas, promovendo o incentivo a entrada no mercado de trabalho,
capacitação profissional voltada ao atendimento dos portadores de TEA e a produção de pesquisas
para agregar conhecimento sobre o TEA. Entende-se a Lei Berenice Piana como sendo uma garantidora dos direitos humanos das
pessoas com TEA, ao assegurar que estes não serão submetidos a tratamento desumano ou
degradante, não serão privados de sua liberdade ou do convívio familiar, nem sofrerá discriminação
por motivo da deficiência (Brasil, 2012). Nesse viés, é nítido que a Lei nº 12.764 trouxe respaldo jurídico de modo a assegurar os direitos
das pessoas com autismo, oportunizando aos portadores do TEA a conquista daquilo que tanto fora
almejado, depois de muito lutar para garantir a efetivação dos direitos na esfera legislativa. Com o desenvolvimento natural da sociedade e sua consequente evolução, o ordenamento
jurídico deve ser atualizado e ampliado. Nesse sentido, a Lei Berenice Piana teve alguns dispositivos
originários alterados pela Lei nº 13.977/2020, conhecida como Lei Romeo Mion. A Lei Romeo Mion, dentre várias atribuições, garante aqueles que não possuem fenótipo
aparente do autismo a prioridade no atendimento em todos os serviços de saúde, sejam privados ou
públicos, garantindo, conforme já previsto na Lei Berenice Piana, atenção adequada e especializada
aqueles que tanto necessitam, incentivando, ademais, pesquisas sobre o autismo, promovendo ações
de conscientização para combater a discriminação e o preconceito relacionados ao tema. 3. DA TUTELA ESPECÍFICA No ordenamento jurídico brasileiro, existem várias Leis e Políticas Nacionais que versam
especificamente sobre os direitos das pessoas com Transtorno do Espectro Autista. A legislação sobre
o autismo, é uma garantia do Estado, cabendo a ele e seus órgãos assegurar às pessoas portadoras
de autismo o pleno exercício dos direitos sociais basilares, decorrentes da Constituição Federal de
1988. A Política Nacional de Proteção dos Direitos da Pessoa com Transtorno do Espectro Autista,
Lei nº 12.764, instituída em 27 de dezembro de 2012, conhecida como Lei Berenice Piana é um marco
na história de lutas dos portadores do Espectro Autista, ao garantir uma qualidade de vida igualitária e
melhor aos portadores. A referida lei, foi a primeira legislação federal sobre os autistas, os equiparando as pessoas
com deficiência, estendendo àqueles as garantias que estes já possuíam e atribuindo outras. Sancionada numa época em que pouco se conhecia do assunto no Brasil, esta determina o acesso a
um diagnóstico precoce, tratamentos específicos, terapias e medicamentos fornecidos pelo SUS, à
educação e ao ensino profissionalizante, à residência protegida, ao mercado de trabalho, à previdência
social e à assistência social, além dos direitos sociais já previstos na CF/88, que propiciem a igualdade
de oportunidades. Nesse ínterim, as pessoas com TEA foram abrigadas também pela Lei nº 13.146/2015 -
Estatuto da Pessoa com Deficiência e pela Convenção das Nações Unidas sobre os Direitos das
Pessoas com Deficiência, promulgada por meio do Decreto nº 6.949/2009. De acordo com os ensinamentos de Carlos Eduardo Amaral (2016), a Lei Berenice Piana, é
fruto do projeto de lei do Senado Federal nº 168/2011, proposta pela Comissão de Direitos Humanos e
Legislação Participativa, decorrente da sugestão apresentada pela Associação em Defesa do Autista,
a qual, Berenice Piana é diretora, ocupando esse cargo em razão de ser mãe do autista Dayan, tendo
estudado sozinha e chegando a conclusão do diagnóstico do garoto, pois no Rio de Janeiro, local em
que vivia, não haviam médicos especialistas em autismo. O Artigo 1º, § 2º, da referida Lei, dispõe, de imediato, que a pessoa com Transtorno do Espectro
Autista é considerada pessoa com deficiência para todos os efeitos legais (Brasil, 2012), se tornando 12 v.4, n.11, 2023 I - Nome completo, filiação, local e data de nascimento, número da carteira de
identidade civil, número de inscrição no Cadastro de Pessoas Físicas (CPF), tipo
sanguíneo, endereço residencial completo e número de telefone do identificado; RECIMA21 - Ciências Exatas e da Terra, Sociais, da Saúde, Humanas e Engenharia/Tecnologia DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento II - Fotografia no formato 3 (três) centímetros (cm) x 4 (quatro) centímetros (cm) e
assinatura ou impressão digital do identificado;
III - nome completo, documento de identificação, endereço residencial, telefone e e-
mail do responsável legal ou do cuidador;
IV - Identificação da unidade da Federação e do órgão expedidor e assinatura do
dirigente responsável. II Fotografia no formato 3 (três) centímetros (cm) x 4 (quatro) centímetros (cm) e
assinatura ou impressão digital do identificado;
III - nome completo, documento de identificação, endereço residencial, telefone e e-
mail do responsável legal ou do cuidador; p
g
III - nome completo, documento de identificação, endereço residencial, telefone e e-
mail do responsável legal ou do cuidador; p
g
;
IV - Identificação da unidade da Federação e do órgão expedidor e assinatura do
dirigente responsável. Com a CIPTEA, permite-se a contagem das pessoas com TEA no território nacional, no
entanto, não com precisão, devido ao fato de serem expedidas somente mediante requerimento e ao
fato de que, infelizmente, nem todas as famílias terem conhecimento sobre a legislação vigente. A Lei Romeo Mion assegura o reconhecimento prioritário de todos os indivíduos portadores do
autismo, inclusive aos imigrantes detentores de visto temporário ou de autorização de residência,
residente fronteiriço ou solicitante de refúgio (Brasil, 2020). Deste modo, a Lei Romeo Mion, ao implantar a garantia da expedição da “carteirinha dos
autistas”, auxiliando na identificação dos portadores, além de contemplar, não só os nacionais, como
também alguns estrangeiros, evita vários constrangimentos às famílias e aos portadores e os assegura
a prioridade no atendimento, em diversos locais, exibindo apenas um único documento que comprova
a sua qualidade. Além das Leis específicas mencionadas, que tutelam sobre os direitos aos autistas mais
abrangentes, existem leis esparsas que asseguram mais direitos aos autistas, a saber: a Lei
8.899/1994, que assegura a gratuidade no transporte coletivo interestadual às pessoas portadoras de
deficiência que façam prova dispor de apenas dois salários-mínimos para subsistência mensal; a Lei
10.098/2000, que estabelece normas gerais e critérios básicos para a promoção da acessibilidade das
pessoas portadoras de deficiência ou com mobilidade reduzida; a Lei 10.048 que dá prioridade de
atendimento às pessoas autistas; a Lei 14.626/2023 que assegura que as empresas públicas de
transporte e as concessionárias de transporte público reservarão assentos identificados às pessoas
com transtorno do espectro autista. RECIMA21 - REVISTA CIENTÍFICA MULTIDISCIPLINAR
ISSN 2675-6218 Além disso, a referida legislação assegura atendimento preferencial em estabelecimentos
privados e repartições públicas. Para tanto, a Lei Romeo Mion ao adicionar o Art. 3º-A à Lei Berenice
Piana, instituiu a criação da Carteira de Identificação da Pessoa com Transtorno do Espectro Autista –
CIPTEA, que permite aos portadores, que se reitera,
não
possuem
garantia
visual
de
reconhecimento do autismo, a possibilidade de serem identificados com um único documento de
expedição gratuita (Brasil, 2020). A CIPTEA, deve ser expedida pelos órgãos responsáveis pela execução da Política Nacional
de Proteção dos Direitos da Pessoa com Transtorno do Espectro Autista dos Estados, do Distrito
Federal e dos municípios, mediante requerimento, que deverá ser acompanhado do relatório médico
contendo a CID do TEA e conter informações como: 13 v.4, n.11, 2023 DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento Outrossim, para lá das leis pátrias, existem, em todo o território nacional, Leis Estaduais que
asseguram a isenção tributária de alguns impostos às pessoas com autismo. No Estado do Rio Grande
do Norte, por exemplo, a Lei nº 10.632/2019 assegura aos autistas isenção no tocante ao IPVA de
veículos de passeio, adquiridos diretamente ou pelos representantes legais dos autistas, limitando o
benefício a 01 (um) veículo por beneficiário, porém, sem limitar o modelo ou cilindradas do veículo. (Rio
Grande do Norte, 2019) RECIMA21 - REVISTA CIENTÍFICA MULTIDISCIPLINAR
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Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento de benefício à pessoa com deficiência e ao idoso que comprovem não possuir meios de prover a própria
manutenção ou tê-la provida por sua família (Brasil, 1993). de benefício à pessoa com deficiência e ao idoso que comprovem não possuir meios de prover a própria
manutenção ou tê-la provida por sua família (Brasil, 1993). Diferentemente do que muitas pessoas costumam falar, o Benefício de Prestação Continuada
(BPC) não é um benefício previdenciário, mas sim um benefício assistencial que visa proteger
justamente aqueles que por alguma razão não conseguiram ou são incapazes de contribuir com o
Instituto Nacional do Seguro Social – INSS e não pode ser cumulado com qualquer outro benefício da
seguridade social, como aposentadoria ou pensão. Em razão da sua natureza, caso o beneficiário
faleça, sua família ou até mesmo seus dependentes não terão direito ao recebimento de pensão por
morte. Assim, podemos definir o BPC às pessoas com deficiência, em especial aos autistas, como
sendo um auxílio financeiro destinado aqueles que necessitam de uma atenção maior por parte do
Estado, por necessitarem de tratamentos multidisciplinares e muitas vezes necessitarem da supervisão
diária dos seus genitores ou responsáveis, que não conseguem auferir renda própria. O valor do
benefício é de um salário-mínimo vigente, não tendo nenhum acréscimo como o 13º salário. Para fazer jus ao recebimento do BPC a pessoa com autismo pode ter qualquer idade de vida,
mas não basta apenas a condição de pessoa com Transtorno do Espectro Autista. Conforme acima
transcrito, mesmo que estas pessoas detenham o direito inconteste ao benefício assistencial em
comento, elas precisam cumprir alguns requisitos, dentre os quais podem se extrair o requisito
econômico, qual seja, o de não conseguir prover ou tê-lo provido o seu sustento, evidenciando a
vulnerabilidade socioeconômica, com renda mensal per capita inferior a ¼ do salário-mínimo vigente. Ademais, além da vulnerabilidade econômica, deve a pessoa interessada estar cadastrada no
Cadastro Único para Programas Sociais do Governo Federal, o CadÚnico e possuir laudos médicos
fundamentados que indiquem a limitação que ela possui de realizar atividades cotidianas, ainda que
seja em grau leve, o que pode tornar o pedido burocrático, pois mesmo com toda a documentação
probatória, o requerente ainda passará por perícia social e médica que comprove suas condições. 4. DO DIREITO AO BENEFÍCIO DE PRESTAÇÃO CONTINUADA (LEI Nº 8.742/93) A pessoa com TEA, conforme já fora mencionado, assim como todos os cidadãos, tomando
por base o princípio constitucional da igualdade material, é detentora de alguns benefícios
previdenciários e protegidos, especialmente assegurados e assistidos pela Lei Brasileira de Inclusão
da Pessoa com Deficiência, Lei nº 13.146/2015. Nesse ínterim, ao dispor sobre a assistência social, o legislador brasileiro, através da Lei nº
8.742/1993 – Lei Orgânica da Assistência Social, garantiu, por objetivo, a garantia de um salário-mínimo RECIMA21 - Ciências Exatas e da Terra, Sociais, da Saúde, Humanas e Engenharia/Tecnologia 14 v.4, n.11, 2023 RECIMA21 - Ciências Exatas e da Terra, Sociais, da Saúde, Humanas e Engenharia/Tecnologia RECIMA21 - REVISTA CIENTÍFICA MULTIDISCIPLINAR
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CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento Infelizmente, nem todas as famílias de autistas possuem conhecimento acerca das Defensorias
Públicas, advogados dativos ou até mesmo de Núcleos de Prática Jurídica, que realizam atendimento
ao público de forma gratuita e acabam deixando os seus direitos “de lado” por entender que não tem
condições sequer de arcar com um advogado para ingressar com um processo no judiciário. Na cidade de Caicó, interior do Rio Grande do Norte, durante o ano 2022, a genitora de um
menor autista (D.R.D.A.D.) buscou o Núcleo de Prática Jurídica (NPJ) da UFRN – campus Caicó, por
enfrentar o problema supramencionado, acrescido do fato de o seu filho ter sofrido abandono
socioafetivo e financeiro pelo genitor, como também ao fato de o menor ser inteiramente dependente
dela para todas as suas atividades, pois apresenta agressividade a terceiros, impedindo que ela exerça
qualquer labor remunerado, visto que executa o trabalho de cuidado do filho diariamente. A genitora do menor D.R.D.A.D. informou e comprovou que ingressou com um processo
administrativo perante o INSS em fevereiro daquele ano. Instruiu o processo com todos os meios
probatórios que estavam ao seu alcance, inclusive o laudo médico datado de abril do ano de 2021 que
diagnosticou a criança com Transtorno do Espectro Autista e Transtorno de Déficit de Atenção com
Hiperatividade. Entretanto, após a perícia médica realizada pela autarquia, seu pedido inconteste, lhe
veio negado sob a alegação de que o menor não atendia ao critério de deficiência para ter acesso ao
BPC-LOAS. Dito desse contexto, não restou a genitora do menor uma alternativa senão ajuda do Núcleo
de Prática Jurídica para recorrer desta decisão no Judiciário, o que resultou no processo que tramitou
perante a 9ª Vara Federal da cidade de Caicó/RN. DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento O Benefício de Prestação Continuada deve ser solicitado, inicialmente, junto ao Instituto
Nacional do Seguro Social, pelo telefone da autarquia (135) ou pelo portal (https://meu.inss.gov.br),
devendo o requerente juntar todos os seus documentos pessoais, comprovantes de ganhos e gastos,
inscrição no CadÚnico, laudos e atestados e demais documentos pertinentes (Beschizza, 2023) Após o pedido, o INSS poderá conceder ou não o benefício, podendo a análise do pedido variar
entre 45 (quarenta e cinco) dias até 02 (dois) anos. Caso o pedido seja negado e a pessoa entenda
que a análise não foi justa, pode apresentar, com argumentos justificáveis, recurso administrativo ou
judicial para que tenha seu direito assegurado. Com isso, observa-se que, por vezes, mesmo que a parte burocrática seja cumprida, com muita
dificuldade pelos autistas e suas famílias, estes ainda têm os seus pedidos administrativos negados
pelo INSS, por motivos, que muitas vezes giram em torno de afirmar, mesmo com todos os laudos e
documentos comprobatórios que os necessitados não possuem nenhum grau de deficiência, os
levando a recorrer ao judiciário para ter seu direito assegurado. CIMA21 - Ciências Exatas e da Terra, Sociais, da Saúde, Humanas e Engenharia/Tecnologia 15 v.4, n.11, 2023 RECIMA21 - REVISTA CIENTÍFICA MULTIDISCIPLINAR
ISSN 2675-6218 No judiciário, a perícia médica realizada constatou
que de fato o menor era portador do Transtorno do Espectro Autista, conforme já era sabido e a
autarquia, que administrativamente negou o pedido do menor, após o laudo judicial, já no mês de
dezembro de 2022, ofereceu proposta de acordo para a implementação do Benefício de Prestação
Continuada ao menor, retroagindo a data de início do benefício à data que este havia sido solicitado
inicialmente. Por meio do caso concreto acima referido, é possível constatar que o menor autista ficou quase
um ano sem o recebimento daquilo que lhe era devido pelo fato de o INSS entender que todos aqueles
documentos comprobatórios da sua deficiência eram insuficientes. O que acaba demonstrando a
grande dificuldade dos autistas e suas famílias para subsistirem, face as suas demandas cotidianas,
tratando-se de uma situação de marginalização social, marcando a inclusão social dessas pessoas de
uma forma desafiadora e, por vezes, incerta, mesmo que estes provem que detém aquele direito. RECIMA21 - Ciências Exatas e da Terra, Sociais, da Saúde, Humanas e Engenharia/Tecnologia 6. CONSIDERAÇÕES E DISCUSSÃO Após a análise do tema, visualiza-se que o desenvolvimento do presente trabalho possibilitou,
após minuciosa análise doutrinária, o conhecimento sobre o que é o Transtorno do Espectro Autista,
concepções, seu tratamento, diagnóstico e quais direitos são assegurados a essas pessoas, se
revelando um universo particular, instigante, relevante e sempre atual. Por conseguinte, no tocante à análise jurisprudencial e legislativa, constatou-se que após o
aumento do número de autistas, devido aos diagnósticos mais precisos, houve a promulgação da Lei
Berenice Piana e da Lei Romeo Mion, as quais aos autistas brasileiros e até mesmo aos estrangeiros
residentes neste país, diversos benefícios e garantias foram assegurados. Assim, percebeu-se que, no Brasil, existe vasto ordenamento e estrutura legislativa garantidora
dos direitos das Pessoas com Transtorno do Espectro Autista. Entretanto, algumas vezes, as políticas
públicas asseguradoras dos direitos dessas pessoas não são efetivadas, o que evidencia que, na
prática, a situação das famílias com autistas ainda está aquém daquilo que é previsto legalmente. Apenas as normas escritas e formalizadas com aquilo que é desejável ao público em comento
não são suficientes. É necessária a efetivação e concretização das políticas públicas e o cumprimento
integral daquilo que é previsto, para que as famílias com autistas sejam incluídas no rol de pessoas
com vida digna, princípio basilar da Constituição da República Federativa do Brasil de 1988. É imprescindível que o poder público de fato forneça, gratuitamente, as condições necessárias
para que as pessoas com Transtorno do Espectro Autista tenham acesso ao estudo, ao trabalho, a
saúde, ao lazer, a segurança, a moradia, a assistência social e tudo aquilo que seja fundamental para
o desenvolvimento intelectual destas, garantindo a isonomia e não distinção entre as pessoas. Mesmo com o advento das Leis Berenice Piana e Romeo Mion terem trazidos enormes avanços
e melhorias em relação ao que era oferecido anteriormente aos autistas e seus familiares, ainda há um
longo caminho para que o ordenamento jurídico brasileiro trilhe em prol dos direitos e garantias de
pessoas autistas, visto que no Brasil, a primeira lei específica sobre o tema – a Lei Berenice Piana –
teve seu advento somente em 2012, tornando o autista pessoa com deficiência para todos os efeitos
legais. Nesse ínterim, no tocante a Assistência Social especificamente, notou-se, ainda mais, que é
uma questão que merece ser revista. 5. MÉTODOS A produção deste trabalho tomou como base a abordagem qualitativa, buscando compreender
o direito dos autistas em todos os âmbitos, inclusive no da seguridade social, por meio de uma
investigação doutrinária, legislativa e jurisprudencial. Para isso, pode-se mencionar que é uma pesquisa
descritiva. No tocante aos procedimentos técnicos de pesquisa, é possível afirmar que foi realizada
uma revisão bibliográfica inicial, com o intuito de compreender o autismo, partindo do seu conceito até RECIMA21 - Ciências Exatas e da Terra, Sociais, da Saúde, Humanas e Engenharia/Tecnologia 16 16 v.4, n.11, 2023 DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
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CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento o diagnóstico do TEA. Em momento posterior, é feita uma análise legislativa, explorando o ordenamento
jurídico voltado aos autistas, partindo da Constituição Federal de 1988 e chegando à Lei Orgânica nº
8.742/1993, que concede aos autistas o Benefício de Prestação Continuada. Em oportunidade
conclusiva, foi utilizado, de modo explicativo, o método dedutivo, partindo da generalização do tema,
para chegar à conclusão, com a finalidade de estabelecer uma relação entre o direito dos autistas e a
seguridade social no Brasil. RECIMA21 - Ciências Exatas e da Terra, Sociais, da Saúde, Humanas e Engenharia/Tecnologia RECIMA21 - REVISTA CIENTÍFICA MULTIDISCIPLINAR
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CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento multidisciplinares, que embora legalmente sejam obrigações do Sistema Único de Saúde brasileiro, por
vezes não são oferecidos adequadamente. Conforme pôde ser visto no primeiro capítulo do presente trabalho, o tratamento do Transtorno
do Espectro Autista exige médicos pediatras, nutricionistas, psicólogos, psiquiatras, terapeutas,
pedagogos, dentre vários outros profissionais que sejam capacitados o suficiente para que
semanalmente, ou até mesmo diariamente, acompanhe cada autista que deles necessitar e
infelizmente, não são todos os municípios brasileiros que contam com atendimento constante de tais
profissionais sem que se enfrente uma longa fila de espera. Além disso, existem inúmeros casos de abandono socioafetivo por parte de um dos genitores
quanto as crianças portadoras de autismo, ficando somente o outro responsável pelo sustento da
família, dedicando todo o seu tempo em prol daquela criança, o que nos leva a crer que somente um
salário-mínimo, ofertado a título de Benefício de Prestação Continuada, não é o suficiente para ajudar
na assistência social dessas referidas famílias, face a todos os motivos já expostos. Constatou-se que o procedimento administrativo além de extremamente burocrático ainda
apresenta negligência em algumas vezes, ocasionando em prejuízos, marginalização, isolamento
social e até mesmo angústia e sofrimento somente aos autistas e suas famílias, o que necessita de
mudança urgentemente. Ante o exposto, constata-se que os objetivos específicos e gerais dessa pesquisa foram
alcançados, pois ao longo do seu desenvolvimento fora possível analisar quais direitos o ordenamento
jurídico brasileiro assegura às pessoas com Transtorno do Espectro Autista e a atuação dos órgãos
que efetivam essas garantias, observando principalmente essa atuação perante a seguridade social. Deste modo, conclui-se que o acesso ao Benefício de Prestação Continuada por parte das
pessoas com TEA, enquanto garantia Constitucional e Social, deve ser contínuo e facilitado,
especialmente para as famílias que já possuem o diagnóstico do Transtorno do Espectro Autista em
laudos multidisciplinares fundamentados, haja vista que estes chegam a um diagnóstico depois de
meses de acompanhamento àquele autista em específico, não sendo uma perícia ofertada pelo Instituto
Nacional do Seguro Social em poucos minutos capaz de desfazer aquilo que levou dias para ser
construído. 6. CONSIDERAÇÕES E DISCUSSÃO Isso porque, conforme o próprio exemplo citado no capítulo 4 do
presente trabalho, não são raras as vezes em que os trabalhadores celetistas, pais de autistas, são
demitidos ou pedem demissão para cuidar dos seus filhos, que necessitam de tratamentos 17 v.4, n.11, 2023 RECIMA21 - Ciências Exatas e da Terra, Sociais, da Saúde, Humanas e Engenharia/Tecnologia RECIMA21 - REVISTA CIENTÍFICA MULTIDISCIPLINAR
ISSN 2675-6218 As fontes basilares usadas no presente trabalho apontam que a legislação, a doutrina e a
jurisprudência pátria seguem o mesmo caminho de efetivação dos direitos dos autistas previstos
legalmente, mas a prática legal ainda deixa algumas lacunas a serem preenchidas, as quais pretende-
se serem sanadas doravante. Portanto, conclui-se que o ordenamento jurídico brasileiro, embora passível de avanços e
melhorias, vem caminhando numa vertente crescente com responsabilidade no tocante ao direito das
pessoas com autismo, legislando no sentindo de proporcionar aos portadores de TEA uma maior
isonomia perante os demais brasileiros. 18 18 v.4, n.11, 2023 RECIMA21 - REVISTA CIENTÍFICA MULTIDISCIPLINAR
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CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL DIREITO DAS PESSOAS COM AUTISMO: UMA ANÁLISE SOBRE O TEMA À LUZ DA
CONSTITUIÇÃO FEDERAL E DA SEGURIDADE SOCIAL
Dh l
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dezembro de 1990, para estender o direito a horário especial ao servidor público federal que tenha
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Dhelman Salete Melo de Medeiros, Carlos Francisco do Nascimento DECLARAÇÃO DE SALAMANCA: sobre princípios, políticas e práticas na área das necessidades
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rio.br/25540/25540_1.PDF. Acesso em: 12 set. 2023. FERREIRA, Beatriz Soares de Araújo. Um estudo sobre as conexões afetivas no autismo. 2014. Dissertação (mestrado) - Pontifícia Universidade Católica. Rio de Janeiro, Departamento de Psicologia,
2014. Disponível em https://web.archive.org/web/20180719203801/https://www.maxwell.vrac.puc-
rio.br/25540/25540_1.PDF. Acesso em: 12 set. 2023. GAIATO, Mayra. Níveis de gravidade e os principais sintomas do autismo. [S. l.: s. n.], 2016. Disponível em: https://www.youtube.com/watch?v=AXOdHF3MBNM. Acesso em: 12 set. 2023. OLDSTEIN, Ariela. O autismo sob o olhar da terapia ocupacional. Minas Gerais: UFMG, 200 MAENNER, M. J.; WARREN, Z.; WILLIAMS, A. R. et al. Prevalência e características do transtorno
do espectro autista entre crianças de 8 anos / Autism and Developmental Disabilities Monitoring
Network, 11 Sites, Estados Unidos, 2020. MMWR Súmula 2023;72 (nº. SS-2):1–14. Disponível
em: http://dx.doi.org /10.15585/mmwr.ss7202a1. Acesso em: 12 set. 2023. MELICIO, Rose Kelly Irene Santos Da Conceição; VENDRAMETTO, Oduvaldo. Autista no mercado
de trabalho. São Paulo: Blucher, 2021. https://openaccess.blucher.com.br/download-pdf/495. Acesso
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15 set. 2023. 21
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Are Clinical Practice Guidelines for Low Back Pain interventions of high quality and updated? A systematic review using the AGREE II instrument
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Research Square (Research Square)
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Are Clinical Practice Guidelines of Low Back Pain interventions of high quality
and updated? A systematic review using the AGREE II instrument
Greta Castellini
IRCCS Istituto Ortopedico Galeazzi Are Clinical Practice Guidelines of Low Back Pain interventions of high quality
and updated? A systematic review using the AGREE II instrument
Greta Castellini
IRCCS Istituto Ortopedico Galeazzi Are Clinical Practice Guidelines of Low Back Pain interventions of high quality
and updated? A systematic review using the AGREE II instrument Are Clinical Practice Guidelines of Low Back Pain interventions of high quality
and updated? A systematic review using the AGREE II instrument Background The worldwide point prevalence of Low Back Pain (LBP) is 9.4% (95% CI, 9.0-9.8) in 2010. (1) Next to the common cold, it is one of the commonest reasons
why people seek their physician, with a substantial medical social and economic impact for individuals, families, and society due to its high direct and indirect
costs. (2-4) Back pain is a leading cause of years lived with disability and the first cause of activity limitation and absence from work. (1) The overall burden
of LBP arising from ergonomic exposures at work was estimated at 21.8 million [95% Confidence Interval (CI) 14.5–30.5] disability adjusted life years (DALYs)
in 2010. (5) In response to the global burden, numerous CPGs have been issued by medical societies and working groups, providing recommendations for its
diagnosis and management. (6, 7) While the principles for developing CPGs are well established, their proliferation has raised concern about quality. Published
CPGs appraisals report that the quality is generally poor, though it appears to have recently improved, and that their applicability is generally low. (8, 9)
Appraisals of CPGs for LBP (9-14) do not take into account the most recently published guidelines. Since CPGs provide a bridge between scientific literature
and clinical decision making, their implementation in clinical practice should be based on recent evidence, and consider as much as possible a wide range of
therapeutic choices. (15) But because 1 out of 5 recommendations in clinical guidelines go out of date within 3 years, the validity of recommendations beyond 3 years is potentially
questionable. (16) As a general rule, CPGs should be reviewed every 3 years after their issue. (17) The National Institute of Clinical Excellence (NICE), the
benchmark in guidelines production, has stated that “A formal review of the need to update a guideline is usually undertaken by NICE 3 years after its
publication”. (18) This is warranted by the time span between the year of running the systematic search strategy during guideline production and the year of
publication in a systematic review. (19) This time span is further stretched because guidelines production and dissemination need to be based on systematic
reviews. The use of guidelines older than 3 years would be considered unethical in clinical decision making and mistaken in identifying high quality guidelines
with not the most recent-update, available and reliable evidence (16, 17, 20). REGISTRATION PROSPERO DETAILS: CRD42019127619. REGISTRATION PROSPERO DETAILS: CRD42019127619. 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 on October 22nd, 2020. See the published version at https://doi.org/10.1186/s12913-020-05827-w. Page 1/13 Page 1/13 Background Moreover, existing appraisals of guidelines for LBP do not rely on a comprehensive search of the many possible therapeutic options (rehabilitative,
pharmacological or surgical) for treating acute and chronic LBP. (21) The scope is an important item in the AGREE II favoring guidelines that are broad in
scope rather than those focusing on a particular set of interventions for a specific condition. (22) With this study, we critically appraised only the most recent evidence-based CPGs for LBP interventions by means of the AGREE (Appraisal of Guidelines
Research and Evaluation) II instrument, the gold standard for critical appraisal of guidelines (22, 23), consistent with the assumption that time can influence
CPG reliability. Also, we evaluated the inter-rater reliability of AGREE II and recorded the time span as the years between the date of last search and period
covered by the search and guideline publication date. Abstract BACKGROUND: Clinical practice guidelines (CPGs) provide recommendations for practice, but the proliferation of CPGs issued by multiple organisations in
recent years has raised concern about their quality. The aim of this study was to systematically appraise CPGs quality for low back pain (LBP) interventions
and to explore inter-rater reliability (IRR) between quality appraisers. The time between systematic review search and publication of CPGs was recorded. METHODS: Electronic databases (PubMed, Embase, PEDro, TRIP), guideline organisation databases, websites, and grey literature were searched from January
2016 to January 2020 to identify GPCs on rehabilitative, pharmacological or surgical intervention for LBP management. Four independent reviewers used the
Appraisal of Guidelines for Research and Evaluation II (AGREE II) tool to evaluate CPGs quality and record the year the CPGs were published and the year the
search strategies were conducted. RESULTS: A total of 21 CPGs met the inclusion criteria and were appraised. Seven (33%) were broad in scope and involved surgery, rehabilitation or
pharmacological intervention. The score for each AGREE II item was: Editorial Independence (median 67%, interquartile range [IQR] 31 – 84%), Scope and
Purpose (median 64%, IQR 22 – 83%), Rigour of Development (median 50%, IQR 21 – 72%), Clarity and Presentation (median 50%, IQR 28 – 79%), Stakeholder
Involvement (median 36%, IQR 10 – 74%), and Applicability (median 11%, IQR 0 – 46%). The IRR between the assessors was nearly perfect (interclass
correlation 0.90; 95% confidence interval 0.88 – 0.91). The median time span was 2 years (range, 1-4), however, 38% of the CPGs did not report the coverage
dates for systematic searches. CONCLUSIONS: We found methodological limitations that affect CPGs quality. In our opinion, a universal database is needed in which guidelines can be
registered and recommendations dynamically developed through a living systematic reviews approach to ensure that guidelines are based on updated
evidence. LEVEL OF EVIDENCE: 1 LEVEL OF EVIDENCE: 1 Methods The reporting of this systematic review fulfils the Preferred Reporting Items for Systematic Reviews and Meta-Analyses. (24, 25). No ethics committee
approval was needed. The protocol is registered in PROSPERO (CRD42019127619). Inclusion and exclusion criteria Page 2/13 Selection of clinical practice guidelines Search results were uploaded to Endnote software and duplicates were removed. (27),(28) Two independent reviewers (SG, VI) screened the titles and
abstracts according to the eligibility criteria. Full texts were retrieved when abstracts gave insufficient information or in case of disagreement between the two
reviewers. When disagreement persisted, a third reviewer was consulted (GC). Rayyan software (https://rayyan.qcri.org/) was used to manage screening and
selection. (29) Reasons for study exclusion are reported. Appraisal of clinical practice guidelines Four independent researchers (MB, GC, SG, VI) appraised each CPG using the AGREE II instrument and recorded with a self-chronometer the time taken for
each assessment. The researchers received training in the use of AGREE II. They completed the AGREE II Online Training Tool Four independent researchers (MB, GC, SG, VI) appraised each CPG using the AGREE II instrument and recorded with a self-chronometer the time taken for
each assessment. The researchers received training in the use of AGREE II. They completed the AGREE II Online Training Tool
(http://www.agreetrust.org/resource-centre/agree-ii-training-tools/) and participated in two calibration rounds with a sample of four relevant CPGs of varying
quality from a previous overview of clinical guidelines for chronic LBP restricted to 2012. (30) The original AGREE tool was published in 2003 has since then
been revised in an updated version. The AGREE II instrument (22) consists of 23 items organized into six quality domains: scope and purpose, stakeholder
involvement, rigour of development, clarity of presentation, applicability, and editorial independence. Supplementary Digital Content 2 shown the items and
domains of the AGREE II instrument. (31) Answers to items are graded on a 7-point scale from 1 (strongly disagree) to 7 (strongly agree). A standardized score
(range, 0 to 100%) was calculated for each domain. The appraisers completed the first global rating item on a 7-point scale (1=lowest possible quality, 7= highest possible quality) and the second global rating
item of recommending the guidelines for use in practice, with one of three options (Yes, Yes, with modifications, and No). One author (VI) calculated the
standardised domain score for each of the six domains as recommended by AGREE II. (22, 32) The general data from each CPG were collected: i) authors and
year of publication; ii) ex novo, update or adoption/adolopment CPG status; iii) continent of origin; iv) organization/society/association, funding source,
conflict of interest. We also extracted content information such as target population, target interventions (i.e., surgery, physical therapy, pharmaceutics,
educational / behavioural, alternative medicine), rating methods for the quality of evidence (e.g., the Grading of Recommendations Assessment, Development
and Evaluation - GRADE), presence of a multidisciplinary panel (as defined by AGREE II: potential candidates for a panel group include clinicians, content
experts, researchers, policy makers, clinical administrators, and funders; at least one methodology expert), and patient involvement (as defined by AGREE II: to
capture patient/public views and preferences). Supplementary Digital Content 2. Information sources and search strategy We systematically searched the PubMed, Embase, PEDro, and TRIP databases using the adapted terms and keywords derived from the scoping search
outlined in the search strategy. We checked guideline organisation databases (e.g., National Institute for Clinical Excellence) and guideline websites (e.g.,
eGuidelines). Supplementary Digital Content 1 illustrates the search strategy. Two reviewers (SG, GC) with a solid background in clinical epidemiology ran the
search strategy in March 2019 and updated the results in January 2020. Grey literature was searched using Google Scholar and reference lists were screened
for further eligible CPGs. Page 2/13 Page 2/13 In line with the World Health Organization, we defined a CPG as a document containing “systematically developed evidence-based statements that assist
providers, patients, policy makers and other stakeholders to make informed decisions on health care and public health policy”. (26) In line with the World Health Organization, we defined a CPG as a document containing “systematically developed evidence-based statements that assist
providers, patients, policy makers and other stakeholders to make informed decisions on health care and public health policy”. (26) Inclusion criteria were: (i) the systematic process evaluated the recommendations; (ii) the CPG was focused on rehabilitation, pharmacological or surgical
therapeutic intervention for LBP management; (iii) the full text was published in the last 4 years (2016-2020). We used the most up-to-date version and its
supplementary documents. No language restrictions were applied. Exclusion criteria were: (i) not primarily focused on LBP, such as national/international
guidelines in which LBP was briefly mentioned in the context of a more comprehensive disease evaluation; (ii) not issued by a national or international society
(e.g., designed for local use); (iii) declaration of recommendations was based exclusively on consensus statements or systematic reviews or commentary
editorials related to published CPGs; (iv) focus on interventions other than therapeutic (e.g., prevention, diagnosis); (v) based on population subgroups (e.g.,
pregnant women), specific causes (e.g. spondyloarthritis) or mixed/generic population (e.g., musculoskeletal chronic pain). Data synthesis We used descriptive statistics to summarize the characteristics of CPGs deemed eligible for inclusion. Data are summarized as frequency number
(percentage) or median and interquartile range (IQR). We calculated a quality score for each of the six domains of CPGs using the formula presented in the
AGREE II User’s Manual. (32) The appraisers added notes and completed the two global rating items at the end of each AGREE II assessment. The first global
rating item asks appraisers to rate the overall quality of the guideline on a 7-point scale (1=lowest possible quality and 7= highest possible quality). Domain
scores are calculated by summing up the appraisers’ scores of the individual items in a domain and then scaling the total as a percentage of the maximum
possible score for that domain, which is then automatically generated on the platform My AGREE PLUS. (33) The second global rating item asks whether the appraiser would recommend the guideline for use in practice and to respond with one of three options (Yes,
Yes, with modifications, and No). The first global rating was adopted to formulate the agreement on the overall assessment between the four appraisers measuring the intraclass correlation
coefficient (ICC) with 95% confidence interval (CI). The degree of agreement was graded according to Landis and Koch (34): slight (0.01-0.2); fair (0.21-0.4);
moderate (0.41-0.6); substantial (0.61-0.8); and almost perfect (0.81-1). Statistical significance was a P value < 0.05. All tests were two-sided. (34) All data
analyses were performed using STATA (StataCorp. 2017. Stata Statistical Software: Release 15. College Station, TX, USA: StataCorp LLC). Page 3/13 Page 3/13 Search results The systematic search retrieved 2502 citations; additional 30 citations were retrieved from the grey literature. A total of 70 CPGs and related documents
underwent full-text screening, 25 of which met the inclusion criteria. Four are awaiting assessment (Figure 1). Finally, we appraised 21 CPGs using AGREE II
(Supplementary Digital Content 1 and 3). Characteristics of CPGs Characteristics of CPGs Table 1 presents the main characteristics of the 21 CPGs: 10 (47.6%) addressed multiple interventions. Rating of evidence quality was planned in 76% of the
guidelines and reported in 67%. More than half (52%) had a multidisciplinary panel and less than half (38%) reported patient involvement (Supplementary
Digital Content 3). Table 1. Characteristics of CPGs. Clinical Practice Guideline
Publication
year
Country
Status
Topic
Publication dates of
systematic search
strategy
American College of Physician (ACP) (35)
2017
USA
Update
Educational / behavioural,
physical therapy, pharmaceutics
2008 - 2015
American Imaging Management (AIM)
Specialty Health (36)
2019
USA
Update
Surgery
Not reported
American Osteopathic Association (AOA)(37)
2016
USA
Update
Physical therapy
2003 - 2014
American Society of Interventional Pain
Physicians (ASIPP) (38)
2019
USA
New
Pharmaceutics
Not reported
Brazilian Medical Association (BMA)(39)
2018
Brazil
New
Educational / behavioural
Not reported
China Association of Acupuncture-
Moxibustion (CAAM)(40)
2016
China
New
Alternative medicine
Not reported
Canadian Chiropractic Guideline Initiative
(CCGI) (41)
2018
Canada
New
Educational / behavioural,
physical therapy
2015 - 2017
Council on Chiropractic Guidelines and
Practice Parameters (CCGPP) (42)
2016
USA
Adoption1
Physical therapy
2009 - 2014
Change Pain Latin America (CPLA) (43)
2018
Latin
America
Adoption2
Physical therapy, pharmaceutics
2004 - 2014
Dutch Society of Anesthesiologists (DSA)(44)
2016
Netherlands
New
Surgery
1990 - 2011
Global Spine Care Initiative (GSCI)(45)
2018
International
New
Surgery
Not reported
Institute for Clinical Systems Improvement
(ICSI)(46)
2018
USA
Update
Educational / behavioural,
physical therapy, pharmaceutics
2000 - 2017
Belgian Health Care Knowledge Centre (KCE)
(47)
2017
Belgium
Adoption3
Educational / behavioural,
physical therapy, pharmaceutics,
surgery
2010 - 2015
Korea Institute of Oriental Medicine (KIOM)
(48)
2017
Korea
New
Physical therapy, pharmaceutics,
alternative medicine
start date not reported -
2015
Korean Society of Spine Surgery (KSSS) (49)
2017
Korea
Adoption4
Educational / behavioural,
physical therapy, pharmaceutics
2000 - 2016
Labor & Industries (L&I), Washington
State(50)
2016
USA
Update
Surgery
Not reported
National Institute for Health and Care
Excellence (NICE)(51)
2016
UK
Update
Educational / behavioural,
physical therapy, pharmaceutics,
surgery
2013 - 2015
Polish Society of Physiotherapy (PSP)(52)
2017
Poland
New
Physical therapy
Not reported
Polish Spine Surgery Society (PSSS)(53)
2016
Poland
New
Surgery
Not reported
Toward Optimized Practice (TOP) Low Back
Pain Working Group. Legend 1 Three evidence-based clinical practice guidelines for the surgical and interventional management of persistent and disabling spine disorders were selected
by consensus (Globe et al. J Manipulative Physiol Ther 2008; Baker et al. Top Integr Health Care, 2012; Farabaugh RJ, J Manipulative Physiol Ther 2010). 2 Eleven evidence-based clinical practice guidelines were selected according to the field of interest. Table 4 of original publication contains all references. 3 One evidence-based clinical practice guidelines was selected (de Campos, NICE 2016); 3 One evidence-based clinical practice guidelines was selected (de Campos, NICE 2016); 4 Three evidence-based clinical practice guidelines were selected according to the field of interest (Dagenais et al. Spine J 2010; Koes et al. Eur Spine J 2010;
Chou et al. Ann Intern Med 200). Note: For full details of CPG see Supplement Digital Content 3. Note: For full details of CPG see Supplement Digital Content 3. Characteristics of CPGs (54)
2017
USA
Update
Educational / behavioural,
physical therapy, pharmaceutics,
surgery
2010 - 2014
Veterans Affairs/Department of Defense
(VADoD) Collaboration Office(55)
2017
USA
Update
Educational / behavioural,
physical therapy, pharmaceutics
2006 - 2016 Page 4/13 Table 2. Overall domain assessment of CPGs. AGREE II domains assessment Overall, the highest rating AGREE II domain was Editorial Independence (median 67%, interquartile range [IQR] 31 – 84%), followed by Scope and Purpose
(median 64%, IQR 22 – 83%), Rigour of Development (median 50%, IQR 21 – 72%), Clarity and Presentation (median 50%, IQR 28 – 79%), Stakeholder
Involvement (median 36.1%, IQR 10 – 74%), and Applicability (median 11%, IQR 0 – 46%). In the overall guideline assessment, the median of the overall
quality item was 42% (IQR 15 – 67%) and the most frequent recommendation regarding the use of the guideline was “No” (Table 2). The NICE guideline (51) had the highest quality (96%) in the area of Educational/behavioural, physical therapy, pharmaceutical interventions. The Belgian
Healthcare Knowledge Centre (KCE) (83%) guideline (56) had high quality and covered the same interventions plus surgery with a short time span (1 and 2
years, respectively) for searching evidence (Supplementary Digital Content 3). Table 2. Overall domain assessment of CPGs. Table 2. Overall domain assessment of CPGs. Page 5/13 Clinical Practice Guideline
Scope
and
Purpose
Stakeholder
Involvement
Rigour of
Development
Clarity of
Presentation
Applicability
Editorial
Independence
OVERALL
first
global
rating
(Personal
rating)
second
global rating
(I would
recommend?)
National Institute for
Health and Care
Excellence (NICE)(51)
2016
98,61%
95,83%
90,10%
100,00%
65,63%
93,75%
95,83%
Yes
Canadian Chiropractic
Guideline Initiative
(CCGI) (41)
2018
90,28%
84,72%
88,54%
90,28%
73,96%
85,42%
87,50%
Yes
Belgian Health Care
Knowledge Centre
(KCE) (47)
2017
88,89%
77,78%
83,85%
69,44%
86,46%
95,83%
83,33%
Yes
American College of
Physicians (ACP) (35)
2017
97,22%
75,00%
76,56%
80,56%
22,92%
93,75%
75,00%
Yes
Veterans
Affairs/Department of
Defense
(VADoD) Collaboration
Office (55)
2017
84,72%
90,28%
80,21%
87,50%
41,67%
60,42%
70,83%
Yes, with
mod. Institute for Clinical
Systems Improvement
(ICSI) (46)
2018
80,56%
72,22%
68,23%
86,11%
51,04%
79,17%
62,50%
Yes, with
mod. Discussion Here we report the results of quality appraisal using AGREE II of the most recent CPGs for LBP interventions (published January 2016 to January 2020) that
we retrieved by systematic search of electronic medical databases and guidelines websites. A key finding was the variability in the quality of the CPGs across
all six AGREE II domains; the highest average scores (>60%) were recorded for Domain 6 - Editorial Independence and the Domain 1 - Scope and Purpose and
the lowest (<15%) for Domain 5 - Applicability. The overall quality was rated low and the most frequent response for guideline recommendation was “No” (15
out of 21 CPGs). Our findings are shared by previous appraisals of CPGs for rehabilitation (57) and other contexts (8, 58, 59) that suggest room for improvement regarding
rigour of development, stakeholder involvement, and applicability (8, 58, 59). While only half of the CPGs were noted to have acceptable rigour of development
(Domain 3 - Rigour of Development), the variability in this domain was considerable. A low score for this domain is worrying, as it has been identified as a
strong predictor of quality by the AGREE instrument. (8) Regression analysis showed a statistically significant influence of the assessment of the items in this
domain on overall guideline quality. (60) The item assessing the systematic search can have great importance (i.e., “Item 7: Systematic methods were used to
search for evidence”) because CPGs ought to be based on recently updated evidence. However, we found that less than half did not report the time coverage
of systematic search and, when reported, it ranged from 1 to 4 years before publication. Two-thirds of the CPGs in our sample adequately planned and judged
the body of the evidence linked to recommendations (e.g., GRADE). However, because the application of a system for grading the evidence (i.e., GRADE)
cannot always ensure inclusion of the most updated evidence within an acceptable time span, reliability should be evaluated with caution. The validity of each recommendation, and of the CPG, is determined by the methodological quality and the transparency of its development and by the “living
evidence” on which it is based. As suggested by Garcia et al., waiting more than 3 years to review a guideline is potentially too long, in which case the
recommendations may be outdated by the time of guideline publication. Discussion (16) This critical issue has been addressed by the living CPGs concept, (61) which
draws inspiration from the established model of living systematic reviews, where evidence is continuously updated and incorporated as soon as available in
the literature through a process of continuous surveillance. (62) Accordingly, AGREE II should place importance on timing and rate CPG a high-quality score
when the search is conducted within 2 years of completion of the review. (63) Less than one third of the CPGs in this sample met the AGREE II criterion for participation of patients and their advocate (Domain 2 - Stakeholder Involvement). Guideline developers need to prioritize patient and stakeholder involvement starting from the early stages of CPG development. They should be actively
involved as members on guideline panels and their comments and inputs included in the draft guideline. (64) Furthermore, evidence suggests that
involvement of patients and stakeholders leads to the inclusion of patient-relevant topics and enhances CPG implementation. (65) Unfortunately, development
and implementation are erroneously considered as separate activities. (8) In our appraisal, the poorest score was recorded for CPGs applicability (Domain 5 -
Applicability), with results similar to other CPGs in rehabilitation (57) and other conditions. (8, 12, 66-68) CPGs can provide healthcare professionals with the
necessary guidance to access the best research evidence efficiently. Nonetheless, they have little effect on changing clinical behavior. Only half of the CPGs in our sample were rated satisfactory for adequacy of the reporting of recommendations and options for management (Domain 4 -
Clarity of Presentation). This may be related to the purpose of AGREE II: the current version makes no distinction between quality of reporting and quality of
conduct of a CPG. Despite good reporting, the methodological conduct underlying a guideline can still be weak. (69) Quality of conduct and reporting should
be judged separately, just as for all other study designs. (70, 71) In systematic reviews, for instance, PRISMA and the AMSTAR assess the quality of reporting
and the quality of conduct, respectively. (72) We recorded high compliance of the CPGs with the overall aim of the guideline, the clinical question, and the target population (Domain 1 - Scope and
Purpose). This could be explained by the focus on LBP, which is the most prevalent musculoskeletal condition for which guidelines are needed in view of the
years lived with disability in most countries. Discussion (73) Lastly, we recorded high compliance of the CPGs with the reporting of sources of support (Domain 6 -
Editorial Independence). Given the global socioeconomic burden of LBP and the need for care, CPGs must report the presence and management of conflict of
interests. Inter-rater reliability and time for AGREE II appraisal Inter-rater agreement was nearly perfect (ICC 0.90; 95% CI 0.88 – 0.91). Guidelines appraisal took 42 minutes on average to complete (95% CI 35-50). Inter-rater agreement was nearly perfect (ICC 0.90; 95% CI 0.88 – 0.91). Guidelines appraisal took 42 minutes on average to complete (95% CI 35-50). Time to publication Inter-rater agreement was nearly perfect (ICC 0.90; 95% CI 0.88 – 0.91). Guidelines appraisal took 42 minutes on average to complete (95% CI 35-50). Time to publication Time to publication Overall, 38.1% of the CPGs did not report the dates of systematic search strategy, whereas less than half (47.6%) reported a median of 2 years (IQR 1 - 4) from
search to publication. Only half provided a search within 1 year after publication (Table 1). AGREE II domains assessment Toward Optimized
Practice (TOP) Low
Back Pain Working
Group (54)
2017
69,44%
48,61%
66,15%
77,78%
71,88%
75,00%
58,33%
No
Korea Institute of
Oriental Medicine
(KIOM) (48)
2017
72,22%
40,28%
47,92%
59,72%
1,04%
83,33%
45,83%
No
China Association of
Acupuncture-
Moxibustion (CAAM)
(40)
2016
63,89%
44,44%
35,94%
63,89%
0,00%
29,17%
45,83%
No
Global Spine Care
Initiative (GSCI) (45)
2018
52,78%
11,11%
25,00%
70,83%
11,46%
75,00%
41,67%
No
American Society of
Interventional Pain
Physicians (ASIPP)
(38)
2019
66,67%
25,00%
57,29%
33,33%
6,25%
81,25%
41,67%
No
American Osteopathic
Association (AOA)
(37)
2016
76,39%
36,11%
52,60%
25,00%
33,33%
58,33%
41,67%
No
Change Pain Latin
America (CPLA) (43)
2018
26,39%
13,89%
27,60%
27,78%
23,96%
93,75%
33,33%
No
Council on
Chiropractic
Guidelines and
Practice Parameters
(CCGPP) (42)
2016
43,06%
33,33%
53,13%
47,22%
16,67%
66,67%
29,17%
No
Dutch Society of
Anesthesiologists
(DSA) (44)
2016
63,89%
56,94%
50,52%
50,00%
4,17%
45,83%
29,17%
No
Brazilian Medical
Association (BMA)
(39)
2018
22,22%
0,00%
34,90%
29,17%
0,00%
29,17%
20,83%
No
Korean Society of
Spine Surgery (KSSS)
(49)
2017
15,28%
2,78%
17,71%
20,83%
0,00%
29,17%
8,33%
No
Polish Spine Surgery
Society (PSSS) (53)
2016
20,83%
2,78%
12,50%
29,17%
0,00%
62,50%
8,33%
No
American Imaging
Management (AIM)
Specialty Health (36)
2019
18,06%
8,33%
6,77%
27,78%
0,00%
0,00%
4,17%
No y
Physiotherapy (PSP)
(52)
Labor & Industries
(L&I), Washington
State (50)
2016
12,50%
13,89%
0,00%
29,17%
0,00%
0,00%
0,00%
No Strengths and limitations Strengths and limitations Page 7/13 Page 7/13 Page 7/13 Our appraisal has several strengths. We performed an exhaustive search that included explicit eligibility criteria and independent duplicate assessment of
eligibility. Four reviewers were involved in the appraisal, with a nearly perfect inter-rater reliability. While all appraisers were trained in the use of AGREE II, it
should be acknowledged that the appraisers shared a similar background (methodology and rehabilitation), which may partially explain the high overall
agreement. Indeed, our team included clinical experts and methodologists with experience in clinical epidemiology, including systematic reviews and CPGs. Even after receiving the same training however, guideline appraisers from different areas may still interpret the items and the scoring system differently. (74)
Furthermore, it is possible that the appraisers, basing the assessment on their own experience, paid more attention to assessing the quality of reporting than
the quality of conduct and vice versa. We analysed a reliable subset of CPGs restricted to LBP in order to ensure consistency of appraisal, while avoiding
discrepancies in item judgement due to different clinical contexts (e.g., AGREE II to assess CPGs in oncology differs from orthopaedics). We focused on the
most recent guideline versions in order to offer stakeholders, policy makers, clinicians, and patients the latest evidence for the effectives of interventions. However, selecting the CPGs was a challenge, since the definition of guidelines is not universally established and the meaning of consensus and that of
evidence-based CPG are sometimes confused. The rigour of methods and panel of experts have to be simultaneously considered in a CPG, but the current
definition does not explicate these elements. A possible limitation of our work is linked to characteristics of the AGREE II itself. It focuses on the quality of the development of CPGs, but this is not
sufficient to ensure implementation of single clinical recommendations and improvement in health outcomes. (75) While high-quality CPGs can guarantee
rigour in the production of recommendations, their implementation depends largely on how health care professionals decide whether or not to implement a
single recommendation in the balance between content (strength and direction of a recommendation), clinical expertise, patients’ values and resources
available. The implementation of a single clinical recommendation cannot be disjointed from overall CPG quality. Future spin for research At the time of its publication, a CPG can already be outdated and so will not reflect the most recent evidence. Indeed, time can influence its reliability: (a)
during the conduction of systematic reviews for the production of the body of the evidence needed during CPG development; (b) between finalization of a CPG
and its publication. In order to avoid waste of effort and of resources due to duplication of CPGs or CPGs outdated before their time, we urge for the creation
of a universal database in which guidelines can be registered and updated along the lines of registers for RCTs (e.g., WHO or clincialtrials.gov) and systematic
reviews (e.g., PROSPERO) but for CPGs. In this way, a “living and dynamic” development of recommendations can be better recognized by identifying the most
recent literature. (76) Conclusion We found methodological limitations affecting CPG quality. Our work highlights the importance of adoption of high quality and updated CPGs to guarantee
the validity of a single recommendations, notwithstanding the possibility that implementation of each single recommendation may be the result of a balanced
decision between content (strength and direction of a recommendation), clinical expertise, and available resources. We call for a universal database in which
guidelines can be registered and recommendations dynamically developed through a living systematic reviews approach to ensure that CPGs are based on
recent evidence. Abbreviations American College of Physicians (ACP); American College of Physicians (ACP); Appraisal of Guidelines for Research and Evaluation II (AGREE II); American Osteopathic Association (AOA); Clinical Practice Guidelines (CPGs); Belgian Health Care Knowledge Centre (KCE); Council on Chiropractic Guidelines and Practice Parameters (CCGPP); Page 8/13 Disability Adjusted Life Years (DALYs); Dutch Society of Anesthesiologists (DSA); Global Spine Care Initiative (GSCI); Global Spine Care Initiative (GSCI); Grading of Recommendations Assessment, Development and Evaluation (GRADE); Intraclass correlation (ICC); Intraclass correlation (ICC); Institute for Clinical Systems Improvement (ICSI); Korea Institute of Oriental Medicine (KIOM); Korean Society of Spine Surgery (KSSS); Labor & Industries (L&I); Labor & Industries (L&I); Low Back Pain (LBP); National Institute for Health and Care Excellence (NICE); Physiotherapy Evidence Database (PEDro); Polish Society of Physiotherapy (PSP); Polish Spine Surgery Society (PSSS); Preferred Reporting Intervention for Systematic Review and Meta-analysis (PRISMA); Toward Optimized Practice Low Back Pain Working Group (TOP); Turning Research into Practice (TRIP); Veterans Affairs/Department of Defense Collaboration Office (VADoD); Veterans Affairs/Department of Defense Collaboration Office (VADoD); World Health Organization (WHO). Availability of data and materials: All data generated or analysed during this study are included in this publis
are stored at the following link: https://osf.io/xwbu2/?view_only=d3aa81b467874b468bd1207d96df7376 Availability of data and materials: All data generated or analysed during this study are included in this published article with all additional materials. Row data
are stored at the following link: https://osf.io/xwbu2/?view_only=d3aa81b467874b468bd1207d96df7376 Authors' Contributions: SG, CG provided the idea and concept development for the research; SG, CG, VI planned the study design; SG, CG, VI, MB performed
data collection; SG, CG, VI performed data analysis; VI, SG, GC, MB, DC interpreted the data; VI, SG, GC drafted the work or substantively revises it. DC, GB, LMS
provided critical review (revised manuscript for intellectual content; this does not relate to spelling and grammar checking). All authors approved the
submitted version. All authors agreed accuracy and integrity of any part of the work. Declarations Ethics approval and consent to participate: Not applicable. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Consent for publication: Not applicable. Funding: The work was supported by the Italian Ministry of Health “Linea 3 – Valutazione della qualità delle attuali linee guida in ortopedia e in riabilitazione”
L3042. The funding sources had no controlling role in the study design, data collection, analysis, interpretation or report writing. Acknowledgments: The authors wish to thank Kenneth Adolf BRITSCH, Avicenna snc, the external English service for language revision. Acknowledgments: The authors wish to thank Kenneth Adolf BRITSCH, Avicenna snc, the external English servic authors wish to thank Kenneth Adolf BRITSCH, Avicenna snc, the external English service for language revision. Competing interest: The authors declare no conflict of interest regarding the publication of this paper. The manuscript does not contain information about
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Belief in Information Conspiracy and Personality Traits of Generations X and Y
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Belief in Information Conspiracy and Personality Traits of Generations X
and Y Vlada I. Pishchik1*
1Don State Technical University, Department of General and Consultative Psychology, Rostov-on-Don, Russian Federation,
e-mail: vladaph@yandex.ru Vlada I. Pishchik1*
1Don State Technical University, Department of General and Consultative Psychology, Rostov-on-Don, Russian Federation,
e-mail: vladaph@yandex.ru Vlada I. Pishchik1*
1Don State Technical University, Department of General and Consultative Psychology, Rostov-on-Don, Russian Federation,
e-mail: vladaph@yandex.ru Abstract: The article presents the results of comparing the belief in information conspiracies in connection with
personality traits among representatives of Generations Y and X, nationalities – the Russians and the Kumyks. We call information
conspiracies those that are presented in the information space and are presented as close to reality. Therefore, users who
get acquainted with information conspiracies do not doubt and believe in them. The sample was presented by the Russian
university students and middle-aged working people. The questionnaire of conspiracy mentality (CMQ) (Bruder, Haffke) and
the FPI technique (Farenberg, Zarg and Gampel) have been applied. It was revealed that the older the generation, the more it
believes in the state conspiracy; generations have stable beliefs about the presence of a political conspiracy; the representatives
of the Kumyk group are more prone to exaggeration of the importance of conspiracy than the group of the Russians generation
Y; the personality trait “irritability” correlates with the scale of belief in public conspiracy; there was a negative dependence of
emotional lability and belief in a political conspiracy. The results are compared with the data of foreign studies on the samples
of representatives of the USA, Great Britain, Turkey, Germany. It was concluded that it is possible to observe cultural and
intergenerational differences in expression of conspiracy mentality. Representatives of Generation Y believe more in political
and public conspiracies. Representatives of Generation X believe more in public conspiracy and secret organizations.i Keywords: online teaching, English for Specific Purposes, computer-mediated learning, synchronous conferencing,
synchronous online teaching, teaching methods. Original scientific paper Received: September, 05.2022. Revised: October, 27.2022. Accepted: November, 07.2022. Received: September, 05.2022. Revised: October, 27.2022. Accepted: November, 07.2022. Pishchik, V. (2022). Belief in information conspiracy and personality traits of generations X and Y, International Journal of
Cognitive Research in Science, Engineering and Education (IJCRSEE), 10(3), 83-88. Pishchik, V. (2022). Belief in information conspiracy and personality traits of generations X and Y, International Journal of
Cognitive Research in Science, Engineering and Education (IJCRSEE), 10(3), 83-88. Pishchik, V. (2022). Belief in information conspiracy and personality traits of generations X and Y, International Journal of
Cognitive Research in Science, Engineering and Education (IJCRSEE), 10(3), 83-88. UDK:
316.624:159.923.072
10.23947/2334-8496-2022-10-3-83-88 Original scientific paper © 2022 by the authors. 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/). Pishchik, V. (2022). Belief in information conspiracy and personality traits of generations X and Y, International Journal of
Cognitive Research in Science, Engineering and Education (IJCRSEE), 10(3), 83-88. Pishchik, V. (2022). Belief in information conspiracy and personality traits of generations X and Y, International Journal of
Cognitive Research in Science, Engineering and Education (IJCRSEE), 10(3), 83-88. Bruder, et al. (2013) argue that the cross-cultural aspect of conspiracy theory is poorly studiede, except for
the examination of the UK and Austria described by Swami and others in 2011 (Bruder et al., 2013; Swami,
et al., 2011). In this context, Bruder, et al. (2013) created a questionnaire of the conspiratorial mentality
(CMQ), which was tested in a large international study covering the cultures of North America, Western
Europe (the UK, Ireland, Germany) and the Middle East (Turkey). They hypothesized that individual
characteristics and cultural factors interact when a person comes to believe in a particular conspiracy
theory, and this has an impact on his attitudes and behavior. In particular, researchers have shown that
subcultures within the national groups are differently susceptible to belief in conspiracy theories. For
example, African, American, and Latin American communities in the United States are particularly inclined
to endorse conspiracy theories, claiming that HIV is spread to destroy certain ethnic groups (Ross et al.,
2006). )
Modern research has expanded the palette of research. They can distinguish gender, clinical,
cognitive, prognostic, behavioral aspects of the problem. Van der Tempel and Alcock (2015), using the
scale GCB (Brotherton, French and Pickering, 2013), on a group of the Canadian students, showed that
belief in conspiracy and the detection of a hyperactive agent reliably predicted schizotype. At the same
time, it was found out that women are more prone to believe in the supernatural. Shapiro et al. (2016)
questioned 1427 Canadian parents and found out that belief in conspiracy is negatively associated with
the willingness to vaccinate their children. g
Lamberty, Hellmann and Oeberst, (2018) demonstrated in a study that a higher level of conspiracy
mentality can predict voting behavior for a more conservative party candidate. In Italy, researchers Leone
et al. (2018) showed that the style of avoiding attachment due to an emphasis on self-confidence, his
motivation to suppress psychological stresses and a Manichaean view of the world based on a neat
distinction between good and evil would be associated with a belief in conspiracy. g
p
y
In our study (Pishchik, 2017) it was found out that Russian managers have a certain feature. Pishchik, V. (2022). Belief in information conspiracy and personality traits of generations X and Y, International Journal of
Cognitive Research in Science, Engineering and Education (IJCRSEE), 10(3), 83-88. The
less critical thinking they manifest, the more they believe in conspiracies, but at the same time, the ability
to draw logical conclusions and justify their response, is associated with the belief that there is a real
threat from aliens from other planets. Ståhl and Van Prooijen (2018) explain that analytical thinking is
associated with a less tendency to believe in various conspiracy theories and paranormal phenomena, but
only among people who strongly value epistemological rationality. In the study by Wood and Gray (2019)
it was found out a strong correlation of right-wing authoritarianism with the belief in conspiracy theories
and the influence of intergroup support on this belief. l
g
p
pp
Kowalski and Gawęda (2021) highlighted the mediating role of metacognitive escapes, cognitive
attention syndrome in the manifestations of conspiracy beliefs. Dyrendal, Kennair and Bendixen
(2021) proved that schizotypal personality measurements, strange beliefs and paranoid ideas were
predicted by various intermediaries, and their influence on belief in conspiracy theories was completely
mediated. Hattersley et al. (2022) showed how belief in plausible and implausible conspiracy theories is
heterogeneously related to several aspects of reasoning, including information samples, reasoning style
(intuitive versus reflexive reasoning) and confirmation bias. As we can see, the authors are looking for predictive components of belief in conspiracies. The
search continues to build an explanatory model of conspiracy (Beemster, Bijleveld and Treur, 2021) with a
tendency to look more for reasons in the personal qualities of respondents, culture and to a lesser extent
to address to situational factors. The research presents little generational approach to the problem of studying the belief in
conspiracies in respondents belonging to different groups of generations. Each generation has its
own special values. They can influence the adherence of belief in conspiracy. This fact determined the
purpose of our study - to demonstrate the differences in adherence to conspiracy theories among the
representatives of the generations of the two national groups. www.ijcrsee.com Introduction The great interest of scientists to the problems of quality of life, psychological well-being, social
capital stimulates the interest to phenomena, namely, the belief in conspiracies, conspiratorial mentality,
impeding the well-being of people in society. The scientific significance of measuring the indicators of
the belief conspiracy is increased by the fact that it has a prognostic potential. For example belief in
pharmaceutical conspiracy can predict the refusal from vaccination. In today’s world, there is increasing
evidence that there are stable individual differences in people’s propensity to believe in conspiracy theories
(Dyrendal, Kennair and Bendixen, 2021). It is shown that if a person believes in one conspiracy theory, he
will also be more likely to believe in other conspiracy theories (Swami et. al., 2011; Green and Douglas,
2018). This has led some researchers to the assumption that the alignment of specific conspiracy theories
depends more on individual differences with a certain tendency to adopt such a belief and that there is a
general conspiracy mentality or conspiratorial mentality. Belief in conspiracies is belief in an implausible
description of an event or situation. This term was originally formulated by S. Moscovici, who defined
conspiracy as something which unites representatives of different religions, parties or ethnicities by an
indissoluble secret tie (Moscovici, 1987). The object of such an alliance is incitement, revolution in the life
of society, perversion of the values of civil society, exacerbation of crises, promotion of defeat, and so on
(Moscovici, 1987, p. 154). Entin believes that “for people with this mentality, conspiracy is the only model
of change in history, and conspiracy theories are the only form of historical explanation” (Entin, 2000, p. 70). We believe that conspiracy theory is one of the explanatory models of disruption of the stability of
society along with others.l y
g
Swami, et al. (2011), Bruder, et al. (2013) reflect the cross-cultural aspect of concpiracy theory. *Corresponding author: vladaph@yandex.ru © 2022 by the authors. This article is an open access article distributed under the terms and conditions of t
Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). www.ijcrsee.com 83 Pishchik, V. (2022). Belief in information conspiracy and personality traits of generations X and Y, International Journal of
Cognitive Research in Science, Engineering and Education (IJCRSEE), 10(3), 83-88. Materials and Methods In our study we had the following participants: 4 groups of Russian generations (2 groups of
Generation Y – student youth and 2 groups of Generation X – working middle-aged people). They belong
to two nationalities – the Kumyk (Dagestan, Russia) and the Russian (Rostov-on-Don, Russia), the total
number is 350 people aged between 19 and 23 and between 25 and 35. The CMQ questionnaire (Bruder
et al., 2013) and the FPI technique (Farenberg, Hampel, and Selg 2001) were conducted. C Q
f
(
) )
q
(
g
p
g
)
The CMQ method is aimed at identifying ideas (conspiracies) that people believe in. In this method,
there are 5 statements that need to be assigned a coefficient in accordance with how much the subject www.ijcrsee.com 84 Pishchik, V. (2022). Belief in information conspiracy and personality traits of generations X and Y, International Journal of
Cognitive Research in Science, Engineering and Education (IJCRSEE), 10(3), 83-88. Pishchik, V. (2022). Belief in information conspiracy and personality traits of generations X and Y, International Journal of
Cognitive Research in Science, Engineering and Education (IJCRSEE), 10(3), 83-88. agrees or disagrees with this statement. As a result, faith in the state, political, public, in secret actions, in
secret organizations are revealed. g
The Freiburg Personality Questionnaire FPI is designed to diagnose states and personality traits
that are of paramount importance for the process of social adaptation and regulation of behavior. The FPI
questionnaire contains 12 scales; Form B differs from the full form only by half the number of questions. The total number of questions in the questionnaire was 114. q
q
The data were statistically processed using the t–criterion of differences, correlation analysis. We have made assumptions: H1 – it is possible to detect differences in belief in conspiracies, personal
characteristics between students – Generation Y and older working people – Generation X. H2 – it is
possible to find a relationship between certain types of belief in conspiracies and the personality traits of
representatives of Generations X and Y. Results
The results were recorded by types of belief in conspiracy. The results are shown in Figure 1. Figure 1. Indicators of faith in conspiracy of generations Results Results The following results were obtained on personality traits in the groups, which are presented in
Figure 2 The following results were obtained on personality traits in the groups, which are presented in
Figure 2. Figure 2. Expression of personality traits in generation groups Figure 2. Expression of personality traits in generation groups However, these results are different from those of Bruder, et al. (2013). According to their data,
the representatives of the United States (M=6,3; SD=2,0) and Great Britain/Ireland (M=6,3; SD=1,9)
had average indicators of conspiratorial mentality. The Turkish participants (M=7,3; SD=2,1) had a
conspicuously higher conspiratorial mentality than all other groups (DS>0.58), while the German
participants had a lower one than the other groups (M=5,9; SD=2,1). As it can be seen, representatives of
different cultures are committed to conspiratorial mentality. The comparison of personality traits and belief in conspiracies was of interest. In a sample of
respondents, we found out a strong correlation between indicators of emotional lability and scales of belief
in public conspiracy (r=0,52**) and belief in secret societies (r=0,32*). The personality trait of irritability
correlated with the scale of belief in public conspiracy (r=0,62*). In the group of the Kumyk generation X
there was a negative dependence of emotional lability and belief in political conspiracy (r=-0,92**). The
scale balance was negatively correlated with belief in secret societies (r=-0,84*). Discussions The obtained results of differences in belief in conspiracies among the Kumyks and the Russians
differ from the data of Bruder, et al. (2013). According to their data, representatives of the USA (M=6.3;
SD=2.0) and Great Britain/Ireland (M=6.3; SD=1.9) had average indicators of a conspiratorial mentality. The Turkish participants (M=7.3; SD=2.1) had a significantly higher conspiratorial mentality than all other
groups (DS>0.58), while the German participants had it lower than the other groups (M=5.9; SD=2.1). As
you can see, representatives of different cultures are committed to a conspiratorial mentality. y
,
p
p
y
Poise was negatively correlated with the belief in secret societies in the Kumyk group. A study
(Green and Douglas, 2018) found out that a higher degree of anxiety is more likely to adhere to conspiracy
beliefs. y
,
p
p
y
Poise was negatively correlated with the belief in secret societies in the Kumyk group. A study
(Green and Douglas, 2018) found out that a higher degree of anxiety is more likely to adhere to conspiracy
b li f We found a stable correlation between indicators of emotional lability and scales of belief in a public
conspiracy. This result is combined with a study by Georgiou, Delfabbro and Balzan (2019), who showed
that psychopathological factors (schizo-type and tendency to delusion) were the strongest predictors of
belief in conspiracies. p
If our respondents are committed to the belief in conspiracies, this may indicate a manifestation of
bias in reasoning in the general field (Ermakov and Belousova, 2021; Hattersley et al., 2022). g
gi
(
y
)
To reduce the belief in conspiracies of students, it is necessary to develop critical thinking. We can
get acquainted with such an experience in study (Almulla, 2018). www.ijcrsee.com Results Figure 1. Indicators of faith in conspiracy of generations There were significant differences found between the indicators of belief in political conspiracy (t=-
0,857, p>0.34) and secret societies (t=-0,781, p>0.22) between Generations Y of the two national groups. The belief in political conspiracy (t=-0.631, p>0.04) significantly differed in the groups of generations X of
the two nationalities. The results are shown in Table 1. Table 1. Average values and significance of the differences in the types of belief in conspiracies of genera Table 1. Average values and significance of the differences in the types of belief in conspiracies of generations 1. ge values and significance of the differences in the types of belief in conspiracies of generations As you can see, generations have stable beliefs about the existence of political conspiracy. Apparently, the sphere of politics in the views of the studied groups is not so stable and carries a threat.i In the group of the Russian, significant differences were found between generations on the scale of
belief in the state conspiracy (t=-1,375, p>0.01). Thus, the older the generation, the more it believes in the
state conspiracy, perhaps this is the influence of the restructuring experienced in the country. i In the group of the Russian, significant differences were found between generations on the scale of
belief in the state conspiracy (t=-1,375, p>0.01). Thus, the older the generation, the more it believes in the
state conspiracy, perhaps this is the influence of the restructuring experienced in the country. i p
y p
pl
g
p
y
In the Kumyk group, significant differences were found between generations on the scales of
belief in political conspiracy (t=-1,447, p>0,31), belief in state conspiracy (t=-1,105, p>0,01) and public
conspiracy (t=-2,698, p>0,01). The data obtained allow to assert that the Kumyk group (youth), (middle-
aged) are more likely to exaggerate the significance of conservativism than the Russian group (youth),
(middle-aged). It can be assumed that the Kumyk group of youth is more susceptible to doubts in terms
of stability and reliability of stability in life. www.ijcrsee.com 85 Pishchik, V. (2022). Belief in information conspiracy and personality traits of generations X and Y, International Journal of
Cognitive Research in Science, Engineering and Education (IJCRSEE), 10(3), 83-88. Pishchik, V. (2022). Belief in information conspiracy and personality traits of generations X and Y, International Journal of
Cognitive Research in Science, Engineering and Education (IJCRSEE), 10(3), 83-88. Conclusions In our study, we examined the current state of the problem of belief in conspiracies of representatives
of certain generations. We realized that belief in conspiracies is an explanatory model of events in the
world, bringing confidence. There is evidence of a personal and cultural predisposition to believe in www.ijcrsee.com 86 Pishchik, V. (2022). Belief in information conspiracy and personality traits of generations X and Y, International Journal of
Cognitive Research in Science, Engineering and Education (IJCRSEE), 10(3), 83-88. conspiracies. There are methods for determining the belief in conspiracies in various countries. We have
tested our methodology for identifying the types of belief in conspiracies. Thus, it is possible to observe
the cultural and intergenerational differences in expression of conspiratorial belief in conspiracies. The
belief in the conspiracy theory can partly act as an indicator of increasing levels of anxiety in society, social
instability, which is especially evident in situations of various social crises. In the future, we would like to
increase the sample of research in representatives of generations and national groups. conspiracies. There are methods for determining the belief in conspiracies in various countries. We have
tested our methodology for identifying the types of belief in conspiracies. Thus, it is possible to observe
the cultural and intergenerational differences in expression of conspiratorial belief in conspiracies. The
belief in the conspiracy theory can partly act as an indicator of increasing levels of anxiety in society, social
instability, which is especially evident in situations of various social crises. In the future, we would like to
increase the sample of research in representatives of generations and national groups. Conflict of interests Conflict of interests
Author declare no conflict of interest. Conflict of interests
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Cognitive Research in Science, Engineering and Education (IJCRSEE), 10(3), 83-88. Swami, V., Coles, R., Stieger, S., Pietschnig, J., Furnham, A., Rehim, S., & Voracek, M. (2011). Conspiracist ideation in Britain
and Austria: Evidence of a monological belief system and associations between individual psychological differences
and real-world and fictitious conspiracy theories. British Journal of Psychology, 102(3), 443-463. https://doi.org/10.1111/
j.2044-8295.2010.02004.x j
van Der Tempel, J., & Alcock, J. E. (2015). Relationships between conspiracy mentality, hyperactive agency detection, and
schizotypy: Supernatural forces at work?. Personality and Individual Differences, 82, 136-141. https://doi.org/10.1016/j. paid.2015.03.010 p
Wood, M. J., & Gray, D. (2019). Right-wing authoritarianism as a predictor of pro-establishment versus anti-establishment
conspiracy theories. Personality and Individual Differences, 138, 163-166. https://doi.org/10.1016/j.paid.2018.09.036 www.ijcrsee.com www.ijcrsee.com 88
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Edição especial com os egressos em comemoração aos 10 anos de existência
do programa de pós-graduação. Edição especial com os egressos em comemoração aos 10 anos de existência
do programa de pós-graduação. ISSN: 2526-849X
Edição especial com os egressos em comemoração aos 10 anos de existência
do programa de pós-graduação.
Tema: a pluralidade e os diferentes contextos de pesquisa e formação de professores ISSN: 2526-849X
Edição especial com os egressos em comemoração aos 10 anos de existência
do programa de pós-graduação. Tema: a pluralidade e os diferentes contextos de pesquisa e formação de professores ISSN: 2526-849X Revista Devir Educação, Lavras-MG. Edição Especial – Ago./2020.
1 Revista Devir Educação, Lavras-MG. Edição Especial – Ago./2020.
2 Organizadores: Prof.dr. Carlos Betlinski e Profa.dra. Ilsa do Carmo Vieira Goulart O direcionamento das pesquisas em educação pode ser pensado a partir da realidade
investigativa e assegurada pelos cursos de Mestrado Profissional em Educação, Conforme
regulamenta a Capes pela Portaria nº 080, de 16 de dezembro de 1998, a prioridade está na
formação de profissionais pós-graduados competentes a elaborar novas técnicas e processos
de ensino aprendizagem, com vistas ao “desempenho diferenciado de egressos dos cursos de
mestrado que visem preferencialmente um aprofundamento de conhecimentos ou técnicas de
pesquisa científica, tecnológica ou artística”. (BRASIL, 1998, p.1) Instituídos no Brasil em 1995, os mestrados profissionais foram regulamentados pelas
Portaria da Capes nº 47 e nº 80/1998, assumindo o Ministério da Educação a responsabilidade
relacionada à regulamentação e à oferta desses cursos. Deste modo, segundo os estudos de Chisté (2016, p. 790) a proposta de flexibilização
do modelo de Pós-Graduação stricto sensu em nível de mestrado nas modalidades acadêmico
e profissional regulou-se a partir “[...] da necessidade de uma formação universitária que
atendesse as demandas sociais, considerando que as mudanças tecnológicas e as correntes de
transformações econômico-sociais necessitam de profissionais com perfis de especialização
distintos dos tradicionais”. Frente a essa demanda, no ano de 2011 ocorreu a criação do Programa de Pós-
graduação em Educação (PPGE), na modalidade de Mestrado Profissional, integrando os
demais programas de pós-graduação da Universidade Federal de Lavras. O curso de Mestrado
Profissional do PPGE assume por objetivo a qualificação de professores para a atuação
profissional avançada, transformadora de procedimentos e questões inerentes à atuação
docente. O PPGE tem como área de concentração a “Formação de Professores” e conta,
atualmente com três linhas de pesquisa: (1) “Fundamentos da Educação, corpo e cultura:
teoria e prática nos processos educativos”; (2) “Desenvolvimento profissional docente, Revista Devir Educação, Lavras-MG. Edição Especial – Ago./2020. 1 Revista Devir Educação, Lavras-MG. Edição Especial – Ago./2020. 1 ISSN: 2526-849X Revista Devir Educação, Lavras-MG. Edição Especial – Ago./2020.
2 ISSN: 2526-849X Nessa vertente, organizamos um conjunto de textos que resultaram de ações
investigativas desenvolvidas por egressos do PPGE em parceria com os professores
orientadores, que buscaram apresentar diferentes modalidades e/ou procedimentos de
pesquisas e seus desdobramentos em práticas educativas. O Dossiê “A pluralidade e os diferentes contextos de pesquisa e formação de
professores” reuniu diferentes produções com o propósito de discutir sobre a atuação
profissional dos egressos do curso, tomando como referência a realidade vivenciada pelos
mestrandos e tematizada pelas linhas de pesquisas que integram o Programa de Pós-graduação
em Educação, da Universidade Federal de Lavras. Estes estudos trazem como finalidades
descrever elementos fundantes no processo de formação dos mestrandos, e também analisar a
repercussão do mestrado na continuidade dos estudos acadêmicos, na inserção profissional
dos egressos, tanto na educação básica, quanto no ensino superior. O Dossiê “A pluralidade e os diferentes contextos de pesquisa e formação de
professores” reuniu diferentes produções com o propósito de discutir sobre a atuação
profissional dos egressos do curso, tomando como referência a realidade vivenciada pelos
mestrandos e tematizada pelas linhas de pesquisas que integram o Programa de Pós-graduação
em Educação, da Universidade Federal de Lavras. Estes estudos trazem como finalidades
descrever elementos fundantes no processo de formação dos mestrandos, e também analisar a
repercussão do mestrado na continuidade dos estudos acadêmicos, na inserção profissional
dos egressos, tanto na educação básica, quanto no ensino superior. A Edição especial com os egressos do Programa de Pós-graduação em Educação, em
comemoração aos 10 anos de existência do Mestrado Profissional, no qual reunimos dezessete
produções de mestres que compartilham os estudos e as pesquisas desenvolvidas com seus
respectivos orientadores, dando visibilidade às diferentes ações investigativas produzidas na
área de formação de professores. A seguir apresentamos os artigos e a entrevista publicados,
nesta edição, com destaque para a riqueza das abordagens temáticas e de metodologias
científicas utilizadas. O critério para a organização da sequência dos textos foi a ordem
alfabética considerando o nome do primeiro autor. No primeiro artigo “Formação docente continuada: ensino híbrido e sala de aula
invertida como recurso metodológico para o aprimoramento do profissional de educação” as
autoras Agnes Priscila Martins de Morais e Priscila Franciely Souza tomam como objeto
de estudo o uso das tecnologias digitais e das metodologias ativas para analisar a sala de aula
invertida e ensino híbrido como recurso metodológico para a formação docente. ISSN: 2526-849X práticas pedagógicas e inovações” e (3) “Linguagens, diversidade cultural e inovações
pedagógicas”. Segundo o Regulamento do PPGE, a perspectiva pedagógica que distingue o curso de
mestrado profissional de um mestrado acadêmico, encontram-se numa estrutura curricular que
preconiza o aprofundamento da formação técnico-profissional conquistada na graduação; na
produção de um trabalho de conclusão em que o estudante apresente capacidade de articular
os conhecimentos básicos, o domínio teórico e metodológico do objeto de estudo e, por fim,
na aplicabilidade profissional relativa à área de concentração. Nessa perspectiva, o PPGE tem investido em ações formativas que privilegiam a uma
formação docente de qualidade, a partir da reflexão crítica sobre a prática pedagógica, da
apreensão ações investigativas pautadas em métodos científicos e na utilização de recursos
tecnológicos ou técnico-metodológicos referentes ao processo de ensino e aprendizagem,
compreendendo como tais aparatos podem contribuir para as práticas educativas. Ao contemplar uma perspectiva pedagógica distinta do mestrado acadêmico, o PPGE
busca consolidar ações formativas por meio da estrutura curricular dos projetos de pesquisa,
de organização de eventos científicos, de cursos de extensão e de produção de materiais
didáticos. O processo de formação oferecido pelo PPGE procura contribuir, efetivamente,
para um percurso formativo em articulação com os cursos de graduação, com atuação de
docentes voluntários, de modo garantir a formação para a docência no ensino superior e
potencializar aspectos da formação inicial. Outra preocupação encontra-se no desenvolvimento do letramento acadêmico, propiciando
experiências de produção acadêmica, participação em grupos de pesquisa e em projetos de
extensão, produção científica, com a participação em eventos científicos com a apresentação
de trabalhos, escrita de capítulos e de artigos, buscando com isso uma formação profissional
que se prima pela reflexão teórico-prática, alicerçada na crítica e na inovação pedagógica. Durante seus dez anos de existência na Universidade Federal de Lavras, o PPGE vem
desenvolvendo estudos sobre a multiplicidade de saberes, de espaços e de diferentes formas
de produção de conhecimento, contemplando as dimensões estéticas, éticas, políticas e
epistemológicas, culturais, pedagógicas e linguístico-discursivas do processo educativo, de
modo a provocar deslocamentos nos modos de fazer e de conceber o desenvolvimento
profissional e a atuação docente. 2 2 ISSN: 2526-849X O segundo artigo “Educação da audição e valores estéticos na Filosofia da Nova
Música de Theodor Adorno”, de autoria de Alan Barcelos Ribeiro e Carlos Betlinski, faz uma
abordagem teórica sobre o tema da educação da audição e toma como objeto de investigação a
“Filosofia da nova música” de Theodor Adorno. O estudo teve como objetivos a identificação
das principais características estéticas nas obras musicais de Stravinsky e de Schoenberg e
apontar alguns fundamentos filosóficos para diferenciar progresso e regressão na estética
musical em Adorno. Concluiu que uma estética assentada na crítica e na criação dissonante e
autônoma são critérios estéticos para tomar a música como objeto estético de progresso ou
regressão da audição. Adorno amplia as tensões em torno das dissonâncias para demonstrar o
processo de libertação da música das garras do tradicionalismo estático que acorrenta a
produção musical e impede os ouvidos de aceitar o progresso musical ditado pela nova
música. O terceiro artigo “Dimensões formativas decorrentes da congada em Lambari/MG:
entre experiência, saberes e ancestralidade” de autoria de Aline Guerra da Costa, Fabio Pinto
Gonçalves dos Reis, Lívia Nascimento Monteiro e Kleber Tüxen Carneiro refere-se a
pesquisa desenvolvida sobre os aspectos formativos da Congada e teve como lócus
investigativo o município de Lambari/MG. Participaram do estudo oito depoentes com forte
influência e representação que desfrutam no espaço congadeiro da localidade. A investigação
constatou que houve modificações no modo de ensinar e aprender os saberes aos novos
congadeiros que, apesar de alguns aspectos intuitivos no modo de aprender, tem suscitado
preocupação ou desinteresse em relação aos saberes (tradicionais) atinentes à congada, sob
pena da descontinuidade da propagação desse patrimônio cultural (imaterial) da humanidade. Na sequência da publicação, o quarto artigo “A nova reforma do ensino médio
amparada pela Lei 13.415 e suas ameaças para a disciplina de educação física” de autoria de
Bruna Beatriz da Rocha e Márcio Norberto Farias, os autores fazem uma reflexão teórica
sobre a Reforma do Ensino Médio - Lei Federal 13.415 e suas consequências para a
disciplina de Educação Física. Como metodologia ancorou-se na literatura produzida sobre
o tema, especialmente aquelas obras que servem de subsídio para as propostas pedagógicas
disponíveis dentro da Educação Física voltadas para o Ensino Médio. O estudo considerou
que existem várias possibilidades para intervir e aprender agindo diretamente na realidade
social, por meio da disciplina de Educação Física. ISSN: 2526-849X O estudo
teórico concluiu que, apesar de todas as dificuldades e empecilhos vivenciados pelos
professores no cenário nacional, é possível que a escola e a universidade articulem
experiências práticas na formação profissional simulando problemas e situações da realidade,
ensinando aos professores e estudantes a solucionarem questões práticas de suas profissões. Assim, o emprego das TDIC e das metodologias ativas pelos docentes podem favorecer a
autonomia do estudante, despertando a curiosidade, estimulando tomadas de decisões
individuais e coletivas, advindos das atividades essenciais da prática social. Revista Devir Educação, Lavras-MG. Edição Especial – Ago./2020. 3 3 Revista Devir Educação, Lavras-MG. Edição Especial – Ago./2020.
4 ISSN: 2526-849X Os professores possuem um papel
importante, assim sendo, devem buscar uma práxis estabelecendo relação intrínseca entre os Revista Devir Educação, Lavras-MG. Edição Especial – Ago./2020. 4 ISSN: 2526-849X
conhecimentos científicos e individuais da cultura corporal, considerando o aluno como ser
sócio-histórico. ISSN: 2526-849X conhecimentos científicos e individuais da cultura corporal, considerando o aluno como ser
sócio-histórico. conhecimentos científicos e individuais da cultura corporal, considerando o aluno como ser
sócio-histórico. Quanto ao quinto artigo “A educação na contemporaneidade: construindo relações
com os pensamentos de Christoph Türcke a partir da análise de uma sequência do filme a
religiosa, de Jacques Rivette”, as autoras Daniela Aparecida de Melo Costa, Bruna Beatriz da
Rocha e Luciana Azevedo Rodrigues analisam o filme “A Religiosa” à luz dos conceitos de
“choque de imagem do cinema” e a “compulsão à repetição traumática” de Türcke (2016). A
partir das relações com o pensamento destaca-se que o regime global da atenção, o choque de
imagem que a sequência fílmica em destaque propiciou a seus espectadores, hoje acontece de
forma ainda mais intensificada sobre a sociedade. Devido aos avanços tecnológicos da
contemporaneidade, a escola, os currículos e principalmente o papel do professor vem sendo
afetados por essa era do regime global de atenção. O sexto artigo “O minicurso na formação continuada de professores de línguas de uma
escola cooperativa: do processo formativo à efetivação da experiência”, de autoria de Danielle
Cristine Silva e Patrícia Vasconcelos Almeida, a partir de uma abordagem qualitativa discute
sobre as etapas permeadas ao trabalhar com a ferramenta formativa conhecida como
minicurso e produção didático-pedagógica a partir dessa experiência na formação do
professor em exercício. O texto demonstra que o minicurso ministrado contribuiu para a
formação do professor, salientando que a mudança não se encontra no artefato webquest, mas
sim sua utilização como recurso didático. Por sua vez, o sétimo artigo “Por uma pedagogia latino-americana” os autores
Dulcineia Aparecida Ferraz Ribeiro e Vanderlei Barbosa tomam como questão central a
formação humana na perspectiva das praticas educacionais emancipatórias e realizam um
estudo teórico-bibliográfico a partir da obra de Paulo Freire, partindo do livro Pedagogia do
Oprimido (1988), complementando com outras obras mais recentes que descrevem o
pensamento freireano na América Latina. Como resultados afirmam que uma pedagogia
tipicamente latino-americana se fundamenta na formação ética e se integra às dimensões
epistemológica, política, estética, teológica, mística, utópica, além de colocar no centro do
problema educacional a questão do poder. Revista Devir Educação, Lavras-MG. Edição Especial – Ago./2020.
5 Revista Devir Educação, Lavras-MG. Edição Especial – Ago./2020.
5 ISSN: 2526-849X Os processos de emancipação passam pela
afirmação do sujeito de voz, reconhecimento da subjetividade de saberes e de culturas,
incluindo o cuidado com o planeta terra em sua dimensão ecológica. 5 5 Revista Devir Educação, Lavras-MG. Edição Especial – Ago./2020.
6 ISSN: 2526-849X Na sequência Edmilson Francisco traz na oitava publicação uma reflexão sobre “O
impacto do mestrado profissional em educação na formação de um docente da educação
básica: um relato autobiográfico”, a partir de uma escrita que compartilha as experiências de
um professor que ao realizar um curso de mestrado e as buscas incessantes pela reelaboração
de sua prática pedagógica, desencadeadas enquanto mestrando no Programa de Pós-graduação
em Educação, da Universidade Federal de Lavras. No artigo número nove, a partir de uma pesquisa realizada com estudantes do quinto
ano do Ensino Fundamental de uma escola pública do interior de Minas Gerais, os autores
Francislaine Ávila de Souza, José Antônio Araújo Andrade e Francine de Paulo Martins
discutem sobre “As práticas de letramento matemático digital e o papel mediador das
tecnologias digitais: uma experiência com o software superlogo na Educação Básica”. O
artigo tem por finalidade apresentar os resultados de uma pesquisa que buscar analisar as
potencialidades do ambiente computacional Logo em práticas de letramento matemático
digital no desenvolvimento do pensamento algébrico. No artigo “Autobiografia como instrumento no processo identitário do docente: uma
reflexão sobre vivências no PIBID” a autora Jenifer Jully Vilela de Oliveira insere-se nessa
vertente de pesquisa e analisa como estudante de Letras, sua participação no PIBID,
destacando suas contribuições na participação do programa e dificuldades encontradas. Com a
elaboração e análise da autobiografia percebeu que a reflexão acerca da sua prática docente
permitiu que ressignificasse suas ações, transformasse as concepções sobre ensino-
aprendizagem, valorizasse a ajuda do outro, evidenciar que o PIBID influenciou na sua
identidade docente enxergando diversas formas de exercer a prática docente e que tipo de
professora gostaria de ser, uma vez que o programa permitiu uma prática mais reflexiva sobre
as ações e papéis no ambiente escolar. A autora Lara Nascimento Scherrer, no artigo número onze “Contribuições da
gramática do design visual para o ensino da leitura: sinalizações apontadas pela análise de
uma campanha educativa”, busca compreender como os diferentes modos de leitura
influenciam na formação do professor. Partindo da discussão de que a nossa sociedade está
em constante mudança ocasionada pelas Tecnologias Digitais de Informação e Comunicação
(TDIC), de modo a agregar elementos nas formas de comunicação, a autora destaca que os
modos de produzir e ler texto têm se diversificado, surgindo novos gêneros textuais, a partir 6 Revista Devir Educação, Lavras-MG. Edição Especial – Ago./2020.
7 Revista Devir Educação, Lavras-MG. Edição Especial – Ago./2020.
7 ISSN: 2526-849X aproximação entre as ações escolares e sociais, de modo a contribuir para uma
(re)significação do processo de ensino e de aprendizagem da produção de textos na escola. No artigo décimo quinto, Patricia Keli dos Santos aborda o tema do uso das
tecnologias digitais para o ensino de língua espanhola. A autora destaca que ensino mediado
por tecnologias digitais com suas múltiplas possibilidades como variadas práticas linguístico-
culturais, ferramenta de imersão virtual que representa um avanço significativo nas práticas
pedagógicas e pode auxiliar a promover um ensino de qualidade da língua espanhola. Quanto ao décimo sexto artigo “Relação entre ensino e literatura: um olhar para a
dimensão da ação e atuação docente”, as autoras Rita Cássia Oliveira, Ludmila Magalhães
Naves e Ilsa do Carmo Vieira Goulart apresentaram uma reflexão sobre a relação entre
literatura e ensino, pautadas em um relato de experiência das ações e práticas desenvolvidas a
partir da leitura literária, realizadas por uma professora do 2º ano do Ensino Fundamental,
com as crianças em processo de alfabetização, apresenta-se uma análise reflexiva das ações de
leitura literária. O relato procurou aproximar teoria e prática, ressignificando o papel da
leitura literária na escola, ao trazer uma visibilidade à atração docente professor como ação
mediadora no processo de formação de leitores. As autoras Sarah Silva Garcia e Tania Regina de Souza Romero trazem no artigo
número dezessete, “Narrativas autobiográficas e sua presença em periódicos A1”, uma
reflexão sobre a relevância da escrita autobiográfica. Pautado numa discussão sobre a história
da formação docente e sobre a formação docente crítico-reflexiva, o estudo traz o uso das
narrativas autobiográficas como ferramenta para avaliar a formação docente, como uma
possibilidade para se investigar os conflitos, para compreender a diversidade da realidade
social, para se conferir outros sentidos às experiências vividas e para se superar as
dificuldades profissionais e/ou pessoais, demonstrando um recurso que contribui para o
desenvolvimento docente. Na “entrevista com Tania Regina de Souza Romero, professora da Universidade
Federal de Lavras, em homenagem aos 10 anos do Programa de Mestrado Profissional em
Educação da UFLA” a docente do Programa de Pós-Graduação em Educação que marcou
presença desde a implementação do curso de Mestrado Profissional em Educação, período em
que se firmaram parcerias com os departamentos de Letras, de Educação Física e Ciências
Humanas. ISSN: 2526-849X disso busca analisar os efeitos de sentido advindos da composição imagética de uma
campanha educativa veiculada em redes sociais. No artigo “Preditores de permanência e evasão em cursos superiores a distância:
revisão da literatura”, os autores Ludmila de Oliveira Amaral Ferreira, Luanna Cristina de
Souza Paternoster, Ronei Ximenes Martins e Estela Aparecida Oliveira Vieira trazem uma
reflexão sobre os cursos em EAD ao apresentarem um estudo teórico, a partir de uma revisão
da literatura que procurou reunir fatores preditivos para a permanência ou a evasão de
estudantes matriculados em cursos de graduação a distância. O estudo aponta ações necessárias para a EAD como: conhecer os estudantes, para
assim oferecer-lhes metodologias de aprendizagem que possibilitem acionar estratégias de
estudo que favorecem os diferentes estilos de aprendizagem. Maria Betânia de Castro Nunes Santos, autora do artigo número treze que se refere a
uma pesquisa onde analisou a política pública do Pacto Nacional Pela Alfabetização na Idade
Certa (PNAIC) implantada em todo o país em 2013 e teve como principal propósito entender
quais são as percepções das professoras alfabetizadoras em matemática com relação aos temas
abordados e as propostas de atuação em sala de aula apresentadas durante este curso de
formação. Por meio de entrevista semiestruturada, aplicada a um grupo focal de oito professoras
alfabetizadoras participantes do PNAIC/2014, no município de Lavras – MG. A partir da
coleta de dados foi possível identificar que o PNAIC, apesar de colaborar, não é suficiente
para a formação das profissionais da educação, sendo necessária a formação continuada e
efetiva em Matemática e que, cursos como o PNAIC não são, por si só, capazes de mudar a
educação de forma imediata, mas que representam um importante passo de uma longa
caminhada. As Autoras Natany da Silva Avelar e Helena Maria Ferreira, no artigo quatorze “A
pedagogia dos multiletramentos e multiplicidade semiótica no texto infantil”, a partir de uma
reflexão sobre o papel do professor alfabetizador no processo de produção de textos infantis e
sobre os multiletramentos, procuram discutir sobre possibilidades de uso de múltiplas
semioses em retextualizações feitas por crianças em fase de alfabetização. O estudo desenvolvido pelas pesquisadoras demonstrou que o trabalho pedagógico
com recursos tecnológicos e o contato com textos multissemióticos propicia uma 7 Revista Devir Educação, Lavras-MG. Edição Especial – Ago./2020.
8 Revista Devir Educação, Lavras-MG. Edição Especial – Ago./2020.
9 ISSN: 2526-849X A entrevista foi realizada por Márcio Barbosa de Assis, egresso do mestrado e
abordou questões sobre a trajetória profissional e como se deu o direcionamento dos estudos e Revista Devir Educação, Lavras-MG. Edição Especial – Ago./2020. 8 8 8 ISSN: 2526-849X suas pesquisas, com destaque para utilização dos instrumentos como autobiografia e diários
reflexivos para o campo da formação de professores. Sinalizamos que foi prazeroso responsabilizar-se pela organização desta edição e
disponibilizar aos leitores, especialmente ao público que está diretamente relacionado com o
PPGE, uma representação significativa da produção de pesquisas e conhecimentos
diretamente relacionados com a área de concentração do programa que é a formação de
professores. Desejamos a todos uma leitura instigante e crítica. Agradecemos aos autores a
disposição para contribuir com essa construção coletiva que remete ao espírito e ao desafio de
amalgamar o Programa de Pós-Graduação em Educação da Universidade Federal de Lavras. Lavras, agosto de 2020. Dr.Carlos Betlinski Dr.Carlos Betlinski Drª Ilsa do Carmo Vieira Goulart Drª Ilsa do Carmo Vieira Goulart Drª Ilsa do Carmo Vieira Goulart 9
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Bangkok’s existing mixed fruit orchards are tree diversity hotspots for city greening
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Urban ecosystems
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Abstract Bangkok’s Inner Orchard (BIO), located in the west peri-urban area of the city, provides both fresh products and ecologi
cal benefits to its inhabitants. Although this unique forest-like cultivation of mixed fruit trees growing on dikes has the
potential to become an urban forest, it has never been thoroughly investigated. Urban expansion is a threat to this orchard. Sustainable cities must maintain a balance between development and the environment. In this study, six orchards were
surveyed by collecting data on tree species, plant height, crown width, and location and analyzed to identify the orchard
areas covered by forest canopy and clarify the forest structure and tree species diversity in the orchards. Overall, 98.64%
of the forest canopy within the orchard area was evaluated using cover analysis. Analysis of the forest structure of the
BIO revealed that the average tree height was greater than that of common orchard trees. Tree diversity analysis showed
an average Shannon index value of 2.53, indicating mid-range diversity. Older orchards showed a greater diversity of
fruit tree species, whereas newer orchards, replanted after major flood and drought events, showed a lesser diversity of
fruit tree species. In order to protect BIO on the long-term, it is essential to identify and apply appropriate conservation
strategies. Including BIO areas in the Bangkok greening scheme would cause rapid increases in the green area per capita
ratio. Both urban food forest and urban agriculture concepts could be applied to existing BIO orchards, which represent
new urban green area types. BIO thus has the potential to act as an urban forest and tree diversity hotspot for Bangkok. Keywords Bangkok’s Inner Orchard · Geographic information system · Tree diversity · Urban agriculture · Urban forest Keywords Bangkok’s Inner Orchard · Geographic information system · Tree diversity · Urban agriculture · Urban forest Keywords Bangkok’s Inner Orchard · Geographic information system · Tree diversity · Urban agriculture · Urban forest Keywords Bangkok’s Inner Orchard · Geographic information system · Tree diversity · Urban agr Introduction the urban environment is strongly affected by various forms
of urbanization, and the sustainable development of urban
areas is challenging. Therefore, Goal 11—sustainable cities
and communities—is included in the sustainable develop
ment goals of the UN (Division for Sustainable Develop
ment Goals, Department of Economic and Social Affairs,
United Nations 2015). Vudipong Davivongs1
· Sigit Dwiananto Arifwidodo1 Accepted: 25 March 2023 / Published online: 15 April 2023
© The Author(s) 2023 Accepted: 25 March 2023 / Published online: 15 April 2023
© The Author(s) 2023 Urban Ecosystems (2023) 26:991–1005
https://doi.org/10.1007/s11252-023-01352-w Urban Ecosystems (2023) 26:991–1005
https://doi.org/10.1007/s11252-023-01352-w 1
Department of Landscape Architecture, Faculty of
Architecture, Kasetsart University, 50 Ngamwongwan Rd.,
Lat Yao, Bangkok 10900, Thailand Vudipong Davivongs
archvpd@ku.ac.th Bangkok’s existing mixed fruit orchards are tree diversity hotspots for
city greening Vudipong Davivongs1
· Sigit Dwiananto Arifwidodo1 Urban green areas The world population has shown a trend for the movement of
populations from rural to urban areas, with more than half of
the global population already settled in cities (Debnath et al. 2014; Anguluri and Narayanan 2017). The United Nations
(UN) estimated that 68% of the world’s population would
live in urban areas by 2050 (Department of Economic and
Social Affairs, United Nations 2018). Some members of the
urban population live in crowded compact cities, whereas
the remaining live in vast urban sprawls. As a consequence, Urban greening is one of the strategies used to maintain
and improve the urban environment, which affects humans
to large extent. A lack of green areas in the urban environ
ment has become a major problem that influences the quality
of living, health, and well-being of urban dwellers (Bolund
and Hunhammar 1999; De la Barrera et al. 2016; Wolch et
al. 2014; Arifwidodo and Chandrasiri 2021; Arifwidodo et
al. 2022). Green areas provide ecosystem services such as
the mitigation of air pollution, capture of particulate matter
(PM 2.5 and 10), microclimate regulation, urban cooling,
noise reduction, and runoff mitigation, which aid in coping
with the environmental problems resulting from urbaniza
tion (Bolund and Hunhammar 1999; Arifwidodo 2014). 1 3 3 3 992 Urban Ecosystems (2023) 26:991–1005 State Model (Rodrigue 2020). Different types of agricultural
land are located in the form of rings at specific distances
from a city, with the distance based on the perishability and
heaviness of the goods. For instance, perishable products
from market gardens and dairy farms are produced in close
proximity to the city to prevent decay during long periods
of transportation. Firewood and lumber production areas are
located further away from the center in the next ring, but
they remain close to the city for easy transportation of these
heavy products (Rodrigue 2020).i Green areas help people relax and maintain good health
by engaging in physical activity and recreation (Cohen et
al. 2007; De Jong et al. 2012; Pietilä et al. 2015; Wang et al. 2019; Arifwidodo and Chandrasiri 2020). Urban agriculture
as well as urban food forest in green areas also provide food
and various materials, thus they are an essential part of the
urban food system (Tsuchiya et al. 2015; Opitz et al. 2016;
Nogeire-McRae et al. 2018; Park et al. 2019). Urban forest as a green area in Bangkok Urban forest is collectively defined as an urban ecosystem
that consists of forest patches, trees, plants, and associated
animals located in and around the city where people live,
work, and play (Nilsson et al. 2001; Weirsum and Sands
2013; Vogt 2020). From this inclusive urban forest defini
tion, forest patches, wooded areas, and tree-dominated parks
can be counted as urban forests in the same as trees planted
along roads and in gardens (Weirsum and Sands 2013). To
define a forest, the tree canopy cover was a key index used
for forest area identified by the Global Forest Resources
Assessments (FRA) of the Food and Agriculture Organiza
tion (FAO) and the United Nations Framework Convention
on Climate Change (UNFCCC). The conventional design of urban public parks in Bang
kok, as in many developing cities, is primarily focused on
the growing of ornamental plants and lawns (Richards et
al. 2017). However, diverse types of green areas should be
included in the planning. Vegetation in existing semi-natural
green areas aids in improving the urban environment and
ecology, providing ecosystem services that are necessary for
a livable city (Richards et al. 2017). Green areas of agricul
tural lands already exist in the BMR, but thorough inves
tigation on the potential of existing fruit forest patches for
urban greening is still lacking. Urban forests became a global phenomenon that is the
backbone of cities’ green infrastructure. They were used as
bridges and buffers between rural and urban areas in various
cities (FAO 2017). Urban forests enhanced the environmen
tal cities’ footprint which is necessary for urbanites’ well-
being (Weirsum and Sands 2013). Urban green areas Bangkok, the capital city of Thailand, is one of the
fastest-growing cities in the world (Lo et al. 1996). Over
the past four decades, the urbanized area of Bangkok has
expanded to its surrounding provinces, forming a metro
politan region called the Bangkok Metropolitan Region
(BMR). New urban fringe development, in the form of
built-up housing areas, commercial facilities, offices, fac
tories, and warehouses, has taken the place of the green
areas of former agricultural lands, while in the center of the
city, real estate developers invested in former open green
areas in central business districts to develop high-density
high-rise condominiums, hotels, offices, and shopping malls
(Intasen et al. 2017). The Bangkok Metropolitan Adminis
tration (BMA) is concerned about the lack of green areas in
the city and aims to increase such areas to 10 m2/person by
building more new urban public parks in the Green Bang
kok 2030 campaign (C40 Cities 2020; Bangkok Metropoli
tan Administration 2021). The goals of this campaign also
include increasing the urban tree canopy cover in Bangkok
from 8.6 to 30% (Intasen et al. 2017). However, the expan
sion of green areas by building new public parks is a slow
process, and the rate of greening will be insufficient to reach
this goal, particularly because both urban expansion and the
urban population are increasing at an extremely high rate
due to birth and migration. Various types of agricultural lands—rice paddy fields,
aquaculture farms, vegetable gardens, and fruit orchards—
are located around Bangkok. Even before the establishment
of Bangkok as the capital city of Thailand, the primary
function of these agricultural lands has been the provision
of food. Bangkok originated as a small agricultural vil
lage with fertile deltaic soil, growing various tropical fruits
and vegetables to serve Ayudhaya, the former capital city. Although the urbanization of Bangkok led to a lack of green
areas, the existing agricultural land may have good poten
tial to become a multi-functional urban agricultural land,
bringing additional benefits to the urban environment and
population. 1 3 Surrounding agricultural lands In Thailand, forest clearing was previously the main
form of land exploitation. The legislature gave property
rights to the individual who cleared and utilized wildlands. This concept is still rooted in the existing laws. There
fore, most of former Bangkok’s natural forests have been
cut down over time, giving way to agriculture and urban
development (Tejajati et al. 1999; Delang 2005; Asanok et Cities are the primary economic organ of most countries
(Jacobs 1970) and are usually surrounded by agricultural
land, which tends to be fertile, with high-quality soils (Bry
ant et al. 1982). The spatial relationships between urban and
agricultural lands are described in Von Thünen’s Isolated 1 1 3 Urban Ecosystems (2023) 26:991–1005 993 al. 2017). However, mixed fruit trees were planted instead
of natural forests along the Chao Phraya River, forming a
series of multi-storied, forest-like orchards, jointly called
Bangkok’s Inner Orchard (BIO) (Chankrajang and Vech
banyongratana 2020). These mixed fruit orchards, a typical
traditional cultivation system in Southeast Asia, could act as
an urban green area, with the potential to become an urban
forest (Sritongchuay et al. 2019). al. 2017). However, mixed fruit trees were planted instead
of natural forests along the Chao Phraya River, forming a
series of multi-storied, forest-like orchards, jointly called
Bangkok’s Inner Orchard (BIO) (Chankrajang and Vech
banyongratana 2020). These mixed fruit orchards, a typical
traditional cultivation system in Southeast Asia, could act as
an urban green area, with the potential to become an urban
forest (Sritongchuay et al. 2019). spatial distribution, spreading from the northern to the
southern part of the whole BIO (Fig. 1). Orchards that are still operational include both old con
tinuing orchards (OC) and newly planted orchards (NP). The selected orchards were: (1) Orchard A, a NP orchard,
located in the Muang district, Nonthaburi province; (2)
Orchard B, an OC orchard, located in the Talingchan dis
trict, Bangkok; (3) Orchard C, an OC orchard, located in
the Bangkoknoi district, Bangkok; (4) Orchard D, an OC
orchard, located in the Bangkoknoi district, Bangkok; (5)
Orchard E, an OC orchard, located in the Chom Thong dis
trict, Bangkok; and (6) Orchard F, an OC orchard, located
in the Phra Pradaeng district, Samut Prakarn province. More
detailed information on orchard characteristics can be found
in Table 1. Urban forests are urban green areas that contribute
to urban ecology by enhancing the biodiversity in urban
regions (Korhonen et al. 2021). Landscape and vegetation survey Because the study area was located in the urban fringe area
of Bangkok where rapid urbanization of the former orchard
area is continuously happening, pre-surveys were conducted
on-ground across the entire orchard region based on maps
and documented studies to obtain a clear picture of the cur
rent status of the orchards. Urban development encroached
upon the existing fruit orchard area, forming intermixed
urban–rural parcels. As a consequence of this type of urban
ization, the following three types of orchards were found:
(1) old continuing (OC), (2) newly planted (NP), and (3)
abandoned orchards (AO). Surrounding agricultural lands Tree diversity is a key driver
of overall forest-associated biodiversity (Ampoorter et al. 2020). A wide array of ecosystem services is supported by
tree species diversity which is necessary for urban ecosys
tem adaptability and resilience to disturbance (Morgenroth
et al. 2016; Cordonnier et al. 2018). Lack of biodiversity,
reflected in a low diversity of tree species, is an essential
problem associated with deterioration of the urban environ
ment caused by the widening of urban areas (Mirski 2020;
Berthon et al. 2021). This study aims to understand the
potential of BIO as tree diversity hotspots. The objectives
of the study were (1) to identify the orchard areas covered
by forest canopy and (2) to explore forest structure and tree
species diversity. The findings from this study can be used
by the local government to evaluate their potential to be
integrated into green space planning policies in Bangkok. Methods An on-ground survey was conducted on the six selected
orchards. Physical information on these orchards was col
lected, focusing on landscape and vegetation because the
BIO landscape combines natural and human-made systems
for growing fruit trees on low-lying land near the Chao
Phraya River. The land has been rearranged into ditches and
dikes, which provide a higher ground level for growing fruit
trees and a lower ground level for irrigation ditches. The
orchard size and landform of each orchard were measured
with the orchard owners’ guidance for the land boundary. These measured spatial data were mapped in a geographic
information system (GIS) database using ArcGIS 10.4
(Environmental Systems Research Institute, Redlands, CA,
USA). Study area The study area was focused on the BIO located in the west
ern part of the BMR. The area was selected because it had
the most extensive land use changes due to urbanization but
at the same time, it also had the biggest patch area identified
as the BIO. We obtained land-use maps from 1959 to 2000
with aerial photographs in 2022 retrieved from the Royal
Thai Survey Department to understand the overall spatial
patterns of BIO at the macro scale. A total area of 230 km2
was identified as the BIO at the western part of BMR. We then conducted a pre-survey of all areas categorized
as the BIO to select the microscale study areas based on
three criteria: (1) it was still operational, (2) it had clear
boundaries, (3) it was possible to identify the owner of the
orchard (because we needed the owner’s permission to sur
vey the orchards). Using a snowballing sampling method,
six orchards were selected for the study. We could not
expand the number of orchards in the study due to time
constraints and budget limitations. Although the number
of orchards used for the study was small, they had a good Five graduate students majoring in forestry were recruited
for the field survey. We conducted a three-days training for
the surveyors in identifying tree species and measuring
tree dimensions. The following parameters were collected
during the survey: tree location, tree height, crown width,
and species. A handheld global positioning system device
(GPSMAP 62sc; Garmin Ltd.; Olathe, KS, USA) was used
to acquire the exact location of each tree. A laser measuring
tool (Leica Disto D2; Leica Geosystems Ltd., Heerbrugg, 1 3 994 Urban Ecosystems (2023) 26:991–1005 Fig. 1 Location of the six study
orchards in the BMR Fig. Fig. 1 Location of the six study
orchards in the BMR Study area 1 Location of the six study
orchards in the BMR Table 1 Comparison of orchard size, type, average tree canopy cover, average tree height, average tree canopy diameter, and Shannon diversity
index in six orchards
Orchard
Orchard Size
(m2)
Type
Average Tree
Canopy Cover (%)
Average Tree
Height (m)
Average Tree Canopy
Diameter (m)
Shan
non
Index
A
12,000
NP
61.37
4.27
3.44
1.62
B
3,680
OC
57.35
4.46
3.06
2.75
C
4,320
OC
62.35
5.69
3.78
2.57
D
2,176
OC
44.93
4.66
2.70
1.99
E
11,200
OC
58.27
4.37
3.28
3.51
F
1,712
OC
50.66
4.18
2.92
2.73
Orchard types: Old continuing (OC), Newly planted (NP), Abandoned orchard (AO) Table 1 Comparison of orchard size, type, average tree canopy cover, average tree height, average tree canopy diam
ndex in six orchards e, type, average tree canopy cover, average tree height, average tree canopy diameter, and Shannon diversity 1 3 995 Urban Ecosystems (2023) 26:991–1005 Switzerland) was used for measuring tree dimensions. Tree
guidebooks were consulted to correctly identify tree species
(Veesommai and Jenjittikul 2006; Gardner et al. 2007; Vee
sommai and Kaewduangtien 2009). When necessary, leaves
and flowers were collected along with photographs, and
submitted to domain experts for the identification of tree
species. The collected tree information was entered into a
GIS map for further analysis. forest structure analysis. Trees at least 2 m high from the
field survey data were included for the analysis. The aver
age tree height and crown width per orchard were calculated
and compared. The maximum and minimum tree height and
crown width were also included in the comparisons. Scaled
forest transects were drawn on-site to create a forest profile
for each orchard, including the tree canopy layers. Tree pro
file diagrams were drawn at the sides of each orchard. Tree diversity analysis Data on tree species were used for
tree diversity analysis by employing the Shannon diver
sity index. Only trees with a minimum height of 2 m were
included in this analysis. Identification of orchard areas covered by forest
canopy The tree datasets, including tree height, average tree
crown width, and tree location, were used for spatial analy
sis to identify orchard areas covered by forest canopy. Arc
GIS 10.4 was used for data preparation. Data from trees at
least 2 m high were used for further analysis. The crown
area of each tree was generated in a circular shape using
ArcGIS based on the collected average crown width data. The average crown width data came from measurements
of each tree’s crown width in 4 directions from its stem–
north, south, east, and west. These data were used to cre
ate a canopy cover layer map, which was superimposed on
a 10 × 10 m fishnet grid layer map. The percentage of tree
canopy cover in each cell was analyzed using the zonal sta
tistics tool in ArcGIS 10.4. Analysis of the forest canopy based on a 10 × 10 m grid layer
map revealed that nearly all orchard areas were covered by
forest canopy. Out of 147 squares from six studied orchards,
145 were evaluated as forest areas. Tree canopy covered 8,496.6 m2 of the 14,700 m2
(57.80%) covered by all study orchards. However, some
variation in the tree canopy cover ratio was recorded in
each orchard. The highest average tree canopy cover ratio
(62.35%) was observed in Orchard C (max = 99.67%,
min = 30.15%), followed by that in Orchard A, which was
61.37% (max = 84.4%, min = 14.27%). The average tree can
opy cover ratio was the third highest in Orchard E at 58.27%
(max = 95.54%, min = 17.88%), followed by Orchard B at
57.35% (max = 91.21%, min = 1.67%), Orchard F at 50.66%
(max = 85.54%, min = 15.02%), and Orchard D with 44.93%
(max = 63.18%, min = 19.82%) (Figs. 2 and 3) (Table 1). Identification of orchard areas covered by forest canopy H′ = −
S
i=1
PilnPi Tree canopy cover is a metric widely used to evaluate urban
forests (Barron et al. 2016). To estimate the urban affor
estation, we adopted the definition of a forest from FRA
and UNFCCC to appropriately measure forest areas in the
orchard scale. The afforestation scale, tree height, and tree
canopy cover were used to determine the forest attributes. For FRA, forest is a land area of more than 0.5 ha, with a
tree canopy cover of more than 10% of which was covered
by a canopy of trees at least 3 m high (UNEP 2009). For
UNFCCC, forest is a land area of 0.01–0.1 ha, with 10–30%
minimum tree canopy cover of 2–5 m tree height (UNEP
2009). In this study, an orchard area of 10 × 10 m, over 10%
of which was covered by a canopy of trees at least 2 m high
was considered to be a forest area. where H’ is the Shannon index, Pi is the proportion of indi
vidual trees found in i species, S is the total number of spe
cies, and S
i=1 Pi is the sum of Pi of tree species 1 to S. i 1
Values of the Shannon index were compared among the
six orchards. Data analyses Data from the landscape and vegetation surveys were ana
lyzed (1) to identify the orchard areas covered by forest
canopy, and (2) to explore the forest tree structure and tree
diversity in the orchards. The Shannon index (H’) scores species diversity from 0
to 5, with a higher score indicating a higher diversity. The
Shannon index was computed according to the following
formula: Fig. 2 Map of tree canopy cover
age ratio in each of the 10 × 10 m
cells of the Orchard D area Forest structure and tree diversity analysis The lowest tree diversity was found in
Orchard A, which had a Shannon index score of 1.62. There
were 18 species within 208 trees and most were Musa ×
paradisiaca (62 plants), Erythrina fusca Lour. (52), and
Durio zibethinus Moon (45) (Tables 1 and 2). The tree heights from six orchards were measured and com
pared. The average tree height in Orchard C was 5.69 m
(max = 19.40 m, min = 2 m), followed by Orchard D with
4.66 m (max = 13.10 m, min = 2 m). The third highest value
was noted in Orchard B, which had an average tree height
of 4.46 m (max = 14 m, min = 2 m), followed by Orchard E
with 4.37 m (max = 13.20 m, min = 2 m), Orchard A with
4.27 m (max = 12 m, min = 2 m), and Orchard F with 4.18 m
(max = 12 m, min = 2 m) (Figs. 4 and 5) (Table 1). 2.75 for 36 species within 159 trees. Most of them were
Syzygium malaccense (L.) Merr. & L.M.Perry (36 plants),
Musa × paradisiaca L. (27), Carica papaya L. (15), and
Syzygium samarangense (Blume) Merr. & L.M.Perry (15). Orchard F had the third highest tree diversity, with a Shan
non index score of 2.73 for 29 species within 134 plants. Most of them were Cocos nucifera L. (21 plants), Leucaena
leucocephala (Lam.) de Wit (19), and Senna siamea (Lam.)
H.S.Irwin & Barneby (19). Orchard C had the third highest
tree diversity, with a Shannon index score of 2.57 for 39
species within 420 trees. The most prevalent species were
Mangifera indica L. (85 plants), Artocarpus heterophyllus
(78), and Cocos nucifera (77). The fifth was Orchard D with
a Shannon index score of 1.99 for 19 species within 142
trees. Most of them were Musa × paradisiaca (47 plants),
Azadirachta indica A.Juss. (29), Mangifera indica (20), and
S. malaccense (20). The lowest tree diversity was found in
Orchard A, which had a Shannon index score of 1.62. There
were 18 species within 208 trees and most were Musa ×
paradisiaca (62 plants), Erythrina fusca Lour. (52), and
Durio zibethinus Moon (45) (Tables 1 and 2). The canopy diameters of trees from six orchards were
measured and compared. Forest structure and tree diversity analysis Orchard C had the largest average
tree canopy diameter at 3.78 m (max = 12 m, min = 0.60 m),
followed by Orchard A with 3.44 m (max = 8.60 m,
min = 1 m). The third was Orchard E, whose average tree
canopy diameter was 3.28 m (max = 11.60 m, min = 0.60 m),
followed by Orchard B having an average tree canopy
diameter of 3.06 m (max = 8 m, min = 0.46 m), Orchard F
with 2.92 m (max = 7.8 m, min = 0.6 m), and Orchard D
with 2.70 m (max = 7.10 m, min = 0.34 m) (Figs. 4 and 5)
(Table 1). Forest structure and tree diversity analysis Forest structure The structure of a forest is vital to its biodi
versity (Pan et al. 2018). The data collected on tree species,
height, and crown width from six orchards were used for 1 3 1 1 3 Urban Ecosystems (2023) 26:991–1005 996 Forest structure and tree diversity analysis
Forest structure analysis
Fig. 3 Comparison of tree canopy
cover ratio in 10 × 10 m cells
among six orchards
Fig. 2 Map of tree canopy cover
age ratio in each of the 10 × 10 m
cells of the Orchard D area Fig. 2 Map of tree canopy cover
age ratio in each of the 10 × 10 m
cells of the Orchard D area Fig. 2 Map of tree canopy cover
age ratio in each of the 10 × 10 m
cells of the Orchard D area of tree canopy cover
each of the 10 × 10 m
Orchard D area Forest structure and tree diversity analysis
Forest structure analysis
Fig. 3 Comparison of tree canopy
cover ratio in 10 × 10 m cells
among six orchards Fig. 3 Comparison of tree canopy
cover ratio in 10 × 10 m cells
among six orchards Fig. 3 Comparison of tree canopy
cover ratio in 10 × 10 m cells
among six orchards Forest structure and tree diversity analysis
Forest structure analysis 1 3 1 3 997 Urban Ecosystems (2023) 26:991–1005 2.75 for 36 species within 159 trees. Most of them were
Syzygium malaccense (L.) Merr. & L.M.Perry (36 plants),
Musa × paradisiaca L. (27), Carica papaya L. (15), and
Syzygium samarangense (Blume) Merr. & L.M.Perry (15). Orchard F had the third highest tree diversity, with a Shan
non index score of 2.73 for 29 species within 134 plants. Most of them were Cocos nucifera L. (21 plants), Leucaena
leucocephala (Lam.) de Wit (19), and Senna siamea (Lam.)
H.S.Irwin & Barneby (19). Orchard C had the third highest
tree diversity, with a Shannon index score of 2.57 for 39
species within 420 trees. The most prevalent species were
Mangifera indica L. (85 plants), Artocarpus heterophyllus
(78), and Cocos nucifera (77). The fifth was Orchard D with
a Shannon index score of 1.99 for 19 species within 142
trees. Most of them were Musa × paradisiaca (47 plants),
Azadirachta indica A.Juss. (29), Mangifera indica (20), and
S. malaccense (20). Forest structure Forest structure, particularly tree height, is important to for
est biodiversity (Martins et al. 2017; Pan et al. 2018). Tree
height has a strong relationship with tree species richness
(Marks et al. 2016). Our analysis revealed that the aver
age tree height in the BIO was greater than that of common
orchard trees. Tree height in rural areas is usually as low as
1.5–2.25 m because farmers need to trim tree branches to
facilitate harvesting and the distribution of sunlight (Mika
1992; Wagenmakers and Callesen 1995). However, the tree
height recorded in the field survey in the BIO was greater. The average tree height in each orchard ranged from 4.18
to 5.69 m. The tallest tree (19.4 m) was Cocos nucifera,
in Orchard C. Mangifera indica was the tallest tree in
Orchard D (13.1 m), Erythrina fusca (12 m) was the tall
est in Orchard A, Cocos nucifera (12 m) in Orchard F, and
Artocarpus altilis (Parkinson) Fosberg and Litchi chinensis
(13.2 m) in Orchard E. An increase in urban tree canopy (UTC) cover is one of
the indices that the BMA aims to pursue. UTC is a basic
but important index for green space management in cit
ies worldwide (Parmehr et al. 2016). UTC values in some
major cities in the US are: New York City 20.9%, Phila
delphia 15.7%, and Chicago 11%, and in other continents:
London 20%, Tokyo 19%, and Beijing 24% (Nowak and
Dwyer 2007; Endreny et al. 2017). For Bangkok, a study
conducted in 2013 estimated that the urban tree cover was
8.6%, which was relatively low (Intasen et al. 2017). How
ever, agricultural areas were excluded from this study. A tree
cover analysis of BIO revealed that the mixed fruit tree can
opy covered a relatively large area: 57.8% of the orchards
studied. Therefore, inclusion of the BIO as an urban green
area will increase the UTC of both Bangkok and the BMR. Fruit growing in the BIO followed the traditional style
of agricultural practice on small subsistence farms (Kha
okhrueamuang 2014). Various fruit trees occurring here
imitate a natural forest structure with canopy layers at dif
ferent heights. These allowed suitable sunlight conditions
for different kinds of fruit trees, vegetables, and herbs that
can grow together naturally in a compact area. Tree branch
trimming to lower the height of fruit trees is not a common
practice in the BIO. Discussion and conclusions The diversity of the trees in the six orchards was analyzed
using the Shannon index. Orchard E was the richest in terms
of species, with a Shannon index of 3.51. There were 292
trees of 58 species in this orchard. The majority were Litchi
chinensis Sonn. (31 plants), Musa balbisiana Colla (24),
and Artocarpus heterophyllus Lam. (20). Orchard B had
the second highest tree diversity, with a Shannon index of Our results revealed that the BIO had the potential to be an
urban forest as a part of the Bangkok greening scheme. This
BIO green area is composed of large trees that provide tree
canopy cover and tree species diversity. Fig. 4 Comparison of tree height
and tree canopy diameter in six
orchards
1 Fig. 4 Comparison of tree height
and tree canopy diameter in six
orchards 1 Urban Ecosystems (2023) 26:991–1005 998 Fig. 5 Tree profile and photos of Orchard D Fig. 5 Tree profile and photos of Orchard D Forest structure and tree diversity The intermixing of rural and urban areas in Asian cities
has aided their greening evolution from agricultural lands
to a large extent (McGee 1972). Our results revealed that
the existing mixed fruit orchards in Bangkok, the BIO,
can be categorized as urban forests. Based on the FRA and
UNFCCC criteria, most of the study orchard areas (14,500
m2 from a total 14,700 m2 (98.64%)) were regarded as for
ests based on analysis of the tree canopy cover ratio. Forest structure In a previous study, Sommeechai et al. Tree diversity The Shannon index measures species abundance and tree
evenness (Magurran 2004). In the BIO, the Shannon index
score ranged from 1.5 to 3.5. This scoring range is a typical
value in most ecological studies (Kirkhoff 2010). The aver
age Shannon index calculated from the six studied orchards
was 2.53, which falls in the medium range of diversity. The
highest score recorded in Orchard E was 3.51, a relatively
high diversity value. The lowest score recorded in Orchard
A was 1.62, which is considered a low diversity value. How
ever, the present study excluded vegetation having a height
lower than 2 m—shrubs and ground cover—from the analy
sis. Therefore, a Shannon index value including all vegeta
tion present in the orchards should be included in future
studies. (
)
A collection of large trees in the BIO is thus a valuable
resource for improving Bangkok’s urban environment,
especially in expanding suburban residential areas. Large
trees provide shade, and fresh air by significantly reducing
air temperature and air pollution (Nowak and Heisler 2010;
Krul 2015). New residents enjoy the lush green atmosphere
of neighboring orchards by utilizing them as parks for rec
reation and exercise. The orchards have been encroached
upon by new housing estates built by various real estate
development companies. These new housing estates pose
as a threat to the surrounding green areas of the BIO while
clearing large amounts of large trees on their own land. Nev
ertheless, the presence of trees in urbanized areas increases
a property’s real estate value because the quality of life is
enriched by tree diversity, which provides both aesthetic
appeal and microclimate improvement (Singh et al. 2018). Old continuing orchards, represented by Orchards B, C,
D, E, and F, consisted of relatively diverse fruit tree species. In contrast, the new Orchard A, replanted after a major flood
in 2011 and a drought in the following year, had a lower
diversity of fruit tree species. To further investigate tree diversity, we randomly
selected three case studies investigating natural tropical for
ests in Thailand and compared the diversity indices obtained
in these studies. The first study was a tree diversity study
of a mountain evergreen forest in the Chiang Dao Wildlife
Sanctuary in the Chiang Mai province. The Shannon index
scores from four study plots ranged from 1.32 to 2.10. The
average diversity score was 2.06 (Kambai et al. 2016). Forest structure 1 3 1 3 999 Urban Ecosystems (2023) 26:991–1005 Plant Species
Common Name
Number of Plants in Orchard
A
B
C
D
E
F
Acacia pennata
Climbing wattle
0
0
2
0
3
0
Aegle marmelos
Bael
0
2
0
0
0
0
Alocasia sanderiana
Sander’s alocasia
0
1
0
0
0
0
Alpinia galanga
Galanga
0
1
0
2
0
0
Annona muricata
Soursop
0
2
0
0
0
0
Annona squamosa
Sugar apple
3
1
1
0
0
0
Antidesma thwaitesianum
Mao fruit
0
2
0
0
0
0
Ardisia polycephala
Duck’s eyes
0
0
0
0
0
3
Areca catechu
Areca palm
18
0
2
0
12
3
Artocarpus altilis
Breadfruit
1
1
8
0
2
1
Artocarpus heterophyllus
Jackfruit
0
4
78
1
20
1
Averrhoa bilimbi
Bilimbi
0
2
0
0
12
1
Averrhoa carambola
Carambola
0
0
1
0
0
0
Azadirachta indica
Siamese neem tree
1
1
10
29
5
7
Baccaurea ramiflora
Rambeh Bambi
0
2
1
0
1
0
Bambusa bambos
Bamboo
0
0
0
1
0
0
Bambusa multiplex
Hedge bamboo
0
0
6
0
0
0
Barringtonia acutangula
Indian oak
0
0
0
0
9
0
Barringtonia racemosa
Powderpuff tree
0
0
1
0
0
0
Bauhinia acuminata
Snowy orchid tree
0
1
0
0
0
0
Bouea macrophylla
Marian plum
0
3
2
1
2
0
Broussonetia papyrifera
Paper mulberry
0
0
0
0
0
2
Canna indica
Canna lily
0
0
0
0
6
0
Carallia brachiata
Corkwood
0
0
0
0
1
0
Carica papaya
Papaya
1
15
2
2
3
1
Caryota mitis
Fishtail palm
0
0
0
0
1
2
Cerbera odollam
Pong pong
3
0
0
0
0
0
Citrus aurantifolia
Key lime
4
1
0
0
12
0
Citrus hystrix
Kiffir lime
0
2
2
1
0
0
Citrus maxima
Pomelo
1
0
0
1
2
0
Citrus reticulata
Tangerine
13
0
0
0
1
0
Citrus sinensis
Sweet orange
1
0
0
0
1
0
Clerodendrum fragrans
Glory bower
0
0
0
0
1
0
Cocos nucifera
Coconut
0
10
77
0
5
21
Dalbergia cochinchinensis
Siamese rosewood
0
0
0
0
1
0
Dillenia indica
Elephant apple
0
0
0
0
1
0
Diospyros rhodocalyx
Ebony
0
0
1
0
0
0
Dolichandrone serrulata
Trumpet tree
0
0
0
0
0
1
Duranta erecta
Golden dewdrop
0
0
0
0
1
0
Elaeocarpus hygrophilus
Ma Kok Nam
0
0
0
0
2
0
Erythrina fusca
Coral tree
0
0
1
0
12
3
Etlingera eliator
Torch ginger
0
0
0
0
2
0
Fagraea fragrans
Tembusu
0
0
0
0
1
0
Ficus maciellandii
Banana leaf fig
0
0
0
0
2
0
Ficus racemosa
Cluster fig
0
0
6
0
1
0
Garcinia cowa
Cowa
0
0
0
0
2
0
Garcinia mangostana
Mangosteen
1
0
0
0
0
0
Garcinia schomburgkiana
Madan
0
3
1
0
1
1
Table 2 List of plant species found in six orchards Table 2 List of plant species found in six orchards Common Name
Number of Plants in Orchard
A
B
C
D
E
F
Climbing wattle
0
0
2
0
3
0
Bael
0
2
0
0
0
0
Sander’s alocasia
0
1
0
0
0
0
Galanga
0
1
0
2
0
0
Soursop
0
2
0
0
0
0
Sugar apple
3
1
1
0
0
0
Mao fruit
0
2
0
0
0
0
Duck’s eyes
0
0
0
0
0
3
Areca palm
18
0
2
0
12
3
Breadfruit
1
1
8
0
2
1
Jackfruit
0
4
78
1
20
1
Bilimbi
0
2
0
0
12
1
Carambola
0
0
1
0
0
0
Siamese neem tree
1
1
10
29
5
7
Rambeh Bambi
0
2
1
0
1
0
Bamboo
0
0
0
1
0
0
Hedge bamboo
0
0
6
0
0
0
Indian oak
0
0
0
0
9
0
Powderpuff tree
0
0
1
0
0
0
Snowy orchid tree
0
1
0
0
0
0
Marian plum
0
3
2
1
2
0
Paper mulberry
0
0
0
0
0
2
Canna lily
0
0
0
0
6
0
Corkwood
0
0
0
0
1
0
Papaya
1
15
2
2
3
1
Fishtail palm
0
0
0
0
1
2
Pong pong
3
0
0
0
0
0
Key lime
4
1
0
0
12
0
Kiffir lime
0
2
2
1
0
0
Pomelo
1
0
0
1
2
0
Tangerine
13
0
0
0
1
0
Sweet orange
1
0
0
0
1
0
Glory bower
0
0
0
0
1
0
Coconut
0
10
77
0
5
21
Siamese rosewood
0
0
0
0
1
0
Elephant apple
0
0
0
0
1
0
Ebony
0
0
1
0
0
0
Trumpet tree
0
0
0
0
0
1
Golden dewdrop
0
0
0
0
1
0
Ma Kok Nam
0
0
0
0
2
0
Coral tree
0
0
1
0
12
3
Torch ginger
0
0
0
0
2
0
Tembusu
0
0
0
0
1
0
Banana leaf fig
0
0
0
0
2
0
Cluster fig
0
0
6
0
1
0
Cowa
0
0
0
0
2
0
Mangosteen
1
0
0
0
0
0
Madan
0
3
1
0
1
1
Caricature plant
0
1
0
0
0
0
Bitterleaf tree
0
1
0
2
0
0
Heliconia
0
0
0
0
1
0
Hibiscus
0
0
0
0
0
1 1 3 1 1 3 Urban Ecosystems (2023) 26:991–1005 1000 Plant Species
Common Name
Number of Plants in Orchard
A
B
C
D
E
F
Hylocercus undatus
Dragon fruit
0
0
1
0
0
0
Lagerstroemia speciosa
Queen’s flower
0
0
0
0
5
0
Lansium domesticum
Langsat
0
1
0
0
0
0
Lasia spinosa
Spiny Lasia
0
0
1
0
0
0
Lepisanthes fruticosa
Luna nut
0
2
0
0
0
0
Lepisanthes rubiginosa
Mertajam
0
0
0
0
0
2
Leucaena leucocephala
River tamarind
0
0
3
0
15
19
Litchi chinensis
Lychee
1
0
4
0
31
0
Livistona speciosa
Mountain serdang
0
0
0
0
0
1
Malpighia glabra
Barbados cherry
0
1
0
0
0
0
Mangifera indica
Mango
3
9
85
20
14
3
Manilkara zapota
Sapodilla
0
0
1
0
0
0
Mimusops elengi
Asian bulletwood
0
0
0
0
2
0
Morinda citrifolia
Noni
0
0
2
8
9
12
Moringa oleifera
Moringa
4
0
0
0
0
0
Morus alba
Mulberry
0
0
1
0
1
0
Muntingia calabura
Jamaican cherry
0
0
2
0
0
1
Musa balbisiana
Banana
2
0
14
1
24
0
Musa sapientum
Banana
54
27
22
47
8
15
Oroxylum indicum
Broken bones tree
0
0
0
0
6
0
Pandanus amaryllifolius
Pandanus palm
0
0
0
0
1
0
Persicaria odorata
Vietnamese coriander
0
0
0
0
1
0
Phyllanthus acidus
Star gooseberry
0
2
2
0
2
2
Phyllocarpus septentrionalis
Monkey flower tree
0
0
0
0
1
0
Phyllostachys sulphurea
Yellow running bamboo
0
0
0
0
6
0
Pithecellobium dulce
Manila tamarind
0
0
0
0
0
2
Pseuderanthemum atropurpureum
Purple false Eranthemum
0
1
0
0
0
0
Psidium guajava
Guava
0
0
1
2
4
0
Pterocarpus indicus
Burmese rosewood
0
0
0
0
0
1
Ptychesperma macarthuii
MacArthur palm
0
1
0
0
0
0
Punica granatum
Pomegranate
0
0
0
0
0
1
Saccharum officinarum
Sugar cane
0
0
0
1
1
0
Sandoricum koetjape
Santol
0
0
23
1
0
0
Senna siamea
Cassod tree
0
1
0
0
0
19
Sesbania javanica
Sesbania
0
2
0
0
0
0
Solanum ferox
Solanum
0
0
0
0
2
0
Solanum torvum
Turkey berry
0
1
0
0
0
0
Solanum xanthocarpum
Yellow berried night shade
0
0
1
0
0
0
Spathodea campanulata
African tulip tree
0
1
0
0
0
0
Spondias pinnata
Hog plum
0
0
0
0
3
0
Streblus asper
Siamese rough bush
0
2
13
0
2
1
Syzygium jambos
Rose apple
0
0
2
0
0
0
Syzygium malaccense
Malay apple
0
36
26
20
4
0
Syzygium samarangense
Java apple
0
15
11
0
2
0
Tabebuia argentea
Silver trumpet tree
0
0
0
0
1
0
Tabebuia rosea
Pink trumpet tree
0
0
0
0
0
1
Tabernaemontana divaricata
Crepe jasmine
0
0
0
1
0
0
Tamarindus indica
Tamarind
0
0
2
0
1
6
Tecoma stans
Yellow elder
0
0
0
0
1
0
Tectona grandis
Teak
0
0
0
0
11
0
Terminalia catappa
Bengal almond
0
0
1
0
0
0
Theobroma cacao
Cacoa
0
1
0
0
0
0 1 3 1001 Urban Ecosystems (2023) 26:991–1005 Plant Species
Common Name
Number of Plants in Orchard
A
B
C
D
E
F
Thevetia peruviana
Yellow oleander
0
0
0
0
2
0
Thyrsostachys siamensis
Bamboo
0
0
0
0
4
0
Wrightia religiosa
Water jasmine
0
0
0
0
2
0
Table 2 (continued) (2018) identified three layers of tree canopy, whereas Sam
panpanish and Lamroenprucksa (1994) identified four layers
in these mixed fruit tree orchards. Forest structure Because the tree canopies
formed in the six studied orchards were continuous, the
identification of tree canopy layers varied depending on the
methods employed in previous studies (Sampanpanish and
Lamroenprucksa 1994). Nao forest corridor in the Phitsanulok province. The Shan
non index score of dry evergreen forest trees was 2.4, that of
mountain coniferous forest trees was 2.36, that of deciduous
forest trees was 1.15, and that of deciduous dipterocarp for
est trees was 2.04 (Suksawang et al. 2012). Tree diversity
in the BIO was in the same range as that assessed in natural
forests, and higher than that in agricultural lands. The BIO urban forests can be considered as multifunc
tional agricultural land that provides ecosystem services to
the city. The BIO areas also play an essential role as an urban
food forest, producing fresh tropical fruits such as durians,
mangos, mangosteens, rose apples, jackfruits, litchis, coco
nuts, bananas, etc., and local herbs. The species diversity
of fruit trees found in these traditional mixed-fruit orchards
reflected a large variety of fruits and herbs that can be pro
duced from the BIO. They also provide regulating services
that improve the urban environment. Large trees located in
urban forests are a sink for CO2 and can help in offsetting
carbon emissions from urban areas through photosynthesis
(Pasher et al. 2014; Schafer et al. 2019). Tree diversity The
second study assessed the tree diversity of a forest com
plex in the Kui Buri National Park, Prachuap Khiri Khan
province. This study shows that the Shannon index score
depended on the type of the forest: mixed deciduous forest
(1.20–2.60), secondary forest (0.43–1.85), or agricultural
area (0–1.85) (Temchai et al. 2016). The third study was
an assessment of the tree diversity of the Phu Khiao-Nam Taking all these problems into consideration, we suggest
that it is important to conserve the BIO using appropriate
strategies because the ongoing development of civil engi
neering affects the area to a large extent. Protection of this
semi-natural green space at an early stage of urban devel
opment is essential because it is difficult to regenerate this
land in the future (Richards et al. 2017). In terms of plan
ning, these agricultural lands could also act as buffer zones 1 3 1002 Urban Ecosystems (2023) 26:991–1005 Acknowledgements This research was co-funded by the Thailand
Research Fund, Office of the Higher Education Commission, and Kas
etsart University (Grant No. MRG6080249). The authors are grateful
to the editor and anonymous reviewers for their valuable comments
and suggestions. We are also thankful to Kasetsart University Research
and Development Institute for supporting the English language editing
service. We thank the graduate students from the Faculty of Forestry,
Kasetsart University, for assisting with the field surveys. We are grate
ful to all farmers and orchard owners who gave us information and
allowed us to study their orchards. to help avoid conflict between the urban environment and
natural forests (Zhou and Wang 2011). Despite the fact that
suitable greening policies are essential for the sustainabil
ity of urban development (Zhou and Wang 2011), the green
areas of Bangkok are currently only defined as public parks,
according to an open-space plan developed by the BMA
(Taylor and Hochuli 2017). Including these BIO areas in the
Bangkok greening scheme would be a powerful strategy to
improve the green area per capita ratio. Urban food forest
and urban agriculture share concepts of improving the sus
tainability and resilience of urban communities by the use
of food-producing trees and agricultural produce to create
urban edible landscapes (Bailkey and Nasr 2000; Clark and
Nicholas 2013). Tree diversity Both urban food forest and urban agricul
ture concepts could be applied in this greening scheme as
new types of urban green areas in the BIO for long-term
community benefits (Kowalski et al. 2019). Author Contribution Vudipong Davivongs: Conceptualization, Meth
odology, Data acquisition, Data curation, Validation, Formal analysis,
Investigation, Project administration, Writing – original draft, Writing
– review & editing. Sigit Dwiananto Arifwidodo: Writing – review &
editing. Funding This research was co-funded by the Thailand Research Fund,
Office of the Higher Education Commission, and Kasetsart University
(Grant No. MRG6080249). Data Availability The datasets generated and/or analysed during the
study are available from the corresponding author upon request. Effective green urban planning and management that
includes urban forests could provide many benefits to urban
dwellers. The urban forests of the BIO are located primar
ily on private properties. However, Clark et al. (1997) sug
gested that urban forests on both public and private lands
should be included in planning and management with the
aim of addressing the association between the benefits and
disadvantages of urban forest management (Dwyer et al. 1992). Therefore, accurate and quantitative assessment of
data pertaining to these urban forests are vital to determine
the value of services provided and to assess appropriate
management strategies (Pirnat and Hladnik 2016). Ethics approval Not applicable. Competing interests The authors declare that they have no known
competing financial interests or personal relationships that could have
appeared to influence the work reported in this paper. 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/. At present, many BIO orchards have been negatively
affected by urbanization, and an effective conservation plan
does not exist. However, many farmers continue farming
using traditional approaches. Some orchards have been
adapted to urbanization by turning them into semi-public
spaces in which people can hike and undertake various other
activities. These orchards are an example of how conserva
tion and utilization of agricultural land can co-exist in urban
areas. However, all community members should be respon
sible for the management of these urban tree resources
(Kenney et al. 2011). Declarations Ethics approval Not applicable. 1 3 References https://doi.org/10.3390/su141912938 Department of Economic and Social Affairs, United Nations (2018)
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5369 Pietilä M, Neuvonen M, Borodulin K, Korpela K, Sievänen T, Tyrväinen
L (2015) Relationships between exposure to urban green spaces,
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open space on residents’ physical activity in China. BMC Public
Health 19:1093. https://doi.org/10.1186/s12889-019-7416-7 Pirnat J, Hladnik D (2016) Connectivity as a tool in the prioritization
and protection of sub-urban forest patches in landscape conser
vation planning. Landsc Urban Plan 153:129–139. https://doi. org/10.1016/j.landurbplan.2016.05.013 Weirsum KF, Sands R (2013) Social forestry. In: Sands R (ed) Forestry
in a global context. CABI, Boston, pp 185–217 Wolch JR, Byrne J, Newell JP (2014) Urban green space, public
health, and environmental justice: the challenge of making cities’
just green enough. Landsc Urban Plan 125:234–244. https://doi. org/10.1016/j.landurbplan.2014.01.017 Richards DR, Passy P, Oh RRY (2017) Impacts of population density
and wealth on the quantity and structure of urban green space in
tropical Southeast Asia. Landsc Urban Plan 157:553–560. https://
doi.org/10.1016/j.landurbplan.2016.09.005 1 3 Urban Ecosystems (2023) 26:991–1005 1005 Zhou X, Wang Y (2011) Spatial–temporal dynamics of urban green
space in response to rapid urbanization and greening policies. Landsc Urban Plan 100:268–277. https://doi.org/10.1016/j. landurbplan.2010.12.013 Springer Nature or its licensor (e.g. a society or other partner) holds
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Burden of SARS-CoV-2 infection in healthcare workers during second wave in England and impact of vaccines: prospective multicentre cohort study (SIREN) and mathematical model
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What is already known on this topic Trial registration
ISRCTN registry ISRCTN11041050. est. Protected by copyright. Apparent and occupational risk factors for primary SARS-CoV-2 infection in
healthcare workers were established before vaccine rollout, guiding local vaccine
delivery
During rollout, coverage varied between healthcare worker groups, leading to
disparities in exposure and protection across the workforce
What this study adds
The rapid covid-19 vaccine rollout from December 2020 averted infection in a
large proportion of hospital healthcare workers in England
Demographic and occupational risk factors persisted in healthcare workers
despite vaccine rollout and should guide further infection prevention and control
measures Apparent and occupational risk factors for primary SARS-CoV-2 infection in
healthcare workers were established before vaccine rollout, guiding local vaccine
delivery RESEARCH RESEARCH BMJ: first published as 10.1136/bmj-2022-070379 on 20 July 2022. Downloa Main outcome measurei Main outcome measurei SARS-CoV-2 infection confirmed by polymerase
chain reaction. Mixed effects logistic regression
was conducted to determine demographic and
occupational risk factors for infection, and an
individual based mathematical model was used
to predict how large the burden could have been if
vaccines had not been available from 8 December
2020 . Conclusions Cite this as: BMJ 2022;378:e070379
http://dx.doi.org/10.1136/
bmj‑2022‑070379 Cite this as: BMJ 2022;378:e070379
http://dx.doi.org/10.1136/
bmj‑2022‑070379 Cite this as: BMJ 2022;378:e070379
http://dx.doi.org/10.1136/
bmj‑2022‑070379
Accepted: 16 June 2022 The rapid covid-19 vaccine rollout from December
2020 averted infection in a large proportion of
hospital healthcare workers in England: without
vaccines, second wave infections could have been
69% higher. With booster vaccinations being
needed for adequate protection from the omicron
variant, and perhaps the need for further boosters
for future variants, ensuring equitable delivery to
healthcare workers is essential. The findings also
highlight occupational risk factors that persisted
in healthcare workers despite vaccine rollout; a
greater understanding of the transmission dynamics
responsible for these is needed to help to optimise
the infection prevention and control policies that
protect healthcare workers from infection and
therefore to support staffing levels and maintain
healthcare provision. on 14 September 2022 by guest. Protected by copyright. //www.bmj.com/ on 14 September 2022 by guest. Protected by copyright. / Results During England’s second wave, 12.9% (2353/18 284)
of susceptible SIREN participants became infected
with SARS-CoV-2. Infections peaked in late December
2020 and decreased from January 2021, concurrent
with the cohort’s rapid vaccination coverage and
a national lockdown. In multivariable analysis, What is already known on this topic
Apparent and occupational risk factors for primary SARS-CoV-2 infection in
healthcare workers were established before vaccine rollout, guiding local vaccine
delivery
During rollout, coverage varied between healthcare worker groups, leading to
disparities in exposure and protection across the workforce
What this study adds
The rapid covid-19 vaccine rollout from December 2020 averted infection in a
large proportion of hospital healthcare workers in England
Demographic and occupational risk factors persisted in healthcare workers
despite vaccine rollout and should guide further infection prevention and control
measures Burden of SARS-CoV-2 infection in healthcare workers during
second wave in England and impact of vaccines: prospective
multicentre cohort study (SIREN) and mathematical model 1UK Health Security Agency,
London, UK
2The National Institute for
Health Research Health (NIHR)
Protection Research Unit in
Healthcare Associated Infections
and Antimicrobial Resistance at
the University of Oxford, Oxford,
UK
3The National Institute for
Health Research (NIHR) Health
Protection Research Unit in
Healthcare Associated Infections
and Antimicrobial Resistance
at Imperial College London,
London, UK
*Joint first authors: contributed
equally
Correspondence to: E J M Monk
edward.monk@ukhsa.gov.uk
(or @EJMMonk on Twitter :
ORCID 0000-0001-9546-9650)
Additional material is published
online only. To view please visit
the journal online. Cite this as: BMJ 2022;378:e070379
http://dx.doi.org/10.1136/
bmj‑2022‑070379
Accepted: 16 June 2022 Introduction In autumn 2020 England entered its second wave of
the SARS-CoV-2 pandemic. During this eight month
period (1 September 2020 to 30 April 2021), the
National Health Service (NHS) was under considerable
strain, with covid-19 related admissions peaking
at 25 938 during the week of 4 January 2021.1 The
most intense period of the second wave followed the
lifting of England’s second national lockdown on 2
December 2020,1 2 and it was likely amplified by the Participants Clinical, support, and administrative staff enrolled in
the SARS-CoV-2 Immunity and Reinfection Evaluation
(SIREN) study with no evidence of previous infection. Vaccination status was obtained from national
covid-19 vaccination registries and self-reported. Abstract
Objective factors increasing the likelihood of infection in
the second wave were being under 25 years old
(20.3% (132/651); adjusted odds ratio 1.35, 95%
confidence interval 1.07 to 1.69), living in a large
household (15.8% (282/1781); 1.54, 1.23 to 1.94,
for participants from households of five or more
people), having frequent exposure to patients with
covid-19 (19.2% (723/3762); 1.79, 1.56 to 2.06, for
participants with exposure every shift), working in
an emergency department or inpatient ward setting
(20.8% (386/1855); 1.76, 1.45 to 2.14), and being
a healthcare assistant (18.1% (267/1479); 1.43,
1.16 to 1.77). Time to first vaccination emerged as
being strongly associated with infection (P<0.001),
with each additional day multiplying a participant’s
adjusted odds ratio by 1.02. Mathematical model
simulations indicated that an additional 9.9% of all
patient facing hospital healthcare workers would have
been infected were it not for the rapid vaccination
coverage. 1UK Health Security Agency,
London, UK 2The National Institute for
Health Research Health (NIHR)
Protection Research Unit in
Healthcare Associated Infections
and Antimicrobial Resistance at
the University of Oxford, Oxford,
UK To describe the incidence of, risk factors for, and
impact of vaccines on primary SARS-CoV-2 infection
during the second wave of the covid-19 pandemic in
susceptible hospital healthcare workers in England. 3The National Institute for
Health Research (NIHR) Health
Protection Research Unit in
Healthcare Associated Infections
and Antimicrobial Resistance
at Imperial College London,
London, UK National Health Service secondary care health
organisations (trusts) in England between 1
September 2020 and 30 April 2021. Outcome We used primary infection, defined as the first PCR
positive result of a susceptible participant, as the
primary outcome of our statistical analyses. We
defined the date of infection as the specimen date of
the sample. Data collection and sources Participants underwent fortnightly asymptomatic PCR
testing and monthly antibody testing at their site of
enrolment, as per protocol.31 PCR and serology assay
type and threshold for positivity varied according
to local laboratory protocols. In addition, frontline
healthcare workers were able to participate in
twice weekly lateral flow device testing (with PCR
confirmation of positive lateral flow device results),
as per government guidelines and hospital policy.32
Data on demographics and exposures (workplace,
community, and household) were collected in the
enrolment questionnaire. The SIREN database comprises all SARS-CoV-2
PCR and serology results captured by the UK Health
Security
Agency
(UKHSA)
Second
Generation
Surveillance System (SGSS) since the beginning of the
pandemic, whether taken clinically or as part of SIREN,
and questionnaire results. We obtained participants’
vaccination data by questionnaire and linkage on
personal identifiable information to the National
Immunisation Management System (NIMS). In this study, we aimed to describe the incidence
of, risk factors for, and impact of vaccines on primary
SARS-CoV-2 infection during the second wave of the
pandemic in a large cohort of susceptible healthcare
workers in England, enrolled into the SARS-CoV-2
Immunity and Reinfection Evaluation (SIREN) study. Our findings are relevant to inform hospital infection
prevention and control policy for healthcare workers if
further waves of the SARS-CoV-2 pandemic occur and
to guide future winter pressure preparedness in the
NHS, particularly if SARS-CoV-2 begins to contribute
annually. the bmj | BMJ 2022;378:e070379 | doi: 10.1136/bmj-2022-070379 1 RESEARCH 2020 (delayed cohort entry). We excluded participants
from this analysis if they enrolled after the start of the
vaccine rollout (8 December 2020) or did not have
documented and linked PCR tests during the second
wave, defined here as 1 September 2020 to 30 April
2021. emergence and spread of the more transmissible alpha
variant (B.1.1.7).3 4 Fortunately, this period also saw
the delivery of the UK’s first licensed covid-19 vaccine
(Pfizer-BioNTech: 8 December 2020), with further
vaccines introduced shortly after (AstraZeneca: 4
January 2021; Moderna: 7 April 2021).5 6 A rapid
vaccine rollout across England followed, prioritising
frontline healthcare workers and achieving high
population coverage.7 8i on 14 September 2022 by guest. P
http://www.bmj.com/
BMJ: first published as 10.1136/bmj-2022-070379 on 20 July 2022. Downloaded from on 14 September 2022 by guest. Protected by copyright. http://www.bmj.com/
published as 10.1136/bmj-2022-070379 on 20 July 2022. Downloaded from We restricted our analysis to participants recruited
from English sites only to match source datasets for
the modelling component of this study (appendix 1:
mathematical modelling methods). For our risk factor
(regression) analysis, we removed participants who
could not contribute to all demographic, household,
and occupational variables (missing gender, ethnicity,
household, or postcode details). During the first wave, hospital healthcare workers
were observed to be at higher risk of exposure to
and infection with SARS-CoV-2 than the general
population.9-12 Demographic characteristics, such
as ethnicity, and occupational factors, such as
occupational setting, shift work, and frequency of
exposure to patients with covid-19, were associated
with higher risk.13-19 Understanding how these factors
contribute to infection risk within the healthcare
workforce is essential for policy planning, especially
in the context of emerging variants of concern, such
as the omicron variant (B.1.1.529), which may require
vaccine boosters for adequate protection against
primary infection and reinfection.20-27 Staff shortages
due to illness and isolation, particularly during the
peak of the second wave before the vaccine rollout,
compounded the already high clinical burden faced by
the NHS in winter 2020-21.28 29 Protecting healthcare
workers from infection is crucial to not only their
health but also healthcare provision and the safety of
patients. Study design and participants SIREN is a multicentre prospective cohort study
among NHS staff in the UK across 135 healthcare
organisations, investigating immunity to SARS-CoV-2
following infection and vaccination. In England, SIREN
centres represent secondary care health organisations
(NHS trusts) that can operate over several hospital
sites. The full study design and methods have been
described previously.30 31 Statistical analysis
We calculated the weekly incidence of primary
SARS-CoV-2 infection and the weekly cumulative
vaccination coverage (one or more doses) in SIREN
participants susceptible to primary infection both
nationally and regionally. We also estimated the
weekly vaccination coverage for demographic and
occupational subgroups. We used R version 4.1.1 for
these analyses.i guest. Protected by copyright. Our study population was SIREN participants who
entered the second wave susceptible to a primary
SARS-CoV-2 infection, defined as having no record
of SARS-CoV-2 polymerase chain reaction (PCR) or
serological positivity. We included participants in our
statistical analysis if they were susceptible to primary
infection on 1 September 2020 or on enrolment date,
if enrolled between 1 September 2020 and 7 December We stratified primary infection attack rates during
the second wave by demographic, household, and
occupational characteristics (gender, age group,
ethnicity, medical conditions, Index of Multiple
Deprivation
fifth,
household
size,
children
in
household, region, frequency of exposure to patients
with covid-19, occupational setting, and occupation). doi: 10.1136/bmj-2022-070379 | BMJ 2022;378:e070379 | the bmj RESEARCH Participants enrolled in SIREN
Withdrew and requested their data to be removed
Excluded
Laboratory data missing
Vaccine dates are unreliable (pre-2020)
Infection history at enrollment could not be determined
1518
40
4383
252
5941
Participants from devolved administrations
Scotland
Wales
Northern Ireland
555
0
23
Participants not meeting study inclusion criteria*
Previous positive on record
Enrolled aer vaccine rollout
No linked PCR during second wave
11 182
11 314
350
19 491
Participants with missing characteristic data*
Missing gender data
Missing ethnicity data
Missing Index of Multiple Deprivation data
Missing household size data
Missing children in household data
9
37
219
49
32
44 546
Participants involved in SIREN
44 294
Participants with infection history and vaccine data
38 353
Participants susceptible to primary infection during second wave
18 862
Participants meeting criteria for this study
18 284
Participants able to be included in risk factor analysis
17 973
578
311
g 1 | Flow diagram of participant inclusion and exclusion criteria used to define study cohorts for analysis. Study design and participants Crude attack rates by groups of
sceptible participants excluded from risk factor analysis (devolved administrations (n=578) and missing data (n=311)) are available in
pplementary tables A and B. PCR=polymerase chain reaction. *Participants may meet more than one exclusion criterion
on 14 September 2022 by gue
http://www.bmj.com/
BMJ: first published as 10.1136/bmj-2022-070379 on 20 July 2022. Downloaded from We calculated odds ratios and adjusted odds ratios
for primary infection: adjustment used a mixed
effects logistic regression model, reported with 95%
confidence intervals and Wald test results. In this
regression,
all
stratification
characteristics
were
Mathematical model
We assessed the impact of the vaccin
on infection rates by using a mat
with which we simulated a count
in which nobody was vaccinated. W
Participants enrolled in SIREN
Withdrew and requested their data to be removed
Excluded
Laboratory data missing
Vaccine dates are unreliable (pre-2020)
Infection history at enrollment could not be determined
1518
40
4383
252
5941
Participants from devolved administrations
Scotland
Wales
Northern Ireland
555
0
23
Participants not meeting study inclusion criteria*
Previous positive on record
Enrolled aer vaccine rollout
No linked PCR during second wave
11 182
11 314
350
19 491
Participants with missing characteristic data*
Missing gender data
Missing ethnicity data
Missing Index of Multiple Deprivation data
Missing household size data
Missing children in household data
9
37
219
49
32
44 546
Participants involved in SIREN
44 294
Participants with infection history and vaccine data
38 353
Participants susceptible to primary infection during second wave
18 862
Participants meeting criteria for this study
18 284
Participants able to be included in risk factor analysis
17 973
578
311
w diagram of participant inclusion and exclusion criteria used to define study cohorts for analysis. Crude attack rates by
ble participants excluded from risk factor analysis (devolved administrations (n=578) and missing data (n=311)) are avai
entary tables A and B. PCR=polymerase chain reaction. *Participants may meet more than one exclusion criterion on 14 September 2022 by guest. http://www.bmj.com/
BMJ: first published as 10.1136/bmj-2022-070379 on 20 July 2022. Downloaded from Fig 1 | Flow diagram of participant inclusion and exclusion criteria used to define study cohorts for analysis. Crude attack rates by groups of
susceptible participants excluded from risk factor analysis (devolved administrations (n=578) and missing data (n=311)) are available in
supplementary tables A and B. PCR=polymerase chain reaction. *Participants may meet more than one exclusion criterion Mathematical model We calculated odds ratios and adjusted odds ratios
for primary infection: adjustment used a mixed
effects logistic regression model, reported with 95%
confidence intervals and Wald test results. In this
regression, all stratification characteristics were
included as categorical variables (fixed effects) within
organisation level clusters (random effects). We
included continuous fixed effect variables to adjust
for time contributing to the analysis (owing to the
rolling recruitment to the cohort) and for time to first
vaccination. We used Stata Statistical Software (release
15.1) for these analyses. We assessed the impact of the vaccination programme
on infection rates by using a mathematical model,
with which we simulated a counterfactual scenario
in which nobody was vaccinated. We compared this
with modelled output for the scenario representing the
vaccine rollout in England. This individual based model
simulated transmission between and within patient
facing hospital healthcare workers and patients. Full
details and parameterisation of the modelling methods
are given in appendix 1: mathematical modelling
methods. the bmj | BMJ 2022;378:e070379 | doi: 10.1136/bmj-2022-070379 3 the bmj | BMJ 2022;378:e070379 | doi: 10.1136/bmj-2022-070379 RESEARCH Characteristic
Susceptible to primary infection during
second wave (n=18 284)
Gender:
Female
15 528 (84.9)
Male
2730 (14.9)
Non-binary
17 (0.1)
Prefer not to say
9 (0.0)
Age group:
<25
675 (3.7)
25-34
3566 (19.5)
35-44
4544 (24.9)
45-54
5555 (30.4)
55-64
3627 (19.8)
≥65
317 (1.7)
Ethnicity:
Asian
1207 (6.6)
Black
297 (1.6)
White
16 238 (88.8)
Mixed race
296 (1.6)
Other ethnic group
209 (1.1)
Prefer not to say
37 (0.2)
Medical conditions:
No medical conditions
13 595 (74.4)
Immunosuppression
402 (2.2)
Chronic respiratory disease
2365 (12.9)
Chronic non-respiratory disease
1922 (10.5)
Index of Multiple Deprivation fifth*:
1 (most deprived)
1936 (10.6)
2
3243 (17.7)
3
4216 (23.1)
4
4341 (23.7)
5 (least deprived)
4329 (23.7)
Unknown
219 (1.2)
Household size:
1
1828 (10.0)
2
6068 (33.2)
3 or 4
8535 (46.7)
≥5
1804 (9.9)
Prefer not to say
49 (0.3)
Children in household:
No children
10 780 (59.0)
Children
7472 (40.9)
Prefer not to say
32 (0.2)
Region:
East Midlands
1803 (9.9)
East of England
2263 (12.4)
London
2065 (11.3)
North East
373 (2.0)
North West
1976 (10.8)
South East
2398 (13.1)
South West
4182 (22.9)
West Midlands
1791 (9.8)
Yorkshire and the Humber
1433 (7.8)
Frequency of close proximity to patients with
covid-19:
Every shift
3833 (21.0)
Once a week
3050 (16.7)
Once a month
1651 (9.0)
Less than once a month
2646 (14.5)
Never
7104 (38.9)
Occupational setting:
Office
4002 (21.9)
Patient facing (non-clinical)
687 (3.8)
Outpatient
3273 (17.9)
Maternity/labour ward
193 (1.1)
Table 1 | Demographic, household, and occupational characteristics of SIREN
participants susceptible to primary infection in second wave of SARS-CoV-2 in England. Values are numbers (percentages)
(Continued) In summary, the model had a susceptible-exposed-
infected-recovered
(SEIR)
structure:
simulated
individuals
represented
patient
facing
hospital
healthcare workers and patients, all of whom
were all fully susceptible at the start of pandemic,
and who progressed on transmission through an
incubation period before an infectious stage (including
asymptomatic cases) and recovery. A schematic of the
model is presented in appendix 2: supplementary
figure C. The individual based model simulated
transmission to patient facing hospital healthcare
workers via three routes: from the public (community),
from patients in hospital, and from other patient facing
hospital healthcare workers. Participant and public involvement As part of the SIREN study, we have engaged with
our participants throughout via regular newsletters
and webinars. More recently, we have established a
participant and public involvement panel that will
meet every six weeks to ensure that the research we
generate remains relevant. The findings of this work
will be discussed at the next webinar and participant
and public involvement working groups. the bmj | BMJ 2022;378:e070379 | doi: 10.1136/bmj-2022-070379 Nosocomial transmission
to patients could occur via three routes: direct
transmission to susceptible patients from infected
patients, direct transmission from infected patient
facing hospital healthcare workers (for example,
by droplet or aerosol), and indirect transmission
from infected patients (for example, via fomites). We included change in vaccination status by using a
logistic growth curve fitted to data from SIREN-NIMS
linked data (for patient facing hospital healthcare
workers) and NIMS data (for the public). Vaccine
efficacy was included from 21 days after the first
dose, reducing transmission (by 50%) and preventing
infection (at 70% efficacy, rising to 80% after two
doses).33-35 The virulence of each variant circulating
throughout the second wave and the probability of an
individual having each variant were included by using
previously published NHS regional data.36 on 14 September 2022 by guest. Protected by copyright. http://www.bmj.com/
BMJ: first published as 10.1136/bmj-2022-070379 on 20 July 2022. Downloaded from on 14 September 2022 by guest. Prote
http://www.bmj.com/
July 2022. Downloaded from The individual based model was calibrated using
cumulative infection data from the SIREN study
between 1 March 2020 and 8 December 2020, and from
NHS Situational Reports (appendix 2: supplementary
figure D).37 on 14 September 2022 by guest. Protected by copyright. ://www.bmj.com/ (Continued) Results guest. Protected by copyright. We included 18 284 susceptible participants from
England, recruited from 105 secondary care health
organisations (fig 1). Table 1 shows their demographic,
household, and occupational characteristics. Between
the beginning of September 2020 and the end of
April 2021, 2353 new primary infections occurred: a
crude attack rate of 12.9%. Figure 2 shows the weekly
incidence of primary infection in this cohort. Incidence
peaked during the week of 29 December 2020 and then
rapidly decreased, coinciding with vaccine rollout
and England’s third national lockdown beginning
on 6 January 2021 (fig 2). Vaccination coverage (first
dose) was 26.9% (4914/18 284) on 31 December (Continued) doi: 10.1136/bmj-2022-070379 | BMJ 2022;378:e070379 | the bmj RESEARCH on 14 September 2022 by guest. Protected by copyright. http://www.bmj.com/
BMJ: first published as 10.1136/bmj-2022-070379 on 20 July 2022. Downloaded from Characteristic
Susceptible to primary infection during
second wave (n=18 284)
Emergency department†/inpatient ward
1906 (10.4)
Intensive care
1029 (5.6)
Theatres
324 (1.8)
Other
6870 (37.6)
Occupation:
Nurse
6011 (32.9)
Healthcare assistant
1497 (8.2)
Doctor
1890 (10.3)
Midwife
465 (2.5)
Bedside therapist‡
572 (3.1)
Administrator/executive (office based)
2953 (16.2)
Estates/porters/security
170 (0.9)
Pharmacist
280 (1.5)
Healthcare scientist
702 (3.8)
Student§
909 (5.0)
Other
2835 (15.5)
*Residential relative deprivation score according to postcode and English Indices of Deprivation 2019. †Including ambulance setting
‡Physiotherapist, occupational therapist, speech and language therapist. §Medical student, nursing student, midwifery student, student: other. Table 1 | Continued the community (appendix 2: supplementary figure
A).38 Rate of initial growth in vaccination coverage
(26.8% (4825/17 973) by 31 December 2020) varied
by occupational factors and ethnicity (fig 4). It was
fastest among doctors (50.4% (923/1832); P<0.001
for proportion comparison) and staff working in
intensive care settings (41.2% (417/1012); P<0.001)
or in theatres (38.9% (124/319); P<0.001). Growth
in vaccination coverage was slowest in administrators
(15.9% (464/2917); P<0.001), office based staff
(18.6% (736/3953); P<0.001), and participants of
black ethnicity (19.4% (55/284); P=0.006). Table 1 | Continued Risk factors for infection in SIREN participants Risk factors for infection in SIREN participants
Our regression analyses included 17 973 participants
who met our criteria (fig 1). Table 2 shows analyses
of potential demographic and household risk factors
for primary infection, and table 3 shows occupational
risk factors. Table 3 also shows the temporal variables
included in the regression model. Within demographic and household characteristics,
we observed the highest infection rates in younger
participants, participants of Asian or black ethnicity,
and participants living in larger households (table
2). Within occupational characteristics, the highest
infection rates were in participants working in
settings where they were often exposed to patients 2021, 88.3% (16 143/18 284) on 31 January 2021,
and 96.1% (17 576/18 284) on 30 April 2021. The
peak incidence was highest in the East of England,
London, and South East regions (fig 3), which all had
the earliest rise in circulation of the alpha variant in No of susceptible participants
Proportion of susceptible participants
0
Sep
Oct
Nov
Dec
Jan
Feb
Mar
Apr
May
Proportion vaccinated
(1st dose)
No of primary infections
No tested (x0.01)
120
180
300
240
60
0
0.4
0.6
1.0
0.8
0.2
2020
2021 Week commencing Tuesday
No of susceptible participants
Proportion of susceptible participants
0
Sep
Oct
Nov
Dec
Jan
Feb
Mar
Apr
May
Proportion vaccinated
(1st dose)
No of primary infections
Vaccination
Pfizer-BioNTech
LFD testing
Social
distancing
legislation
No tested (x0.01)
Moderna
AstraZeneca
120
180
300
240
60
0
0.4
0.6
1.0
0.8
0.2
National
Lockdown
Local
Step 2
Patient facing HCW
Tiers 1-3
Step 1
Tiers 1-4
Lockdown
National
Regions
Regions
2020
2021
Fig 2 | Weekly incidence of SARS-CoV-2 primary infections, and weekly cumulative vaccination coverage, in SIREN participants susceptible to
primary infection in England, with calendar of England-wide covid-19 interventions during second wave (1 September 2020 to 30 April 2021). HCW=healthcare workers; LFD=lateral flow device
on 14 September 2022 by guest. Protected by copyright. http://www.bmj.com/
from No of primary infections
No tested (x0.01) Week commencing Tuesday Fig 2 | Weekly incidence of SARS-CoV-2 primary infections, and weekly cumulative vaccination coverage, in SIREN participants susceptible to
primary infection in England, with calendar of England-wide covid-19 interventions during second wave (1 September 2020 to 30 April 2021). Risk factors for infection in SIREN participants HCW=healthcare workers; LFD=lateral flow device the bmj | BMJ 2022;378:e070379 | doi: 10.1136/bmj-2022-070379 5 RESEARCH Week commencing Tuesday
South West
West Midlands
Yorkshire and the Humber
Proportion of tested
susceptible participants
Sep Oct Nov Dec
Jan
Feb Mar Apr May
Week commencing Tuesday
Sep Oct Nov Dec
Jan
Feb Mar Apr May
Week commencing Tuesday
Sep Oct Nov Dec
Jan
Feb Mar Apr
North East
North West
South East
Proportion of tested
susceptible participants
East Midlands
East of England
London
Proportion of tested
susceptible participants
0
0.04
0.06
0.10
0.08
0.02
0
0.04
0.06
0.10
0.08
0.02
0
0.04
0.06
0.10
0.08
0.02
May
Date
2020
2021
2020
2021
2020
2021
Fig 3 | Positive polymerase chain reaction results as proportion of SIREN participants susceptible to primary infection in England tested by week,
stratified by region (1 September 2020 to 30 April 2021)
on 14 S
http://www.bmj.com/
BMJ: first published as 10.1136/bmj-2022-070379 on 20 July 2022. Downloaded from East Midlands
Proportion of tested
susceptible participants
0
0.04
0.06
0.10
0.08
0.02 East of England on 14 September
http://www.bmj.com/
BMJ: first published as 10.1136/bmj-2022-070379 on 20 July 2022. Downloaded from London on 14 September 2022 by guest. Protected by copyright. http://www.bmj.com/
BMJ: first published as 10.1136/bmj-2022-070379 on 20 July 2022. Downloaded from on 14 September 2022 by guest. Protected by copyright. http://www.bmj.com/
irst published as 10.1136/bmj-2022-070379 on 20 July 2022. Downloaded from North West South East South East West Midlands Yorkshire and the Humber Proportion of tested
i
i i Week commencing Tuesday Week commencing Tuesday Fig 3 | Positive polymerase chain reaction results as proportion of SIREN participants susceptible to primary infection in England tested by week,
stratified by region (1 September 2020 to 30 April 2021) with covid-19, participants who worked in emergency
department or inpatient ward settings, and participants
working as healthcare assistants, bedside therapists,
porters, and security and estates staff (table 3). We observed regional differences, which remained
after adjustment, with the greatest adjusted odds ratios
being for participants working either in the highest
peak regions or in the Midlands and North West (where
incidence in the autumn was above average). The
factors included in the regression model accounted
for 54% of the variation seen between organisation
level clusters: the median second wave attack rate
by organisation was 12.5% (interquartile range 8.9-
15.9%; n=92 organisations with study sample >50). Risk factors for infection in SIREN participants Before adjustment, univariate analyses suggested
that many demographic, household, and occupational
characteristics had a significant association with
infection, in addition to those with the highest crude
infection rates (table 2; table 3). However, after
adjustment, which included time to first vaccination
as a strongly associated predictor (P<0.001, with each
additional day multiplying a participant’s adjusted
odds ratio by 1.02), many of these associations were
weakened. Characteristics that remained a strong risk
(at P≤0.01) after adjustment were being under 25 years
old, living in a household of three or more people,
having exposure to patients with covid-19 at least
once monthly, working in an emergency department or
inpatient ward setting, and being a healthcare assistant. y guest. Protected by copyright. Estimated impact of vaccines on patient facing
hospital healthcare workers Simulated infection rates in patient facing hospital
healthcare workers under a “vaccine rollout” scenario,
in which vaccination proceeded as observed via NIMS,
began to diverge from a counterfactual “no vaccines
available” scenario after 15 January 2021 (fig 5). Allowing for the latent period of SARS-CoV-2 infection, doi: 10.1136/bmj-2022-070379 | BMJ 2022;378:e070379 | the bmj RESEARCH on 14 September 2022 by guest. Protected by copyright. http://www.bmj.com/
BMJ: first published as 10.1136/bmj-2022-070379 on 20 July 2022. Downloaded from the counterfactual no vaccines available scenario
simulations compared with 42.5% (37.5-46.4%) in
the vaccine rollout scenario; this equates to a 19% cut
in cumulative infections from the start of the pandemic
due to vaccine rollout. As 28.2% (25.4-33.7%) of
simulated patient facing hospital healthcare workers
had been infected before the start of the second wave,
the model results indicate that infections in patient
facing hospital healthcare workers during the second
wave would have been 69% (24.1/14.3) higher had
vaccines not been available. these cases would not be detectable by PCR until
14 days later. In the model, 52.3% (interquartile
46 6 55 9%)
f
i
f
i
h
i
l h
l
Nurse
Healthcare assistant
Doctor
Midwife
Bedside therapist
Administrator/executive
Estates/porters/security
Pharmacist
Asian
Black
White
Mixed race
Other ethnic group
Office
Patient facing (non-clinical)
Outpatient
Maternity/labour ward
Emergency department/
inpatient wards
Intensive care
Theatre
Other
Healthcare scientist
Student
Other
C
Proportion vaccinated
(1st dose)
0
May
Week commencing Tuesday
Dec
Jan
Feb
Mar
Apr
0.4
0.6
1.0
0.8
0.2
B
Proportion vaccinated
(1st dose)
0
0.4
0.6
1.0
0.8
0.2
A
Proportion vaccinated
(1st dose)
0
0.4
0.6
1.0
0.8
0.2
2020
2021
Fig 4 | Weekly cumulative vaccination coverage of SIREN participants susceptible
to primary infection in England, stratified by demographic and occupational
characteristics. Proportion of susceptible SIREN participants with complete
characteristic data (n=17 973) and one or more dose of covid-19 vaccine, by date
dose, stratified by ethnicity (A), occupational setting (B), and occupation (C) Proportion vaccinated
(1st dose) After 8 December 2020, the total attack rate in
susceptible patient facing hospital healthcare workers
in the counterfactual scenario simulations was twice
as high as in the vaccine rollout scenario (fig 5). Estimated impact of vaccines on patient facing
hospital healthcare workers In
these simulations, the greatest absolute effect of
vaccines was seen in East of England, where 16.7%
fewer patient facing hospital healthcare workers
were infected between 8 December 2020 and 30 April
2021 (33.4% (interquartile range 29.9-38.9%) versus
17.1% (14.2-20.4%)), and the smallest absolute effect
was seen in South West (9.5% difference: 17.8%
(16.3-19.8%) versus 8.3% (7.0-9.1%)) (appendix 2:
supplementary figures E and F). This reflects the timing
of the alpha variant’s circulation in those regions
(appendix 2: supplementary figure C). B
0 B
0 Proportion vaccinated
(1st dose) Discussion During the eight months of England’s second wave of
SARS-CoV-2, 12.9% of SIREN participants susceptible
to primary infection became infected. From our
mathematical model, we estimate that an additional
9.9% of all patient facing hospital healthcare workers
would have been infected during this period were it not
for the rapid vaccination coverage, prioritising this group. p
g ,p
g
g
p
After peaking in late December 2020, new primary
infections decreased sharply, concurrent with the
rapid vaccination coverage in the cohort and a
national lockdown. We found the strongest risk
factor for infection in the second wave to be time to
first vaccination: disparities in vaccination coverage
within our cohort are likely to account for the strong
univariate association of infection risk with black
ethnicity disappearing after adjustment. In our
multivariable analysis, risk of infection remained
significantly higher for occupational groups with
frequent exposure to patients with covid-19 and those
working in an emergency department or inpatient
ward setting or working as a healthcare assistant. These findings underscore the importance of research
into the nature of healthcare workers’ role and setting
specific contact with patients and staff, even in the
context of vaccination. Combined, our analyses
highlight the crucial role of rapid covid-19 vaccine
deployment in averting infections in our cohort during
the second wave and the importance of equitable
rollout throughout the healthcare workforce. C
0 y guest. Protected by copyright. Week commencing Tuesday Week commencing Tuesday Fig 4 | Weekly cumulative vaccination coverage of SIREN participants susceptible
to primary infection in England, stratified by demographic and occupational
characteristics. Proportion of susceptible SIREN participants with complete
characteristic data (n=17 973) and one or more dose of covid-19 vaccine, by date
dose, stratified by ethnicity (A), occupational setting (B), and occupation (C) these cases would not be detectable by PCR until two to
14 days later. In the model, 52.3% (interquartile range
46.6-55.9%) of patient facing hospital healthcare
workers were infected by the end of April 2021 in Strengths and weaknesses of study SIREN is a large prospective cohort study that is well
positioned to explore the incidence of SARS-CoV-2 on 14 September 2022 by guest. Protected by copyright.
http://www.bmj.com/
BMJ: first published as 10.1136/bmj-2022-070379 on 20 July 2022. Downloaded from Table 2 | Association of demographic and household characteristics of susceptible SIREN participants with primary infection during second wave of
SARS-CoV-2 pandemic in England (1 September 2020 to 30 April 2021)
Characteristic
Infected
(n=2318)
Total*
(n=17 973)
Infected (%)
Odds ratio (95% CI)
P value
Adjusted odds ratio†
(95% CI)
P value
Gender†:
Female
1950
15 290
12.8
Reference
-
Reference
-
Male
365
2665
13.7
1.08 (0.96 to 1.22)
0.21
1.16 (1.01 to 1.33)
0.03
Non-binary‡
-
≤17
-
1.05 (0.24 to 4.66)
0.95
1.00 (0.20 to 4.90)
01.00
Age group:
<25
132
651
20.3
1.86 (1.51 to 2.29)
<0.001
1.35 (1.07 to 1.69)
0.01
25-34
540
3455
15.6
1.35 (1.20 to 1.53)
<0.001
0.94 (0.82 to 1.08)
0.42
35-44
555
4486
12.4
1.03 (0.92 to 1.16)
0.60
0.90 (0.79 to 1.02)
0.10
45-54
659
5479
12.0
Reference
-
Reference
-
55-64
406
3591
11.3
0.93 (0.82 to 1.06)
0.30
1.00 (0.87 to 1.15)
0.98
≥65
26
311
8.4
0.67 (0.44 to 1.01)
0.05
0.73 (0.48 to 1.12)
0.15
Ethnicity:
Asian
211
1163
18.1
1.57 (1.34 to 1.84)
<0.001
1.23 (1.03 to 1.47)
0.02
Black
65
284
22.9
2.10 (1.59 to 2.79)
<0.001
1.18 (0.86 to 1.62)
0.30
White
1983
16 034
12.4
Reference
-
Reference
-
Mixed race
33
288
11.5
0.92 (0.64 to 1.32)
0.64
0.72 (0.49 to 1.06)
0.10
Other ethnic group
26
204
12.7
1.03 (0.68 to 1.57)
0.87
0.73 (0.47 to 1.13)
0.16
Medical conditions:
No medical conditions
1736
13 355
13.0
Reference
-
Reference
-
Immunosuppression
38
396
9.6
0.71 (0.51 to 1.00)
0.05
0.75 (0.52 to 1.06)
0.10
Chronic respiratory disease
290
2332
12.4
0.95 (0.83 to 1.09)
0.45
1.01 (0.88 to 1.16)
0.90
Chronic non-respiratory disease
254
1890
13.4
1.04 (0.90 to 1.20)
0.60
1.11 (0.96 to 1.29)
0.16
Index of Multiple Deprivation fifth:
1 (most deprived)
302
1922
15.7
1.36 (1.17 to 1.59)
<0.001
1.05 (0.88 to 1.25)
0.58
2
430
3220
13.4
1.13 (0.98 to 1.29)
0.09
1.04 (0.89 to 1.20)
0.64
3
510
4200
12.1
1.01 (0.89 to 1.15)
0.89
0.99 (0.86 to 1.14)
0.90
4
557
4323
12.9
1.08 (0.95 to 1.23)
0.24
1.05 (0.92 to 1.20)
0.48
5 (least deprived)
519
4308
12.0
Reference
-
Reference
-
Household size:
1
189
1801
10.5
Reference
-
Reference
-
2
755
5971
12.6
1.23 (1.04 to 1.46)
0.01
1.21 (1.01 to 1.44)
0.04
3 or 4
1092
8420
13.0
1.27 (1.08 to 1.50)
0.004
1.32 (1.09 to 1.59)
0.004
≥5
282
1781
15.8
1.60 (1.32 to 1.96)
<0.001
1.54 (1.23 to 1.94)
<0.001
Children in household:
Children
1357
10 592
12.8
Reference
-
Reference
-
No children
961
7381
13.0
1.02 (0.93 to 1.11)
0.68
0.92 (0.81 to 1.05)
0.23
Region:
East Midlands
265
1775
14.9
1.87 (1.58 to 2.22)
<0.001
1.66 (1.18 to 2.32)
0.003
East of England
375
2233
16.8
2.15 (1.84 to 2.52)
<0.001
1.92 (1.41 to 2.60)
<0.001
London
316
1990
15.9
2.01 (1.71 to 2.37)
<0.001
1.71 (1.27 to 2.31)
<0.001
North East
41
366
11.2
1.34 (0.95 to 1.89)
0.090
1.48 (0.81 to 2.69)
0.20
North West
309
1956
15.8
2.00 (1.70 to 2.36)
<0.001
1.91 (1.42 to 2.57)
<0.001
South East
267
2364
11.3
1.36 (1.15 to 1.60)
<0.001
1.49 (1.12 to 1.98)
0.006
South West
352
4102
8.6
Reference
-
Reference
-
West Midlands
256
1776
14.4
1.79 (1.51 to 2.13)
<0.001
1.68 (1.23 to 2.29)
0.001
Yorkshire and the Humber
137
1411
9.7
1.15 (0.93 to 1.41)
0.20
1.15 (0.81 to 1.62)
0.43
*311 participants excluded from risk factor analyses owing to missing characteristics data. on 14 September 2022 by guest. Protected by copyright.
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BMJ: first published as 10.1136/bmj-2022-070379 on 20 July 2022. Downloaded from †Adjusted for above characteristics and those in table 3, with secondary care health organisation as random effect. ‡Owing to low absolute number of infections in participants identifying as non-binary, counts within gender categories have either been suppressed or rounded to nearest 5 to mitigate disclosure Table 2 | Association of demographic and household characteristics of susceptible SIREN participants with prim
SARS-CoV-2 pandemic in England (1 September 2020 to 30 April 2021) the bmj | BMJ 2022;378:e070379 | doi: 10.1136/bmj-2022-070379 the bmj | BMJ 2022;378:e070379 | doi: 10.1136/bmj-2022-070379 7 RESEARCH on 14 September 2022 by guest. Pro
http://www.bmj.com/
9 on 20 July 2022. Downloaded from on 14 September 2022 by guest. Protected by copyright. //www.bmj.com/ y guest. Protected by copyright. infection in the hospital workforce. We have well
defined previous exposure history from an enrolment
questionnaire (with negligible impact from missing
data), a frequent PCR testing schedule, and laboratory
records since the pandemic began through SGSS,
regardless of participants’ enrolment date. This study
gains considerable strength from our multifaceted
approach, using both statistical and mathematical
modelling built on data from SIREN and additional
national datasets. and control policies during the second wave
and organisational infrastructure. Disparities in
infrastructure and pandemic response at the level
of the organisation likely contributed to the residual
variance seen between organisations that was not
accounted for by our regression model. on 14 September 2022 by guest. Pro
http://www.bmj.com/
9 on 20 July 2022. Downloaded from This study is
also unable to unpick the behavioural nature of the
demographic and occupational risk factors observed,
which might contribute to the higher adjusted odds
ratio of infection, but not unadjusted odds ratio,
in male compared with female healthcare workers
and the higher adjusted odds ratio in healthcare
assistants, but not nurses, compared with office The main limitation of this study is the lack of
detail at organisational level, which is needed to
explore the impact of particular infection prevention doi: 10.1136/bmj-2022-070379 | BMJ 2022;378:e070379 | the bmj RESEARCH Table 3 | Association of occupational and temporal characteristics of susceptible SIREN participants with primary infection during second wave of
SARS-CoV-2 pandemic in England (1 September 2020 to 30 April 2021)
Characteristic
Infected
(n=2318)
Total*
(n=17 973)
Infected (%)
Odds ratio (95% CI)
P value
Adjusted odds ratio†
(95% CI)
P value
Frequency of close proximity to patients with
covid-19:
Every shift
723
3762
19.2
2.36 (2.10 to 2.65)
<0.001
1.79 (1.56 to 2.06)
<0.001
Once a week
436
2981
14.6
1.70 (1.49 to 1.93)
<0.001
1.45 (1.25 to 1.68)
<0.001
Once a month
219
1620
13.5
1.55 (1.31 to 1.82)
<0.001
1.39 (1.16 to 1.66)
<0.001
Less than once a month
298
2609
11.4
1.28 (1.10 to 1.48)
0.001
1.19 (1.02 to 1.40)
0.03
Never
642
7001
9.2
Reference
-
Reference
-
Occupational setting:
Office
393
3953
9.9
Reference
-
Reference
-
Patient facing (non-clinical)
93
676
13.8
1.45 (1.13 to 1.84)
0.003
1.24 (0.94 to 1.63)
0.12
Outpatient
406
3223
12.6
1.31 (1.13 to 1.51)
<0.001
1.02 (0.86 to 1.22)
0.80
Maternity/labour ward
18
189
9.5
0.95 (0.58 to 1.57)
0.85
0.87 (0.51 to 1.49)
0.61
Emergency department‡/inpatient wards
386
1855
20.8
2.38 (2.04 to 2.78)
<0.001
1.76 (1.45 to 2.14)
<0.001
Intensive care
135
1012
13.3
1.39 (1.13 to 1.72)
0.002
0.90 (0.71 to 1.14)
0.39
Theatres
31
319
9.7
0.98 (0.66 to 1.43)
0.90
0.80 (0.52 to 1.22)
0.30
Other
856
6746
12.7
1.32 (1.16 to 1.49)
<0.001
1.06 (0.91 to 1.23)
0.47
Occupation:
Nurse
821
5918
13.9
1.43 (1.24 to 1.64)
<0.001
1.12 (0.94 to 1.33)
0.20
Healthcare assistant
267
1479
18.1
1.95 (1.63 to 2.34)
<0.001
1.43 (1.16 to 1.77)
0.001
Doctor
222
1832
12.1
1.22 (1.01 to 1.47)
0.03
0.99 (0.78 to 1.24)
0.91
Midwife
46
458
10.0
0.99 (0.71 to 1.37)
0.95
0.76 (0.53 to 1.10)
0.15
Bedside therapist§
96
556
17.3
1.85 (1.44 to 2.38)
<0.001
1.32 (0.99 to 1.76)
0.06
Administrator/executive (office based)
296
2917
10.1
Reference
-
Reference
-
Estates/porters/security
31
170
18.2
1.97 (1.31 to 2.97)
<0.001
1.34 (0.86 to 2.10)
0.20
Pharmacist
34
270
12.6
1.28 (0.87 to 1.86)
0.21
0.89 (0.58 to 1.37)
0.61
Healthcare scientist
55
689
8.0
0.77 (0.57 to 1.04)
0.09
0.73 (0.53 to 1.01)
0.06
Student¶
110
894
12.3
1.24 (0.98 to 1.57)
0.07
1.00 (0.78 to 1.29)
0.10
Other
340
2790
12.2
1.23 (1.04 to 1.45)
0.01
1.02 (0.84 to 1.23)
0.87
Temporal characteristics:
Time to vaccination (days after 8 December)
-
-
-
-
-
1.02 (1.01 to 1.02)
<0.001
Time in cohort (days)
-
-
-
-
-
1.00 (0.99 to 1.00)
<0.001
*311 participants excluded from risk factor analyses owing to missing characteristics data. Characteristic on 14 September 2022 by guest. Protec
http://www.bmj.com/
20 July 2022. Downloaded from workers. Understanding the behavioural elements
contributing to these findings is important in order to
identify people at risk and target appropriate infection
prevention and control measures accordingly. Clearly,
frequent close proximity to patients with covid-19 is
a strong risk factor, and the nature of an individual
healthcare worker’s contact with patients and working
practices is likely to modify this.39 professionals between 1 September 2020 and 7
January 2021. Another study, REACT-1, reported
infection in 2.1% (1.8% to 2.5%) and 0.7% (0.5%
to 0.9%) of healthcare workers with direct patient
contact between 6-22 January and 4-23 February,
respectively.41-43 These findings should be compared
with ours with caution: these studies consider different
periods of the second wave, have less temporally dense
testing protocols, have wider occupational definitions
(for example, including primary care), and do not
consider vaccination or susceptibility status. Our data
are more relevant to hospital healthcare settings and
better suited to guide hospital level policy. A limitation of the mathematical modelling work
included in this study is that we used empirical
data to inform the community prevalence in non-
healthcare workers: it does not incorporate a probable
counterfactual increase in community cases in the
absence of vaccines.40 Hence, in the simulation results,
the contribution of community acquired infection
in patient facing hospital healthcare workers to the
overall infection risk in the “no vaccine” scenario is
underestimated, and the simulated 9.9% reduction is
a conservative figure. guest. Protected by copyright. Although studies before the vaccine rollout have
previously highlighted demographic and occupational
risk factors for SARS-CoV-2 infection,13 15 44 our study
adds significantly to the literature by considering a
period during rapid vaccination coverage, allowing the
identification of risk factors that remain in the context
of vaccination. Our chosen period of observation
is likely the last opportunity in this pandemic to
study a truly susceptible cohort and identify these
demographic
and
occupational
characteristics,
as infection and vaccination patterns since the
emergence of the omicron variant in November 2021 on 14 September 2022 by guest. Pro
http://www.bmj.com/
9 on 20 July 2022. Downloaded from †Adjusted for above characteristics and those in table 2, with secondary care health organisation as random effect. ‡Including ambulance setting. §Physiotherapist, occupational therapist, speech and language therapist. ¶Medical student, nursing student, midwifery student, student: other. p
p
j
y Table 3 | Association of occupational and temporal characteristics of susceptible SIREN participants with primar
SARS-CoV-2 pandemic in England (1 September 2020 to 30 April 2021) Strengths and weaknesses in relation to other
studiesfi Downloaded from B
Date
Cumulative proportion of
pfHCW newly infected
0
0.2
0.3
0.5
0.4
0.6
0.1
Mar Apr May Jun Jul Aug
2020
Sep
Before
wave
28.2%
At 7 Dec
2020
32.7%
At wave end
No vaccine: 52.3%
Vaccine rollout: 42.5%
Oct Nov Dec Jan Feb Mar Apr May
2021 B
6 Proportion of pfHCW newly infected Proportion of pfHCW newly infected Date
C
Cumulative proportion of pfHCW
infected aer 8 Dec 2020
0
0.10
0.15
0.20
0.05
Dec
2010
Jan
2021
Feb
2021
Mar
2021
Apr
2021
May
2021
Date D
Attack rate in susceptible
pfHCW aer 8 Dec 2020
0
0.2
0.3
0.4
0.1
Vaccine
rollout
No
vaccine
Date D
4 Fig 5 | Effect of vaccines on rates of SARS-CoV-2 infection in patient facing hospital healthcare workers (pfHCW) from simulation output. Individual
based model simulation output for “vaccine rollout” and “no vaccines” scenarios. A) Proportion of pfHCW infected per day until 30 April 2021. B)
Daily cumulative proportion of pfHCW infected until 30 April 2021. C) Daily cumulative proportion of pfHCW infected after 8 December 2020 (start of
vaccine rollout). D) Proportion of susceptible pfHCW at 8 December 2020 who were subsequently infected by end of April 2021 have led to very few people in the UK remaining fully
susceptible. Future studies might struggle to recruit
enough susceptible participants to have the statistical
power needed to explore occupational risk factors for
SARS-CoV-2, which are likely to contribute to future
winter pressures. variants of concern, such as the omicron variant, that
may necessitate boosters for suitable protection.26 The
occupational
group
with
the
strongest
association with infection after adjustment was
healthcare assistants (P<0.001), followed by bedside
therapists (physiotherapists, occupational therapists,
and speech and language therapists) (P=0.06),
bringing into question whether the nature of their
interaction with patients carries additional risk. The
maintained risk in certain healthcare settings and roles
after adjustment, likely due to residual confounders
not included in our model, highlight the importance
of understanding the nature of staff-patient contact
and staff-staff mixing in different healthcare worker
groups and settings, including households,45 and
consideration of whether factors such as optimisation
of personal protective equipment could reduce
infection rates in these at-risk groups.46 Meaning of study: possible explanations and
implications for clinicians and policy makersf This study shows a clear effect of vaccines on
the incidence of SARS-CoV-2 infection in SIREN
participants: without the vaccine rollout, which
prioritised frontline healthcare workers, staff absence
due to covid-19 could have been 69% higher during
the second wave, threatening healthcare provision. Patients’ safety could have been affected both directly,
through an increase in downstream nosocomial
infections, and indirectly, through staff shortages,
if the vaccine rollout had not occurred when it did. The differential impact of vaccines on demographic
and occupational groups can, in part, be explained
by variations in speed of coverage; equitable
opportunities for healthcare staff to be vaccinated
must be prioritised, with monitoring of uptake and
targeted encouragement in groups with lower uptake. This is particularly important during the emergence of by guest. Protected by copyright. Strengths and weaknesses in relation to other
studiesfi The Office for National Statistics’ population SARS-
CoV-2 prevalence study reported infection in 4.2%
(95% confidence interval 3.7% to 4.6%) of healthcare 9 the bmj | BMJ 2022;378:e070379 | doi: 10.1136/bmj-2022-070379 RESEARCH Before 8 Dec 2020
No vaccine
Scenario
Vaccine rollout
A
Date
C
Cumulative proportion of pfHCW
infected aer 8 Dec 2020
0
0.10
0.15
0.20
0.05
Dec
2010
Jan
2021
Feb
2021
Mar
2021
Apr
2021
May
2021
D
Attack rate in susceptible
pfHCW aer 8 Dec 2020
0
0.2
0.3
0.4
0.1
Vaccine
rollout
No
vaccine
Date
Proportion of pfHCW newly infected
0
0.002
0.003
0.005
0.004
0.006
0.001
Mar Apr May Jun Jul Aug
2020
Sep Oct Nov Dec Jan Feb Mar Apr May
2021
B
Date
Cumulative proportion of
pfHCW newly infected
0
0.2
0.3
0.5
0.4
0.6
0.1
Mar Apr May Jun Jul Aug
2020
Sep
Before
wave
28.2%
At 7 Dec
2020
32.7%
At wave end
No vaccine: 52.3%
Vaccine rollout: 42.5%
Oct Nov Dec Jan Feb Mar Apr May
2021
Fig 5 | Effect of vaccines on rates of SARS-CoV-2 infection in patient facing hospital healthcare workers (pfHCW) from simulation output. Individual
based model simulation output for “vaccine rollout” and “no vaccines” scenarios. A) Proportion of pfHCW infected per day until 30 April 2021. B)
Daily cumulative proportion of pfHCW infected until 30 April 2021. C) Daily cumulative proportion of pfHCW infected after 8 December 2020 (start of
vaccine rollout). D) Proportion of susceptible pfHCW at 8 December 2020 who were subsequently infected by end of April 2021
p
p
j
y BMJ: first published as 10.1136/bmj-2022-070379 on 20 July 2022. Down Before 8 Dec 2020
No vaccine
Scenario
Vaccine rollout
A
Date
Proportion of pfHCW newly infected
0
0.002
0.003
0.005
0.004
0.006
0.001
Mar Apr May Jun Jul Aug
2020
Sep Oct Nov Dec Jan Feb Mar Apr May
2021 on 14 September 2022 by gues
http://www.bmj.com/
BMJ: first published as 10.1136/bmj-2022-070379 on 20 July 2022. Downloaded from on 14 September 2022 by guest. Protected by copyright. http://www.bmj.com/
BMJ: first published as 10.1136/bmj-2022-070379 on 20 July 2022. Provenance and peer review: Not commissioned; externally peer
reviewed. Joint Committee
on Vaccination and Immunisation: advice on priority groups for
COVID-19 vaccination, 30 December 2020. 2021. https://www. gov.uk/government/publications/priority-groups-for-coronavirus-
covid-19-vaccination-advice-from-the-jcvi-30-december-2020/joint-
committee-on-vaccination-and-immunisation-advice-on-priority-
groups-for-covid-19-vaccination-30-december-2020. 7
Joint Committee on Vaccination and Immunisation. Joint Committee
on Vaccination and Immunisation: advice on priority groups for
COVID-19 vaccination, 30 December 2020. 2021. https://www. gov.uk/government/publications/priority-groups-for-coronavirus-
covid-19-vaccination-advice-from-the-jcvi-30-december-2020/joint-
committee-on-vaccination-and-immunisation-advice-on-priority-
groups-for-covid-19-vaccination-30-december-2020. g
p
8
Mathieu E, Ritchie H, Ortiz-Ospina E, et al. A global database
of COVID-19 vaccinations. Nat Hum Behav 2021;5:947-53. doi:10.1038/s41562-021-01122-8 9
Kua J, Patel R, Nurmi E, et al. healthcareCOVID: a national cross-
sectional observational study identifying risk factors for developing
suspected or confirmed COVID-19 in UK healthcare workers. PeerJ 2021;9:e10891. doi:10.7717/peerj.10891 Funding: The study is funded by the Department of Health and
Social Care (DHSC) and UK Health Security Agency (UKHSA; formally
Public Health England), with contributions from the governments of
Northern Ireland, Scotland, and Wales. Funding was also provided by
the National Institute for Health Research (NIHR) as an Urgent Public
Health Priority Study and through the Health Protection Research
Unit in Healthcare Associated Infections and Antimicrobial Resistance
(NIHR200915), a partnership between UKHSA and the University of
Oxford. The funders of the study had no role in study design, data
collection, data analysis, data interpretation, or writing of the report. The views expressed are those of the authors and not necessarily
those of the DHSC, UKHSA, or NIHR. 10 Mutambudzi M, Niedwiedz C, Macdonald EB, et al. Occupation
and risk of severe COVID-19: prospective cohort study of 120 075
UK Biobank participants. Occup Environ Med 2020;78:307-14. doi:10.1136/oemed-2020-106731 11 Nguyen LH, Drew DA, Graham MS, et al, COronavirus Pandemic
Epidemiology Consortium. Risk of COVID-19 among front-line health-
care workers and the general community: a prospective cohort
study. Lancet Public Health 2020;5:e475-83. doi:10.1016/S2468-
2667(20)30164-X 12 Ward H, Atchison C, Whitaker M, et al. SARS-CoV-2 antibody
prevalence in England following the first peak of the pandemic. Nat
Commun 2021;12:905. doi:10.1038/s41467-021-21237-w Competing interests: All authors have completed the ICMJE uniform
disclosure form at https://www.icmje.org/disclosure-of-interest/ and
declare: support from the Department of Health and Social Care, UK
Health Security Agency, and National Institute for Health Research;
no financial relationships with any organisations that might have an
interest in the submitted work in the previous three years; no other
relationships of activities that could appear to have influenced the
submitted work. 13 Eyre DW, Lumley SF, O’Donnell D, et al, Oxford University Hospitals
Staff Testing Group. on 14 September 2022 by guest. Protecte
http://www.bmj.com/
BMJ: first published as 10.1136/bmj-2022-070379 on 20 July 2022. Downloaded from with participants and the network of SIREN study teams through
regular participant and research site newsletters and webinars. Lay
summaries will be produced and shared through social media and an
upcoming website. healthcare provision. Focused patient and participant
involvement work will be essential in understanding
the barriers to achieving high vaccination coverage
within at-risk demographic and occupational groups,
including those impeding access and increasing
hesitancy. This will be particularly important given
the need for ongoing booster vaccination coverage
to improve protection against the omicron variant,
and the likely continued need for boosters for future
variants of concern. Provenance and peer review: Not commissioned; externally peer
reviewed. Provenance and peer review: Not commissioned; externally peer
reviewed. This is an Open Access article distributed in accordance with the
terms of the Creative Commons Attribution (CC BY 4.0) license, which
permits others to distribute, remix, adapt and build upon this work,
for commercial use, provided the original work is properly cited. See:
http://creativecommons.org/licenses/by/4.0/. 1
UK Health Security Agency. Coronavirus in the UK: Cases in the UK. https://coronavirus.data.gov.uk/details/cases.fi Future work should explore variability in the built
environment, infection prevention and control and
personal protective equipment policy, and ward based
pressures across organisations and their associations
with
infection
risk. Greater
understanding
of
transmission dynamics among healthcare workers,
particularly according to role and setting, will support
NHS trusts in protecting their workforce and patients
from SARS-CoV-2 infection and potentially other
seasonal winter viruses. 2
Office for National Statistics. Coronavirus (COVID-19) Infection Survey:
England. https://www.ons.gov.uk/peoplepopulationandcommunity/
healthandsocialcare/conditionsanddiseases/datasets/
coronaviruscovid19infectionsurveydata. 3
Public Health England. PHE investigating a novel variant of COVID-19. 2020. https://www.gov.uk/government/news/phe-investigating-a-
novel-variant-of-covid-19. 4
Davies NG, Abbott S, Barnard RC, et al, CMMID COVID-19 Working
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transmissibility and impact of SARS-CoV-2 lineage B.1.1.7 in England. Science 2021;372:eabg3055. doi:10.1126/science.abg3055 g
g
5
Medicines and Healthcare products Regulatory Agency. UK medicines
regulator gives approval for first UK COVID-19 vaccine. 2020. https://
www.gov.uk/government/news/uk-medicines-regulator-gives-
approval-for-first-uk-covid-19-vaccine. We acknowledge the contributions of SIREN’s participants, site
teams, and Study Group to this manuscript and thank them for their
continued support. We also acknowledge the contributions of Alex
Bhattacharya, Andre Charlett, Ayoub Saei, and James Stimson. ppi
6
British Broadcasting Corporation. Covid-19 vaccine: First person
receives Pfizer jab in UK. 2020. https://www.bbc.co.uk/news/uk-
55227325. Contributors: DP, EJMM, and SE are joint first authors and contributed
equally: order was decided by first name alphabetical order. CSB,
JR, SH, and VJH conceived the study. AA, CSB, EJMM, EW, JI, SF, SH,
VJH, and the SIREN Study Group delivered the SIREN study, including
recruitment, data collection, and project management. DP and EJMM
did the statistical analyses. DP, JR, RH, and SE contributed to and
conducted the mathematical modelling. DP, EJMM, JI, SE, and VJH
drafted the manuscript and supplementary materials. CSB, JR, SH,
and VJH critically revised the manuscript for intellectual content. All
authors read and approved the final manuscript. The corresponding
author attests that all listed authors meet authorship criteria and that
no others meeting the criteria have been omitted. SH is the guarantor. 7
Joint Committee on Vaccination and Immunisation. Unanswered questions and future research This study reinforces the importance of vaccination
among healthcare workers during a significant wave
of the SARS-CoV-2 pandemic in England: mechanisms
to ensure high vaccination coverage, including
boosters, throughout the workforce must be used to
minimise the impact of future waves on the NHS and 10 10 doi: 10.1136/bmj-2022-070379 | BMJ 2022;378:e070379 | the bmj RESEARCH on 14 September 2022 by guest. Protected by copyright. http://www.bmj.com/
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39 Healthcare Safety Investigation Branch. COVID-19 transmission
in hospitals: management of the risk - a prospective safety
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reports/covid-19-transmission-in-hospitals-management-of-the-
risk/.fi 24 Nafilyan V, Islam N, Mathur R, et al. Ethnic differences in COVID-19
mortality during the first two waves of the Coronavirus Pandemic:
a nationwide cohort study of 29 million adults in England. Eur J
Epidemiol 2021;36:605-17. doi:10.1007/s10654-021-00765-1 25 Elliott P, Haw D, Wang H, et al, COVID-19 Genomics UK (COG-UK)
Consortium11‡. Exponential growth, high prevalence of SARS-
CoV-2, and vaccine effectiveness associated with the Delta variant. Science 2021;374:eabl9551. doi:10.1126/science.abl9551 40 Panovska-Griffiths J, Swallow B, Hinch R, et al. Statistical and
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31-10-december-2021.pdf. 41 Office for National Statistics. Coronavirus (COVID-19) Infection
Survey: characteristics of people testing positive for COVID-19
in England, 22 February 2021. https://www.ons.gov.uk/
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covid19inengland22february2021.i p
27 Pulliam JRC, van Schalkwyk C, Govender N, et al. Increased risk of
SARS-CoV-2 reinfection associated with emergence of Omicron in
South Africa. Science 2022;376:eabn4947. doi:10.1126/science. abn4947 28 Health and Social Care Committee. Workforce burnout and resilience
in the NHS and social care. 2021. https://committees.parliament.uk/
work/494/workforce-burnout-and-resilience-in-the-nhs-and-social-
care/publications/. 42 Riley S, Eales O, Walters CE, et al. REACT-1 round 8 final report: high
average prevalence with regional heterogeneity of trends in SARS-
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data-and-information/publications/statistical/nhs-sickness-absence-
rates. 43 Riley S, Wang H, Eales O, et al. REACT-1 round 9 final report:
Continued but slowing decline of prevalence of SARS-CoV-2 during
national lockdown in England in February 2021.medRxiv 2021. doi:10.1101/2021.03.03.21252856 . 30 Hall VJ, Foulkes S, Charlett A, et al, SIREN Study Group. SARS-CoV-2
infection rates of antibody-positive compared with antibody-negative
health-care workers in England: a large, multicentre, prospective
cohort study (SIREN). Lancet 2021;397:1459-69. doi:10.1016/
S0140-6736(21)00675-9 44 Zheng C, Hafezi-Bakhtiari N, Cooper V, et al. Characteristics and
transmission dynamics of COVID-19 in healthcare workers at
a London teaching hospital. J Hosp Infect 2020;106:325-9. doi:10.1016/j.jhin.2020.07.025 45 Abuown A, Taube C, Koizia LJ. Impact of COVID-19 second
wave on healthcare worker staffing levels. Infect Control Hosp
Epidemiol 2021;42:787. doi:10.1017/ice.2020.353 fi 45 Abuown A, Taube C, Koizia LJ. Impact of COVID-19 second
wave on healthcare worker staffing levels. Infect Control Hosp
Epidemiol 2021;42:787. doi:10.1017/ice.2020.353 fi 31 Wallace S, Hall V, Charlett A, et al. Impact of prior SARS-CoV-2
infection and COVID-19 vaccination on the subsequent incidence
of COVID-19: a multicentre prospective cohort study among UK
healthcare workers - the SIREN (Sarscov2 Immunity & REinfection
EvaluatioN) study protocol. BMJ Open 2022;12:e054336. 46 Ferris M, Ferris R, Workman C, et al. Efficacy of FFP3 respirators
for prevention of SARS-CoV-2 infection in healthcare workers. Elife 2021;10:e71131. doi:10.7554/eLife.71131 32 NHS England. NHS Improvement. Novel coronavirus (COVID-19)
standard operating procedure: NHS England and NHS Improvement
rollout of lateral flow devices for asymptomatic staff testing for
SARS CoV-2 (phase 2: trusts). 2020 https://www.england.nhs.uk/
coronavirus/wp-content/uploads/sites/52/2020/11/C0873_i_
SOP_LFD-rollout-for-asymptomatic-staff-testing_phase-2-trusts-
v1.1_16-nov20.pdf.f Web appendix: Appendix 1: Mathematical modelling
methods Provenance and peer review: Not commissioned; externally peer
reviewed. Differential occupational risks to healthcare
workers from SARS-CoV-2 observed during a prospective
observational study. Elife 2020;9:e60675. doi:10.7554/eLife.60675 f 14 Hanrath AT, Schim van der Loeff I, Lendrem DW, et al. SARS-
CoV-2 Testing of 11,884 Healthcare Workers at an Acute NHS
Hospital Trust in England: A Retrospective Analysis. Front Med
(Lausanne) 2021;8:636160. doi:10.3389/fmed.2021.636160 15 Martin CA, Patel P, Goss C, et al. Demographic and occupational
determinants of anti-SARS-CoV-2 IgG seropositivity in hospital staff. J
Public Health (Oxf) 2020;fdaa199. Ethical approval: The study was approved by the Berkshire Research
Ethics Committee, Health Research Authority (IRAS ID 284460, REC
reference 20/SC/0230) on 22 May 2020; the vaccine amendment
was approved on 12 January 2021. The study is registered with
ISRCTN (trial ID: ISRCTN11041050). Participants gave informed
consent before taking part in the study. 16 Valdes AM, Moon JC, Vijay A, et al. Longitudinal assessment
of symptoms and risk of SARS-CoV-2 infection in healthcare
workers across 5 hospitals to understand ethnic differences in
infection risk. EClinicalMedicine 2021;34:100835. doi:10.1016/j. eclinm.2021.100835 Data sharing: The metadata for this analysis will be available on
reasonable request to researchers through the Health Data Research
UK CO-CONNECT platform and available for secondary analysis. 17 Maidstone R, Anderson SG, Ray DW, Rutter MK, Durrington HJ,
Blaikley JF. Shift work is associated with positive COVID-19 status
in hospitalised patients. Thorax 2021;76:601-6. doi:10.1136/
thoraxjnl-2020-216651 The lead author (the manuscript’s guarantor) affirms that this
manuscript is an honest, accurate, and transparent account of the
study being reported; that no important aspects of the study have
been omitted; and that any discrepancies from the study as planned
(and, if relevant, registered) have been explained. 18 Mathur R, Rentsch CT, Morton CE, et al, OpenSAFELY Collaborative. Ethnic differences in SARS-CoV-2 infection and COVID-19-related
hospitalisation, intensive care unit admission, and death in 17
million adults in England: an observational cohort study using the
OpenSAFELY platform. Lancet 2021;397:1711-24. doi:10.1016/
S0140-6736(21)00634-6 Dissemination to participants and related patient and
public communities: The results of this study will be shared 11 the bmj | BMJ 2022;378:e070379 | doi: 10.1136/bmj-2022-070379 RESEARCH Web appendix: Appendix 1: Mathematical modelling
methods Web appendix: Appendix 2: Supplementary tables
and figures 33 Harris RJ, Hall JA, Zaidi A, Andrews NJ, Dunbar JK, Dabrera G. Effect of
Vaccination on Household Transmission of SARS-CoV-2 in England. N
Engl J Med 2021;385:759-60. doi:10.1056/NEJMc2107717 Web appendix: Appendix 3: SIREN Study Group
members doi: 10.1136/bmj-2022-070379 | BMJ 2022;378:e070379 | the bmj 12 12
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Tata Laksana Infertilitas yang Rasional dan Efisien untuk Mempersingkat "Time to Pregnancy"
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Obgynia
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cc-by-sa
| 2,580
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Abstrak Tujuan: Memberikan pandangan kepentingan melakukan penatalaksanaan infertilitas yang efisien untuk
mempersingkat time to pregnancy. Metode: Telaah pustaka dan basis bukti. Metode: Telaah pustaka dan basis bukti. Hasil: Kunci mempersingkat time to pregnancy pada penanganan infertilitas adalah melakukan work-up yang
efisien dan rasional. Terdapat beberapa pemeriksaan yang mempunyai manfaat jelas berdasarkan basis bukti. Hari-
hari tertentu pada siklus menstruasi seorang wanita dapat dimanfaatkan untuk melakukan beberapa pemeriksaan
terpilih. Fokus pemeriksaan adalah identifikasi latar belakang kesehatan suami dan istri, mengetahui adanya
ovulasi dan cadangan ovarium, tes patensi tuba, evaluasi anatomik uterus dan peritoneum, serta analisis sperma. Setelah data dasar tersebut diperoleh, dilakukan evaluasi komprehensif untuk mengetahui program penanganan
yang sesuai. Saat ini telah terdapat panduan nasional praktik kedokteran penanganan infertilitas sebagai dasar
untuk dapat menangani infertilitas secara efisien. p
g
Kesimpulan: Time to pregnancy dapat dipersingkat dengan proses diagnostik dan penanganan yang berbasis bukti
dengan memanfaatkan waktu-waktu tertentu dari siklus menstruasi. Kata kunci: Infertilitas, time to pregnancy, penanganan infertilitas, rujukan eISSN 2615-496X eISSN 2615-496X Key words: Infertility, time to pregnancy, infertility management, referral system. Tatalaksana Infertilitas yang Rasional dan Efisien untuk Mempersingkat
"Time to Pregnancy" Hartanto Bayuaji
Departemen Obstetri dan Ginekologi Fakultas Kedokteran Universitas Padjadjaran
Rumah Sakit Dr. Hasan Sadikin Bandung
Korespondensi: Hartanto Bayuaji, Email: tantobayuaji@gmail.com Abstract Objective: Provide an insight of the importance of conducting efficient and rational infertility management to
shorten time to pregnancy. Method: Literature and evidence base review. Results: The key to shortening time to pregnancy in infertility management is to do an efficient and rational work-
up. There are several examination that have clear benefits. Certain days of a woman's menstrual cycle can be
used to do some selected examinations. The focus of the examination is identification of the health background of
husband and wife, ovulation documentation and ovarian reserve assay, tubal patency tests, anatomic evaluation
of the uterus and peritoneum, and sperm analysis. After the basic data is obtained, a comprehensive evaluation is
carried out to find out the appropriate management program. Currently there is a national guide to the practice of
medical treatment of infertility as a basis for infertility management efficiently. Conclusion: Time to pregnancy can be shortened by an evidence-based diagnostic and treatment pr
utilizing certain times of the menstrual cycle. Key words: Infertility, time to pregnancy, infertility management, referral system. ey words: Infertility, time to pregnancy, infertility management, referral system. 73 Obgynia, Volume 1 Nomor 2 September 2018 Obgynia, Volume 1 Nomor 2 September 2018 Pendahuluan Infertilitas adalah tidak terjadinya kehamilan setelah periode satu tahun dengan hubungan
seksual teratur tanpa kontrasepsi. Masalah ini terjadi pada sekitar 15% pasangan suami istri usia
reproduktif. 1-3 Walaupun infertilitas merupakan masalah penting dalam kesehatan reproduksi,
sering ditemukan hambatan dalam talaksananya. Hal ini disebabkan beragamnya etiologi yang
mendasari maupun modalitas pemeriksaan yang tersedia. Beragamnya tingkat pemahaman
pengelola kesehatan tentang masalah ini menyebabkan risiko penanganan menjadi berlarut-
larut. Dampak dari hal ini adalah berkurangnya potensi fertilitas seorang wanita akibat lamanya
proses penanganan. Terdapat konsep time to pregnancy, yaitu waktu yang dibutuhkan untuk
didapatkannya kelahiran hidup dari mulai masalah infertilitas ditegakkan.4,5 Time to pregnancy
akan dapat memanjang bila penatalaksanaan infertilitas tidak dilakukan secara efisien dan
rasional. Beberapa hal akan dibahas dalam tulisan ini sebagai upaya untuk mempersingkat time
to pregnancy pada penatalaksanaan infertilitas. Langkah Pertama: Back To Basic Seperti telah disebutkan bahwa terdapat beragam etiologi infertilitas, sehingga dengan
sendirinya pemahaman patofisiologi menjadi beragam serta pemeriksaan yang dibutuhkan
menjadi bertambah. Walaupun demikian, secara praktis fokus penatalaksanaan dapat diarahkan
pada beberapa hal agar lebih efektif dan efisien. Beberapa hal tersebut adalah sebagai berikut:6 1. Pahami proses terjadinya kehamilan secara benar
Pemahaman yang cukup pada dasar-dasar terjadinya kehamilan secara alami. Seperti
diketahui agar terjadi kehamilan dibutuhkan: sperma yang memenuhi syarat, kavum uteri
normal, tuba paten, dan ovulasi yang konsisten. Pengalaman klinis menunjukkan hal-hal
tersebut dirasakan cukup untuk memperkirakan faktor mana yang merupakan penyebab
kelainan infertilitas. 2. Pemeriksaan dasar fokus pada hal-hal berikut:1,7
a. Adanya gangguan ovulasi yang biasanya ditandai dengan oligo atau amenore
b. Dugaan gangguan patensi tuba, kelainan anatomis uterus, dan atau endometriosis. c. Terdapat dugaan penyebab faktor pria. 2. Pemeriksaan dasar fokus pada hal-hal berikut:1,7 . e e
saa dasa
o us pada a
a be
ut:
a. Adanya gangguan ovulasi yang biasanya ditandai dengan oligo atau amenore
b. Dugaan gangguan patensi tuba, kelainan anatomis uterus, dan atau endometrios a. Adanya gangguan ovulasi yang biasanya ditandai dengan oligo atau amenore b. Dugaan gangguan patensi tuba, kelainan anatomis uterus, dan atau endometriosi c. Terdapat dugaan penyebab faktor pria. c. Terdapat dugaan penyebab faktor pria. 3. Manfaatkan hari-hari tertentu pada siklus menstruasi secara efisien. Proses diagnostik infertilitas dimulai dari anamnesis. Anamnesis infertilitas memerlukan
waktu yang cukup agar tergali beberapa aspek penting yang mungkin menjadi etiologi. Selain
itu dengan waktu yang cukup juga memungkinkan memberikan konseling pada pasangan. Informasi yang cukup diharapkan dapat meningkatkan kepatuhan pasangan untuk menjalani
penatalaksanaan infertilitas menjadi lebih optimal. Anamnesis perlu menggali hal-hal sebagai berikut: lama infertilitas, hasil-hasil penanganan
yang mungkin pernah dijalani sebelumnya, karakteristik siklus haid, riwayat obstetri, riwayat
kontrasepsi sebelumnya, frekuensi koitus dan riwayat disfungsi seksual, riwayat pembedahan
sebelumnya, kelainan tiroid, galaktore, hirsutisme, nyeri panggul atau dispareunia, obat-obatan
yang tengah digunakan atau riwayat alergi, riwayat keluarga dengan kelainan kongenital,
retardasi mental, menopause dini, atau kegagalan fungsi reproduktif lain, kemungkinan paparan 74 Hartanto Bayuaji : Tatalaksana Infertilitas yang Rasional dan Efisien untuk Mempersingkat "Time To Pregnancy " Hartanto Bayuaji : Tatalaksana Infertilitas yang Rasional dan Efisien untuk Mempersingkat "Time To Pregnancy " zat berbahaya seperti toksin atau radiasi, dan. penggunaan tembakau atau narkoba. Pemeriksaan
fisik perlu mencari hal-hal yang terkait patologi sistem reproduksi maupun kelainan sistemik
yang berpotensi mengganggu fertilitas.1,6 zat berbahaya seperti toksin atau radiasi, dan. penggunaan tembakau atau narkoba. Langkah Pertama: Back To Basic Pemeriksaan
fisik perlu mencari hal-hal yang terkait patologi sistem reproduksi maupun kelainan sistemik
yang berpotensi mengganggu fertilitas.1,6 Pemeriksaan penunjang, diharapkan dapat dilakukan secara efisien pada waktu yang tepat. Perlu diperhatikan kaitan pemeriksaan dengan siklus menstruasi, karena beberapa pemeriksaan
hanya dapat dilakukan pada hari-hari tertentu siklus menstruasi. Memanfaatkan hari-hari
tertentu pada siklus menstruasi ternyata dapat mempersingkat waktu yang dibutuhkan untuk
memperoleh data awal. Sangat ideal apabila pasien dapat berkunjung pertama kali pada hari
awal menstruasi. Secara lebih terperinci, hal tersebut dapat dilihat pada uraian berikut: a. Hari kedua/ketiga siklus menstruasi: periode ini merupakan saat untuk melakukan
pemeriksaan ultrasonografi transvaginal guna mengevaluasi anatomi uterus dan ovarium. Penghitungan folikel antral juga dilakukan pada saat ini. Demikian pula, endometrium
pada saat ini harus terlihat tipis, yang menandakan rendahnya kadar estrogen di awal
siklus. Selain itu periode ini merupakan waktu yang tepat untuk melakukan pemeriksaan
hormonal (kadar FSH, LH, estradiol, prolaktin). Hasil dari pemeriksaan hormonal tersebut
digunakan untuk mengevaluasi faktor ovulasi. Dalam kunjungan ini direncanakan pula
untuk melakukan analisis semen. b. Hari ke-9/10/11: periode ini merupakan saat yang optimal untuk melakukan evaluasi patensi
tuba, baik secara radiologis maupun ultrasonografis. Selain itu dapat pula dilakukan analisis
semen apabila suami telah mendapat penjelasan yang cukup dalam kunjungan sebelumnya. b. Hari ke-9/10/11: periode ini merupakan saat yang optimal untuk melakukan evaluasi patensi
tuba, baik secara radiologis maupun ultrasonografis. Selain itu dapat pula dilakukan analisis
semen apabila suami telah mendapat penjelasan yang cukup dalam kunjungan sebelumnya. Langkah Kedua: Pilih Pemeriksaan Spesifik yang Efisien Bila telah didapatkan perkiraan kondisi patologi yang ada, maka langkah berikutnya adalah
memilih pemeriksaan lanjutan yang sesuai. Pada tahap ini dapat timbul masalah mengingat
banyaknya modalitas pemeriksaan yang tersedia. Walaupun demikian terdapat berbagai basis
bukti (evidence) yang dapat digunakan sebagai dasar memilih pemeriksaan yang paling efisien. B.
Gangguan Patensi Tuba B. Gangguan patensi tuba menjadi penyebab dari sekitar 30% infertilitas wanita, oleh karena
itu evaluasi faktor ini merupakan hal penting. Pemeriksaan yang dapat dilakukan antara
lain:1,8 Gangguan patensi tuba menjadi penyebab dari sekitar 30% infertilitas wanita, oleh karena
itu evaluasi faktor ini merupakan hal penting. Pemeriksaan yang dapat dilakukan antara
lain:1,8 - Histerosalpingografi (HSG): dapat digunakan untuk mendeteksi sumbatan tuba
proksimal maupun distal, adanya salpingitis ismika nodosa, adhesi perituba,
serta hidrosalping. Nilai prediksi positif HSG adalah sekitar 38% sementara nilai
prediksi negatifnya sekitar 94%. Hal ini berarti bila hasil HSG menunjukkan tuba
paten, maka hasil tersebut dapat dipercaya dengan cukup baik. Sebaliknya bila hasil
HSG menunjukkan adanya obstruksi tuba, maka diperlukan evaluasi lanjut untuk
mengkonfirmasi hal tersebut. - Sonohisterosalpingografi: teknik ini hampir serupa dengan histerosalpingografi
menggunakan sinar X, namun aliran cairan dalam tuba diikuti menggunakan
ultrasonografi Doppler transvaginal. Kadang ditemukan kesulitan untuk
mengidentifikasi tuba mana yang paten. Dengan demikian bila didapatkan adanya
koleksi cairan di kavum Douglas pasca tindakan maka disimpulkan salah satu tuba
adalah paten. -
Kromotubasi laparoskopi: merupakan baku emas untuk penilaian patensi tuba. Teknik ini dilakukan bila ditemukan dugaan obstruksi tuba dari hasil HSG atau
sonohisterosalpingografi. Walaupun merupakan baku emas, teknik ini merupakan
teknik operatif. Dengan demikian dapat timbul potensi penyulit dari teknik operasi
maupun anestesinya. A.
Gangguan Ovulasi A. Gangguan Ovulasi Gangguan ovulasi merupakan penyebab sekitar 40% infertilitas pada wanita. Gejala klinik
yang tampak dapat berupa gangguan menstruasi. Walaupun demikian pada beberapa
kondisi kelainan menstruasi tidak tampak jelas secara klinis. Riwayat menstruasi
merupakan hal penting. Siklus menstruasi yang normal berkisar antara 25−35 hari,
dengan karakteristik perdarahan yang konsisten. Bila siklus haid tidak teratur, biasanya
diagnosis anovulasi dapat ditegakkan dengan baik. Sebaliknya bila siklus teratur, maka
adanya ovulasi harus dibuktikan dengan menggunakan beberapa cara seperti pengukuran
suhu basal badan, pengukuran kadar progesterone serum, deteksi lonjakan LH, dan
ultrasonografi transvaginal serial. 1 Di samping mengetahui adanya anovulasi, salah satu pemeriksaan yang penting adalah
penilaian cadangan ovarium. Cadangan ovarium merupakan gambaran potensi fertilitas
yang dilihat dari jumlah oosit yang masih tersedia. Pemeriksaan ini dilakukan secara
selektif pada beberapa golongan berikut: usia di atas 35 tahun, riwayat keluarga dengan
menopause dini, pernah menjalani operasi pada satu atau kedua ovarium, pernah
menjalani kemoterapi atau radioterapi pelvis, mengalami infertilitas yang tak terjelaskan 75 Obgynia, Volume 1 Nomor 2 September 2018 (unexplained infertility), respon yang buruk terhadap stimulasi ovarium sebelumnya,
dan rencana mengikuti program teknologi reproduksi berbantu. Beberapa tes yang dapat
dilakukan adalah:1,6,7 kadar FSH dan estradiol hari ke-3, hitung folikel antral (antral follicle
count), dan kadar Anti Mullerian Hormone (AMH). Beberapa ulasan menunjukan bahwa
ketiga teknik di atas termasuk dapat diterapkan secara klinis dan memiliki kemampuan
diagnostik yang cukup baik. B. Gangguan Patensi Tuba B. Gangguan Patensi Tuba D.
Kelainan Sperma Sama halnya dengan pemeriksaan pada wanita, pemeriksaan pada pria juga diawali dengan
investigasi mengenai karakteristik hubungan seksual, lama infertilitas, penyakit yang
pernah dialami, kondisi medis sistemik saat ini, riwayat pembedahan sebelumnya, obat
yang digunakan dan riwayat alergi, riwayat penyakit menular seksual, dan paparan zat yang
toksik terhadap gonad. Analisis semen harus dilakukan sejak awal dan tidak menunggu
pemeriksaan faktor wanita selesai. Hal ini karena faktor pria merupakan penyebab dari
sekitar 30-40% infertilitas.6,9 Dalam menginterpretasi hasil analisis semen diperlukan
kehati-hatian. Bila hasil analisis semen abnormal, dianjurkan untuk mengidentifikasi
faktor yang mungkin dapat dikoreksi seperti gaya hidup sebelum melakukan analisis
ulang. Analisis ulang dapat dilakukan sekitar 2 bulan kemudian. Dalam satu penelitian
ditemukan bahwa subfertilitas pria diprediksi terjadi apabila didapat konsentrasi sperma
kurang dari 13,5 juta/mL, motilitas kurang dari 32%, dan morfologi normal kurang dari
9%.10 Langkah Ketiga: Analisis Komprehensif Semua Data yang Terkumpul Setelah hasil-hasil pemeriksaan terkumpul, maka dilakukan analisis komprehensif. Tergantung
dari kesimpulan yang didapat, penatalaksanaan dapat berupa induksi ovulasi pada anovulasi,
koreksi kelainan anatomik pada uterus maupun tuba, koreksi kelainan yang dapat diubah
(modifiable) pada kelainan sperma, aplikasi teknologi reproduksi berbantu (assisted reproductive
technology).11 mioma submukosa, dan sinekia uteri. mioma submukosa, dan sinekia uteri. -
Histeroskopi: merupakan metode definitif pemeriksaan kavum uteri. Walaupun
demikian aplikasinya terkendala ketersediaan peralatan dan biaya pemeriksaan
yang masih tinggi. -
Histeroskopi: merupakan metode definitif pemeriksaan kavum uteri. Walaupun
demikian aplikasinya terkendala ketersediaan peralatan dan biaya pemeriksaan
yang masih tinggi. C.
Kelainan Uterus Kelainan uterus sebagai penyebab infertilitas difokuskan pada kemungkinan terjadinya
gangguan pada proses fertilisasi dan implantasi. Dalam hal ini integritas kavum uteri
berperan penting. Beberapa penyebab infertilitas akibat kelainan uterus antara lain
kelainan kongenital (uterus unikornis, bikornis, atau septatus), maupun kelainan didapat
(polip endometrium, mioma submukosa, atau sinekia uteri). Untuk mengetahui adanya
kelainan tersebut, dapat dipilih beberapa teknik pemeriksaan yaitu:6 -
Histerosalpingografi (HSG): merupakan teknik yang telah lama digunakan. Walaupun demikian, teknik ini mempunyai sensitivitas dan nilai prediksi positif
yang rendah (50% dan 30%) untuk mendeteksi kelainan yang sering terjadi seperti
polip endometrium serta mioma submukosa. -
Histerosalpingografi (HSG): merupakan teknik yang telah lama digunakan. Walaupun demikian, teknik ini mempunyai sensitivitas dan nilai prediksi positif
yang rendah (50% dan 30%) untuk mendeteksi kelainan yang sering terjadi seperti
polip endometrium serta mioma submukosa. -
Ultrasonografi transvaginal dan sonohisterosalpingografi: dengan menggunakan
teknik sonohisterosalpingografi dapat diperoleh pencitraan yang lebih baik dari
kavum uteri. Teknik sonohisterosalpingografi mempunyai nilai prediksi positif
yang tinggi (sekitar 90%) untuk mendeteksi kelainan seperti polip endometrium, Ultrasonografi transvaginal dan sonohisterosalpingografi: dengan menggunakan
teknik sonohisterosalpingografi dapat diperoleh pencitraan yang lebih baik dari
kavum uteri. Teknik sonohisterosalpingografi mempunyai nilai prediksi positif
yang tinggi (sekitar 90%) untuk mendeteksi kelainan seperti polip endometrium, 76 Hartanto Bayuaji : Tatalaksana Infertilitas yang Rasional dan Efisien untuk Mempersingkat "Time To Pregnancy " Hartanto Bayuaji : Tatalaksana Infertilitas yang Rasional dan Efisien untuk Mempersingkat "Time To Pregnancy " Langkah Keempat: Ketahui Saat Merujuk yang Tepat Mengetahui saat yang tepat merujuk pasangan dapat membantu meningkatkan keberhasilan
penanganan. Tidak dapat dipungkiri bahwa tidak semua tempat pelayanan kesehatan reproduksi
memiliki sarana yang memadai untuk menangani infertilitas hingga tahap teknologi reproduksi
berbantu. Walaupun demikian, hampir di tiap propinsi talah berdiri pusat-pusat pelayanan
tersebut sehingga yang harus dioptimalkan adalah kemitraan dengan sejawat yang bertugas
di daerah. Beberapa tindakan seperti investigasi dasar, induksi ovulasi sederhana, bahkan
inseminasi intrauterin memungkinkan untuk dilakukan di tempat praktik. Beberapa jenis kasus
yang membutuhkan rujukan segera antara lain azoospermia, obstruksi tuba bilateral, periode
infertilitas yang terlalu lama dan usia wanita 23-39 tahun.11 Dari pemaparan ini dapat disimpulkan bahwa mempersingkat time to pregnancy pada
penanganan infertilitas tergantung pada pemilihan waktu dan jenis pemeriksaan yang tepat,
mengidentifikasi kemampuan penanganan oleh klinisi, dan melakukan proses rujukan yang
optimal. Diharapkan dengan pemahaman ini, tidak hanya keberhasilan proses reproduksi yang
menjadi tujuan namun juga kualitas individu yang dihasilkan melalui penatalaksanaan ini. 77 77 Obgynia, Volume 1 Nomor 2 September 2018 Daftar Pustaka 7. Practice Committee of the American
Society for Reproductive M. Testing
and interpreting measures of ovarian re-
serve: a committee opinion. Fertil Steril. 2015;103(3):e9-e17. 1. Practice Committee of the American
Society for Reproductive M. Diagnos-
tic evaluation of the infertile female:
a committee opinion. Fertil Steril. 2015;103(6):e44-50. 8. Saunders RD, Shwayder JM, Nakajima
ST. Current methods of tubal patency as-
sessment. Fertil Steril. 2011;95(7):2171-
9. 2. Zegers-Hochschild F, Adamson GD,
Dyer S, Racowsky C, de Mouzon J, So-
kol R, et al. The International Glossary on
Infertility and Fertility Care, 2017. Fertil
Steril. 2017;108(3):393-406. 9. Practice Committee of the American Soci-
ety for Reproductive M. Diagnostic eval-
uation of the infertile male: a committee
opinion. Fertil Steril. 2015;103(3):e18-
25. 3. American College of O, Gynecologists
Committee on Gynecologic P, Practice
C. Female age-related fertility decline. Committee Opinion No. 589. Fertil Ster-
il. 2014;101(3):633-4. 10. Guzick DS, Overstreet JW, Factor-Litvak
P, Brazil CK, Nakajima ST, Coutifaris C,
et al. Sperm morphology, motility, and
concentration in fertile and infertile men. N Engl J Med. 2001;345(19):1388-93. 4. Sozou PD, Hartshorne GM. Time to
pregnancy: a computational method for
using the duration of non-conception
for predicting conception. PLoS One. 2012;7(10):e46544. 11. Himpunan Endokrinologi Reproduksi
dan Fertilitas Indonesia, Perhimpunan
Fertilisasi In Vitro Indonesia, Ikatan Ahli
Urologi Indonesia, Perkumpulan Obste-
tri & Ginekologi Indonesia. Konsensus
Penanganan Infertilitas.Jakarta. 2013. 5. Cooney MA, Buck Louis GM, Sundaram
R, McGuiness BM, Lynch CD. Validity
of self-reported time to pregnancy. Epi-
demiology. 2009;20(1):56-9. 6. Balen AH. Infertility in Practice. edition
4. Boca Raton, Florida, USA: CRC Press
Taylor & Francis Group; 2014. 78
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Whole-Genome Sequence and Comparative Analysis of Trichoderma asperellum ND-1 Reveal Its Unique Enzymatic System for Efficient Biomass Degradation
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Citation: Zheng, F.; Han, T.; Basit, A.;
Liu, J.; Miao, T.; Jiang, W. Whole-Genome Sequence and
Comparative Analysis of Trichoderma
asperellum ND-1 Reveal Its Unique
Enzymatic System for Efficient
Biomass Degradation. Catalysts 2022,
12, 437. https://doi.org/10.3390/
catal12040437 Academic Editors: Antonio Zuorro,
Yung-Chuan Liu and Jose M. Guisan Keywords: Trichoderma asperellum ND-1; whole-genome sequencing; secretome; comparative genomics;
lignocellulolytic enzymes; biomass degradation Received: 12 March 2022
Accepted: 6 April 2022
Published: 13 April 2022 Fengzhen Zheng 1, Tianshuo Han 2, Abdul Basit 3, Junquan Liu 2, Ting Miao 2 and Wei Jiang 2 1
College of Biological and Environmental Engineering, Zhejiang Shuren University, 36 Zhoushan E Rd,
Hangzhou 310015 China; 18811068358@163 com 1
College of Biological and Environmental Engineering, Zhejiang Shuren University, 36 Zhoushan E Rd,
Hangzhou 310015, China; 18811068358@163.com
2
State Key Laboratory of Agro-Biotechnology, College of Biological Sciences, China Agricultural University,
Yuan Ming Yuan West Road No. 2, Haidian, Beijing 100193, China; hants@cau.edu.cn (T.H.);
ljq0802040421@163.com (J.L.); 15501017617@163.com (T.M.)
3
Department of Microbiology, University of Jhang, Jhang 35200, Pakistan; abdul_9090@yahoo.com
*
Correspondence: jiangwei01@cau.edu.cn; Tel./Fax: +86-10-62731440 g
g
g
g
j
g
y
Hangzhou 310015, China; 18811068358@163.com
2
State Key Laboratory of Agro-Biotechnology, College of Biological Sciences, China Agricultural University,
Yuan Ming Yuan West Road No. 2, Haidian, Beijing 100193, China; hants@cau.edu.cn (T.H.);
ljq0802040421@163.com (J.L.); 15501017617@163.com (T.M.)
3
Department of Microbiology, University of Jhang, Jhang 35200, Pakistan; abdul_9090@yahoo.com
*
Correspondence: jiangwei01@cau.edu.cn; Tel./Fax: +86-10-62731440 Hangzhou 310015, China; 18811068358@163.com
2
State Key Laboratory of Agro-Biotechnology, College of Biological Sciences, China Agricultural University,
Yuan Ming Yuan West Road No. 2, Haidian, Beijing 100193, China; hants@cau.edu.cn (T.H.);
ljq0802040421@163.com (J.L.); 15501017617@163.com (T.M.) *
Correspondence: jiangwei01@cau.edu.cn; Tel./Fax: +86-10-62731440 Abstract: The lignocellulosic enzymes of Trichoderma asperellum have been intensely investigated
toward efficient conversion of biomass into high-value chemicals/industrial products. However, lack
of genome data is a remarkable hurdle for hydrolase systems studies. The secretory enzymes of newly
isolated T. asperellum ND-1 during lignocellulose degradation are currently poorly known. Herein, a
high-quality genomic sequence of ND-1, obtained by both Illumina HiSeq 2000 sequencing platforms
and PacBio single-molecule real-time, has an assembly size of 35.75 Mb comprising 10,541 predicted
genes. Secretome analysis showed that 895 proteins were detected, with 211 proteins associated
with carbohydrate-active enzymes (CAZymes) responsible for biomass hydrolysis. Additionally,
T. asperellum ND-1, T. atroviride IMI 206040, and T. virens Gv-298 shared 801 orthologues that were
not identified in T. reesei QM6a, indicating that ND-1 may play critical roles in biological-control. In-depth analysis suggested that, compared with QM6a, the genome of ND-1 encoded a unique
enzymatic system, especially hemicellulases and chitinases. Moreover, after comparative analysis of
lignocellulase activities of ND-1 and other fungi, we found that ND-1 displayed higher hemicellulases
(particularly xylanases) and comparable cellulases activities. Our analysis, combined with the whole-
genome sequence information, offers a platform for designing advanced T. asperellum ND-1 strains
for industrial utilizations, such as bioenergy production. catalysts catalysts catalysts Fengzhen Zheng 1, Tianshuo Han 2, Abdul Basit 3, Junquan Liu 2, Ting Miao 2 and Wei Jiang 2,* huo Han 2, Abdul Basit 3, Junquan Liu 2, Ting Miao 2 and Wei Jiang 2,* catalysts catalysts 1. Introduction Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. Lignocellulose from agricultural wastes, such as corn stover and sugarcane bagasse,
serves as a widespread, renewable, and available resource [1–3]. Its components contain
abundant and complex polysaccharides, including hemicellulose, cellulose, and lignin [4–6]. Particularly, hemicellulose and cellulose are becoming potential biomass feedstocks in
the generation of high-value chemicals or bioenergy products [7–9]. Efficiently catalytic
conversion of lignocellulose is mainly dependent on the availability of carbohydrate-
active enzymes (CAZymes) [10,11], typically glycoside hydrolases (GHs), which degrade
lignocellulosic biomass into simple sugars [12], a critical process for the production of
second-generation bioethanol [13]. In spite of remarkable progress that has been achieved
in enzymatic biodegradation of lignocellulosic materials [14,15], the high production cost
of lignocellulases is still a major hurdle that must be solved prior to commercial-scale
implementation of cellulosic ethanol [16]. 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/). In nature, the complete hydrolysis of biomass polysaccharides is usually carried out
by synergetic action of various CAZymes (hemicellulases, cellulases, and lignin-modifying https://www.mdpi.com/journal/catalysts Catalysts 2022, 12, 437. https://doi.org/10.3390/catal12040437 Catalysts 2022, 12, 437 2 of 19 2 of 19 enzymes) rather than individuals [17–20]. Moreover, the discovery of lytic polysaccharide
monooxygenases (LPMOs) has profoundly changed the way in which we view the enzy-
matic conversion of polysaccharides, particularly recalcitrant materials, such as cellulose
and chitin [21]. LPMOs have been classified in the CAZymes database, within the Auxiliary
Activity (AA) families AA9-11 and AA13-16, on account of their sequences [22–24]. The
most widely investigated LPMO families are AA9 and AA10 [25]. LPMOs are currently
known to be encoded in genomes across all kingdoms of life, especially fungi, and catalyze
cleavage of various substrates [26–29]. In addition, LPMOs may be subjected to various
post-translational modifications depending on their origin with effects on protein function
and stability [30]. y
Due to their high specific enzyme activities and relatively strong protein secretion
ability, several Trichoderma species and their cultivation on diverse agricultural wastes
to yield polysaccharide-degrading enzymes have been intensely studied in the past two
decades [31–33]. For example, T. reesei has been widely utilized in industrial fields and
is regarded as a major source of commercial cellulases [34–36]. 1. Introduction However, the enzymatic
mixtures produced by T. reesei is deficient in high efficiency hemicellulases and other acces-
sory enzymes, which facilitated investigations of other enzymes and/or fungi [37]. Among
the numerous filamentous fungi that secreted hemicellulases and cellulases, T. asperellum
is also known for its strong lignocellulosic hydrolysis ability [38–40]. Moreover, most of
the investigations on T. asperellum were concentrated on the selection and expression of
specific genes related to biomass-degrading enzymes, such as xylanases, β-glucanases, and
cellobiohydrolases [11,38,41]. However, the genome sequence information of T. asperellum
to further understand secretory proteins and carbohydrate-active enzymes remains to be
fully investigated. Rapid developments in sequencing strategies have facilitated the improvement of
reference genomes in distinct microorganisms and the analysis of genome-scale varia-
tions [42]. The achievements have accelerated investigation for sequencing several Tricho-
derma genomes (including T. reesei, T. virens, and T. atroviride), utilizing genome shotgun
approach, which provided a platform for detection of genome-wide differences and un-
derstanding degradation mechanisms of plant biomass polysaccharides [43,44]. Moreover,
the PacBio RS sequencing platform has emerged to be the most advanced third-generation
sequencer in the market in 2011 [45,46]. The system utilizes a unique and novel single
molecule real-time (SMRT) detection technology that promotes the production of sequences
with longer reads and decreases the level of bias [47–49]. Therefore, the application of
PacBio sequencing technology provides a promising strategy to obtain advanced and
accurate assemblies for Trichoderma genomes. The goal of this study is to detect potentially significant enzymes and obtain an
in-depth understanding of the lignocellulose-degrading mechanism from T. Asperellum
ND-1. Therefore, the whole genome sequencing and comparative analysis of T. Asperellum
ND-1 were performed by PacBio RS sequencing technology, with particular emphasis on
biomass hydrolysis-related genes. Here, we described lignocellulolytic enzyme charac-
teristics of T. asperellum ND-1 and compared them with other fungi (particularly T. reesei). Secretome analysis of T. asperellum ND-1 was also carried out to identified extracellular
CAZymes. In addition, the diversity comparative analysis of the CAZymes in the genome
of T. asperellum ND-1 and other fungi were achieved to provide novel insights into the
biomass-decomposing enzymatic system of this fungus. 2. Results and Discussion Genome features of T. asperellum ND-1. Whole-genome sequence of T. asperellum
ND-1 was determined using PacBio and Illumina Hiseq × 10 platforms. De novo assembly
using SOAPdenovo (version 2.04) and CANU (version 1.7) resulted in 32 scaffolds with
N50 size of 2,032,888 bp (each with a length >1000 bp and N90 value of 792,243 bp). The
largest scaffold size was 3.7 Mb. The sum of the assembly length was 35.75 Mb, with a
coverage of 99.3% (Table S1). Protein-coding genes, using the MAKER annotation method, Catalysts 2022, 12, 437 3 of 19 yielded 10,541 genes for T. asperellum ND-1 and 12,802 genes for T. asperellum CBS 433.97,
respectively, both greater than the estimate for T. asperellum IC-1 (8803) (Table S1). p
y
g
p
The average gene density in the T. asperellum ND-1 genome was 290 genes per Mb. The average gene length was 1.86 (kb) and consisted of an average of 0.52 (kb) of the
coding region and 0.16 (kb) of the non-coding region (Table 1), which were similar to other
Trichoderma fungi [43]. The overall G+C content of the predicted genes was approximately
52.8%. tRNAScan-SE [50,51] identified a sum of 246 tRNAs containing 21 types of tRNAs
in the genome (Table 1). Gene Ontology (GO) mapping was performed to detect GO terms
for BLASTP functionally analyzed ORFs. BLASTP generated 6669 genes according to GO
(Figure 1). Among them, 5092 genes belonged to molecular function (MF) (Figure 1A),
4325 genes were assigned to cellular component (CC) (Figure 1B), and 4903 genes were dis-
tributed into the biological process (BP) (Figure 1C). The major GO terms were constructed
by the following groups: metabolic process (56.6%), catalytic activity (50.8%), cellular
process (45.4%), binding (42.2%), cell (41.4%), cell part (41.2%), single-organism process
(33.8%), organelle (33.7%), membrane (33.2%), and membrane part (30.5%). The genome
size (35.75 Mb), total number of predicted genes (10, 541), and % (G+C) contents (48.65%)
(Table 1) of T. asperellum ND-1 are comparable to the hypocreales mesophilic ascomycete
fungus T. reesei (33.9 Mb) [44]. In addition, PFAM domains (7281) and proteases (82) of T. asperellum ND-1 genome were identified. Table 1. Genome assembly and annotation statistics of T. asperellum ND-1. Featuers
Trichoderma asperellum ND-1
Coverage
99.3%
Protein length, amino acids
516.18
Avg. Gene Density (genes/kb)
0.29
Avg. 2. Results and Discussion Gene length (bp)
1.86 kb
Repeat Content %
1.66
tRNAs
246
Secreted Proteins
895
PHI genes
2340
Proteases
82
Average exons per gene
2.98
Average exon length (bp)
0.52 kb
Average introns per gene
1.98
Average intron length (bp)
0.16 kb
Supported by homology, Swissprot
6746 (64%)
Supported by homology, NR
9496 (90%)
Has PFAM domain
7281 (69%) Table 1. Genome assembly and annotation statistics of T. asperellum ND-1. Mobile elements. Repetitive DNA elements and transposable elements (TEs) play
critical roles related to the gene functions, the evolution, and genome structure of the
filamentous fungi [52]. The repeated sequences of the T. asperellum ND-1 genome were
identified to be approximately 591,590 bp, including simple repeats, low complexity, small
RNA, interspersed repeats, and satellites (Table S2). The repeated sequences represent
1.66% of the genome. Moreover, 78% of the TEs were simple repeats, whereas LINEs was
just estimated to be 2% (Figure S1, Supplementary Materials). Notably, low complexity and
small RNA account for 16 and 4%, respectively. Mobile elements. Repetitive DNA elements and transposable elements (TEs) play
critical roles related to the gene functions, the evolution, and genome structure of the
filamentous fungi [52]. The repeated sequences of the T. asperellum ND-1 genome were
identified to be approximately 591,590 bp, including simple repeats, low complexity, small
RNA, interspersed repeats, and satellites (Table S2). The repeated sequences represent
1.66% of the genome. Moreover, 78% of the TEs were simple repeats, whereas LINEs was
just estimated to be 2% (Figure S1, Supplementary Materials). Notably, low complexity and
small RNA account for 16 and 4%, respectively. 4 of 19 Catalysts 2022, 12, 437 Figure 1. Gene ontology (GO)-based functional annotation of genes present in the T. asperellum ND-1
genome. (A) Molecular function domain (MF); (B) cellular process domains (CC); (C) biological
process domains (BP). Figure 1. Gene ontology (GO)-based functional annotation of genes present in the T. asperellum ND-1
genome. (A) Molecular function domain (MF); (B) cellular process domains (CC); (C) biological Figure 1. Gene ontology (GO)-based functional annotation of genes present in the T. asperellum ND-1
genome. (A) Molecular function domain (MF); (B) cellular process domains (CC); (C) biological
process domains (BP). Prediction and analysis of the T. asperellum ND-1 secretome. The secretory pro-
teome is believed to have an important role in identifying the capacity of the fungi to
interact with distinct nature environment [38]. 2. Results and Discussion According to online software SignalP
(version 4.1), the total number of 895 (represent 8.5% of the protein-coding genes) secreted
proteins were predicted and annotated in T. asperellum ND-1 genome, which was higher
than that of T. reesei Rut C30 (636 proteins) [53]. From this, GO terms were identified into
529 putative secreted proteins in the GO groups, namely, biological process (730), molecular
function (561), and cellular component (675) (Figure S2). In the cellular component group, Catalysts 2022, 12, 437 5 of 19 secretory proteins for membrane and membrane part, cell and cell part, organelle and
organelle part, extracellular region, and macromolecular complex were highly abundant. Within the biological process, including the metabolic process, cellular process, localiza-
tion, single-organism process, biogenesis or cellular component organization, biological
regulation, and regulation of biological process, responses to the stimulus were highly
represented. Under molecular function category, proteins related to binding, nucleic acid
binding transcription factor activity, transporter activity, catalytic activity, electron carrier
activity, and antioxidant activity were most abundant. As for potential pathogenesis-related proteins of T. asperellum ND-1 secretome, 175 secreted
proteins identified within the PHI database were assigned to various categories. Among
them, 81 (41%) proteins were associated with reduced virulence, (76) 38% proteins were
of unaffected pathogenicity, (18) 9% proteins were related to increased virulence (hyper-
virulence), and (18) 9% were related to loss of pathogenicity (Figure 2). Cytochrome P450
(CYP450) monooxygenase superfamily is involved in numerous metabolisms of the fila-
mentous fungi, including secondary metabolites, lifestyle, and pathogenicity [54–56]. In
T. asperellum ND-1, 163 CYP proteins were confirmed, of which 99 showed homologous
counterparts in the PHI database. Figure 2. Summary of different phenotypic categories of orthologs of T. asperellum ND-1 secretome
genes in the pathogen-host interactions (PHI-base) database. Figure 2. Summary of different phenotypic categories of orthologs of T. asperellum ND-1 secretome
genes in the pathogen-host interactions (PHI-base) database. Figure 2. Summary of different phenotypic categories of orthologs of T. asperellum ND-1 secretome
genes in the pathogen-host interactions (PHI-base) database. A large number of extracellular enzymes secreted from T. asperellum have been recog-
nized, many of which are involved in the degradation of complex biomass carbohydrates
in various environments [38,41]. 2. Results and Discussion Using the CAZy database and carrying out a HMMER
(version 3.3) scan, according to the profile compound in dbCAN release 2.0, we identi-
fied the presence of 67% GHs, 12% auxiliary activities (AAs), 10% carbohydrate esterases
(CEs), 6% glycosyl transferases (GTs), 3% polysaccharide lyases (PLs), and 2% carbohy-
drate binding modules (CBMs) in T. asperellum ND-1 secretome (Figure 3A). LPMOs are
the monocopper enzymes widely distributed in nature that catalyze the hydroxylation of
glycosidic bonds in most abundant available polysaccharide in nature, i.e., cellulose [57,58]. Secretomic analysis revealed that T. asperellum ND-1 encode two predicted LPMOs from
AA9 and AA11, respectively. Moreover, the AA9 family could have important roles as
copper dependent LPMOs, cleaving oxidatively biomass cellulose [38]. Additionally, this
work contributes to the broader mapping of enzyme activity in the Auxiliary Activity
family (particularly AA9, AA11, and AA14) and provides new biocatalysts for potential
applications in biomass modification. The analysis of the CAZy categories was performed for the biomass hydrolysis en-
zymes families. Results showed that 141 genes encoding glycosyl hydrolases enzymes were
divided into 49 families. The GH families possessing three or more genes had 18 in T. as-
perellum ND-1 secretome, with GH18 being the largest family (17 genes), followed by GH16
(11 genes), GH55 (8 genes), GH3 (7 genes), GH92 (7 genes), and GH5 (6 genes) (Figure 3F). PL7 (3 genes), PL20 (2 genes), PL1 (1 genes), and PL8 (1 genes) of the PL families were also
identified (Figure 3C). A previous study reported that members of the Trichoderma fungi
(particularly T. atroviride and T. harzianum) are widely utilized as agricultural biocontrol
agents [59,60], and both secondary metabolites and GH18 (chitinases) could play critical
roles in growth and attacking pathogens [61]. Additionally, out of seven CEs families Catalysts 2022, 12, 437 6 of 19 confirmed, members of family CE10 contained the maximum genes [7], followed by CE5
(4 genes), CE8 (3 genes), CE4 (2 genes), CE3 (2 genes), and CE1 (1 gene) (Figure 3E). The
enzymatic activities of carboxylesterases were displayed in both CE10 and CE1 families [62]. Further, the enzymes providing auxiliary functions for degradation of polysaccharides were
represented by four families of carbohydrate binding modules (CBM6, CBM24, CBM42,
CBM66), glycosyl transferases (9 families) (Figure 3B), and auxiliary activities (9 families)
(Figure 3D). Among them, the numbers of CBM42, AA7, and GT90/GT22/GT15 genes were
significantly higher. Moreover, the secretory proteins of T. 2. Results and Discussion asperellum ND-1 also contained
an assortment of proteases, transferases, and chitinases. These results imply that T. asperel-
lum ND-1 secretome consists of various functional proteins and the major components
associated with proteolytic and cellulolytic enzymes, which are crucial for promoting the
hydrolysis of the host plant to obtain essential nutrients and adapt various environments. Figure 3. CAZymes identified in the secretome of T. asperellum ND-1. (A) Summary of the six CAZyme
categories: auxiliary activities (AAs), carbohydrate- binding modules (CBMs), polysaccharide lyases
(PLs), glycoside hydrolases (GHs), glycosyl transferases (GTs), and carbohydrate esterases (CEs). (B) Distinct summaries of the CAZyme GTs. (C) Distinct summaries of the CAZyme PLs. (D) Distinct
summaries of the CAZyme AAs. (E) Distinct summaries of the CAZyme CEs. (F) Distinct summaries
of the CAZyme GHs. Figure 3. CAZymes identified in the secretome of T. asperellum ND-1. (A) Summary of the six CAZyme
categories: auxiliary activities (AAs), carbohydrate- binding modules (CBMs), polysaccharide lyases
(PLs), glycoside hydrolases (GHs), glycosyl transferases (GTs), and carbohydrate esterases (CEs). (B) Distinct summaries of the CAZyme GTs. (C) Distinct summaries of the CAZyme PLs. (D) Distinct
summaries of the CAZyme AAs. (E) Distinct summaries of the CAZyme CEs. (F) Distinct summaries
of the CAZyme GHs. Phylogenetic relationships. The evolutionary relationships of T. asperellum ND-1 and
other selected fungi species were evaluated using the proteomes of these fungi. Accord-
ing to phylogenetic analysis results, all the selected Trichoderma were distributed into a
single primary cluster (Figure 4). Majority of Trichoderma species are commonly applied
in agriculture as effective agents for biological control against many phytopathogenic
microorganisms; examples are T. asperellum T203 [63], T. harzianum [59], and T. asperellum
SKT-1 [64]. The isolated lignocellulolytic fungus T. asperellum ND-1 is evolutionally close
to T. asperellum CBS 433.97 (Figure 4). T. asperellum ND-1 is also close to the other two
biological species, T. atroviride IMI 206040 and T. gamsii T6085, suggesting that T. asperellum
ND-1 may have biocontrol functions applied in agriculture. Moreover, T. asperellum ND-1
and T. reesei QM6a (a representational producer of plant biomass degrading enzymes) were
distributed into different subclusters (Figure 4). In addition, all the Asperellus species were
grouped into another single clade that is distantly related to T. asperellum ND-1. Grifola
frondosa 9006-11 was served as an outgroup in the phylogenomic analysis. 7 of 19 7 of 19 Catalysts 2022, 12, 437 Figure 4. Whole-genome phylogenetic analysis of T. asperellum ND-1 sequences. 2. Results and Discussion The tree of selected
genome sequences was constructed by using the neighbor-joining (NJ) method with the Poisson
model, as implemented in Mega software 7.0. T. asperellum ND-1 was labeled with diamonds. Figure 4. Whole-genome phylogenetic analysis of T. asperellum ND-1 sequences. The tree of selected
genome sequences was constructed by using the neighbor-joining (NJ) method with the Poisson
model, as implemented in Mega software 7.0. T. asperellum ND-1 was labeled with diamonds. Comparative analysis of orthologous genes between different Trichoderma species. The annotated proteome of T. asperellum ND-1 was further compared with the other three
biological species, T. reesei QM6a, T. virens Gv-298, and T. atroviride IMI 206040, by using
orthoMCL [65]. Among the four Trichoderma species, a total of 7073 common clusters
were identified (Figure 5). The common clusters accounted for 60–80% of the four fungal
proteomes, respectively, which indicated that the vast majority of the genes were conserved
in the Trichoderma group. However, T. asperellum ND-1, T. atroviride IMI 206040, and T. virens Gv-298 contained about 1381, 1618, and 1991 species-specific clusters, respectively,
but the T. reesei QM6a had only 520 unique clusters (Figure 5), consistent with a previous
study, showing that T. reesei contained fewer exclusive orthologous genes than other
sequenced fungus [43,44]. Moreover, T. virens and T. atroviride are probably the most
popular investigated biocontrol agents utilized in various agriculture fields [60]. In this
study, we found that T. asperellum ND-1, T. atroviride IMI 206040, and T. virens Gv-298
shared 801 orthologues that were not detected in T. reesei QM6a (Figure 5), which may be
partial factors that resulted in a T. asperellum ND-1 biological control function [63,64]. In
addition, we identified that 7250 orthologous genes were present between T. asperellum
ND-1 and T. reesei QM6a (Figure 5), indicating that T. asperellum ND-1 may have strong
biomass degradation ability [38,41]. A number of 7889, 8179, and 8957 common clusters
were also predicted between Trichoderma species (Figure 5) when comparing T. reesei QM6a
vs. T. atroviride IMI 206040, T. reesei QM6a vs. T. virens Gv-298, and T. atroviride IMI 206040
vs. T. virens Gv-298, respectively. Diversity of carbohydrate-active enzymes in T. asperellum ND-1 and other fungi. The components of lignocellulosic biomass contain structural polysaccharides, e.g., cel-
lulose, xylan, and mannan [6,13,16]. CAZymes that hydrolyzed the poly- and oligosac-
charides play a critical role in the biology of filamentous fungi [38,40]. The T. 2. Results and Discussion asperellum
ND-1 genome harbored 438 genes encoding for CAZymes, the members of which were
identified with the presence of 83 candidate GTs from 29 families, 59 AAs from 11 families,
41 CEs from 8 families, and 14 CBMs from 9 families, in addition to 7 PLs from 4 families
(Figure S3). The largest group of CAZymes were GHs (234 genes), which were categorized
into 57 various families. The size of the GHs in T. asperellum ND-1 was close to T. atroviride
IMI 206040 (242 genes) and T. virens Gv-298 (250 genes) (Table S3). Catalysts 2022, 12, 437 8 of 19 Figure 5. Distribution of orthologues of T. atroviride IMI 206040, T. virens Gv-298, T. reesei QM6a, and
T. asperellum ND-1. Figure 5. Distribution of orthologues of T. atroviride IMI 206040, T. virens Gv-298, T. reesei QM6a, and
T. asperellum ND-1. To further evaluated biomass-degrading abilities of T. asperellum ND-1, the diver-
sity of CAZymes in T. asperellum ND-1 was compared with other fungi (particularly T. reesei and T. asperellum CBS 433.97) (Figure 6). The number of CAZyme-encoding genes
and GH class distribution among the five Trichoderma species were remarkably different
(Figure 6). T. reesei QM6a, a well-known biomass polysaccharide degrader, possesses a
variety of genes encoding GHs [44]. However, with a total of 189 GH encoding genes,
it has fewer GHs than the T. asperellum ND-1 (234 genes) (Table S3). Analysis of the
T. asperellum ND-1 for CAZymes predicted various proteins involved in xylan degrada-
tion. For example, three endo-1,4(3)-β-xylanases (representing families GH10) and four
endo-1,4(3)-β-xylanases (GH11) were identified for T. asperellum ND-1, as opposed to
one endo-1,4(3)-β-xylanase (GH10) and three endo-1,4(3)-β-xylanases (GH11) in T. reesei
QM6a (Figure 6). T. asperellum CBS 433.97 also contained two endo-1,4(3)-β-xylanases
(representing families GH10) and four endo-1,4(3)-β-xylanases (GH11) (Figure 6). Xy-
lanase adding to cellulase mixtures has significant improvement for complete degradation
of lignocellulosic biomass, on account of enhancing the cellulase’s accessibility to cellu-
lose [17,19,66]. However, the component of xylan in lignocellulosic polymers has a back-
bone of xylose units, which can be connected with various residues [20]. Complete degra-
dation of heteroxylan needs a battery of side-chain-degrading enzymes [67], including GH2
(β-glucuronidase or β-mannanase), GH5 (endo-β-1,4-mannase), GH27 (α-galactosidase),
GH28 (α-L-rhamnosidase), GH62 (α-L-arabinofuranosidase), GH67 (α-glucuronidase),
GH76 (α-1,6-mannanase or α-glucosidase), GH78 (α-L-rhamnosidase), GH92 (α-1,2(3)-
mannosidase), GH125 (exo-α-1,6-mannosidase), and GH154 (β-glucuronidase), which
were predicted in all five Trichoderma species (Figure 6). 2. Results and Discussion Catalysts 2022, 12, 437 10 of 19 10 of 19 Degradation of complex cellulose polysaccharides depends on synergistic actions of
three representative cellulases: β(α)-glucosidases, exo-1,4-β-glucanases, and endo-1,4-β-
glucanases [69,70]. A much higher number of enzymes associated with cellulose degrada-
tion were identified in the genome of T. virens Gv-298, T. asperellum ND-1, and T. atroviride
IMI 206040 (54, 53, and 50 proteins, respectively), compared with that of T. reesei QM6a and
T. asperellum CBS 433.97 (Figure 6). Three endoglucanases (GH12), five β(α)-glucosidases
(GH17 and GH63), and three exo-1,4-β-glucanases (GH6/7) were predicted in the T. asperel-
lum ND-1 genome, and three endoglucanases (GH12), four β(α)-glucosidases (GH17 and
GH63), and three exo-1,4-β-glucanases (GH6/7) were identified in the genome of T. atro-
viride IMI 206040 (Figure 6). Particularly in T. virens Gv-298, 17 β-glucosidases (GH3), four
endoglucanases (GH12), and three exo-1,4-β-glucanases (GH6/7) were the most abundant
hydrolases (Figure 6), compared to the other described fungi. Moreover, 17 endo-1,3(4)-
β-glucanase (GH16) and four endo-1,3-β(α)-glucosidase (GH17) were identified in the
genome of T. asperellum CBS 433.97. In contrast, detected cellulolytic enzymes of T. reesei
QM6a contained only two endoglucanases (GH12), two β(α)-glucosidases (GH17 and
GH63), and three exo-1,4-β-glucanases (GH6/7) (Figure 6), consistent with a previous
study, which showed that the genome of T. reesei contained fewer cellulase-related genes
than other Trichderma fungi [38,43,44]. In addition, the number of CAZyme-encoding
genes and GH class distribution among the different Trichoderma species were remarkably
different. T. reesei Rut-C30 had at least two exo-1,4-β-glucanases (Cel6a: GH6 and Cel7a:
GH7), four endo-1,4-β- glucanases (Cel5a: GH5, Cel7b: GH7, Cel12b: GH12, and Cel45a:
GH45), and one β-glucosidase (Cel3a: GH3) [71]. In recent years, the characteristics of
11 β-glucosidases (two GH1 enzymes and nine GH3 enzymes) has been analyzed [68,72]. Moreover, a high number of GH3, GH16, and GH128 enzymes suggested that T. asperellum
ND-1 has a larger substrate range, which can be applied for various applications, including
biomass conversion and biofuel production. All five Trichoderma species also produced a large series of enzymes, the majority
of which were known to be associated with chitin degradation. For example, the GH18
family, containing various enzymes linked to chitin hydrolysis [61], was remarkably ex-
panded in the genomes of T. virens Gv-298, T. atroviride IMI 206040, T. asperellum CBS
433.97, and T. asperellum ND-1 (32, 27, 27, and 26 genes, respectively), relative to T. ree-
sei QM6a (19 genes) (Figure 6). 2. Results and Discussion In addition, GH1 was mainly
composed of β-glucosidase and β-galactosidase, and the two GH1 enzymes of T. reesei
QM9414 were β-glucosidase [68], which was similar with the present study. In T. asperel-
lum ND-1 and T. asperellum CBS 433.97, α-mannosidases, α-L-arabinofuranosidases, and
β-xylosidases were identified in higher abundances (Figure 6). For example, the genome
of T. asperellum ND-1 and T. asperellum CBS 433.97 contained eight genes encoding GH31
enzymes with α-mannosidase or α-xylosidase activities (Figure 6). A number of GH43
(β-xylosidase or α-L-arabinofuranosidase) were also remarkably expanded in T. asperellum
CBS 433.97, T. asperellum ND-1, and T. atroviride IMI 206040. Enzymes from other GH Catalysts 2022, 12, 437 9 of 19 families, such as GH32 (arabinosidase), GH93 (exo-α-L-1,5-arabinanase), and GH114 (endo-
α-1,4-polygalactosaminidase), were detected in the genome of T. asperellum CBS 433.97, T. atroviride IMI 206040, T. virens Gv-298, and T. asperellum ND-1 (alongside T. reesei QM6a)
(Figure 6). The diversity of hemicellulases in T. asperellum ND-1 was much larger in some
respects than other biomass-degrading fungi (especially T. reesei), which were similar to
previous studies [38,44]. Moreover, hydrolases from GH51 (α-L-arabinofuranosidase or β-
xylosidase), GH127 (β-L-arabinofuranosidase), and GH142 (β-L-arabinofuranosidase) were
predicted in the T. asperellum CBS 433.97, T. asperellum ND-1, and T. atroviride IMI 206040
genome only (Figure 6). In addition, the genome of T. asperellum CBS 433.97 contained
nine genes encoding GH27 enzymes with α-galactosidase activities, which was three-fold
higher than that of T. asperellum ND-1 (Figure 6). These findings, taken together, revealed
that T. asperellum ND-1 generated more diversity of CAZymes relevant to hemicellulose
hydrolysis than those of T. reesei QM6a. Figure 6. Heat map showing the distribution of glycoside hydrolases (GHs) in the genomes of T. atroviride IMI 206040 T virens Gv-298 T reesei QM6a T asperellum ND-1 and T asperellum CBS 433 97 Figure 6. Heat map showing the distribution of glycoside hydrolases (GHs) in the genomes of T. atroviride IMI 206040, T. virens Gv-298, T. reesei QM6a, T. asperellum ND-1, and T. asperellum CBS 433.97. CAZymes families are grouped according to their activities in major components of plant cell walls. Figure 6. Heat map showing the distribution of glycoside hydrolases (GHs) in the genomes of T. atroviride IMI 206040, T. virens Gv-298, T. reesei QM6a, T. asperellum ND-1, and T. asperellum CBS 433.97. CAZymes families are grouped according to their activities in major components of plant cell walls. 2. Results and Discussion The component of fungal cell walls was comprised of
substantial chitin and chitinolytic enzymes and was therefore an indispensable part of
mycoparasitic attack [48]. Moreover, hydrolases from GH75 (chitosanases) and GH18
(endo-β-N-acetylglucosaminidases) also play a critical role in the degradation of fungal cell
walls [43,61]. The most abundant of all glycoside hydrolases in T. asperellum ND-1 genome
was GH18 comprised of 26 chitinolytic enzymes (Figure 6), which is consistent with a
previous study [73]. Therefore, the T. asperellum ND-1 may be served as an effective and
environmentally friendly bio-control agent, similar to T. virens Gv-298 and T. atroviride IMI
206040, against numerous phytopathogenic microorganisms [43]. In addition, identified
amylolytic enzymes of T. asperellum ND-1 comprised six α-amylase (GH13), and three
glucoamylase (GH15) were detected (Figure 6). Consequently, T. asperellum ND-1 could
have great application potentials in the production of value-added biomolecules maltose
from α-glucan like starch. Comparative analysis of lignocellulolytic enzyme activities. Efficiently catalytic
degradation of lignocellulose is dependent on the synergistic action of various enzymes
that hydrolyze lignocellulolytic biomass into fermentable sugars [17,19,20]. The present
results show that T. asperellum ND-1 and other filamentous fungi displayed different time
course profiles of lignocellulase activities (Figure 7). 11 of 19 Catalysts 2022, 12, 437 Figure 7. Comparison of lignocellulolytic enzyme activities produced by T. asperellum ND-1 and other
fungi. The activities of hemicellulases (endoxylanase, β-xylosidase, and α-L- arabinofuranosidase)
are shown in (A–C), respectively. The activities of cellulases (endoglucanase, cellobiohydrolase, and
β-glucosidase) are shown in (D–F), respectively. Figure 7. Comparison of lignocellulolytic enzyme activities produced by T. asperellum ND-1 and other
fungi. The activities of hemicellulases (endoxylanase, β-xylosidase, and α-L- arabinofuranosidase)
are shown in (A–C), respectively. The activities of cellulases (endoglucanase, cellobiohydrolase, and
β-glucosidase) are shown in (D–F), respectively. Figure 7. Comparison of lignocellulolytic enzyme activities produced by T. asperellum ND-1 and other
fungi. The activities of hemicellulases (endoxylanase, β-xylosidase, and α-L- arabinofuranosidase)
are shown in (A–C), respectively. The activities of cellulases (endoglucanase, cellobiohydrolase, and
β-glucosidase) are shown in (D–F), respectively. Among hemicellulases, xylanase activity in T. asperellum ND-1 extract improved
sharply over time and obtained the highest level of 173.25 ± 3.14 U/mL after 5 days
of cultivation (Figure 7A). Xylanases produced by T. asperellum ND-1 were identified
to efficiently hydrolyze xylan into major product xylobiose [11]. For P. decumbens, the
corresponding activity increased slowly to reach the maximum value (80.83 ± 4.55 U/mL)
on day 3 (Figure 7A). 2. Results and Discussion Xylanase activity produced by T. reesei increased gradually until the
end of the cultivation and a maximum of 68.84 ± 2.98 U/mL was achieved on day 6, while
it was much lower in G. frondosa, F. solani, A. tamarii, A. niger ND-1, and M. thermophila
extract (Figure 7A). β-xylosidase activity of T. asperellum ND-1 increased over time, and a
peak (0.54 ± 0.08 U/mL) displayed on day 3 (Figure 7B). For A. niger ND-1 and P. decumbens,
the activity was up to a maximum (0.43 ± 0.003 U/mL and 0.31 ± 0.02 U/mL, respectively)
after 4 days (Figure 7B) and then remained relative stable. In contrast, the enzyme activity
of T. reesei, M. thermophila, and F. solani fluctuated at a low level between 0.046 ± 0.003 and
0.12 ± 0.02 U/mL during the cultivation time (Figure 7B). A minimal β-xylosidase activity
was observed in the G. frondosa and A. tamarii extract (Figure 7B). In addition, reports reveal Catalysts 2022, 12, 437 12 of 19 12 of 19 that side-chain-degrading enzymes play a crucial role in the degradation of biomass [17]. α-L-arabinofuranosidase activities were found and showed the maximum level on day 5 in
all selected fungi. In T. asperellum ND-1, the enzyme activity reached a maximum value of
1.58 ± 0.06 U/mL (Figure 7C), with a 5.5-fold, 4-fold, and 1.3-fold higher level, respectively,
compared with that of M. thermophila, T. reesei, and P. decumbens. These results, taken
together, reveal that T. asperellum ND-1 produced various hemicellulases with significantly
higher activities, which can be utilized in various fields, particularly in the production of
valuable biomolecules (prebiotics, xylooligosaccharides). In terms of cellulolytic enzymes, endoglucanases from T. asperellum ND-1 displayed
the highest level (6.33 ± 0.11 U/mL) on day 5, and the activity exhibited was comparable
with that of T. reesei (8.78 ± 0.03 U/mL) (Figure 7D). The corresponding enzyme activity in P. decumbens and M. thermophila increased rapidly to the maximum value (17.71 ± 0.03 U/mL
and 11.20 ± 0.84 U/mL, respectively) on day 5 (Figure 7D), but it was too low in A. niger ND-1, A. tamarii, F. solani, and G. frondosa during the overall cultivation period. In
addition, another two major cellulase activities (cellobiohydrolase (0.23 ± 0.01 U/mL) and
β-glucosidase (0.087 ± 0.01 U/mL)), produced by T. asperellum ND-1, were much higher
than those of other selected fungi (Figure 7E,F), including A. niger ND-1, F. solani, and G. frondosa. 3. Conclusions The whole genome sequence and lignocellulases activities of the newly isolated T. asperellum ND-1 were determined for the first time. A high-quality genomic sequence of
ND-1 has an assembly size of 35.75 Mb comprising 10,541 predicted genes. Secretome anal-
ysis showed that 895 proteins were detected, with 211 proteins associated with CAZymes,
possessing remarkable potential for utilization in biomass decomposition. Comparative
genome analysis suggested that the genome of ND-1 contained many genes involved in
biological-control, which would be useful to investigate Trichoderma species as biocontrol
agents. Furthermore, the genome of ND-1 encoded a higher diversity of polysaccharide-
degrading enzymes, especially those associated with hemicellulose deconstruction. Com-
pared with T. reesei (CICC 40932), ND-1 produced higher hemicellulases (particularly
xylanase) and similar cellulases activities. These results will help us understand the unique
hydrolytic enzyme system of T. asperellum ND-1 and promote the investigation of more
efficient and cost-effective enzymes for the degradation of lignocellulosic biomass. 2. Results and Discussion The maximum exoglucanase activity (1.01 ± 0.06 U/mL) in P. decumbens extract
appeared on day 4 (Figure 7E), and the β-glucosidase secreted by A. tamatrii obtained a
maximum activity of 0.14 ± 0.01 U/mL on day 6 (Figure 7F). T. reesei was well known for
its involvement in the degradation of complex biomass carbohydrates and was used as
the main industrial producers of cellulases [34,35]. These lignocellulase activities profiles
indicated that T. asperellum ND-1 generated an enzyme mixture with enhanced cellulose
hydrolysis capability similar to that of T. reesei. Moreover, genome sequencing and analysis
of the biomass-degrading fungus T. asperellum ND-1 were performed to pave the way
for designing enhanced T. asperellum ND-1 strains toward a more rapid conversion of
lignocellulose into soluble sugars for bioenergy production. 4. Materials and Methods The purity and concentration of genomic
DNA were quantified the with NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA,
USA) and TBS-380 (Turner BioSystems Inc., Sunnyvale, CA, USA) methods, respectively. )
(
y
,
y
,
,
)
,
p
y
Sequencing and assembly. T. asperellum ND-1 genome was sequenced using a com-
bination of PacBio sequel single molecule real-time (SMRT) [42] and Illumina sequencing
platforms (MajorBio Co., Shanghai, China). DNA libraries containing ~400 bp and 10-kb
inserts were prepared. The 400-bp library was constructed according to NEXTflex™Rapid
DNA-Seq Kit, including fragmentation of genomic DNA, end repair, adaptor ligation,
and PCR amplification. The 400-bp library was used for paired-end Illumina sequenc-
ing (2 × 150 bp) by Illumina HiSeq 2000 and assembled with SOAPdenovo version 2.04
(http://soap.genomics.org.cn/, accessed on 5 April 2022). The 10-kb library was prepared
using PacBio’s standard methods. DNA fragments were purified, end-repaired, and lig-
ated with SMRTbell sequencing adapters following the manufacturer’s instruction (Pacific
Biosciences, Menlo Park, CA, USA). The 10-kb library was evaluated with 2100 Bioanalyzer
(Agilent, Santa Clara, CA, USA), sequenced by SMRT, and the sequencing results (filtered
reads: 4.92 G, sequencing depth: 123×) were assembled into contigs through CANU
(version 1.7) with default parameters [74]. Furthermore, error correction of the PacBio as-
sembly results was performed using the Illumina reads and gap filling with GAPCLOSER
version 1.12 [75]. Finally, quality assessment of genome assembly was carried out using
CEGMA (version 2.5) and BUSCO (version 3.0) softwares. (
)
(
)
Gene prediction and annotation. Genome prediction of protein-encoding sequences
(opening reading frames, ORFs) were carried out by a combination of four independent
softwares, GeneMark-ES (version 2.3a) [76], SNAP [77], MAKER (version 2.31.9) (http://
www.yandell-lab.org/software/maker.html, accessed on 5 April 2022), and Augustus
(version 2.5.5) (http://augustus.gobics.de/, accessed on 5 April 2022). The tRNAscan-SE
version 2.0 was used for tRNA prediction (50,51). Gene annotations for predicted ORFs
were carried out by various databases, including the Non-Redundant Protein database (NR)
(ftp://ftp.ncbi.nlm.nih.gov/blast/db/, accessed on 5 April 2022), Swiss-Prot database (https:
//web.expasy.org/docs/swiss-prot_guideline.html, accessed on 5 April 2022) [78], COG
(http://www.ncbi.nlm.nih.gov/COG/, accessed on 5 April 2022) [79], and KEGG database
(http://www.genome.jp/kegg/, accessed on 5 April 2022) [80] using blastP with E-values
of ≤l × 10−5. Proteins coding for proteases were classified by conducting Blastp (batch)
against the MEROPS database (http://merops.sanger.ac.uk, accessed on 5 April 2022). 4. Materials and Methods Strains, reagents, and media. The T. asperellum ND-1 (GenBank accession number
MH496612) and A. niger ND-1 (GenBank Accession number MH137707) strains were
isolated from soil samples collected in Chifeng, Inner Mongolia, China, and preserved
in the laboratory. The T. reesei (CICC 40932), Grifola frondosa (CICC 14078), Penicillium
decumbens (CICC 40674), Fusarium solani (CICC 2618), and A. tamarii (CICC 40233) were
obtained from the China Center of Industrial Culture Collection. Myceliophthora thermophila
ATCC 42464 was from the American Type Culture Collection. P-nitrophenyl (pNP)-D-β-
glucopyranoside (pNPG), pNP-L-α-arabinofuranoside (pNPAf), pNP-β-D-xylopyranoside
(pNPX), pNP-D-β-cellobiose (pNPC), and sodium carboxymethyl cellulose (CMC-Na) were Catalysts 2022, 12, 437 13 of 19 13 of 19 from Sigma-Aldrich (St. Louis, MO, USA). Beechwood xylan (BWX) was purchased from
Megazyme (Wicklow, Ireland). from Sigma-Aldrich (St. Louis, MO, USA). Beechwood xylan (BWX) was purchased from
Megazyme (Wicklow, Ireland). All fungi were precultured on potato dextrose agar (PDA) at 28 ◦C for 4 days. In total,
5 g of unpretreated, dry corn stover (milling to 2 cm), 0.2 g tryptone, 0.2 g yeast extract, and
100 mL of a basal salt solution (0.5 g/L MgSO4·7H2O, 1 g/L K2HPO4·3H2O, 2 g/L NH4Cl,
0.5 g/L KCl, 0.02 g/L FeSO4·7H2O, 0.03 g/L CaCl2, and 0.02 g/L ZnSO4·7H2O) were
added to 250 mL Erlenmeyer flasks, and the mixtures were sterilized at 121 ◦C for 30 min
used as inducing medium. For lignocellulases (cellulases, hemicellulases) activity analysis,
suspensions of T. asperellum ND-1 and other fungi were inoculated onto sterile inducing
medium at approximately 5 × 108 spores, which were cultured with agitation at 200 rpm,
28 ◦C, for 6 days. Culture samples were taken every day and centrifuged at 12,000× g for
10 min to collect the supernatant. The supernatant containing the crude enzymes was then
used directly for enzyme assays. Experiments were performed in triplicate. Genomic DNA preparation and quality assessment. The T. asperellum ND-1 (MH496612)
strain cultured on PDA medium at 28 ◦C for 2 days was inoculated in potato dextrose broth
(PDB) and incubated at 28 ◦C for 3 days, 200 rpm. Fungal biomass (3.5 g) of 500 mL was
acquired via centrifugation for 15 min, at 4000 rpm, and maintained in liquid nitrogen. Ge-
nomic DNA of T. asperellum ND-1 was isolated using the Omega Fungal DNA Kit D3390-02,
according to fungal DNA extraction protocol. 4. Materials and Methods Domain identification of predicted protein-encoding sequences were analyzed according
to the Pfam database (http://pfam.xfam.org/, accessed on 5 April 2022) [81] and HMMER
version 3.3 (http://www.hmmer.org/, accessed on 5 April 2022) [82]. The enrichment Catalysts 2022, 12, 437 14 of 19 14 of 19 analysis of gene ontology (GO) was obtained by using Blast2GO version 2.5 (https://www. blast2go.com/, accessed on 5 April 2022) [83]. Identification of transposable elements. Transposable elements (TEs) containing
various classes (LTRs (long terminal repeats), LINEs (long interspersed nuclear elements),
DNA transposons, etc.) were determined strictly using three methods. We firstly detected
the T. asperellum ND-1 with a de novo software Repeat Modeler (http://repeatmasker.org/
RepeatModeler/, accessed on 5 April 2022), then the predicted repetitive elements were
identified by BLASTP searches against the Repeat Protein Masker (www.repeatmasker.org/
cgi-bin/RepeatProteinMaskRequest, accessed on 5 April 2022) and Repeat Masker (version
4.0.7) database-based softwares (http://www.repeatmasker.org/, accessed on 5 April 2022). All the parameters were set as default. Secretome prediction and analysis. The identification of putative secreted proteins
was carried out by SignalP version 4.1 (http://www.cbs.dtu.dk/services/SignalP, ac-
cessed on 5 April 2022). The Blast2GO version 2.5 (https://www.blast2go.com/, accessed
on 5 April 2022) [83] and BLASTP analysis with E-values of ≤1 × 10−5 were used for
the functional annotations of predicted secretome, according to the terms “cellular com-
ponent”, “biological process”, and “molecular function” in the GO database. Potential
pathogenicity-related genes (related to reduced virulence, unaffected pathogenicity, lethal,
loss of pathogenicity, etc.) were analyzed by detecting against the pathogen-host interac-
tion (PHI) database (http://www.phi-base.org/, accessed on 5 April 2022) by Diamond
(version 0.8.35) with E-values of ≤1 × 10−5 [84]. Phylogenetic analyses. The evolutionary relationships of T. asperellum ND-1 and other
selected fungi species were evaluated using the proteomes of these fungi. Protein sequence
alignment was performed using ClustalW software [85], and the phylogenetic tree was
constructed by MEGA version 7.0 [86] with the UPGMA method. In addition to T. asperellum
ND-1, the proteomes of other selected fungi available on DOE Joint Genome Institute [87]
were contained: T. asperellum CBS 433.97 (GenBank assembly accession GCA_003025105.1), T. reesei QM6a (GCF_000167675.1), T. virens Gv-298 (GCF_000170995.1), T. harzianum CBS 226.95
(GCA_003025095.1), T. longibrachiatum ATCC 18648 (GCA_003025155.1), T. atroviride IMI
206040 (GCF_000171015.1), T. guizhouense NJAU 4742 (GCA_002022785.1), T. gamsii T6085
(GCF_001481775.2), T. parareesei CBS 125925 (GCA_001050175.1), T. citrinoviride TUCIM 6016
(GCA_00302 5115.1), A. niger CBS 513.88 (GCF_000002855.3), A. oryzae 3.042 (GCA_000269785.2),
A. terreus NIH2624 (GCF_000149615.1), A. 4. Materials and Methods The
reaction system (150 µL of 1.0% (w/v) substrate with 50 µL crude enzyme) was incubated
in 50 mM sodium acetate buffer (pH 5.0) for 10 min at 50 ◦C, and the reaction was stopped
by adding 50 µL of 1 M NaOH. A mixture without enzymes was used as the control. After
boiling at 100 ◦C for 5 min, the amount of reducing sugar was assayed at absorbance
540 nm, with one activity unit defined as the enzyme (endoglucanase or endoxylanase)
amount that liberated 1 µmol of reducing sugar (equivalent to glucose or xylose) per min
from CMC-Na or BWX (equivalent to glucose or xylose) per min under assay conditions. The respective standard curves were obtained with 0.1–0.7 mg/mL glucose and xylose. All
enzyme activities were performed in triplicate. Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/catal12040437/s1, Figure S1. Percentage distribution of different
types of repetitive elements in the genome of T. asperellum ND-1.; Figure S2. Functional annotation of
the T. asperellum ND-1 secretome showing top 20 hits of different category. MF, molecular function;
CC, cellular component; BP, biological process; Figure S3. Statistical analysis of CAZymes of T. asperellum ND-1 genome. Different colors of the pie chart represent different CAZy classifications,
and their areas represent the proportion of genes in the classification; Table S1. Genome features of
T. asperellum ND-1, T. asperellum IC-1 and T. asperellum CBS 433.97.; Table S2. Repetitive elements
identified in the T. asperellum ND-1 genome. Table S3. Glycoside hydrolases (GHs) identified in the
genome of T. atroviride IMI 206040, T. virens Gv-298, T. reesei QM6a and T. asperellum ND-1. Author Contributions: F.Z. designed the experiments. F.Z., T.H., and J.L. performed the experi-
ments. F.Z. and T.M. analyzed the data. F.Z. and A.B. wrote the original and revised manuscripts. W.J. supervised the present study. All authors have read and agreed to the published version of
the manuscript. Author Contributions: F.Z. designed the experiments. F.Z., T.H., and J.L. performed the experi-
ments. F.Z. and T.M. analyzed the data. F.Z. and A.B. wrote the original and revised manuscripts. W.J. supervised the present study. All authors have read and agreed to the published version of
the manuscript. Funding: This research was funded by National Natural Science Foundation of China, grant
number 31570067. 4. Materials and Methods Reaction mixture containing 100 µL crude enzyme
and 100 µL of 5 mM pNPX, pNPC, pNPG, and pNPAf substrates were incubated in 50 mM
sodium acetate buffer (pH 5.0) at 50 ◦C for 10 min. The reaction was terminated using
100 µL sodium carbonate (1.0 M). A mixture without enzymes was used as the control. An
amount of liberated pNP was quantified by determining the absorbance at 405 nm, and
one unit was defined as the number of enzymes required to release 1 µmol pNP per min. hydrolase activities were evaluated [91]. Reaction mixture containing 100 µL crude enzyme
and 100 µL of 5 mM pNPX, pNPC, pNPG, and pNPAf substrates were incubated in 50 mM
sodium acetate buffer (pH 5.0) at 50 ◦C for 10 min. The reaction was terminated using
100 µL sodium carbonate (1.0 M). A mixture without enzymes was used as the control. An
amount of liberated pNP was quantified by determining the absorbance at 405 nm, and
one unit was defined as the number of enzymes required to release 1 µmol pNP per min. Endoxylanase and endoglucanase activities were assayed using the 3,5-dinitrosalicylic
acid (DNS) method [92], with 1% (w/v) of CMC-Na and BWX as substrate, respectively. The
reaction system (150 µL of 1.0% (w/v) substrate with 50 µL crude enzyme) was incubated
in 50 mM sodium acetate buffer (pH 5.0) for 10 min at 50 ◦C, and the reaction was stopped
by adding 50 µL of 1 M NaOH. A mixture without enzymes was used as the control. After
boiling at 100 ◦C for 5 min, the amount of reducing sugar was assayed at absorbance
540 nm, with one activity unit defined as the enzyme (endoglucanase or endoxylanase)
amount that liberated 1 µmol of reducing sugar (equivalent to glucose or xylose) per min
from CMC-Na or BWX (equivalent to glucose or xylose) per min under assay conditions. The respective standard curves were obtained with 0.1–0.7 mg/mL glucose and xylose. All
enzyme activities were performed in triplicate. y
q
µ
p
p
Endoxylanase and endoglucanase activities were assayed using the 3,5-dinitrosalicylic
acid (DNS) method [92], with 1% (w/v) of CMC-Na and BWX as substrate, respectively. 4. Materials and Methods glaucus CBS516.65 (GCF_001890805.1), A. aculea-
tus ATCC16872 (GCF_001890905.1), A. flavus NRRL3357 (GCF_00000627 5.2), G. frondosa
9006-11 (GCA_001683735.1), P. decumbens IBT 11843 (GCA_002072245.1), Thermothelomyces
thermophila 42464 (GCF_000226095.1), F. graminearum PH-1 (GCF_000240135.3), Aureobasid-
ium pullulans EXF-150 (GCA_000721785.1). The neighbor joining method with a Poisson
model was used for phylogenetic evaluation, and the reliability of branching order was
evaluated by 1000 bootstrap replications. y
p
p
Comparison analysis of orthologous gene families. In order to identify the ortholo-
gous genes of the four Trichoderma species (T. asperellum ND-1, T. reesei QM6a, T. atroviride
IMI 206040, T. virens Gv-298), we used orthoMCL for the similar pairwise matches to
confirm that the groups were orthologous in the Trichoderma genomes [65,88]. The genes
that were defined as orthologs from clusters of paralogs were subtracted, then the rest of
species-specific gene sets of the cluster group expanded because of the most recent common
ancestor (MRCA) of the four Trichoderma genomes [43]. Carbohydrate-active enzymes identification and analysis. For the detection of CAZymes,
the families of structurally related catalytic (glycosyltransferases (GTs), carbohydrate es-
terases (CEs), glycoside hydrolases (GHs), auxiliary activities (AAs), polysaccharide lyases
(PLs)), and carbohydrate-binding modules (CBMs)) in the four Trichoderma species (T. as-
perellum ND-1, T. reesei QM6a, T. atroviride IMI 206040, and T. virens Gv-298) were analyzed
exactly based on the CAZymes database (http://www.cazy.org/, accessed on 5 April 2022). A HMMER version 3.3 scan (http://www.hmmer.org/, accessed on 5 April 2022) was
performed for annotated CAZyme domain boundaries according to the dbCAN CAZyme Catalysts 2022, 12, 437 15 of 19 15 of 19 domain HMM database [89,90]. DIAMOND (http://www.diamondsearch.org/, accessed
on 5 April 2022) and Hotpep were used for fast blast hits in the CAZy database and short
conserved motifs in the Peptide Pattern Recognition (PPR) library, respectively. The Hotpep
combined with peptide patterns was performed to acquire a stand-alone software for
functional prediction and annotation of protein models corresponding to CAZymes. domain HMM database [89,90]. DIAMOND (http://www.diamondsearch.org/, accessed
on 5 April 2022) and Hotpep were used for fast blast hits in the CAZy database and short
conserved motifs in the Peptide Pattern Recognition (PPR) library, respectively. The Hotpep
combined with peptide patterns was performed to acquire a stand-alone software for
functional prediction and annotation of protein models corresponding to CAZymes. Enzyme assays. β-glucosidase, β-xylosidase, α-L-arabinofuranosidase, and cellobio-
hydrolase activities were evaluated [91]. 4. Materials and Methods Data Availability Statement: The whole genome sequence data reported in this paper has been
deposited in the Genome Warehouse [93] at the National Genomics Data Center, Beijing Institute of
Genomics (China National Center for Bioinformation), Chinese Academy of Sciences, under accession
number GWHAOQT00000000, which is publicly accessible at https://bigd.big.ac.cn/gwh, accessed
on 5 April 2022. Conflicts of Interest: The authors declare no competing interest. Conflicts of Interest: The authors declare no competing interest. 3.
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Postcollisional cooling history of the Eastern and Southern Alps and its linkage to Adria indentation
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International journal of earth sciences
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Postcollisional cooling history of the Eastern and Southern Alps
and its linkage to Adria indentation Bianca Heberer1 · Rebecca Lee Reverman2,3 · Maria Giuditta Fellin4 ·
Franz Neubauer1 · István Dunkl5 · Massimiliano Zattin6 · Diane Seward7 ·
Johann Genser1 · Peter Brack2 Received: 20 February 2016 / Accepted: 28 June 2016 / Published online: 13 July 2016
© The Author(s) 2016. This article is published with open access at Springerlink.com Apatite (U–Th–Sm)/He ages from the Karawanken Moun-
tains range between 12 and 5 Ma and indicate an episode of
fault-related exhumation leading to the formation of a posi-
tive flower structure and an associated peripheral foreland
basin. In the Southern Alps, apatite (U–Th–Sm)/He and fis-
sion-track data combined with previous data also indicate
a pulse of mainly Late Miocene exhumation, which was
maximized along thrust systems, with highly differential
amounts of displacement along individual structures. Our
data contribute to mounting evidence for widespread Late
Miocene tectonic activity, which followed a phase of major
exhumation during strain localization in the Tauern Win-
dow. We attribute this exhumational phase and more dis-
tributed deformation during Adriatic indentation to a major
change in boundary conditions operating on the orogen,
likely due to a shift from a decoupled to a coupled system,
possibly enhanced by a shift in convergence direction. Abstract Indentation of rigid blocks into rheologically
weak orogens is generally associated with spatiotempo-
rally variable vertical and lateral block extrusion. The
European Eastern and Southern Alps are a prime example
of microplate indentation, where most of the deformation
was accommodated north of the crustal indenter within the
Tauern Window. However, outside of this window only the
broad late-stage exhumation pattern of the indented units
as well as of the indenter itself is known. In this study we
refine the exhumational pattern with new (U–Th–Sm)/He
and fission-track thermochronology data on apatite from
the Karawanken Mountains adjacent to the eastern Peri-
adriatic fault and from the central-eastern Southern Alps. Electronic supplementary material The online version of this
article (doi:10.1007/s00531-016-1367-3) contains supplementary
material, which is available to authorized users. Keywords Southern and Eastern Alps · Low-temperature
thermochronology · Adria indentation · Exhumation * Bianca Heberer
bianca.heberer@sbg.ac.at * Bianca Heberer
bianca.heberer@sbg.ac.at 1
Department of Geography and Geology, University
of Salzburg, Hellbrunner Str. 34, 5020 Salzburg, Austria 1
Department of Geography and Geology, University
of Salzburg, Hellbrunner Str. 34, 5020 Salzburg, Austria Int J Earth Sci (Geol Rundsch) (2017) 106:1557–1580
DOI 10.1007/s00531-016-1367-3 ORIGINAL PAPER ORIGINAL PAPER Introduction PO - Pohorje
TW - Tauern Window
WTD - W Tauern dome
Faults
LB
RA
TW
GÖ
PO
KB
TO
MeM
15°
12°
48°
46°
12° E
15° E
46° N LA
ID
Adriatic
indenter
Molasse basin
KA
SV
Styrian
basin
Sava folds
MM
VAL
KF
BF
SEMP
FE-SA
GIU
Medvenica
Fig. 3
Fig. 2a
Po basin
GB
AD
VT
PAF
BAS-VAL - Bassano
Valdobiadene t. BF - Brenner f. CAN-MAN - Cansiglio-
Maniagio f. FE-SA - Fella-Sava f. GIU - Giudicarie f. GÖ - Görtschitz f. HF - Hochstuhl f. ID - Idrija f. KF - Katschberg f. LA - Lavant / Labot f. MOE - Möll Valley f. MON - Montello--Friuli
MeM - Meran-Mauls f. MM - Mur-Mürz f. PAF - Periadriatic f. RA - Ravne f. SEMP - Salzach-Enns-
Mariazell-Puchberg f. SV - Schio Vicenza f. TO - Tonale f. VAL - Valsugana t. VT - Val Trompia t. WTD
ETD
BAS-VAL
BEL
Veneto-Friuli
basin
MON
CAN-MAN
MOE
KAR
HF
Pannonian
basin
AD - Adamello batholith
ETD - E Tauern dome
GB - Giudicarie belt
KB - Klagenfurt basin
LB - Lessini block
KAR - Karawanken Mts. PO - Pohorje
TW - Tauern Window
WTD - W Tauern dome
Faults
LB
RA
TW
GÖ
PO
KB
TO
MeM
15°
12°
48°
46°
12° E
15° E
46° N 48° S
Faults 15°
15° E Pannonian
basin Adriatic
indenter Fig. 1 DEM of the Southern Alps, Eastern Alps and northern Dinarides displaying major faults. The dark red signature within the Tauern Win-
dow denotes outcropping “Zentralgneiss” of the Tauern subdomes. Frames denote outline of Figs. 2a and 3 crustal depths via orogen-parallel extension (e.g., Behr-
mann 1988; Scharf et al. 2013) (Figs. 1, 2). Exhumation
continued until the Late Miocene leading to the formation
of large-amplitude folds and domes in the Tauern Window
(e.g., Schmid et al. 2013). South of the PAF system, lim-
ited exhumation from shallow crustal levels (Zanchetta
et al. 2015; Zattin et al. 2006) occurred, and throughout
the Oligo-Miocene most deformation was accommodated
by progressive southward thrust propagation and transpres-
sional shear along the indenter margin (Picotti et al. 1995;
Pomella and Stipp 2012; Prosser 1998; Viola et al. 2001). Introduction 2
Geological Institute, ETH Zürich, Sonneggstrasse 5,
8092 Zurich, Switzerland 2
Geological Institute, ETH Zürich, Sonneggstrasse 5,
8092 Zurich, Switzerland Late-orogenic indentation by rigid lithospheric plates
and microplates into a weaker continent leads to postcol-
lisional shortening, lithospheric thickening, vertical and
lateral extrusion and erosion (e.g., Robl and Stüwe 2005;
Tapponnier et al. 1986). The European Eastern Alps are a
prime example of microplate indentation (Ratschbacher
et al. 1991). Their Late Neogene geodynamic framework
is influenced primarily by the ca. NW-ward motion of
the Adriatic plate and its counterclockwise rotation with
respect to Europe, which resulted in an oblique, dextral
transpressional setting (e.g., Caputo and Poli 2010; Scharf 3
Department of Earth and Planetary Sciences, University
of California, Berkeley, Berkeley, CA 94720, USA 3
Department of Earth and Planetary Sciences, University
of California, Berkeley, Berkeley, CA 94720, USA 4
Institute for Geochemistry and Petrology, ETH Zürich,
Clausiusstrasse 25, 8092 Zurich, Switzerland 4
Institute for Geochemistry and Petrology, ETH Zürich,
Clausiusstrasse 25, 8092 Zurich, Switzerland 5
Geoscience Center, University of Göttingen,
Goldschmidtstrasse 3, 37077 Göttingen, Germany 6
Department of Geosciences, University of Padua,
Via G. Gradenigo 6, 35131 Padua, Italy 7
School of Geography, Environment and Earth Sciences,
Victoria University of Wellington, PO Box 600,
Wellington 6012, New Zealand 7
School of Geography, Environment and Earth Sciences,
Victoria University of Wellington, PO Box 600,
Wellington 6012, New Zealand 1 3 1 1558 Int J Earth Sci (Geol Rundsch) (2017) 106:1557–1580 LA
ID
Adriatic
indenter
Molasse basin
KA
SV
Styrian
basin
Sava folds
MM
VAL
KF
BF
SEMP
FE-SA
GIU
Medvenica
Fig. 3
Fig. 2a
Po basin
GB
AD
VT
PAF
BAS-VAL - Bassano
Valdobiadene t. BF - Brenner f. CAN-MAN - Cansiglio-
Maniagio f. FE-SA - Fella-Sava f. GIU - Giudicarie f. GÖ - Görtschitz f. HF - Hochstuhl f. ID - Idrija f. KF - Katschberg f. LA - Lavant / Labot f. MOE - Möll Valley f. MON - Montello--Friuli
MeM - Meran-Mauls f. MM - Mur-Mürz f. PAF - Periadriatic f. RA - Ravne f. SEMP - Salzach-Enns-
Mariazell-Puchberg f. SV - Schio Vicenza f. TO - Tonale f. VAL - Valsugana t. VT - Val Trompia t. WTD
ETD
BAS-VAL
BEL
Veneto-Friuli
basin
MON
CAN-MAN
MOE
KAR
HF
Pannonian
basin
AD - Adamello batholith
ETD - E Tauern dome
GB - Giudicarie belt
KB - Klagenfurt basin
LB - Lessini block
KAR - Karawanken Mts. 1 3 Introduction West of the Giudicarie fault, thermochronological data
from the Adamello complex, the largest Tertiary intrusion
of the Alps, record rapid uplift and exhumation from a shal-
low depth (~2 km) between 8 and 6 Ma (Reverman et al. 2012) (Fig. 3). Much less is known about the exhumation
pattern at the eastern and northeastern immediate front/rim
of the eastern Adriatic indenter. For the indented units north
of the PAF, in particular SE and E of the Tauern Window,
age data are sparse and only the broad exhumation pattern et al. 2013). The northern edge of the Adriatic indenter
roughly corresponds to the Periadriatic fault system (PAF,
Fig. 1), the largest, most important discontinuity and the
main structural divide of the European Alps. The PAF sys-
tem is offset by the sinistral NE–SW trending transpres-
sive Giudicarie fault, which defines the western border of
the eastern Adriatic indenter (sensu Handy et al. 2014), i.e.,
the still northward moving triangular northeastern part of
the Southalpine block that indented the Eastern Alps (also
labeled North Adriatic indenter by, e.g., Massironi et al. (2006), Southalpine indenter by, e.g., Pomella and Stipp
(2012), or Dolomites indenter by Frisch et al. (2000),
among others). The onset of indentation occurred at ca. 23–21 Ma (Pomella and Stipp 2012; Scharf et al. 2013). The bulk of Adriatic plate motion was transferred into
differential deformation north and south of the PAF sys-
tem. Exhumation and deformation north of the PAF primar-
ily occured in the axial zone of the orogen in the Tauern
Window, where between 23 and 21 Ma deep, high-pressure
units (the Penninic nappes) started to exhume to shallow 1 3 1 3 1559 Int J Earth Sci (Geol Rundsch) (2017) 106:1557–1580 is known (Hejl 1997; Staufenberg 1987; Wölfler et al. 2012) (Fig. 2a), although the Eastern Alps are tectonically
i
h
h W
d C
l Al
d
thermochronological data exist for the eastern se
of the PAF system, the first focus of this study. Th
li h d
d i
b
d
f
h
a
b
Fig. 2 a Geological map of the Eastern and Southern Alps border-
ing the easternmost PAF and the Karawanken Mts. illustrating major
faults and summarized low-temperature thermochronology data. AFT
ages were compiled from Bertrand (2013), Hejl (1997), Staufenberg
(1987), Wölfler et al. (2008, 2010, 2012). Introduction DMB Drau-Möll block, FE
Fella fault, GÖ Görtschitz fault, HA Hochalm–Ankogel subdome, HF
Hochstuhl fault, IF Isel fault, KA Katschberg fault, LA Lavant/Labot
fault, MM Mur-Mürz fault, MOE Möll Valley fault, PAF Peri
fault, PG Pusteria–Gailtal fault segment of the PAF, PO P
RB Rieserferner block, SA Sava fault, SB Sonnblick su
SEMP Salzach–Enns–Mariazell–Puchberg fault, SO Sostan
Frame denotes outline of (b). b New AHe ages with 2σ error
Karawanken Mountains. Referenced AFT age from 1Nemes (1 a Fig. 2 a Geological map of the Eastern and Southern Alps border-
ing the easternmost PAF and the Karawanken Mts. illustrating major
faults and summarized low-temperature thermochronology data. AFT
ages were compiled from Bertrand (2013), Hejl (1997), Staufenberg
(1987), Wölfler et al. (2008, 2010, 2012). DMB Drau-Möll block, FE
Fella fault, GÖ Görtschitz fault, HA Hochalm–Ankogel subdome, HF
Hochstuhl fault, IF Isel fault, KA Katschberg fault, LA Lavant/Labot fault, MM Mur-Mürz fault, MOE Möll Valley fault, PAF Periadriatic
fault, PG Pusteria–Gailtal fault segment of the PAF, PO Pohorje,
RB Rieserferner block, SA Sava fault, SB Sonnblick subdome,
SEMP Salzach–Enns–Mariazell–Puchberg fault, SO Sostanj fault. Frame denotes outline of (b). b New AHe ages with 2σ error for the
Karawanken Mountains. Referenced AFT age from 1Nemes (1996) thermochronological data exist for the eastern segment
of the PAF system, the first focus of this study. The pub-
lished age record is more abundant for the western part of
the leading edge of the eastern Adriatic indenter, but mostly
limited to FT data. Along the NW corner of the indenter
fission-track data on apatites and zircons (ZFT) portray is known (Hejl 1997; Staufenberg 1987; Wölfler et al. 2012) (Fig. 2a), although the Eastern Alps are tectonically
more active than the Western and Central Alps due to ongo-
ing indentation (e.g., Battaglia et al. 2004). Except for a
single apatite fission-track (AFT) age from the Karawanken
tonalite (Nemes 1996) (Fig. 2b), no low-temperature 1 1560 Int J Earth Sci (Geol Rundsch) (2017) 106:1557–1580 Geological background and tectonic setting of the
study areas AFT dating of Southalpine crystalline basement in the
hanging wall of a major thrust (Valsugana thrust, Fig. 3)
yielded evidence for prominent Late Miocene cooling and
exhumation (Zattin et al. 2006), but no constraints exist for
other major structural units and new low-temperature ther-
mochronological data are needed to bridge this gap. Thus,
our second focus is on the Southern Alps, from where we
present new AFT and apatite (U–Th–Sm)/He (AHe) ages
that complement and enhance previous work. In particular,
we focus on the Giudicarie belt and surrounding regions
corresponding to the western boundary zone of the eastern
Adriatic indenter. The more than 700-km-long PAF is the most outstand-
ing fault system of the Alps. It juxtaposes the N-vergent
part of the orogen to the Southalpine retrowedge and sepa-
rates terrains with distinct paleogeographic, magmatic and
metamorphic development (e.g., Laubscher 1983; Schmid
et al. 1987), which were, however, closely coupled since
the Miocene (e.g., Massironi et al. 2006). The PAF delimits
the SSE-vergent fold and thrust belt of the eastern South-
ern Alps to the north. The Giudicarie belt and the relatively
undeformed Lessini foreland block represent the western,
the NW–SE trending dextral Idria and Ravne faults (Fig. 1)
its eastern boundary. The Southalpine fold and thrust belt
east of the Adamello complex mainly developed during
polyphase Neogene contraction and inversion of the Adri-
atic passive margin (Zampieri and Massironi 2007). The
most important tectonic features of the eastern Southern
Alps are from N to S the Valsugana, Belluno, Bassano
and Montello thrust sheets, involving both basement and
Permo-to-Cenozoic cover rocks. Frontal thrusts are linked
via the NW–SE trending Schio-Vicenza and N–S trending
Trento-Cles strike-slip faults to the Giudicarie belt (Mas-
sironi et al. 2006) (Figs. 1, 3). Our goal is to capture the spatial and temporal variabil-
ity of the vertical component of extrusion associated with
microplate indentation and ongoing convergence during
the late stages of Alpine orogeny. A better understanding
of the magnitude and timing of uplift will also shed light
on the discussion on the topographic evolution of the Alps
(e.g., Hergarten et al. 2010; Robl et al. 2015) and its poten-
tial coupling to climate and deep-seated tectonic processes
beneath the Alps (e.g., Baran et al. 2014; Cederbom et al. 2004; Fox et al. 2015; Herman et al. 2013). Geological background and tectonic setting of the
study areas Fig. 3 Geological map of the Southalpine units targeted for low-
temperature thermochronology. New and published AFT and AHe
ages with errors given as 2σ are shown. Labeled published ages are
from 1Zattin et al. (2003, 2006); 2Pomella et al. (2011) and 3Rever-
man et al. (2012). #-Sample failed Chi-square test indicating sig-
nificant dispersion in ages. ZFT ages were compiled from Pomella
et al. (2011, 2012), Stipp et al. (2004), Viola (2000), Viola et al. (2001, 2003). Compiled AHe data (unlabeled) come from Reverman
et al. (2012). Geology is based on the geological map of Italy, scale
1:1,250,000 (2005) and the structural model of Italy, scale 1:500.000
(Bigi et al. 1990). BAS-VAL Bassano–Valdobiadene thrust, BC Bas-
sano–Cornuda thrust, BEL Belluno line, N GIU North Giudicarie line,
PE Pejo fault, S GIU South Giudicarie line, SV Schio–Vicenza fault,
TB Thiene–Bassano thrust, TC Trento–Cles fault, TO Tonale fault,
VAL Valsugana thrust, VT Val Trompia thrust
◂ After the Cretaceous-to-early-Paleogene subduction and
closure of the Piemontais-Ligurian (“Penninic”) ocean, the
Eocene-to-Oligocene collision of the stable European con-
tinent and the Adriatic microplate resulted in the building
of the European Alps (e.g., Handy et al. 2010). The Adri-
atic indenter, i.e., the northern promontory of the Adriatic
microplate (Southern Alps) had only experienced lower-
greenschist-facies Alpine overprint (e.g., Spalla and Gosso
1999) and acted as a strong indenter (Robl and Stüwe 2005;
Willingshofer and Cloetingh 2003). Intra-orogenic N–S
shortening was largely compensated by orogenic thicken-
ing due to nappe stacking, e.g., by thrusting along a crus-
tal-scale shear zone known as sub-Tauern ramp (Gebrande
et al. 2002; Lammerer et al. 2008), followed by large-scale
doming and exhumation (e.g., Favaro et al. 2015). In the
Eastern Alps maximum amounts of collisional shortening
occurred in the western part of the Tauern Window (Rosen-
berg et al. 2015). ZFT ages mostly range between 18 and
12 Ma in the Tauern Window (Bertrand et al. 2015 and ref-
erences therein) and are older only in its SE corner (Dunkl
et al. 2003; Staufenberg 1987). There, AFT and AHe
ages are between 23 and 7 and 15 and 5 Ma, respectively
(Foeken et al. 2007; Wölfler et al. 2012) (Fig. 2a). a complex cooling pattern with a corridor of young Mio-
cene ZFT ages (Pomella and Stipp 2012; Viola et al. 2001). Thermochronological data from within the indenter are
again scarce due to limited availability of suitable litholo-
gies. 1 3 1561 Int J Earth Sci (Geol Rundsch) (2017) 106:1557–1580 Geological background and tectonic setting of the
study areas By recombin-
ing our new age information with the published record
from the Eastern and Southern Alps and the clastic record
from the peripheral foreland basin of the Karawanken
Mountains we find a young, latest Mid-Miocene but pre-
dominantly Late Miocene exhumation pulse as a first-
order feature of the cooling pattern. This adds a second
phase of exhumation and shortening to the postcollisional
evolution of the Eastern Alps, following an initial, well-
known stage of Early to Middle Miocene stationary, large-
amplitude exhumation concentrated in the Tauern Window
(e.g., Luth and Willingshofer 2008). The spatial evolution
of exhumation through time is discussed in light of Adri-
atic indentation. The PAF system comprises various segments, i.e., from
W to E the Canavese, Tonale, Giudicarie and Pusteria–Gail-
tal faults and its extension into the Karawanken Mountains
(Fig. 1). This study targets two key areas in the vicinity of 1 3 Int J Earth Sci (Geol Rundsch) (2017) 106:1557–1580 1562 2000 m
1000 m
-1000 m
-2000 m
-3000 m
-4000 m
-5000 m
0 m
S
Southalpine Units
Austroalpine Units
Magmatic belts
North Karawanken Mts. South Karawanken Mts. Klagenfurt
Basin
N
Platform deposits
(Upper - Middle Triassic)
Lower Paleozoic basement
Sattnitz conglomerate
(Tortonian - Pliocene)
Rosenbach coal fm. (Upper Serravallian)
N Karawanken plutonic
belt (Permian - Triassic)
Sandstones, Shales
(Permian, Carboniferous)
Upper Austroalpine units
(Upper - Middle Triassic)
Karawanken tonalite
(Oligocene)
Austroalpine basement
Eisenkappel diabase
lamella (Paleozoic)
AHe samples (this study)
5 km
A
Fig. 4 S–N section across the Karawanken flower structure (modified after Nemes et al. 1997). Trace of the section is indicated in Fig. 2 2000 m
1000 m
-1000 m
-2000 m
-3000 m
-4000 m
-5000 m
0 m
S
Austroalpine Units
Magmatic belts
North Karawanken Mts. South Karawanken Mts. Klagenfurt
Basin
N
5 km
A North Karawanken Mts. N N Southalpine Units Fig. 4 S–N section across the Karawanken flower structure (modified after Nemes et al. 1997). Trace of the section is indicated in Fig. 2 oss the Karawanken flower structure (modified after Nemes et al. 1997). Trace of the section is indicated in Fig. 2 system is buried beneath the Tertiary sediments of the Pan-
nonian basin (Fodor et al. 1998). the PAF system at the leading northern and western edge of
the eastern Adriatic indenter (Fig. 1): (1) the easternmost
PAF segment within the Karawanken Mountains and (2)
the central-eastern Southern Alps. Geological background and tectonic setting of the
study areas In the following, we give
a brief overview for those major units targeted for low-tem-
perature thermochronological dating. The brittle dextral Hochstuhl–Möll Valley fault system
links the eastern PAF with the Tauern Window (Figs. 1, 2). Structural and metamorphic basement domes of Variscan
granitoids (“Zentralgneis” of the Sonnblick and Hochalm
subdomes, Figs. 1, 2) within the eastern Tauern window are
transected by the Möll Valley fault, which also transects the
eastern PAF system (Figs. 1, 2) (Kurz and Neubauer 1996;
Polinski and Eisbacher 1992; Scharf et al. 2013). During
the Early-to-Middle Miocene extrusion the Möll Valley
fault accommodated up to 25 km of displacement (Kurz
and Neubauer 1996; Scharf et al. 2013; Wölfler et al. 2008). Eastern Periadriatic fault/Karawanken Mountains 3 Pejo fault
Tonale
fault zone
Cima Presanella
N
B
S
11
11
30
25
52
11
VST4
VST5
3
8
9
7
14
18
13
20
12
11
22
8
19
7
15
7
16
23
79
AFT
ZFT
20
51
22
84
29
Sample #
Published age
Published age, projected
TL1
VS3
VS13
VS11
10
VS4
VS7
12
21
3000
m
1000
m
2000
m S Cima Presanella B Tonale
fault zone Fig. 5 N–S cross section of the Tonale fault. Published ages are from 1Viola et al. (2003); 2Stipp et al. (2004); 3Reverman et al. (2012). Cross
section modified after Dal Piaz et al. (2007). Trace of the section (B) is indicated in Fig. 3 Blanckenburg and Davies 1995), are aligned along the PAF
system. The segment of the Karawanken Mountains tar-
geted for sampling displays narrow ca. E-W trending bands
of Permotriassic Eisenkappel granite (North Karawanken
plutonic belt), Paleozoic metasedimentary rocks and duc-
tilely deformed Oligocene Karawanken tonalite in the
immediate vicinity of the PAF system (Fig. 4) (Cliff et al. 1974; Exner 1976; Miller et al. 2011; Scharbert 1975; von
Gosen 1989). Campani et al. 2010; Mahéo et al. 2013). The eastern sec-
tor, instead, runs north of the Adamello complex and sepa-
rates it from the Austroalpine units, where the alpine over-
print is confined to greenschist shear zones and cooling to
temperatures ≥300 °C is Variscan (e.g., Viola et al. 2003). In this sector only 5 km of estimated north side up vertical
displacement occurred during dextral strike-slip movement
between 32 and 30 Ma (Schmid et al. 1996). Pure dextral
strike-slip movement continued until ~20 Ma, after which
the mylonites of the eastern Tonale fault were offset by sin-
istral shearing presumably due to activity along the Giudi-
carie fault (Stipp et al. 2002). Based on paleomagnetic data, mapping, stratigraphy
and sedimentological studies, Fodor et al. (1998) provided
a detailed structural framework and kinematic sequence
for the Miocene–Pliocene evolution of the Slovenian part
of the Periadriatic fault, just east of our study area. They
differentiated an Early Miocene compression, a Karpatian
transtension and a Middle Miocene-to-Quaternary com-
pressional event. They also found a complex pattern of
block rotations in the vicinity of the PAF. Giudicarie belt The transpressive Giudicarie belt is a broad region of ESE-
vergent thrusts and N–S trending sinistral strike-slip faults,
bounded to the east by the sinistral Trento-Cles line and
to the west by the sinistral transpressive Giudicarie line
(Fig. 3), which are partially or totally the result of reacti-
vation of Permian-Mesozoic normal faults (Castellarin et al. 1993; Picotti et al. 1995; Prosser 1998). The Giudicarie
region was mainly affected by two phases of deformation
during the Neogene: a Mid-Miocene phase, typified by the
Valsugana thrust discussed below, and a younger, Late Mio-
cene–Pliocene event, possibly associated with the initiation
of the Montello–Friuli belt to the southeast (Caputo and Poli
2010; Castellarin and Cantelli 2000; Castellarin et al. 1992;
Martin et al. 1998; Massironi et al. 2006; Viola et al. 2001). Eastern Periadriatic fault/Karawanken Mountains The eastern PAF system is trisected (Fig. 2): (1) A straight
western segment coincides with the easternmost Puste-
ria–Gailtal fault. (2) The central segment is limited by the
Hochstuhl–Möll Valley fault system and Lavant (Labot)
fault which both offset the PAF system (ca. 4–6 km dis-
placement, respectively, ca. 10–15 km). A positive flower
structure straddles the PAF, which separates the distinct
North and South Karawanken units (Laubscher 1983;
Polinski and Eisbacher 1992; Tollmann 1985) (Fig. 4). (3)
In the eastern segment, east of the Lavant fault, the PAF
juxtaposes the Pohorje basement in the north and the Sava
fold area in the south. The Sava folds were deformed by
N–S shortening during Middle and Late Miocene times,
leading to cooling below 110 °C from 10 to 15 Ma (Sach-
senhofer et al. 2001). East of the Lavant fault, the PAF In the central segment, the North Karawanken unit was
overthrust onto the flexural Klagenfurt basin (Figs. 2, 4). This intra-orogenic basin comprises a more than 1000-m
clastic sequence ranging from fine-grained, coal-bearing
early Sarmatian (Serravalian) deposits (Klaus 1956; Toll-
mann 1985) to coarse-grained Pontian (Late Messinian)
deposits and Pliocene to possible Pleistocene conglom-
erates (for details see Nemes et al. 1997 and references
therein). Several large and small calc-alkaline intru-
sions and mafic dike swarms of Oligocene age, that were
emplaced due to the break-off of the European slab (von 1 3 3 Int J Earth Sci (Geol Rundsch) (2017) 106:1557–1580 1563 Pejo fault
Tonale
fault zone
Cima Presanella
N
B
S
11
11
30
25
52
11
VST4
VST5
3
8
9
7
14
18
13
20
12
11
22
8
19
7
15
7
16
23
79
AHe
AFT
ZFT
20
51
22
84
29
Sample #
Published age
Published age, projected
Age from this study
Age from this study, projected
TL1
VS3
VS13
VS11
10
VS4
VS7
12
21
3000
m
1000
m
2000
m
Fig. 5 N–S cross section of the Tonale fault. Published ages are from 1Viola et al. (2003); 2Stipp et al. (2004); 3Reverman et al. (2012). Cross
section modified after Dal Piaz et al. (2007). Trace of the section (B) is indicated in Fig. Tonale fault The Tonale fault is the central-eastern segment of the PAF
system (Figs. 3, 5). Its Cenozoic kinematic history is com-
plex and records competing effects of terminal oblique con-
vergence and rotation of the Adriatic indenter and simul-
taneous orogen-parallel extension (Mancktelow 1992). The
western sector of the Tonale fault, adjacent to the Bergell
intrusion, is primarily a greenschist-facies mylonite zone
separating the Southern Alps from the amphibolite facies
units of the Central Alps (Lepontine dome), which were
exhumed to shallow crustal levels till the Pliocene (e.g., The NNE-SSW-trending Giudicarie structural belt
is oblique to the strike of the Southern Alps (Figs. 1, 3). 1 3 Int J Earth Sci (Geol Rundsch) (2017) 106:1557–1580 1564 Along this belt there is evidence of a structural boundary at
crustal scale (Spada et al. 2013), whose surface expressions
are major differences between the sectors east and west of
the Giudicarie belt. West of the Giudicarie belt south-ver-
gent structures are sealed by the Early Oligocene Adamello
complex (Figs. 1, 3) (Brack 1981), whereas to the east
there is little pre-Adamello deformation (Bigi et al. 1990)
limited to the Eocene Dinaric deformation from the Dolo-
mites eastwards (Doglioni and Bosellini 1987). During the
Miocene the south-vergent structures, both west and east
of the Giudicarie belt, propagated into the Po Plain fore-
land. Subsequently, the deformed Miocene sediments to the
west were partially or completely eroded or buried beneath
younger sediments (Pieri and Groppi 1981) and the internal
parts of the fold and thrust belt have undergone little to no
recent deformation (D’Adda et al. 2011; Wolff et al. 2012). To the east, instead, the foreland sediments continued to
be involved in the deformation throughout the Pliocene–
Pleistocene (Venzo 1977; Massari et al. 1986). At present,
the direction of maximum horizontal compressive stress
derived from focal mechanism is roughly perpendicular to
the thrust fronts along the Giudicarie belt compatible with
its dextral strike-slip reactivation (Vigano et al. 2008). radioactive spontaneous fission of 238U. At temperatures
above ca. 110 °C tracks are annealed, whereas at tempera-
tures below 60 °C tracks are retained. The range between
these two temperatures is called the partial annealing zone
(Carlson et al. 1999; Naeser 1979). AHe geochronology is
based on the ingrowth of α-particles produced during the
decay of U, Th and Sm. Tonale fault At temperatures exceeding 80 °C
He is rapidly diffused and lost from the system, while at
temperatures below 40 °C He is quantitatively retained
(House et al. 2002; Wolf et al. 1996). The cooling rate,
radiation damage and grain size control the temperature
range for helium retention (Reiners and Farley 2001; Shus-
ter and Farley 2009). AHe ages are corrected for He loss
generated by α-ejection using a geometric correction factor
(Ft). The total analytical error was computed as the rela-
tive standard error of weighted uncertainties on U, Th, Sm
and He measurements. Ft corrections were made following
Farley (2002). The uncertainty of the age of a single-grain aliquot was
calculated by the Gaussian error propagation from the
U, Th, Sm and He measurements and from the estimated
uncertainty of the Ft. The sample average is the unweighted
arithmetic mean of the aliquot ages; the error is given as 2σ
in the text and in Figs. 2 and 3. The scatter of the single-
aliquot apparent ages derives mostly from submicroscopic
inclusions, zoning of the alpha-emitting elements and from
the differences between the sizes (diffusion domains) of the
crystals (see, e.g., Fitzgerald et al. 2006). AHe analyses of
the Austroalpine Karawanken samples were carried out in
the Thermochronology Laboratory at Geoscience Center,
University of Göttingen, Germany. The Southalpine sam-
ples were analyzed at the Noble Gas Lab, ETH Zürich. Thermochronology results Table 1 presents AHe results from the Karawanken Moun-
tains, Table 2 the new AFT data and Table 3 AHe data from
the Southern Alps. AHe ages range from 2.9 to 28.4 Ma,
with a majority of samples falling between 6 and 12 Ma. AFT ages from the Southern Alps range from 7.9 to
29.7 Ma. The low number of spontaneous tracks in our
samples prohibited the systematic measurement of confined
track lengths, necessary for a detailed assessment of the
thermal history. Below we discuss the ages in the context
of each of the main structural features associated with the
sampling. Errors are given as 2σ. The Val Trompia thrust borders the Adamello complex
to the south, and it terminates to the east against the Giu-
dicarie belt (Picotti et al. 1995) (Figs. 1, 3). It is part of a
fold and thrust belt where AHe ages in both the footwall
and hanging wall are within 1σ error indicating the fault
has been inactive since the Mid-Miocene (Reverman et al. 2012). Valsugana and Val Trompia thrusts Two of the main structures of the Southern Alps are the
Valsugana and Val Trompia thrusts that uplifted and
exposed crystalline basement (Castellarin et al. 1988, 1993,
Picotti et al. 1995) (Fig. 3). The timing of the deformation
is well established along the Valsugana thrust, where AFT
ages from the hanging wall of the thrust and detrital AFT
data from the preserved syntectonic basin lying directly
to the south constrain a period of intense activity between
12 and 8 Ma (Zattin et al. 2003, 2006). Clasts of Permian
intrusives from the hanging wall appear in basin deposits
by the Messinian, further arguing for intense uplift and ero-
sion along the structure during the Late Miocene (Zattin
et al. 2003). 3 Karawanken Mountains The methodology is briefly outlined here; details are given
in a supplementary file. Fission tracks in apatite (AFT)
are damage zones in the crystal lattice formed during the Samples for AHe analysis were taken from the gabbroic
to granitic members of the Permian to Triassic North
Karawanken plutonic and the Oligocene tonalitic southern 3 3 Int J Earth Sci (Geol Rundsch) (2017) 106:1557–1580 1565 Table 1 (U–Th–Sm)/He analytical data from the eastern Periadriatic fault
Sample
Grain
ID
Lithology
Location
He
U238
Th232
Th/U
ratio
Sm
Ejection
correct. (Ft)
Uncorr. He age
(Ma)
Ft-Corr. He age
(Ma)
2σ (Ma) Sample
unweighted
aver. ± 1σ
(Ma)
Lat (°N)
Long (°E)
Alti-
tude
(m)
Vol. (ncc)
1σ
Mass
(ng)
1σ
Conc. (ppm)
Mass (ng)1σ
Conc. (ppm)
Mass (ng)1σ
Conc. Karawanken Mountains (ppm)
KA10
a1
Hbl-granite 46°28.480′
14°35.623′
581
0.078
2.6
0.058
2.1
40.8
0.143
2.5
100.0
2.45
0.498
6
347
0.66
6.7
10.1
1.2
a2
0.020
4.7
0.023
3.2
16.8
0.068
2.5
50.4
3.00
0.310
7
230
0.79
4.0
5.1
0.6
a3
0.034
3.3
0.031
2.7
19.2
0.053
2.6
32.3
1.68
0.379
7
231
0.64
6.1
9.5
1.3
8.2
2.7
KA16
a1
Tonalite
46°32.680′
13°50.737′
653
0.177
1.6
0.094
1.9
65.8
0.079
2.5
55.0
0.84
0.241
7
169
0.63
12.8
20.5
2.5
a2
0.209
1.5
0.105
1.9
51.2
0.128
2.5
62.0
1.21
0.398
7
194
0.61
12.5
20.3
2.5
20.4
0.1
KA17
a1
Granodi-
orite
46°28.492′
14°37.341′
655
0.023
4.1
0.024
3.2
17.3
0.054
2.5
39.3
2.27
0.202
8
147
0.65
5.0
7.7
1.1
a2
0.041
3.4
0.038
2.4
18.2
0.121
2.5
57.2
3.14
0.374
7
177
0.72
4.9
6.8
0.8
a3
0.092
2.4
0.101
1.9
39.8
0.115
2.5
45.1
1.13
0.548
7
215
0.77
5.8
7.4
0.7
7.3
0.4
KA18
a1
Diorite
46°28.621′
14°36.452′
619
0.063
2.6
0.070
2.0
31.7
0.159
2.4
72.0
2.27
0.905
6
410
0.67
4.6
6.8
0.8
a2
0.029
4.0
0.042
2.3
32.7
0.044
2.6
33.7
1.03
0.361
7
278
0.73
4.3
5.9
0.7
a3
0.078
2.4
0.081
2.0
33.9
0.146
2.5
61.3
1.81
0.847
7
356
0.63
5.3
8.4
1.1
7.1
1.3
KA19
a1
Gabbro
46°28.553′
14°35.318′
605
0.165
1.7
0.064
2.0
13.2
0.354
2.4
73.0
5.52
0.505
7
104
0.78
9.0
11.5
0.9
a2
0.220
1.8
0.087
1.9
24.6
0.369
2.4
104.0
4.23
0.441
7
124
0.82
10.2
12.4
0.9
a3
0.245
1.6
0.119
1.9
17.6
0.478
2.4
71.1
4.03
0.887
6
132
0.82
8.5
10.3
0.7
11.4
1.1
KA27
a1
Metabasite
46°28.857′
14°38.605′
706
0.007
7.7
0.005
14.5
4.3
0.029
2.7
25.4
5.96
0.292
8
254
0.54
3.9
7.3
1.7
a2
0.028
3.8
0.017
4.1
2.7
0.088
2.5
13.9
5.19
1.552
6
246
0.72
4.6
6.3
0.8
a3
0.014
5.5
0.011
6.5
3.4
0.046
2.6
14.1
4.09
0.890
6
270
0.74
3.9
5.3
0.8
6.3
1.0
KA7
a1
Tonaliteg-
neis
46°27.967′
14°36.967′
645
0.162
1.8
0.110
1.9
18.7
0.219
2.4
37.2
1.99
0.999
6
169
0.77
7.9
10.2
0.8
a2
0.115
2.1
0.105
1.9
22.9
0.204
2.4
44.6
1.95
0.736
6
161
0.74
6.0
8.1
0.8
a3
0.170
1.7
0.129
1.9
21.7
0.236
2.4
39.6
1.83
1.046
6
175
0.75
7.3
9.6
0.8
9.3
1.1
KS14
a3
Tonalite
46°25.954′
14°58.373′
982
0.079
2.4
0.075
2.0
20.3
0.148
2.5
39.9
1.97
0.637
7
172
0.76
5.7
7.5
0.7
a4
0.070
2.4
0.089
1.9
33.0
0.059
2.5
21.7
0.66
0.454
7
168
0.66
5.4
8.2
1.0
a5
0.147
1.9
0.135
1.9
23.4
0.209
2.4
36.1
1.54
0.974
6
169
0.76
6.3
8.4
0.7
8.0
0.5
KS-11
a1
Tonalite
46°27.055′
14°49.973′
708
0.084
5.3
0.149
1.9
30.3
0.245
2.4
49.80
1.64
0.834
8.4
169.85
0.69
3.25
4.7
0.68
a2
0.152
2.7
0.154
1.9
21.4
0.241
2.4
33.48
1.57
1.040
8.4
144.65
0.76
5.73
7.5
0.71
a3
0.079
3.1
0.096
1.9
27.4
0.131
2.5
37.45
1.36
0.532
8.4
151.67
0.72
4.98
6.9
0.75
6.4
1.5
KAR-25
a2
Tonalite
N46°
35.799′
E14°
08.624′
800
0.138
2.9
0.039
2.4
5.5
0.026
2.7
3.7
0.68
0.518
8
73
0.80
23.1
28.9
2.7
a3
0.026
6.3
0.008
7.7
4.777
0.0
2.8
13.0
2.715
0.2
8.5
101.0
0.56
14.6
26.274
5.3
27.6
1.8 1 3 Int J Earth Sci (Geol Rundsch) (2017) 106:1557–1580 1566 Table 2 AFT analytical data from the Southern Alps Table 2 AFT analytical data from the Southern Alps Ns: number of spontaneous tracks counted on internal mineral surface; ρs: spontaneous track density (×104 cm−2); Ni: number of induced tracks
counted on external mica detector; ρi: induced track density (×104 cm−2); Nd: number of dosimeter tracks counted on external mica detector;
ρd: dosimeter track density (×104 cm−2); P(χ)2: probability of obtaining Chi-square value for n degrees of freedom (where n = number of crys-
tals − 1); a probability >5 % is indicative of a homogenous population. Valsugana thrust AHe ages were obtained from samples that were previously
dated by AFT analysis from the hanging wall of the Vals-
ugana thrust (Zattin et al. 2003) (Figs. 3, 6, Section C). The
highest elevation sample (CDA1) has AHe and AFT ages
that are essentially the same, indicating rapid cooling at
~11 Ma, in agreement with previous studies (Zattin et al. 2003, 2006). While AFT ages are invariant at lower eleva-
tions, the AHe ages get slightly younger (~7 Ma). Two AHe
ages were obtained on samples from basement highs that
were never buried by more than 4 km of sediments since
the Cretaceous (Bosellini and Doglioni 1986) (Fig. 3). These samples (FRI and CAL11) yield Oligocene AHe (27
and 28 Ma) and Mesozoic AFT ages (77 and 202 Ma). In
sample FRI two grains (a1 and a4) yield anomalously older
ages and the remaining two grains yield Oligocene ages Karawanken Mountains Seward
is 360 ± 5, for R. Reverman (*) 257 ± 14, for M. Zattin (**) 346 ± 18. Ages given in this table and the text are central ages. Error is quoted as
2σ belt along the eastern part of the PAF system and from the
Paleozoic basement (Figs. 2b, 4; Table 1). (ca. 50–20 Ma) and youngest ages south of the Tonale
fault (Fig. 3). In contrast, AFT and our new AHe ages from
samples in the same area do not show differential cooling
(Figs. 3, 5) but imply Miocene cooling as a coherent block
across the Tonale fault. A Pliocene (2.9 Ma, TL1) AHe age
is found within the Austroalpine basement north of the line
(Figs. 3, 5). Given that all other samples in the immediate
area yield Miocene ages, we interpret this sample to have
been recently reset (i.e., due to localized fluid flow), rather
than indicating significant recent exhumation. The α-ejection corrected mean AHe ages from within
the positive flower structure of the Karawanken vary from
11 to 6 (±2) Ma without any systematic trends (Table 1). The most obvious change occurs outside the flower struc-
ture where AHe ages are substantially older. A site along
the PAF but west of the flower structure and HMV fault
yields an AHe age of 20 Ma (KA16) (Fig. 2b). A further
AHe age of 28 ± 4 Ma (KAR25) was derived for the Oli-
gocene Reifnitz tonalite north of the Karawanken Moun-
tains, which is located in the basement of the Neogene Kla-
genfurt basin, in the footwall of the North Karawanken unit
(Fig. 2b). Karawanken Mountains AFT ages were obtained using the standard external detector method and
the zeta calibration approach. The zeta value, obtained on Durango and Fish Canyon apatite standards (Hurford and Green 1983) for D. Seward
is 360 ± 5, for R. Reverman (*) 257 ± 14, for M. Zattin (**) 346 ± 18. Ages given in this table and the text are central ages. Error is quoted as
2σ
Sample number Long. (°E) Lat. (°N) Altitude (m) No. of crystals Spontane-
ous
Induced
Dosimeter
U (ppm) P(χ)2 Age (Ma) ± 2σ
Ns
ρs
Ni
ρi
Nd
ρd
VT2-10
10.3664
45.8446
2000
8
45 10.3
630 144
3658 114
16
64
14.5 ± 4.6
PBFT6
10.2185
45.8954
240
20
124
4.87 2211
86.8 3658 111
10
46
11.1 ± 2.2
PBFT8
10.1669
45.8820
280
20
98 10.1 1027 106
2859 110
12
84
18.9 ± 4.0
PBFT9
10.1659
45.8820
270
20
45
4.57
655
63.7 2859 107
7.4
92
13.8 ± 4.2
PBFT10
10.2810
45.9400
300
20
43
4.63
981 106
3658 102
13
53
7.9 ± 2.4
PBFT11
10.4157
45.8115
1990
20
91
9.09
574
57.4 2859 104
6.9
39
29.7 ± 7.0
PBFT12
10.6045
45.9473
600
20
68
7.68 1227 137
3658 110
16
3
11.3 ± 3.4
PBFT13
10.6045
45.9473
730
20
173 16.1 3476 323
3658 107
38
3.5
10.1 ± 2.2
BG72.2
10.4686
45.8185
640
20
57
4.65
806
65.8 3904
14.82
3.3
99
17.7 ± 4.9
VT1-10*
10.3783
45.8364
1935
20
177 26.4 3194
47.7 4454 143
40.7
86.9
9.9 ± 3.5
CDA1**
11.6051
46.1701
2130
19
58
8
1212 166
4910 103
20.6
97.8
9.1 ± 2.4
FRI**
11.2160
45.9677
1089
23
325
9.3
366 105
5924 125
5.1
39.4
202.5 ± 33.8 Ns: number of spontaneous tracks counted on internal mineral surface; ρs: spontaneous track density (×104 cm−2); Ni: number of induced tracks
counted on external mica detector; ρi: induced track density (×104 cm−2); Nd: number of dosimeter tracks counted on external mica detector;
ρd: dosimeter track density (×104 cm−2); P(χ)2: probability of obtaining Chi-square value for n degrees of freedom (where n = number of crys-
tals − 1); a probability >5 % is indicative of a homogenous population. AFT ages were obtained using the standard external detector method and
the zeta calibration approach. The zeta value, obtained on Durango and Fish Canyon apatite standards (Hurford and Green 1983) for D. Tonale fault Samples across the Tonale fault were taken as close as pos-
sible to previous AFT and ZFT samples but only a few
locations provided suitable apatites for AHe dating. AHe
ages across the Tonale fault record mostly Late Miocene
cooling, though discrepancies occur (Figs. 3, 5). AHe ages
north of the line are ~11 Ma (VST4 and VST5), while to
the south ages at similar elevations show a larger spread
between 6 and 12 Ma. Interestingly, ZFT analyses revealed
a significant difference in cooling ages (Viola et al. 2003)
with the oldest ages in the footwall of the Pejo fault (ca. 80 Ma), younger ages between the Pejo and Tonale faults 1 3 3 3 Int J Earth Sci (Geol Rundsch) (2017) 106:1557–1580 1567 1 3
y
p
Sample
Grain
ID
Lithology
Long
(°E)
Lat
(°N)
Alti-
tude
(m)
Mass
(μg)
4He
(fmol)
238U
(fmol)
232Th
(fmol)
147Sm
(fmol)
eU
(ppm)
Uncorr. He age
(Ma)
Analyti-
cal error
(Ma)
Ejection
correct. (Ft)
Ft-Corr. Tonale fault He age
(Ma)
Mean
age (Ma)
σ
(Ma)
Tonale fault (S)
VS13
a1
Tonalite,
Pre-
sanella
unit
10.6556
46.2556
1935
2.86
2.13
298.49
359.44
467.18
35.18
4.30
0.06
0.71
6.03
6.7
0.6
a2
1.87
2.35
313.08
361.35
429.38
55.78
4.57
0.07
0.68
6.72
a3
2.29
1.97
239.57
298.48
421.77
35.67
4.90
0.08
0.68
7.24
VS4
a2
Tonalite,
Pre-
sanella
unit
10.6393
46.2275
2916
1.52
2.03
178.64
289.13
297.62
43.71
6.37
0.11
0.65
9.86
9.8
0.1
a3
2.11
2.51
215.98
313.81
431.67
36.58
6.69
0.10
0.69
9.67
VS11
a3
Tonalite,
Pre-
sanella
unit
10.6565
46.2429
2300
1.42
1.53
183.76
195.84
227.66
42.12
5.14
0.10
0.65
7.89
8.5
1.1
a2
5.11
6.56
669.20
819.63
1138.40
44.42
5.89
0.07
0.76
7.77
a1
2.23
3.34
363.68
81.04
233.24
42.04
6.75
0.09
0.70
9.71
VS7
a3
Tonalite,
Pre-
sanella
unit
10.6431
46.2316
2703
4.61
5.83
414.27
716.61
903.06
34.14
7.73
0.09
0.74
10.45
11.6
1.2
a2
4.73
4.81
320.32
277.10
474.96
20.99
9.64
0.11
0.75
12.86
a1
4.60
7.94
514.08
832.86
1024.97
41.51
8.65
0.10
0.75
11.60
Fault zone
VS3
a1
Schist,
Tonale
unit
10.6553
46.2744
1362
5.31
0.26
13.04
145.73
74.24
2.82
4.36
0.21
0.76
5.72
8.3
3.8
a2
3.89
0.39
44.26
74.13
134.25
4.27
4.93
0.17
0.75
6.56
a3
3.59
0.34
9.15
80.35
50.81
2.43
9.32
0.32
0.73
12.72
DIM1
a1
Tonalite,
Pre-
sanella
unit
10.8650
46.3202
850
3.80
32.38
145.46
2756.35
475.00
48.67
32.05
1.15
0.71
45.09
6.4
0.8
a2
2.24
6.55
768.46
1669.20
579.30
122.93
4.39
0.08
0.66
6.62
a3
2.68
7.69
842.78
1604.24
473.47
108.03
4.91
0.08
0.69
7.08
a4
2.24
2.62
418.89
540.82
231.68
58.02
3.73
0.07
0.67
5.58
Tonale fault (N)
TL1
a1
Schist,
Tonale
unit
10.6677
46.2881
1376
15.97
6.90
1690.26
22.28
2251.55
25.49
3.13
0.04
0.83
3.76
2.9
0.8
a2
46.16
6.46
2506.07
135.16
2795.57
13.25
1.96
0.02
0.88
2.24
a3
7.48
1.27
458.49
5.10
581.37
14.74
2.13
0.04
0.78
2.71
VST4
a1
Schist,
Tonale
unit
10.6062
46.2960
2276
1.17
0.63
56.63
3.89
57.58
11.84
8.46
0.24
0.64
13.28
10.8
2.1
a2
11.51
8.61
753.25
50.19
599.49
16.05
8.68
0.11
0.83
10.42
a3
10.48
0.53
43.77
b.d. 209.95
0.99
9.26
0.22
0.83
11.17
a4
1.61
0.20
27.61
b.d. Tonale fault 37.29
4.06
5.52
0.37
0.68
8.17
VST 5
a1
Schist,
Tonale
unit
10.6375
46.2792
1624
5.30
10.98
951.52
173.88
1379.23
44.82
8.51
0.10
0.78
10.89
11.3
0.5
a2
4.68
11.68
1039.44
149.75
1237.88
54.96
8.37
0.10
0.75
11.18
a3
6.20
15.60
1256.30
189.61
1501.70
50.22
9.24
0.11
0.78
11.82
a4 1 3 Int J Earth Sci (Geol Rundsch) (2017) 106:1557–1580 1568 3
Sample
Grain
ID
Lithology
Long
(°E)
Lat
(°N)
Alti-
tude
(m)
Mass
(μg)
4He
(fmol)
238U
(fmol)
232Th
(fmol)
147Sm
(fmol)
eU
(ppm)
Uncorr. He age
(Ma)
Analyti-
cal error
(Ma)
Ejection
correct. (Ft)
Ft-Corr. Tonale fault He age
(Ma)
Mean
age (Ma)
σ
(Ma)
Giudicarie
belt
01RZ12
a1
Eocene,
Ponte Pia
Fm
10.9726
46.1307
1023
20.52
25.90
1532.75
3700.64
3234.05
27.75
8.35
0.09
0.84
9.94
10.4
0.6
a2
5.98
7.11
472.32
719.57
773.48
25.51
8.58
0.12
0.77
11.16
a3
1.62
1.82
126.90
363.92
308.44
30.99
6.64
0.13
0.65
10.24
02RZ12
a1
Eocene,
Ponte Pia
Fm
10.9761
46.1307
1023
4.73
4.85
298.03
773.99
750.73
24.00
7.82
0.11
0.75
10.43
9.9
0.5
a2
6.03
7.14
454.71
1214.65
925.29
29.08
7.48
0.09
0.77
9.76
a3
4.60
5.14
361.08
861.52
757.27
29.01
7.06
0.10
0.75
9.44
05RZ12
a1
Eocene,
Ponte Pia
Fm
11.0653
46.2723
332
9.57
33.44
1027.03
2469.96
952.81
39.79
16.17
0.18
0.79
20.51
17.0
3.3
a2
10.62
31.38
1040.17
3448.75
1264.08
41.18
13.20
0.15
0.80
16.56
a3
4.63
9.71
407.81
1364.50
508.90
37.19
10.38
0.13
0.74
14.04
vd 11
a1
Tonalite,
Western
Adamello
unit
10.5644
46.0859
1908
3.16
4.50
418.12
689.39
737.49
43.62
6.00
0.08
0.72
8.35
7.9
0.6
a2
2.40
2.51
291.39
449.86
513.95
39.31
4.89
0.08
0.67
7.27
a3
6.63
7.72
693.18
1150.45
1346.53
34.53
6.20
0.08
0.76
8.21
VM5-10
a1
Tonalite,
Western
Adamello
unit
10.4713
46.1278
2522
8.99
8.26
973.46
1569.69
1804.27
35.47
4.76
0.06
0.79
6.02
5.9
0.1
a2
1.98
2.14
309.70
506.95
492.90
51.37
3.86
0.07
0.66
5.85
a3
2.86
3.09
422.43
672.11
652.42
48.22
4.12
0.07
0.71
5.85
VM6-10
a1
Tonalite,
Western
Adamello
unit
10.4420
46.1306
2070
7.01
7.82
863.04
90.33
813.56
30.21
6.82
0.10
0.77
8.80
7.8
1.0
a2
3.91
5.13
201.54
270.14
231.38
16.13
14.99
0.31
0.73
20.62
a4
2.06
2.21
299.94
284.29
241.77
42.42
4.67
0.07
0.68
6.85
a5
4.37
5.36
584.63
586.77
498.40
39.43
5.75
0.08
0.76
7.60
Valsugana
CDA1
a1
Cima
D’Asta
granite
11.6051
46.1701
2480
1.19
1.03
154.64
336.14
794.26
46.56
3.39
0.07
0.57
5.90
9.5
3.2
a2
1.58
2.79
239.39
328.61
472.20
47.75
6.77
0.13
0.64
10.57
a3
0.92
1.03
76.34
182.81
442.09
30.86
6.61
0.19
0.55
11.99
CAL1
a1
Cima
D’Asta
granite
11.4955
46.0876
722
4.54
4.86
574.34
858.45
1384.39
40.68
4.83
0.07
0.74
6.53
6.9
0.3
a2
1.68
1.70
228.36
283.57
494.99
41.86
4.44
0.10
0.63
7.05
a3
1.35
1.35
184.12
193.63
356.27
40.67
4.52
0.11
0.64
7.07
CAL11
a4
Paragneiss
11.2985
46.0514
1640
3.19
4.52
79.34
219.64
551.61
9.71
26.33
0.40
0.71
36.94
28.4
8.5
a2
7.27
12.31
359.93
1090.09
2633.63
20.05
15.27
0.20
0.77
19.87
a1
4.04
7.08
158.26
464.34
1553.48
15.65
20.07
0.30
0.71
28.35 1 3 Int J Earth Sci (Geol Rundsch) (2017) 106:1557–1580 1569 Sample
Grain
ID
Lithology
Long
(°E)
Lat
(°N)
Alti-
tude
(m)
Mass
(μg)
4He
(fmol)
238U
(fmol)
232Th
(fmol)
147Sm
(fmol)
eU
(ppm)
Uncorr. Tonale fault He age
(Ma)
Analyti-
cal error
(Ma)
Ejection
correct. (Ft)
Ft-Corr. He age
(Ma)
Mean
age (Ma)
σ
(Ma)
CAL2
a5
Cima
D’Asta
granite
11.5009
46.0966
875
2.75
5.73
721.42
628.22
926.00
75.32
5.10
0.07
0.70
7.25
9.0
2.3
a6
1.96
3.76
318.12
199.45
368.61
44.36
7.95
0.11
0.68
11.64
a4
2.01
4.88
575.03
572.45
904.85
84.23
5.32
0.12
0.66
8.04
FRI
a1*
Paragneiss
11.2160
45.9677
1089
1.42
81.92
73.26
346.66
1010.41
25.64
392.46
5.90
0.58
678.15
27.2
4.5
a2
1.78
6.28
141.14
413.33
1075.33
31.68
20.10
0.27
0.66
30.44
a3
1.11
2.45
71.31
259.30
794.13
28.19
14.04
0.26
0.58
24.01
a4*
1.56
11.66
86.05
324.98
1185.22
24.60
53.96
0.74
0.66
82.33
Val Trom-
pia-Val
Camonica
PBFT6
a1
Permian,
Auccia
Volcanics
10.2185
45.8954
240
1.22
0.31
38.01
76.75
394.17
10.90
4.13
0.18
0.61
6.73
7.0
0.4
a2
3.90
2.36
226.52
437.14
1408.81
20.04
5.46
0.07
0.75
7.30
a3*
1.68
0.79
37.15
104.41
497.14
8.71
9.55
0.23
0.66
14.37
a4*
1.75
2.79
91.50
83.53
227.10
15.16
19.31
0.27
0.69
28.01
* Grain rejected from age calculation (see text for details) 1 3 1570
Int J Earth Sci (Geol Rundsch) (2017) 106:1557–1580
Valsugana
thrust
Paleogene-Messinian Sediments
Cretaceous
Jurassic
Dolomia Principale
Triassic
Paleozoic
Crystalline basement
Permian
Post-Messinian Sediments
Southern Alps
10 km
SSE
H=V
Tonale line
D
Val Trompia thrust
9
CDA1
9
11
PBFT6
7
15
VT2-10
16
10
VT1-10
17
11
CAL2
9
12
CAL1
7
N
S
C
N
NNW
D’’
ESE
WNW
E
AHe
AFT
Sample #
Published age
Published age, projected
Age from this study
Age from this study, projected
30
PBFT11
14
2000 m
0 m
S. Giudicarie
Line
VM6-10
8
VM5-10
6
22
9
01&02RZ12
10
9
8
6
17
2000
0m
3000 m
0 m
1000 m
2000 m
Fig. 6 Cross sections of the central Southern Alps. Section C: N–S
cross section of the Valsugana thrust. Published ages are from 1Zat-
tin et al. (2003). Cross section modified after Caputo and Bosellini
(1994), Selli (1998) and Barbieri and Grandesso (2007). Section D
and D″: N–S and NNW–SSE cross section of the Val Trompia thrust. Published ages are from 2Reverman et al. (2012). Cross section modi-
fied after Schoenborn (1992) and Picotti et al. (1995). Section E: Ca. W–E cross section of the Giudicarie belt. Published ages are from
2Reverman et al. 2012; 3Viola 2000, 4Viola et al. 2001. Cross section
after Picotti et al. Tonale fault 1995. Traces of the sections are indicated in Fig. 3 1570 Int J Earth Sci (Geol Rundsch) (2017) 106:1557–1580 Valsugana
thrust
Paleogene-Messinian Sediments
Cretaceous
Jurassic
Dolomia Principale
Triassic
Paleozoic
Crystalline basement
Permian
Post-Messinian Sediments
Southern Alps
10 km
SSE
H=V
Tonale line
D
Val Trompia thrust
9
CDA1
9
11
PBFT6
7
15
VT2-10
16
10
VT1-10
17
11
CAL2
9
12
CAL1
7
N
S
C
N
NNW
D’’
S
30
PBFT11
14
2000 m
0 m
2000
0m Valsugana
thrust
Paleogene-Messinian Sediments
Cretaceous
Jurassic
Dolomia Principale
Triassic
Paleozoic
Crystalline basement
Permian
Post-Messinian Sediments
Southern Alps
9
CDA1
9
11
CAL2
9
12
CAL1
7
N
S
C
2000 m
0 m S Cretaceous
Jurassic
Triassic
Permian 10 km
SSE
H=V
Tonale line
D
Val Trompia thrust
11
PBFT6
7
15
VT2-10
16
10
VT1-10
17
N
NNW
D’’
30
PBFT11
14
2000
0m D
Val Trompia thrust
VT1 10
NNW N SSE H=V
ESE
WNW
E
AHe
AFT
Sample #
Published age
Published age, projected
Age from this study
Age from this study, projected
S. Giudicarie
Line
VM6-10
8
VM5-10
6
22
9
01&02RZ12
10
9
8
6
17
3000 m
0 m
1000 m
2000 m
Fig. 6 Cross sections of the central Southern Alps. Section C: N–S
cross section of the Valsugana thrust. Published ages are from 1Zat-
tin et al. (2003). Cross section modified after Caputo and Bosellini
(1994), Selli (1998) and Barbieri and Grandesso (2007). Section D
and D″: N–S and NNW–SSE cross section of the Val Trompia thrust. Published ages are from 2Reverman et al. (2012). Cross section modi-
fied after Schoenborn (1992) and Picotti et al. (1995). Section E: Ca. W–E cross section of the Giudicarie belt. Published ages are from
2Reverman et al. 2012; 3Viola 2000, 4Viola et al. 2001. Cross section
after Picotti et al. 1995. Traces of the sections are indicated in Fig. 3 ESE
WNW
E
AHe
AFT
Sample #
Published age
Published age, projected
Age from this study
Age from this study, projected
S. Giudicarie
Line
VM6-10
8
VM5-10
6
22
9
01&02RZ12
10
9
8
6
17
3000 m
0 m
1000 m
2000 m Age from this study
Age from this study, projected Published ages are from 2Reverman et al. (2012). Cross section modi-
fied after Schoenborn (1992) and Picotti et al. (1995). Section E: Ca. W–E cross section of the Giudicarie belt. Val Trompia thrust Our new AHe cooling ages ranging from 13 to 4 Ma from
the Karawanken flower structure in conjunction with the
clastic record from the Klagenfurt basin (Nemes et al. 1997 and references therein) indicate a hitherto undated
late Neogene exhumation pulse along the eastern segment
of the PAF. This pulse was associated with surface uplift
and topography growth during the formation of the flower
structure and thrusting of the North Karawanken unit onto
proximal clastic deposits of the Klagenfurt basin, the only
flexural basin inside the Alps. The coarsening-upward
sequence of the basin fill derived from the Karawanken
clearly reveals the initiation of uplift and fast-growing
relief of the chain (Polinski and Eisbacher 1992 and ref-
erences therein). Further evidence for rapid cooling comes
from an AFT age of 12.8 ± 1.8 (Nemes 1996) from the
Karawanken tonalite (Fig. 2b). This age is only slightly
older than the AHe ages presented herein, suggesting rapid
upper crustal cooling and unroofing on the order of 3–5 km
since latest Mid-Miocene time. We present ten new AFT ages and one new AHe age from
the Val Trompia area (Figs. 3, 6, Section D″/D). All ages
record Middle to Late Miocene cooling. The AFT data
show a discrepancy in ages across the fault, with 29.7 Ma
(PBFT11) in the footwall and 14.5 Ma (VT2-10) and
9.9 Ma (VT1-10) in the hanging wall. However, no major
discrepancies in the AHe ages occur across the fault, where
the ages from the hanging wall are slightly older, but within
error, of those in the footwall, 16.4 and 13.5 Ma, respec-
tively (Reverman et al. 2012). A noticeable problem in the hanging wall is that the
AFT ages are younger than the AHe ages. Inversion of AFT
and AHe ages has been identified in several studies and has
yet to be fully explained, though it is often argued that the
problem lies either in zonation of the parent nuclides, or He
implantation from neighboring grains with high U content,
or in an imperfect understanding of helium diffusion kinet-
ics for samples with complex and slow cooling histories
(Ault and Flowers 2012; Flowers et al. 2009; Green and
Duddy 2006; Shuster et al. 2006; Spiegel et al. 2009), all of
which are applicable to these samples. Tonale fault Published ages are from
2Reverman et al. 2012; 3Viola 2000, 4Viola et al. 2001. Cross section
after Picotti et al. 1995. Traces of the sections are indicated in Fig. 3 Published ages are from 2Reverman et al. (2012). Cross section modi-
fied after Schoenborn (1992) and Picotti et al. (1995). Section E: Ca. W–E cross section of the Giudicarie belt. Published ages are from
2Reverman et al. 2012; 3Viola 2000, 4Viola et al. 2001. Cross section
after Picotti et al. 1995. Traces of the sections are indicated in Fig. 3 Fig. 6 Cross sections of the central Southern Alps. Section C: N–S
cross section of the Valsugana thrust. Published ages are from 1Zat-
tin et al. (2003). Cross section modified after Caputo and Bosellini
(1994), Selli (1998) and Barbieri and Grandesso (2007). Section D
and D″: N–S and NNW–SSE cross section of the Val Trompia thrust. 1 3 Int J Earth Sci (Geol Rundsch) (2017) 106:1557–1580 1571 exhumation in the frame of the broader Neoalpine evolu-
tion and Adriatic indentation. similar to those found in sample CAL11 (Table 1). Both
samples have a high dispersion of individual grain ages,
which suggests, along with the relatively large discrepancy
with their AFT ages, very slow cooling since the Mesozoic. Val Trompia thrust Importantly, AHe ages derived from outside the
Karawanken flower structure are distinctly older: Along
the western segment of the eastern PAF, no similar Late
Neogene shortening occurred, as evidenced by an older
AHe age of 20 Ma (KA16) from west of the Hochstuhl–
Möll Valley fault system (Fig. 2b). Combining our new
age with published AFT ages of 26 ± 2 Ma from the same
locality and 25 ± 2 Ma further west (Hejl 1997) attests to
an earlier phase of Late Oligocene to Early Miocene cool-
ing. A further AHe age of 28 ± 4 Ma from the Reifnitz
tonalite (KAR25) corroborates that Late Miocene cool-
ing has not seriously affected the Austroalpine lid from
the immediate surroundings of the Karawanken flower
structure. Giudicarie belt In the Giudicarie belt samples were collected with the aim
of quantifying vertical offsets across this major boundary. However, suitable apatite bearing lithologies are scarce. Three samples were taken from the Ponte Pià formation,
reworked intermediate tuffs of the Eocene Giudicarie-
Insubric Flysch (Castellarin et al. 1993, 2006; Sciun-
nach 1994) (Figs. 2, 6, Section E). The northern sample
(05RZ12) records an AHe age of 17 Ma, while the two
southern samples (01RZ12 and 02RZ12) yield ages of
~10 Ma. An earlier cooling phase may be related to Late Oligo-
cene to earliest Miocene NNW-ward advancement of the
eastern Adriatic indenter (Pomella et al. 2011, 2012), and
possibly its incipient northward subduction (Handy et al. 2014; Lippitsch et al. 2003) leading to a phase of major
extrusion, mainly in the Tauern Window. In its western part
rapid exhumation (2–4 mm/a) was bracketed between 20
and 12–10 Ma (Fügenschuh et al. 1997). During this early
extrusion phase strain concentration in front of the tip of
the indenter within the Tauern Window was influenced
primarily by the occurrence of wedge-shaped blocks of
Austroalpine units at the leading edge of the eastern Adri-
atic indenter (Scharf et al. 2013). These blocks may have Discussion In the following, we will first discuss evidence for Late
Miocene exhumation along the eastern PAF segment in
the Karawanken Mountains, then discuss late-stage cool-
ing in the Southern Alps, followed by a synthesis of evi-
dence for widespread latest Mid-Miocene to Late Miocene
foci of deformation and exhumation. We will proceed with
a discussion of the potential engine driving Late Miocene 1 3 3 1572 Int J Earth Sci (Geol Rundsch) (2017) 106:1557–1580 the deepest units of the Southern Alps along the main
thrusts but locally also the top of the sedimentary pile
with removal of up to 2 km of overburden between dep-
osition and the Mid- to Late Miocene, when exhumation
occurred. The overburden of the Eocene units located in
the core of the Giudicarie belt could have been partly due
to thrusting (samples 01RZ12 and 02RZ12) as suggested
by their location between imbricated thrust sheets and
their AHe age of 10 Ma (Fig. 6, Section E). Nevertheless
for the sample from the Eocene units with an AHe age of
17 Ma (05RZ12) located to the east of the Trento-Cles
fault, there is no unequivocal evidence of overthrusting
suggesting that the nature of the overburden there could
have been sedimentary. The 2-km estimate of sedimentary
overburden is well in line with vitrinite reflectance and
solid bitumen reflectance measurements from the Dolo-
mites indicating about 1800 ± 200 m of eroded thickness
(Grobe et al. 2015) as well as the preservation of Oligo-
cene/Miocene coastal sediments at ca. 2600 m altitude in
the Eastern Dolomites (Keim and Stingl 2000). Moreover,
the location of the older sample supports the argument for
a general southward advance of deformation for the east-
ern Southern Alps (Mellere et al. 2000; Zattin et al. 2006). This argument is based on the structural evolution of the
belt and of its foreland basin. In fact, an increase in sub-
sidence in the Venetian Friuli basin is also recorded at
17 Ma (Mellere et al. 2000), while maximum subsidence
occurred from the Serravallian to the Messinian in relation
to tectonic loading due to the advancing Southalpine thrust
belt. Given the petrologic evolution upsection of the fore-
land basin sediments, from carbonates (Cretaceous-Juras-
sic), to dolomites (Triassic), to metamorphic and volcanics
(Permian), rapid progressive unroofing of the Southalpine
basement must have occurred during the Serravallian to
Messinian (Massari et al. 1986; Mellere et al. Late‑stage exhumation in the Southern Alps Throughout most of the Southern Alps exhumation of
deep crustal material did not occur and erosion of less
than 10 km is evident from the Mesozoic ZFT ages and
the Mesozoic-to-Eocene AFT ages recorded in the Orobic
Alps (Bertotti et al. 1999; Zanchetta et al. 2011, 2015), the
Giudicarie region (Zattin et al. 2006) and the Dolomites
(Emmerich et al. 2005). Shortening during Alpine orog-
eny led to significant exhumation mainly in localized areas
in and around the Giudicarie belt. We find Late Miocene
ages (AFT and AHe) as a first-order feature of the cool-
ing and exhumation pattern of the central-eastern Southern
Alps. Major Late Miocene exhumation along the Valsugana
thrust, already indicated by Tortonian AFT ages from the
Valsugana thrust (Zattin et al. 2006) and its southern fore-
land (Monegato and Stefani 2010), is corroborated by our
new AHe data. Unroofing of ca. 4 km of sediment along the
thrust and exposure of the crystalline basement correlates
with rapid growth, migration and erosion of the Southal-
pine fold-and-thrust belt. AFT ages to the north and imme-
diately to the west of the Valsugana thrust (e.g., CAL11
77.4 Ma, Fig. 2) are Mesozoic (Emmerich et al. 2005; Zat-
tin et al. 2003) indicating that Miocene exhumation was
limited to and focused along the fault. Moreover, the Oligo-
cene AHe ages in the western sector of the Valsugana thrust
indicate differential amounts of exhumation along this
structure, which cannot be explained simply by differential
thickness of Mesozoic sediments due to inherited structural
highs and lows (Bosellini and Doglioni 1986). This pattern
of focused exhumation can be extended to the Val Trompia
fault, where Middle to Late Miocene AFT ages are found
juxtaposed with Oligocene ages across the fault (Fig. 6,
Section D″/D). In summary, major S-directed thrusting and rapid growth
of the Southalpine fold-and-thrust belt within the eastern
Adriatic indenter was active during the Serravallian (Cas-
tellarin and Cantelli 2000) or the Tortonian (e.g., Doglioni
and Bosellini 1987; Schoenborn 1992), corroborated by
thermochronological data evidencing Mid-Late Miocene
exhumation. Accordingly, the eastern Adriatic indenter
must have behaved in a rather rigid manner for ca. 8 myr
with only little internal deformation in its northernmost
sector (Caputo and Poli 2010 and references therein) after
initial indentation at ca. 21 Ma. Discussion 2000; Zattin
et al. 2003). The timing of basin subsidence and aggrada-
tion fits well with our results about the overall timing of
exhumation in the Southern Alps and suggested geody-
namic models (Bressan et al. 1998; Massironi et al. 2006). In summary, major S-directed thrusting and rapid growth
of the Southalpine fold-and-thrust belt within the eastern
Ad i ti
i d
t
ti
d
i
th
S
lli
(C undergone internal fragmentation into the Drau–Möll and
Rieserferner blocks (Fig. 2a) at the end of the Oligocene
triggering ESE-ward migration of exhumation in the East-
ern Tauern subdome (Favaro et al. 2015). 1 3 Further evidence for late‑stage exhumation The Hochstuhl–Möll Valley fault system delimits the
North Karawanken block to the west and shortening by
thrusting disappears within a few kilometers, as also evi-
denced by narrowing of the westernmost Klagenfurt basin. W of the Hochstuhl fault, the basin fill is composed of
about 150 m of gently folded Neogene strata, which die
out laterally after a few kilometers, as well as a few tens
of meters of Quaternary deposits. In contrast, to the east of
the fault more than 600 m of flat-lying Tertiary clastics are
overlain by up to 150 m of Quaternary deposits (Polinski
and Eisbacher 1992). No flower structure nor thrust fault is
known further west along the Pusteria–Gailtal fault though
a subvertical ductile strike-slip duplex (Eder unit) with
Early Oligocene cooling ages has been reported from the
Carnian Alps (Läufer et al. 1997). Our new thermochronological data from the Southern
Alps and the Karawanken Mountains clearly indicate that
latest Middle to Late Miocene unroofing is a widespread
phenomenon at the leading and the western edge as well
as within the eastern Adriatic indenter. Further evidence
for NNW–SSE shortening and related exhumation exists
at the northeastern edge of the indenter: east of the Lavant
fault vitrinite reflectance and AFT data (10–14 Ma) from
the northern Sava fold (Fig. 1) (Boc anticline) indicate
Late Miocene shortening, basin inversion and exhumation
on the order of 2.5–3 km south of the PAF (Sachsenhofer
et al. 2001; Tomljenović and Csontos 2001). Folded latest
Early and Middle Miocene rocks east of the Lavant fault
also suggest that pronounced dextral transpression and
deformation occurred during the Late Miocene (Fodor
et al. 1998). According to these authors, dextral transpres-
sion along the Slovenian part of the PAF system culminated
during the Late Miocene, in line with the evolution of the
Karawanken flower structure. Evidence for yet younger,
Plio-Pleistocene activity of the PAF system and the Sava
fault associated with uplift of the Slovenian Kamnik Moun-
tains has recently been shown in a study on cave sediments
(Häuselmann et al. 2015). This might indicate a migration
of Late Miocene exhumation from the North Karawanken
Mts. southward into the Kamnik Mts. during Pliocene
times during ongoing or renewed transpression. The young ages reported here contrast with Paleogene
AHe ages from Austroalpine units east of the Tauern Win-
dow (Wölfler et al. 2011) (Fig. 2a). Further evidence for late‑stage exhumation There, hilly and mod-
erately shaped remnants of Miocene planation surfaces
are widespread (“cold spots” according to Hejl 1997) as
opposed to the steep relief, at places in excess of 1500 m
of the Karawanken Mts. and the Tauern window, without
such paleosurfaces. This part of the Eastern Alps has expe-
rienced significantly less postcollisional shortening and
exhumation and no upright folding in contrast to the Tauern
Window with high-amplitude folding, major exhumation
and erosion (e.g., Rosenberg et al. 2015). Our AHe data
in conjunction with published age constraints allow one to
construct an improved pattern of spatial heterogeneity of
exhumation of the Austroalpine unit, which is more com-
plex than previously thought. Distinct blocks in the Eastern
Alps and northernmost Dinarides experienced a Late Mio-
cene widely distributed deformation phase accompanied by
significant rock uplift (Fig. 7). As outlined above (“Late-stage exhumation at the lead-
ing edge of the indenter in the Karawanken Mountains”
section) the push of the eastern Adriatic indenter initiated
rapid exhumation within the Tauern Window in the Early
Miocene (Foeken et al. 2007; Fügenschuh et al. 1997;
Scharf et al. 2013). Processes related to indentation per-
sisted throughout the Miocene and may be ongoing today
(e.g., Caporali et al. 2013; Massironi et al. 2006), but their
rates slowed down during Middle to Late Miocene time
(e.g., Fügenschuh et al. 1997; Schneider et al. 2015). Based
on thermochronology data from tunnel and surface samples
from the Penninic Hochalm-Ankogel dome, that yielded
slightly older AFT and AHe ages than those reported here
(Figs. 2, 4), Foeken et al. (2007) suggested that the present-
day topography evolved between 10 and 7 Ma during a
phase of slow cooling (2–4 °C/Ma), after the major exhu-
mation phase (ca. 22-16 Ma) associated with rapid cooling
(40 °C/Ma). This Late Miocene phase of relief evolution in
the SE Tauern Window is in line with palinspastic restora-
tions that denote a Late Miocene age to the evolution of the
Eastern Alps topography into a more mountainous land-
scape and a switch from a N–S-directed to a W–E-directed
drainage system (Brügel et al. 2003; Frisch et al. 1998;
Robl et al. 2008). Wrench deformation along the eastern PAF system
reveals a peculiar pattern of strain localization/concentra-
tion: Strain was accommodated over a wide area in the SE
Alps and NE Dinarides between Medvenica Mts./Karlovac
basin (van Gelder et al. Late‑stage exhumation in the Southern Alps In “Driving forces for Late
Miocene deformation and exhumation” section we will dis-
cuss in more detail how and why strain was accommodated
differently in response to indentation. The Late Miocene ages found in the Giudicarie belt and
Adamello area are ascribed to uplift associated with base-
ment ramps that folded the earlier/older thrust sheets in the
area (Fig. 6, Section D″/D) during thickening of the wedge. The uplift of the entire Adamello complex likely occurred
due to it acting as a rigid block, whereas elsewhere in the
Southern Alps an inherited network of Mesozoic faults
allowed for strain partitioning and localization of deforma-
tion. A similar Tortonian pulse of exhumation has also been
reported for the northern sectors of the central Southern
Alps based on AFT data (Zanchetta et al. 2015). Along the Giudicarie belt, the Miocene AHe ages
from Eocene sedimentary units (Ponte Pia Fm) indi-
cate that locally significant erosion affected not only 1 3 Int J Earth Sci (Geol Rundsch) (2017) 106:1557–1580 1573 Further evidence for late‑stage exhumation 2015) and the PAF system south of
the Pohorje Mts. (Sava folds) (Fig. 1). In contrast, in the
Karawanken Mountains, strain localization was most effi-
cient and strain was concentrated within the narrow belt of
the flower structure, with mainly NW thrusting and forma-
tion of the flexural Klagenfurt basin (Nemes et al. 1997). We attribute different deformation styles to contrasting
preceeding thermal evolution and resulting lateral differ-
ences in crustal strengths: Pervasive Sava folding resulted
from thermal crustal weakening due to addition of heat dur-
ing syncollisional Early Miocene magmatism and extreme
extension in the Early-Middle Miocene (Sachsenhofer et al. 2001; Fodor et al. 2008 and references therein) (Fig. 7). Weak lithosphere has also been reported for the Tauern 1 3 3 Int J Earth Sci (Geol Rundsch) (2017) 106:1557–1580 1574 Fig. 7 Summary of episodes of major deformation, cooling, exhuma-
tion as well as sedimentation and volcanic activity for the Eastern and
Southern Alps. Chronostratigraphic correlation chart for the Mediter-
ranean and Paratethyan stages according to Piller et al. (2007). South-
alpine thrust systems: VAL-BEL Valsugana and Belluno, CAN-MAN
Cansiglio-Maniago, BAS-VALD Bassano-Valdobbiadene, FR. THR
frontal thrusts Fig. 7 Summary of episodes of major deformation, cooling, exhuma-
tion as well as sedimentation and volcanic activity for the Eastern and
Southern Alps. Chronostratigraphic correlation chart for the Mediter-
ranean and Paratethyan stages according to Piller et al. (2007). South- alpine thrust systems: VAL-BEL Valsugana and Belluno, CAN-MAN
Cansiglio-Maniago, BAS-VALD Bassano-Valdobbiadene, FR. THR
frontal thrusts of smaller magnitude, indicating that convergence between
the eastern Adriatic indenter and Europe is less focused
and thus accommodated differently. Based on brittle fault
analyses yielding predominant extensional and strike-slip
states of stress in the Tauern Window, Bertrand et al. (2015)
argued for such a switch in accommodation mechanism
with initial vertical extension followed by strike-slip and
normal faulting under constant N–S compression. This shift
was triggered after orogen-scale folds were nearly isoclinal
and the push of the indenter needed to be accommodated
differently, leading to normal faulting and E–W extension
within the Tauern Window. Importantly, this second stage
initiated in the Late Miocene (Bertrand et al. 2015), coe-
val with more widespread exhumation in the Southern and
Eastern Alps outlined above. Window (Genser et al. Further evidence for late‑stage exhumation 2007; Willingshofer and Cloetingh
2003 and references therein), where northward movement
of the eastern Adriatic indenter was initially accommodated
by major ductile folding, exhumation and erosion as well as
lateral extrusion. In contrast, such folding did not occur in
the Karawanken Mountains and in the Southern Alps. Here
mechanically stronger crust, which had experienced only
weak Alpine metamorphic overprint, may have promoted
localized strain concentration along faults (Karawanken
flower structure; Valsugana thrust) instead. Driving forces for Late Miocene deformation
and exhumation Based on our new low-temperature thermochronology ages
and published data we observe a shift of strain distribution
during the Miocene. Early Miocene exhumation is clearly
focused within the Tauern Window in front of the East-
ern Adriatic indenter and the wedge-shaped Austroalpine
blocks (Rieserferner and Drau-Möll blocks in Fig. 2a) (e.g.,
Fügenschuh et al. 1997; Scharf et al. 2013) (Fig. 7). Late
Miocene exhumation is, however, more distributed, but also A possible mechanism leading to the observed exhuma-
tion pattern may lie in a shift of the mechanical coupling
state and rheological behavior between the orogenic wedge
and adjacent plates. Based on lithosphere-scale analogue
modeling, Willingshofer and Sokoutis (2009) demonstrated
that coupling is a key process for stress transmission and
thus for the resulting deformation pattern. The amount of 1 3 Int J Earth Sci (Geol Rundsch) (2017) 106:1557–1580 1575 Glotzbach et al. 2011; Fox et al. 2015). Only few studies
have addressed this point for the Eastern Alps (Wölfler
et al. 2012), which are tectonically more active than the
Western and Central Alps, where convergence stopped at
ca. 6 Ma (Battaglia et al. 2004; Grenerczy et al. 2005). In
the Eastern Alps, an increasing body of data is testifying to
a yet younger stage of uplift and/or erosion (e.g., Wagner
et al. 2010). Genser et al. (2007) report late-stage surface
uplift of the eastern Molasse basin on the order of 400 m
since ca. 6 Ma. Gusterhuber et al. (2012) find evidence for
even more intense erosion of 1–2 km of the Alpine fore-
land basin since Late Miocene times. Pliocene-Quater-
nary uplift also occurred in the Styrian basin (Sachsen-
hofer et al. 1997), the western part of the Pannonian basin
(Bada et al. 2001) and the easternmost unglaciated part of
the Eastern Alps (Legrain et al. 2014, 2015; Wagner et al. 2010) (Figs. 1, 2). The cause of this uplift is controversially
debated, being associated with either (1) crustal delamina-
tion and/or convective removal of thickened lithosphere
(e.g., Genser et al. 2007), (2) the coeval major reorganiza-
tion of the external stress field in the ALCAPA region (e.g.,
Horváth and Cloetingh 1996; Peresson and Decker 1997). Our new AHe data with the majority of ages ranging from
11 to 6 Ma clearly corroborate that not enough tectonic
and/or erosional exhumation has occurred since then to be
recorded by low-temperature thermochronology. Driving forces for Late Miocene deformation
and exhumation Thus, our
findings do not support an orogen-wide drastic increase in
denudation during Pliocene and Pleistocene times. coupling will increase during continental collision follow-
ing subduction. They correlated their experimental results
with the pattern of deformation in the Eastern Alps and
observe a switch from north-directed to south-directed
thrusting as well as initiation of internal deformation of
the indenter between ca. 12 and 10 Ma. Based on numeri-
cal modeling Robl and Stüwe (2005) argue for a strong
decrease in rheology contrast between the initially much
stiffer Adriatic indenter into the softer European margin. Interestingly, substantial strengthening of the indented
Eastern Alpine orogenic wedge occurred since ca. 13 Ma
(Robl and Stüwe 2005). These modeling results are well
in line with our findings of a latest Mid- to Late Miocene
shift from focused exhumation outside of the indenter
(mainly Tauern Window) to more widespread exhumation
within and along the rims of the indenter (Fig. 7). Major
coupling of the Alpine wedge and the South Alpine fold
and thrust belt is also evidenced by the fault framework
and fault linkages between the Austroalpine and Southal-
pine domains, which were fully developed during the latest
Miocene–early Pliocene (Bartel et al. 2014a, b; Massironi
et al. 2006). A further plausible mechanism may have been a reor-
ganization of the stress field leading to a change in bound-
ary conditions operating on the orogen. Neogene conver-
gence rates based on shortening values are 0.6–1 cm/year
(Rosenberg and Berger 2009). However, magnetic anoma-
lies and structural analyses indicate a change in relative
plate motion of Africa with respect to Europe from NE
to NNW 16 Ma ago and from NNW to NW 8.5 Ma ago
(Caputo and Poli 2010 and references therein). Such rota-
tions may have governed the compressional stress field in
the study area and may have intensified strain transfer and
exhumation. Conclusions • AHe data from the eastern Periadriatic fault reveal a
Late Miocene phase of activity and exhumation leading
to the formation of the Karawanken flower structure and
the infill of the Klagenfurt basin. In summary, we suggest that a major change of the cou-
pling state of the orogen, possibly enhanced by a reorgani-
zation of the large-scale stress field between Africa and
Europe initiated widespread latest Mid- to Late Miocene
deformation, thrusting and exhumation at various sites
within, at the rim and at the tip of the indenter. • New thermochronological data from the central-eastern
Southern Alps constrain a coeval phase of uplift and
erosion during the latest Mid- to Late Miocene. • Mid- to Late Miocene AHe cooling ages from the Vals-
ugana thrust, the Val Trompia thrust, the Tonale fault
and the Giudicarie belt in the Southern Alps indicate at
least 2 km of exhumation since then. Along thrust sys-
tems (i.e., Valsugana and Val Trompia thrusts) uplift and
erosion were larger as demonstrated by Miocene AFT
ages. Young uplift in the Eastern Alps? Triggered by the detection of a dramatic increase in sedi-
ment flux to circum-Alpine basins since ca. 5 Ma (Kuh-
lemann et al. 2002) a large number of studies focused on
the question, whether there has been a coeval, possibly
climatically triggered increase in exhumation and erosion
(e.g., Cederbom et al. 2004; Willett et al. 2006). In situ as
well as detrital thermochronological data challenge sedi-
ment budget calculations and instead imply earlier phases
of rapid exhumation or long-term steady-state conditions
for the Western and Central Alps (e.g., Bernet et al. 2009; • However, even along the thrusts exhumation can be
highly differential, as shown for the western sector of
the Valsugana thrust. • Exhumation and deformation related to Adria indenta-
tion was initially confined within the orogenic core of
the Eastern Alps, the Tauern Window, but became more
widespread during Mid- to Late Miocene times. 1 3 1576 Int J Earth Sci (Geol Rundsch) (2017) 106:1557–1580 geochronological and modeling study of the Mesozoic South
Alpine rifted margin. Tectonics 18:185–200. doi:10.1029/199
8TC900028 • This shift from focused exhumation outside the indenter
to exhumation within, and deformation of the indenter,
is ascribed to a major shift in the coupling state, from a
decoupled to a coupled system. Bertrand A (2013) Exhuming the core of collisional orogens, the Tau-
ern Window (Eastern-Alps). Dissertation, FU Berlin Bertrand A, Rosenberg C, Garcia S (2015) Fault slip analysis and late
exhumation of the Tauern Window, Eastern Alps. Tectonophys-
ics 649:1–17. doi:10.1016/j.tecto.2015.01.002 Acknowledgments Open access funding provided by Paris Lodron
University of Salzburg. We acknowledge support by Grant P22110 of
the Austrian Science Fund (FWF) and grant 120537 from the Swiss
National Science Foundation (SNF) as part of the ESF Thermo-
Europe Collaborative Research Project of the TOPO-Europe Eurocore
program. Samuel Graf is thanked for preparation of the samples from
the Southern Alps. We thank Andreas Wölfler for his comments on an
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ISSN: 2615-0891 (Media Online) Jayapangus Press Volume 5 Nomor 3 (2022)
ISSN: 2615-0891 (Media Online) Volume 5 Nomor 3 (2022)
ISSN: 2615-0891 (Media Online) Abstract This research looks at education in adult development and how it affects education. This study is motivated by the rapid advancement of science, technology, and
communication at this time, which necessitates the development and improvement of
human quality as a solution to face the current of modernization; thus, an educational
process is required that can play an active role in following changes and developments
that exist both now and in the future. Recognize that adulthood is a time when you have
reached a mature and complete view of life, which can be achieved through consistent
decision-making. However, it frequently appears when people fail to recognize the
significance of adulthood and middle age. The goal of this study was to examine and
analyze education during the adult development period, as well as its surprise in education. This study employs an approach approach in data processing through literature analysis,
particularly literature descriptions. Helping people face life's problems objectively,
assisting adults with problem-solving skills, assisting people in changing social
circumstances, and assisting people in acquiring or skills required to complete their life
needs are all realizations of adult education practices that are frequently overlooked. Education in The Period of Adult Development and
Its Implications on Education Ari Mawarni1, Muhammad Halim Munandar2, Kasinyo Harto3, Ermis Suryana4
1234Raden Fatah State Islamic University Palembang
1arimawarni.mts@gmail.com, 2mhalimmmunandar@gmail.com,
3masyo71@yahoo.com, 4ermissuryana@radenfatah.ac.id Abstrak Jurnal ini mengkaji tentang pendidikan pada masa perkembangan dewasa dan
implikasinya pada pendidikan. Kajian ini dilatar belakangi oleh pesatnya kemajuan ilmu
pengetahuan, teknologi, dan komunikasi pada saat ini yang mengharuskan pengembangan
dan peningkatan kualitas manusia sebagai solusi untuk mengimbangi arus moderenisasi,
dengan demikian diperlukan proses pendidikan yang dapat berperan aktif dalam
mengikuti perubahan dan perkembangan yang ada baik di masa sekarang maupun di masa
yang akan datang. Perlu diketahui bahwa masa dewasa adalah masa dimana sudah
mencapai pandangan hidup yang matang dan utuh yang dapat menjadi dasar dalam
membuat keputusan yang konsisten. Berarti seseorang telah memiliki tanggung jawab
terhadap kehidupan dan keputusan yang diambil, mampu melakukan peran-peran sosial
yang biasa berlaku di masyarakat. Namun permasalahan seringkali muncul, mana kala
masyarakat kurang menyadari pentingnya pada masa dewasa awal dan madya. Tujuan
penelitian ini adalah untuk mengkaji dan menganalisis pendidikan pada masa
perkembangan dewasa dan implikasinya pada pendidikan. Penelitian ini menggunakan
pendekatan kualitatif, sehingga dalam melakukan olah data melalui analisis literatur
khususnya literatur deskripsi. Membantu masyarakat menghadapi sesuatu persoalan
hidup mereka secara objektif, memfasilitasi orang dewasa dengan keterampilan
memecahkan masalah, membantu masyarakat dalam merubah kondisi sosial, dan https://jayapanguspress.penerbit.org/index.php/cetta 235 membantu masyarakat memperoleh pengetahuan atau keterampilan yang diperlukan guna
melengkapi kebutuhan hidup mereka adalah realisasi dari praktik pendidikan orang
dewasa yang sering dilupakan. membantu masyarakat memperoleh pengetahuan atau keterampilan yang diperlukan guna
melengkapi kebutuhan hidup mereka adalah realisasi dari praktik pendidikan orang
dewasa yang sering dilupakan. Introduction Human development can be classified into several categories. Physical
development (motor development), the process of changing the structure and functions of
the human body, as well as the addition of varied physical flesh. Cognitive development
is associated with the process of altering one's way of thinking. Changes occur throughout
development, leading to growth and development (maturity). Specific development is a term that refers to the process of changing an existing
personality. Meanwhile, social and moral developments refer to changes in how humans
interact with other people or their own fields as individuals and as groups. Human beings have four boundaries, according to Islamic psychology: the
physical-natural dimension, the internal-psychic dimension, the socio-artistic dimension,
and the spiritual dimension. The physical-natural dimension is the simplest to understand
because it can be seen directly with the mortal eye's senses. According to QS Al-Tin: 4,
the mortal constitution is shaped by a stylized construction by God to be grateful. Surely
we have created man in the best possible form. Human life is heavily reliant on the reality of the spirit, because when the spirit
departs from man (body or physical), life ends. Nonetheless, the understanding of reason
is the thinking power contained in the human soul, not the brain. In other words, the bond
between the three bases, namely permissible, feelings, and wills, emerges later. While lust
(shahwat) refers to the human impulse and fermentation. Socio-cultural boundaries are
those that are almost inextricably linked to the environment and nature around them. Human life in the world is like God's caliph on the face of the earth. As a result, this
dimension is unavoidable in man. While the spiritual dimension is defined by the concept
of fitrah, which inherently glorifies God as his God. The various dimensions that a child
possesses are fundamental abilities that must be fully developed in order to achieve a
perfect personality (Suralaga, 2021). Tohirin (2008) in general, all proses individual
development until becoming oneself (person) occurs in three stages, namely: 1. The stage of conception, which is the process of meeting sperm cells in men and
ovum in women, thus forming new individuals that uniquely occur changes in
order to undergo reproductive tasks. 1. The stage of conception, which is the process of meeting sperm cells in men and
ovum in women, thus forming new individuals that uniquely occur changes in
order to undergo reproductive tasks. 2. 1. Understanding and Classification of The Adult Phase 1. Understanding and Classification of The Adult Phase In general, when a person has reached adulthood, he has a variety of knowledge
and experience. While in addition they are directly dealing with work problems,
community problems and marriage. On that basis, the education given in adulthood should
be in accordance with the situation and conditions. Islamic religious education for adults
is still needed. Adulthood begins from the end of the psychiatric shocks that afflict
adolescence. Thus, adulthood can be said to be peace of mind, determination and firm
faith. In line with the level of age development, Jalaluddin said religious attitudes in adults
have characteristics, namely: a. Accepting religious truth based on careful consideration is not just a follow-up. a. Accepting religious truth based on careful consideration is not just a follow-up. b. Tends to be realistic so that religious norms are more widely applied in attitudes and
behaviors. c. Be kind to religious teachings and norms and strive to study and deepen religion. d. The level of religious observance takes precedence over self-consideration and
responsibility, until religiousness is the realization of life's attitude. d. The level of religious observance takes precedence over self-consideration and
responsibility, until religiousness is the realization of life's attitude. f. Being more critical of the core of religious teachings so that religious steadiness in
addition to being based on thought considerations is also based on conscience
considerations. g. Religious attitudes are more towards their respective personality types so that they can
see the influence of personality in accepting, understanding and implementing the
teachings of the religion they believe in (Suralaga, 2021). g. Religious attitudes are more towards their respective personality types so that they can
see the influence of personality in accepting, understanding and implementing the
teachings of the religion they believe in (Suralaga, 2021). In the development of adolescence Elizabeth B. Hurlock divides adulthood into
three phases, namely: early adulthood, intermediate adulthood, old age. The same
grouping is also made clear by Lewis Sherril who divides adolescence as follows: In the development of adolescence Elizabeth B. Hurlock divides adulthood into
three phases, namely: early adulthood, intermediate adulthood, old age. The same
grouping is also made clear by Lewis Sherril who divides adolescence as follows: a. Early Adulthood, this time a teenager tends to choose the direction of life by facing
the temptation of various possible choices. a. Method In this study, the method used is a qualitative approach. The literature method used
is literature description, by means of collecting and compiling a list of the literature studied
and then making a description or description of each literature in detail. In the description
it is stated and explained the state of the literature, notes on the contents of the literature
and the main contents of the literature. Therefore, this method describes in detail and
analyzes an event or phenomenon, social strata, nature or attitude, and personal opinion or
group opinion on an ongoing problem. The research process begins with preparing and
compiling hypotheses of basic conjectures and thinking rules used in research. Then,
collect data and then review it in the study. The object of qualitative research covers all
fields and aspects of human life. Data analysis in this study is defined as one way of
compiling and searching regularly and orderly writings of research results. Therefore, this
study uses literature analysis to reveal various data that have been scattered in various
references that are already available. Introduction The stage of birth is that the baby is born from the mother's womb to the world. 3. Stages of the process of individual development into a typical person (development
of selfhood). Other experts divide it into six developmental stages: infants, children, adolescents,
early adults, intermediate adults, and late adulthood (Tohirin, 2008). Based on this development period, there are several educations that need to be
carried out so that children's development can be done properly. So, this research will
discuss education during the development of adulthood and its implications on education. In this paper, the method used in this research is the method of literature study (library
research). Literature or literature study is an activity of collecting data from various
reading sources. The data used in completing this research comes from sources in the form
of books, journals and research that are in accordance with this research (Siregar &
Harahap, 2019). https://jayapanguspress.penerbit.org/index.php/cetta 236 2. Understanding Adult Education In the great dictionary of Indonesian, education is defined by the process of
changing the attitudes and manners of a person or group of people in an effort to mature
people through teaching and training efforts. Then the term adult is interpreted by a human
being who is not a child or teenager anymore, meaning (Nasional, 2008) an individual who
is in the phase after adolescence. In the context of this study, adult education in question
is a process in which learning interactions are found between educators and learners who
are mature, both in the scope of formal and non-formal education. According to Mustofa Kamil, the definition of adult education is based on the
condition of adult learners both from the physical (biological), psychological, and social
dimensions. A person is considered biologically mature if he has been able to reproduce. Then psychologically mature, means that a person already has responsibility for life and
decisions taken. Furthermore, sociologically mature, means that a person has been able to
carry out social roles that are generally applicable in society. Thus, the term adult is based
on the completeness of physical condition as well as age, and psyche, in addition to being
able to play a role in accordance with the demands of the task of the status possessed
(Kamil, 2007). In the Islamic perspective, a person is considered an adult when he has entered the
age of puberty, which is the age that has ended childhood and has reached adulthood in
shari'a so that he has a responsibility intended for him to assume the legal obligations of
syar'i (taklif) (Manzur, 1997). When entering puberty, a person is considered to be able to
distinguish good and bad and also has a broader view or thought than childhood. This
period is characterized by starting to experience wet dreams for men and coming months
(menstruation) for women. Regarding the age details of men and women at the beginning
of experiencing these signs are relative, some are fast and some are slow, and usually occur
between the age range of 12-16 years. g
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y
Adult education is a systematic and continuous learning process in people who are
adults with the aim of achieving changes in knowledge, attitudes, values, and skills. 2. Understanding Adult Education The
conditions that can be derived from that definition are adults are motivated to learn
according to their needs and interests, learning orientation for adults is life-centered,
experience as a source of wealth for adult learning, adults expect to relate themselves to
the right needs, individual differences among individuals develop according to their age. In its practice, adult education is carried out in the form of formal and non-formal
education. The form of adult education in formal form is carried out at the level of
education of advanced schools / high schools (SLTA) to higher education (PT). Furthermore, in non-formal form, it is carried out in the form of out-of-school education
by the community (PLSM), courses, health guidance and counseling, religious studies
activities or taklim assemblies, training organizations, community development programs,
and so on. These forms of adult education open up learning opportunities for every adult
citizen, both disabled and healthy and the program is implemented continuously. 1. Understanding and Classification of The Adult Phase Early Adulthood, this time a teenager tends to choose the direction of life by facing
the temptation of various possible choices. b. Middle Adulthood, at this time already life challenges began to be faced. This is the
time when you have achieved a mature and complete outlook on life that can be the
basis for making consistent decisions. b. Middle Adulthood, at this time already life challenges began to be faced. This is the
time when you have achieved a mature and complete outlook on life that can be the
basis for making consistent decisions. c. Late Adulthood which has the main characteristic, namely surrender. https://jayapanguspress.penerbit.org/index.php/cetta 237 3. Principles of Adult Education According to the perspective of adult education, independence is an important
benchmark in any development of learning models. Therefore, the concept of learning in
the context of andragogy, more specifically has a basic core that refers to developing the
values of independence for all its learners (learning citizens). Without that goal every
learning in the context of andragogy becomes meaningless and the same as with other
learning models. This assumption is a special limitation that is able to distinguish the
concept of andragogy learning from other learning concepts (Kamil, 2007). https://jayapanguspress.penerbit.org/index.php/cetta 238 Independence according to the concept of andragogy means also self directed
learning. Knowles describes in detail the definition of self directed learning as a process
in which individuals take the initiative, whith or without the help of others, in diagnosing
their learning needs, formulating learning goals, identifying human and other resources for
learning, choosing and implementing learning strategies, and evaluating learning
outcomes self directed learning provide reference to how learners have the initiative to
learn, write their own learning needs, find their own learning resources, formulate their
own learning goals, choose and implement learning strategies and self-evaluating. These
components are a dimension of how andragogy forms the character of self-reliance in
learners (aoutonomous learning) (Knowles, 2014). In the principle of andragogy, teaching and learning activities are centered on
learners. Adult learning is in principle carried out and arranged together between learning
resources (teachers, tutors, coaches) and learners (learning citizens, students, trainees) is
valid until the evaluation stage, then also in the development of learning with the principle
of andragogy learners are given the right in develop, and carry out learning programs, and
evaluate the program independently. The basic principle that is used as a handle is in line
with the concept of from, by, and for learners, so that the role of learning resources
(teachers, coaches, pamong, tutors, facilitators) acts as individuals providing guidance,
encouragement and direction when needed. This concept shows that learners compile
programs on the basis of their own activities and abilities with the capital of knowledge,
skills and resources that exist and can be used (Jarvis, 2012). The above guidelines accept that training with andragogy standards will survey the
potential and independence of students. With that goal becomes the main reason for any
preparation and development of educational projects. 3. Principles of Adult Education Logically speaking, this view
corresponds to the student's thoughts and responsibilities namely: human instincts are
usually great, opportunity and independence, exclusive and probability, self-confidence
and self, self-fulfillment, insight, obligation and humanity (Elias & Merriam, 2005). This view provides clarity that the motivation for the application of andragogy
standards in the advancement of education is to create the potential and possibilities of
students as ideally as possible, so that eventually they can improve and help themselves,
besides that they can build their current abilities. Circumstances, society, and more broadly
can take a functioning part in building the country and the country. With regard to the
expansion of freedom of learning, andragogy has a rule that the idea of learning should be
a side interest and the need to make self active learning propelling (dynamic progress
without the help of others). In certain situations, acquiring desires and needs arises from
within oneself (intrinsic motivation) and is not energized or suggested by others (extrinsic
motivation). This attitude arises when students feel that by learning they believe that they
have gained information, add incentives for self-improvement, and can overcome
problems that appear without the help of others and others. Furthermore, by learning,
students (people) feel an increase in thinking capacity, expanding perspectives, developing
capture power, abilities, personal satisfaction and their lives and professions. Perceived
learning provides reasonable meaning and good value for life, the urge to complete a job,
and makes it easier to overcome life's problems, so that learning can drive a cycle of change
towards freedom (Kamil, 2007). Furthermore, it is important to understand that the overall idea of adults in daily
life and regular communication needs to be considered. As we age, an adult's ability and
development in thinking, acting relentlessly also increases. For this situation, adults have
the choice to decide and decide on choices related to themselves and their current situation. Regarding this capacity, adults need to be given the opportunity to determine mentality
and choices, including those related to learning. Furthermore, Edward Lindeman, an 239 https://jayapanguspress.penerbit.org/index.php/cetta andragogyist stated that adults are encouraged to come forward by confessional demands. In learning exercises, they do not need a mentality that underestimates their privileges and
investments, but believes that their capacity should be cared for and felt, so that they can
make a significant commitment to the educational experience (Kamil, 2007). 3. Principles of Adult Education The principle of adult education as knowles explains, also attaches importance to
the intellectual and emotional interconnectedness of learners in the learning process. In
this case, learners are not only given the opportunity to express opinions and ideas of
thought in enriching learning resources and experiences, but also participate emotionally
in learning, such as attitudes and behaviors to support and be responsible in achieving
learning success. In order to realize the intellectual and emotional involvement of students,
then in the implementation of adult education it is necessary to apply participatory
planning, which includes students in formulating learning plans, so that learning programs
and objectives can be adjusted to their needs (Suprijanto & Arikunto, 2016). The process of adult education is in line with the implementation of the concept of
education in the paradigm of freedom as Paulo Freire explained that educators must
practice co-intentional education, meaning educators and learners, both being subjects
interconnected to discover and create knowledge. Thus, students are given the opportunity
to develop their potential to the maximum, unlike pseudo-learners, but become learners
who have full involvement in forming a commitment to the educational process (Soeharto,
2012). In implementing andragogy-based learning there are several principles and
strategies of adult learning that need to be considered. These principles are: a. Adults have a self-idea. Adults have an understanding that they are fit to simply
decide, can face the gamble of the choices made, and can handle their lives
independently. Self-confidence is very important for adults, and he really wants the
recognition of others for his value. Behavior that seems dismissive, in general will be
responded negatively by adults. As a result, if adults are valued and invited to work
together by educators, they will include themselves in learning. Learning exercises
will create expected learning (arranged in the future) and participatory learning (with
others) by thinking and acting in and in the universe of their lives. b. y
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b. Adults have a great experience. Every adult has a wide range of knowledge of
circumstances, connections, and selves to each other that are demonstrated by various
foundations throughout their daily lives and current circumstances. Knowledge of
circumstances is the development of circumstances that adults have faced in the past
that can be used to answer the current state. 3. Principles of Adult Education Adults are interested in learning, as they answer subject matter
and cycles related to its parts throughout everyday life. Learning exercises generally
lie in the real world (reality). Therefore, it advances the need to encourage capacity
building to address the problems it faces in its requirements. The pragmatic
consequence is that progress must be placed to address issues related to adult work in
their lives. Opportunities to thrive should be planned taking into account the needs
and problems seen by adults, such as needs and problems in work, socio-social work,
and the economy. The regulated ability gained turned into a field of strength for adult
learning (Sudjana, 2007). d. Adults need to have the choice to take advantage of the consequences of their
immediate progress. Adults are interested in learning, as they answer subject matter
and cycles related to its parts throughout everyday life. Learning exercises generally
lie in the real world (reality). Therefore, it advances the need to encourage capacity
building to address the problems it faces in its requirements. The pragmatic
consequence is that progress must be placed to address issues related to adult work in
their lives. Opportunities to thrive should be planned taking into account the needs
and problems seen by adults, such as needs and problems in work, socio-social work,
and the economy. The regulated ability gained turned into a field of strength for adult
learning (Sudjana, 2007). g (
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)
In adult education principles, learners are grouped by interests, talents, and
abilities, where educators provide support to assist learners in determining their learning
needs. Therefore, in adult education, learners participate in involving themselves in
determining their planning, learning process, and evaluation. In addition, in adult education, learning orientation is directed to solve problems,
namely learning while working on current problems to be used in the present. Unlike the
learning orientation for children, all subjects learned by students at this time are directed
to provide for future life. According to Mukhlis, the principle of adult education is also used as a foundation
in practicing the concept of critical education that hasthe following ideas: a. Learn from reality or experience. The material in question is not speculation, but rather
the individual's experience or the actual circumstances of the individual associated
with the learning cycle. So, there is no higher information authority than others. 3. Principles of Adult Education The experience of cooperation leads to
the development of the ability of adults to coordinate attention to see themselves
according to the point of view of others. Self-experience is the capacity of adults to
introduce themselves to past circumstances. Functional consequences in learning,
adults will want to continue learning to remember the encounters they experienced. Normal experience can be utilized as a rich asset to be utilized in learning. Adults who
acquire some useful new knowledge will be more often deciphered by utilizing old
meetings. Correspondingly, adult students should be involved as a source of learning. The presentation and utilization of new ideas will be more straightforward with the
assumption of departing from the adult experience. b. Adults have a great experience. Every adult has a wide range of knowledge of
circumstances, connections, and selves to each other that are demonstrated by various
foundations throughout their daily lives and current circumstances. Knowledge of
circumstances is the development of circumstances that adults have faced in the past
that can be used to answer the current state. The experience of cooperation leads to
the development of the ability of adults to coordinate attention to see themselves
according to the point of view of others. Self-experience is the capacity of adults to
introduce themselves to past circumstances. Functional consequences in learning,
adults will want to continue learning to remember the encounters they experienced. Normal experience can be utilized as a rich asset to be utilized in learning. Adults who
acquire some useful new knowledge will be more often deciphered by utilizing old
meetings. Correspondingly, adult students should be involved as a source of learning. The presentation and utilization of new ideas will be more straightforward with the
assumption of departing from the adult experience. Adults have preparation for learning. The preparation of adults to learn will be in line
with the work that the person does both in the public arena and in the task / work. The
consequence is that the grouping of learning programs should be organized based on
the arrangement of tasks played by adults, not given the consistent order of subjects. Changes in learning materials and exercises must be applicable to the needs and efforts
/ work of more advanced learners. c. https://jayapanguspress.penerbit.org/index.php/cetta https://jayapanguspress.penerbit.org/index.php/cetta 240 d. Adults need to have the choice to take advantage of the consequences of their
immediate progress. 3. Principles of Adult Education A
person's legitimacy is still airborne by proving in the truth of activity or direct insight,
not with knowledge in discourse. g
b. Not Patronizing. Every individual involved in the educational process is treated
equally, educators are also learners. c. Dialogical. The continuous learning cycle to date is not a one-way educational and
educational experience, but is a correspondence interaction in different types of
exercises, for example, group conversations, role playing and so on. d. Reorder (reconstruction). Uncovering details, such as facts, elements, sequences of
events and so on from the reality. This stage can also be called the process of
experiencing; because this process always starts with an experience call by doing
direct activities. In this process learners engage and act following a certain pattern. What he does and experiences is to practice, observe, see, and say things. It is this
experience that ultimately becomes the starting point of the next learning process. e. Phrase. After the stage experiences, the next phase is the process of expressing by
restating what he has experienced, how to respond, impression of the experience. e. Phrase. After the stage experiences, the next phase is the process of expressing by
restating what he has experienced, how to respond, impression of the experience. f. Review (analysis). Discuss the causes and heterogeneity of the problems that exist in
this reality, namely the order, rules, systems that are the root cause. f. Review (analysis). Discuss the causes and heterogeneity of the problems that exist in
this reality, namely the order, rules, systems that are the root cause. g. Conclusion. Formulate the meaning or essence of this reality as a lesson and a new
understanding that is better, in the form of principles, or in the form of general
conclusions from the results of the study of the experience. Thus, what is experienced
and learned will help summarize, detail, and clarify the things that have been obtained. h. Action. It is the final phase of the critical education process, which is to decide and
implement new actions that are better based on the results of understanding the reality,
making it possible to create new, better realities. This step can be more actualized by
planning actions in the implementation of the principles that have been concluded
(Tatang, 2016). https://jayapanguspress.penerbit.org/index.php/cetta 241 Adults need prerequisites, in particular educational experiences that can foster
components of adult mentality and behavior (mature individuals). 3. Principles of Adult Education Adults generally have a variety of assessments, insights, and learning approaches. q. Learning for adults focuses more on problems than content. r. Learning for adults can sometimes be a troublesome process. s. Learning is a process of progress and development. The various different concepts that have been expressed above are then used as
rules and references to investigate the repetition of the Qur'an which provides a
fundamental stance and commitment to adult training. a. Change from depending on others to living freely. b. The change from a latent mentality and a way of behaving into a dynamic perspective
and way of behaving. b. The change from a latent mentality and a way of behaving into a dynamic perspective
and way of behaving. c. Change from an abstract mentality to a true character. c. Change from an abstract mentality to a true character. d. Change from disposition and data retrieval behavior to data-giving mentality and
behavior. e. Change from low-ability to higher ability. f. Change from a limited liability to a broader obligation. g. Change from a unique interest to a different interest. h. A change from an individualist attitude towards paying attention to others. h. A change from an individualist attitude towards paying i. Change from the attitude of rejecting one's own statements to accepting self-
realization. j. The change from a diverse personality to self-honesty. k. Change from special thinking to standard reasoning. l. The change from a horizontal view to a deep view. m. The change from modeling mentality and way of behaving towards improving
perspective and way of behaving. n. The change from a consistency mentality to an attitude of capacity to bear contrast o. The change from a sloppy mentality to a wise attitude (Sudjana, 2007). o. The change from a sloppy mentality to a wise attitude (Sudjana, 2007). From some of the opinions that have been described above, it can be conclud From some of the opinions that have been described above, it can be concluded that the principles of adult learning are as follows: a. The inspiration for success comes from itself. c. Adults realize when it's valuable to them. d. Adults realize when their perspective is considered. e. There must be mutual trust among educators and learners. f. Adults should be associated with organizing and assessing their education. g. Want a fun and challenging learning atmosphere. h. 3. Principles of Adult Education The adult aspect
proposed by Harry Overstreet which was later developed by Malcolm Knowlesl as
follows: Adults need prerequisites, in particular educational experiences that can foster
components of adult mentality and behavior (mature individuals). The adult aspect
proposed by Harry Overstreet which was later developed by Malcolm Knowlesl as
follows:
a. Change from depending on others to living freely. b. The change from a latent mentality and a way of behaving into a dynamic perspective
and way of behaving. c. Change from an abstract mentality to a true character. d. Change from disposition and data retrieval behavior to data-giving mentality and
behavior. e. Change from low-ability to higher ability. f. Change from a limited liability to a broader obligation. g. Change from a unique interest to a different interest. h. A change from an individualist attitude towards paying attention to others. i. Change from the attitude of rejecting one's own statements to accepting self-
realization. j. The change from a diverse personality to self-honesty. k. Change from special thinking to standard reasoning. l. The change from a horizontal view to a deep view. m. The change from modeling mentality and way of behaving towards improving
perspective and way of behaving. n. The change from a consistency mentality to an attitude of capacity to bear contrast. o. The change from a sloppy mentality to a wise attitude (Sudjana, 2007). From some of the opinions that have been described above, it can be concluded
that the principles of adult learning are as follows:
a. The inspiration for success comes from itself. b. Adults have a willingness to learn. c. Adults realize when it's valuable to them. d. Adults realize when their perspective is considered. e. There must be mutual trust among educators and learners. f. Adults should be associated with organizing and assessing their education. g. Want a fun and challenging learning atmosphere. h. Adults are looking for ways to find out their advantages and disadvantages. i. The direction of adult learning is fixated on reality. j. The source of learning materials for adults is in the individual himself. k. Prioritizing the role of adults as learners. l. Learning is a profound and scientific cycle. m. Learning for adults is a consequence of facing something. n. Learning is the result of human participation. o. There may be mutual communication and exchange of thoughts. p. 4. Adult Education Goals The purpose of teaching for adults is not exactly the same as the purpose of school
for children. In children's schools, learning goals are not fully determined before the
implementation of learning exercises, but in adult training, learning goals can be adjusted,
which means that teachers and students can decide together as needed which is considered
more important for adults study meeting. On this premise, Suprijanto states that the reason
for adult teaching lies in the purpose of learning whose approach is heavier on expanding
capacity and pragmatic abilities in the most limited opportunities to meet their daily needs
(Suprijanto & Arikunto, 2016). Similarly adult education is also regulated to foster the soul and administrative
mentality found in every adult student. Individuals who are in this age range of adults are
known to be capable people, given their reality that they cannot be separated from their
obligations as the head of a family, association, or certain situations in their work. That is
why the direction of adult training cannot be separated from the increased perspective of
authority, so that the educational experience is coordinated to think fundamentally, make
choices and move, supervise the main administration, overcome problems (problem
solving) and so on. Another part of the goal of adult education is to help adult learners understand and
understand the desperation of the requirements for deep-rooted teaching. Adults are largely
persuaded to expand their expected information or abilities to work on their government
assistance. In addition, the authority requirements a certain type of ability to build
compensation or pay at work. Clearly this urges adults to participate in non-formal
education that takes place in society. In this way, training is still needed by adults as long
as longevity is still lived. g
y
In addition, adult education is also expected to help adult learners achieve progress
in the process of learning, deep, and otherworldly personal development. In following the
learning cycle, adults are prepared and accustomed to creating ideal models of reasoning,
attention, encouragement, and obligation, social considerations, and having a person who
is commended as a person who believes in God. Through information, experience, and
abilities obtained through teaching, adults can carry out improvements in the community. Another goal of adult education is to prepare for the expected ability to find and
deal with problems. 3. Principles of Adult Education Adults are looking for ways to find out their advantages and disadvantages. i. The direction of adult learning is fixated on reality. j. The source of learning materials for adults is in the individual himself. l. Learning is a profound and scientific cycle. m. Learning for adults is a consequence of facing something. There may be mutual communication and exchange of thoughts. Adults generally have a variety of assessments, insights, and learning approaches. q. Learning for adults focuses more on problems than content. r. Learning for adults can sometimes be a troublesome process. s. Learning is a process of progress and development. The various different concepts that have been expressed above are then used as
rules and references to investigate the repetition of the Qur'an which provides a
fundamental stance and commitment to adult training. https://jayapanguspress.penerbit.org/index.php/cetta 242 4. Adult Education Goals The ability to find and deal with issues is important to adults, as it
relates to the ability to answer and track answers to life's problems, as well as the ability
to make appropriate and insightful choices and activities. Thus, the direction of adult
training underlines the problem and the effort to solve it, not set to the problem. In addition, coordinated education for adults is also intended to provide assistance
for adults to become free, independent, and independent human beings. The evolving
experience created for adults does not depend on the requests and directions of educators,
but can be done at the instigation of the learners themselves, and learners are given the
opportunity to choose and find out who the appointed teacher or staff is. Therefore, adult
education educators are required to have self-freedom in making views and actions. Based on the explanation above, it can be concluded that adult education has
several main objectives, namely: a. Helping adult learners have the knowledge, experience and skills to improve well-
being for their lives; a. Helping adult learners have the knowledge, experience and skills to improve well-
being for their lives; b. To help adult learners understand themselves, their talents, their limitations, and their
interpersonal relationships; c. Developing the spirit and attitude of leadership that is found in every adult learner c. Developing the spirit and attitude of leadership that is found in every adult learner;
d
Help adult learners know and understand the urgency of lifelong education needs d. Help adult learners know and understand the urgency of lifelong education needs. 243 https://jayapanguspress.penerbit.org/index.php/cetta e. Help adult learners achieve progress in the maturation process intellectually,
emotionally, and spiritually. e. Help adult learners achieve progress in the maturation process intellectually,
emotionally, and spiritually. f. Complete the skills necessary to find and solve problems. f. Complete the skills necessary to find and solve problems. a. Physical Factors Actual physical factors determine the success or disappointment of adults in
learning. There is a negative relationship between getting older and adult learning capacity. That is, the more established a person develops, the more difficult it will be for him to
learn (given the fact that all parts of his actual capacity are reduced). For example, hearing,
vision, body strength, thinking capacity, ability to focus, etc. all show age-appropriate
declines. 5. Factors Affecting Adult Education In general, there are two factors that can affect adult education, especially internal
factors and external factors. Internal factors are factors that come from within learners who
are learning, such as physical factors and psychic factors. Physical factors combine
hearing, vision, and other physiological states, while mental elements include
requirements, insights, inspiration, consideration, thinking, memory, and neglect. External factors are factors that come from outside the actual learner, for example studying
environmental elements and event framework factors. For this situation, learning
environment factors include normal, physical, and social environments, while presentation
system factors include educational programs, performance materials, and learning
strategies. More clearly, the factors that affect the education or learning process of adults can
e described as follows: a. Physical Factors b. Psychic Factors b. Psychic Factors In addition to the physical aspect, psychic factors also have a big influence in
determining the success or failure of adults in the learning process. Psychic factors that
affect the process of adult learning interaction in the outline can be grouped on the aspects
of intelligence / talent, motivation, attention, thinking, memory / forget, and so on. 1) Intelligence/talent
Intelligence / talent is one of the important elements that determine the success or
disappointment of a person in taking an interest in a particular learning movement. The task of educators is to create potential insights / abilities of citizens to learn in
concentrating on a teaching material as ideally as possible. Regarding the formation
of this intelligence potential, the critical period theory developed by Bloom explains
that ± 50% of the intelligence possessed by adults is obtained at the age of ± 4 years
and ± 80% obtained by the age of ± 8 years. This means that the period of education
in kindergarten and in elementary school is the most important learning experience
in one's life. Other experts suggest that a difficult environment at a young age can
cause a person to experience a loss or slow intelligence of approximately 2.5 IQ per
year (Basleman & Mappa, 2020). Intelligence increases sharply from birth to age
approximately 20 years ago began to decline at the age of 35 to 60 years, then
decreased slightly in line with the retreat of one's health in old age. George J. Mouly
suggested that human differences can be demonstrated by a person's level of
intelligence and age. The difference between those with high IQs and those with low
IQs tends to increase as they age (Mouly, 1965). Intelligence / talent is one of the important elements that determine the success or
disappointment of a person in taking an interest in a particular learning movement. 2) Motivation
According to Mark R. Douglas, the main part of motivation is hope. Hope is an
amazing desire, which is the dream torealize what is desired and develop beliefs and 2) Motivation
According to Mark R. Douglas, the main part of motivation is hope. Hope is an
amazing desire, which is the dream torealize what is desired and develop beliefs and 244 https://jayapanguspress.penerbit.org/index.php/cetta plans to achieve goals (Douglas, 2010). Motivation can lead a person to obtain the
right learning goals. 3) Think Thinking is one of the psychic factors that add to the progress of adults in learning. Thinking is a psychological movement as a thought in an information-driven view
by producing cause-and-effect relationships that are calculated and organized
logically and plausibly. In general, thinking activities include steps such as the
following: a) Formation of key understanding as the initial stage of thinking. b) Understanding/evidence that can be recognized from the problem to be thought
of. c) Preparation of complaints for the preparation of assessments or critical thinking. ) Preparation of complaints for the preparation of assessments or critical thinking. ) Withdrawal of conclusions (Idri, 2009). d) Withdrawal of conclusions (Idri, 2009). 4) Memory/Forgotten
Memory is a mental action that allows an individual to repeat the information he has. Remembering is the capacity to repeat information or experience gained. Memory
has the following stages: a) Fixation, which is remembering something vital that happened deliberately and
related to the experience experienced. A decent obsession is a quick and
intensive expression. b) Retention, which is an attempt to save an impression without acknowledging it
that can be raised assuming the circumstances requested / needed. c) Evocation or reproduction, that is, being a special solution or familiarity with
the impressions that are removed (Idri, 2009). c. Learning Environment Factors b. Psychic Factors With good motivation, one can study diligently and passionately
so that it is easy to understand the science material learned (Idri, 2009). plans to achieve goals (Douglas, 2010). Motivation can lead a person to obtain the
right learning goals. With good motivation, one can study diligently and passionately
so that it is easy to understand the science material learned (Idri, 2009). Students who are less successful in completing learning tasks often seem miserable
d
i
l
d F
thi
it
ti
th
i
t
t
/ f
ilit t
d
t
id plans to achieve goals (Douglas, 2010). Motivation can lead a person to obtain the
right learning goals. With good motivation, one can study diligently and passionately
so that it is easy to understand the science material learned (Idri, 2009). Students who are less successful in completing learning tasks often seem miserable
and uninvolved. For this situation, the instructor / facilitator needs to provide
inspiration to fortify his learning soul. This inspiration can begin by providing
persuasive solidarity to the learning citizens, then guiding them to conduct learning
exercises/ meetings, and then deciding on activities that are considered common to
achieve the goal. c. Learning Environment Factors 2) Off-campus learning environment/place of study
The natural environment outside the campus includes topography, flora and fauna as
well as the community and the type of livelihood of the population around the
campus can be a source of teaching materials and a source of motivation for residents
and learning to support the ongoing process of passionate teaching and learning. Physical environments, including buildings, offices, public housing, factories,
installations, projects, roads, bridges, ports, amusement venues or parks and
environmental sanitation around the campus can also be a source of teaching
materials and a source of inspiration for learning and learning residents. The social
environment includes social structures, customs, local culture, cooperation,
sympathy and kinship towards young generations who learn, can also add passion to
learning generayoung person. c. Learning Environment Factors The learning environment can be divided into an on-campus environment where
you study and an off-campus environment / place of study. These two environments can
also be further differentiated into natural, physical, and social environments. The learning environment can be divided into an on-campus environment where
you study and an off-campus environment / place of study. These two environments can
also be further differentiated into natural, physical, and social environments. 1) The learning environment within the campus where you study. The on-campus learning environment combines conditions, temperature, humidity
and indoor air and light trading, all of which include ventilation arrangements and
structural lighting. This classification combines plants that are nearby. Meanwhile,
the physical climate includes structures, furniture, buildings, nurseries, water and
sewage systems as well as the hardware and learning materials used, given the
development and design of all items for the land. In addition, the social climate also
includes a learning climate, including an atmosphere of equal relations between all
citizens, learning assets, and guides around. A charming natural environment can
increase the determination and energy to take part in the learning association process. The utilization of tables, chairs and whiteboards on wheels considers changing
learning collaborations and supports learning exercises and brings learning to life. https://jayapanguspress.penerbit.org/index.php/cetta 245 Close proportional relationships between residents, learning assets, and guides can
strengthen the recognition of local areas of learning. Close proportional relationships between residents, learning assets, and guides can
strengthen the recognition of local areas of learning. 2) Off-campus learning environment/place of study
The natural environment outside the campus includes topography, flora and fauna as
well as the community and the type of livelihood of the population around the
campus can be a source of teaching materials and a source of motivation for residents
and learning to support the ongoing process of passionate teaching and learning. Physical environments, including buildings, offices, public housing, factories,
installations, projects, roads, bridges, ports, amusement venues or parks and
environmental sanitation around the campus can also be a source of teaching
materials and a source of inspiration for learning and learning residents. The social
environment includes social structures, customs, local culture, cooperation,
sympathy and kinship towards young generations who learn, can also add passion to
learning generayoung person. 6. Innovative Strategies in Adult Education The main purpose of introducing innovative strategies and methods is to promote
personal and professional development amongst the adults. In the implementation of
innovative strategies, there is a new type of learning activity that has been introduced with
the group or category of learners, who have not experienced it before and use of a learning
activity that has some new elements, e.g. a new set of materials such as, hand-outs or
worksheets, a different form of presentation of the new input, a new way of grouping
learners, different sequencing of steps to complete the activity, and a different focus on
some targeted learning behaviour (Kapur, 2018). g
g
p
Adult education has developed innovative strategies and methods with a focus on
self-direction (autonomy), shared ownership of training organizations (autonomy and
relatedness), active engagement in learning (relatedness), immediate applicability of
learning (competence), recognition of learner achievement (competence, relatedness), and
supportive emotional environment (relatedness). Innovative strategies and methods are
used to create an environment in which students feel motivated and encouraged. Conditions are created that support students' experiences of autonomy, competence, and
relatedness, as well as those that foster the highest levels of motivation and engagement,
such as self-regulation for learning, improved academic performance, determination in
learning, creativity, resourcefulness, and well-being (Boldisevica & Dislere, 2015). More specifically, the following are the types of innovative strategies that affect
the education or learning process of adults (Kapur, 2018). a. Biographic Work Biographic work is a novel approach to actively involving learners from deprived,
marginalized, and socioeconomically disadvantaged communities in the development of
cultural awareness and expression. Biography is a personal approach; it is an opportunity
to learn more about oneself, to identify and connect the past, present, and future, one's
social environment and personal life, personal viewpoints and objectives. The primary
goal of this work is to identify the purpose of life and learning and to implement measures
to actively initiate them. Working with these subject areas allows one to make resources
available and learn about the possibilities for redesigning concepts relevant to one's own
life. b. Online Café in Language Learning https://jayapanguspress.penerbit.org/index.php/cetta c. Community Mapping c. Community Mapping The new purpose of community mapping is to evaluate the use of a popular
community development activity. Community mapping is a strategy that uses maps to gain
understanding of a local situation and to facilitate discussion within a diverse group. It
serves as the foundation for developing a shared understanding of the limitations and
features of a community or neighborhood, as well as how all participants in the discussion
fit into it. Participants collaborate to create a map of their community that depicts the
locations of various capitals, resources, activities, and opportunities. Everyone's input is
required to create a detailed description of the community. It is a novel approach to utilizing learning materials. The innovative strategy,
stepping into the picture, is a learning activity that involves the use of a picture depicting
a real-life, typically difficult situation. It takes active participation from the participants to
expose their understanding of the specific theme or issue represented in the image and to
share their knowledge and experience with the topic. The strategy aims to improve the
participants critical and creative thinking, decision-making, and problem-solving abilities. It also helps participants develop their social and civic skills, which has the potential to
motivate them to continue their education. It is applicable to a variety of adult groups and
frameworks. e. Reading with Predictions Reading with predictions is a novel strategy used by parents with their young
children. Reading with prediction is based on posing open questions to a group of readers
who are all reading the same text and stopping at specific points to discuss and reflect on
the text's meanings. Reading books, listening to stories, experiences, and examples, and
participating in book discussions all help to achieve better academic results later in life. This innovative strategy promotes the development of reflective learners; it is founded on
purposeful inquiry and includes a variety of features. It allows readers to set their own
reading objectives, it encourages readers to frame their own questions, it keeps readers
actively engaged in the reading process, raises motivation for reading, generates
interesting discussions, encourages readers to express their opinions, it creates a thoughtful
and considerate learning environment, individuals are allowed to express their viewpoints
and perspectives, and readers acquire understanding of conceptual knowledge. f. Basic Skills for Group Discussions In adult education, group discussions are a common activity in which adult learners
participate. b. Online Café in Language Learning An online café in language learning is a new medium for practicing structured
communication in a foreign language. The online café is essentially a forum. Both
institutions used it to motivate language students during the CreMoLe project's piloting 246 https://jayapanguspress.penerbit.org/index.php/cetta and cross-testing phases. Blended learning includes the online café. Blended learning is a
synthesis of the benefits of face-to-face instruction and technology-based e-learning. During the e-learning phase, the trainer must provide instructions to the students. An e-
learning platform should ideally be used for this purpose, where the trainer can provide
students with information or additional materials, quizzes, open online forums, and so on
at any time and from any location. The implementation of blended learning was a lengthy
process. Various types of exercises were developed and tested during this phase. Both
institutions used the open source e-learning platform moodle during the cross-testing
phase. g. Film as a Tool for Motivation A number of educational researchers have discovered that when film is used in the
classroom, students are more motivated to engage in learning. Some educators see film as
a form of public pedagogy that engages students in dialogue. Individuals become more
motivated to learn after watching a film that piques their interest and enthusiasm. The main
goal of developing this innovative strategy is to capitalize on the power of film as a
potential motivator for long-term participation in lifelong learning. h. Active Participation in Occupational Safety and Health Education After more than 20 years of providing workforce with the necessary training to
lead to their professional development, it has been observed that there is a significant lack
of motivation in trainees participating in mandatory training activities mandated by
legislation, which workers must complete in order to perform their job well. When
introducing the innovative training strategy, the main goal was to motivate low-skilled
workers to continue with job-related training and improve their engagement in learning. The following strategy was tested on two different target groups. c. Community Mapping These take place with their classmates, supervisors, and instructors. There are
times when adult learners are apprehensive and unable to participate in group discussions. This is especially prevalent among people from deprived, marginalized, and
socioeconomically disadvantaged backgrounds. Innovative strategies for developing basic
skills for group discussions allow individuals to hone their communication skills so that
they can participate effectively. This innovative strategy assists adult learners in
developing their communication skills. 247 https://jayapanguspress.penerbit.org/index.php/cetta https://jayapanguspress.penerbit.org/index.php/cetta g. Film as a Tool for Motivation 7. Use of Innovative Teaching and Learning Strategies on Adult Education https://jayapanguspress.penerbit.org/index.php/cetta https://jayapanguspress.penerbit.org/index.php/cetta 248 One can obtain a wide range of knowledge and information from the internet. Other
functions, such as mathematical calculations, spreadsheet preparation, letter preparation,
notice preparation, pamphlet preparation, and so on, can be easily performed using
technology. gy
The primary goal of incorporating innovative strategies and methods into the
teaching-learning process is to impart, support, and improve on the activities and methods
associated with the teaching and learning processes. For example, if an instructor is
teaching adult learners about using digital technology, it is critical that he perform the task
practically in front of the students and provide them with the same equipment to help them
learn effectively. In most cases, when students practice the concept after their class times,
they will not encounter any problems or difficulties. When information about using
computer technology is provided, computers are given to each student, and the teacher
gives instructions while all of the students perform the task alongside him. Students can
thus improve their understanding and feel satisfied with the teaching and learning
processes. The verbal explanations provided by the instructors become more clear to the
learners, allowing them to gain an adequate understanding. The main advantages of the
innovative strategies and methods are that they remove impediments to learners'
participation in group discussions and other class activities. To improve the quality and
efficiency of adult education, proper performance appraisal methods must be
implemented. These methods aid in identifying flaws and inconsistencies and in initiating
measures that lead to improvement to accelerate the process of assessing skills and
competencies, as well as their validation and recognition. To ensure that adequate funds
are available because they are regarded as critical to effectively monitor the adult education
sector (Boldisevica & Dislere, 2015). Innovative strategies and methods contribute to more creative and ingenious
teaching and learning processes. It is critical for teachers to have adequate knowledge and
understanding of the subject they are teaching in order for the teaching-learning processes
to function. There have been instances where teachers arrive to class unprepared and are
unable to provide appropriate answers to questions posed by students. As a result, the
internet is regarded as one of the critical areas that contribute to teacher knowledge
enhancement. Teachers can improve their knowledge and understanding by using
innovative strategies and methods. Students, on the other hand, can learn more effectively
by employing novel strategies and methods. 7. Use of Innovative Teaching and Learning Strategies on Adult Education When implementing innovative strategies and methods in any field, it is critical to
analyze them to ensure that they are beneficial and worthwhile. The following are some
examples of innovative strategies and methods used in the teaching-learning process. Students are more likely to remember information with which they can agree or
relate, and to forget information with which they disagree or that is of no use to them. Disagreement or disharmony is investigated objectively. Adult learners can improve their
writing skills, particularly sentence structure, thanks to the innovative strategies and
methods. Then, perspectives can be reinforced or altered. These techniques make learning
more manageable. The diversity of the students should be the basis for questioning and
discussion. These strategies have improved adult learners' interactions with their
instructors (Baxter, 2007). Individuals frequently complain about finding lectures boring, especially when the
topics are abstract in nature. These students work better by themselves and gain a better
understanding of the concepts by working independently, so they are less expected to
perform well in groups. Diverger learners learn from case studies and actively participate
in group discussions, but they may struggle to separate personal values from the issue. These students are frequently inspirational group leaders. Individuals with the assimilator
personality influence ideas effectively, so they will participate in group discussions or
write comprehensive papers. These students, however, may be less practical and struggle
with some of the realism of nurturing practice. Case studies, novel or unusual teaching
strategies, skill labs, and an attempt to comprehend new equipment are typical interests of
accommodators. These students are more likely to respond to difficult and complicated
situations (Baxter, 2007). Technology is the first and foremost innovative technique that has facilitated not
only learning among adults, but also learning among children. Computers and laptops are
widely used in technology. Adults of all backgrounds and categories are taught how to use
technology to conduct research and prepare assignments and reports. Adults, particularly
those who have never used technology before, are initially hesitant to use it. However,
once they have a thorough understanding of it, they can use it effectively. When writing
on a computer, errors and mistakes are easily corrected. Individuals' primary need is to
improve their writing abilities. Power point presentations can be prepared in an appealing
manner. Through the internet, one can expand his or her knowledge and understanding. 7. Use of Innovative Teaching and Learning Strategies on Adult Education For example, if the teacher is teaching about
environmental pollution, students will be able to gain a better understanding of the concept
if they can view pictures of the polluted environment on the internet. https://jayapanguspress.penerbit.org/index.php/cetta 8. Implications of Adult Education in a Learning Society The practice of adult education in order to practice the concept of lifelong
education (lifelong education) has developed rapidly in the midst of people's lives. Adult
education not only takes place in formal institutions such as universities, but also takes
place in community institutions, such as courses, counseling, exercises, or taklim
assemblies. Adult education is carried out to meet learning needs throughout life as long
as the community exists. People gain knowledge, skills and other understandings not only
enough formally, but the community also needs to receive other education as
complementary, both through informal and non-formal education. The implementation of adult education in society with the principle of lifelong
education (lifelong education) generally aims to help the community in facing life
problems objectively, facilitate adults with the power to solve problems, help the 249 https://jayapanguspress.penerbit.org/index.php/cetta community in changing their social situation, and help the community acquire the
necessary knowledge or skills to complete their living needs. Adults in every country need learning exercises. Teaching is needed for people of
all ages and ages as a result of the demands of social change and improvement along with
the advancement of science and innovation today. As stated by Soelaiman Joesoef, in the
midst of the rapid advancement of science and innovation, adults feel the absence of their
abilities and at the same time need important new abilities (Idri, 2009). To answer this need, there has been established a course organization or similar
type of training locally that has a project to prepare adults to master skills in a particular
type of work as well as about the type of work in a very wide scope. Preparatory courses
and programs like these are supervised in a coordinated manner to aid adult learning
exercises as a type of implementation of entrenched training guidelines. In addition to courses and training organizations, local adult learning exercises are
also created as taklim assemblies. Practically speaking, the taklim assembly is the most
suitable place for strict Islamic teaching or schooling and is not limited by the overall
arrangement. Silaturahmi taklim is available for all ages, layers or social layers and
orientations. The adaptation of the taklim assembly makes the field of power for it can be
obtained and is the closest islamic educational foundation to the ummah (community). 8. Implications of Adult Education in a Learning Society Therefore, taklim gatherings become the educational platform of choice for assemblies
(especially guardians) that require more manpower, time, and opportunities to obtain
rigorous information through proper training (Ridwan & Ulwiyah, 2020). Community-based educational institutions (learning society) such as this taklim
assembly have a leading role in realizing lifelong learning (lifelong education). The
function of such a taklim assembly is one of the solutions for the community to add and
complete the knowledge that is lacking and they have not had time to get in formal
educational institutions. In learning activities in society, adults need to be aware of their
capacity to concentrate on meetings. For adults there is a tendency to need to know their
assets and shortcomings. In this way, it is important that there is a joint assessment by all
individuals gathered, so that it tends to be perceived the value of his judgment as a
reflection material that can judge himself from the perspective of others whose insights
may contrast. To answer the above, it is important to grow the idea of a learning society. The
idea of learning society advocates that learning outcomes be obtained through joint efforts
with others. Whenever a person knows how to play a musical instrument, he asks his friend
how to play the instrument. Then, at that moment, his friend, who was used to him, told
him the best way to play the instrument. The association of two individuals has framed a
learning society. Things like this become significant as a fundamental idea in learning in
nonformal training. To realize the learning community, the implementation of adult schools can be
created in accordance with the idea of learning support points sparked by UNESCO. Learning supporting points ideas include: 1) learning to know, 2) learning to do 3) learning
to be, 4) learning to life together and 5) learning to believe in God. These five support
points are a collection of various information and abilities obtained by people from youth
to adulthood. Adults who have been equipped with basic knowledge will have a variety of
knowledge and thinking skills. This blend of information and figuring ability can be
shaped into the capacity to act, work on one's own nature, the capacity to help others, and
work on personal satisfaction as a strict being. https://jayapanguspress.penerbit.org/index.php/cetta 8. Implications of Adult Education in a Learning Society In addition, the mainstay utilization of
figuring out how to do it in life inspires adult students to be imaginative, overcome
problems, and solve various developments. This premise withdrew from the information 250 https://jayapanguspress.penerbit.org/index.php/cetta he had and the application to his character and the advantages of many individuals with
respect to the beliefs he had. Then, at that time, the principle of learning how to be, makes
adult students live independently without relying on other groups. In this view, people
have the opportunity to get something or act. he had and the application to his character and the advantages of many individuals with
respect to the beliefs he had. Then, at that time, the principle of learning how to be, makes
adult students live independently without relying on other groups. In this view, people
have the opportunity to get something or act. On this premise, the individual is allowed to choose what information he needs to
secure, it is permissible to decide to cooperate with others based on his standards or
religious lessons. In addition, the pillar of learning to live together gives awareness to adult
learners to realize harmony in life in the midst of society. Together with society, adults are
able to gain knowledge, able to live together while still appreciating individual differences
and each other's potential in the pattern of working together. All of this work can be
accounted for to God Almighty. On the other hand, the pillar of learning to believe in God encourages adult learners
to have a universal handle in relating to their environment and connecting with their
creators. This implies that the information one seeks should have the choice to benefit the
goods in nature itself, and how to supervise it for the benefit of all people on an ongoing
basis who are fully responsible to God almighty. All of the above supporting points are important systems that can be created in
adult learning to empower the recognition of the design and culture of a durable learning
society, so that everyone will have personal satisfaction. This is in accordance with the
order of law no. 8. Implications of Adult Education in a Learning Society 20 of 2003 concerning the national education system research 3 which
states: education the ability of the community to foster abilities and form a noble person
and civilization of the state in order to educate the life of the state, planning the
development of the ability of students to become a person who accepts and obeys God
Almighty. Strong, learned, capable, innovative, free, and a majority ruler and a reliable
population (Nasional, 2003). Conclusion Adulthood is a time when you have achieved a mature and complete outlook on
life that can be the basis for making consistent decisions. Adult education is a systematic
and continuous learning process in people who are adults with the aim of achieving
changes in knowledge, attitudes, values, and skills. The educational goals of adults rotate
on their learning goals which approach is heavier on improving abilities and practical skills
in the shortest possible time to meet the needs of their lives. In addition, adult education is
also oriented towards developing the spirit and leadership attitude found in every adult
learner and directs learners to have an autonomous right to take attitudes and actions. Broadly speaking, there are two factors that can affect adult education, namely
internal factors and external factors. Internal factors are all factors that come from within
learners who learn, such as physical factors and psychic factors. Meanwhile, adult
education in order to realize the concept of lifelong education (lifelong education) has
developed rapidly in the midst of people's lives. It generally aims to help people deal with
their problems in life objectively, facilitate adults with problem-solving skills, assist
communities in changing their social conditions, and help people acquire the knowledge
or skills necessary to complement their living needs. References
Basleman, A., & Mappa, S. (2020). Teori belajar orang dewasa. Bandung: PT Remaja
Rosdakarya.
Baxter, D. (2007). Teaching strategies for adult learners. Rivier Academic Journal, 3(2),
1–3. References References
Basleman, A., & Mappa, S. (2020). Teori belajar orang dewasa. Bandung: PT Remaja
Rosdakarya. Baxter, D. (2007). Teaching strategies for adult learners. Rivier Academic Journal, 3(2),
1–3. https://jayapanguspress.penerbit.org/index.php/cetta 251 Boldisevica, M., & Dislere, V. (2015). The use of learning video in handicraft technologies
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English
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Combining disparate data sources for improved poverty prediction and mapping
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Proceedings of the National Academy of Sciences of the United States of America
| 2,017
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cc-by
| 11,700
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Neeti Pokhriyala,1,2 and Damien Christophe Jacquesb,1 Neeti Pokhriyala,1,2 and Damien Christophe Jacquesb,1 aComputer Science and Engineering, State University of New York, Buffalo, NY 14221; and bEarth and Life Institute–Envir
Louvain, 1348 Louvain-la-Neuve, Belgium ring, State University of New York, Buffalo, NY 14221; and bEarth and Life Institute–Environment, Universit´e Catholique d
Belgium Edited by Anthony J. Bebbington, Clark University, Worcester, MA, and approved . Bebbington, Clark University, Worcester, MA, and approved September 26, 2017 (received for review January 9, 2017) Edited by Anthony J. Bebbington, Clark University, Worcester, MA, and approved September 26, 2017 (received for review Recently, there has been a growing interest in realizing the
potential of “Big Data” to understand societal development in
Africa. However, most current studies are limited to using sin-
gle source datasets, such as mobile phone data (7) or satellite
imagery (8). Since poverty is a complex phenomenon, under-
standing it using multiple lenses obtained from diverse datasets
will help to chart more accurate maps for poverty. More than 330 million people are still living in extreme poverty in
Africa. Timely, accurate, and spatially fine-grained baseline data
are essential to determining policy in favor of reducing poverty. The potential of “Big Data” to estimate socioeconomic factors
in Africa has been proven. However, most current studies are
limited to using a single data source. We propose a computa-
tional framework to accurately predict the Global Multidimen-
sional Poverty Index (MPI) at a finest spatial granularity and cov-
erage of 552 communes in Senegal using environmental data
(related to food security, economic activity, and accessibility to
facilities) and call data records (capturing individualistic, spatial,
and temporal aspects of people). Our framework is based on
Gaussian Process regression, a Bayesian learning technique, pro-
viding uncertainty associated with predictions. We perform model
selection using elastic net regularization to prevent overfitting. Our results empirically prove the superior accuracy when using
disparate data (Pearson correlation of 0.91). Our approach is used
to accurately predict important dimensions of poverty: health,
education, and standard of living (Pearson correlation of 0.84–
0.86). All predictions are validated using deprivations calculated
from census. Our approach can be used to generate poverty maps
frequently, and its diagnostic nature is, likely, to assist policy mak-
ers in designing better interventions for poverty eradication. Neeti Pokhriyala,1,2 and Damien Christophe Jacquesb,1 p
p
p
y
Several studies highlight that significant spatial variation of
poverty may be due to a variety of geographic factors, includ-
ing agrometeorological conditions, accessibility and proximity to
markets, access to land, and so forth (9, 10) (see Table S3). Earth
Observation Satellites collect data on metrics such as night-
time lights, vegetation cover, and meteorological conditions. The unique features of such datasets are their global coverage,
high revisit capability, and free availability. A complementary
resource lies in Geographic Information Systems (GIS) analysis. In particular, proximity to important services (schools, hospitals)
and density of infrastructure (such as roads) are all factors that
might contribute to alleviating poverty (11). While satellite and GIS data are apt to observe and under-
stand the availability of and access to natural resources and
manmade structures, they lack information about population
structure, especially the socioeconomic ties, cultural interac-
tions, and micro- and macrobehavior that is essential to under-
standing poverty. One way to study societal interactions is pro-
vided by the widespread use of digital technologies (12). The
Internet is still finding ground in sub-Saharan Africa. However,
mobile phones are a prevalent technology, with adoption rates
of more than 70%, even with 43% of population living in abject
poverty (13). Such widespread use of mobile phones generates an poverty mapping | Gaussian process | mobile phone | remote sensing poverty mapping | Gaussian process | mobile phone | remote sensing M M
ore than 330 million people are still living in extreme
poverty in Africa (1). Consequently, the goal to “eradicate
extreme poverty for all people everywhere by 2030” tops the list
of the 17 Sustainable Development Goals adopted by world lead-
ers at the United Nations summit in September 2015. The lack
of good-quality and fine-grained data to assess poverty regularly
features in discussions of the development agenda for Africa (2,
3). Timely measurement and availability of data are vital in end-
ing poverty. COMPUTER SCIENCES www.pnas.org/cgi/doi/10.1073/pnas.1700319114 Significance Spatially finest poverty maps are essential for improved diag-
nosis and policy planning, especially keeping in view the Sus-
tainable Development Goals. “Big Data” sources like call data
records and satellite imagery have shown promise in provid-
ing intercensal statistics. This study outlines a computational
framework to efficiently combine disparate data sources, like
environmental data, and mobile data, to provide more accu-
rate predictions of poverty and its individual dimensions for
finest spatial microregions in Senegal. These are validated
using the concurrent census data. SUSTAINABILITY SUSTAINABILITY
SCIENCE Despite the nature of the strategies used to reduce poverty,
governments and development agencies need a baseline depic-
tion. Poverty maps provide such a spatial distribution of the
socioeconomic deprivations and help policy makers assess the
impact of interventions. For efficient targeting of policies at
microregions and specific demographics, poverty maps should
be made available at the finest administrative unit of planning. Also, these values should be disaggregated into individual dimen-
sions of poverty, like deprivations in education, standard of liv-
ing, health, and so forth (4). Author contributions: N.P. and D.C.J. designed research, performed research, analyzed
data, and wrote the paper. Currently, the most reliable way to estimate poverty is
through intensive socioeconomic household surveys. However,
this approach is costly and time consuming and can only be real-
istically carried out for a small sample of households. The extrap-
olation of the local poverty estimation to a larger scale is tra-
ditionally done by exploiting links between census (wide area)
and survey (smaller area coverage) data through small area esti-
mation methods (5, 6). These techniques depend on the timely
availability of census, which is typically collected every 10 y and
whose analysis is delayed for poorer economies by years, making
timely updates of poverty challenging. The authors declare no conflict of interest. This article is a PNAS Direct Submission. This article is a PNAS Direct Submission. This open access article is distributed under Creative Commons Attribution License 4.0
(CC BY). This open access article is distributed under Creative Commons Attribution License 4.0
(CC BY). Data deposition: The environmental data used in this manuscript is publicly available at
https://doi.org/10.6084/m9.figshare.4910099.v1. Data deposition: The environmental data used in this manuscript is publicly available at
https://doi.org/10.6084/m9.figshare.4910099.v1. 1N.P. and D.C.J. contributed equally to this work. 1N.P. and D.C.J. contributed equally to this work. 1N.P. and D.C.J. contributed equally to this work. PNAS PLUS PNAS PLUS Significance 2To whom correspondence should be addressed. Email: neetipok@buffalo.edu. Thisarticlecontainssupportinginformationonlineat www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1700319114/-/DCSupplemental. Thisarticlecontainssupportinginformationonlineat www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1700319114/-/DCSupplemental. PNAS
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Published online October 31, 2017
|
E9783–E9792 Table 1. Summary statistics and characteristics of the data used—CDRs, environment, census, and MPI
Summary statistics
CDRs
Environment data
Census
Poverty index
Timeline
January–December 2013
1960–2014
2013
2013
Number of total calls and text
11 billion
N/A
N/A
N/A
Number of unique individuals
9.54 M
N/A
1.4 M
N/A
Spatial granularity of available data
Antenna level (1666)
Vector data—100 m−1·km
Household level
Region level (14)
Cost incurred in data collection
Low/no cost
Low/no cost
US$29 million
Very high cost,
and preparation
(data exhaust)
(data exhaust)
and human expertise
Frequency of update of data
Real time
∼1 y
3–5 y
3–5 y cs and characteristics of the data used—CDRs, environment, census, and MPI Summary statistics and characteristics of the data used—CDRs, environment, census, and MPI Table 1. Summary statistics and characteristics of the data used—CDRs, environment, census, and MPI
Summary statistics
CDRs
Environment data
Cens Our objective is to present a computational framework that
integrates disparate data sources to accurately predict the Global
MPI and its individual dimensions at the finest level of spa-
tial granularity. This framework consists of models trained inde-
pendently on each data source. Each source-specific model
uses Gaussian process (GP) regression (GPR) (15) to infer
poverty values. GP falls under the class of kernel methods,
where the choice of different kernel functions enables one to
learn different nonlinear relationships between the indepen-
dent and target variables. Each GP-based model provides a
probabilistic estimate of poverty for a given commune, includ-
ing the mean and variance of the estimates. The variance pro-
vides a measure of uncertainty, which allows us to combine
the predictions from the multiple data sources. An impor-
tant advantage of this methodology is that the different data
ecosystems need not share any data between them. The indi-
vidual datasets remain private within their specific ecosystems,
and only the output predictions and the associated variances
are shared. unprecedented volume of data called call data records (CDRs). CDRs capture how, when, where, and with whom individuals
communicate. These data, traditionally used by the telecommu-
nication companies for billing purposes, capture both micro- and
macropatterns of human interaction, while preserving the indi-
vidual anonymity via spatial and temporal aggregation. Results p
p
p
p
The study focuses on Senegal, a sub-Saharan country that
suffers from persistently high poverty. This study uses mobile
phone data in the form of CDRs, and data related to food secu-
rity (availability and access components), economic activity, and
access to services are grouped together as environmental data
(Table 1). The CDR variables capture not only the basic phone
use statistics of a user but also the regularity, diversity, and spa-
tiotemporal variability in the user’s mobile interactions. Tables
S1 and S2 detail the variables extracted from CDR and envi-
ronment data, respectively. The poverty maps are produced at
the spatially finest level of policy planning, called “communes,”
and validated at that level using the concurrent census data. Current poverty maps, based on Global MPI (see Fig. 1) and
consumption-based measures (14), do not exist uniformly for
all communes of Senegal. The map produced by our analysis is
available for all 552 communes (see Fig. 2). Such maps can be
generated frequently in between cycles of surveys and census,
since CDR and environmental data are available at fine temporal
granularity. GP Model for Predicting Poverty from a Single Data Source. To pre-
dict poverty for a commune from a single data source (CDR or
environment), the following model is assumed: yi = β⊤xi + f (xi) + ϵ
[1] [1] where yi is the target poverty value and xi is a vector of indepen-
dent variables derived from the particular data source for the ith
commune. The first term is a linear combination of the indepen-
dent variables. The function f () models the nonlinear relation-
ship between yi and xi. The residual term, ϵ, models the remain-
ing unexplained noise and is modeled as a zero-mean Gaussian
random variable—that is, ϵ ∼N(0, σ2
n). Without the nonlinear term, f () in Eq. 1, the model is equiva-
lent to ordinary linear regression. However, a linear model is not
rich enough to capture the relationships between the target and
the independent variables (see Fig. S6), thus motivating the need
for a nonlinear term. Table 2. Spatially cross-validated results of the predictions of MPI, headcount of poverty (H), and intensity of poverty (A), along with
the individual indicators for poverty given by our model using disparate datasets
Multisource data
CDR
Environment
Poverty indicators
and dimensions
Corr. Rank corr. RMSE
Corr. Rank corr. RMSE
Corr. Rank corr. Significance Poverty has traditionally been measured in one dimension,
usually income or consumption, called income poverty. Another
internationally comparable measure is the Global Multidi-
mensional Poverty Index (MPI), which is used in this study. Global MPI is a composite of 10 indicators across three critical
dimensions—education (years of schooling, school enrollment),
health (malnutrition, child mortality), and standard of living
conditions (see Global MPI). Throughout the paper, “poverty”
refers to the Global MPI, and “dimensions” refers to education,
health, and standard of living. MPI is calculated as a product of
the incidence or headcount of poverty (H) and the average inten-
sity (A) across the poor. H is the proportion of the population
that is multidimensionally poor. A is the average proportion of
indicators in which poor people are deprived. Results RMSE
MPI
0.91 (0.06)
0.88 (0.06)
0.08 (0.01)
0.89 (0.07)
0.86 (0.07)
0.08 (0.01)
0.84 (0.09)
0.80 (0.10)
0.10 (0.02)
H
0.91 (0.07)
0.85 (0.08)
10.79 (3.96)
0.90 (0.08)
0.84 (0.08)
10.76 (2.60)
0.83 (0.11)
0.75 (0.11)
13.65 (4.86)
A
0.86 (0.05)
0.85 (0.07)
4.71 (0.96)
0.83 (0.07)
0.82 (0.08)
4.98 (1.14)
0.81 (0.07)
0.79 (0.08)
5.36 (0.75)
Education
0.86 (0.05)
0.84 (0.05)
11.84 (1.88)
0.82 (0.05)
0.81 (0.07)
13.08 (1.68)
0.76 (0.07)
0.74 (0.07)
14.98 (3.03)
Health
0.49 (0.15)
0.50 (0.16)
12.76 (2.12)
0.50 (0.12)
0.52 (0.12)
12.91 (1.92)
0.36 (0.23)
0.35 (0.23)
13.91 (2.32)
Standard of living
0.83 (0.11)
0.75 (0.13)
14.82 (3.92)
0.81 (0.11)
0.74 (0.11)
15.24 (3.45)
0.73 (0.18)
0.64 (0.20)
17.88 (4.50)
The results are compared when single source data are available. Corr., Pearson’s r correlation; rank corr., Spearman’s rank correlation; RMSE, rms error. For
both types of correlations, all P values were less than 10−20. An SD associated with the multiple runs for each measurement is reported within parentheses. Table 2. Spatially cross-validated results of the predictions of MPI, headcount of poverty (H), and intensity of poverty (A), along with
the individual indicators for poverty given by our model using disparate datasets
Multisource data
CDR
Environment Table 2. Spatially cross-validated results of the predictions of MPI, headcount of poverty (H), and inten
the individual indicators for poverty given by our model using disparate datasets
Multisource data
CDR results of the predictions of MPI, headcount of poverty (H), and intensity of poverty (A), along with
rty given by our model using disparate datasets
tisource data
CDR
Environment The results are compared when single source data are available. Corr., Pearson’s r correlation; rank corr., Spearman’s rank correlation; RMSE, rms error. For
both types of correlations, all P values were less than 10−20. An SD associated with the multiple runs for each measurement is reported within parentheses. E9784
|
www.pnas.org/cgi/doi/10.1073/pnas.1700319114
Pokhriyal and Jacques E9784
|
www.pnas.org/cgi/doi/10.1073/pnas.1700319114 PNAS PLUS Fig. 1. Details about the target country, Senegal. On the Left is a composite map of Senegal. Black dots depict the location of mobile towers (antennas). The Voronoi tessellation formed by these towers is shown in gray. The commune (which is the finest administrative unit in Senegal) boundaries are shown
in red. There are 552 communes with 431 rural communes and 121 urban centers. Results The navy blue boundaries are those of regions, which are the coarsest
administrative units in Senegal. There are 14 regions that are named in the map. On the Right is the current (2016) map of Global MPI for four divisions of
the country (West, North, South, and Center). Fig. 1. Details about the target country, Senegal. On the Left is a composite map of Senegal. Black dots depict the location of mobile towers (antennas). The Voronoi tessellation formed by these towers is shown in gray. The commune (which is the finest administrative unit in Senegal) boundaries are shown
in red. There are 552 communes with 431 rural communes and 121 urban centers. The navy blue boundaries are those of regions, which are the coarsest
administrative units in Senegal. There are 14 regions that are named in the map. On the Right is the current (2016) map of Global MPI for four divisions of
the country (West, North, South, and Center). Instead of assuming a fixed parametric form for f (), we adopt
a nonparametric approach, by assuming a GP prior on f (). The
generative process thus becomes: is, K[i, j] = k(xi, xj )—and k is a vector of the kernel compu-
tation between each training input and the test input—that is,
k[i] = k(x∗, xi), k∗= k(x∗, x∗)—and I is an identity matrix. f (x) ∼GP(m(x), k(x, x′))
[2]
yi ∼N(β⊤xi + f (xi), σ2
n), ∀i
[3] [2] Choice of Kernel Function. The role of the kernel function is to
specify how the function values f (x) and f (x′) vary as the func-
tion of their corresponding inputs x and x′. We use the following
kernel function: [3] A GP is a stochastic process, indexed by x∈Rd. Any finite sam-
ple generated from it is jointly multivariate normal (15). m(x) is
the mean of f (x) and k(x, x’) is a kernel function that defines
the covariance between any two evaluations of f (x)—that is,
m(x) = E[f (x)], and k(x, x′) = E[(f (x) −m(x))(f (x′) −m(x′))]. For model simplicity, we assume that m(x) = 0, which is a stan-
dard practice in GP-based methods (15). Results k(x, x′) = σ2
f exp
−∥x −x′∥2
2ℓ2
exp
−∥xs −x′
s∥2
2ℓ2s
[6] [6] where xs and x′
s are the spatial coordinates (latitude, longitude)
of the commune centers corresponding to x and x′, respectively. The first exponent term captures nonlinear dependencies in the
feature space. The second exponent term plays the same role,
but in the geographic space and models, the spatial autocorrela-
tion is a continuous function, which is same as Kriging, a widely
used method in geostatistics (16). The parameter σ2
f is the vari-
ance of the stochastic process f , l is the process length scale for
the feature space part, and ls is the process length scale for the
spatial part. 2
2 Given a training set of examples, D = {xi, yi}N
i=1, the GP prior
on f (), and other terms in Eq. 1, the posterior distribution of y∗
(for an unseen input vector, x∗) is a Gaussian distribution, with
the following mean and variance (see GP Regression Model for
details): ¯y∗:= E[y∗] = β⊤x + k⊤(K + σ2
nI )
−1y
[4]
σ2
∗:= var[y∗] = k∗−k⊤(K + σ2
nI )
−1k + σ2
n
[5] [4] [4] p
p
The quantities β, ℓ, ℓs, σ2
n, and σ2
f are estimated by maxi-
mizing the marginalized log-likelihood of the training data, as
discussed in Materials and Methods. To remove the effect of
spurious features, we couple the GP model with elastic net [5] Here, y = [y1, y2, . . .]⊤, and K is a matrix that contains the ker-
nel function evaluation on each pair of training inputs—that Here, y = [y1, y2, . . .]⊤, and K is a matrix that contains the ker-
nel function evaluation on each pair of training inputs—that Fig. 2. Quantiles of predicted (Left) and actual (Right) MPI at the commune level. The urban centers are depicted by small circles on the map. The communes
in the Dakar and Thi`es regions are shown enlarged. Fig. 2. Quantiles of predicted (Left) and actual (Right) MPI at the commune level. The urban centers are depicted by small circles on the map. The communes
in the Dakar and Thi`es regions are shown enlarged. 2. Quantiles of predicted (Left) and actual (Right) MPI at the commune level. Results wic =
1
σ2
ic
1
σ2
ic +
1
σ2
ie
, wie =
1
σ2
ie
1
σ2
ic +
1
σ2
ie
[7] [7] The weights assign greater importance to the source that pro-
vides a smaller predictive variance, signifying higher confidence
in the prediction for the particular commune. The mean and the
variance for the combined poverty estimate are (see Estimating
Moments of a Mixture Distribution): E[yi] = wic¯yic + wie¯yie
var[yi] = wicσ2
ic + wieσ2
ie + wicwie(¯yic −¯yie)2
[8] E[yi] = wic¯yic + wie¯yie [8] ar[yi] = wicσ2
ic + wieσ2
ie + wicwie(¯yic −¯yie)2 Predicted MPI Poverty Values. The predicted map of MPI for
microregions—that is, 552 communes of Senegal—is depicted
in Fig. 2, Left. Compared with the current poverty map in Fig. 1, our map highlights heterogeneity in the existence of poverty
within each macroregion. The communes toward the interior of
the country have more poverty compared with the rest. The west
regions, containing the capital city Dakar, and communes neigh-
boring the coastal boundary are less poor than the rest of the
country. Of special interest is the spatially large division in the
south, consisting of the regions of Tambacounda, Kedougou, and
Kolda, which are depicted as one color on the current map in Fig. 1 but have communes of varying poverty values spread through-
out. Interestingly, the communes in the Kedougou region in the
extreme southeast corner of Senegal are predicted as wealth-
ier than other communes within the region. The communes
in the region of Ziguinchor, in the southwest corner, are
wealthier compared with other communes in the south. This is
attributed to the fact that Ziguinchor is the second largest city
in Senegal, with the economic advantage of being a port and a
tourist center. As a comparative study of how our model performs using
multisource and single-source data, we experimented with three
datasets—Multisource, CDR, and Environment—to predict H,
A, and MPI at the commune level (see Table 2). We report highly
accurate results for all three targets (H, A, and MPI). Rank cor-
relations are preserved, as we report a Spearman’s correlation
of 0.85 for both H and A. The values of Pearson’s r correla-
tion are much higher than rank correlation, across all prediction
tasks, indicating the linear correspondence of the poverty val-
ues with the predicted ones. Results We report significantly low P values
(< 10−34) for spatial CV compared with standard CV, signify-
ing more stable performance. For detailed results, see Table S5. Table 2 shows that combining multiple data sources (CDRs and
environmental data) results in a consistent improvement of accu-
racy over using the individual data sources. The improvement is
more pronounced in detailed results for all of the indicators of
poverty and given in Table S5. Fig. 3, Left plots the relationship between MPI values pre-
dicted by our model and those estimated from census. We
observe a linear relationship, in general, for MPI, with lower val-
ues for urban areas (shown in red) and higher values for rural
areas (shown in blue). Predominantly urban communes of Dakar
and a few urban centers are underestimated for poverty (i.e.,
they are predicted richer than they are). Likewise, there are very
few rural communes, where poverty is overestimated. We also
observe that for communes with lower population densities, the
predicted variance is comparatively higher than it is for com-
munes with higher densities, signifying that lesser numbers of
data points in the vicinity of a given commune contribute to its
higher variance (see Fig. S5). The 121 urban centers are shown as small circles on the map
and, in general, have less poverty values compared with rural
communes. The population in urban centers is generally richer
than the population living in adjacent rural communes. This is
true even for very poor communes of Senegal in the regions of
Kaffrine and Tambacounda in the center, for which the contrast
is even higher. The urban centers bordering with the neighbor-
ing country Mauritania, in the northeast, are wealthier; this could
be attributed to the economy of the Senegal river basin and to
cross-border trade. The predominantly urban areas in Dakar are
shown enlarged in the map. All communes in Dakar are more
well-off than the rest of Senegal because of the concentration of
economic activity over the years. Predicted Values for the Dimensions of Poverty. Global MPI con-
sists of 10 individual deprivation indicators grouped along
three dimensions: (i) education (indicators—years of schooling
and school attendance), (ii) health (indicators—child mortality
and nutrition), and (iii) standard of living (indicators—cooking
fuel, sanitation, access to drinking water, electricity, and floor
and asset ownership). Results The urban centers are depicted by small circ
he Dakar and Thi`es regions are shown enlarged. PNAS
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Published online October 31, 2017
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E9785 Pokhriyal and Jacques regularization (17) during the model learning phase. This allows
for automatic relevant feature selection and learning a parsimo-
nious model that improves interpretability. 90% of communes to the training set and evaluate the remain-
ing 10% of communes. This procedure is repeated 250 times to
provide a robust assessment of the variability of model param-
eters and prediction statistics. Using standard CV, the model
gives a Pearson’s correlation of 0.94, with a P value of <0.0001. Though training and evaluation data are selected randomly, the
above-described method of validation may prove to be insuf-
ficient, as the poverty deprivations tend to be spatially corre-
lated. Thus, a model may appear to perform well when eval-
uated this way, even though it may have poor extrapolation
power in the spatial sense. The above results are provided for
comparison. Combining Source-Specific Models. To predict poverty for a com-
mune, we use two independently trained models specified in Eq. 1, corresponding to the two data sources of CDRs and environ-
mental data. Each model produces a posterior Gaussian distri-
bution, denoted by yic ∼N(¯yic, σ2
ic) and yie ∼N(¯yie, σ2
ie) for
the CDR and environmental data, respectively. The combined
poverty estimate, yi, is assumed to be a mixture distribution con-
sisting of two Gaussians, defined above, and the mixing weights
defined as: p
To measure the extrapolation ability of the model to spatial
areas that were not represented in the training data, we use a
spatial CV procedure (18) (details in Materials and Methods). Here, the training and evaluation sets are sampled from geo-
graphically distinct regions ensuring that the model is tested
rigorously. The experiments were repeated 250 times with ran-
dom samples of training and evaluation sets, while ensur-
ing that all communes are represented in the evaluation. We
report Pearson’s and Spearman’s correlations, and rms error
(RMSE), averaged over the multiple CV runs. The predictions
in Fig. 2, Left have a spatially cross-validated Pearson’s cor-
relation of 0.91 and rank correlation of 0.87, with P values
less than 10−20 for both tests, indicating strong significance. This emphasizes the efficacy of our model in predicting poverty
values accurately at the finest spatial granularity, using multi-
source data. E9786
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www.pnas.org/cgi/doi/10.1073/pnas.1700319114 Pokhriyal and Jacques Results Referring to Fig. S2, nighttime lights appear to be the most
important feature regardless of the predicted dimensions, con-
forming to the current research (8, 20). Nighttime lights show
a strong correlation with MPI (Spearman correlation of −0.66). Urban areas and road density, two other important indicators
of economic activity, are relevant but to a lesser extent. Even
though the coefficient values of each dimension are not directly
comparable, since each dimension was taken as a separate tar-
get, it is interesting to note that the weights of nighttime lights
intensity for electricity and asset ownership deprivation are the
highest. This result confirms previous findings (21) that access to
electricity is correlated with nighttime lights (Spearman correla-
tion of −0.67). Additional observations regarding water depriva-
tion, food security (access component), and climate are given in
Interpretation of Weights—Along the Dimensions of Poverty. Indicators related to education—years of schooling and school
attendance—are predicted well, because use of short message
service (SMS) is indicative of literacy (19). The environmental
data also perform well, because they capture the distance to
schools, main roads, and urban centers, all of which facilitate
access to educational attainment. Fig. 3, Bottom Right shows that
all areas of Senegal are deprived in education, as the rural (in
blue) and urban (in red) points are spread evenly on the plot. However, rural areas tend to dominate at the very high depriva-
tion index, while very low deprivation areas are urban. p
f
g
g
f
y
A similar analysis for the CDR features reveals several inter-
esting insights regarding the relationship between poverty and
the individual characteristics captured in CDR features. While
we considered CDR features for each month individually, for the
ease of visualization (see Fig. S3), we average the monthly values
of the weights associated with each feature. The model performs poorly for the indicators within the
health dimension—that is, child mortality and nutrition. This is
attributed to the fact that our data are not representative of the
children population, and thus, the features extracted from CDR
data do not capture this deprivation. A similar inference can be
drawn for poorer correlations for nutrition. Moreover, the vali-
dation of deprivation values computed from the census for nutri-
tion indicators are based on two hunger-related questions, as
detailed nutritional information is not available to us (see Table
S7 for details). Results Each individual deprivation indicator is
taken as the target of our model, and the averaged spatially
cross-validated results, along the three dimensions, are reported A quantitative validation of the predictions is provided against
commune-level poverty values estimated from census data (see
Fig. 2, Right) using cross-validation (CV) procedures (details in
Materials and Methods). A standard CV is often performed to
ensure that the model generalizes to out-of-sample data. We per-
formed a standard 10-fold CV, where the data are randomly split
into 10-folds. Each time, ninefolds are used for training, and
singlefold is used for evaluation, meaning we randomly assign E9786
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www.pnas.org/cgi/doi/10.1073/pnas.1700319114 Pokhriyal and Jacques PNAS PLUS PNAS PLUS Fig. 3. Predictive power of the Gaussian process model. Left denotes the comparison of actual and predicted MPI values for all communes and urban
areas of Senegal. The rural and urban areas are differentiated using blue and red colors, respectively. The size of the circle denotes the variance of the MPI
prediction for that commune. Top Right shows how the actual and predicted values compare for asset ownership, while Bottom Right shows the comparison
for years of schooling. Fig. 3. Predictive power of the Gaussian process model. Left denotes the comparison of actual and predicted MPI values for all communes and urban
areas of Senegal. The rural and urban areas are differentiated using blue and red colors, respectively. The size of the circle denotes the variance of the MPI
prediction for that commune. Top Right shows how the actual and predicted values compare for asset ownership, while Bottom Right shows the comparison
for years of schooling. in Table 2. Detailed results for each of the 10 indicators are given
in Table S5. for these two indicators. The following interpretations are given
for information purposes. These are, by no means, indicators of
causality. Referring to Table S5, we note that the accuracy of the model
is high for some deprivations and good for most deprivations. All deprivations are better predicted using CDR data, probably
because they characterize the individual behavior while environ-
mental data depict conditions that might have an influence on
poverty (see Tables S1 and S2). Fig. 3, Top Right compares our
predictions for asset ownership with those estimated from the
census. Rural communes depicted (by blue) are clustered closely
toward high deprivation. The urban areas have, generally, lower
deprivation than rural areas, though it is spread out. Pokhriyal and Jacques PNAS
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E9787 Discussion The technological advances over the past decade have led to
building of communication devices (like phones) and sensors
(like satellites and weather and ground sensors) that produce and
store a myriad of data. In this work, we show how these novel
sources of data, which are characterized by their volume, vari-
ety, and associated uncertainty, can be used to generate accurate
poverty maps. An important consideration is the number of features ex-
tracted from the data. Recent work (20) has used four features—
namely, call volume and mobile ownership per capita, night-
lights, and population density—to estimate the MPI of sectors
in Rwanda using a linear regression model. As a baseline for
our model, we used the same features and model to predict
MPI values at the commune level in Senegal. A spatially cross-
validated Pearson’s correlation of 0.84 was achieved with a
significant P value (<0.0001) (see Table S8 for comparison). Although less features provide computational tractability of
analysis, they offer no insight into other features that could be
useful in understanding poverty. Also, linear models are lim-
ited in their ability by the linearity assumption and sensitivity to
outliers. We outline several challenges that lie in establishing relation-
ships between auxiliary data sources (that are not collected to
directly measure socioeconomic deprivations) and poverty. The
first challenge occurs due to the varying spatial granularity at
which the different datasets are available; this requires an aggre-
gation mechanism to link them. CDR data are available for each
subscriber, while environmental data have mixed spatial resolu-
tion, from very accurate vector data to low-resolution satellite
imagery (1 km). On the other hand, census data are available for
individuals or households, depending on the response variable. However, given that the individual information is anonymized
for both CDRs and census data, there is no obvious way to link
the records across these two datasets. In this work, we localize
the individuals and/or households to their respective communes,
or urban centers, by using their census information (details in
Materials and Methods). This lets us calculate the commune-level
deprivations. For CDRs, the individuals are localized to their
home antennas based on their most frequent night location. The
CDR and environmental data are aggregated to commune levels. Though we have taken a commune as the level of aggregation,
the framework allows for the same analysis at even finer spatial
resolutions. Results g
Here we discuss the CDR features that were selected by
the model as the strongest predictors for the various targets. These features are listed in Table S6. One of the strongest
negative predictors for most of the targets is the number of
active days (for call and text), which characterizes that indi-
viduals in wealthier communes have monetary resources to
recharge their phone and make/receive calls. The ratio of calls
vs. text shows the preference for calls and emerges as an impor-
tant factor to predict education-based deprivations. The feature
“interevent time call” measures the irregularity in responding
to calls/text and emerges as a positive predictor for depriva-
tions. Features that indicate diversity in communication, such
as entropy of contacts and interactions per contact (call and
text), report a negative relationship to poverty. These results con-
firm previous findings (7, 22, 23) that diversity of an individual’s Dimensions of Poverty—Interpretation of Weights. Figs. S2 and S3
display the features deemed important by our model for the
environment and CDR data, respectively. The important fea-
tures are those for which the corresponding entries in the coef-
ficient vector, β, are high in magnitude. We ignore child mortal-
ity and nutrition, as our model does not perform very accurately PNAS
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E9787 (like elevation, soil types, etc.) or long-term dynamic phenom-
ena (like climate). The second type includes human-induced
aspects, like urban areas, roads, access to facilities, and so forth. Though the natural environment acts as a constraint in design-
ing poverty eradication plans, effective policies and sustainable
approaches should be made an integral part of policy planning. Environmental features derived from satellite images (nighttime
lights, NDVI, etc.) have the potential to be computed in near
real-time to monitor the impact of shocks such as natural haz-
ards, armed conflicts, or crop pests that can rapidly cause serious
deprivations. However, for reliability, these variables need to be
aggregated for a longer period, typically at an annual level for
nighttime lights and for the growing season for NDVI. Open-
StreetMap (OSM) data, which are used to map facilities and
roads, are crowd-sourced and therefore have the (theoretical)
potential to be updated in near real time. This capability could
be limited in African countries. Results Due to the above constraints,
1 y is probably the relevant period for consistent monitoring of
poverty with our method (compare with 3–5 y for a detailed and
costly census). relationships is positively correlated with his or her economic
wellbeing. However, for features such as percent pareto inter-
actions and balance of contacts, which are proportional to an
individual’s diversity in communication, we report a positive rela-
tionship with poverty. This counterintuitive relationship needs to
be further studied in the context of telecommunication patterns
in Senegal. g
We observe a negative relationship between the “activeness”
of an individual in his or her mobile interactions and poverty. For instance, the delay in responding to text has a positive rela-
tionship to poverty. Interestingly, the feature of percent initi-
ated interactions (calls) has, again, a positive relationship to
poverty, signifying that in Senegal individuals living in more
deprived communes are more likely to initiate calls (for request
of resources, etc.) than those living in less deprived communes. The mobility patterns of individuals, captured using spatial fea-
tures such as number of frequent antennas, entropy of anten-
nas, and total number of antennas used by an individual, indi-
cate a negative relationship to poverty. Thus, individuals living
in more deprived communes tend to move fewer antennas than
those living in less deprived communes. This observation should
be viewed cautiously because of sparse antenna density in rural
communes. Another challenge is the ease of availability of data. Environ-
mental datasets are available to researchers for free and typi-
cally have no privacy constraints, especially at the resolution at
which it is analyzed here. CDR data are collected by commercial
telecommunication entities and might suffer from lack of acces-
sibility to researchers due to sharing constraints between differ-
ent organizations. However, our methodology requires no raw
data to be shared between different data-owning entities; only
the output predictions from each individual model and the asso-
ciated uncertainties are combined at the final step. E9788
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www.pnas.org/cgi/doi/10.1073/pnas.1700319114 Pokhriyal and Jacques Discussion An important advantage of our GPR model is that each pre-
dicted poverty value is associated with an uncertainty (gen-
erated by the model). This highlights the strength of confi-
dence in the predictions and can be used as guidance by policy
makers. Comparing these source-specific uncertainties can
reveal which data hold a better signal for a specific predic-
tion (see Fig. 4). We note that for predicting A, the pre-
dictions of CDRs and environment data are comparable for
most of the communes. For predicting H, CDRs perform with
lower uncertainties than environmental data. These variations
may be attributed to multiple reasons, including resolution
and concurrency of data, demographics and mobile penetration
of the cellular provider, and spatial heterogeneity of poverty
deprivations. A key concern associated with using CDR data for population-
level analyses is the selection bias arising from mobile phone
ownership. In Senegal, however, there were 92.93 mobile phone
subscriptions per 100 inhabitants in 2013, which implies that most
of the population owns cell phones (24). The second challenge is
the bias arising when using data from only one provider. How-
ever, the provider of the data used here, Sonatel, had nearly 62%
of the cell phone market in 2013 (25). The third concern is that
some demographic subgroups like children and the ultra poor are
left out by the analysis while only using CDR data. Also, results
may be biased toward urban regions, rather than rural regions,
because of factors like lack of electricity in rural areas. Though we have discussed the methodology for predictions
at the commune level, our predictions of MPI and associated
dimensions can be successfully aggregated to coarser adminis-
trative units, if needed, for policy planning. Since we use global
MPI as the poverty index, its limitations, as noted by global MPI
researchers (26), are applicable to our study as well. In particu-
lar, global MPI does not include characteristics such as parents’
education, social norms and beliefs, empowerment, etc. y
Here, we used two distinct types of environment data. The
first type includes static natural/physical environment variables p
Additionally, it will be interesting to see how well this method-
ology can be used to predict other indicators of deprivation and PNAS PLUS Fig. 4. Feature Extraction. CDRs. We have access to more than 11 billion mobile phone transactions
involving calls and texts for a year in Senegal. Each time a call or text is
placed, it is logged as a transaction. Missed, forwarded, and other undeliv-
ered calls were removed from the logs. Lastly, though GPR model uncertainty is impacted by the bias
and inaccuracy of each data source (quality of soil type map,
interpolation of climatic data, missing facilities, mobile opera-
tor’s market share), a higher resolution and accuracy of the input
data should benefit the modeling relevance and quality. To extract important features that quantify the mobile use pattern of
a subscriber, we focus on well-studied metrics capturing the individualis-
tic, spatial, and temporal patterns of the subscriber (28–30). The individual
aspects quantify the typical use pattern of an individual. Some of the metrics
that belong to this category are the number of active days, the number of
contacts, the average call duration, percent nocturnal, and so forth. Spatial
metrics are the ones that quantify the typical movement pattern of an indi-
vidual. Examples of spatial metrics for a subscriber include radius of gyra-
tion, entropy of antennas, and so forth. There are 43 core features (briefly
described in Table S2), extracted using the Bandicoot toolbox (31). All fea-
tures were calculated at monthly granularity capturing the temporal aspect
of a subscriber, resulting in 43 × 12 CDR-based features. Materials and Methods Target Country. Senegal is a sub-Saharan country that ranks 170 on the
Human Development Index with a score of 0.466 and a population of 14.5
million (with 43.5% urban population) (27). As one of the poorest coun-
tries in the world, it has 52% of the population living in multidimensional
poverty (27). On the other hand, there are 98.8 mobile phone subscrip-
tions per 100 people (24). Senegal is composed of 14 coarsest administra-
tive units called regions, which are further divided into 45 administrative
units called departments. The finest level of administrative units is called a
commune. There are 552 communes (121 as urban centers and 431 rural)
(Fig. 1). The second step is to localize each subscriber, i, to his or her home
antenna. A home antenna, hi, is calculated as one from where the subscriber
makes the most nocturnal calls (from 7 PM to 7 AM) during each month. We
filtered out individuals who made less than five calls during each month and
who were not active for at least half of the year within the range of their
home antennas. This ensures that individuals are reliably allocated to their
home antennas. After the filtering step, the sample contained 6.19 million
individuals (65% of the original subscriber population). Discussion The uncertainty associated with each dataset evidenced by the most accurate one (denoted as CDR and ENV) for the average intensity of poverty
(A) (Left) and prediction of the headcount of poverty (H) (Right). Fig. 4. The uncertainty associated with each dataset evidenced by the most accurate one (denoted as CDR and ENV
(A) (Left) and prediction of the headcount of poverty (H) (Right). Fig. 4. The uncertainty associated with each dataset evidenced by the most accurate one (denoted as CDR and ENV) for the average intensity of poverty
(A) (Left) and prediction of the headcount of poverty (H) (Right). with each dataset evidenced by the most accurate one (denoted as CDR and ENV) for the average intensity of poverty
dcount of poverty (H) (Right). inequality, like the GINI index, at the microregional level. Apart
from being useful in producing interim statistics in between long
cycles of census and surveys, such methodology can also be
extended to places of conflict or remote areas that are difficult
to reach by census takers. a 10% sample of the 2013 census [called RGPHAE (Recensement General
de la Population de l’Habitat de l’Agriculture et de l’Elevage)]. The data are
evenly sampled across the entire population of Senegal and are from 1.4 mil-
lion individuals, spread across 150,000 households, characterizing informa-
tion related to demographic statistics (mortality, fertility, migration, literacy,
occupation, etc.), along with habitat features, such as type of roof, floor,
access to drinking water, sanitation, and agriculture practices. The advan-
tage of the census is that it represents important national statistics at the
level of individuals. Brief statistics of the data sources are given in Table 1. y
As described in the results, the interpretation of the model
coefficients provides some insights on the dimensions of MPI. However, due to the number of variables, this interpretation is
still complex and not necessarily straightforward for policy inter-
vention. Conversely, the MPI dimensions are well-known fac-
tors for which policy planning is feasible (26). As an illustration,
Fig. S4 shows the highest predicted deprivation for each com-
mune within each dimension. The mobile phone data used in this study can be obtained for repli-
cation purposes by contacting Zbigniew Smoreda (zbigniew.smoreda@
orange.com). Pokhriyal and Jacques Data Sources.
CDR
A CDR The L2 penalty,
imposed by Ridge regression, ensures shrinkage of regression coefficients to
avoid overfitting. On the other hand, the L1 penalty imposed by Lasso forces
the coefficients to be sparse, thereby providing feature selection. However,
neither of the two regularization methods have been found to universally
dominate the other (38). For instance, in the presence of groups of corre-
lated features, Lasso tends to select only one feature within each group,
which leads to poor interpretability of the estimated coefficients. Elastic net
regularization (17) is a weighted addition of L1 and L2 penalties and com-
bines the strengths of both Lasso and Ridge regression. It is known to select
a greater number of influential features than Lasso and has a lower false-
positive rate than ridge regression. The economic activity corresponds to the intensity of urbanization. Among the studied features, the nighttime lights are the most fre-
quently used to describe poverty using remote-sensing data (20). More-
over, a clear link between household wealth and the level of night
light emissions has been shown before (32). The underlying hypothesis
is that economic activity and urbanization are strong indicators of living
standards. Finally, the access to services can help to predict some of the individual
indicators of poverty. In particular, the proximity to school, water towers,
and hospitals can be used to determine the deprivation in education, water,
and health, respectively. We used elastic net regularization to penalize complexity of the solu-
tion and to avoid overfitting on the limited training dataset. The elastic net
penalty is computed as: The raw environmental data are available either in raster grid (at differ-
ent spatial resolutions) or in vector format. As a first step, all vector data
were converted into raster grid format. Then, all data layers were resam-
pled (using the nearest neighbor approach) at a spatial resolution of 100 m. Pixel values falling within each commune’s boundary were averaged to give
a unique value for that commune. αλ∥β∥2
2 + (1 −α)λ|β|
[14] [14] Our empirical results show that elastic net regularization results in better
prediction accuracy, compared with ordinary least squares, Ridge, and Lasso
regression. All environmental data are available at high spatial resolution, with the
exception of crop production and millet prices (see Table S1 for the data
sources). Millet prices were available in 56 local markets, potentially miss-
ing some of the local heterogeneity. Data Sources.
CDR
A CDR Production estimation features were
derived from the Direction de l’Analyze, de la Pr´evision et des Statistiques
Agricoles (DAPSA) database. The granularity of these features is at the
department level. Cultivated areas were masked using the 2005 1:100,000
Scale Senegal Land Cover Map produced by the Global Land Cover Net-
work based on the GlobCover 2005 map (33), which is the most accu-
rate map for Senegal (34). Since reliable information on the spatial dis-
tribution of each crop is unavailable, we made an assumption that they
were grown evenly within the cultivated areas of a specific department. Therefore, the production of a specific department was distributed evenly
among all of the 100-m pixels that fell within the cropland of this depart-
ment. This raster was then used to aggregate the production estimations by
communes. Model Validation. This section details the steps followed to validate our
model, namely creating commune-level poverty statistics from census data
and methodology for spatial CV. Creating commune poverty statistics from census. The 10% sample of
the 2013 RGPHAE census, used here, has survey responses for 150,000
households and 1.4 million individuals pertaining to their socioeconomic
indicators (literacy, birth and death in the family, etc.) and habitat (type
of house, access to electricity and drinking water, etc.). Some survey
responses are individualistic (like literacy and profession), while others
are associated with the entire household (like type of roof, sanitation,
electricity). The first step is to assign the individuals to their respective households
using information from the fields in the census. The second step is to calcu-
late per-household deprivations in the poverty indicators of interest. Global
MPI computation (26) requires deprivations along three dimensions (with
10 indicators)—namely, health (child mortality, nutrition), education (child
school attendance, years of schooling), and standard of living (electricity,
sanitation, drinking water, flooring, cooking fuel, assets). The Normalized Difference Vegetation Index (NDVI) is used as a proxy of
potential agricultural production within a department. The NDVI, defined
as the difference between near-infrared and red reflectances normalized
by the sum of the two parameters, is a useful yield proxy in regions where
water or soil fertility are the main limiting factors, such as Sahel (35, 36). For each pixel within cultivated areas, NDVI values above 0.2 during the
growing season (July to November) were integrated (TNDVI), which limited
the contribution of bare soil to the signal. Data Sources.
CDR
A CDR To study
how well the Voronoi-based approach has performed in assigning people
to their communes, we study the correlation of the commune population
estimated by this approach and that calculated from census. The Pearson’s
correlation is reported as 0.85 with a P value of < 0.00001, thus ensuring
the validity of our approach. log p(y|X) = −1
2 (y −Xβ)⊤(K + σ2
nI)
−1(y −Xβ)
−1
2 log |K + σ2
nI| −N
2 log 2π
[12]
he number of training examples [12] Environmental features. In this study, we focus on three broad cate-
gories of environmental features: food security (divided into the availabil-
ity and access components), economic activity, and access to services (see
Table S1). These three categories cover most of the features that have
been shown to be significantly related to poverty in the literature (see
Table S3). where N is the number of training examples. where N is the number of training examples. where N is the number of training examples. To regularize the coefficients in β, we apply elastic net regularization on
the marginalized log likelihood to obtain the following objective function: To regularize the coefficients in β, we apply elastic net regularization on
the marginalized log likelihood to obtain the following objective function: J(β, ℓ, ℓs, σ2
n, σ2
f ) = log p(y|X) −(αλ∥β∥2
2 + (1 −α)λ|β|)
[13 [13] Food security is mainly described by agrometeorological measurements
(temperature, precipitation, slope, elevation, soil type) that drive agricul-
tural production (crop production), one of the most important inputs, along
with livestock and fishing, of food availability in the country. On the other
hand, access to staple food can be approximated by the average millet
prices observed in the markets (retail prices in 56 local markets). Millet
serves as the main local staple food crop in the country, making it a poten-
tially good indicator of poverty. In addition, proximity to main road and
urban centers was also computed to describe the connectivity to major
markets. The function J is maximized to estimate the hyperparameters using conju-
gate gradient descent (37). All codes used to replicate the results can be obtained by writing to the
corresponding author. Regularization. Regularization techniques, such as those used in Lasso (38)
or Ridge regression (39), are often used to improve model performance,
especially when the data contain several irrelevant features. Data Sources.
CDR
A CDR CDRs. A CDR consists of an identifier with the caller and callee, the antenna
location of the caller, the time of the call, duration of the class, and a
flag indicating if the record is a text or a call. A CDR is generated each
time a call or text is placed. The data belong to the subscribers of Sonatel,
Orange, which is the dominant telecom provider in Senegal. The data are
anonymized and span a period from January 1 to December 31, 2013. They
contain more than 9.54 million unique aliased mobile phone subscribers. The population of Senegal in 2013 was 14.13 million. Additionally, the
geographical coordinates of the mobile antennas are known, and shown
in Fig. 1. We then computed the average feature value for each antenna site by
computing the average of the feature values for all individuals who consider
that antenna as their home: m(f)
a
=
1
Na
X
i:hi=a
m(f)
i
[9] [9] Environmental Features. Based on literature, several environmental fea-
tures that may have a relationship with poverty have been explored (see
Table S1). They are either based on Geographical Information System (GIS),
Earth Observation data, or weather stations. where m(f)
i
is the fth feature value. Finally, we compute the feature value for each commune as the weighted
average of all antennas whose voronoi polygon intersects with the com-
mune boundary as: Census. The Agence Nationale de la Statistique et de la Demographie
(ANSD), which is the National Statistics Office of Senegal, provided us with PNAS
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E9789 PNAS
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E9789 m(f)
c
=
1
P wc,a
X
wc,am(f)
a
[1 p(y|X) =
Z
p(y|f,X)p(f|x)df
[11 p(y|X) =
Z
p(y|f,X)p(f|x)df [10] [11] where the matrix X contains the training input vectors as rows and f is a
vector containing the latent function values for the inputs in X. The GP
prior means that p(f|X) ∼N (0, K) and the likelihood is a Gaussian—that
is, p(y|f, X) ∼N (Xβ + f, σ2
nI). The integration of Eq. 11 yields the following
marginalized log likelihood (15) of the training data: The weight wc,a is the ratio Area(c∩a)
Area(a) , which is a measure of how much of the
voronoi cell for antenna a falls within the boundary of commune c. Pokhriyal and Jacques Data Sources.
CDR
A CDR Jerven M (2013) Comparability of GDP estimates in Sub-Saharan Africa: The effect
of revisions in sources and methods since structural adjustment. Rev Income Wealth
59:S16–S36. 4. Alkire S, et al. (2014) Mobilising the Household Data Required to Progress toward
the Sdgs (University of Oxford, Oxford). Available at unsdsn.org/wp-content/uploads/
2014/09/Mobilising-the-household-data-required-to-progress-toward-the-SDGs-
WEB.pdf. Accessed October 24, 2017. 27. World Bank (2014) Overview. Available at www.worldbank.org/en/country/senegal/
overview. Accessed October 21, 2017. 4. Alkire S, et al. (2014) Mobilising the Household Data Required to Progress toward
the Sdgs (University of Oxford, Oxford). Available at unsdsn.org/wp-content/uploads/
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tices on spatial variation of soil fertility and millet yields in the Sahel of Mali. Agric
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58:267–288. 18. Bahn V, McGill BJ (2013) Testing the predictive performance of distribution models. Oikos 122:321–331. 39. Hoerl A, Kennard R (1988) Ridge regression. Encyclopedia Stat Sci 8:129–136. 19. Sundsøy P (2016) Can mobile usage predict illiteracy in a developing country? arXiv:1607.01337. 40. Alkire S, Foster J (2011) Counting and multidimensional poverty measurement. J Pub-
lic Econ 95:476–487. 20. Njuguna C, McSharry P (2017) Constructing spatiotemporal poverty indices from Big
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ness and scope of the multidimensional poverty index. Data Sources.
CDR
A CDR We thank Nicolas de Cordes and Stephanie de Pre-
voisin (Orange) and the other winner teams of the Data 4 Development
Challenge (D4D), and we thank the two anonymous reviewers for their use-
ful comments. We thank Orange Sonatel, Senegal, and Orange Laboratories,
Paris, for providing the raw CDRs and the National Statistics Office of Sene-
gal (ANSD) for the census. N.P. and D.C.J. are both funded by Bill & Melinda
Gates Foundation Grant OPP1114791. D.C.J.’s work is also partly funded by
the Belgian National Fund for Scientific Research through Fonds pour la For-
mation `a la Recherche dans l’Industrie et dans l’Agriculture Grant 5211815F. ACKNOWLEDGMENTS. We thank Nicolas de Cordes and Stephanie de Pre-
voisin (Orange) and the other winner teams of the Data 4 Development
Challenge (D4D), and we thank the two anonymous reviewers for their use-
ful comments. We thank Orange Sonatel, Senegal, and Orange Laboratories,
Paris, for providing the raw CDRs and the National Statistics Office of Sene-
gal (ANSD) for the census. N.P. and D.C.J. are both funded by Bill & Melinda
Gates Foundation Grant OPP1114791. D.C.J.’s work is also partly funded by
the Belgian National Fund for Scientific Research through Fonds pour la For-
mation `a la Recherche dans l’Industrie et dans l’Agriculture Grant 5211815F. The value of threshold θ is taken as 0.3. We varied θ from 0.2 to 0.75,
and the H and A values obtained in each run were correlated with region-
level H and A, available from University of Oxford’s MPI calculation [Oxford
Poverty & Human Development Initiative (OPHI)]. The results were stable
and peaked at 0.3, which is also the threshold value taken by OPHI for its
calculations. 1. Beegle K, Christiaensen L, Dabalen A, Gaddis I (2016) Poverty in a Rising Africa (World
Bank Publications, Washington, DC). 1. Beegle K, Christiaensen L, Dabalen A, Gaddis I (2016) Poverty in a Rising Africa (World
Bank Publications, Washington, DC). 25. Autorite de R´egulation des Telecommunications et des Postes (2013) Observatoire de
la telephonie mobile: Tableau de bord au 31 decembre 2013 (Autorit´e de R´egulation
des T´el´ecommunications et des Postes, Dakar, Senegal), Technical Report. 2. Devarajan S (2013) Africa’s statistical tragedy. Rev Income Wealth 59:S9–S15. 2. Devarajan S (2013) Africa’s statistical tragedy. Rev Income Wealth 59:S9–S15. 26. Oxford Poverty and Human Development Initiative (2013) Country briefing: Sene-
gal. Available at www.ophi.org.uk/wp- content/uploads/Senegal- 2013.pdf?79d835. Accessed October 21, 2017. 3. Data Sources.
CDR
A CDR We follow the procedure similar to the widely used Alkire–Foster
methodology for computing MPI (40). First, we create a deprivation vec-
tor depveci,d corresponding to each household i in poverty indicators d =
1, . . . , D. Each vector entry is either 1 if yi,d ≤zd, where yi,d is the achieve-
ment of household i in indicator d and zd is the cutoff score in indicator d, or
0 otherwise. A value of 0 for depveci,d implies nondeprivation of the house-
hold in that particular indicator. For the values of cutoff scores for different
indicators, see Table S7. We aggregate all households that are deprived in a
particular indicator, for each commune, and divide by the total number of
households in that commune. This score gives the proportion of households
deprived in a particular indicator within a commune. Model Training. The unknown parameters of each source-specific model
in Eq. 1 are as follows: the parameter β of the linear component, the
hyperparameters of the kernel function ℓ, ℓs, σ2
f , and the variance of the
error term σ2
n. These are estimated by maximizing the marginalized like-
lihood of the target poverty values in the training data y. The marginal-
ized likelihood is obtained by taking the integral of the likelihood times
the prior: Since MPI is a multiplicative combination of H and A—that is, MPI = H ×
A—we first calculate H and A. For H, we introduce a weight, wd, for E9790
|
www.pnas.org/cgi/doi/10.1073/pnas.1700319114 Pokhriyal and Jacques PNAS PLUS each indicator d. For each household, we compute a weighted deprivation
score, ci = PD
d=1 wddepveci,d. The weights wd are assigned as follows. The
education- and health-related indicators are given a weight of 1
6 , while each
of the six standard of living indicators are given a weight of
1
18 . Thus, each
dimension has a weight of 1
3 . each indicator d. For each household, we compute a weighted deprivation
score, ci = PD
d=1 wddepveci,d. The weights wd are assigned as follows. The
education- and health-related indicators are given a weight of 1
6 , while each
of the six standard of living indicators are given a weight of
1
18 . Thus, each
dimension has a weight of 1
3 . Spatial CV. Data Sources.
CDR
A CDR To measure the extrapolation capacity of the model on out-
of-sample data, spatial CV techniques, where training and evaluation sets
are sampled from geographically distinct regions, are more robust (18, 41). The following spatial CV strategy was adopted: For each CV run, we first
randomly sampled a region r from the set of 14 regions and then randomly
sampled a commune c belonging to r. All communes that lie within distance
d of the commune c are included in the training dataset. The remaining
communes are included in the evaluation dataset. Spatial CV. To measure the extrapolation capacity of the model on out-
of-sample data, spatial CV techniques, where training and evaluation sets
are sampled from geographically distinct regions, are more robust (18, 41). The following spatial CV strategy was adopted: For each CV run, we first
randomly sampled a region r from the set of 14 regions and then randomly
sampled a commune c belonging to r. All communes that lie within distance
d of the commune c are included in the training dataset. The remaining
communes are included in the evaluation dataset. 3
Hj, which is the relative headcount of poor households in commune j, is
calculated as: Hj = 1
Nj
Nj
X
i=1
I(ci > θ)
[15] This strategy ensures that communes from all regions of Senegal are repre-
sented in the training and evaluation datasets during CV. To ensure that the
training dataset has enough examples, we forced at least 40% of the com-
munes (225) to be included in the training dataset. To achieve this, d is initially
set to 100 km and is increased by 50 km until the size of the training dataset
meets the threshold. CV is repeated 250 times. We report the mean predictive
performance (using Pearson’s and Spearman’s correlation and RMSE values)
on the evaluation dataset, along with the SD across multiple runs. [15] where θ is a cutoff, whose higher values mean a higher cutoff for household
achievement, and I(ci > θ) is the indicator function. Nj is equal to the total
number of households in the jth commune. To calculate A, we count only the poor households, and their depriva-
tions, as follows: Aj =
1
PNj
i=1 I(ci > θ)
Nj
X
i=1
I(ci > θ) ∗ci
[16] [16] ACKNOWLEDGMENTS. 1. Beegle K, Christiaensen L, Dabalen A, Gaddis I (2016) Poverty in a Rising Africa (World
Bank Publications, Washington, DC). 2. Devarajan S (2013) Africa’s statistical tragedy. Rev Income Wealth 59:S9–S15. Data Sources.
CDR
A CDR World Dev 59:251–274. 22. Eagle N, Macy M, Claxton R (2010) Network diversity and economic development. Science 328:1029–1031. 43. von Mises R (1964) Chapter IX—Analysis of statistical data. Mathematical Theory of
Probability and Statistics, ed von Mises R (Academic, Cambridge, MA), pp 431–493. 23. Soto V, Fr´ıas-Mart´ınez V, Virseda J, Fr´ıas-Mart´ınez E (2011) Prediction of socio-
economic levels using cell phone records. Proceedings of the 19th International Con-
ference on User Modeling, Adaption and Personalization (Springer, Gerona, Spain),
pp 377–388. lity and Statistics, ed von Mises R (Academic, Cambridge, MA), pp 431–4 44. Kim TH, White H (2003) On More Robust Estimation of Skewness and Kurtosis: Simu-
lation and Application to the S&P500 Index (Department of Economics, University of
California, San Diego, La Jolla, CA). lation and Application to the S&P500 Index (Department of Economics, University of
California, San Diego, La Jolla, CA). 45. Hijmans RJ, Cameron SE, Parra JL, Jones PG, Jarvis A (2005) Very high reso-
lution interpolated climate surfaces for global land areas. Int J Climatol 25:
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World
Telecommunication
(2016)
Key
ICT
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for
developed
and
developing countries and the world. Available at www.itu.int/en/ITU-D/Statistics/
Pages/default.aspx. Accessed October 21, 2017. PNAS
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Published online October 31, 2017
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E9791 PNAS
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Published online October 31, 2017
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E9791 Pokhriyal and Jacques 46. Direction de l’Analyse, de la Pr´evision et des Statistiques Agricoles (2013) Rap-
port de Pr´esentation des R´esultats D´efinitifs de la Campagne Agricole 2012-
2013 (Minist`ere de l’Agriculture et de l’´Equipement Rural, Dakar, Senegal), Tech-
nical Report. Washington, DC). Available at siteresources.worldbank.org/INTPGI/Resources/342674-
1092157888460/Minot.PovertyInequalityVietnam.pdf. Accessed October 24, 2017. Washington, DC). Available at siteresources.worldbank.org/INTPGI/Resources/342674-
1092157888460/Minot.PovertyInequalityVietnam.pdf. Accessed October 24, 2017. 53. Rogers D, Emwanu T, Robinson T (2006) Poverty mapping in Uganda: An analysis using
remotely sensed and other environmental data (Pro-Poor Livestock Policy Initiative, 53. Rogers D, Emwanu T, Robinson T (2006) Poverty mapping in Uganda: An analysis using
remotely sensed and other environmental data (Pro-Poor Livestock Policy Initiative,
Food and Agriculture Organization, Rome), Working Paper No. 36. p
47. Jacques D, et al. (2015) Genesis of millet prices in Senegal: The role of produc-
tion, markets and their failures. Fourth Conference on the Analysis of Mobile Phone
Datasets, NetMob 2015 (MIT Media Labs, Boston). Food and Agriculture Organization, Rome), Working Paper No. 36 54. E9792
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www.pnas.org/cgi/doi/10.1073/pnas.1700319114 Data Sources.
CDR
A CDR Benson T, Chamberlin J, Rhinehart I (2005) An investigation of the spatial determi-
nants of the local prevalence of poverty in rural Malawi. Food Pol 30:532–550. 48. Leonardi U (2008) Senegal Land Cover Mapping (Food and Agriculture Organization,
Rome), Technical Report. 55. Kam SP, Hossain M, Bose ML, Villano LS (2005) Spatial patterns of rural poverty
and their relationship with welfare-influencing factors in Bangladesh. Food Pol 30:
551–567. 49. Dasgupta S, Deichmann U, Meisner C, Wheeler D (2005) Where is the poverty–
environment nexus? Evidence from Cambodia, Lao PDR, and Vietnam. World Devel-
opment 33:617–638. 56. Watmough GR, Atkinson PM, Saikia A, Hutton CW (2016) Understanding the evidence
base for poverty–environment relationships using remotely sensed satellite data: An
example from Assam, India. World Dev 78:188–203. 50. Vista BM, Murayama Y (2011) Spatial determinants of poverty using GIS-based
mapping. Spatial Analysis and Modeling in Geographical Transformation Process
(Springer, New York), pp 275–296. 57. Smith C, Mashhadi A, Capra L (2013) Ubiquitous sensing for mapping poverty in devel-
oping countries. Third Conference on the Analysis of Mobile Phone Datasets, NetMob
2013 (MIT Media Labs, Boston). 51. Amarasinghe U, Samad M, Anputhas M (2005) Spatial clustering of rural poverty and
food insecurity in Sri Lanka. Food Pol 30:493–509. 58. Pokhriyal N, Dong W (2015) Virtual network and poverty analysis in Senegal. Fourth
Conference on the Analysis of Mobile Phone Datasets, NetMob 2015 (MIT Media Labs,
Boston). 52. Minot N, et al. (2006) Poverty and inequality in Vietnam: Spatial patterns and geo-
graphic determinants (International Food Policy Research Institute, World Bank, Pokhriyal and Jacques E9792
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https://openalex.org/W2993994311
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https://europepmc.org/articles/pmc6890929?pdf=render
|
English
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Variability in the Response of Bacterial Community Assembly to Environmental Selection and Biotic Factors Depends on the Immigrated Bacteria, as Revealed by a Soil Microcosm Experiment
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MSystems
| 2,019
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cc-by
| 11,373
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Variability in the Response of Bacterial Community Assembly
to Environmental Selection and Biotic Factors Depends on the
Immigrated Bacteria, as Revealed by a Soil Microcosm
Experiment Xiaogang Wu,a,b Yun Wang,a,b Ying Zhu,a,b Hao Tian,a,b Xianchao Qin,a,b Changzheng Cui,d Liping Zhao,a,b Pascal Simonet,c
Xiaojun Zhanga,b aState Key Laboratory of Microbial Metabolism, School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai, China
bJoint International Research Laboratory of Metabolic & Developmental Sciences, School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai,
China cEnvironmental Microbial Genomics Group, Laboratoire AMPERE, CNRS, Ecole Centrale de Lyon, Université de Lyon, Ecully, France
dSchool of Resource and Environmental Engineering, East China University of Science and Technology, Shanghai, China ABSTRACT
Exploring the assembly mechanism of microbiota is critical for under-
standing soil ecosystem functions. However, the relative importance of different bi-
otic and abiotic factors in determining the bacterial community has not been prop-
erly clarified. In this study, the effects of inocula and recipients on the assembly of
the soil community were investigated to evaluate their importance by inoculation
experiments with sterile soil. Two distinct soils, conventional nitrogen-fertilized soil
and aromatic-compound-contaminated soil, were sterilized, cross inoculated, and in-
cubated for 2 months under different inoculation doses and oxygen conditions. The
results showed that the greatest variation in community structure emerged in the
samples inoculated with distinct inocula rather than in the samples of different soil
recipients. The phylogenies in the two inocula were diverse and dissimilar, although
there were many ecologically equivalent bacteria. When the inocula with dissimilar
ecologically equivalent species were used, the assembled communities were primar-
ily determined by the inocula as indicated by the beta diversity and variation parti-
tioning analyses. In contrast, environmental selection dominated the process when
ecologically equivalent species in the inocula were similar, as when only one type of
inoculum was used, where the soil habitat selected the most adaptive bacteria from
the defined inoculum pool. These results indicate that inoculated bacteria are domi-
nant over environmental selection if they are sufficiently dissimilar, although the ef-
fect of environmental selection is more obvious when similar bacteria are inoculated
in the soil for community assembly. Our findings suggest that the immigration of
exotic bacteria could be a primary factor impacting community assembly. Citation Wu X, Wang Y, Zhu Y, Tian H, Qin X,
Cui C, Zhao L, Simonet P, Zhang X. 2019. Variability in the response of bacterial
community assembly to environmental
selection and biotic factors depends on the
immigrated bacteria, as revealed by a soil
microcosm experiment. mSystems
4:e00496-19. https://doi.org/10.1128/
mSystems.00496-19. RESEARCH ARTICLE
Ecological and Evolutionary Science RESEARCH ARTICLE
Ecological and Evolutionary Science Citation Wu X, Wang Y, Zhu Y, Tian H, Qin X,
Cui C, Zhao L, Simonet P, Zhang X. 2019.
Variability in the response of bacterial
community assembly to environmental
selection and biotic factors depends on the
immigrated bacteria, as revealed by a soil
microcosm experiment. mSystems
4:e00496-19. https://doi.org/10.1128/
mSystems.00496-19. November/December 2019
Volume 4
Issue 6
e00496-19 KEYWORDS bacterial community, community assembly, microcosms, selection,
sterile soil T
he soil environment harbors the greatest bacterial diversity (1). These bacteria play
critical roles in numerous biological functions, including soil fertility regulation,
pollutant degradation, plant health, and global cycling of carbon, nitrogen, and other
nutrients (2). Understanding soil biodiversity and ecosystem function is critical for
deciphering the mechanisms that sustain the assembly of different soil bacterial
populations into functional communities and planning management strategies for their
activities further to improve ecosystem functions. Thus far, the mechanisms underlying
the generation of bacterial communities are not clear and require further study. For
example, immigration of bacteria and environmental selection are both reported as
important ecological factors in determining community assembly (3–5), although their
relative importance is not consistent among the published references (4–7). T The community-environment interaction has been extensively studied in commu-
nity ecology (8). Some authors claim that environmental selection is extremely impor-
tant for structuring microbiota and that the community structures in each niche are
primarily determined through deterministic partitioning of resources among organisms
(9–11). Habitat and environmental filtering perspectives suggest that community as-
sembly is a specific and predicable process (12–14). For example, the famous statement
proposed by Baas Becking is that “everything is everywhere, the environment selects.”
Thus, there might be a one-to-one match between niche availability (or environmental
condition) and community structure. In soil environments, the soil type in particular is
critical for determining the structures of bacterial communities (15). This conclusion
was supported by Griffiths et al. (16), who seeded sterilized sandy and clay soil samples
with soil bacterial communities and found that the structures of the newly developed
microbiota were largely influenced by the soil type, rather than the initial inoculum. Similar conclusions were drawn by another group, who used two soils sampled from
England and Italy that were sterilized, reinoculated, and incubated prior to DNA
extraction and sequencing (17), and by Xun et al. (5), who developed the same
experiment with two soil samples from the same origin but differing fertilization
treatments. However, other studies have reported that the role of biotic factors is more
important for structuring the microbiota. According to the neutral theory, the influence
of environmental factors on each species in the habitat is neutral and neither promotes
nor inhibits change (18–20). Stochastic factors, such as immigration, dispersal birth/
death processes, drift, and speciation, are the primary drivers of ecological diversity and
community structure (20). November/December 2019
Volume 4
Issue 6
e00496-19 Variability in the Response of Bacterial Community Assembly
to Environmental Selection and Biotic Factors Depends on the
Immigrated Bacteria, as Revealed by a Soil Microcosm
Experiment IMPORTANCE The soil microbiota conducts important biological ecosystem func-
tions, but the mechanism underlying community-environment interactions for soil
microbiota remains unclear. By using two distinct soils for cross inoculation, we suc-
cessfully simulated the assembly of the bacterial community in sterile soil. Thus, the
reasons why inoculum and recipient have dominated community assembly in previ-
ous investigations are investigated in this study. We found that inoculated bacteria
presided over environmental selection for community assembly due to the varied
difference of ecological equivalent bacteria, either divergent or convergent. The sig-
nificance of neutrality for the ecologically equivalent bacterial species that immi-
grated into the recipients should be emphasized in exploring the mechanisms of
community assembly. Our finding is helpful for understanding the community- Editor Nick Bouskill, Lawrence Berkeley
National Laboratory Copyright © 2019 Wu et al. This is an open-
access article distributed under the terms of
the Creative Commons Attribution 4.0
International license. Address correspondence to Xiaojun Zhang,
xjzhang68@sjtu.edu.cn. Address correspondence to Xiaojun Zhang,
xjzhang68@sjtu.edu.cn. Received 15 August 2019
Accepted 13 November 2019
Published 3 December 2019 msystems.asm.org
1 November/December 2019
Volume 4
Issue 6
e00496-19 Wu et al. environment interaction, a basic question in ecology, and it would shed light on this
issue that has perplexed scientists for many years. RESULTS Changes in soil biomass after incubation. Regular plating of the uninoculated
sterilized soil samples on growth media did not yield bacterial or fungal colonies, which
confirmed the effectiveness of the irradiation treatment used to kill the indigenous
microorganisms. Additionally, the effectiveness of irradiation was monitored by mea-
suring the amount of DNA extracted from the soil samples. DNA quantities of 1.45 and
32.90 g · g1 soil were obtained from the aromatic-contaminated soil (ACS) and
optimized nitrogen-fertilized soil (ONS), respectively, before irradiation and decreased
to 0.084 and 0.098 g · g1 after irradiation, thus confirming the strong lethal effect
and denaturation of most of the DNA (Fig. 1A). Persisting extracellular DNA was
gradually degraded until completely eliminated by DNase enzymes during the incu-
bation. The decrease in DNA and the lack of microorganisms growing from the
microcosms excluded the possibility that any indigenous microorganisms survived the
sterilization treatment (Fig. 1A). Bacterial growth in the inoculated samples was assessed by quantifying the total
amount of DNA and the 16S rRNA gene copy numbers (see Table S1 in the supple-
mental material). These two parameters indicated the recovery of the microbiota after
2 months of incubation at a quantitative level similar to the level detected in the
samples prior to sterilization (P 0.1707 for DNA content, P 0.8404 for increased
DNA, and P 0.0694 for 16S rRNA gene copies by the Mann-Whitney U test) (Fig. 1A
and B). In addition, the growth for the total extracted DNA and the 16S rRNA gene copy
number reached a plateau, confirming that the microbiota reached its biotic capacity
in both soils within 2 months (Fig. 1A and B). The numbers of bacterial cells (abun-
dance) that developed from a subsample inoculated into previously sterilized soil
samples were higher under low pH than under high pH conditions (Fig. 1C, R2 0.2053,
P 0.0003) and under high dissolved nitrogen (DN) consumption than under low DN
consumption conditions (Fig. 1D, R2 0.3936, P 0.0001). Overall structural shift of bacterial communities after incubation. A total of
4,214,140 quality-controlled sequences for the amplicons of the amplified V3-V4 region
of the 16S rRNA gene were obtained (median, 47,058 sequences; range, 20,379 to
88,416 sequences). These sequences were clustered into 5,338 OTUs (operational
taxonomy units) (97% similarity). KEYWORDS bacterial community, community assembly, microcosms, selection,
sterile soil This theory has been reported to accurately predict the
future structures of bacterial communities (21, 22). Empirical evidence has demon-
strated that the local species pool controls community composition due to dispersal
limitations for bacteria, which is consistent with the neutral process (19, 23). Thus,
microcosm experiments using sterilized media recolonized by microbes provide a
suitable system for examining the importance of inocula versus environmental factors
for the initial assembly of bacterial communities (24). In these experiments, several
natural microbe sources are seeded as inocula into sterilized environmental samples
and incubated under laboratory conditions to elucidate the resulting community
assembly and the factors that affect it (4, 6, 7). Langenheder et al. (6) performed batch
culture experiments in which bacteria from eight distinct aquatic habitats were re-
grown under identical conditions to determine the extent to which similar communities
would develop under the same selective pressures. These results demonstrated that the
inocula had a greater impact than the environmental conditions (6). Pagaling et al. (7)
drew the same conclusions from their experiments with sediment and water samples
collected from seven sources colonized by significantly different bacterial communities. msystems.asm.org
2 Community Assembly in Sterile Soil The taxonomic structure of the developed bacterial communities in the incubated
microcosms clearly demonstrated source dependence (7). The results were not due to
the laboratory effect because the same assembly theory was applied under field
conditions. Different sterilized liquid growth media processed into microcosms were
spread over nine field locations across three spatial scales in the Inner Mongolian
grassland to be naturally reinoculated by indigenous bacteria. The structures of the
bacterial communities that assembled in the microcosms were dictated by the different
inocula instead of by the growth media due to their separate field locations and
dissimilar immigration of species (4). In general, inoculum and recipient dominated community assembly in previous
investigations. However, the variability in all investigations has perplexed scientists and
obscured community assembly mechanisms. From these varied conclusions arose the
question of what is the relative importance of inoculum and recipient in the determi-
nation of community structure? To answer this question, in this study, we simulated the
bacterial community assembly in soil by incubating microcosms of sterilized soil
reinoculated with two distinct soil samples. The impacts of the inoculation dosage and
aeration condition were also checked. November/December 2019
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e00496-19 KEYWORDS bacterial community, community assembly, microcosms, selection,
sterile soil After incubation, microbiota analyses were
performed to determine the relative importance of inocula and recipient conditions
when comparing all developed microbiota derived from the different soil inocula
versus separately comparing the microbiota developed from each inoculum. Wu et al. FIG 1 Recovery of bacterial biomass after incubation. (A) Box plot reflecting the quantity of extracted DNA from soil samples. Increased DNA of inoculated
samples is the weight of recovered DNA from the sample after incubation minus the weight of DNA introduced by the inoculum. (B) Box plot reflecting the
quantity of 16S RNA gene copies within soil. The quantity in the irradiated soil was under the detection limit (negative). (C) Relationship between pH and 16S
rRNA gene copies (log transformed). (D) Relationship between dissolved nitrogen (DN) consumption and 16S rRNA gene copies (log transformed). The box plots
represent the smallest observation, lower quartile, median, upper quartile, and largest observation. Values are not significantly different (ns) according to the
Mann-Whitney U test (nonparametric test). Linear regression was used to describe the relationships among pH, DN consumption, and bacterial abundance. FIG 1 Recovery of bacterial biomass after incubation. (A) Box plot reflecting the quantity of extracted DNA from soil samples. Increased DNA of inoculated
samples is the weight of recovered DNA from the sample after incubation minus the weight of DNA introduced by the inoculum. (B) Box plot reflecting the
quantity of 16S RNA gene copies within soil. The quantity in the irradiated soil was under the detection limit (negative). (C) Relationship between pH and 16S
rRNA gene copies (log transformed). (D) Relationship between dissolved nitrogen (DN) consumption and 16S rRNA gene copies (log transformed). The box plots
represent the smallest observation, lower quartile, median, upper quartile, and largest observation. Values are not significantly different (ns) according to the
Mann-Whitney U test (nonparametric test). Linear regression was used to describe the relationships among pH, DN consumption, and bacterial abundance. from the inocula in the samples of sterilized soil. The MANOVA (multivariate analysis of
variance) test separated the resulting bacterial communities into two groups according
to aerobic or anaerobic incubation status (Fig. 2A and B). This result was confirmed by
the Bray-Curtis principal-coordinate analysis (PCoA) based on the OTU data, which
showed divergent development of the bacterial communities driven by aeration during
incubation (Fig. 2D). However, the inoculum was another factor that contributed to the taxonomic
structuration of the soil microbiota that developed in the soil microcosms, as evidenced
by the significant differences in the MANOVA between the ONS- and ACS-inoculated
samples detected by the unweighted UniFrac PCoA plots for the OTUs (Fig. 2C). November/December 2019
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e00496-19 RESULTS Rarefaction and Shannon diversity curves revealed
that the ONS-inoculated samples had higher diversity (alpha diversity index) than the
ACS-inoculated samples (see Fig. S1 and Table S2 in the supplemental material). The
aeration conditions significantly influenced the bacterial communities that developed msystems.asm.org
3 The
results also showed that the samples separated according to the recipient type along
the principal component 1 (PC1) axis and according to the inoculum dosage along the
PC2 axis when the microcosms were seeded with different amounts of the same
inoculum (Fig. 2E to H). MANOVA showed significant differences between the recipi-
ents, which indicated a recipient effect on the community profile when the same
inoculum was inoculated with both high and low inoculum dosage and incubated
under aerobic and anaerobic conditions. Bacterial community structuration in soil microcosms. Compared with the un-
sterilized soil microcosms, the inoculated sterile soil developed significantly different
bacterial communities depending on the inoculum, recipient, and incubation condi-
tions. Aerobic incubation of the microcosms led to systematic development of Actino-
bacteria, particularly representatives of Nocardioidaceae and Streptomycetaceae, which
were found at higher quantitative levels than those observed in the controls, regardless
of the inoculum used. When the microcosms were incubated under anaerobic condi- November/December 2019
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4 Community Assembly in Sterile Soil FIG 2 Long-term soil incubation shows significant inoculum-associated and environment-associated structural segregation of the bacterial community. (A)
Bray-Curtis principal-coordinate analysis (PCoA) of the soil bacterial community structure based on the family data. (B) Clustering of the soil microbiota based
on Bray-Curtis distances calculated with multivariate analysis of variance (MANOVA). Values that are significantly different (P 0.0001) are indicated by four
asterisks. (C) Unweighted UniFrac PCoA of the soil bacterial community structure based on operational taxonomic unit (OTU) data. (D) Bray-Curtis PCoA of the
soil bacterial community structure based on OTU data. (E) Bray-Curtis PCoA of the ONS inoculum bacterial community structure under aerobic conditions based
on OTU data. (F) Bray-Curtis PCoA of the ONS inoculum sample bacterial community structure under anaerobic conditions based on OTU data. (G) Bray-Curtis
PCoA of the ACS inoculum sample bacterial community structure under aerobic conditions based on OTU data. (H) Bray-Curtis PCoA of the ACS inoculum
sample bacterial community structure under anaerobic conditions based on OTU data. MANOVA analysis showed significant differences between the circled
clusters (P 0.0001). (I) Phylum-level community structures after the different treatments. The distributions presented at different levels are based on the 80%
similarity clusters of the OTUs. (J) Family-level community structures of the different treatments. The distributions presented at different levels are based on
the 80% similarity clusters of the OTUs. November/December 2019
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5 (K) OTU-level community structures of the different treatments recovered from sterile soil. Two sterilized soils (rONS
and rACS) were inoculated with a subsample of their initial inoculum and a subsample from the other soil (ONS and ACS) with different inoculation doses (heavy
[H] and light [L]) and oxygen conditions (aerobic [ae] and anaerobic [an]). In all PCoA plots, the percentage of the variation explained by the plotted principal
coordinates (PCs) is shown in parentheses. FIG 2 Long-term soil incubation shows significant inoculum-associated and environment-associated structural segregation of the bacterial community. (A)
Bray-Curtis principal-coordinate analysis (PCoA) of the soil bacterial community structure based on the family data. (B) Clustering of the soil microbiota based
on Bray-Curtis distances calculated with multivariate analysis of variance (MANOVA). Values that are significantly different (P 0.0001) are indicated by four
asterisks. (C) Unweighted UniFrac PCoA of the soil bacterial community structure based on operational taxonomic unit (OTU) data. (D) Bray-Curtis PCoA of the
soil bacterial community structure based on OTU data. (E) Bray-Curtis PCoA of the ONS inoculum bacterial community structure under aerobic conditions based
on OTU data. (F) Bray-Curtis PCoA of the ONS inoculum sample bacterial community structure under anaerobic conditions based on OTU data. (G) Bray-Curtis
PCoA of the ACS inoculum sample bacterial community structure under aerobic conditions based on OTU data. (H) Bray-Curtis PCoA of the ACS inoculum
sample bacterial community structure under anaerobic conditions based on OTU data. MANOVA analysis showed significant differences between the circled
clusters (P 0.0001). (I) Phylum-level community structures after the different treatments. The distributions presented at different levels are based on the 80%
similarity clusters of the OTUs. (J) Family-level community structures of the different treatments. The distributions presented at different levels are based on
the 80% similarity clusters of the OTUs. (K) OTU-level community structures of the different treatments recovered from sterile soil. Two sterilized soils (rONS
and rACS) were inoculated with a subsample of their initial inoculum and a subsample from the other soil (ONS and ACS) with different inoculation doses (heavy
[H] and light [L]) and oxygen conditions (aerobic [ae] and anaerobic [an]). In all PCoA plots, the percentage of the variation explained by the plotted principal
coordinates (PCs) is shown in parentheses. tions, the proportions of Firmicutes and Proteobacteria increased significantly, with a
particularly significant increase in representatives of Halomonadaceae (Fig. 2I and J). tions, the proportions of Firmicutes and Proteobacteria increased significantly, with a
particularly significant increase in representatives of Halomonadaceae (Fig. 2I and J). However, the taxonomic analysis also indicated that the two inocula differentially
impacted the bacterial community structure of the developed microbiota. At the However, the taxonomic analysis also indicated that the two inocula differentially
impacted the bacterial community structure of the developed microbiota. At the
phylum level, the microbiota was dominated by Acidobacteria when the microcosms
were inoculated with the ONS inoculum (Fig. 2I). An impact of the inoculum was also
noted at the family level when the microcosms were incubated anaerobically, with a
prevalence of Bacillaceae 1 and Clostridiales_Incertae Sedis XI (both Firmicutes) in the msystems.asm.org
5 November/December 2019
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e00496-19 Wu et al. samples with the ONS samples being used as inoculum and a prevalence of Micromono-
sporaceae (Actinobacteria) for these samples in which the ACS was used as inoculum
(Fig. 2J). The development of bacterial communities in previously sterilized soils was im-
pacted less by the physical and chemical characteristics of the recipient soils than by
the inoculum or the incubation conditions, including aeration (Fig. 2K). The resulting
community structure suggested that the species influenced by the recipient type also
depended on the inoculum, with several species specifically enriched in samples
according to the inoculum used. For instance, after inoculation with the ONS inoculum,
we detected 2,390 OTUs in the ONS-inoculated samples, whereas 801 OTUs were
detected in the ACS-inoculated samples (Fig. S1). Several OTUs were positively influ-
enced by the recipient soils when the ONS inoculum was used, including OTU4
(Streptomyces) and OTU17 (Ohtaekwangia), which were specifically enriched by the
combination of ONS as the donor and recipient (H-ONS-rONS-ae and L-ONS-rONS-ae)
(treatments explained in “Soil microcosms” in Materials and Methods), and OTU11
(Kribbella), which was enriched by the combination of ONS as the donor and ACS as the
recipient (H-ONS-rACS-ae and L-ONS-rACS-ae). The relative abundance of OTU_3 (Ha-
lomonas) depended on a recipient soil with a higher proportion of ACS (H-ONS-rACS-an
and L-ONS-rACS-an) than ONS (H-ONS-rONS-an and L-ONS-rONS-an). Several OTUs
were positively quantitatively influenced by the recipient soil when the ACS inoculum
was used, including OTU1 (Nocardioides) and OTU11 (Kribbella), which were enriched in
H-ACS-rACS-ae and L-ACS-rACS-ae. OTU94 (unclassified Kitasatospora), OTU4 (Strepto-
myces), and OTU17 (Ohtaekwangia) were enriched in H-ACS-rONS-ae and L-ACS-rONS-
ae. OTU3 (Halomonas) was more abundant in H-ACS-rACS-an and L-ACS-rACS-an. November/December 2019
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e00496-19 OTU2
(unclassified Janibacter) was enriched in H-ACS-rONS-an and L-ACS-rONS-an (Fig. 2K). Impact of biotic and abiotic factors on shaping of soil bacterial communities. Redundancy analysis (RDA) (Fig. 3A) assessed three pairs of nominal variables (oxygen
condition, inoculum, and recipient soil) and four nonnominal variables (inoculation
dose, pH, dissolved organic carbon [DOC], and DN) demonstrated that the bacterial
community composition was significantly (r 0.887, P 0.0001) shaped by several key
soil environmental variables. Bacterial communities were well separated by RDA1 (24%)
according to the major variable oxygen condition and by RDA2 (22%) according to the
major variable inoculum. However, RDA did not show clear clustering of the soil
samples based on the recipient soils. A variation partitioning analysis (VPA) was performed to calculate the relative
contributions of the inoculum (type and dosage), oxygen, and recipient basic soil
characteristics on the taxonomic composition of the bacterial community after
2 months of incubation (Fig. 3B). These variables explained 63.7% of the observed
variation, with inoculum type accounting for the largest proportion (17.2%; P 0.001),
followed by oxygen availability (15.6%; P 0.001). The characteristics of the recipient
soil, including the type of recipient soil, explained 9.5% of the observed variation (P
0.001), followed by the inoculum density (dosage) (7.9%; P 0.001). y
y
g
Correlation of OTUs with the inoculum. The RDA approach was used to identify
the OTUs driven by the inoculum and to determine the main contributor to the
establishment of the community structure in the incubated soil samples (Fig. 4A and C). Under aerobic incubation conditions, 46 significantly different OTUs were found in the
developed bacterial communities inoculated with either the ONS or ACS subsamples
(Fig. 4A and B). Twenty-three OTUs were enriched following inoculation with ONS and
were affiliated with 15 different genera, and 23 OTUs were enriched after the micro-
cosms were inoculated with ACS and affiliated with 17 different genera. Under anaer-
obic incubation conditions, the number of different OTUs in the developed bacterial
communities according to the inoculum type was 49 (RDA selection, Fig. 4C and D),
with 28 OTUs enriched in the ONS-inoculated samples affiliated with 19 different
genera, including 5 OTUs in Gp4 and Gp6. The 21 OTUs enriched in the ACS-inoculated
samples were affiliated with 18 different genera, with the 3 OTUs with the highest
abundance belonging to Pseudomonas. November/December 2019
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6 Community Assembly in Sterile Soil FIG 3 Ecological effects of biotic and abiotic factors on bacterial communities. (A) Redundancy analysis (RDA) plot of the
microbiota composition resulting from different treatments based on the sequencing data. Nonnominal environmental
variables are indicated by brown arrows, and nominal environmental variables are represented by brown triangles. Different management strategies are represented by circles. Blue circles represent ONS inoculum samples, and red circles
represent ACS inoculum samples. Circles containing small white circles represent aerobic samples, and solid circles
represent anaerobic samples. (B) Variation partitioning analysis (VPA) was used to determine the effects of the inoculum
factor, oxygen availability, soil basic characteristics, receptor and dosage, and interactions between these parameters on
the bacterial community structure. The Venn diagram shows the percentage of variation explained by the factors alone and
in combination. The unexplained variation is depicted as an ellipse at the bottom of panel B. FIG 3 Ecological effects of biotic and abiotic factors on bacterial communities. (A) Redundancy analysis (RDA) plot of the
microbiota composition resulting from different treatments based on the sequencing data. Nonnominal environmental
variables are indicated by brown arrows, and nominal environmental variables are represented by brown triangles. Different management strategies are represented by circles. Blue circles represent ONS inoculum samples, and red circles
represent ACS inoculum samples. Circles containing small white circles represent aerobic samples, and solid circles
represent anaerobic samples. (B) Variation partitioning analysis (VPA) was used to determine the effects of the inoculum
factor, oxygen availability, soil basic characteristics, receptor and dosage, and interactions between these parameters on
the bacterial community structure. The Venn diagram shows the percentage of variation explained by the factors alone and
in combination. The unexplained variation is depicted as an ellipse at the bottom of panel B. FIG 3 Ecological effects of biotic and abiotic factors on bacterial communities. (A) Redundancy analysis (RDA) plot of the
microbiota composition resulting from different treatments based on the sequencing data. Nonnominal environmental
variables are indicated by brown arrows, and nominal environmental variables are represented by brown triangles. Different management strategies are represented by circles. Blue circles represent ONS inoculum samples, and red circles
represent ACS inoculum samples. Circles containing small white circles represent aerobic samples, and solid circles
represent anaerobic samples. (B) Variation partitioning analysis (VPA) was used to determine the effects of the inoculum
factor, oxygen availability, soil basic characteristics, receptor and dosage, and interactions between these parameters on
the bacterial community structure. The Venn diagram shows the percentage of variation explained by the factors alone and
in combination. The unexplained variation is depicted as an ellipse at the bottom of panel B. FIG 3 Ecological effects of biotic and abiotic factors on bacterial communities. (A) Redundancy analysis (RDA) plot of the
microbiota composition resulting from different treatments based on the sequencing data. Nonnominal environmental
variables are indicated by brown arrows, and nominal environmental variables are represented by brown triangles. Different management strategies are represented by circles. Blue circles represent ONS inoculum samples, and red circles
represent ACS inoculum samples. Circles containing small white circles represent aerobic samples, and solid circles
represent anaerobic samples. (B) Variation partitioning analysis (VPA) was used to determine the effects of the inoculum
factor, oxygen availability, soil basic characteristics, receptor and dosage, and interactions between these parameters on
the bacterial community structure. The Venn diagram shows the percentage of variation explained by the factors alone and
in combination. The unexplained variation is depicted as an ellipse at the bottom of panel B. msystems.asm.org
7 November/December 2019
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e00496-19 Wu et al. FIG 4 Redundancy analysis (RDA) plot of the bacterial community composition determined by the inoculum factor and heat map of the RDA-identified key
OTUs responding to the inoculum factor. The inoculum sources were used as environmental variables. OTUs are indicated by blue arrows. P values were
obtained by the Monte Carlo permutation procedure (MCPP) (P 0.0001). (A) Aerobic conditions. (C) Anaerobic conditions. (B and D) Key OTUs responding
to the inoculum factor under aerobic (B) and anaerobic (D) incubation. The color of the spot corresponds to the log-transformed relative abundance of the OTU. The OTUs are organized according to Spearman’s correlation of the OTU abundance. The family and genus names of the OTUs are shown to the right. OTUs
whose names are in red in panels B and D are rare species in the control ACS sample but enriched in the ACS inoculum sample. OTUs whose names are in
blue in panels B and D are rare species in the control ONS sample but enriched in the ONS inoculum sample. All of these OTUs were enriched after incubation, and some became
predominant after incubation, including OTU74 (Dyadobacter), OTU5094 (unclassified
Phytohabitans), OTU4024 (unclassified Promicromonospora), OTU4151 (unclassified Ac-
tinosynnema), OTU1177 (unclassified Citricoccus), OTU13 (Promicromonospora), OTU59
(Nocardia), OTU1651 (Saccharothrix), OTU70 (Pseudonocardia), OTU3050 (unclassified
Aeromicrobium), OTU156 (unclassified Aciditerrimonas), OTU86 (unclassified Luteimo-
nas), OTU395 (Ohtaekwangia), OTU125 (Aliidiomarina), OTU15 (unclassified Fervidicella),
OTU1610 (Cellulomonas), OTU103 (unclassified Pelotomaculum), OTU94 (unclassified
Kitasatospora), OTU1372 (unclassified Marmoricola), OTU72 (unclassified Azonexus), and
OTU117 (Cupriavidus) (Fig. 4B and D). OTUs associated with the recipient soils. The soil bacterial community structure
was primarily impacted by the inoculum. However, differences were also detected
when the same inoculum was used to seed different soils, indicating that differences in
soil physical and chemical properties also influenced the growth of some bacteria. OTUs
that were specifically impacted by the recipient ONS (rONS) and ACS (rACS) soils were
identified using RDA (Fig. 5). When only the ONS inoculum samples were taken into
account, a large number of OTUs were significantly positively impacted by the recipient
type (Fig. 5A and B). This finding was confirmed when only the ACS inoculum samples
were taken into account (Fig. 5C and D). FIG 4 Redundancy analysis (RDA) plot of the bacterial community composition determined by the inoculum factor and heat map of the RDA-identified key
OTUs responding to the inoculum factor. The inoculum sources were used as environmental variables. OTUs are indicated by blue arrows. P values were
obtained by the Monte Carlo permutation procedure (MCPP) (P 0.0001). (A) Aerobic conditions. (C) Anaerobic conditions. (B and D) Key OTUs responding
to the inoculum factor under aerobic (B) and anaerobic (D) incubation. The color of the spot corresponds to the log-transformed relative abundance of the OTU. The OTUs are organized according to Spearman’s correlation of the OTU abundance. The family and genus names of the OTUs are shown to the right. OTUs
whose names are in red in panels B and D are rare species in the control ACS sample but enriched in the ACS inoculum sample. OTUs whose names are in
blue in panels B and D are rare species in the control ONS sample but enriched in the ONS inoculum sample. Interestingly, all of these bacteria, which could be distinguished by the inoculum
and aeration conditions, behaved (with regard to growth and survival) similarly regard-
less of the recipient soil. Furthermore, among the OTUs correlated with the inoculum
factor, most were abundant in the initial inoculum (with a relative abundance of 0.1%
in the original soil), whereas few of them were rare (with a relative abundance of 0.1%
in the original soil). All of these OTUs were enriched after incubation, and some became
predominant after incubation, including OTU74 (Dyadobacter), OTU5094 (unclassified
Phytohabitans), OTU4024 (unclassified Promicromonospora), OTU4151 (unclassified Ac-
tinosynnema), OTU1177 (unclassified Citricoccus), OTU13 (Promicromonospora), OTU59
(Nocardia), OTU1651 (Saccharothrix), OTU70 (Pseudonocardia), OTU3050 (unclassified
Aeromicrobium), OTU156 (unclassified Aciditerrimonas), OTU86 (unclassified Luteimo-
nas), OTU395 (Ohtaekwangia), OTU125 (Aliidiomarina), OTU15 (unclassified Fervidicella),
OTU1610 (Cellulomonas), OTU103 (unclassified Pelotomaculum), OTU94 (unclassified
Kitasatospora), OTU1372 (unclassified Marmoricola), OTU72 (unclassified Azonexus), and
OTU117 (Cupriavidus) (Fig. 4B and D). Interestingly, all of these bacteria, which could be distinguished by the inoculum
and aeration conditions, behaved (with regard to growth and survival) similarly regard-
less of the recipient soil. Furthermore, among the OTUs correlated with the inoculum
factor, most were abundant in the initial inoculum (with a relative abundance of 0.1%
in the original soil), whereas few of them were rare (with a relative abundance of 0.1%
in the original soil). November/December 2019
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e00496-19 DISCUSSION Identifying the mechanisms by which bacterial communities are generated and
maintained in the soil is essential for understanding the biological functions of soil (2). msystems.asm.org
8 November/December 2019
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e00496-19 Community Assembly in Sterile Soil FIG 5 Redundancy analysis (RDA) plot of the bacterial community composition determined by recipient
type. The recipient types were used as environmental variables. The OTUs are indicated by blue arrows. P values were obtained by the Monte Carlo permutation procedure (MCPP). (A and B) ONS inoculum
under aerobic (A) and anaerobic (B) conditions. (C and D) ACS inoculum under aerobic (C) and anaerobic
(D) conditions. FIG 5 Redundancy analysis (RDA) plot of the bacterial community composition determined by recipient
type. The recipient types were used as environmental variables. The OTUs are indicated by blue arrows. P values were obtained by the Monte Carlo permutation procedure (MCPP). (A and B) ONS inoculum
under aerobic (A) and anaerobic (B) conditions. (C and D) ACS inoculum under aerobic (C) and anaerobic
(D) conditions. Numerous studies have attempted to elucidate the main ecological processes by which
the microbiota was assembled (4, 24–27). Among all attempts, an approach often used
is to investigate the microbiota structures developed in microcosms of which the
well-referenced inocula were inoculated into sterilized samples (24, 25). However,
significant discrepant conclusions were drawn among previous studies (5, 16, 17). From
these seemingly contradictory conclusions arose the question of which factor is more
important for the determination of community structure. Here, our results suggest that
the inoculum is a more important factor for community assembly. However, we found
that inoculum and recipient were demonstrated as the dominant factor for community
assembly in sterile recipients because of the varied differences among the inocula used
in these experiments. First, well-designed experiments are necessary to determine the reasons for the
variation existing in the explanation of the role of inoculum and environment selection
in community assembly. For this purpose, the completeness of the sterility of the
recipient soil is an important issue to truly reflect microbial community assembly in the
inoculated soil microcosms. November/December 2019
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e00496-19 DISCUSSION Ecologically equivalent bacteria adapt to different recip-
ient soils; their presence and development in the community are primarily determined
by their immigration, although different soils may have different abiotic environments. The prevailing heterogeneous microenvironments inside the soil may create various
niches that could provide a wide space to host extremely diverse microorganisms. Thus,
the assembly of a microbial community in sterile soil depends on the species immi-
grating from the exotic environment. The two inocula (ONS and ACS) contained both
shared and specific “ecologically equivalent species,” which were not selected by the
soil type and proliferated in the recipient soil. They might also be functionally equiv-
alent and perform fundamental biological functions in different soil habitats, such as
maintaining the magnitude and stability of nutrient cycling. Thus, they can participate
in carbon and nitrogen metabolism in ONS soil or degrade organic pollutants in ACS
soil. Most of the “ecologically equivalent OTUs” in our study belonged to the abundant
members in the inocula, although others were members of the rare biosphere, such as
OTU13 (Promicromonospora), OTU15 (unclassified Fervidicella), and OTU59 (Nocardia). Some of the rare OTUs multiplied rapidly to become predominant in the newly
developed soil bacterial communities. We speculate that these rare bacteria were
predominant in the history of the source community. The development of equivalent
species in the recipient soils led to a significant correlation of the community structure
with the inoculum, the most important factor shaping the bacterial community struc-
ture. Clearly, when dissimilar ecologically equivalent species immigrated into the
recipient soils, they survived or were enriched in sterile environments. Even if
the recipient soils were the same, the dissimilar equivalent species provided by the
different inocula resulted in distinct community structures. Therefore, the effect of the
inoculum on the community structure profile was obvious, and the immigration of
equivalent species dominates community assembly in different recipients (summarized
in Fig. 6). Although our data demonstrated that the assembly of the bacterial community in
soil was determined by bacteria that immigrated from the inocula, the analysis also
suggested that the filtering role of the environment was not negligible, particularly
when comparing the microbiota developed from one inoculum (either ONS or ACS). DISCUSSION In our study, the absence of bacterial and fungal colonies
on solidified media after plating diluted irradiated soil subsamples as well as the
constant decrease in extracted DNA during incubation of the control microcosms
confirmed the effectiveness of the soil sterilization and excluded any bias due to the
persistence of surviving cells in the recipient soils. The results also showed that the
2-month incubation time permitted the restoration of a bacterial biomass that was at
least quantitatively similar to that estimated in the original soils or in the nonirradiated
controls, confirming the development and assembly of a new bacterial community. According to the perspective of microbial biogeography, the dispersal of microor-
ganisms is limited, and the local species pools in different habitats are dissimilar (28, 29,
53). Dispersal limitation and microbial biogeography can result in different species
pools that behave as different inocula immigrating into the recipients. The two inocula
from distinct soil habitats in our investigation represented dissimilar local species pools,
which determined the bacterial community structuration of the newly developed
microbiota. These results indicate that inoculating soil bacteria into sterilized soil
microcosms behave similarly when introduced into other habitats, such as liquid media
and freshwater microcosms (4, 6, 7), where the assembly of bacterial community in
each of these sterilized environments is determined by inoculum. In these cases, the
bacteria from the inoculum dominated community assembly (4, 24, 25). msystems.asm.org
9 Wu et al. Interestingly, some bacterial species in this study acted as indispensable contribu-
tors to the developing microbiota and behaved similarly in the two widely different
recipient soils (rONS and rACS). These species survived and colonized similarly in an
agricultural soil and a telluric environment contaminated by aromatic compounds. For
example, 35 genera from the ACS inoculum are known to be common and abundant
agricultural soil colonizers (see Text S1 in the supplemental material). In addition, 33
genera (except Gaiella) in the ONS inoculum have been reported to possess aromatic-
and organic-compound-degrading functions or were detected in sites polluted with
aromatic and organic compounds (Text S1). Although these taxa belonged to phylo-
genetically remote taxa with markedly different functional traits, they exhibited strong
involvement in the community reassembly process regardless of the ambient resources
available in the two sterilized soil types. The observation that the species exhibited
equal fitness in the two environmental conditions is in agreement with the ecological
equivalence concept (20, 22). November/December 2019
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10 DISCUSSION In
that situation, the finite ecologically equivalent species would occupy corresponding
niches in both recipients; however, other nonequivalent species in the inoculum would
be differentially selected by the physical and chemical properties of the recipient soils;
thus, the niche-adapted bacteria would be selected from the inoculum (4, 24). The
sterilized soil provides ambient resources for species immigrating from the inoculum. The aromatic compound contamination in the recipient of ACS (rACS) soil implied that
bacteria having specific degrading functions should have higher fitness than other taxa. Similarly, bacteria adapted genetically to agricultural conditions would develop pref-
erentially in the recipient of ONS (rONS) soil. Then, the selection of recipients for
nonequivalent species would dominate the differentiation of community assembly,
although the different recipients might enrich and share some predominant ecologi-
cally equivalent species. Different fertilization treatments in agricultural land that msystems.asm.org
10 Community Assembly in Sterile Soil FIG 6 Scheme of community assembly driven by inocula and recipient in soil. (A to C) Community assembly with two
inocula containing completely different bacteria (A), with two inocula containing both different and identical bacteria (B),
and with two inocula with the identical bacterial community (C). The colorful ovals, rectangles, and stars in the inocula and
recipients represent different bacteria. Ecologically equivalent bacteria (EEB) are represented by stars in the scheme. The
horizontal axis represents the degree of difference of inocula, either divergence or convergence. The deeper the color, the
more dissimilar the inocula. The vertical axis represents the richness of EEB. The deeper the color, the higher the richness
of EEB. FIG 6 Scheme of community assembly driven by inocula and recipient in soil. (A to C) Community assembly with two
inocula containing completely different bacteria (A), with two inocula containing both different and identical bacteria (B),
and with two inocula with the identical bacterial community (C). The colorful ovals, rectangles, and stars in the inocula and
recipients represent different bacteria. Ecologically equivalent bacteria (EEB) are represented by stars in the scheme. The
horizontal axis represents the degree of difference of inocula, either divergence or convergence. The deeper the color, the
more dissimilar the inocula. The vertical axis represents the richness of EEB. The deeper the color, the higher the richness
of EEB. shared the same original species pool equate to the situation of inoculating the similar
or same inoculum into different recipients as performed in our investigation. November/December 2019
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e00496-19 DISCUSSION On the basis of the results of our study, we may
deduce that once the sources of inoculated bacteria are homologous and their composi-
tions are similar, environmental selection would dominate the assembly processes. If
inoculated bacteria are heterologous and phylogenetically dissimilar, they would dominate
over the selection effect of environmental conditions. Here, we touched upon the basic and
eternal ecological topic about the community-environment interaction. Speculatively, the
types of inocula impact the community-environment interactions and thus act as the most
important factor for the community assembly. The similarity between inocula is also
important for the assembly of the bacterial community. In extreme situations, the inocula
have totally different or identical bacteria, as the cases of our experiment with two type of
distinctly different inocula or only with one type of inoculum (Fig. 6A and C). However, in
many natural situations, the differences of inocula are between above-mentioned situa-
tions, in which both inocula and environment selection could be important factors (Fig. 6B). Furthermore, our study showed that there are two types of immigrated bacteria, ecologi-
cally equivalent bacteria (EEB) or nonequivalent bacteria, in the inocula. The more ecolog-
ically equivalent bacteria are in the inoculum, the more similar bacterial communities
assembled in the recipients. The less similar inocula containing much more different
ecologically equivalent bacteria would assemble more distinct communities. In that case,
the inoculated bacteria would be the predominant factor for determining the bacterial
community. Conversely, if the inoculated bacteria are more similar, the assembled bacterial
communities would be determined by either ecologically equivalent bacteria or recipient-
selected nonecologically equivalent bacteria depending on the community structure of the
inoculum (Fig. 6). Therefore, in general, the role of inoculum and recipient environment
factors relies on the inocula being used in the experiment. However, soils are complex environments that dictate the use of artificial conditions
to address these questions. The use of sterilized soil microcosms that are artificially
reinoculated and incubated under lab conditions is far from a simulation of natural
conditions. Therefore, other approaches are required to verify the conclusions drawn in
this study. Nevertheless, by using cross inoculation with two distinct soils, we explored
the relative importance of inoculum and recipient during community assembly and
revealed the reason for the variability in the explanation of the role of these factors. DISCUSSION If sterilized
soil recipients are inoculated with a similar equivalent bacterial species pool, taxonomic
analysis of the resulting microbiota after incubation would indicate the selection of
“ecologically nonequivalent species” that colonize specific niches. This finding supports
the idea that environmental selection is the determining factor, as shown in a previous
study by Xun et al. (5), who reported that the taxonomic structures of the microbiota
developing in sterilized microcosms were mainly shaped by the recipient soil properties
when the inocula originated from the similar soil source. Under these experimental
conditions, the inocula contained similar immigrating species, some of which were
ecologically equivalent for all recipients, whereas other nonequivalent species were
specifically selected by the specific niches in each soil habitat. Clearly, selection of
nonequivalent species by the recipient soils dominated the differentiation of soil
microbiota structuration and excluded a role of ecologically equivalent species (sum-
marized in Fig. 6). According to our results, we found that the community structures are shaped by
multiple factors (both inoculated bacteria and environmental factors) (Fig. 3). The
findings are consistent with an integrated perspective for the explanation of commu-
nity assembly, such as the concept of meta-community (30), which emphasizes differ-
ent processes and their potential importance (31). The local microbiota is considered to
be a product of random events during the recruitment of ecologically equivalent
microorganisms from the source community or meta-community, which explains why
there were differences in the roles of immigrated bacteria and environmental selection
for dominating the community assembly processes in previously published studies. The
variability in different studies might be caused by the degree of differences among
the inocula used for these experiments. The key point for these two factors to establish
the determinative role in community assembly might lie in the similarity of species
pools that were used for immigration. In conclusion, we distinguished the relative importance between inoculum and recipi-
ent environment in the determination of community structure. We found that inoculum
was more important in the process of community assembly. Our investigation also ex-
plained the reason for the variability in previous studies about the role of biotic factors and
environmental selection. Discrepancy in the determinative factors for community assem- msystems.asm.org
11 Wu et al. Wu et al. blies was associated with the differences between inocula that contained either similar or
dissimilar ecological equivalent bacteria. DISCUSSION This
study would be a step forward toward realizing the prediction and manipulation of the
composition and dynamics of naturally occurring microbial communities, which would
be beneficial to engineering, medical, agricultural, and environmental science. November/December 2019
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e00496-19 MATERIALS AND METHODS For example, the four parts of H-ONS-rONS-ae represent the dosage, inoculum, recipient
soil, and aeration conditions, respectively. “H” and “L” indicate high- (3 g) or low-dosage (0.3 g) soil
inoculum; “ae” and “an” indicate that the soil was incubated under aerobic or anaerobic conditions,
respectively; “r” in the recipient indicates that the soil had been sterilized by radiation. To eliminate any
effects of exotic soil particles, the soil microcosm was mixed vigorously. Analysis of the soil biomass and bacterial community. DNA was extracted from 0.5 g of soil as
described previously (35, 36). Each treatment had triplicate samples, and DNA was extracted from each
sample in triplicate to eliminate the influence of soil heterogeneity. The three triplicate extracts of each
soil sample were pooled as one DNA sample. The quality of the DNA was assessed based on the
260-nm/280-nm absorbance ratio as measured by a BioDrop LITE (Biochrom, UK), and the quantity of
DNA was measured with a Quan-It PicoGreen double-stranded DNA (dsDNA) assay kit (Invitrogen, USA). Quantitative real-time PCR was conducted on a Light cycler 96 (Roche, Switzerland) using SYBR green as
a fluorescent dye to determine the relative abundance of the 16S rRNA gene (37) (Text S1). The extracted DNA was used as the template to amplify the V3-V4 region of the 16S rRNA gene to
construct the sequencing library. The library was constructed according to the manufacturer’s instruc-
tions (38) with some modifications as previously described (39) and sequenced on the Illumina MiSeq
platform (Illumina Inc., USA) as previously described with minor modifications (40). The preparation of
the sequencing library, including DNA extraction and PCR amplification, was conducted as previously
described (41). Specifically, the V3-V4 hypervariable region of the bacterial 16S rRNA gene was amplified
from the genomic DNA using the universal primer set B341F/B785F (42). The purified amplicons were
sequenced using an Illumina MiSeq system (41). Bioinformatics and sequencing data analysis. Both the forward and reverse ends of the read were
trimmed at the base which gave a q value (false-discovery rate) of less than 20. If a pair of reads had a
minimum overlap of 50 bp, they were merged into a complete read, which was retained only if it was
longer than 399 bp, and the expected error was no more than 1 (43). The quality-filtered reads were
dereplicated into unique sequences and sorted by decreasing abundance, and singletons were dis-
carded. MATERIALS AND METHODS Nonchimeric OTU (operational taxonomy unit) representative sequences were picked afterwards
by Uparse’s default (44). Further reference-based chimera detection was performed using UCHIME (45)
against an RDP classifier training database (v9) (46) as the reference database. The OTU table was
finalized by mapping quality-filtered reads to the remaining OTUs with the Usearch (43) global alignment
algorithm at a cutoff of 97%. The number of high-quality reads was greater than 20,000 for all samples. Therefore, the sequences
of each sample were randomly reextracted 20,000 reads per time (1,000 permutations) for rarefaction to
equalize the differences in sequencing depth using QIIME (47). Representative sequences for each OTU
were subjected to the RDP classifier to determine the phylogeny with a bootstrap cutoff of 80% (RDP
database version 2.10). The alpha diversity and beta diversity of the bacterial community structure were
calculated with QIIME (47). The phylogenetic tree for the UniFrac analysis (48) was constructed with the
maximum-likelihood method by FastTree (49). The statistical significance of community structural similarity between different clusters was assessed
by multivariate analysis of variance (MANOVA) with MATLAB 2014a (MathWorks Inc., Natick, MA, USA) as
described by Xu et al. (50) and Tong et al. (51). A redundancy analysis (RDA) performed with CANOCO
for Windows 4.5 (Microcomputer Power, Ithaca, NY, USA) was used to investigate the most significant
environmental variables that shifted their composition and structure (52). For the RDA, a Monte Carlo
permutation test was used to examine the correlations between community structure and each variable
(P 0.05). Statistical significance was assessed by the Monte Carlo permutation procedure (MCPP) with
9,999 random permutations under the full model. Variation partitioning analysis (VPA) was performed to
partition the total variation of the dependent variable into various portions as described by Xun et al. (5). Data availability. The data on natural ONS and ACS samples have been reported in reference 33. Th 16S RNA
i
thi
t d
b
itt d t
th G
B
k S
R
d A hi Data availability. The data on natural ONS and ACS samples have been reported in reference 33. The 16S rRNA gene sequences in this study were submitted to the GenBank Sequence Read Archive
(SRA) database in the National Center for Biotechnology Information (NCBI) under accession number
SRP153935. MATERIALS AND METHODS Soil sampling. Two different types of soil were used in this investigation. One was sampled from
cultivated farmland with optimized nitrogen fertilization with straw management (optimized nitrogen
fertilized soil [ONS]) at a cropland in Quzhou (36°86=N, 115°02=E), Hebei Province, northern China. The
field at this sampling site consists of intensively managed agricultural soils, where a winter
wheat-summer maize rotation is the dominant crop production system and urea- and NH4-based
fertilizers are the most commonly applied nitrogen fertilizers (32). The soil is a calcareous fluvo-aquic soil
(calcareous Cambisols according to the FAO Classification). The other soil sample (aromatic-compound-
contaminated soil [ACS]) was collected from a severely organic-compound-contaminated site at a
chemical plant in Shanghai (31°41=N, 121°46=E), eastern China. The parent material of this soil is mainly
sediments transported by the Yangtze River that have separated into light loam and medium loam after
long-term tillage. The representative characteristic of this ACS soil sample was contamination by
polycyclic aromatic hydrocarbons. ONS soil samples were collected from the top 20 cm by sterile manual
corers (10-cm diameter), and ACS soil samples were sampled at 150 cm below ground with GeoProbe
Systems. Both soil samples were transported and preserved as described previously (5, 17). The two soil
samples had distinct physicochemical properties and bacterial community structures, which were
reported in our previous study (33). Soil microcosms. After the soil samples were sieved, both subsamples were sterilized by gamma
irradiation (50 kGy; Shanghai Heming Radiation Technology Co. Ltd., China), and the irradiated soil
samples were stored as in reference 34. The details of the irradiation can be found in the supplemental
material (Text S1). To evaluate the efficiency of sterilization, soil suspensions were spread for plate
counting with beef extract peptone medium, actinomycetes culture medium, Martin medium, and 1/10
tryptic soy agar medium. Sterile water was added to maintain a constant moisture level (the water
content of soil was 19% for ONS and 26% for ACS). The inocula were prepared with nonsterile soil
preincubated at 25°C in regularly aerated bags for 1 week. The two sterilized soils (rONS and rACS) were msystems.asm.org
12 Community Assembly in Sterile Soil inoculated and mixed with inocula of nonsterile ONS and ACS soils at different inoculation doses (heavy
[H] and light [L]) and oxygen conditions (aerobic [ae] and anaerobic [an]) (Text S1). November/December 2019
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e00496-19 MATERIALS AND METHODS Sterile soil (30 g) was
randomly inoculated with one of the inocula at different dosages (0.3 g of soil for the low dose or 3 g of
soil for the high dose). In addition, 30 g of nonsterile ONS and ACS soil were incubated in serum bottles
as controls. Headspace vials (100 ml;, CNW Technologies, Germany) with soil were sealed to retain
moisture. The aerobic incubation bottles were sealed directly in a laminar flow bench (Shanghai, China),
and the vials were not supplemented with extra oxygen during the incubation. The gas in the anaerobic
incubation vials was replaced with helium by a pumping ventilation system (Shanghai, China). All vials
were incubated at 25°C in the dark for 2 months in triplicate for each treatment. There were a total of
20 treatments (60 samples) in this investigation, and each treatment was named according to its
management. For example, the four parts of H-ONS-rONS-ae represent the dosage, inoculum, recipient
soil, and aeration conditions, respectively. “H” and “L” indicate high- (3 g) or low-dosage (0.3 g) soil
inoculum; “ae” and “an” indicate that the soil was incubated under aerobic or anaerobic conditions,
respectively; “r” in the recipient indicates that the soil had been sterilized by radiation. To eliminate any
effects of exotic soil particles, the soil microcosm was mixed vigorously. inoculated and mixed with inocula of nonsterile ONS and ACS soils at different inoculation doses (heavy
[H] and light [L]) and oxygen conditions (aerobic [ae] and anaerobic [an]) (Text S1). Sterile soil (30 g) was
randomly inoculated with one of the inocula at different dosages (0.3 g of soil for the low dose or 3 g of
soil for the high dose). In addition, 30 g of nonsterile ONS and ACS soil were incubated in serum bottles
as controls. Headspace vials (100 ml;, CNW Technologies, Germany) with soil were sealed to retain
moisture. The aerobic incubation bottles were sealed directly in a laminar flow bench (Shanghai, China),
and the vials were not supplemented with extra oxygen during the incubation. The gas in the anaerobic
incubation vials was replaced with helium by a pumping ventilation system (Shanghai, China). All vials
were incubated at 25°C in the dark for 2 months in triplicate for each treatment. There were a total of
20 treatments (60 samples) in this investigation, and each treatment was named according to its
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mSystems.00496-19. Supplemental material for this article may be found at https://doi.org/10.1128/
mSystems.00496-19. y
TEXT S1, PDF file, 0.4 MB. FIG S1, TIF file, 2.9 MB. TABLE S1, PDF file, 0.1 MB. TABLE S2, PDF file, 0.1 MB. TEXT S1, PDF file, 0.4 MB. FIG S1 TIF file 2 9 MB TEXT S1, PDF file, 0.4 MB. TEXT S1, PDF file, 0.4 MB. FIG S1, TIF file, 2.9 MB. FIG S1, TIF file, 2.9 MB. TABLE S1, PDF file, 0.1 MB. TABLE S2, PDF file, 0.1 MB. TABLE S1, PDF file, 0.1 MB. TABLE S2, PDF file, 0.1 MB. msystems.asm.org
13 November/December 2019
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e00496-19 Wu et al. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (NSFC
31670105, 31861133018, and Key Project 41230856) and the National Key Research and
Development Program of China (2017YFD0200102). We thank Xiaotang Ju for his help in collecting the ONS soil samples. We declare that we have no competing interests. November/December 2019
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.1038/nmeth.2604. msystems.asm.org
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Lower glycolysis carries a higher flux than any biochemically possible alternative
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Nature communications
| 2,015
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cc-by
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ARTICLE Received 27 Mar 2014 | Accepted 21 Aug 2015 | Published 29 Sep 2015 ARTICLE Reaction step Reaction step Figure 1 | The trunk pathway of glycolysis and gluconeogenesis. The Figure 1 | The trunk pathway of glycolysis and gluconeogenesis. The
end points are glyceraldehyde 3-phosphate (G3P) and pyruvate (PYR);
intermediate metabolites are 1,3-bisphosphoglycerate (1,3-BPG);
3-phosphoglycerate (3-PG); 2-phosphoglycerate (2-PG) and
phosphoenolpyruvate (PEP). For each reaction, the external metabolites
involved and the first three numbers from the EC number classification
are indicated. The inset shows thermodynamic profiles for the trunk
pathway in the glycolytic and gluconeogenenic (pps) directions
(see Methods for details). Figure 1 | The trunk pathway of glycolysis and gluconeogenesis. The
end points are glyceraldehyde 3-phosphate (G3P) and pyruvate (PYR);
intermediate metabolites are 1,3-bisphosphoglycerate (1,3-BPG);
3-phosphoglycerate (3-PG); 2-phosphoglycerate (2-PG) and
phosphoenolpyruvate (PEP). For each reaction, the external metabolites
involved and the first three numbers from the EC number classification
are indicated. The inset shows thermodynamic profiles for the trunk
pathway in the glycolytic and gluconeogenenic (pps) directions
(see Methods for details). p
p
g y
3-phosphoglycerate (3-PG); 2-phosphoglycerate (2-PG) and
phosphoenolpyruvate (PEP). For each reaction, the external metabolites
involved and the first three numbers from the EC number classification
are indicated. The inset shows thermodynamic profiles for the trunk
pathway in the glycolytic and gluconeogenenic (pps) directions
(see Methods for details). Here, we perform an exhaustive computational search for all
possible biochemically feasible alternatives to one of the most
ancient and most highly conserved sections of central carbon
metabolism, the trunk pathway of glycolysis and gluconeogenesis. Glycolysis breaks down glucose to pyruvate, generating ATP,
NADH and biosynthetic precursors, whereas gluconeogenesis
uses ATP and NADH to generate glucose from pyruvate. The glycolytic-gluconeogenic pathway is almost linear and can
be divided into two parts: an ‘upper’ chain of reactions involving
6-carbon molecules, which connects glucose to glyceraldehyde-
3-phosphate (G3P), and a ‘lower’ chain of reactions involving
3-carbon molecules, which connects G3P to pyruvate (Fig. 1). This lower reaction chain is known as the trunk pathway. In
prokaryotes, the glycolytic and gluconeogenic trunk pathways
consist of almost the same set of five reactions, differing only
in one step. Results A network of all possible biochemical reactions. We used a
computer program to generate the network of all possible bio-
chemically feasible pathways between G3P and pyruvate. A network of all possible biochemical reactions. We used a
computer program to generate the network of all possible bio-
chemically feasible pathways between G3P and pyruvate. Metabolites. Our program first systematically generates all possible
relevant internal metabolites, that is, molecules intermediate
between G3P and pyruvate, including those that are not found in
nature (see Methods for details). Central carbon metabolism
consists exclusively of reactions between ‘CHOPN’ molecules:
those composed of carbon, hydrogen, oxygen, phosphorus and
nitrogen atoms, with phosphorus being present only in phosphate
groups21,22. Moreover,
the
trunk
pathway
contains
only
unbranched
aliphatic
3-carbon
CHOP
molecules. For
completeness, we therefore include in our analysis all possible
unbranched aliphatic CHOPN molecules containing up to four
carbon atoms. We consider only molecules that are negatively
charged
(that
is,
include
carboxyl
or
phosphate
groups). This condition is motivated by the need to avoid leakage
through the negatively charged phospho-lipid membrane, and is
satisfied by almost all molecules in core metabolism23. Applying
these
criteria
results in
almost
1,500
different
molecules,
including all the internal metabolites in the real trunk pathway. We computed the free energy of formation DfG for all our
internal metabolites, using existing experimental data where
possible24 or, in the absence of such data, using a variant of the
group contribution method25–28 (see Methods for details). Although the upper part of the glycolytic/gluconeogenic path-
way exists in several very distinct variants, most notably the
Embden–Meyerhof–Parnas (EMP) and Entner–Doudoroff (ED)
pathways (Supplementary Fig. 1)8,18, the trunk pathway is near-
ubiquitous, and contains enzymes which are highly conserved and
universally distributed across the three domains of life19,20. Only a
very small number of variations are observed in nature (see
Supplementary Discussion for details). The existence of such an
ancient and universal pathway suggests three possible scenarios: (i)
the trunk pathway is the only biochemical possibility, (ii)
alternatives exist but the extant pathway is evolutionarily optimal
and (iii) alternatives are possible but have not been found by
evolution. Distinguishing these scenarios lies at the heart of much
of evolutionary biology. Here, we address this question directly
using a computational approach. By systematically constructing
and exploring the full space of biochemically feasible metabolites
and reactions, many of which are not currently exploited by any Reactions. ARTICLE In glycolysis, the exergonic conversion of phos-
phoenolpyruvate (PEP) to pyruvate is coupled to the phosphor-
ylation of ADP to ATP, whereas in gluconeogenesis the reverse
reaction is driven by the hydrolysis of ATP to AMP, either with
release
of
inorganic
phosphate
(the
phosphoenolpyruvate
synthase
(pps)
route)
or
via
consumption
of
inorganic
phosphate and release of pyrophosphate (the phosphate dikinase
(ppdk) route). known organism, we find that hundreds of alternative trunk
pathways are possible. The one observed in nature, however,
carries the maximal biochemical flux, under reasonable constraints
on the intermediate metabolite and enzyme concentrations. Our
results suggest that the trunk pathway represents an optimal
solution among many possible alternatives. Lower glycolysis carries a higher flux than any
biochemically possible alternative Steven J. Court1, Bartlomiej Waclaw1 & Rosalind J. Allen1 The universality of many pathways of core metabolism suggests a strong role for evolutionary
selection, but it remains unclear whether existing pathways have been selected from a large
or small set of biochemical possibilities. To address this question, we construct in silico all
possible biochemically feasible alternatives to the trunk pathway of glycolysis and
gluconeogenesis, one of the most highly conserved pathways in metabolism. We show that,
even though a large number of alternative pathways exist, the alternatives carry lower flux
than the real pathway under typical physiological conditions. We also find that if physiological
conditions were different, different pathways could outperform those found in nature. Together, our results demonstrate how thermodynamic and biophysical constraints restrict
the biochemical alternatives that are open to evolution, and suggest that the existing trunk
pathway of glycolysis and gluconeogenesis may represent a maximal flux solution. 1 SUPA, School of Physics and Astronomy, University of Edinburgh, James Clerk Maxwell Building, Peter Guthrie Tait Road, Edinburgh EH9 3FD, UK. Correspondence and requests for materials should be addressed to R.J.A. (email: rallen2@staffmail.ed.ac.uk). NATURE COMMUNICATIONS | 6:8427 | DOI: 10.1038/ncomms9427 | www.nature.com/naturecommunications 1 & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | 6:8427 | DOI: 10.1038/ncomms9427 | www.nature.com/naturecommunications
& 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE ARTICLE 0
0
–10
–20
–30
1
2
3
4
5
G3P
NAD, Pi
NADH
1,3-BPG
ADP
ADP
ATP
ATP
3PG
2PG
1.2.1
1.2.1
1.2.1
2.7.2
2.7.2
2.7.2
2.7.1
2.7.1
2.7.9
2.7.9
5.4.2
5.4.2
5.4.2
4.2.1
4.2.1
4.2.1
AMP,
Pi/PPi
ATP,
H2O/Pi
H2O
PEP
PYR
Free energy (kJ mol–1)
Reaction step
Figure 1 | The trunk pathway of glycolysis and gluconeogenesis. The
end points are glyceraldehyde 3-phosphate (G3P) and pyruvate (PYR);
intermediate metabolites are 1,3-bisphosphoglycerate (1,3-BPG);
3-phosphoglycerate (3-PG); 2-phosphoglycerate (2-PG) and
phosphoenolpyruvate (PEP). For each reaction, the external metabolites
involved and the first three numbers from the EC number classification
are indicated. The inset shows thermodynamic profiles for the trunk
pathway in the glycolytic and gluconeogenenic (pps) directions
(see Methods for details). 0
0
–10
–20
–30
1
2
3
4
5
G3P
NAD, Pi
NADH
1,3-BPG
ADP
ADP
ATP
ATP
3PG
2PG
1.2.1
1.2.1
1.2.1
2.7.2
2.7.2
2.7.2
2.7.1
2.7.1
2.7.9
2.7.9
5.4.2
5.4.2
5.4.2
4.2.1
4.2.1
4.2.1
AMP,
Pi/PPi
ATP,
H2O/Pi
H2O
PEP
PYR
Free energy (kJ mol–1)
Reaction step T
he biochemical pathways of central carbon metabolism are
highly conserved across all domains of life, and largely
control the productivity of life on the Earth1,2. Yet it
remains unknown whether these pathways are the result of
historical contingency during early evolution3,4, or are instead
optimal solutions to the problem of energy and biomass
production5–10. Put simply, are there alternative biochemically
feasible pathways that could perform the same function, and if so,
how do they perform compared with those found in nature? T y p
p
Previous studies have mainly addressed this question either
by constructing simplified artificial metabolic networks5–7, or by
mining databases of biochemical compounds and reactions for
known organisms4,8,9,11–13. Both of these approaches have
drawbacks: the former does not capture real biochemistry,
whereas the latter is limited to metabolites and reactions
found in well-studied organisms. Nevertheless, this work has
led to suggested optimality principles including maximizing
biochemical flux or yield6,7, minimizing biochemical steps or
protein costs5,8 and ensuring that function is maintained in a
changing environment11. Only a few studies have attempted
to explore the full universe of possible metabolic pathways,
using realistic rules of biochemistry10,14–17. In particular, using
this approach, Noor et al.10 recently suggested that central
carbon metabolism, taken as a whole, uses the minimal number
of enzymatic steps needed to generate a predefined set of
biochemical precursors. Results Although we show non-ionic formulae, all compounds are assumed to exist as an equilibrium
mixture of protonated and deprotonated species in solution (Supplementary Methods). Phosphate groups -PO2
4
are denoted by a ‘p’. Not all variants of each reaction class are listed here; for a complete
list, see Supplementary Table 3. The real paths maximize flux under physiological conditions. We next evaluated the performance of the alternative trunk
pathways
generated
by
our
network,
by
comparing
their
steady-state metabolic flux. For glycolytic pathways, the metabolic
flux corresponds to the rate of ATP production, whereas for
gluconeogenic pathways, it corresponds to the rate of production
of G3P (and ultimately of glucose). This flux depends not only on
the total free energy change across a given pathway, but also on the
distribution of the individual reaction free energies (see, for
example, Fig. 1), which in turn depends on the intracellular
environment via the concentrations of the external metabolites. For
linear pathways, the flux can be calculated analytically, assuming
linear kinetics with diffusion-limited enzymes7,30,31 (see Methods
for details). We also assume that, for each pathway, the individual
enzyme concentrations are optimized to maximize pathway flux,
for a fixed total amount of enzyme (see Methods for details). reaction classes29, which encompass all the reactions between
CHOPN molecules in core metabolism, of length four carbon
atoms or less (Table 1). Many of these reactions involve cofactors
such as ATP and NAD þ; we refer to these as external metabolites
and we assume that they have fixed concentrations, which define
the cellular (physiological) conditions. The full set of external
metabolites in our reaction network is shown in Supplementary
Table 1 and includes the energy currency metabolite ATP, the
redox cofactor NAD þ, the amino-group donor glutamate, as well
as inorganic phosphates and CO2. reaction classes29, which encompass all the reactions between
CHOPN molecules in core metabolism, of length four carbon
atoms or less (Table 1). Many of these reactions involve cofactors
such as ATP and NAD þ; we refer to these as external metabolites
and we assume that they have fixed concentrations, which define
the cellular (physiological) conditions. The full set of external
metabolites in our reaction network is shown in Supplementary
Table 1 and includes the energy currency metabolite ATP, the
redox cofactor NAD þ, the amino-group donor glutamate, as well
as inorganic phosphates and CO2. Our network generates many alternative trunk pathways. Results Our
network reveals a huge number of alternative pathways con-
necting G3P and pyruvate, which are consistent with the rules of
biochemistry. Enumerating these alternatives, we find 2, 29, 555
and 5,859 pathways of length 3, 4, 5 and 6, respectively, with the
number of pathways increasing exponentially with path length
(Supplementary Fig. 2). Some of these alternative pathways are
found to use the same set of reaction types as the natural trunk
pathway, but execute them in a different order (for example, left
pathway of Fig. 2a), whereas others make use of a different set of
reaction types (right pathway of Fig. 2a). y
Importantly, when calculating the metabolic flux, we impose
constraints on the intermediate internal metabolite concentra-
tions. For the bacterium Escherichia coli, metabolite concentra-
tions range from 0.1 mM to 100 mM, with the total intracellular
metabolite pool being around 300 mM (ref. 32). We expect that
very low metabolite concentrations are undesirable because of
vanishing fluxes, whereas very high concentrations are precluded
by osmotic considerations. In our initial comparison, we therefore
set the flux to zero for a given pathway if any of its intermediate
metabolite concentrations falls outside the range of 0.1 mM–
100 mM. We wish to investigate how many of these alternative trunk
pathways are feasible under typical physiological conditions. First, in the glycolytic direction, we demand that candidate
pathways
produce
at
least
two
ATP
molecules. This
is
required so that the full glycolytic pathway produces a net
ATP yield, as the two dominant forms of glycolysis, the EMP
and ED pathways, consume one ATP molecule per G3P in their
upper halves. In addition, motivated by considerations of
simplicity5,10 and cost of enzyme production8, we further
restrict our analysis to those pathways with a near-minimal
number of steps. This results in a set of 1,787 candidate glycolytic
pathways of length 4, 5 and 6 that produce at least two ATP
molecules,
and
a
set
of
6,445
biochemically
plausible
gluconeogenic pathways of lengths 3–6 (not taking into account
thermodynamics). Imposing this constraint, we calculated the metabolic flux of all
our candidate pathways, across a wide range of intracellular
conditions, as defined by the external metabolite concentrations. NATURE COMMUNICATIONS | 6:8427 | DOI: 10.1038/ncomms9427 | www.nature.com/naturecommunications
& 2015 Macmillan Publishers Limited. All rights reserved. & 2015 Macmillan Publishers Limited. All rights reserved. Results Our program generates all possible reactions among our set
of internal metabolites, based on the 20 Enzyme Commission (EC) NATURE COMMUNICATIONS | 6:8427 | DOI: 10.1038/ncomms9427 | www.nature.com/naturecommunications
& 2015 Macmillan Publishers Limited. All rights reserved. 2 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9427 Table 1 | The set of enzymatic reaction types used in generating the network of reactions. EC class
Example reaction
1.1.1
Oxidation
CH3-CH2(OH) þ NAD þ"CH3-CHO þ NADH
1.2.1
phosphorylation
CH3-CHO þ NAD þ þ Pi"CH3-COp þ NADH
1.3.1
COOH-CH2-CH2-COOH þ NAD þ"COOH-CH ¼ CH-COOH þ NADH
1.4.1
Deamination
COOH-CH(NH2)-CH3 þ NAD þ þ H2O"COOH-CO-CH3 þ NADH þ NH3
2.6.1
Transamination
COOH-CH(NH2)-CH3 þ 2-oxoglutarate"COOH-CO-CH3 þ glutamate
2.7.1
Phosphate transfer
CHO-CH2(OH) þ ATP"CHO-CH2p þ ADP
2.7.2
CH3-COOH þ ATP"CH3-COp þ ADP
2.7.9
CH3-CO-COOH þ ATP þ H2O/Pi"CH2 ¼ Cp-COOH þ AMP þ Pi/PPi
3.1.3
Hydrolysis
CH3-CH2p þ H2O"CH3-CH2(OH) þ Pi
3.5.1
CH3-CO(NH2) þ H2O"CH3-COOH þ NH3
3.6.1
CH3-COp þ H2O"CH3-COOH þ Pi
4.1.1
Decarboxylation
CH3-CH(OH)-COOH þ H2O"CH3-CH2(OH) þ CO2(aq)
4.2.1
Dehydration
CH3-CH(OH)-COOH"CH2 ¼ CH-COOH þ H2O
4.3.1
Ammonia-lyase
COOH-CH(NH2)-CH3"COOH-CH ¼ CH2 þ NH3
5.3.1
Isomerization
CH3-CO-CH2(OH)"CH3-CH(OH)-CHO
5.3.2
COOH-CH2-CO-COOH"COOH-CH ¼ C(OH)-COOH
5.4.2
CH3-CH(OH)-CH2p"CH3-CHp-CH2(OH)
5.4.3
COOH-CH(NH2)-CH2-CH3"COOH-CH2-CH(NH2)-CH3
6.3.1
ATP-driven amine ligase
R-COOH þ ATP þ NH3"R-CO(NH2) þ ADP þ Pi
6.4.1
ATP-driven carboxylation
CH3-CH2(OH) þ CO2(aq) þ ATP"CH3-CH(OH)-COOH þ ADP þ Pi
The set of reaction types included in our analysis, defined by the first three numbers of the EC classification. Although we show non-ionic formulae, all compounds are assumed to exist as an equilibrium
mixture of protonated and deprotonated species in solution (Supplementary Methods). Phosphate groups -PO2
4
are denoted by a ‘p’. Not all variants of each reaction class are listed here; for a complete
list, see Supplementary Table 3. Table 1 | The set of enzymatic reaction types used in generating the network of reactions. The set of reaction types included in our analysis, defined by the first three numbers of the EC classification. Although we show non-ionic formulae, all compounds are assumed to exist as an equilibrium
mixture of protonated and deprotonated species in solution (Supplementary Methods). Phosphate groups -PO2
4
are denoted by a ‘p’. Not all variants of each reaction class are listed here; for a complete
list see Supplementary Table 3 The set of reaction types included in our analysis, defined by the first three numbers of the EC classification. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9427 (a) Two alternative glycolytic pathways; left-hand
on classes, defined by EC number, as the real trunk pathway, whereas the right-hand pathway uses a different set. (b–d) Figure 2 | The real glycolytic and gluconeogenic trunk pathways represent maximal flux solutions. (a) Two alternative glycolytic pathways; left-hand
pathway uses the same set of reaction classes, defined by EC number, as the real trunk pathway, whereas the right-hand pathway uses a different set. (b–d)
Results of pathway comparison. (b,d) Alternative pathways generated in our analysis ordered by their comparative flux (see text for details), averaged
across the whole parameter space. The top 25 paths are shown. (b) Glycolytic pathways; the black bar (indicated by the arrow) is the natural pathway. (d) Gluconeogenic paths; the red bar (indicated by the arrow) is the natural pps route. (c,e) Relative pathway performance as a function of the intracellular
environment. Each dot represents a randomly sampled point in parameter space; the colour of the dot indicates the candidate pathway, which had the
highest flux at that point in parameter space with colours corresponding to b and d. For parameter sampling procedure, see Methods. The axes correspond
to two important characteristic measures of a cell, the redox state and energy state, via [NAD][Pi]/[NADH] and [ATP]/[ADP], respectively. The red boxes
indicate the typical physiological state of the cell32,43: [ATP]/[ADP] ¼ 3–20, [NAD][Pi]/[NADH] ¼ 0.01–10 M (based on [NAD]/[NADH] ¼ 10–100 and
[Pi] ¼ 1–100 mM). (c) Natural glycolytic pathway (black) tends to perform best under typical physiological conditions. (e) Natural pps gluconeogenic
pathway (red) tends to perform best under typical cellular conditions. See Supplementary Data 1 and 2 for pathway structures. As a simple metric, we first compare the performance of our
candidate pathways, averaged over the entire parameter space. To this end, we compute the comparative flux (CF), which is the
flux of a given pathway, divided by the maximum flux obtained
among all pathways, at a given point in parameter space. Averaging this quantity across the whole parameter space gives a
measure of relative performance for each candidate path. Based
on this metric, we find that the real glycolytic and gluconeogenic
pathways perform remarkably well, compared with the many
alternatives (Fig. 2b,d). For glycolysis (Fig. 2b and Supplementary
Data 1), the natural trunk pathway (in black, indicated by the
arrow) outperforms all the alternative pathways; whereas for
gluconeogenesis (Fig. Results To explore the performance of our candidate pathways over a
wide range of possible intracellular conditions, we randomly
selected 10,000 points from the parameter space consisting of the
concentrations of the 11 external metabolites, G3P and pyruvate,
sampling each parameter logarithmically over several orders of
magnitude above and below its typical physiological concentra-
tion (see Methods for details). For each point in parameter space,
we evaluated the flux of each candidate pathway. 3 3 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9427 NATURE COMMUNICATIONS | 6:8427 | DOI: 10.1038/ncomms9427 | www.nature.com/naturecommunications
& 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9427 2d, Supplementary Data 2), one of the real
pathways (the pps variant) is also ranked first (in red, indicated
by the arrow). These results strongly suggest that the natural
trunk pathways carry a high flux compared with alternatives;
however, this metric is dependent on the range of parameter
space that is sampled. remarkably, the natural trunk pathway (black dots) outperforms
all the alternatives in the region of parameter space close to that
found in living cells (red box). This suggests that the glycolytic
trunk pathway represents a maximal flux solution for the
conversion of G3P to pyruvate, under typical intracellular
conditions. Similarly, for the gluconeogenic pathways (Fig. 2d),
the pps pathway found in nature (red dots) outperforms the
alternatives under typical physiological conditions (red box). Alternative flux calculations. The flux calculation used above
makes the simplifying assumption that all enzymes are perfect
catalysts6,7,30,31 and thus it neglects the important physical feature
of substrate saturation. Any attempt at applying a generic choice of
kinetic equations to a system of reactions is problematic, and
more complicated forms give rise to a greater number of
parameters that must be specified. However, one can test the
effect that using alternative forms has on the overall outcome, and
we find that the above results still hold when two forms of
reversible Michaelis–Menten kinetics are assumed (Supplementary
Figs 3, 4 and Methods), capturing the behaviour of substrate and
product inhibition. Thus, our results are not dependent on the
detailed assumptions made in modelling the enzyme kinetics. To overcome this issue and to investigate in more detail, we
analysed which of our candidate pathways achieved the highest
flux at different points in the parameter space. This allows us to
understand how the performance of a given pathway depends on
the intracellular environment. Our results show that different
candidate pathways perform best in different regions of the
parameter space. In particular, pathway performance is very
sensitive to the cellular energy state, as measured by the ratio of
the ATP and ADP concentrations ([ATP]/[ADP]), and redox
state, represented by the ratio [NAD][Pi]/[NADH] (Fig. 2c,e). Focusing on the glycolytic pathways (Fig. 2b) we see that, Constraints on metabolite concentrations are important. Imposing constraints on the intermediate internal metabolite
concentrations is crucial to our analysis. NATURE COMMUNICATIONS | 6:8427 | DOI: 10.1038/ncomms9427 | www.nature.com/naturecommunications
& 2015 M
ill
P bli h
Li
it d All i ht
d NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9427 0.6
0.5
0.4
0.3
0.2
0.1
0
1 3 5 7 9 11 13 15 17 19 21 23 25
CF
Pathway
Glycolysis
b G3P
3PG
2PG
G3P
CHO-CH(OH)-CH2p
CHO-CHp-CH2(OH)
CHO-Cp=CH2
COp-Cp=CH2
COOH-Cp=CH2
COOH-Cp=CH2
COOH-CH(OH)-CH2p
COOH-CHp-CH2(OH)
COOH-CO-CH2-C
COOH-CO-CH3
COOH-CO-CH3
CHO-CH(OH)-CH2p
5.4.2
5.4.2
NAD, H2O
NADH
NAD, Pi
NADH
1.2.1
1.2.1
2.7.2
2.7.1
4.2.1
4.1.1
4.2.1
6.4.1
H2O
H2O
ADP
ADP, Pi
ADP
ADP, CO2
ATP
ATP
ATP, CO2
ATP, H2O
PEP
PYR
PYR
PEP
a 5
100
100
10
10
1
1
0.1
0.1
0.01
0.001
0.0001
NAD.Pi/NADH
ATP/ADP
c b 0.6
0.7
0.5
0.4
0.3
0.2
0.1
0
1 3 5
7 9 11 13 15 17 19 21 23 25
CF
Pathway
Pathway
Gluconeogenesis
OH
d
XA 1
100
10
1
0.1
100
10
0.1
0.01
0.001
0.0001
NAD.Pi/NADH
ATP/ADP
e d e e ATP/ADP Pathway Figure 2 | The real glycolytic and gluconeogenic trunk pathways represent maximal flux solutions. (a) Two alternative glycolytic pathways; left-hand
pathway uses the same set of reaction classes, defined by EC number, as the real trunk pathway, whereas the right-hand pathway uses a different set. (b–d)
Results of pathway comparison. (b,d) Alternative pathways generated in our analysis ordered by their comparative flux (see text for details), averaged
across the whole parameter space. The top 25 paths are shown. (b) Glycolytic pathways; the black bar (indicated by the arrow) is the natural pathway. (d) Gluconeogenic paths; the red bar (indicated by the arrow) is the natural pps route. (c,e) Relative pathway performance as a function of the intracellular
environment. Each dot represents a randomly sampled point in parameter space; the colour of the dot indicates the candidate pathway, which had the
highest flux at that point in parameter space with colours corresponding to b and d. For parameter sampling procedure, see Methods. The axes correspond
to two important characteristic measures of a cell, the redox state and energy state, via [NAD][Pi]/[NADH] and [ATP]/[ADP], respectively. The red boxes
indicate the typical physiological state of the cell32,43: [ATP]/[ADP] ¼ 3–20, [NAD][Pi]/[NADH] ¼ 0.01–10 M (based on [NAD]/[NADH] ¼ 10–100 and
[Pi] ¼ 1–100 mM). (c) Natural glycolytic pathway (black) tends to perform best under typical physiological conditions. (e) Natural pps gluconeogenic
pathway (red) tends to perform best under typical cellular conditions. See Supplementary Data 1 and 2 for pathway structures. gluconeogenic trunk pathways represent maximal flux solutions. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9427 0.6
0.5
0.4
0.3
0.2
0.1
0
0.6
0.7
0.5
0.4
0.3
0.2
0.1
0
1 3 5 7 9 11 13 15 17 19 21 23 25
1 3 5
7 9 11 13 15 17 19 21 23 25
CF
CF
Pathway
Pathway
100
100
10
10
1
1
1
0.1
0.1
100
10
1
0.1
0.01
0.001
0.0001
100
10
0.1
0.01
0.001
0.0001
Glycolysis
Gluconeogenesis
NAD.Pi/NADH
NAD.Pi/NADH
ATP/ADP
ATP/ADP
G3P
3PG
2PG
G3P
CHO-CH(OH)-CH2p
CHO-CHp-CH2(OH)
CHO-Cp=CH2
COp-Cp=CH2
COOH-Cp=CH2
COOH-Cp=CH2
COOH-CH(OH)-CH2p
COOH-CHp-CH2(OH)
COOH-CO-CH2-COOH
COOH-CO-CH3
COOH-CO-CH3
CHO-CH(OH)-CH2p
5.4.2
5.4.2
NAD, H2O
NADH
NAD, Pi
NADH
1.2.1
1.2.1
2.7.2
2.7.1
4.2.1
4.1.1
4.2.1
6.4.1
H2O
H2O
ADP
ADP, Pi
ADP
ADP, CO2
ATP
ATP
ATP, CO2
ATP, H2O
PEP
PYR
PYR
PEP
a
b
d
e
c
OXA
Figure 2 | The real glycolytic and gluconeogenic trunk pathways represent maximal flux solutions. (a) Two alternative glycolytic pathways; left-hand
pathway uses the same set of reaction classes, defined by EC number, as the real trunk pathway, whereas the right-hand pathway uses a different set. (b–d)
Results of pathway comparison. (b,d) Alternative pathways generated in our analysis ordered by their comparative flux (see text for details), averaged
across the whole parameter space. The top 25 paths are shown. (b) Glycolytic pathways; the black bar (indicated by the arrow) is the natural pathway. (d) Gluconeogenic paths; the red bar (indicated by the arrow) is the natural pps route. (c,e) Relative pathway performance as a function of the intracellular
environment. Each dot represents a randomly sampled point in parameter space; the colour of the dot indicates the candidate pathway, which had the
highest flux at that point in parameter space with colours corresponding to b and d. For parameter sampling procedure, see Methods. The axes correspond
to two important characteristic measures of a cell, the redox state and energy state, via [NAD][Pi]/[NADH] and [ATP]/[ADP], respectively. The red boxes
indicate the typical physiological state of the cell32,43: [ATP]/[ADP] ¼ 3–20, [NAD][Pi]/[NADH] ¼ 0.01–10 M (based on [NAD]/[NADH] ¼ 10–100 and
[Pi] ¼ 1–100 mM). (c) Natural glycolytic pathway (black) tends to perform best under typical physiological conditions. (e) Natural pps gluconeogenic
pathway (red) tends to perform best under typical cellular conditions. See Supplementary Data 1 and 2 for pathway structures. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9427 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9427 large DrG values, and the real glycolytic pathway falls to number 87
with
respect
to
its
CF
averaged
over
parameter
space
(Supplementary Note 1 and Supplementary Data 3). It is possible,
however, that our limits on the concentrations are too severe, as in
reality the internal structure of a cell, as well as phenomena such as
substrate channelling, can give rise to effective concentrations
much larger or smaller than any global limit. Nevertheless,
Supplementary Fig. 5 along with Supplementary Data 4 and 5
demonstrates that our results still hold when we repeat our analysis
with a more generous allowed concentration range of 1 nM–
500 mM. Path B is a pathway of length 6 and corresponds to the second-
highest performing pathway (green) in Fig. 2b. This pathway
follows the first four steps of the natural pathway before diverging
at PEP to pass through oxaloacetate. This shunt first involves the
carboxylation and dephosphorylation of PEP via PEP carboxylase
EC
4.1.1.31,
followed
by
a
coupled
decarboxylation
and
ATP-generation via pyruvate carboxylase EC 6.4.1.1. Path C is similar to the real glycolytic trunk path, except that
1,3-bisphosphoglycerate (1,3-BPG) is first isomerized (to 2,3-BPG
via EC 5.4.2.4) and then dephosphorylated (to 2-phosphoglyce-
rate (2-PG), with ATP generation), rather than being first
dephosphorylated and then isomerized as in the natural pathway. Interestingly, a similar pathway exists in red blood cells, where
2,3-BPG is produced from 1,3-BPG via the Rapoport–Leubering
shunt33. In red blood cells, however, the 2,3-BPG is hydrolysed to
either
3-phosphoglycerate
(3-PG)
or
2-PG
without
ATP
generation33, thus sacrificing one ATP compared with the usual
glycolytic pathway. Although no enzyme has been characterized
that generates ATP from 2,3-BPG, this reaction does actually
appear in the KEGG database, as R02664. Other paths can perform better under different conditions. We
expect the concentrations of the external metabolites to vary
across organisms and across different environmental habitats. Hence, an interesting question to ask is could any alternative
pathways outperform the real trunk pathways, if the physiological
conditions were different? Indeed, we find that for some values of
the external metabolite concentrations, other pathways outper-
form the natural ones (Fig. 2). Of particular interest is the fact
that the pyrophosphate concentration is an important factor in
our comparison of the gluconeogenic pathways. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9427 Figure 3 shows
that at low PPi concentrations, the natural ppdk variant of the
gluconeogenic
pathway
becomes
more
thermodynamically
favourable and can outperform the more common pps pathway
under typical physiological conditions. In nature, cellular pyr-
ophosphate concentrations can be kept at sub-mM levels through
the highly efficient inorganic diphosphatase enzyme, EC 3.6.1.1. Path D differs from the natural pathway in that it converts G3P
directly to 3-PG without the production of ATP, via EC 1.2.1.9. This large exorgonic initial step gives path D a favourable
thermodynamic profile and results in the two ATP-generating
steps being the final two reactions, catalysed by PEP carbox-
ykinase EC 4.1.1.49 and pyruvate carboxylase EC 6.4.1.1. These
two enzymes are usually associated with a gluconeogenic role in G3P
CHO-CH(OH)-CH2p
CH2p-CH(OH)-COp
CH2(OH)-CHp-COOH
CH2=Cp-COOH
CH2(OH)-CH(OH)-COOH
COOH-CH(OH)-CH2p
COOH-CH2-CO-COOH
COOH-CHp-CH2p
1.2.1.12
1.2.1.9
1,3-BPG
3-PG
2-PG
2,3-BPG
5.4.2.4
5.4.2.1
4.2.1.11
4.1.1.31
6.4.1.1
4.2.1
4.1.1.49
ADP
ADP
ADP
ADP,
CO2
ADP
ATP
ATP
ATP
ATP
ATP,
Pi
ATP,
CO2
ATP
2.7.2.3
2.7.2.-
2.7.1.31
2.7.1.40/
2.7.9.1
Pi
CO2
PEP
GLYC
AMP,
PPi
ADP,
Pi
OXA
PYR
CH3-CO-COOH
Flux
Path A (real)
Path B
Path C
Path D
Path E
Path F
Figure 4 | Thermodynamically feasible alternative glycolytic pathways
utilizing compounds found in KEGG35 (see Supplementary Note 2 for
more details). Reactions are indicated by coloured lines; full EC numbers in
black correspond to enzymes present in KEGG, incomplete numbers are not
found in KEGG. All of the enzymes for alternative Paths B, D and E are
known to exist in nature. GLY, glycerate; OXA, oxaloacetate; 2,3-BPG ¼ 2,3-
bisphosphoglycerate; all others as in Fig. 1. G3P
CHO-CH(OH)-CH2p
CH2p-CH(OH)-COp
CH2(OH)-CHp-COOH
CH2=Cp-COOH
CH2(OH)-CH(OH)-COOH
COOH-CH(OH)-CH2p
COOH-CH2-CO-COOH
COOH-CHp-CH2p
1.2.1.12
1.2.1.9
1,3-BPG
3-PG
2-PG
2,3-BPG
5.4.2.4
5.4.2.1
4.2.1.11
4.1.1.31
6.4.1.1
4.2.1
4.1.1.49
ADP
ADP
ADP
ADP,
CO2
ADP
ATP
ATP
ATP
ATP
ATP,
Pi
ATP,
CO2
ATP
2.7.2.3
2.7.2.-
2.7.1.31
2.7.1.40/
2.7.9.1
Pi
CO2
PEP
GLYC
AMP,
PPi
ADP,
Pi
OXA
PYR
CH3-CO-COOH
Flux
Path A (real)
Path B
Path C
Path D
Path E
Path F Alternative trunk pathways. It is interesting to investigate the
alternative pathways revealed by our analysis in more detail. Our
understanding of even the most well-studied metabolic pathways
is incomplete, with new features being discovered in glycolysis33
and the pentose phosphate pathway34 in recent years. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9427 Hence, it is
interesting to consider the alternatives found that most closely
resemble the natural pathway, as it is possible that some of these
exist in nature, but may be as yet uncharacterized. Figure 4 shows
five thermodynamically feasible alternative glycolytic pathways
found in our network, which utilize only chemical compounds
found
in
the
KEGG
COMPOUND
database
of
known
metabolites35 (Supplementary Fig. 6 and Supplementary Data
6). In fact, in three of these pathways (B, D and E), the relevant
enzyme
for
each
reaction
is
known
to
exist
in
nature
(Supplementary Note 2). Flux 0.1
1
10
100
1,000
10,000
100,000
1e+06
1e+07
10
100
Pi/PPi
ATP/AMP
Figure 3 | The sampled parameter ranges are an important factor in our
analaysis. Repeating the comparison of candidate gluconeogenic paths, but
now sampling [PPi] from 10 8 to 10 2 M instead of from 10 4 to 10 2
shows that the real ppdk pathway (black) becomes favourable compared
with the pps pathway (red), when the ratio of orthophosphate to
pyrophosphate concentrations is increased. Colours as in Fig. 2. 0.1
1
10
100
1,000
10,000
100,000
1e+06
1e+07
10
100
Pi/PPi
ATP/AMP ATP/AMP Figure 4 | Thermodynamically feasible alternative glycolytic pathways
utilizing compounds found in KEGG35 (see Supplementary Note 2 for
more details). Reactions are indicated by coloured lines; full EC numbers in
black correspond to enzymes present in KEGG, incomplete numbers are not
found in KEGG. All of the enzymes for alternative Paths B, D and E are
known to exist in nature. GLY, glycerate; OXA, oxaloacetate; 2,3-BPG ¼ 2,3-
bisphosphoglycerate; all others as in Fig. 1. Figure 3 | The sampled parameter ranges are an important factor in our
analaysis. Repeating the comparison of candidate gluconeogenic paths, but
now sampling [PPi] from 10 8 to 10 2 M instead of from 10 4 to 10 2
shows that the real ppdk pathway (black) becomes favourable compared
with the pps pathway (red), when the ratio of orthophosphate to
pyrophosphate concentrations is increased. Colours as in Fig. 2. Figure 3 | The sampled parameter ranges are an important factor in our
analaysis. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9427 When these constraints
are neglected, a very different set of pathways dominate over the
parameter space, many of which contain individual reactions with NATURE COMMUNICATIONS | 6:8427 | DOI: 10.1038/ncomms9427 | www.nature.com/naturecommunications
& 2015 Macmillan Publishers Limited. All rights reserved. 4 Discussion
h In this study, we have limited our analysis to pathways that
start and end at G3P and pyruvate. Relaxing this requirement
would certainly lead to many more alternative pathways for the
generation of energy and for biosynthesis. Although it is also
important to consider other factors, including the need for
integration within a wider metabolic network, our analysis
suggests that key principles underlying the structure of core
metabolism may emerge from simple biochemical, thermody-
namic and biophysical considerations. Despite the huge variety and complexity of life on Earth, the
biochemistry of core metabolism is remarkably universal. Our
analysis shows that this universality does not arise from an absence
of other possibilities. Using a systematic approach, we have
identified many alternatives to perhaps the most highly conserved
set of metabolic reactions, the glycolytic and gluconeogenic trunk
pathways. Our alternative pathways obey the rules of biochemistry,
carry positive flux under reasonable intracellular conditions, and
satisfy
reasonable
constraints
on
metabolite
concentrations. Remarkably, of all these alternatives, we find that the trunk
pathway observed in nature carries the highest biochemical flux in
both the glycolytic and gluconeogenic directions, for parameters
that represent typical intracellular physiological conditions. Of the
two variants of the prokaryotic gluconeogenic pathway that are
found in nature, the pps route is the best performer across a wide
parameter range, whereas the ppdk route is more sensitive to
environmental conditions, requiring a low concentration of
pyrophosphate (Fig. 3). The fact that our analysis identifies the
natural pathways using only flux maximization combined with
constraints on intermediate metabolite concentrations suggests
that these factors are likely to have been important driving forces
in the evolution of metabolism. Importantly, our key results are
also independent of the detailed assumptions made in modelling
the enzyme kinetics. p y
Finally, we briefly discuss broader evolutionary implications of
our work. Assuming that the trunk pathway can be replaced by
any of our alternative pathways without affecting the rest of the
metabolic network too much, the ATP flux could be used as a
proxy for fitness, that is, the ability of the organism to outcompete
other, similar to organisms. Endowed with a mutational graph of
possible mutations between different pathways (different geno-
types), this would create a fitness landscape in which the highest
peaks correspond to local maxima of the ATP flux. ARTICLE Although the final reaction of this pathway is not present in the
KEGG database, this putative reaction is chemically rather
simple—involving the dehydration of glycerate to enolpyruvate
followed by a spontaneous tautomerization to pyruvate—and so it
seems plausible that it might be catalysed by the uncharacterized,
non-primary action of an existing enzyme. Path E is identical to Path A with the exception that the final
step generates ATP from AMP and pyrophosphate (via pyruvate
phosphate dikinase, EC 2.7.9.1) rather than from ADP and
Pi—that is, it is the exact reverse of the natural ppdk
gluconeogenic pathway. This may result in an effectively greater
energetic yield than the natural glycolytic pathway, as an ATP is
recovered from an AMP directly, rather than through the action
of the adenylate kinase enzyme (EC 2.7.4.3), which involves the
consumption of another ATP molecule. This pathway actually
corresponds to a glycolytic variant observed in some anaerobic
eukaryotes, in which glycolysis is the primary mode of ATP
production36. Its flux and feasibility will depend on the cellular
[AMP] and [PPi] concentrations, which is not the case for the
natural glycolytic pathway (which does not involve AMP or PPi). Finally, Path F represents a pathway of length 4 that
produces two ATP molecules. It converts 3-PG to glycerate with
the production of ATP, via glycerate 3-kinase, EC 2.7.1.31. Although the final reaction of this pathway is not present in the
KEGG database, this putative reaction is chemically rather
simple—involving the dehydration of glycerate to enolpyruvate
followed by a spontaneous tautomerization to pyruvate—and so it
seems plausible that it might be catalysed by the uncharacterized,
non-primary action of an existing enzyme. Our analysis also reveals alternative trunk pathways that can
perform better than the natural one under different physiological
conditions. Although some of these alternatives involve com-
pounds and reactions that are not found in biochemical
databases, others use enzymes that are known to exist in nature. The latter pathways (Fig. 4) are clearly plausible biochemically
and thermodynamically. It could be that some of these pathways
do operate glycolytically under some circumstances, and may be
revealed by future biochemical investigations (see Supplementary
Note 2 for details). Discussion
h An interesting
question is whether the global maximum (the real pathway) is
accessible, that is, can it be reached from anywhere in the
landscape? Such fundamental evolutionary questions have been
addressed recently using the full set of known biochemical
reactions4. It would be interesting to repeat this analysis using our
data, and to compare the fitness landscape obtained for our model
to the landscapes of other evolutionary models38–41. This could
show to what extent different computer models of artificial life are
similar to one another, and perhaps even shed light on the
structure of real fitness landscapes. Flux maximization is widely recognized as an important
concept in the study of metabolism; both from the perspective of
glycolysis and more broadly37. Previous works addressing this
issue for glycolysis5–7 have arrived at similar conclusions but have
remained abstract in nature. For the first time, we attempt to
expand on these fundamental and influential studies through
the use of a realistic set of generic enzymatic rules along with the
inclusion of thermodynamics. Our results add support to the
picture
that
has
been
developing,
and
also
suggest
that
evolutionary pressures on metabolic fluxes have to operate
within the context of reasonable constraints on metabolite
concentrations,
and
that
neglecting
these
constraints
can
produce dramatically
different
outcomes. Our
results also ARTICLE ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9427 animals and their feasibility in the glycolytic direction is sensitive
to the intracellular pH; it becomes increasingly thermodynami-
cally favourable to produce two ATP molecules from PEP with
decreasing pH (see Supplementary Note 2 for details). expand on the recent suggestion of Noor et al.10 that central
carbon metabolism can be understood as a minimal walk between
the set of metabolites essential for growth. We find that the
requirement to produce a set of essential biochemical precursors
is not sufficient to explain the biochemical structure of the natural
trunk pathway, as alternative pathways are possible which
produce the essential precursors with the same number of steps
(for example, Path D, Fig. 4). Also, many of our alternative
pathways produce very similar, but not identical, intermediates to
those of the real trunk pathway and it is conceivable that these
could be used as alternative precursors. Our results show that flux
maximization provides a criterion by which these alternative
minimal-length pathways may be distinguished. We also note
that this criterion is not affected by ATP yield because alternative
pathways producing three or more ATP molecules are never
thermodynamically
feasible
under
typical
physiological
conditions (Supplementary Fig. 7), hence there cannot be any
trade-off between the yield and the flux in our model. decreasing pH (see Supplementary Note 2 for details). Path E is identical to Path A with the exception that the final
step generates ATP from AMP and pyrophosphate (via pyruvate
phosphate dikinase, EC 2.7.9.1) rather than from ADP and
Pi—that is, it is the exact reverse of the natural ppdk
gluconeogenic pathway. This may result in an effectively greater
energetic yield than the natural glycolytic pathway, as an ATP is
recovered from an AMP directly, rather than through the action
of the adenylate kinase enzyme (EC 2.7.4.3), which involves the
consumption of another ATP molecule. This pathway actually
corresponds to a glycolytic variant observed in some anaerobic
eukaryotes, in which glycolysis is the primary mode of ATP
production36. Its flux and feasibility will depend on the cellular
[AMP] and [PPi] concentrations, which is not the case for the
natural glycolytic pathway (which does not involve AMP or PPi). Finally, Path F represents a pathway of length 4 that
produces two ATP molecules. It converts 3-PG to glycerate with
the production of ATP, via glycerate 3-kinase, EC 2.7.1.31. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9427 Repeating the comparison of candidate gluconeogenic paths, but
now sampling [PPi] from 10 8 to 10 2 M instead of from 10 4 to 10 2
shows that the real ppdk pathway (black) becomes favourable compared
with the pps pathway (red), when the ratio of orthophosphate to
pyrophosphate concentrations is increased. Colours as in Fig. 2. Figure 3 | The sampled parameter ranges are an important factor in our
analaysis. Repeating the comparison of candidate gluconeogenic paths, but
now sampling [PPi] from 10 8 to 10 2 M instead of from 10 4 to 10 2
shows that the real ppdk pathway (black) becomes favourable compared
with the pps pathway (red), when the ratio of orthophosphate to
pyrophosphate concentrations is increased. Colours as in Fig. 2. 5 NATURE COMMUNICATIONS | 6:8427 | DOI: 10.1038/ncomms9427 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | 6:8427 | DOI: 10.1038/ncomms9427 | www.nature.com/naturecommunications
& 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9427 This expression assumes that the enzyme acts as a
perfect catalyst, and is used to derive an expression for pathway flux and metabolite
concentrations (see Supplementary Methods for details). We then use Powell’s
method42 to find the set of enzyme concentrations that maximize the flux subject
to the constraints that (i) all steady-state intermediate concentrations are within the
prescribed range and (ii) the total enzyme concentration is fixed. We also repeat
our analysis using reversible Michaelis–Menten kinetics, see Supplementary
Methods for details of calculation. where kd is the diffusion-controlled rate constant, [Ei] is the enzyme concentration,
[Si 1] and [Si] represent substrate and product concentrations and qi is the
thermodynamic constant. This expression assumes that the enzyme acts as a
perfect catalyst, and is used to derive an expression for pathway flux and metabolite
concentrations (see Supplementary Methods for details). We then use Powell’s
method42 to find the set of enzyme concentrations that maximize the flux subject
to the constraints that (i) all steady-state intermediate concentrations are within the
prescribed range and (ii) the total enzyme concentration is fixed. We also repeat
our analysis using reversible Michaelis–Menten kinetics, see Supplementary
Methods for details of calculation. 20. Verhees, C. H. et al. The unique features of glycolytic pathways in Archaea. Biochem. J. 375, 231–246 (2003). 21. Morowitz, H. J., Kostelnik, J. D., Yang, J. & Cody, G. D. The origin of
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reductive chemoautotroph. Biol. Bull. 217, 222–232 (2009). 24. Alberty, R. A. Biochemical thermodynamics: applications of mathematica. Methods Biochem. Anal. 48, 1–458 (2006). Sampling the parameter space. We randomly selected 10,000 points from the
parameter space corresponding to the concentrations of 11 external metabolites and
the G3P and pyruvate concentrations. Each parameter was sampled logarithmically
over a range covering several orders of magnitude above and below its typical
physiological concentrations (see Supplementary Table 4 for details). For each of
these points, we calculated the optimized flux Ji of each candidate pathway as detailed
above, and computed the CF of path i as CFi ¼ Ji/max{Jk}, by dividing its flux by the
highest flux obtained across all pathways at the given point in parameter space. 25. Mavrovouniotis, M. L. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9427 Estimation of standard Gibbs energy changes of
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that combines accuracy and coverage. Bioinformatics 28, 2037–2044 (2012). Robustness of our results to small free energy changes. Using the group
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for details). To check the robustness of our results to such errors, our entire
analysis was repeated using DfG values computed using different sets of training
molecules, consisting of 80% of the molecules from the original training set, chosen
at random. The qualitative results using such networks were identical from those
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colytic pathways obtained from the reduced set contained 23 out of the 25 path-
ways from the original analysis. 29. Webb, E. Enzyme Nomenclature 1992: Recommendations of the Nomenclature
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f
y
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active site occupancy in Escherichia coli. Nat. Chem. Biol. 5, 593–599 (2009). NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9427 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9427 systematically whether the reactions from Table 1 (see Supplementary Table 3 also
for details) could transform one molecule into another, allowing for all possible
couplings with the external metabolites. In this way, a network of 7,940 reactions
was generated. systematically whether the reactions from Table 1 (see Supplementary Table 3 also
for details) could transform one molecule into another, allowing for all possible
couplings with the external metabolites. In this way, a network of 7,940 reactions
was generated. 8. Flamholz, A., Noor, E., Bar-Even, A., Liebermeister, W. & Milo, R. Glycolytic
strategy as a tradeoff between energy yield and protein cost. Proc. Natl Acad. Sci. USA 110, 10039–10044 (2013). 9. Barve, A. & Wagner, A. A latent capacity for evolutionary innovation through
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bi
h
i
l
lk b t
f
bi
d
M l C ll 39 10. Noor, E., Eden, E., Milo, R. & Alon, U. Central carbon metabolism as a minimal
biochemical walk between precursors for biomass and energy. Mol. Cell 39,
809–820 (2010). Free energies of compounds and reactions. For those internal metabolites that
are known biochemical species, standard free energies of formation DfG were taken
from the literature24. For other internal metabolites, for which such data does not
exist, we employed a variant of the group contribution method25–28, which Free energies of compounds and reactions. For those internal metabolites that
are known biochemical species, standard free energies of formation DfG were taken
from the literature24. For other internal metabolites, for which such data does not
exist, we employed a variant of the group contribution method25–28, which
accounts for the fact that molecules exist in solution as an equilibrium mixture of
different ionic species. For each such molecule g1g2ygn, composed from building
blocks {gi}, we calculated DfG using 11. Samal, A., Wagner, A. & Martin, O. C. Environmental versatility promotes
modularity in genome-scale metabolic networks. BMC Syst. Biol. 5, 135 (2011). p y
g
p
accounts for the fact that molecules exist in solution as an equilibrium mixture of
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blocks {gi}, we calculated DfG using 12. Bar-Even, A., Noor, E., Lewis, N. E. & Milo, R. Design and analysis of synthetic
carbon fixation pathways. Proc. Natl Acad. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9427 Sci. USA 107, 8889–8894 (2010). 13. Handorf, T., Ebenho¨h, O. & Heinrich, R. Expanding metabolic networks: Scopes
of compounds, robustness, and evolution. J. Mol. Evol. 61, 498–512 (2005). Df G ¼ E0 þ
X
j
E1 gj
þ
X
oj;k4
E2 gj; gk
;
ð1Þ ð1Þ 14. Mele´ndez-Hevia, E., Waddell, T. G., Heinrich, R. & Montero, F. Theoretical
approaches to the evolutionary optimization of glycolysis: chemical analysis. Eur. J. Biochem. 244, 527–543 (1997). where E0 is a constant, E1(gj) is the contribution of group gj and E2(gj,gk) is a small
correction due to neighbouring group–group interactions. The values of E0, the
vector E1 and matrix E2 are determined by performing a least-squares fit to a
training set of molecules with known DfGs that correspond most closely to the
linear CHOPN molecules of our network (see Supplementary Methods for details). 15. Mittenthal, J. E., Yuan, A., Clarke, B. & Scheeline, A. Designing metabolism:
Alternative connectivities for the pentose phosphate pathway. Bull. Math. Biol
60, 815–856 (1998). 16. Hatzimanikatis, V. et al. Exploring the diversity of complex metabolic
networks. Bioinformatics 21, 1603–1609 (2005). Flux calculation. We used the method mentioned in ref. 7 to calculate the flux
carried by a linear pathway. This method assumes that the flux through reaction i is
given by7,30 17. Henry, C. S., Broadbelt, L. J. & Hatzimanikatis, V. Discovery and analysis
of novel metabolic pathways for the biosynthesis of industrial chemicals:
3-hydroxypropanoate. Biotechnol. Bioeng. 106, 462–473 (2010). vi ¼ kd Ei
½
Si 1
½
qi Si
½
ð
Þ
1 þ qi
;
ð2Þ 18. Bar-Even, A., Flamholz, A., Noor, E. & Milo, R. Rethinking glycolysis: on the
biochemical logic of metabolic pathways. Nat. Chem. Biol. 8, 509–517 (2012). vi ¼ kd Ei
½
Si 1
½
qi Si
½
ð
Þ
1 þ qi
;
ð2Þ ð2Þ 19. Ronimus, R. S. & Morgan, H. W. Distribution and phylogenies of enzymes of the
Embden-Meyerhof-Parnas pathway from archaea and hyperthermophilic bacteria
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‘building blocks’ shown in Supplementary Table 2. Each of the building blocks was
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1,477 of which are electrostatically charged in solution, that is, containing at least
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& 2015 Macmillan Publishers Limited. All rights reserved. 6 NATURE COMMUNICATIONS | 6:8427 | DOI: 10.1038/ncomms9427 | www.nature.com/naturecommunications
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complex features. Nature 423, 139–144 (2003). 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/ Additional information 41. Ullrich, A., Rohrschneider, M., Scheuermann, G., Stadler, P. F. & Flamm, C. In
silico evolution of early metabolism. Artif. Life 17, 87–108 (2011). Supplementary Information accompanies this paper at http://www.nature.com/
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Recipes: The Art of Scientific Computing 3rd edn (Cambridge Univ., 2007). Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. 43. Berg, J., Hung, Y. P. & Yellen, G. A genetically encoded fluorescent reporter of
ATP:ADP ratio. Nat. Methods 6, 161–166 (2009). Reprints and permission information is available online at http://npg.nature.com/
reprintsandpermissions/ References 7 NATURE COMMUNICATIONS | 6:8427 | DOI: 10.1038/ncomms9427 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | 6:8427 | DOI: 10.1038/ncomms9427 | www.nature.com/n & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | 6:8427 | DOI: 10.1038/ncomms9427 | www.nature.com/naturecommunications
& 2015 Macmillan Publishers Limited. All rights reserved. Acknowledgements B.W. and R.J.A. contributed equally to this work. We thank Patrick Warren, Vincent
Danos and Oliver Ebenho¨h for insightful discussions. This work was funded by EPSRC
under Grant No. EP/J007404/1 and by HFSP under grant RGY0081/2012. S.J.C. was
supported by a Carnegie/Caledonian Scholarship from the Carnegie Trust for Scotland,
B.W. was supported by a Leverhulme Trust Early Career Fellowship and by a Royal
Society of Edinburgh Personal Research Fellowship, and R.J.A. was supported by a Royal
Society University Research Fellowship. How to cite this article: Court, S. J. et al. Lower glycolysis carries a higher flux than any
biochemically possible alternative. Nat. Commun. 6:8427 doi: 10.1038/ncomms9427
(2015). Author contributions S.C., B.W. and R.J.A. designed the study. S.C. and B.W. generated the network, B.W. performed the thermodynamic calculations, S.C. conducted the simulations and analysis. All authors contributed to writing the manuscript. 8
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Water quality and sustainability of Merdada Lake, Dieng, Indonesia
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1 Introduction also identify the human interaction between local
communities and surrounding lake environment. Research on the remote lake that has a big impact from
anthropogenic is now categorized as a hot issue in
environmental change research. Most of them revealed
that the environmental degradations of the lakes were the
results from human activities, some of which were
agriculture and tourism. Those phenomena caused the
ecosystems
on
the
lake
changed
due
to
the
environmental degradation in their catchment area [1]. A
big degradation of invertebrates found on Lake Chaouhu
Basin, China is caused by intense agricultural practices
in its catchment area [2]. Agricultural practices also lead
some problems to environmental degradation and need
obligation to rule good agricultural and environmental
conditions, which is protection soil from erosion by
maintenance soil organic matters and soil structure [3]. In Indonesia, the trophic status of Sudirman Reservoir
and Tondano Lake has been examined and revealed that
the water quality deteriorations in both lakes were
caused by human activities [4,5]. Another study on a
small lake in Tolire Lake, Ternate, mentioned that the
water quality is inevitably influenced by the volcanic
activity [6]. The research on small lakes is also
interesting because of the interaction between local
community and environmental management, which
usually considers the principle of local wisdom. Only
view studies conducted in a volcanic lake, beside that In Java, there are many volcanic lakes with different
characteristic, most of them are maar lake. This study try
to examine the role of human interaction on lake water
use and its water quality in the selected volcanic lake. The study was conducted in Merdada Volcanic Lake and
the surrounding areas, which presumably affect the
condition of the lake. Merdada Volcanic Lake is located
in Karangtengah Village, Batur District, Banjarnegara
District and one of the largest lakes in Dieng Volcanic
Complex. Merdada Volcanic Lake formed from
stratocone Merdada which ages approximately 0.37 Ma
[7]. It has no springs and rivers that supply water into the
lake. The water input is mostly from rainwater and only
few shares of it is from the seepage in the surrounding
areas. Therefore, the water level of the lake descends
rapidly during the dry season. However, its current
condition is much different from the past. The natural
beauty, fertile land, and abundant water resource no
longer exist in the lake. Water quality and sustainability of
Merdada Lake, Dieng, Indonesia Sudarmadji1,2,*, Slamet Suprayogi1,2, Sri Lestari3, and Mukhamad Ngainul Malawani2 1Hydrology Study Group, Department of Environmental Geography, Faculty of Geography, Universitas Gadjah Mada, Indonesia
2Department of Environmental Geography, Faculty of Geography, Universitas Gadjah Mada, Indonesia
3Study Program on Environmental Science, the Graduate School of Universitas Gadjah Mada, Indonesia Abstract. The volcanic lakes in Dieng Plateau offer some unique phenomena, which interest not only
tourists but also scientists. However, as the land use changes, the lakes are now facing environmental
degradation especially from agricultural practices. This research aims to study the impact of agricultural
practices on the environmental degradation of the lakes especially the water quality and analyze the
sustainability of the lakes to support water needs. Merdada Volcanic Lake, one of the lakes in Dieng Plateau
was selected as the focus of this research. Method conducted on this research are fields survey to collect the
physical parameters temperature and conductivity also chemical parameters like pH, Nitrate, BOD,
phosphate, and coliform. A complementary data was acquired using interviews method for defining the
sustainability of the lake. Based on observation, water level of the lake is gradually decreased, especially
during the dry season. At the same time, the agricultural practices in the surrounding area led to soil erosion,
which involved surface runoff that transporting sediments into the lake. This research found that the
practices of potato cultivation pumped out the water from Merdada Volcanic Lake and distributed over the
plantation area. This practice caused a dramatic decrease in the water surface. Besides that, the agricultural
practices had several adverse effects on the volcanic lakes in Dieng Plateau especially eutrophication and
decrease of water quality in Merdada Lake where nitrate is over from water quality standard class II. Coliform also has an over expected value, range from 3–11 MPN/100 ml. © 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/). *Corresponding author: sudarmadji@ugm.ac.id E3S Web of Conferences 76, 02003 (2019) E3S Web of Conferences 76, 02003 (2019) https://doi.org/10.1051/e3sconf/20197602003 E3S Web of Conferences 76, 02003 (2019)
ICST 2018 2 Methods The research utilized both primary and secondary data. The primary data was collected from fieldwork, while
the secondary data was derived from the previous studies
on Merdada Volcanic Lake. The research used several
tools and instruments for data collection, including,
maps (i.e., the administrative maps of Wonosobo and
Banjarnegara District), satellite images, GPS for plotting
the location of collected data and samples, a set of
questionnaire for in-depth interviews with local leaders,
and digital camera for documentation. Primary data
collected in field are several sample for water quality
analysis. Water samples then analyzed through six
different
parameters;
they
are
temperature,
pH,
conductivity, NO3, COD, and phosphate. The locations
of the samples were plotted into the maps based on the
GPS reading. The location of the research showed by
Figure 1. Fig. 1. (A) Location of Merdada Volcanic Lake in Dieng Volcanic Area Jawa Tengah, showing in district border. (B) Located on
mountainous area on Dieng Volcanic Complex, showing in sub-district border. (C) Merdada Volcanic Lake has area of 2.5 hectares,
over its catchment used as cultivated area. Fig. 1. (A) Location of Merdada Volcanic Lake in Dieng Volcanic Area Jawa Tengah, showing in district border. (B) Located on
mountainous area on Dieng Volcanic Complex, showing in sub-district border. (C) Merdada Volcanic Lake has area of 2.5 hectares,
over its catchment used as cultivated area. water pumping for irrigation is quite excessive. Beside
positive impact for economic background, the negative
impact of agricultural practices on the environment was
clearly shown by the contribution of potato plantation to
soil erosion. The surrounding land on Merdada Volcanic
Lake was originally used as forest before converted to
the agricultural area. The series change of area
surrounding Merdada Volcanic Lake showed by Figure
2. 1 Introduction Due to the presence of various
changes and interventions, Merdada Volcanic Lake is no
longer interesting from the perspective of tourism [8]. This lake has a thick mud layer, as a result of high
sedimentation. The erosion take place in the surrounding
area which sediments transported by runoff into the lake. From those presence conditions, Merdada Volcanic Lake
is quite difficult for restoration practice because *Corresponding author: sudarmadji@ugm.ac.id E3S Web of Conferences 76, 02003 (2019)
ICST 2018 https://doi.org/10.1051/e3sconf/20197602003 lake water. Therefore, the impact of agricultural
activities on the water quality and sustainability of
Merdada Volcanic Lake becomes necessary to analyse. peleolimnological study is important key for the success
of lake restoration [9]. But this research tries to evaluate
the existing condition as a benchmark for future
development. Water availability is very useful for farming,
especially in Merdada Volcanic Lake. One of intensive
farming cultivated around in Merdada Volcanic Lake is
potato (Solanum tuberosum L.). Merdada water has been
exploited for agricultural purposes, i.e., irrigating the
potato plantation by pumping method. Beside that, the
fertilizer need for potato is very high and susceptible for
water quality in lake during rainy season. High runoff
always happen during rainy day and transported much
nutrient into the lake and expected lead to algae bloom
or eutrophication. Trophic status of Merdada Volcanic
Lake categorize as hypertrophic, but when measured by
amount of chlorophyll status of Merdada Lake
categorize as oligotrophic [10]. Other research mention
that lake surface water temperature is main factor of
algae blooming beside other factors like COD and total
nitrogen [11]. However, the catchment area of Merdada
Volcanic Lake has a steep slope that can create a higher
rate of erosion and support for high runoff rate. The use
of fertilizers and pesticides also potentially pollutes the 3 Results The water of Merdada Volcanic Lake is utilized as the
source
of
irrigation
water
for
the
surrounding
agricultural land, i.e., potato plantation, which gives an
enormous impact on the economy of the local
communities. The economic improvement is evidenced
by the extent of the agricultural land in the area. The 2 2 E3S Web of Conferences 76, 02003 (2019)
ICST 2018 E3S Web of Conferences 76, 02003 (2019) https://doi.org/10.1051/e3sconf/20197602003 Fig. 2. [A] Merdada lake form RBI Map 1999, area surrounding lake covered by dryland farming. [B] Imagery from Google Earth
showed settlement and building on the eastern part of the lake converted into dryland farming. [C] Imagery from 2015 showed that
area of the lake shrinking by euthropication processes. Fig. 2. [A] Merdada lake form RBI Map 1999, area surrounding lake covered by dryland farming. [B] Imagery from Google Earth
showed settlement and building on the eastern part of the lake converted into dryland farming. [C] Imagery from 2015 showed that
area of the lake shrinking by euthropication processes. The inland area of the lake that was flattening
gradually was utilized as agricultural land. This process
would reduce the depth of the lake or, in other words, the
lake would become narrower. The erosion and
sedimentation processes are clearly observed during the
rainy season in which precipitation acts as the agent of
erosion. The runoff that flows down from the
surrounding areas brings sediments into the lake. The
dramatic water decrease on Merdada Volcanic Lake shown in Figure 3 (left) clearly showed how farmer tries
hard to use water lake for irrigation even the water
almost drain. However, many farmers depend on the
availability of water from Merdada Volcanic Lake
because of no others water source for irrigating
accessible for their land. There were more than 100
water pumps with an estimated pumping duration of 3
hours per suction day. Fig. 3. Water decreases extremely by over pumping activities, the picture was taken in 2015, a dozens water pump together toke up
water for cultivating (left). Samples location for water quality analysis taken from western part of the lake (right). decreases extremely by over pumping activities, the picture was taken in 2015, a dozens water pump together toke up
vating (left). Samples location for water quality analysis taken from western part of the lake (right). must be highlighted on sample D because its value has
large differences than other samples. 4 Discussion different season showed that the deepest point on
Merdada lake is 4.2 meters [10]. This is clearly showed
that Merdada Volcanic Lake has high fluctuation water
level between rainy season and dry season. Merdada Volcanic Lake with an area of 25 hectares is a
potential source for irrigation water. The main water
input of the lake is from rainwater, meaning that the
water availability of the lake relies solely on the
precipitation in the rainy season. The catchment area of
the lake is particularly small, which implies a limited
amount of water potential. Since Merdada Volcanic Lake
receives most of its water from the rain, the water quality
is presumed to be similar to the quality of the rainwater. In other words, the lake is expected to contain no
excessive substances. The shape of Merdada lake was initially been round
(circular), it is indicated from topography showed by
DEM data, but the development and human occupation
on the west side, including the former mushroom nursery
in 1950–1988, changed it into nearly half-round. The
erosion and sedimentation in the surrounding area
contributed significantly to the changes in the shape of
the lake. Morphometric measurement on Merdada lake
catchment showed that eastern slope of the lake has a
longest slope. The shortest slope in western part of the
lake with just below 100 m. Highest different elevation
also conducted in eastern slope, ranging about 100 m
above
lake
water
level. Other
morphometric
measurement showed resembled result with this research
[10]. This morphometric characteristic made Merdada
Volcanic Lake very vulnerable to lake degradation. Fig. 4. Merdada Volcanic Lake has an approximately depth 2
meter. The deepest point is 5.7 m [12]. y
g
The water scarcity endangered Merdada Volcanic
Lake, especially in dry season. Water use for irrigation is
main cause the water scarcity of the lake. From the
interview with farmers, they used different capacity of
water pumping, but it can be estimated that average of
water discharge is 40 L/min per water pump, similar
with 240 L/h—other measurement of water pumping
taken from a lake near Merdada Volcanic Lake,
conducted in Pengilon and Warna Lake [13]. Pengilon
and Warna Lake also have water use for irrigation. During potato cultivation, pumping is done for 3 hours
every suctioning day. There were 28 time of suctioning
during cultivation. In this case, Pengilon and Warna
release 12600 m3 for irrigation in every cultivating
season. 3 Results Sample D expected
rice phosphate (0.332 m/L) when other samples has a
very small amount of phosphate (<0.02 mg/L). Its
indicated that sample D located near inlet from erosion
path moving down from the agricultural slope or
supposed to be point of accumulating. Coliform
measurement also has different value, ranging form 3–11
MPN/100mL. Location of sampling sites and its water
quality showed in Figure 3 (right) and Table 2. must be highlighted on sample D because its value has
large differences than other samples. Sample D expected
rice phosphate (0.332 m/L) when other samples has a
very small amount of phosphate (<0.02 mg/L). Its
indicated that sample D located near inlet from erosion
path moving down from the agricultural slope or
supposed to be point of accumulating. Coliform
measurement also has different value, ranging form 3–11
MPN/100mL. Location of sampling sites and its water
quality showed in Figure 3 (right) and Table 2. Several parameters of water quality measured
through laboratory analysis, where conducted in the
Laboratory of Environment Hidroclimatology, Faculty of
Geography, UGM. Fourth sample taken from western
part of lake, ranging from north to south. Merdada lake
water quality showed that from the four samples value of
each parameter has a different result. Nitrate on samples
C has larget value, consist of 2.217 mg/L while others
have values below 2 mg/L. Other parameters with
dramatic different are COD. Sample C has lowest COD
than other samples. From various parameters, phosphate 3 3 E3S Web of Conferences 76, 02003 (2019) E3S Web of Conferences 76, 02003 (2019)
ICST 2018 https://doi.org/10.1051/e3sconf/20197602003 Table 2. Water quality of samples has quite different value for each parameter. Parameters
Sample A
Sample B
Sample C
Sample D
Temperature (C)
27.3
27.2
25.8
27.2
Conductivity (µmhos/cm)
276.48
272.37
188.67
161.57
pH
6.85
7.30
7.71
8.43
NO3 (mg/L)
1.328
1.105
2.127
1.744
COD (mg/L)
21.28
12.86
10.86
17.50
Phosphate (mg/L)
<0.02
<0.02
<0.02
0.332 Table 2. Water quality of samples has quite different value for each parameter. 4 Discussion By those scenario, Merdada Lake has more water
release for cultivation than Pengilon and Warna Lake. On every season water irrigated from Merdada Lake
counting about 14976 m3. In similar scenario, Pengilon
and Warna Lake wasted about 360 liters oil from 120
water pump for every cultivating season. But on
Merdada Lake oil wasted counting less than them, about
300 liters oil wasted direct or indirectly to Merdada
Volcanic Lake water and influenced to its water quality. However, field observation found that the position of the
water pumps, which were exactly on the side of the lake,
potentially introduced oil spills into the lake and caused
contamination like showed in Figure 3 (left). Fig. 4. Merdada Volcanic Lake has an approximately depth 2
meter. The deepest point is 5.7 m [12]. Merdada Volcanic Lake has no inlet from the river. Thereby, in normal condition, the water is mainly added
by rainwater. However, since Merdada Volcanic Lake is
extensively used as the source of irrigation water in the
surrounding agricultural land, a considerable amount of
its water is extracted continuously. Because the
excessive use of water, Merdada Volcanic Lake now has
an average depth approximately 2 meters and the deepest
point is 5.7 meters (Figure 4). By its morphometric
measurement, indicated that Merdada Volcanic Lake has
an average water volume 0.5 × 106 m3. Other bathimetric
measurement conducted in Merdada Volcanic Lake on 4 E3S Web of Conferences 76, 02003 (2019)
ICST 2018 https://doi.org/10.1051/e3sconf/20197602003 is the most unaffected in term of water quality because
volcanic activity is still active on that site [14]. Previous research mentions that the water of
Merdada Volcanic Lake was suitable for agricultural
practices [12]. Those research mentioned that TDS
higher on the rainy season than dry season indicated that
much of nutrient contained in soil carried by overland
flow from cultivated land. Others fact is sediment grain
contained on Merdada Volcanic Lake has a similar
texture with the topsoil on the cultivated slope, tasted by
hand texture observation method. Samples were taken on
the dry season (Table 2) also showed that water quality
of Merdada Volcanic Lake included adequate low water
quality. Compared to Pengilon and Warna lake, Merdada
Volcanic Lake is the most affected lake by agricultural
impact, indicated by the high amount of nitrate and
phosphate. Table 2 showed the comparison between
three lakes. 4 Discussion In order the effect of agriculture, Warna lake Another water quality measurement on volcanic lake
in Azores Archipelagos also showed that physical
condition of water quality on volcanic lake is quite
different with research area. From 13 volcanic lake from
that certain area, pH is ranging from 4.2 to 9.9. Besides
that, conductivity on those lakes is lower than research
area. The lowest conductivity is 6 and the biggest is 159
µmhos/cm [15]. The differences of physical condition
from those water quality influenced mostly by the
geological setting and human activities on two sites. On
Merdada Volcanic Lake, the use of fertilizers for
farming triggered the nutrient enrichment in lake and
create an environment where aquatic vegetation thrived
in the lake. Showed in Figure 3C that new algae was
bloom covered water surface compared to Figure 3B. Table 2. Comparison water quality between Merdada Lake and others lake
Parameters
Sample A
Sample B
Sample C
Sample D
Average (1)
Average (2)
Average (3)
Temperature (°C)
27.3
27.2
25.8
27.2
26.88
26.9
25.77
Conductivity
(µmhos/cm)
276.48
272.37
188.67
161.57
224.77
1144
184.6
pH
6.85
7.30
7.71
8.43
7.57
3.47
8.51
NO3 (mg/L)
1.328
1.105
2.127
1.744
1.58
0.381
0.99
COD (mg/L)
21.28
12.86
10.86
17.50
15.63
2.96
9.95
Phosphate (mg/L)
<0.02
<0.02
<0.02
0.332
<0.02
<0.02
0.153
Average water quality (1) Merdada Lake, (2) Pengilon Lake, (3) Warna Lake; 2 and 3 [14]. Table 2. Comparison water quality between Merdada Lake and others lake The ecosystem of the lake is prone to significant
shocks and stresses despite the implementation of some
conservation efforts. The conservation is however
different from the one that is implemented in a large
lake. A
large
lake
requires
much-complicated
conservation. International
Lake
Environment
Committee (ILEC) Foundation has introduced several
guidelines of the conservation efforts [16]. However,
some adjustments are required in order to suit the
condition in Indonesia. The conservation efforts will not
work properly when they focus only on the lake itself
because the water in the lake is also influenced by the
condition of the catchment area. They have to consider
the participation of the local community. Therefore, a
comprehensive concept of conservation in Merdada
Volcanic Lake becomes necessary in order to sustain the
existence of the lake especially focusing on water use
regulation on irrigation. to excessive pumping. 4 Discussion The water quality is altered by the
unabsorbed fertilizers in agricultural lands, beside that
the great number of water pumps can contribute to the
decrease in water quality by their oils spilling. Those
processes lead Merdada Volcanic Lake to become most
vulnerable to environmental degradation compared to
others lake in Dieng Area due to agricultural activities. In order to maintain the sustainability of the lake, many
efforts need to do especially on the role of water use. Limitation of water pumping is the most important
regulation for water conservation in Merdada Lake. The research was sponsored by The Graduate School of
Universitas Gadjah Mada. Authors would like to thank the
Vice Director of the Graduate School, Universitas Gadjah
Mada for providing many supporting facilities during the
research. High appreciation is also addressed to the students of
the following study programs: Environmental Science, tourism,
and Disaster Management, for their assistance in data
collection and during the research. 5 Conclusion The potato farming adversely affects the condition of
Merdada Lake both directly and indirectly. The
sedimentation and siltation of the materials from
agricultural land reduce the environmental capacity and,
even change the shape of the lake. The water level of
Merdada Volcanic Lake will continuously descend due 5 E3S Web of Conferences 76, 02003 (2019)
ICST 2018 E3S Web of Conferences 76, 02003 (2019) https://doi.org/10.1051/e3sconf/20197602003 References 1. Sudarmadji. Proceeding Seminar on Limnology. (Faculty of Biology UGM, 2004) 2. Y. Zhang, L. Cheng, K.E. Tolonen, Y. Hongbin, J. Gao, Z. Zhang, K. Li, and Y. Cai. Science of Total
Environment. 627. 57–66 (2018) 3. P. Borrelli, K. Paustian, P. Panagos, A. Jones, B. Schütt, E. Lugato. Land Use Policy. 50. 408–421
(2016) 4. A.S. Piranti. Dissertation. (Universiitas Gadjah
Mada, Yogyakarta, 2012) 5. S. Wantasen. 2009. Dissertation. (Sekolah
pascasarjana UGM. Yogyakarta, 2009) 6. S. Setiawan, H. Wibowo, A..B. Santosa, S. Nomosatryo, I. Yuniarti. Limnotek. 21. 103–114
(2014) 7. A. Harijoko, R. Uruma, H.E. Wibowo, L.D. Setijadji, A. Imai, K. Yonezu, K.J. Watanabe. J. of
Volcanology and Geothermal Research. 310. 209–
224 (2016) 8. M. Rusiah, N. Satya, A. Wahyudin. PELITA. 1. (2005) 9. H. Bennion, G.L. Simpson, B.J. Goldsmith. Frontiers in Ecology and Evolution. 3 (2015) 10. A. Kusumastuti, L.W. Santosa. Majalah Geografi
Indonesia. 24 (2010) 11. K. Yang, Z. Yu Y. Luo, Y. Yang, L. Zhao, X. Zhuo. Science of the Total environment. 624. 859–871
(2018) 12. M. Widyastuti, L. Fadilah, F.A. Rasyadi. Research
Report. (Fact. Geography UGM, Yogyakarta, 2014) 13. C.Y. Lastiantoro, P.B. Putra, S.A. Cahyono. Prosiding. 241–254. (Seminar Nasional Geografi
UMS, 2017) 14. Sudarmadji,
H. Pudjiastuti. E3S
Web
of
Conferences. 31 (ICENIS, 2017) 15. P. Antunes, F.C. Rodrigues. Proceedings of
symposium IUGG. 106–114 (2011) 16. ILEC. Integrated lake basin management: an
introduction. (Kusatsu, Japan, 2007) 6 6
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English
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Quasi-combinatorial energy landscapes for nanoalloy structure optimisation
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Physical chemistry chemical physics/PCCP. Physical chemistry chemical physics
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1 Introduction distance metric. From the viewpoint of basic topology, the
two metrics induce two local neighbourhood structures: one
continuous and the other discrete. It is clear that the global
minimum of a model nanoalloy ought to be a local minimum
with respect to both metrics/neighbourhoods and, therefore,
must be a biminimum. In fact, the global minimum in a specified
domain ought to be a local minimum in any subdomain that
retains the global minimum and admits a definition of ‘‘locality’’. Hence, one could generalise further still and define multiminima
by invoking more subdomains with a different metric and/or local
neighbourhood structure. Alloy nanoparticles (nanoalloys1) exhibit structure-dependent
properties with potential applications in catalysis,2 sensing,3
plasmonics,4 etc.5,6 However, unlocking the full potential of
this class of materials requires reliable structure prediction at
the atomistic level, which, from a theoretical viewpoint, often
amounts to finding the ground state (the global minimum) of a
model system described by a highly nonlinear potential energy
function. For homogeneous metal clusters the number of meta-
stable states (local minima) is expected to grow exponentially
with the number of atoms,7,8 but for nanoalloys the growth is
significantly more rapid due to the presence of homotops9 –
local minima with a similar geometric motif but a different
ordering of atomic species. As a result, the exponential growth in
the number of non-degenerate minima can be further multiplied
by a combinatorial factor. We tackle this formidable global optimi-
sation problem using a mixed-variable approach that targets
multiminima – configurations that are local minima in more
than one sense of the word ‘‘local’’. Whether incorporating another neighbourhood structure
will make a global search more effective or not will depend
on two competing factors: (i) the additional cost associated
with multiminimisation and (ii) the consequent reduction in
the number of multiminima. In the present study we show
that targeting biminima15 can lead to a significant net effi-
ciency gain, which is explained by the topography of what we
call quasi-combinatorial energy landscapes. These effective
landscapes may not have a well-defined global combinatorial
structure, but can still be explored and (to some degree)
characterised using local combinatorial constructions. For
model nanoalloys with small lattice mismatch, representing a
scenario where global combinatorial counting arguments are
applicable, we find that the energetic preference for mixing/
segregation is linked to the fraction of locally optimal homo-
tops (with respect to the interchange distance). University Chemical Laboratories, Lensfield Road, Cambridge CB2 1EW, UK.
E-mail: Dmitri.Schebarchov@gmail.com, dw34@cam.ac.uk Open Access Article. Published on 08 May 2015. Downloaded on 23/11/2015 16:31:45.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Cite this: Phys.Chem.Chem.Phys.,
2015, 17, 28331 D. Schebarchov* and D. J. Wales We formulate nanoalloy structure prediction as a mixed-variable optimisation problem, where the homotops
can be associated with an effective, quasi-combinatorial energy landscape in permutation space. We survey
this effective landscape for a representative set of binary systems modelled by the Gupta potential. In
segregating systems with small lattice mismatch, we find that homotops have a relatively straightforward
landscape with few local optima – a scenario well-suited for local (combinatorial) optimisation techniques
that scale quadratically with system size. Combining these techniques with multiple local-neighbourhood
structures yields a search for multiminima, and we demonstrate that generalised basin-hopping with a
metropolis acceptance criterion in the space of multiminima can then be effective for global optimisation
of binary and ternary nanoalloys. Received 28th February 2015,
Accepted 8th May 2015
DOI: 10.1039/c5cp01198a
www.rsc.org/pccp Received 28th February 2015,
Accepted 8th May 2015 PCCP
PAPER
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View Journal | View Issue PCCP PCCP Phys. Chem. Chem. Phys., 2015, 17, 28331--28338 | 28331 This journal is ©the Owner Societies 2015 2.2
Quasi-combinatorial landscapes and biminima A locally optimal multiset can be defined as one with the lowest
‘‘energy’’ in its local neighbourhood. This definition can also be
combined with the conventional notion of a local minimum
(in coordinate space) to define biminima,15 which, in the
context of nanoalloys, correspond to homotops that are (meta)-
stable with respect to both the Euclidean and the interchange
distance metrics. Open Access Article. Published on 08 May 2015. Downloaded on 23/11/2015 16:31:45.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The only difference is that now one b has been transmuted into
g, which results in the domain doubling in size, while the size
of a local neighbourhood has increased by one. In general, if Na
specifies the multiplicity of atomic component a, then the size
of the permutation domain is given by N! Q
a
Na!;
(1) (1) (1) 2
Framework where N ¼ P
a
Na, and the size of the local neighbourhood
induced by the interchange distance metric is given by where N ¼ P
a
Na, and the size of the local neighbourhood
induced by the interchange distance metric is given by We formulate nanoalloy structure prediction as a mixed-
variable optimisation problem, where each atom i is assigned
coordinates xi A R3 and a categorical label li A aM
a=1, with M
being the number of constituent atomic species. The objective
is to minimise a specified energy function E(X,L) with respect to
X = {xi}N
i=1 and L = {li}N
i=1, where N ¼ P
M
a¼1
Na is the total number of
atoms and each Na (the population of species a) is fixed. Both
X and L are treated as variables, in spite of the obvious
redundancy (i.e. permuting labels has the same effect as
permuting the corresponding coordinates), with L being parti-
cularly useful for exploiting (quasi-)combinatorial features of
E(X,L). We formulate nanoalloy structure prediction as a mixed-
variable optimisation problem, where each atom i is assigned
coordinates xi A R3 and a categorical label li A aM
a=1, with M
being the number of constituent atomic species. The objective
is to minimise a specified energy function E(X,L) with respect to
X = {xi}N
i=1 and L = {li}N
i=1, where N ¼ P
M
a¼1
Na is the total number of 1 þ 1
2
X
a
X
baa
NaNb:
(2) (2) Hence, while the size of the entire domain grows combinatorially
(BN!), the size of the induced local neighbourhood scales quad-
ratically (BN2). In the optimisation techniques considered below,
the local neighbourhood size plays a central role. Hence, while the size of the entire domain grows combinatorially
(BN!), the size of the induced local neighbourhood scales quad-
ratically (BN2). In the optimisation techniques considered below,
the local neighbourhood size plays a central role. atoms and each Na (the population of species a) is fixed. Both
X and L are treated as variables, in spite of the obvious
redundancy (i.e. permuting labels has the same effect as
permuting the corresponding coordinates), with L being parti-
cularly useful for exploiting (quasi-)combinatorial features of
E(X,L). 2.1
Multiset permutations and the interchange distance It is helpful to think of L as an indexed multiset whose distinct
permutations span the domain of nanoalloy homotops. For
example, consider all distinct permutations of a multiset of
N = 4 atoms, with half of the elements characterised as a-type
and the other half as b-type: Fig. 1 illustrates how an energy function can exhibit local
minima in the two metric domains. It also illustrates how a
sequence of swaps at fixed geometry may differ from a sequence
where each swap is accompanied by a local geometry relaxation
(i.e. a quench). Local minima in permutation space may be
shifted, created or destroyed as a result of quenching (local
minimisation in coordinate space), and the net effect can be fa; a; b; bgðP1Þ;
fa; b; a; bgðP2Þ;
fb; a; a; bgðP3Þ;
fa; b; b; agðP4Þ;
fb; a; b; agðP5Þ;
fb; b; a; agðP6Þ: With this domain we can associate an interchange distance
metric16 – the Hamming21 distance restricted to multiples of
two – giving the minimum number of label interchanges (or
swaps) required to arrive from one point in the domain to
another. In the example above, the distance between permuta-
tions P1 and P2 is one interchange (‘‘swap’’), and that between
P1 and P6 is two swaps (which is the maximum for N = 4). This
simple metric induces a local neighbourhood structure for each
permutation: it contains the permutation in question and all
others that can be reached from it by a single swap. Recalling
the above example again, the local neighbourhood of P1 con-
tains all other permutations apart from P6. Fig. 1
A schematic diagram illustrating how the energy of a nanoalloy
might vary along a continuous coordinate (a) or a sequence of atom
swaps/interchanges (b). In (b), the filled symbols correspond to a swap
sequence at fixed geometry, whereas the open symbols illustrate the
effect of quenching. Fig. 1
A schematic diagram illustrating how the energy of a nanoalloy
might vary along a continuous coordinate (a) or a sequence of atom
swaps/interchanges (b). In (b), the filled symbols correspond to a swap
sequence at fixed geometry, whereas the open symbols illustrate the
effect of quenching. Fig. 1
A schematic diagram illustrating how the energy of a nanoalloy
might vary along a continuous coordinate (a) or a sequence of atom
swaps/interchanges (b). 28332 | Phys. Chem. Chem. Phys., 2015, 17, 28331--28338 1 Introduction In strongly
segregating systems this fraction is found to be minuscule,
and it appears to grow with the preference for mixing –
consistent with our earlier findings for binary Lennard-Jones
clusters.15 Furthermore, the fraction of locally optimal homo-
tops appears to decrease with system size for segregating While most methods currently used for nanoalloy structure
prediction tend to focus on local minima in coordinate space,10–14
one could argue that it might be more effective to systematically
target a smaller subspace that is somehow guaranteed to
retain the global minimum. This reasoning motivated the
definition of biminima15 – configurations that are local minima
in two different metric domains: (i) in the usual coordinate
domain, measured by the Euclidean distance metric, and (ii)
in the permutation domain, measured by the interchange16 Phys. Chem. Chem. Phys., 2015, 17, 28331--28338 | 28331 This journal is ©the Owner Societies 2015 28331 View Article Online View Article Online PCCP Paper For further illustration consider a (slightly) more compli-
cated example: nanoalloys and increase for mixing nanoalloys. We are unable
to detect similarly useful trends for lattice mismatched nano-
alloys, largely due to the breakdown of global combinatorial
structure. However, our benchmark calculations for selected
binary (CunAu38n) and ternary (Cu13AgnAu42n) systems clearly
show that systematic multiminima-targeting provides a highly
effective strategy for global optimisation. fa; a; b; gg;
fa; b; a; gg;
fb; a; a; gg;
fa; a; g; bg;
fa; b; g; ag;
fb; a; g; ag;
fa; g; a; bg;
fa; g; b; ag;
fb; g; a; ag;
fg; a; a; bg;
fg; a; b; ag;
fg; b; a; ag: Incidentally, the idea of using multiple definitions of
‘‘locality’’ in global optimisation is not new: it has been
exploited by Mladenovic´ et al. in the so-called variable neigh-
bourhood search metaheuristic.17–20 To the best of our knowledge,
this approach has been applied to either purely combinatorial17
or (to a lesser extent) purely continuous19 problems. Here we
apply it within a less restrictive and relatively unexplored realm
of mixed-variable optimisation, where the constituent degrees of
freedom can be different: continuous, discrete, categorical, etc. Open Access Article. Published on 08 May 2015. Downloaded on 23/11/2015 16:31:45.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Since biminimisation (and, more generally, multiminimisation)
inherently constitutes a local search, we sample different multi-
minima using a generalisation of the basin-hopping (BH) global
optimisation technique.10,11 While BH involves steps in the space of
(conventional) minima, the new generalised variant (GBH) considers
the reduced space of multiminima. In both cases one has to specify
the temperature parameter for the metropolis acceptance criterion
and also provide an appropriate random-move set.34 We choose to
adaptively adjust the temperature to maintain the (rolling average)
acceptance ratio close to 0.5, and our move set consists of just two
operations: (i) random permutation of atomic labels and (ii) random
Cartesian displacement of atomic coordinates. Both operations are
applied at each GBH step, but for homotop optimisation the effect
of random Cartesian moves can be rendered ineffectual by setting
the maximum displacement to zero. Finally, when Nres GBH steps
fail to produce a lower-energy multiminimum, the search is restarted
from a completely random configuration/permutation. We note
that such large steps in configuration space can be used, since
no detailed balance condition is required. One obvious approach to biminima searching is to execute a
monotonically decreasing sequence of quenched swaps15,22–24
until the sequence becomes stuck at a biminimum. That is, if
we use DEij to denote swap gain24 – the energy change due to a
quenched swap of labels between atoms i and j – the policy of
accepting only swaps with DEij o 0 will guarantee that a sequence
will converge when DEij Z 0, 8i, j, which is precisely the definition
of a biminimum.15 Note that finding a swap with DEij o 0 is
usually easier than verifying that no such swap exists, because the
verification requires an exhaustive search through the entire local
neighbourhood in permutation domain. Without a reliable way of
bypassing this computational bottleneck, the best case scenario
for the cost of biminima searching is a linear scaling with the size
of the local neighbourhood. Finally, it is worth noting that the concepts of quasi-
combinatorial landscapes and biminima are also applicable when
one of the constituent species represents vacancies (quasiparticles),
and so the framework of mixed-variable optimisation could sub-
sume the approach of dynamic-lattice searching.25,26 However,
since vacancies are usually not intrinsically defined in off-lattice
atomistic models, they have to be generated by some additional
means, and it may require introducing another, distinctly different
local neighbourhood structure. 2.1
Multiset permutations and the interchange distance In (b), the filled symbols correspond to a swap
sequence at fixed geometry, whereas the open symbols illustrate the
effect of quenching. Fig. 1
A schematic diagram illustrating how the energy of a nanoalloy
might vary along a continuous coordinate (a) or a sequence of atom
swaps/interchanges (b). In (b), the filled symbols correspond to a swap
sequence at fixed geometry, whereas the open symbols illustrate the
effect of quenching. 28332 | Phys. Chem. Chem. Phys., 2015, 17, 28331--28338 This journal is ©the Owner Societies 2015 View Article Online PCCP Paper difficult to track. It is even conceivable that a quenched
swap may not always be a reversible operation. This potential
irreversibility implies that a revisited permutation may be mapped
to different energy values, and so the total number of distinct
configurations that could be generated by a sequence of quenched
swaps is not necessarily bounded above by the combinatorial
number in (1). It is precisely for this reason that we refer to the
effective energy landscape in the permutation domain as quasi-
combinatorial. The term locally combinatorial may also be
appropriate, because we can still rely on (2) to enumerate the
local neighbourhood. in the sorted list, which does not guarantee that our definition
of biminima will necessarily be fulfilled. Also note that there
are multiple ways of scanning the local neighbourhood,15 which
could involve the use of point-group symmetry,32,33 though it is
reasonable to expect the relative performance of these different
schemes to be system specific. 3.3
Surface refinement Results from Lai et al.23 indicate that incorporating a surface
refinement stage into a global structure search can be beneficial. Hence, when performing a global search with random Cartesian
moves, we combine biminimisation with another procedure akin
to a dynamic-lattice search.25,26 This procedure relies on a nearest-
neighbour analysis to generate an appropriate set of vacancies
(see below) for a given biminimum. These vacancies and the
subset of least-coordinated atoms (irrespective of their chemical
identity) define an ad hoc local neighbourhood, and we sequentially
attempt quench-assisted swaps between atoms and vacancies
within this neighbourhood. If a swap leads to a reduction in energy
then it is immediately accepted and the local neighbourhood
structure is rebuilt. By analogy with biminimsation, the surface
refinement procedure terminates when an exhaustive scan of the
entire neighbourhood fails to produce a lower energy structure. Note that, unlike biminimisation, the local neighbourhood struc-
ture used for surface refinement does not have a predefined
size. Finally, biminimisation and surface refinement are repeated
in tandem until one of them fails to produce a lower energy
structure, in the end converging on a triminimum. Open Access Article. Published on 08 May 2015. Downloaded on 23/11/2015 16:31:45.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. In that case the term triminima
would be more appropriate for describing the local optima. 3 Methods Throughout this study, nanoalloys are modelled using the
Gupta potential27 with standard parameter values.28–30 All search
techniques described below have been implemented in the
GMIN program.31 Phys. Chem. Chem. Phys., 2015, 17, 28331--28338 | 28333 † The hit rate, or relative encounter probability, is equal to the number of times
the lowest-encountered biminimum has been hit, divided by the total number of
convergences to a biminimum. 3.1
Biminimisation We perform biminima searching (‘‘biminimisation’’) using a
scheme similar to that of Lai et al.,23 which exploits correlations
between DEij (see Section 2.2) and DEij* – the energy change
evaluated immediately after the swap but before quenching. Systematically attempting quenched swaps within a local neigh-
bourhood sorted by DEij* (from lowest to highest) is a cost-effective
way of finding DEij o 0. The first encountered swap with DEij o 0
is immediately accepted, and then the local neighbourhood is reset
and sorted by the new DEij*. The procedure terminates when a
complete scan of the entire local neighbourhood fails to produce
DEij o 0. Note that Lai et al.23 consider only the first N 1 entries To generate the vacancies we first identify the nearest neigh-
bours for each atom (i) and, if its coordination number is less
than twelve, we apply local inversion symmetry (about xi) on each of
neighbour’s coordinates to generate a local set of candidate
vacancies. We discard candidates that are within d = 0.8rNN
min from
another atom, where rNN
min denotes the shortest interatomic distance
in the current structure. The union of all local candidate sets is Phys. Chem. Chem. Phys., 2015, 17, 28331--28338 | 28333 This journal is ©the Owner Societies 2015 View Article Online Paper Fig. 2
Hit rates for the (putative) lowest-lying biminimum and total biminima counts accumulated from up to B103 independent biminimisation runs for
PtnPdNn (top), AunAgNn (middle) and CunAgNn (bottom) with N = 38 (left), 55 (centre) and 75 (right). The runs were initiated from a uniform random
sample of distinct starting points in permutation domain. Paper
PCCP Open Access Article. Published on 08 May 2015. Downloaded on 23/11/2015 16:31:45. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 2
Hit rates for the (putative) lowest-lying biminimum and total biminima counts accumulated from up to B103 independent biminimisation runs for
PtnPdNn (top), AunAgNn (middle) and CunAgNn (bottom) with N = 38 (left), 55 (centre) and 75 (right). The runs were initiated from a uniform random
sample of distinct starting points in permutation domain. Cartesian moves, i.e. a converged biminimum is escaped solely
by virtue of randomly permuting the labels. 3.1
Biminimisation The intention is to
(i) initiate biminimisation from a uniform sample of starting
points in the permutation domain, (ii) accumulate representa-
tive count statistics for energetically distinct biminima (using a
threshold of 106 eV), and (iii) estimate the relative encounter
probabilities of the (putative) lowest-lying biminimum. The results
are presented in Fig. 2. then sorted by atomic coordination number, and all but
the highest coordinated vacancies are discarded. If any of the
remaining candidate vacancies are separated by less than d,
they are merged and the corresponding coordinates are averaged. In the end, we proceed with the surface refinement scheme only
if the coordination of remaining vacancies exceeds the lowest
coordination amongst the atoms. While both PtnPdNn and AunAgNn represent lattice-matched
systems, the former favours segregation and the latter favours
mixing.38 In spite of this qualitative difference, the corresponding
count statistics from biminima sampling are comparable for
N = 38 and 55. In both cases the number of sampled biminima
is consistently orders of magnitude smaller than the number of
sampled homotops, even for compositions when the size of the
permutation domain is at its peak (50 : 50 stoichiometry). Furthermore, the relative occurrence (‘‘hit rate’’†) for the most
elusive biminimum at a given stoichiometry often exceeds 0.5,
suggesting that in many cases one or two biminimisations can
be sufficient to find the lowest-lying homotop. This observation
can be interpreted in terms of the effective landscape topography
comprising only a few significant optima, which would make This journal is ©the Owner Societies 2015 Open Access Article. Published on 08 May 2015. Downloaded on 23/11/2015 16:31:45.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 3
Ball-and-stick
representation
of
biminima
for
Cu13Ag25
(a),
Cu34Ag4 (b) and Cu44Ag11 (c)–(g). Cu atoms are smaller and coloured
red, Ag atoms are larger and coloured grey. Arrows in (e) indicate the
vertex sites from which two Ag atoms have been dislodged. The subtle
geometric differences between (f) and (g) are highlighted by a circle
around a most shifted Ag atom and an arrow pointing to a destroyed/
created nearest-neighbour ‘‘bond’’. These rearrangements often involve one or two Ag atoms being
dislodged from a vertex site and moved to a different position
on the cluster surface, as illustrated in Fig. 3c–e. In the same
system we also find configurations that look almost identical to
the naked eye (and could be mistakenly regarded as the same
homotop) but are actually different biminima. Snapshots in
Fig. 3f and g show two such biminima, illustrating how lattice
strain effects can lead to very subtle consequences that may be
difficult to detect. These observations also highlight the inher-
ent coupling of the combinatorial and the continuous sub-
problems in nanoalloy structure prediction, and untangling
these sub-problems in lattice mismatched systems may be
particularly difficult (and perhaps unnecessary). These rearrangements often involve one or two Ag atoms being
dislodged from a vertex site and moved to a different position
on the cluster surface, as illustrated in Fig. 3c–e. In the same
system we also find configurations that look almost identical to
the naked eye (and could be mistakenly regarded as the same
homotop) but are actually different biminima. Snapshots in
Fig. 3f and g show two such biminima, illustrating how lattice
strain effects can lead to very subtle consequences that may be
difficult to detect. These observations also highlight the inher-
ent coupling of the combinatorial and the continuous sub-
problems in nanoalloy structure prediction, and untangling
these sub-problems in lattice mismatched systems may be
particularly difficult (and perhaps unnecessary). We now shift focus to CunAgNn – a system with a relatively
large degree of lattice mismatch (more than 10%). Results for
N = 38 and 75 show strikingly large biminima counts, which
can be attributed to our sampling procedure spanning multiple
geometries that are distinctly different from the initial trun-
cated octahedron (N = 38) or Marks decahedron (N = 75). As an
illustrative example, in Fig. 3a and b we visualise the lowest-
lying biminima for Cu13Ag25 (a) and Cu34Ag4 (b). 4 Results and discussion We start by attempting to characterise the quasi-combinatorial
energy landscape associated with nanoalloy homotops for a
representative set of binary systems: PtnPdNn, AunAgNn,
CunAgNn for N = 38, 55 and 75. The initial coordinates are
derived from the (single-component) Lennard-Jones global
minima:11,35 truncated octahedron for N = 38, Mackay icosa-
hedron36 for N = 55, and Marks decahedron37 for N = 75. To
impose appropriate nearest-neighbour separation while preser-
ving the overall symmetry, these coordinates are multiplied by a
scaling factor (f) equal to the arithmetic average of the nearest-
neighbour distances (raa
0 ) for each of the constituent metals (a),
i.e. f ¼ P
a
raa
0
M, where M is the number of species. The labels are initialised randomly but consistent with a specified stoi-
chiometry. The resulting configurations are then used for GBH
at high temperature (kBT = 1.0 eV) and without random 28334 | Phys. Chem. Chem. Phys., 2015, 17, 28331--28338 This journal is ©the Owner Societies 2015 Fig. 3
Ball-and-stick
representation
of
biminima
for
Cu13Ag25
(a),
Cu34Ag4 (b) and Cu44Ag11 (c)–(g). Cu atoms are smaller and coloured
red, Ag atoms are larger and coloured grey. Arrows in (e) indicate the
vertex sites from which two Ag atoms have been dislodged. The subtle
geometric differences between (f) and (g) are highlighted by a circle
around a most shifted Ag atom and an arrow pointing to a destroyed/
created nearest-neighbour ‘‘bond’’. Paper
View Article Online View Article Online View Article Online PCCP Paper local (combinatorial) search techniques particularly effective. It
is also worth noting that biminima counts for PtnPdNn and
AunAgNn do not always peak at n = N/2, indicating that the size of
the permutation domain (1) is not the only determining factor. p
( )
y
g
Notable differences in biminima counts emerge for PtnPd75n
and AunAg75n, and the decrease in counts for PtnPd75n
(compared to N = 55) is particularly surprising. It indicates that
the effective landscape topography may become ‘‘simpler’’ as
N increases. To corroborate this hypothesis, we applied the
same homotop sampling procedure to PtnPd98n Leary tetra-
hedra,39,40 in the range 43 r n r 55, and in each case found
that a uniform random sample of about 2000 homotops was
mapped onto a single biminimum. This result suggests some-
thing quite remarkable: it is possible that, out of 98!/49!49! Open Access Article. Published on 08 May 2015. Downloaded on 23/11/2015 16:31:45.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. In both cases
the initial geometry is a truncated octahedron, which remains
intact throughout biminima sampling for Cu34Ag4, but gradu-
ally changes for Cu13Ag25 solely by virtue of quenching, and
eventually we find the structure illustrated in Fig. 3a. In fact,
the biminimum in Fig. 3a closely resembles (but not quite
matches) the putative global minimum,42 and it is noteworthy
that it has been found without random Cartesian moves, espe-
cially in view of the differences between the initial and the final
geometric motifs. This result implies that, for systems with large
lattice mismatch, quench-assisted local combinatorial optimisa-
tion (in the permutation domain) can provide an additional and
potentially significant guiding force towards the globally optimal
geometry. Hence, even if the notion of nanoalloy homotops loses
its global combinatorial character, the use of a local combina-
torial search will still remain a viable option. Due to a growing interest in trimetallic nanoalloys,43,44 we have
also sampled biminima for selected PtlPdmAun and CulAumAgn
Mackay icosahedra with l + m + n = 55 and the Gupta parameters
taken from ref. 30 and 45. Compared with the binary counter-
parts, these ternary systems proved more susceptible to geometric
distortion during the quasi-combinatorial sampling procedure. This complication precludes further characterisation of the effec-
tive quasi-combinatorial landscape, but it suggests that strain
effects may become more pronounced as the number of species
grows (at fixed total atom count). Having demonstrated the (albeit limited) utility of biminima
in characterising the topography of quasi-combinatorial land-
scapes, we now consider the utility of bi- and multiminima for
the purpose of global optimisation. Fig. 4 shows that, for all
binary and ternary systems not containing both Cu and Au, the
average number of quenches required to find a biminimum
is only marginally larger than the local neighbourhood size. While the reasons why Cu–Au-based systems stand out are not
entirely clear, the scaling for all other systems shows that most
of the computational effort is spent on verifying biminima
as opposed to locating them. This observation suggests that
developing more efficient ways of verifying biminima will be
worthwhile. It is worth noting that CunAg55n yields total biminima counts
and hit rates that are comparable to PtnPd55n and AunAg55n
(see Fig. 2). This congruence is possibly due to the relative
robustness of closed-shell icosahedra, which evidently remain
(largely) intact for all three systems. This journal is ©the Owner Societies 2015 Phys. Chem. Chem. Phys., 2015, 17, 28331--28338 | 28335 4 Results and discussion E
2.5 1028 homotops, only one is locally optimal with respect to
the interchange distance in the permutation domain. To check
if similar results can be expected for other systems, we sampled
biminima for PdnAuNn (using the ‘‘average’’ Gupta parameters
from ref. 41) and again found biminima counts decreasing
with N, similar to PtnPdNn. We suspect that this trend is a
general characteristic of segregating systems, where the number
of significant optima in the permutation domain appears to
diminish with system size. In contrast, the increase in biminima
counts for AunAg75n (compared to N = 55) suggests that the
exact opposite may be true for systems that favour mixing, which
is consistent with our previous observations for binary Lennard-
Jones clusters.15 Open Access Article. Published on 08 May 2015. Downloaded on 23/11/2015 16:31:45.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 4
Mean biminimisation cost quantified by the number of quenches
for all the model nanoalloys considered in this study, each with a particular
local neighbourhood size. Note that the symbol ‘‘A’’ spans Pt, Pd, and Au,
while ‘‘B’’ stands for Pd, Au and Ag, respectively. y
g
g
It is worth reiterating that GBH1 carries a significant com-
putational overhead due to biminima verification. This over-
head is reduced in GBH2, often (but not always) yielding
significantly lower MFETs. The efficiency gain varies due to
variable correlation between exact (DEij) and approximate
(DEij*) swap gains. Nonetheless, it is clear that reducing the
local neighbourhood structure can lower the computational
cost by an order of magnitude. Although the particular approxi-
mation in GBH2 (adapted from Lai et al.23) is not easily
tractable and does not guarantee convergence to a biminimum,
biminima candidates could be verified in a separate post-
processing step if required. Alternatively, one could redefine
biminima by restricting interchanges to atoms that are near-
neighbours in coordinate space, which will make the size of the
local neighbourhood scale roughly linearly with the number of
particles (N), though it may also produce more biminima. Linear
scaling with N can also be achieved by relaxing the constraint of
fixed stoichiometry and using the Hamming distance,21 which
measures the minimum number of substitutions (as opposed to
swaps) required to obtain one multiset from another. We intend
to explore these possibilities in future studies. As a final demonstration, in Fig. 5 we present mean first-
encounter times (MFETs) for global optimisation of CunAu38n
and Cu13AgnAu42n, with the target energies for the putative
global minima taken from ref. 29 and 30, respectively. We consider
three different variants of GBH as a global search strategy, each
with random displacement and permutation moves. The first
variant (GBH1) includes the surface refinement scheme described
in Section 3.3 and it involves complete scanning of the local
neighbourhood to verify biminima each time. In the second
variant (GBH2) we chose not to verify biminima and limit the
sorted neighbourhood scan just to the first ten entries (recall
Section 3.1). In the third variant (GBH3) we also disable the
surface refinement scheme to gauge its effectiveness. Open Access Article. Published on 08 May 2015. Downloaded on 23/11/2015 16:31:45.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Finally, the
results are compared with previously reported benchmarks for
the adaptive immune optimisation algorithm (AIOA).29,30 Note that MFETs for GBH1 were averaged over a variable
number of hits, ranging from 10 to 103. Also, the benchmarking
procedure comes in two slightly different flavours: in one
the temperature parameter is reset to the initial value of
kBT = 0.05 eV each time the specified target energy is hit, and
in the other the temperature is not reinitialised during the Finally, MFETs obtained using GBH3 show that the effective-
ness of our surface refinement scheme is system-dependent. For CunAu38n with n Z 13 the scheme can either increase or
decrease MFET by up to an order of magnitude, while for
Cu13AgnAu42n with n Z 21 it consistently improves the effi-
ciency by more than an order of magnitude. To elucidate the
general utility of surface refinement we consider MFETs for
single-component CuN and AuN clusters with N = 38 and 55,
focusing on the effect of random step size. Fig. 6 clearly shows
that, without surface refinement, choosing a random displace-
ment step that is too small or too large can severely hamper the
search performance, and there appears to be an optimal step
size of around 1.1 Å for all the cases considered (and it was used
in GBH3). The sweet spot exists because an overly small step
decreases the probability of escape from the catchment basin of
a current minimum, while an overly large step decreases the
probability of reaching a low-energy minimum. Evidently, the
use of surface refinement consistently reduces the penalty
associated with overly large steps, which can be advantageous
when an optimal step size is not known in advance and has to
be chosen arbitrarily. Fig. 5
Mean first-encounter times (MFETs), quantified by the number of
quenches, for (a) CunAu38n and (b) Cu13AgnAu42n versus n. The red
squares correspond to data from (a) ref. 29 and (b) ref. 30. Black circles
(GBH1), blue triangles (GBH2) and green crosses (GBH3) correspond to
data sets for three different variants of generalised basin-hopping. The
dashed and solid black circles represent Monte Carlo protocols with
slightly different handling of the temperature parameter. Fig. 5
Mean first-encounter times (MFETs), quantified by the number of
quenches, for (a) CunAu38n and (b) Cu13AgnAu42n versus n. The red
squares correspond to data from (a) ref. 29 and (b) ref. 30. This journal is ©the Owner Societies 2015 Open Access Article. Published on 08 May 2015. Downloaded on 23/11/2015 16:31:45.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. However, subtle morphologi-
cal distortions still occur in CunAg55n in the range 23 r n r 44. Phys. Chem. Chem. Phys., 2015, 17, 28331--28338 | 28335 View Article Online PCCP Paper Fig. 4
Mean biminimisation cost quantified by the number of quenches
for all the model nanoalloys considered in this study, each with a particular
local neighbourhood size. Note that the symbol ‘‘A’’ spans Pt, Pd, and Au,
while ‘‘B’’ stands for Pd, Au and Ag, respectively. entire run. In both cases the random Cartesian displacement
was capped at 1.5 Å, while Nres (recall Section 3.2) was set to
50 for CunAu38n and 10 for Cu13AgnAu42n. These minor
differences in the Monte Carlo protocol are somewhat arbitrary,
but the agreement between them provides some reassurance
that the statistics are meaningful. Hence, from Fig. 5 we infer
that GBH1 generally outperforms AIOA, by more than an order
of magnitude in some cases, and is slower only in four
instances: CunAu38n with n = 11, 17, 35 and 36. For
Cu13AgnAu42n the superiority of GBH1 appears to be more
convincing. In a few cases we have checked that the relative
efficiency is not overly sensitive to the choice of Nres or the
initial temperature, and so the superiority of GBH1 can be
attributed to systematic multiminima-targeting. Open Access Article. Published on 08 May 2015. Downloaded on 23/11/2015 16:31:45. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 5 Summary 14 A. R. Oganov and C. W. Glass, J. Chem. Phys., 2006,
124, 244704. We have outlined a general strategy for global optimisation for
nanoalloys based on a mixed-variable approach and systematic
targeting of multiminima configurations that are local minima
with respect to multiple metrics. The strategy was incorporated
into a global search method, namely generalised basin-hopping
(GBH), which was used to explore and characterise the quasi-
combinatorial energy landscape associated with nanoalloy homo-
tops. The number of biminima in these effective landscapes was
shown to systematically vary with the preference for mixing/
segregation, whereas the computational cost of finding biminima
scaled quadratically with system size in most cases. Based on
these observations, we conclude that biminima targeting is likely
to be more effective for model nanoalloys with a higher preference
for segregation. GBH was also demonstrated to be effective for
global optimisation of binary and ternary nanoalloys that feature a
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This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Black circles
(GBH1), blue triangles (GBH2) and green crosses (GBH3) correspond to
data sets for three different variants of generalised basin-hopping. The
dashed and solid black circles represent Monte Carlo protocols with
slightly different handling of the temperature parameter. 28336 | Phys. Chem. Chem. Phys., 2015, 17, 28331--28338 This journal is ©the Owner Societies 2015 View Article Online PCCP Paper Fig. 6
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A COVID–19-pandémia mentálhigiénés következményei. Hogyan tudunk felkészülni a pszichodémiás krízisre?
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ÖSSZEFOGLALÓ KÖZLEMÉNY ÖSSZEFOGLALÓ KÖZLEMÉNY A COVID–19-PANDÉMIA ORVOSSZAKMAI KÉRDÉSEI A COVID–19-pandémia mentálhigiénés
következményei. Hogyan tudunk
felkészülni a pszichodémiás krízisre? Osváth Péter dr. Pécsi Tudományegyetem, Általános Orvostudományi Kar, Pszichiátriai és Pszichoterápiás Klinika, Pécs Pécsi Tudományegyetem, Általános Orvostudományi Kar, Pszichiátriai és Pszichoterápiás Klinika, Pécs Az elmúlt hónapokban életünket alapvetően megváltoztatta a COVID–19-pandémia, melynek egészségügyi, gazda
sági és társadalmi hatásai egyelőre szinte felbecsülhetetlenek. A vírusfertőzés akut következményei mellett egyre több
adat bizonyítja a teljes népességre kifejtett hatásait: a pszichológiai distressz, a depressziós és szorongásos tünetek,
valamint az addiktív viselkedésformák gyakoriságának növekedését. Az is nagyon fontos kérdés, hogy a globális vál
sághelyzet hogyan befolyásolja az öngyilkossági arányszámokat. Írásomban az elmúlt időszak legjelentősebb pszichi
átriai szakirodalma alapján foglalom össze a vírusfertőzés akut és krónikus hatásait, valamint a járványhelyzet általános
és specifikus pszichológiai-pszichopatológiai következményeit, kiemelt figyelmet fordítva a suicidiumrizikóra és a
leginkább veszélyeztetett csoportokra. A vizsgálatok arra utalnak, hogy a pandémia következtében kialakuló mentális
gondok és a suicid viselkedés egyre fontosabb népegészségügyi problémává válnak. Bár napjainkban még a vírusfer
tőzöttek gyógyítása és a fertőzés terjedésének lassítása a legfontosabb cél, mindannyiunknak fel kell készülnünk a
járvány hosszú távú következményeire. A pandémia várható negatív mentálhigiénés hatásainak megelőzésére és eny
hítésére általános és specifikus módszerek kidolgozása és alkalmazása szükséges. Ebben az egészségügyi, mentálhigi
énés és közösségi ellátórendszerek mellett szerepet kell vállalniuk a politikai és gazdasági döntéshozóknak, a társadal
mi szervezeteknek és a média munkatársainak is. Hatékony együttműködésük kulcsfontosságú az egyéni, közösségi
és társadalmi szinten is alkalmazható prevenciós stratégiák megvalósításában, hiszen csak így válik lehetővé a súlyo
sabb mentálhigiénés problémák járványszerű elterjedésének, a „pszichodémiának” a megelőzése. Orv Hetil. 2021; 162(10): 366–374. Kulcsszavak: COVID–19-vírus, lelki egészség, pszichopatológia, öngyilkosság, prevenció Kulcsszavak: COVID–19-vírus, lelki egészség, pszichopatológia, öngyilkosság, prevenció Osváth P. [Psychological outcome of COVID-19 pandemic. How can we prepare for a psychodemic crisis?] Orv
Hetil. 2021; 162(10): 366–374. Psychological outcome of COVID-19 pandemic.
How can we prepare for a psychodemic crisis? In recent months, our lives have been fundamentally changed by the COVID-19 pandemic, the health, economic, and
social impacts of which are almost invaluable for the time being. In addition to the acute consequences of viral infection,
more and more data are proving its effects on the entire population: an increase in the incidence of psychological dis
tress, depressive and anxiety symptoms, and addictive behaviours. It is also a very important question, how the global
crisis is affecting suicide rates. In my paper, I summarize the acute and chronic effects of viral infection and the general
and specific psychological-psychopathological consequences of the epidemic based on the most significant psychiatric
literature of the recent period, paying special attention to suicidal risk and the most vulnerable groups. Studies suggest
that mental troubles and suicidal behaviour resulting from a pandemic are becoming an increasingly important public
health problem. Although the treatment of viral infections and slowing the spread of the infection are still the most
important goals today, we all need to be prepared for the long-term consequences of the epidemic. In order to prevent
and mitigate the expected negative mental health effects of a pandemic, it is necessary to develop and apply general and
specific methods. In addition to health care, mental health and community care systems, political and economic deci
sion-makers, civil society organizations and the media must also play a role. Their effective cooperation is key to the
implementation of prevention strategies that can be applied at the individual, community and social levels, as this is the
only way to prevent the epidemic spread of more serious mental health problems, the “psychodemia”. Keywords: COVID-19 virus, mental health, psychopathology, suicide, prevention Osváth P. [Psychological outcome of COVID-19 pandemic. How can we prepare for a psychodemic crisis?] Orv
Hetil. 2021; 162(10): 366–374. (Beérkezett: 2020. december 21.; elfogadva: 2021. január 15.) (Beérkezett: 2020. december 21.; elfogadva: 2021. január 15.) DOI: 10.1556/650.2021.31141 ■ © Szerző(k) 366 2021 ■ 162. évfolyam, 10. szám ■ 366–374. Unauthenticated | Downloaded 10/24/24 05:16 AM UTC Unauthenticated | Downloaded 10/24/24 05:16 AM UTC ÖSSZEFOGLALÓ KÖZLEMÉNY Rövidítések
COVID–19 = (coronavirus disease 2019) koronavírus-beteg
ség 2019; WHO = (World Health Organization) Egészségügyi
Világszervezet organikus pszichoszindrómák (különböző tudatzavarok,
delírium stb.), melyek kórokai között egyre jobban kör
vonalazódik a vírusfertőzésnek, illetve a következménye
sen kialakuló gyulladásos és immunológiai mechanizmu
soknak a szerepe [6]. A vírusfertőzés hosszú távú hatásai A vírusfertőzés okozta késői (krónikus) gyulladás megje
lenési formáinak hátterében is több különféle kórélettani
mechanizmust valószínűsítenek. A cardiovascularis és
pulmonalis szövődmények mellett neurológiai és pszi
chológiai következményei is lehetnek [9]. A központi
idegrendszeri tünetek hátterében a vírusfertőzés direkt
hatásának (encephalitis), a szisztémás gyulladásnak és az
ennek következtében kialakuló perifériás funkciózava
roknak (máj, vese, tüdő), valamint a cerebrovascularis
változásoknak a komplex interakciója állhat [9]. A króni
kus szakasz – más vírusfertőzéshez hasonlóan – sokszor
hónapokig is elhúzódhat, és olyan bizonytalan testi tü
netekkel járhat, mint például kimerültség, nehezített lég
zés, ízületi vagy akár mellkasi fájdalmak. Bár ma még el
sősorban az akut fertőzés megelőzési és kezelési
lehetőségeinek fejlesztése áll a kutatások középpontjá
ban, fel kell készülni arra is, hogy egyre inkább a króni
kus formák okozta problémák kerülnek majd előtérbe,
ezért nagy jelentőséggel bír a hosszú távú következmé
nyek mechanizmusának pontosabb felderítése [4].i Psychological outcome of COVID-19 pandemic.
How can we prepare for a psychodemic crisis? A delirózus tudatzavar a hatodik
leggyakoribb akut tünet, mely az idős fertőzöttek közel
egyharmadánál megjelenik. Ez különösen azokra a mul
timorbid páciensekre jellemző, akik sokféle gyógyszert
(köztük pszichotrop szereket) is szednek. Ezen tudatza
var jelentőségét hangsúlyozza, hogy növeli az intenzív
osztályos kezelés időtartamát és a halálozási arányt is [7]. Mivel gyakran ez a fertőzés elsődleges tünete egyéb
– a vírusfertőzésre típusos – eltérés nélkül, klinikai szem
pontból fontos szereppel bír az infekcióhoz társuló delí
rium jeleinek és típusának felismerése, mivel a különböző
megjelenési formák eltérő betegséglefolyással bírnak. Rozzini és mtsai [8] a dementiához társuló hipokinetikus
forma esetében észlelték a legmagasabb mortalitást. Így
az akut szakaszban megjelenő pszichopatológiai tünetek
adekvát értékelése a prognózis előrejelzésében és a páci
ens kezelésének pontosabb megtervezésében is segítsé
get nyújt [7, 8]. Az elmúlt év orvostudományi szakirodalmának legfonto
sabb témakörét a COVID–19-járvány képezte; az akut
és (a várható) hosszú távú egészségügyi következménye
ivel foglalkozó közlemények sokaságával találkozhatunk,
melyek zöme a vírus biológiai-infektológiai hatásaival és
kezelési lehetőségeivel foglalkozik. Szerencsére ma már
egyre többet olvashatunk a vírusfertőzés idegrendszeri
és mentálhigiénés-pszichológiai következményeiről is. A 21. század eddigi legnagyobb hatású, a modern társa
dalmak életét a legváltozatosabb módon és mértékben
befolyásoló világjárvány gazdasági-kulturális-társadalmi
hatásai egyelőre szinte felbecsülhetetlenek. Az elmúlt
hónapokban a járvány korábban sosem látott sebességgel
terjedt el az egész világon, így ezen összefoglaló írásának
idején (2020 decemberében) a járvány második hullámá
nak emelkedő szakaszában vagyunk, és még közel sem
körvonalazódik a vége, bár a védőoltások alkalmazásának
lehetősége óvatos optimizmusra ad okot. A kutatók már
a tavaszi időszakban felhívták a figyelmet a járvány akut
és elhúzódó pszichológiai és mentálhigiénés hatásaira és
a mentális zavarok gyakoriságának várható emelkedésére
[1–3]. Az elmúlt hónapokban végzett epidemiológiai
felmérések ezt az akkor még talán pesszimistának tűnő
előrejelzést maximálisan visszaigazolták, hiszen a pszi
chológiai distressz, a depressziós és szorongásos tünetek,
valamint az addiktív viselkedésformák gyakoriságának
növekedését észlelték. A járvány következtében kialakuló
megbetegedések ellátása egyre nagyobb kihívás elé állítja
a „békeidőben” is túlterhelt egészségügyi ellátórendsze
reket és más segítő szolgálatokat [2]. Az egyik legfonto
sabb kérdés az, hogy milyen közvetett és közvetlen hatá
sokra számíthatunk, és hogyan készülhetünk fel ezek
megelőzésére és hatékony kezelésére. Írásomban az el
múlt hónapok legjelentősebb pszichiátriai szakirodalma
alapján szeretném bemutatni a vírusfertőzés és a pandé
mia akut és elhúzódó pszichológiai-pszichopatológiai
következményeit, kiemelt figyelmet fordítva a mentálhi
giénés ellátás és a megelőzés lehetőségeire. A vírusfertőzés akut szövődményei Az idegrendszeri patofiziológiai mechanizmusok ter
mészetesen ilyenkor is szövődnek a fertőzéssel kapcsola
tos stresszélmény következményeivel, ezek között említ
hetjük például a fertőzöttséggel kapcsolatos aggodalma
kat, a mások megfertőzésétől való félelmet, a tünetek
okozta gyötrelmeket, a kórházi (különösen az intenzív
osztályos) kezeléssel kapcsolatos korlátozásokat [2]. A súlyos – akár intenzív osztályos kezelést indokló –
vírusfertőzés túlélői között gyakoribbnak találták a
poszttraumás tünetcsoportot is. Tehát a gyógyult bete
gek esetében jelentős pszichológiai distresszre kell szá
mítani. Janiri és mtsai felmérésében [10] ez a nők és az Bár a vírusfertőzés pontos patofiziológiáját ma sem is
merjük teljesen, a járvány exponenciálisan emelkedő ter
jedése miatt egyre többet tudunk a COVID–19-vírus
okozta fertőzés szerteágazó tünettanáról és az ezek hát
terében álló immunológiai és neurobiológiai változások
ról [4]. Jól ismert a vírusfertőzés akut és posztakut
(hyperinflammatiós) szakasza, mely sok esetben az im
munrendszer súlyos szabályozási zavarát (multisziszté
más gyulladást) idézi elő [5]. Az akutan kialakuló pszi
chopatológiai szövődmények között a leggyakoribbak az 367 2021 ■ 162. évfolyam, 10. szám ORVOSI HETILAP Unauthenticated | Downloaded 10/24/24 05:16 AM UTC ÖSSZEFOGLALÓ KÖZLEMÉNY idősebbek körében gyakoribb volt, és pozitívan korrelált
az impulzivitással és bizonyos temperamentumjellem
zőkkel (például a depressziós vagy a ciklotim típusokkal). Ezen eltérések hátterében az emocionális diszreguláció
mediáló szerepét emelik ki. A szerzők az adekvát érzelmi
szabályozás szerepét hangsúlyozzák a pszichológiai
distressz leküzdésében és így a teljes felépülésben [10]. Az eddigi legszélesebb körben elvégzett észak-amerikai
retrospektív kohorszvizsgálatban a fertőzés túlélői kö
rében a mentális problémák (különösen a szorongásos és
depressziós zavarok, a dementia és az insomnia) emelke
dett arányát találták, mely folyamatosan növekedett a fel
gyógyulást követő időszakban. Gyakoriságuk lényegesen
meghaladta az influenzán vagy más, felső légúti megbe
tegedésen átesettek körében észlelt arányszámokat, ez
pedig szintén a COVID–19-vírus okozta specifikus neu
robiológiai-neuroimmunológiai eltérések etiológiai sze
repére hívja fel a figyelmet [11]. alvászavar vagy akár a suicid gondolatok megjelenése [5,
12]. Mivel a járvány még napjainkban is tombol, és hos�
szabb távú hatása alig becsülhető meg pontosan, nem
könnyű megjósolni azt sem, hogy ez milyen hatással lesz
az öngyilkossági gyakoriságra. Bár számos, vírusfertőzés
sel összefüggő tragikus öngyilkosságról számoltak be
[2], sem a járvány első időszakában végzett epidemioló
giai felmérések [15–18], sem a klinikai tapasztalatok
nem utalnak a járvánnyal összefüggésben a suicid maga
tartás gyakoribbá válására. Ez nem meglepő, hiszen a
nagy társadalmi tragédiák, például háborúk vagy diktatú
rák esetében inkább csökkenni szoktak a suicidium-
arányszámok. A vírusfertőzés akut szövődményei A korábbi hasonló események utóhatása
alapján azonban feltételezhető, hogy a pandémia súlyos
gazdasági és mentálhigiénés következményei hosszabb
távon növelni fogják az öngyilkos viselkedés előfordulá
sát [2, 12, 16, 19–23]. A pszichés problémák hátterében az általános kataszt
rófahangulat, az életkörülmények és életfeltételek kedve
zőtlen és szokatlan megváltozása, valamint a szociális
izoláció mellett kiemelt szerepet játszik a gazdasági vál
ság következtében kialakuló létbizonytalanság és az eg
zisztenciális helyzet romlásától való félelem is [2, 12, 13,
19, 21–25]. A szociális izoláció és a munkahely elveszté
se suicidiumrizikót növelő hatásának hátterében két
olyan, jól ismert lélektani jellemző szerepét igazolták,
mint az összetartozás hiánya és annak érzése, hogy az
egyén terhet jelent mások számára [26]. Tehát a vírusfertőzés hosszú távú hatásai között olyan
pszichológiai-pszichopatológiai maradványtünetek je
lentkezhetnek, mint például a szorongásos vagy depres�
sziós zavar, valamint a poszttraumás stressz zavar [6]. A pszichológiai distressz és a társuló tünetek indirekt
módon járulhatnak hozzá a suicidiumrizikó növekedésé
hez a fertőzésből felgyógyultak körében. Kiemelésre ér
demes, hogy a legújabb neurobiológiai kutatási ered
mények arra utalnak, hogy a vírus bizonyos központi
idegrendszeri hatásai (renin–angiotenzin rendszer, gyul
ladásos faktorok) és az okozott immunológiai diszregu
láció direkt és indirekt módon járulhatnak hozzá a suici
diumrizikó emelkedéséhez [9, 12]. Ráadásul a fertőzés
gyógyulását követően sok esetben észlelnek tartós fájda
lommal és funkcióromlással járó maradványtüneteket,
melyek szintén növelhetik a suicidiumkockázatot [2]. j
A gyakran megjelenő alvászavar önmagában is hozzá
járul a suicidiumrizikó növekedéséhez, a szorongásos és
a depressziós tünetekkel való sajátos kétirányú kapcsolata
révén egymás hatását erősítve súlyosbíthatják az öngyil
kossági rizikót [2]. A járványhelyzettel szükségszerűen
együtt járó korlátozások a társadalom minden tagjának
életét alapvetően megváltoztatják. A negatív hatások kö
zött említhetjük például a szociális kapcsolatok beszűkü
lését [19]. Ez különösen fontos rizikófaktora lehet a sui
cid viselkedésnek [2, 22], mivel a járvány okozta
krízishelyzetben a szociális és érzelmi támogatásra külö
nösen nagy szükség lenne. Az izolált helyzet ráadásul az
esetlegesen kialakuló suicidiumkrízis jeleinek korai felis
merését sem teszi lehetővé. A pandémia általános mentálhigiénés
hatásai Ezek
az aggodalmak a fiatal felnőttek negyedét érintették, és
az is kiderült, hogy a mintában szereplők 35%-a nem ka
pott megfelelő egészségügyi ellátást a panaszai miatt
[34]. Bár a bizonytalanságtól való félelem és szorongás
szinte általános tünetnek tekinthető, a sérülékeny egyé
nekben ez jelentősen megnövelheti a poszttraumás
stressz zavar rizikóját, így közvetve is hozzájárulhat a sui
cid viselkedéshez. Kiemelendő, hogy a járvány okozta
korlátozások és pszichés szövődmények következtében
gyakoribbá válhatnak a különböző addiktív viselkedés
formák is, mint például az alkohol- és drogfüggőség, ami
szintén hozzájárulhat az emelkedő öngyilkossági arány
számokhoz [2, 22]. sokkal veszélyeztetettebbek a megfertőződés szempont
jából, körükben – a betegség típusától függetlenül – a
vírusfertőzés rizikója több mint másfélszeresére nőtt
[11]. Ez az eredmény a mentális zavarokkal összefüggő
immunológiai tényezőkre, a hátrányos szocioökonómiai
helyzetre és bizonyos viselkedési problémákra irányítja a
figyelmet, melyek a pszichiátriai betegek csoportjában
jelentősen megnehezítik a járvány elleni védekezést [11]. A vírusfertőzés közvetlen, valamint a fertőzöttséggel
együtt járó korlátozások indirekt hatásának következté
ben sokkal nagyobb a kedvezőtlen betegségkimenetel
veszélye, mind a szomatikus, mind a pszichés szövődmé
nyek szempontjából. Ráadásul a járvány következtében
beszűkült a pszichiátriai ellátórendszer kapacitása, és
romlanak a segítségnyújtás lehetőségei [22], s ezek szin
tén hozzájárulhatnak a mentális alapbetegség relapsusá
hoz [5, 19]. Éppen ezért a mentális zavarban szenvedők
esetében speciális segítő beavatkozások szükségesek az
állapotromlás és a társuló szövődmények (például ön
gyilkos magatartás) megelőzésére [37]. Ezek között ter
mészetesen az a legfontosabb, hogy a korlátozások elle
nére is biztosítsuk számukra a rendszeres pszichiátriai
kezelést és gondozást. Ebben egyre nagyobb szerepet
kapnak az online, telepszichiátriai módszerek [22, 25,
36]. Itt kell megemlíteni, hogy a mentális betegségben
szenvedők esetében különösen fontos az adekvát és sze
mélyre szabott információközlés a fertőzés megelőzésé
nek lehetőségeiről és a fertőzés kockázatairól, valamint
az egészséges életmód jelentőségéről és összetevőiről
(táplálkozás, testmozgás, napirend stb.) [38]. i Az idős populáció speciális helyzete is figyelmet érde
mel, akik talán a leginkább veszélyeztetettek a szomati
kus és pszichológiai következmények szempontjából [5,
12, 13, 19]. A pandémia miatti szorongás és aggodalom
miatt különösen nagy szükségük lenne a szociális és ér
zelmi támogatásra, miközben a megfertőződés kockáza
tának csökkentése érdekében elengedhetetlen a szociális
távolságtartás [19]. Emiatt körükben is egyre gyakoribbá
válhatnak a különböző pszichiátriai megbetegedések,
melyek – különös tekintettel a depressziós hangulatza
varra – jelentősen növelik az öngyilkossági rizikót [39]. A pandémia általános mentálhigiénés
hatásai A pandémia következtében kialakuló általános bizonyta
lanságérzés és fenyegetettség – a fertőzéssel vagy a fertő
zöttekkel való közvetlen vagy közvetett érintkezés nélkül
is – komoly stresszt okoz a társadalom tagjainak [13]. Ezeket az általános és elhúzódó pszichológiai szövődmé
nyeket ma már külön elnevezéssel, a „koronafóbia” név
vel illetik. Ez olyan, sajátos alkalmazkodási zavarnak te
kinthető, melynek fő tünetei: szédülés, alvászavarok,
izomfeszülés, étvágycsökkenés, hányinger és hasi disz
komfort. Nagyon fontos ezeknek a tüneteknek a pontos
felmérése annak érdekében, hogy ezt az állapotot el tud
juk különíteni a jól definiálható mentális zavaroktól és a
populációra általánosságban jellemző és a helyzetnek
megfelelő, de funkcióromlást nem okozó adekvát stressz
reakciótól [14]. A kínai felmérések már a járvány kezdetén kimutatták,
hogy a populáció közel felénél alakultak ki a járványhely
zet okozta pszichológiai distressz tünetei, míg közel egy
harmaduknál közepes vagy súlyos szorongásos, közel
egyhatoduknál pedig közepes vagy súlyos depressziós
tüneteket észleltek [27]. Más vizsgálatok pedig még ma
gasabb arányokat találtak [28]. A pandémia európai el
terjedését követően hasonló arányszámokról számoltak
be olasz, spanyol és cseh vizsgálatok is: a populáció közel
harmada súlyos stressztünetektől szenvedett, és jelentős
növekedést észleltek a szorongásos-depressziós tünetek
és a poszttraumás stressz zavar gyakoriságában is [3, 29,
30]. Az Egyesült Államokban tavasszal végzett reprezen
tatív felmérések is a pszichológiai distressz és a szorongá
sos-depressziós tünetek növekvő gyakoriságát igazolták A járványhelyzet közvetlen pszichoszociális következ
ményeként nagyon gyakori lehet az akut stresszreakció,
illetve a krízishelyzet következtében kialakuló szorongás,
lehangoltság, kilátástalanság és reménytelenségérzés, az 2021 ■ 162. évfolyam, 10. szám 368 ORVOSI HETILAP Unauthenticated | Downloaded 10/24/24 05:16 AM UTC Unauthenticated | Downloaded 10/24/24 05:16 AM UTC ÖSSZEFOGLALÓ KÖZLEMÉNY [31], egyenes arányban a vírusfertőzés terjedésével [32]. A szintén még a járvány tavaszi időszakában végzett
észak-amerikai populációs felmérés a depressziós tünetek
több mint háromszoros emelkedését mutatta a járványt
megelőző időszakhoz képest (27,8% vs. 8,5%) [33]. Egy
júliusi vizsgálat még magasabb arányszámokat mutatott
az Egyesült Államokban, nemcsak a mentális zavarok, de
a szerhasználat és a suicid gondolatok vonatkozásában is. Ezen eredmények szerint a depresszió négyszer, a szo
rongás háromszor volt gyakoribb, mint a megelőző év
ben, a suicid gondolatok aránya pedig a kétszeresére nőtt
[34]. Kiemelésre érdemes, hogy a depressziós tünettan
különösen az alacsony jövedelmű, hátrányos helyzetű,
aktuális veszteséget (például állásvesztést) elszenvedett
embereket érintette [28, 33]. A pszichológiai distressz
hátterében a leggyakrabban a vírusfertőzés miatti aggo
dalom (65,9%), a munkahely elvesztésétől való félelem
(65,1%) és az anyagi gondok (60,6%) szerepeltek. A pandémia általános mentálhigiénés
hatásai Az idős populáció esetében különösen fontosnak tűnik
olyan suicidogen tényezők szerepe, mint amilyen az ös�
szetartozás hiánya és annak érzése, hogy terhet jelente
nek hozzátartozóik és a tágabb közösség számára [40]. Érdekes módon az idősek egy része sokkal jobban viseli
a korlátozásokat, és kevésbé jelentkeznek náluk a pszi
chológiai distressz következményei [36]. Ez főleg azok
ra volt jellemző, akik olyan tulajdonságokkal bírtak, mint
a fejlettebb érzelemszabályozás és megküzdőmechaniz
musok, a resilientia, a bölcsesség, az empátia és az
együttérzés, valamint lakóotthonban éltek, így kevésbé
hatott rájuk a szociális izoláció. A hatékony stresszoldást
biztosító faktorok ismerete kiemelt fontossággal bír az
idősek számára nyújtott pszichológiai segítségnyújtás
megtervezése szempontjából [41]. A kognitív hanyatlás
sal küzdő idősek speciális rizikócsoportot jelentenek [5]. A kedvezőtlen hatások által különösen
veszélyeztetett csoportok A fenti tényezők mellett ki kell emelni a
napi ritmus felborulását, az alvásminőség romlását és az
alulinformáltságból eredő információéhséget is, mely
gyakran vezet hírfüggőséghez. A fentiek arra hívják fel a
figyelmet, hogy nagyon pontosan meg kell határozni a
karanténhelyzet pontos kritériumait és ellátási protokoll
ját, annak érdekében, hogy a korlátozások csak a leg
szükségesebb időtartamban kerüljenek alkalmazásra, és
az egyének a számukra leginkább megfelelő támogatást
kapják [13]. Nagyon fontos szerephez jut a családi és
közösségi támogatás is [47]. Fontos hangsúlyozni a ka
ranténban lévők altruista magatartását, hiszen ők jelen
tős áldozatot hoznak annak érdekében, hogy a közösség
más tagjai egészségesek maradhassanak [48]. A kutatók
arra is felhívják a figyelmet, hogy a karantén feloldása
sem oldja meg az összes problémát, mivel gyakran ta
pasztalható a karantén feloldását követő („visszatérési”)
szorongásos reakció, mely megnehezítheti a hétköznapi
életbe való visszailleszkedést [49]. j
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Az idősek mellett a fiatalok életét is alapvetően meg
változtatták a korlátozások, hiszen az online oktatásra
való áttérés nemcsak nehezíti a tanulást, de fokozhatja a
képzési előmenetellel kapcsolatos aggodalmakat [13,
38], ráadásul jelentősen beszűkíti a kortárs kapcsolatokat
is, ami csökkenti a szociális támogatás esélyét [28]. Ezt
egy francia felmérés eredményei is igazolták, melyek sze
rint a karanténban lévő egyetemi hallgatók jelentős
arányban számoltak be súlyos pszichológiai distresszről,
szorongásos-depressziós tünetekről és suicid intenciók
ról [43]. Ennek hátterében a hangulati homeosztázis ön
szabályozásának zavarát igazolták, mely különösen kife
jezett volt a pszichiátriai betegségben szenvedők
esetében. Ennek alapján körvonalazódott, hogy az érzel
mi szabályozást javító módszerek elsajátítása fontos sze
reppel bír a mentálhigiénés prevencióban [44]. pp
g
p
Az egyik legmagasabb suicidiumrizikóval bíró csopor
tot a vírusfertőzés szövődményeitől szenvedőket ellátó
egészségügyi személyzet képezi, hiszen a súlyos vagy akár
életveszélyes állapotban lévő fertőzöttek ellátása koráb
ban szinte elképzelhetetlen fizikai és érzelmi megterhe
lést jelent [5, 13, 19, 20, 45]. Ráadásul sokszor nem
állnak rendelkezésükre még a szükséges krízisoldást és
tehermentesítést biztosító mentálhigiénés támogató
szolgálatok sem. A segítségnyújtásra pedig óriási szükség
van, hiszen az egészségügyi dolgozók között különösen
magas a szorongásos-depressziós tünetek és a kiégés elő
fordulása. Egy széles körű metaanalízis adatai szerint a
fertőzöttek ellátásában közreműködő egészségügyi dol
gozók közel negyede szenved szorongásos vagy depres�
sziós tünetektől, közel 40%-uknál pedig alvászavar
jelentkezett. A negatív pszichés következmények szem
pontjából a nővérek számítanak a legveszélyeztetettebb
nek [46]. A kedvezőtlen hatások által különösen
veszélyeztetett csoportok Nagyon fontos hangsúlyozni, hogy a pandémia negatív
hatásai különböző mértékben hatnak a populáció tagjai
ra. Az eddigi kutatások alapján számos olyan rizikócso
port azonosítható, akiknél a pandémia közvetlen és köz
vetett következményei különösen negatívan befolyá
solhatják a mentális egészséget, és növelhetik a suicid
viselkedés gyakoriságát [22, 24]. A megelőzés hatékony
ságának növeléséért nagyon fontos ezeknek a rizikócso
portoknak az azonosítása és a válság hatásának ponto
sabb vizsgálata, annak érdekében, hogy célzott és az
egyes rizikócsoportok sajátosságaihoz illesztett prevenci
ós és terápiás beavatkozásokat tudjunk kialakítani [22]. A veszélyeztetett csoportok között elsőként a mentális
zavarban szenvedőket kell megemlíteni [13], hiszen őket
különösen kedvezőtlenül érintik a korlátozások, a szoci
ális izoláció, valamint a gazdasági és a munkaerőpiaci
helyzet romlása [20, 35, 36]. Az izoláció és a szociális
visszahúzódás – az önpusztításra hajlamosító tüneteket
(például szorongás, depresszió vagy alvászavar) provoká
ló direkt hatás mellett [32, 37] – az alapbetegség relap
susán keresztül indirekt módon is növelheti az öngyil
kossági rizikót [22]. A mentális zavarokban szenvedők A veszélyeztetett csoportok között elsőként a mentális
zavarban szenvedőket kell megemlíteni [13], hiszen őket
különösen kedvezőtlenül érintik a korlátozások, a szoci
ális izoláció, valamint a gazdasági és a munkaerőpiaci
helyzet romlása [20, 35, 36]. Az izoláció és a szociális
visszahúzódás – az önpusztításra hajlamosító tüneteket
(például szorongás, depresszió vagy alvászavar) provoká
ló direkt hatás mellett [32, 37] – az alapbetegség relap
susán keresztül indirekt módon is növelheti az öngyil
kossági rizikót [22]. A mentális zavarokban szenvedők 2021 ■ 162. évfolyam, 10. szám 369 ORVOSI HETILAP Unauthenticated | Downloaded 10/24/24 05:16 AM UTC Unauthenticated | Downloaded 10/24/24 05:16 AM UTC ÖSSZEFOGLALÓ KÖZLEMÉNY Körükben a járványhoz kapcsolódóan a dementiához
társuló tünetek (szorongás, depresszió, alvászavar, irrita
bilitás és más viselkedészavarok) romlását észlelték, ami
jelentősen növelte a gondozók terheit [42]. azt is, hogy az érintettek megfelelő segítséget kapjanak. Egyre több adat bizonyítja, hogy a karanténban való tar
tózkodás vírusfertőzés nélkül is tovább súlyosbíthatja a
negatív pszichés következményeket, melyek akár hos�
szabb ideig fennállhatnak [25]. Ezek között a szorongás
érzelmi és vegetatív tüneteinek fokozódásáról, a poszttra
umás stressz tüneteiről, zavartságról és indulatkitörésről
egyaránt beszámoltak [47]. Ennek hátterében a karan
ténban való tartózkodás elhúzódása, a megfertőződéstől
való félelem, az alulinformáltság, a megfelelő érzelmi és
szociális támogatás hiánya, a kényszerű bezártság követ
keztében kialakuló unalom és frusztráció, az anyagi-eg
zisztenciális problémák felerősödése és az esetleges fer
tőzéssel kapcsolatos stigmatizáció egyaránt szerepet
játszhat [22]. 2021 ■ 162. évfolyam, 10. szám A kedvezőtlen hatások által különösen
veszélyeztetett csoportok Gyakran tapasztalható az ellátásban részt vevő
egészségügyi dolgozókkal kapcsolatban a stigmatizáció
[12] és az ebből fakadó elutasító magatartás a környeze
tük részéről, ami szintén megnehezíti, hogy megfelelő
érzelmi támogatást kaphassanak [5]. Pedig az egészség
ügyi személyzet problémáinak gyors megoldása különö
sen fontos, hiszen munkavégzésük hatékonyságának
megőrzése vagy helyreállítása alapvető jelentőségű a pá
ciensek kezelésében és a járvány következményeinek le
küzdésében. A családok kényszerű bezártsága nemcsak a meg
szokott életritmus megváltozását és a családi szerepek
felbomlását okozhatja, de felerősítheti a családi konflik
tusokat, valamint a családon belüli erőszak és a gyermek
bántalmazás előfordulásának gyakoriságát is növelheti
[5, 13, 20]. Ezen tényezők összességében jelentős mér
tékben hozzájárulhatnak a traumatizált családtagok akut
vagy hosszú távú suicid veszélyeztetettségének növeke
déséhez [22]. A család izolált helyzete tehát a diszfunk
cionális folyamatok felerősödését idézheti elő, miközben
ilyenkor a külső segítség igénybevételének lehetősége is
jelentősen beszűkül. j
A pandémia közvetett negatív hatásai között kell meg
említeni a gazdasági válságot, mely az előrejelzések sze
rint akár súlyosabb lehet, mint a 2008. évi krízis. Bár a
negatív gazdasági következmények a népesség széles kö
rét érintik, ezek a folyamatok különösen kedvezőtlenül
hatnak a hátrányos helyzetű, eleve rossz szociális körülmé
nyek között élőkre [12]. A gazdasági-financiális válság a
legtöbbször számos olyan kedvezőtlen hatással jár (elbo
csátások, munkanélküliség, anyagi és egzisztenciális
problémák stb.), melyek az öngyilkossági magatartás ri
zikófaktorait jelentik, így a pandémiát követően a gazda
sági helyzet elhúzódó romlásával összefüggésben a men Kiemelésre érdemes a tünetmentes fertőzöttek és a fer
tőzöttekkel kapcsolatban álló kontaktszemélyek – egyre
növekvő számú – populációja is, hiszen őket érinti a leg
jobban a megbetegedéstől való állandó rettegés és a tü
netekkel való túlzott foglalkozás, ami komoly rizikót je
lent a mentális betegségek kialakulása szempontjából. Különösen veszélyeztetettek azok, akik a vírusfertőzés
következtében elvesztették hozzátartozójukat [2, 13]. Állapotukat tovább ronthatják a fertőzésveszély miatt al
kalmazott korlátozások, melyek jelentősen megnehezítik 370 ORVOSI HETILAP Unauthenticated | Downloaded 10/24/24 05:16 AM UTC Unauthenticated | Downloaded 10/24/24 05:16 AM UTC ÖSSZEFOGLALÓ KÖZLEMÉNY tális betegségek és a suicid viselkedés gyakoribbá válása
prognosztizálható [19]. fejlesztése, valamint a financiális problémák kezelését és a
munkanélküliség csökkentését célzó munkapiaci progra
mok kidolgozása [12, 20–24, 34]. Mivel a pandémia
mentálhigiénés következményeinek elhúzódó jelentke
zése várható, olyan, nemzeti öngyilkosság-megelőző
stratégia kidolgozása látszik szükségesnek, melyben az
általános suicidiumprevenciós alapelvek mellett a regio
nális jellemzők is szerepet kapnak [16, 20, 22]. A kedvezőtlen hatások által különösen
veszélyeztetett csoportok Az ön
gyilkosságokkal foglalkozó jövőbeli kutatások fontos te
rületét képezi a prevenciós és terápiás módszerek alkal
mazhatóságának folyamatos vizsgálata ezen módszerek
eredményességének növelésére [2, 22, 25]. Napjainkban a pandémiával kapcsolatos információs
robbanásnak lehetünk szem- és fültanúi. A WHO által
találóan infodémiának nevezett információáramlásban a
médiumok legkülönbözőbb formái – a hírportáloktól
kezdve a közösségi médiáig – ontják a híreket. Az adek
vát és hasznos információkon túl gyakoriak az álhírek és
a rémhírek, sok esetben jellemző a félelemkeltő és a
szenzációhajhász stílus. Mivel a járvány okozta fenyege
tettség következtében jelentősen megnőtt a hírolvasás
gyakorisága, az ellenőrizetlen és kaotikus információ
áradat tovább növeli a szorongást, és ez a distressz és a
médiahasználat fokozódásának egymást erősítő ördögi
körét képezi. Közismert, hogy a járvány negatív követ
kezményeit túlhangsúlyozó, szenzációkeltő, stigmatizá
ló és katasztrofizáló médiaközlések nemcsak fokozzák az
aggodalmakat és a szorongást, de akár suicidiumot is
provokálhatnak [22]. A pandémiához kapcsolódó öngyilkos viselkedés sike
res megelőzésében is a komplex módszerek alkalmazása
szükséges, az univerzális, a szelektív és a célzott megkö
zelítésnek egyaránt szerepet kell kapnia [2, 20, 22, 25]. szükséges, az univerzális, a szelektív és a célzott megkö
zelítésnek egyaránt szerepet kell kapnia [2, 20, 22, 25]. Az univerzális megközelítésben nagy jelentőségük van
az általános mentálhigiénés és egészségprevenciós mód
szereknek. Idetartoznak például a szorongás és a félelem
csökkentésére alkalmazott hatékony és egyszerű stressz
oldó technikák, melyeknek természetesen indirekt suici
diumprevenciós hatásuk is van [22]. Nagy szerepük van
a hagyományos és a szociális média által közvetített
egészségpromóciós alapelveknek is [12, 25]. Az öngyil
kosság-megelőzés szempontjából nagy jelentőséggel bír
az öngyilkossággal kapcsolatos médiaközléseket szabá
lyozó irányelvek alkalmazása [12, 16, 19, 20, 22, 24]. Így egyrészt megelőzhető a suicid modellkövetés negatív
hatása, másrészt a segítségnyújtás lehetőségeiről való tá
jékoztatás révén a suicid krízisben lévők időben megfele
lő ellátást kaphatnak [50]. A járvány következményeivel
és kezelésének lehetőségeivel foglalkozó, adekvát és hite
les információközlés mellett fontos a beszűkült és meg
változott életkörülményekhez társuló problémák meg
oldási lehetőségeinek bemutatása is. Ez elősegíti a
pszichológiai distressz oldását, mely a saját élet feletti
kontroll elvesztéséhez és a szociális izolációhoz társul. A korlátozások következményeinek enyhítését célzó
életmódbeli változások bemutatása is elengedhetetlen. Ennek elemei közé tartozik a meghatározott napirend
kialakítása, a túlzott hírolvasás vagy hírfüggőség kerülé
se, a rendszeres fizikai aktivitás és a szabadtéri tevékeny
ségek, valamint az egyénre szabott stresszkezelő mód
szerek alkalmazása is [25]. Általános és célzott prevenciós lehetőségek Egy ilyen világméretű válsághelyzetben sokszor megfi
gyelhető, hogy a közösség tagjai összefognak a bajban, a
szociális kohézió erősödése pedig protektív szereppel
bírhat a negatív pszichológiai következmények enyhítése
szempontjából [22, 25]. Sokat segíthetnek a modern in
fokommunikációs technikák, melyek a különböző virtu
ális platformok, online kapcsolati formák és közösségi
hálózatok révén járulnak hozzá a szociális kapcsolatok
fenntartásához akkor is, amikor a fertőzésveszélyt jelentő
találkozásokra nincs lehetőség [1, 19, 20]. A különböző
mentálhigiénés prevenciós módszerek (mint például az
adekvát stresszoldás és a hatékonyabb érzelemkezelés)
tudatos alkalmazásával [47] még a karantén jelentette
kényszerű bezártságnak is lehetnek előnyei. Kiszakadva a
hétköznapok taposómalmából, a család tagjainak több
ideje lehet egymásra és saját magukra is. A látszólag be
szűkült életkörülmények között egészségesebb és stressz
mentesebb életmód alakítható ki. A napi hajsza helyett
jóval több lehetőség nyílik aktív kikapcsolódásra, pihen
tetőbb alvásra vagy akár meditációra és elmélkedésre, in
tenzívebb fizikális aktivitásra és egészségesebb táplálko
zásra. A passzív (jóga, meditáció) és aktív (éneklés, online
kommunikáció, testmozgás) módszerek egyaránt haté
konynak bizonyultak a szorongásos-depressziós tünetek
megelőzésében [28, 39]. Nemcsak a járvány idején, de az akut időszak lezajlását
követően is kiemelt jelentőségű a mentálhigiénés problé
mák folyamatos szűrése és a rizikófaktorok pontos érté
kelése, hiszen csak így nyílik lehetőség a veszélyeztető
tünetek korai felismerésére és kezelésére [22, 25]. Fon
tos szerephez jutnak a különböző közösségi, szociális és
mentálhigiénés szervezetek, melyek azok számára is biz
tosíthatják a megfelelő érzelmi támogatást, akik egyedül
élnek, és nem számíthatnak a családjukra vagy a környe
zetükben élőkre. Kiemelt jelentőséggel bírnak a minden
ki számára elérhető, azonnali segítséget nyújtó öngyil
kosság-megelőző telefonszolgálatok is [12]. A kliensekkel
közvetlen kapcsolatban lévő segítőknek (szociális mun Szerencsére ma már számos, evidenciákon alapuló
népegészségügyi módszer ismert, melyeknek a jelenlegi
helyzetre való adaptációja esélyt nyújt a járvány követ
keztében kialakuló mentális problémák enyhítésére és a
suicid viselkedés megelőzésére [22]. Elsőként az általá
nos prevenciós beavatkozásokat kell említeni. Idetarto
zik a mentálhigiénés, pszichiátriai és addiktológiai ellátó
rendszerek elérhetőségének és hatékonyságának növelé
se, a szociális és egzisztenciális segítséget nyújtó közössé
gi szervezetek és a krízisintervenciós (telefon)szolgálatok 371 2021 ■ 162. évfolyam, 10. szám ORVOSI HETILAP ORVOSI HETILAP Unauthenticated | Downloaded 10/24/24 05:16 AM UTC ÖSSZEFOGLALÓ KÖZLEMÉNY kások, házigondozók és ápolók, háziorvosok stb.) kivé
teles lehetőségük nyílik arra, hogy a kapcsolattartás során
folyamatosan követni tudják klienseik lelkiállapotát [39]. A számukra szervezett speciális képzések növelik annak
esélyét, hogy felismerjék a suicid veszély figyelmeztető
előjeleit [12]. Általános és célzott prevenciós lehetőségek A
mentális zavarban szenvedők esetében fontos az ellátás
folyamatosságának biztosítása és a páciens igényeihez il
lesztett módszerek alkalmazása. Hangsúlyozzuk, hogy a
többi veszélyeztetett csoport (egészségügyi dolgozók,
idősek és fiatalok, hátrányos szocioökonómiai helyzet
ben lévők stb.) esetében is fontos suicidiumprevenciós
jelentősége van a mentálhigiénés problémák minél ko
rábbi felismerésének és ezek hatékony kezelésének [40]. A különösen magas suicid rizikóval rendelkezők cso
portja (mint például a suicid kísérletet elkövetők, a lélek
tani krízisben lévők) esetében elengedhetetlen a célzott
beavatkozás, a krízisintervenciós technikák azonnali
alkalmazása és a háttérben lévő mentális zavarok felisme
rése és kezelése, szükség esetén a páciens gondozásba
vétele [22, 25]. Figyelmet kell fordítani a tragikus ese
ményeket átéltek poszttraumás tüneteinek, valamint a
fertőzésben elhunytak gyászoló hozzátartozóinak haté
kony kezelésére. A mentális zavarok kezelésében napja
inkban már egyre inkább szerepet kapnak a modern te
lepszichiátriai és telepszichoterápiás lehetőségek is [39,
40], melyek a személyes kontaktushoz hasonló haté
konysággal működnek, így a járvány okozta korlátozások
esetén is eredményesen alkalmazhatók [19]. rábbi felismerésének és ezek hatékony kezelésének [40]. A különösen magas suicid rizikóval rendelkezők cso
portja (mint például a suicid kísérletet elkövetők, a lélek
tani krízisben lévők) esetében elengedhetetlen a célzott
beavatkozás, a krízisintervenciós technikák azonnali
alkalmazása és a háttérben lévő mentális zavarok felisme
rése és kezelése, szükség esetén a páciens gondozásba
vétele [22, 25]. Figyelmet kell fordítani a tragikus ese
ményeket átéltek poszttraumás tüneteinek, valamint a
fertőzésben elhunytak gyászoló hozzátartozóinak haté
kony kezelésére. A mentális zavarok kezelésében napja
inkban már egyre inkább szerepet kapnak a modern te
lepszichiátriai és telepszichoterápiás lehetőségek is [39,
40], melyek a személyes kontaktushoz hasonló haté
konysággal működnek, így a járvány okozta korlátozások
esetén is eredményesen alkalmazhatók [19]. Anyagi támogatás: A közlemény megírása anyagi támo
gatásban nem részesült. A szerző a cikk végleges változatát elolvasta és jóvá
hagyta. Érdekeltségek: A szerzőnek nincsenek érdekeltségei. Érdekeltségek: A szerzőnek nincsenek érdekeltségei. Érdekeltségek: A szerzőnek nincsenek érdekeltségei. A suicid viselkedés multifaktoriális háttérrel bír, mely
számos intrapszichés, interperszonális és környezeti-kul
turális tényező bonyolult összjátékának következtében
alakul ki. A pandémiához és elhúzódó hatásaihoz kap
csolódó suicid rizikó hátterében is számos neurobiológi
ai, pszichológiai, szociális és gazdasági-társadalmi ténye
ző szerepe igazolódott, melyek egymást erősítő hatásuk
révén a suicid ráták növekedését okozhatják [2, 22]. Az
egészségügyi és közösségi mentálhigiénés és segítő szol
gálatoknak minden lehetőséget meg kell ragadniuk a
pandémia okozta válsághelyzet negatív következményei
nek mérséklésére és a pozitív hatások facilitálására [37]. A suicid viselkedés megelőzésében azonban ezen ellátó
rendszerek mellett a döntéshozó politikai-kormányzati
szerveknek, a közösségi támogató szervezeteknek és a
média munkatársainak is fontos részt kell vállalniuk [22]. Az egészségügy túlterhelése miatt kiemelt jelentőséggel
bírnak a különböző, közösségi alapú suicidiumprevenci
ós módszerek is. A rövid, strukturált, akár személyesen, Általános és célzott prevenciós lehetőségek Így a súlyosbodó depresszió, szorongás és
más pszichés tünetek, krízisjegyek vagy akár suicid gon
dolatok észlelése esetén gondoskodni tudnak arról, hogy
a pszichés tünetekkel küzdő vagy krízisben lévő páciens
a lehető legszakszerűbb ellátásban részesüljön [22]. akár online is alkalmazható módszerek segítségével a kö
zösség tagjaival kapcsolatban álló háziorvosok, védőnők,
mentálhigiénikusok vagy szociális munkások is könnyeb
ben tudják azonosítani a suicid veszélyben lévőket, és
hatékonyan tudják alkalmazni az életmentő krízisinter
venciós technikákat [22]. kások, házigondozók és ápolók, háziorvosok stb.) kivé
teles lehetőségük nyílik arra, hogy a kapcsolattartás során
folyamatosan követni tudják klienseik lelkiállapotát [39]. A számukra szervezett speciális képzések növelik annak
esélyét, hogy felismerjék a suicid veszély figyelmeztető
előjeleit [12]. Így a súlyosbodó depresszió, szorongás és
más pszichés tünetek, krízisjegyek vagy akár suicid gon
dolatok észlelése esetén gondoskodni tudnak arról, hogy
a pszichés tünetekkel küzdő vagy krízisben lévő páciens
a lehető legszakszerűbb ellátásban részesüljön [22]. [
]
A vizsgálatok arra utalnak, hogy a pandémia következ
tében kialakuló krónikus pszichológiai distressz, a men
tális zavarok és a suicid viselkedés egyre fontosabb
közegészségügyi problémát jelentenek. Bár jelenleg a
vírusfertőzöttek gyógyítása és a járvány akut következ
ményeinek mérséklése áll az egészségügyi ellátás közép
pontjában, mindannyiunknak fel kell készülnünk a hos�
szú távú következményekre is. A pandémia várható
negatív mentálhigiénés hatásainak megelőzésére és eny
hítésére általános és specifikus, lokális szinten is alkal
mazható hatékony stratégiák kidolgozása és alkalmazása
szükséges [22, 24]. Az egyéni, közösségi és társadalmi
szinten alkalmazott prevenciós módszerek segítségével
megelőzhető a súlyosabb mentálhigiénés problémák jár
ványszerű terjedésének (a „pszichodémiának”) a kialaku
lása. Ezek korai felismerésével és adekvát kezelésével
nemcsak pácienseink gyötrelmeit tudjuk enyhíteni, de a
sokszor életveszélyes szövődmények – mint például a
suicid viselkedésformák – is megelőzhetővé válnak. A
krízisparadigma értelmében a válsághelyzet eredményes
megoldása nemcsak a pandémia előtti helyzetbe való
visszatérést eredményezi, de esélyt nyújt az egyének és a
közösségek fejlődésére is. g
j
Az emelkedett suicid rizikóval bíró csoportok esetében
a szelektív módszerek alkalmazása indokolt [22, 25]. A
mentális zavarban szenvedők esetében fontos az ellátás
folyamatosságának biztosítása és a páciens igényeihez il
lesztett módszerek alkalmazása. Hangsúlyozzuk, hogy a
többi veszélyeztetett csoport (egészségügyi dolgozók,
idősek és fiatalok, hátrányos szocioökonómiai helyzet
ben lévők stb.) esetében is fontos suicidiumprevenciós
jelentősége van a mentálhigiénés problémák minél ko
rábbi felismerésének és ezek hatékony kezelésének [40]. Az emelkedett suicid rizikóval bíró csoportok esetében
a szelektív módszerek alkalmazása indokolt [22, 25]. Irodalom [1] Galea S, Merchant RM, Lurie N. The mental health consequenc
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Pécs, Rét u. 2., 7623
e-mail: osvath.peter@pte.hu) „Lupus pilum mutat, non mentem.”
(A farkas csak a szőrét váltja, nem a gonosz szándékát.) 2021 ■ 162. évfolyam, 10. szám 374 ORVOSI HETILAP Unauthenticated | Downloaded 10/24/24 05:16 AM UTC
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Two Lives: The Immigrant Odyssey of Vikram Seth
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International Journal For Multidisciplinary Research
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Dr Sangeeta Dr Sangeeta Independent Scholar, Lucknow Abstract: Plunge into the riveting narrative of Vikram Seth’s literary masterpiece, Two Lives an evocative expedition
through the labyrinth of the immigrant odyssey. This scholarly inquiry meticulously dissects the novel’s
core themes, navigating the intricacies of cultural assimilation, the dynamic interplay of dual identity, and
the formidable challenges inherent in carving a new existence in uncharted realms. With consummate
artistry, Seth crafts a vibrant tableau of the immigrant experience, seamlessly interweaving personal
anecdotes with a rich historical tapestry. This examination affords a tantalizing glimpse into the profound
and universally resonant revelations encapsulated in Seth’s magnum opus, Two Lives extending readers
an entrancing voyage through the convoluted landscapes of immigration and self-discovery. Keywords: Two Lives, immigration, cultural assimilation, identity, self-discovery. Embarking on the exploration of immigrant literature is akin to peering through a profound and
resonant lens that captures the intricate tapestry of human migration. This literary realm, diverse in genres
and cultures, unveils a distinctive journey of challenges, triumphs, and a myriad of experiences woven
into the fabric of immigration. From Jhumpa Lahiri’s exploration of Indian-American identity in The
Namesake to Chimamanda Ngozi Adichie’s vivid portrayal of Nigerian immigrants in Americanah these
narratives offer a rich tapestry of insights. Immigrant literature, through its rich narratives and nuanced
storytelling, provides a window into the profound dynamics of cultural assimilation, identity negotiation,
and the universal pursuit of belonging in unfamiliar territories. Join us on an immersive journey through
the pages of works like Edwidge Danticat’s Brother, I’m Dying and Isabel Allende’s The House of the
Spirits where the shared human experience effortlessly transcends borders, fostering empathy and
understanding across the diverse landscapes of global literature. Vikram Seth, a luminary in contemporary literature, is renowned for his significant contributions
as a novelist. Born in Kolkata in 1952, Seth’s literary prowess spans diverse genres, earning particular
acclaim for his magnum opus, A Suitable Boy. This monumental work, set against the backdrop of post-
colonial India, showcases Seth’s mastery in crafting intricate narratives that delve into the complexities of
relationships, societal dynamics, and the human experience. With a distinctive storytelling style marked
by eloquence and depth, Vikram Seth has indelibly shaped the literary landscape, garnering critical
acclaim and a devoted readership worldwide. In his literary opus, Seth skilfully intertwines the narratives of the Indian and Jewish communities
spanning two millennia, echoing themes of survival, hope, compassion, and devotion. ational Journal for Multidisciplinary Research (IJFMR) challenges posed by colonial dominance, exile, migration, and the intricate dynamics of dual migrations,
Seth, as a diasporic writer, navigates the fractures arising from conflicting affiliations. Unlike conventional
postcolonial narratives, Seth’s work, particularly exemplified in his magnum opus Two Lives transcends
expected trajectories. This literary masterpiece unfolds as a unique fusion of biography, memory,
autobiography, documentary, history, fiction, and essay-like explorations, resisting confinement to themes
of cultural resistance and instead embracing a narrative seeking global reconciliation. Set against the backdrop of the Second World War, Two Lives serves as a poignant reminder of
the war’s harrowing realities and trauma. Departing from expected norms in Indian writing in English,
Seth’s work defies categorization, emerging as a cosmopolitan tale narrated through innovative
approaches and narrative techniques. This transcendent work extends beyond regional labels, offering a
compelling narrative that breaks cultural boundaries, encapsulating Vikram Seth’s autobiographical
journey in a truly unique and globally resonant manner. The novel unfolds through four distinct segments, initiating with the author’s relational threads
with central characters. From initial encounters at ages two and a half, nine, and seventeen to the author’s
habitation with them during his educational stint in Tonbridge, the narrative weaves a poignant tale. Despite scholarly diversions, the narrator consistently gravitates back to London in 1989, assuming a
poignant presence at his aunt’s final moments. The second segment delves into Shanti uncle’s academic
odyssey in 1930s Berlin, his exile, requalification in Edinburgh, and resettlement in London, set against
the backdrop of his dedicated service in the British army. Part Three explores the clandestine history
encapsulating Helga Gerda Caro and her familial milieu. The narrative arc, rendered feasible by the
discovery of Henny’s treasured documents, unveils a profound sense of depth, perspective, intimacy, and
a psychological connection. The concluding part meticulously chronicles the protracted denouement of
Shanti Uncle’s life. Vikram Seth’s departure from conventional chronological narrative structures in Two Lives is
illuminated through his contemplations on his position as “an anomalous third braid.” He justifies this
deviation by underscoring the impracticality of oscillating between protagonists in their formative two
decades. Seth’s commitment to a more expansive human perspective surpasses ethical reservations,
unveiling his aunt’s private documents for public scrutiny. In 1969, immersed in his uncle and aunt’s abode, Seth perceives their residence as familial mooring
in an uncharted milieu. Abstract: Addressing the Volume 5, Issue 6, November-December 2023 IJFMR23069655 1 International Journal for Multidisciplinary Research E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com Seth’s unease appears rooted in unconscious guilt, triggered by an awareness of the complex Indo-
German connection. Shanti’s narrative unfolds as a confrontation with “the invincible singularity of the
verbal body.” His experience in Berlin is marked by disorientation, propelled more by necessity than
passion, with his exile as a racial outsider in Germany eventually leading to acceptance. Shanti’s student life in Berlin, witnessing Hitler’s rise and the Nazis’ isolation of Jews, is pivotal. His escape to Britain becomes a compelling narrative within the broader tapestry of “Two Lives.” As the
war unfolds, Shanti, having requalified in Edinburgh, enlists and faces the battlefield’s ravages, enduring
the life-altering loss of his right arm. Undeterred, he navigates adaptation, acquiring skills in documentary
filmmaking and becoming a lecturer. Amid uncertainties about marriage and providing for Henny, Shanti, signing letters as “yours most
unfortunate,” transforms vulnerability into resilience. Encouragement from dentist confidant Henry
Edwards propels Shanti to perform dental procedures with his left hand, establishing a distinct practice
and acquiring a house in London. Seth juxtaposes Shanti’s fortitude against the perverse Nazi ideology,
highlighting the chilling reality of extermination. Henny’s role in Shanti’s life is determined by complex dynamics and the unpredictable turns of
history. Her premonitory advice about not taking the black man and her strange insight during her
engagement to Hans Mahnert reveal a complex web of relationships. Henny’s move to England, various
jobs, and adoption of a new English identity, encapsulated in the nickname “the friendly lady,” depict a
resilient example of Jewish and feminist resistance. The union of Shanti, a small, black-skinned, disabled Indian man, and Henny, an impressively tall,
fair, and attractive woman, stands as a triple traumatizing yet resilient example of Jewish and feminist
resistance. In patriarchal societies where marriage often symbolizes banishment, their unconventional
union defies norms and serves as a testament to their enduring love and strength. Against the ominous backdrop of the Third Reich’s fascist ideology, special marriage laws sought
to enforce rigid racial classifications. Within this draconian framework, Hans, classified as a Mischling,
strategically chose to marry Wanda, a Catholic girl, preserving his status. The narrative unfolds with
Henny’s profound distress, stemming from Hans’ poignant choice to renounce his Jewish heritage. ational Journal for Multidisciplinary Research (IJFMR) Confronted with assimilating years’ worth of German language proficiency in six
months, Seth embraces the foreignness of the German language. The distinctive bond shared by Aunt
Henny and Uncle Shanti is palpable, with Shanti endearingly labeling Seth as his “Söhnchen.” As Seth
traverses Germany, Austria, and Switzerland, the robust connections bridging Indian and German cultures
are underscored. Seth’s odyssey entails a profound cultivation of admiration for German literature and music,
forming intimate connections with the cultural tapestry. Despite familial bonds, Seth’s encounter with the
bureaucratic German language kindles a visceral anger, embodying a paradoxical realm for him—
simultaneously endearing and repulsive. Volume 5, Issue 6, November-December 2023 2 ational Journal for Multidisciplinary Research (IJFMR) a primary right and the zero degree of dignity. By deriving solace and purpose from their respective
vocations, Shanti and Henny navigate the intricate challenges of exile, forging a collective sense of
belonging and dignity through their unwavering commitment to their work. a primary right and the zero degree of dignity. By deriving solace and purpose from their respective
vocations, Shanti and Henny navigate the intricate challenges of exile, forging a collective sense of
belonging and dignity through their unwavering commitment to their work. Henny, devoid of tranquillity, consciously selects Shanti, the symbolic harbinger of peace, as her
life partner—a important relation to her earlier. While Hans aligns himself with patriarchal norms and
embraces whiteness, Henny intentionally embraces blackness to honour her mother. Shanti’s physical
handicap provides her with a means to confront “the anxiety of her own vulnerability, her own incapacity,
and the death of her own body or mind” (Kristeva 2003, 33). Their love story, devoid of passionate
intensity, is characterized by Shanti’s endearing terms of affection, such as calling her Hennerle. He turns
into her “kuckuck,” and she calls him Henny’s little and black spot (125). Shanti is resolute in keeping the
delicate balance between the kuckuck of his dreams and the material world, and Henny is adamant about
not taking Shanti’s extended family ties personally. Henny, who is unfamiliar with the customs of Indian
extended families, finds it offensive when strangers enter at any time and will not go to India because she
is afraid of germs, she is afraid of crowds, and she believes that family ties are consuming. He turns into
her “kuckuck,” and she calls him Henny’s little and black spot (125). Shanti is resolute in keeping the
delicate balance between the kuckuck of his dreams and the material world, and Henny is adamant about
not taking Shanti’s extended family ties personally. Henny, who is unfamiliar with the customs of Indian
extended families, finds it offensive when strangers enter at any time and will not go to India because she
is afraid of germs, she is afraid of crowds, and she believes that family ties are consuming. Henny’s correspondence with longstanding friends lays bare her persistent endeavors to trace her
mother and sister. International Journal for Multidisciplinary Research Despite
engaging in polite correspondence with Hans’ father, Henny consciously opts for silence when confronted
with Hans’ attempts to elucidate his decisions and profess enduring love. Shanti continues to correspond with Hans, unaware of the depths of Henny’s muted anguish. This
restrained suffering on Henny’s part functions as a poignant symbolic counterpart to Shanti’s phantom
hand—a constant reminder of the lost arm’s memory that persists, demanding endurance throughout his
daily life and work. Vikram Seth astutely observes that within each other’s company, Shanti and Henny discover “a
home” and a profound “sense of belonging” through their individual professional endeavors. This
conclusion echoes Julia Kristeva’s assertion that work remains a vital necessity for the foreigner, offering IJFMR23069655 Volume 5, Issue 6, November-December 2023 3 International Journal for Multidisciplinary Research (IJFMR)
E ISSN: 2582 2160
●
Website: www ijfmr com
●
Email: editor@ijfmr com E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ij Representation of Jews in Germany in 1935 to Reich Association of Jews in Germany in 1935. Despite
being politically disempowered, this organization unknowingly worked with the Gestapo, sharing and
gathering information that allowed for the mass expulsion, deportation, and murder of its members. It also
worked in tandem with the Jewish Order Service, which was established in 1942. This partnership is a
prime example of the kind of Jewish self-hatred that Theodore Lessing described in the 1930s as a result
of a guilt complex over the bad luck they felt they had caused themselves. Born and raised in Berlin,
Henny finds that the barriers that once divided her old pals have now been replaced by a postwar Berlin
wall as well as other obstacles like distance, restricted transit, currency reforms, and intersectoral passes. In 1979, forty years after her departure, she revisits Germany. However, the biographer cannot confirm
whether she laid eyes on Berlin again. Vikram Seth avoids taking a clear stance on Israel, acknowledging German Jewish contributions
to science and communism. He critiques the partitioning of Palestine, disapproving of Israel’s approach
to Arabs. Seth explores the complex nature of forgiveness and the pragmatic imperative for peace. Shanti's
neutrality is seen in his 1938 passport specifying 'Hindu Aryan.' Seth reflects on Shanti's behavior and the
potential unsettling memories of his Hindu funeral. Drawing on Derrida, Seth discusses 20th-century upheavals as re-enactments of primal violence. Shanti and Henny's ability to embrace each other's uncanniness guides communities in the contemporary
era. "Two Lives" skillfully explores immigrant complexities, resonating with universal themes of love and
resilience. The novel transcends narrative boundaries, offering insights into identity, belonging, and the
quest for understanding. Vikram Seth's masterpiece stands as a testament to storytelling's power,
prompting reflection on the past, present challenges, and the hope for a more inclusive future. Work Cited ational Journal for Multidisciplinary Research (IJFMR) Through the preservation of photographs, the Jewish Bible, and her sister’s prayer book
from her tenure at the Gemeinde (Jewish Community Organization), she conscientiously keeps the flame
of her familial memories alive. Of particular significance are the poignant last postcards sent by her mother
and sister in 1942 through the Red Cross and a mutual friend in 1943, serving as haunting markers of their
tragic fate in Theresienstadt and Birkenau. Even after Henny finds out that her mother passed away in
October 1945 at the age of seventy, the details of her mother’s passing are still unknown. Her search for
Lola becomes more intense; she puts an ad in the Aufbau newspaper in New York for Missing Persons
and contacts several Jewish organizations in Britain. Henny dies of urticaria pigmentosa, a psychosomatic
ailment that serves as the only external sign of her extreme distress and intolerance, while friends recount
the heartbreaking details of her mother’s and sister’s last days and counsel her not to hold out hope for her
sister’s survival. Henny’s pursuit of restitution from the German state for lost property and the detriment to her
career may be construed as a gesture aimed at overcoming the paradoxical challenge of grieving from afar
and within a temporally deferred framework—a dilemma emblematic of the destinal disparities
experienced by migrants. Vikram Seth meticulously explores the notions of fatherland, hometown, and
community. When orchestrating Henny’s emigration, Franz Mahnert conveys in a letter, “The world is
beautiful even outside your fatherland... You will soon find out how wonderfully one can live in another
country, and that there are good people there as well” (109). At first, progressive Jews identified Berlin as
their hometown and Germany as their fatherland. But because of Hitler, their 1933-founded organization
changed its name three times in as many years: from The Reich Representation of German Jews to Reich Volume 5, Issue 6, November-December 2023 4 6. Seth, Vikram. Two Lives. Little, Brown & Co., 2019. 6. Seth, Vikram. Two Lives. Little, Brown & Co., 2019. Work Cited 1. Hron, Madelaine. “‘Perversely through Pain’: Immigrants and Immigrant Suffering.” Translating
Pain: Immigrant Suffering in Literature and Culture, University of Toronto Press, 2009, pp. 3–32. JSTOR, http://www.jstor.org/stable/10.3138/9781442689497.5. 2. Hron, Madelaine. “‘Suffering Matters’: The Translation and Politics of Pain.” Translating Pain:
Immigrant Suffering in Literature and Culture, University of Toronto Press, 2009, pp. 33–62. JSTOR,
http://www.jstor.org/stable/10.3138/9781442689497.6. 3. Kawahatsu, Carrie Natsu. Demystifying Immigrant Youth Representations : A Look at Mainstream and
Immigrant
Perspectives
within
Literature
and
Media,
2021,
https://doi.org/10.32920/ryerson.14662587.v1. 4. Phillips, John. Contested Knowledge: A Guide to Critical Theory. Zed Books, 2000 5. Pourjafari, Fatemeh, and Abdolali Vahidpour. “Migration Literature: A Theoretical Perspective - the
Dawn Journal.” The Dawn Journal, 2014, www.thedawnjournal.in/wp-content/uploads/2013/12/2-
Fatemeh-Pourjafari.pdf. 6. Seth, Vikram. Two Lives. Little, Brown & Co., 2019. IJFMR23069655 Volume 5, Issue 6, November-December 2023 5
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Evaluation of propiconazole operational treatments of oaks for oak wilt control
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public-domain
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Jordan Eggers, Jennifer Juzwik, Shawn Bernick,
and Lori Mordaunt Oak wilt is a significant problem in both urban and
suburban landscapes where trees are of higher value than
those in forested settings. Increasing development of
wooded areas in major urban centers of the Upper Midwest
has led to increases in oak wilt (Juzwik and Schmidt
2000). Various oak wilt control tools and integrated oak
wilt management regimes are used in such settings (Juzwik
et al. 2004). One such tool is the fungicide propiconazole
(PPZL). ABSTRACT.—Oaks commercially treated with
propiconazole on 29 sites in Minnesota in 1998 were
evaluated for efficacy in controlling oak wilt. Root graft
spread occurred in 39 percent of preventively treated red
oaks over 5 years; spread in white oaks occurred only once. Propiconazole generally prevented further disease symptom
development in white oaks. KEY WORDS: Ceratocystis fagacearum, oak wilt,
propiconazole. The first studies involving intravascular injection of oaks
with PPZL for oak wilt control were conducted in 1987 on
live oaks (Quercus virginiana Mill. and Q. fusiformis Small)
in Texas (Appel 2001). A few published studies exist on
effective use of PPZL on oaks in the Northern United States
(Johnson 2001, Nair 1995, Osterbauer and French 1992,
Osterbauer et al. 1994). In the Northern States, current
operational use of PPZL by commercial arborists is based
on observation and experience, as well as on these
published reports. Arborists desire documentation of their
operational treatments to either test or validate their
application practices for PPZL. Oak wilt, caused by the fungus Ceratocystis fagacearum
(Bretz) Hunt, affects oaks (Quercus spp.) in both the red
(Lobatae) and white (Quercus) oak groups. Multiple
species of both groups are common in urban and rural
forested landscapes of the Midwestern United States. Following infection, red oaks wilt within weeks to months
and die in the same or the next summer. In white oaks,
infection causes scattered wilting of crown branches or
major limb or fork dieback in one to several years. Death of
white oaks does not usually occur until several years later. In 1998, evaluation studies of operational treatments of red
and white oaks in the Minneapolis-St. Paul, Minnesota,
metropolitan area began in a cooperative effort of the USDA
Forest Service and a local arboricultural research company. The objectives of the 5-year project were to 1) evaluate
efficacy of operational PPZL treatments of oaks used in an
effort to prevent belowground spread of C. Forest Service
North Central
Research Station
Research Note NC-390
2005 Forest Service
North Central
Research Station
Research Note NC-390
2005 Study Area and Trees The trees monitored in these studies were located on 29
sites in 20 suburbs surrounding the Minneapolis—St. Paul,
Minnesota, area. The trees were commercially treated by
the cooperating arboricultural company for clients
requesting the service. Treated species of the red oak group
were northern red oak (Quercus rubra L.) and northern pin
oak (Q. ellipsoidalis Hill). Treated species of the white oak
group were bur oak and white oak. Data Collection and Tree Condition Assessment Diameter of the tree stem at 1.4 m height (diameter at
breast height – d.b.h.) was recorded for each treated tree at
the time of PPZL injection. In the two preventive studies,
the distance between each treated tree and the nearest oak
with wilt symptoms was also determined and recorded. The crown condition of each tree was assessed at the time
of PPZL treatment in 1998 and once during the summer in
1999 (first year), in 2000 (second year), and in 2003 (fifth
year) after treatment. The arborist firm developed a rating
system, which determined a score for each of the following
categories: characteristics of crown development, trunk
quality, branch structure, twig growth rate, foliage color,
incidence of insect and disease organism damage or
presence, and evidence of visible root problems. The
possible values of the rating system ranged from 5
(indicating best condition) to 0 (poorest condition) for
each category. These individual scores were then added to
produce an overall condition index for each tree, with 35
being the maximum value possible. Extent of crown wilt
present in the tree crown was estimated separately. The
percentage of wilt observed in the crown, as determined
from four different locations around the tree (cardinal
directions), was recorded once each summer during 1999,
2000, and 2003. In cases where landowners removed trees
that died in subsequent years, the year of tree death and
year of removal were recorded for each. Red oaks that
developed > 60 percent wilt in the last year of this study
were considered dead in the data summarization. Study Design Three related studies were defined based on the intended
purpose of the chemical treatment. In the red oak
preventive study, operational treatment was intended to
protect apparently healthy red oaks located within root
grafting distance of recently wilted red oaks from
developing oak wilt symptoms. The intent of operational
treatment in the white oak preventive study was similar,
except the treated trees and the nearby C. fagacearum-
infected trees exhibiting active wilt were bur and/or white
oaks. In the white oak therapeutic study, all white and bur
oaks on a site diagnosed with oak wilt were treated with
PPZL. Diagnosis of oak wilt was based on visual presence
of symptomatic foliage or crown wilting and, in some cases,
laboratory isolation of the pathogen. The extent of wilting
in the crowns of affected oaks ranged from 5 to 50 percent
at the time of PPZL treatment. Treatment All study trees were injected with PPZL at the standard
lower rate stated on the product label (Novartis Crop
Protection, Greensboro, NC): 10 ml product (1.4 g a.i or
.045 oz.) per 2.5 cm (1 in.) diameter of main tree stem at
1.4 m (4 ft) height added to the appropriate amount of
water (1.0 liter water for each 10 ml of PPZL). Established
protocols for macro-infusion were followed (Prosser et al. not dated). At least four crews, each consisting of two
technicians and one crew leader, performed the injections. A total of 14 individuals were assigned to the various crews Jordan Eggers, Jennifer Juzwik, Shawn Bernick,
and Lori Mordaunt fagacearum from
wilting red oak(s) to healthy red oaks; 2) evaluate efficacy
of PPZL operational treatments of bur (Quercus macrocarpa
Michx.) and white oak (Quercus alba L.) for preventive
treatment as in 1; and 3) evaluate efficacy of therapeutic,
operational treatments of bur and white oaks with PPZL. This observational study involved re-visiting treatment sites JORDAN EGGERS and JENNIFER JUZWIK are a biological
technician and research plant pathologist, respectively, with
the North Central Research Station, USDA Forest Service,
St. Paul, MN 55108; SHAWN BERNICK is a scientist with
Rainbow Treecare Scientific Advancements, 2239
Edgewood Ave. S., Minneapolis, MN 55426; and LORI
MORDAUNT is a section head with Rainbow Treecare,
2239 Edgewood Ave. S., Minneapolis, MN 55426. JJ is
corresponding author: to contact, call (651) 649-5114 or
e-mail at jjuzwik@fs.fed.us. during the treatment period. In the spring before treatment,
all crews received training in standard PPZL macro-infusion
techniques. The trees were treated between June 17 and
September 28, 1998, which corresponded to the period
between full leaf expansion (late spring) and onset of fall
coloration. In the therapeutic white oak study in
subsequent years, selective pruning was performed on the
treated trees during winter to remove dead wood from the
tree crowns. three times over 5 years to determine long-term efficacy of
PPZL in controlling oak wilt. No controls were included in
this evaluation study. Red Oak Preventive Study The 46 PPZL-treated red oaks ranged in size from 13 to 89
cm (5 to 35 in.) d.b.h. They were located from 4.6 to 15.2
m (15 to 50 ft) from the nearest C. fagacearum-infected 2 tree. Oak wilt was first observed in the treated trees in
1999 when one (2%) began wilting late in the growing
season and died the following year (table 1). An additional
17 trees (37%) wilted between early September 2000 and
late August 2003. Three of these were observed with active
wilt in 2003. Mortality due to C. fagacearum was higher in
trees < 9 m (30 ft) from previously wilted trees compared
to treated trees between 9 and 15 m (50 ft) from the
nearest pathogen-killed tree (P = 0.08). Of the 18 trees that
died over the 5 years, 13 (72%) were less than 9 m (30 ft)
from the nearest C. fagacearum-killed tree in 1998 when
PPZL was injected. Tree mortality due to C. fagacearum was
also higher (44%, or 8 of 18 trees) in larger diameter trees
(d.b.h. > 48 cm or 19 in.) (P = 0.10). The remaining live
trees showed no change to slight improvement in tree
condition when ratings between 2003 and 1998 were
compared. tree in 1999 and 2000, but no new symptoms were
observed in 2003. The condition index for all of the other
trees did not change appreciably over the 5-year period. White Oak Therapeutic Study The eight bur oaks injected with PPZL ranged in size from
42 to 86 cm (17 to 34 in.) d.b.h. The initial percentages of
tree crowns exhibiting wilt symptoms ranged from 5 to 30
(table 2). During the 5-year period, only one tree exhibited
new oak wilt symptoms (5% increase) and this occurred
only in 2000. The tree was injected with PPZL again in
2000 and dead branches were removed; no evidence of
new wilt was found in 2003. Dead branches were pruned
from tree crowns during winters in subsequent years after
injection. Dead branch pruning of the tree crowns in 1998
resulted in slightly decreased (< 1%) crown size (table 2). Tree condition index over the 5 years differed by tree size. The index slightly decreased for bur oaks < 56 cm (22 in.)
d.b.h., but increased for those > 56 cm. 2 Wilting crown observed late in growing season, but did not result in mortality in 1999. Year trees were treated.
2 Wilting crown observed late in growing season, but did not result in mortality in 1999. 3 White Oak Preventive Study The 26 PPZL-treated white and bur oaks ranged in size
from 14 to 89 cm (6 to 34 in.) d.b.h. They were located
from 0.6 to 13.7 m (2 to 45 ft) from the nearest C. fagacearum-infected white or bur oak. Only one tree (a
white oak) exhibited oak wilt symptoms over the 5-year
period, and none died. A few symptomatic branches
(< 10% of the crown) were observed on the one affected The 13 white oaks (Q. alba) treated with PPZL ranged in
size from 25 to 69 cm (12 to 27 in.) d.b.h. The initial
percentages of tree crowns exhibiting wilt symptoms
ranged from 5 to 45 (table 2). Of eight oaks < 56 cm (22
in.) d.b.h., one exhibited new oak wilt symptoms in 1999,
but none of the trees exhibited symptoms in 2000 and Table 1.—Mortality observed in red oaks operationally treated with propiconazole in 1998 for prevention of root graft spread of
Ceratocystis fagacearum
Distance to
Tree
No. treated
Tree mortality by year
Cumulative mortality (5 yrs)
diseased tree
diameter
trees
19981 1999 2000 2001-2003 Number Percent
(m)
(cm)
≤ 9
≤ 33
11
0
0
0
4
4
36
> 33 to 48.3
5
0
0
0
3
3
60
> 48.3
10
0
02
1
5
6
60
> 9 to 15
<33.02
9
0
0
0
2
2
22
> 33 to 48.3
7
0
0
0
1
1
14
> 48.3
4
0
0
0
2
2
50
Total
46
0
0
1
17
18
39
1Year trees were treated. 2 Wilting crown observed late in growing season, but did not result in mortality in 1999. 2 Wilting crown observed late in growing season, but did not result in mortality in 1999. 3 Table 2.—Incidence and extent of crown wilting in operationally treated bur and white oaks infected with Ceratocystis fagacearum prior to and following propiconazole treatment
Oak species
D.b.h. No. Red Oak Preventive Treatment Comparing these earlier results to our study suggests that
re-treatment with PPZL two growing seasons after initial
treatment may be beneficial. For example, if a tree is
treated in year 1, re-treatment would seem appropriate just
before fall leaf coloration of year 2, or soon after full leaf
expansion in year 3. The observed 100-percent survival rate of red oaks over 2
years and the 61-percent survival rate over the total 5 years
of this study suggest that preventive macro-infusion
treatments with the PPZL product at 1.4 g a.i./ 2.54 cm
d.b.h. (0.045 oz/1 in.) provides limited suppression of oak
wilt spread via root grafts. It appears that the ability of the
chemical treatment to suppress disease progression
decreases over time (years). Many factors likely influence
efficacy of PPZL in preventing root graft spread of the
pathogen. Such factors include distances over which root
grafts can be expected, frequency of root graft transmission,
rapidity with which C. fagacearum moves through
connected root systems, longevity of PPZL activity in trees,
and PPZL application rate. Chemical application rate is an additional factor to
consider. Red oaks injected with 0.42, 0.56, 0.7, and 0.84
g PPZL / 2.54 cm d.b.h. (0.015, 0.020, 0.024, and 0.030
oz / in.) to prevent root graft transmission of the pathogen
had a combined survival rate of 100 percent at < 14
months and 76 percent at 24 months (Johnson 2001). Trees treated with the highest rate (0.84 g PPZL / 2.54 cm
d.b.h.) had a 95-percent survival rate after 2 years. Results
of our study can be compared to those of Johnson (2001). Treatment with PPZL at 0.84 g a.i. / 2.54 cm d.b.h. (Johnson 2001) is as successful over a 2-year period as 1.4
g a.i./ 2.54 cm d.b.h. (0.045 oz /1 in.) (our study), while
lower rates (Johnson 2001) had lower success rate in
preventing wilt 2 years after injection. Perhaps increasing
the application rate may lengthen the time of disease
suppression. Root graft transmission of the oak wilt pathogen to healthy
oaks commonly occurs within 15 m (50 ft) of a diseased
oak of the same species. Heimlick and Fox (1961) reported
pathogen spread and subsequent death in trees up to 15 m
(50 ft) from a disease source. White Oak Preventive Study Comparing these earlier results to our study suggests that
re-treatment with PPZL two growing seasons after initial
treatment may be beneficial. For example, if a tree is
treated in year 1, re-treatment would seem appropriate just
before fall leaf coloration of year 2, or soon after full leaf
expansion in year 3. Red Oak Preventive Treatment In another study in which
trees were injected with radioisotopes, radioisotopes were
detected in trees up to ~15 m (50 ft) from the injected tree,
presumably translocated between trees through root grafts
(Kuntz and Riker 1955). The rate of spread of oak wilt
through root grafts, especially in red oaks, can be as much
as 12 m (40 ft) per year (Bruhn et al. 1991). In our study,
all preventively treated red oaks were less than 15 m (50 ft)
from diseased trees and thus had a high probability of
becoming infected with C. fagacearum within several years
after the death of the adjacent oak wilt-killed tree. White Oak Preventive Study trees
Intitial percent wilt (1998)
Trees exhibiting new crown wilt
Cumulative percent of crown
(cm)
evaluated
Average Range
Year Number Crown wilt
removed by 2003
(percent)
Average
Range
Bur
42-86
8
16
5-30
1999
0
0
16
5-30
2000
1
5
17
5-30
2001-2003
0
0
17
5-30
White
< 56
8
14
5-35
1999
1
5
14
5-35
2000
0
0
14
5-35
2001-2003
0
0
14
5-35
≥ 56
5
21
5-45
1999
1
25
26
5-45
2000
2
5, 5
28
5-50
2001-2003
0
0
28
5-50 Himelick and Fox (1961) found that while the majority of
oak wilt centers remained active year after year, a few other
centers remained dormant for up to 6 years and then re-
activated, i.e., newly wilted trees were observed. With such
a wide time span over which transmission may occur, it is
possible that the pathogen can spread after the PPZL is
degraded or no longer active in a treated tree. Osterbauer
and French (1992) injected trees with PPZL at a rate of
0.43 g a.i./ 2.54 cm d.b.h. (0.014 oz / in.). They were able
to detect the PPZL activity at 1, 2, 8, and 12 months after
injection, but not 20 or 23 months after injection. 2003. Of five trees > 56 cm d.b.h., one tree had new wilt in
1999 and two had new wilt in 2000. Pruning of wilting
and other dead branches from tree crowns resulted in a
slight (2%) reduction in crown size. Tree condition index
over the 5 years differed by tree size. The index slightly
decreased for white oaks < 56 cm (22 in.) d.b.h., but
slightly increased for those >56 cm. Himelick and Fox (1961) found that while the majority of
oak wilt centers remained active year after year, a few other
centers remained dormant for up to 6 years and then re-
activated, i.e., newly wilted trees were observed. With such
a wide time span over which transmission may occur, it is
possible that the pathogen can spread after the PPZL is
degraded or no longer active in a treated tree. Osterbauer
and French (1992) injected trees with PPZL at a rate of
0.43 g a.i./ 2.54 cm d.b.h. (0.014 oz / in.). They were able
to detect the PPZL activity at 1, 2, 8, and 12 months after
injection, but not 20 or 23 months after injection. Bruhn, J.N.; Pickens, J.B. 1991. Bruhn, J.N.; Pickens, J.B. 1991. Probit analysis of oak wilt transmission through root grafts in
red oak stands. Forest Science. 37: 28-44. Osterbauer, N.K.; French, D.W. 1992. Propiconazole as a treatment for oak wilt in Quercus rubra
and Q. ellipsoidalis. Journal of Arboriculture. 18: 221-226. White and Bur Oak Preventive Treatments Preventive treatment of white and bur oaks with PPZL has
been proven to be very effective in protecting these trees
from oak wilt symptom development, perhaps by
preventing pathogen spread of C. fagacearum through root
grafts. Although preventive treatment is effective, the
current viewpoint of arborists is to not treat preventively
due to the observed success of operational therapeutic
treatments. An arborist will usually not treat a white or bur
oak unless the tree is in an area where disease pressure and
aesthetic value are very high and/or the client requests
treatment. The length of time required for transmission of the fungus
from infected to adjacent healthy trees varies. By some
accounts the transmission from an oak wilt-killed tree to a
healthy tree via root grafted roots is rapid due to the loss of
upward vascular movement caused by vascular plugging in
the diseased tree (Menges and Kuntz 1985). However, 5 5 Appel, D.N. 2001. Osterbauer, N.K. 1991. Evaluation of propiconazole as a treatment for oak wilt in two
species of Quercus. St. Paul, MN: University of Minnesota. 41 p. M.S. thesis. The use of Alamo for oak wilt management. In: Ash, Cynthia
L., ed. Shade tree wilt diseases. St. Paul, MN: APS Press:
101-106. Bruhn, J.N.; Pickens, J.B. 1991. Probit analysis of oak wilt transmission through root grafts in
red oak stands. Forest Science. 37: 28-44. Himelick, E.B.; Fox, H.W. 1961. Osterbauer, N.K.; Salisbury, T.; French, D.W. 1994. Propiconazole as a treatment for oak wilt in Quercus alba and
Q. macrocarpa. Journal of Arboriculture. 20: 202. Experimental studies on control of oak wilt disease. Illinois
Agricultural Experiment Station Bulletin. 680: 1-48. Experimental studies on control of oak wilt disease. Illinois Experimental studies on control of oak wilt disease. Illinois
Agricultural Experiment Station Bulletin. 680: 1-48. Agricultural Experiment Station Bulletin. 680: 1-48. Johnson, J. 2001. Effects of propiconazole fungicide on fungal mat formation by
Ceratocystis fagacearum and the long distance spread of oak
wilt from infected black and northern pin oaks. Green Bay, WI:
University of Wisconsin–Green Bay. 111 p. M.S. thesis. White and Bur Oak Therapeutic Treatments Juzwik, J.; Schmidt, T. 2000. Oak wilt and oak decline in the Upper Midwest USA. In:
Oszako, T.; Delatour, C., eds. Recent advances on oak
health in Europe. Warsaw, Poland: Forest Research
Institute: 139-145. PPZL applications provided very good control of oak wilt
in white and bur oaks already infected at time of treatment. This finding supports the commonly recommended
treatment of these species. Although preventive treatment
of white and bur oaks at high risk for oak wilt is valid,
therapeutic treatment of infected white and bur oaks, when
done promptly, arrests disease progression with minimal
crown loss. In a previous study, trees with up to 50-percent
affected crown were successfully treated with PPZL
(Osterbauer 1991, Osterbauer et al. 1994). Juzwik, J.; Cook, S.; Haugen, L.; Elwell, J. 2004. Oak wilt: people and trees, a community approach to
management. Gen. Tech. Rep. NC-238. St. Paul, MN: U.S. Department of Agriculture, Forest Service, North Central
Research Station. CD-Rom version 2004 v 1.3. Kuntz, J.E.; Riker, A.J. 1955. The use of radioactive isotopes to ascertain the role of root
grafting in the translocation of water, nutrients, and disease-
inducing organisms among forest trees. In: Proceedings
International conference on the Peaceful uses of atomic
energy. New York, NY: United Nations. 12: 144-148. Concluding Remarks Results of our studies provide valuable documentation on
the efficacy of PPZL in operational preventive treatment of
red and white oaks and in therapeutic treatment of white
oaks. However, further studies with untreated controls and
randomly assigned treatments are needed to provide
additional efficacy information on preventive treatments
particularly for red oak species. Such studies are currently
underway at the University of Minnesota and at the North
Central Research Station, USDA Forest Service. Menges, E.S.; Kuntz, J.E. 1985. Predictive equations for local spread of oak wilt in southern
Wisconsin. Forest Science. 31: 43-51. Nair, V.M.G. 1995. Chemotherapeutic control of oak wilt by the use of “Alamo”
propiconazole. Alamo Research Symposium, Bloomington,
MN. 28 p. Johnson, J. 2001. Johnson, J. 2001. Effects of propiconazole fungicide on fungal mat formation by
Ceratocystis fagacearum and the long distance spread of oak
wilt from infected black and northern pin oaks. Green Bay, WI:
University of Wisconsin–Green Bay. 111 p. M.S. thesis. Prosser, T.; Zwack, J.; Johnson, B. not dated. Macro-infusion guide. St. Louis Park, MN: Rainbow Treecare
Scientific Advancements. Prosser, T.; Zwack, J.; Johnson, B. not dated. 6 6
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IOP Conference Series: Earth and Environmental Science IOP Conference Series: Earth and Environmental Science PAPER • OPEN ACCESS
Extraction and accuracy assessment of DTMs
derived from remotely sensed and field surveying
approaches in GIS framework
To cite this article: Hossein Mojaddadi Rizeei and Biswajeet Pradhan 2018 IOP Conf. Ser.: Earth
Environ. Sci. 169 012009
View the article online for updates and enhancements
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IGRSM 2018
IOP Publishing
IOP Conf. Series: Earth and Environmental Science 169 (2018) 012009
doi :10.1088/1755-1315/169/1/012009 1234567890 ‘’“”
IGRSM 2018
IOP Publishing
IOP Conf. Series: Earth and Environmental Science 169 (2018) 012009
doi :10.1088/1755-1315/169/1/012009 1234567890 ‘’“”
Conf. Series: Earth and Environmental Science 169 (2018) 012009
doi :10.1088/1755-1315/169/1/0120 1
Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution
of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.
Published under licence by IOP Publishing Ltd Hossein Mojaddadi Rizeei and Biswajeet Pradhan* 1School of Systems, Management and Leadership, Faculty of Engineering and IT,
University of Technology Sydney, CB11.06.217, Building 11, 81 Broadway, Ultimo NSW
2007, Australia. 1School of Systems, Management and Leadership, Faculty of Engineering and IT,
University of Technology Sydney, CB11.06.217, Building 11, 81 Broadway, Ultimo NSW
2007, Australia. biswajeet24@gmail.com, Biswajeet.Pradhan@uts.edu.au Abstract. Generating a high precision continuous surface is a key capability required in most
geographic information system (GIS) applications. In fact the most commonly used surface
type is a digital elevation model (DEM). Recently, there are some sources of remote sensing
data that provide DEM information such as; LiDAR, InSAR and ASTER GDEM which
ranged from very high to low spatial resolution. However, new methods of topographic field
surveying still highly on demand e.g. Differential GPS and Total station devices. In both
method of capturing the terrain elevation the post processing need to be applied to create a
continuous surface from point clouds. Geostatistical analysis were used to interpolate the taken
sample points from site into continuous surface. In current research, we examined the height
accuracy of LiDAR point clouds and total station dataset with three non-adoptive interpolation
models including, invers distance weightage (IDW), nearest neighbour (NN) and radial basis
function (RBF) based on referenced DGPS points. RMSE and R square regression analysis
were conducted to reveal the most accurate approaches in pilot study area. The results showed
Lidar surveying (less than 0.5 meter RMSE) has higher height accuracy compared to Total
station surveying (above 1 meter in RMSE) to extract DTM in flat area; while consumed less
computational processing time. Moreover, IDW was the best and accurate interpolation model
in both datasets to generate raster cautious terrain model. 1. Introduction Remote sensing methods have been efficiently utilized in various fields for decision making due to
their display capabilities and spatial analysis. The efficacy of analysis procedures is indeed
meaningfully enhanced by using 3D geospatial models since they simplify interpretation and
visualization. Digital Elevation Model (DEM) is the adopted data structure to store topographical
information and is usually needed to be interpolated to create the height values for entire terrain
areas[1]. DEM is a arithmetical demonstration of topography, typically create up of same-sized cells
that indicate the value of elevation [2]. Generally, DEM might be derived from field surveys,
topographic contour lines, radar interferometry, photogrammetry methods and laser scanning [3]. Since, topography is an important parameter that can controls the behave of natural systems, DEM is
greatly valuable to cope with environmental phenomena [4]. Numerous Geographic Information
Systems (GIS) applications are relying on DEM; for instance, hydrologic modelling [5], urban
assessment studies [6, 7] soil distribution and erosion analysis [8]. There are some factors which
effect on the quality of DEM negatively. Some of the existing surveying approaches can be classified 1 IGRSM 2018 IOP Publishing doi :10.1088/1755-1315/169/1/012009 1234567890 ‘’“”
IOP Conf. Series: Earth and Environmental Science 169 (2018) 012009
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IOP Conf. Series: Earth and Environmental Science 169 (2018) 012009
d into ground-based and airborne laser scanner which is suitable for moderately small terrain with very
high resolution result [9]; while traditional topographic contour maps, high-resolution satellite images
and common stereoscopic air-photos are being used for large areas surveying [10]. Light detection
and ranging (LIDAR) knowledge is an active remote sensing surveying which has a great capability
of concurrently mapping the terrain and surface of lands with sub-metre height vertical accuracy. The
airborne LiDAR normally engages a laser scanner pulses to an inertial measurement unit (IMU) with
global positioning system (GPS) for exact surveying [2]. Earlier investigation has revealed that the
accuracy of DEM differs in different terrain and land use/land cover types [11]. Basically, LiDAR
surveying errors can be into four modules included, surveying error, LiDAR system measurements
uncertainty, interpolation errors and horizontal displacement malfunction [2]. This study experiments
on interpolation errors and their techniques. As, LiDAR point clouds are not correspondingly
distributed, the interpolation technique should be used to create unidentified points by using the
magnitude and location of the known points [12]. 1. Introduction Many interpolation methods have been
implemented on geospatial application including, kriging, Spline, IDW and so one. Though LiDAR
and Total station surveyed points has been broadly used in GIS frameworks, still a few researches
were certainly discovered which interpolation methods is precise on discrete surveyed points with
respect to mixed, hilly and flat trains [13]. Hence, in this research Lidar surveying using different non-
adaptive interpolation methods were compared with field station surveying based on referenced
definitional global positioning system (DGPS).The experiments were implemented on flat terrain
where methods compared together in terms of height accuracy and computational time. 2. Materials and Methods 2. Materials and Methods
Figure 1. Computational flowchart applied in this study area
2.1. Study Area
Point clouds to LAS format
Stratified random sampling
234 Topology points
Terrain Points
Surface Points
Geostatistical
Interpolation Models
IDW
NN
RBF
LiDAR RFB
DTM
Station
RBF DTM
RMSE & R2
Accuracy
Assessment by
Referenced
DGPS
Noise Removal
Ground and Non-Ground Filtering
Field surveying by Total Station
Airborne Lidar Imagery
Terrain Points
LiDAR
NN DTM
Station
NN DTM
LiDAR
IDW DTM
Station
IDW DTM Field surveying by Total Station Airborne Lidar Imagery Stratified random sampling Ground and Non-Ground Filtering Terrain Points Surface Points Terrain Points Geostatistical
Interpolation Models LiDAR
NN DTM LiDAR
IDW DTM Station
NN DTM Station
IDW DTM Figure 1. Computational flowchart applied in this study area 2.1. Study Area 2.1. Study Area 2.1. Study Area
The study area is a part of University Putra Malaysia (UPM) which covers an area of 4.34 hectare. It
located at Selangor state of Malaysia where has longitude of 101° 42’ 55’’ to 101° 43’ 09’’ E and y
The study area is a part of University Putra Malaysia (UPM) which covers an area of 4.34 hectare. It
located at Selangor state of Malaysia where has longitude of 101° 42’ 55’’ to 101° 43’ 09’’ E and 2 IOP Publishing 1234567890 ‘’“”
Conf. Series: Earth and Environmental Science 169 (2018) 012009
doi :10.1088/1755-1315/169/1/01200 latitude 2° 59’ 17’’ to 2° 59’ 24’’ N. The study area is basically covered by mixed land cover
including; railways, pasture, bare land and trees. latitude 2° 59’ 17’’ to 2° 59’ 24’’ N. The study area is basically covered by mixed land cover
including; railways, pasture, bare land and trees. Figure 2. Location of the study area 2.2.3. Reference dataset. Sixteen height DGPS points were collected from study area. They cover
the entire study area and was collected following a stratified random sampling strategy. DGPS 2.3. Methods Three geostatistical interpolation method namely invers distance weightage (IDW), radial based
function (RBF) and nearest neighbour [4] were performed on both LiDAR and field station surveying
points to create elevation model. Derived DTMs were evaluated with the DGPS reference points to
given study area. Adopted methodology is illustrated in Figure 2. In this study, 252 ground Points
were collected by total station surveying within 18 hours at every changes of ground surfaces and/or
every 10m depending on the surface of the ground. The more points per area or densely collected
points will result in a much better accuracy. 2.3.1. LiDAR Pre-processing. Clouds points should be first converted to LAS format and then
filtered into first return and ground return to separate surface from train model, respectively [14]. Next
step is Lidar Noise removal which is detect and remove likely noise points from loaded or selected
Lidar data. Unclassified Point Clouds were selected to Find Likely Noise Points In. Then, apply
Maximum Allowed Variance from Local Average in order to highlight Outside Elevation Range
(noise) and eliminate them [11]. All the pre-processing steps were done in Global mapper v.19 in
WGS-84 datum. After filtering and noise removal, point clouds were sampled into a regular grid, and
ready for next step which is interpolation. 2.2. Materials 2.2. Materials
2.2.1. Remotely sensed dataset. LiDAR point clouds were surveyed on 2015 using an airborne laser
terrain mapper 3100 instrument in 1510 meter flying height. The density of points are almost 8 points
per square meter with a 25,000Hz pulse rate frequency. The data accuracy or spatial resolution is 0.5
meter, regarding to point spacing. LiDAR data was collected in four different pulse returns (Table 1). 2.2.1. Remotely sensed dataset. LiDAR point clouds were surveyed on 2015 using an airborne laser
terrain mapper 3100 instrument in 1510 meter flying height. The density of points are almost 8 points
per square meter with a 25,000Hz pulse rate frequency. The data accuracy or spatial resolution is 0.5
meter, regarding to point spacing. LiDAR data was collected in four different pulse returns (Table 1). Table 1. LiDAR point clouds characteristics
Category
Points
count
Percentage
Height
Minimum
(meter)
Height
Maximum
(meter)
First Returns
197904
94.71
44.79
667.62
Second Returns
9951
4.76
45.44
79.05
Third Returns
1042
0.50
45.54
77.47
Fourth Returns
55
0.03
46.92
67.90
All Returns
208952
100.00
44.79
667.62 2.2.2. Field practical survey. Field surveying was conducted by using Total station TOPCPN GTS-
230N Series. GTS-230N Series have their basic functions for distance and angle measurement in
addition to maintaining superb durability against the environment. Basically, a total station device
combines a digital theodolite and an Electronic Distance Measure (EDM) that work together with a
microprocessor to rapidly and accurately perform tasks. 2.2.3. Reference dataset. Sixteen height DGPS points were collected from study area. They cover
the entire study area and was collected following a stratified random sampling strategy. DGPS 2.2.3. Reference dataset. Sixteen height DGPS points were collected from study area. They cover
the entire study area and was collected following a stratified random sampling strategy. DGPS 3 IGRSM 2018 1234567890 ‘’“”
IOP Conf. Series: Earth and Environmental Science 169 (2018) 012009
doi :10.1088/1755-1315/169/1/012009 doi :10.1088/1755-1315/169/1/0120 1234567890 ‘’“”
IOP Conf. Series: Earth and Environmental Science 169 (2018) 012009 provides differential corrections to a GPS receiver in order to improve navigation accuracy and
monitors the integrity of GPS satellite transmissions. The CHC X91 GNSS Receiver was used to take
DGPS sampling points with less than 2 cm accuracy which uses 220 Channel, Enhanced GNSS
technology supports L2C, L5, SBAS, GLONASS and Galileo, Advanced Multipath Mitigation and
Low Noise Carrier Phase Measurement. 2.3.2. Interpolation methods a) Inverse Distance Weighting concept. The IDW mathematical concept is assumed things that
are nearby to another are more similar rather those that are farther apart and obtain higher
weightage [15]. To calculate a value for unexampled area, IDW uses the inverse measured values
surrounding the prediction place. where, is estimation value of variable z in point I; stands as sample value in point I; is
distance of sample point to estimated point and n shows the total number of predictions. where, is estimation value of variable z in point I; stands as sample value in point I; is
distance of sample point to estimated point and n shows the total number of predictions. b) Radial Basis Functions concept. The RBFs are equivalent to fitting out a rubber membrane by
the observed sample values, while reducing the sum of the curvature of the surface [16]. The
selected basis function specifies, how the rubber membrane will match between the measures. Radial basis functions are typically used to build up function approximations of the form: Here is the sum of the number of radial basis functions, where each item related to
distinct centre xi and weighted by suitable factor of . Here is the sum of the number of radial basis functions, where each item related to
distinct centre xi and weighted by suitable factor of . c) Nearest Neighbour Interpolation concept. The NN interpolation was developed by Sibson [17],
which use Voronoi & Delaunay graph which is a discrete diagram of spatial points. For
interpolation, it exert weight on the nearest points based on their proportional areas. Relevant
equation which is used for NN interpolation method is: p where: G(x, y): Evaluated value of nearest neighbour in (x, y);N: Number of nearest
neighbours used at interpolation; Shows observed values at and : Associated where: G(x, y): Evaluated value of nearest neighbour in (x, y);N: Number of nearest
neighbours used at interpolation; Shows observed values at and : Associated 4 IGRSM 2018 1234567890 ‘’“”
IGRSM 2018
IOP Publishing
IOP Conf. Series: Earth and Environmental Science 169 (2018) 012009
doi :10.1088/1755-1315/169/1/012009 1234567890 ‘’“”
onf. Series: Earth and Environmental Science 169 (2018) 012009
doi :10.1088/1755-1315/169/1/0120 weight. The weights are estimated while deciding what portion of neighbouring area needs to be
taken when creating diagrams [13]. 2.3.3. Accuracy Assessment. Statistical parameters and visual analysis have been used for comparative
assessment of the interpolated areas. 2.3.2. Interpolation methods During visual analysis via field visiting, DEM generated
elevations were controlled via GPS and observational methods. Statistical analysis has been
accomplished by measuring the standard deviations of interpolated elevation values from
commensurate observed values according RMSE (Root Mean Square Error)[18]. The RMSE is
defined as the square root of the mean squared error: (4) (4) where is referenced values and is interpolated values at time/place i. where is referenced values and is interpolated values at time/place i. where is referenced values and is interpolated values at time/place i. 3.2. Comparative analysis of interpolation methods 3.1. Surveyed DGPS Data for Elevations bench mark Basically, 50 GNSS reference stations are located in Peninsular Malaysia though which surveyed
DGPS points can be corrected either real time or post processed one. They computes the correction
for their positions based on geodetic network measurements and atomic horologe [19]. Accordingly,
in this study, the used DGPS were corrected by post processing approach. Sixteen DGPS ground truth
points were selected based on the topographic variability between receivers without any obstruction
with accuracies of ± 1 cm to ± 2 cm. These points were uniformly distributed over the study area. The
amount of GDOP (geometric dilution of precision) and PDOP (position dilution of precision)
uncertainties, which describes error caused by the relative position of the GPS satellites, are very low
among all collected DGPS points. Also, recorder heights were measured based on both mean see
levels (MSL) or Geoid and Ellipsoid which are projected in UTM WGS19984 coordinate system. Figure 3. Visual presentation on derived DTM from LiDAR dataset
3.2. Comparative analysis of interpolation methods 3.2. Comparative analysis of interpolation methods Figure 3. Visual presentation on derived DTM from LiDAR dataset 5 IGRSM 2018 IGRSM 2018 1234567890 ‘’“”
IGRSM 2018
IOP Publishing
IOP Conf. Series: Earth and Environmental Science 169 (2018) 012009
doi :10.1088/1755-1315/169/1/012009 1234567890 ‘’“”
Conf. Series: Earth and Environmental Science 169 (2018) 012009
doi :10.1088/1755-1315/169/1/01200 Elevation points from Lidar and Total station were investigated regarding to different
interpolation methods with reference to numerous terrain circumstances. Visual comparisons as well
as mathematical accuracy analyses have been conducted. Figure 4. Visual presentation on derived DTM from Total Station field surveying Figure 4. Visual presentation on derived DTM from Total Station field surveying Visual comparison of elevation models generated using different interpolation techniques is
presented in Fig. 2. As, LIDAR data has holes so, non-adaptive IDW, RBF and NN interpolation are
applied to automatically identify and fill the gaps to acquire a higher and smooth resolution grid
surface. In LiDAR visual presentation there was no significant differences in terms of pattern and
features between the terrain elevations among different interpolation methods (Fig. 3); however, the
elevation maximum and minimum range were fluctuated from one to another model. For example in
RBF model the maximal was 57.7405 meter and low altitude was 44.7358 meter which showed the
highest and lowest interpolated elevation n compared with IDW and NN. 3.1. Surveyed DGPS Data for Elevations bench mark Basically, extracted terrain
characteristics by Total station surveying with 252 points are not as precise as 208952 Lidar point
clouds. However, DTMs which derived by IDW and RBF methods are more similar to LiDAR pattern
terrain (Fig. 4). DGPS Ellipsoid elevation value is much closer to measure height rather MSL for height
assessment (Table 2). In order to discover the most accurate interpolation models, we need to apply
detailed statistics tests including R square regression and RMSE. DTMs were compared to ground
truth DGPs in different location randomly. 6 6 IGRSM 2018 1234567890 ‘’“”
g
IOP Conf. Series: Earth and Environmental Science 169 (2018) 012009
doi :10.1088/1755-1315/169/1/012009 Table 2. Compared extracted DTMs with DGPS Reference points
No
Referenced DGPS
Height points (meter)
Extracted DTM from LiDAR
(meter)
Extracted DTM from Total
Station (meter)
MSL
Ellipsoid
IDW
RBF
NN
IDW
RBF
NN
1
59.352
57.385
57.078
56.870
56.867
56.283
56.002
55.985
2
59.655
57.690
57.377
57.178
57.182
56.574
56.262
57.146
3
59.126
57.161
56.975
56.895
56.807
54.421
54.680
54.312
4
57.960
55.994
55.732
55.517
55.506
55.029
54.733
55.628
5
57.448
55.482
55.202
55.013
55.007
54.179
53.989
54.072
6
55.898
53.931
53.897
53.730
53.691
53.227
53.122
53.116
7
54.320
52.353
52.071
51.851
51.851
51.550
51.086
51.982
8
54.194
52.227
52.749
51.456
51.467
51.568
50.528
50.480
9
53.085
51.117
51.211
51.092
51.049
50.198
50.093
50.798
10
52.340
50.370
49.981
49.769
49.778
49.375
49.125
48.968
11
50.158
48.187
47.982
47.809
47.800
46.700
46.623
47.397
12
52.736
50.767
50.509
50.300
50.300
49.845
49.543
48.741
13
56.431
54.464
54.121
53.920
53.927
52.534
52.809
52.446
14
54.636
52.667
52.286
52.099
52.083
51.294
51.174
50.977
15
51.553
49.583
49.227
48.999
49.018
49.204
48.244
49.404
16
51.327
49.355
49.107
48.908
48.929
49.145
48.795
49.112
h
d d d
i i
i i
d
i
h i h
l
d
d Table 2. Compared extracted DTMs with DGPS Reference points The standard deviation, average, minimum and maximum height values were extracted and
mathematically can prove which model has received the lowest error and uncertainties (Table 3). The standard deviation, average, minimum and maximum height values were extracted and
mathematically can prove which model has received the lowest error and uncertainties (Table 3). The standard deviation, average, minimum and maximum height values were extracted and
mathematically can prove which model has received the lowest error and uncertainties (Table 3). Figure 5. Height differentiation between interpolated models and referenced DGPS points. 4. Conclusion Current study examined two different high quality dataset that have been collected by remotely and on
filed approaches namely, LiDAR point clouds and Total station surveying over a flat terrain. We also
investigated the comparison experiments on various geostatistical interpolators such as IDW, RBF
and NN. Extremely accurate DGPS was used as bench mark to compare methods and datasets. The
results showed even though both collected data are very reliable, LiDAR dataset is not only more
accurate but also is a fast surveying method to collect height information. Moreover, IDW was the
best and accurate interpolation model in both datasets to generate DTM due to its mathematical
concept and distribution of samples on study area. IGRSM 2018 1234567890 ‘’“”
IOP Conf. Series: Earth and Environmental Science 169 (2018) 012009 1234567890 ‘’“”
Conf. Series: Earth and Environmental Science 169 (2018) 012009
doi :10.1088/1755-1315/169/1/0120 constant and accurate than Total station surveyed points (Fig. 5). Some techniques have been
suggested for DEM quality assessment to achieve its accuracy and precision. Precision is typically
assessed by equations without spatial dimension e.g. the mean absolute error (MAE) or the root mean
square error (RMSE) and R square regression method. Table 3. Accuracy assessment and statistical test results
Test
IDW -LiDAR
RBF-LiDAR
NN -LiDAR
IDW-Station
RBF-Station
NN-Station
Standard
deviation
3.056
3.079
3.068
2.835
2.920
2.914
RMSE
0.301
0.488
0.491
1.247
1.329
1.473
R-square
0.997
0.996
0.994
0.980
0.960
0.922 Table 3. Accuracy assessment and statistical test results Table 3. Accuracy assessment and statistical test results It is shown that Total station surveying is low accurate (above 1 meter in RMSE) than Lidar
surveying (less than 0.5 meter RMSE) to extract DTM in flat area while consumed more time. In fact,
it took several hours to complete rather than several minute for Lidar mapping. Table 3 shows that
IDW showed more precise result (0.301 and 0.997 RSME and R2) which followed by RBF and NN. Additionally, the same ranking of interpolation models were observed in Total station where IDW
performed with highest accuracy (1.25 meter in RMSE and 0.980 R2) to interpolate DTM with respect
to referenced DGPS truth points. 3.1. Surveyed DGPS Data for Elevations bench mark -0.7
-0.4
-0.1
0.2
0.5
0.8
1.1
1.4
1.7
2.0
2.3
2.6
2.9
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
HEIGHT DIFFERENTIATION
DGPS POINTS
IDW -LiDAR
RBF-LiDAR
NN -LiDAR
IDW-Station
RBF-Statio
NN-Statio DGPS POINTS Figure 5. Height differentiation between interpolated models and referenced DGPS points. The LiDAR-derived DTM as well as total station-derived DTM for the entire study under-
predicted the ground elevation when validated with the DGPS ground truth data, with an average
overall mean error of 0.4 and 2.01 meter, respectively (Table 3). LiDAR cloud points are more 7 References [1]
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and age estimation from WorldView-3 imagery and LiDAR data using an integrated
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* Статья представляется в рамках проекта по поддержке публикаций авторов российских образо-
вательных и научных организаций.
Работа опубликована при поддержке Программы Университетское партнерство у
р
Работа опубликована при поддержке Программы «Университетское партнерство». 196
RUSSIAN SOCIOLOGICAL REVIEW. 2023. Vol. 22. No. 3 doi: 10.17323/1728-192x-2023-3-196-218 doi: 10.17323/1728-192x-2023-3-196-218 Почему граждане доверяют правительству?
Истоки политического доверия
в современной России* 1 В более длительной перспективе доверие необходимо для решения долгосрочных
социальных проблем, таких как изменение климата, старение населения и авто-
матизация труда. Доверие способствует экономическому росту и экономической
эффективности, предоставлению общественных благ, социальной интеграции, со-
трудничеству и гармонии, удовлетворенности личной жизнью, демократической
стабильности и развитию и даже хорошему здоровью и долголетию (Hethering-
ton, Husser, 2012). Кроме того, социальное доверие в целом и политическое доверие
в частности являются ключевым компонентом социального капитала и обычно
используется в качестве его основного показателя (Патнэм, 1996; Фукуяма, 2004). ность политической системы. Оно имеет решающее значение для поддержания
участия в политической жизни и социальной сплоченности (Seligman, 1997: 13). В более длительной перспективе доверие необходимо для решения долгосрочных
социальных проблем, таких как изменение климата, старение населения и авто-
матизация труда. Доверие способствует экономическому росту и экономической
эффективности, предоставлению общественных благ, социальной интеграции, со-
трудничеству и гармонии, удовлетворенности личной жизнью, демократической
стабильности и развитию и даже хорошему здоровью и долголетию (Hethering-
ton, Husser, 2012). Кроме того, социальное доверие в целом и политическое доверие
в частности являются ключевым компонентом социального капитала и обычно
используется в качестве его основного показателя (Патнэм, 1996; Фукуяма, 2004). Необходимо отметить, что настоящая статья не является первой работой, кото-
рая стремится получить ответ на вышеназванный вопрос. В научной литературе
можно встретить пул работ, авторы которых стремятся объяснить уровень дове-
рия к политическим институтам в целом и к правительству в частности на данных
отдельно взятых стран или группы государств: Ганы (Godefroidt, Langer, Meule-
man, 2017), африканских стран (Hutchison, Johnson, 2011), Китая (Chen, 2017; Shi,
2001), государств АТР (Chang, Chu, 2006; Choi, Woo, 2016), стран Карибского бас-
сейна (Stoyan, Niedzwiecki, Morgan, Hartlyn, Espinal, 2016), западноевропейских
государств (Fitzgerald, Wolak, 2016), а также посткоммунистического пространства
(Lühiste, 2006; Mishler, Rose, 2001). Доверие к российским политическим институ-
там становится важной темой отечественных исследователей (Давыборец, 2016;
Киселев, 2014; Козырева, Смирнов, 2015; Малкина, Овчинников, Холодилин, 2020;
Терин, 2018). Целью данной статьи является выявление факторов, определяющих доверие
граждан к политическим институтам в России. В этом исследовании основное
внимание уделяется трем детерминантам институционального доверия. В ходе
работы планируется проверить, как 1) межличностное и социальное доверие,
2) участие в деятельности неправительственных организаций, 3) эффективность
политических институтов и 4) просмотр телевизионных новостей влияют на по-
литическое доверие. Для этого используются данные 7-й волны исследований Все-
мирного обзора ценностей (WVS-7). Хотя литература о детерминантах политического доверия обширна, данные
о его истоках в России недостаточны. Основной вклад настоящей статьи в лите-
ратуру по институциональному доверию имеет несколько оснований. Почему граждане доверяют правительству?
Истоки политического доверия
в современной России* 1 Руслан Мухаметов
Кандидат политических наук, доцент, Уральский федеральный университет
Адрес: пр. Ленина, д. 51, г. Екатеринбург, Российская Федерация, 620083
E-mail: muhametov.ru@mail.ru Политическое доверие занимает центральное место в изучении политических режи-
мов. Оно является одним из наиболее важных показателей политической легитим-
ности и стабильности, указывая на степень поддержки государства (правительства
или партии) народом. В статье проводится исследование количественных показателей
влияния социально-психологических, гражданских, институциональных и информа-
ционных факторов на политическое доверие. Целью этого исследования было внести
вклад в понимание детерминант доверия к правительству. Анализ основан на данных
социологического опроса 7-й волны исследований Всемирного обзора ценностей
(2017–2022 гг.). Используя набор данных по России, автор оценил влияние межлич-
ностного и обобщенного доверия, участия в работе некоммерческих неправитель-
ственных организаций, восприятия гражданами своего материального положения,
безопасности и коррупции, просмотра ТВ новостей на доверие к правительству. Дан-
ное исследование показало, что более высокий уровень межличностного и социально-
го доверия оказывает положительное влияние на политическое доверие. Выдвигается
и подтверждается гипотеза о положительном влиянии просмотра политических ТВ
программ на доверие к правительству. Статья дополняет существующие эмпириче-
ские исследования институционального доверия, а также адаптирует известные тео-
рии, объясняющие происхождение доверия, к российской политической реальности. Ключевые слова: доверие, политическое доверие, социальный капитал, политические
институты, правительство, «школа демократии», некоммерческие организации Ключевые слова: доверие, политическое доверие, социальный капитал, политические
институты, правительство, «школа демократии», некоммерческие организации Почему одни граждане больше доверяют политическим институтам, чем другие? За последние время этот вопрос стал занимать важное место в области политиче-
ской социологии и сравнительной политологии. Политическое доверие является
одной из главных тем для обсуждения политологами и социологами. Считается,
что его наличие или отсутствие оказывает важное влияние на политическую
стабильность и эффективность правительства (Zmerli, Newton, 2008). Доверие
к политическим институтам важно для поддержания стабильности режима (Van
der Meer, Hakhverdian, 2017). Доверие к основным институтам — президенту,
правительству, СМИ, военным — занимает центральное место в функциониро-
вании общества. Когда граждане не доверяют государственным органам, таким
как правительство и парламент, стабильность политического режима подверга-
ется сомнению. Доверие — это фундамент, на котором основывается легитим- * Статья представляется в рамках проекта по поддержке публикаций авторов российских образо-
вательных и научных организаций. б
б RUSSIAN SOCIOLOGICAL REVIEW. 2023. Vol. 22. No. 3 196 RUSSIAN SOCIOLOGICAL REVIEW. 2023. Vol. 22. No. 3 197 ность политической системы. Оно имеет решающее значение для поддержания
участия в политической жизни и социальной сплоченности (Seligman, 1997: 13). Почему граждане доверяют правительству?
Истоки политического доверия
в современной России* 1 Она при-
звана, во-первых, систематизировать основные теоретические подходы, лежащие
в основе подобной работы, во-вторых, проверить эти теоретические положения
на соответствие последнему исследованию 7-й волны Всемирного обзора цен-
ностей (2017–2022 гг.). В результате предполагается расширить понимание факто-
ров, оказывающих влияние на доверие граждан к институту правительства, про-
должив традицию изучения политического доверия в России (Mishler, Rose, 2005;
Shlapentokh, 2006). 198 СОЦИОЛОГИЧЕСКОЕ ОБОЗРЕНИЕ. 2023. Т. 22. № 3 Структура статьи представлена следующим образом. Первая часть посвящена
систематизации различных подходов к определению происхождения политиче-
ского доверия. На основе базовых теорий будут сформулированы гипотезы для
дальнейшей эмпирической проверки. Во второй части представлены источники
данных и методы исследования, используемые в работе. Результаты регрессионно-
го анализа рассматриваются в третьем разделе. Наконец, в заключительной части
будут подведены основные итоги. Теоретические подходы к объяснению происхождения политического
доверия Доверие — это многогранное понятие, означающее для людей несколько вещей. Это сложная концепция, которую исследователи из разных дисциплин пытались
четко и последовательно определить. Для целей настоящей работы доверие опре-
деляется как вера субъекта в то, что в худшем случае другие люди сознательно
или добровольно не причинят ему вреда, а в лучшем случае — будут действовать
в его интересах (Newton, 2001: 202). Политическое доверие можно определить как
«доверие отдельных лиц к государственно-правовым и политическим институтам
и субъектам» (Berg, Hjerm, 2010: 391). Оно в своей основе является отношенческим
и ситуативным (Van der Meer, Zmerli, 2017). Политическое доверие может менять-
ся с течением времени, отражая краткосрочные и/или долгосрочные результаты,
а также ожидания общественности по отношению к правительствам и институтам
(Bauer, Freitag, 2017). Это определение не включает доверие к конкретным долж-
ностным лицам или политикам, но относится к очень обобщенной, «абстрактной»
форме доверия к политическим институтам (Гидденс, 2011: 208). Политическое
доверие можно рассматривать как форму диффузной поддержки политической
системы и ее основных институтов в целом. Оно не обязательно должно отражать
согласие с конкретными политическими решениями (Easton, 1965). Более того,
можно даже утверждать, что демократическое устройство представляет собой
«институционализированное недоверие», когда разные ветви власти и группы
акторов уравновешивают влияние друг друга, составляя систему сдержек и про-
тивовесов (Штомпка, 2012). Частным случаем политического доверия выступает
доверие к правительству. Мы следуем за большинством ученых в определении до-
верия к правительству, как соотношения оценки людьми деятельности правитель-
ства по отношению к их нормативным ожиданиям того, как должно действовать
правительство (Hetherington, Husser, 2012: 313). Под правительством в работе пони-
мается совокупность государственных учреждений в целом, которые занимаются
реализацией политики, а также регулированием и предоставлением государствен-
ных услуг. В научной литературе отсутствует консенсус относительно истоков политиче-
ского доверия. В этом разделе рассматриваются четыре основных теоретических
подхода к возникновению политического доверия, которые предполагается эмпи- RUSSIAN SOCIOLOGICAL REVIEW. 2023. Vol. 22. No. 3 199 рически проверить, — социально-психологические теории, теория добровольных
организаций, теория институциональной эффективности, а также авторитарная
теория массовой коммуникации. Представители социально-психологической школы мысли утверждают, что до-
верие является основной чертой личности индивидов (Эриксон, 1996; Rosenberg,
1957; Kramer, 1999). Оно возникает в раннем детстве и, как правило, сохраняется
в дальнейшей жизни, меняясь медленно в результате последующего опыта, осо-
бенно травматического. По мнению исследователей, политическое доверие есть
часть более широкого синдрома личностных характеристик, которые включают
оптимизм, веру в сотрудничество и уверенность в том, что люди могут разрешить
свои разногласия и жить удовлетворительной социальной жизнью вместе (Del-
hey, Newton, 2003). Теоретические подходы к объяснению происхождения политического
доверия Родители влияют на отношение и нормы своих детей тремя
основными способами. Во-первых, дети, которые социализируются в доверитель-
ном и открытом родительском окружении, в атмосфере толерантности, с большей
вероятностью будут доверять и отвечать взаимностью. Во-вторых, родители учат
своих детей, как судить других и с кем сотрудничать. В-третьих, семьи функцио-
нируют как реальные арены обучения, где дети непосредственно сталкиваются
с эпизодами сотрудничества или дезертирства (Stolle, 2003). Весомый вклад в раз-
витие этого подхода внес Э. Усланер, который утверждает, что дети рано учатся
доверию у родителей, которое основано на двух других ключевых характеристиках
личности: оптимизме и способности контролировать мир или, по крайней мере,
собственную жизнь (Uslaner, 1999: 138). Таким образом, в рамках этого подхода по-
литическое доверие воспринимается как пролонгация межличностного доверия. Связь межличностного доверия и доверия к правительству в этом случае заклю-
чается в том, что высокий уровень частного доверия может улучшить доверие
граждан к правительству: индивиды с высоким уровнем межличностного дове-
рия склонны доверять другим лицам и верить, что они действуют не только в сво-
их интересах, но и в интересах третьих лиц; это приводит к тому, что они будут
склонны доверять правительству как институту, который представляет интересы
всех граждан. Стоит отметить, что описание причинно-следственного механизма,
связывающего межличностное доверие и абстрактное доверие системам и органи-
зациям, требует проведения отдельного исследования. Второй подход — теория добровольных организаций. Он заключается в том,
что политическое доверие есть результат участия индивидов в социальных и по-
литических институтах, которые поощряют развитие кооперативных отношений
(Morales, De Ulzurrun, 2002). Эта теория утверждает, что политическое доверие
порождается личным участием в добровольных ассоциациях, что создает взаим-
ные нормы в отношениях между гражданами (Van Der Meer, Van Ingen, 2009). В силу этих обстоятельств НКО в целом рассматриваются как «школы низовой
демократии» (Stolle, Rochon, 1998). Данная теория восходит к идеям А. де Токвиля,
который считал, что личное взаимодействие в рамках добровольных ассоциаций
является доминирующим способом создания социального капитала. Участие в по- СОЦИОЛОГИЧЕСКОЕ ОБОЗРЕНИЕ. 2023. Т. 22. № 3 200 литических объединениях Токвиль рассматривает в качестве школы, в которой
каждый гражданин изучает общую теорию ассоциаций. Гражданские объедине-
ния подготавливают почву для создания политических ассоциаций; политические
ассоциации способствуют развитию и совершенствованию гражданских союзов. Иными словами, участие в деятельности НКО вызывает желание объединяться
и обучает искусству создания союзов массы людей, которые в противном случае
всегда жили бы сами по себе. Теоретические подходы к объяснению происхождения политического
доверия Демократической страной является то государство,
в котором индивиды достигли наивысшего совершенства в искусстве сообща до-
биваться цели, отвечающей их общим желаниям, и чаще других применяют этот
новый метод коллективного действия (Токвиль, 2002). Третий подход — теория эффективности институтов. Данный подход берет
свое начало в основополагающей работе Э. Даунса (Downs, 1957). Этот тип объяс-
нения институционального доверия опирается на теорию рационального выбора. Ее центральное утверждение состоит в том, что рациональный избиратель — это
тот, кто принимает решение голосовать только в том случае, если ожидаемая по-
лезность голосования превышает стоимость действия. Акторы совершают поступ-
ки и/или принимают решения, которые максимизируют полезность в достижении
целей. Институциональная теория эффективности предполагает, что политиче-
ское доверие является следствием деятельности институтов. Доверие к институ-
там рационально обоснованно: институты, которые хорошо работают, порождают
доверие; ненадежные институты порождают скептицизм и недоверие (Kaasa, An-
driani, 2021). Другими словами, доверие к правительству является следствием эф-
фективного управления, политические институты должны продемонстрировать
компетентность, чтобы создать/вызвать доверие. Теория институциональной эф-
фективности основывается на модели когнитивной оценки, которая изображает
политическое доверие как результат постоянных субъективных оценок гражда-
нами деятельности политических субъектов, политических институтов или поли-
тической системы в целом (Hetherington, 1998). Данный подход предполагает, что
политическое доверие основывается на оценках гражданами результативности
политического института (Paxton, 2007). Люди, которые являются безработными
или чьи личные финансы пострадали от того, что они считают государственной
политикой, скорее всего, будут меньше доверять политическим институтам, чем
те, кто находится в лучших или улучшающихся экономических условиях. Инсти-
туциональное доверие изменяется в соответствии с индивидуальными оценками
экономических и социальных условий и зависит от способности институтов удо-
влетворять и представлять чисто экономические потребности, а также запросы
граждан, которые в основном коренятся в социально-экономических интересах
(Stolle, 2008). Таким образом, доверие к правительству отражает краткосрочные
результаты, оценки институтов и их способности предоставлять качественные
государственные услуги, реагировать на потребности, запросы граждан. Четвертый подход — авторитарная теория массовой коммуникации. Согласно
ему, СМИ должны уважать то, чего хотят власти, и работать в соответствии с по- RUSSIAN SOCIOLOGICAL REVIEW. 2023. Vol. 22. No. 3 201 желаниями властей. СМИ не могут работать независимо. Свободное распростра-
нение информации может поставить под удар национальную безопасность и стать
угрозой. Данная теория исходит из того, что государство должно контролировать
средства массовой информации. Свобода слова в целом бросает вызов авторитету,
а свобода слова, которая подразумевает критику тех, кто находится у власти, рас-
сматривается как подрывная. Теоретические подходы к объяснению происхождения политического
доверия Власти имеют все права разрешать или запрещать
любые СМИ, предоставляя или отзывая лицензию и устанавливая определен-
ные ограничения на звучащие в СМИ мнения. Средства массовой информации
в рамках данной теории являются рупором правительства (Siebert et al., 1956). Вос-
приятие спонсируемых государством новостей как весьма достоверных облегчает
некритическое принятие большинством населения проправительственной поли-
тической повестки (Сироткина, Завадская, 2016; Sirotkina, 2020). Таким образом, основываясь на предыдущих исследованиях и их результатах,
мы ожидаем, что: H1: Чем больше уровень межличностного доверия, тем сильнее доверие гра-
ждан к политическим институтам. H2: Чем активнее граждане принимают участие в деятельности НКО, тем выше
уровень политического доверия. H3-1: Более высокий уровень материального положения граждан приводит
к более высокому уровню политического доверия. H3-2: Чем выше уровень восприятия коррупции, тем ниже уровень политиче-
ского доверия. H3-3: Чем выше уровень восприятия безопасности, тем выше уровень доверия
граждан к политическим институтам. H4: Граждане, которые чаще подвергаются воздействию информации из кон-
тролируемых властями СМИ, должны больше доверять правительству. Источники данных и методы исследования Данные гипотезы делают конкурирующие теоретические основы проверяемыми. Для их эмпирического тестирования нужны количественные данные, содержащие
показатели институционального и межличностного доверия, гражданской актив-
ности, восприятия коррупции и безопасности, а также экономические индикаторы. Для проверки этих теоретических подходов необходимо определить институ-
циональное доверие и уточнить способ его измерения. Зависимая переменная —
доверие граждан к правительству. Доверие — это суждение, которое может быть
понято дихотомически (либо доверяет, либо не доверяет) или более градуирован-
ным образом (доверяю или не доверяю в определенной степени) (Levi, Stoker, 2000:
476). Доверие можно измерить тремя способами: Данные гипотезы делают конкурирующие теоретические основы проверяемыми. Для их эмпирического тестирования нужны количественные данные, содержащие
показатели институционального и межличностного доверия, гражданской актив-
ности, восприятия коррупции и безопасности, а также экономические индикаторы. Д
б Для проверки этих теоретических подходов необходимо определить институ-
циональное доверие и уточнить способ его измерения. Зависимая переменная —
доверие граждан к правительству. Доверие — это суждение, которое может быть
понято дихотомически (либо доверяет, либо не доверяет) или более градуирован-
ным образом (доверяю или не доверяю в определенной степени) (Levi, Stoker, 2000:
476). Доверие можно измерить тремя способами: 1) субъективным и прямым с помощью опросов населения; 2) объективным и косвенным, используя прокси-переменные (например, дове-
рие, измеряемое как соотношение совпадения между рекомендацией городского 202 СОЦИОЛОГИЧЕСКОЕ ОБОЗРЕНИЕ. 2023. Т. 22. № 3 правительства по вопросу, поставленному на голосование, и фактическим резуль-
татом голосования) (Kucher, Götte, 1998); 3) с помощью экспериментальных измерений доверия с использованием пове-
денческих игр (Habyarimana et al., 2009). 3) с помощью экспериментальных измерений доверия с использованием пове-
денческих игр (Habyarimana et al., 2009). По мнению исследователей, опросы восприятия являются самым популярным
методом измерения политического доверия на сегодняшний день (Kumagai, Ilorio,
2020: 7–8). В этих условиях источником данных выступили результаты 7-го опро-
са о мировых ценностях, который проводится в рамках проекта «Обзор мировых
ценностей» (World Values Survey, Wave 7, 2017–2022) (Haerpfer et al., 2022). В России
опрос проведен в 2017 году. Основным методом сбора данных в опросе WVS-7 было
личное интервью у респондента дома/по месту жительства. Выборка составила 1465
респондентов. В WVS-7 доверие к институтам измерялось с помощью следующего
вопроса: «Я перечислю Вам сейчас некоторые организации и общественные инсти-
туты. Скажите, насколько Вы доверяете каждому из них — полностью доверяете,
в некоторой степени доверяете, не очень доверяете или совсем не доверяете?» Выбор независимых переменных определялся теоретической рамкой иссле-
дования и поставленными гипотезами. В рамках гипотез H1 проверяется влия-
ние переменной, которая отражает межличностное доверие. Источники данных и методы исследования Для его измерения
были выбраны ответы респондентов на вопрос о том, насколько Вы доверяете 1)
людям, с которыми вы лично знакомы, 2) людям, живущим с вами по соседству. Вариантами ответов были: «полностью доверяю», «в некоторой степени доверяю»,
«не очень доверяю» или «совсем не доверяю этим людям». Вторая гипотеза пред-
полагает тестирование теории добровольных организаций/«школы демократии». Для проверки этого теоретического подхода переменная была операционализи-
рована через уровни участия граждан в деятельности НКО. Источником данных
стали ответы на вопрос: «Сейчас я назову различные общественные организации,
а Вы скажете по каждой из них — Вы в них активно участвуете; состоите, но ак-
тивно не участвуете; не состоите в этой организации или группе?» В вопроснике
респондентам предлагалось определить уровень своего участия в разных типах
НКО — религиозных, спортивных, профессиональных, экологических, гумани-
тарных. Переменная «Уровень участия в деятельности НКО» представляет собой
агрегированную переменную, которая получалась путем объединения ответов
опрошенных. В случае, если респондент принимает активное участие, например,
в работе организации по защите прав потребителей (кодировалось как 2), а в орга-
низации по защите прав женщин состоит, но активность не проявляет (кодирова-
лось как 1), то переменная получала значение 2. Для тестирования теории инсти-
туциональной эффективности и сформулированных на ее основе рабочих гипотез
был использован ряд переменных. Способом, с помощью которого исследователи
из WVS измерили уровень финансовой удовлетворенности (H3-1), был вопрос:
«Насколько Вы удовлетворены финансовым положением Вашей семьи?» Респон-
дентам предлагалось выбрать ответ на шкале от 1 до 10, где 1 — совершенно не удо-
влетворен, а 10 — полностью удовлетворен. Переменная «коррупция» в рамках RUSSIAN SOCIOLOGICAL REVIEW. 2023. Vol. 22. No. 3 203 гипотезы H3-2 была операционализирована через ответы на следующий вопрос
в WVS-7: «Теперь мне хотелось бы узнать Ваше мнение о коррупции — ситуациях,
когда люди дают взятки, дарят подарки или делают одолжения другим для того,
чтобы были решены их вопросы или оказаны услуги, на которые они и так име-
ют право. В какую точку Вы поместили бы Россию на этой шкале, где 1 означает,
что в стране “совсем нет коррупции”, а 10 — что “коррупция в России повсемест-
на”». Операционализация переменной «безопасность» в рамках гипотезы H3-3
была осуществлена через ответы на такой вопрос, как: «Насколько Вы чувствуете
себя в безопасности в наши дни?» (в полной безопасности; довольно безопасно;
не очень безопасно; совсем небезопасно). Наконец, переменная «Частота просмо-
тра ТВ новостей» операционализирована через ответы респондентов на следую-
щий вопрос из анкеты: «Люди узнают о происходящем в России и в мире из раз-
ных источников. Источники данных и методы исследования По каждому из следующих источников скажите, пользуетесь Вы
им каждый день, каждую неделю, каждый месяц, реже, чем раз в месяц, или нико-
гда?» В качестве источника информации были взяты телевизионные новости. Для повышения достоверности результатов исследования необходимо контро-
лировать влияние смежных факторов и альтернативных объяснений. В этой связи
в качестве управляемой переменной использовались показатели, отражающие фи-
нансовый и образовательный уровни респондентов. Теоретически они опираются
на концепцию модернизации С. М. Липсета, которая утверждает, что рост доходов
и образования приведет к поддержке демократии и неприятию авторитарных режи-
мов (Lipset, 1959). Необходимо оговориться, что ряд исследователей считает, что уро-
вень экономического развития не влияет на вероятность перехода к демократии, а из-
обилие делает демократические режимы более стабильными, шансы на выживание
демократии выше, когда страна богаче (Przeworski, Limongi, 1997). Исходя из этого,
следует ожидать, что более образованные и более состоятельные граждане демон-
стрируют более низкий уровень доверия к правительству авторитарного государства. р ру
ур
р
р
у
р
р
у
р
Объясняющие переменные, которые могут быть условно разделены на основ-
ные и контрольные, представлены в таблице 1. Таблица 1. Переменные, используемые в регрессионном анализе
Переменные
Тип переменной
Теоретический подход
Влияние в рамках
гипотезы
Доверие
к правительству
Зависимая
Уровень
межличностного
доверия
Независимая
Социально-
психологический
+
Степень доверия
к соседям
Независимая
+
Уровень участия
в деятельности НКО
Независимая
Теория добровольных
организаций
+ Таблица 1. Переменные, используемые в регрессионном анализе СОЦИОЛОГИЧЕСКОЕ ОБОЗРЕНИЕ. 2023. Т. 22. № 3 204 Уровень финансовой
удовлетворенности
Независимая
Теория
институциональной
эффективности
+
Уровень восприятия
коррупции в стране
Независимая
–
Уровень восприятия
безопасности
Независимая
+
Частота просмотра ТВ
новостей
Независимая
Авторитарная
теория массовой
коммуникации
+
Уровень доходов
Контрольная
Теория модернизации
–
Уровень образования
Контрольная
–
Описательная статистика выборки представлена в таблице 2. Таблица 2. Описательная статистика по переменным (n = 1465)
Переменная
Среднее
Медиана
Станд. отклон. Источники данных и методы исследования Минимум
Максимум
Доверие
к правительству
2,48
2
0,930
1
4
Уровень
межличностного
доверия
1,69
2
0,598
1
2
Степень доверия
к соседям
2,1
2
0,861
1
4
Уровень участия
в деятельности НКО
0,112
0
0,384
0
2
Уровень
финансовой
удовлетворенности
5,49
6
2,28
1
10
Уровень
восприятия
коррупции в стране
7,36
8
2,52
1
10
Уровень
восприятия
безопасности
2,2
2
0,811
1
4
Частота просмотра
ТВ новостей
1,71
1,00
1,21
1
5
Уровень доходов
4,59
5
2,19
2
10
Уровень
образования
4,85
5
1,84
1
8
В силу того, что зависимая переменная представляет собой порядковую (ранго-
вую) шкалу (варианты ответов
полностью доверяю в некоторой степени дове Уровень финансовой
удовлетворенности
Независимая
Теория
институциональной
эффективности
+
Уровень восприятия
коррупции в стране
Независимая
–
Уровень восприятия
безопасности
Независимая
+
Частота просмотра ТВ
новостей
Независимая
Авторитарная
теория массовой
коммуникации
+
Уровень доходов
Контрольная
Теория модернизации
–
Уровень образования
Контрольная
– Описательная статистика выборки представлена в таблице 2. Описательная статистика выборки представлена в таблице 2. Таблица 2. Описательная статистика по переменным (n = 1465)
Переменная
Среднее
Медиана
Станд. отклон. Минимум
Максимум
Доверие
к правительству
2,48
2
0,930
1
4
Уровень
межличностного
доверия
1,69
2
0,598
1
2
Степень доверия
к соседям
2,1
2
0,861
1
4
Уровень участия
в деятельности НКО
0,112
0
0,384
0
2
Уровень
финансовой
удовлетворенности
5,49
6
2,28
1
10
Уровень
восприятия
коррупции в стране
7,36
8
2,52
1
10
Уровень
восприятия
безопасности
2,2
2
0,811
1
4
Частота просмотра
ТВ новостей
1,71
1,00
1,21
1
5
Уровень доходов
4,59
5
2,19
2
10
Уровень
образования
4,85
5
1,84
1
8 Таблица 2. Описательная статистика по переменным (n = 1465) В силу того, что зависимая переменная представляет собой порядковую (ранго-
вую) шкалу (варианты ответов — полностью доверяю, в некоторой степени дове- RUSSIAN SOCIOLOGICAL REVIEW. 2023. Vol. 22. No. 3 205 ряю, не очень доверяю и совсем не доверяю), то методом анализа количественных
данных была порядковая логистическая регрессия. Анализ данных был осущест-
влен в прикладном программном пакете GRETL. *** p < 0,01; ** p < 0,05. Результаты исследования В данном разделе статьи мы тестируем влияние четырех объясняющих пере-
менных, отражающих уровень межличностного доверия, степень участия гра-
ждан в деятельности НКО, эффективность правительства, частоту просмотра
телевизионных новостей. Для проверки установленных в ходе исследования ги-
потез и определения наиболее значимых факторов, влияющих на доверие к пра-
вительству, была построена порядковая логистическая регрессионная модель
(см. табл. 3). После построения модели регрессии был сделан тест на обнаруже-
ние мультиколлинеарности. Для проверки регрессионной модели в программе
GRETL был использован метод инфляционных факторов (VIF). Самое большое
значение VIF фактора у переменной «Уровень дохода» 1,229. Таким образом,
можно сделать вывод об отсутствии мультиколлинеарности в построенной мо-
дели. Таблица 3. Результаты регрессионного анализа
Переменные
Коэффициенты
Стандарт. ошибка
t
P-значение
Уровень
межличностного
доверия
0,140
0,044
3,182
0,001***
Уровень доверия
к соседям
0,177
0,031
5,710
0,00***
Уровень участия
в деятельности НКО
0,029
0,067
0,428
0,668
Уровень финансовой
удовлетворенности
−0,054
0,012
−4,353
0,00***
Уровень восприятия
коррупции в стране
0,097
0,01
9,191
0,00***
Уровень восприятия
безопасности
0,023
0,032
0,730
0,464
Частота просмотра ТВ
новостей
0,098
0,021
4,552
0,00***
Уровень доходов
−0,000
0,013
−0,056
0,954
Уровень образования
0,031
0,014
2,199
0,027**
R-квадрат — 0,43
*** p < 0,01; ** p < 0,05. Таблица 3. Результаты регрессионного анализа Таблица 3. Результаты регрессионного анализа СОЦИОЛОГИЧЕСКОЕ ОБОЗРЕНИЕ. 2023. Т. 22. № 3 206 Из полученных коэффициентов регрессионных моделей, оценивающих взаи-
мосвязь между уровнем межличностного доверия и степенью политического дове-
рия, видно, что гипотеза о прямой связи между данными показателями в моделях
подтверждается. Иными словами, тезис о том, что политическое доверие — это
своего рода расширение межличностного доверия на институциональный объ-
ект, является верным. Данный вывод соответствует результатам предшествующих
исследований (Schifman, Telen, Sherman, 2010). Согласно результатам регрессионного анализа, неотоквилианский подход
не подтвердился, гипотеза была опровергнута. Этот вывод нуждается в опре-
деленной интерпретации. Важно сказать, что данный результат согласуется с ря-
дом предыдущих работ российских исследователей, где утверждается, что рос-
сийские НКО трудно назвать «школами демократии». Так, Л. Якобсон отмечает,
что «взаимоотношения внутри НКО… зачастую далеки от идеала “гражданских
добродетелей”. Третий сектор, выступая “ареалом” опережающей группы, сам на-
ходится в лучшем случае на полпути в “обучении демократии”» (Якобсон, 2014:
104). Ю. Скокова подчеркивает, что ряд отечественных НКО способствуют раз-
витию социального капитала их участников, имеют потенциал для взращивания
нового «демократического» поколения граждан, но экстраполировать это на весь
российский третий сектор преждевременно (Скокова, 2016: 65). Результаты исследования Исследователи
утверждают, что большинство НКО в авторитарных государствах нельзя назвать
«школами демократии» а-ля Токвиль, они с равным успехом могут быть и сторон-
никами автократий. НКО являются «органами-амфибиями», которые связывают
общество и государство. Если автократическим режимам удается кооптировать
их лидеров, ассоциации превращаются из противников в защитников существую-
щих режимов (Wischermann, Bunk, Köllner, Lorch, 2018). Для обозначения квази-
общественных организаций, работающих на интересы власти, был придуман
термин ГОНГО (GONGO, Government Organized Non-Government Organization) —
спонсируемые правительством неправительственные организации (Naim, 2009). Данный феномен «отражает реалии существования имитационных форм демо-
кратических институтов и соответствующих организаций в рамках незападных
политий» (Лушников, 2019: 138). Важно подчеркнуть, что гражданские ассоциации
могут способствовать формированию устойчивости авторитарного порядка и его
легитимации. В научной литературе отмечается, что гражданское общество и его
институты порой используются в качестве каналов опосредованного взаимодей-
ствия с населением как механизм обратной связи между гражданами и властью
(Бедерсон, 2020). Недавние исследования показывают, что не все добровольные
объединения производят положительный эффект. Как отмечают исследователи,
работы, выполненные в неотоквилианской традиции, рассматривают все добро-
вольные ассоциации одинаково, что некорректно, т.к. определенные типы добро-
вольных ассоциаций лучше способствуют достижению просоциальных результа-
тов, в то время как некоторые типы ассоциаций наносят ущерб тем же результатам
(Rothstein, Stolle, 2008). Участие в деятельности НКО может приводить к росту 207 RUSSIAN SOCIOLOGICAL REVIEW. 2023. Vol. 22. No. 3 межличностного доверия, доверия к членам группы, но не обобщенному доверию
к политическим институтам (Paxton, 2007). Необходимо обсудить принципиаль-
ную возможность не просто присутствия двусторонней направленности между
переменными, но и, особенно в современном российском контексте, рассмотреть
участие в деятельности НКО как следствие (а вовсе не причину) низкого уровня
доверия к правительству. Исследования показывают, что одним из мотивирующих
факторов, который стимулирует молодых людей оказывать помощь и поддержку
незащищенным гражданам России через неправительственные организации, —
это недоверие к государству в вопросах решения социальных проблем (Нежина
и др., 2014: 64). В рамках группы факторов, отобранных на основании теории институцио-
нальной эффективности, проверялись три гипотезы: 1) о положительном влиянии
уровня финансовой удовлетворенности; 2) о негативном воздействии коррупции;
3) о положительном влиянии восприятия безопасности на уровень доверия к пра-
вительству. Прямая связь между финансовым благополучием и политическим
доверием чаще всего воспринимается как прямое следствие хорошо известной
закономерности, согласно которой личный экономический успех усиливает вос-
приятие легитимности, лояльности и доверия к существующим политическим
институтам. Данная логика подтверждается предыдущими исследованиями (Gi-
ustozzi, Gangl, 2021; Lee, Chang, Hur, 2020). Существует устойчивая положительная
корреляция между социально-экономическим статусом и социальным доверием,
т. е. Результаты исследования бедные граждане сообщают о меньшем социальном доверии, чем их коллеги
с более высоким социально-экономическим статусом (Alesina, La Ferrara, 2002). Эти результаты работы опровергают теоретические ожидания, что обусловлено
спецификой российского «среднего класса» (среди ученых нет единого мнения
о том, какие критерии следует использовать при определении принадлежности
к этому классу, обычно применяют три широких критерия: доход и собственность,
образование и профессия, самоидентификация). Исследования показывают, что
многие представители данного класса в материальном плане зависят от государ-
ства и его бюджета (Gontmakher, Ross, 2015). Большинство участников российского
среднего класса, при всей спорности этого понятия, — это государственные служа-
щие, менеджеры высшего и среднего звена государственных корпораций, а также
предприниматели, принимающие активное участие в государственных тендерах
и закупках (Rosenfeld, 2017). Кроме того, полученные результаты регрессионного
анализа показывают, что коррупция повышает лояльность и политическое дове-
рие граждан, что противоречит рабочей гипотезе H3-2. Полученные результаты
представляют интерес с точки зрения интерпретации. Коррупцию можно рас-
сматривать как один из описанных ранее в социальной науке механизмов умень-
шения «сложности» и снижения рисков, что повышает уверенность одного лица
в ожиданиях будущих действий другого человека (Luhmann, 2018). Кроме того,
коррупция может быть выгодна гражданам из-за искажений, вызванных плохо
функционирующими институтами. Согласно теории «смазывания колес», кор- 208 СОЦИОЛОГИЧЕСКОЕ ОБОЗРЕНИЕ. 2023. Т. 22. № 3 рупция может обеспечить гражданам определенные выгоды, которые повышают
их доверие к политическим институтам. Коррупция — это способ избежать бюро-
кратических препятствий, особенно в странах с часто меняющимся законодатель-
ством (Bayley, 1967; Lef, 1964; Leys, 1965; Хантингтон, 2004). y y
y
В результате регрессионного анализа была обнаружена положительная связь
между просмотром новостей и политическим доверием. Иными словами, чем
чаще граждане смотрят новостные программы по ТВ, тем выше уровень дове-
рия российскому правительству. Это подтверждает четвертую гипотезу. Данный
вывод укладывается в доминирующий тезис, который существует в научной ли-
тературе, о том, что использование традиционных СМИ оказывает прямое поло-
жительное влияние на доверие к правительству (Marcinkowski, Starke, 2018). Что
касается влияния использования Интернета и социальных сетей на доверие к пра-
вительству, то оценки исследователей разные. Так, одни находят убедительные
доказательства значительного положительного влияния использования Интер-
нета на доверие пользователей сети к правительству (Lu et al., 2020), в то время как
другие — отрицательное (Im et al., 2014). Таким образом, первая и четвертая гипотезы были подтверждены, а вторая
и третья не прошли эмпирическую проверку. Такой результат можно объяснить
двумя взаимосвязанными причинами. Во-первых, некоторые вопросы в анкете
могут являться чувствительными для респондентов. Результаты исследования Страх перед государствен-
ными институтами может вызывать существенную и значительную предвзятость
в ответах, особенно на вопросы, касающиеся отношений между гражданами
и властью. Респонденты, как правило, дают неправдивые ответы на деликатные
вопросы. Если истинное мнение отклоняется от общепринятой в обществе нормы
и точки зрения, то опрашиваемый может сообщить социально одобряемый ответ
(Tannenberg, 2017). Респонденты могут почувствовать необходимость подверг-
нуть цензуре свои ответы, если (а) это вторжение в частные/интимные вопросы
(например, сексуальность или личный доход); (б) это вызывает ответы, которые
могут быть социально нежелательными или политически некорректными; (в) ре-
спондент опасается, что его ответ может иметь последствия в случае раскрытия
(Tourangeau, Yan, 2007). Во-вторых, в любом обществе люди склоны хранить мол-
чание, когда они чувствуют, что их взгляды противоречат мнению большинства
по какому-либо вопросу. Это явление в научной литературе получило название
«спирали молчания». Согласно существующей теории, граждане хранят молчание
по нескольким причинам: 1) страх изоляции, когда группа или общественность
осознает, что мнение отдельного человека расходится со сложившимся положе-
нием вещей; 2) страх репрессий, когда высказывание мнения может привести
к негативным последствиям, выходящим за рамки простой изоляции, например,
к потере работы (Ноэль-Нойман, 1996). В ходе исследования при работе с контрольными переменными было получено
несколько интересных результатов, которые требуют дальнейшей интерпретации. Во-первых, в ходе исследования выявлено, что уровень дохода граждан не явля- RUSSIAN SOCIOLOGICAL REVIEW. 2023. Vol. 22. No. 3 209 ется достаточным фактором, который мог бы объяснить уровень политического
доверия. С учетом того, что гипотеза H3-1 о положительном влиянии восприятия
финансовой удовлетворенности на институциональное доверие показала проти-
воположный эффект, представляется необходимым проведение дополнительных
научных изысканий. Во-вторых, модель, оценивающая влияние уровня образова-
ния на политическое доверие, значима. В то же время она показывает положи-
тельную взаимосвязь между переменными: граждане с более высоким уровнем
образования больше доверяют правительству. Это противоречит нашим теорети-
ческим ожиданиям. Заключение Доверие к другим лежит в основе социального, политического и экономическо-
го функционирования современного общества. Несмотря на его важность для
поддержания социальной структуры, истоки политического доверия остаются
неясными. В этой статье предпринята попытка проверить ряд объяснений того,
почему уровни доверия граждан к правительственным институтам в целом раз-
личаются. Эта работа началась с изложения четырех теоретических подходов,
которые преобладают в качестве объяснений политического доверия. Согласно
социально-психологическому подходу, семья является потенциальным источ-
ником таких установок, как доверие и нормы взаимности. В литературе по соци-
альной психологии отмечается, что семейный опыт будет оказывать значитель-
ное влияние на уровень доверия индивидов, отношение родителей к открытости
незнакомцам передается ребенку. Доверие является базовой личностной ха-
рактеристикой, приобретаемой в основном в раннем возрасте. Второй подход
опирается на теорию добровольных организаций. Следуя ее логике, участие
в гражданских объединениях порождает определенный тип межличностных
отношений, который лежит в основе доверия к политическим институтам. Су-
ществует подход, который утверждает, что институциональное доверие — это
положительная обратная связь от восприятия гражданами эффективности эко-
номики, политическое доверие есть продукт эгоистичных оценок институцио-
нальной продукции, интерпретируемых в терминах экономических показате-
лей. Наконец, авторитарная теория массовой коммуникации подразумевает, что
СМИ, большинство из которых находится под фактическим контролем государ-
ства, не разрешается печатать или транслировать что-либо, что могло бы подо-
рвать авторитет существующей политической системы, уменьшив тем самым ее
легитимность. Эти конкурирующие объяснения формирования институционального доверия
редко подвергались эмпирической проверке. В этом и заключается цель данной
статьи. Для этого мы обратились к данным всероссийского социологического
опроса, который был выполнен в рамках 7-й волны исследований Всемирного об-
зора ценностей в 2017–2022 гг. 210 СОЦИОЛОГИЧЕСКОЕ ОБОЗРЕНИЕ. 2023. Т. 22. № 3 Удалось достичь ряда результатов, определяющих новизну представленной
работы. Во-первых, осуществлено исследование взаимосвязи межличностного
и социального доверия с политическим доверием. Во-вторых, немногие из про-
шлых исследований проводились на узких выборках. В данной работе использова-
ны данные Всемирного обзора ценностей с выборкой в 1465 респондентов. Таким
образом, представленное исследование становится важным шагом к пониманию
влияния восприятия материального положения, коррупции, безопасности на до-
верие граждан РФ к правительству РФ. Результаты подтверждают гипотезу о том, что уровень доверия граждан к по-
литическим институтам в целом и к правительству в частности в значительной
степени положительно зависит от уровня межличностного и социального дове-
рия. Вопреки теоретическим ожиданиям, было установлено, что российские НКО
не являются «школами демократии», а также «инкубаторами» социального капи-
тала в целом. Иными словами, участие граждан в деятельности НКО не оказывает
влияния на институциональное доверие. Заключение Эмпирическая проверка теории инсти-
туциональной эффективности также не нашла своего подтверждения. Исследова-
ние показало, что просмотр ТВ новостей и политических программ положитель-
ным образом влияет на доверие к правительству. В ходе работы были даны ответы на все исследовательские вопросы, сформу-
лированные в начале исследования, а также проверены все гипотезы. Несмотря
на это, представленное исследование имеет ограничения. Следует отметить ис-
пользование данных социологического опроса только 7-й волны. Общественное
мнение имеет такое свойство, как изменчивость и динамичность. Общественные
настроения могут меняться под воздействием определенных факторов. Исходя
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Address: Lenin Avenue, 51, Ekaterinburg, Russian Federation 620083
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Distributed Faulty Node Detection in Delay Tolerant Networks: Design and Analysis
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IEEE transactions on mobile computing
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To cite this version: Wenjie Li, Laura Galluccio, Francesca Bassi, Michel Kieffer. Distributed Faulty Node Detection in
Delay Tolerant Networks: Design and Analysis. IEEE Transactions on Mobile Computing, 2018, 17
(4), pp.831-844. 10.1109/TMC.2017.2743703. hal-01576587 1
INTRODUCTION can still be detected, but identifying directly which node
produces erroneous measurements is not possible. Delay Tolerant Networks (DTN) are challenging networks
characterized by dynamic topology with frequent discon-
nections [1]. Examples of DTNs include Vehicular DTNs
(VDTNs) [2] where two nodes can communicate with each
other only when they are closely located. This connection is
intermittent as the nodes are moving vehicles. Due to this
sparse and intermittent connectivity, inference and learning
over DTNs is much more complicated than in traditional
networks, see, e.g., [3]–[8]. This paper presents a fully distributed and easily im-
plementable algorithm to allow each node of a DTN to
determine whether its own sensors are defective. We assume
as in [9] that nodes are not aware of the status (good or
defective) of their sensors, while their computation and
communication capabilities remain fine, even if some of
their sensors are defective. Most of the nodes of the DTN
are assumed to behave in a rational way and are willing to
know the status of their sensors. Some nodes, however, may
be misbehaving, trying to perturb the detection process. This paper considers the problem of distributed faulty
node detection (DFD) in DTNs. A node is considered as
faulty when one of its sensors frequently reports erroneous
measurements. The identification of such faulty nodes is
very important to save communication resources and to
prevent erroneous measurements polluting estimates pro-
vided by the DTN. This identification problem is quite
complicated in DTNs when interactions are mainly between
pairs of encountering nodes. Most of the classical DFD
algorithms are using measurements of spatially-correlated
physical quantities collected by many nodes to determine
the presence of outliers and identify the nodes producing
these outliers. In case of pairwise interactions, mismatch
between measurements provided by two different nodes As in [9]–[13], a Local Outlier Detection Test (LODT)
is assumed to be able to detect the presence of outliers in
a set of measurements, without necessarily being able to
determine which are the outliers. This is a typical situation
when only pairwise interactions are considered, where mea-
surements from sensors of only two nodes are compared. The generic LODT is characterized by its probabilities of
detection and false alarm. When two nodes meet, they
exchange their local measurements and use them to perform
the same LODT. •
W. Li, F. Bassi, and M. Kieffer are with Laboratoire des Signaux et
Systemes (L2S, UMR CNRS 8506) CNRS-CentraleSupelec-Universit¨ı¿œ
Paris-Sud 3, rue Joliot Curie 91192 Gif-sur-Yvette, France.
E-mail: firstname.name@lss.supelec.fr
•
L. Galluccio is with Dipartimento di Ingegneria Elettrica, Elettronica e
Informatica, University of Catania, Catania, Italy.
E-mail: laura.galluccio@dieei.unict.it
•
M. Kieffer is also with LTCI Telecom ParisTech, 75013 Paris, France and
Institut Universitaire de France, 75005 Paris, France
•
F. Bassi is also with ESME-Sudria, 94200 Ivry-sur-Seine, France Distributed Faulty Node Detection in
Delay Tolerant Networks: Design and Analysis Wenjie Li, Student Member, IEEE, Laura Galluccio, Member, IEEE,
Francesca Bassi, Member, IEEE, and Michel Kieffer, Senior Member, IEEE Abstract—Propagation of faulty data is a critical issue. In case of Delay Tolerant Networks (DTN) in particular, the rare meeting events
require that nodes are efficient in propagating only correct information. For that purpose, mechanisms to rapidly identify possible faulty
nodes should be developed. Distributed faulty node detection has been addressed in the literature in the context of sensor and
vehicular networks, but already proposed solutions suffer from long delays in identifying and isolating nodes producing faulty data. This
is unsuitable to DTNs where nodes meet only rarely. This paper proposes a fully distributed and easily implementable approach to
allow each DTN node to rapidly identify whether its sensors are producing faulty data. The dynamical behavior of the proposed
algorithm is approximated by some continuous-time state equations, whose equilibrium is characterized. The presence of misbehaving
nodes, trying to perturb the faulty node detection process, is also taken into account. Detection and false alarm rates are estimated by
comparing both theoretical and simulation results. Numerical results assess the effectiveness of the proposed solution and can be
used to give guidelines for the algorithm design. Index Terms—Delay Tolerant Networks; Fault detection; Iterative algorithms; Distributed estimation; Equilibrium analysis. Index Terms—Delay Tolerant Networks; Fault detection; Iterative algorithms; Distributed estimation; Equilibrium ! HAL Id: hal-01576587
https://centralesupelec.hal.science/hal-01576587v1
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INTRODUCTION A distributed ranking
among nodes is performed according to the performance of
their sensor. The proposed solution allows an identification
of defective nodes with sensors producing measurements
of high bias or high variance. Nevertheless, the proposed
solution highly relies on the measurement models and on
the communication conditions. A problem related to DFD in DTNs has been considered
in [21] in the context of VDTN. A large number of sensor
nodes are fixed and some vehicles, called mobile carriers
(MC) collect data from these sensors. The sensor nodes can
only communicate with the MCs in their vicinity. A MC
needs to collect enough measurements to perform a test
to decide which have been produced by defective sensors. Once a node is deemed defective by a MC, it is added to
its blacklist. The MC provides information to sensors about
their status. MCs also exchange their blacklists to accelerate
the faulty node detection. p
p
y
p
g
The rest of the paper is organized as follows. Section 2
discusses some related work. Section 3 presents the system
model and basic assumptions. Section 4 details the DFD
algorithm for DTNs. Section 5 introduces the Markov model
describing the behavior of the DFD algorithm and describes
the transition probabilities between the node states. Sec-
tion 6 develops the theoretical analysis of the macroscopic
evolution of the proportion of nodes in different states. Section 7 analyzes the properties of the equilibrium obtained
from the continuous-time state equations by approximating
the stochastic evolution of the proportions of nodes with
similar beliefs. Section 8 discusses the effect of having
misbehaving nodes in the system. Section 9 provides some
numerical results as well as a comparison with an alterna-
tive DFD algorithm and Section 10 concludes this paper. Notations are presented in Table 1. Proofs of propositions
and lemmas are available in the Appendix. y
In [22], a related problem of distributed malware detec-
tion in DTNs is addressed. Each node evaluates after a meet-
ing with another node whether the latter has performed
suspicious actions (malware transmission trial). When after
several meetings with Node j, Node i detects for a given
number of times suspicious activities, a cut-off decision
is performed against Node j, which is ignored in next
meetings. The main drawback of this approach is the long
time required to identify and isolate misbehaving nodes. 1
INTRODUCTION The LODT results help both nodes to
update their estimate of the status of their own sensors. When, for a given node, the proportion of meetings during
which the LODT suggests the presence of outliers is larger
than some threshold, this node decides its sensors may be
defective. In this case, it becomes silent. Accordingly, it does
not transmit any more its measurements to its neighbors,
but keeps collecting measurements from nodes met and
updates the estimate of the status of its sensors. It may then
have the opportunity to change its estimate and communi-
cate again. Although the LODT considered here are those
of [9], this work differs significantly from [9] due to the
communication conditions of DTNs, which require a totally 2 different DFD algorithm. The analysis of the properties of
the algorithm is also totally different. This paper shows
that the behavior of the proposed DFD algorithm can be
described using Markov models and tools borrowed from
control theory and population dynamics. these measurements. Applying directly classical DFD algo-
rithms in DTNs may thus be quite ineffective. Moreover,
usually the performance of DFD algorithms is characterized
experimentally. A theoretical analysis of the equilibrium
and convergence properties of these algorithms is seldom
performed. More in depth, the belief of each node about the status
of its sensors is quantized. The evolution of these quan-
tized beliefs are then shown to follow two Markov chains. A continuous-time approximation of the evolution of the
proportion of nodes with similar beliefs is then derived. Sufficient conditions on the decision parameters to ensure
the existence and uniqueness of an equilibrium of the DFD
algorithm are then provided. Given the characteristics of
the LODT, upper and lower bounds of the detection rate, i.e.,
proportion of nodes which have effectively identified their
sensors as defective, and of the false alarm rate, i.e., propor-
tion of nodes which believe that their good sensors are in
fact defective, are also obtained. The impact of misbehaving
nodes, trying to perturb the results of the DFD algorithm, is
also taken into account. These results provide guidelines to
properly choose the parameters of the DFD algorithm. In the context of distributed estimation via consensus in
a WSN, [17]–[20] have considered the simultaneous estima-
tion of a common quantity from measurements corrupted by
different levels of bias or of variance. 1
INTRODUCTION Misbehavior detection in DTNs is also considered in [6],
[23], where the DTN is perturbed by routing misbehavior
caused by selfish or malicious nodes. The identification
approach in [6] is not distributed, since a trusted author-
ity periodically checks the forwarding history of nodes
to identify possible misbehavior. A collaborative approach
is proposed in [23], where each node can detect whether
the encountered node is selfish using a local watchdog. The detection result is disseminated over the network to
increase the detection precision and to reduce the delay. Trust/Reputation management is another important aspect
to help DTNs to resist various potential threats. For exam-
ple, [24] provides an iterative trust management mechanism
to fight against Byzantine attacks in which several nodes
are totally controlled by the adversary. In [25], a defense
against Sybil attacks [26] is introduced, which is based on
the physical feature of the wireless propagation channel. A
trust model in the scenario of underwater acoustic sensor
networks is presented in [27] to take into account several
trust metrics such as link trust, data trust, and node trust. 2
RELATED WORK DFD is a well-investigated topic when considering Wire-
less Sensor Networks (WSNs) (see [14]–[16] and references
therein). The WSNs considered in most of the papers are
dense and have a static topology. DFD in DTNs is much
less investigated. Classical DFD algorithms usually consist
of two phases. First, an LODT is performed using data
collected from neighboring nodes. LODTs (based on ma-
jority voting [10], the median [11], or the mean [12] of the
measurements, the modified three-sigma edit test [13], etc.)
aim to decide which data is erroneous. Second, the outcomes
of the LODTs are disseminated to improve the decision
accuracy. Nevertheless, when LODTs have to process measure-
ments from two or three nodes only, it becomes difficult
to identify the defective nodes. It may, however, still be
possible to detect inconsistencies among measurements due
to the presence of a node producing outliers. This is a
typical situation in DTNs when there are mainly pairwise
interactions: two nodes meet, take measurements, and share In this paper, differently from previous works in the
field, we consider that in a distributed way each node
performs a self-determination on whether its sensors are
producing outliers in the context of DTNs. In this case, new
issues arise, mainly related to the limited proximity time 3 TABLE 1
Symbols used in this paper
S0, S1, S2
sets of good, defect., and misbehav. 2
RELATED WORK nodes
nS
number of nodes
θi
status of node i
bθi
estimate of θi
nθ
number of nodes with status θ
pθ
proportion of nodes with status θ
pθbθ
proportion of nodes with status θ and
estimating their status as bθ
pθbθ
value of pθbθ at equilibrium
epθbθ
approximate value of pθbθ at equilibrium
λ
inter-contact rate
ν
decision threshold
t
time
yi
outcome of a LODT performed by node i
qD
detection probability of a LODT
qFA
false alarm probability of a LODT
cm,i
number of LODTs performed by node i
cd,i
number of LODTs by node i resulting in a
detection of outliers
M
number of previous LODT results
considered for the decision
xi
state of node i, containing θi, cm,i, and cd,i
πδm,δd
θ
(t, cm, cd)
transition probability from state (θ, cm, cd)
to state (θ, cm + δm, cd + δd)
Xcm,cd
θ
proportion of nodes of actual status θ with
state xi = (θ, cm, cd)
Xcm,cd
θ
expected value of Xcm,cd
θ
X
cm,cd
θ
value of Xcm,cd
θ
at equilibrium
e
Xcm,cd
θ
approximate value of Xcm,cd
θ
at equilibrium TABLE 1
Symbols used in this paper their neighbors indicating that their sensors are good. More-
over, they try to provide measurements to the encountered
nodes so that the LODTs performed by these nodes lead to
the outcome of identifying outliers. Our aim is (i) to design a distributed algorithm so that
each node i rapidly evaluates an accurate estimate bθi of its
own status θi despite the presence of misbehaving nodes,
(ii) to provide a theoretical analysis of the behavior of this
algorithm. 3.2
Local outlier detection test As in [9], we consider a family of LODTs able to detect
the presence of outliers in a set of n data measurements
M = {m1, . . . , mn} but unable to identify which data is
an outlier. Denote y (M) the outcome of the LODT, i.e.,
y (M) = 1 if data corresponding to outliers are detected
within M, otherwise, y (M) = 0. LODTs can take various forms (see [9] and Example 1
below). The LODT is characterized by a false alarm probability
qFA (the probability of having y (M) = 1 under the condi-
tion that none of the data in M are produced by defective
sensors) and by a detection probability qD (the probability of
having y (M) = 1 under the condition that some data in M
are really produced by defective sensors). In M, let n0 be
the number of data produced by good sensors and n1 be the
number of data coming from defective sensors. We further
assume that both qD and qFA in average depend only on the
number of data involved in the LODT. As a consequence,
we can denote qFA as qFA (n0) and qD as qD (n0, n1). Each
node performing a LODT on a set of data has not to know
n0 and n1, but the performance of the LODT will depend
on the actual values of n0 and n1, which are used in the
analysis of the DFD algorithm. of DTN nodes and the sporadic contacts which call for the
consideration of the history of contacts in the identification
process. Also, we provide a mathematical characterization
of the problem and prove the convergence of the algorithm. 3
SYSTEM MODEL Consider a set S of nS moving nodes equipped with sensors. S can be partitioned into three subsets, S0, S1, and S2. S0 contains all nodes with good sensors. S1 is the subset
of nodes with defective sensors producing outliers, i.e., mea-
surements corrupted by a noise which has characteristics
significantly different from those of the noise corrupting
measurements provided by good sensors. Finally, S2 rep-
resents the set of misbehaving nodes, trying to voluntarily
perturb the behavior of the network. Example 1. This example introduces a LODT inspired from
bounded-error parameter estimation problem (see, e.g., [30]–
[32]). It assumes that only pairwise interactions occur. Con-
sider some sensor nodes taking temperature measurements
in the same room, e.g., with actual value t = 25◦C. For a
non-defective sensor, suppose that its measurement error
is bounded, e.g., ±1◦C. Assume that two sensors provide
t1 = 25.6◦C and t2 = 23.5◦C respectively. Assuming
that none of the sensors is defective, and considering the
bounded measurement noise, one deduces that t ∈t1 =
[t1 −1, t1 + 1] = [24.6, 26.6] and t ∈t2 = [22.5, 24.5]. Since
t1 ∩t2 = ∅, there exists at least one outlier, but one is unable
to determine which sensor has produced an outlier. The status of node i is θi(t) = 0 (good node) if all its
sensors are good, θi(t) = 1 (defective node) if at least one of
them is defective, and by convention θi(t) = 2 (misbehaving
node). The proportion and number of nodes with status θ
are respectively pθ and the number of nodes in status θ
is nθ = pθnS, with p0 + p1 + p2 = 1. All nodes, except
misbehaving nodes, are not aware of their own status. In
what follows, we assume that over the time horizon of
the experiment, the status of sensors does not change, i.e.,
θi(t) = θi. Misbehaving nodes aim at disrupting network opera-
tions by causing congestion along paths, unreliable packet
delivery, or erroneous data delivery [6], [24], [28]. Here, we
assume that misbehaving nodes always transmit data to 3.1
Communication model Nodes can exchange information only during the limited
time interval in which they are in vicinity. As in [7], [8], [23],
[29], we assume that the time interval between two succes-
sive meetings follows an exponential distribution with an
inter-contact rate λ. Moreover, we assume that each meeting
involves only two nodes. When more than two nodes meet
simultaneously, processing is performed pair-by-pair. 4
DFD ALGORITHM a)
µ = µ + 1. Perform a LODT with outcome
yµ
i . b)
Update cm,i and cd,i as
(
cm,i(tκ
i ) = min {µ, M} ,
cd,i(tκ
i ) = Pµ
m=max{1,µ−M+1} ym
i . (5) a)
µ = µ + 1. Perform a LODT with outcome
yµ
i . yi
b)
Update cm,i and cd,i as (
cm,i(tκ
i ) = min {µ, M} ,
cd,i(tκ
i ) = Pµ
m=max{1,µ−M+1} ym
i . (5) (5) c)
Update bθi using (4). 6)
κ = κ + 1. Algorithm 2 is analyzed in the next sections. Algorithm 2 is analyzed in the next sections. node is (M + 1) (M + 2) /2. The evolution of xi (t), condi-
tioned by its status θi, follows a Markov model with state
transition diagram of the kind shown in Figure 1 for M = 4. 3.3
Detection scenario 2)
Do
bθi (t) = bθi
tκ−1
i
,
cm,i (t) = cm,i
tκ−1
i
,
cd,i (t) = cd,i
tκ−1
i
,
(1)
t = t + δt
(2) (1) (2) until the κ-th meeting occurs at time t = tκ
i with
Node jκ ∈S \ {i}. j
\ { }
3)
Perform local measurement of data mi (tκ
i ). b j
\ { }
3)
Perform local measurement of data mi (tκ
i ). b 4)
If bθi (tκ
i ) = 0, then transmit mi (tκ
i ) to node jκ. 4)
If bθi (tκ
i ) = 0, then transmit mi (tκ
i ) to node jκ. 5)
If data mjκ have been received from node jκ, then 5)
If data mjκ have been received from node jκ, then a)
Perform a LODT with outcome yi (tκ
i ). b)
U d
d
di a)
Perform a LODT with outcome yi (tκ
i ). b)
d
d
d 4
DFD ALGORITHM ( i )
b)
Update cm,i and cd,i according to ( i )
b)
Update cm,i and cd,i according to In the proposed DFD algorithm, each (good or defective)
node i manages two counters cm,i(t) and cd,i(t) initialized
at 0 at t = 0. Using cm,i(t), node i counts the number of
meetings during which it has received data from its neighbor,
and has been able to perform a LODT. Using cd,i(t), it counts
the number of LODT resulting in the detection of outliers. When cd,i(t)/cm,i(t) ⩾ν, where ν is some constant deci-
sion threshold, node i considers itself as carrying defective
sensors, i.e., it sets its own estimate bθi (t) = 1. Otherwise, it
considers that its sensors are good, i.e., bθi (t) = 0. (
cm,i(tκ
i ) = cm,i(tκ−1
i
) + 1
cd,i(tκ
i ) = cd,i(tκ−1
i
) + yi (tκ
i )
(3) (3) c)
Update bθi as follows c)
Update bθi as follows bθi(tκ
i ) =
(
1
if cd,i(tκ
i )/cm,i(tκ
i ) ⩾ν,
0
else. (4) bθi(tκ
i ) =
(
1
if cd,i(tκ
i )/cm,i(tκ
i ) ⩾ν,
0
else. (4) (4) When a node with bθi (t) = 1 encounters another node, it
still takes measurements, but it does not send these data to
the other node to avoid infecting the network with outliers. Any node, upon receiving data from another node, performs
a LODT and updates cm,i(t) and cd,i(t). As a consequence,
a node which meets another node considering itself as
defective, transmits its data, but since it does not receive
any data, it does not update cm,i(t) and cd,i(t) at the end
of the meeting. Algorithm 1 summarizes the proposed DFD
technique for an arbitrary reference node i. Algorithm 2 Sliding-Window DFD algorithm for node i Algorithm 2 Sliding-Window DFD algorithm for node i
1)
Initialize t0
i = 0, bθi
t0
i
= 0, cm,i(t0
i ) = cd,i(t0
i ) = 0,
κ = 1, and µ = 0. 2)
Do (1)-(2) until the κ-th meeting occurs at time tκ
i
with Node jκ ∈S \ {i}. 3)
Perform local measurement of data mi (tκ
i ). 4)
If bθi (tκ
i ) = 0, then transmit mi (tκ
i ) to node jκ. 5)
If data mjκ have been received from node jκ, then
a)
µ = µ + 1. 4
DFD ALGORITHM Perform a LODT with outcome
yµ
i . b)
Update cm,i and cd,i as
(
cm,i(tκ
i ) = min {µ, M} ,
cd,i(tκ
i ) = Pµ
m=max{1,µ−M+1} ym
i . (5)
c)
Update bθi using (4). 6)
κ = κ + 1. 7)
Go to 2. 1)
Initialize t0
i = 0, bθi
t0
i
= 0, cm,i(t0
i ) = cd,i(t0
i ) = 0,
κ = 1, and µ = 0. 1)
Initialize t0
i = 0, bθi
t0
i
= 0, cm,i(t0
i ) = cd,i(t0
i ) = 0,
κ = 1, and µ = 0. κ = 1, and µ = 0. 2)
Do (1)-(2) until the κ-th meeting occurs at time tκ
i
with Node jκ ∈S \ {i}. 3)
Perform local measurement of data mi (tκ
i ). 4)
If bθi (tκ
i ) = 0, then transmit mi (tκ
i ) to node jκ. 5)
If data mjκ have been received from node jκ, then
a)
µ = µ + 1. Perform a LODT with outcome
yµ
i . b)
Update cm,i and cd,i as
(
cm,i(tκ
i ) = min {µ, M} ,
cd,i(tκ
i ) = Pµ
m=max{1,µ−M+1} ym
i . (5)
)
d
bθ
( ) µ
2)
Do (1)-(2) until the κ-th meeting occurs at time tκ
i
with Node jκ ∈S \ {i}. \ { }
3)
Perform local measurement of data mi (tκ
i ). b 4)
If bθi (tκ
i ) = 0, then transmit mi (tκ
i ) to node jκ. 5)
If data mjκ have been received from node jκ, then The vector xi(t) = (θi, cm,i(t), cd,i(t)) represents the
(microscopic) state of each node i. As t →∞, one has
cm,i(t) →∞, which leads to an infinite number of possible
values for xi(t) and the global (macroscopic) behavior of
the algorithm is difficult to analyze. To limit the number of
possible states, we have chosen to consider the evolution
of cm,i(t) and cd,i(t) over a sliding time window containing
the time instants of the last M meetings during which node i
has performed a LODT. Algorithm 2 is a modified version of
Algorithm 1 accounting for this limited horizon. It involves
an additional counter µ for the number of LODT performed
by node i. As long as µ < M, (5) is equivalent to (3). 3.3
Detection scenario We assume that during each meeting of a pair of nodes
(i, j) ∈S, the nodes collect data with their sensors. Each 4 Algorithm 1 DFD algorithm for node i
1)
Initialize at t0
i = 0, bθi
t0
i
= 0, cm,i(t0
i ) = cd,i(t0
i ) =
0, κ = 1. 2)
Do
bθi (t) = bθi
tκ−1
i
,
cm,i (t) = cm,i
tκ−1
i
,
cd,i (t) = cd,i
tκ−1
i
,
(1)
t = t + δt
(2)
until the κ-th meeting occurs at time t = tκ
i with
Node jκ ∈S \ {i}. 3)
Perform local measurement of data mi (tκ
i ). 4)
If bθi (tκ
i ) = 0, then transmit mi (tκ
i ) to node jκ. 5)
If data mjκ have been received from node jκ, then
a)
Perform a LODT with outcome yi (tκ
i ). b)
Update cm,i and cd,i according to
(
cm,i(tκ
i ) = cm,i(tκ−1
i
) + 1
cd,i(tκ
i ) = cd,i(tκ−1
i
) + yi (tκ
i )
(3)
c)
Update bθi as follows
bθi(tκ
i ) =
(
1
if cd,i(tκ
i )/cm,i(tκ
i ) ⩾ν,
0
else. (4)
6)
κ = κ + 1. 7)
Go to 2. Algorithm 1 DFD algorithm for node i node may or may not transmit its data to the other node,
as detailed in the DFD algorithm description. If a node has
received data from its neighbor, it may run a LODT involv-
ing its own data and those received from its neighbor. We
assume that the spatial and temporal correlation between
data is such that only data collected during the meeting of
two nodes can be exploited by a LODT. Therefore, previ-
ously collected data are not exploited. As a consequence,
contrary to [9], where n0 and n1 may be large, in the
DTN scenario, a LODT will involve n0, n1 ∈{0, 1, 2}, with
n0 + n1 = 2. In this paper, one furthermore assumes that
qFA (2) < qD (1, 1) ⩽qD (0, 2), which is reasonable, since the
outcome of a LODT is more likely to be 1 as the number of
outliers involved increases. 1)
Initialize at t0
i = 0, bθi
t0
i
= 0, cm,i(t0
i ) = cd,i(t0
i ) =
0, κ = 1. where Similarly, one may introduce the random event Eθ
3(t) =
n
Yi (t) = 0 | θi = θ, bθJ (t) = 0
o
, and show that Icm,cd
θ
(t) = {i : i ∈Sθ, θi = θ, cm,i(t) = cm, cd,i(t) = cd} , and | · | denotes the cardinality of a set. and | · | denotes the cardinality of a set. P
n
Eθ
3 (t)
o
=
( p0(1−qFA(2))p00(t)+p1(1−qD(1,1))p10(t)
p0p00(t)+p1p10(t)
,
if θ = 0,
p0(1−qD(1,1))p00(t)+p1(1−qD(0,2))p10(t)
p0p00(t)+p1p10(t)
,
if θ = 1. (15) | |
y
Assuming that the nodes are randomly spread, the
probability that node J believes it has only good sensors
conditioned to its true status is (15) pθ0 (t) = P
bθJ (t) = 0|θJ (t) = θ
,
(8)
P {E1(t)} = p0p00 (t) + p1p10 (t) . (9) (8) and then P {E1(t)} = p0p00 (t) + p1p10 (t) . (9) 5.1.2
Probability of detecting the presence of an outlier 5.1.2
Probability of detecting the presence of an outlier Since node i performs an LODT only when it meets a node J
with bθJ (t) = 0, one introduces the random event Eθ
2(t) =
n
Yi (t) = 1 | θi = θ, bθJ (t) = 0
o
, for the reference node with
actual status θ. As discussed in Section 3.2, the statistical
properties of the outcome Yi (t) of the LODT depend only
on θi and θj. For example, when node i has good sensors,
one has Fig. 1. Example of Markov model for the evolution of the state
θ, cm,i (t) , cd,i (t)
of a node when M = 4. 5.1
Analysis of some random events P
E0
2(t)
=
1
X
ϕ=0
P
n
Yi (t) = 1, θJ = ϕ | θi = 0, bθJ (t) = 0
o
(a)
=
1
X
ϕ=0
P {Yi (t) = 1 | θi = 0, θJ = ϕ} P
n
θJ = ϕ | bθJ (t) = 0
o
(b)
= p0qFA (2) p00 (t) + p1qD (1, 1) p10 (t)
p0p00 (t) + p1p10 (t)
. (12) In order to estimate the transition probability from a generic
state of the Markov chain to another, one first calculates
the probability a given node meets an other node believing
its status good or its status is bad. One also evaluates the
probability to perform a LODT outcome concluding in the
absence or in the presence of outliers. (12) In (12-a), one uses the fact that the LODT outcome is not
influenced by the estimate of the status of a node and that
in P
n
θJ = ϕ | θi = 0, bθJ (t) = 0
o
, the status of node J, does
not depend on θi. In (12-b), In (12-a), one uses the fact that the LODT outcome is not
influenced by the estimate of the status of a node and that
in P
n
θJ = ϕ | θi = 0, bθJ (t) = 0
o
, the status of node J, does
not depend on θi. In (12-b), 5.1.1
Probability of meeting a node believing its status is
good/defective Assume that at time t, some reference node i meets an other
node which index is represented by the random variable J
and define the random event E1(t) =
n
bθJ (t) = 0
o
, repre-
senting the event that the node met believes its status is
good. As illustrated in (4), among the nodes with status θ,
the proportion of nodes that believe themselves as good is1 (
P {Yi (t) = 1 | θi = 0, θJ = 0} = qFA (2) ,
P {Yi (t) = 1 | θi = 0, θJ = 1} = qD (1, 1) . (13) (13) Moreover, 5
EVOLUTION OF THE STATE OF A NODE In this section, to simplify the presentation, the presence of
misbehaving nodes is not taken into account. The impact of
such nodes on the behavior of Algorithm 2 will be detailed
in Section 8. In particular, there are two chains, one conditioned
by θi = 0 and the other conditioned by θi = 1. Both
are characterized by a transient phase for state values
with cm,i(t) < M; then, a permanent regime starts when
cm,i(t) = M. With cm,i (t) = cm and cd,i (t) = cd, the tran-
sitions from State (θ, cm, cd) to State (θ, c′
m, c′
d) are analyzed
in the following. Consider the state xi (t) = (θi, cm,i (t) , cd,i (t)) of node i. Since cm,i (t) ∈{0, . . . , M} and cd,i (t) ∈{0, . . . , cm,i (t)},
the number of values that may be taken by the state of a 5 5 0
0
1
2
3
4
1
2
3
4
cd
cm
Fig. 1. Example of Markov model for the evolution of the state
θ, cm,i (t) , cd,i (t)
of a node when M = 4. 0
0
1
2
3
4
1
2
3
4
cd
cm where p01 (t) and p11 (t) represent the false alarm rate (FAR)
and the detection rate (DR) respectively. From (10), one gets (11) 1. For the sake of simplicity, the dependency of pθ0 (t) in ν is omitted,
as ν is constant during the DFD algorithm. Moreover, P
n
θJ = ϕ | bθJ (t) = 0
o
=
P
n
bθJ (t)=0|θJ =ϕ
o
P{θJ = ϕ}
P1
φ=0P
n
bθJ (t)=0|θJ =φ
o
P{θJ = φ}
=
pϕpϕ0 (t)
p0p00 (t) + p1p10 (t). pθ0 (t) = X0,0
θ
(t) +
X
cm>0,cd:cd/cm<ν
Xcm,cd
θ
(t) ,
(6) (6) where p10 (t) is in fact the non-detection rate (NDR) of the
nodes with defective sensors at time t and Xcm,cd
θ
(t) repre-
sents the proportion of nodes in state (θ, cm, cd) defined as If node i has defective sensors, one has P
E1
2(t)
= p0qD (1, 1) p00 (t) + p1qD (0, 2) p10 (t)
p0p00 (t) + p1p10 (t)
. (14) Xcm,cd
θ
(t) = |Icm,cd
θ
(t) |/nθ,
(7) Xcm,cd
θ
(t) = |Icm,cd
θ
(t) |/nθ, (7) (14) where 5.2.1
Case I, cm,i(t) < M Considering the same assumption used to get (21), one has ,
In the transient regime, when cm,i(t) < M, cm,i(t) and
cd,i(t) are updated according to (3) whenever node J with
bθJ (t) = 0 is met. The only possibility that leads to δm = 0
is the event E∗
1 , i.e., node i meets node J with bθJ (t) = 1. As
a consequence, no LODT is performed by node i. Therefore,
for any θ ∈{0, 1}, In the transient regime, when cm,i(t) < M, cm,i(t) and
cd,i(t) are updated according to (3) whenever node J with
bθJ (t) = 0 is met. The only possibility that leads to δm = 0
is the event E∗
1 , i.e., node i meets node J with bθJ (t) = 1. As
a consequence, no LODT is performed by node i. Therefore,
for any θ ∈{0, 1}, In the transient regime, when cm,i(t) < M, cm,i(t) and
cd,i(t) are updated according to (3) whenever node J with
b In the transient regime, when cm,i(t) < M, cm,i(t) and
cd,i(t) are updated according to (3) whenever node J with
b P
E0
4 (t)
= 1 −P
E1
4 (t)
≈M −cd
M
. (23) (23) bθJ (t) = 0 is met. The only possibility that leads to δm = 0
is the event E∗
1 , i.e., node i meets node J with bθJ (t) = 1. As
a consequence, no LODT is performed by node i. Therefore,
for any θ ∈{0, 1}, Assume that the (µ −M)-th LODT performed by node i
occurred at time ˜t, then yµ−M
i
can also be denoted as yi
˜t
and the transition related to cd,i is such that δd = yi (t) −
yi
˜t
∈{−1, 0, 1} . Assume that the (µ −M)-th LODT performed by node i
occurred at time ˜t, then yµ−M
i
can also be denoted as yi
˜t
and the transition related to cd,i is such that δd = yi (t) −
yi
˜t
∈{−1, 0, 1} . 5.2.2
Case II, cm,i(t) = M 5.2.2
Case II, cm,i(t) = M In this section, we have so far completely characterized
the transition probabilities between any possible pair of
states in the Markov chain. Accordingly, we are now able
to completely describe the evolution of the DTN state com-
ponents and, thus, the expected proportion of nodes in a
specific state. In the permanent regime, cm,i(t) = M and does not increase
any more, thus δm = 0. In Algorithm 2, µ is the number of
LODTs performed by node i up to time t. When µ ⩾M, only
the last M LODT outcomes are considered: LODT outcomes
ym
i
with m ⩽µ −M are no more considered. yi
µ
To determine the value taken by δd ∈{−1, 0, 1} after the
µ-th LODT, consider the random event E1
4 (t) =
(
Y µ−M
i
= 1 |
µ−1
X
m=µ−M
Y m
i
= cd
)
,
(20) 5.2
Transition probabilities One evaluates now the transition probabilities for the
state
of
a
node. More
specifically,
define
as
πδm,δd
θ
the
transition
probability
from
State
(θ, cm, cd)
to
State (θ, cm + δm, cd + δd), where θ
∈
{0, 1}. One has
δm ∈{0, 1} since cm may either increase (δm = 1) in the
transient regime or remain constant (δm = 0) in the perma-
nent regime. One has δd ∈{−1, 0, 1}, depending on the
value of the last LODT outcome and on the value of the
M + 1-th last LODT outcome, which is no more considered
in the permanent regime. Similarly, introduce E∗
1 (t) = {bθJ (t) = 1}, representing
the event that the node met believes its status is defective. Among the nodes with sensors in status θ, the proportion of
d
ith bθ (t)
1 i g
nodes with bθj (t) = 1 is g
nodes with bθj (t) = 1 is pθ1 (t) =
X
cm>0,cd:cd/cm⩾ν
Xcm,cd
θ
(t) ,
(10) (10) 1. For the sake of simplicity, the dependency of pθ0 (t) in ν is omitted,
as ν is constant during the DFD algorithm. 6 )}.are independently distributed over the time horizon corre-
sponding to m = µ −M, . . . , µ −1. One obtains then Thus, (δm, δd) ∈{(0, 0) , (0, 1) , (0, −1) (1, 0) , (1, 1) , (1, −1
Note that πδm,δd
θ
depends on the current state of the reference
node, but also on the current proportion of active (good
and defective) nodes. Therefore, the transition probabilities
are denoted as πδm,δd
θ
(t, cm, cd), where t is the time instant,
cm,i(t) = cm, and cd,i(t) = cd. Depending on the value of
cm, two different cases are considered in Section 5.2.1 and
in Section 5.2.2, respectively corresponding to the transient
and permanent regimes. P
E1
4 (t)
= cd
M . (21) (21) This approximation is exact in steady-state, when the Xcm,cd
θ
s
do not vary any more. Similarly, define y
y
Similarly, define E0
4 (t) =
(
Y µ−M
i
= 0 |
µ−1
X
m=µ−M
Y m
i
= cd
)
. (22) (22) 5.2.1
Case I, cm,i(t) < M 5.2.1
Case I, cm,i(t) < M Thus, the transition probability is π1,1
θ (t, cm, cd)=
(
p0qFA (2) p00 (t) + p1qD (1, 1) p10 (t) ,
if θ = 0,
p0qD (1, 1) p00 (t) + p1qD (0, 2) p10 (t) , if θ = 1. (18)
Finally, π1,0
θ
(t, cm, cd) = P
n
Yi (t) = 0, bθJ (t) = 0|θi = θ
o
is
obtained similarly from (15) π1,1
θ (t, cm, cd)=
(
p0qFA (2) p00 (t) + p1qD (1, 1) p10 (t) ,
if θ = 0,
p0qD (1, 1) p00 (t) + p1qD (0, 2) p10 (t) , if θ = 1. (18) π0,−1
θ
(t, M, cd) = P{E1 (t)}P{Eθ
3 (t)}P{E1
4 (t)}
=
( p0(1−qFA (2)) p00 (t) + p1(1−qD (1, 1)) p10 (t)
cd
M , if θ = 0,
p0(1−qD (1, 1)) p00 (t) + p1(1−qD (0, 2)) p10 (t)
cd
M ,if θ = 1. (26) Finally, π1,0
θ
(t, cm, cd) = P
n
Yi (t) = 0, bθJ (t) = 0|θi = θ
o
is
obtained similarly from (15) π1,0
θ
(t, cm, cd) = (
p0 (1 −qFA (2)) p00 (t) + p1 (1 −qD (1, 1)) p10 (t) ,
if θ = 0,
p0 (1 −qD (1, 1)) p00 (t) + p1 (1 −qD (0, 2)) p10 (t) , if θ = 1. (19) Finally, by substituting eqs. (25-26) it is possible to calcu-
late π0,0
θ
(t, M, cd) which is given by
π0,0
θ
(t, M, cd) = 1 −π0,1
θ
(t, M, cd) −π0,−1
θ
(t, M, cd) . (27) Finally, by substituting eqs. (25-26) it is possible to calcu-
late π0,0
θ
(t, M, cd) which is given by π0,0
θ
(t, M, cd) = 1 −π0,1
θ
(t, M, cd) −π0,−1
θ
(t, M, cd) . (27) (27) 5.2.2
Case II, cm,i(t) = M 5.2.1
Case I, cm,i(t) < M π0,0
θ
(t, cm, cd) = P {E∗
1 (t)} = p0p01 (t) + p1p11 (t) ,
(16) π0,0
θ
(t, cm, cd) = P {E∗
1 (t)} = p0p01 (t) + p1p11 (t) ,
(16)
where pθ1 (t) is defined by (10) (16) y
{
, , }
To have (δm, δd) = (0, 1), three independent events have
to occur: 1) the encountered node J believes it is good at
time t, i.e., E1 (t); 2) yi (t) = 1, i.e., Eθ
3 (t) (t); 3) yi
˜t
= 0,
i.e., E0
4 (t). Thus the transition probability may be expressed
as where pθ1 (t) is defined by (10). ( )
y (
)
A state transition occurs with (δm, δd) = (1, 1) when
node i with status θi = θ meets node J with bθJ (t) = 0
and when the LODT yields yi (t) = 1. Since the two events
are independent, one has as
π0,1
θ
(t, M, cd) = P{E1 (t)}P{Eθ
3 (t)}P{E0
4 (t)}. (24)
Using (9), (12), (14), and (21) in (24), one gets (24) π1,1
θ
(t, cm, cd) = P
n
Yi (t) = 1, bθJ (t) = 0|θi = θ
o
= P {E1 (t)} P
n
Eθ
2 (t)
o
. (17) π0,1
θ
(t, M, cd)
( (17) (17) =
( p0qFA(2) p00 (t) + p1qD(1, 1) p10 (t)
M−cd
M
,
if θ = 0,
p0qD(1, 1) p00 (t) + p1qD(0, 2) p10 (t)
M−cd
M
, if θ = 1. (25) =
( p0qFA(2) p00 (t) + p1qD(1, 1) p10 (t)
M−cd
M
,
if θ = 0,
p0qD(1, 1) p00 (t) + p1qD(0, 2) p10 (t)
M−cd
M
, if θ = 1. (25) (25) Depending on the value of θi, using (9), (12), and (14), one
may rewrite (17) as Depending on the value of θi, using (9), (12), and (14), one
may rewrite (17) as Consider now (δm, δd) = (0, −1). To have such transi-
tion, the three following independent events should occur:
1) E1 (t); 2) yi (t) = 0, i.e., Eθ
3 (t) (t); 3) yi
˜t
= 1, i.e., E1
4 (t). 7.2
Existence and uniqueness of the equilibrium point As a consequence, Xθ (t) converges in probability to Xθ (t)
as nθ goes to infinity. This is typically the approximation
performed in the seminal work [35] where the SIR model
was proposed. This model is the one used to characterize
most widely studied classes of epidemic models. Accord-
ingly, analogously to what was presented for example in
[7], [35]–[40], the proposed system consists of ordinary dif-
ferential equations approximating jump Markov processes. δ
δ Now we investigate the existence and the uniqueness of the
solution of (34), which is rewritten in detail in (36) at the top
of the next page. p g
For that purpose, using fixed-point theorems, one may
alternatively show that for all p (0) =
p00 (0) , p10 (0)
∈
P0, the discrete-time system
p00 (n + 1) = F0
p00 (n) , p10 (n)
,
p10 (n + 1) = F1
p00 (n) , p10 (n)
. (39) (39) The state equations in (28) are nonlinear, since each πδm,δd
θ
depends on Xcm,cd
θ
, see (6) and (10). converges to a unique equilibrium point
p00, p10
, which is
then solution of (36). 7
ANALYSIS OF THE DTN STATE EQUATIONS One first shows the existence of an equilibrium using
Brouwer’s fixed-point theorem [41] in the following propo-
sition. In what follows, the asymptotic behavior of the DTN state
equations (28) is characterized. Algorithm 2 may drive
Xcm,cd
θ
to an equilibrium X
cm,cd
θ
at which the proportions
of nodes in different states Xcm,cd
θ
(t) do not vary any more. As a consequence, pθ0 (t) defined in (6) also tends to an
equilibrium pθ0. Proposition 5. For any ν ∈[0, 1], (36) always admits a solution,
which is an equilibrium point of the dynamical system (28). Before proving Proposition 5, one first shows that p00 (n)
and p10 (n) are contained in intervals with lower (and
upper) bounds increasing (resp. decreasing) with n. and consider the functions , M−1, with the initial conditions X0,0
θ
(0) =
1 and Xcm,cd
θ
(0) = 0, ∀cm, cd ̸= 0. (28)
for any cm = 1, . . . , M−1, with the initial conditions X0,0
θ
(0) =
1 and Xcm,cd
θ
(0) = 0, ∀cm, cd ̸= 0. (28)
for any cm = 1, . . . , M−1, with the initial conditions X0,0
θ
(0) =
1 and Xcm,cd
θ
(0) = 0, ∀cm, cd ̸= 0. p = F (p) ,
(34) (34) and for any θ ∈{0, 1} and cd ⩽cm, Proof: See Appendix A X
cm,cd
θ
=
(
0,
∀cm < M,
M
cd
(hθ (p))cd (1 −hθ (p))M−cd ,
cm = M. (35) Kurtz’s theorem [33], [34] can then be used to show that
for all ε > 0, there exists α1 ⩾0 and α2 (ε) > 0 such that (35) Pr
max
t∈[0,T ] ∥Xθ (t) −Xθ (t)∥> ε
⩽α1 exp (−α2 (ε) nθ) . Proof: See Appendix C. Proposition 3. For any cm < M and cd ⩽cm, lim
t→∞Xcm,cd
θ
(t) =
0 Proposition 3. For any cm < M and cd ⩽cm, lim
t→∞Xcm,cd
θ
(t) = (
pθ0
min (n + 1) = Fθ
p00
min (n) , p10
max (n)
,
∀n ∈N+,
pθ0
max (n + 1) = Fθ
p00
max (n) , p10
min (n)
,
∀n ∈N+. (40) (40) Proof: See Appendix B. Moreover, and consider the functions Proposition 2. The evolution of the DTN state components, i.e.,
the expected proportion of nodes Xcm,cd
θ
(t) in the states (θ, cm, cd),
with θ ∈{0, 1}, cm = 0, . . . , M, and cd ⩽cm is described by h0 (p) = p0qFA (2) p00 + p1qD (1, 1) p10
p0p00 + p1p10
,
(31)
h1 (p) = p0qD (1, 1) p00 + p1qD (0, 2) p10
p0p00 + p1p10
,
(32) (31)
dX0,0
θ
dt
(a)
= −λX0,0
θ
π1,0
θ
(0, 0) + π1,1
θ (0, 0)
,
dXcm,0
θ
dt
(b)
= λ
−Xcm,0
θ
π1,0
θ
(cm, 0) + π1,1
θ
(cm, 0)
+Xcm−1,0
θ
π1,0
θ
(cm −1, 0)
,
dXcm,cm
θ
dt
(c)
= λ
−Xcm,cm
θ
π1,0
θ
(cm, cm) + π1,1
θ
(cm, cm)
+Xcm−1,cm−1
θ
π1,1
θ
(cm −1, cm −1)
,
dXM,0
θ
dt
(d)
= λ
−XM,0
θ
π0,1
θ
(M, 0) + XM−1,0
θ
π1,0
θ
(M −1, 0)
+XM,1
θ
π0,−1
θ
(M, 1)
,
dXM,M
θ
dt
(e)
= λ
−XM,M
θ
π0,−1
θ
(M, M)+XM,M−1
θ
π0,1
θ
(M, M −1)
+ XM−1,M−1
θ
π1,1
θ
(M −1, M −1)
,
(28) (32) Fθ (p) =
⌈Mν⌉−1
X
cd=0
M
cd
! (hθ (p))cd (1 −hθ (p))M−cd ,
(33) (33) and F (p) = (F0 (p) , F1 (p)). The following proposition
provides a non-linear equation that has to be satisfied by p. The various X
M,cd
θ
at equilibrium are easily deduced from
the solutions of the mentioned equation. and F (p) = (F0 (p) , F1 (p)). The following proposition
provides a non-linear equation that has to be satisfied by p. The various X
M,cd
θ
at equilibrium are easily deduced from
the solutions of the mentioned equation. Proposition 4. Assume that the dynamic system described by
(28) admits some equilibrium X
cm,cd
θ
, then p ∈P0 is the solution
f (28) (28)
for any cm = 1, . . . 6
MACROSCOPIC EVOLUTION OF THE DTN STATE (20) All node state transition probabilities evaluated in Section 5
are now used to determine the evolution of the proportion
of nodes in state θ, i.e. which corresponds to a situation where one knows that
cd LODTs where positive among the last M tests and the
LODT that will be ignored, once the new LODT outcome
is available, also concluded in the presence of defective
sensors. P
E1
4 (t)
is relatively complex to evaluate, since
P {Y n
i = 1} is time-varying according to (12-14). In what
follows, we assume that LODT outcomes with Y m
i
= 1 which corresponds to a situation where one knows that
cd LODTs where positive among the last M tests and the
LODT that will be ignored, once the new LODT outcome
is available, also concluded in the presence of defective
sensors. P
E1
4 (t)
is relatively complex to evaluate, since
P {Y n
i = 1} is time-varying according to (12-14). In what
follows, we assume that LODT outcomes with Y m
i
= 1 Xθ (t)=
X0,0
θ
(t) , X1,0
θ
(t) , X1,1
θ
(t) , . . . , XM,0
θ
(t) , . . . , XM,M
θ
(t)
and the corresponding expected values and the corresponding expected values Xθ (t)=
X0,0
θ
(t) , X1,0
θ
(t) , X1,1
θ
(t) , . . . , XM,0
θ
(t) , . . . , XM,M
θ
(t) Xθ (t)=
X0,0
θ
(t) , X1,0
θ
(t) , X1,1
θ
(t) , . . . , XM,0
θ
(t) , . . . , XM,M
θ
(t)
7 and consider the functions Moreover, f
pp
From Proposition 3, the only possible value at equilib-
rium of Xcm,cd
θ
(t) when cm < M is 0. Thus pθ0 may be
written as
M p00
min (n + 1) > p00
min (n) ,
p00
max (n + 1) < p00
max (n) . (41) pθ0 =
X
cd:cd/M<ν
X
M,cd
θ
. (29) 7.1
Equilibrium of Xcm,cd
θ Lemma 6. For any n ∈N∗and θ ∈{0, 1}, one has Lemma 6. For any n ∈N∗and θ ∈{0, 1}, one has Lemma 6. For any n ∈N∗and θ ∈{0, 1}, one has
pθ0
min (n) ⩽pθ0 (n) ⩽pθ0
max (n) , One investigates first the evolution of Xcm,cd
θ
(t) when cm <
M. As shown in the following proposition, the DTN state
always reaches the permanent regime. pθ0
min (n) ⩽pθ0 (n) ⩽pθ0
max (n) , pθ0
min (n) ⩽pθ0 (n) ⩽pθ0
max (n) , with pθ0
min (0) = 0, pθ0
max (0) = 1, and with pθ0
min (0) = 0, pθ0
max (0) = 1, and Proposition 3. For any cm < M and cd ⩽cm, lim
t→∞Xcm,cd
θ
(t) =
0. 8
INFLUENCE OF MISBEHAVING NODES A LODT involving data coming from a misbehaving node
will always result in the detection of an outlier. Thus, when
a node i with state xi(t) = (θ, cm,i (t) , cd,i (t)) meets a
misbehaving node, the possible transitions are such that A LODT involving data coming from a misbehaving node
will always result in the detection of an outlier. Thus, when
a node i with state xi(t) = (θ, cm,i (t) , cd,i (t)) meets a
misbehaving node, the possible transitions are such that
•
(δm, δd) = (1, 0) or (δm, δd) = (1, 1) if cm,i (t) < M Proof: See Appendix D. (29) Using Lemma 6, one can now prove Proposition 5. g
p
p
Proof: F0 and F1 are both continuous functions. For
some n > 0, consider the set Pn =
p00
min (n) , p00
max (n)
×
p10
min (n) , p10
max (n)
, where pθ0
min (n) and pθ0
max (n) are defined Denote p =
p00, p10 ∈P0 with Denote p =
p00, p10 ∈P0 with P0 = {(x, y) ∈[0, 1] × [0, 1] and (x, y) ̸= (0, 0)}
(30) 8
p00 = F0
p00, p10 = P
cd:cd/M<ν
M
cd
p0qFA(2)p00+p1qD(1,1)p10
p0p00+p1p10
cd
p0(1−qFA(2))p00+p1(1−qD(1,1))p10
p0p00+p1p10
M−cd ,
p10 = F1
p00, p10 = P
cd:cd/M<ν
M
cd
p0qD(1,1)p00+p1qD(0,2)p10
p0p00+p1p10
cd
p0(1−qD(1,1))p00+p1(1−qD(0,2))p10
p0p00+p1p10
M−cd . (36)
c0(qFA(2), qD(0,2),qD(1,1), p1,M, ν, n)=
M (qD (1,1) −qFA (2)) p0p1p00
max (n) p10
max (n)
(p0p00
min(n)+p1p10
min(n))((1 −qFA(2))p0p00
min(n)+(1 −qD(1,1))p1p10
min(n)),
(37)
c1(qFA(2), qD(0,2),qD(1,1), p1,M, ν, n)=
M (qD (0,2) −qD (1,1)) p0p1p00
max (n) p10
max (n)
(p0p00
min(n)+p1p10
min(n))((1 −qD(1,1))p0p00
min(n)+(1 −qD(0,2))p1p10
min(n)),
(38) (36) (37) (38) 0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0.4
0.5
0.6
0.7
0.8
0.9
1
p1
νmax
M=4, qD(1,1)=0.8
M=4, qD(1,1)=0.5
M=10, qD(1,1)=0.8
M=10, qD(1,1)=0.5
Fig. 2. Upper bounds of ν to satisfy (42), with qFA (2) = 0.05, qD (0, 2) =
0.9, qD (1, 1) ∈{0.5, 0.8}, M ∈{4, 10}, and p1 ∈[0.05, 0.5]. 0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0.4
0.5
0.6
0.7
0.8
0.9
1
p1
νmax
M=4, qD(1,1)=0.8
M=4, qD(1,1)=0.5
M=10, qD(1,1)=0.8
M=10, qD(1,1)=0.5 properly chosen, the probabilities of false alarm and non-
detection tend to zero, as shown in Proposition 9. Proposition 9. If qFA (2) < ν < qD (1, 1), then (36) has a
unique solution and lim
M→∞p00 = 1,
lim
M→∞p10 = 0. (43)
Proof: See Appendix F. (43) 7.4
Approximations of the Equilibrium Closed-form expressions for p00 and p10 are difficult to
obtain from (36). This section introduces an approximation
of (36) from which some insights may be obtained on the
way ν should be chosen. Fig. 2. Upper bounds of ν to satisfy (42), with qFA (2) = 0.05, qD (0, 2) =
0.9, qD (1, 1) ∈{0.5, 0.8}, M ∈{4, 10}, and p1 ∈[0.05, 0.5]. in (40). For any p =
p00, p10 ∈Pn, one can prove using
Lemma 6 that F (p) ∈Pn. Thus F maps Pn to Pn. Applying
Brouwer’s fixed-point theorem, F admits a fixed point and
Proposition 5 is proved. Since p10 represents the expected proportion of nodes
with defective sensors that have not detected their status,
the value of p10 should be small. From (31-32) one sees that
limp10→0 h0 = qFA (2) and limp10→0 h1 = qD (1, 1), thus one
may consider the following approximations Sufficient conditions on p0, p1, qD, qFA, M and ν are then
provided to ensure the uniqueness of this equilibrium by
applying Banach’s fixed-point theorem [42]. h0 ≈eh0 = qFA (2) ,
h1 ≈eh1 = qD (1, 1) . (44)
Therefore, (36) may be rewritten as (44) Therefore, (36) may be rewritten as Proposition 7. If there exists some N ′, such that ∀θ ∈{0, 1}
and ∀n > N ′, one has (
ep00 = P
cd:cd/M<ν
M
cd
(qFA (2))cd (1 −qFA (2))M−cd ,
ep10 = P
cd:cd/M<ν
M
cd
(qD (1, 1))cd (1 −qD (1, 1))M−cd . ( cθ(qFA(2), qD(0, 2),qD (1, 1), p1,M, ν, n)< 1,
(42) (42) (45) (45)
from which one deduces approximate values
e
XM,cd
0
of
XM,cd
0
at equilibrium from eq. (35) where c0 and c1 are defined in (37-38), then the discrete-time
system (39) converges to a unique equilibrium point and the
solution of (36) is unique. where c0 and c1 are defined in (37-38), then the discrete-time
system (39) converges to a unique equilibrium point and the
solution of (36) is unique. (
e
XM,cd
0
=
M
cd
(qFA (2))cd (1 −qFA (2))M−cd ,
e
XM,cd
1
=
M
cd
(qD (1, 1))cd (1 −qD (1, 1))M−cd . (46) (46) Proof: See Appendix E. f
pp
Due to the monotonicity of pθ0
min (n) and pθ0
max (n) shown
in Lemma 6, cθ decreases with n. Hence, if a given ν satisfies
(42) for some N ′, then ν will satisfy (42) for all n ⩾N ′
and the equilibrium is unique. If the values of p1, qD, qFA,
and M are fixed, then one may deduce sufficient conditions
on the value of ν to have a unique equilibrium point. See
Example 8. For any fixed value of M, qFA (2), and qD (1, 1), the
values of detection rate (p11) and false alarm rate (p01) at equi-
librium can be predicted using (45), since p01 = 1 −p00 and
p11 = 1−p10. Consider for example M = 10, qFA (2) = 0.05,
and qD (1, 1) = 0.8. Figure 3 presents ep11 as a function of ep01
for different values of ν. This figure is helpful to choose the
value of ν to meet different performance requirements. The
actual values of p11 and p01 are also shown in Figure 3,
which are very close to ep11 and ep01, in the region where p11
is close to 1. Example 8. Consider qFA (2) = 0.05, qD (0, 2) = 0.9,
qD (1, 1) ∈{0.5, 0.8}, M ∈{4, 10}, and p1 ∈[0.05, 0.5]. One verifies whether (42) is satisfied considering n = 10
for different values of ν. One obtains that (42) holds if
0 < ν ⩽νmax, where νmax depends on the values of p1,
qD, qFA, and M. See Figure 2 for the numerical values of
νmax in each case. 7.3
Equilibrium point as M →∞ For the nodes with θ = 1, only X4,4
1
, X4,3
1
, and X4,2
1
are
relatively large as compared to the other states. Consider
the two sets of cds for which XM,cd
0
and XM,cd
1
are large. These sets have no common cd and it is thus easy to choose
a decision threshold to distinguish both sets. The accuracy
of the algorithm is then very good. With ν = 0.4, one has
p00 = 0.985 and p10 = 0.027. Only 1.5% of the good nodes
believe they are carrying defective sensors. Less than 3% of
the nodes with defective sensors have not been detected. Then, in the evaluation of the probability of the events
E∗
1 (t), Eθ
2(t), and Eθ
3(t) introduced in Section 5, one has to
account for the probability of meeting a misbehaving node. For example, (11) can be rewritten as Then, in the evaluation of the probability of the events
E∗
1 (t), Eθ
2(t), and Eθ
3(t) introduced in Section 5, one has to
account for the probability of meeting a misbehaving node. For example, (11) can be rewritten as P {E∗
1 (t)} = p0p01 (t) + p1p11 (t) + p2. (47) (47) The transition probabilities introduced in Sections 5.2.1
and 5.2.2 have to be updated accordingly. The form of the
DTN state equations (28) remains the same. q
(
)
Finally, the effect misbehaving nodes can be taken into
account in (45) for the computation of the approximate
expressions of ep00 and ep10. More specifically, Consider now a set S of nS = 1000 moving nodes
uniformly distributed over a square of unit area. In the
first displacement model (jump motion model): node i
randomly chooses its location at time instant (k + 1) ∆t,
independently from its previous location at time k∆t. Two
nodes communicate only at discrete time instants k∆t when
their distance is less than r0. Node i has its neighbors in the
set Ni = {j ∈S : 0 < ri,j ≤r0}, where ri,j is the distance
between Nodes i and j. Furthermore, if |Ni| > 1, we assume
that node i communicates only with its closest neighbor. De-
note ρ = πr2
0nS as the average value of |Ni|. 9
NUMERICAL RESULTS In this Section we provide results aimed at assessing the
convergence of the theoretical framework (Section 10.1),
the appropriateness and accuracy of the framework also
in case of specific mobility models such as the Brownian
motion (Section 10.2) or other more realistic mobility models
derived from user traces (Section 10.3), as well as to compare
the proposed DFD methodology to other state-of-the-art so-
lutions (Section 10.4). Finally, in Section 10.5 we investigate
on the stability and accuracy of the Algorithm upon varying
some key parameters. λ∆t = P {|Ni| = 1} = ρ exp (−ρ) . In the Monte-Carlo simulations, we set r0 = 0.014, so
that ρ ≈0.6 and λ∆t ≈0.33. Using the same values of pθ,
M, ν, qD, and qFA as in Figure 4, the simulation results for
this jump motion model are shown in Figure 5. Comparing
Figure 4 and Figure 5, one remarks that the state evolution
in the transient phase has similar shape but with different
convergence speed, which depends mainly on λ. Figure 6
shows a good match between theory and simulation for the
proportions of states at equilibrium. The approximation of
X
4,cd
θ
using (46) is also presented in Figure 6, which is very
close to its actual value. Note that the difference between
the approximated value and those obtained through theory
and simulation is less than 0.1%. 7.3
Equilibrium point as M →∞ The cardinality
of Ni approximately follows a Poisson distribution as nS is
large enough, the inter-contact probability is thus
ep00 =P
cd:cd/M<ν
M
cd
p0qFA(2)+p2
p0+p2
cd
1 −p0qFA(2)+p2
p0+p2
M−cd ,
ep10 =P
cd:cd/M<ν
M
cd
p0qD(1,1)+p2
p0+p2
cd
1 −p0qD(1,1)+p2
p0+p2
M−cd
(49) 9.1
Numerical verification of theoretical results This section presents first the solution of the state equation
(28) describing the evolution of the proportion of nodes
in various states. Algorithm 2 is simulated considering a
random displacement of nodes without any constraint on
their speed. This allows to verify the correctness of the
theoretical results presented in this paper. p
p p
Consider a LODT where qFA (0, 2) = 0.05, qD (1, 1) =
0.8, and qD (0, 2) = 0.9. Besides p0 = 0.9, p1 = 0.1, p2 = 0,
M = 4, ν = 0.4, and λ = 1. Figure 4 presents the evolution
of the proportion of nodes with good sensors (left part) and
defective sensors (right part) in different states, obtained 7.3
Equilibrium point as M →∞ Both p00 and p10 can be seen as functions of M. As M →∞,
Algorithm 2 turns into Algorithm 1. In this situation, if ν is •
(δm, δd) = (1, 0) or (δm, δd) = (1, 1) if cm,i (t) < M 9 9
5
10
15
20
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
t/∆t
θ=0
5
10
15
20
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
t/∆t
θ=1
X00
θ
X10
θ
X11
θ
X20
θ
X21
θ
X22
θ
X30
θ
X31
θ
X32
θ
X33
θ
X40
θ
X41
θ
X42
θ
X43
θ
X44
θ
Fig. 4. Evolution of Xcm,cd
0
(t) (left) and Xcm,cd
1
(t) (right) obtained from
(28), when qFA (0, 2) = 0.05, qD (1, 1) = 0.8, qD (0, 2) = 0.9, M = 4,
ν = 0.4, and λ = 1. 9
5
10
15
20
0
1
2
3
4
5
6
7
8
9
1
t/∆t
θ=1
X00
θ
X10
θ
X11
θ
X20
θ
X21
θ
X22
θ
X30
θ
X31
θ
X32
θ
X33
θ
X40
θ
X41
θ
X42
θ
X43
θ
X44
θ 9
10
−15
10
−10
10
−5
10
0
0
0.2
0.4
0.6
0.8
1
p01=1−p00
p11=1−p10
qD(1.1)=0.8, actual value
qD(1.1)=0.8, approximation
qD(1.1)=0.5, actual value
qD(1.1)=0.5, approximation
ν=0.2
ν=0.4
ν=1
Fig. 3. Approximate p11 as a function of approximate p01, for various ν
and fixed M = 10 . 5
10
15
20
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
t/∆t
θ=0
5
10
15
20
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
t/∆t
θ=1
X00
θ
X10
θ
X11
θ
X20
θ
X21
θ
X22
θ
X30
θ
X31
θ
X32
θ
X33
θ
X40
θ
X41
θ
X42
θ
X43
θ
X44
θ
Fig. 4. Evolution of Xcm,cd
0
(t) (left) and Xcm,cd
1
(t) (right) obtained from
(28), when qFA (0, 2) = 0.05, qD (1, 1) = 0.8, qD (0, 2) = 0.9, M = 4,
ν = 0.4, and λ = 1. 10
−15
10
−10
10
−5
10
0
0
0.2
0.4
0.6
0.8
1
p01=1−p00
p11=1−p10
qD(1.1)=0.8, actual value
qD(1.1)=0.8, approximation
qD(1.1)=0.5, actual value
qD(1.1)=0.5, approximation
ν=0.2
ν=0.4
ν=1
Fig. 3. 7.3
Equilibrium point as M →∞ Approximate p11 as a function of approximate p01, for various ν
and fixed M = 10 . 10
−15
10
−10
10
−5
10
0
0
0.2
0.4
0.6
0.8
1
p01=1−p00
p11=1−p10
qD(1.1)=0.8, actual value
qD(1.1)=0.8, approximation
qD(1.1)=0.5, actual value
qD(1.1)=0.5, approximation
ν=0.2
ν=0.4
ν=1 5
10
15
20
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
t/∆t
θ=0
0
0
0
0
0
0
0
0
0 Fig. 4. Evolution of Xcm,cd
0
(t) (left) and Xcm,cd
1
(t) (right) obtained from
(28), when qFA (0, 2) = 0.05, qD (1, 1) = 0.8, qD (0, 2) = 0.9, M = 4,
ν = 0.4, and λ = 1. Fig. 3. Approximate p11 as a function of approximate p01, for various ν
and fixed M = 10 . •
(δm, δd) = (0, 0) or (δm, δd) = (0, 1) if cm,i (t) = M
and 0 < cd,i (t) < M
•
(δm, δd) = (0, 0) if cm,i (t) = cd,i (t) = M. by solving (28). Note that ∆t represents the duration of a
unit time slot used in the simulation. One observes that the
proportion of nodes in each state becomes almost constant
as t/∆t ⩾15. For the nodes with θ = 0, only X4,0
0
and
X4,1
0
are larger than 0.05, while the others are very close to
0. For the nodes with θ = 1, only X4,4
1
, X4,3
1
, and X4,2
1
are
relatively large as compared to the other states. Consider
the two sets of cds for which XM,cd
0
and XM,cd
1
are large. These sets have no common cd and it is thus easy to choose
a decision threshold to distinguish both sets. The accuracy
of the algorithm is then very good. With ν = 0.4, one has
p00 = 0.985 and p10 = 0.027. Only 1.5% of the good nodes
believe they are carrying defective sensors. Less than 3% of
the nodes with defective sensors have not been detected. by solving (28). Note that ∆t represents the duration of a
unit time slot used in the simulation. One observes that the
proportion of nodes in each state becomes almost constant
as t/∆t ⩾15. For the nodes with θ = 0, only X4,0
0
and
X4,1
0
are larger than 0.05, while the others are very close to
0. 9.3
Simulations with real databases 0
1
2
3
4
0
0.2
0.4
0.6
0.8
1
cd
X0
4,cd, theory
X0
4,cd, simulation
X0
4,cd, approximation
X1
4,cd
X1
4,cd
X1
4,cd, approximation
, theory
, simulation 0
1
2
3
4
0
0.2
0.4
0.6
0.8
1
cd
X0
4,cd, theory
X0
4,cd, simulation
X0
4,cd, approximation
X1
4,cd
X1
4,cd
X1
4,cd, approximation
, theory
, simulation
Fig. 6. Comparison of X
4,cd
θ
at the equilibrium. In this section, Algorithm 2 is executed using some ex-
perimental databases instead of motion models. These
databases, provided by the MIT Reality Mining Project [43]
and the Haggle Project [44], have been used in several
previous works, e.g., [4]. In this work, we use the following
databases: •
Reality, where nS = 97, lasts more than 200 days with
about 111 inter-contacts per day. Fig. 6. Comparison of X
4,cd
θ
at the equilibrium. p
y
•
Infocom05, where nS = 41, lasts 3 days with approxi-
mately 312 inter-contacts every hour. More specifically, one is interested in the inter-contact trace,
i.e., which pair of nodes have a meeting at which time. The
traces were taken from [45], which are converted from the
original databases [43], [44]. orientation when it reaches the boundary of the unit square. Let χi =
χi
1, χi
2
be the location of Agent i. Consider a
first-order Brownian mobility model orientation when it reaches the boundary of the unit square. Let χi =
χi
1, χi
2
be the location of Agent i. Consider a
first-order Brownian mobility model g
Consider again the following parameters: qFA (2) = 0.05,
qD (1, 1) = 0.8, qD (0, 2) = 0.9, M = 10, and ν = 0.4. Monte-Carlo simulations are performed 500 times for each
database. In each test, n1 nodes with random index are
chosen to be defective. One sets n1 = 10 in Infocom05 and
n1 = 20 in Reality. Two cases are considered. First, no
misbehaving node is introduced, i.e., n2 = 0. In a second
case, n2 = 1 in Infocom05 and n2 = 2 in Reality. At the
top of Figure 8, the index of the active nodes (which have
contact with the others) are presented at each time to show
the frequency of the inter-contacts at different epochs. The
evolution of p10 and p01 is plotted at the bottom of Figure 8. 9.2
Simulations with Brownian motion model
p00 = P
cd:cd/M<ν
M
cd
p0qFA(2)p00+p1qD(1,1)p10+p2
p0p00+p1p10+p2
cd
p0(1−qFA(2))p00+p1(1−qD(1,1))p10
p0p00+p1p10+p2
M−cd ,
p10 = P
cd:cd/M<ν
M
cd
p0qD(1,1)p00+p1qD(0,2)p10+p2
p0p00+p1p10+p2
cd
p0(1−qD(1,1))p00+p1(1−qD(0,2))p10
p0p00+p1p10+p2
M−cd . (48) (48) 0
20
40
60
80
100
10
−3
10
−2
10
−1
10
0
t/∆t
σ=1
p01, Brownian,
p01, Brownian,
σ=0.1
p10, Brownian, σ=1
p01, Jump Model
p10, Brownian,
p10, Jump Model
σ=0.1
Fig. 7. Evolution of p01 and p10 for the two moving models, with σ ∈
{0.1, 1}, qFA (2) = 0.05, qD (1, 1) = 0.8, qD (0, 2) = 0.9, M = 10 and
ν = 0.4. 0
5
10
15
20
25
30
35
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
t/∆t
θ=0
0
5
10
15
20
25
30
35
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
t/∆t
θ=1
Xθ
00
Xθ
10
Xθ
11
Xθ
20
Xθ
21
Xθ
22
Xθ
30
Xθ
31
Xθ
32
Xθ
33
Xθ
40
Xθ
41
Xθ
42
Xθ
43
Xθ
44
Fig. 5. Evolution of Xcm,cd
0
(t) (left) and Xcm,cd
1
(t) (right) by simulations
with the jump model, when qFA (0, 2) = 0.05, qD (1, 1) = 0.8, qD (0, 2) =
0.9, M = 4, ν = 0.4, and λ∆t = 0.33. 5
0
5
10
15
20
25
30
35
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
t/∆t
θ=1
Xθ
00
Xθ
10
Xθ
11
Xθ
20
Xθ
21
Xθ
22
Xθ
30
Xθ
31
Xθ
32
Xθ
33
Xθ
40
Xθ
41
Xθ
42
Xθ
43
Xθ
44 0
20
40
60
80
100
10
−3
10
−2
10
−1
10
0
t/∆t
σ=1
p01, Brownian,
p01, Brownian,
σ=0.1
p10, Brownian, σ=1
p01, Jump Model
p10, Brownian,
p10, Jump Model
σ=0.1 0
5
10
15
20
25
30
35
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
t/∆t
θ=0 Fig. 7. Evolution of p01 and p10 for the two moving models, with σ ∈
{0.1, 1}, qFA (2) = 0.05, qD (1, 1) = 0.8, qD (0, 2) = 0.9, M = 10 and
ν = 0.4. Fig. 5. 9.2
Simulations with Brownian motion model Evolution of Xcm,cd
0
(t) (left) and Xcm,cd
1
(t) (right) by simulations
with the jump model, when qFA (0, 2) = 0.05, qD (1, 1) = 0.8, qD (0, 2) =
0.9, M = 4, ν = 0.4, and λ∆t = 0.33. 9.2
Simulations with Brownian motion model Consider now a Brownian motion model where each node
is moving with a random speed. Each node changes its 10
p0qFA(2)p00+p1qD(1,1)p10+p2
p0p00+p1p10+p2
cd
p0(1−qFA(2))p00+p1(1−qD(1,1))p10
p0p00+p1p10+p2
M−cd ,
p0qD(1,1)p00+p1qD(0,2)p10+p2
p0p00+p1p10+p2
cd
p0(1−qD(1,1))p00+p1(1−qD(0,2))p10
p0p00+p1p10+p2
M−cd . (48)
15
20
25
30
35
t/∆t
θ=1
Xθ
00
Xθ
10
Xθ
11
Xθ
20
Xθ
21
Xθ
22
Xθ
30
Xθ
31
Xθ
32
Xθ
33
Xθ
40
Xθ
41
Xθ
42
Xθ
43
Xθ
44
cd (t) (right) by simulations
qD (1, 1) = 0.8, qD (0, 2) =
0
20
40
60
80
100
10
−3
10
−2
10
−1
10
0
t/∆t
σ=1
p01, Brownian,
p01, Brownian,
σ=0.1
p10, Brownian, σ=1
p01, Jump Model
p10, Brownian,
p10, Jump Model
σ=0.1
Fig. 7. Evolution of p01 and p10 for the two moving models, with σ ∈
{0.1, 1}, qFA (2) = 0.05, qD (1, 1) = 0.8, qD (0, 2) = 0.9, M = 10 and
ν = 0.4. 10 10
p00 = P
cd:cd/M<ν
M
cd
p0qFA(2)p00+p1qD(1,1)p10+p2
p0p00+p1p10+p2
cd
p0(1−qFA(2))p00+p1(1−qD(1,1))p10
p0p00+p1p10+p2
M−cd ,
p10 = P
cd:cd/M<ν
M
cd
p0qD(1,1)p00+p1qD(0,2)p10+p2
p0p00+p1p10+p2
cd
p0(1−qD(1,1))p00+p1(1−qD(0,2))p10
p0p00+p1p10+p2
M−cd . (48)
0
5
10
15
20
25
30
35
t/∆t
θ=0
0
5
10
15
20
25
30
35
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
t/∆t
θ=1
Xθ
00
Xθ
10
Xθ
11
Xθ
20
Xθ
21
Xθ
22
Xθ
30
Xθ
31
Xθ
32
Xθ
33
Xθ
40
Xθ
41
Xθ
42
Xθ
43
Xθ
44
g. 5. Evolution of Xcm,cd
0
(t) (left) and Xcm,cd
1
(t) (right) by simulations
th the jump model, when qFA (0, 2) = 0.05, qD (1, 1) = 0.8, qD (0, 2) =
9, M = 4, ν = 0.4, and λ∆t = 0.33. 0
20
40
60
80
100
10
−3
10
−2
10
−1
10
0
t/∆t
σ=1
p01, Brownian,
p01, Brownian,
σ=0.1
p10, Brownian, σ=1
p01, Jump Model
p10, Brownian,
p10, Jump Model
σ=0.1
Fig. 7. Evolution of p01 and p10 for the two moving models, with σ ∈
{0.1, 1}, qFA (2) = 0.05, qD (1, 1) = 0.8, qD (0, 2) = 0.9, M = 10 and
ν = 0.4. 9.3
Simulations with real databases Interestingly, in absence of misbehaving nodes, both p10 and
p01, obtained with both databases, decrease to 10−3 after a
sufficiently long time. One also observes that the conver-
gence speed of p10 and p01 is highly related to the inter-
contact rate (reflected by the density of points in the sub-
figures at the top). Considering Infocom05, Table 2 further
shows the influence of misbehaving nodes for various n2. In presence of misbehaving nodes, the performance of the
DFD algorithms worsens, but remains satisfying if the value
of ν is properly chosen. χi
1 ((k + 1) ∆t) = χi
1 (k∆t) + ηi
1 (k∆t)
χi
2 ((k + 1) ∆t) = χi
2 (k∆t) + ηi
2 (k∆t) where
ηi
1 (k∆t)
∼
N
0, (σr0)2
and
ηi
2 (k∆t)
∼
N
0, (σr0)2
.
Consider σ ∈{0.1, 1}, qFA (2) = 0.05, qD (1, 1) = 0.8,
qD (0, 2) = 0.9, M = 10, and ν = 0.4. Figure 7 compares
the evolution of p01 and p10 as functions of time for the
jump motion model and the Brownian motion model, with
fixed ρ ≈0.6. At equilibrium, the performance obtained
for both models is quite close. However, the convergence
speed depends on the inter-contact rate λ. When σ = 0.1,
the algorithm converges slowly in the Brownian motion
model. When σ = 1, which results in a larger value of λ, the
evolution of p01 and p10 with the Brownian motion model
are close to the jump motion model. At the beginning of the algorithm, each node believes
that its sensors are good, thus p01(0) = 0 and p10(0) = 1. During the algorithm, p10(t) decreases in the transient phase
until it reaches the equilibrium. Whereas, p01(t) increases at
first and then decreases to the equilibrium. This comes from
the fact that p10(t) is large at the beginning and the LODT
performed on a good node often detects outliers. 9.3
Simulations with real databases When n2 = 0, Figure 9 represents the states at equilib-
rium X
M,cd
θ
obtained with the databases Reality and Info- 11 TABLE 2
Values of p01 and p10 at the end of simulations for different number of
misbehaving nodes
n2
1
2
3
4
5
6
ν
0.5
0.5
0.5
0.5
0.6
0.6
p01(%)
0.3
1.4
2.7
8.0
3.4
7.2
p10(%)
0.7
0.5
0.3
0.3
1.3
1.4
0
2
4
6
8
10
0
0.2
0.4
0.6
0.8
1
cd
X0
10,cd, Reality
X1
10,cd, Reality
X0
10,cd, Infocom05
X1
10,cd, Infocom05
X0
10,cd, approximation
X1
10,cd, approximation
Fig. 9. Comparison of X
10,cd
θ
at the equilibrium obtained using the
Reality database, the Infocom05 database, and predicted by the ap-
proximation (46). 0
20
40
60
80
10
−4
10
−3
10
−2
10
−1
10
0
time (hours)
classification error
0
20
40
60
80
10
−2
10
−1
10
0
time (hours)
estimation error
DFD, multi
DFD, single
reference, multi
reference, single
0
20
40
60
80
10
−3
10
−2
10
−1
10
0
time (hours)
classification error
0
20
40
60
80
10
−2
10
−1
10
0
time (hours)
estimation error
DFD, multi
DFD, single
reference, multi
reference, single
Fig. 10. Comparison of the DFD part of the estimation algorithm pro-
posed in [18] with the proposed DFD algorithm, when σ = 0.2 (top) and
σ = 0.3 (bottom) 0
20
40
60
80
10
−4
10
−3
10
−2
10
−1
10
0
time (hours)
classification error
0
20
40
60
80
10
−2
10
−1
10
0
time (hours)
estimation error
DFD, multi
DFD, single
reference, multi
reference, single 0
20
40
60
80
10
−4
10
−3
10
−2
10
−1
10
0
time (hours)
classification error 0
20
40
60
80
10
−2
10
−1
10
0
time (hours)
estimation error
DFD, multi
DFD, single
reference, multi
reference, single estimation error 0
2
4
6
8
10
0
0.2
0.4
0.6
0.8
1
cd
X0
10,cd, Reality
X1
10,cd, Reality
X0
10,cd, Infocom05
X1
10,cd, Infocom05
X0
10,cd, approximation
X1
10,cd, approximation (
)
0
20
40
60
80
10
−3
10
−2
10
−1
10
0
time (hours)
classification error
DFD, multi
DFD, single
reference, multi
reference, single 0
20
40
60
80
10
−2
10
−1
10
0
time (hours)
estimation error estimation error Fig. 9. 9.3
Simulations with real databases Comparison of X
10,cd
θ
at the equilibrium obtained using the
Reality database, the Infocom05 database, and predicted by the ap-
proximation (46). com05, and also considering the approximation (46). There
is an excellent match between the values at equilibrium
predicted by theory and those obtained in practice. Note
that the difference between the approximated value and
those obtained through theory and simulation is less than
0.05%. Fig. 10. Comparison of the DFD part of the estimation algorithm pro-
posed in [18] with the proposed DFD algorithm, when σ = 0.2 (top) and
σ = 0.3 (bottom) where δ is a threshold that results in different values of
probabilities qFA(2), qD(1, 1), and qD(0, 2); then the proper
value of ν can be set accordingly. (50) (50) of a common quantity c, where vi. are realizations of
independent zero-mean Gaussian random variables with
variance σ2 and θi. ∈{0, 1} denotes the bias of each node. Each node is interested in the joint estimation of c and θi. Since the measurements produced by the sensors with non-
zero bias are more likely to have larger values, [18] proposes
a estimator of θi based on a distributed ranking of the nodes
according to their measurement mi . Nodes with a large
rank get an estimate bθi = 1, while nodes with a small rank
have bθi = 0. 9.4
Comparison with state-of-the-art solutions This section compares the proposed DFD algorithm to some
closely related scheme in the literature. As mentioned in
Section 2, classical DFD algorithms are difficult to apply in
the context of DTN and no solutions have been presented
so far in the literature for this specific scenario. Accordingly,
in order to perform a meaningful comparison between
our algorithm and a state-of-the-art approach, we have
considered the gossip algorithm discussed in [18] which
represents the most robust and efficient methodology in
the context of classification and distributed estimation in
dynamic scenarios like DTNs. Consider again Infocom05 for the simulation with n1 =
10 nodes chosen randomly with θ = 1 and without misbe-
having node. Two scenarios are considered. In the first case,
all nodes take a single measurement of c at initialization. In
the second case, nodes take measurements at each meeting. Results are obtained as the average of 200 independent
Monte-Carlo simulations. Figure 10 compares the results
when σ = 0.2 and σ = 0.3. The classification error and
the estimation error are defined as Ec = P θi −bθi
/nS and
Ee = P |c −bci| /nS. If nodes take a single measurement,
the performance of the proposed algorithm is close to the
reference method in terms of Ec and Ee. When nodes take
new measurements at each meeting, the proposed DFD
algorithm performs better than the reference method: the
value of Ec decreases faster and turns to be much smaller. This is mainly due to the node ranking algorithm used
in [18], which becomes less efficient when nodes update
at each meeting the quantity according to which they are
ranked. In [18] , nS nodes are assumed to get a measurement mi = c + θi + vi, ∀i ∈S,
(50) 9.5
Influence of the parameters Indexes of active nodes (having met another node) at different time (top) and evolution of p01 and p10 (bottom) obtained by using the Reality
database (left) and the Infocom05 database (right), with qFA (2) = 0.05, qD (1, 1) = 0.8, qD (0, 2) = 0.9, M = 10, and ν = 0.4. Fig. 8. Indexes of active nodes (having met another node) at different time (top) and evolution of p01 and p10 (bott
database (left) and the Infocom05 database (right), with qFA (2) = 0.05, qD (1, 1) = 0.8, qD (0, 2) = 0.9, M = 10, p1=0.1, M=10
p1=0.1, M=20
p1=0.5, M=4
p1=0.5, M=10
p1=0.5, M=20
p1=0.1, M=4
0
50
100
150
10
−4
10
−3
10
−2
10
−1
10
0
p10
t/∆t
0
50
100
150
10
−4
10
−3
10
−2
10
−1
10
0
p01
t/∆t
Fig. 11. Evolution of p10 and p01 for various M ∈{4, 10, 20} and p1 ∈
{0.1, 0.5}, with qFA (2) = 0.05, qD (1, 1) = 0.8, qD (0, 2) = 0.9. 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
qD(1,1)
qFA(2)
qFA(2)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
qD(1,1)
p01>10-1 or p10>10-1
p01<10-1 and p10<10-1
p01<10-2 and p10<10-2
p01<10-3 and p10<10-3
p1=0.1
p1=0.5
Fig. 12. Achievable p10
or p01
for different values of the pair
(qD (1, 1) , qFA (2)) when p1 = 0.1 (left) and for p1 = 0.5 (right) and
p2 = 0. p1=0
p1=0.5, M=4
p1=0
p1=0.1, M=4
0
50
100
150
10
−4
10
−3
10
−2
10
−1
10
0
p10
t/∆t
01 p1=0.1, M=10
p1=0.1, M=20
p1=0.5, M=4
p1=0.5, M=10
p1=0.5, M=20
p1=0.1, M=4
0
50
100
150
10
−4
10
−3
10
−2
10
−1
10
0
p10
t/∆t
0
50
100
150
10
−4
10
−3
10
−2
10
−1
10
0
p01
t/∆t
Fig. 11. Evolution of p10 and p01 for various M ∈{4, 10, 20} and p1 ∈
{0.1, 0.5}, with qFA (2) = 0.05, qD (1, 1) = 0.8, qD (0, 2) = 0.9. 9.5
Influence of the parameters When M = 10, both p10
and p01 are around 10−3. The proportion of the nodes with
defective sensors has also an impact on the convergence
speed of the algorithm. For example, when p1 is large, more
time is needed to achieve a given level of performance (in
terms of p10 and p01). on the performance at the equilibrium, even when 50% of
nodes are defective. This assesses the robustness of the approach. 9.5
Influence of the parameters This section characterizes the influence of the parameters,
such as p1, qD (1, 1) , and M, on the performance of Algo-
rithm 2. The jump motion model is used throughout this
section to describe the displacement of the nodes. Consider fixed qFA (2) = 0.05, qD (1, 1) = 0.8, qD (0, 2) =
0.9, and p2 = 0. The evolution of p10 and p01 for various
p1 ∈{0.1, 0.5} and M ∈{4, 10, 20} is shown in Figure 11. This section characterizes the influence of the parameters,
such as p1, qD (1, 1) , and M, on the performance of Algo-
rithm 2. The jump motion model is used throughout this
section to describe the displacement of the nodes. In order to apply the proposed DFD algorithm to this
problem, consider the following LODT Consider fixed qFA (2) = 0.05, qD (1, 1) = 0.8, qD (0, 2) =
0.9, and p2 = 0. The evolution of p10 and p01 for various
p1 ∈{0.1, 0.5} and M ∈{4, 10, 20} is shown in Figure 11. yi,j = yj,i =
(
1
if |mi −mj| > δ
0
else
, 12 0
20
40
60
80
100
120
140
160
180
200
0
50
100
time (days)
0
10
20
30
40
50
60
70
80
0
20
40
time (hours)
0
20
40
60
80
100
120
140
160
180
200
10
−3
10
−2
10
−1
10
0
time (days)
p10, no misbehaving nodes
p01, no misbehaving nodes
p10, 2 misbehaving nodes
p01, 2 misbehaving nodes
0
10
20
30
40
50
60
70
80
10
−3
10
−2
10
−1
10
0
time (hours)
p10, no misbehaving nodes
p01, no misbehaving nodes
p10,
misbehaving node
p01, misbehaving node
1
1
Fig. 8. Indexes of active nodes (having met another node) at different time (top) and evolution of p01 and p10 (bottom) obtained by using the Reality
database (left) and the Infocom05 database (right), with qFA (2) = 0.05, qD (1, 1) = 0.8, qD (0, 2) = 0.9, M = 10, and ν = 0.4. 0
10
20
30
40
50
60
70
80
0
20
40
time (hours) Fig. 8. 9.5
Influence of the parameters 1, M=10
p1=0.1, M=20
5, M=10
p1=0.5, M=20
0
50
100
150
10
−4
10
−3
10
−2
10
−1
10
0
p
t/∆t 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
qD(1,1)
qFA(2)
(2)
p01>10-1 or p10>10-1
p01<10-1 and p10<10-1
p1=0.1 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
qD(1,1)
qFA(2)
qFA(2)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
qD(1,1)
p01>10-1 or p10>10-1
p01<10-1 and p10<10-1
p01<10-2 and p10<10-2
p01<10-3 and p10<10-3
p1=0.1
p1=0.5
Fig. 12. Achievable p10
or p01
for different values of the pair
(qD (1, 1) , qFA (2)) when p1 = 0.1 (left) and for p1 = 0.5 (right) and
p2 = 0. qFA(2)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
qD(1,1)
p01<10-2 and p10<10-2
p01<10-3 and p10<10-3
p1=0.5 p01<10-1 and p10<10-1 p01<10-3 and p10<10-3 p01>10-1 or p10>10-1 p01<10-2 and p10<10-2
p01<10-3 and p10<10-3 p01<10-2 and p10<10-2
p0 p01<10-2 and p10<10-2 Fig. 12. Achievable p10
or p01
for different values of the pair
(qD (1, 1) , qFA (2)) when p1 = 0.1 (left) and for p1 = 0.5 (right) and
p2 = 0. Fig. 11. Evolution of p10 and p01 for various M ∈{4, 10, 20} and p1 ∈
{0.1, 0.5}, with qFA (2) = 0.05, qD (1, 1) = 0.8, qD (0, 2) = 0.9. For each case, the value of ν is chosen such that it minimizes
ep01 + ep10. One observes that a large M leads to a better
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defective sensors has also an impact on the convergence
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time is needed to achieve a given level of performance (in
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CONCLUSION This paper presents a fully distributed algorithm allowing
each node of a DTN to estimate the status of its own sensors
using LODT performed during the meeting of nodes. The
DFD algorithm is analyzed considering a Markov model
of the evolution of the proportion of nodes with a given
belief in their status. This model is then used to derive
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Area 3: both p10 and p01 are less than 10−3 ; •
Area 2: both p10 and p01 are less than 10−2 ; p
p
•
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nodes with defective sensors p1. Nevertheless, p1 has not
a significant impact on the non-detection and false alarm
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d
d
k REFERENCES degree in
Advanced Systems of Wireless Communication
from the University of Paris-Sud, France, in
2013. He is currently pursuing the Ph.D. degree
at the Laboratoire des Signaux et Systemes,
France. He has been visiting Ph.D. student at
the University of Bologna, Italy, in 2014 and
2015. His research interests lie in the areas of
distributed signal processing over networks and Laura Galluccio received her laurea degree in
Electrical Engineering from University of Cata-
nia, Catania, Italy, in 2001. In March 2005
she got her Ph.D. in Electrical, Computer and
Telecommunications Engineering at the same
university. Since 2002 she is also at the Ital-
ian National Consortium of Telecommunications
(CNIT), where she worked as a Research Fel-
low within the VICOM (Virtual Immersive Com-
munications) and the SATNEX Projects. Since
November 2010 she is Assistant Professor at
University of Catania. Her research interests include ad hoc and sensor
networks, protocols and algorithms for wireless networks, and network
performance analysis. From May to July 2005 she has been a Visiting
Scholar at the COMET Group, Columbia University, NY. In Septem-
ber 2015 she has been Visiting Professor at CentraleSupelec, Gif-
sur-Yvette, France. She is member of the IEEE, Comsoc and ACM
N2Women Group. Francesca Bassi received the Master of Sci-
ence degree in telecommunications engineering
from the University of Bologna, Bologna, Italy,
in 2006, and the Ph.D. degree from University
Paris-Sud, Orsay, France, and from the Univer-
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Gif-sur-Yvette, France. Between 2011 and 2012,
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expected proportions of nodes Xcm,cd
θ
(t), one will consider
an inter-contact rate λ and a well-mixed population of
nodes. During a short time interval [t, t + δt] the number
of nodes with state (θ, cm, cd) that will meet another node is
λpθnSXcm,cd
θ
(t)δt. lim
t→∞
ˆ t
0
p0p00 (τ) + p1p10 (τ)
dτ = ∞
(54)
then p0p00 (t) + p1p10 (t) > 0 for all t ∈R+. Proof: Since p0 > 0, p1 > 0, p00 ⩾0, and p10 ⩾0, it
suffices to prove that (54) θ
( )
When 0
⩽cm
< M, and thus also cd
⩽cm
<
M, nodes with state (θ, cm, cd) will switch to the states
(θ, cm + δm, cd + δd), with (δm, δd) ∈{(0, 0) , (1, 0) , (1, 1)}
with a probability πδm,δd
θ
(t, cm, cd), see Figure 13. p00 (t) + p10 (t) ̸= 0
∀t > 0. (55) (55) Assume that there exists a time instant t∗> 0, such that
p00 (t∗)+p10 (t∗) = 0. As a consequence, at time t∗, all nodes
in the network believe themselves as carrying defective
sensors. As a consequence, no node will transmit its data
to its neighbors. No LODTs will be performed after time
t∗and the state of nodes will remain constant. Hence, if
p00 (t∗) + p10 (t∗) = 0 for some t∗, then p00 (t) + p10 (t) = 0
for all t > t∗. Consequently, Assume that there exists a time instant t∗> 0, such that
p00 (t∗)+p10 (t∗) = 0. As a consequence, at time t∗, all nodes
in the network believe themselves as carrying defective
sensors. As a consequence, no node will transmit its data
to its neighbors. No LODTs will be performed after time
t∗and the state of nodes will remain constant. Hence, if
p00 (t∗) + p10 (t∗) = 0 for some t∗, then p00 (t) + p10 (t) = 0
for all t > t∗. APPENDIX B At time t, remind that Xcm,cd
θ
(t) is the proportion of nodes
in state (θ, cm, cd). For θ ∈{0, 1} , the process At time t, remind that Xcm,cd
θ
(t) is the proportion of nodes
in state (θ, cm, cd). For θ ∈{0, 1} , the process REFERENCES Michel Kieffer is co-author of more than 150
contributions in journals, conference proceedings, or books. He is one
of the co-authors of the book Applied Interval Analysis published by
Springer-Verlag in 2001, and of the book Joint source-channel decod-
ing: A crosslayer perspective with applications in video broadcasting
published by Academic Press in 2009. He serves as associate editor
of Signal Processing since 2008 and of the IEEE Transactions on
Communications from 2012 to 2016. From 2011 to 2016, Michel Kieffer
was junior member of the Institut Universitaire de France. 15 15
𝜋𝜃
0,−1
𝜋𝜃
0,−1
𝜃, 𝑀, 𝑐𝑑+ 1
𝜃, 𝑀, 𝑐𝑑−1
𝜃, 𝑀, 𝑐𝑑
𝜃, 𝑀−1, 𝑐𝑑
𝜃, 𝑀−1, 𝑐𝑑−1
𝜋𝜃
1,0
𝜋𝜃
1,1
𝜋𝜃
0,0
𝜋𝜃
0,1
𝜋𝜃
0,1
Fig. 14. Permanent regime: Possible state transitions from and to State
(θ, M, cd) when 0 < cd < M 𝜋𝜃
1,0
𝜋𝜃
1,1
𝜃, 𝑐𝑚+ 1, 𝑐𝑑+ 1
𝜃, 𝑐𝑚+ 1, 𝑐𝑑
𝜃, 𝑐𝑚, 𝑐𝑑
𝜃, 𝑐𝑚−1, 𝑐𝑑
𝜃, 𝑐𝑚−1, 𝑐𝑑−1
𝜋𝜃
1,0
𝜋𝜃
1,1
𝜋𝜃
0,0
Fig. 13. Transient regime: Possible state transitions from and to state
(θ, cm, cd) when 0 < cm < M and 0 < cd < cm Fig. 14. Permanent regime: Possible state transitions from and to State
(θ, M, cd) when 0 < cd < M Fig. 13. Transient regime: Possible state transitions from and to state
(θ, cm, cd) when 0 < cm < M and 0 < cd < cm Fig. 13. Transient regime: Possible state transitions from and to state
(θ, cm, cd) when 0 < cm < M and 0 < cd < cm Similar derivations can be made for the remaining DTN
state components to obtain (28). PROOF OF PROPOSITION 3 For the proof, one considers first the following lemmas. Xθ (t)=
X0,0
θ
(t) , X1,0
θ
(t) , X1,1
θ
(t) , . . . , XM,0
θ
(t) , . . . , XM,M
θ
(t)
APPENDIX A
PROOF OF PROPOSITION 2 PROOF OF PROPOSITION 2 Lemma 10. If Consequently, p
y
θ
( ,
m,
d)
g
As a consequence, at time t + δt, the number of nodes in
State (θ, cm, cd) may be expressed as follows pθnSXcm,cd
θ
(t + δt) = pθnSXcm,cd
θ
(t)
+ λδtpθnS
−Xcm,cd
θ
(t)
π1,0
θ
(t, cm, cd)+π1,1
θ
(t, cm, cd)
+Xcm−1,cd−1
θ
(t)π1,1
θ (t, cm−1,cd−1)+Xcm−1,cd
θ
(t)π1,0
θ (t,cm−1,cd)
. (51) lim
t→∞
ˆ t
0
p0p00(τ)+p1p10(τ)
dτ =
ˆ t∗
0
p0p00(τ)+p1p10(τ)
dτ, The evolution of the expected value Xcm,cd
θ
(t) of Xcm,cd
θ
(t) is
then described by the following differential equation2 The evolution of the expected value Xcm,cd
θ
(t) of Xcm,cd
θ
(t) is
then described by the following differential equation2 which contradicts (54). Lemma 11. The property (54) is always satisfied. Proof: From (28-a), one has dXcm,cd
θ
dt
= −λXcm,cd
θ
π1,0
θ
(cm, cd) + π1,1
θ
(cm, cd)
+ λXcm−1,cd−1
θ
π1,1
θ
(cm−1,cd−1) + λXcm−1,cd
θ
π1,0
θ
(cm−1,cd) . (52) Lemma 11. The property (54) is always satisfied.
Proof: From (28-a), one has Proof: From (28-a), one has X0,0
θ
(t) = exp
−λ
ˆ t
0
p0p00 (τ) + p1p10 (τ)
dτ
. (56) (56) When cm
=
M
and 0
<
cd
<
M, nodes in
state (θ, M, cd) will switch to the states (θ, M, cd + δd),
δd ∈{−1, 0, 1} with a probability π0,δd
θ
(t, M, cd). Nodes
in the states (θ, M −1, cd −1) and (θ, M −1, cd) that
have met an other node in the time interval [t, t + δt]
may reach state (θ, M, cd), respectively with a probability
π1,1
θ
(t, M −1, cd −1) and π1,0
θ
(t, M −1, cd), see Figure 14. As a consequence, the evolution of the expected value
XM,cd
θ
(t) of XM,cd
θ
(t) can be described by Assume that there exists C∗> 0 such that lim
t→∞
ˆ t
0
p0p00 (τ) + p1p10 (τ)
dτ ⩽C∗
(57) (57) then ∀t ⩾0, one has ˆ t
0
p0p00 (τ) + p1p10 (τ)
dτ ⩽C∗. (58) (58) g (56) and (58), one gets
X0,0
θ
(t) ⩾exp (−λC∗) > 0. (59) dXM,cd
θ dXM,cd
θ
dt
= −λXM,cd
θ
π0,1
θ
(M, cd) + π0,−1
θ
(M, cd)
+ λXM−1,cd−1
θ
π1,1
θ
(M −1, cd −1) + λXM−1,cd
θ
π1,0
θ
(M −1, cd)
+ λXM,cd−1
θ
π0,1
θ
(M, cd −1) + λXM,cd+1
θ
π0,−1
θ
(M, cd + 1) . (53) X0,0
θ
(t) ⩾exp (−λC∗) > 0. (59) (59) Moreover, from (6), one has p00 (τ) ⩾X0,0
θ
(τ), leading to
ˆ t
0
p0p00(τ)+p1p10(τ)
dτ ⩾
ˆ t
0
p0X0,0
θ
(τ)dτ >p0 exp (−λC∗) t. (60) (53) (60) 2. Notice that to lighten notations, time dependency is omitted. Moreover, πδm,δd
θ
(cm, cd) and pθbθ in the rest of the paper represent the
expected values, as they can be represented as functions of Xcm,cd
θ
(t) or
Xcm,cd
θ
(t). Since exp (−λC∗) t →∞as t →∞, (60) leads to a violation
of the hypothesis (57). Hence, one always has (54). 16 where using (25) and (26), one has where using (25) and (26), one has where using (25) and (26), one has The proof of Proposition 3 is then by induction. Starting
with (28-a), one has (56). Lemma 11. The property (54) is always satisfied.
Proof: From (28-a), one has Since (54) is satisfied according to
Lemma 11, for any ξ > 0, there exists t00 > 0 such that
t > t00 implies X0,0
θ (t) < ξ and limt→∞X0,0
θ (t) = 0.
η0 =
p0qFA(2)p00+p1qD(1,1)p10
p0(1−qFA(2))p00+p1(1−qD(1,1))p10 ,
η1 =
p0qD(1,1)p00+p1qD(0,2)p10
p0(1−qD(1,1))p00+p1(1−qD(0,2))p10 . (64) (64) p
θ
θ
Then, assume that for any cm ⩽M −1, and ξ > 0, there
exists t(cm−1)0 > · · · > t00 such that t > t(cm−1)0 implies
Xj,0
θ (t) < ξ for j = 0, . . . , cm −1. One has to show now that
there exists tcm0 > t(cm−1)0 such that Xcm,0
θ
(t) < ξ for all
t > tcm0. Define Zcm,0
θ
(t) = Pcm
j=0 Xj,0
θ
(t). From (28a) and (28b),
one has
dZcm 0 with p00 and p10 defined in (29). From (62) and (63), one deduces with p00 and p10 defined in (29). From (62) and (63), one deduces with p00 and p10 defined in (29). p
p
(
)
From (62) and (63), one deduces X
M,cd
θ
= X
M,0
θ
cd−1
Y
j=0
ηθ M −j
j + 1
= X
M,0
θ
ηcd
θ
M · (M −1) · (M −cd + 1)
1 · 2 · · · · cd
=
M
cd
! ηcd
θ X
M,0
θ
. (65) dZcm,0
θ
dt
= −λ
v (t) Zcm−1,0
θ
(t) +
p0p00 (t) + p1p10 (t)
Xcm,0
θ
(t)
, dZcm,0
θ
dt
= −λ
v (t) Zcm−1,0
θ
(t) +
p0p00 (t) + p1p10 (t)
Xcm,0
θ
(t)
(65) Since PM
cd=0 X
M,cd
θ
= 1, one has Since PM
cd=0 X
M,cd
θ
= 1, one has where v(t) = π1,1
θ
(t, cm, cd), since π1,0
θ
and π1,1
θ
do not
depend on cm and cd when cm < M. Using (55) one has
dZcm,0
θ
/dt < 0 for any Xcm0
θ
> 0. As a consequence, Zcm,0
θ
(t)
decreases until Xcm,0
θ
(t) reaches 0. Hence, for any ξ > 0,
there exists tcm,0 > t(cm−1)0, such that Xcm,0
θ
< ξ and
limt→∞Xcm,0
θ
(t) = 0. Since PM
cd=0 X
M,cd
θ
= 1, one has
1 =
M
X
cd=0
M
cd
! PROOF OF LEMMA 6 M and cd ⩽cm. To evaluate X
M,cd
θ
, one thus considers the
following simplified dynamics derived from (28) for θ ∈
{0, 1}, To prove Lemma 6, one needs first to investigate the mono-
tonicity of Fθ. To lighten the notations, let α = qFA (2),
β = qD (1, 1) and γ = qD (0, 2). Then h0 and h1 defined
in (31-32) can be rewritten as
dXM,0
θ
dt
=λ
−XM,0
θ
π0,1
θ (M, 0)+XM,1
θ
π0,−1
θ
(M, 1)
,
dXM,M
θ
dt
=λ
−XM,M
θ
π0,−1
θ
(M, M)+XM,M−1
θ
π0,1
θ (M, M −1)
,
dXM,cd
θ
dt
=λ
−XM,cd
θ
π0,−1
θ
(M, cd) + π0,1
θ (M, cd)
+XM,cd+1
θ
π0,−1
θ
(M, cd + 1) + XM,cd−1
θ
π0,1
θ (M, cd−1)
(61) h0 (x, y) = αp0x + βp1y
p0x + p1y
, h1 (x, y) = βp0x + γp1y
p0x + p1y ,
(68) (68) with (x, y) ∈P0. One starts showing some monotonicity
properties. with (x, y) ∈P0. One starts showing some monotonicity
properties.
(61) Lemma 12. If α < β < γ, then h0 and h1 are decreasing with
x and increasing with y, for all (x, y) ∈P0. If β = γ, then
h1 = β = γ is a constant. (61)
At equilibrium, one has dXM,cd
θ
(t)/dt = 0 for all cd ⩽M. Moreover, the transition probabilities do not vary any more. T p
y
y
Let X
M
θ
=
X
M,1
θ
, . . . , X
M,M
θ
T
, aθ (cd) = π0,1
θ
(M, cd),
and bθ (cd) = π0,−1
θ
(M, cd). From (61), one deduces that
the vector X
M
θ
should satisfy Ψθ · X
M
θ = 0 where Proof: Since α < β ⩽γ, one has Proof: Since α < β ⩽γ, one has ∂h0
∂x = (α −β) p0p1y
(p0x + p1y)2 ⩽0,
∂h0
∂y = (β −α) p0p1x
(p0x + p1y)2 ⩾0,
∂h1
∂x = (β −γ) p0p1y
(p0x + p1y)2 ⩽0,
∂h1
∂y = (γ −β) p0p1x
(p0x + p1y)2 ⩾0. Ψθ =
−aθ (0)
bθ (1)
aθ (0)
−aθ(1) −bθ (1)
bθ (2)
... ... Lemma 11. The property (54) is always satisfied.
Proof: From (28-a), one has ηcd
θ X
M,0
θ
= (ηθ + 1)M X
M,0
θ
. (66)
From (65) and (66), ∀cd = 0, . . . , M,
X
M,cd
θ
=
M
cd
!
ηθ
ηθ + 1
cd
1
ηθ + 1
M−cd
=
M
cd
! (hθ)cd(1 −hθ)M−cd
(67) 1 =
M
X
cd=0
M
cd
! ηcd
θ X
M,0
θ
= (ηθ + 1)M X
M,0
θ
. (66) (66) From (65) and (66), ∀cd = 0, . . . , M, θ
In the same way, using (28c) and the previous results
that Xcmcd
θ
(t) →0 with cd = 1, . . . , M −2 and cm =
cd, . . . , M −2, one can prove that for any cd = 1, . . . , M −1,
Xc′
m,(cd+1)
θ
(t) tends to zero as t →∞, with any c′
m =
cd + 1, . . . , M −1. θ
In the same way, using (28c) and the previous results
that Xcmcd
θ
(t) →0 with cd = 1, . . . , M −2 and cm =
cd, . . . , M −2, one can prove that for any cd = 1, . . . , M −1,
′ (
) (67) Xc′
m,(cd+1)
θ
(t) tends to zero as t →∞, with any c′
m =
cd + 1, . . . , M −1. with hθ =
ηθ
ηθ+1. Introducing (67) into (29), one obtains (34)
with Fθ defined in (33). Thus one needs to solve (34) to
determine p, which is then used to deduce X
M,d
θ
using (67). with hθ =
ηθ
ηθ+1. Introducing (67) into (29), one obtains (34)
with Fθ defined in (33). Thus one needs to solve (34) to
determine p, which is then used to deduce X
M,d
θ
using (67). θ
( )
cd + 1, . . . , M −1. PROOF OF PROPOSITION 4 PROOF OF PROPOSITION 4 PROOF OF LEMMA 6 aθ (M − Ψθ =
−aθ (0)
bθ (1)
aθ (0)
−aθ(1) −bθ (1)
bθ (2) bθ (2)
... ... aθ (M −1)
−bθ (M)
. then Lemma 12 can be proved. APPENDIX D According to Proposition 3, one has X
cm,cd
θ
= 0, for all cm <
M PROOF OF LEMMA 6 then Lemma 12 can be proved. In (71), each i in the
sum is such that
i
M < ν ⩽z. From Lemma 13, fi (z) is
a decreasing function for any
i
M < z, thus g(z) is also
decreasing with z. have module less than 1. The eigenvalues of A are the
solutions of g
Now, consider z ∈[0, ν], one rewrites (71) as z2 −
∂F0
∂x + ∂F1
∂y
z +
∂F0
∂x
∂F1
∂y −∂F0
∂y
∂F1
∂x
= 0. (75) (75) g(z) = 1 −
X
i:i/M⩾ν
M
i
! fi (z) ,
(72) (72) As in Appendix D, denote α = qFA (2), β = qD (1, 1) and
γ = qD (0, 2). First, one evaluates in which each i in the sum is such that z < ν ⩽
i
M . Applying
again Lemma 13, since fi (z) is an increasing function for
any z ⩽
i
M , the sum in (72) is also increasing with z and
g (z) is decreasing. Thus g(z) decreases over [0, 1]. ∂F0
∂x
∂F1
∂y −∂F0
∂y
∂F1
∂x = ∂g
∂h0
∂g
∂h1
∂h0
∂x
∂h1
∂y −∂h0
∂y
∂h1
∂x
(a)
= 0, where (a) comes from ∂h0
∂x
∂h1
∂y = ∂h0
∂y
∂h1
∂x , using the partial
derivatives calculated in the proof of Lemma 12. Then, the
solutions of (75) are z1 = ∂F0
∂x + ∂F1
∂y and z2 = 0. Hence, it
suffices to prove that |z1| < 1. ( )
g
( )
[
]
Considering the functions hθ and g, then one may
rewrite Fθ as Fθ (x, y) = g (hθ (x, y)), ∀θ ∈{0, 1}. The
monotonicity of F0 and F1 is shown in the following lemma. Lemma 15. If α < β < γ, then F0 and F1 are increasing
functions of x and decreasing functions of y, for all (x, y) ∈P0. If β = γ, then F1 = g(β) = g(γ) is a constant. p
|
|
We begin with the evaluation of an upper bound of the
partial derivative of F0 (x, y) with respect to x ∂F0 (x, y)
∂x
= ∂g (h0 (x, y))
∂x
= ∂g
∂h0 · ∂h0
∂x
(a)
= (β −α) p0p1y
(p0x + p1y)2
X
i:i/M<ν
M
i
! then Lemma 12 can be proved. hi
0 (1 −h0)M−i
h0M −i
h0 (1 −h0)
(b)
⩽(β −α) p0p1y
(p0x + p1y)2 F0 (x, y)
M
1 −h0 ⩽c0 (α, β, γ, M, ν, n) ,
(76) Proof:
The
proof
of
obtained
by
combining
Lemma 12 and Lemma 14. Proof:
The
proof
of
obtained
by
combining
Lemma 12 and Lemma 14. The proof of Lemma 6 is by induction. At the beginning,
one has 0 ⩽pθ0(0) ⩽1, thus pθ0
min (0) = 0 and pθ0
max (0) = 1. Using Lemma 15, one has Fθ(0, 1) ⩽Fθ
p00 (0) , p10 (0)
⩽
Fθ (1, 0), thus
p00
min (1) = F0 (0, 1) = g (β) > 0 = p00
min (0) ,
p00
max (1) = F0 (1, 0) = g (α) < 1 = p00
max (0) ,
p10
min (1) = F1 (0, 1) = g (γ) > 0 = p10
min (0) ,
p10
max (1) = F1 (1, 0) = g (β) < 1 = p10
max (0) ,
(73)
pmin (1) = F0 (0, 1) = g (β) > 0 = pmin (0) ,
p00
max (1) = F0 (1, 0) = g (α) < 1 = p00
max (0) ,
p10
min (1) = F1 (0, 1) = g (γ) > 0 = p10
min (0) ,
p10
max (1) = F1 (1, 0) = g (β) < 1 = p10
max (0) ,
(73) where (a) is obtained using (70), (b) comes from i ⩾0, and
c0 is defined in (37). Meanwhile, from Lemma 15, one has
∂F0 (x, y) /∂x ⩾0, as F0 is an increasing function of x. Similarly, (73) thus (40) and (41) are true for n = 1. thus (40) and (41) are true for n = 1. ∂F1 (x, y)
∂y
= ∂g (h1 (x, y))
∂y
= ∂g
∂h1 · ∂h1
∂y
= (γ −β) p0p1x
(p0x + p1y)2
X
i:i/M<ν
M
i
! hi
1 (1 −h1)M−i
i −h1M
h1 (1 −h1)
⩾(γ −β) p0p1x
(p0x + p1y)2 F1 (x, y) −M
1 −h1 ⩾−c1 (α, β, γ, M, ν, n) , (77 Consider then an arbitrary n ∈N∗and n > 1. then Lemma 12 can be proved. .. .. aθ (M −1)
−bθ (M)
.. .. aθ (M −1)
−bθ (M)
Lemma 13. For z ∈[0, 1], the family of functions 3. For z ∈[0, 1], the family of functions
fi (z) = zi (1 −z) M−i, i = 0, . . . , M. (69) Summing Lines 1 to cd + 1, for all cd = 0, . . . , M −1, one
obtains aθ (cd) X
M,cd
θ
= bθ (cd + 1) X
M,cd+1
θ
, which leads to fi (z) = zi (1 −z) M−i, i = 0, . . . , M. (69) (69) X
M,cd
θ
= X
M,0
θ
cd−1
Y
j=0
a0 (j)
b0 (j + 1). (62) are increasing over [0,
i
M ] and decreasing over [ i
M , 1]. are increasing over [0,
i
M ] and decreasing over [ i
M , 1]. (62) Proof: Consider three possible situations: 1) If i = 0,
f0 (z) = (1 −z)M is decreasing over [0, 1]. 2) If i = M,
fM (z) = zM is increasing over [0, 1]. 3) If 1 ⩽i ⩽M −1, One evaluates aθ (j)
bθ (j + 1) =
π0,1
θ
(M, j)
π0,−1
θ
(M, j + 1)
= ηθ M −j
j + 1 ,
(63) dfi
dz = zi−1 (1 −z) M−i−1 (i −Mz) ,
(70) (63) (70) 17 and dfi/dz ⩾0 when z ∈[0,
i
M ] and dfi/dz ⩽0 when
z ∈[ i
M , 1]. Therefore, Lemma 13 holds ∀i = 0, . . . , M. F is contracting, i.e., that for any pairs p = (x, y) ∈Pn and
p + δ = (x + δx, y + δy) ∈Pn, one has Lemma 14. If 0 < ν < 1, the function Lemma 14. If 0 < ν < 1, the function |F (p + δ) −F (p)| < |δ| . (74) (74) g(z) =
X
i:i/M<ν
M
i
! fi (z) =
X
i:i/M<ν
M
i
! zi (1 −z) M−i,
(71) A sufficient condition to have (74) is that the eigenvalues
of the matrix is decreasing for all z ∈[0, 1]. is decreasing for all z ∈[0, 1]. A =
∂F0(x,y)
∂x
∂F0(x,y)
∂y
∂F1(x,y)
∂x
∂F1(x,y)
∂y
! Proof: First, consider z ∈[ν, 1]. PROOF OF PROPOSITION 7 PROOF OF PROPOSITION 7 and and ∂F1 (x, y) /∂y ⩽0 as F1 is a non-decreasing function of
y. One concludes that and ∂F1 (x, y) /∂y ⩽0 as F1 is a non-decreasing function of
y. One concludes that pθ0
max (n) = Fθ
p00
max (n −1) , p10
min (n −1)
< Fθ
p00
max (n −2) , p10
min (n −2)
= pθ0
max (n −1) , −c1 ⩽∂F0 (x, y)
∂x
+ ∂F1 (x, y)
∂y
⩽c0, Similarly, one gets p10
min (n) > p10
min (n −1) and pθ0
max (n) <
pθ0
max (n −1) . Similarly, one gets p10
min (n) > p10
min (n −1) and pθ0
max (n) <
pθ0
max (n −1) . thus c0 < 1 and c1 < 1 lead to |z1| < 1, which ensures the
uniqueness of the equilibrium. then Lemma 12 can be proved. Assume
that (40) and (41) are satisfied for any n′ < n and n′ ∈N∗,
one needs to see whether (40) and (41) are still satisfied for
n. Applying Lemma 15 again, one obtains pθ0
min (n) = Fθ
p00
min (n −1) , p10
max (n −1)
> Fθ
p00
min (n −2) , p10
max (n −2)
= pθ0
min (n −1) , and APPENDIX F
PROOF OF PROPOSITION 9 As seen in the proof of Proposition 5, ∀n
∈
N∗,
F (p) maps Pn to Pn, with Pn =
p00
min (n) , p00
max (n)
×
p10
min (n) , p10
max (n)
. In order to apply Banach’s fixed-point
theorem [42] to prove Proposition 7, it suffices to show that PROOF OF PROPOSITION 9 First, one shows that if ν < qD (1, 1), then for any ε > 0,
there exists M > M ′, such that p10 < ε. 18 From Lemma 6, p10 can be bounded as From Lemma 6, p10 can be bounded as As a conclusion, for any ε > 0, if χ (ε) + ε < ν <
qD (1, 1) −ε, then p00
⩾
1 −ε and p10
<
ε. Since
limε→0 χ (ε) = qFA (2), one concludes that if qFA (2) < ν <
qD (1, 1), one obtains (43). p10 = F1
p00, p10
<
X
cd:cd/M<ν
M
cd
! (qD (1, 1))cd (1 −qD (1, 1))M−cd
(78 (78) (78) Consider Φ1, Φ2, . . . an infinite sequence of i.i.d. binary
random variables with P {Φm = 1} = qD (1, 1). For any
ϱ ∈[0, 1] such that ϱM ∈N+, one has P
(PM
m=1 Φm
M
=ϱ
)
=
M
ϱM
! (qD (1,1))ϱM(1−qD (1,1))M(1−ϱ). According to the weak law of large numbers [46], for ε > 0,
there exists M ′, such that for any M > M ′, one has P
(
PM
m=1 Φm
M
−qD (1, 1)
> qD (1, 1)
)
< ε. (79) (79) From (79), one also has From (79), one also has X
cd:cd/M<(qD(1,1)−ε)
(qD (1, 1))cd (1 −qD (1, 1))M−cd
=P
(PM
m=1Φm
M
−qD(1,1)<−ε
)
⩽P
(
PM
m=1Φm
M
−qD(1,1)
>ε
)
< ε. (80) < ε. If ν < qD (1, 1) −ε, then using (80), the bound of p10 in (78)
may be further written as If ν < qD (1, 1) −ε, then using (80), the bound of p10 in (78)
may be further written as p10 <
X
cd:cd/M<ν
M
cd
! (qD (1, 1))cd(1−qD (1, 1))M−cd
⩽
X
cd:cd/M<(qD(1,1)−ε)
(qD (1, 1))cd(1−qD (1, 1))M−cd<ε. (81) (81) From Lemma 12 and the fact that qFA (2) ⩽p10 ⩽
qD (1, 1) and 0
⩽
p10
<
ε, one has h0
p00, p10
∈
[qFA (2) , χ (ε)], with χ (ε) = p0 (qFA (2))2 + p1qD (1, 1) ε
p0qFA (2) + p1ε
. (82) (82) Thus, according to Lemma 14, p00 = F0
p00, p10
= g
h0
p00, p10
⩾g (χ (ε)) =
X
cd:cd/M<ν
M
cd
! (χ (ε))cd (1 −χ (ε))M−cd . PROOF OF PROPOSITION 9 (83) (83) Using derivations similar to those leading to (80), one gets Using derivations similar to those leading to (80), one gets X
cd:cd/M>(χ(ε)+ε)
M
cd
! (χ (ε))cd (1 −χ (ε))M−cd < ε,
(84) which leads to which leads to X
cd:cd/M⩽(χ(ε)+ε)
M
cd
! (χ (ε))cd (1 −χ (ε))M−cd ⩾1 −ε. (85) If ν > χ (ε) + ε, then If ν > χ (ε) + ε, then If ν > χ (ε) + ε, then p00 ⩾
X
cd:cd/M<ν
M
cd
! (χ (ε))cd (1 −χ (ε))M−cd ⩾
X
cd:cd/M⩽(χ(ε)+ε)
(χ (ε))cd (1 −χ (ε))M−cd ⩾1 −ε. (86) ⩾
X
cd:cd/M⩽(χ(ε)+ε)
(χ (ε))cd (1 −χ (ε))M−cd ⩾1 −ε. (86)
|
https://openalex.org/W3003922378
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https://europepmc.org/articles/pmc7064027?pdf=render
|
English
| null |
Association between DNA damage repair gene somatic mutations and immune‐related gene expression in ovarian cancer
|
Cancer medicine
| 2,020
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cc-by
| 7,731
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K E Y W O R D S DNA damage repair, immunotherapy, mutation, ovarian cancer O R I G I N A L R E S E A R C H O R I G I N A L R E S E A R C H Correspondence Correspondence
Huaying Wang, Department of Gynecologic
Oncology, Fudan University Shanghai
Cancer Center; Department of Oncology,
Shanghai Medical College, Fudan
University, Shanghai, People’s Republic of
China, 200032. Conclusions: DDR deficiency was associated with specific immune signatures in OvCa. Our findings emphasize the urgent need for biomarker-guided rational immune combi-
nation therapy to maximize the OvCa patients who could benefit from immunotherapy. Email: wanghuayingfducc@163.com Wenjuan Tian1 | Boer Shan1 | Yuzi Zhang2 | Yulan Ren1 | Shanhui Liang1 |
Jing Zhao2 | Zhengyi Zhao2 | Guoqiang Wang2 | Xiaochen Zhao2 | Dongxian Peng3 |
Rui Bi4
| Shangli Cai2 | Yuezong Bai2 | Huaying Wang1 Wenjuan Tian1 | Boer Shan1 | Yuzi Zhang2 | Yulan Ren1 | Shanhui Liang1 |
Jing Zhao2 | Zhengyi Zhao2 | Guoqiang Wang2 | Xiaochen Zhao2 | Dongxian Peng3 |
Rui Bi4
| Shangli Cai2 | Yuezong Bai2 | Huaying Wang1 Wenjuan Tian1 | Boer Shan1 | Yuzi Zhang2 | Yulan Ren1 | Shanhui Liang1 |
Jing Zhao2 | Zhengyi Zhao2 | Guoqiang Wang2 | Xiaochen Zhao2 | Dongxian Peng3 |
Rui Bi4
| Shangli Cai2 | Yuezong Bai2 | Huaying Wang1 1Department of Gynecologic Oncology,
Fudan University Shanghai Cancer Center,
Department of Oncology, Shanghai Medical
College, Fudan University, Shanghai,
People’s Republic of China 1Department of Gynecologic Oncology,
Fudan University Shanghai Cancer Center,
Department of Oncology, Shanghai Medical
College, Fudan University, Shanghai,
People’s Republic of China
2The Medical Department, 3D Medicines
Inc, Shanghai, People’s Republic of China
3Department of Obstetrics and Gynecology,
Zhujiang Hospital, Southern Medical
University, Guangdong, People’s Republic
of China Abstract
Background: Defects in DNA damage repair (DDR) system may lead to genomic insta-
bility and manifest as increased immunogenicity. DDR deficiency is prevalent in ovar-
ian cancer (OvCa); however, the association of DDR mutation with immune profiles in
OvCa remains largely unknown. This knowledge will provide an essential basis to the
rational design of biomarker-guided immune combination therapy of OvCa in the future. Methods: Whole-exome sequencing data of 587 OvCa from The Cancer Genome
Atlas (TCGA) were used to determine the expression profiles of 47 immune-related
genes and the abundance of tumor-infiltrating immune cells. A Chinese OvCa cohort
(n = 220) tested by next-generation sequencing (NGS) was used to validate the as-
sociation between DDR status and tumor mutation burden (TMB). Results: A total of 19.3% in TCGA cohort and 25.9% in Chinese cohort harbored
at least one DDR somatic mutation. DDR deficiency exhibited a distinct immune
profile with significant higher expression levels of PTPRCAP, CCL5, IFI16, LAG3,
IL15RA, and GBP1 in OvCa in the TCGA cohort. Different DDR pathway defi-
ciency displayed various immune profiles. Increased levels of Th1 cells, TMB, and
neoantigen were also observed in DDR-deficient tumors. Conclusions: DDR deficiency was associated with specific immune signatures in OvCa. Our findings emphasize the urgent need for biomarker-guided rational immune combi-
nation therapy to maximize the OvCa patients who could benefit from immunotherapy. K E Y W O R D S
DNA damage repair, immunotherapy, mutation, ovarian cancer Received: 27 November 2019 |
Revised: 2 January 2020 |
Accepted: 2 January 2020 Received: 27 November 2019 |
Revised: 2 January 2020 |
Accepted: 2 January 2020 Received: 27 November 2019 |
Revised: 2 January 2020 |
Accepted: 2 January 2020
DOI: 10.1002/cam4.2849 Abstract Abstract
Background: Defects in DNA damage repair (DDR) system may lead to genomic insta-
bility and manifest as increased immunogenicity. DDR deficiency is prevalent in ovar-
ian cancer (OvCa); however, the association of DDR mutation with immune profiles in
OvCa remains largely unknown. This knowledge will provide an essential basis to the
rational design of biomarker-guided immune combination therapy of OvCa in the future. Methods: Whole-exome sequencing data of 587 OvCa from The Cancer Genome
Atlas (TCGA) were used to determine the expression profiles of 47 immune-related
genes and the abundance of tumor-infiltrating immune cells. A Chinese OvCa cohort
(n = 220) tested by next-generation sequencing (NGS) was used to validate the as-
sociation between DDR status and tumor mutation burden (TMB). 2The Medical Department, 3D Medicines
Inc, Shanghai, People’s Republic of China
3Department of Obstetrics and Gynecology,
Zhujiang Hospital, Southern Medical
University, Guangdong, People’s Republic
of China 2The Medical Department, 3D Medicines
Inc, Shanghai, People’s Republic of China 3Department of Obstetrics and Gynecology,
Zhujiang Hospital, Southern Medical
University, Guangdong, People’s Republic
of China 4Department of Pathology, Fudan
University Shanghai Cancer Center,
Department of Oncology, Shanghai Medical
College, Fudan University, Shanghai,
People’s Republic of China Results: A total of 19.3% in TCGA cohort and 25.9% in Chinese cohort harbored
at least one DDR somatic mutation. DDR deficiency exhibited a distinct immune
profile with significant higher expression levels of PTPRCAP, CCL5, IFI16, LAG3,
IL15RA, and GBP1 in OvCa in the TCGA cohort. Different DDR pathway defi-
ciency displayed various immune profiles. Increased levels of Th1 cells, TMB, and
neoantigen were also observed in DDR-deficient tumors. 2.1 | Characterization of DDR gene status in
TCGA cohort OvCa carrying DNA damage repair (DDR) deficiency ex-
hibits a high sensitivity to platinum-based chemotherapy.10
Various DDR pathways dealing with DNA damage were
identified, such as mismatch repair (MMR), base excision re-
pair (BER), checkpoint factors (CPF), Fanconi anemia (FA),
and homologous recombination repair (HRR).11 DDR system
is required to maintain the genomic integrity and stability.12
Further alteration in DDR may induce a hypermutated phe-
notype, with a higher tumor mutation burden (TMB), which
has been established as a predictive biomarker of ICIs treat-
ment.13,14 As an example, defects in MMR (MLH1, MSH2,
MSH6, and PMS2) pathway or BER (POLE) pathway, which
are responsible for the extraordinary fidelity of human ge-
nome, typically exhibited an ultra-mutated phenotype and
further resulted in a durable clinical benefit with ICIs.15,16
Melanoma patients with a response to ICIs commonly har-
bored mutations in BRCA2, a well-known gene in HRR
pathway.17 Most recently, a study has revealed that co-muta-
tions in specific DDR pathways might predict clinical bene-
fit from ICIs treatment.18 Another study further verified the
association between alterations in 34 DDR genes exhibited
a higher TMB level and improved clinical benefit from ICIs
in urothelial cancer.19 Previous studies have also illustrated
the interaction of DDR with immune system, and immune-re-
lated genes such as GZMA may also have an impact on the
function of DDR system.20 A recent study has revealed that OvCa carrying DNA damage repair (DDR) deficiency ex-
hibits a high sensitivity to platinum-based chemotherapy.10
Various DDR pathways dealing with DNA damage were
identified, such as mismatch repair (MMR), base excision re-
pair (BER), checkpoint factors (CPF), Fanconi anemia (FA),
and homologous recombination repair (HRR).11 DDR system
is required to maintain the genomic integrity and stability.12
Further alteration in DDR may induce a hypermutated phe-
notype, with a higher tumor mutation burden (TMB), which
has been established as a predictive biomarker of ICIs treat-
ment.13,14 As an example, defects in MMR (MLH1, MSH2,
MSH6, and PMS2) pathway or BER (POLE) pathway, which
are responsible for the extraordinary fidelity of human ge-
nome, typically exhibited an ultra-mutated phenotype and
further resulted in a durable clinical benefit with ICIs.15,16 suffered recurrence. Unfortunately, effective options are lim-
ited for these patients and therefore novel therapeutic strate-
gies are under urgent need. biallelic inactivation of DDR gene mutations exhibits de-
creased expression of immune regulatory gene in bladder
cancer.21 Furthermore, DDR inactivation was associated with
higher levels of TMB without a higher tumor-infiltrating im-
mune cell abundance in bladder cancer.21 Immune checkpoint inhibitors (ICIs) have revolution-
ized the area of anticancer therapy and been approved
for the treatment across a broad range of solid tumors.2-4
Unfortunately, objective response rate (ORR) is relatively
low with ICIs monotherapy in unselected OvCa patients,
which was ranging from 9.7% to 15%.5-7 Current efforts
have been focused on the development of new predictive
biomarkers to optimize patient benefit from ICIs. PD-L1
immunohistochemistry diagnostic assay was approved by
FDA as a companion to pembrolizumab treatment in non-
small cell lung cancer (NSCLC) patients. However, results
from Keynote-028 have demonstrated that OvCa patients
with positive PD-L1 expression status (levels ≥ 1%) de-
rived limited benefit from pembrolizumab monotherapy
(an ORR of 11.5%),7 not to mention of challenges such as
antibody uniformity, expression heterogeneity, and unstable
expression level, which limited the wide use of PD-L1 in
clinical practice.8 Although FDA has also approved mis-
match repair deficient (dMMR)/microsatellite instability
high (MSI-H) as a biomarker for pembrolizumab treatment
in solid tumors, only 3.2% of OvCa harbored dMMR/MSI-
H.9 Therefore, promising biomarkers and rational immune
combination therapies are needed to maximize patients with
OvCa who could benefit from immunotherapy. However, a comprehensive view of DDR mutation-as-
sociated immunogenicity in OvCa is still unclear. We
hypothesize that DDR mutations may exhibit a distinct
immune profile in OvCa. This knowledge will provide
essential basis to the design of biomarker-guided rational
immune combination therapy in the future. To validate this
hypothesis, we interrogated the expression profiles of 47
immune-related gene panel using the sequencing data from
The Cancer Genome Atlas (TCGA) (n = 587) to charac-
terize the pattern of DDR mutations and investigate their
association with the expression profiles of immune-related
genes in OvCa. 1 |
INTRODUCTION patients were diagnosed as advanced stage disease with a
5-year relative survival of only around 40%.1 Despite the
standard combined approach based on cytoreductive surgery
and platinum-taxane chemotherapy, most patients ultimately Ovarian cancer (OvCa) remains the leading cause of death
among gynecological cancers.1 More than 60% of OvCa 2190 |
Cancer Medicine. 2020;9:2190–2200. wileyonlinelibrary.com/journal/cam4
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original
work is properly cited. © 2020 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. Drs. Wenjuan Tian and Boer Shan contributed equally to this work. Cancer Medicine. 2020;9:2190–2200. 2191 TIAN et al. 2.1 | Characterization of DDR gene status in
TCGA cohort We utilized cBioPortal to retrieve the web-based whole-ex-
ome sequencing (WES), mRNA expression, and clinical data
of 587 OvCa patients from TCGA data portal (https://portal. gdc.cancer.gov). A total of 579 patients with DNA muta-
tion data were included into the analysis. Demographic and
clinicopathologic characteristics are shown in Table S1. To
identify the DDR inactivation mutation status, the DNA data
of copy number variant and single-nucleotide variants of 21
DDR genes (Table S2) were retrieved and combined. We de-
fined alternations in DDR pathway as any non-synonymous
somatic alteration (including missense, nonsense, insertion,
deletion, and splice) in protein-coding region or the presence
of homozygous deletions of at least one gene involved in the
corresponding DDR pathways. 2.7 | Associations between DDR
mutations and clinical prognosis To determine the association between DDR status and progno-
sis, clinical data were downloaded from TCGA. Overall survival
(OS) was defined as the time from initial surgery to the date of
death or last contact (censored). Progression-free survival (PFS)
was defined as the time from initial surgery to the date of pro-
gressed or recurrence disease or last contact (censored). (CAP)-certified and Clinical Laboratory Improvement
Amendments (CLIA)-certified 3D Medicines Library. of 22 immune cell types for TCGA cohort.29 The association
between DDR status and the immune cells of T helper type
1 (Th1), Th2, and Th17 was also investigated. Data for the
three cell types were obtained from a published study.30 2.6 | Associations between DDR
mutations and PD-L1 expression The immune gene signature was defined by 47 immune genes. The immune gene list is shown in Table S3. After data filter-
ing, the mRNA expression data of 40 immune genes were
available in the TCGA cohort. The mRNA expression and
DNA mutation data were used and the relationships between
DDR status and immune gene signature were analyzed. To
evaluate the association between immune-related gene sig-
nature and DDR status, we also performed gene set enrich-
ment analysis (GSEA) analysis by javaGSEA 3.0 Desktop
Application
(http://software.broadinstitute.org/gsea/index. jsp). The enrichment score (ES) was the primary result of
GSEA and the ES for the gene set was the score at the peak. The positive normalized enrichment score (NES) indicated
an association between the DDR status and the gene set en-
richment results. A gene set with significant enrichment was
identified at false discovery rate (FDR) < 0.05. To determine the association between DDR status and PD-L1
protein expression level, a total of 175 OvCa with PD-L1 pro-
tein expression data from the Chinese cohort were analyzed. The expression of PD-L1 protein on tumor cells was evalu-
ated with an immunohistochemical assay (IHC, Ventana,
SP263). PD-L1 expression positive was defined as at least
1% PD-L1 expression detected on tumor cells or in tumor
stroma. 2.4 | Association between DDR gene
mutations and genomic changes To investigate if DDR gene mutations are associated with
TMB, DNA somatic mutation data were downloaded from
TCGA. TMB was defined as the total number of non-syn-
onymous somatic mutation in the coding region. We also
used the Chinese cohort to validate the association between
DDR status and TMB. Germline variants were excluded. The TMB estimated by 381 cancer-genes NGS panel was
strongly associated with TMB estimated by WES.23 To de-
termine the association between DDR status and neoanti-
gen, we downloaded the neoantigen data per tumor sample
ID from a published study.24 The association between DDR
status and genomic instability was also explored. Data of
genomic instability in terms of number of telomeric al-
lelic imbalances (NtAI), large-scale state transitions (LST),
HRD-LOH, weighted genome integrity index (wGII), and
ploidy status were downloaded from five previously pub-
lished studies.25-28 2.8 Data were analyzed with GraphPad Prism (version 7.01,
GraphPad Software, USA), SPSS statistical software (version
20.0, SPSS, IBM Corporation, USA), and R (version 4.3.1, R
Development Core Team). Mann–Whitney U test was used to
determine the difference between two groups. The Kaplan–
Meier method was used to estimate PFS and OS. Differences
in PFS or OS were assessed with log-rank test. Hazard ratios
(HR) and associated 95% CIs were determined by Cox's re-
gression. All reported P values were two-sided and considered
statistically significant at P < .05, unless otherwise specified. 2.2 | Chinese cohort tested by next-
generation sequencing (NGS) Melanoma patients with a response to ICIs commonly har-
bored mutations in BRCA2, a well-known gene in HRR
pathway.17 Most recently, a study has revealed that co-muta-
tions in specific DDR pathways might predict clinical bene-
fit from ICIs treatment.18 Another study further verified the
association between alterations in 34 DDR genes exhibited
a higher TMB level and improved clinical benefit from ICIs
in urothelial cancer.19 Previous studies have also illustrated
the interaction of DDR with immune system, and immune-re-
lated genes such as GZMA may also have an impact on the
function of DDR system.20 A recent study has revealed that A total of 220 ovarian tumors from Fudan University Shanghai
Cancer Center (FDCC) and Zhujiang Hospital (ZJH) received
next-generation sequencing (NGS) during January 2016
to July 2019. Genomic DNA from formalin-fixed paraffin-
embedded (FFPE) tumor specimens or fresh tumor tissues
and matched blood sample were used for sequence analy-
sis of 381 cancer-associated genes (including the 21 DDR
genes in Table S2) in the NGS platform illumina Nextseq
500 to > 500X coverage as previously described.22 The test-
ing was performed in the College of American Pathologists 2192 |
(CAP)-certified and Clinical Laboratory Improvement
Amendments (CLIA)-certified 3D Medicines Library. 2192 TIAN et al. Among the 40 immune-related genes, mRNA expression of
PTPRCAP, CCL5, IFI16, LAG3, IL15RA, and GBP1 were
significantly higher in the DDR mutation group than the
DDR wild-type group (P < .05) (Figure 2A). The expression
of VEGFA was significantly lower in DDR mutation group
compared to DDR wild-type group. By averaging z-score
expression levels per tumor, tumors with DDR mutation
had lower expression levels of CTLA-4, IFNG, TNF, FAS,
and VTCN1, and higher expression levels of IL6, IL1B,
and IL12A compared with the DDR wild-type ones (Figure
2B). Across the five DDR pathway, FA and HRR pathway
mutations shared lower expression levels of CTLA-4 and
TBX21, and higher expression levels of IL6, IFI16, and
IL12A. Checkpoint, MMR, and BER pathway mutations
shared lower expression levels of PDCDILG2, IL6, and
CD27. Furthermore, BRCA1 mutation tumors showed lower
expression levels of TNF, IL18, and PDCDILG2 compared
to BRCA1 wild-type tumors. However, BRCA2 mutation
was associated with a higher expression level of TNF, IL18,
and PDCD1LG2 compared with BRCA2 wild-type tumors
(Figure S1). (23/579), 2.9% (17/579), and 1.2% (7/579), respectively. The
frequencies of every DDR gene mutation are summarized in
Figure 1. The most frequently mutated genes were BRCA1
(3.8%, 22/579), FANCA (3.8%, 22/579), BRCA2 (3.1%,
18/579), RAD51 (2.9%, 17/579), and ATM (1.7%, 10/579)
(Figure 1A). (23/579), 2.9% (17/579), and 1.2% (7/579), respectively. The
frequencies of every DDR gene mutation are summarized in
Figure 1. The most frequently mutated genes were BRCA1
(3.8%, 22/579), FANCA (3.8%, 22/579), BRCA2 (3.1%,
18/579), RAD51 (2.9%, 17/579), and ATM (1.7%, 10/579)
(Figure 1A). In the Chinese cohort, a total of 25.9% (57/220) OvCa
carried at least one DDR gene somatic mutation. The fre-
quencies of mutations in FA, HRR, Checkpoint, MMR, and
BER pathways were 12.3% (27/220), 10.9% % (24/220), 7.3%
(16/220), 4.5% (10/220), and 1.4% (3/220), respectively. The
most frequently mutated genes were BRCA1 (6.8%, 15/220),
BRCA2 (4.1%, 9/220), ATM (4.1%, 9/220), FANCA (4.1%,
9/220), and ATR (3.6%, 8/220) (Figure 1B). 2.5 | Association between DDR
mutations and immune cell abundance A total of 19.3% (112/579) OvCa in TCGA cohort tested by
WES harbored at least one DDR gene somatic mutation. The
frequencies of mutation in FA, HRR, Checkpoint, MMR, and
BER pathways were 10.2% (59/579), 8.5% (49/579), 3.9% To determine the association between DDR status and im-
mune cell abundance, the CIBERSORT algorithm (https://
cibersort.stanford.edu) was used to calculate the abundance 2193 TIAN et al. 3.3 | DDR somatic mutations exhibit
increased TMB As expected, tumors with DDR somatic mutations also
had higher levels of genomic instability, represented by num-
ber of synonymous and non-synonymous exome mutations
(Nmut) (P = .002) and LST (P = .021), which is in line with
the increased TMB levels with DDR mutations (Figure 4). In concordance with the previous results, a significantly higher
level of TMB was observed in OvCa harboring DDR somatic
mutations compared to DDR wild-type OvCa in TCGA cohort
(P < .0001) (Figure 3). Mutations in one single DDR gene
(RAD50, FANCC, BRCA2, PMS2, or BRCA1) also showed
a higher TMB level (Figure 3A). More specifically, there was
a higher level of TMB in FA, MMR, HR, and Checkpoint
pathway mutations than the wild-type group (Figure 3C). 3.2 | Immune-related gene expression
pattern associates with DDR somatic mutation We further identified 512 OvCa tumors from TCGA, for
whom RNAseq and DNAseq data were both available. FIGURE 1
Mutation frequencies of 21 DNA damage repair genes in TCGA cohort and Chinese cohort. (A, B) Alteration frequency of 21
DDR genes in TCGA cohort (A) and Chinese cohort (B) FIGURE 1
Mutation frequencies of 21 DNA damage repair genes in TCGA cohort and Chinese cohort. (A, B) Alteration frequency of 21
DDR genes in TCGA cohort (A) and Chinese cohort (B) FIGURE 1
Mutation frequencies of 21 DNA damage repair genes in TCGA cohort and Chinese cohort. (A, B) Alteration frequency of 21
DDR genes in TCGA cohort (A) and Chinese cohort (B) 2194 |
TIAN et al. TIAN et al. 2194 FIGURE 2
Expression profiles of immune-related genes in OvCa patients with different DDR deficiency status. (A) DDR deficiency OvCa
(red) exhibited significantly higher expression levels of PTPRCAP, CCL5, IFI16, LAG3, IL15RA, and GBP1 compared with DDR wild-type OvCa
(blue). (B) Heatmap depicting the mean difference in immune-related gene mRNA expression between DDR deficiency and DDR wild-type in
each DDR pathway. (C, D) The GSEA analysis showed prominent enrichment of signatures related to the genes upregulated in IL6-JAK-STAT3
signaling (C) and inflammatory response (D) in DDR deficiency OvCa FIGURE 2
Expression profiles of immune-related genes in OvCa patients with different DDR deficiency status. (A) DDR deficiency OvCa
(red) exhibited significantly higher expression levels of PTPRCAP, CCL5, IFI16, LAG3, IL15RA, and GBP1 compared with DDR wild-type OvCa
(blue). (B) Heatmap depicting the mean difference in immune-related gene mRNA expression between DDR deficiency and DDR wild-type in
each DDR pathway. (C, D) The GSEA analysis showed prominent enrichment of signatures related to the genes upregulated in IL6-JAK-STAT3
signaling (C) and inflammatory response (D) in DDR deficiency OvCa We further did the GSEA analysis. The results revealed
prominent enrichment of signatures related to the genes upregu-
lated in IL6-JAK-STAT3 signaling and inflammatory response in DDR mutation group (Figure 2C,D). However, the GSEA-
based analysis did not show a significant prominent enrichment
of immunologic signatures in DDR mutation group (Figure S2). 2195 TIAN et al. 4 |
DISCUSSION 4 In this study, we revealed that OvCa with somatic DDR mu-
tation manifested as a distinct immune profile with higher ex-
pression levels of PTPRCAP, CCL5, IFI16, LAG3, IL15RA,
and GBP1. Increasing levels of Th1 cell abundance, TMB,
and neoantigen were also observed in OvCa with somatic
DDR mutation. 3.4 | DDR somatic mutations exhibit
increased neoantigen (a) Comparison of FLOH, HRD_LOH, LST, Nmut, NtAI, Ploidy,
and wGII between DDR deficiency (red) and DDR wild-type (blue) in TCGA cohort. DDR deficiency showed higher levels of LST and Nmut 3.5 | Association between DDR
mutation and immune cell infiltration pattern better OS (median, 51.9 months vs 38.4 months; HR = 0.67,
95%CI 0.54-0.83; P = .0002) and disease-free survival (DFS,
median, 18.1 months vs 16.7 months; HR = 0.74, 95%CI
0.56-0.91; P = .005) in the TCGA cohort (Figure 6A,B). More specifically, patients with mutations in FA pathway
had even better OS (median, 51.0 months vs 44.3 months;
HR = 0.73, 95%CI 0.58-0.93; P = .019) and DFS (median,
18.1 months vs 17.5 months; HR = 0.75, 95%CI 0.60-0.95;
P = .024) (Figure S5I,J). Patients with Checkpoint pathway
mutations obtained a significantly prolonged OS (median,
49.6 months vs 44.3 months; HR = 0.76, 95%CI 0.59-0.98;
P = .005) (Figure S5C,D). We further assessed the effect of
TMB on clinical prognosis. Similar to DDR, patients with
a higher TMB level (we used the median as cutoff value)
showed prolonged OS (median, 48.3 months vs 44.8 months;
HR = 0.81, 95%CI 0.65-0.99; P = .045) (Figure 6). Since higher levels of TMB and neoantigen were observed
in DDR mutations, a higher immune cell abundance in DDR
somatic mutation was expected. However, based on the
CIBERSORT analysis, no association was observed between
DDR deficiency and immune cell abundance (Figure 5A,B). Furthermore, DDR somatic mutation exhibited a higher
abundance of Th1 immune cells (P = .0012) without higher
levels of Th2 or Th17 immune cells (Figure 5C). 3.4 | DDR somatic mutations exhibit
increased neoantigen Given the higher TMB levels in OvCa with DDR mutations, we
further investigated whether there was an association between
tumor-specific neoantigen and DDR somatic mutations in OvCa. DDR somatic mutations did show a higher level of neoantigen
(Figure S3). Mutations in FANCC, PSM2, PALB2, and RAD51
genes exhibited significantly increased neoantigens. Tumors with
FA, Checkpoint, or HRR pathway mutations displayed higher
levels of neoantigen compared to tumors with DDR wild-type. Similar phenomenon was obtained from the NGS cohort
(Figure 3B,D). Although not observed in MMR, BER, or
HRR pathway, mutations in FA and Checkpoint pathways
displayed a significantly higher TMB level (Figure 3D). Moreover, mutations in ATR gene manifested a significantly
increased TMB level in the Chinese cohort (Figure 3B), which
was not observed in terms of other DDR gene mutations. FIGURE 3
Correlations between DDR gene mutations and tumor mutation burden. (A, B) Comparison of TMB between DDR gene wild-
type (blue) and DDR gene mutation (red) from TCGA (A) and Chinese (B) cohorts. (C, D) Comparison of TMB between each DDR pathway
mutation from TCGA (C) and Chinese (D) cohorts. *P < .05, **P < .01, ***P < .001, ****P < .0001, NS, not significant FIGURE 3
Correlations between DDR gene mutations and tumor mutation burden. (A, B) Comparison of TMB between DDR gene wild-
type (blue) and DDR gene mutation (red) from TCGA (A) and Chinese (B) cohorts. (C, D) Comparison of TMB between each DDR pathway
mutation from TCGA (C) and Chinese (D) cohorts. *P < .05, **P < .01, ***P < .001, ****P < .0001, NS, not significant FIGURE 3
Correlations between DDR gene mutations and tumor mutation burden. (A, B) Comparison of TMB between DDR gene wild-
type (blue) and DDR gene mutation (red) from TCGA (A) and Chinese (B) cohorts. (C, D) Comparison of TMB between each DDR pathway
mutation from TCGA (C) and Chinese (D) cohorts. *P < .05, **P < .01, ***P < .001, ****P < .0001, NS, not significant 2196 |
TIAN et al. FIGURE 4
Correlations between DDR deficiency and genomic instability. (a) Comparison of FLOH, HRD_LOH, LST, Nmut, NtAI, Ploidy,
and wGII between DDR deficiency (red) and DDR wild-type (blue) in TCGA cohort. DDR deficiency showed higher levels of LST and Nmut TIAN et al. 2196 TIAN et al. FIGURE 4
Correlations between DDR deficiency and genomic instability. 3.6 | Association between DDR
mutation and PD-L1 expression The association between DDR status and PD-L1 expression
was assessed in 175 tumors with PD-L1 expression data. A
PD-L1 tumor proportion score of 1% or greater was observed
for 46.8% (22/47) in the DDR mutation group and 44.5%
(57/128) in the DDR wild-type group without reaching a sig-
nificant difference level (P = .88) (Figure S4). 3.7 | DDR mutation or higher TMB level
shows favorable clinical prognosis The presence of DDR mutation has been established
as a promising biomarker of immunotherapy in urothelial
cancers.19 OvCa shows similar characteristics to urothelial
cancers, such as an optimistic sensitivity to platinum-based
chemotherapy and a high prevalence of DDR mutations.10,31 Of particular interest, we next investigated whether the 21
DDR genes somatic mutations were associated with im-
proved survival. As expected, the presence of any DDR
genes somatic mutation was associated with significantly |
2197 TIAN et al. |
2197
TIAN et al. FIGURE 5
Subtype of immune infiltrates in OvCa. (A) Comparison of leukocyte fraction in each DDR pathway deficiency from TCGA
cohort. No significant difference was observed. (B) Comparison of Th1, Th2, and Th17 cells between DDR deficiency (red) and DDR wild-type
(blue) OvCa. OvCa with DDR deficiency (red) showed low abundance of Th1 immune cells compared with DDR wild-type (blue). (C) Comparison
of 22 types of immune cells between DDR mutation (red) and DDR wild-type (blue) in the CIBERSORT-based analysis. No significant association
was observed FIGURE 5
Subtype of immune infiltrates in OvCa. (A) Comparison of leukocyte fraction in each DDR pathway deficiency from TCGA
cohort. No significant difference was observed. (B) Comparison of Th1, Th2, and Th17 cells between DDR deficiency (red) and DDR wild-type
(blue) OvCa. OvCa with DDR deficiency (red) showed low abundance of Th1 immune cells compared with DDR wild-type (blue). (C) Comparison
of 22 types of immune cells between DDR mutation (red) and DDR wild-type (blue) in the CIBERSORT-based analysis. No significant association
was observed bladder cancer.21 Similarly, we did not identify a significantly
higher level of abundance in 22 immune cell types calculated
by CIBERSORT. Paradoxically, an increased Th1 cell level
was observed in OvCa with DDR mutation, which indicated
an immunologically hot state. Th1 cells, which could secrete
IL-2, TNF-α, and INF-γ, evolved from CD4 + T cells and
stimulated the function of other immune cells, such as macro-
phage cells and CD8 + T cells.30,34 Consistent with the higher
levels of Th1 cell abundance, a trend of higher level of macro-
phage cells was observed in the DDR mutation group. Based
on these findings, we speculated that DDR deficiency in
OvCa might modulate the response to ICIs to a certain extent,
but it might not play a key role. These results also suggested However, whether DDR mutation could serve as a predictive
biomarker of immunotherapy remains unclear in OvCa. 3.7 | DDR mutation or higher TMB level
shows favorable clinical prognosis In this
comprehensive analysis, we explored the association between
DDR mutation and immune-related gene expression in OvCa. Previous studies suggested that DDR deficiency could prime
the activation of type I interferons (IFN) system and induce
the secretion of chemokines recruiting immune cells, such as
CCL5 and CXCL10.32,33 Our analyses revealed a higher ex-
pression level of CCL5 in DDR mutation OvCa and a higher
level of CXCL10 in BER mutation OvCa. DDR mutations
were also associated with the activation of IL6-JAK-STAT3
pathway. One previous study has shown that DDR deficiency
did not exhibit a significantly higher level of immune cells in Previous studies suggested that DDR deficiency could prime
the activation of type I interferons (IFN) system and induce
the secretion of chemokines recruiting immune cells, such as
CCL5 and CXCL10.32,33 Our analyses revealed a higher ex-
pression level of CCL5 in DDR mutation OvCa and a higher
level of CXCL10 in BER mutation OvCa. DDR mutations
were also associated with the activation of IL6-JAK-STAT3
pathway. One previous study has shown that DDR deficiency
did not exhibit a significantly higher level of immune cells in 2198 | 2198 |
TIAN et al. FIGURE 6
OvCa patients with DDR deficiency or high TMB levels showed favorable clinical outcomes. (A, B) Kaplan–Meier survival
curves of PFS (A) and OS (B) comparing DDR mutation (red) with DDR wild-type (blue) in OvCa patients from TCGA cohort. (C, D) Kaplan–
Meier survival curves of PFS (C) and OS (D) comparing high TMB level (red) with low TMB level (blue) groups in OvCa patients from TCGA
cohort TIAN et al. TIAN et al. FIGURE 6
OvCa patients with DDR deficiency or high TMB levels showed favorable clinical outcomes. (A, B) Kaplan–Meier survival
curves of PFS (A) and OS (B) comparing DDR mutation (red) with DDR wild-type (blue) in OvCa patients from TCGA cohort. (C, D) Kaplan–
Meier survival curves of PFS (C) and OS (D) comparing high TMB level (red) with low TMB level (blue) groups in OvCa patients from TCGA
cohort that the expression of proteins such as CCL5, IFI16, LAG3,
and others should be evaluated simultaneously, and provided
the rationale for future immune combination therapy trials in
OvCa with DDR deficiency. 3.7 | DDR mutation or higher TMB level
shows favorable clinical prognosis Given that PARP inhibitors have
shown promising antitumor activity in patients with HRR-
deficient OvCa,35 clinical trials aimed at evaluating the effi-
cacy of PARP inhibitor and ICIs combination therapy, such as
KEYNOTE-162, are well expected. this study, we observed a trend of greater TMB in tumors
harboring BRCA2 mutations. Besides, the associations be-
tween TMB and DDR gene mutations varied between the
two cohorts. The baseline characteristics, such as race and
tumor stage, may contribute to the differences. Given that
DDR mutations were associated with a higher TMB level and
were correlated with improved clinical outcomes in urothelial
cancer patients receiving platinum-based chemotherapy,19,39
TMB may be a good prognostic factor in urothelial cancer. In
this analysis, we also found that higher TMB was associated
with better OS in OvCa, which was consistent with the prog-
nostic role of DDR deficiency in OvCa. It should be noted
that, the TMB level of OvCa ranked low in the pan-cancer
analysis.40 In other words, although DDR deficiency was cor-
related with higher TMB, the TMB level in DDR-deficient
OvCa was still far behind the immunologically hot tumors
such as lung cancer and melanoma. Further studies are still
warranted to investigate whether DDR deficiency is a predic-
tive biomarker for ICIs in OvCa. this study, we observed a trend of greater TMB in tumors
harboring BRCA2 mutations. Besides, the associations be-
tween TMB and DDR gene mutations varied between the
two cohorts. The baseline characteristics, such as race and
tumor stage, may contribute to the differences. Given that
DDR mutations were associated with a higher TMB level and
were correlated with improved clinical outcomes in urothelial
cancer patients receiving platinum-based chemotherapy,19,39 TMB may be a good prognostic factor in urothelial cancer. In
this analysis, we also found that higher TMB was associated
with better OS in OvCa, which was consistent with the prog-
nostic role of DDR deficiency in OvCa. It should be noted
that, the TMB level of OvCa ranked low in the pan-cancer
analysis.40 In other words, although DDR deficiency was cor-
related with higher TMB, the TMB level in DDR-deficient
OvCa was still far behind the immunologically hot tumors
such as lung cancer and melanoma. Further studies are still
warranted to investigate whether DDR deficiency is a predic-
tive biomarker for ICIs in OvCa. REFERENCES 1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal
A. Global cancer statistics 2018: GLOBOCAN estimates of inci-
dence and mortality worldwide for 36 cancers in 185 countries. CA
Cancer J Clin. 2018;68:394-424. 2. Bellmunt J, de Wit R, Vaughn DJ, et al. Pembrolizumab as sec-
ond-line therapy for advanced urothelial carcinoma. New Engl J
Med. 2017;376:1015-1026. 3. Reck M, Rodríguez-Abreu D, Robinson AG, et al. Pembrolizumab
versus chemotherapy for PD-L1-positive non-small-cell lung can-
cer. New Engl J Med. 2016;375:1823-1833. 4. Steven Lemery MHS, Keegan P, Pazdur R. First FDA approval ag-
nostic of cancer site — when a biomarker defines the indication. New Engl J Med. 2017;377:1409-1412. One limitation of our study is that we did not distinguish
the biallelic or monoallelic inactivation of DDR genes, which
might exhibit different degrees of immunogenicity in OvCa. Most of the samples in TCGA cohort were obtained before
treatment and the changes of immune-related gene expression
after platinum-based chemotherapy were ignored. Besides,
we defined mutations as non-silent genomic changes in the
coding region including deleterious mutations and other
mutations of unknown significance. One prior study has in-
dicated that protein function could still be affected by the be-
nign mutation.42 However, we may not be able to identify the
specific protein function in this study. Furthermore, germline
mutations were not included in this study, which may also
have influences on the results. 5. Hamanishi J, Mandai M, Ikeda T, et al. Safety and antitumor activ-
ity of anti-PD-1 antibody, nivolumab, in patients with platinum-re-
sistant ovarian Cancer. J Clini Oncol. 2015;33:4015-4022. 6. Disis ML, Patel MR, Pant S, et al. Avelumab (MSB0010718C; an-
ti-PD-L1) in patients with recurrent/refractory ovarian cancer from
the JAVELIN solid tumor phase Ib trial: safety and clinical activity. J Clin Oncol (R Coll Radiol). 2016;34:5533. 7. Varga A, Piha-Paul SA, Ott PA, et al. Antitumor activity and safety
of pembrolizumab in patients (pts) with PD-L1 positive advanced
ovarian cancer: interim results from a phase Ib study. J Clin Oncol
(R Coll Radiol). 2015;33:5510. 8. Gibney GT, Weiner LM, Atkins MB. Predictive biomarkers
for checkpoint inhibitor-based immunotherapy. Lancet Oncol. 2016;17:e542-e551. 9. Cortes-Ciriano I, Lee S, Park WY, Kim TM, Park PJ. A molecular
portrait of microsatellite instability across multiple cancers. Nat
Commun. 2017;8:15180. In conclusion, DDR mutations are prevalent in OvCa
and exhibited a distinct immune profile. REFERENCES Our findings may
provide insights into the biomarker development for further
stratification of OvCa patients and better rational design of
immune combination therapies in OvCa in trials. 10. Pennington KP, Walsh T, Harrell MI, et al. Germline and somatic
mutations in homologous recombination genes predict platinum
response and survival in ovarian, fallopian tube, and peritoneal
carcinomas. Clin Cancer Res. 2014;20:764-775. ORCID Rui Bi
https://orcid.org/0000-0003-1217-1838
Huaying Wang
https://orcid.org/0000-0002-3780-4497 in the previous studies. The germline or somatic mutation fre-
quency of 12 DDR gene set provided by cBioPortal (CHEK1,
CHEK2, RAD51, BRCA1, BRCA2, MLH1, MSH2, ATM,
ATR, MDC1, PARP1, and FANCF) was 39.3% (322/820) in
OvCa patients in the public dataset from cBioPortal (www. cbioportal.org). TCGA has showed a high HRR deficiency
of ~ 50% in high-grade serous OvCa, including more alter-
ation forms such as BRCA1 hypermethylation, RAD51C
hypermethylation, and BRCA1 methylation, which were not
assessed in this study.41 One previous study has identified the
somatic mutations of 13 HRR genes (BRCA1, BRCA2, ATM,
BARD1, BRIP1, CHEK1, CHEK2, FAM175A, MRE11A,
NBN, PALB2, RAD51C, and RAD51D) in 8.7% (32/367)
OvCa patients based on the massively parallel sequencing.10
Another study demonstrated a 9.9% (32/324) somatic muta-
tion frequency of 16 HRR genes (ATM, ATR, BARD1, BLM,
BRCA1, BRCA2, BRIP1, CHEK2, MRE11A, NBN, PALB2,
RAD51C, RAD51D, RBBP8, SLX4, and XRCC2) in OvCa
patients from GOG-0218 clinical trial based on BROCA, a
massively parallel sequencing test.31 Overall, we showed that
19.3% in TCGA cohort and 25.9% in Chinese cohort harbored
at least one somatic mutation in 21 DDR genes in our anal-
ysis. Several reasons may explain the difference of mutation
frequencies between two cohorts. First, most patients in the
TCGA cohort were Caucasian race; however, all patients in
our cohort were Asian. Second, most of the tumors in TCGA
cohort were stage III and nearly half of the tumors in Chinese
cohort were stage IV. Third, previous treatments may exert an
effect on the mutation frequency. in the previous studies. The germline or somatic mutation fre-
quency of 12 DDR gene set provided by cBioPortal (CHEK1,
CHEK2, RAD51, BRCA1, BRCA2, MLH1, MSH2, ATM,
ATR, MDC1, PARP1, and FANCF) was 39.3% (322/820) in
OvCa patients in the public dataset from cBioPortal (www. cbioportal.org). DATA AVAILABILITY STATEMENT The data that support the findings of this study are available
on request from the corresponding author. The data are not
publicly available due to privacy or ethical restrictions. TCGA has showed a high HRR deficiency
of ~ 50% in high-grade serous OvCa, including more alter-
ation forms such as BRCA1 hypermethylation, RAD51C
hypermethylation, and BRCA1 methylation, which were not
assessed in this study.41 One previous study has identified the
somatic mutations of 13 HRR genes (BRCA1, BRCA2, ATM,
BARD1, BRIP1, CHEK1, CHEK2, FAM175A, MRE11A,
NBN, PALB2, RAD51C, and RAD51D) in 8.7% (32/367)
OvCa patients based on the massively parallel sequencing.10
Another study demonstrated a 9.9% (32/324) somatic muta-
tion frequency of 16 HRR genes (ATM, ATR, BARD1, BLM,
BRCA1, BRCA2, BRIP1, CHEK2, MRE11A, NBN, PALB2,
RAD51C, RAD51D, RBBP8, SLX4, and XRCC2) in OvCa
patients from GOG-0218 clinical trial based on BROCA, a
massively parallel sequencing test.31 Overall, we showed that
19.3% in TCGA cohort and 25.9% in Chinese cohort harbored
at least one somatic mutation in 21 DDR genes in our anal-
ysis. Several reasons may explain the difference of mutation
frequencies between two cohorts. First, most patients in the
TCGA cohort were Caucasian race; however, all patients in
our cohort were Asian. Second, most of the tumors in TCGA
cohort were stage III and nearly half of the tumors in Chinese
cohort were stage IV. Third, previous treatments may exert an
effect on the mutation frequency. The authors thank all the patients for participating in this
study. The authors thank all the patients for participating in this
study. CONFLICT OF INTEREST Drs. Yuzi Zhang, Jing Zhao, Zhengyi Zhao, Guoqaing Wang,
Xiaochen Zhao, Shangli Cai, and Yuezong Bai declare that
they are employees of 3D Medicines Inc Other authors de-
clare no potential conflict of interests. 3.7 | DDR mutation or higher TMB level
shows favorable clinical prognosis DDR system plays an important role in maintaining ge-
nome stability.12 TMB, which is considered to be a predictive
biomarker of ICIs,13,36 may reflect the degree of genomic
instability at the nucleotide level. In support of this notion,
we discovered a significantly higher level of TMB, Nmut,
and LST in DDR-deficient tumors, which was consistent with
previous studies in urothelial carcinoma and melanoma.17,19
TMB as a potential predictor may generate different effects
on various tumor types. A higher TMB level was closely as-
sociated with more neoantigen loads, which have been proven
to be the target of ICIs.8,20 BRCA1/2 deficiency has been
shown to result in a specific signature with higher mutation
burden and more tumor-specific neoantigens in OvCa.37,38 In DDR system plays an important role in maintaining ge-
nome stability.12 TMB, which is considered to be a predictive
biomarker of ICIs,13,36 may reflect the degree of genomic
instability at the nucleotide level. In support of this notion,
we discovered a significantly higher level of TMB, Nmut,
and LST in DDR-deficient tumors, which was consistent with
previous studies in urothelial carcinoma and melanoma.17,19 TMB as a potential predictor may generate different effects
on various tumor types. A higher TMB level was closely as-
sociated with more neoantigen loads, which have been proven
to be the target of ICIs.8,20 BRCA1/2 deficiency has been
shown to result in a specific signature with higher mutation
burden and more tumor-specific neoantigens in OvCa.37,38 In Given the methodological differences, it is difficult to
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Single cell analysis revealed that two distinct, unique CD4+ T cell subsets were increased in the small intestinal intraepithelial lymphocytes of aged mice
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OPEN ACCESS EDITED BY
Oscar Medina-Contreras,
Mexico Children’s Hospital, Mexico
REVIEWED BY
Vu L. Ngo,
Georgia State University, United States
Bin Bao,
Harvard Medical School, United States
*CORRESPONDENCE
Yasuhiro Nemoto
ynemoto.gast@tmd.ac.jp
RECEIVED 19 November 2023
ACCEPTED 02 January 2024
PUBLISHED 22 January 2024
CITATION
Yonemoto Y, Nemoto Y, Morikawa R,
Shibayama N, Oshima S, Nagaishi T,
Mizutani T, Ito G, Fujii S and Okamoto R
(2024) Single cell analysis revealed that two
distinct, unique CD4+ T cell subsets were
increased in the small intestinal intraepithelial
lymphocytes of aged mice. Front. Immunol. 15:1340048. doi: 10.3389/fimmu.2024.1340048 EDITED BY
Oscar Medina-Contreras,
Mexico Children’s Hospital, Mexico
REVIEWED BY
Vu L. Ngo,
Georgia State University, United States
Bin Bao,
Harvard Medical School, United States 1Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental
University (TMDU), Tokyo, Japan, 2Institute of Research, Tokyo Medical and Dental University (TMDU),
Tokyo, Japan, 3Department of Advanced Therapeutics for Gastrointestinal Diseases, Tokyo Medical
and Dental University (TMDU), Tokyo, Japan, 4Advanced Research Institute, Tokyo Medical and Dental
University (TMDU), Tokyo, Japan CITATION
Yonemoto Y, Nemoto Y, Morikawa R,
Shibayama N, Oshima S, Nagaishi T,
Mizutani T, Ito G, Fujii S and Okamoto R
(2024) Single cell analysis revealed that two
distinct, unique CD4+ T cell subsets were
increased in the small intestinal intraepithelial
lymphocytes of aged mice. Front. Immunol. 15:1340048. doi: 10.3389/fimmu.2024.1340048 Recent advances in research suggest that aging has a controllable chronic
inflammatory disease aspect. Aging systemic T cells, which secrete pro-
inflammatory factors, affect surrounding somatic cells, and accelerate the
aging process through chronic inflammation, have attracted attention as
potential therapeutic targets in aging. On the other hand, there are few reports
on the aging of the intestinal immune system, which differs from the systemic
immune system in many ways. In the current study, we investigated the age-
related changes in the intestinal immune system, particularly in T cells. The most
significant changes were observed in the CD4+ T cells in the small intestinal IEL,
with a marked increase in this fraction in old mice and reduced expression of
CD27 and CD28, which are characteristic of aging systemic T cells. The
proliferative capacity of aging IEL CD4+ T cells was significantly more reduced
than that of aging systemic T cells. Transcriptome analysis showed that the
expression of inflammatory cytokines was not upregulated, whereas Cd8a, NK
receptors, and Granzymes were upregulated in aging IEL CD4+ T cells. TYPE Original Research
PUBLISHED 22 January 2024
DOI 10.3389/fimmu.2024.1340048 TYPE Original Research
PUBLISHED 22 January 2024
DOI 10.3389/fimmu.2024.1340048 1 Introduction fractions such as gdT cells, CD8ab-CD4-TCRab+ T cells, and CD8aa+
T cells that are rarely found in peripheral blood. The intestinal immune
system is the first line of defense against foreign antigens and maintains
the homeostasis of the organism by maintaining a delicate balance
between inflammation and tolerance (19, 20). Today, the world is facing an unprecedented super-aging
society, and medical and social countermeasures are an urgent
issue (1). Several mechanisms have been proposed to explain aging,
including limits on programmed cell division due to telomeres and
other mechanisms (2), impaired gene repair mechanisms due to the
accumulation of errors at the cellular level (3), mitochondrial
degradation due to reactive oxygen species or oxidative stress
(4–8), and stem cell deterioration (9), although these are not yet
fully understood (10). The intestinal tract is a relatively unaffected organ by aging. For
example, progeria syndrome causes phenotypes in the skin, hair,
cardiovascular system, brain, etc., but the gut is less affected (21). On the other hand, colorectal cancer and gastrointestinal infections
increase with age, while inflammatory bowel diseases; IBD and
allergies decrease, suggesting that some changes are occur in the
intestinal immune system (22), but there are few reports on the
phenotype of intestinal aging. Furthermore, while immune aging
has been extensively studied in the systemic immune system, there
are few reports on age-related changes in the intestinal
immune system. In addition to these mechanisms, the immune system plays an
important role in the aging process by promoting it through chronic
inflammation (11, 12). In immune aging, which begins in the 20s
(13), the aging of long-lived T cells is more important than that of
short-lived myeloid cells such as granulocytes, monocytes, and
macrophages. At the individual level of aging, thymic atrophy
disrupts the supply of fresh naive T cells, and antigen exposure
leads to an increase in effector/memory cells (14). At the cellular
level, it is also known that senescent T cells, like other somatic cells,
have a low proliferative capacity and secrete pro-inflammatory
humoral factors, such as TNF-a/IFN-g (this phenotype is termed
senescence-associated secretory phenotype; SASP) (14–18). In mice
in which senescence was specifically induced in T cells, age-related
changes such as atherosclerosis were accelerated (4), and
administration of a peptide vaccine targeting senescent T cells
ameliorated the pathology of a mouse model of diabetes (18),
suggesting that senescent T cells are an important therapeutic
target in systemic aging. OPEN ACCESS Functional
analysis showed that aging IEL T cells had a higher cytotoxic function against
intestinal tumor organoids in vitro than young IEL T cells. scRNAseq revealed that
splenic T cells show a transition from naïve to memory T cells, whereas intestinal
T cells show the emergence of a CD8aa+CD4+ T cell fraction in aged mice,
which is rarely seen in young cells. Further analysis of the aging IEL CD4+ T cells
showed that two unique subsets are increased that are distinct from the systemic
CD4+ T cells. Subset 1 has a pro-inflammatory component, with expression of
IFNg and upregulation of NFkB signaling pathways. Subset 2 does not express
IFNg, but upregulates inhibitory molecules and nIEL markers. Expression of © 2024 Yonemoto, Nemoto, Morikawa,
Shibayama, Oshima, Nagaishi, Mizutani, Ito, Fujii
and Okamoto. 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. 01 Frontiers in Immunology frontiersin.org Yonemoto et al. 10.3389/fimmu.2024.1340048 granzymes and Cd8a was common to both. These fractions were in opposite
positions in the clustering by UMAP and had different TCR repertoires. They
may be involved in the suppression of intestinal aging and longevity through
anti-tumor immunity, elimination of senescent cells and stressed cells in the
aging environment. This finding could be a breakthrough in aging research. intraepithelial lymphocyte, T cell aging, intestinal immunity, CD4 T cell, scRNAseq intraepithelial lymphocyte, T cell aging, intestinal immunity, CD4 T cell, scRNAseq 1 Introduction In this study, we investigated the role of the intestinal immune
system in aging by analyzing the qualitative, quantitative, and
functional changes associated with aging in the intestinal immune
system, in particular in T cells, which are long-lived and important
therapeutic targets in the aging process, and by comparing them
with aging T cells in the systemic immune system. Frontiers in Immunology KEYWORDS
intraepithelial lymphocyte, T cell aging, intestinal immunity, CD4 T cell, scRNAseq Yonemoto et al. 2.7 RT-qPCR Total RNA from SI-IELs and SP from 5 to 6-weeks old or 80 to
95 weeks-old C57BL/6 mice was isolated as described above and
converted to cDNA using QuantiTect reverse transcription kit
(QIAGEN) according to the manufacturer’s instructions. Real-
time PCR was performed with QuantiTect SYBRgreen PCR kits
(QIAGEN) using the StepOnePlus real-time PCR system and
StepOne software (Thermo Fisher Scientific). Gene expression
was normalized to the housekeeping gene b-actin and was
expressed as the relative expression. Primers used for RT-qPCR
are listed in Supplementary Table 1. 2.5 Microarray analysis SI-IELs and SP cells were collected from 5-weeks old or 87weeks
old C57BL/6 mice, stained with anti- CD3e-PerCP-Cy5.5 and anti-
CD4-PE-Cy7, and then sorted by the FACS Melody as the CD4
(CD3+CD4+) fractions. More than 98% purity of each fraction was
confirmed by analysing post-sorted cells with the FACS Canto II. Total RNA was collected from each fraction with a RNeasy Mini Kit
(QIAGEN). Microarray analysis was outsourced to KAMAKURA
TECHNO-SCIENCE INC. Samples were analysed with a 3D gene
chip (Mouse oligo 24k). Enrichment analysis was performed with
DAVID (https://david.ncifcrf.gov/). Enriched genes were defined by
more than 2-fold change and more than 100 signals after the
global normalization. 2.1 Mice We selected 5- to 9-weeks old and 18-months old male mice as
the young and aged models based on previous papers (23–26). The
general condition and organ appearance of aged mice were
observed before the experiments, and mice with obvious diseases
were excluded and not used in the studies. C57BL/6 mice were
purchased from Jackson Laboratory or Charles River or CLEA in
Japan. Both male and female mice were used for experiments. All
mice were maintained under specific pathogen-free conditions in
the Center for Experimental Animals in Tokyo Medical and Dental
University (TMDU). All experiments were approved by animal
study committees and performed in accordance with institutional
guidelines and Home Office regulations. The gut is the one of the largest reservoirs of immune cells,
particularly T cells. The intestinal lumen contains a large number of
foreign antigens such as dietary antigens and gut microbiota. The
intestinal lumen fulfils the conflicting roles of tolerating harmless
antigens and inducing inflammation in response to harmful
pathogens from the sea of antigens (19, 20). The intestinal tract
therefore has a highly developed and unique intestinal mucosal
immune system that is distinct from the systemic immune system. The intestinal immune system has its own lymphoid tissues such as
gut-associated lymphoid tissue; GALT, and in addition to the
conventional CD4 and CD8+ T cells, there are many other unique Frontiers in Immunology 02 frontiersin.org Yonemoto et al. Yonemoto et al. 10.3389/fimmu.2024.1340048 More than 98% purity of each fraction was confirmed by post-sort
purity checking with the FACS Canto II. More than 98% purity of each fraction was confirmed by post-sort
purity checking with the FACS Canto II. 2.6 Cell proliferation assay 1 x 105 of sort-purified IEL- or SP CD4+ cells were labelled with
2.5mM CFSE (CellTrace CFSE Cell Proliferation Kit, ThermoFisher
SCIENTIFIC), and then stimulated with PMA+ionomycin PMA
+ionomycin (PMA 50ng/ml, ionomycin 500ng/ml) in the complete
medium with 100 U/mL recombinant human IL2 (Roche), 50 ng/
mL mouse IL7 (Peprotech), and 50 ng/mL mouse IL15 (Peprotech)
under the condition of 37°C, 5% CO2. After 72 hours, cells were
harvested and stained with appropriate antibodies and analyzed by
FACS Canto II (BD Bioscience). To isolate SP, MLN, and PP cells, each organ was mashed and
passed through a nylon mesh. To lyse red blood cells among
splenocytes, Ack buffer was used. To isolate small intestinal IELs
or LPLs, we used modified protocols in previous reports (27). After
the removal of PPs, a half-length of the distal ileum, proximal ileum,
distal jejunum, and proximal jejunum was opened longitudinally,
washed with Hank’s balanced salt solution (HBSS), and cut into
small pieces. Dissected mucosae were gently inverted (50–60 rpm)
on a rotator for 10–15 min at 37°C in 40 ml HBSS with 2 mM
EDTA. Then, the supernatant and remaining mucosae were
separated with a nylon mesh. The supernatant was centrifuged
and resuspended with a 40% isotonic Percoll (GE Healthcare)
solution and then subjected to Ficoll-Hypaque density gradient
centrifugation (40%/75%). Collected cells were IELs. Remaining
mucosae were gently inverted (50–60 rpm) on the rotator for 15–20
min at 37°C in 40 ml HBSS with 0.5 mg/ml collagenase D (Roche)
and 25 mg/ml DNase I (Roche). Then, they were filtered,
centrifuged, and separated with the Percoll system as described
above. To isolate colonic mucosal cells, the same method was used,
but the incubation time in EDTA and for digestion was 60 min. 2.2 Antibodies The following antibodies were used: anti-mouse CD3e-FITC,
-PerCP-Cy5.5, or -APC-Cy7 (clone 145-2C11; BioLegend), CD4-
APC or -PE-Cy7 (clone RM4-5; BioLegend), anti-mouse CD8a-
Alexa Fluor 488 (clone 53-6.7; BioLegend), anti-mouse CD8b-PE
(clone YTS156.7.7; BioLegend), anti-mouse CD27-Brilliant Violet
510 (clone LG.3A10; BioLegend), anti-mouse CD28-Brilliant Violet
421 (clone 37.51; BioLegend), anti-mouse CD45- Brilliant
Violet 421 (clone 30-F11; BioLegend), anti-mouse CD274 (PD-
L1)-PE (clone 10F.9G2; BioLegend), anti-mouse epithelial cell
adhesion molecule (EpCAM)-APC (clone G8.8; BioLegend), anti-
mouse MHC-II-FITC (clone M5/114.15.2; eBioscience), anti-mouse
TCRb- APC-Cy7 (clone H57-597; BioLegend) or anti-mouse
gdTCR -PE-Cy7 (clone GL3; BioLegend). For scRNAseq analysis,
anti-mouse Hashtags 1 to 4 (M1/42; 39-F11, Biolegend, 155861,
155863, 155865, 155867) were used. Frontiers in Immunology 2.8 Preparation of small
intestinal organoids In some experiments, CD4+ cells or CD45+ cells were enriched
with CD4 (L3T4) microbeads or CD45 microbeads (Miltenyi
Biotec) according to official protocols before the cell sorting by
flow cytometer. To stain surface molecules, the single cell
suspension isolated from each organ was incubated with specific
antibodies for 20 min at 4°C. For analysis, cells were resuspended
with PBS and analysed by a FACS Canto II (BD Bioscience). Data
were analysed by FlowJo software (FlOWJO LLC). For cell sorting,
stained cells were sorted using a FACS Melody (BD Bioscience). Crypts were isolated from the small intestine of male WT mice
aged 7–11 weeks or small intestinal tumors of female APCmin mice
aged 21 weeks as previously described (27). One mouse was used for
each isolation. The crypts were embedded in Matrigel (Corning). Advanced DMEM/F12 (Gibco) containing 50 ng/mL mEGF
(Peprotech), 100 ng/mL mNoggin (R&D Systems), 100 U/mL Frontiers in Immunology 03 frontiersin.org 10.3389/fimmu.2024.1340048 Yonemoto et al. penicillin/streptomycin (Nacalai), 10 mM HEPES (Nacalai), 2 mM
GlutaMAX-1 (Gibco), 1 mM N-acetylcysteine (Sigma), 1×N2
supplement (Gibco), 1×B27 supplement (Gibco) and 10 mM Y-
27632 (Wako) were added to each well (complete medium). Organoids from APCmin mice were passaged at least twice before
co-culture with IECs. cells and CD3+CD4+ cells were mixed as 1:1 in young and old IEL. The single cell suspension was stained with Trypan Blue and the
number of cells was determined by manual cell count. The following procedures were outsourced to AZENTA,
GENEWIZ (Tokyo, Japan). Cell suspension (6,000 cells each) was
loaded in a 10x Chromium microfluidics system based on the
manufacturer’s guidelines. One set of libraries were obtained from
the 10x loaded samples: a 5’ gene expression messenger RNA library
and a single-cell TCR library, using primers for amplification as per
the manufacturer’s instructions. Library were pooled together and
run separate lanes of 150 base-paired, paired-end, flow cell using the
Illumina Novaseq6000. 5668 cells for young SP, 5538 cells for old
SP, 4348 cells for young IEL and 5811 cells for old IEL were
sequenced and reported in the datasets. 2.12 Statistical analysis Organoids and IELs were collected and centrifuged for 3 min at
100 g in experiments. The pellet was shaken (500 rpm) in the
trypLE express (Invitrogen) for 15 min at 37°C. After pipetting, cells
were centrifuged for 3 min at 400 g, and the pellet was suspended
with RPMI-medium (Sigma). Numbers of live single epithelial cells
and IELs were counted with FACSCanto II (BD Bioscience) and
Count Bright Absolute counting Beads (Invitrogen). The exact number of samples is described in the figure legends. Statistical analyses were performed using GraphPad Prism 10 for
Windows 64-bit (GraphPad Software). Normality of the
distribution of results was examined in each group. Differences
between two groups were assessed using Student’s t-test for equal
variance and Welch’s t-test for unequal variance. Non-parametric
tests, such as the Mann-Whitney U test, were used for comparison
between two groups without normality. For comparison among 3 or
more groups was evaluated with one-way ANOVA followed by
parametric multiple comparisons test. For comparison among 3 or
more groups without normality, one-way ANOVA followed by
non-parametric test was used. Results are expressed as the mean ±
S.E.M. Differences were considered significant at P < 0.05. 2.9 Co-culture of IELs and small intestinal
tumor organoids We followed a modified protocol from a previous report (27). Briefly, intestinal organoids were cultured for 2 days prior to the co-
culture with IELs. On the first day of co-culture, SI-IELs collected
from the WT mice aged 9-weeks old or 89 weeks old were stained
with anti-mouse CD3e-APC-Cy7, anti-mouse CD4-PE-Cy7, and
the CD3+ (CD3+) and CD4+ (CD3+ CD4+) -IELs were sorted using
FACSMelody (BD Bioscience). Cultured organoids released from
Matrigel were washed and counted. We combined 200 organoids
and 2.0 × 105 IELs and centrifuged the samples for 3 min at 200 g. In
the control group, the same number of organoids as the co-culture
group was centrifuged. The pellet was suspended in 20 mL of
Matrigel and placed in 24-well plates. After Matrigel
polymerization, 500 mL of the complete medium with 100 U/mL
recombinant human IL-2 (Roche), 10 ng/mL mouse IL-7
(Peprotech) and 10 ng/mL mouse IL-15 (Peprotech) were
supplemented. The medium was refreshed every 1–2 days. Images
of organoids were taken with a BZ-X710 microscope (Keyence). The Cell Ranger software (10x Genomics) was used to perform
barcode counting and unique molecular identifier counting after
filtering and alignment to the mouse/mm10 reference genome to
generate the feature-barcode matrix and determine clusters. Dimensionality reduction was performed using principal
component analysis, and first ten principal components were
used to generate clusters by K-means algorithm and graph-based
algorithm, respectively. Data analysis was performed through the
Loupe Cell Browser software (10x genomics). Next, the differential expression genes for each cluster were
imported into Metascape (https://metascape.org/) for Kyoto
Encyclopedia of Gene and Genomes (KEGG) pathway analysis
was performed with a false discovery rate (FDR) <0.05 as the cut-
off value. We used the Loupe V(D)J browser to analyse the TCR
clonotypes, and the V and J genes. 2.11 Single-cell 5’ transcription and T cell
receptor sequencing SI-IELs or SP cells were collected and pooled from young (6-
weeks old) or old C57BL/6 mice (80-weeks old), the cells from 3
young mice and 3 old mice were mixed and pooled as young SP,
young IEL, old SP and old IEL. CD45+ cells were enriched from
each group using CD45 microbeads (Miltenyi Biotec), followed by
cell hashing with TotalSeq-C anti-mouse hashtag antibodies
(hashtag1-young IEL, hashtag2-old IEL, hashtag3-young SP,
hashtag4-old SP). After the Fc blocking, cells were stained with
anti-CD3e-FITC mAbs and anti-CD4-APC mAbs, and the CD3+
cells and CD3+CD4+ cells were sorted from each group by
FACSMelody. Dead cells removal was performed with 7-AAD. To
enrich CD4+ cells, which are rare population in young IEL, CD3+ Frontiers in Immunology 3.1 CD27-CD28-CD4+ T cells increased in
the small intestine of aged mice To investigate the quantitative changes in T cells with age, we
examined changes in T cell fractionation in the spleen (SP), Frontiers in Immunology 04 frontiersin.org Yonemoto et al. 10.3389/fimmu.2024.1340048 TCRb+TCRgd-), CD8+ T cells (CD3+CD4-CD8b+TCRb+TCRgd-),
DNT; double negative T cells (CD3+CD4-CD8b-TCRb+TCRgd-),
and gd T cells (CD3+TCRb-TCRgd+). As seen in Figure 1B, SP, a
reservoir organ for systemic T cells, showed little fractional change,
whereas MLN showed a decrease in CD4+ T cells and an increase in mesenteric lymph nodes (MLN), Peyer’s patches (PP), small
intestinal IELs (IEL), small intestinal LPLs (LPL), and colon (CL)
of young mice (5-6 weeks old) and old mice (87-105 weeks old)
(Figure 1A). T cells were CD3+TCR+ cells, which included
the following fractions: CD4+ T cells (CD3+CD4+CD8b- B
C
D
E
F
A
FIGURE 1
CD4+ CD27- CD28- CD8a+ T cells increased in the small intestine of aged mice. (A) Experimental design. T cells in the spleen (SP), mesenteric
lymph nodes (MLN), Peyer’s patches (PP), small intestinal IELs (IEL), small intestinal LPLs (LPL), and colon (CL) of young mice (5-6 weeks old) and old
mice (87-105 weeks old) were analyzed. Pooled data from 5 similar independent experiments were analyzed (n=7-11 mice/group). In this figure, “Y”
indicates young mice and “O” indicates old mice. (B) Changes in the percentage of each fraction of T cells in each organ between young and old
mice. Graph shows mean ± SEM. (C) Expression of CD27 and CD28 on CD4+ T cells in each organ of young and old mice. Representative dot plots
are shown. The numbers in each plot reflect the percentage of CD27-CD28- cells. (D) Percentage of CD27-CD28- cells in each T-cell fraction of
each organ from young and old mice. Graph shows mean ± SEM. *P<0.05. (E) Expression of CD8a on each T-cell fraction in each organ of young
and old mice. Representative histograms are shown. The number on each histogram reflects the percentage of CD8a+ cells. (F) Percentage of
CD8a+ cells in each fraction of each organ from young and old mice. Graphs show mean ± SEM. ∗P < 0.05. B
C
A D
E
F
FIGURE 1
CD4+ CD27- CD28- CD8a+ T cells increased in the small intestine of aged mice. (A) Experimental design. 3.1 CD27-CD28-CD4+ T cells increased in
the small intestine of aged mice T cells in the spleen (SP), m
lymph nodes (MLN), Peyer’s patches (PP), small intestinal IELs (IEL), small intestinal LPLs (LPL), and colon (CL) of young mice (5-6 wee
mice (87-105 weeks old) were analyzed. Pooled data from 5 similar independent experiments were analyzed (n=7-11 mice/group). In
indicates young mice and “O” indicates old mice. (B) Changes in the percentage of each fraction of T cells in each organ between yo
mice. Graph shows mean ± SEM. (C) Expression of CD27 and CD28 on CD4+ T cells in each organ of young and old mice. Represent
are shown. The numbers in each plot reflect the percentage of CD27-CD28- cells. (D) Percentage of CD27-CD28- cells in each T-cel
each organ from young and old mice. Graph shows mean ± SEM. *P<0.05. (E) Expression of CD8a on each T-cell fraction in each or
and old mice. Representative histograms are shown. The number on each histogram reflects the percentage of CD8a+ cells. (F) Perce
CD8a+ cells in each fraction of each organ from young and old mice. Graphs show mean ± SEM. ∗P < 0.05. E FIGURE 1
CD4+ CD27- CD28- CD8a+ T cells increased in the small intestine of aged mice. (A) Experimental design. T cells in the spleen (SP), mesenteric
lymph nodes (MLN), Peyer’s patches (PP), small intestinal IELs (IEL), small intestinal LPLs (LPL), and colon (CL) of young mice (5-6 weeks old) and old
mice (87-105 weeks old) were analyzed. Pooled data from 5 similar independent experiments were analyzed (n=7-11 mice/group). In this figure, “Y”
indicates young mice and “O” indicates old mice. (B) Changes in the percentage of each fraction of T cells in each organ between young and old
mice. Graph shows mean ± SEM. (C) Expression of CD27 and CD28 on CD4+ T cells in each organ of young and old mice. Representative dot plots
are shown. The numbers in each plot reflect the percentage of CD27-CD28- cells. (D) Percentage of CD27-CD28- cells in each T-cell fraction of
each organ from young and old mice. Graph shows mean ± SEM. *P<0.05. (E) Expression of CD8a on each T-cell fraction in each organ of young
and old mice. Representative histograms are shown. The number on each histogram reflects the percentage of CD8a+ cells. (F) Percentage of
CD8a+ cells in each fraction of each organ from young and old mice. Graphs show mean ± SEM. ∗P < 0.05. 3.1 CD27-CD28-CD4+ T cells increased in
the small intestine of aged mice 05 Frontiers in Immunology frontiersin.org Yonemoto et al. 10.3389/fimmu.2024.1340048 CD8+ T cells. Interestingly, the greatest change was seen in the IEL. The major fraction, gd, DNT and CD8+ T cells showed little change,
whereas the minor fraction, CD4+ T cells, more than doubled (6.55
± 0.570 to 15.4 ± 2.35). Similar changes were observed in LPL. completely different characteristics from the previously reported aging
T cells. Therefore, we performed a comprehensive transcriptional assay
as a further investigation. SP CD4+ and IEL CD4+ T cells from young
and aged mice were sorted by flow cytometer and total RNA was
collected for microarray analysis of expressed genes. ± 0.570 to 15.4 ± 2.35). Similar changes were observed in LPL. As shown in Figures 1C, D, IEL CD4+ T cells showed a marked
downregulation of CD27 and CD28, co-stimulatory molecules
known to be downregulated with age, and a marked increase in the
proportion of CD27-CD28- cells (15.5 ± 1.60→60.5 ± 3.74). LPL
CD4+ T cells also showed a similar increase in CD27-CD28- cells. CD27-CD28- cells were increased in both IEL and LPL CD8+ T cells,
but the greatest change was seen in CD4+ T cells, with a 37-fold
increase in cell number (Supplementary Figure 1). On the other hand,
CD27-CD28- cells were only partially altered in SP, MLN and PP. The intestinal tract is known to contain many unique T cells
expressing CD8aa homodimers rarely found in the systemic
immune system, but interestingly, the percentage of CD8aa+ cells
was also increased in the small intestinal IEL, LPL CD4+ T cell
fraction of aged mice (Figures 1E, F). As shown in Figures 1C, D, IEL CD4+ T cells showed a marked
downregulation of CD27 and CD28, co-stimulatory molecules
known to be downregulated with age, and a marked increase in the
proportion of CD27-CD28- cells (15.5 ± 1.60→60.5 ± 3.74). LPL
CD4+ T cells also showed a similar increase in CD27-CD28- cells. CD27-CD28- cells were increased in both IEL and LPL CD8+ T cells,
but the greatest change was seen in CD4+ T cells, with a 37-fold
increase in cell number (Supplementary Figure 1). On the other hand,
CD27-CD28- cells were only partially altered in SP, MLN and PP. There was no marked upregulation of known senescence markers
in old IEL CD4+ T cells (Figures 2C, D). 3.1 CD27-CD28-CD4+ T cells increased in
the small intestine of aged mice As previously reported, SP
CD4+ T cells from aged mice showed increased expression of
inflammatory cytokines such as Tnf, Ifng, Il18, Il21, and Il4,
whereas IEL-CD4+ cells showed similar levels of Infg and Tnf
expression as young mice, but low levels of Il21, and Il4 expression. Interestingly, the expression of Il10, an inhibitory cytokine, was high
in IEL CD4+ from young mice, but decreased with age. On the other
hand, the expression of chemokines such as Ccl3, Ccl4, and Cxcl2 was
increased as in the spleen (Figures 2C, E). Interestingly, the
expression of Cd8a and NK receptors such as, Klrd1, and Cd244,
which are rarely expressed on SP CD4+ cells, was upregulated, and
these cells had features similar to gut-specific fractions such as gd T
cells and DNT cells. They also had unique features that differed from
those of systemic senescent T cells, such as high expression of
cytotoxic markers such as Gzma (Figures 2C, F). The intestinal tract is known to contain many unique T cells
expressing CD8aa homodimers rarely found in the systemic
immune system, but interestingly, the percentage of CD8aa+ cells
was also increased in the small intestinal IEL, LPL CD4+ T cell
fraction of aged mice (Figures 1E, F). 3.4 Analysis of anti-tumor cytotoxic
function of bulk-old IEL CD4+ T cells SP CD4+ and IEL CD4+ T cells from young and aged mice were
sorted by flow cytometry, labelled with CFSE and stimulated with
PMA+ionomycin, and cell division after 72 hours was assessed by
flow cytometry. Although IEL have been reported to have a lower
proliferative capacity than systemic T cells, even the positive control
IEL CD4+ T cells from young mice showed little cell division under
normal conditions (data not shown). When IL-2, IL-7, and IL-15
were added as T cell maintenance factors, as shown in Figures 2A, B,
SP CD4+ T cells showed only a slight decrease in cell division with
age (97.5 ± 0.29→92.7 ± 1.05), whereas IEL-CD4 showed a marked
decrease in proliferative capacity (62.0 ± 4.82→26.5 ± 5.60). As mentioned above, the incidence of food allergy and IBD is
known to decrease with age, while the incidence of colorectal
tumors increases. Transcriptional analysis showed increased
expression of cytotoxic genes such as NK markers and Granzyme
A in intestinal IEL CD4+ T cells in aged mice, suggesting that
intestinal CD4+ T cells in aging individuals may respond to the
increased tumor risk by increasing their cytotoxic activity. To
investigate the role of old IEL CD4+ T cells in intestinal tumor
immunity, we examined their anti-tumor cellular immunity in vitro. In our previous report, we successfully recapitulated the anti-tumor
cellular immune response of IELs in vitro by co-culturing IELs with
intestinal tumor-derived organoids (27). In the present study, we
used a similar model to examine the anti-tumor immune potential
of old IEL CD4+ T cells. Organoids were generated from intestinal
tumors of APCmin mice, a spontaneous model of multiple intestinal
tumors, and co-cultured with CD4+ T cells and CD3+ T cells from
SP and IEL of young and old mice to investigate the cytotoxic
potential (Figure 3A). As shown in Figures 3B, C, although IEL
CD4+ T cells alone showed low cytotoxic activity, old IEL CD3+ T 3.2 Old IEL-CD4+ T cells had a much lower
cell proliferative capacity than young IEL-
CD4+ or old SP-CD4+ T cells KEGG pathway analysis revealed that although the NK-kB
signaling, TCR signaling, TNF signaling, Th17, and cytokine-
cytokine receptor pathways were enriched in old SP CD4+ T cells
compared to young SP CD4+ T cells, the NK cell-mediated cytotoxicity
and chemokine signaling pathways were enriched in old IEL CD4+ T
cells compared to young IEL CD4+ T cells (Figures 2G, H). As described above, the number of CD4+ T cells in the small
intestinal mucosa doubled with age, and surface markers suggested
that CD27-CD28- had characteristics as aging T cells. On the other
hand, the expression rate of CD8aa, a marker specific for intestinal
T cells, also increased, suggesting that they may have different
characteristics from those of the systemic immune system. To validate the microarray results, qPCR of some representative
genes were performed. As shown in Supplementary Figure 2, we
could confirm that higher expression of Cd8a, Gzma, Cd200r2,
Fcer1g and Lag3 in old IEL-CD4 and no age-related increase of Ifng
and Ctla4 in IEL-CD4. We therefore focused on IEL CD4+ T cells to analyze functional
changes associated with aging. Since senescent T cells in systemic
immunity are known to express senescence markers, have low
proliferative capacity, and secrete inflammatory humoral factors
such as TNF-a, IFN-g, and other cytokines, a phenotype known as
SASP, we investigated these points. 3.3 Comprehensive transcriptional analysis
of SP CD4+ and IEL CD4+ T cells revealed a
unique change in the characteristics of IEL
CD4+ T cells with age As mentioned above, IEL CD4+ T cells from aged mice differed
from systemic CD4+ T cells in many ways, suggesting that they have 06 Frontiers in Immunology frontiersin.org 10.3389/fimmu.2024.1340048 Yonemoto et al. B
C
D
E
F
G
H
A
FIGURE 2
Comparison of cell proliferation capacity and comprehensive transcriptional analysis of systemic and intestinal T cells between young and old mice. (A, B) The proliferation capacity of SP CD4+ T cells and IEL CD4+ T cells from young (8-weeks old) and old mice (85-86-weeks old) was assessed by
CFSE assay. Sort-purified IEL- or SP CD4+ cells were labelled with CFSE, and stimulated with PMA+ionomycin in the complete medium with IL-2, IL-
7, and IL-15 for 72 hours. Pooled data from 2 similar independent experiments were analyzed (n=6/group). (A) Expression of CFSE after the 72 hours
of stimulation Representative histograms are shown The number on each histogram is the percentage of CFSE-negative divided cells (B) B
C
A A A B B D FIGURE 2
Comparison of cell proliferation capacity and comprehensive transcriptional analysis of systemic and intestinal T cells between young and old mice. (A, B) The proliferation capacity of SP CD4+ T cells and IEL CD4+ T cells from young (8-weeks old) and old mice (85-86-weeks old) was assessed by
CFSE assay. Sort-purified IEL- or SP CD4+ cells were labelled with CFSE, and stimulated with PMA+ionomycin in the complete medium with IL-2, IL-
7, and IL-15 for 72 hours. Pooled data from 2 similar independent experiments were analyzed (n=6/group). (A) Expression of CFSE after the 72 hours
of stimulation. Representative histograms are shown. The number on each histogram is the percentage of CFSE-negative divided cells. (B)
Percentage of CFSE-negative divided cells in CD4+ T cells of SP or IELs from young (8-weeks old) and old (85- to 86-weeks old) mice. Graph shows
mean ± SEM. *P<0.05. (C-H) Microarray analysis of sort purified CD4 IELs or SP T cells in young (5-weeks old) or old (89-weeks old) mice. The
expression value shows the log2 of the signals after global normalization. (C) Heat maps of the top 30 genes enriched in old SP CD4+ T cells (left
column) and old IEL CD4+ T cells (right column) compared to young ones. (D) Heat maps of senescent associated genes in young and old SP or IEL
CD4+ T cells. 3.3 Comprehensive transcriptional analysis
of SP CD4+ and IEL CD4+ T cells revealed a
unique change in the characteristics of IEL
CD4+ T cells with age 10.3389/fimmu.2024.1340048 B
C
D
A
FIGURE 3
Analysis of anti-tumor cytotoxic function of bulk-old IEL CD4+ T cells. To investigate the role of old IEL CD4+ T cells in intestinal tumor immunity,
we examined their anti-tumor cellular immunity in vitro. Pooled data from 2 similar independent experiments were analyzed (n=6 mice/group). “Org” means organoid in this figure. (A) Experimental design of this experiment. Organoids were generated from intestinal tumors of APCmin mice,
and co-cultured with CD4+ T cells and CD3+ T cells from SP and IEL of young and old mice. After 7 days of co-culture, they were harvested, and
the numbers tumor cells and effector T cells were assessed by flow cytometry. (B) Representative images of organoids and IELs after 7 days of co-
culture. (C) The number of live epithelial cells (7-AAD-EpCAM+CD45- cells) collected from organoids in each group. Graphs show mean ± SEM. ∗P <
0.05. (D) Number of IELs (7-AAD-EpCAM-CD45+ cells) in each group (n = 6). Graphs show mean ± SEM. ∗P < 0.05. B
A B B C
D C D C C D FIGURE 3
Analysis of anti-tumor cytotoxic function of bulk-old IEL CD4+ T cells. To investigate the role of old IEL CD4+ T cells in intestinal tumor immunity,
we examined their anti-tumor cellular immunity in vitro. Pooled data from 2 similar independent experiments were analyzed (n=6 mice/group). “Org” means organoid in this figure. (A) Experimental design of this experiment. Organoids were generated from intestinal tumors of APCmin mice,
and co-cultured with CD4+ T cells and CD3+ T cells from SP and IEL of young and old mice. After 7 days of co-culture, they were harvested, and
the numbers tumor cells and effector T cells were assessed by flow cytometry. (B) Representative images of organoids and IELs after 7 days of co-
culture. (C) The number of live epithelial cells (7-AAD-EpCAM+CD45- cells) collected from organoids in each group. Graphs show mean ± SEM. ∗P <
0.05. (D) Number of IELs (7-AAD-EpCAM-CD45+ cells) in each group (n = 6). Graphs show mean ± SEM. ∗P < 0.05. cells showed higher cytotoxic activity against tumor organoids than
that of young IEL CD3+ T cells. Interestingly, the number of old IEL
CD3+ T cells harvested after the co-incubation was much higher
than that of young IEL CD3+ T cells (Figure 3D). 3.3 Comprehensive transcriptional analysis
of SP CD4+ and IEL CD4+ T cells revealed a
unique change in the characteristics of IEL
CD4+ T cells with age (E) Heat map of cytokine or chemokine genes in young and old SP or IEL CD4+ T cells. (F) Heat maps of genes of NK or cytotoxic
markers or IEL markers in young and old SP or IEL CD4+ T cells. (G) KEGG pathway analysis of genes enriched in old SP CD4+ T cells compared to
young SP CD4+ T cells. (H) KEGG pathway analysis of genes enriched in old IEL CD4+ T cells compared to young IEL CD4+ T cells. FIGURE 2
Comparison of cell proliferation capacity and comprehensive transcriptional analysis of systemic and intestinal T cells between young and old mice. (A, B) The proliferation capacity of SP CD4+ T cells and IEL CD4+ T cells from young (8-weeks old) and old mice (85-86-weeks old) was assessed by
CFSE assay. Sort-purified IEL- or SP CD4+ cells were labelled with CFSE, and stimulated with PMA+ionomycin in the complete medium with IL-2, IL-
7, and IL-15 for 72 hours. Pooled data from 2 similar independent experiments were analyzed (n=6/group). (A) Expression of CFSE after the 72 hours
of stimulation. Representative histograms are shown. The number on each histogram is the percentage of CFSE-negative divided cells. (B)
Percentage of CFSE-negative divided cells in CD4+ T cells of SP or IELs from young (8-weeks old) and old (85- to 86-weeks old) mice. Graph shows
mean ± SEM. *P<0.05. (C-H) Microarray analysis of sort purified CD4 IELs or SP T cells in young (5-weeks old) or old (89-weeks old) mice. The
expression value shows the log2 of the signals after global normalization. (C) Heat maps of the top 30 genes enriched in old SP CD4+ T cells (left
column) and old IEL CD4+ T cells (right column) compared to young ones. (D) Heat maps of senescent associated genes in young and old SP or IEL
CD4+ T cells. (E) Heat map of cytokine or chemokine genes in young and old SP or IEL CD4+ T cells. (F) Heat maps of genes of NK or cytotoxic
markers or IEL markers in young and old SP or IEL CD4+ T cells. (G) KEGG pathway analysis of genes enriched in old SP CD4+ T cells compared to
young SP CD4+ T cells. (H) KEGG pathway analysis of genes enriched in old IEL CD4+ T cells compared to young IEL CD4+ T cells. 07 Frontiers in Immunology frontiersin.org Yonemoto et al. 3.3 Comprehensive transcriptional analysis
of SP CD4+ and IEL CD4+ T cells revealed a
unique change in the characteristics of IEL
CD4+ T cells with age We also
confirmed the induction of high levels of MHC-II and PD-L1 on
the EpCAM+ tumor epithelial cells co-cultured with IEL CD4 or
CD3+ T cells (Supplementary Figure 3). These results suggest that
IEL T cells exerted the strong immune response against tumor
organoids, and that old IEL CD4+ T cells exert their anti-tumor
activity through MHC-II dependent antigen presentation with the
help of other T cell fractions such as gdT or CD8+ T cells. systemic T cells, such as reduced expression of co-stimulatory
molecules and reduced proliferative capacity, they also have
characteristics that are clearly distinct from those of aging
systemic T cells, such as low SASP, high cytotoxicity and CD8aa
expression. On the other hand, functional analysis of the bulk IEL
CD4+ T cells showed limited results. Therefore, we considered it difficult to understand the
characteristics of old IEL CD4+ T cells by bulk CD4+ T cell
analysis, and therefore, single cell analysis was performed with
the aim of subdividing aging T cells and precisely studying their
characteristics at the single cell level. CD3+ T cells were collected
from the SP and IEL of young and old mice and sorted by flow
cytometry, and clustering by scRNAseq, expressed gene analysis
and T cell receptor repertoire analysis were performed. In young
IEL CD4+ T cells, which are the focus of this study, are a minor
fraction, and in order to analyze a sufficient number of cells, CD4+ T
cells were enriched in both young and old IEL. 3.5 Two distinct, unique CD4+ T cell
subsets were increased in the small
intestinal intraepithelial lymphocytes of
aged mice 3.5 Two distinct, unique CD4+ T cell
subsets were increased in the small
intestinal intraepithelial lymphocytes of
aged mice In the UMAP of all T cells, IEL T cells were classified into 9 clusters
(Figure 4A). Based on the expression pattern of each marker gene, it
was confirmed that all IEL T cells analyzed were CD3+ T cells, and each These results showed that although aging intestinal CD4+ T
cells have similar characteristics to previously reported aging Frontiers in Immunology frontiersin.org 08 10.3389/fimmu.2024.1340048 Yonemoto et al. B
C
D
E
F
A
FIGURE 4
Single-cell gene expression analysis of SP and IEL T cells. SI-IELs or SP cells were collected and pooled from young (6-weeks old) or old (80-weeks
old) C57BL/6 mice. The cells from 6 young mice and 3 old mice were mixed and pooled as young SP, young IEL, old SP and old IEL. To enrich CD4+
cells, which are a rare population in young IEL, CD3+ cells and CD3+CD4+ cells were mixed as 1:1 in young and old IEL. (A) UMAP projection of
young and old IEL T cells. (B) The proportion of each cluster in IEL T cells. (C) Expression of canonical marker genes to define each cluster in IEL T
cells. (D) UMAP projection of young and old SP T cells. The definition of each cluster is described in the first column. (E) The proportion of each
cluster in SP T cells. (F) Expression of canonical marker genes to define each cluster in SP T cells. D
E A A D D E B C C F F C F FIGURE 4
Single-cell gene expression analysis of SP and IEL T cells. SI-IELs or SP cells were collected and pooled from young (6-weeks old) or old (80-weeks
old) C57BL/6 mice. The cells from 6 young mice and 3 old mice were mixed and pooled as young SP, young IEL, old SP and old IEL. To enrich CD4+
cells, which are a rare population in young IEL, CD3+ cells and CD3+CD4+ cells were mixed as 1:1 in young and old IEL. (A) UMAP projection of
young and old IEL T cells. (B) The proportion of each cluster in IEL T cells. (C) Expression of canonical marker genes to define each cluster in IEL T
cells. (D) UMAP projection of young and old SP T cells. The definition of each cluster is described in the first column. (E) The proportion of each
cluster in SP T cells. 3.5 Two distinct, unique CD4+ T cell
subsets were increased in the small
intestinal intraepithelial lymphocytes of
aged mice (F) Expression of canonical marker genes to define each cluster in SP T cells. cluster was subdivided into CD4, CD8, gd and DNT; CD4 was further
subdivided into CD8aa+CD4 and CD8aa-CD4 (Figures 4A–C and
Supplementary Figure 4). SP T cells were grouped into 6 clusters
(Figures 4D–F). Based on the expression of each marker gene, splenic T
cells were classified as naïve CD4, naïve CD8, central memory CD8,
effector memory CD8, effector memory CD4 and gd T cells
(Figures 4D–F and Supplementary Figure 4). As previously reported,
young SP were predominantly naïve, whereas old SP showed a marked increase in memory CD4 and CD8 (Figures 4D, E). Interestingly,
comparison of old and young IEL showed that they were quite
different, with young IEL being dominated by CD4, CD8 and gd T
cells, whereas old IEL showed the appearance of age-specific
CD8aa+CD4+ T cell clusters, whereas in SP, most of the fractions
were overlapped between young and old T cells (Figures 4A, B). To further analyze the aging-related changes in the IEL CD4
fraction, re-clustering was performed and revealed that IEL CD4+ T Frontiers in Immunology 09 frontiersin.org 10.3389/fimmu.2024.1340048 Yonemoto et al. cells fell into five clusters (Figure 5A). Old IEL CD4+ T cells were
broadly subdivided into two clusters, C1 and C2; C2 was also
present in young IEL CD4+ T cells, albeit at a lower proportion,
whereas C1 was found to be an old IEL CD4+ T cell specific fraction
(Figures 5A, B). According to the pathway analysis, C1 and C2 are
independent of each other, whereas C4 and C5 or C3 and C4 had
some association (Figure 5C). Pathway analysis also revealed that
although the NK-kB signaling, Th17, and chemokine signaling
pathways were enriched in C1 subset compared to C2, the NK cell-mediated cytotoxicity and JAK-STAT signaling pathways were
enriched in C2 subset compared to C1 (Figures 5D–F). We therefore defined C1 as age-related IEL CD4+ T cell subset
1, and C2 as age-related IEL CD4+ T cell subset 2, and noticed their
gene expression. In terms of age-related genes, although both didn’t
express Cd28, subset 2 expressed low levels of Cd27. Subset 1 and
other clusters expressed Ctla4, whereas subset 2 didn’t express it but
expressed high levels of Lag3 and Tigit (Figure 6A and
Supplementary Figure 5A). 3.5 Two distinct, unique CD4+ T cell
subsets were increased in the small
intestinal intraepithelial lymphocytes of
aged mice B B
C
D
E
F
A
FIGURE 5
Two distinct, unique CD4+ T cell subsets were increased in the small intestinal intraepithelial lymphocytes of aged mice. To further analyze the age-
related changes in the IEL CD4 fraction, re-clustering was performed. (A) UMAP projection of sub-clustered IEL CD4+ T cells from young and old
mice. (B) The proportion of each cluster in IEL CD4+ T cells from young and old mice. (C) The Circos plot shows how genes from the input gene
lists overlap. Inside, each arc represents a gene list, with each gene member of that list is assigned a point on the arc. Dark orange color represents
the genes that are shared by multiple lists and light orange color represents genes that are unique to that gene list. Purple lines connect the same
gene that is shared by multiple gene lists. (D) KEGG pathway analysis of genes enriched in each cluster. Heatmap cells are colored based on the P-
values of the enriched terms, and white cells indicate the lack of enrichment for that term. (E) KEGG pathway analysis of genes enriched in cluster1
compared to cluster2. (F) KEGG pathway analysis of genes enriched in cluster2 compared to cluster1. A B A B C
D
E D C E
F E E
F F FIGURE 5
Two distinct, unique CD4+ T cell subsets were increased in the small intestinal intraepithelial lymphocytes of aged mice. To further analyze the age-
related changes in the IEL CD4 fraction, re-clustering was performed. (A) UMAP projection of sub-clustered IEL CD4+ T cells from young and old
mice. (B) The proportion of each cluster in IEL CD4+ T cells from young and old mice. (C) The Circos plot shows how genes from the input gene
lists overlap. Inside, each arc represents a gene list, with each gene member of that list is assigned a point on the arc. Dark orange color represents
the genes that are shared by multiple lists and light orange color represents genes that are unique to that gene list. Purple lines connect the same
gene that is shared by multiple gene lists. (D) KEGG pathway analysis of genes enriched in each cluster. Heatmap cells are colored based on the P-
values of the enriched terms, and white cells indicate the lack of enrichment for that term. (E) KEGG pathway analysis of genes enriched in cluster1
compared to cluster2. 3.5 Two distinct, unique CD4+ T cell
subsets were increased in the small
intestinal intraepithelial lymphocytes of
aged mice (F) KEGG pathway analysis of genes enriched in cluster2 compared to cluster1. 10 Frontiers in Immunology frontiersin.org Yonemoto et al. 10.3389/fimmu.2024.1340048 B
C
D
A
FIGURE 6
The expression level of age-related genes, cytokines/chemokines, cytotoxic markers and NK cells/nIELs related genes in each cluster of young and
old IEL CD4+ T cells. Gene expression of each cluster of young and old IEL CD4+ T cells defied in Figure 5. (A) Age-related genes. (B) Cytokine/
Chemokine genes. (C) Cytotoxic marker genes. (D) NK cells/nIELs related genes and others. B B C D The expression level of age-related genes, cytokines/chemokines, cytotoxic markers and NK cells/nIELs related genes in each cluster of young and
old IEL CD4+ T cells. Gene expression of each cluster of young and old IEL CD4+ T cells defied in Figure 5. (A) Age-related genes. (B) Cytokine/
Chemokine genes. (C) Cytotoxic marker genes. (D) NK cells/nIELs related genes and others. subset 2 expressed very unique genes such as Cd200r1, Cd200r2,
klrd1, klre1, Tnfrsf9 and Fcer1g, which are features of natural IEL
(Figure 6D and Supplementary Figure 5C). Both subsets didn’t
express Treg marker genes such as foxp3 and Il10, or Th17 marker
genes such as Il17a and Il21 (Supplementary Figure 5D). Regarding cytokines and chemokines, subset 1 expressed Ifng,
whereas subset 2 didn’t express it. Both subsets didn’t express Tnf,
which is consistent with the result of the bulk CD4+ T cell
analysis (Figure 2E). Interestingly, both subsets expressed the
chemokines Ccl3, Ccl4 and Ccl5 (Figure 6B). TCR repertoire analysis was performed to determine age-related
changes in T cell clonality in IEL and SP. Diversity was predominantly
reduced at SP (Figures 7A–C and Supplementary Figure 6), as
previously reported (28, 29). In the gut, diversity was also
significantly reduced, with less than 22 of the top chronotypes In terms of cytotoxic markers, both subsets expressed high levels of
Gzma, Gzmb, but subset 2 expressed higher levels of them than subset In terms of cytotoxic markers, both subsets expressed high levels of
Gzma, Gzmb, but subset 2 expressed higher levels of them than subset
1. Both expressed Fasl (Figure 6C and Supplementary Figure 5B). Although both subsets expressed high levels of Cd8a, subset 2
expressed higher levels of it than subset 1 (Figure 6D). Interestingly, 1. Both expressed Fasl (Figure 6C and Supplementary Figure 5B). Frontiers in Immunology 3.5 Two distinct, unique CD4+ T cell
subsets were increased in the small
intestinal intraepithelial lymphocytes of
aged mice Although both subsets expressed high levels of Cd8a, subset 2
expressed higher levels of it than subset 1 (Figure 6D). Interestingly, 11 frontiersin.org 10.3389/fimmu.2024.1340048 Yonemoto et al. B
C
D
E
A
GURE 7
wo distinct age-related IEL CD4+ T cell subsets showed very specific TCR chronotypes that differed each other. TCR repertoire analysis was
erformed to determine age-related changes in T cell clonality in IEL and SP. (A) Heatmaps show the expression frequency of V-J gene pairs in
oung IEL (Y-IEL), old IEL (O-IEL), young SP (Y-SP) and old SP T cells (O-SP). The x and y axes indicate the combination of TRA + BV and TRA + BJ
enes. (B) The graphs show the percentage of each TCR clonotype in each group. (C) The frequency of the top 10 TCR clonotypes in each group. D) UMAP shows the distribution of the top 3 clonotypes in each group. (E) The graphs show the numbers of the top 3 clonotypes in each group. B
A B C
D C D D E E E FIGURE 7
Two distinct age-related IEL CD4+ T cell subsets showed very specific TCR chronotypes that differed each other. TCR repertoire analysis was
performed to determine age-related changes in T cell clonality in IEL and SP. (A) Heatmaps show the expression frequency of V-J gene pairs in
young IEL (Y-IEL), old IEL (O-IEL), young SP (Y-SP) and old SP T cells (O-SP). The x and y axes indicate the combination of TRA + BV and TRA + BJ
genes. (B) The graphs show the percentage of each TCR clonotype in each group. (C) The frequency of the top 10 TCR clonotypes in each group. (D) UMAP shows the distribution of the top 3 clonotypes in each group. (E) The graphs show the numbers of the top 3 clonotypes in each group. Frontiers in Immunology 4 Discussions The intestinal tract is an organ that is relatively unaffected by
aging compared to the skin, hair and vasculature. Although reduced
barrier function (30), increased susceptibility to DSS enteritis (31),
reduced stem cell function (32–35) and shortened villi (34) have
been reported, there are few reports of changes in the
immune system. Old IEL CD4+ T cells expressed high levels of the gut-specific
marker CD8aa homodimer. CD8aa+CD4+ T cells have been
reported to play an immunosuppressive role in IBD (42–45). It is
known that there are fewer Foxp3+ regulatory T cells (Treg) in the
small intestine than in the large intestine, and pathways for Treg
differentiation into CD4+ IELs have also been reported (46). Old
IEL CD4+ T cells, particularly subset 2, which express high levels of
tolerogenic markers and CD8aa may induce immune tolerance, in
contrast to systemic aged T cells that secrete pro-inflammatory
cytokines and induce chronic inflammation. In the present study, we found a marked increase in intestinal T
cells, particularly CD4+ T cells in the small intestinal IEL. Previous
reports have suggested that nIELs decrease and iIELs increase with
age. However, in aged mice, there was no significant change in the
number of gd and DNT cells, while CD4+ T cells increased but
CD8+ T cells did not increase. These aging IEL CD4+ T cells were
CD27-CD28- like so-called aged systemic T cells, but differed in
many ways. Their proliferative capacity was significantly lower than
that of SP CD4+ T cells, they had a reduced capacity to produce
inflammatory cytokines such as TNF-a, and they expressed surface
markers similar to nIELs, such as NK receptors, granzymes
and CD8aa. Although the present study did not examine humans, cytotoxic
CD4+ T cells are known to be increased in the peripheral blood of
human long-lived human subjects (39, 40), suggesting an
association with intestinal CD4+ T cells. Comparisons of the
proportion of subset 1/2 in different elderly people are also
of interest. In the present study, although we attempted to deplete IEL
CD4+ T cells with an anti-CD4 depletion antibody to verify the
physiological role of aging IEL CD4+ T cells, they could not be
deleted by commonly used clones. Adoptive transfer into RAG
deficient mice was also attempted. Although young IEL CD4+ T
cells were successfully engrafted, it was difficult to engraft old IEL
CD4+ T cells with their low proliferative capacity. 4 Discussions Based on these characteristics, we hypothesized that old IEL
CD4, like nIELs and CD8aa+CD4+ T cells, may play an important
role in anti-tumor immunity and intestinal immune homeostasis
via cytotoxic activity, and performed functional analysis in vitro. Although IEL CD4+ T cells alone showed low cytotoxic activity, old
IEL CD3+ T cells showed higher cytotoxic activity against tumor
organoids than that of young IEL CD3+ T cells. The results of the
scRNAseq analysis showed that the aged intestinal CD4+ T cells,
which had been analyzed together were actually an aggregate of two
fractions with completely different properties. Subset 1 has a strong
pro-inflammatory component, with secretion of Ifng and Ccl4, and
TCR and NFkB signals being extracted in pathway analysis, while
expression of inhibitory molecules such as Ctla4 is also observed. Subset 2 has no Ifng expression, high expression of inhibitory
molecules such as Lag3 and Tigit, nIEL markers such as NKR,
Cd200r and Fcer1g, and a strong tolerogenic element, while Ccl5
expression is high, and granzyme, Cd8a, which is characteristic of
aging intestinal CD4+ T cells expression was common to both
fractions. These two fractions were in opposite positions in the
clustering by UMAP and indeed had different repertoires. This may
explain why the cytotoxic capacity of the bulk CD4+ T cells alone
was weak. Although it is difficult to infer the function of each
fraction simply from the genes expressed, the results suggest that
they at least have properties that are very different from the aging T
cells of the systemic immune system. Although we have also tried to sort subset 1 and subset 2 from
SI-IEL of aged mice, we could not separate them due to a large
discrepancy between the scRNAseq results and the flow cytometry
results, with some mRNAs expressed but no surface markers
stained, and others with low mRNA expression but surface
markers stained (data not shown). In the future, we would like to
investigate the physiological role of these cells by generating mice
specifically deficient in subset 1 and subset 2 by creating appropriate
conditioned KO mice or appropriate reporter mice. In the current study, we discovered for the first time that two
unique CD4+ T cell subsets, distinct from the aging systemic T cells,
are increased in the intestinal mucosa of aged mice. 4 Discussions showing diversity in young IEL, whereas in old IEL, two chronotypes
formed the majority, and the frequency of the others was significantly
reduced (Figures 7A–C and Supplementary Figure 6). Interestingly, the
most abundant chronotype (830) was found to be expressed in most of
subset 1, whereas the second most abundant chronotype (439) was
found to be expressed only in subset 2. In contrast, the old SP
chronotype was concentrated in one clonotype, and this TCR was
concentrated in the central memory CD8 (Figures 7D, E). showing diversity in young IEL, whereas in old IEL, two chronotypes
formed the majority, and the frequency of the others was significantly
reduced (Figures 7A–C and Supplementary Figure 6). Interestingly, the
most abundant chronotype (830) was found to be expressed in most of
subset 1, whereas the second most abundant chronotype (439) was
found to be expressed only in subset 2. In contrast, the old SP
chronotype was concentrated in one clonotype, and this TCR was
concentrated in the central memory CD8 (Figures 7D, E). The intestinal tract, the frontline of enormous foreign antigens
such as food antigens and gut microbiota, is always in a dynamic
balance between inflammation and tolerance and has a highly
developed and unique immune system to maintain homeostasis,
particularly unique are the IELs; the number of IELs is comparable
to the total number of T cells in the spleen and includes T cell Frontiers in Immunology 12 frontiersin.org Yonemoto et al. 10.3389/fimmu.2024.1340048 fractions unique to the gut, gd T cells and DNT cells. These
fractions, also known as natural IELs (nIELs) because they are
abundant immediately after birth, have a limited TCR repertoire
and express many innate immune markers such as NK receptors
and are thought to maintain epithelial cell homeostasis. On the
other hand, major fractions in the peripheral blood, such as CD4+ T
cells and CD8+ T cells, are also referred to as induced IELs (iIELs). immunity (41). As subset 2, the aging- specific subset, expresses
high levels of granzymes and Fcer1g, they may have cytotoxic
potential in tumor immunity with an increased risk of developing
tumors with age. In addition, as they express nIEL-like surface
markers such as NK receptors, they may play an important role not
only in anti-tumor immunity but also in epithelial cell quality
control, such as eliminating stress cells and maintaining barrier
function in aged individuals. Frontiers in Immunology Funding The Supplementary Material for this article can be found online at:
https://www.frontiersin.org/articles/10.3389/fimmu.2024.1340048/
full#supplementary-material. The author(s) declare financial support was received for the
research, authorship, and/or publication of this article. JSPS
KAKENHI grants 21K07938ZA, 22K08051ZA, 22KK0126ZA, 4 Discussions Subset 1 has a
strong pro-inflammatory component, with expression of Ifng and
Ccl4 and upregulation of TCR and NFkB signaling pathways. Subset
2, on the other hand, does not express Ifng, but upregulates
inhibitory molecules such as Lag3 and Tigit, and nIEL markers
such as NKR, Cd200r and Fcer1g. Expression of granzymes and
Cd8a was common to both. These two fractions were in opposite
positions in the clustering by UMAP and indeed had different
TCR repertoires. CD4+ T cells with cytotoxic properties have recently attracted
attention (36–38). These cells are increased in the peripheral blood
of super-centenarians, suggesting that they play an important role
in the immune system of long-lived individuals (39, 40). In
addition, innate T cell fractions expressing high levels of Fcer1g
have recently been reported to play an important role in tumor Although the detailed functions of these fractions are still
unknown, they may be involved in the suppression of intestinal
aging and longevity through anti-tumor immunity, elimination of
senescent cells and stressed cells in the aging environment. This
finding could be a breakthrough in aging research. Frontiers in Immunology frontiersin.org 13 Yonemoto et al. 10.3389/fimmu.2024.1340048 22K16035ZA, 22H00465ZA, 21K19476ZC, 21H02897ZA,
23H02888ZA, 21H02895ZA, 21KK0146ZA, 23K15005ZA Naoki
Tsuchida Research Grant. The data presented in this study are deposited in the GEO
repository under the following accession numbers (microarray:
“GSE249501”; scRNAseq: “GSE252360”). Author contributions 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. YY: Conceptualization, Investigation, Writing – review &
editing. YN: Conceptualization, Investigation, Funding
acquisition, Writing – original draft. RM: Investigation, Writing –
review & editing. NS: Writing – review & editing. SO:
Conceptualization, Funding acquisition, Writing – review &
editing. TN: Conceptualization, Funding acquisition, Writing –
review & editing. TM: Conceptualization, Writing – review &
editing. GI: Conceptualization, Writing – review & editing. SF:
Conceptualization, Writing – review & editing. RO:
Conceptualization, Funding acquisition, Supervision, Writing –
review & editing. 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. Ethics statement We thank all the members of the Okamoto Lab at TMDU for
their discussions and advice. We thank medical student, Kohsuke
Yamakoshi for helping some experiments. The animal study was approved by Center for Experimental Animals
in Tokyo Medical and Dental University. The study was conducted in
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المستخمص نفذت ىذه الدراسة في مزرعة الزيتون الواقعة في الحبانية والتي تبعد30
كم شرق الرمادي مركز محافظة األنبار في العراق خالل موسم النمو
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في الزيتون. ا:لكممات المفتاحية الزيتون، حامض البوريك، مستخمص الطحالب البحرية. 1 Researcher, Department of Horticulture and Landscape Gardening, College of Agriculture, College of Agriculture,
Iraq. 2 Prof., Department of Horticulture and Landscape Gardening, College of Agriculture, College of Agriculture, Iraq. Iraqi Journal of Desert Studies 2021, 11 (1): 11-28 Iraqi Journal of Desert Studies 2021, 11 (1): 11-28 Iraqi Journal of Desert Studies 2021, 11 (1): 11-28 Original Article إستجابة أشجار الزيتون صنف شماللي لمتغذية الورقية بحامض البوريك ومستخمص الطحالب البحرية في
بعض الصفات الخضرية والمحتوى الكيميائي
عمر صائب سرحان الخفاجي*
1، حميد حمدان العمي2
1 باحث، قسم البستنة وىندسة الحدائق، كمية الزراعة، جامعة االنبار، العراق. 2
أ تاذ
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ال اق إستجابة أشجار الزيتون صنف شماللي لمتغذية الورقية بحامض البوريك ومستخمص الطحالب البحرية في
بعض الصفات الخضرية والمحتوى الكيميائي
عمر صائب سرحان الخفاجي*
1، حميد حمدان العمي2 Omer S. S. Al-Khafaji* 1, Hameed H. Al-Ali 2 1 Researcher, Department of Horticulture and Landscape Gardening, College of Agriculture, College of Agriculture,
Iraq. Keywords: Olive, Boric acid, Seaweed extract. Keywords: Olive, Boric acid, Seaweed extract. الخاليا في القمم النباتية الخضرية والجذرية وفي األوراق
والبراعم الحديثة حيث أن نقصو يسبب فشل نمو النبات
كما يعمل عمى تكوين معقدات مع المركبات السكرية
ويسيل حركتيا وا نتقاليا داخل النبات وا لى مواقع التخزين
في الثمار، ولو دور تنظيمي من خالل أ ثره في تنظيم
( اليرمونات النباتيةBlevins
وLukaszewski
،
1998
). كما يتحد البورون مع المركبات الييدروكسيمية
الداخمة في تركيب جدران الخمية ولو تأثير كبير عمى
صفاتيا ونوعيتيا وفعاليتيا (
Al-sahhaf
،
1989
.)
تعتبر مستخمصات الطحالب البحرية من بين المصادر
العضوية المستخدمة في اإلنتاج الزراعي وىي مكممة
( لألسمدة وليست بديالً عنياVerkleij
،
1992
)،
إذ
تحتوي الطحالب البحرية عمى عدد من العناصر الغذائية
وبعض منظمات النمو مثل األوكسينات والجبرلينات
واألحماض االمينية التي تعمل عمى تحسين النمو
الخضري والجذري . كما تسيم مستخم صات الطحالب
البحرية في زيادة قوة النبات وزيادة قدرتو عمى إمتصاص
العناصر وبالتالي زيادة مقاومتو لألمراض األمر الذي
( يؤدي إلى إنتاجيتو وتحسين نوعيتوSpinelli
،وآخرون
2010
). بينSantana
( وآخرون2006
) أن ىذه
المستخمصات تزيد من كفاءة إمتصاص المغذيات ومحتوى Abstract This study was carried out at an olive farm located in Habbaniyah, 30 km east of Ramadi, the center of Anbar
province in Iraq during the 2020 growing season to study the effect of foliar spraying with boric acid with three
concentration levels 0, 0.5 and 1.0 g L-1 and seaweed extract under the trade name Alga600 with four concentration Corresponding author. Corresponding author. Email: oma19g5002@uoanbar.edu.iq
https:// doi.org/10.36531/ijds/21110102
Received 18 March 2021; Accepted 04 April 2021 Al-Khafaji & Al-Ali, 2021 0, 1.0, 1.5 and 2.0 g L-1 and their interaction in some of the vegetative and chemical characteristics of the olive c.v
"Chemlali". The trees were sprayed till run-off on three dates. The first spray in the beginning of March before
flowering and fruit set, the second spray after 1 month from the first spray after flowering and before the fruit set
and the third spray after 2 month from the first spray after flowering and fruit set. The results showed the positive
effect of foliar spraying with boric acid on all the vegetative growth characteristics of the olives, where the
concentration 1.0 g L-1 significantly exceeded the rate of increase in branch length, number of leaves, total
chlorophyll, dry matter ratio, carbohydrates, nitrogen, C/N and boron in the leaves compared with the control
treatment (distilled water), spraying with seaweed extract at a concentration 2.0 g L-1 significantly outperformed
most of the traits compared to the comparison treatment , while the interaction treatment (1 g L-1 Boric Acid + 2 g L-
1 seaweed extract) was superior in the rate of increase of branch length and total chlorophyll, the percentage of
leaves dry weight, total carbohydrates, leaves content of nitrogen and boron, compared to the comparison treatment,
(1.0 g L-1 Boric Acid + 1.0 g L-1 seaweed extract) was significantly superior in the number of leaves compared to the
treatment (0.5 g L-1 Boric acid + 1.0 g L-1 seaweed extract), which gave the lowest value in the number of leaves in
olives. المقدمة الخاليا في القمم النباتية الخضرية والجذرية وفي األوراق
والبراعم الحديثة حيث أن نقصو يسبب فشل نمو النبات
كما يعمل عمى تكوين معقدات مع المركبات السكرية
ويسيل حركتيا وا نتقاليا داخل النبات وا لى مواقع التخزين
في الثمار، ولو دور تنظيمي من خالل أ ثره في تنظيم
( اليرمونات النباتيةBlevins
وLukaszewski
،
1998
). كما يتحد البورون مع المركبات الييدروكسيمية
الداخمة في تركيب جدران الخمية ولو تأثير كبير عمى
صفاتيا ونوعيتيا وفعاليتيا (
Al-sahhaf
،
1989
.) تعد شجرة الزيتون شجرة مباركة
، إرتبطت بتاريخ
الحضارات التي سادت حوض المتوسط ، إذا لطالما
إعتبروىا
شجرة مقدسة ومثاالً لمخصب والبقاء لقدرتيا عمى
العيش والتكاثر واإلثمار في الظروف القاسية والترب
الفقيرة، أتت عمى ذكرىا كل الكتب السماوية وكانت عمى
مر العصور منذ بدء اإلنسانية رمزاً لمخير والسالم ، وىي
من األشجار الدائمة الخضرة التي تعمر لمئات السنين
(
Mahdi
،
2011
). إن أغـمب الدراســات تشير إلى أن
الميد األصمي لشجرة الزيتون ىــــو منطقة اليالل الخصيب
من الوطن العربي وعمى وجو التحديد الخط الوىمي المار
من شمال غرب العراق وجنوب تركيا وسوريا ولبنان
وفمسطين، ومما يؤيد ذلك وجود بساتين طبيعية برية نامية
في المنطقة ال( جبمية شمال غرب العراقDarwish
،
2015
.)
أ ي ي
ي و و ي ي و(
)
تعتبر مستخمصات الطحالب البحرية من بين المصادر
العضوية المستخدمة في اإلنتاج الزراعي وىي مكممة
( لألسمدة وليست بديالً عنياVerkleij
،
1992
)،
إذ
تحتوي الطحالب البحرية عمى عدد من العناصر الغذائية
وبعض منظمات النمو مثل األوكسينات والجبرلينات
واألحماض االمينية التي تعمل عمى تحسين النمو
الخضري والجذري . كما تسيم مستخم صات الطحالب
البحرية في زيادة قوة النبات وزيادة قدرتو عمى إمتصاص
العناصر وبالتالي زيادة مقاومتو لألمراض األمر الذي
( يؤدي إلى إنتاجيتو وتحسين نوعيتوSpinelli
،وآخرون
2010
). بينSantana
( وآخرون2006
) أن ىذه
المستخمصات تزيد من كفاءة إمتصاص المغذيات ومحتوى يعد البورون من أحد العناصر الصغرى األساسية غير
المتحركة داخل النبات ويعد حامض البوريك الصورة
الجاىزة لإلمتصاص من النبات وقد أثبتت أىمية ىذا
العنصر لنمو النبات منذ عام1910
(
Havlin
،وآخرون
2005) وتعود أىميتو إلى دوره ا ألساسي في إنقسام Iraq J Desert Studies • 2021 • Vol. 11 • Iss. 1 12 Al-Khafaji & Al-Ali, 2021 العراق التي تقع عمى خط طول43.5
°
وعرض33.3
°
خالل موسم2020
لدراسة تأثير الرش الورقي بحامض
البوريك ومستخمص الطحالب البحرية في نمو وحاصل
الزيتون صنف شماللي. المقدمة المعامالت ومستوياتيا
ت الرمز المعاممة
7
B0 S0
المقارنة رش بالماء المقطر فقط
8
B0 S1
1.0
غم لتر-
1 مستخمص طحالب بحرية
9
B0 S2
1.5
غم لتر-
1 مستخمص طحالب بحرية
:
B0 S3
.0
2
غم لتر-
1 مستخمص طحالب بحرية
;
B1 S0
0.5
غم لتر-
1 حامض البوريك
<
B1 S1
0.5
غم لتر-
1
+ حامض البوريك.0
1
غم لتر-
1 مستخمص طحالب بحرية
=
B1 S2
0.5
غم لتر-
1
+ حامض البوريك1.5
غم لتر-
1 مستخمص طحالب بحرية
>
B1 S3
0.5
غم لتر-
1
+ حامض البوريك2.0
غم لتر-
1 مستخمص طحالب بحرية
? B2 S0
1.0
غم لتر-
1 حامض البوريك
72
B2 S1
1.0
غم لتر-
1
+ حامض البوريك1.0
غم لتر-
1 مستخمص طحالب بحرية
77
B2 S2
1.0
غم لتر-
1
+ حامض البوريك1.5
غم لتر-
1 مستخمص طحالب بحرية
78
B2 S3
1.0
غم لتر-
1
+ حامض البوريك2.0
غم لتر-
1 مستخمص طحالب بحرية
الصفات المدروسة
تم إختيار خمسة فروع بعمر سنة عشوائياً في بداية الدراسة
في شير شباط من جميع جوانب الشجرة ومن ثم تم إجراء
:القياسات التالية م
توسط
)الزيادة في طول الفرع (سم
قيست أطوال الفروع المعممة قبل عممية الرش بتاريخ
(
1
/
2
/
2020) وأستعمل شريط القياس بالسنتمتر وتم قياسو
( مرة أخرى في نياية الموسم بتاريخ25
/
10
/
2020
)
. والفرق يمثل معدل الزيادة في طول الفرع جدول2
. محتويات مستخمص الطحالب البحريةAlga 600
. Alga 600
ىو مستخمص أعشاب بحرية100 من سرجسوم٪
أعشاب بحرية بنية المون. ويحتوي عمى
ىرمونات نباتية طبيعية ومختمف المواد المغذية الطبيعية والمعادن النزرة والكربوىيدرات مثل
األحماض األلجينية والسكريات المتعددة وغيرىا. المكونات
مادة عضوية
حامض األلجنيك
أوكسيد البوتاسيوم
الرقم الييدروجيني
35-40 %
12-15%
17%
9-11
توصية الشركة لمرش الورقي
مخففة بنسبة1
:
2000
-
2200
. 550
جرام-
750
جرام ىكتار. ضع3
-
4
مرات. عمى
فترات15
-
20
.يومًا خالل موسم النمو جدول3. المقدمة 1 13 Al-Khafaji & Al-Ali, 2021 3
، وبثالث مواعيد األول بتاريخ1
/
3
/
2020
قبل اإلزىار
والعـَـقـِـد والموعد الثاني بتاريخ1
/
4
/
2020
بعد اإلزىار
وقبل العَـقِـد والموعد الثالث بتاريخ1
/
5
/
2020
بعد اإلزى ار
والعَـقِـد، إذ رشت األشجار حتى البمل الكامل في الصباح
الباكر باستخدام مرشة ظيرية وأضيفت مادة (الصابون
السائل) كمادة ناشرة بتركيز0.1
وذلك لتقميل الشد%
السطحي لجزيئات الماء لضمان
ا لتصاق المحاليل المغذية
عمى األوراق وبقائيا لفترة أطول مما يزيد من كفاءة عم ل
( محمول الرشChang
،
2002
). أما أشجار المقارنة فقد
.رشت بالماء المقطر فقط 3
، وبثالث مواعيد األول بتاريخ1
/
3
/
2020
قبل اإلزىار
والعـَـقـِـد والموعد الثاني بتاريخ1
/
4
/
2020
بعد اإلزىار
وقبل العَـقِـد والموعد الثالث بتاريخ1
/
5
/
2020
بعد اإلزى ار
والعَـقِـد، إذ رشت األشجار حتى البمل الكامل في الصباح
الباكر باستخدام مرشة ظيرية وأضيفت مادة (الصابون السائل) كمادة ناشرة بتركيز0.1
وذلك لتقميل الشد%
السطحي لجزيئات الماء لضمان
ا لتصاق المحاليل المغذية
عمى األوراق وبقائيا لفترة أطول مما يزيد من كفاءة عم ل
( محمول الرشChang
،
2002
). أما أشجار المقارنة فقد
.رشت بالماء المقطر فقط السائل) كمادة ناشرة بتركيز0.1
وذلك لتقميل الشد%
السطحي لجزيئات الماء لضمان
ا لتصاق المحاليل المغذية
عمى األوراق وبقائيا لفترة أطول مما يزيد من كفاءة عم ل
( محمول الرشChang
،
2002
). أما أشجار المقارنة فقد
.رشت بالماء المقطر فقط ي ب ريخ
و و
ِ
و/
/إلز ر
ب
وقبل العَـقِـد والموعد الثالث بتاريخ1
/
5
/
2020
بعد اإلزى ار
والعَـقِـد، إذ رشت األشجار حتى البمل الكامل في الصباح
الباكر باستخدام مرشة ظيرية وأضيفت مادة (الصابون
ن
يز
ي
ي
ي
ق
عمى األوراق وبقائيا لفترة أطول مما يزيد من كفاءة عم
ل
( محمول الرشChang
،
2002). أما أشجار المقارنة فقد
.رشت بالماء المقطر فقط
جدول2
. محتويات مستخمص الطحالب البحريةAlga 600
. Alga 600
ىو مستخمص أعشاب بحرية100 من سرجسوم٪
أعشاب بحرية بنية المون. ويحتوي عمى
ىرمونات نباتية طبيعية ومختمف المواد المغذية الطبيعية والمعادن النزرة والكربوىيدرات مثل
األحماض األلجينية والسكريات المتعددة وغيرىا. المكونات
مادة عضوية
حامض األلجنيك
أوكسيد البوتاسيوم
الرقم الييدروجيني
35-40 %
12-15%
17%
9-11
توصية الشركة لمرش الورقي
مخففة بنسبة1
:
2000
-
2200
. 550
جرام-
750
جرام ىكتار. ضع3
-
4
مرات. عمى
فترات15
-
20
.يومًا خالل موسم النمو
جدول3. المقدمة تم إختيار36
شجرة بعمر15
سنة متجانسة في نموىا الخضري قدر اإلمكان ومزروعة
( بمسافة5
×
5
) م نفذت عممية الخدمة والمتضمنة الري
بالتنقيط وا زالة األدغال حول األشجار المختارة. وقد أخذت
عينة من تربة الموقع لغرض تحميل بعض الصفات
الفيزيائية والكيميائية والعضوية مختبرياً وكانت كما في
( الجدول1
.) األو راق من الكموروفيل فضالً عن زيادة نشاط عمميتي
. التركيب الضوئي والتنفس تيدف الدراسة إلى بيان تأثير التغذية الورقية لمزيتون
بحامض البوريك ومستخمص الطحالب البحرية عمى
أشجار عالية الزيت كالصنف شماللي والمزروعة ضمن
مناطق مختمفة في العراق ومن أجل تحسين صفات النم و
ًالخضري والحالة الغذائية لمشجرة التي ستنعكس إيجابيا
.عمى الحاصل المواد
والطرائق
تمَ تنفيذ التجربة في مزرعة زيتون في قضاء الحبانية والتي
تبعد30
كم شرق الرمادي مركز محافظة األنبار في 13 Iraq J Desert Studies • 2021 • Vol. 11 • Iss. 1
جدول1. خصائص التربة الفيزيائية والكيميائية والعضوية. الخصائص
)العمق (سم الخصائص
)العمق (سم
0
-
30
سم
30
-
60
سم
0
-
30
سم
30
-
60
سم
مفصوالت
التربة
(غم.كغم-
1)
Sand
818
808
التىصيل الكهزبائي(
EC)
Ds.m-1
86<>
8692
Silt
150
180
درجة تفاعل التزبة
pH
=6;7
=6:>
Clay
32
12
األيىنات
المىجبة
والسالبة
الذائبة
(ملي
6مكافئ
لتز-
7 )
Ca++
8>6<8
8<6;2
صنف النسجة
Loamy
Sand
Loamy
Sand
Mg++
7:6>? 96?>
الكثافة الظاىرية (غم.سم-
3)
1.588
1.468
Na+
2692
267? معادن الكاربونات
CaCO3
(غم.كغم-
1)
309.32
345.80
K+
26;2
267>
المادة العضوية
O.M
(غم.كغم-
1)
0.804
0.168
CO-
2
Nil
Nil
النتروجين الكمي (ممغم.كغم-
1)
0.466
0.083
HCO-
3
26:2
26<2
الفسفور الجاىز (ممغم.كغم-
1)
1.744
1.319
Cl-
=6>:
26<2
البوتاسيوم الجاىز (ممغم.كغم-
1)
110.22
83.16
SO4
=
7;6;>
796?:
البورون الجاىز (ممغم.كغم-
1)
0.62
0.44
تضمنت التجربة الرش الورقي بحامض البوريكBoric
Acid
والذي فيو نسبة البورون17
وبثالث مستويات%
بتركيز0
. 0
،
0.5
،
1.0
غم لتر-
1
ورمز لياB0
،
B1
،
B2
بالتتابع وكــذلك الرش بمستخـمص الطحـــالــب البحـــريـــة
Seaweed
نـــــوعAlga600
المذكورة مكوناتيا في
الجدول
2
وبأربع مستويات بتـركيــز0
،
1.0
،
1.5
،
2.0
غم لتر-
1
ورمز لياS0
،
S1
،
S2
،
S3
بالتتابع وكذلك
الخميط بين تمك المركبات والموضحة تفاصيميا في الجدول تضمنت التجربة الرش الورقي بحامض البوريكBoric
Acid
والذي فيو نسبة البورون17
وبثالث مستويات%
بتركيز0
. 0
،
0.5
،
1.0
غم لتر-
1
ورمز لياB0
،
B1
،
B2
بالتتابع وكــذلك الرش بمستخـمص الطحـــالــب البحـــريـــة Iraq J Desert Studies • 2021 • Vol. 11 • Iss. المقدمة المعامالت ومستوياتيا
ت الرمز المعاممة
7
B0 S0
المقارنة رش بالماء المقطر فقط
8
B0 S1
1.0
غم لتر-
1 مستخمص طحالب بحرية
9
B0 S2
1.5
غم لتر-
1 مستخمص طحالب بحرية
:
B0 S3
.0
2
غم لتر-
1 مستخمص طحالب بحرية
;
B1 S0
0.5
غم لتر-
1 حامض البوريك
<
B1 S1
0.5
غم لتر-
1
+ حامض البوريك.0
1
غم لتر-
1 مستخمص طحالب بحرية
=
B1 S2
0.5
غم لتر-
1
+ حامض البوريك1.5
غم لتر-
1 مستخمص طحالب بحرية
>
B1 S3
0.5
غم لتر-
1
+ حامض البوريك2.0
غم لتر-
1 مستخمص طحالب بحرية
? B2 S0
1.0
غم لتر-
1 حامض البوريك
72
B2 S1
1.0
غم لتر-
1
+ حامض البوريك1.0
غم لتر-
1 مستخمص طحالب بحرية
77
B2 S2
1.0
غم لتر-
1
+ حامض البوريك1.5
غم لتر-
1 مستخمص طحالب بحرية
78
B2 S3
1.0
غم لتر-
1
+ حامض البوريك2.0
غم لتر-
1 مستخمص طحالب بحرية الصفات المدروسة
تم إختيار خمسة فروع بعمر سنة عشوائياً في بداية الدراسة
في شير شباط من جميع جوانب الشجرة ومن ثم تم إجراء
:القياسات التالية م
توسط
)الزيادة في طول الفرع (سم
قيست أطوال الفروع المعممة قبل عممية الرش بتاريخ
(
1
/
2
/
2020) وأستعمل شريط
القياس بالسنتمتر وتم قياسو
( مرة أخرى في نياية الموسم بتاريخ25
/
10
/
2020
)
. والفرق يمثل معدل الزيادة في طول الفرع قيست أطوال الفروع المعممة قبل عممية الرش بتاريخ
(
1
/
2
/
2020) وأستعمل شريط
القياس بالسنتمتر وتم قياسو
( مرة أخرى في نياية الموسم بتاريخ25
/
10
/
2020
)
. والفرق يمثل معدل الزيادة في طول الفرع الصفات المدروسة
تم إختيار خمسة فروع بعمر سنة عشوائياً في بداية الدراسة
في شير شباط من جميع جوانب الشجرة ومن ثم تم إجراء
:القياسات التالية م
توسط
)الزيادة في طول الفرع (سم الصفات المدروسة 14 Iraq J Desert Studies • 2021 • Vol. 11 • Iss. 1 Al-Khafaji & Al-Ali, 2021 2
حامض البوريك ثم سححت النماذج بحامض%
( الييدروكموريك0.01
ع) وجرى تقدير نسبة ا لنتروجين
بتطبيق المعادلة اآلتية:
( ) العيارية حامض حجم
(غم)النموذج وزن ( )النايتروجين
نسبة الكاربوىيدرات عمى النتروجين : إعتماداً عمى نتائج
النسبة المئوية لمكربوىيدرات ونتائج النسبة المئوية
لمنتروجين بالمعادلة التالية:
C/N
=
لمكاربوىيدرات المئوية النسبة
لمنايتروجين المئوية النسبة أ
×
100 م
توسط عدد األوراق في الفرع (ورقة فرع-
1
ً): وتم
ذلك
بحساب عدد األوراق النامية عمى الفروع المعممة قبل
المعاممة بتاريخ
1
/
2
/
2020
وكذلك في نياية التجربة
بتاريخ25
/
10
/
2020
وحاصل الفرق يمثل معدل الزيادة
. في عدد األوراق م
توسط عدد األوراق في الفرع (ورقة فرع-
1
ً): وتم
ذلك
بحساب عدد األوراق النامية عمى الفروع المعممة قبل
المعاممة بتاريخ
1
/
2
/
2020
وكذلك في نياية التجربة
بتاريخ25
/
10
/
2020
وحاصل الفرق يمثل معدل الزيادة
. في عدد األوراق ( ) العيارية حامض حجم
(غم)النموذج وزن ( )النايتروجين
نسبة الكاربوىيدرات عمى النتروجين : إعتماداً عمى نتائج
النسبة المئوية لمكربوىيدرات ونتائج النسبة المئوية
لمنتروجين بالمعادلة التالية:
×
100 (غم)النموذج وزن( )ي روجين
نسبة الكاربوىيدرات عمى النتروجين : إعتماداً عمى نتائج
النسبة المئوية لمكربوىيدرات ونتائج النسبة المئوية
لمنتروجين بالمعادلة التالية:
100 محتوى الكموروفيل الكمي في األوراق (ممغم غم-
1
وزن
طري): تم قياس الكموروفيل الكمي حسب طريقة
(
Ranganna
،
1977
) C/N
=
لمكاربوىيدرات المئوية النسبة
لمنايتروجين المئوية النسبة أ Iraq J Desert Studies • 2021 • Vol. 11 • Iss. 1 C/N
=
لمكاربوىيدرات المئوية النسبة
لمنايتروجين المئوية النسبة أ 1 Al-Khafaji & Al-Ali, 2021 مشتركة في معدل الزيادة في طول الفرع، إذ أعطت
المعاممةB2
والمتمثمة بالرش بحامــض البوريـك بتركيز1
غم لتر-
1،
إذ أعطت معــدل زيـادة بمغت46.379
سم
متفوقة معنوياً عمى المعامالت األخرى تمتيا المعاممةB1
0.5
غم لتر-
1
والتي سجمت معدل زيادة39.125
سم
والتي بدورىا تفوقت معنوياً عمى معاممة المقارنةB0
والتي
سجمت أقل معدل ز يادة في طول الفرع بمغ33.042
سم. جدول4
. تأثير الرش الورقي بحامض البوريك ومستخمص الطحالب البحرية والتداخل بينيما في معدل زيادة طول الفرع في
)شجرة الزيتون صنف شماللي (سم
معاملة البىرون
معاملة مستخلص
الطحالب البحزية
B0
B1
B2
متىسط مستخلص
الطحالب البحزية
S0
8<6822
926<99
:=6999
9:6=88
S1
9862<=
9<6899
:86><=
9=62;<
S2
9:6<22
:86;22
::6<;2
:26;>9
S3
9?6922
:=6799
;26<<=
:;6=22
متىسط البىرون
9962:8
9?678;
:<69=? L.S.D
%5
البىرون
مستخلص
الطحالب البحزية التذاخل
967>2
96<=8
<69<2 ويبين الجدول4
أن معامالت الرش الورقي بمستخمص
الطحالب البحرية أدت إلى حصول فروق معنوية في معدل
زيادة طول الفرع إذ تفوقت المعاممةS3
معنوياً عمى
جميع المعامالت األخرى بتسجيميا أعمى معدل نمو بمغ
45.700
سم تمتيا المعاممةS2
وS1
ًوالتي تفوقت معنويا
عن المعاممتين األخريتين
بينما لم تختمف المعاممتانS1
و
S0
معنوياً بينيما وسجمت المعاممةS0
أقل معدل زيادة
لطول الفرع بمغ34.722
.سم
أما فيما يخص معامالت التداخل الثنائي بين معامالت
الدراسة كما في الجدول3
فأظيرت فروق معنوية فيما
بينيا، إذ أعطت معاممة التداخل الثنائيB2S3
أعمى
مع دل زيادة في طول الفرع بمغ50.667
سم ، فيما
سجمت معاممة المقارنةB0S0
أقل معدل زيادة في طول
الفرع بمغ26.200
.سم
وقد يرجع السبب في زيادة طول
الفرع عند الرش بحامض البوريك إلى الدور الذي يمعبو ويبين الجدول4
أن معامالت الرش الورقي بمستخمص
الطحالب البحرية أدت إلى حصول فروق معنوية في معدل
زيادة طول الفرع إذ تفوقت المعاممةS3
معنوياً عمى
جميع المعامالت األخرى بتسجيميا أعمى معدل نمو بمغ
45.700
سم تمتيا المعاممةS2
وS1
ًوالتي تفوقت معنويا
عن المعاممتين األخريتين
بينما لم تختمف المعاممتانS1
و
S0
معنوياً بينيما وسجمت المعاممةS0
أقل معدل زيادة
لطول الفرع بمغ34.722
.سم البورون بتأثيره عمى بعض العمميات الحيوية والفسمجية
كإمتصاص
الماء والمغذيات والتركيب الضوئي وحركة
إنتقال المغذيات في النبات ودوره في إنقسام الخاليا
وا ستطالتيا لتأثيره اإليجابي في األوكسينات وبشكل خاص
IAA
(
Goldbach
،وآخرون1990
). C/N
=
لمكاربوىيدرات المئوية النسبة
لمنايتروجين المئوية النسبة أ ( [ =الكموروفيل الكمي7.12
×
القراءة عمى طول660
( + ) نانومتر16.8
×
القراءة عمى642.5
] ) نانومتر
×
الحجم النيائي لممحمول المستخمص / وزن العينة ×
1000 ( [ =الكموروفيل الكمي7.12
×
القراءة عمى طول660
( + ) نانومتر16.8
×
القراءة عمى642.5
] ) نانومتر
×
الحجم النيائي لممحمول المستخمص / وزن العينة ×
1000 محتوى البورون في( األوراقppm
:)
قدرت نسبة البورون
الكميـة وذلك بأخذ.0
1
غـم مـن العينـة ووضعـو فــي أنبـوبة
اليضم بطريقـةSemi - micro kjeldal AOAC
(
1980
) مع إضافة1
غم من العامل المساعدCuSo4
،
ثم أضيف5
مل حامض الكبريتيك المركز98
%
ووضعت أنابيب اليضم عمى السخان لغرض ىضم العينة
، وبعد إن أصبح المزيج رائقا بردت العينات وتم تخفيف
المحاليل إلى100
ممميتر بالماء المقطر ثم تقدير تراكيز
البورون بجيازFlame photometer
نوعPGI 2000
Automatic flame photometer
إنكميزي المنشأ
الذي أعطى تراكيز البورون بصورة مباشرة بعد تسقيط
القراءة عمى المنحنى القياسي لمبورون ، بعده جرى ضرب
التركيز النيائي في نسبة التخفيف وتقسيم الناتج عمى وزن
. العينة الجافة ): تم قياس محتوى%( نسبة المادة الجافة في األوراق
المادة الجافة حسب ماجاء فيA.O.A.C
(
1980
)
األمريكية من
خالل أخذ عينات من األوراق من فروع عمر
سنة بدءاً من العقدة الرابعة والخامسة مكتممة النمو
موضعياً من القمة ، ثم وزنت بميزان كيربائي حساس بعد
ذلك وضعت بفرن كيربائيOVEN
في درجة70
درجة
مئوية ولحين ثبات الوزن ثم حسبت النسبة المئوية
لممادة
: الجافة وفق المعادلة التالية الجاف الوزن
الرطب الوزن ( )الجافة لممادة المئوية النسبة ): تم تقدير%( نسبة الكربوىيدرات في األوراق
الكربوىيدرات الكمية حسب طريقةJoslyn
(
1970
. )
:)%( نسبة النتروجين في األوراق
قدرت نسبة النتروجين
بطريقةSemi-micro kjeldal A.O.A.C
(
1980
)
وذلك
بأخذ0.2
غم من العينة ووضعو في أنبوبة اليضم
مع إضافة1
غم من العامل المساعدCuSo4
ثم
أضيف5
مل حامض الكبريتيك المركز98
ووضعت%
أنابيب اليضم عمى السخان لغرض ىضم العينة ، وبعد ان
أصبح المزيج رائقا بردت العينات ثم أضيف25
مل من
الماء المقطر و10
مل من ىيدروكسيد الصوديوم ، وتم
تقطير الناتج وأستمم غاز األمونيا في25
مل من محمول التصميم التجريبي
( نفذت التجربة عاممية3
×
4
) حسب تصميم القطاعات
ال( عشوائية الكاممةRCBD
) بحيث تضمنت التجربة12
.معاممة بثالثة مكررات وعدت كل شجرة وحدة تجريبية
حممت النتائج إحصائياً حسب التصميم المستخدم
وباستعمال الحاسوب اإللكتروني بإستخدام برنامج
GENSTAT
، وتم إختبار المتوسطات بإستعمال اختبار
L.S.D
عند مستوى إحتمال5
%
(
Al-Muhammadi
وAl-Muhammadi
،
2012
). 15 Iraq J Desert Studies • 2021 • Vol. 11 • Iss. Iraq J Desert Studies • 2021 • Vol. 11 • Iss. 1 C/N
=
لمكاربوىيدرات المئوية النسبة
لمنايتروجين المئوية النسبة أ ويتفق ىذا مع
وجدتوMuhamed
( وآخرون2013
) عند رش أشجار
الزيتون صنفي دان ونبالي محسن، وم ع ماوجدهJasrotia
( وآخرون2014
) عمى أشجار الزيتون صنفFrontoio
.حيث أدى الرش بالبورون إلى زيادة في نمو الفرع
يعزى تأثير الرش بمستخمص الطحالب البحرية في صفات
النمو الخضري ألشجار الزيتون إلحتواء ىذه المستخمصات
عمى العديد من العناصر الكبرى والصغرى واليرمونات
النباتية كاألوكسينات والجبرلينات ممايؤدي إلى تحفيز
وا نقسام وا ستطالة الخاليا ونمو األنسجة النباتية كما تنظم
العمميات الحيوية والفسمجية مما يؤدي إلى تحفيز وزيادة لطول الفرع بمغ34.722
.سم
أما فيما يخص معامالت التداخل الثنائي بين معامالت
الدراسة كما في الجدول3
فأظيرت فروق معنوية فيما
بينيا، إذ أعطت معاممة التداخل الثنائيB2S3
أعمى
مع دل زيادة في طول الفرع بمغ50.667
سم ، فيما
سجمت معاممة المقارنةB0S0
أقل معدل زيادة في طول
الفرع بمغ26.200
.سم
وقد يرجع السبب في زيادة طول
الفرع عند الرش بحامض البوريك إلى الدور الذي يمعبو 16 Iraq J Desert Studies • 2021 • Vol. 11 • Iss. 1 Al-Khafaji & Al-Ali, 2021 كفاءة التمثيل الضوئي وبالتالي تحسن صفات النمو
( الخضري لمنباتStephenson
،
1968
) األمر الذي
يؤدي إ لى زيادة طول الفرع ، وىذا يتفق مع ما توصل إليو
Sharif
( وآخرون2010
) عمى أشجار الزيتون صنف
بعشيقي ، وAbd El-Migeed
واخرون(
2018
) عمى
أشجار الزيتون صنفKoroneiki
حيث وجدوا تفوق
معنوي في أطوال الفروع
عند الرش بالبورون. تأثير الرش الورقي بحامض البوريك ومستخمص
الطحالب البحرية والتداخل بينهما في معدل عدد األوراق
في الفرع (ورقة فرع-
1) كفاءة التمثيل الضوئي وبالتالي تحسن صفات النمو
( الخضري لمنباتStephenson
،
1968
) األمر الذي
يؤدي إ لى زيادة طول الفرع ، وىذا يتفق مع ما توصل إليو
Sharif
( وآخرون2010
) عمى أشجار الزيتون صنف
بعشيقي ، وAbd El-Migeed
واخرون(
2018
) عمى
أشجار الزيتون صنفKoroneiki
حيث وجدوا تفوق
معنوي في أطوال الفروع
عند الرش بالبورون. تأثير الرش الورقي بحامض البوريك ومستخمص
الطحالب البحرية والتداخل بينهما في معدل عدد األوراق
في الفرع (ورقة فرع-
1) بينت النتائج في الجدول5
تأثير الرش الورقي بحامض
البوريك في معدل عدد األوراق في الفرع في أشجار
الزيتون حيث أعطت المعاممةB2
أعمى معدل في عدد
األوراق في الفرع بمغ89.067
ورقة فرع-
1
ًمتفوقة معنويا
عمى المعاممتين األخريتين والمتان لم تختمفا معنوياً بينيما
وسجمت المعاممةB1
أقل معدل عدد األوراق في الفرع بمغ
72.517
ورقة فرع-
1. ع
ي ي
تأثير الرش الورقي بحامض البوريك ومستخمص
الطحالب البحرية والتداخل بينهما في معدل عدد األوراق
في الفرع (ورقة فرع-
1) جدول5. C/N
=
لمكاربوىيدرات المئوية النسبة
لمنايتروجين المئوية النسبة أ تأثير الرش الورقي بحامض البوريك
ومستخمص الطحالب البحرية في معدل عدد األوراق في الفرع في شجرة
الزيتون صنف شماللي (ورقة فرع-
1). معاملة البىرون
معاملة مستخلص
الطحالب البحزية
B0
B1
B2
م
تىسط
مستخلص
الطحالب البحزية
S0
<86<22
<?6?99
>;6799
=86;;<
S1
=96><=
<26:<=
?<6799
=<6>88
S2
>26>22
=96=99
><6:22
>26977
S3
>:6:22
>;6?99
>>6<22
><6977
م
تىسط البىرون
=;6:7=
=86;7=
>?62<=
L.S.D
5 %
البىرون
مستخلص
الطحالب البحزية التذاخل
;6>8;
<6=8<
776<;2 جدول5. تأثير الرش الورقي بحامض البوريك
ومستخمص الطحالب البحرية في معدل عدد األوراق في الفرع في شجرة
الزيتون صنف شماللي (ورقة فرع-
1). معاملة البىرون
معاملة مستخلص
الطحالب البحزية
B0
B1
B2
م
تىسط
مستخلص
الطحالب البحزية
S0
<86<22
<?6?99
>;6799
=86;;<
S1
=96><=
<26:<=
?<6799
=<6>88
S2
>26>22
=96=99
><6:22
>26977
S3
>:6:22
>;6?99
>>6<22
><6977
م
تىسط البىرون
=;6:7=
=86;7=
>?62<=
L.S.D
5 %
البىرون
مستخلص
الطحالب البحزية التذاخل
;6>8;
<6=8<
776<;2
وفي الجدول5
يظير تأثير المستخمص البحري حصول
اختالفات
معنوية بين المعامالت في عدد األوراق في الفرع
السيما المعاممتانS3
وS2
عمى معاممة المقارنةS0
،
إذ
بمغتا86.311
و80.311
ورقة فـرع-
1
بالتتابع بينما
سجمت معاممة المقارنة أقل معدل في عدد األوراق في
الفرع بمغ72.556
ورقة فرع-
1
والتي لم تختمف معنوياً مع
المعاممةS1
التي بمغت76.822
ورقة فرع-
1
والتي لم
تختمف معنوياً مع المعاممةS2
. كما يشير الجدول5
تأثير التداخل بين عوامل الدراسة في
معدل عدد األوراق في الفرع وجود فروق معنوية إذ تفوقت
المعاممةB2 S1
التي بمغ أعمى معدل في عدد األو راق في
الفرع96.133
ورقة فرع-
1
بينما أظيرت المعاممةB1 S1
أقل معدل لعدد األوراق في الفرع بمغ60.467
ورق
ــــــ ة
فرع-
1. قد يرجع السبب في الزيادة الحاصمة في عدد األوراق في
الزيتون عند الرش بحامض البوريك إلى دور عنصر
البورون في تسييل حركة وا نتقال
نواتج التركيب الضوئي
من األوراق إلى المناطق الفعالة في النبات ولو دور في
تحفيز تكوين البروتينات من خالل تأثيره في عممية تكوين
الحامض النوويRNA
فضالً عن دوره في تنشيط بعض
( األنزيماتShaaban
،
2010
) وبالتالي زيادة عدد
األوراق في الفرع . وىذا يتفق مع ماوجدهIsmail
(
2011
) عمى أشجار الزيتون صنف "صوراني" ومع جدول5. تأثير الرش الورقي بحامض البوريك
ومستخمص الطحالب البحرية في معدل عدد األوراق في الفرع في شجرة
الزيتون صنف شماللي (ورقة فرع-
1). C/N
=
لمكاربوىيدرات المئوية النسبة
لمنايتروجين المئوية النسبة أ 1 Al-Khafaji & Al-Ali, 2021 تأثير الرش الورقي بحامض البوريك ومستخمص
الطحالب البحرية وا لتداخل بينهما في محتوى الكموروفيل
الكمي في األوراق (ممغم غم-
1
)
أوضحت النتائج في الجدول6
أن الرش الورقي بحامض
البوريك عمى أشجار الزيتون لو تأثير معنوي في محتوى
الكموروفيل الكمي حيث أعطت المعاممةB2
أعمى فرق
معنوي التي بمغت87.417
ممغم غم-
1
متفوقة معنوية
عمى باقي المعامالت تمتيا المعاممةB1
التي سجمت
81.000
ممغم غم-
1
والتي بدورىا تفوقت معنوياً عمى
معاممة المقارنةB0
التي أعطت أقل محتوى لمكموروفيل
بمغت
الكمي76.333
غم
ممغم-
1. Hassan
(
2017
) عمى أشجار الزيتو "ن صنف "أشرسي عند الرش بالبورون. يعزى تأثير مستخمصات الطحالب البحرية في زيادة عدد
األوراق من خالل مايحتويو
من محفزات لمنمو وأحماض
أمينية وعضوية والتي ليا دور ميم في النمو الخضري
ونشوء جزيئة الكموروفيل التي تعد األساس لعممية البناء
الضوئي ممايزيد من نواتجيا في األوراق وتوفير الطاقة
( الالزمة لمنمو والبناءSalisbury
وRoss
،
1978
.)
إتفقت النتائج مع ماوجدهIsmail
وGhazai
(
2012
) أن
معاممة الرش الورقي لشتالت الزيتون صنفيK18
وخضيري بالمستخمص الطحالب البحرية قد أدت إلى زيادة
معنوية في عدد األوراق وكذلك مع ماوجدتوAl-
Shabani
وAl-Janabi
(
2017
.) عمى شتالت النارنج جدول6. تأثير الرش الورقي بحامض البوريك
ومستخمص الطحالب البحرية في محتوى الكموروفيل الكمي في األوراق في
شجرة الزيتون صنف شماللي (ممغم غم-
1). معاملة البىرون
معاملة مستخلص
الطحالب البحزية
B0
B1
B2
م
تىسط
مستخلص
الطحالب البحزية
S0
=86222
>76222
>;6999
=?6:::
S1
=<6999
>26222
>=6999
>76888
S2
=>6999
>76999
>>6222
>86;;<
S3
=>6<<=
>76<<=
>?6222
>96777
م
تىسط البىرون
=<6999
>76222
>=6:7=
L.S.D
5 %
البىرون
مستخلص
الطحالب البحزية التذاخل
26>>:
76287
76=<> دول6. تأثير الرش الورقي بحامض البوريك
ومستخمص الطحالب البحرية في محتوى الكموروفيل الكمي في األوراق في
شجرة الزيتون صنف شماللي (ممغم غم-
1). كما أوضح الجدول6
أن معامالت الرش الورقي
بمستخمص الطحالب البحرية أدت إلى حصول فروق
معنوية في محتوى الكموروفيل الكمي في األوراق حيث
أعطت المعاممةS3
أعمى فرق معنوي بمغت83.111
ممغم غم-
1
والتي لم تختمف معنوياً مع المعاممةS2
حيث
بمغت82.556
ممغم غم-
1
والتي تفوقت عمى المعاممة
S1
(
81.222ممغم غم-
1
)
بينما أعطت معاممة المقارنة
أقل القيم بمغت79.444
ممغم غم-
1. أما فيما يخص
معامالت التداخل الثنائي بين معامالت الدراسة كما في الجدول5
أظيرت فروق معنوية فيما بينيما حيث أعطت
معاممة التداخل الثنائيB2 S3
أعمى محتوى لمكموروفيل
الكمي في ا ألوراق التي بمغت89.000
ممغم غم-
1
بينما
سجمت معاممة المقارنةB0 S0
أقل محتوى لمكموروفيل
الكمي في األوراق بمغت72.000
ممغم غم-
1. C/N
=
لمكاربوىيدرات المئوية النسبة
لمنايتروجين المئوية النسبة أ معاملة البىرون
معاملة مستخلص
الطحالب البحزية
B0
B1
B2
م
تىسط
مستخلص
الطحالب البحزية
S0
<86<22
<?6?99
>;6799
=86;;<
S1
=96><=
<26:<=
?<6799
=<6>88
S2
>26>22
=96=99
><6:22
>26977
S3
>:6:22
>;6?99
>>6<22
><6977
م
تىسط البىرون
=;6:7=
=86;7=
>?62<=
L.S.D
5 %
البىرون
مستخلص
الطحالب البحزية التذاخل
;6>8;
<6=8<
776<;2
وفي الجدول5
يظير تأثير المستخمص البحري حصول
اختالفات
معنوية بين المعامالت في عدد األوراق في الفرع
السيما المعاممتانS3
وS2
عمى معاممة المقارنةS0
،
إذ
بمغتا86.311
و80.311
ورقة فـرع-
1
بالتتابع بينما
سجمت معاممة المقارنة أقل معدل في عدد األوراق في
الفرع بمغ72.556
ورقة فرع-
1
والتي لم تختمف معنوياً مع
المعاممةS1
التي بمغت76.822
ورقة فرع-
1
والتي لم
تختمف معنوياً مع المعاممةS2
. كما يشير الجدول5
تأثير التداخل بين عوامل الدراسة في
معدل عدد األوراق في الفرع وجود فروق معنوية إذ تفوقت
المعاممةB2 S1
التي بمغ أعمى معدل في عدد األو راق في
الف96 133قة ف-
1ال ا مة
ا أظB S
أقل معدل لعدد األوراق في الفرع بمغ60.467
ورق
ــــــ ة
فرع-
1. قد يرجع السبب في الزيادة الحاصمة في عدد األوراق في
الزيتون عند الرش بحامض البوريك إلى دور عنصر
البورون في تسييل حركة وا نتقال نواتج التركيب الضوئي
من األوراق إلى المناطق الفعالة في النبات ولو دور في
تحفيز تكوين البروتينات من خالل تأثيره في عممية تكوين
الحامض النوويRNA
فضالً عن دوره في تنشيط بعض
( األنزيماتShaaban
،
2010) وبالتالي زيادة عدد
األوراق في الفرع . وىذا يتفق مع ماوجدهIsmail
(
2011) عمى أشجار الزيتون صنف "صوراني" ومع وفي الجدول5
يظير تأثير المستخمص البحري حصول
اختالفات
معنوية بين المعامالت في عدد األوراق في الفرع
السيما المعاممتانS3
وS2
عمى معاممة المقارنةS0
،
إذ
بمغتا86.311
و80.311
ورقة فـرع-
1
بالتتابع بينما
سجمت معاممة المقارنة أقل معدل في عدد األوراق في
الفرع بمغ72.556
ورقة فرع-
1
والتي لم تختمف معنوياً مع
المعاممةS1
التي بمغت76.822
ورقة فرع-
1
والتي لم
تختمف معنوياً مع المعاممةS2
. كما يشير الجدول5
تأثير التداخل بين عوامل الدراسة في
معدل عدد األوراق في الفرع وجود فروق معنوية إذ تفوقت
المعاممةB2 S1
التي بمغ أعمى معدل في عدد األو راق في
الفرع96.133
ورقة فرع-
1
بينما أظيرت المعاممةB1 S1 17 Iraq J Desert Studies • 2021 • Vol. 11 • Iss. C/N
=
لمكاربوىيدرات المئوية النسبة
لمنايتروجين المئوية النسبة أ يعزى زيادة الكموروفيل الكمي مع زيادة تركيز البورون إلى
زيادة نواتج عممية البناء الضوئي وتنشيط عممية تكوين
الكاربوىيدرات من خالل زيادة إمتصاص العناصر الغذائية
( ماسبب زيادة بناء صبغة الكموروفيلKumar
و كما أوضح الجدول6
أن معامالت الرش الورقي
بمستخمص الطحالب البحرية أدت إلى حصول فروق
معنوية في محتوى الكموروفيل الكمي في األوراق حيث
أعطت المعاممةS3
أعمى فرق معنوي بمغت83.111
ممغم غم-
1
والتي لم تختمف معنوياً مع المعاممةS2
حيث
بمغت82.556
ممغم غم-
1
والتي تفوقت عمى المعاممة
S1
(
81.222ممغم غم-
1
)
بينما أعطت معاممة المقارنة
أقل القيم بمغت79.444
ممغم غم-
1. أما فيما يخص
معامالت التداخل الثنائي بين معامالت الدراسة كما في Iraq J Desert Studies • 2021 • Vol. 11 • Iss. 1 18 Iraq J Desert Studies • 2021 • Vol. 11 • Iss. 1 Al-Khafaji & Al-Ali, 2021 Bhushan
،
1978
). كما وجدهMohamed
و آخرون
(
2015
) عمى المانجو السكرية حيث الرش بالبورون أدى
إلى تفوق في الكموروفيل الكمي، وكذلكSharif
وMuhammad
(
2016
.) عمى شتالت الخروب
يعزى تأثير مستخمصات الطحالب البحرية في زيادة
محتوى الكموروفيل في األوراق فقد يعود السبب إلى دور
مكونات ىذا المستخمص في بناء البروتينات المرتبطة
ًبصبغة الكموروفيل وكذلك األنزيمات الخاصة بيا فضال
عن دورىا في حماية جزيئات الكموروفيل من التحمل
(
McHugh
،
2003). وىذا يتفق م ع ماوجدهAl-
Hawezy
(
2014
) عمى أشجار الينكي الدنيا ومع
ماوجدهAmro
(
2015
) عمى أشجار البرتقال الصيفي عند الرش بالمستخمص الطحالب البحرية حيث أدت إلى
.تفوق معنوي في محتوى الكموروفيل الكمي
تأثير الرش الورقي بحامض البوريك ومستخمص
الطحالب البحرية والتداخل بينهما في نسبة المادة الجافة
)%( في األوراق عند الرش بالمستخمص الطحالب البحرية حيث أدت إلى
.تفوق معنوي في محتوى الكموروفيل الكمي
تأثير الرش الورقي بحامض البوريك ومستخمص
الطحالب البحرية والتداخل بينهما في نسبة المادة الجافة
)%( في األوراق ي
ورو
و
وي ي
وق
تأثير الرش الورقي بحامض البوريك ومستخمص
الطحالب البحرية والتداخل بينهما في نسبة المادة الجافة
)%( في األوراق أكدت النتائج في الجدول7
عند الرش الورقي بحامض
البوريك أدى إلى فروق معنوية في نسبة المادة الجافة في
األوراق حيث أعطت المعاممةB2
أعمى نسبة لممادة الجافة
في األوراق التي بمغت45.883
وتمتيا المعاممة%
B1
التي بمغت45.508
والتي تفوقت معنوياً بدورىا عمى%
المعاممةB0
التي سجمت أقل نسبة لممادة الجافة في
األوراق بمغت44.825
%
. جدول7
. تأثير الرش الورقي بحامض البوريك ومستخمص الطحالب البحرية في نسبة المادة الجافة في األوراق في شجرة
الزيتون صنف)%( شماللي. C/N
=
لمكاربوىيدرات المئوية النسبة
لمنايتروجين المئوية النسبة أ الطحالب ال بحرية عمى شتالت الزيتون صنفيK18
وخضيري وكذلك مع ماوجدهIbrahim
(
2013
) عمى
شتالت الزيتون بعمر سنتين صنفHojBlanca
. ا
تأثير الرش الورقي بحامض البوريك ومستخمص
الطحالب البحرية والتداخل بينهما في نسبة الكاربوهيدرات
)%( في األوراق أ
أظيرت النتائج في الجدول8
أن الرش الورقي بحامض
البوريك أعطى تأثيراً معنوياً إذ سجمت المعاممةB2
أعمى
نسبة لمكاربوىيدرات في األواق حيث بمغت6.934
%
والتي تفوقت عمى بقية المعامالت وتمتيا المعاممةB1
التي
بمغت6.536
والتي تفوقت معنوياً عمى معاممة المقارنة%
التي أعطت أقل القيم بمغت6.303
%
. جدول8
. تأثير الرش الورقي بحامض البوريك ومستخمص الطحالب البحرية في نسبة الكاربوهيدرات في األوراق في شجرة
)%( الزيتون صنف شماللي. معاملة البىرون
معاملة مستخلص
الطحالب البحزية
B0
B1
B2
م
تىسط
مستخلص
الطحالب البحزية
S0
<68>=
<69?=
<6=<=
<6:>9
S1
<68>=
<6:;9
<6>=9
<6;9>
S2
<692=
<6<29
=628=
<6<:<
S3
<6999
<6<?2
=62=2
<6<?>
م
تىسط البىرون
<6929
<6;9<
<6?9:
L.S.D
5 %
البىرون
مستخلص
الطحالب البحزية التذاخل
2628? 26299
262;>
ويبين الجدول
8
أن معامالت الرش الورقي بمستخمص
الطحالب البحرية أدت إلى حصول فروق معنوية في نسبة
الكاربوىيدرات في األوراق إذ تفوقت المعاممةS3
ًمعنويا
عمى جميع المعامالت التي بمغت6.698
وتمتيا%
المعاممةS2
التي بمغت6.646
بينما بمغت معاممة%
المقارنة أقل قيمة بمغت6.483
ً والتي إختمفت معنويا%
مع المعاممةS1
التي سجمت6.538
%
. أما بالنسبة
لمتداخل الثنائي بين معامالت الدراسة كما في الجدول8
حيث أعطت المعاممةB2 S3
أعمى فرق معنوي بمغت
7.070 بينما لم تختمف مع الم%
عاممةB2 S2
التي بمغت
7.027
بينما بمغت معاممة المقارنة%
B0 S0
والمعاممة
B0 S1
أقل القيم حيث أعطت نفس القيمة بمغت
6.287
.% جدول8
. تأثير الرش الورقي بحامض البوريك ومستخمص الطحالب البحرية في نسبة الكاربوهيدرات في األوراق في شجرة
)%( الزيتون صنف شماللي. معاملة البىرون
معاملة مستخلص
الطحالب البحزية
B0
B1
B2
م
تىسط
مستخلص
الطحالب البحزية
S0
<68>=
<69?=
<6=<=
<6:>9
S1
<68>=
<6:;9
<6>=9
<6;9>
S2
<692=
<6<29
=628=
<6<:<
S3
<6999
<6<?2
=62=2
<6<?>
م
تىسط البىرون
<6929
<6;9<
<6?9:
L.S.D
5 %
البىرون
مستخلص
الطحالب البحزية التذاخل
2628? 26299
262;> ويبين الجدول
8
أن معامالت الرش الورقي بمستخمص
الطحالب البحرية أدت إلى حصول فروق معنوية في نسبة
الكاربوىيدرات في األوراق إذ تفوقت المعاممةS3
ًمعنويا
عمى جميع المعامالت التي بمغت6.698
وتمتيا%
المعاممةS2
التي بمغت6.646
بينما بمغت معاممة%
المقارنة أقل قيمة بمغت6.483
ً والتي إختمفت معنويا%
مع المعاممةS1
التي سجمت6.538
%
. C/N
=
لمكاربوىيدرات المئوية النسبة
لمنايتروجين المئوية النسبة أ معاملة البىرون
معاملة مستخلص
الطحالب البحزية
B0
B1
B2
م
تىسط
مستخلص
الطحالب البحزية
S0
::6=99
:;68<=
:;6;<=
:;67>? S1
::6>22
:;6:22
:;6?22
:;69<=
S2
::6><=
:;6<<=
:<62<=
:;6;99
S3
::6?22
:;6=22
:<6222
:;6;99
م
تىسط البىرون
::6>8;
:;6;2>
:;6>>9
L.S.D
5 %
البىرون
مستخلص
الطحالب البحزية التذاخل
262>9
262?<
267<= ويبين الجدول
7
أن ىناك فروق معنوية عند الرش
بمستخمص الطحالب البحرية عمى أشجار الزيتون حيث
أعطت المعاممتانS3
وS2
أعمى فروق معنوية بمغتا
45.533
و45.533
بالتتابع والتي لم تختمفا فيما%
بينيما وتفوقتا عمى باقي المعامالت وأعطت معاممة
المقارنةS0
أقل قيمة حيث سجمت45.189
والتي%
إختمفت معنوياً مع المعاممةS1
التي بمغت45.367
.% بينما فيما يخص التداخل الثنائي بين عوامل الدر اسة كما
في الجدول6
في نسبة المادة الجافة في األوراق إذ تفوقت
المعاممةB2 S2
التي بمغت أعمى قيمة ليا46.067
%
بينما بمغت معاممة المقارنةB0 S0
أقل نسبة لممادة الجافة
في األوراق التي بمغت44.733
.% أ
يعود سبب زيادة نسبة المادة الجافة في األوراق عند الرش
بحامض ا لبوريك إلى دور البورون في زيادة محتوى
الكموروفيل ثم زيادة كمية الكربوىيدرات المخزونة فييا 19 Iraq J Desert Studies • 2021 • Vol. 11 • Iss. 1 Al-Khafaji & Al-Ali, 2021 ممايؤدي إلى زيادة الوزن الجاف في األوراق ، وقد يعزى
أيضاً إلى دور البورون في تحفيز عممية التمثيل الضوئي
ممايؤدي إلى إنتقال منتجاتيا إلى األجزاء النباتية
(
Wajcik
وWajcik
،
2006
)،
وىذا يتفق مع ماوجده
Al-Hadethi
( وآخرون2019
) عمى أشجار الزيتون
صنف "أشرسي" ومعSharif
وMuhammad
(
2016
)
.عمى شتالت الخروب عند الرش بالبورون
يعزى السبب وراء زيادة نسبة المادة الجافة في األوراق عند
الرش بمستخمص الطحالب البحرية إلى زيادة العناصر
( الكبرىN
و P
وK
) في األوراق وبالتالي تنشيط إنقسام
الخاليا وزيادة التخميق الحيوي لممنتجات العضوية التي
تؤدي إلى تراكم الكربوىيدرات والبروتينات في األوراق
(
Mancuso
،وآخرون2006
). كما يتفق مع ماوجداه
Ismail
وGhazai
(
2012
) عند رش بالمستخمص K18 الطحالب ال بحرية عمى شتالت الزيتون صنفيK18
وخضيري وكذلك مع ماوجدهIbrahim
(
2013
) عمى
شتالت الزيتون بعمر سنتين صنفHojBlanca
. تأثير الرش الورقي بحامض البوريك ومستخمص
الطحالب البحرية والتداخل بينهما في نسبة الكاربوهيدرات
)%( في األوراق
أظيرت النتائج في الجدول8
أن الرش الورقي بحامض
البوريك أعطى تأثيراً معنوياً إذ سجمت المعاممةB2
أعمى
نسبة لمكاربوىيدرات في األواق حيث بمغت6.934
%
والتي تفوقت عمى بقية المعامالت وتمتيا المعاممةB1
التي
بمغت6.536
والتي تفوقت معنوياً عمى معاممة المقارنة%
التي أعطت أقل القيم بمغت6.303
%
. C/N
=
لمكاربوىيدرات المئوية النسبة
لمنايتروجين المئوية النسبة أ كما أوضح الجدول
9
حصول تأثير معنوي عند الرش
بمستخمص الطحالب البحرية فقد أظيرت المعاممةS3
تفوقاً معنوياً عمى باقي المعامالت في إعطائيا أعمى نسبة
لمنتروجين بمغت1.829
تمتيا المعاممة%
S2
والتي بدورىا
تفوقت معنوياً عمى المعاممتينS1
وS0
وا ختمفتS1
عن
S0
معنوياً أيضاً بينما سجمت المعاممةS0
أقل قيمة
1.777
%
. كمـا يشيـر الجدول8
التداخل بين عـوامل
الدراسة وجود فروق معنوية بين المعامالت إذ سجمت
المعاممةB2 S3
أعمى نسبة مئوية لمنتروجين في أورا قيا
بمغت1.920
بينما سجمت معاممة المقارنة%
B0 S0
أقل
نسبة مئوية لمنتروجين في األوراق بمغت1.713
%
يعزى
سبب زيادة النتروجين في األوراق إلى دور البورون في
تحفيز وتنشيط أنزيم مختزل النتراتNitrate reductase
( الذي يعمل عمى تمثيل النتروجين في األوراقBonilla جدول9
. تأثير الرش الورقي بحامض البوريك ومستخمص الطحالب البحرية في النسبة المئوية لمنتروجين في األوراق في
)%( شجرة الزيتون صنف شماللي. معاملة البىرون
معاملة مستخلص
الطحالب البحزية
B0
B1
B2
م
تىسط
مستخلص
الطحالب البحزية
S0
76=79
76=>2
76>9=
76===
S1
76=82
76=?9
76>==
76=?=
S2
76=89
76>89
76>?9
76>79
S3
76=89
76>:9
76?82
76>8? م
تىسط البىرون
76=82
76>72
76>>8
L.S.D
5 %
البىرون
مستخلص
الطحالب البحزية التذاخل
2622:
2622;
2622? جدول9
. تأثير الرش الورقي بحامض البوريك ومستخمص الطحالب البحرية في النسبة المئوية لمنتروجين في األوراق في
)%( شجرة الزيتون صنف شماللي. معاملة البىرون
معاملة مستخلص
الطحالب البحزية
B0
B1
B2
م
تىسط
مستخلص
الطحالب البحزية
S0
76=79
76=>2
76>9=
76===
S1
76=82
76=?9
76>==
76=?=
S2
76=89
76>89
76>?9
76>79
S3
76=89
76>:9
76?82
76>8? م
تىسط البىرون
76=82
76>72
76>>8
L.S.D
5 %
البىرون
مستخلص
الطحالب البحزية التذاخل
2622:
2622;
2622? كما أوضح الجدول
9
حصول تأثير معنوي عند الرش
بمستخمص الطحالب البحرية فقد أظيرت المعاممةS3
تفوقاً معنوياً عمى باقي المعامالت في إعطائيا أعمى نسبة
لمنتروجين بمغت1.829
تمتيا المعاممة%
S2
والتي بدورىا
تفوقت معنوياً عمى المعاممتينS1
وS0
وا ختمفتS1
عن
S0
معنوياً أيضاً بينما سجمت المعاممةS0
أقل قيمة
1.777
%
. كمـا يشيـر الجدول8
التداخل بين عـوامل كما أوضح الجدول
9
حصول تأثير معنوي عند الرش
بمستخمص الطحالب البحرية فقد أظيرت المعاممةS3
تفوقاً معنوياً عمى باقي المعامالت في إعطائيا أعمى نسبة
لمنتروجين بمغت1.829
تمتيا المعاممة%
S2
والتي بدورىا
تفوقت معنوياً عمى المعاممتينS1
وS0
وا ختمفتS1
عن
S0
معنوياً أيضاً بينما سجمت المعاممةS0
أقل قيمة
1.777
%
. C/N
=
لمكاربوىيدرات المئوية النسبة
لمنايتروجين المئوية النسبة أ كما أوضح الجدول
9
حصول تأثير معنوي عند الرش
بمستخمص الطحالب البحرية فقد أظيرت المعاممةS3
تفوقاً معنوياً عمى باقي المعامالت في إعطائيا أعمى نسبة
لمنتروجين بمغت1.829
تمتيا المعاممة%
S2
والتي بدورىا
تفوقت معنوياً عمى المعاممتينS1
وS0
وا ختمفتS1
عن
S0
معنوياً أيضاً بينما سجمت المعاممةS0
أقل قيمة
1 777
%ال د ل
ك ا ش8ا ل
ن
التداخل
الدراسة وجود فروق معنوية بين المعامالت إذ سجمت
المعاممةB2 S3
أعمى نسبة مئوية لمنتروجين في أورا قيا
بمغت1.920
بينما سجمت معاممة المقارنة%
B0 S0
أقل
نسبة مئوية لمنتروجين في األوراق بمغت1.713
%
يعزى
سبب زيادة النتروجين في األوراق إلى دور البورون في
تحفيز وتنشيط أنزيم مختزل النتراتNitrate reductase
(األ اق
ن ف
ت ث ل النت
م
ل
الذBonilla قد يرجع السبب في زيادة محتوى األوراق من
الكاربوىيدرات عند الرش بحامض البوريك نتيجة تأثير
عنصر البورون في زيادة محتواىا من الكموروفيل كم ا في
الجدول5
وىذا بدوره إنعكس إيجابياً عمى كفاءة عممية
التمثيل الضوئي وزيادة المواد المصنعة في األوراق كما
وجدهIbrahim
( وآخرون2018
) عمى أشجار السدر
.صنف تفاحي Abd El-Moniem
، وآخرون2008
)،
وىذا يتفق مع
ماوجدهFaissal
( وآخرون2013
) عمى أشجار المانجو
صنفEwaise
وكما وجدهAhmed
( وآخرون2019
)
.عمى أشجار النخيل صنف سكوتي
تأثير الرش الورقي بحامض البوريك ومستخمص
الطحالب البحرية والتداخل بينهما في النسبة المئوية
لمنتروجين في ا )%( ألوراق
أوضحت النتائج في الجدول9
إن الرش بحامض
البوريك أدت في زيادة النسبة المئوية لمنتروجين في األوراق
إذ تفوقت المعاممةB2
تفوقاً معنوياً عمى المعاممتين
األخرتين بمغت قيمتيا1.882
تمتيا المعاممة%
B1
والتي
بدورىا تفوقت معنوياً المعاممةB0
والتي سجمت أقل نسبة
مئوية لمنتروجين في األوراق بمغت1.720
%
. Abd El-Moniem
، وآخرون2008
)،
وىذا يتفق مع
ماوجدهFaissal
( وآخرون2013
) عمى أشجار المانجو
صنفEwaise
وكما وجدهAhmed
( وآخرون2019
)
.عمى أشجار النخيل صنف سكوتي Abd El-Moniem
، وآخرون2008
)،
وىذا يتفق مع
ماوجدهFaissal
( وآخرون2013
) عمى أشجار المانجو
صنفEwaise
وكما وجدهAhmed
( وآخرون2019
)
.عمى أشجار النخيل صنف سكوتي
تأثير الرش الورقي بحامض البوريك ومستخمص
الطحالب البحرية والتداخل بينهما في النسبة المئوية
لمنتروجين في ا )%( ألوراق
أوضحت النتائج في الجدول9
إن الرش بحامض
البوريك أدت في زيادة النسبة المئوية لمنتروجين في األوراق
إذ تفوقت المعاممةB2
تفوقاً معنوياً عمى المعاممتين
األخرتين بمغت قيمتيا1.882
تمتيا المعاممة%
B1
والتي
بدورىا تفوقت معنوياً المعاممةB0
والتي سجمت أقل نسبة
مئوية لمنتروجين في األوراق بمغت1.720
%
. C/N
=
لمكاربوىيدرات المئوية النسبة
لمنايتروجين المئوية النسبة أ ي
تأثير الرش الورقي بحامض البوريك ومستخمص
الطحالب البحرية والتداخل بينهما في النسبة المئوية
لمنتروجين في ا )%( ألوراق ي)
(أ
أوضحت النتائج في الجدول9
إن الرش بحامض
البوريك أدت في زيادة النسبة المئوية لمنتروجين في األوراق
إذ تفوقت المعاممةB2
تفوقاً معنوياً عمى المعاممتين
األخرتين بمغت قيمتيا1.882
تمتيا المعاممة%
B1
والتي
بدورىا تفوقت معنوياً المعاممةB0
والتي سجمت أقل نسبة
مئوية لمنتروجين في األوراق بمغت1.720
%
. يعزى سبب زيادة الكاربوىيدرات عند الرش بمستخمص
الطحالب البحرية لتأثير المستخمص بشكل أساسي من
خال ل التأثير الفسيولوجي لممغذيات والفيتامينات ومنظمات
النمو . كما أن زيادة محتوى الكموروفيل الكمي لألوراق
إنعكس في زيادة معدل التمثيل الضوئي مما أدى إلى
التأثير اإليجابي في زيادة الكاربوىيدرات في األوراق
(
Abdel-Maguid
،وآخرون2004
و جدول9
. تأثير الرش الورقي بحامض البوريك ومستخمص الطحالب البحرية في النسبة المئوية لمنتروجين في األوراق في
)%( شجرة الزيتون صنف شماللي. معاملة البىرون
معاملة مستخلص
الطحالب البحزية
B0
B1
B2
م
تىسط
مستخلص
الطحالب البحزية
S0
76=79
76=>2
76>9=
76===
S1
76=82
76=?9
76>==
76=?=
S2
76=89
76>89
76>?9
76>79
S3
76=89
76>:9
76?82
76>8? م
تىسط البىرون
76=82
76>72
76>>8
L.S.D
5 %
البىرون
مستخلص
الطحالب البحزية التذاخل
2622:
2622;
2622? كما أوضح الجدول
9
حصول تأثير معنوي عند الرش
بمستخمص الطحالب البحرية فقد أظيرت المعاممةS3
تفوقاً معنوياً عمى باقي المعامالت في إعطائيا أعمى نسبة
لمنتروجين بمغت1.829
تمتيا المعاممة%
S2
والتي بدورىا
تفوقت معنوياً عمى المعاممتينS1
وS0
وا ختمفتS1
عن
S0
معنوياً أيضاً بينما سجمت المعاممةS0
أقل قيمة
1.777
%
. كمـا يشيـر الجدول8
التداخل بين عـوامل
الدراسة وجود فروق معنوية بين المعامالت إذ سجمت
المعاممةB2 S3
أعمى نسبة مئوية لمنتروجين في أورا قيا
بمغت1.920
بينما سجمت معاممة المقارنة%
B0 S0
أقل
نسبة مئوية لمنتروجين في األوراق بمغت1.713
%
يعزى
سبب زيادة النتروجين في األوراق إلى دور البورون في
تحفيز وتنشيط أنزيم مختزل النتراتNitrate reductase
( الذي يعمل عمى تمثيل النتروجين في األوراقBonilla جدول9
. تأثير الرش الورقي بحامض البوريك ومستخمص الطحالب البحرية في النسبة المئوية لمنتروجين في األوراق في
)%( شجرة الزيتون صنف شماللي. معاملة البىرون
معاملة مستخلص
الطحالب البحزية
B0
B1
B2
م
تىسط
مستخلص
الطحالب البحزية
S0
76=79
76=>2
76>9=
76===
S1
76=82
76=?9
76>==
76=?=
S2
76=89
76>89
76>?9
76>79
S3
76=89
76>:9
76?82
76>8? م
تىسط البىرون
76=82
76>72
76>>8
L.S.D
5 %
البىرون
مستخلص
الطحالب البحزية التذاخل
2622:
2622;
2622? C/N
=
لمكاربوىيدرات المئوية النسبة
لمنايتروجين المئوية النسبة أ أما بالنسبة لمتداخل الثنائي بين معامالت الدراسة كما في الجدول8
حيث أعطت المعاممةB2 S3
أعمى فرق معنوي بمغت
7.070 بينما لم تختمف مع الم%
عاممةB2 S2
التي بمغت
7.027
بينما بمغت معاممة المقارنة%
B0 S0
والمعاممة
B0 S1
أقل القيم حيث أعطت نفس القيمة بمغت
6.287
.% 20 Iraq J Desert Studies • 2021 • Vol. 11 • Iss. 1 Al-Khafaji & Al-Ali, 2021 j
,
قد يرجع السبب في زيادة محتوى األوراق من
الكاربوىيدرات عند الرش بحامض البوريك نتيجة تأثير
عنصر البورون في زيادة محتواىا من الكموروفيل كم ا في
الجدول5
وىذا بدوره إنعكس إيجابياً عمى كفاءة عممية
التمثيل الضوئي وزيادة المواد المصنعة في األوراق كما
وجدهIbrahim
( وآخرون2018
) عمى أشجار السدر
.صنف تفاحي
يعزى سبب زيادة الكاربوىيدرات عند الرش بمستخمص
الطحالب البحرية لتأثير المستخمص بشكل أساسي من
خال ل التأثير الفسيولوجي لممغذيات والفيتامينات ومنظمات
النمو . كما أن زيادة محتوى الكموروفيل الكمي لألوراق
إنعكس في زيادة معدل التمثيل الضوئي مما أدى إلى
التأثير اإليجابي في زيادة الكاربوىيدرات في األوراق
(
Abdel-Maguid
،وآخرون2004
و
Abd El-Moniem
، وآخرون2008
)،
وىذا يتفق مع
ماوجدهFaissal
( وآخرون2013
) عمى أشجار المانجو
صنفEwaise
وكما وجدهAhmed
( وآخرون2019
)
.عمى أشجار النخيل صنف سكوتي
تأثير الرش الورقي بحامض البوريك ومستخمص
الطحالب البحرية والتداخل بينهما في النسبة المئوية
لمنتروجين في ا )%( ألوراق
أوضحت النتائج في الجدول9
إن الرش بحامض
البوريك أدت في زيادة النسبة المئوية لمنتروجين في األوراق
إذ تفوقت المعاممةB2
تفوقاً معنوياً عمى المعاممتين
األخرتين بمغت قيمتيا1.882
تمتيا المعاممة%
B1
والتي
بدورىا تفوقت معنوياً المعاممةB0
والتي سجمت أقل نسبة
مئوية لمنتروجين في األوراق بمغت1.720
%
. جدول9
. تأثير الرش الورقي بحامض البوريك ومستخمص الطحالب البحرية في النسبة المئوية لمنتروجين في األوراق في
)%( شجرة الزيتون صنف شماللي. معاملة البىرون
معاملة مستخلص
الطحالب البحزية
B0
B1
B2
م
تىسط
مستخلص
الطحالب البحزية
S0
76=79
76=>2
76>9=
76===
S1
76=82
76=?9
76>==
76=?=
S2
76=89
76>89
76>?9
76>79
S3
76=89
76>:9
76?82
76>8? م
تىسط البىرون
76=82
76>72
76>>8
L.S.D
5 %
البىرون
مستخلص
الطحالب البحزية التذاخل
2622:
2622;
2622? C/N
=
لمكاربوىيدرات المئوية النسبة
لمنايتروجين المئوية النسبة أ كمـا يشيـر الجدول8
التداخل بين عـوامل الدراسة وجود فروق معنوية بين المعامالت إذ سجمت
المعاممةB2 S3
أعمى نسبة مئوية لمنتروجين في أورا قيا
بمغت1.920
بينما سجمت معاممة المقارنة%
B0 S0
أقل
نسبة مئوية لمنتروجين في األوراق بمغت1.713
%
يعزى
سبب زيادة النتروجين في األوراق إلى دور البورون في
تحفيز وتنشيط أنزيم مختزل النتراتNitrate reductase
( الذي يعمل عمى تمثيل النتروجين في األوراقBonilla 21 Iraq J Desert Studies • 2021 • Vol. 11 • Iss. 1 Al-Khafaji & Al-Ali, 2021 ،وآخرون1980
). كما بينAl-Hadethi
وآخرون
(
2019
،) عند الرش بالبورون عمى الزيتون صنف أشرسي
وكذلكFarhat
(
2017
) عمى أشجار البرتقال السرة حيث
.وجدوا تفوق معنوي في محتوى النتروجين في األوراق
يعود سبب زيادة محتوى النتروجين في األوراق عند الرش
بمستخمصات الطحالب ا لبحرية إلحتواءه عمىbetaine
الذي يعتبر مصدر لمنتروجين في التراكيز القميمة ومنظم
( لألزموزية في التراكيز العاليةMorales-Payan
و
Norrie
،
2010
). ويتفق مع ما وجدتوAl-Tamimi
( وآخرون2015
) عمى شتالت الزيتون صنف خضيري
ومع ماوجدهAhmed
( وآخرون2014
) عمى أشجار
ال نخيل صنف زغمول حيث أدى الرش بالمستخمص
الطحالب البحرية إلى زيادة في نسبة النتروجين في
.األوراق ،وآخرون1980
). كما بينAl-Hadethi
وآخرون
(
2019
،) عند الرش بالبورون عمى الزيتون صنف أشرسي
وكذلكFarhat
(
2017
) عمى أشجار البرتقال السرة حيث
.وجدوا تفوق معنوي في محتوى النتروجين في األوراق
يعود سبب زيادة محتوى النتروجين في األوراق عند الرش
بمستخمصات الطحالب ا لبحرية إلحتواءه عمىbetaine
الذي يعتبر مصدر لمنتروجين في التراكيز القميمة ومنظم
( لألزموزية في التراكيز العاليةMorales-Payan
و
Norrie
،
2010
). ويتفق مع ما وجدتوAl-Tamimi
( وآخرون2015
) عمى شتالت الزيتون صنف خضيري
ومع ماوجدهAhmed
( وآخرون2014
) عمى أشجار
ال نخيل صنف زغمول حيث أدى الرش بالمستخمص
الطحالب البحرية إلى زيادة في نسبة النتروجين في
.األوراق تأثير الرش الورقي بحامض البوريك ومستخمص
نسبة
في
بيتهما
والتداخل
البحرية
الطحالب
الكاربوهيدرات/النتروجين في األوراق
أن النسبة بين الـC/N
تعبر عن الحالة الغذائية ل ألشجار
وتراكم الخزين الغذائي كذلك تعد عامالً محدداً لتمايز
وتكشف البراعم الزىرية ومصدراً ميماً لمد النموات الحديثة
( بالغذاء الالزمAl-Ali
،
2002
) أظيرت النتائج في
الجدول10
ًأن الرش الورقي بحامض البوريك أعطى تأثيرا
ًمعنويا،
المعاممة
سجمت
إذB2
نسبة
أعمى
لمكاربوىيدرات/النتــروجين فـي األواق حيث بمغت3.685
والتي تفوقت عمى بقية المعامالت وتمتيا المعاممةB0
التي
بمغت3.664
والتي تفوقت عمى معاممةB1
التــي أعطت
أقـــل القيــم بمغت3.610
. جدول10. تأثير الرش الورقي بحامض البوريك ومستخمص الطحا
لب البحرية في نسبة الكاربوهيدرات/ن ال تروجين في
األوراق في شجرة الزيتون صنف شماللي. C/N
=
لمكاربوىيدرات المئوية النسبة
لمنايتروجين المئوية النسبة أ معاملة البىرون
معاملة مستخلص
الطحالب البحزية
B0
B1
B2
م
تىسط
مستخلص
الطحالب البحزية
S0
96<<? 96;?9
96<>:
96<:? S1
96<;:
96;?>
96<<8
96<9>
S2
96<;? 96<87
96=77
96<<:
S3
96<=;
96<8? 96<>8
96<<8
م
تىسط البىرون
96<<:
96<72
96<>;
L.S.D
5 %
البىرون
مستخلص
الطحالب البحزية التذاخل
2627? N.S
N.S
أما بالنسبة لمستخمص الطحالب البحرية كما في الجدول
10
نسبة
في
معنوية
فروق
ىناك
يكن
لم
الكاربوىيدرات/النتروجين في األوراق. أيضاً في الجدول9
لم تكن ىناك فروق معنوية في نسبة
الكاربوىيدرات/
النتروجين في األوراق عند التداخل الثنائي
بين ع
اممي الدراسة عمى شجرة الزيتون . يعزى سبب زيادة
النسبة مابين الكاربوىيدرات عمى النتروجين عند الرش
بحامض البوري ك وذلك لدور البورون في زيادة الكموروفيل
حيث أدى إلى تكوين زيادة في المادة الجافة وبالتالي زيادة
نسبة الكاربوىيدرات والنتروجين ونرى زيادة نسبة
الكربوىيدرات عمى حساب نسبة النتروجين فتزيد نسبة
C/N
، كما وجدهAl-Rawi
و
Al-Ali
(
2013) عمى
"أشجار الزيتون صنف "نبالي محسن. جدول10. تأثير الرش الورقي بحامض البوريك ومستخمص الطحا
لب البحرية في نسبة الكاربوهيدرات/ن ال تروجين في
األوراق في شجرة الزيتون صنف شماللي. معاملة البىرون
معاملة مستخلص
الطحالب البحزية
B0
B1
B2
م
تىسط
مستخلص
الطحالب البحزية
S0
96<<? 96;?9
96<>:
96<:? S1
96<;:
96;?>
96<<8
96<9>
S2
96<;? 96<87
96=77
96<<:
S3
96<=;
96<8? 96<>8
96<<8
م
تىسط البىرون
96<<:
96<72
96<>;
L.S.D
5 %
البىرون
مستخلص
الطحالب البحزية التذاخل
2627? N.S
N.S جدول10. تأثير الرش الورقي بحامض البوريك ومستخمص الطحا
لب البحرية في نسبة الكاربوهيدرات/ن ال تروجين في
األوراق في شجرة الزيتون صنف شماللي. معاملة البىرون
معاملة مستخلص
الطحالب البحزية
B0
B1
B2
م
تىسط
مستخلص
الطحالب البحزية
S0
96<<? 96;?9
96<>:
96<:? S1
96<;:
96;?>
96<<8
96<9>
S2
96<;? 96<87
96=77
96<<:
S3
96<=;
96<8? 96<>8
96<<8
م
تىسط البىرون
96<<:
96<72
96<>;
L.S.D
5 %
البىرون
مستخلص
الطحالب البحزية التذاخل
2627? N.S
N.S
أما بالنسبة لمستخمص الطحالب البحرية كما في الجدول
10
نسبة
في
معنوية
فروق
ىناك
يكن
لم
الكاربوىيدرات/النتروجين في األوراق. أيضاً في الجدول9
لم تكن ىناك فروق معنوية في نسبة
الكاربوىيدرات/
النتروجين في األوراق عند التداخل الثنائي
بين ع
اممي الدراسة عمى شجرة الزيتون . يعزى سبب زيادة
النسبة مابين الكاربوىيدرات عمى النتروجين عند الرش
بحامض البوري ك وذلك لدور البورون في زيادة الكموروفيل
حيث أدى إلى تكوين زيادة في المادة الجافة وبالتالي زيادة
نسبة الكاربوىيدرات والنتروجين ونرى زيادة نسبة
الكربوىيدرات عمى حساب نسبة النتروجين فتزيد نسبة
C/N
، كما وجدهAl-Rawi
و
Al-Ali
(
2013) عمى
"أشجار الزيتون صنف "نبالي محسن. Iraq J Desert Studies • 2021 • Vol. 11 • Iss. 1 C/N
=
لمكاربوىيدرات المئوية النسبة
لمنايتروجين المئوية النسبة أ أما بالنسبة لمستخمص الطحالب البحرية كما في الجدول
10
نسبة
في
معنوية
فروق
ىناك
يكن
لم
الكاربوىيدرات/النتروجين في األوراق. أيضاً في الجدول9
لم تكن ىناك فروق معنوية في نسبة
الكاربوىيدرات/
النتروجين في األوراق عند التداخل الثنائي
بين ع
اممي الدراسة عمى شجرة الزيتون . يعزى سبب زيادة
النسبة مابين الكاربوىيدرات عمى النتروجين عند الرش بحامض البوري ك وذلك لدور البورون في زيادة الكموروفيل
حيث أدى إلى تكوين زيادة في المادة الجافة وبالتالي زيادة
نسبة الكاربوىيدرات والنتروجين ونرى زيادة نسبة
الكربوىيدرات عمى حساب نسبة النتروجين فتزيد نسبة
C/N
، كما وجدهAl-Rawi
و
Al-Ali
(
2013) عمى
"أشجار الزيتون صنف "نبالي محسن. 22 Iraq J Desert Studies • 2021 • Vol. 11 • Iss. 1 Iraq J Desert Studies • 2021 • Vol. 11 • Iss. 1 Al-Khafaji & Al-Ali, 2021 تأثير الرش الورقي بحامض البوريك ومستخمص
الطحالب البحرية والتداخل بينهما في نسبة البورون في
( األوراقppm
)
أظيرت المعطيات المدونة في الجدول11
أن الرش
الورقي بحامض البوريك قد أعطى زيادة في نسبة البورون
في األوراق إذ تفوقت المعاممةB2
تفوقاً معنوياً عمى بقية
المعامالت حيث بمغت قيمتيا82.583
ppm
تمتيا
المعاممةB1
بمغت53.417
ppm
والتي بدروىا تفوقت
معنوياً عمى المعاممةB0
والتي سجمت أقل نسبة لمبورون
بمغت
األوراق
في16.500
ppm
. جدول11
. تأثير الرش الورقي بحامض البوريك ومستخمص الطحالب البحرية في نسبة البورون في األوراق في شجرة
( الزيتون صنف شمالليppm
). معاملة البىرون
معاملة مستخلص
الطحالب البحزية
B0
B1
B2
م
تىسط
مستخلص
الطحالب البحزية
S0
7=6222
:?6<<=
>26999
:?6222
S1
7=6222
;96999
=?6<<=
;26222
S2
7:6<<=
;:6999
>96222
;26<<=
S3
7=6999
;<6999
>=6999
;96<<=
م
تىسط البىرون
7<6;22
;96:7=
>86;>9
L.S.D
5 %
البىرون
مستخلص
الطحالب البحزية التذاخل
76=<:
8629=
96;8> تأثير الرش الورقي بحامض البوريك ومستخمص
الطحالب البحرية والتداخل بينهما في نسبة البورون في
( األوراقppm
)
أظيرت المعطيات المدونة في الجدول11
أن الرش
الورقي بحامض البوريك قد أعطى زيادة في نسبة البورون
في األوراق إذ تفوقت المعاممةB2
تفوقاً معنوياً عمى بقية
المعامالت حيث بمغت قيمتيا82.583
ppm
تمتيا
المعاممةB1
بمغت53.417
ppm
والتي بدروىا تفوقت
معنوياً عمى المعاممةB0
والتي سجمت أقل نسبة لمبورون
بمغت
األوراق
في16.500
ppm
. جدول11
. تأثير الرش الورقي بحامض البوريك ومستخمص الطحالب البحرية في نسبة البورون في األوراق في شجرة
( الزيتون صنف شمالليppm
). REFERENCES REFERENCES
A.O.A.C. 1980. Official Methods of Analysis. 13th
Ed. Association
of
Official
Analytical
Chemists. Washington, D.C6
Abd-El Migeed M.M.M., M.A.A. El-Naggar, A.A. Afifi and M.A. El- Shawadfy. 2018. Response
of olive trees (cv. Koroneiki) to algae extract
sprays and its impact on growth and
productivity under saline conditions. Middle
East Journal of Agriculture Research, 7(1):
34-40 . Ahmed, M.M.A, A. Ali, H. Ali and E.M. Elbakry. 2019. Effect of Spraying Seaweed Extract on
Fruiting of Sakkoti Date Palms. Stem Cell,
10(2):127-132. REFERENCES
A.O.A.C. 1980. Official Methods of Analysis. 13th
Ed. Association
of
Official
Analytical
Chemists. Washington, D.C6 Abd-El Migeed M.M.M., M.A.A. El-Naggar, A.A. Afifi and M.A. El- Shawadfy. 2018. Response
of olive trees (cv. Koroneiki) to algae extract
sprays and its impact on growth and
productivity under saline conditions. Middle
East Journal of Agriculture Research, 7(1):
34-40 . Al-Ali, H.H. 2002. A study of the use of some
treatments in reducing the phenomenon of
alternating pregnancy in the two olive
varieties, Bashiqa and Manzanillo, Olea
europaea L. PhD thesis - College of
Agriculture - University of Baghdad - Iraq. (In
Arabic) Abd El-Moniem, E.A. and A.S.E. Abd-Allah. 2008. Effect of green algae cells extract as foliar
spray on vegetative growth, yield and berries
quality of superior grapevines. American-
Eurasian Journal of Agriculture and Environ. Sciences, 4 (4): 427-433. Al-Hadethi, M.E., U.Y. Salih,, S. H. J. Al-Hgemi,
and A.O. Janabi, 2019. Effect of Boron and
Gibberellins spray on leaves chemical content
in Olive trees . International Journal of
Agricultural and Statistical Sciences. Vol,
15(1): 237-241. Abdel-Maguid, A.A., A.B. El-Sayed and H.S. Hassan. 2004. Growth enhancement of olive
transplants by broken cells of fresh green
algae as soil application. Minufiya Journal of
Agriculture Research 29: 723-733. Al-Hawezy, S.M.N. 2014. The Use of Kelpak to
Seedlings Loquat (Eriobotya Jappanica L.). International
Journal
of
Scientific
and
Research Publications, 4: 85-886 Al-Muhammadi, S.M. and F.M. Al-Muhammadi. 2012. Statistics and experimental design. Osama House for Publishing and Distribution. Amman - Jordan P.O. p. 376. (In Arabic) Ahmed, F.F., H. A. A. Mohamed, and M.R. Jad El-
Kareem. 2014. The promotive effect of
seaweed extract in fruiting of Zaghloul date
palms
grown
under
Minia
region. 5
International Date Palm Conf., 16-18 Mar. Emirates Palace Abu Dhabi United Arab
Emirates6 Al-Rawi, A.M., H.H. Al-Ali. 2013. C/N
=
لمكاربوىيدرات المئوية النسبة
لمنايتروجين المئوية النسبة أ معاملة البىرون
معاملة مستخلص
الطحالب البحزية
B0
B1
B2
م
تىسط
مستخلص
الطحالب البحزية
S0
7=6222
:?6<<=
>26999
:?6222
S1
7=6222
;96999
=?6<<=
;26222
S2
7:6<<=
;:6999
>96222
;26<<=
S3
7=6999
;<6999
>=6999
;96<<=
م
تىسط البىرون
7<6;22
;96:7=
>86;>9
L.S.D
5 %
البىرون
مستخلص
الطحالب البحزية التذاخل
76=<:
8629=
96;8> العناصر من جية أ ّخرى، فعمى الرغم من أن البورون عد
( من العناصر قميمة الحركة خالل مدة طويمةDugger
،
1983
) إال أنو أثبتت حركية البورون عند كثير من
( أشجار الفاكية مثل التفاح والخوخ والكمثرىHanson
،
1991
( ) والزيتونDelgado
،وآخرون1994
) بناء عمى
نوعية السكر الناقل عبر المحاء و ًىذا يعد البورون عنصرا
متحركاً في األشجار التي تحتوي عمى السوربيتول أو
المانيتول أو الفركتوز كسكر أساسي يتنقل عبر المحاء
(
Hu
وBrown
،
1997
)،
المانيتول عبارة عن
( كاربوىيدرات نقل أولي في الزيتونDrossopoulos
و
Niavis
،
1988
) ، كما أن بعض األحماض كحامض
الماليك
( لو القدرة عمى حمل ىذا العنصرDembitsky
، وآخرون2002
) وىذا يفسر سبب زيادة تركيز عنصر
البــورون فــي األوراق. كمــا وجـدهHegazi
(
2018
) عـمى
أشجــار الـزيتــون صنـفFrantoio
ًوأيضـاKhudair
وAl-Mousawi
(
2014
) عمى أشجار الزيتون صنف
خستاوي
.وجدوا فروق معنوية عند الرش بالبورون أما فيما يخص مستخمص الطحالب
البحرية فيظير
الجدول
11
أن المعاممةS3
ًفقد سجمت تفوقاً معنوياً أيضا
عمى باقي المعامالت األخرى بنسبة البورون بمغت
53.667
ppm
ولم تختمف المعامالت األخرى الباقية
فروق معنوية فيما بينيا وسجمت المعاممةS0
أقل نسبة
لمبورون في األوراق بمغت49.000
ppm
. كما يظير الجدول11
نتائج التداخل بين عوامل
الدراسة في نسبة البورون في األوراق إذ تفوقت المعاممة
B2 S3
معنوياً عمى جميع المعامالت األخرى التي بمغت
أعمى قيمة ليا87.333
ppm
وعمى العكس من ذلك
أظيرت المعاممةB0 S2
أقل نسبة لمبورون في األوراق
بمغت14.667
ppm
. في ما يخص محتوى األوراق من عنصر البورون بينت
بعض الدراسات إزدياد محتوى أوراق النبات من العناصر
المعدنية الكبرى والصغرى عند الرش الورقي وأرتبط ذلك
بزيادة محتواىا من السكريات ، وربطHan
وآخرون
(
2008
) ىذه الزيادة بزيادة الوزن الجاف من جية وبحركة 23 Iraq J Desert Studies • 2021 • Vol. 11 • Iss. 1 Al-Khafaji & Al-Ali, 2021 إن ىذه المستخمصات تزيد من كفاءة إمتصاص
المغذيات ومحتوى األوراق من الكموروفيل فضالً عن زيادة
( نشاط عمميتي التركيب الضوئي والتنفسSantana
،وآخرون2006
) وبالتالي زيادة إحتياج األوراق من
العناصر الغذائية وبا لتالي زيادة إمتصاص عنصر
البورون. Iraq J Desert Studies • 2021 • Vol. 11 • Iss. 1 اإلستنتاج
ات ج
لإ
أثّـر الرش الورقي بحامض البوريك ومستخمص
الطحالب البحرية إيجابياً في النمو الخضري حيث أظيرت
زيادة في
أ ا طو
ل األ فرع وعدد األوراق ومحتوى الكموروفيل
الكمي ونسبة المادة الجافة في األوراق ونسبة الكاربوىيدرات
ونسبة ال نتروجين
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Rapidly fatal metastatic melanoma arising from a congenital nevus in a young female
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Nasza Dermatologia Online
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How to cite this article: Kassel J, Elloudi S, Soussy K, Chhiti S, Baybay H, Douhi Z, Bouhafa T, Mernissi FZ. Rapidly fatal metastatic melanoma arising from a
congenital nevus in a young female. Our Dermatol Online. 2023;14(1):119-120. Case Letter Case Letter Sir, Melanoma is an aggressive and potentially fatal tumor
of melanocytic origin. It may occur at any age yet more
rarely at a young age [1]. Melanomas in young patients
have overall a more favorable prognosis than in older. However, progression to the metastatic stage and the
death of the patient are not exceptional [2]. One of the
major risk factors for the development of melanoma in
children and young adults is the congenital melanocytic
nevus (CMN) [3]. The risk of malignant transformation
of all congenital nevi ranges from 0.05% to 10.7%. The
risk of malignant degeneration is correlated with size
and location [4]. The size of the CMN above 40 cm as
well as the presence of satellite nevi and the location in
the trunk seem to increase the risk of developing MM. The role of surgical removal in inducing melanomas
is controversial. In anatomopathology, melanomas
arising from CMNs are usually located in the dermis
and hypodermis, while melanocytic proliferation in a
melanoma without a CMN starts in the epidermis [3]. Given the differences in the anatomical involvement
of the disease, melanoma arising from congenital
nevi may be considered a separate entity from the
conventional case of melanoma and management may
differ. Large excision may not be sufficient to remove all
neoplastic cells from the nevi, and adjuvant aggressive
systemic therapies may be essential to avoid a fatal
outcome [4]. A recent study revealed that congenital
nevi preferentially harbor NRAS mutations rather than
BRAF mutations commonly seen in other types of nevi,
indicating an altered molecular basis of nevogenesis
in congenital nevi [3]. Herein, we report the case of a Melanoma is an aggressive and potentially fatal tumor
of melanocytic origin. It may occur at any age yet more
rarely at a young age [1]. Melanomas in young patients
have overall a more favorable prognosis than in older. However, progression to the metastatic stage and the
death of the patient are not exceptional [2]. One of the
major risk factors for the development of melanoma in
children and young adults is the congenital melanocytic
nevus (CMN) [3]. The risk of malignant transformation
of all congenital nevi ranges from 0.05% to 10.7%. The
risk of malignant degeneration is correlated with size
and location [4]. Jihad Kassel1, Sara Elloudi1, Kaoutar Soussy2, Soukaina Chhiti1, Hanane Baybay1,
Zakia Douhi1, Touria Bouhafa2, Fatima-Zahra Mernissi1 1Department of Dermatology, University Hospital Hassan II, Fes, Morocco, 2Radiation oncology department, Oncology
hospital, University Hospital Hassan II, Fes, Morocco Corresponding author: Jihad Kassel, MD, E-mail: kassel.jihad@gmail.com rapidly fatal metastatic melanoma in a young female
arising from a congenital nevus of the trunk. Rapidly fatal metastatic melanoma arising from a
Rapidly fatal metastatic melanoma arising from a
congenital nevus in a young female
congenital nevus in a young female Jihad Kassel1, Sara Elloudi1, Kaoutar Soussy2, Soukaina Chhiti1, Hanane Baybay1,
Zakia Douhi1, Touria Bouhafa2, Fatima-Zahra Mernissi1 How to cite this article: Kassel J, Elloudi S, Soussy K, Chhiti S, Baybay H, Douhi Z, Bouhafa T, Mernissi FZ. Rapidly fatal metastatic melanoma arising from a
congenital nevus in a young female. Our Dermatol Online. 2023;14(1):119-120.
Submission: 06.01.2022; Acceptance: 03.03.2022 Submission: 06.01.2022; Acceptance: 03.03.2022
DOI: 10.7241/ourd.20231.33 Consent The examination of the patient was conducted according to the
principles of the Declaration of Helsinki. The authors certify that they have obtained all appropriate patient
consent forms, in which the patients gave their consent for images
and other clinical information to be included in the journal. The
patients understand that their names and initials will not be
published and due effort will be made to conceal their identity,
but that anonymity cannot be guaranteed. a Sir, A total body CT scan was
performed showing metastases of the brain, lungs, soft
tissue, pancreas, and bone with peritoneal calcinosis. The patient was transferred to the oncology and
radiotherapy department and deceased two weeks later. © Our Dermatol Online 1.2023 Sir, The size of the CMN above 40 cm as
well as the presence of satellite nevi and the location in
the trunk seem to increase the risk of developing MM. The role of surgical removal in inducing melanomas
is controversial. In anatomopathology, melanomas
arising from CMNs are usually located in the dermis
and hypodermis, while melanocytic proliferation in a
melanoma without a CMN starts in the epidermis [3]. Given the differences in the anatomical involvement
of the disease, melanoma arising from congenital
nevi may be considered a separate entity from the
conventional case of melanoma and management may
differ. Large excision may not be sufficient to remove all
neoplastic cells from the nevi, and adjuvant aggressive
systemic therapies may be essential to avoid a fatal
outcome [4]. A recent study revealed that congenital
nevi preferentially harbor NRAS mutations rather than
BRAF mutations commonly seen in other types of nevi,
indicating an altered molecular basis of nevogenesis
in congenital nevi [3]. Herein, we report the case of a A young female 24 years of age presented with a
pigmented, congenital, 5 cm lesion of the abdomen. The patient underwent surgical excision of the
lesion without histological assessment. A painful
angiomatous nodule appeared over the existing lesion
evolving for the last year (Fig. 1). The evolution was
then marked three months later by the appearance of
numerous erythematous and angiomatous cutaneous
and subcutaneous nodules disseminated over the
entire body (Figs. 2a and 2b). We also noted inguinal
and axillary bilateral lymph nodes associated with
asthenia, dyspnea, headaches, and dizziness. A biopsy
of the angiomatous nodule adjacent to the nevus Figure 1: The pigmented angiomatous nodule above an asymmetric
pigmented macule in the abdominal area. Figure 1: The pigmented angiomatous nodule above an asymmetric
pigmented macule in the abdominal area. Submission: 06.01.2022; Acceptance: 03.03.2022
DOI: 10.7241/ourd.20231.33 Submission: 06.01.2022; Acceptance: 03.03.2022
DOI: 10.7241/ourd.20231.33 119 © Our Dermatol Online 1.2023 www.ode
Figure 2: (a) Multiple subcutaneous nodules on the back. (b) The
angiomatous nodule at the abdominal level. b
a www.odermatol.com HMB45 and Melan A. A total body CT scan was
performed showing metastases of the brain, lungs, soft
tissue, pancreas, and bone with peritoneal calcinosis. The patient was transferred to the oncology and
radiotherapy department and deceased two weeks later. HMB45 and Melan A. REFERENCES 1. Bebe FN, Hu S, Brown TL, Tulp OL. Metastatic melanoma in Florida,
1996-2010: Racial, demographic, occupational and tumor characteristics,
and burden of metastasis. Our Dermatol Online. 2018;9:369-79. 2. Limam SAM, Erebih CE, Beyrouk A, Boye KI, Didi EH, Ely SO, et
al. [Acral melanoma of the foot: A study of 9 cases and guidelines
update]. Our Dermatol Online. 2019;10:23-9. 2. Limam SAM, Erebih CE, Beyrouk A, Boye KI, Didi EH, Ely SO, et
al. [Acral melanoma of the foot: A study of 9 cases and guidelines
update]. Our Dermatol Online. 2019;10:23-9. 3. Lacoste C, Avril MF, Frassati-Biaggi A, Dupin N, Chrétien-Marquet B,
Mahé E, et al. Malignant melanoma arising in patients with a large
congenital melanocytic naevus: Retrospective study of 10 cases with
cytogenetic analysis. Acta Derm Venereol. 2015;95:686-90. 3. Lacoste C, Avril MF, Frassati-Biaggi A, Dupin N, Chrétien-Marquet B,
Mahé E, et al. Malignant melanoma arising in patients with a large
congenital melanocytic naevus: Retrospective study of 10 cases with
cytogenetic analysis. Acta Derm Venereol. 2015;95:686-90. b 4. Wei CH, Shoo BA, Zedek DC, Kashani-Sabet M, Sagebiel RW,
Leong SP. Rapidly lethal metastatic melanoma arising
from a large congenital melanocytic naevus. BMJ Case
Rep. 2009;2009:bcr09.2008.0981. 4. Wei CH, Shoo BA, Zedek DC, Kashani-Sabet M, Sagebiel RW,
Leong SP. Rapidly lethal metastatic melanoma arising
from a large congenital melanocytic naevus. BMJ Case
Rep. 2009;2009:bcr09.2008.0981. Figure 2: (a) Multiple subcutaneous nodules on the back. (b) The
angiomatous nodule at the abdominal level. Copyright by Jihad Kassel, 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. was performed and showed a predominant dermal
proliferation with some intraepidermal nests of
atypical melanocytic cells with foci of tumor necrosis
and ulceration. IHC showed a positive marking of Source of Support: Nil, Confl ict of Interest: None declared. 120 © Our Dermatol Online 1.2023
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Ability of Different Measures of Adiposity to Identify High Metabolic Risk in Adolescents
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Journal of obesity
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cc-by
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Hindawi Publishing Corporation
Journal of Obesity
Volume 2011, Article ID 578106, 5 pages
doi:10.1155/2011/578106
Research Article
Ability of Different Measures of Adiposity to Identify High
Metabolic Risk in Adolescents
Carla Moreira,1 Rute Santos,1 Susana Vale,1 Paula C. Santos,1, 2 Sandra Abreu,1
Ana I. Marques,1 Luı́sa Soares-Miranda,1 and Jorge Mota1
1 Research
Centre in Physical Activity, Health and Leisure, Faculty of Sport, University of Porto, Rua Dr. Plácido Costa 91,
4200-450 Porto, Portugal
2 Department of Physiotherapy, School of Health Technology of Porto, IPP, Portugal
Correspondence should be addressed to Carla Moreira, carla m moreira@sapo.pt
Received 21 March 2011; Revised 13 May 2011; Accepted 16 May 2011
Academic Editor: Gianluca Iacobellis
Copyright © 2011 Carla Moreira et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Introduction. This study aimed to evaluate the screening performance of different measures of adiposity: body mass index (BMI),
waist circumference (WC), and waist-to-height ratio (WHtR) for high metabolic risk in a sample of adolescents. Methods. A crosssectional school-based study was conducted on 517 adolescents aged 15–18, from the Azorean Islands, Portugal. We measured
fasting glucose, insulin, total cholesterol (TC), HDL-cholesterol, triglycerides, and systolic blood pressure. HOMA and TC/HDLC ratio were calculated. For each of these variables, a Z-score was computed by age and sex. A metabolic risk score (MRS) was
constructed by summing the Z-scores of all individual risk factors. High risk was considered when the individual had ≥1SD of
this score. Receiver-operating characteristics (ROC) were used. Results. Linear regression analyses showed that, after adjusting for
age and pubertal stage, all different measures of adiposity are positively and significantly associated with MRS in both sexes, with
exception of WHtR for boys. BMI, WC, and WHtR performed well in detecting high MRS, indicated by areas under the curve
(AUC), with slightly greater AUC for BMI than for WC and WHtR in both sexes. Conclusion. All measures of adiposity were
significantly associated with metabolic risk factors in a sample of Portuguese adolescents.
1. Introduction
The prevalence of overweight and obesity is high in most
parts of the world, and this is particularly alarming not only
for the increasing risk of multiple comorbidities [1], but also
due to the tendency of childhood overweight and obesity to
track into adulthood [2].
The metabolic syndrome (MetS), a cluster of several
cardiovascular disease risk factors, is a complex entity of
metabolic disorders that significantly increases the risk of
type II diabetes and cardiovascular disease beyond that of its
individual components [3]. The emergence of MetS parallels
the rising rates of overweight and obesity observed in youth
worldwide [4].
A variety of anthropometric indices have been used
as a proxy for total and abdominal fat to assess risk for
diseases, particularly cardiovascular diseases (CVD) and
diabetes [5]. Epidemiologic studies have shown that body
mass index (BMI), the most widely recognised measure of
obesity, is a powerful predictor of CVD [6]. Nevertheless,
waist circumference (WC) and waist-to-height ratio (WHtR)
measures of central obesity have yielded important insight as
well and also provide information on the risk of CVD [7].
Although different anthropometrical measures of obesity
have been proposed, it remains unclear which measures of
adiposity best predict the role of metabolic risk factors. The
aim of this study was to determine the ability of different
measures of adiposity, namely, BMI, WC, and WHtR to
discriminate between low/high metabolic risk using receiveroperating characteristic (ROC) curves in a sample of Portuguese adolescents from the Azorean Archipelago.
2. Methods
2.1. Study Design and Sampling. Data for the present study
derived from a longitudinal school-based study, the Azorean
2
Physical Activity and Health Study II, aimed to evaluate physical activity, physical fitness, overweight/obesity prevalence,
health-related quality of life, and related factors. Details
on the study design and sampling strategy are reported
elsewhere [8]. The final sample included in this crosssectional analysis was comprised of 517 adolescents (297 girls
and 220 boys) aged 15 to 18.
All participants in this study were informed about the
objectives of the work, and the parent or guardian of each
participant provided written informed consent. The study
was approved by the faculty and the Portuguese Foundation
for Science and Technology ethics committee and conducted
in accordance with the Declaration of Helsinki for Human
Studies of the World Medical Association.
2.2. Anthropometric Measures. Height was measured to the
nearest millimeter in bare or stocking feet with the adolescent standing upright against a stadiometer (Holtain Ltd.,
Crymmych, Pembrokeshire, UK). Weight was measured to
the nearest 0.10 kg, with adolescents lightly dressed using a
portable electronic weight scale (Tanita Inner Scan BC 532).
BMI was calculated as body weight (kg) divided by body
height (m2 ). WC measurements were taken as described
by Lohman et al. [9]. The waist and height were used to
compute the WHtR.
2.3. Pubertal Stage. To determine the pubertal stage (ranging
from stage 1 to 5), each subject was asked to self-assess
his/her stage of secondary sex characteristics. Stages of breast
development in girls and genital development in boys were
evaluated according to the criteria of Tanner and Whitehouse
[10].
2.4. Blood Sampling. Blood samples were collected from the
antecubital vein between 8:00 and 10:00 a.m., in a sitting
position after ten hours of fasting. Blood samples were
drawn in vacuum tubes gel (Sarstedt) in order to obtain
values of plasmatic total cholesterol (TC), high-density
lipoprotein cholesterol (HDL-C), triglycerides (TG), glucose,
and insulin. The following analyses were measured on a
Cobas Integra 400 Plus (ROCHE Diagnostics, Indianapolis,
Ind, USA): TC, HDL-cholesterol, TG, and glucose. The
fasting serum insulin was measured on an Immulite 2000,
(Diagnostic Products Corporation, Los Angeles, Calif). The
ratio of total cholesterol to HDL-C was calculated as an
index of atherogenic lipid profile [11]. The homeostatic
model assessment (HOMA), calculated as the product of
basal glucose and insulin levels divided by 22.5, was used as
a proxy measure of insulin resistance [12]. The biochemical
evaluation of all participants from the different islands was
conducted in the same laboratory.
2.5. Blood Pressure. Blood pressure (BP) was measured using
the Dynamap adult/pediatric vital signs monitors, model
BP 8800 (Critikon, Inc., Tampa, Fla, USA). Measurements
were taken by trained nurses, and with all adolescents were
required to sit and rest for at least five minutes prior to the
BP test. The participants were in a seated, relaxed position
Journal of Obesity
with their feet resting flat on the ground. Two measurements
in the right arm were taken after five and ten minutes of
rest. The mean of these two measurements was considered. If
the two measurements differed by 2 mmHg or more, a third
measure was taken.
2.6. Metabolic Risk Score. Since there is no consensus
regarding the establishment of a universal criterion for
definition of the MetS in adolescents, we decided to compute
a continuous metabolic risk score (MRS) from the following
measurements: TC/HDL-C ratio, TG, HOMA, and systolic
BP. For each of these variables, a Z-score was computed by
age and sex. Then, an MRS was constructed by summing
the Z-scores of all individual risk factors. High risk was
considered when the individual had ≥1 SD of this score.
The score only applies to this study population. A similar
Z-score approach has been used previously in children and
adolescents [13].
2.7. Statistical Analysis. Comparisons between groups
involved Student t-test for continuous variables. Linear
regression analyses were used to study the relationship
between different measures of adiposity and low and
high MRS, adjusting for age and pubertal stage. Receiveroperating characteristic (ROC) curve analyses were used to
analyse the potential diagnostic accuracy of the different
measures of adiposity to discriminate between low and high
MRS. The area under the curve (AUC) and 95% confidence
interval (CI) were calculated. The AUC represents the ability
of the test to correctly classify adolescents having a low and
high MRS. The values of AUC range between 1 (perfect
test) and 0.5 (worthless test). Data were analyzed using the
PASW Statistic v.18 (SPSS, Chicago, Ill, USA) and Med Calc
software v.10.4.5 (MedCalc Software, Mariakerke, Belgium).
A P value under 0.05 denoted statistical significance.
3. Results
Descriptive characteristics of the adolescents are shown in
Table 1. Girls had lower height, weight, WC, systolic BP,
glucose, and TC/HDL-C levels and higher TC and HDL-C
than boys (P < 0.05 for all). All adolescents reported to be in
Tanner stage 4 or 5.
Linear regression analyses, adjusted for age and pubertal
stage, showed that BMI (girls: β = 0.291, 95% CI: 0.219–
0.364, P < 0.001; boys: β = 0.396, 95% CI: 0.324–0.468,
P < 0.001), WC (girls: β = 0.086, 95% CI: 0.060–0.111,
P < 0.001; boys: β = 0.121, 95% CI: 0.093–0.150, P < 0.001),
and WHtR (girls: β = 1.738, 95% CI: 0.555–2.922, P =
0.004; boys: β = 1.220, 95% CI: −0.209–2.648, P = 0.09)
were positively and significantly associated with MRS in both
sexes, with exception of WHtR for boys.
ROC curve analysis showed that all measures of adiposity
performed well on average in identifying high MRS, as
indicated by AUC greater than 0.7. The ROC performance
of BMI showed a better discriminatory accuracy than WC
and WHtR in predicting high MRS in both sexes. In boys,
the ROC performance of BMI was slightly better than in
Journal of Obesity
3
Table 1: Descriptive characteristics of the study sample.
Variables
Age, years
Height, cm
Weight, kg
BMI, kg/m2
Waist circumference, cm
Systolic BP, mmHg
Diastolic BP, mmHg
Total cholesterol, mg/dL
HDL-cholesterol, mg/dL
Triglycerides, mg/dL
Total cholesterol/HDL-C ratio
Fasting glucose, (mg/dL)
Fasting insulin, (uU/mL)
HOMA
Metabolic risk scorea
Total (n = 517)
16.5 ± 0.9
165.0 ± 13.6
63.1 ± 12.5
22.9 ± 3.7
79.3 ± 10.7
115.2 ± 15.3
66.4 ± 9.4
161.9 ± 32.3
55.6 ± 13.4
70.7 ± 35.1
3.0 ± 0.7
86.8 ± 9.2
9.1 ± 6.0
1.9 ± 1.3
0.1 ± 3.1
Girls (n = 297)
16.5 ± 1.0
160.0 ± 11.0
58.6 ± 10.0
22.7 ± 3.5
78.3 ± 10.3
111.8 ± 13.8
66.3 ± 10.0
169.3 ± 33.6
59.3 ± 13.0
72.5 ± 34.6
2.9 ± 0.8
84.6 ± 8.7
9.3 ± 5.1
1.9 ± 1.1
0 ± 3.1
Boys (n = 220)
16.4 ± 0.8
170.2 ± 1.0∗
69.3 ± 13.1∗
22.7±3.5
80.7 ± 11.1∗
120.0 ± 16.1∗
66.5 ± 8.6
151.8 ± 27.5∗
50.5 ± 12.3∗
68.1 ± 35.6
3.1 ± 0.7∗
89.7 ± 9.1∗
8.7 ± 7.0
1.9 ± 1.6
0.1 ± 3.2
Data are means ± standard deviations. ∗ P < 0.05 for sex comparisons (one-tailed t-test). BMI, body mass index; BP, blood pressure; HDL, high-density
lipoprotein; a obtained by summing individual risk factors (total cholesterol/HDL-C ratio, triglycerides, HOMA, and systolic blood pressure) age- and sexstandardized scores.
girls. The AUC of BMI were significantly different from WC
(P < 0.05) for the whole sample. In girls, the AUC of WC was
significantly different from WHtR (P < 0.05).
A BMI of 23.7 kg/m2 for girls and 27.0 kg/m2 for boys,
a WC of 83 cm for girls and 92 cm for boys, and a WHtR
of 0.55 for girls and 0.49 for boys were found to be optimal
cutoffs for defining high MRS in this adolescent population
(Table 2).
4. Discussion
The main findings of this study suggested that BMI provides
a marginally superior tool for discriminating high MRS
compared with WC and WHtR, for both sexes. Slightly
higher pooled AUC were observed in boys compared to
girls, suggesting that discrimination is more precise, on
average, in male. Linear regression analyses showed that
all different measures of adiposity were positively and
significantly associated with MRS in both sexes, with the
exception of WHtR for boys.
Jung et al. showed that BMI had the best predictive
power to identify metabolic syndrome, its components,
and markers for low-grade inflammation [14], which is in
agreement with our results. The BMI and WC are widely
used to define overweight and obesity across populations
[15]. The BMI has been used to predict body composition
and health risk [16], whereas WC indicates visceral adipose
tissue and can predict health risks in children [17]. Both BMI
and WC are simple measures to use and interpret, yet they
have some limitations. The BMI does not distinguish fat mass
from fat-free mass or between different body fat distributions
[18], and for WC, there are currently no agreements about
a health-related classification for children and adolescents.
Several previous studies considered the 90th percentile as a
cut-off point for high WC, whereas other studies consider
the 75th or 70th percentile as a cutoff point. Both of these
measures are also age and-sex dependent.
The WHtR has been significantly associated with
cardiovascular risk factors, due to abdominal obesity both in
adults and children [19]. The higher AUC value for WHtR
than for WC could be due to the fact that WHtR takes into
account differences in body height. Contrary to our findings,
some studies have shown that WHtR is better for classifying
obesity related to cardiovascular risk than BMI and WC
[5, 20]. In girls, the AUC value for WHtR is slightly better
than AUC value for WC. Despite the AUC value for BMI was
slightly higher than the other two measures in girls, the AUC
value for WHtR showed higher sensibility than AUC value
for BMI.
There is no agreement for which anthropometrical
measures of adiposity best predict a role of unfavorable
cardiovascular risk factors. Studies carried out among
children and adolescents in Cyprus [21] and Japan [5]
concluded that both WC and WHtR are better predictors of TC, TG, HDL-cholesterol, low-density lipoprotein
cholesterol, and systolic and diastolic BP levels than BMI.
Also, the Bogalusa study reported that WC and WHR
were related to adverse levels of TG and HDL-cholesterol,
independently of race, sex, age, weight, and height [22].
Conversely, BMI is the measure of obesity most used and
has been shown to be extremely effective when used in
longitudinal studies [7].
Nevertheless, we were unable to draw cause-effect
conclusions and to make observations over the time
because of the cross-sectional nature of our data and
the multifactorial etiology of high MRS. However, the
association between these measures of adiposity and the
clustering of metabolic risk factors has not been studied
in this population, making it one of the strengths of this
4
Journal of Obesity
Table 2: Cut-off values, sensitivity, and specificity for the association of different measures of adiposity with metabolic risk score by sex.
BMI
BMI cut-off (kg/m2 )
Sensitivity (%)
Specificity (%)
AUC
WC
WC cut-off (cm)
Sensitivity (%)
Specificity (%)
AUC
WHtR
WHtR cut-off
Sensitivity (%)
Specificity (%)
AUC
All
Girls
Boys
> 23.6
79.4
72.8
0.807 (0.770–0.840)
P < 0.001‡
> 23.7
76.3
71.8
0.772 (0.720–0.818)
P < 0.001
> 27.0
66.7
94.7
0.852 (0.798–0.896)
P < 0.001
> 84
66.2
79.7
0.760 (0.721–0.796)
P < 0.001
> 83
65.8
75.3
0.714 (0.659–0.764)
P < 0.001†
> 92
60.0
94.7
0.827 (0.770–0.874),
P < 0.001
> 0.54
61.8
89.5
0.794 (0.756–0.828)
P < 0.001
> 0.55
60.5
90.0
0.767 (0.715–0.814)
P < 0.001
> 0.49
80.0
80.0
0.834 (0.778– 0.881),
P < 0.001
AUC: area under the curve; 95% CI in parentheses; ‡ AUC significantly different from WC (P < 0.05); † AUC significantly different from WHtR (P < 0.05).
study. Further studies are needed to confirm or contrast our
findings.
[4]
5. Conclusion
In conclusion, BMI, WC, and WHtR are all predictors of high
MRS. Despite the small differences in the discriminatory
capabilities among measures of adiposity, making it difficult
to recommend the best measure of obesity, BMI seems to
have the best trade-off between sensitivity and specificity to
screen for high MRS in both sexes.
[5]
[6]
[7]
Conflict of Interests
All the authors declare not to have any conflict of interests.
Acknowledgments
This study was supported by FCT-MCTES Grants nos.
BD/44422/2008, BPD/65180/2009, BSAB/1025/2010, and
PTDC/DES/098309/2008 and by Azorean Government.
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Accelerated Exploration for Long-Term Urban Water Infrastructure Planning through Machine Learning
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Received: 15 October 2018; Accepted: 1 December 2018; Published: 5 December 2018 Abstract: In this study, the neural network method (Multi-Layer Perceptron, MLP) was integrated
with an explorative model, to study the feasibility of using machine learning to reduce the exploration
time but providing the same support in long-term water system adaptation planning. The specific
network structure and training pattern were determined through a comprehensive statistical
trial-and-error (considering the distribution of errors). The network was applied to the case study in
Scotchman’s Creek, Melbourne. The network was trained with the first 10% of the exploration data,
validated with the following 5% and tested on the rest. The overall root-mean-square-error between
the entire observed data and the predicted data is 10.5722, slightly higher than the validation result
(9.7961), suggesting that the proposed trial-and-error method is reliable. The designed MLP showed
good performance dealing with spatial randomness from decentralized strategies. The adoption
of MLP-supported planning may overestimate the performance of candidate urban water systems. By adopting the safety coefficient, a multiplicator or exponent calculated by observed data and
predicted data in the validation process, the overestimation problem can be controlled in an acceptable
range and have few impacts on final decision making. Keywords: urban planning; water infrastructure; adaptation planning; artificial neural network;
multi-layer perception Sustainability 2018, 10, 4600; doi:10.3390/su10124600 Article
Accelerated Exploration for Long-Term Urban Water
Infrastructure Planning through Machine Learning Junyu Zhang 1,2, Dafang Fu 1,2,*, Christian Urich 1,3 and Rajendra Prasad Singh 1,2 1
Joint Research Centre for Water Sensitive Cities, Southeast University-Monash University Joint Gr
School (Suzhou), Southeast University, Suzhou 215123, China; junyu.zhang@monash.edu (J.Z.);
christian.urich@monash.edu (C.U.); rajupsc@seu.edu.cn (R.P.S.)
2
Department of Civil Engineering, Southeast University, #2Sipailou, Nanjing 210096, China
3
Department of Civil Engineering, Monash University, Clayton, VIC 3800, Australia
*
Correspondence: fdf@seu.edu.cn; Tel.: +86-185-5182-4285 1
Joint Research Centre for Water Sensitive Cities, Southeast University-Monash University Joint Graduate
School (Suzhou), Southeast University, Suzhou 215123, China; junyu.zhang@monash.edu (J.Z.);
christian.urich@monash.edu (C.U.); rajupsc@seu.edu.cn (R.P.S.)
2
Department of Civil Engineering, Southeast University, #2Sipailou, Nanjing 210096, China
3
Department of Civil Engineering, Monash University, Clayton, VIC 3800, Australia
*
Correspondence: fdf@seu.edu.cn; Tel.: +86-185-5182-4285 sustainability sustainability sustainability sustainability 1. Introduction Long-term strategic planning on urban infrastructures is often obsessed with future uncertainties
such as the state of the world (e.g., economic situation, climate) or state of the city (e.g., population
growth). These uncertainties are not statistical in nature which makes them hard to predict. One of the
most convincing examples is the “Shrinking City” event in Dresden since 1990, where 7 predictions
have been made during 15 years to predict the population growth and guide the city planning but
none of them turned out to be right [1,2]. To deal with this issue, computational tools have been developed to look into more future scenarios
and offer more reliable plans, such as Adaptation tipping points [3], Robust decision making [4],
Info-gap [5]. The adaptation tipping points offered shifting between different strategies and plans
but no guarantee of success adaptation due to lack of system performance evaluation. The robust
decision-making and info-gap both aim to explore as much future as possible and evaluate the
robustness of candidate plans by trade-off on the target. As an improvement exploring planning tools have been developed to model the performance
of different infrastructure plans under different scenarios, such as Adaptive policy making [6], Sustainability 2018, 10, 4600; doi:10.3390/su10124600 www.mdpi.com/journal/sustainability 2 of 16 Sustainability 2018, 10, 4600 Adaptation pathways [7] and Dynamic adaptive policy making [8]. The adaptation pathways are able
to simulate the dynamic of different infrastructure and the adaptation among them under relatively
small range of future scenarios. Meanwhile, the adaptive policy making looks into wide range of future
scenarios without lack of infrastructure adaptation. As the improvement of them the dynamic adaptive
policy making tries to consider both but could only work out plans for independent strategies. The limitation of the current tools is they are not able to evaluate the adaptation of a real-world
combined system (centralized + decentralized) as such simulation is excessively time-consuming. More precisely, one of the major challenges on reducing the time consumption in such exploration
planning tools is the robustness problem. The more detailed designs to be modelled (especially spatial
distributed decentralized systems) and the more scenarios to be considered, the more time it will take,
the more robust the plan can be. Unfortunately, there are only few methods or tools that could reduce the exploration time while
maintain the exploration range. 1. Introduction This problem is being addressed in this paper by integrating the
neural network method (multi-layer perceptron) with an explorative model that simulates possible
urban infrastructure adaptation, to study the feasibility of using machine learning to reduce the
computational time in such exploration. In recent years, Artificial Neural Networks (ANNs), as a data-drive, self-adaptive and
non-linear forecasting tool was applied in various fields such as natural resource management [9–11],
pattern recognition [12,13], medical diagnosis [14] and decision making [15,16]. As a matter of factor,
the methods and its derivative tool are often used in short-term decision makings or predictions
(event scale) rather than long-term planning (strategy scale). To cope with the exploration model,
the machine learning algorithm was designed and trained to predict urban water infrastructure
performance for individual events while the decision on planning was made based on microscopic
strategy performance distribution. In this paper, the above accelerated explorative long-term planning method was proposed and
tested. The following works have been conducted: (1) a comprehensive statistical trial-and-error
analysis method is proposed and tested to avoid local optimization of network structure. (2) a neural
network was integrated in the explorative adaptation planning to significantly reduce the simulation
time, performance was tested and analyzed; (3) a correction method was proposed and tested to
minimize the overestimation problem of the designed exploration framework. 2.1. Site Description and the Exploration With the initial city scenario established based on the
real-world catchment in 2015, DAnCE4Water ran in a 5-year interval to simulate the transformation of
the city and assess the urban water system performance with different drainage infrastructure updates
under all possible development scenarios. Sustainability 2019, 11 FOR PEER REVIEW
3
uptake ratio and drainage pipe system upsizing. With the initial city scenario established based on
the real-world catchment in 2015, DAnCE4Water ran in a 5-year interval to simulate the
transformation of the city and assess the urban water system performance with different drainage
infrastructure updates under all possible development scenarios The development scenario consists of two parameters: the population growth rate (PGR) and the
climate change factor (CCF). The 5-year population growth rate is ranged in [0.03,0.06] which calculated
based on the maximum annual growth rate (0.012 per year) in the area according to the 1990–2015
census data from the Australian Bureau of Statistics. DAnCE4Water would replace old buildings and
construct new ones according to the increased population through its urban development module
(UDM) [17,18]. The 5-year climate change factor is a coefficient used to magnify the 5-year designed
storm. Initialized to 1.00, CCF is assumed to change every 5 years within three rates: 0.95X, 1.00X
or 1.05X. infrastructure updates under all possible development scenarios. The development scenario consists of two parameters: the population growth rate (PGR) and
the climate change factor (CCF). The 5-year population growth rate is ranged in [0.03,0.06] which
calculated based on the maximum annual growth rate (0.012 per year) in the area according to the
1990–2015 census data from the Australian Bureau of Statistics. DAnCE4Water would replace old
buildings and construct new ones according to the increased population through its urban
development module (UDM) [17, 18]. The 5-year climate change factor is a coefficient used to magnify
the 5-year designed storm. Initialized to 1.00, CCF is assumed to change every 5 years within three
rates: 0 95X 1 00X or 1 05X Three drainage update options were tested in this paper: (1) business as usual, (2) uptake rainwater
harvesting tanks and (3) upsize drainage pipes. “Business as usual (BAU)” maintained the existing
infrastructures from the previous step. The more BAU was taken, the less contribution would be done
in reducing flooded junctions. “Uptake rainwater harvesting tank (RWHT)” increased the current
probability of households installing rainwater harvesting tanks by 5%. 2.1. Site Description and the Exploration The case was carried out in Scotchman’s Creek catchment, locates at the southeast of Melbourne
CBD. The catchment is mostly located within Monash City council but a part of the catchment (6%) is
situated within Whitehorse City council. It has an area of approximately 10.36 km2 and a population
of approximately 25,000 residents. The council started to introduce rainwater tanks to households since 2005 to deal with the
unpredictable rainfall events (e.g., reduce peak flow during highly intensive rainfall event, store rain
water during drought season). Although the council tried to set up a progressive goal of rainwater tank
uptake rate in the area, there were several obstacles in making such a plan: (1) The spatial distribution
of rainwater tanks will largely influence the flood resistance in the catchment resulting from them. Thus, the promoting of higher rainwater tank uptake rate cannot be easily determined compared to
upsizing pipe systems; (2) The population growth in the area could infect the construction of houses
and buildings which increases the impervious surfaces in the catchment as well as the opportunity
for uptake rainwater tanks; (3) The flood-resistance robustness of the combined drainage system
(under different rainwater tank uptake ratio and pipe system capacity) was unclear. Thus, a long-term (2015–2035) evolution of the urban development, climate change and water
infrastructure adaptation were simulated by DAnCE4Water (Dynamic Adaptation for enabling City Sustainability 2018, 10, 4600 3 of 16 Evolution for Water) [17,18] to set up a robust plan of progressive goals for both rainwater take
uptake ratio and drainage pipe system upsizing. With the initial city scenario established based on the
real-world catchment in 2015, DAnCE4Water ran in a 5-year interval to simulate the transformation of
the city and assess the urban water system performance with different drainage infrastructure updates
under all possible development scenarios. Sustainability 2019, 11 FOR PEER REVIEW
3
uptake ratio and drainage pipe system upsizing. With the initial city scenario established based on
the real-world catchment in 2015, DAnCE4Water ran in a 5-year interval to simulate the
transformation of the city and assess the urban water system performance with different drainage
infrastructure updates under all possible development scenarios. Evolution for Water) [17,18] to set up a robust plan of progressive goals for both rainwater take
uptake ratio and drainage pipe system upsizing. 2.1. Site Description and the Exploration The more RWHT was taken,
the more decentralized systems would be built to reduce the runoff and peak flow. “Upsize drainage
system (PIPE)” upgrades the drainage network, which was divided into 4 groups according to their
diameters. Each upgrade enlarged one group of pipes, from the large one to the small one. The more
PIPE was taken, the higher capacity of the drainage network would be. rates: 0.95X, 1.00X or 1.05X. Three drainage update options were tested in this paper: (1) business as usual, (2) uptake
rainwater harvesting tanks and (3) upsize drainage pipes. “Business as usual (BAU)” maintained the
existing infrastructures from the previous step. The more BAU was taken, the less contribution would
be done in reducing flooded junctions. “Uptake rainwater harvesting tank (RWHT)” increased the
current probability of households installing rainwater harvesting tanks by 5%. The more RWHT was
taken, the more decentralized systems would be built to reduce the runoff and peak flow. “Upsize
drainage system (PIPE)” upgrades the drainage network, which was divided into 4 groups according
to their diameters. Each upgrade enlarged one group of pipes, from the large one to the small one. The more PIPE was taken, the higher capacity of the drainage network would be. The exploration randomly selected a PGR, a CCF and a drainage infrastructure update within the
available range and applied to the base city scenario. The UDM would then generate a future scenario
of the city while the performance of the combined system (the number of flooded junctions in the
catchment area along the drainage network) would be evaluated by SWMM. The result city scenario
was saved as the base city scenario for the next 5-year decision (see Figure 1). The more PIPE was taken, the higher capacity of the drainage network would be. The exploration randomly selected a PGR, a CCF and a drainage infrastructure update within
the available range and applied to the base city scenario. The UDM would then generate a future
scenario of the city while the performance of the combined system (the number of flooded junctions
in the catchment area along the drainage network) would be evaluated by SWMM. The result city
scenario was saved as the base city scenario for the next 5-year decision (see Figure 1). Figure 1. Designed exploration of the Scotchman’s Creek catchment area. Figure 1. Designed exploration of the Scotchman’s Creek catchment area. Figure 1. 2.1. Site Description and the Exploration Designed exploration of the Scotchman’s Creek catchment area
Figure 1. Designed exploration of the Scotchman’s Creek catchment area. Figure 1. Designed exploration of the Scotchman’s Creek catchment area. Figure 1. Designed exploration of the Scotchman’s Creek catchment area. The result scenarios were classified by the drainage infrastructure status (e.g., how many steps
of BAU, RWHT and PIPE were adopted respectively). The corresponding distribution of system
f
(fl
d d j
ti
) f
h t t
l
l t d A
l
t
t
t k
i
The result scenarios were classified by the drainage infrastructure status (e.g., how many steps
of BAU, RWHT and PIPE were adopted respectively). The corresponding distribution of system 4 of 16 Sustainability 2018, 10, 4600 performance (flooded junctions) for each status was calculated. As only one strategy was taken in each
decision step, the status contains the year information as well. If the number of flooded junctions of a
status was below the target (110 in 2020, 100 in 2025, 90 in 2030 and 80 in 2035, which is 100%, 91%, 82%,
73% of the flooded junctions in 2015) in over 95% of the cases, the status would be consider “robust.”
The “robust” statuses were connected in a time line to form a drainage infrastructure implementation
pathway as the long-term plan in this case study. To compare the proposed acceleration exploration method, the plan was first explored through
the above traditional exploration. The 20-year planning took 2.93 million simulations including
1.73 million explorations with uniformed input values and 1.2 million with random input values for
the last two decision steps. The uniformed input values were listed in Figure 1, with 36 scenarios in
2020 (4 PGRs * 3 CCFs * 3 add-on strategies), 362 in 2025, 363 in 2030 and 364 in 2035. The random
explorations selected result scenarios in 2025 and 2030, PGR and CCF within range of [1.03,1.06] and
[0.95,1.05]. The whole exploration took 1 year and 4 months with 32 instances in the DAnCE4Water
cloud server while the result was saved in a SQLite database containing the input values and output
values for every simulation. 2.2. The Accelerated Exploration and ANN Design The proposed accelerated exploration started with a normal exploration and paused when a
certain amount of simulation had been finished. These simulations would be used as the training set
to train an ANN while the exploration continued. The exploration then stopped when another certain
amount simulation had been finished. These extra simulations would be used for validation. The ANN
would be trained with different structures and settings and tested on the validation simulations. The errors of the validation would be used to choose the best structure and setting, and the ANN
would do the rest of exploration by predicting with the scheduled PGR, CCF and add-on strategies (as
the normal exploration) but skipping the UDM and SWMM process. The results in the reference exploration (the scenarios as well as the evaluated system performance)
were classified into three sets: the training set (size: 0.1%, 1% or 10%), the validation set (size: 10%)
and the test set (size: the remaining data). The training set was used to train the network (e.g., weights) while the validation set was for
adjusting the structure of the network (e.g., number of nodes) [4]. The test set was used to assess the
performance of a trained and validated network. In most literature [14,19–25], as the network structure
are usually pre-defined or tested by trial-and-error, the validation sets are usually disused or replaced
by the test sets. Under such substitution, the performance of the network is only meaningful for certain
sets (the ‘test sets’), which have been optimized during the training, rather than for the untrained data
which we expect more precise predictions. 2.2.2. The Structure of MLP Network
between this node and the jth node in
h l
f
b
h
f The MLP usually consists of nodes(units) arranged in three types of layer: the input layer,
the hidden layer(s) and the output layer. As Figure 2 shows, each node (unit) has its own output value
y and is connected by real-valued weights w to all (and only) the nodes of the subsequent layer. For the
ith node in the lth layer nil, let Sil be the set of nodes that connect to nil, f(x) be the activation function
of nil, the output value is calculated using Formula (1):
mth layer; f(x) be the activation function of this node. The input layer receives the input data while the output of output layer refers to the predicted
results. Thus, both only requires only 1 layer to fulfill the task. The number of nodes in these layers
are determined according to the number of input variables and target variables [37]. In some cases,
the input and output variables are linearly normalized to (0,1) or (−1,1), to avoid computational
problems or to meet algorithm requirement [24 38 39] In this study such methods were not applied ynl
i = f ( ∑
nm
j ∈Sl
i
wml
ji ynm
j )
(1)
tinues, the input variables will always exceed the range of the
able also has the chance. (2) the weights may undo the scaling. nd its nodes has a significant impact on MLP training[37, 40]. (1)
the
ing where ynl
i is the output value the ith node in the lth layer; wml
ji is the weight of the connection between
this node and the jth node in the mth layer; ynm
j is the output value of the jth node in the mth layer; f(x)
be the activation function of this node. Simple networks maybe less accurate in learning the problem while complex networks may take
excessively long training time. one hidden layer is usually sufficient in most cases [14, 19–25, 33, 41–
43] while sometimes multiple hidden layers shows better learning on certain problems [35]. Figure 2. Structure and value propagation of MLP. Figure 2. Structure and value propagation of MLP. Figure 2. Structure and value propagation of MLP. Figure 2. Structure and value propagation of MLP. The number of nodes in hidden layer is usually determined through trial-and-error method [19,
23, 43]. 2.2.2. The Structure of MLP Network
between this node and the jth node in
h l
f
b
h
f The range of attempts is usually within 1 to 20 [14, 19–25], or 3 times the number of input
variables [43]. The best number of nodes was the one having the smallest mean-square error (MSE)
and root-mean-square error (RMSE) and the highest correlation coefficient (r) for the validation data
set. [11]
In this paper, the designed MLP consists 1 input layer, 1 hidden layer and 1 output layer. There
will be 5 nodes in the input layer representing climate change factor, population, the number of
The input layer receives the input data while the output of output layer refers to the predicted
results. Thus, both only requires only 1 layer to fulfill the task. The number of nodes in these layers
are determined according to the number of input variables and target variables [37]. In some cases,
the input and output variables are linearly normalized to (0,1) or (−1,1), to avoid computational
problems or to meet algorithm requirement [24,38,39]. In this study, such methods were not applied
because: (1) with the exploration continues, the input variables will always exceed the range of the
existing records while the output variable also has the chance. (2) the weights may undo the scaling. p
y
p
g
g
p p
decision take for BAU, RWHT and PIPE within the 20 years and 1 node in the output layer referring
to the flooded junctions. No variables will be normalized. The number of nodes in the hidden layer
will be determined within 1 to 20 through trail-and-error method. The number of hidden layers and its nodes has a significant impact on MLP training [37,40]. Simple networks maybe less accurate in learning the problem while complex networks may take
excessively long training time. one hidden layer is usually sufficient in most cases [14,19–25,33,41–43]
while sometimes multiple hidden layers shows better learning on certain problems [35]. The number of nodes in hidden layer is usually determined through trial-and-error method [19,23,43]. The range of attempts is usually within 1 to 20 [14,19–25], or 3 times the number of input variables [43]. The best number of nodes was the one having the smallest mean-square error (MSE) and root-mean-square
error (RMSE) and the highest correlation coefficient (r) for the validation data set. [11] In this paper, the designed MLP consists 1 input layer, 1 hidden layer and 1 output layer. 2.2.1. Type of ANN There are several groups of networks such as Feedforward Networks (e.g., Multi-layer
Perceptron [26], the Probabilistic Neural Network [27], the Dynamic Neural Network [28]),
Recurrent Networks (e.g., Elman Network [29], Autoregressive Networks [30]), Polynomial
Networks (e.g., Ridge Polynomial Networks [31], Function Link Network [32]), Modular Networks,
Support Vector Machine and so forth. [33]. Among these extensive types of ANNs and their derivations, The multi-layer perceptron (MLP),
a feedforward multilayer network with non-linear node functions, is the most commonly encountered
one [33,34]. Practically, MLP shows successful generalization capability, effectiveness and efficiency in
forecasting time series [10,11,19,23], as well as great compatibility coping with different optimization
methods or existing models [19,35]. Although MLP is usually the better choice or at least the same
performance with respect to other proposal networks [33], there remain certain delimitations that have
a remarkable impact on the training accuracy and efficiency. Such aspects include the structure of
the network, the activation function of nodes, the existence of bias units, the quality and quantity of 5 of 16
nction Sustainability 2018, 10, 4600
ith node in the lth laye
of nil, the output value training and validation datasets, the choice of training algorithm and parameters and so forth. In this
paper, the MLP network will be adopted while the design process of these aspects will be investigated
and adapted to the case study. The network will be established using PyBrain [36], a modular Machine
Learning Library for Python. 𝑦
= 𝑓( 𝑤
𝑦ೕ
ೕ
∈ௌ
)
(1)
where 𝑦 is the output value the ith node in the lth layer; 𝑤
is the weight of the connection 2.2.3. The Activation Functions The role of activation function (AF) in MLP is to non-linearize the linear combination of weights
and node values passing through from the previous layer. Practically, there are three types of AFs:
(1) the analytic AFs, which are classic functions such as Gaussian, Sigmoid and Tanh; (2) the fuzzy
AFs, which has faster convergence in training; and (3) the adaptive AFs, which improves the nonlinear
response of the network [40]. Although the fuzzy AFs perform better on specific problems [44], there is
little evidence on the advantage of such AFs in practice. On the other hand, the adaptive AFs also
suffer from a more complex and error-prone training algorithm [40]. Thus, only classic analytic AFs
are considered in this study. For nodes in the hidden layer,
most commonly used AFs are the logistic sigmoid
function [34,38,41], the tanh function [35,43,45]. These two functions are similar in shape while
different in output ranges (sigmoid: [0,1], tanh: [–1,1]). For the output layer, most researchers adopt
linear function [11,35,41,45]. In this paper, the log-sigmoid function has been used for the hidden layer nodes while linear
function has been applied in the output layer to test their performance on handling random noise. 2.2.2. The Structure of MLP Network
between this node and the jth node in
h l
f
b
h
f There will
be 5 nodes in the input layer representing climate change factor, population, the number of decision
take for BAU, RWHT and PIPE within the 20 years and 1 node in the output layer referring to the Sustainability 2018, 10, 4600 6 of 16 flooded junctions. No variables will be normalized. The number of nodes in the hidden layer will be
determined within 1 to 20 through trail-and-error method. flooded junctions. No variables will be normalized. The number of nodes in the hidden layer will be
determined within 1 to 20 through trail-and-error method. 2.2.4. Bias Unit The bias unit is an extra set of nodes added to all layers but the output layer, which helps to get a
better and quicker learning of the network. The output value of a bias unit is fixed value while the
weights of connection from the bias unit to the subsequent nodes are still adjustable. The addition of
bias unit introduces a threshold value that may influence the activation of the subsequent nodes [24,37],
or, from another perspective, helps to move the AF in the subsequent nodes along the x-axis for better
learning results. Thus, in most cases, bias units always contribute positively to the network. 2.2.5. Learning Algorithm and Parameter Setting 2.2.5. Learning Algorithm and Parameter Setting The traditional and most commonly used training method for MLP is the two-step
error-backpropagation method [14,19,24]. Firstly, the input vector is fed into the input layer,
propagating forward through hidden layer(s) to the output layer. Then, the error is calculated in the
gradient descent and propagated backward from the output layer through the hidden layer(s) to the
input layer, which modifies the weights for every connection between nodes. The training repeats until
the network’s overall error are less than a predefined learning rate, or until the number of maximum
epochs is reached. Learning rate is a damping factor applied to weights correction during training [40],
indicating the amount that the weights are updated. Epoch is a measure of the number of times all of
the training vectors are used once to update the weights. Obviously, when dealing with huge datasets,
it is super time consuming if all the weights are recomputed for each training vector. Thus, there is
also a batch-learning term for the backpropagating method, which feeds multiple training samples in
one forward/backward pass. The number of samples in one pass is called batch size while such one
forward/backward process is count as one iteration. As the original backpropagation method is likely to be slow [41], improved strategies such as
Second-order On-Line training methods have been developed. Although these second-order training
algorithms are likely to converge significantly faster than first-ordered backpropagation [37], they require
more complex data preprocessing as well as more storage and computational costs. Luckily, there are also
several improved first-order backpropagation methods. The most commonly used is the Backpropagation
with Momentum [22,24], which significantly speed up the training process. The momentum is an inertial
factor applied to the weights during the back propagate process, which aims to maintain the direction of
weight changing [40]. The addition of momentum accelerates convergence where the learning quality is
good while precisely reduces the number of oscillations where bad [37]. 7 of 16 Sustainability 2018, 10, 4600 The settings of training parameters are more likely to be empirical and case-dependent. In most
cases, the start/fixed learning rate will be in the range of [0.01,0.3] [21,22,25,34] while the end learning
rate within [0.00013,0.001] [19,21]. The number of epochs usually depends on the training data size and
the computational capacity, ranging from 200 to 15,000 [19,21,22,24,34,35,42]. Momentum is typically
set to 0.9 [22], although the optimal value might be task-specific [21,24,34]. 2.3. Trial and Error The performance of learning results was assessed by the root-mean-square error (RMSE), which is
a commonly used index in machine learning [14,20,21,34]. The lower RMSE it is, the better prediction
the module makes [19]. RMSE is defined as the absolute value of the estimated error between the predicted result and the
observed result, calculated by:
s RMSE =
s
∑n
i=1(Oi −Pi)2
n
(2) (2) where Oi is the observed result; Pi is the predicted result. where Oi is the observed result; Pi is the predicted result. As the unit of RMSE is case-dependent, the correlation coefficient (r) [14,20,21,34] was adopted to
compare the training performance with other studies. r =
∑n
i=1
Pi −P
Oi −O
q
∑n
i=1
Pi −P
2 ∑n
i=1
Oi −O
2
(3) (3) where Oi is the observed result; Pi is the predicted result; O is the mean value of the observed result;
P is the mean value of the predicted result. Practically, as the decision in long-term infrastructure implementation planning is not
scenario-based but strategy-based, the distribution of predict results for each strategy combination
should be more convincible than RMSE. Thus, the prediction distribution of outputs was also adopted
in this study as the other performance indicator 2.2.5. Learning Algorithm and Parameter Setting The designed network structure and learning parameters are shown in Table 1. All combinations
of structure and learning parameters were tested with the first 0.1% of data and validated with the
following 0.05% data. After the best structure was determined, the network was again tested with
different size of training set size to find the best application pattern. The validation set size is half of
the training set. The best performing structure and application patter were applied to the case study to
study the feasibility of ANN in supporting long-term planning. Table 1. Designed Neural Network Parameters. Type
Structure
Activation Function
Bias Units
Learning Settings
Name
Layer
Node
MLP
input
1
5
-
True
training size 1
0.1%, 1%, 10%
batch size
1
hidden
1
1–20
sigmoid
True
learning rate
0.01, 0.1, 0.3
learning rate decay
1.0
output
1
1
linear
False
momentum
0.1–0.9
epoch
500, 1000, 5000
1 Training size is the percentage of total data used as the training set, tested after the ANN structure
being determined. Table 1. Designed Neural Network Parameters. Table 1. Designed Neural Network Parameters. 3.1. ANN Structure and Training Parameters As mentioned in the previous section, all combinations of structure (number of hidden nodes)
and learning parameters (learning rate, momentum and number of epochs) were tested with the first 8 of 16
is no
g, the Sustainability 2018, 10, 4600
nodes to 20 nodes, th
significant difference 8 of 16
is no
g
the 0.1% of all data (training size = 0.1) and validated with the following 10% of data. For each parameter,
the distributions of RMSE for each candidate value under all possible combinations are shown in
Figure 3. distributions of RMSE still have dramatic and irregular variations. These distributions are
characterized by the minimum, maximum, Q1, Q3 and mid-values, which indicates 100%, 75%, 50%,
25%, 0% chance of getting a higher RMSE than the given value, respectively. Thus, the lower these
values are, the better performance of the network we will get. Figure 3. RMSE Distributions under different manipulated variables. Figure 3. RMSE Distributions under different manipulated variables. Figure 3. RMSE Distributions under different manipulated variables. Figure 3. RMSE Distributions under different manipulated variables. As shown in Table 2, the MLP network with 15 nodes was always in the top 5 well-performed
structure and has significant advantages in low mid-value compared to others. The 17 nodes network
is slightly better than the 15 nodes one on minimum, Q3 and maximum as well as slightly poor on
Q1 and mid-value. Thus, the network of 15 and 17 hidden nodes are selected as the candidate
structure for the following studies. Table 2. Comparison of performance distribution for different number of hidden nodes. 1st
RMSE
2nd
RMSE
3rd
RMSE
4th
RMSE
5th
RMSE
Min
12
10.97
14/17
11.17
19
11.18
9
11.25
15
11.26
Q1
19
11.95
18
12.04
16
12.05
15
12.08
13
12.16
d
By adopting ANN(MLP) in urban water infrastructure performance prediction, the RMSE of
such method ranges from 10.97–19.33 nodes with the observed flooded junctions ranging from 20 to
146. For the number of hidden nodes, setting 1 node caused the highest average RMSE (16.62) which
may due to the strongest linearity of the network. With the number of hidden nodes rises to 4 nodes,
the average RMSE drops gradually to 15.46 where the non-linearity starts to develop effect. From 4
nodes to 20 nodes, the average RMSE keeps stable within (15.13,15.56). 3.1. ANN Structure and Training Parameters Although there is no significant
difference in the average RMSE with the number of hidden nodes changing, the distributions of RMSE
still have dramatic and irregular variations. These distributions are characterized by the minimum,
maximum, Q1, Q3 and mid-values, which indicates 100%, 75%, 50%, 25%, 0% chance of getting a higher
RMSE than the given value, respectively. Thus, the lower these values are, the better performance of
the network we will get. Mid
15
14.02
17
14.20
8
16.67
19
16.79
10
16.98
Q3
17
18.15
5
18.17
10/12
18.19
13/14
18.20
15
18.21
Max
17
18.37
8
18.39
9
18.41
6
18.42
15/16
18.43
As shown in Table 2, the MLP network with 15 nodes was always in the top 5 well-performed
structure and has significant advantages in low mid-value compared to others. The 17 nodes network
is slightly better than the 15 nodes one on minimum, Q3 and maximum as well as slightly poor on Q1
and mid-value. Thus, the network of 15 and 17 hidden nodes are selected as the candidate structure
for the following studies. Following the same process, the rest parameters are then determined: momentum = 0.1, learning
rate = 0.01, epoch = 5000. The candidate network was again tested with different size of training set size to find the best
application pattern (see Table 3). The result indicates that network with 15 nodes performs better than
the 17 nodes one under the select learning parameter, which is within 3 times the number of input
variables [38]. Training with the first 10% data will have a significant improvement in reducing the
RMSE while maintaining an acceptable time-saving capacity (reduce 80% of the time). Sustainability 2018, 10, 4600
ut variables [38]. Train 9 of 16
n reduc The best performing structure and application pattern (Table 3) were then applied to the case
study. The overall RMSE for the whole observed data and the predicted data is 10.5722 and the detailed
performance of MLP prediction is shown in Figure 4. The overall RMSE is slightly higher than the
validation result (9.7961). The best performing structure and application pattern (Table 3) were then applied to the c
dy. The overall RMSE for the whole observed data and the predicted data is 10.5722 and
ailed performance of MLP prediction is shown in Figure 4. The overall RMSE is slightly hig
n the validation result (9.7961). gure 4. 3.1. ANN Structure and Training Parameters ANN performance for different strategy combinations (supported by Matplotlib
Figure 4. ANN performance for different strategy combinations (supported by Matplotlib [46] ure 4. ANN performance for different strategy combinations (supported by Matplotlib [4
Figure 4. ANN performance for different strategy combinations (supported by Matplotlib [46]). 10 of 16 Sustainability 2018, 10, 4600 Table 2. Comparison of performance distribution for different number of hidden nodes. 1st
RMSE
2nd
RMSE
3rd
RMSE
4th
RMSE
5th
RMSE
Min
12
10.97
14/17
11.17
19
11.18
9
11.25
15
11.26
Q1
19
11.95
18
12.04
16
12.05
15
12.08
13
12.16
Mid
15
14.02
17
14.20
8
16.67
19
16.79
10
16.98
Q3
17
18.15
5
18.17
10/12
18.19
13/14
18.20
15
18.21
Max
17
18.37
8
18.39
9
18.41
6
18.42
15/16
18.43
Table 3. ANN performance under different training set sizes. Training Size
Hidden Nodes
Learning Rate
Momentum
Epoch
RMSE
Validation set
0.001
15
0.01
0.1
5000
11.5051
0.01
11.8653
0.1
9.7961
0.001
17
12.2593
0.01
12.5760
0.1
11.9862
Test set
0.1
15
0.01
0.1
5000
10.5722 Table 2. Comparison of performance distribution for different number of hidden nodes. Table 3. ANN performance under different training set sizes. Table 3. ANN performance under different training set sizes. Training Size
Hidden Nodes
Learning Rate
Momentum
Epoch
RMSE
Validation set
0.001
15
0.01
0.1
5000
11.5051
0.01
11.8653
0.1
9.7961
0.001
17
12.2593
0.01
12.5760
0.1
11.9862
Test set
0.1
15
0.01
0.1
5000
10.5722 The correlation coefficient (r) of the test set was 0.821, which was preferable compared to rs
in the other close applications of ANN (flood discharge: 0.683–0.851 [47], open-channel junction
velocity field: 0.035–0.884 [48], drought effects on surface water quality:0.819–0.922 [49], BOD in river:
0.505–0.821 [19]). Taking account of the tremendous amount of data in this case study, the above result suggested
the proposed statistical trial-and-error method for determining network parameters is feasible and
reliable on selecting the best structures. 3.2. Performance on Supporting Long-Term Planning To analyze the performance variations of different implementation strategy combinations for the
urban water system in the case study, boxplots are again used while the upper end of the whiskers is
set to 95th percentile (Figure 4). In other word, the probability of a certain system performing better
than this upper end is 95%. Thus, the accuracy on the 95th percentile and Q3 is practically more
important than that of mid-value, Q1 and minimum. For strategies containing only rainwater tanks ([0,5.0,0], [0,10.0,0], [0,15.0,0] and [0,20.0,0]), the first
two combinations are all included in the training set and share the same distribution with the observed
results. For the latter two strategies, the 95th percentile errors are −0.24% and −1.26% respectively
while the Q3 errors being −2.28% and −5.68%. This suggests the designed MLP network is effective
and has relatively good performance in predicting strategies with spatial randomness. The performance
of purely decentralized systems may have stronger and more linear relation with the rainfall events
and urban permeability (related to buildings/population), which makes the prediction of these purely
decentralized strategies better than mix strategies. For the same reason, the purely business as usual strategies also have good predictions: for
[3,0.0,0], Q3 = −0.22% and 95th = 0.18%; for [4,0.0,0], Q3 = −0.77% and 95th= −0.88%. As no additional
systems were implemented in these scenarios, the designed network performs well in generalizing the
relation between water system performance and rainfall events and urban permeability. For the overall performance, the MLP result has similar minimum, Q1 and mid-value compared
to the observed result (min: 20, 20; Q1: 48.1, 47.0; mid: 58.3, 60.0). Whereas the predicted values have
a narrower range (20.0–88.44) than the observed ones (20–93) despite the outliers. Such phenomena
indicate that the prediction in the high-value events (poorly performed water system in practice)
tend to aggregate to the Q3. This suggests that, from an overview perspective, the adoption of ANN
supported planning may raise the chance of overestimating the performance of urban water systems. 11 of 16
11 Sustainability 2018, 10, 4600
S
t i
bilit 2019 11 FOR P To make this proposed method applicable and reliable in practice, the error distributions of the
result are investigated to solve the overestimating problem. As shown in Figure 5, all errors of Q3 lie
between (−10.56%,8.76%) and 95th percentile between (−18.91%,14.95%). 3.2. Performance on Supporting Long-Term Planning The majority of these errors
are negative, indicating universal overestimations of the urban water system. investigating the observed data and the predicted data in the validation set, a multiplicator or
exponent can be calculated out and applied for the test set. As the 95th percentile is the dominant
factor of this case study, the safety coefficient also comes from the 95th percentile of the validation
(multiplicator:1.0910, exponent:1.0272). (a)
(b)
(c)
(d)
(e)
(f)
Figure 5. Error distribution of MLP predicted result and corrected result ((a,b) observed errors for
95th percentile and Q3; (c,d) corrected errors for 95th percentile and Q3 by multiplication; (e,f)
corrected errors for 95th percentile and Q3 by exponent). Figure 5. Error distribution of MLP predicted result and corrected result ((a,b) observed errors for 95th
percentile and Q3; (c,d) corrected errors for 95th percentile and Q3 by multiplication; (e,f) corrected
errors for 95th percentile and Q3 by exponent). (a) (b) (a)
(c) (d) (d) (c)
(e) (f) (f) (e) Figure 5. Error distribution of MLP predicted result and corrected result ((a,b) observed errors for
95th percentile and Q3; (c,d) corrected errors for 95th percentile and Q3 by multiplication; (e,f)
corrected errors for 95th percentile and Q3 by exponent). Figure 5. Error distribution of MLP predicted result and corrected result ((a,b) observed errors for 95th
percentile and Q3; (c,d) corrected errors for 95th percentile and Q3 by multiplication; (e,f) corrected
errors for 95th percentile and Q3 by exponent). Table 4. Mean ± SD error of adopting the safety coefficient. Observed Error
Multiplicator
Exponent
Q3
−2 29% ± 4 28%
3 38% ± 4 73%
3 43% ± 4 72%
As Table 4 shows, the adoption of safety coefficient could effectively raise the error from negative
to positive (from overestimation to under estimation) while slightly enlarge the standard deviation of
the errors. Table 4. Mean ± SD error of adopting the safety coefficient. Observed Error
Multiplicator
Exponent
Q3
−2 29% ± 4 28%
3 38% ± 4 73%
3 43% ± 4 72%
As Table 4 shows, the adoption of safety coefficient could effectively raise the error from negative
to positive (from overestimation to under estimation) while slightly enlarge the standard deviation of
the errors. Q
95th percentile
−3.13% ± 6.34%
2.63% ± 7.15%
2.96% ± 7.32%
The result of correction is shown in Figure 5. There is no obvious difference between correcti
ith multiplicator and exponent. 3.2. Performance on Supporting Long-Term Planning The corrected errors of Q3 lied in −3.05% to 18.24% (multiplicato
nd −2.96% to 17.87% (exponent) while that of the 95th percentile in −11.69% to 25.41% (multiplicato
Table 4. Mean ± SD error of adopting the safety coefficient. Observed Error
Multiplicator
Exponent
Q3
−2.29% ± 4.28%
3.38% ± 4.73%
3.43% ± 4.72%
95th percentile
−3.13% ± 6.34%
2.63% ± 7.15%
2.96% ± 7.32% (
p
)
As shown in Table 5, the accelerated exploration identified all robust drainage infrastructure
status in the reference exploration while overestimated three. The corrected accelerated exploration
identified most robust drainage infrastructure status in the reference exploration while
d
ti
t d
Th
d
ti
t d
h
i fl
th
l
ti
th
i
As these errors are related to the network structure and its final status, a safety coefficient, which
comes from the validation process, is adopted to adjust the final output of the network. By investigating
the observed data and the predicted data in the validation set, a multiplicator or exponent can be Sustainability 2018, 10, 4600 12 of 16 calculated out and applied for the test set. As the 95th percentile is the dominant factor of this case
study, the safety coefficient also comes from the 95th percentile of the validation (multiplicator:1.0910,
exponent:1.0272). The result of correction is shown in Figure 5. There is no obvious difference between correction
with multiplicator and exponent. The corrected errors of Q3 lied in −3.05% to 18.24% (multiplicator)
and −2.96% to 17.87% (exponent) while that of the 95th percentile in −11.69% to 25.41% (multiplicator)
and −11.60% to 25.36% (exponent). p
As shown in Table 5, the accelerated exploration identified all robust drainage infrastructure
status in the reference exploration while overestimated three. The corrected accelerated exploration
identified most robust drainage infrastructure status in the reference exploration while underestimated
one. The underestimated one has no influence on the plan generation as there is no connectable route
in the previous decision year. Thus, the correction is essential and effective to raise the robustness of
the proposed accelerated exploration. Table 5. Robust progressive goal for Scotchman’s Creek. Reference Exploration
Accelerated Exploration
Corrected Accelerated Exploration
2020
[0,0,1]1
[0,0,1]
[0,0,1]
2025
[0,0,2]
[0,0,2]
[0,0,2]
2030
[0,0,3]
[0,5,2]
[0,10,1]
[0,15,0]
[1,0,2]
-
[0,0,3]
[0,5,2]
[0,10,1]
[0,15,0]
[1,0,2]
[1,5,1]
[0,0,3]
[0,5,2]
[0,10,1]
-
[1,0,2]
-
2035
[0,0,4]
[0,5,3]
[0,10,2]
-
-
[0,0,4]
[0,5,3]
[0,10,2]
[1,5,2]
[2,0,2]
[0,0,4]
[0,5,3]
[0,10,2]
-
-
1 [BAU,RHWT(%),PIPE]. 3.2. Performance on Supporting Long-Term Planning Table 5. Robust progressive goal for Scotchman’s Creek. Table 5. Robust progressive goal for Scotchman’s Creek. 1 [BAU,RHWT(%),PIPE]. Notably, for 95th percentile, the majority of errors are controlled within ±10%. The two outliers
represent the two pure strategies of upgrading pipes, [0,0.0,3] and [0,0.0,4]. Although there are great
errors on these two strategies (underestimation of water system), the origin system performance of
them is good enough that the errors have no influence on identifying them as good strategies (not
influencing decision). This error also indicates that different from purely decentralized strategies, such
purely centralized strategies which have only relations with rainfall events, do not have a preferable
prediction at all. p
Such a result indicates that when using the MLP to predict a black box problem, such as the urban
water system in the case study, there should be at least two related input factors for each variable (the
candidate infrastructure, e.g., pipe, rwht) to ensure reliable prediction. 4. Conclusions In this study, an accelerated exploration planning method was proposed by integrating
the neural network method (multi-layer perceptron) with an explorative model (DAnCE4Water),
to significantly reduce the simulation time of generating a robust long-term water system adaptation
plan. The proposed method was applied to a case study in Scotchman’s Creek, Melbourne, Australia. Results showed the proposed method can cut down 80% of the simulation time while offering the
same plan. Instead of modifying the network parameters, the network structure and settings in this paper
were determined through a comprehensive statistical trial-and-error analysis (evaluating for all possible 13 of 16 Sustainability 2018, 10, 4600 13 of 16 parameter combination). With 10% of the training data, the validation error (10% data) was 9.7961,
the overall prediction RMSE was 10.5722 (80% data) and the correlation coefficient (r) was 0.821. This suggests that the ANN could have stable and reliable with good designed network and low
proportion of training data. It also emphasis the necessity of network design which did take time in
the trial-and-error analysis but having promising return in total time saving and accuracy. The ANN showed diverse capacity on predicting the performance of different type of
flood-resisting strategies. The estimation of purely decentralized strategies (scenarios with RWHT only)
and purely BAU strategies is far more accurate than that of mixed strategies. Meanwhile, the purely
centralized strategies (scenarios with PIPE only) had the worst prediction. Considering the input
variables related to the strategy, it is obvious that the performance estimation would be more accurate
if more flood-related input variables are related to it (two for RWHT and BAU while one for PIPE). Thus, more flood-related input variables should be considered (for each strategy) in future studies. The proposed exploration method raised the chance of overestimating the performance of urban
drainage systems (−3.13% ± 6.34% flooded junctions than observed). By adopting the safety coefficient,
a multiplicator or exponent calculated by observed data and predicted data in the validation process,
the overestimation problem was controlled in an acceptable range and have very limited impacts on
final decision making (2.63% ± 7.15% flooded junctions than observed). Such correction is effective
in practice as the real-world goal for planning is either above or below a certain target. 4. Conclusions Instead of
reducing the error which is a tough task, the correction shifts the error along one direction (to more
underestimate side) to ensure the reliability of the given plan. As the error came from the method,
the safety coefficient calculated by the validation data could be reasonable to some extent. Although the proposed accelerated exploration method was proved to be efficiency in time saving
(saved 80% of exploration) and effective (offered similar decisions after correction), there are still
several aspects requires further studies. (1) The training set used in this study followed a “real-world
exploration” time sequence, which means there were much more simulations in the later decision steps
than in the earlier steps. Such setting may have influence on the network performance. Further studies
have to be conducted on the composition of the training set to ensure efficient and effective training;
(2) Further investigation in the cause of the universal overestimation have to be conducted to optimize
the algorithm or training pattern. (3) More case studies should be carried out to further validate and
improve the proposed accelerated exploration method. Author Contributions: Conceptualization, J.Z., D.F., C.U. and R.P.S.; Methodology, J.Z. and C.U.; Software, J.Z.;
Validation, J.Z.; Formal Analysis, J.Z.; Data Curation, J.Z. and C.U.; Writing-Original Draft Preparation, J.Z.;
Writing-Review & Editing, R.P.S.; Visualization, J.Z.; Supervision, D.F.; Project Administration, D.F.; Funding
Acquisition, D.F. ding: The research was co-funded by National Key R&D Program of China (Grant No. 2018YFC0809900) and
Priority Academic Program Development of the Jiangsu Higher Education Institution. Funding: The research was co-funded by National Key R&D Program of China (Grant No. 2018YFC0809900)
he Priority Academic Program Development of the Jiangsu Higher Education Institution. Acknowledgments: Thanks to Professor Dafang Fu and Christian Urich for the creative leadership and for
motivation to finish the research. Thanks to Chenli Wu, who continuously supports us to finish the work. Acknowledgments: Thanks to Professor Dafang Fu and Christian Urich for the creative leadership and for
motivation to finish the research. Thanks to Chenli Wu, who continuously supports us to finish the work. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References 1. Moss, T. ‘Cold spots’ of Urban Infrastructure: ‘Shrinking’ Processes in Eastern Germany and the Modern
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English
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Antioxidant effects of Dendropanax morbifera Léveille extract in the hippocampus of mercury-exposed rats
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BMC complementary and alternative medicine
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cc-by
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© 2015 Kim et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly credited. 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. Kim et al. BMC Complementary and Alternative Medicine (2015) 15:247
DOI 10.1186/s12906-015-0786-1 Kim et al. BMC Complementary and Alternative Medicine (2015) 15:247
DOI 10.1186/s12906-015-0786-1 RESEARCH ARTICLE
Open Access
Antioxidant effects of Dendropanax
morbifera Léveille extract in the
hippocampus of mercury-exposed rats
Woosuk Kim1†, Dae Won Kim2†, Dae Young Yoo1, Hyo Young Jung1, Jong Whi Kim1, Dong-Woo Kim3,
Jung Hoon Choi4, Seung Myung Moon5, Yeo Sung Yoon1 and In Koo Hwang1* * Correspondence: vetmed2@snu.ac.kr
†Equal contributors
1Department of Anatomy and Cell Biology, College of Veterinary Medicine,
and Research Institute for Veterinary Science, Seoul National University, Seoul
151-742, South Korea
Full list of author information is available at the end of the article Abstract Background: Dendropanax morbifera Léveille has been employed for the treatment of infectious diseases using folk
medicine. In this study, we evaluated the antioxidant effects of a leaf extract of Dendropanax morbifera Léveille in
the hippocampus of mercury-exposed rats. Methods: Seven-week-old Sprague–Dawley rats received a daily intraperitoneal injection of 5 μg/kg dimethylmercury
and/or oral Dendropanax morbifera Léveille leaf extract (100 mg/kg) for 4 weeks. Animals were sacrificed 2 h after the
last dimethylmercury and/or leaf extract treatment. Mercury levels were measured in homogenates of hippocampal
tissue, a brain region that is vulnerable to mercury toxicity. In addition, we measured reactive oxygen species
production, lipid peroxidation levels, and antioxidant levels in these hippocampal homogenates. Results: Treatment with Dendropanax morbifera Léveille leaf extract significantly reduced mercury levels in
hippocampal homogenates and attenuated the dimethylmercury-induced increase in the production of reactive
oxygen species and formation of malondialdehyde. In addition, this leaf extract treatment significantly reversed the
dimethylmercury-induced reduction in the hippocampal activities of Cu, Zn-superoxide dismutase, catalase, glutathione
peroxidase, and glutathione-S-transferase. Conclusion: These results suggest that a leaf extract of Dendropanax morbifera Léveille had strong antioxidant effects
in the hippocampus of mercury-exposed rats. Keywords: Dendropanax morbifera extract, Mercury, Hippocampus, Reactive oxygen species, Protei endropanax morbifera extract, Mercury, Hippocampus, Reactive oxygen species, Protein modification RESEARCH ARTICLE Open Access Antioxidant effects of Dendropanax
morbifera Léveille extract in the
hippocampus of mercury-exposed rats Woosuk Kim1†, Dae Won Kim2†, Dae Young Yoo1, Hyo Young Jung1, Jong Whi Kim1, Dong-Woo Kim3,
Jung Hoon Choi4, Seung Myung Moon5, Yeo Sung Yoon1 and In Koo Hwang1* Background and animals is the consumption of fish containing MeHg
[7, 8]. Although MeHg has low lipid solubility, the
ingested MeHg easily permeates the blood–brain barrier
and accumulates in the hippocampus [9], a brain region
that is vulnerable to acute MeHg exposure [10]. In a re-
cent study, MeHg was found to induce the accumulation
of amyloid-β in the brain and facilitate the progression of
Alzheimer’s disease [11]. Recently, several attempts have been made to develop ap-
proaches that facilitate the removal of heavy metals from
the body, because these metals have been found to accu-
mulate in the body over time [1–4]. The accumulation of
heavy metals such as mercury (Hg), lead, and cadmium
can cause dangerous conditions, including neurological
dysfunction and metabolic disorders [5]. Organic Hg can
accumulate due to exposure to dimethylmercury (MeHg)
and this is the most common cause of intoxication in
humans [6]. The main cause of Hg exposure in humans Strong chelating agents such as ethylenediaminetetra-
acetic acid (EDTA) can efficiently remove heavy metals
from
contaminated
soils. However,
the
toxicity
of
EDTA limits its application in animals or humans. * Correspondence: vetmed2@snu.ac.kr
†Equal contributors
1Department of Anatomy and Cell Biology, College of Veterinary Medicine,
and Research Institute for Veterinary Science, Seoul National University, Seoul
151-742, South Korea
Full list of author information is available at the end of the article Recently, there have been many attempts to exploit
the antioxidant potential of compounds identified in
plants [12–16]. We previously demonstrated that a
stem extract of Dendropanax morbifera facilitated the Kim et al. BMC Complementary and Alternative Medicine (2015) 15:247 Page 2 of 7 physiological saline, 3) a MeHg group was administered
with oral distilled water and intraperitoneal injections of
5 μg/kg MeHg, and 4) a DML-MeHg group received
100 mg/kg oral DML and intraperitoneal injections of
MeHg. MeHg was administered intraperitoneally and
DML was administered orally to 7-week-old rats once a
day for 4 weeks. excretion of cadmium and increased antioxidant levels
in the hippocampus [17]. In addition, Dendropanax mor-
bifera extract increased the activities of antioxidant en-
zymes and showed protective effects against diabetes,
cancer,
atherosclerosis,
and
kidney
toxicity
[18–21]. However, there are no reports on the effects of Den-
dropanax morbifera extracts on the hippocampal anti-
oxidant status after MeHg intoxication. Background Therefore,
this study examined the effects of an extract of Den-
dropanax morbifera leaves (DML) on oxidative stress
and the status of antioxidant enzymes in the hippo-
campus of rats exposed to MeHg. Hg levels in hippocampal homogenates
Rats in the control, DML, MeHg, and MeHg-DML
groups (n = 7 from each group) were anesthetized with
1 g/kg urethane (Sigma-Aldrich) and the hippocampi
were dissected out to measure the accumulation of Hg in
the
hippocampal
homogenates. Hippocampi
were
weighed in glass vessels, and tissues were digested by
adding 3-8 mL of HNO3 for 3 h, after which 2 mL of
H2O2 was added and the samples were heated for 1 h. Digested hippocampal samples were transferred to poly-
propylene flasks for Hg determination, which was per-
formed
using
inductively
coupled
plasma
mass
spectrometry (ICP-MS; PerkinElmer Sciex, Thornhill,
Canada). Preparation of DML Fresh Dendropanax morbifera Léveille was purchased
from a local market on Jeju Island in Korea. The plant
was authenticated by two practitioners of traditional
Asian medicine, and a voucher specimen was depos-
ited with Egreen Co. Ltd. (deposition number: 2013–
002). Leaves from the plant samples (100 g) were
chopped, blended, soaked in 2 L 80 % ethanol, and
then refluxed three times at 20 °C for 2 h. The insol-
uble
materials
were
removed
by
centrifugation
at
10,000 × g for 30 min, and the resulting supernatant
was concentrated and freeze-dried to yield a powder. Before each experiment, the dried extract was dis-
solved in distilled and deionized water. Measurement of reactive oxygen species (ROS)
production and lipid peroxidation in the hippocampus production and lipid peroxidation in the hippocampus
The effects of DML and MeHg on ROS production and
lipid peroxidation were determined in hippocampal ho-
mogenates from rats in the control, DML, MeHg, and
MeHg-DML groups (n = 7 from each group) using the
fluorescent
probe,
2ʹ,7ʹ-dichlorofluorescin
diacetate
(DCFH-DA) [22], and by measuring malondialdehyde
(MDA) formation, respectively. Intracellular ROS oxidize
DCFH-DA to dichlorofluorescein (DCF), an intensely
fluorescent chemical. The rats were deeply anesthetized
with urethane and euthanized by decapitation after treat-
ment for 4 weeks. Bilateral hippocampi were dissected
out and the left and right parts were used to measure
ROS production and lipid peroxidation, respectively. Hippocampal mitochondria were obtained as described
previously [23]. Mitochondrial protein was quantified by
the Bradford method [24] using bovine serum albumin
(BSA)
as
the
standard. The
isolated
mitochondria
(0.5 mg protein/mL) were incubated with 10 μM DCFH-
DA at 37 °C for 60 min, and the fluorescence intensity
of DCF was measured at an excitation wavelength of
488 nm and emission wavelength of 527 nm in a micro-
plate reader (SpectraMax M5, Molecular Devices LLC,
Sunnyvale, CA). Experimental animals Male Sprague–Dawley rats were purchased from Orient
Bio Inc. (Seongnam, South Korea). Rats were housed in
a conventional animal facility at 23 °C with 60 % humid-
ity, a 12 h/12 h light/dark cycle, and free access to food
and tap water. The handling and care of the animals
conformed to the guidelines established in order to com-
ply with current international laws and policies (NIH
Guide for the Care and Use of Laboratory Animals, NIH
Publication No. 85–23, 1985, revised 1996). Ethical
and experimental protocol approvals were obtained
from the Institutional Animal Care and Use Commit-
tee (IACUC) of Seoul National University (Approval
number: SNU-130522-1). All of the experiments were
conducted with an effort to minimize the number of
animals used and the suffering caused by the proce-
dures employed. Administration of MeHg and DML g
MeHg was purchased from Sigma-Aldrich (St. Louis,
MO, USA). Animals were divided into 4 treatment
groups (n = 21 in each group): 1) a control group re-
ceived oral distilled water and intraperitoneal injections
of physiological saline, 2) a DML group was treated with
100 mg/kg oral DML and intraperitoneal injections of To measure MDA production, the hippocampal tissues
were homogenized in 20 mM phosphate buffered saline
(pH 7.4) containing 5 mM butylated hydroxytoluene. After centrifugation of the homogenates at 3000 × g for
10 min at 4 °C, the supernatants were collected. For each
reaction, 10 μL of probucol and 640 μL of diluted R1 Page 3 of 7 Kim et al. BMC Complementary and Alternative Medicine (2015) 15:247 potassium phosphate buffer (pH 7.7), 1 mM EDTA,
0.4 mM sodium azide, 2 mM glutathione, 0.1 mM
NADPH, 0.62 U of glutathione reductase, and 50 μL
of homogenate. reagent
(1:3
of
methanol:N-methyl-2-phenylindole)
were added and mixed with 150 μL of 12 N HCl. Each reaction was incubated at 45 °C for 60 min and
then centrifuged at 10,000 × g for 10 min. The super-
natant was collected and MDA formation was deter-
mined by measuring the absorbance at 586 nm. MDA
data were normalized to the protein concentration of
each sample. Total sulfhydryl (TSH) content was determined using
the 5,5’-dithiobis (2-nitrobenzoic acid) method (Sigma)
reported by Riddles et al. [29]. GST activity was determined spectrophotometrically
using 1-chloro-2,4-dinitrobenzene as a substrate [30]. GR, which has been shown to utilize NADPH to convert
oxidized glutathione (GSSG) to the reduced form (GSH),
was assayed using the method reported by Horn and
Burns [31]. Effects of MeHg and DML on hippocampal ROS formation
and lipid peroxidation Similar DCF fluorescence intensity and MDA levels were
detected in the DML and control groups. In the MeHg
group, DCF fluorescence intensity and MDA levels were
significantly increased to 202.5 % and 231.3 % of the con-
trol values, respectively. In the DML-MeHg group, the
DCF fluorescence intensity and MDA levels were signifi-
cantly decreased, as compared with those in the MeHg
group (Fig. 1). CAT activity was assayed at 25 °C by determining
the rate of H2O2 degradation in 10 mM potassium
phosphate buffer (pH 7.0), according to the method
described by Aebi [27]. An extinction coefficient of
43.6 mM/cm was used for the calculations. One unit
was defined as consuming 1 pmol of H2O2 per min
and the specific activity was reported as units/mg
protein. Results Effects of MeHg and DML on hippocampal Hg levels Effects of MeHg and DML on hippocampal Hg levels
Similar hippocampal Hg concentrations were observed
in the DML and control groups. The Hg concentration
in the MeHg group was significantly increased by
5.57-fold, as compared with the control group. In the
DML-MeHg group, the Hg concentration was signi-
ficantly decreased to 75.4 % of that observed in the
MeHg group, although it remained significantly ele-
vated by 4.20-fold, as compared with the control
group (Fig. 1). SOD1 activity was measured by monitoring its capacity
to
inhibit
the
reduction
of
ferricytochrome
c
by
xanthine/xanthine oxidase, as described by McCord and
Fridovich [25]. Protein samples were electrophoresed in
10 % native polyacrylamide gels prior to SOD1 activity
visualization, as described by Beauchamp and Fridovich
[26]. Briefly, the gel was soaked in 2.45 mM nitroblue
tetrazolium solution for 15 min, followed by 30 min in
28 mM N,N,N′′,N′′-tetramethylethylene diamine and
28 μM riboflavin in 0.36 mM potassium phosphate buffer
(pH 7.8). The gel was then exposed to a fluorescent light
source until the bands showed maximum resolution. Effects of MeHg and DML on hippocampal ROS formation
and lipid peroxidation Effects of MeHg and DML on hippocampal ROS formation
and lipid peroxidation Measurement of antioxidant enzyme activity in
hippocampal homogenates To elucidate the effects of DML and MeHg on Cu, Zn-
superoxide dismutase 1 (SOD1), catalase (CAT), glutathi-
one peroxidase (GPx), and glutathione-related enzymes
such as glutathione-S-transferase (GST), and glutathione
reductase (GR), the activities of these enzymes were mea-
sured in rats from the control, DML, MeHg, and DML-
MeHg groups (n = 7 from each group). Animals were
deeply anesthetized with urethane and euthanized by de-
capitation after treatment for 4 weeks. Bilateral hippo-
campi were dissected out and the left part was used to
measure SOD1, CAT, and GPx activities, while the
right part was used to measure glutathione-related
enzyme activities. Left and right hippocampal tissues
were homogenized in 10 mM Tris buffer containing
1 mM EDTA or 1 mM phenylmethanesulfonylfluor-
ide, respectively. The homogenates were centrifuged
at
600 × g
for
10
min,
and
then
centrifuged
at
13,000 × g for 20 min at 4 °C. Statistical analysis All data are expressed as mean ± standard error of the
mean (SEM). Differences between the means were sta-
tistically analyzed using two-way analysis of variance
(ANOVA) with repeated measures and Bonferroni’s
post hoc test. Effects of MeHg and DML on hippocampal SOD1, CAT,
and GPx activities Effects of MeHg and DML on hippocampal SOD1, CAT,
and GPx activities In the control group, the mean SOD1, CAT, and GPx ac-
tivities were 10.57 U/mg protein, 88.51 U/mg protein,
and 197.37 U/mg protein, respectively. In the DML
group, SOD1 and GPx activities were higher than those
in the control group, but these differences were not sta-
tistically significant. In contrast, CAT activity in the DML
group was similar to that observed in the control group. In the MeHg group, SOD1, CAT, and GPx activities were GPx activity was assayed by measuring nicotinamide
adenine dinucleotide phosphate (NADPH) oxidation
using t-butyl-hydroperoxide as a substrate, as described
by Maral et al. [28]. Briefly, the reaction was carried out
at 25 °C in 600 μL of a solution containing 100 mM Page 4 of 7 Kim et al. BMC Complementary and Alternative Medicine (2015) 15:247 Fig. 1 Serum mercury (Hg) levels, hippocampal production of
reactive oxygen species as determined by 2′,7’-dichlorofluorescein
(DCF) levels, and malondialdehyde (MDA) levels in rats. * Indicates
a significant difference between the control and dimethylmercury
(MeHg) groups or between the Dendropanax morbifera leaf extract
(DML) and DML-MeHg groups (P < 0.05); # indicates a significant
difference between the control and DML groups or between the
MeHg and DML-MeHg groups (p < 0.05; n = 7 per group). Serum
Hg, hippocampal DCF, and MDA levels are significantly increased
in the MeHg group and are reduced by DML treatment. The data
represent means ± standard error of the mean (SEM) significantly decreased to 60.3 %, 46.7 %, and 57.7 % of
the control values, respectively. In the DML-MeHg
group, SOD1 and GPx activities were significantly in-
creased to 94.9 % and 93.8 % of the control values, re-
spectively. However, CAT activity only showed a slight
increase, compared with the control group (Fig. 2), and
this change was not statistically significant. Effects of MeHg and DML on hippocampal level of TSH
and activities of GST and GR The control and DML groups showed similar levels of
TSH and activities of GST and GR. In the MeHg group,
the TSH level and activities of GST and GR were signifi-
cantly changed, as compared with the control group. The TSH level and GR activity in hippocampal homoge-
nates were significantly decreased to 50.3 % and 51.4 %
of the control values, respectively. In contrast, GST ac-
tivity was significantly increased to 170.3 % of the value
observed in the control group. In the DML-MeHg
group, the TSH level was slightly increased compared
with that in the MeHg group, while GST activity was de-
creased. However, these changes were not statistically
significant. GR activity in the DML-MeHg group was
significantly increased to 148.8 % of the value in the
MeHg group (Fig. 3). Discussion MeHg is a highly neurotoxic organometallic cation that
poses a great risk to human health. Several lines of evi-
dence show that its main neurotoxic mechanism involves
induction of oxidative stress [32–34]. The hippocampus
is particularly vulnerable to MeHg and shows detri-
mental changes in response to MeHg exposure [35,
36]. It has been reported that subchronic (20 days) ex-
posure to a low concentration of Hg (1-2 mg/kg) had
no direct toxic effects on the reproductive system of
rats. In contrast, subcutaneous treatment of rats with
0.6 μg/g MeHg on postnatal day 7 caused spatial mem-
ory deficits on postnatal day 21, by reducing hippo-
campal neurogenesis [37]. In the present study, we
treated rats with 5 μg/kg MeHg daily for 4 weeks and
observed the effects of DML against MeHg-induced Kim et al. BMC Complementary and Alternative Medicine (2015) 15:247 Page 5 of 7 Fig. 2 Rat hippocampal activities of Cu, Zn-superoxide dismutase 1
(SOD1), catalase (CAT), and glutathione peroxidase (GPx). * Indicates a
significant difference between the control and dimethylmercury
(MeHg) groups or between the Dendropanax morbifera leaf extract
(DML) and DML-MeHg groups (p < 0.05); # indicates a significant
difference between the control and DML or between the MeHg and
DML-MeHg groups (p < 0.05; n = 7 per group). The activities of SOD1,
CAT, and GPx are significantly decreased in the MeHg group, but
DML administration significantly attenuates these effects. The data
represent means ± standard error of the mean (SEM)
Fig. 3 Levels of total sulfhydryl (TSH), glutathione-S-transferase (GST),
and glutathione reductase (GR) in the hippocampi of control, DML,
MeHg, and DML-MeHg groups following 4 weeks of DML and/or
MeHg treatment. * Indicates a significant difference between the
control and MeHg groups or DML and DML-MeHg groups (p < 0.05);
# indicates a significant difference between the control and DML or
between MeHg and DML-MeHg groups (p < 0.05; n = 7 per group). TSH levels and GR activity are significantly decreased in the MeHg
group, while GST activity is significantly increased. The administration
of DML reduces the changes of these parameters. The data
represent means ± standard error of the mean (SEM) Fig. 3 Levels of total sulfhydryl (TSH), glutathione-S-transferase (GST),
and glutathione reductase (GR) in the hippocampi of control, DML,
MeHg, and DML-MeHg groups following 4 weeks of DML and/or
MeHg treatment. Discussion * Indicates a significant difference between the
control and MeHg groups or DML and DML-MeHg groups (p < 0.05);
# indicates a significant difference between the control and DML or
between MeHg and DML-MeHg groups (p < 0.05; n = 7 per group). TSH levels and GR activity are significantly decreased in the MeHg Fig. 2 Rat hippocampal activities of Cu, Zn-superoxide dismutase 1
(SOD1), catalase (CAT), and glutathione peroxidase (GPx). * Indicates a
significant difference between the control and dimethylmercury
(MeHg) groups or between the Dendropanax morbifera leaf extract
(DML) and DML-MeHg groups (p < 0.05); # indicates a significant
difference between the control and DML or between the MeHg and
DML-MeHg groups (p < 0.05; n = 7 per group). The activities of SOD1,
CAT, and GPx are significantly decreased in the MeHg group, but
DML administration significantly attenuates these effects. The data
represent means ± standard error of the mean (SEM) Fig. 3 Levels of total sulfhydryl (TSH), glutathione-S-transferase (GST),
and glutathione reductase (GR) in the hippocampi of control, DML,
MeHg, and DML-MeHg groups following 4 weeks of DML and/or
MeHg treatment. * Indicates a significant difference between the
control and MeHg groups or DML and DML-MeHg groups (p < 0.05);
# indicates a significant difference between the control and DML or
between MeHg and DML-MeHg groups (p < 0.05; n = 7 per group). TSH levels and GR activity are significantly decreased in the MeHg
group, while GST activity is significantly increased. The administration
of DML reduces the changes of these parameters. The data
represent means ± standard error of the mean (SEM) Page 6 of 7 Kim et al. BMC Complementary and Alternative Medicine (2015) 15:247 oxidative stress in the hippocampus. DML significantly
reduced the MeHg-induced accumulation of Hg in hippo-
campal homogenates. protective effects through its antioxidant activity, protec-
tion of mitochondria, and anti-apoptotic actions [49]. The
administration of DML attenuated the changes in TSH
levels and GR and GST activities in MeHg-exposed rats. This ameliorative effect of DML may reflect the increased
GPx and GST activities, facilitating detoxification of the
H2O2 produced by MeHg [50]. Next, we measured ROS production in hippocampal
homogenates, because some studies demonstrated that
the main mechanism of Hg toxicity in biological systems
was related to the production of ROS [5, 32, 33]. Acknowledgements
Thi R
h This Research was supported by High Value-added Food Technology
Development Program, Ministry for Agriculture, Food and Rural Affairs,
Republic of Korea (112106-022-HD020). Discussion In the
present study, we observed that administration of
MeHg significantly increased ROS formation in the
hippocampus. This result was consistent with previous
studies showing that MeHg significantly increased ROS
production in brain synaptosomes [38] and mitochondria
[39, 40]. DML administration significantly attenuated the
MeHg-induced ROS production in hippocampal homoge-
nates. This may be associated with the antioxidant proper-
ties of DML. In a previous study, we demonstrated that
DML strongly reduced cadmium-induced ROS produc-
tion in the hippocampus [17]. In addition, a study re-
ported that a methanol extract of the debarked stem of
Dendropanax morbifera had strong antioxidant activ-
ities based on its 1,1-diphenyl-2-picrylhydrazyl (DPPH)
scavenging activity and ferric-reducing ability, as com-
pared with a control material (butylated hydroxy-
toluene) [20]. In addition, this methanol extract of
Dendropanax
morbifera
debarked
stem
contained
abundant phenolic compounds, and the total flavonoid
content was higher than that observed in extracts of
the branches, bark, or yellow leaves of Dendropanax
morbifera [20]. Authors’ contributions WK, DWK, DYY, JHC, HYJ, SMN, JWK, YSY, and IKH conceived the study. WK
and IKH designed the study. WK, DYY, HYJ, SMN, JWK conducted the animal
experiments and DWK and JHC conducted the biochemical experiments. DWK made the Dendropanax morbifera Leveille extract. JHC, SMM, and YSY
participated in designing and discussing the study. All authors have read
and approved the final manuscript. Received: 19 March 2015 Accepted: 17 July 2015 Received: 19 March 2015 Accepted: 17 July 2015 Received: 19 March 2015 Accepted: 17 July 2015 Competing interests
Th
h
d
l
h Competing interests
The authors declare that they have no competing interests. Conclusions In conclusion, DML significantly reduced MeHg-induced
oxidative stress in the rat hippocampus by directly scav-
enging free radicals or by increasing the activities of anti-
oxidant enzymes such as SOD1 and GPx. Author details
1 1Department of Anatomy and Cell Biology, College of Veterinary Medicine,
and Research Institute for Veterinary Science, Seoul National University, Seoul
151-742, South Korea. 2Department of Biochemistry and Molecular Biology,
Research Institute of Oral Sciences, College of Dentistry, Kangneung-Wonju
National University, Gangneung 210-702, South Korea. 3Central Research
Center, Egreen Co. Ltd, Seongnam 463-862, South Korea. 4Department of
Anatomy, College of Veterinary Medicine, Kangwon National University,
Chuncheon 200-701, South Korea. 5Department of Neurosurgery, Dongtan
Sacred Heart Hospital, College of Medicine, Hallym University, Hwaseong
445-170, South Korea. In the present study, we also measured the levels of
antioxidant and glutathione-related enzyme activities
because MeHg-induced oxidative damage reportedly
decreased the levels of endogenous non-enzymatic an-
tioxidants and inhibited antioxidant enzymes [41–46]. Conversely, depletion of glutathione facilitates MeHg
accumulation and enhances MeHg-induced oxidative
stress [47]. In the present study, MeHg exposure sig-
nificantly decreased the activities of SOD1, CAT, and
GPx in hippocampal homogenates. The reduction of
antioxidant enzyme activity was most marked for CAT. The
administration
of
DML
reversed
the
MeHg-
induced depletion of SOD1 and GPx activity to nearly
the same levels as those observed in the control group. However, we did not observe any significant recovery
of CAT activity. MeHg exposure significantly decreased
the rat hippocampal TSH level and GR activity, while
GST activity was significantly lower than that of the
control group. This result was consistent with previous
studies indicating that sulfhydryl groups represented
the main target of MeHg in biological systems [34]
and that MeHg decreased glutathione levels in the
cerebellum [48]. This result was also consistent with a
previous study showing that the CHCl3 fraction of
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www.biomedcentral.com/submit References Dendropanax
morbifera Léveille extract facilitates cadmium excretion and prevents
oxidative damage in the hippocampus by increasing antioxidant levels in
cadmium-exposed rats. BMC Complement Altern Med. 2014;14:428. 42. Lucena GM, Franco JL, Ribas CM, Azevedo MS, Meotti FC, Gadotti VM, et al. Cipura paludosa extract prevents methyl mercury-induced neurotoxicity in
mice. Basic Clin Pharmacol Toxicol. 2007;101:127–31. 18. Park BY, Min BS, Oh SR, Kim JH, Kim TJ, Kim DH, et al. Isolation and
anticomplement activity of compounds from Dendropanax morbifera. J Ethnopharmacol. 2004;90:403–8. 43. Chang JY, Tsai PF. Prevention of methylmercury-induced mitochondrial
depolarization, glutathione depletion and cell death by 15-deoxy-delta-
12,14-prostaglandin J2. Neurotoxicology. 2008;29:1054–61. 19. Chung IM, Kim MY, Park SD, Park WH, Moon HI. In vitro evaluation of the
antiplasmodial activity of Dendropanax morbifera against chloroquine-
sensitive strains of Plasmodium falciparum. Phytother Res. 2009;23:1634–7. 44. Farina M, Campos F, Vendrell I, Berenguer J, Barzi M, Pons S, et al. Probucol
increases glutathione peroxidase-1 activity and displays long-lasting
protection against methylmercury toxicity in cerebellar granule cells. Toxicol
Sci. 2009;112:416–26. y
20. Hyun TK, Kim MO, Lee H, Kim Y, Kim E, Kim JS. Evaluation of anti-oxidant
and anti-cancer properties of Dendropanax morbifera Léveille. Food Chem. 2013;141:1947–55. 21. Moon HI. Antidiabetic effects of dendropanoxide from leaves of
Dendropanax morbifera Leveille in normal and streptozotocin-induced
diabetic rats. Hum Exp Toxicol. 2011;30:870–5. 45. Farina M, Aschner M, Rocha JB. Oxidative stress in MeHg-induced
neurotoxicity. Toxicol Appl Pharmacol. 2011;256:405–17. 46. Farina M, Rocha JB, Aschner M. Mechanisms of methylmercury-induced
neurotoxicity: evidence from experimental studies. Life Sci. 2011;89:555–63. 46. Farina M, Rocha JB, Aschner M. Mechanisms of methylmercury-induced
neurotoxicity: evidence from experimental studies. Life Sci. 2011;89:555–63. 22. Lebel CP, Ali SF, McKee M, Bondy SC. Organometal-induced increases in
oxygen reactive species: the potential of 2′,7’-dichlorofluorescin diacetate as
an index of neurotoxic damage. Toxicol Appl Pharmacol. 1990;104:17–24. 47. Kaur P, Aschner M, Syversen T. Glutathione modulation influences methyl
mercury induced neurotoxicity in primary cell cultures of neurons and
astrocytes. Neurotoxicology. 2006;27:492–500. 23. Hwang IK, Yoo KY, Kim DW, Lee CH, Choi JH, Kwon YG, et al. Changes in
the expression of mitochondrial peroxiredoxin and thioredoxin in neurons
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damage. Free Radic Biol Med. 2010;48:1242–51. 48. Zimmermann LT, dos Santos DB, Colle D, dos Santos AA, Hort MA, Garcia
SC, et al. Methionine stimulates motor impairment and cerebellar mercury
deposition in methylmercury-exposed mice. J Toxicol Environ Health A. 2014;77:46–56. Kim et al. BMC Complementary and Alternative Medicine (2015) 15:247 Submit your next manuscript to BioMed Central
and take full advantage of: 27. Aebi H. Catalase in vitro. Methods Enzymol. 1984;105:121–6. 28. Maral J, Puget K, Michelson AM. Comparative study of superoxide
dismutase, catalase and glutathione peroxidase levels in erythrocytes of
different animals. Biochem Biophys Res Commun. 1977;77:1525–35. 28. Maral J, Puget K, Michelson AM. Comparative study of superoxide
dismutase, catalase and glutathione peroxidase levels in erythrocytes of
different animals. Biochem Biophys Res Commun. 1977;77:1525–35. 29. Riddles PW, Blakeley RL, Zerner B. Reassessment of Ellman’s reagent. Methods Enzymol. 1983;91:49–60. • Convenient online submission 29. Riddles PW, Blakeley RL, Zerner B. Reassessment of Ellman’s reagent. Methods Enzymol. 1983;91:49–60. • Thorough peer review 30. Habig WH, Jakoby WB, Guthenberg C, Mannervik B, Vander Jagt DL. 2-Propylthiouracil does not replace glutathione for the glutathione
transferases. J Biol Chem. 1984;259:7409–10. • No space constraints or color figure charges 31. Horn HD, Burns FH. Methods of enzymatic analysis. (ed. Bergmeyer HV). New York: Academic; 1978. p. 875–9. 31. Horn HD, Burns FH. Methods of enzymatic analysis. (ed. Bergmeyer HV). New York: Academic; 1978. p. 875–9. 32. Allen JW, Shanker G, Tan KH, Aschner M. The consequences of
methylmercury exposure on interactive functions between astrocytes and
neurons. Neurotoxicology. 2002;23:755–9. 32. Allen JW, Shanker G, Tan KH, Aschner M. The consequences of
methylmercury exposure on interactive functions between astrocytes and
neurons. Neurotoxicology. 2002;23:755–9. Submit your manuscript at
www.biomedcentral.com/submit
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1
Konferensen erhöll stöd från en rad insti
tutioner: Historiska museet (Stockholm),
Goetheinstitutet, Södertörns högskola,
Uppsala universitet, Hochschule für
jüdische Studien, Heidelberg, Sveriges
Utrikesdepartement, Ukrainska institutet i
Sverige, Judisk Kultur, Universität Potsdam,
the Europe for Citizens Programme of the
European Union och Tjeckiens ambassad i
Sverige. DOI: https://doi.org/ 10.30752/nj.91948 Editorial D
etta nummer av Nordisk judaistik –
Scandinavian Jewish Studies inleds med
två artiklar som behandlar svensk-judisk
historia. Maja Hultman beskriver hur två i
Stockholm bosatta judiska kvinnor under
första delen av 1900-talet använde sina hem
för att manifestera sina olika judiska ideal. Därefter följer Jens Carlesson Magalhães
artikel om den i Stockholm och Göteborg
i mitten av 1800-talet verksamma organisa
tionen ”Samfundet I.I: Judiska intresset” och
dess kamp för judiskt likaberättigande i det
svenska samhället. D av Paideia – The European Institute for Jewish
Studies in Sweden i Stockholm 31 januari–1
februari, 2019.1 Alla dessa texter diskuterar
återuppbyggnaden av judiska gemenskaper
och identiteter i ett Europa efter kommunis
mens fall. Mer specifikt diskuterar artiklarna
olika sätt att bygga ett meningsfullt judiskt liv
i den postkommunistiska perioden som tog
sin början 1989. 1
Konferensen erhöll stöd från en rad insti
tutioner: Historiska museet (Stockholm),
Goetheinstitutet, Södertörns högskola,
Uppsala universitet, Hochschule für
jüdische Studien, Heidelberg, Sveriges
Utrikesdepartement, Ukrainska institutet i
Sverige, Judisk Kultur, Universität Potsdam,
the Europe for Citizens Programme of the
European Union och Tjeckiens ambassad i
Sverige. Återstoden av numret utgörs av en tema
helhet redigerad av gästredaktörerna Lukasz
Gorniok och Lena Roos. Det består av bear
betade versioner av föredrag och presentationer
som hållits vid konferensen “1989–2019: Jews in
Post-Communist Europe” som organiserades Attribution 4.0 International (CC BY 4.0) Nordisk judaistik • Scandinavian Jewish Studies | Vol. 31, No. 1 förnyelse. För dessa var 1989 inte bara en mil
stolpe i europeisk och global historia, utan även
ur judiskt perspektiv. Som Zvi Gitelman skrev
i en essä från slutet av 1990-talet: ”Sedan 1989
har det blivit möjligt att återskapa judiskt liv
överallt i regionen och även för individer att
åter göra anspråk på och praktisera judiskt
liv. Som många judar på annat håll ofta har
påpekat, så har judisk identitet i Östeuropa
blivit en fråga om val, och detsamma gäller för
återuppbyggandet av gemenskaper. […] Judar
kan nu välja både sina egna föreställningar om
vilka de är och hur de vill explicit uttrycka dessa
i det offentliga”.2 Men när dessa möjligheter
nu existerar, så har processerna för att återskapa
judiskt liv sett olika ut i olika länder. Artiklarna
i detta specialnummer vill uppmuntra läsarna
att reflektera över dessa förändringar. Att arbeta för förnyelse av europeiskt
judiskt kulturellt och intellektuellt liv efter
kommunismens fall har varit ett av Paideias
huvudfokus ända sen grundandet. Editorial Paideia
grundades år 2000 genom anslag från
svenska regeringen och har som uppdrag att
ge näring åt en renässans för europeisk judisk
kultur, att stötta tvärkulturell dialog och att
främja en positiv modell för minoritetskultur
i en europeisk kontext. För att stärka judisk
kultur i ett bredare europeiskt sammanhang
anordnar Paideia en rad utbildningsprogram:
det ettåriga programmet i judiska studier som
fokuserar på att studera och tolka judiska
texter i ett tvärvetenskapligt sammanhang,
Project-Incubator, som stöttar nya initiativ för
judiskt liv i Europa och Paradigm Program,
som fokuserar på ett nära och personligt
förhållningssätt till judisk identitet. l
Numret innehåller två sorters texter. Texterna som härrör från Paideia-konferensen
är av två slag: den referentgranskade artikeln
av Christina von Braun följs av fem artiklar
som snarare är personliga reflektioner över
temat, av Israel Bartal, Konstanty Gebert, Alix
Landgrebe, Eli Reich och Mina Pasajlic. Hur judiska sammanhang och identiteter
har formats i Europa har också behandlats
i tidigare Paideiakonferenser. Teman som
”exil”, ”migration”, ”mångkultur” och “hem” har
belysts i relation till judisk identitet i samtidens
Europa och globalt. Detta övergripande tema präglade även
den senaste konferensen som markerade att
trettio år hade gått sedan Berlinmurens fall. Talarna diskuterade utmaningar och orosmo
ment som hänger samman med ny frihet, men
även möjligheter till kulturell kreativitet och
ett förändrat förhållande till historien, allt
genom föredrag och diskussioner om judiskt liv
i Polen, Tyskland, Tjeckien, Ungern, Ryssland
och på andra platser. Dessutom diskuterades
frågor rörande judiskt ledarskap och judisk
gemenskap. Bland talarna fanns representanter
från universitet i Europa och Israel, men även
aktivister som är verksamma inom europeiska
judiska sammanhang, många av dem är tidigare
Paideiastudenter. Vissa teman återkommer i många av
artiklarna, till exempel erfarenheter av anti
semitism och förtryck men också av förnyelse
och pånyttfödelse, både av judiskt liv och ett
intresse för judiska studier. Christina von Braun ger en bred översikt
över de senaste decenniernas utvecklingar inom
fältet judiska studier i det postkommunistiska
Europa, med särskilt fokus på de tysktalande
länderna, särskilt det återförenade Tyskland. Hon diskuterar återfödelsen av Wissenschaft
des Judentums som hade upphört att existera 2
Zvi Gitelman, “Reconstructing Jewish
Communities and Jewish Identities in Post-
Communist East Central Europe”, A. Kovacs,
Jewish Studies at the CEU, 1 Yearbook (Public
lectures, 1996–1999) (Budapest, 2001), 35–50. Editorial <http://web.ceu.hu/jewishstudies/pdf/01_
gitelman.pdf> Konferensen fokuserade på gemenska
per, individer, aktivister, migranter och andra
personer som drivit förändring och kulturell 2 Nordisk judaistik • Scandinavian Jewish Studies | Vol. 31, No. 1 förnyade intresset för judisk kultur och histo
ria och pånyttfödelsen av judiskt liv i Polen. Landgrebe diskuterar ett urval kontroversiella
fall och hur de kan förstås i relation till dessa
parallella processer. som forskningsfält, först under nationalso
cialismen och sedan kommunismen, och hur
denna återfödelse inte bara ses inom det strikt
akademiska forskningsfältet utan även inom
rabbinutbildning. Denna utveckling har även
stärkts av en judisk invandring till Tyskland
under de senaste decennierna, främst från forna
sovjetrepubliker och från Israel.l Rabbinutbildning i Berlin/Potsdam är
ämnet för Eli Reichs artikel där han dis
kuterar Abraham Geiger Kolleg (reform) and
Zacharias Frankel Rabbinical College (kon
servativ/masorti) som startade 2001 respek
tive 2013. Reich skisserar de olika rörelsernas
historiska rötter och hur dessa rötter, såväl som
den samtida kontexten, påverkar hur studierna
utformas idag. j
p
Avdelningen med personliga reflektioner
börjar med en artikel av Israel Bartal som
reflekterar över de ambivalenta känslor som
många judiska migranter i Israel, men även
deras barn som är födda där, har känt gente
mot sina forna hemländer. Han beskriver hur
föräldrarna kunde behålla polskan, men talad
med tydlig jiddischbrytning, som ett sätt att
hålla kvar något av det gamla, medan deras
barn såg judisk kultur i Östeuropa som något
som skulle avvisas och undvikas, genom att
det betraktades genom haskalans ögon, som
de hade blivit lärda i sina israeliska skolor. Som historiker diskuterar han även de senaste
decenniernas förändringar i hur man skriver
de östeuropeiska judarnas historia, och hur
detta har påverkats av vågor av migration och
förnyade kontakter mellan israeliska och euro
peiska forskare. Avslutningsvis diskuterar den före detta
Paideiastudenten Mina Pasajlic det interna
tionella judiska ungdomslägret Szarvas his
torik och betydelse. Lägret grundades för tre
decennier sedan och tar varje sommar emot
cirka 1 600 barn från mer än fyrtio försam
lingar i tjugofem länder. Med utgångspunkt
i sina egna erfarenheter som deltagare och
programansvarig, men även på basis av andra
deltagares erfarenheter, reflekterar Pasajlic över
vilken betydelse lägret har haft när det gäller
att främja och stärka judiskt liv i Central- och
Östeuropa. Editorial T The remainder of the issue is dedicated to
a unified theme edited by the guest editors
Lukasz Gorniok and Lena Roos. It primarily
contains revised versions of lectures and pres
entations given at the conference ‘1989–2019:
Jews in Post-Communist Europe’ organised by
Paideia – The European Institute for Jewish
Studies in Sweden, in Stockholm from 31
January to 1 February 2019.1 These texts dis
cuss the reconstruction of Jewish communities
and identities in post-Communist Europe. In
particular, the essays deliberate on different
ways in which it has become possible to create
meaningful Jewish life in the specific context
of the post-Communist era since 1989. The processes of shaping European Jewish
communities and identities have also been
addressed during past conferences organised
by Paideia. Subjects such as ‘exile’, ‘migra
tion’, ‘multiculturalism’ and ‘home’ have been
examined in relation to Jewish identity within
contemporary Europe, and globally.h This most recent conference, marking thirty
years since the fall of the Berlin Wall, was no
different. Through a series of lectures and dis
cussions on Jewish life in Poland, Germany, the
Czech Republic, Hungary, Russia and other
locations, presenters debated the challenges and
concerns of new freedoms, cultural creativity
and the realignment of memory. In addition,
questions of Jewish leadership and community
were raised and discussed. Conference present
ers included representatives from universities
from Europe and Israel, educators and activists
working in the field of European Jewish life and
culture, many of whom were Paideia alumni.h The subject of the revival of European
Jewish cultural and intellectual life in the
wake of Communism has been at the heart of
Paideia since its very foundation. The insti
tute, founded in 2000 through grants from the 1
The conference was organized with the sup
port of a broad spectrum of institutions:
The Swedish History Museum, the Goethe
Institute Schweden, Södertörn University,
Uppsala University, Hochschule für jüdische
Studien, Heidelberg, the Swedish Ministry
for Foreign Affairs, the Ukrainian Institute in
Sweden, Judisk Kultur, Universität Potsdam,
the Europe for Citizens Programme of the
European Union and the Embassy of the
Czech Republic. The conference focused on communities,
individuals, activists, migrants, agents of change
and cultural awakening. For them, 1989 was a
milestone not only in European and global
history, but in Jewish history as well. Editorial I och med detta nummer sker också ett
personbyte inom redaktionen: Karin Hedner
Zetterholm stiger år sidan från uppdraget
som redaktör och vi välkomnar istället Svante
Lundgren som ny redaktör tillsammans med
Ruth Illman. Svante Lundgren är docent i
judaistik vid Åbo Akademi, men arbetar nu
som forskare vid Centrum för Teologi och
Religionsvetenskap och vid Centrum för
Mellanösternforskning vid Lunds universitet. Hans forskning har huvudsakligen handlat
om modernt judiskt tänkande, judisk–kristna
relationer, antisemitism och Förintelsen samt
kristna i och från Mellanöstern, framför allt
armenier och assyrier. Hedner Zetterholm
kommer att fortsätta sitt engagemang för NJ
som medlem av redaktionsrådet. Konstanty Gerberts berättelse tar sin
början tidigare, redan på 1970-talet, när han
återger en återfödelse av intresset för judiska
ämnen, och som en parallell, ett uppsving för
antisemitism under samma period. Han skil
drar även den personliga erfarenheten hos en
grupp polska judar som var födda efter kriget,
uppvuxna som polacker, och deras nyvaknade
intresse för judendom och ansträngningar
för att hitta sätt att förena polsk och judisk
identitet under kommunismens sista decennier
och tiden därefter. Även i Alix Landgrebes artikel är det Polen
som står i fokus för diskussionen om vilken
roll antisemitismen spelade i återskapandet
av en polsk nationell identitet efter kom
munismens fall. Artikeln diskuterar även det Nordisk judaistik • Scandinavian Jewish Studies | Vol. 31, No. 1 3 T
his issue of Nordisk judaistik –
Scandinavian Jewish Studies opens with
two articles dealing with Swedish Jewish
history. Maja Hultman writes about how
two Jewish women in Stockholm during the
first half of the twentieth century used their
bourgeois homes to establish their individual
practices of Jewishness. The article by Jens
Carlesson Magalhães deals with the organisa
tion ‘Samfundet I.I: Judiska intresset’ (Society
I.I: the Jewish Cause) and its struggle in the
middle of the nineteenth century for equal
rights for Jews in Sweden. Swedish government, has the mandate to nur
ture the renewal of European Jewish culture,
to support cross-cultural dialogue and to pro
mote a positive paradigm of a minority culture
within European societies. Dedicated to the
strengthening of Jewish culture within wider
European society, Paideia organises various
educational programmes, including the One-
Year Jewish Studies Programme concentrating
on the study and interdisciplinary interpreta
tion of textual sources, the Paideia Project-
Incubator, supporting the development of new
initiatives for European Jewish life and the
Paradigm Programme, with a personal and
intimate approach to Jewish identity. 1
The conference was organized with the sup
port of a broad spectrum of institutions:
The Swedish History Museum, the Goethe
Institute Schweden, Södertörn University,
Uppsala University, Hochschule für jüdische
Studien, Heidelberg, the Swedish Ministry
for Foreign Affairs, the Ukrainian Institute in
Sweden, Judisk Kultur, Universität Potsdam,
the Europe for Citizens Programme of the
European Union and the Embassy of the
Czech Republic. Editorial As Zvi
Gitelman noted in an essay published in late
1990s: ‘Since 1989, it has become possible to rec
reate Jewish life everywhere in the region and for 4 Nordisk judaistik • Scandinavian Jewish Studies | Vol. 31, No. 1 from the former Soviet Union and Israel. individuals to claim or reclaim Jewishness and
practice Judaism. As has often been remarked
of Jews elsewhere, Jewish identity in Eastern
Europe has become a matter of choice, as has
communal reconstruction. […] Jews are able
to choose both their own conceptions of who
they are and the more public and explicit ways
in which they want to express them.’2 Yet, while
these new opportunities have been available,
the processes of recreating Jewish life have
taken different forms in different countries. The papers in this issue encourage readers to
reconsider many of these developments. The section of personal reflections opens
with an article by Israel Bartal, who reflects on
the ambivalent feelings many Jewish migrants
to Israel, but also their Israeli-born children,
have harboured towards their old home coun
tries. He describes how the parents could retain
Polish, spoken with a thick Yiddish accent,
as a way of claiming something of the old,
while their children regarded Jewish culture
in eastern Europe as something to be rejected
and avoided, when seen through the lens of the
haskala, as they had been taught in their Israeli
schools. As a historian, he also discusses the
development of the historiography of the Jews
of eastern Europe over recent decades, and
how it has been influenced by waves of migra
tion and renewed contacts between Israeli and
European scholars. This issue contains two types of contribu
tions. The texts derived from to the Paideia
conference are of two types: the peer-reviewed
academic article by Christina von Braun is
followed by five articles that are rather per
sonal reflections on the theme, by Israel Bartal,
Konstanty Gebert, Alix Landgrebe, Eli Reich
and Mina Pasajlic. Konstanty Gebert’s account starts earlier, as
far back as the early 1970s, when he sketches a
rebirth of an interest in Jewish matters, and as
a parallel, a rebirth of antisemitism during the
same period. Editorial He also relates the personal expe
rience of a group of Polish Jews born after the
war, raised Polish, and their awakened interest
in Judaism and efforts to find ways of reconcil
ing Polish and Jewish identities during the final
decades of Communism and the beginning of
the post-Communist era. Certain themes reoccur in many of the arti
cles: experiences of antisemitism and oppres
sion, but also of renewal and rebirth, of Jewish
life and an interest in Jewish studies. Christina von Braun gives a broad survey of
recent developments of Jewish studies in post-
Communist Europe, with special emphasis on
German-speaking countries, especially the reu
nited Germany. She discusses the renaissance of
the Wissenschaft des Judentums that had come to
a standstill first as a result of National Socialism
and then of Communism, and how this renais
sance is seen both in purely academic studies of
Judaism, and in rabbinical studies. This develop
ment has also been nourished by a migration of
Jews to Germany over the last decades, mainly Poland is also the focus of Alix Landgrebe’s
article, which discusses the role antisemitism
played in the construction of Polish national
identity after the fall of Communism, but also
the renewed interest in Jewish culture and his
tory, and the revival of Jewish life in Poland. Landgrebe discusses a selection of controver
sial cases and how they can be understood in
relation to these parallel processes. 2
Zvi Gitelman, “Reconstructing Jewish
Communities and Jewish Identities in Post-
Communist East Central Europe”, A. Kovacs,
Jewish Studies at the CEU, 1 Yearbook (Public
lectures, 1996–1999) (Budapest, 2001), 35–50. <http://web.ceu.hu/jewishstudies/pdf/01_
gitelman.pdf> Rabbinical studies in Berlin/Potsdam is the
topic of Eli Reich’s article, in which he dis
cusses the Abraham Geiger College (reform)
and the Zacharias Frankel Rabbinical College
(conservative/masorti), which opened in 2001 5 Nordisk judaistik • Scandinavian Jewish Studies | Vol. 31, No. 1 and 2013 respectively. Reich outlines the histor
ical roots of each tradition and how those roots
as well as the contemporary setting impacts
how the studies are conducted today. This issue marks a transition in the editorial
team: Karin Hedner Zetterholm is stepping
down from the position as editor, and instead,
we are welcoming Svante Lundgren from
Lund University as the new editor together
with Ruth Illman. Hedner Zetterholm will
continue her engagement with NJ through her
membership of the editorial board. LUKASZ GORNIOK
LENA ROOS
Gästredaktörer – Guest Editors
KARIN HEDNER ZETTERHOLM
RUTH ILLMAN
SVANTE LUNDGREN
Chefredaktörer – Editors-in-Chief LUKASZ GORNIOK
LENA ROOS
Gästredaktörer – Guest Editors KARIN HEDNER ZETTERHOLM
RUTH ILLMAN
SVANTE LUNDGREN
Chefredaktörer – Editors-in-Chief Lukasz Gorniok is dean of
academic studies of the
One-Year Jewish Studies
Programme and director
of Paideia Project-Incubator
at Paideia – The European
Institute for Jewish Studies
in Sweden. He holds a doc
torate in history from Umeå
University, Sweden. His
research concerned the migration of Polish Jews
and Czechoslovaks to Sweden in the late 1960s. Lena Roos is professor of
the study of religions at
Södertörn University. Her
research has covered top
ics such as medieval and
contemporary Judaism,
religious literature in
Yiddish, teaching religion,
religion and popular cul
ture, religion and sexuality,
religion and food. She is currently working on a
book on religion and gardening. Karin Hedner
Z e t t e r h o l m
is
Associate
Professor of
Jewish Studies
at Lund Uni
versity. Her
fields of inter
est include early Jewish bibli
cal interpretation, interaction
between rabbinic Jews and
Jesus-oriented Jews in anti
quity and Jewish-Christian rela
tions. She is currently involved
in a multidisciplinary project
on identity formation pro
cesses within early Judaism and
Christianity. S v a n t e
Lundgren is
the new editor
together with
Ruth Illman. He
is a researcher
at the Centre
for Theology
and Religious
Studies and the Centre for Middle
Eastern Studies at Lund University. He has done research on modern
Jewish thought, Jewish–Christian
relations, antisemitism and the
Holocaust, and Christians in and
from the Middle East, primarily
Armenians and Assyrians. Ruth Illman is
the Director
of the Donner
Institute
for
Research
in
Religion and
Culture
in
Å b o / Tu r k u ,
Finland and a docent of compar
ative religion at Åbo Akademi
University and of the history of
religions at Uppsala University. Her main research interests
include contemporary Judaism,
interreligious dialogue and cul
tural encounters as well as reli
gion and the arts. Editorial Finally, the Paideia alumna Mina Pasajlic pre
sents the history and impact of the International
Jewish Youth Camp Szarvas. Established three
decades ago, the camp hosts approximately 1600
children from more than forty communities from
twenty-five countries each summer. Based on her
own experience as a participant and programme
director, as well as other participants’ experiences,
Pasajlic reflects on the importance of the camp
in promoting and strengthening Jewish life in
central and eastern Europe. LUKASZ GORNIOK
LENA ROOS Nordisk judaistik • Scandinavian Jewish Studies | Vol. 31, No. 1 6 Nordisk judaistik • Scandinavian Jewish Studies | Vol. 31, No. 1
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https://www.emerald.com/insight/content/doi/10.1108/JHOM-08-2020-0353/full/pdf?title=i-feel-i-have-no-voice-hospital-doctors-workplace-silence-in-ireland
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“I feel I have no voice”: hospital doctors' workplace silence in Ireland
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Journal of health organization and management
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“I feel I have no voice”:
hospital doctors’ workplace
silence in Ireland
Jennifer Creese and John-Paul Byrne
Royal College of Physicians of Ireland, Dublin, Ireland
Anne Matthews
School of Nursing, Psychotherapy and Community Health, DCU, Dublin, Ireland
Aoife M. McDermott
Cardiff Business School, Cardiff University, Cardiff, UK
Edel Conway
DCU Business School, Dublin City University, Dublin, Ireland, and
Niamh Humphries
Royal College of Physicians of Ireland, Dublin, Ireland
JHOM
35,9
178
Received 31 August 2020
Revised 8 March 2021
Accepted 26 March 2021 178 Received 31 August 2020
Revised 8 March 2021
Accepted 26 March 2021 Edel Conway
DCU Business School, Dublin City University, Dublin, Ireland, and Niamh Humphries p
Royal College of Physicians of Ireland, Dublin, Ireland Abstract Purpose – Workplace silence impedes productivity, job satisfaction and retention, key issues for the hospital
workforce worldwide. It can have a negative effect on patient outcomes and safety and human resources in
healthcare organisations. This study aims to examine factors that influence workplace silence among hospital
doctors in Ireland. Design/methodology/approach – A national, cross-sectional, online survey of hospital doctors in Ireland
was conducted in October–November 2019; 1,070 hospital doctors responded. This paper focuses on responses
to the question “If you had concerns about your working conditions, would you raise them?”. In total, 227
hospital doctor respondents (25%) stated that they would not raise concerns about their working conditions. Qualitative thematic analysis was carried out on free-text responses to explore why these doctors choose to opt
for silence regarding their working conditions. Findings – Reputational risk, lack of energy and time, a perceived inability to effect change and cultural norms
all discourage doctors from raising concerns about working conditions. Apathy arose as change to working
conditions was perceived as highly unlikely. In turn, this had scope to lead to neglect and exit. Voice was seen as
risky for some respondents, who feared that complaining could damage their career prospects and workplace
relationships. Originality/value – This study highlights the systemic, cultural and practical issues that pressure hospital
doctors in Ireland to opt for silence around working conditions. It adds to the literature on workplace silence
and voice within the medical profession and provides a framework for comparative analysis of doctors’ silence
and voice in other settings. Keywords Silence, Working conditions, Health workforce, Doctors, Ireland Paper type Research paper Journal of Health Organization and
Management
Vol. 35 No. 9, 2021
pp. 178-194
Emerald Publishing Limited
1477-7266
DOI 10.1108/JHOM-08-2020-0353 © Jennifer Creese, John-Paul Byrne, Anne Matthews, Aoife M. McDermott, Edel Conway and Niamh
Humphries. Published by Emerald Publishing Limited. This article is published under the Creative
Commons Attribution (CC BY 4.0) licence. Anyone may reproduce, distribute, translate and create
derivative works of this article (for both commercial and non-commercial purposes), subject to full
attribution to the original publication and authors. The full terms of this licence may be seen at http://
creativecommons.org/licences/by/4.0/legalcode The authors would like to thank all respondents who took part in the survey and the Medical Council
of Ireland for assistance with distribution. This research was funded by the Health Research Board
(HRB) via an Emerging Investigator Award (EIA-2017–022). The current issue and full text archive of this journal is available on Emerald Insight at:
https://www.emerald.com/insight/1477-7266.htm The current issue and full text archive of this journal is available on Emerald Insight at:
https://www.emerald.com/insight/1477-7266.htm Journal of Health Organization and
Management
Vol. 35 No. 9, 2021
pp. 178-194
Emerald Publishing Limited
1477-7266
DOI 10.1108/JHOM-08-2020-0353 Introduction Organisational voice and silence are of vital importance in healthcare settings, impacting
staff experiences, organisational capabilities and patient safety (Dixon-Woods et al., 2019;
Martin et al., 2018; Wilkinson et al., 2015). The right for health workers to express concerns
about working conditions is enshrined in the Conventions of the International Labour
Organisation (Wiskow, 2017). Learning from the voices of frontline staff is key for
responsive health services and competitive healthcare organisations and is particularly
important in the context of the COVID-19 pandemic where healthcare systems and
organisations need to act and learn as fast as possible (Bohmer et al., 2020; Daphna-Tekoah
et al., 2020). However, studies of health workers across the world show that their concerns
can often go unheard or unexpressed (Hughes, 2019; Landgren et al., 2016; Lock et al., 2017). This research builds on existing work on organisational voice in a healthcare context,
examining the reasons why hospital doctors in Ireland are reluctant to voice their concerns
about their working conditions. Importantly, voice and silence are distinct behaviours, such
that enablers of voice may not be sufficient to overcome silence (Knoll and Redman, 2016). This has resulted in calls for research to focus on why employees withhold their voice and
engage in silence. 179 g g
This study sheds light on the organisational, professional and cultural factors
contributing to that silence in Irish hospital medicine and provides insights that should be
relevant to hospital medicine in other country contexts. Previous studies of doctors in Ireland
and Irish emigrant doctors overseas have touched on issues of voice in relation to unfair
treatment (Crowe et al., 2017) or in the use of exit over voice (Humphries et al., 2019). This
paper builds on the issues these studies introduce, offering a more detailed analysis of
doctors’ silence in the Irish setting, with wider relevance to studies of silence on working
conditions in health outside Ireland. Reducing silence and enhancing voice mechanisms may
help the Irish health system improve learning and support its workforce and reduce the
likelihood of emigration and burnout. The Republic of Ireland operates a two-tiered public–private health system, with the
delivery of public health services under the remit of the Health Service Executive (HSE). Abstract The funders had no role in the design of the
study, in the collection, analysis and interpretation of data, or in writing the manuscript. Introduction Ireland’s public health system has been severely strained and underfunded for decades,
particularly since the global financial crisis of 2008 and subsequent austerity measures,
which have cut public service funding (Burke et al., 2014; McGowan et al., 2013; Turner, 2018). This has resulted in deteriorating job quality for hospital doctors: reduced resourcing, heavier
workloads and longer working hours. In turn, these issues have driven high rates of doctor
emigration (Brugha et al., 2016; Humphries et al., 2019a, b). Amongst those doctors who
remain in Ireland, burnout has been found to be high (Hayes et al., 2019; McNicholas et al.,
2020; O’Dea et al., 2017). At the time of writing, the Irish health system also faces significant
changes and challenges due to the introduction of the Slaintecare healthcare system reforms
scheduled for the 2018–2028 period (Burke et al., 2018) and, though after the data collection
period of this research, the COVID-19 pandemic (Kennelly et al., 2020). Workplace silence and voice p
In organisational literature, silence has a variety of different definitions and interpretations. Silence can be understood at an organisational or group level, stemming from a systemic lack
of formal voice mechanisms within an organisation (Morrison and Milliken, 2000), a
workplace culture of contempt for voice (Morrison and Milliken, 2000; Wilkinson et al., 2015)
or poor “psychological safety”, the belief that the work environment is unsafe for personal
risk-taking (Edmondson, 2019). Ineffective or lack of organisational change can cause a “deaf
effect” (Jones and Kelly, 2014; Martin et al., 2018) where voices calling for change are neither
heard nor actioned and the status quo remains. Silence can also be understood on a personal, individual level, as a conscious withholding
of voice from the organisation by an individual employee (Pinder and Harlos, 2001; Van Dyne
et al., 2003). Silence may be driven by apathy, labelled “acquiescent silence” (Pinder and
Harlos, 2001; Van Dyne et al., 2003), a passive form of silence where voice is desired by
workers but not expected to bring about change, leading to disengagement. Silence may also
stem from fear, labelled “quiescent” (Pinder and Harlos, 2001) or “defensive” (Van Dyne et al.,
2003) silence, where the individual employee perceives negative consequences for themselves
if they speak out. This is an active form of silence, where risks of using voice are consciously
weighed and silence chosen as the best personal strategy. 180 g
p
gy
Silence is shaped by a range of contextual and individual factors that inform how workers
perceive the efficacy and safety of speaking out or remaining silent (Morrison, 2011; Withey
and Cooper, 1989). Working lives are mediated by a range of “conversion factors” (Hobson,
2013) that enable or constrain workers’ agency: societal and regulatory factors like laws or
socio-cultural norms (macro), organisational and departmental factors like policies and
institutional norms (meso) and individual dispositions, beliefs and domestic factors like
family or financial needs (micro) (Wilkinson et al., 2018a, b). These “conversion factors” and
forms of “capital” come together in different ways for different workers; some are silent and
have limited opportunities to use their voices to achieve the change they desire, while others
are enabled and empowered to use their voices for change. Factors causing silence and
enabling voice may also overlap for some individuals (Knoll and Redman, 2016). Workplace silence and voice The concept of voice has been identified and characterised in a variety of ways across
organisational literature. Voice can be understood as discretionary, informal communication
upwards within the organisation, aiming to bring about change when employees are
dissatisfied (Hirschman, 1970; Morrison, 2011). Hirschman positions voice alongside exit and
loyalty as options for dissatisfied employees; Rusbult et al. (1982) add neglect to this list of
options. Voice is also understood as a system-level phenomenon, rather than an individual
one; the process through which employees make actions and appeals for change, and
contribute to organisational decision-making, through the employer or a union (Pyman et al.,
2006; Wilkinson et al., 2004; Wilkinson et al., 2018a, b). In justice terms, voice is understood as
the provision of testimony or whistleblowing to uncover serious organisational problems
(Kenny, 2019; Pinder and Harlos, 2001). Good voice experiences – being able to raise concerns at work and have them actioned
with no negative repercussions – are linked positively to employees’ commitment,
engagement, sense of belonging, self-esteem and job satisfaction (Edmonson, 2009;
Morrison and Milliken, 2000; Ryan and Oestreich, 1991). Working without effective
mechanisms to voice concerns can negatively influence psychological and physiological well-
being (Morrison and Milliken, 2000). Conversely, silence, stemming from an inability to safely
raise concerns at work and have them effectively actioned, has been shown to render
employees less motivated, satisfied and engaged at work, less adaptable to organisational
change and less committed to remain part of the organisation (Morrison and Milliken, 2000;
Ryan and Oestreich, 1991). Ultimately, silence can generate apathy, making employees more
likely to exit their organisation or system, or to be neglectful of themselves or their work,
calling in sick or late, losing interest, making errors and processing problems poorly (Ryan
and Oestreich, 1991; Withey and Cooper, 1989). Silence and voice in the healthcare context Voice has a unique dimension in the healthcare context: as Wilkinson et al. (2020, p. 562) state,
“
i
i
h
lth
k
h
l t
ifi
ll t
ti
t Voice has a unique dimension in the healthcare context: as Wilkinson et al. (2020, p. 562) state,
“any given concern or issue a healthcare worker has may relate specifically to patient care or
their own well-being and working conditions” Healthcare workers including doctors have q
(
, p
)
,
“any given concern or issue a healthcare worker has may relate specifically to patient care or
their own well-being and working conditions”. Healthcare workers, including doctors, have
been actively trained and encouraged to voice concerns directly related to patient care (see, e.g. Ward et al., 2019), and most reportedly will do so (Landgren et al., 2016; Schwappach and
Richard, 2018). Much of the literature on voice in the healthcare context focuses on voice in
patient welfare (Wilkinson et al., 2020). However, doctors are often silent when it comes to
issues relating to their own professional conditions (Martinez et al., 2017; Wilkinson et al.,
2020). The health sector has begun to take the turn towards “fostering climates and cultures
of respect—in part encouraged by the realization that high-reliability organizations in other
sectors do likewise” (Dixon-Woods et al., 2019, p. 579), but progress has been slow and
difficult. e.g. Ward et al., 2019), and most reportedly will do so (Landgren et al., 2016; Schwappach and
Richard, 2018). Much of the literature on voice in the healthcare context focuses on voice in
patient welfare (Wilkinson et al., 2020). However, doctors are often silent when it comes to
issues relating to their own professional conditions (Martinez et al., 2017; Wilkinson et al.,
2020). The health sector has begun to take the turn towards “fostering climates and cultures
of respect—in part encouraged by the realization that high-reliability organizations in other
sectors do likewise” (Dixon-Woods et al., 2019, p. 579), but progress has been slow and
difficult. 181 While, on paper, many hospitals have adopted staff voice mechanisms (Wilkinson et al.,
2018a, b), in practice, poor implementation of staff suggestions or “organisational deafness”
has been identified as rife in healthcare settings (Attree, 2007; Pope and Burnes, 2013;
Wilkinson et al., 2018), leading to apathy amongst doctors. Avgar et al. Silence and voice in the healthcare context (2016) suggest that
hospitals where doctors and administrators work collaboratively are more likely to have less
silence. Yet, the cultural divides between doctors and administrators are widespread in
contemporary hospitals, which create a barrier to engagement and collaboration (Keller et al.,
2019). Voice and union effectiveness in hospitals are also closely linked (Avgar et al., 2016), yet
Lipset et al. (2004), studying professional union participation in both Canada and the USA,
suggest that physicians are largely reluctant to join unions or use them for industrial action
as they consider them “unprofessional”. Professional culture in medicine is a key factor influencing silence around working
conditions. The medical profession sets norms that govern professional identity, conduct and
belonging, which are communicated to new entrants to the profession via the formal and
“hidden curriculum” (Mahood, 2011; Monrouxe, 2010). Several international studies of medical
culture argue that the expectation of silence about negative work experiences is central to the
“hidden curriculum” within medicine (Helmreich and Merritt, 1998; Wear and Zarconi, 2010). Professional medical culture may also be seen to constitute a “field” in the sense of Bourdieu
(1982), where those at the core with high cultural and social capital set and emphasise norms,
while those on the periphery with less cultural and social capital have less legitimacy in the
field (Anheier et al., 1995). In medicine, the stratified power structures and power bases within
the field dictate who does not have the “capital” to be able to use voice safely – those lower in
the professional hierarchy, women and those from overseas (Luke, 2003). The health sector, then, has several factors that foster different reasons and outcomes for
silence than other sectors. It is governed by powerful cultural forces and values, often
dissociated from the administrative structures that manage doctors as workers. Unlike in
other sectors, the nature of healthcare work is also not focused on productivity or economic
output, but on treating all patients to the extent of their abilities and treatments. The
outcomes of silence – exit, neglect, workplace error, reduced engagement and adaptability,
and poor well-being – are of particular significance in healthcare. However, while these
outcomes have been well documented and publicised, comparatively little attention has been
paid to the reasons why doctors remain silent. Silence and voice in the healthcare context This prompted the focus in this paper on those
respondents who would not voice concerns about working conditions and exploration of the
factors behind this decision. JHOM
35,9 Method This paper analyses voice-related closed and open-ended questions from a national cross-
sectional survey of hospital doctors in Ireland. Using the Qualtrics online survey tool, the
survey was distributed to a grade-stratified sample of 5,356 hospital doctors via the national
Medical Register, with the assistance of the Medical Council of Ireland (for more information
on methods, see Humphries et al., 2020). An overall response rate of 1,070 responses (20%)
was achieved – in line with similar surveys of hospital doctors (O’Brien et al., 2019; Shanafelt et al., 2019) – which was reduced to 990 after filtering out incomplete responses. The survey
included a mix of quantitative and open-response qualitative questions. In total, 895
respondents answered the question “If you had concerns about your working conditions,
would you raise them?”; 450 stated they would, 227 stated they would not and 208 were
unsure. et al., 2019) – which was reduced to 990 after filtering out incomplete responses. The survey
included a mix of quantitative and open-response qualitative questions. In total, 895
respondents answered the question “If you had concerns about your working conditions,
would you raise them?”; 450 stated they would, 227 stated they would not and 208 were
unsure. Survey respondents who answered that they would not raise concerns about their working
conditions were given a follow-up open-text question asking them to elaborate why not. Respondents who answered “yes” or “unsure” to raising working conditions were not
prompted to elaborate. In total, 200 “no” respondents elaborated on their responses, and these
were analysed utilising a thematic textual analysis approach (Braun and Clarke, 2006). This is
a method used effectively in other mixed methods studies in health services research
(Humphries et al., 2015, 2020; Popping, 2015). A combination of inductiveand deductivecoding
was employed (Fereday and Muir-Cochrane, 2006). Deductive themes of apathy and fear
(i.e. acquiescent and quiescent silence) were generated, in accordance with Pinder and Harlos
(2001). Broader inductive themes and subthemes were also identified in the responses and
coded (Figure 1). Coding was completed by the first author and revised through iterations. Verbatim quotes from respondents are used throughout the paper, and respondents are
referred to as R (Respondent), XXX (survey number), followed by their grade (i.e. Intern, Senior
House Officer (SHO), Registrar, Specialist Registrar (SpR), Consultant). 182 House Officer (SHO), Registrar, Specialist Registrar (SpR), Consultant). The 227 respondent doctors who would not voice concerns about their working conditions
(i.e. Method silent doctors) came from all levels of seniority and from all backgrounds. Table 1 shows a The 227 respondent doctors who would not voice concerns about their working conditions
(i.e. silent doctors) came from all levels of seniority and from all backgrounds. Table 1 shows a SILENCE
Apathy
System won't
change
Strain
Powerlessness
No me to
change
Norms
Societal
Organisaonal
Professional
Fear
Workplace
treatment
Gender
Naonality
Seniority
Vulnerability
Precarious
employment
Career
Progress
Total respondents
227 (%)
Gender
Male
108 (48)
Female
117 (51)
Prefer not to say
2 (1)
Citizenship country
Irish
172 (76)
EU country
17 (7)
Non-EU country
38 (17)
Grade
Consultant
35 (15)
Senior/SpR
43 (19)
Registrar
42 (19)
SHO
52 (23)
Intern
55 (24)
Status
Non-consultant hospital doctor (NCHD) (yes)
192 (85)
Locum contract (yes)
15 (7)
Figure 1. Themes and sub-
themes from open-
ended responses to
question “Why would
not you raise those
concerns?”
Table 1. Demographic
characteristics of silent
respondents Status
Table 1. Demographic
characteristics of silent
respondents SILENCE
Apathy
System won't
change
Strain
Powerlessness
No me to
change
Norms
Societal
Organisaonal
Professional
Fear
Workplace
treatment
Gender
Naonality
Seniority
Vulnerability
Precarious
employment
Career
Progress
Figure 1. Themes and sub-
themes from open-
ended responses to
question “Why would
not you raise those
concerns?” Norms Professional No me to
change demographic profile of these by gender, country of citizenship, country of qualification, grade
and locum status. “Working conditions” were not explicitly defined in the survey and were open to
respondents’ own interpretations. The majority of respondent doctors did not specify any
particular “working conditions” they had concerns about in their free-text responses; specific
issues and conditions raised by some respondent doctors included staffing and health service
funding levels, working hours, conflict between healthcare workers and conflict with hospital
administration and the HSE systems. These align with working conditions that hospital
doctors in Ireland, and expatriate Irish doctors in other countries, have identified as
problematic in previous studies (Humphries et al., 2015; Humphries et al., 2019a, b; McGowan
et al., 2013). Findings Apathy: the improbability of change Apathy: the improbability of change
Among silent doctors, silence driven by apathy was more common than fearful silence. In the
200 text responses analysed, there were 140 apathetic responses and 78 fearful responses; 18
respondents gave multiple reasons both apathetic and fearful for their silence. Apathetic
respondents saw no personal risk inherent in speaking out, but no potential for change. They
had little hope that the hospital or health system, already under strain, was likely to change: Management are well aware of these concerns, and yet have no willingness to do anything about
them. Raising them again would not be of benefit (R334, Consultant). Raising concerns about lack of resources in the HSE would be like raising my concern about global
warming (R165, Intern). Raising concerns about lack of resources in the HSE would be like raising my concern about global
warming (R165, Intern). Some felt powerless in effecting change: It’s easier to get on with it than put time and effort into changing things that are not in my power to
change (R391, SpR). [I’m] not sure that it would help or that anybody would listen to me (R357, Intern). [I’m] not sure that it would help or that anybody would listen to me (R357, Intern). Respondents also indicated that from a practical point of view, their heavy workloads left
them with a limited amount of time to push for improved working conditions: Respondents also indicated that from a practical point of view, their heavy workloads left
them with a limited amount of time to push for improved working conditions: I do not have the time or energy to try and promote that change on top of my full-time job (R39,
Intern). I do not have the time or energy to try and promote that change on top of my full-time job (R39,
Intern). There’s no time in the working week to fight for change (R153, SHO). There’s no time in the working week to fight for change (R153, SHO). Normalising silence
Culture heavily influenced the silence of many respondent doctors. This was reported as a
mix of an Irish cultural attitude towards complaint, a feature of the organisational culture of
the health system, and a professional culture within medicine, all of which encouraged a silent
acceptance of working conditions: [There’s] an Irish attitude of “get on with it” (R969, SpR). [I] could be labelled as difficult (R85, Female SpR). Findings [There’s] an Irish attitude of “get on with it” (R969, SpR). Lots of talking but no action, no political will. It’s the Irish way unfortunately (R987, Intern). Raising these concerns achieves no positive outcome. It is considered the “norm” in most. . .hospita
in Ireland (R386, SHO). For new entrants, messaging about voice norms came from their peers and superiors: I’ve had multiple doctors, of various levels, express that “this is what working as a doctor is” (R57,
Intern). [There’s an] atmosphere of we went through this so you should too. Feels like a “rite of passage”
(R365, Intern). 184 Silence is safer for the vulnerable
For many hospital doctors, career stage and grade were important factors in dictating
whether they considered it appropriate to raise concerns about their working conditions. Many non-consultant hospital doctor respondents were reluctant to speak up about working
conditions out of concern for the risk to their career. These doctors are employed on
temporary six-month contracts until they progress to consultant grade, which takes a
minimum of nine years. Many felt that they lacked the security and seniority to voice
concerns and make change happen: NCHDs are essentially told to put up with the way things are (R207, SpR). Junior docs are powerless. . .nobody wants to hear it, consultants do not support [us]
(R188, SHO). Junior docs are powerless. . .nobody wants to hear it, consultants do not support [us]
(R188, SHO). Interns felt particularly reluctant to voice concerns. They remained silent because they were
new to the hospital and to the realities of life as a hospital doctor, but also because of their lack
of seniority. Some established these voice norms themselves: As an intern I am new and feel I do not deserve to [speak up] yet because I have not worked as long as
others (R58, Intern). As an intern I am new and feel I do not deserve to [speak up] yet because I have not worked as long as
others (R58, Intern). I do not feel comfortable as an intern asking a locum consultant [or] complaining to [a] registrar
(R142, Intern). Non-consultant hospital doctors were highly conscious of the reputational risks of
voice, and often saw speaking up as a career risk outweighing any benefit they
might gain. Findings Non-consultant hospital doctors were highly conscious of the reputational risks of
voice, and often saw speaking up as a career risk outweighing any benefit they
might gain. It would risk earning me a reputation as being difficult to work with (R183, Reg). I would be known as someone who caused a problem (R1014, Intern). I would be known as someone who caused a problem (R1014, Intern). I would be known as someone who caused a problem (R1014, Intern). Non-consultant hospital doctors felt like they needed good references from senior hospital
doctors to progress their careers and faced a risk of stalled or blocked career progression if
they voiced concerns: Non-consultant hospital doctors felt like they needed good references from senior hospital
doctors to progress their careers and faced a risk of stalled or blocked career progression if
they voiced concerns: The consultants would not be happy [with me] and I need their reference (R785, SHO). Respondents pointed out that as a small country, Ireland has a relatively small medical
profession who are highly interconnected. This can be beneficial for training and collegiality
but heightens reputational concerns and may discourage voice: In a system as small as Ireland, the consultants you complain to will be the same ones writing your
references [or] sitting on the [training] interview panels, [it’s] better and wiser to just put up with it
(R354, SHO). Some female doctors argued that speaking out would change nothing and would attract the
sort of negative gendered stereotyping that might damage their professional reputation: I would worry about being seen as a nag or troublemaker (R779, Female SpR). I would worry about being seen as a nag or troublemaker (R779, Female SpR). As a non-EU citizen. . . I feel I have no voice and my work and abilities [are] not recognised
(R879, Reg). 185 In addition, some non-European doctors felt that speaking out might damage their
reputations, career progression and job security: As I’m a non-Irish doctor the administration does not care about my concerns, they feel like I’m easy
to replace (R410, Reg). I need to have my contract and work permit renewed every 6 months [so] I do not want to be seen as
being difficult or not hardworking (R553, SHO). Implications of perceived need for silence When lacking the desire or ability to voice, respondents were left to consider their options. For
many, there was little choice but to continue working on in silence, blocking out any concerns
about their working conditions: Everyone has the same concerns, it’s just [become] white noise (R158, Intern). This is tolerable when you know you are only going to be in a centre for a year max. . . disincentive to
change (R171, SpR). This is tolerable when you know you are only going to be in a centre for a year max. . . disincentive to
change (R171, SpR). However, for others, their concerns were significant enough to mean that continuing in
silence was not an option. For these respondents who maintained that voice was impossible
and ineffective, the only remaining option was exit, either leaving the job, the country or the
medical profession: However, for others, their concerns were significant enough to mean that continuing in
silence was not an option. For these respondents who maintained that voice was impossible
and ineffective, the only remaining option was exit, either leaving the job, the country or the
medical profession: [I] would rather leave [the] job, [I] do not think the system has room to change working conditions
(R53, SHO). [I] would rather leave [the] job, [I] do not think the system has room to change working conditions
(R53, SHO). I’m considering leaving this profession because of the conditions and I do not think the change will
come quickly enough to stay working as a doctor in Ireland (R528, Intern). I’m considering leaving this profession because of the conditions and I do not think the change will
come quickly enough to stay working as a doctor in Ireland (R528, Intern). Silence for self, voice for others
While respondent doctors would not speak up about their own working conditions, many
drew a distinction between speaking up for themselves and speaking up on issues directly
related to patient safety: Silence for self, voice for others Silence for self, voice for others
While respondent doctors would not speak up about their own working conditions, many
drew a distinction between speaking up for themselves and speaking up on issues directly
related to patient safety:
I have raised legitimate clinical concerns in the past. . . [but] I have little hope for concerns regarding
NCHD working conditions (R68, SpR). No point raising concerns about our own working conditions because nothing will change. [But] I
would immediately raise concerns if they related to a patient’s care, and have done so in the past
(R354, SHO). While senior doctors were less likely to be silent than junior doctors, some who did choose
silence drew a distinction between speaking up for themselves and speaking up for those
under their supervision:
I would and have for junior doctors and nurses. For myself. . .I think I would not take the mental
strain [of speaking up] (R349, Consultant). I have raised legitimate clinical concerns in the past. . . [but] I have little hope for concerns regarding
NCHD working conditions (R68, SpR). I have raised legitimate clinical concerns in the past. . . [but] I have little hope for concerns regarding
NCHD working conditions (R68, SpR). No point raising concerns about our own working conditions because nothing will change. [But] I
would immediately raise concerns if they related to a patient’s care, and have done so in the past
(R354, SHO). No point raising concerns about our own working conditions because nothing will change. [But]
would immediately raise concerns if they related to a patient’s care, and have done so in the pas
(R354, SHO). While senior doctors were less likely to be silent than junior doctors, some who did choose
silence drew a distinction between speaking up for themselves and speaking up for those
under their supervision: I would and have for junior doctors and nurses. For myself. . .I think I would not take the mental
strain [of speaking up] (R349, Consultant). Discussion
These findings suggest that there are many factors that influence workplace silence among
hospital doctors in Ireland. Among these, improvements in Irish state finances (2013–2019)
have not translated into improvements in working conditions for hospital doctors (Burke
et al., 2014; Humphries et al., 2019a, b). Discussion 186 Silence and apathy
While studies from the corporate world suggest that employees are more likely to keep silent
out of fear rather than apathy (Milliken et al., 2003; Ryan and Oestreich, 1991), respondent
doctors in in this study reported being more likely to remain silent about working conditions
due to apathy than fear, and this was identified across all demographics of those who said
they would not voice. This is similar to findings by Pope and Burnes (2013) in the UK’s
National Health Service (NHS), suggesting that silence in the healthcare sector might stem
more from organisational deafness and stagnation than from a culture of silence. The danger
for the Irish health system is that apathy among hospital doctors may further compound the
problem of silence and dissuade voice (Pinder and Harlos, 2001), allowing problems with
working conditions to proliferate and deteriorate. Findings from the responses suggest that junior hospital doctors in Ireland, particularly
interns, feel they have less cultural capital within the profession, that their concerns are
perceived as less professionally legitimate and are more silent than others. This aligns with
studies of junior doctors in other countries (Landgren et al., 2016; Luke, 2003; Martinez et al.,
2017). Non-consultant hospital doctors are also on temporary contracts, and regularly change
workplaces, which reflects Garsten’s (1999) findings on the liminal and peripheral status of
temporary workers in the workplace. If apathy towards voice becomes endemic among junior
doctors, this may further entrench a status quo of silence and make it more difficult to
encourage voice. Silence and vulnerability Silence for self, voice for others The survey responses suggest that this lack of
improvement has bred a sense of apathy among hospital doctors and an acceptance of poor
working conditions. Yet, as Edmondson (2019, p. 26) argues, “psychological safety is essential
to unleashing talent” across the entire workforce. Responding to workplace silence,
encouraging voice without stigma and using feedback to inform health system
improvements will require system and cultural solutions. Silence and vulnerability Without access to training, these doctors are unable to progress their career; this may
encourage them to stay silent to ensure good reputations and references and improve their
chances of selection for further contracts or training posts. As part of their migration
permission, these doctors are required to maintain consistent employment contracts to have
their visas renewed every six months (Department of Justice and Equality, 2015), which leads
them to report feeling particularly vulnerable. Non-European doctors’ reluctance to voice is
consistent with previous studies of silence and voice, which have found that ethnic minorities
in western settings may be more likely to choose silence (Meares et al., 2004). 187 Exit, voice, loyalty or neglect If reluctant to use voice in response to dissatisfaction with workplace conditions, as the 227
respondents in this study were, doctorsare left with three other options according to Hirschman
(1970) and Rusbult et al. (1982) – exit, loyalty or neglect. They may continue working within the
system (loyalty). They may leave the Irish health system to practise medicine elsewhere, leave
hospitalmedicineforadifferentspeciality or for another profession,orretire (exit). Equally,they
may remain within the system but neglect parts of their work or their non-work lives. Studies
from the corporate world indicate that neglect of work duties is an often-used alternative to
voice (Naus et al., 2007; Withey and Cooper, 1989). Professional and ethical obligations to avoid
patient harm are likely to dissuade doctors from workplace neglect, but apathy and
disengagement, as demonstrated in these findings, may be regarded as a form of neglect. g g
,
g ,
y
g
g
Many respondent doctors indicated, without prompting, that their silence led directly to
plans or hopes for exit. Of the 227 silent respondents in this study, 181 mentioned considering
either leaving Ireland to practice medicine or leaving medicine altogether. When voice poses a
high risk, high energy cost and only a slight possibility of improvement, but exit almost
guarantees improvement, exit may come to be seen as a better use of energy (Hirschman,
1970). This may be especially true of early career doctors, for whom the social and emotional
costs of exit are lower and the potential cost of voice is high, as Humphries et al. (2019a, b)
suggest. Ireland already has a high rate of doctor emigration; studies suggest this is influenced
by health system factors (Byrne et al., 2021; Humphries et al., 2019a, b). Silence and vulnerability Another group prone to workplace silence are those who feel vulnerable within their
professional culture and worry that voice might damage their professional reputation,
particularly non-consultant hospital doctors. They reported feeling vulnerable on multiple
fronts: they are temporary employees, they are trainees or aspiring trainees and most aspire
to achieving a secure consultant status in the Irish health system. These factors interact to
encourage silence. Competition for places in basic and higher specialist training programmes is intense, and
professional connections and references from supervising consultants or clinical directors
can be highly influential in the selection progress. These structures make trainees vulnerable
to reputational capital and the negative consequences of voice, motivating silence; many non-
consultant hospital doctor respondents felt that voicing their concerns about working
conditions might harm their future career opportunities. It is also difficult for non-consultant
hospital doctors to voice through professional lobby groups (Hendrick, 2017): while health is
one of the most prominent issues raised to the Irish government by lobby groups every year
(Register of Lobbying Ireland, 2019), the most vocal medical lobby groups are made up of
consultant doctors only, such as the Irish Hospital Consultants Association (IHCA) and the
Consultants’ Committee of the Irish Medical Association (IMO). Other factors on a demographic level, such as gender and citizenship, may further
compound this vulnerability. Some female doctors and non-European doctors also felt they
had less cultural capital within Irish medicine and were peripheral to those who set the norms
and have legitimacy within the profession to be able to use voice safely. Many female doctors
were particularly concerned about acquiring reputations that played into gender stereotypes,
echoing similar findings from studies of women’s workplace voice (Babcock and Laschever,
2003; Meares et al., 2004; Pinder and Harlos, 2001; Ryan and Oestreich, 1991). Although the
medical profession in Ireland has become increasingly “feminized” in recent decades,
McAleese (2013, p. 12) argues that there remains “a gender bias against female doctors”,
which may make some feel vulnerable and less confident in voicing concerns. Many non-
European doctors were also concerned about the reputational risks of voice. The Irish health
system employs almost 2,500 non-consultant hospital doctors in non-training positions, 40%
of the total cohort of 6,500 non-consultant hospital doctors; over 70% of these non-trainee
non-consultant hospital doctors are non-European doctors (Medical Council of Ireland, 2018). Silence and vulnerability High emigration rates
perpetuate more exit, as people “underestimate the effectiveness of voice when exit is
dominant” (Hirschman, 1970, p. 125). Therefore, workplace silence and the unwillingness or
inability to voice are key factors in the issue of retention for the medical workforce in Ireland. Further research and implications
F
i
h
fi di
ff
l
bl For practice, these findings offer valuable insights into the organisational, professional and
cultural challenges to effective voice for hospital doctors. In particular, they indicate that
many doctors across all grades are silent due to apathy and a lack of faith in the ability to
change the system. Reputational risk, lack of energy and time, a perceived inability to effect
change and cultural norms all discourage doctors from raising concerns about working
conditions. Furthermore, many would prefer to make plans to exit rather than voice their
concerns. This poses a real risk to hospital administrations and health services, especially in
the COVID-19 environment where healthcare worker recruitment is highly competitive and
destination countries are actively pursuing trained doctors with a mind to emigrate (Graham-
McLay, 2020). A “polyphonic” approach (Hujala and Rissanen, 2012), where multiple voices,
including critical voices, throughout all levels of the hospital workforce are sought and given
space, can promote innovation and development and contribute to organisational success for
the hospital and health system. Another approach may be to follow the model of the “freedom
to speak up guardian” in place in the UK’s NHS, which emerged in the wake of the Francis
report demonstrating the horrific impact of employee silence on patient safety issues
(Hughes, 2019). The guardian is an independent officer within the healthcare organisation
who encourages doctors to voice their concerns in confidence, escalates them confidentially to
responsible stakeholders and ensures voicing doctors are aware of actions relating to their
concerns; this may help provide a safe alternative route through which to voice concerns. ;
y
p p
g
The policy implications of these findings reinforce the need for health systems across the
world, including the Irish system, to encourage organisational and professional cultures,
which are open to hearing and learning from frontline staff, without professional risk for
those staff. Such a culture needs to be created and nurtured collaboratively by multiple
stakeholders up, down and across the hierarchies and silos of the system (Edmondson, 2019). As the 2015 Francis inquiry into the NHS found, when staff voice is ignored or seen as
professionally unsafe within a health system, changes to culture and practice need to be made
not only at a workplace level, but at a broader system level (Hughes, 2019). Working conditions and patient safety: a consistent need for voice Working conditions and patient safety: a consistent need for voice
E
bli
i
ff
i
l f
ll h
i l
k
i
i Working conditions and patient safety: a consistent need for voice
Enabling voice effectively for all hospital workers at an organisational level has been shown
to have positive outcomes both for patients’ safety (Schwappach and Richard, 2018; Enabling voice effectively for all hospital workers at an organisational level has been shown
to have positive outcomes both for patients’ safety (Schwappach and Richard, 2018; Wilkinson et al., 2018a, b) and clinical outcomes (Edmondson, 2019), as well as reducing
burnout and improving retention and motivation (Hujala and Rissanen, 2012; Tucker and
Edmondson, 2003; Wilkinson et al., 2018). Respondent doctors understood the importance of
using their voices to speak up about direct patient safety issues. However, the same
consideration for voice relating to their own working conditions was not evident, indicating a
disconnect in doctors’ minds between their own conditions at work and the care they are able
to provide patients. Doctors are trained and encouraged to voice patient safety concerns
(Ward et al., 2019), but are not similarly encouraged to voice concerns about their working
conditions. Doctors may be weighing the “cost” of voice in each situation (Hirschman 1970),
where silence is costly for patient safety and may cause death, but it is voice that is costly for
their own conditions when there may be severe professional repercussions. Yet, Avgar et al. (2016) and Wilkinson et al. (2020) provide clear links between patient safety and hospital
working conditions. Likewise, West et al. (2018) show that patient safety and satisfaction are
linked to physician well-being, which is negatively affected by silence. Initiatives to train and
encourage hospital doctors to voice patient safety concerns are certainly positive steps
towards high-quality healthcare, but to fully optimise patient safety, voice regarding doctors’
working conditions needs to be encouraged and resolved too. 188 JHOM
35,9 Further research and implications
F
i
h
fi di
ff
l
bl Hospital
administrators, health services, medical training colleges and governments need to
understand and temper the factors that lead hospital doctors to remain silent about
problematic working conditions, to build a safe, open system for enabling voice and
implementing solutions. Not doing so poses a substantial risk to service delivery, patient safety and staff motivation and retention, particularly important in the rapidly changing
frontline context of COVID-19 where apathy and fear may have severe consequences
(Bohmer et al., 2020; Daphna-Tekoah et al., 2020). safety and staff motivation and retention, particularly important in the rapidly changing
frontline context of COVID-19 where apathy and fear may have severe consequences
(Bohmer et al., 2020; Daphna-Tekoah et al., 2020). Ho These findings have opened the door to many potential avenues of future research on
voice among hospital doctors. Quantitatively, an in-depth exploration of whether doctors’
likelihood of choosing voice or silence is linked to other factors in their evaluations and
experiences of their workplaces may be fruitful. Qualitatively, asking doctors who would
choose voice what factors make them feel confident to do so, or asking doctors unsure
whether they would voice concerns what factors might positively or negatively influence
their decision, and whether these vary by group, may shed further light on voice behaviours
and motivating factors in healthcare organisations. While Humphries et al. (2019) touch on
the role of lack of voice in emigration decisions of Irish doctors, future studies could more
explicitly examine the connection between lack of voice and the pursuit of loyalty or neglect
(Allen and T€uselmann, 2009) amongst hospital doctors in Ireland. Studies could also explore
empirically whether the psychological and physiological effects of lack of voice mentioned by
Morrison and Milliken (2000) are experienced by hospital doctors. Studies of the use of social
media at work as a form of workplace voice by doctors may shed light on whether silent
doctors are raising concerns about working conditions in other ways, particularly the new
generation of doctors (Holland et al., 2019). Comparative studies between Ireland and other
nations may illuminate whether the factors limiting voice are specific to Irish cultural and
health system phenomena, or are indicative of wider professional norms. 189 Limitations
Th fi di The findings of this study may not be representative of the experience of all hospital doctors
for several reasons. Firstly, the low response rate, while similar to previous studies of doctors
(O’Brien et al., 2019; Shanafelt et al., 2019), may mean that the response trends within the
cohort of respondents are not representative of the wider profession. The response rate and
the self-selecting nature of participation may also mean the possibility of bias, as respondents
may be made up of doctors with more negative experiences wishing to express their views
anonymously. Alternatively, some doctors who experience very high levels of fear at
speaking out about conditions may have declined the opportunity to participate in the survey
over fears of disclosure and repercussions, despite clear messaging around the anonymity
and security of responses. Open-ended responses within the survey may also not have
captured the experiences of all doctors, or even all respondents, as open-ended questions
often have higher non-response rates, and some respondent groups are more likely to provide
responses in this format than others (Miller and Lambert, 2014). Specifying a definition of
“working conditions”, or providing a list of potential issues from which respondents could
select, may have shed more light on the particular concerns they had but chose to remain
silent about. Finally, the survey also only provided the option for silent doctors to explain
why they would not raise concerns; asking voicing doctors for the reasons why they do raise
concerns, or unsure doctors what factors might influence their choice, may have provided
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hi
d h This study has highlighted why hospital doctors may opt to remain silent about their work-
related concerns. Silence places doctors at risk of self-neglect, burnout and exit. This poses
risks in particular for the Irish hospital system, given its existing issues with stress and
scarcity, precarity, competitive progression and heavy reliance on migrant and temporary
workers. There is a need for thoughtful solutions to encourage and protect voice for those hospital doctors who are silent out of fear. There is also a need to encourage a culture of
change and improvement informed by frontline voice, to re-engage those hospital doctors
who are silent out of apathy. This is increasingly pertinent in world where COVID-19
continues to challenge the capacities of healthcare systems and healthcare organisations, as
well as the working lives and well-being of the medical workforce. Significant changes to both
the operating environment and organisational culture of hospital systems, and the
professional culture of medicine, can help address the factors preventing doctors from
exercising their rights to workplace voice mechanisms and raising concerns. 190 JHOM
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Platelet-rich plasma as a treatment for DE Quervain's Disease
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Al-Qadisiah medical journal
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Platelet-rich plasma as a treatment for DE Quervain's Disease Iskandar Mahdi AL-Ardi , college of medicine,Al-Qadisiyah Universi ABSTRUCT: platelet-rich-plasma is increasingly used in De Quervain's Disease. This study show the
clinical outcomes of a single PRP injections.my study was done between Dec. 2015 and Oct. 2016, thirty patient receive single injection of platelet-rich –plasma All patients underwent
prospective clinical evaluation, including visual analog scale (VAS) for pain for six month
follow up .most of the patient (VAS) significantly improve from 5.9,to 2.0 (p > 0.05) after
injection with platelet-rich –plasma . PRP injections in De Quervain's Disease showed better improvement in outcomes . ct o ;
0.52% of them is males and 1.32% in
females.[8] xDe Quervain’s typically
present in the xfifth and sixth decades of
xlife, and xcommon in pregnantx and
lactatingx females.[4] Fritz de Quervainz first describedz
Dex Quervain’s tenosynovitis in 18952.[1,2]
Dez Quervain’s diseasex is defined as
soreing xstenosing tenosynovitis of the first
xdorsal compartment of the hand.[3,4] It is
generally xcaused by abuse or an increasex
in repetitive activityx, resultant in crop
xmicrotrauma from monotonous glidingz of
the first dorsal xcompartment tendons
(zabductor pollicis longusz or APL, and
zextensor pollicis xbrevis or EPB) under the
xsheath of the first compartmentz above the
styloid of the xradius cousing to xthickening
of the extensor retinaculumx of cwrist.[2,3,5]
.Influencing xmovements enclose powerful
xgrasping with ulnar deviance or xrepetitive
usage of the xthumb (like sporty xpursuits,
such
as
golfx,
fly-fishing
and
row
xsports)[.3] The generalx management process is as
xfollows: conservative measures such as
limitingx usage of xthumb, supports and
intra-sheath xsteroid injections, and, if those
procedures arex unsuccessful, tendon
xsheath of the first xdorsal compartment is
xsurgically freed [9, 13]. usually xsurgery is
often done without xexhausting all these
conservative xtreatments. Though there are
new xreports on the effectiveness of the
xintra-sheath injection of triamcinolone
acetonide (TC), which is a long-acting and
xlyophobic
steroid,to
the
patients
complaining snapping digits [9, 11, 12], no
xcomprehensive reports telling the xclinical
results of intra-sheath injectionz in the
management of de Quervain’s zdisease. p
Patientsa frequentlyxcomplaining of radialx
wrist discomfort with xthumb activities and
tenderness
above
the
first
xdorsal
compartment.[2–4 ]Diagnosis is typically
xconcluded by a positive xFinkelstein’s
exam (that result in a reproduction of the
aching
at
the
radial
xstyloid),
The
reliabilityx,
validity,
xspecificity
and
sensitivity of thisx test has not been
xreported,.[5–7] It has also been xsuggested
that a patient must have xpain four days out
of xseven.[8] AL-Qadisiyah Medical Journal AL-Qadisiyah Medical Journal AL-Qadisiyah Medical Journal Methods Our study was zdone at the zOrthopaedic
Department
of
zal
qadissyia
teaching
hospitalz, Iraq, This study was done between
Dec. 20151 and Oct. 20161. After approvala
from ethical committee wthirty patients were
qincluded in study after screening &
qexclusion from diagnosed criteriax. The patientsa involved in the studya were
greater than twentyz years, develop hurt
with
thumbz
movements
that
cause
tenderness
above
the
first
dorsal
compartmentz. Our study was zdone at the zOrthopaedic
Department
of
zal
qadissyia
teaching
hospitalz, Iraq, This study was done between
Dec. 20151 and Oct. 20161. After approvala
from ethical committee wthirty patients were
qincluded in study after screening &
qexclusion from diagnosed criteriax. The patientsa involved in the studya were
greater than twentyz years, develop hurt
with
thumbz
movements
that
cause
tenderness
above
the
first
dorsal
compartmentz. The patientsa involved in the studya were
greater than twentyz years, develop hurt
with
thumbz
movements
that
cause
tenderness
above
the
first
dorsal
compartmentz. The incidence of xDe Quervain’s is not
well-known in xprimary care, but the
prevalencex that found in people of sUK as 197 Vol. 13 No.23 2017 AL-Qadisiyah Medical Journal There are many kindz of systems used to
preparez the PRPz. In our study we used the
Ycellbio
prpz
systemz
. For
PRPz
preparation, 20 mL venous bloodz was
drawn from the antecubitalz vein using an
aseptic ztechnique and mixed with the
anticoagulant zcitrate phosphate dextrose
adenine (CPDA-1) 1.51 cc. The blood was
then aplaced into the PRPz kit and
centrifuged for 43 minutes at 3,4003 rpm to
separate it into platelet-poora plasma, red
cellsz, and PRP. After blood was collectedz,
1.52-23 mL PRPw was made and used for
winjection. A sterile field was set upa and
ensure
throughout
the
zprocedure. Dependingz upon the clinical examinationz
the injection inserted into one apoint nearly
above the indurated ztendon sheath in the
first zdorsal compartment of the wrist. whichz was before zconfirmed with clinical
xexamination. (Table 2). Those patients who are treated
with cPRP therapy xVAS score decreases at
three zmonths and remnants xconstant until
six xmonths. At xone, three and six xmonths
those who are on PRP therapy xVAS score
remnants considerably lesser xthan befor the
xtreatment. Discussion These
zfindings
are
consistent
with
zprevious
prospective
xstudies
that
demonstrate
zbenefits
conferred
byx
intratendinous
xPRP
insertional
tendinopathiesx. [14]
and
xprovides
satisfactory
results
in
young
subjects
recalcitrantz non-insertional xtendinopathy
reducing xpain and improving functionx[15]. And Thesex findings also are in xagreement
with previous literature data in patients
xsuffering from xAchilles, cpatellar, and
elbow tendinopathiesx . These
zfindings
are
consistent
with
zprevious
prospective
xstudies
that
demonstrate
zbenefits
conferred
byx
intratendinous
xPRP
insertional
tendinopathiesx. [14]
and
xprovides
satisfactory
results
in
young
subjects
recalcitrantz non-insertional xtendinopathy
reducing xpain and improving functionx[15]. And Thesex findings also are in xagreement
with previous literature data in patients
xsuffering from xAchilles, cpatellar, and
elbow tendinopathiesx . the PRPa treatment should be xadapted as a
best of ztherapy for relief zsymptoms . Though this must be zadvised merely next
other ztype of nonsurgical ztreatment failed
xbecause
lower
involvement
ofx
tools/technologies & fewer contact toz blood
xproducts in other type of therapiesx
.[16,17,18,19] the PRPa treatment should be xadapted as a
best of ztherapy for relief zsymptoms . Though this must be zadvised merely next
other ztype of nonsurgical ztreatment failed
xbecause
lower
involvement
ofx
tools/technologies & fewer contact toz blood
xproducts in other type of therapiesx
.[16,17,18,19] To all xpatient used a xpovidone iodine
dressing at the xinjection site after the
procedurec. Patient was watchedx for 153
minute , at that time they xdischarged with
advicex to takings the rest in xnext 243
houre. The patient cadvised not takings an
anti-inflammatoryx
pain-relieving
drugsx.The xparacetamol was the only
painkiller xwhich was givenx. all xpatients
assessed with VASx score earlier the
procedurex & later one, cthree & sixc
months ainterval. The main xfindings of this xstudy are that
xPRP injection xresulted in better pain
control and the improvementx in functional
xoutcome was stable and xmaintained up to
a mid-term xfollow-up. p
It is current xopinion that the therapeutic
xactivity of PRP is xmainly due to the
release of many growth factorsx (GFsx),
which can act on xmany aspects of tendon
xrepair,
including
angiogenesisx,
xchemotaxis, and cell xproliferation by
activating intracellular xsignal-transduction It is current xopinion that the therapeutic
xactivity of PRP is xmainly due to the
release of many growth factorsx (GFsx),
which can act on xmany aspects of tendon
xrepair,
including
angiogenesisx, Results The xresults evaluated on the xbasis of
VASx score. We asked the xpatients to
degree their resultsx dependent on the
xscoring scheme of VASx (Tables 1).The
patient xfollow up complete at one xmonth,
three & six months. All Patients xasked
about the xpain by VASx score. All patients
xexamined
for
existence
of
any
xcomplication of thez injection xsite like
xinfection, loss of functionz and existence of
zstiffness and tendon zrupture. In our study
no one of patient developz complication like
xmentioned above. activating intracellular xsignal-transduction
xpathways . [18,19] In the wshort sterm (1–33 months) xeffect ,
xGFs can directly sstimulate tenocytes to
xproduce
extracellularx
matrix,
and
xpromote
neofibrils
xformation
and
remodelingx. Insulinlike GF-12 stimulates
sproduction of xcollagen.[19] remodelingx. Insulinlike GF-12 stimulates
sproduction of xcollagen.[19] in elong-term (62– 122 months), depend on a
direct astimulation, probably relies on the in elong-term (62– 122 months), depend on a
direct astimulation, probably relies on the After one xmonth of treatment xVAS score
significantly ( p < 0.0012 ) in PRPx therapy 198 Vol. 13 No.23 2017 AL-Qadisiyah Medical Journal activationx
of
resident
wtendon
stem/xprogenitor cells (xTSPCs), which have
been recentlyz identified in xtendons tissue
from xdifferent animal species. Like stem
xcells found in adult tissues, TSPCs are
zbelieved to be the source of xrecent
differentiated xtenocytes, responsible for
maintainingx adequate xtenocyte numbers in
the xtissue throughout life and zreplenishing
them after injuryx.[20] activationx
of
resident
wtendon
stem/xprogenitor cells (xTSPCs), which have
been recentlyz identified in xtendons tissue
from xdifferent animal species. Like stem
xcells found in adult tissues, TSPCs are
zbelieved to be the source of xrecent
differentiated xtenocytes, responsible for
maintainingx adequate xtenocyte numbers in
the xtissue throughout life and zreplenishing
them after injuryx.[20] Conclusions Althoughz it is an invasive xmethod, it has a
lot of cadvantages in that. It is less time
consuming and has an sautologous nature
with easy sapplication. We believe that xPRP
injection should be offeredx to all patients
with xDe Quervain’s diseasex after xfailure
of other conservative treatmentx . Further
xcomparative studies xwith other type of
xinjection or zsurgery are requiredx to
evaluate the long-term xoutcomes . Regarding the zamount of injectionx ,
Although smaller xvolume (2-32 ml) of
PRPx was injected in present xstudy or even
1.52 ml such as in previous xstudy, the
proportion of spread beyond ztendon was
little.so the amount of PRPx is even 1.52 ml
is adequate to achieve xgood result. on the
xother hand the xgreater volume of xPRP
could be an xoption. However, large
volumex can lead to further diffusionx and
require much more zblood collectionx, which
is undesirable. References [1] Huang TH, Feng CK, Gung YW, Tsai MW, Chen
CS, Liu CL. Optimization design of thumb spica
splint using finite element method. Med Bio Eng
Comput. 2006; 44(12):1105–1111. [2] Peters-Veluthamaninal C, Winters JC, Groenier
KH, Mayboom-deJong B. Randomized controlled trial
of local corticosteroid injections for de Quervain’s
tenosynovitis in general practice. BMC Musculoskel
Disorders. 2009; 10:131. [3] Retig AC. Athletic injuries of the wrist and hand. Part II: overuse injuries of the wrist and traumatic
injuries to the hand. Am J Sports Med. 2004;
32(1):262–273. In our xstudy, we not used xultrasonographic
injectionx technique and the accuracy of
injectionx was not to be xguaranteed. Therefore we increase the volumex of
injectionx up to 33ml so we can get
xmaximum distribution of wPRP in the area
of xmaximal tenderness ,in comprising with
ultrasoundx injection whichx use 1.52 ml . In our xstudy, we not used xultrasonographic
injectionx technique and the accuracy of
injectionx was not to be xguaranteed. Therefore we increase the volumex of
injectionx up to 33ml so we can get
xmaximum distribution of wPRP in the area
of xmaximal tenderness ,in comprising with
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nonsurgical treatment measures for de Quervain’s
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musculoskeletal disorders, 2nd ed. St. Louis, MO:
Elsevier; 2004: 331. Other
ztherapies
modalities
want
fewxexpertise in contrast to injection PRP
therapyx. all xstaff should be good xtrained
to make xPRP from blood while these is not
required in xsteroid injections or others. xCorticosteroid injections have also been
used xextensively for this problem, but
studies showed that there is controversy
xabout their xefficacy.[22] [8 ]Crawford JO, Laiou E. Conservative treatment of
workrelated upper limb disorders-a review. Occup
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1989–2006 There is essential of xlong time trials to
found xPRP as a best of treatment for long
term permanentx heal from xtendinitis due
to mechanical ccauses. References the study wasx limitedx by a minor sample
csize and absence of a ccontrol group. Larger-scale
randomizedx
controlled
xstudies are required to assistance celucidate
cPRP as a goodx management for this
xmusculoskeletal injuryx. There is essential of xlong time trials to
found xPRP as a best of treatment for long
term permanentx heal from xtendinitis due
to mechanical ccauses. [11]. Lapidus PW (1953) Stenosing tenosynovitis. Surg Clin North Am33:1317–1347 [11]. Lapidus PW (1953) Stenosing tenosynovitis. Surg Clin North Am33:1317–1347
[12]Newport L, Lane LB, Stuchin SA (1990)
Treatment of trigger finger by steroid injection. J
Hand Surg [Am] 15:748–750
[13]Ta KT, Eidelman D, Thomson JG (1999) Patient
satisfaction and outcomes of surgery for de
Quervain’s tenosynovitis. J Hand Surg[Am] 24:1071–
1077 [12]Newport L, Lane LB, Stuchin SA (1990)
Treatment of trigger finger by steroid injection. J
Hand Surg [Am] 15:748–750 the study wasx limitedx by a minor sample
csize and absence of a ccontrol group. Larger-scale
randomizedx
controlled
xstudies are required to assistance celucidate
cPRP as a goodx management for this
xmusculoskeletal injuryx. [13]Ta KT, Eidelman D, Thomson JG (1999) Patient
satisfaction and outcomes of surgery for de
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based therapies provide additional benefit beyond
physical therapy in resistant elbow tendinopathy: a 199 Vol. 13 No.23 2017 AL-Qadisiyah Medical Journal 2017 prospective,
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autologous blood injections versus platelet rich
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injections: their use and abuse. J Am Acad Orthop
Surg. 1994;2:133e140 [19] Fardale PD, Wiggens ME. Corticosteroid
injections: their use and abuse. J Am Acad Orthop
Surg. 1994;2:133e140 prospective,
single-blind,
randomized
trial
of
autologous blood injections versus platelet rich
plasma injections. Br J Sports Med. 2011;45:966-971
[15] Brox JL. Regional musculoskeletal conditions:
shoulder pain. Best Pract Res Clin Rheumatol. 2003;17:33-56 [20] de Mos M, van der Windt AE, Jahr H, et al. Can
platelet-rich plasma enhance tendon repair? A cell
culture study. Am J Sports Med. 2008;36:1171e1178. y
p
[21] Dohan Ehrenfest DM, Rasmusson L, Albrektsson
T. Classification of platelet concentrates: from pure
platelet-rich plasma (P-PRP) to leucocyte- and
platelet-rich fibrin (L-PRF). Trends Biotechnol. 2009;27:158e167 [16] Cook JL, Purdam CR. Is tendon pathology a
continuum? A pathology model to explain the clinical
presentation of load-induced tendinopathy. References Br J Sports
Med. 2009;42:409-16. [17] Wilson JJ, Best TM. Common overuse tendon
problems: a review and recommendations for
treatment. Am Fam Physician. 2005;72:811-8 [17] Wilson JJ, Best TM. Common overuse tendon
problems: a review and recommendations for
treatment. Am Fam Physician. 2005;72:811-8 [22] Alsousou J, Thompson M, Hulley P, Noble A,
Willett K. The biology of platelet-rich plasma and its
application in trauma and orthopaedic surgery: a
review of the literature. J Bone Joint Surg Br. 2009;91:987e996. [22] Alsousou J, Thompson M, Hulley P, Noble A,
Willett K. The biology of platelet-rich plasma and its
application in trauma and orthopaedic surgery: a
review of the literature. J Bone Joint Surg Br. 2009;91:987e996. y
[18] Sun Y, Feng Y, Zhang CQ, Chen SB, Cheng XG
(2010) The regenerative effect of platelet-rich plasma
on healing in large osteochondral defects. Int Orthop
34(4):589–97 Table 1 : VASx pain xscore in patients with PRPx therapyx. VASx score before
prpx therapy
VASx score at
xone monthz
VASx score at three
monthz
VASx score at six
monthz
7
2
2
1
7
2
3
1
6
1
1
1
7
1
1
1
7
1
1
1
6
2
3
1
5
2
2
2
6
2
2
2
7
2
2
2
5
1
1
1
6
2
2
2
7
2
1
1
7
1
1
1
5
2
2
3
6
2
2
3
5
3
2
2
7
2
2
2
6
1
2
2
5
2
2
2
6
2
2
2
7
1
1
1
7
3
3
3
8
3
3
3
7
3
3
3
5
1
1
1
7
3
3
3
6
1
1
1
7
1
2
2
5
2
1
2
6
3
2
2 Table 1 : VASx pain xscore in patients with PRPx therapyx 200 Table 2 zVAS score zbefore and after ztreatment with zPRP
Before
treatmentx
Mean (SDx)
10 Month after
treatment
Mean (SDx)
30 Months after
treatment
Mean (SDx)
60 Months after
treatment
Mean (SDx)
PRPx
therapy
(n ¼ 30)
5.92 (0.76x)
2.11 (1.0x)
2.01 (0.45x)
2.01 (0.452) 201
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Early efficacy and safety of obinutuzumab with chemotherapy in previously untreated patients with follicular lymphoma: A real-world retrospective report of the Polish Lymphoma Research Group
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Advances in Clinical and Experimental Medicine
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Early efficacy and safety of obinutuzumab with chemotherapy
in previously untreated patients with follicular lymphoma:
A real-world retrospective report of the Polish Lymphoma Research Group 2023;32(1):131–136 Address for correspondence
Ewa Paszkiewicz-Kozik
E-mail: Ewa.Paszkiewicz-Kozik@pib-nio.pl
Funding sources
None declared Address for correspondence
Ewa Paszkiewicz-Kozik
E-mail: Ewa.Paszkiewicz-Kozik@pib-nio.pl Research letters Research letters Early efficacy and safety of obinutuzumab with chemotherapy
in previously untreated patients with follicular lymphoma:
A real-world retrospective report of the Polish Lymphoma Research Group Abstract Background. The first-line obinutuzumab-based immunochemotherapy improves the outcome of patients
with follicular lymphoma (FL) compared with rituximab-based regimens. However, infusion-related reactions
occur in almost half of patients during the 1st obinutuzumab administration. Objectives. The study aimed to evaluate the early effectiveness and safety of obinutuzumab-based induc
tion regimens in a real-world setting. Early efficacy and safety of obinutuzumab with chemotherapy
in previously untreated patients with follicular lymphoma:
A real-world retrospective report of the Polish Lymphoma Research Group Ewa Paszkiewicz-Kozik1,A–F, Iwona Hus2,A,C,E,F, Monika Palka3,B,F, Małgorzata Dębowska4,5,A–C,E,F,
Agnieszka Końska2,B,F, Martyna Kotarska1,B,F, Agata Tyczyńska6,B,E,F, Monika Joks7,B,F, Maja Twardosz8,B,F, Agnieszka Giza9,B,F,
Ewa Wąsik-Szczepanek10,B,F, Elżbieta Kalicińska11,B,F, Anna Wiśniewska12,B,F, Marta Morawska13,B,F, Barbara Lewicka14,B,F,
Marcin Szymański1,B,F, Łukasz Targoński1,B,F, Joanna Romejko-Jarosińska1,B,F, Joanna Drozd-Sokołowska15,B,E,F, Edyta Subocz16,B,F,
Ryszard Swoboda8,B,E,F, Monika Długosz-Danecka3,B,F, Ewa Lech-Maranda2,B,F, Jan Walewski1,A,C–F 1 Department of Lymphoid Malignancies, Maria Sklodowska-Curie National Research Institute of Oncology, Warszawa, Poland
2 Department of Hematology, Institute of Hematology and Transfusion Medicine, Warszawa, Poland
3 Department of Lymphoid Malignancies, Maria Sklodowska-Curie National Research Institute of Oncology, Kraków, Poland
4 Department of Mathematical Modeling of Physiological Processes, Nalecz Institute of Biocybernetics and Biomedical Engineering, Polish Academy of Sciences, Warszawa, Poland
5 Department of Computational Oncology, Maria Sklodowska-Curie National Research Institute of Oncology, Warszawa, Poland
6 Department of Hematology and Transplantology, Medical University of Gdańsk, Poland
7 Department of Hematology and Marrow Transplantation, Poznan University of Medical Sciences, Poland
8 Department of Bone Marrow Transplantation and Oncohematology, Maria Sklodowska-Curie National Research Institute of Oncology, Gliwice, Poland
9 Department of Hematology, Jagiellonian University Medical College, Kraków, Poland
10 Department of Hematooncology and Bone Marrow Transplantation, Medical University in Lublin, Poland
11 Department of Hematology, Wroclaw Medical University, Poland
12 Department of Oncology and Chemotherapy, Nicolas Copernicus State Hospital, Koszalin, Poland
13 Department of Hematology, St. John’s Cancer Center, Lublin, Poland
14 Department of Hematology, Ludwik Rydygier Hospital, Kraków, Poland
15 Department of Hematology, Transplantation and Internal Diseases, Medical University of Warsaw, Poland
16 Department of Hematology, Warmian-Masurian Cancer Center of the Ministry of the Interior and Administration Hospital, Olsztyn, Poland
A – research concept and design; B – collection and/or assembly of data; C – data analysis and interpretation;
D – writing the article; E – critical revision of the article; F – final approval of the article
Advances in Clinical and Experimental Medicine ISSN 1899–5276 (print) ISSN 2451–2680 (online)
AdvClinExpMed 2023;32(1):131–136 1 Department of Lymphoid Malignancies, Maria Sklodowska-Curie National Research Institute of Oncology, Warszawa, Poland A – research concept and design; B – collection and/or assembly of data; C – data analysis and interpretation;
D – writing the article; E – critical revision of the article; F – final approval of the article Advances in Clinical and Experimental Medicine, ISSN 1899–5276 (print), ISSN 2451–2680 (online) Adv Clin Exp Med. 2023;32(1):131–136 Adv Clin Exp Med. Background sites. The Independent Ethics Committee of the Maria
Sklodowska-Curie National Research Institute of Oncol
ogy in Warsaw approved the study (approval No. 84/2021),
which was conducted in accordance with the Declaration
of Helsinki and Good Clinical Practice Guidelines. Follicular lymphoma (FL) is the 2nd most common
type of B-cell non-Hodgkin lymphoma. The median age
at diagnosis is 65 years, and the median overall survival
is 20 years.1 There are still controversies about the most
efficient first-line treatment for FL and the time when
it should be initiated. The watch-and-wait strategy is rec
ommended for advanced FL without symptoms or organ
impairment.2,3 All patients included in the study had a histological di
agnosis of FL based on the World Health Organization
(WHO) classification.8 Patients were treated with obinu
tuzumab in combination with CVP, CHOP or BENDA
at the physician’s discretion. Obinutuzumab was admin
istrated at a fixed dose of 1000 mg, in up to eight 21-day
cycles, in combination with CVP (O-CVP), up to six 21-day
cycles with CHOP (O-CHOP), followed by 2 additional
cycles of obinutuzumab monotherapy or up to six 28-day
cycles in combination with bendamustine (O-BENDA). In the 1st cycle, obinutuzumab was administered on days 1,
8 and 15, then only on the 1st day of every next cycle.9
However, physicians modify the treatment schedule, e.g.,
split the 1st dose over 2 days instead of 1 in individual cases. Patients received standard premedication before each in
fusion. Intravenous infusions of obinutuzumab were ad
ministered at the standard recommended infusion rates.9
Seventeen patients received corticosteroid dose the day
before obinutuzumab at 1 participating center. Statistical analyses Descriptive statistics was used to evaluate variables
including demographic, pathologic, clinical, laboratory,
type of treatment, response to the therapy, and deaths
of patients receiving obinutuzumab-based immunoche
motherapy regimens. The χ2 test or the exact Fisher’s test
was used to compare categorical variables. Survival curves
were estimated using the Kaplan–Meier method, and log-
rank test was used to compare time-to-event distributions. Statistical analyses were performed using Matlab v. 2021a Objectives The aim of the study was to evaluate the efficacy and
adverse events, especially IRRs, following obinutuzumab-
based induction therapy in patients with FL in need
of treatment. Conflict of interest
None declared
Acknowledgemets
The authors want to thank Marcin Balcerzak for medical
writing advice.
Received on August 3, 2022
Reviewed on September 14, 2022
Accepted on December 9, 2022
Published online on January 5, 2023 over 2 days (100 mg/900 mg). Infusion-related reactions were reported only during the 1st administration
of obinutuzumab in 9.1% of patients, with a similar incidence in those receiving the total dose on a single
day or split over 2 days (p = 0.458). The most common adverse events were hematological. Five patients
died from coronavirus disease 2019 (COVID-19). Acknowledgemets
The authors want to thank Marcin Balcerzak for medical
writing advice. Acknowledgemets
The authors want to thank Marcin Balcerzak for medical
writing advice. Conclusions. The early responses to induction regimens and adverse events profile were similar for
every type of induction treatment. The infusion-related reactions were rare and limited to the 1st dose
of obinutuzumab. Received on August 3, 2022
Reviewed on September 14, 2022
Accepted on December 9, 2022
Published online on January 5, 2023 Received on August 3, 2022
Reviewed on September 14, 2022
Accepted on December 9, 2022 Key words: follicular lymphoma, chemotherapy, obinutuzumab, induction treatment Published online on January 5, 2023 Background The standard of care for initial therapy of patients with
FL in need of treatment is rituximab with one of the fol
lowing regimens of chemotherapy: CVP (cyclophospha
mide, vincristine and prednisone), CHOP (CVP with doxo
rubicin) or BENDA (bendamustine), with rituximab with
CVP (R-CVP) representing the least toxic and R-BENDA
the most active regimen.3,4 The type II anti-CD20 antibody, obinutuzumab, when
combined with chemotherapy, resulted in prolonged
progression-free survival (PFS) compared to rituximab
with chemotherapy in a randomized Gallium study, al
though grade 3 or 4 neutropenia, thrombocytopenia and
infusion-related reactions (IRRs) were more frequent.5,6
Effective measures for reducing incidence of IRRs are
generally adopted, including splitting the dose of the 1st
infusion, slowing down infusion rates and routine use
of pre-medications.7 The response to treatment was evaluated with positron
emission tomography/computed tomography (PET/CT)
in 81.3% of patients (n = 109/134), and with computed to
mography (CT) in 18.7% of patients (n = 25/134), according
to response criteria for non-Hodgkin’s lymphomas or re
vised response criteria when PET/CT was available.10,11
Adverse events (AEs) were described and graded according
to the National Cancer Institute Common Terminology
Criteria for AEs (NCI-CTCAE v. 5).12 Cite as Paszkiewicz-Kozik E, Hus I, Palka M, et al. Early efficacy and
safety of obinutuzumab with chemotherapy in previously
untreated patients with follicular lymphoma: A real-world
retrospective report of the Polish Lymphoma Research Group. Adv Clin Exp Med. 2023;32(1):131–136. doi:10.17219/acem/157290 Materials and methods. Outcomes of patients diagnosed with FL and treated with obinutuzumab between
January 2020 and September 2021 were analyzed. Results. The study group included 143 treatment-naïve patients with FL. The median age was 52 years
(range: 28–89 years); 45.1% of patients had a high-risk disease as assessed using the Follicular Lymphoma
International Prognostic Index (FLIPI). Induction chemotherapy included: O-CVP (obinutuzumab, cyclophos
phamide, vincristine, prednisolone) in 49.0% of patients, O-CHOP (O-CVP plus doxorubicin) in 28.7% and
O-BENDA (obinutuzumab, bendamustine) in 22.4%. Complete response (CR) and partial response (PR) rates
were 69.9% and 26.5%, respectively. There was no difference in response rates between different regimens
(p = 0.309). Maintenance was started in 115 patients (85.2%). In the 1st cycle, obinutuzumab was adminis
tered as a single 1000-milligram infusion in 47.9% of patients, whereas in 52.1%, initial infusions were split Copyright
Copyright by Author(s)
This is an article distributed under the terms of the
Creative Commons Attribution 3.0 Unported (CC BY 3.0)
(https://creativecommons.org/licenses/by/3.0/) E. Paszkiewicz-Kozik et al. Induction obinutuzumab based treatment in FL 132 Conflict of interest
None declared
Acknowledgemets
The authors want to thank Marcin Balcerzak for medical
writing advice. Received on August 3, 2022
Reviewed on September 14, 2022
Accepted on December 9, 2022
Published online on January 5, 2023 Discussion Follicular lymphoma patients in this real-world study
were younger than FL patients in general population
in Poland (median age: 52 years compared to 61 years,
respectively),14 and younger than patients treated in Gal
lium study (median: age 59 years) that established supe
riority of obinutuzumab over rituximab (both combined
with chemotherapy). Briefly, 45.1% and 36.5% of patients
had high-risk FL according to Follicular Lymphoma In
ternational Prognostic Index (FLIPI) and PRIMA prog
nostic index (PRIMA PI) assessment, respectively. These
patient characteristics may suggest a preferred choice
of obinutuzumab for younger patients with more aggres
sive disease. The O-CVP regimen was used across the analyzed group
the most often (49.0%). The majority of patients responded
to the induction treatment. Progression of disease (PD)
was reported at the end of immunochemotherapy in less
than 3% of patients. We did not find differences in re
sponse rates between used regimens (Fisher’s exact test,
p = 0.309; Table 2). Clinical and laboratory characteristics
of patients treated according to different regimens are pre
sented in Table 1. The median follow-up was 15 months and the median
number of treatment cycles was 6. Maintenance treat
ment was started in 115 patients (85.2%) at the discretion
of treating physicians. We did not find differences between
regimens regarding progression-free survival (PFS; log-
rank test, p = 0.327) and overall survival (OS; log-rank
test, p = 0.897) (Fig. 1). During the follow-up, 5 patients
exhibited disease progression. No lymphoma-related
deaths were reported. Five patients died from coronavirus
disease 2019 (COVID-19) during the induction therapy
of lymphoma. In our study, we focused on evaluation of response and
safety. The median follow-up time was 15 months. We pre
sented objective response rate of 96.3%. The limitations
of our analysis are retrospective design and small group
of patients, but our overall response rate (ORR) is superior
to the 88.5% of ORR in the Gallium study.5 y
The choice of chemotherapy regimen combined with
the antibody was consistent with the European Society
for Medical Oncology (ESMO) guidelines and practice
in the particular health center.3 So far, it is not clear which
of the recommended induction chemotherapy regimens
is the most effective for FL – CVP, CHOP or bendamus
tine – but except for Gallium trial, they were analyzed
in combination with rituximab. Materials and methods The study included patients aged ≥18 years diagnosed
with FL in need of treatment. The study was conducted
in the Polish Lymphoma Research Group (PLRG) sites
(participating health centers). Clinical and laboratory
characteristics of the patients were identified retro
spectively in electronic records of 14 contributing PLRG Adv Clin Exp Med. 2023;32(1):131–136 133 on a single day or split over 2 days experienced IRRs – 11.8%
and 6.8%, respectively (χ2 test statistics = 0.551, df = 1,
p = 0.458). A median duration of the 1st infusion measured
in 69 patients was 4 h and 52 min (range: 1.30–9.45). (https://www.mathworks.com) and R v. 4.2.0 (R Foundation
for Statistical Computing, Vienna, Austria).13 The χ2 test,
the Fisher’s exact test and the Kaplan–Meier method were
performed using ‘chisq.test’, ‘fisher.test’ and ‘survfit’ func
tions from R, whereas figures were plotted using Matlab. Effects were considered statistically significant for p < 0.05. (https://www.mathworks.com) and R v. 4.2.0 (R Foundation
for Statistical Computing, Vienna, Austria).13 The χ2 test,
the Fisher’s exact test and the Kaplan–Meier method were
performed using ‘chisq.test’, ‘fisher.test’ and ‘survfit’ func
tions from R, whereas figures were plotted using Matlab. Effects were considered statistically significant for p < 0.05. The most common grade 3 to 4 AEs in patients receiv
ing obinutuzumab-containing regimens was neutropenia
(46.1%). Granulocyte-colony stimulating factor (G-CSF)
was used in 94 patients (65.7%), including 69 patients
(48.2%) who received G-CSF as a primary prophylaxis
of neutropenic fever. Grade 3 or 4 thrombocytopenia
occurred in 4 patients (2.8%). The COVID-19 infection
occurred in 19 patients (13.3%), and other infections (pneu
monia, upper respiratory infections, zoster, contagious
impetigo) in 6 patients (4.2%). The incidence of AEs was
similar across chemotherapy regimens used. Results A total of 143 consecutive patients who started treatment
after obinutuzumab was made available for use in Poland
on January 1, 2020, were included in the analysis. The last
analyzed patients began treatment in September 2021. The characteristics of patients are presented in Table 1. According to the Groupe d’Étude des Lymphomes Fol
liculaires (GELF) criteria, the median tumor burden was 2
(range: 0–7). Four patients with GELF = 0 received treat
ment on their explicit request. Discussion In FOLL05 trial, response
rates after induction therapies R-CVP, R-CHOP, R-FM
(rituximab, fludarabine, mitoxantron) were similar (88%,
93% and 91%, respectively, p = 0.247), but improved PFS
of R-CHOP and R-FM over R-CVP were demonstrated.15
The PLRG4 trial compared R-CVP to R-CHOP followed
by rituximab maintenance for the treatment of indolent
lymphoma in need of induction therapy (including FL). Overall response rate (ORR) in the R-CVP arm was 97.1%,
and in the R-CHOP arm – 94.5% (p = 0.218). No differences
is PFS or OS were demonstrated.16 The 1st obinutuzumab dose was administered as a single
infusion on day 1 of the 1st cycle in 68 patients (47.9%)
or split in 2 infusions in 74 patients (52.1%), i.e., 100 mg
on day 1 and 900 mg on day 2 of the 1st cycle. The major
ity of patients (n = 136, 95.1%) received all planned infu
sions in the 1st cycle. Doses were omitted in 7 patients due
to COVID-19 (n = 4), neutropenia (n = 2) or pneumonia
with neutropenia (n = 1). In 27 patients (19.3%), doses were
postponed. In 11 patients, day 8 dose was delayed, and in 20
– day 15 dose. Delays occurred due to grade 3 or 4 neutro
penia (n = 18), admission restrictions due to the pandemic
(n = 5), infections other than COVID-19 (n = 4), COVID-19
(n = 1), and other reasons (n = 3). The IRRs were reported during the 1st administration
of obinutuzumab only, and occurred in 13 patients (9.1%)
(grade 1 or 2 in 11 patients and grade 3 in 2 patients). A similar proportion of patients receiving the 1st infusion E. Paszkiewicz-Kozik et al. Induction obinutuzumab based treatment in FL 134 Table 1. Discussion Baseline characteristics of patients with newly diagnosed FL (number and percentages or median (IQR))
Variable
All
O-CVP
O-CHOP
O-BENDA
Number of patients
143
70
41
32
Gender, male
59 (41.3%)
30 (42.9%)
18 (43.9%)
11 (34.4%)
Age (IQR)
52 (41–63)
55 (43–65)
47 (40–58)
52 (41–67)
FL grade (n = 137)
G1
30 (21.9%)
17 (24.6%)
3 (7.7%)
10 (34.5%)
G2
61 (44.5%)
31 (44.9%)
20 (51.3%)
10 (34.5%)
G3
46 (33.6%)
21 (30.4%)
16 (41.0%)
9 (31.0%)
ECOG Performance
Score (n = 142)
0
42 (29.6%)
22 (31.4%)
15 (37.5%)
5 (15.6%)
1
77 (54.2%)
33 (47.1%)
19 (47.5%)
25 (78.1%)
2
20 (14.1%)
13 (18.6%)
5 (12.5%)
2 (6.2%)
3
3 (2.1%)
2 (2.9%)
1 (2.5%)
0 (0.0%)
Clinical stage
1
1 (0.7%)
1 (1.4%)
0 (0.0%)
0 (0.0%)
2
12 (8.4%)
6 (8.6%)
3 (7.3%)
3 (9.4%)
3
34 (23.8%)
21 (30.0%)
6 (14.6%)
7 (21.9%)
4
96 (67.1%)
42 (60.0%)
32 (78.0%)
22 (68.8%)
B symptoms (n = 141)
63 (44.7%)
34 (49.3%)
18 (45.0%)
11 (34.4%)
Extranodal disease
96 (67.1%)
43 (61.4%)
30 (73.2%)
23 (71.9%)
HGB < 12 g/dL
36 (25.2%)
22 (31.4%)
9 (22.0%)
5 (15.6%)
Elevated LDH
50 (35.0%)
23 (32.9%)
14 (34.1%)
13 (40.6%)
Serum β2-microglobulin >3 mg/L (n = 99)
35 (35.4%)
21 (38.9%)
9 (31.0%)
5 (31.2%)
Bone marrow involvement (n = 134)
73 (54.5%)
36 (58.1%)
24 (58.5%)
13 (41.9%)
Involvement of at least 3 nodal sites (n = 142)
87 (61.3%)
43 (61.4%)
21 (51.2%)
23 (74.2%)
Organ dysfunction
41 (28.7%)
21 (30.0%)
13 (31.7%)
7 (21.9%)
Exudation
19 (13.3%)
11 (15.7%)
5 (12.2%)
3 (9.4%)
Splenomegaly (n = 142)
20 (14.1%)
10 (14.5%)
8 (19.5%)
2 (6.2%)
FL cells in blood (n = 60)
11 (18.3%)
6 (18.2%)
3 (14.3%)
2 (33.3%)
Bulky disease >7 cm diameter
63 (44.1%)
28 (40.0%)
18 (43.9%)
17 (53.1%)
Lymph nodes >3 cm diameter
73 (51.0%)
38 (54.3%)
21 (51.2%)
14 (43.8%)
GELF criteria
(n = 109)
0
4 (3.7%)
1 (1.6%)
2 (5.4%)
1 (11.1%)
1
23 (21.1%)
13 (20.6%)
8 (21.6%)
2 (22.2%)
2
37 (33.9%)
24 (38.1%)
11 (29.7%)
2 (22.2%)
3
22 (20.2%)
14 (22.2%)
6 (16.2%)
2 (22.2%)
4
12 (11.0%)
6 (9.5%)
5 (13.5%)
1 (11.1%)
5
8 (7.3%)
4 (6.3%)
4 (10.8%)
0 (0.0%)
6
1 (0.9%)
1 (1.6%)
0 (0.0%)
0 (0.0%)
7
2 (1.8%)
0 (0.0%)
1 (2.7%)
1 (11.1%)
FLIPI (n = 142)
low risk
29 (20.4%)
15 (21.4%)
12 (29.3%)
2 (6.5%)
intermediate risk
49 (34.5%)
24 (34.3%)
12 (29.3%)
13 (41.9%)
high risk
64 (45.1%)
31 (44.3%)
17 (41.5%)
16 (51.6%)
PRIMA PI (n = 96)
low risk
31 (32.3%)
15 (29.4%)
9 (31.0%)
7 (43.8%)
intermediate risk
30 (31.2%)
15 (29.4%)
11 (37.9%)
4 (25.0%)
high risk
35 (36.5%)
21 (41.2%)
9 (31.0%)
5 (31.2%)
Total number of patients (n) = 143, otherwise indicated in brackets. Discussion There were no differences in overall survival (C) and progression-free survival (D) between obinutuzumab combined with CHOP, CVP and
bendamustine (log-rank test)
O-CVP – obinutuzumab, cyclophosphamide, vincristine, prednisolone; O-CHOP – obinutuzumab, cyclophosphamide, doxorubicin, vincristine,
prednisolone; O-BENDA – obinutuzumab, bendamustine. Table 2. Response to induction regimens
Response/induction regimen
O-CVP
O-CHOP
O-BENDA
All regimens
CR
48 (73.8%)
28 (70.0%)
19 (61.3%)
95 (69.9%)
PR
15 (23.1%)
9 (22.5%)
12 (38.7%)
36 (26.5%)
SD
1 (1.5%)
0 (0.0%)
0 (0%)
1 (0.7%)
PD
1 (1.5%)
3 (7.5%)
0 (0%)
4 (2.9%)
Total
65 (100%)
40 (100%)
31 (100%)
136 (100%)
Not evaluated
5
1
1
7
CR – complete response; PR – partial response; SD – stable disease; PD – progression of disease; O-CVP – obinutuzumab, cyclophosphamide, vincristine,
prednisolone; O-CHOP – obinutuzumab, cyclophosphamide, doxorubicin, vincristine, prednisolone; O-BENDA – obinutuzumab, bendamustine; p = 0.309
(Fisher’s exact test, for CR+PR and SD+PD compared to O-CVP, O-CHOP and O-BENDA).Fig. 1. Overall survival (A) and progression-free survival (B) of patients with newly diagnosed follicular lymphoma treated with obinutuzumab-based regimens. There were no differences in overall survival (C) and progression-free survival (D) between obinutuzumab combined with CHOP, CVP and bendamustine (log-rank test) CR – complete response; PR – partial response; SD – stable disease; PD – progression of disease; O-CVP – obinutuzumab, cyclophosphamide, vincristine,
prednisolone; O-CHOP – obinutuzumab, cyclophosphamide, doxorubicin, vincristine, prednisolone; O-BENDA – obinutuzumab, bendamustine; p = 0.309
(Fisher’s exact test, for CR+PR and SD+PD compared to O-CVP, O-CHOP and O-BENDA).Fig. 1. Overall survival (A) and progression-free survival (B) of patients with newly diagnosed follicular lymphoma treated with obinutuzumab-based regimens. There were no differences in overall survival (C) and progression-free survival (D) between obinutuzumab combined with CHOP, CVP and bendamustine (log-rank test) Fig. 1. Overall survival (A) and progression-free survival (B) of patients with newly diagnosed follicular lymphoma treated with obinutuzumab-based
regimens. There were no differences in overall survival (C) and progression-free survival (D) between obinutuzumab combined with CHOP, CVP and
bendamustine (log-rank test)
O-CVP – obinutuzumab, cyclophosphamide, vincristine, prednisolone; O-CHOP – obinutuzumab, cyclophosphamide, doxorubicin, vincristine,
prednisolone; O-BENDA – obinutuzumab, bendamustine. (Fisher s exact test, for CR+PR and SD+PD compared to O CVP, O CHOP and O BENDA).Fig. 1. Overall survival (A) and progression-free survival (B) of patients with newly diagnosed follicular lymphoma treated with obinutuzumab-based regimens. Discussion IQR – interquartile range; FL – follicular lymphoma; ECOG – Eastern Cooperative
Oncology Group; clinical stage – Ann Arbor clinical stage; HGB – hemoglobin; LDH – lactate dehydrogenase; GELF – Groupe d’Étude des
Lymphomes Folliculaires; FLIPI – Follicular Lymphoma International Prognostic Index; IQR – interquartile range; PRIMA PI – PRIMA prognostic index;
O-CVP – obinutuzumab, cyclophosphamide, vincristine, prednisolone; O-CHOP – obinutuzumab, cyclophosphamide, doxorubicin, vincristine, Total number of patients (n) = 143, otherwise indicated in brackets. IQR – interquartile range; FL – follicular lymphoma; ECOG – Eastern Cooperative
Oncology Group; clinical stage – Ann Arbor clinical stage; HGB – hemoglobin; LDH – lactate dehydrogenase; GELF – Groupe d’Étude des
Lymphomes Folliculaires; FLIPI – Follicular Lymphoma International Prognostic Index; IQR – interquartile range; PRIMA PI – PRIMA prognostic index;
O-CVP – obinutuzumab, cyclophosphamide, vincristine, prednisolone; O-CHOP – obinutuzumab, cyclophosphamide, doxorubicin, vincristine,
prednisolone; O-BENDA – obinutuzumab, bendamustine. Adv Clin Exp Med. 2023;32(1):131–136 135 Table 2. Response to induction regimens
Response/induction regimen
O-CVP
O-CHOP
O-BENDA
All regimens
CR
48 (73.8%)
28 (70.0%)
19 (61.3%)
95 (69.9%)
PR
15 (23.1%)
9 (22.5%)
12 (38.7%)
36 (26.5%)
SD
1 (1.5%)
0 (0.0%)
0 (0%)
1 (0.7%)
PD
1 (1.5%)
3 (7.5%)
0 (0%)
4 (2.9%)
Total
65 (100%)
40 (100%)
31 (100%)
136 (100%)
Not evaluated
5
1
1
7
CR – complete response; PR – partial response; SD – stable disease; PD – progression of disease; O-CVP – obinutuzumab, cyclophosphamide, vincristine,
prednisolone; O-CHOP – obinutuzumab, cyclophosphamide, doxorubicin, vincristine, prednisolone; O-BENDA – obinutuzumab, bendamustine; p = 0.309
(Fisher’s exact test, for CR+PR and SD+PD compared to O-CVP, O-CHOP and O-BENDA).Fig. 1. Overall survival (A) and progression-free survival (B) of patients with newly diagnosed follicular lymphoma treated with obinutuzumab-based regimens. There were no differences in overall survival (C) and progression-free survival (D) between obinutuzumab combined with CHOP, CVP and bendamustine (log-rank test) The Gallium study was not designed to compare differ
ences within chemotherapy groups combined with ritux
imab or obinutuzumab but ORR and PFS were superior
they occurred only during the 1st obinutuzumab infusion. We assume that this phenomenon can be associated with
the underreporting of adverse events in retrospective stud
Fig. 1. Overall survival (A) and progression-free survival (B) of patients with newly diagnosed follicular lymphoma treated with obinutuzumab-based
regimens. Discussion There were no differences in overall survival (C) and progression-free survival (D) between obinutuzumab combined with CHOP, CVP and bendamustine (log-rank test) Fig. 1. Overall survival (A) and progression-free survival (B) of patients with newly diagnosed follicular lymphoma treated with obinutuzumab-based
regimens. There were no differences in overall survival (C) and progression-free survival (D) between obinutuzumab combined with CHOP, CVP and
bendamustine (log-rank test)
O-CVP – obinutuzumab, cyclophosphamide, vincristine, prednisolone; O-CHOP – obinutuzumab, cyclophosphamide, doxorubicin, vincristine,
prednisolone; O BENDA
obinutuzumab bendamustine Fig. 1. Overall survival (A) and progression-free survival (B) of patients with newly diagnosed follicular lymphoma treated with obinutuzumab-based
regimens. There were no differences in overall survival (C) and progression-free survival (D) between obinutuzumab combined with CHOP, CVP and
bendamustine (log-rank test) O-CVP – obinutuzumab, cyclophosphamide, vincristine, prednisolone; O-CHOP – obinutuzumab, cyclophosphamide, doxorubicin, vincristine,
prednisolone; O-BENDA – obinutuzumab, bendamustine. they occurred only during the 1st obinutuzumab infusion. We assume that this phenomenon can be associated with
the underreporting of adverse events in retrospective stud
ies. The initial dose of obinutuzumab was split in 2 in half
of the patients; however, the incidence of IRRs was simi
lar no matter if the dose was split or not. An amendment
of the CLL11 study protocol changing the administration
of the 1st obinutuzumab infusion to patients with chronic
lymphocytic leukemia (CLL) from 1 day to 2 days resulted
in a decreasing grade 3 or 4 IRRs to 20%.18 The practice
adapted from the CLL therapy probably does not need
to be extended to patients with FL. The occurrence of IRRs
in FL and CLL patients beyond the 1st infusion are only
occasional; thus, shortened duration of obinutuzumab they occurred only during the 1st obinutuzumab infusion. We assume that this phenomenon can be associated with
the underreporting of adverse events in retrospective stud
ies. The initial dose of obinutuzumab was split in 2 in half
of the patients; however, the incidence of IRRs was simi
lar no matter if the dose was split or not. An amendment
of the CLL11 study protocol changing the administration
of the 1st obinutuzumab infusion to patients with chronic
lymphocytic leukemia (CLL) from 1 day to 2 days resulted
in a decreasing grade 3 or 4 IRRs to 20%.18 The practice
adapted from the CLL therapy probably does not need
to be extended to patients with FL. Discussion The occurrence of IRRs
in FL and CLL patients beyond the 1st infusion are only
occasional; thus, shortened duration of obinutuzumab The Gallium study was not designed to compare differ
ences within chemotherapy groups combined with ritux
imab or obinutuzumab, but ORR and PFS were superior
with obinutuzumab to rituximab, with consistent effects
across chemotherapy regimens.5 In any of these trials, OS
difference was demonstrated. The events of special interest for safety evaluation were
IRRs, which usually occur during the 1st administration
of obinutuzumab.9 In a phase IV GAZELLE study, where
safety of 90-minute infusion of obinutuzumab from C2 was
evaluated in 101 patients,17 IRRs were observed in the 1st
cycle of treatment with a standard infusion duration. A single patient experienced grade >3 IRRs beyond the 1st
cycle. In our study, the rate of IRRs was even lower and E. Paszkiewicz-Kozik et al. Induction obinutuzumab based treatment in FL 136 infusions starting from the 2nd can be used as a routine,
and since October 2021, obinutuzumab is approved for
induction treatment of FL.9,14 2. Freedman A, Jacobsen E. Follicular lymphoma: 2020 update on diag
nosis and management. Am J Hematol. 2020;95(3):316–327. doi:10.1002
/ajh.25696 3. Dreyling M, Ghielmini M, Rule S, et al. Newly diagnosed and relapsed
follicular lymphoma: ESMO Clinical Practice Guidelines for diagnosis,
treatment and follow-up. Ann Oncol. 2021;32(3):298–308. doi:10.1016
/j.annonc.2020.11.008 Neutropenia was the most common AE that required
therapeutic use of G-CSF and delay of subsequent cy
cles of treatment. Unfortunately, 5 patients died from
COVID-19; there were no other deaths in this group
of patients. Therefore, the protection of patients requir
ing treatment with anti-CD20 antibodies against severe
acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2)
is a high priority, with measures including appropriate vac
cination and/or pre-exposure treatment with anti-SARS-
CoV-2 antibodies, as well as early treatment with anti-viral
agents in case the infection occurs. 4. Salles G, Seymour JF, Offner F, et al. Rituximab maintenance for
2 years in patients with high tumour burden follicular lymphoma
responding to rituximab plus chemotherapy (PRIMA): A phase 3, ran
domised controlled trial. Lancet. 2011;377(9759):42–51. doi:10.1016/
S0140-6736(10)62175-7 5. Marcus R, Davies A, Ando K, et al. Obinutuzumab for the first-line treat
ment of follicular lymphoma. N Engl J Med. 2017;377(14):1331–1344. doi:10.1056/NEJMoa1614598 6. Amitai I, Gafter-Gvili A, Shargian-Alon L, Raanani P, Gurion R. Obinutu
zumab-related adverse events: A systematic review and meta-analysis. Hematol Oncol. 2021;39(2):215–221. doi:10.1002/hon.2828 7. Discussion Byrd JC, Flynn JM, Kipps TJ, et al. Randomized phase 2 study of obinu
tuzumab monotherapy in symptomatic, previously untreated chronic
lymphocytic leukemia. Blood. 2016;127(1):79–86. doi:10.1182/blood-
2015-03-634394 ORCID iDs Ewa Paszkiewicz-Kozik https://orcid.org/0000-0003-2629-3932
Iwona Hus https://orcid.org/0000-0002-6096-4093
Monika Palka https://orcid.org/0000-0002-8558-4214
Małgorzata Dębowska https://orcid.org/0000-0001-8741-7930
Martyna Kotarska https://orcid.org/0000-0003-3918-9704
Monika Joks https://orcid.org/0000-0002-7965-9873
Agnieszka Giza https://orcid.org/0000-0001-9095-9749
Ewa Wąsik-Szczepanek https://orcid.org/0000-0002-9715-3745
Elżbieta Kalicińska https://orcid.org/0000-0002-4797-8001
Marta Morawska https://orcid.org/0000-0001-6836-2903
Łukasz Targoński https://orcid.org/0000-0002-6155-8791
Joanna Romejko-Jarosińska https://orcid.org/0000-0003-4603-1112
Joanna Drozd-Sokołowska https://orcid.org/0000-0002-4562-6264
Edyta Subocz https://orcid.org/0000-0001-5225-3921
Ryszard Swoboda https://orcid.org/0000-0002-5897-3919
Monika Długosz-Danecka https://orcid.org/0000-0002-8927-4125
Ewa Lech-Marańda https://orcid.org/0000-0001-9592-0851
Jan Walewski https://orcid.org/0000-0003-4247-2674 13. R Core Team. R: A language and environment for statistical computing. https://www.R-project.org/. Accessed May 2, 2022. 14. Szumera-Ciećkiewicz A, Wojciechowska U, Didkowska J, et al. Popu
lation-based epidemiological data of follicular lymphoma in Poland:
15 years of observation. Sci Rep. 2020;10(1):14610. doi:10.1038/s41598-
020-71579-6 15. Federico M, Luminari S, Dondi A, et al. R-CVP versus R-CHOP versus
R-FM for the initial treatment of patients with advanced-stage follicu
lar lymphoma: Results of the FOLL05 trial conducted by the Fondazi
one Italiana Linfomi. J Clin Oncol. 2013;31(12):1506–1513. doi:10.1200/
JCO.2012.45.0866 16. Walewski J, Paszkiewicz-Kozik E, Michalski W, et al. First-line R-CVP
versus R-CHOP induction immunochemotherapy for indolent lym
phoma with rituximab maintenance: A multicentre, phase III ran
domized study by the Polish Lymphoma Research Group PLRG4. Br J Haematol. 2020;188(6):898–906. doi:10.1111/bjh.16264 17. Hübel K, Buchholz TA, Izutsu K, et al. Obinutuzumab can be admin
istered as a 90-minute short-duration infusion (SDI) in patients with
previously untreated follicular lymphoma: GAZELLE end of induc
tion analysis. Hematol Oncol. 2021;39(S2):hon.30_2880. doi:10.1002/
hon.30_2880 Conclusions 8. Swerdlow SH, Campo E, Pileri SA, et al. The 2016 revision of the World
Health Organization classification of lymphoid neoplasms. Blood. 2016;127(20):2375–2390. doi:10.1182/blood-2016-01-643569 The first-line treatment of follicular lymphoma pa
tients with obinutuzumab combined with cytotoxic
chemotherapy is efficacious and well-tolerated. The re
sponse to treatment with 3 different chemotherapy regi
mens was similar. Low grade and incidence of infusion-
related reactions that exclusively occurred in the 1st
administration of obinutuzumab support the use of ab
breviated 90-minute infusions starting from the 2nd
treatment cycle. 9. European Medicines Agency (EMA). Gazyvaro. Summary of product
characteristics. Updated May 10, 2022. May 2022. https://www.ema. europa.eu/en/medicines/human/EPAR/gazyvaro. Accessed May 7,
2022. 10. Cheson BD, Pfistner B, Juweid ME, et al. Revised response criteria for
malignant lymphoma. J Clin Oncol. 2007;25(5):579–586. doi:10.1200/
JCO.2006.09.2403 11. Cheson BD, Horning SJ, Coiffier B, et al. Report of an international work
shop to standardize response criteria for non-Hodgkin’s lymphomas. J Clin Oncol. 1999;17(4):1244–1244. doi:10.1200/JCO.1999.17.4.1244 12. National Cancer Institute (NCI). Common Terminology Criteria for
Adverse Events (CTCAE). April 2021. https://ctep.cancer.gov/proto
coldevelopment/electronic_applications/ctc.htm. Accessed May 7,
2022. 1.
Mounier M, Bossard N, Remontet L, et al. Changes in dynamics
of excess mortality rates and net survival after diagnosis of follicular
lymphoma or diffuse large B-cell lymphoma: Comparison between
European population-based data (EUROCARE-5). Lancet Haematol.
2015;2(11):e481–e491. doi:10.1016/S2352-3026(15)00155-6 References 1. Mounier M, Bossard N, Remontet L, et al. Changes in dynamics
of excess mortality rates and net survival after diagnosis of follicular
lymphoma or diffuse large B-cell lymphoma: Comparison between
European population-based data (EUROCARE-5). Lancet Haematol. 2015;2(11):e481–e491. doi:10.1016/S2352-3026(15)00155-6 18. Goede V, Fischer K, Busch R, et al. Obinutuzumab plus chlorambucil
in patients with CLL and coexisting conditions. N Engl J Med. 2014;
370(12):1101–1110. doi:10.1056/NEJMoa1313984
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The Compilation of a Shona Children's Dictionary: Challenges and Solutions
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Lexikos
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The Compilation of a Shona
Children's Dictionary: Challenges
and Solutions Peniah Mabaso, African Languages Research Institute (ALRI), University of
Zimbabwe, Harare, Zimbabwe (penmabaso@arts.uz.ac.zw) Abstract: This article outlines the challenges encountered by the African Languages Research
Institute (ALRI) team members in the compilation of the monolingual Shona Children's Dictionary. The focus is mainly on the problems met in headword selection. Solutions by the team members
when dealing with these problems are also presented. Keywords: SHONA CHILDREN'S DICTIONARY, LOANWORDS, TABOO, HEADWORD
SELECTION, SLANG, TONE MARKING, ILLUSTRATIONS, SYNONYMS, VARIANTS Opsomming: Die samestelling van 'n Sjonakinderwoordeboek: Uitdagings
en oplossings. Hierdie artikel skets die uitdagings waarvoor die spanlede van die African
Languages Research Institute (ALRI) te staan gekom het by die samestelling van die eentalige
Sjonakinderwoordeboek. Die fokus is hoofsaaklik op die probleme ondervind by die keuse van
trefwoorde. Oplossings deur die spanlede by die benadering van hierdie probleme word ook
aangebied. Sleutelwoorde: SJONAKINDERWOORDEBOEK, LEENWOORDE, TABOE, TREFWOORD-
KEUSE, SLENG, TOONAANDUIDING, ILLUSTRASIES, SINONIEME, VARIANTE Challenges encountered in headword selection Headword selection is an important stage in the compilation of any dictionary
as it determines the lexical items to include in and exclude from a dictionary. Svensén (1993: 40) argues that the selection of headwords must be preceded by
a number of decisions regarding the size and aim of the dictionary. For the
SCD compilers, headword selection was difficult mainly because of the speci-
fied nature of the dictionary, which is meant for school children. As a result not
all Shona words could find their way into the dictionary. Only words which
the compilers and reference groups agreed were suitable for the targeted age
group were entered as headwords. In dictionary compilation, headwords have to be carefully selected and
defined in a special way to satisfy the needs of the target users. This can only
be achieved when the users can access lexical items which they anticipate and
which are helpful to them. The selection process for the SCD headwords was
designed according to well-informed principles. The compilers selected head-
words specifically suited for children. In this case, the problem was how to
determine whether a headword was commonly used by children. The problem was partially solved when the compilers agreed that Shona
primary school textbooks should be used as sources for headwords. However,
from all the Shona textbooks, the compilers could not collect enough head-
words for the dictionary. As a result, they decided that words from other pri-
mary school textbooks for subjects such as mathematics and general publica-
tions encompassing subjects such as science, biology, geography, religious
studies and agriculture should also be used as headword sources. The head-
words were then adopted in and adapted to Shona. This also implies that the
dictionary incorporated loanwords to make provision for new concepts which
previously did not exist in Shona. Introduction This article outlines the challenges encountered by ALRI researchers in the
compilation of the monolingual Shona Children's Dictionary (henceforth SCD)
which is the first of its kind in Shona. The compilers adopted some of the tech-
niques used in compiling the advanced Shona dictionary Duramazwi Guru
reChiShona (henceforth DGC). Two of the SCD compilers and editors were also
part of the compilers and editors of DGC. However, the dictionary is different
from the DGC in many respects such as the target users, the structure and the
contents. The SCD is mainly meant for primary school children, the seven to
thirteen years age group and their teachers. Student teachers at teacher training
colleges and their lecturers are also some of the potential target users. On the
contrary, the DGC targeted high school learners and students at tertiary institu-
tions like colleges and universities. g
The main challenge of this project was what exactly such a specified dic-
tionary should contain. Another challenge the compilers faced was the issue of Lexikos 19 Supplement (AFRILEX-reeks/series 19: 2009): 112-119 113 The Compilation of a Shona Children's Dictionary: Challenges and Solutions language dynamism. The question was if slang, colloquial and loanwords
should be included in the dictionary. Should taboo or obscene words be given
as well? Is it important to mark tone? It had to be decided which illustrations
should be included in the dictionary, considering the different urban and rural
backgrounds of the main target users. The compilers agreed on possible solu-
tions to some of the challenges for the dictionary to be user-friendly, satisfying
its users after publication. language dynamism. The question was if slang, colloquial and loanwords
should be included in the dictionary. Should taboo or obscene words be given
as well? Is it important to mark tone? It had to be decided which illustrations
should be included in the dictionary, considering the different urban and rural
backgrounds of the main target users. The compilers agreed on possible solu-
tions to some of the challenges for the dictionary to be user-friendly, satisfying
its users after publication. Loanwords in Shona: Why are there so many? The use of loanwords is not unique to Shona speakers. Shona has not been
immune to language contact and its resultant effect of cultural and language
borrowing. In Zimbabwe, English has always enjoyed a prestigious position in
both urban and rural areas, mainly because Zimbabwe is a former British col- 114 Peniah Mabaso ony. Despite the fact that Zimbabwe has enjoyed political independence for 28
years, English has remained the official language. It is still obligatory for social
promotion, being associated with prestige. Many children from well-to-do
families are sent to Group A schools or former white-only schools where Eng-
lish is used as medium of instruction. In Zimbabwe where many Shona and
Ndebele speakers find themselves in a diglossic situation with English in the
most prominent position, borrowing therefore mainly occurs from English. Chitiga (1995: 6) who discusses the politics of language in Zimbabwe, asserts
that English has assumed the most important position in the life of Zimbabwe-
ans in all formal sectors like education, commerce, media and technology. Owing to the prevailing socio-linguistic situation in Zimbabwe, the Shona lexi-
con largely contains loanwords from English. ony. Despite the fact that Zimbabwe has enjoyed political independence for 28
years, English has remained the official language. It is still obligatory for social
promotion, being associated with prestige. Many children from well-to-do
families are sent to Group A schools or former white-only schools where Eng-
lish is used as medium of instruction. In Zimbabwe where many Shona and
Ndebele speakers find themselves in a diglossic situation with English in the
most prominent position, borrowing therefore mainly occurs from English. Chitiga (1995: 6) who discusses the politics of language in Zimbabwe, asserts
that English has assumed the most important position in the life of Zimbabwe-
ans in all formal sectors like education, commerce, media and technology. Owing to the prevailing socio-linguistic situation in Zimbabwe, the Shona lexi-
con largely contains loanwords from English. Basing themselves on the above historical, social, technological and eco-
nomic factors, the compilers included loanwords as headwords in the SCD. To
exclude loanwords from the SCD was to ignore the actual language situation,
as some loanwords have in fact become part of the Shona lexicon. All lan-
guages develop through borrowing, as Ndlovu (1998: 33) argues: Language change is a universally accepted and attested principle that has long
been established by historical linguistics. Loanwords in Shona: Why are there so many? No language is fixed; all languages
undergo constant change over time. New words are constantly coming into use
and at the same time old words are gradually dropping out of use. This assertion accentuates that the lexicons of all languages are continuously
changing. These language changes are due to a number of factors, among oth-
ers, multilingual interactions as well as the continued spreading of English and
other languages inherited from former colonial powers. In Zimbabwe, literate
Shona speakers use both English and Shona in general conversation causing
English sounds, grammar and vocabulary to exercise a strong influence on
Shona. Criteria used for the selection of loanwords Kipfer (1984: 35) argues that lexicographers have to keep up with new words
and meanings as well as the development of language in general. The SCD
compilers saw it unwise to exclude loanwords since they now constitute a sig-
nificant percentage of the Shona lexicon. They considered the historical, social,
technological and economic factors to justify their selection of loanwords in the
SCD. From the July 2005 outreach programme held by the SCD compilers, dif-
ferent views were received from target users on the issue of loanwords. There
were some Shona language conservatives who were against the inclusion of
loanwords, while 'modernists' argued for their inclusion. Svensén (1993: 47)
asserts that one of the principal recommendations of publishers is that their
new dictionaries contain the latest material. Taking Svensén's point of view 115 The Compilation of a Shona Children's Dictionary: Challenges and Solutions into consideration for the SCD to appeal to its target users, the latest material in
the form of loanwords relating to science education and technology were
included as headwords. Where there is an original Shona term for some words,
that Shona term was used. However, there were some cases where it was found
that loanwords were more popular, even though there were indigenous Shona
terms. For example, it is now rare to hear speakers refer to mathematics/maths
as masvomhu rather than metsi, bicycle as bhizautare rather than bhasikoro, and
sugar as tsvigiri rather than shuga. Faced with such a problem, the SCD compilers (and other ALRI team
members) agreed that not every loanword should find its way into the diction-
ary. Systematic and well-defined ways were followed in selecting loanwords. The SCD compilers chose loanwords from the viewpoint of their stability and
acceptance within the language. It was agreed that loanwords incorporated
into the language and used in formal speech, showing that they have stood the
test of time, should be included as headwords. Another problem for the SCD compilers was their dealing with words
containing letters or digraphs not recognised in Shona orthography. Some let-
ters and digraphs like q, x and hl are prevalent in Shona speech, especially in
the Karanga and Ndau dialects. Although they are found in the major Shona
dialects, they are not part of the standard Shona alphabet. Criteria used for the selection of loanwords Magwa (2002) notes: From 1967 onwards, speakers of different dialects were experiencing certain dif-
ficulties arising from the defective alphabet and the spelling and word division
system. The current orthography is linguistically constricting in a number of
ways. For example, the standard alphabet does not have symbols representing
the sounds [l] and [x], which are found in ChiKaranga, ChiNdau and ChiKore-
kore dialects. The compilers agreed that English loanwords should be adapted to conform to
the Shona CV syllable structure. Most English loanwords are written as they
are pronounced in Shona. Many technical terms such as fax, diskette and com-
puter were entered respectively as fakisi, dhisiketi and kombiyuta in the SCD. Most Shona writers make sure that loanwords from English adhere to the
Shona phonetic system. As has been mentioned earlier, in the Shona phono-
logical alphabet, there is no lateral /l/. The closest equivalent of /l/ in Shona is
the trill /r/. Loanwords like loan and leave were respectively rendered as roni
and rivhi in Shona. However, the use of /r/ in place of /l/ does not apply to
all English loanwords. There are exceptions such as kukala (to colour) and yelo
(yellow) (Chitauro-Mawema 2000: 211). When r replaces l, the meaning of
words changes, for example kukala (to colour) will become kukara (to be
greedy). This shows that there is a limit to replacing loanwords with the letter l
with those with the letter r. It seems that nowadays new words with l are
incorporated into Shona. At present it is rare to hear someone using the ac-
cepted trill r in these loanwords. Shona speakers no longer say raki (luck), rika
(leak) or roni (loan). In the SCD, all loanwords were included and represented 116 Peniah Mabaso as they are pronounced using the letters, digraphs and trigraphs not recognised
in standard Shona orthography. The compilers hope that these considerations
will compel those responsible for changes in the Shona orthography, like the
Shona Language and Culture Association (SLCA) and the Ministry of Educa-
tion, to allow the use of some of the letters which are not recognised in the cur-
rent Shona orthography. Taboo, obscene and offensive words In Shona, it is a cultural taboo to use derogatory words, especially those de-
scribing excretory and sexual organs and acts publicly in spoken or written
form. The compilers had problems in deciding whether to include these ob-
scene and offensive words in the dictionary. A few examples of such words are
gongo (clitoris), jende (testicle) and beche (vagina). Since it is a taboo in Shona
culture to use these words in all public communication, the Shona people have
developed euphemistic terms to refer to these organs or acts. The anus is re-
ferred to as kushure (the back) and the penis as nhengo yomurume (a man's
private part). The use of such euphemistic terms and phrases when referring to
human anatomy and sexual behaviour shows how sensitive the Shona people
are to these obscene and offensive words. The biggest problem likely to be
caused by the use of these euphemistic terms in the SCD is that the children
would not understand them since they appear ambiguous. Including obscene
and offensive words in a dictionary meant for such a young age group would
be a matter of licensing their use in everyday speech. The dictionary would be
the first-ever Shona publication meant for children to use obscene and offen-
sive words. However, excluding them from the dictionary would have been to
ignore the actual language situation. It would also have been to overlook the
educational value that the correct reference to these human anatomical parts
can have for teaching children about sexual diseases and abuse. The SCD compilers also faced many problems in dealing with offensive
words describing certain negative features of people, for instance cripples and
albinos. Shona words like zvirema for cripples, mukawu/musupe/murungu-
dunhu for albinos, hure for a prostitute and ngomwa for a barren woman are
often avoided, especially in the presence of the affected people themselves,
who might feel offended. The use of such terms is against the Shona culture
that protects the socially disadvantaged. Nevertheless, some of these offensive
words were entered into the dictionary. Slang and colloquial words Shona speakers use many slang and colloquial words. According to Tullock
(1994: 1454), slang refers to words, phrases and language use regarded as in-
formal and often restricted to special contexts or peculiar to specific classes of 117 The Compilation of a Shona Children's Dictionary: Challenges and Solutions users. The use of slang and colloquial words varies with age groups. Among
Shona speakers, children and youths seem to be the main age groups that often
use slang. Some slang and colloquial words are only used for a short period
while others are well established in the language. In Shona, slang words like
bhoo (okay) are now part of everyday speech, especially among children. Some
slang words like chibhanzi (money) and shuzura (to go) are transitory. Chim-
hundu (2001: xxxv) likens such transitory slang words to fashion which comes
and goes, and therefore they were not included in the SCD. The SCD compilers agreed that slang words that have become permanent
in the Shona vocabulary should be treated as headwords and given the style
marker "manje" (an abbreviation of "chimanjemanje") to show that they are
colloquial words. Transitory slang words should not be included in the dic-
tionary. The challenge for the SCD compilers was to distinguish between slang
words that are established in Shona and those that are transitory. Tone marking In the DGC, tone was marked. Tone marking in Shona is very important as it
distinguishes between words spelt the same but pronounced differently. For
example, if tone is not marked, there will be no difference between nzara low
tone (hunger) and nzara low, high tone (finger nail). However, primary school
teachers and lecturers consulted during outreach programmes argued that tone
should not be marked in such a dictionary, as it will confuse children. The dic-
tionary should be very simple and concentrate on the meaning of words. Linguistic details The compilers agreed upon the marking of all parts of speech in the dictionary
to give linguistic detail which was thought necessary for such a dictionary. It is
indicated whether a headword is a noun, pronoun, adverb, adjective, preposi-
tion and so on. Idioms Idioms are also important in the SCD since they reflect the culture of the Shona
speech community, so just like the DGC, the SCD includes and explains them. However, not all Shona idioms are included and explained in the SCD but only
those suitable for children. To this purpose, the compilers used Shona primary
school textbooks as sources for the idioms. Synonyms and variants Synonyms are lexical items which have the same meaning or are so close in
meaning that they can be used interchangeably in certain contexts. In Shona the
word baboon can be translated with bveni, dede, diro, gudo and mutiro,
depending on one's dialect. According to Crystal (1997: 408), a variant can be
defined as a set of alternatives in a given context. Shona has many variants,
mainly because of its different dialects. For example, the Karanga people in
some instances use zh and gw where the other dialects like Zezuru and Manyi-
ka use nz and rw respectively as can be seen in words like zhara and nzara
(hunger), -gwara and -rwara (to be sick). The problem regarding synonyms and 118 Peniah Mabaso variants is their treatment in the dictionary, whether to define them separately
or not. In this connection, the compilers followed the procedure of the DGC com-
pilers. The synonym or variant that is commonly used, carries the definition
and all the others that are less common are cross-referenced to it. For example,
for the synonyms bveni, dede, diro, gudo and mutiro, gudo carries the defini-
tion to which the others are cross-referenced, as shown below: bveni D- z 5. ONA gudo 5. gudo D- z 5. Mhuka yemusango ine mahobi neuso hwakada kufanana
nehwetsoko. FAN bveni 5, dede 5, diro 5, mutiro 3. gudo D- z 5. Mhuka yemusango ine mahobi neuso hwakada kufanana
nehwetsoko. FAN bveni 5, dede 5, diro 5, mutiro 3. Variants are defined as follows: Variants are defined as follows: zhara D- z 9. ONA nzara 9. nzara [zhara] D- z 9. Kunzwa kuda kudya nokuti mudumbu munenge
musisina chikafu. The SCD like the DGC contains as much information on the grammar in the
front matter as is necessary for the target users to understand the abbreviations
used. Conclusion The article discussed some of the major problems unique to the SCD, when
compared to other general dictionaries ALRI had compiled and published. Headword selection has proved to be the greatest challenge for the SCD com-
pilers. Different word categories found in Shona like loanwords, offensive
words, taboo or obscene words and slang words made headword selection dif-
ficult. Shona culture and the inadequacy of the Shona orthography caused a
dilemma for the SCD compilers. Adhering to the current Shona orthography
would have meant excluding some important headwords common in speech. The compilers explicitly state the challenges in the front matter and the com-
promises and solutions decided and agreed on. Illustrations Since the dictionary is meant for children in both rural and urban areas, it has
to include illustrations to explain some unfamiliar headwords in the dictionary. The major problem was to determine which headwords are unfamiliar to the
target users, especially considering their different backgrounds. Some things
familiar to urban children might not be familiar to rural children. Trying to
satisfy the expectations of both the rural and the urban target users might have
caused the dictionary to become nothing more than a book of illustrations. The
compilers therefore agreed to include group illustrations as part of the back
matter of the dictionary. 119 The Compilation of a Shona Children's Dictionary: Challenges and Solutions References ALRI. 2002. Shona Children's Dictionary Style Manual. Unpublished Document. Harare: University
of Zimbabwe. Chabata, E. 1998. Using the Predictability Criteria for Selecting Extended Verbs for Shona Diction-
aries. Lexikos 8: 140-153. Chimhundu, H. (Ed.). 2001. Duramazwi Guru reChiShona. Harare: College Press. Chitauro-Mawema, M.B. 2002. Challenges Encountered in the Compilation of an Advanced Shona
Dictionary. Lexikos 10: 209-224. Chitiga, M. 1995. Codeswitching in the Classroom. Unpublished M.A. Thesis. Harare: University of
Zimbabwe. Crystal, D. 1997. The Cambridge Encyclopedia of Language. Second Edition. Cambridge/New York:
Cambridge University Press. Kipfer, B.A. 1984. Workbook on Lexicography: A Course for Dictionary Users with a Glossary of English
Lexicographical Terms. Exeter: University of Exeter. Magwa, W. 2002. The Shona Writing System: An Analysis of its Problems and Possible Solutions. Zambezia 29(1): 1-11. Ndlovu, F. 1998. Considering Factors that are Relevant to Adopting and Adapting COBUILD Defining
Formats for the General Ndebele Dictionary. Unpublished M.A. Thesis. Harare: University of
Zimbabwe. Svensén, B. 1993. Practical Lexicography: Principles and Methods of Dictionary-Making. New York:
Oxford University Press. Tullock, S. 1994. Reader's Digest: Complete Word Finder. Oxford: Reader's Digest.
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METAMORFOSIS IN THE LIVES OF ELDERLY PEOPLE CARING FOR DEPENDENT ELDERLY IN BRAZIL
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Original A
METAMORFOSIS IN THE LIVES
OF ELDERLY PEOPLE CARING
FOR DEPENDENT ELDERLY IN BRAZIL
Girliani Silva de Sousa1
Raimunda Magalhães da Silva2
Amanda Márcia dos Santos Reinaldo3
Christina Cesar Praça Brasil2
Maria Odete Pereira3
Maria Cecilia de Souza Minayo4
1Universidade Federal de São Paulo, Escola Paulista de Enfermagem, Departamento de Enfermagem Clínica e Cirúrgica.
São Paulo, São Paulo, Brasil.
2Universidade de Fortaleza, Programa de Pós-Graduação em Saúde Coletiva e de Enfermagem. Fortaleza, Ceará, Brasil.
3Universidade Federal de Minas Gerais, Escola de Enfermagem. Belo Horizonte, Minas Gerais, Brasil.
4Fundação Oswaldo Cruz, Escola Nacional de Saúde Pública, Departamento de Estudos sobre Violência e Saúde Jorge Careli.
Rio de Janeiro, Rio de Janeiro, Brasil. Original Article METAMORFOSIS IN THE LIVES
OF ELDERLY PEOPLE CARING
FOR DEPENDENT ELDERLY IN BRAZIL 1Universidade Federal de São Paulo, Escola Paulista de Enfermagem, Departamento de Enfermagem Clínica e Cirúrgica. São Paulo, São Paulo, Brasil. 2Universidade de Fortaleza, Programa de Pós-Graduação em Saúde Coletiva e de Enfermagem. Fortaleza, Ceará, Brasil. 3Universidade Federal de Minas Gerais, Escola de Enfermagem. Belo Horizonte, Minas Gerais, Brasil. 4Fundação Oswaldo Cruz, Escola Nacional de Saúde Pública, Departamento de Estudos sobre Violência e Saúde Jorge Careli. Rio de Janeiro, Rio de Janeiro, Brasil. ABSTRACT Objective: to understand the phenomenon concerning the change in life and routine of elderly family caregivers
who care for dependent elderly family members in Brazil. Method: qualitative, exploratory and descriptive study, developed with 33 elderly family caregivers, from
June to September 2019, in the cities of Belo Horizonte, Rio de Janeiro, Porto Alegre, Araranguá, Manaus,
Fortaleza and Teresina. Semi-structured interviews were conducted based on the theme of care, facilitating
circumstances and difficulties. The analysis of the information was guided by the theoretical-methodological
framework of hermeneutics-dialectics. Results: a comprehensive model of the experience of being an elderly caregiver of a dependent elderly
family member. This process was part of two categories: “Assuming care” and “Unmet needs”. The facilitating
circumstances to assume care were positive emotions and bonds with the older person, acceptance of the
disease, stable clinical status and support from health professionals, formal caregivers, domestic employees
and family support. The difficulties were when the behavior of the dependent elderly becomes aggressive,
experiencing loneliness, poor health, social isolation, financial restrictions and absence of social and health
support. Conclusion: elderly caregivers are important agents in the exercise of care for the dependent elderly. When
they have support, they can perform the care activities and have time to take care of themselves. However, for
the most part, these people give up their lives, withdraw from the labor market, isolate themselves and suffer
from the lack of material resources and support from health services. TORS: Qualitative research. Family caregiver. Fragile elderly. Elderly health. Geriatric nursing. 1/14 RESUMO Objetivo: compreender o fenômeno da mudança de vida e rotina de idosos familiares que passam a cuidar
de idosos dependentes no Brasil. Método: estudo qualitativo, exploratório e descritivo, desenvolvido com 33 cuidadores idosos familiares, de
junho a setembro de 2019, nas cidades de Belo Horizonte, Rio de Janeiro, Porto Alegre, Araranguá, Manaus,
Fortaleza e Teresina. Realizaram-se entrevistas semiestruturadas norteadas pelo tema de assumir o cuidado,
circunstâncias facilitadoras e dificuldades. A análise das informações foi guiada pelo referencial teórico-
metodológico da hermenêutica-dialética. Resultados: constituiu-se um modelo compreensivo da experiência de ser cuidador idoso de um membro
familiar idoso. Esse processo integrou duas categorias: “Assumindo o cuidado” e “Necessidades não
atendidas”. As circunstâncias facilitadoras para assumir o cuidado foram as emoções positivas e os vínculos
com a pessoa idosa, a aceitação da doença, o quadro clínico estável e o suporte de profissionais de saúde,
cuidadores formais, colaboradores domésticos e apoio familiar. As dificuldades ocorrem, principalmente,
quando o comportamento do idoso dependente é agressivo, pois há vivência da solidão, saúde fragilizada,
isolamento social, restrições financeiras e ausência de suporte social e de saúde. Conclusão: os cuidadores idosos são importantes agentes no exercício de cuidado à pessoa idosa dependente. Quando possuem apoio, conseguem conciliar as atividades do cuidado e têm tempo para cuidar de si. Em sua
maioria, essas pessoas, entretanto, renunciam às suas vidas, se retiram do mercado de trabalho, se isolam e
sofrem com a falta de recursos materiais e de apoio dos serviços de saúde. DESCRITORES: Pesquisa qualitativa. Cuidador familiar. Idoso fragilizado. Saúde do idoso. Enfermagem
geriátrica. METAMORFOSIS EN LA VIDA DE LOS ANCIANOS QUE CUIDAN A ANCIANOS
DEPENDIENTES EN BRASIL DESCRIPTORES: Investigación cualitativa. Cuidador familiar. Ancianos frágiles. Salud de los ancianos.
Enfermería geriátrica. RESUMEN Objetivo: comprender el fenómeno del cambio en la vida y la rutina de los familiares ancianos que comienzan
a cuidar a las personas mayores dependientes en Brasil. Método: estudio cualitativo, exploratorio y descriptivo, desarrollado con 33 cuidadores familiares ancianos,
de junio a septiembre de 2019, en las ciudades de Belo Horizonte, Río de Janeiro, Porto Alegre, Araranguá,
Manaus, Fortaleza y Teresina. Se realizaron entrevistas semiestructuradas, guiadas por el tema de cuidar,
facilitar circunstancias y dificultades. El análisis de la información estuvo guiado por el marco teórico-
metodológico de la hermenéutica-dialéctica. Resultados: Se constituyó un modelo integral de la experiencia de ser cuidador anciano de un familiar anciano. Este proceso integró dos categorías: “Asumir cuidados” y “Necesidades insatisfechas”.Las circunstancias
facilitadoras para el cuidado fueron emociones positivas y vínculos con el anciano, aceptación de la
enfermedad, estado clínico estable y apoyo de profesionales de la salud, cuidadores formales, trabajadores
domésticos y apoyo familiar. Las dificultades se presentan principalmente cuando el comportamiento del
anciano dependiente es agresivo, ya que existe una experiencia de soledad, salud frágil, aislamiento social,
restricciones económicas y falta de apoyo social y sanitario. Conclusión: los cuidadores de ancianos son agentes importantes en el ejercicio del cuidado del anciano
dependiente. Cuando cuentan con apoyo, logran conciliar las actividades de cuidado y tienen tiempo para
cuidarse. La mayoría de estas personas, sin embargo, renuncian a sus vidas, se retiran del mercado laboral,
se aíslan y padecen la falta de recursos materiales y apoyo de los servicios de salud. Texto & Contexto Enfermagem 2021, v. 30:e20200608
ISSN 1980-265X DOI https://doi.org/10.1590/1980-265X-TCE-2020-0608 2/14 Texto & Contexto Enfermagem 2021, v. 30:e20200608
ISSN 1980-265X DOI https://doi.org/10.1590/1980-265X-TCE-2020-0608 INTRODUCTION The phenomenon of aging is rapidly evolving at the global level. In Brazil, population projections
tend to reduce young people and the progression of the elderly contingent in the coming decades1. Due to this trend, the occurrence of diseases and the need for long-term care are incre In Brazil, care for dependent elderly traditionally takes place in the home context and is an
assignment assumed by a family member, who becomes the caregiver and, consequently, embraces
responsibility for the provision of care2–4. Demographic transition and changes in family structure,
however, lead elderly people to be representative in the segment of caregivers of dependent family
components, also elderly2,4. Becoming a caregiver imposes a challenge for an old person, because it means reconciling
one’s own well-being and self-care with high loads of tasks that the family’s health status requires. Studies indicate that elderly caregivers denote fragile health, physical, emotional, financial overload,
social isolation and greater self-perception of stress4–11. In this context, elderly family caregivers
deserve attention in health services, because health problems can precipitate an interruption of care
and result in hospitalizations or admissions to Long-Term Care Institutions (LSI). The role of the caregiver is greatly explored by research in the areas of Gerontology and
Nursing. However, there is a gap in knowledge regarding the elderly family caregiver, mainly from
the perspective of this social agent. In addition, the lack of public policies and the limited resources
and services available to support elderly people who care for elderly family members in Brazil make
this reflection necessary2–3. In light of the above, recognizing the needs, challenges and circumstances that involve the
process of becoming an elderly caregiver of dependent elderly people is a way for the elaboration
of systematic care actions. In these actions, care should be included for the elderly who assist their
sick family member. In this study, by listening to their experiences, we attempt to understand life and routine changes
of elderly family members who start caring for dependent elderly people in Brazil. METHOD This is a qualitative, exploratory and descriptive study, carried out from June to September
2019. This investigation is part of a multicentric research called “Situational Study of Dependent Elderly
People Who Live with Their Families Aiming to Support a Care Policy and Support for Caregivers”. We sought to understand the situation of elderly people who care for dependent family members, who
are also elderly. The hypothesis is portrayed in the idea that a better understanding supports actions
that support them in their daily routine. Thirty-three elderly family caregivers participate in this study - in eight municipalities in the
five Regions of Brazil: Rio de Janeiro (RJ), Belo Horizonte (MG), Porto Alegre (RS), Araranguá (SC),
Brasília (DF), Fortaleza (CE), Teresina (PI) and Manaus (AM), and was called: “Situational Study of
Dependent Elderly living with their Families Aiming to Support a Care Policy and Support to Caregivers”. The participants had to be the main caregiver of the elderly person; exercise full-time care for at least
one month and be oriented in time and space in order to report their experience. This investigation
found 43 elderly caregivers, but only 33 were able to describe their situation. The participant identification occurred upon indication of the health professionals of the Family
Health Strategy (ESF) and the geriatric outpatient clinics of these municipalities. The researchers
met with health services and explained that the elderly person was dependent on another in order
to perform Basic Activities of Daily Living (ADL) and/or Instrumental Activities of Daily Living (IADL). 3/14 The semi-structured interview script that served as an instrument for the conversation consisted
of open and free questions. It was previously elaborated and improved by researchers with experience
in the theme. With the support of this device, the participants were invited to talk about their experiences
of taking care of the dependent elderly person, report the changes in their lives due to the care routine,
the facilitating circumstances and the difficulties involved in the tasks they perform on a daily basis. A sociodemographic questionnaire was also included to characterize the participants: age; kinship;
marital status; length of care; illness and dependence of the elderly. The interview was previously scheduled with the participants and conducted individually in their
homes, with an average duration of 60 minutes. This time was sufficient to establish a comfortable
situation and a relationship of mutual trust between researcher and interviewee. METHOD The Informed Consent
Form (TCLE) was read and signed by the participants, which included the consent for recording on
digital media and respect for the stories told by the interviewees, without judgment or criticism. The theoretical-methodological framework of hermeneutics-dialectics was adopted was the
purpose of data analysis12. In this framework, the statements must be understood as part of the
context in which they are produced. The data interpretation considered the concrete situations that
were placed in a reflexive and critical perspective, valuing both empirical data and national and
international literature on the subject.i The evaluation of the statements was made by first grouping the interviews, observing similarities
and differences in the statements separately by each municipality studied, then grouping them,
according to the finding that, regardless of the place of residence, the situations narrated coincided. The analysis occurred based on levels of interpretation and the data were previously organized,
taking into account the following steps: 1) the main question of the analysis - the act of assuming
the care of the dependent old person and the facilitating and hindering circumstances of this task;
2) selection of excerpts from the interviews in which the caregivers talked about these aspects; 3)
interpretative synthesis of each point, considering all the statements; and 4) interpretative synthesis
of the set of data and aspects. As a result of this synthesis, the representative categories described
and discussed here were chosen. In order to protect the identities of the participants, the abbreviation “CI” is used which refers
to “elderly caregiver”, followed by the numbers from one to 33, in addition to this information: kinship,
involvement of the cared person and municipality. Therefore, CI1 represents the first interviewee and
so on. The research was approved by the Research Ethics Committee of the Oswaldo Cruz F Texto & Contexto Enfermagem 2021, v. 30:e20200608
ISSN 1980-265X DOI https://doi.org/10.1590/1980-265X-TCE-2020-0608 RESULTS According to the above statement, it is inferable that the elderly caregiver was in a marriage and
shared dreams, desires and a family, which is the driving force for being a caregiver. Consequently, he
associated the attention he paid to his wife with his admiration for her, which made him more resilient
and adapted to the situation he experienced. In general, the statements showed that harmonious experiences are also related to the personal,
social and economic conditions of the family. Thus, the participants reported several facilitating aspects,
among them: 1) it concerns the social order, because, when there is family support, the caregiver
has to share the multiple tasks he/she performs; 2) the elements of emotional content, when the
caregiver manages to balance the care of the elderly and their personal desires, that is, care is not
the only source of personal fulfillment for the caregiver; 3) Finally, the financial content, because,
when the family has the possibility of contracting formal caregivers, rehabilitation professionals and
other employees for household duties, the family caregiver’s load decreases. There’s a speech therapist and a physiotherapist who do home visits. Physical therapy every
weekday. She has a caregiver, but I help with the dishes, the clothes, the care, the maintenance of
the whole house. I realize that it makes a difference to help the caregiver change my mother’s diaper,
there is a way that she holds onto my arm, then she holds my hand and is calm (IC2, 65 years old,
son, takes care of the mother affected by stroke, Rio de Janeiro). With the various types of support, the family caregiver is able to manage the tasks, without
neglecting the loved one. It is important to highlight that the formal caregiver does not replace the
care offered by the family member, but is a fundamental support for the latter to preserve their quality
of life, have some time and space for themselves and with a view to socializing, as this participant
points out: I work with artistic restoration to distract myself (IC23) and others: I can visit my friends,
have an afternoon tea, I go to the country house to rest (IC17). However, not all family caregivers
have economic and financial conditions and support from other relatives. Thus, for many interviewees, the exercise of care is something heavy, unsettling and
unimaginable. RESULTS Twenty-eight women and five men were interviewed. From the total amount, 18 were between
60 and 69 years old;11, between 70 and 79 years; and four were over 80 years of age. The degrees
of kinship of the respondents were: 15 wives, six daughters, three sisters, three husbands, two sons,
two ex-wives, a mother and the wife of a nephew. Regarding the time of care, 18 reported that they
performed this activity for between two months to five years; five worked between five to ten years;
and ten between ten to 30 years. According to the participants’ reports, all the elderly relatives were
dependent on ADL’s and IADL’s. Regarding the illnesses and dependence, the caregivers reported that 18 elderly people had
been affected by stroke, six had developed Alzheimer’s disease, four suffered stroke with physical
sequelae of alcoholism. The other suffered from multisystemic atrophy, infantile paralysis, head trauma,
cancer and depression. Only 12 elderly caregivers alternated the care of their elderly relatives with
another person, as five also had a formal caregiver and cleaner. The others had the help of other
family members. The majority of the interviewees (28) accumulated the functions of family caregivers 4/14 in addition to the execution of domestic activities. Most lived with the dependent elderly person and
only five of them lived on their own property. The category “assuming care” unveils that, for some of the participants, care acquires meaning
in marital and filial responsibility and is impregnated with positive emotions - such as dedication, love
and gratitude. Thus, the lightness with which the care of the other is attributed to the harmonious
relationship with the extension of life is surprising, observing an understanding and acceptance of
both elderly people - caregivers and dependents - of the new life situation. I give full-time care, I don’t see it as an obligation or a burden. Today she sees me as a person
who takes care of her, no longer recognizes me as a husband and I am aware that today this is the
reality and I seek to live in the best possible way, it is not something she chose and because she
knows that she has always been a wonderful person, I care with a lot of love and dedication (IC1, 81
years old, husband, takes care of the wife with Alzheimer’s disease, Brasilia). Texto & Contexto Enfermagem 2021, v. 30:e20200608
ISSN 1980-265X DOI https://doi.org/10.1590/1980-265X-TCE-2020-0608 RESULTS The category “unmet needs” gathers the adaptations in life for the exercise of care that lead
to physical and mental health problems, associated with tiredness, which impels the elderly caregiver
to feel sad, to lose the pleasure of life and to feel isolate socially. My routine doubled, I already have several health problems, so it’s one patient taking care of
the other. At first, I thought I’d take care of it. He’s my husband, I’ve been taking care of him for 54
years, how could I not take care of him now? I didn’t think it was going to happen like this and it would
be so heavy, to do it all, but after two months I couldn’t do it anymore. One day my daughter came
and saw me crying, because he was all wet with pee and I could not help, I had no more strength
to carry him. Today, I don’t live anymore, my life is to live with him indoors. I do not go out, I don’t
do anything else (IC5, 70 years old, wife, takes care of her husband affected by stroke, Araranguá). Several elderly caregivers revealed the worsening of previous health problems, such as chronic-
degenerative diseases, hypertension, diabetes, arthritis and the onset of more diseases, such as:
back, arm pain and depressive symptoms. The elderly caregiver handles another sick elderly person
despite feeling pain, performing basic care with difficulties - such as moving between rooms, bathing
and changing diapers. This reality has a course in all the regions surveyed and reflects the need for
family, social and governmental support for the exercise of care. Most participants (28) mentioned restrictions or no longer having a social life to participate in
culture and leisure programs, go to church or travel. The loneliness of elderly caregivers is related to
home confinement. Some even expressed shame for the situation they experience: I miss, sometimes, traveling. I’m sad because I have a daughter who lives in Brasilia. I wanted
to go there to see her, [...] she calls me several times, but he (the husband) doesn’t like me to travel
alone, because he only trusts me to take care of him. If I go out and take longer than planned, he’s
already in agony. He doesn’t want to travel with me either because he doesn’t like to go out. RESULTS The dark side of this task is experienced as a solitary duty, because there is a lack
of support and sharing, and the caregiver suffers the distancing, coldness and neglect of the other
family members. Additionally, many of these elderly caregivers report difficulties in the relationship
with the person they assist, particularly when they have aggressive behavior. I split up with him before he got sick. He was very womanizing. I came home to help take care
of his mother. Then he had a stroke and I took care of him. He’s the father of my daughter. I think it’s
my duty, despite the disagreement, I wouldn’t let him go without anything. Our coexistence is difficult,
because it seems that he does not accept the disease. He gets angry, is very stubborn, complains,
throws things at us, when he wants things on time and I cannot give and that’s how we go on living,
I keep going reluctantly (IC3, 62 years old, ex-wife, takes care of the ex-husband affected by stroke,
Belo Horizonte). 5/14 The second type of situation is when the dependent old person does not accept his or her
clinical condition. I assumed the role of the main and only family caregiver when he had health problems, was
unable to perform the activities, did not walk alone and was diagnosed with Parkinson’s disease. My daughter worked and soon after she got married. He went on to get very nervous about his life
condition, he was very active and now finds himself unable [...] he became stubborn and very angry,
it’s hard to deal with him, I’m tired of taking care of him alone, I’m on 24 hours, but what can I do? [...]
only have me to take care of him (IC4, 64 years old, wife, takes care of her husband witch Parkinson’s
disease, Brasília) Although the dark side of care was not directly explained by the participants, they openly
discussed the physical, emotional and relational problems they experience. Elderly people who
care for the elderly alone have many difficulties. This fact occurs because, in addition to personal
renunciations to devote oneself to care, there is a self-demanding factor in the sense of not making
mistakes in the exercise of their routine. Texto & Contexto Enfermagem 2021, v. 30:e20200608
ISSN 1980-265X DOI https://doi.org/10.1590/1980-265X-TCE-2020-0608 RESULTS She
demonstrates it, saying that well-being and pleasure-like leisure activities are no longer part of her life.fi The following are some difficulties experienced by elderly caregivers and their family components:
financial restrictions to give some kind of comfort to the patient; problems for care in the health system,
to have access to medicines, ambulance, among others. Although Brazil maintains policies for the supply of medicines, equipment, consultations and
surgeries, these resources are often not guaranteed on time and according to people’s needs. Many
complain about bureaucracy to receive them and the lack of public funding for funding: He went blind. I took care of him for four years and decided to make a loan to his account to
do his surgery privately, because the SUS never worked. They said they’d do it, but they never called. There are nights when he sleeps and has nights that he does not sleep, he is shouting: “oh my God,
oh my God” (IC8, 71 years old, wife, takes care of the blind husband, Manaus). Many of the interviewees reported an increase in expenses due to the need to purchase
equipment and supplies, including hospital beds; wheelchairs and bathing chairs; diapers; medicines,
serum and gauze; adaptations at home with ramps and support bars; and in some cases; the cost
of surgeries and private consultations with specialists. These facts lead family members to become
indebted to bank loans and car sales (among other goods and objects), when there is no other means. Regarding health care, caregivers highlight the limitations of home visits by health agents or
family health physicians, delay in getting appointments for consultations with specialists, unavailability
of medications and ambulances for transportation. All this, at the time of crisis, entails significant
costs for the family. The health center doesn’t have physiotherapy. What they have given is medicine for blood
pressure, gauze, serum, glove, but other remedies and diapers, they have not. The transportation
from the ambulance to the doctor, we are covering everything. I just feel like all of a sudden you can
give me something and then how does it look? What’s going to happen to her? I have to have surgery
on my arm, but how am I going to do it? Then there’s physiotherapy, this whole thing, how am I going
to do it? Who’s going to take care of her? RESULTS So it is
difficult (IC6, 71 years old, wife, takes care of her husband affected by stroke, Teresina). Some situations are likely to impose embarrassment on the sick elderly people, such as urinary
incontinence, oxygen use, wheelchair and others, which restricts them to their home. Dependence on
the dependent elderly-caregiver- binomial was also reported as one of the reasons for social isolation. It is important to emphasize that simply leaving the home requires planning and logistics, either due
to the difficulty of locomotion of the sick elderly, due to the unfeasibility of using public transport or the
fear that the place outside the home does not offer the necessary comfort to the dependent person. Among the elderly daughters and sons caregivers, the total renunciation of their lives for their
parents is also a burden. They are people who withdraw from the labor market and try to reconcile
social functions with the activities of the home. 6/14 We give up our lives in everything. We can’t plan anything. We live for them [...], I already
have my family, I already have my husband, so it’s a difficulty. We have no leisure time. We don’t
have time to go out, go to a beach I like, get out of this routine. We are not able to pay caregivers
so we became the caregivers. We had to make sacrifices between us, we had to bear each other. We dedicate ourselves with love, but in the midst of love, we have stress too, we get tired [...] one
thing that I gets to me is having left work, I miss it, it was my personal satisfaction (IC7, 60 years old,
daughter, takes care of parents affected by stroke and physical sequelae due to alcoholism, Fortaleza). This woman reports the alternation of care between the four daughters and all support,
instrumentally or in relay on duty, the care to the parents. These women gave up their professional
activities to take care of their parents. The participant of the research commented that, when she
is not with her parents, she is concerned with the organization of her home and the attention to her
husband, showing the feeling of renunciation of personal life. The exit from the labor market is also
a circumstance that causes suffering to IC7, reflecting a common reality within several families. Texto & Contexto Enfermagem 2021, v. 30:e20200608
ISSN 1980-265X DOI https://doi.org/10.1590/1980-265X-TCE-2020-0608 Taking over the care For many elderly caregivers, the positive emotions involved in the feelings of gratitude and
pleasure in caring for others results in harmonious affective bonds with the sick elderly person16. This
is reflected in the increase in the self-esteem of these people15. It is positive experience, particularly
when the clinical picture of the dependent elderly is stable8 and care must be exercised with gestures
of affection, also counting on the collaboration of the sick person himself, overcoming the fulfillment
of basic life needs. When there is support from the team of health professionals for the provision of physical and
mental care, elderly caregivers feel more security in their routine and perceive greater stability in
the health status of the dependent elderly relative13–14. Regarding this support, however, only those
who experience it are a minority of the Brazilian population that has a financial condition to fund
professionals with their own resources. In Belgium, care for basic life activities, such as bathing and
dressing, are generally provided by a nurse at home15, which helps elderly caregivers. Similarly, in
other European countries, public policies support elderly caregivers, which is done as part of social
security systems, although support modalities are different in each location3.fil The difficulties that caregivers of the elderly encounter are mainly related to the conflicting and
aggressive behavior of the cared person, a previous dysfunctional family experience with the sick
elderly and their own challenges in meeting the needs of the dependent elderly - such as turning them
in bed, lifting them up, bathing and others. Research conducted in Canada and China relates to the
fact that managing and controlling the aggressive behaviors of the dependent elderly and the challenge
of dealing with the highest levels of dependence represent the main burden among caregivers8,13. The
literature indicates that diseases such as dementia, solid tumors, physical disabilities or comorbidities
have a direct effect on informal caregiver burden6,11. Particularly in Brazil, the poor social support network weighs on the routine of elderly caregivers. The modification of family arrangements and the migration of elderly people from rural to urban areas
tend to distance them from relatives and close friends who would be fundamental at the time of
reduction of functional or mental autonomy. Many only have their sick spouses as company at home5. DISCUSSION The literature shows that elderly caregivers are mostly women, with a mean age of around 70
years and mean care time provided by ten years4,8–9,13–14. Research in Belgium, however, identified
that the average age of caregivers is 80 years15, which signals the growing reality of spouses and
elderly family members as caregivers of older people. In this context, ensuring the health of elderly
caregivers should be a priority for all health professionals who care for the elderly. Texto & Contexto Enfermagem 2021, v. 30:e20200608
ISSN 1980-265X DOI https://doi.org/10.1590/1980-265X-TCE-2020-0608 RESULTS (IC9, 76 years old, husband, takes care of his wife with
sequela of stroke, Porto Alegre). Among the elderly caregivers living in the various places surveyed, there were complaints
about the lack of certain primary care services, such as rehabilitative physiotherapy. In addition,
a number of deficiencies were pointed out by elderly caregivers: lack of public funding to support
formal caregivers, financial help for expenses due to the consequences of illness, support for home
maintenance and leisure activities. 7/14 Unmet needs The elderly caregivers under study reported health alterations, expressing, in greater proportions,
physical and mental health problems, when compared with adult caregivers4,6. A research with elderly
caregivers identified the fact that 89.1% express moderate, intense and unbearable pain; and 38.7%
highlighted moderate, severe and severe physical and mental burden9. The prevalence of depressive
symptoms among them is, on average, 30%9,15. Elderly people who feel alone and those who express
depressive symptoms are more likely to become vulnerable because of increased physical vulnerability
and, above all, malnutrition15,18. The stress perceived by elderly caregivers is associated with high
levels of pain, difficulty sleeping and poor self-assessment in relation to health10. Caregivers with poor health or anxiety symptoms experience greater burden10–11. Studies
conducted in Brazil and Belgium indicate that elderly caregivers have, on average, six chronic-
degenerative diseases; they use, on average, three to five medications, including anxiolytics; and have
chronic pain in the lumbar region, lower limbs, dorsal, upper limbs and cervical/abdominal/thoracic
regions9–10,15. In general, the occurrence of depressive symptoms in elderly caregivers is associated
with family income, number of diseases, number of medications used, pain intensity, overload and
perceived stress9–10. One of the most difficult aspects of the spouse caregiver’s experience is loneliness, social
isolation, as already mentioned6,8,13. The socially constituted identity of family members concerning
the dependence of the elderly relative contributes to the real and symbolic removal of children,
grandchildren and other relatives. This affective absence results in psychological suffering and lower
quality of life, especially for elderly caregivers, when they do not have time for themselves and in
order to perform other activities that give them pleasure and joy4,10–11,13,19.fi The difficult routine and the accumulation of functions also weigh on the health condition of
elderly caregivers, when there is no one to help them, as they need to arrange care for the dependent
elderly person as well as perform the household chores4,10. The attribution of additional responsibilities
is certainly more costly for the informal caregiver than the demand for time with care10–11. These
problems were also found in Canada, where caregivers of the elderly complain of excessive effort
and overload, as well as the involvement of multiple chronic diseases8. In Portugal, in addition to
being overloaded, older caregivers consider themselves less competent to assist the sick person
in aspects such as food, mobility, transfers, medication and symptom control and communication20. Taking over the care What was observed here was also observed in the Netherlands, where elderly caregivers have less
social support than adult caregivers6 and those who care for their spouses alone feel the weight of
loneliness, express less hope for the future and enjoyment in life, when compared to independent
elderly husbands and wives8,17. Accordingly, the belief and culture, socially imposed in Brazil, that the responsibility of caring
for the elderly is family, lead husbands and wives to embrace their function as a social protocol and
conformism. Most do not question the negligence and refusal of other family members and public
authorities to support this hard, daily and routine task. There was only one case in which the sister of
a dependent elderly woman reported to the Public Prosecutor’s Office a complaint of mistreatment
caused by her husband and daughter, who had assumed care. However, this surveillance is rare and
it is known that the main victims of violence in old age are dependent elderly people2. 8/14 Even when elderly caregivers point out feelings of reward, they associate the act of caring
with exhaustion8. Positive and negative feelings are part of their daily lives, particularly the weight
of the daily routine, when the sick person needs long-term care or has cognitive limitations6,8. It is
necessary to take into account the notion that these elderly caregivers have almost no time to rest
when they are the only ones responsible for the sick person4,7–8. The constant fear caused by the
advance of the disease of the loved one, as well as the fear of not being able to handle the heavy
routine, lead them to neglecting their own needs and physical and emotional pains. What was found
in this research was also observed among elderly Canadian caregivers, who feel pressured to bear
such complex responsibilities in relation to their patients. Texto & Contexto Enfermagem 2021, v. 30:e20200608
ISSN 1980-265X DOI https://doi.org/10.1590/1980-265X-TCE-2020-0608 Unmet needs The physical barriers in the home, the existence of stairs and the lack of accessible public or
private transport were also mentioned as limiting factors for leaving the home and for the access of
the elderly to health related appointments8. In addition, financial difficulties represent a social burden for elderly caregivers in the care of
their loved ones4–11,13–14, because both are exposed by them to severe social vulnerability and the risk
of worsening diseases5. Several studies point to this problem2–9,21 and to the way some countries have
dealt with it. In Germany, for example, there is financial support and a type of reimbursement for the 9/14 costs of the dependent person, according to the classification of the severity of the dependency21. This type of support is of high relevance and would be very welcome in Brazil. When the needs are not met by health and social services, the caregiver’s burden increases,
particularly when he or she is elderly8–11,13,20–21. In Canada, which has a policy very focused on the
informal caregiver, evaluations show that the elderly, caregivers and health professionals consider
that institutional support for this service is fragmented, disjointed and with little coordination8. These
social groups demonstrate the need for care centered on the person and on better communication
between primary, secondary and home care in order to obtain information and provide services in a
timely manner8,20–21. Research in 18 European countries found that those with policies to support family caregivers,
with training or counseling on the disease and its consequences for care; provision of equipment such
as beds and wheelchairs; financial support for unemployed caregivers; and the provision of formal
and professional caregivers to perform more complex care all reduce the stress on caregivers19. In Brazil, there is an urgent need to strengthen primary care, geriatric outpatient clinics and
home programs for people who are in need of effective coverage. Professionals in the home visit, the
flow of referral of exams and the availability of medications are fundamental for elderly caregivers,
dishonoring the weight they carry and creating bonds with the services21. Few participants pointed
out the importance of learning about the disease, while in some countries13,20–21 this is a recognized
and disseminated urgency.i The finitude of life is also a theme highlighted by the elderly caregivers participating in the
study. CONCLUSIONS The change in the lives of elderly family members who start to exercise the care of their elderly
loved one involves facilitating circumstances that are: rewarding feelings, such as dedication, love and
gratitude; harmonious experiences with the sick old person; stability of the patient’s clinical status; their
acceptance of the disease; alternation between family components in care and having purchasing power
to fund a team of health professionals, formal caregivers and domestic collaborators. In this context,
elderly caregivers are able to care, have time for their activities and also take care of themselves. However, for many elderly caregivers the exercise of care is seen as something exhausting
and heavy. The difficulties in the routine refer, first, to the degree of physical and cognitive impairment
of the elderly person, which causes physical and mental health problems, associated with tiredness,
back pain, worsening of preexisting diseases, such as hypertension, diabetes and arthritis. From
the emotional point of view, loneliness and social isolation are circumstances pointed out by elderly
caregivers who give up their social relationships, withdraw from the labor market and from the previous
life context, and begin to devote themselves entirely to the sick relative, without space and time for
themselves. Financial restriction is a reality in which the majority, in addition to caring for the sick as
well as performing domestic activities. This article shows that elderly family caregivers are important agents of the care network in
the world and in Brazil. However, they urgently need support for the effective exercise of this function. Future research is necessary in order to understand how care is exercised by elderly caregivers from
the perspective of health professionals, managers and the dependent elderly themselves and how
these social actors are inserted in the agendas of health services. The results of this study are valuable for nursing care practices, as they indicate that nurses
play a very important role in providing comprehensive care, centered on the person and the family,
surpassing those that focus on diseases and their repercussions. As a final point, it is important to express the argument that Brazilian society needs a public
policy that covers the large number of people who care for the elderly and other dependent patients
in their homes, doing an indisputably necessary job, however invisible and undervalued. CONCLUSIONS In particular,
women deserve this support, who make up more than 90% of the total of this segment of workers,
most of whom are elderly people taking care of elderly people. Unmet needs Many said they were afraid of being absent a little and their loved one had a serious problem
or passed away. In turn, spouse caregivers represent the most important figure of attachment for the
sick and dependent elderly7–8,15. For many of these segments of caregivers, the involvement of children
is not mandatory, as they have obligations to their families. This finding is quite unique in comparison
with other cultures, such as Chinese, in which caregivers in general have the social support of close
family members as well as the more extensive family13. Elderly caregivers who receive help from
relatives denote greater well-being and have fewer negative consequences on their personal, social
and professional lives in the exercise of care13,19. The results of this study highlight the reality that the elderly caregivers interviewed passively
accept the role of caregiver as natural to the female gender and are overburdened11, understanding other
family members as people who collaborate, visiting the sick elderly on weekends. Women caregivers
who are married or in a stable union have a lower quality of life, because of the additional concern
of balancing between the difficulties of providing care and personal investment so that the caregiver
routine does not affect their relationship4,15. The current Oxfam22 document, released at the World
Economic Forum in Davos in 2020, highlights the invisibility of the work of women caregivers around
the world, a work that financially impoverishes them and enriches the capitalist and patriarchal world. Among the limitations of the study, we highlight the fact that a very small sample is used that
does not intend to generalize the results, although there is a broad agreement of local information
with the national and international literature. The limited number of participants per studied cities is
related to the difficulty in locating these elderly caregivers in the official health services records, which
contributes to the invisibility of people and the theme in the Brazilian context. Texto & Contexto Enfermagem 2021, v. 30:e20200608
ISSN 1980-265X DOI https://doi.org/10.1590/1980-265X-TCE-2020-0608 10/14 Texto & Contexto Enfermagem 2021, v. 30:e20200608
ISSN 1980-265X DOI https://doi.org/10.1590/1980-265X-TCE-2020-0608 REFERENCES 1. Instituto Brasileiro de Geografia e Estatística (Brasil). Sinopse do censo demográfico. Projeção
da pop. Brasil por sexo e idade 2000-2030; Projeção da população das UF por sexo e idade
2000-2030. Rio de Janeiro, RJ(BR): IBGE; 2013 [cited 2020 Oct 21]. Available from: http://www. ibge.gov.br/home/estatistica/populacao/projecao_da_populacao/2013/ 2. Minayo MCS. The imperative of caring for the dependent elderly person. Ciên Saúde Colet
[Internet]. 2019 [cited 2020 Oct 21]; 24(1):247-52. Available from: https://www.scielo.br/scielo. php?pid=S1413-81232019000100247&script=sci_arttext 2. Minayo MCS. The imperative of caring for the dependent elderly person. Ciên Saúde Colet
[Internet]. 2019 [cited 2020 Oct 21]; 24(1):247-52. Available from: https://www.scielo.br/scielo. php?pid=S1413-81232019000100247&script=sci_arttext 3. Minayo MCS, Mendonça JMB, Sousa GS, Pereira TFS, Mangas NMR. Policies to support the
elderly in a situation of dependence: Europe and Brazil. Cien Saude Colet [Internet]. 2021 [cited
2020 Oct 21];26(1):137-46. Available from: https://doi.org/10.1590/1413-81232020261.30262020 4. Araujo MGO, Dutra MOM, Freitas CCSL, Guedes TG, Souza FS, Baptista RS. Caring for the
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carer: quality of life and burden of female caregivers. Rev Bras Enferm [Internet]. 2019 [cited
2020 Oct 21];72(3):728-36. Available from: https://doi.org/10.1590/0034-7167-2018-0334. 11/14 Texto & Contexto Enfermagem 2021, v. 30:e20200608
ISSN 1980-265X DOI https://doi.org/10.1590/1980-265X-TCE-2020-0608 5. Rossetti ES, Terassi M, Ottaviani AC, Santos-Orlandi AA, Pavarini SCI, Zazzetta MS. Frailty,
depressive symptoms and overload of elderly caregivers in a context of high social vulnerability. Texto Contexto Enferm [Internet]. 2018 [cited 2020 Oct 21];27(3):e3590016. Available from:
https://doi.org/10.1590/0104-07072018003590016 6. Oldenkamp M, Hagedoorn M, Slaets J, Stolk R, Wittek R, Smidt M. Subjective burden among
spousal and adult-child informal caregivers of older adults: results from a longitudinal cohort study. BMC Geriatr [Internet] 2016 [cited 2020 Oct 21];16:208. Available from: https://doi.org/10.1186/
s12877-016-0387-y 7. Pinquart M, Sörensen S. Spouses, adult children, and children-in-law as caregivers of older
adults: A meta-analytic comparison. Psychol Aging [Internet]. 2011 [cited 2020 Oct 21];26(1):1–14. Available from: https://doi.org/10.1037/a0021863 8. Ploeg J, Matthew-Maich N, Fraser K, Dufour S McAiney C, Kaasalainen S, et al. Managing
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ISSN 1980-265X DOI https://doi.org/10.1590/1980-265X-TCE-2020-0608 REFERENCES Factors associated
with depressive symptoms in elderly caregivers with chronic pain. Rev Bras Enferm [Internet]. 2020 [cited 2020 Oct 21];73(1):e20170782. Available from: https://doi.org/10.1590/0034-7167-
2017-0782 10. Luchesi BM, Souza EN, Gratão ACM, Oliveira Gomes GA, Inouye K, da Silva Alexandre T, et al. The evaluation of perceived stress and associated factors in elderly caregivers. Arch Gerontol
Geriatr [Internet]. 2016 [cited 2020 Oct 21];67:7-13. Available from: https://doi.org/10.1016/j. archger.2016.06.017 11. Lindt N, Van Berkel J, Mulder BC. Determinants of overburdening among informal carers: a
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Hucitec Publishing House; 2006. 13. Xiao LD, Wang J, He G, Bellis AD, Verbeeck J, Kyriazopoulos H. Family caregiver challenges in
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transition of family members who care of elderly people with parkinson’s disease. Texto
Contexto Enferm [Internet]. 2019 [cited 2020 Oct 21];28:e20170438. Available from: https://doi. org/10.1590/1980-265x-tce-2017-0438 15. Potier F, Degryse J, Bihin B, Chainiaux FD, Martens H, Saint-Hubert MS. Health and frailty among
older spousal caregivers: an observational cohort study in Belgium. BMC Geriatr [Internet]. 2018. [cited 2020 Oct 21];18:291. Available from: https://doi.org/10.1186/s12877-018-0980-3 16. Greenwood N, Smith R. Motivations for being informal carers of people living with dementia:
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21];19(1):169. Available from: https://doi.org/110.1186/s12877-019-1185-0 17. Adams KB. Specific effects of caring for a spouse with dementia: differences in depressive
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caregivers of the elderly: frailty, loneliness and depressive symptoms. Rev Bras Enferm [Internet]. 2019 [cited 2020 Oct 21];72(Suppl 2):88-96. Available from: https://doi.org/10.1590/0034-7167-
2018-0137 Texto & Contexto Enfermagem 2021, v. 30:e20200608
ISSN 1980-265X DOI https://doi.org/10.1590/1980-265X-TCE-2020-0608 12/14 19. Verbakel E. Informal caregiving and well-being in Europe: What can ease the negative
consequences for caregivers? J Eur Soc Policy [Internet]. 2014 [cited 2020 Oct 21];4:424-41. Available from: https://doi.org/10.1177/0958928714543902 20. Dixe MDACR, Conceição Teixeira LF, Areosa TJTCC, Frontini RC, Peralta TJA, Querido AIF. Needs and skills of informal caregivers to care for a dependent person: a cross-sectional study. Texto & Contexto Enfermagem 2021, v. 30:e20200608
ISSN 1980-265X DOI https://doi.org/10.1590/1980-265X-TCE-2020-0608 REFERENCES BMC Geriatr [Internet]. 2019 [cited 2020 Oct 21];19:255 Available from: https://doi.org/10.1186/
s12877-019-1274-0 21. Plöthner M, Schmidt K, de Jong L, Damm K. Needs and preferences of informal caregivers
regarding outpatient care for the elderly: a systematic literature review. BMC Geriatr [Internet]. 2019 [cited 2020 Oct 21];19:82. Available from: https://doi.org/10.1186/s12877-019-1068-4 22. Oxfam. Report on us and the inequality “time to care”. World Economic Forum 2020, Davos
[Internet]. 2020 [cited 2020 Oct 21] Available from: https://oxfam.org.br/justica-social-e-economica/
forum-economico-de-davos/tempo-de-cuidar/ Texto & Contexto Enfermagem 2021, v. 30:e20200608
ISSN 1980-265X DOI https://doi.org/10.1590/1980-265X-TCE-2020-0608 13/14 NOTES ORIGIN OF THE ARTICLE Extracted from the multicentric research – Situational Study of Dependent Elderly living with their
families Aiming to Support a Policy of Care and Support to Caregivers. The research was carried
out by the Department of Studies on Violence and Health Jorge Careli CLAVES /ENSP/FIOCRUZ in
partnership with universities of Fortaleza, Teresina, Manaus, Porto Alegre, Araranguá, Rio de Janeiro
and Belo Horizonte, from 2018 to 2020. EDITORS Editor-in-chief: Roberta Costa. HISTORICAL
Received: December 17, 2020. Approved: June 15, 2021. Texto & Contexto Enfermagem 2021, v. 30:e20200608
ISSN 1980-265X DOI https://doi.org/10.1590/1980-265X-TCE-2020-0608 CONTRIBUTION OF AUTHORITY Discussion of the results: Sousa GS, Silva RM, Reinaldo AMS, Pereira MO, Brasil CCP. Writing and/or critical review of the content: Sousa GS, Silva RM, Reinaldo AMS, Brasil CCP, Pereira
MO, Minayo MCS. Writing and/or critical review of the content: Sousa GS, Silva RM, Reinaldo AMS, Brasil CCP, Pereira
MO, Minayo MCS. Writing and/or critical review of the content: Sousa GS, Silva RM, Reinaldo AMS, Brasil CCP, Pereira
MO, Minayo MCS. Review and final approval of the final version: Sousa GS, Silva RM, Reinaldo AMS, Brasil CCP,
Pereira MO, Minayo MCS. HISTORICAL 14/14
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Drivers of atmospheric deposition of polycyclic aromatic
hydrocarbons at European high-altitude sites 1Institute of Environmental Assessment and Water Research (IDÆA-CSIC), Jordi Girona 18,
08034 Barcelona, Catalonia, Spain p
2Environmental Change Research Centre, University College London, Gower Street,
London, WC1E 6BT, UK p
2Environmental Change Research Centre, University College London, Gower Street,
London, WC1E 6BT, UK 3Department of Atmospheric and Cryospheric Sciences, University of Innsbruck,
Innrain 52, 6020 Innsbruck, Austria
4 3Department of Atmospheric and Cryospheric Sciences, University of Innsbruck,
Innrain 52, 6020 Innsbruck, Austria 4Institute of Interdisciplinary Mountain Research, Austrian Academy of Sciences,
Technikerstrasse 21a, 6020 Innsbruck, Austria 4Institute of Interdisciplinary Mountain Research, Austrian Academy of Sciences,
T
h ik
21
6020 I
b
k A
i 5Biology Centre, Czech Academy of Science, Institute of Hydrobiology, Na Sadkach 7,
37005 Ceske Budejovice, Czech Republic 6Centre for Advanced Studies of Blanes (CEAB-CSIC), Accés a la Cala St. Francesc 14,
17300 Blanes, Catalonia, Spain 7Centre for Ecological Research and Forestry Applications (CREAF), Campus UAB, Edifici C,
08193 Cerdanyola, Catalonia, Spain 7Centre for Ecological Research and Forestry Applications (CREAF), Campus UAB, Edifici C,
08193 Cerdanyola, Catalonia, Spain Correspondence: Pilar Fernández (pilar.fernandez@cid.csic.es) Correspondence: Pilar Fernández (pilar.fernandez@cid.csic.es) Received: 21 June 2018 – Discussion started: 24 July 2018
Revised: 21 September 2018 – Accepted: 15 October 2018 – Published: 8 November 2018 Gossenköllesee and Skalnate Pleso, showing higher transfer
from gas phase to particles of the more volatile PAHs. The
sites with lower insolation, e.g. those located at lower alti-
tude, were those with a higher proportion of photooxidable
compounds such as benz[a]anthracene. Abstract. Polycyclic aromatic hydrocarbons (PAHs) were
analysed in bulk atmospheric deposition samples collected
at four European high-mountain areas, Gossenköllesee (Ty-
rolean Alps), Redon (Central Pyrenees), Skalnate Pleso
(High Tatra Mountains), and Lochnagar (Grampian Moun-
tains) between 2004 and 2006. Sample collection was per-
formed monthly in the first three sites and biweekly in
Lochnagar. The number of sites, period of study and sam-
pling frequency provide the most comprehensive description
of PAH fallout in high mountain areas addressed so far. p
[ ]
According to the data analysed, precipitation is the main
driver of PAH fallout. However, when rain and snow depo-
sition were low, particle settling also constituted an efficient
driver for PAH deposition. Redon and Lochnagar were the
two sites receiving the highest amounts of rain and snow and
the fallout of PAH fluxes was related to this precipitation. Drivers of atmospheric deposition of polycyclic aromatic
hydrocarbons at European high-altitude sites No significant association was observed between long-range
backward air trajectories and PAH deposition in Lochnagar,
but in Redon PAH fallout at higher precipitation was essen-
tially related to air masses originating from the North At-
lantic, which were dominant between November and May
(cold season). In these cases, particle-normalised PAH fall-
out was also associated with higher precipitation as these air
masses were concurrent with lower temperatures, which en- The average PAH deposition fluxes in Gossenkölle-
see, Redon and Lochnagar ranged between 0.8 and
2.1 µg m−2 month−1,
and
in
Skalnate
Pleso
it
was
9.7 µg m−2 month−1, showing the influence of substan-
tial inputs from regional emission sources. The deposited
distributions of PAHs were dominated by parent phenan-
threne, fluoranthene and pyrene, representing 32 %–60 % of
the total. The proportion of phenanthrene, the most abundant
compound, was higher at the sites of lower temperature, Atmos. Chem. Phys., 18, 16081–16097, 2018
https://doi.org/10.5194/acp-18-16081-2018
© Author(s) 2018. This work is distributed under
the Creative Commons Attribution 4.0 License.
Drivers of atmospheric deposition of polycyclic aromatic
hydrocarbons at European high-altitude sites
Lourdes Arellano1, Pilar Fernández1, Barend L. van Drooge1, Neil L. Rose2, Ulrike Nickus3, Hansjoerg Thies4,
Evzen Stuchlík5, Lluís Camarero6, Jordi Catalan7, and Joan O. Grimalt1
1Institute of Environmental Assessment and Water Research (IDÆA-CSIC), Jordi Girona 18,
08034 Barcelona, Catalonia, Spain
2Environmental Change Research Centre, University College London, Gower Street,
London, WC1E 6BT, UK
3Department of Atmospheric and Cryospheric Sciences, University of Innsbruck,
Innrain 52, 6020 Innsbruck, Austria
4Institute of Interdisciplinary Mountain Research, Austrian Academy of Sciences,
Technikerstrasse 21a, 6020 Innsbruck, Austria
5Biology Centre, Czech Academy of Science, Institute of Hydrobiology, Na Sadkach 7,
37005 Ceske Budejovice, Czech Republic
6Centre for Advanced Studies of Blanes (CEAB-CSIC), Accés a la Cala St. Francesc 14,
17300 Blanes, Catalonia, Spain
7Centre for Ecological Research and Forestry Applications (CREAF), Campus UAB, Edifici C,
08193 Cerdanyola, Catalonia, Spain
Correspondence: Pilar Fernández (pilar.fernandez@cid.csic.es)
Received: 21 June 2018 – Discussion started: 24 July 2018
Revised: 21 September 2018 – Accepted: 15 October 2018 – Published: 8 November 2018
Abstract. Polycyclic aromatic hydrocarbons (PAHs) were
analysed in bulk atmospheric deposition samples collected
at four European high-mountain areas, Gossenköllesee (Ty-
rolean Alps), Redon (Central Pyrenees), Skalnate Pleso
(High Tatra Mountains), and Lochnagar (Grampian Moun-
Gossenköllesee and Skalnate Pleso, showing higher transfe
from gas phase to particles of the more volatile PAHs. Th
sites with lower insolation, e.g. those located at lower alti
tude, were those with a higher proportion of photooxidabl
compounds such as benz[a]anthracene. Atmos. Chem. Phys., 18, 16081–16097, 2018
https://doi.org/10.5194/acp-18-16081-2018
© Author(s) 2018. This work is distributed under
the Creative Commons Attribution 4.0 License. L. Arellano et al.: Drivers of atmospheric deposition 16082 (Golomb et al., 2001; Halsall et al., 2001; Feng et al., 2017). Deposited PAHs may revolatilise, be transported over long
distances and be deposited again on soil and water surfaces
far from the emission sources (Fernández et al., 2003; Singh
et al., 2017), e.g. in high-altitude areas (Fernández et al.,
2000, 2002; Vilanova et al., 2001; Vives et al., 2004; Gri-
malt et al., 2001, 2004; Halsall et al., 2001; Arellano et al.,
2011; van Drooge et al., 2010; Yang et al., 2016). hanced gas to particle partitioning transfer. In the warm sea-
son (June–October), most of the air masses arriving at Re-
don originated from the south and particle deposition was
enhanced as consequence of Saharan inputs. In these cases,
particle settling was also a driver of PAH deposition despite
the low overall PAH content of the Saharan particles. In Gossenköllesee, the site receiving lowest precipitation,
PAH fallout was also related to particle deposition. The
particle-normalised PAH fluxes were significantly negatively
correlated to temperature, e.g. for air masses originating from
central and eastern Europe, showing a dominant transfer
from gas phase to particles at lower temperatures, which en-
hanced PAH fallout, mainly of the most volatile hydrocar-
bons. Atmospheric deposition is one of the main mechanism
of transport of PAHs to all ecosystems (Leister and Baker,
1994). Direct measurements of the atmospheric deposition
of these pollutants have mostly been performed in indus-
trial/urban areas (Halsall et al., 1997; Franz et al., 1998;
Golomb et al., 2001; Park et al., 2001; Garban et al., 2002;
Gigliotti et al., 2005; Gocht et al., 2007; Esen et al., 2008;
Wang et al., 2011b; Bari et al., 2014). Only in a few cases
has deposition been considered in remote areas such as high-
altitude regions (Fernández et al., 2003; Offenthaler et al.,
2009; Foan et al., 2012). However, high mountain areas pro-
vide the reference background information of the pollution
impact, in this case PAHs, of the overall anthropogenic ac-
tivities in large regions. The study of the processes of trans-
port and pollutant incorporation into these remote ecosys-
tems shows the basic mechanisms of action of these com-
pounds to be far from direct human influence. Understand-
ing of these processes provides basic knowledge for descrip-
tion of the air-to-soil transfer mechanisms in all ecosystems. 1
Introduction Polycyclic aromatic hydrocarbons (PAHs) are semi-volatile
organic compounds (SOCs) originating from incomplete
combustion and pyrolysis of carbonaceous materials. In ad-
dition to natural sources, the global emissions of these com-
pounds are dominated by anthropogenic activity such as fos-
sil fuel combustion (Wild and Jones, 1995; Pacyna et al.,
2003), which are responsible for their ubiquitous occurrence
in the environment, especially in urban/industrial regions. In
Europe, the emission of PAHs in 2004 was estimated about
5.1×104 tons (calculated for 16 PAHs), with residential com-
bustion representing 88 % of the total contribution, followed
by industrial activities (van der Gong et al., 2007). To improve the knowledge on how PAHs move through
the atmosphere and partition between air and the terres-
trial and aquatic environments, bulk atmospheric deposi-
tion was collected at four high-altitude sites over 2 years,
covering different European climatic and source regions:
Redon (2235 m above sea level, Pyrenees), Gossenköllesee
(2413 m, Alps), Skalnate Pleso (1787 m, Tatras) and Lochna-
gar (790 m, Grampian Mountains). Bulk atmospheric deposi-
tion (dry and wet) was collected monthly in the three former
sites and biweekly in Lochnagar. The samples were analysed
to determine seasonal, spatial and temporal trends of atmo-
spheric PAH inputs and to identify the main processes deter-
mining PAH fallout at each site. Backward air mass trajec-
tories were calculated using the Hybrid Single-Particle La-
grangian Integrated Trajectory (HYSPLIT) model for assess-
ment of the air sources during sampling. To the best of our
knowledge, the number of sites and sampling frequency and
period of study constitute the most comprehensive approach
performed so far on PAH deposition in high mountain areas. The widespread distribution of PAHs is of great environ-
mental concern, since several parent (non-methylated) com-
pounds of this group of hydrocarbons are human carcino-
gens and priority pollutants (Baek et al., 1991; IARC, 1983;
Armstrong et al., 2004). Moreover, the Convention on Long-
range Transboundary Air Pollution included PAHs in the list
of persistent organic pollutants (POPs) (UNECE, 1998), the
emissions of which should be reduced to 1990 levels (EC,
2001). Implementation of regulatory activity caused an ini-
tial decrease in PAH emissions (Meijer et al., 2008) but re-
cent studies showed that PAHs have increased globally as a
consequence of a higher urban population, energy consump-
tion and vehicle use (van Metre and Mahler, 2005). L. Arellano et al.: Drivers of atmospheric deposition Furthermore, these remote areas may also act as secondary
sources of toxic substances as a consequence of re-emission
from terrestrial and aquatic ecosystems by air–water or air–
soil exchange or ice/glacier melting (Ma and Cao, 2010; Ma
et al., 2011; Kirchgeorg et al., 2016). Comparison of PAH atmospheric deposition and lacustrine
sedimentary fluxes showed much higher values in the lat-
ter case of 24–100 µg m−2 yr−1 vs. 120–3000 µg m−2 yr−1. A strong significant correlation was observed between these
two fluxes, which is consistent with a dominant origin related
to atmospheric deposition at each site. 2.1
Sampling Bulk atmospheric deposition samples were regularly col-
lected at four high mountain European areas (Table 1)
over the same period: monthly between 2004 and 2006 in
Gossenköllesee (Tyrolean Alps), Redon (Central Pyrenees)
and Skalnate Pleso (Tatra Mountains) and biweekly between
2004 and 2007 in Lochnagar (Grampian Mountains) (Fig. 1). All lakes are located in remote areas under special protec-
tion. Lake Redon is situated within the Aigüestortes area
belonging to the Natura 2000 European network. Lochna-
gar is located within the Deeside and Lochnagar National
Scenic Area, which define protected landscapes in Scotland
in the same category as national parks. Gossenköllesee and
its catchment area constituted one of the UNESCO Biosphere
Reserve from 1977 to 2014, while Skalnate Pleso is situated
in the area of the Tatra National Park. Meteorological param-
eters, i.e. air temperature and precipitation, were provided by
automatic weather stations (AWSs) located at each site ex-
cept in Skalnate Pleso where data were provided by the me-
teorological observatory of the Earth Science Institute of the
Slovak Academy of Science. In summer, precipitation was
collected with a polyethylene funnel (different diameter de-
pending on the sampling site) and connected to stainless steel
or Teflon-coated reservoirs by a tube. In winter, precipitation
occurs as snow and sampling was performed either with a
wider tube assembled to the top of a tank or with cylindrical
collectors. These devices were equipped with windscreens 1
Introduction Average temperature, atmospheric precipitation and particle (mg m−2 month−1) and PAH (µg m−2 month−1) monthly deposition
fluxes in the European mountain areas considered in the present study. a Metres above sea level. b Mean temperature for the whole sampling period. c Sum of all PAHs analysed from acenaphthylene to coronene. d Sum from fluorene to pyrene. e Sum from benz[a]anthracene to coronene. f Sum of PAHs included in the EPA list (excluding naphthalene). 2
Materials and methods around the collector’s mouth to prevent wind disturbance. The samplers were placed 1.5 m above ground level. Sam-
ples were filtered on site using pre-weighed Whatman glass
fibre filters (GF/B, 45 mm diameter, 1 µm pore size) at each
field station and the filtrates were solid-phase-extracted with
C18 Empore disks (47 mm diameter, 0.5 mm thickness) as
described elsewhere (Carrera et al., 1998). After sample re-
moval, the bulk collectors were rinsed with Milli-Q water,
which was filtered and solid-phase-extracted with the same
disk used for the corresponding deposition sample. Glass fi-
bre filters and disks were wrapped in aluminium foil and
transported frozen to the laboratory. More details on sam-
pling procedures are reported elsewhere (Arellano et al.,
2015). 2.2
Extraction and clean-up Glass-fibre filters were freeze-dried and weighed for mea-
suring total particle content in bulk atmospheric depo-
sition. PAHs were extracted from the filters by soni-
cation with dichloromethane:methanol (2 : 1) (3 × 10 mL,
20 min each). The pollutants adsorbed in the C18 disks
were eluted sequentially with methanol, cyclohexane and
dichloromethane (Carrera et al., 1998). Both phases were
combined and purified by column adsorption chromatogra-
phy with aluminium oxide after adding a recovery standard
mixture of perdeuterated anthracene-d10, benz[a]anthracene-
d10, benzo[b]fluoranthene-d12 and benzo[ghi]perylene-d12
(Dr. Ehrenstorfer GmbH; Augsburg, Germany). Prior to in-
strumental analysis, samples were spiked with an internal 1
Introduction Once in the atmosphere, PAHs may remain in the gas
phase or associate with particles (Gustafson and Dickhut,
1997; Park et al., 2001; Simcik et al., 1998), be degraded by
direct and/or indirect photolysis (Wang et al., 2011a, Zhang
et al., 2018) and be deposited by wet and dry processes www.atmos-chem-phys.net/18/16081/2018/ Atmos. Chem. Phys., 18, 16081–16097, 2018 L. Arellano et al.: Drivers of atmospheric deposition
16083
Table 1. Average temperature, atmospheric precipitation and particle (mg m−2 month−1) and PAH (µg m−2 month−1) monthly deposition
fluxes in the European mountain areas considered in the present study. Lochnagar
Redon
Gossenköllesee
Skalnate Pleso
Sampling period
Jun 2004–Mar 2007
May 2004–Sep 2006
Jun 2004–Aug 2006
May 2004–May 2006
Mountain range
Grampian Mountains
Pyrenees
Tyrolean Alps
Tatra Mountains
Latitude (N)
56.95914
42.64208
47.22528
49.189933
Longitude (E)
−3.23128
0.77951
11.01390
20.234217
Altitude (m a.s.l.)a
790
2235
2413
1787
Temperature (◦C)b
4.9
5.3
−1.4
2.2
Mean precipitation (mm month−1)
129
79
66
120
Particle flux(min–max)
130 (15–1600)
320 (14–2800)
145 (18–590)
330 (43–1800)
PPAHc (min–max)
2.1 (0.53–10)
0.80 (0.24–1.4)
1.3 (0.19–3.7)
9.7 (2.3–32)
Warm
1.8 (0.53–4.4)
0.89 (0.24–1.4)
1.3 (0.22–3.7)
8.4 (2.3–15)
Cold
2.25 (0.74–10)
0.63 (0.24–1.2)
1.3 (0.19–3.7)
8.8 (3.6–32)
LMW-PAHd (min–max)
1.1 (0.25–7.6)
0.30 (0.06–0.74)
0.89 (0.09–3.1)
5.6 (1.4–22)
Warm
0.94 (0.25–2.2)
0.30 (0.07–0.74)
0.86 (0.10–3.1)
5.7 (1.4–10)
Cold
1.2 (0.39–7.6)
0.28 (0.06–0.66)
0.90 (0.09–2.8)
4.2 (1.7–22)
HMW-PAHe (min–max)
0.97 (0.08–2.6)
0.49 (0.18–0.94)
0.40 (0.10–0.84)
3.9 (0.73–11)
Warm
0.82 (0.20–2.6)
0.57 (0.21–0.94)
0.42 (0.11–0.98)
2.3 (0.73–4.1)
Cold
1.1 (0.08–2.5)
0.34 (0.18–0.55)
0.41 (0.10–0.84)
4.5 (1.6–11)
PAH (P15 PAH-EPA)f (min–max)
1.9 (0.48–9.6)
0.70 (0.22–1.3)
1.2 (0.17–3.5)
8.8 (2.2–29)
a Metres above sea level. b Mean temperature for the whole sampling period. c Sum of all PAHs analysed from acenaphthylene to coronene. d Sum from fluorene to pyrene. e Sum from benz[a]anthracene to coronene. f Sum of PAHs included in the EPA list (excluding naphthalene). L. Arellano et al.: Drivers of atmospheric deposition
16083
Table 1. Average temperature, atmospheric precipitation and particle (mg m−2 month−1) and PAH (µg m−2 month−1) monthly deposition
fluxes in the European mountain areas considered in the present study. L. Arellano et al.: Drivers of atmospheric deposition 16083 Table 1. Average temperature, atmospheric precipitation and particle (mg m−2 month−1) and PAH (µg m−2 month−1) monthly deposition
fluxes in the European mountain areas considered in the present study. Table 1. 2.5
Back trajectory air mass calculations Three-day back trajectories were calculated using the Hy-
brid Single-Particle Lagrangian Integrated Trajectory (HYS-
PLIT data available at http://ready.arl.noaa.gov/HYSPLIT. php) modelling system developed by the National Oceanic
and Atmospheric Administration (NOAA) Air Resources
Laboratory (ARL) (Draxler and Hess, 1998; Draxler and
Rolph, 2013). www.atmos-chem-phys.net/18/16081/2018/ Atmos. Chem. Phys., 18, 16081–16097, 2018 16084 L. Arellano et al.: Drivers of atmospheric deposition L. Arellano et al.: Drivers of atmospheric deposition Figure 1. Location of the high-altitude areas included in this study. Lake identification: LN is Lochnagar, RDN is Lake Redon, GKS is
Gossenköllesee and SKP is Skalnate Pleso. Situation of the Black Triangle between Germany, Poland and Czech Republic is indicated by a
shaded area. Figure 1. Location of the high-altitude areas included in this study. Lake identification: LN is Lochnagar, RDN is Lake Redon, GKS is
Gossenköllesee and SKP is Skalnate Pleso. Situation of the Black Triangle between Germany, Poland and Czech Republic is indicated by a
shaded area. standard mixture of pyrene-d10 and perylene-d12 dissolved
in isooctane. Field and procedural blanks were collected at each sam-
pling site and processed together with the samples. For field
blanks, Milli-Q water was filtered and solid-phase adsorbed,
transported and stored for subsequent analysis. In general,
blank values represented less than 10 % of bulk deposition
sample concentrations. These values were used to determine
method detection limits (MDLs) that were established as av-
erage blank values plus 3 times the standard deviation. These
limits ranged between 0.45 and 28 pg depending on the com-
pound. 2.3
Instrumental analysis PAHs were analysed by gas chromatography–mass spec-
trometry (GC–MS; Trace DSQ II Instrument Thermo,
Austin, USA.) using a 60 m HP-5MS column (0.25 mm i.d. ×0.25 µm film thickness). The oven temperature programme
started at 90 ◦C (held for 1 min) and increased to 120 ◦C at
10 ◦C min−1, and then to 310 ◦C at 6 ◦C min−1 (final holding
time 25 min). Injector, transfer line and ion source temper-
atures were 280, 270 and 250 ◦C. Helium was used as the
carrier gas (1.2 mL min−1). The injector operated in splitless
mode. Data were acquired in electron impact and selective
ion monitoring modes. Further details on the ions selected
for quantification and mass spectrometric conditions are re-
ported elsewhere (Fernández et al., 1999). 2.4
Quality control and assurance The meteorological data used to compute the 6-hourly
back trajectories were obtained from the National Centre
for Environmental Prediction (NECP) Global Data Assimi-
lation System (GDAS) and from Eta Data Assimilation Sys-
tem (EDAS). The trajectories were calculated for those days
with precipitation (rain or snow) and every 2 days when no
precipitation was recorded by the AWS. Quantification was performed by the internal standard
method. Recoveries of the analytical procedure were eval-
uated using surrogate standards. The reported values were
corrected by these recoveries, which varied between 76 % for
benz[a]anthracene-d10 and 53 % for benzo[ghi]perylene-d12. Atmos. Chem. Phys., 18, 16081–16097, 2018 www.atmos-chem-phys.net/18/16081/2018/ 16085 L. Arellano et al.: Drivers of atmospheric deposition
16085
Table 2. Comparison of PAH deposition fluxes with those reported in the literature. Sampling site
Sampling period
Site type
P15 PAH-EPA
Benzo[a]pyrene
Reference
(µg m−2 d−1)
(ng m−2 month−1)
Skalnate Pleso
May 2004–May 2006
remote
0.28
280
This study
Lochnagar
Jun 2004–Mar 2007
remote
0.06
61
This study
Gossenköllesee
Jun 2004–Aug 2006
remote
0.04
20
This study
Redon
May 2004–Sep 2006
remote
0.02
51
This study
Gossenköllesee
Oct 1996–Oct 1998
remote
0.038
22
Fernandez et al. (2003)
Redon
Mar 1997–Oct 1998
remote
0.052
59
Fernandez et al. (2003)
Øvre Neadalsvatn
Dec 1997, Apr–Aug 1998
remote
0.063
41
Fernandez et al. (2003)
Chesapeake Bay
Jun 1990–Dec 1991
remote
0.55
500
Leister and Baker (1994)
Chesapeake Bay
1991
remote
0.4–0.55
500
Dickhut and Gustafson (1995)
Siskiwit Lake (Lake Superior, USA)
1983–1984
remote
0.46
225
McVeety and Hites (1988)
Natural Park (northern Spain)
Jun 2010–May 2011
remote
0.18
Foan et al. (2012)
Southern Ontario (Canada)
Oct 2001–Dec 2002
remote
0.41
345
Su et al. (2007)
Southern Italy
Dec 2003–Jan 2004
rural
0.09 (0.07–0.15)
140
Menichini et al. (2006)
Southern Germany
Aug 2001–Aug 2002
rural
0.55
Gocht et al. (2007)
Beijing–Tianjin region
Jun 2007–May 2008
rural
3.9
Wang et al. (2011b)
Pleumeur-Bodou (France)
Oct 1999–Oct 2000
rural
0.14
15 000
Garban et al. (2002)
Balaton Lake (Hungary)
1996–1997
rural
0.51–0.81
1000–1500
Kiss et al. (2001)
Abeschviller (France)
Oct 1999–Oct 2000
forest
0.19
15 000
Garban et al. (2002)
Bayreuth (Germany)
May 1995–Apr 1996
forest
0.42–2.4
620–1700
Horstmann and Mclachlan (1998)
Gardsjon (Sweden)
1991–1994
forest
0.51
Brorstrom-Lunden et al. (1998)
Western Mediterranean Sea
1989–1990
coastal
0.13
183
Lipiatou et al. (1997)
Eastern Mediterranean
Nov 2000–Jul 2002
coastal
0.46
Tsapakis et al. 3
Results and discussion Different altitudes were used to calculate the trajectories. Usually, they did not show differences; thus altitudes repre-
senting the air mass above the sampling point were selected
at 3000 m above sea level (a.s.l.) in Redon and Skalnate
Pleso, 3500 m a.s.l. for Gossenköllesee and 2000 m a.s.l. for
Lochnagar. The total number of backward trajectories was
1968, being 335, 475, 598 and 560 for Redon, Gossenkölle-
see, Lochnagar and Skalnate Pleso. More details on air
mass trajectory determination can be found in Arellano et
al. (2014). 2.4
Quality control and assurance (2006)
Atlantic Ocean (Canada)
1998–2000
coastal
0.12
25–75
Brun et al. (2004)
New England coast
Dec 1998–May 2000
coastal
0.22
Golomb et al. (2001)
Galvesston Bay (Texas, USA)
Feb 1995–Aug 1996
coastal
0.63
Park et al. (2001)
New Jersey
Jun 1999–Aug 2002
coastal
1.0
1.6
Gigliotti et al. (2005)
New Jersey
Feb 1998–Jan 2003
coastal
0.4–6.1
1.3–24
Gigliotti et al. (2005)
Tampa Bay
May–Aug 2002
coastal
6.8
120
Poor et al. (2004)
Beijing–Tianjin region
Jun 2007–May 2008
background
0.82
Wang et al. (2011b)
Western Greece
Jan 2001–Oct 2002
background
0.19
Terzi and Samara (2005)
New Jersey
Feb 1998–Aug 2002
suburban
0.81–3.1
1.7–12
Gigliotti et al. (2005)
Evreux (France)
Mar 2001–Feb 2002
suburban
0.13
Motelay-Massei et al. (2003)
Yangsuri, Yangpyoung
Feb–May 2000
suburban
5.5–24
Bae et al. (2002)
Taichung (Taiwan)
Aug–Dec 2002
suburban
39
Fang et al. (2004)
Izmit Bay (Turkey)
Sep 2002–Jul 2003
urban
8.3
1500
Pekey et al. (2007)
Manchester
1991–1992
urban
5.2
9000
Halsall et al. (1997)
Cardiff
1991–1992
urban
4.1
6600
Halsall et al. (1997)
Taichung (Taiwan)
Aug–Dec 2002
urban
49
Fang et al. (2004)
Beijing–Tianjin region
Jun 2007–May 2008
urban
8.3
Wang et al. (2011b)
Paris
Oct 1999–Oct 2000
urban
0.63
Garban et al. (2002)
Paris (France)
Nov 1999–Oct 2000
urban
0.64 (0.2–2.0)
Ollivon et al. (2002)
Seoul and Inchon
Feb–May 2000
urban
5.5–24
Bae et al. (2002)
New Jersey
Feb 1998–Aug 2002
urban
11–16
42–54
Gigliotti et al. (2005)
Bursa (Turkey)
Jul 2004–Mar 2005
industrial
0.3–19.5
Esen et al. (2008) L. Arellano et al.: Drivers of atmospheric deposition Table 2. Comparison of PAH deposition fluxes with those reported in the literature. 3
Results and discussion L. Arellano et al.: Drivers of atmospheric deposition e.g. remote, rural, coastal, industrial, suburban and urban ar-
eas. In addition, the deposition fluxes of benzo[a]pyrene are
also included in this Table for comparison. This hydrocar-
bon is a known carcinogen taken for reference in regula-
tory pollution values of PAH concentrations. As observed
for total PAHs, the benzo[a]pyrene mean deposition fluxes in
Skalnate Pleso, 280 ng m−2 month−1, are much higher than
those observed in Lochnagar, Gossenköllesee and Redon,
20–61 ng m−2 month−1 (Table 2). Skalnate Pleso and 66–129 mm month−1 in the other sites)
(Table 1) or particle flux (330 mg m−2 month−1 in Skalnate
Pleso and 145–320 mg m−2 month−1 in the other lakes) (Ta-
ble 1). The higher PAH deposition in Skalnate Pleso com-
pared to the other sites is in agreement with atmospheric PAH
concentrations and probably reflects regional contributions
from industrial emissions by factories located in southern
Poland and other areas such as the Black Triangle (Fig. 1)
(van Drooge et al., 2010). This geographical difference is
consistent with PAH concentrations reported in air on a Eu-
ropean scale, which identified high PAH emissions in eastern
Europe (Jaward et al., 2004). g
(
)
The observed PAH deposition fluxes in these high moun-
tain European areas, 0.02–0.28 µg m−2 d−1, range among
the lowest values reported in the literature (Table 2). Thus,
PAH deposition fluxes of 0.40–0.55 µg m−2 d−1 have been
recorded at remote sites from the USA (Su et al., 2007;
McVeety and Hites, 1988; Leister and Baker, 1994; Dickhut
and Gustafsson, 1995), whereas Foan et al. (2012) reported
mean deposition fluxes of PAHs of 0.18 µg m−2 d−1 in re-
mote areas of northern Spain. The reported PAH depositions
in background areas are again higher than those observed
in the high mountain European sites, 0.19–0.82 µg m−2 d−1
(Brorstrom-Lunden et al., 1998; Horstmann and McLach-
lan, 1998; Garban et al., 2002). Higher PAH deposition has
also been measured in rural areas, 0.09–3.9 µg m−2 d−1 (Kiss
et al., 2001; Garban et al., 2002; Menichini et al., 2006;
Gocht et al., 2007; Wang et al., 2011b) or in coastal sites,
0.4–6.8 µg m−2 d−1 (Lipiatou et al., 1997; Golomb et al.,
2001; Park et al., 2001; Brun et al., 2004; Poor et al., 2004;
Gigliotti et al., 2005; Tsapakis et al., 2006). L. Arellano et al.: Drivers of atmospheric deposition However, the values reported for different types
of sites do not show uniform trends such as those ob-
served when considering total PAH deposition. Thus,
benzo[a]pyrene deposition fluxes ranged between 225 and
500 ng m−2 month−1 in remote sites (Su et al., 2007;
McVeety and Hites, 1988; Leister and Baker, 1994; Foan et
al., 2012). In forested and rural areas, the benzo[a]pyrene
deposition has been measured to account between 140
and 15 000 ng m−2 month−1 (Horstmann and McLachan,
1998; Garban et al., 2002; Kiss et al., 2001; Menichini
et al., 2006). Finally, benzo[a]pyrene fluxes between 42
and 9000 ng m−2 month−1 have been reported in urban sites
(Halsall et al., 1997; Gigliotti et al., 2005; Pekey et al., 2007). The differences between Lochnagar, Gossenköllesee, and
Redon, and separately at Skalnate Pleso cannot be explained
by differences in precipitation (e.g. 120 mm month−1 in L. Arellano et al.: Drivers of atmospheric deposition As expected,
the PAH deposition fluxes measured in industrial areas, 0.3–
19.5 µg m−2 d−1 (Esen et al., 2008), in suburban areas, 0.13–
39 µg m−2 d−1 (Bae et al., 2002; Motelay-Massei et al., 2003;
Fang et al., 2004; Gigliotti et al., 2005), and in urban areas
0.63–49 µg m−2 d−1 (Halsall et al., 1997; Bae et al., 2002;
Garban et al., 2002; Ollivon et al., 2002; Fang et al., 2004;
Gigliotti et al., 2005; Pekey et al., 2007; Wang et al., 2011b),
are higher than those in the remote European mountains. p (
,
)
Comparison
of
the
PAH
deposition
measured
in
Gossenköllesee and Redon in 2004–2006 with those found
in 1996–1998 (Fernandez et al., 2003) shows nearly the same
values at Gossenköllesee (Table 2) but statistically signifi-
cant differences in Redon (p>0.05), involving lower fluxes
in the 2004–2006 period. In both cases, the deposition fluxes
range among the lowest measured in remote sites (Table 2),
which suggest that they correspond to background concen-
trations of long-range-transported PAHs and not to direct
pollution inputs. A very similar deposition of total particles
has been measured in Gossenköllesee in the two sampling
periods, 145 mg m−2 month−1 in 4 June–6 August (Table 1)
and 130 mg m−2 month−1 in October 1996–October 1998
(Fernandez et al., 2003), whereas total precipitation shows
a strong difference, with lower values in 2004–2006 of
66 mm month−1 (Table 1) vs. 110 mm month−1 (Fernandez
et al., 2003) in the 1996–1998 period. Precipitation in
the recent period is quite low, suggesting that the PAH
fallout fluxes at Gossenköllesee were mainly determined by
particle deposition. By contrast, in Redon both decreases
in particle deposition of 320 mg m−2 month−1 (Table 1)
from 420 mg m−2 month−1 (Fernandez et al., 2003) and
atmospheric precipitation of 79 mm month−1 (Table 1) from
110 mm month−1 (Fernandez et al., 2003) are observed
when comparing the recent (4 May–6 September) and
previous sampling periods (March 1997–October 1998). The differences in the deposition of particles and wet
precipitation are consistent with the lower PAH fallout from
the atmosphere observed at this site in 2004–2006. g
p
Concerning the benzo[a]pyrene deposition fluxes, again
those observed in the European high mountains, 20–
280 ng m−2 month−1, range among the lowest described (Ta-
ble 2). 3.1
Atmospheric PAH deposition fluxes. Spatial and
temporal variability Mean, minimum and maximum atmospheric deposition
fluxes of the PAHs at the four sampling sites during the stud-
ied period are summarised in Table 1. A strong contrast is
found between the total PAH mean fluxes found in Lochna-
gar, Gossenköllesse and Redon of 0.80–2.1 µg m−2 month−1,
and in Skalnate Pleso of 9.7 µg m−2 month−1, the latter
showing PAH deposition fluxes between 5 and 10 times
higher than the others. In Table 2, these deposition fluxes
are compared with those observed in different environments, www.atmos-chem-phys.net/18/16081/2018/ Atmos. Chem. Phys., 18, 16081–16097, 2018 16086 L. Arellano et al.: Drivers of atmospheric deposition Figure 2. Qualitative distribution of PAH mixtures in atmospheric deposition samples. Compound identification: Acnphlene
is acenaphthylene, Acnphtene is acenaphthene, Flu is fluorene, Phe is phenanthrene, Ant is anthracene, Fla is fluoranthene,
Acephe is acephenanthrilene, Pyr is pyrene, B[a]A is benz[a]anthracene, Chrys + Triph is chrysene + triphenilene, B[b + j]Fla is
benzo[b]fluoranthene + benzo[j]fluoranthene, B[k]Fla, benzo[k]fluoranthene, B[e]P is benzo[e]pyrene, B[a]P is benzo[a]pyrene, Per is
perylene, IndChrys is indeno[7,1,2,3-cdef]chrysene, IndPyr is indeno[1,2,3-cd]pyrene, B[ghi]Per is benzo[ghi]perylene, DB[ah]A is
dibenz[a,h]anthracene, Cor is coronene. Figure 2. Qualitative distribution of PAH mixtures in atmospheric deposition samples. Compound identification: Acnphlene
is acenaphthylene, Acnphtene is acenaphthene, Flu is fluorene, Phe is phenanthrene, Ant is anthracene, Fla is fluoranthene,
Acephe is acephenanthrilene, Pyr is pyrene, B[a]A is benz[a]anthracene, Chrys + Triph is chrysene + triphenilene, B[b + j]Fla is
benzo[b]fluoranthene + benzo[j]fluoranthene, B[k]Fla, benzo[k]fluoranthene, B[e]P is benzo[e]pyrene, B[a]P is benzo[a]pyrene, Per is
perylene, IndChrys is indeno[7,1,2,3-cdef]chrysene, IndPyr is indeno[1,2,3-cd]pyrene, B[ghi]Per is benzo[ghi]perylene, DB[ah]A is
dibenz[a,h]anthracene, Cor is coronene. Table 3. Average PAH isomeric ratios in atmospheric deposition. Table 3. Average PAH isomeric ratios in atmospheric deposition. Pyr / (Fla + Pyr)
BaA / (BaA + Chrys)
BaP / (BaP + BeP)
Gossenköllesee
Mean
0.40 ± 0.03
0.18 ± 0.04
0.38 ± 0.10
Warma
0.41 ± 0.04
0.19 ± 0.04
0.31 ± 0.13
Coldb
0.39 ± 0.02
0.17 ± 0.04
0.39 ± 0.06
Redon
Mean
0.43 ± 0.02
0.17 ± 0.04
0.42 ± 0.05
Warm
0.42 ± 0.02
0.18 ± 0.03
0.41 ± 0.05
Cold
0.43 ± 0.02
0.17 ± 0.05
0.43 ± 0.05
Skalnate Pleso
Mean
0.40 ± 0.02
0.28 ± 0.07
0.45 ± 0.12
Warm
0.41 ± 0.01
0.22 ± 0.04
0.51 ± 0.18
Cold
0.40 ± 0.02
0.32 ± 0.05
0.42 ± 0.07
Lochnagar
Mean
0.41 ± 0.06
0.31 ± 0.06
0.33 ± 0.12
Warm
0.42 ± 0.08
0.32 ± 0.06
0.36 ± 0.10
Cold
0.40 ± 0.04
0.30 ± 0.06
0.31 ± 0.11
a June–October, b November–May. a June–October, b November–May. were those with highest proportion of this compound (31 %–
32 %), whereas the lowest was observed in Redon (12 %,
5.3 ◦C). Lochnagar (4.9 ◦C) also showed a low but intermedi-
ate proportion of phenanthrene (18 %). Higher local temper-
atures likely involve lower condensation of the most volatile
PAHs and therefore lower deposition fluxes. L. Arellano et al.: Drivers of atmospheric deposition 2007; Fernández et al., 2003; Cetin et al., 2016), America
(Gigliotti et al., 2005; Brun et al., 2004) and Asia (Li et al.,
2009; Wang et al., 2011b; Sharma et al., 2018). In the present study, no significant differences in quali-
tative composition between sites were observed, except for
the relative proportion of phenanthrene, the most volatile
and abundant compound. The PAH distributions from the
sites with lower annual mean temperature, Gossenköllesee
and Skalnate Pleso at −1.4 and 2.2 ◦C respectively (Table 1), Consistently with these differences in phenanthrene con-
tent, the coldest sites, Gossenköllesee and Skalnate Pleso,
were those with the highest deposition of low and high Atmos. Chem. Phys., 18, 16081–16097, 2018 3.2
PAH composition Overall, the PAH composition in the atmospheric depo-
sition samples was dominated by parent compounds and,
among these, the low molecular weight fraction (LMW-
PAHs, from acenaphthene to pyrene). Phenanthrene, fluoran-
thene and pyrene were the most abundant, representing be-
tween 32 % and 60 % of the total PAH mixture (Fig. 2). These
compounds are also the most abundant in the atmosphere,
namely in the gas phase (Fernández et al., 2002). Dominance
of LMW-PAHs in atmospheric deposition and, particularly,
phenanthrene, fluoranthene and pyrene, is a common feature
of studies in Europe (Halsall et al., 1997; Holoubek et al., The differences between Lochnagar, Gossenköllesee, and
Redon, and separately at Skalnate Pleso cannot be explained
by differences in precipitation (e.g. 120 mm month−1 in Atmos. Chem. Phys., 18, 16081–16097, 2018 www.atmos-chem-phys.net/18/16081/2018/ L. Arellano et al.: Drivers of atmospheric deposition 16087 L. Arellano et al.: Drivers of atmospheric deposition Normalisation
of PAH fallout to particle deposition also showed significant
positive correlations with precipitation (p<0.01), which cor-
roborated the dominant role of rain and snow in the transfer
of PAHs from the atmosphere to the soil. These results are
also consistent with reports indicating that particle scaveng-
ing processes are significant mechanisms of PAH removal
from the atmosphere (Gocht et al., 2007; Li et al., 2016). In Gossenköllesee, the site receiving lowest precipitation,
66 mm month−1 (Table 1), PAH fallout was significantly cor-
related to particle deposition for the higher molecular weight
PAHs (p<0.05; Table 4) and no correlation was observed
between PAH fluxes and precipitation. Normalisation of the
PAH fluxes to particle deposition showed significant negative
correlations with precipitation for some compounds in the
low molecular weight range, e.g. fluoranthene, pyrene and
chrysene (p<0.05; Table 4). The particle-normalised depo-
sition flows of these compounds were also negatively corre-
lated to temperature. Lower temperatures enhance the PAH
transfer from gas phase to particles and this effect is stronger
among the PAHs of higher volatility, such as fluoranthene,
pyrene and chrysene. This association with the particulate
matter increases the deposition of these compounds. Previous studies considering the gas-particle partitioning
of these compounds in Gossenköllesee, Redon and Skalnate
Pleso showed that the observed distributions in Gossenkölle-
see and Skalnate more closely followed the theoretical
slope constants of the correlations between gas-phase par-
tition coefficients and octanol–air constants (Fernandez et
al., 2002; van Drooge et al., 2010), which was consistent
with a temperature-dependent reversible transfer between
gas phase and particle adsorption. Moreover, the significant
negative correlations of the particle-normalised deposition
fluxes of these compounds in Gossenköllesee with precipi-
tation may be explained by the significant positive correla-
tion between precipitation and temperature at this site. Ac-
cordingly, in the cold period lower precipitation and higher
particle-normalised PAH deposition were observed. The
average
benzo[a]pyrene / (benzo[e]pyrene
+
benzo[a]pyrene) ratios found in Gossenköllesee, Redon
and Skalnate Pleso were also consistent with insolation
differences, since the proportion of benzo[a]pyrene, the
compound more labile to photooxidation, was higher at the
lower-altitude sampling sites, at 0.38, 0.42 and 0.45 (Ta-
ble 3). However, this trend was not observed in Lochnagar,
the lowest-altitude site, which showed the lowest average
benzo[a]pyrene / (benzo[e]pyrene + benzo[a]pyrene)
ra-
tio. L. Arellano et al.: Drivers of atmospheric deposition molecular weight PAHs, of 0.89 and 0.40 µg m−2 month−1
and
5.6
and
3.9 µg m−2 month−1
(Table
1),
whereas
this proportion was inverse in Redon, with 0.30 and
0.49 µg m−2 month−1, and about the same in Lochnagar,
with 1.1 and 0.97 µg m−2 month−1 (Table 1). vestigated. Pearson correlation coefficients between the log-
transformed PAH monthly deposition fluxes and these vari-
ables were calculated at each site. The statistically signifi-
cant correlations found at 95 % (p<0.05) and 99 % (p<0.01)
confidence levels are shown in Table 4. µg
(
)
Further assessment on the qualitative changes in PAH
composition between samples can be obtained from the
study of diagnostic ratios (Table 3; Sicre et al., 1987). The
pyrene / (fluoranthene + pyrene)
ratios
showed
a
uniform composition between samples and therefore no
environmental dependence. In contrast, the sites located
at lower altitude, e.g. Lochnagar (790 m) and Skalnate
(1787 m) were those with a higher relative proportion
of benz[a]anthracene to its less labile to photooxida-
tion isomer, chrysene (Ding et al., 2007; Xing et al.,
2016). The lower-altitude sites receive less insolation than
those located at higher altitude. Accordingly, the aver-
age
benz[a]anthracene / (chrysene + benz[a]anthracene)
ratios
were
higher
in
Skalnate
(1787 m a.s.l.)
and
Lochnagar
(790 m a.s.l.),
between
0.28
and
0.31,
than
in
Gossenköllesee
(2413 m a.s.l.)
and
Redon
(2230 m a.s.l.),
0.17–0.18
(Table
3). Furthermore,
the
average benz[a]anthracene / (chrysene + benz[a]anthracene)
ratio in Skalnate Pleso (0.28; 1787 m) was even lower than
the one in Lochnagar (0.31; 790 m). Thus, insolation and
not local temperature was the main driver of the relative
changes between benz[a]anthracene and chrysene in all
sites. Differentiation between the cold (November–May)
and the warm (June–October) sampling periods, involving
higher insolation in the latter, did not show significant
differences in this ratio in Gossenköllesee, Redon and
Lochnagar (Table 3). However, in Skalnate Pleso higher
benz[a]anthracene / (chrysene + benz[a]anthracene)
ratio
was observed in the cold season (Table 3), which confirmed
a predominant insolation effect in the relative concentrations
of these two PAHs. In Redon and Lochnagar, the two sites receiving highest
precipitation, the fallout fluxes of most of these hydrocar-
bons were directly correlated to wet deposition, e.g. higher
precipitation involving higher PAH fallout. At these two
sites, no significant correlations were observed between PAH
fluxes and particle deposition or temperature. L. Arellano et al.: Drivers of atmospheric deposition In general, the benzo[a]pyrene / (benzo[e]pyrene +
benzo[a]pyrene) ratio is less sensitive to photooxidation than
the
benz[a]anthracene / (chrysene
+
benz[a]anthracene)
ratio (Behymer and Hites, 1988). Skalnate Pleso has some specific features since this lo-
cation receives enhanced PAH inputs from regional emis-
sions. These emissions involve the release of large amounts
of particulate matter containing PAHs (van Drooge et al.,
2010). This site received the highest particle deposition
flux, 330 µg m−2 month−1 (Table 1), and PAH fallout was
significantly correlated with particle deposition (p<0.05; www.atmos-chem-phys.net/18/16081/2018/ 16088 3.3
Meteorological drivers of PAH atmospheric
deposition The influence of particle deposition, precipitation and tem-
perature on PAH fallout fluxes at these remote sites was in- Atmos. Chem. Phys., 18, 16081–16097, 2018 www.atmos-chem-phys.net/18/16081/2018/ 16089 L. Arellano et al.: Drivers of atmospheric deposition Table 5. Pearson correlation analysis between PAH deposition fluxes and percentage of air mass trajectories recorded during sample collection. Otherwise
statistically significant at 95 % or 99 % confidence level. Table 4). Some PAHs, e.g. benzo[a]pyrene (R2 = 0.585),
benzo[ghi]perylene (R2 = 0.516 at p<0.001) and coronene
(R2 = 0.561), also showed significant negative correlations
with temperature (p<0.05; Table 4), which may reflect the
effects of higher winter production of electric power or other
combustion processes in the region. These results are consis-
tent with previous studies in urban and rural areas (Schif-
man and Boving, 2015; Birgul et al., 2011; Gaga et al.,
2009; Gocht et al., 2007; Brun et al., 2004; Shahpoury et al.,
2015), which also found higher PAH fluxes in winter. The
observed seasonal trend in Skalnate Pleso could reflect direct
impacts of regional sources, while Redon, Lochnagar and
Gossenköllesee received PAH mixtures from more distant ar-
eas and no significant seasonal influences were recorded. Table 5. Pearson correlation analysis between PAH deposition fluxes and percentage of air mass trajectories recorded during sample collection. Otherwise note that all correlations are
statistically significant at 95 % or 99 % confidence level. nb
Particles
Precipitation
T (◦C)
PPAH
LMW-PAH
HMW-PAH
PPAH / partc
LMW-PAH / partc
HMW-PAH / partc
Gossenköllesee
465
North Atlantic (17 %)a
−0.493
−0.580
−0.573
−0.558
Central–eastern Europe (11 %)
0.573
0.573
0.479
Redon
339
North Atlantic (38 %)
−0.4401
0.491
−0.601
0.558
0.564
0.513
South (51 %)
0.498
0.805
−0.483
−0.524
−0.4311
Central–eastern Europe (11 %)
−0.541
−0.611
Skalnate Pleso
560
South (26 %)
−0.493
−0.534
a Percentages of air mass trajectories from the specific origin with respect to total air mass trajectories calculated during the sampling periods (Table 1). Only the air mass trajectory origins involving significant correlations with PAH
deposition are included. A full description of the origins of the air mass trajectories is available in Arellano et al. (2014). b Total air mass trajectories calculated during the sampling period. c Normalised to particle deposition. 1 Statistically significant at 90 % confidence level. s. Chem. Phys., 18, 16081–16097, 2018 Normalisation of the PAH fallout to particle deposition
showed that precipitation is not a main driver of the transfer
of these hydrocarbons from atmosphere to soil in Skalnate
Pleso. However, temperature appeared as a significant pa-
rameter that was negatively correlated with PAH deposition
fluxes (p<0.05; Table 4). L. Arellano et al.: Drivers of atmospheric deposition As in the case of Gossenköllesee,
these negative correlations may reflect the higher adsorption
of PAHs to particles at lower temperatures, involving higher
particle PAH content and therefore higher deposition fluxes
of these hydrocarbons. Lower temperatures enhance the ca-
pacity of settling particles for the transfer of PAHs from at-
mosphere to soil. L. Arellano et al.: Drivers of atmospheric deposition Table 4. Pearson correlation analysis between atmospheric deposition fluxes of PAHs and environmental variables. Gossenköllesee
Redon
Skalnate Pleso
Lochnagar
Particles
Precipitation
T (◦C)
Particles
Precipitation
T (◦C)
Particles
Precipitation
T (◦C)
Particles
Precipitation
T (◦C)
Particles
–
–
−0.495a
0.460a
–
0.550b
0.529b
–
Precipitation
–
0.559a
−0.495a
–
0.550b
–
0.613b
–
Temperature
0.559a
–
0.460a
–
0.529b
0.613b
–
–
PPAH
0.720b
0.481a
LMW-PAH
0.616b
0.509a
HMW-PAH
0.547a
0.635b
0.416a
Phe
0.472b
0.531a
Fla
0.679b
0.444a
Pyr
0.661b
0.457a
Chrys
0.644a
0.645b
0.409a
BaP
0.562b
0.482a
−0.466a
0.378a
IndPyr
0.521a
0.510b
−0.448a
0.559a
BghiPer
0.619a
0.557b
0.475a
Cor
0.624a
0.603b
−0.488a
−0.390a
0.444a
Normalised by particle deposition
PPAH
−0.449a
−0.492a
0.544b
−0.608b
0.394a
LMW-PAH
−0.471a
0.564b
−0.490a
HMW-PAH
−0.488a
0.517b
−0.731b
0.443a
Phe
0.537b
Fla
−0.531a
−0.485a
0.556b
−0.654b
Pyr
−0.522a
−0.489a
0.553b
−0.637b
Chrys
−0.440a
−0.597a
0.510b
−0.684b
0.411a
BaP
0.484b
−0.765b
0.418a
IndPyr
0.594b
−0.738b
0.530b
BghiPer
0.497b
−0.407a
−0.718b
0.508b
Cor
0.562b
−0.433a
−0.749b
0.501b
All data have been log-transformed except precipitation and temperature. Only those correlations statistically significant at 95 % (a) and 99 % (b) are shown. chem-phys.net/18/16081/2018/
Atmos. Chem. Phys., 18, 160 www.atmos-chem-phys.net/18/16081/2018/ Atmos. Chem. Phys., 18, 16081–16097, 2018 16090 www.atmos-chem-phys.net/18/16081/2018/ 3.4
Correlation with air mass back trajectories Determination of the PAH source regions for each site was
performed by 72 h backward trajectory analyses using the
HYSPLIT model for the entire sampling periods (Table 1). A detailed description of the back trajectory analyses per-
formed in each study site is reported elsewhere (Arellano
et al., 2014). Air mass back trajectories and their contribu-
tions to the total trajectories measured in each sampling pe-
riod were assigned to each monthly deposition sample. The
number of trajectories calculated in each sampling site is in-
dicated in Table 5. The relationship between air mass trajectories and PAH
deposition was investigated for each sample. No trend was
observed at Lochnagar, indicating diffuse PAH inputs from
unspecific sources in this site. In Skalnate Pleso, a negative dependence between particle-
normalised PAH fluxes and air masses from the south was
observed, which may indicate that particles from this origin
were depleted in PAH content (Table 5). In Gossenköllesee, total particle mass was negatively cor-
related with air masses from the North Atlantic (Table 5). These air masses were also negatively correlated with to-
tal PAHs, including LMW-PAHs and HMW-PAHs (from
benzo[a]anthracene to coronene) (Table 5), which is consis-
tent with the predominant role of particle deposition at this
site (Table 4). Normalisation of PAH fallout to particle de- Atmos. Chem. Phys., 18, 16081–16097, 2018 www.atmos-chem-phys.net/18/16081/2018/ L. Arellano et al.: Drivers of atmospheric deposition 16091 Table 6. Comparison between atmospheric deposition and lacustrine sediment fluxes of PAHs in the study regions (µg m−2 yr−1). Lochnagar
Redon
Gossenköllesee
Skalnate Plesoa
Atmospheric deposition
Sampling period
Jun 2004–Mar 2007
May 2004–Sep 2006
Jun 2004–Aug 2006
May 2004–May 2006
PPAH
25
9.6
16
120
Sediment fluxes
Sampling year
2001
1991
1998
1991
2001
Sampling site
Lochnagar
Redón
Gossenköllesee
Starolenianske Pleso
Ladove Pleso
Catchment area (ha)
109
155
85
3.2
14.4
Lake area (ha)
10.4
24
1.7
0.71
1.7
PPAH concentration (ng g−1 dw)
4000
680
780
18 000
8700
PPAH total fluxes
480
120
125
3000
1400
PPAH total fluxes (estimated)b
260
62
780
452
850
PPAH ratio (measured / calculated)c
0.54
0.52
6.2
0.18
0.73
a Sediment data from Skalnate Pleso were not available. Sedimentation rates from two nearby lakes in the Tatra Mountains are shown for comparison. b Calculated considering that all atmospheric
inputs in the lake and its catchment area accumulate in the lake sediments (atmospheric deposition × catchment area)/lake area. 3.4
Correlation with air mass back trajectories c Ratio between measured sediment fluxes and those calculated from
the atmospheric deposition considering the lake and its catchment area. a Sediment data from Skalnate Pleso were not available. Sedimentation rates from two nearby lakes in the Tatra Mountains are shown for comparison. b Calculated considering that all atmospheric
inputs in the lake and its catchment area accumulate in the lake sediments (atmospheric deposition × catchment area)/lake area. c Ratio between measured sediment fluxes and those calculated from
the atmospheric deposition considering the lake and its catchment area. Figure 3. Prevalent back trajectories in Redon from 1 to
29 June 2006, mainly originating from southern areas. Southern air masses in this site are characterised by high
temperature and particle content (positive correlation with
T and particle deposition flows, Table 5), which is consis-
tent with Saharan dust inputs and the well-known geograph-
ical characteristics of the northern African regions. In this
case, a significant negative association was observed between
particle-normalised PAH flows and air masses from this ori-
gin, mainly for the LMW-PAHs (Table 5), which may re-
spond to both a gas-phase–aerosol displacement towards the
former and a low PAH content of the Saharan dust in compar-
ison to the PAH concentrations in particles from other areas. Despite this, the overall result is an increase in PAH loads to
Redon during the warm periods due to the high amount of
particles arriving at this site in spring–summer (Table 1). Air
masses originating from the North Atlantic involved higher
precipitation and lower particle content (significant positive
and negative correlation with these variables respectively). Particle-normalised PAH fallout was positively correlated
with air masses of this origin (Table 5), which was consis-
tent with the dependence of PAH deposition on precipitation
at this site (Table 4), likely enhanced by gas phase-aerosol
partitioning of PAHs at low temperatures and increased scav-
enging efficiency of snow compared to rain (Fernandez et al.,
2002; Arellano et al., 2011). Finally, air masses from central
and eastern Europe are negatively correlated with PAH depo-
sition fluxes, mainly for HMW-PAHs (Table 5). Air masses
from this origin were therefore not significant for PAH fall-
out. Figure 3. Prevalent back trajectories in Redon from 1 to
29 June 2006, mainly originating from southern areas. position showed a positive correlation with air masses from
central and eastern Europe (Table 5). 3.4
Correlation with air mass back trajectories The two main air mass trajectories arriving at Redon are
clearly different in terms of meteorological characteristics
and PAH deposition fluxes. As reported elsewhere, back-
wards air mass trajectories in Redon showed a well-defined
seasonal pattern (Arellano et al., 2014). Southern trajecto-
ries were dominant during the warm season (June–October;
59 %; see Fig. 3 as an example), while North Atlantic trajec-
tories prevailed during cold periods (November–May; 48 %). The central and eastern European trajectories identified in
this site were less frequent and did not show any seasonal
trend. 3.5
Atmospheric PAH deposition and lacustrine
sedimentary fluxes The atmospheric deposition samplers are located close
to high mountain lakes. Previous studies of sediment
cores in these lakes allowed the average PAH sediment Atmos. Chem. Phys., 18, 16081–16097, 2018 L. Arellano et al.: Drivers of atmospheric deposition The overall mass
balance involved pollutant incorporation into the lake waters
due to atmospheric precipitation and a substantial degassing
to the atmosphere. Thus, in these lakes the air–water transfer
processes cannot explain the higher sedimentation fluxes in
comparison to atmospheric precipitation. Figure 4. Average PAH sediment and atmospheric deposition
fluxes at the high-altitude lakes considered in this study. Units in
µg m−2 yr−1. atmospheric PAH origin in all studied sites. In Gossenkölle-
see, calculated sediment fluxes are 6-fold higher than mea-
sured values, which indicates that a small fraction of the
PAHs deposited in the lake watershed accumulate in the sur-
face sediments. On the contrary, calculated sediment fluxes
in Starolesnianske Pleso are 1 order of magnitude lower
than measured fluxes. The difference between Ladove and
Starolesnianske Pleso, both situated in the same region could
be related to orographic and hydrological characteristics of
the latter that enhanced the PAH inputs and accumulation
in Staroleniasnke Pleso sediments. This would explain the
high concentration of PAHs found in this site in comparison
with other lakes from the same area (Fernández et al., 1999,
2000). Another possibility for these differences could be re-
lated to a decrease in PAH atmospheric concentrations in
this region during the last decades. Atmospheric deposition
fluxes were measured between 2004 and 2006, whereas sed-
imentary fluxes correspond to 2001 in Ladove, but 10 years
before in the case of Starolesnianske Pleso (1991). Representation of the PAH sedimentary settling fluxes and
average atmospheric deposition from each high mountain site
showed a strong significant correlation (R2 = 0.99; p<0.01;
Fig. 4). The standardised residual of this correlation fulfilled
the normality conditions according to the Shapiro–Wilk test. Representation of the same data set excluding the area of
highest PAH deposition, High Tatras, also showed a strong
correlation (R2 = 0.86; p<0.20) but in this case there was
no statistical significance given the small statistical freedom
resulting from the number of cases considered. In any case,
the values of both data sets (Table 6) followed parallel distri-
butions. Regarding the contributions of PAH inputs from the wa-
tershed, an estimated sediment flux was calculated consid-
ering that the total amount of atmospheric PAHs deposited
in the lake and its catchment area were accumulated in the
lake sediments (Table 6). Interestingly, in the case of Lochna-
gar, Redon and Ladove, the ratio between calculated and
measured sediment fluxes varied between 0.5 and 0.7. L. Arellano et al.: Drivers of atmospheric deposition 16092 Figure 4. Average PAH sediment and atmospheric deposition
fluxes at the high-altitude lakes considered in this study. Units in
µg m−2 yr−1. fluxes to be determined in the top cores from Redon
and Gossenköllesee, 120 and 125 µg m−2 yr−1 respec-
tively (Fernandez et al., 1999). The sedimentation flux in
Lochnagar was calculated from the PAH concentrations
(4000 ng g−1 dw; data not published) using a sedimentation
rate of 0.012 g cm−2 yr−1 (Rose, 2001), which resulted in a
PAH flux of 480 µg m−2 yr−1. No lake sediment was avail-
able for Skalnate Pleso. Therefore, PAH fluxes from two
nearby lakes in the Tatra Mountains were considered, e.g. Starolesnianske Pleso with 3000 µg m−2 yr−1 (Fernandez et
al., 1999) and Ladove Pleso with 1400 µg m−2 yr−1 (Drooge
et al., 2011). ,
)
Comparison of the PAH atmospheric deposition and lacus-
trine sedimentary fluxes showed much higher values in sed-
iments, i.e. 24–100 and 120–3000 µg m−2 yr−1 respectively
(Table 6). All lakes considered in this comparison are lo-
cated in high mountain areas and their hydrological regime
is determined by atmospheric precipitation into the water-
shed. The strong difference in flux values of the direct PAH
atmospheric and sedimentary measurements may respond to
processes such as the sediment focusing (Rowan et al., 1995)
or lake sediment concentration of these hydrocarbons falling
into the surface of the lake catchment. Studies at low alti-
tude (seawater) have reported that air–water exchange is the
most important process for low molecular weight PAH inputs
into aquatic systems, exceeding the wet and dry deposition
(Tsapakis et al., 2006; Ruge et al., 2015). In these high moun-
tain lakes, the average concentrations of volatile PAHs are
lower than in low-altitude aquatic systems, e.g. air and water
concentrations of phenanthrene 0.99 ng m−3 (Fernandez et
al., 2003; van Drooge et al., 2010) and 180 ng m−3 (Vilanova
et al., 2001) respectively vs. 3.3–16 and 450–5600 ng m−3 in
marine systems (Gigliotti et al., 2002; Tsapakis et al., 2006). The smaller concentrations in high mountains should involve
lower gas–water transfer gradients (Nelson et al., 1998). Ir-
respective of these values, flux calculations in some of these
high mountain lakes for compounds with properties similar
to those of PAH showed that the main transfer essentially oc-
curs from water to air (Meijer et al., 2009). 4
Summary and conclusions and eastern Europe. Lower deposition of total particles and
PAHs were observed in this site for air masses originating
from the north. The PAH and benzo[a]pyrene deposition fluxes recorded
in the studied high mountain areas of Europe (790–
2413 m) range among the lowest described in remote,
rural, coastal, suburban, urban or industrial areas. Nev-
ertheless, a strong contrast is observed between the
PAH deposition fluxes in Lochnagar, Gossenköllesee and
Redon,
0.80–2.1 µg m−2 month−1,
and
Skalnate
Pleso,
9.7 µg m−2 month−1, the latter showing PAH deposition
fluxes between 5 and 10 times higher than the others, likely
as a consequence of inputs from southern Poland and other
areas. Comparison of PAH atmospheric deposition and lacustrine
sedimentary fluxes showed much higher values for the latter,
24–100 µg m−2 yr−1 compared with 120–3000 µg m−2 yr−1
respectively. However, the representation of the PAH set-
tling fluxes and average atmospheric deposition at each site
showed a strong significant correlation. Moreover, estimated
sediment fluxes calculated from the PAH atmospheric depo-
sition measured in each site, taking into account inputs from
the lake catchment, showed slightly lower values than mea-
sured fluxes, which indicates that the PAHs accumulated in
the lacustrine sediments of high mountains reflect the atmo-
spheric fallout of these hydrocarbons. Low molecular weight compounds from acenaphthene to
pyrene dominate the atmospheric deposition of PAHs, with
phenanthrene, fluoranthene and pyrene representing 32 %–
60 % of the total. The proportion of phenanthrene, the most
abundant compound in the PAH distribution of each sample
was higher at the sites of lower temperature, Gossenköllesee
and Skalnate Pleso, indicating higher transfer from gas phase
to particles of the more volatile PAHs. Data availability. The PAH deposition data are available from the
public repository digital.CSIC (http://hdl.handle.net/10261/171874,
Arellano et al., 2018). Insolation was another local property determining the
deposited PAH distributions. The sites with lower insola-
tion, e.g. those located at lower altitude, were those with
a higher proportion of photooxidable compounds such as
benz[a]anthracene. Author contributions. LA carried out sample processing and OC
analysis of the samples taken between 2004 and 2007 and per-
formed the backward air mass trajectory calculations. Sampling and
other field work were designed and performed at each site by NLR
(Lochnagar), UN and HT (Gossenköllesee), ES (Skalnaté), and LC
(Redón). PF performed the data interpretation and prepared the pa-
per with contributions from all co-authors, especially JOG. L. Arellano et al.: Drivers of atmospheric deposition This
consistency between atmospheric and sedimentary PAHs in
high-altitude areas situated in different areas of Europe is re-
markable, taking into account that it has been considered that
sediment area equals lake area, and confirms the predominant www.atmos-chem-phys.net/18/16081/2018/ Atmos. Chem. Phys., 18, 16081–16097, 2018 16093 Competing interests. The authors declare that they have no conflict
of interest. Competing interests. The authors declare that they have no conflict
of interest. Acknowledgements. This paper is dedicated to the memory of the
mountain guides Unai Pérez de Arenaza and Gaspar Giner-Abati,
who lost their lives because of an avalanche that occurred when
they were accompanying a scientific expedition to take samples in
Lake Redon (Pyrenees, 22 March 2018), and to Rober Sánchez,
who was seriously injured. We thank Patricia Alabart, Roser Chaler,
Dori Fanjul, and María Comesaña for their technical assistance in
GC and GC-MS analysis. We also thank the meteorological obser-
vatory in Skalnate Pleso (Division of Geophysics, Earth Science
Institute of Slovak Academy of Science) for providing of working
facilities, help with sampling and for meteorological data. Financial
support was provided by the CUANTOX (CTM2015-71832-P) and
GRACCIE-REDES (CTM2014-59111-REDC) projects from the
Spanish Ministry of Economy and Competitiveness and the EU
project EUROLIMPACS (GOCE-CT-2003-505540). Lourdes Arel-
lano is thankful for a grant provided jointly by Banco Santander
Central Hispano. In Gossenköllesee, the site receiving lowest precipitation,
PAH fallout was related to particle deposition, namely in the
case of higher molecular weight homologues. No correlation
was observed with precipitation and PAH deposition. How-
ever, the particle-normalised PAH deposition fluxes were sig-
nificantly negatively correlated to temperature according to
the transfer of these compounds from gas phase to particles
at lower temperatures, which enhanced PAH fallout, mainly
for the most volatile hydrocarbons. These dependences were
observed to occur when air masses originated from central Edited by: Kimitaka Kawamura
Reviewed by: two anonymous referees Edited by: Kimitaka Kawamura
Reviewed by: two anonymous referees Atmos. Chem. Phys., 18, 16081–16097, 2018 4
Summary and conclusions Precipitation was the main driver of PAH fallout. However,
when rain and snow deposition were low, particle settling
also constituted an efficient driver for PAH deposition. Ac-
cordingly, in Redon and Lochnagar, the two sites receiving
background long-range transported PAHs and highest precip-
itation, the fallout PAH fluxes were related to precipitation. Whereas no significant association was observed between
long-range backward air trajectories and PAH deposition in
Lochnagar, enhanced PAH fallout at higher precipitation es-
sentially occurred for air masses originating from the North
Atlantic in Redon. In these cases, particle-normalised PAH
fallout was also associated with higher precipitation as these
air masses were concurrent with lower temperatures, which
enhanced gas to particle partitioning transfer. In the warm
season (June–October), most of the air masses arriving at
Redon originated from the south and particle deposition was
enhanced as a consequence of Saharan inputs. In these cases,
particle settling was also a driver of PAH deposition despite
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Changing Human Visual Field Organization from Early Visual to Extra-Occipital Cortex
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See next page for additional authors Follow this and additional works at: https://digitalcommons.wustl.edu/open_access_pubs Follow this and additional works at: https://digitalcommons.wustl.edu/open_access_pubs Part of the Medicine and Health Sciences Commons i Part of the Medicine and Health Sciences Commons Part of the Medicine and Health Sciences Commons i Please let us know how this document benefits you. Please let us know how this document benefits you. Jack, Anthony I.; Patel, Gaurav H.; Astafiev, Serguei V.; Snyder, Abraham Z.; Akbudak, Erbil; Shulman,
Gordon L.; and Corbetta, Maurizio, "Changing human visual field organization from early visual to extra-
occipital cortex." PLoS One. 2, 5. 1-16. (2007).
https://digitalcommons.wustl.edu/open_access_pubs/414 Washington University School of Medicine
Washington University School of Medicine
Digital Commons@Becker
Digital Commons@Becker
Open Access Publications
2007
Changing human visual field organization from early visual to
Changing human visual field organization from early visual to
extra-occipital cortex
extra-occipital cortex
Anthony I. Jack
Washington University School of Medicine in St. Louis
Gaurav H. Patel
Washington University School of Medicine in St. Louis
Serguei V. Astafiev
Washington University School of Medicine in St. Louis
Abraham Z. Snyder
Washington University School of Medicine in St. Louis
Erbil Akbudak
Washington University School of Medicine in St. Louis
See next page for additional authors
Follow this and additional works at: https://digitalcommons.wustl.edu/open_access_pubs
Part of the Medicine and Health Sciences Commons
Please let us know how this document benefits you. Washington University School of Medicine
Washington University School of Medicine
Digital Commons@Becker
Digital Commons@Becker
Open Access Publications
2007
Changing human visual field organization from early visual to
Changing human visual field organization from early visual to
extra-occipital cortex
extra-occipital cortex
Anthony I. Jack
Washington University School of Medicine in St. Louis
Gaurav H. Patel
Washington University School of Medicine in St. Louis
Serguei V. Astafiev
Washington University School of Medicine in St. Louis
Abraham Z. Snyder
Washington University School of Medicine in St. Louis
Erbil Akbudak
Washington University School of Medicine in St. Louis
See next page for additional authors
Follow this and additional works at: https://digitalcommons.wustl.edu/open_access_pubs
Part of the Medicine and Health Sciences Commons
Please let us know how this document benefits you. Recommended Citation
Recommended Citation Jack, Anthony I.; Patel, Gaurav H.; Astafiev, Serguei V.; Snyder, Abraham Z.; Akbudak, Erbil; Shulman,
Gordon L.; and Corbetta, Maurizio, "Changing human visual field organization from early visual to extra-
occipital cortex." PLoS One. 2, 5. 1-16. (2007). https://digitalcommons.wustl.edu/open_access_pubs/414 This Open Access Publication is brought to you for free and open access by Digital Commons@Becker. It has been
accepted for inclusion in Open Access Publications by an authorized administrator of Digital Commons@Becker. For more information, please contact vanam@wustl.edu. Authors
Authors
Anthony I. Jack, Gaurav H. Patel, Serguei V. Astafiev, Abraham Z. Snyder, Erbil Akbudak, Gordon L. Shulman, and Maurizio Corbetta Authors
Authors
Anthony I. Jack, Gaurav H. Patel, Serguei V. Astafiev, Abraham Z. Snyder, Erbil Akbudak, Gordon L.
Shulman, and Maurizio Corbetta This open access publication is available at Digital Commons@Becker: https://digitalcommons.wustl.edu/
open_access_pubs/414 Background Both single-unit studies in macaques and BOLD imaging studies
in humans indicate that early visual areas show a precise
topographic organization, such that a large portion of occipital
cortex consists of a series of smooth and continuous representa-
tions of the contralateral visual field [1–3]. Outside occipital
cortex, both single-unit studies in macaques [4–8] and BOLD
imaging studies in humans [9–19] provide evidence for areas in
parietal and frontal cortices that prefer contralateral to ipsilateral
visual locations. However, these studies provide different perspec-
tives on the visual field organization of these higher areas. Single unit studies in macaques have clearly demonstrated
changes in visual field organization moving from early visual to
higher visual areas. Felleman and Van Essen [20] distinguish four
categories of topographic organization in the monkey, varying
from extremely precise and regular (V1), through intermediate
(V2/V3), course and irregular (e.g. V3A, V4), and finally little or
no discernible topography. The exact categorization of topo-
graphic organization of areas in macaque extra-occipital cortex
with visual receptive fields remains to be definitively determined;
however recent studies suggest these regions fall in the last two
categories. Thus, while most neurons in lateral intraparietal,
arcuate, and principal sulci (LIP, FEF and area 46) respond more
strongly to stimuli presented in the contralateral visual field, i.e. show a contralateral preference [but see 21], neurons representing
any given polar angle within the contralateral field are relatively
evenly distributed across the cortical surface. Therefore, at best
very coarse polar angle topography exists in these areas, with
a tendency for some grouping of neurons that represent similar
parts of the visual field, and/or a modest skew in the distribution of
receptive fields across the cortical surface [4–8]. Academic Editor: Nava Rubin, New York University, United States of America Academic Editor: Nava Rubin, New York University, United States of America
Received October 11, 2006; Accepted April 4, 2007; Published May 16, 2007 Received October 11, 2006; Accepted April 4, 2007; Published May 16, 2007 Copyright: 2007 Jack 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. Changing Human Visual Field Organization from Early
Visual to Extra-Occipital Cortex ony I. Jack1, Gaurav H. Patel2,3, Serguei V. Astafiev3, Abraham Z. Snyder1,3, Erbil Akbudak3, Gordon L. Shulman1, Patel2,3, Serguei V. Astafiev3, Abraham Z. Snyder1,3, Erbil Akbudak3, Gordon L. Shulman1, Maurizio Corbetta1,2,3* 1 Neurology, Washington University in St. Louis Medical School, St. Louis, Missouri, United States of America, 2 Anatomy and Neurobiology,
Washington University in St. Louis Medical School, St. Louis, Missouri, United States of America, 3 Radiology, Washington University in St. Louis
Medical School, St. Louis, Missouri, United States of America Background. The early visual areas have a clear topographic organization, such that adjacent parts of the cortical surface
represent distinct yet adjacent parts of the contralateral visual field. We examined whether cortical regions outside occipital
cortex show a similar organization. Methodology/Principal Findings. The BOLD responses to discrete visual field locations
that varied in both polar angle and eccentricity were measured using two different tasks. As described previously, numerous
occipital regions are both selective for the contralateral visual field and show topographic organization within that field. Extra-
occipital regions are also selective for the contralateral visual field, but possess little (or no) topographic organization. A
regional analysis demonstrates that this weak topography is not due to increased receptive field size in extra-occipital areas. Conclusions/Significance. A number of extra-occipital areas are identified that are sensitive to visual field location. Neurons
in these areas corresponding to different locations in the contralateral visual field do not demonstrate any regular or robust
topographic organization, but appear instead to be intermixed on the cortical surface. This suggests a shift from processing
that is predominately local in visual space, in occipital areas, to global, in extra-occipital areas. Global processing fits with a role
for these extra-occipital areas in selecting a spatial locus for attention and/or eye-movements. Citation: Jack AI, Patel GH, Astafiev SV, Snyder AZ, Akbudak E, et al (2007) Changing Human Visual Field Organization from Early Visual to Extra-
Occipital Cortex. PLoS ONE 2(5): e452. doi:10.1371/journal.pone.0000452 fiev SV, Snyder AZ, Akbudak E, et al (2007) Changing Human Visual Field Organization from Early Visual to Extra-
e452. doi:10.1371/journal.pone.0000452 itation: Jack AI, Patel GH, Astafiev SV, Snyder AZ, Akbudak E, et al (2007) Changing Human Visual Field Organizatio
Occipital Cortex. PLoS ONE 2(5): e452. doi:10.1371/journal.pone.0000452 between areas (but see [27–29]). The reason is that studies that
have compared more than two visual locations in humans have
tended to rely on a model-based approach called ‘phase-encoding’. Changing Human Visual Field Organization from Early
Visual to Extra-Occipital Cortex In phase-encoding studies, the BOLD response at each voxel is
measured as the location of a stimulus is cyclically varied at a fixed
frequency. The phase of the response then reflects the stimulus
position that evokes the strongest response. Two limitations of
phase-encoding, as compared to single unit studies, are: (i) phase-
encoding only measures the part of the BOLD response that varies
with visual location. In contrast, single units both measure signals
that vary with visual location and signals that are independent of
visual location. (ii) phase-encoding studies do not distinguish
different profiles of responses across visual locations. For instance,
single unit studies indicate quite different profiles of response for
early visual and extra-occipital areas in the macaque. Summing
unit responses over a small patch of cortex in macaque V1 would
produce a strong response to stimuli at one visual location in the
contralateral field and greatly diminished responses to all other
visual locations. In contrast, summing unit responses over a small
patch of cortex in macaque principal sulcus (area 46) would Academic Editor: Nava Rubin, New York University, United States of America
Received October 11, 2006; Accepted April 4, 2007; Published May 16, 2007 PLoS ONE | www.plosone.org Experimental Design and Tasks The problem is that the maps show the best
fitting location in a ‘winner take all’ fashion, even when the differences
between locations are insignificant. A more in-depth examination
requires looking at how the magnitude of BOLD response varies as we
move across the cortical surface. The dotted black line illustrates
a trajectory across the cortical surface. The graphs in the bottom panel
plot BOLD magnitudes along that trajectory. The topographic and non-
topographic cases can now be clearly distinguished. The investigations
we report here employ measures that take account of the relative
magnitude of BOLD response to distinct visual locations. doi:10.1371/journal.pone.0000452.g001 Figure 1. Different methods for assessing visual field organization. The top two panels show two simulated distributions of neurons
overlaid on a portion of the right hemisphere’s cortical surface. Note
that all the neurons prefer locations in the contralateral (left) visual field
(see figure key in top left corner). Electrophysiological studies
consistently show that most neurons prefer contralateral locations. In
the top left panel, the neurons are topographically organized, such that
neurons preferring nearby visual locations tend to lie close to each
other on the cortical surface. In the top right panel, there is no
topographic organization, such that neurons preferring different parts
of contralateral visual field are randomly intermixed on the cortical
surface. The middle panels show simulated BOLD maps in which voxels
are colored to indicate the preferred visual location. The topographic
organization of the neurons on the left is accurately reflected in the
surface map. However, the map on the right also produces an illusory
impression of topography. The problem is that the maps show the best
fitting location in a ‘winner take all’ fashion, even when the differences
between locations are insignificant. A more in-depth examination
requires looking at how the magnitude of BOLD response varies as we
move across the cortical surface. The dotted black line illustrates
a trajectory across the cortical surface. The graphs in the bottom panel
plot BOLD magnitudes along that trajectory. The topographic and non-
topographic cases can now be clearly distinguished. The investigations
we report here employ measures that take account of the relative
magnitude of BOLD response to distinct visual locations. doi:10.1371/journal.pone.0000452.g001 A total of four subjects were tested. Their participation in
different tasks is detailed in methods (Table 1). Experimental Design and Tasks p
g
We measured whole-brain BOLD response during performance of
two different tasks. The primary task was a delayed saccade task,
shown in Figure 2A. This task was similar to that used in recent
phase-encoding studies of parietal cortex (with just one substantial
difference, see below), in which subjects perform memory-guided
saccades to different visual field locations [9,10]. The second task
was a visual oddball task, shown in Figure 2B. A stream of
standard targets presented at the fovea was interrupted by
unexpected and low frequency ‘oddball’ stimuli presented at
different peripheral locations. This second task was originally
chosen for its ability to activate a network of areas which are
preferentially engaged when unexpected events occur and/or
subjects are required to reorient attention [for a review see 32]. Hence, we expected this task to reveal visual field organization in
additional areas, however the results turned out to be highly
consistent across the two tasks. As a result, in our analyses the
oddball task serves the role of an independent data set that is used
to replicate findings from the delayed saccade task, while
conveniently controlling for a variety of task-specific factors. Figure 1. Different methods for assessing visual field organization. The top two panels show two simulated distributions of neurons
overlaid on a portion of the right hemisphere’s cortical surface. Note
that all the neurons prefer locations in the contralateral (left) visual field
(see figure key in top left corner). Electrophysiological studies
consistently show that most neurons prefer contralateral locations. In
the top left panel, the neurons are topographically organized, such that
neurons preferring nearby visual locations tend to lie close to each
other on the cortical surface. In the top right panel, there is no
topographic organization, such that neurons preferring different parts
of contralateral visual field are randomly intermixed on the cortical
surface. The middle panels show simulated BOLD maps in which voxels
are colored to indicate the preferred visual location. The topographic
organization of the neurons on the left is accurately reflected in the
surface map. However, the map on the right also produces an illusory
impression of topography. The problem is that the maps show the best
fitting location in a ‘winner take all’ fashion, even when the differences
between locations are insignificant. A more in-depth examination
requires looking at how the magnitude of BOLD response varies as we
move across the cortical surface. Background Funding: This investigation was supported by National Institutes of Health,
National Eye Institute Institutional National Research Service Award 5-T32-
EY13360-04, National Institute of Mental Health R01 MH71920-06, National
Institute of Neurological Disorders and Stroke R01 NS48013. The authors declare
no conflicts of interest. In humans, studies of visual field organization have tended to
emphasize the presence of topographic organization in early visual
[1–3], higher occipital [22–26] and extra-occipital areas [9–12]. There has been less focus on differences in visual field organization Competing Interests: The authors have declared that no competing interests
exist. * To whom correspondence should be addressed. E-mail: mau@npg.wustl.edu * To whom correspondence should be addressed. E-mail: mau@npg.wustl.edu PLoS ONE | www.plosone.org May 2007 | Issue 5 | e452 May 2007 | Issue 5 | e452 1 Visual Field Organization The ability to characterize visual field organization in this way is
significant because it sheds light on the distribution of neuronal
inputs to these areas [30]. This, in turn, has implications for the
function of these areas [31]. It is also significant for methodological
reasons. The ability to identify visual areas on the basis of their
visual field organization has proven a key tool for neuroscientific
research on the human visual system. The most efficient method
for identifying specific visual areas will clearly depend on the
nature of their visual field organization. A further motivation for
this study is to help resolve an apparent discrepancy in current
characterizations of visual field organization in human parietal
lobe [9–11]. To address this, we initially focus our analysis by
examining changes in visual field organization from early visual to
parietal cortex. We then widen our net to examine other areas. In
total we were able to identify seven extra-occipital regions sensitive
to visual field location, all but one in cortical locations similar to
where previous studies have found either topographic maps or
a contralateral preference [10,12–19]. produce nearly equal responses to all locations in the contralateral
visual field, and diminished responses for locations in the ipsilateral
visual field. Phase encoding measurements cannot easily distin-
guish between these two profiles of response. Goals and Significance Figure 1 illustrates the sensitivity of different methods for detecting
topographic organization. The goal of the current study is to use
a methodology suited to revealing differences in visual field
organization between areas. Images generated by phase encoding
have created the impression of clear topographic organization in
human extra-occipital cortex, akin to that seen in occipital visual
areas. We wanted to assess whether this impression is correct. To
do this, we obtained independent measurements of the BOLD
response associated with discrete locations in the contralateral and
ipsilateral visual fields, relative to a no-stimulus control. This
technique allows us to measure (i) the magnitude of responses that
depend on visual location vs. the magnitude of responses that do
not; and (ii) measure the profile of response to different visual
locations. Experimental Design and Tasks The dotted black line illustrates
a trajectory across the cortical surface. The graphs in the bottom panel
plot BOLD magnitudes along that trajectory. The topographic and non-
topographic cases can now be clearly distinguished. The investigations
we report here employ measures that take account of the relative
magnitude of BOLD response to distinct visual locations. doi:10.1371/journal.pone.0000452.g001 Since an overall goal of the experiment was to compare visual
field organization in occipital and extra-occipital areas, we wanted
to measure location specific responses in all areas using the same
task. Previous studies have suggested a gradient of sensitivity, such
that occipital regions are more sensitive to visual stimulation and
extra-occipital regions are more sensitive to top-down factors,
while all regions appear to show at least some sensitivity to both
types of process [10,11,33,34]. Thus, in the present tasks, visual
stimulation and top-down processes were varied in tandem,
allowing us to map both occipital and extra-occipital regions with
maximal sensitivity using the same task. The trade-off is that no
claims are made regarding the specific signal (e.g. sensory or
attentional) that was mapped. The inclusion of visual stimulation,
specific to the region of visual space as the saccade target,
represented the only substantial difference between the delayed
saccade task used here, and the tasks used in previous investiga-
tions [9,10,33]. Figure 1. Different methods for assessing visual field organization. The top two panels show two simulated distributions of neurons
overlaid on a portion of the right hemisphere’s cortical surface. Note
that all the neurons prefer locations in the contralateral (left) visual field
(see figure key in top left corner). Electrophysiological studies
consistently show that most neurons prefer contralateral locations. In
the top left panel, the neurons are topographically organized, such that
neurons preferring nearby visual locations tend to lie close to each
other on the cortical surface. In the top right panel, there is no
topographic organization, such that neurons preferring different parts
of contralateral visual field are randomly intermixed on the cortical
surface. The middle panels show simulated BOLD maps in which voxels
are colored to indicate the preferred visual location. The topographic
organization of the neurons on the left is accurately reflected in the
surface map. However, the map on the right also produces an illusory
impression of topography. Experimental Design and Tasks Subject
Delayed
saccade
Oddball task
Delayed
saccade
Passive
retinotopy
polar angle
polar angle
eccentricity
A
3
3
3
1
B
3
3
-
1
C
3
-
3
1
D
3
3
-
-
doi:10.1371/journal.pone.0000452.t001
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Table 1. Number of scanning sessions (separate days) in
which subjects participated in each task
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . doi:10.1371/journal.pone.0000452.t001
. . . documented, we sought to verify that we could reproduce the
typical findings (Figure 3). In order to explicitly compare our active
tasks with a traditional passive viewing of checkerboard wedges,
we conducted meridian mapping in three of our subjects (see
methods). The results are illustrated in Figure 3A. The solid and
dotted black lines were drawn to mark the horizontal and vertical
meridians on the basis of the passive data, and then overlaid on the
data from the two active tasks (Figure 3B&C) to allow a close
comparison. The horizontal meridian (solid line) from the
meridian mapping task was centered within the middle-location
stimuli from the active tasks, coded in dark blue. The vertical
meridian (dotted line) from the meridian mapping task was slightly
offset from the upper (green) and lower field (red) stimuli from the
active tasks. This is exactly as would be predicted, since the upper
and lower locations in the active tasks subtended 30 deg from the
vertical meridian. The high degree of consistency between the Figure 3. Correspondence between passive retinotopy and active
mapping tasks for polar angle topography. A flattened representation
of occipital cortex of subject A is shown. (A) shows data from a passive
viewing task in which contrast reversing checkerboards were displayed
in alternating blocks along the horizontal and vertical meridians. Experimental Design and Tasks Lines
corresponding to the most robust representations of the horizontal and
vertical meridians were drawn on the basis of this data set, and are
reproduced in the other panels for comparison. (B) shows data from the
polar angle version of the delayed saccade paradigm. Voxels showing
a preference for the contralateral field were colored according to which
of the three contra-lateral locations produced the greatest response. Note the close correspondence for the horizontal meridian, and the
slight gap in activated representation on the vertical meridian, due to
the stimuli lying 30 degrees from the vertical in this paradigm. (C)
shows data from the oddball task, derived in the same way. doi:10.1371/journal.pone.0000452.g003 Figure 2. The two behavioral tasks used in the study. (A) In the
delayed saccade task subjects maintained fixation while a target dot
was briefly presented. This was followed by a variable delay period
during which flickering dots appeared in the same sector of the visual
field as the target. When the screen went black, subjects made a rapid
saccade to the remembered target location, then back to the center. Trials occurred in blocks of three, with small variations in target
location, within the same sector, between trials (see methods). (B) The
oddball task required subjects to categorize each stimulus, presented
approx. once per second, as either ‘standard’ or ‘oddball’ using a manual
response. On most trials the same ‘standard’ object was presented at
the center, while on 12% of trials a novel object was presented in one of
7 locations (fovea or 6 peripheral locations as in the polar angle version
of the delayed saccade task). Subjects maintained fixation throughout. (C) In the oddball task and the polar angle version of the delayed
saccade task, the six sector centers were evenly distributed around
a circle with a radius of approx 7 degrees visual angle. In the
eccentricity version of the delayed saccade task, the sector centers lay
on the horizontal meridian, at 2, 13 or 24 degrees eccentricity. doi:10.1371/journal.pone.0000452.g002 Figure 3. Correspondence between passive retinotopy and active
mapping tasks for polar angle topography. A flattened representation
of occipital cortex of subject A is shown. (A) shows data from a passive
viewing task in which contrast reversing checkerboards were displayed
in alternating blocks along the horizontal and vertical meridians. Experimental Design and Tasks Since this
study focuses on fine-grained functional anatomy, the data was
not intentionally spatially smoothed at any point. Unavoidable
smoothing due to co-registration of images and atlas registration
was
minimized
by
re-sampling
the
data
only
once
(see
methods). May 2007 | Issue 5 | e452 May 2007 | Issue 5 | e452 PLoS ONE | www.plosone.org 2 Visual Field Organization Figure 2. The two behavioral tasks used in the study. (A) In the
delayed saccade task subjects maintained fixation while a target dot
was briefly presented. This was followed by a variable delay period
during which flickering dots appeared in the same sector of the visual
field as the target. When the screen went black, subjects made a rapid
saccade to the remembered target location, then back to the center. Trials occurred in blocks of three, with small variations in target
location, within the same sector, between trials (see methods). (B) The
oddball task required subjects to categorize each stimulus, presented
approx. once per second, as either ‘standard’ or ‘oddball’ using a manual
response. On most trials the same ‘standard’ object was presented at
the center, while on 12% of trials a novel object was presented in one of
7 locations (fovea or 6 peripheral locations as in the polar angle version
of the delayed saccade task). Subjects maintained fixation throughout. (C) In the oddball task and the polar angle version of the delayed
saccade task, the six sector centers were evenly distributed around
a circle with a radius of approx 7 degrees visual angle. In the
eccentricity version of the delayed saccade task, the sector centers lay
on the horizontal meridian, at 2, 13 or 24 degrees eccentricity. doi:10.1371/journal.pone.0000452.g002 Table 1. Number of scanning sessions (separate days) in
which subjects participated in each task
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Experimental Design and Tasks Lines
corresponding to the most robust representations of the horizontal and
vertical meridians were drawn on the basis of this data set, and are
reproduced in the other panels for comparison. (B) shows data from the
polar angle version of the delayed saccade paradigm. Voxels showing
a preference for the contralateral field were colored according to which
of the three contra-lateral locations produced the greatest response. Note the close correspondence for the horizontal meridian, and the
slight gap in activated representation on the vertical meridian, due to
the stimuli lying 30 degrees from the vertical in this paradigm. (C)
shows data from the oddball task, derived in the same way. doi:10.1371/journal.pone.0000452.g003 PLoS ONE | www.plosone.org Path of the cortical trajectory j
y
The
single
continuous
‘cortical
trajectory’
is
illustrated
in
Figures 4A–C. This trajectory was drawn by hand to optimally
capture visual field organization in lower and higher occipital
areas, and to focus on extra-occipital parietal regions investigated
in earlier studies. The color-coded map in Figures 4A–C shows
significantly greater activity for contralateral than ipsilateral
locations, and is color-coded according to which of the three
locations within the contralateral field produced the greatest
BOLD response. Green corresponds to the upper visual field
location, blue to the horizontal meridian location, and red to the
lower visual field location (as in Figure 3B&C). The cortical
trajectory started at the horizontal meridian representation of V1
in the fundus of the calcarine sulcus (Figures 4A and 4C),
continued through dorsal visual areas V2, V3, V3A & V7, and the
medial bank of IPS (Figure 4B). In visual areas, the trajectory was
drawn to best capture the polar-angle topography that defines the
borders between regions. It was then carefully extended into
medial IPS to cross-sect those parts of cortex that showed the
greatest evidence of spatial selectivity. Thus, this trajectory moved
through the area of cortex between V7 and IPS previously
reported to contain topographic maps IPS1 and IPS2 [9,11]. Figure 4C shows this cortical trajectory on a flattened represen-
tation of cortex. Figures 4D&E shows BOLD responses to discrete
visual field locations in the polar-angle version of the delayed
saccade task, as a function of distance along the cortical surface. These are explained in further detail in the following sections. Figure 4. Visual field organization of dorsal visual areas and medial
intra-parietal sulcus, from the polar angle version of the delayed
saccade task. A and B: Medial and lateral views of an inflated
representation of occipital cortex. C: flat representation of dorsal occipital
and parietal cortex. Maps show preferred visual field location thresholded
by contra-lateral preference, as in Figure 3 B&C. The cortical trajectory
(shown in black) was drawn from the horizontal meridian of V1, in the
calcarine sulcus, through V2, V3, V3A, V7 and then through those parts of
medial intraparietal sulcus showing the greatest sensitivity to visual field
location. D: Magnitude of BOLD activity associated with the three contra-
lateral visual field positions along the trajectory (colors match maps in
panels A–C). E: Mean magnitude associated with contra-lateral (pink) and
ipsi-lateral (blue) visual field positions. Progressive shift in organization from V1 to parietal
cortex We next aim to illustrate how visual field organization changes
from early visual areas through to extra-occipital areas. The extra-
occipital area we initially focus on is the medial bank of intra-
parietal sulcus (IPS), since this has been a focus of prior
investigations. Sereno et al [10] found evidence of an isolated
region containing a map of the contralateral visual hemisphere
some distance dorsal/anterior to V7, the most dorsal part of the
retinotopic belt. This finding is supported by other studies that
have found evidence of an isolated region of contralateral
preference in a similar cortical location [e.g. 18,19]. This region
is sometimes referred to as the putative human homologue of
monkey lateral intraparietal sulcus, or hLIP. In contrast, Silver et
al [11] and Schluppeck et al [9] reported evidence for two
topographic maps, IPS1 and IPS2, that tile the region extending
anteriorly from V7 along the medial bank of IPS. Atlas
coordinates appear to place hLIP anterior to both IPS1 and
IPS2. One possibility, which might help account for these
discrepancies, is that the functional organization of parietal
regions sensitive to visual location may primarily reflect larger
responses for contralateral than ipsilateral stimuli, i.e. contralateral
organization,
rather
than
differential
responses
within
the
contralateral field, i.e. topographic organization. If this were the
case, the apparent topographic organization revealed by phase
encoding maps would likely prove unreliable and misleading,
causing different groups to reach divergent conclusions when
drawing the borders between areas on the basis of those maps. To
assess this possibility, we measured responses along a cortical
trajectory from occipital cortex to IPS. Data from one hemisphere
is illustrated in Figure 4. Data from all hemispheres can be found
in figures S1 and S2. Figure 4. Visual field organization of dorsal visual areas and medial
intra-parietal sulcus, from the polar angle version of the delayed
saccade task. A and B: Medial and lateral views of an inflated
representation of occipital cortex. C: flat representation of dorsal occipital
and parietal cortex. Maps show preferred visual field location thresholded
by contra-lateral preference, as in Figure 3 B&C. The cortical trajectory
(shown in black) was drawn from the horizontal meridian of V1, in the
calcarine sulcus, through V2, V3, V3A, V7 and then through those parts of
medial intraparietal sulcus showing the greatest sensitivity to visual field
location. Progressive shift in organization from V1 to parietal
cortex D: Magnitude of BOLD activity associated with the three contra-
lateral visual field positions along the trajectory (colors match maps in
panels A–C). E: Mean magnitude associated with contra-lateral (pink) and
ipsi-lateral (blue) visual field positions. F: Compass plots illustrating the
BOLD magnitude associated with each of the six polar angles. Each plot
comes from a cortical area demonstrating a preference for the contra-
lateral horizontal meridian (blue regions in panels A–C). Data shown
comes from the right hemisphere of subject A. Supplementary figures S1
and S2 show panels C–E for all eight hemispheres investigated. doi:10.1371/journal.pone.0000452.g004 Topographic maps of early visual areas We first show that our mapping technique yields the expected
topography in early visual cortex (Figure 2). This provides
preliminary support for the reliability of our methods. Our
methods were designed to reveal visual field organization in both
early and higher visual areas, by combining bottom-up and top-
down influences. Since the topographic organization of early
visual areas using passive viewing techniques has already been well PLoS ONE | www.plosone.org May 2007 | Issue 5 | e452 3 Visual Field Organization Figure 4. Visual field organization of dorsal visual areas and medial
intra-parietal sulcus, from the polar angle version of the delayed
saccade task. A and B: Medial and lateral views of an inflated
representation of occipital cortex. C: flat representation of dorsal occipital
and parietal cortex. Maps show preferred visual field location thresholded
by contra-lateral preference, as in Figure 3 B&C. The cortical trajectory
(shown in black) was drawn from the horizontal meridian of V1, in the
calcarine sulcus, through V2, V3, V3A, V7 and then through those parts of
medial intraparietal sulcus showing the greatest sensitivity to visual field tasks provides a validation of the underlying method and confirms
that subjects maintained fixation during the active tasks. tasks provides a validation of the underlying method and confirms
that subjects maintained fixation during the active tasks. Responses to contralateral and ipsilateral locations Table 2. Summary of extra-occipital cortical areas preferring the contra-lateral visual field. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Area
Hemi-
sphere
Mean
volume
(mm3)
Talaraich coordinates mean(s.d.)
Mean peak contra-lateral
z score
Number passing
whole brain correction
for delay saccade
Replication in oddball task
x
y
z
Delay sacc. Oddball
t
p
MIPSv
L
347
230(5)
258(4)
47(4)
15.2
7.0
4/4
4.8
0.002
R
261
27(1)
259(6)
52(3)
15.7
5.7
4/4
PCuv
L
171
27(2)
260(4)
50(2)
11.1
7.2
4/4
3.1
0.013
R
117
5(3)
262(11)
52(4)
9.8
3.8
4/4
STv
L
110
248(3)
251(9)
6(4)
6.6
3.6
2/4
3.8
0.006
R
65
56(6)
239(8)
16(5)
6.6
3.2
3/4
IFEFv
L
234
234(5)
211(4)
50(5)
8.8
3.5
3/4
2.5
0.027
R
214
32(4)
211(3)
52(3)
9.9
3.9
4/4
SFEFv
L
122
223(3)
211(6)
55(6)
6.2
3.4
3/4
4.0
0.005
R
104
21(1)
211(8)
60(6)
7.3
2.7
3/4
MPCev
L
203
246(3)
25(4)
41(4)
10.5
4.4
4/4
3.8
0.006
R
97
48(7)
26(5)
38(7)
7.8
3.0
2/4
IFSv
L
180
240(5)
16(6)
28(5)
7.7
3.0
3/4
3.2
0.011
R
131
39(4)
12(4)
29(5)
8.4
3.8
3/4
doi:10.1371/journal.pone.0000452.t002
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Responses to distinct contralateral locations Evidence for progressive changes in topography is shown in
Figure 4D, which plots the magnitude of the BOLD response to
the three contralateral visual field locations at each point along the
cortical trajectory. In early visual areas (V1, V2, V3) there was
very little overlap in the activity due to different locations in the
contralateral field, reflecting the fine topography in these areas. For instance, throughout dorsal V1–V3 there was very little
activation for the upper field quadrant (green line); starting from
the fundus of the calcarine sulcus (position 0 on the x-axis), high
activity was measured for the horizontal meridian location (blue
line) but this activity decreased as the stimulus moved toward the
lower quadrant and the vertical meridian representation (red);
correspondingly, the response for the lower quadrant (red line)
increased to the maximal value (position 20 on x-axis). Similar
reversals were seen in V2 and V3, which also contain quarter-field
representations. As the cortical trajectory proceeded through V3A
and V7, the responses to each location overlapped more, reflecting
the coarser topography in those regions [20]. Finally, in parietal
cortex (the swath of cortex between V7 and medial IPS, MIPS
included) the BOLD responses to discrete contralateral locations
were very similar, reflecting very coarse or absent topography. Even when the response functions separated; the spatial profile of
activity remained very similar, indicating that the magnitudes
associated with distinct contralateral field positions were highly
correlated across the cortical surface. Path of the cortical trajectory F: Compass plots illustrating the
BOLD magnitude associated with each of the six polar angles. Each plot
comes from a cortical area demonstrating a preference for the contra-
lateral horizontal meridian (blue regions in panels A–C). Data shown
comes from the right hemisphere of subject A. Supplementary figures S1
and S2 show panels C–E for all eight hemispheres investigated. doi:10.1371/journal.pone.0000452.g004 May 2007 | Issue 5 | e452 PLoS ONE | www.plosone.org May 2007 | Issue 5 | e452 4 Visual Field Organization cortex, reflecting an increase in the component of the BOLD
signal that was independent of visual location. In parietal cortex
immediately anterior/dorsal to V7, the ipsilateral response
reached the
level
of the contralateral
response,
indicating
a complete insensitivity to visual location; however a clear
separation between contralateral and ipsilateral responses was
observed for a small region in medial intraparietal sulcus. The
location of this region in atlas space (Table 2) and relative to
anatomical landmarks (supplementary text S2) suggests it was the
same region identified by Sereno et al (2001), often referred to as
hLIP. We refer to it as MIPS to avoid assumptions about human-
monkey homology. Compass plots of spatial tuning Figure 4F provides an overall plot of spatial tuning for polar angle
in 4 areas. Within each area, the BOLD response is plotted across
the six visual locations for a region that demonstrated a preference
for the middle contralateral sector (colored blue in Figure 4A–C). Although these four regions showed the same overall location
preference, the compass plots illustrated a progressive decrease in
spatial tuning, with tight location tuning in V1, intermediate
tuning in V3A and much coarser tuning in MIPS. PLoS ONE | www.plosone.org
5
May 2007 | Issue 5 | e452 Summary of findings from cortical trajectory The findings illustrated in Figure 4 were highly consistent across
subjects (see figures S1 and S2). These analyses indicate a pro-
gressive change in visual field organization from early visual
occipital areas to higher-order occipital areas to parietal cortex. Early visual areas responded highly selectively to stimuli presented
at a specific contralateral location, i.e. good topography, with weak
or negative responses to stimuli presented at ipsilateral locations. By IPS, however, topography was much weaker and ipsilateral
responses were much stronger, indicating that a larger component
of the BOLD signal was independent of visual location. Nonethe-
less, a region in IPS (MIPS) demonstrated a clear contralateral
organization. A quantitative and statistical treatment of the visual impressions
described here was derived by calculating correlation coefficients. This analysis fully supports the findings as described here and can
be found in the supplementary text S1. Responses to contralateral and ipsilateral locations
Figure 4E plots the mean magnitudes of the response averaged
over the three contralateral locations (pink line) and the response
averaged over the three ipsilateral locations (light blue line). The
ipsilateral response was negative or zero in V1–V3, but began to
rise in V3A and V7 and was maintained throughout parietal doi:10.1371/journal.pone.0000452.t002
. . . Additional extra-occipital areas sensitive to visual
location We used the replication approach shown above for MIPS to
identify six additional areas sensitive to visual location. In the first
step, regions were selected in each subject’s left and right
hemisphere based on the conservative multiple-comparisons
corrected data from the polar-angle version of the delayed saccade
task. If
these
maps
revealed
evidence
for
a
region
with
a contralateral preference in the same general area, as defined
by proximity to anatomical landmarks (see supplementary text S2),
in more than half of the eight hemispheres examined, we used
a less stringent statistical threshold to define the region in the
remaining hemispheres. This criterion revealed evidence for six
additional regions outside occipital cortex with a significant
preference for the contralateral visual field. In the second step,
we tested the reliability of these regions against data from the
oddball task (see above). All six additional areas were significant
using this random-effects test on an independent data set (Table 2). The six additional regions were named based on their
anatomical proximity to gyral or sulcal landmarks, with the
exception of the FEF region, which was already defined in prior
work [35]: PCu, precuneus; ST, superior temporal; SFEF, superior
FEF; IFEF, inferior FEF; MPCe, middle precentral; IFS, inferior
frontal sulcus. They are shown schematically in Figure 5. The atlas
co-ordinates of these regions are listed in Table 2 and their Figure 5. Schematic illustration of regions sensitive to visual field
location on the PALS atlas. A shows a flat representation of the cortex
of the right hemisphere. B shows three different views of an inflated
representation of the same surface. PCu, precuneus; ST, superior
temporal; SFEF, superior FEF; IFEF, inferior FEF; MPCe, middle precentral;
IFS, inferior frontal sulcus. The atlas co-ordinates of these regions are
listed in Table 2. doi:10.1371/journal.pone.0000452.g005 location relative to anatomical landmarks are described in
Supplementary text S2. Figure S3 shows cortical trajectory
analyses of these six extra-occipital regions, illustrating the absence
of any clear topographic organization. All but one of these regions,
ST (Superior Temporal), has also been observed in phase
encoding studies [12]. Responses to contralateral and ipsilateral locations . . . . Table 2. Summary of extra-occipital cortical areas preferring the contra-lateral visual field. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . May 2007 | Issue 5 | e452 5 5 Visual Field Organization Statistical reliability of MIPS across subjects and
tasks Figure 5. Schematic illustration of regions sensitive to visual field
location on the PALS atlas. A shows a flat representation of the cortex
of the right hemisphere. B shows three different views of an inflated
representation of the same surface. PCu, precuneus; ST, superior
temporal; SFEF, superior FEF; IFEF, inferior FEF; MPCe, middle precentral;
IFS, inferior frontal sulcus. The atlas co-ordinates of these regions are
listed in Table 2. doi:10.1371/journal.pone.0000452.g005 PLoS ONE | www.plosone.org tasks While previous studies of visual field organization in intraparietal
cortex have reported partial and/or qualitative replications, they
did not involve any formal test that the reported regions could be
reliably identified. We regard this as an important step. We
established the reliability of MIPS across hemispheres as follows. Statistical maps were computed for the contrast contralateral
minus ipsilateral using data from the polar-angle version of the
delayed saccade task and thresholded according to a conservative
multiple-comparisons correction for the whole brain. We found
a candidate region in the proximity of MIPS in every hemisphere
(see mean coordinates in Table 2, and supplementary text S2 for
a description of the anatomical landmarks). The average peak z-
score in the delayed saccade task obtained by averaging the
individual z-scores for the contra-ipsi contrast across hemispheres
was 15.2 (Table 2). We then tested the reliability of the MIPS regions across tasks
by using data from the oddball task to test the reliability of the
regions identified from the delayed saccade data. We computed for
each individual the peak z-score for the contrast contralateral
minus ipsilateral in the oddball task, i.e. oddball targets appearing
in the contralateral vs. ipsilateral visual field, across all the voxels
of the region, and then averaged this peak z-score over subjects
and hemispheres. The mean replication z-score in the oddball task
was 7.0 (Table 2). In addition, we performed a random effects
analysis to establish that these apriori defined MIPS areas were
reliable across the population of hemispheres as a whole, and were
not driven by one or two robust examples. We computed response
magnitudes for each region for the horizontal contralateral and
ipsilateral stimulus positions in the oddball task. Six pairs of
observations, one pair per hemisphere, were entered into a paired-
t test. In this random-effect analysis, MIPS showed significantly
stronger contralateral responses in the oddball task (t = 4.8
p = .002; see Table 2). These results demonstrate the statistical
significance
over
hemispheres
of
a
region
in
MIPS
with
a preference for the contralateral visual field. Eccentricity organization Voxels were selected on the basis of contralateral
preference, and so this measure can be considered as a baseline for
comparison. The red bars reflect the difference between preferred and
non-preferred within field locations. This measure reflects the degree of
topography present in the region. In early visual areas, the red and
black bars are identical, indicating a degree of topographic specificity as
great as the contralateral preference. The blue bars indicate the
difference between the non-preferred visual field location and the
ipsilateral location. This measure reflects the degree to which the region
possesses a non-topographic preference for the contra-lateral field. In
extra-occipital areas, the black and blue bars are identical, indicating
that these regions demonstrate a preference for the contra-lateral visual
field but no detectable topography. Higher visual areas demonstrate
a transition between these two types of organization. doi:10.1371/journal.pone.0000452.g006 Figure 7A plots responses along cortical trajectories to targets at
different eccentricities in the contralateral visual field using the
eccentricity version of the delayed saccade task. The cortical
trajectories for V1–V3A followed the representations of the
horizontal meridian revealed by passive retinotopy (the solid lines
shown in Figure 3). In V1–V3, the maximum response occurred in
more eccentric locations as one moves along the cortical
trajectory, reflecting the well-defined topography of these areas. In contrast MIPS showed similar responses to all eccentricities at
all points along the cortical trajectory. Responses to the three
eccentricities in the ipsilateral field were also highly similar (not
shown). Therefore,
MIPS
was
most
reliably
identified
by
a preference for the contralateral field, irrespective of whether
stimulus location was varied by polar angle or by eccentricity (see
also figure S5). Figure 7B shows regional responses. The figure plots the
responses to the 2, 13, and 24 deg eccentricity in previously
defined regions. It is important to bear in mind that these regions
were defined by activity in the polar angle task (see methods), in
which the targets were at 7 degrees eccentricity. Areas with
topographic eccentricity organization should show their largest
response to the 13 degree target, since this is the closest to the
reference eccentricity of 7 degrees. This is what was observed for
areas V1–V3. Figure 6B, the black bars plot the difference score when the
vertical meridian is crossed (230 vs 90), the red bars when the
meridian is not crossed (230 vs 2150). In extra-occipital areas
(e.g. Eccentricity organization Up until this point, we have focused on polar-angle organization. In this final section, we examine the effect of varying the
eccentricity of the target (Figure 7). The same progressive trends in
visual field organization that were observed for polar angle
responses were also observed for responses to stimuli at different
eccentricities. Figure 6. Quantification of visual field organization. A shows how the
measures are calculated. The analysis was restricted to voxels which
preferred either the upper or lower contralateral visual field locations. Each measure involves a subtraction of BOLD magnitudes associated
with two visual field positions. The measures are comparable in the
sense that the visual distance between locations was the same for each
comparison. Importantly, increases in receptive field size should
influence all measures equally. The black bars show the difference in
BOLD response between the preferred visual field location and an
ipsilateral location. Voxels were selected on the basis of contralateral
preference, and so this measure can be considered as a baseline for
comparison. The red bars reflect the difference between preferred and
non-preferred within field locations. This measure reflects the degree of
topography present in the region. In early visual areas, the red and
black bars are identical, indicating a degree of topographic specificity as
great as the contralateral preference. The blue bars indicate the
difference between the non-preferred visual field location and the
ipsilateral location. This measure reflects the degree to which the region
possesses a non-topographic preference for the contra-lateral field. In
extra-occipital areas, the black and blue bars are identical, indicating
that these regions demonstrate a preference for the contra-lateral visual
field but no detectable topography. Higher visual areas demonstrate
a transition between these two types of organization. doi:10.1371/journal.pone.0000452.g006 Figure 6. Quantification of visual field organization. A shows how the
measures are calculated. The analysis was restricted to voxels which
preferred either the upper or lower contralateral visual field locations. Each measure involves a subtraction of BOLD magnitudes associated
with two visual field positions. The measures are comparable in the
sense that the visual distance between locations was the same for each
comparison. Importantly, increases in receptive field size should
influence all measures equally. The black bars show the difference in
BOLD response between the preferred visual field location and an
ipsilateral location. Region-based quantitative assessment of
organization This measure reflects the degree of
topography present in the region. In early visual areas, the red and
black bars are identical, indicating a degree of topographic specificity as
great as the contralateral preference. The blue bars indicate the
difference between the non-preferred visual field location and the
ipsilateral location. This measure reflects the degree to which the region
possesses a non-topographic preference for the contra-lateral field. In
extra-occipital areas, the black and blue bars are identical, indicating
that these regions demonstrate a preference for the contra-lateral visual
field but no detectable topography. Higher visual areas demonstrate
a transition between these two types of organization. doi:10.1371/journal.pone.0000452.g006 A corollary of the first property of contralateral organization is
that any contralateral location should yield a larger response than
any ipsilateral location. A 120 deg change in polar angle that
crosses the vertical meridian should yield a similar change in
response whether the starting point is the preferred contralateral
location (black arrow in Figure 6A) or a non-preferred contralat-
eral location (blue arrow in Figure 6A). Figure 6B shows that
responses
in
extra-occipital
areas
changed
equivalently
for
preferred (black bars) and non-preferred (blue bars) starting
points. Conversely, in early visual areas preferred starting points
(black bars) yielded much larger changes in BOLD response than
non-preferred starting points (blue bars). These results were highly
consistent over the two tasks. Therefore, both measures of contralateral dominance indicated
low dominance in early visual areas (red = black&blue) and high
dominance in extra-occipital areas (red%black = blue). The pre-
dominant functional organization of visual responses in extra-
occipital areas to be a broad selectivity for the contralateral visual
field with a significant response to ipsilateral stimuli (about half of
the contralateral response, PCu excepted). Topographic signals
may be present but represent a small fraction of the total signal
measured in these areas. In addition to this region-based analysis, we computed separate
normalized measures of contralateral preference and topography
(see figure S3). These findings were highly consistent with those
shown in Figure 6. Region-based quantitative assessment of
organization The results of the cortical trajectory analyses might depend on the
exact cortical trajectory chosen (but see figure S4). To overcome
this concern, and to provide a more thorough quantitative
assessment of visual field organization across areas, we devised
a separate region based analysis (see methods). This analysis is also
significant since it controls for effects of receptive field size. An area that shows a largely contralateral organization should
demonstrate two related characteristics. First, responses should
change much more strongly to fixed changes in polar angle that
cross the vertical meridian than to changes that do not. For
example, Figure 6A illustrates a voxel that responds preferentially
to a visual target at 230 deg as opposed to the other five locations. If that preference mainly reflects a contralateral bias, then
responses should be much weaker to a location that is 120 deg
distant in polar angle and crosses the vertical meridian (i.e. the
location at 90 deg, shown by the black arrow) than to a location
that is 120 deg distant but remains within the contralateral visual
field (i.e. the location at 2150 deg, shown by the red arrow). In PLoS ONE | www.plosone.org May 2007 | Issue 5 | e452 May 2007 | Issue 5 | e452 May 2007 | Issue 5 | e452 6 Visual Field Organization Figure 6B, the black bars plot the difference score when the
vertical meridian is crossed (230 vs 90), the red bars when the
meridian is not crossed (230 vs 2150). In extra-occipital areas
(e.g. MIPS) the scores were much larger when the vertical
Figure 6. Quantification of visual field organization. A shows how the
measures are calculated. The analysis was restricted to voxels which
preferred either the upper or lower contralateral visual field locations. Each measure involves a subtraction of BOLD magnitudes associated
with two visual field positions. The measures are comparable in the
sense that the visual distance between locations was the same for each
comparison. Importantly, increases in receptive field size should
influence all measures equally. The black bars show the difference in
BOLD response between the preferred visual field location and an
ipsilateral location. Voxels were selected on the basis of contralateral
preference, and so this measure can be considered as a baseline for
comparison. The red bars reflect the difference between preferred and
non-preferred within field locations. PLoS ONE | www.plosone.org Eccentricity organization Results from the eccentricity version of the delayed saccade task, in which targets lay at one of three eccentricities (2, 13 and 24
degrees visual angle) on the horizontal meridian. A illustrates topographic organization in dorsal visual and parietal areas. The graphs plot the
magnitude of BOLD response to three locations of varying eccentricity in the contralateral visual field, by distance across the cortical surface. The
dotted black lines show the mean response to ipsilateral locations. Cortical trajectories were drawn using data from the passive retinotopy task. The
trajectories for V1, V2/V3 and V3A each followed representations of the horizontal meridian in dorsal occipital cortex (these are the solid lines shown
in Figure 3). The trajectory for MIPS was drawn at the same orientation on the flat surface. B shows mean responses for regions. The regions were
defined using data from the polar angle version of the delayed saccade task, in which the eccentricity of the targets was approx. 7 degrees visual
angle. The bars plot the BOLD response to contra-lateral locations, with the mean response to ipsi-lateral targets subtracted. These values are
averaged across hemispheres (n = 4), with error bars showing the standard error. Note that in early visual areas there is a clear preference for the
middle (13 deg.) contralateral location. This location corresponded most closely to the location of targets used to define these regions, and thus this
preference reflects the topographic organization of these areas. This effect is not present in intermediate and extra-occipital areas. However, a few
areas (V6/POS, PCu, ST) showed a preference for more peripheral stimuli. doi:10.1371/journal.pone.0000452.g007 locations, regardless of whether non-preferred locations lay within
the contralateral or ipsilateral visual field. In contrast, extra-
occipital areas showed a much more robust difference in response
when non-preferred locations lay in the ipsilateral visual field as
opposed to the contralateral visual field. Therefore, visual field
organization showed a progressive change from topographic in
early visual areas to contralateral in extra-occipital areas. that more peripheral visual areas are preferentially represented in
these areas (although they do not appear to have any topographic
organization, see also figure S4). We conducted regional contrasts
(most eccentric minus least eccentric contralateral location) to
assess the reliability of these preferences (see methods). Eccentricity organization MIPS) the scores were much larger when the vertical
meridian was crossed, while in occipital areas (e.g. V1) the two
scores were much more equivalent, reflecting the fact that the
relative BOLD responses to two locations in these areas were
much less affected by whether the meridian was crossed. These
results were highly consistent over the two tasks. Areas lacking topography and in which all eccentricities are
represented approximately equally should show approximately
equal responses to the three eccentricities. This was observed for
most regions, including MIPS. Regions V6/POS, PCu, and ST demonstrated a graded
preference for more eccentric visual field locations. This indicates May 2007 | Issue 5 | e452 PLoS ONE | www.plosone.org 7 Visual Field Organization Figure 7. Results from the eccentricity version of the delayed saccade task, in which targets lay at one of three eccentricities (2, 13 and 24
degrees visual angle) on the horizontal meridian. A illustrates topographic organization in dorsal visual and parietal areas. The graphs plot the
magnitude of BOLD response to three locations of varying eccentricity in the contralateral visual field, by distance across the cortical surface. The
dotted black lines show the mean response to ipsilateral locations. Cortical trajectories were drawn using data from the passive retinotopy task. The
trajectories for V1, V2/V3 and V3A each followed representations of the horizontal meridian in dorsal occipital cortex (these are the solid lines shown
in Figure 3). The trajectory for MIPS was drawn at the same orientation on the flat surface. B shows mean responses for regions. The regions were
defined using data from the polar angle version of the delayed saccade task, in which the eccentricity of the targets was approx. 7 degrees visual
angle. The bars plot the BOLD response to contra-lateral locations, with the mean response to ipsi-lateral targets subtracted. These values are
averaged across hemispheres (n = 4), with error bars showing the standard error. Note that in early visual areas there is a clear preference for the
middle (13 deg.) contralateral location. This location corresponded most closely to the location of targets used to define these regions, and thus this
preference reflects the topographic organization of these areas. This effect is not present in intermediate and extra-occipital areas. However, a few
areas (V6/POS, PCu, ST) showed a preference for more peripheral stimuli. doi:10.1371/journal.pone.0000452.g007 Figure 7. Eccentricity organization For all
three areas the effect was significant in each of the four
hemispheres tested: V6/POS, p,0.001 for all cases (A left
z = 17.1; A right z = 7.3; C left z = 9.1 ; C right z = 12.4). PCu,
p,0.001 for all cases (A left z = 7.5; A right z = 3.4; C left z = 6.1;
C right z = 9). ST, p,0.05 for all cases (A left z = 4.9; A right
z = 4.75; C left z = 1.7; C right z = 4.8). Higher-order frontal, parietal, and temporal areas
sensitive to visual location In addition to MIPS, six extra-occipital regions showed signals that
depended on visual location and demonstrated considerably more
robust contralateral selectivity than topographic organization. Aside from PCu, each region also showed clear ipsilateral
activations. Signals that depend on location have been previously
reported in each region, with the exception of ST [10,12]. Area
ST on the posterior portion of the superior temporal gyrus
matches a functional region active for reorienting attention to
unattended
visual
targets
(STG,
57,245,12
vector
distan-
ce = 7.2 mm; Corbetta, 2000; 2002), and for detecting salient
sensory
multimodal
changes
(54,242,13
vector
distan-
ce = 4.7 mm; Downar et al., 2000). Previous studies involving
group-averaged data have failed to reveal contralateral organiza-
tion. Interestingly, this region showed a preference for peripheral
stimuli, consistent with its putative role in spatial re-orienting. It is possible that the areas identified by Schluppeck et al. and
Silver et al. have contralateral preferences and topography that are
too weak to be detected with the current methods/hardware. A
second possibility is that MIPS matches IPS2. However, the
Talairach coordinates for MIPS are considerably anterior to IPS2
(Silver et al report the average center of IPS2 as lying at 19, 275,
48, some 19 mm distance from the center of MIPS) and the
topographic maps we observed for MIPS in 3/8 hemispheres had
the reverse orientation to those reported for IPS2. A third, related
possibility is that MIPS constitutes a part of IPS2, consistent with
the pattern observed in the right hemisphere of subject D (Figure
S2). It would be surprising if Schluppeck et al. and Silver et al. did
not identify voxels in MIPS, since our results indicate that they can
be detected more easily than voxels in IPS1 and IPS2. A fourth
possibility is that, in some cases, regions on the medial surface have
been inadvertently included in IPS1 and IPS2. While this does not
appear to hold for Silver et al [11], close examination of
Schluppeck et al [9], who use a task very similar to the delayed
saccade task used here, appears to show this is true for half the Area IFEF and SFEF are located at the intersection of superior
frontal and precentral sulci, the putative human homologue of
monkey FEF [35,40–42]. DISCUSSION The current results indicate that as one moves from early visual
areas to extra-occipital areas, topographic signals become difficult
to detect while contralateral preferences remain robust and extend
throughout the entire contralateral field. These results suggest that
extra-occipital areas whose response depends on visual location
are more reliably determined by measuring contralateral prefer-
ence than by searching for a complete, ordered phase represen-
tation of polar angle. The present results are not inconsistent with
the presence of topography in previous phase-encoding studies of
extra-occipital cortex, since our study may have had lower signal- We investigated visual field organization in occipital and extra-
occipital cortex by comparing BOLD responses to discrete
stimulus locations. The results indicated progressive changes in
visual field organization from early visual areas to extra-occipital
areas. First, there was a tendency for spatial selectivity to decrease,
as demonstrated by increases in the BOLD response associated
with ipsilateral locations. Second, the spatial profile of the selective
signal changed. Early visual areas showed a large difference in
response
to
preferred
compared
with
non-preferred
visual PLoS ONE | www.plosone.org May 2007 | Issue 5 | e452 May 2007 | Issue 5 | e452 8 Visual Field Organization cases examined (S1 left, S2 left, S2 right, S3 right). However,
intraparietal sulcus can be distinguished from regions on the
medial surface both on the basis of anatomical location and the
tendency for medial regions to prefer more eccentric visual
locations (see Pitzalis et al [37] and figure S6). A final possibility is
that our method of combining bottom-up signals due to visual
stimulation with top-down signals due to attention or eye-
movement planning obscured responses in IPS1 and IPS2. However, Silver et al [11] showed modest but reliable activation
of IPS1 and IPS2 in response to passive stimulation. to-noise, for instance because we used a head coil [but see 12 who
also used a head coil for some subjects]. Nonetheless, any absolute
difference in sensitivity does not bear on the progressive changes in
visual field organization noted above (i.e. multiplying all responses
by a factor of two would not affect the relative changes in field
organization documented in Figure 6). The organization of regions sensitive to visual
location in the intraparietal sulcus The current literature on this topic is unsettled since the two
principal groups that have studied IPS using phase encoding
methods have reported divergent results. Sereno et al. [10]
reported a topographic region in medial IPS that (as suggested by
Silver et al. [11]) may lie anterior to two IPS regions, IPS1 and
IPS2, discovered by Schluppeck et al. [9] and Silver et al. [11]. These latter two regions extended from V7, indicating a contin-
uous tiling along an axis of occipital-parietal cortex. In contrast,
the area of Sereno et al was isolated from the retinotopic belt. It is
unclear why Schluppeck et al. and Silver et al. did not find Sereno
et al.’s area or why Sereno did not find the areas of Schluppeck et
al. and Silver et al. The task used by Schluppeck et al was the same
as that used by Sereno et al. A further issue is that Sereno et al
reported that the orientation of the topographic map varied
somewhat across subjects in IPS [10], as well as in other regions
[12]. In contrast, Silver et al and Schluppeck et al report that IPS1
and IPS2 were oriented consistently across subjects, akin to the
highly stereotyped organization seen for maps in early visual areas. Ipsilateral BOLD signals are present in extra-
occipital areas sensitive to visual location p
Most extra-occipital regions sensitive to visual location neverthe-
less showed substantial ipsilateral activations, roughly 50% of the
signal evoked by contralateral stimuli (see figure S3). The one
exception was PCu, which also showed a preference for very
peripheral stimuli. Ipsilateral activations may reflect neurons
within these areas that have very large receptive fields, i.e. many
neurons in these regions may be insensitive to visual location, or
neurons that have receptive fields centered in the ipsilateral visual
field. In addition, the BOLD response may average signals in
a neuron that occur at different times and show a different
dependence on spatial location. In LIP, many neurons respond
during the period immediately after a saccade has occurred, and
this response has been found to be less spatially specific than
earlier responses in the same neurons [38]. The current study
mixed sensory and attentional signals, each of which may produce
more or less ipsilateral activation. A final possibility is that
ipsilateral BOLD activations reflect inhibitory inputs to these areas
rather than excitatory neuronal responses. For example, if these
regions are involved in selectively attending to a location and this
process is implemented competitively between the two visual fields
[e.g. as in biased competition, 39], they may receive inhibitory
inputs from regions representing the other visual field. Inhibitory
inputs are known to produce a positive BOLD response [30]. The present results show that these studies measured topo-
graphic signals which were either very small in magnitude, or
entirely absent. Therefore, inconsistency of results across studies
may not be surprising. Figure S6 (see also discussion in
supplementary text S1) shows that there was little evidence of
either contralateral preference or of topographic organization in
the area separating V7 and MIPS. The current results in IPS appear most consistent with those of
Sereno et al. since: (i) area MIPS was clearly separated from V7 (ii)
the location relative to anatomical landmarks (see supplementary
text S2) and Talairach coordinates were broadly similar (MIPS lay
approx 7 mm distance from Sereno et al’s area, whose coordinates
were 32, 264, 46 after MNI to Talaraich conversion [36]) (iii) in
those subjects/hemispheres in which we found evidence of
topographic maps, these were oriented in a manner consistent
with the orientation described as predominant by Sereno et al
[10]. Comparison with monkey electrophysiology The progressive changes in human topography observed here are
similar to that observed in the macaque using electrophysiological
methods. Felleman and Van Essen (1991) distinguish four
categories of topographic organization in the monkey. The first
visual areas, V1, V2, V3 and VP all fall into the first two
categories, with well defined topography. Similarly we find a high
within-field index of at least 70% for these areas in the human (see
figure S6). This means that BOLD response to non-preferred
locations within the contralateral field was 70% smaller than the
response to the preferred location, on average. Felleman and Van
Essen (1991) describe the topography of more anterior areas on
the ventral, lateral and dorsal occipital surfaces, including V3A,
V4 and MT as coarse and irregular. Correspondingly we measure
a more modest within-field index of 20–50% for these areas in the
human (50% for V3A, 30% for V4/VO, and 20% for MT/LO). The reliability of the results demonstrated by the replication
approach extended to the measures of visual field organization
presented here (Figures 6 and S3). The two tasks produced highly
similar estimates of visual field organization. All of the trends in
that were evident with one task - e.g. in extra-occipital areas, the
predominance of contralateral over topographic organization -
were clearly evident with the other task. The replication approach also answers a possible criticism of the
current approach, namely, that since the voxels comprising each
region were defined by contralateral preference, noise within those
voxels biased the results toward greater contralateral preference
than topography. However, this difference was demonstrated in
a dataset that was separate from that used to define the regions,
eliminating any bias in the noise. Very crude topography has been observed in monkey LIP [4],
corresponding to the low within-field value of ,10% observed in
human MIPS. The same single-unit study of monkey LIP found
that, while some neurons preferred the ipsilateral visual field, the
majority of neurons preferred the contralateral visual field. Again
these findings are in accord with the current results for MIPS [but
see 21 who did not see a tendency for neurons to prefer the
contralateral field]. In relation to the quantitative measures of topography used
(Figures 6 and S3), there may a concern relating to the fact that we
used one task to determine the preferred contralateral location in
order to calculate an estimate of topography in the other task. Higher-order frontal, parietal, and temporal areas
sensitive to visual location The cortex that is responsive to visually
guided eye movements consists of a strip of cortex that starts at the
junction of superior frontal sulcus and precentral sulci (about
z = 50–60 mm), moves laterally along the horizontal ramus of the
precentral sulcus (from x = 620 to x = 640 mm), and extends
ventrally along the ventral ramus of the precentral sulcus (from
z = 50–60 mm to z = 30–40 mm). Petit and Beauchamp identified May 2007 | Issue 5 | e452 May 2007 | Issue 5 | e452 PLoS ONE | www.plosone.org 9 Visual Field Organization a dorsal precentral sulcus region that generally matches the
location of SFEF and IFEF (Beauchamp: 226,214,53 vector
distance from left SFEF = 4.6 mm; 31,28,52 vector distance from
right SFEF = 13 mm; from right IFEF = 3.1 mm). Corbetta et al
1998 distinguished a dorsal precentral sulcus region (41,27,46
vector distance from right IFEF = 13 mm; 235,29,46 vector
distance from left IFEF = 4.5 mm), and a precentral-superior
frontal sulcus region (29,23,60 vector distance from right
SFEF = 11 mm;
227,213,46
vector
distance
from
left
SFEF = 12.7 mm). These findings are consistent with the evidence
of two dorsal frontal eye movement areas that show a robust
contralateral preference. degree of topographic organization. However, if it turned out that
topographic organization was task dependent then this procedure
would produce artificially reduced estimates of sensitivity to
location within the contralateral field. This explanation of our
findings is implausible for a number of reasons. First, there is no
evidence of task dependent topography. Topographic organization
clearly remains constant across tasks in occipital areas (e.g. as
illustrated in Figure 3). Previous studies suggest that topographic
organization also remains constant across tasks in extra-occipital
areas [9–11]. Similarly, we found consistent topographic organi-
zation in one case for MIPS (figure S7). Second, it is unclear why
contralateral organization should remain constant while topo-
graphic organization changes. Third, the qualitative analyses
presented illustrate that contra-lateral selectivity was far more
robust than topographic organization even when we look just at
data from a single task, as illustrated in Figures 4, 7, S1, S2, S4, S5,
S7. Area
MPCe
corresponds
to
the
ventral
precentral
eye
movement region defined by Beauchamp et al (244,214,40
vector distance with left MPCe = 9.6 mm; 47,26,40 vector
distance with right MPCe = 2.2 mm). Reliability of the results and possible artifacts Because signals in extra-occipital areas that depend on visual
location can be small in magnitude and quite variable, it is
important to demonstrate the statistical significance of the results,
not only within subjects, but also across subjects or hemispheres. The statistical significance within a subject of the contralateral
preference of each of the 7 regions sensitive to visual location was
established using the delayed-saccade data with voxel-level z-
statistics that were multiple-comparison corrected over the entire
brain. The statistical significance across subjects of the contralat-
eral preference of each region defined by the delayed-saccade data
was determined by conducting a regional random-effects group t-
test on the data from the oddball task, an independent data set,
with subjects/hemispheres as the random factor. A final methodological concern is that the results may be partly
due to the limited spatial resolution of fMRI, and in particular to
partial volume averaging. Data relevant to this issue is presented in
supplementary text S1 and figure S7. It is likely that partial volume
effects influence our results to some degree. Nonetheless, as
discussed in the supplementary text S1, the progressive changes in
organization observed cannot be easily explained by this factor
alone. The across-tasks replication approach not only rigorously
demonstrated the across-subject/hemisphere reliability of the
results, but also eliminated potential explanations of the results
that focused on the particular characteristics of one or the other
tasks, since the two tasks involved different stimuli (randomly
positioned dots vs, colored pictures), judgments (delayed saccade
vs. identification of visual oddballs), baseline conditions (fixation
vs. rapid serial presentation of standard objects), responses (eye
movements vs. key-press) and attentional states (endogenous vs. stimulus-driven). PLoS ONE | www.plosone.org Higher-order frontal, parietal, and temporal areas
sensitive to visual location The IFS area was the only
area that has not been reliably activated in studies of eye
movements and attention, and has instead been associated with
storage in working memory [43]. Another concern is that the results were affected by the limited
sampling of locations in each hemifield. In polar-angle scans of
phase encoding procedures, the stimulus sweeps continuously
through every location in the field. However, in V1–V3, the main
effect of limited sampling in the ‘cortical-trajectory’ analyses of
Figures 4 and 5 was to restrict the number of voxels that showed
strong within-field indices, not to eliminate those voxels. In
contrast, no voxels in extra-occipital areas showed strong or even
moderate within-field indices. Increasing the number of spatial
samples would not have changed this result. The functional significance of contralateral
organization The changing nature of visual field organization may provide
some insight into the function of these areas. The organization of
brain areas into topographic maps is a recurring feature of early
sensory areas, not just in vision but also in audition, touch and
olfaction. Why do early visual areas possess a well defined
topographic organization, and why does this organization change
for higher visual areas? A recent inactivation study reports evidence of well defined
topographic organization in macaque principal sulcus, as assessed
by deficits in performance [6]. It is possible that a small patch of
cortex whose inputs and/or neurons represent locations across the
visual field nonetheless has outputs and/or a functional role
specific to a particular portion of the visual field. There is also
evidence of topographic organization by eccentricity in monkey
frontal eye fields, as assessed by the amplitude of saccades elicited
by microstimulation [44]. We looked for evidence of similar
organization
in
human
frontal
cortex,
however
any
such
organization was too weak for us to detect. We can not rule out
the presence of crude eccentricity topography in this area,
however the topography revealed by microstimulation in the
monkey appears to be well defined. One possibility is that these
eccentricity maps would be evident during saccade execution. The
delayed saccade task used here and in previous studies [9,10] has
limited ability to detect activity associated with the act of making
a saccade, since saccades occur infrequently and it is not possible
to separate out the BOLD response associated with outbound and
return saccades. Rather, the most significant aspect of this task, at
least in relation to visual field dependent responses in parietal
cortex, appears to be the need to remember the intended saccade
location during the delay period (see [10,18,19,33,45]). An
alternative possibility, similar to our discussion of principal sulcus
above, is that there is topographic organization of outputs from
FEF, but not of inputs. There is evidence of eccentricity
topography in projections from macaque FEF to visual areas &
LIP [46], and superior colliculus [47]. Finally, there is always
a possibility that these areas are not homologous between
macaque and human. A simple account might hold that the early visual areas merely
preserve the spatial arrangement present in the retina, and that
this organization becomes progressively more diluted at each
synapse along the chain of processing. The functional significance of contralateral
organization However, topographic
maps need not match the spatial arrangement of incoming
projections [48], and specific developmental mechanisms have
been identified that aid the formation of topographic maps [for
a review see 49]. The leading theoretical account is that topographic organiza-
tion minimizes wiring costs for connecting neurons within an area
that predominantly analyzes local spatial relationships [for a review
see 31]. In early visual areas, neurons representing nearby visual
locations need to combine information to aid the identification of
visual features. An ordered retinotopic map minimizes wiring
length by placing neurons with adjacent receptive fields as close to
each other as possible. Chklovskii and Koulakov [31] suggest that
the principle of wiring optimization is sufficiently robust to allow
an inference from visual field organization to function: ‘‘If the
representation of the visual field in that cortical area is…nonre-
tinotopic, then the processing is not likely local in the visual
space’’. Following Chklovskii and Koulakov’s reasoning, the present
findings may indicate that processing becomes progressively less
local in visual space moving from early visual areas, through
higher occipital areas, to extra-occipital regions. The processing in
these latter regions may still be highly spatial, but not emphasize
local spatial relationships over distant relationships. The absence
of a local processing bias in these regions could be consistent with
their involvement in saccade planning and/or the allocation of
attention, since the selection of a single target location might
reflect competition between neurons that represent all parts of the
visual field. Comparison with monkey electrophysiology The
advantage of this approach is that it provides an unbiased method
to estimate the preferred location and thus a true estimate of the These data indicate that as one moves from early to higher
visual areas in the human, just as in the monkey, contralateral
preference remains clearly identifiable while topographic signals
become difficult to detect. May 2007 | Issue 5 | e452 May 2007 | Issue 5 | e452 10 Visual Field Organization However, a detailed comparison of monkey and human maps
will require measurements involving the same technique, since
single-unit recording and BOLD imaging differ along many
dimensions. For example, single unit recordings involve post-hoc
reconstructions of the position of receptive fields where sampling
biases and inaccuracies in reconstruction may be present. Interestingly, two recent studies of monkey parietal cortex using
optical imaging, which allows a bird’s eye view of an entire area,
have found some evidence of topography. However, the mapped
variable was eye position rather than retinal location (Raffi and
Siegel, 2001; 2002), again consistent with the current results. Another difference is that BOLD signals may predominately
reflect afferent inputs to areas, rather than the firing of neurons in
those areas [30]. dominance on phase encoding measurements remains to be
determined. Our findings raise a concern about the typical phase-
encoding approach, and suggest that contralateral preference
provides a more robust criterion than topography for identifying
the borders of extra-occipital areas. Methodological significance for phase-encoding
studies If these
inter-area connections are sufficiently prevalent relative to intra-
area connections, then locating these neurons in the right
hemisphere would minimize wiring costs. Therefore, minimizing
inter-area connection lengths would produce a contralateral
organization for area X, even in the absence of a local processing
tendency sufficient to create a robust topographic organization. Under this model, neurons in area X would have equal numbers
of intra-area connections within and across hemispheres; however
their inter-area connections would be strongly biased towards
areas in the same hemisphere. Why should an ipsilateral bias occur
for inter-area connections? One possibility is that neurons in area
X are very strongly connected to neurons in early visual areas that
represent the same location in visual space. For instance, they may
output directly to early visual areas in order to modulate their
activity. Alternatively, these neurons may be strongly connected to
motor neurons that control effectors on the same side of space. position, and back to the center. The fixation point then re-
appeared. Trials occurred in blocks of three, with the three targets
appearing at different locations within the sector (in random order
at 25, 0, and +5 degrees polar angle from the sector center, and at
a randomly determined eccentricity between 6 and 7.5 degrees). Within blocks, trials were separated by a 0.7 second fixation
interval. Successive blocks were separated by a randomly selected
variable fixation interval of 4, 6.5 or 9 secs. These prolonged and
variable fixation intervals allowed estimation of the main effect of
the task as compared to a fixation baseline. The delay durations
for the three trials within each block were randomly selected from
permutations that allowed for a fixed total delay length of 9 sec per
block (25% of blocks had three delays of 3 sec, in the remaining
blocks one of the six possible permutations of 1.5, 3 and 4.5 sec
was selected at random). Sector location varied pseudo-randomly
(counterbalanced within each scanner run) from block to block,
with the constraint that sector side (left or right) alternated from
one block to the next. The fixation-cross subtended 0.225 degrees
visual angle, as did each of the square dots, which marked the
target location and acted as delay-period distracters. Methodological significance for phase-encoding
studies Phase-encoding studies aim to reveal topographic organization in
order to identify the borders of distinct visual areas. Our findings
support this method of identifying early visual areas. However,
they also suggest caution when using phase-encoding methods to
identify higher areas, particularly outside occipital cortex. The
topographic signal is weaker in these areas, and thus may not
prove reliable. Further, the finding that contra-lateral selectivity
accounts for far more BOLD modulation than topographic
organization raises specific methodological concerns for phase
encoding. The typical approach in phase-encoding studies is to
threshold phase maps using a statistical test that is sensitive to
periodic activity at the frequency of the stimulus. However, contra-
lateral organization alone will be sufficient to produce significant
periodic activity. Thus, contrary to what is commonly assumed,
the statistical test does not entail the presence of reliable
topographic organization. The full effect of contralateral pre- However, this account does not explain why contralateral
organization persists, even when topographic organization sub-
sides. Contralateral organization might reflect a fundamental
division between the left and right visual fields, such that
processing within each field differs from processing that spans
the vertical meridian. Individuals with damage to the corpus
callosum show evidence of independent processing of attentional
cues relevant to each hemisphere [50] and an increased cost for
attentional shifts between visual fields [51]. These results indicate
that compromised connectivity between neurons representing
different
visual
fields
does
produce
observable
effects
on
attentional performance. However, since these effects were not
observed in control groups with normal connectivity, neurons
representing different visual fields appear just as well connected as
neurons representing the same field, despite the expensive wiring
costs of callosal connections. Rather than postulating a fundamen- PLoS ONE | www.plosone.org May 2007 | Issue 5 | e452 May 2007 | Issue 5 | e452 May 2007 | Issue 5 | e452 11 Visual Field Organization tal division in processing between visual fields, for which there is
little evidence, a more parsimonious explanation for the pre-
dominance of contralateral over topographic organization is the
need to minimize the wiring costs associated with connections
between areas. Suppose, for example, that neurons in area X that
represent the left visual field have a high degree of connectivity
with other cortical areas in the right hemisphere, but few
connections to other cortical areas in the left hemisphere. Subjects Four healthy right-handed subjects (one female, ages 19–26) with
normal vision were recruited. Informed consent was obtained
according to procedures approved by the local human studies
committee. Table 1 details subject participation. Apparatus Stimuli for the delayed saccade task were generated using an
Apple G4 Macintosh computer. A PC was used to generate stimuli
and collect responses in the oddball task. In both cases the visual
image was projected onto a screen at the head of the bore by
a Sharp LCD projector. Subjects viewed the stimuli through
a mirror attached to the head coil. Manual responses in the
oddball task were obtained using an MRI-compatible fiber-optic
keypad held in the right hand. Oddball paradigm (polar angle only) Stimuli were presented against a plain bright white background. A
colored picture stimulus was presented twice during each fMRI
frame (every 1+/20.25 sec for subject A, every 1.25+/20.25 sec
for subjects B & D). On most trials the same ‘standard’ picture was
presented at the fovea, while on ,12% of trials a new picture was
presented in one of 7 locations (fovea or 6 peripheral locations). All
picture stimuli were presented for a duration of 0.15 sec. Each trial
had an equal probability of being an oddball trial (p = 1/7), except
that the trial after an oddball trial was always a standard trial. Each of the seven possible oddball target locations occurred with
equal probability. A grey fixation diamond subtending 0.05
degrees visual angle was visible in the center of the screen at all
times except when a central picture stimulus was present. Subjects
were asked to maintain central fixation throughout. Subjects were
required to respond to every picture stimulus, categorizing it either
as ‘standard’ or ‘oddball’ using a key-press. Oddball stimuli were
randomly selected with replacement from a pool of 100 pictures of
everyday objects. Since approximately 500 oddball targets were
presented in each scanner session, the same object was presented We did not record eye movements in the scanner. At the start of
each scanning session, subjects practiced the task in the scanner
control room, at which time the experimenter monitored the
subject’s eye-movements. Delayed saccade paradigm - eccentricity version Delayed saccade paradigm - eccentricity version
In the eccentricity version of the delayed saccade paradigm the
sector centers all lay on the horizontal meridian. There were three
sectors per side, at 2, 13 and 24 degrees visual angle from fixation. The size of each sector and component stimuli was scaled linearly
with distance from fixation, scaled to the same sizes used in the
polar angle version. The wider field of view was achieved by using
a larger mirror and a projector screen that was placed inside the
bore of the scanner, much closer to the subject. Sector location
was
varied
pseudo-randomly
(counterbalanced
within
each
scanner run) without any constraint on alternating side. In all
other respects the design was identical to the polar angle version of
the delayed saccade task. Methodological significance for phase-encoding
studies During the
delay period, on average slightly fewer than 6 dots were visible at
any one time within a wedge of 30 degrees polar angle and
between 6 and 7.5 degrees eccentricity. The screen was updated
every 0.1 sec, at which time each dot had a 60% probability of
being randomly re-generated. Overlapping and/or touching dots
were avoided by setting dot locations discretely rather than
continuously, such that there were 180 possible polar angles and 6
possible eccentricities, uniformly distributed. In conclusion, our finding of clear contralateral selectivity but
weak or absent topography in extra-occipital areas may indicate that
these areas are engaged in processing that is global across visual
space and that these areas have strong direct connections to areas
that are engaged in local processing. These features are consistent
with a role for these areas in the control of spatial attention. Delayed saccade paradigm - polar angle version Delayed saccade paradigm - polar angle version
All stimuli were bright white against a black background. Subjects
fixated a central crosshair while a peripheral target location was
briefly (0.5 sec) presented within one of six sectors (sectors were
centered at 30, 90, 150, 230, 230, 2150 polar degrees from the
upper vertical meridian, and 6.75 degrees visual angle eccentric-
ity). After target offset, fixation was maintained for a variable
duration (1.5, 3 or 4.5 sec), during which random flickering dots
were presented throughout the target sector (e.g., 15–45 degrees
polar angle, 6–7.5 degrees eccentricity). A blank screen (0.7 sec)
signaled the time for the saccade to the remembered target May 2007 | Issue 5 | e452 May 2007 | Issue 5 | e452 PLoS ONE | www.plosone.org 12 Visual Field Organization were corrected for multiple comparisons by thresholding at z.4
and only including clusters of 5 or more voxels. This z-score/
cluster size criterion was conservative, corresponding to a multiple-
comparison corrected threshold of p = 0.025. only 5 times over a session, out of a total of approximately 4000
stimulus presentations. The peripheral locations were centered at
the same polar angles as for the delayed saccade paradigm, with
stimuli occupying from 6.25 to 11.25 degrees visual angle from the
fovea. Stimuli presented at the center subtended approx. 2.3
degrees visual angle. For each subject, three anatomical MPRAGE images were
averaged to produce a high-resolution structural image. Surefit
and Caret (Van Essen et al., 2001) (http://brainmap.wustl.edu/
caret) were used for surface generation & flattening, visual
inspection, drawing & re-embedding of regions. Topographic maps and definition of regions p g
p
p
g
All regions, occipital and extra-occipital, used in analyses were
defined on the basis of data from the polar angle version of the
delayed saccade task, and comprised voxels that showed above
threshold contralateral preference in that task (with the exception of
IPS1&2, see below). For the purposes of delineating retinotopic
areas, images were created in which each voxel was given one of
three colors depending on which of the three contra-lateral stimulus
positions produced the greatest BOLD response (Figure 3). Thus one
color marked the horizontal meridian, one color indicated a 30
degree position from the upper vertical meridian, and the third color
indicated 30 degrees from the lower vertical meridian. This is
analogous to coloring voxels according to phase in the phase-
encoding methodology. This image was thresholded by the whole
brain multiple comparison corrected contrast contralateral minus
ipsilateral. Regions V1, V2, V3, VP, V3A & V7 were drawn by
reference to the established correspondences between their borders
and the horizontal and vertical meridians. On the ventral occipital
surface, we grouped visuotopic voxels anterior to the VP border as
V4/VO, as we could not confidently separate V4 from other regions. Similarly on the lateral surface, in the absence of other localizers
involving motion and/or presentation of specific objects, we could
not confidently determine the location of different regions, and
grouped all voxels showing evidence of lateralization as LO/MT. Voxels preferring the contralateral visual field which lay medial to
V3/V3A/V7 were labeled V6/POS. These voxels primarily lay
within the parieto-occipital sulcus, the described location of V6
(Pitzalis et al., 2006). Identification of extra-occipital areas Because mapping studies are often based on a relatively small
number of subjects in whom an extensive amount of data is
collected, formal statistical tests of the reliability of results across
subjects are not always provided. Because of the intrinsic
anatomical variability of functional areas, a standard approach
for assessing reliability across subjects is to qualitatively compare
patterns of activation that are diagnostic of an area (e.g. polar
angle topography) in approximately the same location or in
relation to other functional markers (e.g. anterior to functionally
defined area V7, Schluppeck et al., 2005; Silver et al., 2005). Image acquisition and pre-processing g
q
p
p
g
An asymmetric spin-echo echoplanar imaging sequence was used
to measure blood oxygenation-level-dependent (BOLD) contrast
on a Siemens Allegra 3T scanner. In the delayed saccade
paradigm 156 whole brain volume images were collected in each
scanner run. In the oddball task there were 150 volumes per run. Either 39 or 40 contiguous 3.25 mm slices were acquired with
3.2563.25 mm in-plane resolution, a slice TR of 0.0641 sec
(volume TR of 2.5 or 2.564 sec), TE = 25, flip angle = 90u. Except
for the images acquired for subject A in the oddball task, where 31
contiguous
4 mm
slices
were
acquired,
464 mm
in-plane
resolution,
a
slice
TR
of
0.0645 sec
(volume
TR = 2 sec),
TE = 25, flip angle = 90u. In the delayed saccade paradigm
subjects A, B, C and D participated in 36, 36, 36 & 35 scanner
runs of 156 consecutive volume images over three sessions. In the
oddball task, subjects A, B & D participated in 44, 42 & 42 scanner
runs of 150 consecutive volume images over three sessions. Realignment parameters for the functional data were calculated
first within run, then across runs within a single session, then
across sessions. The functional data was re-sampled once directly
to atlas space with a uniform voxel size of 3 mm3. The strategy of
using a single re-sampling of the functional data to simultaneously
correct for head movement within and across runs as well as effect
an atlas transformation was found in previous comparisons to
substantially improve signal to noise and reduce smoothing due to
repeated resampling. For each peripheral location in the delayed
saccade paradigm, subjects experienced ,320 target location
presentations of 0.5 sec, were required to remember those target
locations while distracters were presented for ,3 sec, and then
made a rapid saccade to and from the target location. For each
peripheral location in the oddball task, subjects experienced ,200
stimulus presentations of 0.15 sec, and gave an immediate
response to indicate that the standard stimulus, which always
appeared in the center, had not occurred. PLoS ONE | www.plosone.org Graphs of BOLD magnitude along a cortical
trajectory j
y
An important tool for the qualitative assessment of visual field
organization was the use of graphs that traced activity along
a trajectory drawn on the cortical surface. These are shown in
figures 4, 7, S1, S2, S4, S5, S7. These graphs were generated using
Matlab programs written to operate on data files generated by
Caret. Volume files were generated using the general linear model
described
above
which
provided
estimates
of
the
BOLD
magnitude associated with discrete visual locations. These images
then were projected on to the surface using Caret, a process that
involves assigning a magnitude to each surface node depending on
the value of the voxel in which the node lies in volume space. A
transformation is calculated by virtually cutting and warping the
cortical surface so that each node has defined coordinates both in
talairach volume space and in a flat (two dimensional) coordinate
system. Cortical trajectories are drawn by hand in the flat
coordinate system, and consist of a series of points spaced
approximately 2 mm apart. While distances on the flat surface are
prone to distortion due to the warping that occurs as part of the
flattening process, they are normalized over the whole surface so
that the total cortical surface area is preserved. The magnitude of Image analysis and statistics Separate responses for each of the six peripheral locations (and
a further response for the central oddball stimulus in the oddball
task) were estimated at the voxel level using the general linear
model. For the delayed saccade task, we assumed a sustained
response over the three trials, modeled by convolving a gamma
function with a delay of 2 sec and time constant of 1.25 sec
(Boynton et al., 1996) with the duration of the block. In the oddball
task we assumed an impulse response modeled using a gamma
function. The design matrix was defined using impulse-basis
functions such that at each frame, the data were modeled as the
sum of the overlapping hemodynamic responses produced by each
task effect plus a linear trend. Fixation periods in the delayed
saccade paradigm and responses to ‘standard’ stimuli in the oddball
task were not separately modeled but served as the baseline against
which responses to peripheral stimuli were estimated. In this study, we formally replicate the existence of extra-
occipital areas showing a contralateral visual field preference using
a two-step (hypothesis-test) approach, in which: 1) we selected
a region (e.g. MIPS) with a significant contralateral preference in
the delayed saccade task that showed a relatively consistent
anatomical location across subjects and hemispheres, and 2) we
replicated
the
contralateral
preference
of
this
area
in an
independent data set on the oddball task, including a random
effects group statistic across hemispheres/subjects. To identify voxels with a contralateral preference, the response
to sectors in the contralateral visual field minus the response to
sectors in the ipsilateral visual field was first computed. The
resulting t-statistic for each subject was converted to equally
probable z-statistics prior to threshold and display. Statistical maps In the first step, the statistical map for the contrast contralateral
minus ipsilateral, thresholded according to a conservative multi-
ple-comparisons correction for the whole brain, was computed
using data from the polar-angle version of the delayed saccade PLoS ONE | www.plosone.org May 2007 | Issue 5 | e452 13 Visual Field Organization task. If these maps revealed evidence of a visual field-selective
region in the same region, as defined by proximity to anatomical
landmarks (see Supplementary text S2 for a description of the
landmarks for each region), in more than half of the eight
hemispheres examined, we used a less stringent statistical threshold
to define the region in the remaining hemispheres. Quantitative measures of visual field organization Quantitative measures of visual field organization
An important goal of this experiment was to extend our qualitative
examinations of visual field organization by quantifying visual field
organization. The measures of within field, preferred across field
and non-preferred across field BOLD modulation shown in
Figure 6 were calculated as follows. For the delayed saccade
(oddball) task estimates, the preferred polar angle for each voxel
was calculated using data from the oddball (delayed saccade) task
data. This ensured an unbiased estimate of preferred location. Only voxels that preferred either the upper or lower contra-lateral
visual field positions were included. Each measure was calculated
separately for each voxel, as described in Figure 6. An estimate of
each measure was calculated separately for each hemisphere/
subject and for each region by averaging across all included voxels. The graphs show the mean and standard error of these estimates
over subjects/hemispheres. The lateralization and within-field indices shown in figure S3
provided estimates of contra-lateral preference and topographic
modulation that were normalized across areas, making it easier to
compare areas. The lateralization index was calculated using the
formula: Lateralization index = (contra2ipsi)/contra. The within
field
index
was calculated
using
the
formula: Within-field
index = (preferred2mean (non-preferred))/preferred. The pre-
ferred location used to calculate the within field index for the
delayed saccade task was established using data from the oddball
task, and vice-versa. In each case, the normalization was
accomplished by dividing by the BOLD response associated with
the preferred visual field location. However, when this de-
nominator is small it leads to unstable estimates. To compensate
for this problem, we calculated the mean value associated with
numerator and denominator separately for each subject/hemi-
sphere by averaging those values over all voxels with above
baseline responses to the preferred location(s). The value of the
index was then calculated for each subject/hemisphere using these
corrected mean values. Graphs show the mean and standard error
of these estimates across subjects/hemispheres. Including voxels in
which the BOLD response to the preferred location(s) was below
zero produced a qualitatively similar but noisier pattern. Image analysis and statistics This criterion
revealed evidence for seven regions (see Table 2 and Supplemen-
tary text S2) outside occipital cortex with a significant preference
for the contralateral visual field. In the second step, we tested the
reliability of these regions against data from the oddball task using
two separate analyses. We computed for each individual the peak
z-score for the contrast contralateral minus ipsilateral in the
oddball task, i.e. oddball targets appearing in the contralateral vs. ipsilateral visual field, across all the voxels of the region, and then
averaged this peak z-score over subjects and hemispheres. In
addition, we performed a random effects analysis to establish that
these a-priori areas were reliable across the population of
hemispheres as a whole, and were not driven by one or two
robust examples. We computed response magnitudes for each
region for the horizontal contralateral and ipsilateral stimulus
positions in the oddball task. BOLD response at each point along the trajectory was established
by taking the mean BOLD magnitude associated with every node
which (i) lay within 2 mm of that point on the flat surface (ii) was
closer to that point than to neighboring points on the trajectory. Passive retinotopy py
We collected passive retinotopy data for subjects A, B & C. Full
field vertical and horizontal meridians, 4 Hz contrast reversing
black and white checkerboards, were presented for 12.8 secs in
alternating blocks; 10, 8 & 6 scanner runs for subjects A, B & C;
176 whole-brain volumes per scanner run; 40 contiguous 3.25 mm
slices
with 3.2563.25 mm
in-plane
resolution,
volume
TR
2.564 sec). Rings were also presented at four different eccentric-
ities (4 Hz contrast reversing black and white checkerboards,
12.8 second stimulus blocks alternating with 12.8 seconds fixation,
random stimulus order; 4 scanner runs per subject, 145 whole
brain volumes per scanner run, other parameters as for meridians). These data were used to verify the areal boundaries drawn on the
basis of the delayed saccade data. The correspondence was
excellent (see Figure 3). Passive presentation of horizontal and
vertical meridians has previously been shown to produce results
consistent with phase-encoding methods most commonly em-
ployed in investigations of visuotopic organization [27,52]. PLoS ONE | www.plosone.org Supplementary Text S2 Supplementary Text S2
Found
at:
doi:10.1371/journal.pone.0000452.s009
(0.03
MB
DOC) ACKNOWLEDGMENTS The authors would like to thank John Harwell, Donna Hanlon and David
Van Essen for their help with Surefit and Caret, and Mark McAvoy for
assistance with statistics. Author Contributions Conceived and designed the experiments: MC AJ GP SA GS. Performed
the experiments: AJ GP SA. Analyzed the data: MC AJ GS. Contributed
reagents/materials/analysis tools: AJ AS EA. Wrote the paper: MC AJ GS. Found at: doi:10.1371/journal.pone.0000452.s006 (0.86 MB TIF) Found at: doi:10.1371/journal.pone.0000452.s006 (0.86 MB TIF) Figure S7
Comparison of best example of MIPS topography
(Subject C, right hemisphere) with area V3A in the same
hemisphere. The figure illustrates that the reduced topographic
organization of area MIPS cannot be accounted for by partial
volume effects or by noise-induced spatial smoothing. A shows an
inflated representation of the cortical surface with trajectories
drawn to optimally capture topographic organization in MIPS and
V3A. In area MIPS there is a high degree of correlation between
the three contralateral locations. The topography in area V3A is
much more clearly defined. The difference between the two areas
cannot be attributed to distance, as illustrated by the x-axis of the
graphs. In B the graphs trace 5 face-connected voxels that follow
the cortical surface (indicated by a white line). Again, response
profiles for different visual field positions are highly correlated in
MIPS and clearly dissociate in area V3A. The contrast between
areas is even more striking in the majority of cases, in which MIPS
had no discernable topography. Figure S7
Comparison of best example of MIPS topography
(Subject C, right hemisphere) with area V3A in the same
hemisphere. The figure illustrates that the reduced topographic
organization of area MIPS cannot be accounted for by partial
volume effects or by noise-induced spatial smoothing. A shows an
inflated representation of the cortical surface with trajectories
drawn to optimally capture topographic organization in MIPS and
V3A. In area MIPS there is a high degree of correlation between
the three contralateral locations. The topography in area V3A is
much more clearly defined. The difference between the two areas
cannot be attributed to distance, as illustrated by the x-axis of the
graphs. In B the graphs trace 5 face-connected voxels that follow
the cortical surface (indicated by a white line). Again, response
profiles for different visual field positions are highly correlated in
MIPS and clearly dissociate in area V3A. The contrast between
areas is even more striking in the majority of cases, in which MIPS
had no discernable topography. Figure S4
Visual field organization of other extra-occipital
regions. We took the most robust example of each region from the
two subjects (A and C) who participated in both polar-angle and
eccentricity versions of the delayed saccade. 1. DeYoe EA, Carman GJ, Bandettini P, Glickman S, Wieser J, et al. (1996)
Mapping striate and extrastriate visual areas in human cerebral cortex. Proc
Natl Acad Sci U S A 93: 2382–2386. 5. Constantinidis C, Franowicz MN, Goldman-Rakic PS (2001) Coding specificity
in cortical microcircuits: a multiple-electrode analysis of primate prefrontal
cortex. J Neurosci 21: 3646–3655. 6. Sawaguchi T, Iba M (2001) Prefrontal cortical representation of visuospatial
working memory in monkeys examined by local inactivation with muscimol.
J Neurophysiol 86: 2041–2053. 4. Ben Hamed S, Duhamel JR, Bremmer F, Graf W (2001) Representation of the
visual field in the lateral intraparietal area of macaque monkeys: a quantitative
receptive field analysis. Exp Brain Res 140: 127–144. 4. Ben Hamed S, Duhamel JR, Bremmer F, Graf W (2001) Representation of the
visual field in the lateral intraparietal area of macaque monkeys: a quantitative
receptive field analysis. Exp Brain Res 140: 127–144.
5. Constantinidis C, Franowicz MN, Goldman-Rakic PS (2001) Coding specificity
in cortical microcircuits: a multiple-electrode analysis of primate prefrontal
cortex. J Neurosci 21: 3646–3655.
6. Sawaguchi T, Iba M (2001) Prefrontal cortical representation of visuospatial
working memory in monkeys examined by local inactivation with muscimol.
J Neurophysiol 86: 2041–2053. 2. Sereno MI, Dale AM, Reppas JB, Kwong KK, Belliveau JW, et al. (1995)
Borders of multiple visual areas in humans revealed by functional magnetic
resonance imaging. Science 268: 889–893. PLoS ONE | www.plosone.org Found at: doi:10.1371/journal.pone.0000452.s005 (7.40 MB TIF) Found at: doi:10.1371/journal.pone.0000452.s005 (7.40 MB TIF) Found at: doi:10.1371/journal.pone.0000452.s005 (7.40 MB TIF) Found at: doi:10.1371/journal.pone.0000452.s005 (7.40 MB TIF) oddball task. The lateralization index compares the mean response
to contra-lateral visual locations with the mean response ipsi-
lateral visual locations. The within-field index compares upper,
middle and lower field targets in the preferred (contra-lateral)
visual field. The measures are based on the BOLD response
observed to a target in the preferred location, and indicate the
average percentage decrease in BOLD response for targets in non-
preferred locations (see methods). Error bars show standard error
of the mean value, computed across hemispheres. V4/VO - voxels
preferring the contralateral field anterior to VP on the ventral
surface. LO/MT - voxels preferring the contralateral field anterior
to the foveal confluence and early visual areas on the lateral
surface. V6/POS - voxels preferring the contralateral field medial
to V3A/V7, primarily located within the parietal-occipital sulcus. Found
at:
doi:10.1371/journal.pone.0000452.s003
(0.26
MB
DOC) Figure S6
Visuotopic organization of intraparietal sulcus and
surrounding cortex. A shows the abrupt change in the degree of
topographic organization that occurs between V7 to MIPS. See
Figure 6 for further explanation of the graphs. Previous studies
have claimed a continuous retinotopic organization stretching
along intraparietal sulcus. However the cortical area seperating V7
from MIPS, indicated here as IPS1/2, shows little evidence of
contralateral
preference
or
of
topographic
organization. B
illustrates eccentricity organization in intraparietal sulcus and
more medial regions. See figure 7 for further explanation of
graphs. Regions within intraparietal cortex can be clearly
distinguished from more medial regions on the basis of eccentricity
preference. Found at: doi:10.1371/journal.pone.0000452.s006 (0.86 MB TIF) We drew two
trajectories through each region, and plotted BOLD activity
corresponding the three contra-lateral locations in the polar angle
(top graph) and eccentricity (middle graph), with mean BOLD
response to ipsi-lateral shown by a dotted black line. The bottom
of the three graphs for each area shows the mean difference
between contralateral and ipsilateral positions for the two data sets
(polar angle and eccentricity), with the scale normalized for
comparison. F
d t d i 10 1371/j
l
0000452 004 (9 28 MB TIF) Found at: doi:10.1371/journal.pone.0000452.s004 (9.28 MB TIF) Figure S5
Profiles of activity for cortical trajectories cross-secting
area MIPS at three different orientations. An inflated represen-
tation of the left hemisphere of subject A is shown, overlayed with
a statistical map showing voxels that prefer the contralateral visual
field. Trajectories were drawn through area MIPS at three
different orientations. The graphs below show the profile of
activity along the trajectories, labeled 1–3. (A) shows data from the
polar angle version of the delayed saccade task. The three contra-
lateral locations are color coded as shown in the key. The dotted
black line shows the mean activity due to ipsi-lateral locations. (B)
shows data from the oddball task, displayed in the same format (C)
shows data from the eccentricity version of the delayed saccade
task, with contralateral locations color coded as shown in the key. (D)
shows
the
mean
difference
between
contralateral
and
ipsilateral field locations for the three tasks, with the scale
normalized for comparison. Note that there is evidence of polar
angle topography along trajectory (1), with the lower field
represented more anterior and the upper field more posterior. This topographic organization was consistent across the two tasks. Nonetheless, BOLD modulation associated with topography was
slight compared with the contralateral preference seen for this
area. Note the highly consistent profile of contralateral preference
for the three tasks illustrated in (D). The dotted circle in the top
figure shows the location of area ST in the left hemisphere. Found at: doi:10.1371/journal.pone.0000452.s007 (6.56 MB TIF) SUPPORTING INFORMATION Figure S1
Visual field organization of dorsal visual areas and
medial intra-parietal sulcus - data for left and right hemispheres of
subjects A and B. See figure 4 for details. The lower two panels
quantify the degree of topographic organization (within max) and
contralateral preference (laterality), allowing the reader to visualize
the relative magnitude of these features as we move from early
visual to parietal cortex. They are described further in the
supplementary text. Found at: doi:10.1371/journal.pone.0000452.s001 (6.28 MB TIF) Found at: doi:10.1371/journal.pone.0000452.s001 (6.28 MB TIF) Figure S2
Visual field organization of dorsal visual areas and
medial intra-parietal sulcus - data for left and right hemispheres of
subjects C and D. See figure 4 and supplementary text for details. Found at: doi:10.1371/journal.pone.0000452.s002 (5.67 MB TIF) Figure S3
Lateralization and within-field indices for both tasks. Both indices measure the change in BOLD response magnitude
due to variation in retinotopic stimulus location, derived from data
from the polar angle version of the delayed saccade task and the PLoS ONE | www.plosone.org May 2007 | Issue 5 | e452 14 Visual Field Organization Supplementary Text S1 Found
at:
doi:10.1371/journal.pone.0000452.s008
(0.05
MB
DOC) Found
at:
doi:10.1371/journal.pone.0000452.s008
(0.05
MB
DOC) Found
at:
doi:10.1371/journal.pone.0000452.s008
(0.05
MB
DOC) Supplementary Text S2
Found
at:
doi:10.1371/journal.pone.0000452.s009
(0.03
MB
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https://openalex.org/W3194745484
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https://www.frontiersin.org/articles/10.3389/fmed.2021.713733/pdf
|
English
| null |
The Risk of Neuraxial Anesthesia-Related Hypotension in COVID-19 Parturients Undergoing Cesarean Delivery: A Multicenter, Retrospective, Propensity Score Matched Cohort Study
|
Frontiers in medicine
| 2,021
|
cc-by
| 6,858
|
ORIGINAL RESEARCH
published: 19 August 2021
doi: 10.3389/fmed.2021.713733 Yuan Zhang 1,2†, Rong Chen 1,2†, Chen Cao 3†, Yuan Gong 4†, Qin Zhou 1,2, Min Wei 5,
ZhongYuan Xia 1,2, XiangDong Chen 6‡ and QingTao Meng 1,2*‡ ‡These authors have contributed
equally to this work and share senior
authorship Specialty section:
This article was submitted to
Intensive Care Medicine and
Anesthesiology,
a section of the journal
Frontiers in Medicine
Received: 24 May 2021
Accepted: 19 July 2021
Published: 19 August 2021 Specialty section:
This article was submitted to
Intensive Care Medicine and
Anesthesiology,
a section of the journal
Frontiers in Medicine Specialty section:
This article was submitted to
Intensive Care Medicine and
Anesthesiology,
a section of the journal
Frontiers in Medicine Received: 24 May 2021
Accepted: 19 July 2021
Published: 19 August 2021 Yuan Zhang 1,2†, Rong Chen 1,2†, Chen Cao 3†, Yuan Gong 4†, Qin Zhou 1,2, Min Wei 5,
ZhongYuan Xia 1,2, XiangDong Chen 6‡ and QingTao Meng 1,2*‡ Yuan Zhang 1,2†, Rong Chen 1,2†, Chen Cao 3†, Yuan Gong 4†, Qin Zhou 1,2, Min Wei 5,
ZhongYuan Xia 1,2, XiangDong Chen 6‡ and QingTao Meng 1,2*‡ Reviewed by:
Cristian Deana,
Azienda Sanitaria Universitaria
Integrata di Udine, Italy
Francesco Meroi,
University of Udine, Italy
Federico Barbariol,
Azienda Sanitaria Universitaria Friuli
Centrale (ASU FC), Italy 1 Department of Anaesthesiology, Renmin Hospital of Wuhan University, Wuhan, China, 2 Department of Anaesthesiology,
East Hospital, Renmin Hospital of Wuhan University, Wuhan, China, 3 Department of Medical Center, Renmin Hospital of
Wuhan University, Wuhan, China, 4 Department of Anaesthesiology, Yichang Central People’s Hospital, The First College of
Clinical Medical Science, China Three Gorges University, Yichang, China, 5 Department of Obstetrics, East Hospital, Renmin
Hospital of Wuhan University, Wuhan, China, 6 Department of Anaesthesiology, Union Hospital, Tongji Medical College,
Huazhong University of Science and Technology, Wuhan, China Background: SARS-CoV-2 infection was referred to sympathetic hyperactivity, which
might increase the susceptibility of neuraxial anesthesia-related hypotension resulted
from sympathetic inhibition. We conducted a multicenter, retrospective, propensity score
matched (PSM) cohort study to determine whether COVID-19 parturients have an
increased risk of hypotension after neuraxial anesthesia for cesarean delivery. †These authors have contributed
equally to this work and share first
authorship Methods:
Clinical data of COVID-19 parturients were collected from the electronic
medical records from 1th January to 31th May, 2020 in three hospitals of Hubei Province,
China. Information of Control parturients (without COVID-19) were obtained at the same
institutions over a similar period in 2019. All American Society of Anaesthesiologists (ASA)
Physical Status II full termed pregnant women who received cesarean delivery under
neuraxial anesthesia were included. The primary objective was to obtain and compare
the incidence of neuraxial anesthesia-related hypotension. Secondary objectives were
the analysis of anesthetic implementation and administration, intraoperative maternal
vital signs and adverse reactions, and neonatal Apgar scores at 1 and 5 min after
delivery. The clinical characteristics of COVID-19 parturients were also analyzed. PSM
was derived to balance the predictors for neuraxial anesthesia-related hypotension based
on previous studies. Edited by:
Luigi Vetrugno,
University of Udine, Italy METHODS Ethical approval for this study (No. WDRY2020-K077) was
provided by the Institutional Review Board at Renmin Hospital
of Wuhan University, Wuhan, China (Chairperson Prof. Hong
Chen) on 29 February 2020. The study received exemption from
informed consent. All personal information were removed from
the database to protect patients’ confidentiality. Although
the
clinical
characteristics
of
COVID-19
in
pregnant
women
are
similar
with
non-pregnant
women
(4), COVID-19 results in additional challenges for obstetric
anesthesia as reported in previous study (5, 6). The most
preferred method for cesarean delivery is neuraxial anesthesia
[epidural anesthesia (EA), spinal anesthesia (SA), and combined
epidural–spinal anesthesia (CES)] which allows parturients to
remain respiration and avoids the effects of general anesthetic
on newborns. Meanwhile, sympathetic blockade induced by
neuraxial anesthesia can provoke maternal hypotension which
has a potential risk for transient tachypnea of newborns (7). Several studies have examined the incidence and associated
factors of hypotension after neuraxial anesthesia in healthy
parturients undergoing cesarean delivery (8–11). However,
parturients
with
COVID-19
have
not
been
included
in
those studies. A multicenter, retrospective, propensity score matched cohort
study was designed to compare the incidence of neuraxial
anesthesia-related hypotension in parturients with and without
COVID-19 undergoing cesarean delivery. The methodology
reported in this study was accordance with the recommendations
of the Strengthening the Reporting of Observational studies in
Epidemiology (STROBE) statement (16). Renmin Hospital of Wuhan University, Union Hospital
Affiliated to Tongji Medical College of Huazhong University
of Science and Technology, and Yichang Central People’s
Hospital were designated as the diagnosis and treatment
center for COVID-19 patients (including pregnant women)
in Hubei province during the pandemic. SARS-CoV-2 nucleic
acid test was used to screen COVID-19 in all parturients. And
the
chest
CT
scan
was
performed
on
parturients
after delivery. We planned to collect all of the available
COVID-19 parturients undergoing cesarean delivery who met
the inclusion/exclusion criteria in three hospitals from 1th
January to 31th May, 2020. And, information of control
parturients (without COVID-19) were obtained at the same
institutions over a similar period in 2019. We identified
patients with a primary International Classification of Diseases,
Tenth Revision (ICD-10) diagnosis code of cesarean delivery
from the electronic medical records to establish the study
cohort. All ASA Physical Status II full termed pregnant
women who received cesarean delivery (ICD-10 codes O82.0–
O82.9, and O84.2 for cesarean delivery) under neuraxial
anesthesia were eligible for inclusion in the study. Citation: Zhang Y, Chen R, Cao C, Gong Y,
Zhou Q, Wei M, Xia Z, Chen X and
Meng Q (2021) The Risk of Neuraxial
Anesthesia-Related Hypotension in
COVID-19 Parturients Undergoing
Cesarean Delivery: A Multicenter,
Retrospective, Propensity Score
Matched Cohort Study. Front. Med. 8:713733. doi: 10.3389/fmed.2021.713733 Results: In present study, 101 COVID-19 parturients and 186 Control parturients were
derived from 1,403 cases referenced to propensity score matching. The incidence of
neuraxial anesthesia-related hypotension was 57.4% in COVID-19 parturients and 41.9%
in Control parturients with an incidence risk ratio (IRR) of 1.37 (95% CI 1.08–1.74;
P = 0.012; post-hoc Cramér’s V = 0.15) in the PSM cohort. The incidences of nausea,
vomiting, dizziness, and shaking were significantly higher in the COVID-19 group than August 2021 | Volume 8 | Article 713733 Frontiers in Medicine | www.frontiersin.org Zhang et al. Risk of Hypotension in COVID-19 Parturients Control group (48.5 vs. 17.2%, P < 0.001; 10.9 vs. 4.3%, P = 0.03; 18.8 vs. 3.2%,
P < 0.001; 51.5 vs. 18.3%, P < 0.001; respectively). The Apgar scores at 1 min
was significantly lower in newborns from COVID-19 parturients than that in Control
babies (P = 0.04). Conclusions:
An increased risk of neuraxial anesthesia-related hypotension in
COVID-19 parturients undergoing cesarean delivery should be stressed. Keywords: neuraxial anesthesia, hypotension, COVID-19, cesarean delivery, propensity score matching INTRODUCTION In
the
present
study,
we
conducted
a
retrospective
analysis
to
obtain
and
compare
the
prevalence
of
neuraxial
anesthesia-related
hypotension
during
cesarean
delivery in COVID-19 parturients and control parturients
(without COVID-19). A novel coronavirus (SARS-CoV-2) infection disease (COVID-
19) has devastated the global community since the end of
2019 (1). More importantly, mutations in SARS-CoV-2 might
(partly) escape the immune response which led to a large
drop in efficacy of vaccine. Several studies have forecasted a
second rebound of COVID-19 would be manifested in countries
with outbreaks (2, 3). We have to pay more attention to the
current COVID-19 pandemic to cope with the unexpected
medical scenarios. METHODS Parturients
who had a cesarean delivery after failed vaginal delivery
were also included. The exclusion criteria included severe
complicated births, significant bleeding (intraoperative bleeding Angiotensin-converting enzyme 2 (ACE2) has recently been
identified as the SARS-CoV-2 receptor. Binding by SARS-CoV-
2 attenuated the cardiovascular protection of ACE2 system,
which closely linked with sympathetic overactivation and renin-
angiotensin system overflow (12, 13). Besides, both psychological
stress after being diagnosed with COVID-19 and emotional
components associated with clinical isolation may further
hasten sympathetic excitation (13). Enhanced sympathetic
activity combined with hypoxemia induced by pulmonary
inflammation would add significant stress to cardiovascular
system. A latest study found the incidence of autonomic
dysfunction (sympathetic, parasympathetic, or both) was 78.0%
in mild COVID-19 patients, which resulted in a higher risk
of orthostatic hypotension when compared with un-COVID-19
patients (14). Cardiovascular system complications in COVID-
19 patients increasingly become a concern (15). However, the
effects of SARS-CoV-2 infection on hemodynamics of parturients
who underwent neuraxial anesthesia for cesarean delivery are
still unclear. August 2021 | Volume 8 | Article 713733 Frontiers in Medicine | www.frontiersin.org 2 Risk of Hypotension in COVID-19 Parturients Zhang et al. parturients (without COVID-19). Hypotension was defined
as the systolic blood pressure (SBP) below 100 mmHg, or the
mean arterial blood pressure (MAP) below 80% of the baseline
value (the mean of repeated measurements before commencing
anesthesia) (17, 18). Given the administration of vasopressors
and fluid for prophylactic or treatment of hypotension depended
heavily on anaesthesiologists, infusion volume over 1,000 ml or
vasoconstrictor utilization were also considered as the presence
of hypotension. Neuraxial anesthesia-related hypotension was
based on a single episode of defined hypotension from the time
of local anesthetic injection until 15 min after delivery of the
newborn. The secondary objective was the analysis of anesthetic
implementation and administration (full stomach, antiemetic
utilization, infusion volume, site of puncture, approach of
puncture, frequency of puncture, dose of local anesthetic,
vasoconstrictor,
and
oxytocin
utilization),
intraoperative
vital signs (SBP, MAP, heart rate, and SaPO2), intraoperative
adverse reactions (hypoxia (SaPO2 <95%), arrhythmia, nausea,
vomiting, dizziness, and shaking), and neonatal Apgar scores
at 1 and 5 min after delivery. The clinical characteristics
(COVID-19 diagnosis, signs and symptoms, and preoperative
laboratory
parameters)
of
COVID-19
parturients
also
be analyzed. over 1,000 ml), inadequate blockade (requiring addition of a
general anesthetic administration) or incomplete data. The same
inclusion and exclusion criteria were used for both two groups in
present study. METHODS A post-hoc estimated effect size (Cramér’s V) for the study
was assessed by comparing the incidence of neuraxial anesthesia-
related hypotension between parturients with and without
COVID-19 before and after propensity score matching (PSM). The clinical data including patient demographics, anesthesia
management, and intraoperative records were independently
collected using prefabricated forms and cross checked by two
investigators in each institution in order to maintain the quality
and consistency of data. Neuraxial anesthesia protocols for cesarean delivery are
similar in three institutions. Before initiation of anesthesia,
an intravenous line, ECG, pulse oximetry, and non-invasive
automatic blood pressure monitors (1- or 2-min interval) were
placed. The puncture procedure was performed in the left lateral
decubitus position. Block height was assessed bilaterally using
loss of cold sensitivity to alcohol at an interval of 3–5 min. All patients were delivered oxygen by face masker and placed
in the supine position with a right hip wedge after puncture
procedure until the end of the surgical procedure. All BP
recordings in this study were performed with the patient in the
supine position. We derived PSM to balance the predictors for neuraxial
anesthesia-related hypotension based on previous studies (8–
11, 19–25). Maternal age, body mass index (BMI), baseline
heart rate, baseline MAP, baseline SBP, urgency of surgery,
anesthetic technique (including EA, SA, and CES), sensory block The main objective of this study was to obtain and compare
the prevalence of neuraxial anesthesia-related hypotension
during cesarean delivery in COVID-19 parturients and control FIGURE 1 | The study flowchart. Frontiers in Medicine | www.frontiersin.org
3
August 2021 | Volume 8 | Article 713733 FIGURE 1 | The study flowchart. FIGURE 1 | The study flowchart. Frontiers in Medicine | www.frontiersin.org August 2021 | Volume 8 | Article 713733 3 Risk of Hypotension in COVID-19 Parturients Zhang et al. TABLE 1 | The characteristics of predictors for neuraxial anesthesia-related hypotension in parturients before and after propensity score matching (PSM). METHODS Before PSM
After PSM
COVID-19
(n = 102)
Control
(n = 1,301)
P-value
ASD (%)
COVID-19
(n = 101)
Control
(n = 186)
P-value
ASD (%)
Age (years)
28.3 ± 5.9
29.1 ± 5.4
0.16
13.8
28.4 ± 5.9
29.0 ± 5.3
0.39
3.4
BMI (kg/m2)
25.7 ± 4.7
25.5 ± 4.4
0.80
−2.5
25.6 ± 4.7
25.3 ± 4.3
0.61
8.9
Baseline heart rate (bpm)
92 ± 14
92 ± 13
0.76
3.1
92 ± 14
93 ± 13
0.45
−0.9
Baseline MAP (mmHg)
83 ± 9
82 ± 9
0.11
−16
83 ± 9
83 ± 10
0.77
6.5
Baseline SBP (mmHg)
117 ± 11
116 ± 11
0.42
−8.5
117 ± 11
11 7± 12
0.84
3.7
Urgency of surgery
<0.01a
29.8
0.71
−4.2
Emergency
47 (46.1%)
412 (31.7%)
46 (45.5%)
89 (47.8%)
Elective
55 (53.9%)
944 (72.6%)
55 (54.5%)
97 (52.2%)
Anesthetic technique
<0.01a
−27.4
0.89
1.9
EA
48 (47.1%)
789 (60.6%)
48 (47.5%)
90 (48.4%)
CES
54 (52.9%)
512 (39.4%)
53 (52.5%)
96 (51.6%)
Sensory block height
−17.4
−2.2
EA
0.38
0.37
T2
0
1 (0.1%)
0
0
T4
16 (33.3%)
239 (30.3%)
16 (33.3%)
20 (22.2%)
T6
31 (64.6%)
545 (69.1%)
31 (64.6%)
68 (75.6%)
T8
1 (2.1%)
3 (0.4%)
1 (2.1%)
2 (2.2%)
CES
0.05
0.21
T2
2 (3.7%)
36 (7.0%)
2 (3.8%)
5 (5.2%)
T4
7 (13.0%)
143 (27.9%)
7 (13.2%)
4 (4.2%)
T6
45 (83.3%)
331 (64.6%)
44 (83.0%)
72 (75.0%)
T8
0
2 (0.4%)
0
0
Block- surgery time (min)
9.9
−3.9
EA
19.7 ± 6.5
17.9 ± 5.1
0.02a
19.7 ± 6.5
18.7 ± 5.6
0.35
CES
6.4 ± 3.1
6.4 ± 2.7
1.00
6.3 ± 3.0
6.5 ± 2.6
0.67
Newborn weight (kg)
3.5 ± 0.4
3.5 ± 0.5
0.58
6.1
3.5 ± 0.4
3.5 ± 0.5
0.40
−4
Anaesthesiologist experience (years)
5.9 ± 4.0
5.0 ± 3.7
0.02a
−22.7
5.8 ± 4.0
5.6 ± 4.0
0.66
−5.1
Values are Number (proportion) or Mean ± SD. Block- surgery time, the time interval between neuraxial anesthesia block and start of surgery. ASD, absolute standardized difference,
the difference in means or proportions divided by the mutual standard deviation and reported as percentages; BMI, body mass index; MAP, mean arterial blood pressure; SBP, systolic
blood pressure; EA, epidural anesthesia; CES, combined epidural–spinal anesthesia. The t-test was used to compare COVID-19 group vs. METHODS Control group for continuous variables, and
χ2-test or Fisher’s exact test for categorical variables. aComparing COVID-19 group with Control group, P < 0.05. Values are Number (proportion) or Mean ± SD. Block- surgery time, the time interval between neuraxial anesthesia block and start of surgery. ASD, absolute standardized difference,
the difference in means or proportions divided by the mutual standard deviation and reported as percentages; BMI, body mass index; MAP, mean arterial blood pressure; SBP, systolic
blood pressure; EA, epidural anesthesia; CES, combined epidural–spinal anesthesia. The t-test was used to compare COVID-19 group vs. Control group for continuous variables, and
χ2-test or Fisher’s exact test for categorical variables. aComparing COVID-19 group with Control group, P < 0.05. Frontiers in Medicine | www.frontiersin.org RESULTS height, the time interval between neuraxial anesthesia block
and start of surgery (block-surgery time), newborns weight and
experience of anaesthesiologists were included in the calculation
of propensity scores with multivariate logistic regression in our
study. Matching was performed without replacement with a 1:
2 matching protocol and a caliper width of 0.03. The quality of
the PSM was assessed in two ways. First, we visually assessed
propensity score histograms for two groups before and after PSM. Second, absolute standardized difference (ASD) <10% for a given
covariate was considered well-balanced. The study flowchart was shown in Figure 1. Among 1,538 eligible
subjects, we included 1,403 subjects [102 parturients infected
with SARS-CoV-2 (COVID-19 group), and 1,301 parturients
without (Control group)] for PSM after exclusion of individuals
with inadequate blockade (84 cases) or incomplete data (51
cases). After PSM, 287 patients were created, with 101 cases
in COVID-19 group and 186 cases in Control group. The
characteristics of predictors for neuraxial anesthesia-related
hypotension before and after PSM were shown in Table 1. There were statistically significant differences (P < 0.05) between
COVID-19 and Control groups across several variables before
PSM, including urgency of surgery (P < 0.01), anesthetic
technique (P < 0.01), the time interval between EA initiation
and surgery start (P = 0.02), and anaesthesiologist experience
(P = 0.02). Parturients with preoperative comorbidities (such as All statistical analyses were performed using Stata/SE 15.1
(Stata Corp, College Station, TX, USA). Continuous variables
are expressed as Mean ± standard deviation (SD) or Median
(inter-quartile range, IQR). Categorical variables are expressed as
Number (proportion). The Mann-Whitney U-test, χ2 or Fisher’s
exact test were used on appropriate. A two-tailed P < 0.05 was
considered as statistical significance. August 2021 | Volume 8 | Article 713733 4 Risk of Hypotension in COVID-19 Parturients Zhang et al. TABLE 3 | Anesthetic implementation and administration in COVID-19 group vs. Control group after propensity score matching (PSM). Frontiers in Medicine | www.frontiersin.org RESULTS After PSM
COVID-19
(n = 101)
Control
(n = 186)
P-value
Full stomach
32 (31.7%)
50 (26.9%)
0.39
Antiemetic utilization
0.45
Metoclopramide
74 (73.3%)
169 (90.7%)
Metoclopramide and
5-HT3 receptor
antagonist
27 (26.7%)
17 (9.3%)
Infusion volume (ml)
0.08
>1,000
12 (11.9%)
10 (5.4%)
500–1,000
74 (73.2%)
155 (83.3%)
<500
15 (14.9%)
21 (11.3%)
Type of fluids
0.15
Crystalloids
68 (67.3%)
109 (58.6%)
Crystalloids and Colloids
33 (32.7%)
77 (41.4%)
Site of puncture
0.94
EA
L2-3
41 (40.6%)
78 (41.9%)
L3-4
7 (6.9%)
12 (6.5%)
CES
L3-4
38 (37.6%)
73 (39.2%)
L4-5
15 (14.9)
23 (12.4%)
Approach of puncture
0.89
EA
Median
45 (44.5%)
86 (46.2%)
Lateral
3 (3.0%)
4 (2.2%)
CES
Median
53 (52.5%)
96 (51.6%)
Lateral
0
0
Frequency of puncture
<0.001a
1
75 (74.3%)
173 (93.0%)
2
26 (25.7%)
11 (5.9%)
3
0
2 (1.1%)
CES
Dose of lidocaine (mg)
0
0
Dose of ropivacaine (mg)
12.4 ± 1.8
12.1 ± 1.7
0.50
EA
Dose of lidocaine (mg)
330 ± 38
340 ± 50
0.42
Dose of ropivacaine (mg)
55.0 ± 13.9
59.1 ± 11.6
0.28
Vasoconstrictor utilization
0.02a
Methoxamine
17 (16.8%)
22 (11.8%)
Metaraminol
8 (7.9%)
8 (4.3%)
Phenylephrine
12 (11.9%)
13 (7.0%)
Oxytocin utilization
Peripheral intravenous
101 (100%)
186 (100%)
Intramyometrial
27 (26.7%)
51 (27.4%)
0.90
Values are Number (proportion) or Mean ± SD. EA, epidural anesthesia; CES, combined
spinal-epidural anesthesia. The t-test was used to compare COVID-19 group vs. Control group for continuous variables, and chi-square test or Fisher’s exact test for
t
i
l
i bl TABLE 2 | The incidences of neuraxial anesthesia-related hypotension in
COVID-19 group vs. Control group before and after propensity score matching
(PSM). Hypotension
Before PSM
After PSM
COVID-19
59 (57.8%)
58 (57.4%)
Control
509 (39.1%)
78 (41.9%)
P-value
<0.001a
0.01a
χ2
13.76
6.30
IRR
1.48 (95% CI 1.24–1.77)
1.37 (95% CI 1.08–1.74)
Cramér’s V
0.10
0.15
Values are Number (proportion). IRR, incidence risk ratio; CI, confidence interval. The
χ2-test was used to compare COVID-19 group vs. Control group for categorical variables. aComparing COVID-19 group with Control group, P < 0.05. TABLE 3 | Anesthetic implementation and administration in COVID-19 group vs. Control group after propensity score matching (PSM). TABLE 2 | The incidences of neuraxial anesthesia-related hypotension in
COVID-19 group vs. Control group before and after propensity score matching
(PSM). Values are Number (proportion). IRR, incidence risk ratio; CI, confidence interval. RESULTS After PSM
COVID-19
(n = 101)
Control
(n = 186)
P-value
SBP (mmHg)
113 ± 7
114 ± 8
0.29
MAP (mmHg)
80 ± 8
78 ± 7
0.03a
Heart rate (bpm)
84 ± 13
85 ± 11
0.49
SaPO2 (%)
99 ± 1
99 ± 1
1.00
Hypoxia (SaPO2 <95%)
0
0
Arrhythmia
Tachycardia (heart rate >100 bpm)
18 (17.8%)
40 (21.5%)
0.46
Nausea
49 (48.5%)
32 (17.2)
<0.001a
Vomiting
11 (10.9%)
8 (4.3%)
0.03a
Dizziness
19 (18.8%)
6 (3.2%)
<0.001a
Shaking
52 (51.5%)
34 (18.3%)
<0.001a
Values are Number (proportion). The χ2-test or Fisher’s exact test was used to compare
COVID-19 group vs. Control group for categorical variables. aComparing COVID-19 group with Control group, P < 0.05. TABLE 4 | Intraoperative maternal vital signs and adverse reactions in COVID-19
group vs. Control group after propensity score matching (PSM). Values are Number (proportion). The χ2-test or Fisher’s exact test was used to compare
COVID-19 group vs. Control group for categorical variables. aComparing COVID-19 group with Control group, P < 0.05. might experience neuraxial anesthesia-related hypotension more
frequently than without. TABLE 5 | Neonatal Apgar scores in COVID-19 group vs. Control group after
propensity score matching (PSM). After PSM
COVID-19
(n = 101)
Control
(n = 186)
P-value
1 min
0.04a
10
80 (79.2%)
165 (88.7%)
9
16 (15.8%)
20 (10.8%)
8
4 (4.0%)
1 (0.5%)
7
1 (1%)
0
5 min
0.36
10
99 (98.0%)
185 (99.5%)
9
1 (1.0%)
1 (0.5%)
8
1 (1.0%)
0
Values are Number (proportion). The Fisher’s exact test was used to compare COVID-19
group vs. Control group for categorical variables. aComparing COVID-19 group with Control group, P < 0.05. TABLE 5 | Neonatal Apgar scores in COVID-19 group vs. Control group after
propensity score matching (PSM). Previous studies showed that the incidences of hypotension
in parturients who underwent a cesarean section were 32–52%
after EA, 43–64% after SA, and 32–70% after CES (8–11, 20–
22, 26). In present study, the incidences of hypotension after
neuraxial anesthesia were ∼58 and 40% in parturients with and
without COVID-19, respectively. These different results might be
attributed to several reasons, such as definition of hypotension,
methods and interval time of measurement, and experience
of anaesthesiologist (10, 11, 23, 24). Additionally, the short-
lived hypotension might also be missed or rapidly recovered by
vasoconstrictor pre-treatment. RESULTS Given the underestimation of risk
of hypotension in the retrospective study design, we adopted a
loosen hypotension definition in present study. yp
p
y
A recent study found that baby weight, baseline SBP, sensory
block height, the time interval between spinal induction and skin
incision, and experience of anaesthesiologists were associated
with SA-related hypotension in obstetric surgery (11). Maternal
age, BMI, weight gain, gravidity, history of hypotension, baseline
heart rate, fluid preloading, and anesthetic adjuvant were also
demonstrated to be predictive factors for SA-related hypotension
in cesarean delivery (10). Chronic alcohol consumption, history
of hypertension, ASA physical status, urgency of surgery, surgical
department, amount of local anesthetic, and preload liquid were
found to have a critical predictive value for the incidence of
hypotension in SA and CES for non-obstetric surgery (23, 24). According to evidences above, maternal age, BMI, baseline
heart rate, MAP, SBP, urgency of surgery, anesthetic technique,
sensory block height, block- surgery time, newborn weight,
and anaesthesiologist experience were employed to calculate
the propensity scores in present study. However, significant
differences were only found in urgency of surgery, anesthetic
technique, block- surgery time of EA, and anaesthesiologist
experience between COVID-19 group and Control group in un-
PSM cohort of present study. The effects of COVID-19 epidemic Body temperature over 37.4◦C (38.6%) and cough (32.7%) were
the main signs and symptoms, respectively. The other signs
and symptoms included fatigue (9.9%), chest distress (14.6%),
dyspnea (8.9%), and diarrhea (6.9%). Significant difference of
neonatal Apgar scores at 1 min (P = 0.046), but not 5 min
(P = 0.35), was observed in COVID-19 parturients with
neuraxial anesthesia-related hypotension vs. without (Table 6). RESULTS The
χ2-test was used to compare COVID-19 group vs. Control group for categorical variables. aComparing COVID-19 group with Control group, P < 0.05. chronic hypertension and hypertensive disorders of pregnancy)
which were known to impact maternal blood pressure were not
found in the PSM cohort. There were no significant differences
between two groups for any of the measured variables after PSM. Meanwhile, ASD for all variables were <10%, indicating that the
propensity scores were well-matched (Table 1). As shown in Table 2, the incidence of neuraxial anesthesia-
related hypotension was higher in COVID-19 group than that in
Control group before and after PSM [57.8 vs. 39.1% (P < 0.001);
57.4 vs. 41.9% (P = 0.01), respectively]. The incidence risk ratio
for neuraxial anesthesia-related hypotension in parturients were
1.48 (95% CI 1.24–1.77) and 1.37 (95% CI 1.08–1.74) before
and after PSM, respectively. At the 0.05 significance level, the
un-PSM cohort had lower Cramér’s V-value (0.10 vs. 0.15) than
PSM cohort. In PSM cohort, COVID-19 parturients had more times
of puncture (P < 0.001) and higher rate of vasoconstrictor
utilization (P = 0.02) than Control parturients. However, there
were no significant differences between two groups across those
variables, including the rate of full stomach, antiemetic and
oxytocin utilization, infusion volume and type of fluids, site
and approach of puncture, and the dose of local anesthetic
(Table 3). COVID-19 group showed significantly higher than
Control group in MAP, but not in SBP, heart rate, and SaO2%. The incidences of nausea, vomiting, dizziness, and shaking were
significantly higher in COVID-19 group than Control group
(48.5 vs. 17.2%, P < 0.001; 10.9 vs. 4.3%, P = 0.03; 18.8
vs. 3.2%, P < 0.001; 51.5 vs. 18.3%, P < 0.001; respectively). There was no significant difference in the incidence of maternal
tachycardia (P = 0.46) between two groups (No other arrhythmia
was observed). And intraoperative hypoxia wasn’t observed in
two groups (Table 4). The Apgar score of newborns at 1 min
(P = 0.04) after birth was significantly lower in COVID-19
group than that in Control group (Table 5), while no significant
difference at 5 min (P = 0.36). Among COVID-19 parturients, there were no significant
differences in the signs, symptoms, and preoperative laboratory
parameters between parturients with neuraxial anesthesia-related
hypotension and without (all P > 0.05, Supplementary Table 1). RESULTS August 2021 | Volume 8 | Article 713733 Frontiers in Medicine | www.frontiersin.org 5 Risk of Hypotension in COVID-19 Parturients Zhang et al. TABLE 4 | Intraoperative maternal vital signs and adverse reactions in COVID-19
group vs. Control group after propensity score matching (PSM). After PSM
COVID-19
(n = 101)
Control
(n = 186)
P-value
SBP (mmHg)
113 ± 7
114 ± 8
0.29
MAP (mmHg)
80 ± 8
78 ± 7
0.03a
Heart rate (bpm)
84 ± 13
85 ± 11
0.49
SaPO2 (%)
99 ± 1
99 ± 1
1.00
Hypoxia (SaPO2 <95%)
0
0
Arrhythmia
Tachycardia (heart rate >100 bpm)
18 (17.8%)
40 (21.5%)
0.46
Nausea
49 (48.5%)
32 (17.2)
<0.001a
Vomiting
11 (10.9%)
8 (4.3%)
0.03a
Dizziness
19 (18.8%)
6 (3.2%)
<0.001a
Shaking
52 (51.5%)
34 (18.3%)
<0.001a
Values are Number (proportion). The χ2-test or Fisher’s exact test was used to compare
COVID-19 group vs. Control group for categorical variables. aComparing COVID-19 group with Control group, P < 0.05. TABLE 5 | Neonatal Apgar scores in COVID-19 group vs. Control group after
propensity score matching (PSM). After PSM
COVID-19
(n = 101)
Control
(n = 186)
P-value
1 min
0.04a
10
80 (79.2%)
165 (88.7%)
9
16 (15.8%)
20 (10.8%)
8
4 (4 0%)
1 (0 5%) TABLE 6 | Neonatal Apgar scores from COVID-19 parturients with or without
hypotension. COVID-19 parturients
With hypotension
(n = 58)
Without hypotension
(n = 43)
P-value
1 min
0.046a
10
41 (70.7%)
39 (90.6%)
9
14 (24.1%)
2 (4.7%)
8
2 (3.5%)
2 (4.7%)
7
1 (1.7%)
0
5 min
0.35
10
57 (98.3%)
42 (97.7%)
9
0
1 (2.3%)
8
1 (1.7%)
0
Values are number (proportion). The Fisher’s exact test was used to compare COVID-19
group vs. Control group for categorical variables. aComparing COVID-19 group with Control group, P < 0.05. TABLE 6 | Neonatal Apgar scores from COVID-19 parturients with or without
hypotension. natal Apgar scores from COVID-19 parturients with or without TABLE 4 | Intraoperative maternal vital signs and adverse reactions in COVID-19
group vs. Control group after propensity score matching (PSM). Frontiers in Medicine | www.frontiersin.org ETHICS STATEMENT The studies involving human participants were reviewed and
approved by Institutional Review Board at Renmin Hospital
of Wuhan University, Wuhan, China (Chairperson Prof. Hong
Chen). Written informed consent for participation was not
required for this study in accordance with the national legislation
and the institutional requirements. Consistent with previous studies, our data also revealed
anaesthesiologist experience in association with the risk of
neuraxial anesthesia-related hypotension (11). An experienced
anaesthesiologist can protect parturients with low baseline
BP or high level of sensory blockade from high risk of
hypotension. Intriguingly, there was a significantly higher
incidence of hypotension in COVID-19 group than that in
Control group before PSM, although those anaesthesiologists
had more experience. In the PSM cohort, experience of
anaesthesiologists was comparable in two groups, but COVID-19
parturients still had a higher incidence of neuraxial anesthesia-
related hypotension and vasoconstrictor utilization. Those results
supported COVID-19 parturients were at an increased risk for
neuraxial anesthesia-related hypotension. Additionally, COVID-
19 parturients suffered more times of puncture in present study. Part of this difference seems due to the personal protective
equipment of anaesthesiologists. DISCUSSION In this study, we conducted a retrospective analysis of
the incidence of neuraxial anesthesia-related hypotension in
parturients with or without COVID-19 undergoing cesarean
delivery. Our data suggested that parturients with COVID-19 In this study, we conducted a retrospective analysis of
the incidence of neuraxial anesthesia-related hypotension in
parturients with or without COVID-19 undergoing cesarean
delivery. Our data suggested that parturients with COVID-19 August 2021 | Volume 8 | Article 713733 Frontiers in Medicine | www.frontiersin.org 6 Zhang et al. Risk of Hypotension in COVID-19 Parturients on urgency of surgery and the choice of anesthetic technique
should be taken into account during anesthesia and investigated
in future studies. after PSM would be another limitation. However, an increased
risk of neuraxial anesthesia-related hypotension in COVID-19
parturients undergoing cesarean delivery should be stressed. There was a higher incidence of hypotension in obese
parturients
during
neuraxial
anesthesia
due
to
vascular
compression by hypertrophic uterus (27). A left-tilt position
has been commonly used in parturients, particularly in obese
parturients. Nevertheless, a recent study declared that the
hemodynamic parameters derived from a non-invasive cardiac
output monitoring system were not statistically different
between the left-tilt and supine position (28). Pre-administration
of vasoconstrictor and volume most likely concealed the
appearance of hypotension (18, 29, 30). FUNDING This work was supported by grants from the National Natural
Science Foundation of China (81671948 and 81801085). This work was supported by grants from the National Natural
Science Foundation of China (81671948 and 81801085). q p
g
ACE2 dysfunction induced by SARS-CoV-2 infection was
referred to virus toxicity, hypoxia status, inflammation, and
sympathetic hyperactivity (31), which might increase the
susceptibility
of
neuraxial
anesthesia-related
hypotension
resulted from sympathetic inhibition in COVID-19 parturients. Although clinical practices have demonstrated that neuraxial
anesthesia is a safe technique for obstetric anesthesia in COVID-
19 parturients (5, 6, 32, 33), higher incidences of hypotension
and discomforts, and lower Apgar scores at 1 min were observed
in COVID-19 parturients and their babies in present study. More
evidences derived from a larger sample size and randomized
controlled trails are also needed to determine the effects and
mechanisms of COVID-19 on hemodynamics of parturients
undergoing neuraxial anesthesia for cesarean delivery. ACKNOWLEDGMENTS Assistance with the study: We would like to thank Prof. Daqing
Ma, Ph.D., FRCA for his assistance with the study. 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. AUTHOR CONTRIBUTIONS QM and XC designed the study. YZ, RC, CC, YG, and
QZ collected the data. CC and YG analyzed and interpreted
the work. YZ and RC drafted the manuscript. ZX and MW
revised it critically for important intellectual content. All authors
contributed to the manuscript and approved the final version. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found
online
at:
https://www.frontiersin.org/articles/10.3389/fmed. 2021.713733/full#supplementary-material Supplementary Figure 1 | Summaries of the balance of covariates before and
after propensity score matching. Absolute standardized difference <10% for a
given covariate was considered well balanced. Block- surgery time, the time
interval between neuraxial anesthesia block and start of surgery; BMI, body mass
index; SBP, systolic blood pressure; MAP, mean arterial blood pressure. Clinical practice, such as anesthetic technique, local anesthetic
concentration, vasopressor, and volume administration, were
not sufficiently standardized in this multicenter retrospective
study in order to draw conclusive conclusions. And, a post-hoc
estimated power value 0.71 and effect size (Cramér’s V-value) 0.15 Supplementary Table 1 | Clinical characteristics of COVID-19 parturients with or
without hypotension. Values are Number (proportion) or Median (IQR). 2. Malki Z, Atlam E-S, Ewis A, Dagnew G, Alzighaibi AR, ELmarhomy G,
et al. ARIMA models for predicting the end of COVID-19 pandemic
and the risk of second rebound. Neural Comput Appl. (2020) 18:833–
41. doi: 10.1007/s00521-020-05434-0
3. Panovska-Griffiths J, Kerr CC, Stuart RM, Mistry D, Klein DJ, Viner RM, et al.
Determining the optimal strategy for reopening schools, the impact of test 3. Panovska-Griffiths J, Kerr CC, Stuart RM, Mistry D, Klein DJ, Viner RM, et al.
Determining the optimal strategy for reopening schools, the impact of test 2. Malki Z, Atlam E-S, Ewis A, Dagnew G, Alzighaibi AR, ELmarhomy G,
et al. ARIMA models for predicting the end of COVID-19 pandemic
and the risk of second rebound. Neural Comput Appl. (2020) 18:833–
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S, et al. Assessment of autonomic nervous system dysfunction in the
early phase of infection with SARS-CoV-2 virus. Front Neurosci. (2021)
15:640835. doi: 10.3389/fnins.2021.640835 32. Sun
X,
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Y,
Mei
W. Safety
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studies. Int J Surg. (2014) 12:1495–9. doi: 10.1016/j.ijsu.2014.07.013 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. 17. Klöhr S, Roth R, Hofmann T, Rossaint R, Heesen M. Definitions of
hypotension after spinal anaesthesia for caesarean section: literature search
and application to parturients. Acta Anaesthesiol Scand. (2010) 54:909–
21. doi: 10.1111/j.1399-6576.2010.02239.x 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. 18. Singh PM, Singh NP, Reschke M, Ngan Kee WD, Palanisamy A, Monks
DT. REFERENCES Vasopressor drugs for the prevention and treatment of hypotension
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107. doi: 10.1016/j.bja.2019.09.045 19. Garrido MM. Covariate adjustment and propensity score. JAMA. (2016)
315:1521–2. doi: 10.1001/jama.2015.19081 Copyright © 2021 Zhang, Chen, Cao, Gong, Zhou, Wei, Xia, Chen and Meng. 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. 20. Klimek M, Rossaint R, van de Velde M, Heesen M. Combined spinal-epidural
vs. spinal anaesthesia for caesarean section: meta-analysis and trial-sequential
analysis. Anaesthesia. (2018) 73:875–88. doi: 10.1111/anae.14210 21. McDonnell NJ, Paech MJ, Muchatuta NA, Hillyard S, Nathan EA. A
randomised double-blind trial of phenylephrine and metaraminol infusions August 2021 | Volume 8 | Article 713733 Frontiers in Medicine | www.frontiersin.org Frontiers in Medicine | www.frontiersin.org 8
|
https://openalex.org/W4235530907
|
https://www.biodiversitylibrary.org/partpdf/72531
|
English
| null |
January 15, 1901
|
Proceedings of the Zoological Society of London
| 1,901
|
public-domain
| 11,196
|
1901,
Vol.
I.
(January
to
April). January
15,
1901. January
15,
1901. Proc.
Zoou.
Soc.—1901,
Vou.
I. January
15,
1901. Prof. G. B. Howzs,
LL.D.,
F.R.S.,
Vice-President,
in the Chair. Prof. G. B. Howzs,
LL.D.,
F.R.S.,
Vice-President,
in the Chair. The
Secretary
read
the
following
report
on
the
additions
to
the
Society’s
Menagerie
during
the
month
of
December
1900
:— The
registered
additions
to
the
Society’s
Menagerie
during
the
month
of
December
1900
were
211
in
number. Of
these
91
were
acquired
by
presentation
and
16
by
purchase,
and
104
were
received
on
deposit. The
total
number
of
departures
during
the
same period, by death and removals, was 158. Amongst
the
additions
attention
may
be
specially
directed
to
the
seven
specimens
of
Verreaux’s
Guinea-fowl
(Guttera
edouardi)
presented
on
December
31st
by
Mr. J. F. Walker
of
Bulawayo. Mr. Walker
states
that
this
Guinea-fowl
is
found
only,
so
far
as
he
knows,
in
the
Wankie
Hills,
a
district
due
north
of
Bulawayo,
and
about
midway
between
that
city
and
the
Zambesi. Only
one
specimen
of
it
had
previously
reached
the
Society’s
aviaries
(see
P.Z. 8. 1890,
p. 86). I
wish
also
to
direct
attention
to
the
valuable
series
of
Indian
birds
lately
presented
to
the
Society
by
Mr. E. W. Harper,
F.Z.S.,
of
Calcutta,
consisting
of
examples
of
twenty
species,
all
new
to
the
Society’s
Collection,
of
which
the
following
is
a
list. 1 Proc. Zoou. Soc.—1901,
Vou. I. Proc. Zoou. Soc.—1901,
Vou. I. MR. W. E. DE WINTON ON CYNICTIS SELOUSI. (Jan, 15, bo 1
Indian
Roller
(Coracias
indica). 1 Bengal Weaver-bird (Ploceus bengalensis) 3 . 1
Manyar
Weaver-bird
(Ploceus
many’)
oe
l
k h
d
bi d
( /
i
l ) 4
Black-throated
Weaver-birds
(P/oceus
atriqula),
2
3,
2) ON Second consignment (September 21st, 1900) :— 2
Western
Yellow-winged
Laughing-Thrushes
(7'rochalo-
pterum
nigrimentum)
3
OF
“
1
R f
- hi
d
L
hi
-Th
h
(J
th
i
l
fi- )
1
Rufous-chinned
Laughing-Thrush
(Janthocincla
rufi-
gularis). 2
( )
1
Slaty-headed
Scimitar
Babbler
(Pomatorhinus
schis-
ticeps). p )
1
Black-throated
Ouzel
(Turdus
atrigularis)
¢
. 2 Tickell’s Ouzels (Turdus unicolor ). (
)
1
Spotted-wing
(Psaroglossa
spiloptera). 1
Spotted-wing
(Psaroglossa
spiloptera). Third
consignment
(November
27th,
1900)
:— Third
consignment
(November
27th,
1900)
:— 4
Ashy-crowned
Finch-Larks
(Pyrrhulauda
grisea),
3
3. Oe 2
Singing
Bush-Larks
(Mirafra
cantillans)
Is)
Oe 2
Slaty-headed
Parrakeets
(Palwornis
schisticeps)
3
Q. 1
Burmese
Slaty-headed
Parrakeet
(Palwornis
finschi)
3
. 1
Burmese
Slaty-headed
Parrakeet
(Palwornis
finschi)
3
. 1
Golden-eyed
Fruit-Pigeon
(Carpophaga
concinna). 1
Golden-eyed
Fruit-Pigeon
(Carpophaga
concinna). Fourth
consignment
(January
Ist,
1901). Fourth
consignment
(January
Ist,
1901). Fourth
consignment
(January
Ist,
1901). 2
Blue-winged
Sivas
(Siva
cyanoptera). 1
Silver-eared
Mesia
(Mesia
argentauris). 1
White-capped
Redstart
(Ruticilla
leucocephala). 1
Rufous-bellied
Niltava
(Niltava
sundara), 1
Burmese
Roller
(Coracias
affinis). Mr. W. E. de
Winton
exhibited
a
specimen
of
the
large
Grey
Meerkat
(Cynictis
selousi
de
Winton),
described
in
the
*
Annals
and Magazine of Natural History,’ ser. 6, vol. xviii. 1896, p. 469,
hitherto known from a skull only, obtained near Bulawayo. The
skin
exhibited
(see
Plate
I.),
together
with
a
skull,
had
been obtained by Mr. P. C. Reid on the west bank of the Linyanti
River
on
the
5th
July,
1899. The
following
description
was
given :— Size
about
half
as
large
again
as
the
Bay
Meerkat
(C. peni-
cillata) ; body-colour grizzled drab-grey ; hairs of the tail broadly
white-tipped
;
both
fore
and
hind
feet
black;
belly
buff. The
grizzling of the head and body is much coarser than in the Bay
Meerkat,
owing
to
the
broader
annulations
on
the
hair,
but
the
pattern on the hairs of the tail is similar in both species. There is
an entire absence of rufous in the colouring of the Grey Meerkat,
the
tips
of
the
under-fur
and
the
broad
subbasal
band
of
the Mi
mp. IP,
oS),
AON
sv-oll
IU
PLA, Mintern
Bros.imp. J.Smit
del,
et
hth. CM
INUCTIS
SELOW
Si: CM
NUCT
SELO
Si: 3 1901.] N
FISHES
FROM
LAKES
TANGANYIKA
AND
K coarser
fur
being
dull
buff,
and
the
tail
whitish
instead
of
orange
colour. The
hands
and
feet
of
the
larger
species
are
black,
while
in
the
smaller
they
are
golden-fawn. coarser
fur
being
dull
buff,
and
the
tail
whitish
instead
of
orange
colour. The
hands
and
feet
of
the
larger
species
are
black,
while
in
the
smaller
they
are
golden-fawn. Measurements
taken
from
the
dried
skin:—Head
and
body
400
millim.,
tail
230,
hind
foot
90,
ear
30;
all
these
figures
must
be
considered
only
approximate. Mr. Lydekker
exhibited
the
skull
of
an
English
Fox
(Canis
vulpes)
with
two
perfect
canine
teeth
on
each
side
of
the
upper
jaw
(see
text-fig. 1). A
dog’s
skull
with
the
upper
canine
of
each
side partially divided had been figured on p. 211 of Mr. Bateson’s Text-fig. 1. Text-fig. 1. Skull of Fox showing double canine teeth. Skull of Fox showing double canine teeth. (Plates
I1.-LV.") Whilst
recently
staying
at
Sapelle
Station,
at
the
junction
of
the
Ethiop
and
Jamieson
Rivers,
Dr. Ansorge
has,
at
my
request,
made a small collection of the Fishes, which proves to be of quite
an exceptional interest, from the fact that through it representatives
of two families are added to the African freshwater fish-fauna, one
being
even
entirely
new,
an
event
that
has
not
happened
since
1873, when the late Professor Peters described the genus Pantodon,
the
type
of
the
family
Pantodontide. I
feel
extremely
grateful
to
Dr. Ansorge
for
the
trouble
he
has
taken,
under
difficult
circumstances
and
without
better
preserving-fluid
than
common
trade-gin. The
collection
was
made
in
August
and
September
1900. Some
of
the
small
Perch-like
fishes
(Cichlidw)
were
caught
with
hook
and
line
baited
with
worms. But
most
of
the
fishes,
including
Calanuchthys,
the
Mormyrs,
and
the
new
Phractolemus,
were
captured
in
creels
bailed
with
the
orange-red
fleshy
nut
of
the
oil-palm, set at the mouth of the Ethiop River, close to the bank,
by
Dr. Ansorge’s
native
boy. All
these
fishes
are
considered
good-eating by the blacks. T
am
pleased
to
add
that
the
examples
of
the
new
species
have
been
generously
presented
to
the
British
Museum
by
Dr. Ansorge. Fourth
consignment
(January
Ist,
1901). Study
of
Variation,
and
the
present
specimen
would
seem
to
indicate
a
fuller
development
of
the
same
feature. An
instance
of
the
full
duplication
of
the
corresponding
teeth
of
both
sides
was
afforded by the skull of a Cat figured on p. 225 of the work cited. Study
of
Variation,
and
the
present
specimen
would
seem
to
indicate
a
fuller
development
of
the
same
feature. An
instance
of
the
full
duplication
of
the
corresponding
teeth
of
both
sides
was
afforded by the skull of a Cat figured on p. 225 of the work cited. The
Fox
to
which
the
skull
belonged
had
been
killed
by
the
South
Oxfordshire
Hounds. The
skull
itself
was
the
property
of
Mr. H. G. Pease. In
describing
the
collection
of
Fishes
brought
home
from
Lakes
Tanganyika
and
Kivu
by
the
Tanganyika
Exploring
Expedition,
under
the
leadership
of
Mr. J. E. S. Moore,
Mr. G. A. Boulenger
pointed
out
that
the
study
of
this
important
collection
did
not
modify
the
conclusions
embodied
in
his
first
report
published
in
1898. The
exploration
of
Lake
Kivu
had
thrown
no
light
on
the
origin
of
the
Tanganyikan
fauna;
the
smaller
lake
had
proved
to
be
very
thinly
populated
with
Fishes,
which
all
belonged
to
widely
distributed
genera,
the
species
showing
a
mixture
of
Nile
and
Tanganyika
elements,
with
two
that
might
prove
to
be
endemic. The
list
of
the
Fishes
from
the
two
lakes
comprised
91
species,
74
of
which
had
been
named
by
the
author. The
collection
now
described
consisted
of
examples
of
50
species,
26
of
which
were
* 1* n. 15, A. BOULENGER
ON 4 new to science, two being made the types of additional genera of
the
family
Oichhde. y
This
Memoir
will
be
published
in
full
in
the
Society’s
‘
Trans-
actions.’ ’ For an explanation of the Plates, see p. 10. The following papers were read :— The following papers were read :— 1. On
the
Fishes
collected
by
Dr. W. J. Ansorge
in
the
Niger
Delta. By
G. A. Boutencer,
F.R.S.,
F.Z.S. [Received January 4, 1901.]
(Plates
I1.-LV.") 1.
On
the
Fishes
collected
by
Dr.
W.
J.
Ansorge
in
the
Niger
Delta.
By
G.
A.
Boutencer,
F.R.S.,
F.Z.S.
[Received January 4, 1901.]
( l
") 1. On
the
Fishes
collected
by
Dr. W. J. Ansorge
in
the
Niger
Delta. By
G. A. Boutencer,
F.R.S.,
F.Z.S. [Received January 4, 1901.] [Received January 4, 1901.] 1.
CALAMICHTHYS
CALABARICUS
J.
A.
Smith. The
single
specimen
contained
in
the
collection,
a
female
measuring
345
millimetres,
with
11
dorsal
spines,
is
extremely
remarkable
for
having
a
very
small,
but
perfectly
distinct
sub-
operculum. The
absence
of
this
bone,
verified
on
a
considerable
number
of
specimens,
had
been
regarded
as
one
of
the
generic
characters
distinguishing
Calamichthys
from
Polypterus. The
coloration
of
the
specimen
is
a
dark
olive-brown
above,
gradually
shading
into
a
bright
yellow
beneath;
a
large
deep-black
spot
on
the pectoral fin. Hyery possibility of the presence of a suboperculum indicating a sibility of the presence of a suboperculum indicating a ’ For an explanation of the Plates, see p. 10. MMS
W
TO
SZ
ai UDSH
SOND
IOLOVYHA
‘hat
soiguresuy
“AIL
Tep
aS
pg UDS
SOND
YHA
hat
igures
“A
T
aS
pg TNC
VT
OGL
SZ
vat ‘VWIVI
SIS
“SITVNV
SQINAS
1
‘Har
sorgussquy
CGR
ID
SMS)
ach
TNC
VT
OGL
SZ
at le o74 1S), LOO oll Ede IW PJ.Smut
de]
et
lth. Mintern
Bros
TL
PELMATOCHROMIS
ANSORGII. 21>
J
UILCIEBIEIR
. 3.P. TANIATUS. 5 OF
THE
NIGER 1901.] species
distinct
from
Calamichthys
seems
to
me
removed
by
the
fact
that
I
have
carefully
compared
this
specimen
with
others
like-
wise from the Niger delta, without being able to detect any other
important
difference. I
look
upon
it
as
an
atavistic
individual
anomaly. 3.
MARCUSENIUS
LONGIANALIS,
sp.
n.
(Plate
IIT.
fig.
1.) Depth of body 5 to 5% times in total length, length of head 6 to
63
times. Head
17
as
long
as
deep;
snout
convex,
+
length
of
head,
slightly
projecting
beyond
the
mouth;
latter
small,
sub-
inferior,
below
level
of
eye,
its
width
1
length
of
head;
teeth
feebly notched, 5 in the upper jaw, 6 in the lower; nostrils nearly
equally distant from end of snout and from eye, anterior on a level
with
centre
of
latter,
posterior
with
its
lower
border;
eye
small,
4
length
of
snout,
2
interorbital
width. Dorsal
15-16,
its
length
4
its
distance
from
the
head,
originating
above
16th
or
17th
ray
of
anal. Anal
32-33,
thrice
as
long
as
dorsal,
nearer
base
of
caudal
than
base
of
ventral. Pectoral
obtusely
pointed,
a
little
shorter
than
head,
14
length
of
ventral,
reaching
base
of
latter. Caudal
scaly
at
the
base,
with
pointed
lobes. Candal
peduncle
34
times
as long as deep, nearly as long as head. 63 to 66 scales in the
lateral
line,
5
in
a
transverse
line
on
the
body,
=
between
dorsal
and
anal,
12
round
caudal
peduncle. Purplish
brown,
more
or
less
profusely
speckled
with
blackish
;
fins
dark
brown. Total
length
145
millim. Two
specimens. Closely
allied
to
M. brachyhistius
Gill. Distinguished
by
the
more
elongate
form,
the
more
slender
caudal
peduncle,
the
longer
anal fin, and the higher number of scales in the lateral line. 2, IsSICHTHYS HENRYI Guill. The
single
specimen
is
more
elongate
than
any
on
record,
the
depth
of
the
body
being
contained
13
times
in
the
total
length,
the
length
of
the
head
8}
times. D.53;
A. 52;
lat. 1. 140. Total
length
210
millim. PHRACLOLEZMUS, gen. n. Body
elongate,
subcylindrical,
covered
with
large
striated
scales
‘
lateral line complete, formed of a series of straight tubes extending
along
the
entire
length
of
the
exposed
part
of
the
seales. Head
small,
strongly
ossified,
covered
with
thin
skin;
mouth
small,
proboscidiform,
capable
of
being
thrust
forward,
when
at
rest
folded
over
and
received
into
a
depression
on
the
upper
surface
of
the
head;
a
single
narial
orifice,
preceded
by
a
barbel;
eyes
small,
lateral. Gill-openings
narrow,
restricted
to
the
sides;
gular
region
protected
by
the
interopercles,
that
on
one
side
(usually
the
right)
overlapping
that
on
the
other
side. Four
gills;
no
pseudobranchie. Pectoral
fins
small,
with
18
rays
;
ventrals
far
back,
with
6
rays;
dorsal
short,
with
6
rays,
opposite
to the space between the ventrals and the anal ; latter short, with
6
rays;
caudal
fan-shaped,
with
18
to
20
rays;
all
the
fin-rays
articulated. Air-bladder
very
large,
extending
as
far
back
as
the
anal
fin. Stomach
with
3
pyloric
appendages
;
intestine
extremely
long and much convoluted. PHRACTOLEZMID 2. The
highly
remarkable
fish
discovered
by
Dr. Ansorge,
which
I
here
describe
under
the
name
of
Phractolemus
ansorgii,
cannot
be
incorporated
into
any
of
the
families
known
at
present. It
falls
into the suborder Malacopterygii as restricted and defined by me },
and
occupies
a
position
intermediate
between
the
Ostcoglosside
and
the
Clupeide. The
family
Phractolemide
may
be
characterized
as
follows
:— Mouth
edentulous,
projectile,
bordered
by
the
very
slender Mouth
edentulous,
projectile,
bordered
by
the
very
slender
' Poissons du Bassin-du Congo, p. 44 (1901). n. 15, A. BOULENGER
ON
‘ 6 premaxillaries
and
maxillaries. Supraoccipital
in
contaey
ee
ee
trontals,
widely
separating
the
small
ecg
amen
ae
ua
br
suboperculum
well
developed;
preoperculum
small;
in
es
um
enormous,
covering
the
gular
region
and
overlapping
its
fe
Lows
symplectic
absent
;
only
three
slender
bn
rays
ee
pharyngeal
teeth. Ribs
stout,
sessile,
nearly
completely
encire
ing
the
body;
slender
epineurals
;
no
epipleurals
:
caudal
eles
short. No
postelavicle. Pectoral
fins
inserted
low
down,
folding
like the ventrals ; latter with 6 rays. 5.
PHRACTOLEMUS
ANSORGII,
sp.
n.
(Plate
LI.) Depth of body 5 to 6 times in total length, length of head 63
to
7%
times. Head
depressed,
with
very
broad,
slightly
convex
interorbital region ; diameter of eye 43 to 53 times in length of
head, 3 to 33 times in interorbital width ; barbel nearly 4 length
of
head. Dorsal
with
the
two
anterior
rays
simple,
the
other
four
bifid;
the
first
ray
equally
distant
from
the
head
and
from
the
root
of
the
caudal;
second
ray
longest,
14
length
of
head. Anal
similar
to
dorsal,
but
rays
shorter,
the
second
or
longest
only
#
length
of
head. Pectoral
rounded,
a
little
shorter
than
head,
as
long
as
ventral,
which
is
pointed
and
equally
distant
from
head
and
from
anal. Caudal
rounded. Caudal
peduncle
compressed, nearly as long as deep, as long as head. Scales large,
longitudinally
striated,
35
to
37
in
a
longitudinal
series,
2
ima
transverse
series. On
the
caudal
region
the
scales
of
the
lateral
line and those of the series above it may bear a central sclerous
tubercle
(probably
a
seasonal
character). Uniform
olive-grey. The vertebra, in a male specimen of which a skeleton has been
prepared, number 34, 26 precaudal and 8 caudal, the last bearing
6
hypurals
to
support
the
homocereal
fin;
the
ribs
are
subequal, ff 1901.] OF
'HH
NIGHR very
thick,
with
a
wing-like
expansion
behind
at
the
base,
and
begin on the second vertebra, there being besides a strong occipital
rib. The
frontals
are
very
large
and
the
right
extends
with
its
curved border beyond the median line, as if overlapping its fellow;
two
supraorbital
bones
on
each
side;
the
parietals
are
very
small,
and
completely
separated
by
the
broad
and
short
supraoccipital,
which does not bear a crest. The pair of large bones covering the
throat,
the
right
overlapping
the
left,
and
which
at
first
suggest
the
gular
plates
of
the
Polypteride,
are
to
be
identitied
as
inter-
operculum
;
above
the
interoperculum
two
very
large
suborbitals,
covering
the
hyomandibular
and
quadrate,
which
are
thrust
forward
for
the
suspension
of
the
feeble
mandibular
rami,
which
are
dis-
connected
at
the
symphysis;
the
premaxillary
and
maxillary
bones
more
slender
still
and
connected
by
ligament
with
the
mandible. ALESTES
LONGIPINNIS
Gthr. ALESTES
LONGIPINNIS
Gthr. 5.
PHRACTOLEMUS
ANSORGII,
sp.
n.
(Plate
LI.) The
shoulder-girdle
is
suspended
from
the
posttemporal
close
to
the
operculum
;
it
includes
the
ordinary
elements
(clavicular,
supraclavicular,
coracoid,
scapula),
but
a
postclavicular
is
absent:
the mesocoracoid arch is present, slender ; the coracoids are much
smaller than the claviculars, and do not meet on the median line ;
4
pterygials
support
the
pectoral
fin-rays. Four
specimens
of
this
extraordinary
fish,
measuring
from
50
to
150
millim.,
were
brought
home
by
Dr. Ansorge,
to
whom
it
gives me great pleasure to dedicate the species. OPHIOCEPHALID
&. 13. OPpHIOCEPHALUS
OBSCURUS
Gthr. SILURID 2. S,
CEARIAS
ANGOLENSIS
Stdr. S,
CEARIAS
ANGOLENSIS
Stdr. 9,
SCHILBE
DISPILA
Gthr. 10. CHRYSICHTHYS
NIGRODIGIVATUS
Lacép, 11. MaALopreRuURUS
ELECTRICUS
Gm. CYPRINODONTID &. 12. HAPLOGHILUS
INFRAFASCIATUS
Gthr. OPHIOCEPHALID
&. 13. OPpHIOCEPHALUS
OBSCURUS
Gthr. ANABANTID ©. 14,
ANABAS
KINGSLEY
Gthr. NANDID#. The
Nandide
(ineluding
the
Polycentride)
ave
a
small
family
of 12.
HAPLOGHILUS
INFRAFASCIATUS
Gthr. 12. HAPLOGHILUS
INFRAFASCIATUS
Gthr. ANABANTID ©. 14,
ANABAS
KINGSLEY
Gthr. CHARACINID®. 6, SARCODACES ODOo# BI. 6, SARCODACES ODOo# BI. 6, SARCODACES ODOo# BI. CYPRINODONTID &. 12. HAPLOGHILUS
INFRAFASCIATUS
Gthr. NANDID#. NANDID#. The
Nandide
(ineluding
the
Polycentride)
ave
a
small
family
of an. 15, an. 15, A. BOULENGER
ON 8 freshwater
fishes
from
8.E. Asia
and
South
America,
apparently
most nearly allied to the Centrarchide, but distinguished from them
by
the
absence
of
the
entopterygoid. =
e y
p
yg
The
new
genus
here
described
is
its
first-known
African
representative. * Nandus marmoratus has also 23 vertebree, but 138+10. POLYCENTROPSIS, gen. Nn. Body
short,
elevated,
very
strongly
compressed
;
scales
mode-
rately
lmge,
ciliated. Lateral
line
incomplete,
reduced
to
a
few
tubes. Mouth
large,
extremely
protractile,
the
ascending
processes
of the premaxillaries extremely long and extending to the occipital
region
;
villiform
bands
of
very
small
teeth
in
the
jaws,
on
the
vomer,
and
on
the
palatines;
head
for
the
greater
part
covered
with
scales;
preorbital,
preopercle,
and
interopercle
serrated
;
opercle
ending
ina
spine. Gill-membranes
separate
;
six
branchi-
ostegals
;
no
pseudobranchie. Dorsal
and
anal
fins
nearly
equally
developed,
with
numerous
strong
spines
and
the
soft
portion
much
reduced. Ventrals
below
the
pectorals,
close
together,
with
a
strong
spine. Vertebre
23
(10+13)?. 15.
PoLYCENTROPSIS
ABBREVIATA,
sp.n.
(Plate
III.
fig.
2.) Depth of body twice in total length, length of head twice and a
half. Snout
acutely
pointed,
chin
slightly
projecting;
diameter
of eye a little longer than snout or interorbital width ; nearly one
third
length
of
head;
maxillary
extending
to
below
posterior
third
of eye; suborbital arch very slender; 6 or7 series of scales on the
cheek. 10
gill-rakers
on
lower
part
of
anterior
arch,
the
longest
nearly
as
long
as
gill-filaments. Dorsal
XV-XVI
11;
spines
in-
creasing in length to the fourth and decreasing from the seventh
or eighth, the longest half length of head and a little longer than
the
soft
rays. Anal
similar,
X
9. Pectoral
obtusely
pointed,
half
length
of
head. Ventral
longer,
produced
in
a
filament,
extending
beyond
origin
of
anal. Caudal
truncate. Caudal
peduncle
extremely
short. Sq. 32-35
a3
lat. 1. 5-6. Pinkish
brown,
warbled
with
darker;
spinous
dorsal
and
anal
dark
brown,
with
darker and ligliter spots and edged with black ; ventrals blackish ;
base of soft dorsal, anal, and caudal blackish, edged with pink. Two
specimens, CICHLID®. 16. Hemicnuromis
rascratus
Peters. 17. H»pMicHRomis
BIMACULATUS
Gill. 18. PELMATOCHROMIS
GUENTHERI
Sauv. (Hemichromis
volte
Stdr.;
H. tersquamatus
Gt 19,
PELMATOCHROMIS
ANSORGII,
sp. n. (Plate
IV. fig. 1.)
Teeth
in
2
or
3
series
in
each
jaw,
outer
largest
but
rather
small. Depth
of
body
2! to
24
times
in
total
length,
length
of Teeth
in
2
or
3
series
in
each
jaw,
outer
largest
but
rather
small. Depth
of
body
2! to
24
times
in
total
length,
length
of 9 1901.] OF
THE
NIGER head
24
or
3
times. Snout
broad,
rounded,
with
straight
or
slightly
convex
upper
profile,
as
long
as
the
diameter
of
the
eye,
which is contained 34 to 32 times in length of head and 13 times
in
interorbital
width;
maxillary
extending
to
below
anterior
border
of eye; 3 or 4 series of scales on the cheek; large scales on the
opercle. Gill-rakers
short,
10
or
11
on
lower
part
of
anterior
arch. Dorsal
XV-XVI
10-11;
spines
subequal,
not
quite
3
length
of
head; middle soft rays somewhat produced, 2? or + length of head. Pectoral
2
or
3
length
of
head. Ventral
produced
into
a
filament,
reaching
origin
of
anal
or
a
little
beyond. Anal
III
8;
third
spine
as
long
as
dorsals. Caudal
rounded. Caudal
peduncle
much
deeper
than
long. Scales
smooth,
with
very
distinct
concentric
striation,
28-29
a3
lat. 1. —-
Dark
olive-brown
above,
yellowish
beneath
;
a
blackish
opercular
spot;
three
or
four
vertically
elongate
large
dark
spots
on
each
side
of
the
body,
below
the
upper
lateral
line;
fins
greyish,
soft
dorsal,
anal,
and
caudal
chequered
with
small
darker
and
lighter
spots. Total
length
90
millim. Four
specimens. Allied
to
the
preceding,
from
which
it
differs
chiefly
in
the
shorter
snout,
the
smaller
mouth,
and
the
more
rounded
caudal. 22,
TILAPIA
MARIE
Bley. 23. Tinapra
bara
Gthr. 20.
PELMATOCHROMIS
PULCHER,
sp.
n.
(Plate
LV.
fig.
2.) Teeth
in
4
or
5
series
in
each
jaw,
outer
largest. Depth
of
body 23 to 3 times in total length, length. of head 3 to 32 times. Snout broad, rounded, with convex upper profile, as long as the eye,
which is contained 33 times in length of head and does not quite
equal
interorbital
width
;
maxillary
extending
to
between
nostril
and eye; 2 or 3 series of scales on the cheek ; large scales on the
opercle. Gull-rakers short, 10 to 12 on lower part of anterior arch. Dorsal
XVI
9-10;
spines
gradually
increasing
in
length
to
the
last,
which
measures
half
length
of
head;
some
of
the
soft
rays
more
or
less
produced,
often
longer
than
the
head. Pectoral
3
or
*
length
of
head. Ventral
more
or
less
produced
into
a
filament,
reaching
origin
of
anal,
or
beyond. Anal
III
7-8;
third
spine
as
long
as
longest
dorsal. Caudal
rounded
or
subacuminate. Cauda]
pedunele
as
long
as
deep. Scales
smooth,
27-29
ae
late
1
a
Olive,
with
two
darker
or
blackish
longitudinal
bands
on
each
side, the upper from the occiput to the base of the soft dorsal, the
lower from the eye, over the opercle, to the extremity of the caudal
fin;
sides
of
body
below
lower
lateral
band
and
between
pectorals
and
ventrals
of
a
beautiful
rose-colour;
spinous
dorsal
grey,
black
at
the
base,
the
black
area
gradually
rising
to
cover
nearly
the
whole
of
the
soft
dorsal;
pectoral,
outer
side
of
ventral,
and
extremity
of
anal
blackish;
caudal
grey,
with
an
oblique
white
streak above in the males. Total
length
95
millim. Several
specimens. p
Most
nearly
related
to
P. swbocellatus
Gthr.,
from
the
Gaboon,
but
easily
distinguished
by
the
proportions
of
the
dorsal
spines. 1 Jan. 15, Jan. 15, E
FISHES
OF
THE
NIGER
DELT 10 21,
PELMATOCHROMIS
THNIATUS,
sp. n. (Plate
IV. fig. 3.) (FOBILD &. 24,
ELEOVRIS
SENEGALENSIS
Stdr. 24,
ELEOVRIS
SENEGALENSIS
Stdr. EXPLANATION
OF
THE
PLATES. EXPLANATION
OF
THE
PLATES. Prats II. Phractoleius ansorgii, with upper, lower, and side views of head,
and skeleton, p. 6. Prats
TT,
Fig. 1. Marcusenius longianalis, p. 5. 2. Polycentropsis abbreviata, and skeleton, p-3. Puars LY. Fig. 1. Pelmatochromis ansorgit, p. 8. »
y
A
Nar
Q
2. 5
pulcher,
p. 9. 3. 3
teniatus,
p. 10. 21,
PELMATOCHROMIS
THNIATUS,
sp.
n.
(Plate
IV.
fig.
3.) Teeth in 2 series in the upper jaw, in 3 in the lower, outer largest. Depth of body 3 times in total length, length of head 34 times. Snout
broad,
rounded,
with
convex
upper
profile,
as
long
as
the
eye,
which
is
contained
33
times
in
length
of
head
and
nearly
equals
interorbital
width;
maxillary
extending
slightly
beyond
vertical of anterior border of eye; 2 series of scales on the cheek ;
large
seales
on
the
opercle. Gill-rakers
short,
11
on
lower
part
of
anterior
arch. Dorsal
XVIII
7;
spines
gradually
increasing
in
length
to
the
last,
which
measures
half
length
of
head;
longest
soft
rays
produced
into
a
filament,
as
long
as
head. Pectoral
4
length
of
head. Ventral
produced
into
a
filament,
extending
beyond
origin
of
anal. Anal
III
7;
third
spine
as
long
as
longest
dorsal. Caudal
rounded. Caudal
peduncle
a
little
deeper
than
long. Scales
smooth,
28
:
alateale
ae
Brownish
above,
yellowish
beneath;
two
blackish
longitudinal
bands
on
each side, the upper from the occiput to the base of the soft dorsal,
the lower from the eye, over the opercle, to the root of the caudal ;
fins greyish, ventrals white with a black outer border; oblique dark
streaks
on
the
soft
dorsal
;
small
blackish
spots
on
the
caudal
and two larger ones edged with white on its upper border,
Total
length
75
millim Also
nearly
allied
to
P. subocellutus. Readily
distinguished
from
it, and from P. ansorgii, by the dorsal formula. 22,
TILAPIA
MARIE
Bley. This species was described from a single specimen from Azumine
Creek,
Opobo
River,
in
Miss
Kingsley’s
collection. Three
specimens
are
in
Dr. Ansorge’s
collection,
the
largest
measuring
135
millim. The
caudal
fin
is
rounded
rather
than
truncate. :
:
A
Gyn
oS
20-2
D. XVI
12-13;
A. 111
10;
Sq. 29-30
3;
tat. 1
22d. 22,
TILAPIA
MARIE
Bley. This species was described from a single specimen from Azumine
Creek,
Opobo
River,
in
Miss
Kingsley’s
collection. Three
specimens
are
in
Dr. Ansorge’s
collection,
the
largest
measuring
135
millim. The
caudal
fin
is
rounded
rather
than
truncate. :
:
A
Gyn
oS
20-2
D. XVI
12-13;
A. 111
10;
Sq. 29-30
3;
tat. 1
22d. Hab.
Salisbury,
Mashonaland,
8.
Africa,
5000
feet,
Nov.
1898
to Jan. 1899 (G@. A. K. Marshall). * For an explanation of the Plate, see p. 16. Puars LY. ©
7%. S,
19Ol,
voll. ELV. ©
7%. S,
19Ol,
voll. ELV. ATHollick
delet
lith. West,
Newman
chromo,
NEW
EXOTIC
SPIDERS ATHollick
delet
lith. ATHollick
delet
lith. ATHollick
delet
lith. West,
Newman
chromo, NEW
EXOTIC
SPIDERS. NEW
EXOTIC
SPIDERS. 1901.} 1901.} 1] Y. 0. PICKARD-CAMBRIDGE
ON
EXOTIC
SPIDE 2,
On
some
new
and
interesting
Exotic
Spiders
collected
by
Messrs. G. A. K. Marshall
and
R. Shelford. By
the
Rev. Octavius
Pickarp-CamBrines,
M.A.,
F.R.S.,
&e. 2,
On
some
new
and
interesting
Exotic
Spiders
collected
by
Messrs. G. A. K. Marshall
and
R. Shelford. By
the
Rev. Octavius
Pickarp-CamBrines,
M.A.,
F.R.S.,
&e. 2,
On
some
new
and
interesting
Exotic
Spiders
collected
by
Messrs. G. A. K. Marshall
and
R. Shelford. By
the
Rev. Octavius
Pickarp-CamBrines,
M.A.,
F.R.S.,
&e. Gen. nov. TITUS. Cephalothorax
elongate-oval,
rounded
behind,
broadly
and
a
little
roundly
truncate
before
;
lateral
marginal
impressions
at
the
caput
gradual
but
distinct
;
upper
surface
strongly
convex
;
from
the fore part of the caput to the hinder slope the rise 1s strong, a
little
curved
and
even,
with
a
very
slight
dip
at
the
thoracic
junction. The sides of the cephalothorax project over the bases of
the legs, making them appear to be articulated on the same plane
as
the
sternum. ‘The
thoracic
indentation
is
very
minute,
and
the
other
normal
ones
obsolete;
hinder
slope
steep;
height
of
the
clypeus,
which
projects,
is
half
that
of
the
facial
space,
its
fore
margin overhanging the base of the falces. Eyes
moderate
and
not
greatly
unequal
in
size;
in
two
trans-
verse
curved
rows;
the
hinder
row
considerably
longest,
its
eyes
ave very nearly equally separated, and the conyexity of its curve is
directed
forwards,
while
that
of
the
anterior
row
is
backwards. The
hind-lateral
eyes
are
larger
than
the
hind-centrals
and
are
placed outside a strong tubercle ; those of the anterior row on a
well-marked
transverse
prominence
or
ridge. The
fore-centrals
are
yery
nearly
if
not
quite
of
equal
size,
the
interval
between
them being about double that which separates each from the fore-
lateral
eye
on
its
side. ‘The
central
quadrangle
is
slightly
broader
than long, and its anterior side shortest. Legs
short,
rather
slender,
4,
1,
2,
3;
the
femora
strongly
elavate
or
tumid
at
their
posterior
end,
furnished
with
hairs
and
spines
;
two
pairs
of
these
are
beneath
the
metatarsi
and
three
pairs beneath tibie of the first pair. ‘T'arsi end with 2 claws. Palpi
(@). The
digital
joint
is
double
the
length
of
the
radial,
rather
claviform,
and
ending
with
a
very
minute,
slightly
curved
single claw. g
Falces
moderate
in
length,
powerful,
subconical. Mawille
rather
short,
strong,
straight,
but
inclined
to
the
labium;
rounded
at
their
outer
extremity,
and
a
little
impressed
and
obliquely
truncate
at
their
inner
extremity. Labium
short,
broader
than
long,
narrowest
at
the
apex,
the
outer
corners
of
which
are
rounded,
and
the
middle
a
little
im-
pressed. Sternum
longer
than
broad,
oval,
slightly
hollow-truncate
in
front,
bluntish
pointed
behind,
and
its
margins
strongly
indented
by the basal joints of the legs. V.
0.
PICKARD-CAMBRIDGE
ON
EXOTIC
SPIDERS.
[Jan.
15, 12 Gen.
Prosruzsima
L.
Koch. PROSTHESIMA
ALBOMACULATA,
sp. n. (Plate
V. figs. 2-2
¢.) Adult
female,
length
23
lines
(4°5
mm.). Cephalothorax
flattish,
oval,
truncate
at
each
end,
fore
end
rather
the
narrowest,
lateral
marginal
impressions
at
caput
very
slight,
profile-line
nearly
level. Colour
deep
black-brown,
softening
to
yellowish
brown
round
the
thoracic
indentation
;
surface
thinly
covered
with
grey
adpressed
hairs. Eyes
in
two
transverse
rows
of
very
nearly
equal
length. Curve
of
posterior
row
slight
and
its
convexity
directed
backwards. Anterior
row
almost
straight,
laterals
of
this
row
largest
of
the
eight,
the
two
centrals
being
placed
on
a
slight
prominence,
and
further
from
each
other
than
from
the
laterals. The
two
centrals
of
the
posterior
row
are
much
further
from
each
other
than
from
the
laterals
and
are
slightly
the
largest. The
four
centrals
form
a
quadrangle
as
long
as
broad,
the
fore
side
being
shortest. Legs
moderate
in
length
and
strength,
4,
1,
2,
3. Colour
yellow
to
yellow-brown
;
the
tibiz,
femora,
and
genue
of
the
first
pair
black-brown,
these
joints
of
the
second
pair
yellow-brown,
and
of
the
third
and
fourth
pairs
more
or
less
deeply
marked
longitudinally
and
suffused
with
black
and
brown,
furnished
with
coarse
hairs
and-
spines,
the
latter
most
numerous
and
strongest
on the tibie and metatarsi of the third and fourth pairs. Falees,
maaille,
and
labiwm
deep
brown. Falees,
maaille,
and
labiwm
deep
brown. Sternum
oval,
pointed
behind
;
colour
reddish
yellow-brown. Abdomen
oval,
somewhat
flattened,
black,
with
four
conspicuous
white spots forming a quadrangle on the fore half of the upperside,
the
two
hinder
spots
largest
and
nearly
round,
the
anterior,
near
the
fore
margin,
oval
or
subtriangular
and
forming
a
shorter
transverse
line
than
the
hinder
spots. On
each
side
of
the
under-
side,
about
the
middle,
is
a
large
somewhat
irregular
triangular-
shaped
white
patch,
whose
inner
angles
are
nearly
contiguous
a
little
way
behind
the
middle. Spinners
of
the
inferior
pair
much
wider
apart
than
the
superiors. Genital
aperture
simple
but
characteristic
in
form. Hab. Salisbury,
Mashonaland,
8. Africa,
5000
feet,
Nov. 1898
to Jan. 1899 (G@. A. K. Marshall). V. 0. PICKARD-CAMBRIDGE
ON
EXOTIC
SPIDERS. [Jan. 15, Gen. nov. TITUS. Salisbury,
Mashonaland,
8. Africa,
5000
feet,
Nov. 1898
to
Jan. 1899
(GA. K. Marshall), NEPHILENGYS
MALABARENSIS
Walck. An
adult
female
of
this
common
and
widely
dispersed
Epeirid
from
Karkloof,
Natal
(@. 4. K. Marshall). Genus
NEPHILENGYs
L.
Koch. Genus
NEPHILENGYs
L. Koch. NEPHILENGYS
MALABARENSIS
Walck. NEPHILENGYS
MALABARENSIS
Walck. Subfam.
Eurycomin». Subfam. Eurycomin». Fam.
GASTERACANTHID®. Subfam. Eurycomin». Fam.
HPEIRID
4, Genus
NEPHILENGYs
L. Koch. Gen. nov. TITUS. From the hinder end a chitinous
plate
runs
between
the
coxe
of
the
fourth
pair
of
legs
and
spreading
out
behind
them
joins
in
with
the
upperside
of
the
cephalothorax. Abdomen
short,
broad,
its
upper
surface
covered
with
a
strong
kind
of
granulose
coriaceous
shield
furnished
with
plumose
and
other
hairs;
sides,
especially
backwards,
protuberant
and
tumid,
these
parts
connected
behind
by
transverse
ruge
or
folds,
in
the
midst
of
which
the
spinners
are
placed
and
almost
hidden
in
a
circular
cavity. 1901. REV. 0. PICKARD-CAMBRIDGE
ON
EXOTIC
SPIDE 13 TITUS
LUGENS,
sp.n. (Plate
V. figs. 3-3
¢.) TITUS
LUGENS,
sp.n. (Plate
V. figs. 3-3
¢.) Adult
female,
length
21
lines. Cephalothoraxz
bright
red-brown,
suffused
with
a
darker
hue
on
the sides and on the caput, the fore part of which is nearly black ;
the
surface
is
thickly
covered
with
small
round
shining
tubercles
or
granulosities,
and
it
is
thinly
clothed
with
hairs,
of
which
some
on the sides and hinder part are white and of a plumose nature. Legs
yellow
tinged
with
brown;
the
femora
much
strongest,
granulose, as also are the uppersides of coxze. Colour of the femora
of
Ist
pair
black-brown,
of
the
second
pair
not
so
dark,
of
the
third
and
fourth
pairs
paler
and
indistinctly
banded
with
darker. The tarsi are enlarged slightly and gradually to the ends, which are
furnished
with
two
claws
and
a
compact
claw-tuft. Falces
deep
reddish
black-brown,
paler
at
the
fore
extremity,
furnished
in
front
with
bristly
hairs. Masille
and
labium
yellow-brown. Masille
and
labium
yellow-brown. Sternum
yellow-red,
covered
thickly
with
small
granulosities
like
the
cephalothorax. Abdomen
coriaceous,
covering
of
the
upperside
black
with
a
central
triangular
patch
of
white
plumose
hairs,
two
patches
of
the
same on the lateral margins, and one at the hinder extremity, sides
and
underside
of
a
paler
browner
hue. The
fore
extremity
on
the
underside
is
covered
with
a
coriaceous
granulose
integument
(the
granulosities
much
strongest
and
becoming
tubercular
at
the
fore
end),
which
forms
a
short
sheath,
covering
most
of
the
connecting
pedicle as well as the spiracular openings and the genital aperture,
For
the
peculiar
form
of
the
abdomen,
see
generic
characters
above
;
but
whether
this
is
only
specific
or
whether
generic,
it
is
hard to say in the absence of allied species. Hab. Salisbury,
Mashonaland,
8. Africa,
5000
feet,
Nov. 1898
to
Jan. 1899
(GA. K. Marshall), Hab. CYRTARACHNE
CONICA,
sp.
n.
(Plate
V.
figs.
1-1.) CYRTARACHNE
CONICA,
sp. n. (Plate
V. figs. 1-1.) CYRTARACHNE
CONICA,
sp. n. (Plate
V. figs. 1-1.) Adult
female,
length
rather
over
3
lines,
or
8
mm.;
leneth
of
abdomen
23
lines,
width
34
lines. ; Cephalothorax
short,
slightly
longer
than
broad,
broadest
and Cephalothorax
short,
slightly
longer
than
broad,
broadest
and V,
0. PIOKARD-CAMBRIDGE
ON
EXOTIC
SPIDERS. [Jan. 15, 14 rounded
behind,
truncate
before;
the
profile-line
forms
a
con-
tinuous
curve;
the
lateral
marginal
impressions
at
the
caput
are
yery
slight. Colour
yellowish
brown. ay y
y
g
y
y
Eyes
small,
in
the
ordinary
Epeirid
position
;
the
four
centrals
form
as
nearly
as
possible
a
square,
its
posterior
eyes
slightly
largest. The
lateral
pairs
are
close
to
the
anterior
corners
of
the
caput, minute; those of each pair are contiguous to each other and
form
nearly
a
straight
line
with
the
anterior
pair
ot
the
central
quadrangle. Pies)
ae quad a g e. es)
ae
Legs short, not very strong, 1 2) Ares devoid of spines, furnished
with
fine
hairs
only;
colour
brownish
yellow,
tinged
with
orange. Falces short, strong, subconical ; colour like that of the cephalo-
thorax. Maxille
and
!abium
normal
in
form,
and
similar
in
colour
to
the
cephalothorax,
perhaps
rather
paler. q
g
)
Legs short, not very strong, 1 2) Ares devoid of spines, furnished
with
fine
hairs
only;
colour
brownish
yellow,
tinged
with
orange. Falces short, strong, subconical ; colour like that of the cephalo-
thorax. Maxille
and
!abium
normal
in
form,
and
similar
in
colour
to the cephalothorax, perhaps rather paler. Sternum similar in colour to the legs. Abdomen
coriaceous,
large,
subtriangular,
broader
than
long,
rounded in front, the fore corners rounded, though scarcely to be
described
as
forming
distinct
prominences
;
upper
surface
consider-
ably
elevated
in
a
subconical
form;
colour
yellowish
white,
that
of
the
cone
tinged
with
yellow-brown. Near
the
middle
of
the
anterior
margin,
quite
visible
but
not
very
distinct,
are
three
sigilliform
markings
with
two
others
behind,
halfway
to
the
summit
of
the
conical
abdomen;
behind
these
last,
and
one
on
either side of the base of the cone, are two others similar, in a
transverse line, and forming a line equal in length to that formed
by the three anterior sigillz ; the upper part of the cone is encircled
by
some
indistinct
fine
darker
concentric
lines. CYRTARACHNE
CONICA,
sp.
n.
(Plate
V.
figs.
1-1.) The
underside
is dark dull yellow-brown, and from the outer margins of it sundry
fine dark lines issue upwards in groups of two or three, converging
until they meet about one-third of the way towards the top of the
cone. Genital
process
broad
and
very
characteristic
m
form. (
) Hab. Singapore
(R. Shelford). Hab. Singapore
(R. Shelford). Fam.
SALTICID
&.: Genus
Sanricus
Latr. (sensu
restricto). V,
0.
PICKARD-CAMBRIDGE
ON
EXOTIC
SPIDER The
legs
have
a
longitudinal
red-brown
streak
on
the
outer
side
of
the
femora
of
the
first
pair,
and
a
blackish
one
on
the inner side of those of the fourth pair, a white line also runs
along
the
side
of
the
tibie
and
metatarsi;
the
tarsi
of
the
first
and second pairs are white; the general colour of the legs is dull
orange-yellow. The abdomen is joined to the thorax by a distinct jointed pedicle ;
it
is
of
an
oval
form,
broadest
behind
and
pointed
in
front,
and
without
any
lateral
transverse
constriction. It
is
of
a
dull
yellowish hue tinged with reddish ; on either side towards the hinder
extremity is a large black spot ; along the middle of the upperside
on
the
hinder
half
are
two
converging
rows
of
small
white
spots,
with some other white ones towards the fore extremity ; on each
side also of the fore half are two broadish, but not very strongly
defined,
oblique
brownish
stripes,
the
hinder
ones
meeting
at
an
angle in the middle and continued in the median line to the fore
end. The
genital
aperture
is
well
marked
and
of
a
very
charac-
teristic
form. Hab. Singapore. Sent
to
Mr. Shelford
by
Mr. H. N. Ridley,
Director
of
the
Botanic
Gardens,
Singapore. The
type
of
the
genus,
A. forticeps
Cambr. (Ceylon),
has
two
similar spots on the abdomen. The
type
of
the
genus,
A. forticeps
Cambr. (Ceylon),
has
two
similar spots on the abdomen. This
Spider
was
found
in
company
with
the
ant
Cecophylla
smaragdina,
the
habits
of
which
have
been
descrited
by
Mr. Ridley
(Journ. Asiat. Noc.,
Straits
Branch,
1890,
No. 42,
p. 345). This
Spider
was
found
in
company
with
the
ant
Cecophylla
smaragdina,
the
habits
of
which
have
been
descrited
by
Mr. Ridley
(Journ. Asiat. Noc.,
Straits
Branch,
1890,
No. 42,
p. 345). Genus
Amycrma
Sim.
(Amycle
Cambr.). AMYCIEA
LINEATIPES,
sp. n. (Plate
V. figs. 4-4
d.) AMYCIEA
LINEATIPES,
sp. n. (Plate
V. figs. 4-4
d.) AMYCIEA
LINEATIPES,
sp. n. (Plate
V. figs. 4-4
d.) Adult
female,
length
24
lines. g
This
Spider
is
nearly
allied
to
A. forticeps
Cambr. (P. Z. 38. Lond. 1873,
p. 122)
from
Ceylon,
and
bears
a
close
general
resemblance to it; but it may be distinguished by the shorter legs,
by the area of the four larger outer eyes, of which the anterior is of
the same length as the posterior side, and the four anterior eyes —
forming
a
straight
transverse
line. The
markings
on
the
legs,
palpi, and abdomen, and the form also of the abdomen, differ from
those of A. forticeps, though it is possible that this last character
may only be sexual. The palpi have a longitudinal black streak on their inner sides. 15 1901.] EV,
0. PICKARD-CAMBRIDGE
ON
EXOTIC
SPIDE Genus
Sanricus
Latr.
(sensu
restricto). Sternum
elongate,
narrow;
the
basal
joints
of
the
legs
are
articulated
around
it
on
the
same
plane,
the
first
two
pairs
with
their coxe almost contiguous on their inner sides. Abdomen
narrow,
elongate-oval,
strongly
and
broadly
constricted
towards
the
fore
extremity;
pedicle
as
long
as
the
caput,
two-
jointed,
the
posterior
joint
longest
and
set
in
a
circular
cavity
or
socket
at
the
extremity
of
the
abdomen. Colour
black,
a
little
paler at the constricted part, just below the sides of the constriction
white. Abdomen
narrow,
elongate-oval,
strongly
and
broadly
constricted
towards
the
fore
extremity;
pedicle
as
long
as
the
caput,
two-
jointed,
the
posterior
joint
longest
and
set
in
a
circular
cavity
or
socket
at
the
extremity
of
the
abdomen. Colour
black,
a
little
paler at the constricted part, just below the sides of the constriction
white. Hab. Singapore. Sent
by
Mr. H. N. Ridley
to
Mr. R. Shelford. EXPLANATION
OF
PLATE
V. Fig. 1. Cyrtarachne conica, 9 (p. 13). 1a, profile; 1%, eyes and falces from
in front; 1c, genital aperture. 2. Prosthesima albomaculata, 2 (p.11). 2a, profile; 24, eyes and falces
from in front; 2¢, genital aperture. (
) Fig. 1. Cyrtarachne conica, 9 (p. 13). 1a, profile; 1%, eyes and falces from
in front; 1c, genital aperture. 2. Prosthesima albomaculata, 2 (p.11). 2a, profile; 24, eyes and falces
from in front; 2¢, genital aperture. (
) 3. Titus lugens, 2 (p.18). 34a, profile; 54, eyes and falces from in front ;
3c, maxille, labium, and sternum; 3d, cephalothorax and eyes from
above and behind; 3¢, genital aperture. (
) g
p
4. Amyciea lineatipes, 9 (p. 14). 4a, profile; 44, eyes and falces from
in front; 4¢, eyes and cephalothorax from above and behind; 4d,
genital aperture. g
p
5. Ccophylla smaragdina (p. 15). (Ant with which Amyciea lineatipes
lives. )
(
) 6. Salticus attenuatus, 2 (p.15). 64a, profile; 66, cephalothorax and
connecting pedicle from above; 6c, genital aperture. (It is doubtful
whether this example is quite adult.) 6. Salticus attenuatus, 2 (p.15). 64a, profile; 66, cephalothorax and
connecting pedicle from above; 6c, genital aperture. (It is doubtful
whether this example is quite adult.) 3. Notes
on
the
Anatomy
of
Picarian
Birds—No. IV. On
the
Skeletons
of
Bucorvus
cafer
and
B. abyssinicus
;
with
Notes
on
other
Hornbills. By
Frank
E. Bepparp,
M.A.,
F.R.S.,
Prosector
and
Vice-Secretary
of
the
Society. Genus
Sanricus
Latr.
(sensu
restricto). SALTICUS
ATTENUATUS,
sp. n. (Plate
V,
figs. 6-6
c.) SALTICUS
ATTENUATUS,
sp. n. (Plate
V,
figs. 6-6
c.) SALTICUS
ATTENUATUS,
sp. n. (Plate
V,
figs. 6-6
c.) (
)
g
(
g
)
Cephalothoraxv
oblong,
narrowing
gradually
to
the
posterior
end,
which
is
truncate. Caput
flat,
rather
longer
than
the
thorax
and
rather longer than broad, divided from the thorax by a deep inden-
tation
or
constriction. Colour
deep
black-brown
on
the
thorax
;
eaput black; in the constriction are three short lines or patches of
white hairs, one on each side and one in the middle. (
)
g
(
g
)
Cephalothoraxv
oblong,
narrowing
gradually
to
the
posterior
end,
which
is
truncate. Caput
flat,
rather
longer
than
the
thorax
and
rather longer than broad, divided from the thorax by a deep inden-
tation
or
constriction. Colour
deep
black-brown
on
the
thorax
;
eaput black; in the constriction are three short lines or patches of
white hairs, one on each side and one in the middle. Hyes normal, ocular area longer than broad. Legs
rather
short,
furnished
with
short
hairs,
and
a
few
fine
spines
in
pairs
beneath
the
tibie
of
the
first
and
second
pairs
;
these are of a pale yellow colour; the outer side of the tibie, meta-
tarsi, and tarsi of the first pair, and of the tibie and metatarsi of the
second,
marked
with
a
longitudinal
black
stripe;
the
coxee,
femora,
tibiz,
and
base
of
the
metatarsi
of
the
third
pair
black,
the
rest
pale
yellow;
the
fourth
pair
have
the
coxze
pale
yellow,
with
an
exterior
longitudinal
black
line
on
the
outer
side,
and
the
femora
and
tibiz
black,
the
metatarsi
and
tarsi
being
yellow. Palpt
yellowish
;
radial
joint
blackish
;
digital
joint
large,
oval,
flattish
and
tumid. n. 15, HE,
BEDDARD
ON 16 Fulees vather shorter than the caput, strong, prominent, of a dull
yellow-brown
colour. es yellow brown
colour. es
Mazille
dull
blackish,
extremities
pale
yellowish. y
Mazille
dull
blackish,
extremities
pale
yellowish. Labium
dull
black,
apex Labium
dull
black,
apex Sternum
elongate,
narrow;
the
basal
joints
of
the
legs
are
articulated
around
it
on
the
same
plane,
the
first
two
pairs
with
their coxe almost contiguous on their inner sides. 1 “ On some Points in the Structure of the Hornbills,” P. ZS. 1889, p. 587.
‘Untersuchung. zur Morph. d. Vogel,’ Amsterdam, 1888.
Bronn’s ‘ Ordnungen des Tierreichs, Aves.
‘The Structure and Classification of Birds,’ Longmans, 1896.
Proc.
Zoou.
Soc.—1901,
Vou.
I.
No.
II.
2
ro
Nn (Text-figures
2-5.) The opportunity of comparing the two known species of Ground-
Hornbills, Bucorvus cafer and B. abyssinicus, has been afforded me
by the death of an example of each of them during the past year
in
the
Society’s
Gardens. I
have
taken
the
opportunity
of
com-
paring
the
structure
of
the
genus
Bucorvus
with
several
forms
of arboreal Hornbills, of which I possess skeletons, with a view of
separating
from
a
general
description
of
Bucorvus
those
features
in which it is different from other Hornbills, and which are there-
fore
distinctive
characters
of
the
genus,
or
subfamily
as
some
would prefer to regard it. T
limit
myself
in
the
present
communication
to
the
skeleton, —
17 ANATOMY
OF
PICARIAN 1901. since
L
have
nothing
new
to
add
to
my! earlier
account
of
the
muscles and the viscera of Bucorvus and other genera of Hornbills,
or
to
Prof. Fiirbringer’s*
almost
contemporaneous
investigations
upon
the
same
subject. The
latter
work
contains,
naturally,
a
number of facts relating to the skeleton of the Hornbills in general,
as
well
as
of
Bucorvus;
but
these,
as
might
be
expected,
deal
chiefly
with
the
shoulder-girdle. Another
source
of
information
concerning the bones ot the Bucerotide is Mr. Hyton’s ‘ Osteologia
Avium,’
which
work
includes
figures
of
the
skeletons
of
Bucorvus
and of a few other forms together with some quite brief notes in
the
text. The
family
is
of
course
not
neglected
in
the
general
works
of
Dr. Gadow
°
and
myself*
upon
bird-anatomy. There is, however, at least so far as | am aware, no account of
the bones of the two species with which I deal here—no comparison
of the two forms. Vertebral
Column.—Only
two
features
in
the
vertebral
column
distinguish
the
two
species
of
Bucorvus. In
the
first
place,
the
relative
lengths
of
the
several
regions
differ:
in
Bucorvus
cafer
the
cervical
series
(13
vertebre
in
both
birds)
is
shorter
than
that
of
B. abyssinicus. The
total
difference
of
length
is
rather
more
than
an
inch,
and
each
individual
vertebra
is
distinctly
shorter
than
the
corresponding
one
of
the
other
species. This
is
not
an
expression
of a smaller-sized bird, since the dorsal vertebre are of exactly the
same
length
collectively
and
individually
in
the
two
species. Nor
is
there
any
difference
except
the
very
minutest
in
the
lengths
of
the
sacral
and
caudal
series. The
last
cervical
vertebra
of
B. (Text-figures
2-5.) cafer
has a transverse process which is slightly more rib-like than is that
of
B. abyssinicus. Though
firmly
welded
to
its
vertebra,
the
homologue of the rib is more slender, as is the case in those birds
where it is a free structure. The
second
point
of
difference
concerns
the
presence
of
an
additional
rib
in
B. cafer
at
the
end
of
the
series. The
vertebra
bearing
that
rib
is
not,
however,
free
itself. The
rib
is
long
and
slender. Vertebrul
Column
of
Bucorvus
compared
with
other
Hornbills.—
The
great
breadth
and
excavation
below
of
the
cervical
vertebree
distinguish
Bucorvus
from
Buceros. There
are,
moreover,
thirteen
of them, while in Buceros the thirteenth vertebra bears a small but
movable
rib
on
each
side. In
Bucorvus
there
are
no
closely
approximated
catapophyses
;
in
Buceros
the
1ith
vertebra
has
a
pair
of
these. The
remaining
salient
characteristic
of
Bucorvus
is
the
slenderness
of
the
pygostyle,
which
might
be
expected
in
a
eround-living
bird. g
_
Sternum—The
only
difference
that
I
could
detect
between
the
sterna
of
the
two
species
was
that
in
B. abyssinicus
the
lateral
incision of the xiphisternum is not nearly so deep as in B. cafer. 2 [Jan. 15, MR. F. BE. BEDDARD 1(9
0) There
is
no
need
to
enter
into
comparisons
between
this
and
of
the
shoulder-girdle
in
the
Ground-Hornbills
and
the
arboreal
forms,
since
the
structure
and
relations
are
as
nearly
as
possible identical. This seems to show that use is a more important
factor
than
disuse
in
the
modification
of
organs,
since
the
hind
limbs
show
noteworthy
differences. other parts Skull.—Very
slight,
but
still
perfectly
recognizable
and
defin-
able,
differences
distinguish
the
skulls
of
the
two
species
of
Bucorvus (ef. text-figs. 2, 3). (
g
)
The most striking difference is, however, possibly a sexual one: in
B. cafer the bony prominence on which sits the casque of the bird
is much lower than it is in B. abyssinicus, and at the same time its
texture is decidedly more solid ; in B. abyssinacus this part of the
skull
is
formed
of
very
delicate
cancellated
bony
tissue
which
immediately
underlies
the
horny
casque. My
specimen
of
B. afer, Text-fig. 2. Text-fig. 2. Text-fig. 2. Skull of Bucorvus abyssinicus, 6. (X 4.) Skull of Bucorvus abyssinicus, 6. (X 4.) however,
is
a
female
bird;
the
skeleton
of
B. abyssinicus
belongs
to a male. (Text-figures
2-5.) however,
is
a
female
bird;
the
skeleton
of
B. abyssinicus
belongs
to a male. _ When the two skulls are viewed from above, they can be readily
distinguished
by
the
greater
breadth
of
that
of
B. cafer. The
widest
part
is
Just
behind
the
orbits. The
measurements
in
the
two species are as follows: Bucorvus
cafer...... Length
206
mm.;
breadth
63
mm. LB HOSSHMOUIS <4 8 6 Length 203 mm. ; breadth 59 mm. A very small fragment of the tip of the beak in B. abyssinicus
was,
however,
broken
off
and
lost. This
would
therefore
increase
the length of the skull in that species, and thus render the propor-
tions a little more striking than is apparent from the measurements
The greater breadth of the skull in B. cafer can, however, be well
appreciated
without
any
measurements
at
all. h d
f
h h
h
k ll
f
h
d
b ll A
third
feature
in
which
the
skulls
of
the
two
Ground-Hornbills 19 1901. ANATOMY
OF
PICARTAN differs
is
in
the
form
of
the
occipital
condyle. In
B. cafer
it
is
a
little
more
elongated
transversely
than
in
B. abyssinicus. As
will
be
seen
from
an
inspection
of
the
accompanying
drawings
(text-figs. 2, 3), the outline of the orbit is a little different in the
two
species. p
In
other
respects
the
two
skulls
can
hardly
be
distinguished. Characteristics
of
the
Skull
of
Bucorvus.—These
can
be
arrived
at
frem a comparison of the two species of Bucorvus with a skull of
Buceros
rhinoceros,
which
I
shall
take
as
a
type
of
the
arboreal
Hornbills,
indicating
at
the
same
time
such
divergences
as
are
exhibited
by
other
arboreal
Hornbills. In
comparing
the
skulls
of
the
two,
the
first
striking
difference
between
the
two
genera
is
that
shown
by
the
cancellated
bone
which
fills
the
casque. This,
in Buceros, is solid behind where it projects back considerably over
the root of the skull; anteriorly it ends abruptly in a steep declivity Text-fig. 3. Text-fig. 3. Skull of Bucorvus cafer, 2. (x 4.) but
the
pterygoids
themselves
are
bowed
inwards opposite to these processes ; at the place where they should,
so
to
speak,
articulate
with
the
basipterygoid
processes
they
bear
a roughened outgrowth which seems to suggest the remains of a
pterygoid
articular
facet. A comparison of the dorsal aspect of the skull in the two genera
shows
several
points
of
divergence
in
the
two
types. The
greater
breadth of the cranium of Bucorvus is apparent, this bemg mainly
due to the projecting shelf of bone over the orbit, already referred
to. Furthermore
the
“
lacrymal”
ring
in
front
of
the
orbit
which
is absent or at least not so fully developed in Buceros, causes a
very sharp demarcation between the cranium and the face in Bu-
corvus, a distinction which is wanting in Buceros, where one region
gradually
fades
into
the
other. The
commencement
of
the
beak
region is quite as wide asthe anterior part of the orbit in Buceros ;
in
Bucorvus
it
is
very
plainly
much
narrower. The
contrast
1s
so
great that measurements are unnecessary to express the differences. g
y
p
The basal aspect of the skull of Bucorvus is in some respects
different
from
that
of
Buceros. In
the
first
place,
the
foramen
magnum in Bucorvus is much more distinctly upon the ventral sur-
face than in Buceros, where this foramen looks partly backwards. It
thus happens that the dorsal wall of the foramen is more apparent
on
a
dorsal
view
in
Buceros
than
it
is
in
Bucorvus. The
palatal
region too shows differences which are not without a certain interest
in
relation
to
the
connection
between
the
two
types
of
Hornbill. As
has
been
already
recorded
by
Fiirbringer,
the
Bucerotide
possess
basiptervgoid
processes,
These
are,
however,
rudimentary,
and are far from being in contact with the pterygoids. In Buceros
not only are there present a pair of somewhat jagged rudimentary
basipterygoid
processes. but
the
pterygoids
themselves
are
bowed
inwards opposite to these processes ; at the place where they should,
so
to
speak,
articulate
with
the
basipterygoid
processes
they
bear
a roughened outgrowth which seems to suggest the remains of a
pterygoid
articular
facet. So
exactly
does
the
position
of
this
facet
correspond
to
the
Dasipterygoid
process,
that
if
the
bones
could
be
forcibly
pushed
together
they
would
meet
at
those
points. Skull of Bucorvus cafer, 2. (x 4.) Skull of Bucorvus cafer, 2. (x 4.) Skull of Bucorvus cafer, 2. (x 4.) which
is
formed
of
finely
and
beautifully
cancellated
bone. In
Bucorvus,
cn
the
contrary,
whether
the
cancellated
bone
shows
exteriorly
or
not,
the
whole
bony
process
slopes
gradually,
first
upwards
and
then
downwards
in
an
even
curve,
there
being
no
abrupt
demarcation
between
it
and
the
maxille
in
front. In
Buceros
a
delicate
shelf
of
bone
slightly
projecting
marks
the
an-
terior
boundary
of
the
bony
part
of
the
casque. When
the
skulls
of
the
two
Hornbills
are
viewed
laterally,
notable
differences
are
obvious. The
walls
of
the
brain-case
are
seen
to
arise
in
Buceros
to a considerable distance above the orbit. The top of the skuli is
in
fact
swollen
and
convex. In
Bucorvus,
on
the
other
hand,
the
top
of
the
skull
is
almost
flat
and
it
is
contimued
to
form
a
projecting
shelf
over
the
orbit,
which
thus
stands
out
more
con-
spicuously
from
the
sides
of
the
head
than
im
Buceros. The
prominence
of
the
orbit
in
Bucorvus
is
further
emphasized
by
the
prolongation
downwards
in
front
of
the
lacrymal
region
of
a
plate
Ox which
is
formed
of
finely
and
beautifully
cancellated
bone. In
Bucorvus,
cn
the
contrary,
whether
the
cancellated
bone
shows
exteriorly
or
not,
the
whole
bony
process
slopes
gradually,
first
upwards
and
then
downwards
in
an
even
curve,
there
being
no
abrupt
demarcation
between
it
and
the
maxille
in
front. In
Buceros
a
delicate
shelf
of
bone
slightly
projecting
marks
the
an-
terior
boundary
of
the
bony
part
of
the
casque. When
the
skulls
of
the
two
Hornbills
are
viewed
laterally,
notable
differences
are
obvious. The
walls
of
the
brain-case
are
seen
to
arise
in
Buceros
to a considerable distance above the orbit. The top of the skuli is
in
fact
swollen
and
convex. In
Bucorvus,
on
the
other
hand,
the
top
of
the
skull
is
almost
flat
and
it
is
contimued
to
form
a
projecting
shelf
over
the
orbit,
which
thus
stands
out
more
con-
spicuously
from
the
sides
of
the
head
than
im
Buceros. The
prominence
of
the
orbit
in
Bucorvus
is
further
emphasized
by
the
prolongation
downwards
in
front
of
the
lacrymal
region
of
a
plate
Ox Ox n. 15, EH. BEDDARD
ON 20 of bone to form a projecting ridge which joins the jugal arch behind. Skull of Bucorvus cafer, 2. (x 4.) his
renders
the
margins
of
the
orbit
perfectly
visible
when
the
skull
is
viewed
directly
from
the
front. In
Buceros
there
is
no
such ridge, and the orbit is invisible when the skull is looked at
from
in
front. ies
RJ J
The narial aperture is double on each side in Bucorvus as rt 1s in,
for
example,
the
Toucans. Hach
of
the
two
apertures
into
which
the originally single aperture is divided in this genus 1s of a rather
elongated
oval
outline. In
Buceros
the
single
narial
orifice
is
circular
in
outline. A; A comparison of the dorsal aspect of the skull in the two genera
shows
several
points
of
divergence
in
the
two
types. The
greater
breadth of the cranium of Bucorvus is apparent, this bemg mainly
due to the projecting shelf of bone over the orbit, already referred
to. Furthermore
the
“
lacrymal”
ring
in
front
of
the
orbit
which
is absent or at least not so fully developed in Buceros, causes a
very sharp demarcation between the cranium and the face in Bu-
corvus, a distinction which is wanting in Buceros, where one region
gradually
fades
into
the
other. The
commencement
of
the
beak
region is quite as wide asthe anterior part of the orbit in Buceros ;
in
Bucorvus
it
is
very
plainly
much
narrower. The
contrast
1s
so
great that measurements are unnecessary to express the differences. The basal aspect of the skull of Bucorvus is in some respects
different
from
that
of
Buceros. In
the
first
place,
the
foramen
magnum in Bucorvus is much more distinctly upon the ventral sur-
face than in Buceros, where this foramen looks partly backwards. It
thus happens that the dorsal wall of the foramen is more apparent
on
a
dorsal
view
in
Buceros
than
it
is
in
Bucorvus. The
palatal
region too shows differences which are not without a certain interest
in
relation
to
the
connection
between
the
two
types
of
Hornbill. As
has
been
already
recorded
by
Fiirbringer,
the
Bucerotide
possess
basiptervgoid
processes,
These
are,
however,
rudimentary,
and are far from being in contact with the pterygoids. In Buceros
not only are there present a pair of somewhat jagged rudimentary
basipterygoid
processes. Skull of Bucorvus cafer, 2. (x 4.) Bucorvus
shows
a
further stage of degeneration, which fits in well with the presump-
tion
that
it
is
a
later
type
than
Buceros. The
basipterygoid
pro-
cesses
are
distinctly
more
rudimentary,
and,
indeed,
they
are
only
just
recognizable
in
B. abyssinicus. The
pterygoids
are
straight,
and
are
not
at
all
bowed
inwards
towards
the
basipterygoid
pro-
cesses. In
the
place
of
what
I
have
regarded
as
an
articular
facet upon the pterygoids in Buceras, there is in Bucorvus a thin,
large,
upwardly
directed
lamellar
process
of
bone. This,
however, 1901. ANATOMY
OF
PICARIAN 21 is only to be-seen in B. cafer. I consider this plate of bone arising
from
the
pterygoid
to
be
the
homologue
of
the
rudimentary
arti-
cular facet of Buceros, but increased in a different direction. As is
sometimes seen with degenerating Oinen, it has as it were run to
seed. Thatit
does
not
point
toward
the
basipterygoid
may
perhaps
be
put
down
to
the
straightening
and
consequent
rotation
of
the
pterygoid. The majority of these differences also hold good for other genera
of
arboreal
Hornbills. The
distinction
between
the
cancellated
bone which fills the casque, the maxilla, and between the posterior
and
anterior
regions
of
the
core
of
the
casque
is
apparent
even
in
the
almost
casque-less
Aceros. The
really
casque-less
Voccus
may
be
left
out
of
consideration. The
principal
feature
in
which
the skulls of other Hornbills are less marked than Buceros are the
lower
elevation
of
the. brain-case
and
the
comparative
straightness
of the pterygoids. Pelvis
of
Bucorvus
and
Buceros.—The
pelves
of
the
two
species
of Bucorvus agree exactly in the proportions of the pre-and of the
post-acetabular
regions. But
when
the
genus
is
compared
with
Buceros,
differences
appear. In
the
latter
genus
the
two
regions
of
the
pelvis
which
are
separated
by
the
antitrochanter
are
as
nearly as possible equal in length; in Bucorvus the posterior region
of
the
pelvis
is
rather
the
longer. This
difference
is
coupled
with
another,
viz.,
the
greater
depth
of
the
ischia
of
Buceros,
and
the
consequently
more
acute
angle
formed
by
the
pubes
with
the
longitudinal
axis. In
Bucorvus
the
pubes
slope
more
nearly
parallel
to
the
long
axis
ot
the
pelvis. One
cannot
but
put
down
this
difference
to
the
difference
in
mode
of
life
exhibited
by
the
two genera. Skull of Bucorvus cafer, 2. (x 4.) Hind
Limb.—Measurements
of
the
proportions
of
the
several
sections
of
the
hind
limb
in
the
two
species
show
some
slight
differences
which
are
perhaps
worthy
of
being
recorded. “Tn
Bucorvus
ubyssinicus
the
measurements
were
as
follows:
femur
110 mm. ; tibia 200 mm.; metatarsus 157 mm.; middle toe 90 mm. Of
B,
cafer
the
corresponding
figures
were
100;
SHS
Nee
Te
‘ Hind
limb
of
Bucorvus
and
Buceros.—It
‘is
of
course
well
enough
known
that
the
Ground-Hornbills
have
longer
legs
than
the
arbore
al
genera;
but
nevertheless
a
few
exact
measurements
may
be
useful. I
append
therefore
a
number
of
such
measure-
ments
(in
millim.),
which
have
been
taken
in
every
case
from
the
dried
skeleton
:— dried
skeleton
:
Femur. Tibia. Metatarsus. Middle
toe. Buceros
rhinoceros
...... 90
1D5;
62
Tht
Kthytidiceros
plicatus
.... 74
102
49
62
Dichoceros
bicornis
...... 108
141
al
80 It
is
plain
from
these
measurements
that
the
tibia
is
shorter
relatively
to
the
femur
in
the
flying
Horubills,
and
that
the
meta-
tarsus
in
the
same
birds
is
shorter
relatively
to
the
tibia
than
in
the
Ground-Hornbills. Taking
the
femur
in
all
cases
as
1,
the (Jan. 15, MR. F, E. BEDDARD ON THE Text-fie. 4. L ft f
t
f B
b
i i
(N t
i
) Text-fie. 4. Left foot of Bucorvus abyssinicus. (Nat. size_) 23 1091.] ANATOMY OF PICARIAN BIRDS. proportions
of
the
segments
of
the
hind
limb
in
Bucorvus
and
Buceros
will
be
(quite
roughly)
these
:— Bucorvus...... 1
2
We
1
IBCEROS
a
my
IL
13
=
1 Bucorvus...... 1
2
We
1
IBCEROS
a
my
IL
13
=
1 5. Left foot of Buceros rhinoceros. (Nat. size.) 5. Left foot of Buceros rhinoceros. (Nat. size.) Jan,
15, E
ANATOMY
OF
PICARIAN
BIRDS. 24 A final point of some little interest concerns the bones of the
foot. Perching
and
walking
on
the
ground
are
clearly
two
very
different modes of using the feet, and we should therefore expect
to find some corresponding differences in the structure of the foot. As a matter of fact, such differences do occur in the two series of
Hornbills. In
Buceros
the
middle
metacarpal
is
if
anything
slightly
longer
than
that
of
the
second
toe,
while
the
fourth
metacarpal
is
about
one
half
of
the
length
of
the
two
metatarsals
of
the
middle
toes. Skull of Bucorvus cafer, 2. (x 4.) In
Bucorvus
the
second
metatarsal
is
slightly
longer,
and
also
rather
stouter,
than
the
third
metatarsal,
while
the
fourth
metatarsal
is
not
so
much
reduced
as
in
Buceros. It
is
clear
then,
that,
apart
from
the
differences
in
length
which
distinguish
the
genera,
the
prevailing
toe
in
the
bipedal
Bucorvus
is the second, which is really functionally the first toe, for the true
first toe is of course turned backwards. It is true that the third
toe is the longest ; but nevertheless the increased length of the
second
metatarsal
gives
to
that
toe
a
preponderance
in
the
foot. This
state
of
affairs
contrasts
with
that
observable
in
the
quadru-
pedal Ungulates, where it is the middle toe (or the two middle toes )
that
is
the
prevailing
one. In
correspondence
with
the
greater
length
of
the
second
metatarsal,
the
tibio-tarsus
is
more
strongly
developed on that side and projects beyond the rest of the bone,
the
articular
surface
of
which
is
therefore
oblique
to
the
trans-
verse axis of the leg. In Buceros the line of articulation is exactly
transverse. This
will
be
apparent
from
the
drawings
exhibited
(text-figs. 4 & 5, pp. 22 & 23). The last-mentioned feature is not,
however,
distinctive
of
Bucorvus;
for
in
Rhytidiceros
the
same
obliquity
at
the
end
of
the
tibio-tarsus
occurs. In
Voccus,
more-
over,
in
addition
to
the
obliquity,
the
second
metatarsal
is
the
longest. From
this
description
of
certain
features
in
the
anatomy
of
Bucorvus, the osteological characters of the genus and of the twe
subspecies B. cafer and B. abyssinicus can be formulated :— Genus
Bucorvus.—Cerviecal
vertebrae
18,
short
and
broad,
with
concave
centra
and
transverse
processes
forming
a
gutter
beneath. No
catapophysial
canal
or
approach
towards
one. Pygostyle
comparatively
rudimentary. Skull
flat
above,
with
marked
shelf-like
supraorbital
plates. Foramen
magnum
ventral
in
position. Pterygoids
straight. Basipterygoid
processes
rudimentary. Bony
core
of
casque
not
sharply
marked
off
from
maxilla
in
front. ;
Second
metatarsal
the
stoutest
and
longest:
end
of
tibio-
tarsus
oblique. ‘Tibia
twice
as
long
as
femur;
tibio-tarsus
one and a half times as long. B. cafer.—Neck
comparatively
short. |
Sternum
rather
deeply
notched
with
one
incision. Skull
broad
in
proportion
to
length. B. cafer.—Neck
comparatively
short. |
Sternum
rather
deeply
notched
with
one
incision. Skull
broad
in
proportion
to
length. B. cafer.—Neck
comparatively
short. |
Sternum
rather
deeply
notched
with
one
incision. Howes, George Bond. 1901. "January 15, 1901." Proceedings of the Zoological
Society of London 1901, 1–34. https://doi.org/10.1111/j.1469-7998.1901.tb08535.x. View This Item Online: https://www.biodiversitylibrary.org/item/98562
DOI: https://doi.org/10.1111/j.1469-7998.1901.tb08535.x
Permalink: https://www.biodiversitylibrary.org/partpdf/72531
Holding Institution
Smithsonian Libraries and Archives
Sponsored by
Biodiversity Heritage Library
Copyright & Reuse
Copyright Status: Public domain. The BHL considers that this work is no longer under
copyright protection. This document was created from content at the Biodiversity Heritage Library, the world's
largest open access digital library for biodiversity literature and archives. Visit BHL at
https://www.biodiversitylibrary.org. Howes, George Bond. 1901. "January 15, 1901." Proceedings of the Zoological
Society of London 1901, 1–34. https://doi.org/10.1111/j.1469-7998.1901.tb08535.x. View This Item Online: https://www.biodiversitylibrary.org/item/98562
DOI: https://doi.org/10.1111/j.1469-7998.1901.tb08535.x
Permalink: https://www.biodiversitylibrary.org/partpdf/72531 Skull of Bucorvus cafer, 2. (x 4.) Skull
broad
in
proportion
to
length. B. abyssinicus.—Neck
comparatively
long. Sternum
not
deeply
notched. Skull
narrower
in
proportion
to
length. B. abyssinicus.—Neck
comparatively
long. Sternum
not
deeply
notched. Skull
narrower
in
proportion
to
length. B. abyssinicus.—Neck
comparatively
long. Sternum
not
deeply
notched. Skull
narrower
in
proportion
to
length. This file was generated 1 April 2024 at 04:03 UTC Holding Institution Smithsonian Libraries and Archives Copyright & Reuse Copyright & Reuse
Copyright Status: Public domain. The BHL considers that this work is no longer under
copyright protection. This document was created from content at the Biodiversity Heritage Library, the world's
largest open access digital library for biodiversity literature and archives. Visit BHL at
https://www.biodiversitylibrary.org. This file was generated 1 April 2024 at 04:03 UTC
|
https://openalex.org/W2137872210
|
https://pure.au.dk/ws/files/50646749/1471_2156_13_44.pdf
|
English
| null |
Partitioning additive genetic variance into genomic and remaining polygenic components for complex traits in dairy cattle
|
BMC genomic data
| 2,012
|
cc-by
| 8,554
|
Abstract Background: Low cost genotyping of individuals using high density genomic markers were recently introduced as
genomic selection in genetic improvement programs in dairy cattle. Most implementations of genomic selection
only use marker information, in the models used for prediction of genetic merit. However, in other species it has
been shown that only a fraction of the total genetic variance can be explained by markers. Using 5217 bulls in the
Nordic Holstein population that were genotyped and had genetic evaluations based on progeny, we partitioned
the total additive genetic variance into a genomic component explained by markers and a remaining component
explained by familial relationships. The traits analyzed were production and fitness related traits in dairy cattle. Furthermore, we estimated the genomic variance that can be attributed to individual chromosomes and we
illustrate methods that can predict the amount of additive genetic variance that can be explained by sets of
markers with different density. Results: The amount of additive genetic variance that can be explained by markers was estimated by an analysis of
the matrix of genomic relationships. For the traits in the analysis, most of the additive genetic variance can be
explained by 44 K informative SNP markers. The same amount of variance can be attributed to individual
chromosomes but surprisingly the relation between chromosomal variance and chromosome length was weak. In
models including both genomic (marker) and familial (pedigree) effects most (on average 77.2%) of total additive
genetic variance was explained by genomic effects while the remaining was explained by familial relationships. Conclusions: Most of the additive genetic variance for the traits in the Nordic Holstein population can be
explained using 44 K informative SNP markers. By analyzing the genomic relationship matrix it is possible to predict
the amount of additive genetic variance that can be explained by a reduced (or increased) set of markers. For the
population analyzed the improvement of genomic prediction by increasing marker density beyond 44 K is limited. Keywords: Genomic variance, Polygenic variance, Chromosomes, Complex traits, Dairy cattle genetic markers selection (GS), can reduce the cost of running an intensive
breeding program due to potential reductions in the num-
ber of individuals tested for own or progeny performance,
a shorter generation interval [2], and simultaneously
greatly enhance the genetic gain from the program without
a concomitant extra increase in the accumulation of
inbreeding [3]. © 2012 Jensen 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. RESEARCH ARTICLE Open Access Abstract Currently, this has led to implementation of
GS methods in many large scale commercial dairy cattle
breeding programs [4-6]. Implementation in other animal
and plant species are being initiated and is expected to in-
crease considerably in the near future [7]. Just Jensen*, Guosheng Su and Per Madsen Just Jensen*, Guosheng Su and Per Madsen Jensen et al. BMC Genetics 2012, 13:44
http://www.biomedcentral.com/1471-2156/13/44 * Correspondence: Just.Jensen@agrsci.au.dk
Department of Molecular Biology and Genetics, Centre for Quantitative
Genetics and Genomics, Aarhus University, Research Centre Foulum,
DK-8830, Tjele, Denmark Background Low cost genotyping of individuals or families using gen-
omic markers with constantly increasing density is cur-
rently being introduced in genetic improvement programs
for agricultural animal and crop species. Use of dense gen-
omic markers can increase the accuracy of predicting addi-
tive genetic merit especially for selection of candidates that
do not yet have own or progeny records [1]. Such applica-
tion of dense genomic markers, usually called genomic * Correspondence: Just.Jensen@agrsci.au.dk
Department of Molecular Biology and Genetics, Centre for Quantitative
Genetics and Genomics, Aarhus University, Research Centre Foulum,
DK-8830, Tjele, Denmark The current industry standard in dairy cattle breeding
is use of 50 K chips such as the Illumina Bovine SNP50 Page 2 of 9 Page 2 of 9 Page 2 of 9 Jensen et al. BMC Genetics 2012, 13:44
http://www.biomedcentral.com/1471-2156/13/44 If not all genetic variance can be explained by markers
then, in order to ensure optimal predictions, the remaining
genetic variance should be accounted for in other ways. A
simple approach is to combine predicted breeding values
based on genomic information with traditional breeding
values based on pedigree using selection index theory [16]. An alternative method is to include both genomic and
pedigree relationships in the analysis simultaneously. Previ-
ous studies have report that a model including a residual
polygenic effect slightly increases reliability and reduce bias
in prediction of future records [17,18]. Such a method
requires the partitioning of genetic variance accounted for
by genomic information and remaining genetic variance
accounted for by pedigree relationships. BeadChip [8] but other options, ranging from low density
3 K chips to high density 800 K chips, are also commer-
cially available. Increasing the density of genetic markers
is expected to increase the amount of genetic variance that
can be explained by markers due to increased linkage dis-
equilibrium between markers and causative loci [9]. A fur-
ther step is the use of complete sequencing of individual
genomes either based on direct sequence data or based on
sequence data for selected individuals and imputing geno-
types for animals that has been genotyped with lower mar-
ker density In human genetics very high density chips have been
used in large scale studies [10]. However, genome wide as-
sociation studies (GWAS) have generally not been able to
identify individual genes that can explain a large propor-
tion of total genetic variance of complex traits recorded in
humans. Background A typical model trait in humans is height that
has been investigated in many studies. This is a trait mea-
sured with an accuracy that is comparable to the accuracy
of daughter yield deviations of dairy sires that are
progeny-tested using large daughter groups. Height in
humans is known to have a heritability around 0.8 [11]. In
several well designed large scale studies with the use of
very high density SNPs, numerous loci have been identi-
fied that are significantly associated with human height,
yet each of them typically only account for a very small
fraction of total phenotypic variance. Collectively these
loci are only responsible for up to 5% of the total pheno-
typic variance in human height [11,12]. This has lead to
discussions among human geneticists about the missing
heritability of complex traits [13]. Suggested explanations
have included dominance and epistatic interactions, geno-
type by environment interactions and common epigenetic
factors causing resemblance between relatives [14]. Recently, [19,20], proposed a one-step method combining
marker based genomic relationships and pedigree-based
relationships into a single relationship matrix. An import-
ant factor in this procedure is that marker-based and
pedigree-based relationship coefficients must be expressed
on the same scale, i.e. the variance of the genomic and the
classical additive genetic effects must refer to the same base
population, and the proportion of total genetic variance
explained by markers and the remaining genetic variance
must be known. Currently implemented multistep proce-
dures may also need to be on the same scale in order to en-
sure derivation of optimum combined predictions. The purpose of this study was to evaluate the amount of
additive genetic variation in production and fitness related
traits in dairy cattle, to quantify the amount of additive
genetic variation that can be explained using genomic
markers with different density, and to quantify the amount
of genomic variance that can be ascribed to individual
chromosomes. The value of increasing the density of mar-
ker information for predicting genetic merit was also
assessed using subsets of available markers. As mentioned above the total variance explained by pre-
viously identified causal loci is usually only a small fraction
of total genetic variance in the populations investigated. In
GWAS very stringent significance thresholds are necessary
due to the very large number of statistical tests that are con-
ducted when searching the whole genome using high dens-
ity SNP marker panels. Background This will only allow loci with large
effects to become statistically significant. However, [10]
showed that a considerable proportion of additive genetic
variance can be explained by a very large number of SNPs
and their effects can be predicted simultaneously using ap-
propriate statistical models. Such models also includes the
SNPs with small effects as long as they are associated with
the trait of interest This association may be either due to
SNPs being located within causative loci, being in linkage
disequilibrium (LD) with causative genes, or due to markers
tracing parts of familial additive genetic relationships among
genotyped animals [15]. These results are well in line with
results from dairy cattle where a large proportion of additive
genetic can be explained using dense markers. Methods Data on deregressed proofs (DRP) were used as response
variable in the present study, which were derived from the
Nordic Holstein genetic evaluations official spring 2011
run. Production traits included milk production (milk), fat
production (fat), protein production (protein). Fitness
related traits included female fertility (fertility), other dis-
eases than mastitis (health), and mastitis (mastitis). The
specific trait definitions and recording procedures are
detailed
in
http://www.nordicebv.info/Forside.htm
and
[21]. The Nordic Genetic Evaluation (NAV) system for
dairy cattle includes all recorded dairy cattle in Denmark,
Sweden and Finland and published proofs is standardized
so that the variance of predicted breeding values for bulls
belonging to a recent two birth year cohort was 100. Since
the reliabilities of published proofs vary among traits the
variance of the deregressed proofs also vary among the Page 3 of 9 Jensen et al. BMC Genetics 2012, 13:44
http://www.biomedcentral.com/1471-2156/13/44 accounted for by markers on a specific autosomal chromo-
some and go is the vector of additive genetic effects
accounted for by markers on all remaining chromosomes. 1 is a vector of ones and Za and Zg are incidence matrices
relating observations in y to additive genetic effects in a
and in gx, gc or go, respectively. Subscript x on vectors in
different models indicates that definitions vary with model. Due to computational constraints specific effects of mar-
kers on single chromosomes was estimated for only one
chromosome at a time, and each analysis included effects
of markers on the specific chromosome and the combined
effects of markers on all other chromosomes. Therefore
model (4) was run one time for each trait and for each
chromosome. traits analyzed. The deregression removes the shrinkage of
the published proofs and the DRP, therefore, is similar to
progeny group means corrected for all non-genetic effects
influencing the traits analyzed. The ratios of variance com-
ponents are dependent of the accuracy of the individual
DRP. All estimates of variance ratios etc. are shown at the
average reliability of the bulls included in the study. The deregressed proofs were merged with marker
records of individual bulls that were typed using Illumina
Bovine SNP50 BeadChip (Illumina, San Diego, California,
US). Only bulls with both genotype records and dereg-
ressed proof for at least one trait in the database were
included in the analysis. A summary of the data used are
shown in Table 1. Methods In total there were 5217 bulls which
were born in the period from 1989 to 2006. The parameter μ was considered as a fixed effect in all
models and all other effects were assumed random nor-
mally distributed effects with variances var ax
ð
Þ ¼ Aσ2
a ,
where A is the additive genetic relationship matrix com-
puted from the full pedigree, and var gx
ð
Þ ¼ Gσ2
g, where A total of 47152 SNP markers were available in the raw
marker data, and after removing markers with minor allele
frequency (MAF) < 0.01 and non-informative markers that
were a simple linear function of another marker, a total of
44012 markers remained for analysis. A sire-dam pedigree
of all bulls included in the analysis was constructed from
the official pedigree file from NAV (http://www.nordicebv. info). The pedigree file included a total of 42144 animals
and most bulls were traced to the middle of the previous
century or earlier. G ¼ M % P
ð
Þ M % P
ð
Þ
0
2 P
m
j¼1
pj 1 % pj
"
#
ð5Þ ð5Þ In (5) M is an allele sharing matrix containing the num-
ber of copies of the second allele, and P is a matrix con-
taining twice the population frequency of the second
allele, i.e.2pj, and m is the number of marker loci included
in computation of G. The division with 2Pm
j¼1pj 1 % pj
"
#
in (5) is included to ensure that the scale of A and G are
comparable but will otherwise not influence the predic-
tions from the model but only the scale of model para-
meters. Finally, var e
ð Þ ¼ Dσ2
e
where D is a diagonal
matrix containing weights proportional to the effective
number of records in each DRP. The linear models (1)-(4)
are based on different ways of describing the relationship
among animals. For genomic relationships the methods
used are detailed in [16]. In short, the relationship matrix
A, based on pedigree information uses probability of iden-
tity by descent, whereas the genomic relationship matrix
G based on marker information use probability of identity
by state. Analysis of genomic relationships Randomly sample 2 N SNPs across the genome and
divide them in two groups of equal size. 2. Calculate Gm using then SNPs in group one and
calculate Gt using the SNPs in group two, assuming
that the SNPs in group two are the causal variants. 3. Use linear regression ð6Þ gtjk ¼ α þ βgmjk þ e
ð6Þ for j ≤k. The diagonal dominance of Gt and Gm is
removed by subtracting 1.0 from the diagonal elements
before estimating α and β. In order to separate effects of polygenic/familial gen-
etic relationships from genomic relationships model (3)
were run. In this model the covariance among animals
due to additive genetic relationships and due to genomic
relationships (markers) both were included. Averaged
across all traits total genetic variance estimated in model
(3) was 101.7% of total genetic variance estimated in the
animal model (1) with a range from 98.4% for protein to
106.3% for mastitis (computed from results in Table 2). Model (3) is thus able to explain all genetic variance in
the population and estimates of total genetic variance
are essentially identical to estimates from the classical
individual animal model, which is expected to yield un-
biased estimates of additive genetic variance. Averaged This procedure is repeated for different N and the re-
lation between β and N can be determined empirically. To obtain an unbiased estimate of Gt we want E
Gt G'
m
$$
#
¼ G'
m
"
. This can be accomplished by comput-
ing: G'
m ¼ β Gm % I
ð
Þ þ I
ð7Þ ð7Þ The genomic covariance matrices are diagonally dom-
inant with diagonal elements close to unity. If all diag-
onal elements in Gmis unity the adjustment in (7)
corresponds to adjusting estimated variance components
by β. In other words the estimate of genetic variance
obtained from model (2) is biased downwards with an
amount proportional to β. Models The data were analyzed using the following models: y ¼ 1μ þ Zaa1 þ e1
ð1Þ
y ¼ 1μ þ Zg g2 þ e2
ð2Þ
y ¼ 1μ þ Zg g3 þ Za a3 þ e3
ð3Þ
y ¼ 1μ þ Zg gc þ Zg go þ Za a4 þ e4
ð4Þ y ¼ 1μ þ Zaa1 þ e1
ð1Þ
y ¼ 1μ þ Zg g2 þ e2
ð2Þ
y ¼ 1μ þ Zg g3 þ Za a3 þ e3
ð3Þ
y ¼ 1μ þ Zg gc þ Zg go þ Za a4 þ e4
ð4Þ y ¼ 1μ þ Zaa1 þ e1
ð1Þ
y ¼ 1μ þ Zg g2 þ e2
ð2Þ
y ¼ 1μ þ Zg g3 þ Za a3 þ e3
ð3Þ
y ¼ 1μ þ Zg gc þ Zg go þ Za a4 þ e4
ð4Þ ð1Þ ð4Þ where μ is the general mean, ax is the vectors of additive
genetic effects not accounted for by genetic markers (for
model 1 this reduces to the classical individual animal
model since no markers are included in the model), gx is
the vector of the additive genetic effects accounted for
by markers, gc is the vector of additive genetic effects Table 1 Mean and standard deviation of deregressed bull
proofs Table 1 Mean and standard deviation of deregressed bull
proofs
Abbreviation
Trait
n
mean
Sd
Milk
Milk yield
4398
97.41
13.21
Fat
Fat yield
4398
96.99
12.23
Protein
Protein yield
4398
95.44
14,55
Fertility
Female fertility
4415
99.44
16.90
Health
Health index
4240
96.69
19.22
Mastitis
Mastitis resistance
4398
95.98
11.70 The number of markers included in computation of G
in models (2) and (3) were 44012. The same markers
were used in model (4) but markers were split into mar-
kers for one chromosome at a time and the markers on
all other chromosomes pooled such that two genomic
relationship of same size were computed. Model (4) was
used in this way for all 29 autosomal chromosomes. All analysis including estimation of variance compo-
nents using Restricted Maximum Likelihood were con-
ducted using the DMU software [22,23]. Page 4 of 9 Jensen et al. BMC Genetics 2012, 13:44
http://www.biomedcentral.com/1471-2156/13/44 Jensen et al. BMC Genetics 2012, 13:44
http://www.biomedcentral.com/1471-2156/13/44 Analysis of genomic relationships Table 2 Additive genetic and genomic variances for
production and fitness traits estimated in models 1, 2, 3
and 4 Table 2 Additive genetic and genomic variances for
production and fitness traits estimated in models 1, 2, 3
and 4 Model 1
Model 2
Model 3
Model 4
Trait
^σa2
VR1
aÞ
^σg2
VR1
gÞ
^σg2
^σa;2
VR1
aþgÞ
P^σa2
Milk
138.24
0.92
134.18
0.88
119.49
20.87
0.93
115.3
Fat
113.10
0.91
109.33
0.87
93.61
22.36
0.94
90.5
Protein
143.16
0.97
132.99
0.88
106.67
34.26
0.96
103.0
Fertility
151.74
0.78
142.42
0.74
110.38
40.10
0.78
106.5
Health
141.57
0.65
136.70
0.63
101.84
42.60
0.66
98.4
Mastitis
99.19
0.82
97.30
0.79
81.77
23.67
0.85
79.0
1) Ratio of genetic variance in model over total variance. Analysis of genomic relationships used as reference in the standardization predicted breed-
ing values. The ratio of additive genetic variance over
the total variance (VR) corresponds to a heritability of
the DRP. DRP are basically progeny group means so it
would be expected that the proportion of additive gen-
etic variance in DRP increases with increasing progeny
group size. In general dairy bulls in the Nordic countries
are tested on large groups of progeny. For the produc-
tion traits, VR were in the range 0.92 for milk to 0.97
for protein, which clearly indicate that the DRP have
high accuracy. For the fitness traits VR were between
0.75 for health and 0.82 for mastitis. The lower level of
VR for the fitness traits is due to the lower underlying
heritability of such traits since they are based on the
same progeny groups as the production traits. Additive genetic differences between individuals are due
to, generally unknown, causative genes. If the genotypes
at all causative loci were known, the true genomic rela-
tionship matrix (Gt) with regard to the trait of interest
could be computed based on all the causative loci. In
practice this is not possible and instead we compute G
based on the marker data only and here we name this
Gm. The accuracy of Gm to describe the genetic covari-
ance among individuals sharing the same causative genes
(Gt) depends on the linkage disequilibrium between the
markers and the causative genes. The accuracy of the
genomic relationship matrix can be assessed using the
procedure of [10] who regard Gm as an estimate of Gt. The procedure includes the following steps: For all traits analyzed, the genetic (total genomic) vari-
ance estimated in model (2) was lower than the additive
genetic variance estimated using the classical individual
animal model (1). The reason for this is that the genomic
relationship matrix (G) do not trace all relationships due
to sharing of causative alleles. However, the difference is
small and generally the genomic information accounts for
between 92% and 98% of the total additive genetic vari-
ance depending on the trait in question. These results are
well in line with results of [17] and [16] who used regres-
sions on future data to validate their models. 1. Randomly sample 2 N SNPs across the genome and
divide them in two groups of equal size. 1. Variance components A summary of the estimated variance components from
all models (1) to (4) are shown in Table 2. Estimates of
additive genetic variance using a classical individual ani-
mal model (model (1)) ranged from 99.19 to 151.74 on
the deregressed scale from the Nordic Genetic Evalu-
ation of Dairy Cattle. The scale of the deregressed proofs
are somewhat arbitrary since it depends on the accuracy
of breeding values for the bulls included in the cohort Jensen et al. BMC Genetics 2012, 13:44
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http://www.biomedcentral.com/1471-2156/13/44 Jensen et al. BMC Genetics 2012, 13:44
http://www.biomedcentral.com/1471-2156/13/44 over the six traits 77.2% of total genetic variance esti-
mated in model (3) is attributed to genomic relation-
ships among bulls and the remaining 22.8% is attributed
to familial genetic relationships among bulls traced via
the additive genetic relationship matrix. For the produc-
tion traits this percentage varied between 75% and 85%
and for the fitness related traits it varied between 71%
and 78% (percentages computed from Table 2). This
indicates that the genomic relationship matrix G is able
to catch a large proportion of the genetic variation in
the population when based on the SNP density used in
the current study. The estimates of variance due to individual chromosomes
were then summed over the models. For all traits the sum
of variances due to individual chromosomes is slightly
smaller than the total genomic variance for each trait in
model (3). The sum of chromosomal variance as a per-
centage of the total genomic variance estimated in model
(3) varied between 96% and 97% for all traits (Results not
shown). This indicates that covariance between genomic
effects on different chromosomes is positive but weak. Estimates of variance components due to individual
chromosomes are shown in Figure 1. The estimates are
plotted against the length of individual chromosomes
measured as the number of nucleotides (in Mb) between
first and last marker on each chromosome. Note that
the scale of the estimated variances varies by trait in Fig-
ure 1. Very large variances for milk and fat were found
on chromosome 14 where it is known that a gene of
very large effect segregates in the Holstein population
[24]. As can clearly be seen from the figure the relation
between chromosomal variance and chromosome length
is weak. Discussion and conclusions Figure 2 Expected proportion of total additive genetic variance
traced by increasing number of markers. The records analyzed in this paper were DRPs which were
derived from the routine genetic evaluations of dairy cattle
in Denmark, Sweden and Finland. Such DRPs are similar
to progeny group means adjusted for non-genetic effects. Therefore, a very large proportion of phenotypic variance
in analyzed DRP was due to additive genetic effects. For
all traits analyzed more than 92% of all additive genetic
variance could be explained using 44 K SNP markers. For
models including both polygenic additive genetic (pedi-
gree) effects and genomic (marker) effects, the latter
accounted for between 71% and 85% of all additive genetic
variance. Estimation of genomic variance of each individ-
ual chromosome showed that 96%–97% of all genomic
(marker) variance could be attributed to individual chro-
mosomes. Inclusion of polygenic familial effects in the
models ensured that potential linkage disequilibrium
across chromosomes was already taken into account. Most
of the additive genetic variance in the population analyzed
could be explained by genetic markers. The effect of redu-
cing (increasing) the number of genetic markers on gen-
omic prediction could be predicted by estimating the
accuracy of the genomic relationship matrix. it would be expected that chromosomal variance was
closely related to chromosome length. The R2 values for a
linear regression model of chromosomal variance on
chromosome length varied between 0.11 and 0.21 (Figure 1). This indicates that QTL are not evenly distributed over the
genome. This is clearly the case for milk and fat that had
very low R2 due to the major gene segregating as men-
tioned above. In general the relation between variance due
to individual chromosomes might also be dependent on the
model and method used as well as the underlying popula-
tion structure in the data analyzed. Variance components The estimates of total genomic variance followed
the expectation (proportional to β) quite closely when the
numbers of markers included in the genomic relationship
matrix were varied. Variance components If QTL were evenly distributed over the genome Additive genetic variance due to individual chromosomes
Results from analysis using model (4), where genomic
variance due to individual chromosomes were estimated,
is also summarized in Table 2. Only the genomic variance
summed over all chromosomes is shown. The chromo-
somal variance was estimated in 29 individual models
where the effect of an individual chromosome and the
combined effect of all other chromosomes were estimated. Figure 1 Estimates of genomic variance (y axis) due to individual chromosomes in relation to chromosome length (x axis) in Mb. Figure 1 Estimates of genomic variance (y axis) due to individual chromosomes in relation to chromosome length (x a Jensen et al. BMC Genetics 2012, 13:44
http://www.biomedcentral.com/1471-2156/13/44 Page 6 of 9 expected proportion of additive genetic variance explained
by markers reaches 0.96 when all 44 K markers were used. The additive genetic variance explained by different
number of markers were investigated using model (2) by
varying the number of markers used to compute the gen-
omic relationship matrix. Results averaged over all traits
are shown in Table 3. Results using all 44 K markers are
the same as in the analysis presented in Table 2 and are
repeated here in relative form for reference. As the num-
ber of markers included in the analysis decreases the pro-
portion of total additive genetic variance estimated as
genomic variance also decreases. As can be seen from
Figure 2. The estimates of total genomic variance followed
the expectation (proportional to β) quite closely when the
numbers of markers included in the genomic relationship
matrix were varied. Figure 2 Expected proportion of total additive genetic variance
traced by increasing number of markers. expected proportion of additive genetic variance explained
by markers reaches 0.96 when all 44 K markers were used. The additive genetic variance explained by different
number of markers were investigated using model (2) by
varying the number of markers used to compute the gen-
omic relationship matrix. Results averaged over all traits
are shown in Table 3. Results using all 44 K markers are
the same as in the analysis presented in Table 2 and are
repeated here in relative form for reference. As the num-
ber of markers included in the analysis decreases the pro-
portion of total additive genetic variance estimated as
genomic variance also decreases. As can be seen from
Figure 2. Amount of variance explained depending on number of
SNPs The procedure of [10] were used to estimate the proportion
of additive genetic variance that can be explained by mar-
kers. This expected proportion (β) is shown in (Figure 2) as
a function of number of markers included in the genomic
relationship matrix. The expected proportion of additive
genetic variance explained by markers is less than 0.85 when
the number of markers is below 5 K but increases rapidly
until between 15 K and 20 K markers is used in the estima-
tion of genomic variance. Further increases in the number of
markers only increase the expected proportion of additive
genetic variance explained by markers marginally. The Variance components Clearly more research on partitioning of genomic
variance into effects of individual chromosomes, chromo-
some segments or grouping of markers that are expected
to be located near causative genes etc. is needed. The amount of additive genetic variance that can be
explained by markers obviously cannot exceed the total
additive genetic variance in the population. The classical
individual animal model (1) yields unbiased estimates of
population additive genetic variance. Comparing results
from model (2) (genomic model) with results from model
(1) (animal model) clearly illustrates this. For the traits
milk and fat 97% of the additive genetic variance can be
explained by markers. The estimates of additive genetic
variance due to individual chromosomes clearly show that
these traits are influenced by a major gene that contributes
to the large proportion of additive genetic variance. Statis-
tical models fitting effects of individual marker genes
might be a better alternative in these cases. For protein and fertility the amount of additive genetic
variance that can be explained by markers is somewhat
lower than for milk and fat. This indicate that models used
for these traits should include an effect to account for addi-
tive genetic effects not accounted for by markers or alterna-
tively more markers are needed to ensure that most of the
additive genetic variance can be explained by markers. This
is clearly seen from the results of model (3) where both mar-
ker effects and classical polygenic familial additive genetic
effects were included in the model. However, in general, a
large proportion of the total additive genetic variance was
explained by markers in this study. This is in considerable
contrast to [10] who found that only 45% of additive genetic
variance in human height was explained using a marker
panel of 294,831 SNPs. One major difference between that
study and the current study is that the individuals in the
human study were nearly unrelated whereas the bulls in the
current study have very many relationships due to the highly
structured breeding programs used in commercial dairy cat-
tle. Consequently, the genomic variance (σ2
g ) include three
different sources of variation: segregation of observed SNPs
that cause functional differences in the genes affecting the
trait analyzed, co-segregation of markers and causative genes
due to physical linkage at population level and linkage dis-
equilibrium at family level. Variance components Most of the total phenotypic variance in the traits analyzed
was additive genetic due to the use of deregressed proofs,
which average out any dominance deviations across mul-
tiple daughters. Such proofs, of course, are functions of the Table 3 Expected (β) and estimated relative amount of additive genetic variance explained by different number of
markers
No of Markers
β
Estimated proportion of genetic variance explained by markers
44012
0.960
0.936
22006
0.930
0.918
11003
0.909
0.880 Table 3 Expected (β) and estimated relative amount of additive genetic variance explained by different number of
markers
No of Markers
β
Estimated proportion of genetic variance explained by markers
44012
0.960
0.936
22006
0.930
0.918
11003
0.909
0.880 cted (β) and estimated relative amount of additive genetic variance explained by different number of Jensen et al. BMC Genetics 2012, 13:44
http://www.biomedcentral.com/1471-2156/13/44 Page 7 of 9 Jensen et al. BMC Genetics 2012, 13:44
http://www.biomedcentral.com/1471-2156/13/44 Additive genetic variance due to individual chromosomes
When summing over chromosomes the estimates of gen-
omic variance due to individual chromosomes yielded total
genomic variances that were similar to the total genomic
variance in model (2) where this quantity was estimated
directly. The method therefore seems able to yield esti-
mates of genomic variance due to individual chromosomes. Surprisingly the estimates of variance due to individual
chromosomes only showed a weak relationship with
chromosome length (Figure 1). The results on individual
chromosomes are in contrast to results of [26] who found
strong
relations
between
chromosomal
variance
and
chromosome length. These authors used a matrix of kin-
ships between individuals as genomic covariance matrix
and estimated the variance due to individual chromosomes
as the difference between models using all markers and
models using all markers but the ones on the specific
chromosome in question. Such an indirect procedure is
necessary when using kinship matrices, because such
matrices normally are singular if they are based on a lim-
ited number of markers. We repeated our analysis using
methods as in [26]. Generally variance estimates due to
genetic similarity matrices are larger than variances due to
the genomic relationship matrix used in this paper because
of different scales. However, the correlations between esti-
mates of variances due to individual chromosomes and
chromosome length were even lower than those presented
in this paper and several estimates were negative! This
method, therefore, was not further pursued [27]. Variance components Also esti-
mate genomic variance due to individual chromosomes. They used a Bayesian approach that allocated equal prior
variance to each SNP. Chromosomes with many SNPs,
therefore, received more prior variance than chromosomes
with few(er) SNPs. They were able to identify genomic
regions with larger contribution to genomic variance due
to known major genes. However, most SNPs had small
effects and therefore there were strong associations be-
tween the amount of variance per chromosome and
chromosome length or equally number of SNPs per
chromosome. Effects of individual SNPs are composed of
effects due to co-segregation with closely linked QTL and
effects due to LD with QTL elsewhere in the genome, the
latter generated by familial relationships in the population. This effect tends to smooth all genomic variance over all
SNPs and may therefore give an unclear picture of how
much genomic variance can be ascribed to each chromo-
some. Clearly more research on partitioning of genomic
variance into effects of individual chromosomes, chromo-
some segments or grouping of markers that are expected
to be located near causative genes etc. is needed. Amount of variance explained by genomic markers procedures and definitions used in the recording system
and methods used in the genetic evaluation. For produc-
tion traits (milk, fat, and protein) the additive genetic vari-
ance was between 91% and 97% of total phenotypic
variance. The high level is expected because of the large
sizes of progeny group used in the testing procedures for
dairy bulls in all the Nordic countries and because of the
high heritability of the underlying traits. For the fitness
related traits (fertility, health, and mastitis) the relative
amount of additive genetic variance is lower reflecting the
lower accuracy of fitness related DRP. procedures and definitions used in the recording system
and methods used in the genetic evaluation. For produc-
tion traits (milk, fat, and protein) the additive genetic vari-
ance was between 91% and 97% of total phenotypic
variance. The high level is expected because of the large
sizes of progeny group used in the testing procedures for
dairy bulls in all the Nordic countries and because of the
high heritability of the underlying traits. For the fitness
related traits (fertility, health, and mastitis) the relative
amount of additive genetic variance is lower reflecting the
lower accuracy of fitness related DRP. Variance components Additive genetic variance due to individual chromosomes
When summing over chromosomes the estimates of gen-
omic variance due to individual chromosomes yielded total
genomic variances that were similar to the total genomic
variance in model (2) where this quantity was estimated
directly. The method therefore seems able to yield esti-
mates of genomic variance due to individual chromosomes. Surprisingly the estimates of variance due to individual
chromosomes only showed a weak relationship with
chromosome length (Figure 1). The results on individual
chromosomes are in contrast to results of [26] who found
strong
relations
between
chromosomal
variance
and
chromosome length. These authors used a matrix of kin-
ships between individuals as genomic covariance matrix
and estimated the variance due to individual chromosomes
as the difference between models using all markers and
models using all markers but the ones on the specific
chromosome in question. Such an indirect procedure is
necessary when using kinship matrices, because such
matrices normally are singular if they are based on a lim-
ited number of markers. We repeated our analysis using
methods as in [26]. Generally variance estimates due to
genetic similarity matrices are larger than variances due to
the genomic relationship matrix used in this paper because
of different scales. However, the correlations between esti-
mates of variances due to individual chromosomes and
chromosome length were even lower than those presented
in this paper and several estimates were negative! This
method, therefore, was not further pursued [27]. Also esti-
mate genomic variance due to individual chromosomes. They used a Bayesian approach that allocated equal prior
variance to each SNP. Chromosomes with many SNPs,
therefore, received more prior variance than chromosomes
with few(er) SNPs. They were able to identify genomic
regions with larger contribution to genomic variance due
to known major genes. However, most SNPs had small
effects and therefore there were strong associations be-
tween the amount of variance per chromosome and
chromosome length or equally number of SNPs per
chromosome. Effects of individual SNPs are composed of
effects due to co-segregation with closely linked QTL and
effects due to LD with QTL elsewhere in the genome, the
latter generated by familial relationships in the population. This effect tends to smooth all genomic variance over all
SNPs and may therefore give an unclear picture of how
much genomic variance can be ascribed to each chromo-
some. Jensen et al. BMC Genetics 2012, 13:44
http://www.biomedcentral.com/1471-2156/13/44 Page 8 of 9 Jensen et al. BMC Genetics 2012, 13:44
http://www.biomedcentral.com/1471-2156/13/44 Jensen et al. BMC Genetics 2012, 13:44
http://www.biomedcentral.com/1471-2156/13/44 population. The analysis of the genomic relationship
matrices revealed that a large proportion of the total addi-
tive genetic variance in the Nordic Holstein population
was expected to be explained by a set of 44 K markers. Analysis of both production and fitness related traits
showed that the amount of variance accounted for by
markers in the Nordic Holstein population was close to
the expectations from regression based analysis of the gen-
omic relationship matrix. Estimates of genomic variance
closely followed expectation when the number of markers
included in computation of genomic relationship matrix
was varied. The amount of additive genetic variance that
can be explained by genomic markers depends on several
factors: Number of markers on causative sites, markers in
linkage disequilibrium with causative genes due to close
“historical” linkage at population level, and finally linkage
disequilibrium among markers and genes at family level,
due to the family structure in the population. With 44 K
markers spread over the genome the number of markers
within causative sites probably is limited. The linkage dis-
equilibrium between markers and causative genes is very
dependent on effective population size [28]. The Holstein
cattle population has a low effective population size and,
therefore, there will be relatively few recombination events
in the recent history of the breed. In practice this means
that there will be considerable linkage disequilibrium be-
tween markers and causative genes. This is also supported
by the fact that most of the total additive genetic variation
was explained by genomic relationships and not by addi-
tive genetic relationships based on pedigree when the
model includes both relationship matrices. The family
structure in dairy cattle populations creates linkage dis-
equilibrium between markers and causative genes even if
they are on different chromosomes and this also helps the
markers in being able to explain most of the additive gen-
etic variance in the population. Analysis of the structure of the genomic relationship
matrices might be of considerable value in deciding on
avenue for future development of typing strategies when
using genomic markers. Such analysis also could give
extra insight in the effects of population structure and
population history on effectiveness of future selection
programs using genomic selection in other breeds or in
other species. Competing interests g
The authors declare no competing interests. Authors’ contributions
JUJ
d h
d JUJ conceived the study and conducted all analysis, GS developed and
implemented algorithms for computing genomic relationship matrices and
PM maintained the DMU software package used in the statistical analysis. JUJ edited the manuscript based on extensive input from all authors who
have read and approved the final manuscript. The analysis of genomic relationship matrices showed
that a high proportion of additive genetic variance can be
expected to be explained using 44 k genomic markers in
this population of dairy cattle. This leaves limited room
for further improvements of predictive ability of genomic
models by including more markers. One of the current
trends in use of genomic markers is to move from 50 K
marker chips to 800 K marker chips or even complete se-
quencing of whole genomes for individual animals. Our
results indicate that the advantages of this route may be
limited. In fact including several orders of more markers
than used in this study may turn out to be counterpro-
ductive. Extremely dense markers will include more mar-
kers on most causative sites and given knowledge of
variation in the causative genes there is no extra informa-
tion in the remaining markers. Alternative models that
better can distinguish between causative genes and non in-
formative markers might be of great value in future Jensen et al. BMC Genetics 2012, 13:44
http://www.biomedcentral.com/1471-2156/13/44 In summary we estimated the amount of additive genetic
variance that can be explained using dense SNP marker
panels. In the Holstein population analyzed, almost all the
additive genetic variance could be explained using 44 K
SNP markers. The amount of additive genetic variance that
is expected to be explained by markers could be predicted
from analysis of the genomic relationship matrix. Further
increases in marker density will have limited effects on pre-
dictive accuracy unless better methods distinguishing be-
tween markers with real effects and markers with no effect
are used. Results presented in this study can be used to de-
termine the weight given to marker relationships and to fa-
milial relationships in one step prediction methods where
these sources of relationships are combined and in two
step methods where information based on genomic rela-
tionships must be combined with information form poly-
genic relationships. Abbreviations
GS G
l GS: Genomic selection; DRP: Deregressed proof; Milk: Milk production;
Fat: Fat production; Protein: Protein production; Fertility: Female fertility;
Health: Veterinary treatments for diseases other than mastitis;
Mastitis: Mastitis. Acknowledgments The Danish Cattle Federation, FaBa Co-Op, Swedish Dairy Association and
Nordic Genetic evaluation is thanked for providing the data. External funding
for this study, including the extensive genotyping, were provided by the
Danish Ministry for Food, Agriculture and Fisheries project “Genomic
Selection – from function to efficient utilization in cattle breeding” Grant no
3405-10-0137 and the Milk Levy Fund, Viking Genetics and Nordic Genetic
Evaluation. The first author was funded through a grant from Aarhus
University. Received: 1 March 2012 Accepted: 31 May 2012
Published: 13 June 2012 Received: 1 March 2012 Accepted: 31 May 2012
Published: 13 June 2012 Variance components A second difference is that the
effective population size in dairy cattle is small with esti-
mates in Danish Holstein of Ne=49 [25], whereas the effect-
ive population size in humans is expected to be several
orders larger than in dairy cattle. Amount of variance explained by genomic markers
Obviously genetic markers cannot explain more than all
the total (additive) genetic variation present in the Obviously genetic markers cannot explain more than all
the total (additive) genetic variation present in the 1.
Meuwissen TH, Hayes BJ, Goddard ME: Prediction of total genetic value
using genome-wide dense marker maps. Genetics 2001, 157:1819–1829.
2.
Schaeffer LR: Strategy for applying genome wide selection in dairy cattle.
Journal of Animal Breeding and Genetics 2006, 123:218–223.
3.
Buch LH, Sørensen MK, Berg P, Pedersen LD, Sørensen AC: Genomic
selection strategies in dairy cattle: Strong positive interaction between References 1. Meuwissen TH, Hayes BJ, Goddard ME: Prediction of total genetic value
using genome-wide dense marker maps. Genetics 2001, 157:1819–1829. 2. Schaeffer LR: Strategy for applying genome wide selection in dairy cattle. Journal of Animal Breeding and Genetics 2006, 123:218–223. 3. Buch LH, Sørensen MK, Berg P, Pedersen LD, Sørensen AC: Genomic
selection strategies in dairy cattle: Strong positive interaction between Jensen et al. BMC Genetics 2012, 13:44
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http://www.biomedcentral.com/1471-2156/13/44 26. Pimentel ECG, Erbe M, Koenig S, Simianer H: Frontiers: Genome Partitioning
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Cattle. Genomics: Frontiers in Livestock; 2011:2. 27. Cole JB, VanRaden PM, O’Connell JR, Van Tassell CP, Sonstegard TS,
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of long term response. Genetica 2009, 136:245–257. doi:10.1186/1471-2156-13-44
Cite this article as: Jensen et al.: Partitioning additive genetic variance
into genomic and remaining polygenic components for complex traits
in dairy cattle. BMC Genetics 2012 13:44. use of genotypic information and intensive use of young bulls on
genetic gain. Journal of Animal Breeding and Genetics 2012, 129:138-151. 4. Mc Hugh N, Meuwissen THE, Cromie AR, Sonesson AK: Use of female
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Submit your manuscript at
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and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
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and take full advantage of:
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www.biomedcentral.com/submit Submit your next manuscript to BioMed Central
and take full advantage of: Submit your next manuscript to BioMed Central
and take full advantage of: Submit your next manuscript to BioMed Central
and take full advantage of: 24. Grisart B, Coppieters W, Farnir Fdr, Karim L, Ford C, Berzi P, et al: Positional
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Breeds. Journal of dairy science 2005, 88:1865–1872. Submit your manuscript at
www.biomedcentral.com/submit
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Development and Implementation of Stre@mline, a Locally Developed Electronic Health Platform in Uganda (Preprint)
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JMIR FORMATIVE RESEARCH JMIR FORMATIVE RESEARCH Liang et al Original Paper A Locally Developed Electronic Health Platform in Uganda:
Development and Implementation of Stre@mline Li Liang1, BEng; Matthew O Wiens2, PhD; Phaisal Lubega3, BS; Ian Spillman4, MBBS; Samuel Mugisha5, BS, MBA Li Liang1, BEng; Matthew O Wiens2, PhD; Phaisal Lubega3, BS; Ian Spillman4, MBBS; Samuel Mugisha5, BS, MBA
1 Corresponding Author:
Samuel Mugisha, BS, MBA
Innovation Streams Limited
Plot 76/78 High Street
Mbarara La Grand Mall Room L2-03
Mbarara, PO Box 1340
Uganda
Phone: 256 704899935
Email: samuel.gisha@gmail.com Corresponding Author:
Samuel Mugisha, BS, MBA
Innovation Streams Limited
Plot 76/78 High Street
Mbarara La Grand Mall Room L2-03
Mbarara, PO Box 1340
Uganda
Phone: 256 704899935
Email: samuel.gisha@gmail.com Abstract Background: Electronic health records (EHRs) are especially important in low-resource settings due to their potential to address
unique challenges such as a high number of patients requiring long-term treatments who are lost to follow-up, the frequent
shortages of essential drugs, poor maintenance and storage of records, and inefficient clinical triaging. However, there is a lack
of affordable and practical EHR solutions. Stre@mline is an EHR platform that has been locally developed by Ugandan clinicians
and engineers in Southwestern Uganda. It is tailored to the specific context and needs of low-resource hospitals. It operates
without internet access, incorporates locally relevant standards and key patient safety features, has a medication inventory
management component, has local technical support available, and is economically sustainable without funding from international
donors. Stre@mline is currently used by over 60,000 patients at 2 hospitals, with plans to expand across Uganda. Objective: The purpose of this article is to describe the key opportunities and challenges in EHR development in sub-Saharan
Africa and to summarize the development and implementation of a “Made-for-Africa” EHR, Stre@mline, and how it has led to
improved care for over 60,000 vulnerable patients in a rural region of Southwestern Uganda. Methods: A quantitative user survey consisting of a set of 33 questions on usability and performance was conducted at Kisiizi
Hospital. Users responded to each question through a Likert scale with the values of strongly disagree, disagree, agree, and
strongly agree. Through purposive sampling, 30 users were identified and 28 users completed the survey. Results: We found that users were generally very satisfied with the ease of use of Stre@mline, with 96% (27/28) finding it easy
to learn and 100% (28/28) finding it easy to use. Users found that Stre@mline was helpful in improving both clinical efficiency
and enhancing patient care. Conclusions: The partnership of local clinicians and developers is crucial to the design and adoption of user-centered technologies
tailored to the specific needs of low-resource settings. The EHR described here could serve as a model for the development of
future technologies suitable for developing countries. (JMIR Formativ Res 2018;2(2):e20) doi: 10.2196/formative.9658 (JMIR Formativ Res 2018;2(2):e20) doi: 10.2196/formative.9658 JMIR Formativ Res 2018 | vol. 2 | iss. 2 | e20 | p. 1
(page number not for citation purposes) http://formative.jmir.org/2018/2/e20/ Background Although often viewed as a resource only available to
well-funded health facilities in developed countries, electronic
health records (EHRs) are especially important for low-resource
settings such as those in sub-Saharan Africa due to the unique
challenges they can address. A well-developed EHR system
can fill critical gaps common to such environments, such as
assisting busy frontline workers to treat diseases, especially
those requiring long-term treatment and monitoring (such as
HIV and diabetes), and to anticipate and prevent frequently
reported shortages of essential drugs. Furthermore, limited
human resource capacity often leads to poorly developed and
maintained medical records as well as poor triaging of patients
presenting to hospitals. An optimized EHR can efficiently help
address these ubiquitous issues. While EHRs can, and should,
be instrumental in helping resource-poor countries meet the UN
Sustainable Goals, many unique challenges have prevented the
development and scaling of EHR systems in many resource-poor
environments, including Uganda. The purpose of this article is
to describe the key opportunities and challenges in EHR
development in sub-Saharan Africa and to summarize the
development and implementation of a “Made-for-Africa” EHR
and how it has led to improved care for over 60,000 vulnerable
patients in a rural region of Southwestern Uganda. Liang et al Liang et al Liang et al Despite the many potential advantages of EHR systems, several
important implementation challenges specific to developing
countries have been identified. High initial costs associated with
computer purchase and infrastructure setup is a critical barrier
[8]. However, this can, at least, be partially mitigated by
potential cost saving from reductions in record-keeping costs
[9] and significantly improved staff efficiency [10]. Arguably,
the most critical challenge to the success of existing EHR
systems is the lack of involvement of local staff in the design
and testing of systems to ensure that they are designed according
to local needs and workflows. Other significant challenges
include the purchase cost of the EHR software, the lack of local
and inexpensive technical support to maintain EHR systems,
frequent power and internet outages, the lack of computer
literacy, and the fact that most EHRs are not sustainable without
funding from international partnerships [8,11,12]. Overcoming
these challenges will be crucial to ensure successful
implementation and scaling of any EHR in a low-resource
setting. Opportunities and Challenges With Electronic Health
Records One of the biggest public health challenges to the success of
managing diseases like HIV/AIDS and tuberculosis is the high
rates of patients who are “lost to follow-up” [1]. EHRs have
been proposed as one of the top solutions [2,3], with preliminary
evidence suggesting EHRs can significantly reduce the estimated
24% of all HIV patients lost to follow-up [4]. In addition, EHRs
are useful for the long-term follow-up of chronic illnesses like
cancer, high blood pressure, and diabetes and to ensure that
patients are treated following the correct guidelines. Moreover,
accurate and thorough data collection using EHRs can also
inform targeted allocation of limited resources by recognizing
trends and helping define priorities. JMIR Formativ Res 2018 | vol. 2 | iss. 2 | e20 | p. 2
(page number not for citation purposes) http://formative.jmir.org/2018/2/e20/ KEYWORDS ectronic health record; locally developed technology; appropriate technology; eHealth in low-resource sett http://formative.jmir.org/2018/2/e20/ JMIR FORMATIVE RESEARCH Local Setting In Uganda, there are currently 1.5 million people living with
HIV/AIDS and 79,000 new cases of active tuberculosis are
reported every year. In addition, as many as 50% of essential
drugs are not available at public hospitals [13]. Moreover, there
is a significant shortage of qualified health workers, with only
0.12 physicians and 1.3 nurses per 1000 people [14]. Uganda
is one of the poorest countries in the world, with about one-third
of Ugandans living on less than US $1.90 purchasing power
parity per day. The region in which the described EHR was
implemented (Kisiizi, a rural region in Southwestern Uganda)
is among the poorest regions in Uganda, thus, providing an ideal
context for the development of a system ideal for rural and
remote health facilities in sub-Saharan Africa. The EHR system,
Stre@mline, was codeveloped by Ugandan software developers
at a technology startup named istreams (acronym for Innovation
Streams) and a team of physicians from the Kisiizi Hospital, a
private not-for-profit hospital in Southwestern Uganda. Opportunities and Challenges With Electronic Health
Records Unique Features of Stre@mline In addition to the standard features such as interfaces for
clinicians from different departments to communicate with one
another, Stre@mline has a pharmacy interface that can be seen
by prescribers, and it continuously updates stock, expiry date,
and price of all drugs in real time. Stre@mline enrolls the
patients’ journey through a system of unique identifiers. If a
patient’s folder name is not present in the system, a unique
identifier specific to the hospital is generated for the patient (eg,
KH-60570 for one patient and KH-60571 for the subsequent
patient). In addition, identifier information including the
patient’s first and last name, village name, phone number, sex,
and age is also recorded in the database because oftentimes,
there are multiple patients with the same name in the hospital’s
database. For returning patients, at triage, a patient’s folder will
be searched based on the abovementioned identifiers. When the
patient’s folder is found, a new file will be created for the current
visit; this file will be time stamped and appended to the patient’s
previous files in the folder. Another key sustainability feature of the platform is that istreams
offers local technical support to promptly solve technical issues
and make improvements according to user needs. Through
continuous and consistent user input, Stre@mline incorporates
many locally relevant standards and guidelines, such as the
locally developed Kissizii Early Warning System and the World
Health Organization Emergency Triage Assessment and
Treatment guidelines, local drug guidelines, and built-in linkage
to the Uganda National Drug Authority adverse drug reactions
reporting system. These systems are described in more detail
in Table 1. Codesigning with local clinicians ensures simplicity
of training and general use. Clinicians who have basic
knowledge of using a computer can learn the key features of
Stre@mline in less than 30 minutes. Furthermore, Stre@mline
can be customized to integrate with local insurance plans, such
as the Kisiizi Hospital Health Insurance Scheme used by 40,000
users, to monitor health-seeking behaviors. Finally, Stre@mline
is affordable, with funding for development of this program
coming entirely from within Uganda. The Stre@mline platform is also unique as it does not require
internet access to operate. This is very important as internet
access is very unreliable and expensive in Uganda. Instead, the
program operates through a local area network consisting of 30
computers connected with each other and a local central server
run through Ethernet cables. Partnerships Inventory management and triaging challenges are also
especially important for low-resource settings. Severe shortage
of essential drugs as well as the dispensing of expired drugs are
the major problems faced by patients in developing countries
[5,6]. Poor patient record storage as a result of staffing shortages
is another key challenge in low-resource hospitals. Thus, EHRs
can play a significant role in allowing hospitals to better track
and plan resources to ensure that essential medications are in
stock and not expired and in ensuring that medical records are
available and organized. Both of these are expected to result in
improved patient care. Another intriguing advantage offered by
EHRs is that supportive tools and guidelines for triaging, which
are especially important given severe staffing shortages in
low-resource settings [6], can be embedded within EHRs to
focus resources on the most vulnerable patients. Improved
triaging has been demonstrated to improve outcomes, including
mortality, without the need for additional staffing [7]. The partnership between istreams and the Kisiizi Hospital was
initiated in 2013 when physicians from the hospital approached
istreams to assist in developing a very simple EHR to overcome
several specific challenges faced by the hospital that could not
be adequately addressed by commercially available software
applications. Over the following 2 years, the team met regularly
to develop and pilot a platform tailored to the specific constraints
and criteria of a busy Ugandan hospital. The resulting product
is a sustainable and scalable EHR system that can address many
of the shortcomings in the existing EHR platforms described
earlier. The development cost for Stre@mline was funded by
a National Science & Technology Improvement Grant (NSTIP)
from Uganda National Council for Science & Technology
(UNCST) as well as from the Kisiizi Hospital. XSL•FO
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RenderX XSL•FO
RenderX JMIR FORMATIVE RESEARCH Liang et al Liang et al inverter system designed to prevent data loss during power
outages. Unique Features of Stre@mline All data are backed up on the
central server that is backed up through a battery-powered Currently, Stre@mline has been implemented in 2 hospitals in
Southwestern Uganda and has been used for over 60,000
patients. Furthermore, the system is being set up in 3 additional
hospitals in the region. istreams hopes to expand the system to
50 hospitals in Uganda over the next 2 years. Table 1. Key local health care challenges addressed by Stre@mline. Stre@mline response
Issue and problem
Follow-up for long-term treatments
Monitoring of follow-up attendance, facilitation of contacting patients to ensure good
on-going care in place
High number of patients lost to follow-up
Medicines
Live monitoring of stock levels of medicines and triggering ordering in good time to
avoid stock-outs
Severe shortages of drugs
Warns pharmacists of drugs due to expire in 2 months, facilitating better resource
planning by pharmacists and prescribers
Drugs often expire in storage, wasting valuable resources
Facilitates 100% capture of prescribing errors through built-in linkage to the Uganda
National Drug Authority drug reactions reporting system
Auditing of drug prescribing errors is often poor or erratic
Triage
Incorporates the World Health Organization Emergency Triage, Assessment, and
Treatment (ETAT) tool and the locally developed Kisiizi Early Warning System
Triage often poorly done, especially in children
Ensures that 100% children are properly triaged using ETAT tool as it uses mandatory
fields. Users rapidly learn the new routine and comply happily as they see the benefits. Paper-based triage systems were often omitted or only
partially done
Medical records
Captures key data relating to a patient’s symptoms, investigations, treatment, and
follow-up
Often incomplete, poor quality records were kept
Allows files to be stored (eg, x-rays, clinical letters, and photographs, for immediate
access in future)
Patients often forget to bring previous notes, images, etc,
and may end up undergoing unnecessary duplicate tests
Customization
Stre@mline is designed to allow free, easy, and comprehensive customization by local
institutions to ensure that the system is optimal for the local environment
Commercial systems are often difficult and expensive to
customize to local requirements
JMIR Formativ Res 2018 | vol. 2 | iss. 2 | e20 | p. 3
http://formative.jmir.org/2018/2/e20/
(page number not for citation purposes)
FO
erX Table 1. Key local health care challenges addressed by Stre@mline. Monitoring of follow-up attendance, facilitation of contacting patients to ensure good
on-going care in place JMIR Formativ Res 2018 | vol. 2 | iss. 2 | e20 | p. 3
(page number not for citation purposes) Medicines Severe shortages of drugs JMIR Formativ Res 2018 | vol. 2 | iss. 2 | e20 | p. 3
(page number not for citation purposes) http://formative.jmir.org/2018/2/e20/ XSL•FO
RenderX JMIR FORMATIVE RESEARCH Liang et al Liang et al has ensured the achievement of maximum benefits for patient
care. In contrast, many health information technology programs
fail when the software use is difficult for the intended users,
thus, reducing the efficiency of the provider. Thus, it is critical
to understand the clinical workflows, the patient journey, and
key clinical data points needed. has ensured the achievement of maximum benefits for patient
care. In contrast, many health information technology programs
fail when the software use is difficult for the intended users,
thus, reducing the efficiency of the provider. Thus, it is critical
to understand the clinical workflows, the patient journey, and
key clinical data points needed. Principal Findings Designing the EHR according to the specific needs of Ugandan
hospitals has been critical to the successful implementation of
Stre@mline. Having senior clinical input throughout the process Results The results of this survey showed that users were generally very
satisfied with the ease of use of Stre@mline, with 96% (27/28)
finding Stre@mline easy to learn and 100% (28/28) finding it
easy to use. The Stre@mline platform has addressed several
problems at the hospital. First, Stre@mline has allowed
physicians at the Kisiizi Hospital to reliably access patients’
past medical records and investigations, which was generally
not possible with the prior paper-based system. This feature has
also increased workflow efficiency, with 80% (8/20) of users
agreeing that it has allowed them to “see more patients in a day”
as well as increased apparent trust in physicians by patients (as
identified by clinicians). The embedded guidelines and triage
assistance within Stre@mline has also substantially improved
patient care, with 100% (20/20) of respondents agreeing that it
has improved their “decision making.” Limitations Limitations of the current system include a lack of data
portability between different hospitals because data are currently
stored on local area networks. In addition, although the system
is low cost, it may still be cost prohibitive for small public
hospitals and clinics within Uganda and other African countries. Finally, quantitative data on cost savings and patient safety are
yet to be collected and analyzed, limiting the ability to generate
any cost metrics. These data are, however, currently being
collected and will be incorporated into a cost-benefit analysis
in the future. Furthermore, clinicians interviewed at the Kisizi Hospital
generally agreed that Stre@mline has allowed clinicians to
prescribe alternatives if one drug is not in stock, and this has,
in turn, “improved patient care and compliance rates.”
Stre@mline has also improved resource planning by allowing
pharmacists to track their drug stocks in real time, thus,
improving consistent stocking and availability of essential
medications at the hospital, with 96% of clinicians agreeing
that it has “provided better mechanism for drug availability”
and 100% agreeing that system has led to “safer and more
reliable prescriptions.” Conclusions Stre@mline is a locally developed EHR system tailored to the
specific needs of resource-constrained settings. It is unique in
being entirely locally developed through a partnership between
a local hospital and a local technology company; it has been
developed and is sustainable without funding from outside
Uganda. The EHR system is currently being used by over 60,000
patients at 2 hospitals across Uganda with plans for further
scaling. The process described here could serve as a model for
the development of future appropriate technologies in
developing countries. Methods A quantitative user survey was conducted at the Kisiizi Hospital
by a Masters student at the Mbarara University of Science and
Technology, who is not affiliated with istreams, between January
and June 2017. It was a paper-based survey consisting of a set
of 33 questions on system usability and performance. Users
responded to each question through a Likert scale with the
values of strongly disagree, disagree, agree, and strongly agree. Through purposive sampling, 30 users were identified to
complete the survey. In total, 28 users (6 doctors, 3 clinical
officers, 6 nurses, 4 other health professionals, 6 administrative
staff, and 3 support staff) consented and completed the survey. The analysis of survey data was performed primarily through
descriptive statistics using Microsoft Excel (Redmound, USA). The key findings are described below. Partnerships between local technology entrepreneurs and
clinicians have the potential to not only create well-designed,
but more importantly, sustainable and scalable technological
solutions for these settings. Such technologies are more likely
to have access to effective local support for maintenance and
further development through an intimate understanding of local
needs. Furthermore, these solutions tend to be more
economically sustainable, with less external donor funding
needed. In addition, leveraging local pride can be an important
contributor to the adoption of any technology and has certainly
been well leveraged in the development and application of
Stre@mline. Supporting local developers of health care
technologies financially and technologically is, therefore, a
model that may be far more sustainable and impactful than
developing such technologies in Western countries. Finally,
another key to the success of Stre@mline in Kisiizi has been
strong leadership from clinician-administrators who ensured
and mandated computer workshops for all hospital employees
and organized piloting the EHR sequentially, one department
at a time, until it was scaled across each department. The
approach described could thereby serve as a model for the
development
of
future
appropriate
technologies
for
research-limited settings. http://formative.jmir.org/2018/2/e20/ JMIR Formativ Res 2018 | vol. 2 | iss. 2 | e20 | p. 4
(page number not for citation purposes) Acknowledgments We would like to acknowledge the support for initial development of Stre@mline received from NSTIP of the UNCST in 2016. We would also like to thank Amos Baryashaba from the Mbarara University of Science and Technology for providing preliminary
data from his MSc thesis and Dr Mark Ansermino from the University of British Columbia for helpful feedback on the manuscript. JMIR Formativ Res 2018 | vol. 2 | iss. 2 | e20 | p. 4
(page number not for citation purposes) http://formative.jmir.org/2018/2/e20/ XSL•FO
RenderX JMIR FORMATIVE RESEARCH Liang et al Conflicts of Interest None declared. References Improved triage and emergency care for children reduces inpatient mortality in a
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in developing countries. Inform Prim Care 2005;13(2):83-95 [FREE Full text] [Medline: 15992493] 11. Fraser HSF, Biondich P, Moodley D, Choi S, Mamlin BW, Szolovits P. Implementing electronic medical record systems
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systematic review. J Am Med Inform Assoc 2015 Mar;22(2):479-488. [doi: 10.1093/jamia/ocu038] 12. Fritz F, Tilahun B, Dugas M. Success criteria for electronic medical record implementations in low-resource settings: a
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loss to follow-up: survey of antiretroviral therapy programmes in resource-limited settings. Bull World Health Org 2008
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countries: a survey of surgical and anesthesia capacity in Uganda's public hospitals. World J Surg 2012 May;36(5):1056-1065. [doi: 10.1007/s00268-012-1482-7] [Medline: 22402968] 5. Linden AF, Sekidde FS, Galukande M, Knowlton LM, Chackungal S, McQueen KAK. Challenges of surgery in developing
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resource-constrained setting. Bull World Health Organ 2006 Apr;84(4):314-319 [FREE Full text] [Medline: 16628305] 7. Molyneux E, Ahmad S, Robertson A. References J Am Med Inform Assoc 2015 Mar;22(2):479-488. [doi: 10.1093/jamia/ocu038] 13. Oluka P, Ssennoga F. Tackling supply chain bottlenecks of essential drugs: A case of Uganda local government health
units. 4th Int Procure Conf 2013:26-28 [FREE Full text] 13. Oluka P, Ssennoga F. Tackling supply chain bottlenecks of essential drugs: A case of Uganda local government health
units. 4th Int Procure Conf 2013:26-28 [FREE Full text] 14. World Bank Group. 2017. Physicians (per 1,000 people): World Health Organization's Global Health Workforce Statistics,
OECD, supplemented by country data URL: https://data.worldbank.org/indicator/SH.MED.NUMW.P3?locations=UG
[accessed 2018-08-22] [WebCite Cache ID 71sBprIm4] 14. World Bank Group. 2017. Physicians (per 1,000 people): World Health Organization's Global Health Workforce Statistics,
OECD, supplemented by country data URL: https://data.worldbank.org/indicator/SH.MED.NUMW.P3?locations=UG
[accessed 2018-08-22] [WebCite Cache ID 71sBprIm4] Abbreviations Edited by G Eysenbach; submitted 15.12.17; peer-reviewed by B Tilahun, J Buttolph; comments to author 18.01.18; revised version
received 14.03.18; accepted 26.07.18; published 24.09.18
Please cite as: G Eysenbach; submitted 15.12.17; peer-reviewed by B Tilahun, J Buttolph; comments to author 18.01.18; revised version
4.03.18; accepted 26.07.18; published 24.09.18 JMIR Formativ Res 2018 | vol. 2 | iss. 2 | e20 | p. 5
(page number not for citation purposes) http://formative.jmir.org/2018/2/e20/ Liang et al ©Li Liang, Matthew O Wiens, Phaisal Lubega, Ian Spillman, Samuel Mugisha. Originally published in JMIR Formative Research
(http://formative.jmir.org), 24.09.2018. This is an open-access article distributed under the terms of the Creative Commons
Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction
in any medium, provided the original work, first published in JMIR Formative Research, is properly cited. The complete
bibliographic information, a link to the original publication on http://formative.jmir.org, as well as this copyright and license
information must be included. JMIR Formativ Res 2018 | vol. 2 | iss. 2 | e20 | p. 6
(page number not for citation purposes) JMIR FORMATIVE RESEARCH Liang et al ©Li Liang, Matthew O Wiens, Phaisal Lubega, Ian Spillman, Samuel Mugisha. Originally published in JMIR Formative Research
(http://formative.jmir.org), 24.09.2018. This is an open-access article distributed under the terms of the Creative Commons
Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction
in any medium, provided the original work, first published in JMIR Formative Research, is properly cited. The complete
bibliographic information, a link to the original publication on http://formative.jmir.org, as well as this copyright and license
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Evaluation of insulation systems for the optimal design of high voltage pulse transformers
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CERN-ACC-2015-0005
Davide.Aguglia@cern.ch CERN-ACC-2015-0005 Evaluation of Insulation Systems for the Optimal
Design of High Voltage Pulse Transformers Sylvain Candolfi1, Sebastian Blume2, Davide Aguglia3, Philippe Viarouge1, Jürgen Biela2, Jérôme
Cros1
1LEEPCI Lab, Québec, Canada, 2ETH Zürich, Zurich, Switzerland, 3CERN, Geneva, Switzerland Keywords: High voltage, insulation system, pulse transformer Abstract This paper presents a study for the design of the insulation systems of high voltage pulse transformer
based on experimental tests and numerical simulations. Data of high voltage tests on solid and liquid
insulation materials are presented and discussed. The understanding of each part of the insulation is
supported by electrostatic fields simulations. CERN-ACC-2015-0005
16/01/2015 CERN-ACC-2015-0005
16/01/2015 1 INTRODUCTION At CERN high voltage (HV) pulse transformers for klystron
modulators are currently under study for the Compact Linear
Collired (CLIC). The insulation distances and materials are key parameters
that determine the dynamic performances. They must be as
small as possible but ensure that no breakdown will occur
during the lifespan of the transformer. Figure 1. Transformer’s cross-sectional view showing the structure of the
insulation system. Design directions for the insulation systems are presented. The study consists in classical destructive HV tests [1]
combined with in Finite-Element Analyses (FEA) for the
identification of the weak insulation point. Safety factors on
breakdown fields, for each insulation system, shall be
considered to cope with ageing and imperfections in the
insulation material. The impact on the performances of the
pulse transformer versus the selected safety factor is presented
considering a transformer which meets CLIC specifications. ABSTRACT This paper presents a study for the design of the insulation
systems of high voltage pulse transformer based on
experimental tests and numerical simulations. Data of high
voltage tests on solid and liquid insulation materials are
presented and discussed. The understanding of each part
of the insulation is supported by electrostatic fields
simulations. Figure 1. Transformer’s cross-sectional view showing the structure of the
insulation system. Kernel
Primary winding
Field stabilizer
Secondary winding
Coils supports Index Terms — High voltage, insulation system, pulse
transformer Sylvain Candolfi1, Sebastian Blume2, Davide Aguglia3, Philippe Viarouge1, Jürgen Biela2,
Jérôme Cros1 1LEEPCI Lab., Electrical and Computer Eng. Dept., Laval University, G1V 0A6 Quebec (QC), Canada
Emails: sylvain.candolfi.1@ulaval.ca, philippe.viarouge@gel.ulaval.ca, jerome.cros@gel.ulaval.ca
2ETH Zürich, Laboratory for High Power Electronics (HPE), ETL F13 Physikstrasse 3, 8092 Zürich, Switzerland
Emails: blume@hpe.ee.ethz.ch, jbiela@ethz.ch 3CERN - European Organization for Nuclear Research, Technology Dept., Electric Power Converter Group
CH-1211 Geneva 23, Switzerland Email: davide.aguglia@cern.ch a half-pipe shaped conductor is added on the top of the HV
coil to increase the radius of curvature. The whole transformer
is immersed in oil. a half-pipe shaped conductor is added on the top of the HV
coil to increase the radius of curvature. The whole transformer
is immersed in oil. 3.1 LIQUID MATERIAL 3.1 LIQUID MATERIAL Two types of synthetic insulating oils have been compared:
MIDEL 7131 and RHODORSIL silicon oil. Used silicon oil
has been tested and a comparison between used and new
MIDEL oil has been carried out. The used oil was removed
from transformers in operation at CERN. The different oils
were characterized by their breakdown voltage and dispersion
of the measurements. Rogowski electrodes with a gap of 1.8
mm or 3 mm have been used (Figure 2a). A linearly increasing
voltage of 500 V/s is applied on the sample until breakdown. Results are presented in Table 1. Only a low decrease of the
breakdown field between old and new MIDEL oil is observed
but the standard deviation is larger because the impurities Presented at: IEEE IPMHVC, 1-5 June 2014, Santa Fe, US Presented at: IEEE IPMHVC, 1-5 June 2014, Santa Fe, US Presented at: IEEE IPMHVC, 1-5 June 2014, Santa Fe, US Geneva, Switzerland
January,2015 Evaluation of Insulation Systems for the Optimal Design of
High Voltage Pulse Transformers Sylvain Candolfi1, Sebastian Blume2, Davide Aguglia3, Philippe Viarouge1, Jü
Jérôme Cros1 4.1 INVESTIGATED SETUP To develop a better understanding of the electrical field
distribution at the field stabilizer surface, the geometry was
investigated in a simplified setup, as shown in Figure 4b,
where the high voltage is applied to two tubes with a fixed
distance with respect to a grounded plate, representing the
distance of the two field stabilizers to the top of core window. A DC ramp (1000V/s) is used to obtain the average
breakdown voltages with the two mentioned new transformer
oils for various distances, each with at least 6 samples. The
geometric setup was then simulated in 2D-FEM to determine
the electrical peak field (Table 3) for each distance at the
respective breakdown voltage. Additionally, the field path
with the most critical electrical field was identified, which is
displayed in Figure 5a for RHODORSIL and 5b for MIDEL. 2 INSULATION STRUCTURE An oil-based insulation is selected for its ability to
efficiently evacuate the transformer’s heat-losses. Coils are
fixed on solid supports (Error! Reference source not
found.). The top secondary-winding’s wire of the high voltage
coil is located near the grounded magnetic core. In order to
reduce the peak electric field (related to the Schweiger factor), make the oil less homogeneous and trigger breakdown. We
can deduce from those tests that the breakdown field in new
MIDEL oil is about 20 kVmm-1 and decrease to 18 kVmm-1
with used oil. Used silicon and MIDEL oils cannot be fairly
compared as they did not experience the same degradation
conditions. Figure 3. Setup for the tests of wires. Wire under test
Copper braid (a)
(b)
Figure 2. Rogowski electrodes for oil testing (a) and Setup for solid materials
testing (b). Surface arcs around the PEEK plate are observed. (a)
(b) (b) (b) (a)
(b)
Figure 2. Rogowski electrodes for oil testing (a) and Setup for solid materials
testing (b). Surface arcs around the PEEK plate are observed. Figure 3. Setup for the tests of wires. 3.2 SOLID INSULATION MATERIALS A plate of PolyEther Ether Ketone (PEEK) of 20 cm by 20
cm and 1 mm thickness has been tested between two 50 mm
diameter electrodes (Figure 2b) applying a linearly increasing
voltage. The breakdown voltage could not be determined
because electric arcs at the surface occurred before breakdown
through the plate. Breakdown occurred with an electrical field
(inside the PEEK) of 80 kVmm-1. Therefore the PEEK
breakdown field is greater than this value. 4 TEST OF THE FIELD STABILIZER Table 1. Result of tests on oil. All averages are issued of 5 tests. Table 1. Result of tests on oil. All averages are issued of 5 tests. Type of oil
Average
breakdown
voltage Vbr
[kV]
Standard
deviation
Average
breakdown
field
[kV/mm]
Used RHODORSIL (gap 3 mm)
39.0
8.9
13.0
Used MIDEL (gap 1.8 mm)
34.3
13.6
19.1
Used MIDEL (gap 3 mm)
54.4
7.2
18.1
New MIDEL (gap 1.8 mm)
36.3
6.3
20.17
New MIDEL (gap 3 mm)
61.1
5.0
20.7 In high voltage pulse transformers field stabilizers are used
to reduce the electrical peak field occurring at the secondary
turn with the highest potential. The transformer geometry
investigated here considers the secondary turn with the highest
potential directly as field stabilizer by increasing its radius
(Figure 4a) [4]. 5.2 RESULTS The results of the breakdown voltage for the different
configurations of the assembly are summarized in Table 4. The time to breakdown from the beginning of the pulse is
generally included between 10 µs and 60 µs. Figure 6. Assembly used for the destructive tests on the insulation assembly. Table 4 Breakdown voltages for different configurations of the assembly Figure 5. Field path with the most critical electrical field for different
distances between the two tubes and the grounded plate for a) MIDEL 7131 b)
for RHODORSIL 604 V 50. Figure 5. Field path with the most critical electrical field for different
distances between the two tubes and the grounded plate for a) MIDEL 7131 b)
for RHODORSIL 604 V 50. Figure 6. Assembly used for the destructive tests on the insulation assembly. Figure 6. Assembly used for the destructive tests on the insulation assembly. Table 4. Breakdown voltages for different configurations of the assembly. Table 4. Breakdown voltages for different configurations of the assembly. Table 3. Result field shape ring geometry setup. Type of oil
Distance
d [mm]
Mean
breakdown
voltage Vbr
[kV]
Standard
deviation
[kV]
Eav=
Vbr/d
[kV/mm]
Peak
breakdown
field
simulated
[kV/mm]
RHODORSIL
2
2.5
5
8
31.44
32.85
50.44
67.16
3.77
3.24
3.59
2.33
15.72
13.14
10.09
8.40
20.15
16.00
15.58
15.43
MIDEL
2
2.5
5
8
41.77
52.49
64.47
79.275
1.14
2.8
7.97
8.00
20.88
21.00
12.89
9.91
26.80
25.56
19.92
17.9
5 TEST OF AN INSULATION SYSTEM
COMBINING SEVERAL INSULATION
MATERIALS
5.1 TESTED ASSEMBLY DESCRIPTION Table 3. Result field shape ring geometry setup. Type of oil
Distance
d [mm]
Mean
breakdown
voltage Vbr
[kV]
Standard
deviation
[kV]
Eav=
Vbr/d
[kV/mm]
Peak
breakdown
field
simulated
[kV/mm]
RHODORSIL
2
2.5
5
8
31.44
32.85
50.44
67.16
3.77
3.24
3.59
2.33
15.72
13.14
10.09
8.40
20.15
16.00
15.58
15.43
MIDEL
2
2.5
5
8
41.77
52.49
64.47
79.275
1.14
2.8
7.97
8.00
20.88
21.00
12.89
9.91
26.80
25.56
19.92
17.9 Oil
Support
Wire
Breakdown
voltage
[kV]
Std. deviation
[kV]
Used MIDEL
PEEK
Enameled
71.3
7.4
One layer mica
80.0
1.6
Two layers mica
79.0
12.6
G11
One layer mica
74.0
2.8
New MIDEL
PEEK One layer mica
91.8
15.7
G11
95.0
0
Used
RHODORSIL
PEEK One layer mica
67.8
4.3
G11
71.9
8.2 4.2 RESULTS ANALYSIS plate by a layer of 2 mm of oil. The assembly is depicted in
Figure 6. The results, displayed in Table 3 verify the higher
performance of MIDEL in comparison to RHODORSIL
previously shown in Table 1. They also show that the peak
field is a key factor both in relatively homogeneous as well as
in inhomogeneous electrical fields. The peak electrical field
decreases from around 20.1 kV/mm to 15.5 kV/mm for
RHODORSIL and from 26.8 kV/mm to 19.7 kV/mm for
MIDEL with increasing distance. The decrease in electrical
peak field is significantly smaller than for an assumed average
electrical field (
) (Table 1). Therefore it is crucial to
consider in the design process the maximal electrical peak
field rather than referring to an average electrical field for
larger distances, which occur in a high voltage transformer. In order to investigate the effect of pulse length on the
breakdown voltage, a double exponential with a front time of
1 µs and a time to half-value of 900 µs is used. The voltage is
recorded on an oscilloscope to measure the time before
breakdown. 3.3 WIRES Wires have their own layer of insulation. Enamelled wire
and enamelled wire covered with one and two layers of mica
strips have been tested in a copper braid connected to the
ground (Figure 3). All the system is immersed in oil to avoid
arcs between connectors, wire and grounded parts outside the
copper braid. Figure 4. a) Transformer geometry with turn at the highest potential designed
as field stabilizer. b) Simplified geometry representing the distance between
the two field shape rings to the top of the core window. The mica insulation enhance the breakdown voltage by
more than a factor 10 (Table 2) but it does not depend on the
number of layers of mica. The first layer of mica sheet is the
most important as the electric field is larger near the wire
where the curvature radius is the smallest. Furthermore inter-
layers air bubbles could have been trapped (no vacuum filling
process) increasing partial discharges and consequent arc
discharges. Table 2. Breakdown voltage for different types of wires. Type of wire
Breakdown voltage [kV]
Enamelled wire
3.4
Mica, one layer
42
Mica, two layers
35 Figure 4. a) Transformer geometry with turn at the highest potential designed
as field stabilizer. b) Simplified geometry representing the distance between
the two field shape rings to the top of the core window. 5.3 RESULTS ANALYSIS The speed of streamer in oil for low breakdown voltages is
in the order of 1 km s-1 [2]. The time from the beginning of the
pulse to the breakdown is compatible with the time that the
streamer takes to propagate into the 2 mm of oil gap. Once the
streamer has propagated through the oil, the electrical field in
the support is high enough to cause the breakdown of the
support insulator. 5 TEST OF AN INSULATION SYSTEM
COMBINING SEVERAL INSULATION
MATERIALS
5.1 TESTED ASSEMBLY DESCRIPTION The type and quality of oil is the main factor determining
the breakdown voltage. For RHODORSIL oil the breakdown
voltage is the lowest, between 67.8 and 71.9 kV, for used
MIDEL between 71.3 and 80 kV and the highest breakdown
voltage is held from for new MIDEL oil, between 91.8 and 95
kV. The electrical field at breakdown voltage in the assembly
with a PEEK support, one layer mica insulation wire and used
MIDEL oil is simulated with finite element methods (Figure
7). The electrical field inside the support is lower than the field 5.1 TESTED ASSEMBLY DESCRIPTION When combining different insulation material with known
electrical characteristics, it becomes difficult to clearly
identify the weak point of the insulation system. For this
purpose a prototype assembly combining the solid, liquid and
wire insulation has been built for destructive tests purposes. The assembly is composed of a wire in contact with a solid
insulation plate of 1 mm thickness separated from a grounded The safety factor in the solid material is higher than the
safety factor in oil (higher breakdown consequences). achieved during the test of solid material. The field in the oil
between the grounded plate and the support is equal to the
breakdown field measured for the oil. It confirms that the
streamer initiate and propagate first in the oil. The safety factor forces the insulation thickness. The other
dimensions of the transformer will be changed in order to
meet the specifications. The relative volume is expressed in
percentage of the volume of a transformer with a safety factor
of 1. The influence of one particular component of the insulation
can be assessed by exchanging it for a component of another
type without replacing the other materials. Figure 8. Influence of the insulation safety factor on the overall transformer
volume. 100
200
300
400
500
600
700
1
2
3
4
5
Relative volume [%]
Safety factor [-]
G11
PEEK The three first tests with used MIDEL oil and PEEK
support can be used to examine the influence of the type of
wire insulation. The results are similar to the test of the wires
only. The breakdown voltage increases by roughly 10 %
between enameled and mica insulated wire and there is no
significant difference between wires with one or two layers of
mica. The measurement incertitude is too high compared to the
influence of the support. The streamer starts in the oil, the only
impact of the support is its permittivity which modifies the
electric field distribution between oil and the solid support. Figure 8. Influence of the insulation safety factor on the overall transformer
volume. Figure 7. Simulation showing the electrical field on a section of the assembly. 7 CONCLUSION Combining experimental breakdown tests and Finite
element Analyses of the same set-up, allows to easily
determining the actual electrical field distribution inside the
insulation material under evaluation. As soon as the electrical
field distribution becomes inhomogeneous because of a
complex geometry, and when an insulation system is
composed of different materials, numerical field’s simulations
are necessary to identify the weak point in the insulation
system/assembly. Alternatively,
insulation
materials
datasheets can be directly used for FEA. In the specific case of
HV pulse transformers the weakest point where a breakdown
phenomenon starts is in the oil. The selected insulation safety
factor greatly influences the pulse transformer optimization in
terms of volume (and cost). Figure 7. Simulation showing the electrical field on a section of the assembly. 6 EFFECTS OF SAFETY FACTORS ON
TRANSFORMER PERFORMANCES Once the breakdown field has been determined, a safety
factor is considered to ensure that no breakdown will occur. The determination of this factor is based on experience and
empirical rules. An environment used for the optimal design
of pulse transformer described in [3] is used to perform a
sensitivity analysis of the performances versus the insulation
safety factor selected for a high voltage pulse transformer. The considered pulse transformer must produce 29 MW
pulses of 140 µs length at 180 kV secondary, 15 kV primary. The voltage is considered as stabilized when the voltage at
secondary remains within ±0.45\% of the nominal voltage. The time between the beginning of the pulse and stabilization
must be lower than 8 µs and the maximum allowed secondary
voltage overshoot is 1%. ACKNOWLEDGMENT This work is supported by CERN, Switzerland where the
high voltage tests have been realized. 5.1 TESTED ASSEMBLY DESCRIPTION Density Plot: |E|, V/m
3.810e+007 : >4.000e+007
3.620e+007 : 3.810e+007
3.430e+007 : 3.620e+007
3.240e+007 : 3.430e+007
3.050e+007 : 3.240e+007
2.860e+007 : 3.050e+007
2.670e+007 : 2.860e+007
2.480e+007 : 2.670e+007
2.290e+007 : 2.480e+007
2.100e+007 : 2.290e+007
1.910e+007 : 2.100e+007
1.720e+007 : 1.910e+007
1.530e+007 : 1.720e+007
1.340e+007 : 1.530e+007
1.150e+007 : 1.340e+007
9.600e+006 : 1.150e+007
7.700e+006 : 9.600e+006
5.800e+006 : 7.700e+006
3.900e+006 : 5.800e+006
<2.000e+006 : 3.900e+006
Solid support
Oil
Wire
Mica layer
Plane electrode
40
20
10
2
|E| kV/mm Figure 8 shows the relation between the safety factor and the
volume of the pulse transformer. The support material does
not influence significantly the size. For low safety factor the
relation is approximately proportional. For safety factor above
3, the volume of the transformer increase more rapidly. REFERENCES [1] H. Murase, S. Okabe, T. Kumai, H. Takakura, M. Takahashi and H. Okubo, ”Systematization of insulation design technology for various
electric power apparatus,” Dielectrics and Electrical Insulation, IEEE
Transactions on , vol.13, no.2, pp.400,407, April 2006 [1] H. Murase, S. Okabe, T. Kumai, H. Takakura, M. Takahashi and H. Okubo, ”Systematization of insulation design technology for various
electric power apparatus,” Dielectrics and Electrical Insulation, IEEE
Transactions on , vol.13, no.2, pp.400,407, April 2006 The pulse transformer that has the smallest volume is found
by an optimization procedure. pp
p
[2] R.E. HEBNER, ”Measurement of electrical breakdown in liquids”,
liquids”, in The liquid state and Its Electrical Properties, 1987, p. 519-
537, Plenum Press, New York and London. The safety factor is defined as the ratio between the
breakdown field in the oil Ebreakdown and the maximum
electrical field in the oil insulation Emax considered in the
design phases. ,
,
[3] S. Candolfi, P. Viarouge, D. Aguglia and J. Cros, “Finite element based
optimal design approach for high voltage pulse transformers” , Pulsed
Power Conference (PPC), San Francisco 2013, pp.1-6. [3] S. Candolfi, P. Viarouge, D. Aguglia and J. Cros, “Finite element based
optimal design approach for high voltage pulse transformers” , Pulsed
Power Conference (PPC), San Francisco 2013, pp.1-6. [4] S. Blume, and J. Biela, Optimal Transformer Design for Ultraprecise
Solid State Modulators, IEEE Transactions on Plasma Science,
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Oct. 2013, pp. 2691-2700
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https://openalex.org/W2772091186
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https://europepmc.org/articles/pmc5756874?pdf=render
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English
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Nature versus nurture? Consequences of short captivity in early stages
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Ecology and evolution
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cc-by
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K E Y W O R D S K E Y W O R D S
domestication, fitness, isotope, migration, population restoration, reintroduction, Salmo salar,
zoo K E Y W O R D S
domestication, fitness, isotope, migration, population restoration, reintroduction, Salmo salar,
zoo K E Y W O R D S
domestication, fitness, isotope, migration, population restoration, reintroduction, Salmo salar,
zoo Correspondence Correspondence
Jose L. Horreo, Department of Biodiversity
and Evolutionary Biology, National Museum of
Natural Sciences (CSIC), Madrid, Spain. Email: horreojose@gmail.com This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited. Jose L. Horreo1
| America G. Valiente2 | Alba Ardura2 | Aida Blanco2 |
Claudia Garcia-Gonzalez2 | Eva Garcia-Vazquez2 Jose L. Horreo1
| America G. Valiente2 | Alba Ardura2 | Aida Blanco2 |
Claudia Garcia-Gonzalez2 | Eva Garcia-Vazquez2 1Department of Biodiversity and Evolutionary
Biology, National Museum of Natural Sciences
(CSIC), Madrid, Spain Ecology and Evolution. 2018;8:521–529. © 2017 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Funding information g
J. L. Horreo was supported by a MINECO
Spanish postdoctoral grant IJCI-2015-23618. This study was funded by the Asturias
Regional Government, the INTERREG project
AARC, and the Grant GRUPIN-2014-093. This
is a contribution from the Marine Observatory
of Asturias and the Research Group ARENA. Abstract Abstract
Biological changes occurring as a consequence of domestication and/or captivity are
not still deeply known. In Atlantic salmon (Salmo salar), endangered (Southern Europe)
populations are enhanced by supportive breeding, which involves only 6 months of
captive rearing following artificial spawning of wild-collected adults. In this work, we
assess whether several fitness-correlated life-history traits (migratory behavior, stray-
ing rate, age at maturity, and growth) are affected by early exposure to the captive
environment within a generation, before reproduction thus before genetic selection. Results showed significant differences in growth and migratory behavior (including
straying), associated with this very short period of captivity in natural fish populations,
changing even genetic variability (decreased in hatchery-reared adults) and the native
population structure within and between rivers of the species. These changes ap-
peared within a single generation, suggesting very short time of captivity is enough for
initiating changes normally attributed to domestication. These results may have po-
tential implications for the long-term population stability/viability of species subjected
to restoration and enhancement processes and could be also considered for the man-
agement of zoo populations. 2Department of Functional Biology, University
of Oviedo, Oviedo, Spain eceived: 12 August 2017 | Revised: 14 September 2017 | Accepted: 15 September 2017 eceived: 12 August 2017 | Revised: 14 September 2017 | Accepted: 15 September 2017 Received: 12 August 2017 | Revised: 14 September 2017 | Accepted: 15 September 2017
DOI: 10.1002/ece3.3555 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited In this study, we focused on different key life-history traits that
have been documented to change significantly with domestication
in Atlantic salmon: migratory behavior (determined through isotope
analysis of scale tissue), straying rate (measured with genetic infor-
mation), age at maturity (also determined from scales), and growth—
therefore potential fitness (determined from condition factor [CF]). Stable isotope analysis offers the potential to reconstruct food webs
(Hutchinson & Trueman, 2006; Syväranta, Vesala, Trask, Ruuhijärvi, &
Jones, 2008), assess changes in trophic level (Wainwright, Fogarty,
Greenfield, & Fry, 1993) and diet preference (Pruell, Taplin, & Cicchelli,
2003), and analyze ecosystem responses to decadal climate forcing
cycles (Satterfield & Finney, 2002), to name some applications. Here, it
was employed to asses changes in trophic level and to infer differences
in the marine-growing region (MacKenzie et al., 2011). Our hypothesis
was these traits may be altered by captivity rearing (nurture) within a
generation, before reproduction thus before genetic selection (nature). North Iberian populations were selected as a case study. In this region,
supportive breeding is based on artificial spawning of wild mature
individuals and rapid release of their offspring—after only 6 months
in hatchery—for an average age at maturity of 3 years (Horreo et al.,
2012). Therefore, most of their life they are in the wild environment. One example of a recently domesticated species with co-occurring
wild populations is Atlantic salmon (Salmo salar): domestic stocks are
so different from wild populations that the name Salmo domesticus
has been proposed for the former (Gross, 1998). As the first steps
of captivity, changed temperature during egg maturation influences
egg production of their offspring (Jonsson & Jonsson, 2016), as well
as changes in growth in both gonadal (Jonsson, Jonsson, & Finstad,
2014) and body sizes (Finstad & Jonsson, 2012) in later stages. Reduced genetic diversity, rapid growth, and advanced age at matu-
rity (e.g., Fleming & Einum, 1997; Jonsson & Jonsson, 2006), as well
as salmon migratory behavior (Jonsson, Jonsson, & Hansen, 1990,
2003a, 2003b), are other of the changes known to be induced by cap-
tivity in Atlantic salmon. Footprints of selection throughout the ge-
nome were already detected in early stages of domestication (after
5–6 generations of captivity; Mäkinen, Vasemägi, McGinnity, Cross,
& Primmer, 2015). Whatever the causative mechanisms and genes involved, species
that adapt to captivity experience genetic changes, and the number of
generations in captivity is directly associated with the magnitude of
genetic differences between wild types and domesticates (Frankham,
2008). Divergence of captive populations from wild populations is of
great importance to conservation programs. In an attempt to save en-
dangered populations, managers, conservationists, and scientists have
developed strategies of supportive breeding, which can include arti-
ficial crosses in captivity, as well as release of offspring into the wild. However, these well-intentioned initiatives can promote maladaptive
traits as natural selection and can be distorted by human interven-
tion (e.g., Bestgena, Zelaskoa, Comptona, & Chartb, 2008; Jónás et al.,
2010; Levin, Zabel, & Williams, 2001; Massaro et al., 2013). Reducing
the number of captivity generations is expected to reduce the extent
of adaptation to captivity (Frankham, 2008) and potentially increase
the success of reintroduction of populations into the wild from cap-
tivity. However, the maximum number of captivity generations that
can occur without hindering future adaptation in the wild is not clear. Some authors postulate that changes from wild to domestic type can
occur quite rapidly (Jensen, 2014; Trut et al., 2009), even within only
one generation of domestication (Christie, Marine, French, & Blouin,
2011). This includes heritable changes in the expression of hundreds
of genes, probably involved in adaptation to high densities in hatchery
conditions compared to the wild environment (Christie, Marine, Fox,
French, & Blouin, 2016). In summary, it appears that durable genetic
changes in captive populations can occur in as little as one generation
and that these changes can have effects across multiple generations
(e.g., epigenetic and heritable changes; Jonsson & Jonsson, 2014). processes of inadvertent selection in the captive environment (Horreo
et al., 2008), and changes induced in the population structure (Horreo,
Machado-Schiaffino, Ayllon, et al., 2011), among others. Hatchery-
reared and wild individuals differ for smolt run time (Petersson & Järvi,
2006) and straying (higher than usual values; Horreo, de la Hoz, Pola,
Machado-Schiaffino, & Garcia-Vazquez, 2012; Jonsson et al., 2003a,
2003b; Moran, Pendas, Garcia-Vazquez, & Izquierdo, 1994; Vasemägi
et al., 2001). Regional-scale studies in the Baltic Sea have also demon-
strated that both ages at return and distribution in the Baltic main
basin differ between hatchery-reared and wild Atlantic salmon (e.g.,
Jutila, Jokikokko, Kallio-Nyberg, Saloniemi, & Pasanen, 2003). After 10 generations of captivity, genes involved in
growth seemed to be upregulated while immune-related and environ-
mental information processing systems were downregulated in juve-
niles (Bicskei, Bron, Glover, & Taggart, 2014). Reduced responsiveness
to stress was also reported after 10 hatchery generations (Solberg,
Skaala, Nilsen, & Glover, 2013). 1 | BACKGROUND consequences that would be anomalous in the wild but are success-
ful in domestic populations, including, for example, fertile offspring in
crosses between related taxa (Dobney & Larson, 2006). Many genes
of moderate effect would be involved in the differences between do-
mesticated stocks and their wild-type progenitors (Albert et al., 2011;
Kukekova et al., 2011; Trut, Oskina, & Kharlamova, 2009), and different
genomic imprinting (inheritable epigenetic change) in domestic and wild
individuals would contribute to explain at least a part of the changes oc-
curring in domestication processes (e.g., O’Doherty, MacHugh, Spillane,
& Magee, 2015; Trut et al., 2009; Wilkins, Wrangham, & Fitch, 2014). The genetic and physiological changes occurring as a consequence of
domestication are not easy to generalize because they obviously de-
pend on the particular species, rearing system, and domestication time. The genetic and physiological changes occurring as a consequence of
domestication are not easy to generalize because they obviously de-
pend on the particular species, rearing system, and domestication time. Most domestication processes involve behavioral, morphological, and
physiological changes (e.g., faster growth), and generally a loss of genetic
diversity in domesticated stocks (Horreo, 2015; Lacy, 1987). Increased
genetic and phenotypic flexibilities in domesticates (as compared to
wild counterparts) have been proposed to explain behaviors and their Most domestication processes involve behavioral, morphological, and
physiological changes (e.g., faster growth), and generally a loss of genetic
diversity in domesticated stocks (Horreo, 2015; Lacy, 1987). Increased
genetic and phenotypic flexibilities in domesticates (as compared to
wild counterparts) have been proposed to explain behaviors and their | 521
www.ecolevol.org www.ecolevol.org 521 Ecology and Evolution. 2018;8:521–529. 522 HORREO et al. 2.6 | Statistical analysis For determining the variables contributing more to the dataset vari-
ance, an exploratory principal component analysis (PCA) was carried
out, with correlation matrix and 9,999 bootstrap. Eigenvalue cutoff
was 0.7. This preliminary inspection of the dataset served to identify
the main component variables and their relationships. Normality was
tested in the dataset by analysis of residuals and a correlation test, and
variables were transformed for normalization when required. The PCA
was performed in the whole dataset, without excluding any datum. 2.1 | Study populations and sample collection North Iberian Atlantic salmon populations, located at the natural south-
ern edge of the species’ distribution, are among the most affected by en-
vironmental changes (Horreo, Machado-Schiaffino, Griffiths, et al., 2011). Asturian salmonids are managed by the Regional Government of Asturias
in collaboration with fishermen associations (especially in terms of popu-
lation enhancement activities). Supportive breeding in these populations
has produced up to ten million juveniles released in the rivers between
1992 and 2008 (Castillo et al., 2008). All of the breeders employed for
supportive breeding in Cares and Sella rivers in 2005 were sampled (via
adipose fin clips) and genotyped for analysis. In the hatcheries, located
close to the rivers of origin, breeders are crossed via pooling of the ova
of each female with the sperm of three males and juveniles are released
6 months later into the river from which their parents were taken. Scale
samples of a portion of the returning adults (498 from River Cares and
313 from River Sella, rivers with census sizes in the year 2009 of 1,073
and 1,464 individuals, respectively) were kindly provided by sport anglers Wild anadromous Atlantic salmon populations are declining
throughout all its natural distribution (Chaput, 2012). In this spe-
cies, supportive breeding has resulted in introgression of hatchery/
foreign genes into wild populations (Horreo et al., 2014) through the 523 HORREO et al. from legal salmon catches in 2007–2009. The scales were preserved dry
in paper envelops. Fish length and weight were also recorded. From pedi-
gree analysis, the early rearing of each adult (short hatchery rearing or
wild) was determined (Horreo et al., 2012). the natural age distribution of returning adults in these populations
(Juanes et al., 2007). Acid pretreatment of scales was not performed as such pretreat-
ment results in biologically insignificant changes in the bulk isotopic
composition (Sinnatamby et al., 2009). Scales were manually cleaned
using forceps to remove adherents (lipids and guanine). The last sum-
mer of growth at sea was excised as source sample to measure iso-
topes. In 1SW (one-sea-winter) fish, the summer section from the edge
of the scale was sampled. In MSW (multisea-winter) fish, the summer
immediately before the final winter at sea was sampled (MacKenzie
et al., 2011). Regenerated scales were not employed for analyses. 2.3 δ(‰)=103 [Rsample∕Rstandard −1] In a previous work (Horreo et al., 2012), pedigree tests were carried out
with seven microsatellite loci in more than 800 salmon individuals in both
rivers and the breeders employed for their production in hatcheries in
order to identify hatchery descendants and their hatchery (thus river) of
origin. The mentioned microsatellite information was here employed to
estimate genetic variability and population differentiation between the
group of individuals issued from supportive stock and the group of wild
individuals. Effective number of alleles, observed and expected heterozy-
gosity, and population differentiation (FST value) among groups were cal-
culated with Genodive (Merimans & Van Tienderen, 2004). Straying rate
of wild individuals between rivers was estimated from between-river
gene flow, measured as the number of migrants per generation through
the mean frequency of private alleles, with the online GENEPOP soft-
ware (http://genepop.curtin.edu.au). Resulting δ13 C values in the collagen are related with the isoto-
pic composition of other tissues under different conditions of diet and
growth rates (Satterfield & Finney, 2002; Sinnatamby et al., 2009). More importantly for this study, carbon isotopes are associated with
marine feeding areas in Atlantic salmon (MacKenzie et al., 2011); dif-
ferences between groups of the same age could be considered indi-
cators of different diets. Nitrogen is more strongly fractionated than
carbon during dietary assimilation (MacKenzie et al., 2011), being
therefore an indicator of trophic level. 2.2 | Age determination The age of anadromous salmonids is given as X.Y, X, and Y being the
number of years in freshwater and at sea, respectively. Y can be one
(one-sea-winter or grilse, 1SW) or more (multisea-winter, MSW) years. Adult Atlantic salmon in the study region are predominantly 1.2 plus a
variable proportion of 1.1 grilse and a few 1.3, 2.1, and 2.2 individuals
(Juanes, Perez, & Garcia-Vazquez, 2007). Therefore, young of the year
released in 2005 are expected to return to the river as adults in 2007,
2008, and 2009 at the ages 1.1, 1.2/2.1, and 1.3/2.2, respectively. Age reading was performed from dried scales based on scale growth
circuli and double-checked by different researchers, as published by
(Horreo et al., 2012). Samples were cut into small pieces and weighed to approximately
0.60 mg in 5 × 3.5 mm diameter tin cups (several scales of each in-
dividual were needed to obtain enough amount of sample mate-
rial). Isotopes ratios were determined by elemental analysis using
l-glutamic acid, sugar, wheat flour, USGS40 and USGS41 as calibra-
tion standards. The resulting isotope ratios of carbon (13C/12C) and
nitrogen (15N/14N) of the analyzed samples were reported versus
the Pee Dee Melemnite (PDB) (carbon) and atmospheric air (nitrogen)
standards [26] as: 2.4 | Condition factor As an indicator of growth, we have chosen CF for all the samples with
available length and weight data because it is an indicator of the size
and shape of fish and principally of growth rate (Gjedrem, 2005). It is
also considered to indicate salmon health and fitness potential (Miller,
Miller, Mills, & Sheehan, 2014). Its formula is Very divergent outliers were removed from further analysis. When
it was not possible to assume normality, nonparametric tests were car-
ried out. Comparison between multiple groups of samples was carried
out using analysis of variance (ANOVA) tests or their nonparametric
equivalent for medians Kruskal–Wallis. Post hoc tests were performed
after significant ANOVA, as pairwise comparison between means em-
ploying t tests with the corresponding correction in case of unequal
variances. Levene’s test was applied for checking variance equality. Analyses were performed with PAST software (Hammer, Harper, &
Ryan, 2001). 3.2 | Principal component analysis Carbon (δ13C) and nitrogen (δ15N) values for wild and early cap-
tive Atlantic salmon from Sella and Cares rivers are shown in Figure 1. For the marine-growing area in the last summer (it leaves a signa-
ture on carbon isotopes; MacKenzie et al., 2011), we tested if the
free individuals from different rivers and years had significantly dif-
ferent mean δ13C values (Table 3). Two-way ANOVA showed signifi-
cant differences between rivers (F = 6.355, p = .014) and among age
classes (F = 48.29, p ≪ .001), with significant interaction (F = 10.31,
p = .0001). Differences between ages were expected from highly sig-
nificant correlation between δ13C and salmon age in the whole dataset
(r = .491, df = 122, p ≪ .0001). In contrast, from two-way ANOVA, the
early captives did not differ significantly between rivers (F = 1.256,
p = .268), but did, as expected differ significantly in early captives
among ages (F = 19.19, p ≪ .001), while there was no significant inter-
action between rivers and ages. Thus comparisons of free individuals
with early captives were made independently for each year in post
hoc paired tests. The wild individuals of the two rivers were pooled
together when no significant differences were found between them. The difference in mean values of carbon isotopes (δ13C; Table 3) be-
tween wild individuals caught in 2007 in Sella and Cares rivers was
significant (t = 4.514, p = .0009 for unequal variances), thus they In the whole dataset of isotope content (carbon as δ13C and nitro-
gen as δ15N) and life-history traits (age, CF, living type as early hatch-
ery rearing in months), three principal components were significant
(Eigenvalue > 0.7; Table 2). The two main components contributed a
total of 69% variance (Table 2). The variables contributing more to
each component were age and δ15N in PC1, and living type and CF
in PC2. 524 |
3 | RESULTS 524 HORREO et al. (r = −.212, df = 122, p = .017), and no other variable correlated signifi-
cantly with the type of early living (r = −.083, −.023, and −.114 with
age, δ15N, and δ13C respectively, all not significant). Highly significant
correlations between the two isotopes and the variables age and CF
were found (data are shown below). 3.1 | Genetic variation The genetic variability of early captive and wild individuals (Table 1)
was relatively high, with mean effective number of alleles (Eff_Na) of
5.105, mean observed heterozygosity (Ho) of 0.787, and mean ex-
pected heterozygosity (He) of 0.786. Assessing differences between
origins and rivers (thus four groups were employed: hatchery sella,
hatchery cares, wild sella, wild cares), significant differences in Eff_Na
occurred between early captive and free individuals (t test = −4.33,
p < .05), being fewer in early captive (mean Eff_Na = 4.66) than in free
ones (mean Eff_Na = 5.55). Significant differences were not found
between these groups either for Ho (t test = −0.51, p = .66) or He (t
test = −2.68, p = .11). 3.3 | Migratory behavior data Significant differences were not found between early captive and free
individuals for the number of years at sea, neither among rivers (two-
way ANOVA for the factors age and living type: F = 0.05 and p = .83
for river, F = 1.00 and p = .32 for living type, no interaction between
the two factors p = .99). Because no genetic differences were found between rivers, stray-
ing rate of free individuals was here accounted through Nm, that is,
the number of migrants per generation, estimated from the mean
frequency of private alleles present on each dataset. The result was
8.04%, clearly lower than 49% found for early captive individuals in
the same populations and years (Horreo et al., 2012). For the number
of individuals analyzed, the difference between these percentages was
highly significant (z = −5.92, p = .00). FST values revealed significant genetic differentiation (p < .05) be-
tween early captive individuals of River Cares and all the other groups
(free Cares and both free and early captive Sella individuals). No ge-
netic differences were found between rivers even when analyzing only
the wild fish (FST = 0.00, p = .170). 2.5 | Isotope analysis For reasons of saving material for the collection of Atlantic salmon
scales for Asturias Principality, isotope measurements were per-
formed only for the samples with many scales, because the analytical
protocol destroys the scale. A total of 63 and 62 samples from Cares
and Sella rivers respectively were taken, in proportions that reflected 3.2 | Principal component analysis Rearing environment and CF loadings on PC2 displayed posi-
tive and negative signs and were significantly negatively correlated TABLE 1 Genetic variability of early captive (EC) and wild (W)
individuals of Sella and Cares rivers, measured as the effective
number of alleles (Eff_Na), the observed heterozygosity (Ho) and
expected heterozygosity (He) Population
Eff_Na
Ho
Hs
EC-Sella
4.741
0.794
0.781
EC-Cares
4.578
0.768
0.763
W-Sella
5.361
0.774
0.795
W-Cares
5.739
0.811
0.807 PC 1
PC 2
PC 3
PC 4
PC 5
Eigenvalue
(% var)
2.42 (48.48)
1.03 (20.54)
0.83 (16.55)
0.48 (9.64)
0.24 (4.79)
Age
0.57379
0.13866
−0.023808
−0.32655
0.73779
CF
0.36419
−0.32926
0.76361
0.4192
−0.011167
N
0.55643
0.20754
0.059087
−0.4458
−0.66715
C
0.45908
0.099205
−0.55228
0.68268
−0.091336
Rearing
−0.13331
0.90523
0.32837
0.22989
0.045893
Bold values show the variables contributing more to each component 525 HORREO et al. FIGURE 1 Plot of carbon (δ13C) on nitrogen (δ15N) values for wild
and early captive Atlantic salmon from Sella and Cares rivers. Green:
wild Sella salmon; blue: wild Cares salmon; purple: early captive in
Sella; orange: early captive in Cares differences in δ15N mean values both between rivers (F = 4.43, p = .04)
and among years (F = 54.1, p ≪ .001). In 2009, differences among the
wild group and the groups of early captives entering River Sella and
River Cares were not significant (t = 0.097, p = .923). In 2008, the dif-
ferences were not significant either (t = 2.04, p = .05), as they were
not in 2007 (t = 0.542, p = .616). Therefore, we compared mean val-
ues of δ15N between wild and early captives joining the two rivers
each year. None of the comparisons were statistically significant (data
not shown), thus differences in trophic level between wild and early
captive individuals were not found any year. Condition factor mean values of wild individuals (Table 3) did not
differ significantly between rivers, although they did clearly among
ages (Two-way ANOVA: F = 0.049 with p = .826, N.S., for the fac-
tor river; F = 9.054, p = .0003 for the factor living type; F = 2.198,
p = .120, N.S., for the interaction). CF correlated positively and signifi-
cantly with age in the whole dataset (r = .402, df = 122, p = .003). 3.2 | Principal component analysis For
early captive individuals, however, no significant difference was found
between ages (neither between rivers), with F = 0.75 and p = .39 for
river effect, F = 3.06, p = .06 for living, not significant interaction
(F = 0.37, p = .69). Without differences between rivers for any type
of early living, the data of the two rivers were pooled together for a
two-factor ANOVA for testing early captivity and age as factors. The
results showed significant differences between the two living types
(F = 4.15, p = .04) as well as among ages, indeed (F = 10.45, p ≪ .001),
with no significant interaction (F = 0.49, p = .61). From significant
negative correlation between early captivity months and CF reported
above for the whole dataset, we expected lower CF for early hatchery-
reared individuals. Accordingly, individuals raised in captivity during
their first months exhibited generally lower CF than wild individuals
of the same age (0.942 and 0.965 for early captive and wild grilse, re-
spectively; 1.01 and 1.06 respectively for 2-sea-winter individuals; the
few 3-sea-winter individuals, however, exhibited the opposite trend
with 1.046 for wild and 1.064 for early captives), although pairwise
differences between living types were significant only for the most
abundant 2-sea-age class (test for samples with unequal variance,
t = 2.12, p = .03). FIGURE 1 Plot of carbon (δ13C) on nitrogen (δ15N) values for wild
and early captive Atlantic salmon from Sella and Cares rivers. Green:
wild Sella salmon; blue: wild Cares salmon; purple: early captive in
Sella; orange: early captive in Cares were tested separately against early captives. In the River Sella, they
were significantly different (t = 2.371, p = .037) but not in River Cares
(t = 1.05, p = .315). For those caught in 2008, there were no significant
differences between the wild of the two rivers (t = 1.49, p = .144) and
the two rivers were joined together. The difference between the two
groups (wild vs. early captive) was significant (t = 2.07, p = .04). Finally,
for the individuals caught in 2009, there was no significant difference
between Cares and Sella wild individuals (t = 0.581, p = .567), and the
difference between wild and early captive for mean δ13C values was
highly significant (t = 3.26, p = .004). 3.4 | Feeding and growth Regarding nitrogen isotopes (Table 3), indicators of trophic level,
for wild individuals, the two-way ANOVA did not reveal differences
between rivers (F = 1.84, p = .179), but highly significant difference
among years (F = 54, p ≪ 0.001), as expected from significant correla-
tion between δ15N and age in the whole dataset (r = .755, df = 122,
p ≪ .001). For early captives, however, there were significant TABLE 3 Number of individuals (n) employed for salmon condition factor and isotope analyses depending on their river (Sella and Cares),
origin (early captive: EC, wild: W), and ages (river age.sea age). Condition factor (CF), carbon (δ13C), and nitrogen (δ15N) means are given with
standard deviations in brackets TABLE 3 Number of individuals (n) employed for salmon condition factor and isotope analyses depending on their river (Sella and Cares),
origin (early captive: EC, wild: W), and ages (river age.sea age). Condition factor (CF), carbon (δ13C), and nitrogen (δ15N) means are given with
standard deviations in brackets Sella
Cares
Age
n
δ15N
δ13C
CF
n
δ15N
δ13C
CF
Origin
W
1.1
10
10.123 (0.823)
−16.318 (0.683)
0.995 (0.104)
11
10.089 (0.620)
−16.366 (0.217)
0.937 (0.127)
1.2
16
11.803 (0.593)
−16.782 (0.254)
1.054 (0.064)
16
11.769 (0.578)
−16.929 (0.500)
1.065 (0.071)
2.2
2
11.925 (1.152)
−17.675 (0.134)
0.953 (0.037)
6
11.785 (0.887)
−16.985 (0.605)
1.106 (0.043)
1.3
5
12.770 (0.607)
−18.942 (0.426)
1.036 (0.033)
1
13.060 (0.000)
−16.900 (0.000)
1.097 (0.000)
EC
1.1
10
10.062 (0.435)
−16.464 (0.324)
0.949 (0.079)
10
10.168 (0.439)
−16.470 (0.223)
0.927 (0.148)
1.2
16
12.011 (0.573)
−16.770 (0.330)
1.017 (0.119)
16
11.609 (0.736)
−16.607 (0.482)
0.999 (0.111)
2.2
1
11.910 (0.000)
−16.830 (0.000)
1.075 (0.000)
2
11.745 (0.007)
−16.685 (0.276)
1.098 (0.116)
1.3
2
12.800 (0.579)
−17.830 (1.159)
1.108 (0.082)
1
12.820 (0.000)
−18.680 (0.000)
0.977 (0.000) 526 |
FIGURE 2 Correlation between δ15N and condition factor (CF)
at individual level, for the region. Green: wild Sella salmon; blue: wild
Cares salmon; purple: early captive in Sella; orange: early captive
in Cares. δ15N and CF values were highly significantly correlated
(p < .01) 526 | HORREO et al. 526 of the species with increased gene flow between rivers. Thus, sup-
portive breeding could not only affect the river where it is done, but
also the whole region through extremely high straying rates. 3.4 | Feeding and growth Carbon isotope analysis also suggests differences between early
captive and wild Atlantic salmon in the marine areas where they stayed
at least the last summer before entering the river. Significant differ-
ences were found between the two life styles in 2008 and 2009, and
for River Sella in 2007. MacKenzie et al. (2011) found differences
between marine areas based on carbon isotopes between individu-
als of different ages, and here, the data also suggest the occurrence
of differences between captive and wild Atlantic salmon, so relatively
short exposure to the captive rearing environment appears to have
the potential to affect adult behavior in the populations studied. Early
nurture would therefore influence adult behavior in natural popula-
tions. The main novelty of our study is that these changes appeared
within a generation, suggesting very short time of captivity produces
either epigenetic changes or selection of phenotypes, which may lead
to evolve differences between hatchery and wild individuals if the trait
has a genetic basis, and that it is enough for initiating changes nor-
mally attributed to domestication in this species (Christie et al., 2011;
Jonsson et al., 2003a, 2003b; Jutila et al., 2003; Moran et al., 1994;
Vasemägi et al., 2001). FIGURE 2 Correlation between δ15N and condition factor (CF)
at individual level, for the region. Green: wild Sella salmon; blue: wild
Cares salmon; purple: early captive in Sella; orange: early captive
in Cares. δ15N and CF values were highly significantly correlated
(p < .01) Despite significant differences between living types (free vs. cap-
tive during the first months) for CF but not for δ15N, CF was highly sig-
nificantly correlated with δ15N in this dataset (Figure 2; p < .01). This
result was the same even after outliers removal (CF values lower than
0.8 and higher than 1.3; p < .01). Atlantic salmon size is directly related to fitness (Garant, Dodson, &
Bernatchez, 2003; Jonsson, Jonsson, & Fleming, 1996). In other stud-
ies, differences in size between captive and wild juveniles occurred
after the river stage, captive-origin Atlantic salmon released as juve-
niles were observed to be generally smaller than wild counterparts at
the smolt life stage (De Mestral, O’Reilly, Jones, Flanagan, & Herbinger,
2013). Also reduced fitness of hatchery individuals have been found
after one generation of captive breeding (Jonsson & Jonsson, 2006). 3.4 | Feeding and growth In our study, we found significant differences between early captive
and wild individuals for CF, which is a fish growth indicator (Gjedrem,
2005); it was significantly higher in free individuals than in the others,
suggesting that wild individuals return healthier (Miller et al., 2014). Nitrogen isotopes, a signal of trophic level (Jutila et al., 2003), did not
reveal differences between the two life types here compared; there-
fore, differences in CF would not be likely due to different feeding be-
havior but perhaps to some early modifications by captive rearing, as
suggested by Christie et al. (2011) after one generation of domestica-
tion. Christie et al. (2016) suggested such rapid modifications could be
due to adaptation to high density in hatchery conditions or, in our case,
even due to differential mortality during the first months of captivity. On the other hand, nitrogen and CF were highly positive correlated
(Figure 2), indeed, principally due to highly significant differences
among ages for trophic level and CF occurring for this species. CF is an
heritable trait that affects salmon fitness (Carlson & Seamons, 2008). Decreased CF in early captive individuals that are released in the rivers
to supplement natural populations should be taken as an alert signal
regarding this management practice. Especially in threatened popu-
lations such as the ones here studied (Horreo, Machado-Schiaffino,
Griffiths, et al., 2011), negative consequences such as reduced natural
egg production in the artificially enhanced populations would be ex-
pected via CF decrease. REFERENCES These results may have potential implications not only regard-
ing evolution and conservation issues of this species, but also of
other species subjected to population restoration and/or enhance-
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domestication-related changes, thus early rearing in conditions as nat-
ural as possible should be seriously considered if the animals are going
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& Garcia-Vazquez, E. (2008). Jose L. Horreo
http://orcid.org/0000-0002-9851-7651 Jose L. Horreo
http://orcid.org/0000-0002-9851-7651 AUTHOR CONTRIBUTIONS The results above discussed were obtained for microsatellite loci
and logically are not expected to be related with natural selection due
to early captivity as they correspond to two fractions of the same wild
populations; it should be recalled here that the supplementary stock
is produced every year from wild individuals. Nature, as represented
by genetics, cannot be changed within a generation. Nurture, in this
case, a short period of captivity during their early development, has
notwithstanding produced significant changes in Atlantic salmon
that can be seen in their adulthood, after the marine period of life. These changes may have potential implications for long-term popu-
lation stability and, for this reason, alternative methods of population
enhancement such as recovering lost spawning areas (Horreo, De La
Hoz, Machado-Schiaffino, Pola, & Garcia-Vazquez, 2011) should be
explored and would be strongly recommended for conservation of the
wild remnants of this valuable species. JLH, AGV, and EGV designed and performed both data and statistical
analyses. AGV, AA, and CG-G contributed with data generation. JLH
and EGV wrote the manuscript. Significant reductions in the effective number of alleles were found
in the individuals identified as released in the river after early captivity
(Table 1), which is logical because only a fraction of the population is
employed for supplementary breeding (Horreo et al., 2012). Such re-
duced genetic variability in supplementary stocks had been previously
detected in juveniles in this region (Horreo et al., 2008) and is here
reported for adults as well. Significant differences in allele frequencies
(FST) were also found between the early captive and free individuals of
River Cares. If early captive individuals represented the majority of the
population, these subtle differences would be highly risky. Together
with altered migratory behavior, these differences are altering the
population structure in the region (no genetic differences between
rivers were here found, so they are being diluted via straying) and all
the subjacent evolutionary processes implicated on it (Millar & Libby,
1991). Evolutionary genetic adaptation of species provides them a
resistance capacity to adverse environmental conditions (Frankel &
Soulé, 1981), so the observed changes would put at risk these popula-
tions, especially in adverse ecological conditions. Zoo populations are recommended to maintain their evolutionary in-
tegrity through more natural mating systems that include mate choice,
especially when they are employed for reintroductions in the wild
(Schulte-Hostedde & Mastromonaco, 2015). Considering the impor-
tance of early stages in domestication processes, it could be advisable
as well for zoo population management to maintain individuals in wild-
like conditions since very early stages, preferably since the birth, which
could increase the success of captive-born individuals in the wild. Zoo populations are recommended to maintain their evolutionary in-
tegrity through more natural mating systems that include mate choice,
especially when they are employed for reintroductions in the wild
(Schulte-Hostedde & Mastromonaco, 2015). Considering the impor-
tance of early stages in domestication processes, it could be advisable
as well for zoo population management to maintain individuals in wild-
like conditions since very early stages, preferably since the birth, which
could increase the success of captive-born individuals in the wild. ACKNOWLEDGMENTS J. L. Horreo was supported by a MINECO Spanish postdoctoral grant
IJCI-2015-23618. This study was funded by the Asturias Regional
Government, the INTERREG project AARC, and the Grant GRUPIN-
2014-093. This is a contribution from the Marine Observatory of
Asturias and the Research Group ARENA. CONFLICT OF INTEREST None declared. None declared. ORCID Jose L. Horreo
http://orcid.org/0000-0002-9851-7651 4 | DISCUSSION This work shows significant differences in both growth and migratory
behavior associated with a very short period of captivity (6 months)
in natural fish populations, in line with previous findings found with
smolts (Jonsson et al., 2003b). The straying rate of early captive
Atlantic salmon in Asturias was extremely high (49%; Horreo et al.,
2012), but in comparison with it, estimated straying of wild individuals
of the same populations and years was 8%. The methods employed for
estimating straying were completely different because one was based
on counting of strayed individuals and the other was based on the
presence of allelic differences between rivers. Although there are no
allele frequency differences between free-living individuals and early
captive ones, from these data it could be deduced that as a maximum
only roughly 8% of the strayed individuals reproduced. An alternative
(or concomitant) explanation could be that, at least in the last part of
the marine migration (coastal waters nearby the natal rivers), the route
may differ between early captive and free Atlantic salmon. It is known
that stocked salmonids have poor homing behavior (e.g., Jonsson
et al., 2003a, 2003b; Quinn, 1993); however, the enormous difference
found in this study, whether due to poor reproduction of early captive
or to direct differences in straying, would be attributable to only a few
months of captivity. This is surprising and suggests an impact of very
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ysis. Biological Conservation, 141, 355–363. https://doi.org/10.1016/j. biocon.2007.11.007 HORREO et al. 529 Genetic variation among Atlantic salmon in six Spanish rivers. Journal
of Fish Biology, 45, 831–837. https://doi.org/10.1111/jfb.1994.45. issue-5 Wilkins, A. S., Wrangham, R. W., & Fitch, W. T. (2014). The “domestica-
tion syndrome” in mammals: A unified explanation based on neural
crest cell behavior and genetics. Genetics, 197, 795–808. https://doi. org/10.1534/genetics.114.165423 O’Doherty, A. M., MacHugh, D. E., Spillane, C., & Magee, D. A. (2015). Genomic imprinting effects on complex traits in domesticated an-
imal species. Frontiers in Genetics, 6, 156. https://doi.org/10.3389/
fgene.2015.00156 How to cite this article: Horreo JL, Valiente AG, Ardura A,
Blanco A, Garcia-Gonzalez C, Garcia-Vazquez E. Nature versus
nurture? Consequences of short captivity in early stages. Ecol
Evol. 2018;8:521–529. https://doi.org/10.1002/ece3.3555 Petersson, E., & Järvi, T. (2006). Anti-predator response in wild and sea-
ranched brown trout and their crosses. Aquaculture, 253, 218–228. https://doi.org/10.1016/j.aquaculture.2005.08.012 Pruell, R. J., Taplin, B. K., & Cicchelli, K. (2003). Stable isotope ra-
tios in archived striped bass scales suggest changes in trophic
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1. Introduction The issue of media environment is very interesting, but too
complicated to be mapped and analyzed in its absolute entirety. Nowadays, the questions dealing with the penetrating of the info-
tainment into the area of news service are raised, together with the
questions of increasing interconnections with some areas of mar-
keting (advertising, public relations). To reach the aim we set five elementary hypotheses and to
increase the relevance of the research we used three research
methods. The results of the research could be consequently com-
pared and interpreted in wider connection. The basic pillar of the
research was the content analyses of texts (press releases and selec-
ted media contents). We used also questionnaires and non-structured
qualitative interviews with PR workers to ensure the complexity of
information. Regarding the fact that the dynamic marketing progress disturbs
the traditional procedures in the area of journalism, it has become
the subject matter of wide professional and academic interest, and
vice versa, the marketing workers are interested in the attributes
of media environment-functioning and creation of media content. The knowledge and perceptive procedures of the functioning
of media and marketing environment are the precondition to the
determination of the level of the usage of marketing (or PR) con-
tents in seemingly journalistic manifestations. Press releases and their reflection (media contents in the form
of editorial contributions) in the segment of print media (yellow
and serious journalism) and their online versions on the Internet,
as well as the editorial contributions of press agencies are the
subject matter of our research. The further stage of the research
was based on questionnaires and interviews with PR workers and
journalists. The article presents particular results of the research which has
been motivated by the need to explore the level of influence (and
impact) of public relations worker on journalism. This need was
accompanied by the attempt to look into the issue both from the
aspects of journalist and public relations worker. We focused on the
exploration of media environment (in relation to media market)
with respect to possibilities of being influenced by marketing, speci-
fically by the area of public relations. We focused on the exploration of media environment stressing
the borderline connections with marketing, primarily with the area
of public relations. 1. Introduction The aim included several points: – To confirm or confute the possibility of the penetrating of infor-
mation from the press releases to the area of news service. – To determine the influence of press release on the number of
media outputs. Slavka Pitonakova * PUBLIC RELATIONS AND JOURNALISM
PUBLIC RELATIONS AND JOURNALISM The paper offers the findings of possible interconnection between journalism and public relations. It was created as a reaction to the need
to explore the level of influence (and impact) of public relations worker on journalism. This need was accompanied by the attempt to look into
the issue both from the aspects of journalist and public relations worker. We focused on the exploration of media environment (in relation to
media market) with respect to possibilities of being influenced by marketing, specifically by the area of public relations. Keywords: public relations, journalism, interconnection Keywords: public relations, journalism, interconnection Keywords: public relations, journalism, interconnection An important inspiration for the research was one of the jour-
nalistic rules that a journalist should use information from different
sources at his/her work and the feeling that this rule is often for-
gotten and overlooked and PR materials have become an essential
source medium for journalism. * Slavka Pitonakova
Faculty of Humanities, University of Zilina, Slovakia, E-mail: slavka.pitonakova@fpv.uniza.sk 49
C O M M U N I C A T I O N S 1 / 2 0 1 2 G https://doi.org/10.26552/com.C.2012.1.49-54 https://doi.org/10.26552/com.C.2012.1.49-54 The research realization was conducted in several phases: with the media outputs in print media. We explored whether the
selected press releases in newspapers, magazines and online media
are published in: The first phase of the research: the study of relevant texts with
the aim to understand and analyze up to now knowledge relevant
for the research theme. – identical version, The second phase of the research: the determination of hypothe-
ses and the proposal of methodology (including data gathering from
subjective enunciations). – partially changed, not appended version, – partially changed and appended version, – not published. The third phase of the research: The research realization (con-
tent analysis, questionnaire, qualitative interviews). We also explored the possible interconnections between the
themes of press releases and their attractiveness to journalists (i. e. which of the press releases are the most interesting for the journa-
lists). When choosing the press releases we focused on the follow-
ing areas: politics, economy, culture and edifying activities. The fourth phase of the research: The analysis of data, inter-
pretation of findings and evaluation of the research. The selection of research sample The selection of research sample reflected the current situation
in Slovak media environment. Selected press releases and media
contents (technologically quite simple which allowed the realization
of their content analysis), the answers of respondents in question-
naires and interviews with PR workers were the subject matter of
the research. The content analysis of the products required the specifying of
documents for the research. We concentrated on written, publically
available institutional documents (print, online media and press
releases), which were examined in the context of preset themes
without regard to periodicity of their occurrence. We mainly focused
on press releases, and then print and online media published in
Slovakia. The underlying condition for the subsuming of an article
(in printed or electronic form) to the research was its particular
or general equivalence with examined press release published on
the Internet. Before the research realization five research hypotheses were
stated: Hypothesis No. 1: More than one third of published press
releases are the basic information source for the creation of jour-
nalistic contributions. Hypothesis No. 2: Journalists use press releases for their jour-
nalistic contributions without adequate verification of information
from different sources The selection of press releases was based on the method of
coincidental selection in respect to the formerly chosen thematic
fields and the subject matter of further analysis were only the press
releases which were completely or partially transformed into the
form of a particular media output (article in printed or electronic
media). Hypothesis No. 3: The theme of press release influences the
level of its importance for journalists (public). Hypothesis No. 4: PR workers economically pressure jour-
nalists. Hypothesis No. 4: PR workers economically pressure jour-
nalists. 30 press releases were the subject matter of our research. 14
articles published in different media with the name of their redac-
tors (redaction, agency) met our requirements for detailed analysis
(disclosure of similarity or identity with the prototext of press
release). Hypothesis No. 5: Positive relationships between PR workers
and journalists are the main reason of the press release penetrating
into the area of news service. 2. Research – To characterize the relationship between PR workers and jour-
nalists. The aim of the research was to map mutual similarities, diffe-
rences and interlinks between both areas (journalism and public
relations) and to set the level and causes of the penetrating of infor-
mation from press releases into the area of journalism. – To analyze the use of press release as an information source. [2] * Slavka Pitonakova
Faculty of Humanities, University of Zilina, Slovakia, E-mail: slavka.pitonakova@fpv.uniza.sk 49
C O M M U N I C A T I O N S 1 / 2 0 1 2 G 50
G
C O M M U N I C A T I O N S 1 / 2 0 1 2 The specific focus was laid on the attempt to find connections
between press releases and news service in so called serious daily
papers in Slovakia. No direct connection was found. Surprisingly,
we have found out that in the case of similarities between press
releases and media response the only sources used by the editors
of specific media was from the prepaid service of press agencies. – press release focus, – press release theme, – number of words, – media response of press release (frequency): yes (description)/
no (x) the use of press release in media: percentage – media response of press release (frequency): yes (description)/
no (x) the use of press release in media: percentage We added following elements to the analysis of media outputs: We added following elements to the analysis of media outputs: From the total number of examined press releases 18 press
releases had no media response. The other 13 press releases were
primary (and almost the unique) information source for the news
contributions. All articles state as their main information sources
press releases or speaker (i. e. a PR worker is an information). In
no case other information source was specified. – media response of an article (in the case if a press release was
published by a press agency), – main source of information: specified (specification)/non-spec-
ified, – other sources of information: yes (description)/no (x), – the prevalence percentage of a press release in an article (exten-
sity): percentage of the number of identical words with proto-
text of the press release. When analyzing the texts we focused on the facts mostly inter-
esting for press release editors. We found out that the press releases
from the area of politics are the most attractive (five from the total
number of seven press releases are from the area of politics). The
second place is occupied by the press releases focused on the edify-
ing activities and health campaigns (three from five press releases). This fact may be explained from two points of view. First, an ambi-
tion of editors to help with the advertising of positive activities, and
second, the journalist outcome corresponds to the theory of gatekee-
ping, which means that journalists prefer the news having so called
human touch. 3. Methodology of research We considered following as a classification unit: We considered following as a classification unit: – integrated contribution specified by the title “press release”, We used the combination of three methods in the methodology
of our research – content analysis, questionnaire and qualitative
interview. The reason was an attempt to increase the relevance of
the research. – integrated contribution tagged by a title. We defined a word as a coding unit and selected following ele-
ments for the classification: – media output: yes (number)/no (x), The questionnaire was validated by means of questionnaire for
PR workers and content analysis. The answers in questionnaires
were compared with the results of analysis of particular media
outputs. – frequency (media response of a press release) – the description
of media which published the articles inspired by the particular
press release, – extensity (the percentage of a press release in media output) –
the prevalence of the code – the number of the words in an
article which are identical with the words of the compared press
release. We tried to increase the reliability of content analysis by the
rigorous determination of percentage exploitability of press releases
and percentage of press releases in media outputs. Only the identi-
cal (not paraphrased) words, sentences and paragraphs were con-
sidered as a relevant unit for the analysis in each case. Following elements were evaluated in the analysis of press
releases [according to 1]: – title, – title, The aim of the analysis was to determine the level of equivalence
(expressed in the percentage of press releases on media response) – author of the press release, – publication date, 50
G
C O M M U N I C A T I O N S 1 / 2 0 1 2 The press releases from the area of culture were not as
interesting (three from seven press releases) and the press releases
from the field of economy were the least interesting [2]. Following criteria and quantification method were chosen for
the comparison of the selected press release with particular media
output (the article): Exploring the equivalence of the press releases with the media
outputs we focused on the measurement of the number of identical
words, sentences and paragraphs, as well as on the frequency of
the prevalence of a word (coding unit) in the text. Regarding the fact that it is necessary to adjudicate the texts
in their context, we assumed some controversies in analyses. For
the minimizing of different aberrations (specifically subjective eval-
uation) and the improvement of data quantification we specified
that power and intensity of examined phenomenon would be expres-
sed in percentage (percentage of press release in media output
and percentage of the press release use.) It is worthy to mention the composition aspect of the texts as
well. If the editors are inspired only by the press releases they rarely
change the texts and usually keep the original composition. The
titles are only a transformation of press release title or its main
idea. Creativity and original asset of an author are not usual; we
may say that it is very rare. After the realization of the content analysis we can state several
important findings. In the preparatory phase of the research we
assumed that the researches conducted by several professionals in
the area would correspond to the outcomes of our analysis. The
mapping and analyzing of 30 different press releases allowed us to
submit that the editors of print media in news service did not use
press releases in any case. The research followed the aim to explore particular phenom-
ena also from the perspective of a PR worker. From his/her per-
spective we must conclude that we may consider PR as successful
if more than one third of published press releases appear in media
in an almost original form. 51
C O M M U N I C A T I O N S 1 / 2 0 1 2 G
Fig. The answers do not correspond to the results
of the content analysis from which resulted that if a journalist uses
a TS he/she does not verify it in other sources. This discrepancy
may be explained by the fact that there were more print media
editors than editors from press agencies involved in our research
and their answers increased the number of positive answers. The questionnaire had 14 items which were divided into four
groups as they could correspond to the need to verify preset hypothe-
ses. The groups of questions aimed to map the level of press release
publishing in print and online media as well as the verification of
information in press releases and other sources, relationships bet-
ween PR workers and journalists and possible pressure on journal-
ists caused by PR workers. The questionnaire was created in a way
to ensure respondents’ comfort when answering the questions. We
assumed that both target groups have time-consuming and stress-
ful jobs, which means that in the case of a complicated question-
naire they would not have answered at all. PR workers are convinced that they do not put pressure on
journalists (27), but 12 from 40 confessed that PR agencies intrude
media workers. The journalists think differently. 13 journalists think
that PR agencies put pressure on them. It is necessary to complete
the information that 23 PR workers and 13 journalists think that
a present a journalist is offered does not influence the content of
media outcome. Simultaneously, a smaller part of PR workers and
journalists thinks the opposite. The average age of the PR workers was 35.28 years; the youngest
participant was 26 years old, the oldest 59 years old. The average
age of the journalists was lower – 31.76 years; the youngest partici-
pant was only 19 years old and the oldest 59 years old. The results
show that in both areas (PR and journalism) mostly young people
work. The professional experience of the journalists is less than
five years and experience of the PR workers is up to 10 years. The
differences are clear mainly in connection with the last indicator
(period of work experience is 10 – 15 years) which adverts to the
smaller number of PR workers. This fact may be explained by the
young tradition of PR profession. On the basis of content analysis we may state that the hypothe-
ses No. 1, 2 and 3 were proven true. The facts were further veri-
fied by means of a questionnaire. rial rules. Surprisingly, 5 journalists could not give clear answer
yes/no. More than half of the PR workers (25) are convinced that
journalists publish PR materials as editorial contributions. On the
contrary, more than half journalists (11 from 21) think that it does
not happen. Most journalists (4) who think that their colleagues
work this way have been in the area of journalism less than 5 years. 35 from 40 people working in PR confessed that their PR material
had been published in its original form, whilst the most positive
experience with this have the PR workers who have worked in PR
less than 5 years. None of them has had an experience with at least
one unpublished PR material or TS. The journalists had slightly
different attitude. 18 journalists answered clear “no”, only three of
them answered “yes”. From these three two have worked as jour-
nalists less than 10 – 15 years, one more than 15 years. These find-
ings enable us to conclude that the higher age of a journalist, the
more opportunities (and possibly mistakes as well) may influence
him/her at their work. While realizing our research we sent e-mails to 100 journalists
and to the same number of PR workers who could answer the
questions in questionnaires anonymously by means of independent
web application. In accordance with our assumption more PR
workers than journalists joined the research (the return of ques-
tionnaires was 40%). The return of questionnaires from the jour-
nalists was only 20%. The similar situation repeated when we tried
to realize qualitative interviews with journalists. While all of the
three addressed PR workers agreed on the interviews (the interview
was conducted by means of phone call or e-mail), no journalist
agreed to participate. By means of particular items in the first part of the question-
naires we explored necessary demographic data. We were interested
in the age of the respondents and the time (in years) they had
spent in spotted areas (PR, journalism). Other demographic data
(gender, …) were considered in our research as irrelevant. 17 journalists from the total number of 21 answered that they
verify information. In the sixth part of the questionnaire both target groups mention
different kinds of smaller or bigger presents, which are offered or
accepted. From among them the most valuable are notebooks, soft-
ware applications, electronics, jewelry, holidays, mobile phones,
wellness stays, trips abroad, invitations to cultural or sport events,
alcohol (luxury wine), beer barrel, food and cosmetics, exclusive
pens, ski tickets, 500 EUR, … Some of the PR workers and journalist think that they haven’t
had any experience with presents, but one of them admitted an
offer of a trip to Monza F1. Regarding considerable benefits (ques-
tion No. 10), which might lead to concealing information, the PR
workers (39 from 40) adduced that they had never been offered
any of them and the journalists unanimously answered that they
had never accepted any of them. 31 PR workers from the total number of 40 think that journal-
ists follow internal editorial rules for their work with PR materials. Only five of them are convinced that journalists ignore the rules. Regarding the fact that in our questionnaire we explored how long
the respondents had worked in the professional area, we were inte-
rested in the answers of the most experienced PR workers. Those
who have worked in PR more than 15 years (only two) concurred in
the fact that journalist rather respect editorial rules. This finding
correlated to the outcomes of our content analysis. 1 The share of press releases editorial contributions in relation to the number of articles 51
C O M M U N I C A T I O N S 1 / 2 0 1 2 G 52
G
C O M M U N I C A T I O N S 1 / 2 0 1 2 the problem is not the “step over” of journalists toward PR workers,
but also back to media environment). them. Some of the PR workers provided further explanations for
publishing their press releases. For example, three PR workers think
that the reason is the quality of sources, professional PR and serious
information (“well-taken and informative value of PR”). One PR
worker said the following about journalists’ tendency to subscribe
PR: “They are not as good at a certain area, they are too busy to
study the topic, there is no investigative journalism in Slovakia”. Regarding the theme several researches have been realized
showing some models of relationships between journalism and PR. Some of them point out the fact that PR determined journalism
and that there is a mutual interconnection between PR and journa-
lism, which has also been proven in our research (mainly by means
of the interviews and the last question in questionnaire, in which
the respondents openly expressed their opinion on the question-
naire and relationships between PR workers and journalists). A major part of PR workers does not admit their dependence
on journalists (30), only 8 of them think that PR workers depend
on journalists; two expressed that journalists depend on PR mate-
rials (workers). It is necessary to realize the status of both disciplines in media
space and to intensify our perception of reality proving the fact
that there is no partnership (in its real meaning). The questions
such as how often, how much and in what way media use advertis-
ing materials and press releases must be relevantly answered. The
reason is that PR is a specific area as its results are immeasurable. There are some specialists who document the results by the quan-
tification of the spatium in media (the calculation in squared cen-
timeters), but these numbers may not be (and are not) authoritative,
as the spatium of a PR material does not correspond to the publi-
city reached by it [3] (and finally, PR itself is not just about “media
relations”). The journalists think the opposite regarding PR workers and
journalists. 10 from 21 respondents answered that the relationship
is equivalent, but almost the same number of them think (11) that
PR workers depend on journalists. No journalist expressed an
opinion that journalists depend on PR workers. The journalists’ view on the publishing of TS as editorial con-
tributions is also interesting. More than half of them (11) mentioned
non-professionalism of their colleagues; the second place is taken
by busyness of journalists. Positive relationships with PR workers
as the main reason for this are stated (the same as in the evaluation
of the PR workers) as the least relevant reason. A detailed view on
the respondents’ answers is also interesting. Three journalists admit-
ted that they had published a PR material as their own contribu-
tion. As the reason for this they stated their busyness and positive
relationships with PR workers. One of them openly confessed non-
professional attitude. All of them have been in the area of journa-
lism more than 10 years. One of the respondents commented the
situation: “it is a usual thing, that is why it is a PR article – the
point is the quality. The problem is not in the content, but in the
form”, another one stated: “I don’t think that journalists sign PR
with their own names”. The differentness illustrates the controversy
of the theme and inconsistence of reactions [2]. In our research we have noticed that if journalists use a TS as
a source, they do not change its original text. They paraphrase the
title, copy particular parts (some of them are slightly amended after-
wards) and modify the structure. The equivalence with the proto-
text is more than 30 %. This finding corresponds to the attempt of PR workers to use
the methods of journalists which results in qualitatively high-written
and professional texts (attributes of the inverted pyramid, “human
touch”, story). This is proven by the journalists’ opinions from the
questionnaire, as well as by the interviews with PR workers. Press releases are interesting also from their thematic point of
view. The influence of a theme on TS selection as an informative
source is essential regarding the theory of gatekeeping (with an em-
phasis on “human touch”). Commonly used press releases are from
political environment (specifically those having the character of
scandals and suspicions). These are followed by the TS from the
area of edifying activities and culture. The least common TS have
economic character. Hypothesis No. 4 not proven Regarding the last hypothesis we may conclude that 38 PR
workers admit benefits of positive relationships with journalists in
the time of critical communication. Concerning the TS publishing
as editorial contributions, more PR workers think that the main
reason is busyness of journalists, their non-professional attitude to
work and the last place is occupied by positive relationships with The journalists conveyed the same – 15 journalists from the
total number of 20 answered that journalists follow internal edito- 52
G
C O M M U N I C A T I O N S 1 / 2 0 1 2 [1] KOLLARIK, T., SOLLAROVA, E.: Methods of Social-psychological Practice, Bratislava: Ikar, p. 264, ISBN 80-551-0756-3.
[2] PITONAKOVA, S.: Dynamics of Media Space in Slovakia, Dissertation thesis, Nitra: UKF, 2011.
[3] L’ETANG, J.: Public Relations – Basic Theory, Practice, Critical Approach, Praha: Portal, 2009, p. 338, ISBN 978-80-7367-596-7. have more precise methodological research and should provide not
only the answers to solve practical problems (media relations, media
trainings, techniques and tools of PR, …). informative source for journalism. On the other hand, media space
and journalists is a very dynamic organism, full of people with
ambitions to work professionally, and therefore there is a high
probability that the tries to set the clear rules for the work with PR
materials remain a crucial theme of journalists. For these reasons, it is necessary in this period when the impor-
tance of PR is growing (the number of PR workers is increasing,
we may talk about so called marketing communicative mix), to deal
with this practical discipline also from the academic perspective. Likewise, it will be important to identify limits between PR and
journalism, as some of the journalists’ answer and PR workers indi-
cate that some practitioners consider PR publishing as standard
editorial method. The research may be an inspiration and background for further
– more detailed-conceived researches, for the character of this kind
of researches requires higher personal and financial provisions. Even from this point of view our research represents an insight
into the media environment. Considering the situation of media,
journalism and public relations in Slovakia, as well as the fact that
the issue has been worked out only from the perspective of parti-
cular disciplines and not interdisciplinary, our research may be even
more inspirational. Moreover, some of the professionals remind
us that even the research from the field of public relations should PR and journalism are in a very interesting and equal relation-
ship of their interdependence and competition and PR is and will
be a meaningful, but a specific source. Professional level of a jour-
nalist remains his/her essential attribute, as well as his/her ability
to distinguish credible and objective information [2]. 54
G
C O M M U N I C A T I O N S 1 / 2 0 1 2 4. Conclusion The contribution offers a view on the issue of dynamic deve-
lopment of media environment with regard to possibility to use PR
materials as a specific informative tool. The challenge was to write
a text providing interdisciplinary perspective on the problem, which
has been (because of many reasons) unnoticed. Accepting the uniqueness of media space we remind ourselves
that journalism has been traditionally profiled as an area with high
professional ideals, which are not in accordance with practical
achievements of journalists. Public, professionals, as well as prac-
titioners from the area of media point out non-standard examples
of the activities of journalists and media workers. Together with
the poor quality of texts and the lack of investigation, they criticize
non-standard relationships with PR people. To advocate journal-
ism one may say that in comparison with PR, journalism is con-
siderably financially undersized. This may lead to different kinds
of “cooperation” between journalists and PR workers (sometimes According to PR workers and journalists (self-critically), the
most common reasons of the penetrating of PR information into
the area of media are busyness and non-professionalism of journa-
lists. By means of the questionnaire and interviews and concerning
different theories, we have explored the opinions of practitioners
on the relationship between PR and journalism. None of the jour-
nalists thinks that journalists depend on PR, 8 PR workers think
that journalists are dependent on PR, but both professions usually
agree on the fact that their relationship is equal. Nowadays, it is necessary for both professions to realize that
PR is at the peak of its expansion and it is and will be an important 53
C O M M U N I C A T I O N S 1 / 2 0 1 2 G 53
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Current Status In Kedir City Towards 90-90-90 Unaids Target (A Case study of Pesantren 1 Health Center)
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Current Status In Kedir City Towards 90-90-90
Unaids Target (A Case study of Pesantren 1 Health
Center)
Geofrey Ssekalembe ( gsekalembe@gmail.com )
The University of Manchester Faculty of Biology Medicine and Health
Muhammad Atoillah Isfandiari
Universtas Airlangga Faculty of Public Health
Research article
Keywords: AIDS, ART, HIV, Suppression, Viral, Pesantran, Kediri
Posted Date: December 17th, 2019
DOI: https://doi.org/10.21203/rs.2.15289/v2
License: This work is licensed under a Creative Commons Attribution 4.0 International License.
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Screening Kandungan Plastik pada Minyak Goreng yang Digunakan pada Jajanan Pecel Lele
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Abstract The food has a benefit if it is considered from the quality (microbiologycal and physical) and the number of
nutrient. The food can be harmful if it is contained by additionl substances. The additional substance frequently used is
plastic. The objective of this study was to find out
type of plastic compound and the percentage. study was taken from October 2014 until January 2015 using 5 sample
been analyzed by GC-MS QP2010 type RT
injection of 200ºC, split mode of injection, mode flow control with linear velocity, pressure 162.2kPa, tot
255.8mL/min, the flow field 2.50 mL/min, a li
cooking oils contained plastics with the percentag
plastic compounds were benzene (phthalates), acrylic acid, a
Keywords: screening, plastic, cooking oil, GC
is considered from the quality (microbiologycal and physical) and the number of
nutrient. The food can be harmful if it is contained by additionl substances. The additional substance frequently used is
he objective of this study was to find out plastic content in cooking oil which is used in
the percentage.This was a descriptive qualitative study with total sampling method. This
taken from October 2014 until January 2015 using 5 samples of 100 ml cooking oil in Jati Padang which has
MS QP2010 type RT-5MS column with temperature column oven 80ºC, the temperature
injection of 200ºC, split mode of injection, mode flow control with linear velocity, pressure 162.2kPa, tot
255.8mL/min, the flow field 2.50 mL/min, a linear speed of 58.3 cm/sec. The results showed that 3 of the 5 s
with the percentages were the 1st (7.88%), the 2nd (1.62%), and the 3
benzene (phthalates), acrylic acid, and cyclopentene. screening, plastic, cooking oil, GC-MS
is considered from the quality (microbiologycal and physical) and the number of
nutrient. The food can be harmful if it is contained by additionl substances. The additional substance frequently used is
plastic content in cooking oil which is used in pecel lele and to the
s a descriptive qualitative study with total sampling method. This
s of 100 ml cooking oil in Jati Padang which has
5MS column with temperature column oven 80ºC, the temperature
injection of 200ºC, split mode of injection, mode flow control with linear velocity, pressure 162.2kPa, total flow of
results showed that 3 of the 5 samples of
(1.62%), and the 3rd(15.65%). The makanan yang dikonsumsi. Abstract justru akan lebih banyak
manfaatnya karena adanya
makanan didalamnya.1 Bahan
(BTP) adalah bahan untuk
warna, dan komponen mutu
pengolahan pangan.2
Makanan yang dikonsumsi
merugikan dibandingkan
adanya zat aditif atau tambahan
Bahan Tambahan Pangan
untuk memperbaiki tekstur,
mutu lainnya kedalam proses 76
http://jurnal.fk.unand.ac.id 76
http://jurnal.fk.unand.ac.id http://jurnal.fk.unand.ac.id Artikel Penelitian Artikel Penelitian Screening Kandungan Plastik pada Minyak Goreng yang
Digunakan pada Jajanan Pecel Lel
Screening Kandungan Plastik pada Minyak Goreng yang
Digunakan pada Jajanan Pecel Lele
Screening Kandungan Plastik pada Minyak Goreng yang Merisca Gayatri Ryosa1, Yustini Alioes
, Yustini Alioes2, Husnil Kadri2 Merisca Gayatri Ryosa1, Yustini Alioes
, Yustini Alioes2, Husnil Kadri2 Abstrak Makanan bermanfaat jika ditinjau dari kualitas (aspek mikrobiologis dan fisik) dan nilai gizinya. Makanan dapat
merugikan jika mengandung bahan tambahan pangan. Bahan tambahan pangan yang kerap digunakan adalah plastik. Tujuan penelitian ini adalah untuk mengetahui ada tidaknya k
pada jajanan pecel lele serta jenis dan persentase kandungan
kualitatif dengan metode pengambilan sampel secara
2015 terhadap 5 sampel minyak goreng jajanan pecel lele di kelurahan Jati kota Padang
dianalisis dengan GC-MS QP2010 jenis kolom RT
mode injeksi split, mode kontrol aliran dengan kecepatan linear, tekanan 162.2 kPa, total alirn 255.8 mL/menit, aliran
kolom 2.50 mL/menit, kecepatan linear 58.3 cm/detik. mengandung derivat plastik dengan persentase
plastik yang ditemukan adalah benzene (phthalate), acrylic, dan cyclopentene. Kata kunci: screening, plastik, minyak goreng, GC
ditinjau dari kualitas (aspek mikrobiologis dan fisik) dan nilai gizinya. Makanan dapat
merugikan jika mengandung bahan tambahan pangan. Bahan tambahan pangan yang kerap digunakan adalah plastik. mengetahui ada tidaknya kandungan plastik pada minyak goreng yang digunakan
jenis dan persentase kandungan derivat plastik didalamnya. kualitatif dengan metode pengambilan sampel secara total sampling. Penelitian dilakukan dari Oktober 2014
5 sampel minyak goreng jajanan pecel lele di kelurahan Jati kota Padang. QP2010 jenis kolom RT-5MS pada temperatur kolum oven 80ºC, temperatur injeksi 200ºC,
jeksi split, mode kontrol aliran dengan kecepatan linear, tekanan 162.2 kPa, total alirn 255.8 mL/menit, aliran
kolom 2.50 mL/menit, kecepatan linear 58.3 cm/detik. Hasil penelitian ini menunjukkan 3 dari 5 sampel minyak goreng
engan persentase: sampel 1 (7,88%), sampel 2 (1,62%) dan sampel 3
plastik yang ditemukan adalah benzene (phthalate), acrylic, dan cyclopentene. screening, plastik, minyak goreng, GC-MS
ditinjau dari kualitas (aspek mikrobiologis dan fisik) dan nilai gizinya. Makanan dapat
merugikan jika mengandung bahan tambahan pangan. Bahan tambahan pangan yang kerap digunakan adalah plastik. andungan plastik pada minyak goreng yang digunakan
Studi ini bersifat deskriptif
n dari Oktober 2014 - Januari
. Sebanyak 100 ml sampel
temperatur kolum oven 80ºC, temperatur injeksi 200ºC,
jeksi split, mode kontrol aliran dengan kecepatan linear, tekanan 162.2 kPa, total alirn 255.8 mL/menit, aliran
ini menunjukkan 3 dari 5 sampel minyak goreng
(1,62%) dan sampel 3 (15,65%). Derivat Kata kunci: screening, plastik, minyak goreng, GC
screening, plastik, minyak goreng, GC-MS Affiliasi penulis: 1. Prodi Profesi Dokter FK Unand (
Kedokteran Universitas Andalas Padang), 2. Bagian Biokimia FK
Unand.
FK Unand (Fakultas
, 2. Bagian Biokimia FK Jurnal Kesehatan Andalas. 201
Jurnal Kesehatan Andalas. 2017; 6(1) PENDAHULUAN Makanan merupakan kebutuhan
tidak mungkin ditinggalkan. Salah satu
mempengaruhi kesehatan manusia adalah
kebutuhan pokok yang
Salah satu faktor yang
adalah nilai gizi Plastik merupakan bahan baru
berkembang. Plastik merupakan suatu polimer sintetik
Plastik merupakan bahan baru yang semakin
Plastik merupakan suatu polimer sintetik Jurnal Kesehatan Andalas. 201
Jurnal Kesehatan Andalas. 2017; 6(1) Jurnal Kesehatan Andalas. 201
Jurnal Kesehatan Andalas. 2017; 6(1) http://jurnal.fk.unand.ac.id atau polimer termoplastik (polimer yang akan melunak
jika dipanaskan) berbahan baku gas yang kemudian
akan mengalami polimerisasi. Pada proses ini, tidak
semua gas dapat terbentuk menjadi polimer sehingga
sebagian gas teperangkap dalam keadaan tidak
berikatan dan tidak dapat keluar. Zat ini mudah
menguap apabila bersentuhan dengan benda bersuhu
tinggi. Hasil penguapan kemudian masuk kedalam air,
minyak, atau apa saja yang langsung bersinggungan
dengan plastik. Apabila zat tersebut masuk ke dalam
metabolisme
dan
menumpuk
di
dalam
tubuh,
kemungkinan terbentuk kanker akan semakin besar. Saat ini plastik digunakan dalam penggorengan
makanan. Plastik dimasukkan kedalam minyak yang
panas kemudan digunakan untuk menggoreng, maka
sifat plastik yang mudah menguap akan masuk ke
dalam makanan dan menimbulkan masalah dalam
tubuh.3 METODE Penelitian ini menggunakan metode deskriptif
kualitatif yang dilakukan di kelurahan Jati Kota Padang
dari Oktober 2014 sampai Januari 2015. Metode
pengambilan sampel yang digunakan adalah total
sampling dengan jumlah sampel akhir sebanyak 5
sampel. Sampel yang digunakan berupa minyak
goreng yang didapatkan dari penjual jajanan pecel lele
sebanyak 100 ml dengan kriteria sampel yang
digunakan merupakan minyak baru ataupun minyak
jelantah yang diambil sewaktu proses penggorengan
sedang
berlansung. Instrumen
penelitian
yang
digunakan
adalah
Gas
Chromatography
Mass
Spectrometry
(GC-MS)
untuk
menentukan
ada
tidaknya derivate plastik pada sampel dan mengetahui
jenis
derivat
plastik
tersebut
beserta
dengan
persentasenya. Pengukuran dengan GC-MS dilakukan
dengan kondisi : jenis kolom RT-5MS dengan kolum
oven 80ºC, temperatur injeksi 200ºC, mode injeksi
split, mode control aliran dengan kecepatan linear,
tekanan 162.2 kPa, total alirn 255.8 mL/menit, aliran
kolom 2.50 mL/menit, kecepatan linear 58.3 cm/detik. Hasil ukur yang didapat berupa persentase dengan
skala ukur rasio. Data yang diperoleh diolah secara
deskriptif yang disertai tabel, narasi dan pembahasan
serta diambil kesimpulan apakah terdapat derivat
plastik pada 5 sampel minyak goreng. Berdasarkan data survey Badan Pengawasan
Obat dan Makanan (BPOM) seperti yang dikutip oleh
Hartanto (2012) dari sampling yang dilaksanakan 30
Balai BPOM di Indonesia dengan sampel 886 Sekolah
Dasar yang tersebar di 30 kota di Indonesia,
didapatkan sebanyak 35% sampel tidak memenuhi
syarat.4
Dugaan
adanya
pangan
gorengan
mengandung plastik, salah satunya dapat diketahui
dari bentuk bahan pangan goreng yang tetap tampak
terlihat
garing
meskipun
sesudah
berjam-jam
dimasak.5 Kelurahan Jati adalah salah satu kawasan
mahasiswa yang ramai akan jajanan kaki lima. Telah
ditemukan
sembilan
titik
dari
sepuluh
sampel
pedagang gorengan di kelurahan Jati yang menjadi
sampel penelitian tentang penggunaan plastik pada
minyak goreng positif menggunakan plastik dalam
penggorengannya.6 HASIL
Sampel 1:
Gambar 1. Grafik analisa GC-MS sampel 1 Sampel 1: Jajanan kaki lima yang juga ramai ditemukan di
kawasan Jati adalah pecel lele. Pecel lele pada
dasarnya adalah jajanan sehat karena terdiri dari ikan
dan lalapan. Pemakaian minyak goring yang tidak baik
tentu akan meningkatkan kadar produk sisa berupa
peroksida. Oleh karena itu, perlu dilakukan penelitian
dengan tujuan screening kandungan plastik pada
minyak goreng yang terdapat pada jajanan pecel lele
di kelurahan Jati Padang. Gambar 1. Grafik analisa GC-MS sampel 1 Jurnal Kesehatan Andalas. 2017; 6(1) Tabel 1. Senyawa hasil analisa GC-MS sampel 1
Puncak
(peak)
Waktu
Retensi
(min)
Senyawa
%
Ket
A
8.823
Thiosulfuric
acid
(H2S2O3), S-
(2-aminoethyl)
ester
4.13
Senyawa
normal
B
9.412
Octadecanoid
acid, (2-
phenyl-1,3-
dioxolan-4-
yl)methyl ester
8.53
Senyawa
normal
C
10.027
Benzene, (1,2-
dimethylpropyl
)-(CAS) 2-
PHENYL 3-
METHYLBUT
ANE
4.55
Derivat
plastik
D
10.449
1,2-
Benzenedicar
boxylic acid,
bis(2-
ethylhexyl)
ester (CAS)
Bis (2-
ethylhexyl)
phthalte
3.33
Derivat
plastik
E
10.541
Cinnamylcinna
mate
11.22
Senyawa
normal
F
12.412
Thiosulfuric
acid
(H2S2O3), S-
(2-aminoethyl)
ester
4.42
Senyawa
normal
Sampel 2:
Gambar 2. Grafik analisa GC-MS sampel 2 http://jurnal.fk.unand.ac.id Tabel 1. Senyawa hasil analisa GC-MS sampel 1
Puncak
(peak)
Waktu
Retensi
(min)
Senyawa
%
Ket
A
8.823
Thiosulfuric
acid
(H2S2O3), S-
(2-aminoethyl)
ester
4.13
Senyawa
normal
B
9.412
Octadecanoid
acid, (2-
phenyl-1,3-
dioxolan-4-
yl)methyl ester
8.53
Senyawa
normal
C
10.027
Benzene, (1,2-
dimethylpropyl
)-(CAS) 2-
PHENYL 3-
METHYLBUT
ANE
4.55
Derivat
plastik
D
10.449
1,2-
Benzenedicar
boxylic acid,
bis(2-
ethylhexyl)
ester (CAS)
Bis (2-
ethylhexyl)
phthalte
3.33
Derivat
plastik
E
10.541
Cinnamylcinna
mate
11.22
Senyawa
normal
F
12.412
Thiosulfuric
acid
(H2S2O3), S-
(2-aminoethyl)
ester
4.42
Senyawa
normal
Sampel 2:
Gambar 2. Grafik analisa GC-MS sampel 2 Tabel 2. Senyawa hasil analisa GC-MS sampel 2
Puncak
(peak)
Waktu
Retensi
(min)
Senyawa
%
Ket
A
13.233
Pentanoic acid,
4-nitro-, methyl
4-
nitropentanoate
9.97
Senyawa
normal
B
13.308
Hexadecanoic
acid, 2-hydroxy-
1,3-propanedyl
ester (CAS)
GLYCEROL 1,3-
DIHEXADECAN
OATE
0.68
Senyawa
normal
C
13.549
2,6-DIMETHYL -
3-OCTANOL
2.28
Senyawa
normal
D
14.718
2,6,10,14,18,22-
Tetracosahexae
ne,
2,6,10,15,19,23-
hexamethyl-
(CAS) Squalene
9.84
Senyawa
normal
E
18.057
Acylic acid decyl
ester
1.62
Derivat
plastik
Sampel 3:
Gambar 3. Grafik analisa GC-MS sampel 3 Tabel 2. Jurnal Kesehatan Andalas. 2017; 6(1) Sampel 1: Senyawa hasil analisa GC-MS sampel 2
Puncak
(peak)
Waktu
Retensi
(min)
Senyawa
%
Ket
A
13.233
Pentanoic acid,
4-nitro-, methyl
4-
nitropentanoate
9.97
Senyawa
normal
B
13.308
Hexadecanoic
acid, 2-hydroxy-
1,3-propanedyl
ester (CAS)
GLYCEROL 1,3-
DIHEXADECAN
OATE
0.68
Senyawa
normal
C
13.549
2,6-DIMETHYL -
3-OCTANOL
2.28
Senyawa
normal
D
14.718
2,6,10,14,18,22-
Tetracosahexae
ne,
2,6,10,15,19,23-
hexamethyl-
(CAS) Squalene
9.84
Senyawa
normal
E
18.057
Acylic acid decyl
ester
1.62
Derivat
plastik
Sampel 3:
Gambar 3. Grafik analisa GC-MS sampel 3 Tabel 2. Senyawa hasil analisa GC-MS sampel 2
Puncak
(peak)
Waktu
Retensi
(min)
Senyawa
%
Ket
A
13.233
Pentanoic acid,
4-nitro-, methyl
4-
nitropentanoate
9.97
Senyawa
normal
B
13.308
Hexadecanoic
acid, 2-hydroxy-
1,3-propanedyl
ester (CAS)
GLYCEROL 1,3-
DIHEXADECAN
OATE
0.68
Senyawa
normal
C
13.549
2,6-DIMETHYL -
3-OCTANOL
2.28
Senyawa
normal
D
14.718
2,6,10,14,18,22-
Tetracosahexae
ne,
2,6,10,15,19,23-
hexamethyl-
(CAS) Squalene
9.84
Senyawa
normal
E
18.057
Acylic acid decyl
ester
1.62
Derivat
plastik Tabel 1. Senyawa hasil analisa GC-MS sampel 1
Puncak
(peak)
Waktu
Retensi
(min)
Senyawa
%
Ket
A
8.823
Thiosulfuric
acid
(H2S2O3), S-
(2-aminoethyl)
ester
4.13
Senyawa
normal
B
9.412
Octadecanoid
acid, (2-
phenyl-1,3-
dioxolan-4-
yl)methyl ester
8.53
Senyawa
normal
C
10.027
Benzene, (1,2-
dimethylpropyl
)-(CAS) 2-
PHENYL 3-
METHYLBUT
ANE
4.55
Derivat
plastik
D
10.449
1,2-
Benzenedicar
boxylic acid,
bis(2-
ethylhexyl)
ester (CAS)
Bis (2-
ethylhexyl)
phthalte
3.33
Derivat
plastik
E
10.541
Cinnamylcinna
mate
11.22
Senyawa
normal
F
12.412
Thiosulfuric
acid
(H2S2O3), S-
(2-aminoethyl)
ester
4.42
Senyawa
normal Tabel 2. Senyawa hasil analisa GC-MS sampel 2 Tabel 1. Senyawa hasil analisa GC-MS sampel 1 Sampel 3:
Gambar 3. Grafik analisa GC-MS sampel 3 Sampel 2: Gambar 3. Grafik analisa GC-MS sampel 3 Gambar 3. Grafik analisa GC-MS sampel 3 Tabel 3. Senyawa hasil analisa GC-MS sampel 3
Puncak
(peak)
Waktu
Retensi
(min)
Senyawa
%
Ket
A
0.925
Ethanol (CAS)
Ethylalcohol
35.43
Senyawa
normal
B
1.101
Trans-3,5-
Dideutero
hydroxycyclopen
tene
15.65
Derivat
plastik
C
15.360
9-Octdecenoic
acid (Z)-(CAS)
Oleic acid
4.73
Senyawa
normal
D
16.377
Hexadecanoic
acid,1-
(hydroxymethyl)-
1,2-ethanediyl
ester (CAS) 1,2-
Dipalmitin
2.34
Senyawa
normal
E
16.933
DODECANOIC
ACID, 12-
HYDROXY
3.32
Senyawa
normal Tabel 3. Senyawa hasil analisa GC-MS sampel 3 Gambar 2. Grafik analisa GC-MS sampel 2 http://jurnal.fk.unand.ac.id 79 Sampel 4:
Gambar 4. Grafik analisa GC-MS sampel 4 Tabel 5. Senyawa hasil analisa GC-MS sampel 5
Puncak
(peak)
Waktu
Retensi
(min)
Senyawa
%
Ket
A
1.215
Ethanol (CAS) Ethyl
alcohol
2.16
Senyawa
normal
B
15.363
9-Octadecenoic acid
(Z)-(CAS) oleic acid
5.34
Senyawa
normal
C
15.676
Decanoic acid (CAS)
Capric acid
0.92
Senyawa
normal
D
16.093
Oxiraneundecanoic
acid, 3-penthyl-,
methyl ester, cis-
(CAS) METHYL
CIS-12,13-
EPOXISTEARATE
0.84
Senyawa
normal
E
17.393
Hexadecanoic acid,
2-hydroxy-1,3-
propanedyl ester
(CAS) GLYCEROL
1,3-
DIHEXDECANOATE
1.04
Senyawa
normal Tabel 5. Senyawa hasil analisa GC-MS sampel 5 Sampel 4: Gambar 4. Grafik analisa GC-MS sampel 4 Tabel 4. Senyawa hasil analisa GC-MS sampel 4
Puncak
(peak)
Waktu
Retensi
(min)
Senyawa
%
Ket
A
12.237
Hexadecanoic acid
(CAS) Palmitic
acid
0.94
Senyawa
normal
B
14.739
2,6,10,14,18,22-
Tetracosahexaene,
2,6,10,15,19,23-
hexamethyl- (CAS)
Squalene
4.56
Senyawa
normal
C
15.351
9-Octadecenoic
acid (Z)-(CAS)
Oleic acid
2.03
Senyawa
normal
D
17.821
9-Octadecanoic
acid, (2-phenyl-
1,3-dioxolan-4-
yl)methyl ester. 0.98
Senyawa
normal
E
19.325
9-Octadecenoic
acid (Z)-(CAS)
Oleic acid
3.36
Senyawa
normal Tabel 4. Senyawa hasil analisa GC-MS sampel 4
Puncak
(peak)
Waktu
Retensi
(min)
Senyawa
%
Ket
A
12.237
Hexadecanoic acid
(CAS) Palmitic
acid
0.94
Senyawa
normal
B
14.739
2,6,10,14,18,22-
Tetracosahexaene,
2,6,10,15,19,23-
hexamethyl- (CAS)
Squalene
4.56
Senyawa
normal
C
15.351
9-Octadecenoic
acid (Z)-(CAS)
Oleic acid
2.03
Senyawa
normal
D
17.821
9-Octadecanoic
acid, (2-phenyl-
1,3-dioxolan-4-
yl)methyl ester. 0.98
Senyawa
normal
E
19.325
9-Octadecenoic
acid (Z)-(CAS)
Oleic acid
3.36
Senyawa
normal
Sampel 5:
Gambar 5. Grafik analisa GC-MS sampel 5. Tabel 4. Senyawa hasil analisa GC-MS sampel 4 Berdasarkan hasil dari analisis minyak goreng
yang digunakan pada jajanan pecel lele di kelurahan
Jati kota Padang, sampel yang positif menggunakan
plastik disajikan dibawah ini. Tabel 6. Jurnal Kesehatan Andalas. 2017; 6(1) Sampel 2 Ada 30 senyawa yang teridentifikasi dengan
komponen utama adalah pentanoic acid, 4-nitro-,
methyl ester (CAS) Methyl 4-nitropentanoate (9,97%)
dan 2,6,10,14,18,22-tetracosahexaena, 2,6,10,15,19,
23-hexamethyl- (CAS)Squalene (9,84%). Sampel 2 ini
juga mengandung senyawa berbahaya acrylic acid
decyl ester (1,62%). Sampel 3 Identifikasi senyawa plastik yang terkandung
dalam
minyak
dianalisis
menggunakan
GC-MS
menunjukkan peak (puncak) senyawa yang terdeteksi
kemudian diidentifikasi melalui data massa spektrum
yang ada pada instrumen. Ada 30 senyawa yang teridentifikasi dengan
komponen utama adalah ethanol (CAS) ethyl alcohol
(35.43%), 9-Octadecenoic acid (Z)- (CAS) Oleic acid
(24,97%), Trans-3,5-Dideutero hydroxy cyclopentene
(15,65%). Ethanol
(CAS)
ethyl
alcohol
merupakan
senyawa yang ditemukan pada pemakaian minyak
jelantah yang lazim digunakan oleh pedagang.8 Oleic
acid atau asam oleat adalah kandungan normal pada
minyak.13
Cyclopentene
adalah
senyawa
yang
terdapat pada crude oil yang merupakan hasil dari
dekomposisi limbah plastik jenis polypropylene (PP).14
Komponen utama Sampel 3 dapat disimpulkan
mengandung produk dari plastik. Sampel 1 Ada 30 senyawa yang teridentifikasi dengan
komponen
utama
adalah
cinnamyl
cinnamate
(11,22%), thiosulfuric acid (H2S2O3), S-(2-aminoethyl)
ester (8,55%), benzene,(1,2-dimethylpropyl)-(CAS),
1,2-Benzenedicarboxylic
acid,bis(2-ethylhexyl)ester
(CAS) bis (2-ethylhexyl) phthalate (7,88%). Cinnamyl
cinnamate
merupakan
senyawa
asam lemak aromatik yang normal terdapat pada
minyak goreng.7 Asam tiosulfat (H2S2O3) adalah
komponen yang lazim ditemukan pada minyak curah
yang dalam bentuk garamnya digunakan untuk
penentuan bilangan peroksida.8 Phthalate merupakan
senyawa tambahan pada pembuatan plastik yang
berfungsi sebagai plasticizer yaitu memberikan sifat
lembut dan fleksibel pada polimer Polivinil Klorida
(PVC).9
Komponen
utama
Sampel
1
dapat
disimpulkan mengandung produk dari plastik. Sampel 4 Ada 30 senyawa yang teridentifikasi dengan
komponen utama adalah 9-Octadecenoic acid (Z)-
(CAS) Oleic acid (6,37%), dan 2,6,10,14,18,22-
tetracosahexaene, 2,6,10,15,19,23-hexamethyl- (CAS)
Squalene (4,56%). Oleic acid atau asam oleat adalah kandungan
normal pada minyak.13 Squalene adalah senyawa
bukan minyak karena tidak memiliki gugusan COOH,
namun dapat terkandung dalam jumlah minimal pada
kelapa
sawit.11
Komponen
utama
Sampel
4
disimpulkan senyawa yang normal dalam minyak. Sampel 2: Hasil analisis sampel dengan GC-MS
No
Sampel
Derivat Plastik
Konsentrasi
1
benzene,(1,2-
dimethylpropyl)-(CAS), 1,2-
Benzenedicarboxylic
acid,bis(2-ethylhexyl)ester
(CAS) bis (2-ethylhexyl)
phthalate
7,88%
2
acrylic acid decyl ester
1,62%
3
Trans-3,5-Dideutero
hydroxycyclopentene
15,65% Sampel 5:
Gambar 5. Grafik analisa GC-MS sampel 5. Gambar 5. Grafik analisa GC-MS sampel 5. Jurnal Kesehatan Andalas. 2017; 6(1) http://jurnal.fk.unand.ac.id 80 Jurnal Kesehatan Andalas. 2017; 6(1) DAFTAR PUSTAKA 1. Kristianingrum S. Pengawet makanan yang aman
bagi kesehatan (skripsi). Yogyakarta: Universitas
Negeri Yogyakarta; 2006. 2. Hardiansyah, Sumali. Pengendalian mutu dan
keamanan pangan. Jakarta: Koswara; 2001. Persentase minyak goreng yang mengandung
senyawa plastik adalah: Sampel 1(7.88%), 2(1.62%),
dan 3(15.65%). Persentase yang didapatkan memang
relatif tidak tinggi, namun plastic akan tetap menjadi
toksik pada tubuh meskipun dalam kadar yang sangat
rendah karena sifat plastik yang tidak dapat diuraikan
oleh enzim. Meskipun demikian penelitian ini memiliki
keterbatasan,
namun
diharapkan
tetap
dapat
memberikan manfaat dalam upaya pendeteksian dan
perlindungan konsumen terhadap kasus kecurangan
produsen sehingga resiko terjadinya efek plastic
terhadap kesehatan dalam jangka waktu yang lama
seperti kanker, cacat lahir, perubahan genetik,
bronkiitis
kronik,
penyakit
kulit,
tuli,
gangguan
penglihatan, gangguan pencernaan, disfungsi hati,
asma, perubahan hormon, gangguan sistem imun, dan
reproduksi dapat diturunkan. 3. Fatimah A. Hidup kita dekat dengan senyawa kimia
berbahaya. [serial
online]
2012
(diunduh
6
September
2014). Tersedia
dari:
URL:
HYPERLINK http://humaniora.kompasiana.com 4. Hartanto D. Masih ditemukan gorengan bercampur
plastik di jaksel. [serial online] 2012 (diunduh 6
September
2014). Tersedia
dari:
URL:
HYPERLINK http://ikebayoran.com 5. Burhani R. Masyarakat diminta waspadai gorengan
mengandung plastik. [serial online] 2013 (diunduh
6
September
2014). Tersedia
dari:
URL:
HYPERLINK http://www.antaranews.com 5. Burhani R. Masyarakat diminta waspadai gorengan
mengandung plastik. [serial online] 2013 (diunduh
6
September
2014). Tersedia
dari:
URL:
HYPERLINK http://www.antaranews.com 6. Sari AR. Screening kandungan plastik pada
minyak goreng yang terdapat pada gorengan di jati
padang (skripsi). Padang: Fakultas Kedokteran
Universitas Andalas; 2013. 6. Sari AR. Screening kandungan plastik pada
minyak goreng yang terdapat pada gorengan di jati
padang (skripsi). Padang: Fakultas Kedokteran
Universitas Andalas; 2013. 7. Opdyke. Monographs on fragrance raw material. Australia: Pergamon Press (Aust); 2011. 7. Opdyke. Monographs on fragrance raw material. Australia: Pergamon Press (Aust); 2011. Ketelitian
masyarakat
sebelum
membeli
makanan menjadi salah satu kunci agar dapat
terhindar dari makanan yang mengandung plastik. Makanan yang mengandung plastik akan tampak dan
terasa gurih dan garing walaupun sudah berjam-jam
dimasak
dibandingkan
dengan
makanan
yang
digoreng dengan minyak biasa. Terkadang akan
tampak bercak putih mengkilap plastic pada makanan. Namun,
bercak
ini
baru akan terlihat
apabila
konsentrasi plastic dalam minyak goring sangat tinggi. Penelitian ini diharapkan dapat memberikan ide dan
dapat
menjadi
pembanding
bagi
penelitian
selanjutnya. 8. Aminah S. Bilangan peroksida minyak goreng
curah
dan
sifat
organoleptik
tempe
pada
pengulngan penggorengan. Jurnal Pangan dan
Gizi Universitas Muhammadiyah Semarang. 2010;
1(1): 7-14. 9. Irawan S, Supeni G. DAFTAR PUSTAKA Krakterisasi migrasi kemasan
dan peralatan rumah tangga berbasis polimer. Jurnal Kimiadan Kemasan Balai Besar Kimia dan
Kemasan.2013; 2(35):105-12. 10. Winarni, Sumarto W, Mntini S. Penetralan dan
adsorbsi minyak goreng bekas menjadi minyak
goreng layak pakai konsumsi. Jurnal Universitas
Negeri Semarang.2010; 8(1):46-56. Sampel 5 Ada 30 senyawa yang teridentifikasi dengan
komponen utama adalah 9-Octadecenoic acid (Z)-
(CAS) Oleic acid (5,34%) dan Ethanol (CAS) ethyl
alcohol (2,16%). Pentanoic acid (asam pentanoat) yang memiliki
nama trivial asam valerat adalah jenis asam lemak
jenuh rantai lurus dengan atom C ganjil yang
merupakan komponen yang normal ada pada minyak
goreng.10 Squalene adalah senyawa bukan minyak
karena tidak memiliki gugusan COOH, namun dapat
terkandung dalam jumlah minimal pada kelapa sawit.11
Acrylic
merupakan
produk
dari
plastik
jenis
polypropylene (PP).12Salah satu komponen sampel 2
dapat disimpulkan mengandung produk dari plastik. Oleic acid atau asam oleat adalah kandungan
normal pada minyak.13 Ethanol (CAS) ethyl alcohol
adalah senyawa yang ditemukan pada pemakaian
minyak
jelantah
yang
lazim
digunakan
oleh
pedagang.8 Komponen utama Sampel 5 disimpulkan
senyawa yang normal dalam minyak. Penelitian tentang praktik penggunaan plastik
juga telah dilakukan di kawasan Jati Padang dengan
hasil berupa ditemukannya 9 dari 10 sampel positif
menggunakan plastik sebagai bahan tambahan Jurnal Kesehatan Andalas. 2017; 6(1) http://jurnal.fk.unand.ac.id 81 pangan.6
Hasil penelitian tersebut hampir sama
dengan penelitian ini yaitu adanya penggunaan plastik
yang ditambahkan ke minyak goreng dengan hasil 3
dari 5 sampel yang diambil dari penjual jajanan pecel
lele di kelurahan Jati kota Padang mengandung
senyawa plastik. 13. Mursalin, Hariyadi P, Purnomo EH, Fardiaz D,
Andarwulan H. Pengaruh laju pendinginan, suhu,
dan lama kristalisasi pada produk triasilgliserol dan
sifat pelelehan produk fraksionasi minyak kelapa.
Jurnal Ilmu Pertanian Indonesia. 2013;18(1):6-14. 14. Naimah S, Nuraeni C, Rumonday I, Jati BN,
Ermawati
R.
Dekomposisi
limbah
plastik
polypropylene dengan metode pirolisis. Journal of
Material Science Balai Besar Kimia dan Kemasan.
2012;13(3):226-9. Jurnal Kesehatan Andalas. 2017; 6(1) SIMPULAN 11. Gunstone F. Vegetable oils in food technology:
composition, properties, and uses. Edisi ke-2. United States: John Wiley & Sons; 2011. Sampel minyak goreng yang mengandung
senyawa plastik adalah sampel 1, 2, dan 3. Persentase konsentrasi senyawa plastik pada minyak
goreng adalah 7.88% pada sampel 1, 1.62% pada
sampel 2 dan 15.65% pada sampel 3. 12. Wang JC. Polymeric materials. Dalam: Wang JC,
Liu CM, Zhao QC, Dong X. Materials design,
processing, and applications. 2013.hlm. 690(693). Jurnal Kesehatan Andalas. 2017; 6(1) Jurnal Kesehatan Andalas. 2017; 6(1) http://jurnal.fk.unand.ac.id 82 14. Naimah S, Nuraeni C, Rumonday I, Jati BN,
Ermawati
R. Dekomposisi
limbah
plastik
polypropylene dengan metode pirolisis. Journal of
Material Science Balai Besar Kimia dan Kemasan. 2012;13(3):226-9. Jurnal Kesehatan Andalas. 2017; 6(1)
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https://openalex.org/W2929294594
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https://europepmc.org/articles/pmc6521218?pdf=render
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English
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Characterization of Two Pseudomonas aeruginosa Viruses vB_PaeM_SCUT-S1 and vB_PaeM_SCUT-S2
|
Viruses
| 2,019
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cc-by
| 12,743
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Received: 5 January 2019; Accepted: 28 March 2019; Published: 1 April 2019 Abstract: The sophisticated antibiotic resistance mechanism of Pseudomonas aeruginosa has urged the
development of alternative antibacterial strategies. Phage therapy has been proven successful for the
treatment of multidrug-resistant infections. In this study, we reported two virulent P. aeruginosa phages,
vB_PaeM_SCUT-S1 (S1) and vB_PaeM_SCUT-S2 (S2), which were characterized at morphological,
genomic, and proteomic levels. Phages S1 and S2 were assigned to the Myoviridae family. The genome
sequencing showed that the genome size of Phage S1 was 66,046 bp and that of Phage S2 was
94,434 bp. The phylogenetic tree indicated that the two phages were distantly related to each other
and were classified in the genera Pbunavirus and Pakpunavirus respectively. Thirty-one proteins were
identified for each phage by mass spectrometry and were used to substantiate the function of the
predicted coding genes. The two phages inhibited the growth of P. aeruginosa strain PAO1 at low
multiplicity of infection levels and had good performance both on preventing biofilm formation and
eradicating preformed biofilms. They were also stable over a wide range of temperature and pH
values, supporting their potential use in the treatment of P. aeruginosa infections. Keywords: Pseudomonas aeruginosa; phage therapy; Myoviridae; complete genome; proteomics;
one-step growth curve; lysis kinetics; biofilm www.mdpi.com/journal/viruses Viruses 2019, 11, 318; doi:10.3390/v11040318 viruses viruses viruses Characterization of Two Pseudomonas aeruginosa
Viruses vB_PaeM_SCUT-S1 and vB_PaeM_SCUT-S2 Yangyijun Guo, Ping Chen, Zhanglin Lin * and Tingting Wang * Yangyijun Guo, Ping Chen, Zhanglin Lin * and Tingting Wang * School of Biology and Biological Engineering, South China University of Technology, 382 East Outer Loop Road,
Higher Education Mega Centre, Guangzhou 510006, China; 201620133950@mail.scut.edu.cn (Y.G.);
chenp211@scut.edu.cn (P.C.) * Correspondence: zhanglinlin@scut.edu.cn (Z.L.); wangtt@scut.edu.cn (T.W.); Tel.: +86-20-3839-0639 (T.W
Received: 5 January 2019; Accepted: 28 March 2019; Published: 1 April 2019 1. Introduction The recent emergence and the expanding distribution of multidrug-resistant (MDR), extensively
drug-resistant, and pandrug-resistant bacterial strains have been a great challenge for public health
due to the lack of effective antibiotic treatments. In particular, the ESKAPE organisms (Enterococcus
faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa,
and Enterobacter species) are the dominant causes of serious nosocomial infections [1–3]. As a ubiquitous
Gram-negative opportunistic bacterium, P. aeruginosa can cause life-threatening infections in patients
suffering from cystic fibrosis, severe burns, and other immunocompromising conditions, leading to
considerable morbidity and mortality [4,5]. Unfortunately, MDR P. aeruginosa is difficult to eradicate by
conventional antibiotics, owing to its sophisticated antibiotic resistance mechanisms, which includes
intrinsic and acquired drug resistance, and its capacity to form biofilms [6–8]. Thus, there is an urgent
demand for the development of alternative antibacterial strategies to combat these superbugs [9]. p
g
p
g [ ]
The interest in phages, which have been recognized as antimicrobial therapeutics for nearly a
century, has recently resurged because of their high specificity and abundance [10–13]. Bacteriophages
are natural predators of bacteria and are classified as virulent (lytic) phages or temperate (lysogenic)
phages depending on their distinct life cycle. However, only virulent phages have been explored for
phage therapy, mainly because temperate phages are associated to potential problems originating
from their ability of transferring DNA between different host bacteria and the possible alteration of 2 of 19 Viruses 2019, 11, 318 their host pathogenicity when shifting from quiescent state to lytic state [14,15]. Phage therapy has
been proved a successful treatment option, not only in animal models [16] but also in human clinical
trials [17]. Due to the high specificity of phages for a determined host, phage cocktails are often utilized
in order to broaden the antibiotic spectrum of the therapy, which makes a request for exploiting and
characterizing more therapeutic phages. Recent advances in genome sequencing and biotechnology have greatly promoted the discovery
and identification of numerous novel phages [18]. As for December 2018, the Genebank database
has deposited 323 Pseudomonas phage genome sequences, among which more than two-thirds were
P. aeruginosa phages. In order to be considered eligible for their use in phage therapy, P. 1. Introduction aeruginosa-specific
phages must be fully characterized to ensure that they are safe, i.e., their genomes must be studied
in detail to prove the lack of genes encoding for toxins, virulence factors or other undesirable genes,
through complete genome sequencing [19,20]. In this study, we characterized two P. aeruginosa phages,
vB_PaeM_SCUT-S1 (Phage S1) and vB_PaeM_SCUT-S2 (Phage S2), at morphological, genomic, and
proteomic levels. The two phages were new members of the genera Pbunavirus and Pakpunavirus,
respectively, and their therapeutic potentials were investigated for the growth inhibitory effects on the
planktonic cells and biofilm cells. 2.1. Bacterial Strains and Culture Conditions The bacterial strains used in this study are listed in Table S1. P. aeruginosa ATCC 9027, ATCC
15442, ATCC 27853, and Stenotrophomonas maltophilia ATCC 51331 were purchased from Guangdong
Microbial Culture Center, China (GDMCC). The strains PALWL1.001, PALWL1.002, and PALWL1.003
were collected by our laboratory. Strain PAO1 was a gift from Prof. Hu (Third Military Medical
University, Chongqing, China). All bacterial strains and phages were routinely cultured at 37 ◦C in
lysogeny broth (LB) containing 2 mM CaCl2. Phage plating was performed using the overlay agar
method, with LB containing 0.6% and 1.5% agar used for the top and the base agar, respectively. 2.2. Bacteriophage Isolation, Propagation, and Purification Phages were isolated by following the enrichment method using strain PAO1 as the host [21,22]. Briefly, aqueous samples were collected from a small pond in Guangzhou, China (N 23◦03′7.12”,
E 113◦04′5.35”). After centrifugation for 10 min at 9000× g, the supernatant was filtered through
a 0.45 µm filter and incubated with the host strains for 24 h at 37 ◦C. The bacterial cell debris was
removed by centrifugation and filtration, and the supernatant was plated using the overlay agar
method to check the host lysis. Twelve candidate plaques obtained were scraped with pipette tips
and resuspended in SM buffer (100 mM NaCl, 8 mM MgSO4, and 50 mM Tris-HCl, pH 7.5), were then
diluted 1000-fold in the same buffer, and purified by repeated co-culturing with the host strains using
the overlay agar method (three rounds). The uniqueness of the phages was confirmed by analysis of
the genome restriction maps obtained using three endonucleases (HindIII, EcoRI or XbaI) individually. Only two different phages were identified. The phage propagation was performed according to the classic procedure [23]. An aliquot of
500 mL of PAO1 culture (oa. OD 0.4–0.6 at 600 nm) was infected by the pure phage stocks and grown
overnight. Subsequently, 10 mL of chloroform were added, and the cells were incubated with shaking
for 10 min at 37 ◦C to obtain the lysates. The genomic DNA of the host was removed by the treatment of
1 µg/mL DNase I and RNase for 30 min at room temperature. The lysate was then supplemented with
a final concentration of 1 M NaCl and incubated for 1 h at 4 ◦C. After incubation and centrifugation, the
supernatant was supplemented with 10% (w/v) PEG 8000 and stored at 4 ◦C overnight to precipitate
the phage particles. After centrifugation (11,000× g, 10 min), the pellets were suspended in SM buffer
and an equal volume of chloroform was added to extract the PEG and the bacterial debris. After
centrifugation at 3000× g for 15 min at 4 ◦C, the suspension was adjusted by adding CsCl reagent to a 3 of 19 Viruses 2019, 11, 318 density of 1.15 g/mL and carefully loaded on a CsCl density gradient consisting of the step gradient
1.35 g/mL, 1.5 g/mL, and 1.7 g/mL. 2.4. Electron Microscopy For electron microscopy, the CsCl-purified phages were spotted onto 400 mesh carbon-coated
grids and negatively stained with 2% phosphotungstic acid (pH 6.5) [25]. The grids were observed by
a Hitachi transmission electron microscope at 80 kV. The dimensions of three viral particles of each
phage was measured, and the values were averaged. 2.2. Bacteriophage Isolation, Propagation, and Purification After ultracentrifugation at 87,000× g for 2 h at 4 ◦C, the band of
phage particles was collected and dialyzed twice for 1 h at 4 ◦C against a 500-fold volume of SM buffer
to remove the remaining CsCl. 2.3. Host Range Analysis Host range analysis was performed using the spot testing method [24] on three biological replicates. Briefly, 10 µL of phage suspensions of six concentrations ranging from 104 to 109 pfu/mL were spotted
onto bacterial lawns and incubated at 37 ◦C for 24 h. After incubation, the spot morphology was
observed and classified as “C+++”, a large lysis zone at 102 pfu; “C++”, individual plaques at 102–103
pfu; “C+”, individual plaques at 104–105 pfu; “T”, a turbid lysis zone at 106–107 pfu; “-”, no lysis. 2.5. Temperature and pH Stability The thermal stability testing was performed by incubating the phages (106 pfu/mL) at different
temperature for 1 h [26], the phage titers were then determined by using the double-layer overlay
method. The relative titer was calculated as the ratio of phage titers at a different temperature to
those stored at 4 ◦C. For the pH stability analysis, the phages (106 pfu/mL) were diluted 100-fold with
SM buffer of different pH values and incubated for 1 h at room temperature. The relative titer was
calculated as the ratio of phage titers treated with different pH levels to those by the original SM buffer
(pH 7.5). Three independent experiments were performed. 2.6. Genome DNA Extraction and Sequencing 2.9. One-Step Growth Curve and Lysis Kinetics A 0.1 mL aliquot of phage suspension (107 pfu/mL) was added to 9.9 mL of mid-log-phase bacterial
culture of host strain PAO1 (ca. OD600 ~ 0.5) and incubated for 5 min [42]. Phage titers were detected
by collecting samples at 5- or 10-min intervals and plating them immediately by the overlay method. The average burst size was quantified as the difference between the final and the initial phage titer
divided by the initial phage titer. To measure the lytic kinetics, phages with varying multiplicity of infection (MOI) in the range
0.01–100 were incubated with mid-log-phase bacterial cultures of host strain PAO1 (ca. OD600 ~ 0.5) in
96-well microtiter plates at 37 ◦C, 180 rpm. The kinetic data were obtained by monitoring the change
of absorbance at 600 nm for 20 h with intervals of 30 min using a microplate reader (Tecan infinite
M200PRO, Zürich, Switzerland). 2.8. Proteomics Analysis The structural proteins of the phages were analyzed after removing the DNA, as previously
described [41]. Briefly, the purified phage particles were mixed with an equal volume of 10 M LiCl
and incubated for 10 min at 46 ◦C. Next, the mixture was diluted 10-fold with SM buffer, followed
by 10 mM MgCl2 and 50 U DNase I per 1012 pfu. After 2 h incubation at 37 ◦C, the “ghost” particles
were precipitated by ultracentrifugation at 100,000× g at 4 ◦C for 30 min. Subsequently, the phage
pellet was suspended in protein loading buffer and denatured at 95 ◦C for 10 min. The phage proteins
were separated on a 12% SDS-PAGE gel and stained with Coomassie Brilliant Blue. For the protein
identification, the whole gel lane was cut, and the peptide mixture obtained by in-gel trypsin digestion
was analyzed by nanoLC-MS/MS (Eksigent nanoLC-Ultra 2D and TripleTOF 5600, AB SCIEX). The
MS/MS data were analyzed by MASCOT (http://www.matrixscience.com/) using the predicted proteins
of Phage S1 or S2 as the databases. The phage proteins were identified using a minimum threshold of
two peptides per protein. 2.6. Genome DNA Extraction and Sequencing The genomic DNA was extracted as follows [23,27]. The purified phage was first treated with
2.5 U/mL DNase I and incubated at 37 ◦C for 1 h to remove the host DNA. Next, 0.1 volumes of 2 M
Tris-HCl (pH 8.5)/0.2 M EDTA, 0.01 volumes of 0.5 M EDTA (pH 8.0), and 1 volume of formamide
were added, and the solution was incubated for 30 min at room temperature. The raw genomic
DNA was precipitated adding 2 volumes of 100% ethanol followed by centrifugation at 9000× g for
20 min at 4 ◦C. The DNA pellet was washed twice with 70% ethanol and suspended in 567 µL of TE
buffer (10 mM Tris-HCl, 1 mM EDTA) and added with 30 µL of 10% (w/v) SDS and 3 µL of 20 mg/mL
proteinase K. The mixture was incubated for 1 h at 50 ◦C. Subsequently, 100 µL of 5 M NaCl and 80 µL
of CTAB (cetyltrimethylammonium bromide)/NaCl solution were added and incubated for 10 min
at 65 ◦C. Next, the mixture was sequentially treated with one volume of chloroform, one volume
of phenol/chloroform/isoamyl alcohol (25:24:1), and one volume of chloroform, to gradually purify
the genomic DNA. The aqueous phase was collected, and 0.7 volumes of isopropanol were added to
precipitate the DNA. After centrifugation at 13,000× g for 15 min at 4 ◦C, the purified DNA pellet was
washed twice with 500 µL of ice-cold 70% ethanol and left to dry. The air-dried pellets were suspended
in 20 µL TE. The DNA concentration was measured by a Nanodrop2000 (Thermo Fisher Scientific,
Waltham, MA, USA). The purified DNA was used for the whole genome sequencing by Personal
Biotechnology Corp. (Shanghai, China). A DNA library with an insert size of 400 bp was prepared for
each sample. The two phage samples were sequenced by an Illumina MiSeq platform using the PE
250 bp strategy. Viruses 2019, 11, 318 4 of 19 2.7. Genome Assembly, Annotation, and Comparison 2.7. Genome Assembly, Annotation, and Comparison After filtering the raw reads to remove the adaptor contamination (i.e., low-quality reads
with ‘N’ bases), the remaining high-quality reads were assembled by ABYSS 2.0.2 [28] and MIRA
4.0 [29]. The contigs were manually assembled based on overlaps of more than 40 bp to obtain
the final scaffold. Next, the coverage was visualized using Geneious 10.2.3 to identify the
genome termini, and the results were confirmed using PhageTerm [30], which is available on the
public Galaxy server (http://galaxy.pasteur.fr/). After the whole genome sequences were obtained,
the annotation was carried out using RAST [31–33], Glimmer [34], and GeneMarkS [35], then
was subsequently manually confirmed. In addition, tRNAs were predicted by tRNA-Scan [36]
and ARAGORN (http://mbio-serv2.mbioekol.lu.se/ARAGORN/). The complete genome sequences
of Phages S1 and S2 were searched for similarity against the reported genomes by BLASTN
https://www.ncbi.nlm.nih.gov/BLAST/). Next, the genomes of the five most similar phages were
re-annotated by the same procedure as that of the two phages, and the transcribed ORF sequences were
compared by BLASTP. Comparative analysis of the whole genomes was performed by MAFFT [37]. The conserved sequences were extracted by Gblocks [38] and then used to construct a maximum
likelihood tree by RAxML [39]. The phylogenetic trees based on the amino acid sequences of the
terminase large subunit were constructed using Molecular Evolutionary Genetic Analysis (MEGA)
7.0 [40]. All the phylogenetic trees were visualized by MEGA 7.0. 2.10. Biofilm Inhibition Assays Biofilms were grown in 96-well microplates as previously described [43], with slight modifications. Briefly, an overnight-grown PAO1 culture was diluted 1:100 with fresh tryptic soy broth medium (TSB,
17 g of pancreatic digest of casein, 3 g of papaic digest of soya bean, 5 g of NaCl, 2.5 g of K2HPO4, and 5 of 19 Viruses 2019, 11, 318 2.5 g of glucose monohydrate for a 1 L solution, with a final pH adjusted to 7.3), and 150 µL aliquots of
this diluted culture were transferred into the wells of round-bottomed microplates (polystyrene) and
incubated under a static condition at 37 ◦C. To analyze the capacity of preventing biofilm formation,
50 µL of phage suspensions (approximately 108 pfu/well) in TSB medium or 50 µL of TSB medium
without phages as the controls were added to each well, and the plates were incubated for different
time periods (4, 8, and 24 h). To analyze the capacity of removing preformed biofilms, the plates were
incubated for 24 h to form the biofilms and rinsed three times with 0.9% NaCl to remove the planktonic
cells. Next, 200 µL of phage suspensions (approximately 108 pfu/well) in TSB medium, or 200 µL of
TSB medium without phages as the controls, were added to each well and were incubated at 37 ◦C for
different time periods (4, 8, and 24 h). The plates were all washed three times with 0.9% NaCl for the
next assays. y
CV (crystal violet) staining or XTT assay was performed to evaluate the biofilm conditions. For
the CV staining, the biofilms were stained with 220 µL of 0.1% crystal violet solution for 10 min, then
washed three times with 0.9% NaCl to remove the excess of CV, and left to dry in the air. Next, 220 µL
of 30% acetic acid were added to dissolve the bound CV. The eluted stain was transferred into another
microplate, and the absorbance was measured at 590 nm [44]. For the XTT assay, 220 µL of a solution
containing 0.5 mg/mL XTT and 50 µM menadione were added to the wells containing the rinsed
biofilms. After incubation in the dark for 2 h at 37 ◦C, the solution was then transferred into another
microplate to measure the absorbance at 460 nm [45]. 2.11. Accession Number The whole genome sequences of phage vB_PaeM_SCUT-S1 and vB_PaeM_SCUT-S2 were deposited
in GenBank under the accession number MK340760 and MK340761, respectively. 3.2. Basic Characteristics of the Genomes
3.2. Basic Characteristics of the Genomes
were about 93% of the whole genome w
tRNA prediction tools used was able to After performing the genome sequencing and data processing, high-quality, 578 and 424 Mbp
sequences were obtained for Phages S1 and S2, and the average genome coverage was about ~8700
and ~4500, respectively. After performing the assembly and manually refinement, we obtained the
After performing the genome sequencing and data processing, high-quality, 578 and 424 Mbp
sequences were obtained for Phages S1 and S2, and the average genome coverage was about ~8700
and ~4500, respectively. After performing the assembly and manually refinement, we obtained the
complete genomes of the two phages. For Phage S1, the genome had a size of 66,089 bp and a G +
C content of 55.43%. The genome termini were identified as circularly permuted by assessing the
sequence coverage using PhageTerm. The predicted Pac site was located in an AT-rich region, which
could be a replication origin (Figure 2). The genome was tightly organized, and the coding regions
were about 93% of the whole genome with 94 predicted ORFs (Figure 2 and Table S2). None of the
tRNA prediction tools used was able to identify any tRNA genes, indicating that Phage S1 is likely to
exploit the host tRNA machinery for its protein synthesis. For Phage S2, the complete genome size
was 94,434 bp with direct repeats (DRs) of 1189 bp at the ends. There were 197 ORFs identified for
Phage S2 (Figure 3 and Table S3). Phage S2 had a much lower G + C content (49.34%) than that of the
host strain PAO1 (66.3%), which was indicative of differences in the codon usage between the phage
and its host. Accordingly, a tRNA cluster of 11 tRNA genes was predicted (Figure 3). exploit the host tRNA machinery for its protein synthesis. For Phage S2, the complete genome size
was 94,434 bp with direct repeats (DRs) of 1189 bp at the ends. There were 197 ORFs identified for
Phage S2 (Figure 3 and Table S3). Phage S2 had a much lower G + C content (49.34%) than that of the
host strain PAO1 (66.3%), which was indicative of differences in the codon usage between the phage
and its host. Accordingly, a tRNA cluster of 11 tRNA genes was predicted (Figure 3). 3.1. Isolation and Characterization of the Phages Using P. aeruginosa PAO1 as the host strain, we isolated two phages from aquatic environment
samples. Both the phages formed clear plaques in the double agar layer lawn (Figure S1), which
indicated they were virulent phages. The spotting test showed that the two phages could infect most
P. aeruginosa strains with large and clear lysis zones (Table S1). From the electron microscopic imaging,
it was observed that both phages had icosahedral heads and contractile tails (Figure 1a,b), which
suggested that they both belonged to the order Caudovirales and the Myoviridae family. Thus, we
designated them as vB_PaeM_SCUT-S1 and vB_PaeM_SCUT-S2 (hereinafter referred to as S1 and S2,
respectively) according to the ICTV nomenclature for virus. Phage S1 had a capsid size of 77 ± 2 nm in
diameter and a tail length of 154 ± 2 nm, while Phage S2 had a capsid size of 85 ± 3 nm in diameter
and a tail length of 136 ± 3 nm (Figure 1a,b). 6 of 19
nm in Viruses 2019, 11, 318
nm in diameter a Figure 1. Transmission electron microscopy images of phages (a) vB_PaeM_SCUT-S1 and (b) vB_
PaeM_SCUT-S2 negatively stained by 2% phosphotungstic acid. Figure 1. Transmission electron microscopy images of phages (a) vB_PaeM_SCUT-S1 and
(b) vB_ PaeM_SCUT-S2 negatively stained by 2% phosphotungstic acid. uses 2019, 11, x FOR PEER REVIEW
6 of
mplete genomes of the two phages. For Phage S1, the genome had a size of 66,089 bp and a G +
ntent of 55.43%. The genome termini were identified as circularly permuted by assessing th
quence coverage using PhageTerm. The predicted Pac site was located in an AT-rich region, whi
uld be a replication origin (Figure 2). The genome was tightly organized, and the coding regio
re about 93% of the whole genome with 94 predicted ORFs (Figure 2 and Table S2). None of th 19, 11, x FOR PEER REVIEW
te genomes of the two phages. For Phage S
of 55.43%. The genome termini were id Figure 1. Transmission electron microscopy images of phages (a) vB_PaeM_SCUT-S1 and (b) vB_
PaeM_SCUT-S2 negatively stained by 2% phosphotungstic acid. Figure 1. Transmission electron microscopy images of phages (a) vB_PaeM_SCUT-S1 and
(b) vB_ PaeM_SCUT-S2 negatively stained by 2% phosphotungstic acid. uence coverage using PhageTerm. The predicted Pac site was located in an AT-rich region, wh
d be a replication origin (Figure 2). 3.1. Isolation and Characterization of the Phages The genome was tightly organized, and the coding regio
e about 93% of the whole genome with 94 predicted ORFs (Figure 2 and Table S2) None of t evolutionary level. Phage S1 ha
isolated in Poland and phage LBL
3.3. Comparative Genome Analysis to Burkholderia ambifaria phage BcepF1 (EF153632.1), isolated in the USA, and Escherichia phage FEC19
(MH816966.1), isolated in China (Figure 4). For the phages of Pakpunavirus, two main distant clades
were clustered, the smallest of which consisted of five phages. To better illustrate the phylogeny of
Phage S2, the members of the largest clade were selected and used to construct the maximum
likelihood tree. Phage S2 was located at a distinct branch, with a close relationship to phage C11
(KT804923.1) isolated in China and distant to the other phages, which is indicative of the low
homology of Phage S2 to the other Pakpunavirus (Figure 5). Since the comparative genome analysis
indicated that Phages S1 and S2 are new members of the genera Pbunavirus and Pakpunavirus,
respectively, the phylogenetic relationships among Pseudomonas phages of the Myoviridae family were
explored based on the terminase large subunit (Figure S3). Most phages clustered in five groups,
especially the Pbunavirus group (Green) and Pakpunavirus group (Brown) (Figure S3). Pbunaviruses
showed a distant relationship to the other groups, separated by some phages in the scattered clades
(Figure S3). The BLASTN results indicated that Phages S1 and S2 were homologous to the viruses of genera
Pbunavirus and Pakpunavirus, with the nucleotide similarity to congeneric phages varying from
68.56–94.00% and 94.97–97.99%, respectively. Based on these results, we compared the amino acid
identity of the two phages to their five most similar phages by BLASTP analysis. Phage S1 harbored
two or more unique ORFs, and Phage S2 had six or more unique ORFs (Table S4). There were 50 and 22
complete genome sequences for genus Pbunavirus (taxid:1198980) and Pakpunavirus (taxid:1921407) in
the Genbank database (as to December 2018), respectively. Thus, the intra-genus phylogenetic location
was explored at a genomic scale. For Phage S1, no phages were found at the same evolutionary level. Phage S1 had the highest homology with phage vB_Pae_PS44 (KM434184.1) isolated in Poland and
phage LBL3 (FM201281.1) isolated in Belgium, and was more distantly related to Burkholderia ambifaria
phage BcepF1 (EF153632.1), isolated in the USA, and Escherichia phage FEC19 (MH816966.1), isolated in
China (Figure 4). For the phages of Pakpunavirus, two main distant clades were clustered, the smallest
of which consisted of five phages. To better illustrate the phylogeny of Phage S2, the members of
the largest clade were selected and used to construct the maximum likelihood tree. 3.2. Basic Characteristics of the Genomes
3.2. Basic Characteristics of the Genomes
were about 93% of the whole genome w
tRNA prediction tools used was able to The arrows indicate the predicted open reading
frames and the different colors indicate the diverse functions of the coded proteins DR: direct repeat
Figure 3. Genome map of phage vB_PaeM_SCUT-S2. The arrows indicate the predicted open reading
frames, and the different colors indicate the diverse functions of the coded proteins. DR: direct repeat. 3.3. Comparative Genome Analysis
The BLASTN results indicated that Phages S1 and S2 were homologous to the viruses of genera
Pbunavirus and Pakpunavirus, with the nucleotide similarity to congeneric phages varying from 68.56–
94.00% and 94.97–97.99%, respectively. Based on these results, we compared the amino acid identity
of the two phages to their five most similar phages by BLASTP analysis. Phage S1 harbored two or
more unique ORFs, and Phage S2 had six or more unique ORFs (Table S4). There were 50 and 22
complete genome sequences for genus Pbunavirus (taxid:1198980) and Pakpunavirus (taxid:1921407)
in the Genbank database (as to December 2018), respectively. Thus, the intra-genus phylogenetic
location was explored at a genomic scale. For Phage S1, no phages were found at the same
The proteins identified for the two phages could be categorized into five functional classes:
nucleotide metabolism and DNA replication (including DNA repair and modification), virion structure
(including capsid and tail morphogenesis), DNA packaging, host lysis, and hypothetical proteins
(Figures 2 and 3; Tables S2 and S3). The structural protein genes were well clustered, and the nucleotide
metabolism and replication-related genes neighbored each other. In addition, three and five clusters of
unknown function were annotated for Phages S1 and S2 (i.e., ORF7-19, ORF71-76, and ORF78-93 for
Phage S1 and ORF1-21, ORF34-49, ORF79-99, ORF105-121, and ORF129-179 for Phage S2), respectively. No integrase, excisionase, and repressor genes, which are considered indicative of potential for a
lysogenic cycle, were found in the two genomes, supporting the conclusion that both S1 and S2 are
lytic phages. 3.2. Basic Characteristics of the Genomes
3.2. Basic Characteristics of the Genomes
were about 93% of the whole genome w
tRNA prediction tools used was able to The proteins identified for the two phages could be categorized into five functional classes:
nucleotide metabolism and DNA replication (including DNA repair and modification), virion
structure (including capsid and tail morphogenesis), DNA packaging, host lysis, and hypothetical
proteins (Figures 2 and 3; Tables S2 and S3). The structural protein genes were well clustered, and
the nucleotide metabolism and replication-related genes neighbored each other. In addition, three
and five clusters of unknown function were annotated for Phages S1 and S2 (i.e., ORF7-19, ORF71-
76, and ORF78-93 for Phage S1 and ORF1-21, ORF34-49, ORF79-99, ORF105-121, and ORF129-179 for
Phage S2), respectively. No integrase, excisionase, and repressor genes, which are considered
indicative of potential for a lysogenic cycle, were found in the two genomes, supporting the
l
i
th t b th S1
d S2
l ti
h y
p
g
Figure 2. Genome map of phage vB_PaeM_SCUT-S1. The arrows indicate the predicted open reading
frames, and the different colors indicate the diverse functions of the coded proteins. Figure 2. Genome map of phage vB_PaeM_SCUT-S1. The arrows indicate the predicted open reading
frames, and the different colors indicate the diverse functions of the coded proteins. Figure 2. Genome map of phage vB_PaeM_SCUT-S1. The arrows indicate the predicted open reading
frames, and the different colors indicate the diverse functions of the coded proteins. Figure 2. Genome map of phage vB_PaeM_SCUT-S1. The arrows indicate the predicted open reading
frames, and the different colors indicate the diverse functions of the coded proteins. Figure 2. Genome map of phage vB_PaeM_SCUT-S1. The arrows indicate the predicted open reading
frames, and the different colors indicate the diverse functions of the coded proteins. Figure 2. Genome map of phage vB_PaeM_SCUT-S1. The arrows indicate the predicted open reading
frames, and the different colors indicate the diverse functions of the coded proteins. 7 of 19
7 of 19 7 of 19
7 of 19 Viruses 2019, 11, 318
Viruses 2019, 11, x FO Figure 3. Genome map of phage vB_PaeM_SCUT-S2. The arrows indicate the predicted open reading
frames and the different colors indicate the diverse functions of the coded proteins DR: direct repeat
Figure 3. Genome map of phage vB_PaeM_SCUT-S2. The arrows indicate the predicted open reading
frames, and the different colors indicate the diverse functions of the coded proteins. DR: direct repeat. Figure 3. Genome map of phage vB_PaeM_SCUT-S2. evolutionary level. Phage S1 ha
isolated in Poland and phage LBL
3.3. Comparative Genome Analysis Phage S2 was
located at a distinct branch, with a close relationship to phage C11 (KT804923.1) isolated in China
and distant to the other phages, which is indicative of the low homology of Phage S2 to the other 8 of 19 Viruses 2019, 11, 318 9 of 19
ng Viruses 2019, 11, 318
y
[
1000 replicates Figure 5. Maximum likelihood tree showing the phylogenetic relationships among phages of genus
Pakpunavirus. The whole genome sequences were aligned by MAFFT [37] and the tree was visualized
by MEGA 7 [40]. The value at the nodes indicated the bootstrap support scores as calculated using
1000 replicates. Phage vB_PaeM_SCUT-S2 was colored red. Figure 5. Maximum likelihood tree showing the phylogenetic relationships among phages of genus
Pakpunavirus. The whole genome sequences were aligned by MAFFT [37] and the tree was visualized
by MEGA 7 [40]. The value at the nodes indicated the bootstrap support scores as calculated using
1000 replicates. Phage vB_PaeM_SCUT-S2 was colored red. Viruses 2019, 11, 318 Viruses 2019, 11, 318 Viruses 2019, 11, 318 Pakpunavirus (Figure 5). Since the comparative genome analysis indicated that Phages S1 and S2 are
new members of the genera Pbunavirus and Pakpunavirus, respectively, the phylogenetic relationships
among Pseudomonas phages of the Myoviridae family were explored based on the terminase large
subunit (Figure S3). Most phages clustered in five groups, especially the Pbunavirus group (Green)
and Pakpunavirus group (Brown) (Figure S3). Pbunaviruses showed a distant relationship to the other
groups, separated by some phages in the scattered clades (Figure S3). Viruses 2019, 11, x FOR PEER REVIEW
8 of 19 Pakpunavirus (Figure 5). Since the comparative genome analysis indicated that Phages S1 and S2 are
new members of the genera Pbunavirus and Pakpunavirus, respectively, the phylogenetic relationships
among Pseudomonas phages of the Myoviridae family were explored based on the terminase large
subunit (Figure S3). Most phages clustered in five groups, especially the Pbunavirus group (Green)
and Pakpunavirus group (Brown) (Figure S3). Pbunaviruses showed a distant relationship to the other
groups, separated by some phages in the scattered clades (Figure S3). Viruses 2019, 11, x FOR PEER REVIEW
8 of 19 Figure 4. Maximum likelihood tree showing the phylogenetic relationships among phages of genus
Pbunavirus. The whole genome sequences were aligned by MAFFT [37], and the tree was visualized
by MEGA 7 [40]. The value at the nodes indicated the bootstrap support scores as calculated using
1000 replicates. Phage vB_PaeM_SCUT-S1 was colored red. Figure 4. Maximum likelihood tree showing the phylogenetic relationships among phages of genus
Pbunavirus. The whole genome sequences were aligned by MAFFT [37], and the tree was visualized by
MEGA 7 [40]. The value at the nodes indicated the bootstrap support scores as calculated using 1000
replicates. Phage vB_PaeM_SCUT-S1 was colored red. Figure 4. Maximum likelihood tree showing the phylogenetic relationships among phages of genus
Pbunavirus. The whole genome sequences were aligned by MAFFT [37], and the tree was visualized
by MEGA 7 [40]. The value at the nodes indicated the bootstrap support scores as calculated using
1000 replicates. Phage vB_PaeM_SCUT-S1 was colored red. Figure 4. Maximum likelihood tree showing the phylogenetic relationships among phages of genus
Pbunavirus. The whole genome sequences were aligned by MAFFT [37], and the tree was visualized by
MEGA 7 [40]. The value at the nodes indicated the bootstrap support scores as calculated using 1000
replicates. Phage vB_PaeM_SCUT-S1 was colored red. 3.4. Structural Proteins of the Two Phages To better understand the functions of the annotated genes, we performed a proteomic analysis of
the two phages. After the genomic DNAs of the purified phage particles were released by LiCl, the
phages proteins were separated by SDS-PAGE on a 12% gel. The protein profiles of the two phages
were different due to their distinct genomes (Figure S2). For Phage S1, at least 16 protein bands were
detected with a molecular mass ranging from 12 to 100 kDa (Figure S2a). About 14 protein bands
within the range from 14 to 90 kDa were detected for Phage S2 (Figure S2b). In order to identify low
abundance proteins that might not be detected by Coomassie blue staining, the whole lane of the gels
was cut and analyzed by nanoLC-MS/MS instead of each stained band. Using the predicted ORFs as
the searching database, 31 proteins with at least two detected peptides were identified for the two
phages (Tables 1 and 2). For most of the identified proteins (i.e., 27/31 for Phage S1 and 20/31 for Phage
S2), the sequence coverage was above 20% (Tables 1 and 2), which indicated the high confidence of
the peptides. We speculated that ORF22, ORF27, ORF31, and ORF54 of Phage S1, and ORF57, ORF59,
ORF63, ORF68, ORF69, ORF70, ORF72, ORF74, and ORF80 of Phage S2 could be virion structural
proteins, based on the vicinity of their genes to genes coding for structural proteins. For ORF11, ORF72,
and ORF84 of Phage S1, and ORF14, ORF80, and ORF140 of Phage S2, no function could be assigned. 10 of 19 Viruses 2019, 11, 318 Table 1. Genes encoding virion proteins in vB_PaeM_SCUT-S1 identified by mass spectrometry. No. Predicted Function
Gene No. Mol. Mass
(kDa)
No. 3.4. Structural Proteins of the Two Phages of
Peptides
Coverage
1
putative tail protein containing
transglycosylase
ORF41
94.39
72
81%
2
putative tail fiber protein
ORF47
103.16
44
63%
3
putative minor head protein
ORF20
84.53
44
59%
4
putative structural protein
ORF46
54.81
23
60%
5
putative major structural protein
ORF26
41.59
20
49%
6
putative structural protein
ORF32
53.82
19
47%
7
putative minor head protein
ORF21
31.72
16
56%
8
putative structural protein
ORF24
52.10
14
38%
9
putative tail fiber protein
ORF33
15.91
12
70%
10
putative structural protein
ORF42
32.60
12
58%
11
putative structural protein
ORF31
21.48
12
77%
12
putative baseplate protein
ORF45
43.51
11
41%
13
putative endolysin
ORF49
24.35
11
48%
14
putative structural protein
ORF25
21.57
9
59%
15
putative structural protein
ORF38
19.94
9
60%
16
putative structural protein
ORF54
32.21
8
25%
17
putative structural protein
ORF37
17.80
7
67%
18
putative structural protein
ORF28
16.96
7
30%
19
putative structural protein
ORF22
7.46
7
77%
20
putative tail fiber protein
ORF39
21.70
6
44%
21
putative structural protein
ORF36
14.46
6
50%
22
putative structural protein
ORF35
12.74
5
29%
23
unknown function protein
ORF84
8.61
4
47%
24
putative holin
ORF53
21.13
4
25%
25
putative structural protein
ORF27
16.37
4
22%
26
putative DNA helicase
ORF56
59.43
4
7%
27
putative structural protein
ORF34
12.36
3
42%
28
putative ATP-dependent exonuclease V
ORF70
45.54
3
7%
29
putative baseplate protein
ORF44
24.17
3
13%
30
unknown function protein
ORF72
29.47
3
10%
31
unknown function protein
ORF11
15.52
2
21% Table 1. Genes encoding virion proteins in vB_PaeM_SCUT-S1 identified by mass spectrometry. The first step of bacteria elimination by phages is their successful invasion into the host cells [46,47]. During this process, the tail fiber proteins are believed to play an important role in host recognition,
and a single amino acid mutation can change the host specificity and give the phage the capacity of
infecting different species of bacteria [48,49]. Thus, we compared the predicted tail fiber proteins of the
five closest phages based on the terminase large subunit (Figures 4 and 5). For the tail fiber proteins of
Phage S1 and related phages, ORF33 presented only few amino acid differences and ORF47 showed
some variations at the C-terminus (Figure S4a). 3.4. Structural Proteins of the Two Phages Genes encoding virion proteins in vB_PaeM_SCUT-S2 identified by mass spectrometry. 3.4. Structural Proteins of the Two Phages In the case of Phage S2 and related phages, ORF75
presented low identity at the C-terminus and ORF77 showed some amino acid mutations (Figure S4b). Interestingly, several proteins involved in nucleotide metabolism and DNA modification were detected
in the phages particles, such as putative DNA helicase (ORF56 of S1), putative RNA polymerase (ORF58
of S2), putative ATP-dependent exonuclease V (ORF70 of S1), putative ribonucleoside-diphosphate
reductase alpha and beta subunits (ORF127 and ORF128 of S2), putative 3’-phosphatase (ORF123 of S2),
putative nictotinate phosphoribosyltransferase (ORF20 of S2), and putative methyltransferase (ORF53
of S2) (Tables 1 and 2), which indicate a potential role of these proteins in the phage infection mechanism. 11 of 19 Viruses 2019, 11, 318 Table 2. Genes encoding virion proteins in vB_PaeM_SCUT-S2 identified by mass spectrometry. No. Predicted Function
Gene No. Mol. Mass
(kDa)
No. of
Peptides
Coverage
1
putative tape measure protein
ORF67
85.86
55
66%
2
putative structural protein
ORF52
54.49
38
77%
3
putative structural protein
ORF61
46.35
31
51%
4
major capsid protein
ORF56
39.41
30
72%
5
putative tail fiber protein
ORF75
71.97
29
69%
6
putative tail fiber protein
ORF77
53.15
23
58%
7
putative baseplate component
ORF73
52.37
17
36%
8
putative structural protein
ORF74
26.64
17
80%
9
putative structural protein
ORF55
14.85
14
90%
10
putative baseplate protein
ORF71
26.84
11
53%
11
putative structural protein
ORF60
21.27
10
43%
12
putative structural protein
ORF70
34.17
10
35%
13
putative structural protein
ORF68
28.60
10
49%
14
putative structural protein
ORF57
18.24
9
40%
15
putative structural protein
ORF62
18.95
8
55%
16
putative structural protein
ORF63
18.29
8
40%
17
putative structural protein
ORF72
14.07
7
32%
18
putative structural protein
ORF59
14.46
7
73%
19
unknown function protein
ORF108
37.10
6
17%
20
putative RNA polymerase
ORF58
15.51
5
15%
21
putative structural protein
ORF80
16.06
4
32%
22
putative endolysin
ORF78
20.89
4
20%
23
putative ribonucleoside-diphosphate
reductase alpha chain
ORF128
67.42
4
6%
24
putative ribonucleoside-diphosphate
reductase beta subunit
ORF127
40.62
4
8%
25
putative 3’-phosphatase
ORF123
35.43
3
9%
26
putative nictotinate
phosphoribosyltransferase
ORF20
63.05
3
5%
27
unknown function protein
ORF14
14.86
3
6%
28
putative structural protein
ORF64
17.75
2
12%
29
putative methyltransferase
ORF53
17.27
2
14%
30
putative structural protein
ORF69
13.98
2
20%
31
unknown function protein
ORF140
37.78
2
4%
3 5 St bilit A
l
i Table 2. 3.5. Stability Analysis After the characterization at genomic and proteomic level, the physiological properties of Phages
S1 and S2, in terms of pH and temperature stability, were investigated. Phage S1 was more robust to
pH changes than Phage S2 was. Their infectivity both remained almost intact when exposed for 1 h to
a pH ranging from 4 to 10 (Figure 6a,b). However, the performance changed when exposed to the cruel
pH conditions. The infectivity of Phage S1 slightly decreased at pH 3 and pH 11 (Figure 6a), while
that of Phage S2 decreased significantly at pH 3 and nearly lost at pH 11 (Figure 6b). The two phages
were both inactivated when exposed to pH 12 (Figure 6a,b). Regarding the temperature stability, the
two phages also showed different properties. The infectivity of Phage S1 decreased gradually upon
increasing the temperature when exposed for 1 h to temperatures ranging from 30 to 60 ◦C, dropped
significantly at 70 ◦C, and was completely lost at 80 ◦C (Figure 6c). Conversely, the infectivity of Phage
S2 showed a slight decrease upon increasing the temperature up to 60 ◦C but was completely lost at
70 ◦C or above (Figure 6d). The different response of the two phages to the temperature could result
from their distinct capsid proteins or structures. 12 of 19
12 of 19 Viruses 2019, 11, 318
Viruses 2019, 11, x FOR ses 2019, 11, 318
12 of
ses 2019, 11, x FOR PEER REVIEW
12 of
Figure 6. Relative amount of infectious phage particles after different treatments. (a) vB_PaeM_SCUT-
S1 and (b) vB_PaeM_SCUT-S2 incubated for 1 h at different pH levels; (c) vB_PaeM_SCUT-S1 and (d)
vB_PaeM_SCUT-S2 incubated for 1 h at different temperatures. * No plaques were detected at pH 12
for either Phages S1 and S2 (a, b), at 80 °C for Phage S1 (c) and at 70 °C or 80 °C for Phage S2 (d),
indicating that the phages were completely inactive in these conditions. Three independent
experiments were performed. Figure 6. Relative amount of infectious phage particles after different treatments. (a) vB_PaeM_SCUT-S1
and (b) vB_PaeM_SCUT-S2 incubated for 1 h at different pH levels; (c) vB_PaeM_SCUT-S1 and
(d) vB_PaeM_SCUT-S2 incubated for 1 h at different temperatures. 3.5. Stability Analysis * No plaques were detected at
pH 12 for either Phages S1 and S2 (a,b), at 80 ◦C for Phage S1 (c) and at 70 ◦C or 80 ◦C for Phage
S2 (d), indicating that the phages were completely inactive in these conditions. Three independent
experiments were performed. Figure 6 Relative amount of infectious phage particles after different treatments (a) vB PaeM SCUT
Figure 6. Relative amount of infectious phage particles after different treatments. (a) vB_PaeM_SCUT-S1 Figure 6. Relative amount of infectious phage particles after different treatments. (a) vB_PaeM_SCUT-
S1 and (b) vB_PaeM_SCUT-S2 incubated for 1 h at different pH levels; (c) vB_PaeM_SCUT-S1 and (d)
vB_PaeM_SCUT-S2 incubated for 1 h at different temperatures. * No plaques were detected at pH 12
for either Phages S1 and S2 (a, b), at 80 °C for Phage S1 (c) and at 70 °C or 80 °C for Phage S2 (d),
indicating that the phages were completely inactive in these conditions. Three independent
experiments were performed
Figure 6. Relative amount of infectious phage particles after different treatments. (a) vB_PaeM_SCUT-S1
and (b) vB_PaeM_SCUT-S2 incubated for 1 h at different pH levels; (c) vB_PaeM_SCUT-S1 and
(d) vB_PaeM_SCUT-S2 incubated for 1 h at different temperatures. * No plaques were detected at
pH 12 for either Phages S1 and S2 (a,b), at 80 ◦C for Phage S1 (c) and at 70 ◦C or 80 ◦C for Phage
S2 (d), indicating that the phages were completely inactive in these conditions. Three independent
experiments were performed. S1 and (b) vB_PaeM_SCUT-S2 incubated for 1 h at different pH levels; (c) vB_PaeM_SCUT-S1 and (d)
vB_PaeM_SCUT-S2 incubated for 1 h at different temperatures. * No plaques were detected at pH 12
for either Phages S1 and S2 (a, b), at 80 °C for Phage S1 (c) and at 70 °C or 80 °C for Phage S2 (d),
indicating that the phages were completely inactive in these conditions. Three independent
experiments were performed
and (b) vB_PaeM_SCUT S2 incubated for 1 h at different pH levels; (c) vB_PaeM_SCUT S1 and
(d) vB_PaeM_SCUT-S2 incubated for 1 h at different temperatures. * No plaques were detected at
pH 12 for either Phages S1 and S2 (a,b), at 80 ◦C for Phage S1 (c) and at 70 ◦C or 80 ◦C for Phage
S2 (d), indicating that the phages were completely inactive in these conditions. Three independent
experiments were performed. Viruses 2019, 11, 318 13 of 19 Viruses 2019, 11, 318 The effect of S1 and S2 against P. aeruginosa PAO1 planktonic cultures were determined by using
the phages alone or in combination. Phages S1 and S2 efficiently inhibited the growth of the host
bacteria at the exponential phase. The number of phages had a great influence on bacterial growth in
the early stage, and at the highest titer (i.e., MOI = 100) the phages could kill the host cells directly
without needing a latent period to produce further virions (Figure 8a,b). As the growth time elapsed,
this advantage of higher phage titers gradually disappeared. The bacterial growth was totally inhibited
by Phage S2 after 3.5 h for the different treatments, including the lowest MOI = 0.01 (Figure 8b). The inhibitory effect of Phage S1 was slightly lower than Phage S2 (Figure 8a). The host lysis became
inefficient after 12 h of culture, and the higher the MOI used at the beginning, the higher the bacteria
grown at 20 h (Figure 8a,b), which suggested the emergence of phage-insensitive or phage-resistant
strains. This phenomenon was not alleviated by using the two phages in combination (Figure 8c). The lysis curve observed using a 1:1 mixture of Phages S1 and S2 was very similar to that obtained
using Phage S2 alone, indicating that the lysis induced by Phage S1 was nearly covered by that of
Phage S2. Viruses 2019, 11, x FOR PEER REVIEW
14 of 19 Figure 8. Growth curves of PAO1 strains infected with different phages. (a) vB_PaeM_SCUT-S1 of
different MOIs; (b) vB_PaeM_SCUT-S2 of different MOIs; (c) vB_PaeM_SCUT-S1 and
vB_PaeM_SCUT-S2 were combined in a 1:1 ratio to the indicated final MOIs. Figure 8. Growth curves of PAO1 strains infected with different phages. (a) vB_PaeM_SCUT-S1 of
different MOIs; (b) vB_PaeM_SCUT-S2 of different MOIs; (c) vB_PaeM_SCUT-S1 and vB_PaeM_SCUT-S2
were combined in a 1:1 ratio to the indicated final MOIs. Figure 8. Growth curves of PAO1 strains infected with different phages. (a) vB_PaeM_SCUT-S1 of
different MOIs; (b) vB_PaeM_SCUT-S2 of different MOIs; (c) vB_PaeM_SCUT-S1 and
vB_PaeM_SCUT-S2 were combined in a 1:1 ratio to the indicated final MOIs. Figure 8. Growth curves of PAO1 strains infected with different phages. (a) vB_PaeM_SCUT-S1 of
different MOIs; (b) vB_PaeM_SCUT-S2 of different MOIs; (c) vB_PaeM_SCUT-S1 and vB_PaeM_SCUT-S2
were combined in a 1:1 ratio to the indicated final MOIs. p
p
3 6 G
th Ch
t i ti
d L
i Ki
ti
3.6. Growth Characteristics and Lysis Kinetics 3.6. Growth Characteristics and Lysis Kinetics
The one-step growth curve analysis revealed that Phages S1 and S2 had distinctive life cycles
(Figure 7a,b). Phage S1 had a latent period of 40 min and a rise period of 10 min, and generated about
134 virion progenies per infected cell (Figure 7a). Phage S2 had a shorter latent period and a
prolonged rise period, both being about 25 min, and the average burst size was much lower (i.e., 40
progenies per infected cell) than that of Phage S1 (Figure 7b). These differences in growth kinetics
lt i
diff
t h
t l
i
ffi i
The one-step growth curve analysis revealed that Phages S1 and S2 had distinctive life cycles
(Figure 7a,b). Phage S1 had a latent period of 40 min and a rise period of 10 min, and generated about
134 virion progenies per infected cell (Figure 7a). Phage S2 had a shorter latent period and a prolonged
rise period, both being about 25 min, and the average burst size was much lower (i.e., 40 progenies
per infected cell) than that of Phage S1 (Figure 7b). These differences in growth kinetics can result in
different host lysis efficiency. Viruses 2019, 11, x FOR PEER REVIEW
13 of 19 n result in different host lysis efficiency. Figure 7. One step growth curve of (a) vB_PaeM_SCUT-S1 and (b) vB_PaeM_SCUT-S1. Data are
presented as the mean (± standard deviations) titers measured at the indicated infection time obtained
from three independent experiments. Figure 7. One step growth curve of (a) vB_PaeM_SCUT-S1 and (b) vB_PaeM_SCUT-S1. Data are
presented as the mean (± standard deviations) titers measured at the indicated infection time obtained
from three independent experiments. Figure 7. One step growth curve of (a) vB_PaeM_SCUT-S1 and (b) vB_PaeM_SCUT-S1. Data are
presented as the mean (± standard deviations) titers measured at the indicated infection time obtained
from three independent experiments. Figure 7. One step growth curve of (a) vB_PaeM_SCUT-S1 and (b) vB_PaeM_SCUT-S1. Data are
presented as the mean (± standard deviations) titers measured at the indicated infection time obtained
from three independent experiments. Viruses 2019, 11, 318 4. Discussion
4. Discussion i
u
io
The identification and characterization of novel phages can enhance our understanding of phage
biology. Phages S1 and S2, which were assigned to the Myoviridae family, have genome sizes of 66.1
kb and 94.4 kb encoding 94 and 179 putative proteins, respectively. Both phages showed genetic
mosaicism, a typical feature of tailed phages [50], with several functional modules clustered
throughout their genomes (Figures 2 and 3). In addition to a number of clustered genes coding for
proteins with predictable function, several ORFs could only be annotated as coding for proteins with
unknown function (Tables S2 and S3), a common problem in phage studies [51]. Based on our
proteomic analysis, 23 and 21 ORFs were identified as structural protein coding genes for Phages S1
and S2, respectively (Tables 1 and 2), a result that contributes to the enrichment of phage proteome
collection. Although Phages S1 and S2 were isolated from the same location, they belonged to two
different genera, Pbunavirus and Pakpunavirus, respectively. Based on the phylogenetic analysis, they
were found to be distantly related to each other (Figure S3), suggesting that the two phages evolved
independently and might have different roles in regulating bacterial communities. This is consistent
with a recent study in which several phages where isolated from a single environmental source [52]. Interestingly, Phage S2 presented a dominant advantage over Phage S1 in infecting PAO1 strain and
in reducing its biofilm, which inspires for a better exploration of the relationships between the other
h
i
th
Pb
i
d P k
i
The identification and characterization of novel phages can enhance our understanding of phage
biology. Phages S1 and S2, which were assigned to the Myoviridae family, have genome sizes of
66.1 kb and 94.4 kb encoding 94 and 179 putative proteins, respectively. Both phages showed genetic
mosaicism, a typical feature of tailed phages [50], with several functional modules clustered throughout
their genomes (Figures 2 and 3). In addition to a number of clustered genes coding for proteins with
predictable function, several ORFs could only be annotated as coding for proteins with unknown
function (Tables S2 and S3), a common problem in phage studies [51]. Based on our proteomic
analysis, 23 and 21 ORFs were identified as structural protein coding genes for Phages S1 and S2,
respectively (Tables 1 and 2), a result that contributes to the enrichment of phage proteome collection. 3.7. Biofilm Eradication
3.7. Biofilm Eradication Viruses 2019, 11, x FOR PEER REVIEW
15 of 19 biofilm properties: the biofilm biomass (tested by CV staining) and the cell metabolic activity (tested
by XTT assay) (Figure 9). When the 24-h-grown PAO1 biofilms were treated with the phages for
different time periods (4, 8, and 24 h), the biofilm was effectively eradicated by either phage used
alone as well as by the combination of the two phages (Figure 9). The biomass of the biofilm was
reduced more effectively by prolonged treatment. The relative reduction was 5% at 4 h, 10% at 8 h,
and 44% at 24 h for Phage S1 and 50% at 4 h, 61% at 8 h, and 69% at 24 h for Phage S2 (Figure 9a). The combination of Phages S1 and S2 showed nearly the same reduction observed when using Phage
S2 alone (Figure 9a). Regarding the cell metabolic activity, the 8 h treatment provided the most effective
inhibition (i.e., 79% for Phage S1 and 97% for both Phage S2 and the combination of Phages S1 and
S2, Figure 9b), while upon 24 h treatment, a significant recovery of metabolic activity was observed
(Figure 9b). The observed biofilm metabolic activity was more consistent with the measured lysis
kinetics than the biofilm biomass (see paragraph 3.6). Overall, Phage S2 showed a better performance
on growth inhibition and biofilm reduction than Phage S1, and the combination of Phages S1 and S2
did not show an obvious synergistic effect in either planktonic cells or biofilm eradication. Viruses 2019, 11, x FOR PEER REVIEW
15 of 19 Figure 9. Effect of phage treatment on 24-h-grown biofilms. (a) Biomass evaluation by CV staining;
(b) metabolic activity evaluation by XTT assay. Control: without any phages; S1: treated with 108 pfu
of phage vB_PaeM_SCUT-S1; S2: treated with 108 pfu phage vB_PaeM_SCUT-S2; S1 + S2: treated with
a mixture of vB_PaeM_SCUT-S1 and vB_PaeM_SCUT-S2 (0.5 × 108 pfu of each phage). The different
treatment duration is indicated by different bar colors. Figure 9. Effect of phage treatment on 24-h-grown biofilms. (a) Biomass evaluation by CV staining;
(b) metabolic activity evaluation by XTT assay. Control: without any phages; S1: treated with 108 pfu
of phage vB_PaeM_SCUT-S1; S2: treated with 108 pfu phage vB_PaeM_SCUT-S2; S1 + S2: treated with
a mixture of vB_PaeM_SCUT-S1 and vB_PaeM_SCUT-S2 (0.5 × 108 pfu of each phage). The different
treatment duration is indicated by different bar colors. Figure 9. 3.7. Biofilm Eradication
3.7. Biofilm Eradication Effect of phage treatment on 24-h-grown biofilms. (a) Biomass evaluation by CV staining;
(b) metabolic activity evaluation by XTT assay. Control: without any phages; S1: treated with 108 pfu
of phage vB_PaeM_SCUT-S1; S2: treated with 108 pfu phage vB_PaeM_SCUT-S2; S1 + S2: treated with
a mixture of vB_PaeM_SCUT-S1 and vB_PaeM_SCUT-S2 (0.5 × 108 pfu of each phage). The different
treatment duration is indicated by different bar colors. Figure 9. Effect of phage treatment on 24-h-grown biofilms. (a) Biomass evaluation by CV staining;
(b) metabolic activity evaluation by XTT assay. Control: without any phages; S1: treated with 108 pfu
of phage vB_PaeM_SCUT-S1; S2: treated with 108 pfu phage vB_PaeM_SCUT-S2; S1 + S2: treated with
a mixture of vB_PaeM_SCUT-S1 and vB_PaeM_SCUT-S2 (0.5 × 108 pfu of each phage). The different
treatment duration is indicated by different bar colors. 3.7. Biofilm Eradication
3.7. Biofilm Eradication f
We first tested whether the phages could prevent the biofilm formation based on the biofilm
biomass, which was tested by CV staining. The results showed that the phages alone or in
combination could effectively inhibit the biofilm growth upon incubation for 4, 8, and 24 h (Figure
S5). Next, the biofilm-degradation capacity of Phages S1 and S2 was evaluated by investigating two
We first tested whether the phages could prevent the biofilm formation based on the biofilm
biomass, which was tested by CV staining. The results showed that the phages alone or in combination
could effectively inhibit the biofilm growth upon incubation for 4, 8, and 24 h (Figure S5). Next, the
biofilm-degradation capacity of Phages S1 and S2 was evaluated by investigating two complementary 14 of 19 Viruses 2019, 11, 318 biofilm properties: the biofilm biomass (tested by CV staining) and the cell metabolic activity (tested
by XTT assay) (Figure 9). When the 24-h-grown PAO1 biofilms were treated with the phages for
different time periods (4, 8, and 24 h), the biofilm was effectively eradicated by either phage used
alone as well as by the combination of the two phages (Figure 9). The biomass of the biofilm was
reduced more effectively by prolonged treatment. The relative reduction was 5% at 4 h, 10% at 8 h,
and 44% at 24 h for Phage S1 and 50% at 4 h, 61% at 8 h, and 69% at 24 h for Phage S2 (Figure 9a). The combination of Phages S1 and S2 showed nearly the same reduction observed when using Phage
S2 alone (Figure 9a). Regarding the cell metabolic activity, the 8 h treatment provided the most effective
inhibition (i.e., 79% for Phage S1 and 97% for both Phage S2 and the combination of Phages S1 and
S2, Figure 9b), while upon 24 h treatment, a significant recovery of metabolic activity was observed
(Figure 9b). The observed biofilm metabolic activity was more consistent with the measured lysis
kinetics than the biofilm biomass (see paragraph 3.6). Overall, Phage S2 showed a better performance
on growth inhibition and biofilm reduction than Phage S1, and the combination of Phages S1 and S2
did not show an obvious synergistic effect in either planktonic cells or biofilm eradication. Viruses 2019, 11, 318 Viruses 2019, 11, 318 15 of 19 The two phages could be suitable candidates to be further developed for phage therapy. In fact,
Phages S1 and S2 are virulent phages with a conventional genome size and no genes coding for
toxins, virulence factors, or lysogeny-related proteins were discovered by genome annotation and
proteomic analysis (Figures 2 and 3, Tables 1 and 2 and Tables S2 and S3), in contrast to lysogenic
phages, which are not recommended for therapy [53]. The infectivity of the phages remained stable
in a temperature range of 4–37 ◦C and a pH range of 4–10 (Figure 6), indicating that they could be
stored at room temperature and the activity could be maintained in a human physiological condition. Furthermore, they inhibited the growth of planktonic cells effectively alone or together at a low MOI
for a 12 h treatment (Figure 8) and had good performances on preventing the biofilm formation
(Figure S5) and eradicating biofilms (Figure 9). However, particular attention should be paid to the
potential development of phage resistance in clinical settings since the bacterial growth could be
partially recovered with a 24 h treatment in vitro (Figures 8 and 9). Although the development of
bacterial resistance considered inevitable in phage therapy, it is believed to occur less infrequently
in vivo than in vitro [54]. In addition, phage adaptation or training performed by in vitro procedures
could be utilized to solve this problem [54,55]. For example, the short-term antagonistic evolution of
the P. aeruginosa strain PAO1 and the Pbunaviruses phage KPP22 resulted in the formation of KPP22
mutants with recovered infectivity towards phage-resistant PAO1 clones [56]. Members of genera Pbunavirus and Pakpunavirus have been studied for their suitability as
therapeutics, and several phages have already been proved to be effective in controlling P. aeruginosa
MDR infections in animal models [57–62]. For example, Pbunaviruses ΦNH-4 could effectively clear
the Pseudomonas infection of murine lungs in 6 h [59], and a cocktail of three Pbunaviruses DL52, DL60,
DL68 and other three Podoviridae phages could prolong survival of Galleria mellonella infected by clinical
strains of P. aeruginosa [60]. Pakpunavirus PAK_P1 was effective in treating acute lung infection in a
mouse model [61] and six closely related Pakpunavirus phages gave high survival rates, between 75
and 100%, with a 13-day treatment in a mouse lung infection model [62]. Acknowledgments: We thank the members of Lin’s lab, South China University of Technology, for the helpful
discussions and Marco Pistolozzi, South China University of Technology, for revising the manuscript. We thank
Ming Li of the Third Military Medical University for generously providing us with the PAO1 strain. Author Contributions: Conceptualization: T.W. Formal analysis: Y.G., P.C. and T.W. Investigation: Y.G. Project
administration: T.W. Supervision: Z.L. Visualization: Y.G. and T.W. Writing—original draft: Y.G. and T.W.
Writing—review & editing: P.C., Z.L. and T.W. 4. Discussion
4. Discussion Although Phages S1 and S2 were isolated from the same location, they belonged to two different genera,
Pbunavirus and Pakpunavirus, respectively. Based on the phylogenetic analysis, they were found to be
distantly related to each other (Figure S3), suggesting that the two phages evolved independently and
might have different roles in regulating bacterial communities. This is consistent with a recent study in
which several phages where isolated from a single environmental source [52]. Interestingly, Phage S2
presented a dominant advantage over Phage S1 in infecting PAO1 strain and in reducing its biofilm,
which inspires for a better exploration of the relationships between the other phages in the genera
Pbunavirus and Pakpunavirus. Viruses 2019, 11, 318 Viruses 2019, 11, 318 Phages S1 and S2 harbored
distinctive fiber proteins (Figure S4) and might give rise to unique host recognition ranges [48,49]. Consequently, Phages S1 and S2 can have a host range different from that of previously reported
phages [21,63,64] (e.g., both phages could infect 6/7 host P. aeruginosa strains in our study (Table S1)),
which makes them potential candidates for their inclusion in phage therapy cocktails. Further research
will study their efficacy in controlling clinical P. aeruginosa MDR infections and confirm the safety of
the two phages as therapeutic tools. Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4915/11/4/318/s1. Figure S1: Plaques formed by phage vB_PaeM_SCUT-S1 (a) and vB_PaeM_SCUT-S2 (b) infecting P. aeruginosa
PAO1. Figure S2: Identification of the structural proteins by SDS–PAGE analysis. Figure S3: Neighbor-joining
tree showing the phylogenetic relationships among Pseudomonas phages of Myoviridae family. Figure S4: The
comparison of the tail fiber proteins of phage vB_PaeM_SCUT-S1 (a) and vB_PaeM_SCUT-S2 (b) to the five most
closely related phages. Figure S5. The inhibitory effect of phage treatment on biofilm formation based on the
biomass tested by CV staining. Table S1: Host range of phage vB_PaeM_SCUT-S1 and vB_PaeM_SCUT-S2. Table
S2: The genome annotation of Pseudomonas phage vB_PaeM_SCUT-S1. Table S3: The genome annotation of
Pseudomonas phage vB_PaeM_SCUT-S2. Table S4: Amino acid identity between phage vB_PaeM_SCUT-S1 (a) and
phage vB_PaeM_SCUT-S2 (b) with five most similar members indicated by BLASTN analysis. Author Contributions: Conceptualization: T.W. Formal analysis: Y.G., P.C. and T.W. Investigation: Y.G. Project
administration: T.W. Supervision: Z.L. Visualization: Y.G. and T.W. Writing—original draft: Y.G. and T.W. Writing—review & editing: P.C., Z.L. and T.W. Funding: This study was funded by the National Natural Science Founding of China (Grant No. 31800718),
the Doctoral Scientific Research Foundation of China (Grant No. 2017M612649), the National Natural Science
Founding of Guangdong province (Grant No. 2018A030310361) and the Fundamental Research Funds for the
Central Universities of South China University of Technology (Grant No. x2sw-D2172890, x2sw-D2172900 and
x2sw-D2182030). Acknowledgments: We thank the members of Lin’s lab, South China University of Technology, for the helpful
discussions and Marco Pistolozzi, South China University of Technology, for revising the manuscript. We thank
Ming Li of the Third Military Medical University for generously providing us with the PAO1 strain. 16 of 19 Viruses 2019, 11, 318 16 of 19 Conflicts of Interest: The authors declare no conflict of interest. References 1. Boucher, H.W.; Talbot, G.H.; Bradley, J.S.; Edwards, J.E.; Gilbert, D.; Rice, L.B.; Scheld, M.; Spellberg, B.;
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