1
Supplementary Material
2
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Evidence for carry-over effects of predator exposure on pathogen transmission potential
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Olivier Roux, Amélie Vantaux, Benjamin Roche, Koudraogo B. Yameogo, Roch K. Dabiré,
8
Abdoulaye Diabaté, Frederic Simard and Thierry Lefèvre
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Insect collections
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Mosquitoes - Experiments were conducted with F1 to F3 mosquito larvae obtained
12
from wild, gravid Anopheles gambiae s.l. females were collected while at rest in inhabited
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human dwellings in Bama, Burkina Faso (11°23'14"N, 4°24'42"W) during the rainy season
14
(May-October 2011). Females were placed individually in oviposition cups containing spring
15
water and maintained under controlled conditions (27±1°C, 80±10% relative humidity,
16
12L:12D). After oviposition, they were identified to species level by routine PCR-RFLP
17
based on segregating SNP polymorphisms in the X-linked ribosomal DNA InterGenic Spacer
18
region following the procedure described in [1]. Only larvae of An. coluzzii were kept for this
19
study. The larvae were reared in spring water exposed to ambient conditions in the insectaries
20
(27±1°C, 80±10% relative humidity, 12L:12D) and fed with Tetramin® Baby Fish Food ad
21
libitum. Adults were reared in mesh cages (30x30x30cm) and provided with 5% glucose and
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water on imbibed cotton wool pads ad libitum. Females were allowed to take three successive
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blood meals on rabbits for egg maturation and were then provided with an egg-laying site (ie,
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plastic cup Ø=45mm; h=40mm) for oviposition.
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Experimental design
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Exposure to predator – 300 first instar larvae and one predator were placed together in
28
a plastic container (L:28cm, l:18cm, h:5.5cm) filled with 1L of spring water. The predator
29
was free to feed upon mosquito larvae during the entire period of larval mosquito
30
development. To standardize hunger levels in the predators, they were starved for 48h and
31
then fed with 20 third instar mosquito larvae 12h before the beginning of the experiment.
32
Indeed, fully starved predators were not adequate because they ate all the first instar larvae
33
within the first 48h of exposure at the beginning of the experiment. For each experimental
34
container, its equivalent without a predator was set up as a control. Two replicates were run,
35
one on 26 containers (13 experimental and 13 controls) and a second one on 32 containers (16
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experimental and 16 controls). To avoid a potential density effect on adult life-history traits,
37
larval densities were equalized only one time when larvae reached the late second instar.
38
Indeed, most of the predation occurs during early development (on first and second instar
39
larvae). Later, predation rate decreases as larvae become bigger (third and fourth instar) and
40
densities are therefore less quick to change (see results on emergence rate).
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Experimental infections
43
We used Direct Membrane Feeding Assays (DMFA) whereby gametocyte-infected
44
blood is drawn from naturally-infected patients and on which mosquitoes feed through a
45
membrane [2]. Plasmodium falciparum gametocyte carriers were recruited by examining
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thick blood smears from school children aged between five and ten years, who live and attend
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school in Soumousso and Dande. Malaria positive individuals were treated according to
48
national recommendations (Artésunate + Amodiaquine). Eight ml of venous blood were taken
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from selected carriers with at least 20 gametocytes/ml of blood (estimated based on an
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average of 8000 white blood cells/ml). Four- to-six-day-old female mosquitoes were allowed
51
to feed for up to 30 minutes through a Parafilm membrane. As a negative control (uninfected
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mosquitoes), females were fed on the same blood in which the gametocytes were heat-
53
inactivated for 20 minutes at 45°C. This heat-inactivation inhibits the infection and does not
54
affect the blood nutritive quality [3]. This procedure is used routinely in our lab [3-14].
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Measurements of infection intensity
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Seven days post-blood meal, the females were dissected. Their midguts were removed
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under a stereomicroscope (magnification 35x, Leica EZ4D, Wetzlar, Deutschland) and stained
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with a 2% Mercurochrome® solution. Oocysts were counted under a light microscope
60
(magnification 400x, Leica ICC50, Wetzlar, Deutschland).
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At day 14 post-blood meal, the heads and thoraces of mosquitoes were dissected under
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a stereomicroscope (magnification 35x, Leica EZ4D, Wetzlar, Deutschland) and frozen at -
63
80°C until molecular analysis. The quantification of P. falciparum sporozoites in salivary
64
glands was determined by qPCR using 7500 Fast Real time PCR System (Applied
65
Biosystems, Foster City CA, USA). The mosquito heads and thoraxes were crushed
66
individually and DNA extracted as previously described in Morlais et al. [15]. For sporozoite
67
quantification, we targeted the fragment of subunit 1 of the mitochondrial cytochrome c
68
oxidase gene (cox 1) using the forward and reverse primer sequences, qPCR-PfF 5’-
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TTACATCAGGAATGTTATTGC-3’ and qPCR-PfR 5’-ATATTGGATCTCCTGCAAAT-3,
70
respectively. The reaction was conducted in a 10µL final volume containing: 1µL of DNA
71
template, 1x HOT Pol EvaGreen qPCR Mix Plus ROX, and 600nM of each primer.
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Amplification started by initial activation step at 95°C for 15min and 40 cycles of
73
denaturation at 95°C for 15s and annealing / extension at 58°C for 30s. Detection was
74
conducted during the last step [16].
75
Quantification was based on a standard curve built from four serial dilutions (12%) of
76
an asexual parasite culture. We made dilutions ranging from 60 to 60,000 genome/µl of
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DNAs from a standard culture. The first dilution (10-1) was used as a positive control. The
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standard curve (y= -3.384X +35.874) was obtained by linear regression analysis of Ct values
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(Cycle threshold) versus log10 genome copy number of parasite culture.
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Fecundity and wing size measurement
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Seven days post-blood meal, the ovaries were dissected and the eggs counted and
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photographed for each female under stereomicroscope equipped with a camera (magnification
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35x, Leica EZ4D, Wetzlar, Deutschland). Five eggs per female were randomly chosen and
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measured from tip-to-tip. One wing per female was also dissected, photographed and
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measured from the alular notch to the apical margin tip, excluding fringe scales. All
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measurements were made with the ImageJ software (V1.47, Wayne Rasband National
88
Institute of Health, USA).
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Mathematical model
We provide here the details of the mathematical model used to quantify the
epidemiological outcomes in the presence or absence of predators.
𝑑𝐿𝑚
= [𝑏𝑆 𝑆𝑚 + 𝑏𝐸 𝐸𝑚 + 𝑏𝐼 𝐼𝑚 ]𝜃 − 𝜖𝛾𝐿𝑚
𝑑𝑡
𝑑𝑆𝑚
𝑆𝑚
= 𝜖𝛾𝐿𝑚 − 𝑑𝑆 𝑆𝑚 − 𝑎𝑏𝐼ℎ
𝑑𝑡
𝑆𝑚 + 𝐸𝑚 + 𝐼𝑚
𝑑𝐸𝑚
𝑆𝑚
= 𝑎𝑏𝐼ℎ
− (𝑑𝐸 + 𝜎)𝐸𝑚
𝑑𝑡
𝑆𝑚 + 𝐸𝑚 + 𝐼𝑚
𝑑𝐼𝑚
= 𝜎𝐸𝑚 − 𝑑𝐼 𝐼𝑚
𝑑𝑡
𝑑𝑆ℎ
𝑆ℎ
= −𝑎𝑐𝐼𝑚
𝑑𝑡
𝑆ℎ + 𝐼ℎ + 𝑅ℎ
98
𝑑𝐼ℎ
𝑆ℎ
= 𝑎𝑐𝐼𝑚
− 𝜔𝐼ℎ
𝑑𝑡
𝑆ℎ + 𝐼ℎ + 𝑅ℎ
99
𝑑𝑅ℎ
= 𝜔𝐼ℎ
𝑑𝑡
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The different categories are presented in detail in the main text and the parameters are
101
described in table S1. It is worth pointing out that we did not consider the demography for the
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human population. Indeed, since we focused on the outbreak size over one season, human
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demography, definitely slower than mosquito demography, is not expected to significantly
104
influence this epidemiological outcome. Similarly, we did not consider an “Exposed” class for
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human, since this latency period in humans is considered very short compared to the human
106
lifespan. Finally, the Latin hypercube sampling was conducted within the confidence interval
107
of each parameter detailed in Table S1 under Matlab®.
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To estimate the relative contribution of consumptive versus non-consumptive effects
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to malaria transmission potential, we used our model and changed some values of the
110
parameters described in Table S1. First, we ran our model including consumptive effects only.
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We assumed that control and predation-exposed mosquitoes had similar adult life-history
112
traits but different larval mortality. Thus, we replaced in the predation group the values of all
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parameters but the Y parameter (i.e. the percentage of mosquito developmental success, see
114
Table S1) by those of the control group. In so doing, the observed difference in pathogen
115
transmission intensity can only be due to differences in larval mortality (i.e. consumptive
116
effect of predators). Second, we ran the model including non-consumptive effects only. We
117
assumed that control and predation-exposed mosquitoes had similar larval mortality but
118
different adult life-history traits. Thus, we replaced the value of the Y parameter of the
119
predation group by that of the control group to capture the carry-over effects of predation
120
exposure only. In so doing, the difference in pathogen transmission intensity can only be due
121
to differences in adult life-history traits (i.e. carry-over effects of predation stress). All these
122
simulations were performed over both one and ten seasons (short and long term effects).
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Table S1: Parameters used in the mathematical model.
Parameter
Unit
Control IC
Predation IC
bS (% of gravid Sm)
%
0.534-0.747
0.454-0.677
bE (% of gravid Em)
%
0.542-0.762
0.502-0.711
bI (% of gravid Im)
%
0.542-0.762
0.502-0.711
θS (Mean number of eggs produced by Sm)
133.58-145.18
137.09-149.31
θE (Mean number of eggs produced by Em)
132.44-144.82
118.75-128.27
θI (Mean number of eggs produced by Im)
132.44-144.82
118.75-128.27
ε (Mosquito development time)
days.ind-1
10.52-10.54
11.09-11.11
Υ (% of mosquito developmental success)
%
0.684-0.740 (a)
0.361-0.381
dS (Mean longevity of Sm)
days.ind-1
8.19-9.37
7.14-8.14
dE (Mean longevity of Em)
days.ind-1
8.44-9.4
7.49-8.11
dI (Mean longevity of Im)
days.ind-1
8.44-9.4
7.49-8.11
b (Mosquito competence)
%
0.45-0.62
0.52-0.69
σ (Duration of parasite development in mosquito)
days.ind-1
10-18
10-18
a (Biting rate)
days.ind-1
1/4
1/4
c (Human susceptibility)
%
0.5
0.5
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Sm: Susceptible individuals, Em: Exposed individuals, Im: Infectious individuals. (a) Due to
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predator consumption, mosquito larval densities varied over time between experimental and
130
control containers. To avoid potential density effects on adult life-history traits, larval density
131
was homogenised by randomly removing supernumerary larvae from control containers. For
132
this reason, the emergence rates observed in the control containers could not be used in the
133
model. To obtain an emergence rate for the control containers, we additionally exposed 200
134
larvae per container (N=5) to control conditions as explained in the main text. No density
135
homogenization was performed on these containers.
136
137
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138
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Cameroon. PLoS ONE 8, e54820. (doi:doi:10.1371/journal.pone.0054820).
195
196
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Supplementary Figures
198
199
200
a.
b.
30
70
Oocyst intensity (mean se)
Oocyst prevalence (% 95IC)
80
60
50
40
30
20
10
134
138
control
predation
0
20
15
10
5
62
84
control
predation
68
44
control
predation
0
c.
d.
5.0
100
80
60
40
20
77
51
control
predation
0
Sporozoite intensity (mean se)
Sporozoite prevalence (% 95IC)
120
201
202
203
204
205
206
207
208
209
25
4.0
3.0
2.0
1.0
0.0
Figure S1: Effects of predator exposure on infection at day 7 and 14 post-blood meal (a-b and
c-d, respectively). a. Oocyst prevalence in females exposed to an infectious blood meal; b.
Oocyst intensity (mean number of oocysts) in infected females; c. Sporozoite prevalence in
females exposed to an infectious blood meal; d. Sporozoite intensity (log of nb of copy of
gene cox1) in infected females. Numbers in bars indicate sample size.
0.6
0.4
0.0
0.2
Infection prevalence
0.8
1.0
210
211
3000
3200
3400
3600
Wing size (µm)
212
213
214
215
Figure S2: Predictive curves obtained from the model showing the effect of wing size and
predator exposure on oocyst prevalence. Red = females exposed to predator as larvae; Blue =
control.
Figure S3: Predicted mean number of eggs (±95% IC) produced as a function of oocyst
intensity.
150
50
100
Number of eggs
200
216
217
218
219
0
20
40
60
80
100
120
Number of oocysts
220
221
222
223
Figure S4: Mean number of eggs in infected females as a function of oocyst intensity and
larval predator exposure. In red: larval predator-exposed, infected females; in blue: control
infected females.
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225
226
227
228
Figure S5: Predicted mean number of eggs (±95% IC) produced as a function of wing size.
a)
Theoretical distribution of mosquito wing size under the selection scenario
(preferential predation on bigger prey)
Eaten by predators
survivors
b)
Observed distribution of mosquito wing size
predation
control
New phenotype
Histogram of our data
Frequency
20
30
40
c)
control
0
10
predation
2800
229
230
231
232
233
234
235
236
237
238
239
3000
3200
3400
Wing size (µm)
3600
Figure S6: Confrontation of the selection scenario with our data for mosquito wing size.
a) Distribution which should be observed if predators selected the bigger prey. Only the
smaller individuals from control should be observed in predator containers and reaction norms
of the two treatments are overlaid. b) Observed distributions. Distribution of wing size from
mosquitoes that survived to predation exposure is shifted toward smaller size than those of
control containers. The two reaction norms are not overlaid. This is probably due to the tradeoff between anti-predator behaviour and feeding behaviour. Indeed, under predation risk,
larvae tend to rest more and to feed less to be less conspicuous. c) Histogram of our data for
mosquito wing size.
240
241
242
243
244
Statistical Results
Larval development duration
Predator exposure
Sex
Predation : Sex
245
246
247
X²
df
P value
652
27.25
0.004
1
1
1
<0.001
<0.001
0.94
Oocyst prevalence
Predator exposure
Wing size
Gametocytemia
Predation : Wing
Predation : Gametocytemia
Wing : Gametocytemia
Predation : Wing : Gametocytemia
248
249
250
0.44
0.011
0.008
0.42
0.73
0.01
0.33
X²
df
P value
0.07
1.19
6.36
1.17
0.07
0.91
0.02
1
1
1
1
1
1
1
0.78
0.27
0.01
0.28
0.78
0.34
0.88
X²
df
P value
0.23
0.58
1.29
1
1
1
0.62
0.44
0.25
X²
df
P value
0.45
0.50
0.11
1
1
1
0.50
0.47
0.73
Sporozoite intensity
Predator exposure
Gametocytemia
Predation : Gametocytemia
257
258
259
260
261
P value
1
1
1
1
1
1
1
Sporozoite prevalence
Predator exposure
Gametocytemia
Predation : Gametocytemia
254
255
256
df
Oocyst intensity
Predator exposure
Wing size
Gametocytemia
Predation : Wing
Predation : Gametocytemia
Wing : Gametocytemia
Predation : Wing : Gametocytemia
251
252
253
X²
0.58
6.33
6.83
0.64
0.11
5.51
0.92
262
Egg prevalence
Predator exposure
Wing size
Infection status
Predation : Wing
Predation : Infection status
Wing : Infection status
Predation : Wing : Infection status
263
264
265
P value
1
1
2
1
2
2
2
0.009
0.37
0.06
0.92
0.06
0.73
0.75
X²
df
P value
0.002
1.05
0.76
0.40
0.003
6e-4
0.03
1
1
1
1
1
1
1
0.95
0.30
0.38
0.52
0.95
0.98
0.84
Egg load
Predator exposure
Wing size
Infection status
Egg size
Predation : Wing
Predation : Infection status
Predation : Egg size
Wing : Infection status
Wing : Egg size
Infection status : Egg size
Predation : Wing : Infection status
Predation : Wing : Egg size
Predation : Infection status : Egg size
Wing : Infection status : Egg size
Predation : wing : Infection status : Egg size
268
269
df
Egg prevalence in infected mosquitoes only
Predator exposure
Wing size
Oocysts
Predation : Wing
Predation : Oocysts
Wing : Oocysts
Predation : Wing : Oocysts
266
267
X²
6.63
0.78
5.56
0.008
5.41
0.60
0.56
X²
df
P value
0.38
17.03
5.44
1.51
2.12
4.83
0.04
4.38
6e-4
1.44
0.31
0.21
4.92
0.21
1.64
1
1
2
1
1
2
1
2
1
2
2
1
2
2
2
0.53
<0.001
0.06
0.21
0.14
0.08
0.83
0.11
0.98
0.48
0.85
0.64
0.08
0.89
0.44
X²
df
P value
4.06
16.31
6.73
1.32
0.59
10.54
2.49
0.59
0.07
6.29
2.28
2.14
0.04
0.03
0.05
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0.04
<0.001
0.009
0.24
0.44
0.001
0.11
0.44
0.77
0.01
0.13
0.14
0.82
0.85
0.82
Egg load in infected mosquitoes only
Predator exposure
Wing size
Oocysts
Egg size
Predation : Wing
Predation : Oocysts
Predation : Egg size
Wing : Oocysts
Wing : Egg size
Oocysts : Egg size
Predation : Wing : Oocysts
Predation : Wing : Egg size
Predation : Oocysts : Egg size
Wing : Oocysts : Egg size
Predation : wing : Oocysts : Egg size
270
Egg size
Predator exposure
Wing size
Infection status
Egg load
Predation : Wing
Predation : Infection status
Predation : Egg load
Wing : Infection status
Wing : Egg load
Infection status : Egg load
Predation : Wing : Infection status
Predation : Wing : Egg load
Predation : Infection status : Egg load
Wing : Infection status : Egg load
Predation : wing : Infection status : Egg load
271
272
273
274
275
276
Predator exposure
Wing size
Oocysts
Egg load
Predation : Wing
Predation : Oocysts
Predation : Egg load
Wing : Oocysts
Wing : Egg load
Oocysts : Egg load
Predation : Wing : Oocysts
Predation : Wing : Egg load
Predation : Oocysts : Egg load
Wing : Oocysts : Egg load
Predation : wing : Oocysts : Egg load
<0.001
0.35
0.23
0.23
0.36
0.15
0.48
0.60
0.46
0.42
0.84
0.93
0.08
0.50
0.32
X²
df
P value
6.77
0.29
0.43
0.17
0.21
1.40
0.05
0.19
1.26
2.30
0.009
1.69
0.66
0.12
0.14
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0.009
0.58
0.51
0.97
0.64
0.23
0.80
0.65
0.26
0.12
0.92
0.19
0.41
0.71
0.70
X²
df
P value
8.75
0.34
0.06
1
1
1
0.003
0.55
0.79
Survival during oocyst phase
X²
df
P value
0.41
10.25
0.005
1
1
1
0.39
0.001
0.93
X²
df
P value
4.09
4.74
0.01
1
1
1
0.04
0.02
0.89
Survival during sporozoite phase
Predator exposure
Infection
Predation : Infection
281
P value
1
1
2
1
1
2
1
2
1
2
2
1
2
2
2
Survival
Predator exposure
Infection
Predation : Infection
279
280
df
Egg size in infected mosquitoes only
Predator exposure
Exposure to infection
Predation : Exposure
277
278
X²
23.32
0.84
2.93
1.38
0.81
3.76
0.49
0.99
0.54
1.69
0.33
0.007
4.96
1.35
2.25