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Online Appendices
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Sarah A. Orlofske, Robert C. Jadin, Pieter T.J. Johnson. It's a predator-eat-parasite
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world: how characteristics of predator, parasite and environment affect consumption.
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Appendix A. Collection of infected snails and molecular identification of parasite
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species.
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Naturally infected snails were collected from field sites in June–August 2009, 2010, and
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2011. Snails infected with Ribeiroia ondatrae were collected from sites in the San
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Francisco Bay area of California. Cephalogonimus americanus infected snails were
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collected from wetlands in Montana, while “Magnacauda” infected snails were collected
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from wetlands in Washington. Finally, snails infected with Echinostoma trivolvis were
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collected from wetlands in Wisconsin.
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We identified cercariae using a compound microscope to observe distinguishing
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morphological features from the literature (e.g., Schell 1985; Johnson and McKenzie
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2009; Szuroczki and Richardson 2009; Fig. 1). Samples of cercariae were also vouchered
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for molecular analysis to verify morphological identification because some species have
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not yet been described or cannot be distinguished using morphology alone. Briefly, we
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obtained genomic DNA of individual cercaria using a Qiagen DNeasy extraction kit and
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protocol. The internal transcribed spacer region of ribosomal DNA (ITS 1 and ITS 2)
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gene fragments were independently PCR amplified using GoTaq® Green master mix by
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Promega, Madison, WI, USA, and the primer pairs: BD1 + 4S (ITS 1) and 3S + ITS2.2
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(ITS 2). Protocols for amplification and primer sequence are as described in (Bowles and
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McManus 1993; Bowles et al. 1995; Hugall et al. 1999; Wilson et al. 2005). Sequencing
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was performed in both forward and reverse directions using the PCR primers on a
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Beckman Coulter automated capillary sequencer, and sequence chromatographs were
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edited using Sequencher 4.2, Gene Codes Corporation, Ann Arbor, MI, USA. Sequences
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for each gene fragment were aligned separately, first automatically using the program
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MUSCLE (Edgar 2004) and, then manually rechecked using Se-Al v2.0a11. Using these
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data we were able to identify Cephalogonimus americanus, Echinostoma trivolvis, and
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Ribeiroia ondatrae to species with 99–100% maximum identity through BLAST searches
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in GenBank as well as by making Bayesian phylogenetic analyses incorporating the
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datasets of Razo-Mendivil and Pérez-Ponce de León (2011), Detwiler et al. (2010), and
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Wilson et al. (2005), respectively. Sequences for “Magnacauda” were not available in
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GenBank, but our phylogenetic analysis placed it in the family Echinostomatidae.
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To obtain measurements of cercariae body and tail lengths, we digitally
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photographed between three and nine unique specimens on a compound microscope
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(Olympus SZX10) at 10X or 20X magnification. These photos were then analyzed using
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Image J (Image Processing and Analysis in Java, National Institutes of Health) for
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obtaining cercarial measurements.
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Appendix B. Laboratory animal collection and maintenance
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We collected mosquitofish (Gambusia affinis) in June–August 2009, 2010, and 2011
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from ponds in central California that were known to be free of the snail host for the
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trematodes used in our feeding trials. Furthermore, we dissected a subset of fish (n = 10)
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prior to conducting our experiments to quantify any preexisting infection with the
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trematode species used in out experiments. Fish were shipped specimens from California
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to the laboratory at University of Colorado Boulder where we conducted the experiments.
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Prior to the experiments mosquitofish were maintained in 10-gallon glass aquaria with
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constant aeration and fed commercial fish food once per day (TetraMin, Tetra®).
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Damselflies (Enallagma spp.) were collected from two ponds in eastern Colorado.
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Damselflies are unsuitable hosts for any of the parasites used in our experiments. We
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maintained damselflies individually in 1-L containers with dechlorinated tap water
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(AmQuel plus and NovAqua plus, Kordon®) and fed Daphnia middendorffiana ad lib.
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Prior to all experiments, body length of each predator was measured to the nearest
0.1 mm with digital calipers and the data are included in Table B1.
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Appendix C. Methods for vertebrate and invertebrate necropsy
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Mosquitofish were euthanized with buffered MS-222 (Tricaine methanesulfonate,
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Western Chemical Inc.) and preserved in 10% buffered formalin until necropsy, where
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we inspected all external surfaces and removed and examined all muscle tissue and
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organs using a dissecting microscope. Metacercariae were examined under a compound
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microscope to observe distinguishing features to allow for species identification (Schell
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et al. 1985; Johnson and McKenzie 2009; Szuroczki and Richardson 2009). Damselflies
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were dissected following preservation in 70% ethanol. All external and internal surfaces
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were removed and examined using a dissecting microscope.
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Appendix D. Additional analysis of the relationship of parasite body size on predation
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Methods
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To examine the relationship of parasite body size on consumption by predators we
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performed a series of single linear regressions with parasite total length (mm) as the
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independent variable and the number of parasites consumed as a response. To test for
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non-linear relationships between the two variables we also preformed non-linear
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regressions and compared the two models using ANOVA. For damselflies, data were
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pooled across predator body sizes but light and dark trials were analyzed separately. For
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mosquitofish, the data for small and large fish were analyzed separately as well as light
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and dark trials.
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Results
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For damselflies feeding on parasites in the light there was significantly better fit of the
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non-linear regression compared to the linear regression (ANOVA: F1 = 65.5, P <
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0.0001). The non-linear regression indicated a significant hump-shaped relationship
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between parasite length and numbers consumed (F2,57 = 36.7, R2 = 0.56, Parasite Size2: P
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< 0.0001, Parasite Size: P < 0.0001, Fig. D1a). Although total parasites consumed were
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lower in the dark trials, there was a similar non-linear relationship due to parasite length
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that was significantly more accurate than the linear regression (ANOVA: F1 = 37.8, P <
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0.0001). There was a significant hump-shaped relationship between parasite length and
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consumption (F2,57 = 24.4, R2 = 0.46, Parasite Size2: P < 0.0001, Parasite Size: P <
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0.0001, Fig. D1a).
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The relationship of parasite size to mosquitofish consumption was strongly
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influenced by fish size. For the small fish (<10 mm body length), there was no
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statistically significant effect of parasite length on consumption for either the light (F1,32 =
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0.0, R2 = 0.0, P = 0.912, Fig. D1b) or dark trials (F1,33 = 3.4, R2 = 0.09, P = 0 .074, Fig
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XB). For large fish (>25 mm body length), there was a positive, linear relationship of
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consumption with parasite length (F1,33 = 9.3, R2 = 22, P = 0 .005, Fig. D1c). In contrast,
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the relationship of consumption of the large fish in the dark with parasite length was
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significantly non-linear (ANOVA: F1= 9.1, P = 0.005), showing a downward curve
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between the two smallest parasite species and a rapid increase in consumption for two
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larger parasites (F2,32 = 16.8, R2 = 0.51, Parasite Size2: P = 0.005, Parasite Size: P =
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0.017, Fig. D1c). Infection by Riberoia ondatrae may have reduced fish consumption
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leading to the less than linear relationship, while consumption of Magnacauda remained
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high in both light and dark trials likely due to the behavior that likely increased predator
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detection.
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Appendix E: Supplementary tables.
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References
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Bowles J, McManus DP (1993) Rapid discrimination of Echinococcus species and strains
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using a polymerase chain reactionbased RFLP method. Mol Biochem Parasitol
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57:231–240.
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111
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Bowles J, Blair D, McManus DP (1995) A molecular phylogeny of the human
schistosomes. Mol Phylogenet Evol 4:103–109.
Detwiler JT, Bos DH, Minchella DJ (2010) Revealing the secret lives of cryptic species:
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examining the phylogenetic relationships of echinostome parasites in North
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America. Mol Phylogenet Evol 55:611–620.
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Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high
throughput. Nucleic Acids Res 32:1792–1797.
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Hugall A, Stanton J, Moritz C (1999) Reticulate evolution and the origins of ribosomal
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internal transcribed spacer diversity in apomictic Meloidogyne. Mol Biol Evol
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16:157–164.
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Johnson PTJ, McKenzie VJ (2009) Effects of environmental change on helminths
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infections in amphibians: exploring the emergence of Ribeiroia and Echinostoma
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infections in North America. Pages 249–280 in B. Fried and R. Toledo, editors. The
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Biology of Echinostomes. Springer, New York, NY.
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Razo-Mendivil U, Pérez-Ponce de León G (2011) Testing the evolutionary and
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biogeographical history of Glypthelmins (Digenea: Plagiorchiida), a parasite of
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anurans, through a simultaneous analysis of molecular and morphological data. Mol
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Phylogenet Evol 59:331–341.
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Schell SC (1985) Trematodes of North America North of Mexico. University of Idaho
Press, Moscow, ID), pp. 263.
Szuroczki D, Richardson JML (2009) The role of trematode parasites in larval anuran
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communities: an aquatic ecologist’s guide to the major players. Oecologia 161:371–
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385.
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Wilson WD, Johnson PTJ, Sutherland DR, Moné H, Loker ES (2005) A molecular
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phylogenetic study of the genus Ribeiroia (Digenea): trematodes known to cause
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limb malformations in amphibians. J Parasitol 91:1040–1045.
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Tables
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Table B1 The number (n) of each predator species examined for the ability to reduce the
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number of four species of trematode cercariae, and characterize functional response with
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the average body size of the specimens.
Predation Bioassay
Cephalogonimus
americanus
Echinostoma
trivolvis
“Magnacauda”
10
20
Predator Species
Mosquitofish
Damselfly nymphs
Body
n
Body
n
Body
Range
Length
Length
Length
(mm) ±
(mm)
(mm) ±
SE
± SE
SE
26.3 ± 0.5 10
9.2 ±
30
9.3 ± 0.5
4.7–
0.1
15.0
26.0 ± 0.8 20
9.7 ±
30
9.0 ± 0.5
4.6–
0.1
14.6
27.2 ± 0.5 20 9.5 ± 0.3 30
9.0 ± 0.5 4.6–13.5
Ribeiroia ondatrae
20
26.1 ± 0.6
Parasite Density
Experiment
Echinostoma
trivolvis
Ribeiroia ondatrae
12
11.8 ± 0.3
15
5.6 ± 0.2
9
10.5 ± 0.2
15
6.4 ± 0.2
Parasite Species
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145
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147
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149
150
151
152
153
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n
20
20
8.9 ±
0.1
30
9.1 ± 0.5
5.1–14.1
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Table E1 Results of generalized linear mixed models examining infection of
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mosquitofish by two parasite species under light and dark conditions.
Variable
“Magnacauda”
Intercept
Light condition
Predator size class
Ribeiroia ondatrae – All trials
Intercept
Light condition
Predator size class
Light Condition*Predator size class
Ribeiroia ondatrae – Light
Intercept
Predator size class
Ribeiroia ondatrae – Dark
Intercept
Predator size class
* Bold = Significant at P = 0.05
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158
159
160
161
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165
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Z-value P-value*
BIC
AIC
n
–3.65
0.25
–1.87
0.000
0.801
0.062
10.28
–3.00
0.42
1052 1170
–5.57
–0.84
–0.19
–3.22
2.63E-08
0.404
0.848
0.001
27.95
–2.38
–3.04
7.31
806.6 1200
–5.35
–3.37
9.00E-08
<0.001
25.80
8.58
260.7
600
–6.17
–0.27
6.74E-10
0.791
35.29
–2.71
546.6
600
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Table E2 Results of generalized linear mixed models of two different predator species
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and two different parasite species as prey provided at three densities. Data for damselfly
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predators were analyzed with both parasite species combined, but then subsequently
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analyzed for each parasite species separately to examine the significant interaction.
Variable
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174
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Z-value
P-value*
Damselflies - Both parasite species
Intercept
–1.383
Parasite Density
2.222
Parasite Species
0.267
Density*Species
–2.061
Ribeiroia ondatrae
Intercept
–0.96
Parasite Density
–0.682
Echinostoma trivolvis
Intercept
–1.423
Parasite Density
2.29
Fish - Both parasite species
Intercept
–0.822
Parasite Density
2.013
Parasite Species
1.403
Density*Species
–1.83
* Bold = Significant at P = 0.05
BIC
AIC
n
0.167
0.026
0.790
0.039
–1.34
1.68
–3.18
0.99
2023
1800
0.337
0.495
–2.03
–2.49
985.1
900
0.155
0.022
–0.93
2.29
1040
900
0.411
0.044
0.161
0.067
–2.42
0.80
–1.29
0.09
907.7
1260
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Figure legends
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Fig. D1 Regression plots depicting the relationship between parasite (prey) size and the
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number of parasites consumed by A) Damselfly nymphs in either light (open squares and
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dashed line) or dark (filled circles and solid line) with the best fit regression line (light:
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F2,57 = 36.7, R2 = 0.56, Parasite Size2: P < 0.0001, Parasite Size: P < 0.0001; dark: F2,57 =
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24.4, R2 = 0.46, Parasite Size2: P < 0.0001, Parasite Size: P < 0.0001). B) Mosquitofish
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(<10 mm body length) in either light (open squares) or dark (filled circles). C)
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Mosquitofish (>25 mm, body length) in either light (open squares and dashed line) or
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dark (filled circles and solid line) with the best fit regression line (light: F1,33 = 9.3, R2 =
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22, P = 0 .005; dark: F2,32 = 16.8, R2 = 0.51, Parasite Size2: P = 0.005, Parasite Size: P =
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0.017).
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Fig. D1