Bottom-up and trait-mediated effects of resource quality on amphibian parasitism

Transcription

1 Journal of Animal Ecology 2017, 86, doi: / Bottom-up and trait-mediated effects of resource quality on amphibian parasitism Jeffrey P. Stephens*,1, Karie A. Altman 2, Keith A. Berven 2, Scott D. Tiegs 2 and Thomas R. Raffel 2 1 Wayne State School of Medicine, Detroit, MI, USA; and 2 Oakland University, 2200 N. Squirrel Rd., Rochester, MI, USA Abstract 1. Leaf litter subsidies are important resources for aquatic consumers like tadpoles and snails, causing bottom-up effects on wetland ecosystems. Recent studies have shown that variation in litter nutritional quality can be as important as litter quantity in driving these bottom-up effects. Resource subsidies likely also have indirect and trait-mediated effects on predation and parasitism, but these potential effects remain largely unexplored. 2. We generated predictions for differential effects of litter nutrition and secondary polyphenolic compounds on tadpole (Lithobates sylvatica) exposure and susceptibility to Ribeiroia ondatrae, based on ecological stoichiometry and community-ecology theory. We predicted direct and indirect effects on key traits of the tadpole host (rates of growth, development and survival), the trematode parasite (production of the cercaria infective stages) and the parasite s snail intermediate host (growth and reproduction). 3. To test these predictions, we conducted a large-scale mesocosm experiment using a natural gradient in the concentrations of nutrients (nitrogen) and toxic secondary compounds (polyphenolics) of nine leaf litter species. To differentiate between effects on exposure vs. susceptibility to infection, we included multiple infection experiments including one with constant per capita exposure. 4. We found that increased litter nitrogen increased tadpole survival, and also increased cercaria production by the snail intermediate hosts, causing opposing effects on tadpole per capita exposure to trematode infection. Increased litter polyphenolics slowed tadpole development, leading to increased infection by increasing both their susceptibility to infection and the length of time they were exposed to parasites. 5. Based on these results, recent shifts in forest composition towards more nitrogen-poor litter species should decrease trematode infection in tadpoles via density- and trait-mediated effects on the snail intermediate hosts. However, these shifts also involve increased abundance of litter species with high polyphenolic levels, which should increase trematode infection via trait-mediated effects on tadpoles. Future studies will be needed to determine the relative strength of these opposing effects in natural wetland communities. [Correction added after online publication on 5 January 2017: wording changed to which should increase trematode infection via trait-mediated effects on tadpoles.] Key-words: amphibian disease, decomposition, frog, larvae, leaf litter, Rana sylvatica, reciprocal subsidy Introduction The among-ecosystem transfer of energy and nutrients, referred to as spatial subsidies, is vital to the ecology of many ecosystems (Polis, Anderson & Holt 1997). Studies *Correspondence author. jestephe@med.wayne.edu of spatial subsidies have generally focused on bottom-up effects on communities and ecosystems via increased primary and secondary productivity (Earl & Semlitsch 2013), leading to reduced competition between primary consumers (Leroux & Loreau 2012) and changes in nutrient cycling (Capps, Berven & Tiegs 2014; Tiegs et al. 2015). However, spatial subsidies might also influence organisms 2016 The Authors. Journal of Animal Ecology 2016 British Ecological Society

2 306 J. P. Stephens et al. via indirect top-down effects, by changing rates of predation or parasitism (Loreau, MOuquet & Holt 2003; Tylianakis, Didham & Wratten 2004; Stoler & Relyea 2013a). In addition to serving as an allochthonous food resources to aquatic organisms [a bottom-up effect (Motomori, Mitsuhashi & Nakano 2001; Stoler et al. 2015)], leaf litter also leaches secondary compounds that might cause direct toxicity to organisms or their parasites (Earl & Semlitsch 2015). Here we investigate the potential for the quality of wetland leaf litter subsidies to influence rates of trematode infection in larval amphibians, via direct and indirect effects on trematodes, their amphibian hosts and their snail intermediate hosts. Understanding which ecological factors drive disease transmission is fundamental for disease ecology and conservation biology. Despite extensive research of spatial subsidies on recipient ecosystems, little is known about how spatial subsidies influence parasitism (an indirect topdown effect). Researchers know that host nutrition can influence host parasite dynamics by affecting host traits like body size, population density and immunocompetance (Coop & Kyriazakis 2001; Pulkkinen & Ebert 2004; Frost, Ebert & Smith 2008; Vale, Choisy & Little 2013). While studies have focused on the total quantity of food available to hosts (Civitello et al. 2015), the nutritional quality of food (e.g. carbon : nitrogen ratios) can also influence the outcome of parasitic infection (Hall et al. 2009b; Penczykowski et al. 2014). Food quality effects can be predicted by the theory of ecological stoichiometry (ES), a framework derived from first principles of chemistry and physics to predict rates of organism growth, assimilation and excretion based on nutrient imbalances between resources and consumers (Sterner & Elser 2002; Stephens, Berven & Tiegs 2013; Stephens et al. 2015; Stoler et al. 2015). ES theory predicts increased rates of growth, development and survival in consumers that eat food with higher concentrations of their limiting nutrients, and which nutrients are limiting should depend on the relative concentrations of these nutrients in consumer body tissues, relative to their food. More recently, ES theory has been used to predict rates of parasite production and direct transmission in zooplankton (Hall et al. 2009b; Vale, Choisy & Little 2013; Penczykowski et al. 2014), suggesting that ES might provide a powerful framework for predicting subsidy effects on host parasite systems. Parasitic infection is ultimately determined by two factors: (i) host exposure to the parasite [i.e. the rate at which hosts encounter parasite infective stages (Dumont et al. 2007)] and (ii) host susceptibility to infection [i.e. the proportion of parasites that successfully infect the host postexposure (Telfer et al. 2010)]. These two processes might be influenced by spatial subsidies in multiple ways. Highquality food resources might lead to improved host immune responses, thereby reducing susceptibility to infection (Coop & Kyriazakis 1999). However, high-quality food resources might also allow a parasite s prior host (in this case the snail intermediate host) to produce more parasite infective stages, thereby increasing exposure to infection (Bernot 2013). Distinguishing these potentially conflicting effects requires experiments that tease apart exposure vs. susceptibility to infection (Civitello et al. 2013; Civitello & Rohr 2014) and that simultaneously assess multiple mechanisms by which resource subsidies can influence hosts and their parasites. This is challenging because many well-studied spatial subsidies have little natural variation in nutrient content, making it difficult to manipulate subsidy quality. Forested wetlands are ideal systems to explore effects of resource quality on consumers, because many receive almost all their energy and nutrients from deciduous tree leaf litter (Fisher & Likens 1973) that can vary dramatically in quality based on the tree species of origin (Ostrofsky 1997). Studies have shown that tadpoles of wood frogs (Lithobates sylvaticus), which can reproduce in heavily forested ponds, consume leaf litter as a primary food resource and respond to increased leaf litter quality (i.e. increased litter N) with improved rates of survival, growth and development (Stephens, Berven & Tiegs 2013; Stoler & Relyea 2013b, 2016). In this study, we investigated how leaf litter characteristics influence infection rates of the trematode parasite Ribeiroia ondatrae in wood frog tadpoles. Ribeiroia ondatrae infects developing tadpoles and is best known for causing limb deformities in metamorphic amphibians (Johnson et al. 1999). This parasite uses the snail Helisoma trivolvis as its first intermediate host, which releases freeliving cercariae that seek out, penetrate and encyst in amphibian larvae (Rohr et al. 2008). Ribeiroia ondatrae cercariae are strictly aquatic, so wood frogs are only exposed while in the pond as tadpoles. Anthropogenic nutrient subsidies (fertilizers) are known to increase snail population densities via bottom-up effects on their algal food resources, with subsequent effects on trematode infection in tadpoles and frogs (Johnson & Sutherland 2003). Helisoma trivolvis snails consume leaf litter as an important primary food source (Stoler et al. 2015), so variation in litter quality might influence the biology of the snail intermediate host, potentially causing indirect effects on trematode infection in tadpoles. Herbivores- and detritivores-like tadpoles and snails tend to be limited by the low concentrations of nitrogen (N) and/or phosphorus (P) in their food (Sterner & Elser 2002), with N typically being the more important limiting nutrient for these consumers (Stoler et al. 2015). ES theory therefore predicts increased physiological performance (e.g. growth, development, survival and/or immune competence) in these species when fed leaf litter with higher N concentrations (Stephens et al. 2015). We tested multiple competing hypotheses for how leaflitter nutrient content should influence R. ondatrae infection in tadpoles. Based on ES theory and prior studies, we hypothesized that litter with higher N content would increase rates of tadpole development, growth and survival (Stephens, Berven & Tiegs 2013; Stephens et al. 2015; Stoler & Relyea 2016). These effects should in turn:

3 Resource quality affects amphibian parasitism 307 (1) decrease exposure to infection by shortening the larval period of developing tadpoles, decrease tadpole susceptibility to infection by (2) accelerating development such that the animal spends less time in the more susceptible early stages of development (Raffel et al. 2010) and (3) improving body condition (Coop & Kyriazakis 1999) and (4) decrease per capita exposure to infection (cercariae per tadpole) by increasing the number of surviving tadpoles. Based on ES theory and prior studies, litter nutrient content could also have important effects on the snail intermediate hosts (Stoler et al. 2015), leading to indirect effects on tadpole exposure. We hypothesized that high litter quality would (5) increase cercaria release (and tadpole exposure to infection) due to snails having larger energy budgets (Bernot 2013; Paull et al. 2015) and (6) increase snail reproduction, which in a natural setting should lead to more infected snails and ultimately greater exposure to tadpoles. Alternatively, (7) better nutrition might increase snail immune performance, resulting in less parasite production (but see Bernot 2013). Leaf litter also contains secondary plant compounds, such as polyphenolics, that can negatively influence aquatic organisms (Earl & Semlitsch 2015). Studies have shown that dissolved polyphenolics can slow larval amphibian development and increase mortality (Stephens, Berven & Tiegs 2013). Such toxic effects of dissolved chemicals (such as pesticides) on aquatic organisms are analogous to the top-down effects of predation (Rohr, Kerby & Sih 2006). We therefore hypothesized that polyphenolics would delay tadpole maturation and thus increase both (8) exposure and (9) susceptibility to trematode parasites. These nine predictions are summarized in Table 1. These hypothesized mechanisms are not mutually exclusive and could be operating simultaneously, with the outcome depending on the relative strength of various effects. We therefore sought to distinguish mechanisms by testing separately for effects of leaf litter traits on either tadpole exposure or susceptibility to infection, and by using path analysis to assess support for competing hypotheses. Materials and methods experimental design We conducted a fully factorial randomized block mesocosm experiment at the Oakland University Biological Research Station (Rochester, MI, USA) in 2013, crossing nine leaf litter species with three infection treatments ( Uninfected, Susceptibility, and Exposure ). These infection treatments will be referred to as infection experiments hereafter. In the exposure experiment, tadpoles were exposed to one R. ondatrae-infected snail that was rotated through the mesocosms every 2 days, allowing rates of exposure to vary depending on each snail s rate of cercaria production and each tadpole s time to metamorphosis. In the susceptibility experiment, four tadpoles per mesocosm were each individually exposed to 20 cercariae in floating containers, so variation in infection was driven solely by tadpole susceptibility. Uninfected mesocosms received no parasites. This design allowed us to independently assess the effects of leaf litter on tadpole exposure or susceptibility to infection, similar to the experimental design of Raffel et al. (2010). Litter species were green ash (Fraxinus pennsylvanicus), reed grass (Phalaris arundinacea), common reed (Phragmites australis), white oak (Quercus alba), black willow (Salix nigra), eastern cottonwood (Populus deltoides), tussock sedge (Carex stricta), red maple (Acer rubrum), and cattail (Typha latifolia). Each litter 9 infection treatment combination was replicated twice, for a total of 54 mesocosms (9 leaf litter species 9 3 treatments 9 2 replicates = 54 mesocosms). Treatments were then randomly assigned to each mesocosm. Fifty-four mesocosms (110-L plastic wading pools) were filled with tap water, allowed to off-gas chlorine for 1 week, inoculated with 1 L pond water from a local wetland, and dosed with 130 g (air-dried) of respective leaf litter in the December of The mesocosms were then covered with 25% shade cloth lids, and allowed to overwinter until April of 2013 when they received tadpoles and snails. See Appendix S1, Supporting Information for more detailed experimental methods. Litter N and polyphenolics were assayed as described in Table S1. Table 1. A complete summary of all predictions, and whether or not there was experimental support generated from the experiment Prediction # Prediction Experimental support 1 Increased litter quality will decrease exposure to infection by shortening the larval period. No 2 Increased litter quality will decrease susceptibility to infection by accelerating development. No 3 Increased litter quality will decrease susceptibility to infection by increasing body condition. No 4 Increased litter quality will decrease per capita exposure to infection by increasing the number Yes of surviving tadpoles. 5 Increased litter quality will increase the cercaria release (and tadpole exposure to infection) Yes due to snails having larger energy budgets. 6 Increased litter quality will increase snail reproduction. No 7 Increased litter quality (better nutrition) will increase snail immune performance resulting in No less parasite production. 8 Increased litter polyphenols will delay tadpole maturation and thus increase tadpole exposure Yes to parasites. 9 Increased litter polyphenols will delay tadpole maturation and thus increase tadpole susceptibility to parasites. Yes

4 308 J. P. Stephens et al. tadpole and snail addition to mesocosms Helisoma trivolvis snails were raised in outdoor mesocosms from stock populations originally collected from Pinchot Lake in Pennsylvania. Snails used for experiments were 3 5 generations removed from the wild-caught snails. Nine infected snails were generated by exposing them to faeces of laboratory-infected rats, as described previously (Johnson et al. 2007; Paull et al. 2015). Thirty recently hatched tadpoles [Gosner stage 25 (Gosner 1960)] and five uninfected H. trivolvis snails (diameter c. 1 cm) were added to each mesocosm in May of Snails were weighed prior to addition to obtain average snail wet biomass. On 1 June 2013, one snail infected with R. ondatrae was introduced into one randomly selected replicate of the exposure experiment for each leaf litter species. Infected snails were housed in cages constructed of 5-inch diameter PVC pipe screened at either end with fibreglass window screen. Ample (c. 1 g) leaf litter from the corresponding treatment was also placed in the cages as a source of food. Each of the nine caged infected snails was rotated every other day between the two replicates of each litter treatment, and received new leaf litter food from the new mesocosm each time it was transferred. Thus, each snail (or litter species) acted as a single unit of replication for litter effects on cercaria production, while each exposure mesocosm acted as a unit of replication for litter effects on tadpole time to metamorphosis and infection levels. The susceptibility experiment allowed us to isolate the effects of leaf litter quality and polyphenolics on tadpole susceptibility to infection by experimentally controlling for trematode exposure following Raffel et al. (2010). Midway through the experiment, four tadpoles (out of the 30 initially added) from each mesocosm were randomly selected and removed, placed into 75 ml of mesocosm-specific water, and exposed to 20 R. ondatrae cercariae for 24 h. After exposure, these tadpoles were developmentally staged, euthanized in a 01% benzocaine solution, and preserved in neutrally buffered formalin for later microscopic examination. data collection At three different time points after addition of infected snails, we assessed snail cercaria production. Infected snails were removed from the mesocosms and placed into 75 ml tubes containing mesocosm water. The tubes were floated in the mesocosms for 24 h. We then returned snails to their cages and added Lugol s iodine to the tubes to preserve and stain cercariae for counting. Snail reproduction was assessed by counting the number of egg masses that were visible around the walls of the mesocosms on the day that the infected snails were introduced into the mesocosms. At the end of the experiment, the remaining uninfected snails initially placed into the mesocosm were re-weighed to estimate snail growth through the experiment. Once the first tadpole with emerging forearms was found, all mesocosms were checked daily for metamorphosing individuals following the protocol of Stephens, Berven & Tiegs (2013). Metamorphosed frogs were removed and placed in mesocosmspecific plastic containers with 1 cm aged tap water until the tail fully resorbed. They were then euthanized in 01% benzocaine and preserved individually in 10% buffered formalin for later analysis. Trematode metacercariae were enumerated for all exposed tadpole individuals from the susceptibility experiment and from all metamorphosed individuals from the exposure experiment following the procedure of Raffel et al. (2010). Briefly, we removed the digestive tract of each tadpole, bleached pigment by immersion in a basic hydrogen peroxide solution, and rendered tissues transparent by immersion in glycerine. Specimens were then placed between two sheets of glass and examined under a compound microscope (see Fig. 1). Mesocosms with higher rates of tadpole survival should have had higher tadpole densities at the start of the exposure period. This would lead to lower rates of exposure per tadpole and was therefore a potentially important predictor of infection in the Exposure experiment. We therefore estimated the proportion of tadpoles surviving to the beginning of the exposure period based on the initial number of tadpoles (30), the number surviving to metamorphosis, and the mean time of metamorphosis in each mesocosm. We then estimated the surviving number of tadpoles present in each mesocosm at the beginning of the exposure period by assuming an exponential decline (i.e., constant mortality rate) from the start of the experiment (30 tadpoles) until the mean date of metamorphosis (# surviving to metamorphosis). The resulting numbers were highly correlated with the final proportion of tadpoles surviving to metamorphosis, so we used them both as a proxy for tadpole survival in Path models (proportion surviving to exposure) and a proxy for tadpole density (# tadpoles) to estimate per capita exposure in each mesocosm (see below). statistical analyses All analyses were conducted using the statistical software R (R Core Team 2012). We tested effects of infection experiments ( exposure and unexposed ) on tadpole mass at metamorphosis, proportion surviving to metamorphosis and time to metamorphosis using a separate one-way ANOVAs (analysis of variance). We also tested for differences in leaf litter %N and % Phenolics using one-way ANOVAs. The susceptibility experiment was terminated after tadpoles were removed; thus they were not part of this analysis. We analysed effects of leaf litter traits (%N and % Phenolics) on trematode infection in tadpoles with path analysis (a type of structural equation model) to compare the predictive power of hypothesized direct and indirect effects, using the package lavaan (Rosseel 2012). In all analyses, %N and %Phenolics were treated as continuous predictor variables. Path analysis is a valuable way to explore a priori mechanistic relationships, allowing one to simultaneously compare the predictive power of hypothesized direct vs. indirect treatment effects (Civitello et al. 2015). While path analysis cannot directly demonstrate causation, it can help to distinguished among competing causal hypotheses when combined with empirical evidence. See Table S2 for variables and transformations. Path analyses were conducted as follows. To test predictions in the exposure mesocosms, we tested whether litter N and polyphenolics influenced the number of cercariae produced per infected snail, tadpole exposure duration and the proportion of tadpoles surviving at exposure, with subsequent effects of these variables on the number of metacercariae per metamorphosed frog. To test predictions in the susceptibility mesocosms, we tested whether litter N and polyphenolics influenced tadpole developmental rates (i.e. tadpole Gosner stage), with subsequent effects on the proportion of cercariae that successfully encysted in each tadpole (number of cercariae encysted/20). We also included direct paths from N and polyphenolics to the proportion

5 Resource quality affects amphibian parasitism 309 Fig. 1. The independent effects of (a) cercaria emergence rate (snail shedding rate), (b) proportion of tadpoles surviving at exposure and (c) days of exposure on metamorph infection levels from the exposure experiment. (d) The relationship between metamorph infection level and the composite predictor (predicted per capita exposure) for infection level created by multiplying the cercaria emergence rate times the tadpole days of exposure, then dividing that number by the tadpole number at exposure. Each point represents the mesocosm mean. Linear trend lines are fit to relationships with P values <01. encysted, to test whether litter traits affect tadpole exposure or susceptibility directly after controlling for indirect effects (e.g. via toxic effects on the cercariae). Our experimental design led to different predictor variables having different levels of replication (i.e. N = 9 litter species vs. N = 18 mesocosms in each infection experiment), and we wanted to use mixed-effects models to assess the effects of each predictor at the most appropriate level of replication. However, it is not yet possible to incorporate nested mixed-effects models into structural equation models using the lavaan package. We therefore conducted general linear mixed-effects models to determine whether individual arrows in the path models remained significant when using the appropriate level of replication for each predictor. For the exposure mesocosms, we tested for effects of the cercaria production rate, days of tadpole exposure and estimated tadpole number at the beginning of exposure on metacercaria number in metamorphosed frogs, including litter species and mesocosm as nested random-effects variables. We also created a composite variable (predicted per capita exposure) indicating the predicted per capita exposure for tadpoles in the Exposure mesocosms ([snail cercaria production per day 9 days of exposure] estimated number of tadpoles present), and assessed this as an alternative predictor of metacercaria abundance. We then included predicted per capita exposure in a combined model with cercaria production rate, days of tadpole exposure and tadpole number at the beginning of exposure, to determine if any of these individual predictors explained additional variation not explained by the predicted exposure level. We also tested whether litter N and polyphenolics were significant predictors of the cercaria production rate, days of tadpole exposure, or tadpole survival using separate linear mixed-effects models. We compared mixed-effects models using Delta AIC (DAIC) and used F-statistics to determine if variables contributed significantly to each model. All models met the assumptions of linear regression. Results There were significant differences in N (F 8,18 = 387, P < 0001) and polyphenolics (F 8,18 = 407, P < 0001) among the leaf litter species used in the experiment. There were no significant differences in tadpole mass at metamorphosis, proportion surviving to metamorphosis, or time to metamorphosis between the control vs. exposure (all P > 005). We therefore focused on effects of litter traits on levels of infection in the Exposure and Susceptibility experiments. exposure experiment When cercaria emergence rate, proportion of tadpoles surviving at exposure and days of tadpole exposure were included as predictors of metacercaria counts (metacercariae per metamorph) in a linear mixed-effects model, cercaria emergence rate and days of tadpole exposure were significant positive predictors of infection level (metacercariae per metamorph), while estimated tadpole survival was a significant negative predictor (Table S4, DAIC = 213). However, the composite predicted per capita exposure variable was by itself a significant positive predictor of metacercariae per metamorph, and

6 310 J. P. Stephens et al. according to AIC this simpler model was a substantial improvement over the initial model with three separate predictors (Table S4; Fig. 1d; DAIC = 0). When predicted per capita exposure was added to the initial model (along with the three individual predictors), cercaria emergence rate and the proportion of tadpoles surviving at exposure became non-significant, whereas days of tadpole exposure explained less but still a significant amount of variation (Table S4, DAIC = 224). Taken together, these results indicate that the composite predicted per capita exposure variable explained most of the variation in infection due to the cercaria emergence rate and the proportion of tadpoles surviving at exposure, but that it only explained part of the variation due to days of tadpole exposure. Figure 1a d demonstrates the independent effects of the predictor variables on metacercariae per metamorph. Tadpole mass at metamorphosis was not a significant predictor of tadpole infection in this model (P > 005). See Table S3 for the proportion of tadpoles infected and mean metacercariae load per tadpole. Path analysis supported the hypothesis that litter traits indirectly influenced metamorph infection level (metacercariae per metamorph) by affecting cercaria emergence rate, days of tadpole exposure, and the proportion of tadpoles surviving at exposure (Fig. 2). Specifically, increased litter polyphenolics were associated with decreased cercaria emergence rates (near significant) and increased days of tadpole exposure (Table S5). Higher litter N was associated with an increase in the proportion of tadpoles surviving at exposure, and cercaria emergence rates, but had no apparent influence on tadpole days of exposure (Table S5). Consistent with the mixed model analysis (above), cercaria emergence rate and days of tadpole exposure were positively related to tadpole infection level (metacercariae per metamorph), whereas the proportion of tadpoles surviving at exposure was negatively related to tadpole infection level (Table S5). Both direct paths from litter traits to metacercariae per metamorph were non-significant (Fig. 2). The proposed mechanistic paths explained a large proportion of the data with an overall R 2 = 081 (Fig. 2). susceptibility experiment Path analysis also provided support for significant direct negative effects of the litter traits N and polyphenolics on tadpole susceptibility (proportion of parasites encysted), and for an indirect negative effect of polyphenolics on tadpole susceptibility (proportion of parasites encysted) via a negative effect on tadpole development (Fig. 3, Table S5). litter effects on snail survival, growth and reproduction There were no differences in egg mass count among litter types (P > 005). The proportion of uninfected snails Fig. 2. Path analysis examining the mechanism hypothesized to affect tadpole infection level. Solid lines and dashed lines represent positive and negative relationships, respectively. Line thickness represents the magnitude of the path coefficient. We also report the R 2 for the whole model. surviving to the end of the experiment was high (x = ) and did not differ among leaf litter treatments (F 8,26 = 0782, P = 0621). However, increased litter N tended to increase snail mass (F 1,6 = 5594, P = 0055). Discussion The results of this study indicated that leaf litter nutritional quality has complex effects on trematode parasitism in tadpoles, mediated by both bottom-up effects on tadpole traits (time to metamorphosis, survival and susceptibility) and top-down effects caused by changes in parasite production by the snail intermediate host for R. ondatrae. Many of our proposed hypotheses, derived from predictions of ES theory, were supported by results from the mesocosms with continuous exposure to R. ondatraeinfected snails (exposure experiment). Mesocosms with increased litter N had increased cercaria production by infected snails (increasing total exposure per mesocosm), and also had greater tadpole survival, leading to higher tadpole densities and thus lower per capita exposure rates (i.e. fewer cercaria per tadpole). Mesocosms with higher polyphenolic levels had reduced cercaria production by snails (decreasing daily exposure rates), and also had tadpoles with delayed development and thus longer exposure durations to R. ondatrae (increasing total exposure per tadpole). Using path analysis, we found support for many of our proposed mechanisms contributing to per capita tadpole infection levels (see Table 1). There were proposed mechanisms that were not supported by our

7 Resource quality affects amphibian parasitism 311 However, it seems unlikely that polyphenolic compounds would have such an immune-enhancing effect on tadpoles, given the usually toxic nature of polyphenolic compounds (Maerz et al. 2005). Therefore, the most likely explanation for this direct negative effect on parasite infection is that polyphenolic compounds reduce the ability of R. ondatrae cercariae to infect tadpoles. While it has been documented that certain plants contain secondary compounds that are cercaricidal (Warren & Peters 1968; Saleh et al. 1985), this is to our knowledge the first evidence that plant secondary compounds might have negative effects on trematode cercariae infectivity in freshwater systems, which could have important implications for trematode parasitism in forested wetlands. It would be interesting in the future to test directly for effects of temperate-deciduous litter phenolics on survival or performance of trematode cercariae, particularly for trematodes that infect humans (e.g. schistosomes that cause swimmers itch). litter-mediated effects on parasite exposure Fig. 3. Path analysis examining the mechanism hypothesized to affect tadpole susceptibility to trematode infection. Solid lines and dashed lines represent positive and negative relationships respectively. Line thickness represents the magnitude of the path coefficient. We also report the R 2 for the whole model. experiment. Specifically, there was no evidence suggesting that better-fed snails had stronger resistance to the parasite, which should have led to lower parasite production in the higher N mesocosms. Additionally, there was no difference in snail reproduction or survival among litter species, contrary to our prediction that higher N species should increase snail reproduction and survival (see Table 1). Higher levels of litter polyphenolics also delayed tadpole development in the susceptibility mesocosms, and less-developed tadpoles at the time of parasite exposure explained much of the variation in tadpole susceptibility to infection. This is consistent with the results of prior studies that found greater susceptibility to trematode infection in less-developed tadpoles (Schotthoefer et al. 2003; Raffel et al. 2010; Rohr, Raffel & Hall 2010). However, it should be noted that stage-dependent susceptibility can also be dependent on the species of tadpole host and parasite examined (Rohr, Raffel & Hall 2010). After accounting for this developmental effect, however, it appears that higher levels of both litter N and polyphenolics both had significantly negative effects on tadpole susceptibility to infection. These apparent direct effects on susceptibility might indicate positive effects of polyphenolics on the host immune system, or perhaps higher levels of polyphenolics at the time of parasite exposure had negative effects on the parasite infective stage (cercaria). High litter N might plausibly lead to increased tadpole resistance to infection, due to improved tadpole nutrition. Increased litter N led to increased production of cercariae by infected snails, despite a low number of replicates for this response variable (N = 9 infected snails). This result supports our prediction that cercaria production would be directly limited by snail energetics, instead of indirectly limited by the immune system of the host snail. Immune systems are costly to maintain, so increased nutrients should have resulted in an increased snail immune response (Lochmiller & Deerenberg 2000). Such an immune-mediated effect would counteract the direct benefits of increased energy stores to parasites (Aalto, Ketola & Pulkkinen 2014). Our results support previous findings suggesting that the direct benefits to trematodes of increased energy stores in their snail intermediate hosts outweigh any potential increases in snail immune competence (Johnson et al. 2007; Bernot 2013; Narr & Krist 2015; Paull et al. 2015). Snail production of parasite cercaria is energetically and nutrient demanding (Sterner & Elser 2002; Hall et al. 2009a), such that trematode parasites in better-provisioned snails have more nutrients available for the production of infective stages. Our results also reinforce prior studies suggesting that resource quality is as important to snail energetics as resource quantity (Bernot 2013; Narr & Krist 2015; Stoler et al. 2015). Increased parasite production by hosts is one of the most important drivers of disease transmission among organisms (Hudson, Rizzoli & Bryan 2002). For example, Johnson et al. (2007) demonstrated that snails infected with R. ondatrae were larger in nutrient-enriched mesocosms and released more cercariae per snail, leading to higher tadpole infection rates. Similarly, Civitello et al. (2015) found that Daphnia spore production rates of Metschnikowia bicuspidata (a fungal parasite) were the strongest predictor of fungal epidemic size in natural

8 312 J. P. Stephens et al. populations of Daphnia dentifera. In our study, both path analysis and a linear mixed-effects model indicated that the cercaria production rate was an important predictor of tadpole infection in the Exposure experiment, despite the low number of true replicates (N = 9 infected snails). Litter N was positively associated with increased tadpole survival and the estimated number of tadpoles present during exposure, which was negatively associated with tadpole infection level. Positive effects of litter N on tadpole survival have been demonstrated in other mesocosm studies with multiple species of amphibians (Maerz, Cohen & Blossey 2010; Stephens, Berven & Tiegs 2013; Stoler & Relyea 2016). This positive effect can be explained by ES theory, which predicts that food with elemental ratios closer to those of the consumer s body tissues should result in improved consumer performance (Sterner & Elser 2002). In parasite host systems in which hosts can directly transmit parasites to other individuals of the population (i.e. direct transmission), host density is often positively associated with infection level (Burdon & Chilvers 1982; Civitello et al. 2015). However, in this system where only infected snails can transmit the parasite to tadpoles, increasing the tadpole population density actually decreases the per capita exposure to trematode cercariae (Raffel et al. 2010; Rohr et al. 2015). When we added the predicted per capita exposure composite variable to the model, tadpole survival became a non-significant predictor of infection in the Exposure experiment, indicating that the effect of tadpole density on per capita exposure fully accounted for the observed effect of survival on infection. Litter polyphenolics increased the tadpole time to metamorphosis, leading to a longer period of exposure to trematodes and thus higher infection levels. These results resemble those of a prior study of tadpole trematode infection, in which increased density slowed toad tadpole development resulting in longer exposure to Echinostoma trivolvis cercariae (Raffel et al. 2010). Thus, our study adds support to previous findings suggesting that any ecological factor increasing the exposure period of tadpoles to trematode parasites will likely increase trematode infection. Litter-derived dissolved polyphenolics have consistently been shown to reduce the developmental rate and survival of wood frog tadpoles (Stephens, Berven & Tiegs 2013; Stoler & Relyea 2016). The mechanism by which these compounds act remains unclear, but possibilities include damage to gill membranes or effects on neuroendocrine pathways (Lutz et al. 2005; Maerz et al. 2005). However, we advise caution in extrapolating these results to other tadpole species, because polyphenolic effects on tadpoles have been found to vary depending on both the litter species of polyphenolic origin and the tadpole species examined (Maerz et al. 2005; Martin & Blossey 2013; Stephens, Berven & Tiegs 2013; Stoler & Relyea 2013a; Earl & Semlitsch 2015). litter-mediated effects on tadpole susceptibility to infection In this study, we predicted that low-quality litter (low N or high polyphenolics) would lead to slower tadpole development. Such an effect might influence both tadpole exposure and susceptibility to R. ondatrae infection, by increasing time to metamorphosis and causing tadpoles to be exposed at earlier developmental stages. We sought to disentangle these effects by holding per capita parasite exposure constant in the susceptibility experiment. Using path analysis, we found support for a negative effect of litter-derived polyphenolics on tadpole development, leading to lower tadpole developmental stages during exposure. This effect was not significant in a mixed-effects model using litter species as the unit of replication (N = 9 instead of N = 18). However, we think it is likely a real pattern based on findings of prior studies (Maerz et al. 2005; Stephens, Berven & Tiegs 2013; Stephens et al. 2015; Stoler & Relyea 2016) and the significant effect of litter polyphenolics on tadpole time to metamorphosis (or exposure duration ) in the exposure experiment. Both path analysis and a mixed-effects model found that lessdeveloped tadpoles were significantly more susceptible to R. ondatrae encystment. This result is consistent with prior studies documenting stage-dependent susceptibility of other Ranid and Hylid species to R. ondatrae (Rohr, Raffel & Hall 2010; Johnson, Kellermanns & Bowerman 2011), and it helps to explain the effect of time-to-metamorphosis (exposure duration) on trematode infection in the exposure experiment, which remained significant even after accounting for the predicted number of cercariae per tadpole in each mesocosm. After controlling for the indirect effects of stage-dependent susceptibility in the path analysis, we found significant direct effects of increased N and polyphenolics on the proportion of cercariae that successfully encysted in tadpoles. However, these results might be spurious because neither of these paths remained significant in the mixed-effects model using the most appropriate unit of replication (N = 9 litter species). Nevertheless, there are plausible mechanisms that could explain each direct path, assuming they might be real effects. First, high dietary N has been found to increase tadpole resistance to infection after controlling for the effects of tadpole developmental stage, apparently due to upregulation of the immune response (Venesky et al. 2012). A similar effect would account for reduced infection in tadpoles from the high-n litter treatments. Second, plant-derived polyphenolics have been shown to possess antihelminth properties (Fukai, Ishigami & Hara 1991; Abozeid, Shohayeb & Ismael 2012). It is therefore plausible that higher levels of polyphenolics in the water decreased cercaria swimming performance during the 24-h exposure period for each tadpole. Alternatively, polyphenolics have been shown to have antioxidant properties and upregulate host immune systems via multiple mechanisms (Cuevas et al. 2013),

9 Resource quality affects amphibian parasitism 313 which might result in increased host resistance and thus reduced cercaria penetration. consequences for changing forests Deciduous forests across the globe are changing due to human related drivers, including emerging infectious disease (Flower, Knight & Gonzalez-Meler 2013), invasive species spread (Martin 1999) and climate change (Prentice, Sykes & Cramer 1993). The consequences of such changes to forests communities might cause dramatic effects on ecosystems including both bottom-up and top-down processes (Ellison et al. 2005; Kominoski et al. 2013). Thus, future field-based studies should address how such shifts in forest tree species composition could influence disease transmission between parasites and their hosts in ponds via alterations to leaf litter chemistry. Prior studies found that dissolved, litter-derived polyphenols inhibited growth of the amphibian chytrid fungus (Davidson, Larsen & Palmer 2012; Stoler, Berven & Raffel 2016), but to our knowledge, there have been no such studies investigating polyphenolic effects on trematode infection. A recent shift in forest leaf litter chemistry that warrants investigation is the replacement of nutrient-rich and phenolic-poor tree species [e.g. Ash and Elm (Herms & McCullough 2014)] by nutrient-poor and phenolic-rich species [e.g. Red maple and Norway maple (Abrams 1998)]. Based on the results of this study, it could be hypothesized that shifts in forest composition from phenolic-poor to phenolic-rich tree species might increase tadpole infection due to increased time to metamorphosis and decreased resistance to infection. Conversely, shifts in forest community composition from nutrient-rich to nutrient-poor tree species could cause a net decrease in R. ondatrae infection in tadpoles, due to decreases in cercaria production rates by infected snails (the strongest path in the exposure experiment). It is important to note that decreasing litter quality might also lead to reduced snail population sizes, which should reinforce this effect by reducing the density of infected snails in a given wetland. Our path analysis showed that the effects of litter N on exposure were stronger than the effects of litter phenolics on susceptibility in our experiment. However, there is no general relationship between N and lignin (a fundamental structural polyphenol) across litter species (Ostrofsky 1997), and the strength of these conflicting forces in natural wetlands will depend on the specific plant species composition in a particular area. Given that many amphibian populations are threatened by anthropogenic change and disease (Stuart et al. 2004; Beebee & Griffiths 2005; Albert et al. 2015), future studies of how litter quality influences trematode parasitism in natural wetlands are clearly warranted. Authors contributions J.P.S. wrote the manuscript. J.P.S., K.A.B., S.D.T. and T.R.R. conceived and designed the experiment. T.R.R. provided funding. K.A.A. aided in field work, sample processing and statistical analysis. J.P.S., K.A.A., K.A.B., S.D.T. and T.R.R. helped to revise the manuscript. Acknowledgements We thank University of Michigan for allowing us to collect animals on their property. E. Fetzer, M. Golembieski and J. Hite assisted in mesocosm sampling, L. Goriel and K. Milko aided in microscopy. We also thank Peter Johnson and his laboratory at the University of Colorado for sending us infected rat faeces for use in generating infected snails. This research was funded by an Oakland University (OU) Graduate Student Research Award to J.P.S. and an NSF grant to T.R.R. (IOS ). Animal work was approved by the OU IACUC committee and a collecting permit was issued by the MI-DNR. Data accessibility Data are accessible through Dryad Data Repository /dryad.v8r1p (Stephens et al. 2016). References Aalto, S.L., Ketola, T. & Pulkkinen, K. (2014) No uniform associations between parasite prevalence and environmental nutrients. Ecology, 95, Abozeid, K., Shohayeb, M. & Ismael, A. (2012) In vitro tests for efficacy of tannins extracted from pomegranate (punica granatum) against schistosoma mansoni miracidia. Journal of Science and Technology, 13, Abrams, M.D. (1998) The red maple paradox. BioScience, 48, Albert, E.M., Fernandez-Beaskoetxea, S., Godoy, J.A., Tobler, U., Schmidt, B.R. & Bosch, J. (2015) Genetic management of an amphibian population after a chytridiomycosis outbreak. Conservation Genetics, 16, Beebee, T.J. & Griffiths, R.A. (2005) The amphibian decline crisis: a watershed for conservation biology? Biological Conservation, 125, Bernot, R.J. (2013) Parasite host elemental content and the effects of a parasite on host-consumer-driven nutrient recycling. Parasite, 32, Burdon, J. & Chilvers, G. (1982) Host density as a factor in plant disease ecology. Annual Review of Phytopathology, 20, Capps, K.A., Berven, K.A. & Tiegs, S.D. (2014) Modelling nutrient transport and transformation by pool-breeding amphibians in forested landscapes using a 21-year dataset. Freshwater Biology, 60, Civitello, D.J. & Rohr, J.R. (2014) Disentangling the effects of exposure and susceptibility on transmission of the zoonotic parasite Schistosoma mansoni. Journal of Animal Ecology, 83, Civitello, D.J., Pearsall, S., Duffy, M.A. & Hall, S.R. (2013) Parasite consumption and host interference can inhibit disease spread in dense populations. Ecology Letters, 16, Civitello, D.J., Penczykowski, R.M., Smith, A.N., Shocket, M.S., Duffy, M.A. & Hall, S.R. (2015) Resources, key traits and the size of fungal epidemics in daphnia populations. Journal of Animal Ecology, 84, Coop, R. & Kyriazakis, I. (1999) Nutrition parasite interaction. Veterinary Parasitology, 84, Coop, R.L. & Kyriazakis, I. (2001) Influence of host nutrition on the development and consequences of nematode parasitism in ruminants. Trends in Parasitology, 17, Cuevas, A., Saavedra, N., Salazar, L.A. & Abdalla, D.S. (2013) Modulation of immune function by polyphenols: possible contribution of epigenetic factors. Nutrients, 5, Davidson, E.W., Larsen, A. & Palmer, C.M. (2012) Potential influence of plant chemicals on infectivity of Batrachochytrium dendrobatidis. Diseases of Aquatic Organisms, 101, Dumont, M., Mone, H., Mouahid, G., Idris, M., Shaban, M. & Boissier, J. (2007) Influence of pattern of exposure, parasite genetic diversity and sex on the degree of protection against reinfection with schistosoma mansoni. Parasitology Research, 101, Earl, J.E. & Semlitsch, R.D. (2013) Spatial subsidies, trophic state, and community structure: examining the effects of leaf litter input on ponds. Ecosystems, 16,

10 314 J. P. Stephens et al. Earl, J.E. & Semlitsch, R.D. (2015) Effects of tannin source and concentration from tree leaves on two species of tadpoles. Environmental Toxicology and Chemistry, 34, Ellison, A.M., Bank, M.S., Clinton, B.D. et al. (2005) Loss of foundation species: consequences for the structure and dynamics of forested ecosystems. Frontiers in Ecology and the Environment, 3, Fisher, S.G. & Likens, G.E. (1973) Energy flow in bear brook, New Hampshire: an integrative approach to stream ecosystem metabolism. Ecological Monographs, 43, Flower, C.E., Knight, K.S. & Gonzalez-Meler, M.A. (2013) Impacts of the emerald ash borer (Agrilus planipennis Fairmaire) induced ash (Fraxinus spp.) mortality on forest carbon cycling and successional dynamics in the eastern United States. Biological Invasions, 15, Frost, P.C., Ebert, D. & Smith, V.H. (2008) Responses of a bacterial pathogen to phosphorus limitation of its aquatic invertebrate host. Ecology, 89, Fukai, K., Ishigami, T. & Hara, Y. (1991) Antibacterial activity of tea polyphenols against phytopathogenic bacteria. Agricultural and Biological Chemistry, 55, Gosner, K.L. (1960) A simplified table for staging anuran embryos and larvae with notes on identification. Herpetologica, 16, Hall, S.R., Simonis, J.L., Nisbet, R.M., Tessier, A.J. & Caceres, C.E. (2009a) Resource ecology of virulence in a planktonic host-parasite system: an explanation using dynamic energy budgets. The American Naturalist, 174, Hall, S.R., Knight, C.J., Becker, C.R., Duffy, M.A., Tessier, A.J. & Caceres, C.E. (2009b) Quality matters: resource quality for hosts and the timing of epidemics. Ecology Letters, 12, Herms, D.A. & McCullough, D.G. (2014) Emerald ash borer invasion of North America: history, biology, ecology, impacts, and management. Annual Review of Entomology, 59, Hudson, P.J., Rizzoli, A. & Bryan, G. (2002) The Ecology of Wildlife Diseases. Oxford University Press, Oxford, UK. Johnson, P.T., Kellermanns, E. & Bowerman, J. (2011) Critical windows of disease risk: amphibian pathology driven by developmental changes in host resistance and tolerance. Functional Ecology, 25, Johnson, P.T. & Sutherland, D.R. (2003) Amphibian deformities and Ribeiroia infection: an emerging helminthiasis. Trends in Parasitology, 19, Johnson, P.T., Lunde, K.B., Ritchie, E.G. & Launer, A.E. (1999) The effect of trematode infection on amphibian limb development and survivorship. Science, 284, Johnson, P.T., Chase, J.M., Dosch, K.L., Hartson, R.B., Gross, J.A., Larson, D.J., Sutherland, D.R. & Carpenter, S.R. (2007) Aquatic eutrophication promotes pathogenic infection in amphibians. Proceedings of the National Academy of Sciences of the USA, 104, Kominoski, J.S., Shah, J.J.F., Canhoto, C. et al. (2013) Forecasting functional implications of global changes in riparian plant communities. Frontiers in Ecology and the Environment, 11, Leroux, S.J. & Loreau, M. (2012) Dynamics of reciprocal pulsed subsidies in local and meta-ecosystems. Ecosystems, 15, Lochmiller, R.L. & Deerenberg, C. (2000) Trade-offs in evolutionary immunology: just what is the cost of immunity? Oikos, 88, Loreau, M., MOuquet, N. & Holt, R.D. (2003) Meta-ecosystems: a theoretical framework for a spatial ecosystem ecology. Ecology Letters, 6, Lutz, I., Jie, Z., Opitz, R., Kloas, W., Ying, X., Menzel, R. & Steinberg, C.E. (2005) Environmental signals: synthetic humic substances act as xeno-estrogen and affect the thyroid system of Xenopus laevis. Chemosphere, 61, Maerz, J.C., Cohen, J.S. & Blossey, B. (2010) Does detritus quality predict the effect of native and non-native plants on the performance of larval amphibians? Freshwater Biology, 55, Maerz, J.C., Brown, C.J., Chapin, C.T. & Blossey, B. (2005) Can secondary compounds of an invasive plant affect larval amphibians? Functional Ecology, 19, Martin, P.H. (1999) Norway maple (Acer platanoides) invasion of a natural forest stand: understory consequence and regeneration pattern. Biological Invasions, 1, Martin, L.J. & Blossey, B. (2013) Intraspecific variation overrides origin effects in impacts of litter-derived secondary compounds on larval amphibians. Oecologia, 173, Motomori, K., Mitsuhashi, H. & Nakano, S. (2001) Influence of leaf litter quality on the colonization and consumption of stream invertebrate shredders. Ecological Research, 16, Narr, C.F. & Krist, A.C. (2015) Host diet alters trematode replication and elemental composition. Freshwater Science, 34, Ostrofsky, M. (1997) Relationship between chemical characteristics of autumn-shed leaves and aquatic processing rates. Journal of the North American Benthological Society, 16, Paull, S.H., Raffel, T.R., LaFonte, B.E. & Johnson, P.T. (2015) How temperature shifts affect parasite production: testing the roles of thermal stress and acclimation. Functional Ecology, 29, Penczykowski, R.M., Lemanski, B.C., Sieg, R.D., Hall, S.R., Housley Ochs, J., Kubanek, J. & Duffy, M.A. (2014) Poor resource quality lowers transmission potential by changing foraging behaviour. Functional Ecology, 28, Polis, G.A., Anderson, W.B. & Holt, R.D. (1997) Toward an integration of landscape and food web ecology: the dynamics of spatially subsidized food webs. Annual Review of Ecology and Systematics, 28, Prentice, I.C., Sykes, M.T. & Cramer, W. (1993) A simulation model for the transient effects of climate change on forest landscapes. Ecological Modelling, 65, Pulkkinen, K. & Ebert, D. (2004) Host starvation decreases parasite load and mean host size in experimental populations. Ecology, 85, R Core Team (2012) R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. Available at: (accessed 1 September 2013). Raffel, T.R., Hoverman, J.T., Halstead, N.T., Michel, P.J. & Rohr, J.R. (2010) Parasitism in a community context: trait-mediated interactions with competition and predation. Ecology, 91, Rohr, J.R., Kerby, J.L. & Sih, A. (2006) Community ecology as a framework for predicting contaminant effects. Trends in Ecology & Evolution, 21, Rohr, J.R., Raffel, T.R. & Hall, C.A. (2010) Developmental variation in resistance and tolerance in a multi-host parasite system. Functional Ecology, 24, Rohr, J.R., Raffel, T.R., Sessions, S.K. & Hudson, P.J. (2008) Understanding the net effects of pesticides on amphibian trematode infections. Ecological Applications, 18, Rohr, J.R., Civitello, D.J., Crumrine, P.W., Halstead, N.T., Miller, A.D., Schotthoefer, A.M., Stenoien, C., Johnson, L.B. & Beasley, V.R. (2015) Predator diversity, intraguild predation, and indirect effects drive parasite transmission. Proceedings of the National Academy of Sciences of the United States of America, 112, Rosseel, Y. (2012) Lavaan: an R package for structural equation modeling. Journal of Statistical Software, 48, Saleh, M., Zwaving, J., Malingre, T.M. & Bos, R. (1985) The essential oil of Apium graveolens var. secalinum and its cercaricidal activity. Pharmaceutisch Weekblad, 7, Schotthoefer, A.M., Koehler, A.V., Meteyer, C.U. & Cole, R.A. (2003) Influence of Ribeiroia ondatrae (Trematoda: Digenea) infection on limb development and survival of northern leopard frogs (Rana pipiens): effects of host stage and parasite-exposure level. Canadian Journal of Zoology, 81, Stephens, J.P., Berven, K.A. & Tiegs, S.D. (2013) Anthropogenic changes to litter input affect the fitness of a larval amphibian. Freshwater Biology, 58, Stephens, J.P., Berven, K.A., Tiegs, S.D. & Raffel, T.R. (2015) Ecological stoichiometry quantitatively predicts the response of tadpoles to a food quality gradient. Ecology, 96, Stephens, J.P., Altman, K.A., Berven, K.A., Tiegs, S.D. & Raffel, T.R. (2016) Data from: Bottom-up and trait-mediated effects of resource quality on amphibian parasitism. Dryad Digital Repository, doi.org/ /dryad.v8r1p. Sterner, R.W. & Elser, J.J. (2002) Ecological Stoichiometry: The Biology of Elements from Molecules to the Biosphere. Princeton University Press, Princeton, NJ, USA. Stoler, A.B., Berven, K.A. & Raffel, T.R. (2016) Leaf litter inhibits growth of an amphibian fungal pathogen. EcoHealth, 13, Stoler, A.B. & Relyea, R.A. (2013a) Bottom-up meets top-down: leaf litter inputs influence predator prey interactions in wetlands. Oecologia, 173, Stoler, A.B. & Relyea, R.A. (2013b) Leaf litter quality induces morphological and developmental changes in larval amphibians. Ecology, 94, Stoler, A.B. & Relyea, R.A. (2016) Leaf litter species identity alters the structure of pond communities. Oikos, 125, Stoler, A.B., Golembieski, M., Stephens, J.P. & Raffel, T.R. (2015) Differential consumption and assimilation of leaf litter by wetland