Chronic Exposure to a PFAS Mixture Resembling AFFF-Impacted Surface Water Decreases Body Size in Northern Leopard Frogs (Rana pipiens).

Per- and polyfluoroalkyl substances (PFAS) occur in the environment as mixtures, yet mixture toxicity remains poorly understood. Aqueous film-forming foams (AFFFs) are a common source of PFAS. Our objective was to examine chronic effects of a complex PFAS mixture on amphibian growth and development. We tested toxicity of a five-chemical PFAS mixture summing to 10 µg/L and that accounts for >90% of the PFAS in AFFF-affected surface waters: perfluorooctane sulfonate (PFOS, 40%), perfluorohexane sulfonic acid (PFHxS, 30%), perflurooctanoic acid (PFOA, 12.5%), perfluorohexanoic acid (PFHxA, 12.5%), and perfluoropentanoic acid (PFPeA, 5%). We also included treatments to determine whether PFOS drove mixture toxicity and whether PFOS and mixture components act additively. We exposed Northern leopard frog (Rana pipiens) larvae through metamorphosis (∼130 d) in outdoor mesocosms. After 21 days of exposure, the larval body condition fell ∼5% relative to controls in the 4 µg/L PFOS treatment and mixtures lacking PFOS. At metamorphosis, the full 5-component 10 µg/L PFAS mixture reduced mass by 16% relative to controls. We did not observe effects on development. Our results indicate that toxicity of PFOS and other PFAS mixtures typical of AFFF sites act additively and that PFOS is not more inherently toxic than other mixture components.

Toxicities of Legacy and Current-Use PFAS in an Anuran: Do Larval Exposures Influence Responses to a Terrestrial Pathogen Challenge?

Legacy polyfluoroalkyl substances (PFAS) [perfluorooctanesulfonate (PFOS) and perfluorooctanoic acid (PFOA)] are being replaced by various other fluorinated compounds, such as hexafluoropropylene oxide dimer acid (GenX). These alternatives are thought to be less bioaccumulative and, therefore, less toxic than legacy PFAS. Contaminant exposures occur concurrently with exposure to natural stressors, including the fungal pathogen Batrachocytrium dendrobatidis (Bd). Despite evidence that other pollutants can increase the adverse effects of Bd on anurans, no studies have examined the interactive effects of Bd and PFAS. This study tested the growth and developmental effects of PFOS, PFOA, and GenX on gray treefrog (Hyla versicolor) tadpoles, followed by a Bd challenge after metamorphosis. Despite PFAS exposure only occurring during the larval stage, carry-over effects on growth were observed post metamorphosis. Further, PFAS interacted with Bd exposure to influence growth; Bd-exposed animals had significantly shorter SVL [snout-vent length (mm)] with significantly increased body condition, among other time-dependent effects. Our data suggest that larval exposure to PFAS can continue to impact growth in the juvenile stage after exposure has ended. Contrary to predictions, GenX affected terrestrial performance more consistently than its legacy congener, PFOA. Given the role of Bd in amphibian declines, further investigation of interactions of PFAS with Bd and other environmentally relevant pathogens is warranted.

Acute aquatic toxicity of two commonly used fungicides to midwestern amphibian larvae.

Fungicide usage has increased globally in response to the rise in fungal pathogens, especially in the agricultural sector. However, research examining the toxicity of fungicides is still limited for many aquatic species. In this study, we examined the acute toxicity of two widely used fungicides, chlorothalonil and pyraclostrobin, on six North American larval amphibian species across multiple families using 96-h LC50 tests. We found that pyraclostrobin was approximately 3.5x more toxic than chlorothalonil; estimated LC50 values ranged from 5-18 µg/L for pyraclostrobin and 15-50 µg/L for chlorothalonil. Comparing across amphibian groups, we found that salamanders were 3x more sensitive to pyraclostrobin than anuran species and equally as sensitive to chlorothalonil. Notably, our estimated LC50 values within the range of the expected environmental concentration for these fungicides suggesting environmental exposures could lead to direct mortality in these species. Given the widespread and increasing usage of fungicides, additional work should be conducted to assess the general risk posed by these chemicals to amphibian and their associated aquatic habitats.

Pesticides alter ecosystem respiration via phytoplankton abundance and community structure: Effects on the carbon cycle?

Freshwater systems are critical to life on earth, yet they are threatened by the increasing rate of synthetic chemical pollution. Current predictions of the effects of synthetic chemicals on freshwater ecosystems are hampered by the sheer number of chemical contaminants entering aquatic systems, the diversity of organisms inhabiting these systems, the myriad possible direct and indirect effects resulting from these combinations, and uncertainties concerning how contaminants might alter ecosystem metabolism via changes in biodiversity. To address these knowledge gaps, we conducted a mesocosm experiment that elucidated the responses of ponds composed of phytoplankton and zooplankton to standardized concentrations of 12 pesticides, nested within four pesticide classes, and two pesticide types. We show that the effects of the pesticides on algae were consistent within herbicides and insecticides and that responses of over 70 phytoplankton species and genera were consistent within broad taxonomic groups. Insecticides generated top-down effects on phytoplankton community composition and abundance, which were associated with persistent increases in ecosystem respiration. Insecticides had direct toxic effects on cladocerans, which led to competitive release of copepods. These changes in the zooplankton community led to a decrease in green algae and a modest increase in diatoms. Herbicides did not change phytoplankton composition but reduced total phytoplankton abundance. This reduction in phytoplankton led to short-term decreases in ecosystem respiration. Given that ponds release atmospheric carbon and that worldwide pesticide pollution continues to increase exponentially, scientists and policy makers should pay more attention to the ways pesticides alter the carbon cycle in ponds via changes in communities, as demonstrated by our results. Our results show that these predictions can be simplified by grouping pesticides into types and species into functional groups. Adopting this approach provides an opportunity to improve the efficiency of risk assessment and mitigation responses to global change.

Effects of Per- and Polyfluoroalkyl Substance Mixtures on the Susceptibility of Larval American Bullfrogs to Parasites.

Per- and polyfluoroalkyl substances (PFAS) are persistent environmental contaminants known to adversely affect health and development in many taxa. Although PFAS generally occur as mixtures in the environment, little is known about the effects of PFAS mixtures on organisms compared to single chemical exposures. Moreover, PFAS exposure in nature occurs alongside biotic factors such as parasitism. Even though host-parasite interactions are common in natural systems, there is little information about how PFAS affect these interactions. Here, we examined the effects of PFAS mixtures on the susceptibility of larval American bullfrogs (Rana catesbeiana) to echinostomes. Our PFAS treatments included perfluorooctanesulfonic acid (PFOS) at 4 and 10 ppb, two mixtures without PFOS as a component at 6 and 10 ppb total PFAS, and a mixture containing PFOS at 10 ppb total PFAS. We found that a 62-day PFAS exposure increased parasite loads by 42-100% in all treatments relative to the control. Additionally, we found that the singular exposure to PFOS increased parasite loads by ∼40% compared to a mixture containing PFOS suggesting antagonism among PFAS in mixtures. Our results highlight the need for further investigation into the effects of PFAS mixtures on organisms and how PFAS affect common ecological interactions.

Acute Toxicity of Eight Aqueous Film-Forming Foams to 14 Aquatic Species.

Researchers have developed numerous per- and polyfluoroalkyl substances (PFAS)-free aqueous film-forming foam (AFFF) formulations to replace PFAS-containing AFFF used for fire suppression. As part of the Department of Defense's Strategic Environmental Research and Development Program (SERDP), we examined the direct lethal effects of seven PFAS-free AFFF and a PFAS-containing AFFF on 14 aquatic species using a series of lethal concentration (LC50) tests. We assessed the LC10, LC50, and LC90 values using log-logistic and logit analyses. Across all aquatic species tested, we discovered that exposure to at least one PFAS-free AFFF was more or as toxic as exposure to the PFAS-containing AFFF. For most cases, National Foam Avio F3 Green KHC 3% and Buckeye Platinum Plus C6MILSPEC 3% were the most and least toxic formulations, respectively. Moreover, we found consistency among results from multiple experiments using the same minnow species (Pimephales promelas) and among closely related taxa (e.g., daphnids, amphibians). Lastly, the LC50 values for AFFF formulations trended lower for tested marine species as compared to those of freshwater species. These results dramatically increase the current knowledge on the potentially toxic effects of AFFF but also highlight the need for additional research and the development of new PFAS-free AFFF that are more "ecologically friendly" than those containing persistent PFAS.

Pathogens and predators: examining the separate and combined effects of natural enemies on assemblage structure.

Natural enemy ecology strives to unify predator-prey and host-pathogen interactions under a common framework to gain insights into community- and ecosystem-level processes. To address this goal, ecologists need a greater emphasis on: (1) quantifying pathogen-mediated effects on community structure to enable comparisons with predator-mediated effects and (2) determining the interactive effects of combined natural enemies on communities. We conducted a mesocosm experiment to assess the individual and combined effects of predators (dragonfly larvae and adult water bugs) and a pathogen (ranavirus) on the abundance and composition of a larval amphibian assemblage. We found that our three natural enemies structured victim assemblages in unique ways, producing distinct assemblages. Additionally, we found that in combination treatments, predators mainly drove assemblage structure such that the assemblages most closely resembled their respective predator treatments. We also found that predators reduced infection prevalence in combination treatments, and that the magnitude of this effect was dependent on predator identity. Compared to virus-alone treatments, the presence of dragonflies and water bugs reduced infection prevalence by 79% and 63%, respectively. Additionally, the presence of dragonflies eliminated ranavirus infection in two species, which demonstrates the prominent role of predators in disease dynamics in this system. Overall, this work demonstrates the importance of considering natural enemies in community ecology, as each enemy can elicit a unique structural change. Additionally, this study provides a unique empirical test of the healthy herds hypothesis for multi-species assemblages and underscores the importance of advancing our understanding of multi-enemy interactions within communities.

Population-level variation in pesticide tolerance predicts survival under field conditions in mayflies.

An increasing number of studies have found tolerance variation in populations consistently exposed to contaminants, but few studies have examined whether these laboratory-derived estimates of tolerance have survival implications in field conditions. We examined four populations of the mayfly Stenacron interpunctatum for variation in tolerance to the common agricultural insecticide clothianidin. Using laboratory bioassays, we found a 2.3× range in 96 h EC50 tolerance values to clothianidin between our four populations. We then conducted a common-garden experiment with nymphs from each population placed into the collection stream most heavily impacted by upstream agricultural activities to assess whether our laboratory tolerance estimates predict survival under field conditions. We monitored survival and growth in situ for three weeks during the spring planting season, when clothianidin is applied to croplands upstream of our study site. While growth was similar across all groups, the most tolerant population, which was native to the impacted stream, had higher survival than the more sensitive populations. This suggests that population-level variation in contaminant tolerance as measured in laboratory bioassays could have real-world survival implications for sensitive aquatic macroinvertebrates in contaminated streams.

Predator- and competitor-induced responses in amphibian populations that evolved different levels of pesticide tolerance.

Exposure to agrochemicals can drive rapid phenotypic and genetic changes in exposed populations. For instance, amphibian populations living far from agriculture (a proxy for agrochemical exposure) exhibit low pesticide tolerance, but they can be induced to possess high tolerance following a sublethal pesticide exposure. In contrast, amphibian populations close to agriculture exhibit high, constitutive tolerance to pesticides. A recent study has demonstrated that induced pesticide tolerance appears to have arisen from plastic responses to predator cues. As a result, we might expect that selection for constitutive pesticide tolerance in populations near agriculture (i.e., genetic assimilation) will lead to the evolution of constitutive responses to natural stressors. Using 15 wood frog (Rana sylvatica) populations from across an agricultural gradient, we conducted an outdoor mesocosm experiment to examine morphological (mass, body length, and tail depth) and behavioral responses (number of tadpoles observed and overall activity) of tadpoles exposed to three stressor environments (no-stressor, competitors, or predator cues). We discovered widespread differences in tadpole traits among populations and stressor environments, but no population-by-environment interaction. Subsequent linear models revealed that population distance to agriculture (DTA) was occasionally correlated with tadpole traits in a given environment and with magnitudes of plasticity, but none of the correlations were significant after Bonferroni adjustment. The magnitudes of predator and competitor plasticity were never correlated with the magnitude of pesticide-induced plasticity that we documented in a companion study. These results suggest that while predator-induced plasticity appears to have laid the foundation for the evolution of pesticide-induced plasticity and its subsequent genetic assimilation, inspection of population-level differences in plastic responses show that the evolution of pesticide-induced plasticity has not had a reciprocal effect on the evolved plastic responses to natural stressors.

Timing and order of exposure to two echinostome species affect patterns of infection in larval amphibians.

The study of priority effects with respect to coinfections is still in its infancy. Moreover, existing coinfection studies typically focus on infection outcomes associated with exposure to distinct sets of parasite species, despite that functionally and morphologically similar parasite species commonly coexist in nature. Therefore, it is important to understand how interactions between similar parasites influence infection outcomes. Surveys at seven ponds in northwest Pennsylvania found that multiple species of echinostomes commonly co-occur. Using a larval anuran host (Rana pipiens) and the two most commonly identified echinostome species from our field surveys (Echinostoma trivolvis and Echinoparyphium lineage 3), we examined how species composition and timing of exposure affect patterns of infection. When tadpoles were exposed to both parasites simultaneously, infection loads were higher than when exposed to Echinoparyphium alone but similar to being exposed to Echinostoma alone. When tadpoles were sequentially exposed to the parasite species, tadpoles first exposed to Echinoparyphium had 23% lower infection loads than tadpoles first exposed to Echinostoma. These findings demonstrate that exposure timing and order, even with similar parasites, can influence coinfection outcomes, and emphasize the importance of using molecular methods to identify parasites for ecological studies.

Pesticide tolerance induced by a generalized stress response in wood frogs (Rana sylvatica).

Increasing evidence suggests that phenotypic plasticity can play a critical role in ecotoxicology. More specifically, induced pesticide tolerance, in which populations exposed to a contaminant show increased tolerance to the contaminants later, has been documented in multiple taxa. However, the physiological mechanisms of induced tolerance remain unclear. We hypothesized that induced pesticide tolerance is the result of a generalized stress response based on previous studies showing that both natural stressors and anthropogenic stressors can induce tolerance to pesticides. We tested this hypothesis by first exposing larval wood frogs (Rana sylvatica) to either an anthropogenic stressor (sublethal carbaryl concentration), a natural stressor (cues from a caged predator), or a simulated stressor via exogenous exposure to the stress hormone corticosterone (125 nM). We also included treatments that inhibited corticosterone synthesis with the compound metyrapone (MTP). We then exposed the larvae to a lethal carbaryl treatment to assess time to death. We found that prior exposure to 125 nM of exogenous CORT and predator cues induced tolerance to a lethal concentration of carbaryl through a slight delay in time to death. Pre-exposure to sublethal carbaryl, as well as MTP alone or in combination with predator cues, did not induce tolerance to the lethal carbaryl concentration relative to the ethanol vehicle control treatment. Our study provides evidence that pesticide tolerance can be induced by a generalized stress response both in the presence and absence (exogenous CORT) of specific cues and highlights the importance of considering physiological ecology and environmental context in ecotoxicology.

Effects of pesticides on exposure and susceptibility to parasites can be generalised to pesticide class and type in aquatic communities.

Pesticide pollution can alter parasite transmission, but scientists are unaware if effects of pesticides on parasite exposure and host susceptibility (i.e. infection risk given exposure) can be generalised within a community context. Using replicated temperate pond communities, we evaluate effects of 12 pesticides, nested in four pesticide classes (chloroacetanilides, triazines, carbamates organophosphates) and two pesticide types (herbicides, insecticides) applied at standardised environmental concentrations on larval amphibian exposure and susceptibility to trematode parasites. Most of the variation in exposure and susceptibility occurred at the level of pesticide class and type, not individual compounds. The organophosphate class of insecticides increased snail abundance (first intermediate host) and thus trematode exposure by increasing mortality of snail predators (top-down mechanism). While a similar pattern in snail abundance and trematode exposure was observed with triazine herbicides, this effect was driven by increases in snail resources (periphytic algae, bottom-up mechanism). Additionally, herbicides indirectly increased host susceptibility and trematode infections by (1) increasing time spent in susceptible early developmental stages and (2) suppressing tadpole immunity. Understanding generalisable effects associated with contaminant class and type on transmission is critical in reducing complexities in predicting disease dynamics in at-risk host populations.

Healthy but smaller herds: Predators reduce pathogen transmission in an amphibian assemblage.

Predators and pathogens are fundamental components of ecological communities that have the potential to influence each other via their interactions with victims and to initiate density- and trait-mediated effects, including trophic cascades. Despite this, experimental tests of the healthy herds hypothesis, wherein predators influence pathogen transmission, are rare. Moreover, no studies have separated effects mediated by density vs. traits. Using a semi-natural mesocosm experiment, we investigated the interactive effects of predatory dragonfly larvae (caged or lethal [free-ranging]) and a viral pathogen, ranavirus, on larval amphibians (grey treefrogs and northern leopard frogs). We determined the influence of predators on ranavirus transmission and the relative importance of density- and trait-mediated effects on observed patterns. Lethal predators reduced ranavirus infection prevalence by 57%-83% compared to no-predator and caged-predator treatments. The healthy herds effect was more strongly associated with reductions in tadpole density than behavioural responses to predators. We also assessed whether ranavirus altered the responses of tadpoles to predators. In the absence of virus, tadpoles reduced activity levels and developed deeper tails in the presence of predators. However, there was no evidence that virus presence or infection altered responses to predators. Finally, we compared the magnitude of trophic cascades initiated by individual and combined natural enemies. Lethal predators initiated a trophic cascade by reducing tadpole density, but caged predators and ranavirus did not. The absence of a virus-induced trophic cascade is ostensibly the consequence of limited virus-induced mortality and the ability of infected individuals to continue interacting within the community. Our results provide support for the healthy herds hypothesis in amphibian communities. We uniquely demonstrate that density-mediated effects of predators outweigh trait-mediated effects in driving this pattern. Moreover, this study was one of the first to directly compare trophic cascades caused by predators and pathogens. Our results underscore the importance of examining the interactions between predators and pathogens in ecology.

Biodiversity decreases disease through predictable changes in host community competence.

Accelerating rates of species extinctions and disease emergence underscore the importance of understanding how changes in biodiversity affect disease outcomes. Over the past decade, a growing number of studies have reported negative correlations between host biodiversity and disease risk, prompting suggestions that biodiversity conservation could promote human and wildlife health. Yet the generality of the diversity-disease linkage remains conjectural, in part because empirical evidence of a relationship between host competence (the ability to maintain and transmit infections) and the order in which communities assemble has proven elusive. Here we integrate high-resolution field data with multi-scale experiments to show that host diversity inhibits transmission of the virulent pathogen Ribeiroia ondatrae and reduces amphibian disease as a result of consistent linkages among species richness, host composition and community competence. Surveys of 345 wetlands indicated that community composition changed nonrandomly with species richness, such that highly competent hosts dominated in species-poor assemblages whereas more resistant species became progressively more common in diverse assemblages. As a result, amphibian species richness strongly moderated pathogen transmission and disease pathology among 24,215 examined hosts, with a 78.4% decline in realized transmission in richer assemblages. Laboratory and mesocosm manipulations revealed an approximately 50% decrease in pathogen transmission and host pathology across a realistic diversity gradient while controlling for host density, helping to establish mechanisms underlying the diversity-disease relationship and their consequences for host fitness. By revealing a consistent link between species richness and community competence, these findings highlight the influence of biodiversity on infection risk and emphasize the benefit of a community-based approach to understanding infectious diseases.

Parasite-induced vulnerability to predation in larval anurans.

Within communities, pathogens and parasites have the potential to indirectly influence predator-prey interactions. For instance, prey that exhibit pathology or altered traits (e.g. behavioral shifts) following infection could be more prone to predation, which is known as parasite-induced vulnerability to predation (PIVP). PIVP has been frequently documented for pathogens with trophic transmission, because predators are often critical in the pathogen's life cycle. However, for pathogens without trophic transmission, PIVP can lead to a healthy herds effect, thereby reducing transmission in the system. In this study, we explored whether the pathogen ranavirus (family Iridoviridae) enhances vulnerability of 4 species of larval amphibians (spring peepers Pseudacris crucifer, gray treefrogs Hyla versicolor, American toads Anaxyrus americanus, and northern leopard frogs Lithobates pipiens) to 2 common tadpole predators (larval green darners Anax junius [hereinafter Anax] and adult water bugs Belostoma flumineum [hereinafter Belostoma]). For each anuran species, we conducted short-term microcosm experiments to assess predation rates on individuals that were or were not exposed to virus. For 3 of the 4 species, we found that exposure to ranavirus decreased survival rates with Anax between 2- and 9-fold. However, we did not see the same trend with Belostoma, which indicates that predator identity is important in this interaction. More specifically, the higher efficiency of Anax in capturing and consuming prey, relative to Belostoma, may allow Anax to capitalize on trait changes induced by virus exposure and enhance the PIVP effect. Our results indicate that trait-mediated indirect effects could play a role in creating healthy herds in amphibian communities.

Larval amphibians rapidly bioaccumulate poly- and perfluoroalkyl substances.

Poly- and perfluoroalkyl substances (PFAS) are ubiquitous contaminants that can bioaccumulate in aquatic taxa. Amphibians are particularly vulnerable to contaminants and sensitive to endocrine disruptors during their aquatic larval stage. However, few studies have explored PFAS uptake rates in amphibians, which is critical for designing ecotoxicology studies and assessing the potential for bioaccumulation. Uptake rates of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) were measured for larval northern leopard frogs (Rana pipiens), American toads (Anaxyrus americanus), and eastern tiger salamanders (Ambystoma tigrinum) during a 240-h exposure to 10 and 1000 µg/L concentrations. We measured body burden and calculated bioconcentration factor (BCF) every 48 h during the experiments. For all species and exposures, body burdens often reached steady state within 48-96 h of exposure. Steady-state body burdens for PFOA and PFOS ranged from 3819 to 16,481 ng/g dry weight (BCF = 0.46-2.5) and 6955-489,958 ng/g dry weight (47-259 BCFs), respectively. Therefore, PFAS steady state occurs rapidly in the larval amphibians we studied and particularly for PFOS. This result reflects a high potential for PFAS trophic transfer because amphibians are often low in trophic position and are important prey for many aquatic and terrestrial species.

Of poisons and parasites-the defensive role of tetrodotoxin against infections in newts.

Classical research on animal toxicity has focused on the role of toxins in protection against predators, but recent studies suggest these same compounds can offer a powerful defense against parasites and infectious diseases. Newts in the genus Taricha are brightly coloured and contain the potent neurotoxin, tetrodotoxin (TTX), which is hypothesized to have evolved as a defense against vertebrate predators such as garter snakes. However, newt populations often vary dramatically in toxicity, which is only partially explained by predation pressure. The primary aim of this study was to evaluate the relationships between TTX concentration and infection by parasites. By systematically assessing micro- and macroparasite infections among 345 adult newts (sympatric populations of Taricha granulosa and T. torosa), we detected 18 unique taxa of helminths, fungi, viruses and protozoans. For both newt species, per-host concentrations of TTX, which varied from undetectable to >60 µg/cm2 skin, negatively predicted overall parasite richness as well as the likelihood of infection by the chytrid fungus, Batrachochytrium dendrobatidis, and ranavirus. No such effect was found on infection load among infected hosts. Despite commonly occurring at the same wetlands, T. torosa supported higher parasite richness and average infection load than T. granulosa. Host body size and sex (females > males) tended to positively predict infection levels in both species. For hosts in which we quantified leucocyte profiles, total white blood cell count correlated positively with both parasite richness and total infection load. By coupling data on host toxicity and infection by a broad range of micro- and macroparasites, these results suggest that-alongside its effects on predators-tetrodotoxin may help protect newts against parasitic infections, highlighting the importance of integrative research on animal chemistry, immunological defenses and natural enemy ecology.

Co-exposure to multiple ranavirus types enhances viral infectivity and replication in a larval amphibian system.

Multiple pathogens commonly co-occur in animal populations, yet few studies demonstrate how co-exposure of individual hosts scales up to affect transmission. Although viruses in the genus Ranavirus are globally widespread, and multiple virus species or strains likely co-occur in nature, no studies have examined how co-exposure affects infection dynamics in larval amphibians. We exposed individual northern red-legged frog Rana aurora larvae to 2 species of ranavirus, namely Ambystoma tigrinum virus (ATV), frog virus 3 (FV3), or an FV3-like strain isolated from a frog-culturing facility in Georgia, USA (RCV-Z2). We compared single-virus to pairwise co-exposures while experimentally accounting for dosage. Co-exposure to ATV and FV3-like strains resulted in almost twice as many infected individuals compared to single-virus exposures, suggesting an effect of co-exposure on viral infectivity. The viral load in infected individuals exposed to ATV and FV3 was also higher than the single-dose FV3 treatment, suggesting an effect of co-exposure on viral replication. In a follow-up experiment, we examined how the co-occurrence of ATV and FV3 affected epizootics in mesocosm populations of larval western chorus frogs Pseudacris triseriata. Although ATV did not generally establish within host populations (<4% prevalence), when ATV and FV3 were both present, this co-exposure resulted in a larger epizootic of FV3. Our results emphasize the importance of multi-pathogen interactions in epizootic dynamics and have management implications for natural and commercial amphibian populations.

The benefits of coinfection: trematodes alter disease outcomes associated with virus infection.

Coinfections are increasingly recognized as important drivers of disease dynamics. Consequently, greater emphasis has been placed on integrating principles from community ecology with disease ecology to understand within-host interactions among parasites. Using larval amphibians and two amphibian parasites (ranaviruses and the trematode Echinoparyphium sp.), we examined the influence of coinfection on disease outcomes. Our first objective was to examine how priority effects (the timing and sequence of parasite exposure) influence infection and disease outcomes in the laboratory. We found that interactions between the parasites were asymmetric; prior infection with Echinoparyphium reduced ranaviral loads by 9% but there was no reciprocal effect of prior ranavirus infection on Echinoparyphium load. Additionally, survival rates of hosts (larval gray treefrogs; Hyla versicolor) infected with Echinoparyphium 10 days prior to virus exposure were 25% greater compared to hosts only exposed to virus. Our second objective was to determine whether these patterns were generalizable to multiple amphibian species under more natural conditions. We conducted a semi-natural mesocosm experiment consisting of four larval amphibian hosts [gray treefrogs, American toads (Anaxyrus americanus), leopard frogs (Lithobates pipiens) and spring peepers (Pseudacris crucifer)] to examine how prior Echinoparyphium infection influenced ranavirus transmission within the community, using ranavirus-infected larval wood frogs (Lithobates sylvaticus) as source of ranavirus. Consistent with the laboratory experiment, we found that prior Echinoparyphium infection reduced ranaviral loads by 19 to 28% in three of the four species. Collectively, these results suggest that macroparasite infection can reduce microparasite replication rates across multiple amphibian species, possibly through cross-reactive immunity. Although the immunological mechanisms driving this outcome are in need of further study, trematode infections appear to benefit hosts that are exposed to ranaviruses. Additionally, these results suggest that consideration of priority effects and timing of exposure are vital for understanding parasite interactions within hosts and disease outcomes.

Immediate and lag effects of pesticide exposure on parasite resistance in larval amphibians.

Across host-parasite systems, there is evidence that pesticide exposure increases parasite loads and mortality following infection. However, whether these effects are driven by reductions in host resistance to infection or slower rates of parasite clearance is often unclear. Using controlled laboratory experiments, we examined the ability of larval northern leopard frogs (Lithobates pipiens) and American toads (Anaxyrus americanus) to resist and clear trematode (Echinoparyphium sp.) infections following exposure to the insecticide carbaryl. Northern leopard frogs exposed to 1 mg L-1 of carbaryl had 61% higher parasite loads compared with unexposed individuals, while there was no immediate effect of carbaryl on parasite encystment in American toads. However, when tadpoles were exposed to carbaryl and moved to freshwater for 14 days before the parasite challenge, we recovered 37 and 63% more parasites from carbaryl-exposed northern leopard frogs and American toads, respectively, compared with the control. No effects on clearance were found for either species. Collectively, our results suggest that pesticide exposure can reduce the ability of amphibians to resist parasite infections and that these effects can persist weeks following exposure. It is critical for researchers to incorporate species interactions into toxicity studies to improve our understanding of how contaminants affect ecological communities.