Population-level variation in insecticide tolerance across three life stages of the trematode Echinostoma trivolvis.

Ecotoxicological studies using single test populations may miss the inherent variation of natural systems and limit our understanding of how contaminants affect focal species. Though population-level variation in pesticide tolerance is commonly observed in host taxa, few studies have assessed population-level differences in the tolerance of parasites to different contaminants. We investigated population-level variation in insecticide tolerance of three Echinostoma trivolvis life stages (egg, miracidium, and cercaria) to three insecticides (carbaryl, chlorpyrifos, and diazinon). We tested two relevant metrics of insecticide tolerance-baseline and induced-across up to eight different parasite populations per life stage. Across all life stages, the insecticide treatments tended to reduce survival, but the magnitude of their effects often varied significantly among populations. Surprisingly, we found that exposure to chlorpyrifos increased the hatching success of echinostome eggs relative to the control treatment in three of six tested populations. We also found that cercariae shed from snails previously exposed to a sublethal concentration of chlorpyrifos had a significantly lower mortality rate when subsequently exposed to a lethal concentration of chlorpyrifos relative to individuals from snails that were not previously exposed; this suggests inducible tolerance in cercariae. We found no evidence that insecticide tolerance is correlated across parasite life stages within a population. Together the findings of our study demonstrate that single-population toxicity assays may greatly over- or underestimate the effects of pesticides on the survival of free-living parasite stages, insecticide tolerance levels may not be predictable from one parasite life stage to the next, and insecticides can have both expected and counterintuitive effects on non-target taxa.

Short and long-term phytoremediation capacity of aquatic plants in Cu-polluted environments.

Freshwater ecosystems face numerous threats from human populations, including heavy metal contamination. Phytoremediation, the use of plants to remediate contaminated soils and sediments, is an effective and low-cost means of removing chemical contaminants, including heavy metals, from polluted environments. However, key questions remain unanswered in the application of this technology in aquatic environments, such as the long-term fate of pollutants following plant uptake. In this study, using two common wetland plant species (duckweed and tape grass), we first examined the capacity of plants to remove copper (Cu) from polluted water. Next, we evaluated the leaching potential of plant tissues following decomposition and how it is affected by a simulated freeze-thaw cycle. Using phytoremediated water and leachates from senesced plants we assessed phytoremediation success and Cu leaching potential by conducting standard toxicity assays using pond snails (Physa acuta), a species with known Cu sensitivity. We found that duckweed outperformed tape grass as a phytoremediator at low Cu concentrations. In addition, for plants grown in low concentrations of Cu, leaching from decaying plant material did not negatively impact snail survival, while at high concentrations of Cu, leaching did result in toxicity. Lastly, we found that a simulated freeze-thaw cycle increased the release of Cu from plant tissue in the presence of high Cu concentrations only, resulting in reduced snail survival. Our results indicate that in moderately Cu-polluted environments, some aquatic plants can remove contaminants without a long-term risk of leaching. In contrast, phytoremediation in highly polluted environments will likely require removal of plant tissue to prevent leaching of previously accumulated metals. Land managers must not only consider plant species and degree of contamination, but also geographic location, as freeze-thaw cycles may enhance plant decomposition and increase the likelihood of contaminant leaching following phytoremediation efforts in aquatic ecosystems.

Life stage and proximity to roads shape the skin microbiota of eastern newts (Notophthalmus viridescens).

Host-associated microbiomes play an essential role in the health of organisms, including immune system activation, metabolism and energy uptake. It is well established that microbial communities differ depending on the life stage and natural history of the organism. However, the effects of life stage and natural history on microbial communities may also be influenced by human activities. We investigated the effects of amphibian life stage (terrestrial eft vs. aquatic adult) and proximity to roadways on newt skin bacterial communities. We found that the eft and adult life stages differed in bacterial community composition; however, the effects of roads on community composition were more evident in the terrestrial eft stage compared to the aquatic adult stage. Terrestrial efts sampled close to roads possessed richer communities than those living further away from the influence of roads. When accounting for amplicon sequence variants with predicted antifungal capabilities, in the adult life stage, we observed a decrease in anti-fungal bacteria with distance to roads. In contrast, in the eft stage, we found an increase in anti-fungal bacteria with distance to roads. Our results highlight the need to consider the effects of human activities when evaluating how host-associated microbiomes differ across life stages of wildlife.

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.

Amphibian Host and Skin Microbiota Response to a Common Agricultural Antimicrobial and Internal Parasite.

Holistic approaches that simultaneously characterize responses of both microbial symbionts and their hosts to environmental shifts are imperative to understanding the role of microbiotas on host health. Using the northern leopard frog (Lithobates pipiens) as our model, we investigated the effects of a common trematode (family Echinostomatidae), a common agricultural antimicrobial (Sulfadimethoxine; SDM), and their interaction on amphibian skin microbiota and amphibian health (growth metrics and susceptibility to parasites). In the trematode-exposed individuals, we noted an increase in alpha diversity and a shift in microbial communities. In the SDM-treated individuals, we found a change in the composition of the skin microbiota similar to those induced by the trematode treatment. Groups treated with SDM, echinostomes, or a combination of SDM and echinostomes, had higher relative abundances of OTUs assigned to Flavobacterium and Acinetobacter. Both of these genera have been associated with infectious disease in amphibians and the production of anti-pathogen metabolites. Similar changes in microbial community composition between SDM and trematode exposed individuals may have resulted from stress-related disruption of host immunity. Despite changes in the microbiota, we found no effect of echinostomes and SDM on host health. Given the current disease- and pollution-related threats facing amphibians, our study highlights the need to continue to evaluate the influence of natural and anthropogenic stressors on host-associated microbial communities.

Local adaptation of the MHC class IIß gene in populations of wood frogs (Lithobates sylvaticus) correlates with proximity to agriculture.

Major histocompatibility complex (MHC) genes code for membrane-embedded proteins that are involved in parasite/pathogen recognition. The link between the MHC and immunity makes these genes important genetic markers to evaluate in systems where infectious disease is associated with population declines. As human impacts on wildlife populations continue to increase, it is also essential to evaluate the role of MHC and immunity in the context of anthropogenic change. Amphibians are an ideal model to test the role of the MHC in infectious disease resistance, as parasites and anthropogenic disturbances currently threaten populations worldwide. We characterized the diversity of MHC class IIß peptide binding region alleles, 13 microsatellite loci, and population-level trematode resistance in 14 populations of wood frogs (Lithobates sylvaticus) in northwestern Pennsylvania with varying geographic distances to agriculture. To assess local adaptation in the MHC IIß, we compared genetic differentiation of MHC IIß and microsatellite markers (FST). We also tested for an effect of isolation by distance on genetic differentiation of MHC IIß and microsatellite markers. In addition, we evaluated whether population-level MHC IIß diversity and common allele frequencies correlate with distance to agriculture and trematode resistance. We found no evidence for genetic structure based on microsatellite analysis nor an effect of isolation by distance on neutral and immunogenetic markers. However, we did detect structure based on the MHC IIß locus, suggesting that it is under local selection. The MHC IIß allele Lisy-DAB*1 was more common in populations living near agricultural sites. Populations with higher MHC IIß diversity showed increased resistance to trematodes. Our results suggest that wood frog populations experience immunogenetic differences at a small scale. In addition, agriculture may disturb natural associations between hosts and parasites through its influence on immunocompetence, underscoring the importance of examining the effects of environmental context on host-parasite interactions.

Inducible pesticide tolerance in Daphnia pulex influenced by resource availability.

Pesticides are a ubiquitous contaminant in aquatic ecosystems. Despite the relative sensitivity of aquatic species to pesticides, growing evidence suggests that populations can respond to pesticides by evolving higher baseline tolerance or inducing a higher tolerance via phenotypic plasticity. While both mechanisms can allow organisms to persist when faced with pesticides, resource allocation theory suggests that tolerance may be related to resource acquisition by the organism. Using Daphnia pulex, we investigated how algal resource availability influenced the baseline and inducible tolerance of D. pulex to a carbamate insecticide, carbaryl. Individuals reared in high resource environments had a higher baseline carbaryl tolerance compared to those reared in low resource environments. However, D. pulex from low resource treatments exposed to sublethal concentrations of carbaryl early in development induced increased tolerance to a lethal concentration of carbaryl later in life. Only individuals reared in the low resource environment induced carbaryl tolerance. Collectively, this highlights the importance of considering resource availability in our understanding of pesticide tolerance.

Evolved pesticide tolerance influences susceptibility to parasites in amphibians.

Because ecosystems throughout the globe are contaminated with pesticides, there is a need to understand how natural populations cope with pesticides and the implications for ecological interactions. From an evolutionary perspective, there is evidence that pesticide tolerance can be achieved via two mechanisms: selection for constitutive tolerance over multiple generations or by inducing tolerance within a single generation via phenotypic plasticity. While both mechanisms can allow organisms to persist in contaminated environments, they might result in different performance trade-offs including population susceptibility to parasites. We have identified 15 wood frog populations that exist along a gradient from close to agriculture and high, constitutive pesticide tolerance to far from agriculture and inducible pesticide tolerance. Using these populations, we investigated the relationship between evolutionary responses to the common insecticide carbaryl and host susceptibility to the trematode Echinoparyphium lineage 3 and ranavirus using laboratory exposure assays. For Echinoparyphium, we discovered that wood frog populations living closer to agriculture with high, constitutive tolerance experienced lower loads than populations living far from agriculture with inducible pesticide tolerance. For ranavirus, we found no relationship between the mechanism of evolved pesticide tolerance and survival, but populations living closer to agriculture with high, constitutive tolerance experienced higher viral loads than populations far from agriculture with inducible tolerance. Land use and mechanisms of evolved pesticide tolerance were associated with susceptibility to parasites, but the direction of the relationship is dependent on the type of parasite, underscoring the complexity between land use and disease outcomes. Collectively, our results demonstrate that evolved pesticide tolerance can indirectly influence host-parasite interactions and underscores the importance of including evolutionary processes in ecotoxicological studies.

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.