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.

Temperature affects the toxicity of pesticides to cercariae of the trematode Echinostoma trivolvis.

Global climate change is predicted to have significant impacts on ecological interactions such as host-parasite relationships. Increased temperatures may also interact with other anthropogenic stressors, such as chemical contaminants, to exacerbate or reduce parasite transmission. However, studies on the effects of pesticides on non-target species are typically conducted at one standard temperature, despite the toxicity of many agrochemicals being temperature-dependent. Furthermore, most studies assessing the effects of temperature on pesticide toxicity have been conducted on host organisms, limiting our understanding of how temperature affects the toxicity of pesticides to free-living parasite stages as they move through the environment in search of a host. Using the free-swimming cercariae stage of the trematode Echinostoma trivolvis, we examined how the toxicities of three different pesticides (a carbamate insecticide, strobilurin fungicide, and triazine herbicide) vary by temperature by monitoring cercarial swimming activity over time. Our three main findings were: 1) a strong main effect of temperature across all pesticide trials - higher temperatures caused cercariae to cease swimming activity earlier, likely due to an increased rate of energy expenditure, 2) atrazine, azoxystrobin, and carbaryl were directly toxic to cercariae to some degree, but not in a predictable dose-dependent manner, and 3) the temperature at which pesticide exposure occurs could affect its toxicity to cercariae. The interaction between pesticide and temperature was most evident in the azoxystrobin exposure; azoxystrobin caused cercariae to cease swimming activity earlier at 30 °C but caused cercariae to maintain swimming activity longer at 18 °C relative to their respective pesticide-free control treatments. These findings highlight the importance of conducting toxicity assays at multiple temperatures and suggest that the combined effects of pesticides and temperature on host-parasite interactions may be difficult to generalize.

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.

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.