Understanding Amphibian Decline: the Role of Pesticides and the Pathogenic Chytrid Fungus on Amphibians and Aquatic Communities

Amphibians are the most threatened taxon on the planet. Declines have been associated with over-exploitation, habitat loss, pollution, and pathogenic diseases, but of these factors, pollution and disease have been relatively under-studied. Here, I investigated: 1) the impacts of commonly used pesticides on aquatic communities, 2) the effect of these pesticides on amphibian susceptibility to the pathogenic chytrid fungus, Batrachochytrium dendrobatidis (Bd), and 3) whether there are non-amphibian hosts of Bd and 4) how to best quantify the survival of Bd through ontogeny of the host. In my first research chapter, I quantified the effects of environmentally relevant concentrations of the mot commonly used synthetic fungicide in the US, chlorothalonil, on 34 species-, 2 community- and 11 ecosystem-level responses in a multitrophic-level system. Chlorothalonil increased mortality of amphibians, gastropods, zooplankton, algae, and a macrophyte (reducing taxonomic richness), reduced decomposition and water clarity, and elevated dissolved oxygen and net primary productivity. These ecosystem effects were indirect but were predictable based on changes in taxonomic richness. A path analysis suggests that chlorothalonil-induced reductions in biodiversity and top-down and bottom-up effects facilitated algal blooms that shifted ecosystem functions. In my second chapter, I investigated how a wide range of ecologically relevant concentrations of chlorothalonil affected four species of amphibians (Osteopilus septentrionalis, Rana sphenocephala, Hyla squirella and H. cinerea). I also evaluated the effects of chlorothalonil on liver tissue, immune cell density, and the stress hormone, corticosterone. Chlorothalonil killed nearly every amphibian at the expected environmental concentration (EEC) and, at concentrations to which humans are commonly exposed (up to the EEC), it was associated with elevated corticosterone levels and changes in immune cells. Three species (O. septentrionalis, R. sphenocephala, and H. cinerea) showed a non-monotonic dose-response, with low and high concentrations causing significantly greater mortality than intermediate concentrations and controls. Corticosterone exhibited a similar non-monotonic dose response and chlorothalonil concentration was inversely associated with liver tissue and immune cell densities. These studies on chlorothalonil emphasize the need to re-evaluate its safety and to further link anthropogenic-induced changes in biodiversity to altered ecosystem functions. In my third research chapter, I investigated the effects of chlorothalonil and atrazine, one of the most commonly used herbicides in the US, on amphibian susceptibility to Bd, a leading cause of amphibian extinctions. Relative to controls, atrazine monotonically reduced Bd growth in culture and on tadpoles. In contrast, chlorothalonil non-monotonically reduced Bd growth in culture and on tadpoles, with low and high concentrations causing significantly greater mortality than intermediate concentrations and controls. This study is one of only a handful of studies to document a non-monotonic dose response of an invertebrate (Bd) to a pesticide. Although both pesticides reduced Bd growth on tadpoles and in culture, neither eliminated Bd entirely, and because we know little about the long-term effects of the pesticides on hosts (e.g., immunosuppression), I do not recommend using these chemicals to control Bd. In my fourth research chapter, I investigated whether there are non-amphibian hosts for Bd. Non-amphibian hosts could explain how Bd is able to persist in the environment after amphibians are extirpated, and the extreme virulence and distribution of Bd. In laboratory and field studies, I found that crayfish, but not mosquitofish, were hosts for Bd. I found that crayfish could be infected with Bd, could maintain that infection long term (at least 3 months) and could transfer that infection to susceptible amphibians. I also revealed that exposure to water that previously held Bd caused significant crayfish mortality and gill recession, suggesting that Bd releases a chemical that can cause host pathology in the absence of infection. Most efforts to conserve and restore amphibian populations challenged by Bd have been unsuccessful, but managing alternative hosts offers a new and potentially more effective approach to managing Bd. Likewise, identifying the specific pathology-inducing chemical released by Bd might facilitate the development of new strategies to reduce the risk posed by this pathogen. The fifth and sixth research chapters are aimed to improve the quality and efficiency of Bd research. During amphibian development, Bd infections transition from the mouthparts of tadpoles to the skin of post-metamorphic frogs but this transition has never been quantified and thus researchers might be sampling the wrong parts of amphibian bodies to detect Bd. I showed that Bd abundance in O. septentrionalis mouthparts declined from Gosner stages 35-42 and increased on epidermis from Gosner stages 38-46. Assuming our findings are general across species, I recommend sampling mouthparts of amphibians less than Gosner stage 41 and hind limbs of amphibians greater than Gosner stage 41. This should provide researchers with guidance on where to sample to maximize detection of Bd. I also investigated whether Trypan blue dye could be used to determine the viability of Bd. I showed that the proportion of zoospores stained with Trypan blue dye matched the proportion of known dead zoospores added to cultures. In contrast, all of the zoosporangia stage (including known dead zoosporangia) of Bd stained blue. These results demonstrate that Trypan blue can be used to determine the viability of Bd zoospores but not zoosporangia. I recommend using Trypan blue to report the number of live zoospores to which hosts are exposed and to help determine whether factors have lethal or sublethal effects on Bd. My work demonstrates that managing exposure to contaminants and biological reservoirs for Bd might provide new hope for imperiled amphibians. Further exploring how pesticides and pathogens are contributing to amphibian declines will allow us to formulate crucial management and conservation plans to begin remediation