Multiple stressor effects on ranavirus transmission and susceptibility

This thesis consists of three chapters that explore the effects of stressors on the amphibian disease, ranavirus, and the existence of ranavirus in southern Illinois. While these three chapters are distinct from one another, they are related in that they all seek to elucidate factors that allow the transmission and persistence of disease and how stressors may influence disease dynamics. Chapter one is a review of the effects of atrazine on amphibian physiology and behavior and seeks to summarize the existing literature on the subject into one succinct document. I reviewed all available literature and extracted the results while also reviewing the methods to ensure that the studies were reliable. I was mainly interested in direct effects of the pesticide atrazine on amphibians in order to elucidate how direct chemical contamination can influence disease susceptibility and spread. Atrazine is an herbicide used to control broadleaf weeds in agricultural fields. It is mainly used for weed reduction in cornfields with the heaviest use occurring in the Midwestern states. However, atrazine can be very persistent and highly mobile. Given the intensity and wide distribution of corn agriculture in the United States, atrazine is likely ubiquitous throughout North American waterways during the spring and summer. Amphibians are likely to come in to contact with atrazine during critical early life stages because atrazine is sprayed in the spring and summer when amphibian larvae are within eggs and developing as tadpoles. This is especially true in the Midwest where the heaviest atrazine use occurs. This suggests atrazine could impact larval development and immunity, thus worth considering as a possible factor in recent disease outbreaks. In my review, I discovered that the most devastating effect of atrazine on amphibians is endocrine disruption, specifically the feminization of male frogs. However, many other effects on physiology and behavior have been observed such as reduced immune function, developmental abnormalities, increased behavior, and decreased anti-predator behavior all of which could have profound effects on disease dynamics and survival. My second chapter sought to detect ranavirus in southern Illinois using Environmental DNA (eDNA) techniques. Environmental DNA is a fairly recent technology that allows researchers to nondestructively detect DNA within the environment using only water or soil samples. This is a great way to detect species that are rare or hard to find but also disease because you do not have to actually find a diseased animal to know if it is present. While there is evidence of ranavirus in southern Illinois, most of this is in box turtles with no positive identifications in amphibians. For this reason, using eDNA is beneficial because we can sample ponds where amphibians are known or expected to be present even if a diseased animal cannot be found and still be able to detect ranavirus. For this chapter, I collected water samples from southern Illinois water bodies were I knew or expected amphibians to be present. Water samples were filtered and analyzed using standard DNA extraction and qPCR protocols. I also validated the use of eDNA to detect ranavirus by making serial dilutions of the virus in water and analyzing them in the same manner as the samples. Ranavirus was detected in southern Illinois water bodies and I was able to validate the use of eDNA for ranavirus detection. My third chapter is the main portion of my thesis and explains an experiment that seeks to elucidate the role that multiple stressors such as thermal stress and chemical contamination play in disease dynamics. As climate change progresses, it is imperative that we understand how wildlife populations will respond and how their response will shape communities and ecosystems going forward. An increase in thermal stress coupled with the stressors that are already present, such as chemical pollution, could have unforeseen effects if scientists only look at one stressor at a time. That is why research exploring how stressors work in tandem is crucial if we are to understand what is actually happening in the environment as few wildlife populations are subjected to only one stressor at a time. This experiment used mesocosms to test the effects of atrazine and thermal stress on the transmission of ranavirus through alterations in behavior and physiology. I did this by setting up four treatments each with ten tubs and ten individuals per tub for a total of 400 animals. The treatments were Control-Ambient, Control-Heat, Atrazine-Ambient, and Atrazine-Heat. One focal animal from each tub was infected with ranavirus and placed in with its conspecifics to test differences in transmission. Animals were then placed into individual tubs and allowed to progress naturally. I found that atrazine and thermal stress increased survival of ranavirus infection and increased developmental rate. While this is the opposite of what might be expected it could be explained by critical windows of disease which in this case is metamorphosis. Animals that proceed through metamorphosis at a faster rate have a better chance of surviving ranavirus infection. We also found that animals in the atrazine treatments were much smaller at metamorphosis than animals from the control treatments so while atrazine may increase survival of ranavirus infection there is a substantial tradeoff. Animals in the Atrazine-Heat treatment also had much lower viral loads than the other treatments which likely also contributed to their increased survival. Viral loads may have been lower in this treatment because of the effect that multiple stressors would have on the secretion of glucocorticoids. Temporary elevation of glucocorticoids can induce a temporary boost to immune function and provide individuals in this treatment with the ability to fight off the virus