MERCURY BIOACCUMULATION AND EFFECTS IN THE BROWN WATERSNAKE (NERODIA TAXISPILOTA) by DAVID LEE HASKINS (Under the Direction of Tracey D. Tuberville and Robert B. Bringolf) ABSTRACT Mercury (Hg) is a global pollutant of concern capable of contaminating aquatic and terrestrial environments. Unlike many other pollutants, Hg is well-known due to its propensity to bioaccumulate, biomagnify, and maternally transfer in vertebrates. The detrimental effects of Hg exposure in vertebrates include impaired growth, decreased survival and reproductive success, and altered behavior. Although these effects have been described in mammals and birds, little is known about the bioaccumulation and effects of Hg in reptiles, especially snakes. All snakes are predators, typically occupy upper-level trophic positions in their respective food webs, and many are long-lived. These charateristics suggest snakes may be effective biomonitors for Hg contamination. In this dissertation, I explored how a common watersnake species, the brown watersnake (Nerodia taxispilota), may be used to evaluate spatial and temporal trends in Hg contamination in a major riverine system in the southeastern United States. Next, I optimized an in vitro immune-based assay in N. taxispilota, providing a framework for the investigation of how mitogens, contmainants, and other stressors may impact their immune response. Using Hg data from previous studies, I then examined how in vitro exposure to Hg affected lymphocyte proliferation in N. taxispilota. Overall, my results demonstrate that N. taxispilota are effective biomonitors for Hg contamination in a riverine system, providing risk assessors with novel data that will aid in future investigations of snake ecotoxicology. In addition, my results suggest that N. taxispilota in my study system are at low risk of Hg immunotoxicity. However, Nerodia spp. in heavily contaminated systems in other areas of the United States could be subject to negative impacts due to Hg exposure. INDEX WORDS: Squamate, Bioindicator, Pollution, Immunology, Toxicology, Snake MERCURY BIOACCUMULATION AND EFFECTS IN THE BROWN WATERSNAKE (NERODIA TAXISPILOTA) by DAVID LEE HASKINS B.S., Maryville College, 2014 M.S., University of Georgia, 2016 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2021 © 2021 David Lee Haskins All Rights Reserved MERCURY BIOACCUMULATION AND EFFECTS IN THE BROWN WATERSNAKE (NERODIA TAXISPILOTA) by DAVID LEE HASKINS Major Professors: Tracey D. Tuberville Robert B. Bringolf Committee: Robert M. Gogal, Jr. Travis C. Glenn Melissa A. Pilgrim Electronic Version Approved: Ron Walcott Vice Provost for Graduate Education and Dean of the Graduate School The University of Georgia May 2021 DEDICATION This dissertation is dedicated to all the loved ones I have lost along the way. iv ACKNOWLEDGEMENTS I am incredibly grateful to Tracey Tuberville for serving as my major advisor during my doctoral program. I am thankful for her support, wisdom, and patience, as I know I stopped in her office countless times with so many random thoughts (especially early on in my master’s program). Tracey leads by example, is a phenomenal scientist (her passion for herpetofauna is infectious) and is a fantastic person. Throughout my time as her student, her advice and support always encouraged me to be a better scientist. I am going to miss going to her and Kurt’s place for pizza nights. Thank you and Kurt for always supporting me in and outside of the lab. I am extremely thankful for my co-advisor, Robert Bringolf, for his invaluable contributions to my time at the University of Georgia. As a student that was stationed at the Savannah River Ecology Laboratory (SREL; Aiken, SC), it is often hard to stay connected to campus. From the beginning, Robert made it easy to do this and always made time for me. While Robert is a fantastic, well-accomplished scientist, what always struck me is how humble and open he is with his students and colleagues. His mentorship, kindness, and wisdom have helped me make great strides as a scientist and as a person. His examples of kindness, work-life balance, and faith have been invaluable. I am proud to call him a mentor, colleague, and true friend. I am indebted to the other members of my advisory committee, including Robert ‘Bob’ Gogal, Melissa Pilgrim, and Travis Glenn. I am extremely lucky that John Finger recommended that I connect with Bob prior to the beginning of my PhD program. Bob is v exactly the kind of scientist that you want on your committee as a graduate student. He always is willing to working in new systems or with new species. His expertise (and patience) in the laboratory, inquisitive nature, and passion truly made it so much enjoyable to conduct our research. Bob taught me much (in and outside of the lab) that will serve me well as I move forward into a postdoctoral position. I thank him profusely for his willingness to work with a loud, young man catching wild snakes for his dissertation. I am thankful that my research with Tracey allowed me to work with Melissa and for her agreeing to be on my committee. Melissa always has challenged me to remember to think about all of our work in a broad context. This one piece of advice has helped me tremendously. While Melissa is well-versed in snake physiology and was a valuable academic mentor, I also really appreciate her taking the time to chat with me about life outside work. Finally, I would like to thank Travis for sticking with me as a committee member (master’s and doctoral). I have learned from Travis in the classroom and in meetings. Travis is a brilliant but shows humility, and his willingness to help students understand topics is something I truly admire. I am very excited about our RNA- seq and microbiome side projects and working together in the coming years. I would also like to take the opportunity to thank Mark Mills for all his help during my doctoral program. I am fortunate that Mark was so willing to share his expertise on brown watersnakes with me. The field sampling for this species is unique, and I thank Mark for coming to the lab multiple times to help us remember to just “take the bite.” I am also indebted to Sue Blas, who took the time to hear our watersnake research proposal and support our project. Thank you for helping with my numerous vi requests for fish data and for sending pictures of all the snakes you see on the Savannah River Site! I am thankful to the entire SREL community and the folks there that made it a wonderful place to learn and grow as a scientist. First, I would like to thank Gene Rhodes for agreeing to help support me financially as a doctoral student during my time at SREL. I also thank him for coordinating our basketball group while at the lab—this was a much- needed outlet for many of us and I enjoyed getting to know everyone. I would like to thank Larry Bryan for allowing me to work as a technician in his lab the summer before my doctoral program started. Larry always made me feel welcome at the lab and I thoroughly enjoyed working with him in the field and the lab. I also thank Angela Lindell for providing help with our lab’s mercury analyses. I want to thank the entire SREL maintenance crew for their help over the last few years, and I’d especially like to thank Dennis Frasier for helping us make sure we always had a working boat. Thanks to my friends and lab mates that I spent so much time with at SREL and on campus, especially Austin Coleman, Kyle Brown, Matthew Hale, Amelia Russell, Alexis Korotasz, Pearson McGovern, Rebecca McKee, Becca Cozad, Carmen Candal, Lonnie Helton, Wes Flynn, Ben Thesing, Tyler Carter, Heather Gaya, Chris Leaphart, Sarah Chinn, Laura Kojima, Jackie Newbold, and Emma Browning. A very special shout out to Kyle Brown for spending so many hours with me in the field and snagging as many watersnakes as possible. I would like to thank University of Georgia’s Interdisciplinary Toxicology Program and the U.S. Department of Energy for supporting my research at SREL. In addition, I thank the University of Georgia’s Graduate School for supporting my research vii and travel during my program. (the Dissertation Completion Award, annual travel grants, Interdisciplinary Research awards). I also thank Riverbanks Zoo and Garden and the American Society of Herpetologists for their support to our numerous projects. Lastly, thank you to the numerous conferences (e.g., TWS, SICB, SETAC, SEPARC, TSA) that supported my travel as a graduate student to present my research at their meetings. This project was partially funded by the Department of Energy under award number DE- EM0004391 to the University of Georgia Research Foundation and by Savannah River Nuclear Solutions – Area Completions Project. A special thanks to the professors at Maryville College that prepared me for graduate school. I thank Dr. Dave Unger for taking a chance on a wild, curious undergraduate student. To Drs. Drew Crain, Jerilyn Swann, and Josh Ennen, thank you for mentoring and supporting me during my time at MC. I would like to thank my family and close friends for all their support throughout my program. I especially would like to thank my parents for always pushing me to work hard. To my father, thank you for leading by example, teaching me that things can always be improved, and the wisdom of “all things are finite.” I thank my mother for always fostering my inquisitive nature and supporting me even when I did not believe in myself.