Old Dominion University ODU Digital Commons Biological Sciences Theses & Dissertations Biological Sciences Summer 2006 Chemical Evidence for Dietary Toxin Sequestration in the Asian Snake Rhabdophis tigrinus Deborah A. Hutchinson Old Dominion University Follow this and additional works at: https://digitalcommons.odu.edu/biology_etds Part of the Biochemistry Commons, Ecology and Evolutionary Biology Commons, and the Zoology Commons Recommended Citation Hutchinson, Deborah A.. "Chemical Evidence for Dietary Toxin Sequestration in the Asian Snake Rhabdophis tigrinus" (2006). Doctor of Philosophy (PhD), dissertation, Biological Sciences, Old Dominion University, DOI: 10.25777/d4ng-k993 https://digitalcommons.odu.edu/biology_etds/61 This Dissertation is brought to you for free and open access by the Biological Sciences at ODU Digital Commons. It has been accepted for inclusion in Biological Sciences Theses & Dissertations by an authorized administrator of ODU Digital Commons. For more information, please contact [email protected]. CHEMICAL EVIDENCE FOR DIETARY TOXIN SEQUESTRATION IN THE ASIAN SNAKE RHABDOPHIS TIGRINUS by Deborah A. Hutchinson M.S. August 2001, Old Dominion University B.A. May 1999, University of San Diego A Dissertation Submitted to the Faculty of Old Dominion University in Partial Fulfillment of the Requirement for the Degree of DOCTOR OF PHILOSOPHY ECOLOGICAL SCIENCES OLD DOMINION UNIVERSITY August 2006 Approved by: Alan H. Savjlzk/'fbirector) Mark J. Buuer IV (Member) Akira Mori (Member) Rose (Member) Frank C. Schroeder (Member) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT CHEMICAL EVIDENCE FOR DIETARY TOXIN SEQUESTRATION IN THE ASIAN SNAKE RHABDOPHIS TIGRINUS Deborah A. Hutchinson Old Dominion University, 2006 Director: Dr. Alan H. Savitzky Rhabdophis tigrinus (Colubridae: Natricinae) is an oviparous, bufophagous (toad-eating) snake from eastern Asia that possesses defensive integumentary glands on the neck known as nuchal glands. These glands are used in antipredator displays and typically contain bufadienolide toxins. Whereas toads are known to synthesize bufadienolide steroids from cholesterol precursors, we found that chemically undefended R. tigrinus must sequester bufadienolides from ingested toads in order to exhibit these compounds in their nuchal glands. Chemically defended females are capable of provisioning their embryos with these toxins so their unfed hatchlings possess defensive bufadienolides prior to consuming toads themselves. All of the hatchling R. tigrinus from an island with a dense population of toads were chemically defended regardless of their diet in the laboratory. In contrast, none of the hatchlings from an island lacking toads possessed bufadienolides in their nuchal glands until after they consumed bufonid prey. Proton NMR and HPLC analyses demonstrated that newly acquired bufadienolides from ingested toads can be transferred from the dam to the embryos as late as 12 days prior to oviposition, suggesting that at least some transport of toxins occurs within the oviduct. Although hatchlings are provisioned with most of the bufadienolide compounds possessed by the dam, there may be chemical Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. selectivity to this process. In a feeding experiment with gravid R. tigrinus from various localities, most clutches had been provisioned with moderate to large quantities of bufadienolides. In the provisioned clutches, the most abundant bufadienolide in the unfed hatchlings was almost always the same compound, although it generally was not the most abundant bufadienolide in the dam. R. tigrinus is the first amniote vertebrate known to have evolved specialized defensive structures dependent upon sequestered dietary compounds, either obtained directly from prey or provisioned by the dam. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. This dissertation is dedicated to my parents, Emilie M. Hutchinson and Thomas W. Hutchinson, both of whom taught me the importance of education. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. VI ACKNOWLEDGMENTS Many people were necessary for the successful completion of this dissertation. First and foremost, I wish to thank my advisor Alan Savitzky who provided me with years of positive influence, unique research opportunities, and emotional and financial support during the completion of my Master’s and Doctoral Degrees. Additionally, this project would not have been possible without our collaboration with Akira Mori of Kyoto University, who first hypothesized that Rhabdophis tigrinus sequesters its defensive bufadienolides from dietary toads. Dr. Mori was also directly involved in the feeding experiments presented in this dissertation. I was honored to conduct research in Japan with Dr. Mori and in Jerrold Meinwald’s laboratory at Cornell University. I am grateful for all of the time invested by Frank Schroeder to train me on the use and interpretation of NMR spectroscopy and HPLC at Cornell. Sven Possner also assisted me in this regard and graciously shared his lab space and equipment with me. Dr. Schroeder and Xiaogang Wu spent many hours determining the structures of bufadienolides in our samples. I also thank my wonderful family (Trevor Adams, Thomas and Emilie Hutchinson, Judy and Isabella Fercioni, and Maggie) for their continuous support during this rewarding and often challenging journey. I extend gratitude to the remaining members of my dissertation committee, as well as other individuals for their support. Ian Bartol, Mark Butler IV, and Robert Rose contributed valuable comments on the writing of this dissertation. Daniel Sonenshine graciously donated his time, advice, and laboratory equipment to me in the early stages of our HPLC work. Gordon Burghardt assisted in the design of initial feeding Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. vii experiments. I benefited from discussions on herpetology and experimental design with William Resetarits, Jr., Christopher Binckley, Barbara Savitzky, and Victor Townsend, Jr. I also thank Jackie Grant for supplying us with a transcutaneous amphibian stimulator and advising us on its use. John Daly and Thomas Spande kindly donated the telocinobufagin for our dilution curves. I am indebted to many individuals (mainly students) who helped me care for research animals and provided intellectual stimulation and emotional support. I extend special gratitude to the past and present students in the Herpetology Laboratory at Old Dominion University. Specifically, I thank Julie Ray, Angie and Gabe Rivera, John Allsteadt, Kathy Roberts, Chris Petersen, Scott Goetz, Kelsey Holzman, Shannon Davis, Dalyn Kuklock, Monica McGarrity, Gabrielle Mueller, Erica Schmutzler, and Kira White. I am also grateful to numerous additional individuals in Japan and the United States who assisted with the collection and husbandry of animals as well as the collection of samples and data entry. This list includes Trevor Adams, Jay Bolin, Jennifer Bradford, Gordon Burghardt, Matthew Close, Takashi Haramura, Masami Hasegawa, Azusa Hayano, Tatsuya Hishida, Isami Ikeuchi, Yohei Kadota, Akira Katayama, Noriko Kidera, Toshiro Kuroki, Alan Lemmon, Taku Mizuta, Koji Mochida, Yoshihisa Mori, Emily Moriarty Lemmon, Eiko Nagata, Aya Nakadai, Sumio Okada, Taku Okamoto, Hidetoshi Ota, Julie Ray, Barbara Savitzky, Kunio Sekiya, Tomohiko Shimada, Hirohiko Takeuchi, Koji Tanaka, Mamoru Toda, Victor Townsend Jr., and Ken Yoda. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. viii Due to the collaborative nature of this project, I used the pronoun “we” throughout my three main dissertation chapters. This was done to reflect the contributions of all authors of these chapters, which will be converted to manuscripts for publication. The authors of Chapter II are myself, Akira Mori, Alan Savitzky, Gordon Burghardt, Xiaogang Wu, Jerrold Meinwald, and Frank Schroeder. The authors of Chapter III are the same as those of Chapter II, with the exception of Xiaogang Wu. For Chapter IV, the authors include those of Chapter II, with the exception of Xiaogang Wu and Gordon Burghardt. Funding for this project was provided by the National Science Foundation (IBN-0429223 and IOB-0519458 to Alan Savitzky and Jerrold Meinwald); Old Dominion University (Dissertation Fellowship and Dominion Graduate Scholarship to Deborah Hutchinson); the Society for Integrative and Comparative Biology (Grant-in- Aid of Research to Deborah Hutchinson); Sigma Xi, The Scientific Research Society (Grant-in-Aid of Research to Deborah Hutchinson); Kyoto University (Grant for Biodiversity Research of the 21st Century COE A14 to Akira Mori and Dept, of Zoology); the Kyoto University Museum (Visiting Faculty Award to Alan Savitzky); the Japan-US Cooperative Science Program (Japan Society for the Promotion of Science to Akira Mori and NSF INT-9513100 to Gordon Burghardt); and the Fujiwara Natural History Foundation (10th Annual Grant for Scientific Research to Akira Mori). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 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