Western Michigan University ScholarWorks at WMU Dissertations Graduate College 4-2005 Bufadienolides In The Chemical Defenses Of The Toads, Bufo Americanus and Bufo Fowleri Catherine E. Merovich Western Michigan University Follow this and additional works at: https://scholarworks.wmich.edu/dissertations Part of the Animal Sciences Commons, Biochemistry, Biophysics, and Structural Biology Commons, and the Biology Commons Recommended Citation Merovich, Catherine E., "Bufadienolides In The Chemical Defenses Of The Toads, Bufo Americanus and Bufo Fowleri" (2005). Dissertations. 1049. https://scholarworks.wmich.edu/dissertations/1049 This Dissertation-Open Access is brought to you for free and open access by the Graduate College at ScholarWorks at WMU. It has been accepted for inclusion in Dissertations by an authorized administrator of ScholarWorks at WMU. For more information, please contact [email protected]. BUFADIENOLIDES IN THE CHEMICAL DEFENSES OF THE TOADS, BUFO AMERICANUS AND BUFO FOWLERI b y Catherine E. Merovich A Dissertation Submitted to the Faculty of The Graduate College in partial fulfillm ent of the requirements for the Degree of Doctor of Philosophy Department of Biological Sciences Western Michigan University Kalamazoo, Michigan A p r il 2005 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. BUFADIENOLIDES IN THE CHEMICAL DEFENSES OF THE TOADS, BUFO AMERICANUS AND BUFO FOWLERI Catherine E. Merovich, Ph.D. Western Michigan University, 2005 I investigated the steroidal chemical defenses (bufadienolides) of Bufo americanus and Bufo fowleri. By the nature of their complex, biphasic life cycles, toads, like other amphibians are im portant components of aquatic and terrestrial habitats and are prey to numerous invertebrates and vertebrates. Bufadienolides are presumed to be important anti-predatory compounds although much of their chemical ecology is poorly understood. I investigated (1) ontogenetic variation in bufadienolides, (2) bufadienolides from adult parotoid secretion, (3) effectiveness of bufadienolides against a terrestrial predator, and (4) effectiveness of bufadienolides against an aquatic predator. I hypothesized that B. americanus would have a more extensive bufadienolide profile and a more effective suite of chemical defenses than B. fowleri and th a t this could account for distributional differences in these toads. Results showed variability in numbers and concentrations of bufadienolides among toad developmental stages, but cumulatively no difference in total mean Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. concentrations. Bufadienolide concentrations across developmental stages appeared to fit Brodie and Formanowicz's (1987) model, but were more pronounced in eggs. Bufadienolides did not appear to be inducible following metamorphosis. More bufadienolides were detected in adult B. americanus b u t their mean total concentrations were not larger than in B. fowleri suggesting that distributional differences m ight be explained by variation in bufadienolide types rather than by bufadienolide concentrations. However, seven bufadienolides were statistically different between toad species. In B. americanus there was much overlap in bufadienolide types and concentrations from all collection sites. Repeated expressions of parotoid glands revealed highly variable individual responses among toads. In tongue-flick bioassays, terrestrial snake predators responded to chemical stim uli from both B. americanus an d B. fowleri w ith more tongue-flicks and greater tongue-flick attack scores than snakes exposed to distilled water. Because snakes showed elevated tongue-flick rates w ith parotoid secretions than w ith toad skin stim uli, parotoid chemicals may present a more concentrated toad stimulant and are not necessarily deterrents to predation. Also, aquatic Dytiscid beetle predators equally consumed both B. americanus an d B. fowleri suggesting no discrim ination between species or between toad developmental stages. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. NOTE TO USERS This reproduction is the best copy available. ® UMI Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 3164169 INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. ® UMI UMI Microform 3164169 Copyright 2005 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. © 2005 Catherine E. M erovich Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGMENTS West-Eberhard (2001) wrote that a truly taxon-centered biologist is a person who possesses several im portant attitudes that include (1) recognition of the organism as an individual, (2) proprietary recognition of the species or group, (3) respect for the organism as a living thing, (4) recognition of the organism as a beautiful and aesthetically pleasing entity, and (5) a taxon- centered biologist has a genuine feeling for the organism. For me, anuran amphibians are the reason I became a biologist. I have found myself immersed in an amphibian bias that urges me to learn all I can about my taxon. As West-Eberhard also wrote, a taxon-centered biologist is a person "who lets the organism suggest the im portant questions." In my dissertation, I have examined several im portant questions about toad chemical defenses and I certainly have many people to thank for helping me get to this point on my journey. I first wish to acknowledge and express my sincere appreciation to the Department of Biological Sciences and to the Graduate College at Western Michigan University for support as a Doctoral Associate and for the grants that I received in support of my research. Being a Doctoral Associate in the ii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Acknowledgments—continued Department of Biological Sciences at Western Michigan University also helped me to learn how to become a good teacher through experiences as a lab instructor as w ell as a lecturer. I especially thank Dr. Alexander Enyedi for giving me such wonderful teaching tools and for remaining a source of valuable advice. Many have encouraged me throughout my academic pursuits and helped to shape my development as a taxon-centered biologist—whether they realized it or not. Dr. Stephen B. Malcolm excited me about ecology while I was an undergraduate so much so that I came back to become his Ph.D. student. I owe him tremendous thanks for his advice, guidance, wisdom, and especially for his patience. In staying true to the attitudes of a taxon-centered biologist, w ith my desire to learn all about frogs, Steve always had a knack of reminding me about the nature of food webs and the place of amphibians in them—namely as prey. It's no wonder that I choose to focus my dissertation on toad anti-predator defenses. I am also sincerely grateful to the other members of my committee, Dr. David Cowan, Dr. Charles Ide, D r. D a v id K arowe, and Dr. Steven Kohler. I also received tremendous help w ith field collections and kindly thank all those involved, especially to Mike Franz, Steve Malcolm, George i i i Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Acknowledgments—continued Merovich, Guy Link, Heather Link, and a young, budding herpetologist, Miss Katherine Schwallier. Sandy Tavema also permitted access to her property rich in wetlands and encouraged me to study hard and "save the frogs." Tammi Roberts and Jim Fordyce assisted me w ith HPLC methods and I owe them many thanks. Also, Dr. Robert Eversole kindly used his contacts at MSU to prepare and stain my parotoid gland tissues. Kurtis Hunsberger was a tremendous help to me in this regard. Thanks also to Dr. Carlos Jared for his valuable comments on the parotoid gland histology of my toads and to Dr. Christy Foran and Dr. Sarah Farris for their comments on microscopy and for the use of their laboratories at West Virginia University. Dr. Steve Kohler and Julie Ryan helped w ith insect identification. I received wonderful support from many graduate students and friends and especially thank M arla Fisher, Julie Stahlhut, Eric Bushrow, Bobbie Heard, Daryl Arkwright-Keeler, and Celene and Chris Jackson. Finally, I thank my family for their continuous love and support, especially my mom, Suzanne Link, and my sister, Heather, who aided me in many ways — especially by proof reading my document over and over again. My dad, Guy Link, gave me