EVOLUTION of VENOM VARIATION in the FLORIDA COTTONMOUTH, Agkistrodon Piscivorus Conanti

EVOLUTION OF VENOM VARIATION IN THE FLORIDA COTTONMOUTH, Agkistrodon piscivorus conanti By RYAN JAMES ROBERT MCCLEARY A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009 1 © 2009 Ryan James Robert McCleary 2 To my parents, Karen Rogers, Dr. Ken McCleary, and Dr. Pamela Weaver, without whose support none of this would have been possible 3 ACKNOWLEDGMENTS I thank my parents and extended family, who supported me throughout this long endeavor. I also wish to thank all those intrepid individuals who forayed into the wild with me to search for venomous snakes, whether during hot, steamy Florida days, or pitch black, hot, insect-infested Florida nights. Special thanks go to Leslie Babonis for absolutely (almost) everything, Dr. Kelly Hyndman for unfailing encouragement and numerous reviews, and Kevin Neal for extreme field support and continued fascination with venom. I would further like to thank all the collaborators and mentors on the various projects that came out of this work, including my major professor, Dr. Harvey Lillywhite; committee members Drs. Martin Cohn, Marta Wayne, David Steadman, and Darryl Heard; all the staff (past and present) and the University of Florida College of Veterinary Medicine, including Dr. Rolando Quesada and Dahlonega Peck; A. M. “Ginger” Clark and everyone at the UF Interdisciplinary Center for Biotechnology Research (including Dr. D. “Gigi” Ostrow, Angela Gomez, Dr. Margaret Kellogg, and Dr. Kimberly Pause); Irvy Quitmyer and the UF Zooarcheology section of the Florida Museum of Natural History (FLMNH); Drs. Kenney Krysko and Max Nickerson of the Herpetology Department at FLMNH; Dr. Rob Robins of the FLMNH Ichthyology Department; Drs. David Julian, Kent Vliet, and Charlie Baer; my wonderful undergraduate students throughout the years, especially Vanessa Trujillo and Kristy Staudenmaier; Dr. Stephen Mackessy of the University of Northern Colorado; Dr. Nget- Hong Tan of the University of Malaya; various and diverse individuals with whom I have interacted at scientific meetings; and last, but definitely not least, all of the excellent people in the Departments of Zoology and Biology, including the staff and faculty, but especially graduate students, both past and present. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................. 4 LIST OF TABLES............................................................................................................ 8 LIST OF FIGURES.......................................................................................................... 9 ABSTRACT ................................................................................................................... 11 CHAPTER 1 INTRODUCTION .................................................................................................... 13 Venoms, Poisons, and Toxins ................................................................................ 13 Snake Venoms ....................................................................................................... 14 Venom Delivery Systems........................................................................................ 23 Variables That May Affect Snake Venom Evolution................................................ 28 Studies of Venom Variation—General .................................................................... 29 Studies of Venom Variation—Evolutionary Implications ......................................... 30 The Florida Cottonmouth—A Model for Study of Venom Evolution ........................ 33 2 THE VENOM OF THE COTTONMOUTH ............................................................... 36 Introduction ............................................................................................................. 36 Human Envenomations by Cottonmouths............................................................... 38 Demographics of Human Envenomation.......................................................... 38 General Physiological Effects (Human)............................................................ 39 Specific Physiological Effects (Comparative) ................................................... 40 Epithelial tissue .......................................................................................... 40 Subdermal tissue ....................................................................................... 40 Blood—hemolysis ...................................................................................... 40 Blood—coagulation.................................................................................... 42 Blood—anti-coagulation............................................................................. 43 Muscle ....................................................................................................... 44 Nervous tissue ........................................................................................... 45 Respiratory system .................................................................................... 46 Circulatory system ..................................................................................... 46 Immune system.......................................................................................... 48 Lymphatic system ...................................................................................... 49 Excretory system ....................................................................................... 49 Treatment of Cottonmouth Bite ........................................................................ 50 Medical treatment ...................................................................................... 50 Antivenom research ................................................................................... 50 Venom-Associated Anatomy................................................................................... 52 Venom Characteristics............................................................................................ 53 5 Basic Description.............................................................................................. 53 Volume....................................................................................................... 53 Specific gravity........................................................................................... 54 Protein content........................................................................................... 54 Glycoprotein content .................................................................................. 54 Dry mass.................................................................................................... 54 Spectrophotometry..................................................................................... 55 Stability ...................................................................................................... 55 Variation..................................................................................................... 56 Toxicity....................................................................................................... 56 Separation techniques ............................................................................... 58 Specific Components ....................................................................................... 61 Enzymes .................................................................................................... 61 Other enzymatic activities .......................................................................... 76 Nonenzymatic proteins/peptides ................................................................ 80 Small organic compounds.......................................................................... 82 Toxins ........................................................................................................ 83 Vascular endothelial growth factor ............................................................. 83 Potential Components................................................................................ 84 Venom Studies ....................................................................................................... 84 Human Disease Clinical Research ................................................................... 84 Immunology...................................................................................................... 86 Conclusion .............................................................................................................. 88 3 VENOM EXTRACTION FROM ANESTHETIZED FLORIDA COTTONMOUTHS... 93 Introduction ............................................................................................................. 93 Materials and Methods............................................................................................ 95 Animals............................................................................................................. 95 Anesthesia........................................................................................................ 96 Venom Extraction ............................................................................................. 97 Demographic Data ........................................................................................... 98 Results...................................................................................................................

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