DESCRIPTION OF THE CHEMICAL SENSES OF THE FLORIDA MANATEE, TRICHECHUS MANATUS LATIROSTRIS, IN RELATION TO REPRODUCTION By MEGHAN LEE BILLS 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 2011 1 © 2011 Meghan Lee Bills 2 To my best friend and future husband, Diego Barboza: your support, patience and humor throughout this process have meant the world to me 3 ACKNOWLEDGMENTS First I would like to thank my advisors; Dr. Iskande Larkin and Dr. Don Samuelson. You showed great confidence in me with this project and allowed me to explore an area outside of your expertise and for that I thank you. I also owe thanks to my committee members all of whom have provided valuable feedback and advice; Dr. Roger Reep, Dr. David Powell and Dr. Bruce Schulte. Thank you to Patricia Lewis for her histological expertise. The Marine Mammal Pathobiology Laboratory staff especially Drs. Martine deWit and Chris Torno for sample collection. Thank you to Dr. Lisa Farina who observed the anal glands for the first time during a manatee necropsy. Thank you to Astrid Grosch for translating Dr. Vosseler‟s article from German to English. Also, thanks go to Mike Sapper, Julie Sheldon, Kelly Evans, Kelly Cuthbert, Allison Gopaul, and Delphine Merle for help with various parts of the research. I also wish to thank Noelle Elliot for the chemical analysis. Thank you to the Aquatic Animal Health Program and specifically: Patrick Thompson and Drs. Ruth Francis-Floyd, Nicole Stacy, Mike Walsh, Brian Stacy, and Jim Wellehan for their advice throughout this process. To Dr. Bob Bonde, Cathy Beck and everyone at USGS, thank you not only for your help with my project but those of sirenian researchers everywhere. I also owe much gratitude to Sally O‟Connell who kept me on track with the paperwork and requirements of a PhD student and Dr. Charles Courtney who has provided support and advice for the achievement of my career goals. Thank you very much to the facilities that allowed me to work with their captive manatees. Specifically: Marilyn Margold at the South Florida Museum‟s Parker 4 Aquarium, Joe Gaspard of Mote Marine Laboratory, Dr. Andy Stamper and Kim O‟Dell of Disney‟s Epcot, The Living Seas. Finally, I would like to thank my Mom, Dad and brother for their encouragement and support not only during my PhD but for my entire college career. I would also like to thank Heather Maness, Maggie Hunter, Gretchen Henry, Shannon Skevakis, Noel Takeuchi and Jen McGee for their feedback, critiques and friendship. I also could not have been successful without the love and support of my fiancé, Diego Barboza. Funding was provided through the Florida Fish and Wildlife Conservation Commission, Whitney Marine Laboratory for Marine Bioscience and the University of Florida Aquatic Animal Health Program, the University of Florida College of Veterinary Medicine Consolidated Faculty Research Development Grant and Sigma Xi-Grant in Aid of Research. Research completed under US Fish and Wildlife permit #: MA067116-1 and UF IACUC Study #: 200902684. 5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 8 LIST OF FIGURES .......................................................................................................... 9 LIST OF ABBREVIATIONS ........................................................................................... 12 ABSTRACT ................................................................................................................... 15 CHAPTER 1 INTRODUCTION .................................................................................................... 17 Chemoreception ..................................................................................................... 17 Olfaction ........................................................................................................... 17 Vomeronasal Organ ......................................................................................... 20 Taste ................................................................................................................ 21 Chemical Communication ....................................................................................... 25 Pheromones ..................................................................................................... 26 Mammalian Reproductive Chemical Communication ....................................... 31 Paenungulata ................................................................................................... 33 Aquatic Mammals ................................................................................................... 37 Olfaction ........................................................................................................... 38 Vomeronasal Organ ......................................................................................... 42 Taste ................................................................................................................ 43 The Florida Manatee ............................................................................................... 47 2 ANAL GLANDS OF THE FLORIDA MANATEE, TRICHECHUS MANATUS LATIROSTRIS: A POTENTIAL SOURCE OF CHEMOSENSORY SIGNALS ......... 52 Background ............................................................................................................. 52 Materials and Methods............................................................................................ 54 Light Microscopy .............................................................................................. 55 Transmission Electron Microscopy ................................................................... 55 Results .................................................................................................................... 56 Light Microscopy .............................................................................................. 58 Electron Microscopy ......................................................................................... 61 Discussion .............................................................................................................. 61 3 TASTE BUDS IN THE ORAL CAVITY OF THE FLORIDA MANATEE, TRICHECHUS MANATUS LATIROSTRIS, WITH ESTROGEN RECEPTOR Β WITHIN THE LINGUAL ROOT TASTE BUDS ........................................................ 66 6 Background ............................................................................................................. 66 Materials and Methods............................................................................................ 70 Gross and Microanatomy ................................................................................. 71 Taste Bud Size and Quantification ................................................................... 72 Immunohistochemistry...................................................................................... 74 Transmission Electron Microscopy ................................................................... 76 Results .................................................................................................................... 76 Estrogen Receptor β ........................................................................................ 80 Ultrastructure .................................................................................................... 83 Discussion .............................................................................................................. 84 4 THE NASAL CAVITY AND OLFACTORY EPITHELIUM OF THE FLORIDA MANATEE, TRICHECHUS MANATUS LATIROSTRIS .......................................... 91 Background ............................................................................................................. 91 Materials and Methods............................................................................................ 94 Light Microscopy .............................................................................................. 96 Immunohistochemistry...................................................................................... 97 Results .................................................................................................................... 97 Light Microscopy ............................................................................................ 100 Immunohistochemistry.................................................................................... 103 Discussion ............................................................................................................ 104 5 MALE FLORIDA MANATEE, TRICHECHUS MANATUS LATRIROSTIS, BEHAVIORAL RESPONSE TO FEMALE MANATEE URINE FROM DIFFERENT REPRODUCTIVE TIMEPOINTS ..................................................... 109 Background ........................................................................................................... 109 Methods ................................................................................................................ 112 Results .................................................................................................................. 117 Discussion ...........................................................................................................