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University of Florida Thesis Or Dissertation Formatting THE ROLE OF ZINC AND COPPER ACCUMULATION IN QUEEN CONCH, Strombus gigas, REPRODUCTIVE DEFICIENCY AT NEARSHORE SITES IN THE FLORIDA KEYS By DANIEL JAMES SPADE 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 Daniel James Spade 2 To my parents, for your love and support 3 ACKNOWLEDGMENTS I thank my chair, Dr. Nancy Denslow, and supervisory committee, Dr. David Barber, Dr. Gustav Paulay, and Dr. Madan Oli, for their excellent mentorship and support. I thank current and former members of the Denslow and Barber laboratories for help and guidance. In particular, I thank Dr. Christopher Martyniuk and Dr. R. Joseph Griffitt for training and guidance in microarray processing and data analysis, and Dr. Martyniuk for training in mRNA cloning and real-time RT-PCR analysis. I thank Kevin Kroll for assistance in setting up and carrying out conch exposure experiments. I thank April Feswick for training in ICP-MS sample processing and analysis. Additionally, I thank everyone in the laboratory who helped care for conchs or process samples, or who discussed my project with me, improving my thought process, my methodological approach, and my understanding of the project. I thank collaborators who were essential to the work performed for this dissertation, particularly Robert Glazer and Gabriel Delgado at the Florida Fish and Wildlife Conservation Commission, for their expertise and guidance in working with queen conch, for generously providing conch tissue samples, and for allowing me to participate in the 2009 field sampling effort. I thank Megan Davis and Amber Shawl at the Harbor Branch Oceanographic Institute, for generously providing Florida fighting conchs and for guidance in setting up recirculating saltwater holding tanks and caring for conchs. I thank Nancy Brown-Peterson at the University of Southern Mississippi for guidance and training in interpretation of conch gonadal histology. I thank the staff of the UF Interdisciplinary Center for Biotechnology Research, particularly Dr. Li Liu, Dr. D. Gigi Ostrow, and Dr. Yanping Zhang, all of whom provided guidance and assistance in working with the queen conch microarray. I thank Irvy Quitmyer from the Florida 4 Museum of Natural History for guidance in developing methods and processing samples for conch shell metal analysis. I thank the University of Florida for awarding me the Alumni Fellowship, as well as sources of funding for the work presented here, including the UF College of Veterinary Medicine for a Consolidated Faculty Research Development Award, Mote Marine Laboratory (grant no. POR-2007-36), The United States Environmental Protection Agency (grant no. #X7974799-03), and the Florida Fish and Wildlife Conservation Commission (grants no. #F2410 and #NG06-106). I thank the UF Graduate Student Council, the Veterinary Graduate Student Association, the Department of Physiological Sciences, the Army Corps of Engineers, and the Society of Toxicology for providing travel funds that allowed me to attend scholarly meetings, present my research, and participate in a Pellston workshop on ecotoxicology in the 21st century. I thank my family: Alison for always listening, Nathan for always challenging me, Mom and Dad for putting us ahead of everything else. It was only because of their loving support, their guidance, and the example of their hard work and enjoyment of learning that I pursued this opportunity. Finally, I thank Michelle for her love and encouragement, for getting me through the toughest parts of this project, and above all for putting up with me while I wrote this dissertation. 5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES .......................................................................................................... 11 LIST OF FIGURES ........................................................................................................ 13 LIST OF OBJECTS ....................................................................................................... 15 LIST OF ABBREVIATIONS ........................................................................................... 16 CHAPTER 1 LITERATURE REVIEW .......................................................................................... 20 Background ............................................................................................................. 20 Significance of Queen Conch in the Florida Keys ................................................... 22 Economic Significance of Queen Conch in the Florida Keys ............................ 22 Ecological Significance of Queen Conch in the Florida Keys ........................... 22 Queen Conch Life History ....................................................................................... 23 Queen Conch Demographics and Reproductive Stressors in the Florida Keys ...... 27 Copper and Zinc Exposures Lead to Reduced Reproductive Success in Gastropods .......................................................................................................... 30 Copper and Zinc in South Florida ........................................................................... 37 Related Work with Queen Conch ............................................................................ 39 Queen Conch Genetics .................................................................................... 39 Production of an Oligonucleotide Microarray for Queen Conch ........................ 39 Metals in Queen Conch .................................................................................... 40 Overall Hypotheses ................................................................................................ 41 Significance of this Dissertation .............................................................................. 41 2 METHODS .............................................................................................................. 43 Strombus gigas Sample Collection ......................................................................... 43 Algae Sample Collection ......................................................................................... 45 In Vivo Exposures of Strombus alatus to Copper and Zinc ..................................... 45 Sources, Care, and Maintenance of Conchs .................................................... 45 Preliminary In Vivo Exposures .......................................................................... 47 Production of an Alternative Diet ...................................................................... 48 50-Day In Vivo Time-Course Exposure of Fighting Conchs to High Oral Doses of Copper and Zinc ............................................................................. 49 Source of Montastraea faveolata Tissue Samples .................................................. 52 Histological Analysis ............................................................................................... 52 Preparation and Purification of RNA from Tissue Samples ..................................... 54 Reagents and Solutions for RNA Extraction ..................................................... 54 6 RNA Preparation Method A: Preparation of RNA Samples Using Guanidinium/Phenol/Chloroform Extraction and Isopropanol Precipitation ... 54 RNA Preparation Method B: Preparation of RNA Samples Using a CsCl Gradient Centrifugation Step ......................................................................... 55 Further Processing and Purification of RNA ..................................................... 56 RNA Quantification and Quality Analysis.......................................................... 56 Microarray Experimental Processing ...................................................................... 57 Cloning of Strombus gigas and Strombus alatus Partial Transcripts ...................... 59 Real-Time RT-PCR ................................................................................................. 59 18S Real-Time RT-PCR to Validate Performance of RNA Samples Prepared by Method B .................................................................................. 63 Validation of 18S rRNA as an Internal Reference Gene for Real-Time RT- PCR in Testis Samples ................................................................................. 64 Collection of Samples for Conch Shell Metal Testing ............................................. 64 Validation Study ............................................................................................... 64 Historical Shell Metal Study .............................................................................. 65 Metal Analysis by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) ....... 65 Determination of Blood Total Protein ...................................................................... 67 Statistical Analyses ................................................................................................. 67 3 CESIUM CHLORIDE GRADIENT CENTRIFUGATION IMPROVES THE QUALITY OF TOTAL RNA PREPARATIONS FROM THE GASTROPOD Strombus gigas AND THE CORAL Montastraea faveolata ..................................... 75 Background ............................................................................................................. 75 Results ...................................................................................................................
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