THE HISTOLOGY, MICROBIOLOGY, AND MOLECULAR ECOLOGY OF TISSUE-LOSS DISEASES AFFECTING ACROPORA CERVICORNIS IN THE UPPER FLORIDA KEYS by Katheryn W. Patterson A Dissertation Submitted to the Graduate Faculty of George Mason University in Partial Fulfillment of The Requirements for the Degree of Doctor of Philosophy Environmental Science and Public Policy Committee: _________________________________________ Dr. Esther C. Peters, Dissertation Director _________________________________________ Dr. Cara Frankenfeld, Committee Member _________________________________________ Dr. Patrick Gillevet, Committee Member _________________________________________ Dr. Robert Jonas, Committee Member _________________________________________ Dr. E.C.M. Parsons, Committee Member _________________________________________ Dr. Albert Torzilli, Graduate Program Director _________________________________________ Dr. Robert Jonas, Department Chairperson _________________________________________ Dr. Donna M. Fox, Associate Dean, Office of Student Affairs & Special Programs, College of Science _________________________________________ Dr. Peggy Agouris, Dean, College of Science Date: __________________________________ Summer Semester 2015 George Mason University Fairfax, VA The Histology, MicrobiOlogy, and Molecular Ecology of Tissue-Loss Diseases Affecting Acropora cervicornis in the Upper Florida Keys A Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at George Mason University by Katheryn W. Patterson Master of Science George Mason University, 2010 Director: Esther C. Peters, Professor Department of Environmental Science and Public Policy Summer Semester 2015 George Mason University Fairfax, VA This work is licensed under a creative commons attribution-noderivs 3.0 unported license. ii DEDICATION This dissertation is in memory of and dedicated to two professors, mentors, and friends that helped inspire and challenge me to be the person I am today. Thank you for encouraging me to stay on the path to become a marine biologist, Dr. Fr. Gerald Regan, S.J., and thank you for everything you did to help me achieve my dream of going to graduate school, Dr. Shawn B. Allin. I will always hold you both close in my heart. iii ACKNOWLEDGEMENTS I would first like to thank my Mom, my brothers—Stephen and Brian—and all of my family members and friends for always supporting, encouraging, and believing in me throughout this entire experience. My dissertation would not have been possible without the expertise, guidance, support, and patience of my committee members: Drs. Peters, Frankenfeld, Gillevet, Jonas, and Parsons. I am forever grateful to all of my committee members for having confidence in me throughout this journey even when I lacked it at times. To my advisor, Dr. Peters, thank you for bringing me into your collaborative network and introducing me to coral disease research in the Florida Keys. I would also like to thank Dr. Peters for welcoming me into her histology lab, volunteering so much of her time to train me in what I now truly appreciate as the art of histology, for her astute reviews of this work, and for her never-ending encouragement. Special thanks are also owed to: Dr. Margaret Miller (NOAA/NMFS Southeast Fisheries Science Center), GMU’s MicroBiome Analysis Center (MBAC) and Ken Nedimyer (Coral Restoration Foundation), for their collaboration, logistical support, and in-kind donations. I also thank Dr. Masoumeh Sikaroodi for her endless help and support throughout all steps of my molecular work, from troubleshooting PCRs to all of the time, help, and patience she offered me with my analyses. I am forever grateful for all of the friendships and experiences I gained while conducting this research. Thank you to Dr. David Allen for your support in the field, and thank you to all of my lab mates at GMU’s Histology Lab and MBAC for their encouragement, advice, and pizza parties. I am forever thankful to Dr. Lorelei and Andrew Crerar for opening their home to me, and always supporting me through the many adversities life brought me during this process. I would like to give a special thanks to my dear friend Suzanne Hough for always having a vote of confidence in me, and for always finding the silver lining in situations when I needed it most. I am forever grateful to Bryan LeBlanc, Jason O’Bryhim, Lisa Schreffler, and Zach Siders for their unwavering support and encouragement and for always being there for me. I am very thankful for the Pham, Reinecke-Keller, and McAliley families, who welcomed me into their homes, entrusted me to watch their children, and contributed to my financial livelihood, which helped me keep my sanity. Finally, a heartfelt thank you goes to Sharon Bloomquist, Joanne Zimmerman, Roslyn Cress, and Sally Evans for their assistance and support throughout this process. This research would not have been possible without the financial support from the Cosmos Club Foundation, NOAA Coral Reef Conservation Program via the Aquarius Reef Base Program, and GMU’s Environmental Science and Policy Department. The study was conducted under Florida Keys National Marine Sanctuary Permit #FKNM-2011-032-A1. iv TABLE OF CONTENTS Page List of Tables ................................................................................................................... viii List of Figures ..................................................................................................................... x List of Abbreviations ....................................................................................................... xiii Abstract ............................................................................................................................. xv Preface................................................................................................................................. 1 The biology and ecology of Acropora cervicornis ......................................................... 1 Coral reef ecosystems...................................................................................................... 3 Coral diseases .................................................................................................................. 5 Tissue-loss diseases affecting Acropora cervicornis ...................................................... 8 An overview of coral disease research .......................................................................... 11 Goals and hypotheses ........................................................................................................ 14 Chapter 1: Histological differences were observed between apparently healthy and tissue- loss affected Acropora cervicornis ................................................................................... 20 Introduction ................................................................................................................... 20 Methods ......................................................................................................................... 24 Field site .................................................................................................................... 24 Sample collection 2011 ............................................................................................. 27 Sample collection 2012 ............................................................................................. 28 Histopathology........................................................................................................... 29 Results ........................................................................................................................... 31 Histopathological summary ....................................................................................... 31 Histopathological comparisons among sample types ................................................ 43 Histopathological comparisons between white-band disease and rapid tissue loss .. 50 Discussion ..................................................................................................................... 54 Chapter 2: Molecular approaches for characterizing microbial communities associated with apparently healthy and tissue-loss disease-affected acroporids ................................ 59 v Introduction ................................................................................................................... 59 Methods ......................................................................................................................... 63 Sample collection ...................................................................................................... 63 Genomic DNA extraction .......................................................................................... 64 Polymerase chain reaction ......................................................................................... 67 Length heterogeneity polymerase chain reaction ...................................................... 68 Next-generation sequencing ...................................................................................... 70 Multitag pyrosequencing and emulsion PCR ............................................................ 70 Ion Personal Genome Machine™ for next-generation sequencing ........................... 72 Chapter 3: Characterization and comparison of bacterial communities associated with apparently healthy and tissue-loss disease-affected Acropora cervicornis tissues