Bacterial Communities Associated with Healthy and Diseased
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Nova Southeastern University NSUWorks HCNSO Student Theses and Dissertations HCNSO Student Work 7-21-2017 Bacterial Communities Associated with Healthy and Diseased Acropora cervicornis (Staghorn Coral) Using High-Throughput Sequencing Charles Walton Nova Southeastern University, [email protected] Follow this and additional works at: https://nsuworks.nova.edu/occ_stuetd Part of the Animal Diseases Commons, Environmental Microbiology and Microbial Ecology Commons, Marine Biology Commons, Oceanography and Atmospheric Sciences and Meteorology Commons, and the Other Genetics and Genomics Commons Share Feedback About This Item NSUWorks Citation Charles Walton. 2017. Bacterial Communities Associated with Healthy and Diseased Acropora cervicornis (Staghorn Coral) Using High- Throughput Sequencing. Master's thesis. Nova Southeastern University. Retrieved from NSUWorks, . (449) https://nsuworks.nova.edu/occ_stuetd/449. This Thesis is brought to you by the HCNSO Student Work at NSUWorks. It has been accepted for inclusion in HCNSO Student Theses and Dissertations by an authorized administrator of NSUWorks. For more information, please contact [email protected]. HALMOS COLLEGE OF NATURAL SCIENCES AND OCEANOGRAPHY BACTERIAL COMMUNITIES ASSOCIATED WITH HEALTHY AND DISEASED ACROPORA CERVICORNIS (STAGHORN CORAL) USING HIGH-THROUGHPUT SEQUENCING BY CHARLES J. WALTON SUBMITTED TO THE FACULTY OF NOVA SOUTHEASTERN UNIVERSITY OCEANOGRAPHIC CENTER IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE WITH A SPECIALTY IN: MARINE BIOLOGY AND COASTAL ZONE MANAGEMENT NOVA SOUTHEASTERN UNIVERSITY JULY 21, 2017 Thesis of CHARLES J. WALTON Submitted in Partial Fulfillment of the Requirements for the Degree of Masters of Science: Marine Biology and Coastal Zone Management Nova Southeastern University Halmos College of Natural Sciences and Oceanography July 2017 Approved: Thesis Committee Major Professor :______________________________ David S. Gilliam, Ph.D. Committee Member :___________________________ Jose V. Lopez, Ph.D. Committee Member :___________________________ Aurelien Tartar, Ph.D. TABLE OF CONTENTS Acknowledgements ................................................................................................................. II Abstract ..................................................................................................................................... III List of Figures ............................................................................................................................ V List of Tables ........................................................................................................................... VII List of Appendix Figures .................................................................................................... VIII List of Appendix Tables ......................................................................................................... IX Introduction ............................................................................................................................... 1 Methods ....................................................................................................................................... 7 Sample Collection ............................................................................................................................... 7 DNA Extraction .................................................................................................................................... 8 16S rRNA Gene Amplification and Sequencing ........................................................................ 8 Quality Checking and Taxonomic Assignments ....................................................................... 9 Microbial Community Statistical Analyses ............................................................................. 12 Community Diversity (Alpha Diversity) ............................................................................................ 12 Community Composition (Beta Diversity) ....................................................................................... 13 Taxonomic Differential Abundance ..................................................................................................... 15 Results ........................................................................................................................................ 17 Microbial Community Diversity (Alpha diversity) .............................................................. 17 Microbial Community Composition (Beta diversity) .......................................................... 19 All Sample Types ......................................................................................................................................... 19 Tissue ............................................................................................................................................................... 25 Taxonomic Differential Abundance .......................................................................................... 31 Discussion ................................................................................................................................. 46 Conclusions .............................................................................................................................. 54 Citations ..................................................................................................................................... 56 Appendix: Figures .................................................................................................................. 78 Appendix: Tables .................................................................................................................... 85 I Acknowledgements Thank you to my advisor Dr. David S. Gilliam for the opportunity to work in the Coral Reef Restoration and Monitoring lab and for having the confidence in my abilities to take on a project beyond the scope of the laboratory. Additionally, thank you Dave for the all of the excellent field and travel opportunities that allowed me to experience great places and meet many wonderful people. Thank you to Dr. Jose V. Lopez for his guidance and allowing me to use his laboratory for my research. I also thank Dr. Aurelien Tarter for his assistance in the project development and excellent and insightful comments. Thank you to the Nova Southeastern University President’s Faculty Research and Development Grant for providing funding for this research. Thank you to the members of the CRRAM lab past and present for their assistance with the fieldwork. Thank you to members of the Microbiology and Genetics laboratory for their assistance during my lab work and accepting me into their lab. A special thank you to Drs. Andia Chaves-Fonnegra and Cole Easson for their long chats and guidance throughout my research and their excellent comments. A huge thank you to my wife Heather for her patience, understanding, and support throughout this experience. Finally, thank you to my family for their never ending support. II Abstract Coral diseases were first noted in the 1960s and 1970s and have had major impacts globally on coral reef community structures. In the Caribbean, a major outbreak of white band disease has been considered responsible for the drastic decline of Caribbean Acroporids since the 1970s. In addition to white band disease, another more recently described condition known as rapid tissue loss (RTL) has had major impacts on Acropora cervicornis populations, specifically offshore Broward County Southeast Florida. While these diseases have contributed to the population decline, determining their etiologies has been elusive. Coral diseases have been characterized by shifts in their microbial counterparts within many levels of the coral host. While some coral diseases have had specific pathogens identified, research has not been able to determine pathogens for most. Evidence points toward bacterial causes for many diseases, but due to the complexity of the coral holobiont and the interaction with the environment, elucidating the causes has proven difficult. Many studies have examined the microbiomes of specific diseases and determined some potential pathogens or at least taxa playing important roles in the disease, although none have looked at RTL. Recognizing the local affect of RTL on A. cervicornis, this study set out to gain a baseline understanding of the healthy and RTL affected microbiome of A. cervicornis. 16S rRNA gene sequencing was used to examine the microbiome of completely healthy colonies, healthy regions of diseased colonies, and the disease margin of diseased colonies. Analysis of four microbial diversity metrics revealed marked increases in diversity with respect to declining health states. Additionally, community dissimilarity analysis and analysis of differentially abundant taxa exhibited distinct microbial community structures due to coral health. Several highly abundant (Rickettsiales, Rhodobacteraceae) and a few low abundance (Bdellovibrionales) taxa were identified as primary drivers of the differences. Additionally, Piscirickettsiaceae, a known fish pathogen, was consistently associated with RTL and warrants further investigation. All of the taxa identified with in RTL have been associated with other Acroporid and non- Acroporid diseases throughout the Caribbean and the rest of the world. The consistent III association of similar taxa for coral