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University of Florida Thesis Or Dissertation Formatting Template PATHOGEN, HOST, AND ENVIRONMENTAL DYNAMICS: A CASE STUDY OF PANULIRUS ARGUS VIRUS 1 IN CARIBBEAN SPINY LOBSTERS By ABIGAIL S. CLARK 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 2017 © 2017 Abigail S. Clark To Jason, Mom, Dad, and Grandma ACKNOWLEDGMENTS This work would not have been successful without the support of several individuals and funding sources. I would first like to thank my supervisory chair, Donald Behringer, and my supervisory co-chair, Thomas Waltzek, without whom this work would not have possible. I am sincerely grateful to them for their leadership, intellect, and endless encouragement. The following personnel assisted with sample collection: Angelo Jason Spadaro, Mark Butler, Joshua Anderson, Jack Butler, Gaya Gnanalingam, Danielle Puls, Nathan Berkebile, Erica Ross, David Ousley, Evan Hill, and Alyssa Thompson. Additional help was provided by Tom Matthews, Casey Butler, and Gabby Renchen of Florida Fish and Wildlife Conservation Commission who supplied postlarval lobsters. Assistance with molecular work was provided by Linda Archer, Natalie Stilwell, Patrick Thompson, Jason Ferrante, Galaxia Cortés-Hinojosa, Gabriel Diaz, Anna Swigris, and Jaime Haggard. I would like to acknowledge the following faculty members, students, and support staff: Kuttichantran Subramaniam, Andrew Kane, Ross Brooks, Ruth Francis-Floyd, James Wellehan, James Colee, Debra Murie, Daryl Parkyn, Larry Tolbert, Justin Stilwell, Samantha Koda, Mameow Preeyanan, Kamonchai Imnoi, Elizabeth Scherbatskoy, Nelmarie Landrau Giovannetti, Rachel Henriquez, Jared Freitas, Pedro Henrique Viadanna, and Maria Jose Robles Malagamba. These individuals were instrumental in various aspects of my research, ranging from laboratory logistics to lobster dissections. Finally, I would like to recognize my committee members, Samantha Wisely, Jessica Moss Small, and Robert Swett for their unending support and mentorship. This project was funded by a Florida Sea Grant Scholars program grant (ASC), a University of Florida Opportunity Seed Fund grant (DCB, JFXW, and TBW), and National Science Foundation – Biological Oceanography grant OCE-0928398 (DCB). 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES ...........................................................................................................................7 LIST OF FIGURES .........................................................................................................................8 LIST OF ABBREVIATIONS ..........................................................................................................9 ABSTRACT ...................................................................................................................................10 CHAPTER 1 LITERATURE REVIEW .......................................................................................................12 Disease Ecology ......................................................................................................................12 Environmental Effects on Crustacean Health .........................................................................15 Crustacean Immune Response ................................................................................................16 Pathogens in Crustaceans .......................................................................................................17 2 VALIDATION OF A TAQMAN REAL-TIME QUANTITATIVE PCR ASSAY FOR THE DETECTION OF PANULIRUS ARGUS VIRUS 1 .....................................................20 Introduction .............................................................................................................................20 Materials and Methods ...........................................................................................................21 In silico TaqMan qPCR Primer and Probe Design ..........................................................21 Detection of PaV1 DNA Using the qPCR Assay ............................................................21 Estimation of the qPCR Assay Slope, Y-Intercept, Correlation Coefficient (R2), Efficiency, Dynamic Range, Analytical Sensitivity, Repeatability, Reproducibility, and Analytical Specificity .................................................................22 Estimation of the qPCR Assay Diagnostic Sensitivity and Specificity ...........................23 Results.....................................................................................................................................25 In silico TaqMan qPCR Primer and Probe Design ..........................................................25 Estimation of the qPCR Assay Slope, Y-Intercept, Correlation Coefficient (R2), Efficiency, Dynamic Range, Analytical Sensitivity, Repeatability, Reproducibility, and Analytical Specificity .................................................................25 Estimation of the qPCR Assay Diagnostic Sensitivity and Specificity ...........................26 Discussion ...............................................................................................................................26 3 VIABILITY OF PANULIRUS ARGUS VIRUS 1 IN SEAWATER .....................................39 Introduction .............................................................................................................................39 Materials and Methods ...........................................................................................................41 Lobster Screening ............................................................................................................41 Viral Purification and Quantitation .................................................................................42 5 Sampling and Experimental Setup ..................................................................................42 Results.....................................................................................................................................44 Viral Inocula and Concentrations ....................................................................................44 PaV1 Viability .................................................................................................................45 Discussion ...............................................................................................................................46 4 THE RELATIONSHIP BETWEEN VIRAL LOAD AND INFECTION IN CARIBBEAN SPINY LOBSTERS EXPOSED TO PANULIRUS ARGUS VIRUS 1 ..........55 Introduction .............................................................................................................................55 Materials and Methods ...........................................................................................................58 Lobster Collection and Screening ...................................................................................58 Inoculation .......................................................................................................................58 Sampling ..........................................................................................................................60 Data Analyses ..................................................................................................................60 Results.....................................................................................................................................60 Viral Load ........................................................................................................................60 Lobster Mortality .............................................................................................................61 Discussion ...............................................................................................................................62 5 SPONGES AND THE SPATIAL EPIDEMIOLOGY OF PANULIRUS ARGUS VIRUS 1 IN CARIBBEAN SPINY LOBSTERS THROUGHOUT THE FLORIDA KEYS, USA ........................................................................................................................................74 Introduction .............................................................................................................................74 Materials and Methods ...........................................................................................................77 Sample Collection ...........................................................................................................77 Data Analyses ..................................................................................................................78 Results.....................................................................................................................................79 PaV1 Prevalence ..............................................................................................................79 Community Characteristics .............................................................................................79 PaV1 Prevalence vs. Community Characteristics ...........................................................80 Discussion ...............................................................................................................................81 6 CONCLUSIONS ....................................................................................................................95
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