W&M ScholarWorks Dissertations, Theses, and Masters Projects Theses, Dissertations, & Master Projects 2007 Characterization of exotic pathogens associated with the suminoe oyster, Crassostrea ariakensis Jessica A. Moss College of William and Mary - Virginia Institute of Marine Science Follow this and additional works at: https://scholarworks.wm.edu/etd Part of the Fresh Water Studies Commons, Genetics Commons, and the Oceanography Commons Recommended Citation Moss, Jessica A., "Characterization of exotic pathogens associated with the suminoe oyster, Crassostrea ariakensis" (2007). Dissertations, Theses, and Masters Projects. Paper 1539616784. https://dx.doi.org/doi:10.25773/v5-9p8d-g426 This Dissertation is brought to you for free and open access by the Theses, Dissertations, & Master Projects at W&M ScholarWorks. It has been accepted for inclusion in Dissertations, Theses, and Masters Projects by an authorized administrator of W&M ScholarWorks. For more information, please contact [email protected]. CHARACTERIZATION OF EXOTIC PATHOGENS ASSOCIATED WITH THE SUMINOE OYSTER, CRASSOSTREA ARIAKENSIS A Dissertation Presented to The Faculty of the School of Marine Science The College of William and Mary In Partial Fulfillment Of the Requirements for the Degree of Doctor of Philosophy by Jessica A. Moss 2007 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. APPROVAL SHEET This dissertation is submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy <3 Jessica A. Moss Approved, August 2007 ___ Kimberly S. Reece, Ph.D. Advisor £ ^ 7 4 ^ Eugene M. Burreson, Ph.D. J/ltmmett Duffy, Ph.D./ / J £ Gs John Bl Graves, Ph.D. Kehnedj<r7pW n^ j Univeristy of^aryland College Park, Maryland li Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DEDICATION To my parents, whose love and support continue to guide me through all of my endeavors and to Hamish for his love, generosity, and laughter. iii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS Page ACKNOWLEDGEMENTS vi LIST OF TABLES vii LIST OF FIGURES ix ABSTRACT xi INTRODUCTION 2 CHAPTER 1. Survey ofCrassostrea ariakensis and other oyster 15 species in Asia for Perkinsus spp., oyster herpes-virus and other parasites: implications for non-native oyster introduction to Chesapeake Bay Abstract 15 Introduction 16 Materials and Methods 20 Results 26 Discussion 53 DISCLAIMER 59 CHAPTER 2. Description of Perkinsus beihaiensis n. sp. 60 Abstract 60 Introduction 61 Materials and Methods 63 Results 73 Discussion 99 CHAPTER 3. Perkinsus olseni experiments 109 iv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Abstract 109 Introduction 110 Experiment 1 Materials and Methods 112 Experiment 1 Results 119 Experiment 2 Materials and Methods 135 Experiment 2 Results 144 Experiment 3 Materials and Methods 158 Experiment 3 Results 161 Discussion 164 CHAPTER 4. Perkinsus beihaiensis experiments 179 Abstract 179 Introduction 180 Experiment 1 Materials and Methods 182 Experiment 1 Results 187 Experiment 2 Materials and Methods 190 Experiment 3 Results 195 Discussion 202 CONCLUSION 210 LITERATURE CITED 217 VITA 230 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGEMENTS I acknowledge and thank my advisor, Dr. Kimberly S. Reece, for her guidance, patience, and enthusiasm, and for allowing me to travel and gain experience outside of my specific project. I thank the members of my committee, including Dr. Eugene Burreson, Dr. Emmett Duffy, Dr. John Graves and Dr. Kennedy Paynter for their advice, expertise and critical appraisal of this research as it evolved. Dr. Burreson has additionally provided critical review of manuscripts, technical advice, and vocal support for my scientific career. Dr. Gwynne Brown and Alanna MacIntyre have provided many hours of laboratory help, patient technical assistance and advice. I also thank other laboratory members, Jie Xiao, Gail Scott, Bobbi Rawlings, Georgeta Constantin, Elizabeth Francis, Sharon Furiness, Dr. Elamish Small, Julie Stubbs and Dr. Jens Carlsson for assistance in wet lab maintenance. Bill Jones has assisted me with real time PCR and Dr. Jan Cordes and Jie Xiao have been instrumental with oyster species identification. Kelsey Everton enthusiastically helped with wet lab maintenance, experimental sampling, and data analysis during the many hours she spent at VIMS during her mentorship. Dr. Ryan Carnegie and Ms. Nancy Stokes provided technical advice and assistance. Dr. Corinne Audemard assisted in wet lab construction, she taught me real time PCR and offered advice on experimental design and analysis. I also acknowledge Rita Crockett and Susan Denny for teaching me histology techniques and assisting in oyster sample processing. I sincerely thank Mr. Christopher Dungan for assistance with Perkinsus spp. isolation and culture, for providingPerkinsus spp. media, for histological sample preparation, for technical advice, and for critical assistance with manuscript preparation. Rosalee Hamilton assisted with sample processing, histological analysis, Perksinsus spp. culture, and provided Perkinsus spp. culture supplies. I acknowledge Dr. Fu-Lin Chu and Dr. Eric Lund for use of laboratory equipment and for providing me with Perkinsus sp. culture material. Dr. Erin Bromage and Dr. Steve Kaattari allowed me to use their laboratory equipment, provided me with various reagents and taught me protein quantification. Dr. Pete VanVeld allowed me to use his laboratory equipment and Barb Rutan graciously helped me with SDS PAGE gel electrophoresis. I thank Dr. Jeffery Shields for use of laboratory equipment, and Kersten Wheeler for teaching me histology techniques. Dr. David Gauthier helped me with statistical analyses and in situ hybridization. John Vigliotta, Dr. Antonio Villalba, Junya Higano, and Dr. Aimin Wang provided shellfish for this work. vi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES Page CHAPTER 1. Table 1. Sample locations, sizes, bivalve species present, analysis 28 performed and general results Table 2. Prevalence data for Perkinsus spp. and molluscan 34 herpesvirus pathogens Table 3. Histopathological analysis of oysters from 2002 37 Table 4. GenBank accession numbers 43 Table 5. Pairwise distance analysis of Perkinsus spp. ITS rDNA 49 CHAPTER 2. Tablet. Sampling locations 66 Table 2. Pairwise distance analysis of Perkinsus spp. ITS rDNA 96 Table 3. Pairwise distance analysis of Perkinsus spp. LSU rDNA 97 Table 4. Pairwise distance analysis of Perkinsus spp. type 1 actin 98 Table 5. GenBank accession numbers 101 CHAPTER 3. Table 1. RFTM and PCR data for C. ariakensis inoculated with 122 P. olseni or SASW in inoculation experiment 1 Table 2. RFTM and PCR data for unnotched or uninoculated C. 123 ariakensis in inoculation experiment 1 Table 3. RFTM and PCR data for C. virginica in inoculation 124 experiment 1 Table 4. RFTM and PCR data for M. mercenaria in inoculation 126 vii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. experiment 1 Table 5. Mortality data for inoculation experiment 1 127 Table 6. PCR and RFTM data for inoculation experiment 2 151 Table 7. PCR and RFTM data for C. virginica mortality in 155 experiment 2 Table 8. PCR and RFTM data for M. mercenaria in the bath 163 challenge experiment CHAPTER 4. Table 1. PCR and RFTM data for cohabitation experiment 1 188 Table 2. Histological data for a subset of Chinese oysters from 192 cohabitation experiment 1 Table 3. PCR and RFTM data for cohabitation experiment 2 199 viii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES Page CHAPTER 1. Figure 1. Map of sampling locations 31 Figure 2. Parasites of oysters observed by histopathological analysis 38 Figure 3. H&E and ISH images of Perkinsus sp. in Crassostrea 40 hongkongensis Figure 4. Phylogenetic relationships among Perkinsus spp. derived 45 from ITS region neighbor joining analysis Figure 5. Phylogenetic relationships among Perkinsus spp. derived 47 from ITS region maximum parsimony analysis Figure 6. Sequence alignment of molluscan herpesvirus ‘A’ region 51 CHAPTER 2. Figure 1. Map of sampling locations 64 Figure 2. Hoffman modulation contrast image ofP. beihaiensis 75 n. sp. zoosporangia Figure 3. H&E stained tissue section of a C. ariakensis with a 78 P. beihaiensis n. sp. lesion Figure 4. ISH image of a C. ariakensis showing P. beihaiensis n. 78 sp. lesion Figure 5. H&E stained tissue section of a C. hongkongensis with a 78 P. beihaiensis n. sp. lesion Figure 6. ISH image of a C. hongkongensis showing P. beihaiensis n. 78 sp. lesion ix Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 7. H&E stained tissue section showing a P. beihaiensis n. sp. 80 trophozoite Figure 8. H&E stained tissue section showing aP. behaiensis n. sp. 80 schizont Figure 9. H&E stained tissue section showing aP. behaiensis n. sp. 82 trophozoite within the stomach lumen of a C. ariakensis Figure 10. H&E stained tissue section showingP. behaiensis n. sp. 82 trophozoites and schizonts near gonoducts of a C. hongkongensis Figure 11. A heavyP. beihaiensis n. sp. infection visualized by RFTM 84 Figure 12. ISH