IMMUNOGENETIC VARIATION AND TUMOR INCIDENCE OF JUVENILE ENGLISH SOLE (PAROPHRYS VETULUS) ACROSS AN ESTUARINE GRADIENT OF CONTAMINANTS A Thesis submitted to the faculty of San Francisco State University In partial fulfillment of ■2. 0 1 8 - the requirements for p i e u the Degree Master of Science In Biology: Marine Biology by Calvin Yin Tat Lee San Francisco, California May 2018 Copyright by Calvin Yin Tat Lee 2018 CERTIFICATION OF APPROVAL I certify that I have read Immunogenetic variation and tumor incidence of juvenile English sole (Parophrys vetulus) across an estuarine gradient of contaminants by Calvin Yin Tat Lee, and that in my opinion this work meets the criteria for approving a thesis submitted in partial fulfillment of the requirement for the degree Master of Science in Biology: Marine Biology at San Francisco State University. C. Sarah Cohen, Ph.D. Professor, Biology Eric Routman, Ph.D. Professor, Biology Wim Kimmerer, Ph.D. Adjunct Professor, Biology IMMUNOGENETIC VARIATION AND TUMOR INCIDENCE OF JUVENILE ENLGISH SOLE (PAROPHRYS VETULUS) ACROSS AN ESTUARINE GRADIENT OF CONTAMINANTS Calvin Yin Tat Lee San Francisco, California 2018 Chemical contaminants in estuaries may interfere with an organism’s ability to ward against parasites and disease which can shape patterns of genetic diversity in populations, including at the Major Histocompatibility Complex (MHC) which helps recognize foreign pathogens. Here, we examine genetic variation in the MHC, in microsatellite repeats, and in mitochondrial control region (D-Loop) sequences to assess relationships between a tumor-causing pathogen known as X-cell disease, juvenile English sole (Parophrys vetulus), and contaminants in the San Francisco Estuary (SFE), CA. Functional molecular analysis was used to compare fish regionally and by infection status. Supertypes analysis showed no difference in supertype distributions regionally or by infection status, nor was there evidence of differentiation at microsatellite loci and D- loop. These results suggest contaminant distributions may be more fine-grained than patterns of English sole movement in SF Bay, possibly explaining the incidence of tumorous individuals throughout the bay. I certify that the Abstract is a correct representation of the content of this thesis. ACKNOWLEDGEMENTS I would like to thank Kathy Hieb, Max Fish, Jennifer Messineo and Kristine Lesyna (CA Dept of Fish and Wildlife) for help in collecting the fish. I also thank past and current members of the Cohen lab and committee members Wim Kimmerer and Eric Routman for their input. Shared molecular facilities were provided by NSF FSML 0435033 (CSC), SFSU EOS and COSE. Funds for this work came from Myers Ocean Trust (CL), SFSU COSE IRA (CL), and SFSU startup funds (CSC). TABLE OF CONTENTS List of Tables............................................................................................................................vi List of Figures......................................................................................................................... vii List of Appendices.................................................................................................................viii Introduction................................................................................................................................ 1 Methods...................................................................................................................................... 5 Sampling methods.........................................................................................................5 Molecular methods.......................................................................................................5 Data analysis methods...................................................................................................8 Results.......................................................................................................................................11 X-cell Pathogen Phylogenetic analysis......................................................................11 Mitochondrial Control Region (D-Loop) analysis....................................................12 Microsatellite analysis................................................................................................12 MHC analysis...............................................................................................................13 Selection.......................................................................................................................13 Supertypes....................................................................................................................14 Supertype frequency...................................................................................................14 Discussion................................................................................................................................ 16 X-cell pathogen in San Francisco Bay.......................................................................16 Population differentiation in San Francisco Estuary............................................... 16 English sole MHC IIB ................................................................................................18 MHC IIB Supertype Distribution...............................................................................19 Correlation Between Supertypes and Disease......................................................... 20 Conclusion References Appendices LIST OF TABLES Table Page 1. Region and infection status of fish samples..................................................... 38 2. Number of fish used in each part of study....................................................... 3 9 3. MHC IIB primer sets used.................................................................................39 4. Polymorphism statistics for D-Loop data......................................................... 40 5. D-loop Fst and Dest comparisons.......................................................................41 6. Genetic variation of microsatellite loci by region and infection status.........42 7. Microsatellite Fst and Dest comparisons........................................................... 43 8. Summary of MHC dN/dS analysis....................................................................44 9. Positively selected codon sites determined by analyses...................................45 10. Odds ratio and significance of PAST supertypes.............................................46 11. Odds ratio and significance of DAPC supertypes............................................47 LIST OF FIGURES Figures Page 1. Sampling stations...................................................................................................48 2. X-cell 18s Sequence Bayesian Tree....................................................................49 3. X-cell 18s Parsimony Tree...................................................................................50 4. Supertype cluster denedrogram............................................ 51 5. PAST clustering method regional supertype frequenecy................................. 52 6. PAST clustering method infection status supertype frequenecy......................52 7. PAST clustering method South Bay supertype frequency by infection status..53 8. PAST clustering method North Bay supertype frequency by infection status..53 9. DAPC clustering method regional supertype frequency....................................54 10. DAPC clustering method infection status supertype frequency..........................54 LIST OF APPENDICES Appendix Page 1. BIC value versus number of clusters........................................................ 55 2. A-score optimization..................................................................................................56 3. Assignment plot of alleles to supertype cluster ............................................57 4. Assignment plot of alleles to supertype cluster.................................................... 58 1 Introduction Estuaries are important nursery grounds for fish although they are often heavily altered by anthropogenic effects such as habitat loss and pollution (Nichols et al 1986, Boehlert and Mundy 1988, Lotze et al 2006). Juvenile fish are particularly vulnerable to contamination (Arkoosh et al 1998, Arkoohs et al 2001, Moles and Norcross 1998). Non- lethal contamination effects range from chronic physiological stress to adaptive evolutionary responses such as genetic and allelic differences (Moles and Norcross 1998, Meyer et al 2003, Guinand et al 2013, Nacci et al 1999, Nacci et al 2002). Pollution can work in tandem with selective forces such as parasites and disease to shape fish population variation (Arkoosh et al 1998, Arkoosh et al 2001, Cohen 2002, Cohen et al 2006, Nacci et al 2009). Longer-term impacts of pollution and disease may be evaluated at the population genetic level by choosing candidate genes that are broadly responsive to immune challenges and involved in individual fitness (Hoffmann and Willi 2008). Multiple studies show varied patterns of genetic diversity in candidate loci among populations of estuarine killifish in highly contaminated regions of the eastern US coast (Burnett et al 2007, Cohen et al 2006, Whitehead et al 2017). As one of the most genetically diverse gene complexes, the MHC mediates pathogen recognition in vertebrates and is influenced by evolutionary forces such as pathogens