SSStttooonnnyyy BBBrrrooooookkk UUUnnniiivvveeerrrsssiiitttyyy The official electronic file of this thesis or dissertation is maintained by the University Libraries on behalf of The Graduate School at Stony Brook University. ©©© AAAllllll RRRiiiggghhhtttsss RRReeessseeerrrvvveeeddd bbbyyy AAAuuuttthhhooorrr... Molecular characterization of clam (Mercenaria mercenaria) immune responses against Quahog Parasite Unknown (QPX): Effect of host and environmental factors A Dissertation Presented by Kailai Wang to The Graduate School in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Marine Sciences (Concentration – Marine biology) Stony Brook University May 2016 Stony Brook University The Graduate School Kailai Wang We, the dissertation committee for the above candidate for the Doctor of Philosophy degree, hereby recommend acceptance of this dissertation. Bassem Allam – Dissertation Advisor Professor, School of Marine and Atmospheric Sciences Anne McElroy - Chairperson of Defense Professor, School of Marine and Atmospheric Sciences Jackie L. Collier, Associate Professor School of Marine and Atmospheric Sciences Susan E. Ford, Professor Emeritus Department of Marine and Coastal Sciences Haskin Shellfish Research Laboratory, Rutgers University Arnaud Tanguy, Associate Professor Université Pierre et Marie Curie (Paris VI) Station Biologique de Roscoff, France This dissertation is accepted by the Graduate School Charles Taber Dean of the Graduate School ii Abstract of the Dissertation Molecular characterization of clam (Mercenaria mercenaria) immune responses against Quahog Parasite Unknown (QPX): Effect of host and environmental factors by Kailai Wang Doctor of Philosophy in Marine and Atmospheric Science (Concentration –Marine Biology) Stony Brook University 2016 Quahog Parasite Unknown (QPX) is a fatal protistan parasite that infects wild and cultured hard clams (Mercenaria mercenaria) along the northeastern coast of the US. The severity of clam mortality events and resulting economical losses caused by QPX emphasize the urgent need for the development of effective disease control measures, which require a good understanding of disease pathobiology and host resistance mechanisms. This dissertation explored clam immunity and host and environmental factors that affect M. mercenaria resistance against QPX infection. The investigation of molecular immune mechanisms using multiple high-throughput transcriptomic methods revealed an extraordinary complexity of M. mercenaria’s defense system, featured by highly diversified immune recognition receptors and pathways involved in immune cell activation, signal transduction, wound repair and apoptosis. Results also showed that QPX infection induces a tailored immune response in clams with focal up regulation of variable pattern recognition receptors (e.g. lectin, fibrinogen-related proteins, complement-1q-domain containing proteins) and signaling networks involved in the activation of focal adhesion, inflammation, apoptosis and extracellular killing, in contrast to a systemic response characterized by an overall immune suppression and up regulation of stress proteins. Comparative analysis between resistant and susceptible clam strains highlighted a transcriptome-wide deficiency in QPX-responsive immune factors in susceptible clams, suggesting a primary role of host genetic make-up in QPX resistance. Furthermore, results showed that temperature influences disease development mainly through the alteration of the expression of immune genes. Low temperatures resulted in the suppression of the clam immunome thus facilitating the iii establishment of QPX infection, while higher temperatures supported much higher levels of immune gene expression, which were correlated with host resistance. Finally, given the importance of temperature in regulating disease development, the study evaluated the role of heat shock treatments on the progress of pre-established infections. Results showed a reduction of QPX disease in clams submitted to temperature shocks. Best mitigation results were found in conditions with short exposures to moderately elevated (27ºC for 2 hours) temperatures. The heat shock treatment has great potential for field-application in hard clam fisheries. Overall, the findings of this dissertation represent a valuable addition to the currently limited molecular data on M. mercenaria, and contribute to a better understanding of the mechanisms of clam resistance to QPX. The results provide solid guidelines and preliminary data that facilitate future research targeting clam selective breeding and the development of disease mitigation strategies. iv Table of Contents List of Figures ............................................................................................................................. viii List of Tables ................................................................................................................................. x Acknowledgments ........................................................................................................................ xi Chapter 1 Introduction ................................................................................................................ 1 1.1. The hard clam ..................................................................................................................... 2 1.2. QPX disease in hard clams ................................................................................................ 2 1.3. Variability of hard clam susceptibility to QPX ............................................................... 4 1.4. Effects of environment factors on QPX disease ............................................................... 5 1.5. Host-pathogen interactions during QPX infection .......................................................... 7 1.6. Objectives of this study ...................................................................................................... 8 Chapter 2 High-throughput sequencing unravels the diversity of immune transcripts in the hard clam Mercenaria mercenaria ............................................................................................... 9 Abstract .................................................................................................................................... 10 2.1. Introduction ...................................................................................................................... 11 2.2. Materials and methods ..................................................................................................... 12 2.2.1. Mercenaria mercenaria ............................................................................................... 12 2.2.2. QPX and bacterial cultures .......................................................................................... 12 2.2.3. Immune stimulation and M. mercenaria RNA extraction ........................................... 13 2.2.4. Construction of normalized cDNA library and sequencing ........................................ 13 2.2.5. Assembly and functional annotation ........................................................................... 13 2.2.6. Identification of immune-related transcripts ............................................................... 14 2.2.7. Sequence diversity analysis of fibrinogen related proteins (FREPs) .......................... 14 2.3. Results and discussion ...................................................................................................... 15 2.3.1. Transcriptome analysis ................................................................................................ 15 2.3.2. Functional annotation .................................................................................................. 16 2.3.3. Immune-related transcripts in M. mercenaria ............................................................. 17 2.4. Conclusions ....................................................................................................................... 28 Chapter 3 Clam focal and systemic immune responses to QPX infection revealed by RNA- seq technology.............................................................................................................................. 40 Abstract .................................................................................................................................... 41 3.1. Background ....................................................................................................................... 42 3.2. Methods ............................................................................................................................. 43 3.2.1. Clam tissue and RNA samples preparation ................................................................. 43 3.2.2. RNA sequencing, de novo assembly and annotation ................................................... 44 3.2.3. Differential gene expression analysis .......................................................................... 44 v 3.3. Results and discussion ...................................................................................................... 45 3.3.1 Illumina sequencing and de novo transcriptome assembly .......................................... 45 3.3.2. Transcriptome functional annotation ........................................................................... 45 3.3.3. Identification of differentially expressed transcripts ..................................................