THE EASTERN OYSTER, CRASSOSTREA VIRGINICA IS A FILTER-FEEDING MOLLUSK WITH AN ECONOMIC VALUE OF MORE THAN $175 MILLION USD IN 2016, AND THE INDUSTRY IS GROWING RAPIDLY. SEVERAL PATHOGENIC DISEASES HAVE EMERGED THAT CAN CAUSE OUTBREAKS IN FARMS AND HATCHERIES WITH UP TO 90% MORTALITY AND HAVE THE POTENTIAL TO DEVASTATE THE AQUACULTURE INDUSTRY. DERMO DISEASE, CAUSED BY THE PROTOZOAN PARASITE PERKINSUS MARINUS, FIRST EMERGED IN THE GULF OF MEXICO IN THE 1940'S AND IS THE PRIMARY CAUSE OF MORTALITY OF CULTURED OYSTERS ON THE EAST COAST OF THE U.S. DISEASE CAUSED BY SEVERAL VIBRIO SPP. ARE THE CAUSE OF SEVERE LOSSES IN BIVALVES HATCHERIES. TRADITIONAL SELECTIVE BREEDING FOR DISEASE RESISTANCE HAS DEVELOPED SEVERAL LINES OF EASTERN OYSTERS WITH HERITABLE RESISTANCE, SUBSTANTIALLY REDUCING LOSS FROM DISEASE. HOWEVER, RESISTANCE TO ONE DISEASE DOES NOT NECESSARILY CONFER RESISTANCE TO OTHERS, AND THE PRECISE INTERACTIONS BETWEEN PATHOGENS AND EASTERN OYSTER IMMUNITY ARE NOT FULLY UNDERSTOOD. TO THIS END, THIS RESEARCH IS FOCUSED ON C. VIRGINICA INNATE IMMUNITY TO PATHOGENS, WITH THE LONG TERM GOAL OF DISEASE MITIGATION VIA IMPROVEMENT OF SELECTIVE BREEDING. SPECIFICALLY, THIS RESEARCH WILL DETERMINE THE POTENTIAL FOR AN ADAPTIVE INNATE IMMUNE RESPONSE TO DISEASE THROUGH IMMUNE GENE DIVERSIFICATION, WITH A FOCUS ON GENE FAMILIES INVOLVED IN THE PROCESS OF APOPTOSIS (PROGRAMMED CELL DEATH). TRANSCRIPTOMIC AND GENOMIC STUDIES OF THE EASTERN OYSTER AND THE PACIFIC OYSTER, C. GIGAS, REVEAL LARGE DUPLICATED AND DIVERSIFIED FAMILIES OF RECEPTORS, REGULATORS, AND EFFECTOR MOLECULES INVOLVED IN IMMUNITY. EXPOSURE OF OYSTERS TO DIVERSE BIOTIC AND ABIOTIC STRESSORS IDENTIFIES HIGHLY SPECIFIC AND ORCHESTRATED RESPONSES OF MULTIPLE IMMUNE GENE FAMILIES, AND VARIABLE EXPRESSION OF INDIVIDUAL MEMBERS TO PARTICULAR STRESSORS. GENE FAMILIES ARE COMPOSED OF SEVERAL GENES GENERALLY WITH SIMILAR FUNCTIONS, AND THEY CAN EXPAND IN GENE NUMBER, OFTEN THROUGH DUPLICATION. OVERTIME, FUNCTIONALLY REDUNDANT DUPLICATED GENE COPIES CAN MUTATE AND ACQUIRE NEW FUNCTIONS OR SIMILAR, SPECIALIZED FUNCTIONS. IN IMMUNITY, THIS ALLOWS FOR FUNCTIONAL DIVERSIFICATION OF GENE FAMILIES, WHERE DIFFERENT MEMBERS PLAY SPECIFIC AND DIVERSE ROLES. EXPANSION OF IMMUNE GENE FAMILIES IN OYSTERS, COMBINED WITH SPECIFIC TRANSCRIPTIONAL RESPONSES TO STRESSORS, SUGGEST THAT THESE FAMILIES MAY HAVE UNDERGONE FUNCTIONAL DIVERSIFICATION ALLOWING FOR SPECIFIC INNATE IMMUNE RESPONSES TO PARTICULAR STRESSORS. THE APOPTOTIC PROCESS INVOLVES TWO CRUCIAL FAMILIES OF REGULATORY MOLECULES THAT BOTH HAVE EXPANDED GENOMIC COPY NUMBER AND DIFFERENTIAL TRANSCRIPTIONAL RESPONSES TO DISEASE, CALLED THE GTPASE OF THE IMMUNE ASSOCIATED PROTEINS (GIMAPS) AND THE INHIBITOR OF APOPTOSIS PROTEINS (IAPS). APOPTOSIS, ALSO CALLED PROGRAMMED CELL DEATH, IS A CRITICAL COMPONENT OF INNATE IMMUNITY, INVOLVED IN REGULATION OF IMMUNE CELL PROLIFERATION AND DEVELOPMENT, AS WELL AS IN THE REMOVAL OF CELLS INFECTED BY INTRACELLULAR PATHOGENS. SPECIALIZATION OF THE APOPTOTIC RESPONSE MAY BE VITAL TO SUCCESSFULLY COMBATING DIFFERENT DISEASES. GIMAPS ARE VITAL IMMUNE RESPONSE REGULATORS AND APOPTOSIS INHIBITORS, WHILE IAP GENES ARE CONSERVED SUPPRESSORS OF APOPTOSIS. GIMAP AND IAP GENES SHOW DIVERSE TRANSCRIPTIONAL ACTIVITY ACROSS PARASITE/PATHOGEN CHALLENGES, LEADING TO THE HYPOTHESIS THAT GENOMIC DIVERSITY OF THESE FAMILIES MAY ALLOW FOR FUNCTIONAL SPECIALIZATION OF APOPTOTIC RESPONSES. THE PROPOSED RESEARCH TESTS THE HYPOTHESIS THAT OYSTERS TAILOR APOPTOTIC RESPONSES TO PARASITES AND PATHOGENS WITH DIFFERENT MODES OF INFECTION THROUGH MODULATION OF EXPRESSION OF GENES IN EXPANDED GENE FAMILIES TO INCREASE SURVIVAL. SPECIFICALLY, THIS PROJECT AIMS TO: 1) EVALUATE THE ROLE OF HEMOCYTE APOPTOSIS IN OYSTER IMMUNE RESPONSE TO PARASITES/PATHOGENS WITH DIFFERING MODES OF INFECTION, AND 2) INVESTIGATE THE POTENTIAL ROLE OF EXPANDED APOPTOSIS GENE FAMILY MEMBERS, PARTICULARLY GIMAPS AND IAPS, ON HEMOCYTE APOPTOSIS FOLLOWING CHALLENGE. TO ADDRESS THESE AIMS, JUVENILE OYSTERS WILL BE EXPOSED TO THE INTRACELLULAR PARASITE PERKINSUS MARINUS, AND THE EXTRACELLULAR BACTERIA V. CORALLIILYTICUS RE22. DERMO IS A FACULTATIVE INTRACELLULAR PARASITE THAT INVADES HEMOCYTES AND SURVIVES INSIDE BY NEUTRALIZING RADICAL OXYGEN SPECIES, AND MOST PROBABLY INHIBITING APOPTOSIS. V. CORALLIILYTICUS IS AN OPPORTUNISTIC PATHOGEN INTERACTING EXTRACELLULARLY WITH HEMOCYTES, CAUSING DISEASE IN THE MORE SUSCEPTIBLE LARVAL LIFE STAGES AND DURING STRESS IN ADULTS. FOLLOWING CHALLENGE, DISEASE PHENOTYPE WILL BE ASSESSED AS OYSTER SURVIVAL AND PARASITE/PATHOGEN LOAD, APOPTOSIS GENE EXPRESSION BY TRANSCRIPTOME ANALYSIS, AND FUNCTIONAL APOPTOSIS PHENOTYPE BY FLOW CYTOMETRY. ANALYSIS OF RELATIONSHIPS BETWEEN APOPTOSIS PHENOTYPE, GENE EXPRESSION, AND DISEASE WILL BE EVALUATED IN ORDER TO IDENTIFY SPECIFIC GENES INVOLVED IN FUNCTIONAL RESPONSES TO THE PATHOGENS, AND THEIR POTENTIAL ROLE IN DISEASE RESISTANCE/SUSCEPTIBILITY. THE PROPOSEDPREDOCTORAL FELLOWSHIP RESEARCH DIRECTLY SUPPORTS THE ANIMAL HEALTH AND PRODUCTION AND ANIMAL PRODUCTS AFRI FARM BILL PRIORITY BY INCREASING KNOWLEDGE OF HOW OYSTERS RESPOND TO DISEASES THAT NEGATIVELY IMPACT AQUACULTURE. IDENTIFYING SPECIFIC IMMUNE MECHANISMS TO DIFFERENT DISEASES ENABLES THE FUTURE DEVELOPMENT OF PUTATIVE MARKERS FOR DISEASE RESISTANCE THAT CAN BE USED TO SCREEN OYSTER FAMILIES FOR SELECTIVE BREEDING, THUS ENABLING MORE TARGETED, GENETIC IMPROVEMENT TO DECREASE AQUACULTURE LOSSES TO DISEASE. CONTINUED AQUACULTURE INDUSTRY GROWTH IMPROVES U.S. FOOD SECURITY AND ECONOMIC PROSPERITY. THE PROPOSAL SUPPORTS AFRI EWD GOALS BY PROVIDING ROBERTS WITH PROFESSIONAL SKILLS TO DESIGN INNOVATIVE RESEARCH IN HER CAREER STUDYING THE COMPLEXITY OF INNATE IMMUNITY TO DISEASE IN EASTERN OYSTERS AND HOW GENETICS AND BREEDING AND CAN UTILIZE THIS INFORMATION TO IMPROVE THE AQUACULTURE INDUSTRY.