1.1 OBJECTIVES AND EXPECTED SIGNIFICANCE OF PROPOSED RESEARCH ONE OF THE MAIN QUESTIONS OF METAZOAN EVOLUTION IS HOW SUCH ORGANISMS EVOLVED IN A MICROBIAL DOMINATED WORLD(1). SINCE THE FIRST MICROBES EMERGED 3.5 BILLION YEARS AGO (BYA) THERE HAS BEEN A VAST ARRAY OF DIVERSITY DISPLAYED BY UNICELLULAR ORGANISMS IN BOTH BACTERIA AND ARCHAEAL DOMAINS(2). NONETHELESS THE FIRST EUKARYA AROSE ROUGHLY 1.8-1.4 BYA WITH THE FIRST ANIMALS EMERGING APPROXIMATELY 800-600 MYA (MILLION YEARS AGO)(3). THUS AS LONG AS ORGANISMS IN THE EUKARYA DOMAIN HAVE EXISTED THERE HAS ALWAYS BEEN THE PRESENCE OF BACTERIA IN OR AROUND THEIR HABITATS(4). THE EVENTUAL FORMATION OF A HOLOBIOME (HOST + BACTERIUM OR SYMBIONT ) TO ACCOMMODATE PHYSIOLOGICAL OR METABOLIC CAPABILITIES THAT CANNOT BE ACHIEVED BY EITHER PARTNER ALONE IS PARTICULARLY INTERESTING SINCE THESE ASSOCIATIONS CANNOT EXIST WITHOUT THE INTACT SYMBIOSIS(5). THE IMPACT THAT BENEFICIAL BACTERIA HAVE ON ENHANCING HOST FITNESS BY EITHER ALLOWING THE INFILTRATION INTO NOVEL HABITATS OR INITIATING PHYSIOLOGICAL OR METABOLIC CAPABILITIES THAT DID NOT EXIST IN THE HOST BEFORE THE PRESENCE OF THE SYMBIONT IS IMMENSE. THUS THE MAIN GOAL OF THIS PROPOSAL IS TO UNDERSTAND THE PROCESSES THAT LED TO THE EVOLUTION OF BENEFICIAL ANIMALBACTERIAL ASSOCIATIONS THROUGH THE WELL-STUDIED BOBTAIL SQUID-VIBRIO MUTUALISM. WE WILL DETERMINE WHETHER ABIOTIC FACTORS INFLUENCE HOST CHOICE COLONIZATION AND THE SURVIVAL OF ENVIRONMENTALLY TRANSMITTED VIBRIO BACTERIA IN SQUID HOSTS. ADDITIONALLY WE WILL EXAMINE THE MOLECULAR MECHANISMS THAT HAVE EVOLVED IN ANIMALS TO RECOGNIZE AND ESTABLISH AN ASSOCIATION WITH ONLY BENEFICIAL VIBRIOS. OUR THREE OBJECTIVES ADDRESS FUNDAMENTAL QUESTIONS OF HOW MULTICELLULAR EUKARYA (METAZOANS) EVOLVED IN A BACTERIAL LADEN EARTH: I) EXAMINE WHETHER BACTERIA EVOLVED UNDER DIFFERENT ABIOTIC CONDITIONS ARE CAPABLE OF COLONIZING NATIVE AND NOVEL SQUID HOSTS II) MEASURE HOST RESPONSE TO NATIVE NON-NATIVE AND EXPERIMENTALLY EVOLVED VIBRIO BACTERIA TO DETERMINE HOW THE HOST S IMMUNE SYSTEM RECOGNIZES AND DIFFERENTIATES CLOSELY RELATED SYMBIOTIC ADAPTED AND NON-SYMBIOTIC BACTERIA III) DETERMINE WHETHER SPECIFIC ABIOTIC VARIABLES AFFECT THE FITNESS OF THE SYMBIOSIS BY ASSESSING HOW SQUID HOST AND INTRODUCED VIBRIO PERSIST OVER TIME UNDER CHANGING CONDITIONS. ONE OF THE MOST SIGNIFICANT DRIVERS(6) IN THE EVOLUTION OF ANIMAL LIFE ON EARTH IS THE INFLUENCE OF BACTERIA ON THEIR BEHAVIOR PHYSIOLOGY DEVELOPMENT IMMUNITY AND CELLULAR PROCESSES. CELL ADHESION IN SPONGES AND CHOANOFLAGELLATES NUTRITION AND GUT DEVELOPMENT IN MAMMALS METABOLIC CAPABILITIES IN TUBEWORMS AT HYDROTHERMAL VENTS AND PROTECTION AGAINST PATHOGENS THROUGH METABOLITES OR OTHER COMPOUNDS IN INVERTEBRATE EGGS DURING DEVELOPMENT ARE ALL ACCOMPLISHED THROUGH THE PRESENCE OF BACTERIA(1 7 8). WE PROPOSE TO EXAMINE WHETHER ENVIRONMENTALLY TRANSMITTED VIBRIOS ARE AFFECTED BY VARIOUS ABIOTIC FACTORS AND WHETHER THESE FACTORS IMPACT THE LIKELIHOOD OF A SUCCESSFUL SYMBIOTIC ASSOCIATION IN A MULTICELLULAR EUKARYOTE. LIKEWISE WE WILL ALSO EXAMINE HOST RESPONSE TO VIBRIO THAT ARE EVOLVED UNDER DIFFERENT ENVIRONMENTAL REGIMES TO DETERMINE HOW THE HOST IMMUNE SYSTEM ACCOMMODATES CHANGES IN THESE EVOLVED BACTERIA. SINCE METAZOANS DEPEND ON BACTERIA OR THEIR PRODUCTS TO INITIATE SPECIFIC DEVELOPMENTAL PATHWAYS OR SHAPE THE MATURATION OF IMMUNE DIGESTIVE NERVOUS AND CIRCULATORY SYSTEMS INSIGHT LEADING TO THE EFFECTS THAT ENVIRONMENTAL CHANGE HAS ON BACTERIAL SYMBIONTS AND HOST RESPONSE CAN SHED LIGHT INTO THE GENETIC FACTORS AND ASSOCIATED MECHANISMS IMPORTANT FOR DEVELOPMENTAL PLASTICITY AND THE HOLOBIONT(9) AS A UNIT OF EVOLUTION(10-12).