BENEFICIAL MICROBES PLAY A SIGNIFICANT ROLE IN THE NORMAL DEVELOPMENT OF ANIMAL TISSUES. THROUGH THE FORMATION OF HEALTHY STABLE ASSOCIATIONS BETWEEN HOST AND SYMBIONT MICROBES CAN INDUCE A WIDE RANGE OF IMMUNOLOGICAL PHYSIOLOGICAL AND DEVELOPMENTAL PATHWAYS. IN THIS EXPERIMENTAL RESEARCH PLAN WE PROPOSE TO EXAMINE THE IMPACT OF SPACEFLIGHT ON THE MOLECULAR INTERACTIONS BETWEEN HOST AND SYMBIONT THAT LEAD TO NORMAL BACTERIA-INDUCED ANIMAL DEVELOPMENT. TO ADDRESS THIS GOAL WE PROPOSE TO USE THE ESTABLISHED MODEL SYSTEM BETWEEN THE BOBTAIL SQUID EUPRYMNA SCOLOPES AND THE LUMINESCENT BACTERIUM VIBRIO FISCHERI. THE BACTERIUM COLONIZES A SPECIALIZED TISSUE REFERRED TO AS THE LIGHT ORGAN ENABLING THE HOST TO USE THE LUMINESCENCE IN AN ANTI-PREDATOR BEHAVIOR. FOR MORE THAN A QUARTER-CENTURY THIS SQUID-VIBRIO SYSTEM HAS SERVED AS AN IMPORTANT MODEL TO EXAMINE THE CELL-CELL INTERACTIONS BETWEEN MUTUALISTIC BACTERIA AND THEIR ANIMAL HOSTS. OUR PREVIOUS GROUND-BASED STUDIES HAVE IDENTIFIED SEVERAL BACTERIA-INDUCED DEVELOPMENTAL PHENOTYPES UNDER MODELED MICROGRAVITY CONDITIONS; HOWEVER THE MECHANISMS UNDERLYING THESE PHENOTYPES HAVE YET TO BE FULLY ELUCIDATED. OUR PREVIOUS RESULTS ALSO DETERMINED THAT THE PRESENCE OF THE MUTUALISTIC V. FISCHERI MODULATES THE HOST IMMUNE RESPONSE OF THE SQUID BY DOWN REGULATING SEVERAL STRESS AND IMMUNE GENES. HERE IN THIS STUDY WE PROPOSE TO REPLICATE AND EXPAND UPON THE PREVIOUS GROUND-BASED STUDIES BY PARTNERING WITH TECHSHOT AND USING THEIR FLUID PROCESSING CASSETTE HARDWARE FOR THE MANIPULATION OF BOTH THE ANIMAL AND SYMBIOTIC PARTNER IN SITU DURING SPACE FLIGHT. SPECIFICALLY WE PROPOSE THE FOLLOWING: OBJECTIVE 1. CHARACTERIZE THE CHANGES IN THE TRANSCRIPTOME OF THE HOST SQUID IN THE PRESENCE AND ABSENCE OF SYMBIOTIC BACTERIA EXPOSURE UNDER MICROGRAVITY CONDITIONS. WE PROPOSE TO GENERATE TRANSCRIPTOMES TO HOST E. SCOLOPES LIGHT ORGANS IN THE PRESENCE AND ABSENCE OF THE MUTUALISTIC V. FISCHERI AT SEVERAL KEY DEVELOPMENTAL TIME POINTS (0 2 6 12 H) POST-COLONIZATION. THE TRANSCRIPTOMES OF SYMBIOTIC AND APOSYMBIOTIC ANIMALS EXPOSED TO SPACEFLIGHT AT 0 AND 12 H WILL BE COMPARED TO PREVIOUSLY GENERATED TRANSCRIPTOMES (I.E. GROUNDBASED) TO ASCERTAIN WHETHER TRANSCRIPTIONAL DIFFERENCES ARE OBSERVED BETWEEN MODELED AND ACTUAL MICROGRAVITY CONDITIONS. OBJECTIVE 2. ASCERTAIN CHANGES IN THE METABOLOME OF HOST SQUID LIGHT ORGAN IN THE PRESENCE AND ABSENCE OF SYMBIOTIC BACTERIA UNDER SPACEFLIGHT CONDITIONS. USING THE SAME FLIGHT SAMPLES AS DESCRIBED IN OBJECTIVE 1 AN UNTARGETED METABOLOMICS APPROACH WILL BE USED TO REVEAL DIFFERENTIAL METABOLIC ACTIVITY IN SYMBIOTIC AND APOSYMBIOTIC LIGHT ORGANS. OBJECTIVE 3. BUILD INTEGRATIVE NETWORKS OF TRANSCRIPTOMES AND METABOLOMES UNDER DIFFERENT FLIGHT AND GROUND-BASED TREATMENTS TO ELUCIDATE CRUCIAL PATHWAYS THAT DIFFERENTIALLY RESPOND TO THE SPACE FLIGHT ENVIRONMENT. THE NETWORKS GENERATED FROM THE RESULTS OF OBJECTIVES 1 AND 2 WILL BE COMPARED TO GROUND CONTROLS TO ASCERTAIN HOW THE PRESENCE OF V. FISCHERI ALTERS THE HOST TRANSCRIPTIONAL AND METABOLIC RESPONSE TO SPACEFLIGHT CONDITIONS DURING EARLY DEVELOPMENT. SIGNIFICANCE OF PROJECT - THE PROPOSED WORK HAS SEVERAL IMPLICATIONS FOR THE FIELD OF SPACE BIOLOGY AND DIRECTLY RELATES TO NASA SPACE BIOLOGY SCIENCE PLAN AND DECADAL SURVEY SPECIFICALLY THE MICROBIOLOGY (MB) AND CELL MOLECULAR BIOLOGY (CMB) PROGRAM ELEMENT. FIRST BY EXAMINING HOW THE NORMAL BACTERIA-INDUCED DEVELOPMENT OF ANIMALS IS MEDIATED UNDER SPACEFLIGHT CONDITIONS WE CAN BEGIN TO DETERMINE THE SENSITIVITY OF THESE ASSOCIATIONS TO PERTURBATIONS IN THE SPACE ENVIRONMENT. ADDITIONALLY BY REMOVING GRAVITY AS A CONSTANT WE CAN ADDRESS THE QUESTION OF WHETHER GRAVITY OBSCURES ASPECTS OF BACTERIA-INDUCED ANIMAL DEVELOPMENT THAT MAY OTHERWISE GO UNDETECTED UNDER TERRESTRIAL CONDITIONS.