MULTIPLE SPECIES OF FISH HAVE BEEN GENETICALLY ENGINEERED (GE) TO INCORPORATE ADVANTAGEOUS TRAITS FOR AQUACULTURE OR TO PRODUCE VALUABLE PHARMACEUTICAL PROTEINS. UNFORTUNATELY, WIDESPREAD ADOPTION OF THESE GE FISH IS HAMPERED BY CONCERNS OVER THEIR ACCIDENTAL RELEASE TO NATURAL ECOSYSTEMS. FAILPROOF STERILIZATION TECHNIQUES WOULD BE INVALUABLE FOR MINIMIZING THE IMPACT OF GE FISH LINES. STERILE FISH HAVE BEEN PRODUCED BY TRIPLOIDIZATION, TRANSGENIC APPROACHES, AND A VARIETY OF TRANSIENT GENE SILENCING METHODOLOGIES. HOWEVER, THESE APPROACHES OFTEN REMAIN TECHNICALLY DIFFICULT, COSTLY AND INSUFFICIENTLY ROBUST OR SCALABLE TO BE USED IN COMMERCIAL SETTINGS. TO DISCOVER NOVEL AND EFFECTIVE MEANS OF STERILIZING FISH, WE PROPOSE TO INVESTIGATE INDUCED MUTATIONS ACROSS A COMBINATION OF GENES NECESSARY FOR SPERMIOGENESIS AND STEROID HORMONE SYNTHESIS; RESULTING IN MALE STERILITY AND MASCULINIZATION RESPECTIVELY. MORE SPECIFICALLY, DOUBLE MUTANT COMBINATIONS OF GENES SHOULD PRODUCE ALL-MALE STERILE POPULATIONS. LIKEWISE, DOUBLE MUTANT COMBINATIONS OF GENES RESPONSIBLE FOR MALENESS AND FOLLICULOGENESIS SHOULD RESULT IN STERILE ALL-FEMALE POPULATIONS. THUS, OUR PROPOSED PROJECT WILL GENERATE AND CHARACTERIZE 14 DIFFERENT LINES OF TILAPIA CARRYING NULL MUTATIONS IN TWO GENETIC PATHWAYS SIMULTANEOUSLY, ONE GOVERNING SEX DIFFERENTIATION AND THE OTHER SEX/GAMETE COMPETENCY. WE WILL ALSO DEVELOP STRATEGIES TO GENERATE BROODSTOCK CAPABLE OF PROPAGATING THESE MUTANT LINES, THROUGH USE OF SEX-REVERSAL AND TRANSPLANTATION APPROACHES AIMED RESPECTIVELY AT RESCUING THE MISSING GENDER AND RESTORING GAMETE PRODUCTION. FINALLY, WE WILL TEST THE PERFORMANCE OF THESE LINES IN GROW-OUT TRIALS. IF SUCCESSFUL, THIS PROJECT WILL ENABLE SAFE AND EFFECTIVE UTILIZATION OF GE FISH IN A WIDE RANGE OF COMMERCIAL APPLICATIONS. THUS, OUR PROJECT ALIGNS WELL WITH USDA BRAG PROGRAM AREA #1: MANAGEMENT PRACTICES TO MINIMIZE ENVIRONMENTAL RISK OF GE ORGANISMS.