WIDESPREAD USE OF RNAI-BASED PEST CONTROL STRATEGIES IS LIKELY IN THE NEAR FUTURE, YET WE ARE POORLY PREPARED TO ASSESS RISKS TO NON-TARGET ORGANISMS (NTOS). WE BRING TOGETHER EXPERTISE IN RNAI MECHANISMS, INSECT MOLECULAR GENETICS, AND INTEGRATED PEST MANAGEMENT TO TACKLE THIS PROBLEM. OUR GOAL IS TO DEVELOP AND STANDARDIZE A SET OF BIOASSAYS TO IDENTIFY SPECIES AT-RISK DUE TO DSRNA IN THE ENVIRONMENT, WHILE DETERMINING WHETHER BIOSYNTHESIS OF NEW SHORT RNAS WITHIN THE INSECT IS A COMPOUNDING RISK FACTOR, ESPECIALLY FOR TROPHIC EXPOSURE TO PREDATORS. OUR EXPERIMENTS WILL USE SPECIES FROM THREE INSECT ORDERS (DIPTERA, COLEOPTERA AND HEMIPTERA) THAT VARY IN THEIR SUSCEPTIBILITY TO RNAI. THIS WILL PROVIDE INFORMATION FOR GROWERS, RESEARCHERS AND REGULATORY AGENCIES ABOUT POTENTIAL UNINTENDED CONSEQUENCES OF THE USE OF RNAI ON INSECTS FROM DIFFERENT ORDERS.OUR SPECIFIC OBJECTIVES ARE TO: 1) ESTABLISH A SET OF GENES PRODUCING HIGHLY CHARACTERISTIC DEFECTS FOR SENSITIVE BIOASSAYS OF RNAI SUSCEPTIBILITY, INCLUDING LETHAL AND SUBLETHAL EFFECTS, TO IDENTIFY HAZARDS AND PRIORITIZE RISK ASSESSMENT; 2) DETERMINE POTENTIAL FOR BIOSYNTHESIS OF SECONDARY RNAS; AND 3) EVALUATE RISKS TO NTOS FROM TROPHIC EXPOSURE TO GENE SILENCING MOLECULES. THIS WORK WILL CONTRIBUTE TO OUR LONG-TERM GOAL OF IDENTIFYING MECHANISM-BASED PARAMETERS THAT EFFECTIVELY PREDICT SPECIES-LEVEL RNAI SUSCEPTIBILITY. WE FOCUS ON MECHANISMS BECAUSE THE WORK WILL BE APPLICABLE BROADLY TO GENE SILENCING INSECTICIDES RATHER THAN SPECIFIC SEQUENCES OR PRODUCTS. OUR APPROACH MAXIMIZES RISK ASSESSMENT OUTCOMES BY DEVELOPING BIOASSAYS AND GENERATING DATA TO BETTER UNDERSTAND THE LIKELIHOOD, SEVERITY, AND EXTENT OF HAZARD PRESENTED BY RNAI-BASED PEST MANAGEMENT TECHNOLOGIES. THEREFORE, OUR INFORMATION WILL FACILITATE SAFE AND ENVIRONMENTALLY-SOUND REGISTRATION AND USE OF RNAI-BASED PEST MANAGEMENT STRATEGIES TO PROTECT OUR FOOD SUPPLY AGAINST INSECT PESTS. TOGETHER, THESE OBJECTIVES WILL ADDRESS PRIORITY AREA 5A, RESEARCH FOCUSED ON THE ENVIRONMENTAL EFFECTS OF INTRODUCING RNAI TRANSGENES OR OTHER GENE SILENCING MECHANISMS.