THE IMPACT OF INSECTS ON AGRICULTURE, BOTH POSITIVE AND NEGATIVE, CANNOT BE OVERSTATED. ANNUAL POSITIVE VALUE PROVIDED BY U.S. INSECT POPULATIONS IS ESTIMATED TO EXCEED $70 BILLION. NEGATIVE IMPACTS IN THE U.S. ALONE INCLUDE MILLIONS OF DOLLARS IN LOSSES DUE TO STORED GRAIN PESTS AND OVER $1.7 BILLION IN LIVESTOCK LOSSES DUE TO PEST FLIES. CROP LOSS AND ASSOCIATED EXPENSES FOR PEST CONTROL REACHES INTO TENS OF BILLIONS OF DOLLARS WORLDWIDE EACH YEAR.AS A RESULT, USDA HAS BEEN AN AGGRESSIVE SPONSOR OF RESEARCH INTO INSECT BIOLOGY AND METHODS OF INSECT CONTROL. THIS HAS INCLUDED SEQUENCING THE GENOMES OF INSECTS OF AGRICULTURAL RELEVANCE, WHICH HAS ALREADY STARTED TO PAY OFF IN TERMS OF INCREASED UNDERSTANDING OF BASIC INSECT BIOLOGY. OUR PROPOSAL BUILDS ON THESE SEQUENCING PROJECTS IN A DIRECT AND ESSENTIAL WAY BY FACILITATING THE DISCOVERY AND ANNOTATION OF GENE REGULATORY SEQUENCES (ENHANCERS), WHICH ARE CURRENTLY UNIDENTIFIED BUT WHICH COMPRISE A SIGNIFICANT PERCENTAGE OF THE GENOME. KNOWLEDGE OF THESE SEQUENCES IS EXPECTED TO HAVE VALUE AT LEAST EQUAL TO THAT OF KNOWING THE PROTEIN-CODING GENES. FOR EXAMPLE, REGULATORY SEQUENCES IN INSECTS AFFECT RESISTANCE TO INSECTICIDES; CHARACTERIZATION OF ENHANCER SEQUENCES CAN THEREFORE ALLOW FOR THE STUDY AND DIRECT MANIPULATION OF GENES IMPORTANT FOR INSECTICIDE RESISTANCE. ENHANCER SEQUENCES CAN ALSO PROVIDE TOOLS TO STUDY AND MANIPULATE GENE EXPRESSION IN TISSUES VITAL FOR TRANSMISSION OF PLANT PATHOGENS, , AND TO STUDY GENES INVOLVED IN PLANT PEST BEHAVIORS. REGULATORY SEQUENCES HAVE ALSO BEEN IMPLICATED IN INSECT MATING SUCCESS, AND ARE INSTRUMENTAL IN DEVELOPING METHODS TO IMPROVE THE EFFECTIVENESS AND ECONOMICS OF THE STERILE INSECT TECHNIQUE OF POPULATION CONTROL. ENHANCER DISCOVERY IS CRITICAL FOR THE ABILITY TO DEVELOP SAFE BUT EFFECTIVE GENE-DRIVE SYSTEMS TO REDUCE OR ELIMINATE CERTAIN LOCAL INSECT POPULATIONS, AS WELL AS FOR NUMEROUS OTHER BIOTECHNOLOGY APPLICATIONS. THE COMMON ELEMENT IN THESE POWERFUL STRATEGIES IS THAT THEY ALL REQUIRE PRECISE ENGINEERED GENE REGULATION USING TISSUE-SPECIFIC REGULATORY SEQUENCES. SUCH SEQUENCES HAVE SO FAR REQUIRED CONSIDERABLE TIME AND EFFORT TO IDENTIFY AND ISOLATE WITH THE RESULT THAT CURRENT OPTIONS FOR REGULATING TRANSGENE EXPRESSION IN INSECTS OTHER THAN THE FRUIT FLY DROSOPHILA ARE LIMITED TO ONLY A HANDFUL OF KNOWN ENHANCERS. ADDITIONAL RESOURCES ARE THUS CRITICALLY NEEDED.WE DEMONSTRATED PREVIOUSLY THAT OUR COMPUTATIONAL SCRMSHAW METHOD WORKS FOR ENHANCER DISCOVERY IN DIVERGED INSECT SPECIES. HERE, WE WILL UNDERTAKE EXTENSIVE PREDICTION OF ENHANCERS IN THE MAJORITY OF SEQUENCED INSECT SPECIES OF AGRICULTURAL IMPORTANCE, ALONG WITH IN VIVO VALIDATION EXPERIMENTS IN DROSOPHILA AND IN THE GRAIN PEST TRIBOLIUM CASTANEUM. THIS CONTRIBUTION IS HIGHLY SIGNIFICANT BECAUSE THERE IS CURRENTLY NO OTHER METHOD AVAILABLE FOR RAPID, EFFICIENT, AND COST-EFFECTIVE ENHANCER DISCOVERY IN SEQUENCED SPECIES. OUR ENHANCER DISCOVERY EFFORTS WILL LEAD TO NEW INSIGHTS ABOUT GENE REGULATORY NETWORKS CONTROLLING PROCESSES SUCH AS INSECT-HOST RECOGNITION, PESTICIDE RESISTANCE, DISEASE SUSCEPTIBILITY, REPRODUCTION, AND POLLINATOR BIOLOGY, AND THEY WILL ALLOW FOR IMMEDIATE ANNOTATION OF NUMEROUS REGULATORY GENOMES. IN ADDITION TO PROVIDING A DIRECT INCREASE IN OUR UNDERSTANDING OF INSECT BASIC BIOLOGY, THEY WILL PROVIDE POTENTIAL TOOLS FOR TRANSGENIC AND OTHER MANIPULATIONS THAT WILL SIGNIFICANTLY FACILITATE INSECT STUDY AND BIOCONTROL. OVER THE LONG TERM, WE ANTICIPATE THAT OUR RESULTS WILL HAVE A SIGNIFICANT IMPACT ON OUR ABILITY TO UNDERSTAND AND MANIPULATE THE BIOLOGY OF BOTH HARMFUL AND BENEFICIAL INSECTS TO IMPROVE STRATEGIES AND APPROACHES FOR INSECT MANAGEMENT AND CONTROL WITH CONCOMITANT IMPROVEMENTS IN AGRICULTURAL YIELD, SUSTAINABILITY, AND PROFITABILITY.