** AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** THE SPOTTED WING DROSOPHILA, DROSOPHILA SUZUKII, IS A WORLDWIDE CROP PEST OF SOFT-SKINNED FRUITS. CURRENT METHODS TO CONTROL MANY AGRICULTURAL PESTS, SUCH AS D. SUZUKII, RELY ON EXPENSIVE, BROAD-SPECTRUM INSECTICIDES, WHICH HAVE VARIABLE EFFICACY, ARE CHALLENGING TO USE DUE TO THE TIMING OF FRUIT INFESTATION AND INCREASE THE RISK D. SUZUKII EVOLVING RESISTANCE TO THE INSECTICIDE. HOWEVER, THERE ARE NO PRACTICAL ALTERNATIVES TO MANAGING D. SUZUKII INFESTATION, AND IT IS LIKELY THAT, UNLESS MORE EFFECTIVE CONTROL MEASURES ARE DEVELOPED, THIS PEST WILL CONTINUE TO SPREAD AND NEGATIVELY IMPACT FRUIT PRODUCTION.GENETIC TECHNOLOGIES CAN COMPLEMENT EXISTING PEST MANAGEMENT STRATEGIES FOR D. SUZKUII AND OTHER AGRICULTURAL PESTS. THIS PROPOSAL AIMS TO GENERATE TOOLS TO MONITOR, IMPROVE, AND BETTER UNDERSTAND THE BEHAVIOR OF A NEW GENETIC TECHNOLOGY, PRECISION-GUIDED STERILE INSECT TECHNIQUE (PGSIT), AND OTHER CRISPR-BASED TECHNOLOGIES IN THE FIELD. THE FIRST TOOL, SENSITIVE ENZYMATIC NUCLEIC ACID SEQUENCE REPORTER (SENSR), IS A DIAGNOSTIC SYSTEM DESIGNED TO RAPIDLY DETECT PGSIT-SPECIFIC AND CRISPR-ASSOCIATED DNA. RAPID IDENTIFICATION OF THE DNA OF THESE TECHNOLOGIES IS VITAL FOR ASSESSING THE RISK AND PERFORMANCE OF GENETIC BIOCONTROL TOOLS. THEY CAN BE USED AS A RAPID TOOL TO MONITOR THE BEHAVIOR OF GENETICALLY ENGINEERED (GE) TECHNOLOGIES IN THE FIELD AND THEIR POTENTIAL SPREAD INTO NEARBY AREAS.ANOTHER TECHNOLOGY WE AIM TO DEVELOP IN D. SUZUKII IS SEPARATOR (SEXING ELEMENT PRODUCED BY ALTERNATIVE RNA-SPLICING OF A TRANSGENIC OBSERVABLE REPORTER). SEPARATOR CAN IMPROVE THE QUALITY AND ACCURACY OF FIELD RELEASES OF D. SUZUKII. FOR EXAMPLE, ONLY D. SUZUKII FEMALES CAUSE DAMAGE TO FRUIT, SO WE WANT TO REDUCE CROP DAMAGE WE ONLY WANT TO RELEASE MALES IN GENETIC CONTROL PROGRAMS. ADDITIONALLY, IF THERE ARE FREQUENT ERRORS IN THE SEX-SORTING OF PGSIT LINES PRIOR TO MATING IN THE LABORATORY, THEN THERE IS A HIGHER RISK OF RELEASING FERTILE FLIES. WE WILL, THEREFORE, ENGINEER AND OPTIMIZE A SEPARATOR SYSTEM IN D. SUZUKII THAT SHOULD MINIMIZE OR ELIMINATE THE RISK OF MATING ERRORS THAT MIGHT RESULT IN THE RELEASE OF FEMALES THAT DAMAGE CROPS OR FERTILE INDIVIDUALS.WE WILL ALSO EVALUATE THE BEHAVIOR OF PGSIT IN RESPONSE TO CHANGES IN ENVIRONMENTAL CONDITIONS AND WHETHER PGSIT COULD HELP MITIGATE THE GENE FLOW OR SPREAD OF GENE TECHNOLOGIES. THESE STUDIES WILL ENSURE WE UNDERSTAND HOW PGSIT WILL PERFORM IN A CHANGING ENVIRONMENT AND HOW IT CAN BE USED TO REMOVE OTHER GE TECHNOLOGIES FROM THE FIELD. THESE STUDIES WILL BE PERFORMED IN THE LABORATORY AND MODELED TO UNDERSTAND HOW THEY WILL BEHAVE ON A LARGER SCALE.STUDIES OF THESE EMERGING TECHNOLOGIES ARE ESSENTIAL FOR THE RESPONSIBLE DEVELOPMENT AND MANAGEMENT OF GENETICALLY ENGINEERED (GE) TECHNOLOGIES FOR AGRICULTURAL PEST CONTROL. THIS WORK WILL FOCUS ON A MORE COMPREHENSIVE EVALUATION OF THE CRISPR-BASED SIT TECHNOLOGY, PGSIT, FOR D. SUZUKII CONTROL AND TECHNOLOGIES THAT COMPLEMENT ITS MONITORING AND SUCCESS IN,THE FIELD. THIS WILL ALLOW US TO BUILD TOOLS AND KNOWLEDGE TO SUPPRESS PEST POPULATIONS AND BETTER CONFINE AND MANAGE MORE INVASIVE GENETIC TECHNOLOGIES.