**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 VITAL SIGNIFICANCE OF HONEY BEES (APIS MELLIFERA) TO WORLD FOOD PRODUCTION AND THE CRUCIAL ROLE OF VARROA DESTRUCTOR IN HONEY BEE COLONY LOSSES MAKE IT ONE OF THE MOST SEVERE ECTOPARASITES. VARROOSIS, THE MITE INFESTATION, IS A SIGNIFICANT THREAT TO APICULTURE IN THE UNITED STATES OF AMERICA. WITH UNREGULATED PESTICIDE USE, CLIMATIC CHANGES, VARROA DESTRUCTOR MITE INFESTATION, AND OVER A DOZEN VIRAL INFECTIONS, BEES HAVE THE CHALLENGE OF SURVIVAL. THE CONTROL AND PREVENTION OF VARROA REMAIN PRIMARILY BASED ON CHEMICAL ACARICIDES. EVEN MORE WORRYING OBSERVATIONS INCLUDE INCREASING RESISTANCE IN VARROA TO AVAILABLE ACARICIDES, AND ITS VECTORED VIRUSES ARE BECOMING MORE VIRULENT, INCLUDING DEFORMED WING VIRUS (DWV). CURRENT CONTROL STRATEGIES ARE NOT ENOUGH; THEREFORE, INNOVATIVE CONTROL STRATEGIES ARE NEEDED TO PREVENT VARROA INFESTATION AND VARROA-BORNE VIRAL DISEASES OF BEES. THIS SEED GRANT BUILDS ON OUR RECENTLY PUBLISHED WORK AND EXPERIENCE CHARACTERIZING THE VARROA MIRNAS AND DEFORMED WING VIRUS INFECTION. MICRORNAS ARE ~22-25 NUCLEOTIDE LONG NON-CODING SMALL RNAS DERIVED FROM LARGER HAIRPIN RNA PRECURSORS INVOLVED IN SEVERAL PHYSIOLOGICAL AND PATHOLOGICAL PROCESSES THROUGH POST-TRANSCRIPTIONAL REGULATION OF SEVERAL TARGET GENES. THIS STUDY AIMS TO BUILD TECHNOLOGIES BY REALIZING THE POTENTIAL OF EXPLOITING HEMOCYTE BIOLOGY IN VARROA MITES.THE PROPOSED GRANT APPLICATION WILL TEST THE HYPOTHESIS THAT VARROA MITE MIRNAS CAN BE DEVELOPED AS AN ACARICIDE TO PREVENT MITE INFESTATION AND VIRAL TRANSMISSION TO THE BEES. A SYSTEM BIOLOGY APPROACH WILL REVEAL TARGETS THAT CAN BE EXPLOITED TO DISRUPT THE VITAL VARROA PHYSIOLOGICAL PATHWAYS USING NOVEL MIRNA INHIBITORS. THE HYPOTHESIS WILL BE TESTED BY PURSUING THE FOLLOWING AIMS: 1) TO GENERATE SINGLE-CELL SMALL RNA SEQUENCING FROM VARROA DESTRUCTOR HEMOCYTES FOR DOWNSTREAM ANALYSIS, AND 2) TO FUNCTIONALLY CHARACTERIZE THE IDENTIFIED MIRNAS USING MIRNA INHIBITORS TO CONTROL THE VARROA INFESTATION AND VIRAL TRANSMISSION TO THE HONEY BEES. THESE SPECIFIC AIMS WILL PROVIDE CRITICAL REAGENTS, TOOLS, AND DATA TO ADDRESS CRITICAL GAPS IN FUNDAMENTAL KNOWLEDGE OF MITE IMMUNE CELL BIOLOGY AND VECTOR COMPETENCE. THIS PROJECT WILL ALSO OFFER THE OPPORTUNITY TO DEVELOP NEW TOOLS TO STUDY MITE HEMOCYTE BIOLOGY. ADDITIONALLY, THE OUTCOME OF THIS PROJECT COULD BE EXTRAPOLATED TO OTHER MITE SPECIES OF IMPORTANCE TO U. S. AGRICULTURE AND IS EXPECTED TO PROVIDE A CRITICAL COMPARATIVE UNDERSTANDING OF INVERTEBRATE IMMUNOLOGY.