THE OVERALL GOAL OF THIS PROJECT IS TO IDENTIFY NOVEL INSECTICIDAL TARGETS THAT DISRUPT THE INSECT ECDYSIS PROCESS. RECENTLY, WE DISCOVERED THAT A NICOTINIC ACETYLCHOLINE RECEPTOR (NACHR) AGONIST, IMIDACLOPRID, ARRESTED PUPAL ECDYSIS (AE) IN LEPIDOPTERA, WHICH LED TO FAILURE OF LARVAE TO COMPLETE PUPATION. ELUCIDATION OF THIS POTENT MODE OF ACTION WILL ADVANCE OUR UNDERSTANDING OF MECHANISMS REGULATING ECDYSIS BEHAVIOR AND POTENTIALLY RESULT IN DEVELOPMENT OF NEW INSECTICIDES TO HELP MANAGE PEST RESISTANCE WITH REDUCED ENVIRONMENTAL FOOTPRINTS.OUR PRELIMINARY DATA SHOW SIX OF EIGHT LEPIDOPTERA SPECIES EXHIBITED AE WHEN TREATED WITH SUBLETHAL DOSES OF IMIDACLOPRID. THROUGH THE PROPOSED SEED GRANT, WE AIM TO IDENTIFY PUTATIVE SUBUNITS OF NACHRS MEDIATING AE AND EXPLORE DIFFERENCES IN INTERSPECIES SUSCEPTIBILITY BY EMPLOYING MULTIPLE ACETYLCHOLINE RECEPTOR AGONISTS AND ANTAGONISTS AND CONDUCTING DOSIMETRY STUDIES OVER TIME IN AE-SENSITIVE AND AE-INSENSITIVE LARVAE. WE WILL ALSO UNDERTAKEANTIBODY BINDING EXPERIMENTS IN LEPIDOPTERA TO TEST OUR HYPOTHESIS THAT DYSREGULATION OF CRUSTACEAN CARDIOACTIVE PEPTIDE NEURONS LEADS TO AE. FINALLY, WE WILL INVESTIGATE IF COLEOPTERA AND DIPTERA LARVAE EXHIBIT AE WHEN TREATED WITH IMIDACLOPRID. RESULTS OF THESE EXPERIMENTS WILL HELP EXPLAIN THE ROLE OF ACETYLCHOLINE SIGNALING IN REGULATION OF PUPAL ECDYSIS ACROSS THREE MAJOR PEST ORDERS THAT IMPACT AGRICULTURAL AND LIVESTOCK PRODUCTION.BASED ON THE RESULTS OBTAINED, A FUTURE STANDARD GRANT PROPOSAL WILL BE PREPARED TO ELUCIDATE THE TOXICITY PATHWAY(S) LEADING TO AE THROUGH IN-DEPTH MECHANISTIC STUDIES IN THESE AND ADDITIONAL INSECT PESTS. THE RESULTS GENERATED WILL HELP INFORM INSECTICIDE DISCOVERY, RESEARCH, AND DEVELOPMENT EFFORTS IN ACADEMIC, GOVERNMENT, AND INDUSTRIAL LABORATORIES.