This seed grant will support pursuit of a new investigator's research project that has a long-term goal to identify new insecticide mechanisms that disrupt pupal ecdysis in a diverse suite of agriculturally important insect pests. Results of the overall project can lead to development of new, novel insecticides that reduce environmental loading and support insecticide resistance management. Specifically, this project will advance fundamental knowledge of insect ecdysis behavior and its regulation, which will support a future standard grant that will elucidate the mechanisms through which excitatory and inhibitory neurotransmitters, in concert with neuroendocrinal hormones, regulate pupal ecdysis in Lepidoptera, Coleoptera, and Diptera pests. Research described in this seed proposal is designed to characterize the role of acetylcholine signaling pathways in ecdysis and the extent to which disruption of this signaling by specific insecticides can block insect development. Disruption of acetylcholine signaling that is required for ecdysis has not been previously reported as a basis for any current insecticide modes of action. Our new, novel finding that neonicotinoids at sublethal concentrations can readily disrupt pupal ecdysis in several Lepidoptera and Coleoptera pests forms the basis of this proposal.This seed grant proposal addresses the Pests and Beneficial Species in Agricultural Production Systems program area priority through four supporting objectives that characterize a new mode of action for an insecticide:Identify putative acetylcholine receptor subtypes that mediate Lepidoptera pupal ecdysis.Understand the extent to which interspecies variability in Lepidoptera pupal ecdysis behavior is due to toxicokinetic differences.Characterize if interference of acetylcholine signaling during Lepidoptera pupal ecdysis disrupts the function of crustacean cardioactive peptide (CCAP) neurons.Document the disruption of acetylcholine signaling leading to arrested pupal ecdysis in Coleoptera and Diptera pest species.Meeting these objectives will provide preliminary data to support a new investigator standard grant that will undertake a detailed study on insecticidal modes of action that elicit arrested pupal ecdysis across multiple insect orders.