Elucidating the regulation of insect ecdysis through nicotinic acetylcholine receptors in the model organism Drosophila melanogaster

Ecdysis is a complex, well-conserved, multi-step pathway involving a suite of interacting neurons, peptides, and receptors. Despite learning much about the ecdysis process, several of its underlying molecular-level regulations are still unknown. Neonicotinoids are a class of insecticides that act on the nicotinic acetylcholine receptors (nAChRs). At high doses, neonicotinoid exposure results in paralysis and death. At lower doses, they disrupt the larval to pupal ecdysis process, preventing the insect from completely shedding its previous cuticle. We termed this phenotype arrested ecdysis (AE), and observed it in Lepidoptera and Coleoptera. However, as ecdysis regulation has been best studied in Drosophila melanogaster, we treated this species with low doses of neonicotinoids Our preliminary data shows the AE phenotype presents differently in Drosophila, potentially due to puparium formation after their final larval instar. This phenotype aligns with previous CRISPR-Cas9 studies that knocked-down or knocked-out crustacean cardioactive peptide (CCAP) neurons in Drosophila. To test our hypothesis of nAChR regulation of CCAP neurons, we undertook immunostaining studies and determined levels and timing of release of various CCAP neuronal peptides in control and AE larvae. We also undertook a detailed analysis of ecdysis disruption in AE larvae and compared it with CCAP knockdown larvae. Future studies will involve knocking down various nAChR subtypes in Drosophila CCAP neurons. These findings can help guide the development of more targeted pesticides that have minimal non-target effects and help inform pest management and conservation strategies for both pest and beneficial insects exposed to currently registered pesticides targeting nAChRs.