Abstract
Nicotine is a plant-derived pyridine alkaloid with potent neurotoxic properties. A major pathway for detoxification of nicotine in mammals is via glucuronidation to produce nicotine N -glucuronide, but this process in insects remains poorly understood. Using mass spectrometry, we demonstrate that Drosophila melanogaster detoxifies nicotine through glycosylation, producing nicotine N -glycoside. Given that many new agrochemicals contain pyridine rings, we also investigated the metabolism of flonicamid and imidacloprid. We detected glycosylation of flonicamid, but not imidacloprid. A targeted RNAi screen across 21 UDP-glycosyltransferases ( Ugt s) identified Ugt35B1 as important for survival of nicotine exposure. CRISPR-based knockout of Ugt35B1 increases sensitivity to nicotine and flonicamid, but not to imidacloprid, nor to a structurally distinct neonicotinoid (thiamethoxam). Mass spectrometry of knockout and control flies confirms that Ugt35B1 glycosylates nicotine, its metabolite cotinine, and flonicamid. Together these findings establish Ugt35B1 as the principal UGT mediating nicotine detoxification in D. melanogaster , revealing a previously uncharacterized insect glycosylation pathway with potential implications for herbivory, insecticide detoxification and toxicology. HIGHLIGHTS: Drosophila detoxifies nicotine by glycosylation into nicotine N -glycoside. A targeted RNAi screen identifies Ugt35B1 as critical for nicotine survival. Ugt35B1 knockout sensitizes flies to nicotine and flonicamid, but not to imidacloprid or thiamethoxam. First demonstration of an insect UGT mediating in vivo glycosylation of nicotine and cotinine.