Functional and structural insights into P450-mediated resistance: The role of Cyp6g1 and Cyp6g2 in the metabolism of neonicotinoids in Drosophila melanogaster

Insecticide-driven selection pressures have accelerated the evolution and widespread emergence of resistance in insect populations. A major mechanism underlying this resistance is the enhanced metabolic detoxification of insecticides, often mediated by the overexpression or increased activity of cytochrome P450 enzymes. Evidence indicates that the ability of these enzymes to confer resistance may have evolved from their native role in metabolising environmental xenobiotics. This suggests that insect populations may harbor multiple metabolic enzymes capable of conferring resistance, even if not specifically adapted for insecticide metabolism. To investigate this hypothesis, we examined the well-characterised resistance gene Cyp6g1 and five closely related cytochrome P450s in Drosophila melanogaster. Using transgenic overexpression driven by the Accord promoter—responsible for elevated Cyp6g1 expression in natural populations—we found that only Cyp6g1 and Cyp6g2 conferred resistance to the neonicotinoids imidacloprid and nitenpyram. Metabolic assays confirmed that imidacloprid resistance was mediated by the conversion of imidacloprid into 5-hydroxyimidacloprid. Additionally, field-resistant haplotypes promoting Cyp6g1 overexpression were also found to increase Cyp6g2 expression, suggesting that Cyp6g2 contributes to resistance in natural populations. Structural analysis of CYP6G1, using molecular docking and site-directed mutagenesis, identified residues Phe123 and Phe124 as critical for imidacloprid metabolism. These findings contribute to our understanding of the evolutionary pathways leading to metabolic resistance and offer insights that could improve strategies for managing and mitigating insecticide resistance.