Populations of many insect species have evolved a variety of resistance mechanisms in response to insecticide selection. Current knowledge about mutations responsible for insecticide resistance is largely achieved from studies on target-site resistance, while much less is known about metabolic resistance. Although it is well known that P450 monooxygenases are one of the major players involved in insecticide metabolism and resistance, understanding mutation(s) responsible for CYP-mediated resistance has been a big challenge. In this study, we used the house fly to pursue a better understanding of P450 mediated insecticide resistance at the molecular level. Metabolism studies illustrated that CYP6G4 had a broad-spectrum metabolic activity in metabolizing insecticides. Population genotyping revealed that the CYP6G4v1 allele harboring a DNA insertion (MdIS1) had been selected in many house fly populations on different continents. Dual luciferase reporter assays identified that the MdIS1 contained a CncC/Maf binding site, and electrophoretic mobility shift assay confirmed that transcription factor CncC was involved in the MdIS1-mediated regulation. This study highlights the common involvement of the CncC pathway in adaptive evolution, and provides an interesting case supportive of parallel evolution in P450-mediated insecticide resistance in insects.
The house fly ( Musca domestica L.) (Diptera: Muscidae) is a global vector that can transmit >250 human and animal diseases. The control of house flies has heavily relied on the application of various chemical insecticides . The carbamate insecticide propoxur has been widely used for the control of house flies, and resistance to propoxur has been documented in many house fly populations worldwide. Previous studies have identified several propoxur resistance-conferring mutations in the target protein acetylcholinesterase ; however, the molecular basis for metabolic resistance to propoxur remains unknown. In this study, we investigated the involvement of CYP6G4, a cytochrome P450 overexpressed in many insecticide resistant populations of Musca domestica , in propoxur metabolism and resistance by using combined approaches of recombinant protein-based insecticide metabolism and the Drosophila GAL4/UAS transgenic system. The recombinant CYP6G4 and its redox partners (NADPH-dependent cytochrome P450 reductase and cytochrome b 5) were functionally expressed in Escherichia coli . Metabolism experiments showed that CYP6G4 was able to transform propoxur with a turnover rate of around 0.79 min −1 . Six metabolites were putatively identified, suggesting that CYP6G4 could metabolize propoxur via hydroxylation , O-depropylation and N -demethylation. Moreover, bioassay results showed that ectopic overexpression of CYP6G4 in fruit flies significantly increased their tolerance to propoxur. Our in vivo and in vitro data convincingly demonstrate that CYP6G4 contributes to propoxur metabolism and resistance.
The use of various insecticides has resulted in insecticide resistance in many populations of pest insects. CYP6D1 and CYP6G4 are two cytochrome P450s that have been recruited to confer insecticide resistance in the house fly populations, yet the biochemical basis for their roles in insecticide metabolism is not fully understood. Here, we used recombinant enzymes to explore the capacity of the two field-evolved resistance-conferring P450s from house flies in the metabolism of 16 commonly used insecticides. The chemical structure of the detectable metabolite was identified by liquid chromatography coupled with mass spectrometry. Moreover, we analyzed the structural features associated with substrate selectivity between the two P450s through AlphaFold-based modeling and substrate docking. We found that both CYP6D1 and CYP6G4 were able to metabolize multiple classes of structurally diverse insecticides via substrate-dependent catalytic reactions including dealkylation, desulfurization and hydroxylation. Molecular modeling predicted that CYP6D1 possesses a larger solvent-accessible V-shaped pocket that accommodates bulky substrates such as esfenvalerate, whereas CYP6G4 has a more compact architecture that favors smaller substrates like acetamiprid and methomyl. We propose that the intrinsically broad substrate spectra of CYP6D1 and CYP6G4, together with their regulatory evolvability and negligible fitness costs, collectively predispose these two P450s to recurrent selection among the 146 P450 genes in the housefly genome in field populations. These findings advance our understanding of the evolutionary principles governing P450-mediated insecticide resistance, and provide a biochemical framework for designing evidence-based pest management strategies.
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