The Dual Resistance\nMechanism of CYP325G4 and CYP6AA9\nin Culex pipiens pallens Legs According\nto Transcriptome and Proteome Analysis

Mosquitoes within the Culex pipiens complex play a crucial role in human disease transmission. Insecticides,\nespecially pyrethroids, are used to control these vectors. Mosquito\nlegs are the main entry point and barrier for insecticides to gain\ntheir neuronal targets. However, the resistance mechanism in mosquito\nlegs is unclear. Herein, we employed transcriptomic analyses and isobaric\ntags for relative and absolute quantitation techniques to investigate\nthe resistance mechanism, focusing on Cx. pipiens legs. We discovered 2346 differentially expressed genes (DEGs) between\ndeltamethrin-resistant (DR) and deltamethrin-sensitive (DS) mosquito\nlegs, including 41 cytochrome P450 genes. In the same comparison,\nwe identified 228 differentially expressed proteins (DEPs), including\nsix cytochrome P450 proteins. Combined transcriptome and proteome\nanalysis revealed only two upregulated P450 genes, <i>CYP325G4</i> and <i>CYP6AA9</i>. The main clusters of DEGs and DEPs\nwere associated with metabolic processes, such as cytochrome P450-mediated\nmetabolism of drugs and xenobiotics. Transcription analysis revealed\nhigh <i>CYP325G4</i> and <i>CYP6AA9</i> expression\nin the DR strain at 72 h posteclosion compared with that in the DS\nstrain, particularly in the legs. Mosquitoes knocked down for <i>CYP325G4</i> were more sensitive to deltamethrin than the controls. <i>CYP325G4</i> knockdown reduced the expression of several chlorinated\nhydrocarbon (CHC)-related genes, which altered the cuticle thickness\nand structure. Conversely, <i>CYP6AA9</i> knockdown increased\nCHC gene expression without altering cuticle thickness and structure.\nP450 activity analysis demonstrated that CYP325G4 and CYP6AA9 contributed\nto metabolic resistance in the midgut and legs. This study identified\nCYP325G4 as a novel mosquito deltamethrin resistance factor, being\ninvolved in both metabolic and cuticular resistance mechanisms. The\npreviously identified CYP6AA9 was investigated for its involvement\nin metabolic resistance and potential cuticular resistance in mosquito\nlegs. These findings enhance our comprehension of resistance mechanisms,\nidentifying P450s as promising targets for the future management of\nmosquito vector resistance, and laying a theoretical groundwork for\nmosquito resistance management.