The Dual Resistance Mechanism of CYP325G4 and CYP6AA9 in <i>Culex pipiens pallens</i> Legs According to Transcriptome and Proteome Analysis

Mosquitoes within the <i>Culex pipiens</i> complex play a crucial role in human disease transmission. Insecticides, especially pyrethroids, are used to control these vectors. Mosquito legs are the main entry point and barrier for insecticides to gain their neuronal targets. However, the resistance mechanism in mosquito legs is unclear. Herein, we employed transcriptomic analyses and isobaric tags for relative and absolute quantitation techniques to investigate the resistance mechanism, focusing on <i>Cx. pipiens</i> legs. We discovered 2346 differentially expressed genes (DEGs) between deltamethrin-resistant (DR) and deltamethrin-sensitive (DS) mosquito legs, including 41 cytochrome P450 genes. In the same comparison, we identified 228 differentially expressed proteins (DEPs), including six cytochrome P450 proteins. Combined transcriptome and proteome analysis revealed only two upregulated P450 genes, <i>CYP325G4</i> and <i>CYP6AA9</i>. The main clusters of DEGs and DEPs were associated with metabolic processes, such as cytochrome P450-mediated metabolism of drugs and xenobiotics. Transcription analysis revealed high <i>CYP325G4</i> and <i>CYP6AA9</i> expression in the DR strain at 72 h posteclosion compared with that in the DS strain, 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 hydrocarbon (CHC)-related genes, which altered the cuticle thickness and structure. Conversely, <i>CYP6AA9</i> knockdown increased CHC gene expression without altering cuticle thickness and structure. P450 activity analysis demonstrated that CYP325G4 and CYP6AA9 contributed to metabolic resistance in the midgut and legs. This study identified CYP325G4 as a novel mosquito deltamethrin resistance factor, being involved in both metabolic and cuticular resistance mechanisms. The previously identified CYP6AA9 was investigated for its involvement in metabolic resistance and potential cuticular resistance in mosquito legs. These findings enhance our comprehension of resistance mechanisms, identifying P450s as promising targets for the future management of mosquito vector resistance, and laying a theoretical groundwork for mosquito resistance management.