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

Mosquitoes within the Culex pipiens 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 Cx. pipiens 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, CYP325G4 and CYP6AA9. 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 CYP325G4 and CYP6AA9 expression
in the DR strain at 72 h posteclosion compared with that in the DS
strain, particularly in the legs. Mosquitoes knocked down for CYP325G4 were more sensitive to deltamethrin than the controls. CYP325G4 knockdown reduced the expression of several chlorinated
hydrocarbon (CHC)-related genes, which altered the cuticle thickness
and structure. Conversely, CYP6AA9 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.