Overexpression of multiple cytochrome P450 genes with and without knockdown resistance mutations confers high resistance to deltamethrin in Culex quinquefasciatus.

Abstract

Background: The cytochrome P450s-mediated metabolic resistance and the target site insensitivity caused by the knockdown resistance (kdr) mutation in the voltage-gated sodium channel (vgsc) gene were the main mechanisms conferring resistance to deltamethrin in Culex quinquefasciatus from Thailand. This study aimed to investigate the expression levels of cytochrome P450 genes and detect mutations of the vgsc gene in deltamethrin-resistant Cx. quinquefasciatus populations in Thailand.

Methods: Two field-collected strains of Cx. quinquefasciatus, Cq_SP and Cq_NiH, were selected with deltamethrin to generate the resistant strains Cq_SP-R and Cq_NiH-R, respectively. Bioassays were tested on larvae and adults of each strain according to WHO methods. Eight cytochrome P450 genes were analyzed for the expression level using quantitative real time-PCR. The cDNA of mosquitoes was amplified and sequenced for four fragments of vgsc gene. The kdr L1014F mutation and the haplotype of the CYP9M10 gene were detected in survivor and dead mosquitoes after exposure to the deltamethrin WHO test paper. Statistical analyses were performed using Fisher's exaction test.

Results: Bioassay tests revealed a significantly higher resistance level in Cq_SP-R than in Cq_NiH-R strains in both larvae and adults. All eight cytochrome P450 genes were significantly overexpressed in larvae of Cq_NiH-R strain compared to the parent and susceptible Cq_Sus strains. The CYP6AA7 and CYP9J34 genes had the highest expression ratios, exceeding 24-fold in Cq_NiH-R larvae. In Cq_SP-R strain, the CYP4H34 and CYP9J34 genes were overexpressed in both stages. The kdr L1014F mutation was found in Cq_SP-R and its parent Cq_SP strains with a significantly higher mutant allele frequency in the survivor mosquitoes than in dead mosquitoes (P < 0.0001). The V240M and novel L925F mutations were found only in Cq_SP-R strain. Heterozygous genotype for the D-Cu( +)/Cu(-) of CYP9M10 gene was detected in Cq_NiH and Cq_NiH-R strains but other strains were mostly homozygous for the Cu(-)/Cu(-).

Conclusions: Overexpression of multiple cytochrome P450 genes alone has a relatively minor impact on resistance. The combined mechanisms of cytochrome P450- and kdr-mediated resistance result in significantly higher resistance to deltamethrin in Cx. quinquefasciatus. This study supports sustainable public health initiatives in Thailand to address the evolving challenges of insecticide resistance.