Molecular Characterization of Cytochrome P450 Genes (CYP9M10 and CYP4H34) in Insecticide-Resistant and Susceptible Strains of Culex pipiens

Abstract

Background: Culex pipiens is widespread in Iran and is an important vector of several diseases. Although phenotypic resistance to insecticides such as DDT and pyrethroids has been reported using WHO assays, sequence‑level infor­mation on metabolic resistance genes, particularly cytochrome P450 genes, remains limited. This study examined varia­tion in two P450 genes, CYP9M10 and CYP4H34, in deltamethrin‑ and DDT‑resistant versus susceptible strains of Cx. pipiens, and assessed the potential impact of these differences on predicted protein structures.
Methods: Target fragments of CYP9M10 and CYP4H34 were amplified by PCR and sequenced using the Sanger meth­od. Edited nucleotide sequences were aligned with CLUSTAL OMEGA, and amino acid sequences were generated us­ing ExPASy Translate. Comparisons were conducted at both nucleotide and amino acid levels. Representative sequenc­es were submitted to GenBank. Phylogenetic relationships among strains were inferred via maximum-likelihood (ML) anal­ysis in MEGA6 with 1000 bootstrap replicates. Predicted amino acid substitutions were examined for structural relevance.
Results: Four nucleotide differences were detected at positions 1344, 1347, 1396 and within 1428–1442. Previously published permethrin‑ and pyrethroid‑resistant reference sequences were identical across this region, whereas sequences from this study showed distinctions from those references and between resistant and susceptible strains. Some nucleo­tide substitutions led to amino acid changes, though their structural effects were only inferred computationally.
Conclusion: This study provides new sequence-level insights into variation in Cx. pipiens P450 genes and highlights po­tential genetic differences that may contribute to resistance to DDT and deltamethrin, warranting further functional in­ves­tigation.