The overexpression and overactivity of key cytochrome P450s (CYP450) genes are major drivers of metabolic resistance to insecticides in African malaria vectors such as Anopheles funestus s.s. Previous RNAseq-based transcription analyses revealed elevated expression of CYP325A specific to Central African populations but its role in conferring resistance has not previously been demonstrated. In this study, RT-qPCR consistently confirmed that CYP325A is highly over-expressed in pyrethroid-resistant An. funestus from Cameroon, compared with a control strain and insecticide-unexposed mosquitoes. A synergist bioassay with PBO significantly recovered susceptibility for permethrin and deltamethrin indicating P450-based metabolic resistance. Analyses of the coding sequence of CYP325A Africa-wide detected high-levels of polymorphism, but with no predominant alleles selected by pyrethroid resistance. Geographical amino acid changes were detected notably in Cameroon. In silico homology modelling and molecular docking simulations predicted that CYP325A binds and metabolises type I and type II pyrethroids. Heterologous expression of recombinant CYP325A and metabolic assays confirmed that the most-common Cameroonian haplotype metabolises both type I and type II pyrethroids with depletion rate twice that the of the DR Congo haplotype. Analysis of the 1 kb putative promoter of CYP325A revealed reduced diversity in resistant mosquitoes compared to susceptible ones, suggesting a potential selective sweep in this region. The establishment of CYP325A as a pyrethroid resistance metabolising gene further explains pyrethroid resistance in Central African populations of An. funestus. Our work will facilitate future efforts to detect the causative resistance markers in the promoter region of CYP325A to design field applicable DNA-based diagnostic tools.

关键细胞色素 P450s (CYP450) 基因的过度表达和过度活动是非洲疟疾载体（如Anopheles funestus ss）中对杀虫剂的代谢抗性的主要驱动因素先前基于 RNAseq 的转录分析显示CYP325A特定于中非人群的表达升高，但其在赋予以前没有表现出抵抗力。在这项研究中，RT-qPCR 一致证实CYP325A在拟除虫菊酯抗性An 中高度过度表达。来自喀麦隆的funestus ，与对照品系和未接触杀虫剂的蚊子进行比较。使用 PBO的协同生物测定显着恢复了对氯菊酯和溴氰菊酯表明基于 P450 的代谢抗性。对非洲范围内CYP325A编码序列的分析检测到高水平的多态性，但没有通过拟除虫菊酯抗性选择的主要等位基因。在喀麦隆发现了明显的地理氨基酸变化。计算机同源建模和分子对接模拟预测CYP325A结合并代谢 I 型和 II 型拟除虫菊酯。重组 CYP325A 的异源表达和代谢测定证实，最常见的喀麦隆单倍型代谢 I 型和 II 型拟除虫菊酯，其消耗率是刚果民主共和国单倍型的两倍。CYP325A 1 kb 推定启动子的分析揭示了与易感蚊子相比，抗性蚊子的多样性降低，表明该地区可能存在选择性清除。建立CYP325A为拟除虫菊酯性代谢性基因进一步解释的中部非洲国家的人口拟除虫菊酯抗性的。福利。我们的工作将有助于未来检测CYP325A启动子区域的致病抗性标记物，以设计现场适用的基于 DNA 的诊断工具。