A rapidly selected 4.3kb transposon-containing structural variation is driving a P450-based resistance to pyrethroids in the African malaria vector Anopheles funestus

Deciphering the evolutionary forces controlling insecticide resistance in malaria vectors remains a prerequisite to designing molecular tools to detect and assess resistance impact on control tools. Here, we demonstrate that a 4.3kb transposon-containing structural variation drives pyrethroid resistance in central/eastern African populations of the malaria vector Anopheles funestus. In this study, we analysed Pooled template sequencing data and direct sequencing to identify an insertion of 4.3kb containing putative transposons in the intergenic region of two P450s CYP6P5-CYP6P9b in mosquitoes of the malaria vector Anopheles funestus from Uganda. We then designed a PCR assay to track its spread temporally and regionally and decipher its role in insecticide resistance. The insertion originates in or near Uganda in East Africa, where it is fixed and has spread to high frequencies in the Central African nation of Cameroon but is still at low frequency in West Africa and absent in Southern Africa. A strong association was established between this SV and pyrethroid resistance in field populations (SV+ vs SV-; OR=29, P< 0.0001) and is reducing pyrethroid-only nets’ efficacy. Genetic crosses and qRT-PCR revealed that this SV enhances the overexpression of CYP6P9a/b but not CYP6P5. A marked and rapid selection was observed with the 4.3kb-SV frequency increasing from 3% in 2014 to 98 % in 2021 in Cameroon. Our findings highlight the underexplored role and rapid spread of SVs in the evolution of insecticide resistance and provide additional tools for molecular surveillance of insecticide resistance.