The E205D mutation in CYP6P3 drives pyrethroid insecticide resistance in the African malaria mosquito vector <i>Anopheles gambiae</i>

Deciphering the molecular drivers of insecticide resistance is paramount to extending the effectiveness of malaria vector control tools. Here, we demonstrate that a P450 haplotype spanning a Glu²⁰⁵→Asp (E205D) amino acid point mutation in the <i>CYP6P3</i> gene drives pyrethroid insecticide resistance in the mosquito malaria vector <i>Anopheles gambiae</i>. Pooled whole-genome DNA sequencing data from <i>A. gambiae</i> mosquitoes detected a major P450-linked locus (CYP6 haplotype) on chromosome 2R adjacent to the locus encoding a sodium channel. In vitro metabolism assays with recombinantly expressed CYP6P3 protein revealed that the catalytic efficiency of the 205D variant for the pyrethroid insecticide permethrin was 3.7 times higher than that of the E205 variant. Similar findings were made for the related insecticide α-cypermethrin. Overexpression of the 205D variant in transgenic flies conferred higher resistance to pyrethroids compared with flies expressing the susceptible E205 variant. A DNA-based assay confirmed that the <i>CYP6P3</i>-E205D variant correlates with pyrethroid resistance in field mosquito populations [odds ratio (OR): 26.4; <i>P</i> < 0.0001] and reduces the efficacy of pyrethroid-only long-lasting insecticide bed nets. The homozygous resistance genotype of <i>A. gambiae</i> exhibited higher survival after exposure to the PermaNet 3.0 bed net compared with the susceptible SS genotype (OR: 6.1; <i>P</i> = 0.011). Furthermore, the <i>CYP6P3</i>-E205D variant together with the <i>kdr</i> target-site resistance mechanism exacerbated the loss of bed net efficacy. The 205D variant is predominant in West and Central Africa but less abundant or absent in East and South Africa, with signs of introgression with <i>Anopheles coluzzii</i> in Ghana.