A single mutation G454A in P450 CYP9K1 drives pyrethroid resistance in the major malaria vector Anopheles funestus reducing bed net efficacy

Metabolic resistance to pyrethroids is jeopardizing the effectiveness of insecticide-based interventions against malaria. The complexity of the Africa-wide spatio-temporal evolution of the molecular basis of this resistance, the major genetic drivers should be detected to improve resistance management. Here, we demonstrated that a single amino acid change G454A in the cytochrome P450 CYP9K1 drives pyrethroid resistance in Anopheles funestus vector in East and Central Africa. Polymorphism analysis revealed drastic reduction of diversity of the CYP9K1 gene in Uganda (2014) with the selection of a predominant haplotype (90%), exhibited a G454A mutation. However, 6 years later (2020) the Ugandan 454A-CYP9K1 haplotype was also predominant in Cameroon (84.6%), but absent in Malawi (Southern Africa) and Ghana (West Africa). In vitro comparative heterologous metabolism assays revealed that the mutant-type 454A-CYP9K1 (R) allele metabolises type II pyrethroid (deltamethrin) better than the wild-type G454-CYP9K1 (S) allele. Transgenic Drosophila melanogaster flies expressing the mutant-type 454A-CYP9K1 allele were significantly more resistant to both type I and II pyrethroids than the flies expressing the wild-type G454-CYP9K1 allele. Genotyping with a newly designed DNA-based diagnostic assay targeting the G454A replacement revealed that this mutation is strongly associated with pyrethroid resistance as mosquitoes surviving pyrethroid exposure were significantly more homozygote resistant (Odds ratio = 567, P<0.0001). Furthermore, Cone test and experimental hut trials showed that 454A-CYP9K1 reduces the efficacy of LLINs. The resistant allele (454A) is under directional selection in Eastern and Central Africa, present but not strongly selected in Southern Africa and at very low frequency in West Africa. This study reveals the rapid spread of P450-based metabolic pyrethroid resistance driven by CYP9K1, greatly reducing the efficacy of pyrethroid-based control tools. The new DNA-based assay designed here will add to the toolbox to monitor resistance in the field and improve resistance management strategies. Author SummaryThe complex molecular basis and genetic drivers of metabolic resistance in malaria vectors should be detected to improve resistance management. Here, we established that allelic variation by a single mutation G454A in P450 CYP9K1 enzyme drives pyrethroid resistance in Anopheles funestus. Drastic reduction of diversity was noted in Ugandan female An. funestus samples collected in 2014, with a major haplotype (454A) already fixed but absent in other African regions. However, this Ugandan 454A-CYP9K1 haplotype was highly selected within 6 years in An. funestus samples from Cameroon (Central Africa), but still absent in Ghana (West Africa) and Malawi (Southern Africa). Metabolism assays revealed that the 454A-resistant allele metabolized pyrethroid better than the susceptible G454 allele and driving higher pyrethroid resistance in transgenic Drosophila melanogaster flies. DNA-based diagnostics designed around the G454A-CYP9K1 marker strongly correlates with pyrethroid resistance, reducing bed net efficacy indicating that this assay should be added to the toolbox to monitor this 454A-CYP9K1 resistance which is rapidly spreading in An. funestus mosquito populations from Eastern and Central Africa.