Two highly selected mutations in the tandemly duplicated CYP6P4a and CYP6P4b drive pyrethroid resistance in Anopheles funestus

Gaining a comprehensive understanding of the genetic mechanisms underlying insecticide resistance in malaria vectors is crucial for optimising the effectiveness of insecticide-based vector control methods and developing diagnostic tools for resistance management. Considering the heterogeneity of metabolic resistance in major malaria vectors, the implementation of tailored resistance management strategies is essential for successful vector control. In this study, we provide evidence demonstrating that two highly selected mutations in the tandemly duplicated cytochrome P450 genes namely CYP6P4a and CYP6P4b , are driving pyrethroid insecticide resistance in the major malaria vector Anopheles funestus, in West Africa. Through a continent-wide polymorphism survey, we observed heightened indications of directional selection in both genes between 2014 and 2021. By conducting in vitro insecticide metabolism assays with recombinant enzymes expressed from both genes, we established that mutant alleles under selection exhibit higher metabolic efficiency compared to their wild-type counterparts. Furthermore, using the GAL4-UAS transgenic system, we demonstrated that transgenic Drosophila melanogaster flies overexpressing mutant alleles displayed an increased resistance to pyrethroids. These findings were in agreement with in silico characterisation, which highlighted changes in enzyme active site architecture that enhance the affinity of mutant alleles for type I and II pyrethroids. Furthermore, we developed two DNA-based assays capable of detecting the CYP6P4a-M220I and CYP6P4b-D284E mutations, showing their current confinement to West Africa. Genotype/phenotype correlation analyses revealed that these markers are strongly associated with resistance to types I and II pyrethroids and combine to drastically reduce the efficacy of pyrethroid bednets. Overall, our study makes available two field-applicable insecticide resistance molecular markers that will help in the monitoring and better management of insecticide resistance in West Africa.