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Pyrethroid resistance in  Anopheles funestus  is a potential obstacle to malaria control in Africa. Tools are needed to detect resistance in field populations. We have been using a positional cloning approach to identify the major genes conferring pyrethroid resistance in this vector. A quantitative trait locus (QTL) named  rp1  explains 87% of the genetic variance in pyrethroid susceptibility in two families from reciprocal crosses between susceptible and resistant strains. Two additional QTLs of minor effect,  rp2  and  rp3 , were also detected. We sequenced a 120-kb BAC clone spanning the  rp1  QTL and identified 14 protein-coding genes and one putative pseudogene. Ten of the 14 genes encoded cytochrome P450s, and expression analysis indicated that four of these P450s were differentially expressed between susceptible and resistant strains. Furthermore, two of these genes,  CYP6P9  and  CYP6P4 , which are 25 and 51 times overexpressed in resistant females, are tandemly duplicated in the BAC clone as well as in laboratory and field samples, suggesting that P450 gene duplication could contribute to pyrethroid resistance in  An. funestus . Single nucleotide polymorphisms (SNPs) were identified within  CYP6P9  and  CYP6P4 , and genotyping of the progeny of the genetic crosses revealed a maximum penetrance value f 2  = 1, confirming that these SNPs are valid resistance markers in the laboratory strains. This serves as proof of principle that a DNA-based diagnostic test could be designed to trace metabolic resistance in field populations. This will be a major advance for insecticide resistance management in malaria vectors, which requires the early detection of resistance alleles.

Two duplicated P450 genes are associated with pyrethroid resistance in Anopheles funestus, a major malaria vector