Identification of a rapidly-spreading triple mutant for high-level metabolic insecticide resistance inAnopheles gambiaeprovides a real-time molecular diagnostic for anti-malarial intervention deployment

AbstractInsecticide resistance provides both an increasingly pressing threat to the control of vector-borne diseases and insights into the remarkable capacity of natural populations to show rapid evolutionary responses to contemporary selection. Malaria control remains heavily dependent on deployment of pyrethroid insecticides, primarily in long lasting insecticidal nets (LLINs), but resistance in the major malaria vectors has increased over the last 15 years in concert with dramatic expansion of LLIN distributions. Identifying genetic mechanisms underlying high-level resistance in mosquitoes, which may almost entirely overcome pyrethroid efficacy, is crucial for the development and deployment of potentially resistance-breaking tools. Using theAnopheles gambiae1000 genomes (Ag1000g) data we identified a very recent selective sweep in mosquitoes from Uganda which localized to a cluster of cytochrome P450 genes, including some commonly implicated in resistance. Further interrogation revealed a haplotype involving a trio of mutations, a nonsynonymous point mutation inCyp6p4(I236M), an upstream insertion of a partial Zanzibar-like transposable element (TE) and a duplication of theCyp6aa1gene. The mutations appear to have originated recently inAn. gambiaefrom the Kenya-Uganda border region around Lake Victoria, with stepwise replacement of the double-mutant (Zanzibar-like TE andCyp6p4-236M) with the triple-mutant haplotype (includingCyp6aa1duplication), which has spread into the Democratic Republic of Congo and Tanzania. The triple-mutant haplotype is strongly associated with increased expression of genes able to metabolise pyrethroids and is strongly predictive of resistance to pyrethroids most notably deltamethrin, a commonly-used LLIN insecticide. Importantly, there was increased mortality in mosquitoes carrying the triple-mutation when exposed to nets co-treated with the synergist piperonyl butoxide (PBO). Frequencies of the triple-mutant haplotype remain spatially variable within countries, suggesting an effective marker system to guide deployment decisions for limited supplies of PBO-pyrethroid co-treated LLINs across African countries. Duplications of theCyp6aa1gene are common inAn. gambiaeacross Africa and, given the enzymes metabolic activity, are likely to be a useful diagnostic for high levels of pyrethroid resistance.