Carbamates and pyrethroid cross-resistance in the African malaria vector Anopheles funestus

Cross-resistance to multiple insecticides in malaria vectors is hampering resistance management. Understanding its underlying molecular basis is critical to successful implementation of insecticide-based control interventions. Here, we established that the of tandemly duplicated cytochrome P450s, CYP6P9a/b are driving carbamate and pyrethroid cross-resistance in southern African populations of Anopheles funestus. RNAseq-based transcription analyses revealed that cytochrome P450s are the most over-expressed genes in bendiocarb and permethrin resistant mosquitoes. In southern Africa (Malawi) CYP6P9a and CYP6P9b are over-expressed, versus CYP6P4a and CYP6P4b in West Africa (Ghana). Other up-regulated genes include several additional cytochrome P450s (e.g. CYP9J5, CYP6P2, CYP6P5), glutathione-S transferases, ATP-binding cassette transporters, digestive enzymes, microRNA and transcription factors. Targeted enrichment sequencing strongly linked a known major pyrethroid resistance locus (rp1) to carbamate resistance centering around CYP6P9a/b. In bendiocarb resistant mosquitoes this locus had reduced nucleotide diversity, lowest p-values when comparing allele frequencies, and most non-synonymous substitutions. Recombinant enzyme metabolism assays with native An. funestus cytochrome P450 reductase showed that both CYP6P9a/b metabolize carbamates. GAL4/UAS transgenic expression of CYP6P9a/b in Drosophila melanogaster revealed that flies expressing both genes were significantly more resistant to carbamates than controls. Furthermore, a strong correlation was observed between their genotypes and carbamate resistance with homozygote resistant mosquitoes exhibiting a greater ability to withstand carbamate exposure than homozygote susceptible mosquitoes and heterozygotes. Double homozygote resistant genotype (RR/RR) were even more able to survive than any other genotype combination showing an additive effect. This study highlights the risk that resistance escalation to pyrethroids poses to distinct classes of insecticides. Available metabolic resistance DNA-based diagnostic assays should be used by control programs to monitor cross-resistance between insecticides before implementing new interventions.