Abstract
Scale up of Long Lasting Insecticide Nets (LLINs) has massively contributed to reduce malaria mortality across Africa. However, resistance to pyrethroid insecticides in malaria vectors threatens its continued effectiveness. Deciphering the detailed molecular basis of such resistance and designing diagnostic tools is critical to implement suitable resistance management strategies. Here, we demonstrated that allelic variation in two cytochrome P450 genes is the most important driver of pyrethroid resistance in the major African malaria vector Anopheles funestus and detected key mutations controlling this resistance. An Africa-wide polymorphism analysis of the duplicated genes CYP6P9a and CYP6P9b revealed that both genes are directionally selected with alleles segregating according to resistance phenotypes. Modelling and docking simulations predicted that resistant alleles were better metabolizers of pyrethroids than susceptible alleles. Metabolism assays performed with recombinant enzymes of various alleles confirmed that alleles from resistant mosquitoes had significantly higher activities toward pyrethroids. Additionally, transgenic expression in Drosophila showed that flies expressing resistant alleles of both genes were significantly more resistant to pyrethroids compared with those expressing the susceptible alleles, indicating that allelic variation is the key resistance mechanism. Furthermore, site-directed mutagenesis and functional analyses demonstrated that three amino acid changes (Val109Ile, Asp335Glu and Asn384Ser) from the resistant allele of CYP6P9b were key pyrethroid resistance mutations inducing high metabolic efficiency. The detection of these first DNA markers of metabolic resistance to pyrethroids allows the design of DNA-based diagnostic tools to detect and track resistance associated with bednets scale up, which will improve the design of evidence-based resistance management strategies.
Highlights
Despite the recent decrease in malaria mortality (47%) [1], the disease remains a serious public health burden in the tropical world, with 584,000 deaths globally in 2013, of which 90% occurred in WHO African region, and mostly in children under the age of 5
The relative genetic homogeneity in each region is shown by (i) the fact that haplotypes from each country and region cluster together on the maximum likelihood phylogenetic tree (Fig 1A and 1B), notably those from the three southern African countries; (ii) the fact that only few amino acid changes (5 to 7) are observed within regions in contrast to the higher number of replacements between them (22 to 51) (S1 Table)
DNA-based diagnostic tools are essential for this purpose, but the diagnostics require a thorough understanding of the molecular basis of insecticide resistance
Summary
Despite the recent decrease in malaria mortality (47%) [1], the disease remains a serious public health burden in the tropical world, with 584,000 deaths globally in 2013, of which 90% occurred in WHO African region, and mostly in children under the age of 5. Resistance to insecticides, especially pyrethroids (the only class approved by WHO for LLINs [3]), in major malaria vectors such as and An. funestus [4,5,6] and An. gambiae [7, 8] is threatening to derail these intervention tools [9]. An. funestus is widely, geographically distributed across Sub-Saharan Africa [10], and it has high vectorial capacity in some places surpassing even that of An. gambiae [11]. It reaches maximal abundance in the dry season when the density of An. gambiae and An. arabiensis have declined, thereby extending the period of malaria transmission [12]. The design of DNA-based diagnostic tools requires a thorough understanding of the molecular basis of the resistance
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