Abstract
Carbamates are increasingly used for vector control notably in areas with pyrethroid resistance. However, a cross‐resistance between these insecticides in major malaria vectors such as Anopheles funestus could severely limit available resistance management options. Unfortunately, the molecular basis of such cross‐resistance remains uncharacterized in An. funestus, preventing effective resistance management. Here, using a genomewide transcription profiling, we revealed that metabolic resistance through upregulation of cytochrome P450 genes is driving carbamate resistance. The P450s CYP6P9a,CYP6P9b and CYP6Z1 were the most upregulated detoxification genes in the multiple resistant mosquitoes. However, in silico docking simulations predicted CYP6Z1 to metabolize both pyrethroids and carbamates, whereas CYP6P9a and CYP6P9b were predicted to metabolize only the pyrethroids. Using recombinant enzyme metabolism and inhibition assays, we demonstrated that CYP6Z1 metabolizes bendiocarb and pyrethroids, whereas CYP6P9a and CYP6P9b metabolize only the pyrethroids. Other upregulated gene families in resistant mosquitoes included several cuticular protein genes suggesting a possible reduced penetration resistance mechanism. Investigation of the target‐site resistance in acetylcholinesterase 1 (ace‐1) gene detected and established the association between the new N485I mutation and bendiocarb resistance (odds ratio 7.3; P < 0.0001). The detection of multiple haplotypes in single mosquitoes after cloning suggested the duplication of ace‐1. A TaqMan genotyping of the N485I in nine countries revealed that the mutation is located only in southern Africa with frequency of 10–15% suggesting its recent occurrence. These findings will help in monitoring the spread and evolution of carbamate resistance and improve the design of effective resistance management strategies to control this malaria vector.
Highlights
Malaria burden remains high in the tropical world with around 584 000 deaths globally in 2013 alone and mostly in African children under the age of 5 (WHO2014)
Priority was given to the list of probes significantly upregulated in the bendiocarb-resistant vs. control mosquitoes (Rb-C) (Tables 1 and S1, Supporting information) as this comparison directly compares mosquitoes with a same genetic background which only differ in the resistance phenotype
The cytochrome P450 gene CYP6P9a appears to be the best candidate detoxification gene associated with bendiocarb resistance in the Anopheles funestus from Chikwawa, as two out of three probes of this gene were consistently overexpressed in the three Rb-C, resistant and the susceptible FANG strain (Rb-S) and C-S comparisons (Table 1)
Summary
Malaria burden remains high in the tropical world with around 584 000 deaths globally in 2013 alone and mostly in African children under the age of 5 (WHO2014). Resistance to pyrethroids and carbamates is spreading rapidly in Anopheles mosquitoes across Africa. In the major vector Anopheles funestus, both pyrethroid and carbamate resistances are increasingly reported in southern and West Africa with the fear that such resistance could disrupt malaria control (Brooke et al 2001; Casimiro et al 2006; Cuamba et al 2010; Hunt et al 2010; Djouaka et al 2011; Wondji et al 2012). The molecular basis of carbamate resistance in An. funestus remained uncharacterized, whereas significant progress has been made in elucidating the mechanisms of pyrethroid resistance (Wondji et al 2009; Riveron et al 2013; Ibrahim et al 2015). A detailed characterization of the molecular basis of carbamate resistance is a prerequisite for the implementation of suitable resistance management strategies to control this malaria vector
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