Association of Reduced Long-Lasting Insecticidal Net Efficacy and Pyrethroid Insecticide Resistance With Overexpression of CYP6P4, CYP6P3, and CYP6Z1 in Populations of Anopheles coluzzii From Southeast Côte d'Ivoire.

BACKGROUND: Resistance to major public health insecticides in Côte d'Ivoire has intensified and now threatens the long-term effectiveness of malaria vector control interventions. METHODS: This study evaluated the bioefficacy of conventional and next-generation long-lasting insecticidal nets (LLINs), determined resistance profiles, and characterized molecular and metabolic mechanisms in wild Anopheles coluzzii from Southeast Côte d'Ivoire in 2019. RESULTS: Phenotypic resistance was intense: >25% of mosquitoes survived exposure to 10 times the doses of pyrethroids required to kill susceptible populations. Similarly, the 24-hour mortality rate with deltamethrin-only LLINs was very low and not significantly different from that with an untreated net. Sublethal pyrethroid exposure did not induce significant delayed vector mortality effects 72 hours later. In contrast, LLINs containing the synergist piperonyl butoxide, or new insecticides clothianidin and chlorfenapyr, were highly toxic to A. coluzzii. Pyrethroid-susceptible A. coluzzii were significantly more likely to be infected with malaria, compared with those that survived insecticidal exposure. Pyrethroid resistance was associated with significant overexpression of CYP6P4, CYP6P3, and CYP6Z1. CONCLUSIONS: Study findings raise concerns regarding the operational failure of standard LLINs and support the urgent deployment of vector control interventions incorporating piperonyl butoxide, chlorfenapyr, or clothianidin in areas of high resistance intensity in Côte d'Ivoire.

The P450 CYP6Z1 confers carbamate/pyrethroid cross-resistance in a major African malaria vector beside a novel carbamate-insensitive N485I acetylcholinesterase-1 mutation.

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.