A 6.5‐kb intergenic structural variation enhances P450‐mediated resistance to pyrethroids in malaria vectors lowering bed net efficacy

Abstract

Elucidating the complex evolutionary armory that mosquitoes deploy against insecticides is crucial to maintain the effectiveness of insecticide‐based interventions. Here, we deciphered the role of a 6.5‐kb structural variation (SV) in driving cytochrome P450‐mediated pyrethroid resistance in the malaria vector, Anopheles funestus. Whole‐genome pooled sequencing detected an intergenic 6.5‐kb SV between duplicated CYP6P9a/b P450s in pyrethroid‐resistant mosquitoes through a translocation event. Promoter analysis revealed a 17.5‐fold higher activity (p < .0001) for the SV− carrying fragment than the SV− free one. Quantitative real‐time PCR expression profiling of CYP6P9a/b for each SV genotype supported its role as an enhancer because SV+/SV+ homozygote mosquitoes had a significantly greater expression for both genes than heterozygotes SV+/SV− (1.7‐ to 2‐fold) and homozygotes SV−/SV− (4‐to 5‐fold). Designing a PCR assay revealed a strong association between this SV and pyrethroid resistance (SV+/SV+ vs. SV−/SV−; odds ratio [OR] = 2,079.4, p < .001). The 6.5‐kb SV is present at high frequency in southern Africa (80%–100%) but absent in East/Central/West Africa. Experimental hut trials revealed that homozygote SV mosquitoes had a significantly greater chance to survive exposure to pyrethroid‐treated nets (OR 27.7; p < .0001) and to blood feed than susceptible mosquitoes. Furthermore, mosquitoes homozygote‐resistant at the three loci (SV+/CYP6P9a_R/CYP6P9b_R) exhibited a higher resistance level, leading to a far superior ability to survive exposure to nets than those homozygotes susceptible at the three loci, revealing a strong additive effect. This study highlights the important role of structural variations in the development of insecticide resistance in malaria vectors and their detrimental impact on the effectiveness of pyrethroid‐based nets.

Multi-omics analysis identifies a CYP9K1 haplotype conferring pyrethroid resistance in the malaria vector Anopheles funestus in East Africa

“…This locus contains the CYP6P9a and CYP6P9b cytochrome P450 genes, which confer pyrethroid-resistance and are under strong directional selection in southern African populations of An. funestus [5,11,19]. Although statistically attractive, the replicated PoolSeq offers us little extra over inter-country comparisons of pooled-sequencing as demonstrated by detection of the rp1 locus here and prior work [5,9].…”

“…Such genes were also over-expressed in resistant An. funestus mosquitoes in previous studies [5,11,19,20].…”

Experimental Hut Trials Reveal That CYP6P9a/b P450 Alleles Are Reducing the Efficacy of Pyrethroid-Only Olyset Net against the Malaria Vector Anopheles funestus but PBO-Based Olyset Plus Net Remains Effective

“…Recent studies have focused on elucidating the genetic factors which cause the increased expression of detoxification genes associated with insecticide resistance in malaria vectors such as An. funestus, leading to the development of simple molecular assays used to detect metabolic resistance [28][29][30][31].…”

“…The recent design of molecular assays for the duplicated CYP6P9a/b and the associated 6.5 kb structural variant (SV), refs. [28,29,31] now provides a great opportunity to assess the impact of P450-based pyrethroid resistance on the efficacy of various LLINs, including pyrethroid-only (Olyset) and PBO (Olyset Plus) nets. This will provide evidence-based information on their effectiveness in the presence of a growing and escalading resistance to pyrethroids.…”

“…This will provide evidence-based information on their effectiveness in the presence of a growing and escalading resistance to pyrethroids. Previous assessment of this impact on PermaNet nets made with type II pyrethroid deltamethrin has been performed [28,29,31], but this remains to be carried out for type I pyrethroid nets, such as the common permethrin-based Olyset nets.…”