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.
Highlights
Malaria control programmes rely heavily on insecticide-based vector control interventions including the mass distribution of long-lasting insecticidal nets (LLINs) impregnated with pyrethroids (Bhatt et al, 2015)
This study investigated the role that structural variation (SV) in cis-regulatory regions play in cytochrome P450 monooxygenases (P450s)-mediated metabolic resistance to pyrethroids in mosquitoes by focusing on a 6.5-kb intergenic insertion
The whole-genome Pool-seq confirmation of the 6.5-kb insertion in field populations of A. funestus supported the previous report in the laboratory pyrethroid-resistant FUMOZ-R strain (Weedall et al, 2019)
Summary
Malaria control programmes rely heavily on insecticide-based vector control interventions including the mass distribution of long-lasting insecticidal nets (LLINs) impregnated with pyrethroids (Bhatt et al, 2015). The widespread resistance to insecticides in major malaria vectors including Anopheles gambiae and Anopheles funestus is probably one of the main factors behind the recent increase in malaria cases across the world, from 214 million in 2015 to 219 million in 2017 (WHO, 2018) or the stagnation of such control efforts (WHO, 2019). Such growing resistance reports call for urgent action to implement suitable resistance management strategies to reduce the impact on the effectiveness of current and future insecticide-based tools, as highlighted by the WHO global plan for insecticide resistance management (WHO, 2012). We designed a PCR (polymerase chain reaction)-based diagnostic that allows us to track this SV-based resistance and use it to show that this 6.5-kb insertion acts as an enhancer significantly contributing to increase pyrethroid resistance and to exacerbate the loss of efficacy of long-lasting insecticidal nets against malaria vectors
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