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 6.5kb structural variation (SV) in driving cytochrome P450-mediated pyrethroid resistance in the malaria vector, Anopheles funestus . Whole genome pooled sequencing detected an intergenic 6.5kb SV between duplicated CYP6P9a/b P450s in pyrethroid resistant mosquitoes through a translocation event. Promoter analysis revealed a 17.5-fold higher activity (P&lt;0.0001) for the SV-carrying fragment than the SV-free one. qRT-PCR expression profiling of CYP6P9a/b for each SV genotype supported its role as an enhancer since SV+/SV+ homozygote mosquitoes had significantly greater expression for both genes than heterozygotes SV+/SV- (1.7-2-fold) and homozygotes SV-/SV- (4-5-fold). Designing a PCR assay revealed a strong association between this SV and pyrethroid resistance (SV+/SV+ vs SV-/SV-; OR=2079.4, P =&lt;0.001). The 6.5kb 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 significantly greater chance to survive exposure to pyrethroid-treated Nets (OR 27.7; P &lt; 0.0001) and to blood feed than susceptible. Furthermore, triple homozygote resistant (SV+/CYP6P9a_R/CYP6P9b_R) exhibit a higher resistance level leading to a far superior ability to survive exposure to nets than triple susceptible mosquitoes, 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.

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