Genetic mapping of resistance: A QTL and associated polymorphism conferring resistance to alpha-cypermethrin inAnopheles funestus

The heavy reliance on pyrethroid-based interventions has accelerated the spread of resistance malaria vectors includingAnopheles funestus, jeopardising control efforts. The efficacy of Insecticide-based interventions, especially insecticide-treated nets (ITNs), the cornerstone of malaria control and management, is threatened by the widespread resistance complicating malaria control. Alpha-cypermethrin, a type II pyrethroid, is increasingly utilised in various ITN formulations, including those combined with piperonyl butoxide (PBO) and chlorfenapyr-based Interceptor® G2 (IG2) nets, to enhance effectiveness against resistant mosquito populations. Therefore, understanding the molecular basis of resistance is essential to monitor and track resistance trends for an effective malaria control program. In this study, we identified a 1.4 Mb QTL on the telomeric end of the left arm of chromosome 2, conferring resistance to α-cypermethrin (rap1QTL). Different crossing schemes and sequencing approaches were explored to determine the most effective strategy. Individual-based QTL mapping performed on segregating individuals from an isofemale family identified a QTL at the F₇generation. Higher recombination density relative to the physical genome in the F₇isofemale family, with a recombination every 240 kb, facilitated the detection of a QTL compared to the F₂family (335 kb/cM). Additionally, we exploited bulk segregate analysis (BSA) between susceptible and resistant phenotypes from the F₇isofemale family and an F₇mixed cross-family to perform cost-effective and rapid QTL-mapping discovery. The strongest signal in both independent BSA analyses overlaps with therap1QTL, further supporting its role in α-cypermethrin resistance. The known resistant alleles of the cytochrome P450CYP6P9aand 6.5-kb structural variant within therap1QTL strongly correlate with survival to α-cypermethrin. In this study, we validated that previously developed DNA-based assays, originally designed to monitor permethrin resistance, are effective for tracking resistance to α-cypermethrin as well. Additionally, we identified candidate variants that can serve as reliable markers for monitoring α-cypermethrin resistance.