Signature of resistance gene evolution and pyrethroid resistance escalation in the major malaria vector Anopheles funestus across the Kenyan Rift Valley

Background Landscape features such as the Rift Valley (RV) can restrict gene flow in malaria vectors and influence resistance patterns. Here, we assessed resistance alleles and profiles in Anopheles funestus s.s. populations across Kenyan malaria-endemic regions separated by the RV. Methods Anopheles funestus s.s. populations in western, coastal and Kerio Valley (KV, within the RV) were assessed for key resistance markers and their association with Plasmodium sporozoite infection. Phenotypic resistance using F1 progeny was also assessed. Results The 4.3Kb-SV and G454A-Cyp9k1 alleles were nearly fixed in western Kenya but declined towards the RV and coast, whereas L119F-GSTe2 increased across a west-KV-coast gradient with a novel haplotype distinct from known African variants detected at the coast. There were lower odds of Plasmodium infection in mosquitoes with L119F-GSTe2-RR than RS genotype (OR = 0.2, p = 0.046). Likewise, mosquitoes harboring the R allele of the 4.3kb marker had higher Plasmodium infection rates than the S allele (OR = 5.7, p = 0.049). An. funestus populations exhibited a high degree of pyrethroid resistance with intensity higher in KV compared to western Kenya, a traditional malaria hotspot. Pre-exposure to PBO increased mortality for type II (deltamethrin, alpha-cypermethrin), than I (permethrin) pyrethroids, yet recovery remained lower in KV, suggesting non-P450-mediated resistance. Coastal mosquitoes showed extreme permethrin resistance (< 10% mortality at 10× dose). DDT resistance was widespread, while all populations remained fully susceptible to bendiocarb, pirimiphos-methyl, clothianidin, and chlorfenapyr. Conclusions Region-specific selection may drive varying resistance profiles in An. funestus across the RV, with implications for malaria transmission and insecticide resistance management.