WHO insecticide susceptibility assay

Insecticide susceptibility tests were conducted on both adults captured as well as larvae raised to adults. Adults captured (all females) were immediately transported to the laboratory and sugar fed. Insecticide susceptibility tests using 0.75% permethrin-impregnated papers, following standard insecticide susceptibility monitoring guidelines [48], were conducted 3–4 h following capture, irrespective of abdominal (blood-fed) status. Larvae sampled from Ahero were raised to adults in the insectary following standard larval rearing methodologies [47]. Three-day-old adult, non-blood fed females raised from larvae were exposed to 0.75% permethrin-impregnated papers [48]. Anopheles gambiae (Kisumu Strain) permethrin-susceptible mosquitoes were used in paired controls. Negative controls using only carrier oil and susceptible controls were not conducted. For all specimens, knockdown rates were recorded at 10-min intervals for 1 h and mortality recorded 24 h post-exposure. Mosquitoes that remained alive after 24 h of exposure were killed at − 20 °C. All mosquitoes were labeled by assay phenotype (knocked down/alive) and morphologically identified to species before being individually preserved in Eppendorf tubes containing silica gel. Phenotypic insecticide resistance status was determined based on mortality rates according to WHO [48], where mortality rates between 98 and 100% indicated susceptibility, 90–97% suggested possible resistance that needed additional analysis and < 90% indicated resistance.