Gyimah_2026_Sci.Rep__

The evolution of insecticide resistance in mosquitoes is shaped by direct insecticide exposure and environmental conditions encountered during development. This study used an experimental evolution approach to investigate how larval habitats select for insecticide resistance profiles in An. gambiae s.l. over 10 successive generations. Mosquito larvae were collected from a single site in Accra, Ghana, and reared separately in three water types: field-collected water (FW), dechlorinated tap water (TW), and distilled water (DW) for 10 filial generations. At each generation, phenotypic susceptibility to four classes of insecticides was assessed using WHO bioassays, including synergist assays with piperonyl butoxide (PBO) and, the frequencies of the kdr-w (L995F) and ace-1 (G119S) target-site mutations were determined molecularly. Activity of detoxification enzymes, P450 monooxygenases, alpha- and beta-carboxylesterases, and insensitive acetylcholinesterase were measured biochemically. Key selected physicochemical properties of the rearing waters were also characterized to provide contextual information on potential larval environmental influences. kdr-w mutation rapidly increased to fixation by generation F(2) in mosquitoes reared in dechlorinated tap water, suggesting a strong, water-mediated selection advantage. Across the entire experiment, kdr-w mutation declined from 90-100% at F(0) to ~ 63% by F(10), whereas ace-1 mutation increased steadily from 60-90%. Mosquitoes reared in the nutrient and ion-rich field water consistently exhibited significantly higher detoxification enzyme activity, especially alpha-esterases and mixed-function oxidases, with oxidase levels elevated by as much as 32% relative to TW and DW indicating phenotypic plasticity induced by natural environmental co-factors. These findings demonstrate that larval aquatic conditions strongly influence both the genetic and biochemical foundations of insecticide resistance in adult An. gambiae s.l. Environmental co-factors within breeding habitats can shape resistance trajectories, highlighting larval source management as a potentially important strategy not only for reducing vector populations but also for mitigating the selection and persistence of resistant phenotypes.