Larval breeding water drives differential selection pressures on genetic insecticide resistance and metabolic enzyme plasticity in Anopheles gambiae s.l

Background The control of mosquito-borne diseases is heavily reliant on insecticide-based interventions. The evolution of insecticide resistance is a complex process driven by both direct chemical exposure and indirect environmental pressures. While the larval environment is known to influence adult mosquito traits, its long-term impact on the evolution of multiple resistance mechanisms is poorly understood. This study used an experimental evolution approach to investigate how larval aquatic environments select for insecticide resistance profiles in An. gambiae s.l. over 10 successive generations. Methods Anopheles gambiae s.l. larvae were collected from a single site in Accra, Ghana, and colonized in the laboratory for 10 filial generations. The larvae were reared in three distinct water types: field-collected water (FW), dechlorinated tap water (TW), and distilled water (DW). At each generation, phenotypic susceptibility to four classes of insecticides was assessed using WHO bioassays, including synergist assays with piperonyl butoxide (PBO). The frequencies of the kdr-w ( L 995 F ) and ace-1 ( G 119 S ) target-site mutations were determined using molecular analysis. The activity of key metabolic enzymes, P450 monooxygenases, carboxylesterases (α and β), and insensitive acetylcholinesterase was quantified through biochemical assays. Selected physicochemical properties of the rearing waters were also characterized. Results kdr-w mutation rapidly increased to fixation by generation F ₂ in mosquitoes reared in dechlorinated tap water, a trend not observed in the other two water types, suggesting a strong, water-mediated selective advantage provided by tap water chemistry. There was an overall significant decline in the frequency of the kdr-w mutation from 90–100% at F0 to ~ 63% by F ₁₀ . Conversely, the frequency of the ace-1 mutation increased steadily from approximately 60% to 90% over the 10 generations. Mosquitoes reared in the nutrient and ion-rich field water consistently exhibited significantly elevated levels of detoxification enzymes, particularly ⍺-esterases and mixed-function oxidases (up to 32% for oxidases), compared to those reared in tap and distilled water indicating phenotypic plasticity induced by natural environmental co-factors. Conclusion The larval aquatic environment fundamentally shapes the genetic and biochemical basis of insecticide resistance in adult Anopheles gambiae s.l.. The physicochemical composition of breeding water induces metabolic detoxification systems and influences the rate of fixation of target-site mutations. These findings suggest that environmental co-factors play a critical role in the persistence of resistance genes, providing a new evolutionary framework for integrated vector management. Larval source management can serve not only to reduce vector populations but also be a critical tool for managing insecticide resistance by modifying the environmental pressures that select for resistant phenotypes.