Biochemical and molecular characterization of organophosphate resistance in Aedes aegypti in the sub-Himalayan region of West Bengal, India.

Continuous use of organophosphate for dengue vector management has increased the risk of resistance development in Aedes aegypti, threatening the effectiveness of current control programmes. This study evaluated susceptibility and mechanisms associated with organophosphate resistance in Ae. aegypti populations from five dengue-endemic districts of sub-Himalayan West Bengal, India. Larvae and adults were subjected to insecticide bioassays, synergist assays, biochemical assays, gene expression analysis, partial sequencing of the acetylcholinesterase gene (ace-1), and molecular docking. Bioassays confirmed malathion resistance (mortality<90%) in DAR, JAL and ALI populations, whereas UTT and COO exhibited possible resistance (mortality=90-98%). High temephos resistance (RR50>10) was detected in DAR, JAL and ALI, while UTT showed moderate resistance (RR50=7.31) and COO remained susceptible (RR50=2.52). Pre-exposure to triphenyl phosphate restored susceptibility in most populations, indicating possible carboxylesterase-mediated metabolic resistance. Resistant populations exhibited significantly elevated alpha and beta carboxylesterase activity. Stage-specific overexpression was observed, with CCEae3a (13.68-39.21-fold) overexpressed in resistant larvae, whereas CCEae6a (12.25-33.99-fold) was overexpressed in resistant adults and both positively correlating with resistance phenotypes. Enzyme activity and gene expression were significantly correlated with reduced malathion mortality and increased temephos LC50. Most individuals from JAL, COO and UTT did not exhibit altered AChE activity, while altered activity was detected in < 25% of individuals from DAR and ALI. Partial ace-1 sequence analysis identified no organophosphate resistance-associated polymorphisms within analysed region, suggesting limited role of target-site insensitivity. Molecular docking suggested possible interactions of malathion and temephos with carboxylesterase. These findings provide biochemical and molecular markers for resistance surveillance and support integrated vector management.