Establishment of a Stable Anopheles melas Laboratory Colony for the First Time and Characterization of Its Pyrethroid...

In Senegal, scaling up vector control has significantly reduced malaria, but coastal transmission remains a challenge due to secondary vectors like Anopheles melas. This exophilic and zoophilic species evades conventional indoor interventions, yet its bionomics and insecticide resistance profiles remain poorly characterized due to difficulties in laboratory colonization. Anopheles melas larvae were collected from brackish breeding sites in Mbine Coly (Mbour District). A laboratory colony was established from 26 PCR-confirmed founder females and stabilized up to the F17 generation by optimizing water salinity (10 g/L) and transitioning to communal oviposition. Standardized WHO tube bioassays were conducted on generations F2 to F9 to evaluate susceptibility to pyrethroids, organophosphates, and carbamates, alongside synergist tests using 4% piperonyl butoxide (PBO). The colony showed confirmed resistance to alpha-cypermethrin (81.7% mortality) and suspected resistance to deltamethrin (91.3%) and permethrin (94.3%). Pre-exposure to PBO fully restored susceptibility to deltamethrin and permethrin (100%), and significantly increased alpha-cypermethrin mortality to 96.3% (p < 0.001). While metabolic resistance typically involves multiple enzyme families including cytochrome P450s, glutathione S-transferases (GSTs), and esterases, these synergist results demonstrate a predominant role of cytochrome P450-mediated detoxification pathways in this population. Moderate-intensity resistance was confirmed for alpha-cypermethrin at 5× diagnostic doses. Conversely, the population was 100% susceptible to pirimiphos-methyl and bendiocarb. This study reports the successful long-term colonization of a wild An. melas strain for the first time and provides the first comprehensive evidence of multi-insecticide resistance in this population. The presence of high-intensity, metabolic pyrethroid resistance strongly justifies the immediate deployment of next-generation PBO-LLINs and the integration of organophosphates or carbamates in indoor residual spraying rotations to manage resistance in coastal Senegal.