Abstract
INTRODUCTION: Control of Stegomyia aegypti (St. aegypti), a major vector of arboviruses, has been increasingly challenged by insecticide resistance, driven by genetic, metabolic, behavioral, and environmental factors. While extensively studied in other contexts, the role of gut microbiota in insecticide resistance remains largely unexplored. Therefore, this study assessed the α-cypermethrin susceptibility status of St. aegypti from Puducherry by characterizing the culturable gut bacteria from resistant and susceptible mosquito populations and evaluating their role in mediating resistance. MATERIALS AND METHODS: Mosquito larvae were collected from Puducherry, India, reared, and emerged as female St. aegypti, which were tested for α-CP (0.05%) susceptibility following World Health Organisation (WHO) guidelines, along with laboratory strains as controls. Larval and adult guts were dissected, and culturable bacteria were isolated and identified using MALDI-TOF MS and 16S rRNA sequencing. The resistant populations were treated with vancomycin, gentamicin, and streptomycin to suppress the gut microbiota and reassessed for α-CP susceptibility. The gut bacterial load was quantified by qPCR, and bacterial isolates were evaluated for in vitro degradation ability in minimal salt medium containing α-CP. RESULTS: The St. aegypti populations tested from three locations in Puducherry exhibited confirmed resistance to α-CP. A total of 35 gut bacterial isolates were obtained (α-CP-resistant -26 and α-CP-susceptible -9). Compared to susceptible mosquitoes, resistant populations showed greater gut bacterial diversity and higher bacterial load. The resistant mosquitoes were dominated by Bacillota (adults 46.6%, larvae 54.5%) and Pseudomonadota (adults 46.6%, larvae 45.4%), with Bacillaceae and Enterobacteriaceae being the most abundant. The antibiotic-mediated microbiota suppression increased mortality (vancomycin 97%, gentamicin 95%, streptomycin 92%) and reduced LT50 following α-CP exposure. Further, isolates such as Enterobacter hormaechei and Bacillus spp. demonstrated growth in α-CP supplemented minimal salt media. DISCUSSION: These findings suggest a strong association between gut microbiota and α-CP resistance in St. aegypti. The higher microbial diversity, evenness, and bacterial load observed in resistant populations, along with increased mortality following antibiotic treatment and the ability of certain isolates to grow in α-CP-supplemented media, indicate a potential symbiont-mediated mechanism contributing to insecticide resistance.