First report of the pyrethroid-resistance-associated V1016I mutation in Aedes aegypti at the seaport of Cotonou, Benin, West Africa.

BACKGROUND: Monitoring insecticide resistance in disease vectors is a key strategy to anticipate emerging and re-emerging diseases, particularly in the context of interventions such as vector control interventions. This study aims to assess the insecticide resistance profile of three major mosquito species in the Port of Cotonou and to characterize the molecular mechanisms associated with resistance, with a focus on mutations in Aedes aegypti and Anopheles gambiae s.l. METHODS: This study was conducted at the Port of Cotonou from June 2023 to November 2024. Standard WHO susceptibility tube tests were performed using several insecticides: deltamethrin, permethrin, and alpha-cypermethrin (pyrethroids), bendiocarb (carbamate), and pirimiphos-methyl (organophosphate). The potential role of metabolic resistance mechanisms was investigated through synergist assays using piperonyl butoxide (PBO). In addition, molecular analyses were performed: SINE-PCR was used to differentiate Anopheles gambiae from Anopheles coluzzii, while allele-specific PCR (AS-PCR) assays were employed to detect the mutations L1014F and L1014S in Anopheles gambiae s.l., as well as the F1534C, S989P, and V1016I mutations in Aedes aegypti. RESULTS: The three mosquito species tested showed confirmed resistance to most pyrethroids (rate mortality < 90%). The addition of PBO significantly restored the efficacy of alpha-cypermethrin, with mortality rates of 93% in Culex, 100% in Aedes, and 98.97% in Anopheles, compared to 45, 89.80, and 69.39% respectively, when alpha-cypermethrin was used alone. This difference was statistically significant, with p-values < 0.05. Pirimiphos-methyl remained effective, inducing 100% mortality across all species. Suspected resistance to bendiocarb was observed in Aedes aegypti and Anopheles gambiae, with respective mortality rates of 92.78 and 95.83%, while resistance to bendiocarb was confirmed in Culex quinquefasciatus, with a mortality rate of 85.86%. The analysis of kdr mutations in Anopheles gambiae s.s. and Anopheles coluzzii revealed a high prevalence of the L1014F mutation in both species, with allele frequencies of f(F) = 0.8421 in An. gambiae s.s. and f(F) = 0.9292 in An. coluzzii. No individuals carried the wild-type homozygous genotype (SS) for this mutation, indicating a high level of fixation of the resistant L1014F allele, particularly in An. coluzzii. The L1014S mutation, previously detected in northern Benin, was identified for the first time in the southern region in heterozygous Anopheles gambiae s.s. INDIVIDUALS: This mutation was absent in An. coluzzii but was detected at a very low frequency of f(S) = 0.052 in An. gambiae s.s.. For the F1534C mutation, the frequency of the mutant C allele was f(C) = 0.7284. Regarding the V1016I mutation, the mutant I allele had a frequency of f(I) = 0.1935. Notably, this is the first detection of this mutation in Benin. Finally, the S989P mutation showed an allele frequency of f(P) = 0.637 for the mutant P allele. These findings reveal a high prevalence of the F1534C and S989P mutations, while the V1016I mutation appears at a more moderate frequency in the tested population. CONCLUSION: This study confirms widespread resistance to pyrethroids among major mosquito vectors in the Port of Cotonou. The detection of L1014S and V1016I mutations highlights an evolving dynamic of kdr-mediated resistance. These findings underscore the urgent need for strengthened insecticide resistance monitoring within an integrated vector management framework, taking into account the target species, underlying resistance mechanisms, and local ecological contexts.