Widespread pyrethroid resistance and high kdr L995F allele frequencies in Anopheles gambiae sensu lato populations from Southern Benin, West Africa

BACKGROUND: Pyrethroid resistance in Anopheles gambiae sensu lato threatens the efficacy of long-lasting insecticidal nets (LLINs) in Benin. District-level resistance data and evaluations of next-generation insecticides are needed to inform insecticide resistance management (IRM). METHODS: We assessed insecticide susceptibility in An. gambiae s.l. populations from three southern Beninese districts (Porto-Novo, Akpro-Missérété, Lokossa) via WHO tube bioassays with pyrethroids (deltamethrin, permethrin, alpha-cypermethrin), pirimiphos-methyl, clothianidin, and chlorfenapyr. Molecular species identification and genotyping of the kdr L995F and Ace-1R G119S mutations were performed via allele-specific PCR. RESULTS: Confirmed pyrethroid resistance was recorded at all sites, with permethrin mortality as low as 17.1% (95% CI 10.6-26.2%) in Porto-Novo. The kdr L995F allele was detected at near-fixation frequencies ranging from 82 to 93% across both An. coluzzii and An. gambiae s.s. Ace-1R G119S remained rare (3-7%), which is consistent with its retained susceptibility to pirimiphos-methyl. The species composition ranged from exclusive to An. coluzzii in Porto-Novo to near-equal distribution in Lokossa. Clothianidin was associated with ≥ 99% cumulative mortality by day 7, and chlorfenapyr was associated with ≥ 98.8% cumulative mortality within 24-48 h across all populations. CONCLUSIONS: Pyrethroid resistance is entrenched in southern Benin, driven by the near-fixation of kdr L995F along an urbanization gradient. The fully preserved efficacy of clothianidin and chlorfenapyr against these resistant populations supports the urgent scale-up of next-generation LLINs as a core IRM strategy in this high-burden region.