Substrate promiscuity of key resistance P450s confers clothianidin resistance whilst increasing chlorfenapyr potency in malaria vectors

Abstract

Novel insecticides were recently introduced to counter pyrethroid resistance threats in African malaria vectors. To prolong their effectiveness, potential cross-resistance from promiscuous pyrethroids metabolic resistance mechanisms must be elucidated. Here, we demonstrate that the duplicated P450s, CYP6P9a/b, proficient pyrethroid metabolizers, are reducing neonicotinoid efficacy in Anopheles funestus while exacerbating the potency of Chlorfenapyr. Clothianidin resistance was strongly linked to CYP6P9a/-b genotypes with homozygote resistant individuals significantly surviving more than other genotypes. An additive effect of both genes was observed as double homozygote resistant genotypes were more resistant to clothianidin than other genotype combinations. However, homozygote resistant mosquitoes were more susceptible to chlorfenapyr than homozygote susceptible using laboratory and field tests. Transgenic expression of CYP6P9a/-b in Drosophila confirmed that flies expressing both genes were significantly more resistant to neonicotinoids than controls whereas the contrasting pattern was observed for chlorfenapyr. This phenotype was also confirmed by RNAi knock-down experiments. In vitro expression of recombinant CYP6P9a and metabolism assays established that it significantly depletes both clothianidin and chlorfenapyr, with metabolism of chlorfenapyr producing the insecticidally active intermediate, metabolite tralopyril. This study highlights the risk of cross-resistance between pyrethroid and neonicotinoid and reveals that chlorfenapyr-based control interventions such as Interceptor G2 could remain efficient against some P450-based resistant mosquitoes.