Sub-lethal exposure to chlorfenapyr reduces the probability of developingPlasmodium falciparumparasites in survivingAnophelesmosquitoes

AbstractPyrethroid resistance in the key malaria vectors threatens the success of pyrethroid-treated nets. To overcome pyrethroid resistance, Interceptor® G2 (IG2), a ‘first-in-class’ dual insecticidal net that combines alpha-cypermethrin with chlorfenapyr was developed. Chlorfenapyr is a pro-insecticide, requiring bio-activation by oxidative metabolism within the insect’s mitochondria, constituting a mode of action preventing cross-resistance to pyrethroids. Recent epidemiological trials conducted in Benin and Tanzania confirm IG2’s public health value in areas with pyrethroid-resistantAnophelesmosquitoes. As chlorfenapyr might also interfere with the metabolic mechanism of thePlasmodiumparasite, we hypothesised that chlorfenapyr may provide additional transmission-reducing effects even if a mosquito survives a sub-lethal dose. Therefore, we tested the effect of chlorfenapyr netting to reducePlasmodium falciparumtransmission using a modified WHO tunnel test with a dose yielding sub-lethal effects. Pyrethroid-resistantAnopheles gambiae s.s. with established mixed-function oxidases andVgsc-L995F knockdown resistance alleles were exposed to untreated netting and netting treated with 200 mg/m3chlorfenapyr for 8 hours overnight and then fed on gametocytemic blood meals from naturally infected individuals. Prevalence and intensity of oocysts and sporozoites were determined on day 8 and day 16 after feeding. Both prevalence and intensity ofP. falciparuminfection in the surviving mosquitoes were substantially reduced in the chlorfenapyr-exposed mosquitoes compared to untreated nets. The odds ratios in the prevalence of oocysts and sporozoites were 0.33 (95% confidence interval; 95% CI: 0.23-0.46) and 0.43 (95% CI: 0.25-0.73), respectively, while only the incidence rate ratio for oocysts was 0.30 (95% CI: 0.22-0.41). We demonstrated that sub-lethal exposure of pyrethroid-resistant mosquitoes to chlorfenapyr substantially reduces the proportion of infected mosquitoes and the intensity of theP. falciparuminfection. This will likely also contribute to the reduction of malaria in communities beyond the direct killing of mosquitoes.Author summaryMalaria remains a serious problem in many tropical and sub-tropical areas, affecting the welfare and health of many individuals. Since 2016, malaria has increased and the emergence of mosquitoes that are resistant to different classes of insecticides used in vector control tools may have contributed to some of this increase. Therefore, insecticides with a different mode of action are required to manage vector resistance to insecticides used for public health vector control. One of the main resistance mechanisms is metabolic resistance where mosquitoes upregulate detoxification enzymes to break down insecticides. Chlorfenapyr is a pyrrole-pro-insecticide that is metabolised by these detoxification enzymes from chlorfenapyr to tralopyril that disrupts mitochondrial function in mosquitoes. We therefore hypothesized that the metabolites of chlorfenapyr may also have an effect onPlasmodiasince they, too possess mitochondria and this could reduce the development ofPlasmodiumin mosquitoes that survived a sub-lethal dose of chlorfenapyr. In this study we established and evaluated a modified WHO tunnel assay to investigate the effect of chlorfenapyr inPlasmodium-infectedAnophelesmosquitoes. In this bioassay, we found that chlorfenapyr substantially reduces the proportion ofPlasmodium-infected mosquitoes at doses sub-lethal to mosquitoes. Our findings demonstrate that chlorfenapyr provides additional benefits beyond mosquito killing although the mechanism of action requires further elucidation.