Sub-lethal exposure to chlorfenapyr reduces the probability of developing Plasmodium falciparum parasites in surviving Anopheles mosquitoes

Pyrethroid resistance in the key malaria vectors threatens the success of pyrethroid-treated nets. To overcome pyrethroid resistance, Interceptor(R) 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 insects mitochondria, constituting a mode of action preventing cross-resistance to pyrethroids. Recent epidemiological trials conducted in Benin and Tanzania confirm IG2s public health value in areas with pyrethroid-resistant Anopheles mosquitoes. As chlorfenapyr might also interfere with the metabolic mechanism of the Plasmodium parasite, 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 reduce Plasmodium falciparum transmission using a modified WHO tunnel test with a dose yielding sub-lethal effects. Pyrethroid-resistant Anopheles gambiae s.s. with established mixed-function oxidases and Vgsc-L995F knockdown resistance alleles were exposed to untreated netting and netting treated with 200 mg/m3 chlorfenapyr 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 of P. falciparum infection 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 the P. falciparum infection. 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 on Plasmodia since they, too possess mitochondria and this could reduce the development of Plasmodium in 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 in Plasmodium-infected Anopheles mosquitoes. In this bioassay, we found that chlorfenapyr substantially reduces the proportion of Plasmodium-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.