The voltage-gated sodium channel, para, limits Anopheles coluzzii vector competence in a microbiota dependent manner

Abstract

The voltage-gated sodium channel, para, is a target of DDT and pyrethroid class insecticides. Single nucleotide mutations in para, called knockdown resistant or kdr, which contribute to resistance against DDT and pyrethroid insecticides, have been correlated with increased susceptibility of Anopheles to the human malaria parasite Plasmodium falciparum. However, a direct role of para activity on Plasmodium infection has not yet been established. Here, using RNA-mediated silencing, we provide in vivo direct evidence for the requirement of wild-type (wt) para function for insecticide activity of deltamethrin. Depletion of wt para, which is susceptible to insecticide, causes deltamethrin tolerance, indicating that insecticide-resistant kdr alleles are likely phenocopies of loss of para function. We then show that normal para activity in An. coluzzii limits Plasmodium infection prevalence for both P. falciparum and P. berghei. A transcriptomic analysis revealed that para activity does not modulate the expression of immune genes. However, loss of para function led to enteric dysbiosis with a significant increase in the total bacterial abundance, and we show that para function limiting Plasmodium infection is microbiota dependent. In the context of the bidirectional "enteric microbiota-brain" axis studied in mammals, these results pave the way for studying whether the activity of the nervous system could control Anopheles vector competence.

Figure 1

Depletion of para expression reduces deltamethrin sensitivity. (A) Daily mortality rates between dsGFP and dsPara mosquito groups were monitored for 14 days after dsRNA injection. No difference in mortality was found between dsGFP and dsPara. (B) Sensitivity to the pyrethroid deltamethrin (0.05%) was measured in dsGFP and dsPara by exposing the mosquitoes to the impregnated papers for 7 min, and 24h post-exposure, the mortality rate was counted in each group. The 7 min time exposure choice was made to avoid saturation of the para channels by the insecticide, and for which the control group is reaching close to 100% death. Mortality rate was significantly reduced in dsPara background at 4 and 6-days post-exposure, but not at 10 days.

Figure 2

Para normal activity limits Plasmodium infection prevalence. Panel (A) and panel (B) show results of infection prevalence (proportion of infected mosquitoes) in An. coluzzii infected with P. berghei and P. falciparum, respectively. Grey color shows proportion of infected mosquitoes. Infection prevalence between dsPara and dsGFP was measured at d-8 post-infection and showed statistically significant increase in dsPara as compared to dsGFP control, for both Plasmodium species. Combined p-value (Fisher method) from the 3 independent biological replicates was indicated in each graph represented in both A and B panels. n = Total number of dissected mosquitoes. Values obtained in every biological replicate are depicted by dots and dotted lines.

Figure 3

Para activity limiting Plasmodium infection is microbiota dependent. (A) 16srDNA relative fold change was measured by qPCR in samples collected from antibiotic treated and non-treated backgrounds. In the non-treated background, Para silencing leads to increased level of the total abundance of An. coluzzii bacteria as compared to the dsGFP control group (dotted line), whereas in antibiotic treated background this effect is abolished. The ratio of the normalized 16srDNA in dsPara/dsGFP was calculated using triplicates. *: Statistical p-value related to the deltaCt distribution between dsPara and dsGFP on 3 independent biological replicates are mentioned in the graph. (B) Infection prevalence between dsPara and dsGFP in antibiotic (ATB)-treated background was measured at d-8post infection and showed no statistical difference between the two conditions dsPara and dsGFP. Combined p-value (Fisher method) from the 3 independent biological replicates was obtained for the infection prevalence. n = Total number of dissected mosquitoes. Values obtained in every biological replicate are depicted by dots and dotted lines.