The presence of the wAnD strain of Wolbachia is correlated with lower levels of Plasmodium sporozoites and a less diverse microbiome in wild Anopheles demeilloni mosquito cephalothoraxes

Background: The increasing insecticide resistance of malaria vectors is an urgent concern for disease control and novel vector control strategies are needed. Wolbachia are endosymbiotic bacteria that can invade mosquito populations and reduce transmission of human pathogens. Wolbachia strains in wild Anopheles (An.) malaria vectors are rare, with only two known genuine symbioses; An. moucheti with wAnM and An. demeilloni with wAnD. In this study, we set out to determine if there was a correlation between wAnD in different An. demeilloni mosquito body parts, infective stage Plasmodium (Pl.) falciparum malaria sporozoites in cephalothoraxes and the mosquito microbiome. 

Methods: We undertook a combination of quantitative PCR, 16S rRNA amplicon sequencing and sanger sequencing of the Wolbachia surface protein (wsp) gene after isolating An. demeilloni female body parts from wild caught individuals collected in 2021 and 2024 from the Sud Kivu region of Democratic Republic of Congo. 

Results Wolbachia prevalence rates were significantly higher in abdomens compared to cephalothoraxes and density was also significantly higher in abdomens (P<0.0001). Overall sporozoite prevalence was 1.3% (9/704) which was not significantly different between Wolbachia-positive and Wolbachia-negative cephalothoraxes (P=0.3630) despite Pl. falciparum only detected in Wolbachia-negative cephalothoraxes. However, Wolbachia-positive abdomens were associated with a lower sporozoites rate compared to Wolbachia-negative abdomens (P=0.0329). 16S rRNA amplicon sequencing revealed no significant difference in alpha/beta diversities between abdomens and cephalothoraxes but the cephalothorax microbiome composition between Wolbachia-positive and Wolbachia-negative was significantly different (P<0.05). 

Conclusions: Our findings indicate a significant effect of the wAnD strain on the cephalothorax microbiome and potentially the ability of sporozoites to reach Salivary glands in mosquitoes with Wolbachia-infected abdomens. Further studies are needed to determine the mechanisms in which the wAnD strain interacts with Plasmodium sporozoites in An. demeilloni and if this strain could be used for malaria biocontrol through transinfection of major malaria vectors.