Prevalence, Genetic Diversity of Microsporidia MB, and Correlation to Insecticide Resistance in Anopheles gambiae s.l. and Anopheles funestus Mosquitoes in Busia, Kenya

Background: The discovery of Microsporidia MB , a malaria-impairing symbiont in Anopheles arabiensis , suggests its potential for malaria control. This study investigated its prevalence and diversity in An. gambiae s.l. and An. funestus in Busia, Kenya. The study also explored its association with insecticide resistance and environmental factors. Methods: Mosquito larvae and adults were collected from three sub-counties in Busia, Kenya. Species were identified morphologically, and DNA was extracted. PCR was used to determine species distribution and Microsporidia MB prevalence. Insecticide resistance markers were identified using TaqMan genotyping. Microsporidia MB -positive samples underwent whole-genome sequencing and phylogenetic analysis. Statistical tests, including chi-square, ANOVA, and regression, were used to assess relationships between Microsporidia MB , insecticide resistance, and ecological variables. Results: There was notable variation in the distribution of species in Busia, where An. gambiae s.l. emerged as the most prevalent species in Teso South and Butula sub-counties, whereas An. funestus was the most prevalent in Budalangi sub-county. Microsporidia MB was observed at a low to moderate occurrence of 0 to 6.4%, with the highest prevalence noted in An. gambiae s.s. Despite the significant fluctuations in temperature, pH, and dissolved oxygen levels across different ecological habitats, only the variation in pH was associated with the prevalence of Microsporidia MB . There was no correlation between Microsporidia MB infection and molecular markers of insecticide resistance. Phylogenetic analysis revealed significant genetic diversity in Microsporidia MB , with geographic location influencing lineage divergence. Conclusion: The present study highlights the occurrence of Microsporidia MB in multiple Anopheles vectors associated with malaria and highlights the potential role of ecological factors in sustaining the prevalence of Microsporidia MB . Future studies will tease out whether malaria-protective phenotypes are conserved traits among the distinct evolutionary lineages to enhance our understanding of critical considerations that are necessary for the successful implementation of this novel malaria control strategy in areas with varying strains and ecological conditions. Geographic location significantly shapes the genetic diversity of Microsporidia MB in mosquitoes, revealing distinct evolutionary lineages and dispersal patterns.