Microbes reduce susceptibility of Aedes aegypti to Zika virus by enhancing blood digestion and limiting midgut cell infection

The worldwide expansion of mosquito-borne pathogens necessitates improved control measures, including approaches to restrict infection and transmission by mosquito vectors. Reducing transmission is challenging because determinants of vector competence for viruses like Zika (ZIKV) are poorly understood. Our previous work established that Aedes (Ae.) aegypti larvae reared in microbe-rich environmental water are less susceptible to ZIKV as adults compared to cohorts reared in microbe-deficient laboratory tap water. Here, we explain the association by identifying a mechanism by which environment-derived microbes reduce susceptibility of Ae. aegypti for ZIKV. Provided that the midgut represents the first barrier to mosquito infection, we hypothesized that microbial exposure modulates midgut infection by ZIKV. Since mosquitoes live in water as larvae and pupae and then transition to air as adults, we also define the stage in the life of a mosquito when microbial exposure reduces ZIKV susceptibility. Ae. aegypti larvae were reared in microbe-rich water and then treated with antibiotics during the pupal and adult stages, adult stage only, or provided no antibiotics at any stage. Vector competence was next evaluated in mosquitoes that ingested ZIKV-spiked bloodmeals. Antibiotic treatment enhanced ZIKV infection and dissemination rates, especially in Ae. aegypti treated as both pupae and adults. Antibiotic treated adult mosquitoes also had increased midgut epithelium permeability, higher numbers of ZIKV-infected midgut cells, and impaired bloodmeal digestion. Consistent with these changes, Ae. aegypti treated with antibiotics as pupae and adults that ingested ZIKV in bloodmeals showed reduced expression of genes associated with bloodmeal digestion and metabolism relative to mosquitoes that were not antibiotic treated. Together, these data show that exposure to microbes throughout the life of Ae. aegypti restricts ZIKV dissemination by facilitating blood digestion and limiting midgut cell infection. Understanding the roles mosquito microbiota play in determining midgut physiology and arbovirus susceptibility can lead to novel approaches to decrease mosquito transmission and will improve understanding of vector competence in microbe-rich environmental habitats. ### Competing Interest Statement The authors have declared no competing interest.