Abstract
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 environmental water containing microbes are less susceptible to ZIKV as adults compared to cohorts reared in laboratory tap water with fewer species and lower abundance of microbes. Here, we identify a process 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 water containing microbes 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 treated mosquitoes with reduced microbiota showed enhanced ZIKV infection rates in Ae. aegypti treated as both pupae and adults. Antibiotic treatment to reduce microbes in pupal and adult mosquitoes also resulted in increased midgut epithelium permeability, higher numbers of ZIKV-infected midgut cells, and impaired bloodmeal digestion. Parallel control experiments with antibiotic-treated or gnotobiotic mosquitoes reared in laboratory water showed that the dysbiotic state created by antibiotic use does not influence ZIKV vector competence or midgut permeability and that more than the single bacterial species in gnotobiotic mosquitoes is responsible for reducing ZIKV vector competence. Ae. aegypti with depleted microbes via antibiotic treatment as pupae and adults that ingested ZIKV in bloodmeals showed reduced expression of genes associated with bloodmeal digestion and metabolism relative to mosquitoes whose microbes were not reduced with antibiotics. Together, these data show that exposure to microbes throughout the life of Ae. aegypti restricts ZIKV infection by facilitating blood digestion and reducing midgut cell infection. Understanding the connections between mosquito microbiota, midgut physiology, and arbovirus susceptibility can lead to novel approaches to decrease mosquito transmission and will improve understanding of vector competence in environmental habitats containing microbes.
Author Summary Mosquito-transmitted viruses like Zika continue to threaten human health. Absent vaccines or treatments, controlling mosquitoes or limiting their ability to transmit viruses represents a primary way to prevent mosquito-borne viral diseases. The role mosquito microbiota play in shaping transmission of Zika virus has been limited to association-based studies. Our prior work showed that Aedes aegypti mosquito larvae that develop in water containing bacteria are less susceptible to Zika virus compared to larvae reared in laboratory tap water with fewer numbers and species of bacteria. Here we identify a process that explains this association. Since mosquitoes are aquatic as larvae and pupae and terrestrial as adults, we also define the life stage when microbes need be present to reduce Zika virus susceptibility. We used antibiotics to reduce environmental water-derived microbes at pupal and adult or only adult stages and observed that microbe depletion via antibiotic treatment increases Zika virus infection and midgut permeability and impairs bloodmeal digestion. These data show that microbial exposure throughout the life of a mosquito restricts Zika virus infection by facilitating blood digestion and restricting midgut cell infection. These findings advance understanding of microbiota-mosquito-virus interactions by defining how microbes reduce susceptibility of Aedes aegypti to Zika virus.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Additional studies were performed to show that the dysbiotic state created by antibiotic use does not influence ZIKV vector competence or midgut permeability. Use of gnotobiotic mosquitoes show that exposure to more than the single bacterial species is responsible for reducing ZIKV vector competence. Vector competence data were reanalyzed and dissemination data are reported as the fraction of infected mosquitoes (viral RNA in bodies) with disseminated infection (viral RNA in legs/wings). Transmission data are reported as the fraction of mosquitoes with disseminated infection that also transmitted (viral RNA in saliva expectorates).