Simple SummaryRNA interference (RNAi) is a widely conserved antiviral mechanism whereby double-stranded RNA (dsRNA) is identified and degraded in cells through the RNAi pathway. Since the RNAi pathway uses dsRNA sequences to degrade a target, researchers can introduce dsRNA sequences that match genes of interest to study the impact of a gene. In mosquitoes, researchers introduce dsRNA to study an array of genes at different life stages and in different species, but the destination of dsRNAs in mosquitoes has yet to be characterized. In this work, dsRNA sequences were fluorescently labeled and tracked in mosquito species of medical significance, includingAedes aegypti,Anopheles gambiae, andCulex pipiens.Different exposure routes were trialed, including injection (peritoneal), feeding (per os), and topical application. Following injection, dsRNAs accumulate in a subset of cells associated with phagocytosis functions such as hemocytes and ovaries, but were not internalized followingper osor topical routes. Additionally, Northern blotting was used to monitor the degradation and clearance of dsRNA following exposure inAe. aegypti, identifying dsRNA in tissues up to a week post exposure, but were cleared from most individual tissues more rapidly. Overall, these findings describe the distribution of introduced dsRNAs in mosquitoes and may direct future research using this technique.AbstractIn mosquitoes, the utilization of RNAi for functional genetics is widespread, usually mediated through introduced double-stranded RNAs (dsRNAs) with sequence identity to a gene of interest. However, RNAi in mosquitoes is often hampered by inconsistencies in target gene knockdown between experimental setups. While the core RNAi pathway is known to function in most mosquito strains, the uptake and biodistribution of dsRNAs across different mosquito species and life stages have yet to be extensively explored as a source of variation in RNAi experiments. To better understand mosquito-RNAi dynamics, the biodistribution of a dsRNA to a heterologous gene, LacZ (iLacZ), was tracked following various routes of exposure in the larval and adult stages ofAedes aegypti,Anopheles gambiae, andCulex pipiens. iLacZ was largely limited to the gut lumen when exposed per os, or to the cuticle when topically applied, but spread through the hemocoel when injected. Uptake of dsRNA was noted in a subset of cells including: hemocytes, pericardial cells of the dorsal vessel, ovarian follicles, and ganglia of the ventral nerve cord. These cell types are all known to undergo phagocytosis, pinocytosis, or both, and as such may actively take up RNAi triggers. InAe. aegypti, iLacZ was detected for up to one week post exposure by Northern blotting, but uptake and degradation drastically differed across tissues. The results presented here reveal that the uptake of RNAi triggers is distinct and specific to the cell type in vivo.