Using RNAi and CRISPR to improve sex-sorting in mosquitoes

Disease vectoring Aedes aegypti mosquitoes infect millions of people worldwide, transmitting deadly diseases like yellow fever, dengue, and chikungunya. Current methods of mosquito control rely largely on chemical insecticides that harm many non-target species, which has prompted the search for environmentally safer methods to prevent the spread of mosquito-borne-diseases. The sterile insect technique (SIT) is a non-chemical strategy that involves releasing large numbers of sterile male insects into natural populations to reduce offspring in subsequent generations through competitive mating. SIT has been used to control several insect species but has limited success in Aedes aegypti due in part to inadequate sorting of female mosquitoes from sterile males for large-scale release efforts. Current sex separation methods use automated sorting to selectively remove larger female pupae from smaller males prior to sterilization, however these strategies still exhibit high levels of female contamination which may exacerbate disease spread. Size differences between male and female Aedes aegypti are a result of differential growth rates during larval development, which are likely determined by a subset of sex-biased developmental genes. Using transcriptomic analyses, we have identified differentially expressed genes during the larval stages in both sexes of Aedes aegypti. Using RNA interference (RNAi) and CRISPR/Cas9 technologies, we are identifying genes that contribute to the differential size of the two sexes and are exploiting RNAi techniques to improve on the sex sorting processes, with the goal to enhance the efficacy of SIT for Aedes mosquitoes.