A knockout screen of genes expressed specifically in Aedes aegypti pupae reveals a critical role for stretchin in mosquito flight

Aedes aegypti is a critical vector for transmitting Zika, dengue, chikungunya, and yellow fever viruses to humans. Genetic strategies to limit mosquito survival based upon sex distortion or disruption of development may be valuable new tools to control Ae. aegypti populations. We identified six genes with expression limited to pupal development; osi8 and osi11 (Osiris protein family), CPRs and CPF (cuticle protein family), and stretchin (a muscle protein). Heritable CRISPR/Cas9-mediated gene knockout of these genes did not reveal any defects in pupal development. However, stretchin-null mutations (strn^∆35/∆41) resulted in flightless mosquitoes with an abnormal open wing posture. The inability of adult strn^∆35/∆41 mosquitoes to fly restricted their escape from aquatic rearing media following eclosion, and substantially reduced adult survival rates. Transgenic strains which contain the EGFP marker gene under the control of strn regulatory regions (0.8 kb, 1.4 kb, and 2.2 kb upstream, respectively), revealed the gene expression pattern of strn in muscle-like tissues in the thorax during late morphogenesis from L₄ larvae to young adults. We demonstrated that Ae. aegypti pupae-specific strn is critical for adult mosquito flight capability and a key late-acting lethal target for mosquito-borne disease control.