RNAi-based Gene Silencing in Mosquitoes

RNAi experiments were performed as previously described (Kwon et al., 2017 (link); Kwon and Smith, 2019 (link); Reynolds et al., 2020 (link); Smith et al., 2016 (link); Smith et al., 2015 (link)). T7 primers for lozenge (Lz; AGAP002506) were designed using the E-RNAi web application (http://www.dkfz.de/signaling/e-rnai3/idseq.php) and listed in Supplementary file 7. T7 templates for dsRNA synthesis were prepared from amplified cDNA from 4 day old whole naïve mosquitoes. PCR amplicons were purified using the DNA Clean and Concentration kit (Zymo Research), and dsRNAs were synthesized using the MEGAscript RNAi kit (Life Technologies). Subsequent dsRNA targeting GFP (control) or Lz was resuspended in nuclease-free water to 3 µg/µl after ethanol precipitation. Injections were performed in 3- to 4-day-old cold anesthetized mosquitoes by intrathoracic injection with 69 nl (~200 ng) of dsRNA per mosquito using a Nanoject III. The effects of gene silencing were measured at 3 days post-injection in whole mosquitoes (n=15) by qRT-PCR as previously described (Kwon and Smith, 2019 (link)).