RNAi Trigger Delivery into <em>Anopheles gambiae</em> Pupae

Abstract

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory for functional genomic studies. The ability to knockdown individual genes selectively via this reverse genetic technique has allowed many researchers to rapidly uncover the biological roles of numerous genes within many organisms, by evaluation of loss-of-function phenotypes. In the major human malaria vector Anopheles gambiae, the predominant method used to reduce the function of targeted genes involves injection of double-stranded (dsRNA) into the hemocoel of the adult mosquito. While this method has been successful, gene knockdown in adults excludes the functional assessment of genes that are expressed and potentially play roles during pre-adult stages, as well as genes that are expressed in limited numbers of cells in adult mosquitoes. We describe a method for the injection of Serine Protease Inhibitor 2 (SRPN2) dsRNA during the early pupal stage and validate SRPN2 protein knockdown by observing decreased target protein levels and the formation of melanotic pseudo-tumors in SRPN2 knockdown adult mosquitoes. This evident phenotype has been described previously for adult stage knockdown of SRPN2 function, and we have recapitulated this adult phenotype by SRPN2 knockdown initiated during pupal development. When used in conjunction with a dye-labeled dsRNA solution, this technique enables easy visualization by simple light microscopy of injection quality and distribution of dsRNA in the hemocoel.

RNAi by Soaking Aedes aegypti Pupae in dsRNA

“…We hypothesize that the dsRNA intake is due to indirect uptake instead of feeding because pupae do not feed and still showed significant knockdown. The pupal mortality was higher in our experiments compared to Regna et al [ 13 ]; however, the mortality was not significantly different between the control and experimental sets ( Table 1 ). The higher mortality could be due to mechanical injury to the pupae during transfer by the paint brush.…”

“…We exposed individuals right after they molt (4th instar to pupae) when the cuticle is possibly more permissive. Another study in A. gambie pupal RNAi by injection also suggested that the early pupal stage, before cuticle tanning was complete, resulted in optimum RNAi efficiency and pupal survival post-injection [ 13 ]. We hypothesize that the dsRNA intake is due to indirect uptake instead of feeding because pupae do not feed and still showed significant knockdown.…”

“…In mosquitoes, adult injections have been most successful. While microinjection in mosquito larvae [ 10 , 11 , 12 ] and pupae [ 13 ] has been shown to work, it is time-consuming and has little field application. An alternative to the microinjection technique is an uptake of dsRNA through ingestion (reviewed in [ 14 ]).…”

Delivery Methods for RNAi in Mosquito Larvae

“…Injection ensures delivery into the hemocoel, distribution of dsRNA through body organs and tissues, and bypassing some body barriers such as cuticle and gut. Regarding mosquitoes, dsRNA delivery through injection is common in pupal and adult stages ( Regna et al 2016 , Isoe et al 2019 ); however, it was used in few cases in larval stages. In general, the injection process includes anaesthetizing larvae in cold environment followed by using lab-made capillary glass needle for injecting few nanoliters of the dsRNA solution into the thoracic region.…”

RNA Interference Is Enhanced by Knockdown of Double-Stranded RNases in the Yellow Fever Mosquito Aedes aegypti

“…In mosquitoes, many researchers choose to deliver dsRNA by direct hemocoel injections, to ensure precise dsRNA doses and consistent knockdown of the genes under study [15][16][17][18][19]. DsRNA feeding in mosquitoes has proven effective in inducing RNAi, but, typically, only partial reductions in the target transcripts or limited knockdown phenotypes have been described [20][21][22].…”