Abstract
Simple SummaryEven after decades of control interventions, mosquito-borne diseases still pose a huge threat to humans. Understanding gene functions is important for discovering new targets for mosquito and mosquito-borne disease control. One of the well-established and widely used methods for understanding gene function is RNA-interference (RNAi). The use of RNAi is, however, restricted mostly to adult mosquitoes. A few studies have shown its applicability in mosquito larvae, and just one in pupae. The current double-stranded RNA (dsRNA) delivery methods for RNAi are labor-intensive and require microinjections into mosquitoes (adults, larvae, or pupae). In this study, we present a simple, fast, and less labor-intensive technique for RNAi in the pupal stage by soaking pupae in water containing dsRNA. This method will be useful in studying genes expressed in immature life stages of the mosquitoes and will hopefully open new avenues for identifying mosquito control targets in early life stages.RNA-interference (RNAi) is a standard technique for functional genomics in adult mosquitoes. However, RNAi in immature, aquatic mosquito stages has been challenging. Several studies have shown successful larval RNAi, usually in combination with a carrier molecule. Except for one study in malaria mosquito, Anopheles gambiae, none of the previous studies has explored RNAi in mosquito pupae. Even in the study that used RNAi in pupae, double stranded RNA (dsRNA) was introduced by microinjection. Here, we describe a successful method by soaking pupae in water containing dsRNA without any carrier or osmotic challenge. The knockdown persisted into adulthood. We expect that this simple procedure will be useful in the functional analysis of genes that highly express in pupae or newly emerged adults.
Highlights
The introduction of exogenous double-stranded RNA into the cells of diverse eukaryotic organisms, the mechanism known as RNA interference (RNAi), has been shown to induce rapid and sustained degradation of mRNAs containing sequences complementary to the dsRNA [1]
The CYP4G subfamily is evolutionarily conserved across insects [22] but is absent in other orders such as Crustacea and Chelicerata
Previous work in other insects and mosquito A. gambiae has shown that the CYP4G enzymes, in association with its reductase partner (Cytochrome P450 reductase, CPR), catalyze the last two steps in cuticular hydrocarbon biosynthesis and are known for this alcohol oxidase and aldehyde decarbonylase activity [23]
Summary
The introduction of exogenous double-stranded RNA (dsRNA) into the cells of diverse eukaryotic organisms, the mechanism known as RNA interference (RNAi), has been shown to induce rapid and sustained degradation of mRNAs containing sequences complementary to the dsRNA [1]. The most common method for delivering the dsRNA in insects is by injection [3,4,5,6,7,8,9]. 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]).
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