Involvement of LvSID-1 in dsRNA uptake in Litopenaeus vannamei

Abstract

In the past decade, RNA interference (RNAi) has emerged as a successful tool for functional genomics and anti-viral applications in shrimp. However, the mechanism of extracellular dsRNA uptake into shrimp cells has not been determined. Systemic RNA interference defective-1 protein (SID-1) is a transmembrane protein which is required for dsRNA transport between cells in several organisms including Caenorhabditis elegans but not in Drosophila and some insects. Recently, a SID-1 homolog (LvSID-1) was identified in whiteleg shrimp (Litopenaeus vannamei). Therefore, this study aimed to evaluate whether LvSID-1 is involved in the uptake of dsRNA into shrimp cells. LvSID-1 mRNA expression was up-regulated in gills and muscles but not in hepatopancreas of the shrimp that received long dsRNA introduced by injection. To elucidate the role of LvSID-1 in dsRNA uptake, a strategy of sequential introduction of dsRNAs was employed. Shrimp were initially injected with a long dsRNA to induce LvSID-1 mRNA expression. In a first experiment hemocytes of the LvSID-1 induced shrimp were then collected and seeded in a chamber slide before incubation with Cy3-labeled dsRNA to monitor cellular uptake. Under a confocal microscope, the Cy3 signal in the LvSID-1 induced hemocytes was significantly higher than the signal in naïve hemocytes. In a second experiment, LvSID-1 induced shrimp were separately injected with a second dsRNAs specific to a shrimp endogenous gene (signal transduction and transcription protein (STAT) or clathrin heavy chain (CHC)). Levels of STAT or CHC suppression in the LvSID-1 induced-shrimp compared with the control shrimp reflected the efficiency of uptake of the second dsRNA. Significantly, improved suppression of STAT and CHC was found in gills of the LvSID-1 induced shrimp. These results suggest that LvSID-1 participates in the uptake of injected dsRNA into shrimp cells. This study reports for the first time the involvement of the LvSID-1 in dsRNA uptake in shrimp, which could help to improve the potency of RNAi mediated anti-viral approaches in shrimp in the future.