Involvement of endocytosis in cellular uptake of injected dsRNA into hepatopancreas but not in gill of Litopenaeus vannamei

Abstract

RNA interference (RNAi) technology has been widely applied to shrimp research for functional genomics, as well as for investigation of its potential anti-viral applications. While the integral membrane protein SID-1 of Litopenaeus vannamei has been reported to participate in the uptake of injected dsRNA into shrimp cells, this may not be the only uptake mechanism. Therefore the possible involvement of endocytosis in the delivery of injected dsRNA into shrimp hepatopancreatic and gill cells was evaluated. Clathrin-mediated endocytosis was inhibited through the injection of shrimp with two pharmacological endocytosis inhibitors (chlorpromazine and bafilomycin-A1) before injection of long dsRNAs directed against STAT (dsSTAT) or Rab7 (dsRab7). Levels of STAT or Rab7 suppression in the treated shrimp as compared to the control shrimp reflect the capability of cells to take up dsRNAs (dsSTAT or dsRab7). Inhibition of clathrin-mediated endocytosis showed a reduction of specific mRNA suppression in the shrimp hepatopancreas. In contrast, neither chlorpromazine nor bafilomycin-A1 effectively blocked dsRNA mediated inhibition of STAT or Rab7 in gill tissue. These results support our conclusion that endocytosis is required in cellular uptake of injected dsRNA into shrimp hepatopancreas but is not participated in the process in gills. This is the first report of the involvement of different pathways of cellular dsRNA uptake into different tissues of shrimp.