Injection of ectopic naked siRNAs does not induce antiviral immunity or gene silencing. (a) Shrimp (1 to 2 g) were injected with either saline (no-virus and no-dsRNA controls) or 2 μg of dsRNA as indicated. vp19 siRNAs are an equimolar cocktail of three synthetic siRNAs targeting vp19 (Table 1). WSSV-containing tissue was fed at 10% biomass daily for 3 consecutive days, starting 4 h after dsRNA injection. A collective challenge system was used (n = 32 to 33). vp19 long dsRNA afforded significant antiviral immunity 10 dpi compared to duck Igυ dsRNA, vp19 siRNA, vp19 siRNA plus duck Igυ dsRNA, and no-dsRNA treatments (P < 0.0001 in every case). (b) All controls were as described above for a. In experiment 1 (Exp. 1), shrimp were injected with 2 μg of dsRNA and challenged per os in a collective challenge system (n = 32 to 33). In experiment 2, shrimp were injected with 2.5 μg of dsRNA and challenged per os in the individual challenge system (n = 28 to 30). The inset on the right shows long vp19 dsRNA and diced vp19 dsRNA resolved by polyacrylamide gel electrophoresis and stained with ethidium bromide. M, 10-bp dsDNA ladder. (c) Shrimp (1 to 2 g) were injected with either saline (no-virus and no-dsRNA controls) or 5 μg of the indicated dsRNAs 72 h prior to infection by intramuscular injection of WSSV-homogenate (diluted 4 × 10⁻⁸, wt/vol). The control siRNA is of arbitrary sequence (Table 1). An individual challenge system was used (n = 30 to 32). Duck Igυ dsRNA afforded significant antiviral immunity 12 dpi compared to siRNAs and the virus-only control (P < 0.0001 in both cases). (d) Individual shrimp were injected with 5 μg of dsRNA as indicated, and hemocyanin (Hem) expression was analyzed by Northern blot in the hepatopancreas 48 h after injection. Expression of ribosomal protein S20 (NCBI accession number CK591746) is shown as a reference. Hemocyanin siRNAs are an equimolar cocktail of three siRNAs targeting the hemocyanin mRNA (Table 1); the same siRNA of arbitrary sequence used in panel c was used as a control. Each lane represents RNA from a single shrimp.