Fig. 2.

DsRNA activates Dicer-2/Ampk/FoxO axis for transcriptional response. (A) Effect of RNAi-components knockdown on dsGFP-induced antiviral immunity. Presilenced shrimp were stimulated with dsGFP/siGFP. Infection was performed 24 h later to determine viral load another 24 h later, n = 4 biological replicates. (B–D) Inhibition of dsGFP-induced FoxO-nuclear localization by Dicer-2-knockdown. Presilenced shrimp were stimulated with dsGFP/siGFP. Hemocytes were sampled 6 h later to extract nuclear protein for blotting assay (B), and immunocytochemical assay. Scale bar, 10 μm (C). FoxO-nuclei colocalization was digitalized from eight randomly selected field of view, n > 100 cells (D). (E) Inhibition of dsGFP-induced gene transcription by Dicer-2-knockdown. Gene expression was detected at 24 h after stimulation, n = 4 biological replicates. (F) Induction of ADP/ATP ratio by dsGFP. Hemocytes ADP/ATP ratio was determined at 3 h after dsGFP (1, 2, 5 μg/g) or siGFP (5 μg/g) stimulation, n = 4 biological replicates. (G) Inhibition of dsGFP-induced increase in ADP/ATP ratio by Dicer-2-knockdown. Presilenced shrimp were stimulated with dsGFP/siGFP. ADP/ATP ratio was determined 3 h later, n = 4 biological replicates. (H) Induction of hemocytes Ampkα-phosphorylation by dsGFP. Botting assay was performed at 6 h after stimulation. (I) Inhibition of dsGFP-induced Ampkα-phosphorylation by Dicer-2 knockdown. Presilenced shrimp were stimulated with dsGFP/siGFP. Botting assay was performed 6 h later. (J–M) Inhibition of dsGFP-induced FoxO-nuclear localization and gene transcription by Ampkα-knockdown. Experimental procedures are similar to (B–E). (N) Working model. Energy consumption caused by Dicer-2-mediated dsRNA-cleavage activates Ampk/FoxO axis to induce RNAi components and antiviral effectors. Images are representative of three independent replicates. ^∗∗∗P < 0.001, ns, not significant, Student’s t test.