dsRNA mediated silencing of trypsin affects the digestive capacity in the Pacific whiteleg shrimp (Penaeus vannamei)

Digestive peptidases in the Pacific whiteleg shrimp Penaeus vannamei comprise a set of at least three trypsins, two chymotrypsins, and one metallopeptidase (Mpc1), with trypsin and chymotrypsin performing 60% of the digestive function. In this exploratory study, we investigated the relative importance of digestive trypsin by silencing trypsin-2 (Try2) using RNA interference (RNAi) and assessing its impact on the expression and activity of other peptidases and the muscle amino acid profile. RNAi was induced by injecting double-stranded RNA of trypsin-2 (dsRNATry2) into the hepatopancreas (also known as the digestive gland), with controls including non-injected specimens and those injected with dsRNA-GFP or NaCl. The bioassays evaluated the effect of single and multiple doses of dsRNA in the gene expression of trypsins, chymotrypsin-B1, and Mpc1, as well as total and specific proteolytic activities. Results showed that, at 120 h post-injection, trypsin-2 expression was significantly lower in the dsRNA-Try2 group compared with non-injected and NaCl-injected controls, whereas no significant difference was detected relative to the dsRNA-GFP group, indicating that a non-specific dsRNA effect cannot be excluded. Other genes (e.g., trypsin-1, trypsin-3, chymotrypsin B1, Mpc1) and proteolytic activity measures showed directionally consistent but non-significant trends. Zymography qualitatively supported reductions in trypsin-associated bands. With repeated dosing, specific trypsin activity decreased, whereas changes in total and chymotrypsin activities varied. Muscle amino acid patterns and epithelial changes in the hepatopancreas suggested possible systemic responses; however, limited replication and pooling precluded statistical inference for these endpoints. No compensatory mechanism for reduced trypsin transcripts was observed.