Identification of potential roles of cathepsin L1 in the response to alkaline treatment in Macrobrachium nipponense

Cathepsin L (CTSL) is an important cysteine protease, which plays an important role in the process of environmental stress in aquatic animals. Previous study reported that CTSL of Macrobrachium nipponense (MnCTSL1) may be involved in the alkaline stress response. To further elucidate its potential function under alkali treatment in M. nipponense, this study employed integrated approaches including quantitative real-time PCR (qPCR), in situ hybridization (ISH), and RNA interference (RNAi) to systematically investigate the response of MnCTSL1 to alkaline treatment. qPCR analysis revealed ubiquitous MnCTSL1 expression across all tested tissues of M. nipponense, with the expression level in the hepatopancreas significantly surpassing those in other tissues in both sexes. Significant upregulation of MnCTSL1 expression was observed in hepatopancreas and gills under the alkalinity of both 5 mmol/L and 10 mmol/L, suggesting its involvement in alkaline stress response. ISH results showed that MnCTSL1 mRNA signals were detected in the basement membrane of hepatopancreas, and in the hemolymph vessels and membranes of gills in both sexes. After knockdown of MnCTSL1 expression using RNAi, the cumulative mortality of prawns in the dsCTSL1-injected group was 2.50- and 2.52-fold higher than that in the dsGFP-injected group at 5 mmol/L and 10 mmol/L alkalinity, respectively, suggesting that MnCTSL1 plays an important role in regulation of alkaline acclimatization of M. nipponense. These findings demonstrate that MnCTSL1 serves as a crucial regulator of alkaline adaptation in this commercially important crustacean species.