Identification of Potential Roles of Cathepsin B-like in the Response to Alkali Treatment in Macrobrachium nipponense

Cathepsin B is a member of the cysteine protease family and plays an important role in the innate immunity of aquatic invertebrates. A previous study identified that Cathepsin B-like (<i>CTSB-l</i>) may be involved in the response of alkali treatment in <i>Macrobrachium nipponense</i>. The present study aims to identify the potential regulatory roles of <i>CTSB-l</i> in the response of alkali treatment in <i>M. nipponense</i> through performing the quantitative real-time PCR analysis (qPCR), in situ hybridization (ISH) analysis, and RNA interference (RNAi) analysis. The full length of the <i>MnCTSB-l</i> cDNA was 1272 bp with an open reading frame of 987 bp, encoding 328 amino acids. Phylogenetic tree analysis indicated that the amino acid sequence of <i>MnCTSB-l</i> is highly homologous to those of crustacean cathepsin B-like. qPCR analysis showed that <i>MnCTSB-l</i> mRNA is expressed in all tested tissues with the highest level of expression in hepatopancreas in both male and female prawns. The expressions of <i>MnCTSB-l</i> were significantly stimulated in gills under the alkali concentration of both 5 mmol/L and 10 mmol/L, predicting that this gene may be involved in the response of alkali treatment in <i>M. nipponense</i>, which was consistent with the previous study. ISH showed that <i>MnCTSB-l</i> signals were mainly observed in the hemolymph vessels and membranes of gills, as well as in the basement membranes of hepatopancreas, in both male and female prawns. RNAi analysis revealed that the injection of double-stranded RNA of <i>CTSB</i> (<i>dsCTSB</i>) resulted in a significant decrease in <i>MnCTSB-l</i> expressions. In addition, prawn cumulative mortality was significantly higher in the <i>dsCTSB</i>-injected group, compared to that of <i>dsGFP</i>-injected group, under alkali treatments of both 5 mmol/L and 10 mmol/L, indicating <i>CTSB-l</i> plays an essential role in regulating alkalinity acclimation in <i>M. nipponense</i>. The present study identifies the regulatory functions of <i>CTSB-l</i> in the response of alkali treatment in <i>M. nipponense</i>, promoting the survival rate and aquaculture of this species in a water environment with high alkalinity.