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Cathepsin L genes, which belonged to cysteine proteases, were a series of multifunctional protease and played important roles in a lot of pathological and physiological processes. In this study, we analyzed the characteristics a cathepsin L (named Mn-CL2) in the female oriental river prawn, Macrobrachium nipponense which was involved in ovary maturation. The Mn-CL2 was1,582 bp in length, including a 978 bp open reading frame that encoded 326 amino acids. The Mn-CL2 was classified into the cathepsin L group by phylogenetic analysis. Real-time PCR (qPCR) analysis indicated that Mn-CL2 was highly expressed in the hepatopancreas and ovaries of female prawns. During the different ovarian stages, Mn-CL2 expression in the hepatopancreas and ovaries peaked before ovarian maturation. In situ hybridization studies revealed that Mn-CL2 was localized in the oocyte of the ovary. Injection of Mn-CL2 dsRNA significantly reduced the expression of vitellogenin. Changes in the gonad somatic index also confirmed the inhibitory effects of Mn-CL2 dsRNA on ovary maturation. These results suggest that Mn-CL2 has a key role in promoting ovary maturation.

Legumain, also called aspartic endopeptidase (AEP), is a member of the cysteine protease family and is involved in various physiological processes. In this study, we analyzed the characteristics of a novel legumain-like (named Mn-Lel) in the female oriental river prawn, Macrobrachium nipponense, which is involved in ovary maturation. The Mn-Lel is 1,454 bp in length, including a 1,290-bp open reading frame that encodes 430 amino acids. qPCR analysis indicated that Mn-Lel is specifically highly expressed in the hepatopancreas and ovaries of female prawns. It is rarely expressed in embryogenesis, weakly expressed in early larval development stages, and then significantly increased after metamorphosis, which indicated that Mn-Lel is not a maternal gene and mainly plays a role in adults. During the different ovarian stages, Mn-Lel expression in the hepatopancreas had no obvious rules, while its expression in the ovaries had a significant peak in stage III. In situ hybridization studies revealed that Mn-Lel is localized in the oocyte of the ovary. Changes in the gonadosomatic index confirmed the inhibitory effects of Mn-Lel dsRNA on ovary maturation. These results suggest that Mn-Lel has a key role in promoting ovary maturation.

The oriental river prawn Macrobrachium nipponense is an economically important aquacultural species. However, its aquaculture is negatively impacted by the rapid sexual maturation of female M. nipponense. The fast sexual maturation produces a large number of offspring which leads to a reduction in resilience, a low survival rate, and an increased risk of hypoxia, this in turn, seriously affects the economic benefits of prawn farming. Cathepsin D is a lysosomal protease involved in the ovarian maturation of M. nipponense. In the current study, the cDNA of the gene encoding cathepsin D (Mn-CTSD) was cloned from M. nipponense. The total length was 2391 bp and consisted of an open reading frame (ORF) of 1158 bp encoding 385 amino acids. Sequence analysis confirmed the presence of conserved N-glycosylation sites and characteristic sequences of nondigestive cathepsin D. The qPCR analysis indicated that Mn-CTSD was highly expressed in all tissues tested, most significantly in the ovaries, whereas in situ hybridization showed that expression occurred mainly in oocyte nuclei. Analysis of its expression during development showed that Mn-CTSD peaked during the O-IV stage of ovarian maturation. For the RNAi interference experiment, female M. nipponense specimens in the ovary stage I were selected. Injection of Mn-CTSD double-stranded (ds)RNA into female M. nipponense decreased the expression of Mn-CTSD in the ovaries, such that the Gonad Somatic Index (GSI) of the experimental group was significantly lower than that of the control group (1.79% versus 4.57%; p < 0.05). Ovary development reached the O-III stage in 80% of the control group, compared with 0% in the experimental group. These results suggest that Mn-CTSD dsRNA inhibits ovarian maturation in M. nipponense, highlighting its important role in ovarian maturation in this species and suggesting an approach to controlling ovarian maturation during M. nipponense aquaculture.

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