Molecular Characterization of a Novel Cathepsin L in Macrobrachium nipponense and Its Function in Ovary Maturation

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.

Integrated Metabolomics and Transcriptomic Analysis of Hepatopancreas in Different Living Status Macrobrachium nipponense in Response to Hypoxia

As the basic element of aerobic animal life, oxygen participates in most physiological activities of animals. Hypoxia stress is often the subject of aquatic animal research. Macrobrachium nipponense, an economically important aquatic animal in southern China, has been affected by hypoxia for many years and this has resulted in a large amount of economic loss due to its sensitivity to hypoxia; Metabolism and transcriptome data were combined in the analysis of the hepatopancreas of M. nipponense in different physiological states under hypoxia; A total of 108, 86, and 48 differentially expressed metabolites (DEMs) were found in three different comparisons (survived, moribund, and dead shrimps), respectively. Thirty-two common DEMs were found by comparing the different physiological states of M. nipponense with the control group in response to hypoxia. Twelve hypoxia-related genes were identified by screening and analyzing common DEMs. GTP phosphoenolpyruvate carboxykinase (PEPCK) was the only differentially expressed gene that ranked highly in transcriptome analysis combined with metabolome analysis. PEPCK ranked highly both in transcriptome analysis and in combination with metabolism analysis; therefore, it was considered to have an important role in hypoxic response. This manuscript fills the one-sidedness of the gap in hypoxia transcriptome analysis and reversely deduces several new genes related to hypoxia from metabolites. This study contributes to the clarification of the molecular process associated with M. nipponense under hypoxic stress.

MnFtz-f1 Is Required for Molting and Ovulation of the Oriental River Prawn Macrobrachium nipponense

Molting and ovulation are the basic processes responsible for the growth and reproduction of Macrobrachium nipponense; however, the molecular mechanisms of molting and ovulation in M. nipponense are poorly understood. The present study aimed to use MnFtz-f1 as the starting point to study the molting and ovulation phenomena in M. nipponense at the molecular level. The full-length MnFtz-f1 cDNA sequence was 2,198 base pairs (bp) in length with an open reading frame of 1,899 bp encoding 632 amino acids. Quantitative real-time PCR analysis showed that MnFtz-f1 was highly expressed in the ovary at the cleavage stage and on the fifth day after hatching. In vivo administration of 20-hydroxyecdysone (20E) showed that 20E effectively inhibited the expression of the MnFtz-f1 gene, and the silencing of the MnFtz-f1 gene reduced the content of 20E in the ovary. In situ hybridization (ISH) analysis revealed the localization of MnFtz-f1 in the ovary. Silencing of MnFtz-f1 by RNA interference (RNAi) resulted in significant inhibition of the expression of the vitellogenin (Vg), Spook, and Phantom genes, thus confirming that MnFtz-f1 had a mutual regulatory relationship with Vg, Spook, and Phantom. After RNAi, the molting frequency and ovulation number of M. nipponense decreased significantly, which demonstrated that MnFtz-f1 played a pivotal role in the process of molting and ovulation.