Quantitative real-time PCR (qPCR) is widely used in molecular biology, although the accuracy of the quantitative results is determined by the stability of the reference genes used. Recent studies have investigated suitable reference genes for some crustaceans under various conditions, but studies in Macrobrachium nipponense are currently lacking. In this study, we selected the following seven genes from among 35 commonly used housekeeping genes as candidate qPCR reference genes for temporal and spatial expression: EIF (eukaryotic translation initiation factor 5A), 18S (18S ribosomal RNA), EF-1α (elongation factor-1α), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), TUB (α-tubulin), β-act (β-actin), and RPL18 (Ribosomal protein L18). The stability of each reference gene was evaluated by GeNorm, NormFinder, BestKeeper, and comparative ∆C _t methods, and was comprehensively ranked using RefFinder. RPL18 was shown to be the most suitable reference gene for adult M. nipponense tissues, while EIF was the most stable in different ovarian and embryo stages and in white spot syndrome virus infection, and β-act was the most stable reference gene under hypoxia stress. The reliability of the rankings was confirmed by RNA interference experiments. To the best of our knowledge, this represents the first systematic analysis of reference genes for qPCR experiments in M. nipponense, and the results will provide invaluable information for future research in closely related crustaceans.

The doublesex and mab-3 related transcription factor (DMRT) gene family involvement in sex development is widely conserved from invertebrates to humans. In this study, we identified a DM (Doublesex/Mab-3)-domain gene in Macrobrachium nipponense, which we named MniDMRT11E because it has many similarities to and phylogenetically close relationships with the arthropod DMRT11E. Amino acid alignments and structural prediction uncovered conservation and putative active sites of the DM domain. Real-time PCR analysis showed that the MniDMRT11E was highly expressed in the ovary and testis in both males and females. Cellular localization analysis showed that DMRT11E was mainly located in the oocytes of the ovary and the spermatocyte of the testis. During embryogenesis, the expression level of MniDMRT11E was higher at the cleavage stage than at other stages. During the different stages of ovarian development, MniDMRT11E expression gradually increased from OI to OIII and decreased to the lowest level at the end of OIV. The results indicated that MniDMRT11E probably played important roles in embryonic development and sex maturity in M. nipponense. MniDMRT11E dsRNA injection also significantly reduced vitellogenin (VG) expression and significantly increased insulin-like androgenic gland factor (IAG) expression, indicating a close relationship in gonad development.

Gem-associated protein 2-like isoform X1 (GEM) was previously predicted to be involved in the sexual development of male Macrobrachium nipponense. In this study, we analyze the GEM functions in depth using quantitative polymerase chain reaction (qPCR), in situ hybridization, and RNA interference (RNAi). The full-length Mn-GEM cDNA sequence was 1018 base pairs (bp) long with an open reading frame of 777 bp encoding 258 amino acids. qPCR analysis of Mn-GEM in different tissues and developmental stages showed that Mn-GEM was highly expressed in the gonad and from post-larval developmental stage day 5 (PL5) to PL15, which indicated that GEM has potential roles in gonad differentiation and development in M. nipponense. In situ hybridization and qPCR analysis of various stages of the reproductive cycle of the testis and ovary indicated that GEM may promote spermatid development and gametogenesis in M. nipponense. After injecting with double-stranded RNA (dsRNA) of Mn-GEM, mRNA expression of Mn-insulin-like androgenic gland hormone (Mn-IAG) and the content of testosterone increased with the decrease of Mn-GEM expression, indicating that GEM has negative effects on the male sexual differentiation and development in M. nipponense. Results of this study highlight the functions of GEM in M. nipponense, which can be applied to future studies of male sexual development in M. nipponense and other crustacean species.

Metabolic adaption to hypoxic stress in crustaceans implies a shift from aerobic to anaerobic metabolism. Lactate dehydrogenase (LDH) is a key enzyme in glycolysis in prawns. However, very little is known about the role of LDH in hypoxia inducible factor (HIF) pathways of prawns. In this study, full-length cDNA of LDH (MnLDH) was obtained from the oriental river prawn Macrobrachium nipponense, and was characterized. The full-length cDNA is 2267-bp with an open reading frame of 999 bp coding for a protein of 333 amino acids with conserved domains important for function and regulation. Phylogenetic analysis showed that MnLDH is close to LDHs from other invertebrates. Quantitative real-time PCR revealed that MnLDH is expressed in various tissues with the highest expression level in muscle. MnLDH mRNA transcript and protein abundance in muscle, but not in hepatopancreas, were induced by hypoxia. Silencing of hypoxia-inducible factor 1 (HIF-1) α or HIF-1β subunits blocked the hypoxia-dependent increase of LDH expression and enzyme activity in muscle. A series of MnLDH promoter sequences, especially the full-length promoter, generated an increase in luciferase expression relative to promoterless vector; furthermore, the expression of luciferase was induced by hypoxia. These results demonstrate that MnLDH is probably involved a HIF-1-dependent pathway during hypoxia in the highly active metabolism of muscle.

The glutathione-S-transferase (GST) superfamily includes seven classes, and different classes have different functions. GST superfamily members function in various processes including detoxification of xenobiotics, protection against oxidative damage, and intracellular transport of hormones, endogenous metabolites, and exogenous chemicals. Herein, to elucidate the tissue-specific expression pattern of GSTs in response to hypoxia stress, which induces cell death, we investigated the expression of GSTs in response to hypoxia and reoxygenation in oriental river prawn, Macrobrachium nipponense. Full-length cDNAs of two δ class GSTs were cloned from the hepatopancreas, and named MnGST-1 and MnGST-2 based on the established GST nomenclature system. Expression profiles of both GSTs in various tissues were different under acute and chronic experimental hypoxia stress conditions, suggesting that both respond strongly to hypoxia-induced oxidative stress. However, the intensity of responses to hypoxia and reoxygenation were different in different tissues. During acute hypoxia stress, MnGST-1 responds earlier than MnGST-2 in the hepatopancreas and gill, but more slowly in muscle. By contrast, during chronic hypoxia stress, MnGST-2 plays a more important role in the hepatopancreas and gill than MnGST-1.