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Glutathione peroxidase (GPx) has been the focus of increased research because of its important role as an antioxidant and in reactive oxygen species (ROS) induced damage repair. Studies on GPxs have relevance with Macrobrachium nipponense because it has poor tolerance to hypoxia in Macrobrachium nipponense. The two subunits named as MnGPx-3 and MnGPx-4 according to the glutathione peroxidase nomenclature system. Both full-length cDNAs were cloned from the hepatopancreas. In this study, we analyzed the expression of two GPxs in Macrobrachium nipponense in response to changes in environmental oxygen. Expression levels of MnGPx-3 and MnGPx-4 indicated that both have strong responses to hypoxia. In situ hybridization showed that MnGPx-3 and MnGPx-4 were located in secretory and storage cells in hepatopancreas. These results suggest that GPx gene is expressed and released by secretory cells and released response to hypoxia. In the gill tissue, however, GPxs are located in blood cells, suggesting that they perform different functions in different tissues or organs. The results of in situ hybridization were consistent with those of quantitative Real-time PCR. This study provides a basis for understanding the oxidative stress response in M. nipponense under hypoxia. [ABSTRACT FROM AUTHOR]

Abstract: Forkhead box protein L2 (Foxl2) has dramatic effects on early differentiation and development of the female gonad in mammals and fish through the repression of SOX9 expression. In this study, we aimed to isolate a full‐length cDNA sequence encoding Foxl2 from the Macrobrachium nipponense (Mn‐Foxl2) and investigated its gene function. The full‐length cDNA sequence of Mn‐Foxl2 was 2097 bp with an open reading frame of 1740 bp, encoding 579 amino acids. Phylogenetic analysis revealed considerable evolutionary divergence between Mn‐Foxl2 and Foxl2 from fish. Mn‐Foxl2 may be involved in gonadal sex differentiation, based on its dramatically high expression during the sex‐differentiation sensitive period. Tissue distributions indicated that Mn‐Foxl2 mRNA expression was higher in the testis than that in the ovary and higher in males than in females in the same tissues, implying that Foxl2 may promote both male and female sexual development in M. nipponense. In situ hybridization analysis and quantitative polymerase chain reaction analysis in the gonad during the reproductive cycle further confirmed the important roles of Foxl2 in early gonad development in M. nipponense. This study advances our understanding of the role of Foxl2 in M. nipponense and provides the basis for further studies of Foxl2 in crustaceans. [ABSTRACT FROM AUTHOR]

Abstract: A full‐length complementary DNA of the crustacean cardioactive peptide (CCAP) was cloned from the eyestalk of the oriental river prawn, Macrobrachium nipponense, and named Mn‐CCAP. Experimental treatment was set to decrease dissolved oxygen levels and generate hypoxic culture conditions. The Mn‐CCAP expression profile was different in the brain and eyestalk. Results suggested Mn‐CCAP responds strongly to hypoxia‐induced oxidative stress. Different expression profiles in the brain and eyestalk indicate that these two tissues may respond in different ways to hypoxia. [ABSTRACT FROM AUTHOR]

The transformer-2 gene ( tra-2) plays a key role in the sexual differentiation regulatory hierarchy. In this study, tra-2 gene homologs designated as Mntra-2 was cloned and characterized from Macrobrachium nipponense. The full-length cDNA of Mntra-2 consists of 1724 bp with an open reading frame ( ORF) encoding 192 amino acids, an 827 bp 5′-untranslated region ( UTR) and a 318 bp 3′- UTR. The predicated molecular mass of Mntra-2 was 20.805 kDa with an estimated theoretical isoelectric point of 10.36. The deduced amino acid sequence shares high homology with Penaeus monodon. Real-time quantitative polymerase chain reaction ( RT-q PCR) analyses demonstrated that the expression levels of Mntra-2 varied significantly during different developmental stages of embryogenesis, larvae, and post-larvae and in various adult tissues. During embryogenesis, the expression level of Mntra-2 was slightly higher at the cleavage stage than at the blastula stage, and reached the highest level at the nauplius stage. During the larvae, the Mntra-2 expression gradually increased from 1 d larvae post hatch ( L1) to L10 and decreased to a lowest level at the end of metamorphosis. During the post-larvae, the Mntra-2 expression was higher level at the 5 d after the metamorphosis ( P5). RT-q PCR showed the Mntra-2 mRNA was expressed in ovary, testis, muscle, heart, abdominal ganglion, brain, and intestine with the highest level of expression in muscle and intestine. The results indicate that Mntra-2 is an arthropods tra-2 homolog and probably plays important roles in embryonic development and sex differentiation of M. nipponense. [ABSTRACT FROM AUTHOR]

• A cDNA sequence of Mn-cyclin A was identified and characterized from oriental river prawn, Macrobrachium nipponense. • The high expression in ovary and ISH suggest that Mn-cyclin A is closely related to ovarian development. • The expression patterns of Mn-cyclin A mRNA in different developmental stages of embryo, larva and post-larval were specific. • The injection of dsRNA against Cyclin A in the pericardial cavity leads to delayed ovary development. Macrobrachium nipponense has the characteristics of fast ovarian development cycle, which leads to the coexistence of multiple generations, the reduction of commodity specifications and the low economic benefit. Therefore, the study on the mechanism of ovarian development is of great significance to the development of industry. Cyclin A (CycA)is a key gene regulating ovarian development in vertebrates, but little information was available for its function in crustaceans. In this study, the full-length cDNA of Mn-CycA was obtained from the ovary. The full-length cDNA (2033 bp) with an open reading frame of 1368 bp, encoded a 456-amino acid protein. qRT-PCR revealed tissue-specific expression pattern of Mn-CycA , with abundant expression in the ovary. Results in different developmental stages of ovary indicated that Mn-CycA expression is positively correlated with ovarian maturation. qRT-PCR In different developmental stages, the expression of Mn-CycA mRNA gradually increased during the embryonic stage and decreased significantly on the first day of the hatching stage. At the 25th day of the metamorphosis stage, the expression level of Mn-CycA mRNA in female shrimp was 3.5 times higher than that in male shrimp, which may be related to the proliferation of oogonia and the formation of oocytes. In situ hybridization (ISH) of ovary showed Mn-CycA was examined in all stages and was mainly located in oogonia and oocytes. Compared with the control group, the obvious change of gonad somatic index (GSI) proved that injection of Mn-CycA dsRNA could delay the ovarian development cycle, which provided strong evidence for the involvement of Mn-CycA in ovarian maturation and oogenesis, and expanded a new perspective for studying the fast ovarian development cycle in M. nipponense. [ABSTRACT FROM AUTHOR]

In this study, a full-length cDNA sequence of SoxE (subgroup E within the Sox family of transcription factors) was cloned from Macrobrachium nipponense and named MnSoxE1. The full-length cDNA of MnSoxE1 is 1748 bp, consisting of a 110 bp 5′ UTR, a 105 bp 3′ UTR, and a 1533 bp ORF that encodes 510 amino acids. Conserved domains showed that MnSoxE1 has a high similarity to the SoxE gene of Penaeus vannamei. Phylogenetic tree analysis classified that MnSoxE1 with the SoxE gene of other arthropods into one clade. These results suggested that MnSoxE1 belongs to the SoxE subgroup. During embryonic development, MnSoxE1 was mainly expressed in the gastrula stage, implicating its involvement in tissue cell differentiation and formation. In the post-larval stages, the expression of MnSoxE1 continued to increase on days 1–10. The expression level in males was significantly higher than that in females. Males are clearly distinguishable from females on post-larval day 25, showing that MnSoxE1 may play a role in promoting early development and germ cell and gonadal differentiation, especially for males. qPCR analysis showed that MnSoxE1 may also be involved in oogonium proliferation during ovary development. Further in situ hybridization analysis revealed that MnSoxE1 was mainly located in oocytes and spermatocytes, especially in sertoli cells, and implies that it may be involved in the development of oocytes and spermatocytes, as well as the maintenance of testes in mature prawns. These results indicate that MnSoxE1 is involved in gonadal differentiation and development in M. nipponense, especially testis development. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The goal of this study was to analyze the functions of DNA polymerase zeta catalytic subunit (Rev3) in the oriental river prawn Macrobrachium nipponense (Mn-Rev3) with a focus on its potential roles in sex differentiation and development. The full length of Mn-Rev3 cDNA sequence was 6832 base pairs (bp) with an open reading frame of 6102 bp encoding 2033 amino acids. Mn-Rev3 showed the closest evolutionary relationship with Penaeus vannamei. The highest expression level of Mn-Rev3 occurred in the hepatopancreas and strong signals were observed in hepatopancreas cells, suggesting that Mn-Rev3 played a role in the immune system. Expression levels of Mn-Rev3 also were relatively high in the androgenic gland and testis, suggesting its potential roles in male sexual differentiation and development. During development, expression of Mn-Rev3 was highest on larval day 15 and relatively high from post-larval day 1 (PL1) to PL15, indicating that it played essential roles in promoting metamorphosis and gonad differentiation and development in M. nipponense. Strong Mn-Rev3 signals were detected in spermatids, spermatocytes, and sperm in the testes, and Mn-Rev3 expression was higher in the testes during the reproductive season than in the non-reproductive season. This result indicated that Rev3 promoted whole testis development, and especially sperm development, in M. nipponense. The expression level of Mn-Rev3 was high from ovary V to ovary II stages, indicating that Rev3 may be involved in yolk deposition. The expression level of Mn-insulin-like androgenic gland hormone (Mn-IAG) and the content of testosterone showed the same expression pattern as that of Mn-Rev3 after injection of double-stranded RNA of Mn-Rev3, which indicated that Rev3 had positive effects on male sexual differentiation and development in M. nipponense. The results of this study advance our understanding of male sexual development in M. nipponense and provide the basis for further studies of male sexual differentiation and development in crustaceans. [ABSTRACT FROM AUTHOR]

In this study, we isolated a full-length cDNA sequence from Macrobrachium nipponense and investigated its gene function. We named the gene Mntra-2a because of high similarities and close evolutionary divergence with arthropod tra-2. The full-length cDNA of Mntra-2a was 1293 bp, consisting of a 212 bp 5′ UTR, a 268 bp 3′ UTR, and an ORF of 813 bp encoding 270 amino acids. It contained an RNA recognition motif and a linker region. Real-time PCR analysis showed that Mntra-2a was highly expressed in the gonads of both males and females. Further in situ hybridization analysis showed that Mntra-2a was mainly located in oocytes and spermatocytes. During embryogenesis, Mntra-2a expression was higher in the cleavage and nauplius stages. During the ovarian reproductive cycle, Mntra-2a expression reached a peak at OvaryV and decreased to the lowest level at OvaryIV. These results indicated that Mntra-2a probably played important roles in embryonic development and early gonad development in M. nipponense. Our results provide basic information for further functional studies of tra-2 in M. nipponense. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The circadian clock is crucial for sustaining rhythmic biochemical, physiological, and behavioral processes in living creatures. In this study, we isolated and characterized two circadian clock genes in Macrobrachium nipponense , period ( Mnper ) and timeless ( Mntim ). The complete Mnper cDNA measures 4283 bp in length with an open reading frame encoding 1292 amino acids, including functional domains such as PER-ARNT-SIM (PAS), cytoplasmic localization domain (CLD), TIM interaction site (TIS), and nuclear localization signal (NLS). The deduced Mntim protein comprises1540 amino acids with functional domains such as PER interaction site (PIS), NLS, and CLD. Tissue distribution analyses showed that the two genes were highly expressed in the eyestalk and brain in both males and females, as well as being expressed in the ovary. The expression profiles of Mnper and Mntim were determined in the eyestalk, brain, and ovary under simulated breeding season and non-breeding season conditions. The expression profiles of both Mnper and Mntim appeared to be unaffected in the eyestalk. However, the expression of both genes exhibited significant seasonal variations in the brain, and thus we assumed the brain to be their functional location. The expression profiles under different simulated seasons and the variations during different ovarian stages indicate that both genes might be involved with female reproduction. Especially the mRNA levels in the brain varied greatly during these stages indicating that the clock function in the brain is closely related to ovarian development and female reproduction. And the reproductive roles of clock genes need to be elucidated. [ABSTRACT FROM AUTHOR]

Most hemoglobins are respiratory proteins and are ubiquitous in animals, bacteria, fungi, protists, and plants. In this study, we describe a membrane-bound hemoglobin in the oriental river prawn Macrobrachium nipponense (MnHb), which also expresses hemocyanin. MnHb cDNA was cloned using the rapid amplification of cDNA ends (RACE) approach, which afforded a 1201 bp gene encoding a 193 amino acid polypeptide. Bioinformatic evaluation suggested MnHb is membrane anchored by N-myristoylation, and immunofluorescence confirmed its location in the membrane of chief cells in the gill. The effect of hypoxia on MnHb expression was investigated, and reverse transcription PCR (RT-PCR) and Western blotting showed that MnHb was expressed almost exclusively in the gill. Quantitative RT-PCR revealed a significant increase in expression after 6 h of hypoxia, and levels peaked at 24 h due to oxidative stress. Exposure of cultured prawns to the stress inducer H 2 O 2 significantly up-regulated the expression of MnHb in a dose-dependent manner. MnHb may have a role in protecting cell membrane lipids from damage by reactive oxygen species. [ABSTRACT FROM AUTHOR]

Caspases are present in the cytosol as inactive proenzymes but become activated when apoptosis is initiated, playing an essential role at various stages of the process. In this study, a caspase-3 (Mncaspase-3c) was cloned from gill of the oriental river prawn Macrobrachium nipponense by reverse-transcription polymerase chain reaction and rapid amplification of cDNA ends, and its properties were characterized. The 1730-bp cDNA contained an open reading frame of 1566 bp, a 123-bp 5′-untranslated region (UTR), and a 41-bp 3′-UTR containing a poly(A) tail. The molecular mass of the deduced amino acid (aa) sequence (521 aa) was 56.3 kDa with an estimated pI of 5.01. The MnCaspase-3c sequence contained a predicted caspase family p20 domain and a caspase family p10 domain at positions 236–367 and 378–468 respectively. Recombinant MnCaspase-3c protein was expressed in Escherichia coli and purified. In vitro activity assays indicated that the recombinant MnCaspase-3c hydrolyzed the substrate Ac-DEVD-pNA, suggesting a physiological role as a caspase-3. Caspase-3c gene transcripts were distributed in all M. nipponense tissues tested by quantitative RT-PCR, being especially abundant in hemocytes. Comet assays in gill tissues showed an obvious time-dependent response to hypoxia. Furthermore, Mncaspase-3c, at both the mRNA and protein levels, was demonstrated to participate in the apoptotic process in gill after stimulation by acute hypoxia. Overall, these results indicate that hypoxia triggers apoptosis in shrimp gill tissues. [ABSTRACT FROM AUTHOR]

The tumor suppressor gene p53 plays a critical role in safeguarding the integrity of the genome in mammalian cells. It acts as a sequence-specific transcription factor. Once p53 is activated by a variety of cellular stresses, it transactivates downstream target genes and regulates the cell cycle and apoptosis. However, little is known about the functions of the p53 pathway in prawns in response to hypoxia. In this study, the cDNA of p53 from the oriental river prawn, Macrobrachium nipponense , (Mnp53) was cloned using a combination of homology cloning and rapid amplification of cDNA ends. The full-length cDNA of Mnp53 has 2130 bp, including an open reading frame of 1125 bp that encodes a polypeptide of 374 amino acids with a predicted molecular weight of 41.9 kDa and a theoretical isoelectric point of 6.9. Quantitative real-time (qRT)-PCR assays revealed that Mnp53 was ubiquitously expressed in all examined tissues, but at high levels in the hepatopancreas. In addition, we studied respiratory bursts and reactive oxygen species (ROS) production in the hepatopancreas of M . nipponense . Our results suggest that oxidative stress occurred in prawns in response to hypoxia and that apoptosis was associated with an increase in caspase-3 mRNA expression. qRT-PCR and western blot results confirmed that hypoxic stress induced the upregulation of Mnp53 at mRNA and protein levels. Furthermore, immunohistochemistry showed remarkable changes in immunopositive staining after the same hypoxic treatment. These results suggest that hypoxia-induced oxidative stress may cause apoptosis and cooperatively stimulate the expression of Mnp53. [ABSTRACT FROM AUTHOR]

A complementary DNA (cDNA) that encodes the vitellogenin receptor (VgR) in the oriental river prawn, Macrobrachium nipponense , was cloned using expressed sequence tag analysis and a rapid amplification of cDNA ends approach. The coding region consists of 5920 base pairs (bp) that encode a 1902 amino acid protein, with a predicted molecular mass of 209 kDa. The coding region is flanked by a 45 bp 5ʹ-untranslated region (UTR) and a 166 bp 3ʹ-UTR. The deduced amino acid sequence of the M. nipponense VgR cDNA had typically conserved domains, such as an extracellular, lipoprotein-binding domain, epidermal growth factor-like and O -glycosylation domains, a transmembrane domain and a short C-terminal, cytosolic tail. Quantitative real-time PCR (qPCR) indicated that Mn-VgR is highly expressed in the female ovary. Expression analysis by qPCR demonstrated the larval and ovarian developmental stage-specific expression pattern. As the ovaries developed, the expression level of Mn-VgR gradually increased during the reproductive cycle (stage I), to reach a peak in stage III. Levels then dropped as a new development cycle was entered after reproduction molting. Eyestalk ablation led to a significant increase in the expression of Mn-VgR during the ovarian development stages ( P < 0.05), when compared with the eyestalk-intact group. The investigation revealed that eyestalk ablation initially affected Mn-VgR expression and then influenced vitellogenesis. In adult females, VgR RNA interference (RNAi) dramatically delayed the maturation of the ovary, in accordance with the gonad somatic index. In addition, Mn-VgR RNAi led to vitellin depletion in the oocytes and the accumulation of vitellin in the hepatopancreas. [ABSTRACT FROM AUTHOR]

This paper reports that Slow-tonic S2 tropomyosin (Sst) and Slow tropomyosin isoform (Sti) was highly expressed in androgenic gland transcriptome of Macrobrachium nipponense , which may play crucial roles in sexual differentiation to maleness. In this study, two Sst and Sti gene homologues designated as Mnsst and Mnsti were cloned and characterized from a freshwater prawn M. nipponense . The full-length cDNA of Mnsst and Mnsti consists of 997 bp and 1926 bp, respectively, with an ORF of 852 bp encoding 284 amino acids, and the similarity in ORF reached to 95.82%. The deduced amino acid sequences of Mnsst and Mnsti shared the highest identity with Slow-tonic S2 tropomyosin and Slow tropomyosin isoform of Homarus americanus . Real-time quantitative RT-PCR showed that the Mnsst and Mnsti genes were expressed in different tissues with the highest level of expression in the androgenic gland, implying that these two genes may be related to sex-determination in M. nipponense . Real-time quantitative RT-PCR revealed that in addition, Mnsst and Mnsti were speculated to be related with embryonic organogenesis of M. nipponense , especially for the formation of complete mouthpart and digestive organ and stimulating larval changes of morphology and initiate metamorphosis, the results of present study implied that the two genes may play complex and important roles in sex differentiation of M. nipponense . Thus, we isolated two candidate genes that may advance the studies of sex-determination mechanism in M. nipponense and even the whole crustacean species, as well as promoting the all-male population culture of M. nipponense . [ABSTRACT FROM AUTHOR]

• Complete Mn-Spook gene was cloned and identified from the ovary of M. nipponense for the first time. • qRT-PCR, ISH, RNAi, ELISA and other techniques were used to systematically analyze Mn-spook gene. • Mn-Spook played a pivotal role in the molting of M. nipponense by participating in 20E production. The aim of this study was to explore the function of the Mn-Spook gene, which was found in the ovary transcriptome of the Oriental river prawn (Macrobrachium nipponense). The Spook gene, which is the precursor gene of 20-hydroxyecdysone (20E), plays an important role in the process of molting in many arthropods, but its function in M. nipponense is unclear. We cloned the full-length Mn-Spook gene from the ovary of M. nipponense and found that it had the same conserved domains as the P450 gene of the Halloween family of genes. The Mn-Spook gene was highly expressed in ovary and gill tissue during the breeding period. During ovarian development, Mn-spook gene expression was highest at the nearly-ripe stage, and it also was highly expressed in the zoea developmental stage. Cellular localization analysis showed that Mn-Spook signals accumulated in the cytoplasmic membrane and nucleus of oocytes. Finally, we used RNA interference to evaluate the function of the Mn-Spook gene. Compared with the control group, in vivo injection of Mn-Spook dsRNA effectively downregulated the expression of Mn-Spook and the content of 20E. The molting frequency of M. nipponense in the experimental group also was significantly inhibited. These results demonstrated that the Mn-Spook gene played an important role in the molting process of M. nipponense. [ABSTRACT FROM AUTHOR]

In invertebrates, neuropeptide F (NPF) has many regulatory functions, similar to NPY, its homologous peptide. In this study, two neuropeptide F genes were identified in Macrobrachium nipponense : Mn-NPF1 and Mn-NPF2. Mn-NPF2 shared the same amino acid sequence with Mn-NPF1, except for a 37 amino acid insert in the middle of the NPF region. The quantitative-PCR (qPCR) results indicated that Mn-NPF1 expression was positively correlated with ovarian maturation, whereas Mn-NPF2 had opposing expression patterns. Both Mn-NPFs were poorly expressed at early embryonic stages, but enhanced expression levels were observed up to day 10 after hatching, when the gonads began to differentiate. Ovary in situ hybridization (ISH) analyses showed that both Mn-NPFs were present at all stages, but were differentially localized to distinct compartments. Temperature gradient studies showed that both Mn-NPFs were implicated in the seasonal regulation of reproduction. A double-stranded (ds) RNA-Mn-NPF2 injection led to a significant 38.5% increase in the vitellogenin (VG) transcript (P < 0.05). These results demonstrated that Mn-NPF2 plays an important role in inhibiting ovarian maturation. Unlabelled Image • Two Neuropeptide F were cloned and characterized from Macrobrachium nipponense. • Mn-NPF1 expression is positively correlated with ovarian maturation and Mn-NPF2 have an opposite expression pattern. • Mn-NPFs expression may involve in seasonal regulation of reproduction. • RNAi of Mn-NPF2 could significantly lead to an apparent 38.5% increase of Vitellogenin (VG) transcript. [ABSTRACT FROM AUTHOR]