Dietary Astragalus polysaccharides improve the growth and innate immune response of giant freshwater prawn Macrobrachium rosenbergii: Insights from the brain-gut axis.

Supplementation with Astragalus polysaccharides (APS) has beneficial effects on aquatic animals. Herein, we aimed to investigate the effects of different doses of APS on the growth, innate immune response, and brain-gut axis of Macrobrachium rosenbergii. The molecular weight and the monosaccharide composition of APS were analyzed. APS were added at concentration of 0 (control), 0.05, 0.10, 0.15, and 0.20 % in practical diets. Growth performance increased significantly under 0.05 to 0.20 % APS, with enhanced lipase and protease activities in intestinal tissues. Prawns receiving APS supplementation had significantly lower amounts of pathogenic intestinal bacteria (Vibrio and Aeromonas) and a markedly different microbial community structure compared with those of the control group. The fecal short chain fatty acid (SCFA) and neurotransmitters γ-aminobutyric acid contents increased in the brains of prawns receiving APS, which was potentially associated with increased Lactobacillus and Bacillus levels. Prawns receiving APS supplementation displayed a significantly enhanced immune function (such as total hemocyte count, total protein concentration, phenoloxidase activity, serum agglutination titer, and lysozyme activity) and improved disease resistance to Vibrio anguillarum compared those in the control group. Thus, dietary APS positively affected the gut-brain axis by altering the microbiota composition, increasing the fecal SCFA content, and enhancing prawn immunity.

Full-Length Transcriptomes and Sex-Based Differentially Expressed Genes in the Brain and Ganglia of Giant River Prawn Macrobrachium rosenbergii.

Macrobrachium rosenbergii is an important aquaculture prawn that exhibits sexual dimorphism in growth, with males growing much faster than females. However, the mechanisms controlling these complex traits are not well understood. The nervous system plays an important role in regulating life functions. In the present work, we applied PacBio RNA-seq to obtain and characterize the full-length transcriptomes of the brains and thoracic ganglia of female and male prawns, and we performed comparative transcriptome analysis of female and male prawns. A total of 159.1-Gb of subreads were obtained with an average length of 2175 bp and 93.2% completeness. A total of 84,627 high-quality unigenes were generated and annotated with functional databases. A total of 6367 transcript factors and 6287 LncRNAs were predicted. In total, 5287 and 6211 significantly differentially expressed genes (DEGs) were found in the brain and thoracic ganglion, respectively, and confirmed by qRT-PCR. Of the 435 genes associated with protein processing pathways in the endoplasmic reticula, 42 DEGs were detected, and 21/26 DEGs with upregulated expression in the male brain/thoracic ganglion. The DEGs in this pathway are regulated by multiple LncRNAs in polypeptide folding and misfolded protein degradation in the different organs and sexes of the prawn. Our results provide novel theories and insights for studying the nervous system, sexual control, and growth dimorphism.

Glucose transporter GLUT1 expression is important for oriental river prawn (Macrobrachium nipponense) hemocyte adaptation to hypoxic conditions.

Crustaceans have an open vascular system in which hemocytes freely circulate in hemolymph. Hemocytes are rich in hemocyanin, a specific oxygen-transport protein in crustaceans; therefore, understanding the response of hemocytes to hypoxia is crucial. Although hemocytes take up glucose during hypoxia, the molecular mechanism of glucose uptake in crustaceans remains unclear. Herein, we identified two highly conserved glucose transporters (GLUT1 and GLUT2) in Macrobrachium nipponense (oriental river prawn) and analyzed their tissue-specific expression patterns. Our immunofluorescence assays showed that GLUT1 and GLUT2 are located on the cell membrane, with a strong GLUT1 signal in primary hemocytes under hypoxia. We found that during acute hypoxia, hypoxia-inducible factor-1α-related metabolic alterations result in decreased mitochondrial cytochrome c oxidase activity, implying a classic glycolytic mechanism. As a proof of concept, we replicated these findings in insect S2 cells. Acute hypoxia significantly induced hypoxia-inducible factor-1α, GLUT1, and pyruvate dehydrogenase kinase isozyme 1 expression in primary hemocytes, and hypoxia-induced increases in glucose uptake and lactate secretion were observed. GLUT1 knockdown induced intracellular reactive oxygen species generation and apoptosis in vitro and in vivo, resulting in increased prawn mortality and more apoptotic cells in their brains, implying a vital function of GLUT1 in hypoxia adaptation. Taken together, our results suggest a close relationship between hypoxia-mediated glycolysis and GLUT1 in hemocytes. These results demonstrated that in crustaceans, adaptation to hypoxia involves glucose metabolic plasticity.

The evolutionarily conserved hif-1/bnip3 pathway promotes mitophagy and mitochondrial fission in crustacean testes during hypoxia.

The hypoxia-inducible factor 1 (HIF-1) cascade is an ancient and strongly evolutionarily conserved signaling pathway that is involved in the hypoxic responses of most metazoans. Despite immense advances in the understanding of the HIF-1-mediated regulation of hypoxic responses in mammals, the contribution of the hif-1 cascade in the hypoxic adaptation of nonmodel invertebrates remains unclear. In this study, we used the oriental river prawn Macrobrachium nipponense for investigating the roles of hif-1-regulated mitophagy in crustacean testes under hypoxic conditions. We identified that the Bcl-2/adenovirus E1B 19-kDa interacting protein (bnip3) functions as a regulator of mitophagy in M. nipponense and demonstrated that hif-1α activates bnip3 by binding to the bnip3 promoter. Hif-1α knockdown suppressed the expression of multiple mitophagy-related genes, and prawns with hif-1α knockdown exhibited higher mortality under hypoxic conditions. We observed that the levels of BNIP3 were induced under hypoxic conditions and detected that bnip3 knockdown inhibited the mitochondrial translocation of dynamin-related protein 1 (drp1), which is associated with mitochondrial fission. Notably, bnip3 knockdown inhibited hypoxia-induced mitophagy and aggravated the deleterious effects of hypoxia-induced reactive oxygen species (ROS) production and apoptosis. The experimental studies demonstrated that hypoxia induced mitochondrial fission in M. nipponense via drp1. Altogether, the study elucidated the mechanism underlying hif-1/bnip3-mediated mitochondrial fission and mitophagy and demonstrated that this pathway protects crustaceans against ROS production and apoptosis induced by acute hypoxia.

Transcriptome Analysis to Study the Molecular Response in the Gill and Hepatopancreas Tissues of Macrobrachium nipponense to Salinity Acclimation.

Macrobrachium nipponense is an economically important prawn species and common in Chinese inland capture fisheries. During aquaculture, M. nipponense can survive under freshwater and low salinity conditions. The molecular mechanism underlying the response to salinity acclimation remains unclear in this species; thus, in this study, we used the Illumina RNA sequencing platform for transcriptome analyses of the gill and hepatopancreas tissues of M. nipponense exposed to salinity stress [0.4‰ (S0, control group), 6‰ (S6, low salinity group), and 12‰ (S12, high salinity group)]. Differentially expressed genes were identified, and several important salinity adaptation-related terms and signaling pathways were found to be enriched, such as "ion transport," "oxidative phosphorylation," and "glycometabolism." Quantitative real-time PCR demonstrated the participation of 12 key genes in osmotic pressure regulation in M. nipponense under acute salinity stress. Further, the role of carbonic anhydrase in response to salinity acclimation was investigated by subjecting the gill tissues of M. nipponense to in situ hybridization. Collectively, the results reported herein enhance our understanding of the mechanisms via which M. nipponense adapts to changes in salinity.

Polystyrene microplastics induced male reproductive toxicity and transgenerational effects in freshwater prawn.

Microplastics have become pervasive environmental pollutants, especially in freshwater rivers and lakes. However, how freshwater prawns' reproductive system is affected by polystyrene microplastics (PS-MPs) remains incompletely understood. Thus, the present study aimed to determine the effect of PS-MPs on the male reproductive system and offspring larval immunity in oriental river prawn. Acute exposure to PS-MPs decreased the survival rate and heart rate of prawn larvae. After chronic exposure to PS-MPs (2 and 20 mg/L) for four weeks, the oxidative stress generation in testis tissue indicated a negative impact on male prawn testicular function. PS-MPs disrupted testicular germ cell quality and caused sex hormone imbalance, leading to reduced hatching success and survival of F1 larvae, despite not being exposed to PS-MPs. Steroidogenic gene expression was altered and apoptosis-related genes had higher expression in the gonads after parental exposure to PS-MPs. Decreased immunity-related enzyme activities were observed in F1 larvae with/without continued PS-MPs exposure, compared with those in untreated prawns. A concentration-dependent increase in bioaccumulation of PS-MPs in different tissues of larval offspring was observed. Thus, PS-MPs had multiple effects on male reproductive dysfunction and transgenerational toxicity in prawns. Our findings provide a novel insight into the reproductive toxicity mechanism of microplastics in freshwater crustaceans.

Occurrence of antibiotics and antibiotic resistance genes in cultured prawns from rice-prawn co-culture and prawn monoculture systems in China.

Antibiotics and antibiotic resistance genes (ARGs) in the aquatic environment have raised great concerns, as the deleterious effects of residual antibiotics and the emergence of ARGs are challenges to aquaculture. This study analyzed feed, water, sediment and prawns' tissues from six culture ponds (integrated culture: rice-prawn pond; monoculture: prawn pond) in Tianjin, Northeast China. Eighteen types of antibiotics were detected in all ponds, which conferring to four classes of antibiotics including sulfonamides, tetracyclines, fluoroquinolones, macrolides. The mean log bioaccumulation factor (BAF) values for five antibiotics were analyzed in the hepatopancreas, muscle, and plasma, and we found the maximum Log BAF (1.45) for enrofloxacin in prawn plasma. Correlation analysis of antibiotic concentrations between the plasma and the other two tissues indicated that enrofloxacin, norfloxacin, and erythromycin levels in the hepatopancreas and muscle can be predicted by their plasma concentrations. We also conducted a hazard quotient analysis and found that the risk to human health of eating antibiotic-exposed prawns from the two types of aquaculture method was relatively low. Compared with monoculture, rice-prawn co-culture could significantly decrease the abundance of ARGs; additionally, significant correlations were detected among ARGs, antibiotics, and non-antibiotic environmental factors (e.g., total nitrogen, total ammonia nitrogen, and chemical oxygen demand) in prawn. The present study indicated that the rice-prawn co-culture system is more effective than monoculture for mitigating the bioaccumulation of antibiotics and the occurrence of ARGs in prawn.

Acupuncture for patients recovering from lacunar infarction: A protocol for systematic review and meta-analysis.

BACKGROUND: Lacunar infarction (LI) is the mild type in the classification of ischemic stroke, mostly occurs in the middle-aged and elderly, with mild hemiplegia and partial sensory disorder as the main manifestations. In the treatment of LI, acupuncture is often regarded as dominant therapy in the convalescence period. However, acupuncture for treatment of LI in the recovery period lacks high-quality reports and evidence-based medical evidence. Thus, we aim to evaluate the curative effect and safety of acupuncture for LI objectively. METHODS: Pubmed, Cochrane Library, Web of Science, EBSCO, Springer, China National Knowledge Infrastructure, Chinese Scientific and Technical Journals Database (VIP), Wan-fang Database, Chinese Biomedical Literature Database, Chinese Science Citation Database, and other electronic databases will be retrieved from the inception to May, 2021. Randomized controlled trials related to this subject will be searched. The inclusion criteria are established and a detailed literature search strategy is designed through discussion. Article retrieval, screening, excluding repetitive studies, assessment of quality, and data processing will be conducted by 2 reviewers independently using EndNote (X9) and Review Manager (5.3.5). The outcome measures include primary outcome measures (total effective rate, National Institute of Health Stroke Scale score, and Fugl-Meyer Assessment score), secondary outcome measures (blood pressure, plasma glucose, and blood lipid), and safety outcome measures. We will perform a meta-analysis, descriptive analysis, and subgroup analysis based on data conditions. RESULTS: The study of total effective rate, National Institute of Health Stroke Scale score, Fugl-Meyer Assessment score, blood pressure, plasma glucose, blood lipid, and adverse effects will provide evidenced outcome for high-quality synthesis and descriptive analysis. CONCLUSION: This systematic review will kindly provide evidence of whether acupuncture is an effective and safe intervention for LI in the recovery period. INPLASY REGISTRATION NUMBER: INPLASY202150060 (DOI:10.37766/inplasy2021.5.0060).

Comparative Phosphoproteomics Reveals a Role for AMPK in Hypoxia Signaling in Testes of Oriental River Prawn (Macrobrachium nipponense).

Hypoxia is one of the major stresses in aquaculture animals. Recently, we reported that hypoxia disrupts the endocrine system and inhibits testicular function of oriental river prawns (Macrobrachium nipponense), but the molecular mechanism of testes responded to hypoxia remains largely unknown. In the present study, we aimed to integrate whole phosphoproteomic profiles of hypoxia-treated testes of the oriental river prawn (Macrobrachium nipponense). We successfully isolated sperm cells and evaluated the mitochondrial morphology and function using laser confocal microscopy, flow cytometry, and biochemical analyses. Quantitative proteomics identified 117 differentially abundant phosphorylated proteins, and these proteins are mainly involved in the pathways related to cellular processes, including autophagy, apoptosis, and the FoxO signaling pathway. Protein-protein interaction analysis clustered these phosphoproteins into three groups, many of which have been suggested to impact carbohydrate metabolism, autophagy, and signal regulation in testes. Western blotting confirmed that phosphorylated proteins including AMPK, ULK1, and TP53 (of the AMPK pathway) may contribute to testicular dysfunction caused by hypoxia. Further, we investigated the potential roles of AMP-activated protein kinase (AMPK)'s in testes mitochondrial autophagy and apoptosis in M. nipponense as induced by hypoxia. Simultaneous knockdown of AMPKα in sperm cells led to a decrease in FOXO3a phosphorylation at Ser413, upregulation of caspase-3 and caspase-9 activities, and an increased apoptosis rate. These results improve our understanding of hypoxia-induced energy metabolism disorders in the testes of M. nipponense.

In vivo visualization assay to evaluate the effects of maternal exposure to mercury on offspring bioaccumulation in the oriental river prawn (Macrobrachium nipponense).

Mercury (Hg) is a persistent pollutant that accumulates in aquatic animals. However, studies related to understand how gonad tissue of this species responds to mercury exposure and elucidation of mercury bioaccumulation in crustacean offspring by cross-generational, are still sparse. The present study aimed to assess the bioaccumulation of Hg2+in vivo in prawn offspring by a specific aggregation-induced emission fluorogen (AIEgen). The 96 h median lethal concentration (LC50) values of mercury to the juveniles were 0.072 mg/L. Hg2+ reduced growth performance, damaged oocyte quality, and inhibited ovary maturation, thus inhibiting gonadal maturation in intact prawns. F1 offspring were exposed to Hg2+ by direct transfer from their F0 parents, as shown by the distribution of mercury in gonads and fertilized eggs. In the medium containing oriental river prawn larvae, the Hg2+ concentration decreased rapidly, indicating fast initial larval uptake of Hg2+. Due to metal ion triggered AIE activity, analysis of fluorescence images showed that prawn offspring accumulated Hg2+ via maternal transfer, and there was a relationship among the photoluminescence intensity, the AIEgen concentration, and mercury levels. The quantitative detection of Hg2+ absorption from prawn larvae by the AIEgen represents a novel analytical technique to understand the dynamics of Hg2+ between maternal and offspring.

Identifying Neuropeptide and G Protein-Coupled Receptors of Juvenile Oriental River Prawn (Macrobrachium nipponense) in Response to Salinity Acclimation.

Neuropeptides and their G protein-coupled receptors (GPCRs) from the central nervous system regulate the physiological responses of crustaceans. However, in crustaceans, our knowledge regarding GPCR expression patterns and phylogeny is limited. Thus, the present study aimed to analyze the eyestalk transcriptome of the oriental river prawn Macrobrachium nipponense in response to salinity acclimation. We obtained 162,250 unigenes after de novo assembly, and 1,392 and 1,409 differentially expressed genes were identified in the eyestalk of prawns in response to low and high salinity, respectively. We used combinatorial bioinformatic analyses to identify M. nipponense genes encoding GPCRs and neuropeptides. The mRNA levels of seven neuropeptides and one GPCR were validated in prawns in response to salinity acclimation using quantitative real-time reverse transcription polymerase chain reaction. A total of 148 GPCR-encoding transcripts belonging to three classes were identified, including 77 encoding GPCR-A proteins, 52 encoding GPCR-B proteins, and 19 encoding other GPCRs. The results increase our understanding of molecular basis of neural signaling in M. nipponense, which will promote further research into salinity acclimation of this crustacean.

Aquatic hypoxia disturbs oriental river prawn (Macrobrachium nipponense) testicular development: A cross-generational study.

Recently, we reported that hypoxia disrupts the endocrine system and causes metabolic abnormalities in prawns. Although transgenerational impairment effects of hypoxia have become a hot topic in vertebrate, it is unknown whether hypoxia could exert cross-generational effects on testicular function crustaceans. The present study aimed to investigate hypoxia's toxic effects on the testicular function of oriental river prawns (Macrobrachium nipponense) and offspring development. Hypoxia disrupted testicular germ cells quality, caused sex hormone imbalance (testosterone and estradiol), and delayed testicular development. The F1 generation derived from male prawns exposed to hypoxia showed retarded embryonic development, and reduced hatching success and larval development, despite not being exposed to hypoxia. Analysis of the transcriptome the F0 generation (exposed to hypoxia) showed that the impaired testicular functions were associated with changes to mitochondrial oxidative phosphorylation, apoptosis, and steroid biosynthesis. Interestingly, quantitative real-time PCR confirmed that hypoxia could significantly suppress the expression of antioxidant and gonad development-related genes in the testis of the F1 generations, with and without continued hypoxia exposures. In addition, paternal exposure to hypoxia could result in a higher production of reactive oxygen species in offspring testis tissue compared with those without hypoxia exposure. The cross-generational effects of testicular function implied that the sustainability of natural freshwater prawn populations would be threatened by chronic hypoxia.

Disentangling the interplay of positive and negative selection forces that shaped mitochondrial genomes of Gammarus pisinnus and Gammarus lacustris.

We hypothesized that the mitogenome of Gammarus lacustris (GL), native to the Qinghai-Tibet Plateau, might exhibit genetic adaptations to the extreme environmental conditions associated with high altitudes (greater than 3000 m). To test this, we also sequenced the mitogenome of Gammarus pisinnus (GP), whose native range is close to the Tibetan plateau, but at a much lower altitude (200-1500 m). The two mitogenomes exhibited conserved mitochondrial architecture, but low identity between genes (55% atp8 to 76.1% cox1). Standard (homogeneous) phylogenetic models resolved Gammaridae as paraphyletic, but 'heterogeneous' CAT-GTR model as monophyletic. In indirect support of our working hypothesis, GL, GP and Gammarus fossarum exhibit evidence of episodic diversifying selection within the studied Gammaroidea dataset. The mitogenome of GL generally evolves under a strong purifying selection, whereas GP evolves under directional (especially pronounced in atp8) and/or relaxed selection. This is surprising, as GP does not inhabit a unique ecological niche compared to other gammarids. We propose that this rapid evolution of the GP mitogenome may be a reflection of its relatively recent speciation and heightened non-adaptive (putatively metabolic rate-driven) mutational pressures. To test these hypotheses, we urge sequencing mitogenomes of remaining Gammarus species populating the same geographical range as GP.

Analysis of testis metabolome and transcriptome from the oriental river prawn (Macrobrachium nipponense) in response to different temperatures and illumination times.

A better understanding of the mechanisms underlying the male sexual differentiation of Macrobrachium nipponense is urgently needed in order to maintain sustainable development of the M. nipponense industry. Environmental factors, especially temperature and illumination, have dramatic effects on gonadal development. The aim of the present study was to identify key genes and metabolites involved in the male sexual differentiation and development of M. nipponense through integrated metabolomics and transcriptome analyses of the testis in response to different temperatures and illumination times. A total of 268 differentially abundant metabolites and 11,832 differentially expressed genes (DEGs) were identified. According to integrated metabolomics and transcriptome analyses, glycerophospholipid and sphingolipid metabolism was predicted to have dramatic effects on the male sexual differentiation and development of M. nipponense. According to the KEGG enrichment analysis of DEGs, oxidative phosphorylation, glycolysis/gluconeogenesis, the HIF-1 signaling pathway, the citrate cycle, steroid hormone synthesis, and the spliceosome complex were predicted to promote male differentiation and development by providing adenosine triphosphate, promoting the synthesis of steroid hormones, and providing correct gene products. Quantitative polymerase chain reaction analysis and in situ hybridization showed that the SDHB, PDE1, HSDL1, CYP81F2, SRSF, and SNRNP40 genes were differentially expressed, suggesting roles in the male sexual differentiation and development of M. nipponense. Strong candidate sex-related metabolic pathways and genes in M. nipponense were identified by integrated metabolomics and transcriptome analyses of the testis in response to different temperatures and illumination times, as confirmed by PCR analysis and in situ hybridization.

Chronic exposure to dietary antibiotics affects intestinal health and antibiotic resistance gene abundance in oriental river prawn (Macrobrachium nipponense), and provokes human health risk.

Antibiotics are employed worldwide to treat diseases of humans and other animals, but most antibiotics and their secondary metabolites are discharged into the aquatic environment, and these pollutants are emerging as a severe threat to human health. However, the systematic effects of dietary antibiotics on intestinal health in crustaceans and the associated risk to human health have not been thoroughly investigated, especially the effects on growth, immune responses, intestinal health, and the abundance of antibiotic residues and antibiotic resistance genes (ARGs). In the present work, two typical antibiotics (sulfamethoxazole and oxytetracycline) were administered orally to juvenile oriental river prawn (Macrobrachium nipponense) for eight weeks to mimic long-term use of antibiotics at legal aquaculture doses. The results indicate that dietary exposure to antibiotics significantly inhibited the growth performance of prawns, suppressed immunological parameters, and caused higher mortality in prawns challenged with Aeromonas hydrophila. Furthermore, prawns fed a diet containing antibiotics displayed a decrease in the number of intestinal goblet cells and lower digestive enzyme activity, as well as impaired intestine antioxidant ability and immune responses. Additionally, redundancy analysis revealed that different dominant bacterial phyla were responsible for increased ARG abundance in the prawn intestinal tract between control and antibiotic diet groups. The health risks posed by antibiotic residues in prawn muscle were also evaluated. Our findings demonstrate the risk posed by long-term use of dietary antibiotics in prawns, and suggest that antibiotics should be administered more carefully during aquaculture.

Molecular cloning, expression, and in situ hybridization analysis of MnGPx-3 and MnGPx-4 from oriental river prawn, Macrobrachium nipponense, in response to hypoxia and reoxygenation.

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.

Identification of neuropeptides from eyestalk transcriptome profiling analysis of female oriental river prawn (Macrobrachium nipponense) under hypoxia and reoxygenation conditions.

The oriental river prawn, Macrobrachium nipponense, is a commercial freshwater prawn species in China. It is highly sensitive to hypoxia, and this has posed a challenge to its intensive culturing. To date, the effects of hypoxia on reproduction in female prawns are not entirely clear, as are the underlying mechanisms of the effects of hypoxia. In this work, comparative transcriptome and gene expression analyses of the eyestalk were performed in M. nipponense females under hypoxia and reoxygenation conditions. Sequencing and de novo assembly of the combined reads yielded 43,583 unigenes with an average length of 1726 bp. A total of 711 genes were found to be differentially expressed in the eyestalk under the hypoxia and reoxygenation conditions. With the help of functional and pathway enrichment analysis of the differentially expressed genes, a novel set of transcripts that were associated with several important functions, such as hormone biosynthesis and progesterone-mediated oocyte maturation, were identified. Additionally, ten neuropeptides were identified based on the differentially expressed transcripts, and they were validated by quantitative real-time polymerase chain reaction (qRT-PCR) and reverse transcription PCR (RT-PCR) analyses. Three neuropeptide genes were expressed in the neural tissue and ovary of the prawns; this indicates that they were involved in reproductive processes. In particular, RNA interference (RNAi) short neuropeptide F dramatically promoted ovary maturation, as indicated by the gonad somatic index. While the present findings do indicate that hypoxia affects reproductive function in M. nipponense females, in-depth functional analyses of the candidate neuropeptides should be conducted in the future to understand their role in hypoxia adaptation and the associated mechanisms that affect the reproductive capacity of this species.

Molecular cloning, expression pattern analysis, and in situ hybridization of a Transformer-2 gene in the oriental freshwater prawn, Macrobrachium nipponense (de Haan, 1849).

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.

Identification and Characterization of the DMRT11E Gene in the Oriental River Prawn Macrobrachium nipponense.

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.

Identification and Characterization of Four Autophagy-Related Genes That Are Expressed in Response to Hypoxia in the Brain of the Oriental River Prawn (Macrobrachium nipponense).

Autophagy is a cytoprotective mechanism triggered in response to adverse environmental conditions. Herein, we investigated the autophagy process in the oriental river prawn (Macrobrachium nipponense) following hypoxia. Full-length cDNAs encoding autophagy-related genes (ATGs) ATG3, ATG4B, ATG5, and ATG9A were cloned, and transcription following hypoxia was explored in different tissues and developmental stages. The ATG3, ATG4B, ATG5, and ATG9A cDNAs include open reading frames encoding proteins of 319, 264, 268, and 828 amino acids, respectively. The four M. nipponense proteins clustered separately from vertebrate homologs in phylogenetic analysis. All four mRNAs were expressed in various tissues, with highest levels in brain and hepatopancreas. Hypoxia up-regulated all four mRNAs in a time-dependent manner. Thus, these genes may contribute to autophagy-based responses against hypoxia in M. nipponense. Biochemical analysis revealed that hypoxia stimulated anaerobic metabolism in the brain tissue. Furthermore, in situ hybridization experiments revealed that ATG4B was mainly expressed in the secretory and astrocyte cells of the brain. Silencing of ATG4B down-regulated ATG8 and decreased cell viability in juvenile prawn brains following hypoxia. Thus, autophagy is an adaptive response protecting against hypoxia in M. nipponense and possibly other crustaceans. Recombinant MnATG4B could interact with recombinant MnATG8, but the GST protein could not bind to MnATG8. These findings provide us with a better understanding of the fundamental mechanisms of autophagy in prawns.