Fujun Xuan

Identification of differentially expressed genes in hepatopancreas of oriental river prawn, Macrobrachium nipponense exposed to environmental hypoxia.

Hypoxia represents a major physiological challenge for prawn culture, and the hepatopancreas plays an important role in these processes. Here, we applied high-throughput sequencing technology to detect the gene expression profile of the hepatopancreas in M. nipponense in response to hypoxia for 3h and hypoxia for 24h. Gene expression profiling identified 1925 genes that were significantly up- or down-regulated by dissolved oxygen availability. Functional categorization of the differentially expressed genes revealed that oxygen transport, electron transport chain, reactive oxygen species generation/scavenging, and immune response were the differentially regulated processes occurring during environmental hypoxia. Finally, quantitative real-time polymerase chain reaction using six genes independently verified the tag-mapped results. Immunohistochemistry analysis revealed, for the first time, hemocyanin protein expression as significant hypoxia-specific signatures in prawns, which opens the way for in depth molecular studies of hypoxia exposure. The analysis of changes in hepatic gene expression in oriental river prawn provides a preliminary basis for a better understanding of the molecular response to hypoxia exposures.

Identification and mRNA expression of antioxidant enzyme genes associated with the oxidative stress response in the Wuchang bream (Megalobrama amblycephala Yih) in response to acute nitrite exposure.

Aquatic organisms possess cellular detoxification systems to deal with pollutants. To explore the influence of reactive oxygen species (ROS) generated in response to nitrite on oxidative stress defenses and the antioxidant system in Megalobrama amblycephala, the full length cDNA sequences were determined for three antioxidant-related genes, namely catalase (MaCAT), selenium-dependent glutathione peroxidase (MaGPx1) and Cu/Zn superoxide dismutase (MaCu/Zn-SOD). Encoded polypeptides that exhibited high identity and similarity with corresponding proteins in other fish species. Expression levels of these antioxidant genes were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) technique. MaCAT, MaGPx1 and MaCu/Zn-SOD expression was greatest in the liver and qRT-PCR was used to assess expression of these genes in juvenile fish during 72 h of exposure to 15 mg/L nitrite. Prolonged nitrite exposure resulted in the formation of excess ROS that caused oxidative damage to lipids and proteins and reduced the activities of antioxidant enzymes. Fish exposed to nitrite also showed liver damage. This study provides transcriptional data for MaCAT, MaGPx1 and MaCu/Zn-SOD that suggest expression is related positively with oxidative stress induced by nitrite exposure, indicating that imbalance between ROS and antioxidant defenses is one mechanism underlying nitrite toxicity in M. amblycephala.

Transciptomic and histological analysis of hepatopancreas, muscle and gill tissues of oriental river prawn (Macrobrachium nipponense) in response to chronic hypoxia

BackgroundOriental river prawn, Macrobrachium nipponense, is a commercially important species found in brackish and fresh waters throughout China. Chronic hypoxia is a major physiological challenge for prawns in culture, and the hepatopancreas, muscle and gill tissues play important roles in adaptive processes. However, the effects of dissolved oxygen availability on gene expression and physiological functions of those tissues of prawns are unknown. Adaptation to hypoxia is a complex process, to help us understand stress-sensing mechanism and ultimately permit selection for hypoxia- tolerant prawns, we performed transcriptomic analysis of juvenile M. nipponense hepatopancreas, gill and muscle tissues by RNA-Seq.ResultsApproximately 46,472,741; 52,773,612 and 58,195,908 raw sequence reads were generated from hepatopancreas, muscle and gill tissues, respectively. A total of 62,722 unigenes were generated, of the assembled unigenes, we identified 8,892 genes that were significantly up-regulated, while 5,760 genes were significantly down-regulated in response to chronic hypoxia. Genes from well known functional categories and signaling pathways associated with stress responses and adaptation to extreme environments were significantly enriched, including genes in the functional categories “response to stimulus”, “transferase activity” and “oxidoreductase activity”, and the signaling pathways “oxidative phosphorylation”, “glycolysis/gluconeogenesis” and “MAPK signaling”. The expression patterns of 18 DEGs involved in hypoxic regulation of M. nipponense were validated by quantitative real-time reverse-transcriptase polymerase chain reactions (qRT-PCR; average correlation coefficient = 0.94). In addition, the hepatopancreas and gills exhibited histological differences between hypoxia and normoxia groups. These structural alterations could affect the vital physiological functions of prawns in response to chronic hypoxia, which could adversely affect growth and survival of M. nipponense.ConclusionsGene expression changes in tissues from the oriental river prawn provide a preliminary basis to better understand the molecular responses of M. nipponense to chronic hypoxia. The differentially expressed genes (DEGs) identified in M. nipponense under hypoxia stress may be important for future genetic improvement of cultivated prawns or other crustaceans through transgenic approaches aimed at increasing hypoxia tolerance.

Nitrite-induced hepatotoxicity in Bluntsnout bream (Megalobrama amblycephala): The mechanistic insight from transcriptome to physiology analysis

Previous studies have investigated the physiological responses to acute nitrite exposure in fish; however, little information is available for the underlying molecular mechanisms of nitrite toxicity in aquatic ecosystems. In an effort to understand the underlying mechanisms of nitrite tolerance and to illuminate global gene expression patterns modulated by nitrite toxicity, we sampled livers from juvenile Megalobrama amblycephala exposed in 0.1, 15 and 30 mg L(-1) nitrite and performed short read (100 bp) next generation RNA sequencing (RNA-seq). The RNA-seq reads from all the exposures (≈24 million reads) were assembled into unigenes datasets according to an available reference transcriptome. Using reads from each nitrite concentration, we performed RNA-seq based gene expression analysis that identified a total of 357 differentially expressed genes. The differentially expressed genes were related to oxidative stress, apoptotic pathway, oxygen transport, immune responses and the metabolism of proteins and fats. Quantitative real-time polymerase chain reaction using six genes independently verified the RNA-seq results, the present study suggests several new candidate genes commonly regulated in liver of M. amblycephala. In addition to liver histology examinations, this study provides important mechanistic insights into nitrite-induced liver toxicity in a whole-animal physiology context, which will help in understanding the syndromes caused by nitrite poisoning.

Molecular characterization and mRNA expression of hypoxia inducible factor-1 and cognate inhibiting factor in Macrobrachium nipponense in response to hypoxia

Hypoxia inducible factors (HIFs) are considered to be the master switches of oxygen-dependent gene expression in mammalian species. Currently, very little is known about the function of this important pathway or the molecular structures of key players in the hypoxia-sensitive Oriental River Prawn Macrobrachium nipponense. In this study, HIFs-1α (HIF-1α), -1β (HIF-1β) and HIF 1-alpha inhibitor (FIH-1) from M. nipponense were cloned. The 4903-bp cDNA of M. nipponense HIF-1α (MnHIF-1α) encodes a protein of 1088 aa, M. nipponense HIF-1β (MnHIF-1β) spans 2042bp encoding 663 aa and the 1163bp M. nipponense FIH-1 (MnFIH-1) specifies a polypeptide of 345 aa. MnHIF-1 and MnFIH-1 homologs exhibit significant sequence similarity and share key functional domains with previously described vertebrate and invertebrate isoforms. Phylogenetic analysis identifies that genetic diversification of HIF-1 and FIH-1 occurred within the invertebrate lineage, indicating functional specialization of the oxygen sensing pathways in this group. Quantitative real-time RT-PCR demonstrated that MnHIF-1 and MnFIH-1 mRNA are expressed in different tissues and exhibit transcriptional responses to severe hypoxia in gill and muscle tissue, consistent with their putative role in oxygen sensing and the adaptive response to hypoxia. The role of HIF-1α in response to hypoxia was further investigated in the gills and muscles of prawns using in situ hybridization. These results suggested that HIF-1α plays an important role in oxygen sensing and homeostasis in M. nipponense.

Comparative proteomic study of the response to hypoxia in the muscle of oriental river prawn (Macrobrachium nipponense)

UNLABELLED Adaptation to hypoxia is a complex process involving up- or down-regulation of numerous different proteins. In order to understand the molecular responses to hypoxia in crustacean muscle tissue, flow cytometry and oxidative stress analysis were used to explore the (hypoxia) physiological response on Macrobrachium nipponense. A 2D-gel-based proteomic approach was performed to compare the muscle proteome of hypoxic and normoxic M. nipponense. MALDI-TOF/TOF identified 15 and five proteins were significantly up- and down-regulated, respectively, in M. nipponense muscle under hypoxic conditions for 24h. Five spots were confirmed as hemocyanin, indicating an important role in environmental regulation. Real-time quantitative PCR confirmed that hemocyanin, heat shock protein 70, glutathione S-transferases, metallothionein, phosphofructokinase, and pyruvate kinase 2 were all up-regulated by hypoxia stress. These results suggest that the cellular response to hypoxia involves regulating proteins that function in maintaining antioxidative capacity, energy levels and muscle structure. Western blotting confirmed that the well-known hypoxic stress markers hemocyanin and heat shock protein 70 were up-regulated. These results increase our understanding of hypoxia-induced proteomic and transcriptional changes in M. nipponense muscle tissue. BIOLOGICAL SIGNIFICANCE This 2-DE proteomic study investigated differentially expressed proteins in the muscle of prawns following hypoxia. Identified proteins may have roles in the response to hypoxia. These results improve our understanding of hypoxic stress in crustaceans and aquatic ecosystems.

Identification and comparative analysis of the oriental river prawn (Macrobrachium nipponense) microRNA expression profile during hypoxia using a deep sequencing approach

Hypoxia refers to a state of oxygen deficiency, which is observed frequently in aquaculture ponds. MicroRNAs (miRNAs) are small non-coding RNAs that are important effectors in regulating gene expression through posttranscriptional mechanisms. They are key elements in the response to hypoxia. The oriental river prawn (Macrobrachium nipponense) is an important commercial aquaculture species, and is sensitive to hypoxia. To date, there are no reports describing M. nipponense miRNAs. In this study, Solexa deep sequencing technology was used for high-throughput analysis of miRNAs in a small RNA library isolated from four M. nipponense tissues (gill, hepatopancreas, muscle and hemocytes). In total, 9,227,356 reads were obtained, 4,293,155 of which were related to 267 unique miRNAs, including 203 conserved and 64 prawn-specific miRNAs. Furthermore, miRNA features including length distribution and end variations were characterized. Annotation of targets revealed a broad range of biological processes and signal transduction pathways regulated by M. nipponense miRNAs. In addition, 880 co-expressed and 39 specific (25 normoxia-specific and 14 hypoxia-specific) miRNAs that may be involved in the response to hypoxia were confirmed using miRNA microarray analysis from the four prawn tissues combined. Real-time quantitative PCR (qPCR) analysis of eight miRNAs in the normoxia and hypoxia groups showed good concordance between the sequencing and qPCR data. This study provides the first large-scale identification and characterization of M. nipponense miRNAs and their potential targets, and represents a foundation for further characterization of their roles in the regulation of the diversity of hypoxia processes.

De novo assembly of the blunt snout bream (Megalobrama amblycephala) gill transcriptome to identify ammonia exposure associated microRNAs and their targets

De novo transcriptome sequencing is a robust method for microRNA (miRNA) target gene prediction, especially for organisms without reference genomes. Following exposure of Megalobrama amblycephala to ammonia (0.1 or 20 mg L−1 ), two cDNA libraries were constructed from the fish gills and sequenced using Illumina HiSeq 2000. Over 90 million reads were generated and de novo assembled into 46, 615 unigenes, which were then extensively annotated by comparing to different protein databases, followed by biochemical pathway prediction. The expression of 2666 unigenes significantly differed; 1961 were up-regulated, while 975 were down-regulated. Among these, 250 unigenes were identified as the targets for 10 conserved and 4 putative novel miRNA families by miRNA target computational prediction. We examined expression of ssa-miRNA-21 and its target genes by real-time quantitative PCR and found agreement with the sequencing data. This study demonstrates the feasibility of identifying miRNA targets by transcriptome analysis. The transcriptome assembly data represent a substantial increase in the genomic resources available for Megalobrama amblycephala and will be useful for gene expression profile analysis and miRNA functional annotation.

Molecular cloning, characterization and expression analysis of caspase-3 from the oriental river prawn, Macrobrachium nipponense when exposed to acute hypoxia and reoxygenation

ABSTRACT 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. HighlightsA capspase‐3 like protein was identified in river prawn Macrobrachium nipponense.Recombinant MnCaspase‐3c hydrolyzed the substrate Ac‐DEVD‐pNA.Mncaspase‐3c participated in apoptosis in gills associated with oxidative stress after stimulation by acute hypoxia.

Dynamic mRNA and miRNA expression analysis in response to hypoxia and reoxygenation in the blunt snout bream (Megalobrama amblycephala)

Adaptation to hypoxia is a complex process involving various pathways and regulation mechanisms. A better understanding of the genetic influence on these mechanisms could permit selection for hypoxia-sensitive fish. To aid this understanding, an integrated analysis of miRNA and mRNA expression was performed in Megalobrama amblycephala under four acute hypoxia and reoxygenation stages. A number of significantly differentially-expressed miRNAs and genes associated with oxidative stress were identified, and their functional characteristics were revealed by GO function and KEGG pathway analysis. They were found to be involved in HIF-1 pathways known to affect energy metabolism and apoptosis. MiRNA-mRNA interaction pairs were detected from comparison of expression between the four different stages. The function annotation results also showed that many miRNA-mRNA interaction pairs were likely to be involved in regulating hypoxia stress. As a unique resource for gene expression and regulation during hypoxia and reoxygenation, this study could provide a starting point for further studies to better understand the genetic background of hypoxia stress.