Hongtuo Fu

Constructing and random sequencing analysis of normalized cDNA library of testis tissue from oriental river prawn (Macrobrachium nipponense)

The oriental river prawn, Macrobrachium nipponense, is an important aquaculture species in China. Sexual precocity is a serious problem because of genetic retrogression, which has negative effects on product quality and dramatically affects price. Culture of all-male populations of this species would be economically advantageous, as the males grow faster and reach a much larger size than females. Developing such a culture scheme will require discovery of sex- or reproduction-related genes that affect sexual maturity and sex determination. In this study, a high-quality normalized testis cDNA library was constructed to identify novel transcripts. Of the 5280 successful sequencing reaction yields, 5202 expressed tagged sequences (ESTs) with an average length of 954 bp. Ultimately, 3677 unique sequences, including 891 contigs and 2786 singletons, were identified based on cluster and assembly analyses. Sixteen hundred (43.5%) genes were novel based on the NCBI protein database, thus these unidentified genes may improve basic molecular knowledge about M. nipponense. Of the novel unigenes, 34.4% (715/2077) were homologous to insects, such as Tribolium castaneum, Drosophila spp. and Apis mellifera. Fifty-two genes were identified as sex- or reproduction-related based on Gene Ontology classification and sequence comparison with data from other publications. These genes can be classified into groups based on different functions, including 10 sex-determination related genes, 8 male-reproductive genes, 5 cathepsin-related genes, 20 ubiquitin-related genes, 5 ferritin-related genes, and 4 LRR genes. The results of this study provide new sequence information about M. nipponense, which will be the basis for further genetic studies of this species and other decapods crustaceans.

Characterization, expression, and function analysis of gonad-inhibiting hormone in Oriental River prawn, Macrobrachium nipponense and its induced expression by temperature.

Gonad-inhibiting hormone (GIH) is a member of crustacean hyperglycemic hormone family and plays a major role in regulating reproduction in crustaceans. In this study, a full-length cDNA of GIH of Oriental River prawn, Macrobrachium nipponense (Mn-GIH) was cloned from the eyestalk. A 1350 bp full-length Mn-GIH cDNA harbored 336 bp of an open reading frame encoding signal peptide of 112 amino acid residues. Sequence analysis revealed that the overall cDNA sequence and specific functional sites of Mn-GIH were highly conserved with those in other crustacean species. Expression analysis by quantitative real-time PCR demonstrated its tissue-specific, larval developmental stage-specific, and ovary developmental stage-specific expression pattern, respectively. The RNAi by GIH-ds-RNA in vivo injection was effective in this study and resulted a 50% (day 1), 83% (day 5) and 63% (day 9) down-regulation compared to control. The obvious changes of gonad somatic index (GSI) rate also provided strong evidence to the inhibition effects of GIH on ovary maturation and spawning. Four temperature gradients (12 °C ± 1 °C, 17 °C ± 1 °C, 22 °C ± 1 °C, 27 °C ± 1 °C) were set to imitate the temperature in breeding and non-breeding seasons. The observed expression profiles suggest that Mn-GIH did not display a high level expression as supposed to maintain an immature ovary state under low temperature (12 °C). The results indicated that GIH was probably activated to concentrating and working by a proper temperature before reaching to breeding season.

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.

Molecular insights into reproduction regulation of female Oriental River prawns Macrobrachium nipponense through comparative transcriptomic analysis

The oriental river prawn, Macrobrachium nipponense, is an important commercial aquaculture resource in China. During breeding season, short ovary maturation cycles of female prawns cause multi-generation reunions in ponds and affect the growth of females representing individual miniaturization (known as autumn -propagation). These reproductive characteristics pose problems for in large - scale farming. To date, the molecular mechanisms of reproduction regulation of M. nipponense remain unclear. To address this issue, we performed transcriptome sequencing and gene expression analyses of eyestalk and cerebral ganglia of female M. nipponense during breeding and non-breeding seasons. Differentially expressed gene enrichment analysis results revealed several important reproduction related terms and signaling pathways, such as “photoreceptor activity”, “structural constituent of cuticle” and “G-protein coupled receptor activity”. The following six key genes from the transcriptome were predicted to mediate environmental factors regulating reproduction of M. nipponense: neuroparsin, neuropeptide F II, orcokinin II, crustacean cardioactive peptide, pigment-dispersing hormone 3 and tachykinin. These results will contribute to a better understanding of the molecular mechanisms of reproduction of oriental river prawns. Further detailed functional analyses of the candidate reproduction regulation related neuropeptides are needed to shed light on the mechanisms of reproduction of crustacean.

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.

Based on the Metabolomic Approach the Energy Metabolism Responses of Oriental River Prawn Macrobrachium nipponense Hepatopancreas to Acute Hypoxia and Reoxygenation

Hypoxia represents a major physiological challenge for prawns and is a problem in aquaculture. Therefore, an understanding of the metabolic response mechanism of economically important prawn species to hypoxia and re-oxygenation is essential. However, little is known about the intrinsic mechanisms by which the oriental river prawn Macrobrachium nipponense copes with hypoxia at the metabolic level. In this study, we conducted gas chromatography-mass spectrometry-based metabolomics studies and assays of energy metabolism-related parameters to investigate the metabolic mechanisms in the hepatopancreas of M. nipponense in response to 2.0 O2/L hypoxia for 6 and 24 h, and reoxygenation for 6 h following hypoxia for 24 h. Prawns under hypoxic stress displayed higher glycolysis-related enzyme activities and lower mRNA expression levels of aerobic respiratory enzymes than those in the normoxic control group, and those parameters returned to control levels in the reoxygenated group. Our results showed that hypoxia induced significant metabolomic alterations in the prawn hepatopancreas within 24 h. The main metabolic alterations were depletion of amino acids and 2-hydroxybutanoic acid and accumulation of lactate. Further, the findings indicated that hypoxia disturbed energy metabolism and induced antioxidant defense regulation in prawns. Surprisingly, recovery from hypoxia (i.e., reoxygenation) significantly affected 25 metabolites. Some amino acids (valine, leucine, isoleucine, lysine, glutamate, and methionine) were markedly decreased compared to the control group, suggesting that increased degradation of amino acids occurred to provide energy in prawns at reoxygenation conditions. This study describes the acute metabolomic alterations that occur in prawns in response to hypoxia and demonstrates the potential of the altered metabolites as biomarkers of hypoxia.

iTRAQ-based quantitative proteomic analysis of embryonic developmental stages in Amur sturgeon, Acipenser schrenckii

The Amur sturgeon, Acipenser schrenckii, is an important aquaculture species in China with annual production of about 150 thousand tons in 2015. In this study, we investigated the regulatory proteins and pathways affecting embryonic development of Amur sturgeon, by analyzing of the differential proteomes among four embryonic developmental stages using isobaric tags for relative and absolute quantitation (iTRAQ), combined with the analysis of effects of microelements and antioxidants on embryonic development. Seventy-four, 77, and 76 proteins were differentially expressed according to iTRAQ analysis between the fertilized egg and blastula, blastula and neurula, and neurula and heart-beat stages, respectively. GO and KEGG enrichment analyses indicated that Gluconeogenesis, Ribosome and Proteasome were the most enriched pathways, which may promote energy formation, immune system protection and protein synthesis process in A. schrenckii. The measurement of microelements indicated that Mn, Cu and Fe were obtained from their parents or water environment in A. schrenckii, while Zn plays vital roles throughout embryonic development. The dramatically high level of malondialdehyde (MDA) across the embryonic development may be the main reason leading to a low hatching rate in A. schrenckii. This study provides the basis for further proteome analysis of embryonic development in A. schrenckii.

Hypoxia induces changes in AMP-activated protein kinase activity and energy metabolism in muscle tissue of the oriental river prawn Macrobrachium nipponense

Hypoxia has important effects on biological activity in crustaceans, and modulation of energy metabolism is a crucial aspect of crustaceans’ ability to respond to hypoxia. The adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) enzyme is very important in cellular energy homeostasis; however, little information is known about the role of AMPK in the response of prawns to acute hypoxia. In the present study, three subunits of AMPK were cloned from the oriental river prawn, Macrobrachium nipponense. The full-length cDNAs of the α, β, and γ AMPK subunits were 1,837, 3,174, and 3,773 bp long, with open reading frames of 529, 289, and 961 amino acids, respectively. Primary amino acid sequence alignment of these three subunits revealed conserved similarity between the functional domains of the M. nipponense AMPK protein with AMPK proteins of other animals. The expression of the three AMPK subunits was higher in muscle tissue than in other tissues. Furthermore, the mRNA expression of AMPKα, AMPKβ, and AMPKγ were significantly up-regulated in M. nipponense muscle tissue after acute hypoxia. Probing with a phospho-AMPKα antibody revealed that AMPK is phosphorylated following hypoxia; this phosphorylation event was found to be essential for AMPK activation. Levels of glucose and lactic acid in hemolymph and muscle tissue were significantly changed over the course of hypoxia and recovery, indicating dynamic changes in energy metabolism in response to hypoxic stress. The activation of AMPK by hypoxic stress in M. nipponense was compared to levels of muscular AMP, ADP, and ATP, as determined by HPLC; it was found that activation of AMPK may not completely correlate with AMP:ATP ratios in prawns under hypoxic conditions. These findings confirm that the α, β, and γ subunits of the prawn AMPK protein are regulated at the transcriptional and protein levels during hypoxic stress to facilitate maintenance of energy homeostasis.