Behavior and toxicity assessment of copper nanoparticles in aquatic environment: A case study on red swamp crayfish.

With the wide use of copper nanoparticles (CuNPs) in various industrial and commercial applications, they inevitably enter the aquatic environment. However, their behavior in the aquatic environment and potential toxicity to aquatic organisms remain little known. In this study, we investigated the behavior of CuNPs in freshwater, as well as the toxicity and bioaccumulation of CuNPs and copper sulfate (CuSO4), used as a positive control for copper ions toxicity, in red swamp crayfish (Procambarus clarkii). The results showed that CuNPs released copper ions into freshwater and aggregated rapidly in freshwater, and their release of copper ions and aggregation slowed down at a higher concentration of CuNPs. The calculated 72-h LC50 values for crayfish were 1.18 and 0.54 mg/L for CuNPs and CuSO4, respectively. Cu accumulation in the gill and hepatopancreas from CuSO4 treatments was significantly higher than that from CuNPs, and the highest Cu bioaccumulation level in crayfish was found in the gill, followed by hepatopancreas and muscle with the exposure of copper. The activities of the antioxidative enzymes in the crayfish significantly decreased after exposure to CuNPs for 48 h, compared to the control (without CuNPs or CuSO4). Histological examination revealed that there was no significant alteration of hepatopancreas in the crayfish exposed to CuNPs. Meanwhile, the growth of crayfish was not significantly inhibited by CuNPs. These results suggested that CuNPs exposure can induce oxidative stress in the crayfish, gill is the main tissue for their accumulation, and their toxicity is mainly caused by the released copper ions.

Reproductive performance and semen characteristics of pond-reared and wild-caught large-sized male broodstock of the Chinese mitten crab Eriocheir sinensis.

There has been a recent emphasis on production of large-sized Eriocheir sinensis broodstock. In China, aquaculturists generally prefer wild-caught (WC) crabs from the Yangtze River as broodstock because offspring performance is superior to that of pond-reared (PR) broodstock. Currently, however, there is a ban on fishing in the Yangtze River, and effects on E. sinensis breeding have not been ascertained. There was comparison in the present study of reproductive performance and semen characteristics of male broodstock of PR and WC groups. After copulation, sperm quantity in the vas deferens of crabs in specimens of both groups was large, although there was a consistent decrease in vaso-somatic index. Although sperm density of PR crabs was less, that of WC specimens remained relatively constant. Specimens of neither group, however, had changes in the hepatopancreas index or condition factor, and sperm survival was close to 100%. Although the acrosome reaction was detected in response to cold-temperature induction, there were differences in extent of reaction to cold temperatures. Importantly, in as many as 98% of sperm from female spermathecae, the reaction was completed, which was considerably greater than 15% for sperm of males post-mating. It is concluded there was no difference between PR and WC crabs with respect to reproductive performance or semen characteristics, and, notably, sperm from PR crabs were of sufficient quality for use in E. sinensis aquaculture enterprises. Accordingly, it is predicted the Yangtze River fishing ban would only have a limited effect on supply of male E. sinensis broodstock.

Chromosome-level genome assembly of Paralithodes platypus provides insights into evolution and adaptation of king crabs.

The blue king crab, Paralithodes platypus, which belongs to the family Lithodidae, is a commercially and ecologically important species. However, a high-quality reference genome for the king crab has not yet been reported. Here, we assembled the first chromosome-level blue king crab genome, which contains 104 chromosomes and an N50 length of 51.15 Mb. Furthermore, we determined that the large genome size can be attributed to the insertion of long interspersed nuclear elements and long tandem repeats. Genome assembly assessment showed that 96.54% of the assembled transcripts could be aligned to the assembled genome. Phylogenetic analysis showed the blue king crab to have a close relationship with the Eubrachyura crabs, from which it diverged 272.5 million years ago. Population history analyses indicated that the effective population of the blue king crab declined sharply and then gradually increased from the Cretaceous and Neogene periods, respectively. Furthermore, gene families related to developmental pathways, steroid and thyroid hormone synthesis, and inflammatory regulation were expanded in the genome, suggesting that these genes contributed substantially to the environmental adaptation and unique body plan evolution of the blue king crab. The high-quality reference genome reported here provides a solid molecular basis for further study of the blue king crab's development and environmental adaptation.

Chromosome-level genome assembly reveals the unique genome evolution of the swimming crab (Portunus trituberculatus).

BACKGROUND: The swimming crab, Portunus trituberculatus, is an important commercial species in China and is widely distributed in the coastal waters of Asia-Pacific countries. Despite increasing interest in swimming crab research, a high-quality chromosome-level genome is still lacking. FINDINGS: Here, we assembled the first chromosome-level reference genome of P. trituberculatus by combining the short reads, Nanopore long reads, and Hi-C data. The genome assembly size was 1.00 Gb with a contig N50 length of 4.12 Mb. In addition, BUSCO assessment indicated that 94.7% of core eukaryotic genes were present in the genome assembly. Approximately 54.52% of the genome was identified as repetitive sequences, with a total of 16,796 annotated protein-coding genes. In addition, we anchored contigs into chromosomes and identified 50 chromosomes with an N50 length of 21.80 Mb by Hi-C technology. CONCLUSIONS: We anticipate that this chromosome-level assembly of the P. trituberculatus genome will not only promote study of basic development and evolution but also provide important resources for swimming crab reproduction.

Evaluating expression of autophagy-related genes in oriental river prawn Macrobrachium nipponense as potential biomarkers for hypoxia exposure.

Autophagy, a crucial process for maintaining cellular homeostasis, is under the control of several autophagy-related (ATG) proteins, and is highly conserved in most animals, but its response to adverse environmental conditions is poorly understood in crustaceans. Herein, we hypothesised that autophagy acts as a protective response to hypoxia, and Beclin 1, ATG7 and ATG8 in oriental river prawn (Macrobrachium nipponense) were chosen as potential biomarkers under hypoxia exposure; thus, their full-length cDNA sequences were cloned and characterised. Open reading frames (ORFs) of 1281, 2076 and 360 bp, encoding proteins of 427, 692 and 120 amino acid residues, respectively, were obtained. Phylogenetic analysis demonstrated the three M. nipponense proteins do not form a clade with vertebrate homologs. Protein and mRNA levels were investigated in different tissues and developmental stages, and all three were significantly upregulated in a time-dependent manner in the hepatopancreas following hypoxia stress. Biochemical and morphological analysis of hepatocytes revealed that hypoxia increased the abundance of hepatic autophagic vacuoles and stimulated anaerobic metabolism. RNA interference-mediated silencing of ATG8 significantly increased the death rate of M. nipponense juveniles under hypoxia stress conditions. Together, these results suggest that Beclin 1, ATG7 and ATG8 contribute to autophagy-based responses against hypoxia in M. nipponense. The findings also expand our understanding of the potential role of autophagy as an adaptive response against hypoxia toxicity in crustaceans. The results showed that hepatic ATG8 levels may be directly indicative of acute hypoxia in prawns, and provide insight into the time at which hypoxia exposure occurs. Autophagy-related genes expression pattern seems to be sensitive and good biomarkers of acute hypoxia exposure.

High-Quality Genome Assembly of Eriocheir japonica sinensis Reveals Its Unique Genome Evolution.

As an important freshwater aquaculture species in China, the Chinese mitten crab (Eriocheir japonica sinensis) has high economic and nutritional value. However, limited genomic information is currently available for studying its basic development and genetic diversity. Here, we performed whole-genome sequencing on Oxford Nanopore Technologies Limited's platform using promethION. The assembled size of E. j.sinensis genome was approximately 1.27 Gb, which is close to the estimated size (1.19 Gb). Furthermore, based on assessment using Benchmarking Universal Single-Copy Orthologs (BUSCO) (Simao et al., 2015), 94.00% of the expected eukaryotic genes were completely present in the genome assembly. In addition, repetitive sequences accounted for ~61.42% of the assembled genome, and 22,619 protein-coding genes were annotated. Comparative genomics analysis demonstrated that the Chinese mitten crab diverged from Penaeus vannamei ~373.6 million years ago, with a faster evolution rate than shrimp. We anticipate that the annotated high-quality genome of E. j. sinensis will promote research on its basic development and evolution and make substantial contributions to comparative genomic analyses of crustaceans.

De novo transcriptome sequencing and analysis of male and female swimming crab (Portunus trituberculatus) reproductive systems during mating embrace (stage II).

BACKGROUND: The swimming crab Portunus trituberculatus is one of the most commonly farmed crustaceans in China. As one of the most widely known and high-value edible crabs, it crab supports large crab fishery and aquaculture in China. Only large and sexually mature crabs can provide the greatest economic benefits, suggesting the considerable effect of reproductive system development on fishery. Studies are rarely conducted on the molecular regulatory mechanism underlying the development of the reproductive system during the mating embrace stage in this species. In this study, we used high-throughput sequencing to sequence all transcriptomes of the P. trituberculatus reproductive system. RESULTS: Transcriptome sequencing of the reproductive system produced 81,688,878 raw reads (38,801,152 and 42,887,726 reads from female and male crabs, respectively). Low-quality (quality <20) reads were trimmed and removed, leaving only high-quality reads (37,020,664 and 41,021,030 from female and male crabs, respectively). A total of 126,188 (female) and 164,616 (male) transcripts were then generated by de novo transcriptome assembly using Trinity. Functional annotation of the obtained unigenes revealed that a large number of key genes and some important pathways may participate in cell proliferation and signal transduction. On the basis of our transcriptome analyses and as confirmed by quantitative real-time PCR, a number of genes potentially involved in the regulation of gonadal development and reproduction of P. trituberculatus were identified: ADRA1B, BAP1, ARL3, and TRPA1. CONCLUSION: This study is the first to report on the whole reproductive system transcriptome information in stage II of P. trituberculatus gonadal development and provides rich resources for further studies to elucidate the molecular basis of the development of reproductive systems and reproduction in crabs. The current study can be used to further investigate functional genomics in this species.

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.

Molecular Cloning and Functional Characterization of a Hexokinase from the Oriental River Prawn Macrobrachium nipponense in Response to Hypoxia.

Metabolic adjustment to hypoxia in Macrobrachium nipponense (oriental river prawn) implies a shift to anaerobic metabolism. Hexokinase (HK) is a key glycolytic enzyme in prawns. The involvement of HK in the hypoxia inducible factors (HIFs) pathway is unclear in prawns. In this study, the full-length cDNA for HK (MnHK) was obtained from M. nipponense, and its properties were characterized. The full-length cDNA (2385 bp) with an open reading frame of 1350 bp, encoded a 450-amino acid protein. MnHK contained highly conserved amino acids in the glucose, glucose-6-phosphate, ATP, and Mg+2 binding sites. Quantitative real-time reverse transcription PCR assays revealed the tissue-specific expression pattern of MnHK, with abundant expression in the muscle, and gills. Kinetic studies validated the hexokinase activity of recombinant HK. Silencing of HIF-1α or HIF-1ß subunit genes blocked the induction of HK and its enzyme activities during hypoxia in muscles. The results suggested that MnHK is a key factor that increases the anaerobic rate, and is probably involved in the HIF-1 pathway related to highly active metabolism during hypoxia.

Molecular cloning, mRNA expression and characterization of membrane-bound hemoglobin in oriental river prawn Macrobrachium nipponense.

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 1201bp 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 6h of hypoxia, and levels peaked at 24h due to oxidative stress. Exposure of cultured prawns to the stress inducer H2O2 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.

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

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.

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 p53 from the oriental river prawn, Macrobrachium nipponense, in response to hypoxia.

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.

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

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.

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), -1ß (HIF-1ß) 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-1ß (MnHIF-1ß) 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.

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.

Characterization of the complete mitochondrial genome of the red crayfish, Procambarus clarkii (Decapoda: Cambaridae).

Here we present the complete mitochondrial (mt) genome of Procambarus clarkii (Decapoda: Cambaridae) and provide its annotation. The complete mt genome was determined to be 15 929 bp and contains 22 tRNA genes, 13 protein-coding genes (PCGs), two rRNA genes, and a D-loop region. The nucleotide composition was biased toward A + T nucleotides (72.91%) and the AT skew of this mt genome was slightly negative. All the 22 tRNA genes displayed a typical clover-leaf structure, with the exception of trnS1 (AGN). About 13 PCGs were initiated by ATN codons, except for cox1 and nad2 genes which were initiated by ACG and GTG, respectively. Six of the 13 PCGs harbor the incomplete termination codon by T or TA. The D-loop region of the mt genome was 1188 bp in length and the A + T content was 81.08%. Phylogenetic analysis showed that the placement of P. clarkii was within the Cambaridae. This mt genome sequence will provide a better understanding for crayfish evolution in the future.

The complete mitochondrial genome of the red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae).

The complete mitochondrial genome (mitogenome) of Tribolium castaneum (Coleoptera: Tenebrionidae) was determined to be 15,883 bp (GenBank accession No. KM009121), which contains 22 tRNA genes, 13 protein-coding genes (PCGs), 2 rRNA genes and a major non-coding A + T-rich region. It has the typical gene organization and order of mitogenomes from ancestral insects. The nucleotide composition was also biased toward A + T nucleotides (71.72%) and the AT skew of this mitogenome was slightly positive. All of the 22 tRNA genes displayed a typical clover-leaf structure, with the exception of trnS1 (AGN). Thirteen PCGs were initiated by ATN codons, except for cytochrome c oxidase subunit 1 (cox1) gene which was initiated by AAT. Eight of the 13 PCGs harbor the incomplete termination codon by T or TA. The A + T-rich region of the mitogenome was 1237 bp in length and the A + T content was 82.30%.

Identification of the complete mitochondrial genome of the pacific white shrimp Litopenaeus vannamei (Decapoda: Penaeidae).

The complete mitochondrial genome (mitogenome) of Litopenaeus vannamei was determined to be 15 989 bp in size, it contains 22 tRNA genes, 13 protein-coding genes (PCGs), two rRNA genes and a D-loop region. The nucleotide composition was biased toward A + T nucleotides (67.8%) and the AT skew of this mitogenome was slightly negative (-0.025). All tRNA genes displayed a typical clover-leaf structure, except for trnS1 (AGN). Thirteen PCGs were initiated by ATN codons, except for cox1 gene which was initiated by ACG. Four of the 13 PCGs had the incomplete termination codon by T. The rrnL was 1371 bp and the rrnS was 853 bp, and the AT content of the combined rRNA genes was 70.9%. The D-loop region of the mitogenome was 985 bp in length and the A + T content was 82.7%, and no tandem repeat was found in this region. Phylogenetic analysis showed that the placement of L. vannamei was within the Penaeidae.

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

BACKGROUND: Oriental 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. RESULTS: Approximately 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. CONCLUSIONS: Gene 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.