AjTGFß alleviates V. splendidus-induced inflammation through SMADs pathway in Apostichopus japonicus.

The inhibition of inflammatory response is an essential process to control the development of inflammation and is an important step to protect the organism from excessive inflammatory damage. As a pleiotropic cytokine, transforming growth factor beta (TGF-ß) plays a regulatory role in inhibiting inflammation in vertebrates. To investigate the role of TGF-ß in the regulation of inflammation in invertebrates, we cloned and characterized the TGF-ß gene from Apostichopus japonicus via rapid amplification of cDNA ends, and the sample was designated as AjTGF-ß. For Vibrio splendidus-challenged sea cucumbers, the expression of AjTGF-ß mRNAs in coelomocytes decreased at 96 h (0.27-fold), which was contrary to the trend of inflammation. AjTGF-ß was expressed in all tissues with the highest expression in the body wall. When AjTGF-ß was knocked down by using small interfering RNA (siRNA-KD) to 0.45-fold, AjSMAD 2/3 and AjSMAD6 were downregulated to 0.32- and 0.05-fold compared with the control group, respectively. Furthermore, when the damaged sea cucumber was challenged by V. splendidus co-incubated with rAjTGF-ß, the damage area had no extensive inflammation, and damaged repair appeared at 72 h compared with the Vs + BSA group, in which the expression of AjSMAD 2/3 was upregulated by 1.35-fold. Under this condition, AjSMAD 2/3 silencing alleviated rAjTGF-ß-induced damage recovery. Moreover, rAjTGF-ß slightly induced the collagen I expression from 6.13 ng/mL to 7.84 ng/mL, and collagen III was upregulated from 6.23 ng/mL to 6.89 ng/mL compared with the Vs + BSA group. This finding indicates that AjTGF-ß negatively regulated the inflammatory progress and accelerated the repair of damage by AjSMADs to regulate the collagens expression.

Combined Effects of Elevated Temperature and Crude Oil Pollution on Oxidative Stress and Apoptosis in Sea Cucumber (Apostichopus japonicus, Selenka).

Currently, global climate change and oil pollution are two main environmental concerns for sea cucumber (Apostichopus japonicus) aquaculture. However, no study has been conducted on the combined effects of elevated temperature and oil pollution on sea cucumber. Therefore, in the present study, we treated sea cucumber with elevated temperature (26 °C) alone, water-accommodated fractions (WAF) of Oman crude oil at an optimal temperature of 16 °C, and Oman crude oil WAF at an elevated temperature of 26 °C for 24 h. Results showed that reactive oxygen species (ROS) level and total antioxidant capacity in WAF at 26 °C treatment were higher than that in WAF at 16 °C treatment, as evidenced by 6.03- and 1.31-fold-higher values, respectively. Oxidative damage assessments manifested that WAF at 26 °C treatment caused much severer oxidative damage of the biomacromolecules (including DNA, proteins, and lipids) than 26 °C or WAF at 16 °C treatments did. Moreover, compared to 26 °C or WAF at 16 °C treatments, WAF at 26 °C treatment induced a significant increase in cellular apoptosis by detecting the caspase-3 activity. Our results revealed that co-exposure to elevated temperature and crude oil could simulate higher ROS levels and subsequently cause much severer oxidative damage and cellular apoptosis than crude oil alone on sea cucumber.

Molecular cloning and functional characterization of theta class glutathione S-transferase from Apostichopus japonicus.

Glutathione S-transferases (GSTs) are the superfamily of multifunctional detoxification isoenzymes and play crucial roles in innate immunity. In the present study, a theta class GST homology was identified from A. japonicus (designated as AjGST-θ) by RACE approaches. The full-length cDNA of AjGST-θ was of 1013 bp encoded a cytosolic protein of 231 amino acids residues. Structural analysis revealed that AjGST-θ processed the characteristic N-terminal GSH-binding site (G-site) and the C-terminal hydrophobic substrate binding site (H-site). Multiple sequence alignment and phylogenetic analysis together supported that AjGST-θ belonged to a new member of theta class GST protein subfamily. Spatial expression analysis revealed that AjGST-θ was ubiquitously expressed in all examined tissues with the larger magnitude in intestine. The Vibrio splendidus challenge in vivo and LPS stimulation in vitro could both significantly up-regulate the mRNA expression of AjGST-θ when compared with control group. The recombinant protein was expressed in Escherichia coli and the purified AjGST-θ showed high activity with GST substrate. Meantime, disc diffusion assay showed that recombinant AjGST-θ protein could markedly improve bacterial growth under Cumene hydroperoxide exposure. More importantly, the recombinant AjGST-θ could effectively prevent primary coelomocytes apoptosis after LPS exposure. Our present findings suggested that AjGST-θ might play significantly roles in the modulation of immune response and protect cells from pathogens infection in A. japonicus.

Characterization and expression analysis of a thioredoxin-like protein gene in the sea cucumber Apostichopus japonicus.

As the most important disulfide bond reducates of intracellular oxidordeuctase, thioredoxin (TRX) plays a crucial role in maintaining reducing state of intracellular proteins to normally perform their function. In this study, a cDNA of TRX-like protein gene from Apostichopus japonicus (denoted as AjTRX) was cloned and characterized. The full-length cDNA of AjTRXwas of 1870 bp, consisting of a 5'-UTR of 101 bp, a long 3'-UTR of 887 bp and a 882 bp open reading frame (ORF) encoding a 293 amino acids. The predicted molecular mass and the theoretical PI of the deduced amino acids of AjTRX were 32.3 kDa and 5.52, respectively. Phylogenetic trees showed that AjTRX had a closer evolution relationship with TRX from Strongylocentrotus purpuratus. AjTRX was found to be ubiquitously expressed in all examined tissues including longitudinal muscle, coelomocytes, tube feet, intestine, respiratory tree and body wall indicating a general role in physiological processes. Temporal expression pattern of AjTRX in coelomocytes showed that AjTRX reached two peak expression levels at 8 h and 48 h after Vibrio splendidus challenge with a 8.6 and 9.3-fold increase compared to their control groups, respectively. The recombinant AjTRX protein (rAjTRX) displayed obvious antioxidant activity in a dose-dependent manner, and the higher reducing activity was detected in 20 µM experimental group. All these results strongly suggested that AjTRX could play an important role as an antioxidant in a physiological context, and might be involved in the process of bacterial challenge.

Cathepsin L of the sea cucumber Apostichopus japonicus-molecular characterization and transcriptional response to Vibrio splendidus infection.

Cathepsin L, a lysosomal endopeptidase, has been noted for its involvement in the innate immune response in invertebrates. Here, the cathepsin L cDNA of the sea cucumber Apostichopus japonicus (AjCatL) is identified from an EST library and then cloned by the rapid amplification of the cDNA ends (RACE) PCR. The full-length cDNA is 1678 bp long containing an open reading frame (ORF) of 1002 bp, an 80 bp 5' UTR and a 599 bp 3' UTR. The cDNA encodes 333 amino acid residues with a predicted molecular mass of 37.07 kDa and a theoretical isoelectric point (pI) of 5.01. The full-length AjCatL contains three active sites of eukaryotic thiol (cysteine) protease at positions 133-144, 278-288 and 295-314. Analysis of the predicted tertiary structure of prepro-CatL (17-333 aa) and mature-CatL (116-333 aa) reveals that the propeptide region (17-115 aa) blocks access to the substrate-binding cleft. Phylogenetic analysis shows that the AjCatL is clustered together with two other CatLs from Strongylocentrotus purpuratus. The enzymatic activity of AjCatL was verified using a substrate hydrolyzing assay with recombinant mAjCatL. Further analysis of real time-PCR demonstrates that the expression of AjCatL mRNA is significantly up-regulated in the coelomocytes in cases of infection with the common bacterial pathogen, Vibrio splendidus. This suggests that the AjCatL is likely to be involved in the immune response.

MIG-seq: an effective PCR-based method for genome-wide single-nucleotide polymorphism genotyping using the next-generation sequencing platform.

Restriction-enzyme (RE)-based next-generation sequencing methods have revolutionized marker-assisted genetic studies; however, the use of REs has limited their widespread adoption, especially in field samples with low-quality DNA and/or small quantities of DNA. Here, we developed a PCR-based procedure to construct reduced representation libraries without RE digestion steps, representing de novo single-nucleotide polymorphism discovery, and its genotyping using next-generation sequencing. Using multiplexed inter-simple sequence repeat (ISSR) primers, thousands of genome-wide regions were amplified effectively from a wide variety of genomes, without prior genetic information. We demonstrated: 1) Mendelian gametic segregation of the discovered variants; 2) reproducibility of genotyping by checking its applicability for individual identification; and 3) applicability in a wide variety of species by checking standard population genetic analysis. This approach, called multiplexed ISSR genotyping by sequencing, should be applicable to many marker-assisted genetic studies with a wide range of DNA qualities and quantities.

Phenoloxidase from the sea cucumber Apostichopus japonicus: cDNA cloning, expression and substrate specificity analysis.

Phenoloxidase (PO) is a crucial component of the immune system of echinoderms. In the present study, the full-length cDNA of PO (AjPO) was cloned from coelomocytes of the sea cucumber Apostichopus japonicus using 3'- and 5'-rapid amplification of cDNA ends (RACE) PCR method, which is 2508 bp, with an open reading frame (ORF) of 2040 bp encoding 679 amino acids. AjPO contains a transmembrane domain, and three Cu-oxidase domains with copper binding centers formed by 10 histidines, one cysteine and one methionine respectively. Phylogenetic analysis revealed that AjPO was clustered with laccase-type POs of invertebrates. Using the isolated membrane proteins as crude AjPO, the enzyme could catalyze the substrates catechol, L-3,4-dihydroxyphenylalanine (L-DOPA), dopamine and hydroquinone, but failed to oxidize tyrosine. The results described above collectively proved that AjPO was a membrane-binding laccase-type PO. The quantitative real-time PCR (qRT-PCR) analysis revealed that AjPO mRNA was expressed in muscle, body wall, coelomocytes, tube feet, respiratory tree and intestine with the highest expression level in coelomocytes. AjPO could be significantly induced by lipopolysaccharide (LPS), peptidoglycan (PGN), Zymosan A and polyinosinic-polycytidylic acid (PolyI:C), suggesting AjPO is closely involved in the defense against the infection of bacteria, fungi and double-stranded RNA viruses.

Molecular cloning, characterization and expression analysis of a C-type lectin (AJCTL) from the sea cucumber Apostichopus japonicus.

For the sea cucumber Apostichopus japonicus, a C-type lectin (AJCTL) was identified using rapid amplification of cDNA ends (RACE) PCR techniques. The full-length cDNA of AJCTL is composed of 710bp with a 618bp open reading frame (ORF) that encodes a polypeptide of 205 amino acids with a N-terminal signal peptide and a C-terminal C-type lectin domain (CTLD). The calculated molecular mass of the whole protein is 22.5kDa and its predicted isoelectric point is 5.59. AJCTL belongs to the group VII of regulatory proteins and it might function as a Ca(2+)-dependent monosaccharide binding lectin specifically and recognizing mannose-type ligands. In situ hybridization demonstrated that the expression of AJCTL was located in the body wall, longitudinal muscles, intestinum and respiratory tree. This became apparent especially in the cytoplasm of epidermal cells and granular haemocytes. Real-time PCR data suggested that AJCTL was mostly synthesized in the longitudinal muscles and intestinum and less pronounced in the respiratory tree and body wall of adults. After 12h stimulation by Vibrio harveyi, at increasing bacterial concentration gradient, the expression of AJCTL in sea cucumber increased as well. This indicated that CTL is related to an innate immune response.

Molecular taxonomy, phylogeny and evolution in the family Stichopodidae (Aspidochirotida: Holothuroidea) based on COI and 16S mitochondrial DNA.

The Stichopodidae comprise a diverse assemblage of holothuroids most of which occur in the Indo-Pacific. Phylogenetic analyses of mitochondrial gene (COI, 16S rRNA) sequence for 111 individuals (7 genera, 17 species) clarified taxonomic uncertainties, species relationships, biogeography and evolution of the family. A monophyly of the genus Stichopus was supported with the exception of Stichopus ellipes. Molecular analyses confirmed genus level taxonomy based on morphology. Most specimens harvested as S. horrens fell in the S. monotuberculatus clade, a morphologically variable assemblage with others from the S. naso clade. Taxonomic clarification of species fished as S. horrens will assist conservation measures. Evolutionary rates based on comparison of sequence from trans-ithmian Isostichopus species estimated that Stichopus and Isostichopus diverged ca. 5.5-10.7Ma (Miocene). More recent splits were estimated to be younger than 1Ma.