Molecular characterization of a cation-dependent mannose-6-phosphate receptor gene in Crassostrea hongkongensis and its responsiveness in Vibrio alginolyticus infection.

The cation-dependent mannose-6-phosphate receptor (CD-M6PR) is a P-type lectin that plays a crucial role in lysosomal enzyme transport, bacterial resistance, and viral entry. In this study, we cloned and analyzed the ORF of the CD-M6PR gene from Crassostrea hongkongensis and named it ChCD-M6PR. We analyzed the nucleotide and amino acid sequence of ChCD-M6PR, its tissue expression pattern and immune response to Vibrio alginolyticus. Our results showed that the ORF of ChCD-M6PR was 801 bp long and encoded a protein of 266 amino acids with a signal peptide at the N-terminus, as well as Man-6-P_recep, ATG27 and transmembrane structural domains. Phylogenetic analysis indicated that Crassostrea hongkongensis shared the highest similarity with Crassostrea gigas in the terms of CD-M6PR. The ChCD-M6PR gene was found to be expressed in various tissues, with the highest expression observed in the hepatopancreas and the lowest in the hemocytes by the fluorescence quantitative PCR. Furthermore, the expression of ChCD-M6PR gene was significantly up-regulated for a short time in response to Vibrio alginolyticus infection in the gill and hemocytes, while it was down-regulated in the gonads. The expression patterns of ChCD-M6PR also varied in the other tissues. The 96 h cumulative mortality rate of Crassostrea hongkongensis infected with Vibrio alginolyticus after knockdown the ChCD-M6PR gene was significantly higher. Overall, our findings suggests that ChCD-M6PR plays a crucial role in the immune response of Crassostrea hongkongensis to Vibrio alginolyticus infection, and its tissue-specific expression patterns may be indicatitive of varied immune responses across tissues.

ERK is involved in the regulation of CpG ODN 2395 on the expression levels of anti-lipopolysaccharide factors in Chinese mitten crab, Eriocheir sinensis.

CpG oligodeoxynucleotides (ODN), as an effective adjuvant or immunopotentiator, activate the immune system and induce various immune responses. Recently, it has also been reported that high dose of CpG ODN can lead to immunosuppression. However, the underlying mechanism of CpG ODN-mediated immune response remains largely unknown in invertebrates. In the present study, the role of ERK in regulating expression levels of anti-lipopolysaccharide factors (ALFs) induced by different doses of CpG ODN 2395 was analyzed in Chinese mitten crab, Eriocheir sinensis. The mRNA expression levels of EsALFs (EsALF1, EsALF2 and EsALF3) and EsERK in haemocytes were observed to increase from 6 h to 48 h post low doses of CpG ODN 2395 (0.5 µg and 2.5 µg) stimulation, while they were suppressed after high dose of CpG ODN 2395 (12.5 µg) injection. Meanwhile, the phosphorylation levels of ERK in haemocytes were significantly promoted after low doses of CpG ODN 2395 injection, and a reduce level of ERK phosphorylation was observed after high dose of CpG ODN 2395 injection. Further investigation showed that the expression levels of EsALFs induced by CpG ODN 2395 were markedly down-regulated after knocking down the expression of EsERK. Similarly, the EsALFs mRNA expression were also inhibited post different doses of CpG ODN 2395 stimulation in PD98059 (ERK inhibitor) injection crabs. These results collectively suggest that ERK is involved in regulating the expression level of EsALFs induced by different dose of CpG ODN 2395 in Chinese mitten crab, which contribute to the understanding of the regulation of CpG ODN involving in immune response in crustaceans.

The involvement of a regucalcin in suppressing hemocyte apoptosis in Pacific oyster Crassostrea gigas.

Regucalcin (RGN), also known as senescence marker protein-30 (SMP30), plays a vital role in the regulation of Ca2+ homeostasis. In the present study, a regucalcin (designated as CgRGN) was identified from Pacific oyster Crassostrea gigas. The complete cDNA sequence of CgRGN was of 1059 bp, containing an open reading frame of 933 bp which encoded a protein of 310 amino acids. The deduced amino acid sequence of CgRGN shared similarity with other RGNs from the genome of C. gigas as well as other species. The mRNA transcripts of CgRGN were universally detected in all tested tissues, with higher level in hepatopancreas, labial palp, and gills. The relative expression level of CgRGN in hemocytes was significantly up-regulated (p < 0.05) at 3, 12, 72, and 96 h after the stimulation of lipopolysaccharide (LPS). After CgRGN expression was interfered by specific CgRGN-dsRNA, the hemocytes apoptosis rate increased dramatically at 12 h post LPS stimulation (1.56 fold, p < 0.01), compared to the control group. The caspase-3 activity in hemocytes and NO concentration in hemolymph increased significantly (p < 0.05) in dsCgRGN injection oysters. These results collectively indicated that CgRGN could suppress LPS-induced apoptosis and be involved in the immune response of oysters.

Transcriptomic profile of oyster Crassostrea gigas hemocyte after short-term cadmium exposure and bacteria stimulation.

Oyster Crassostrea gigas, is considered as a useful environmental indicator since it is widely distributed along the intertidal zone whereby it tends to accumulate cadmium and is always exposed to various pathogen agents. However, its molecular responses to both cadmium and pathogen stimulation remain unclear. In the present study, transcriptome data of hemocytes from oysters were analyzed to reveal specific molecular responses of oyster to cadmium or cadmium/bacteria stimulation. A total of 21591, 22872 and 20107 genes were detected in the BLANK, Cd24h and Cd/Bac24h group, respectively. Among them, there were 685 differentially expressed genes collected in the comparison of Cd24h versus BLANK. GO analysis of these genes found that sixteen terms into the Molecular Function category displayed transporter activities, and were all over-enrichment by cadmium exposure, whereas twelve terms into Biological Process category involved mainly in metabolic process of the various cellular components and two terms into Cellular Component category were all under-enrichment. The 330 immune responsive genes were shared by two gene lists of CdBac24h versus BLANK and CdBac24h versus Cd24h, and seven out of thirty terms in GO analysis were related to the immune process. Further annotation of these genes from the KEGG database revealed fourteen pathways, including two nervous system related pathways, arachidonic acid pathway, four immune pathways, MAPK cascade and other four cell signaling pathways, and two energy related pathways. Twenty-two differentially expressed genes were identified to responsive to both cadmium exposure and bacteria stimulation, but in different expression patterns, suggesting that bilateral responsive genes, such as alkaline phosphatase and sodium and chloride-dependent glycine transporter gene, could be candidate biomarkers for early warning of cadmium pollution. The present results collectively indicated that a profound neuro-endocrine-immune regulatory network was activated in response to cadmium and bacteria stimulation in oyster C. gigas, and the expression pattern of some cadmium responsive genes may be either reversed or strengthened by bacteria stimulation. The results provide knowledge on the transcriptomic response profile of oyster after short-term cadmium exposure and bacteria stimulation, which would be useful for future studies on stress response mechanism of mollusc, and some cadmium-bacteria responsive genes may be explored as potential biomarkers for monitoring marine pollution.

The various components implied the diversified Toll-like receptor (TLR) signaling pathway in mollusk Chlamys farreri.

Toll-like receptor (TLR) signaling pathway, composed of various components, plays pivotal roles in host innate immune defense mechanism. In the present study, twenty-nine TLR signaling pathway components, including receptors, adaptors, transduction molecules and immune effectors, were identified in Zhikong scallop Chlamys farreri via assembling and screening public available transcriptomic data and expression sequence tags (ESTs). These identified TLR signaling pathway components were constitutively expressed and detectable in various tissues, and almost all of them were highly expressed in gill and hepatopancreas. These results indicated the presence of TLR signaling pathways in both MyD88-dependent and MyD88-independent forms in scallop, and implied the diversified TLR signaling pathway in mollusk C. farreri.

CgNrdp1, a conserved negative regulating factor of MyD88-dependent Toll like receptor signaling in oyster Crassostrea gigas.

Toll like receptor (TLR) signaling cascades are under precise regulations to ensure the proper immune responses during various pathogen invasions. The neuregulin receptor degradation protein-1 (Nrdp1) has been demonstrated to be a novel negative regulator of TLR signaling by targeting MyD88 to induce degradation in mammals. In the present study, an Nrdp1 homologue, CgNrdp1, was identified from the genome of Pacific oyster Crassostrea gigas. It contained an open reading frame encoding a polypeptide of 315 amino acids which shared high identities with other homologues from different species. There was a conserved RING domain in CgNrdp1, indicating the functional E3 ubiquitin ligase activity. The bacterially expressed recombinant CgNrdp1 and CgMyD88 showed much stronger affinity compared to control groups in the ELISA assay, showing the interacting ability between CgNrdp1 and CgMyD88. When CgMyD88 or HsMyD88 was co-transfected with CgNrdp1 into HEK293T cells, the luciferase activities of NF-κB were significantly decreased compared to those in MyD88 single-transfection groups, indicating the conserved negative regulating function of CgNrdp1 on the MyD88 induced TLR signaling. These results indicated that CgNrdp1 was a negative regulator of TLR signaling in oyster and the Nrdp1-MyD88 axis was functional and highly conserved from mollusks to mammals in the negative regulation of TLR signaling.

A conserved interferon regulation factor 1 (IRF-1) from Pacific oyster Crassostrea gigas functioned as an activator of IFN pathway.

Interferon regulatory factors (IRFs), a family of transcription factors with a novel helix-turn-helix DNA-binding motif, play important roles in regulating the expression of interferons (IFNs) and IFN-stimulated genes. In the present study, an interferon regulation factor 1 was identified from oyster Crassostrea gigas (designated CgIRF-1), and its immune function was characterized to understand the regulatory mechanism of interferon system against viral infection in invertebrates. The open reading frame (ORF) of CgIRF-1 was 990 bp, encoding a polypeptide of 329 amino acids with a typical IRF domain (also known as DNA-binding domain). The mRNA transcripts of CgIRF-1 were detected in all the tested tissues with the highest expression level in hemocyte. CgIRF-1 protein was distributed in both nucleus and cytoplasm of the oyster hemocyte. The mRNA expression of CgIRF-1 in hemocytes was significantly up-regulated at 48 h after poly (I:C) stimulation (p < 0.05). The recombinant CgIRF-1 (rCgIRF-1) could interact with classically IFN-stimulated response elements (ISRE) in vitro. The relative luciferase activity of interferon-like protein promotor reporter gene (pGL-CgIFNLP promotor) was significantly (p < 0.05) enhanced in HEK293T cell after transfection of CgIRF-1. These results indicated that CgIRF-1 could bind ISRE and regulate the expression of CgIFNLP as a transcriptional regulatory factor, and participated in the antiviral immune response of oysters.

A novel GATA-like zinc finger transcription factor involving in hematopoiesis of Eriocheir sinensis.

GATA transcription factor is a family of DNA-binding proteins that can recognize and bind to sequence of (A/T) GATA (A/G). In the present study, a GATA-like protein (named as EsGLP) was characterized from Eriocheir sinensis, including an 834 bp full length open reading frame of EsGLP, encoding a polypeptide of 277 amino acids. The deduced amino acid sequence of EsGLP contained one conserved GATA-type zinc finger of the form Cys-X2-Cys-X17-Cys-X2-Cys, with four cysteine sites. The EsGLP mRNA transcripts were mainly detected in the hematopoietic tissue, hepatopancreas and gonad. The recombinant EsGLP protein was prepared for the antibody production. The EsGLP protein was mainly distributed in the edge of lobules in the HPT and the cytoplasm of hemocytes. The mRNA transcripts of EsGLP in hemocytes were significantly decreased at 24 h (0.39-fold and 0.27-fold, p < .05) and 48 h (0.35-fold and 0.16-fold, p < .05) after LPS and Aeromonas hydrophila stimulation, respectively. However, one peak of EsGLP mRNA transcripts were recorded at 24 h (8.71-fold, p < .05) in HPT after A. hydrophila stimulation. The expression level of EsGLP mRNA in HPT was significantly up-regulated at 2 h, 2.5 h and 9 h (41.74-fold, 45.38-fold and 26.07-fold, p < .05) after exsanguination stimulation. When EsGLP gene expression was inhibited by the injection of double-stranded RNA, both the total hemocytes counts and the rate of EdU-positive hemocytes were significantly decreased (0.32-fold and 0.56-fold compared to that in control group, p < .05). All these results suggested that EsGLP was an important regulatory factor in E. sinensis which involved in the hemocytes generation and the immune response against invading pathogens.

The involvement of suppressor of cytokine signaling 6 (SOCS6) in immune response of Chinese mitten crab Eriocheir sinensis.

Suppressor of cytokine signaling (SOCS) is a family of cytokine-inducible negative regulators of cytokine signaling and it plays a crucial role in various physiological processes. In the present study, the full-length cDNA of a SOCS (designated as EsSOCS6) was cloned from Chinese mitten crab Eriocheir sinensis. The open reading frame of EsSOCS6 cDNA was of 1266 bp, which encoded a polypeptide of 421 amino acid residues. There were two typically conserved SOCS family domains in EsSOCS6, including a central Src homology 2 (SH2) domain and a C-terminal SOCS box. The deduced amino acid sequence of EsSOCS6 shared 72-76% similarity with those of other SOCS6 family members. EsSOCS6 mRNA was constitutively expressed in all the examined tissues with higher expression levels in the immune-related tissues, such as hepatopancreas, hemocytes and gill. The mRNA expression levels of the EsSOCS6 in hemocytes were significantly up-regulated after the stimulations with lipopolysaccharide (LPS), Aeromonas hydrophila and polyinosinic-polycytidylic acid (poly (I:C)). The mRNA expressions of threonine/serine protein kinase (EsAkt) and EsRelish were dramatically declined after EsSOCS6 was interfered by dsRNA. Collectively, these results demonstrated that EsSOCS6 might regulate the activation of the NF-κB signaling pathway and play an important role in the innate immune responses of E. sinensis.

A novel fucolectin from Apostichopus japonicus with broad PAMP recognition pattern.

F-type lectin (also known as fucolectin) is a newly identified family of fucose binding lectins with the sequence characters of a fucose binding motif and a unique lectin fold (the "F-type" fold). In the present study, a fucolectin was identified from sea cucumber Apostichopus japonicus (designated AjFL-1). The open reading frame (ORF) of AjFL-1 was of 546 bp, encoding a polypeptide of 181 amino acids with a predicted molecular mass of about 20 kDa. The deduced amino acid sequence of AjFL-1 shared 30%-40% similarity with the fucolectins from other animals. There were a typical F-type lectin domain (FLD) (residues 39-180) and a signal peptide (residues 1-24) in AjFL-1. The mRNA transcript of AjFL-1 could be detected by qRT-PCR in various tissues, such as intestinum, coelomocytes, respiratory tree, tentacle, and body wall, while undetectable in the gonads and longitudinal muscle. The mRNA expression level of AjFL-1 in coelomocytes was significantly up-regulated (47.06-fold to that in control group, p < 0.05) at 12 h after Vibrio splendidus challenge. Immunofluorescence assay showed that AjFL-1 protein was mainly distributed on the membrane, while few in cytoplasm of coelomocytes in sea cucumber. The recombinant AjFL-1 (rAjFL-1) could bind lipopolysaccharide (LPS), peptidoglycan (PGN), mannan (MAN) and fucose (FUC), and exhibited a broader binding activities towards Gram-negative bacterium Escherichia coli, Gram-positive bacterium Micrococcus luteus, as well fungus Pichia pastoris. In addition, rAjFL-1 could strongly promote the agglutination of fungus P. pastoris. These results indicated that AjFL-1 was a novel member of fucose-binding lectin family, which functioned as a pattern recognition receptor with broad spectrum of microbial recognition, and involved in innate immune response of sea cucumber.

Functional characterization of hemocytes from Chinese mitten crab Eriocheir sinensis by flow cytometry.

Hemocytes comprise a diversity of cell types with functional and structural heterogeneity, and they play key roles in the host defense of invertebrates. In the present study, the hemocytes from Chinese mitten crab Eriocheir sinensis were directly separated into two groups by flow cytometry. The hemocytes in P1 group were full of round and abundant granules with deeply staining cytoplasm, while P2 hemocytes were more diverse with a wide range of sizes and less granularity. Both P1 and P2 hemocytes exhibited phagocytic ability, but the phagocytic rate of P1 hemocytes increased which was significantly higher than that of P2 hemocytes after LPS stimulations. The levels of ROS production and intracellular Calcium as well as lysosome content were higher in P1 hemocytes than that in P2 hemocytes under both normal and immune-activated situations. The genes involved in phagocytosis, antimicrobial and antioxidant activities were mainly expressed in P1 hemocytes, while the genes involved in proPO activation system were highly expressed in P2 hemocytes. These results collectively suggested that P1 hemocytes were the main immunocompetent hemocytes in Chinese mitten crab and P2 hemocytes mainly participated in proPO activation system.

A galectin from Eriocheir sinensis functions as pattern recognition receptor enhancing microbe agglutination and haemocytes encapsulation.

Galectins are a family of ß-galactoside binding lectins that function as pattern recognition receptors (PRRs) in innate immune system of both vertebrates and invertebrates. The cDNA of Chinese mitten crab Eriocheir sinensis galectin (designated as EsGal) was cloned via rapid amplification of cDNA ends (RACE) technique based on expressed sequence tags (ESTs) analysis. The full-length cDNA of EsGal was 999 bp. Its open reading frame encoded a polypeptide of 218 amino acids containing a GLECT/Gal-bind_lectin domain and a proline/glycine rich low complexity region. The deduced amino acid sequence and domain organization of EsGal were highly similar to those of crustacean galectins. The mRNA transcripts of EsGal were found to be constitutively expressed in a wide range of tissues and mainly in hepatopancreas, gill and haemocytes. The mRNA expression level of EsGal increased rapidly and significantly after crabs were stimulated by different microbes. The recombinant EsGal (rEsGal) could bind various pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide (LPS), peptidoglycan (PGN) and glucan (GLU), and exhibited strong activity to agglutinate Escherichia coli, Vibrio anguillarum, Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus and Pichia pastoris, and such agglutinating activity could be inhibited by both d-galactose and α-lactose. The in vitro encapsulation assay revealed that rEsGal could enhance the encapsulation of haemocytes towards agarose beads. These results collectively suggested that EsGal played crucial roles in the immune recognition and elimination of pathogens and contributed to the innate immune response against various microbes in crabs.

Molecular cloning and characterization of a cytoplasmic manganese superoxide dismutase and a mitochondrial manganese superoxide dismutase from Chinese mitten crab Eriocheir sinensis.

Superoxide dismutase (SOD) functions as the first and essential enzyme in the antioxidant system and is ubiquitously existed in both prokaryotes and eukaryotes. In the present study, both cytoplasmic and mitochondrial manganese SOD were identified from Chinese mitten crab Eriocheir sinensis (designed as EscytMnSOD and EsmtMnSOD). The complete nucleotide sequence of EscytMnSOD comprised 1349 bp and consisted of a 5' untranslated regions (UTR) of 43 bp, a 3' UTR of 445 bp and an open reading frame (ORF) of 861 bp encoding a polypeptide of 286 amino acid residues. The full-length cDNA sequence of EsmtMnSOD comprised 990 bp, containing a 5' UTR of 55 bp, a 3' UTR of 278 bp and an ORF of 657 bp encoding a polypeptide of 218 amino acid residues. The deduced amino acid sequences of EscytMnSOD and EsmtMnSOD contained highly conserved MnSOD signature and typical functional domain, and exhibited high similarity with their reported homologues. In the phylogenetic tree, EscytMnSOD and EsmtMnSOD were clustered with their homologues from the land crab Cardisoma armatum. The EscytMnSOD and EsmtMnSOD transcripts were constitutively expressed in haemocytes, muscle, heart, gill, haepatopancreas and gonad, with the highest expression level in gills and haepatopancreas, respectively. The mRNA expression levels of them were all up-regulated in haemocytes with similar profiles after the stimulation of Vibrio anguillarum, Micrococcus luteus and Pichia pastoris. The EsmtMnSOD with low basal expression level responded to invading microbes intensely, while the EscytMnSOD with high basal expression level exhibited mild responses against stimulating microbes. The purified rEscytMnSOD and rEsmtMnSOD proteins exhibited specific Mn(2+)-dependent enzymatic activities, while rEscytMnSOD with lower basic activity displayed higher stability than rEsmtMnSOD. All these results indicated that EscytMnSOD and EsmtMnSOD were efficiently antioxidant enzymes and potentially involved in the innate immune responses of E. sinensis with different roles, the former might play a routine role in the innate immune system in crabs, while the later might be involved in the immune response against invading microbes specifically.

An EPD/WSD motifs containing C-type lectin from Argopectens irradians recognizes and binds microbes with broad spectrum.

C-type lectins are a superfamily of Ca(2+)-dependent carbohydrate-recognition proteins consisting of at least one carbohydrate-recognition domain (CRD), which play significant roles in nonself-recognition and clearance of invaders. The immune function of a C-type lectin (AiCTL-7) with EPD/WSD motifs from Argopectens irradians was investigated in the present study. The recombinant protein of AiCTL-7 (rAiCTL-7) could bind LPS, PGN, mannan, yeast glucan and poly I:C in vitro, and displayed a broader microbes binding spectrum towards Gram-positive bacteria Staphylococcus aureus, Gram-negative bacteria Escherichia coli, Vibrio anguillarum, as well as fungi Pichia pastoris and Yarrowia lipolytica. Moreover, it could also inhibit the growth of E. coli and significantly (P < 0.01) mediate the cell-cell adhesion in vitro. The results clearly suggested that EPD/WSD motifs containing lectin AiCTL-7 could serve as PRR with wider recognition spectrum, and function both as collectin and selectin participating in the immunity against invaders in scallops. It could be inferred that the diversity and complexity of motifs in Ca(2+) binding site 2 in CRDs endowed C-type lectins with comprehensive recognition spectrum and multiple immune functions against complex living environment.

Sequencing and characterization of the transcriptome of half-smooth tongue sole (Cynoglossus semilaevis).

BACKGROUND: Half-smooth tongue sole (Cynoglossus semilaevis) is a valuable fish for aquaculture in China. This fish exhibits sexual dimorphism, particularly different growth rates and body sizes between two genders. Thus, C. semilaevis is a good model that can be used to investigate mechanisms responsible for such dimorphism, this model can also be utilized to answer fundamental questions in evolution and applied fields of aquaculture. Hence, advances in second-generation sequencing technology, such as 454 pyrosequencing, could provide a robust tool to study the genome characteristics of non-model species. RESULTS: In this study, C. semilaevis was subjected to de novo transcriptome sequencing and characterization. A total of 749,954 reads were generated using a single 454 sequencing run in a full PicoTiter plate. These reads were then assembled into 62,632 contigs with a 10-fold average sequencing coverage. A total of 26,589 sequences were successfully annotated based on sequence similarities; among these sequences, 3,451 transcripts exhibited gene ontology terms and 2,362 showed enzyme commissions associated with 186 pathways from Kyoto Encyclopedia of Gene and Genomes pathways. A search of repetitive elements was performed, and 1,898 transposable elements were identified. Approximately 7,800 simple-sequence repeats and 21,234 single-nucleotide polymorphisms were also detected. CONCLUSIONS: Our data provided an integrated and comprehensive transcriptome resource for C. semilaevis. These data could be used for further research in population genetics, gene function, and tissue-specific gene expressions.

The comparative proteomics analysis revealed the modulation of inducible nitric oxide on the immune response of scallop Chlamys farreri.

Nitric oxide (NO) is an important gasotransmitter which plays a key role on the modulation of immune response in all vertebrates and invertebrates. In the present study, the modulation of inducible NO on immune response of scallop Chlamys farreri was investigated via proteomic analysis. Total proteins from hepatopancreas of scallops treated with lipopolysaccharide (LPS) and/or the inhibitor of vertebrate inducible NO synthase (S-methylisothiourea sulfate, SMT) for 12 h were analyzed via 2-D PAGE and ImageMaster 2D Platinum. There were 890, 1189 and 1046 protein spots detected in the groups treated by phosphate buffered saline (PBS), LPS and LPS+SMT, respectively, and 26 differentially expressed protein spots were identified among them. These proteins were annotated with binding or catalytic activity, and most of them were involved in metabolic or cellular processes. Some immune-related or antioxidant-related molecules such as single Ig IL-1-related receptor, guanine nucleotide-binding protein subunit beta-like protein and peroxiredoxin were identified, and the changes of their expression levels in LPS group were intensified significantly after adding SMT. The decreased expression level of tyrosinase and increased level of glutathione S-transferase 4 in LPS group were diametrically reversed by appending SMT. Moreover, interferon stimulated exonuclease gene 20-like protein and copper chaperone for superoxide dismutase were only induced by LPS+SMT stimulation but not by LPS stimulation. These data indicated that NO could modulate many immunity processes in scallop, such as NF-κB transactivation, cytoskeleton reorganization and other pivotal processes, and it was also involved in the energy metabolism, posttranslational modification, detoxification and redox balance during the immune response.

A high mobility group box 1 (HMGB1) gene from Chlamys farreri and the DNA-binding ability and pro-inflammatory activity of its recombinant protein.

High-mobility group box 1 (HMGB1) protein, a highly conserved DNA binding protein, plays an important role in maintaining nucleosome structures, transcription, and inflammation. In the present research, a cDNA of 1268 bp for the Zhikong scallop Chlamys farreri HMGB1 (designed as CfHMGB1) was cloned via rapid amplification of cDNA ends (RACE) technique and expression sequence tag (EST) analysis. The complete cDNA sequence of CfHMGB1 contained an open reading frame (ORF) of 648 bp, which encoded a protein of 215 amino acids. The amino acid sequence of CfHMGB1 shared 53-57% similarity with other identified HMGB1s. There were two HMG domains, two low complexity regions and a conserved acidic tail in the amino acid sequence of CfHMGB1. The mRNA transcripts of CfHMGB1 were constitutively expressed in all the tested tissues, including haemocytes, muscle, mantle, gill, hepatopancreas, kidney and gonad, with the highest expression level in hepatopancreas. The mRNA expression profiles of CfHMGB1 in haemocytes after the stimulation with different pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide (LPS), peptidoglycan (PGN) and glucan (Glu), were similar with an up-regulation in the early stage and then recovered to the original level. The recombinant CfHMGB1 protein could bind double-stranded DNA and induce the release of TNF-α activity in mixed primary culture of scallop haemocytes. These results collectively indicated that CfHMGB1, with DNA-binding ability and pro-inflammatory activity, could play an important role in the immune response of scallops.

The interaction between 20-hydroxyecdysone and AMPK through PI3K activation in Chinese mitten crab, Eriocheir sinensis.

In crustaceans, the steroid hormone 20-hydroxyecdysone (20E) initiates molting, and the molting process is also regulated by energy metabolism. AMPK is an energy sensor and plays a critical role in systemic energy balance. Here, the regulatory mechanism in the interaction between 20E and AMPK was investigated in Chinese mitten crab, Eriocheir sinensis. The results showed that the 20E concentration and the mRNA expression levels of 20E receptors in hepatopancreas were down-regulated post AMPK activator (AICAR) treatment, and were up-regulated after AMPK inhibitor (Compound C) injection in crabs. Besides, the molt-inhibiting hormone (MIH) gene expression in eyestalk showed the opposite patterns in response to the AICAR and Compound C treatment, respectively. Further investigation found that there was a significant reduction in 20E concentration post PI3K inhibitor (LY294002) treatment, and the phosphorylation level of PI3K was increased in hepatopancreas after AMPK inhibitor injection. On the other hand, the positive regulation of PI3K-mediated activation of AMPK was also observed, the phosphorylation levels of AMPKα, AMPKß and PI3K in hepatopancreas were significantly increased post 20E injection. In addition, the phosphorylation levels of AMPKα and AMPKß induced by 20E were decreased after the injection of PI3K inhibitor. Taken together, these results suggest that the regulatory cross-talk between 20E and AMPK is likely to act through PI3K pathway in E. sinensis, which appeared to be helpful for a better understanding in molting regulation.

The immunomodulation of inducible nitric oxide in scallop Chlamys farreri.

Nitric oxide (NO) is an important signalling molecule which plays an indispensable role in immunity of all vertebrates and invertebrates. In the present study, the immunomodulation of inducible NO in scallop Chlamys farreri was examined by monitoring the alterations of haemocyte behaviours and related immune molecules in response to the stimulations of LPS and/or with S-Methylisothiourea Sulphate (SMT), an inhibitor of inducible NO synthase (NOS). The total activity of NOS and NO concentration in the haemolymph of scallop C. farreri increased significantly at 3, 6 and 12 h after LPS stimulation respectively, whereas their increases were fully repressed when scallops were treated in the collaborating of LPS and SMT. Meanwhile, some cellular and humoral immune parameters were determined after the stimulation of LPS and SMT to investigate the role of inducible NO in innate immunity of scallop. After LPS stimulation, the highest levels of haemocytes apoptosis and phagocytosis were observed at 24 h (38.5 ± 2.5%, P < 0.01) and 12 h (38.6 ± 0.2%, P < 0.01), respectively, and the reactive oxygen species (ROS) level (5.88 ± 0.90%, P < 0.01) of haemocytes and anti-bacterial activity of haemolymph (10.0 ± 2.2%, P < 0.01) all elevated dramatically at 12 h. Although the activity of lysozyme and phenoloxidase (PO) in haemolymph both declined at 48 h (93.0 ± 6.3 U mgprot(-1), 0.40 ± 0.06 U mgprot(-1), P < 0.01), superoxide dismutase (SOD) activity and GSH concentration both increased to the highest level at 24 h post treatment (99.2 ± 8.1 U mgprot(-1), 93.0 ± 6.3 nmol mgprot(-1), P < 0.01). After the collaborating treatment of LPS and SMT, the apoptosis index increased much higher from 48 h, while the increase of haemocytes phagocytosis, ROS level and haemolymph anti-bacteria activities were suppressed completely at 12 h. The declines of lysozyme and PO activity in haemolymph were reversed at 48 h, and the rise of SOD activity and GSH concentration started earlier from 3 h. These results indicated clearly that NO could participate in the scallop immunity and play a crucial role in the modulation of immune response including haemocytes apoptosis and phagocytosis, anti-bacterial activity and redox homeostasis in the haemolymph of scallop.

The CpG ODNs enriched diets enhance the immuno-protection efficiency and growth rate of Chinese mitten crab, Eriocheir sinensis.

CpG oligodeoxynucleotides (ODNs), the well-known vaccine adjuvant in mammals, have been proved to mount innate immune responses in crustaceans. In the present study, CpG ODNs was employed as supplements in diets to fed crab Eriocheir sinensis, and the changes of immune parameters as well as weight gain were investigated to evaluate its possible application in crab farming. After the crabs were fed with 40 mg/kg and 100 mg/kg CpG ODNs containing diets (designated as C40 and C100 group) for four weeks, the lysozyme activities were significantly enhanced (p < 0.01) in both groups, while the catalase activity was only increased (p < 0.01) in the C40 group. When those crabs were subsequently challenged with Aeromonas hydrophila, the cumulative mortalities in C40 and C100 groups were declined by 10.4% and 10.8% (p < 0.05) compared with that of control group, respectively. Interestingly, the final weights of crabs were increased after four weeks' feeding of CpG ODNs, and the percentage of weight gain in C40 group reached 124.5 ± 14.2%, which was significantly higher (p < 0.05) than that of control group (78.1 ± 19.2%) and C100 group (107.3 ± 28.2%). The uptake of CpG ODNs by haemocytes and the possible mechanism of CpG ODNs to active the immune response were investigated by using the laser scanning confocal microscope. CpG ODNs (labeled with 5'-end-FAM) could be internalized by the haemocytes after incubation of 20 min, with strong signals detected at the cell membrane and in the cytoplasm. In the cytoplasm, most of the CpG ODNs were localized in lysosome, and some of them escaped from the lysosomal compartments and aggregated around the nuclear. The results clearly demonstrated that CpG ODNs could be internalized directly by crab haemocytes and mostly located in the late endosome. The enhancements of immuno-protection efficiency and growth rate from CpG ODNs as supplements in diets might depend on the uptaking and locating processes, and they could be used as a potential immunostimulant for the crab aquaculture.