A circadian clock protein cryptochrome inhibits the expression of inflammatory cytokines in Chinese mitten crab (Eriocheir sinensis).

Cryptochrome (Cry), as important flavoprotein, plays a key role in regulating the innate immune response, such as the release of inflammatory cytokines. In the present study, a cryptochrome homologue (EsCry) was identified from Chinese mitten crab Eriocheir sinensis, which contained a typical DNA photolyase domain, a FAD binding domain. The transcripts of EsCry were highly expressed at 11:00, and lowest at 3:00 within one day, while those of Interleukin enhancer binding factor (EsILF), Lipopolysaccharide-induced TNF-alpha factor (EsLITAF), Tumor necrosis factor (EsTNF) and Interleukin-16 (EsIL-16) showed a rhythm expression pattern contrary to EsCry. After EsCry was knocked down by dsEsCry injection, mRNA transcripts of Timeless (EsTim), Cycle (EsCyc), Circadian locomotor output cycles kaput (EsClock), Period (EsPer), and EsLITAF, EsTNF, EsILF, EsIL-16, as well as phosphorylation level of Dorsal significantly up-regulated. The transcripts of EsLITAF, EsTNF, EsILF, and EsIL-16 in EsCry-RNAi crabs significantly down-regulated after injection of NF-κB inhibitor. The interactions of EsCyc and EsCry, EsCyc and Dorsal were observed in vitro. These results indicated that EsCry negatively regulated the expression of the cytokine TNF and IL-16 via inhibiting their transcription factor LITAF and ILF through NF-κB signaling pathway, which provide evidences to better understand the circadian regulation mechanism of cytokine production in crabs.

A pattern recognition receptor CgTLR3 involves in regulating the proliferation of haemocytes in oyster Crassostrea gigas.

Toll-like receptors (TLRs) are expressed on various immune cells as key elements of innate and adaptive immunity, and they also play significant roles in regulating cell proliferation and differentiation. In the present study, the binding activity of CgTLR3 to PAMPs and CgMyD88-2, and its role in mediating the proliferation of haemocytes was investigated. The recombinant proteins of the extracellular six LRR domains (rCgTLR3-LRR) and intracellular TIR domain (rCgTLR3-TIR) of CgTLR3 were obtained respectively. rCgTLR3-LRR exhibited binding activity to lipopolysaccharide (LPS), peptidoglycan (PGN), mannan (MAN) and Poly (I:C), with the highest affinity for LPS. While rCgTLR3-TIR displayed binding activity to the recombinant protein of rCgMyD88-2, with KD value of 7.22 × 10-7 M. The CgTLR3 mRNA and protein were detected in three subpopulations of oyster haemocytes, and they were mainly concentrated in granulocytes, which was 7.27-fold (p < 0.05) of that in semi-granulocytes and 8.51-fold (p < 0.01) of that in agranulocytes. The percentage of CgTLR3 positive cells (FITC+ haemocytes) in granulocytes was 4.45-fold (p < 0.01) and 2.57-fold (p < 0.05) of that in agranulocytes and semi-granulocytes, respectively. After Vibrio splendidus stimulation, the mRNA expression level of CgTLR3 in haemocytes significantly upregulated at 6 h and 12 h, which was 2.93-fold (p < 0.05) and 4.15-fold (p < 0.05) of that in the control group. After the expression of CgTLR3 was inhibited by the injection of si-CgTLR3, the expression levels of transcription factors associated with hematopoiesis (CgGATA, CgRunx), cell cycle-related genes (CgPCNA, CgCDC-45, CgCDK-2), the agranulocyte marker CgCD-9, the granulocyte marker CgAATase, and the inflammatory factor CgIL17-1 significantly decreased (p < 0.05) after the V. splendidus stimulation, which were 0.43-fold, 0.83-fold, 0.48-fold, 0.44-fold, 0.53-fold, 0.7-fold, 0.62-fold, and 0.47-fold of that in NC + V. s group in vivo, respectively. Meanwhile, the percentage of EdU+ haemocytes in si-CgTLR3+V. s group was significantly reduced by 0.54-fold (p < 0.05) compared to the control group (2.7%). These results collectively indicated that CgTLR3 was involved in modulating the proliferation of haemocytes by regulating the expression of proliferation-related genes and inflammatory factor in oyster C. gigas.

CgWnt-1 regulates haemocyte proliferation during immune response of oyster Crassostrea gigas.

Recent findings regarding the immunomodulatory role of Wnt signaling suggest that it is significant in regulating the differentiation and proliferation of immune cells. In the present study, a Wnt-1 homolog (designated as CgWnt-1) with a conserved WNT1 domain was identified from oyster Crassostrea gigas. The transcripts of CgWnt-1 were barely expressed in egg to gastrula stage during early embryogenesis, and up-regulated significantly in the trochophore to juvenile stage. The mRNA transcripts of CgWnt-1 were detected in different tissues of adult oyster, with an extremely high expression level in the mantle, which was 77.38-fold (p < 0.05) of that in labial palp. After Vibrio splendidus stimulation, the mRNA expression levels of CgWnt-1 and Cgß-catenin in haemocytes up-regulated significantly at 3, 12, 24, and 48 h (p < 0.05). After injection of recombinant protein (rCgWnt-1) into oyster in vivo, the expressions of Cgß-catenin, cell proliferation related genes CgRunx-1 and CgCDK-2 in haemocytes significantly up-regulated, which were 4.86-fold (p < 0.05), 9.33-fold (p < 0.05), 6.09-fold (p < 0.05) of those in rTrx group, respectively. The percentage of EDU+ cells in haemocytes also significantly increased (2.88-fold of that in control group, p < 0.05) at 12 h after rCgWnt-1 treatment. When the Wnt signal inhibitor C59 was injected simultaneously with rCgWnt-1, the expressions of Cgß-catenin, CgRunx-1, and CgCDK-2 were significantly reduced, which were 0.32-fold (p < 0.05), 0.16-fold (p < 0.05), and 0.25-fold (p < 0.05) of that in rCgWnt-1 group, respectively, and the percentage of EDU+ cells in haemocytes was also significantly inhibited (0.15-fold compared with that in rCgWnt-1 group, p < 0.05). These results suggested that the conserved CgWnt-1 could modulate haemocytes proliferation via regulating cell cycle related genes and involved in the immune response of oysters.

A C-type lectin from Crassostrea gigas with novel EFG/FVN motif involved in recognition of various PAMPs and induction of interleukin expression.

C-type lectins (CTLs) are a superfamily of Ca2+-dependent carbohydrate-recognition proteins, which participate in the nonself-recognition and triggering the transduction pathways in the innate immunity. In the present study, a novel CTL (designated as CgCLEC-TM2) with a carbohydrate-recognition domain (CRD) and a transmembrane domain (TM) was identified from the Pacific oyster Crassostrea gigas. Two novel EFG and FVN motifs were found in Ca2+-binding site 2 of CgCLEC-TM2. The mRNA transcripts of CgCLEC-TM2 were detected in all tested tissues with the highest expression level in haemocytes, which was 94.41-fold (p < 0.01) of that in adductor muscle. The relative expression level of CgCLEC-TM2 in haemocytes significantly up-regulated at 6 h and 24 h after the stimulation of Vibrio splendidus, which was 4.94- and 12.77-fold of that in control group (p < 0.01), respectively. The recombinant CRD of CgCLEC-TM2 (rCRD) was able to bind lipopolysaccharide (LPS), mannose (MAN), peptidoglycan (PGN), and poly (I: C) in a Ca2+-dependent manner. The rCRD exhibited binding activity to V. anguillarum, Bacillus subtilis, V. splendidus, Escherichia coli, Pichia pastoris, Staphylococcus aureus and Micrococcus luteus in a Ca2+-dependent manner. The rCRD also exhibited agglutination activity to E. coli, V. splendidus, S. aureus, M. luteus and P. pastoris in a Ca2+-dependent manner. The phagocytosis rate of haemocytes towards V. splendidus significantly down-regulated from 27.2% to 20.9% after treatment of anti-CgCLEC-TM2-CRD antibody, while the growth of V. splendidus and E. coli was inhibited compared with the TBS and rTrx groups. After the expression of CgCLEC-TM2 was inhibited by RNAi, the expression level of phospho-extracellular regulated protein kinases (p-CgERK) in haemocytes, and the mRNA expressions of interleukin17s (CgIL17-1 and CgIL17-4) decreased significantly after V. splendidus stimulation, compared with that in EGFP-RNAi oysters, respectively. These results suggested that CgCLEC-TM2 with novel motifs served as a pattern recognition receptor (PRR) involved in the recognition of microorganisms, and induction of CgIL17s expression in the immune response of oysters.

CgBlimp-1 inhibits granulocytes proliferation and interleukin production in the immune response of oyster Crassostrea gigas.

B lymphocyte-inducible maturation protein 1 (Blimp-1) is a SET domain and zinc fingers containing transcriptional repressor, which is necessary for regulating the development of many immune cell lineages and keeping immune homeostasis. In the present study, a Blimp-1 homologue (designated as CgBlimp-1) was identified from oyster Crassostrea gigas, which contained a conserved SET domain and five ZnF_C2H2 domains and shared high homology with Blimp-1 from other species. The mRNA transcripts of CgBlimp-1 were highly expressed in gill and hepatopancreas. CgBlimp-1 protein was detected to be specifically expressed in granulocytes. After V. splendidus stimulation, the mRNA expression level of CgBlimp-1 in haemocytes up-regulated significantly at 24, 48, and 96 h, which was 4.39-fold (p < 0.05), 7.68-fold (p < 0.01) and 2.65-fold (p < 0.05) of that in control group, respectively. When the expression of CgBlimp-1 was knocked-down in vivo by RNAi, the mRNA expressions of downstream transcription factor CgMyc-A (1.63-fold of that in control group, p < 0.05) and cell cycle related gene CgCDK2 (1.70-fold, p < 0.05) increased significantly at 24 h after V. splendidus stimulation. Concomitantly, the ratio of EdU+ haemocytes increased notably (p < 0.01) while the proportion of haemocytes in G0/G1 phase decreased dramatically (p < 0.001), compared to that in control group. More specifically, the proportion of granulocytes in total haemocytes increased apparently (p < 0.05) in CgBlimp-1-RNAi oysters, together with up-regulation (p < 0.05) of the ratio of EdU+ granulocytes and down-regulation (p < 0.001) of the proportion of granulocytes in G0/G1 phase. Furthermore, the mRNA expression levels of CgIL17-1, CgIL17-2 and CgIL17-4 in haemocytes increased significantly in CgBlimp-1-RNAi oysters, which was 1.71-fold (p < 0.05), 144.70-fold (p < 0.01) and 1.93-fold (p < 0.05) of that in control group, respectively. Aforementioned results suggested that CgBlimp-1 could reduce the proliferation of granulocytes by arresting cell cycle in G1/G0 phase and avoid over-expression of interleukin to maintain homeostasis in the immune response of oyster.

CgTNF-2 promotes the proliferation of haemocytes by regulating the expressions of CgRunx and cell cycle related genes in the Pacific oyster Crassostrea gigas.

A TNF-α family member, CgTNF-2, was previously identified from the oyster Crassostrea gigas to involve in the antibacterial response. In the present study, the role of CgTNF-2 in mediating the proliferation of haemocytes was further explored. The mRNA expression of CgTNF-2 in granulocytes was significantly higher than that in semi-granulocytes and agranulocytes, and the percentages of CgTNF-2 antibody labeled cells in agranulocytes, semi-granulocytes and granulocytes were 19.15%, 40.25% and 94.07%, respectively. After the treatment with rCgTNF-2, the percentage of EdU+ cells in haemocytes increased significantly (1.77-fold, p < 0.05) at 6 h compared with that in rGST-treated group, and the mRNA expressions of CgRunx, CgCyclin A, CgCDK2 and CgCDC45 in haemocytes all increased significantly (p < 0.05), which were 1.94-fold, 2.13-fold, 1.97-fold, 1.76-fold of that in rGST-treated group, respectively. Meanwhile, the protein abundance of CgRunx and CgCyclin A in the haemocytes of oysters in the rCgTNF-2-treated group increased, and the percentage of PI+ haemocytes in S phase also increased significantly (2.19-fold, p < 0.05) compared with that in rGST-treated group. These results collectively confirmed that CgTNF-2 was highly expressed in granulocytes and involved in the proliferation of haemocytes by regulating the expressions of CgRunx and cell cycle related genes in C. gigas.

CgDM9CP-5-Integrin-MAPK Pathway Regulates the Production of CgIL-17s and Cgdefensins in the Pacific Oyster, Crassostrea gigas.

DM9 domain containing protein (DM9CP) is a family of newly identified recognition receptors exiting in most organisms except plants and mammals. In the current study, to our knowledge, a novel DM9CP-5 (CgDM9CP-5) with two tandem DM9 repeats and high expression level in gill was identified from the Pacific oyster, Crassostrea gigas. The deduced amino acid sequence of CgDM9CP-5 shared 62.1% identity with CgDM9CP-1 from C. gigas, and 47.8% identity with OeFAMeT from Ostrea edulis. The recombinant CgDM9CP-5 (rCgDM9CP-5) was able to bind d-mannose, LPS, peptidoglycan, and polyinosinic-polycytidylic acid, as well as fungi Pichia pastoris, Gram-negative bacteria Escherichia coli and Vibrio splendidus, and Gram-positive bacteria Staphylococcus aureus. The mRNA transcript of CgDM9CP-5 was highly expressed in gill, and its protein was mainly distributed in gill mucus. After the stimulations with V. splendidus and mannose, mRNA expression of CgDM9CP-5 in oyster gill was significantly upregulated and reached the peak level at 6 and 24 h, which was 13.58-fold (p < 0.05) and 14.01-fold (p < 0.05) of that in the control group, respectively. CgDM9CP-5 was able to bind CgIntegrin both in vivo and in vitro. After CgDM9CP-5 or CgIntegrin was knocked down by RNA interference, the phosphorylation levels of JNK and P38 in the MAPK pathway decreased, and the expression levels of CgIL-17s (CgIL-17-3, -4, -5, and -6), Cg-Defh1, Cg-Defh2, and CgMolluscidin were significantly downregulated. These results suggested that there was a pathway of DM9CP-5-Integrin-MAPK mediated by CgDM9CP-5 to regulate the release of proinflammatory factors and defensins in C. gigas.

The characterization of an agranulocyte-specific marker (CgCD9) in the Pacific oyster Crassostrea gigas.

The agranulocytes in the Pacific oyster Crassostrea gigas are a group of haemocytes that are significantly different from semi-granulocytes and granulocytes on the morphology. Agranulocytes are the smallest haemocytes characterized by a spherical shape, the largest ratio of nucleus to cytoplasm, and no granules in the cytoplasm. The lack of unique cell surface markers impedes the isolation of agranulocytes from total haemocytes. Previous transcriptome sequencing analysis of three subpopulations of haemocytes revealed that a homologue of CD9 (designed as CgCD9) was highly expressed in agranulocytes of oyster C. gigas (data not shown). In the present study, CgCD9 was identified to share a similarity of 60% with other vertebrates CD9s, and it harbored a typical four transmembrane domain and a conserved Cys-Cys-Gly (CCG) motif. The mRNA transcript of CgCD9 was found to be highly expressed in agranulocytes, which was 6.63-fold (p < 0.05) and 3.68-fold (p < 0.05) of that in granulocytes and semi-granulocytes, respectively. A specific monoclonal antibody of CgCD9 (named 3D8) was successfully prepared by traditional hybridoma technology, and a single positive band at 25.2 kDa was detected in the haemocyte proteins by Western Blotting, indicating that this monoclonal antibody exhibited high specificity and sensitivity to CgCD9 protein. The ELISA positive value of 3D8 monoclonal antibody to recognize agranulocytes, semi-granulocytes and granulocytes was 17.35, 4.48 and 1.55, respectively, indicating that monoclonal antibody was specific to agranulocytes. Immunocytochemistry assay revealed that CgCD9 was specifically distributed on the membrane of agranulocytes. Using immunomagnetic beads coated with 3D8 monoclonal antibody, CgCD9+cells with a purity of 94.53 ± 5.60% were successfully isolated with a smaller diameter, a larger N:C ratio and no granules in cytoplasm, and could be primary culture in the modified L-15 medium in vitro. Collectively, these results suggested that CgCD9 was a specific cell surface marker for agranulocytes, which offered a tool for high-purity capture of agranulocytes from total haemocytes in C. gigas.

ROS function as an inducer of autophagy to promote granulocyte proliferation in Pacific oyster Crassostrea gigas.

Hematopoiesis is the biological process to generate new blood cells in the living body and reactive oxygen species (ROS) contribute significantly to the regulation of haematopoietic cell homeostasis. In the present study, the involvement of ROS in the proliferation of haemocytes was examined in Pacific oyster Crassostrea gigas. The ROS content in haemocytes increased significantly after lipopolysaccharide (LPS) treatment, but decreased after the treatment with antioxidant N-Acetyl-L-cysteine (NAC, a scavenger of ROS). The percentage of 5-ethynyl-2'-deoxyuridine labeled (EdU+) granulocytes in total haemocytes significantly increased at 12 h (4.12-fold, p < 0.001) and 24 h (2.36-fold, p < 0.001) after LPS treatment, while decreased at 12 h (0.26-fold, p < 0.001) and 24 h (0.61-fold, p < 0.05) after NAC treatment, respectively. Meanwhile, the percentage of haemocytes with autophagosome positive signals significantly increased at 12 h (1.17-fold, p < 0.01) and 24 h (1.19-fold, p < 0.05) after LPS treatment, but significantly reduced at 12 h (0.41-fold, p < 0.001) and 24 h (0.28-fold, p < 0.001) after the NAC treatment, respectively. After ammonium chloride (NH4Cl) treatment, the percentage of haemocytes with autophagosome and EdU+ granulocytes significantly increased at 12 h, which was 1.27-fold (p < 0.01) and 1.70-fold (p < 0.01) of control group, respectively. These results collectively suggested that ROS produced after LPS treatment could act as an inducer for autophagy and involved in regulating the proliferation of some granulocytes in C. gigas.

CgATP synthase ß subunit involved in the regulation of haemocytes proliferation as a CgAstakine receptor in Crassostrea gigas.

Astakine is considered as an endogenous cytokine-like factor of prokineticin homologue in invertebrate. Recently, an astakine homologue (CgAstakine) has been identified and characterized in oyster Crassostrea gigas. In the present study, a CgATP synthase ß subunit was identified as the receptor of CgAstakine in C. gigas. There was an ATP-synt_ab_N domain and an AAA domain in the CgATP synthase ß subunit protein. The mRNA transcripts of CgATP synthase ß subunit were detected in all tested tissues, with the highest expression level in hepatopancreas and gills, which was 109.11-fold (p < 0.01) and 97.21-fold (p < 0.01) of that in labial palps, respectively. After rCgAstakine stimulation, the mRNA transcripts of CgATP synthase ß subunit in agranulocytes and semi-granulocytes were significantly increased at 24 h (2.44-fold, and 9.01-fold of that in control group, p < 0.01), and those in granulocytes were significantly increased at 6 h (1.83-fold, p < 0.01), 12 h (1.92-fold, p < 0.01) and 24 h (3.47-fold, p < 0.01). The expression level of CgATP synthase ß subunit protein in agranulocytes and granulocytes was also significantly increased after rCgAstakine stimulation, which was 1.64-fold (p < 0.05) and 1.85-fold (p < 0.05) of that in control group, respectively, while there were no significant changes in semi-granulocytes. The immunofluorescence assay showed that CgATP synthase ß subunit positive signals were mainly located on the membrane of haemocytes. The number of haemocytes with EdU positive signals was significantly increased after rCgAstakine stimulation (2.04-fold of seawater group, p < 0.01), while significantly decreased after the RNA interference (RNAi) of CgATP synthase ß subunit, which was 0.28-fold of that in NC group (p < 0.01). Bio-layer interferometry (BLI) assay confirmed in vitro interaction between rCgAstakine and rCgATP synthase ß subunit. There results suggested that CgATP synthase ß subunit acts as the receptor of CgAstakine and plays important roles in CgAstakine induced renewal of haemocytes in C. gigas.

CgRab1 regulates Cgcathepsin L1 expression and participates in the phagocytosis of haemocytes in oyster Crassostrea gigas.

Rab protein plays an important role in a variety of cellular activities, especially the fusion process of the inner membrane during endocytosis. In the present study, a Rab1 protein (CgRab1) was identified from the Pacific oyster Crassostrea gigas. The full-length cDNA sequence of CgRab1 was of 2248 bp with an open reading frame of 618 bp, encoding a polypeptide of 205 amino acids containing a Rab domain. The deduced amino acid sequence of CgRab1 shared 94.2% and 89.3% identity with Rab1 from Pomacea canaliculata and Homo sapiens respectively. In the phylogenetic tree, CgRab1 was firstly clustered with the Rab1s from Aplysia californica and Pomacea canaliculata to form a sister group with Rab1 from invertebrates. The recombinant CgRab1 protein (rCgRab1) was able to bind GTP. The mRNA transcripts of CgRab1 were highly expressed in oyster haemocytes, and its expression level in oyster haemocytes was significantly up-regulated at 24 h after the stimulations with Vibro splendidus, which was 2.43-fold (p < 0.01) of that in the control group. After the expression of CgRab1 was knocked down (0.38-fold of that in EGFP-RNAi experimental group) by RNAi，the protein expression of Cgcathepsin L1 were reduced (0.63-fold, p < 0.01) compared with that in EGFP-RNAi experimental group. The phagocytic rate and phagocytic index of haemocytes in CgRab1-RNAi oysters decreased after V. splendidus stimulation, which was 0.50-fold (p < 0.01) and 0.58-fold (p < 0.01) of that in EGFP-RNAi experimental group. These results indicated that CgRab1 was involved in the process of haemocytes phagocytosis by regulating the expression of Cgcathepsin L1 in oyster C. gigas.

DNA binding protein CgIkaros-like regulates the proliferation of agranulocytes and granulocytes in oyster (Crassostrea gigas).

DNA-binding protein Ikaros is a major determinant of haematopoietic lineage, especially in the development, differentiation and proliferation of lymphocytes. In the present study, a Ikaros homologue (designed as CgIkaros-like) was identified and characterized as a vital determinant in the proliferation of haemocytes during haematopoiesis of Pacific oyster Crassostrea gigas. The complete coding sequence of CgIkaros-like was of 1329 bp encoding a predicted polypeptide of 442 amino acids with four ZnF regions, locating at the C-terminus and N-terminus respectively. The highest expression level of CgIkaros-like mRNA was found in gills, followed by haemocytes and gonad. The mRNA transcripts of CgIkaros-like could be detected in all the haemocytes with higher abundance in semi-granulocytes and agranulocytes. CgIkaros-like protein was localized in both of cytoplasm and nucleus with higher abundance in nucleus of oyster haemocytes. The mRNA and protein expression levels of agranulocyte marker CgCD9, granulocyte marker CgAATase, cell cycle related gene CgCDK2, Notch receptor CgNotch and Notch target gene CgHes1 all increased significantly (p < 0.05) after CgIkaros-like was interfered by siRNAs, which were about 27.33-, 2.63-, 24.34-, 4.45- and 6.08-fold of that in the siRNA-NC control group, respectively. While the transcripts of CgGATA3 and CgRunx did not change significantly after CgIkaros-like was interfered. These results demonstrated that CgIkaros-like functioned as a transcription factor combined with Notch pathway to mediate CgCDK2 and regulate the proliferation of oyster haemocytes.

A membrane-bound dopamine ß-hydroxylase highly expressed in granulocyte of Pacific oyster Crassostrea gigas.

Dopamine ß-hydroxylase (DBH) is one of key rate-limiting enzymes converting dopamine to norepinephrine. It locates not only in catecholaminergic neuron system, but also in immunocytes and plays roles in the immune response of vertebrates. However, the knowledge about the function of DBH in immune system is still very limited in invertebrates. In the present study, the DBH gene family with seven members was screened from Crassostrea gigas genome, and their mRNA expressions in various tissues were recorded. Among them, one DBH (designated CgDBH-1) with high expression level in oyster hemocytes was further characterized. The deduced amino acid sequence of CgDBH-1 was predicted to contain a transmembrane domain and shared 30.1% and 30.9% similarity with that in Mus musculus and Homo sapiens, respectively. CgDBH-1 was closely clustered with DBH from Aplysia californica in the phylogenetic tree. The recombinant protein of CgDBH-1 (rCgDBH-1) exhibited significant enzymatic activity (0.54 ± 0.019 pmol L-1 min-1) to synthesize norepinephrine. Importantly, the mRNA transcript of CgDBH-1 was highly expressed in oyster hemocytes, and the highest expression level was observed in granulocytes among the three types of hemocytes, which was 8.18-fold (p < 0.01) of that in agranulocytes. Moreover, the expression of CgDBH-1 in hemocytes was significantly increased at the late stage of immune response. The CgDBH-1 protein was mainly co-localized with the granules and endoplasmic reticulum (ER) of granulocytes. These results collectively suggested that CgDBH-1, as a novel molluscan norepinephrine synthesizing enzyme highly expressed in granulocytes, involved in the late-stage immune response of oysters, which provided vital insight to understand the crosstalk between neuroendocrine and immune systems in invertebrates.

A C1qDC (CgC1qDC-6) with a collagen-like domain mediates hemocyte phagocytosis and migration in oysters.

Most of the bivalve C1q domain containing proteins (C1qDCs) are either only composed of the globular head domain, or contain an N-terminal coiled-coil domain, presumed to cover a role in oligomerization. On the other hand, collagen regions, widespread in vertebrate C1qDCs, are very uncommon in bivalves. In the present study, a C1qDC with a collagen-like domain (designated CgC1qDC-6) was identified from the Pacific oyster Crassostrea gigas and its possible involvement in immune responses was also characterized. The coding sequence of CgC1qDC-6 was of 756 bp, encoding a peptide of 251 amino acids with an N-terminal signal peptide, a central collagen-like domain, and a C-terminal ghC1q domain. CgC1qDC-6 was clustered with the C1qDCs from several mollusks in the phylogenetic tree. CgC1qDC-6 was detected at both mRNA and protein levels in all tested tissues including hepatopancreas, gonad, gill, mantle, adductor muscle, and hemocytes. The recombinant CgC1qDC-6 protein (rCgC1qDC-6) exhibited binding activity to various pathogen-associated molecular patterns (PAMPs) including LPS, PGN, mannose and Poly I:C, and microorganisms including Gram-negative bacteria (Escherichia coli and Vibrio splendidus), Gram-positive bacteria (Micrococcus luteus and Staphylococcus aureus), and fungus (Pichia pastoris). The phagocytic rates of oyster hemocytes towards V. splendidus pre-incubation with rCgC1qDC-6 were significantly enhanced (p < 0.05). In the chemotaxis assay, rCgC1qDC-6 could mediate the migration of oyster hemocytes in a dose-dependent manner, which exhibited a positive chemotactic effect at low concentration (<10 nM). These results collectively indicated that CgC1qDC-6 could serve as a pattern recognition receptor and mediate the hemocyte phagocytosis and migration to eliminate the invading pathogens.

A new member of the runt domain family from Pacific oyster Crassostrea gigas (CgRunx) potentially involved in immune response and larvae hematopoiesis.

The Runx family is a kind of heteromeric transcription factors, which is defined by the presence of a runt domain. As transcriptional regulator during development and cell fate specification, Runx is best known for its critical roles in hematopoiesis. In the present study, a Runx transcription factor (designed as CgRunx) was identified and characterized from the oyster Crassostrea gigas. The complete coding sequence of CgRunx was of 1638 bp encoding a predicted polypeptide of 545 amino acids with one conserved runt domain, which shared high similarity with other reported Runx proteins. CgRunx was highly expressed in hemocytes, gill and mantle both at the protein and nucleic acid levels. CgRunx protein was localized specifically in the cell nuclei of hemocytes, and distributed at the tubule lumen of gill filament. During the larval developmental stages, the mRNA transcripts of CgRunx gradually increased after fertilization, reached to a relative high level at the 8 cell embryos and the blastula stage of 2-4 hpf (hours post fertilization) (about 40-fold), and peaked at early trochophore larvae (10 hpf) (about 60-fold). Whole-mount immunofluorescence assay further revealed that the abundant immunofluorescence signals of CgRunx distributed through the whole embryo at blastula stage (5 hpf), and progressively reduced with the development to a ring structure around the dorsal region in trochophore larvae (10 hpf). Scattered positive immunoreactivity signals finally appeared in the velum region of D-veliger larvae. After LPS and Vibrio splendidus stimulations, the expression levels of CgRunx mRNA in hemocytes were up-regulated significantly compared with that in the control (0 h), which were 2.98- and 2.46-fold (p < 0.05), 2.67- and 1.5-fold (p < 0.05), 2.36- and 1.38-fold (p < 0.05) at 3 h, 6 h and 12 h, respectively. These results collectively suggested that CgRunx involved in immune response and might participate in larvae hematopoiesis in oyster.

A SPRY domain-containing SOCS box protein 3 (SPSB3) involved in the regulation of cytokine production in granulocytes of Crassostrea gigas.

The sp1A/ryanodine receptor (SPRY) family members have been reported to involve in important biological pathways, including innate immune signaling, cytokine signaling suppression, development, cell growth, and retroviral restriction. In the present study, a SPRY domain-containing SOCS box protein (named as CgSPSB3) was identified and characterized from oyster Crassostrea gigas. The open reading frame of CgSPSB3 gene was of 699 bp, encoding a polypeptide of 232 amino acid residues with a central SPRY domain and a C-terminal SOCS box motif. CgSPSB3 mRNA transcripts could be detected in all the examined tissues with the highest level in hemocytes, which was about 82.72-fold (p < 0.05) of that in gonad. Furthermore, the expression level of CgSPSB3 mRNA in granulocytes was significantly higher than that in semi-granulocytes and agranulocytes, which was about 2.04-fold (p < 0.05) of the average level of hemocytes. Immunofluorescence assay further revealed that CgSPSB3 protein was mainly distributed in the cytoplasm of granulocytes. The mRNA expression of CgSPSB3 in hemocytes was up-regulated after lipopolysaccharide (LPS) and Vibrio splendidus stimulations. The mRNA expression of CgIFNLP, CgIL17-5 and CgTNF-1 decreased significantly (p < 0.05) at 24 h after the CgSPSB3 mRNA was knocked down by RNAi. These results collectively indicated that CgSPSB3 might play an important role in regulating cytokines production in granulocytes of C. gigas.

CgAATase with specific expression pattern can be used as a potential surface marker for oyster granulocytes.

Granulocytes are known as the main immunocompetent hemocytes that play important roles in the immune defense of oyster Crassostrea gigas. In the present study, an alcohol acyltransferase (designed as CgAATase) with specific expression pattern was identified from oyster C. gigas, and it could be employed as a potential marker for the isolation of oyster granulocytes. The open reading frame (ORF) of CgAATase was of 1431 bp, encoding a peptide of 476 amino acids with a typically conserved AATase domain. The mRNA transcripts of CgAATase were highest expressed in hemocytes, lower expressed in hepatopancreas, mantle, gonad, gill, ganglion, adductor muscle, and labial palp. The mRNA expression level of CgAATase in hemocytes was significantly up-regulated at 3-12 h and reached the highest level (27.40-fold compared to control group, p < 0.05) at 6 h after Vibrio splendidus stimulation. The total hemocytes were sorted as granulocytes, semi-granulocytes and agranulocytes by Percoll® density gradient centrifugation. CgAATase transcripts were dominantly observed in granulocytes, which was 8.26-fold (p < 0.05) and 2.80-fold (p < 0.05) of that in agranulocytes and semi-granulocytes, respectively. The monoclonal antibody against CgAATase was produced and employed for the isolation of granulocytes with the immunomagnetic bead. CgAATase protein was mainly detected on the cytomembrane of granulocytes. About 85.7 ±â€¯4.60% of the granulocytes were positive for CgAATase and they could be successfully separated by flow cytometry with immunomagnetic bead coated with anti-CgAATase monoclonal antibody, and 97.7 ±â€¯1.01% of the rest hemocytes (agranulocytes and semi-granulocytes) were negative for CgAATase. The isolated primary granulocytes could maintain cell activity for more than one week in vitro culture that exhibited numerous filopodia. These results collectively suggested that CgAATase was a potential marker of oyster granulocytes, and the granulocytes could be effectively isolated from total circulating hemocytes by immunomagnetic bead coated with the anti-CgAATase monoclonal antibody.

A DM9-containing protein from oyster Crassostrea gigas (CgDM9CP-2) serves as a multipotent pattern recognition receptor.

DM9 is a novel protein domain with unknown function originally discovered in Drosophila melanogaster. Recently, a protein harboring DM9 repeats was identified as mannose-specific lectin (CgCGL1, renamed as CgDM9CP-1) from the Pacific oyster Crassostrea gigas. In the present study, another DM9 containing protein was identified from oyster C. gigas (designated as CgDM9CP-2). The open reading frame of CgDM9CP-2 gene was of 432 bp, encoding a polypeptide of 143 amino acids with two tandem DM9 repeats. The deduced amino acid sequence of CgDM9CP-2 shared 60.8% identity with that of CgDM9CP-1. In the unrooted phylogenetic tree, CgDM9CP-2 was closely clustered with CgDM9CP-1, and then assigned into the branch of invertebrate DM9CPs. The mRNA transcripts of CgDM9CP-2 were expressed in all the tested tissues, including mantle, gonad, gills, adductor muscle, hemocytes, and hepatopancreas, with the highest expression level in gills. CgDM9CP-2 protein was mainly distributed on the cytomembrane of oyster hemocytes. After mannose stimulation, the mRNA expression of CgDM9CP-2 in gills was up-regulated to the peak level (5.90-fold of that in SSW group, p < 0.05) at 24 h, and kept at a significantly higher level compared with that in control group at 6-48 h. It significantly increased at 6 h (2.33-fold, p < 0.05), and 12 h (3.08-fold, p < 0.05) post Vibrio splendidus stimulation, and then gradually decreased from 48 to 72 h (p < 0.05) with significant difference comparing with that in control group. The recombinant CgDM9CP-2 protein (rCgDM9CP-2) displayed higher binding affinity to D-(+)-mannose while lower binding affinity to lipopolysaccharide and peptidoglycan. rCgDM9CP-2 also exhibited binding activity towards fungi (Pichia pastoris and Yarrowia lipolytica), gram-positive bacteria (Staphylococcus aureus and Micrococcus luteus), and gram-negative bacteria (Escherichia coli, Vibrio anguillarum, Aeromonas hydrophila and V. splendidus). It could agglutinate fungi P. pastoris and Y. lipolytica, and inhibit the growth of P. pastoris, S. aureus, V. anguillarum, and V. splendidus. These results collectively indicated that CgDM9CP-2 not only served as a pattern recognition receptor with a broad range of recognition spectrum, but also involved in inhibiting the growth of invading microbe in the innate immune response of oyster, which would provide further evidence for the function of DM9 domain in the innate immune system.

Chinese mitten crab (Eriocheir sinensis) iron-sulphur cluster assembly protein 2 (EsIscA2) is differentially regulated after immune and oxidative stress challenges.

Iron-sulphur clusters (ISCs), one of the oldest and most versatile cofactors of proteins, are involved in catalysis reactions, electron transport reactions, regulation processes as well as sensing of ambient conditions. Iron-sulphur cluster assembly protein (IscA) is a scaffold protein member of ISC formation system, which plays a significant role in the assembly and maturation process of ISC proteins. In the present study, the cDNA sequence of iron-sulphur cluster assembly protein 2 (designated as EsIscA2) was cloned from Eriocheir sinensis. The open reading frame (ORF) of EsIscA2 was of 507 bp, encoding a peptide of 168 amino acids with a typically conserved Fe-S domain. A tetrameric form was predicated by the SWISS-MODEL prediction algorithm, and three conserved cysteine residues (Cys-93, Cys-158, Cys-160) from each IscA monomer were predicted to form a 'cysteine pocket'. The deduced amino acid sequence of EsIscA2 shared over 50% similarity with that of other IscAs. EsIscA2 was clustered with IscA2 proteins from invertebrates and vertebrates, indicating that the protein was highly conservative in the evolution. rEsIscA2 exhibited a high iron binding affinity in the concentration ranging from 2 to 200 µM. EsIscA2 transcripts were detected in all the tested tissues including gonad, hemocytes, gill, muscle, heart, hepatopancreas and eyestalk, and EsIscA2 protein was detected in the mitochondria of hemocytes. The highest mRNA expression level of EsIscA2 was detected in muscle and hepatopancreas, which was about 34.66-fold (p < 0.05) and 27.07-fold (p < 0.05) of that in hemocytes, respectively. After Aeromonas hydrophila and lipopolysaccharide (LPS) stimulations, the mRNA expression of EsIscA2 in hemocytes was down-regulated and reached the lowest level at 24 h (0.31-fold, p < 0.05) and 48 h (0.29-fold, p < 0.05) compared to control group, respectively. And the expression of EsIscA2 mRNA in hepatopancreas was repressed from 6 h to 48 h post stimulation (p < 0.05). When the primary cultured crab hemocytes were incubated with different concentrations of H2O2 for 15 min, the expression level of EsIscA2 mRNA was significantly repressed to the 0.34-0.44-fold of that in the control group. After A. hydrophila stimulation, the mRNA expression of EsGrx2 was up-regulated at 3 h (3.22-fold compared to control group, p < 0.05) and reached the peak at 12 h (4.88-fold, p < 0.05). All these results suggested that EsIscA2 had iron-binding capabilities as observed in IscA proteins from other organisms, supporting the role of EsIscA2 as a mitochondrial iron donor for ISC synthesis in Chinese mitten crab. Its differential mRNA expression after immune and oxidative stress challenges suggested the adaptations of ISC synthesis rates to these stress conditions.

The ancient role for GATA2/3 transcription factor homolog in the hemocyte production of oyster.

Hemocytes, the cellular component of invertebrate hemolymph, are essential for invertebrate immunity, but the hematopoiesis and regulation mechanism are still largely unknown. In the present study, a conserved hematopoietic transcription factor Cg-GATA2/3 was identified in Pacific oyster Crassotrea gigas, which was evolutionarily close to the vertebrate GATA1/2/3. Cg-GATA2/3 was mainly distributed in the immune organs, such as gill, hemocytes, and mantle. After Cg-GATA2/3 was interferenced by dsRNA, the mRNA expressions of hemocytes specific gene (EcSOD) and hematopoietic transcription factor (C-Myb) were all significant down-regulated, and the hemocyte renewal rates also decreased both in hemolymph and gill. During the larval developmental stages, the mRNA transcripts of Cg-GATA2/3 increased immediately after fertilization and kept a high level during blastula and early trochophore larvae stage (4-10 hpf, hours post fertilization), then decreased sharply in early D-veliger larvae stage (15 hpf). Whole-mount immunofluorescence assay further revealed that the abundant immunoreactivity of Cg-GATA2/3 was distributed in the whole body of blastula and gastrula embryos, while specialized gradually to a ring structure around the dorsal region in trochophore larvae. In the D-veliger and umbo larvae, scattered positive signals appeared in the specific sinus structure on the dorsal side and velum region. These results demonstrated that Cg-GATA2/3 was a hematopoietic lineage-specific transcription factor to regulate the hemocyte production, and it could also be used as hematopoietic specific marker to trace potential developmental events of hematopoiesis during ontogenesis of oyster.