Oyster Hemocytes Research Articles

Acute exposure to TiO2 nanoparticles produces minimal apparent effects on oyster, Crassostrea virginica (Gmelin), hemocytes

The response of oyster (Crassostrea virginica) hemocytes was studied following exposure to anatase nanoparticles (ca. 7.4nm), surface-coated rutile nanocomposites (UV–Titan M212, ca. 86nm) and bulk titanium dioxide (TiO2) particles (anatase and rutile crystalline forms; 0.4-0.5μm). Hemocytes were collected from oysters and exposed to one of the four particle types at concentrations of 0.1, 0.5, and 1.0mg/L under dark and environmentally-relevant light conditions for periods of two and four hours. Hemocyte mortality, phagocytosis, and reactive oxygen species (ROS) production were then evaluated using flow-cytometric assays. Bulk and nanoparticulate TiO2 had little effect on viability of oyster hemocytes or on production of ROS. Significant changes in phagocytosis occurred after exposure to anatase nanoparticles for 4h under dark conditions, and UV–Titan for 2h under light conditions. Results demonstrate that TiO2 particles (bulk or nanoscale) produce minimal effects on hemocyte biomarkers examined following acute, in vitro exposures.

LRFN (leucine-rich repeat and fibronectin type-III domain-containing protein) recognizes bacteria and promotes hemocytic phagocytosis in the Pacific oyster Crassostrea gigas

In bivalve mollusks, circulating hemocyte mediated phagocytosis is one of the primary ways to eliminate invading microbes. Here, we have identified one CgLRFN (leucine-rich repeat and fibronectin type-III domain-containing protein) in the Crassostrea gigas as a novel transmembrane LRR (Leucine-rich repeat) domain containing protein in C. gigas, homologous to the jawless fish VLR protein, that plays an important role in recognizing bacteria and promoting hemocytic phagocytosis. Tissue distribution analysis of CgLRFN in Pacific oyster showed that it is widely expressed in various tissues like the gills, adductor muscles, digestive glands, gonads, heart and in the hemocytes. Furthermore, infection of Pacific oysters with two marine Vibrio strains V. alginolyticus and V. parahaemolyticus was found to significantly increase CgLRFN expression in the hemocytes. Analysis of subcellular localization showed that CgLRFN is primarily localized in the cell membrane. Additionally, CgLRFN was found to be able to bind both the bacterial strains, indicating its possible role as a cell surface receptor. Flow cytometry analysis revealed that CgLRFN coated bacteria was phagocytosed by oyster hemocytes at a significantly higher rate compared to the uncoated bacteria. Finally, RNAi mediated knockdown of CgLRFN in vivo resulted in reduced clearance of both the bacterial strains from the oyster hemolymph. Overall, our study demonstrates that CgLRFN acts as a pattern recognition receptor for Vibrio spp. and promotes hemocytic phagocytosis in the Pacific oyster, which is critical for understanding the mechanism of bacterial infection in lower invertebrates, and also contributes to disease management of this economically and ecologically important marine mollusk.

Comparative study of three C1q domain containing proteins from pacific oyster Crassostrea gigas

C1q domain containing proteins (C1qDCs) are a family of proteins containing a globular head C1q domain (ghC1q) in C-terminus, which serve as pattern recognition receptors (PRRs) and mediate a series of immune responses. In the present study, three C1qDC proteins from pacific oyster Crassostrea gigas (CgC1qDC-2, CgC1qDC-3, CgC1qDC-4) were characterized and comparatively investigated to understand their roles in the immune response. All the three recombinant CgC1qDC proteins (rCgC1qDCs) could bind lipopolysaccharide (LPS) significantly but they could not bind lipoteichoic acid (LTA), β-1,3-glucan (GLU), mannan (MAN), and polyinosinic-polycytidylic acid (Poly I:C). Correspondingly, they all exhibited higher binding activities towards Gram-negative bacteria Vibrio anguillarum and V. splendidus. Moreover, they could enhance the phagocytosis of oyster hemocytes, and the enhancements towards Gram-negative bacteria were significantly higher than that towards Gram-positive bacteria (p < 0.01). The LPS binding affinity of rCgC1qDC-3 (KD = 8.74 × 10−7 M) was higher than that of rCgC1qDC-2 (KD = 7.76 × 10−5 M) and rCgC1qDC-4 (KD = 1.09 × 10−5 M). Meanwhile, rCgC1qDC-3 exhibited significantly higher enhancement on phagocytosis of oyster hemocytes towards Gram-negative bacteria than that of rCgC1qDC-2 and rCgC1qDC-4 (p < 0.05). After the secondary challenge with V. splendidus, the up-regulations of CgC1qDC-2 and CgC1qDC-4 mRNA in hemocytes occurred at 6 h, while that of CgC1qDC-3 was observed at 3 h and lasted for 24 h. And CgC1qDC-3 responded with high mRNA level for tested 24 h upon the secondary challenge with V. anguillarum as well. These results collectively suggested that three CgC1qDCs could serve as PRRs to specifically recognize certain Gram-negative bacteria and opsonins to enhance phagocytosis. CgC1qDC-3, with higher binding affinity to LPS, stronger opsonization and more rapid and persistent mRNA expression response upon the secondary challenge with homologous Vibrios, might exert efficient functions in the immune responses against invading pathogens.

The self-activation and LPS binding activity of executioner caspase-1 in oyster Crassostrea gigas

Executioner caspases play important roles in apoptotic pathway and immune defense, which is considered to coordinate the execution phase of apoptosis by cleaving multiple structural and repair proteins. However, the knowledge about the activation mechanism and function of executioner caspases in mollusks, especially marine bivalves is limited. In the present study, the full-length cDNA sequence of caspase-1 was cloned from oyster Crassostrea gigas, which encoded a predicted protein containing a small subunit (p10) and large subunit (p20) with a conserved caspase active site QACRG similar to that of human executioner caspase-3/7. SDS-polyacrylamide gel electrophoresis and western blot results demonstrated that the CgCaspase-1 zymogen could be cleaved into p20p10, p20 and p10 in prokaryotic expression systems, and the C-terminus of CgCaspase-1 was also cleaved into p20 and p10. Both of the recombinant CgCaspase-1 (rCgCaspase-1) and the C-terminus of CgCaspase-1 (rCgCaspase-1-C) exhibited similar caspase activity towards proteolytic substrate Ac-DMQD-pNA and Ac-DEVD-pNA. However, the recombinant N-terminus of CgCaspase-1 (rCgCaspase-1-N) did not display any caspase activity. Moreover, the inhibitor of both caspase-3/7 and pan-caspase could significantly inhibit the proteolytic activity of rCgCaspase-1. The strong binding activities towards lipopolysaccharide (LPS) of both rCgCaspase-1 and rCgCaspase-1-C were revealed by ELISA techniques and western blotting. A high level of CgCaspase-1 mRNA transcripts was detected in the gills and hemocytes by quantitative real-time PCR, and the CgCaspase-1 protein was mainly located in the cytoplasm of oyster hemocytes by immunofluorescence assay. These results collectively suggested that CgCaspase-1 was a homolog of executioner caspase-3/7, which could be self-activated through proteolytic cleavage in prokaryotic expression systems, and performed caspase and LPS binding activities in the innate immune response of oyster.

Two novel LRR and Ig domain-containing proteins from oyster Crassostrea gigas function as pattern recognition receptors and induce expression of cytokines

Leucine-rich repeat (LRR) domain and immunoglobulin (Ig) domain are both competent immune recognition modules, and the immunological roles of LRR and Ig domain containing- proteins (LRRIGs) are speculated to be multifunctional and worth investigating. In the present study, two novel LRRIGs, CgLRRIG-1 and CgLRRIG-2, were identified and characterized from oyster Crassostrea gigas. Both of them contained an N-terminal LRR region, an Ig domain, a transmembrane region, and a C-terminal cytoplasmic tail. The mRNA transcripts of CgLRRIG-1 and CgLRRIG-2 were constitutively expressed in muscle, gill, hepatopancreas, mantle, gonad and hemocytes with the highest expression level in hepatopancreas. Their mRNA expression levels in hemocytes were significantly up-regulated after the stimulations with four PAMPs including peptidoglycan (PGN), lipopolysaccharide (LPS), glucan (GLU) and polyinosinic-polycytidylic acid (poly I:C) and one bacteria Vibrio anguillarum. The recombinant proteins, rCgLRRIG-1 and rCgLRRIG-2, could bind to PGN, LPS, GLU and poly I:C, and rCgLRRIG-2 exhibited higher binding affinity. Additionally, rCgLRRIG-1 and rCgLRRIG-2 could significantly induce the expression of CgTNF-1 and CgIL17-5 in cultured oyster hemocytes, and the activity of rCgLRRIG-2 was higher than that of rCgLRRIG-1. All these results indicated that CgLRRIG-1 and CgLRRIG-2 could function as immune effectors or pro-inflammatory factors as well as PRRs in oyster.

A norepinephrine-responsive miRNA directly promotes CgHSP90AA1 expression in oyster haemocytes during desiccation

Oyster Crassostrea gigas is one model mollusc inhabiting in the intertidal zone and is frequently stressed by desiccation. The adaptation mechanism of oyster to environmental stress involves multiple levels, and miRNA is one of the most important regulators in post-transcriptional level. In the present study, an oyster norepinephrine-responsive miRNA cgi-miR-365 was proved to contribute to the host adaptation against desiccation by directly promoting the expression of CgHSP90AA1. Briefly, a significant increase of cgi-miR-365 was observed from the first day after aerial exposure and the up-regulation was vigorously repressed when oysters were injected with adrenoceptors antagonists. A total of 15 genes involved in biological regulation, metabolic process and response to stimulus were predicted to be modulated by cgi-miR-365. Among these genes, CgHSP90AA1 was up-regulated significantly during desiccation and could be down-regulated after simultaneous injection of adrenoceptors antagonists. The interaction between cgi-miR-365 and CgHSP90AA1 was subsequently verified in vitro, and a significant promotion of CgHSP90AA1 transcripts was observed after overexpressing cgi-miR-365 in either in vitro luciferase reporter assay or primarily cultured haemocytes. Meanwhile, CgHSP90AA1 transcripts decreased in vivo when cgi-miR-365 was repressed by its inhibitor during desiccation. Collectively, it was suggested that cgi-miR-365 could be induced by norepinephrine during desiccation and promote CgHSP90AA1 expression directly after binding to its 3′-UTR, which would provide new evidence in miRNA-mediated adaptation mechanism in oysters against intertidal stress.

The modulation role of serotonin in Pacific oyster Crassostrea gigas in response to air exposure

Serotonin, also known as 5-hydroxytryptamine (5-HT), is a critical neurotransmitter in the neuroendocrine-immune regulatory network and involved in regulation of the stress response in vertebrates and invertebrates. In the present study, serotonin was found to be widely distributed in the tissues of Pacific oyster Crassostrea gigas, including haemolymph, gonad, visceral ganglion, mantle, gill, labial palps and hepatopancreas, and its concentration increased significantly in haemolymph and mantle after the oysters were exposed to air for 1 d. The apoptosis rate of haemocytes was significantly declined after the oysters received an injection of extra serotonin, while the activity of superoxide dismutase (SOD) in haemolymph increased significantly. After the stimulation of serotonin during air exposure, the apoptosis rate of oyster haemocytes and the concentration of H2O2 in haemolymph were significantly decreased, while the SOD activity was significantly elevated. Furthermore, the survival rate of oysters from 4th to 6th d after injection of serotonin was higher than that of FSSW group and air exposure group. The results clearly indicated that serotonin could modulate apoptotic effect and redox during air exposure to protect oysters from stress.

The neuroendocrine immunomodulatory axis-like pathway mediated by circulating haemocytes in pacific oyster Crassostrea gigas.

The neuroendocrine-immune (NEI) regulatory network is a complex system, which plays an indispensable role in the immunity of host. In this study, a neuroendocrine immunomodulatory axis (NIA)-like pathway mediated by the nervous system and haemocytes was characterized in the oyster Crassostrea gigas. Once invaded pathogen was recognized by the host, the nervous system would temporally release neurotransmitters to modulate the immune response. Instead of acting passively, oyster haemocytes were able to mediate neuronal immunomodulation promptly by controlling the expression of specific neurotransmitter receptors on cell surface and modulating their binding sensitivities, thus regulating intracellular concentration of Ca2+. This neural immunomodulation mediated by the nervous system and haemocytes could influence cellular immunity in oyster by affecting mRNA expression level of TNF genes, and humoral immunity by affecting the activities of key immune-related enzymes. In summary, though simple in structure, the ‘nervous-haemocyte’ NIA-like pathway regulates both cellular and humoral immunity in oyster, meaning a world to the effective immune regulation of the NEI network.

The B-cell translocation gene 1 (CgBTG1) identified in oyster Crassostrea gigas exhibit multiple functions in immune response

B-cell translocation gene 1 (BTG1) is a member of the anti-proliferative gene family, which plays important roles in regulation of cell cycle. In the present study, a B-cell translocation gene 1 molecule homologue (designed CgBTG1) are identified and characterized in oyster Crassostrea gigas. CgBTG1 contains a conserved BTG domain with Box A and Box B motifs, and it shares high similarities with both BTG1 and BTG2 proteins in vertebrates. CgBTG1 mRNA is predominantly expressed in hemocytes, and its expression level in hemocytes is significantly up-regulated at 6 h (5.40-fold, p < 0.01) post Vibrio splendidus stimulation. The apoptosis rate of oyster hemocytes is significantly decreased (p < 0.05) after CgBTG1 interfered by dsRNA (dsCgBTG1). This is indicated that CgBTG1 participated in the regulation of oyster hemocytes apoptosis. Furthermore, CgBTG1 could also induce the apoptosis of cancer cells (HeLa, A549 and BEL7402) in vitro. Compared with normal oysters, both vessel-like structures and muscle fibers in CgBTG1 interfered oysters are severely damaged after V. splendidus challenge in paraffin section, considering that CgBTG1 possessed an analogous feature of angiogenesis for maintenance of vessel-like structures in adductor muscle of oyster. The results suggests that CgBTG1 is a multi-functional molecule involved in the immune response of C. gigas against pathogen infection, which provides more clues for intensive studies of BTG family proteins in invertebrates.

The granulocytes are the main immunocompetent hemocytes in Crassostrea gigas

Hemocytes comprise diverse cell types with morphological and functional heterogeneity and play indispensable roles in immunological homeostasis of invertebrates. The morphological classification of different hemocytes in mollusk has been studied since the 1970's, yet the involvement of the different sub-populations in immune functions is far from clear. In the present study, three types of hemocytes were morphologically identified and separated as agranulocytes, semi-granulocytes and granulocytes by flow cytometry and Percoll® density gradient centrifugation. The granulocytes were characterized functionally as the main phagocytic and encapsulating population, while semi-granulocytes and agranulocytes exhibited low or no such capacities, respectively. Meanwhile, the lysosome activity and the productions of ROS and NO were all mainly concentrated in granulocytes under both normal and immune-activated situations. Further, the mRNA transcripts of some immune related genes, including CgTLR, CgClathrin, CgATPeV, CgLysozyme, CgDefensin and CgIL-17, were mainly expressed in granulocytes, lower in semi-granulocytes and agranulocytes. These results collectively suggested that the granulocytes were the main immunocompetent hemocytes in oyster C. gigas, and a differentiation relationship among these three sub-population hemocytes was inferred based on the gradual changes in morphological, functional and molecular features.

Cloning, characterization and comparative analysis of four death receptorTNFRs from the oyster Crassostrea hongkongensis

Apoptosis plays an important role in homeostasis of the immune systems. The tumor necrosis factor receptors (TNFRs) play critical roles in the extrinsic apoptosis pathways and in determining cell fate. In this study, four death receptors (DR) named ChEDAR, ChTNFR27, ChTNFR5, and ChTNFR16 were identified from the oyster Crassostrea hongkongensis. These ChDRs proteins had 382, 396, 414 and 384 amino acids, respectively, with the typical domains of death receptors, such as the signal peptide (SP), transmembrane helix region (TM) and death domains. Phylogenetic analysis showed that the ChDR proteins clustered into three distinct groups, indicating that these subfamilies had common ancestors. mRNA expression of the ChDRs were detected in all 8 of the selected oyster tissues and at different stages of development. Furthermore, expression of all the genes was increased in the hemocytes of oysters challenged with pathogens or air stress. Fluorescence microscopy revealed that the full-length proteins of the ChDRs were located in the plasma membrane of HEK293T cells. Over-expression of the ChDRs activated the NF-κB-Luc reporter in HEK293T cells in a dose-dependent manner. These results indicate that the ChDRs may play important roles in the extrinsic apoptotic pathways in oysters.

An oyster species-specific miRNA scaffold42648_5080 modulates haemocyte migration by targeting integrin pathway

miRNAs are important gene regulators at post-transcriptional level and can modulate diverse biological processes, including immune response. Dozens of species-specific miRNAs have been identified in oyster Crassostrea gigas while their functions remain largely unknown. In the present study, an oyster species-specific miRNA scaffold42648_5080 was found responsive to LPS stimulation and might target a total of 31 oyster genes possibly involved in cell communication, cellular localization and cellular response to stimulus. Besides, in gain-of-function assay of scaffold42648_5080 in vivo, the phagocytosis (30.90% in miRNA group verse 23.20% in miRNA control group), apoptosis (3.10% in miRNA group verse 5.30% in miRNA control group) and migration rate (13.88% in miRNA group verse 21.03% in miRNA control group) of oyster haemocytes were found significantly altered after the injection of scaffold42648_5080 mimics. Among the target genes, integrin-linked kinase (CgILK) was considered crucial in cell migration and its interaction with scaffold42648_5080 was then verified both in vitro and in vivo. Consequently, a significant decrease of relative luciferase ratio was observed in CgILK 3′-UTR luciferase reporter assay after transfection of scaffold42648_5080 mimics (0.70-fold of that in blank group, p < 0.01). Meanwhile, when scaffold42648_5080 was overexpressed in vivo (5.41-fold of miRNA control group, p < 0.01), the expression of CgILK declined significantly to 0.25-fold of miRNA control group (p < 0.01). Comparatively, a significant decrease of the haemocyte migration rate (19.76% verse 34.82% in siEGFP control group, p < 0.01) was observed after knock-down of CgILK in vivo. The present study, as far as we know, for the first time revealed the immunomodulation role of an oyster species-specific miRNA, which might provide new insights into miRNA-mediated adaptation mechanism of oysters.

An invertebrate-specific miRNA targeted the ancient cholinergic neuroendocrine system of oyster

Acetylcholine (ACh) is the main neurotransmitter in the cholinergic neuroendocrine system and plays an indispensable role in modulating diverse immune responses. As important transporters in choline uptake, choline transporter-like proteins (CTLs) can control ACh synthesis and release indirectly in multiple organisms. In this study, cgi-miR-2d, an invertebrate-specific miRNA in oyster Crassostrea gigas, is proved to repress the synthesis/release of ACh by targeting CgCTL1 and choline uptake in haemocytes during the early stage of pathogen infection. In short, an opposite expression pattern between CgCTL1 and cgi-miR-2d is observed during Vibrio splendidus infection, accompanied by changes in haemolymph ACh. In addition, the expression level of CgCTL1 is found to be significantly repressed after cgi-miR-2d overexpression in vivo, while both haemocyte choline and haemolymph ACh are also decreased simultaneously, similar to the finding in CgCTL1 knock-down assay. As a result, the expression of two tumour necrosis factor-like proteins and the bacteriostatic activity of oyster haemocytes are found to be altered significantly by either gain-of-function cgi-miR-2d or knock-down of CgCTL1. To our knowledge, this is the first miRNA identified in invertebrates that can target the ancient cholinergic system and augment immune response during infection.

Flat oyster follows the apoptosis pathway to defend against the protozoan parasite Bonamia ostreae.

The invitro model Ostrea edulis hemocyte - Bonamia ostreae is interesting to investigate host-parasite interactions at the cellular level. Indeed, this unicellular parasite infects the flat oyster Ostrea edulis and multiplies within hemocytes, the central effectors of oyster defenses. Apoptosis is a mechanism used by many organisms to eliminate infected cells. In order to study the potential involvement of this mechanism in the oyster response to B.ostreae, invitro experiments were carried out by exposing hemocytes from the naturally susceptible oyster O.edulis and a resistant oyster species Crassostrea gigas to live and heat-inactivated parasites. Hemocyte apoptotic response was measured using a combination of flow cytometry and microscopy analyses. Whatever the host species was, the parasite was engulfed in hemocytes and induced an increase of apoptotic parameters including intracytoplasmic calcium concentration, mitochondrial membrane potential or phosphatidyl-serine externalization as well as ultrastructural modifications. However, the parasite appears more able to infect flat oyster than cupped oyster hemocytes and the apoptotic response was more important against live than dead parasites in the natural host than in C.gigas. Our results suggest that O.edulis specifically responds to B.ostreae by inducing apoptosis of hemocytes.

Transcriptional activation and translocation of ancient NOS during immune response.

NOS is the key component of the NO system, which plays an indispensable role in many physiologic and immunologic processes; however, the process that underlies the activation of ancient NOSs and their functions remains unclear. Expression of Crassostrea gigas NOS (CgNOS) mRNA in hemocytes was examined after stimulating oysters with LPS and TNF-α. Expression level of CgNOS mRNA was increased significantly, by 2.61-fold (P < 0.05), at 24 h poststimulation. A positive CgNOS signal was detected via immunoprecipitation, and only one protein was detected in oyster hemocytes. Shifting and supershifting bands were observed in EMSAs between the CgNOS promoter and the transcription factors CgNF-κB1 and Cg-signal transducer and activator of transcription (STAT). CgNF-κB1 was detected in the nucleus only at 12 h, whereas CgSTAT was observed in the cytoplasm and nucleus at 12 and 24 h. Expression levels of tyrosine-protein kinase receptor Tie-1, phosphatidylinositide phosphatase SAC2, phosphatidylinositol-4-phosphate 5-kinase type-1α, diacylglycerol kinase θ, LPS-induced TNF-α factor-like protein, cAMP-dependent transcription factor-2, NF-κB1, and STAT6 were significantly elevated in a transcriptome analysis after 12 h of LPS and TNF-α stimulation. An immunoreactive CgNOS signal was observed in both the cell membrane and cytoplasm at 12 h, whereas it was mainly localized to the cytoplasm at 24 h post-LPS and -TNF-α stimulation. These findings revealed that CgNOS could be transcriptionally activated by CgNF-κB1 and CgSTAT via the PI3K-Akt pathway, similar to what occurs for iNOS, but CgNOS translocated to the cytoplasm, similar to neuronal NOS, to modulate downstream signals during an immune defense. These results collectively provide crucial knowledge about the evolution of NOS structure and function.-Jiang, Q., Liu, Z., Zhou, Z., Wang, L., Wang, L., Yue, F., Wang, J., Wang, H., Song, L. Transcriptional activation and translocation of ancient NOS during immune response.

Antiviral Activity of Myticin C Peptide from Mussel: an Ancient Defense against Herpesviruses.

Little is known about the antiviral response in mollusks. As in other invertebrates, the interferon signaling pathways have not been identified, and in fact, there is a debate about whether invertebrates possess antiviral immunity similar to that of vertebrates. In marine bivalves, due to their filtering activity, interaction with putative pathogens, including viruses, is very high, suggesting that they should have mechanisms to address these infections. In this study, we confirmed that constitutively expressed molecules in naive mussels confer resistance in oysters to ostreid herpesvirus 1 (OsHV-1) when oyster hemocytes are incubated with mussel hemolymph. Using a proteomic approach, myticin C peptides were identified in both mussel hemolymph and hemocytes. Myticins, antimicrobial peptides that have been previously characterized, were constitutively expressed in a fraction of mussel hemocytes and showed antiviral activity against OsHV-1, suggesting that these molecules could be responsible for the antiviral activity of mussel hemolymph. For the first time, a molecule from a bivalve has shown antiviral activity against a virus affecting mollusks. Moreover, myticin C peptides showed antiviral activity against human herpes simplex viruses 1 (HSV-1) and 2 (HSV-2). In summary, our work sheds light on the invertebrate antiviral immune response with the identification of a molecule with potential biotechnological applications. Several bioactive molecules that have potential pharmaceutical or industrial applications have been identified and isolated from marine invertebrates. Myticin C, an antimicrobial peptide from the Mediterranean mussel (Mytilus galloprovincialis) that was identified by proteomic techniques in both mussel hemolymph and hemocytes, showed potential as an antiviral agent against ostreid herpesvirus 1 (OsHV-1), which represents a major threat to the oyster-farming sector. Both hemolymph from mussels and a myticin C peptide inhibited OsHV-1 replication in oyster hemocytes. Additionally, a modified peptide derived from myticin C or the nanoencapsulated normal peptide also showed antiviral activity against the human herpesviruses HSV-1 and HSV-2. Therefore, myticin C is an example of the biotechnological and therapeutic potential of mollusks.

A glutamic acid decarboxylase (CgGAD) highly expressed in hemocytes of Pacific oyster Crassostrea gigas

Glutamic acid decarboxylase (GAD), a rate-limiting enzyme to catalyze the reaction converting the excitatory neurotransmitter glutamate to inhibitory neurotransmitter γ-aminobutyric acid (GABA), not only functions in nervous system, but also plays important roles in immunomodulation in vertebrates. However, GAD has rarely been reported in invertebrates, and never in molluscs. In the present study, one GAD homologue (designed as CgGAD) was identified from Pacific oyster Crassostrea gigas. The full length cDNA of CgGAD was 1689 bp encoding a polypeptide of 562 amino acids containing a conserved pyridoxal-dependent decarboxylase domain. CgGAD mRNA and protein could be detected in ganglion and hemocytes of oysters, and their abundance in hemocytes was unexpectedly much higher than those in ganglion. More importantly, CgGAD was mostly located in those granulocytes without phagocytic capacity in oysters, and could dynamically respond to LPS stimulation. Further, after being transfected into HEK293 cells, CgGAD could promote the production of GABA. Collectively, these findings suggested that CgGAD, as a GABA synthase and molecular marker of GABAergic system, was mainly distributed in hemocytes and ganglion and involved in neuroendocrine-immune regulation network in oysters, which also provided a novel insight to the co-evolution between nervous system and immune system.

Characterization of the gene encoding a fibrinogen-related protein expressed in Crassostrea gigas hemocytes.

Four exons of the CgFrep1 gene (3333bplong) encode a putative fibrinogen-related protein (324aa) bearing a single C-terminal FBG domain. Transcripts of the gene obtained from hemocytes of different Pacific oysters show prominent individual variation based on SNP and indels of tandem repeats resulted in polymorphism of N-terminus of the putative CgFrep1 polypeptide. The polypeptide chain bears N-terminal coiled-coil region potentially acting as inter-subunit interface in the protein oligomerization. It is suggested that CgFrep1 gene encodes the oligomeric lectin composed of at least two subunits.

The simple neuroendocrine-immune regulatory network in oyster Crassostrea gigas mediates complex functions.

The neuroendocrine-immune (NEI) regulatory network is a complex system, which plays an indispensable role in the immunity of the host. In the present study, the bioinformatical analysis of the transcriptomic data from oyster Crassostrea gigas and further biological validation revealed that oyster TNF (CgTNF-1 CGI_10018786) could activate the transcription factors NF-κB and HSF (heat shock transcription factor) through MAPK signaling pathway, and then regulate apoptosis, redox reaction, neuro-regulation and protein folding in oyster haemocytes. The activated immune cells then released neurotransmitters including acetylcholine, norepinephrine and [Met5]-enkephalin to regulate the immune response by arising the expression of three TNF (CGI_10005109, CGI_10005110 and CGI_10006440) and translocating two NF-κB (Cgp65, CGI_10018142 and CgRel, CGI_10021567) between the cytoplasm and nuclei of haemocytes. Neurotransmitters exhibited the immunomodulation effects by influencing apoptosis and phagocytosis of oyster haemocytes. Acetylcholine and norepinephrine could down-regulate the immune response, while [Met5]-enkephalin up-regulate the immune response. These results suggested that the simple neuroendocrine-immune regulatory network in oyster might be activated by oyster TNF and then regulate the immune response by virtue of neurotransmitters, cytokines and transcription factors.

A novel siglec (CgSiglec-1) from the Pacific oyster (Crassostrea gigas) with broad recognition spectrum and inhibitory activity to apoptosis, phagocytosis and cytokine release

Sialic acid binding immunoglobulin-type lectin (siglec) belongs to the immunoglobulin superfamily (IgSF), which acts as regulator involved in glycan recognition and signal transduction in the immune and nervous systems. In the present study, a siglec gene (designated CgSiglec-1) was characterized from the Pacific oyster, Crassostrea gigas. The cDNA of CgSiglec-1 was of 1251 bp encoding a predicted polypeptide of 416 amino acids. CgSiglec-1 was composed of two I-set immunoglobulin (Ig) domains, one transmembrane (TM) domain and two ITIM motifs, sharing a sequence similarity with vertebrate CD22 homologs. The mRNA expression of CgSiglec-1 could be detected in all the selected tissues, with the highest level in hemocytes and labial palps. The confocal analysis revealed that CgSiglec-1 mainly distributed on the cytoplasmic membrane of the oyster hemocytes. In addition, the mRNA transcripts of CgSiglec-1 in hemocytes increased significantly (4.29-fold to that of control group, p < 0.05) after Vibrio splendidus stimulation. The recombinant CgSiglec-1 protein (rCgSiglec-1) could bind to poly sialic acid (pSIAS), lipopolysaccharides (LPS) and peptidoglycan (PGN) in a dose-dependent manner. The blockade of CgSiglec-1 by specific polyclonal antibodies could enhance the LPS-induced cell apoptosis, phagocytosis towards V. splendidus and the release of cytokines, such as CgTNF-1, CgIFNLP and CgIL-17. The results collectively indicated that CgSiglec-1 could act as a bridge molecule between invader recognition and signal transduction cascade, and modulate the immune response by inhibiting various important processes of immunity in oyster.