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Norepinephrine (NE) is involved in regulating cytokine expression and phagocytosis of immune cells in the innate immunity of vertebrates. In the present study, the modulation mechanism of NE on the biosynthesis of TNFs in oyster granulocytes was explored. The transcripts of Cg TNF-1, Cg TNF-2 and Cg TNF-3 were highly expressed in granulocytes, and they were significantly up-regulated after LPS stimulation, while down-regulated after NE treatment. The phagocytic rate and apoptosis index of oyster granulocytes were also triggered by LPS stimulation and suppressed by NE treatment. The mRNA expressions of Cg MAPK14 and Cg Relish were significantly induced after NE treatment, and the translocation of Cg Relish from cytoplasm to nucleus was observed. The concentration of intracellular Ca2+ in granulocytes was significantly up-regulated upon NE incubation, and this trend reverted after the treatment with DOX (specific antagonist for NE receptor, Cg A1AR-1). No obvious significance was observed in intracellular cAMP concentrations in the PBS, NE and NE + DOX groups. Once Cg A1AR-1 was blocked by DOX, the mRNA expressions of Cg MAPK14 and Cg Relish were significantly inhibited, and the translocation of Cg Relish from cytoplasm to nucleus was also dramatically suppressed, while the mRNA expression of Cg TNF-1 and the apoptosis index increased significantly to the same level with those in LPS group, respectively. These results collectively suggested that NE modulated TNF expression in oyster granulocyte through A1AR-p38 MAPK-Relish signaling pathway. • Granulocytes were the main sub-population of haecmocytes to synthesize TNFs in response to LPS stimulation. • NE could regulate phagocytosis, apoptosis and TNFs expression in oyster granulocyte. • NE modulated phagocytosis, apoptosis and TNFs expression in granulocyte through A1AR-p38 MAPK-Relish signaling pathway. [ABSTRACT FROM AUTHOR]

• The mRNA of Cg BMP7 was highly expressed in the MF of mantle. • BMP7-BMPR1B pathway could regulate the mRNA expression level and phosphorylation level of Cg Smad1/5/8 in mantle. • Ocean acidification was severely inhibited oyster calcified shell growth and the expression of Cg BMP7, Cg BMPR1B, Cg Smad1/5/8, Cg CaM and Cg engrailed-1. • BMP7 was able to trigger the BMPR-Smad signaling pathway and involved in controlling the formation of oyster calcified shell under OA conditions. Bone morphogenetic proteins (BMPs) are key factors controlling osteoblast differentiation, which have been proved to be involved in the hard tissue formation of marine mollusks. In the present study, a member of BMPs gene (Cg BMP7) was identified from Pacific oyster Crassostrea gigas (C. gigas) with the aim to understand its possible role in the regulation of shell formation under ocean acidification (OA) conditions. The open reading frame (ORF) of Cg BMP7 was of 1254 bp encoding a polypeptide of 417 amino acids. The deduced amino acid sequence of Cg BMP7 was comprised of one signal peptide, one prodomain and one TGF-β domain, which shared 21.69%-61.10% identities with those from other species. The mRNA transcript of Cg BMP7 was ubiquitously expressed in all the tested tissues of adult oysters with a higher expression level in mantle, notably highest in the middle fold (MF) of the three folds of mantle. The expression level of bone morphogenetic protein type I receptor (Cg BMPR1B) mRNA was also highest in the MF and up-regulated dramatically post recombinant BMP7 protein (r Cg BMP7) stimulation. After the blockage of BMPR1B with inhibitor LDN193189 (LDN), the mRNA expression level and phosphorylation level of Cg Smad1/5/8 in mantle were decreased, and the mRNA expression levels of Cg CaM and Cg engrailed-1 were down-regulated significantly. During the oysters were exposed to acidified seawater for weeks, the expression levels of Cg BMP7, Cg BMPR1B and Cg Smad1/5/8 in the MF decreased significantly (p < 0.01) at the 4th week, and Cg CaM and Cg engrailed-1 also exhibited the same variable expression patterns as Cg BMP7. In addition, the growth of shell in the treatment group (pH 7.8) was slower than that in the control group (pH 8.1). These results collectively indicated that BMP7 was able to trigger the BMPR-Smad signaling pathway and involved in controlling the formation of oyster calcified shell under OA conditions. [ABSTRACT FROM AUTHOR]

Bacterial infection and heat stress, as two major environmental threats of marine molluscs, could affect larval development and dramatically promote mortality of oysters. In the present study, next-generation sequencing, together with determinations of mRNA expression and measurements of enzyme activities, were employed to understand the response patterns of oyster larvae under acute heat and bacterial stress. After RNA-seq, a total of 9472 differentially expressed genes including 4895 significantly up-regulated ones and 4577 significantly down-regulated ones were obtained from 12 transcriptome libraries. GO overrepresentation analysis of the up-regulated genes revealed that the neuroendocrine immunomodulation pathway was activated after acute heat and bacterial stimulation, in which the catecholaminergic regulation played an important role. GO overrepresentation analysis of the down-regulated genes suggested that the immune capacity of Crassostrea gigas larvae was suppressed under stress, which was further validated since superoxide dismutase (SOD) and phenoloxidase (PO) activities in the total protein extract of larvae decreased dramatically after stress. Moreover, the shell formation of trochophore was inhibited and severe mortality was caused after acute heat and bacterial stress. These results collectively indicated that acute heat and bacterial stress could significantly inhibit larval development and suppress immune response of oyster C. gigas larvae. And the neuroendocrine immunomodulation, especially the catecholaminergic regulation, played an indispensable role in the stress response of molluscan larvae. [ABSTRACT FROM AUTHOR]

Loss of oxygen in the ocean is accelerating and threatening the coral reef ecosystem. In this study, the impacts of hypoxia on the scleractinian coral Pocillopora damicornis were explored. The algal symbiont density, chlorophyll a + c 2 content, energy consumption of corals, as well as energy available and consumption of their symbionts, decreased significantly post hypoxia stress. Meanwhile, the malondialdehyde contents in corals and symbionts, together with the caspase-3 activation level in corals, increased significantly in response to hypoxia stress. Furthermore, it was revealed that activities such as coral cell division and calcification were inhibited under hypoxia. These results collectively suggest that acute hypoxia stress reduces symbiont density and chlorophyll a + c 2 content in the coral P. damicornis by elevating intracellular oxidative pressure and apoptotic level, which further suppresses energy metabolism in the symbiotic association and negatively affects a series of activities such as coral cell division and calcification. • Acute hypoxia reduces algal symbiont density and chlorophyll a + c 2 content. • Acute hypoxia suppresses energy metabolism in corals and symbionts. • Cell division and calcification of corals are also hampered by acute hypoxia. [ABSTRACT FROM AUTHOR]

Muscarinic receptors, which selectively take muscarine as their ligand, are critical for the immunological and physiological processes in animals. In the present study, the open region frame (ORF) of a homologue of muscarinic acetylcholine (ACh) receptor (mAChR) was amplified from oyster Crassostrea gigas (named as CgmAChR-1), whose full length was 1983 bp and the protein it encoded contained 660 amino acids with a seven transmembrane region. Phylogeny analysis suggested that CgmAChR-1 shared homology with M 5 muscarinic receptor found in invertebrates including Habropoda laboriosa , Acromyrmex echinatior and Echinococcus granulosus . After cell transfection of CgmAChR-1 into HEK293T cells and ACh incubation, the level of intracellular Ca 2+ and cAMP increased significantly ( p < 0.05). Such trend could be reverted with the addition of M 3 and M 5 muscarinic receptor antagonists DAMP and DAR. The CgmAChR-1 transcripts were ubiquitously detectable in seven different tissues with the maximal expression level in adductor muscle. When the oysters received LPS stimulation, CgmAChR-1 mRNA expression in haemocyte was increased to the highest level (6.05-fold, p < 0.05) at 24 h, while blocking CgmAChR-1 using receptor antagonists before LPS stimulation promoted the expression of oyster TNF, resulting in the increase of haemocyte apoptosis index. These results suggested that CgmAChR-1 was the key molecule in cholinergic neuroendocrine-immune system contributing to the regulation of TNF expression and apoptosis process. [ABSTRACT FROM AUTHOR]

Polycyclic aromatic hydrocarbons (PAHs) are highly toxic environmental pollutants and are threatening scleractinian corals. In this study, PAHs treatment did not induce significant physiological responses of the coral Pocillopora damicornis and its algal symbionts, but biological processes including response to toxin, drug metabolic, and oxidation reduction were triggered at the mRNA level. These results implied that PAHs could be a group of slow-acting environmental toxicants, whose effects were moderate but persistent. Besides, it was interesting to find that PAHs activated the neuroendocrine system in the coral by triggering the expression of monoaminergic and acetylcholinergic system related genes, indicating that PAHs might function as environmental hormones. Moreover, the combined treatments of PAHs and heat caused a much obvious effect on the coral and its algal symbionts by elevating antioxidant activity and suppressing photosynthesis in the symbionts. Results from the transcriptome data further indicated that corals might perform stress responses upon PAHs and heat challenges through the TNF and apoptosis pathways, which perhaps was modulated by the neuroendocrine system of corals. Collectively, our survey demonstrates that the PAHs can function as environmental hormones and activate the neuroendocrine regulation in scleractinian corals, which may contribute to the stress responses of symbiotic association by modulating photosynthesis, antioxidation, and apoptosis. [Display omitted] • PAHs imposed a long-term impact on the scleractinian coral and its symbionts. • PAHs activated the neuroendocrine regulation of the coral. • PAHs + heat treatment elevated symbiont antioxidation and suppressed photosynthesis. • PAHs + heat treatment inhibited meiotic process in the coral. • The coral responded to PAHs and heat challenges under neuroendocrine modulation. [ABSTRACT FROM AUTHOR]

Opioid receptors (OR) are a group of G protein-coupled receptors with opioids as ligands, which play an important role in triggering the second messengers to modulate immune response in vertebrate immunocytes. In the present study, the full length cDNA of a homologue of δ-opioid receptor (DOR) for [Met 5 ]-enkaphalin was cloned from oyster Crassostrea gigas (designated as CgDOR), which was 1104 bp encoding a peptide of 367 amino acids containing a conserved 7tm_1 domain. After the stimulation of [Met 5 ]-enkephalin, the concentration of second messengers Ca 2+ and cAMP in the HEK293T cells decreased significantly ( p  <   0.05) with the expression of CgDOR. However, this trend was reverted with the addition of DOR antagonist BNTX. The CgDOR transcripts were ubiquitously detected in the tested tissues including haemocytes, gonad, mantle, kidney, gill, adductor muscle and hepatopancreas, with the highest expression level in the hepatopancreas. After LPS stimulation, the expression level of CgDOR mRNA began to increase (4.05-fold, p  <   0.05) at 6 h, and reached the highest level (5.00-fold, p  <   0.05) at 12 h. Haemocyte phagocytic and antibacterial activities increased significantly after [Met 5 ]-enkephalin stimulation, whereas the increase was repressed with the addition of DOR antagonist BNTX. These results collectively suggested that CgDOR for [Met 5 ]-enkephalin could modulate the haemocyte phagocytic and antibacterial functions through the second messengers Ca 2+ and cAMP, which might be requisite for pathogen elimination and homeostasis maintenance in oyster. [ABSTRACT FROM AUTHOR]

Hexokinase (HK) is generally recognized as a crucial enzyme participating in glycolysis. In the present study, a HK (named Cg HK) was identified as a potential pattern recognition receptor (PRR) from the Pacific oyster Crassostrea gigas. The open reading frame (ORF) of Cg HK was of 1395 bp, encoding a peptide of 464 amino acids with one Hexokinase_1 domain and one Hexokinase_2 domain. The predicted amino acid sequence of Cg HK shared 17%–29% similarities with that of other known HKs. The mRNA transcripts of Cg HK were constitutively detected in all the examined tissues, with relative high expression level in labial palp and haemocytes. Cg HK protein was mainly observed in the cytoplasm of oyster haemocytes. The mRNA expression level of Cg HK in haemocytes was significantly up-regulated and peaked at 3 h after Vibrio splendidus (7.64-fold, p < 0.001) and lipopolysaccharide (LPS) (11.86-fold, p < 0.001) stimulations. The recombinant Cg HK protein (r Cg HK) exhibited Mg2+-dependent adenosine triphosphate (ATP) binding activity in vitro and activity to bind D-(+)-glucose (GLU) and various pathogen-associated molecular pattern (PAMPs) such as LPS and peptidoglycan (PGN) in the absence of Mg2+. It also displayed higher binding activity towards V. splendidus and relatively lower binding activity towards Staphylococcus aureus , Escherichia coli, and Micrococcus luteus. After the mRNA expression of Cg HK in haemocytes was knocked down by dsRNA interference, the expression of Cg IL17-5 mRNA in haemocytes was considerably down-regulated at 3 h after the stimulation with V. splendidus (0.33-fold, p < 0.001). These results collectively indicated that Cg HK was able to recognize various PAMPs and pathogenic bacteria as a PRR apart from being the enzyme to exert ATP binding activity in glycolysis, and activate the anti-bacterial immune response by promoting the expression of pro-inflammatory cytokines Cg IL17-5 in oyster haemocytes. • A HK homologue was identified from oyster with a conserved site to bind ATP coupling with Mg2+. • The mRNA expression of Cg HK in haemocytes was induced significantly by LPS or V. splendidu. • Cg HK exhibited high binding affinity to LPS and PGN as well as pathogenic bacteria. • The expression of Cg IL17-5 was down-regulated after Cg HK was knocked down by RNAi. [ABSTRACT FROM AUTHOR]

Abstract Cathepsin L1 (CTSL1) is a lysosomal cysteine protease with a papain-like structure. It is known to be implicated in multiple processes of immune response against pathogen infection based on the proteolytic activity. In the present study, a CTSL1 homologue (designated as Cg CTSL1) was identified from Crassostrea gigas. It contained a typically single Pept_C1 domain with three conserved catalytically essential residues (Gln25, His135 and Asn178). The mRNA of Cg CTSL1 was ubiquitously expressed in oyster tissues with the highest expression level in important immune tissues such as gill and hemocytes. Cg CTSL1 proteins were mainly detected in gill and hepatopancreas by immunohistochemistry. Recombinant Cg CTSL1 (r Cg CTSL1) exhibited proteolytic activity to cleave the substrate Ac-FR-amino-4-trifluoromethyl coumarin (AFC) in a dose-dependent manner, and the inhibitor could reduce its proteolytic activity. After the interference of Cg CTSL1 mRNA, the proteolytic activity of oyster hemocytes was significantly down-regulated with the released AFC fluorescence value decreasing from 375.84 to 179.21 ( p < 0.05). Flow cytometry analysis revealed that the expression of Cg CTSL1 protein was higher in phagocytes with the mean fluorescence intensity (MFI) value of 21,187 (4.13-fold, p < 0.01) compared to the MFI value of 5,130 in non-phagocytic hemocytes. The further confocal analysis demonstrated that the actively phagocytic hemocytes with green bead signals were co-localized with stronger Cg CTSL1 positive signals. The mRNA expression levels of Cg CTSL1 in phagocyte-like sub-populations of granulocytes and semi-granulocytes were 298.12-fold ( p < 0.01) and 2.75-fold ( p < 0.01) of that in agranulocytes, respectively. Western blotting analysis of the hemocyte proteins revealed that Cg CTSL1 was relatively abundant in granulocytes and semi-granulocytes compared to that in agranulocytes. These results collectively suggested that Cg CTSL1, a CTSL1 homologue highly expressed in phagocyte-like hemocytes, was possibly involved in cellular immune response dependent on its conserved proteolytic activity, which might provide clues for the divergence between phagocytes and non-phagocytic hemocytes as well as the identification of promising molecular markers for phagocytes in oyster C. gigas. Highlights • A lysosomal cysteine protease cathepsin L1 was identified from Crassostrea gigas. • In vitro , r Cg CTSL1 exhibited proteolytic activity in a dose-dependent manner. • The proteolytic activity of oyster hemocytes was significantly decreased after the interference of Cg CTSL1 mRNA. • Transcripts and proteins of Cg CTSL1 were highly expressed in actively phagocytic hemocytes. • The higher content of Cg CTSL1 contributed to the higher proteolytic activity in granulocytes. [ABSTRACT FROM AUTHOR]

Thioester-containing proteins (TEPs), characterized by a unique intrachain β-cysteinyl-γ-glutamyl thioester bond, form an ancient and diverse family of secreted proteins that play central roles in the innate immune response. But the existence form and immune protection mechanism of TEP in invertebrates still remain unclear, especially in the mollusks. The fragmentation and the immune-protective effect of thioester bond in Cf TEP, a previously identified thioester-containing protein in scallop Chlamys farreri , were characterized in the present study. During the early embryonic development of scallop, the mRNA transcript of Cf TEP could be detected in all the stages, and its expression levels in D-larvae, veliger larvae and eye-spot larvae were significantly higher than that in eggs. The Cf TEP protein was also detected in peripheral of D-larvae, veliger larvae and eye-spot larva by immunofluorescence. In the adult scallop, the Cf TEP protein was mainly distributed in the hepatopancreas, gill, kidney, gonad, and mantle. The expression of Cf TEP mRNA in the hemocytes of adult scallop was significantly up-regulated when the scallops were stimulated by LPS, PGN or β-glucan. Two bands (100 and 55 kDa) were detected using anti- Cf TEP-R1 (spanned the C-terminal portion of the thioester, A2M-comp and A2M-recep domain, 942–1472), and a single band (46 kDa) was detected by using anti- Cf TEP-R2 (the N-terminal portion of the following A2M-N-2 domain, 452–496) in the serum of scallop at 12 h after LPS stimulation. When the thioester bond of Cf TEP protein was inactivated by injecting methylamine, the survival rate of scallop was significantly decreased after challenged by Vibrio angulillarum . All these results suggested that Cf TEP protein existed as fragments similar to vertebrate C3, and played central roles in the immune response against pathogen in the innate immunity of scallops. [ABSTRACT FROM AUTHOR]

The scleractinian corals conduct various responses upon heat stress such as bleaching and tissue loss, and colonies from the same coral species can conduct differential physiological activities with the biochemical basis unknown. In the present study, factors that influence the heat stress responses in coral Pocillopora damicornis were investigated. It was observed that P. damicornis conducted three differential physiological responses under heat treatment including tissue loss, bleaching, and polyp bailout. During heat response process, coral colonies conducting tissue loss had significantly higher total antioxidant capacity (T-AOC) level, while the bleached coral colonies exhibited higher caspase-3 activation level. Moreover, the stress response varied based on the energy reserve status. Colonies with higher lipid and sugar reserves were more likely to bleach, while those with lower reserves tended to undergo polyp bailout. We demonstrate that energy reserves influence the heat response patterns of P. damicornis. Colonies with higher lipid and sugar reserves may survive longer under heat stress, suggesting that these energy reserves contribute to their heat resistance. This study suggests that colonies with higher energy reserves prior to thermal stress may have greater thermal resistance, indicating that long-term environmental stressors that deplete energy reserves could increase susceptibility to thermal stress. [Display omitted] • Coral Pocillopora damicornis conducted tissue loss, bleaching and polyp bailout under heat stress. • Colonies conducting tissue loss had higher T-AOC level, while the bleached colonies had higher caspase-3 activation level. • Energy reserve was the major factor influencing the heat response patterns. [ABSTRACT FROM AUTHOR]

Interferons (IFNs) are the key coordinators of antiviral immunity by binding to their receptors to orchestrate a complex transcriptional network in vertebrates. Recently, the existence of molluscan IFN-like system has been certified by the identification of important components in IFN system, such as IFN-like protein (Cg IFNLP) from oyster Crassostrea gigas. In the present study, a novel Cg IFNLP receptor (designed Cg IFNLPR-1) was identified from C. gigas. The open reading frame (ORF) of Cg IFNLPR-1 cDNA was of 1962 bp encoding a peptide of 653 amino acid residues with five fibronectin type III (FNIII) domains and one transmembrane helix region. The mRNA transcripts of Cg IFNLPR-1 were constitutively distributed in all the tested tissues, with the highest level in gonad. After Poly (I:C) stimulation, the mRNA expression of Cg IFNLPR-1 in haemocytes was significantly up-regulated to the highest level at 48 h (4.54-fold of that in control group, p < 0.05). Cg IFNLPR-1 protein was mainly distributed in the cytoplasm and membrane of oyster haemocytes. Cg IFNLP and Cg IFNLPR-1 were able to interact with each other in vitro. After the Cg IFNLPR-1 was knocked down by RNAi, the mRNA expression of IFN-stimulated genes (ISGs), including Cg Mx, Cg Viperin and Cg IFNIP-44, were significantly inhibited after Poly (I:C) stimulation, which was 0.17, 0.31 and 0.53-fold of that in EGFP group, respectively (p < 0.01). These findings suggested that Cg IFNLPR-1 was a novel Cg IFNLP receptor in the oyster to recognize Cg IFNLP and regulate the expressions of Cg ISGs. • A novel Cg IFNLP receptor was identified from C. gigas with conserved domain characteristics. • Cg IFNLPR-1 mRNA widely distributed in oyster tissues and up-regulated after poly (I:C) stimulation in haemocytes. • Cg IFNLPR-1 showed binding capability with Cg IFNLP in vitro. • Cg IFNLPR-1 involved in the Cg IFNLP mediated antiviral immunity through regulating ISG expression. [ABSTRACT FROM AUTHOR]

Tripartite motif (TRIM) proteins are a large family of E3 ubiquitin ligases involved in many biological processes, such as inflammation and antiviral immunity. In the present study, a novel TRIM protein homolog named Cg TRIM1 was identified from Pacific oyster Crassostrea gigas. The open reading frame (ORF) of Cg TRIM1 was of 1914 bp encoding a putative polypeptide of 637 amino acid residues. There were three classical domains in the predicted Cg TRIM1 protein, including one RING domain, two b-box domains and one coiled-coil domain in N-terminal. For the lack of C-terminal domains, the Cg TRIM1 was classified as the member of C–V TRIM subfamily. The mRNA transcripts of Cg TRIM1 were detected in all the tested tissues and haemocytes, with the highest expression level in gill. The mRNA and protein levels of Cg TRIM1 in gill were significantly up-regulated at 6 h after poly (I:C) stimulation. Moreover, the nuclear translocation of Cg TRIM1 was observed in haemocytes of oysters after poly (I:C) stimulation. After IFN-like protein (Cg IFNLP) was knocked down by RNA interference (RNAi), the expression of Cg TRIM1 in gill was markedly inhibited in both mRNA (0.14-fold, p < 0.001) and protein levels after poly (I:C) stimulation. Furthermore, after knocking down of Cg TRIM1, the mRNA expression levels of IFN-stimulated genes, including myxovirus resistance of oyster (Cg Mx) and Interferon-induced protein 44 (Cg IFI44) were significantly down-regulated post poly (I:C) stimulation, while no significant change of the Cg IFNLP expression was observed. These results indicated that Cg TRIM1 participated in the antiviral response of C. gigas by regulating the mRNA expressions of IFN-stimulated genes. • A novel TRIM (Cg TRIM1) was identified from oyster with conserved sequence characteristics. • Cg TRIM1 showed high expression in gills and responded to poly (I:C) stimulation. • Cg TRIM1 involved in the Cg IFNLP mediated antiviral immunity through regulating the expression of ISG. [ABSTRACT FROM AUTHOR]

Microplastics become hotspots for bacteria to trigger a series of ecological effects, but few studies have focused on the potential impacts of microplastic biofilms in coral reef ecosystems. Here, we measured the bacterial communities and antibiotic resistance genes (ARGs) in the seawater and microplastic biofilms. Results showed that microbial biofilms were formed on the surface of microplastics. The alpha diversity of the bacterial community in the microplastic biofilms was lower than that in the seawater, and the bacterial communities were distinct between the two. Further analysis revealed that several bacteria in the microplastic biofilms carried ARGs, and the proportion of which was correlated to the concentration of antibiotics in the seawater. Specifically, Vibrio was positively correlated to sul1 in the microplastic biofilms under higher concentrations of sulfonamides. Pathway analysis reflected significant overrepresentation of human disease related pathways in the bacterial community of microplastic biofilms. These results suggest that the microplastic biofilms could selectively enrich bacteria from the reef environments, causing the development of ARGs under antibiotic driving. This may pose a serious threat to coral reef ecosystems and human health. Our study provides new insights into the ecological impacts of microplastic biofilms in coral reef ecosystems. [Display omitted] • Microplastics could selectively enrich bacteria from seawater in coral reef regions. • Bacteria on microplastics carried ARGs under antibiotic driving. • Microplastics pose a potential threat to coral and human health via pathogens and ARGs. [ABSTRACT FROM AUTHOR]

The phenomena of enhanced protection of innate immunity responding to a pre-exposed pathogen have been reported in invertebrates. The underpinning molecular basis and mechanism for the enhanced immune protection are still missing. In order to explore the possible molecular basis for enhanced immune protection in molluscs, the transcriptomic analysis of oysters Crassostrea gigas hemocytes after twice stimulation of Vibrio splendidus were conducted, and a total of 403 M clean reads and 34254 differentially expressed genes (DEGs) were collected. There were 2964 common DEGs up-regulated in hemocytes after both the first and second immune stimulation, which were mostly enriched in metabolic processes and immune related pathways, such as endocytosis, MAPK signaling pathway and TLR signal pathway. Moreover, 187 and 55 DEGs were higher expressed at resting (0 h after stimulation) and activating state (12 h after stimulation) of the second immune response than that of the first response, respectively, mainly including immune recognition receptor scavenger receptor 2, signal molecule MAPK2, immune regulator IL17-d, apoptosis inhibitor IAP and effector cathepsin. More importantly, 13 DEGs were long-lastingly higher expressed at both the resting and activating state within the second immune response than that of the first, including TLR signal molecule MyD88, anti-virulent tissue inhibitor of metalloproteinase, anti-bacterial proline-rich transmembrane protein, which might play indispensable roles in enhanced immune protection against V. splendidus re-infection. The expression patterns of TLR signals (Cg TLR6 and Cg MyD88) and effector molecules (Cg TIMP and Cg PRTP) were further validated by RT-PCR, which were consistent to transcriptomic results. All the results provided an overall molecular basis of enhanced immune protection for hemocytes defensing against the second stimulation of V. splendidus in oyster, which would be valuable for understanding the protection mechanisms of pre-exposure in invertebrates. • Transcriptomes were conducted to uncover the molecular basis for enhanced immune protection. • Metabolic and immune related genes were up-regulated after both the first and second stimulation. • There were 187 and 55 DEGs highly expressed at resting and activating state of second immune response. • TLR signaling and anti-bacteria effectors were continuously activated for the enhanced immune protection. [ABSTRACT FROM AUTHOR]

Serious juvenile oyster disease induced by pathogenic Vibrio splendidus has resulted in tremendous economic loss, but the molecular mechanisms underlying this killing mechanism remain unclear. The resistance of adult oyster to V. splendidus or its virulence factors might provide a possible access to cognize the interaction between pathogen and host. In the present study, the extracellular products (ECP) from less virulent V. splendidus JZ6 were injected into adult Pacific oyster Crassostrea gigas , and the cellular and humoral immune response induced by ECP were investigated. The phagocytosis rate of hemocytes was significantly up-regulated (30.57%) at 6 h after ECP injection compared with that (21%) of control groups. And significantly high level of ROS production was also observed from 3 h to 12 h in ECP-injected oysters, concomitant with increased apoptosis rate of hemocytes (16.4% in ECP-injected group, p < 0.01) compared with control group (6.7%). By RT-PCR analysis, the expression level of antioxidant Cg SOD in hemocytes significantly increased to 6.41-fold of that in control groups (p < 0.01) at 12 h post ECP injection. The expression levels of anti-toxic metalloprotease inhibitors Cg TIMP629 and Cg TIMP628 were also significantly up-regulated at the early (3–6 h) and late (6–24 h) stage of immune response, respectively. Moreover, after the ECP were incubated with serum proteins isolated from the ECP-injected oysters in vitro , the metalloprotease activity of ECP significantly declined by 21.39%, and less degraded serum proteins were detected by SDS-PAGE. When the primarily cultured hemocytes were stimulated with heat-inactivated ECP or fragments derived from ECP-degraded serum proteins, the expressions of Cg TIMP629 (13.64 and 7.03-fold of that in saline group, respectively, p < 0.01) and Cg TIMP628 (5.07 and 6.08-fold of that in saline group, respectively, p < 0.01) in hemocytes were all significantly induced. All the results indicated that the adult oysters could launch phagocytosis, antioxidant and anti-toxic response to resist the virulence of ECP, possibly by sensing heterologous ECP and ECP-induced endogenous alarm signals. These results provided a possible clue for the resistance mechanism of adult oysters towards the ECP of less virulent V. splendidus , which might be valuable for exploring strategies for the control of oyster disease. • ECPs could significantly induce phagocytosis activity of hemocytes in adult oysters. • Increased Cg SOD could enlighten the ECPs induced high-level ROS and apoptosis rate in hemocytes. • The up-regulated Cg TIMP628 and Cg TIMP628 might help to inhibit the toxic proteolysis of ECPs. • Both inactivated ECPs and ECPs-degraded serum proteins could induce immune response in oysters. [ABSTRACT FROM AUTHOR]

Phagocytosis constitutes a conserved cellular process for multicellular animals to ingest or engulf other cells or particles, which is facilitated by the use of opsonins to bind foreign particles and interact with cell surface receptors. The invertebrate secreted C1q domain-containing proteins (C1qDCs) have been reported to exhibit opsonic activity, while the detailed mechanisms of opsonization still remain unclear. In the present study, a C1qDC (designated as Cg C1qDC-5) with opsonic activity was identified from the hemolymph of oyster Crassostrea gigas. Cg C1qDC-5 exhibited the ability to bind pathogen-associated molecular patterns (PAMPs) of lipopolysaccharides (LPS) and Lipid A. It could also bind and agglutinate Gram-negative bacteria Escherichia coli , Vibrio splendidus and Vibrio anguillarum , whereas the agglutinating activity could be inhibited by LPS. In addition, Cg C1qDC-5 could enhance the phagocytosis of hemocytes toward E. coli , V. splendidus , and V. anguillarum. GST pull-down and surface plasmon resonance assays in vitro revealed that Cg C1qDC-5 could interact with β-integrin (Cg Integrin). In vivo , Cg C1qDC-5 was observed to bind hemocytes and co-localized with Cg Integrin on the cell membrane of hemocytes. Antibody-mediated blockage of Cg Integrin hindered the Cg C1qDC-5-enhanced hemocytic phagocytosis. Cg Integrin also exhibited the ability to bind the Gram-negative bacteria E. coli , V. splendidus , V. anguillarum and Vibrio parahaemolyticus , and PAMP of LPS, but not Lipid A. A phagocytosis assay demonstrated that Cg Integrin could directly mediate phagocytosis toward bacteria as a phagocytic receptor. These results collectively suggested that Cg C1qDC-5 could serve as an opsonin to recognize and bind bacteria, and subsequently interact with Cg Integrin on the hemocyte surface to enhance the Cg Integrin-mediated phagocytosis in oyster. • A oyster C1qDC protein (Cg C1qDC-5) was identified as an opsonin and a lectin. • Cg C1qDC-5 could promote oyster hemocytic phagocytosis via the membrane receptor β-Integrin. • The membrane receptor β-Integrin (Cg Integrin) could mediate the direct phagocytosis via immune recognition. [ABSTRACT FROM AUTHOR]

Abstract P38 mitogen-activated protein kinases are serine/threonine protein kinases reportedly involved in the innate immunity of vertebrates and invertebrates. In the present study, a P38 homolog (Cg P38) was characterized from the Pacific oyster Crassostrea gigas. The full-length cDNA of Cg P38 was of 1515 bp containing a 1101 bp open reading frame. A serine/threonine protein kinase (S_TKc) domain with a conserved Thr–Gly–Tyr motif and an ATRW substrate-binding site was found in the deduced amino acid sequence of Cg P38. Cg P38 shared a close evolutionary relationship with Ch P38 from the Hong Kong oyster Crassostrea hongkongensis. The transcript levels of Cg P38 in hemocytes increased significantly from 12 h to 48 h after lipopolysaccharide (LPS) stimulation and from 12 h to 24 h after Vibrio splendidus stimulation. The phosphorylation level of Cg P38 in oyster hemocytes increased significantly at 2 h after LPS stimulation. Cg P38 positively regulated the expression of interleukins, such as Cg IL17-1, Cg IL17-2, Cg IL17-3, Cg IL17-4 and Cg IL17-6, and tumor necrosis factor Cg TNF after LPS or V. splendidus stimulation. These results suggested that Cg P38 participated in oyster immune response by regulating the expressions of inflammatory cytokines. Highlights • A P38 homolog was identified from oyster Crassostrea gigas. • The expression level of Cg P38 increased significantly after LPS and Vibrio splendidus stimulations. • LPS induced Cg P38 phosphorylation in oyster hemocytes. • Cg P38 regulated the production of Cg IL17s and Cg TNF in oysters after LPS and Vibrio splendidus stimulations. [ABSTRACT FROM AUTHOR]

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 ( Cg CGL1, renamed as Cg DM9CP-1) from the Pacific oyster Crassostrea gigas . In the present study, another DM9 containing protein was identified from oyster C . gigas (designated as Cg DM9CP-2). The open reading frame of Cg DM9CP-2 gene was of 432 bp, encoding a polypeptide of 143 amino acids with two tandem DM9 repeats. The deduced amino acid sequence of Cg DM9CP-2 shared 60.8% identity with that of Cg DM9CP-1. In the unrooted phylogenetic tree, Cg DM9CP-2 was closely clustered with Cg DM9CP-1, and then assigned into the branch of invertebrate DM9CPs. The mRNA transcripts of Cg DM9CP-2 were expressed in all the tested tissues, including mantle, gonad, gills, adductor muscle, hemocytes, and hepatopancreas, with the highest expression level in gills. Cg DM9CP-2 protein was mainly distributed on the cytomembrane of oyster hemocytes. After mannose stimulation, the mRNA expression of Cg DM9CP-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 Cg DM9CP-2 protein (r Cg DM9CP-2) displayed higher binding affinity to D-(+)-mannose while lower binding affinity to lipopolysaccharide and peptidoglycan. r Cg DM9CP-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 Cg DM9CP-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. [ABSTRACT FROM AUTHOR]

Dopamine (DA), a significant member of catecholamines, is reported to induce biomineralization of calcium carbonate vaterite microspheres via dopamine receptor (DR) in bivalves, implying the modulation of dopaminergic system on shell formation during larval development. In this research, a homologue of D1 type DR (CgD1DR-1) was identified from oyster Crassostrea gigas , whose full length cDNA was 1197 bp. It was widely expressed in various tissues of C. gigas , with the significantly higher levels in hepatopancreas, mantle, muscle and gill. During developmental stages, the mRNA transcripts of CgD1DR-1 in D-shape larvae were obviously higher ( p  < 0.05) than those in trochophore and umbo larvae, and CO 2 exposure could inhibit the synthesis of DA and mRNA expression of CgD1DR-1. After cell transfection and DA treatment, intracellular cAMP in cells with the expression of CgD1DR-1 increased significantly ( p  < 0.05). Furthermore, the incubation with SCH 23390 for the blockage of CgD1DR-1 significantly restrained the expressions of six shell formation-related genes including CgTyrosinase-1, CgTyrosinase-3, CgChitinaseLP, CgAMC, CgBMP and CgBMPR in trochophore and D-shape larvae. These results jointly suggested that DA together with its receptor CgD1DR-1 might be involved in shell formation during oyster larval development from trochophore to D-shape larvae, and CO 2 -induced ocean acidification (OA) might influence marine bivalves by inhibiting the DA-D1DR pathway to prohibit their shell formation. [ABSTRACT FROM AUTHOR]

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 Es IscA2) was cloned from Eriocheir sinensis . The open reading frame (ORF) of Es IscA2 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 Es IscA2 shared over 50% similarity with that of other IscAs. Es IscA2 was clustered with IscA2 proteins from invertebrates and vertebrates, indicating that the protein was highly conservative in the evolution. r Es IscA2 exhibited a high iron binding affinity in the concentration ranging from 2 to 200 μM. Es IscA2 transcripts were detected in all the tested tissues including gonad, hemocytes, gill, muscle, heart, hepatopancreas and eyestalk, and Es IscA2 protein was detected in the mitochondria of hemocytes. The highest mRNA expression level of Es IscA2 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 Es IscA2 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 Es IscA2 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 H 2 O 2 for 15 min, the expression level of Es IscA2 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 Es Grx2 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 Es IscA2 had iron-binding capabilities as observed in IscA proteins from other organisms, supporting the role of Es IscA2 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. [ABSTRACT FROM AUTHOR]

Serotonin receptors, including ligand-gated ion channel (LGICs) and G protein-coupled receptors (GPCR), play vital roles in modulating physiological processes and immunoreaction. In the present study, a homologue of serotonin (5-HT) receptor was identified from oyster Crassostrea gigas (designated Cg 5-HTR-1). Its open reading frame (ORF) was of 1239 bp, encoding a polypeptide of 412 amino acids with a seven transmembrane region. Cg 5-HTR-1 shared high similarity with the 5-HTRs from other animals. The cAMP contents in HEK293T cells decreased significantly after Cg 5-HTR-1 transfection and 5-HT incubation ( p  < .05), while blocking Cg 5-HTR-1 with specific receptor antagonist reversed this downtrend. The intracellular Ca 2+ concentrations increased significantly ( p  < .05) after cell transfection and 5-HT incubation, and the antagonist treatment also arrested this process. Cg 5-HTR-1 transcripts were widely distributed in various tissues, with the highest level in hepatopancreas and lowest level in mantle and gill. The mRNA expression of Cg 5-HTR-1 in hemocyte increased significantly after lipopolysaccharide (LPS) stimulation and reached the peak level (6.47-fold, p  < .05) at 6 h post treatment. The inhibition of Cg 5-HTR-1 significantly reduced the expression of tumor necrosis factor (TNF) mRNA in hemocyte, down-regulated the superoxide dismutase (SOD) activity in serum, and induced the apoptosis of hemocyte ( p  < .05). These results suggested that Cg 5-HTR-1 was a novel member of 5-HT 1 receptor family and it mediated serotonergic immunomodulation on both cellular and humoral immune responses. [ABSTRACT FROM AUTHOR]

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 ( Cg C1qDC-2, Cg C1qDC-3, Cg C1qDC-4) were characterized and comparatively investigated to understand their roles in the immune response. All the three recombinant Cg C1qDC proteins (r Cg C1qDCs) 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 r Cg C1qDC-3 ( K D = 8.74 × 10 −7 M) was higher than that of r Cg C1qDC-2 ( K D = 7.76 × 10 −5 M) and r Cg C1qDC-4 ( K D = 1.09 × 10 −5 M). Meanwhile, r Cg C1qDC-3 exhibited significantly higher enhancement on phagocytosis of oyster hemocytes towards Gram-negative bacteria than that of r Cg C1qDC-2 and r Cg C1qDC-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 Cg C1qDCs could serve as PRRs to specifically recognize certain Gram-negative bacteria and opsonins to enhance phagocytosis. Cg C1qDC-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. [ABSTRACT FROM AUTHOR]

Phosphoenolpyruvate carboxykinase (PEPCK) is well known as a key enzyme involved in the metabolic pathway of gluconeogenesis in organisms, but the information about its involvement in immune response is still very limited. In the present study, a novel PEPCK homolog named CgPEPCK was identified from oyster Crassostrea gigas . The deduced amino acid sequence of Cg PEPCK shared 52%–74% similarities with those from other known PEPCKs. There were one conserved guanosine triphosphate (GTP) binding site, one substrate binding site, one metal binding site and one active site in Cg PEPCK. The mRNA transcripts of CgPEPCK were constitutively expressed in all the tested tissues including hemolymph, mantle, gill, muscle, gonad and hepatopancreas. Cg PEPCK proteins were mainly distributed in adductor muscle, gonad, gill and mantle, and rarely detected in hepatopancreas by using immunohistochemical analysis. After the stimulations with lipopolysaccharide (LPS), peptidoglycan (PGN), Vibrio splendidus and V. anguillarum , CgPEPCK transcripts in hemocytes were significantly up-regulated and peaked at 6 h (LPS, 9.62-fold, p < 0.01), 9 h (PGN, 4.25-fold, p < 0.01), 12 h ( V. splendidus , 5.72-fold, p < 0.01), 3 h ( V. anguillarum , 2.87-fold, p < 0.01), respectively. The recombinant Cg PEPCK protein (r Cg PEPCK) exhibited Mn 2+ /Mg 2+ dependent GTP binding activity, and the activities to bind LPS and PGN, but not β-1,3-glucan (GLU), lipoteichoic acid (LTA), mannan (MAN) nor polyinosinic-polycytidylic (Poly I: C). It could also bind Escherichia coli , Staphylococcus aureus , Micrococcus luteus and significantly inhibit their growth. All these results collectively suggested that Cg PEPCK could not only exert GTP binding activity involved in gluconeogenesis, but also mediate the bacteria recognition and clearance in immune response of oysters. [ABSTRACT FROM AUTHOR]

The complement system is one of the major effector mechanisms of immune system, playing essential roles in both the innate and adaptive immune responses. In the present study, the counterparts of vertebrate complement components were identified by screening the sequenced genome of Crassostrea gigas , resulting in the identification of 792 gene models containing complement-related domains. The transcriptome of haemocytes at 6, 12 and 24 h post lipopolysaccharides (LPS) stimulation showed differential expression of 77 C1q domain containing proteins, 53 C-type lectins and 42 fibrinogen-related proteins. mRNAs encoding 18 serine protease domain-containing (SPC) proteins, 4 MACPF-domain containing proteins and 11 C3 receptor-like proteins were up-regulated upon LPS stimulation, and CgC3 mRNA was significantly increased at 12 h. The presence of CgC3 was confirmed in cell free plasma and was present in three subunit chains as expected for the processed mature protein. The complement related PRRs with coiled coil regions and SPC proteins with CUB domains may function in the activation of CgC3, whereas, the C3-like receptors with integrin-α/β domain mediated the phagocytosis of C3-labled pathogens. These PRRs appear to serve as opsonins to promote phagocytosis of opsonized pathogens. The overall results suggested the existence of a potential multi-component complement system in C. gigas . [ABSTRACT FROM AUTHOR]

Glycogen synthase kinase-3 (GSK3) is a serine/threonine protein kinase firstly identified as a regulator of glycogen synthesis. Recently, it has been proved to be a key regulator of the immune reaction. In the present study, a GSK3 homolog gene (designated as EsGSK3) was cloned from Chinese mitten crab, Eriocheir sinensis . The open reading frame (ORF) was 1824 bp, which encoded a predicted polypeptide of 607 amino acids. There was a conserved Serine/Threonine Kinase domain and a DNA binding domain found in EsGSK3. Phylogenetic analysis showed that EsGSK3 was firstly clustered with GSK3-β from oriental river prawn Macrobrachium nipponense in the invertebrate branch, while GSK3s from vertebrates formed the other distinct branch. EsGSK3 mRNA transcripts could be detected in all tested tissues of the crab including haepatopancreas, eyestalk, muscle, gonad, haemocytes and haematopoietic tissue with the highest expression level in haepatopancreas. And EsGSK3 protein was mostly detected in the cytoplasm of haemocyte by immunofluorescence analysis. The expression levels of EsGSK3 mRNA increased significantly at 6 h after Aeromonas hydrophila challenge ( p < 0.05) in comparison with control group, and then gradually decreased to the initial level at 48 h ( p > 0.05). The mRNA expression of lipopolysaccharide-induced tumor necrosis factor (TNF)-α factor ( EsLITAF ) was also induced by A. hydrophila challenge. However, the mRNA expression of EsLITAF and TNF-α production was significantly suppressed after EsGSK3 was blocked in vivo with specific inhibitor lithium, while the phagocytosis of crab haemocytes was significantly promoted. These results collectively demonstrated that EsGSK3 could regulate the innate immune responses of E. sinensis by promoting TNF-α production and inhibiting haemocyte phagocytosis. [ABSTRACT FROM AUTHOR]

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 Cg TLR, Cg Clathrin, Cg ATPeV, Cg Lysozyme, Cg Defensin and Cg IL-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. [ABSTRACT FROM AUTHOR]

Junctional adhesion molecule (JAM), a subfamily of immunoglobulin superfamily (IgSF) with a couple of immunoglobulin domains, can act as regulator in homeostasis and inflammation of vertebrates. In the present study, a structural homolog of JAM-A (designated CgJAM-A-L) was screened out from oyster, Crassostrea gigas , through a search of JAM-A D1 domain (N-terminal Ig domain in JAM-A). The cDNA of CgJAM-A-L was of 1188 bp encoding a predicted polypeptide of 395 amino acids. The immunoreactive area of CgJAM-A-L mainly distributed over the plasma membrane of hemocytes. After Vibro splendidus or tumor necrosis factor (CgTNF-1) stimulation, the mRNA transcripts of CgJAM-A-L in hemocytes increased significantly by 4.46-fold and 9.00-fold ( p < 0.01) of those in control group, respectively. The recombinant CgJAM-A-L protein (rCgJAM-A-L) could bind multiple PAMPs including lipopolysaccharides (LPS), peptidoglycan (PGN), lipoteichoic acid (LTA), mannose (MAN), β-glucan (GLU) and poly(I:C), and various microorganisms including Micrococcus luteus , Staphylococcus aureus , Escherichia coli , Vibro anguillarum , V. splendidus , Pastoris pastoris and Yarrowia lipolytica . The phagocytic rates of oyster hemocytes towards Gram-negative bacteria V. anguillarum and yeast P. pastoris were significantly enhanced after the incubation of rCgJAM-A-L, and even increased more significantly after the pre-incubation of rCgJAM-A-L with microbes ( p < 0.01). The results collectively indicated that CgJAM-A-L functioned as an important pattern recognition receptor (PRR) and opsonin in the immune defense against invading pathogen in oyster. Moreover, as the most primitive specie with homolog of JAMs, the information of CgJAM-A-L in oyster would provide useful clues for the evolutionary study of JAMs and immunoglobulins. [ABSTRACT FROM AUTHOR]

DM9 domain containing protein (DM9CP) is a recently identified pattern recognition molecules exiting in most organisms except plants. In the present study, a novel DM9-containing protein (Cg DM9CP-3) was identified from Pacific oyster Crassostrea gigas with an open reading frame of 438 bp, encoding a polypeptide of 145 amino acids containing two tandem DM9 repeats. The deduced amino acid sequence of Cg DM9CP-3 shared 52.4% and 58.6% identity with Cg DM9CP-1 and Cg DM9CP-2, respectively. The mRNA transcripts of Cg DM9CP-3 were highest expressed in oyster gills and its protein was mainly distributed in cytomembrane of haemocytes. After the stimulations with Vibrio splendidus and mannose, the mRNA expression of Cg DM9CP-3 in oyster gills was significantly up-regulated and reached the peak level at 12 h and 24 h (p < 0.05), which was 7.80-fold (p < 0.05) and 42.82-fold (p < 0.05) of that in the control group, respectively. The recombinant Cg DM9CP-3 protein (r Cg DM9CP-3) was able to bind LPS, PGN and d -Mannose, fungi Pichia pastoris and Yarrowia lipolytica , as well as gram-negative bacteria Escherichia coli , Vibrio anguillarum and V. splendidus in a Ca2+-dependent manner. Moreover, it could enhance the encapsulation of haemocytes and exhibited agglutination activity towards fungi P. pastoris and Y. lipolytica in vitro with Ca2+. These results suggested that Cg DM9CP-3 not only acted as a PRR involved in the pathogen recognition, but also enhanced cellular encapsulation in oyster C. gigas. • A novel DM9-containing protein (Cg DM9CP-3) was identified from Crassostrea gigas. • The mRNA expression of Cg DM9CP-3 mRNA in gills was up-regulated after the stimulation with mannose or V. splendidus. • r Cg DM9CP-3 exhibited significant activities to bind and aggregate fungi and Gram-negative bacteria. • r Cg DM9CP-3 was able to enhance the encapsulation activity of haemocytes. [ABSTRACT FROM AUTHOR]

The enzyme Dicer is best known for its role as an endoribonuclease in the small RNA pathway, playing a crucial role in recognizing viral double-stranded RNA (dsRNA) and inducing down-stream cascades to mediate anti-virus immunity. In the present study, a truncated Dicer-like gene was identified from oyster Crassostrea gigas , and its open reading frame (ORF) encoded a polypeptide (designed as Cg DCL) of 530 amino acids. The Cg DCL contained one N-terminal DEAD domain and a C-terminal helicase domain, but lack the conserved PAZ domain, ribonuclease domain (RIBOc) and dsRNA binding domain. The mRNA transcripts of Cg DCL were detected in all the examined tissues with high expression levels in lip, gills and haemocytes, which were 62.06-fold, 48.91-fold and 47.13-fold (p < 0.05) of that in mantle, respectively. In the primarily cultured oyster haemocytes, the mRNA transcripts of Cg DCL were significantly induced at 12 h after poly(I:C) stimulation, which were 4.04-fold (p < 0.05) of that in control group. The expression level of Cg DCL mRNA in haemocytes was up-regulated significantly after dsRNA and recombinant interferon-like protein (r Cg IFNLP) injection, which was 12.87-fold (p < 0.01) and 3.22-fold (p < 0.05) of that in control group, respectively. Cg DCL proteins were mainly distributed in the cytoplasm of haemocytes. The recombinant Cg DCL protein displayed binding activity to dsRNA and poly(I:C), but no obvious dsRNA cleavage activity. These results collectively suggest that truncated Cg DCL from C. gigas was able to be activated by poly(I:C), dsRNA and Cg IFNLP, and functioned as an intracellular recognition molecule to bind nucleic acid of virus, indicating a potential mutual cooperation between RNAi and IFN-like system in anti-virus immunity of oysters. • A truncated form of Dicer was identified from oyster. • Cg DCL mRNA expression was induced by stimulation of dsRNA, poly(I:C) and Cg IFNLP. • r Cg DCL exhibited binding activity to poly(I:C) and dsRNA while no dsRNA cleavage activity. [ABSTRACT FROM AUTHOR]

Adiponectin receptors (AdipoRs) comprise a seven-transmembrane domain-containing protein family, which specifically recognize adiponectin (APN) and play critical roles in the immunological and physiological processes in vertebrates. In the present study, a novel AdipoR is identified from oyster Crassostrea gigas (designated as Cg AdipoR). The full-length cDNA of Cg AdipoR is of 1209 bp encoding a polypeptide of 343 amino acids. There is an N-terminal domain, a Hly III domain, and a C-terminal domain in Cg AdipoR. After the transfection of Cg AdipoR, the level of intracellular Ca2+ into HEK293T cells increases significantly (1.36-fold, p < 0.05) after APN incubation. The mRNA transcripts of Cg AdipoR are widely distributed in all the tested tissues, with the highest expression level in haemocytes (3.20-fold of that in hepatopancreas, p < 0.05). After lipopolysaccharide (LPS), Vibrio splendidus and polyinosinic-polycytidylic acid (poly (I:C)) stimulations, the mRNA expression of Cg AdipoR in haemocytes is significantly up-regulated and reached the highest level at 24 h (15.07-fold, p < 0.01), 6 h (4.39-fold, p < 0.01) and 24 h (5.62-fold, p < 0.01) compared to control group, respectively. After Cg AdipoR is interfered by specific Cg AdipoR-dsRNA, the expression level of interleukins (Cg IL17-1, Cg IL17-2, Cg IL17-3 and Cg IL17-5) in haemocytes decreases significantly (p < 0.01) at 24 h post LPS stimulation, while the expression level of Cg TNF-1 increases significantly (1.68-fold, p < 0.01), compared to that in the dsEGFP group. In Cg AdipoR dsRNA-injected oysters, the mRNA expressions of anti-apoptotic B-cell lymphoma-2 (Bcl-2) in haemocytes significantly decreases at 24 h after LPS challenge, which is (0.58-fold, p < 0.05) of that in dsEGFP-injected oysters, while the apoptotic rate of haemocytes is significantly up-regulated (1.93-fold of that in dsEGFP group, p < 0.05). These results collectively suggest that Cg AdipoR plays an important role in the immune response of oysters by regulating the expressions of inflammatory cytokines and haemocyte apoptosis. • Cg AdipoR was identified from oyster, which was constitutively expressed in hemocytes. • The expression of Cg AdipoR in the hemocytes was significantly up-regulated after LPS stimulation. • After Cg AdipoR was knockdown, the expressions of some Cg IL17s, were inhibited, while that of Cg TNF-1 increased. • Cg AdipoR regulated apoptosis of hemocyte during immune response in oyster. [ABSTRACT FROM AUTHOR]

• A total of 28 zinc transporter genes were identified in Crassostrea gigas. • Most of the zinc transporter genes were highly expressed in gill or hepatopancreas of C. gigas. • The oyster zinc transporters exhibited divergent response patterns under zinc stress. Zinc transporters play vital roles in regulating zinc content and localization by mobilizing zinc across cellular and intracellular membranes. Pacific oyster Crassostrea gigas is one of the most zinc-rich animals, which has been regarded as an excellent food for zinc supplement. But the information about zinc transporters and their involvements in zinc accumulation in oysters is still limited. In the present study, a total of 28 zinc transporter genes, including nine Zinc transporter genes (Cg ZnTs) and 19 Zrt/Irt-like protein genes (Cg ZIPs), were identified in C. gigas genome using a genome-wide search strategy. There were five ZIP10 homologs in C. gigas , which were much more than those in mammals, fish and other mollusks. Among oyster zinc transporters, immense variations were detected in their gene structure, protein length and physicochemical properties. Phylogenetic analysis showed that most of these transporters were distinctly clustered with their homologs from Homo sapiens , Danio rerio and other mollusks, and the most closely related transporters shared similar motif compositions. The highest zinc content was detected in the oyster mantle and gill, while the lowest level was found in the adductor muscle. The mRNA of all tested Cg ZnTs and Cg ZIPs were constitutively expressed in oyster tissues, and most of them were highly expressed in the gill or hepatopancreas. The analysis of RNA-seq data from gill and hepatopancreas showed that all the transporters exhibited divergent response patterns under zinc stress, except for Cg ZIP4 whose expression was almost undetectable in the two tissues. The results indicated that zinc transporters played important roles in the regulation of zinc homeostasis in C. gigas , which provided a solid foundation for further functional analysis of zinc transporters in oysters and other mollusks. [ABSTRACT FROM AUTHOR]