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The mass mortality of Pacific oyster Crassostrea gigas has become a severe ecological and economic concern to Chinese aquaculture, which is proposed to be linked to the phytoplankton community in the farming waters. In the present study, both field and laboratory experiments were conducted to identify the phytoplankton taxa associated with oyster mortality and explore the molecular mechanism by which they affect the physiological health of oysters. The field experiment showed that more serious mortality of oysters was observed in the North Yellow Sea from July to September in 2018 (average survival rate of 75.11 %) than in 2019 (average survival rate of 85.78 %), with the proportion of Bacillariophyta (diatoms) in the phytoplankton community in 2018 lower than that in 2019. In comparison to 2019, reduced dry weight, lower glycogen and triglyceride contents in hepatopancreas, lower 17β-estradiol and testosterone concentrations in gonad, as well as a generally weaker immune response against Vibrio splendidus stimulation were detected in the oysters sampled in 2018. The treatment of oysters with either starvation (starvation group) or Nitzschia closterium f. minutissima feeding (N. closterium group) was conducted to verify the field findings, with individuals reared in natural seawater as control. After 40 days of N. closterium feeding, dry weight, glycogen and triglyceride contents in hepatopancreas significantly increased, as well as the biosynthesis of sex hormones and gonadal maturation were promoted compared to the control and starvation groups. Moreover, a much stronger immune response against V. splendidus stimulation was observed in the oysters of N. closterium group, with the fold-changes of norepinephrine content in serum, SOD activity in hepatopancreas, and the mRNA expression level of IL17-5 and HSP70 in haemocytes higher than those in the control and starvation groups. Collectively, these results suggested that lack of diatoms in the farming waters suppressed the energy storage and gonadal maturation of adult oysters, and also resulted in a compromised immune response against bacterial infection, which may be a leading cause of the mass mortality of oysters living in diatom-deficient waters during breeding seasons., Competing Interests: Declaration of competing interest The authors declare no competing interests which may be perceived to influence the conduct or analysis of the data., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Inhibitors of NF-κB (IκBs) have been implicated as major components of the Rel/NF-κB signaling pathway, playing an important negative regulatory role in host antiviral immunity such as in the activation of interferon (IFN) in vertebrates. In the present study, the immunomodulatory effect of IκB (CgIκB2) on the expression of interferon-like protein (CgIFNLP) was evaluated in Pacific oyster (Crassostrea gigas). After poly (I:C) stimulation, the mRNA expression level of CgIκB2 in haemocytes was significantly down-regulated at 3-12 h while up-regulated at 48-72 h. The mRNA expression of CgIκB2 in haemocytes was significantly up-regulated at 3 h after rCgIFNLP stimulation. In the CgIκB2-RNAi oysters, the mRNA expression of CgIFNLP, interferon regulatory factor-8 (CgIRF8) and NF-κB subunit (CgRel), the abundance of CgIFNLP and CgIRF8 protein in haemocytes, as well as the abundance of CgRel protein in nucleus were significantly increased after poly (I:C) stimulation. Immunofluorescence assay showed that nuclear translocation of CgIRF8 and CgRel protein was promoted in CgIκB2-RNAi oysters compared with that in EGFP-RNAi group. In the CgRel-RNAi oysters, the mRNA and protein expression level of CgIFNLP significantly down-regulated after poly (I:C) stimulation. The collective results indicated that CgIκB2 plays an important role in regulating CgIFNLP expression through its effects on Rel/NF-κB and IRF signaling pathways., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

DNA methylation, an essential epigenetic alteration, is tightly linked to a variety of biological processes, such as immune response. To identify the epigenetic regulatory mechanism in Pacific oyster (Crassostrea gigas), whole-genome bisulfite sequencing (WGBS) was conducted on C. gigas at 0 h, 6 h, and 48 h after infection with Vibrio alginolyticus. At 6 h and 48 h, a total of 11,502 and 14,196 differentially methylated regions (DMRs) were identified (p<0.05, FDR<0.001) compared to 0 h, respectively. Gene ontology (GO) analysis showed that differentially methylated genes (DMGs) were significantly enriched in various biological pathways including immunity, cytoskeleton, epigenetic modification, and metabolic processes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that transcription machinery (ko03021) is one of the most important pathways. Integrated transcriptome and methylome analyses allowed the identification of 167 and 379 DMG-related DEGs at 6 h and 48 h, respectively. These genes were significantly enriched in immune-related pathways, including nuclear factor kappa B (NF-κB) signaling pathway (ko04064) and tumor necrosis factor (TNF) signaling pathway (ko04668). Interestingly, it's observed that the NF-κB pathway could be activated jointly by TNF Receptor Associated Factor 2 (TRAF2) and Baculoviral IAP Repeat Containing 3 (BIRC3, the homolog of human BIRC2) which were regulated by DNA methylation in response to the challenge posed by V. alginolyticus infection. Through this study, we provided insightful information about the epigenetic regulation of immunity-related genes in the C. gigas, which will be valuable for the understanding of the innate immune system modulation and defense mechanism against bacterial infection in invertebrates., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

CD49d, encoded by the gene Integrin α4, is a significant member of cell adhesion receptors, which is widely expressed in various immune cells to trigger immune responses against invading pathogens. In the present study, the expression of CgCD49d and its regulatory role in TNF expression were investigated in the Pacific oyster Crassostrea gigas. There were five Int-alpha domains, an Integrin&#95;alpha2 region and a unique FG-GAP repeat region inserted identified in CgCD49d. CgCD49d transcript was specifically expressed in haemocytes, and its mRNA expression level in haemocytes increased after LPS and Vibrio splendidus stimulation. After CgCD49d was blocked by using its antibody, the phosphorylation level of CgJNK in the MAPK signaling pathway and CgTNF transcripts decreased significantly post V. splendidus stimulation. After phosphorylation level of CgJNK was inhibited by using its inhibitor, the nuclear translocation of CgRel was restrained and CgTNF transcripts also decreased significantly post V. splendidus stimulation. Furthermore, CgCD49d was found to be mainly expressed in the agranulocyte subpopulation, and Alexa Fluor 488-conjugated CgCD49d antibody labeled agranulocytes with a circle of green fluorescence signals on CgCD49d + agranulocyte surface under Confocal microscopy, which accounted for 24.9 ± 4.53% of total haemocytes. Collectively, these results suggested that CgCD49d promoted TNF expression in oyster haemocytes against bacterial invasion by mediating MAPK pathway, and it could be used as a surface marker to type and sort a subset of agranulocyte subpopulation among haemocytes., (Copyright © 2024. Published by Elsevier Ltd.)

Metabotropic glutamate receptors (mGluRs) play a pivotal role in the neuroendocrine-immune regulation. In this study, eight mGluRs were identified in the Pacific Oyster Crassostrea gigas, which were classified into three subfamilies based on genetic similarity. All CgmGluRs harbor variable numbers of PBP1 domains at the N-terminus. The sequence and structural features of CgmGluRs are highly similar to mGluRs in other species. A uniformly upregulated expression of CgmGluRs was observed during D-shaped larval stage compared to early D-shaped larval stage. The transcripts of CgmGluRs were detectable in various tissues of oyster. Different CgmGluR exhibited diverse expression patterns response against different PAMP stimulations, among which CgmGluR5 was significantly downregulated under these stimulations, reflecting its sensitivity and broad-spectrum responsiveness to microbes. Following LPS stimulation, the mRNA expression of CgmGluR5 and CgCALM1 in haemocytes was suppressed within 6 h and returned to normal levels by 12 h. Inhibition of CgmGluR5 activity resulted in a significant reduction in CgCALM1 expression after 12 h. Further KEGG enrichment analysis suggested that CgmGluR5 might modulate calcium ion homeostasis and metabolic pathways by regulating CgCALM1. This research delivers the systematic analysis of mGluR in the Pacific Oyster, offering insights into evolutionary characteristics and immunoregulatory function of mGluR in mollusks., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Adenosine deaminases acting on RNA 1 (ADAR1) is a dsRNA adenosine (A)-to-inosine (I) editing enzyme that regulates the innate immune response against virus invasion. In the present study, a novel CgADAR1 was identified from the oyster Crassostrea gigas. The open reading frame (ORF) of CgADAR1 was of 3444 bp encoding a peptide of 1147 amino acid residues with two Zα domains, one dsRNA binding motif (DSRM) and one RNA adenosine deaminase domain (ADEAMc). The mRNA transcripts of CgADAR1 were detected in all the examined tissues, with higher expression levels in mantle and gill, which were 7.11-fold and 4.90-fold (p < 0.05) of that in labial palp, respectively. The mRNA transcripts of CgADAR1 in haemocytes were significantly induced at 24 h and 36 h after Poly (A: U) stimulation, which were 6.03-fold (p < 0.01) and 1.37-fold (p < 0.001) of that in control group, respectively. At 48 h after Poly (A:U) stimulation, the mRNA expression of CgRIG-Ⅰ, CgIRF8 and CgIFNLP significantly increased, which were 4.36-fold (p < 0.001), 1.82-fold (p < 0.05) and 1.92-fold (p < 0.05) of that in control group. After CgADAR1 expression was inhibited by RNA interference (RNAi), the mRNA expression levels of CgMDA5, CgRIG-Ⅰ, CgTBK1, CgIRF8 and CgIFNLP were significantly increased, which were 11.88-fold, 11.51-fold, 2.22-fold, 2.85-fold and 2.52-fold of that in control group (p < 0.001), and the phosphorylation level of CgTBK1 was also significantly increased. These results suggested that CgADAR1 played a regulation role in the early stages of viral infection by inhibiting the synthesis of interferon-like protein., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The JAK (Janus kinase)-STAT (Signal transducer and activator of transcription) is a well-known functional signaling pathway that plays a key role in several important biological activities such as apoptosis, cell proliferation, differentiation, and immunity. However, limited studies have explored the functions of STAT genes in invertebrates. In the present study, the gene sequences of two STAT genes from the Pacific oyster (Crassostrea gigas), termed CgSTAT-Like-1 (CgSTAT-L1) and CgSTAT-Like-2 (CgSTAT-L2), were obtained using polymerase chain reaction (PCR) amplification and cloning. Multiple sequence comparisons revealed that the sequences of crucial domains of these proteins were conserved, and the similarity with the protein sequence of other molluscan STAT is close to 90 %. The phylogenetic analyses indicated that CgSTAT-L1 and CgSTAT-L2 are novel members of the mollusk STAT family. Quantitative real-time PCR results implied that CgSTAT-L1 and CgSTAT-L2 mRNA expression was found in all tissues, and significantly induced after challenge with lipopolysaccharide (LPS), peptidoglycan (PGN), or poly(I:C). After that, dual-luciferase reporter assays denoted that overexpression of CgSTAT-L1 and CgSTAT-L2 significantly activated the NF-κB signaling, and, interestingly, the overexpressed CgSTAT proteins potentiated LPS-induced NF-κB activation. These results contributed a preliminary analysis of the immune-related function of STAT genes in oysters, laying the foundation for deeper understanding of the function of invertebrate STAT genes., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

SH2 domain containing inositol polyphosphate5-phosphatase-2 (SHIP2) is a member of the 5-phosphatase family, acting as a vital negative regulator of immune response in vertebrates. In the present study, a SHIP2 homologue (designed as CgSHIP2) was identified from Pacific oyster, Crassostrea gigas. There was a SH2 domain, an IPPc domain and a SAM domain in CgSHIP2. The mRNA transcripts of CgSHIP2 were widely expressed in all the tested tissues with the highest expression in haemolymph. The mRNA expressions of CgSHIP2 in haemocytes increased significantly at 6, 12, 48 and 72 h after Vibrio splendidus stimulation. The positive green signals of CgSHIP2 protein were mainly located in cytoplasm of haemocytes. After the expression of CgSHIP2 was inhibited by RNA interference, the mRNA transcripts of interleukin 17s (CgIL-17-1, CgIL-17-2, CgIL-17-3 and CgIL-17-6) in the haemocytes increased significantly at 24 h after V. splendidus stimulation, which were 8.15-fold (p < 0.001), 3.44-fold (p < 0.05), 2.15-fold (p < 0.01) and 4.63-fold (p < 0.05) compared with that in NC-RNAi group, respectively. Obvious branchial swelling and cilium shedding in gills were observed in CgSHIP2-RNAi group at 24 h after V. splendidus stimulation. The results suggested that CgSHIP2 played an important role in controlling inflammatory response induced by bacteria in oysters., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

LPS induced TNF-α Factor (LITAF) is a transcription factor widely involving in activation of Tumor Necrosis Factor (TNF) and other cytokines in the inflammatory response. In the present study, a homologue of LITAF with a conserved LITAF domain was identified from the Pacific oyster Crassostrea gigas. The transcripts of CgLITAF were detected in all examined tissues with highest expression in hepatopancrease. The immunofluorescence assay and Western blot showed that LPS stimulation induced an obvious nucleus translocation of CgLITAF protein in haemocytes. While the mRNA level of CgLITAF changed slightly after LPS stimulation. When the siRNA of CgLITAF was injected to inhibit its expression, the apoptotic level of haemocytes decreased observably after LPS stimulation. Consistently, the transcripts of CgTNF3 and CgTNF4 (LOC105343080, LOC105341146), the apoptotic-related molecules including CgBax, CgCytochrome c, CgCaspase9 and CgCaspase3, were significantly suppressed in the CgLITAF-RNAi oysters. While the mRNA expression level of CgBcl was enhanced significantly in the CgLITAF-RNAi oysters. These results indicated that CgLITAF promoted haemocyte apoptosis by regulating the expression of apoptotic-related factors, suggesting its important role in the immune response of oysters., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Caspases are cysteinyl aspartate-specific proteinases, playing critical roles in apoptotic pathway to induce apoptosis and inflammatory response. In this study, the expanded repertoire of Caspases was revealed in the Pacific oyster Crassostrea gigas, and a total of 30 Caspases were identified from the genomic and stress-induced transcriptomic databases of the Pacific oyster. They were clustered into CgCaspase-2/9, CgCaspase-8/10, CgCaspase-3/6/7, CgCaspase-Cg, and CgCaspase-L. CgCaspase-Cg subgroup was found to be specifically expanded after a positive selection in oyster with average Ka/Ks of 0.50. The mRNA expression of CgCaspase-Cg-5 was found to be obviously induced against various bacterial and viral stimulations or environmental stresses. The relative expression level of CgCaspase-Cg-5 in haemocytes increased and reached the peak at 6 h after Vibrio splendidus stimulation, which was 5.57-fold of that in the control group (p < 0.01). In the oysters whose CgCaspase-Cg-5 expression was knocked down, the mRNA expression of apoptosis-related genes including CgBcl2, CgBax, CgCaspase3 and CgCaspase9 changed significantly at 12 h after V. splendidus stimulation. The expression of CgBax, CgCaspase3 and CgCaspase9 decreased, which was 0.64-fold (p < 0.05), 0.53-fold (p < 0.05) and 0.62-fold (p < 0.01), while the expression of CgBcl2 increased, which was 2.81-fold (p < 0.01) of that in the EGFP-dsRNA group, respectively. Meanwhile, the apoptotic rate of haemocytes (1.90 ± 0.71%) significantly decreased compared to that in the EGFP-dsRNA group (5.40 ± 0.72%) (p < 0.05), and the histological damages of widened cell spacing, gill filament swelling and loose cytoplasm were observed in the CgCaspase-Cg-5-knockdown oysters after V. splendidus stimulation. Collectively, CgCaspase-Cg subgroup was specifically expanded in oyster and some bivalve species, and species-specific CgCaspase-Cg-5 regulated the mRNA expression of the apoptosis-related genes to induce haemocyte apoptosis in the early stage of immune response. This provided insight into the evolutionary and functional characteristics of Caspase repertoire in the Pacific oyster and highlighted the important role of CgCaspase-Cg-5 in the response to pathogen infection and environmental stresses., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Mannose-binding lectin-associated serine protease (MASP) is a type of central serine protease in the complement lectin pathway. In the present study, a MASP-like was identified from the Pacific oyster Crassostrea gigas, defined as CgMASPL-2. The cDNA sequence of CgMASPL-2 was of 3399 bp with an open reading frame of 2757 bp and encoded a polypeptide of 918 amino acids containing three CUB domains, an EGF domain, two IG domains, and a Tryp&#95;SPC domain. In the phylogenetic tree, CgMASPL-2 was firstly clustered with Mytilus californianus McMASP-2-like, and then assigned into the invertebrate branch. CgMASPL-2 shared similar domains with M. californianus McMASP-2-like and Littorina littorea LlMReM1. CgMASPL-2 mRNA was expressed in all the tested tissues with the highest expression in haemolymph. CgMASPL-2 protein was mainly distributed in the cytoplasm of haemocytes. The mRNA expression of CgMASPL-2 increased significantly in haemocytes after Vibrio splendidus stimulation. The recombinant 3 × CUB-EGF domains of CgMASPL-2 displayed binding activities to diverse polysaccharides (lipopolysaccharide, peptidoglycan and mannose) and microbes (Staphylococcus aureus, Micrococcus luteus, Pichia pastoris, Vibrio anguillarum, V. splendidus and Escherichia coli). In anti-CgMASPL-2 treated oysters, the mRNA expressions of CgIL17-1 and CgIL17-2 in haemocytes decreased significantly after V. splendidus stimulation. The results indicated that CgMASPL-2 could directly sense microbes and regulate the mRNA expressions of inflammatory factors., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

ATP-binding cassette transporter G2 (ABCG2) is a half-transporter of the G subfamily in ATP-binding cassette transporters (ABC transporter), which is involved in the regulation of multidrug-resistant, cell cycle, and cell proliferation. In the present study, a homologue of ABCG2 (named as CgABCG2) with the conserved AAA domain and ABC2 membrane domain was identified from the Pacific oyster Crassostrea gigas. The open reading frame (ORF) of CgABCG2 was of 1956 bp encoding a predicted polypeptide of 652 amino acids, which shared 56.7%-65.7% sequence similarities with previously identified ABCG2s from other animals. The mRNA transcripts of CgABCG2 were detected in all the tested tissues with higher expression levels in gonad and haemocytes (19.31-fold and 11.23-fold of that in adductor muscle respectively, p < 0.05). CgABCG2 was mainly distributed on the cell membrane of the haemocytes with a partial distribution in the cytoplasm and nucleus. After Vibrio splendidus stimulation, the mRNA expression level of CgABCG2 in haemocytes was significantly up-regulated at 3 h and 6 h, which was 5.22-fold and 8.60-fold (p < 0.05) of that in control, respectively. After the expression of CgABCG2 was interfered by RNAi, the number of cells with EdU positive signals was reduced in both haemocytes and the potential hematopoietic sites. And the mRNA expression level of CgPCNA, CgGATA3, CgRunx, CgSCL and CgC-kit decreased significantly (p < 0.05), which were about 0.66-, 0.37-, 0.32-, 0.50-, and 0.50-fold of that in the negative control group, respectively. While the mRNA expression level of CgCDK2 increased significantly (1.84-fold to that in control, p < 0.05) and that of stem cell-related factor CgSOX2 did not change significantly in the si-CgABCG2 oysters. Moreover, the cell cycle of haemocytes was detected by flow cytometry, which was arrested at G0/G1 phase in the si-CgABCG2 oysters. All the results collectively suggested that CgABCG2 might involve the proliferation of haemocytes by regulating the expression of haematopoiesis related transcription factors and the G1/S phase transition of the cell cycle in oyster C. gigas., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The IRF2BP family of transcription regulators act as corepressor molecules by inhibiting both enhancer-activated and basal transcription involving in many biological contexts. In the present study, an IRF2BP homologue (CgIRF2BP) was identified from oyster C. gigas. Its open reading frame is of 1809 bp encoding a polypeptide of 602 amino acids, which contains an IRF-2BP1&#95;2 domain and a RING domain. The mRNA transcripts of CgIRF2BP were detected in all tested tissues with highest level in haemocytes (28.99-fold of that in mantle, p < 0.05). After poly (I:C) stimulation, the expression level of CgIRF2BP was significantly down-regulated at 3 h (0.50-fold of that in control group, p < 0.001) and gradually increased from 6 h to 48 h (2.69-fold of that in control group, p < 0.01). The recombinant protein of CgIRF2BP (rCgIRF2BP) showed high affinity to both rCgIRF1 and rCgIRF8 with Kd value of 1.02 × 10 -7 and 2.09 × 10 -7 , respectively. In CgIRF2BP-RNAi oysters, the mRNA expression of CgIFNLP, CgMx1, CgViperin and CgIFI44L were significantly increased after poly (I:C) stimulation, which were 2.88 (p < 0.01), 1.83 (p < 0.05), 2.47 (p < 0.05), and 1.99-fold (p < 0.01) of that in EGFP group, respectively. These findings suggested that CgIRF2BP negatively regulated CgIFNLP expression by binding with CgIRF1 and CgIRF8., Competing Interests: Declarations of competing interest The authors declare that they have no conflict of interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Ferroptosis is an iron and oxidative dependent form of cell death usually mediated by redox related molecules in vertebrates. In the present study, a glutathione peroxidase 4 (GPX4) and a solute carrier family 7 member 11 (SLC7A11, xCT) homologues were identified from the oyster Crassostrea gigas (designed as CgGPX4 and CgxCT), which contained a GSHPx domain and an AA&#95;permease domain, respectively. The mRNA transcripts of CgGPX4 and CgxCT were expressed in all the examined tissues, including gill, gonad, adductor muscle, labial palp, mantle, hepatopancreas and haemocytes, with the highest expression in haemocytes. After erastin treatment, the rate of cell malformation and cell death increased significantly in haemocytes, and the mitochondrial atrophy, crest loss and fracture were observed in haemocytes. While the amount of Fe 2+ and Malondialdehyde (MDA) increased significantly, the mRNA expressions of CgGPX4, CgxCT and voltage-dependent anion channel 2 (CgVDAC2) in haemocytes decreased significantly after erastin treatment. These results indicated that erastin was able to induce the ferroptosis of oyster haemocytes., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

A TNF-α family member, CgTNF-2, was previously identified from the oyster Crassostrea gigas to involve in the antibacterial response. In the present study, the role of CgTNF-2 in mediating the proliferation of haemocytes was further explored. The mRNA expression of CgTNF-2 in granulocytes was significantly higher than that in semi-granulocytes and agranulocytes, and the percentages of CgTNF-2 antibody labeled cells in agranulocytes, semi-granulocytes and granulocytes were 19.15%, 40.25% and 94.07%, respectively. After the treatment with rCgTNF-2, the percentage of EdU + cells in haemocytes increased significantly (1.77-fold, p < 0.05) at 6 h compared with that in rGST-treated group, and the mRNA expressions of CgRunx, CgCyclin A, CgCDK2 and CgCDC45 in haemocytes all increased significantly (p < 0.05), which were 1.94-fold, 2.13-fold, 1.97-fold, 1.76-fold of that in rGST-treated group, respectively. Meanwhile, the protein abundance of CgRunx and CgCyclin A in the haemocytes of oysters in the rCgTNF-2-treated group increased, and the percentage of PI + haemocytes in S phase also increased significantly (2.19-fold, p < 0.05) compared with that in rGST-treated group. These results collectively confirmed that CgTNF-2 was highly expressed in granulocytes and involved in the proliferation of haemocytes by regulating the expressions of CgRunx and cell cycle related genes in C. gigas., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Yeast cells can play a crucial role in immune activation in fish and shellfish predominantly due to the cell wall component β-1,3-glucan, providing protection against bacterial or viral infections. However, the immunostimulatory capacity of dietary yeast cells remains poorly studied in bivalves. To understand the role of yeast cell wall components (mannan, β-glucan and chitin) as immune activators, this study characterized the surface carbohydrate exposure of the wild-type baker's yeast Saccharomyces cerevisiae (WT) and its Δmnn9 mutant, which presents a defective mannan structure, and compared these profiles with that of β-glucan particles, using fluorescein isothiocyanate (FITC)-labeled lectin binding analysis. Then, a first trial evaluated the immunological response in Crassostrea gigas juveniles after being fed for 24 h with an algae-based diet (100A) and its 50% substituted version (based on dry weight) with WT (50A50WT) and Δmnn9 (50A50Y), and the posterior resistance of the juveniles against Vibrio coralliilyticus infection (trial 1). The mRNA expression was measured for β-glucan-binding protein (CgβGBP), Toll-like receptor 4 (CgTLR4), C-type lectin receptor 3 (CgCLec-3), myeloid differentiation factor 88 (CgMyD88), nuclear factor-kappa B (CgNFκB), lysozyme (CgLys), interleukin 17-5 (CgIL17-5), and superoxide dismutase (CgSOD), in oysters, before and 24 h after the bacterial inoculation. A second trial tested the effect of incorporating Δmnn9 into the 100A diet for 24 h at different substitution levels: 0, 5, 10, 25, and 50% (100A, 95A5Y, 90A10Y, 75A25Y, and 50A50Y), followed by the bacterial challenge with V. coralliilyticus (trial 2). Our findings showed that the outer cell wall surface of WT is largely composed of mannan, while Δmnn9 presents high exposure of β-glucan and chitin, exhibiting similar FITC-lectin binding profiles (fluorescence intensity) to β-glucan particles. A significantly higher survival after the bacterial challenge was observed in oysters fed on 50A50Y compared to those fed 50A50WT and 100A in trial 1. This better performance of 50A50Y was supported by significantly higher gene expressions of CgLys, CgSOD, CgMyD88, and CgβGBP compared to 100A, and CgSOD and CgNFκB in relation to those fed on 50A50WT, prior to the bacterial inoculation. Furthermore, improved survival was observed in oysters fed 50A50Y compared to those offered lower Δmnn9 levels and 100A in trial 2. The superior performance of Δmnn9-fed oysters is mostly associated with the elevated presence of unmasked β-glucans on Δmnn9 cell wall surface, facilitating their interactions with oyster hemocytes. Further studies are needed to evaluate administration dose and frequency of Δmnn9 to develop strategies for long-term feeding., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The protein inhibitor of activated STAT (PIAS) family proteins act as the important negative regulators in janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway, which can be also involved in regulating the expression of interferon-stimulated genes (ISGs). In the present study, a PIAS homologue (designated as CgPIAS) was identified from oyster Crassostrea gigas. The open reading frame (ORF) of CgPIAS cDNA was of 1887 bp encoding a peptide of 628 amino acid residues. The CgPIAS protein contains a conserved scaffold attachment factor A/B/acinus/PIAS (SAP) domain, a Pro-Ile-Asn-Ile-Thr (PINIT) motif, a RING-finger-like zinc-binding domain (RLD) and two SUMO-interaction Motifs (SIMs). The deduced amino acid sequence of CgPIAS shared 74.58-81.36% similarity with other PIAS family members in the RLD domain. The mRNA transcripts of CgPIAS were detected in all the tested tissues with highest level in haemocytes (32.98-fold of mantles, p < 0.001). After poly (I:C) and recombinant Interferon-like protein (rCgIFNLP) stimulation, the mRNA expression of CgPIAS in haemocytes significantly up-regulated to the highest level at 48 h (7.38-fold, p < 0.001) and at 24 h (13.08-fold, p < 0.01), respectively. Moreover, the nuclear translocation of CgPIAS was observed in haemocytes after poly (I:C) stimulation. Biolayer Interferometry (BLI) assay revealed that the recombinant protein CgPIAS-RLD could interact with the recombinant protein CgSTAT in vitro with the K D value of 3.88 × 10 -8  M. In the CgPIAS-RNAi oysters, the green signals of CgSTAT protein in nucleus of haemocytes increased compared with that in NC-RNAi group, and the mRNA expression of myxovirus resistance (CgMx1), oligoadenylate synthase-like proteins (CgOASL), CgViperin and IFN-induced protein 44-like (CgIFI44L-1) in haemocytes significantly increased at 12 h after poly (I:C) stimulation, which were 2.39-fold (p < 0.05), 2.18-fold (p < 0.001), 1.74-fold (p < 0.05), and 2.89-fold (p < 0.01) of that in control group, respectively. The above results indicated that CgPIAS negatively regulated the ISG expression by inhibiting STAT activation in oyster C. gigas., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Cysteinyl aspartate specific proteinase-3 (Caspase-3) is an important protein involved in the apoptosis and gasdermin E (GSDME)-mediated cell pyroptosis pathways in vertebrates. A Caspase-3 homologue (designated as CgCaspase-3) was previously identified as an immune receptor specific for lipopolysaccharide (LPS) to regulate apoptosis in the Pacific oyster Crassostrea gigas. In the present study, the binding activity of CgCaspase-3 to different pathogen associated molecular patterns (PAMPs) and its effects on CgGSDME translocation in haemocytes were further investigated in C. gigas. The mRNA expression of CgCaspase-3 could be detected in all the tested tissues, including hepatopancreas, labial palp, adductor muscle, gonad, gill, mantle and haemocytes, and it was highly expressed in labial palp, gonad, haemocytes, and adductor muscle. The mRNA expression of CgCaspase-3 in haemocytes increased significantly at 3, 24, 48 and 72 h after LPS stimulation, and it increased significantly at 6, 12, 24 and 48 h after Vibrio splendidus stimulation. The recombinant CgCaspase-3 displayed binding activity towards LPS, mannose (MAN), peptidoglycan (PGN), and polyinosinic-polycytidylic acid potassium salt (Poly (I:C)). The positive signals of CgGSDME on haemocyte membrane became stronger at 3 h after V. splendidus stimulation, compared with that of Seawater group, and the co-localization of CgCaspase-3 and CgGSDME was observed in the haemocyte membrane. After the injection of dsCgCaspase-3, the positive signals of CgGSDME on haemocyte membrane became weaker compared with that of EGFP-RNAi group at 24 h after V. splendidus stimulation. The results suggested that CgCaspase-3 was able to bind diverse PAMPs and activate the translocation of CgGSDME in haemocytes of oyster response against pathogen invasion., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Interferon-stimulated genes (ISGs) encoding proteins are the essential executors of interferon (IFN) mediated antiviral defense. In the present study, an ISG member, interferon-induced protein 44-like (IFI44L) gene (designed as CgIFI44L-1) was identified from the Pacific oyster Crassostrea gigas. The ORF of CgIFI44L-1 cDNA was of 1437 bp encoding a polypeptide of 479 amino acids with a TLDc domain and an MMR&#95;HSR1 domain. The mRNA transcripts of CgIFI44L-1 were detected in all the tested tissues with highest level in haemocytes, which was 15.78-fold of that in gonad (p < 0.001). Among the haemocytes, the CgIFI44L-1 protein was detected to be highly expressed in granulocytes with dominant distribution in cytoplasm. The mRNA expression level of CgIFI44L-1 in haemocytes was significantly induced by poly (I:C) stimulation, and the expression level peaked at 24 h, which was 24.24-fold (p < 0.0001) of that in control group. After the treatment with the recombinant protein of an oyster IFN-like protein (rCgIFNLP), the mRNA expression level of CgIFI44L-1 was significantly enhanced at 6 h, 12 h and 24 h, which was 2.67-fold (p < 0.001), 5.44-fold (p < 0.001) and 5.16-fold (p < 0.001) of that in control group, respectively. When the expressions of CgSTAT and CgIFNLP were knocked down by RNA interference (RNAi), the mRNA transcripts of CgIFI44L-1 were significantly down-regulated after poly (I:C) stimulation, which was 0.09-fold (p < 0.001) and 0.06-fold (p < 0.001) of those in EGFP group, respectively. These results suggested that CgIFI44L-1 was a conserved ISG in oyster, which was regulated by CgIFNLP and CgSTAT, and involved in the oyster antiviral immune response., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The stimulator of interferon gene (STING), an intracellular sensor of cyclic dinucleotides, is critical to the innate immune response, especially the induction of type I interferon (IFN) during pathogenic infection. A STING homologue (CgSTING) regulating the expression of IFN-like protein (CgIFNLP) was previously identified in the Pacific oyster Crassostrea gigas, and its involvement in antibacterial immunity was further investigated in the present study. The mRNA transcripts of CgSTING were ubiquitously detected in all the three subpopulations of haemocytes with the highest expression in semi-granulocytes. After the stimulation with Vibrio splendidus, the mRNA expression of CgSTING in haemocytes was significantly up-regulated and peaked at 72 h, which was 12.91-fold of that in control group (p < 0.01). The CgSTING protein was mainly located in the cytoplasm of haemocytes. After the expression of CgSTING was knocked down (0.12-fold of that in control group, p < 0.05) by RNAi, the mRNA expression levels of interleukin17-1 (CgIL17-1), interleukin17-3 (CgIL17-3), interleukin17-4 (CgIL17-4), defensins (Cgdefh1, Cgdefh2), big defensin (CgBigDef1), interferon-like protein (CgIFNLP), tumor necrosis factor (CgTNF) and nuclear factor-κB (CgRel) all decreased significantly at 12 h after V. splendidus stimulation, which was 0.12-fold-0.72-fold (p < 0.05) of that in control group, respectively. The positive signals of CgRel were observed in the haemocyte nucleus after V. splendidus stimulation. The nuclear translocation of CgRel was suppressed in CgSTING-RNAi oysters, and the green signals of CgRel were mainly observed in the haemocyte cytoplasm after V. splendidus stimulation. Furthermore, the number of V. splendidus in the haemolymph of CgSTING-RNAi oysters increased significantly, which was 26.78-fold (p < 0.01) of that in the control group at 12 h after V. splendidus stimulation. These results indicated that CgSTING played important role in the immune defense against bacterial infection by inducing the expressions of cytokines and defensins., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The LGP2 (Laboratory of Genetics and Physiology 2) protein is a member of the retinoic acid-inducible gene I (RIG-I)-like receptor (RLRs) family, which is a class of antiviral pattern recognition receptors located in the cytoplasm. However, few studies have investigated the function of LGP2 in invertebrates. In this study, the complete coding sequence of the LGP2 gene of the Pacific oyster, Crassostrea gigas, was obtained and named CgLGP2-like. Sequence analysis revealed that CgLGP2-like encodes 803 amino acids, and the encoded protein contains a DEXDc, HELICc, and C-terminal regulatory domains. Multiple sequence alignment demonstrated that the sequences of these key protein functional domains were relatively conserved. Phylogenetic analysis revealed that CgLGP2-like was a new member of the animal LGP2 family. Quantitative real-time PCR results showed that CgLGP2-like mRNA was expressed in all tested oyster tissues, with the highest expression observed in the labial palpus and digestive glands. CgLGP2-like expression in gill tissues was significantly induced after the poly(I:C) challenge. Furthermore, multiple IRF and NF-κB binding sites were identified in the CgLGP2-like promoter region, which may be one of the reasons why CgLGP2-like responds to poly(I:C) stimulation. Finally, the results of dual-luciferase reporter gene assays revealed that overexpression of CgLGP2-like may have a regulatory effect on the human IFN, AP-1, and oyster CgIL-17 genes in HEK293T cells. Overall, our results preliminarily elucidate the immune functions of invertebrate LGP2 protein and provide valuable information for the development of comparative immunology., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2022. Published by Elsevier Ltd.)

The agranulocytes in the Pacific oyster Crassostrea gigas are a group of haemocytes that are significantly different from semi-granulocytes and granulocytes on the morphology. Agranulocytes are the smallest haemocytes characterized by a spherical shape, the largest ratio of nucleus to cytoplasm, and no granules in the cytoplasm. The lack of unique cell surface markers impedes the isolation of agranulocytes from total haemocytes. Previous transcriptome sequencing analysis of three subpopulations of haemocytes revealed that a homologue of CD9 (designed as CgCD9) was highly expressed in agranulocytes of oyster C. gigas (data not shown). In the present study, CgCD9 was identified to share a similarity of 60% with other vertebrates CD9s, and it harbored a typical four transmembrane domain and a conserved Cys-Cys-Gly (CCG) motif. The mRNA transcript of CgCD9 was found to be highly expressed in agranulocytes, which was 6.63-fold (p < 0.05) and 3.68-fold (p < 0.05) of that in granulocytes and semi-granulocytes, respectively. A specific monoclonal antibody of CgCD9 (named 3D8) was successfully prepared by traditional hybridoma technology, and a single positive band at 25.2 kDa was detected in the haemocyte proteins by Western Blotting, indicating that this monoclonal antibody exhibited high specificity and sensitivity to CgCD9 protein. The ELISA positive value of 3D8 monoclonal antibody to recognize agranulocytes, semi-granulocytes and granulocytes was 17.35, 4.48 and 1.55, respectively, indicating that monoclonal antibody was specific to agranulocytes. Immunocytochemistry assay revealed that CgCD9 was specifically distributed on the membrane of agranulocytes. Using immunomagnetic beads coated with 3D8 monoclonal antibody, CgCD9 + cells with a purity of 94.53 ± 5.60% were successfully isolated with a smaller diameter, a larger N:C ratio and no granules in cytoplasm, and could be primary culture in the modified L-15 medium in vitro. Collectively, these results suggested that CgCD9 was a specific cell surface marker for agranulocytes, which offered a tool for high-purity capture of agranulocytes from total haemocytes in C. gigas., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Exosomes are 30-150 nm-sized extracellular vesicles of endocytic origin that are released into the extracellular environment and play roles in cell-cell communication. Accumulating research achievements demonstrated that exosomes could act as innate immune effectors that contribute to the host defense mechanism. To better understand the immune functions of exosomes in Crassostrea gigas against bacterial stimulation, the iTRAQ LC-MS/MS approach was applied to identifying differentially expressed proteins (DEPs) of exosomes in oyster post Staphylococcus aureus and Vibrio splendidus stimulation. A total of 9467 unique peptides corresponding to 1634 proteins were identified. Among them, 99 proteins were upregulated and 152 were downregulated after S. aureus infection. After V. splendidus infection, 431 proteins were identified as differentially abundant, including 76 that were upregulated and 355 were downregulated. Several proteins related to apoptosis, including E3 ubiquitin-protein ligase, eukaryotic translation initiation factor 3, and protein kinase C delta type were found up-regulated in the S. aureus stimulation group, indicating that the apoptosis process was involved in the response to S. aureus stimulation. Thirty up-regulated and 123 down-regulated proteins were identified as differentially abundant after both bacterial stimuli. Among them, some proteins related to the actin-myosin cytoskeleton process were down-regulated, indicating that phagocytosis may be inhibited in both bacterial stimuli. This study would enrich the C. gigas proteome database and provide information for further understanding the immune functions of oyster exosomes against bacterial infection., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The RAC-alpha serine/threonine-protein kinase (AKT) is one of the most important protein kinases involved in many biological processes in eukaryotes. In the present study, a novel AKT homologue named CgAKT1 was identified from the Pacific oyster Crassostrea gigas. The open reading frame (ORF) of CgAKT1 cDNA was of 1482 bp encoding a peptide with 493 amino acid residues. There were classical domains in the predicted CgAKT1 protein, including an N-terminal pleckstrin homology domain, a central catalytic domain and a C-terminal hydrophobic domain. The mRNA transcripts of CgAKT1 were detected in all the examined tissues of C. gigas with higher level in gills (8.24-fold of that in mantle, p < 0.05) and haemocytes (3.62-fold of that in mantle, p < 0.05). After poly (I:C) stimulation, the mRNA expression of CgAKT1 decreased significantly in haemocytes from 3 h (0.44-fold of that in the control group, p < 0.001) to 24 h (0.20-fold of that in the control group, p < 0.001), and then increased significantly at 48 h (3.65-fold of that in the control group, p < 0.05). The expression level of CgAKT1 mRNA increased significantly at 6 h after rCgIFNLP stimulation, which was 3.60-fold of that in the control group (p < 0.001). The Alexa Fluor 488 positive signals of CgAKT1 protein were found to be distributed in the cytoplasm and cell membrane of haemocytes, while those in the cytoplasm became weaker after poly (I:C) stimulation. In CgAKT1-RNAi oysters, the mRNA expression of cyclic GMP-AMP synthase (CgcGAS) and TANK-binding kinase 1 (CgTBK1) did not change significantly, but the mRNA expression level of stimulator of interferon gene (CgSTING), interferon regulatory factor-1 (CgIRF-1), interferon regulatory factor-8 (CgIRF-8) and IFN-like protein (CgIFNLP) increased significantly, which was 1.40-fold, 1.53-fold, 1.72-fold and 1.99-fold of that in EGFP-RNAi oysters (p < 0.05), respectively. In CgIFNLP-RNAi oysters, the transcripts of CgAKT1 decreased significantly compared to those in EGFP-RNAi oysters (0.16-fold, p < 0.01). Moreover, the expression of p-CgTBK1, CgSTING and CgIFNLP at the protein level in the oysters treated with p-AKT1 activator (SC-79) was significantly suppressed after poly (I:C) stimulation. After the transfection of CgAKT1, the expression of p-cGAS protein in HEK293T cells increased significantly, while the cyclic GMP-AMP in the cells and the interferon (IFN-β) in the cell culture fluid decreased significantly compared with that in the control group. These results indicated that CgAKT1 might play a negative role in antiviral immunity of oyster by regulating the synthesis of CgIFNLP., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Cortisol is the main stress hormone that plays crucial roles in energy metabolism and immune response in vertebrates. In the present study, the homologues of 11β-hydroxysteroid dehydrogenase type 1 (designated Cg11β-HSD1) and 5α-reductase 1 (designated Cg5αR1), the key enzymes related to cortisol metabolism, were identified from Pacific oyster Crassostrea gigas. The Cg11β-HSD1 harbored a conserved SDR domain, and Cg5αR1 contained a Steroid&#95;dh domain and three transmembrane domains. The mRNA transcripts of Cg11β-HSD1 and Cg5αR1 were constitutively expressed in all the examined tissues of oysters, with the highest expression level in haemocytes and labial palp, respectively. After acute high temperature stress (28 °C), the mRNA expression level of Cg11β-HSD1 in hepatopancreas significantly up-regulated at 6 h and 12 h, and that of Cg5αR1 significantly up-regulated at 6 h, compared with the Blank group (11 °C). The concentration of cortisol and glucose, as well as the activities of superoxide dismutase (SOD) and catalase (CAT) in hepatopancreas all significantly up-regulated after acute high temperature stress, while the glycogen concentration in adductor muscle decreased significantly at 6 h and 12 h. After the blockage of Cg11β-HSD1 with metyrapone, the cortisol concentration and the activities of SOD and CAT significantly decreased after acute high temperature stress, the glucose concentration in hepatopancreas significantly increased at 24 h, and the glycogen concentration in adductor muscle significantly increased at 6 h. These results collectively suggested that cortisol played a crucial role in regulating glucose metabolism and oxidative response in oysters upon acute high temperature stress., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Astakine is considered as an endogenous cytokine-like factor of prokineticin homologue in invertebrate. Recently, an astakine homologue (CgAstakine) has been identified and characterized in oyster Crassostrea gigas. In the present study, a CgATP synthase β subunit was identified as the receptor of CgAstakine in C. gigas. There was an ATP-synt&#95;ab&#95;N domain and an AAA domain in the CgATP synthase β subunit protein. The mRNA transcripts of CgATP synthase β subunit were detected in all tested tissues, with the highest expression level in hepatopancreas and gills, which was 109.11-fold (p < 0.01) and 97.21-fold (p < 0.01) of that in labial palps, respectively. After rCgAstakine stimulation, the mRNA transcripts of CgATP synthase β subunit in agranulocytes and semi-granulocytes were significantly increased at 24 h (2.44-fold, and 9.01-fold of that in control group, p < 0.01), and those in granulocytes were significantly increased at 6 h (1.83-fold, p < 0.01), 12 h (1.92-fold, p < 0.01) and 24 h (3.47-fold, p < 0.01). The expression level of CgATP synthase β subunit protein in agranulocytes and granulocytes was also significantly increased after rCgAstakine stimulation, which was 1.64-fold (p < 0.05) and 1.85-fold (p < 0.05) of that in control group, respectively, while there were no significant changes in semi-granulocytes. The immunofluorescence assay showed that CgATP synthase β subunit positive signals were mainly located on the membrane of haemocytes. The number of haemocytes with EdU positive signals was significantly increased after rCgAstakine stimulation (2.04-fold of seawater group, p < 0.01), while significantly decreased after the RNA interference (RNAi) of CgATP synthase β subunit, which was 0.28-fold of that in NC group (p < 0.01). Bio-layer interferometry (BLI) assay confirmed in vitro interaction between rCgAstakine and rCgATP synthase β subunit. There results suggested that CgATP synthase β subunit acts as the receptor of CgAstakine and plays important roles in CgAstakine induced renewal of haemocytes in C. gigas., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Proliferating cell nuclear antigen (PCNA) is a crucial eukaryotic replication accessory factor in the regulation of DNA synthesis, which is always used as a proliferation marker for haematopoiesis in vertebrates. In the present study, a homologue of PCNA (named as CgPCNA) with a conserved N-terminal PCNA domain and a C-terminal PCNA domain was identified from oyster Crassostrea gigas. The deduced amino acid sequence of CgPCNA shared 85.4% and 86.6% similarities with the PCNAs identified in Mus musculus and Homo sapiens, respectively. CgPCNA was firstly clustered with PCNAs from molluscs, and then with PCNAs from arthropods to form a group falling into the invertebrate clade in the phylogenic tree. The mRNA transcripts of CgPCNA were detected in all tested tissues with higher expression level in gonad, gills and haemolymph. They were also detected in granulocytes, semi-granulocytes and agranulocytes with no significant differences, but the protein level of CgPCNA in agranulocytes was significantly higher (3.67-fold, p < 0.05) than that in granulocytes. In the haemocytes, CgPCNA was mainly distributed in the nucleus and less in the cytoplasm of haemocytes. CgPCNA protein was observed at the tubule lumen regions of gills vessels, and especially colocalized with the EdU signals. After lipopolysaccharide (LPS) and Vibrio splendidus stimulation, the expression level of CgPCNA mRNA in haemocytes was significantly (p < 0.05) up-regulated at 6 h and 12 h, which was 13.87-fold and 3.89-fold of that in control, respectively. In the oysters treated with the recombinant protein CgAstakine (rCgAstakine), the protein abundance of CgPCNA was enhanced in agranulocytes and gills, while no significant change was observed in semi-granulocytes and granulocytes. These results collectively indicated that CgPCNA was highly expressed in the newborn agranulocytes and the potential haematopoietic sites, and it might be applied as a marker for haemocytes proliferation in oysters., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

B cell lymphoma/leukemia 10 (BCL10) is an important member of the caspase recruitment domain-containing (CARD) protein family, which plays crucial roles in mediating the host inflammatory response. In the present study, a BCL10 homologue was identified from Pacific oyster Crassostrea gigas (designed as CgBCL10). The full length cDNA of CgBCL10 was of 897 bp with an open reading frame of 522 bp encoding a polypeptide of 174 amino acids containing a classical CARD domain. The deduced amino acid sequence of CgBCL10 shared low similarity with the previously identified BCL10s from other species. In the phylogenetic tree, CgBCL10 was firstly clustered with CvBCL10 from Crassostrea virginica and then assigned into the branch of invertebrate BCL10s. The mRNA transcripts of CgBCL10 were highly expressed in gonad, gill, adductor muscle, and haemocytes. After Vibrio splendidus stimulation, the mRNA expression level of CgBCL10 in haemocytes increased significantly (p < 0.01) at 24, 72 and 96 h. In CgBCL10-RNAi oysters, the phosphorylation level of mitogen-activated protein kinases (MAPKs), nuclear translocation of NF-κB/Rel and activator protein-1 (AP-1) in haemocytes were inhibited, and the mRNA expressions of inflammatory cytokines including CgIL17-1, CgIL17-2, CgIL17-3, CgIL17-6 and CgTNF all decreased significantly (p < 0.01) at 12 h after V. splendidus stimulation. These results suggested that CgBCL10 regulated the expression of inflammatory cytokines by activating MAPK kinase, and nuclear translocation of NF-κB/Rel and AP-1 to defense pathogen., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Rab protein plays an important role in a variety of cellular activities, especially the fusion process of the inner membrane during endocytosis. In the present study, a Rab1 protein (CgRab1) was identified from the Pacific oyster Crassostrea gigas. The full-length cDNA sequence of CgRab1 was of 2248 bp with an open reading frame of 618 bp, encoding a polypeptide of 205 amino acids containing a Rab domain. The deduced amino acid sequence of CgRab1 shared 94.2% and 89.3% identity with Rab1 from Pomacea canaliculata and Homo sapiens respectively. In the phylogenetic tree, CgRab1 was firstly clustered with the Rab1s from Aplysia californica and Pomacea canaliculata to form a sister group with Rab1 from invertebrates. The recombinant CgRab1 protein (rCgRab1) was able to bind GTP. The mRNA transcripts of CgRab1 were highly expressed in oyster haemocytes, and its expression level in oyster haemocytes was significantly up-regulated at 24 h after the stimulations with Vibro splendidus, which was 2.43-fold (p < 0.01) of that in the control group. After the expression of CgRab1 was knocked down (0.38-fold of that in EGFP-RNAi experimental group) by RNAi，the protein expression of Cgcathepsin L1 were reduced (0.63-fold, p < 0.01) compared with that in EGFP-RNAi experimental group. The phagocytic rate and phagocytic index of haemocytes in CgRab1-RNAi oysters decreased after V. splendidus stimulation, which was 0.50-fold (p < 0.01) and 0.58-fold (p < 0.01) of that in EGFP-RNAi experimental group. These results indicated that CgRab1 was involved in the process of haemocytes phagocytosis by regulating the expression of Cgcathepsin L1 in oyster C. gigas., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The myxovirus resistance (Mx) proteins belong to interferon (IFN)-induced dynamin GTPase and play a pivotal role in the inhibition of replication of numerous viruses. In the present study, an Mx homologue (designated as CgMx1) was identified from oyster Crassostrea gigas. The open reading frame (ORF) of CgMx1 cDNA was of 1689 bp encoding a peptide of 562 amino acid residues. There was an N-terminal dynamin GTPase domain in the predicted peptide, which consisted of a tripartite GTP-binding motif (GDXXSGKS, DLPG and T/NKXD). The deduced amino acid sequence of CgMx1 shared 30-39% similarity with other Mx family members. And CgMx1 was clustered with Mx from H. discus, and then assigned into the invertebrate branch of the phylogenetic tree. The mRNA transcripts of CgMx1 were constitutively distributed in all the tested tissues, with the highest level in haemocytes (1342.45-fold of labial palps, p < 0.05). The mRNA expression of CgMx1 in haemocytes was significantly up-regulated to the highest level at 6 h (13.14-fold, p < 0.001) after poly (I:C) treatment and at 24 h (66.28-fold, p < 0.001) after recombinant IFN-like protein (rCgIFNLP) stimulation, respectively. CgMx1 protein was found to distribute in both the cytoplasm and nucleus of haemocytes. In the oysters with CgIFNLP and signal transducer and activator of transcription (CgSTAT) silenced by RNAi, the mRNA expression of CgMx1 decreased significantly in the haemocytes at 12 h after poly (I:C) stimulation, which was 0.02-fold and 0.04-fold of that in EGFP-RNAi oysters (p < 0.001), respectively. Meanwhile, EMSA assay revealed that CgSTAT was able to transactivate CgMx1 promoter through directly binding to its interferon-stimulated response element (ISRE) and gamma interferon activation site (GAS). The above results indicated that CgMx1 participated in the immune response of C. gigas through the signal pathway mediated by CgIFNLP and CgSTAT., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Caspase-8 has been reported to be involved not only in apoptosis, but also in many other important immune response processes, such as inflammation and autophagy. In the present study, the open reading frame of CgCaspase-8-2 was cloned from the Pacific oyster Crassostrea gigas, which was of 2160 bp encoding 737 amino acids. There were two death effector domains (DEDs) and a cysteine aspartase cysteine structural (CASc) domain in the deduced amino acid sequences of CgCaspase-8-2. The mRNA expressions of CgCaspase-8-2 in haemocytes and gills all increased significantly after Vibrio splendidus stimulation at 3 h, 6 h, and 24 h. The cleaved CgCaspase-8-2 protein was observed in haemocytes at 3 h after V. splendidus stimulation and the expression of CgCaspase-8-2 protein was relatively higher in granulocytes, compared with that in agranulocytes. In CgCaspase-8-2-RNAi oysters, the mRNA expressions of CgIL17s (CgIL17-1, -2, -3, -4, -6), CgTNF, CgIFNLP and CgBigDef1 all decreased significantly at 12 h after V. splendidus stimulation. Meanwhile, the mRNA expressions of CgATG5 and CgBeclin1 decreased significantly at 12 h after V. splendidus stimulation, while CgBcl2 increased significantly. These results indicated that CgCaspase-8-2 was involved in not only the regulation of cytokine and antibacterial peptide production, but also autophagy-related gene expressions., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The apoptosis-inducing factor (AIF) is a phylogenetically old protein with classic function of inducing caspase-independent apoptosis, which extensively present in all primary kingdoms. In the present study, an AIF homologue (designated as CgAIF1) was identified from oyster Crassostrea gigas. The open reading frame of CgAIF1 cDNA was of 1836 bp encoding a peptide of 611 amino acid residues. There are a Pyr&#95;redox&#95;2 domain and an AIF&#95;C domain in the predicted CgAIF1 protein. The deduced amino acid sequence of CgAIF1 shared 35.44%-79.22% similarity with AIF1s from other species. In the phylogenetic tree, CgAIF1 firstly clustered with mollusc AIF1s, and then with insect AIF1s, displaying separation from vertebrate AIF1s. The mRNA transcripts of CgAIF1 were constitutively distributed in all the tested oyster tissues, with the highest level in gills (12.98-fold of that in haemocytes, p < 0.05). After LPS and Poly (I:C) stimulation, the mRNA transcripts of CgAIF1 in gills were significantly increased at 6 h and 24 h (5.79-fold, p < 0.001, and 21.96-fold compared to the control group, p < 0.05), respectively. In immunocytochemical assay, the CgAIF1 positive signals were mainly distributed in the cytoplasm of haemocytes, while after Poly (I:C) stimulation, the increased CgAIF1 positive signals were observed in the nucleus. Moreover, in the HEK293T cells transfected with pcDNA3.1-CgAIF1 recombinant plasmid, green signal of CgAIF1 were observed in both the cytoplasm and nucleus. The cell mortality rate, cell shrinking and the phosphatidylserine (PS) ectropion (Annexin V + /PI - cells and Annexin V + /PI + cells) of CgAIF1 transfected HEK293T cells were significantly increased, compared to the groups with or without pcDNA3.1 transfection. These results collectively suggested that CgAIF1 was a conserved AIF1 member in oysters, and participated in immune response by inducing cell apoptosis., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The family of fibrinogen-related proteins (FREPs) is a group of proteins with fibrinogen-like (FBG) domains, which play important roles as pattern recognition receptors (PRRs) in the innate immune responses. In the present study, a fibrinogen-like protein was identified from the oyster Crassostrea gigas (defined as CgFREP1). The open reading frame of CgFREP1 was of 966 bp that encoded a predicted polypeptide of 321 amino acids comprising a signal peptide and a fibrinogen-like domain. The mRNA expression of CgFREP1 was detected in all the examined tissues. The recombinant CgFREP1 (rCgFREP1) displayed binding activities to lipopolysaccharide (LPS), mannose (MAN), as well as Gram-positive bacteria (Micrococcus luteus and Staphylococcus aureus) and Gram-negative bacteria (Vibrio splendidus and Escherichia coli). The rCgFREP1 displayed the agglutinating activity towards M. luteus, V. splendidus and E. coli in the presence of Ca 2+ . rCgFREP1 was able to enhance the phagocytic activity of haemocytes towards V. splendidus, and exhibited binding activity to the CUB domain of CgMASPL-1. These results suggest that CgFREP1 not only serves as a PRR to recognize and agglutinate different bacteria but also mediates the haemocytes phagocytosis towards V. splendidus., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Regucalcin (RGN), also known as senescence marker protein-30 (SMP30), plays a vital role in the regulation of Ca 2+ homeostasis. In the present study, a regucalcin (designated as CgRGN) was identified from Pacific oyster Crassostrea gigas. The complete cDNA sequence of CgRGN was of 1059 bp, containing an open reading frame of 933 bp which encoded a protein of 310 amino acids. The deduced amino acid sequence of CgRGN shared similarity with other RGNs from the genome of C. gigas as well as other species. The mRNA transcripts of CgRGN were universally detected in all tested tissues, with higher level in hepatopancreas, labial palp, and gills. The relative expression level of CgRGN in hemocytes was significantly up-regulated (p < 0.05) at 3, 12, 72, and 96 h after the stimulation of lipopolysaccharide (LPS). After CgRGN expression was interfered by specific CgRGN-dsRNA, the hemocytes apoptosis rate increased dramatically at 12 h post LPS stimulation (1.56 fold, p < 0.01), compared to the control group. The caspase-3 activity in hemocytes and NO concentration in hemolymph increased significantly (p < 0.05) in dsCgRGN injection oysters. These results collectively indicated that CgRGN could suppress LPS-induced apoptosis and be involved in the immune response of oysters., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The programmed cell death protein 4 (PDCD4) is a newly defined transcriptional and translational inhibitor, which plays a key role in regulating the synthesis of inflammatory cytokines in vertebrates species. In the present study, the full-length cDNA of PDCD4 from oyster Crassostrea gigas (designed as CgPDCD4) was identified to explore its possible involvement in immune response. The open reading frame of pdcd4 gene was of 1344 bp encoding a polypeptide of 447 amino acids with two conserved MA-3 domains. The deduced amino acid sequence of CgPDCD4 shared 60.18% similarity with PDCD4 from Mizuhopecten yessoensis. The mRNA transcripts of CgPDCD4 could be detected in all the tested tissues with a higher expression level in adductor muscle and hemocytes. The mRNA expression of CgPDCD4 in hemocytes was significantly down-regulated at 3 h and 6 h (0.61-fold and 0.42-fold of that in PBS group, p < 0.01, respectively) after LPS stimulation. In hemocytes, CgPDCD4 protein was found to be mainly located in the cytoplasm. After the mRNA expression of CgPDCD4 in hemocytes was knocked down (0.40-fold of that in EGFP-RNAi group) by CgPDCD4 dsRNA (dsCgPDCD4) injection, the CgIL17-5 transcripts were up-regulated (20.11-fold of that in PBS group, p < 0.01) post LPS stimulation, which was significantly higher than that in dsEGFP-injected oysters (7.06-fold of that in PBS group, p < 0.01). Meanwhile, the nuclear translocation of CgRel (homologue of Rel/NF-κB) was significantly enhanced (about 1.36-fold of that in PBS group, p < 0.01), but it was similar as that in EGFP-RNAi group (about 1.52-fold of that in PBS group, p < 0.01) after LPS stimulation. All the results suggested that CgPDCD4 in oysters played the same role as PDCD4 of vertebrates in negatively regulating the production of interleukin in immune response, but the underpinning signal pathway was not conserved during evolution., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The homeostasis of immune cells during immune response is vital for hosts to defend against invaders. Activating transcription factor 6 (ATF6) is an important transcription factor in the unfolded protein response (UPR) to maintaining cellular homeostasis. In the present study, one ATF6 homologue was identified from Pacific oyster Crassostrea gigas (designated as CgATF6β). The full length cDNA of CgATF6β was of 2645 bp with a 1596 bp open reading frame (ORF) encoding a polypeptide of 531 amino acids. The deduced amino acid sequence of CgATF6β was predicted to contain a transmembrane region, a conserved basic leucine zipper (bZIP) domain, a site 1 protease cleavage site, a site 2 protease cleavage site, and a Golgi localization signal. CgATF6β mRNA was constitutively expressed in hemocytes, gill, mantle, gonad, hepatopancreas and labial palp, with a slightly higher expression level in muscle (2.45-fold of that in gill, p < 0.05). After oysters were challenged with Vibrio splendidus, the mRNA expression levels of CgATF6β in hemocytes were significantly up-regulated at 3 h (2.68-fold of that in seawater group, p < 0.01) and peaked at 12 h (3.14-fold of that in seawater group, p < 0.01). The endogenic CgATF6β protein was mainly located in the cytoplasm of oyster hemocytes, and it was significantly transported into the nuclei of hemocytes at 1.5 h after the challenge with V. splendidus. After an injection with CgATF6β dsRNA, the mRNA expression of CgATF6β was knocked down to 0.26-fold of that in dsGFP group (p < 0.01). In CgATF6β dsRNA-injected oysters, the mRNA expressions of glucose-regulated protein 78 (GRP78), calnexin (CNX) and anti-apoptotic B-cell lymphoma-2 (Bcl-2) in hemocytes were significantly decreased at 12 h after V. splendidus challenge, which were 0.65-fold (p < 0.01), 0.54-fold (p < 0.01) and 0.17-fold (p < 0.01) of that in dsGFP-injected oysters, while the apoptotic rate of hemocytes was significantly up-regulated (1.97-fold of that in dsGFP group, p < 0.05). Collectively, these results suggested that CgATF6β was involved in apoptosis inhibition of oyster hemocytes upon V. splendidus challenge by regulating the expression of CgGRP78, CgCNX and CgBcl-2., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Oyster Crassostrea gigas, is considered as a useful environmental indicator since it is widely distributed along the intertidal zone whereby it tends to accumulate cadmium and is always exposed to various pathogen agents. However, its molecular responses to both cadmium and pathogen stimulation remain unclear. In the present study, transcriptome data of hemocytes from oysters were analyzed to reveal specific molecular responses of oyster to cadmium or cadmium/bacteria stimulation. A total of 21591, 22872 and 20107 genes were detected in the BLANK, Cd24h and Cd/Bac24h group, respectively. Among them, there were 685 differentially expressed genes collected in the comparison of Cd24h versus BLANK. GO analysis of these genes found that sixteen terms into the Molecular Function category displayed transporter activities, and were all over-enrichment by cadmium exposure, whereas twelve terms into Biological Process category involved mainly in metabolic process of the various cellular components and two terms into Cellular Component category were all under-enrichment. The 330 immune responsive genes were shared by two gene lists of CdBac24h versus BLANK and CdBac24h versus Cd24h, and seven out of thirty terms in GO analysis were related to the immune process. Further annotation of these genes from the KEGG database revealed fourteen pathways, including two nervous system related pathways, arachidonic acid pathway, four immune pathways, MAPK cascade and other four cell signaling pathways, and two energy related pathways. Twenty-two differentially expressed genes were identified to responsive to both cadmium exposure and bacteria stimulation, but in different expression patterns, suggesting that bilateral responsive genes, such as alkaline phosphatase and sodium and chloride-dependent glycine transporter gene, could be candidate biomarkers for early warning of cadmium pollution. The present results collectively indicated that a profound neuro-endocrine-immune regulatory network was activated in response to cadmium and bacteria stimulation in oyster C. gigas, and the expression pattern of some cadmium responsive genes may be either reversed or strengthened by bacteria stimulation. The results provide knowledge on the transcriptomic response profile of oyster after short-term cadmium exposure and bacteria stimulation, which would be useful for future studies on stress response mechanism of mollusc, and some cadmium-bacteria responsive genes may be explored as potential biomarkers for monitoring marine pollution., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Tumor necrosis factors (TNFs) are a group of multifunctional inflammatory cytokines involved in various pathological and immune processes. Recently, a few primitive TNFs have been characterized from molluscs, which play important roles in modulating cell apoptosis, phagocytosis and production of immune-related enzymes. In the present study, a novel TNF (named as CgTNF-2) with the activity to mediate antibacterial response was identified from the Pacific oyster Crassostrea gigas. The open reading frame of CgTNF-2 was of 783 bp encoding a putative polypeptide of 261 amino acids with a typical TNF domain. The deduced amino acid sequence of CgTNF-2 shared high identity with that of TNFs previously identified from other molluscs, such as 96.1% identity with that in oyster C. hongkongensis, 33.7% identity with that in scallop Mizuhopecten yessoensis and 33.0% identity with CgTNF-1 in oyster C. gigas. There were two distinct TNF branches of vertebrate and invertebrate in the phylogenetic tree, and CgTNF-2 was firstly clustered with TNF-14 from C. hongkongensis, and then clustered with other molluscan TNFs. The mRNA transcripts of CgTNF-2 were widely expressed in various oyster tissues, with the highest expression level in hemocytes. The expression level of CgTNF-2 increased significantly at 6 h (2.45-fold and 6.20-fold, respectively, p < 0.05) after peptidoglycan and lipopolysaccharides treatments, and peaked at 12 h (31.86-fold and 7.90-fold, respectively, p < 0.05). The recombinant protein of CgTNF-2 (rCgTNF-2) inhibited the growth of human alveolar basal epithelial (A549) cells at a concentration of 800 ng/mL. After the oysters received an injection of rCgTNF-2, the serum from those oysters exhibited significantly higher antibacterial activity compared to that from control group, evidenced by inhibiting the growth of Vibrio splendidus. Moreover, the lysozyme activity as well as the contents of nitric oxide in the oyster serum also increased significantly. The above results collectively suggested that CgTNF-2 was a novel member of bivalve TNF-α family, which could prompt the antibacterial activity by inducing the lysozyme activity and the production of nitric oxide in the innate immune response of oyster., (Copyright © 2019. Published by Elsevier Ltd.)

The NF-κB/Rel transcription factors play essential roles in the induction and regulation of innate immune responses. In the present study, the full-length cDNA of CgRel from the Pacific oyster Crassostrea gigas was of 2,647 bp with an RHD and an IPT domain. The mRNA of CgRel was found to be constitutively expressed in all the tested tissues including gills, hepatopancreas, gonad, adductor muscle, labial palps, mantle, hemocytes, and ganglion. After lipopolysaccharide (LPS) stimulation, the expression level of CgRel mRNA in hemocytes was up-regulated to the first peak at 3 h (3.06-fold compared to the control group, p < 0.001) and second peak at 48 h (1.96-fold, p < 0.05). It increased significantly at 3 h (7.68-fold compared to the control group, p < 0.001), 24 h (3.63-fold, p < 0.05) and 48 h (1.99-fold, p < 0.05) post Vibrio splendidus stimulation, respectively. The protein of CgRel was translocated from cytoplasm into nucleus of oyster hemocytes after LPS stimulation. The mRNA expression levels of interleukin17s (CgIL17s) and big defensin (CgBigDef1) in hemocytes were examined after the expression of CgRel was silenced by RNAi. The transcripts of CgIL17-1 (0.25-fold of the control group, p < 0.01), CgIL17-2 (0.12-fold, p < 0.01), CgIL17-4 (0.33-fold, p < 0.01), CgIL17-6 (0.27-fold, p < 0.05) and CgBigDef1 (0.38-fold, p < 0.01) in CgRel-knockdown oysters decreased significantly at 12 h after LPS stimulation. The results indicated that CgRel played important roles in the immune defense against bacteria by regulating the expression of CgIL17 and CgBigDef1., (Copyright © 2019. Published by Elsevier Ltd.)

The activator protein-1 (AP-1) plays an important role in inducing the immune effector production in response to cellular stress and bacterial infection. In the present study, an AP-1 was identified from Pacific oyster Crassostrea gigas (designed as CgAP-1) and its function was investigated in response against lipopolysaccharide (LPS) stimulation. CgAP-1 was consisted of 290 amino acids including a Jun domain and a basic region leucine zipper (bZIP) domain. CgAP-1 shared 98.6% similarities with ChAP-1 from oyster C. hongkongensis, and assigned into the branch of invertebrates in the phylogenetic tree. The mRNA transcripts of CgAP-1 gene were detected in all tested tissues with highest expression level in hemocytes, especially in granulocytes. The mRNA expression level of CgAP-1 gene in hemocytes was significantly up-regulated (8.53-fold of that in PBS group, p < 0.01) at 6 h after LPS stimulation. CgAP-1 protein could be translocated into the nucleus of oyster hemocytes after LPS stimulation. The mRNA transcripts of interleukin17s (CgIL17-4 and CgIL17-5) in the hemocytes of CgAP-1-RNAi oysters decreased significantly at 24 h after LPS stimulation, which were 0.37-fold (p < 0.05) and 0.17-fold (p < 0.01) compared with that in EGFP-RNAi oysters, respectively. The results suggested that CgAP-1 played an important role in the immune response of oyster by regulating the expression of CgIL17s., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

MyD88 is a universal adapter protein for the Toll-like receptor/interleukin-1 receptor (TLR/IL-1R) signaling pathway. Since invertebrates are believed to lack MyD88-independent pathways, MyD88 appears more critical in oyster TLR signaling pathway. In the Pacific oyster (Crassostrea gigas), two complete paralogues, named as CgMyD88-1 and CgMyD88-2, have been identified. In the current study, we indicated that CgMyD88-1 and CgMyD88-2 might act synergistically to increase the efficiency of immune signaling by activating NF-κB transcription factor. However, we found that upon stimulation with lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid [poly (I:C)], CgMyD88-1 and CgMyD88-2 show differences in their response: CgMyD88-1 accumulated as large spots in the cytoplasm, while CgMyD88-2 assembled in the cytoplasm and in the membrane. Our results support the theory that expansion of these immune genes is associated with functional diversity., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Dicer, as a member of ribonuclease III family, functions in RNA interference (RNAi) pathway to direct sequence-specific degradation of cognate mRNA. It plays important roles in antiviral immunity and production of microRNAs. In the present study, a Dicer gene was identified from oyster Crassostrea gigas, and its open reading frame (ORF) encoded a polypeptide (designed as CgDicer) of 1873 amino acids containing two conserved ribonuclease III domains (RIBOc) and a double-stranded RNA-binding motif (DSRM). The deduced amino acid sequence of CgDicer shared identities ranging from 18.5% to 46.6% with that of other identified Dicers. The mRNA transcripts of CgDicer were detectable in all the examined tissues of adult oysters, with the highest expression in hemocytes (11.21 ± 1.64 fold of that in mantle, p < 0.05). The mRNA expression level of CgDicer in hemocytes was significantly up-regulated (36.70 ± 11.10 fold, p < 0.01) after the oysters were treated with double-stranded RNA (dsRNA). In the primarily cultured oyster hemocytes, the mRNA transcripts of CgDicer were significantly induced at 12 h after the stimulation with poly(I:C), which were 2.04-fold (p < 0.05) higher than that in control group. Immunocytochemistry assay revealed that CgDicer proteins were mainly distributed in the cytoplasm of hemocytes. The two most important functional domains of CgDicer, DSRM and RIBOc, were recombinant expressed in Escherichia coli transetta (DE3), and the recombinant DSRM protein displayed significantly binding activity to dsRNA and poly(I:C) in vitro, while the recombinant RIBOc protein exhibited significantly dsRNase activity to cleave dsRNA in vitro. These results collectively suggested that CgDicer functioned as either an intracellular recognition molecule to bind dsRNA or an effector with ribonuclease activity, which might play a crucial role in anti-viral immunity of oyster., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

As a family of negatively feedback regulating factors, the suppressor of cytokine signaling (SOCS) can depress cytokine signal transduction, and eventually modulate growth, development, differentiation, and immune response. In the present study, a SOCS homologue (designated as CgSOCS6) was identified from oyster Crassostrea gigas. The open reading frame of CgSOCS6 cDNA was of 1167 bp encoding a peptide of 388 amino acid residues with a central Src homology 2 (SH2) domain, a conserved C-terminal SOCS box, and a nucleus localization sequence (NLS) in its N-terminus. The deduced amino acid sequence of CgSOCS6 shared 37.9-45.5% similarity with other SOCS6/7 family members. In the unrooted phylogenetic tree, CgSOCS6 was clustered with EsSOCS6 from Chinese mitten crab Eriocheir sinensis and assigned into the SOCS6/7 group. The mRNA transcripts of CgSOCS6 were constitutively distributed in all the tested tissues, with the highest level in hemocytes. After lipopolysaccharide (LPS) stimulation, the mRNA expression of CgSOCS6 in hemocytes was significantly up-regulated to the highest level at 6 h (8.48-fold compared to the control group, p < 0.01), and then kept at a relatively higher level from 12 h to 72 h. CgSOCS6 protein could be translocated into the hemocyte nucleus after LPS stimulation. The mRNA expressions of interleukin 17-4 (CgIL17-4), CgIL17-5, and defensin (CgDefh1) in the hemocytes of CgSOCS6-knockdown oysters increased significantly (2.55-fold, 2.68-fold, 4.68-fold of that in EGFP-RNAi oysters, p < 0.05, p < 0.05, p < 0.001, respectively) after LPS stimulation. These findings suggested that CgSOCS6 was involved in the oyster immune response by regulating the expressions of CgIL17-4, CgIL17-5, and CgDefh1., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

C-type lectins (CTLs), as important pattern recognition receptors (PRRs), are a superfamily of Ca 2+ -dependent carbohydrate-recognition proteins which participate in nonself-recognition and eliminating pathogens. In the present study, a novel CTL (designated as CgCLec-3) was identified from the Pacific oyster Crassostrea gigas. There was only one carbohydrate-recognition domain (CRD) of 151 amino acid residues within the deduced amino acid sequence of CgCLec-3. The deduced amino acid sequence of CgCLec-3 CRD shared low homology with the CRDs of other CTLs in oyster with the identities ranging from 12% to 22%. A novel DIN motif was found in Ca 2+ -binding site 2 of CgCLec-3. The relative expression level of CgCLec-3 in hemocytes was up-regulated significantly after the stimulations of bacteria and Pathogen Associated Molecular Patterns (PAMPs). Immunohistochemistry assay showed that CgCLec-3 protein was mainly distributed in gill and mantle, less in gonad, and could not be detected in adductor muscle and hepatopancreas. The recombinant protein (rCgCLec-3) could bind lipopolysaccharide (LPS), mannose (MAN) and peptidoglycan (PGN), but not poly (I:C). rCgCLec-3 exihibited strong binding ability to Vibrio anguillarum and V. splendidus, moderate binding activities to Escherichia coli, Pichia pastoris and Yarrowia lipolytica, weak binding affinity to Staphylococcus aureus and Micrococcus luteus. rCgCLec-3 could agglutinate microorganisms, in a Ca 2+ -dependent manner and its activity to agglutinate V. splendidus was remarkably higher than that to agglutinate E. coli, S. aureus and P. pastoris. The phagocytic activity of oyster hemocytes was significantly enhanced after incubation with rCgCLec-3. rCgCLec-3 also exhibited antibacterial activity against E. coli and S. aureus. The results clearly suggested that CgCLec-3 in Pacific oyster C. gigas not only served as a PRR involved in the PAMPs recognition and microbes binding, but also functioned as an immune effector participating in the clearance of invaders., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Autophagy, a highly conserved intracellular degradation system, is involved in numerous processes in vertebrate and invertebrate, such as cell survival, ageing, and immune responses. However, the detailed molecular mechanism of autophagy and its immune regulatory role in bivalves are still not well understood. In the present study, an autophagy-related protein ATG10 (designated as CgATG10) was identified from Pacific oyster Crassostrea gigas. The open reading frame of CgATG10 cDNA was of 621 bp, encoding a polypeptide of 206 amino acid residues with an Autophagy&#95;act&#95;C domain (from 96 to 123 amino acid), which shared high homology with that from C. virginica and Octopus bimaculoides. The mRNA transcripts of CgATG10 were widely expressed in all the tested tissues including mantle, gonad, gills, hemocytes and hepatopancreas, with the highest expression level in mantle. After the stimulation with poly (I:C), the mRNA expression level of CgATG10 in the mantle of oysters was significantly up-regulated (4.92-fold of that in Blank group, p < 0.05), and the LC3-conversion from LC3-I to LC3-II (LC3-II/LC3-I) also increased. After an additional injection of dsRNA to knock-down the expression of CgATG10 (0.33-fold and 0.10-fold compared respectively with Blank group and dsGFP group, p < 0.05), the downstream conversion of CgLC3 was inhibited significantly compared with that of the control dsGFP group, while the expression level of autophagy-initiator CgBeclin1 did not change significantly. In addition, the mRNA transcripts of interferon regulatory factor CgIRF-1 increased significantly in CgATG10-knockdown oysters at 12 h post poly (I:C) stimulation. All the results indicated that CgATG10 might participate in the immune response against poly (I:C) by regulating autophagosome formation and interferon system in oysters., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Lectins are carbohydrate-binding proteins with lectin domains, which are extensively studied for their numerous roles in biological recognition. However, the lectin domain containing proteins (LDCPs) chimerized with other non-lectin domains have not received sufficient attention. In the present study, a genome-wide survey of LDCPs in oyster Crassostrea gigas was conducted, and an expansive 640 LDCPs derived from ten lectin domains were identified and functionally explored. In these LDCPs, a total of 282 kinds of domains were predicted, and 90% of the LDCPs contained more than one kind of domain. The lectin domains were frequently fused with non-lectin domains, such as epidermal growth factor domain and peptidase related domains, which supplied LDCPs with more diversity in structures and functions. The C-type lectin domains were the most abundant domains in LDCPs, and they were largely co-existed with non-lectin domains of complement activation-related domains (such as CUB domain and PAN-1 domain) but relative independence with other lectin domains. Furthermore, the C-type lectin domain containing proteins (CTLPs) found to mainly act as pattern immune recognition receptors and were highly expressed in mucosal tissues (digestive gland, male gonad and labial palp) to provide mucosal immune protections. The Concanavalin A-like lectin domains were the second richest domains in LDCPs, and they were mostly constructed into chimeric proteins with epidermal growth factor domain and peptidase related domains. The Concanavalin A-like lectin domain containing proteins (CALPs) were significantly enriched with peptidase activities and mainly expressed in digestive tissues. All the results suggested the mucosal immunity and digestive functions of oyster LDCPs, which provided a fresh idea about the functions of invertebrate lectin family., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

As one of the most important neuropeptides identified only in invertebrates of Mollusca, Annelida and Arthropoda, FMRFamide (Phe-Met-Arg-Phe-NH 2 ) involves in multiple physiological processes, such as mediating cardiac frequency and contraction of somatic and visceral muscles. However, its modulatory role in the immune defense has not been well understood. In the present study, an FMRFamide precursor (designed as CgFMRFamide) was identified in oyster Crassostrea gigas, which could be processed into nineteen FMRFamide peptides. Phylogenetic analysis revealed that CgFMRFamide shared high similarity with other identified FMRFamides in mollusks. The mRNA of CgFMRFamide was mainly concentrated in the tissues of visceral ganglia, hepatopancreas and hemocytes, and a consistent distribution of FMRFamide peptide was confirmed by immunohistochemistry and immunocytochemistry assays. The mRNA expression level of CgFMRFamide in hemocytes was significantly up-regulated after immune stimulation with lipopolysaccharide (LPS). After the concentration of FMRFamide was increased by exogenous injection, the in vivo expressions of pro-inflammatory cytokine CgIL17-5, as well as the apoptosis-related CgCaspase-1 and CgCaspase-3 in hemocytes were promptly increased (p < 0.05), but the concentration of signal molecule nitric oxide (NO) was significantly down-regulated (p < 0.05). Meanwhile, an increased phosphorylation of p38 MAP kinase in hemocytes was also detected after the FMRFamide injection. These results collectively demonstrated that the conserved FMRFamide could not only respond to immune stimulation, but also regulate the expression of immune effectors and apoptosis-related genes, which might be mediated by p38 MAP kinase pathway, thereby effectively involved in clearing pathogens and maintaining homeostasis in oysters., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

A variety of combinations of leucine-rich repeat (LRR) and immunoglobulin-like (Ig) domains have been found and discovered in invertebrates and vertebrates, but the functions remain largely unexplored. In the present study, a novel LRR and Ig domain-containing protein (LRRIG), CgLRRIG-3, was identified and characterized from oyster Crassostrea gigas. It contained two typical LRR motifs, a LRRNT motif and an Ig domain and PSI-BALST and phylogeny analysis revealed that the sequence of CgLRRIG-3 was most related with leucine-rich repeat neuronal 1 proteins from vertebrate. Its mRNA transcripts were constitutively expressed in muscle, gill, hepatopancreas, mantle, gonad and hemocytes with the highest level in hepatopancreas. The mRNA expression level of CgLRRIG-3 in hemocytes could respond to the stimulations of variety PAMPs including lipopolysaccharide (LPS), peptidoglycan (PGN), glucan (GLU) and polyinosinic-polycytidylic acid (poly I:C). The recombinant proteins exhibited a wide PAMP binding repertoire to four typical PAMPs and could significantly induce the expression of CgTNF-1 and CgIL17-5 as well as increase phagocytosis in primary cultured oyster hemocytes. In hepatopancreas, CgLRRIG-3 was mainly distributed in the basolateral membrane of digestive tubule and the hemocoel sinusoid between the digestive tubules. And in hemocytes, the positive signal was mainly distributed in a special group of granulocytes. These results collectively indicated that CgLRRIG-3 could not only function as an immune effector., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Granulocytes are known as the main immunocompetent hemocytes that play important roles in the immune defense of oyster Crassostrea gigas. In the present study, an alcohol acyltransferase (designed as CgAATase) with specific expression pattern was identified from oyster C. gigas, and it could be employed as a potential marker for the isolation of oyster granulocytes. The open reading frame (ORF) of CgAATase was of 1431 bp, encoding a peptide of 476 amino acids with a typically conserved AATase domain. The mRNA transcripts of CgAATase were highest expressed in hemocytes, lower expressed in hepatopancreas, mantle, gonad, gill, ganglion, adductor muscle, and labial palp. The mRNA expression level of CgAATase in hemocytes was significantly up-regulated at 3-12 h and reached the highest level (27.40-fold compared to control group, p < 0.05) at 6 h after Vibrio splendidus stimulation. The total hemocytes were sorted as granulocytes, semi-granulocytes and agranulocytes by Percoll ® density gradient centrifugation. CgAATase transcripts were dominantly observed in granulocytes, which was 8.26-fold (p < 0.05) and 2.80-fold (p < 0.05) of that in agranulocytes and semi-granulocytes, respectively. The monoclonal antibody against CgAATase was produced and employed for the isolation of granulocytes with the immunomagnetic bead. CgAATase protein was mainly detected on the cytomembrane of granulocytes. About 85.7 ± 4.60% of the granulocytes were positive for CgAATase and they could be successfully separated by flow cytometry with immunomagnetic bead coated with anti-CgAATase monoclonal antibody, and 97.7 ± 1.01% of the rest hemocytes (agranulocytes and semi-granulocytes) were negative for CgAATase. The isolated primary granulocytes could maintain cell activity for more than one week in vitro culture that exhibited numerous filopodia. These results collectively suggested that CgAATase was a potential marker of oyster granulocytes, and the granulocytes could be effectively isolated from total circulating hemocytes by immunomagnetic bead coated with the anti-CgAATase monoclonal antibody., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The globular C1q domain containing (C1qDC) proteins are a family of versatile pattern recognition receptors (PRRs) to bind various ligands by their globular C1q (gC1q) domain. In the present study, a novel globular C1qDC (CgC1qDC-7) was characterized from Pacific oyster Crassostrea gigas. The open reading frame of CgC1qDC-7 was of 555 bp, encoding a polypeptide of 185 amino acids. Phylogenetic analysis indicated that CgC1qDC-7 shared high homology with C1qDCs from Crassostrea virginica, Mytilus galloprovincialis, and Mizuhopecten yessoensis. The mRNA transcripts of CgC1qDC-7 were widely expressed in all the tested tissues including mantle, gonad, gills, adductor muscle, hemocytes, hepatopancreas and labial palps, with the highest expression level in hemocytes and gills. The recombinant protein of CgC1qDC-7 (rCgC1qDC-7) exhibited binding activity towards Gram-negative bacteria (Vibrio splendidus, V. anguillarum, Escherichia coli, V. alginolyticus, and Aeromonas hydrophila), Gram-positive bacteria (Micrococcus luteus and Staphylococcus aureus) and fungi (Pichia pastoris and Yarrowia lipolytica), and displayed strongest binding affinity towards Gram-negative bacteria V. splendidus and V. anguillarum. It also exhibited affinity to vital pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS), peptidoglycan (PGN), mannan (MAN) and Poly (I:C) with high affinity towards LPS and PGN, and low affinity to MAN and Poly (I:C). These results collectively indicated that CgC1qDC-7 was a novel PRR in C. gigas with high binding affinity towards LPS and PGN as well as Gram-negative bacteria., (Copyright © 2018 Elsevier Ltd. All rights reserved.)