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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.)

Invertebrate lectins exhibit structural diversity and play crucial roles in the innate immune responses by recognizing and eliminating pathogens. In the present study, a novel lectin containing a Gal&#95;Lectin, a CUB and a transmembrane domain was identified from the Pacific oyster Crassostrea gigas (defined as CgGal-CUB). CgGal-CUB mRNA was detectable in all the examined tissues with the highest expression in adductor muscle (11.00-fold of that in haemocytes, p < 0.05). The expression level of CgGal-CUB mRNA in haemocytes was significantly up-regulated at 3, 24, 48 and 72 h (8.37-fold, 12.13-fold, 4.28-fold and 10.14-fold of that in the control group, respectively) after Vibrio splendidus stimulation. The recombinant CgGal-CUB (rCgGal-CUB) displayed binding capability to Mannan (MAN), peptidoglycan (PGN), D-(+)-Galactose and L-Rhamnose monohydrate, as well as Gram-negative bacteria (Escherichia coli, V. splendidus and Vibrio anguillarum), Gram-positive bacteria (Micrococcus luteus, Staphylococcus aureus, and Bacillus sybtilis) and fungus (Pichia pastoris). rCgGal-CUB was also able to agglutinate V. splendidus, and inhibit V. splendidus growth. Furthermore, rCgGal-CUB exhibited the activities of enhancing the haemocyte phagocytosis towards V. splendidus, and the phagocytosis rate of haemocytes was descended in blockage assay with CgGal-CUB antibody. These results suggested that CgGal-CUB served as a pattern recognition receptor to bind various PAMPs and bacteria, and enhanced the haemocyte phagocytosis towards V. splendidus., (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.)

The interactions induced by RIP homotypic interaction motif (RHIM) are essential for the activation of inflammatory signaling and certain cell death pathways. In the present study, a RHIM-containing protein was identified from Pacific oyster Crassostrea gigas, which harbored a RHIM domain and a Death domain (designated CgRHIM-containing protein). The mRNA transcripts of CgRHIM-containing protein were constitutively expressed in all the examined tissues of oysters, with the highest expression level in mantle. The CgRHIM-containing protein was mainly distributed in the cytoplasm of oyster haemocytes. After high temperature stress, the expression levels of CgRel and CgBcl-2 increased significantly, and reached the peak level at 12 h, then decreased gradually. The transcripts of CgRHIM-containing protein, Cgcaspase-8 and Cgcaspase-3 in haemocytes up-regulated at 12 h after high temperature stress. Moreover, the protein abundance of CgRHIM-containing protein increased significantly, and the ubiquitination level of CgRHIM-containing protein in haemocytes showed an increasing trend at first and then decreased. After the expression of CgRHIM-containing protein was knocked down by siRNA, the mRNA expression levels of CgRel and CgBcl-2 decreased significantly at 6 h after high temperature stress, and those of CgFADD-like, Cgcaspase-8 and Cgcaspase-3, as well as the apoptosis rate of haemocytes also decreased significantly at 24 h. These results indicated that CgRHIM-containing protein might regulate haemocyte apoptosis in oysters upon high temperature stress via mediating the expression of Rel, Bcl-2 and caspase-8/3., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Infections with pathogenic Vibrio strains are associated with high summer mortalities of Pacific oysters Magalana (Crassostrea) gigas, affecting production worldwide. This raises the question of how M. gigas cultures can be protected against deadly Vibro infection. There is increasing experimental evidence of immune priming in invertebrates, where previous exposure to a low pathogen load boosts the immune response upon secondary exposure. Priming responses, however, appear to vary in their specificity across host and parasite taxa. To test priming specificity in the Vibrio - M. gigas system, we used two closely related Vibrio splendidus strains with differing degrees of virulence towards M. gigas. These V. splendidus strains were either isolated in the same location as the oysters (sympatric, opening up the potential for co-evolution) or in a different location (allopatric). We extracted cell-free haemolymph plasma from infected and control oysters to test the influence of humoral immune effectors on bacterial growth in vitro. While addition of haemolypmph plasma in general promoted growth of both strains, priming by an exposure to a sublethal dose of bacterial cells lead to inhibitory effects against a subsequent challenge with a potentially lethal dose in vitro. Inhibitory effects and immune priming was strongest when oysters had been primed with the sympatric Vibrio strain, but inhibitory effects were seen both when challenged with the sympatric as well as against allopatric V. splendidus, suggesting some degree of cross protection. The stronger immune priming against the sympatric strain suggests that priming could be more efficient against matching local strains potentially adding a component of local adaptation or co-evolution to immune priming in oysters. These in vitro results, however, were not reflected in the in vivo infection data, where we saw increased bacterial loads following an initial challenge. This discrepancy might suggests that that it is the humoral part of the oyster immune system that produces the priming effects seen in our in vitro experiments., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Norepinephrine (NE) is involved in regulating cytokine expression and phagocytosis of immune cells in the innate immunity of vertebrates. In the present study, the modulation mechanism of NE on the biosynthesis of TNFs in oyster granulocytes was explored. The transcripts of CgTNF-1, CgTNF-2 and CgTNF-3 were highly expressed in granulocytes, and they were significantly up-regulated after LPS stimulation, while down-regulated after NE treatment. The phagocytic rate and apoptosis index of oyster granulocytes were also triggered by LPS stimulation and suppressed by NE treatment. The mRNA expressions of CgMAPK14 and CgRelish were significantly induced after NE treatment, and the translocation of CgRelish from cytoplasm to nucleus was observed. The concentration of intracellular Ca 2+ in granulocytes was significantly up-regulated upon NE incubation, and this trend reverted after the treatment with DOX (specific antagonist for NE receptor, CgA1AR-1). No obvious significance was observed in intracellular cAMP concentrations in the PBS, NE and NE + DOX groups. Once CgA1AR-1 was blocked by DOX, the mRNA expressions of CgMAPK14 and CgRelish were significantly inhibited, and the translocation of CgRelish from cytoplasm to nucleus was also dramatically suppressed, while the mRNA expression of CgTNF-1 and the apoptosis index increased significantly to the same level with those in LPS group, respectively. These results collectively suggested that NE modulated TNF expression in oyster granulocyte through A1AR-p38 MAPK-Relish signaling pathway., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Tropomyosin was reported as an important allergen in Crassostrea angulata and designated as Cra a 1. The localization of the T cell epitopes and the reduction of the immunoreactivity of Cra a 1 are still lacking. In this study, four T cell epitopes were identified by using wild-type Cra a 1 (wtCra a 1)-immunized mouse splenocytes cultured with synthetic peptides. The immunoreactivity was maintained after chemical denaturation treatment, indicating that the linear epitope is an immunodominant epitope of wtCra a 1. Furthermore, the hypoallergenic derivative (mCra a 1) was developed by the deletion of linear B cell epitopes and retention of T cell epitopes. mCra a 1 could stimulate CD4 + T cell proliferation and upregulate interleukin-10 secretion. Overall, basophil activation by mCra a 1 was low, but its ability to induce T cell proliferation was retained, suggesting that mCra a 1 may serve as a viable candidate for treating oyster allergy.

Phagocytosis is a major cellular mechanism for mollusk granulocytes to eliminate nonself substances and dead cells, and thus to preserve the immune homeostasis. The knowledge of the regulatory mechanisms controlling phagocytic capacity is vital to understanding the immune system. In the present study, an ATF3 homolog (CgATF3) with a typical bZIP domain was identified in the Pacific oyster Crassostrea gigas. Its highly conserved bZIP domain consisted of two structural features, a basic region for DNA binding and a leucine zipper region for dimerization. Its transcript was found to be abundantly expressed in haemocytes, which was induced by Vibrio splendidus stimulation and recombinant CgTNF-2 treatment, along with an increase of its protein content in the nucleus. Moreover, CgATF3 showed a consistent and specific high expression in granulocytes, and CgATF3 + granulocytes were characterized morphologically by the largest diameter, smaller nucleus to cytoplasmic ratio, and abundant cytoplasmic granules, and functionally by a higher capacity for phagocytosis. When CgATF3 expression was inhibited by RNAi, the expression levels of CgRab1, CgRab33 and CgCathepsin L1, as well as the phagocytic rate and index of granulocytes all decreased after V. splendidus stimulation. These results together demonstrated the involvement of CgATF3 in regulating the expressions of Rabs and Cathepsin L1, as well as the phagocytosis of granulocytes in oyster C. gigas., (Copyright © 2024. Published by Elsevier Ltd.)

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.)

RIPK1/TAK1 are important for programmed cell death, including liver death, necroptosis and apoptosis. However, there have been few published reports on the functions of RIPK1/TAK1 in invertebrates. In this study, full-length ChRIPK1 and ChTAK1 were cloned from C. hongkongensis through the rapid amplification of cDNA ends (RACE) technology. ChRIPK1 has almost no homology with human RIPK1 and lacks a kinase domain at the N-terminus but has a DD and RHIM domain. ChTAK1 is conserved throughout evolution. qRT‒PCR was used to analyze the mRNA expression patterns of ChRIPK1 in different tissues, developmental stages, and V. coralliilyticus-infected individuals, and both were highly expressed in the mantle and gills, while ChRIPK1 was upregulated in hemocytes and gills after V. coralliilyticus or S. aureus infection, which indicates that ChRIPK1 is involved in immune regulation. Fluorescence assays revealed that ChRIPK1 localized to the cytoplasm of HEK293T cells in a punctiform manner, but the colocalization of ChRIPK1 with ChTAK1 abolished the punctiform morphology. In the dual-luciferase reporter assay, both ChRIPK1 and ChRIPK1-RIHM activated the NF-κB signaling pathway in HEK293T cells, and ChTAK1 activated ChRIPK1 in the NF-κB signaling pathway. The apoptosis rate of the hemocytes was not affected by the necroptosis inhibitor Nec-1 but was significantly decreased, and ChRIPK1 expression was knocked down in the hemocytes of C. hongkongensis. These findings indicated that ChRIPK1 induces apoptosis but not necroptosis in oysters. This study provides a theoretical basis for further research on the molecular mechanism by which invertebrates regulate the programmed cell death of hemocytes in oysters., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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.)

The exosomal miRNA plays a crucial role in the intercellular communication response to environmental stress and pathogenic stimulation. In the present study, the expression of exosomal miRNAs in the Pacific oyster Crassostrea gigas after high-temperature stress or Vibrio splendidus stimulation was investigated through high-throughput sequencing. The exosomes were identified to be teardrop-like vesicles with the average size of 81.7 nm by transmission electron microscopy. There were 66 known miRNAs and 33 novel miRNAs identified, of which 10 miRNAs were differentially expressed after both high-temperature stress and Vibrio stimulation compared to the control group. A total of 1868 genes were predicted as the putative targets of miRNAs, of which threonine aspartase 1-like was targeted by the highest number of related miRNAs. The robustness and reliability of miRNA expression from the sRNA sequencing data were verified by employing eight miRNAs for qPCR. GO and KEGG clustering analyses revealed that apoptosis was significantly enriched by the target genes of differentially expressed exosomal miRNAs after high-temperature stress, and autophagy and cytokine activity were significantly enriched after Vibrio stimulation. Energy metabolism was found to be significantly shared in the target gene enrichments after both high-temperature stress and Vibrio stimulation. These findings would improve our understanding of the regulatory mechanisms of exosomal miRNAs in C. gigas after high-temperature stress or Vibrio stimulation., 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 © 2024. Published by Elsevier Ltd.)

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.)

Prohibitin2 (PHB2) is recently identified as a novel inner membrane mitophagy receptor to mediate mitophagy. In the present study, the function of CgPHB2 in mediating mitophagy in response to Vibrio splendidus stimulation was investigated in Crassostrea gigas. CgPHB2 protein was mainly distributed in the cytoplasm of three subpopulations of haemocytes. After V. splendidus stimulation, the expressions of CgPHB2 mRNA in haemocytes were up-regulated significantly at 6, 12 and 24 h, and the abundance of CgPHB2 protein was also enhanced at 12-24 h compared to control group. Furthermore, the green signals of CgPHB2 were colocalized respectively with the red signals of mitochondria and CgLC3 in the haemocytes at 12 h after V. splendidus stimulation, and the co-localization value of CgPHB2 and mtphagy Dye was significantly increased. The direct interaction between CgPHB2 and CgLC3 was simulated by molecular docking. In PHB2-inhibitor Fluorizoline-treated oysters, the mRNA expressions of mitophagy-related genes and the ratio of mitophagy were significantly decreased in haemocytes of oysters after V. splendidus stimulation. All the results collectively suggested that CgPHB2 participated in mediating the haemocyte mitophagy in the antibacterial immune response of oysters., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier Ltd.)

Trace amine-associated receptors (TAARs) are a class of G protein-coupled receptors, playing an immunomodulatory function in the neuroinflammatory responses. In the present study, a TAAR homologue with a 7tm&#95;classA&#95;rhodopsin-like domain (designated as CgTAAR1L) was identified in oyster Crassostrea gigas. The abundant CgTAAR1L transcripts were detected in visceral ganglia and haemocytes compared to other tissues, which were 55.35-fold and 32.95-fold (p < 0.01) of those in adductor muscle, respectively. The mRNA expression level of CgTAAR1L in haemocytes significantly increased and reached the peak level at 3 h after LPS or Poly (I:C) stimulation, which was 4.55-fold and 12.35-fold of that in control group, respectively (p < 0.01). After the expression of CgTAAR1L was inhibited by the injection of its targeted siRNA, the mRNA expression levels of interleukin17s (CgIL17-1, CgIL17-5 and CgIL17-6), and defensin (Cgdefh1) significantly decreased at 3 h after LPS stimulation, which was 0.51-fold (p < 0.001), 0.39-fold (p < 0.01), 0.48-fold (p < 0.05) and 0.41-fold (p < 0.05) of that in the control group, respectively. The nuclear translocation of Cgp65 protein was suppressed in the CgTAAR1L-RNAi oysters. Furthermore, the number of Vibrio splendidus in the haemolymph of CgTAAR1L-RNAi oysters significantly increased (4.11-fold, p < 0.001) compared with that in the control group. In contrast, there was no significant difference in phagocytic rate of haemocytes to V. splendidus in the CgTAAR1L-RNAi oysters. These results indicated that CgTAAR1L played an important role in the immune defense against bacterial infection by inducing the expressions of interleukin and defensin., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Interferon regulatory factor 8 (IRF8) is an important transcriptional regulatory factor involving in multiple biological process, such as the antiviral immune response, immune cell proliferation and differentiation. In the present study, the involvement of a previously identified IRF8 homologue (CgIRF8) in regulating haemocyte proliferation of oyster were further investigated. CgIRF8 mRNA transcripts were detectable in all the stages of C. gigas larvae with the highest level in D-veliger (1.76-fold of that in zygote, p < 0.05). Its mRNA transcripts were also detected in all the three haemocyte subpopulations of adult oysters with the highest expression in granulocytes (2.79-fold of that in agranulocytes, p < 0.01). After LPS stimulation, the mRNA transcripts of CgIRF8 in haemocytes significantly increased at 12 h and 48 h, which were 2.04-fold and 1.65-fold (p < 0.05) of that in control group, respectively. Meanwhile, the abundance of CgIRF8 protein in the haemocytes increased significantly at 12 h after LPS stimulation (1.71-fold of that in seawater, p < 0.05). The immunofluorescence assay and Western blot showed that LPS stimulation induced an obvious nucleus translocation of CgIRF8 protein in haemocytes. After the expression of CgIRF8 was inhibited by the injection of CgIRF8 siRNA, the percentage of EdU positive haemocytes, the proportion of granulocytes, and the mRNA expression levels of CgGATA and CgSCL all declined significantly at 12 h after LPS stimulation, which was 0.64-fold (p < 0.05), 0.7-fold (p < 0.05), 0.31-fold and 0.54-fold (p < 0.001) of that in the NC group, respectively. While the expression level of cell proliferation-related protein CgCDK2, CgCDC6, CgCDC45 and CgPCNA were significantly increased (1.99-fold, and 2.41-fold, 3.76-fold and 4.79-fold compared to that in the NC group respectively, p < 0.001). Dual luciferase reporter assay demonstrated that CgIRF8 was able to activate the CgGATA promoter in HEK293T cells after transfection of CgGATA and CgIRF8. These results collectively indicated that CgIRF8 promoted haemocyte proliferation by regulating the expression of CgGATA and other related genes in the immune response of oyster., (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.)

Oyster culture is a sustainable solution to food production. However, this activity can be severely impacted by the presence and proliferation of harmful microalgae such as the benthic dinoflagellates Prorocentrum hoffmannianum and Ostreopsis cf. ovata. This study aimed to evaluate the in vitro effects of P. hoffmannianum and O. cf. ovata on immune system cells (hemocytes) of the native cultured oyster Crassostrea gasar. The direct toxicity of both dinoflagellates was first evaluated assessing hemocyte viability exposed to eight concentrations of each HAB species. No reduction in hemocyte viability was found with the exposure to cell culture or the crude extract of P. hoffmannianum, but O. cf. ovata culture induced hemocyte death in a concentration-dependent manner. Ostreopsis cf. ovata concentration that promoted half of maximal reduction in hemocyte viability (EC 50 ) was 779 cells mL -1 . Posteriorly, hemocytes were exposed to both dinoflagellate cells and crude extracts to investigate their effects on hemocyte functional parameters. Despite no direct toxicity of the dinoflagellate cells, P. hoffmannianum extract caused a threefold increase in ROS production and decreased the phagocytosis rate by less than half. Ostreopsis cf. ovata cells and crude extracts also triggered an increase in ROS production (two-fold), but the phagocytosis rate was reduced (by half) only in response to the two lower cell concentrations. These results indicate a harmful potential of both dinoflagellates through a direct toxicity (only for O. cf. ovata) and functional impairment of hemocytes (both species) which could expose C. gasar oyster to opportunistic infections., 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 © 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.)

Introduction: The culture of Pacific oysters ( Crassostrea gigas ) is of significant socio-economic importance in the U.S. Pacific Northwest and other temperate regions worldwide, with disease outbreaks acting as significant bottlenecks to the successful production of healthy seed larvae. Therefore, the current study aims to describe the mechanisms of a probiotic combination in improving the survival of C. gigas larvae. Specifically, we investigate changes in C. gigas larval gene expression in response to V. coralliilyticus infection with or without a pre-treatment of a novel probiotic combination., Methods: Treatment groups consisted of replicates of Pacific oyster larvae exposed to a) a combination of four probiotic bacteria at a total concentration of 3.0 x 10 5 CFU/mL at 18 hours post-fertilization (hpf), b) pathogenic V. coralliilyticus RE22 at a concentration of 6.0 x 10 3 CFU/mL at 48 hpf, and c) the probiotic combination at 18 hpf and V. coralliilyticus RE22 at 48 hpf. RNA was extracted from washed larvae after 72 hpf, and transcriptome sequencing was used to identify significant differentially expressed genes (DEGs) within each treatment., Results: Larvae challenged with V. coralliilyticus showed enhanced expression of genes responsible for inhibiting immune signaling (i.e., TNFAIP3 , PSMD10 ) and inducing apoptosis (i.e., CDIP53 ). However, when pre-treated with the probiotic combination, these genes were no longer differentially expressed relative to untreated control larvae. Additionally, pre-treatment with the probiotic combination increased expression of immune signaling proteins and immune effectors (i.e., IL-17 , MyD88 ). Apparent immunomodulation in response to probiotic treatment corresponds to an increase in the survival of C. gigas larvae infected with V. coralliilyticus by up to 82%., Discussion: These results indicate that infection with V. coralliilyticus can suppress the larval immune response while also prompting cell death. Furthermore, the results suggest that the probiotic combination treatment negates the deleterious effects of V. coralliilyticus on larval gene expression while stimulating the expression of genes involved in infection defense mechanisms., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Hesser, Mueller, Langdon and Schubiger.)

Based on previous reports，toll-like receptors (TLRs) are recognition molecules common in various aquatic animals and play a vital role in innate immunity. In this study, a novel TLR CgToll-3 with leucine-rich repeats (LRRs) and a TIR (Toll-interleukin 1-resistance) domain was cloned in Crassostrea gigas. CgToll-3 with sixteen potential extracellular N-linked glycosylation sites and shares the closest phylogenic relationship with molluscan TLRs. Alignment of LRRs and TIR domains indicated that CgToll-3 was highly conserved compared to other LRRs of mollusks which could respond against Vibrio or other bacterial molecules, and contained three conserved functionally important motifs (Box 1, Box 2, and Box 3). The Hex Molecular Docking result showed that CgToll-3 could interact with CgMyd88 via the TIR domain. Subcellular Co-localization and BiFC Assay confirmed this interaction, and they could induce NF-κB activation. CgToll-3 was moderately expressed in the digestive gland, and its expression level was significantly up-regulated after Vibrio alginolyticus challenge. Taken together, CgToll-3 might be involved in the innate immune response to V. alginolyticus for C. gigas through a MyD88-dependent TLR mediated signaling pathway., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Oyster is the worldwide aquaculture molluscan and evolves a complex immune defense system, with hemocytes as the major immune system for its host defense. However, the functional heterogeneity of hemocyte has not been characterized, which markedly hinders our understanding of its defense role. Here, we used the single-cell transcriptome profiling (scRNA-seq), which provides a high-resolution visual insight into its dynamics, to map the hemocyte and assess its heterogeneity in a molluscan oyster Crassostrea hongkongensis. By combining with the cell type specific RNA-seq, thirteen subpopulations belonging to granulocyte, semi-granulocyte, and hyalinocyte were revealed. The granulocytes mainly participated in immune response and autophagy process. Pseudo-temporal ordering of granulocytes identified two different cell-lineages. The hematopoietic transcription factors regulated networks controlling their differentiations were also identified. We further identified one subpopulation of granulocytes in immune activate states with the cell cycle and immune responsive genes expressions, which illustrated the functional heterogeneity of the same cell type. Collectively, our scRNA-seq analysis demonstrated the hemocytes diversity of molluscans. The results are important in our understanding of the immune defense evolution and functional differentiation of hemocytes in Phylum Mollusca., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Oysters are an excellent biomonitor of coastal pollution and the hyper-accumulator of toxic metals such as copper and zinc (Zn). One unique feature of molluscs is their hemocytes which are mainly involved in immune defenses. Different subpopulations of hemocytes have been identified, but their functions in metal transport and detoxification are not clear. In this study, we examined the immune responses of different subpopulations of oyster Crassostrea hongkongensis hemocytes under different periods of Zn exposure by using flow cytometer and confocal microscopy. In vitro exposure to Zn resulted in acute immune responses by increasing the reactive oxygen species (ROS) production and phagocytosis and decreased number of granulocytes and mitochondrial membrane potential (MMP) within 3 h. Granulocyte mortality and lysosomal pH increased whereas glutathione (GSH) decreased within 1 h of in vitro exposure, indicating the immune stimulation of granulocytes. Within the first 7 days of in vivo exposure, immunocompetence of granulocytes was inhibited with increasing granulocyte mortality but decreasing ROS production and phagocytosis. However, with a further extension of Zn exposure to 14 days, both phagocytosis and lysosomal content increased with an increasing number of granulocytes, indicating the increase of hemocyte-mediated immunity. Our study demonstrated that granulocytes played important roles in oyster immune defenses while other subpopulations may also participate in immune functions. The degranulation and granulation due to transition between semigranulocytes and granulocytes after Zn exposure were important in metal detoxification. The study contributed to our understanding of the immune phenomena and the adaptive capability of oysters in metal contaminated environments., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Salinity in the oceans is changing due to climate change and global warming. Intense rainfalls and freshwater runoff decrease salinity along the coastal areas. In contrast, intense drought seasons and river damming have certainly increased salinity in lagoons and estuaries. Few studies have focused on aspects of the biology and culture of oyster Crassostrea corteziensis, but until now, physiological and immunological responses in this species have not been assessed under acute hypo- and hypersaline stress conditions. Oysters obtained from a local farm were acclimated for three weeks in laboratory conditions. To avoid closure of oyster valves during salinity induced-stress conditions, a notch was done on each organism shell not only to facilitate oyster tissue exposure to rearing water but also for sampling hemolymph. Oysters (N = 180) were abruptly exposed to three salinity treatments: (HO) hypo-, (C) control, and (HP) hypersaline stress conditions (10, 35, and 50 PSU, respectively). Four oysters per treatment were sampled at 1, 2, 3, 6, 12, 24, and 48 h after exposure. Hemolymph osmolality, water content and total protein concentration in tissues, metabolic and immune responses were assessed for each organism. Oyster survival was not different among treatments and was maintained above 96% at the end of the experimental trial. Hemolymph osmolality reached the value of rearing water at 6 and 48 h of exposure to HP and HO stress conditions, where oysters exposed to salinity increase showed less resilience than those to decrease. Higher glucose levels in plasma and lower ones of hemocyanin were assessed in the oysters exposed to HP compared to HO conditions, suggesting more stressful conditions or susceptibility of oysters during salinity increase. Total hemocyte (THC), hyalinocyte (HC), and granulocyte (GC) counts decreased in oysters exposed to HP condition, while total and differential hemocyte counts were similar among oysters exposed to HO and control conditions. Despite hemocyte phagocytosis was not different among treatments, viability decreased in those exposed to HP condition. Contrastingly, superoxide anion (SOA) production (oxidative capacity) increased in oysters exposed to both induced salinity-stress conditions, which suggest susceptibility increase in oysters, particularly during salinity increase. The results show that HP condition is particularly stressful for C. corteziensis. In turn, this condition could increase both their vulnerability to other environmental stressors, such as temperature and/or acidification or susceptibility to opportunistic pathogenic microorganisms that cause the most common oyster diseases., (Copyright © 2021 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.)

The ancient origin of the lectin pathway of the complement system can be traced back to protochordates (such as amphioxus and tunicates) by the presence of components such as ficolin, glucose-binding lectin, mannose-binding lectin-associated serine protease (MASP), and C3. Evidence for a more primitive origin is offered in the present study on the Pacific oyster Crassostrea gigas. C3 protein in C. gigas (CgC3) was found to be cleaved after stimulation with the bacteria Vibrio splendidus. In addition, we identified a novel C-type lectin (defined as CgCLec) with a complement control protein (CCP) domain, which recognized various pathogen-associated molecular patterns (PAMPs) and bacteria. This protein was involved in the activation of the complement system by binding CgMASPL-1 to promote cleavage of CgC3. The production of cytokines and antibacterial peptides, as well as the phagocytotic ratio of haemocytes in CgCLec-CCP-, CgMASPL-1-, or CgC3-knockdown oysters, decreased significantly after V. splendidus stimulation. Moreover, this activated CgC3 participated in perforation of bacterial envelopes and inhibiting survival of the infecting bacteria. These results collectively suggest that there existed an ancient lectin pathway in molluscs, which was activated by a complement cascade to regulate the production of immune effectors, phagocytosis, and bacterial lysis., Competing Interests: Conflict of interest No potential conflict of interest was reported by the author(s)., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Calmodulin (CaM) is a highly conserved second messenger protein transducing calcium signals by binding and modulating intracellular calcium ions (Ca 2+ ), and involves in the Ca 2+ -dependent physical processes including host defense in vertebrates. In the present study, a CaM homologue (designated as CgCaM) was identified from Pacific oyster Crassostrea gigas. The open reading frame of CgCaM cDNA was of 471 bp encoding a polypeptide of 156 amino acid residues. There were four EFh domains predicted in CgCaM, which shared high homologies with those in CaMs from oyster C. virginica and other invertebrates. The mRNA transcripts of CgCaM were constitutively expressed in all the tested tissues including labellum, mantle, gonad, gills, adductor muscle, haemocytes and hepatopancreas, with the highest expression level in haemocytes. The mRNA expression level of CgCaM in haemocytes decreased significantly (0.31-fold of that in blank, p < 0.05) at 3 h after LPS stimulation, while the intracellular Ca 2+ (1.57-fold of that in blank, p < 0.05) and the mRNA expression of cytokine CgIL17-1 (4.87-fold of that in blank, p < 0.05) both increased in haemocytes. Meanwhile, an oyster miRNA scaffold659&#95;26519 was identified, and it was proved to target the 3'-untranslated regions (3'-UTR) of CgCaM mRNA by luciferase reporter assay. The expression of scaffold659&#95;26519 increased significantly at 3 h (43.523-fold of that of blank, p < 0.05) and 6 h (55.91-fold of that of blank, p < 0.05) after LPS stimulation. When the expression of scaffold659&#95;26519 was inhibited by transfection with its inhibitor in vitro, the expression of CgIL17-1 declined significantly to 0.58-fold of that in LPS stimulation group. These findings indicated that the miRNA scaffold659&#95;26519 targeted CaM was involved in the early inflammatory response of oyster immunity, and provided a new evidence for CaM-mediated immune mechanism in molluscs., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Interferons (IFNs) are the key coordinators of antiviral immunity by binding to their receptors to orchestrate a complex transcriptional network in vertebrates. Recently, the existence of molluscan IFN-like system has been certified by the identification of important components in IFN system, such as IFN-like protein (CgIFNLP) from oyster Crassostrea gigas. In the present study, a novel CgIFNLP receptor (designed CgIFNLPR-1) was identified from C. gigas. The open reading frame (ORF) of CgIFNLPR-1 cDNA was of 1962 bp encoding a peptide of 653 amino acid residues with five fibronectin type III (FNIII) domains and one transmembrane helix region. The mRNA transcripts of CgIFNLPR-1 were constitutively distributed in all the tested tissues, with the highest level in gonad. After Poly (I:C) stimulation, the mRNA expression of CgIFNLPR-1 in haemocytes was significantly up-regulated to the highest level at 48 h (4.54-fold of that in control group, p < 0.05). CgIFNLPR-1 protein was mainly distributed in the cytoplasm and membrane of oyster haemocytes. CgIFNLP and CgIFNLPR-1 were able to interact with each other in vitro. After the CgIFNLPR-1 was knocked down by RNAi, the mRNA expression of IFN-stimulated genes (ISGs), including CgMx, CgViperin and CgIFNIP-44, were significantly inhibited after Poly (I:C) stimulation, which was 0.17, 0.31 and 0.53-fold of that in EGFP group, respectively (p < 0.01). These findings suggested that CgIFNLPR-1 was a novel CgIFNLP receptor in the oyster to recognize CgIFNLP and regulate the expressions of CgISGs., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Oyster is a common food that causes allergy. However, little information is available about its allergens and cross-reactivity. In this study, arginine kinase (AK) was identified as a novel allergen in Crassostrea angulata . The primary sequence of AK was cloned which encoded 350 amino acids, and recombinant AK (rAK) was obtained. The immunodot results, secondary structure and digestive stability showed that native AK and rAK had similar IgG/IgE-binding activity and physicochemical properties. Serological analysis of 14 oyster-sensitive individuals demonstrated that AK exhibited cross-reactivity among oysters, shrimps, and crabs. Furthermore, nine epitopes in oyster AK were verified using inhibition dot blots and inhibition enzyme linked immunosorbent assay, six of which were similar to the epitopes of shrimp/crab AK. The most conserved epitopes were P5 (121-133) and P6 (133-146), which may be responsible for the cross-reactivity caused by AK. These findings will provide a deeper understanding of oyster allergens and cross-reactivity among shellfish.

Tripartite motif (TRIM) proteins are a large family of E3 ubiquitin ligases involved in many biological processes, such as inflammation and antiviral immunity. In the present study, a novel TRIM protein homolog named CgTRIM1 was identified from Pacific oyster Crassostrea gigas. The open reading frame (ORF) of CgTRIM1 was of 1914 bp encoding a putative polypeptide of 637 amino acid residues. There were three classical domains in the predicted CgTRIM1 protein, including one RING domain, two b-box domains and one coiled-coil domain in N-terminal. For the lack of C-terminal domains, the CgTRIM1 was classified as the member of C-V TRIM subfamily. The mRNA transcripts of CgTRIM1 were detected in all the tested tissues and haemocytes, with the highest expression level in gill. The mRNA and protein levels of CgTRIM1 in gill were significantly up-regulated at 6 h after poly (I:C) stimulation. Moreover, the nuclear translocation of CgTRIM1 was observed in haemocytes of oysters after poly (I:C) stimulation. After IFN-like protein (CgIFNLP) was knocked down by RNA interference (RNAi), the expression of CgTRIM1 in gill was markedly inhibited in both mRNA (0.14-fold, p < 0.001) and protein levels after poly (I:C) stimulation. Furthermore, after knocking down of CgTRIM1, the mRNA expression levels of IFN-stimulated genes, including myxovirus resistance of oyster (CgMx) and Interferon-induced protein 44 (CgIFI44) were significantly down-regulated post poly (I:C) stimulation, while no significant change of the CgIFNLP expression was observed. These results indicated that CgTRIM1 participated in the antiviral response of C. gigas by regulating the mRNA expressions of IFN-stimulated genes., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Vertebrate interleukin-12 (IL-12) is a heterodimeric cytokine composing of two subunits (p35 and p40). In the present study, a p35-like subunit homolog of vertebrate IL-12 was identified from oyster Crassostrea gigas (designated as CgIL12p35L), with an open reading frame of 411 bp encoding a putative peptide of 136 amino acids. There was a long four-helix chain in CgIL12p35L, which was similar as that in vertebrate IL-12 p35. Comparative genomic analysis showed that there were conservative kinds of syntenic genes flanked CgIL12p35L. The mRNA transcripts of CgIL12p35L were constitutively expressed in various tissues and its mRNA expression level in haemocytes increased significantly after bacteria challenge. The activity of haemolymph to eliminate bacteria from the oysters treated with recombinant CgIL12p35L protein (rCgIL12p35L) in vivo increased significantly. The results collectively indicated that the homolog of vertebrate IL-12 p35 subunit existed in oysters, and it was involved in immune defense against bacteria challenge., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Ubiquitination is involved in the regulation of granulocyte proliferation in vertebrate, and E3 ubiquitin ligase WWP1 has been reported to play an essential role in this process. In the present study, an HECT type E3 ubiquitin ligase (CgWWP1) was identified from oyster Crassostrea gigas, which contained a N-terminal C2 domain, four WW domains, and a C-terminal HECT domain. CgWWP1 was able to bind the activated ubiquitin (Ub) and formed CgWWP1-Ub complex in vitro. The mRNA transcripts of CgWWP1 were expressed in granulocytes, semi-granulocytes and agranulocytes, with the highest expression level in granulocytes. The expressions of potential granulocyte markers CgSOX11 (0.18-fold, p < 0.05) and CgAATase (0.2-fold, p < 0.01) in haemocytes were significantly down-regulated at 24 h after the treatment with Indole-3-carbinol (I3C), a WWP1 inhibitor. The proportions of EdU + granulocytes reduced significantly at 12 h (8.1% ± 1.4%) and 24 h (9.7% ± 2.8%) after I3C treatment, which were significantly lower than that in the sterile seawater treatment (SW) group at 12 h (15.8% ± 4.2%) and 24 h (17.6% ± 0.8%), respectively. Meanwhile, the green EdU signals observed by confocal scanning microscopy in granulocytes of oysters treated by I3C became weaker compared to that in the SW group. These results indicated that CgWWP1 was involved in the regulation of granulocyte proliferation as a ubiquitin-protein ligase., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

DNA methyltransferase, a key enzyme mediating DNA methylation, is involved in numerous processes including genomic imprinting, X chromosome inactivation, transposable element suppression, and immune defense in vertebrates. In the present study, a DNA cytosine-5-methyltransferase 3 was identified from oyster Crassostrea gigas (designed as CgDNMT3). There were a PWWP domain, a PHD domain and a DNA-methylase domain in the deduced amino acid sequences of CgDNMT3, and the conserved motifs I, IV, VI, Ⅷ, IX and X were identified in its C-terminal catalytic DNA-methylase domain. The mRNA transcripts of CgDNMT3 were detected in haemocytes, mantle, gill, adductor muscle, digestive gland and labial palp, with higher expression level in haemocytes (6.54 folds of those in gill, p < 0.01). The expression level of CgDNMT3 mRNA in haemocytes increased significantly after LPS primed (2.87 folds of that in control group, p < 0.05) in vitro or Vibrio splendidus challenging (1.94 folds of that in control group, p < 0.05) in vivo. Immunocytochemical analysis revealed that CgDNMT3 protein was distributed mainly in cytoplasm and partial in nucleus of oyster haemocytes. After CgDNMT3 was transfected and expressed in HEK293T cells, the DNA 5-methylcytosine (5-mc) level in the transfected group was significantly increased, which was 1.22 folds (p < 0.05) of the pcDNA-3.1 group. The expressions of oyster CgIL17-1, CgIL17-2 and CgIL17-5 in haemocytes increased (13.05 folds, 4.78 folds and 9.41 folds of that in control group, respectively) at 12 h after V. splendidus challenging, but the increase were significantly inhibited when the oysters were pre-treated with DNA methyltransferase inhibitor 5-Azacytidine, which were 9 folds, 1.93 folds and 3.22 folds of that in control group, respectively. These results collectively suggested that CgDNMT3 was a conserved member of DNA methyltransferase 3 family in oyster, and participated in regulating the expression of cytokines during immune response., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Vibrio species are ubiquitously distributed in marine environments, with important implications for emerging infectious diseases. However, relatively little is known about defensive strategies deployed by hosts against Vibrio pathogens of distinct virulence traits. Being an ecologically relevant host, the oyster Crassostrea hongkongensis can serve as an excellent model for elucidating mechanisms underlying host- Vibrio interactions. We generated a Vibrio alginolyticus mutant strain ( V. alginolyticus △ vscC ) with attenuated virulence by knocking out the vscC encoding gene, a core component of type III secretion system (T3SS), which led to starkly reduced apoptotic rates in hemocyte hosts compared to the V. alginolyticus WT control. In comparative proteomics, it was revealed that distinct immune responses arose upon encounter with V. alginolyticus strains of different virulence. Quite strikingly, the peroxisomal and apoptotic pathways are activated by V. alginolyticus WT infection, whereas phagocytosis and cell adhesion were enhanced in V. alginolyticus △ vscC infection. Results for functional studies further show that V. alginolyticus WT strain stimulated respiratory bursts to produce excess superoxide (O2 •- ) and hydrogen peroxide (H 2 O 2 ) in oysters, which induced apoptosis regulated by p53 target protein (p53tp). Simultaneously, a drop in sGC content balanced off cGMP accumulation in hemocytes and repressed the occurrence of apoptosis to a certain extent during V. alginolyticus △ vscC infection. We have thus provided the first direct evidence for a mechanistic link between virulence of Vibrio spp. and its immunomodulation effects on apoptosis in the oyster. Collectively, we conclude that adaptive responses in host defenses are partially determined by pathogen virulence, in order to safeguard efficiency and timeliness in bacterial clearance., Competing Interests: The authors declare that their research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mao, Liu, Wong, Zhang, Yi, Xiang, Xiao, Yu and Zhang.)

Sarcoplasmic-calcium-binding protein (SCP) has been investigated as a novel allergen in Crassostrea angulata . Nevertheless, knowledge of its effector-cell-based allergic relevance and epitopes is limited. In this study, the heat-resistant allergen SCP was able to induce significant upregulation of CD63 and CD203c ( p < 0.05), which showed obvious allergenicity in a basophil activation test. Furthermore, immunoinformatic tools, a one-bead-one-compound peptide library, and phage display technology were combined to analyze the allergenic epitopes of SCP. Five linear epitopes named L-SCP-1 (AA 22-33 ), L-SCP-2 (AA 64-75 ), L-SCP-3 (AA 80-90 ), L-SCP-4 (AA 107-116 ), and L-SCP-5 (AA 144-159 ) were verified using serological tests. Additionally, two conformational epitopes (C-SCP-1 and C-SCP-2) were determined, and C-SCP-1 was located at one of the calcium-binding sites (AA 106-117 ). Moreover, SCP showed weaker typical α-helical features and higher hydrophobicity after Ca 2+ depletion, which reduced its IgE-binding capacity. Overall, these epitope data could enhance our understanding of oyster allergens, which could be used to develop hypoallergenic shellfish products.

Hexokinase (HK) is generally recognized as a crucial enzyme participating in glycolysis. In the present study, a HK (named CgHK) was identified as a potential pattern recognition receptor (PRR) from the Pacific oyster Crassostrea gigas. The open reading frame (ORF) of CgHK was of 1395 bp, encoding a peptide of 464 amino acids with one Hexokinase&#95;1 domain and one Hexokinase&#95;2 domain. The predicted amino acid sequence of CgHK shared 17%-29% similarities with that of other known HKs. The mRNA transcripts of CgHK were constitutively detected in all the examined tissues, with relative high expression level in labial palp and haemocytes. CgHK protein was mainly observed in the cytoplasm of oyster haemocytes. The mRNA expression level of CgHK in haemocytes was significantly up-regulated and peaked at 3 h after Vibrio splendidus (7.64-fold, p < 0.001) and lipopolysaccharide (LPS) (11.86-fold, p < 0.001) stimulations. The recombinant CgHK protein (rCgHK) exhibited Mg 2+ -dependent adenosine triphosphate (ATP) binding activity in vitro and activity to bind D-(+)-glucose (GLU) and various pathogen-associated molecular pattern (PAMPs) such as LPS and peptidoglycan (PGN) in the absence of Mg 2+ . It also displayed higher binding activity towards V. splendidus and relatively lower binding activity towards Staphylococcus aureus, Escherichia coli, and Micrococcus luteus. After the mRNA expression of CgHK in haemocytes was knocked down by dsRNA interference, the expression of CgIL17-5 mRNA in haemocytes was considerably down-regulated at 3 h after the stimulation with V. splendidus (0.33-fold, p < 0.001). These results collectively indicated that CgHK was able to recognize various PAMPs and pathogenic bacteria as a PRR apart from being the enzyme to exert ATP binding activity in glycolysis, and activate the anti-bacterial immune response by promoting the expression of pro-inflammatory cytokines CgIL17-5 in oyster haemocytes., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The Methyltransf&#95;FA domain is well-known as a key protein domain of enzyme synthesizing juvenile hormone, and Methyltransf&#95;FA domain containing proteins (MFCPs) are widely existed in vertebrates and invertebrates. In the present study, a CgMFCP with a single Methyltransf&#95;FA domain was screened from oyster Crassostrea gigas, and its open reading frame of CgMFCP was of 1128 bp, encoding a polypeptide of 376 amino acids with a signal peptide, a Methyltransf&#95;FA domain and a transmembrane region. CgMFCP was clustered with FAMeTs from insecta and crustacea of arthropod. The mRNA transcripts of CgMFCP were detected in different tissues, with the extremely high expression level in haemocytes, which was 131.36-fold (p < 0.05) of that in gills. The expression level of CgMFCP protein was verified to be highly expressed in haemocytes. The expression level of CgMFCP mRNA in primarily cultured haemocytes significantly up-regulated at 3 h, 24 h and 48 h post LPS stimulation, which was 3.25-fold (p < 0.01), 2.04-fold (p < 0.05) and 3.59-fold (p < 0.01) compared to that in blank group. After the oysters were stimulated with Vibrio splendidus in vivo, the expression level of CgMFCP mRNA in haemocytes was also significantly up-regulated at 3 h, 12 h, and 24 h, which was 4.22-fold (p < 0.05), 4.39-fold (p < 0.05) and 6.35-fold (p < 0.01) of that in control group, respectively. By flow cytometry analysis, anti-rCgMFCP can label 95% of oyster haemocytes. And by fluorescence microscope analysis, CgMFCP was mainly distributed in cytomembrane of haemocytes. The recombinant CgMFCP (rCgMFCP) exhibited higher affinity towards MAN and LPS in a dose-dependent manner, while relatively lower affinity to PGN and poly (I:C). rCgMFCP also displayed binding activities towards Gram-negative bacteria (Vibrio anguillarum and V. splendidus), Gram-positive bacteria (Staphylococcu aureu) and fungi (Pichia pastoris). These results collectively indicated that CgMFCP specifically expressed in haemocytes and functioned as a pattern recognition receptor by binding to various microbes in oyster C. gigas, which provided insight into the function of Methyltransf&#95;FA domain containing proteins., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

C-type lectins (CTLs) are important immune molecules that participate in invertebrate defense response. In the present work, a novel structural CTL (CgLec-4E) was identified from Crassostrea gigas, which encodes 237 amino acids (aa) with an extra long chain of aa and in the C-type CRD domain with EPA, QPG and WHD mutated motifs respectively. rCgLec-4E could agglutinate and inhibit the growth of Vibrio alginolyticus, except Chlorella, which might be relevant to three mutated motifs. CgLec-4E was mainly expressed in digestive gland, and its expression level was significantly up-regulated post V. alginolyticus challenge, indicating that the high expression of CgLec-4E could provide necessary mucosal immune protections and might involve in food particle recognition for C. gigas. Moreover, the subcellular locations indicated that CgLec-4E might play different roles in the immune response. Taken together, our results enrich our understanding of the structures and function of CTLs in invertebrates., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Oyster production in Brazil has been highlighted as an important economic activity and is directly impacted by the quality of the environment, which is largely the result of human interference and climate change. Harmful algal blooms occur in aquatic ecosystems worldwide, including coastal marine environments which have been increasing over the last decades as a result of global change and anthropogenic activities. In this study, the native oysters Crassostrea gasar from Northeast of Brazil were exposed to two toxic benthic dinoflagellate species, Prorocentrum lima and Ostreopsis cf. ovata. Their respective effects on C. gasar physiology and defense mechanisms were investigated. Oyster hemocytes were first exposed in vitro to different concentrations of both dinoflagellate species to assess their effects on hemocyte functions, such as phagocytosis, production of reactive oxygen species, as well as mortality. Results highlighted an alteration of hemocyte phagocytosis and viability in presence of O. cf. ovata, whereas P. lima did not affect the measured hemocyte functions. In a second experiment, oysters were exposed for 4 days in vivo to toxic culture of O. cf. ovata to assess its effects on hemocyte parameters, tissues damages and pathogenic Perkinsus spp. infection. An increase in hemocyte mortality was also observed in vivo, associated with a decrease of ROS production. Histopathological analyses demonstrated a thinning of the epithelium of the digestive tubules of the digestive gland, inflammatory reaction and a significant increase in the level of infection by Perkinsus spp. in oysters exposed to O. cf. ovata. These results indicate that oysters C. gasar seem to be pretty resilient to an exposure to P. lima and may be more susceptible to O. cf. ovata. Furthermore, the latter clearly impaired oyster physiology and defense mechanisms, thus highlighting that harmful algal blooms of O. cf. ovata could potentially lead to increased susceptibility of C. gasar oysters to parasite infections., (Copyright © 2021. Published by Elsevier B.V.)

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.)

Interferon (IFN) system is considered as the first defense line against viral infection, and it has been extensively studied in vertebrates from fish to mammals. In invertebrates, Vagos from arthropod and IFN-like protein ( Cg IFNLP) from Crassostrea gigas appeared to function as IFN-like antiviral cytokines. In the present study, the Cg IFNLP protein in hemocytes was observed to increase after Poly (I:C) stimulation. After Cg IFNLP was knocked down by RNAi, the mRNA expression of IFN-stimulated genes ( Cg ISGs) was significantly inhibited. Both cyclic GMP-AMP synthase ( Cg cGAS) and stimulator of interferon gene ( Cg STING) identified from oyster were able to recognize the double-stranded nucleic acid [Poly (I:C) and dsDNA] and expressed at high level after Poly (I:C) stimulation. The expression of Cg IFNLP and interferon regulatory factors ( Cg IRF1/8) and the nuclear translocation of Cg IRF8 were all suppressed in Cg cGAS-RNAi or Cg STING-RNAi oysters after Poly (I:C) stimulation. The expression level of Cg STING and TANK binding kinase1 ( Cg TBK1) did not decrease in Cg cGAS-RNAi oysters. After Cg STING was knocked down, the high expression of Cg TBK1 induced by Poly (I:C) was prevented significantly. These results indicated that there was a primitive IFN-like antiviral mechanism dependent on the cGAS/STING-TBK1-IRFs regulatory axis in mollusks, which was different from the classic cGAS-STING-TBK1 signal pathway in mammals., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Qiao, Zong, Liu, Wu, Li, Wang and Song.)

Programmed cell death (PCD) is an essential process for the immune system's development and homeostasis, enabling the remotion of infected or unnecessary cells. There are several PCD's types, depending on the molecular mechanisms, such as non-inflammatory or pro-inflammatory. Hemocytes are the main component of cellular immunity in bivalve mollusks. Numerous infectious microorganisms produce toxins that impair hemocytes functions, but there is little knowledge on the role of PCD in these cells. This study aims to evaluate in vitro whether marine toxins induce a particular type of PCD in hemocytes of the bivalve mollusk Crassostrea gigas during 4 h at 25°C. Hemocytes were incubated with two types of marine toxins: non-proteinaceous toxins from microalgae (saxitoxin, STX; gonyautoxins 2 and 3, GTX2/3; okadaic acid/dynophysistoxin-1, OA/DTX-1; brevetoxins 2 and 3, PbTx-2,-3; brevetoxin 2, PbTx-2), and proteinaceous extracts from bacteria ( Vibrio parahaemolyticus , Vp; V. campbellii , Vc). Also, we used the apoptosis inducers, staurosporine (STP), and camptothecin (CPT). STP, CPT, STX, and GTX 2/3, provoked high hemocyte mortality characterized by apoptosis hallmarks such as phosphatidylserine translocation into the outer leaflet of the cell membrane, exacerbated chromatin condensation, DNA oligonucleosomal fragments, and variation in gene expression levels of apoptotic caspases 2, 3, 7, and 8. The mixture of PbTx-2,-3 also showed many apoptosis features; however, they did not show apoptotic DNA oligonucleosomal fragments. Likewise, PbTx-2, OA/DTX-1, and proteinaceous extracts from bacteria Vp, and Vc, induced a minor degree of cell death with high gene expression of the pro-inflammatory initiator caspase-1, which could indicate a process of pyroptosis-like PCD. Hemocytes could carry out both PCD types simultaneously. Therefore, marine toxins trigger PCD's signaling pathways in C. gigas hemocytes, depending on the toxin's nature, which appears to be highly conserved both structurally and functionally., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Estrada, Núñez-Vázquez, Palacios, Ascencio, Guzmán-Villanueva and Contreras.)

Gender differences in individual immune responses to external stimuli have been elucidated in many invertebrates. However, it is unclear if gender differences do exist in the Hong Kong oyster Crassostrea hongkongensis , one of the most valuable marine species cultivated along the coast of South China. To clarify this, we stimulated post-spawning adult C. hongkongensis with Vibrio harveyi and lipopolysaccharide (LPS). Gender-based differences in some essential functional parameters of hemocytes were studied via flow cytometry. Obvious gender-, subpopulation-, and immune-specific alterations were found in the hemocyte immune parameters of C. hongkongensis . Three hemocyte subpopulations were identified: granulocytes, semi-granulocytes, and agranulocytes. Granulocytes, the chief phagocytes and major producers of esterase, reactive oxygen species, and nitric oxide, were the main immunocompetent hemocytes. Immune parameter alterations were notable in the accumulation of granulocyte esterase activities, lysosomal masses, nitric oxide levels, and granulocyte numbers in male oysters. These results suggest that post-spawning-phase male oysters possess a more powerful immune response than females. Gender and subpopulation differences in bivalve immune parameters should be considered in the future analysis of immune parameters when studying the impact of pathogenic or environmental factors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lu, Shi, Yao, Bai, Jiang and Ye.)

DM9 domain containing protein (DM9CP) is a recently identified pattern recognition molecules exiting in most organisms except plants. In the present study, a novel DM9-containing protein (CgDM9CP-3) was identified from Pacific oyster Crassostrea gigas with an open reading frame of 438 bp, encoding a polypeptide of 145 amino acids containing two tandem DM9 repeats. The deduced amino acid sequence of CgDM9CP-3 shared 52.4% and 58.6% identity with CgDM9CP-1 and CgDM9CP-2, respectively. The mRNA transcripts of CgDM9CP-3 were highest expressed in oyster gills and its protein was mainly distributed in cytomembrane of haemocytes. After the stimulations with Vibrio splendidus and mannose, the mRNA expression of CgDM9CP-3 in oyster gills was significantly up-regulated and reached the peak level at 12 h and 24 h (p < 0.05), which was 7.80-fold (p < 0.05) and 42.82-fold (p < 0.05) of that in the control group, respectively. The recombinant CgDM9CP-3 protein (rCgDM9CP-3) was able to bind LPS, PGN and d-Mannose, fungi Pichia pastoris and Yarrowia lipolytica, as well as gram-negative bacteria Escherichia coli, Vibrio anguillarum and V. splendidus in a Ca 2+ -dependent manner. Moreover, it could enhance the encapsulation of haemocytes and exhibited agglutination activity towards fungi P. pastoris and Y. lipolytica in vitro with Ca 2+ . These results suggested that CgDM9CP-3 not only acted as a PRR involved in the pathogen recognition, but also enhanced cellular encapsulation in oyster C. gigas., (Copyright © 2020. Published by Elsevier Ltd.)

The enzyme Dicer is best known for its role as an endoribonuclease in the small RNA pathway, playing a crucial role in recognizing viral double-stranded RNA (dsRNA) and inducing down-stream cascades to mediate anti-virus immunity. In the present study, a truncated Dicer-like gene was identified from oyster Crassostrea gigas, and its open reading frame (ORF) encoded a polypeptide (designed as CgDCL) of 530 amino acids. The CgDCL contained one N-terminal DEAD domain and a C-terminal helicase domain, but lack the conserved PAZ domain, ribonuclease domain (RIBOc) and dsRNA binding domain. The mRNA transcripts of CgDCL were detected in all the examined tissues with high expression levels in lip, gills and haemocytes, which were 62.06-fold, 48.91-fold and 47.13-fold (p < 0.05) of that in mantle, respectively. In the primarily cultured oyster haemocytes, the mRNA transcripts of CgDCL were significantly induced at 12 h after poly(I:C) stimulation, which were 4.04-fold (p < 0.05) of that in control group. The expression level of CgDCL mRNA in haemocytes was up-regulated significantly after dsRNA and recombinant interferon-like protein (rCgIFNLP) injection, which was 12.87-fold (p < 0.01) and 3.22-fold (p < 0.05) of that in control group, respectively. CgDCL proteins were mainly distributed in the cytoplasm of haemocytes. The recombinant CgDCL protein displayed binding activity to dsRNA and poly(I:C), but no obvious dsRNA cleavage activity. These results collectively suggest that truncated CgDCL from C. gigas was able to be activated by poly(I:C), dsRNA and CgIFNLP, and functioned as an intracellular recognition molecule to bind nucleic acid of virus, indicating a potential mutual cooperation between RNAi and IFN-like system in anti-virus immunity of oysters., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

DM9 refers to an uncharacterized protein domain that is originally discovered in Drosophila melanogaster . Two proteins with DM9 repeats have been recently identified from Pacific oyster Crassostrea gigas as mannose-specific binding pattern-recognition receptors (PRRs). In the present study, a novel member of DM9 domain containing protein (designated as Cg DM9CP-4) was identified from C. gigas . Cg DM9CP-4, about 16 kDa with only two tandem DM9 domains, was highly enriched in hemocytes and gill. The transcripts level of Cg DM9CP-4 in circulating hemocytes were decreased after LPS, PGN and Vibrio splendidus stimulations. The recombinant protein of Cg DM9CP-4 (r Cg DM9CP-4) displayed a broad binding spectrum towards various pathogen-associated molecular patterns (PAMPs) (LPS, PGN, β-glucan and Mannose) and microorganisms ( Staphylococcus aureus , Micrococcus luteus , V. splendidus , V. anguillarum , Escherichia coli , Pichia pastoris and Yarrowia lipolytica ). Cg DM9CP-4 was mostly expressed in gill and some of the hemocytes. Flow cytometry analysis demonstrated that the Cg DM9CP-4-positive hemocytes accounted for 7.3% of the total hemocytes, and they were small in size and less in granularity. Cg DM9CP-4 was highly expressed in non-phagocytes (~82% of total hemocytes). The reactive oxygen species (ROS) and the expression levels of cytokines in Cg DM9CP-4-positive hemocytes were much lower than that in Cg DM9CP-4-negative hemocytes. The mRNA expression level of Cg DM9CP-4 in hemocytes was decreased after RNAi of hematopoietic-related factors ( Cg GATA, Cg Runt, Cg SCL, and Cg Notch). In addition, Cg DM9CP-4-positive cells were found to be much more abundant in hemocytes from gill than that from hemolymph, with most of them located in the gill filament. All these results suggested that Cg DM9CP-4 was a novel member of PRR that expressed in undifferentiated pro-hemocytes to mediate immune recognition of pathogens., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Jia, Jiang, Wang, Wang, Liu, Lv, Song, Li, Wang and Song.)

Massive mortality outbreaks affecting Pacific oysters ( Crassostrea gigas ) spat/juveniles are often associated with the detection of a herpesvirus called ostreid herpesvirus type 1 (OsHV-1). In this work, experimental infection trials of C. gigas spat with OsHV-1 were conducted using two contrasted Pacific oyster families for their susceptibility to viral infection. Live oysters were sampled at 12, 26, and 144 h post infection (hpi) to analyze host-pathogen interactions using comparative proteomics. Shotgun proteomics allowed the detection of seven viral proteins in infected oysters, some of them with potential immunomodulatoy functions. Viral proteins were mainly detected in susceptible oysters sampled at 26 hpi, which correlates with the mortality and viral load observed in this oyster family. Concerning the Pacific oyster proteome, more than 3,000 proteins were identified and contrasted proteomic responses were observed between infected A- and P-oysters, sampled at different post-injection times. Gene ontology (GO) and KEGG pathway enrichment analysis performed on significantly modulated proteins uncover the main immune processes (such as RNA interference, interferon-like pathway, antioxidant defense) which contribute to the defense and resistance of Pacific oysters to viral infection. In the more susceptible Pacific oysters, results suggest that OsHV-1 manipulate the molecular machinery of host immune response, in particular the autophagy system. This immunomodulation may lead to weakening and consecutively triggering death of Pacific oysters. The identification of several highly modulated and defense-related Pacific oyster proteins from the most resistant oysters supports the crucial role played by the innate immune system against OsHV-1 and the viral infection. Our results confirm the implication of proteins involved in an interferon-like pathway for efficient antiviral defenses and suggest that proteins involved in RNA interference process prevent viral replication in C. gigas . Overall, this study shows the interest of multi-omic approaches applied on groups of animals with differing sensitivities and provides novel insight into the interaction between Pacific oyster and OsHV-1 with key proteins involved in viral infection resistance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Leprêtre, Faury, Segarra, Claverol, Degremont, Palos-Ladeiro, Armengaud, Renault and Morga.)

Glutaminase, an amidohydrolase enzyme that hydrolyzes glutamine to glutamate, plays crucial roles in various immunomodulatory processes such as cell apoptosis, proliferation, migration, and secretion of cytokines. In the present study, a glutaminase homologue (designated as CgGLS-1) was identified from Pacific oyster Crassostrea gigas, whose open reading frame was of 1836 bp. CgGLS-1 exhibited high sequence identity with vertebrate kidney-type GLS, and closely clustered with their homologues from mollusc C. virginica. The enzyme activity of recombinant CgGLS-1 protein (rCgGLS-1) was estimated to be 1.705 U/mg. CgGLS-1 mRNA was constitutively expressed in all the tested tissues of oysters, with the highest expression level in hemocytes. CgGLS-1 mRNA expression in hemocytes was significantly up-regulated and peaked at 6 h (2.07-fold, p < 0.01) after lipopolysaccharide (LPS) stimulation. The CgGLS-1 protein was mainly distributed in the cytoplasm with a significant co-location with mitochondria in oyster hemocytes. The content of Glu in the oyster serum was significantly decreased after the inhibition of CgGLS-1 using specific inhibitor Bis-2- [5-(phenyl acetamido)-1,3,4-thiadiazol-2-yl] ethyl sulfide (BPTES), and the expression levels of CgmGluR6, CgAP-1, cytokines CgIL17-5 and CgTNF-1 were significantly decreased after BPTES and LPS stimulation. The transcripts of CgCaspase3 as well as the apoptosis index of hemocytes were also decreased. These results collectively suggest that CgGLS-1 is the enzyme to synthesize Glu in oyster, which can modulate anti-bacterial immunity by regulating the secretion of pro-inflammatory cytokines CgIL17-5 and CgTNF-1, as well as hemocyte apoptosis.

The Pacific oyster, Crassostrea gigas , is a mollusk bivalve commercially important as a food source. Pacific oysters are subjected to stress and diseases during culture. The autophagy pathway is involved in numerous cellular processes, including responses to starvation, cell death, and microorganism elimination. Autophagy also exists in C. gigas , and plays a role in the immune response against infections. Although this process is well-documented and conserved in most animals, it is still poorly understood in mollusks. To date, no study has provided a complete overview of the molecular mechanism of autophagy in mollusk bivalves. In this study, human and yeast ATG protein sequences and public databases (Uniprot and NCBI) were used to identify protein members of the C. gigas autophagy pathway. A total of 35 autophagy related proteins were found in the Pacific oyster. RACE-PCR was performed on several genes. Using molecular (real-time PCR) and protein-based (western blot and immunohistochemistry) approaches, the expression and localization of ATG12, ATG9, BECN1, MAP1LC3, MTOR, and SQSTM1, was investigated in different tissues of the Pacific oyster. Comparison with human and yeast counterparts demonstrated a high homology with the human autophagy pathway. The results also demonstrated that the key autophagy genes and their protein products were expressed in all the analyzed tissues of C. gigas . This study allows the characterization of the complete C. gigas autophagy pathway for the first time. Abbreviations: ATG: autophagy related; Atg1/ULK: unc-51 like autophagy activating kinase; ATG7: autophagy related 7; ATG9: autophagy related 9; ATG12: autophagy related 12; BECN1: beclin 1; BSA: bovine serum albumin; cDNA: complementary deoxyribonucleic acid; DNA: deoxyribonucleic acid; GABARAP: GABA type A receptor-associated protein; IHC: immunohistochemistry; MAP1LC3/LC3/Atg8: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NCBI: national center for biotechnology information; ORF: open reading frame; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PtdIns3K: class III phosphatidylinositol 3-kinase; RACE-PCR: rapid amplification of cDNA-ends by polymerase chain reaction; RNA: ribonucleic acid; SQSTM1: sequestosome 1; Uniprot: universal protein resource; WIPI: WD repeat domain, phosphoinositide interacting.