Splendidus Stimulation Research Articles

A transcription factor ATF3 involves in the phagocytosis of granulocytes in oyster Crassostrea gigas

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.

Molecular identification and functional characterization of a C-type lectin gene in Meretrix meretrix

C-type lectins (CTLs) are a kind of Ca2+-dependent immunoreactive factors, which participated in pathogens recognition and defense. The present study identified a new CTL from hard clam Meretrix meretrix (designated as MmCTL4). The full-length of MmCTL4 cDNA was 608 bp, encoding a presumed signal peptide of 19 bp and a carbohydrate recognition domain (CRD) of 131 bp. The tertiary structure of recombinant MmCTL4 protein (rMmCTL4) was the typical long double-ring structure with three conserved disulfide bonds, and the motifs in Ca2+-binding sites of MmCTL4 were QPN and WSD. The SYBR Green real-time PCR analysis indicated that MmCTL4 was widely expressed in the hemocytes, hepatopancreas and mantle of healthy clams. After Vibrio splendidus stimulation, the temporal expression profile of MmCTL4 mRNA in hemocytes and hepatopancreas increased by 7.8-fold at 6 hpi and 3.9-fold at 12 hpi, respectively. The cDNA fragments encoding MmCTL4 were recombined into pET-32a (+) vectors, and transformed into Escherichia coli BL21 (DE3). The rMmCTL4 with the presence of Ca2+ performed obvious hemagglutination activity, and could agglutinate E. coli, Bacillus subtilis, and Staphylococcus aureus, while it only weakly agglutinate Vibrio parahaemolyticus and fungi P. pastoris. The agglutination activity of rMmCTL4 were significantly inhibited by D-mannose, D-xylose, D-lactose, maltose and lipopolysaccharides. These results indicated that MmCTL4, as a class of typical pattern recognition receptors (PRRs), could protect the host against pathogen invasion in the innate immunity of clams.

Preliminary investigation on the effect of Vibrio splendidus stimulation on the intestinal flora of Strongylocentrotus intermedius

To better understand the effect of Vibrio splendidus infection on Strongylocentrotus intermedius, 16S rRNA sequencing was carried out to investigate the intestinal flora of S. intermedius stimulated by 0 CFU/mL (Con), 1.5 × 107 CFU/mL (Vib1) and 1.5 × 108 CFU/mL (Vib2) concentrations of V. splendidus. The results showed that there was significant difference in intestinal flora diversity between Con group and Vib1 group, but no significant difference between Con group and Vib2 group. However, there were significant differences in the composition of intestinal flora among all groups. Bacteroidota, Proteobacteria and Firmicutes were the dominant phylum in the Con group. The abundance of Bacteroidota and Firmicutes decreased and Proteobacteria increased in Vib1 and Vib2 groups. The relative abundance of the potential probiotic bacteria Muribaculaceae and Alloprevotella was significantly lower in the Vib1 and Vib2 groups. In addition, the opportunistic pathogen Desulfovibrio was found in Vib1 and Vib2 groups. It is evident that V. splendidus infection not only alters the composition of the microbial community in the intestinal tract of S. intermedius, but may also lead to the production of opportunistic pathogens, which could be potentially harmful to the health of S. intermedius. The results of this study provide a foundation for exploring the diseases caused by V. splendidus stimulation leading to an imbalance in the intestinal flora of S. intermedius, and contribute to our further understanding of the role of Vibrio on the health of S. intermedius.

A novel surface marker CD49d promotes TNF expression in oyster agranulocytes by mediating the MAPK pathway

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_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.

CgSHIP2 negatively regulates the mRNA expressions of CgIL-17s in response to Vibrio splendidus stimulation in Crassostrea gigas

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.

The expression patterns of exosomal miRNAs in the Pacific oyster after high-temperature stress or Vibrio stimulation

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.

CgPHB2 involved in the haemocyte mitophagy in response to Vibrio splendidus stimulation in Pacific oyster Crassostrea gigas

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.

Molecular cloning, biological description, and functional analysis of Ajfos transcription factor in pathogen-induced Apostichopus japonicus

Activator protein-1 subfamily member c-Fos wields significant influence over cellular activities, such as regulation of cell growth and division, cell death, and immune responses under various extracellular situations. In this study, the full-length c-Fos of sea cucumber, Apostichopus japonicus (Ajfos) was successfully cloned and analyzed. The anticipated 306 amino acid sequences of Ajfos displayed a basic-leucine zipper (bZIP) domain, similar to invertebrate counterparts. In addition, the qPCR results suggested Ajfos expressed in all tissues, with the highest level in coelomocytes from polian vesicle (vesicle lumen cells), followed by coelomocytes from coelom (coelomocytes). Moreover, the expression levels of Ajfos in the coelomocytes and vesicle lumen cells of sea cucumber showed significant changes after the Vibrio splendidus challenge, especially reaching a peak at 6 h. Compared with the silencing negative control RNA interference (siNC) group, silencing Ajfos (siAjfos) in vivo decreased the downstream proliferation-related gene expression of vesicle lumen cells after infection with V. splendidus while no significant influence was observed on coelomocytes. Furthermore, the proliferation proportion of vesicle lumen cells in the siAjfos group was significantly reduced under pathogen stimulation conditions. Finally, based on the fluctuation trend of total coelomocyte density (TCD) from coelom and polian vesicle previously discovered, it is evident that Ajfos played a critical role in facilitating the swift proliferation of vesicle lumen cells in response to V. splendidus stimulation. Altogether, this research provided an initial reference of the function of Ajfos in echinoderms, unveiling its participation in host coelomocyte proliferation of sea cucumbers during bacterial challenges.

A novel DM9-containing protein 7 involved in regulating the expression of CgMyD88 and CgIL-17 in oyster Crassostrea gigas

The DM9-containing proteins have been identified as pattern recognition receptors (PRRs) to recognize invading pathogens and subsequently mediate downstream signal pathways, playing essential roles in innate immune responses of molluscs. In the present study, a novel DM9-containing protein (named as CgDM9CP-7) was identified from Pacific oyster Crassostrea gigas, which contained two tandem DM9 repeats similar to the previously identified CgDM9CPs. The mRNA transcripts of CgDM9CP-7 were found to be constitutively expressed in all the tested tissues including haemolymph, gill, hepatopancreas, mantle, adductor muscle and labial palp. The expression level of CgDM9CP-7 mRNA in haemocytes significantly up-regulated at 3 and 6 h after Vibrio splendidus stimulation, which was 5.67-fold (p < 0.01) and 4.71-fold (p < 0.05) of that in the control group, respectively, and it also increased significantly at 6 h (3.08-fold, p < 0.01) post lipopolysaccharide (LPS) stimulation. The protein of CgDM9CP-7 was mainly detected in membrane and cytoplasm of oyster haemocytes after V. splendidus stimulation. The recombinant CgDM9CP-7 protein (rCgDM9CP-7) displayed binding activities to MAN, LPS, PGN, Poly (I:C) as well as gram-negative bacteria (V. splendidus and Escherichia coli), gram-positive bacteria (Staphylococcus aureus and Micrococcus luteus) and fungi (Pichia pastoris and Yarrowia lipolytica). rCgDM9CP-7 was able to agglutinate Bacillus subtilis, V. splendidus, E. coli and S. aureus, inhibit their growth, and bind the recombinant protein CgMyd88-2 (KD = 5.98 × 10−6 M) and CgMyd88s (KD = 8.5 × 10−7 M) in vitro as well. The transcripts of CgIL17-1 (0.45-fold of the control group, p < 0.01), CgIL17-2 (0.19-fold, p < 0.05), CgIL17-3 (0.54-fold, p < 0.05), CgIL17-5 (0.36-fold, p < 0.05) and CgIL17-6 (0.24-fold, p < 0.01) in CgDM9CP-7-siRNA oysters decreased significantly at 6 h after V. splendidus stimulation. These results collectively indicated that CgDM9CP-7 was involved in the regulation of CgMyD88 and CgIL-17 expression in the immune response of oyster.

A galectin-9 involved in the microbial recognition and haemocyte autophagy in the Pacific oyster Crassostrea gigas

Galectin-9 is a tandem-repeat type member of galectin family participating in various immune responses, such as cell agglutination, phagocytosis, and autophagy. In the present study, a tandem repeat galectin-9 (defined as CgGal-9) was identified from Pacific oyster Crassostrea gigas, which consisted of two conserved carbohydrate recognition domains (CRDs) joined by a linker peptide. CgGal-9 was closely clustered with CaGal-9 from C. angulata, and they were assigned into the branch of invertebrate galectin-9s in the phylogenetic tree. The mRNA transcripts of CgGal-9 were detected in all the tested tissues, with the highest expression level in haemocytes. The mRNA expressions of CgGal-9 in haemocytes increased significantly after lipopolysaccharide (LPS) and Vibrio splendidus stimulation. The recombinant CgGal-9 was able to bind all the examined pathogen-associated molecular patterns (LPS, peptidoglycan, and mannose) and microbes (V. splendidus, Escherichia coli, Micrococcus luteus, Staphylococcus aureus, Bacillus subtilis, and Pichia pastoris), and agglutinated most of them in the presence of Ca2+. In CgGal-9-RNAi oysters, the mRNA expressions of autophagy related genes (CgBeclin1, CgATG5, CgP62 and CgLC3) in haemocytes decreased significantly while that of CgmTOR increased significantly at 3 h after V. splendidus stimulation. The autophagy level and mRNA expressions of autophagy related genes decreased in haemocytes after CgGal-9 was blocked by the corresponding antibody. These results revealed that CgGal-9 was able to bind different microbes and might be involved in haemocyte autophagy in the immune response of oyster.

A neural cell adhesion molecule from oyster Crassostrea gigas: Molecular identification and immune functional characterization

Neural cell adhesion molecules (NCAMs) are large cell-surface glycoproteins playing important roles in cell-cell and cell-extracellular matrix interactions in nervous system. Recent study identified a homologue of NCAM (CgNCAM) from the Pacific oyster Crassostrea gigas. Its ORF was of 2634 bp which encodes a protein (877 amino acids) consisting of five immunoglobulin domains and two fibronectin type III domains. CgNCAM transcripts were broadly distributed in oyster tissues especially in mantle, labial palp and haemolymph. CgNCAM showed up-regulated expression in haemocytes of oysters after Vibrio splendidus and Staphylococcus aureus stimulation. The recombinant CgNCAM protein (rCgNCAM) was able to bind manose, lipopolysaccharide and glucan, as well as different microbes including Gram-negative bacteria and fungi. rCgNCAM displayed bacterial agglutination and hemagglutination activity. CgNCAM improved the phagocytosis of haemocytes towards V. splendidus by regulating the expression of CgIntegrin, CgRho J and CgMAPKK. Moreover, CgNCAM was involved in the extracellular trap establishment of haemocytes after V. splendidus stimulation. The results collectively indicated that CgNCAM acted as a recognition receptor executing multiple immune functions to recognize and eliminate invading microorganisms in innate immunity of oysters.

Species-specific CgCaspase-Cg-5 in the pacific oyster induces haemocyte apoptosis by regulating the mRNA expression of apoptosis-related genes in the early stage of immune response.

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 6h 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 12h 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.

A MASP-like functions as PRR to regulate the mRNA expressions of inflammatory factors in the Pacific oyster Crassostrea gigas.

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_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.

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

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

A autophagy related-like protein 16-1 promotes the formation of autophagosomes and autolysosomes in antibacterial immune response of Pacific oyster Crassostrea gigas

Autophagy related 16-like (ATG16L) protein is a core autophagy protein, which promotes the extension of autophagosome membrane through microtubule-associated protein light chain 3 (LC3). In the present study, an ATG16L was identified from oyster Crassostrea gigas (defined as CgATG16L1). The full-length cDNA of CgATG16L1 was of 3184 bp with an open reading frame of 1650 bp that encoded a polypeptide of 549 amino acids. There was an ATG5-interacting motif (AFIM) domain, a coiled-coil (CC) domain and seven tryptophan-aspartic acid 40 (WD40) repeats in CgATG16L1. ATG16L1 mRNA was expressed in all the examined tissues with the highest expression in haemolymph (11.22-fold of that in hepatopancreas, p < 0.05). The mRNA expressions of CgATG16L1 in haemocytes increased significantly at 3, 6, 12, 24 and 72 h after lipopolysaccharide (LPS) stimulation, which were 81.15-fold, 24.95-fold, 6.02-fold, 3.90-fold and 5.97-fold (p < 0.05) of that in control group, respectively. The green positive signals of CgATG16L1 protein and the red positive signals of CgLC3 protein were dotted in the cytoplasm of agranulocytes, semi-granulocytes and granulocytes. The co-localization of CgATG16L1 and CgLC3 was observed in haemocytes after Vibrio splendidus stimulation. In CgATG16L1-RNAi oysters, the number of autophagosomes and autolysosomes in haemocytes was reduced. All these results suggested that CgATG16L1 participated in the bacteria-induced autophagy process in the haemocytes of oyster response to bacteria invasion.

The different expression patterns of cytokines in Pacific oyster Crassostrea gigas response against bacterial stimulation

Cytokines can be induced by pathogen stimulation for the information exchange between cells, and their expression patterns indicate the response status of cells. In the present study, the expression patterns of ten cytokines (CgIL17–1, −2, −3, −4, −5, −6, CgTNF-1/2, CgTGF-β and CgIFNLP) in haemocytes were analyzed in the Pacific oyster Crassostrea gigas after Vibrio splendidus stimulation in the indoor and outdoor experiments to compare the induction patterns of different cytokines and evaluate the response status of haemocytes. After the indoor V. splendidus immersion stimulation, the mRNA abundances of six cytokines (CgIL17–1, −3, −4, −5, CgTGF-β and CgIFNLP) in haemocytes were significantly downregulated at 48 h compared with that in the control group, among which CgIL17–4 was the most down-regulated with 0.009-fold of that in the control group (p < 0.05). The mRNA abundances of CgIL17–2, CgIL17–6, CgTNF-1 and CgTNF-2 showed no significant change at 48 h compared with that in the control group. The apoptosis rate of haemocytes reached the maximum level of 1.138% at 48 h after the stimulation. In the oysters cultured outdoor from April to September 2021, the mRNA abundances of all ten cytokines in haemocytes were found to decrease in June (0.270-fold on average) and increase in July (5.402-fold on average) compared with those in May. After the in situ V. splendidus immersion stimulation in the outdoor cultured oysters in June and July, seven cytokines (CgIL17–1, CgIL17–2, CgIL17–4, CgIL17–5, CgTNF-1, CgTGF-β and CgIFNLP) exhibited upregulated mRNA abundance in June, while in July, the mRNA abundances of five cytokines (CgIL17–1, CgIL17–3, CgTNF-2, CgTGF-β, and CgIFNLP) remained constant, and four cytokines (CgIL17–2, CgIL17–4, CgIL17–5 and CgTNF-1) were suppressed after the stimulation. CgIL17–4 had the highest fold-change in mRNA abundance among all cytokines in June after the stimulation, which was 85.599-fold compared with that in the control group (p < 0.05). The score of haemocyte response status (SH) was evaluated using the mRNA abundance database of all ten cytokines. It decreased to 124.860 at 48 h after the indoor V. splendidus stimulation, and it decreased to 66.793 in June and increased to 451.092 in July in the cultured oysters prior to the stimulation. Collectively, the expression patterns of ten cytokines in haemocytes varied against bacterial stimulation, among which CgIL17–4 was found to response the most significantly, and SH indicated by the mRNA abundances of cytokines suggested an abnormal response status of haemocytes in the oysters at 48 h after indoor V. splendidus stimulation and the oysters cultured outdoor during June and July. These findings will further our understanding of the cytokine variation and haemocyte response of the Pacific oyster against bacterial stimulation.

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

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

The DUF1943 and VWD domains endow Vitellogenin from Crassostrea gigas with the agglutination and inhibition ability to microorganism.

Vitellogenin (Vg) is the major precursor of the egg-yolk proteins, which mainly acts as an energy reserve molecule for providing nutrients during embryonic development. Vg also plays an immune function in vertebrates such as fish, but there are few studies on the immune function of Vg in invertebrates. In the present study, a Vg homologue (CgVg) was identified and characterized in oyster Crassostrea gigas. There are three domains in the CgVg protein, including a Vitellogenin_N domain, a domain of unknown function 1943 (DUF1943) and a von Willebrand factor type D domain (VWD). The mRNA transcripts of CgVg were detected in all tested tissues with high expression in the gonad, hepatopancreas and haemocytes, which was 466.29-, 117.15- and 57.49-fold (p<0.01) of that in adductor muscle, respectively. After Vibrio splendidus stimulation, the mRNA expression level of CgVg in haemocytes increased significantly at 6, 12 and 24h, which was 1.97-, 3.58- and 1.3-fold (p<0.01) of that in the seawater group, respectively. The immunofluorescence assay showed that positive signals of CgVg protein were mainly located at the cytoplasm of haemocytes. The recombinant protein of DUF1943 domain (rDUF1943) and VWD domain (rVWD) was able to bind lipopolysaccharide (LPS), mannose (MAN), peptidoglycan (PGN) and poly (I:C), as well as Gram-positive bacteria (Staphylococcus aureus and Micrococcus luteus), Gram-negative bacteria (Escherichia coli and V. splendidus) and fungi (Pichia pastoris). rDUF1943 exhibited stronger agglutination activity towards S. aureus, M. luteus, E. coli, V. splendidus and P. pastoris, while agglutination was only observed in the rVWD group towards P. pastoris. The rVWD inhibited the growth of E. coli, S. aureus and V. splendidus, while no antibacterial activity was detected in rDUF1943 group. Collectively, CgVg not only functioned as a pattern recognition receptor (PRR) to bind various microorganisms and PAMPs, but also as an immune effector participating in the clearance of invaders, in which DUF1943 and VWD domain were mainly responsible for agglutinating and inhibiting microorganism respectively.

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

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

An immunoglobulin superfamily member (CgIgIT2) functions as immune inhibitory receptor to inhibit the inflammatory cytokine expressions in Crassostrea gigas

Immune inhibitory receptors are increasingly acknowledged as potent regulators of immune response, which inhibit the overactivation of immune system and play an important role in maintaining immune homeostasis. In the present study, a novel immunoglobulin superfamily member (CgIgIT2) was identified from the Pacific oyster, Crassostrea gigas. The protein sequence of CgIgIT2 contained one signal peptide, four Ig domains, one fibronectin type III domain, one transmembrane domain, and a cytoplasmic tail with two intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and one immunoreceptor tyrosine-based switch motif (ITSM). The mRNA transcripts of CgIgIT2 were widely expressed in all the tested tissues, including haemolymph, gill, mantle, adductor muscle, labial palp, gonad and hepatopancreas, with the highest expression in haemolymph. The mRNA expressions of CgIgIT2 in haemocytes increased significantly at 24, 48 and 72 h after Vibrio splendidus stimulation. The positive green signals of CgIgIT2 protein were mainly detected in granulocytes of haemocytes, which were 1.27-fold and 2.15-fold (p < 0.05) higher than that of semi-granulocytes and agranulocytes, respectively. And CgIgIT2 was mainly located in the membrane and cytoplasm of haemocytes. The recombinant protein of CgIgIT2-4 × Ig (rCgIgIT2-4 × Ig) exhibited binding activity towards multiple pathogen-associated molecular patterns (PAMPs), including lipopolysaccharides (LPS), peptidoglycan (PGN), mannose (MAN) and polyinosinic-polycytidylic acid (Poly (I: C)) with the highest affinity for LPS. rCgIgIT2-4 × Ig could also bind Gram-negative bacteria (V. splendidus, V. anguillarum, Escherichia coli), Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis), and fungi (Pichia pastoris). In the blocking assay with anti-CgIgIT2 antibody, the mRNA expressions of interleukins (CgIL17-1, CgIL17-3 and CgIL17-6) and tumor necrosis factors (CgTNF-1 and CgTNF-2) in haemocytes all increased significantly at 12 h after V. splendidus stimulation. These results suggested that CgIgIT2 could function as an inhibitor receptor to bind different PAMPs and microbes, as well as inhibit the mRNA expressions of multiple inflammatory cytokines in oysters.