Splendidus Stimulation Research Articles

CgBlimp-1 inhibits granulocytes proliferation and interleukin production in the immune response of oyster Crassostrea gigas

B lymphocyte-inducible maturation protein 1 (Blimp-1) is a SET domain and zinc fingers containing transcriptional repressor, which is necessary for regulating the development of many immune cell lineages and keeping immune homeostasis. In the present study, a Blimp-1 homologue (designated as CgBlimp-1) was identified from oyster Crassostrea gigas, which contained a conserved SET domain and five ZnF_C2H2 domains and shared high homology with Blimp-1 from other species. The mRNA transcripts of CgBlimp-1 were highly expressed in gill and hepatopancreas. CgBlimp-1 protein was detected to be specifically expressed in granulocytes. After V. splendidus stimulation, the mRNA expression level of CgBlimp-1 in haemocytes up-regulated significantly at 24, 48, and 96 h, which was 4.39-fold (p < 0.05), 7.68-fold (p < 0.01) and 2.65-fold (p < 0.05) of that in control group, respectively. When the expression of CgBlimp-1 was knocked-down in vivo by RNAi, the mRNA expressions of downstream transcription factor CgMyc-A (1.63-fold of that in control group, p < 0.05) and cell cycle related gene CgCDK2 (1.70-fold, p < 0.05) increased significantly at 24 h after V. splendidus stimulation. Concomitantly, the ratio of EdU+ haemocytes increased notably (p < 0.01) while the proportion of haemocytes in G0/G1 phase decreased dramatically (p < 0.001), compared to that in control group. More specifically, the proportion of granulocytes in total haemocytes increased apparently (p < 0.05) in CgBlimp-1-RNAi oysters, together with up-regulation (p < 0.05) of the ratio of EdU+ granulocytes and down-regulation (p < 0.001) of the proportion of granulocytes in G0/G1 phase. Furthermore, the mRNA expression levels of CgIL17-1, CgIL17-2 and CgIL17-4 in haemocytes increased significantly in CgBlimp-1-RNAi oysters, which was 1.71-fold (p < 0.05), 144.70-fold (p < 0.01) and 1.93-fold (p < 0.05) of that in control group, respectively. Aforementioned results suggested that CgBlimp-1 could reduce the proliferation of granulocytes by arresting cell cycle in G1/G0 phase and avoid over-expression of interleukin to maintain homeostasis in the immune response of oyster.

Syk regulates the haemocyte autophagy through inducing the mRNA expressions of autophagy-related genes and the cleavage of CgLC3 in oyster antibacterial immunity

Spleen tyrosine kinase (Syk) is reported to be involved in activating the autophagy. Recently, a homologue of Syk was identified from Pacific oyster Crassostrea gigas (defined as CgSyk). In the present study, the molecular characteristics of CgSyk and its regulation mechanism in autophagy were investigated in oyster C. gigas. The full-length cDNA of CgSyk was of 4566 bp with an open reading frame (ORF) of 1989 bp. CgSyk encoded a polypeptide of 662 amino acids, containing two Src homology 2 (SH2) domains and one tyrosine kinase catalytic (TyrKc) domain. The deduced amino acid sequence of CgSyk shared low similarity with the previously identified Syks from other species. In the phylogenetic tree, CgSyk was first clustered with Crassostrea virginica CvSyk, and then classified into a branch of invertebrate Syks. In CgSyk-RNAi oysters, the mRNA expressions of CgLC3, CgP62, CgBeclin-1 and CgATG5 in haemocytes decreased significantly at 12 h after Vibrio splendidus stimulation. At the same time, the abundance of CgLC3Ⅱ in haemocytes, and the autophagy rate of haemocytes in CgSyk-RNAi oysters decreased significantly at 12 h after V. splendidus stimulation. All the results collectively suggested that CgSyk regulated the autophagy through inducing the mRNA expressions of autophagy-related genes and the cleavage of CgLC3 to defend against bacterial invasion in oysters.

Histone lysine-specific demethylase 1 regulates the proliferation of hemocytes in the oyster Crassostrea gigas.

Lysine-specific demethylase 1 (LSD1) is an essential epigenetic regulator of hematopoietic differentiation, which can specifically mono-methylate H3K4 (H3K4me1) and di-methylate H3K4 (H3K4me2) as a transcriptional corepressor. Previous reports have been suggested that it participated in hematopoiesis and embryonic development process. Here, a conserved LSD1 (CgLSD1) with a SWIRM domain and an amino oxidase (AO) domain was identified from the Pacific oyster Crassostrea gigas. We conducted a comprehensive analysis by various means to verify the function of CgLSD1 in hematopoietic process, including quantitative real-time PCR (qRT-PCR) analysis, western blot analysis, immunofluorescence assay, RNA interference (RNAi) and flow cytometry. The qRT-PCR analysis revealed that the transcripts of CgLSD1 were widely expressed in oyster tissues with the highest level in the mantle. And the transcripts of CgLSD1 were ubiquitously expressed during larval development with the highest expression level at the early D-veliger larvae stage. In hemocytes after Vibrio splendidus stimulation, the transcripts of CgLSD1 were significantly downregulated at 3, 6, 24, and 48 h with the lowest level at 3 h compared to that in the Seawater group (SW group). Immunocytochemical analysis showed that CgLSD1 was mainly distributed in the nucleus of hemocytes. After the CgLSD1 was knocked down by RNAi, the H3K4me1 and H3K4me2 methylation level significantly increased in hemocyte protein. Besides, the percentage of hemocytes with EdU-positive signals in the total circulating hemocytes significantly increased after V. splendidus stimulation. After RNAi of CgLSD1, the expression of potential granulocyte markers CgSOX11 and CgAATase as well as oyster cytokine-like factor CgAstakine were increased significantly in mRNA level, while the transcripts of potential agranulocyte marker CgCD9 was decreased significantly after V. splendidus stimulation. The above results demonstrated that CgLSD1 was a conserved member of lysine demethylate enzymes that regulate hemocyte proliferation during the hematopoietic process.

An ATP-binding cassette transporter G2 (CgABCG2) regulates the haemocyte proliferation by modulating the G1/S phase transition of cell cycle in oyster Crassostrea gigas

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

CgCaspase-3 activates the translocation of CgGSDME in haemocytes of Pacific oyster Crassostrea gigas

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

The expression profile of a multi-stress inducible transient receptor potential vanilloid 4 (TRPV4) in Pacific oyster Crassostrea gigas

Transient receptor potential vanilloid 4 (TRPV4) is one of the major non-selective cation channel proteins, which plays a crucial role in sensing biotic and abiotic stresses, such as pathogen infection, temperature, mechanical pressure and osmotic pressure changes by regulating Ca2+ homeostasis. In the present study, a TRPV4 homologue was identified in Pacific oyster Crassostrea gigas, designated as CgTRPV4. The open reading frame (ORF) of CgTRPV4 was of 2298 bp encoding a putative polypeptide of 765 amino acid residues with three typical ankyrin domains and six conserved transmembrane domains of TRPV4 subfamily proteins, as well as multiple N-glycosylation sites, cAMP- and cGMP-dependent protein kinase phosphorylation sites, protein kinase C phosphorylation sites, casein kinase II phosphorylation sites, and prokaryotic membrane lipoprotein lipid attachment site. The deduced amino acid sequence of CgTRPV4 shared 20.5%-26.2% similarity with TRPV4s from other species. During the early ontogenesis stages of oyster, the mRNA transcripts of CgTRPV4 were detectable in all the stages with the highest expression level in fertilized eggs and the lowest in D-hinged larvae. In adult oyster, the CgTRPV4 mRNA could be detected in all the examined tissues, including gill, hepatopancreas, adductor muscle, labial palp, mantle and haemocyte, with the highest expression level in gill (45.08-fold of that in hepatopancreas, p < 0.05). In immunocytochemical assay, the CgTRPV4 positive signals were distributed in both endoplasmic reticulum and cytoplasmic membrane of oyster haemocytes. The mRNA expression of CgTRPV4 in gill was significantly up-regulated after high temperature stress at 30°C (p < 0.05) and after Vibrio splendidus stimulation (p < 0.05). These results indicated that CgTRPV4 was a classical member of TRPV4 family in oyster, which was induced by either biotic or abiotic stimulations and involved in mediating the stress response of oysters.

A stimulator of interferon gene (CgSTING) involved in antimicrobial immune response of oyster Crassostrea gigas

The stimulator of interferon gene (STING), an intracellular sensor of cyclic dinucleotides, is critical to the innate immune response, especially the induction of type I interferon (IFN) during pathogenic infection. A STING homologue (CgSTING) regulating the expression of IFN-like protein (CgIFNLP) was previously identified in the Pacific oyster Crassostrea gigas, and its involvement in antibacterial immunity was further investigated in the present study. The mRNA transcripts of CgSTING were ubiquitously detected in all the three subpopulations of haemocytes with the highest expression in semi-granulocytes. After the stimulation with Vibrio splendidus, the mRNA expression of CgSTING in haemocytes was significantly up-regulated and peaked at 72 h, which was 12.91-fold of that in control group (p < 0.01). The CgSTING protein was mainly located in the cytoplasm of haemocytes. After the expression of CgSTING was knocked down (0.12-fold of that in control group, p < 0.05) by RNAi, the mRNA expression levels of interleukin17-1 (CgIL17-1), interleukin17-3 (CgIL17-3), interleukin17-4 (CgIL17-4), defensins (Cgdefh1, Cgdefh2), big defensin (CgBigDef1), interferon-like protein (CgIFNLP), tumor necrosis factor (CgTNF) and nuclear factor-κB (CgRel) all decreased significantly at 12 h after V. splendidus stimulation, which was 0.12-fold–0.72-fold (p < 0.05) of that in control group, respectively. The positive signals of CgRel were observed in the haemocyte nucleus after V. splendidus stimulation. The nuclear translocation of CgRel was suppressed in CgSTING-RNAi oysters, and the green signals of CgRel were mainly observed in the haemocyte cytoplasm after V. splendidus stimulation. Furthermore, the number of V. splendidus in the haemolymph of CgSTING-RNAi oysters increased significantly, which was 26.78-fold (p < 0.01) of that in the control group at 12 h after V. splendidus stimulation. These results indicated that CgSTING played important role in the immune defense against bacterial infection by inducing the expressions of cytokines and defensins.

Proteomic analysis of exosomes in pacific oyster Crassostrea gigas during bacterial stimulation

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

The truncated MyD88s negatively regulates TLR2 signal on expression of IL17-1 in oyster Crassostrea gigas

Toll like receptor (TLR) signaling plays a key role in the innate immune recognition and inflammatory regulation in both vertebrates and invertebrates. The expanded TLR signaling components, including 83 TLRs and 10 MyD88s, have been reported in the genome of the Pacific oyster Crassostrea gigas. In the present study, one endogenous TLR (designated CgTLR2) and two MyD88s (including a full-length CgMyD88-2 containing intact TIR domain and Death-domain, and a truncated CgMyD88s with only TIR domain) were identified from oyster C. gigas. CgTLR2 was highly expressed in haemocytes, especially in granulocytes. The recombinant protein of the extracellular LRR domains of CgTLR2 recognized and bound a variety of PAMPs with the strongest binding capability to LPS. The recombinant protein of intracellular TIR domain of CgTLR2 was able to bind the recombinant proteins of rCgMyD88-2 (KD = 1.96 × 10−9 M) and rCgMyD88s (KD = 4.84 × 10−8 M), with higher affinity towards rCgMyD88-2. After Vibrio splendidus stimulation, the mRNA expression levels of CgTLR2 and CgMyD88-2 were rapidly up-regulated at early stage of immune response (from the 3rd hours after V. splendidus stimulation), while that of CgMyD88s did not change until 24 h post stimulation. When CgTLR2 was knocked-down by siRNA interference, the expression levels of CgMyD88-2 and CgMyD88s decreased significantly, concomitant with the down-regulation of expression of CgIL17-1. After the expression of CgMyD88-2 was interfered, the expressions of CgMyD88s and CgIL17-1 were all decreased. In contrast, after the expression of CgMyD88s was interfered, the expressions of CgMyD88-2 and CgIL17-1 all increased. The results showed that CgMyD88s played a negative role in the regulation of CgTLR2 on inflammatory factor CgIL17-1.

CgHMGB1 functions as a broad-spectrum recognition molecule to induce the expressions of CgIL17-5 and Cgdefh2 via MAPK or NF-κB signaling pathway in Crassostrea gigas

High-mobility group box 1 (HMGB1), a highly conserved nucleoprotein, functions in immune recognition, inflammation and antibacterial immunization in vertebrates. In the present study, the mediation mechanism of CgHMGB1 in activating MAPK and NF-κB/Rel signaling pathways to induce the expressions of immune effectors was investigated. CgHMGB1 mRNA was detected in all tested developmental stages from fertilized egg to D-larvae, with the higher expressions in 4 cells and 8 cells stages. CgHMGB1 proteins were mainly distributed in haemocyte granulocytes. The expressions of CgHMGB1 mRNA in haemocytes increased significantly after Vibrio splendidus stimulation, and CgHMGB1 protein translocated into the haemocyte cytoplasm and release into cell-free haemolymph. The phosphorylation of CgERK and CgP38 were induced, the nuclear translocation of CgRel were promoted, and the mRNA expressions of CgIL17-5 and Cgdefh2 increased significantly after rCgHMGB1 treatment. Obvious branchial swelling and cilium shedding were observed after rCgHMGB1 treatment. rCgHMGB1 exhibited binding activity to different polysaccharides, bacteria, and fungi. rCgHMGB1 also displayed obvious antibacterial activity to V. splendidus and E. coli. These results indicated that CgHMGB1 functioned as an immune recognition molecule to recognize various PAMPs and bacteria to induce the mRNA expressions of CgIL17-5 and Cgdefh2 via the activation of MAPK and NF-κB signaling pathways in oysters.

The receptor CgIL-17R1 expressed in granulocytes mediates the CgIL-17 induced haemocytes proliferation in Crassostrea gigas

Inflammatory cytokine interleukin-17 (IL-17) binds its receptors (IL-17Rs) to activate the downstream immune signals and plays an important role in host defense. In the present study, an IL-17 receptor (designated as CgIL-17R1) was identified from oyster Crassostrea gigas with an open reading frame of 3141 bp encoding 1047 amino acids. The amino acid sequence of CgIL-17R1 with two conserved FN3 domains shared higher similarity with other known IL-17Rs from mollusc species. The recombinant CgIL-17R1 protein (rCgIL-17R1) displayed high binding affinity to the recombinant CgIL-17 protein (rCgIL-17) in vitro. The mRNA transcripts of CgIL-17R1 were significantly higher expressed in haemocytes, especially in granunolyctes, compared with that in other tissues. After the stimulation with Vibrio splendidus or rCgIL17-1 in vivo, the expressions of CgIL-17R1 and cell proliferation related genes (CgRunx-1, CgCDC-6, CgCDC-45, and CgCDK-2) were significantly up-regulated in haemocytes (p < 0.01). When the CgIL-17R1 expression was interfered by specific CgIL-17R1-dsRNA, the expressions of these cell proliferation related genes reduced significantly, and the proliferation rate of haemocytes declined dramatically at 6 h post V. splendidus stimulation (p < 0.01), compared to that of blank group. These results collectively indicated that CgIL-17R1 expressed in granulocytes mediated the CgIL-17 induced haemocytes proliferation during immune response in oyster C. gigas, which provided novel information about the regulation of haemocyte proliferation in invertebrates.

The proliferating cell nuclear antigen (PCNA) is a potential proliferative marker in oyster Crassostrea gigas

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

CgRab1 regulates Cgcathepsin L1 expression and participates in the phagocytosis of haemocytes in oyster Crassostrea gigas

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

BCL10 regulates the production of proinflammatory cytokines by activating MAPK–NF–κB/Rel signaling pathway in oysters

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.

A novel C-type lectin activates the complement cascade in the primitive oyster Crassostrea gigas

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.

The involvement of CgCaspase-8-2 in regulating the expressions of cytokines, antibacterial peptide and autophagy-related genes in oysters

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

CgIL17-5 regulates the mRNA expressions of immune effectors through inducing the phosphorylation of CgMAPKs and the nuclear translocation of CgRel and CgAP-1 in the Pacific oyster Crassostrea gigas

Interleukin-17 (IL-17) is a classic pro-inflammatory cytokine that plays an important role in the immune and inflammatory response. In the present study, the sequence feature of CgIL17-5 and its function as a pro-inflammatory factor in inducing the mRNA expressions of downstream immune effectors were investigated in oyster Crassostrea gigas. There were two tightly folded alpha helixes and two pairs of antiparallel beta-pleated sheet in the amino acid sequence of CgIL17-5. The mRNA transcripts of CgIL17-5 were constitutively distributed in all the tested tissues, with the highest level in haemocytes. The mRNA expression level of CgIL17-5 in haemocytes increased significantly at 24 h after Vibrio splendidus stimulation. CgIL17-5 protein was mainly detected in granulocytes which were the main immunocompetent haemocytes in C. gigas. The phosphorylation of mitogen-activated protein kinases (CgJNK, CgERK and CgP38) and nuclear translocation of the transcription factors (CgRel and CgAP-1) in haemocytes were induced after the oysters received an injection of recombinant CgIL17-5 for 2 h. The mRNA expression levels of CgIL-17s, CgTNF-1, Cgdefh1 and Cgdefh2 increased significantly in haemocytes. At the same time, obvious branchial swelling and cilium shedding in gills were observed at 24 h after the oysters received an injection of rCgIL17-5. All the results collectively suggested that CgIL17-5 promoted the activation of CgMAPKs and the nuclear translocation of CgRel and CgAP-1 to promote the mRNA expressions of cytokines and antibacterial peptides.

A tandem-repeat galectin-1 from Apostichopus japonicus with broad PAMP recognition pattern and antibacterial activity

Galectins belong to the family of carbohydrate-binding proteins and play major roles in the immune and inflammatory responses of both vertebrates and invertebrates. In the present study, one novel galectin-1 protein named AjGal-1 was identified from Apostichopus japonicas with an open reading frame of 1179 bp encoding a polypeptide of 392 amino acids. The deduced amino acids sequence of AjGal-1 contained three carbohydrate recognition domains (CRDs) which shared 34–37% identity with that of other galectin proteins from echinodermata, fishes, and birds. In the phylogenetic tree, AjGal-1 was closely clustered with galectins from Mesocentrotus nudus and Paracentrotus lividus. The mRNA transcripts of AjGal-1 were ubiquitously expressed in all the detected tissues, including gut, longitudinal muscle, gonad, coelomocytes, respiratory tree, tentacle and body wall, with the highest expression level in coelomocytes. After Vibrio splendidus stimulation, the mRNA expression levels of AjGal-1 in coelomocytes were significantly increased at 6 and 12 h (P < 0.01) compared with that in control group, and went back to normal level at 72 h. The recombinant protein of AjGal-1 (rAjGal-1) could bind various PAMPs including d-galactose, lipopolysaccharide (LPS), peptidoglycan (PGN) and mannose (Man), and exhibited the highest affinity to d-galactose. Meanwhile, rAjGal-1 could also bind and agglutinate different kinds of microorganisms, including gram-negative bacteria (V. splendidus and Escherichia coli), gram-positive bacteria (Micrococus leteus), and fungi (Pichia pastoris). rAjGal-1 also exhibited anti-microbial activity against V. splendidus and E. coli. All these results suggested that AjGal-1 could function as an important PRR with broad spectrum of microbial recognition and anti-microbial activity against the invading pathogen in A. japonicas.

CgRel involved in antibacterial immunity by regulating the production of CgIL17s and CgBigDef1 in the Pacific oyster Crassostrea gigas

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

Stomach metabolic alterations in response to AHPND infection in shrimp

Tetraspanins belong to the transmembrane 4 superfamily (TM4SF), and play crucial roles in immune responses. In the present study, a novel tetraspanin gene (designated MmTSPAN) was cloned and characterized from the hard clam Meretrix meretrix. The complete cDNA sequence of MmTSPAN contained an open reading frame (ORF) of 816 bp, which encoded a protein of 271 amino acids. MmTSPAN exhibited highly similarity with previously identified tetraspanins from other species. It contained four transmembrane domains (12–35 aa, 69–92 aa, 99–123 aa and 238–261 aa), characteristic CCG motif and four conservative cysteine residues. The mRNA transcripts of MmTSPAN were ubiquitously detectable in all the tested tissues, with the highest expression level in hepatopancreas. Temporal transcriptional levels in the hepatopancreas revealed significant up-regulation of MmTSPAN by Vibrio splendidus stimulation, with a 3.14-fold increase at 6 h compared to the control, and reaching 32.98-fold at 24 h. These results provide useful information for further study of the function of tetraspanin in the innate immune system of M. meretrix, and may offer a new therapeutic target for diseases of M. meretrix.