Immunomodulatory effects of a probiotic combination treatment to improve the survival of Pacific oyster (Crassostrea gigas) larvae against infection by Vibrio coralliilyticus.

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 105 CFU/mL at 18 hours post-fertilization (hpf), b) pathogenic V. coralliilyticus RE22 at a concentration of 6.0 x 103 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.

A trace amine associated receptor mediates antimicrobial immune response in the oyster Crassostrea gigas.

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_classA_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.

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.

The involvement of interferon regulatory factor 8 in regulating the proliferation of haemocytes in oyster Crassostrea gigas.

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.

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.

Immune responses of oyster hemocyte subpopulations to in vitro and in vivo zinc exposure.

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.

A novel toll-like receptor from Crassostrea gigas is involved in innate immune response to Vibrio alginolyticus.

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.

Effect of hypo- and hypersaline stress conditions on physiological, metabolic, and immune responses in the oyster Crassostrea corteziensis (Bivalvia: Ostreidae).

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.

Functional heterogeneity of immune defenses in molluscan oysters Crassostrea hongkongensis revealed by high-throughput single-cell transcriptome.

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.

Virulence of Vibrio alginolyticus Accentuates Apoptosis and Immune Rigor in the Oyster Crassostrea hongkongensis.

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. alginolyticusWT 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. alginolyticusWT infection, whereas phagocytosis and cell adhesion were enhanced in V. alginolyticus△vscC infection. Results for functional studies further show that V. alginolyticusWT strain stimulated respiratory bursts to produce excess superoxide (O2•-) and hydrogen peroxide (H2O2) 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.

Identification and characterization of Crassostrea angulata arginine kinase, a novel allergen that causes cross-reactivity among shellfish.

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.

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.

IgE epitope analysis of sarcoplasmic-calcium-binding protein, a heat-resistant allergen in Crassostrea angulata.

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 (AA22-33), L-SCP-2 (AA64-75), L-SCP-3 (AA80-90), L-SCP-4 (AA107-116), and L-SCP-5 (AA144-159) 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 (AA106-117). Moreover, SCP showed weaker typical α-helical features and higher hydrophobicity after Ca2+ 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.

A novel CgIFNLP receptor involved in regulating ISG expression in oyster Crassostrea gigas.

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.

An HECT domain ubiquitin ligase CgWWP1 regulates granulocytes proliferation in oyster Crassostrea gigas.

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.

A novel C-type lectin from Crassostrea gigas involved in the innate defense against Vibrio alginolyticus.

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.

The cGAS/STING-TBK1-IRF Regulatory Axis Orchestrates a Primitive Interferon-Like Antiviral Mechanism in Oyster.

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 (CgIFNLP) from Crassostrea gigas appeared to function as IFN-like antiviral cytokines. In the present study, the CgIFNLP protein in hemocytes was observed to increase after Poly (I:C) stimulation. After CgIFNLP was knocked down by RNAi, the mRNA expression of IFN-stimulated genes (CgISGs) was significantly inhibited. Both cyclic GMP-AMP synthase (CgcGAS) and stimulator of interferon gene (CgSTING) 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 CgIFNLP and interferon regulatory factors (CgIRF1/8) and the nuclear translocation of CgIRF8 were all suppressed in CgcGAS-RNAi or CgSTING-RNAi oysters after Poly (I:C) stimulation. The expression level of CgSTING and TANK binding kinase1 (CgTBK1) did not decrease in CgcGAS-RNAi oysters. After CgSTING was knocked down, the high expression of CgTBK1 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.

A calmodulin targeted by miRNA scaffold659_26519 regulates IL-17 expression in the early immune response of oyster Crassostrea gigas.

Calmodulin (CaM) is a highly conserved second messenger protein transducing calcium signals by binding and modulating intracellular calcium ions (Ca2+), and involves in the Ca2+-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 Ca2+ (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_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_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_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_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.

A fibrinogen-related protein mediates the recognition of various bacteria and haemocyte phagocytosis in oyster Crassostrea gigas.

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 Ca2+. 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.