CgDM9CP-5-Integrin-MAPK Pathway Regulates the Production of CgIL-17s and Cgdefensins in the Pacific Oyster, Crassostrea gigas.

DM9 domain containing protein (DM9CP) is a family of newly identified recognition receptors exiting in most organisms except plants and mammals. In the current study, to our knowledge, a novel DM9CP-5 (CgDM9CP-5) with two tandem DM9 repeats and high expression level in gill was identified from the Pacific oyster, Crassostrea gigas. The deduced amino acid sequence of CgDM9CP-5 shared 62.1% identity with CgDM9CP-1 from C. gigas, and 47.8% identity with OeFAMeT from Ostrea edulis. The recombinant CgDM9CP-5 (rCgDM9CP-5) was able to bind d-mannose, LPS, peptidoglycan, and polyinosinic-polycytidylic acid, as well as fungi Pichia pastoris, Gram-negative bacteria Escherichia coli and Vibrio splendidus, and Gram-positive bacteria Staphylococcus aureus. The mRNA transcript of CgDM9CP-5 was highly expressed in gill, and its protein was mainly distributed in gill mucus. After the stimulations with V. splendidus and mannose, mRNA expression of CgDM9CP-5 in oyster gill was significantly upregulated and reached the peak level at 6 and 24 h, which was 13.58-fold (p < 0.05) and 14.01-fold (p < 0.05) of that in the control group, respectively. CgDM9CP-5 was able to bind CgIntegrin both in vivo and in vitro. After CgDM9CP-5 or CgIntegrin was knocked down by RNA interference, the phosphorylation levels of JNK and P38 in the MAPK pathway decreased, and the expression levels of CgIL-17s (CgIL-17-3, -4, -5, and -6), Cg-Defh1, Cg-Defh2, and CgMolluscidin were significantly downregulated. These results suggested that there was a pathway of DM9CP-5-Integrin-MAPK mediated by CgDM9CP-5 to regulate the release of proinflammatory factors and defensins in C. gigas.

Involvement of metabotropic glutamate receptors in regulation of immune response in the Pacific oyster Crassostrea gigas.

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

CgADAR1 involved in regulating the synthesis of interferon-like protein in Crassostrea gigas.

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

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.

An LPS-induced TNF-α factor involved in immune response of oyster Crassostrea gigas by regulating haemocytes apoptosis.

LPS induced TNF-α Factor (LITAF) is a transcription factor widely involving in activation of Tumor Necrosis Factor (TNF) and other cytokines in the inflammatory response. In the present study, a homologue of LITAF with a conserved LITAF domain was identified from the Pacific oyster Crassostrea gigas. The transcripts of CgLITAF were detected in all examined tissues with highest expression in hepatopancrease. The immunofluorescence assay and Western blot showed that LPS stimulation induced an obvious nucleus translocation of CgLITAF protein in haemocytes. While the mRNA level of CgLITAF changed slightly after LPS stimulation. When the siRNA of CgLITAF was injected to inhibit its expression, the apoptotic level of haemocytes decreased observably after LPS stimulation. Consistently, the transcripts of CgTNF3 and CgTNF4 (LOC105343080, LOC105341146), the apoptotic-related molecules including CgBax, CgCytochrome c, CgCaspase9 and CgCaspase3, were significantly suppressed in the CgLITAF-RNAi oysters. While the mRNA expression level of CgBcl was enhanced significantly in the CgLITAF-RNAi oysters. These results indicated that CgLITAF promoted haemocyte apoptosis by regulating the expression of apoptotic-related factors, suggesting its important role in the immune response of oysters.

A DM9-containing protein from crab Eriocheir sinensis functions as a novel multipotent pattern recognition receptor.

DM9-containing protein in invertebrates functions as pattern recognition receptor (PRR) to play significant roles in innate immunity. In the present study, a novel DM9-containg protein (defined as EsDM9CP-1) was identified from the Chinese mitten crab Eriocheir sinensis. EsDM9CP-1 is composed of 330 amino acids containing a Methyltransf_FA domain and two tandem DM9 repeats. The deduced amino acid sequence of EsDM9CP-1 shared low similarity with the previously identified DM9CPs from other species, and it was closely clustered with Platyhelminthes DM9CPs and then assigned into the branch of invertebrate DM9CPs in the unrooted phylogenetic tree. The mRNA transcripts of EsDM9CP-1 were highly expressed in haemocytes, gill, and heart. After Aeromonas hydrophila stimulation, the expression levels of EsDM9CP-1 mRNA in haemocytes increased significantly at 3 h (3.88-fold, p < 0.05) and 6 h (2.71-fold, p < 0.05), compared with that of PBS group, respectively. EsDM9CP-1 protein was mainly distributed in the cytoplasm and membrane of haemocytes. The recombinant EsDM9CP-1 protein (rEsDM9CP-1) exhibited binding affinity to MAN, PGN, LPS and Poly (I:C), and also to Gram-positive bacteria (Staphylococcus aureus, Micrococcus luteus and Bacillus subtilis), Gram-negative bacteria (Escherichia coli, A. hydrophila and Vibrio splendidus) and fungi (Pichia pastoris and Metschnikowia bicuspidata) in a Ca2+-dependent manner. It was able to agglutinate A. hydrophila, S. aureus, M. luteus, M. bicuspidata and P. pastoris, and inhibit the growth of A. hydrophila and M. bicuspidate. These results suggested that EsDM9CP-1 in crab not only functioned as a PRR, but also agglutinated and inhibited the growth of microbes.

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 Modification of H3K4me3 Enhanced the Expression of CgTLR3 in Hemocytes to Increase CgIL17-1 Production in the Immune Priming of Crassostrea gigas.

Increasing evidence confirms that histone modification plays a critical role in preserving long-term immunological memory. Immune priming is a novel form of immunological memory recently verified in invertebrates. Toll-like receptor (TLR) signaling and cytokines have been reported to be involved in the immune priming of the Pacific oyster Crassostrea gigas. In the present study, the expression of Toll-like receptor 3 (CgTLR3), myeloid differentiation factor 88-2 (CgMyd88-2) and interleukin 17-1 (CgIL17-1) was found to be elevated in the hemocytes of C. gigas at 6 h after the secondary stimulation with Vibrio splendidus, which was significantly higher than that at 6 h after the primary stimulation (p < 0.05). A significant increase in histone H3 lysine 4 trimethylation (H3K4me3) enrichment was detected in the promoter region of the CgTLR3 gene at 7 d after the primary stimulation with inactivated V. splendidus (p < 0.05). After the treatment with a histone methyltransferase inhibitor (5'-methylthioadenosine, MTA), the level of H3K4me3 at the promoter of the CgTLR3 gene decreased significantly at 7 d after the primary stimulation with inactivated V. splendidus (p < 0.05), and the expression of CgTLR3, CgMyD88-2 and CgIL17-1 was significantly repressed at 6 h after the secondary stimulation with V. splendidus (p < 0.05). Conversely, the treatment with monomethyl fumarate (MEF, an inhibitor of histone demethylases) resulted in a significant increase in H3K4me3 enrichment levels at the CgTLR3 promoter at 7 d after the primary stimulation (p < 0.05), and the expression of CgTLR3, CgMyD88-2 and CgIL17-1 was observed to increase significantly at 6 h after the secondary stimulation (p < 0.05). These results suggested that H3K4me3 regulated MyD88-dependent TLR signaling in the hemocytes of C. gigas, which defined the role of histone modifications in invertebrate immune priming.

Chromosome-level genome assembly of the deep-sea snail Phymorhynchus buccinoides provides insights into the adaptation to the cold seep habitat.

BACKGROUND: The deep-sea snail Phymorhynchus buccinoides belongs to the genus Phymorhynchus (Neogastropoda: Raphitomidae), and it is a dominant specie in the cold seep habitat. As the environment of the cold seep is characterized by darkness, hypoxia and high concentrations of toxic substances such as hydrogen sulfide (H2S), exploration of the diverse fauna living around cold seeps will help to uncover the adaptive mechanisms to this unique habitat. In the present study, a chromosome-level genome of P. buccinoides was constructed and a series of genomic and transcriptomic analyses were conducted to explore its molecular adaptation mechanisms to the cold seep environments. RESULTS: The assembled genome size of the P. buccinoides was approximately 2.1 Gb, which is larger than most of the reported snail genomes, possibly due to the high proportion of repetitive elements. About 92.0% of the assembled base pairs of contigs were anchored to 34 pseudo-chromosomes with a scaffold N50 size of 60.0 Mb. Compared with relative specie in the shallow water, the glutamate regulative and related genes were expanded in P. buccinoides, which contributes to the acclimation to hypoxia and coldness. Besides, the relatively high mRNA expression levels of the olfactory/chemosensory genes in osphradium indicate that P. buccinoides might have evolved a highly developed and sensitive olfactory organ for its orientation and predation. Moreover, the genome and transcriptome analyses demonstrate that P. buccinoides has evolved a sulfite-tolerance mechanism by performing H2S detoxification. Many genes involved in H2S detoxification were highly expressed in ctenidium and hepatopancreas, suggesting that these tissues might be critical for H2S detoxification and sulfite tolerance. CONCLUSIONS: In summary, our report of this chromosome-level deep-sea snail genome provides a comprehensive genomic basis for the understanding of the adaptation strategy of P. buccinoides to the extreme environment at the deep-sea cold seeps.

Synergistic modulation of neuroendocrine-inflammation pathway by microRNAs facilitates intertidal adaptation of molluscs.

Neuroendocrine-immune system is an evolution-conserved regulatory network in maintaining the homeostasis of animals. While knowledge on the roles of neuroendocrine-immune system in the disease and stress responses of organisms is growing, the ecological roles of neuroendocrine-immune system, especially how it shapes the unique lifestyle of organisms remain insufficiently investigated. As an endemic and dominant mollusc in intertidal region, oysters have evolved with a primitive neuroendocrine-immune system and with a sessile lifestyle. Recently, a novel neuroendocrine-immune pathway, Ca2+/calmodulin (CaM)-nitrite oxide synthase (NOS)/nitrite oxide (NO)-tumor necrosis factor (TNF) pathway, is identified in oysters and found altered dynamically during aerial exposure, one common but challenging stresses for intertidal organisms and a decisive factor shaping their habitat. Since the pathway proves fatal in prolonged aerial exposure, we hypothesized that the activation/deactivation of pathway could be strictly modulated in adaptation to the sessile lifestyle of oysters. Here, a synergistic modulation on the Ca2+/CaM-NOS/NO-TNF pathway by four members of miR-92 family and two oyster-specific miRNAs was identified, which further hallmarks the resilience and survival strategy of oysters to aerial exposure. Briefly, these six miRNAs were down-regulating CgCaM24243 post-transcriptionally and deactivating the pathway during the early-stage of stress. However, a robust recession of these miRNAs occurred at the late-stage of stress, resulting in the reactivation of pathway and overwhelming accumulation of cytokines. These results demonstrated a complicated interaction between miRNAs and ancient neuroendocrine-immune system, which facilitates the environmental adaptation of intertidal oysters and provides novel insight on the function and evolution of neuroendocrine-immune system in ecological context.

The involvement of AMP-activated protein kinase α in regulating glycolysis in Yesso scallop Patinopecten yessoensis under high temperature stress.

AMP-activated protein kinase α subunit (AMPKα), the central regulatory molecule of energy metabolism, plays an important role in maintaining energy homeostasis and helping cells to resist the influence of various adverse factors. In the present study, an AMPKα was identified from Yesso scallop Patinopecten yessoensis (PyAMPKα). The open reading frame (ORF) of PyAMPKα was of 1599 bp encoding a putative polypeptide of 533 amino acid residues with a typical KD domain, a α-AID domain and a α-CTD domain. The deduced amino acid sequence of PyAMPKα shared 59.89-74.78% identities with AMPKαs from other species. The mRNA transcripts of PyAMPKα were found to be expressed in haemocytes and all the examined tissues, including gill, mantle, gonad, adductor muscle and hepatopancreas, with the highest expression level in adductor muscle. PyAMPKα was mainly located in cytoplasm of scallop haemocytes. At 3 h after high temperature stress treatment (25 °C), the mRNA transcripts of PyAMPKα, the phosphorylation level of PyAMPKα at Thr170 and the lactic acid (LD) content in adductor muscle all increased significantly, while the glycogen content decreased significantly. The activity of pyruvate kinase (PyPK) and the relative mRNA expression level of phosphofructokinase (PyPFK) were significantly up-regulated at 3 h after high temperature stress treatment (25 °C). Furthermore, the PyAMPKα activator AICAR could effectively upregulate the phosphorylation level of PyAMPKα, and increase activities of PyPFK and pyruvate kinase (PyPK). Meanwhile the glycogen content also declined under AICAR treatment. These results collectively suggested that PyAMPKα was involved in the high temperature stress response of scallops by enhancing glycolysis pathway of glycogen. These results would be helpful for understanding the functions of PyAMPKα in maintaining energy homeostasis under high temperature stress in scallops.

CgTNF-2 promotes the proliferation of haemocytes by regulating the expressions of CgRunx and cell cycle related genes in the Pacific oyster Crassostrea gigas.

A TNF-α family member, CgTNF-2, was previously identified from the oyster Crassostrea gigas to involve in the antibacterial response. In the present study, the role of CgTNF-2 in mediating the proliferation of haemocytes was further explored. The mRNA expression of CgTNF-2 in granulocytes was significantly higher than that in semi-granulocytes and agranulocytes, and the percentages of CgTNF-2 antibody labeled cells in agranulocytes, semi-granulocytes and granulocytes were 19.15%, 40.25% and 94.07%, respectively. After the treatment with rCgTNF-2, the percentage of EdU+ cells in haemocytes increased significantly (1.77-fold, p < 0.05) at 6 h compared with that in rGST-treated group, and the mRNA expressions of CgRunx, CgCyclin A, CgCDK2 and CgCDC45 in haemocytes all increased significantly (p < 0.05), which were 1.94-fold, 2.13-fold, 1.97-fold, 1.76-fold of that in rGST-treated group, respectively. Meanwhile, the protein abundance of CgRunx and CgCyclin A in the haemocytes of oysters in the rCgTNF-2-treated group increased, and the percentage of PI+ haemocytes in S phase also increased significantly (2.19-fold, p < 0.05) compared with that in rGST-treated group. These results collectively confirmed that CgTNF-2 was highly expressed in granulocytes and involved in the proliferation of haemocytes by regulating the expressions of CgRunx and cell cycle related genes in C. gigas.

Effect of high temperature stress on glycogen metabolism in gills of Yesso scallop Patinopecten yessoensis.

Glycogen is the main energy storage material in mollusc, and the regulation of its metabolism is essential for the response against high temperature stress. In the present study, the alternation of lactic acid (LD) content, glycogen reserves, mRNA expression level of genes encoding glycogen metabolism enzymes and activities of glycogen metabolism enzymes in gills of Yesso scallop Patinopecten yessoensis after an acute high temperature treatment at 25 °C were examined to understand the effect of high temperature on glycogen metabolism. The activity of T-ATPase in gills of scallops presented a gradual increase trend especially at 6 h after the acute high temperature treatment (p < 0.05). The glycogen reserves did not change significantly even there was a downward trend at 24 h after the acute high temperature treatment (p > 0.05). The mRNA transcripts of glycogen synthase (PyGCS) in gills of scallops decreased significantly at 1, 3, 6 and 12 h (p < 0.05), and recovered to normal level at 24 h (p > 0.05) after the acute high temperature treatment, while that of glycogen phosphorylase a (PyGPa) and phosphoenol pyruvate carboxy kinase (PyPEPCK) were both significantly down-regulated from 1 h to 24 h (p < 0.05) after the acute high temperature treatment. The activity of PyGPa at 1, 12 and 24 h and the content of LD at 3 and 24 h in gills of scallops after the acute high temperature treatment both increased significantly (p < 0.05). Furthermore, the mRNA transcripts of hexokinase (PyHK) and pyruvate kinase (PyPK) in gills of scallops increased significantly (p < 0.05) after the acute high temperature treatment, and the response of PyHK was stronger. However, there was no significant difference on the activity of PyPK in gills of scallops between the experimental samples and the blank samples (p > 0.05). In addition, the mRNA transcripts of citrate synthase (PyCS) in gills of scallops were significantly down-regulated at 6 h and 12 h (p < 0.05), and finally returned to normal level at 24 h (p > 0.05) after the acute high temperature treatment. These results collectively indicated acute high temperature stress leaded the alternation of glycogen metabolism in the gills of Yesso scallop, glycogenesis, gluconeogenesis and TCA cycle were inhibited, and the glycolysis pathway of glycogen was enhanced to produce more energy for coping with environmental pressure.

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 6 h after Vibrio splendidus stimulation, which was 5.57-fold of that in the control group (p < 0.01). In the oysters whose CgCaspase-Cg-5 expression was knocked down, the mRNA expression of apoptosis-related genes including CgBcl2, CgBax, CgCaspase3 and CgCaspase9 changed significantly at 12 h after V. splendidus stimulation. The expression of CgBax, CgCaspase3 and CgCaspase9 decreased, which was 0.64-fold (p < 0.05), 0.53-fold (p < 0.05) and 0.62-fold (p < 0.01), while the expression of CgBcl2 increased, which was 2.81-fold (p < 0.01) of that in the EGFP-dsRNA group, respectively. Meanwhile, the apoptotic rate of haemocytes (1.90 ± 0.71%) significantly decreased compared to that in the EGFP-dsRNA group (5.40 ± 0.72%) (p < 0.05), and the histological damages of widened cell spacing, gill filament swelling and loose cytoplasm were observed in the CgCaspase-Cg-5-knockdown oysters after V. splendidus stimulation. Collectively, CgCaspase-Cg subgroup was specifically expanded in oyster and some bivalve species, and species-specific CgCaspase-Cg-5 regulated the mRNA expression of the apoptosis-related genes to induce haemocyte apoptosis in the early stage of immune response. This provided insight into the evolutionary and functional characteristics of Caspase repertoire in the Pacific oyster and highlighted the important role of CgCaspase-Cg-5 in the response to pathogen infection and environmental stresses.

The intestinal bacterial community over seasons and its relationship with physiological status of Yesso scallop Patinopecten yessoensis.

Emerging evidence indicates that the intestinal bacterial communities associated with eukaryotes play critical roles in the physiological activities and health of their hosts. Yesso scallop Patinopecten yessoensis, one of the cold-water aquaculture species in the North Yellow Sea of China, has suffered from massive mortality in recent years. In the present study, P. yessoensis were collected from Zhangzi Island, Dalian from March 2021 to January 2022 to investigate the intestinal bacterial community and physiological indices. 16S rRNA gene sequencing data revealed that the diversity of intestinal bacteria changed significantly over seasons, with the highest Chao1 (237.42) and Shannon (6.13) indices detected in January and the lowest Chao1 (115.44) and Shannon (2.73) indices detected in July. Tenericutes, Proteobacteria and Firmicutes were dominant phyla in the intestinal bacteria of P. yessoensis, among which Firmicutes and Proteobacteria significantly enriched in August and January, respectively. Mycoplasma was the most abundant genus during the sampling period, which exhibited the highest abundance in October (75.26%) and lowest abundance in August (13.15%). The functional profiles of intestinal bacteria also exhibited seasonal variation, with the pathways related to pentose phosphate and deoxyribonucleotides biosynthesis enriched in August while the glycogen biosynthesis pathway enriched in October. Redundancy analysis showed that seawater pH, dissolved inorganic nitrogen and silicate were major environmental factors driving the temporal succession of scallop intestinal bacteria. Correlation clustering analysis suggested that the relative abundances of Endozoicomonas and Vibrio in the intestine were positively correlated with superoxide dismutase activity in hepatopancreas while negatively correlated with malondialdehyde content in hepatopancreas and glycogen content in adductor muscle. All the results revealed that the intestine harbored a lower bacterial diversity and a higher abundance of Vibrio in August, compared to January, which were closely related to the oxidative stress status of scallop in summer. These findings will advance our understanding of the relationship between seasonal alteration in the intestinal bacteria and the physiological status of scallops.

An IRF2BP member (CgIRF2BP) involved in negative regulation of CgIFNLP expression in oyster Crassostrea gigas.

The IRF2BP family of transcription regulators act as corepressor molecules by inhibiting both enhancer-activated and basal transcription involving in many biological contexts. In the present study, an IRF2BP homologue (CgIRF2BP) was identified from oyster C. gigas. Its open reading frame is of 1809 bp encoding a polypeptide of 602 amino acids, which contains an IRF-2BP1_2 domain and a RING domain. The mRNA transcripts of CgIRF2BP were detected in all tested tissues with highest level in haemocytes (28.99-fold of that in mantle, p < 0.05). After poly (I:C) stimulation, the expression level of CgIRF2BP was significantly down-regulated at 3 h (0.50-fold of that in control group, p < 0.001) and gradually increased from 6 h to 48 h (2.69-fold of that in control group, p < 0.01). The recombinant protein of CgIRF2BP (rCgIRF2BP) showed high affinity to both rCgIRF1 and rCgIRF8 with Kd value of 1.02 × 10-7 and 2.09 × 10-7, respectively. In CgIRF2BP-RNAi oysters, the mRNA expression of CgIFNLP, CgMx1, CgViperin and CgIFI44L were significantly increased after poly (I:C) stimulation, which were 2.88 (p < 0.01), 1.83 (p < 0.05), 2.47 (p < 0.05), and 1.99-fold (p < 0.01) of that in EGFP group, respectively. These findings suggested that CgIRF2BP negatively regulated CgIFNLP expression by binding with CgIRF1 and CgIRF8.

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.

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.

The ferroptosis in haemocytes of Pacific oyster Crassostrea gigas upon erastin treatment.

Ferroptosis is an iron and oxidative dependent form of cell death usually mediated by redox related molecules in vertebrates. In the present study, a glutathione peroxidase 4 (GPX4) and a solute carrier family 7 member 11 (SLC7A11, xCT) homologues were identified from the oyster Crassostrea gigas (designed as CgGPX4 and CgxCT), which contained a GSHPx domain and an AA_permease domain, respectively. The mRNA transcripts of CgGPX4 and CgxCT were expressed in all the examined tissues, including gill, gonad, adductor muscle, labial palp, mantle, hepatopancreas and haemocytes, with the highest expression in haemocytes. After erastin treatment, the rate of cell malformation and cell death increased significantly in haemocytes, and the mitochondrial atrophy, crest loss and fracture were observed in haemocytes. While the amount of Fe2+ and Malondialdehyde (MDA) increased significantly, the mRNA expressions of CgGPX4, CgxCT and voltage-dependent anion channel 2 (CgVDAC2) in haemocytes decreased significantly after erastin treatment. These results indicated that erastin was able to induce the ferroptosis of oyster haemocytes.

PDGFRß Recognizes and Binds Bacteria to Activate Src/Stat Pathway in Oysters.

The Stat signaling pathway plays important roles in mediating the secretions of a large number of cytokines and growth factors in vertebrates, which is generally triggered by the growth factor receptor, cytokine receptor, G protein coupled receptor, and receptor protein tyrosine kinase. In the current study, a platelet-derived growth factor receptor (defined as CgPDGFRß) was identified from the Pacific oyster Crassostrea gigas, with a signal peptide, three Ig domains, a transmembrane domain, and an intracellular Ser/Thr/Tyr kinase domain. The two N-terminal Ig domains of CgPDGFRß showed relatively higher binding activity to Gram-negative bacteria and LPS compared with Gram-positive bacteria and peptidoglycan. Upon binding bacteria, CgPDGFRß in hemocytes formed a dimer and interacted with protein tyrosine kinase CgSrc to induce the phosphorylation of CgSrc at Tyr416. The activated CgSrc interacted with CgStat to induce the translocation of CgStat into the nucleus of hemocytes, which then promoted the expressions of Big defensin 1 (CgBigdef1), IL17-4 (CgIL17-4), and TNF (CgTNF1). These findings together demonstrated that the Src/Stat signaling was activated after the binding of CgPDGFRß with bacteria to induce the expressions of CgBigdef1, CgIL17-4, and CgTNF1.