Xiaorui Song

Transcriptomic analysis of oyster Crassostrea gigas larvae illustrates the response patterns regulated by catecholaminergic system upon acute heat and bacterial stress

&NA; Bacterial infection and heat stress, as two major environmental threats of marine molluscs, could affect larval development and dramatically promote mortality of oysters. In the present study, next‐generation sequencing, together with determinations of mRNA expression and measurements of enzyme activities, were employed to understand the response patterns of oyster larvae under acute heat and bacterial stress. After RNA‐seq, a total of 9472 differentially expressed genes including 4895 significantly up‐regulated ones and 4577 significantly down‐regulated ones were obtained from 12 transcriptome libraries. GO overrepresentation analysis of the up‐regulated genes revealed that the neuroendocrine immunomodulation pathway was activated after acute heat and bacterial stimulation, in which the catecholaminergic regulation played an important role. GO overrepresentation analysis of the down‐regulated genes suggested that the immune capacity of Crassostrea gigas larvae was suppressed under stress, which was further validated since superoxide dismutase (SOD) and phenoloxidase (PO) activities in the total protein extract of larvae decreased dramatically after stress. Moreover, the shell formation of trochophore was inhibited and severe mortality was caused after acute heat and bacterial stress. These results collectively indicated that acute heat and bacterial stress could significantly inhibit larval development and suppress immune response of oyster C. gigas larvae. And the neuroendocrine immunomodulation, especially the catecholaminergic regulation, played an indispensable role in the stress response of molluscan larvae. HighlightsThe catecholaminergic system was activated after acute heat and bacterial stress in oyster C. gigas larvae.Acute heat and bacterial stress could inhibit shell formation and cause severe mortality of C. gigas larvae.SOD and PO activities in the total protein extract of larvae decreased dramatically after stress.The immune capacity of C. gigas larvae was suppressed under acute heat and bacterial stress.

A shell-formation related carbonic anhydrase in Crassostrea gigas modulates intracellular calcium against CO2 exposure: Implication for impacts of ocean acidification on mollusk calcification

Ocean acidification (OA) could decrease the shells and skeletons formation of mollusk by reducing the availability of carbonate ions at calcification sites. Carbonic anhydrases (CAs) convert CO2 to HCO3- and play important roles in biomineralization process from invertebrate to vertebrate. In the present study, a CA (designated as CgCA) was identified and characterized in Pacific oyster C. gigas. The cDNA of CgCA was of 927bp encoding a predicted polypeptide of 308 amino acids with a signal peptide and a CA catalytic function domain. The mRNA transcripts of CgCA were constitutively expressed in all tested tissues with the highest levels in mantle and hemocytes. During the early development period, the mRNA transcripts of CgCA could be detected in all the stages with the highest level in D-veliger larvae. Elevated CO2 increased the mRNA transcripts of CgCA in muscle, mantle, hepatopancreas, gill and hemocytes significantly (p<0.05) and induced the translocation of CgCA in hemocytes and mantle. Moreover, elevated CO2 also caused the decrease of intracellular Ca2+ in hemocytes (p<0.05). The inhibition of CA by acetazolamide and suppression of CgCA gene via RNA interference could increase the intracellular Ca2+ in hemocytes (p<0.05). Besides, the decrease of intracellular Ca2+ content caused by Ca2+ reagent ionomycin could affect localization of CgCA in mantle tissue. The results indicated CgCA played essential roles in calcification and elevated CO2 accelerated the mutual modulation between calcium and CgCA, implying reduced calcification rate and dissolved shells under OA.

The modulation role of serotonin in Pacific oyster Crassostrea gigas in response to air exposure

ABSTRACT Serotonin, also known as 5‐hydroxytryptamine (5‐HT), is a critical neurotransmitter in the neuroendocrine‐immune regulatory network and involved in regulation of the stress response in vertebrates and invertebrates. In the present study, serotonin was found to be widely distributed in the tissues of Pacific oyster Crassostrea gigas, including haemolymph, gonad, visceral ganglion, mantle, gill, labial palps and hepatopancreas, and its concentration increased significantly in haemolymph and mantle after the oysters were exposed to air for 1 d. The apoptosis rate of haemocytes was significantly declined after the oysters received an injection of extra serotonin, while the activity of superoxide dismutase (SOD) in haemolymph increased significantly. After the stimulation of serotonin during air exposure, the apoptosis rate of oyster haemocytes and the concentration of H2O2 in haemolymph were significantly decreased, while the SOD activity was significantly elevated. Furthermore, the survival rate of oysters from 4th to 6th d after injection of serotonin was higher than that of FSSW group and air exposure group. The results clearly indicated that serotonin could modulate apoptotic effect and redox during air exposure to protect oysters from stress. HighlightsNeurotransmitter 5‐HT existed extensively in the tissues of oyster C. gigas.The level of 5‐HT in haemolymph and mantle increased significantly against air exposure stress.5‐HT could reduce apoptotic effects of haemocytes and modulate redox in haemolymph after air exposure.5‐HT could decrease the mortality of oysters during air exposure, and protect them from stress.

A norepinephrine-responsive miRNA directly promotes CgHSP90AA1 expression in oyster haemocytes during desiccation

ABSTRACT Oyster Crassostrea gigas is one model mollusc inhabiting in the intertidal zone and is frequently stressed by desiccation. The adaptation mechanism of oyster to environmental stress involves multiple levels, and miRNA is one of the most important regulators in post‐transcriptional level. In the present study, an oyster norepinephrine‐responsive miRNA cgi‐miR‐365 was proved to contribute to the host adaptation against desiccation by directly promoting the expression of CgHSP90AA1. Briefly, a significant increase of cgi‐miR‐365 was observed from the first day after aerial exposure and the up‐regulation was vigorously repressed when oysters were injected with adrenoceptors antagonists. A total of 15 genes involved in biological regulation, metabolic process and response to stimulus were predicted to be modulated by cgi‐miR‐365. Among these genes, CgHSP90AA1 was up‐regulated significantly during desiccation and could be down‐regulated after simultaneous injection of adrenoceptors antagonists. The interaction between cgi‐miR‐365 and CgHSP90AA1 was subsequently verified in vitro, and a significant promotion of CgHSP90AA1 transcripts was observed after overexpressing cgi‐miR‐365 in either in vitro luciferase reporter assay or primarily cultured haemocytes. Meanwhile, CgHSP90AA1 transcripts decreased in vivo when cgi‐miR‐365 was repressed by its inhibitor during desiccation. Collectively, it was suggested that cgi‐miR‐365 could be induced by norepinephrine during desiccation and promote CgHSP90AA1 expression directly after binding to its 3′‐UTR, which would provide new evidence in miRNA‐mediated adaptation mechanism in oysters against intertidal stress. HIGHLIGHTScgi‐miR‐365 was found fast and strongly induced in NE‐dependent way after desiccation stress.Fifteen genes involved in biological regulation and metabolic process were targeted by cgi‐miR‐365.CgHSP90AA1, as one putative target of cgi‐miR‐365, was also found induced after desiccation by NE.Interaction between cgi‐miR‐365 and CgHSP90AA1 was verified by in vitro and in vivo.CgHSP90AA1 mRNA could be promoted directly by cgi‐miR‐365 during desiccation.

The fragmentation mechanism and immune-protective effect of CfTEP in the scallop Chlamys farreri

Abstract Thioester‐containing proteins (TEPs), characterized by a unique intrachain &bgr;‐cysteinyl‐&ggr;‐glutamyl thioester bond, form an ancient and diverse family of secreted proteins that play central roles in the innate immune response. But the existence form and immune protection mechanism of TEP in invertebrates still remain unclear, especially in the mollusks. The fragmentation and the immune‐protective effect of thioester bond in CfTEP, a previously identified thioester‐containing protein in scallop Chlamys farreri, were characterized in the present study. During the early embryonic development of scallop, the mRNA transcript of CfTEP could be detected in all the stages, and its expression levels in D‐larvae, veliger larvae and eye‐spot larvae were significantly higher than that in eggs. The CfTEP protein was also detected in peripheral of D‐larvae, veliger larvae and eye‐spot larva by immunofluorescence. In the adult scallop, the CfTEP protein was mainly distributed in the hepatopancreas, gill, kidney, gonad, and mantle. The expression of CfTEP mRNA in the hemocytes of adult scallop was significantly up‐regulated when the scallops were stimulated by LPS, PGN or &bgr;‐glucan. Two bands (100 and 55 kDa) were detected using anti‐CfTEP‐R1 (spanned the C‐terminal portion of the thioester, A2M‐comp and A2M‐recep domain, 942–1472), and a single band (46 kDa) was detected by using anti‐CfTEP‐R2 (the N‐terminal portion of the following A2M‐N‐2 domain, 452–496) in the serum of scallop at 12 h after LPS stimulation. When the thioester bond of CfTEP protein was inactivated by injecting methylamine, the survival rate of scallop was significantly decreased after challenged by Vibrio angulillarum. All these results suggested that CfTEP protein existed as fragments similar to vertebrate C3, and played central roles in the immune response against pathogen in the innate immunity of scallops. HighlightsCfTEP was expressed in early embryonic development stages and various tissues of adult scallop.PAMP and pathogen stimulations induced the expression of CfTEP in hemocytes.CfTEP could be split at RRKR/RL by endogenous proteases in the serum.The thioester bond played a decisive role in the immune function of CfTEP.

Glycogen synthase kinase-3 (GSK3) regulates TNF production and haemocyte phagocytosis in the immune response of Chinese mitten crab Eriocheir sinensis

&NA; Glycogen synthase kinase‐3 (GSK3) is a serine/threonine protein kinase firstly identified as a regulator of glycogen synthesis. Recently, it has been proved to be a key regulator of the immune reaction. In the present study, a GSK3 homolog gene (designated as EsGSK3) was cloned from Chinese mitten crab, Eriocheir sinensis. The open reading frame (ORF) was 1824 bp, which encoded a predicted polypeptide of 607 amino acids. There was a conserved Serine/Threonine Kinase domain and a DNA binding domain found in EsGSK3. Phylogenetic analysis showed that EsGSK3 was firstly clustered with GSK3‐&bgr; from oriental river prawn Macrobrachium nipponense in the invertebrate branch, while GSK3s from vertebrates formed the other distinct branch. EsGSK3 mRNA transcripts could be detected in all tested tissues of the crab including haepatopancreas, eyestalk, muscle, gonad, haemocytes and haematopoietic tissue with the highest expression level in haepatopancreas. And EsGSK3 protein was mostly detected in the cytoplasm of haemocyte by immunofluorescence analysis. The expression levels of EsGSK3 mRNA increased significantly at 6 h after Aeromonas hydrophila challenge (p < 0.05) in comparison with control group, and then gradually decreased to the initial level at 48 h (p > 0.05). The mRNA expression of lipopolysaccharide‐induced tumor necrosis factor (TNF)‐&agr; factor (EsLITAF) was also induced by A. hydrophila challenge. However, the mRNA expression of EsLITAF and TNF‐&agr; production was significantly suppressed after EsGSK3 was blocked in vivo with specific inhibitor lithium, while the phagocytosis of crab haemocytes was significantly promoted. These results collectively demonstrated that EsGSK3 could regulate the innate immune responses of E. sinensis by promoting TNF‐&agr; production and inhibiting haemocyte phagocytosis. HighlightsA homologue of glycogen synthase kinase‐3 (EsGSK3) gene was identified from Chinese mitten carb, E. sinensis.The EsGSK3 transcripts in haemocytes could be induced significantly by immune challenges.The mRNA expression of EsLITAF and TNF‐&agr; production were suppressed after EsGSK3 was blocked in vivo.The activity of EsGSK3 could regulate the phagocytosis of crab haemocytes.

The cyclin-dependent kinase 2 (CDK2) mediates hematopoiesis through G1-to–S transition in Chinese mitten crab Eriocheir sinensis

&NA; Cyclin‐dependent kinases (CDKs), a family of cell cycle‐related serine/threonine kinases, participate in various biological processes, and play crucial roles in the innate immunity. In the present study, a CDK2 (designed as EsCDK2) with a serine/threonine protein kinase catalytic domain was identified from Chinese mitten crab (Eriocheir sinensis). The full‐length cDNA sequence of EsCDK2 was of 2405 bp with an open reading frame (ORF) of 909 bp. EsCDK2 shared 66%–81% sequence similarities with previously identified CDK2s. It was clustered with the CDK2 from Penaeus monodon in the invertebrate branch of the phylogenetic tree. The mRNA transcripts of EsCDK2 were highly expressed in hematopoietic tissue (HPT) and gonad, while lower in hemocytes, heart, gills, and muscle. EsCDK2 protein distributed in both cytoplasm and nucleus of HPT cells. The expression of EsCDK2 mRNA in HPT was significantly up‐regulated and peaked at 3 h post stimulations with Aeromonas hydrophila (2.31‐fold, p < 0.05) and Lipopolysaccharide (LPS) (2.02‐fold, p < 0.05). After exsanguination, the total hemocyte counts (THC) decreased significantly to 0.42 × 107/ml (0.39‐fold, p < 0.05) at 0.5 h, then returned to a normal level at 6 h, while the mRNA expression of EsCDK2 in HPT cells was up‐regulated at the early phase from 0.5 h to 6 h. After injection of EsCDK2‐dsRNA, the mRNA expression level of EsCDK2 in HPT and THC both decreased to 0.53‐fold (p < 0.01) and 0.78‐fold (p < 0.05) at 24 h, respectively, and the percentage of new‐born hemocytes in HPT also decreased significantly from 37.7% to 16.3% (0.43‐fold, p < 0.01). After knocking down of EsCDK2, THC decreased dramatically at 6 h (0.65‐fold, p < 0.01) post exsanguination, while returned normal at 6 h in PBS group. After interference of EsCDK2 mRNA expression, the percentage of G0‐G1 phase cells significantly increased to 85.01% (1.26‐fold, p < 0.01), while S phase and G2‐M phase cells significantly decreased to 7.92% (0.46‐fold, p < 0.01) and 7.07% (0.43‐fold, p < 0.01) respectively, indicating that the cell cycle of HPT cells arrested at G1 phase. These results collectively demonstrated that EsCDK2 participated in the regeneration of hemocytes or hematopoiesis by regulating the transition from G1 to S phase in the cell cycle, and involves in the innate immune responses of E. sinensis. HighlightsA CDK2 member was identified in crab E. sinensis.EsCDK2 expression profiles varied spatio‐temporally in HPT after LPS and A. hydrophila stimulation and hemolymph exsanguination.The percentage of new‐born hemocytes in HPT decreased significantly after RNAi with EsCDK2‐dsRNA.The cell cycle of HPT cells arrested at G1 phase after knocking down of EsCDK2.

A GTP-dependent Phosphoenolpyruvate Carboxykinase from Crassostrea gigas Involved in Immune Recognition

Phosphoenolpyruvate carboxykinase (PEPCK) is well known as a key enzyme involved in the metabolic pathway of gluconeogenesis in organisms, but the information about its involvement in immune response is still very limited. In the present study, a novel PEPCK homolog named CgPEPCK was identified from oyster Crassostrea gigas. The deduced amino acid sequence of CgPEPCK shared 52%-74% similarities with those from other known PEPCKs. There were one conserved guanosine triphosphate (GTP) binding site, one substrate binding site, one metal binding site and one active site in CgPEPCK. The mRNA transcripts of CgPEPCK were constitutively expressed in all the tested tissues including hemolymph, mantle, gill, muscle, gonad and hepatopancreas. CgPEPCK proteins were mainly distributed in adductor muscle, gonad, gill and mantle, and rarely detected in hepatopancreas by using immunohistochemical analysis. After the stimulations with lipopolysaccharide (LPS), peptidoglycan (PGN), Vibrio splendidus and V.xa0anguillarum, CgPEPCK transcripts in hemocytes were significantly up-regulated and peaked at 6xa0h (LPS, 9.62-fold, pxa0<xa00.01), 9xa0h (PGN, 4.25-fold, pxa0<xa00.01), 12xa0h (V.xa0splendidus, 5.72-fold, pxa0<xa00.01), 3xa0h (V.xa0anguillarum, 2.87-fold, pxa0<xa00.01), respectively. The recombinant CgPEPCK protein (rCgPEPCK) exhibited Mn2+/Mg2+ dependent GTP binding activity, and the activities to bind LPS and PGN, but not β-1,3-glucan (GLU), lipoteichoic acid (LTA), mannan (MAN) nor polyinosinic-polycytidylic (Poly I: C). It could also bind Escherichia coli, Staphylococcus aureus, Micrococcus luteus and significantly inhibit their growth. All these results collectively suggested that CgPEPCK could not only exert GTP binding activity involved in gluconeogenesis, but also mediate the bacteria recognition and clearance in immune response of oysters.

A novel GATA-like zinc finger transcription factor involving in hematopoiesis of Eriocheir sinensis

ABSTRACT GATA transcription factor is a family of DNA‐binding proteins that can recognize and bind to sequence of (A/T) GATA (A/G). In the present study, a GATA‐like protein (named as EsGLP) was characterized from Eriocheir sinensis, including an 834 bp full length open reading frame of EsGLP, encoding a polypeptide of 277 amino acids. The deduced amino acid sequence of EsGLP contained one conserved GATA‐type zinc finger of the form Cys‐X2‐Cys‐X17‐Cys‐X2‐Cys, with four cysteine sites. The EsGLP mRNA transcripts were mainly detected in the hematopoietic tissue, hepatopancreas and gonad. The recombinant EsGLP protein was prepared for the antibody production. The EsGLP protein was mainly distributed in the edge of lobules in the HPT and the cytoplasm of hemocytes. The mRNA transcripts of EsGLP in hemocytes were significantly decreased at 24h (0.39‐fold and 0.27‐fold, p<.05) and 48h (0.35‐fold and 0.16‐fold, p<.05) after LPS and Aeromonas hydrophila stimulation, respectively. However, one peak of EsGLP mRNA transcripts were recorded at 24h (8.71‐fold, p<.05) in HPT after A. hydrophila stimulation. The expression level of EsGLP mRNA in HPT was significantly up‐regulated at 2h, 2.5h and 9h (41.74‐fold, 45.38‐fold and 26.07‐fold, p<.05) after exsanguination stimulation. When EsGLP gene expression was inhibited by the injection of double‐stranded RNA, both the total hemocytes counts and the rate of EdU‐positive hemocytes were significantly decreased (0.32‐fold and 0.56‐fold compared to that in control group, p<.05). All these results suggested that EsGLP was an important regulatory factor in E. sinensis which involved in the hemocytes generation and the immune response against invading pathogens. HIGHLIGHTSEsGLP involved in the hematopoiesis process were identified and characterized in Chinese crabs (Eriocheir sinensis).EsGLP could respond to the LPS and Aeromonas hydrophila stimulating.EsGLP play important role in immune defense against pathogens invasion possibly by hematopoiesis regulation.

A hypervariable immunoglobulin superfamily member from Crassostrea gigas functions as pattern recognition receptor with opsonic activity

Abstract Immunoglobulin superfamily (IgSF), an extensive collection of proteins possessing at least one immunoglobulin‐like (Ig‐like) domain, performs a wide range of functions in recognition, binding or adhesion process of cells. In the present study, a cysteine‐rich motif associated immunoglobulin domain containing protein (designated CgCAICP‐1) was identified in Pacific oyster Crassostrea gigas. The deduced protein sequence of CgCAICP‐1 contained 534 amino acidresidues, with three Ig domains which were designated as IG1, IG2 and IG3, and a cysteine‐rich motif between the first and second Ig domain. The mRNA transcripts of CgCAICP‐1 were highly expressed in hemocytes and up‐regulated significantly (p < 0.05) after the stimulation of lipopolysaccharides (LPS), but not peptidoglycan (PGN). The recombinant CgCAICP‐1 protein (rCgCAICP‐1) exhibited binding activity to various pathogen‐associated molecular patterns (PAMPs) including LPS, PGN, mannose (Man) and D‐galactose (D‐Gal), and microorganisms including Vibrio splendidus, Escherichia coli, Staphylococcus aureus, Micrococcus luteus and Pichia pastoris. The phagocytic rates of oyster hemocytes towards Gram‐negative bacteria V. splendidus and Gram‐positive bacteria M. luteus were significantly enhanced (p < 0.05) after pre‐incubation of microbes with rCgCAICP‐1. Furthermore, the transcripts of CgCAICP‐1 exhibited high level of polymorphism among individuals. The ratio of nonsynonymous and synonymous distances (dN/dS) for AA’BCC’D strands of IG1 (the possible binding sites 1, pbs1) across all allelic variants was 2.09 (p < 0.05), while the ratio for the non‐pbs regions was less than 1.0. The 1248 bp fragment amplified from the 5′ end of CgCAICP‐1 open reading frame (ORF) from 24 transcript variants could be divided artificially into seven regions of 50 elements, and all of the allelic variants might be derived from these elements by point mutation and recombination processes. These results collectively suggested that CgCAICP‐1 might function as an important pattern recognition receptor (PRR) to recognize various PAMPs and facilitated the phagocytosis of oyster hemocytes towards both Gram‐positive and Gram‐negative bacteria. Diverse isoforms of CgCAICP‐1 were generated through point mutation and recombination processes and maintained by balancing selection, which would provide a broader spectrum of interaction surface and be associated with immune resistance of oysters to infectious pathogens. HighlightsCgCAICP‐1 was a novel member of IgSF identified from oyster Crassostrea gigas.The dimer and monomer forms of CgCAICP‐1 distributed in hemocytes and plasma.CgCAICP‐1 mRNA expression was significantly up‐regulated post LPS stimulation.rCgCAICP‐1 exhibited binding activity towards different microbes and opsonin activity.Point mutation and recombination contributed to the CgCAICP‐1 polymorphism.