Search

Microplastics (MPs) and polychlorinated biphenyls (PCBs) are known with high persistence and toxicity, posing urgent threats to food safety and human health. However, little is known about the synergistic effect of MPs on PCBs bioaccumulation on Crassostrea hongkongensis. In the present study, diverse types of MPs were analyzed on sea water and C. hongkongensis sampled from three distinct estuary sites, and film-shaped MPs were discovered to be preferentially ingested by the oysters. Interestingly, the content of MPs and PCBs showed negative correlation (R2 = 0.452, p < 0.001) in the oysters sampled from site 2. Upon MPs and PCBs co-treatment, the in vivo accumulation of PCBs in C. hongkongensis was inhibited by 25.90 % when compared to the group treated with PCBs solely. PCBs stresses significantly induced the expression of genes of CYP2C31 , GST , SOD and HSP70 in C. hongkongensis , while, the elevated state was compromised when co-treated with PCBs. The present research alleviates concerns about the potential effects of MPs on promoting PCBs bioaccumulation and provide a better understanding of the combined impact of MPs and PCBs on C. hongkongensis. [Display omitted] • Monitoring MPs and PCBs content in C. hongkongensis of aquacultural areas of South China. • C. hongkongensis preferentially ingest film-type MP in all three sampling aquacultural areas. • MPs' and PCBs' accumulation in C. hongkongensis onsite was negatively correlated. • PE/PCBs co-treatment aroused mutually inhibited accumulation of the two pollutants in oysters. • PE/PCBs co-presence caused deteriorated oxidative stresses in C. hongkongensis. [ABSTRACT FROM AUTHOR]

Integrins, a family of cell adhesion transmembrane receptors, mediate cell adhesion, migration, proliferation, apoptosis, and phagocytosis. In the present study, an integrin ChIntα 4 from Crassostrea hongkongensis was characterized to investigate its role in defensing against pathogenic bacterium Vibrio alginolyticus. The full-length cDNA sequence of ChIntα 4 was 3572 bp with an open reading frame (ORF) of 3168 bp, which encoded a polypeptide with 1055 amino acids. The mRNA expression of ChIntα 4 in the hemocytes was significantly up-regulated at 6 h and 24 h post V. alginolyticus stimulation (p < 0.01). The recombinant Ch Intα 4 protein could agglutinate the rabbit red blood cells and Gram-negative bacteria V. alginolyticus and Escherichia coli. Moreover, the phagocytic activity of the hemocytes was significantly down-regulated from 46.9% to 32.7% when blocked with anti- Ch Intα 4 antibody, and it was significantly up-regulated from 42.7% to 59.5% post transfection with pCI-neo- ChIntα 4 plasmid (p < 0.05). In conclusion, these findings demonstrated that Ch Intα 4 might be involved in resisting V. alginolyticus infection and regulating phagocytosis as a cell adhesion receptor in C. hongkongensis. • The sequence of ChIntα 4 was firstly cloned from Crassostrea hongkongensis. • The mRNA level of ChIntα 4 showed positive response post Vibrio stimulation. • Ch Intα 4 protein agglutinated the erythrocyte and Gram-negative bacteria. • Ch Intα 4 protein was demonstrated to regulate phagocytosis of the hemocytes. [ABSTRACT FROM AUTHOR]

Hong Kong oyster (Crassostrea hongkongensis) is one of the main species of economic shellfish cultivated in the coastal areas of southern China. The cultivation of this shellfish may be adversely impacted by Vibrio parahaemolyticus , a harmful pathogenic bacterium for many mariculture species, as it usually exists on the surface of Hong Kong oysters. Although previous studies have discovered that oysters rely on non-specific immune system to fight pathogen invasion, the genes corresponding to the complex immune system against Vibrio is still not fully elucidated. Therefore, we conducted a transcriptome analysis on the gill from Hong Kong oysters at two time points (i.e., 12 h and 24 h after V. parahaemolyticus or PBS challenge) to identify potential immune genes against V. parahaemolyticus infection. A total of 61779 unigenes with the average length of 1221 bp were obtained, and the annotation information of 39917 unigenes were obtained from Nr, SwissProt, KEGG and COG/KOG. After a pairwise comparison between V. parahaemolyticus or PBS challenge at the two time points, three groups of differentially expressed genes induced by V. parahaemolyticus were captured and analyzed. GO and KEGG analyses showed that multiple immune-related genes played an important role in pathogen infection, including HSP70, PCDP3 and TLR4. Furthermore, genes annotation indicated that LITAF, TNFSF10, Duox2 and big defensin family are also involved in immune regulation. Our study provides a reference for further exploration the molecular mechanism that defenses the pathogen infection regarding the identified immune-related genes in Hong Kong oysters. • RNA-seq was used to investigate the immune response of Hong Kong oyster against V. parahaemolyticus infection in our study. • We mainly focused on the analysis of three groups of differentially expressed genes induced by V. parahaemolyticus. •Our results reflected potential role of multiple immune-related genes in the immune response of Hong Kong oyster. [ABSTRACT FROM AUTHOR]

Caspase 3 plays an important role in apoptotic pathways and contributes to maintaining the homeostasis of the immune system in organisms. The structure, functions, and characteristics of caspase 3 have been extensively investigated in many species, but the research is scarce when it comes to bivalves, particularly oysters. In this study, we identified and cloned a previously unknown caspase 3 gene, named Ch Cas 3, in C. hongkongensis. The full-length cDNA of Ch Cas 3 was 1562 bp and included a 175 bp 5′-untranslated region (UTR), a 141 bp 3′-UTR and a 1245 bp open reading frame (ORF) that encoded a polypeptide of 415 amino acids. Similar to caspase 3 in other species, Ch Cas 3 has a pro-domain, a conserved cysteine active site, a large p20 subunit and a small p10 subunit. Our findings demonstrated the expression of Ch Cas 3 in all the eight tissues via tissue-specific expression assays with the highest expression in haemocytes. Ch Cas 3 was confirmed to be expressed throughout the larval development stages, and fluorescence from pEGFP-N1- Ch Cas 3 was found to be distributed throughout the entire HEK293T cell. In addition, the relative expression of Ch Cas 3 significantly enhanced in hemocytes post bacterial stimulation, and overexpression of Ch Cas 3 led to upregulation of the transcriptional activity of NF-κB and p53 reporter genes in HEK293T cells, which indicated that it was involved in innate immune responses. Finally, the apoptosis rate of the haemocytes declined considerably compared with that of the control group after the expression of Ch Cas 3 was successfully silenced by dsRNA, corroborating its sentinel role in apoptosis. This study provides comprehensive underpinning evidences, affirming caspase 3 crucial role against bacterial infection and apoptosis in C. hongkongensis. • Ch Cas 3 was identified from Crassostrea hongkongensis with a full length of 1562 bp. • Ch Cas 3 mRNA expressions was significantly increased after bacterial stimulation. • Over-expression of Ch Cas 3 could improve the transcriptional activity of NF-κB and p53. • Successful silencing of Ch Cas 3 could significantly reduce the apoptosis rate. [ABSTRACT FROM AUTHOR]

Phagocytosis is one of the fundamental cellular immune defense parameter that helps in the elimination of the invading pathogens in both vertebrates and invertebrates, which require plenty of energy for functioning. In the present study, we identified the critical energy regulator AMP-activated protein kinase (AMPK) in Crassostrea hongkongensis which is composed of three subunits, named Ch AMPK-α, Ch AMPK-β, and Ch AMPK-γ, and then analyzed the function of AMPK in regulating hemocyte phagocytosis. All the three Ch AMPK subunits mRNA were detected to be expressed at various embryological stages, and also constitutively expressed in multiple tissues with high expression in gill and mantle. The phylogenetic tree showed that the three subunits of AMPK were correspondingly clustered with its orthologue branches. Furthermore Western Blot analysis revealed that the AMPK pharmacological inhibitors Compound C could effectively down-regulate the Thr172 phosphorylation level of AMPK-α, and the hemocyte phagocytosis was inhibited by Compound C (CC), which indicate its existence in the oyster. Our results showed that treatment of AMPK inhibitors significantly attenuated the capacity of hemocytes phagocytosis. Moreover, Compound C could also change the organization of actin cytoskeleton in the oyster hemocytes, demonstrating the crucial role of AMPK signaling in control of phagocytosis. • Three subunits of AMPK were identified from Crassostrea hongkongensis. • Compound C effectively blocked Thr172 phosphorylation of Ch AMPK-α subunit. • Inhibition of AMPK signaling significantly attenuated the capacity of hemocytes phagocytosis. • AMPK signaling regulated phagocytosis via remodeling actin cytoskeleton. [ABSTRACT FROM AUTHOR]

Hemocytes are the first line of defence of the innate immune system of molluscs. For the first time hemocytes of Crassostrea hongkongensis were morphologically and functionally characterized, identifying circulating cell types and studying their involvement in immune responses. In the present study, two main populations, hyalinocytes and granulocytes, were characterized based on the presence or absence of cytoplasmic granules, using light and electron microscopy (TEM), and flow cytometry analyses. Granulocytes are 7–13 μm in diameter and present evident cytoplasmic granules, and hyalinocytes, 6–15 μm in diameter, with a few or no granules. The mean number of circulating hemocytes in the hemolymph was 2.52 × 10 6  cells/mL. Flow cytometry indicated that both granulocytes and hyalinocytes showed cell phagocytosis and reactive oxygen species (ROS) production. However, phagocytosis and spontaneous production of reactive oxygen species (ROS) in granulocytes are much more active compared with hyalinocytes, which demonstrated that the granulocytes are the main hemocytes involved in the immune response of Hong Kong oyster. Moreover, the cell-free hemolymph showed antibacterial activity against Vibrio alginolyticus . Our results provide the basic information of hemocytes population of Hong Kong oyster for further investigations associated with innate immunity. [ABSTRACT FROM AUTHOR]

Gnaq, one of Guanine nucleotide-binding protein α subunits, was isolated from cellular nucleus extracts of oyster Crassostrea hongkongensis gills with biotin-labeled ChHsc70 promoter by means of DNA-affinity purification, and preliminarily identified with mass spectrometry analysis. ChGnaq mRNA depletion by RNAi technique led to clear reduction in ChHsc70 mRNA expression of C. hongkongensis hemocytes. Correspondently, ChGnaq over-expression in heterologous HEK293T cells correlated with elevated expression activation of ChHsc70 promoter. Quantitative real time PCR analysis showed that both ChHsc70 and ChGnaq transcriptions were responsive to external physical/chemical stresses by heat, CdCl 2 and NP. This suggested a plausible association between ChHsc70 and ChGnaq in the stress-induced genetic regulatory pathway. This study discovered a positively regulatory role of Ch Gnaq in controlling ChHsc70 transcription of C. hongkongensis , and conduced to a better understanding of the regulatory mechanisms in control of Hsc70 transcription. [ABSTRACT FROM AUTHOR]

A Y-box binding protein Ch YB-1 was discovered as a ChHsc70 promoter-associated protein in Crassostrea hongkongensis by DNA-affinity purification and mass spectrometry analysis. The overexpression of Ch YB-1 in heterologous HEK293T cells led to clear enhancement of ChHsc70 promoter expression, while ChYB-1 depletion correlated with significant reduction of ChHsc70 transcription in the hemocytes of C. hongkongensis . Quantitative Real-time PCR analysis revealed that both ChHsc70 and ChYB-1 were transcriptionally responsive to external chemical or physical stressors. This indicates a plausible correlation between ChHsc70 and ChYB-1 in the genetic regulatory pathway triggered by external stresses. This study presents the first evidence of positive regulator for Hsc70 transcription and contributes to a better understanding of the regulatory mechanisms governing Hsc70 expression. [ABSTRACT FROM AUTHOR]

The cation-dependent mannose-6-phosphate receptor (CD-M6PR) is a P-type lectin that plays a crucial role in lysosomal enzyme transport, bacterial resistance, and viral entry. In this study, we cloned and analyzed the ORF of the CD-M6PR gene from Crassostrea hongkongensis and named it Ch CD-M6PR. We analyzed the nucleotide and amino acid sequence of Ch CD-M6PR, its tissue expression pattern and immune response to Vibrio alginolyticus. Our results showed that the ORF of Ch CD-M6PR was 801 bp long and encoded a protein of 266 amino acids with a signal peptide at the N-terminus, as well as Man-6-P_recep, ATG27 and transmembrane structural domains. Phylogenetic analysis indicated that C rassostrea hongkongensis shared the highest similarity with Crassostrea gigas in the terms of CD-M6PR. The Ch CD-M6PR gene was found to be expressed in various tissues, with the highest expression observed in the hepatopancreas and the lowest in the hemocytes by the fluorescence quantitative PCR. Furthermore, the expression of Ch CD-M6PR gene was significantly up-regulated for a short time in response to Vibrio alginolyticus infection in the gill and hemocytes, while it was down-regulated in the gonads. The expression patterns of Ch CD-M6PR also varied in the other tissues. The 96 h cumulative mortality rate of Crassostrea hongkongensis infected with Vibrio alginolyticus after knockdown the Ch CD-M6PR gene was significantly higher. Overall, our findings suggests that Ch CD-M6PR plays a crucial role in the immune response of Crassostrea hongkongensis to Vibrio alginolyticus infection, and its tissue-specific expression patterns may be indicatitive of varied immune responses across tissues. • First cloned the cation-dependent mannose-6-phosphate receptor gene in bivalve. • The Ch CD-M6PR gene significantly up-regulated in response to Vibrio alginolyticus infection. • Ch CD-M6PR involved the innate immune of Crassostrea hongkongensis. [ABSTRACT FROM AUTHOR]

Extracellular signal-regulated kinases (ERKs) are a group of highly conserved serine/threonine-specific protein kinases that function as important signaling intermediates in mitogen-activated protein kinase (MAPK) pathways, which are involved in a wide variety of cellular activities, including proliferation, inflammation and cytokine production. However, little is known about the roles of this kinase in mollusk immunity. In this study, we identified a molluscan ERK homolog ( Ch ERK) in the Hong Kong oyster ( Crassostrea hongkongensis ) and investigated its biological functions. The open reading frame (ORF) of Ch ERK encoded a polypeptide of 365 amino acids, with a predicted molecular weight of 41.96 kDa and pI of 6.43. The predicted Ch ERK protein contained typical characteristic motifs of the ERK family, including a dual threonine-glutamate-tyrosine (TEY) phosphorylation motif and an ATRW substrate binding site. Phylogenetic analysis revealed that Ch ERK belonged to the mollusk cluster and shared a close evolutionary relationship with ERK from Crassostrea gigas . In addition, quantitative real-time PCR analysis revealed that Ch ERK expression was detected in all of the examined tissues and stages of embryonic development; its transcript level was significantly induced upon challenge with bacterial pathogens ( Vibrio alginolyticus and Staphylococcus haemolyticus ) in vivo and PAMPs (lipopolysaccharide and peptidoglycan) in vitro . Moreover, Ch ERK was mainly located in the cytoplasm of HEK293T cells. Taken together, these findings may provide novel insights into the functions of molluscan ERKs, especially their roles in response to immune challenge in oyster. [ABSTRACT FROM AUTHOR]

This study investigates the nutritional properties of edible oysters cultivated in Hong Kong (i.e. Deep Bay) to determine their nutritional values compared with other foodstuffs (e.g. land and aquatic-based foods). A total of ∼300 edible oysters were harvested and studied from aquaculture areas in Hong Kong. The Hong Kong oyster (Crassostrea hongkongensis) contained the highest concentrations (up to 32%) of polyunsaturated fatty acids (PUFAs), when compared to the Portuguese (Crassostrea angulata) and the Suminoe (Crassostrea ariakensis) oyster species. The C. hongkongensis also exhibited the best meat yield (∼28%) compared to oyster species from other regions (e.g., Crassostrea gigas). The C. ariakensis showed the highest concentrations (µg/g dwt.) of macro-and micro nutrients, e.g. Mg (∼3600), Na (∼22000), Zn (∼5000); compared to C. hongkongensis and C. angulata. Essential amino acids (EEA; mg/100 g wwt.) were highest in C. angulata (3439), followed by C. ariakensis (3330) and leaving C. hongkongensis (2619) as the poorest species in EAA. The comparison of the nutritional profile of oyster species cultivated in Hong Kong with different seafood species and other foodstuffs showed that oysters are nutrient-rich (or superfoods) shellfish species, which contribute with beneficial compounds (e.g. PUFAs, EAAs) and elements (e.g. Cu, Zn) for a human healthy diet. • The C. hongkongensis contained the highest concentration of PUFAs among oysters. • The C. hongkongensis has the best meat yield compared to oysters from other regions. • The C. ariakensis showed the highest concentrations of macro-and micro nutrients. • EEA were highest in C. angulata , following by C. ariakensis and C. hongkongensis. • Consumption of Hong Kong edible oysters can contribute positively for human health. [ABSTRACT FROM AUTHOR]

Vibrio parahaemolyticus (V. parahaemolyticus) is a salt-loving gram-negative bacterium, and is the leading cause of mortality in cultured shellfish in recent years. Toll-like Receptor 4 (TLR4) is a classical pattern recognition receptor (PRRs) that recognizes pathogen-associated molecular patterns (PAMPs) of pathogenic microorganism and activates the immune response. However, the function and signal pathway of TLR4 in oyster are still unknown. In this study, a new TLR4 gene was identified from the Crassostrea hongkongensis (C. hongkongensis). The ChTLR4 contained an open reading frame of 2643 bp, encoding 880 amino acids with seven leucine-rich repeat (LRR) domains and a Toll/IL-1R (TIR) domain. The ChTLR4 shared the highest sequence identity (83.0%) with TLR4 of Crassostrea gigas. Tissue expression analysis revealed that ChTLR4 showed the highest constitutive expression in the gill and hepatopancreas, and was significantly upregulated in immune tissues post V. parahaemolyticus infection, especially in gill and hemocytes. Moreover, TLR4 silencing significantly inhibited the immune-enzyme activities, including SOD, CAT, ACP, AKP in gill and LZM in hemolymph supernatant, and increased MDA content in hemolymph supernatant. Meanwhile, the antimicrobial activities of the hemolymph supernatant were also significantly inhibited by TLR4 silencing. These data demonstrated that the ChTLR4 involved in innate immune response of C. hongkongensis against V. parahaemolyticus challenge. Finally, qRT-PCR analysis showed that ChTLR4 silencing clearly inhibited the expression of genes in TLR4-MyD88 pathway, indicating that MyD88-dependent pathway played a crucial role in ChTLR4 -mediated immune response against V. parahaemolyticus. • TLR4 was characterized from C. hongkongensis with conserved TIR and LRR domains. • ChTLR4 was activated in gill and hemocytes after V. parahaemolyticus challenge. • ChTLR4 silencing decreased immune enzyme activities and antimicrobial activity. • ChTLR4 silencing suppressed the expression of genes in TLR4-MyD88 pathway. • The data indicated that ChTLR4 involved in immune response by TLR4-MyD88 pathway. [ABSTRACT FROM AUTHOR]

TRIM proteins are a group of highly conserved proteins participating in a variety of biological processes such as regulation of development, apoptosis, and innate immunity. However, the functions of these proteins in the mollusk are still poorly understood. In the present study, a TRIM9 homolog (named ChTRIM9 ) was first identified from a transcript-ome library in the Hong Kong oyster Crassostrea hongkongensis . The full-length cDNA of ChTRIM9 is 2928 bp and has a predicted Open Reading Frame ORF) encoding 721 amino acids, encoding a putative 80.2 kDa protein. SMART analysis indicated that ChTRIM9 contains the three typical TRIM domains, a RING finger, two B-boxes, and a coiled-coil domain in the N-terminal region, whereas the C-terminal region contains a SPRY domain. qRT-PCR analysis revealed a ubiquitous presence of ChTRIM9 , with the highest expression in the gills. Upon bacterial challenge in vivo, the ChTRIM9 transcripts in hemocytes were significantly down-regulated, indicating its involvement in signal transduction in immune response of oysters. Furthermore, ChTRIM9 was found to be localized mainly in the cytoplasm, and its over-expression inhibited the transcriptional activity of the NF-κB gene in HEK293T cells, demonstrating its negative role in regulating NF-κB signaling. [ABSTRACT FROM AUTHOR]

HSP70 acts mostly as a molecular chaperone and plays important roles in facilitating the folding of nascent peptides as well as the refolding or degradation of the denatured proteins. Under stressed conditions, the expression level of HSP70 is upregulated significantly and rapidly, as is known to be achieved by various regulatory factors controlling the transcriptional level. In this study, a high mobility group protein DSP1 was identified by DNA-affinity purification from the nuclear extracts of Crassostrea hongkongensis using the ChHSP70 promoter as a bait. The specific interaction between the prokaryotically expressed ChDSP1 and the FITC-labeled ChHSP70 promoter was confirmed by EMSA analysis. ChDSP1 was shown to negatively regulate ChHSP70 promoter expression by Luciferase Reporter Assay in the heterologous HEK293T cells. Both ChHSP70 and ChDSP1 transcriptions were induced by either thermal or CdCl 2 stress, while the accumulated expression peaks of ChDSP1 were always slightly delayed when compared with that of ChHSP70 . This indicates that ChDSP1 is involved, very likely to exert its suppressive role, in the recovery of the ChHSP70 expression from the induced level to its original state. This study is the first to report negative regulator of HSP70 gene transcription, and provides novel insights into the mechanisms controlling heat shock protein expression. [ABSTRACT FROM AUTHOR]

G-protein-coupled receptors (GPCRs) are the largest class of cell-surface receptors and play crucial roles in virtually every organ system. As one of the major downstream effectors of GPCRs, Akt can acquire information from the receptors and coordinate intracellular responses for many signaling pathways, through which the serine/threonine kinase masters numerous aspects of biological processes, such as cell survival, growth, proliferation, migration, angiogenesis, and metabolism. In the present study, we have characterized the first Akt1 ortholog in mollusks using the Hong Kong oyster, Crassostrea hongkongensis (designed ChAkt1 ). The full-length cDNA is 2223 bp and encodes a putative protein of 493 amino acids that contains an amino-terminal pleckstin homology (PH) domain, a central catalytic domain, and a carboxy-terminal regulatory domain. Quantitative real-time PCR analysis showed that ChAkt1 mRNA is broadly expressed in various tissues and during different stages of the oyster's embryonic and larval development. Upon exposure to two stressors (microbial infection and heat shock), the expression level of ChAkt1 mRNA increases significantly. Furthermore, ChAkt1 is located in the cytoplasm in HEK293T cells, where the over-expression of ChAkt1 regulates the transcriptional activities of NF-κB and p53 reporter genes. Taken together, our results indicate that ChAkt1 most likely plays a central role in response to various stimuli in oysters and has a particular response to microbial pathogens and high temperature. [ABSTRACT FROM AUTHOR]

Apoptosis has been primarily investigated in mammals, and little is known about apoptosis in mollusks. The proteins Bax and Bak play critical roles in the mitochondrial apoptosis pathway and in determining cell fate. In this study, Ch Bax and Ch Bak, homologs of the well-known Bax and Bak proteins, were identified from the oyster Crassostrea hongkongensis . The Ch Bax/Bak proteins consist of 207/232 amino acids with the typical domains found in BCL-2 family members. Ch Bax and Ch Bak mRNA expression were detected in all 8 of the selected oyster tissues and at the different stages of development. Fluorescence microscopy revealed that the full-length proteins of Ch Bax/Bak were located in the cytoplasm and mitochondrial outer membrane, of HEK293T cells, respectively. Furthermore, both of the genes' expression levels were found to increase in the hemocytes of oysters challenged with pathogens. The over-expression of Ch Bax or Ch Bak activates the p53-Luc reporter gene in HEK293T cells in a dose-dependent manner. These results indicate that Ch Bax and Ch Bak may play important roles in the mitochondrial apoptotic pathway in oysters. [ABSTRACT FROM AUTHOR]

Prostaglandin E receptor 4 (PTGER4) is an essential receptor that can detect various physiological and pathological stimuli and has been implicated in a wide variety of biological processes, including the regulation of immune responses, cytokine production, and apoptosis. In this report, the first mollusk PTGER4 , referred to as ChPTGER4 , was cloned and characterized from the Hong Kong oyster Crassostrea hongkongensis . Its full-length cDNA is 1734 bp in length, including 5′- and 3′-untranslated region (UTRs) of 354 bp and 306 bp, respectively, and an open reading frame (ORF) of 1074 bp. Ch PTGER4 comprises 357 amino acids and shares significant homology with its vertebrate homologs. The results of phylogenetic analysis revealed that Ch PTGER4 clusters with PTGER4 from the Pacific oyster. In addition, quantitative real-time PCR analysis revealed that ChPTGER4 was constitutively expressed in all tissues examined and that its expression was significantly up-regulated in hemocytes and gills following challenge by pathogens ( Vibrio alginolyticus , Staphylococcus haemolyticus and Saccharomyces cerevisiae ) and pathogen-associated molecular patterns (PAMPs: lipopolysaccharide (LPS) and peptidoglycan (PGN). Moreover, fluorescence microscopy analysis revealed that Ch PTGER4 localized to the membrane, and its overexpression significantly enhanced NF-κB reporter gene activation in the HEK293T cell line. In summary, this study provides the first experimental evidence of a functional PTGER4 in mollusks, which suggests its involvement in the innate immune response in oyster. [ABSTRACT FROM AUTHOR]

The Akirin protein is a nuclear factor in the innate immune system that is highly conserved from insects to mammals and plays key roles in diverse biological processes, including immunity, myogenesis, development and the cellular stress response. However, the function of Akirins in mollusk, the second most diverse group of animals, is still poorly understood. In this study, we report the discovery of an Akirin2 gene homolog ( ChAkirin2 ) and its biological functions in the Hong Kong oyster Crassostrea hongkongensis . Ch Akirin2 is 189 amino acids in length and shares significant homology with invertebrate homologs. Phylogenetic analysis results revealed that Ch Akirin2 is clustered with invertebrate Akirin2s. A sequence analysis of the 5' flanking regions of ChAkirin2 indicated that it harbors several potential PAMP-activated transcription factor binding sites (TFB), including sites for NF-κB, C/EBPα, AP-1, SRF, Oct-1 and GATA-1. An RT-PCR analysis showed that ChAkirin2 mRNA was ubiquitously expressed in various tissues and at different embryonic and larval stages. Additionally, upon infection by pathogens ( Vibrio alginolyticus, Staphylococcus haemolyticus and Saccharomyces cerevisiae ) and pathogen-associated molecular patterns (PAMPs: LPS, PGN and polyI:C), the expression of ChAkirin2 was significantly up-regulated. Moreover, fluorescence microscopy observations show that Ch Akirin2 is located in the nuclei of HeLa cells, and the overexpression of ChAkirin2 activated the transcriptional activities of the NF-κB reporter gene in HEK293T cells. Altogether, this report provided the first experimental demonstration that mollusks possess a functional Akirin2 that is involved in the innate defense and embryogenesis processes of the oyster. [ABSTRACT FROM AUTHOR]

A total of 1,050 strains of heterotrophic bacteria isolated from farming seawater and the intestines of oyster species Crassostrea hongkongensis were tested for resistance to 10 antibiotics by the Kirby–Bauer diffusion method. The resistant rates of seawater-derived bacteria to chloramphenicol, enrofloxacin, and ciprofloxacin were low (less than 20%), whereas the bacteria obtained from oysters showed low resistance to chloramphenicol and enrofloxacin. Many strains showed high resistant rates (more than 40%) to furazolidone, penicillin G, and rifampin. A total of 285 strains from farming seawater and oysters were resistant to more than three antibiotics. Several strains showed resistance to more than nine antibiotics. Furthermore, the peak resistant rates of the seawater-derived strains to multiple antibiotics overlapped in April, June, September, and November, and those of oyster-derived strains overlapped during April, July, and September. The multi-resistant rate patterns of strains from farming seawater and oyster intestines were similar. [ABSTRACT FROM AUTHOR]

Ficolins are a group of soluble animal proteins with multiple roles in innate immunity. These proteins recognize and bind carbohydrates in pathogens and activate the complement system, leading to opsonization, leukocyte activation, and direct pathogen killing, which have been reported in many animal species but might not be present in the shellfish lineage. In the present study, we identified the first fibrinogen-related protein from the oyster, Crassostrea hongkongensis . This novel ficolin-like protein contains a typical signal peptide and a fibrinogen-related domain (designated Ch FCN) at the N and C termini, respectively, but does not contain the additional collagen-like domain of ficolins. The full-length cDNA of Ch FCN is 1105 bp, encoding a putative protein of 297 amino acids with the molecular weight of 35.5 kD. Ch FCN is ubiquitously expressed in selected tissues, with the highest expression level observed in the gills. The temporal expression of Ch FCN following microbe infection shows that the expression of Ch FCN in hemocytes increases at 3 h post-challenge. The Ch FCN protein expression was also examined, and fluorescence microscopy revealed that deCh FCN (truncated signal peptide) is located in the cytoplasm of HeLa cells. Full-length Ch FCN was detected in the medium supernatant by western blot analysis. Recombinant Ch FCN proteins with the molecular weight about 50 kD bind Saccharomyces cerevisiae , Staphylococcus haemolyticus or Escherichia coli K-12, but not those from Vibrio alginolyticus . Furthermore, the r Ch FCN protein could agglutinate Gram-negative bacteria E. coli K-12 and enhance the phagocytosis of C. hongkongensis hemocytes in vitro . These results indicate that Ch FCN might play an important role in the immunity response of oysters. [ABSTRACT FROM AUTHOR]

Serum amyloid A (SAA), a major evolutionarily conserved acute-phase protein, participates in many biological processes in eukaryotic cells, including innate immunity. However, little information regarding the relationship between SAA and innate immunity in mollusks is currently available. In this report, the first bivalve SAA (referred to as ChSAA) gene was identified and characterized from the Hong Kong oyster Crassostrea hongkongensis. Its full-length cDNA is 623 bp, including a 5'-UTR of 147 bp, a 3'-UTR of 56 bp containing a poly(A) tail and an open reading frame (ORF) of 420 bp that encodes a polypeptide of 139 amino acids. The predicted amino acid sequence of ChSAA comprises characteristic motifs of the SAA family, including a typical signal peptide and a conserved SAA domain. Comparison and phylogenetic analyses suggested that ChSAA shares a high identity to known acute-phase SAA proteins (A-SAAs). In addition, quantitative real-time PCR analysis revealed that ChSAA is constitutively expressed in all tissues examined, with the highest expression level in the mantle, and that its expression was acutely and significantly up-regulated in hemocytes following challenge by Vibrio alginolyticus (G-), Staphylococcus haemolyticus (G+) or Saccharomyces cerevisiae (fungus). Furthermore, over-expression of ChSAA via transfection with a ChSAA expression vector led to significantly increased NF-κB activity in HEK293T cells. These results suggest that ChSAA is likely to constitute a member of the A-SAA family involved in anti-pathogen responses in C. hongkongensis. [ABSTRACT FROM AUTHOR]

Abstract: Apoptosis plays an important pathophysiological role in the homeostasis of immune systems. DNA fragmentation factors (DFFs) have been shown to be essential for DNA fragmentation, and the resultant DNA fragments follow a laddering pattern during apoptosis in vertebrates. In invertebrates, the functions of the DFF orthologs are not well characterized; therefore, we cloned and characterized a bivalve DFFA ortholog from the Hong Kong oyster Crassostrea hongkongensis (designated ChDFFA). The full-length cDNA of ChDFFA is 1186 bp in length and encodes a putative protein of 200 amino acids that contains an N-terminal CAD domain and a DFF-C domain at its C-terminus. Real-time RT-PCR results showed that ChDFFA is ubiquitously expressed in several tissues, and its highest expression is in gill. Following a 3- to 48-h challenge by microbial infection, the expression of ChDFFA increased in hemocytes. Using fluorescence microscopy, ChDFFA was localized in nuclei when exogenously expressed in HeLa cells. In addition, over-expression of ChDFFA inhibited the transcriptional activities of p53/p21-Luc reporter genes in HEK293T cells. These results suggest that ChDFFA may be involved in immune response reactions in the Hong Kong oyster C. hongkongensis. [Copyright &y& Elsevier]

Abstract: Growing evidence suggests that the transcription factor activator protein-1 (AP-1), a downstream target of mitogen-activated protein kinase (MAPK) signaling, plays a major role in stimulating the synthesis of immune effector molecules during innate immune responses. We have characterized ChAP-1, an AP-1-like protein in Crassostrea hongkongensis that is a member of the AP-1 family of proteins. ChAP-1 is composed of 290 amino acid residues with a Jun and bZIP domain at the N- and C-termini, respectively, a structure similar to that of known Ap-1 proteins. ChAP-1 mRNA is expressed in several tissues analyzed, with highest expression in the mantle. Expression of ChAP-1 increases in response to Vibrio alginolyticus, Salmo haemolyticus or Salmo cerevisiae infection and, despite the location of GFP-tagged full-length ChAP-1 protein in the cytoplasm, ChAP-1 activates the transcription of an L8G5-luc reporter gene, and its over-expression can also activate the AP-1-Luc reporter gene in HEK293T cells. [Copyright &y& Elsevier]

Abstract: Serum response factor (SRF) function is essential for transcriptional regulation of numerous growth-factor-inducible genes and triggers proliferation, differentiation and apoptosis of the cells. In this report, the first mollusk serum response factor like homolog gene (designated ChSRF) was identified and characterized from the Hong Kong oyster, Crassostrea hongkongensis. The full-length cDNA of ChSRF was 1716 bp in length and encodes a putative protein of 434 amino acids respectively, and shares the MADS domain at the N-terminal. ChSRF is ubiquitously expressed in various tissues, with the highest expression level observed in muscle. Temporal expression of ChSRF following microbe infection shows that the expression of ChSRF in hemocytes increases from 3 to 24 h post-challenge. As a target gene of SRF, β-actin demonstrates a similar gene expression mode in constitutive tissue and pathogen infection. Furthermore, some protein profiles of ChSRF was revealed, fluorescence microscopy results show that ChSRF located in the nuclei of HeLa cells and over-expression of ChSRF activated the transcriptional activities of MAPK signal pathway in HEK293T cells. These results indicate that ChSRF maybe play an important role in signal transduction in the immunity and development response of oysters. Furthermore, we found that ChSRF could regulate the expression of β-actin gene, which indicate that ChSRF is a muscle differentiation regulator in the oyster and it will help us to improve aquaculture production. [Copyright &y& Elsevier]

Ammonia nitrogen, a major oxygen-consuming pollutant in the environment, can adversely affect aquatic organisms such as fish, bivalves, and crustaceans. We investigated the toxic effects of ammonia nitrogen on the Hong Kong oyster, Crassostrea hongkongensis , using flow cytometry and 1H nuclear magnetic resonance metabolomics. Exposure to ammonia nitrogen caused time- and concentration-dependent alterations in various immune parameters in hemocytes and impaired the metabolic profiles of the gills. We observed changes in the rate of apoptosis, esterase activity, lysosomal mass, hemocyte counts, phagocytic activity, and mitochondrial mass. Exposure affected metabolic pathways involved in energy metabolism, osmotic balance, and oxidative stress. We concluded that ammonia nitrogen induces metabolic and hematological dysfunction in C. hongkongensis , and our findings provide insights into the biochemical defense strategies of bivalves exposed to acute high-concentration ammonia nitrogen. [Display omitted] • The toxic effects of ammonia nitrogen on Crassostrea hongkongensis were studied. • Ammonia nitrogen induces metabolic and hematological dysfunction. • Immune parameters in hemocytes were time- and concentration-dependent altered. • Gill metabolic profiles were altered time- and concentration-specifically. • No sex-specific effects were found. [ABSTRACT FROM AUTHOR]

Abstract: Apoptosis plays a key role in many biological processes, including homeostasis within the immune system. A family of cysteine proteases, the caspases, constitutes the core of the apoptotic machinery. We have characterized the first bivalve caspase-8 ortholog from the Hong Kong oyster Crassostrea hongkongensis (designated ChCaspase-8). The full-length cDNA is 1945 bp in length encoding a putative protein of 557 amino acids that contains two N-terminal DED domains, and a CASc domain at the C-terminus. ChCaspase-8 is ubiquitously expressed in oysters, with highest expression levels in the gonad and labial palps. Following microbial infection, the expression of ChCaspase-8 increased in hemocytes from 12 to 72 h post-challenge. When expressed in HeLa cells, ChCaspase-8 is located in the cytoplasm, while over-expression of ChCaspase-8 in HEK293T cells activates the transcriptional activities of NF-κB. These results indicate that ChCaspase-8 might play an important role in the immune and apoptotic responses of oysters. [Copyright &y& Elsevier]

Abstract: Lectins play an important role in immune recognition and host defense. In the present study, a full-length cDNA encoding a novel sialic acid binding lectin was cloned from Crassostrea hongkongensis (designated Ch-salectin) by rapid amplification of cDNA ends (RACE). It is 531 bp in length, containing a 21 bp 5′ UTR, a 39 bp 3′ UTR and a 468 bp ORF coding for 156 amino acids. The Ch-salectin protein contains a signal peptide and a conserved complement component C1q domain. The purified recombinant MBP-tagged Ch-salectin protein can bind to a sialic acid containing protein fetuin and significantly inhibit the growth of both Gram-negative and Gram-positive bacteria. Furthermore, the transcription of Ch-salectin was inducible and significantly up-regulated during Vibrio alginolyticus infection. Thus, these results highlight the essential roles of Ch-salectin in immune recognition and host defense against bacterial infection in C. hongkongensis. [Copyright &y& Elsevier]

Abstract: Catalase is one of the key antioxidant enzymes and it appears to be involved in protection against immune infection and oxidative stress. Here, two catalase cDNAs (ChCat-1 and ChCat-2) were isolated from hemocytes of Crassostrea hongkongensis using SSH and RACE. The full-length cDNAs of ChCat-1 and ChCat-2 are 1913 and 2466 bp in length, encoding proteins of 515 and 511 amino acids, respectively. Multiple alignments of amino acid sequences revealed that both ChCat-1 and ChCat-2 possess several characteristic features of the catalase family of enzymes, including one proximal active site signature, one heme-ligand signature, and three catalytic amino acid residues (His72, Asn145 and Tyr355). Phylogenetic analysis indicates that these two catalases may share a common ancestral gene and result from a gene duplication event following the divergence of bivalves and gastropods. Constitutive expression of ChCat-1 and ChCat-2 was observed in all tissues studied, with highest levels of expression in gill and muscle, respectively. The expression of both genes was inducible by bacterial infection, and reached the maximum at 8 h (9.0-fold) and 12 h (2.3-fold) post-infection, respectively. Furthermore, both the purified ChCat-1 and ChCat-2 protein displayed a strong catalase activity, and S2 cells carrying ChCat-1 or ChCat-2 showed a higher degree of resistance to H2O2 than that of control cells. In a word, this is the first report of the presence of two catalase genes in a single marine bivalve, and our results highlight the involvement of both ChCat-1 and ChCat-2 in host protection against pathogen infection and oxidative stress in C. hongkongensis. [Copyright &y& Elsevier]

SODs are ubiquitous metalloenzymes that can scavenge superoxides in response to various stresses. In the present study, full-length cDNAs of two SOD genes were isolated from Crassostrea hongkongensis (designated ChMnSOD and ChCuZnSOD). The cDNAs are 997 and 918 bp in length with ORFs of 675 and 468 bp (encoding 225 and 156 amino acids), respectively. Sequence analysis revealed a conserved Sod_Fe domain in ChMnSOD, and a Sod_Cu_Zn domain in ChCuZnSOD. Subcellular localization of ChMnSOD is mitochondrial while intracellular expression of ChCuZnSOD is detected. Although their expression overlaps in a wide range of tissues, ChMnSOD mRNA expression is high in gonad while ChCuZnSOD’s is strong in adductor muscle. After infection by Vibrio alginolyticus, ChMnSOD mRNA was up-regulated 5 fold (p < 0.05) at 4 h, but returned to normal level 6 h post-infection. The expression of ChCuZnSOD gene showed a slight delay to the infection challenge and was elevated roughly 4 fold after 8 h (p < 0.05), returning to normal at 12 h post-infection. The elevated transcript levels of the two SOD genes in response to V. alginolyticus infection highlights their important functions in eliminating toxic reactive oxygen species (ROS) and protecting organisms from bacterial invasion in C. hongkongensis. [ABSTRACT FROM AUTHOR]

Abstract: Heat shock protein 90 (HSP90) is a highly conserved and multi-functional molecular chaperone that plays an essential role in both cellular metabolism and stress response. Here, we report the cloning of the HSP90 homologue in Crassostrea hongkongensis (ChHSP90) through SSH in combination with RACE from cDNA of haemocytes. The full-length cDNA of ChHSP90 is 2459 bp in length, consisting of a 3′, 5′-untranslated region (UTR) and an open reading frame of 2169 bp encoding 722 amino acids. The identity analysis of the amino acid sequence of HSP90 revealed that ChHSP90 is highly conserved. Distribution of ChHSP90 mRNA in gonad, heart, adductor muscle, mantle, gill, digestive gland, and haemocytes suggested that ChHSP90 is ubiquitously expressed. The mRNA levels of ChHSP90 under salinity and bacterial challenges were analyzed by real-time PCR. Under hypo-osmotic treatment, ChHSP90 mRNA in gonad, heart and haemocytes were significantly up-regulated on day 2 and onwards; while in gill, digestive gland and adductor muscle it was significantly down-regulated; the expression in mantle was decreased significantly on day 2 and 3 (P < 0.01), and then up-regulated on day 4 (P < 0.05). Under hyper-osmotic treatment, the mRNA level in gonad, heart, adductor muscle was increased on day 2 and onwards; in gill, it was firstly increased, and then gradually decreased, reaching a minimum on day 3. On day 4, the expression level in gill recovered to pre-treatment level; in mantle and digestive gland, the expression levels were decreased, reaching to the minimum on day 3. During Vibrio alginolyticus challenge, the mRNA level of ChHSP90 increased 3-fold at 4 h post-infection, returned to its pre-challenge level at 6 h post-infection, then was further up-regulated from 8 to 36 h post-infection. These experiments demonstrate that ChHSP90 mRNA is constitutively expressed in various tissues and apparently inducible in haemocytes under salinity and bacterial challenges, suggesting its important role in response to both osmotic stress and bacterial invasion. [Copyright &y& Elsevier]

Abstract: Tumor necrosis factor (TNF) receptor-associated factor 7 (TRAF7) is one of several adaptor proteins that are critically involved in the activation of TLR-dependent NF-κB signaling. In this report, the first mollusk TRAF7 (designated ChTRAF7) homolog was isolated from Crassostrea hongkongensis by screening a suppression subtractive library. The full-length cDNA, 2290 bp in length, encodes a putative protein of 686 amino acids that contains a RING finger domain, an adjacent zinc finger domain, and seven WD40 repeats. ChTRAF7 is ubiquitously expressed in various tissues including digestive gland, mantle, gill, heart, hemocytes, muscle, and gonads, with the highest expression observed in gonads. Temporal expression of ChTRAF7 following bacterial infection shows that expression of ChTRAF7 in hemocytes decreases from 2 to 12 h post-challenge, and then recovered to the original level after 24 h. These results indicate that ChTRAF7 may play an important role in signal transduction in the immune response of oysters. [Copyright &y& Elsevier]