Transcriptomic and Physiological Analysis Reveal Melanin Synthesis-Related Genes and Pathways in Pacific Oysters (Crassostrea gigas).

Shell color is one of the shell traits of molluscs, which has been regarded as an economic trait in some bivalves. Pacific oysters (Crassostrea gigas) are important aquaculture shellfish worldwide. In the past decade, several shell color strains of C. gigas were developed through selective breeding, which provides valuable materials for research on the inheritance pattern and regulation mechanisms of shell color. The inheritance patterns of different shell colors in C. gigas have been identified in certain research; however, the regulation mechanism of oyster pigmentation and shell color formation remains unclear. In this study, we performed transcriptomic and physiological analyses using black and white shell oysters to investigate the molecular mechanism of melanin synthesis in C. gigas. Several pigmentation-related pathways, such as cytochrome P450, melanogenesis, tyrosine metabolism, and the cAMP signaling pathway were found. The majority of differentially expressed genes and some signaling molecules from these pathways exhibited a higher level in the black shell oysters than in the white, especially after L-tyrosine feeding, suggesting that those differences may cause a variation of tyrosine metabolism and melanin synthesis. In addition, the in vitro assay using primary cells from mantle tissue showed that L-tyrosine incubation increased cAMP level, gene and protein expression, and melanin content. This study reveals the difference in tyrosine metabolism and melanin synthesis in black and white shell oysters and provides evidence for the potential regulatory mechanism of shell color in oysters.

Structural and functional characterization of an egg-laying hormone signaling system in a lophotrochozoan - The pacific oyster (Crassostrea gigas).

The egg-laying hormones (ELHs) of gastropod mollusks were characterized more than forty years ago. Yet, they have remained little explored in other mollusks. To gain insights into the functionality of the ELH signaling system in a bivalve mollusk - the oyster Crassostrea gigas, this study investigates the processing of its ELH precursor (Cragi-ELH) by mass spectrometry. Some of the ELH mature peptides identified in this study were subsequently investigated by nuclear magnetic resonance and shown to adopt an extended alpha-helix structure in a micellar medium mimicking the plasma membrane. To further characterize the ELH signaling system in C. gigas, a G protein-coupled receptor phylogenetically related to ecdysozoan diuretic hormone DH44 and corticotropin-releasing hormone (CRH) receptors named Cragi-ELHR was also characterized functionally and shown to be specifically activated by the two predicted mature ELH peptides and their N-terminal fragments. Both Cragi-ELH and Cragi-ELHR encoding genes were mostly expressed in the visceral ganglia (VG). Cragi-ELH expression was significantly increased in the VG of both fully mature male and female oysters at the spawning stage. When the oysters were submitted to a nutritional or hyposaline stress, no change in the expression of the ligand or receptor genes was recorded, except for Cragi-ELHR only during a mild acclimation episode to brackish water. These results suggest a role of Cragi-ELH signaling in the regulation of reproduction but not in mediating the stress response in our experimental conditions.

A novel Fas ligand plays an important role in cell apoptosis of Crassostrea hongkongensis: molecular cloning, expression profiles and functional identification of ChFasL.

Background: Apoptosis regulates normal development, homeostasis, immune tolerance and response to environmental stress by eliminating unwanted or diseased cells, and plays a key role in non-specific immunity of invertebrates. The exogenous pathway mediated by death receptors and death ligands is a very important pathway for cell apoptosis. Death ligands are mainly members of the tumour necrosis factor (TNF) family, of which FasL is an important member. The deep involvement of FasL in vertebrates cell apoptosis and immunity has been reported many times, but there is limited research on the FasL gene in shellfish, and its functional importance in oyster cell apoptosis and immunity remains unclear. Methods: The full length of ChFasL was identified and cloned based on the genome of Crassostrea hongkongensis. Quantitative PCR was used to detect the relative expression of ChFasL in different developmental stages and tissues, as well as the changes of relative expression in hemocytes after bacterial infection. The expression position of ChFasL in HEK293T cells was also located by subcellular localization, and the effect of increased recombinant protein content on the activity of reporter genes p53 and p21 was studied by dual-fluorescence reporter gene. Finally, the changes of apoptosis rate in hemocytes after ChFasL silencing was identified by RNA interference technology. Results: We identified a novel FasL gene from C. hongkongensis and named it ChFasL. We found that ChFasL has potential N-linked glycosylation site, a transmembrane domain and a TNF region, which was a typical characteristics of TNF family. ChFasL was expressed in all developmental stages of larvae and in all tissues of oysters. After stimulation by V. alginolyticus or S. haemolyticus, its relative expression in hemocytes increased significantly, suggesting that ChFasL was deeply engaged in the immune response process of C. hongkongensis to external microbial stimulation. The results of subcellular localization showed that ChFasL was mainly distributed in the cytoplasm of HEK293T cells. With the overexpression of the recombinant protein pcDNA3 1- ChFasL, the activity of p53 and p21 significantly increased, showing a positive regulatory effect. Moreover, after dsRNA successfully reduced the relative expression of ChFasL, the apoptosis rate of hemocytes was significantly lower than that the dsGFP group. Conclusion: These results comprehensively confirmed the important role of ChFasL in the apoptosis process of C. hongkongensis, which provided the basis and premise for the in-depth understanding of the immune function of apoptosis in molluscs, and also contributed to the research on the pathogenic death mechanism and disease resistance breeding of marine bivalves.

Employing molecular, chemical and physiological techniques using Crassostrea virginica to assess ecosystem health along coastal South Carolina and North Carolina, United States.

Natural and anthropogenic environmental impacts can introduce contaminants into sensitive habitats, threatening ecosystems and human health. Consistent monitoring of coastal areas provides critical environmental assessment data. Sediments and Eastern Oyster (Crassostrea virginica) tissues were collected at fourteen South Carolina (SC) and four North Carolina (NC) sites as part of the National Oceanic and Atmospheric Administration's Mussel Watch environmental monitoring program. Cellular and molecular techniques were employed to measure C. virginica stress response, specifically, Lipid Peroxidation (LPx), Glutathione (GSH), and qPCR techniques. Gene specific primers targeted for detecting oxidative stress and cellular death were developed in C. virginica to gauge response to current environmental conditions using gill and hepatopancreas (HP) tissue. In order to validate gene specific markers as additional assessment tools, a 96 h zinc (Zn) laboratory exposure was performed. Cellular biomarker data revealed tissue specific responses. Hepatopancreas data showed C. virginica exhibited stress through the lipid peroxidation assay amongst sampling sites, however, response was managed through glutathione detoxification. Gill tissue data had significantly lower levels of cellular biomarker response compared to hepatopancreas. Molecular biomarkers targeting these cellular stress pathways through qPCR analysis show upregulation of Metallothionein in hepatopancreas and gill tissue with a concurrent > 2-fold upregulation in the detoxification marker Superoxide Dismutase (SOD) at three NC sites. SC sites displayed higher stress levels through LPx assays and down-regulation in GPx gene activity. Laboratory zinc exposure revealed no significance in cellular biomarker results, however, molecular data showed gills responding to zinc treatment through upregulation of Metallothionein, SOD and Cathepsin L, indicating an acute response in gills. Collectively, chemical, cellular and molecular methods clarify sentinel stress response of biological impacts and aid in evaluating environmental health in coastal ecosystems. This combined methodological approach provides a detailed analysis of environmental conditions and improves land-use management decisions.

A bone morphogenetic protein regulates the shell formation of Crassostrea gigas under ocean acidification.

Bone morphogenetic proteins (BMPs) are key factors controlling osteoblast differentiation, which have been proved to be involved in the hard tissue formation of marine mollusks. In the present study, a member of BMPs gene (CgBMP7) was identified from Pacific oyster Crassostrea gigas (C. gigas) with the aim to understand its possible role in the regulation of shell formation under ocean acidification (OA) conditions. The open reading frame (ORF) of CgBMP7 was of 1254 bp encoding a polypeptide of 417 amino acids. The deduced amino acid sequence of CgBMP7 was comprised of one signal peptide, one prodomain and one TGF-ß domain, which shared 21.69%-61.10% identities with those from other species. The mRNA transcript of CgBMP7 was ubiquitously expressed in all the tested tissues of adult oysters with a higher expression level in mantle, notably highest in the middle fold (MF) of the three folds of mantle. The expression level of bone morphogenetic protein type I receptor (CgBMPR1B) mRNA was also highest in the MF and up-regulated dramatically post recombinant BMP7 protein (rCgBMP7) stimulation. After the blockage of BMPR1B with inhibitor LDN193189 (LDN), the mRNA expression level and phosphorylation level of CgSmad1/5/8 in mantle were decreased, and the mRNA expression levels of CgCaM and Cgengrailed-1 were down-regulated significantly. During the oysters were exposed to acidified seawater for weeks, the expression levels of CgBMP7, CgBMPR1B and CgSmad1/5/8 in the MF decreased significantly (p < 0.01) at the 4th week, and CgCaM and Cgengrailed-1 also exhibited the same variable expression patterns as CgBMP7. In addition, the growth of shell in the treatment group (pH 7.8) was slower than that in the control group (pH 8.1). These results collectively indicated that BMP7 was able to trigger the BMPR-Smad signaling pathway and involved in controlling the formation of oyster calcified shell under OA conditions.

Effects of Dietary Cystine and Tyrosine Supplementation on Melanin Synthesis in the Pacific Oyster (Crassostrea gigas).

Melanogenesis is a multistep process to produce melanin for dark pigmentation in skin coloration. Previous studies in vertebrates demonstrated that cystine and tyrosine amino acids are involved in the melanin synthesis. However, very little is known about the melanogenesis in bivalve. In this study, cystine supplementation for 30 days significantly upregulated the expression of CgB-aat1, CgCbs and CgTyr and pheomelanin content in the Pacific oyster Crassostrea gigas. Transmission electron microscope (TEM) results revealed more melanosomes in the connective tissue and melanin granules were secreted in epithelium of mantle. In contrast, tyrosine supplementation had no clear effect on melanogenesis except the gene expression changes of CgB-aat1 and CgCbs. In addition, prolonged supplementation of cystine or tyrosine for 60 days had a negative impact on melanogenesis. Indeed, after 60 days, expression of most of the melanin synthesis-related genes under study was decreased, and melanin content was significantly reduced, indicating that cystine and tyrosine might inhibit production of eumelanin and pheomelanin, respectively. In addition, in vitro analysis using primary cell culture from mantle tissue indicated that incubation with cystine, tyrosine, or B-AAT1 polypeptide, CBS/TYR recombinant proteins induced the increase of CgB-aat1 and CgCbs expression in a dose-dependent manner, suggesting the presence of a regulatory network in response to cystine and tyrosine amino acids intakes in pheomelanin synthesis-related gene expression. Taken together, these data indicate that cystine-CgB-aat1-CgCbs-CgTyr axis is a potential regulator of the pheomelanin biosynthesis pathway, and thus plays an important role in the mantle pigmentation in C. gigas. This work provides a new clue for selective cultivation of oyster strains with specific shell colors in bivalve breeding.

Effects of cadmium and zinc on gene expression of novel molecular biomarkers in the mangrove oyster Crassostrea gasar.

Metal contamination impacts various aquatic species, and mollusk bivalves are appropriate sentinel organisms in coastal pollution assessment. Metal exposure can disrupt homeostasis, alter gene expression, and harm cellular processes. However, organisms have evolved mechanisms to regulate metal ions and counteract their toxicity. This study examined the effect of acute cadmium (Cd) and zinc (Zn) on metal-related gene expression in gills of Crassostrea gasar following 24 and 48 h of laboratory exposure. We focused on Zn transport, metallothionein (MT), glutathione (GSH) biosynthesis, and calcium (Ca) transporter genes to understand the underlying Cd and Zn-accumulating mechanisms that prevent metal toxicity. Our findings revealed increased Cd and Zn levels in oyster gills, with significantly higher accumulation after 48 h. C. gasar accumulated high Cd concentrations even in scarce conditions and increased Zn levels, suggesting a strategy to cope with toxicity. While no significant gene expression differences were observed after 24 h, the increased metal accumulation after 48 h led to upregulation of CHAC1, GCLC, ZnT2, and MT-like genes in oysters exposed to Cd, and increased ZnT2-like expression following exposure to higher Cd/Zn mixtures. We found evidence of oysters may mobilize metal-related genes to mitigate Cd-induced toxicity by both chelating metals and/or reducing their intracellular concentrations. The observed genes upregulation also indicates their sensitivity to changes in metal bioavailability. Overall, this study offers insights into oyster mechanisms for coping with metal toxicity and suggests ZnT2, MT, CHAC1, and GCLC-like as molecular biomarkers for monitoring aquatic metal pollution using C. gasar as sentinel species.

In Silico Prospecting for Novel Bioactive Peptides from Seafoods: A Case Study on Pacific Oyster (Crassostrea gigas).

Pacific oyster (Crassostrea gigas), an abundant bivalve consumed across the Pacific, is known to possess a wide range of bioactivities. While there has been some work on its bioactive hydrolysates, the discovery of bioactive peptides (BAPs) remains limited due to the resource-intensive nature of the existing discovery pipeline. To overcome this constraint, in silico-based prospecting is employed to accelerate BAP discovery. Major oyster proteins were digested virtually under a simulated gastrointestinal condition to generate virtual peptide products that were screened against existing databases for peptide bioactivities, toxicity, bitterness, stability in the intestine and in the blood, and novelty. Five peptide candidates were shortlisted showing antidiabetic, anti-inflammatory, antihypertensive, antimicrobial, and anticancer potential. By employing this approach, oyster BAPs were identified at a faster rate, with a wider applicability reach. With the growing market for peptide-based nutraceuticals, this provides an efficient workflow for candidate scouting and end-use investigation for targeted functional product preparation.

Involvement of B-aat1 and Cbs in regulating mantle pigmentation in the Pacific oyster (Crassostrea gigas).

BACKGROUND: Shell color formation is an important physiological process in bivalves, the molecular genetic basis has potential application in bivalve aquaculture, but there is still remaining unclear about this issue. The cystine/glutamate transporter (Slc7a11) and cystathionine beta-synthase (Cbs) are integral genes in pheomelanin synthesis pathway, which is vital to skin pigmentation. METHODS AND RESULTS: Here, the sequences of b (0, +) -type amino acid transporter 1 (B-aat1) and Cbs in Pacific oyster (Crassostrea gigas) (CgB-aat1, CgCbs) were characterized. Phylogenetically, the deduced amino acid sequences of CgB-aat1 and CgCbs both possessed conserved features. Genes were both ubiquitously expressed in six tested tissues with more abundant expression level in central mantle. Besides, the polyclonal antibodies of CgB-aat1, CgCbs, CgTyr, and CgTyrp2 were successfully prepared. Immunofluorescence analysis revealed that CgB-aat1 and CgCbs proteins were both expressed in gill rudiments of eyed-larvae and concentrated mainly in cytoplasm of epithelial cell and nerve axons in mantle. Additionally, after CgB-aat1 or CgCbs silencing, expressions at mRNA and protein levels of CgB-aat1 and CgCbs involved in pheomelanin synthesis were significantly suppressed, and CgTyr, CgTyrp1 and CgTyrp2 related to eumelanin synthesis were also down-regulated but no apparent differences, respectively. Moreover, micrographic examination found less brown-granules at mantle edge in CgB-aat1 interference group. CONCLUSION: These results implied that pheomelanin synthesis was possible induced by CgB-aat1-CgTyr-CgCbs axis, and it played an essential role on mantle pigmentation in the oysters. These findings provide the useful genetic knowledge and enrich the physiological information for the shell color formation in bivalve aquaculture.

High-Yield Preparation of American Oyster Defensin (AOD) via a Small and Acidic Fusion Tag and Its Functional Characterization.

The marine peptide, American oyster defensin (AOD), is derived from Crassostrea virginica and exhibits a potent bactericidal effect. However, recombinant preparation has not been achieved due to the high charge and hydrophobicity. Although the traditional fusion tags such as Trx and SUMO shield the effects of target peptides on the host, their large molecular weight (12-20 kDa) leads to the yields lower than 20% of the fusion protein. In this study, a short and acidic fusion tag was employed with a compact structure of only 1 kDa. Following 72 h of induction in a 5 L fermenter, the supernatant exhibited a total protein concentration of 587 mg/L. The recombinant AOD was subsequently purified through affinity chromatography and enterokinase cleavage, resulting in the final yield of 216 mg/L and a purity exceeding 93%. The minimum inhibitory concentrations (MICs) of AOD against Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus galactis ranged from 4 to 8 µg/mL. Moreover, time-killing curves indicated that AOD achieved a bactericidal rate of 99.9% against the clinical strain S. epidermidis G-81 within 0.5 h at concentrations of 2× and 4× MIC. Additionally, the activity of AOD was unchanged after treatment with artificial gastric fluid and intestinal fluid for 4 h. Biocompatibility testing demonstrated that AOD, at a concentration of 128 µg/mL, exhibited a hemolysis rate of less than 0.5% and a cell survival rate of over 83%. Furthermore, AOD's in vivo therapeutic efficacy against mouse subcutaneous abscess revealed its capability to restrain bacterial proliferation and reduce bacterial load, surpassing that of antibiotic lincomycin. These findings indicate AOD's potential for clinical usage.

Crassostrea gigas-Based Bioactive Peptide Protected Thrombin-Treated Endothelial Cells against Thrombosis and Cell Barrier Dysfunction.

The activation of thrombin-treated endothelial cells resulted in disruption of the vascular tissues. A novel oyster-derived bioactive dodecapeptide (IEELEELEAER, P-2-CG) was reported to protect the human umbilical vein endothelial cells and their barrier function via the decrease of VE-cadherin disruption and the restoration of the F-actin arrangement. The promotion of the extrinsic pathway in this case triggers the release of tissue factors that occurs on the surface of the endothelial cells, thus changing the antithrombotic to prothrombotic. P-2-CG induced accordingly a prolongation of plasma clotting time and thrombin generation time, following the alteration of the antithrombotic phenotype. Furthermore, the antithrombotic activity of P-2-CG was also supported by the reduction of FXa and the inhibition of other factors release, for instance, inflammation factors, ROS, etc. In addition to its antithrombogenic role, P-2-CG displayed anti-inflammatory and antioxidant properties via the mitogen-activated protein kinase cascades and central signaling pathways as shown in an in vitro model of endothelial dysfunction.

Novel Antioxidant Peptides from Crassostrea Hongkongensis Improve Photo-Oxidation in UV-Induced HaCaT Cells.

Enzymatic hydrolysates from Oysters (OAH) display multiple biological activities. Previously, a 3~5 KDa oyster ultrafiltration component (OUP) showed a high property of preventing skin oxidation. Subsequently, we identified specific peptides with such activity. OUP was fractionated stepwise by Sephadex-G25 and RP-HPLC, and active fractions were screened using UV-irradiated HaCaT cells. The most active fractions (OP5-3) were analyzed by LC-MS/MS and a total of 17 peptides were identified. Results from mass spectrometry showed that OP5-3 consisted of peptides with a molecular weight range of 841.51-1786.92 Da. Six of these peptides were synthesized for validating the activity of resisting skin oxidation in the same cell model. All six peptides showed varying degrees of antioxidant activity, while pretreatment of HaCaT cells with AIVAEVNEAAK alleviated UV cytotoxicity, inhibited metalloproteinase 1 (MMP-1) expression, and showed the highest activity to resist UV-induced skin photo-oxidation among these peptides. In addition, results from molecular docking analysis of MMP-1 with AIVAEVNEAAK showed that AIVAEVNEAAK suppresses its enzymatic activity by directly interacting with MMP-1 and thus exhibit anti-photoaging activity.

An integrin alpha 4 (ChIntα 4) from oyster Crassostrea hongkongensis mediates the hemocytes phagocytosis towards Vibrio alginolyticus.

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 ChIntα 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-ChIntα 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 ChIntα 4 might be involved in resisting V. alginolyticus infection and regulating phagocytosis as a cell adhesion receptor in C. hongkongensis.

Recombinant expression and coexpression of oyster defensin and proline-rich peptide in Komagataella phaffii.

Proline-rich peptide (CgPrp) and defensin (CgDef), oyster (Crassostrea gigas)-originated antimicrobial peptides (AMPs), were produced by the recombinant technique in Komagataella phaffii GS115 cells. For this purpose, the nucleotide sequences encoding the CgPrp and CgDef peptides were synthesized by the recursive PCR technique, and ligated in pPICZaA expression vector. Additionally, the expression cassettes of pPICZαA-CgDef and pPICZαA-CgPrp were combined using in vitro multimer ligation strategy to construct the coexpression vector pPICZaA-CgPrp-CgDef. The expression and coexpression vectors transformed into K. phaffii GS115 cells by electroporation. At the end of the 0.5% methanol-induced expression stage for 96 h, the recombinant peptides were purified from the culture medium. The concentrations of purified peptides were changed between 1.05 and 1.21 mg/L. The recombinant peptides successfully inhibited the growth of tested Gram-positive bacterial strains belonging to Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Listeria monocytogenes, and Bacillus cereus. The minimum inhibitory concentrations (MIC) of recombinant CgPrp, CgDef, and CgPrp-CgDef peptides against tested bacteria were in the range of 12.50-25.00, 18.75-75.00, and 5.80-11.60 pg/µl, respectively. The results of the study proved that the recombinant CgPrp, CgDef, and CgPrp-CgDef peptides expressed in K. phaffii might have good potential for the inhibition of common Gram-positive pathogenic bacteria, including drug-resistant MRSA.

A non-canonical, interferon-independent signaling activity of cGAMP triggers DNA damage response signaling.

Cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), produced by cyclic GMP-AMP synthase (cGAS), stimulates the production of type I interferons (IFN). Here we show that cGAMP activates DNA damage response (DDR) signaling independently of its canonical IFN pathways. Loss of cGAS dampens DDR signaling induced by genotoxic insults. Mechanistically, cGAS activates DDR in a STING-TBK1-dependent manner, wherein TBK1 stimulates the autophosphorylation of the DDR kinase ATM, with the consequent activation of the CHK2-p53-p21 signal transduction pathway and the induction of G1 cell cycle arrest. Despite its stimulatory activity on ATM, cGAMP suppresses homology-directed repair (HDR) through the inhibition of polyADP-ribosylation (PARylation), in which cGAMP reduces cellular levels of NAD+; meanwhile, restoring NAD+ levels abrogates cGAMP-mediated suppression of PARylation and HDR. Finally, we show that cGAMP also activates DDR signaling in invertebrate species lacking IFN (Crassostrea virginica and Nematostella vectensis), suggesting that the genome surveillance mechanism of cGAS predates metazoan interferon-based immunity.

Physiological role of CYP17A1-like in cadmium detoxification and its transcriptional regulation in the Pacific oyster, Crassostrea gigas.

Cadmium (Cd) is one of the most harmful heavy metals due to its persistence and bioaccumulation through the food chains, posing health risks to human. Oysters can bioaccumulate and tolerate high concentrations of Cd, providing a great model for studying molecular mechanism of Cd detoxification. In a previous study, we identified two CYP genes, CYP17A1-like and CYP2C50, that were potentially involved in Cd detoxification in the Pacific oyster, Crassostrea gigas. In this work, we performed further investigations on their physiological roles in Cd detoxification through RNA interference (RNAi). After injection of double-stranded RNA (dsRNA) into the adductor muscle of oysters followed by Cd exposure for 7 days, we observed that the expressions of CYP17A1-like and CYP2C50 in interference group were significantly suppressed on day 3 compared with control group injected with PBS. Moreover, the mortality rate and Cd content in the CYP17A1-like dsRNA interference group (dsCYP17A1-like) was significantly higher than those of the control on day 3. Furthermore, the activities of antioxidant enzymes, including SOD, CAT, GST, were significantly increased in dsCYP17A1-like group, while were not changed in dsCYP2C50 group. More significant tissue damage was observed in gill and digestive gland of oysters in RNAi group than control group, demonstrating the critical role of CYP17A1-like in Cd detoxification. Dual luciferase reporter assay revealed three core regulatory elements of MTF-1 within promoter region of CYP17A1-like, suggesting the potential transcriptional regulation of CYP17A1-like by MTF-1 in oysters. This work demonstrated a critical role of CYP17A1-like in Cd detoxification in C. gigas and provided a new perspective toward unravelling detoxification mechanisms of bivalves under heavy metal stress.

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

The family of fibrinogen-related proteins (FREPs) is a group of proteins with fibrinogen-like (FBG) domains, which play important roles as pattern recognition receptors (PRRs) in the innate immune responses. In the present study, a fibrinogen-like protein was identified from the oyster Crassostrea gigas (defined as CgFREP1). The open reading frame of CgFREP1 was of 966 bp that encoded a predicted polypeptide of 321 amino acids comprising a signal peptide and a fibrinogen-like domain. The mRNA expression of CgFREP1 was detected in all the examined tissues. The recombinant CgFREP1 (rCgFREP1) displayed binding activities to lipopolysaccharide (LPS), mannose (MAN), as well as Gram-positive bacteria (Micrococcus luteus and Staphylococcus aureus) and Gram-negative bacteria (Vibrio splendidus and Escherichia coli). The rCgFREP1 displayed the agglutinating activity towards M. luteus, V. splendidus and E. coli in the presence of Ca2+. rCgFREP1 was able to enhance the phagocytic activity of haemocytes towards V. splendidus, and exhibited binding activity to the CUB domain of CgMASPL-1. These results suggest that CgFREP1 not only serves as a PRR to recognize and agglutinate different bacteria but also mediates the haemocytes phagocytosis towards V. splendidus.

Identification and functional analysis of cadmium-binding protein in the visceral mass of Crassostrea gigas.

The Pacific oyster, Crassostrea gigas, is a traditional food worldwide. The soft body of the oyster can easily accumulate heavy metals such as cadmium (Cd). To clarify the molecular mechanism of Cd accumulation in the viscera of C. gigas, we identified Cd-binding proteins. 5,10,15,20-Tetraphenyl-21H,23H-porphinetetrasulfonic acid, disulfuric acid, tetrahydrate, and Cd-binding competition experiments using immobilized metal ion affinity chromatography revealed the binding of water-soluble high molecular weight proteins to Cd, including C. gigas protein disulfide isomerase (cgPDI). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses revealed two CGHC motifs in cgPDI. The binding between Cd and rcgPDI was confirmed through a Cd-binding experiment using the TPPS method. Isothermal titration calorimetry (ITC) revealed the binding of two Cd ions to one molecule of rcgPDI. Circular dichroism (CD) spectrum and tryptophan fluorescence analyses demonstrated that the rcgPDI bound to Cd. The binding markedly changed the two-dimensional or three-dimensional structures. The activity of rcgPDI measured by a PDI Activity Assay Kit was more affected by the addition of Cd than by human PDI. Immunological analyses indicated that C. gigas contained cgPDI at a concentration of 1.0 nmol/g (viscera wet weight). The combination of ITC and quantification results revealed that Cd-binding to cgPDI accounted for 20% of the total bound Cd in the visceral mass. The findings provide new insights into the defense mechanisms of invertebrates against Cd.

Regulation of apoptosis by Pacific oyster Crassostrea gigas reveals acclimation strategy to CO2 driven acidification.

Ocean acidification (OA) has posed formidable threats to marine calcifiers. In response to elevated CO2 levels, marine calcifiers have developed multiple strategies to survive, such as taking advantage of apoptosis, but its regulation mechanism remains largely unknown. Here, we used the Pacific oyster Crassostrea gigas as model to understand the apoptotic responses and regulation mechanism at short- (7 d) to long-term (56 d) CO2 exposure (pH = 7.50). The apoptosis of hemocytes was significantly induced after short-term treatment (7-21 d) but was suppressed under long-term CO2 exposure (42-56 d). Similarly, caspase-3 and caspase-9 were also increased post short-term exposure and fell back to normal levels after long-term exposure. These data together indicated diverse regulation mechanisms of apoptosis through different exposure periods. Through analysis of the B-cell lymphoma 2 (Bcl-2) family mitochondrial apoptosis regulators, we showed that only CgBcl-XL's expression kept at high levels after 42- and 56-day CO2 exposure. CgBcl-XL shared sequence, and structural similarity with its mammalian counterpart, and knockdown of CgBcl-XL in hemocytes via RNA interference promoted apoptosis. The protein level of CgBcl-XL was significantly increased after long-term CO2 exposure (28-56 d), and its distribution in hemocytes became more concentrated and dense. Therefore, CgBcl-XL serves as an essential anti-apoptotic protein for tipping the balance of cell apoptosis, which may play a key role in survival under long-term CO2 exposure. These results reveal a potential adaptation strategy of oysters towards OA and the variable environment changes through the modulation of apoptosis.

Identification, characterization, and expression profiles of insulin-like peptides suggest their critical roles in growth regulation of the Pacific oyster, Crassostrea gigas.

The insulin/insulin-like growth factor signaling (IIS) pathway is well-known in regulation of cell growth and proliferation in vertebrates, while its role in invertebrates such as mollusks remains largely unknown. In this study, we performed an extensive multi-omics data mining and identified four insulin-like peptide genes, including ILP, MIRP3, MIRP3-like and ILP7, in the Pacific oyster, Crassostrea gigas. Their potential roles in growth regulation were further investigated using the selectively bred fast-growing C. gigas variety "Haida No.1". Expression profiling and in situ hybridization of these insulin-like peptides suggested their distinct tissue-specific expression pattern, with dominant expression in the neural enrichment tissues such as labial palp, visceral ganglia, adductor muscle, and digestive gland. The expressions of insulin-like peptides were significantly altered by food abundance in a gene-specific fashion. The expression of ILP was reduced during fasting and increased after re-feeding, the expressions of MIRP3 and ILP7 were generally induced during fasting and down-regulated after re-feeding, while the expression of MIRP3-like was firstly up-regulated and then down-regulated during the fasting and re-feeding process. Furthermore, the expressions of all four insulin-like peptide genes were significantly suppressed at low temperature, in accordance with the growth inhibition. These results indicated that all four insulin-like peptides would play critical but different roles in regulation of growth in the oysters. This work provides valuable information for further investigation on growth regulation mechanism in mollusks and molecular assisted breeding of growth with other production traits in the Pacific oyster.