Potential Function of 3,5-Dihydroxy-4-Methoxybenzyl Alcohol from Pacific Oyster (Crassostrea gigas) in Brain of Old Mice.

SCOPE: 3,5-Dihydroxy-4-methoxybenzyl alcohol (DHMBA) is found in oyster extracts in recent years and is reported to have antioxidant activity. Although it has been reported to be protective in various models of oxidative stress, the therapeutic effect of DHMBA on neurological damage caused by aging remains to be demonstrated. METHODS AND RESULTS: The present study investigates the potential functions of DHMBA in brain of old C57BL/6J mice and aging cell model. Administration of DHMBA improves working memory, reduces anxiety behavior, decreases the expression levels of cell cycle proteins, cycin-dependent kinase inhibitor 1(P21) and peptidase inhibitor 16(P16)  and inhibits neuronal loss in old mice. The data obtained from the aging cell model are consistent with those from the old mice. The interaction between DHMBA and Kelch-like ECH-associated protein 1 (Keap1) is predicted by molecular docking assay, and then it is verified by co-immunopricipitation (CoIP) that factor red lineage 2-related factor 2 (Nrf2)-Keap1 protein-protein interaction is inhibited by DHMBA. Protein levels of Nrf2 and its target genes, such as glutathione peroxidase 4(GPX4) and heme oxygenase 1 (HO-1), are detected in old mice and aging cell model. CONCLUSION: This study provides new evidence that explores the antioxidant mechanism of DHMBA and implies a potential role of DHMBA on antiaging in brain.

Effects of ocean acidification and polystyrene microplastics on the oysters Crassostrea gigas: An integrated biomarker and metabolomic approach.

The adverse impacts of microplastics (MPs) or ocean acidification (OA) on mollusks have been widely reported, however, little is known about their combined effects on mollusks. The oysters Crassostrea gigas were exposed to two sizes of polystyrene MPs with 1 × 104 particles/L (small polystyrene MPs (SPS-MPs): 6 µm, large polystyrene MPs (LPS-MPs): 50-60 µm) at two pH levels (7.7 and 8.1) for 14 days. The antagonistic effects between MPs and OA on oysters were mainly observed. Single SPS-MPs exposure can induce CAT enzyme activity and LPO level in gills, while LPS-MPs exposure alone can increase PGK and PEPCK gene expression in digestive glands. Ocean acidification can increase clearance rate and inhibit antioxidant enzyme activity, whereas combined exposure of OA and SPS-MPs can affect the metabolomic profile of digestive glands. This study emphasized that the potential toxic effects of MPs under the scene of climate change should be concerned.

Ingestion and depuration of polyester microfibers by Crassostrea gasar (Adanson, 1757).

The study aimed to obtain environmentally relevant microfibers (MFs) from polyester fabric and assess their impact on the oyster Crassostrea gasar. MFs were obtained by grinding the fabric, and their accumulation in oysters gills and digestive glands was analyzed after exposure to 0.5 mg/L for 2 and 24 h. Additionally, a 48 h depuration was conducted on the oysters exposed for 24 h. Sublethal effects were assessed in oysters exposed for 24 h and depurated for 48 h, using biomarkers like Catalase (CAT), Glutathione S-transferase (GST), and Glutathione Peroxidase (GPx), along with histological analyses. Polyester fabric grinding produced significant MFs (average length: 570 µm) with degraded surface and increased malleability. Oysters showed increased MF accumulation in digestive glands post-exposure, with no impact on antioxidant enzymes. Depuration decreased MFs accumulation. Histological analysis revealed accumulation in the stomach and brown cells, possibly indicating inflammation. This raises concerns about MFs bioaccumulation in marine organisms, impacting the food chain and safety.

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.

Effects of ambient UVB light on Pacific oyster Crassostrea gigas mantle tissue based on multivariate data.

Ambient ultraviolet radiation (UVB) from solar and artificial light presents serious environmental risks to aquatic ecosystems. The Pacific oyster, Crassostrea gigas, perceives changes in the external environment primarily through its mantle tissue, which contains many nerve fibers and tentacles. Changes within the mantles can typically illustrate the injury of ambient UVB. In this study, a comprehensive analysis of phenotypic, behavioral, and physiological changes demonstrated that extreme UVB radiation (10 W/m²) directly suppressed the behavioral activities of C. gigas. Conversely, under ambient UVB radiation (5 W/m²), various physiological processes exhibited significant alterations in C. gigas, despite the behavior remaining relatively unaffected. Using mathematical model analysis, the integrated analysis of the full-length transcriptome, proteome, and metabolome showed that ambient UVB significantly affected the metabolic processes (saccharide, lipid, and protein metabolism) and cellular biology processes (autophagy, apoptosis, oxidative stress) of the C. gigas mantle. Subsequently, using Procrustes analysis and Pearson correlation analysis, the association between multi-omics data and physiological changes, as well as their biomarkers, revealed the effect of UVB on three crucial biological processes: activation of autophagy signaling (key factors: Ca2+, LC3B, BECN1, caspase-7), response to oxidative stress (reactive oxygen species, heat shock 70, cytochrome c oxidase), and recalibration of energy metabolism (saccharide, succinic acid, translation initiation factor IF-2). These findings offer a fresh perspective on the integration of multi-data from non-model animals in ambient UVB risk assessment.

Purification, Characterization, cDNA Cloning, and Bioinformatic Analysis of Zinc-Binding Protein from Magallana hongkongensis.

Oysters contain significant amounts of the zinc element, which may also be found in their proteins. In this study, a novel zinc-binding protein was purified from the mantle of the oyster Magallana hongkongensis using two kinds of gel filtration chromatograms. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that its molecular weight was approximately 36 kDa. The protein identified by the Q-Exactive mass spectrometer shared the highest sequence identity with carbonic anhydrase derived from Crassostrea gigas concerning amino acid sequence similarity. Based on homologous cloning and RACE PCR, the full-length cDNA of carbonic anhydrase from Magallana hongkongensis (designated as MhCA) was cloned and sequenced. The cDNA of MhCA encodes a 315-amino-acid protein with 89.74% homology to carbonic anhydrase derived from Crassostrea gigas. Molecular docking revealed that the two zinc ions primarily form coordination bonds with histidine residues in the MhCA protein. These results strongly suggest that MhCA is a novel zinc-binding protein in Magallana hongkongensis.

Transcriptomics, proteomics, and physiological assays reveal immunosuppression in the eastern oyster Crassostrea virginica exposed to acidification stress.

Ocean acidification (OA) is recognized as a major stressor for a broad range of marine organisms, particularly shell-building invertebrates. OA can cause alterations in various physiological processes such as growth and metabolism, although its effect on host-pathogen interactions remains largely unexplored. In this study, we used transcriptomics, proteomics, and physiological assays to evaluate changes in immunity of the eastern oyster Crassostrea virginica exposed to OA conditions (pH = 7.5 vs pH = 7.9) at various life stages. The susceptibility of oyster larvae to Vibrio infection increased significantly (131 % increase in mortality) under OA conditions, and was associated with significant changes in their transcriptomes. The significantly higher mortality of larvae exposed to pathogens and acidification stress could be the outcome of an increased metabolic demand to cope with acidification stress (as seen by upregulation of metabolic genes) at the cost of immune function (downregulation of immune genes). While larvae were particularly vulnerable, juveniles appeared more robust to the stressors and there were no differences in mortality after pathogen (Aliiroseovarius crassostrea and Vibrio spp.) exposure. Proteomic investigations in adult oysters revealed that acidification stress resulted in a significant downregulation of mucosal immune proteins including those involved in pathogen recognition and microbe neutralization, suggesting weakened mucosal immunity. Hemocyte function in adults was also impaired by high pCO2, with a marked reduction in phagocytosis (67 % decrease in phagocytosis) in OA conditions. Together, results suggest that OA impairs immune function in the eastern oyster making them more susceptible to pathogen-induced mortality outbreaks. Understanding the effect of multiple stressors such as OA and disease is important for accurate predictions of how oysters will respond to future climate regimes.

Comparative transcriptome elucidates key genes and pathways related to golden phenotype of Crassostrea gigas.

Marine bivalves are economically important and exhibit a remarkable diversity in shell color. The Pacific oyster Crassostrea gigas stands out as an important economic species, with the successful development of four distinct color strains through selective breeding. While previous studies have shed light on the genetic mechanism underlying color segregation, the precise molecular regulatory mechanisms responsible for shell coloration in oysters remains elusive. In this study, we confirmed that the golden phenotype is primarily attributed to pheomelanin by histological and ultrastructural observations. Additionally, we conducted a comparative transcriptome analysis of the black and golden shell color oysters to explore the potential genes and pathways contributing to the golden phenotype in C. gigas. Our results revealed a significant increase in differentially expressed genes in the golden phenotype associated with pathways such as glutathione metabolism, and calcium signaling pathway, suggesting a potential role in the synthesis of pheomelanin. Of particular note, we highlighted the potential role of two-pore channel 2 (TPC2) in modulating tyrosinase activity and melanosomal pH, ultimately determining the shade of pigmentation. Our study in this work provided a preliminary exploration of the mechanism, shedding light on the melanosome microenvironment and shell color.

Transcriptional responses of Crassostrea hongkongensis under high and low salinity stress.

Salinity, a key limiting factor, affects the distribution and survival of marine species. The Hong Kong oyster (Crassostrea hongkongensis), a euryhaline species found along the coast of the South China Sea, has become a major aquaculture bivalve species. To determine the molecular mechanism by which oysters respond to coastal waters with varying salinity levels, we used RNA-seq to sequence the gill samples of oysters exposed to normal (25 ‰, S25), low (5 ‰, S5) and high (35 ‰, S35) salinity conditions for one month. The results revealed different expression transcriptome levels among oysters living under low and high salinity conditions. Using high-throughput sequencing, we identified 811 up-regulated genes and 769 down-regulated genes. As determined by KEGG pathway mapping, the differentially expressed genes (DEGs) were significantly enriched in the prion diseases, histidine metabolism, arginine and proline metabolism, and beta-alanine metabolism pathways in both the S5 vs. S25 and S35 vs. S25 group comparison. Several DEGs including heat shock 70 kDa protein 12B-like, poly (ADP-ribose) polymerase (PARP), and tripartite motif-containing protein 2 (TRIM2), and low-density lipoprotein receptor-like, as well as KEGG pathways, including arginine and proline metabolism, apoptosis, PPAR signaling pathway, the thyroid hormone signaling pathway, were concerning response to salinity stress. Additionally, eight DEGs involved in salinity adaptation were selected for RT-qPCR validation, and the results confirmed the credibility of the transcriptome sequencing data. Overall, we designed a one-month, medium-term experiment to examine the responses of C. hongkongensis exposed to different levels of salinity stress and performed transcriptome analysis using high-throughput sequencing. Our results enhance current understanding of the molecular mechanisms of salinity stress responses in C. hongkongensis and provided insights into the osmotic biology of oysters.

Ancestors' Gift: Parental Early Exposure to the Environmentally Realistic Pesticide Mixture Drives Offspring Phenotype in a Larger Extent Than Direct Exposure in the Pacific Oyster, Crassostrea gigas.

Marine organisms are threatened by the presence of pesticides in coastal waters. Among them, the Pacific oyster is one of the most studied invertebrates in marine ecotoxicology where numerous studies highlighted the multiscale impacts of pesticides. In the past few years, a growing body of literature has reported the epigenetic outcomes of xenobiotics. Because DNA methylation is an epigenetic mark implicated in organism development and is meiotically heritable, it raises the question of the multigenerational implications of xenobiotic-induced epigenetic alterations. Therefore, we performed a multigenerational exposure to an environmentally relevant mixture of 18 pesticides (nominal sum concentration: 2.85 µg·L-1) during embryo-larval stages (0-48 hpf) of a second generation (F1) for which parents where already exposed or not in F0. Gene expression, DNA methylation, and physiological end points were assessed throughout the life cycle of individuals. Overall, the multigenerational effect has a greater influence on the phenotype than the exposure itself. Thus, multigenerational phenotypic effects were observed: individuals descending from exposed parents exhibited lower epinephrine-induced metamorphosis and field survival rates. At the molecular level, RNA-seq and Methyl-seq data analyses performed in gastrula embryos and metamorphosis-competent pediveliger (MCP) larvae revealed a clear F0 treatment-dependent discrimination. Some genes implicated into shell secretion and immunity exhibited F1:F0 treatment interaction patterns (e.g., Calm and Myd88). Those results suggest that low chronic environmental pesticide contamination can alter organisms beyond the individual scale level and have long-term adaptive implications.

Redox control of antioxidants, metabolism, immunity, and development at the core of stress adaptation of the oyster Crassostrea gigas to the dynamic intertidal environment.

This review uses the marine bivalve Crassostrea gigas to highlight redox reactions and control systems in species living in dynamic intertidal environments. Intertidal species face daily and seasonal environmental variability, including temperature, oxygen, salinity, and nutritional changes. Increasing anthropogenic pressure can bring pollutants and pathogens as additional stressors. Surprisingly, C. gigas demonstrates impressive adaptability to most of these challenges. We explore how ROS production, antioxidant protection, redox signaling, and metabolic adjustments can shed light on how redox biology supports oyster survival in harsh conditions. The review provides (i) a brief summary of shared redox sensing processes in metazoan; (ii) an overview of unique characteristics of the C. gigas intertidal habitat and the suitability of this species as a model organism; (iii) insights into the redox biology of C. gigas, including ROS sources, signaling pathways, ROS-scavenging systems, and thiol-containing proteins; and examples of (iv) hot topics that are underdeveloped in bivalve research linking redox biology with immunometabolism, physioxia, and development. Given its plasticity to environmental changes, C. gigas is a valuable model for studying the role of redox biology in the adaptation to harsh habitats, potentially providing novel insights for basic and applied studies in marine and comparative biochemistry and physiology.

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.

Integrative proteome and metabolome analyses reveal molecular basis underlying growth and nutrient composition in the Pacific oyster, Crassostrea gigas.

In order to comprehend the molecular basis of growth, nutrient composition, and color pigmentation in oysters, comparative proteome and metabolome analyses of two selectively bred oyster strains with contrasting growth rate and shell color were used in this study. A total of 289 proteins and 224 metabolites were identified differentially expressed between the two strains. We identified a series of specifically enriched functional clusters implicated in protein biosynthesis (RPL4, MRPS7, and CARS), fatty acid metabolism (ACSL5, PEX3, ACOXI, CPTIA, FABP6, and HSD17B12), energy metabolism (FH, PPP1R7, CLAM2, and RGN), cell proliferation (MYB, NFYC, DOHH, TOP2a, SMARCA5, and SMARCC2), material transport (ABCB1, ABCB8, VPS16, and VPS33a), and pigmentation (RDH7, RDH13, Retsat, COX15, and Cyp3a9). Integrated proteome and metabolome analyses indicate that fast-growing strain utilize energy-efficient mechanisms of ATP generation while promoting protein and polyunsaturated fatty acid synthesis, activating the cell cycle to increase cell proliferation and thus promoting their biomass increase. These results uncovered molecular mechanisms underlying growth regulation, nutrition quality, and pigmentation and provided candidate biomarkers for molecular breeding in oysters. SIGNIFICANCE: Rapid growth has always been the primary breeding objective to increase the production profits of Pacific oyster (Crassostrea gigas), while favorable nutritional quality and beautiful color add commercial value. In recent years, proteomic and metabolomic techniques have been widely used in marine organisms, although these techniques are seldom utilized to study oyster growth and development. In this study, two C. gigas strains with contrasted phenotypes in growth and shell color provided an ideal model for unraveling the molecular basis of growth and nutrient composition through a comparison of the proteome and metabolome. Since proteins and metabolites are the critical undertakers and the end products of cellular regulatory processes, identifying the differentially expressed proteins and metabolites would allow for discovering biomarkers and pathways that were implicated in cell growth, proliferation, and other critical functions. This work provides valuable resources in assistance with molecular breeding of oyster strains with superior production traits of fast-growth and high-quality nutrient value.

Genome-wide identification of STATs and analysis of their role in sex determination in Pacific oysters (Crassostrea gigas).

STAT (signal transducer and activator of the transcription) proteins, are a group of highly conserved transcription factors and fundamental components of the JAK-STAT signaling pathway. They play crucial roles in a variety of biological processes, such as immunity, proliferation, differentiation, and growth. However, little information is known regarding their role in gonad development and sex determination in mollusks. In this study, we identified 3 STAT genes in Pacific Oyster Crassostrea gigas. Phylogenetic analysis showed that STATs from mollusks were highly conserved, and most of them had four identical motif regions, except for the STAT1 and STAT3 predicted sequences from Crassostrea hongkongensis. Tissue expression analysis indicated CgSTAT1 had a high expression level in most tissues, while CgSTAT3 had a low expression level in most tissues. Expression analysis of early developmental stages showed CgSTAT1 had a higher expression level from egg to D shaped larva and a lower expression level in subsequent stages. In contrast CgSTAT1, CgSTAT2 had a reverse expression pattern. Expression analysis of different developmental stages of diploid gonads indicated that CgSTAT1 had a higher expression level at the S1 and S3 stages relative to the S2 stage in females, while in males the S3 stage had a higher expression than than the S2 stage. The expression level of CgSTAT1 between diploids and triploids in females differed significantly, but there were no significant differences in males. Expression of CgSTAT2 differed significantly between diploid and triploid males. These data suggest an important role for STATs in sex differentiation in diploid and triploid oysters. Our study is the first to explore the role of STATs in sex differentiation and gonadal development in oysters, and will help us better understand the molecular mechanisms of sex differentiation in shellfish.

Mitf involved in shell pigmentation by activating tyrosinase-mediated melanin synthesis in Pacific oyster (Crassostrea gigas).

Pigmentation is frequently observed in the molluscan shells, whereas the molecular regulation about these shell pigments formation is not clear. The microphthalmia-associated transcription factor (Mitf) is an important transactivator in melanin synthesis in vertebrates. Here, the Mitf containing a highly conserved basic helix-loop-helixleucine zipper (bHLH-LZ) domain was identified in an economically important marine bivalve Pacific oyster Crassostrea gigas. The Mitf was found to widespread tissue distribution and the expression was higher in the marginal mantle than in the central mantle. Particularly, the expression level of Mitf was high in black shell color oysters compared with white shell oysters. After injecting siRNA, the expression of Mitf decreased significantly, and the efficiency of RNA interference reached 53%. Besides, knockdown Mitf obviously decreased expression of tyrosinase family genes and tyrosinase activity of mantles, indicating a potential regulatory relationship between Mitf and Tyr or Typs. Simultaneously, there was a sharply reduce in the number of the melanosomes in the outer fold of mantle by silencing of Mitf. Luciferase assays in cell culture further verified that Mitf was involved in transcriptional regulation of Typ-2 and Typ-3 genes through binding to their specific promoter regions. These data argue that Mitf is involved in shell pigmentation through activating tyrosinase-mediated melanin synthesis in C. gigas.

The effects of artificial light at night on behavioral rhythm and related gene expression are wavelength dependent in the oyster Crassostrea gigas.

Artificial light at night (ALAN) constitutes a growing threat to coastal ecosystems by altering natural light cycles, which could impair organisms' biological rhythms, with resulting physiological and ecological consequences. Coastal ecosystems are strongly exposed to ALAN, but its effects on coastal organisms are poorly studied. Besides ALAN's intensity, ALAN's quality exposure may change the impacts on organisms. This study aims to characterize the effects of different ALAN's spectral compositions (monochromatic wavelength lights in red (peak at 626 nm), green (peak at 515 nm), blue (peak at 467 nm), and white (410-680 nm) light) at low and realistic intensity (1 lx) on the oyster Crassostrea gigas daily rhythm. Results reveal that all ALAN's treatments affect the oysters' daily valve activity rhythm in different manners and the overall expression of the 13 studied genes. Eight of these genes are involved in the oyster's circadian clock, 2 are clock-associated genes, and 3 are light perception genes. The blue light has the most important effects on oysters' valve behavior and clock and clock-associated gene expression. Interestingly, red and green lights also show significant impacts on the daily rhythm, while the lowest impacts are shown with the green light. Finally, ALAN white light shows the same impact as the blue one in terms of loss of rhythmic oysters' percentage, but the chronobiological parameters of the remaining rhythmic oysters are less disrupted than when exposed to each of the monochromatic light's treatments alone. We conclude that ALAN's spectral composition does influence its effect on oysters' daily rhythm, which could give clues to limit physiological and ecological impacts on coastal environments.

Expression and Characterization of a ß-Galactosidase from the Pacific Oyster, Crassostrea gigas, and Evaluation of Strategies for Testing Substrate Specificity.

ß-Galactosidases (EC 3.2.1.23) are exoglycosidases that catalyze the cleavage of glycoconjugates with terminal ß-D-galactose residues in ß1,3-, ß1,4- or ß1,6-linkage. Although this family of exoglycosidases has been extensively studied in vertebrates, plants, yeast, and bacteria, little information is available for mollusks. Mollusks are a diverse and highly successful group of animals that play many different roles in their ecosystems, including filter feeders and detritivores. Here, the first ß-galactosidase from the Pacific oyster, Crassostrea gigas was discovered, biochemically characterized, and compared to our previously characterized slug enzyme from Arion vulgaris (UniProt Ref. Nr.: A0A0B7AQJ9). Overall, the mussel enzyme showed similar biochemical parameters to the snail enzyme. The enzyme from C. gigas was most active in an acidic environment (pH 3.5) and at a reaction temperature of 50 °C. Optimal storage conditions were up to 37 °C. In contrast to the enzyme from A. vulgaris, the supplementation of cations (Ni2+, Co2+, Mn2+, Mg2+, Ca2+, Cu2+, Ba2+) increased the activity of the enzyme from C. gigas. Substrate specificity studies of the ß-galactosidases from the mussel, C. gigas, and the slug, A. vulgaris, revealed activity towards terminal ß1,3- and ß1,4-linked galactose residues for both enzymes. Using the same substrates in labeled and unlabeled form, we were able to detect the effect of labeling on the ß-galactosidase activity using MALDI-TOF MS, HPTLC, and HPLC. While lactose was cleaved by the enzymes in an unlabeled or labeled state, galacto-N-biose was not cleaved as soon as a 2-amino benzoic acid label was added. In this study we present the biochemical characterization of the first recombinantly expressed ß-galactosidase from the Pacific oyster, C. gigas, and we compare different analytical methods for the determination of ß-galactosidase activity using the enzyme from C. gigas and A. vulgaris.

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.

Comparative Transcriptome and WGCNA Analysis Reveal Molecular Responses to Salinity Change in Larvae of the Iwagaki Oyster Crassostrea Nippona.

The Iwagaki oyster Crassostrea nippona is an important aquaculture species with significant potential for large-scale oyster farming. It is susceptible to the fluctuated salinity in the coastal area. In this study, we compared the transcriptome of Crassostrea nippona larvae under variant conditions with low-salinity stress (28, 20, 15, 10, and 5 practical salinity units (psu)) for 24 h. KEGG enrichment analysis of differentially expressed genes (DEGs) from pairwise comparisons identified several free amino acid metabolism pathway (taurine and hypotaurine, arginine and proline, glycine, and beta-alanine) contributing to the salinity change adaptation and activated "lysosome" and "apoptosis" pathway in response to the low-salinity stress (10 and 5 psu). Trend analysis revealed sustained upregulation of transmembrane transport-related genes (such as SLC family) and downregulation of ribosomal protein synthesis genes faced with decreasing salinities. In addition, 9 biomarkers in response to low-salinity stress were identified through weighted gene co-expression network analysis (WGCNA) and validated by qRT-PCR. Our transcriptome analysis provides a comprehensive view of the molecular mechanisms and regulatory networks underlying the adaptive responses of oyster larvae to hypo-salinity conditions. These findings contribute to our understanding of the complex biological processes involved in oyster resilience and adaptation to changing environmental conditions.

Combination of RNAseq and RADseq to Identify Physiological and Adaptive Responses to Acidification in the Eastern Oyster (Crassostrea virginica).

Ocean acidification (OA) is a major stressor threatening marine calcifiers, including the eastern oyster (Crassostrea virginica). In this paper, we provide insight into the molecular mechanisms associated with resilience to OA, with the dual intentions of probing both acclimation and adaptation potential in this species. C. virginica were spawned, and larvae were reared in control or acidified conditions immediately after fertilization. RNA samples were collected from larvae and juveniles, and DNA samples were collected from juveniles after undergoing OA-induced mortality and used to contrast gene expression (RNAseq) and SNP (ddRADseq) profiles from animals reared under both conditions. Results showed convergence of evidence from both approaches, particularly in genes involved in biomineralization that displayed significant changes in variant frequencies and gene expression levels among juveniles that survived acidification as compared to controls. Downregulated genes were related to immune processes, supporting previous studies demonstrating a reduction in immunity from exposure to OA. Acclimation to OA via regulation of gene expression might confer short-term resilience to immediate threats; however, the costs may not be sustainable, underscoring the importance of selection of resilient genotypes. Here, we identified SNPs associated with survival under OA conditions, suggesting that this commercially and ecologically important species might have the genetic variation needed for adaptation to future acidification. The identification of genetic features associated with OA resilience is a highly-needed step for the development of marker-assisted selection of oyster stocks for aquaculture and restoration activities.