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Oysters are commercially important intertidal filter-feeding species. Mass mortality events of oysters often occur due to environmental stresses, such as exposure to fluctuating temperatures, salinity, and air, as well as to metal pollution and pathogen infection. Here, RNA-seq data were used to identify shared and specific responsive genes by differential gene expression analysis and weighted gene co-expression network analysis. A total of 18 up-regulated and 10 down-regulated shared responsive genes were identified corresponding to five different stressors. Total 27 stressor-specific genes for temperature, 11 for salinity, 80 for air exposure, 51 for metal pollution, and 636 for Vibrio mediterranei pathogen stress were identified in oysters. Elongin-β was identified as a crucial gene for thermal stress response. Some HSP70s were determined to be shared responsive genes while others were specific to thermal tolerance. The proteins encoded by these stress-related genes should be further investigated to characterize their physiological functions. In addition, the uncharacterized proteins and ncRNAs that were identified may be involved in species-specific stress-response and regulatory mechanisms. This study identified specific genes related to stressors relevant to oyster cultivation. These findings provide useful information for new selective breeding strategies using a data driven method.

The protein inhibitor of activated STAT (PIAS) family proteins act as the important negative regulators in janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway, which can be also involved in regulating the expression of interferon-stimulated genes (ISGs). In the present study, a PIAS homologue (designated as CgPIAS) was identified from oyster Crassostrea gigas. The open reading frame (ORF) of CgPIAS cDNA was of 1887 bp encoding a peptide of 628 amino acid residues. The CgPIAS protein contains a conserved scaffold attachment factor A/B/acinus/PIAS (SAP) domain, a Pro-Ile-Asn-Ile-Thr (PINIT) motif, a RING-finger-like zinc-binding domain (RLD) and two SUMO-interaction Motifs (SIMs). The deduced amino acid sequence of CgPIAS shared 74.58-81.36% similarity with other PIAS family members in the RLD domain. The mRNA transcripts of CgPIAS were detected in all the tested tissues with highest level in haemocytes (32.98-fold of mantles, p 0.001). After poly (I:C) and recombinant Interferon-like protein (rCgIFNLP) stimulation, the mRNA expression of CgPIAS in haemocytes significantly up-regulated to the highest level at 48 h (7.38-fold, p 0.001) and at 24 h (13.08-fold, p 0.01), respectively. Moreover, the nuclear translocation of CgPIAS was observed in haemocytes after poly (I:C) stimulation. Biolayer Interferometry (BLI) assay revealed that the recombinant protein CgPIAS-RLD could interact with the recombinant protein CgSTAT in vitro with the K

Yeast cells can play a crucial role in immune activation in fish and shellfish predominantly due to the cell wall component β-1,3-glucan, providing protection against bacterial or viral infections. However, the immunostimulatory capacity of dietary yeast cells remains poorly studied in bivalves. To understand the role of yeast cell wall components (mannan, β-glucan and chitin) as immune activators, this study characterized the surface carbohydrate exposure of the wild-type baker's yeast Saccharomyces cerevisiae (WT) and its Δmnn9 mutant, which presents a defective mannan structure, and compared these profiles with that of β-glucan particles, using fluorescein isothiocyanate (FITC)-labeled lectin binding analysis. Then, a first trial evaluated the immunological response in Crassostrea gigas juveniles after being fed for 24 h with an algae-based diet (100A) and its 50% substituted version (based on dry weight) with WT (50A50WT) and Δmnn9 (50A50Y), and the posterior resistance of the juveniles against Vibrio coralliilyticus infection (trial 1). The mRNA expression was measured for β-glucan-binding protein (CgβGBP), Toll-like receptor 4 (CgTLR4), C-type lectin receptor 3 (CgCLec-3), myeloid differentiation factor 88 (CgMyD88), nuclear factor-kappa B (CgNFκB), lysozyme (CgLys), interleukin 17-5 (CgIL17-5), and superoxide dismutase (CgSOD), in oysters, before and 24 h after the bacterial inoculation. A second trial tested the effect of incorporating Δmnn9 into the 100A diet for 24 h at different substitution levels: 0, 5, 10, 25, and 50% (100A, 95A5Y, 90A10Y, 75A25Y, and 50A50Y), followed by the bacterial challenge with V. coralliilyticus (trial 2). Our findings showed that the outer cell wall surface of WT is largely composed of mannan, while Δmnn9 presents high exposure of β-glucan and chitin, exhibiting similar FITC-lectin binding profiles (fluorescence intensity) to β-glucan particles. A significantly higher survival after the bacterial challenge was observed in oysters fed on 50A50Y compared to those fed 50A50WT and 100A in trial 1. This better performance of 50A50Y was supported by significantly higher gene expressions of CgLys, CgSOD, CgMyD88, and CgβGBP compared to 100A, and CgSOD and CgNFκB in relation to those fed on 50A50WT, prior to the bacterial inoculation. Furthermore, improved survival was observed in oysters fed 50A50Y compared to those offered lower Δmnn9 levels and 100A in trial 2. The superior performance of Δmnn9-fed oysters is mostly associated with the elevated presence of unmasked β-glucans on Δmnn9 cell wall surface, facilitating their interactions with oyster hemocytes. Further studies are needed to evaluate administration dose and frequency of Δmnn9 to develop strategies for long-term feeding.

Oyster is nutritious shellfish, wildly consumed throughout the world. Its polysaccharide (OPS) has various bioactivity. In the present study, the anti-obesity effect of OPS was evaluated in obese mice induced by a high-fat diet (HFD). The results showed that OPS significantly alleviated weight gain, dyslipidemia, and metabolic endotoxemia of obese mice, and accelerated the production of short-chain fatty acids. OPS also regulated lipid metabolism of adipose and liver by activating the expression of p-AMPKα to further down-regulate the expression of SREBP-1c, PPARγ, and p-ACC-1. 16S rRNA results indicated that OPS corrected HFD-induced gut microbiota dysbiosis by enriching beneficial bacteria (Bifidobacterium, Lactobacillus, Dobosiella, and Faecalibaculum) and decreasing harmful bacteria (Erysipelatoclostridium, Helicobacter, and Mucispirillum). In summary, these results revealed that OPS could serve as a potential prebiotic to improve obesity.

The LGP2 (Laboratory of Genetics and Physiology 2) protein is a member of the retinoic acid-inducible gene I (RIG-I)-like receptor (RLRs) family, which is a class of antiviral pattern recognition receptors located in the cytoplasm. However, few studies have investigated the function of LGP2 in invertebrates. In this study, the complete coding sequence of the LGP2 gene of the Pacific oyster, Crassostrea gigas, was obtained and named CgLGP2-like. Sequence analysis revealed that CgLGP2-like encodes 803 amino acids, and the encoded protein contains a DEXDc, HELICc, and C-terminal regulatory domains. Multiple sequence alignment demonstrated that the sequences of these key protein functional domains were relatively conserved. Phylogenetic analysis revealed that CgLGP2-like was a new member of the animal LGP2 family. Quantitative real-time PCR results showed that CgLGP2-like mRNA was expressed in all tested oyster tissues, with the highest expression observed in the labial palpus and digestive glands. CgLGP2-like expression in gill tissues was significantly induced after the poly(I:C) challenge. Furthermore, multiple IRF and NF-κB binding sites were identified in the CgLGP2-like promoter region, which may be one of the reasons why CgLGP2-like responds to poly(I:C) stimulation. Finally, the results of dual-luciferase reporter gene assays revealed that overexpression of CgLGP2-like may have a regulatory effect on the human IFN, AP-1, and oyster CgIL-17 genes in HEK293T cells. Overall, our results preliminarily elucidate the immune functions of invertebrate LGP2 protein and provide valuable information for the development of comparative immunology.

High-mobility group box 1 (HMGB1), a highly conserved nucleoprotein, functions in immune recognition, inflammation and antibacterial immunization in vertebrates. In the present study, the mediation mechanism of CgHMGB1 in activating MAPK and NF-κB/Rel signaling pathways to induce the expressions of immune effectors was investigated. CgHMGB1 mRNA was detected in all tested developmental stages from fertilized egg to D-larvae, with the higher expressions in 4 cells and 8 cells stages. CgHMGB1 proteins were mainly distributed in haemocyte granulocytes. The expressions of CgHMGB1 mRNA in haemocytes increased significantly after Vibrio splendidus stimulation, and CgHMGB1 protein translocated into the haemocyte cytoplasm and release into cell-free haemolymph. The phosphorylation of CgERK and CgP38 were induced, the nuclear translocation of CgRel were promoted, and the mRNA expressions of CgIL17-5 and Cgdefh2 increased significantly after rCgHMGB1 treatment. Obvious branchial swelling and cilium shedding were observed after rCgHMGB1 treatment. rCgHMGB1 exhibited binding activity to different polysaccharides, bacteria, and fungi. rCgHMGB1 also displayed obvious antibacterial activity to V. splendidus and E. coli. These results indicated that CgHMGB1 functioned as an immune recognition molecule to recognize various PAMPs and bacteria to induce the mRNA expressions of CgIL17-5 and Cgdefh2 via the activation of MAPK and NF-κB signaling pathways in oysters.

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.

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.

We investigated the effects of cryopreservation on the quality of Portuguese oyster (Crassostrea angulata) sperm, which were examined before and after freezing; sperm motility, fertilizing capacity, and ultrastructural morphology were analyzed. The motility percentage and fertilizing capacity of the cryopreserved sperm (mean ± standard error) were 16% ± 1% and 17% ± 8%, respectively. In the pre-freezing sperm, these were 58% ± 2% and 76% ± 4%, respectively. The sperm sustained substantial morphological and ultrastructural damage during cryopreservation. The morphological changes varied considerably in nature and extent, ranging from no apparent damage to virtual disintegration. Sperm were stained with fluorescent dyes to assess viability, plasma membrane integrity, mitochondrial activity, acrosomal membrane integrity, oxidation level, and DNA fragmentation and examined through flow cytometry. The methods used for the flow cytometry assays were slightly modified from those used for evaluating the semen quality of livestock. Relative to the pre-freezing sperm, the frozen-thawed sperm exhibited lower acrosomal membrane integrity (acrosomal damage, 59.86 ± 5.29; P

B cell lymphoma/leukemia 10 (BCL10) is an important member of the caspase recruitment domain-containing (CARD) protein family, which plays crucial roles in mediating the host inflammatory response. In the present study, a BCL10 homologue was identified from Pacific oyster Crassostrea gigas (designed as CgBCL10). The full length cDNA of CgBCL10 was of 897 bp with an open reading frame of 522 bp encoding a polypeptide of 174 amino acids containing a classical CARD domain. The deduced amino acid sequence of CgBCL10 shared low similarity with the previously identified BCL10s from other species. In the phylogenetic tree, CgBCL10 was firstly clustered with CvBCL10 from Crassostrea virginica and then assigned into the branch of invertebrate BCL10s. The mRNA transcripts of CgBCL10 were highly expressed in gonad, gill, adductor muscle, and haemocytes. After Vibrio splendidus stimulation, the mRNA expression level of CgBCL10 in haemocytes increased significantly (p 0.01) at 24, 72 and 96 h. In CgBCL10-RNAi oysters, the phosphorylation level of mitogen-activated protein kinases (MAPKs), nuclear translocation of NF-κB/Rel and activator protein-1 (AP-1) in haemocytes were inhibited, and the mRNA expressions of inflammatory cytokines including CgIL17-1, CgIL17-2, CgIL17-3, CgIL17-6 and CgTNF all decreased significantly (p 0.01) at 12 h after V. splendidus stimulation. These results suggested that CgBCL10 regulated the expression of inflammatory cytokines by activating MAPK kinase, and nuclear translocation of NF-κB/Rel and AP-1 to defense pathogen.

Oyster is the worldwide aquaculture molluscan and evolves a complex immune defense system, with hemocytes as the major immune system for its host defense. However, the functional heterogeneity of hemocyte has not been characterized, which markedly hinders our understanding of its defense role. Here, we used the single-cell transcriptome profiling (scRNA-seq), which provides a high-resolution visual insight into its dynamics, to map the hemocyte and assess its heterogeneity in a molluscan oyster Crassostrea hongkongensis. By combining with the cell type specific RNA-seq, thirteen subpopulations belonging to granulocyte, semi-granulocyte, and hyalinocyte were revealed. The granulocytes mainly participated in immune response and autophagy process. Pseudo-temporal ordering of granulocytes identified two different cell-lineages. The hematopoietic transcription factors regulated networks controlling their differentiations were also identified. We further identified one subpopulation of granulocytes in immune activate states with the cell cycle and immune responsive genes expressions, which illustrated the functional heterogeneity of the same cell type. Collectively, our scRNA-seq analysis demonstrated the hemocytes diversity of molluscans. The results are important in our understanding of the immune defense evolution and functional differentiation of hemocytes in Phylum Mollusca.

The myxovirus resistance (Mx) proteins belong to interferon (IFN)-induced dynamin GTPase and play a pivotal role in the inhibition of replication of numerous viruses. In the present study, an Mx homologue (designated as CgMx1) was identified from oyster Crassostrea gigas. The open reading frame (ORF) of CgMx1 cDNA was of 1689 bp encoding a peptide of 562 amino acid residues. There was an N-terminal dynamin GTPase domain in the predicted peptide, which consisted of a tripartite GTP-binding motif (GDXXSGKS, DLPG and T/NKXD). The deduced amino acid sequence of CgMx1 shared 30-39% similarity with other Mx family members. And CgMx1 was clustered with Mx from H. discus, and then assigned into the invertebrate branch of the phylogenetic tree. The mRNA transcripts of CgMx1 were constitutively distributed in all the tested tissues, with the highest level in haemocytes (1342.45-fold of labial palps, p 0.05). The mRNA expression of CgMx1 in haemocytes was significantly up-regulated to the highest level at 6 h (13.14-fold, p 0.001) after poly (I:C) treatment and at 24 h (66.28-fold, p 0.001) after recombinant IFN-like protein (rCgIFNLP) stimulation, respectively. CgMx1 protein was found to distribute in both the cytoplasm and nucleus of haemocytes. In the oysters with CgIFNLP and signal transducer and activator of transcription (CgSTAT) silenced by RNAi, the mRNA expression of CgMx1 decreased significantly in the haemocytes at 12 h after poly (I:C) stimulation, which was 0.02-fold and 0.04-fold of that in EGFP-RNAi oysters (p 0.001), respectively. Meanwhile, EMSA assay revealed that CgSTAT was able to transactivate CgMx1 promoter through directly binding to its interferon-stimulated response element (ISRE) and gamma interferon activation site (GAS). The above results indicated that CgMx1 participated in the immune response of C. gigas through the signal pathway mediated by CgIFNLP and CgSTAT.

A total of 1338 samples were analyzed by ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to study the toxin profiles of lipophilic marine toxins in bivalve mollusks collected from the southeast coast of China from 2017 to 2020. The most abundant toxin was HomoYTX, followed progressively by YTX and PTX2. Low proportions of OA, DTX-1, and DTX-2 were found. No AZA1, AZA2, and AZA3 were quantified above limit of quantitation (LOQ). The highest concentrations of HomoYTX, YTX, PTX2, OA, DTX-1, and DTX-2 were 429, 98.0, 40.3, 33.0, 22.6, and 26.5 μg/kg, respectively. Mussels (Mytilus galloprovincialis, Perna viridis), scallop (Chlamys farreri) and clam (Atrina pectinate) accumulated higher toxin levels than clams (Sinonovaculla Constricta, Ruditapes philippinarum), oyster (Crassostrea gigas) and scallop (Arca granosa). Homo YTX and PTX2 levels reached the maximum in July and June, respectively, and the OA-group peaked in August. The results provide a reliable basis for monitoring marine toxins and protecting the health of aquatic consumers.

In the present study, we investigated the presence, seasonal distribution, and biomolecular characteristics of Vibrio parahaemolyticus and Vibrio vulnificus in samples of bivalve mollusks (Mytilus galloprovincialis, Crassostrea gigas, and Ruditapes decussatus) harvested and marketed in Sardinia (Italy) between 2017 and 2018. A total of 435 samples were submitted for qualitative determination of Vibrio spp., V. parahaemolyticus, and V. vulnificus. Potentially enteropathogenic isolates were detected with biomolecular methods. The overall prevalence of Vibrio spp. was 7.6%. The highest Vibrio prevalence was found in R. decussatus (8.3%). The prevalences of V. parahaemolyticus and V. vulnificus were 2.7 and 4.8%, respectively. Higher prevalences of V. parahaemolyticus and V. vulnificus were found in R. decussatus (4.2%) and C. gigas (6.2%), respectively. Only two pathogenic V. parahaemolyticus strains were recovered (genotypes: tdh- and trh+; tdh+ and trh-), both from M. galloprovincialis. None of the isolates were tdh+ and trh+. Pathogenic Vibrio infections are often underestimated, and human infections are increasing in Europe. European data on the true distribution of Vibrionaceae are scarce, and the results of the present study highlight the need of constant monitoring to update the distribution of pathogenic vibrios. HIGHLIGHTS

The objective of this study was to evaluate the molecular mechanism by which polysaccharides from Crassostrea gigas (RPS) prevent alcoholic liver injury and to uncover whether the steaming process affects the bioactivities of RPS. Oral administration of RPS or polysaccharides from steamed oyster (SPS) (282 mg/kg b.w.) significantly attenuated alcoholic liver injury in mice. RPS and SPS treatments protected gut functions by significantly enhancing the expression of tight-junction proteins and suppressing inflammatory responses. RPS and SPS treatments also significantly increased Lactobacillus reuteri and Roseburia spp. and decreased the level of Escherichia. Microbial metabolites, especially propionate and butyrate, were also increased in RPS- and SPS-treated mice. Correlation analysis revealed that the beneficial effects of RPS and SPS were strongly correlated with the microbiota composition and SCFAs. These results indicated that oyster polysaccharides alleviated alcoholic liver injury by mediating the gut-liver-metabolite axis, and the steaming process had little influence on the bioactivity.

The present study aimed to observe the expression level of metallothionein by analyzing its density and intensity in the gill and stomach of Crassostrea glomerata and Crassostrea iredalei. The mussels were collected from three sampling locations in the Northern coast of East Java, Indonesia: Mayangan, Probolinggo (Station 1), Kenjeran beach, Surabaya (Station 2), and Gresik Ports (Station 3). The density and intensity of metallothionein were analyzed by using immunohistochemistry technique. The results showed that the highest density of metallothionein in Crassostrea iredalei was at Station 3 (Gresik) i.e. 47.8 × 10−3 (MT/µm2) in gill and 42.2 × 10−3 (MT/µm2) in stomach. While, the highest density value in Crassostrea glomerata was observed at Station 3 (Gresik) i.e. 64 × 10−3 (MT/µm2) in gill and 45.6 × 10−3 (MT/µm2) in stomach. Furthermore, the correlation analysis of heavy metals concentration with the density and intensity of metallothionein showed a significant correlation. This result indicates that the concentrations of the investigated heavy metals (i.e. Pb, Cd and Hg) in seawater correlated with the density and intensity of metallothionein in the gill and stomach of mussels (Crassostrea glomerata and Crassostrea iredalei), which can be used as biomarkers of heavy metal contamination.

Pacific oyster (Crassostrea gigas), the most productive economical bivalve mollusc, is identified as an attractive model for developmental studies due to its classical mosaic developmental pattern. Myosin heavy chain is a structural and functional component of myosin, the key muscle protein of thick filament. Here, full length cDNA of striated myosin heavy chains in C. gigas (CgSmhc) was obtained, and the expression profiles were examined in different development stage. CgSmhc had a high expression level in trochophore and D-shaped stage during embryo-larval stage. In adult, CgSmhc was a muscle-specific gene and primarily expressed in muscle tissues. Then, activity of 5′ flanking region of CgSmhc were examined through an reconstructed EGFP vector. The results indicated that 3098 bp 5′-flanking region of CgSmhc owned various conserved binding sites of myogenesis-related regulatory elements, and the 2000 bp 5′-flanking sequence was sufficient to induce the CgSmhc expression. Subsequently, the CRISPR/Cas9-mediated target disruption of CgSmhc was generated by co-injection of Cas9mRNA and CgSmhc-sgRNAs into one-cell stage embryos of C. gigas. Loss of CgSmhc had a visible effect on the sarcomeric organization of thin filaments in larval musculature, indicating that CgSmhc was required during larval myogenesis to regulate the correct assembly of sarcomere.

Ploidy manipulation, such as triploidy, in farmed oysters has been used as a tool to enhance oyster quality throughout all seasons because triploid oysters allocate less energy to gametogenesis, and therefore are deemed better performers than fertile diploids. Nevertheless, scientific reports describing no differences between ploidies and, in certain conditions, disadvantages of triploids are not uncommon. As the use of triploid oyster seed increases culture cost for growers, consistency in performance of triploids is considered to be an important goal. Thus, research to assess how ploidy affects physiological processes underlying oyster performance is fundamental for the aquaculture industry. This work was undertaken to assess if ploidy-based differences in performance in the two most commonly cultivated commercial species of oyster in the United States, Crassostrea virginica and Crassostrea gigas, are associated with filtration, feeding, and metabolism. To test this hypothesis, biodeposition measurements were made with oysters exposed to ambient water conditions at locations and seasons providing a variety of environmental conditions. Oysters did not show differences in filtration and feeding associated with ploidy, but physiological feeding variables fluctuated with environmental characteristics associated with spatial and seasonal differences. A preliminarily test of the hypothesis that differences in energy metabolism may account for differences in performance among ploidy levels indicates that basal metabolic rates of diploid, triploid, and tetraploid Eastern oysters are not different.

Remote-setting of oyster, Crassostrea virginica, larvae from the disease-resistant Haskin NEH line is performed every other year in Delaware to supply small-scale oyster enhancement efforts, citizen oyster gardening program. Over the past four remote sets (2009, 2011, 2013, 2015), three types of shell containment gear were utilized to monitor and compare the setting efficiency rate and post-set survival. Shell containment gears included diamond, plastic mesh bags (2009, 2011), wire baskets (2011, 2015), and plastic aquaculture trays (2013, 2015). Average setting efficiency was estimated at 28%, 23%, 17%, and 29% in the respective 2009, 2011, 2013, and 2015 remote sets while gear specific settling efficiency (%) include 28% for mesh bags in 2009, 22% for mesh bags and 33% for wire baskets in 2011, 17% for aquaculture trays in 2013, and 30% for aquaculture trays and 22% for wire baskets in 2015. Settling efficiency was lowest in 2013 using aquaculture trays compared to other years while aquaculture trays in 2015 and wire baskets in 2011 resulted equally higher settling efficiency. For small-scale growers, the stacked aquaculture trays are advantageous for several reasons: reducing handling time, uniform shell distribution within tanks, environmentally friendly alternative, and ease of cleaning detritus between shell layers.

Regucalcin (RGN), also known as senescence marker protein-30 (SMP30), plays a vital role in the regulation of Ca2+ homeostasis. In the present study, a regucalcin (designated as CgRGN) was identified from Pacific oyster Crassostrea gigas. The complete cDNA sequence of CgRGN was of 1059 bp, containing an open reading frame of 933 bp which encoded a protein of 310 amino acids. The deduced amino acid sequence of CgRGN shared similarity with other RGNs from the genome of C. gigas as well as other species. The mRNA transcripts of CgRGN were universally detected in all tested tissues, with higher level in hepatopancreas, labial palp, and gills. The relative expression level of CgRGN in hemocytes was significantly up-regulated (p

Maternal immune priming is the transfer of immunity from mother to offspring, which may reduce the offspring's risk of disease from a pathogen that previously infected its mother. Maternal immune priming has been described in at least 25 invertebrate taxa, including Crassostrea gigas. Larvae of C. gigas have improved survival to Ostreid herpesvirus (OsHV-1) if their mothers are either infected with OsHV-1 or were injected with a virus mimic called poly(I:C). However, fitness costs associated with maternal immune priming in C. gigas are unknown. Here, we show C. gigas larvae produced from poly(I:C)-treated mothers are smaller, and have higher total bacteria and Vibrio loads compared to control larvae. These results suggest that the improved offspring survival of C. gigas to OsHV-1 due to maternal immune priming with poly(I:C) is potentially traded off with other important life history traits, such as larval growth rate and destabilisation of the microbiome.

The host:parasite interactions of the 3 serious haplosporidian pathogens of oysters, on which most information exists, are reviewed. They are Bonamia ostreae in Ostrea spp. and Crassostrea gigas; Bonamia exitiosa in Ostrea spp.; and Haplosporidium nelsoni in Crassostrea spp. Understanding the haemocytic response to pathogens is constrained by lack of information on haematopoiesis, haemocyte identity and development. Basal haplospridians in spot prawns are probably facultative parasites. H. nelsoni and a species infecting Haliotis iris in New Zealand (NZAP), which have large extracellular plasmodia that eject haplosporosomes or their contents, lyse surrounding cells and are essentially extracellular parasites. Bonamia spp. have small plasmodia that are phagocytosed, haplosporosomes are not ejected and they are intracellular obligate parasites. Phagocytosis by haemocytes is followed by formation of a parasitophorous vacuole, blocking of haemocyte lysosomal enzymes and the endolysosomal pathway. Reactive oxygen species (ROS) are blocked by antioxidants, and host cell apoptosis may occur. Unlike susceptible O. edulis, the destruction of B. ostreae by C. gigas may be due to higher haemolymph proteins, higher rates of granulocyte binding and phagocytosis, production of ROS, the presence of plasma β-glucosidase, antimicrobial peptides and higher levels of haemolymph and haemocyte enzymes. In B.exitiosa infection of Ostrea chilensis, cytoplasmic lipid bodies (LBs) containing lysosomal enzymes accumulate in host granulocytes and in B. exitiosa following phagocytosis. Their genesis and role in innate immunity and inflammation appears to be the same as in vertebrate granulocytes and macrophages, and other invertebrates. If so, they are probably the site of eicosanoid synthesis from arachidonic acid, and elevated numbers of LBs are probably indicative of haemocyte activation. It is probable that the molecular interaction, and role of LBs in the synthesis and storage of eicosanoids from arachidonic acid, is conserved in innate immunity in vertebrates and invertebrates. However, it seems likely that haplosporidians are more diverse than realized, and that there are many variations in host parasite interactions and life cycles.

The homeostasis of immune cells during immune response is vital for hosts to defend against invaders. Activating transcription factor 6 (ATF6) is an important transcription factor in the unfolded protein response (UPR) to maintaining cellular homeostasis. In the present study, one ATF6 homologue was identified from Pacific oyster Crassostrea gigas (designated as CgATF6β). The full length cDNA of CgATF6β was of 2645 bp with a 1596 bp open reading frame (ORF) encoding a polypeptide of 531 amino acids. The deduced amino acid sequence of CgATF6β was predicted to contain a transmembrane region, a conserved basic leucine zipper (bZIP) domain, a site 1 protease cleavage site, a site 2 protease cleavage site, and a Golgi localization signal. CgATF6β mRNA was constitutively expressed in hemocytes, gill, mantle, gonad, hepatopancreas and labial palp, with a slightly higher expression level in muscle (2.45-fold of that in gill, p 0.05). After oysters were challenged with Vibrio splendidus, the mRNA expression levels of CgATF6β in hemocytes were significantly up-regulated at 3 h (2.68-fold of that in seawater group, p 0.01) and peaked at 12 h (3.14-fold of that in seawater group, p 0.01). The endogenic CgATF6β protein was mainly located in the cytoplasm of oyster hemocytes, and it was significantly transported into the nuclei of hemocytes at 1.5 h after the challenge with V. splendidus. After an injection with CgATF6β dsRNA, the mRNA expression of CgATF6β was knocked down to 0.26-fold of that in dsGFP group (p 0.01). In CgATF6β dsRNA-injected oysters, the mRNA expressions of glucose-regulated protein 78 (GRP78), calnexin (CNX) and anti-apoptotic B-cell lymphoma-2 (Bcl-2) in hemocytes were significantly decreased at 12 h after V. splendidus challenge, which were 0.65-fold (p 0.01), 0.54-fold (p 0.01) and 0.17-fold (p 0.01) of that in dsGFP-injected oysters, while the apoptotic rate of hemocytes was significantly up-regulated (1.97-fold of that in dsGFP group, p 0.05). Collectively, these results suggested that CgATF6β was involved in apoptosis inhibition of oyster hemocytes upon V. splendidus challenge by regulating the expression of CgGRP78, CgCNX and CgBcl-2.

The programmed cell death protein 4 (PDCD4) is a newly defined transcriptional and translational inhibitor, which plays a key role in regulating the synthesis of inflammatory cytokines in vertebrates species. In the present study, the full-length cDNA of PDCD4 from oyster Crassostrea gigas (designed as CgPDCD4) was identified to explore its possible involvement in immune response. The open reading frame of pdcd4 gene was of 1344 bp encoding a polypeptide of 447 amino acids with two conserved MA-3 domains. The deduced amino acid sequence of CgPDCD4 shared 60.18% similarity with PDCD4 from Mizuhopecten yessoensis. The mRNA transcripts of CgPDCD4 could be detected in all the tested tissues with a higher expression level in adductor muscle and hemocytes. The mRNA expression of CgPDCD4 in hemocytes was significantly down-regulated at 3 h and 6 h (0.61-fold and 0.42-fold of that in PBS group, p 0.01, respectively) after LPS stimulation. In hemocytes, CgPDCD4 protein was found to be mainly located in the cytoplasm. After the mRNA expression of CgPDCD4 in hemocytes was knocked down (0.40-fold of that in EGFP-RNAi group) by CgPDCD4 dsRNA (dsCgPDCD4) injection, the CgIL17-5 transcripts were up-regulated (20.11-fold of that in PBS group, p 0.01) post LPS stimulation, which was significantly higher than that in dsEGFP-injected oysters (7.06-fold of that in PBS group, p 0.01). Meanwhile, the nuclear translocation of CgRel (homologue of Rel/NF-κB) was significantly enhanced (about 1.36-fold of that in PBS group, p 0.01), but it was similar as that in EGFP-RNAi group (about 1.52-fold of that in PBS group, p 0.01) after LPS stimulation. All the results suggested that CgPDCD4 in oysters played the same role as PDCD4 of vertebrates in negatively regulating the production of interleukin in immune response, but the underpinning signal pathway was not conserved during evolution.

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 ChCas 3, in C. hongkongensis. The full-length cDNA of ChCas 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, ChCas 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 ChCas 3 in all the eight tissues via tissue-specific expression assays with the highest expression in haemocytes. ChCas 3 was confirmed to be expressed throughout the larval development stages, and fluorescence from pEGFP-N1-ChCas 3 was found to be distributed throughout the entire HEK293T cell. In addition, the relative expression of ChCas 3 significantly enhanced in hemocytes post bacterial stimulation, and overexpression of ChCas 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 ChCas 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.

The activator protein-1 (AP-1) plays an important role in inducing the immune effector production in response to cellular stress and bacterial infection. In the present study, an AP-1 was identified from Pacific oyster Crassostrea gigas (designed as CgAP-1) and its function was investigated in response against lipopolysaccharide (LPS) stimulation. CgAP-1 was consisted of 290 amino acids including a Jun domain and a basic region leucine zipper (bZIP) domain. CgAP-1 shared 98.6% similarities with ChAP-1 from oyster C. hongkongensis, and assigned into the branch of invertebrates in the phylogenetic tree. The mRNA transcripts of CgAP-1 gene were detected in all tested tissues with highest expression level in hemocytes, especially in granulocytes. The mRNA expression level of CgAP-1 gene in hemocytes was significantly up-regulated (8.53-fold of that in PBS group, p

Per- and polyfluoroalkyl substances (PFAS) and phthalate esters (PAEs) are emerging contaminants of higher concern due to their wide industrial and commercial use, toxicity, and potential adverse health effects. In this study, we assessed PFAS and PAEs exposure in American oysters collected in three study sites in Florida, USA. Potential physiological effects of these contaminants were assessed by collecting oyster biometric data, calculating condition indices, and assessing oxidative stress levels in these individuals. Finally, a human health risk assessment was conducted based on the concentrations found in the consumable Tampa Bay (TB) oysters. All PFAS and PAEs compounds assessed in this study were detected in at least one oyster in all study sites. Among all locations, ΣPFAS concentration range was 0.611-134.78 ng·g

Microplastics have been documented in a plethora of marine environments and organisms. These small plastics threaten ecosystem health, with implications for seafood species' health. Oysters are an important cultural and economic aquaculture species globally. Due to their filter feeding mechanisms, they can act as an indicator species and proxy for environmental contamination. This makes them an ideal organism for investigating microplastic pollution. Here, we first systematically reviewed the global literature investigating microplastic in oysters. Globally, 94.4% of all oysters had microplastics, with an average of 1.41 ± 0.33 per gram of soft tissue wet weight (gww). The review showed that wild-caught oysters contained more than double the amount of microplastic than aquaculture raised specimens, likely reflecting the clean and productive waters in which oyster aquaculture systems are commonly located. Second, we quantified microplastic presence and polymer type in commercially farmed oysters (Crassostrea gigas and Saccostrea glomerata) across a broad spatial scale, covering eight sites in southern Australia. Microplastics were present in 49.4% of all sampled oysters, with specimens from all locations containing microplastics. On average, whole oysters contained 0.83 ± 0.08 microplastics per individual or 0.09 ± 0.01 microplastics gww. Using Fourier-Transform Infrared Spectroscopy, we identified that 62% of the verified microplastics were vexar plastic netting, a low-density polyethylene commonly used in aquaculture production. Understanding the abundance and source of microplastic in these key seafood species is essential to determine if oysters are vulnerable to these contaminants and pose a risk to the oyster aquaculture industry as an important food resource.

To investigate the effects of high hydrostatic pressure (HHP) on the conformation and IgG binding capacity, tropomyosin (TM) from Pacific oysters was subjected to high pressures of 300, 450 or 600 MPa. The results showed that the α-helix of TM with HHP-induced was decreased, while β-turn, β-sheet (predominantly) and random coil were increased. The surface hydrophobicity and sulfhydryl group content of TM were increased, while the fluorescence/UV intensity were decreased after HHP treatment. Atomic force microscopy (AFM) result exhibited that the morphology of TM was changed at 600 MPa and formed fibrous structures. The IgG binding capacity of TM and digested TM was markedly reduced when the pressure was increased, especially at 600 MPa. Overall, this study indicated that HHP-induced conformational changes in TM contributed to the reduction in IgG binding capacity. These findings suggested that HHP may be a promising non-thermal technology for producing hypoallergenic oyster products.

Climate change, represented by rising and fluctuating temperature, induces systematic changes in marine organisms and in their bacterial symbionts. However, the role of host-microbiota interactions in the host's response to rising temperature and the underlying mechanisms are incompletely understood in marine organisms. Here, the symbiotic intestinal microbiota and transcriptional responses between diploid and triploid oysters that displayed susceptible and resistant performance under the stress of rising temperature during a summer mortality event were compared to investigate the host-microbiota interactions. The rising and fluctuating temperatures triggered an earlier onset and higher mortality in susceptible oysters (46.7%) than in resistant oysters (17.3%). Correlation analysis between microbial properties and environmental factors showed temperature was strongly correlated with indices of α-diversity and the abundance of top 10 phyla, indicating that temperature significantly shaped the intestinal microbiota of oysters. The microbiota structure of resistant oysters exhibited more rapid changes in composition and diversity compared to susceptible oysters before peak mortality, indicating that resistant oysters possessed a stronger ability to regulate their symbiotic microbiota. Meanwhile, linear discriminant analysis effect size (LefSe) analysis found that the probiotics Verrucomicrobiales and Clostridiales were highly enriched in resistant oysters, and that potential pathogens Betaproteobacteriales and Acidobacteriales were enriched in susceptible oysters. These results implied that the symbiotic microbiota played a significant role in the oysters' adaptation to rising temperature. Accompanying the decrease in unfavorable bacteria before peak mortality, genes related to phagocytosis and lysozymes were upregulated and the xenobiotics elimination pathway was exclusively expressed in resistant oysters, demonstrating the validity of these immunological functions in controlling proliferation of pathogens driven by rising temperature. Compromised immunological functions might lead to proliferation of pathogens in susceptible oysters. This study might uncover a conserved mechanism of adaptation to rising temperature in marine invertebrates from the perspective of interactions between host and symbiotic microbiota.

Oysters are frequently used as sentinel organisms for monitoring effects of contaminants due to their sessile, filtering habits and bioaccumulation capacity. These animals can show elevated body burden of contaminants, such as pyrene (PYR). PYR can be toxic at a molecular level until the whole oyster, which can show reproductive and behavioral changes. Considering that biologic parameters, such as gender or reproductive stage can interfere in the toxic effects elicited by contaminants uptake, the aim of this study was to evaluate some molecular and histological responses in females and males of oyster Crassostrea gasar exposed to PYR (0.25 and 0.5 μM) for 24 h at the pre-spawning stage. PYR concentrations were analyzed in water and in tissues of female and male oysters. Gene transcripts related to biotransformation (CYP3475C, CYP2-like, CYP2AU1, CYP356A, GSTO-like, GSTM-like, SULT-like), stress (HSP70), and reproduction (Vitellogenin, Glycoprotein) were quantified in gills. In addition, histological analysis and histo-localization of CYP2AU1 mRNA transcripts in gills, mantle and digestive diverticulum were carried out. Females and males in pre-spawning stage bioconcentrated PYR in their tissues. Males were more sensitive to PYR exposure. CYP2AU1 transcripts were higher in males (p 0.05), as well as tubular atrophy was observed only in males exposed to PYR (p 0.05). As expected, vitellogenin transcripts were lower in males (p 0.05). Given these results, it is suggested that levels of CYP2AU1 be a good biomarker of exposure to PYR in oyster C. gasar and that it is important to consider the gender for the interpretation of biomarker responses.

The production and supply of triploid oysters Crassostrea gigas has become an increasingly frequent activity in aquaculture companies to satisfy the growing demand of growers. These triploid oysters are usually obtained by crossing male tetraploid oysters with female diploid oysters. Furthermore, studies have shown that it is possible to select oysters for better resistance to infection by OsHV-1 and they are currently produced by commercial hatcheries in France. The effects of ploidy and selection on the physiological functioning of the Pacific cupped oyster are still relatively unknown, especially in tetraploid animals selected for better resistance to infection by OsHV-1. In the framework of sustainable domestication, and by taking into account the natural habitat, it has become necessary to shed more light on the subject. Faced by this lack of knowledge, a physiological study of respiration and clearance rate was performed on 6 stocks of C. gigas oysters whose levels of ploidy (2n, 3n and 4n) and sensitivity to OsHV-1 resistance (selected and non-selected) were different. The experiments were performed in controlled and reproducible habitats where OsHV-1 is absent. The physiological results showed: i) an absence of interaction between ploidy levels and selection; ii) families showed no significant difference as a function of the level of selection to OsHV-1 tolerance; and iii) significant differences in clearance rate and daily weight growth as a function of the oysters' ploidy level. In particular, the results revealed that the clearance rate of tetraploids was lower than that of diploid and triploid oysters with a growth rate equal to that of triploids and higher than diploids. Additional studies are envisaged in the discussion to better understand these results.

Hypoxia caused by global climate change and anthropogenic pollution has exposed marine species to increasing stress. Oxygen sensing mediated by prolyl hydroxylase (PHD) is regarded as the first line of defense under hypoxia exposure; however, the function of PHD in marine molluscan species remains unclear. In this study, we identified two PHD2 gene in the oyster Crassostrea gigas using phylogenetic tree analysis with 36 species, namely, CgPHD2A/B. Under hypoxia, the mRNA and protein expression of CgPHD2A displayed a time-dependent pattern, revealing a critical role in the response to hypoxia-induced stress. Observation of interactions between CgPHD2 and CgHIF-1α proteins under normoxia using co-immunoprecipitation and GST-pull down experiments showed that the β2β3 loop in CgPHD2A hydroxylates CgHIF-1α to promote its ubiquitination with CgVHL. With the protein recombination and site-directed mutagenesis, the hydroxylation domain and two target proline loci (P404A and 504A) in CgPHDs and CgHIF-1α were identified respectively. Moreover, the electrophoretic mobility-shift assay (EMSA) and luciferase double reporter gene assay revelaed that CgHIF-1α could regulate CgPHD2A expression through binding with the hypoxia-responsive element in the promoter region (320 bp upstream), forming a feedback loop. However, protein structure analysis indicated that six extra amino acids formed an α-helix in the β2β3 loop of CgPHD2B, inhibiting its activity. Overall, this study revealed that two CgPHD2 proteins have evolved, which encode enzymes with different activities in oyster, potentially representing a specific hypoxia-sensing mechanism in mollusks. Illustrating the functional diversity of CgPHDs could help to assess the physiological status of oyster and guide their aquaculture.

There is a lack of knowledge about the influence of seasonality on the microbial and physicochemical quality of oysters in Sado and Mira rivers. Water, sediment, and oysters (Crassostrea angulata and Crassostrea gigas) were collected for microbiological, nutritional, and sensory analyses. The microbiological water quality and the oyster shell contamination were better during the warmer months. No seasonal effect was observed on sediments and on oyster meat. A good physicochemical and nutritional quality was also observed, with high content of polyunsaturated fatty acids, including omega-3 fatty acids, resulting in good lipid quality indices. From the sensory evaluation, both oysters' species were well scored and presented the highest scores (4) in parameters such as cream-ivory colour, sea smell, firmness and juiciness. These attributes denote the freshness degree at the time of the tasting, reflecting the quality of the bivalve.

Anthropogenic activities in coastal regions cause risks to the environmental and human health. Due to the carcinogenic and mutagenic potential, polycyclic aromatic hydrocarbons (PAH) are considered priority for monitoring. Most of the Brazilian production of Crassostrea gigas oysters are placed in the Bays of Santa Catarina Island. The aim of this study was to evaluate molecular responses (phase I and II of biotransformation and antioxidant defense) of C. gigas from six oyster farming areas potentially contaminated by sanitary sewage in Florianópolis Metropolitan (SC, Brazil): Santo Antônio de Lisboa, Sambaqui, Serraria, Caieira, Tapera, Imaruim. We evaluated the transcript levels of CYP1A1-like, CYP2-like, CYP2AU2-like, CYP356A1, GSTA1A-like, GSTO.4A-like, SULT-like, SOD-like and CAT-like by qRT-PCR. Only oysters from Caieira showed levels of thermotolerant coliforms allowed by the law. Chemicals analyses in soft tissues of oysters showed low to average levels of PAH in all monitored areas. Enhanced transcript levels of phase I (CYP1A1-like, CYP3564A1-like, CYP2-like and CYP2AU2-like) were observed in oysters from Serraria and Imaruí, suggesting higher biotransformation activity in these farming areas. Regarding phase II of biotransformation, GSTO.4A-like was up-regulated in oysters from Imaruí compared to Caieira and Santo Antônio de Lisboa. An upregulation of SOD-like and CAT-like were observed in oysters from Imaruí and Serraria, suggesting that oysters from these sites are facing higher prooxidant conditions compared to other areas. By integrating the biological and chemical data it is suggested that human-derived contaminants are affecting the oyster metabolism in some farming areas.

The levels of linear alkylbenzenes (LABs) and the occurrence of microplastics (MPs) in the oysters Crassostrea gigas were evaluated in six farming areas in southern Brazil. The results revealed higher concentrations of LABs in oyster tissue from the Serraria (1977 ± 497.7 ng g

Due to the large breeding density and the influence of seasonal changes, the quality of oysters declined. Oysters are often fattened with specific microalgae before sale to improve their quality. Here, the purpose of this study was to study the effects of different microalgae diets on the flavor characteristics of Crassostrea gigas (C. gigas) after three weeks of fattening. The result showed that there was a certain correlation between the fatty acid composition of C. gigas and microalgae. The equivalent umami concentration values of C. gigas fed with Chlorella vulgaris and Pavlova viridis (5.34 and 4.91 g/100 g, respectively) were significantly higher than that of the control group. Gas chromatography-ion mobility spectrometry analysis showed 48 volatile organic compounds, and the principal component analysis plot showed an obvious separation between the C. gigas. These results indicated that diets had a significant effect on the flavor components of oysters.

Color polymorphism in Mollusca is of great interest for consumer preference. Although the heritability of shell color variation has been conducted by experimental crossing, little is known about molecular basis involved in these patterns. Tyrosinase-like proteins are important enzymes which are members of the type-3 copper protein superfamily. In this research, two tyrosinase-like protein genes including CgTyp-1 and CgTyp-3 were identified in the Pacific oyster Crassostrea gigas. Tissue expression analysis showed that CgTyp-1 and CgTyp-3 were dominantly expressed in the mantle. Particularly, they were expressed significantly higher in the edge mantle than that in the central mantle whether on the left or right mantles. Additionally, expressions of CgTyp-1 and CgTyp-3 were mainly found in the black shell color oysters, with relative lower levels in the white shell color oysters. In situ hybridization showed that positive signals for CgTyp-1 and CgTyp-3 were both detected within the outer epithelium of the outer fold either in the black or white shell color oysters. After interference, the expression levels of CgTyp-1 and CgTyp-3 mRNA were significantly attenuated, and the efficiency of RNAi reached 84.72% and 71.58%, respectively. Besides, knockdown CgTyp-1 or CgTyp-3, obviously decreased the tyrosinase activity of mantles. Furthermore, the number of the melanosomes within epithelium of the outer fold was sharply reduced by silencing of each Typ. These findings argue that CgTyp-1 and CgTyp-3 may be involved in the melanin synthesis, which lends insight into regulation mechanism of shell pigmentation in C. gigas.

MyD88 is a universal adapter protein for the Toll-like receptor/interleukin-1 receptor (TLR/IL-1R) signaling pathway. Since invertebrates are believed to lack MyD88-independent pathways, MyD88 appears more critical in oyster TLR signaling pathway. In the Pacific oyster (Crassostrea gigas), two complete paralogues, named as CgMyD88-1 and CgMyD88-2, have been identified. In the current study, we indicated that CgMyD88-1 and CgMyD88-2 might act synergistically to increase the efficiency of immune signaling by activating NF-κB transcription factor. However, we found that upon stimulation with lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid [poly (I:C)], CgMyD88-1 and CgMyD88-2 show differences in their response: CgMyD88-1 accumulated as large spots in the cytoplasm, while CgMyD88-2 assembled in the cytoplasm and in the membrane. Our results support the theory that expansion of these immune genes is associated with functional diversity.

In many spiralians, asymmetry in the first two cleavages is achieved through the formation of a polar lobe (PL), which transiently constricts to sequester vegetal cytoplasm into the CD and D blastomeres. While microtubules and actin filaments are required for polar lobe formation, little else is known regarding the structural and functional similarities with the contractile ring, or how the PL constriction is able to form perpendicular to the cleavage plane. Examination of scallop embryos revealed that while activated myosin II could be detected in both the cleavage furrow and early PL constriction, astral or central spindle microtubules were not observed associated with the PL neck until the constriction was nearly complete. Further, inhibition of Aurora B had no effect on polar lobe initiation, but blocked both contractile ring ingression and PL constriction beyond phase II. The cortex destined for PL sequestration was marked by enrichment of the Arp2/3 complex, which was first detected during meiosis and remained enriched at the vegetal pole through the first two cleavages. Inhibition of Arp2/3 affected PL formation and partitioning of cytoplasm into the two daughter cells, suggesting that Arp2/3 plays a functional role in defining the zone of cortex to be sequestered into the polar lobe. Together, these data offer for the first time a mechanism by which a cytoskeletal specialization defines the polar lobe in this atypical form of asymmetric cell division.

In oysters, nutrients are stored in a special type of cells referred to as vesicular-connective tissue cells (VCT-cells). These cells accumulate and provide nutrient to satisfy various needs of the organism, including gametogenesis. During the annual reproductive cycle, VCT-cells pass through a series of changes in their morphology associated with nutrients mobilization for developing germ cells. The results presented here show an approximately 33-35% increase in the number of autophagic vesicles in cytoplasm of VCT-cells in the gonadal area of C. gigas during the stage of active gametogenesis as compared to the resting stage of reproductive cycle. No destruction of VCT-cells due to autophagy or any other factors was observed, both in males and females. Our results indicate that autophagy does increase in VCT-cells of C. gigas and plays a certain role in nutrient mobilization from these cells.

The present study assessed the spatial and temporal variations on metal bioaccumulation and biochemical biomarker responses in oysters Crassostrea gasar transplanted to two different sites (S1 and S2) at the Laguna Estuarine System (LES), southern Brazil, over a 45-days period. A multi-biomarker approach was used, including the evaluation of lipid peroxidation (MDA) levels, and antioxidant defense enzymes (CAT, GPx, GR and G6PDH) and phase II biotransformation enzyme (GST) in the gills and digestive gland of oysters in combination with the quantification of Al, Cd, Cu, Pb, Fe, Ni and Zn in both tissues. The exposed oysters bioaccumulated metals, especially Al, Cd and Zn in gills and digestive gland, with most prominent biomarker responses in the gills. Results showed that GPx, GR and G6PDH enzymes offered an increased and coordinated response possibly against metal (Zn, Ni, Cd and Cu) contamination in gills. GST was inversely correlated to Cd levels, being its activity significantly lowered over the 45-d exposure periods at S2. On contrary, in digestive gland GST was slightly positively correlated to Cd, revealing a compensatory mechanism between tissues to protect oysters' cells against oxidative damages, since MDA levels also decreased. CAT also appeared to be involved in the cellular protection against oxidative stress, being increased in gills. However, CAT was negatively correlated to Al levels, which might suggest a possible inhibitory effect of this metal in the gills of C. gasar. Differences between tissues were evident by the Integrative Biomarker Responses version 2 (IBRv2) indexes, which showed different pattern between tissues when studying the sites and exposure periods separately. This study provided evidence for the effectiveness of using a multi-biomarker approach in oyster C. gasar to monitor estuarine metal pollution.

C-type lectins (CTLs), as important pattern recognition receptors (PRRs), are a superfamily of Ca2+-dependent carbohydrate-recognition proteins which participate in nonself-recognition and eliminating pathogens. In the present study, a novel CTL (designated as CgCLec-3) was identified from the Pacific oyster Crassostrea gigas. There was only one carbohydrate-recognition domain (CRD) of 151 amino acid residues within the deduced amino acid sequence of CgCLec-3. The deduced amino acid sequence of CgCLec-3 CRD shared low homology with the CRDs of other CTLs in oyster with the identities ranging from 12% to 22%. A novel DIN motif was found in Ca2+-binding site 2 of CgCLec-3. The relative expression level of CgCLec-3 in hemocytes was up-regulated significantly after the stimulations of bacteria and Pathogen Associated Molecular Patterns (PAMPs). Immunohistochemistry assay showed that CgCLec-3 protein was mainly distributed in gill and mantle, less in gonad, and could not be detected in adductor muscle and hepatopancreas. The recombinant protein (rCgCLec-3) could bind lipopolysaccharide (LPS), mannose (MAN) and peptidoglycan (PGN), but not poly (I:C). rCgCLec-3 exihibited strong binding ability to Vibrio anguillarum and V. splendidus, moderate binding activities to Escherichia coli, Pichia pastoris and Yarrowia lipolytica, weak binding affinity to Staphylococcus aureus and Micrococcus luteus. rCgCLec-3 could agglutinate microorganisms, in a Ca2+-dependent manner and its activity to agglutinate V. splendidus was remarkably higher than that to agglutinate E. coli, S. aureus and P. pastoris. The phagocytic activity of oyster hemocytes was significantly enhanced after incubation with rCgCLec-3. rCgCLec-3 also exhibited antibacterial activity against E. coli and S. aureus. The results clearly suggested that CgCLec-3 in Pacific oyster C. gigas not only served as a PRR involved in the PAMPs recognition and microbes binding, but also functioned as an immune effector participating in the clearance of invaders.

Phagocytosis constitutes a conserved cellular process for multicellular animals to ingest or engulf other cells or particles, which is facilitated by the use of opsonins to bind foreign particles and interact with cell surface receptors. The invertebrate secreted C1q domain-containing proteins (C1qDCs) have been reported to exhibit opsonic activity, while the detailed mechanisms of opsonization still remain unclear. In the present study, a C1qDC (designated as CgC1qDC-5) with opsonic activity was identified from the hemolymph of oyster Crassostrea gigas. CgC1qDC-5 exhibited the ability to bind pathogen-associated molecular patterns (PAMPs) of lipopolysaccharides (LPS) and Lipid A. It could also bind and agglutinate Gram-negative bacteria Escherichia coli, Vibrio splendidus and Vibrio anguillarum, whereas the agglutinating activity could be inhibited by LPS. In addition, CgC1qDC-5 could enhance the phagocytosis of hemocytes toward E. coli, V. splendidus, and V. anguillarum. GST pull-down and surface plasmon resonance assays in vitro revealed that CgC1qDC-5 could interact with β-integrin (CgIntegrin). In vivo, CgC1qDC-5 was observed to bind hemocytes and co-localized with CgIntegrin on the cell membrane of hemocytes. Antibody-mediated blockage of CgIntegrin hindered the CgC1qDC-5-enhanced hemocytic phagocytosis. CgIntegrin also exhibited the ability to bind the Gram-negative bacteria E. coli, V. splendidus, V. anguillarum and Vibrio parahaemolyticus, and PAMP of LPS, but not Lipid A. A phagocytosis assay demonstrated that CgIntegrin could directly mediate phagocytosis toward bacteria as a phagocytic receptor. These results collectively suggested that CgC1qDC-5 could serve as an opsonin to recognize and bind bacteria, and subsequently interact with CgIntegrin on the hemocyte surface to enhance the CgIntegrin-mediated phagocytosis in oyster.

Bivalves facilitate microbial nitrogen cycling, which can produce nitrous oxide (N2O), a potent greenhouse gas. Potential N2O production by three marine bivalves (Mytilus edulis, Mercenaria mercenaria and Crassostrea virginica) was measured in the laboratory including responses to nitrogen (N) loading and/or warming over short-terms (up to 14 or 28 days). N additions (targeting 100 μM-N ammonium nitrate) or warming (22 °C) individually and in combination were applied with experimental controls (20 μM-N, 19 °C). N2O production rates were higher with N additions for all species, but warming lacked significant direct effects. Ammonium and nitrate concentrations varied but were consistent with nitrification as a potential N2O source for all bivalves. Highest N2O emissions (7.5 nmol N2O g−1 h−1) were from M. edulis under hypoxic conditions coincident with a drop in pH. Macro-epifauna on M. edulis did not significantly alter N2O production. Thus, under short-term hypoxic conditions, micro-organisms in M. edulis guts may be a particularly significant source of N2O.

Perfluoroalkyl substances (PFASs) were investigated in filter-feeding shellfish collected from 2013 to 2017 along the English Channel, Atlantic and Mediterranean coasts of France. PFOS (perfluorooctane sulfonate), PFTrDA (perfluorotridecanoic acid), PFTeDA (perfluorotetradecanoic acid), PFDoDA (perfluorododecanoic acid) and PFUnDA (perfluoroundecanoic acid) were detected in more than 80% of samples, thus indicating widespread contamination of the French coastal environment by these chemicals. The distribution of PFAS concentrations showed differences according to sampling locations and years. PFOS was the predominant PFAS in most samples collected from English Channel and Atlantic coasts until 2014, but the opposite was observed in 2015, 2016 and 2017, while perfluoroalkyl carboxylic acids (PFCAs) prevailed in Mediterranean samples in all study years. Among PFCAs, PFTrDA showed the highest maximum (1.36 ng g−1 ww) and median (0.077 ng g−1 ww) concentrations in 2016–2017. Other PFAS median concentrations were within the 0.014 (PFNA) - 0.055 (PFTeDA) ng g−1 ww range. The profiles determined each year in most Mediterranean samples suggest distinctive sources. PFOS median concentrations showed a significant decrease over the study years, from 0.118 to 0.126 ng g−1 ww in 2013–2015 to 0.066 ng g−1 ww in 2016 and 2017. ∑PFCAs showed no trends in concentration ranges over the same years. The shift in PFAS profiles from PFOS to long-chain PFCAs over the study period reflects PFOS production phase-out, combined with continuous inputs of PFCAs into the marine environment. These results provide reference data for future studies of the occurrence of contaminants of emerging concern on European coasts.

The Pearl River Estuary (PRE) is the third largest estuary in China, where estuarine organisms are under metal stress at various biological levels. Based on the metal concentrations measured in oyster Crassostrea hongkongensis, we documented a change in dominance of metal contamination from Cd, Cr, Cu, Ni and Zn to Ag, Cd, Cu and Zn. In general, metal concentrations were higher in upstream stations and displayed a clear up-downstream gradient. Compared to the historical values, we noted the reductions in Cd, Cr and Ni concentrations, and the changing inputs due to evolving industrial activities were responsible for shaping the metal contamination profile in the PRE region. Along with metal concentrations, a suite of biomarkers was analyzed. Among the metals measured in the oyster tissues, Ag, Cd, Cu, Ni and Zn showed the strongest associations with pro-oxidant and oxidative stress responses (superoxide dismutase, lipid peroxidation and lysosomal membrane destabilization) and detoxification responses (glutathione and metallothionein), suggesting that the present metal contamination still exerts significant amount of stress in biota in the PRE. Metal contamination in estuaries in China is still severe compared to other countries, therefore continuous efforts should be taken to monitor the changing metal profiles with necessary control and remediation measures.

Autophagy, a highly conserved intracellular degradation system, is involved in numerous processes in vertebrate and invertebrate, such as cell survival, ageing, and immune responses. However, the detailed molecular mechanism of autophagy and its immune regulatory role in bivalves are still not well understood. In the present study, an autophagy-related protein ATG10 (designated as CgATG10) was identified from Pacific oyster Crassostrea gigas. The open reading frame of CgATG10 cDNA was of 621 bp, encoding a polypeptide of 206 amino acid residues with an Autophagy_act_C domain (from 96 to 123 amino acid), which shared high homology with that from C. virginica and Octopus bimaculoides. The mRNA transcripts of CgATG10 were widely expressed in all the tested tissues including mantle, gonad, gills, hemocytes and hepatopancreas, with the highest expression level in mantle. After the stimulation with poly (I:C), the mRNA expression level of CgATG10 in the mantle of oysters was significantly up-regulated (4.92-fold of that in Blank group, p

Growth of the oyster aquaculture industry on the US east coast has accelerated in recent decades due to advances in breeding technologies and grow-out methods. As the industry grows, competition in the market place is likely to increase attention to product quality, for example, shell conformation and meat content. This study investigated how gear type and tidal zone location influenced shell shape and product quality of Crassostrea virginica during a four month “finishing” period at an oyster farm on Fishing Creek MD on the eastern shore of Chesapeake Bay. For these experiments, a mix of sub-market sized, triploid oysters from two local farms were deployed in four gear treatments that varied in water column location and exposure to wave action: OysterGro™ floats, a rack and bag system, a bottom cage placed in the intertidal, and a bottom cage placed subtidally, which represents the ‘control’ treatment (no change of gear type for finishing). Shell height, length, width, and weight (total and wet meat) were measured each month from August to December 2015 and an index of shell shape relative to an idealized 3-2-1 ratio of height to length to width was calculated. Overall, oysters grew well in all gear types (3.0–-4.8 mm increase in shell height per month) and experienced relatively low mortality (4–8% across treatments) – mean height of oysters from each gear treatment exceeded market size by the end of the experiment. Gear treatment had a significant effect on oyster height and weight at the end of the experiment (P

Lectins are carbohydrate-binding proteins with lectin domains, which are extensively studied for their numerous roles in biological recognition. However, the lectin domain containing proteins (LDCPs) chimerized with other non-lectin domains have not received sufficient attention. In the present study, a genome-wide survey of LDCPs in oyster Crassostrea gigas was conducted, and an expansive 640 LDCPs derived from ten lectin domains were identified and functionally explored. In these LDCPs, a total of 282 kinds of domains were predicted, and 90% of the LDCPs contained more than one kind of domain. The lectin domains were frequently fused with non-lectin domains, such as epidermal growth factor domain and peptidase related domains, which supplied LDCPs with more diversity in structures and functions. The C-type lectin domains were the most abundant domains in LDCPs, and they were largely co-existed with non-lectin domains of complement activation-related domains (such as CUB domain and PAN-1 domain) but relative independence with other lectin domains. Furthermore, the C-type lectin domain containing proteins (CTLPs) found to mainly act as pattern immune recognition receptors and were highly expressed in mucosal tissues (digestive gland, male gonad and labial palp) to provide mucosal immune protections. The Concanavalin A-like lectin domains were the second richest domains in LDCPs, and they were mostly constructed into chimeric proteins with epidermal growth factor domain and peptidase related domains. The Concanavalin A-like lectin domain containing proteins (CALPs) were significantly enriched with peptidase activities and mainly expressed in digestive tissues. All the results suggested the mucosal immunity and digestive functions of oyster LDCPs, which provided a fresh idea about the functions of invertebrate lectin family.