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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.

A multiplex quantitative PCR (qPCR) assay for the simultaneous detection of 3 eastern oyster Crassostrea virginica parasites, Perkinsus marinus, Haplosporidium nelsoni, and H. costale, was developed using 3 different fluorescently labeled hydrolysis probes. The primers and probe from a previously validated singleplex qPCR for P. marinus detection were combined with newly designed primers and probes specific for H. nelsoni and H. costale. The functionality of the multiplex assay was demonstrated on 2 different platforms by the linear relationship of the standard curves and similar cycle threshold (CT) values between parasites. Efficiency of the multiplex qPCR assay on the Roche and BioRad platforms ranged between 93 and 101%. The sensitivity of detection ranged between 10 and 100 copies of plasmid DNA for P. marinus and Haplosporidium spp., respectively. The concordance between the Roche and BioRad platforms in the identification of the parasites P. marinus, H. nelsoni, and H. costale was 91, 97, and 97%, respectively, with a 10-fold increase in the sensitivity of detection of Haplosporidium spp. on the BioRad thermocycler. The concordance between multiplex qPCR and histology for P. marinus, H. nelsoni, and H. costale was 54, 57, and 87%, respectively. Discordances between detection methods were largely related to localized or low levels of infections in oyster tissues, and qPCR was the more sensitive diagnostic. The multiplex qPCR developed here is a sensitive diagnostic tool for the quantification and surveillance of single and mixed infections in the eastern oyster.

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.

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.

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.

The Guana-Tolomato-Matanzas (GTM) system is a well-flushed estuary in Northeastern Florida, USA, and characterized as having an extraordinarily high abundance of oysters that resembles the populations described by Euro-American settlers. Historically, dense populations of oysters, such as those found in GTM, are believed to play an important role in water filtration; however, most research teams seeking to simulate this role have not had access to such robust populations to parametrize their models. To quantify the filtration service (FS) of Eastern oysters (Crassostrea virginica) in GTM at several spatial scales (i.e., reef, watershed, estuary), we implemented a model that solves for the hydrodynamics and depletion of particulate matter passing over model oyster populations, the latter of which were derived from detailed bay-wide surveys. The model results suggested that oyster reefs populating the GTM play an important role in water quality by filtering ~60% of the estuary’s volume within its residence time. Our approach teases apart the role of reef size, residence time, particle concentration, and other physical factors on the generation of FS at different spatial scales. Downstream effects were found to be very important for estuary FS, which depend on the spatial distribution of the reefs in the GTM and local and estuarine-scale hydrodynamics. Therefore, the difference between “realized” FS and the “potential” FS of a given reef may be substantial when considering the complex hydrodynamic and connectivity among populations at several scales. Our model results provide clear and actionable information for management of these oyster populations and conservation of their ecosystem services.

Ostreid herpesvirus 1 (OsHV-1) has caused mass mortalities in Pacific oysters (Crassostrea gigas) in Europe, Australia, and New Zealand. While aquaculture-associated movements of infected Pacific oysters are a well-known cause of OsHV-1 spread once established in a region, translocation via biofouling of aquaculture equipment or vessels needs further investigation to explain the more distant spread of OsHV-1. Laboratory experiments were designed to test for transmission of OsHV-1 between infected and naive Pacific oysters via a simulated biofouling translocation scenario. Three common biofouling species [Sydney rock oysters (Saccostrea glomerata), Mediterranean mussels (Mytilus galloprovincialis) and Pacific oysters] were tested as intermediaries using a cohabitation challenge with Pacific oysters infected by injection. Transmission occurred, albeit for one of eight replicates when Pacific oysters were the intermediary species. This demonstrated a possible pathway for pathogen spread via biofouling containing Pacific oysters while highlighting the complexity of OsHV-1 transmission. Such complexities require further investigation to inform future risk assessments and management of fouled aquaculture equipment and vessels.

Understanding the mechanisms of biological responses to environmental change is a central theme in comparative and evolutionary physiology. Here, we analyzed variation in physiological responses to temperature, using 21 full-sibling larval families of the Pacific oyster, Crassostrea gigas. Pedigrees were confirmed with genetic markers for adult broodstock obtained from our breeding program. From these 21 larval families, 41 determinations of thermal sensitivity (Q10 values) were assayed for larvae of different sizes. For respiration, thermal sensitivity was consistent within a larval family during growth, but showed significant differences among families. Different Q10 values were evident among 21 larval families, with family accounting for 87% of variation. Specifically, four larval families maintained an increased thermal sensitivity for respiration (Q10 of 3). This physiology would confer resilience to rising temperature by matching the increased energy demand of protein synthesis (Q10 of 3 previously reported). For protein synthesis, differences in Q10 values were also observed. Notably, a family was identified that had a decreased thermal sensitivity for protein synthesis (Q10 of 1.7 cf. Q10 of 3 for other families), conferring an optimal energy allocation with rising temperature. Different thermal sensitivities across families for respiration (energy supply) and protein synthesis (energy demand) were integrated into models of energy allocation at the whole-organism level. The outcome of these analyses provides insights into the physiological bases of optimal energy allocation with rising temperature. These transgenerational (egg-to-egg) experiments highlight approaches to dissect components of phenotypic variance to address long-standing questions of genetic adaptation and physiological resilience to environmental change.

Environmental history (regimes of water quality to which an organism has been exposed in the past) may influence how the physiology of eastern oysters Crassostrea virginica responds to future environmental conditions caused by climate change. Previous research has examined environmental history in a 1-dimensional framework, failing to capture environmental history complexity through space and time. In this study, we examined environmental history as a multi-faceted parameter, incorporating abiotic water quality components, such as temperature, pH, and salinity, that differ among locations. We also assessed how different lengths of environmental histories, defined as proximal and distal, affected oyster physiology and stress response. Finally, we compared the relative influence of abiotic components of environmental history on oyster physiology. We found that physiology and stress response are differentially affected by proximal and distal environmental history, demonstrating the importance of examining environmental history as a multi-faceted and dynamic parameter. Specifically, distal environmental history primarily influenced condition index and total antioxidant potential, while proximal environmental history primarily influenced glycogen content. Salinity of distal environmental history significantly shaped condition index, establishing salinity as a principal factor when considering acclimatization to variable environments. No water quality components were significant influences on glycogen and total antioxidant potential, providing opportunities for research on other components of environmental history. Identifying the temporal portion of oysters’ environmental history that influences physiology supports future efforts to predict population tolerance to climate change. Additionally, examining multiple abiotic and biotic components of environmental history can elucidate means of acclimatization to future environmental change.

Freshwater-dominated estuaries experience large fluctuations in their physical and chemical environments which may influence larval dispersal, settlement, and connectivity of populations with pelagic larval stages. We used settlement patterns and natural tagging along with numerical hydrodynamic model results to assess settlement and connectivity among oysters across the freshwater-dominated Mobile Bay-eastern Mississippi Sound (MB-EMS) system. Specifically, we (1) tested how freshwater inputs and associated environmental attributes influenced settlement patterns during high and low discharge conditions in 2014 and 2016, respectively, and (2) analyzed trace element (TE) ratios incorporated into multiple shell types (larval and settled shell of spat and adult shells) to determine if shells collected in situ incorporate temporally stable site-specific signatures. We also assessed if TE ratios compared between adult (TE natal signature proxy) and larval shells could infer connectivity. Larval settlement was 4× higher during low discharge than during high discharge when oyster larvae only settled in higher salinity regions (EMS). Spat and adult shells incorporated site-specific TE ratios that varied from weeks to months. Connectivity results (May-June 2016 only) suggest that EMS is an important larval source to EMS and lower MB. While we were able to infer probable connectivity patterns using adult and larval shells, more study is needed to assess the utility of adult shells as proxies for natal-location TE signatures. Results provide a baseline for measuring future larval connectivity and adult distribution changes in the MB-EMS system. Biological and geochemical data demonstrate the potential to identify environmental attributes that spatiotemporally mediate settlement and connectivity in dynamic systems.

Two bag float configurations (on-side; on-top) for the culture of four length classes of Eastern oyster (Crassostrea virginica) were tested at two sites in Tracadie Bay, Canada. An interaction betw...

Ocean acidification is predicted to have significant implications for marine calcifying organisms. However, little is known about the physiological responses of Pacific oyster, Crassostrea gigas, to elevated partial pressure of atmospheric carbon dioxide (pCO2) under natural fluctuations associated with a farm environment. The present study evaluated the effect of two pCO2 levels (i.e. ambient ∼625 μatm and elevated ∼1432 μatm) on the physiological processes and growth of C. gigas in in situ mesocosms that simulated the farm environment. Oysters were exposed for 30 days over a sensitive period during their production cycle when they are first exposed to natural coastal conditions. Despite this being a well-known “bottleneck” in production, it remains understudied with respect to climate change. Results showed that elevated pCO2 levels decreased clearance rate, ingestion rate, absorption efficiency, and oxygen to nitrogen ratio, while increasing oxygen consumption and ammonia-N excretion rates. These physiological responses of oysters resulted in a reduction in energy available for growth (scope for growth). No mortality was observed in the control or elevated pCO2 treatments, indicating that although oyster may survive future coastal acidification, the allocation of energy towards production within aquaculture systems will decrease in the future, affecting the culture of these economically important marine bivalves.

The spat of the scallop Flexopecten glaber were collected in cages with the Pacific oyster Crassostrea gigas cultured on a mussel-and-oyster farm (outer roadstead of Sevastopol Bay). For two years they were reared in plastic cages at a depth of 2-3 m. The cages were periodically withdrawn to measure the size and weight parameters of the molluscs: the length (L, mm), height (H, mm), and width (D, mm) of shells and the total live weight (W, g). The correlations among the parameters under study are presented. The changes in the indices of the frontal (D/L) and sagittal (H/L) curvatures, convexity ((H+D)/L) and conditional volume (H×D×L/1000) of the shells in ontogenesis are shown. Upon reaching a shell length of 30-35 mm, the allometry of the volumetric and weight growth of molluscs changed from positive to negative. The largest values of the shell convexity index were registered in thesame length range. A conclusion about the interval-type growth of F. glaber in linear size and weight is made. It is suggested that the optimal strategy of shell formation in F. glaber in ontogenesis implies the ripening and the first reproduction of the molluscs occurring at the highest volumetric characteristics of the shell. The relationships for the linear size and weight growth ofF. glaber in the first two years of life are obtained. It is concluded that the scallop F. glaber should be considered as a possible element for the diversification of the existing aquaculture of molluscs (mussels and oysters) off the coast of Crimea.

This study investigated seasonal microplastics (MPs) pollution characteristics in oysters and surrounding surface seawater from five aquaculture farms located at the Yellow Sea and Bohai Sea. MPs abundances in oysters were 2.40 ± 0.14 (winter) to 3.28 ± 0.19 (autumn) items/individual, and 0.22 ± 0.02 (spring) to 0.45 ± 0.06 (summer) items/g (ww). In surface seawater, average seasonal MPs abundances were 3.41 ± 1.06-8.86 ± 2.48 items/L. Fibers were dominant shape, and cellophane and polyethylene terephthalate (PET) were dominant polymers in oysters and surface seawater. Positive correlation was found between oysters' MPs abundance (items/individual) and environmental factors (NO

Marine ecosystems with ocean warming and industry pollution threaten the survival and adaptation of organisms. G protein-coupled receptors (GPCRs) play critical roles in various physiological and toxicological processes in vertebrates and invertebrates. The Pacific oyster (Crassostrea gigas) was widely used to study the adaptation of marine molluscs to coastal environments. In this work, we identified a total of 586 GPCRs in C. gigas genome. The C. gigas GPCRs were divided into five classes (including class A, B, C, E and F) with different degrees of expansion. Meta-analysis of multiple RNA-seq datasets revealed that transcriptional expression patterns of GPCRs in C. gigas were distinct in response to high temperature, salinity, air exposure, heavy metal, ostreid herpes virus 1 (OsHV-1) and Vibrio challenge. This work for the first time characterized the GPCR gene family and provided insights into the potential roles of GPCRs in adaptation of marine molluscs to stressful coastal environment.

Cysteinyl aspartate specific proteinase-3 (Caspase-3) is an important protein involved in the apoptosis and gasdermin E (GSDME)-mediated cell pyroptosis pathways in vertebrates. A Caspase-3 homologue (designated as CgCaspase-3) was previously identified as an immune receptor specific for lipopolysaccharide (LPS) to regulate apoptosis in the Pacific oyster Crassostrea gigas. In the present study, the binding activity of CgCaspase-3 to different pathogen associated molecular patterns (PAMPs) and its effects on CgGSDME translocation in haemocytes were further investigated in C. gigas. The mRNA expression of CgCaspase-3 could be detected in all the tested tissues, including hepatopancreas, labial palp, adductor muscle, gonad, gill, mantle and haemocytes, and it was highly expressed in labial palp, gonad, haemocytes, and adductor muscle. The mRNA expression of CgCaspase-3 in haemocytes increased significantly at 3, 24, 48 and 72 h after LPS stimulation, and it increased significantly at 6, 12, 24 and 48 h after Vibrio splendidus stimulation. The recombinant CgCaspase-3 displayed binding activity towards LPS, mannose (MAN), peptidoglycan (PGN), and polyinosinic-polycytidylic acid potassium salt (Poly (I:C)). The positive signals of CgGSDME on haemocyte membrane became stronger at 3 h after V. splendidus stimulation, compared with that of Seawater group, and the co-localization of CgCaspase-3 and CgGSDME was observed in the haemocyte membrane. After the injection of dsCgCaspase-3, the positive signals of CgGSDME on haemocyte membrane became weaker compared with that of EGFP-RNAi group at 24 h after V. splendidus stimulation. The results suggested that CgCaspase-3 was able to bind diverse PAMPs and activate the translocation of CgGSDME in haemocytes of oyster response against pathogen invasion.

Interferon-stimulated genes (ISGs) encoding proteins are the essential executors of interferon (IFN) mediated antiviral defense. In the present study, an ISG member, interferon-induced protein 44-like (IFI44L) gene (designed as CgIFI44L-1) was identified from the Pacific oyster Crassostrea gigas. The ORF of CgIFI44L-1 cDNA was of 1437 bp encoding a polypeptide of 479 amino acids with a TLDc domain and an MMR_HSR1 domain. The mRNA transcripts of CgIFI44L-1 were detected in all the tested tissues with highest level in haemocytes, which was 15.78-fold of that in gonad (p 0.001). Among the haemocytes, the CgIFI44L-1 protein was detected to be highly expressed in granulocytes with dominant distribution in cytoplasm. The mRNA expression level of CgIFI44L-1 in haemocytes was significantly induced by poly (I:C) stimulation, and the expression level peaked at 24 h, which was 24.24-fold (p 0.0001) of that in control group. After the treatment with the recombinant protein of an oyster IFN-like protein (rCgIFNLP), the mRNA expression level of CgIFI44L-1 was significantly enhanced at 6 h, 12 h and 24 h, which was 2.67-fold (p 0.001), 5.44-fold (p 0.001) and 5.16-fold (p 0.001) of that in control group, respectively. When the expressions of CgSTAT and CgIFNLP were knocked down by RNA interference (RNAi), the mRNA transcripts of CgIFI44L-1 were significantly down-regulated after poly (I:C) stimulation, which was 0.09-fold (p 0.001) and 0.06-fold (p 0.001) of those in EGFP group, respectively. These results suggested that CgIFI44L-1 was a conserved ISG in oyster, which was regulated by CgIFNLP and CgSTAT, and involved in the oyster antiviral immune response.

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.

Clionaid boring sponges (Cliona sp.) may affect individual oyster growth and condition while destroying oyster habitats, which can impact restoration efforts and aquaculture for eastern oysters (Crassostrea virginica). Since both oysters and boring sponges are affected by similar abiotic variables (i.e., salinity, tidal exposure), this study explored the potential trade-offs between these abiotic variables and sponge presence on growth, condition index, and survival of oysters using a fully-factorial in situ experiment across 3 sites in coastal GA, USA. Across all sites and tide heights, oysters with sponge grew 20% slower and had 10% lower condition index than oysters without sponge. Savannah State, the site with the lowest salinity (mean ± SD = 24.5 ± 2.1, range 20–28), exhibited growth rates that were 1.4–1.7 times faster than the other two sites, with even sponge-infested oysters exhibiting higher growth rates than uninfested oysters at the other two sites. In addition, growth rates of sponge-infested oysters were reduced by 26% relative to uninfested individuals in the low intertidal tier, but only by 12% in the upper intertidal zone, suggesting the negative impact also varies with tidal location. Therefore, oysters exposed to air for prolonged periods and placed in areas with polyhaline salinity exhibit fast growth and high relative condition index, although salinity may be confounded by other variables (food availability, fouling organisms). This study highlights the importance of considering site and tidal location for oyster restoration and culture.

In recent decades, mortality caused by ostreid herpesvirus (OsHV-1) and a variant (OsHV-1 μvar) has severely impacted the production of Pacific oyster, Crassostrea gigas, chronologically in France, New Zealand (NZ) and Australia (Jenkins et al., 2013; Keeling et al., 2014; Segarra et al., 2010). This study was conceived based on observations made in several commercial hatcheries and research institutes in these countries where vertical transmission of OsHV-1 μvar has been absent or extremely rare. We have collated data from the production of Pacific oysters across these countries to assess the risk associated with breeding oysters from broodstock grown in OsHV-1 μvar-infected waters. The aim is to provide a reference point for regulatory authorities to use when developing or updating biosecurity management policy associated with the hatchery production of Pacific oysters.

Diseases are important drivers of population and ecosystem dynamics. This review synthesizes the effects of infectious diseases on the population dynamics of nine species of marine organisms in the Chesapeake Bay. Diseases generally caused increases in mortality and decreases in growth and reproduction. Effects of diseases on eastern oyster (Crassostrea virginica) appear to be low in the 2000s compared to effects in the 1980s–1990s. However, the effects of disease were not well monitored for most of the diseases in marine organisms of the Chesapeake Bay, and few studies considered effects on growth and reproduction. Climate change and other anthropogenic effects are expected to alter host-pathogen dynamics, with diseases of some species expected to worsen under predicted future conditions (e.g., increased temperature). Additional study of disease prevalence, drivers of disease, and effects on population dynamics could improve fisheries management and forecasting of climate change effects on marine organisms in the Chesapeake Bay.

За последние годы значительно увеличилось количество ферм по выращиванию моллюсков у берегов Крыма и Кавказа. Культивирование тихоокеанской устрицы Crassostrea gigas (Thunberg, 1793) требует проведения мониторинга здоровья моллюсков и паразитологического контроля морских ферм. Целью данной работы было изучить видовой состав эпибионтов и эндобионтов, ассоциированных с раковинами культивируемых устриц C. gigas, а также выявить виды, повреждающие раковины. Товарные устрицы с визуальными повреждениями раковины собраны на морской ферме в заливе Донузлав (Крым, Чёрное море) и доставлены для дальнейшего изучения в лабораторию в живом виде охлаждёнными. В результате обследования 22 устриц обнаружены 14 видов макрозообентоса и экземпляры живых моллюсков-камнеточцев Petricola lithophaga (Retzius, 1788). Размеры моллюсков-камнеточцев варьировали от 9 до 16 мм, возраст составлял около двух лет. Длительное нахождение P. lithophaga внутри створок устриц может вызывать деградацию известкового слоя раковины и даже гибель моллюска-хозяина; данный факт имеет большое значение для марикультуры Чёрного моря. Принимая в расчёт годовой цикл развития камнеточца-петриколы, в период массового оседания его личинок (с июля по октябрь) можно рекомендовать проводить осмотр раковин культивируемых устриц. Выполнение дальнейших детальных исследований позволит разработать мероприятия по профилактике и защите двустворчатых моллюсков от заражения камнеточцем P. lithophaga.

Ecological impacts caused by invasive alien species can be severe but may vary depending upon environmental conditions. Many European populations of the native mussel, Mytilus edulis, have been invaded by the Pacific oyster, Crassostrea (Magallana) gigas. Although widespread invasions have occurred, interactions between M. edulis and C. gigas have largely been investigated with regards to competition for space and food as well as effects on species assemblages. Experimental investigation of competitive interactions on physiological responses of the two species requires further exploration. To this end, we used a 12-month field manipulation experiment to examine growth rates, mortality and condition indices of the two species occurring in monospecific and heterospecific groups. Growth rates and mortality of both species were similar in monospecific and heterospecific groups, whereas condition indices were significantly reduced for both species in heterospecific groups. Growth rates and condition indices also differed amongst experimental sites, potentially due to differing water motion. Shell weight-length relationships did not explain the observed differences in condition for either species. We show that coexistence between the two species may occur but could be detrimental for both species. We also provide a preliminary viewpoint that water motion can mediate competitive interactions between these species.

Biofouling has been reported to be both harmful and innocuous on the growth and survival of cultured bivalves. If harmful, it could increase the operational cost substantially in an attempt to mitigate it. The study investigated the effect of sedentary biofoulers on the growth and survival of Crassostrea tulipa cultured on coconut shell and oyster shell cultches in the Densu Delta, Ghana, from December 2017 to July 2018. Physico-chemical parameters were monitored. Environmental temperature (26.4 ± 0.07 °C–29.5 ± 0.50 °C) and pH (7.2 ± 0.01–8.8 ± 0.01) were found to be within optimum levels for oyster propagation. The low levels of salinity (0.2 ± 0.01 ‰) and DO (0.6 ± 0.01 mg/l) and high turbidity (31.4 ± 2.91 NTU) could explain the observed high mortality of cultured oysters in July 2018. The observed fouling organisms were Fistubalanus pallidus (barnacle), Brachidontes sp. (mussel), Ficopomatus sp. (tube worm), Chaetomorpha antennina (green alga), and sea anemone. Oysters cultured on the undersurface of biofouled oyster shell cultches exhibited significantly faster growth than their cleaned counterparts but comparable on the upper surfaces of the cultches. The growth of cultured oysters on biofouled coconut shell cultches exhibits faster but insignificant growth than the oysters on cleaned cultches. There was no significant difference between the survival of oysters cultured on biofouled and cleaned coconut shell and oyster shell cultches. Marketable size oysters (≥ 6 cm shell height) are attainable in 7 to 8 months for both treatments. It was concluded that biofouling had no deleterious effect on the growth and survival of oysters. Besides, given the preferred shucked oysters in the Ghanaian market, it is inessential to incur further cost to control biofoulers.

Commercial off-bottom aquaculture of the eastern oyster Crassostrea virginica is challenged by repeated summer mortality events that appear to affect triploid oysters disproportionally. Many farmers believe common farming practices, especially when performed during hot summer months, may increase triploid mortality. The goal of this study was to investigate how diploid and triploid oysters react to common stressors imposed by farmers: tumbling during size grading and desiccation to prevent biofouling. Triploid and diploid oysters were deployed in floating cages at 3 farm sites along the US Gulf of Mexico coast. In May and July, oysters in 7 cages were subjected to 1 of 6 stress treatments: 1 of 3 levels of desiccation (18, 24, or 48 h) mixed with 1 of 2 levels of tumbling (tumbled or not tumbled), along with a never handled, submersed control. The mortality and growth rate of oysters were assessed in June, August, and September. Growth rates of both ploidies were affected by compounding stressors; that is, treatments with both tumbling and the longest desiccation period exhibited slower growth. Triploid oysters exhibited a greater increase in mortality in response to farm stress than diploid oysters, and mortality increased in all oysters subjected to extreme stress treatments when compared to the control. Based on this study, farmers should limit the desiccation time of oysters (particularly triploids) in the summer months to avoid any mortality or reduced growth resulting from compounding stressor effects.

The geographic boundaries of many important habitat-building shallow estuarine oyster (Family Ostreidae) species are poorly understood, especially in subtropical and tropical waters. These keystone species often have extensive historical and extant ranges, in part because of their ability to adapt to diverse environmental conditions and the transfer and introduction of a few species worldwide for aquaculture production. In addition, oysters exhibit morphological plasticity additionally confounding species identification and taxonomy. Molecular techniques have led to significant improvements in oyster systematics and taxonomy but have not been applied to oysters from many tropical regions, including the coastal areas of the Indian Ocean such as Bangladesh. Because species' identification based on morphological traits alone, in this case primarily shell characteristics, were inadequate, phylogenetic analyses of mitochondrial cytochrome oxidase subunit I gene fragments was also done to confirm the identity of oyster specimens collected from Bangladesh coastal waters. Phylogenetic analyses of collected oyster samples confirmed the two monophyletic subclades of the Ostreidae family Crassostrea and Saccostrea, and five oyster lineages from the region of Bangladesh were identified: Crassostrea gryphoides, Crassostrea belcheri, Saccostrea cuccullata lineage B, S. cuccullata lineage F, and Saccostrea mordax. A new addition to the list of Crassostrea species, C. belcheri was found in Bangladesh, but C. belcheri, C. gryphoides, and S. cuccullata are considered “common” species in the neighboring states of India and Myanmar indicating a widespread distribution of these species along the entire coast of the Bay of Bengal. However, S. mordax, is a new record for the Bay of Bengal region including the coastal waters of Andaman and Nicobar Islands, and thus extends the geographical distribution of this species.

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.

A new species for the Black Sea, a representative of the Far Eastern fauna, the Korean (dark) rockfish Sebastes schlegelii Hilgendorf, 1880, was found in the coastal zone of Crimea and the Caucasus. The first find of the Korean rockfish took place in 2013; presently, the findings of this species have become more frequent, which suggests a successful introduction. This paper presents the detailed morphological characteristics of the Black Sea individuals and the features of their biotopic distribution. Random introduction of Sebastes schlegelii with ship ballast waters or during the acclimatization of the giant oyster Crassostrea gigas is assumed to be the most probable result of the appearance of the species in the Black Sea.

The stimulator of interferon gene (STING), an intracellular sensor of cyclic dinucleotides, is critical to the innate immune response, especially the induction of type I interferon (IFN) during pathogenic infection. A STING homologue (CgSTING) regulating the expression of IFN-like protein (CgIFNLP) was previously identified in the Pacific oyster Crassostrea gigas, and its involvement in antibacterial immunity was further investigated in the present study. The mRNA transcripts of CgSTING were ubiquitously detected in all the three subpopulations of haemocytes with the highest expression in semi-granulocytes. After the stimulation with Vibrio splendidus, the mRNA expression of CgSTING in haemocytes was significantly up-regulated and peaked at 72 h, which was 12.91-fold of that in control group (p 0.01). The CgSTING protein was mainly located in the cytoplasm of haemocytes. After the expression of CgSTING was knocked down (0.12-fold of that in control group, p 0.05) by RNAi, the mRNA expression levels of interleukin17-1 (CgIL17-1), interleukin17-3 (CgIL17-3), interleukin17-4 (CgIL17-4), defensins (Cgdefh1, Cgdefh2), big defensin (CgBigDef1), interferon-like protein (CgIFNLP), tumor necrosis factor (CgTNF) and nuclear factor-κB (CgRel) all decreased significantly at 12 h after V. splendidus stimulation, which was 0.12-fold-0.72-fold (p 0.05) of that in control group, respectively. The positive signals of CgRel were observed in the haemocyte nucleus after V. splendidus stimulation. The nuclear translocation of CgRel was suppressed in CgSTING-RNAi oysters, and the green signals of CgRel were mainly observed in the haemocyte cytoplasm after V. splendidus stimulation. Furthermore, the number of V. splendidus in the haemolymph of CgSTING-RNAi oysters increased significantly, which was 26.78-fold (p 0.01) of that in the control group at 12 h after V. splendidus stimulation. These results indicated that CgSTING played important role in the immune defense against bacterial infection by inducing the expressions of cytokines and defensins.

The RAC-alpha serine/threonine-protein kinase (AKT) is one of the most important protein kinases involved in many biological processes in eukaryotes. In the present study, a novel AKT homologue named CgAKT1 was identified from the Pacific oyster Crassostrea gigas. The open reading frame (ORF) of CgAKT1 cDNA was of 1482 bp encoding a peptide with 493 amino acid residues. There were classical domains in the predicted CgAKT1 protein, including an N-terminal pleckstrin homology domain, a central catalytic domain and a C-terminal hydrophobic domain. The mRNA transcripts of CgAKT1 were detected in all the examined tissues of C. gigas with higher level in gills (8.24-fold of that in mantle, p 0.05) and haemocytes (3.62-fold of that in mantle, p 0.05). After poly (I:C) stimulation, the mRNA expression of CgAKT1 decreased significantly in haemocytes from 3 h (0.44-fold of that in the control group, p 0.001) to 24 h (0.20-fold of that in the control group, p 0.001), and then increased significantly at 48 h (3.65-fold of that in the control group, p 0.05). The expression level of CgAKT1 mRNA increased significantly at 6 h after rCgIFNLP stimulation, which was 3.60-fold of that in the control group (p 0.001). The Alexa Fluor 488 positive signals of CgAKT1 protein were found to be distributed in the cytoplasm and cell membrane of haemocytes, while those in the cytoplasm became weaker after poly (I:C) stimulation. In CgAKT1-RNAi oysters, the mRNA expression of cyclic GMP-AMP synthase (CgcGAS) and TANK-binding kinase 1 (CgTBK1) did not change significantly, but the mRNA expression level of stimulator of interferon gene (CgSTING), interferon regulatory factor-1 (CgIRF-1), interferon regulatory factor-8 (CgIRF-8) and IFN-like protein (CgIFNLP) increased significantly, which was 1.40-fold, 1.53-fold, 1.72-fold and 1.99-fold of that in EGFP-RNAi oysters (p 0.05), respectively. In CgIFNLP-RNAi oysters, the transcripts of CgAKT1 decreased significantly compared to those in EGFP-RNAi oysters (0.16-fold, p 0.01). Moreover, the expression of p-CgTBK1, CgSTING and CgIFNLP at the protein level in the oysters treated with p-AKT1 activator (SC-79) was significantly suppressed after poly (I:C) stimulation. After the transfection of CgAKT1, the expression of p-cGAS protein in HEK293T cells increased significantly, while the cyclic GMP-AMP in the cells and the interferon (IFN-β) in the cell culture fluid decreased significantly compared with that in the control group. These results indicated that CgAKT1 might play a negative role in antiviral immunity of oyster by regulating the synthesis of CgIFNLP.

Hemolymph cellular composition, morphology and functional properties of the Pacific oyster (Crassostrea gigas) hemocytes were studied. Three hemocyte types (agranulocytes, hyalinocytes and granulocytes) were described in hemolymph. The morphology of each type was characterized by light microscopy and flow cytometry. Agranular cells (agranulocytes and hyalinocytes) were the dominant type of cells in hemolymph; their number was 86.7±2.7% of total cell count. Under hypoxia the number of agranulocytes increased (37.4% for control group versus 95.3% for hypoxic probes), whereas granulocyte and hyalinocyte number decreased up to 3.9% and 0.7% in hypoxic specimens respectively. The spontaneous ROS production decreased in each hemocyte type after exposure to hypoxia. Low dissolved oxygen did not influence hemocyte proliferation and mortality level.

Invasive non-native species and global warming are two of the greatest components of global ecosystem change. The Pacific oyster, Crassostrea gigas, is the world most cultivated shellfish and was introduced throughout the Northwest European Shelf (NWES) under the premise it could not complete its life cycle. Recent warming trends have changed this and wild populations can be found as far north as Nordic Scandinavia. Under the RCP8.5 concentration pathway, we predict that the majority of NWES coastline will be within C. gigas’s thermal recruitment niche by 2100. Given the widespread occurrence of current naturalized C. gigas populations, its large larval dispersal potential and a lack of feasible management solutions, C. gigas will likely undergo a considerable range expansion this century. The time taken to reach maturity is predicted to decrease by up to 60 days, which may lead to precocious spawning events, facilitating expansion further. Crassostrea gigas can form extensive reefs completely transforming native systems. This may compromise native biodiversity, protected habitats, and commercial species. However, naturalization can also deliver a number of beneficial ecosystem goods and services to human society. Whether naturalization is deemed positive or negative will depend on biogeographic context, the perceptions of stakeholders, and the wider management priorities.

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.

By filtering large volumes of water and releasing nutrient-rich biodeposits (feces and pseudofeces), oysters can locally enhance sediment biogeochemical cycling. An active Crassostrea virginica restoration program in Mosquito Lagoon, FL (USA), was leveraged to assess the immediate (first-year) effects of restoration on sediment nutrients. Measurements included extractable and total carbon, nitrogen, and phosphorus on dead, natural, and restored reefs using a before-after-control-impact design. To investigate an observed “age-nutrient paradox” in sediment nutrient concentrations, a laboratory experiment compared feeding rates and biodeposit nutrient content between juvenile and older oysters. The field study documented a 136% increase in ammonium, 78% increase in total nitrogen, 46% increase in total phosphorus, and 75% increase in organic matter concentrations 12 months post-restoration, with extractable nutrients responding more rapidly to restoration than total nutrients. Sediment nutrient increases were positively correlated with oyster density, shell length, and reef height. Moreover, the laboratory study indicated juvenile oysters had higher rates of chlorophyll-a removal and ammonium efflux and produced biodeposits with higher concentrations of dissolved organic carbon, nitrate, and ammonium than older oysters. Overall, this study documented increases in sediment nutrients on intertidal reefs within the first year of restoration, which may be explained by a greater filtration rate and more nutrient-enriched biodeposits contributed by young oysters as compared to older oysters. Sediment total nitrogen and ammonium content may be the most robust monitoring metrics for documenting the ecosystem service of enhanced biogeochemical cycling on restored oyster reefs.

1. Bivalve habitat restoration is growing in geographic extent and scale globally. While addressing the wide-scale loss of these biogenic habitats is still a key motivation behind restoration efforts, stakeholders and funders are increasingly drawn to shellfish restoration for the many ecosystem services these habitats provide. 2. There is clear evidence for the provision of ecosystem services from species targeted for restoration in the USA, in particular Crassostrea virginica. Ecosystem services, however, remain largely unquantified or even undescribed for the majority of other species targeted for restoration. 3. A structured review of the literature was undertaken and supplemented by expert knowledge to identify which ecosystem services are documented in the following other bivalve species targeted for restoration: Ostrea edulis, Ostrea angasi, Crassostrea rhizophorae, Perna canaliculus, Modiolus modiolus, Mytilus edulis, Mytilus platensis, Crassostrea gigas, Ostrea denselamellosa, Crassostrea ariakensis, and Crassostrea sikamea. 4. Key knowledge gaps in quantifying ecosystem services and the ecosystem engineering properties of habitat-building bivalves contributing to the provision of ecosystem services were identified. Ecosystem services with the potential to be widely applicable across bivalve habitat-building species were identified. 5. Though there is evidence that many of the ecosystem engineering properties that underpin the provision of ecosystem services are universal, the degree to which services are provided will vary between locations and species. Species-specific, in situ, studies are needed in order to avoid the inappropriate transfer of the ecosystem service delivery between locations, and to further build support and understanding for these emerging targets of restoration.

In Brazil, the cultivation of Crassostrea tulipa (= C. gasar ) is gaining in importance. However, little is known about the best conditions for farming. Therefore, this study aimed to evaluate the effect of oyster density (50, 100, 200, and 400 mL of seeds/0.15 m 2 ; 100, 200, 300, and 400 ind/0.10 m 2 (basket area) and 30, 60, 120, and 180 ind/0.13 m 2 (lantern floor area) in nursey, juvenile and adult phase, respectively) on the growth and survival of C. tulipa in farm cultivation. In the nursery phase, the 50 mL density resulted in smaller oyster size, as expressed in height and height/length (H/L) ratio (1.31 ± 0.03 mm), compared to other densities, while the length and width was equal among densities. The animals' individual average weight and H/L ratio were negatively influenced by a density of 100 ind/0.10 m 2 or bigger. However, animals grown at the density of 200 ind/0.10 m 2 did not differ statistically in length or width from those grown with 100 ind/0.10 m 2 . A high survival rate (≥98%) was found in the nursery and juvenile phases.

The eastern oyster Crassostrea virginica is a keystone species in estuarine environments but faces threats to shell formation associated with warming temperatures and ocean acidification. Extrapallial fluid (EF), which is responsible for shell formation, harbors diverse and abundant microbial communities. Commensal microbial communities are vital to host health and fitness, yet long-term studies investigating temporal responses of the EF microbiome and its function in oyster fitness are lacking. In this study, bacterial communities of oyster EF and the water column were characterized monthly from October 2010 to September 2011. We investigated the selection, composition, and dynamics of resident and transient community members, evaluated the impact of temperature on EF microbial communities, and examined the functional role of the EF microbiome. Oyster EF communities were significantly different from those of the water column and were enriched for several taxa, including the Deltaproteobacteria, Epsilonproteobacteria, and Gammaproteobacteria. Overall, 94 resident members were identified in oyster EF. These members were persistent and abundant, comprising on average 33% of EF communities. Resident EF communities formed high-temperature and low-temperature groups and were more abundant overall at colder temperatures. Oyster EF resident communities were predicted to be enriched for dissimilatory nitrate reduction, nitrogen fixation, nitrification, and sulfite reductase genes. Sulfate and nitrate reduction may have a synergistic effect on calcium carbonate precipitation and indirectly aid in shell formation. Therefore, the potential role of the oyster EF microbiome in shell formation warrants further investigation as oysters and other shellfish face the future impacts of ocean warming and acidification.