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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

Aims To investigate the relationships between individual health status of oysters, particularly with regard to parasitic infection, and variability in abundance of human-pathogenic Vibrio species. Methods and Results Aquacultured eastern oysters, Crassostrea virginica, were analysed individually for infection by the protozoan parasite Perkinsus marinus through quantitative PCR, and total Vibrio vulnificus and total and pathogenic Vibrio parahaemolyticus abundance was assessed using a most probable number (MPN)-qPCR approach. Additionally, perspective on general oyster health and other parasitic infections was obtained through histopathology. Perkinsus marinus infection and human-pathogenic Vibrio species levels were not correlated, but through histology, analyses revealed that oysters infected by Haplosporidium nelsoni harboured more V. vulnificus. Conclusions The highly prevalent parasite P. marinus had little influence on human-pathogenic Vibrio species levels in eastern oysters, but the less prevalent parasite, H. nelsoni, may influence V. vulnificus levels, highlighting the potential nuances of within-oyster dynamics of Vibrio species. Significance and Impact of the Study Human-pathogenic bacteria continue to be a concern to the oyster industry and causes for individual oyster variation in bacterial levels remain unknown. The major oyster pathogen P. marinus does not appear to affect levels of these bacteria within oysters, suggesting that other factors may influence Vibrio spp. levels in oysters.

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.

Aquatic organisms that inhabit coastal areas are often exposed to several contaminants. It is known that the bioaccumulation of contaminants can be amplified according to the species feeding habits and contaminant properties. As a consequence, species can experience different effects to contaminant exposure even if they inhabit the same area. The present study aimed to investigate the activities of carbonic anhydrase (CA), Ca2+-ATPase, and Mg2+-ATPase in different tissues (soft tissue, mantle, and gill) of three mollusk species (Lottia subrugosa, Stramonita brasiliensis, and Crassostrea brasiliana) with different feeding habits (herbivore, carnivore, and filter-feeder, respectively) which were sampled within a known contamination gradient at Santos Estuarine System (Southeastern Brazil). From the three enzymes tested, only CA was affected by the presence of contaminants within the contamination gradient evaluated. In general, the CA activity from the three species were lower in contaminated sites when compared to the reference site. The contrasting CA activity response observed in S. brasiliensis compared to L. subrugosa and C. brasiliana could be related to the tissue-specificity of this enzyme activity and species feeding habits (filter-feeders can accumulate more contaminants than herbivores and even carnivores). Results indicated that C. brasiliana mantle is the most suitable tissue for the use of CA analysis as a biomarker.

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.

Growth and survival rates of juveniles of pacific oyster Crassostrea gigas are estimated for the first time in conditions of Peter the Great Bay (Japan Sea). The data were collected at artificial hatchery in the Aquaculture Center located on Popov Island in 2019. The juveniles were settled and reared on two types of substrate: 1) perforated plastic plates with the diameter of 30 cm (area 7.1 dm2), and 2) scallop shells with the height about 10 cm (average area 0.8 dm2) mounted in the western Peter the Great Bay (Voevoda Bay) in September 2019. Before placing in this site, the average height of the juveniles’ shells did not exceed 7 mm. In July 2020, after the 9-month exposure of the collectors with spat in the Voevoda Bay, the height increased in 12–18 times and reached 55–90 mm on the substrate 1 and 25–65 mm on the substrate 2. Then the oysters reared on the substrate 1 were replaced from the plastic plates to the cage shelves and the cages were moved to the Stark Strait. Difference in the growth rate became more apparent in September 2020, when the height of shells settled on the plastic plates reached 67 mm, on average (73 % in the range 50–85 mm) and for the shells settled on the scallops — 32 mm, on average (72 % in the range 20–45 mm). The shell height increased to 76.6 and 52.4 mm, respectively, to the end of October 2020. Finally, about 59 % of the oysters reared on the substrate 1 and cage shelves and 9 % of the oysters reared on the substrate 2 reached the commercial size (80–100 mm). Their survival in the period from mid-October 2019 to July 2020 is estimated as 46.9 % (28.8–98.2 %) for the substrate 1 and 33.5 % (4.0–78.3 %) for the substrate 2. On the substrate 1, survival of the mollusks attached to lower surface of the plates was twice higher. The survival had a tendency to decrease with increasing of the juveniles density. The experiment showed good viability for artificially hatched juveniles of pacific oyster in conditions of Peter the Great Bay. Their growth in the Voevoda Bay and the Stark Strait, with necessary technological measures, as replacing, thinning, and substrate cleaning from fouling, allows to produce oysters with the commercial size.

Growth and survival rates for larvae of pacific oyster Crassostrea gigas (Thunberg, 1793) hatched in artificial conditions are estimated for the stages of development from D-veliger to pediveliger. The experiment was conducted in the Mariculture Center located on Popov Island (Peter the Great Bay, Japan Sea) for 2 regimes of feeding and water salinity of 26 and 32 ppt. Microalgae Isochrysis galbana, Chaetoceros muelleri and Phaeodactylum tricornutum cultivated in the Mariculture Center were used as a feed in both regimes, in different concentrations. The maximum concentration was 4-fold higher than the minimum one: daily doses of food were 20 and 5 thousand cells/mL for veligers, 40 and 10 thousand cells/mL for early veliconkhes, and 80 and 20 thousand cells/mL for late veliconkhes, respectively, whereas 80 thousand cells/mL for all larvae in transition to pediveliger stage. Statistically significant difference of the growth rate was found for cases with different food concentration (p < 0.05). The larvae with better feeding had higher growth rate under salinity of both 26 and 32 ppt. Besides, the lowered salinity (26 ppt) had some positive effect for growth in the regime of better feeding. Survival rate of the larvae from D-veliger to pediveliger was high under all regimes of the experiment and was estimated for the minimal diet as 77.4 and 64.7 % under salinity of 26 and 32 ppt, respectively, and for the maximum diet as 81.2 and 80.7 % under salinity of 26 and 32 ppt, respectively. According to the experiment results, deficit of food at early stages of the oyster larval development affects negatively on their growth but does not have significant impact on their survival.

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.

As an emerging pollutant, the presence of microplastics in marine organisms has been concerned increasingly. Shellfish, which are both economically and ecologically important, are of particular concern. In this study, we investigated the microplastic pollution in wild and farmed oysters (Crassostrea gigas) and clams (Ruditapes philippinarum) in the Jiaozhou Bay, China, for the first time. We found the microplastic pollution in shellfish in Jiaozhou Bay was at a moderate level. The abundance of microplastics in shellfish ranged from 0.16 to 12.09 items/g (wet weight, ww) or 1 to 9 items/ind. The average abundance of the ingested microplastics was 1.21 items/g (or 2.17 items/ind.) in all shellfish, 1.51 items/g (or 2.00 items/ind.) in clams and 0.92 items/g (or 2.34 items/ind.) in oysters. The abundance of microplastics in clams was significantly higher than that in oysters. Most microplastics (92.97%) were fibers, followed by fragments. The predominant color of the microplastics was black (42.97%), followed by blue, transparent, and red. Cellophane and polyethylene terephthalate (PET) dominated the microplastic composition. According to shellfish consumption, it can be inferred that the average microplastic consumption through Chinese diet is 1.27×103 items per capita per year.

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.

Eastern oysters in the northern Gulf of Mexico are facing rapid environmental changes and can respond to this change via plasticity or evolution. Plasticity can act as an immediate buffer against environmental change, but this buffering could impact the organism's ability to evolve in subsequent generations. While plasticity and evolution are not mutually exclusive, the relative contribution and interaction between them remains unclear. In this study, we investigate the roles of plastic and evolved responses to environmental variation and Perkinsus marinus infection in Crassostrea virginica by using a common garden experiment with 80 oysters from six families outplanted at two field sites naturally differing in salinity. We use growth data, P. marinus infection intensities, 3' RNA sequencing (TagSeq) and low-coverage whole-genome sequencing to identify the effect of genotype, environment and genotype-by-environment interaction on the oyster's response to site. As one of first studies to characterize the joint effects of genotype and environment on transcriptomic and morphological profiles in a natural setting, we demonstrate that C. virginica has a highly plastic response to environment and that this response is parallel among genotypes. We also find that genes responding to genotype have distinct and opposing profiles compared to genes responding to environment with regard to expression levels, Ka/Ks ratios and nucleotide diversity. Our findings suggest that C. virginica may be able to buffer the immediate impacts of future environmental changes by altering gene expression and physiology, but the lack of genetic variation in plasticity suggests limited capacity for evolved responses.

The widely recognized color polymorphisms of molluscan shell have been appreciated for hundreds of years by collectors and scientists, while molecular mechanisms underlying shell pigmentation are still poorly understood. Tyrosinase is a key rate-limiting enzyme for the biosynthesis of melanin. Here, we performed an extensive multi-omics data mining and identified two tyrosinase genes, including tyrosinase and tyrosinase-like protein 2 (Tyr and Typ-2 respectively), in the Pacific oyster Crassostrea gigas, and investigated the expression patterns of tyrosinase during adults and embryogenesis in black and white shell color C. gigas. Tissue expression analysis showed that two tyrosinase genes were both specifically expressed in the mantle, and the expression levels of Tyr and Typ-2 in the edge mantle were significantly higher than that in the central mantle. Besides, Tyr and Typ-2 genes were black shell-specific compared with white shell oysters. In situ hybridization showed that strong signals for Tyr were detected in the inner surface of the outer fold, whereas positive signals for Typ-2 were mainly localized in the outer surface of the outer fold. In the embryos and larvae, the high expression of Tyr mRNA was detected in eyed-larvae, while Typ-2 mRNA was mainly expressed at the trochophore and early D-veliger. Furthermore, the tyrosinase activity in the edge mantle was significantly higher than that in the central mantle. These findings indicated that Tyr gene may be involved in shell pigmentation, and Typ-2 is more likely to play critical roles not only in the formation of shell prismatic layer but also in shell pigmentation. In particular, Typ-2 gene was likely to involve in the initial non-calcified shell of trochophores. The work provides valuable information for the molecular mechanism study of shell formation and pigmentation in C. gigas.

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.

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

Synopsis The presence of standing genetic variation will play a role in determining a population's capacity to adapt to environmentally relevant stressors. In the Gulf of Mexico, extreme climatic events and anthropogenic changes to local hydrology will expose productive oyster breeding grounds to stressful low salinity conditions. We identified genetic variation for performance under low salinity (due to the combined effects of low salinity and genetic load) using a single-generation selection experiment on larvae from two populations of the eastern oyster, Crassostrea virginica. We used pool-sequencing to test for allele frequency differences at 152 salinity-associated genes for larval families pre- and post-low salinity exposure. Our results have implications for how evolutionary change occurs during early life history stages at environmentally relevant salinities. Consistent with observations of high genetic load observed in oysters, we demonstrate evidence for purging of deleterious alleles at the larval stage in C. virginica. In addition, we observe increases in allele frequencies at multiple loci, suggesting that natural selection for low salinity performance at the larval stage can act as a filter for genotypes found in adult populations.

The adaptive capacity of marine calcifiers to ocean acidification (OA) is a topic of great interest to evolutionary biologists and ecologists. Previous studies have provided evidence to suggest that larval resilience to high pCO2 seawater for these species is a trait with a genetic basis and variability in natural populations. To date, however, it remains unclear how the selective effects of OA occur within the context of complex genetic interactions underpinning larval development in many of the most vulnerable taxa. Here we evaluated phenotypic and genetic changes during larval development of Pacific oysters (Crassostrea gigas) reared in ambient (~400 µatm) and high (~1600 µatm) pCO2 conditions, both in domesticated and naturalized “wild” oysters from the Pacific Northwest, USA. Using pooled DNA samples, we determined changes in allele frequencies across larval development, from early “D‐stage” larvae to metamorphosed juveniles (spat), in both groups and environments. Domesticated larvae had ~26% fewer loci with changing allele frequencies across developmental stages and

Genetic variation and phenotypic plasticity are both important to adaptive evolution. However, how they act together on particular traits remains poorly understood. Here, we integrated phenotypic, genomic, and transcriptomic data from two allopatric but closely related congeneric oyster species, Crassostrea angulata from southern/warm environments and Crassostrea gigas from northern/cold environments, to investigate the roles of genetic divergence and plasticity in thermal adaptation. Reciprocal transplantation experiments showed that both species had higher fitness in their native habitats than in nonnative environments, indicating strong adaptive divergence. The southern species evolved higher transcriptional plasticity, and the plasticity was adaptive, suggesting that increased plasticity is important for thermal adaptation to warm climates. Genome-wide comparisons between the two species revealed that genes under selection tended to respond to environmental changes and showed higher sequence divergence in noncoding regions. All genes under selection and related to energy metabolism exhibited habitat-specific expression with genes involved in ATP production and lipid catabolism highly expressed in warm/southern habitats, and genes involved in ATP consumption and lipid synthesis were highly expressed in cold/northern habitats. The gene for acyl-CoA desaturase, a key enzyme for lipid synthesis, showed strong selective sweep in the upstream noncoding region and lower transcription in the southern species. These results were further supported by the lower free fatty acid (FFA) but higher ATP content in southern species and habitat, pointing to significance of ATP/FFA trade-off. Our findings provide evidence that noncoding variation and transcriptional plasticity play important roles in shaping energy metabolism for thermal adaptation in oysters.

Synopsis Eastern oysters (Crassostrea virginica) have long been recognized as model organisms of extreme environmental tolerance, showing resilience to variation in temperature, salinity, hypoxia, and microbial pathogens. These phenotypic responses, however, show variability between geographic locations or habitats (e.g., tidal). Physiological, morphological, and genetic differences occur in populations throughout a species’ geographical range, which may have been shaped by regional abiotic and biotic variations. Few studies of C. virginica have explored the combined factors of physiological mechanisms of divergent phenotypes between locations and the genetic relationships of individuals between these locations. To characterize genetic relationships of four locations with aquacultured oysters along the North Carolina and Virginia coast, we sequenced a portion of cytochrome oxidase subunit I (COI) that revealed significant variation in haplotype distribution between locations. We then measured mitochondrial physiology and expression of the innate immunity response of hemocytes to lab acclimation and combined stress conditions to compare basal expression and stress response in oysters between these locations. For stress sensing genes, toll-like receptors had the strongest location-specific response to hypoxia and Vibrio, whereas mannose receptor and a stress-receptor were specific to hypoxia and bacteria, respectively. The expression of stress response genes also showed location-specific and stressor-specific changes in expression, particularly for big defensin and the complement gene Cq3. Our results further suggested that genetic similarity of oysters from different locations was not clearly related to physiological and molecular responses. These results are informative for understanding the range of physiological plasticity for stress responses in this commercially important oyster species. They also have implications in the oyster farming industry as well as conservation efforts to restore endangered native oyster beds.

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.

Shell color is regarded as an economic trait in many breeding programs of bivalve mollusks, but the relationship between shell color and production traits remains controversial. In our breeding program of Crassostrea gigas, solid white, black, and orange shell lines were obtained, and second-generation (F2) and testcross families with segregating shell colors were constructed by crossing these three lines. These segregating families provided ideal samples for investigating the relationship between shell color and production traits in C. gigas. The growth and survival of 7-month-old oysters with different shell colors sampled within the same families were compared in seven F2 families and 13 testcrosses. In addition, the growth and survival of oysters from the three shell color lines were compared at 4 and 16 months of age. The growth and survival rates of the orange shell line were significantly lower than those of the white and black shell lines. However, no significant difference in growth between oysters with different shell colors was observed within segregating families, except the testcrosses produced by crossing orange- and white-shelled grandparents, and no significant difference in survival was observed in any family. Overall, no significant correlation was observed between shell color and production traits in C. gigas. These results suggest that shell color cannot be used as a marker to guide the selection of growth and survival traits. Thus shell color and production traits should be selected independently in oyster breeding programs.

In this study, the toxicity of titanium nano-oxide nanoparticles (TiO2) was examined on the immunity and antioxidant system of Pacific oyster, Crassostrea gigas. The oysters were collected from the...

The large geographic distribution of the eastern oyster, Crassostrea virginica, makes it an ideal species to test how populations have adapted to latitudinal gradients in temperature. Despite inhabiting distinct thermal regimes, populations of C. virginica near the species' southern and northern geographic range show no population differences in their physiological response to temperature. In this study, we used comparative transcriptomics to understand how oysters from either end of the species' range maintain enantiostasis across three acclimation temperatures (10, 20, and 30°C). With this approach, we identified genes that were differentially expressed in response to temperature between individuals of C. virginica collected from New Brunswick, Canada and Louisiana, USA. We observed a core set of genes whose expression responded to temperature in both populations, but also an even larger set of genes with expression patterns that were unique to each population. Intriguingly, the genes with population-specific responses to temperature had elevated FST and Ka/Ks ratios compared to the genome-wide average. In contrast, genes showing only a response to temperature were found to only have elevated FST values suggesting that divergent FST may be due to selection on linked regulatory regions rather than positive selection on protein coding regions. Taken together, our results suggest that, despite coarse-scale physiological similarities, natural selection has shaped divergent gene expression responses to temperature in geographically separated populations of this broadly eurythermal marine invertebrate.

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 occurrence of polychaete borers was recorded from the alien clam Anadara kagoshimensis (Tokunaga, 1906) at Chushka Spit near Port Kavkaz in January 2020. On the basis of morphological analysis, the polychaetes were identified as Polydora websteri Hartman in Loosanoff & Engle, 1943, alien to the Azov-Black Sea basin. This species was recorded for the first time from coastal waters of Romania on carbonate rocks in 1997 and, subsequently, coastal areas of Sevastopol and Lake Donuzlav in oysters Crassostrea gigas (Thunberg, 1793) in 2009 and 2019. Polydora websteri was not previously recorded from the Kerch Strait and the Sea of Azov. This is the first report of its occurrence in Anadara kagoshimensis in the Azov-Black Sea region, which is an invasion area for both species.

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.

Oysters are widely distributed worldwide, but are mainly concentrated in tropics and subtropics. Total lipid (TL), fatty acid (FA) composition of TL and polar lipid (PoL) fractions, phospholipid (PL) class, and molecular species composition in soft tissues of Crassostrea lugubris were investigated for the first time from Vietnam. Phosphatidylglycolic acid (PGA) is the new phospholipid class first identified in marine species in general and Crassostrea lugubris in particular. Main eight classes of PL were determined in PoL fraction: diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylserine (PS), ceramide aminoethylphosphonate (CAEP), CAEP with hydroxylated FAs (CAEP‐OH), and lysophosphatidylcholine. PE and PC accounted for approximately 63% of total known PL. Polyunsaturated FAs accounted for more than 30% of TL. Ninety molecular species of glycerophospholipids, including PGA, PE, PC, PS, PI, DPG, and PG, and sphingophosphonolipids (CAEP) were identified in PoL. Alkenyl‐acyl forms of glycerophospholipids were predominated in the molecular species of PGA, PE, and PS. PGA 38:1 (p18:0/20:1), PE 40:6 (p18:0/22:6 and p18:1/22:5), PC 30:0 (14:0/16:0), PS 38:1 (p18:0/20:1), PI 40:5 (20:1/20:4), PG 32:0 (16:0/16:0), DPG 88:24 (22:6/22:6/22:6/22:6), and CAEP 34:2 (d18:2/16:0) were major molecular species., This study presented a first structural characterization and quantification of phospholipid molecular species, in general, and phosphoglycolic acid, in particular, of Vietnamese oyster. Eight molecular species of PGA were found using HPLC‐HRMS. Other types of glycerophospholipid were identified, including PE, PC, PS, PI, DPG, and PG.

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.

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

The present study aimed to evaluate the efficacy of a depuration system equipped with UV-irradiation to control Vibrio vulnificus infection such as septicemia (or sepsis) using alive oysters. After 6 h of bioaccumulation of V. vulnificus, Pacific oyster Crassostrea gigas were found to be contaminated by > 8.0 log MPN/g of V. vulnificus cells. After 60 h of depuration, the V. vulnificus cell number significantly decreased to 3.52 log and

The Delft3D Flow model was applied to simulation of the hydrodynamic regime of Voevoda Bay (the southern Primorye, Russky Island, Peter the Great Bay, Russia). The streamflow and distributed inflow from the territories adjacent to the bay was simulated using the SWAT hydrological model. The simulation results were compared with published recommendations on the technology of the off-bottom and bottom culture of the Pacific oyster (Crassostrea gigas) and yesso scallop (Mizuhopecten yessoensis) and with the distribution of benthic organisms in the bay. The lines of equal probability exceedance of optimal environment conditions for bivalves were used to identify the culture zones and boundaries: for bottom culture, in the near-bottom water layer, for off-bottom culture, in the middle water layer. Zones of bottom culture were limited by 1-m isobaths, for off-bottom culture, by 5-m isobaths. The areas optimal for marine aquaculture were outlined.

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.

This study explored the potential of using hybrid pump dryer (HPD) to utilize overproduction in aquaculture of oysters, especially during winter. HPD-dried oysters maybe used as amendments for kimchi, a traditional Korean side dish, for possible nutrient source and flavor enhancer. Oysters were subjected to different heating treatments and evaluated for proximate composition, quality characteristics, and antioxidant activities. Lower lipid and higher glycogen content were observed in HPD-dried oysters processed than the samples dried with hot air (HAD). HPD-dried oysters also exhibited lesser browning activity, better surface color, and higher antioxidant activities. Ash, protein, and water activity were slightly affected by heating treatment. VBN and TBARS were found to be higher in HAD-dried oysters, indicating faster spoilage. Applying heat pattern in drying resulted to improved quality characteristics and antioxidant activities and slower degradation of dried oyster products compared to their single-temperature-drying counterparts, especially those dried at high temperatures.

Molluscan shell color polymorphism is important in genetic breeding, while the molecular information mechanism for shell coloring is unclear. Here, high-throughput RNA sequencing was used to compare expression profiles of coding and non-coding RNAs (ncRNAs) from Pacific oyster Crassostrea gigas with orange and black shell, which were from an F2 family constructed by crossing an orange shell male with a black shell female. First, 458, 13, and 8 differentially expressed genes (DEGs), lncRNAs (DELs), and miRNAs (DEMs) were identified, respectively. Functional analysis suggested that the DEGs were significantly enriched in 9 pathways including tyrosine metabolism and oxidative phosphorylation pathways. Several genes related to melanin synthesis and biomineralization expressed higher whereas genes associated with carotenoid pigmentation or metabolism expressed lower in orange shell oyster. Then, based on the ncRNA analysis, 163 and 20 genes were targeted by 13 and 8 differentially expressed lncRNAs (DELs) and miRNAs (DEMs), severally. Potential DELs-DEMs-DEGs interactions were also examined. Seven DEMs-DEGs pairs were detected, in which tyrosinase-like protein 1 was targeted by lgi-miR-133-3p and lgi-miR-252a and cytochrome P450 was targeted by dme-miRNA-1-3p. These results revealed that melanin synthesis-related genes and miRNAs-mRNA interactions functioned on orange shell coloration, which shed light on the molecular regulation of shell coloration in marine shellfish.

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.

Macroalgae are the primary source of non-animal sulphated polysaccharides (SPs) in the marine environment with fucoidans derived from brown algae (Phaeophyta) and carrageenans from red algae (Rhodophyta). Much research has been carried out on SP effects on Asian shrimp species (genera Penaeus and Metapenaeus) but their effect on commercially important bivalve mollusc species is limited and in Pacific oyster Crassostrea gigas is unknown. Knowledge of their impact on bivalve pathogens and Palaemon shrimp is unknown. The objectives of this study were to assess the effects of Fucus vesiculosus (Phaeophyta), Mastocarpus stellatus (Rhodophyta) and algal derivatives (fucoidan and κ-carrageenan) on C. gigas performance, and on ostreid herpesvirus-1 microvar (OsHV-1 μVar) and bacteria Vibrio spp. development. Both pathogens have been associated with significant oyster mortalities and economic losses globally. The effects of sulphated galactan from Gracilaria fisheri (Rhodophyta) on European common prawn Palaemon serratus, an important fishery species, was also assessed. Findings indicate a rapid and prolonged increase in total blood cell count, lysozyme (enzyme that destroys pathogens), and a difference in the ratio of blood cell types in treated individuals compared to their control counterparts. A significantly lower OsHV-1 μVar prevalence was observed in treated oysters and κ-carrageenan was found to suppress viral replication (loads), while OsHV-1 μVar was not detected in the fucoidan treated oysters from Day 8 of the 26-day trial. No antibacterial effect was observed however, the oysters did not succumb to vibriosis. These findings contribute further knowledge to macroalgae sulphated polysaccharide biotherapeutic properties, their twofold effect on animal health and viral suppression.

Persistence of wild Pacific oyster, Magallana gigas, also known as Crassostrea gigas, has been increasingly reported across Northern European waters in recent years. While reproduction is inhibited by cold waters, recent warm summer temperature has increased the frequency of spawning events. Although correlation between the increasing abundance of Pacific oyster reefs in Northern European waters and climate change is documented, persistence of wild populations may also be influenced by external recruitment from farmed populations and other wild oyster populations, as well as on competition for resources with aquaculture sites. Our understanding of the combined impact of the spawning frequency, external recruitment, and competition on wild population persistence is limited. This study applied an age-structured model, based on ordinary differential equations, to describe an oyster population under discrete temperature-related dynamics. The impact of more frequent spawning events, external recruitment, and changes in carrying capacity on Pacific oyster density were simulated and compared under theoretical scenarios and two case studies in Southern England. Results indicate that long term persistence of wild oyster populations towards carrying capacity requires a high frequency of spawning events but that in the absence of spawning, external recruitment from farmed populations and other wild oyster populations may act to prevent extinction and increase population density. However, external recruitment sources may be in competition with the wild population so that external recruitment is associated with a reduction in wild population density. The implications of model results are discussed in the context of wild oyster population management.

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.