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Preserving and maximizing genetic diversity in conservation programs, including for restocking, are of high importance. The threatened European flat oyster ( Ostrea edulis ) is currently the subject of several applied conservation and restocking programs, but concerns have been raised over potential negative side effects of these programs, for example due to our limited knowledge about the genetic effects in natural populations of releasing offspring of hatchery origin. Here, we developed an effective, easily applicable and highly reliable method to assess the genetic diversity and parental contributions in flat oyster hatchery production based on analyses of 17 microsatellite loci. We analysed four broodstocks and their hatchery-reared spat (total n  = 354) and compared diversity to that in wild samples of adults and spat from the broodstock source in the Limfjorden (total n  = 138). Based on four hatchery tank experiments with fully resolved parentage assignments, we found that five swarming events (larval releases) were characterized by a single maternal and multiple paternal contributions, and that the number of contributing parents varied greatly both among individual tanks, and between swarming events within tank. On average, the effective number of breeders was only one third of the actual broodstock size. Although the broodstock exhibited high genetic variation, the high reproductive skew resulted in produced offspring representing only a relatively small subset of this variation. The work demonstrates potential impact of hatchery reared offspring on decreasing genetic diversity in wild populations, but also that genetic monitoring can be integrated in conservation programs to minimize negative effects on restoration and supplementary restocking programs that utilize hatchery reared spats to support natural populations.

The European flat oyster ( Ostrea edulis L.) represents an economically important oyster production in Southern Italy, widespread in natural beds along the coast. The practice to be eaten raw is an everlasting concern for possible health risk with a need to stringently monitor the health of aquatic environment. A screening survey using histopathological examination was undertaken by harvesting O. edulis from different sites along the Apulian coast of Italy. Tissue samples of the digestive gland, kidney, gonad, and gill were provided for morphologic study in light microscopy (LM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) analysis. The LM observations revealed spherical cytoplasmic inclusions as basophilic prokaryote colonies in 13/250 oysters. The TEM and SEM confirmed the presence of intracytoplasmic inclusions of Rickettsia-like organisms (RLOs), merely in the epithelial cells of the digestive gland tubule tissues in the 13 oysters. Within intracytoplasmic vacuoles, RLOs exhibited a prokaryotic characteristic ultrastructure with transverse binary fission, a DNA zone full of chromatin fibers and a granular periplasmic ribosome zone. O. edulis were found positive for RLOs in wild oysters from Manfredonia, while the other sites were found free of pathological inclusions. Thus, we present the first report of a Rickettsia-like infection in the Apulian wild oyster ( O. edulis ) from Italy, including an ultrastructural description and pathological characterization.

This study investigates polyunsaturated fatty acids (PUFAs) of European flat oyster ( Ostrea edulis L.) and their composition shift in relation to the interaction of selected factors in the marine environment of the Adriatic Sea, over an annual farming cycle. We advance the hypothesis that fatty acid composition may be predicted regarding the changes of abiotic environment parameters. Novel approach was developed as a tool for interpretation of PUFA ratio using mathematical models and algorithms, providing association and better prediction of observed variables compared to standard statistical techniques. Data analysis using R language package resulted in models that primarily provide the possibility to predict the PUFA value based on certain fluctuations in the environment. In addition to predictions, results present a useful tool for insight into the oysters’ quality and understanding of their basic biology in different environments. This is the first report of using mathematical modeling to investigate interactions between PUFA compositions of oyster meat relative to interactions of particular environmental variables. We furthermore discuss the potential of environmental variables in affecting the oyster PUFA composition and apply an approach providing a better insight into the health status and quality of oysters throughout the season. Model projection showed a nonlinear, parabolic dependence between the tested variables and dissolved oxygen. Both, the n - 3 PUFA and DHA alone, were associated with an open-down parabolic relationship; the maximum levels were reached by 95 % oxygen saturation. The n - 3 PUFAs and n - 3/n - 6 ratio were likely negatively affected by increase in sea water temperature, where a 15 % reduction in n - 3 PUFAs content and 50 % reduction in n - 3/n - 6 proportion were predicted. Also, we detected FA ratios (AA/DHA, AA/EPA) and AA positively affected by increase in sulphate concentration and dissolved oxygen, respectively, and strongly negative prediction of ammonia level and sea water pH with AA, DHA and ARA/EPA ratio, respectively. These models might be applied to other aquatic species, for promotion of sea food quality, improvement of farming conditions and production, as well as for optimizing conditions in controlled trails. Graphical Abstract

The physiological response of two species of mussels ( Mytilus edulis and M. galloprovincialis ) and two species of oysters ( Crassostrea gigas and Ostrea edulis ) to temperature, oxygen levels and food concentration, factors likely to vary as a result of climate change, was determined experimentally. Bivalves of similar size from different origins were exposed to six temperatures (3, 8, 15, 20, 25 and 30 °C) at two food regimes (2 and 10 μg Chl a L −1 ) for 6 weeks. In a parallel running experiment M. edulis from the same batches were exposed to three different temperatures (15, 20 and 25 °C) and three different oxygen levels (30, 50 and 100%) at two food regimes (2 and >8 μg Chl a L −1 ) for 3–4 weeks. Survival during the experiment ranged from 93% to 100% except for the mussels exposed to 30 °C which showed 100% mortality after three to 32 days. Higher food conditions showed higher optimal temperatures for growth of mussels and oysters. In addition, at the high food treatment, reduced O 2 saturation resulted in lower growth of mussels. At the low food treatment there were no differences in growth among the different O 2 levels at the same temperature. At high food concentration treatment, M. edulis growth was higher with low temperature and high oxygen level. Condition index was higher at higher food concentrations and decreased with increasing temperature. In addition, condition was lower at low oxygen saturation. Lower clearance rates were observed at high food concentrations. At 100% saturation of oxygen, mussel clearance rate increased with temperature at High food regime, but not at Low food regime. Mussel clearance rates were significantly reduced with low oxygen concentrations together with high temperature. Oxygen consumption significantly increased with temperature. Oxygen saturation was the main factor affecting mussel clearance rate. High temperature and low oxygen concentration combined significantly reduced clearance rate and increased oxygen consumption. These response curves can be used to improve parameterisation of individual shellfish growth models taking into consideration factors in the context of climate change: temperature, food concentration, oxygen concentration and their interactions. The observation that abiotic factors interact in affecting mussels and oysters is an important result to take into account.

Marine organisms exhibit a multitude of biological rhythms synchronized with the interactions of the sun-, earth-, and moon cycles. However, the biological rhythms in bivalves remain poorly studied. This study focuses on the native European flat oyster ( Ostrea edulis ), an endangered species of coastal ecosystems and a key organism in restoring of biogenic reef habitats. We aim to determine whether a molecular endogenous circadian rhythm exists in O. edulis and to characterize its daily expression. To address these questions, the oysters’ valve behavior, as an output of the circadian clock expression, was recorded under different light conditions and free-running regimes using non-invasive valvometry. This work demonstrates the existence of a circadian clock mechanism that generates a labile behavioral circadian oscillation under free-running conditions. In light: dark conditions, a diel rhythm appears nocturnal, synchronizable to a shift of light phase, and remains unmodified whether the oysters are fed or not. This rhythm anticipates light: dark changes, indicating its endogenous origin. Finally, when exposed to artificial light at night the daily behavior is disrupted. This study characterizes the circadian behavioral rhythm of O. edulis ’s as plastic and labile. This plasticity would be advantageous in terms of ecological adaptability but increases sensitivity to anthropogenic pressures such as light pollution.

Reefs formed by Ostrea edulis , the European native oyster, are among many biogenic habitats that have declined globally. European oyster habitats are now rare, and undisturbed examples have not been described. As more is understood of the ecosystem services provided by the reefs, oyster restoration efforts are on the rise, becoming a more prominent component of Europe’s portfolio of marine conservation practices. It is therefore important to establish the relationship between the development of oyster reefs and their associated biotic community if the biodiversity benefits are to be accurately predicted and the progress of restoration projects assessed. The Loch Ryan oyster fishery in Southwest Scotland is the last of its type and uses a rotational harvest system where different areas are fished each year and then left for six years before they are fished again. This provided an opportunity to study the effect of oyster reef development and biodiversity gain at different stages of habitat recovery. In this study three treatments were surveyed for faunal biodiversity, oyster shell density and oyster shell percentage cover. Treatments were plots that had been harvested one year before, two years before, and six years before the study. The treatments were surveyed with SCUBA using a combination of video transects and photo quadrats. Oyster shell density, oyster shell percent cover and macrofaunal biodiversity differed significantly between treatments, with the highest values observed in the six-year treatment. Shell density was 8.5 times higher in the six-year treatment compared to the one-year treatment, whilst Shannon-Wiener’s diversity was 60.5% higher, and Margalef’s richness 68.8% higher. Shell density and percent cover had a significant positive relationship with macrofaunal biodiversity. This is probably due to the provision of increased structural complexity in the matrix of live and dead oyster shells. Projecting forward the trend of biodiversity increase in relation to time since disturbance indicates that full recovery would take approximately ten years in which time diversity (Shannon-Wiener) would probably have doubled. The findings from the present study indicate the probable biodiversity benefits of oyster habitat restoration and a cost-effective metric (shell density) to judge progress in restoration projects.

European oysters ( Ostrea edulis ) once covered large areas of the North Sea, but have disappeared due to a combination of overexploitation and the destruction of benthic habitats including hard settlement substrate. Offshore wind parks offer an opportunity for oyster restoration as fishing is banned inside these parks and scour protection provides hard settlement substrate. However, ecological restoration of marine systems is capital-intensive. The success of restoration projects is mainly determined by the choice of methods and techniques and consequently costs. Costs and cost-effectiveness information are therefore key in decision making processes concerning the selection of restoration efforts and techniques. So far, economic viability of marine ecosystem restoration have mainly focused on near-shore shallow habitats. The aim of this study was to provide insight into the most cost-effective deployment options to create a European flat oyster reef in an offshore wind farm in the North Sea. Within the current policy and legislation framework, several deployment scenarios were identified based on best practices, expert knowledge, and preliminary results of several pilots. The 9 scenarios included ‘adults placed loose on the seafloor’, ‘adults glued on granite’, ‘spat settled on shells’, ‘spat settled on granite’ and a combined ‘adult and spat’ scenario. Cost-effectiveness of the different scenarios was determined by modelling the expected reef biomass post-deployment both with and without the option to add additional settlement substrate post-deployment. The main conclusions from this exercise were that: 1. based on investment value, the scenarios ‘adult loose on the seafloor’, ‘adults in cages’ and ‘spat on shells’ had the highest revenues per Euro invested; 2. adding substrate in the years post-deployment increased cost-effectiveness in the model for all scenarios, and 3. the time post-deployment to reach a self-sustaining adult oyster population was, with 8–10 yr, shortest for the scenarios 'spat settled on shells' and the combined scenario of 'adults placed loose on the seafloor' and 'spat settled on shells'.

Surveillance and diagnosis of parasitic Bonamia ostreae infections in flat oysters (Ostrea edulis) are prerequisites for protection and management of wild populations. In addition, reliable and non-lethal detection methods are required for selection of healthy brood oysters in aquaculture productions. Here we present a non-lethal diagnostic technique based on environmental DNA (eDNA) from water samples and demonstrate applications in laboratory trials. Forty oysters originating from Limfjorden, Denmark were kept in 30 ppt sea water in individual tanks. Water was sampled 6 days later, after which all oysters were euthanized and examined for infection, applying PCR. Four oysters (10%) were found to be infected with B. ostreae in gill and mantle tissue. eDNA purified from the water surrounding these oysters contained parasite DNA. A subsequent sampling from the field encompassed 20 oysters and 15 water samples from 5 different locations. Only one oyster turned out positive and all water samples proved negative for B. ostreae eDNA. With this new method B. ostreae may be detected by only sampling water from the environment of isolated oysters or isolated oyster populations. This non-lethal diagnostic eDNA method could have potential for future surveys and oyster breeding programs aiming at producing disease-free oysters.

Summary Environmental DNA approaches are increasingly used to detect microorganisms in environmental compartments, including water. They show considerable advantages to study non‐cultivable microorganisms like Bonamia ostreae, a protozoan parasite inducing significant mortality in populations of flat oyster Ostrea edulis. Although B. ostreae development within the host has been well described, questions remain about its behaviour in the environment. As B. ostreae transmission is direct, seawater appears as an interesting target to develop early detection tools and improve our understanding of disease transmission mechanisms. In this context, we have developed an eDNA/eRNA approach allowing detecting and quantifying B. ostreae 18S rDNA/rRNA as well as monitoring its presence in seawater by real‐time PCR. B. ostreae DNA could be detected up to 4 days while RNA could be detected up to 30 days, suggesting a higher sensitivity of the eRNA‐based tool. Additionally, more than 90% of shed parasites were no longer detected after 2 days outside the oysters. By allowing B. ostreae detection in seawater, this approach would not only be useful to monitor the presence of the parasite in oyster production areas but also to evaluate the effect of changing environmental factors on parasite survival and transmission. An eDNA/eRNA approach was developed, allowing detecting and quantifying Bonamia ostreae 18S rDNA/rRNA as well as monitoring its presence in seawater by real‐time PCR. B. ostreae DNA and RNA could be detected up to 4 and 30 days respectively, suggesting a higher sensitivity of the eRNA based tool. More than 90% of shed parasites were no longer detected after 2 days outside the oysters.

Background European flat oysters ( Ostrea edulis ) are sequential hermaphrodites that alternate sex in response to environmental change. Epigenetics, including DNA methylation, are often involved in sex reversal through influencing gene transcription. Knowledge on the epigenetic mechanisms underlying sex reversal in hermaphrodite bivalves is limited to gonadal tissue and previous studies have only compared DNA methylomes of males and females. Therefore, the aim of this study is to assess whether sex-specific DNA methylation can be identified in somatic gill tissue of the flat oyster. Results By comparing whole-genome methylomes of 35 oysters of different sex phenotypes using nanopore sequencing, we demonstrate the presence of sex-specific DNA methylation patterns in somatic gill tissue. A total of 9,654 regions and 2,576 genes were differentially methylated between male, female, and hermaphrodite oysters. Functional analysis of differentially methylated genes indicated an association with energy homeostasis and metabolic processes, implying a remodeling of the energy balance. Conclusions This study is the first to characterize DNA methylomes of hermaphrodite oysters, providing new insights into the epigenetic mechanisms underlying sex reversal in a sequential hermaphrodite invertebrate. Additionally, this study characterizes sex-specific DNA methylation in somatic gill tissue, paving the way for non-lethal sex identification using epigenetic biomarkers.