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Establishing the scientific bases and technical procedures and standards to recover the European flat oyster production through strategies to tackle the main constraint, bonamiosis

Final Report Summary - OYSTERECOVER (Establishing the scientific bases and technical procedures and standards to recover the European flat oyster production through strategies to tackle the main constraint, bonamiosis.)

Executive Summary:
Ostrea edulis has been part of the human diet for many centuries. As early as in the 17th century, oyster spat were collected in the field and deployed into ponds in salt marshes on the French Atlantic coast. During the 18th and 19th centuries, high mortality episodes and fishing effort led to over-exploitation, failing recruitment and destruction of European natural beds. Then, two diseases (due to the parasitic epizooties Bonamia ostreae and Martelia refringens) spread in early 1970s and 1980s, respectively, drastically reducing the production, which has remained low since that time.
The disease caused by B. ostreae has been clearly identified but it is still affecting commercial oyster populations since all the strategies to fight against the parasite, including zootechnical prophylaxis and eradication attempts, have failed. Moreover, according to a FAO report on Ostrea edulis, “The future of European flat oyster production is directly linked to the research capacity to eventually provide a strain resistant to bonamiosis by genetic mass selection”. Therefore, the main issue for the industry remains to develop a disease-tolerant strain to initiate a production rebound. This implies a better understanding of the pathogenic mechanisms concerned, as well as the development of sustainable genetic breeding programmes, based on more efficient selecting criteria, as genetic and phenotypic markers of resistance, than just survival after exposure to disease.
These were the main objectives pursued by the Oysterecover project, which after 42 months have provided very useful results.
In first place, the Oysterecover project has contributed to standarise and to validate Bonamia diagnostic methods. For the first time, the screening of heart smears, histology, PCR and In-situ hybridization (ISH) have been tested simultaneously by three different laboratories. As a result, it has been concluded that at least two methods should be used for the accurate detection and determination of prevalence of infection within a sample. These two methods should be a microscopy based method, to allow for visualisation of the parasite, and a molecular method, to increase sensitivity in low infections. Moreover, these techniques should count on fixed protocols which permit obtaining comparable results among laboratories. These findings are very useful for the zoosanitary control authorities and therefore, they will be shared with them.
As it was mentioned before, bonamiosis was discovered over 30 years ago. However, several aspects of the disease are not yet fully elucidated, such as the initial progression of B. ostreae infection within O. edulis tissue. Moreover, it is not clear whether the parasite life cycle is fully represented by direct transmission or, on the contrary, alternative hosts may be implied as well as the presence of a spore stage for B. ostrae, despite the evidences of infection in oyster beds after several years of fallow. Finally, the ability of B. ostreae to infect larvae via vertical transmission or by free-living stages in the water column has not been proved. Due to their relevance for management purposes, all these aspects have been addressed within the Oysterecover project. The results obtained have been used to compile several management recommendations for the sector, which included:
1. to extreme caution with the movement of equipment, seawater and other invertebrates (including commercial bivalve species that are not considered a host for B. ostreae) from infected areas to non-infected areas;
2. to filtrate seawater being pumped into a hatchery in order to remove additional B. ostreae and thus reducing exposure of larvae to the pathogen and
3. to use appropriate diagnostic methods to detect early or “latent’ infections prior to the movement of shellfish consignments.
Furthermore, in order to provide tools to contribute to an efficient management of both cultured and natural oyster beds, a GIS tool was developed within the project. It compiles information about the limits of the oyster beds, biomass, oyster density, bathymetry, type of bottom and Bonamia status useful for identifying potential restocking sites and for providing management advice.
The use of spat collector is a common practice among oyster growers all around the Atlantic coast. However, their efficiency was tested within the project and recommendations on which kind of device to use and when to deploy them in the field were given by the Oysterecover Consortium.
Nonetheless, the main project outcome is the identification of some genetic and phenotypical markers of resistance to bonamiosis. Although they still need to be definitely validated, they represent the first step that makes the recovery of O. edulis production a closer reality.

Project Context and Objectives:
The flat oyster Ostrea edulis, a native of Europe, has been part of the human diet for many centuries. High mortality episodes in some areas and overfishing in others decimated the populations of O. edulis in Europe through the first half of the XXth century. Then, two diseases (due to Marteilia refringens and Bonamia ostreae) spread in the early 1970s and 1980s, respectively, drastically reducing the production of O. edulis in almost all European traditional rearing areas. Despite new management practices and intensive repletion programmes, the production of O. edulis has remained low since that time.
The disease caused by the parasite Bonamia ostreae has been clearly identified but it is still affecting commercial oyster populations since all the strategies to fight against the parasite, including zootechnical prophylaxis and eradication attempts, carried out in different European countries, have failed. Before the start of Oysterecover project, three selective breeding programmes for bonamiosis resistance carried out in France, Ireland and Galicia (Spain) have produced encouraging results for the oyster industry and highlighted the possibility of growing resistant strains of flat oysters with profitable survival rates in areas affected by bonamiosis. Profitable culture and restoration of beds are subject to the availability of such strains, which should also be adapted to each particular environment in order to assure an acceptable performance.
The recovery of European flat oyster production is seen as an important opportunity for and by the shellfish industry in Europe. Thus this project aims to attain a clear competitive advantage for a number of SME Associations and Groupings, for their members and for the production areas where they are established. A total of five Shellfish Producer Associations and two SMEs in major oyster production countries in Europe (Spain, France, Ireland and the Netherlands), concerned about the above mentioned factors and being aware of recent scientific progress in selective breeding programmes for bonamiosis tolerance, decided to work together with the common general objective of facing the challenge of establishing the scientific and technical bases, procedures and standards that allow the recovery of the European flat oyster production, through the development of strategies to tackle the main constraint, bonamiosis. To successfully achieve this goal, most of the European Research Centres and Universities which have mainly contributed to last years’ scientific progress on O. edulis recovery and selective breeding programmes for bonamiosis resistance, have been hired by the SME-AGs and a group of SMEs involved in the OYSTERECOVER project to carry out the relevant research, with the following specific scientific and technical objectives:

O.1.1. To validate diagnostic methods being used by the three laboratories (UCC, CIMA, and CEFAS) involved in the ongoing use of these methods throughout the project.
O.1.2. To determine if invertebrates other than flat oysters are involved in the life cycle of Bonamia ostreae either as vectors or intermediate hosts or if direct transmission from oyster to oyster represents the full life of this pathogen.
O.2. To understand why the oyster defence cells are not able to destroy Bonamia ostreae, through the analysis of their in vitro interaction, and to look for phenotypical markers that can be used in selective breeding programmes for bonamiosis tolerance.
O.2.1. To assess if there are differences in the ability to destroy B. ostreae and, conversely, in the susceptibility to host B. ostreae proliferation, among the flat oyster haemocyte types.
O.2.2. To characterise the sequence of ultrastructural changes in haemocytes and parasite through their in vitro interaction and, specifically, to assess if lysosomes fuse with the phagocytic vacuoles where the parasite is internalised.
O.2.3. To evaluate if there are differences in the efficacy of intrahaemocytic antiparasite mechanisms (namely production of toxic reactive oxygen intermediates, nitric oxide and non specific esterase activity) among oyster stocks with different susceptibility to bonamiosis.
O.3. Genomic resources both from transcriptomic and proteomic analyses will be obtained to study gene expression profiles in response to bonamiosis challenges to identify candidate genes for tolerance. This information will be used in genetic breeding programmes and for management and conservation of genetic resources of flat oyster.
O.4. Evaluation of the gain of selective breeding programmes for oyster tolerance to bonamiosis.
O.5. Basic characterisation of natural and cultured populations of flat oyster with actual or potential commercial interest along the European coast, which is required to develop long term bed restoration programmes, avoiding loss of genetic diversity and enhancing tolerance to bonamiosis.
O.5.1. Demographic characterisation of flat oyster populations through cartography, demographic analysis, and estimation of occurrence of and susceptibility/tolerance to bonamiosis.
O.5.2. Genetic characterization of flat oyster populations with anonymous genetic markers and markers linked to candidate genes for tolerance to bonamiosis, for both management and conservation purposes, as well as for their application in genetic breeding programmes
O.5.3. Assessing the possibility of taking advantage of natural recruitment (cheap way of obtaining seed).

Additionally, consortium and IPR management, dissemination and training activities have also been defined and considered for the project work plan structure to guarantee the following related goals:
O.6. Ensure that the project results also have the desired impact by implementing an efficient dissemination plan targeting all the relevant agents for this aim.
O.7. Ensure that industrial partners have access to the expertise, training and knowledge required to utilise the results obtained from the project’s developments, incorporating them into more successful business and commercial strategies.
O.8. Ensure the project is developed by all the partners according to what has been agreed with the European Commission in terms of: means and resources dedicated, time and effort allocated, reporting, etc.
O.9. Guarantee that Intellectual Property Rights or any results obtained are managed under total and free access basis to foreground knowledge by the industrial members of the consortium and thus a short term industrial impact of project results is also guaranteed.

Project Results:
For successfully achieving these objectives, five Scientific and Technical work packages were established:
WP1-Standardization of diagnostics and Bonamia ostreae life cycle studies.
As it was mentioned before, the spread of the protozoan parasite Bonamia ostreae is of major concern to the European flat oyster industry. Therefore, to prevent the unintentional movement of infected shellfish consignments to uninfected areas, reliable and accurate diagnostic techniques are required. Currently, the diagnostic methods used for diagnosis of B. ostreae include heart smears, histology, PCR, PCR–RFLP (Restriction Fragment Length Polymorphism) and In Situ Hybridisation, with different methods being prioritised depending on the samples being screened, as recommended by the Worl Organisation for Animal Health (OIE). However, a number of authors have highlighted the relative sensitivities and limitations of each technique currently available. To validate these diagnostic methods, three separate laboratories (UCC, CIMA and CEFAS) examined four of these methods of diagnosis routinely used in laboratories - heart imprints, histology, polymerase chain reaction (PCR) and in-situ hybridisation (ISH). As a result, it was concluded that at least two methods, with fixed protocols, should be used for the accurate detection and determination of prevalence of infection within a sample, a microscopy based technique for visualisation of the parasite, the infection and associated pathology, and a molecular based technique. This combination of methods would allow for a clearer and more precise diagnosis of B. ostreae, preventing further spread of the disease and providing more accurate detection levels and epidemiological information.
Apart from an accurate detection of the infection, a deep knowledge of life cycle of the parasite is crucial for preventing the spread of the disease. This way, although transmission of B. ostreae has been proved to occur directly from oyster to oyster, secondary mechanisms of transfer, even unintentional, have not been completely ruled out and the means by which the parasite is transmitted are not yet fully understood. Suspicion persists that the disease may be spread by more means than just direct transmission between oysters. In several previous studies it was found that even after the removal of all Ostrea edulis and a fallowing period of several years, oysters reintroduced into an area developed bonamiosis, indicating that a vector/reservoir of the disease may exist. To have a complete understanding to life cycle of the parasite, two aspects were studied:
1) To try to understand better how B. ostreae persists and maintains itself in the environment, macroinvertebrates and sediment from an oyster bed infected by the parasite were collected over a one-year period and screened using polymerase chain reaction (PCR) analyses. The results of this piece of work would indicate that the occurrence of B. ostreae within other bivalves may allow for the unintentional spread of the pathogen, especially with species such as Mytilus edulis, which are frequently moved for farming purposes. Regarding the sediment study, no infection was detected in the sediment screened or in the oysters exposed to it under laboratory conditions, which may indicate that B. ostreae does not have a spore stage, it might be one of several species of haplosporidians that have abandoned production of spores, or it may imply that B. ostreae does not have a long living external life stage and thus cannot be transmitted through sediment.
2) To determine if B. ostreae can be transmitted from parent to offspring, vertical transmission from adult oysters to brooding larvae was also examined. Samples of oyster larvae were collected from O. edulis, which were sampled from three B. ostreae-endemic Irish sites over two summers. The infection status of the adult oysters was determined by heart imprints and by PCR. Larvae were washed repeatedly and screened by PCR. Certain larvae samples were confirmed to be infected with B. ostreae, however, the corresponding parents were uninfected. These results indicate that larvae may be able to acquire the pathogen from the water column during the process of filter feeding by the brooding adult or by elimination of infected pseudofaeces.
Moreover, despite being discovered over 30 years ago, the initial progression of B. ostreae infection within O. edulis tissue had not been elucidated. Oysters exposed to the parasite show no clinical sign of infection within their cells for between 4 weeks and several months Therefore, the initial progression of B. ostreae infection within O. edulis tissue was studied to determine what occurs during the “latent” period of infection and to know if the infection can be detected during this period. Oysters were challenged with the parasite in tankls and early stages of the progression of infection were examined in three different oysters stocks with varying exposure to B. ostreae (i.e. long-term, short-term and naïve). Twenty four hours after the challenge, some oysters were carrying the parasite (it was detected using in situ hybridisation on histological sections with a B. ostreae DNA probe), but both heart imprints and PCR failed to detect it. This may have major implications for the movement of stock because a stock that is confirmed negative by both methods may be experiencing the latent period of the disease. The movement of such stock may allow for further transmission of the disease. Therefore, revision of the methods used to screen supposedly uninfected stock may need to occur.
Bearing all these relevant outcomes in mind, several recommendations were given to the flat oyster industry during the accomplishment of the Oysterecover Final Workshop, held in Vigo in October 2013. They were aimed at avoiding bonamiosis spreading to new areas and included:
- Extreme caution is required with the movement of equipment, seawater and other invertebrates (including commercial bivalve species that are not considered a host for B. ostreae) from infected areas to non-infected areas.
- When introduction of oysters into Bonamia-free areas is needed for repopulation purposes, either to support fishery or just for the benefit of the ecosystem, the condition of Bonamia-free of the oysters to be introduced is not enough. The oysters to be introduced should derive from a "genetically compatible" area, in order to guarantee good adaptation and to preserve the local adaptation of the oyster populations, such as it has been demonstrated to occur in task 5.2 of this project (see deliverable D.16).
- The filtration of seawater being pumped into a hatchery should be carried out to remove additional B. ostreae thus reducing exposure of larvae to the pathogen.
- Appropriate diagnostic methods should be selected for the detection of early or “latent’ infections prior to the movement of shellfish consignments. PCR diagnostic must be based on the mix of various organs, including gills, digestive gland, gonad and mantle

WP2-Interaction between the parasite and the host haemocytes.
Bivalve molluscs, oysters among them, have an immune system to face pathogenic organisms. Haemocytes and extracellular haemolymph molecules contribute to control and prevent pathogen proliferation. However, some pathogens are able to overcome the immune system giving rise to disease. This is the case of the agents of bonamiosis, Bonamia ostreae, in the flat oyster. One of the main immune responses of molluscs involves phagocytosis of pathogens by haemocytes, by which pathogens are internalised and then degraded within the haemocytes. B. ostreae is actively phagocytosed by flat oyster haemocytes but they fail to destroy the parasite, which multiplies within the haemocytes causing haemocyte disruption. One objective within this WP was to assess if there are differences in the ability to destroy B. ostreae and, conversely, in the susceptibility to host B. ostreae proliferation, among the flat oyster haemocyte types. The other objective of this WP was the detection of differences in susceptibility to bonamiosis between oyster stocks associated with differences in the intrahaemocytic antiparasite mechanisms. If so, these particular antiparasite mechanisms involved could be the base for phenotypical markers for tolerance to bonamiosis.
The planned experimental design to achieve the first objective had to be changed and the protein expression profile of the two oyster haemocyte types, granulocytes and hyalinocytes, was compared before and after challenge with immune response inducers. To achieve the second objective, several laboratory experiments were conducted using different oysters stocks with varying exposure to B. ostreae (i.e. long-term, short-term and naïve). Different immune parameters were tested such as relative numbers of the two types of haemocytes (granulocytes and hialinocytes), nitric oxide production, lisozyme activity, phagocytosis ability and percentage of live cells in the haemolymph.
The results obtained raise expectations on the possibility of using some of these immune parameters as markers of tolerance or resistance to bonamiosis, namely ratio granulocyte/hyalinocyte or granulocyte abundance, production of nitric oxide and lysozyme activity. Nevertheless, results are not conclusive and further research is needed to confirm or reject the hypotheses. If some of these immune –related parameters were confirmed as marker of tolerance or resistance, then it could be used in selective breeding programmes as criteria to select brood-stock. Estimation of any of these parameters is based on the examination of haemolymph samples; bleeding an oyster has to be traumatic for the oyster but recovery is possible. These presumptive markers could be used to select favourable oyster stocks or lineages, which would not require using the haemolymph-donor oysters as breeders; however, their use to select individual oysters would be limited because the oyster should survive and be functional after bleeding, which is possible if performed most carefully. Estimation of any of these parameters would require some lab-equipment and training.

WP3-Genetic bases for flat oyster tolerance to bonamiosis.
Breeding programmes is likely one of the most appealing approaches to address the problems to flat oyster production posed by Bonamia ostreae. Provided a significant heritability for tolerance to this parasite, selected broodstocks would be more resistant and would transfer this tolerance to the offspring depending on the additive component of the trait. Previous results have demonstrated an increasing tolerance to the parasite in mass selection programmes carried out by different European groups or companies. These selective breeding programmes for tolerance to bonamiosis carried out until now have yielded promising results in the form of oysters with increasing tolerance to the disease and a concomitant increase in survival rate. However, the physiological and genetic bases of the increased tolerance remain unknown, which has prevented the use of refined criteria to design selective breeding programmes and to optimise their efficiency. Therefore, this WP was established, with the objective of studying the genetic bases for flat oyster tolerance to bonamiosis, which could provide the needed knowledge to start Marker Assisted Selective (MAS) breeding programmes.
The most efficient way to develop a breeding programme is to exploit both intra- and inter-family genetic variation, but always accommodating the recommended procedures to usual way of production and the characteristics of the sector. For this, a broodstock with a broad genetic variability and low inbreeding should be founded as starting point considering the available genetic resources and the existence of local environmental conditions that may lead to local adaptation. Second, considering the way of reproduction of flat oyster, in which each spawning event (larvae collected in net screens) usually involves one or two females fecundated by an uncertain number of males, a tool to trace back genealogies is essential to identify the families (full or half-sib families). Third, the existence of genes or genomic regions associated with the tolerance or the response to the parasite identified through gene expression or maker-association analysis would facilitate the development of an efficient marker assistant selection (MAS) programme. This way, first of all, and taking into account that information about the O. edulis genome is very limited, an EST database was constructed for the first time for this species. From this database, the first oligo microarray in O. edulis was designed and validated. A set of genes and proteins relevant for bonamiosis tolerance has been identified and genetic markers associated with these genes will be usable for population genetic analysis and genetic breeding programmes.

WP4-Evaluation of the gain of the selective breeding programmes for bonamiosis tolerance.
The main focus of this component of the work was to examine whether susceptibility to infection with Bonamia was derived from local conditions, from local adaptation or through some other mechanism. In order to study this, plans were drawn up to transplant hatchery reared spat into a Bonamia positive area from three main sources. These were generally a susceptible stock, a tolerant stock, and a naive stock that had not previously been exposed to Bonamia. This approach was successfully demonstrated in Spain, France and the Netherlands through the deployment of hatchery reared spat into the field followed by monitoring of growth rates and prevalence and intensity of Bonamia. However, in Ireland, a different approach through lab experiments was taken, due to the problems arisen with the production of seed in hatcheries and fishermen concerns to deploy spat into the field.
After almost two years of monitoring, some interesting results were obtained in the different countries involved in the study. It is apparent from all of these studies that whilst there may be some evidence of tolerance, the best performing stock appears to be the locally derived ones which appear to be adapted to local conditions. Furthermore, any policies derived from the work regarding restoration and management / exploitation strategies will benefit from this work as it will allow decisions for movement of oysters to be based on whether or not the animals are deemed resistant due to an inherent genetic basis or due to prevailing conditions. This data will be useful to farmers and regulators to ensure best practices are adhered to, maximising the genetic integrity of the oysters.
From a local point of view, particular recommendations were shared with the flat oyster industry during the Oysterecover Final workshop held in Vigo in October 2013. They can be consulted in the meeting recordings available through the project website at https://web.archive.org/web/20130715212555/http://www.oysterecover.eu/ as previously also indicated.
WP5-Basic characterization of natural beds with an actual or potential commercial interest envisaged to evaluate the usefulness of restoration.
The basic characterisation of natural and cultured flat oyster beds with an actual or potential commercial interest provides criteria and information to allow restoration of suitable beds with the aim of keeping a balance between genetic diversity and tolerance to bonamiosis. Different types of study sites were selected: natural beds in the intertidal and the subtidal zones (Spain), fished and undisturbed natural beds (Denmark), fished beds in France, cultured beds and natural beds (Netherlands, France, UK and Ireland). In total 17 populations were studied. They were regularly monitored to provide valuable information on the demography, health status, tolerance/susceptibility to bonamiosis and genetic structure of the oyster beds with actual or potential commercial interest. For this, stock size, size structure and deducted information such as mortality, growth and recruitment were recorded, apart from genetic data.
The information obtained was gathered in a GIS tool, which represents a powerful way to store, manipulate, analyze, manage and present all the gathered data that can be transferred to and used by oyster farmers and regulators. Indeed, some partners have expressed their intention to maintain the tool even after the end of the project in order to allow the identification of potential restocking sites and provide management advice.
Furthermore, as a result of the information obtained within the project and a literature review, some management recommendations for the sector have been established. This way, some previous studies into the dynamics of oyster recruitment revealed that in some locations a clear positive correlation was detected between the presence of adults and spat as well as dead oysters and spat. This observation suggests that the use of sanctuary (protected) areas, where broodstock will provide recruitment over a larger area, should be considered for appropriate sites. In addition, recruitment could be improved by relaying cultch on the oyster beds. In exploited populations an effective management measure to ensure that larger (and therefore older and presumably more Bonamia resistant oysters) will remain as brood stock could be the implementation of Maximum Landing Sizes (i.e. oysters bigger than 120 mm, for instance, could not be fished). Environmental conditions recorded in Oysterecover did not exceed the physiological tolerance range of this species. Physiological requirements of Ostrea edulis and Bonamia ostreae are so close that there are not too many options to develop management strategies regarding environmental conditions.
The genetic study revealed a clear genetic structure of the flat oyster in the studied area. Each country supports at least one genetically differentiated population. Currents are probably much more important than geographic distance to create genetic structure in the flat oyster. There is an obvious large scale geographic structure to the genetic variability with three regions in the studied area. These regions are made up of the Danish and Dutch samples forming an eastern group isolated from other groups by currents in the North Sea and the English Channel. There is a north-western group made up by samples from England, Ireland and France and a third group comprising the Spanish samples. This indicates that previous movements of flat oyster have had no detectable genetic effect in the regions they were translocated to. The effective population sizes seem not to indicate that any population or region is being threatened. The presence of both local and regional genetic structure shows the potential for local adaptation in flat oysters and stresses the need for caution when transplanting oysters (especially from distant geographical regions).
Identification and development of molecular markers associated to differentially expressed genes of resistance to Bonamia are necessary for their implementation in genetic breeding programs. A total of 329 differentially expressed genes between tolerant to bonamiosis and naïve Ostrea edulis strains were detected. A set of 31 markers associated with differently expressed genes (tolerant and naïve) were developed and two makers showed strong signals of adaptive divergence. These markers should be further studied and could be useful for selection programs. Moreover, a large list of candidate genes that might be important for disease selection is available to be exploited in the future.
Another objective of this WP was to assess the possibility of taking advantage of natural recruitment. European oysters are fished from wild beds (Denmark and Ireland) or cultured on bottom plots (The Netherlands and Ireland), cultured in bags on tressels (France and the UK) and cultured on ropes (Spain and France). The oyster fishery in Denmark and Ireland depends on recruitment on to naturally available substrate. In the Netherlands and in some managed beds in Ireland, oyster spat is collected onto empty mussel shells that are sewed onto plots bottom in June – July mostly in densities of 30-60m3/ha. The mussel shells degrade over time while the oyster spat remains on the plot bottom. With the decline of the mussel culture in the Netherlands the price of empty shells has increased. In addition, fouling of collectors by Pacific oyster spat presents a problem. In Ireland, the availability of mussel shell is also an issue. In France, artificial substrate is used to collect spat, or hatchery produced spat is used. When artificial substrate is used various methods to remove the spat from the collectors are available. Oyster spat are directly relaid on-bottom in subtidal grounds at a 50kg-100 kg/ha density, 5 to 10 times less than for cupped oysters (Brittany and Normandy) or on ropes (Medierrranean). In the UK, seed is from a combination of all three sources (fisheries, collectors and hatchery) generally, depending on the area being farmed.
At the present, not all countries involved in this project use spat collectors commercially (Denmark). In addition, there is a need for cheaper collectors in other countries (Netherlands and Ireland). The Oysterecover project also aimed to assess the possibility of taking advantage of natural recruitment in those countries. The use of collectors may present a cheap way of obtaining seed compared to and complementing land based hatcheries’. By reviewing established methods that are in the market and testing the most promising ones in the field, better knowledge on the most appropriate tool and the best way to handle them in each place becomes available. So firstly, a review of the efficiency of different spat collectors was conducted through a farmer questionnaire and a literature search. From the review three spat collectors were selected for further testing in the field. These were mussel stockings, calcified Chinese hats and Vexar mesh. These collectors were tested in the Netherlands, France and Denmark and the results showed that mussel stockings and calcified Chinese hats were successful in collecting oyster spat, while the Vexar mesh proved to be unsuccessful. Timing the deployment of the collectors according to an increasing oyster larvae presence in the water is recommended to minimise the effects of fouling. As a recommendation, it can be said that Chinese hats can best be used when grow-out is in bags and mussel shells in stockings when grow-out is on bottom plots.
Summing up, the Oysterecover project has provided very useful information not only to start Marker Assisted Selection (MAS) breeding programmes but also for a proper management of natural oyster beds.

Potential Impact:
The recovery of European flat oyster production is seen as an important opportunity for the shellfish industry in Europe by producers. More clear advantages foreseen by the involved SME AGs and their members and SME partners are related to:

1st- There’s a chance to diversify production and to increase efficiency in managing production areas (even opening opportunities for the recovery of natural beds). Progress in this field will be contributing to the good environmental status of production areas and to the fixation of population in such areas, where there are normally few alternatives for employment. Thus, the recovery of natural production sites is expected to yield important ecosystem services to production areas.

2nd- The chance to increase business profitability may happen due to the high market value of the product. Traditionally European flat oyster has had a market price significantly higher than that for Pacific cupped oyster. Nowadays the difference has narrowed because of the dramatic loss in production of C. gigas, however this has not fostered the drop down of prices for O. edulis.

3rd- There is an increasing acknowledgement of the environmental positive effects of fostering aquaculture activities based on native species. Moreover, flat oyster is increasingly catching the attention of conservation agencies worldwide, and particularly in Europe, because of its filtering capacity and due also to its capacity to form natural riffs.

4rd- Biotechnology provides tools and procedures to face the oyster industry problems that were not available until recently. Selective breeding programmes assisted with genetic markers (MAS) have proved to yield various advantages for producers such as a significant reduction of costs and a fast market impact compared to traditional selection. It is expected that within about five years, from some of the production areas in the project, if action is taken to develop selective breeding strategies, the recovery of production based on bonamia- resistant oysters is started.

These four key advantages summarise the major impact elements for this project at European level. This can be confirmed according to the technical reports being submitted, according also to the description of the project results and to the consultation about project results’ expected implementation made to Oysterecover industrial partner’s, having answers been provided as part of Deliverable 18.

Flat oyster production in Europe

The issues facing the players involved in the cultivation, harvesting, packing, transportation and other activities associated with the European Oyster are stark. The industry has seen a sector valued at around 150 million Euros in today’s terms – in 1962 – shrink to one of around 21 million Euros in 2011. The single most important factor in this decline has been the rise of the Bonamia parasite which has spread to most of the traditional fisheries throughout Europe since the mid-1980’s. Production volumes in Europe in 2011 was only slightly higher than 2,3 thousand tonnes while in the 60’s it had reached a peak of 30 thousand tonnes, and in the 80’s it was approximately 10 thousand tonnes per year. Much of the value has been taken up, since the 1970’s, with the Pacific ‘cupped’ oyster (Crassostrea gigas) with a value in 2011 (Europe-wide) of around 400 Million Euros (according to FAO FIGIS, fig. 1).
However there are problems building for production of C. gigas in Europe with summer mortalities increasingly prevalent throughout the region. C. gigas mortalities in both juvenile and market-sized oysters have doubled the price for this product in the market. Production of oysters around the world is dominated by this single species and with the ease with which it is possible to transfer eyed larvae and the simultaneous appearance of disease at the opposite ends of the earth, it is possible to envisage further catastrophic decline in production. This should be considered together with pressure from environmental bodies in some member states, which are minded to move C. gigas to the status of an invasive or alien species. The reliance, then on C. gigas to maintain the income of the oyster growing and harvesting industry is, thus, increasingly in jeopardy. There are concerns also that a major mollusc species such as the European Oyster has been almost rendered extinct from beds, which were abundant centuries ago. In the UK, steps have been taken by the environmental agencies. and a search again for a more reliable oyster – in which case a native, Ostrea edulis, if reared resistant to Bonamia, might be extremely useful.

The European oyster sector has a long history of supporting large numbers of family businesses in France, Spain, Ireland, UK and, increasingly, in states like Denmark and the Netherlands. Many of these areas where family business of on-growers developed are economically fragile, in some cases with poor transport links and few employment alternatives. Often the activities of harvesting or cultivation are a vital part of the economic sustainability of the surrounding communities – with linked jobs in depuration, packaging, transport, and associated services. Moreover, in recent years fishing and traditional gastronomy has revealed as interesting factors of attractiveness for tourism, and many of the production areas are making successful efforts to benefit from this (Spain, Denmark, Ireland, Uk (mainly Scotland) France…). Thus, recovering the production of this specie could even represent an indirect opportunity for the improvement of the welfare of these coastal communities.

Oysterecover consortium has concentrated its efforts in building the basis for the recovery of the production of this oyster specie by taking advantage of the available background and of last years’ technology progress regarding molecular biology. “The future of European flat oyster production is directly linked to the research capacity to eventually provide a strain resistant to bonamiosis by genetic mass selection”. OYSTERECOVER project partners agree with this sentence of a FAO report on Ostrea edulis and with this project have demonstrated that significant progress towards recovering such production is possible and now nearer.

Productivity and Sites

O. edulis is widely spread along European coast; it once formed extensive beds, which are nowadays considerably exhausted. This trend is clearly reflected in data on landings of flat oysters in Europe, being in decline since early 1980s and currently keeping in very low rates. One of the main factors for explaining this decline is the introduction of Bonamia and its spread through many oyster-growing areas.
As a cultivated species, flat oyster production in Europe is currently mainly from Spain and France, although data should be taken with care regarding to the uncertainty about to what extent cultivation has been distinguished from landings from wild fisheries where the beds are actively managed.
In terms of individual countries, the following points are worth noting:

In Spain, flat oysters are cultivated mainly in Galicia (NW) using rafts, each of which can support 200.000-300.000 oysters. Even though historically, stocks of O.edulis in Spain were first seen to be decline as early as 1778, it is only in 1960 when its exploitation was no longer profitable due to the lack of the resource. Besides this, attempts to regenerate the resource caused the introduction of Bonamia through oyster imports. Moreover, a programme to develop Bonamia ostreae tolerant strain has been led by CIMA with encouraging results for the last 8 years. If the current difficulties for flat oyster farming were overcome, the economic potential of this species could be considered as a re-emerging business, complementary to the prosperous mussel market. In fact in Galicia there are currently available (in force) nearly 150 production licences for sea rafts and only a few of them are being exploited. If it production was recovered, only with the exploitation of the currently installed capacity, it is estimated that about 150 jobs could be generated and a total increase in turnover of about 15M€/year could be reached. The socio-economic impact in the specific production areas within Galicia (mainly the southern Rías) would be according to the above, remarkable. It has to be highlighted that in most of the markets O. edulis us a highly appreciated product with higher prices than for C. gigas. The production values of the two species in Spain, clearly show this assert (fig. 2).
France has traditionally been the world leader in flat oyster production until the decline caused by diseases in the 1970s. Since then the big demand for oyster has been covered with the production of cupped oysters. Several studies on population genetics at the European scale to understand the genetic structure of this species in its range have been led by French research institutions. Most European flat oyster culture remains based upon the use of spat collectors to obtain wild juveniles in Southern Brittany. Two main types of production techniques are then used to produce flat oysters (off-bottom and on-bottom) mainly in Northern Brittany. However hatchery spat is occasionally cultured, and on-growing activities in nurseries have been developed, which guarantees a rapid transfer of the project results to the French industry .In case of solving the current constraints for flat oyster production, this species would have the opportunity to recover the privileged leading status of French shellfish production. French partners in the project consider that confirming the chance to start an MAS selective breeding programme would definitely contribute to making money. In France, the recapture rate is around 10% to 15% after 3 years of production (sale 10 to 15 oysters at commercial size for 100 disseminated spats on concession). This low rate is generated by high Bonamia mortality. At 15% and more of recapture rate, producing flat oyster is economically interesting for industry. It means an increase of 10% to 20% of survey of flat oyster (obtained with selective breeding or natural resistance identification of population) would be enough to guarantee the interest by the industry.

In Ireland, there is an annual production from the flat oyster beds about 350 tonnes and there is a strong interest in developing flat oyster fisheries in disease free areas by utilising measures to manage the stock. Since the parasite was initially diagnosed, a selective breeding programme for resistance is ongoing and led by one of the partner SMEs in the Oysterecover project, Atlantic Shellfish with a reasonable success. This would allow the availability of an interesting broodstock for selective breeding based genetic markers to accelerate the recovery of production.

Throughout much of the United Kingdom, the native oyster is in a severely depleted state in the wild, having suffered for two centuries with over-exploitation, pests, disease, pollution and harsh winters. It is a Biodiversity Action Plan Species as oyster beds can form a flourishing part of the ecosystem. Managed fisheries supply oysters for both direct consumption and, at a small scale of operation, part grown stock for ongrowing over one season in more sheltered and productive areas. Various proposals have been made for restoration but these are hampered by Bonamia disease, which affects most wild stocks.

In The Netherlands, oyster stocks are located in an estuary on two sides, Yerseke Bank and Grevelingen. Both places are affected by bonamiosis since 1980s. The recorded landings before the epidemic outbreak were around 1.200 tonnes on annual average, and they fell by 50% as a consequence of the disease. Eradication programmes developed in that decade were not successful.

In Denmark, the Danish oyster fishery in the Limfjorden is based on natural recruitment. Historically the supply of new oyster spat has been variable due to climatic conditions. The result of the natural fluctuation means that there are years with low or no oyster catch. In order to compensate for this fluctuation in natural recruitment shellfish hatcheries has been attempting to develop methods for artificial propagation of the species (Ostrea edulis). The hatchery at Danish Shellfish Center has been working with this subject since 1999. The Limfjorden is now declared Bonamia free area and the total catch of natural oysters from the Limfjorden in the last years have been around 1000-1500 tonnes pr. year.

However, in all the production areas, one of the major constraints faced by this industry to accelerate from now on the recovery of production relates to implementing reliable protocols for hatchery production. Oysterecover has accomplished one workshop with representatives of another EU project oriented to yield progress on this part of oyster production, SETTLE. The experience of contact between the two projects, OYSTERECOVER and SETTLE, was very satisfactory and now producers in different European Areas have more clear in mind from where to start if further steps for the recovery of O. edulis production are given.

Project results

Oysterecover results are expected to directly impact on the European shellfish sector through the exploitation of the main project results, which have been designed and packed in order to let the industry clearly identify what they consist in, what they can be used or useful for and to what extent they are available. Attached is provided a summary of such results in pdf (Project Results).
In D.18. Oysterecover industrial partners have provided their views about the applicability of these results and their potential specific impact.

Additional remarks about socioeconomic impact

The socioeconomic impact of the Oysterecover project is however hampered by the fact that in most of the production areas, industry is yet a very fragmented and week sector (in terms of investment capacity). As said before, most of the production is hold by family business. Oysterecover provides probably the most relevant progress up to know to recover this production, however there’s yet an important effort to be made in each of the production areas to bring the whole benefit of this progress to market value. Very few companies in this sector are in a position to start alone a MAS breeding programme, however now, given the availability of this successful background owned by the industry, with a strategic mid-term collaborative effort, again involving all the actors: the industry, the research bodies and also the local authorities, there’s a very important chance to speed up the process to return this activity to a path of competitiveness and profitable business.

In order to guarantee that such results reach the relevant end users in each of the relevant production areas, during the project accomplishment it has been implemented an effective plan for the Use and dissemination of knowledge (See section 4.2).
It has to be noticed that in Denmark, oyster fishermen have already defined and started a national programme using the recommendations given by Oysterecover for the use of natural spat collectors. In Spain, the group of partners from this country involved in the project together with the Coordinator, have already accomplished a number of meetings and contacts with the Galician Administration in order to plan future actions to build, with the involvement of the relevant actors, on ther results of Oysterecover and also on Settle project outcomes (as this Region had also a relevant involvement in this project).

List of Websites:
The project website was made accessible at https://web.archive.org/web/20130715212555/http://www.oysterecover.eu/ since the early start of the project. It has been designed as one of the principal communication elements to provide information about the project to the general audience and, at the same time, to work as a useful instrument for the oyster sector in particular. One of the pillars of the effective use of the results gained in the Project is dissemination both within the Consortium, to SME members of the SME AGs and wider to the industry and potential customers and collaborators, including the academic community.
The website is still available not only in English but also in Spanish and French (some sections), in order to make it more accessible to oyster farmers and harvesters from Spain and France, two of the world’s largest flat oyster producing countries. Despite the involvement of Dutch and Danish partners in the project, the website has not been translated to their local languages due to the general good command of English in the Netherlands and Denmark, and particularly of those with potential interest in the project and its results. The website will be kept available at least during one year after the end of the project accomplishment (after that, and to avoid the domain maintenance costs, contents will be redirected to other address owned by the Coordinator partner, so that all the information will be still available for any interested party).
To provide some evidence of the good functioning of the site, it has to be noticed that visitis have constantly grown from the website launch and it is highly remarkable that it has been during the second half of the project implementation period, when visits and downloads of information have increased more dramatically (corresponding with the periods when other dissemination actions were taking place and when more relevant information about results, news and project activities where uploaded). In the attached pdf, some illustrative statistics on website visits are included (fig. 3-5).