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Bio-engineered micro Encapsulation of Active agents Delivered to Shellfish

Final Report Summary - BEADS (Bio-engineered micro Encapsulation of Active agents Delivered to Shellfish)

Executive Summary:
During the past three decades, European shellfish aquaculture has seen a continuous expansion while providing employment to communities often located in remote coastal areas. Although shellfish contribute to the improvement of water quality by filter-feeding microscopic particles present in the water, they can accumulate faecal material from human or animal sources as well as potent algal toxins produced by harmful algal blooms. A ban on shellfish harvesting in areas affected by those microbiological (bacterial and viral) and toxic events can adversely impact the financial viability of the affected shellfish farm particularly when closures are prolonged and recurring.
To mitigate the negative impact caused to shellfish by those contamination events and also to tackle a parasitic disease (bonamiasis) specifically affecting the native oyster Ostrea edulis, the BEADS project (Bio-engineered micro Encapsulation of Active agents Delivered to Shellfish) was specifically set up to address these challenging issues by seeking to boost shellfish immune response and improve purification efficiency by means of active agents delivered directly to the bivalves through microencapsulation.
The project demonstrated that digestible alginate microcapsules fed to various shellfish species were effectively ingested, and it also found the ingestion rate was linked to the size of the microcapsules, the smaller ones being ingested at a faster rate. Those experiments demonstrated the microcapsule technology was an ingenious tool to introduce biological agents such as bacteria into shellfish.
A large number of bacteria (more than 500) were isolated during the course of this project. Some bacteria were isolated from shellfish contaminated with okadaic acid (OA) and domoic acid (DA) and a number of isolates (32) were selected after in vitro toxin degradation experiments shown increased bacterial growth (at least 40%) in the presence of OA and DA over 72 hours. Those 32 isolates were characterised using molecular sequencing and the results revealed a wide variety of bacteria species.
Further toxin utilisation experiments carried out at 12ºC and 20ºC and monitored by liquid chromatography-tandem mass spectrometry (LC-MS/MS) led to the selection of 4 bacterial isolates with promising in-vitro toxin degradation ability (toxin degradation higher than 20%). These bacteria were microencapsulated and were used in laboratory feeding trials. The results of the trials carried out on king scallops and blue mussels at 12ºC did not overall improve the detoxification rate in the tested shellfish over a 7 days period. These experiments have highlighted the necessity of finding fast acting detoxifying bacteria to limit the confinement of shellfish in holding tanks to avoid stress related shellfish losses.
A large number of bacteria were also screened for antimicrobial activity against a selection of pathogenic bacteria and viruses. One bacterial isolate (lactic acid bacterium) which was identified after DNA sequencing showed antilisterial activity against Listeria monocytogenes CECT 935 and several strains of Listeria monocytogenes isolated from seafood products. The active substance (E. hirae 3M21) was partially characterised and shown to be proteic with a molecular weight of 13.5 kDa. Survival rates of free and encapsulated E. hirae 3M21 were subsequently assessed in seawater at 15ºC and in digestive gland fluids from mussels and oysters. Results showed an absence of viability loss in any of the cases studies. Survival of encapsulated 3M21 in saline buffer was 99% after 70 days in refrigerated storage (4ºC) without any observed loss of antilisterial activity. Finally, the antilisterial isolate 3M21 was microencapsulated and fed to shellfish. Results showed higher bacterial counts (p>0.05) detected in the shellfish digestive glands while antilisterial activity was also confirmed thus validating the utilisation of the microencapsulation technique.
Feeding experiments assessing the ability of Ostrea edulis to ingest alginate microcapsules containing some fluorescent microbeads revealed the oyster’s blood cells had actively taken up the microbeads. Screening also revealed the presence of fluorescent microbeads within the digestive and connective tissue areas known to be infected by Bonamia ostreae, thus validating the use of microencapsulation as a potential tool for the transport of immunostimulants directly to the infected areas. An experiment involving the microencapsulation of candidate immunostimulants was carried out to determine if those active compounds could stimulate the native oysters’ immune system. It was found that a particular dose of each immunostimulants evoked a response in both cellular and non-cellular parts of the blood of the oysters. In a second set of trials using two additional immunostimulants, it was found the onset of oysters’ mortalities had been reduced as well as the impact of the disease. In addition, there was a reduction in the prevalence of infection during the trial not related to mortalities indicating that the infection had been eliminated. The blood cells of the O. edulis had been stimulated resulting in better ability to fight the Bonamia infection.

Project Context and Objectives:
WP1 - Investigate use of detoxifying bacteria delivered by micro-encapsulation to sites of toxins in

1) To characterise and identify toxin degrading bacteria
2) To determine properties which may significantly influence degradation of algal toxins by bacteria
3) To develop a ‘probiotic’ diet of toxin degrading bacteria and microencapsulation delivery system for shellfish consumption with targeted release in the digestive tract to degrade algal toxins

Workpackage 2

WP2 - Investigate the use of administered micro-capsules as an aid to effective and efficient depuration of bacterial and viral contaminents

1) To design protocols using fluorescent microbeads to approve the suitability of shellfish purification systems
2) To define a panel of bacteria with potential activity against bacterial and viral pathogens that might be administered to target shellfish
3) To determine the most appropriate method for microencapsulation of selected bacteria
4) To investigate the feasibility of applying microencapsulated bacterial diets to increase the performance/yield of the depuration process of the target shellfish from bacterial and viral pathogens

Workpackage 3

WP3 - Investigate the use of immuno-stimulants or delivery of direct anti-bonamia agents

1) To determine what size and types of microencapsulated beads can be used to target particular
oyster tissues. And investigate the optimal dose and method of introduction
2) To determine if microencapsulated beads can be used to provide a more targeted delivery of probiotics or immuno-stimulants to native flat oysters (Ostrea edulis) infected with Bonamia ostreae
3) To determine if the beads are targeting relevant tissues and organs where infection is present and if they are enhancing the immune response of the infected oyster
4) To determine if there is a dose-time immune response by O. edulis to encapsulated beads with immuno-stimulants
5) To determine if there is a reduction in the prevalence of infection and/or intensity of infection over time after exposure to probiotics or immuno-stimulants

Workpackage 4

WP4 - Demonstration of results of detoxification/depuration at commercial depuration plants

1) Degradation of algal toxins – To determine the potential of probiotic diets to detoxify shellfish in a commercial setting
2) Shellfish depuration system approval - To design ‘alternative’ protocols using fluorescent microbeads (as a proxy for bacteria and/or viruses) for SME depuration system approval
3) Depuration of bacteria and viruses – To determine the ability of microencapsulated probiotics to increase the efficiency of shellfish depuration of bacteria and viruses

Workpackage 5

WP5 - Evaluation, Assessment of IPR, Dissemination Activities and Training

1) To evaluate the results of the project on a month-by–month basis including consideration of IPR
2) To update the plan for dissemination and exploitation and prepare for implementation-development and management of IPR
3) To initiate primary training targeted at key personnel in the SMEs and others, designed to implement results and technology arising from the project.

Project Results:
R1 Detoxifying – Identification of bacteria , size and nature of micro-encapsulation
R2 Challenge test – size and construction of micro-capsules
R3 Depuration – Identified pro-biotics and size and nature of micro-encapsulation
R4 Bonamia – size and composition of micro-capsules delivering bio-agents to oysters
R5 Bonamia – Identification of immunostimulants/pro-biotics and effective dose and effect

Summary of the Research Outcomes

1) Investigate use of detoxifying bacteria delivered by micro-encapsulation to sites of toxins in
Laboratory feeding trials confirmed that shellfish (pacific oysters, king scallops and surf clams) had the ability to ingest microbeads. Results have shown that, for the three species tested, the smallest microbeads were consumed preferably and faster than the larger size microbeads.

A large number of bacteria (322) were isolated from shellfish contaminated with Okadaic Acid (OA) and Domoic Acid (DA). Approximately 10% of those isolates (32) were selected after in-vitro toxin degradation experiments shown increased bacterial growth (at least 40%) in the presence of OA and DA over 72 hours. These 32 isolates were characterised using molecular sequencing and the results revealed a wide variety of bacteria.
Further toxin utilisation experiments carried out at 12ºC and 20ºC and monitored by liquid chromatography-tandem mass spectrometry (LC-MS/MS) led to the selection of 4 isolates with interesting in-vitro toxin degradation ability (toxin degradation higher than 20%). These bacteria were microencapsulated and were used in laboratory shellfish feeding trials. Results of the trials carried out on king scallops and blue mussels have overall not been successful in improving detoxification in shellfish. These experiments highlighted the necessity of finding fast acting detoxifying bacteria to limit the confinement of shellfish in holding tanks to avoid shellfish losses.
In the second part of the project, the 32 isolates showing by absorbance reading at least 40% growthin the presence of OA and DA at 20ºC were selected and successfully characterised using molecular sequencing. These bacterial isolates were used in experiments as individual and as mixes at 12ºC and 20ºC to investigate the utilisation of OA and DA. The experiments were carried out over 7 days for OA and 14 days for DA and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to quantify the toxins in the extracts. In-vitro toxin degradation results were promising and a number of isolates (2 for OA and 2 for DA) and a bacteria mix (for OA) were selected for microencapsulation. Through cell viability testing, demonstration was made that the selected bacteria and mix did not produce toxic metabolites which could have formed during the degradation of OA and DA.
The 4 bacterial isolates were successfully microencapsulated and laboratory feeding trials were carried out during the last months of the project. King scallops (Pecten maximus) and blue mussels (Mytilus edulis) naturally contaminated with DA and OA respectively were placed in tanks filled with filtered seawater and were fed the microencapsulated bacteria.

2)Investigate the use of administered micro-capsules as an aid to effective and efficient
depuration of bacterial and viral contaminants.

The ingestion of different size alginate capsules by mussels and oysters was firstly assessed.
Results showed both bivalve species ingested more readily the smallest microcapsules (5-10 μm) in a progressive way during a three hours feeding experiment. The fluorescent microbeads where mainly found in the digestive system of the two species, validating the belief that microcapsules have the ability to deliver probiotic bacteria directly in the shellfish digestive region. After 48h depuration, it was found that the concentration of beads in the digestive glands of both species was negligible. This trial highlighted the high variability of depuration between shellfish of the same species confirming the difficulty of performing consistent and reproducible challenge tests. A large number of bacteria isolated at ANFACO-CECOPESCA (277), MSS (215) and during the SPIES-DETOX project (17) were screened for antimicrobial activity against a selection of pathogenic bacteria and viruses. One strain (A) which was identified after DNA sequencing, showed anti-listerial activity against Listeria monocytogenes CECT 935 and several strains of Listeria monocytogenes isolated from seafood products. The active substance produced by (a) was partially characterised and shown to be proteic with a molecular weight of 13.5 kDa. The search for suitable bacteria to microencapsulate is still on-going. However, three bacteria have so far been selected as potential probiotic candidates:
(A) and two other bacteria which have shown some slight anti E. coli activity. Other active substances have been sourced from external research group and are under study.

All parameters related to the construction of the microbeads like carrier medium, size, preparation protocol and encapsulation of bacteria and/or different active agents have been established as to ensure the effective delivery of these agents to the hepatopancreas of oysters and mussels.
The duration of the feeding period, the passage of the beads through the gastrointestinal tract of molluscs and the release and accumulation of the selected bacteria in the hepatopancreas have been successfully confirmed.
On the other hand, by application of the protocols thus elaborated for microbead preparation, specific beads with encapsulated fluorescent dyes have been used to test the efficiency of depuration at industrial level simulating E. coli depuration. The confirmation of the aptitude of such alternative challenge test to substitute standard challenge test carried out currently by authorities in the U.K. (Cefas method) has been done at two different depuration plants in Spain (at Mariscos Ria de Vigo installations) and in Scotland (at East Neuk Shellfish Ltd., St. Monans). Optimal concentration of alginate beads to be supplied to the shellfish, duration of the depuration process and similarity between the fluorescent dye used and E. coli behaviour in mollusc hepatopancreas have been established and well demontsrated. The intercomparative study between both assay sites provided additional confirmation on the possibility to apply fluorescent microbeads for standardized testing of SME depuration efficacy.
In parallel, the determination of the antiviral properties (against hepatitis A (HAV) and Norwalk-like viruses (NOV)) of all bacterial isolates obtained at ANFACO-CECOPESCA for the purposes of the project was accomplished and best final probiotic candidates for encapsulation selected. Characterization of the selected probiotic candidates for viability upon encapsulation and antibiotic resistance was done and these were identified to the species level by biochemical and molecular methods. The bacteria selected were encapsulated separately and fed as a mixture, in a fixed ratio, to mussels and clams, naturally contaminated with E.coli and Norwalk-like viruses, and depuration effectiveness was tested at industrial scale (at Mariscos Ria de Vigo installations). Natural contamination of both oysters and mussels with Nov GI and II has been demonstrated as well as the lack of any naturally contaminated with HAV molluscs. Depuration from pathogens was estimated by microbiological and molecular methods in 63 oyster and 67 mussel samples over a period of 72 to 96h. In order to demonstrate well a depuration effect of the potentially probiotic encapsulated bacteria in shellfish at industrial scale high levels of pathogen contamination (in the case of viruses) are necessary.

3) Investigate the use of immuno-stimulants or delivery of direct anti-bonamia agents

Work in the first 9 months of the project included a comprehensive literature review
focusing on oysters. A feeding experiment was carried out to assess the ability of Ostrea edulis to ingest alginate microcapsules (5-10 μm) containing fluorescent microbeads. Results confirmed oysters were successfully filtering the microcapsules. It was also found that the microbeads were actively taken up by the oysters’ blood cells. Screening revealed also the presence of fluorescent beads within the digestive and connective tissue areas known to be infected by Bonamia ostrea, thus validating the use of microencapsulation as a potential tool for the transport of immunostimulants directly to the infected areas.

In the second part of the project a number of trials were undertaken to assess the ability of the beads to transport immunostimulants to tissues of the European oyster infected with the protistan paprasite Bonamia ostreae. In the first set of trials three candidate immunostimulants were tested to determine if they could stimulate the oyster’s immune system. Oysters were screened over hours and days up to 7 days post-exposure and a range of immune parameters in the oysters measured. A particular dose of each immunostimulant that evoked a response was identified. The immunostimulants evoked response in the cellular and non-cellular parts of the blood of the oyster. In the second set of trials – run over 128 days and with 810 oysters screened, two immunostimulants were further tested to determine how they would impact on the immune system and disease development of an infected population of oysters. A naïve population was used as control animals. The tests indicated that exposure to the immunostimulants delayed the onset of mortalities and reduced their impact. In addition there was a reduction in the prevalence of infection during the trial not related to mortalities indicating that the infection had been eliminated. The blood cells of the oysters were stimulated resulting in better ability to fight infection. This was mirrored at the genetic level with over expression of immune related genes. Overall the results indicated that delivery of encapsulated immunostimulants to infected oysters can stimulate the animals ability to fight the infection resulting in a decrease in infection and mortality levels.

Potential Impact:
The industry consortium is made up of 4 SMEs and 2 Associations (OTH)s and represents 4 member and associated states of the EU. The range of interests within the group encompasses biotech companies on the one hand and fishery and aquaculture interests on the other.
The traditional supply chain is represented by 4 of the partners as single SMEs or groups

There are risks and downward pressure on market values associated with:
• supply problems (through closures due to toxins or disease such as Bonamia in Flat Oysters) or Summer mortality in Gigas Oysters.
• Microbial and quality problems, and associated poor prices for product from areas of lower environmental quality status.
• Product recalls leading to loss of confidence and sales (due to toxic episodes or part failure in depuration

What does the downward pressure amount to in European Terms?


European production of farmed shellfish alone was around 825,000 tonnes in 2006 with a value of over €1bn
Tonnages for Mussels was 644,000t for Oysters 143,000t and for clams and cockles 37,000t
The industry leads to 37,000 direct jobs and 50,000 indirect jobs
(Source – Eurostat and European Association of Mollusc Producers)
The problems
Problems with algal toxins are routine in most member states, which have significant stocks of fished or farmed products such as Mussels, Scallops, Oysters, Clams and Cockles.

The loss of money to the Norwegian aquaculture industry due to contamination with DSP toxins is estimated to be more than NOK 20 000 000 (2,571,660 Euros) during the last 5 years. To date, ASP toxin contamination has not caused any harvesting closures in Norway, but it is envisaged this will become an increasing problem as harvest of king scallops (prone to ASP contamination) from 2009 increases to approximately 1 000 000 kg scallop per year. The current plan is to produce 6 000 000 small scallop in hatcheries every year. Although this could be a problem it is difficult to estimate the monetary loss, When compared to mussels the loss could be as great as NOK 10 000 000,-. (1,285,670. Euros).

Toxic episodes cause serious economical losses to industrial sectors related to shellfish production, such as shellfish producers themselves, canning industry, depuration plants and other associated industries that work with fresh or frozen shellfish, including restaurants and tourism, among others. Galicia, located at the NW of Spain, is where most of the Spanish mussel culture takes place. Up to 84 % from the total mollusc product of Spain in 2008 came from Galicia. The total export of Spain of molluscs for countries from the EU and other countries rose up to 160.815 million tons in 2008. It is, therefore, a very important sector from the Spanish market. The impact of toxic episodes in shellfish production can be illustrated with the following example. In one of the most productive Galician regions (Cangas, Pontevedra) between 1998 and 2006 there were long periods during which the harvesting area was closed due to DSP episodes - from 170 days per year in 1998 to 280 days in 2005. In 2006 the closing days of the harvest area for DPS episodes were reduced to the same value as in 1998. As we can see, almost half of the year or even more, the area was closed. 2005 was a very virulent year with respect to DSP toxicity. In this example, harvesting areas was completely open only two months, three months were closed and open alternatively and the rest of the year, 7 months, the areas were closed. This situation has an economic impact on industrial activity. Therefore, in this example mussel production in 2005 was of 10 Million Kg compared to 16 Million Kg in 2004 – a production and sales drop of almost to 50% was registered in 2005.

In Greece, a major operator has an approximate 25 million Euros gross turnover which is broken down to 20 million Euros from selling shelled unpackaged mussels and another 5 million Euros of shucked-without shell-mussels. One third of this gross turnover is estimated to be the net profit (ca. 8 million Euros). The loss of income due to episodes of HABs is estimated at 5 million Euros per year. This cost is due to closing down mussel cultivation areas leading to lower prices for the end product due to the loss of the ‘time-window’ when prices are high for EU markets and due to fierce market competition. Overall, approximately 20 % of the gross turnover of the company is lost when the Greek mussels industry is faced with algal toxin contamination and HABs
The combined result of these research activities could be expected to increase the competitiveness of Industry groups (SMEs/IAGs) in the sectors of shellfish harvesting/aquaculture and fish aquaculture by a factor, which reflects the losses currently, suffered by toxic events. Thus, if 15 % reflects the current level of annual loss, then this same figure could represent the level of increased competitiveness of SMEs benefiting by exploitation of the results of the project

15% equates to an annual loss of around €150m
Pressure downward on levels of some toxins allowable in shellfiah flesh – more accurate detection levels – allowance for loss off water in cooking for instance –thus higher real levels in meat consumed.
Scallops can remain above the limits for many months whereas mussels and cockles have much faster rates of loss.
This has led to a real impact in Orkney –OFA’s area – of a drop to half the production of scallops from a value of £1.1m in 1996 to £469,000 in 2006. Most of this loss of market involves diver caught – sold as live in the shell at top market value.
The Opportunities
Although it represents a relatively minor part of all eventual sales of scallops, the live in-shell market is the most valuable with prices at first sale often around €4/kg mostly from divers. Though there are currently over 150,000 shells under cultivation in one Norwegian farm.

It is estimated that losses due to restriction of the live scallop market in Europe is around €10m per annum.

For products such as farmed mussels a hold of just a few days due to algal episodes –whilst they naturally depurate can lose operators a lot of money in times when demand and prices are high.

The consortium therefore values the potential market for active detoxification of biotoxins in Europe to be in excess of €50m per annum.

Microbial contamination (including viruses) and Depuration
Several procedures are available to improve the microbiological quality of shellfish. Traditional depuration facilitates reduction of faecal coliforms, enteric bacteria, and viral pathogens, and the process does not kill the molluscs.Depuration times to reduce biological contamination in the usual, flow-through systems can range from 24 to 48 hours. This time period can be extremely limiting with regard to the amount of product that can be processed and sold during peak seasons when the demand for shellfish is often greater than the supply.
This depuration process was improved by actively disinfecting the system water instead of simply waiting for contamination levels to decline. This is achieved primarily by the application of ozone, ultraviolet, and "skimmer" treatment to the system water. The effect of this combined treatment is a greatly accelerated elimination of bacterial contamination. Unfortunately, depuration is not very effective for removal of Vibrio species from shellfish, as they appear to be firmly attached to shellfish tissues.
Alternate methods were also developed, and current decontamination procedures permit shellfish to be treated with high hydrostatic pressure, ultra-low temperature freezing, mild heat treatment, or frozen dry storage as individual processes or in combination. These methods reduce the number of bacteria in the final product but often kill the animals.
This project aims to study using probiotic bacteria in order to improve the traditional depuration
Previous works have demonstrated that success will depend on the ability of probiotics to establish within the host, for example two different probiotic bacteria where used to study its protective effect from Edwarsiella tarda in eels.

For instance, a depuration plant in France processes 2000 tonnes/year at a cost of 92 €/tonne.

Bivalve derived products, when are intended for consumption as raw, alive product or processed and refrigerated have a short shelf life. Hence, harvesting of live mollusc is usually performed on customers’ demand. Nevertheless, a depuration step is necessary, and this step introduces a delay in the production chain. Nowadays, food production requires quick response times and some producers try to predict their customers needs, harvesting the molluscs prior to its market demand. As molluscs die after harvesting, “over prediction” has to be sold at reduced prices, with economic loss for the primary producer. In other cases, prediction of consumption in factories means that part of the shelf life of the product is wasted in factory’s warehouse.

The achievement of a faster depuration protocol would mean shorter response times, and a diminution of the need of predictions and hence economic looses would be smaller. Indeed, it would mean a better management of resources and production areas. This reduction in depuration times would positively affect to the manufacturers also, since they will be able to provide final product to their customers faster too.

On the other hand, a shorter depuration protocol would reduce the associated costs (time, consumption of electricity in UV lights, chlorine, production of ozone and pumping of water).

Regarding marine biotoxins, the achievement of depuration protocols would mean to solve what is probably the main problem of the production of molluscs. After a toxic episode occurs, there is nothing to do unless to wait until the toxic algae bloom finishes and the molluscs loose their toxicity in a natural detoxification process, which, depending on the species, can be extremely a very long period.

Altogether, shorting the depuration times (in a microbial and biotoxins point of view) will improve production times along the production chain, from harvesters to factories, with positive results for our associates.

In a direct manner, the knowledge of the developed technology and the experience in its use, would give operators a new services to offer to end users, which represent a new business area.

The Cost of shellfish recalls in the UK can average £20,000 per shipment!

The consortium estimates conservatively that the entry value of the market for an effective and safe active depuration agent would be worth €20m
Productivity and Sites
As stated in the report “A feasibility study of native oyster (Ostrea edulis) stock regeneration in the United Kingdom”, issued by The Centre for Environment, Fisheries and Aquaculture Science, CEFAS (UK), in which trends in O. edulis production in Europe and the feasibility of recovering stocks in some areas are analysed, 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 to develop Bonamia ostreae tolerant strain is currently underway led by CIMA. 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.

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 IFREMER. Furthermore, a selective breeding programme is ongoing for producing bonamiosis-tolerant strains. . 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.

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.
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.

European Production
In spite of several management practices aimed at limiting mortality and facilitating oyster growth rates, diseases have drastically affected wild and cultured flat oyster populations. 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.
Although research programmes have demonstrated the feasibility and effectiveness of a selective breeding for tolerance to bonamiosis, the resulting oyster strains have not yet been widely transferred to the industry. Moreover, this approach requires a long-term commitment from the industry and private hatcheries, the latter being the main stakeholders in managing the selective breeding programme.
Indeed, the new knowledge gathered with this project on the life cycle of Bonamia out of flat oysters and on previously unknown ways of transmission and the new diagnostic procedures will allow improving bonamiosis control strategies, which hopefully will prevent disease spreading. The disease has been recently (2006-2007) confirmed in some areas in Ireland and Scotland previously reputed as Bonamia-free areas.

As it has been previously stated in this proposal, overcoming the main constraint for European flat oyster recovery represents an interesting opportunity for the European shellfish industry. The main reasons for this are:
1st- It represents a chance to diversify shellfish production, and thus a chance to improve risk management;
2nd- High market value of the product. Although prices can be so high due to the short offer of this product, it is considered that flat oyster market is a niche of market with better prices (tree or four times higher) than those for common cupped oyster production.
The original market for Oysters valued at over 150 Million euros in (today’s terms) in 1962 has now (2006 figure) shrunk to around 25 Million euros. The loss has so far been made up – and increased – by the pacific ‘cupped’ oyster (C gigas) at a value of around 315 Million euros. Whilst nearly all the production of pacific oysters is cultivated and in some places in areas of once high production of the European Oyster, there are other areas where original beds have been abandoned –often due to Bonamia.
The pacific oyster is now under threat with lower productivity expected and some resistance by environmental agencies to natural settlement.
There may be an opportunity to reverse the situation which has pertained since the late 1970’s and have the European Oyster as the target for diversification.
In order to make a successful re-entry into the mass market for oysters – the producers of the European oyster have to give confidence back to the market that they can deliver high volumes on a consistent basis. The work of this project is, thus, vital to be able to bring back this level of confidence.
In recent years the price for the European Oyster has been around double that of the Pacific Oytser – around 5,000 euros per tonne versus around 2750 euros. This however masks a large variation in the price of the European oyster which has varied this year from 5400 euros in 2008 to 2600 in spring 2009. This reflects a sudden increase in landings from the fishery in Denmark, which temporarily saturated the small market share currently occupied by O. edulis.
The project could, if successful, lead to more stable prices for O. edulis as the market expands in response to consistent recovery of key sites.
If we are to look upon the European Community’s contribution into the project as a strategic investment into the industry then it is reasonable to look at a return on that investment. The productivity of the European Oyster sector has stayed around 6000 - 8000 tonnes for the past 15 years. The project, if successful in some of it’s aim at least could see an increase to around 15,000 tomes within 7 years of the end of the project (2020) – thus doubling the market value to around 100 million euros (from around 24 million).
The Opportunities

The Consortium predicts that it would be reasonable to estimate an opening market of around €50m per annum and rising to around €100m within 7 years if an effective agent to aid against bonamia could be administered in bulk to infected oysters and to those in infected areas.
The diagram at the front of this section showed a traditional supply chain and it was conspicuous to leave out the biotech companies in the project. This served to emphasis that it is doubtful if the shellfish industry can mange the extra risks and pressures on it without some real technological breakthroughs. These have to be made on a transnational basis to have any impact on the size of the challenges facing the industry. This project does just that and is cross-cutting, bringing a new slant and new ideas.
Companies like GLY and AET can bring new technologies to the industry on the back of first class research from the RTDs in the project.

Together with the other industry partners they have an estimated first market of up to €120m if all the potential results of the project were to materialise.

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Mary Sugrue