Project Description | Montpellier

Oyster farming: history and economic impact

Oyster farming originated in China about 4000 years ago. Romans, who consumed a lot of seashells, were the first to install oyster beds and developed the European oysters. Over centuries, different oysters species were cultivated and production methods evolved to increase production. Nowadays, oysters are farmed across the globe and production has steadily increased since 1990. Though oysters are always cultivated in either salt or brackish water, there exists a variety of production methods adapted to regional specificities.(1). In France, 130 000 tons of oysters are produced each year (2), which represents a 630 millions euros market and position the country as the leading producer and exporter in Europe. In fact, Frenchs are the leading fresh oysters consumers in Europe with 2 kg per inhabitant and per year (2). Oyster production is distributed over distinct French regions. Although the bulk of the production is distributed over the Atlantic coast, the Mediterranean coast is also an important contributor with its thanseven zones of productions (see figure 2).

*Fig.1 - oyster beds (image by Peter Voyvodic).*

*Fig.2 - French zones of oyster production (image realized on Canva premium).*

Thau lagoon

The Thau Lagoon is located 30 km South West of Montpellier. This brackish water pond of 19 km long and 5 km large is the largest lagoon in Occitanie (3) and is home to an exceptional ecological diversity, welcoming more than 400 vegetal and 100 animal species. This natural resource favored the development of various craft activities such as oyster and mussel farming. In fact, the oyster production delivered by the Thau lagoon represents no less than 10% of the national production. With 9,000 to 10,000 tons of oyster produced each year this industry represents 2,000 direct jobs, 2,000 indirect jobs, 450 companies and a market of 50 millions euros per year.

Oysters and their infections

Oysters feed on plankton that they are actively filtering from the water. As such, they are particularly exposed to a wide range of bacteria, viruses or algae, which can occasionally cause infections. Some filtered microorganisms are pathogenic for oysters, leading to large episodes of mortalites, or are harmful to humans, making them unfit for consumption, which in either case results in sizable economic losses (4). In 2008, a syndrome known as “summer mortality” impacted oyster production on the french coasts. This syndrome has been associated with infections from an ostreid herpes virus called OsHV-1 as well as from a bacteria of the genus Vibrio (5). Ever since then, A OsHV-1 has been recurrently responsible for 60 to 90% juvenile mortality observed (6). Over the years, oyster farmers changed their production techniques to reduce the risks and impacts of OsHV-1 infection. . For example, as the virus replication and circulation depends on the temperature of the water (7), farmers have been putting their oysters in beds outside of the most permissive range (16-24 °C (7)). By doing so, a decrease in oyster mortality could be observed. This strategy, however, only partially mitigates losses--and requires complex and costly logistics to shift oysters location. The ability to know whether the virus is present or not would allow producers to decide if this costly and labor intensive strategy is really necessary in a timely fashion. Vibrios form a genus of gram negative bacteria among which many species are able to survive in different marine environments including marine invertebrates. Numerous Vibrios are known to infect oysters, though many are not harmful to them. Since 2008, episodes of mass mortality have been observed in adult pacific oysters, and linked to the pathogenic effect of Vibrio aestuarianus (8). Nowadays, oyster farmers do not have preventive solutions to decrease Vibrio aestuarianus infections and large productions are lost each year. It is estimated that on average, 30% of the production is lost at the end of the farming cycle (2-3 years). (9) Moreover, the bacteria’s detection is not directly accessible to the producers, as existing tests are performed by experts in laboratories and were developed mostly for fundamentals studies such as epidemiological research (10).

Shell’lock: an autotest for oysters

Current detection methods and limits:

Molecular biology approaches such as Polymerase Chain Reaction (PCR) have been developed to detect vibrio aestuarianus (11), to survey ecosystems in the water column (12) and to detect pathogens in oysters (11). Other techniques such as liquid chromatography mass spectrometry (LC-MS) and Enzyme-Linked ImmunoSorbent Assay (ELISA) tests are used to detect toxic algae blooms (13). Although powerful, these techniques require specific and expensive equipment, reactants and expertise to perform useful diagnostics. Moreover, oyster farmers have to pay for the test each time a company performs one and they only have access to the result without any explanation about the kind of pathogen found in water. As a consequence, detection is not integrated into the farming process: oyster farmers only proceed to have these tests performed once it is almost too late and they start detecting mortality increase. To propose a solution we decided to use the power of synthetic biology to develop a fast, easy,affordable test to accurately detect vibrio aestuarianus in the Thau lagoon and to integrate preventive pathogen scanning of the water column in oyster farming habits.

Description of the method used:

The detection is based on a recently developed technology: Specific High-sensitivity Enzymatic Reporter un-LOCKing (SHERLOCK) (14). SHERLOCK is a CRISPR-based diagnostic that usesCRISPR-Cas specific recognition of DNA or RNA target sequences. Our idea is to use the SHERLOCK principle illustrated in figure 4 to detect pathogens in water samples. Because we want to offer an oyster farmer-friendly test, we focussed on a paper-based test development which combines Cas13a specific and collateral activities with gold nanoparticle attachment visualization (see figure 5). To develop the appropriate detection device, we designed guide RNA sequences complementary to pathogen target sequences we identified through bioinformatic approaches.

Fig4: SHERLOCK principle based on Kellner’s et al.

*Fig.5 - Paper-based test principle. See design page.*

Modeling our system:

Our detection technique is based on an enzymatic reaction (Cas13a). To quantify this process we developed a kinetic model that allowed us to gain more information about our system. The model is based on the law of mass action allowing us to relate the concentration of all the species in our test to the various reaction rates. Using ordinary differential equations for the concentrations of the species in the reaction, we were able to fit our experimental data and extract various parameters. The parameters extracted were used as a comparison metric to further quantify the system. Additionally, our project contains a kinetic model. It allowed us to have a better understanding about the Crispr-Cas13 enzymatic process and quantitatively compare SHERLOCK reactions.

Diffusing our project:

The diffusion of our project and our involvement for the general public was divided in two different parts.

As part of our human practices, we identified two principal stakeholders to interact with. First, we decided to discuss with researchers in the field to learn from them. In fact, we participated in exchanges between oyster farmers and researchers in Bouzigues. It was our starting point to gather useful contacts. After meeting “oyster infection experts” our project evolved to be more scientifically coherent and relevant. On the other hand, we worked with oyster farmers to develop the most suitable test. They offered us a precious help to meet the demand as well as possible. In addition, we visited oyster farmers to obtain feedback for our project and to establish a dialogue “oyster farmers - scientists”.
As part of our education and public engagement, we created a booklet dedicated to oyster farmers, to popularize synthetic biology and to explain to them the scope of our detection test.Furthermore the booklet contains a simplified protocol for the realization of the test. Moreover, to initiate children to biology in general, we worked with them on the concept of synthetic biology through a board game. We also wrote a short flyer dedicated to the general public to explain why oysters are infected by pathogens. Communication also took part in our project through social media, through several oral presentations to explain what iGEM is to undergrads.

*Fig.6 - Visit to Yannick Desplats, oyster farmer at Bouzigues (image by Peter Voyvodic)*

Overview of the project

*Fig.7 - Visit to Yannick Desplats, oyster farmer at Bouzigues (image by Peter Voyvodic)*

References

1. France Naissain. Farming methods [Internet]. Available from: https://www.francenaissain.com/en/the-oyster/the-oyster-in-france/farming-methods/
2. Comité National de la Conchyliculture. Les Chiffres Clés [Internet]. Available from: https://coquillages.com/les-statistiques/
3. Tourisme Sète. L’Etang de Thau: une mer intérieure [Internet]. Available from: https://www.tourisme-sete.com/l-etang-de-thau-une-mer-interieure.html
4. ANSES. Neurological disorders associated with the consumption of shellfish : health professionals still unfamiliar with their diagnosis. 2021 Mar. (The bulletin for all of ANSES’s vigilance schemes).
5. Samain JF, McCombie H. Summer mortality of Pacific oyster Crassostrea gigas. The Morest Project. 2008;
6. Martenot C, Oden E, Travaillé E, Malas JP, Houssin M. Detection of different variants of Ostreid Herpesvirus 1 in the Pacific oyster, Crassostrea gigas between 2008 and 2010. Virus Res [Internet]. 2011 Sep; Available from:10.1016/j.virusres.2011.04.012
7. Renault T, Bouquet AL, Maurice JT, Lupo C, Blachier P. Ostreid Herpesvirus 1 Infection among Pacific Oyster (Crassostrea gigas) Spat: Relevance of Water Temperature to Virus Replication and Circulation Prior to the Onset of Mortality. 2014 Sep; Available from:10.1128/AEM.00484-14
8. Azéma P, Lamy JB, Boudry P, Renault T, Travers MA, Dégremont L. Genetic parameters of resistance to Vibrio aestuarianus, and OsHV-1 infections in the Pacific oyster, Crassostrea gigas, at three different life stages. Article number: 23. 2017 Feb; Available from: 10.1186/s12711-017-0297-2
9. Garcia C, Mesnil A, Tourbiez D, Moussa M, Dubreuil C, Goncalves de Sa A, et al. Vibrio aestuarianus subsp. cardii subsp. nov., pathogenic to the edible cockles Cerastoderma edule in France, and establishment of Vibrio aestuarianus subsp. aestuarianus subsp. nov. and Vibrio aestuarianus subsp. francensis subsp. nov. 2021 Feb; Available from:10.1099/ijsem.0.004654
10. Saulnier D, De Decker S, Haffner P. Real-time PCR assay for rapid detection and quantification of Vibrio aestuarianus in oyster and seawater: a useful tool for epidemiologic studies. 2009 May; Available from:10.1016/j.mimet.2009.01.021
11. Cross I, Rebordinos L, Diaz E. Species Identification of Crassostrea and Ostrea Oysters by Polymerase Chain Reaction Amplification of the 5S rRNA Gene. Volume 89. 2006 Jan; Available from: 10.1093/jaoac/89.1.144
12. E. Asplund M, Rehnstam-Holm AS, Atnus V. Water column dynamics of Vibrio in relation to phytoplankton community composition and environmental conditions in a tropical coastal area. 2011 Sep; Available from:10.1111/j.1462-2920.2011.02545.x
13. Elgarch A, Vale P, Rifai S, Fassouane A. Detection of Diarrheic Shellfish Poisoning and Azaspiracids Toxins in Moroccan Mussels: Comparison of LC-MS Method with the Commercial Immunoassay Kit. 2008 Oct; 10.3390/md6040587
14. J.Kellner M, Koob J, S.Gootenberg J, O.Abudayyeh O, Zhang F. SHERLOCK: Nucleic acid detection with CRISPR nucleases. 2020 Mar; Available from: 10.1038/s41596-019-0210-2