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Introduction Marteilia refringens and Bonamia ostreae are protozoan parasites responsible for mortalities of farmed and wild flat oysters Ostrea edulis in Europe since 1968 and 1979, respectively. Despite almost 40 years of research, the life-cycle of these parasites is still poorly known, especially regarding their environmental distribution. Methods We carried out an integrated field study to investigate the dynamics of M. refringens and B. ostreae in Rade of Brest, where both parasites are known to be present. We used real-time PCR to monitor seasonally over four years the presence of both parasites in flat oysters. In addition, we used previously developed eDNA based-approaches to detect parasites in planktonic and benthic compartments for the last two years of the survey. Results M. refringens was detected in flat oysters over the whole sampling period, sometimes with a prevalence exceeding 90%. It was also detected in all the sampled environmental compartments, suggesting their involvement in parasite transmission and overwintering. In contrast, B. ostreae prevalence in flat oysters was low and the parasite was almost never detected in planktonic and benthic compartments. Finally, the analysis of environmental data allowed describing the seasonal dynamics of both parasites in Rade of Brest: M. refringens was more detected in summer and fall than in winter and spring, contrary to B. ostreae which showed higher prevalence in winter and spring. Discussion The present study emphasizes the difference between M. refringens and B. ostreae ecology, the former presenting a wider environmental distribution than the latter, which seems closely associated to flat oysters. Our findings highlight the key role of planktonic and benthic compartments in M. refringens transmission and storage or potential overwintering, respectively. More generally, we provide here a method that could be useful not only to further investigate non cultivable pathogens life-cycle, but also to support the design of more integrated surveillance programs.

The “International symposium on ecology and evolution of marine parasites and diseases” is a unique meeting combining marine ecology and parasitology. Considering the need to integrate parasitology in ecosystem studies and global change scenarios, this symposium provides a great opportunity to present the latest research findings regarding marines parasites, to gather international experts from various disciplines and to identify next research priorities in this area, both at the European and international levels. The symposium included seven sessions spread over 3 days on parasites affecting marine organisms: parasite adaptation and evolution, biogeopgraphy and macroecology, parasite detection, disease impact and management, epidemiology, environmental parasitology. A workshop focusing on micro-parasites of bivalves was also organised on the 4th day and was opened to interested participants. The symposium was held at "Espace Encan" in La Rochelle, on the 15th-18th of November 2022. The 7 sessions were also available remotely through the Imagina application: https://app.imagina.com/marine-parasitology-symposium A total of 123 participants from 22 differents countries attended the Symposium. The scientific committee : - Dr. Isabelle Arzul, IFREMER, France - Dr. David W. Thieltges, Royal Netherlands Institute for Sea Research, The Netherlands - Dr. Mathias Wegner, Alfred Wegener Institute, Germany - Pr. Xavier de Montaudouin, University of Bordeaux, France The organizing committee: IFREMER, Adaptation and health of marine invertabrates Unit (ASIM), La Tremblade, France - Isabelle Arzul, Isabelle.Arzul@ifremer.fr - Lydie Canier,Lydie.Canier@ifremer.fr - Véronique Betto, Veronique.Betto@ifremer.fr The Sponsors: This event has received funding from Ifremer, the Aquitaine Region, the Communauté d'Agglomération de La Rochelle, UMR EPOC/University of Bordeaux, and the European Union Reference Laboratory for Molluscs Diseases.

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International audience

L’huitre plate, la seule huitre native des côtes européennes, est une espèce emblématique de Bretagne. Convoitée depuis l'Antiquité, sur-pêchée au 19ème siècle, fleuron de l'ostréiculture au 20ème, elle est désormais menacée de disparition à l'aube du 21ème. Sous l'impulsion de la commission OSPAR mais aussi dans un contexte grandissant de crise écologique et d'érosion de la biodiversité imposant une nécessaire conservation et/ou restauration de nos patrimoines naturels, une alliance européenne réunissant biologistes et gestionnaires a été créée en novembre 2017 à Berlin sous l'égide de l'Agence Allemande de Conservation de la Nature (BFN) avec un objectif commun: favoriser le retour de l'huitre plate sur les côtes européennes. Cette alliance pour la restauration de l'huitre plate (NORA) regroupe désormais plus d'une centaine de scientifiques et de gestionnaires, tous au chevet de cette espèce. En France, cette démarche est assurée nationalement par le programme FOREVER ("Flat Oyster Recovery"), démarré en 2018 et financé par le FEAMP pour sa première phase (2018-2020) focalisée en Bretagne. Le présent rapport dresse le bilan du projet. En trois ans, ce projet pionnier a permis : (1) de proposer un premier inventaire des dernières populations relictuelles d'huitres plates encore présentes en Bretagne, en fournissant un diagnostic de leur état écologique, parasitaire et génétique et en affinant la description de son habitat actuel. (2) d'étudier plus précisément les mécanismes écologiques impliqués dans la dynamique d'un banc d'huitres plates : abondance et dispersion larvaire, dynamique du recrutement, facteurs environnementaux, effet de la prédation, impact des maladies parasitaires. L'ensemble de ces processus est étudié à la fois en rade de Brest et en baie de Quiberon. (3) d'initier une démarche unique en France et proposer une méthode adaptée permettant la conservation et la restauration écologique des derniers bancs. Cette démarche inclue des opérations de communication et de sensibilisation actives associées à des actions pilotes concrètes et facilement transférables de restauration écologique : enrichissement coquillier, semis de naissain et développement de récifs artificiels adaptés à l'espèce. Ce rapport apporte aussi de nombreux nouveaux éléments de gestion, utiles aux professionnels de l'ostréiculture, de la pêche, mais aussi aux gestionnaires de l'environnement, notamment aux animateurs du réseau des sites Natura 2000, à l'Office Français de la Biodiversité ainsi qu'à différentes associations impliquées. A titre d'exemple, ci-joint quelques résultats marquants : Pour la profession ostréicole, la meilleure compréhension du recrutement de l'espèce ouvre les voies pour assurer un captage "régulier" pour les années à venir. Les cartes de dispersion expliquent la distribution spatiale du recrutement dans les deux seuls sites de captage français. Les tendances temporelles démontrent l'importance de gérer les populations sauvages, seul moyen d'assurer un captage lorsque des étés frais moins favorables à la reproduction. Le seuil des 18°C/19°C constitue une température critique des masses d'eau en deçà de laquelle le recrutement se fait mal, surtout quand les stocks sont faibles. Pour le secteur de la pêche professionnel (mais aussi récréative), les résultats montrent l'extrême fragilité des bancs résiduels bretons et insistent sur le fait qu'aucune exploitation durable de cette espèce ne pourra être menée sans une gestion très précise des populations résiduelles incluant conservation, restauration et suivis scientifiques. Pour les gestionnaires de l'environnement, ce rapport est l'occasion de mettre en lumière cette espèce-ingénieur, qui après trois siècles de surexploitation, est menacée de disparition. Pourtant une huitrière en bonne santé apporte de nombreux services écosystémiques, comme lutter contre l'érosion de la biodiversité et la dégradation de la qualité de l'eau. De nombreux éléments précis sont fournis pour permettre aux services de l'état, aux gestionnaires Natura 2000 et à l'OFB de protéger et restaurer les dernières populations présentes sur leurs périmètres d'intervention. En savoir plus sur le programme FOREVER et les autres projets européens de conservation et restauration de l'huitre plate : https://noraeurope.eu

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The Pacific oyster, Crassostrea gigas, is a mollusk bivalve commercially important as a food source. Pacific oysters are subjected to stress and diseases during culture. The autophagy pathway is involved in numerous cellular processes, including responses to starvation, cell death, and microorganism elimination. Autophagy also exists in C. gigas, and plays a role in the immune response against infections. Although this process is well-documented and conserved in most animals, it is still poorly understood in mollusks. To date, no study has provided a complete overview of the molecular mechanism of autophagy in mollusk bivalves. In this study, human and yeast ATG protein sequences and public databases (Uniprot and NCBI) were used to identify protein members of the C. gigas autophagy pathway. A total of 35 autophagy related proteins were found in the Pacific oyster. RACE-PCR was performed on several genes. Using molecular (real-time PCR) and protein-based (western blot and immunohistochemistry) approaches, the expression and localization of ATG12, ATG9, BECN1, MAP1LC3, MTOR, and SQSTM1, was investigated in different tissues of the Pacific oyster. Comparison with human and yeast counterparts demonstrated a high homology with the human autophagy pathway. The results also demonstrated that the key autophagy genes and their protein products were expressed in all the analyzed tissues of C. gigas. This study allows the characterization of the complete C. gigas autophagy pathway for the first time. Abbreviations: ATG: autophagy related; Atg1/ULK: unc-51 like autophagy activating kinase; ATG7: autophagy related 7; ATG9: autophagy related 9; ATG12: autophagy related 12; BECN1: beclin 1; BSA: bovine serum albumin; cDNA: complementary deoxyribonucleic acid; DNA: deoxyribonucleic acid; GABARAP: GABA type A receptor-associated protein; IHC: immunohistochemistry; MAP1LC3/LC3/Atg8: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NCBI: national center for biotechnology information; ORF: open reading frame; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PtdIns3K: class III phosphatidylinositol 3-kinase; RACE-PCR: rapid amplification of cDNA-ends by polymerase chain reaction; RNA: ribonucleic acid; SQSTM1: sequestosome 1; Uniprot: universal protein resource; WIPI: WD repeat domain, phosphoinositide interacting.

WOS:000544335600001; International audience; Environmental DNA approaches are increasingly used to detect microorganisms in environmentalcompartments, including water. They show consider-able advantages to study non-cultivable microorgan-isms like Bonamia ostreae,a protozoan parasite inducing significant mortality in populations of flat oyster Ostrea edulis. Although B. ostreae development within the host has been well described, questions remain about its behaviour in the environment. As B. ostreae transmission is direct, seawater appears as an interesting target to develop early detection tools and improve our understanding of disease transmission mechanisms. In this context,we have developed an eDNA/eRNA approach allowing detecting and quantifying B. ostreae 18S rDNA/rRNA as well as monitoring its presence in seawaterby real-time PCR. B. ostreae DNA could be detected up to 4 days while RNA could be detected up to 30 days, suggesting a higher sensitivity of the eRNA-based tool. Additionally, more than 90% of shed parasites were no longer detected after 2 days outside the oysters. By allowing B. ostreae detection in seawater, this approach would not only be useful to monitor the presence of the parasite in oyster production areas but also to evaluate the effect of changing environmental factors on parasite survival and transmission.

Macroautophagy is a mechanism that is involved in various cellular processes, including cellular homeostasis and innate immunity. This pathway has been described in organisms ranging in complexity from yeasts to mammals, and recent results indicate that it occurs in the mantle of the Pacific oyster, Crassostrea gigas. However, the autophagy pathway has never been explored in the hemocytes of C. gigas, which are the main effectors of its immune system and thus play a key role in the defence of the Pacific oyster against pathogens. To investigate autophagy in oyster hemocytes, tools currently used to monitor this mechanism in mammals, including flow cytometry, fluorescent microscopy and transmission electron microscopy, were adapted and applied to the hemocytes of the Pacific oyster. Oysters were exposed for 24 and 48 h to either an autophagy inducer (carbamazepine, which increases the production of autophagosomes) or an autophagy inhibitor (ammonium chloride, which prevents the degradation of autophagosomes). Autophagy was monitored in fresh hemocytes withdrawn from the adductor muscles of oysters using a combination of the three aforementioned methods. We successfully labelled autophagosomes and observed them by flow cytometry and fluorescence microscopy, and then used electron microscopy to observe ultrastructural modifications related to autophagy, including the presence of double-membrane-bound vacuoles. Our results demonstrated that autophagy occurs in hemocytes of C. gigas and can be modulated by molecules known to modulate autophagy in other organisms. This study describes an integrated approach that can be applied to investigate autophagy in marine bivalves at the cellular level.

Emerging pathogens threaten aquaculture industries worldwide. There is urgency to become better prepared and meet these challenges, as aquaculture is growing rapidly and will contribute increasingly to food security. There are important Pacific oyster production areas today as yet unchallenged by emergent OsHV-1 microvariants, notably in the Americas, and to these areas the viruses are an immediate threat.The ICES Workshop on Emerging Mollusc Pathogens addressed fundamental questions regarding capabilities (expertise, infrastructure) and communication (among the pathology community, regulation, and industry). We sought to identify areas warranting investment and support to manage emerging diseases in shellfish aquaculture industries, and to provide justification for resources to be directed to these areas for more effective management of shellfish health. Thirty-six priorities were identified spanning four areas: i) improving communication and frameworks; ii) essential infrastructure and expertise; iii) key research priorities; and iv) the central role of husbandry in shellfish aquaculture health management. Improving communication in every regard was recognized as the greatest need. Better coordinating efforts, improving data sharing, and improving trust on the part of industry to create buy-in and improve reporting were among identified priorities. Regulatory frameworks were recognized as needing to be flexible, given the often urgent need to act with incomplete information when a disease emerges.For essential infrastructure and expertise the workshop emphasized the continued importance of traditional fields such as pathology, bacteriology and virology, which remain foundational to aquaculture health management. Maintaining laboratory capacity is critical, in distribution as well as technologically: there is value in maintaining not just national or pan-national laboratories but provincial governmental and academic labs too, which can be more intimately engaged with local producers, and familiar with local environments. Disease challenge models and facilities were additionally identified as essential for testing hypotheses, as was expertise in epidemiology, which is sharply limited for marine systems.Discussion of key research priorities highlighted the value of long-term studies for understanding infection patterns; for appreciating dynamics like resistance and tolerance evolution versus virulence evolution; and for understanding effects of changing environments. The importance of improving ecological and evolutionary perspective in general was emphasized, as was the need to develop strategies for monitoring the diversity of potential pathogens. Establishment of mol-luscan cell lines for viral work would be a particularly valuable tool.Aquatic animal husbandry offers myriad opportunities to control pathogens. Understanding inter-sections of disease with aquaculture production for different host-pathogen systems requires continued research investment. Focused investment is also required to pursue strategies emerging from this research, including disease resistance breeding. Tighter collaboration among practical aquaculture scientists and breeders and pathologists should be promoted.Finally, funding was recognised as a continuing challenge. Priorities of donors and funding agencies do not always match commercial and health management priorities; basic surveillance is a key area difficult to support. Short funding timelines challenge our ability to address key questions and challenges, including breeding to control diseases. And there is little funding for truly trans-national, collaborative research. Managing diseases to support aquaculture growth is a global problem that demands science that is equally global, which requires the capacity to build broad international collaborations. It would be immensely beneficial for sustainable aquaculture development if trans-national research initiatives in aquatic animal health and other aquaculture related fields could be established to support innovation in this area in the future.

Alignment of Mikrocytos small subunit ribosomal gene sequences obtained in the present study. (DOCX 197 kb)

Transmission electron microscopy of Mikrocytos veneroïdes n. sp. at different stages. (DOCX 1814 kb)

The overarching goal of VIVALDI is to increase the sustainability and competitiveness of the European shellfish industry by improving the understanding of bivalve diseases and by developing innovative solutions and tools for the prevention, control and mitigation of the major pathogens affecting the main European farmed shellfish species: Pacific oyster (Crassostrea gigas), mussels (Mytilus edulis and M. galloprovincialis), European flat oyster (Ostrea edulis), clams (Venerupis philipinarum) and scallops (Pecten maximus ). The project addresses the most harmful pathogens affecting either one or more of these shellfish species: the virus OsHV-1, Vibrio species including V. aestuarianus, V. splendidus, V. harveyi and V. tapetis, as well as the parasite Bonamia ostreae. The project is committed to provide practical solutions based on the most advanced knowledge. VIVALDI will dissect the disease mechanisms associated with pathogen virulence and pathogenesis and host immune responses, develop in vivo and in vitro models, and apply "omic" approaches that will help the development of diagnostic tools and drugs against pathogen targets, and breeding programmes in a collaborative effort with industrial partners. The proposal will include a global shellfish health approach, recognising that cultured bivalves are often exposed to several pathogens simultaneously, and that disease outbreaks can be due to the combined effect of two or more pathogens. The proposal will also investigate advantages and risks of the used of disease-resistant selected animals in order to improve consumer confidence and safety.