Environmental distribution and seasonal dynamics of Marteilia refringens and Bonamia ostreae, two protozoan parasites of the European flat oyster, Ostrea edulis.

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.

Passive Samplers, a Powerful Tool to Detect Viruses and Bacteria in Marine Coastal Areas.

The detection of viruses and bacteria which can pose a threat either to shellfish health or shellfish consumers remains difficult. The current detection methods rely on point sampling of water, a method that gives a snapshot of the microorganisms present at the time of sampling. In order to obtain better representativeness of the presence of these microorganisms over time, we have developed passive sampling using the adsorption capacities of polymer membranes. Our objectives here were to assess the feasibility of this methodology for field detection. Different types of membrane were deployed in coastal waters over 2 years and the microorganisms tested using qPCR were: human norovirus (NoV) genogroups (G)I and II, sapovirus, Vibrio spp. and the species Vibrio alginolyticus, V. cholerae, V. vulnificus, and V. parahaemolyticus, OsHV-1 virus, and bacterial markers of fecal contamination. NoV GII, Vibrio spp., and the AllBac general Bacteroidales marker were quantified on the three types of membrane. NoV GII and OsHV-1 viruses followed a seasonal distribution. All membranes were favorable for NoV GII detection, while Zetapor was more adapted for OsHV-1 detection. Nylon was more adapted for detection of Vibrio spp. and the AllBac marker. The quantities of NoV GII, AllBac, and Vibrio spp. recovered on membranes increased with the duration of exposure. This first application of passive sampling in seawater is particularly promising in terms of an early warning system for the prevention of contamination in oyster farming areas and to improve our knowledge on the timing and frequency of disease occurence.

Current distribution and potential expansion of the harmful benthic dinoflagellate Ostreopsis cf. siamensis towards the warming waters of the Bay of Biscay, North-East Atlantic.

In a future scenario of increasing temperatures in North-Atlantic waters, the risk associated with the expansion of the harmful, benthic dinoflagellate Ostreopsis cf. siamensis has to be evaluated and monitored. Microscopy observations and spatio-temporal surveys of environmental DNA (eDNA) were associated with Lagrangian particle dispersal simulations to: (i) establish the current colonization of the species in the Bay of Biscay, (ii) assess the spatial connectivity among sampling zones that explain this distribution, and (iii) identify the sentinel zones to monitor future expansion. Throughout a sampling campaign carried out in August to September 2018, microscope analysis showed that the species develops in the south-east of the bay where optimal temperatures foster blooms. Quantitative PCR analyses revealed its presence across almost the whole bay to the western English Channel. An eDNA time-series collected on plastic samplers showed that the species occurs in the bay from April to September. Due to the water circulation, colonization of the whole bay from the southern blooming zones is explained by inter-site connectivity. Key areas in the middle of the bay permit continuous dispersal connectivity towards the north. These key areas are proposed as sentinel zones to monitor O. cf. siamensis invasions towards the presumably warming water of the North-East Atlantic.

An eDNA/eRNA-based approach to investigate the life cycle of non-cultivable shellfish micro-parasites: the case of Bonamia ostreae, a parasite of the European flat oyster Ostrea edulis.

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

Is pallial mucus involved in Ostrea edulis defenses against the parasite Bonamia ostreae?

Bonamia ostreae is an intrahemocytic parasite that has been responsible for severe mortalities in the flat oyster Ostrea edulis since the 1970́s. The Pacific oyster Crassostrea gigas is considered to be resistant to the disease and appears to have mechanisms to avoid infection. Most studies carried out on the invertebrate immune system focus on the role of hemolymph, although mucus, which covers the body surface of molluscs, could also act as a barrier against pathogens. In this study, the in vitro effect of mucus from the oyster species Ostrea edulis and C. gigas on B. ostreae was investigated using flow cytometry. Results showed an increase in esterase activities and mortality rate of parasites exposed to mucus from both oyster species. In order to better understand the potential role of mucus in the defense of the oyster against parasites such as B. ostreae, liquid chromatography and tandem mass spectrometry were used to describe and compare mucus protein composition from both species. In all oyster species, pallial mucus contains a high level of proteins; however, O. edulis mucus produced a variety of proteins that could be involved in the immune response against the parasite, including Cu/Zn extracellular superoxide dismutase, thioxiredoxin, peroxiredon VI, heat shock protein 90 as well as several hydrolases. Conversely, a different set of antioxidant proteins, hydrolases and stress related proteins were identified in mucus from C. gigas. Our results suggest an innate immunity adaptation of oysters to develop a specific response against their respective pathogens. The mucosal protein composition also provides new insights for further investigations into the immune response in oysters.

Involvement of apoptosis in the dialogue between the parasite Bonamia ostreae and the flat oyster Ostrea edulis.

The protozoan parasite Bonamia ostreae has been associated with the decline of flat oyster Ostrea edulis populations in some European countries. Control of shellfish diseases mostly relies on prevention measures including transfer restrictions and stock management measures such as breeding programmes. These prevention and mitigation measures require a better understanding of interactions between host and pathogens. Previous in vitro studies allowed identifying apoptosis as a mechanism activated by the flat oyster in response to B. ostreae. However, these experiments also suggested that the parasite is able to regulate apoptosis in order to survive and multiply within hemocytes. By simplifying the conditions of infection, in vitro studies allow identifying most distinct features of the response of the host. In order to appreciate the relative importance of apoptosis in this response at the oyster scale, in vivo trials were carried out by injecting with parasites oysters from two French locations, Quiberon Bay (Brittany) and Diana Lagoon (Corsica). Apoptosis was investigated on pools of hemolymph from oysters collected at early and later times after injection using previously developed tools. Apoptotic cellular activities including intracytoplasmic calcium concentration, mitochondrial membrane potential and phosphatidyl serine externalization were analysed using flow cytometry. Moreover, the expression of flat oyster genes involved in both extrinsic and intrinsic pathways was measured using real time quantitative PCR.