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.

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.

The new insights into the oyster antimicrobial defense: Cellular, molecular and genetic view.

Oysters are sessile filter feeders that live in close association with abundant and diverse communities of microorganisms that form the oyster microbiota. In such an association, cellular and molecular mechanisms have evolved to maintain oyster homeostasis upon stressful conditions including infection and changing environments. We give here cellular and molecular insights into the Crassostrea gigas antimicrobial defense system with focus on antimicrobial peptides and proteins (AMPs). This review highlights the central role of the hemocytes in the modulation and control of oyster antimicrobial response. As vehicles for AMPs and other antimicrobial effectors, including reactive oxygen species (ROS), and together with epithelia, hemocytes provide the oyster with local defense reactions instead of systemic humoral ones. These reactions are largely based on phagocytosis but also, as recently described, on the extracellular release of antimicrobial histones (ETosis) which is triggered by ROS. Thus, ROS can signal danger and activate cellular responses in the oyster. From the current literature, AMP production/release could serve similar functions. We provide also new lights on the oyster genetic background that underlies a great diversity of AMP sequences but also an extraordinary individual polymorphism of AMP gene expression. We discuss here how this polymorphism could generate new immune functions, new pathogen resistances or support individual adaptation to environmental stresses.