Investigating the Environmental Survival of Marteilia refringens, a Marine Protozoan Parasite of the Flat Oyster Ostrea edulis, Through an Environmental DNA and Microscopy-Based Approach

Abstract

Outside-host parasite survival is a key parameter to better understand disease mechanisms, especially for marine pathogens transmitted from one host to another through an environmental stage. For non-cultivable micro-parasites like Marteilia refringens, a protozoan parasite infecting the flat oyster Ostrea edulis, investigating this parameter requires innovative approaches. In the present study, we have developed an Environmental DNA (eDNA)-based method allowing detecting and quantifying up to 25 and 10 parasites DNA in seawater and sediment, respectively. This method was used in combination with light and transmission electron microscopy (TEM) to study experimentally parasite survival in seawater and flat oyster faeces after its release from naturally infected oysters. M. refringens DNA could be detected up to 20 days, in both seawater and oyster faeces with a more stable detection over time in the latter. Light and transmission microscopy confirm that parasites leaving flat oysters are sporangia. We also observed a membrane dissolution of the sporangia over time that could indicate the release of parasite spore. This study not only improves our understanding of M. refringens ecology but also highlights the interest to combine molecular and microscopical analysis to study non-cultivable micro-parasites life cycle outside their host.

Fifty years of research to counter the decline of the European flat oyster (Ostrea edulis): a review of French achievements and prospects for the restoration of remaining beds and revival of aquaculture production

“…This Taqman assay was optimized in order to detect parasite DNA (and RNA for B. ostreae) in environmental samples. These eDNA-based approaches are very promising as they avoid killing oysters, which is important in the context of zone status evaluation, surveillance and restoration programs (Mérou et al, 2020;Mérou, 2021;Mérou et al, 2022).…”

“…Juveniles and older life stages are known to be susceptible (Grizel, 1985). Recently, an eDNA-based approach allowed parasite DNA to be detected in seawater 20 days after release from the flat oysters (Mérou et al, 2022).…”

“…Parasite enrichment protocols have been developed, but the lack of culture method for these parasites prevents us from obtaining their DNA in sufficient quantity and quality. Nevertheless, the recent development of eDNA-based approaches for the detection of B. ostreae and M. refringens (Mérou et al, 2020;Jørgensen et al, 2020;Mérou et al, 2022) will certainly allow their distribution in the ecosystem to be clarified and the health status of flat oyster populations to be monitored without killing oysters. Finally, other pathogens than M. refringens and B. ostreae have also been described in Ostrea edulis (see Colsoul et al, 2021 for a review) and would deserve to be monitored so as to better evaluate their impact on flat oyster populations.…”

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

“…Although the infection from Ostrea edulis and Mytilus galloprovincialis to the copepod has been successfully demonstrated ( Audemard et al., 2002 ; Carrasco et al., 2008 ), the transmission from the copepod to flat oysters or mussels has not been proved yet, leaving its role into the parasite life-cycle unclear. M. refringens enters bivalves through feeding process and then develops in the digestive epithelia by endogenous divisions before being eliminated with faeces as sporangia containing mature spores ( Grizel et al., 1974 ; Perkins, 1976 ; Audemard, 2001 ; Mérou et al., 2022 ). In flat oysters, M. refringens detection frequency peak occurs in summer (between June to September) whereas the parasite is usually absent or found in low numbers in winter and early spring ( Audemard et al., 2001 ).…”

“…In other studies, M. refringens detection frequency showed two peaks in summer and spring ( Carrasco et al., 2007 ; Boyer et al., 2013 ; Arzul et al., 2014 ). More recently, an environmental DNA (eDNA) based study revealed that M. refringens DNA could be detected in seawater and flat oysters faeces at least during 20 days after parasites were released from oysters, with a more stable detection over time in faeces, suggesting a better survival of the parasite in this matrix ( Mérou et al., 2022 ). Although it is now suggested to be a different species, M. pararefringens (previously M-type) transmission also seems restricted to the warmest period of the year as suggested by ( Bøgwald et al., 2022 ) in a study carried out on blue mussel Mytilus edulis in a heliothermic marine oyster lagoon in western Norway.…”