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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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Mérou, Lecadet, Ubertini, Pouvreau and Arzul.)

The Pacific cupped oyster Crassostrea gigas is one of the most 'globalized' marine invertebrates and its production is predominant in many parts of the world including Europe. However, it is threatened by mortality events associated with pathogenic microorganisms such as the virus OsHV-1 and the bacteria Vibrio aestuarianus. C. gigas is also a host for protozoan parasites including haplosporidians. In contrast with Haplosporidium nelsoni previously detected in Europe, H. costale was considered exotic although its presence in French oysters was suggested in the 1980s based on ultrastructural examination. Here, a combination of light and transmission electron microscopy, PCR and sequencing allowed characterizing the presence of the parasite in the context of low mortality events which occurred in 2019 in France. Histological observation revealed the presence of uninucleated, plasmodial and spore stages within the connective tissues of some oysters. Ultrastructural features were similar to H. costale ones in particular the presence of axe-shaped haplosporosomes in spore cytoplasms. Three fragments of the genome including partial small subunit rRNA gene, the ITS-1, 5.8S and ITS-2 array and part of the actin gene were successfully sequenced and grouped with H. costale homologous sequences. This is the first time that the presence of H. costale was confirmed in C. gigas in France. Furthermore, a TaqMan real-time PCR assay was developed and validated [DSe = 92.6% (78.2-99.8) and DSp = 95.5% (92.3-98.6)] to enable the rapid and specific detection of the parasite. The application of the PCR assay on archived samples revealed that the parasite has been present in French oyster populations at least since 2008. Considering the little information available on this parasite, the newly developed TaqMan assay will be very helpful to investigate the temporal and geographic distribution and the life cycle of the parasite in France and more generally in C. gigas geographic range., (© 2022 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH.)

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., (© 2020 The Authors. Microbial Biotechnology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

The flat oyster Ostrea edulis is an oyster species native to Europe. It has declined to functional extinction in many areas of the NE Atlantic for several decades. Factors explaining this decline include over-exploitation of natural populations and diseases like bonamiosis, regulated across both the EU and the wider world and caused by the intracellular protozoan parasite Bonamia ostreae. To date, very limited sequence data are available for this Haplosporidian species. We present here the first transcriptome of B. ostreae. As this protozoan is not yet culturable, it remains extremely challenging to obtain high-quality -omic data. Thanks to a specific parasite isolation protocol and a dedicated bioinformatic pipeline, we were able to obtain a high-quality transcriptome for an intracellular marine micro-eukaryote, which will be very helpful to better understand its biology and to consider the development of new relevant diagnostic tools. [ABSTRACT FROM AUTHOR]

The SAR group (Stramenopila, Alveolata, Rhizaria) is one of the largest clades in the tree of eukaryotes and includes a great number of parasitic lineages. Rhizarian parasites are obligate and have devastating effects on commercially important plants and animals but despite this fact, our knowledge of their biology and evolution is limited. Here, we present rhizarian transcriptomes from all major parasitic lineages in order to elucidate their evolutionary relationships using a phylogenomic approach. Our results suggest that Ascetosporea, parasites of marine invertebrates, are sister to the novel clade Apofilosa. The phytomyxean plant parasites branch sister to the vampyrellid algal ectoparasites in the novel clade Phytorhiza. They also show that Ascetosporea + Apofilosa + Retaria + Filosa + Phytorhiza form a monophyletic clade, although the branching pattern within this clade is difficult to resolve and appears to be model-dependent. Our study does not support the monophyly of the rhizarian parasitic lineages (Endomyxa), suggesting independent origins for rhizarian animal and plant parasites. [ABSTRACT FROM AUTHOR]

Abstract: Bonamia ostreae is an intracellular protozoan parasite, infecting haemocytes of the European flat oyster Ostrea edulis. Oyster defence mechanisms mainly rely on haemocytes. In the present study in vitro interactions between parasites and flat oyster haemocytes were investigated using flow cytometry and light microscopy. Haemocyte parameters including: non specific esterase activity, reactive oxygen species (ROS) production and phagocytosis were monitored using flow cytometry after 2 h cell incubation with live and dead B. ostreae. Two ratios of parasites per haemocyte were tested (5:1 and 10:1), haemocytes alone were used as controls and the experiment was carried out three times. Flow cytometry revealed a decrease of non specific esterase activities and ROS production by haemocytes after incubation with live parasites, while there was little difference in phagocytosis activity when compared with controls. Similarly, dead parasites induced a decrease in haemocyte activities but to a lesser extent compared to live parasites. These results suggest that B. ostreae actively contributes to the modification of haemocyte activities in order to ensure its own intracellular survival. [Copyright &y& Elsevier]

The article presents information on a study which investigated the effect of temperature and salinity of different seawater media on the survival of Bonamia ostreae, a parasite that infects flat oysters Ostrea edulis. A brief overview is given on the protistan parasite the belongs to the phylum Haplosporidia. It discusses the incubation of purified parasites in various seawater media at various temperatures and then subjected to a range of salinities. It explores the purification of Bonamia ostreae. It also relates of the findings of the analysis of viability of the parasite using flow cytometry protocols.

Abstract: The life cycle of the species forming the genus Marteilia (Phylum Paramyxea), which are marine protozoan parasites of molluscs, is still poorly known, although there is evidence of the need for intermediate hosts. To date, only the calanoid copepod Acartia (Paracartia) grani has been demonstrated to be involved in the life cycle of the parasite infecting flat oysters growing in ponds with a low biodiversity. However, no complex natural environments, such as bays or estuaries, have been studied for a similar purpose. Therefore, a survey for the presence of the protozoan Marteilia was conducted by PCR on the zooplankton community of a natural ecosystem, the Alfacs and Fangar bays in the Delta de l''Ebre (NW Mediterranean). Identification of zooplankton species found infected allowed two new Marteilia hosts to be proposed: the cyclopoida Oithona sp. and an indeterminate harpaticoida species. This is the first report of Marteilia in zooplankton species sampled from a natural enzootic area. [Copyright &y& Elsevier]

• M. refringens, B. ostreae and B. exitiosa are regulated parasites of flat oysters. • First validated PCR detecting all regulated pathogens of flat oysters in one step. • Multiplex PCR performances similar or higher than currently recommended PCR assays. • Represents a substantial improvement in time, resources and accuracy. • Useful to monitors parasites prevalence or for demonstration of freedom. Aquaculture including shellfish production is an important food resource worldwide which is particularly vulnerable to infectious diseases. Marteilia refringens, Bonamia ostreae and Bonamia exitiosa are regulated protozoan parasites infecting flat oysters Ostrea edulis that are endemic in Europe. Although some PCR assays have been already developed for their detection, a formal validation to assess the performances of those tools is often lacking. In order to facilitate the diagnosis of flat oyster regulated diseases, we have developed and evaluated a new multiplex Taqman® PCR allowing the detection of both M. refringens and Bonamia sp. parasites in one step. First part of this work consisted in assessing analytical sensitivity and specificity of the new PCR assay. Then, diagnostic performances were assessed by testing a panel of field samples with the new real-time PCR and currently recommended conventional PCR methods for the detection of M. refringens and Bonamia sp. Samples were collected from the main flat oyster production sites in France (N = 386 for M. refringens and N = 349 for B. ostreae). In the absence of gold standard, diagnostic sensitivity and specificity of the new PCR were estimated through Bayesian latent class analysis (DSe 87,2% and DSp 98,4% for the detection M. refringens , DSe 77,5% and DSp 98,4% for the detection of Bonamia sp.). Those results suggest equivalent performances for the detection of Bonamia sp. and an improved sensitivity for the detection of M. refringens compared to commonly used conventional protocols. Finally, the new PCR was evaluated in the context of an inter-laboratory comparison study including 17 European laboratories. Results revealed a very good reproducibility with a global accordance (intra-laboratory precision) >96% and a global concordance (inter-laboratory precision) >93% for both targets, demonstrating that this new tool is easily transferable to different laboratory settings. This is the first assay designed to detect both Marteilia refringens and Bonamia sp. in a single step and it should allow reducing the number of analysis to monitor both diseases, and where relevant to demonstrate freedom from infection. [ABSTRACT FROM AUTHOR]