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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 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., 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 © 2022 Chevignon, Dotto-Maurel, Serpin, Chollet and Arzul.)

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.)

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., (Copyright © 2019. Published by Elsevier Inc.)

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Bonamia ostreae has been associated with the decline of flat oyster Ostrea edulis populations in some European countries. This obligatory intracellular parasite persists and multiplies into hemocytes. Previous in vitro experiments showed that apoptosis is activated in hemocytes between 1 h and 4 h of contact with the parasite. The flat oyster uses the apoptosis pathway to defend against B. ostreae. However, the parasite might be also able to modulate this response in order to survive in its host. In order to investigate this hypothesis the apoptotic response of the host was evaluated using flow cytometry, transmission electron microscopy and by measuring the response of genes involved in the apoptotic pathway after 4 h. In parallel, the parasite response was investigated by measuring the expression of B. ostreae genes involved in different biological functions including cell cycle and cell death. Obtained results allow describing molecular apoptotic pathways in O. edulis and confirm that apoptosis is early activated in hemocytes after a contact with B. ostreae. Interestingly, at cellular and molecular levels this process appeared downregulated after 44 h of contact. Concurrently, parasite gene expression appeared reduced suggesting that the parasite could inhibit its own metabolism to escape the immune response.

The in vitro model Ostrea edulis hemocyte - Bonamia ostreae is interesting to investigate host-parasite interactions at the cellular level. Indeed, this unicellular parasite infects the flat oyster Ostrea edulis and multiplies within hemocytes, the central effectors of oyster defenses. Apoptosis is a mechanism used by many organisms to eliminate infected cells. In order to study the potential involvement of this mechanism in the oyster response to B. ostreae, in vitro experiments were carried out by exposing hemocytes from the naturally susceptible oyster O. edulis and a resistant oyster species Crassostrea gigas to live and heat-inactivated parasites. Hemocyte apoptotic response was measured using a combination of flow cytometry and microscopy analyses. Whatever the host species was, the parasite was engulfed in hemocytes and induced an increase of apoptotic parameters including intracytoplasmic calcium concentration, mitochondrial membrane potential or phosphatidyl-serine externalization as well as ultrastructural modifications. However, the parasite appears more able to infect flat oyster than cupped oyster hemocytes and the apoptotic response was more important against live than dead parasites in the natural host than in C. gigas. Our results suggest that O. edulis specifically responds to B. ostreae by inducing apoptosis of hemocytes., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Apoptosis is a fundamental feature in the development of many organisms and tissue systems. It is also a mechanism of host defense against environmental stress factors or pathogens by contributing to the elimination of infected cells. Hemocytes play a key role in defense mechanisms in invertebrates and previous studies have shown that physical or chemical stress can increase apoptosis in hemocytes in mollusks. However this phenomenon has rarely been investigated in bivalves especially in the flat oyster Ostrea edulis. The apoptotic response of hemocytes from flat oysters, O. edulis, was investigated after exposure to UV and dexamethasone, two agents known to induce apoptosis in vertebrates. Flow cytometry and microscopy were combined to demonstrate that apoptosis occurs in flat oyster hemocytes. Investigated parameters like intracytoplasmic calcium activity, mitochondrial membrane potential and phosphatidyl-serine externalization were significantly modulated in cells exposed to UV whereas dexamethasone only induced an increase of DNA fragmentation. Morphological changes were also observed on UV-treated cells using fluorescence microscopy and transmission electron microscopy. Our results confirm the apoptotic effect of UV on hemocytes of O. edulis and suggest that apoptosis is an important mechanism developed by the flat oyster against stress factors., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Marteilia refringens is a protozoan parasite recognized as a significant pathogen of the European flat oyster Ostrea edulis. The life cycle of this species is still poorly known, although there is evidence of the need for intermediate host(s). In the present study, we have used molecular approaches to identify this parasite in samples of the dwarf oyster Ostrea stentina after reports of massive mortality along the Tunisian coasts. In 2009 we evaluated the status of O. stentina from Monastir and checked if there was an infection with M. refringens, using polymerase chain reaction assays. Of the 103 tested O. stentina, 85 were PCR-positive using a Marteilia genus-specific assay. Additional assays were subsequently carried out on some samples collected in 2010 in Monastir and processed for histology, transmission electron microscopy and complementary molecular analyses. PCR was carried out to amplify the IGS and ITS regions. Histological and transmission electron microscopy analyses allowed us to confirm the presence of this parasite in the digestive gland tissue of O. stentina and to characterize it at the ultrastructural level. This is the first record of the occurrence of M. refringens in the oyster O. stentina along the Tunisian coasts., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Bonamia ostreae is an intracellular protistan parasite affecting flat oysters Ostrea edulis. It can be detected in juveniles but mortalities mainly affect oysters which are more than 2 years old. The parasite is usually observed inside haemocytes and sometimes free, notably in gill epithelia suggesting a parasite release through this organ. However, the infective form and ways of entry and release remain undetermined. Flat oysters incubate their larvae in their pallial cavity for 8-10 days before releasing them into the water column. Flat oysters in Bay of Quiberon in South Brittany (France) are known to be infected with B. ostreae since 1979 and is the most important area in France for O. edulis spat collection. Flat oysters incubating larvae were sampled in this area during summertime between 2007 and 2009. Both adults and larvae were preserved and assayed by PCR and in situ hybridisation (ISH). PCR tests revealed the presence of parasite DNA in some adults and larvae. Specific labelling could be detected by ISH in gills, digestive system, gonad and mantle in adults and in the epithelium surrounding the visceral cavity of some larvae. Our results demonstrate that larvae can be infected with B. ostreae. Larvae might thus contribute to the spread of the parasite during their planktonic life. In addition, their transfer for aquaculture purpose should be controlled especially when they are exported from infected zones., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Bonamia ostreae is a protozoan, affiliated to the order Haplosporidia and to the phylum Cercozoa. This parasite is intracellular and infects haemocytes, cells notably involved in oyster defence mechanisms. Bonamiosis due to the parasite B. ostreae is a disease affecting the flat oyster, Ostrea edulis. The strategies used by protozoan parasites to circumvent host defence mechanisms remain largely unknown in marine bivalve molluscs. In the present work, in vitro experiments were carried out in order to study the interactions between haemocytes from O. edulis and purified parasite, B. ostreae. We monitored cellular and molecular responses of oyster haemocytes by light microscopy, flow cytometry and real-time PCR 1, 2, 4 and 8h p.i. Light microscopy was used to measure parasite phagocytosis by oyster haemocytes. Parasites were observed inside haemocytes 1h p.i. and the parasite number increased during the time course of the experiment. Moreover, some bi-nucleated and tri-nucleated parasites were found within haemocytes 2 and 4h p.i., respectively, suggesting that the parasite can divide inside haemocytes. Host responses to B. ostreae were investigated at the cellular and molecular levels using flow cytometry and real-time PCR. Phagocytosis capacity of haemocytes, esterase activity and production of radical oxygen species appeared modulated during the infection with B. ostreae. Expression levels of expressed sequence tags selected in this study showed variations during the experiment as soon as 1h p.i. An up-regulation of galectin (OeGal), cytochrome p450 (CYP450), lysozyme, omega GST (OGST), super oxide dismutase Cu/Zn (Oe-SOD Cu/Zn) and a down-regulation of the extracellular super oxide dismutase SOD (Oe-EcSOD) were observed in the presence of the parasite. Finally, the open reading frames of both SODs (Oe-SOD Cu/Zn and Oe-EcSOD) were completely sequenced. These findings provide new insights into the cellular and molecular bases of the host-parasite interactions between the flat oyster, O. edulis, and the parasite, B. ostreae., (Copyright © 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Bonamiosis due to the parasite Bonamia ostreae is a disease affecting the flat oyster Ostrea edulis. B. ostreae is a protozoan, affiliated to the order of haplosporidia and to the cercozoan phylum. This parasite is mainly intracellular, infecting haemocytes, cells notably involved in oyster defence mechanisms. Suppression subtractive hybridisation (SSH) was carried out in order to identify oyster genes differentially expressed during an infection of haemocytes with B. ostreae. Forward and reverse banks allowed obtaining 1104 and 1344 clones respectively, among which 391 and 480 clones showed a differential expression between both tested conditions (haemocytes alone versus haemocytes in contact with parasites). ESTs of interest including genes involved in cytoskeleton, respiratory chain, detoxification membrane receptors, and immune system were identified. The open reading frames of two selected genes (galectin and IRF-like) were completely sequenced and characterized. Real time PCR assays were developed to study the relative expression of candidate ESTs during an in vitro infection of haemocytes by live and dead parasites. Haemocyte infection with B. ostreae induced an increased expression of omega glutathione S-transferase (OGST), superoxide dismutase (SOD), tissue inhibitor of metalloproteinase (TIMP), galectin, interferon regulatory factor (IRF-like) and filamin genes., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Bonamia ostreae is an intrahaemocytic protozoan affecting Ostrea edulis. The parasite multiplies within haemocytes without being degraded and involves changes in cellular activities. Studies aiming at better understanding host response to a pathogen at the transcriptome levels are frequently based on the use of real time PCR assays, which require some reference genes. However, very few sequence data is available for O. edulis in public databases. Subtracted cDNA libraries were constructed from the O. edulis haemocytes in order to identify genes involved in host reactions against the parasite and quantitative real time PCR assays were developed to study expression of these genes. In this context, identification of reference genes and study of their relative expression stability were required for quantitative real time PCR normalization. The expression of 5 potential candidate reference genes from O. edulis (ie elongation factor 1 alpha (EF1-α), 60S ribosomal protein L5 (L5), glyceraldehyde 3-phosphate-dehydrogenase (GAPDH), polyubiquitin (Ubiq) and β-actin (ACT)) was studied using RNAs extracted from pools of haemocytes in contact with the parasite B. ostreae and haemocytes alone. Gene expression was quantified by real time PCR and expression stability was analysed with two analytical approaches GeNorm and NormFinder. GAPDH and EF1-α were identified as the most stable genes with the GeNorm analysis. Whatever were the tested conditions, EF1-α was also found as the most stable gene using Normfinder. The less stable gene was β-actin although this gene is commonly used as housekeeping gene in many studies. Our results suggest using GAPDH and EF1-α combined as reference genes when studying expression levels in haemocytes of O. edulis. In addition, the complete ORF of these two genes was characterized., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Bonamia ostreae is an intracellular protozoan which is recognized as a cause of mortality in European populations of flat oysters (Ostrea edulis). Based on the recent characterization of actin genes of B. ostreae, specific primers were designed for real-time PCR using SYBR Green chemistry. Specificity was demonstrated by the unique melting temperature peak observed in positive samples and by the lack of amplification in samples of oysters infected by closely related parasites, including Bonamia exitiosa. A calibration curve using a cloned template was defined to estimate copy number. The assay had a 6 log- dynamic range, mean inter- and intra-assay variation coefficients of <1% and a minimum detection limit of 50 gene copies per reaction. Using infected oyster samples as templates, the assay was at least 10-fold more sensitive than conventional PCR. The quantitative assay was applied to test 132 oysters, and results were compared with the heart imprint method. There was a strong correlation between both techniques, and the results showed that the real-time PCR assay should be useful for studies of the ecology of B. ostreae and its host-parasite relationship.

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.

Bonamiosis due to the intrahaemocytic protistan parasite Bonamia ostreae is a European endemic disease affecting the flat oyster Ostrea edulis. The parasite has been described in various ecosystems from estuaries to open sea, but no clear correlation has yet been demonstrated between disease development and environmental parameters. In this study, the effect of temperature and salinity on the survival of purified parasites maintained in vitro in seawater was investigated by flow cytometry. Purified parasites were incubated in various seawater media (artificial seawater, natural seawater, seabed borewater) at various temperatures (4, 15 and 25 degrees C) and subjected to a range of salinities from 5 to 45 g l(-1). Parasites were collected after 12, 24 and 48 h of incubation for flow cytometry analyses including estimation of parasite mortality and parasite viability through detection of non-specific esterase activities. Artificial seawater appeared unsuitable for parasite survival, and results for all media showed a significantly lower survival at 25 degrees C compared to 4 degrees C and 15 degrees C. Moreover, high salinities (> or = 35 g l(-1)) favoured parasite survival and detection of esterase activities. Flow cytometry appears to be a suitable technique to investigate survival and activities of unicellular parasites like B. ostreae under varied conditions. Although these results contribute to a better understanding of existing interactions between the parasite B. ostreae and its environment, validation through epidemiological surveys in the field is also needed.

An Australian (New South Wales) isolate of Bonamia was characterised at the molecular level by sequencing the 18S-ITS-1 region of the small subunit rRNA operon obtained from flat oysters Ostrea angasi shown to be infected by histological examination. Sequence data alignment with homologous genes from 2 other isolates of Bonamia (New Zealand and France) revealed high levels of nucleotide identity with both isolates. However, the Australian Bonamia is shown to be more closely related to the New Zealand isolate, suggesting the existence of an oceanic subgroup of Bonamia.

The development of molecular diagnostic assays with increased sensitivity compared with conventional histological techniques is highly desirable for effective management of bonamiosis in cultured oyster stocks and wild populations. A real-time TaqMan PCR assay was developed for the specific detection of Bonamia species in infected oyster tissues. The TaqMan assay was shown to be significantly more sensitive than histopathology. Although a real-time TaqMan PCR assay is comparable with conventional PCR in terms of sensitivity, it offers the advantages that it is a rapid test and has a very low risk of sample cross-contamination. Furthermore, it can be optimised to quantify the parasite load in samples. The assay detected Bonamia isolates from Australia, New Zealand, Europe, Canada, Chile and the USA and therefore demonstrated genus specificity as tested in this study.

Bonamiosis is a parasitic disease (causative agent: Bonamia ostreae) affecting the European flat oyster Ostrea edulis, responsible for a drastic decline in its aquaculture production. Selective breeding programs for resistance to bonamiosis have been undertaken to counter this disease. In the present study, a 6-month cohabitation challenge experiment was performed in order to transmit the disease from wild oysters injected with the parasite to two tested families of oysters (20 and 8-month old at the beginning of the experiment, with different genetic backgrounds) originating from a selective breeding program developed by IFREMER in France. Mortalities were checked daily and ventricular heart smears were performed on dying or moribund oysters to detect the level of infection by B. ostreae. Mortality started after 4 months of cohabitation in the tested oysters. The cumulative mortalities after 6 months were 58% for the wild oysters, 9% for Family 1 (20-month old) and 20% for Family 2 (8-month old). In the dying oysters, the parasite could be detected in 67% of the wild oysters, 68% of Family 1 and 89% of Family 2. It was detected in only 11% of the surviving oysters of Family 2. The mortality and the level of infection by the parasite were significantly higher in Family 2 than in Family 1. Our results demonstrate that prespawning oysters as young as 1 year-old can become infected with the parasite and, most importantly, can die from bonamiosis. This result is inconsistent with the commonly accepted critical age of 2 years-old for the disease development. Additionally, no clear relationship between shell length and level of infection was observed. We also review the different methods for infection of the European flat oyster O. edulis with B. ostreae under experimental conditions and their main results. [ABSTRACT FROM AUTHOR]