De Novo Transcriptome Assembly and Analysis of the Flat Oyster Pathogenic Protozoa Bonamia Ostreae.

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.

What do the terms resistance, tolerance, and resilience mean in the case of Ostrea edulis infected by the haplosporidian parasite Bonamia ostreae.

The decline of the European flat oyster Ostrea edulis represents a loss to European coastal economies both in terms of food security and by affecting the Good Environmental Status of the marine environment as set out by the European Council's Marine Strategy Framework Directive (2008/56/EC). Restoration of O. edulis habitat is being widely discussed across Europe, addressing key challenges such as the devastating impact of the haplosporidian parasite Bonamia ostreae. The use of resistant, tolerant, or resilient oysters as restoration broodstock has been proposed by restoration practitioners, but the definitions and implications of these superficially familiar terms have yet to be defined and agreed by all stakeholders. This opinion piece considers the challenges of differentiating Bonamia resistance, tolerance, and resilience; challenges which impede the adoption of robust definitions. We argue that, disease-resistance is reduced susceptibility to infection by the parasite, or active suppression of the parasites ability to multiply and proliferate. Disease-tolerance is the retention of fitness and an ability to neutralise the virulence of the parasite. Disease-resilience is the ability to recover from illness and, at population level, tolerance could be interpreted as resilience. We concede that further work is required to resolve practical uncertainty in applying these definitions, and argue for a collaboration of experts to achieve consensus. Failure to act now might result in the future dispersal of this disease into new locations and populations, because robust definitions are important components of regulatory mechanisms that underpin marine management.

A new multiplex real-time PCR assay to improve the diagnosis of shellfish regulated parasites of the genus Marteilia and Bonamia.

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.

Ephemeral detection of Bonamia exitiosa (Haplosporida) in adult and larval European flat oysters Ostrea edulis in the Solent, United Kingdom.

The haplosporidian parasite Bonamia exitiosa was detected using PCR in four adult and six larval brood samples of the European flat oyster Ostrea edulis from the Solent, UK. This represents the second reported detection of this parasite along the south coast of England. Adult oysters were collected and preserved from seabed populations or restoration broodstock cages between 2015 and 2018. The larvae within brooding adults sampled during 2017 and 2018 were also preserved. Molecular analysis of all samples was performed in 2019. The DNA of B. exitiosa was confirmed to be present within the gill tissue of one oyster within the Portsmouth wild fishery seabed population (n = 48), sampled in November 2015; the congeneric parasite Bonamia ostreae was not detected in this individual. This is the earliest record of B. exitiosa in the Solent. Concurrent presence of both B. ostreae and B. exitiosa, determined by DNA presence, was confirmed in the gill and heart tissue of three mature individuals from broodstock cages sampled in October 2017 (n = 99), two from a location on the River Hamble and one from the Camber Dock in Portsmouth Harbour. B. exitiosa was not detected in the November 2018 broodstock populations. A total of six larval broods were positive for B. exitiosa, with five also positive for B. ostreae. None of the brooding adults were positive for B. exitiosa suggesting that horizontal transmission from the surrounding environment to the brooding larvae is occurring. Further sampling of broodstock populations conducted by the Fish Health Inspectorate at the Centre for Environment, Fisheries and Aquaculture Science in June 2019 did not detect infection of O. edulis by B. exitiosa. These findings together suggest that the pathogen has not currently established in the area.

A Naïve Population of European Oyster Ostrea edulis with Reduced Susceptibility to the Pathogen Bonamia ostreae: Are S-Strategy Life Traits Providing Protection?

European populations of the native flat oyster, Ostrea edulis, have been heavily depleted by two protozoan parasites, Marteila refringens and Bonamia ostreae, with mortalities of up to 90% reported in naïve populations. However, in studies carried out over a 10-year period, researching the parasite-host relationship of B. ostreae and O. edulis in several age cohorts within a naïve O. edulis population from Loch Ryan (LR), Scotland, 1364 specimens were challenged and only 64 (5%), across multiple testing protocols, screened positive for B. ostreae. This article presents a case for the development of S-strategy life traits in the LR population that coincide with enhanced immune function and survival. Oysters are considered typical r-strategists (small in size with fast development and high fecundity) while S-strategists, as outlined in Grime's (1977) competitor-stress tolerant-ruderal (C-S-R) triangle theory, are characterized by slow growth and investment in the durability of individuals. This study hypothesizes that slower growth and reduced reproductive output in LR oysters has resulted in the investment of an enhanced immune function and reduced susceptibility to B. ostreae that is, r-strategists with S-strategy life traits equates to protection from significant pathogens. The findings presented here within provide a strong case study for local adaptation of energy allocation and provides empirical support for the C-S-R triangle theory in a marine organism.

Haplosporidian host:parasite interactions.

The host:parasite interactions of the 3 serious haplosporidian pathogens of oysters, on which most information exists, are reviewed. They are Bonamia ostreae in Ostrea spp. and Crassostrea gigas; Bonamia exitiosa in Ostrea spp.; and Haplosporidium nelsoni in Crassostrea spp. Understanding the haemocytic response to pathogens is constrained by lack of information on haematopoiesis, haemocyte identity and development. Basal haplospridians in spot prawns are probably facultative parasites. H. nelsoni and a species infecting Haliotis iris in New Zealand (NZAP), which have large extracellular plasmodia that eject haplosporosomes or their contents, lyse surrounding cells and are essentially extracellular parasites. Bonamia spp. have small plasmodia that are phagocytosed, haplosporosomes are not ejected and they are intracellular obligate parasites. Phagocytosis by haemocytes is followed by formation of a parasitophorous vacuole, blocking of haemocyte lysosomal enzymes and the endolysosomal pathway. Reactive oxygen species (ROS) are blocked by antioxidants, and host cell apoptosis may occur. Unlike susceptible O. edulis, the destruction of B. ostreae by C. gigas may be due to higher haemolymph proteins, higher rates of granulocyte binding and phagocytosis, production of ROS, the presence of plasma ß-glucosidase, antimicrobial peptides and higher levels of haemolymph and haemocyte enzymes. In B.exitiosa infection of Ostrea chilensis, cytoplasmic lipid bodies (LBs) containing lysosomal enzymes accumulate in host granulocytes and in B. exitiosa following phagocytosis. Their genesis and role in innate immunity and inflammation appears to be the same as in vertebrate granulocytes and macrophages, and other invertebrates. If so, they are probably the site of eicosanoid synthesis from arachidonic acid, and elevated numbers of LBs are probably indicative of haemocyte activation. It is probable that the molecular interaction, and role of LBs in the synthesis and storage of eicosanoids from arachidonic acid, is conserved in innate immunity in vertebrates and invertebrates. However, it seems likely that haplosporidians are more diverse than realized, and that there are many variations in host parasite interactions and life cycles.

Infection dynamics of Bonamia exitiosa on intertidal Ostrea angasi farms.

Bonamia spp. cause epizootics in oysters worldwide. In southern Australia, Bonamia exitiosa Hine, Cochennac and Berthe, 2001 threatens aquaculture of Ostrea angasi Sowerby, 1871. Bonamia spp. infections can display strong seasonality, but seasonal dynamics of B. exitiosa-O. angasi are unknown. Ostrea angasi naïve to B. exitiosa infection were stocked onto farms in three growing regions, and B. exitiosa was monitored seasonally for one year. Environmental parameters we measured did not correlate with B. exitiosa prevalence or infection intensities. Extreme temperatures suggest O. angasi culture systems need development. Bonamia exitiosa prevalence increased over time. After three months, O. angasi had B. exitiosa prevalence of 0.08-0.4, and after one year, the prevalence was 0.57-0.88. At some sites, O. angasi had >0.5 B. exitiosa prevalence in >6 months, but at other sites, >9 months passed before prevalence was >0.5. Bonamia exitiosa infection intensities were low with no seasonal pattern but were affected by the interaction of site, season and oyster meat:shell ratio. Understanding infection and initiating a breeding programme for resistance would provide benefits for O. angasi industry expansion.

Rapid transmission of Bonamia exitiosa by cohabitation causes mortality in Ostrea angasi.

The haplosporidian Bonamia was first detected in Australian shellfish in 1991. Australian isolates in Ostrea angasi Sowerby, 1871 were identified as Bonamia exitiosa Hine, Cochennac and Berthe, 2001, which threatens development of an O. angasi aquaculture industry. European field data suggest that Bonamia ostreae Pichot, Comps, Tigé, Grizel and Rabouin, 1980 infections in Ostrea edulis Linnaeus, 1758 build slowly, but infection dynamics of B. exitiosa in O. angasi are unknown. We investigated B. exitiosa infection in O. angasi by cohabiting uninfected juvenile O. angasi with adults infected with B. exitiosa. Oysters were sampled at 10, 21 and 40 days after cohabitation, and B. exitiosa prevalence and intensity were assessed. Bonamia exitiosa rapidly infected and caused disease in O. angasi. Mortalities began at 12 days, with ˜50% mortality by day 21 and >85% mortality by day 40. Mortalities displayed pathology consistent with clinical B. exitiosa infection. Time to first infection is likely influenced by a combination of parasite infectivity, host exposure and host immune capacity. Host death is not required for transmission, but probably facilitates release of parasites from decaying tissue. Understanding B. exitiosa transmission informs design and interpretation of field studies and aids development of management strategies for oyster aquaculture.

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.

Oyster disease in a changing environment: Decrypting the link between pathogen, microbiome and environment.

Shifting environmental conditions are known to be important triggers of oyster diseases. The mechanism(s) behind these synergistic effects (interplay between host, environment and pathogen/s) are often not clear, although there is evidence that shifts in environmental conditions can affect oyster immunity, and pathogen growth and virulence. However, the impact of shifting environmental parameters on the oyster microbiome and how this affects oyster health and susceptibility to infectious pathogens remains understudied. In this review, we summarise the major diseases afflicting oysters with a focus on the role of environmental factors that can catalyse or amplify disease outbreaks. We also consider the potential role of the oyster microbiome in buffering or augmenting oyster disease outbreaks and suggest that a deeper understanding of the oyster microbiome, its links to the environment and its effect on oyster health and disease susceptibility, is required to develop new frameworks for the prevention and management of oyster diseases.

Bonamia in Ostrea angasi: Diagnostic performance, field prevalence and intensity.

Bonamia spp. parasites threaten flat oyster (Ostrea spp.) farming worldwide. Understanding test performance is important for designing surveillance and interpreting diagnostic results. Following a pilot survey which found low Bonamia sp. intensity in farmed Ostrea angasi, we tested further oysters (n = 100-150) from each of three farms for Bonamia sp. using heart smear, histology and qPCR. We used a Bayesian Latent Class Model to assess diagnostic sensitivity (DSe) and specificity (DSp) of these tests individually or in combination, and to assess prevalence. Histology was the best individual test (DSe 0.76, DSp 0.93) compared to quantitative polymerase chain reaction (qPCR) (DSe 0.69, DSp 0.93) and heart smear (DSe 0.61, DSp 0.60). Histology combined with qPCR and defining a positive from either test as an infected case maximized test performance (DSe 0.91, DSp 0.88). Prevalence was higher at two farms in a high-density oyster growing region than at a farm cultivating oysters at lower density. Parasite intensities were lower than in New Zealand and European studies, and this is probably contributed to differences in the performance of test when compared to other studies. Understanding diagnostic test performance in different populations can support the development of improved Bonamia surveillance programs.

Molecular and cellular characterization of apoptosis in flat oyster a key mechanisms at the heart of host-parasite interactions.

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.

Marteilia refringens and Marteilia pararefringens sp. nov. are distinct parasites of bivalves and have different European distributions.

Marteilia refringens causes marteiliosis in oysters, mussels and other bivalve molluscs. This parasite previously comprised two species, M. refringens and Marteilia maurini, which were synonymized in 2007 and subsequently referred to as M. refringens 'O-type' and 'M-type'. O-type has caused mass mortalities of the flat oyster Ostrea edulis. We used high throughput sequencing and histology to intensively screen flat oysters and mussels (Mytilus edulis) from the UK, Sweden and Norway for infection by both types and to generate multi-gene datasets to clarify their genetic distinctiveness. Mussels from the UK, Norway and Sweden were more frequently polymerase chain reaction (PCR)-positive for M-type (75/849) than oysters (11/542). We did not detect O-type in any northern European samples, and no histology-confirmed Marteilia-infected oysters were found in the UK, Norway and Sweden, even where co-habiting mussels were infected by the M-type. The two genetic lineages within 'M. refringens' are robustly distinguishable at species level. We therefore formally define them as separate species: M. refringens (previously O-type) and Marteilia pararefringens sp. nov. (M-type). We designed and tested new Marteilia-specific PCR primers amplifying from the 3' end of the 18S rRNA gene through to the 5.8S gene, which specifically amplified the target region from both tissue and environmental samples.

Proteomic profile of Ostrea edulis haemolymph in response to bonamiosis and identification of candidate proteins as resistance markers.

European flat oyster Ostrea edulis populations have suffered extensive mortalities caused by bonamiosis. The protozoan parasite Bonamia ostreae is largely responsible for this disease in Europe, while its congener B. exitiosa has been detected more recently in various European countries. Both of these intracellular parasites are able to survive and proliferate within haemocytes, the main cellular effectors of the immune system in molluscs. Two-dimensional electrophoresis was used to compare the haemolymph protein profile between Bonamia spp.-infected and non-infected oysters within 3 different stocks, a Galician stock of oysters selected for resistance against bonamiosis, a non-selected Galician stock and a selected Irish stock. Thirty-four proteins with a presumably relevant role in the oyster-Bonamia spp. interaction were identified; they were involved in major metabolic pathways, such as energy production, respiratory chain, oxidative stress, signal transduction, transcription, translation, protein degradation and cell defence. Furthermore, the haemolymph proteomic profiles of the non-infected oysters of the 2 Galician stocks were compared. As a result, 7 proteins representative of the non-infected Galician oysters selected for resistance against bonamiosis were identified; these 7 proteins could be considered as candidate markers of resistance to bonamiosis, which should be further assessed.

Partial 18S rRNA sequences of apicomplexan parasite 'X' (APX), associated with flat oysters Ostrea chilensis in New Zealand.

Apicomplexa is a large phylum of parasitic protists renowned for significant negative health impacts on humans and livestock worldwide. Despite the prevalence and negative impacts of apicomplexans across many animal groups, relatively little attention has been given to apicomplexan parasites of invertebrates, especially marine invertebrates. Previous work has reported an apicomplexan parasite 'X' (APX), a parasite that has been histologically and ultrastructurally identified from the commercially important flat oyster Ostrea chilensis in New Zealand. This apicomplexan may exacerbate host vulnerability to the infectious disease bonamiosis. In this study, we report 18S rRNA sequences amplified from APX-infected O. chilensis tissues. Phylogenetic analyses clearly established that the 18S sequences were of apicomplexan origin; however, their detailed relationship to known apicomplexan groups is less resolved. Two specific probes, designed from the putative APX 18S rRNA sequence, co-localised with APX cells in in situ hybridisations, further supporting our hypothesis that the 18S sequences were from APX. These sequences will facilitate the future development of inexpensive and sensitive molecular diagnostic tests for APX, thereby assisting research focussed on the biology and ecology of this organism and its role in morbidity and mortality of O. chilensis.

Pooled sample testing for Bonamia ostreae: A tale of two SYBR Green real-time PCR assays.

Pooled testing of samples is a common laboratory practice to increase efficiency and reduce expenses. We investigated the efficacy of 2 published SYBR Green real-time PCR assays when used to detect the haplosporidian parasite Bonamia ostreae in pooled samples of infected oyster tissue. Each PCR targets a different gene within the B. ostreae genome: the actin 1 gene or the 18S rRNA gene. Tissue homogenates (150 mg) of the New Zealand flat oyster Ostrea chilensis were spiked with ~1.5 × 103 purified B. ostreae cells to create experimental pools of 3, 5, and 10. Ten positive replicates of each pool size were assayed twice with each PCR and at 2 different amounts of DNA template. The PCR targeting the actin 1 gene was unable to reproducibly detect B. ostreae in any pool size. Conversely, the 18S rRNA gene PCR could reproducibly detect B. ostreae in pools of up to 5. Using a general linear model, there was a significant difference in the number of pools that correctly detected B. ostreae between each PCR ( p < 0.01) and each pool size ( p < 0.01). It is likely that the single copy actin 1 gene is more likely to be diluted and not detected by pooling than the multi-copy 18S rRNA gene. Our study highlights that validation data are necessary for pooled sample testing because detection efficacy may not be comparable to individual sample testing.

Construction of an Ostrea edulis database from genomic and expressed sequence tags (ESTs) obtained from Bonamia ostreae infected haemocytes: Development of an immune-enriched oligo-microarray.

The flat oyster, Ostrea edulis, is one of the main farmed oysters, not only in Europe but also in the United States and Canada. Bonamiosis due to the parasite Bonamia ostreae has been associated with high mortality episodes in this species. This parasite is an intracellular protozoan that infects haemocytes, the main cells involved in oyster defence. Due to the economical and ecological importance of flat oyster, genomic data are badly needed for genetic improvement of the species, but they are still very scarce. The objective of this study is to develop a sequence database, OedulisDB, with new genomic and transcriptomic resources, providing new data and convenient tools to improve our knowledge of the oyster's immune mechanisms. Transcriptomic and genomic sequences were obtained using 454 pyrosequencing and compiled into an O. edulis database, OedulisDB, consisting of two sets of 10,318 and 7159 unique sequences that represent the oyster's genome (WG) and de novo haemocyte transcriptome (HT), respectively. The flat oyster transcriptome was obtained from two strains (naïve and tolerant) challenged with B. ostreae, and from their corresponding non-challenged controls. Approximately 78.5% of 5619 HT unique sequences were successfully annotated by Blast search using public databases. A total of 984 sequences were identified as being related to immune response and several key immune genes were identified for the first time in flat oyster. Additionally, transcriptome information was used to design and validate the first oligo-microarray in flat oyster enriched with immune sequences from haemocytes. Our transcriptomic and genomic sequencing and subsequent annotation have largely increased the scarce resources available for this economically important species and have enabled us to develop an OedulisDB database and accompanying tools for gene expression analysis. This study represents the first attempt to characterize in depth the O. edulis haemocyte transcriptome in response to B. ostreae through massively sequencing and has aided to improve our knowledge of the immune mechanisms of flat oyster. The validated oligo-microarray and the establishment of a reference transcriptome will be useful for large-scale gene expression studies in this species.

Flat oyster follows the apoptosis pathway to defend against the protozoan parasite Bonamia ostreae.

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.

Bonamia ostreae in the New Zealand oyster Ostrea chilensis: a new host and geographic record for this haplosporidian parasite.

Previous reports of the haplosporidian parasite Bonamia ostreae have been restricted to the Northern Hemisphere, including Europe, and both eastern and western North America. This species is reported for the first time in New Zealand infecting the flat oyster Ostrea chilensis. Histological examination of 149 adult oysters identified 119 (79.9%) infected with Bonamia microcells. Bonamia generic PCR of several oysters followed by DNA sequencing of a 300 bp portion of the 18S rDNA gene produced a 100% match with that of B. ostreae. All DNA-sequenced products also produced a B. ostreae PCR-restriction fragment length polymorphism (PCR-RFLP) profile. Bonamia species-specific PCRs further detected single infections of B. exitiosa (2.7%), B. ostreae (40.3%), and concurrent infections (53.7%) with these 2 Bonamia species identifying overall a Bonamia prevalence of 96.6%. Detailed histological inspection revealed 2 microcell types. An infection identified by PCR as B. ostreae histologically presented small microcells (mean ± SE diameter = 1.28 ± 0.16 µm, range = 0.9-2 µm, n = 60) commonly with eccentric nuclei. A B. exitiosa infection exhibited larger microcells (mean ± SE diameter = 2.12 ± 0.27 µm, range = 1.5-4 µm, n = 60) with more concentric nuclei. Concurrent infections of both Bonamia species, as identified by PCR, exhibited both types of microcells. DNA barcoding of the B. ostreae-infected oyster host confirmed the identification as O. chilensis. A suite of other parasites that accompany O. chilensis are reported here for the first time in mixed infection with B. ostreae including apicomplexan X (76.5%), Microsporidium rapuae (0.7%) and Bucephalus longicornutus (30.2%).