Long-term affected flat oyster (Ostrea edulis) haemocytes show differential gene expression profiles from naïve oysters in response to Bonamia ostreae

European flat oyster (Ostrea edulis) production has suffered a severe decline due to bonamiosis. The responsible parasite enters in oyster haemocytes, causing an acute inflammatory response frequently leading to death. We used an immune-enriched oligo-microarray to understand the haemocyte response to Bonamia ostreae by comparing expression profiles between naïve (NS) and long-term affected (AS) populations along a time series (1 d, 30 d, 90 d). AS showed a much higher response just after challenge, which might be indicative of selection for resistance. No regulated genes were detected at 30?d in both populations while a notable reactivation was observed at 90 d, suggesting parasite latency during infection. Genes related to extracellular matrix and protease inhibitors, up-regulated in AS, and those related to histones, down-regulated in NS, might play an important role along the infection. Twenty-four candidate genes related to resistance should be further validated for selection programs aimed to control bonamiosis.     

Molecular characterisation of TNF,AIF, dermatopontin and VAMP genes of the flat oyster Ostrea edulis and analysis of their modulation by diseases

Bonamiosis and disseminated neoplasia (DN) are the most important diseases affecting cultured flat oysters (Ostrea edulis) in Galicia (NW Spain). Previous research of the response of O. edulis against bonamiosis by suppression subtractive hybridisation yielded a partial expressed sequence tag of tumour necrosis factor (TNF) and allograft inflammatory factor (AIF), as well as the whole open reading frame for dermatopontin and vesicle-associated membrane (VAMP). Herein, the complete open reading frames of TNF and AIF genes were determined by the rapid amplification of cDNA, and the deduced amino acid sequences of the four genes were characterised. Phylogenetic relationships for each gene were studied using maximum likelihood parameters. Quantitative-PCR assays were also performed in order to analyse the modulation of the expression of these genes by bonamiosis and disseminated neoplasia. Gene expression profiles were studied in haemolymph cells and in various organs (gill, gonad, mantle and digestive gland) of oysters affected by bonamiosis, DN, and both diseases with regard to non-affected oysters (control). TNF expression in haemolymph cells was up-regulated at heavy stage of bonamiosis but its expression was not affected by DN. AIF expression was up-regulated at heavy stage of bonamiosis in haemolymph cells and mantle, which is associated with heavy inflammatory response, and in haemolymph cells of oysters affected by DN. AIF expression was, however, down-regulated in other organs as gills and gonads. Dermatopontin expression was down-regulated in haemolymph cells and digestive gland of oysters affected by bonamiosis, but DN had no significant effect on its expression. Gills and gonads showed up-regulation of dermatopontin expression associated with bonamiosis. There were significant differences in the expression of TNF and VAMP depending on the bonamiosis intensity stage whereas no significant differences were detected between light and heavy severity degrees of DN for the studied genes. VAMP expression showed also differences among haemolymph cells and the organs studied. The occurrence of both diseases in oysters involved haemolymph cell gene expression patterns different from those associated to each disease separately: no significant effect was observed in TNF expression, dermatopontin was up-regulated and marked up-regulation of AIF and VAMP was recorded, which suggests a multiplier effect of the combination of both diseases for the latter two genes.     

Cellular and molecular responses of haemocytes from Ostrea edulis during in vitro infection by the parasite Bonamia ostreae

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 8 h p.i. Light microscopy was used to measure parasite phagocytosis by oyster haemocytes. Parasites were observed inside haemocytes 1 h 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 4 h 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 1 h 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.     

Variability of haemocyte and haemolymph parameters in European flat oyster Ostrea edulis families obtained from brood stocks of different geographical origins and relation with infection by the protozoan Bonamia ostreae

A research project to compare productive traits (growth and mortality), disease susceptibility and immune capability between Ostrea edulis stocks was performed. This article reports the results on the immune capability and its relation with infection by the intrahaemocytic protozoan Bonamia ostreae. Four to five oyster spat families were produced from each of four European flat oyster populations (one from Ireland, one from Greece and two from Galicia, Spain) in a hatchery. The spat were transferred to a raft in the Ría de Arousa (Galicia) for on growing for 2 years. Total haemocyte count (THC) and differential haemocyte count (DHC) were estimated monthly through the second year of growing-out. Three types of haemocytes were distinguished: granulocytes (GH), large hyalinocytes (LHH) and small hyalinocytes (SHH). Significant correlations between the mean relative abundance of GH and SHH of the families and the mean prevalence of B. ostreae, the overall incidence of pathological conditions and the cumulative mortality of the families were found; these correlations supported the hypothesis that high %GH and low %SHH would enhance oyster immune ability and, consequently, would contribute to lower susceptibility to disease and longer lifespan. Infection by B. ostreae involved a significant increase of circulating haemocytes, which affected more markedly the LHH type. The higher the infection intensity the higher the %LHH. This illustrates the ability of B. ostreae to modulate the immune responses of the O. edulis to favour its own multiplication. A significant reduction of the phenoloxidase activity in the haemolymph of oysters O. edulis infected by B. ostreae was observed. Nineteen enzymatic activities in the haemolymph of O. edulis and Crassostrea gigas (used as a B. ostreae resistant reference) were measured using the kit api ZYM^®, Biomerieux. Qualitative and quantitative differences in enzyme activities in both haemocyte and plasma fractions between B. ostreae noninfected O. edulis from different origins were recorded. However, no clear positive association between enzyme activity and susceptibility to bonamiosis was found. The only enzyme detected in the resistant species C. gigas that was not found in the susceptible one O. edulis was β-glucosidase (in plasma). B. ostreae infected O. edulis showed significant increase of some enzyme activities and the occurrence of enzymes that were not detected in noninfected oysters. These changes could be due to infection-induced enzyme synthesis by the host or to enzyme synthesis by the parasite.     

Combining Two-Stage Testing and Interval Mapping Strategies to Detect QTL for Resistance to Bonamiosis in the European Flat Oyster Ostrea edulis

We have identified quantitative trait loci (QTL) in the flat oyster (Ostrea edulis) for resistance to Bonamia ostreae, a parasite responsible for the dramatic reduction in the aquaculture of this species. An F₂ family from a cross between a wild oyster and an individual from a family selected for resistance to bonamiosis was cultured with wild oysters injected with the parasite, leading to 20% cumulative mortality. Selective genotyping of 92 out of a total of 550 F₂ progeny (i.e., 46 heavily infected oysters that died and 46 parasite-free oysters that survived) was performed using 20 microsatellites and 34 amplification fragment length polymorphism primer pairs. Both a two-stage testing strategy and QTL interval mapping methods were used. The two-stage detection strategy had a high power with a low rate of false positives and identified nine and six probable markers linked to genes of resistance and susceptibility, respectively. Parent-specific genetic linkage maps were built for the family, spanning ten linkage groups (n = 10) with an observed genome coverage of 69–84%. Three QTL were identified by interval mapping in the first parental map and two in the second. Good concordance was observed between the results obtained after the two-stage testing strategy and QTL mapping.     

Living with bonamiasis: Irish research since 1987

The main oyster species produced in Ireland up to the 1980's was the European flat oyster Ostrea edulis. However, since then, production of this species has been severely affected by the presence of the protistan Bonamia ostreae, which was diagnosed in a population of flat oysters on the south coast following heavy mortalities. Research has been ongoing since the first diagnosis in Ireland and has concentrated on aspects of the biology of both the host and the parasite. In recent years research has concentrated on screening populations of oysters to identify any with reduced susceptibility to the parasite.     

Bonamia exitiosa (Haplosporidia) observed infecting the European flat oyster Ostrea edulis cultured on the Spanish Mediterranean coast

Bonamia exitiosa and Bonamia ostreae are parasites that reproduce within the haemocytes of several oyster species. In Europe, the host species is the flat oyster Ostrea edulis. The parasite B. ostreae has been responsible for mortalities since the late 1970s throughout the European Atlantic coast. B. exitiosa was first detected, in 2007, on this continent in flat oysters cultured in Galicia (NW Spain). Since then, the parasite has also been detected in France, Italy and the United Kingdom. The bays of the Ebro Delta in the south of Catalonia represent the main bivalve culture area in the Mediterranean coast of Spain. Previous information from the area includes reports of several flat oyster pathogens, including the notifiable parasite Marteilia refringens. However, the status with regard to Bonamia parasites was uncertain. In the present study, a Bonamia parasite was observed in flat oysters cultured in the Alfacs Bay of the Ebro Delta by histology and real-time PCR. PCR-RFLP and sequencing suggested the presence of B. exitiosa. Finally, phylogenetic analyses of the studied Bonamia isolates corroborated B. exitiosa infection. M. refringens was also observed in the same oyster batch, and co-infection with both parasites was also detected. This is the first detection of B. exitiosa, in Catalonia and the Spanish Mediterranean coast. The impact of the parasite on the Mediterranean flat oyster activity needs to be urgently addressed.     

Observation of a Bonamia sp. infecting the oyster Ostrea stentina in Tunisia, and a consideration of its phylogenetic affinities

The small non-commercial oyster Ostrea stentina co-occurs with commercially important Ostrea edulis in the Mediterranean Sea, yet its disposition with respect to the destructive pathogens Bonamia ostreae and Marteilia refringens is unknown. We began an evaluation of the Bonamia spp. infection status of O. stentina from Hammamet, Tunisia, in June 2007 using polymerase chain reaction diagnostics followed by histology and in situ hybridization. Of 85 O. stentina sampled, nine were PCR-positive for a Bonamia sp. using a Bonamia genus-specific assay; of these nine, one displayed the uninucleate microcells associated with oyster hemocytes characteristic of Bonamia spp. There was no associated pathology. DNA sequencing of the parasite from this one infected individual revealed it to be of a member of the Bonamia exitiosa/Bonamia roughleyi clade, an identification supported by positive in situ hybridization results with probes specific for members of this clade, and by the morphology of the parasite cells: nuclei were central, as in B. exitiosa, not eccentric, as in B. ostreae. There is no basis for identifying the Tunisian parasite as either B. exitiosa or B. roughleyi, however, as these species are genetically indistinguishable. Likewise, there is no basis for identifying any of the other Bonamia spp. with affinities to the B. exitiosa/B. roughleyi clade, from Argentina, Australia, Spain, and the eastern USA, as one or the other of these named species. Though they are clearly distinct from Bonamia perspora and B. ostreae, justification for drawing species boundaries among the primarily austral microcells with affinities to B. exitiosa and B. roughleyi remains elusive.     

Infection with the protozoan parasite Bonamia ostreae modifies in vitro haemocyte activities of flat oyster Ostrea edulis

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.     

Strategies for the retention of high genetic variability in European flat oyster (<Emphasis Type="Italic">Ostrea edulis</Emphasis>) restoration programmes

The native European flat oyster Ostrea edulis is listed in the OSPAR Convention for the Protection of the Marine Environment of the North-East Atlantic (species and habitat protection) and in the UK Biodiversity Action Plan. Once extremely abundant in the nineteenth century, European stocks of O. edulis have declined during the twentieth century to rare, small, localised populations due to overexploitation, habitat degradation and, most recently, the parasitic disease bonamiosis. Selective breeding programmes for resistance to bonamiosis have been initiated in France and Ireland. High genetic diversity and bonamiosis-resistance would be important features of any sustainable restoration programmes for O. edulis. Oysters were sampled across Europe from four hatchery sources, four pond-cultured sources and four wild, but managed fisheries and were genotyped at five microsatellite loci. Hatchery-produced populations from small numbers of broodstock showed a significant loss of genetic diversity relative to wild populations and pedigree reconstruction revealed that they were each composed of a single large full-sib family and several small full-sib families. This extremely low effective population size highlights the variance in reproductive success among the potential breeders. Pond-cultured oysters were intermediate in genetic diversity and effective population size between hatchery and wild populations. Controlled hatchery production allows the development of bonamiosis-resistant strains, but at the expense of genetic diversity. Large scale pond culture on the other hand can provide a good level of genetic diversity. A mixture of these two approaches is required to ensure a healthy and sustainable restoration programme for O. edulis in Europe.     

Development of a TaqMan PCR assay for the detection of Bonamia species

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.     

Minchinia armoricana sp. nov. (Haplosporida), a parasite of the European flat oyster, Ostrea edulis

Examination of European flat oysters, Ostrea edulis, from the Dutch oyster culture, but originating in France, revealed a new disease due to a protistan parasite. Light and electron microscope studies revealed that the parasite belongs to the haplosporidan genus Minchinia. Since comparison with other Minchinia spp. indicate that it is new, the name Minchinia armoricana nov. sp. is proposed. Thus far, the parasite was very rare; only two diseased oysters were observed among ca. 3700 specimens examined histologically during a 2.5-year period. The diseased oysters showed macroscopically a peculiar brown discoloration and microscopically many sporocysts with spores in the connective tissue. Beside the two diseased oysters, another one was observed with an infection of unidentified plasmodial stages in the connective tissue. These may be developmental stages of the new species M. armoricana.     

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 ecology of Bonamia and decline of bivalve molluscs

Bonamia is a protozoan parasite of the haemocytes of oysters (Tiostrea chilensis), in which it has an annual developmental cycle between November and August each year. The parasite transmits directly, oyster to oyster, and therefore disease spread is related to host stock density. The Foveaux Strait oyster population experiences large mortalities every 20-30 years, and these may be attributable to Bonamia. The parasite appears to become less pathogenic at the end of, and probably between, mass mortalities, and some oysters appear more tolerant of infection than others. On the basis of these observations, and considering other protist pathogen:oyster models, the apparently reduced pathogenicity of Bonamia is discussed in terms of parasite kinetics. The population dynamics and selection of parasite tolerant host stocks, and kinetics of parasite transmission, may explain the cyclic nature of large-scale mortalities in Foveaux Strait, without change in parasite pathogenicity.     

Strong seasonality of Bonamia sp. infection and induced Crassostrea ariakensis mortality in Bogue and Masonboro Sounds, North Carolina, USA

Asian oyster Crassostrea ariakensis is being considered for introduction to Atlantic coastal waters of the USA. Successful aquaculture of this species will depend partly on mitigating impacts by Bonamia sp., a parasite that has caused high C. ariakensis mortality south of Virginia. To better understand the biology of this parasite and identify strategies for management, we evaluated its seasonal pattern of infection in C. ariakensis at two North Carolina, USA, locations in 2005. Small (<50 mm) triploid C. ariakensis were deployed to upwellers on Bogue Sound in late spring (May), summer (July), early fall (September), late fall (November), and early winter (December) 2005; and two field sites on Masonboro Sound in September 2005. Oyster growth and mortality were evaluated biweekly at Bogue Sound, and weekly at Masonboro, with Bonamia sp. prevalence evaluated using parasite-specific PCR. We used histology to confirm infections in PCR-positive oysters. Bonamia sp. appeared in the late spring Bogue Sound deployment when temperatures approached 25 degrees C, six weeks post-deployment. Summer- and early fall-deployed oysters displayed Bonamia sp. infections after 3-4 weeks. Bonamia sp. prevalences were 75% in Bogue Sound, and 60% in Masonboro. While oyster mortality reached 100% in late spring and summer deployments, early fall deployments showed reduced (17-82%) mortality. Late fall and early winter deployments, made at temperatures <20 degrees C, developed no Bonamia sp. infections at all. Seasonal Bonamia sp. cycling, therefore, is influenced greatly by temperature. Avoiding peak seasonal Bonamia sp. activity will be essential for culturing C. ariakensis in Bonamia sp.-enzootic waters.