Lack of detection of a putative retrovirus associated with haemic neoplasia in the soft shell clam Mya arenaria.

Haemic neoplasia (HN) is a leukemia-like disease that affects at least 20 species of marine bivalves including soft shell clam, Mya arenaria. Since the disease was discovered in 1969, the etiology remains unknown. A retroviral etiology has been suggested based on the detection of reverse transcriptase activity and electron microscopic observation of retroviral-like particles using negative staining. To date, however no virus isolate and no retroviral sequence from HN has been obtained. Moreover, transmission of the disease by cell-free filtrate from affected clams has not been reproduced. In the current study, we reinvestigated the association of HN with a putative retrovirus. Sucrose gradient centrifugation followed by assessment of reverse transcriptase activity, electrophoretic analysis of protein and RNA, and electron microscopic examinations of fractions corresponding to retroviral density were employed. Detection of retroviral pol sequences using degenerate RT-PCR approaches was also attempted. Our results showed visible bands at the expected density of retrovirus in HN-positive and HN-negative clam tissues and both with reverse transcriptase activity. Electron microscopy, RNA analysis, protein analysis, and PCR systems targeting the pol gene of retroviruses did not however provide clear evidence supporting presence of a retrovirus. We point out that the retrovirus etiology of HN of Mya arenaria proposed some 25 years ago should be reconsidered in the absence of a virus isolate or virus sequences.

Reverse transcriptase activity in tissues of the soft shell clam Mya arenaria affected with haemic neoplasia.

Since all retroviruses possess reverse transcriptase (RT) enzyme, reverse transcriptase activity has been the main supportive evidence of retroviral etiology of haemic neoplasia (HN) in soft shell clams, Mya arenaria. The objective of the present study was to search for a putative retrovirus in various tissues of diseased clams following quantification of RT activity (biochemical indicator of retroviral infection). The clams were assessed by flow cytometry (FCM) for diagnosis of HN. RT activity was quantified by TaqMan-product enhanced reverse transcriptase (TM-PERT) assay in four different organs, gonad, gills, digestive gland, and mantle, at various stages of HN. The digestive gland, the organ with the highest RT activity, and haemocytes, the target cell of HN, were assessed by EM for presence of retroviruses. All organs were assessed by histology. The results of this study demonstrated that although all organs of healthy clams have some background RT activity, the activity observed in most of organs of diseased clams was significantly increased (p<0.05). An association was observed between the degree of neoplastic cell infiltration and the level of RT activity. Digestive gland showed the highest and most consistent RT activity in both healthy and diseased clams. No evidence for the existence of a retrovirus like particle was found by positive staining EM. The presence of RT activity without indications of retroviral particles in digestive gland and haemocytes suggests a probable endogenous source of RT.

Reverse transcriptase activity associated with haemic neoplasia in the soft-shell clam Mya arenaria.

Reverse transcriptase (RT) activity has been reported in bivalves affected by haemic neoplasia (HN). Since all retroviruses have RT, detection of RT activity was regarded as evidence for the retroviral etiology of HN. This study investigates the relationship between RT levels and the progress of HN as indicated by percentages of tetraploid cells in soft-shell clams Mya arenaria. The percentages of tetraploid cells were estimated by flow cytometry, and the RT levels were quantified using TaqMan product-enhanced RT (TM-PERT) assay. Results demonstrated that the amount of RT was positively correlated with the percentage of tetraploid cells circulating in clam haemolymph (R2 = 0.974, p < 0.001). Compared to HN-negative clams (<5% tetraploid cells), 2 stages with significantly elevated levels of RT activity were observed: the first stage at approximately 10 to approximately 20% tetraploid cells, and the second at approximately 30 to approximately 80% tetraploid cells (p < 0.01). These data support the well established fact from mammalian models that transformed cells express high levels of non-telomeric RT. The observed increase in RT levels at approximately 30% tetraploidy coincides with previously reported p53 gene expression. Taken together, this could indicate that using RT levels as an indicator of HN, > or = 30% tetraploidy is the stage at which the disease process undergoes a change, and perhaps becomes irreversible.

Patterns of p53, p73 and mortalin gene expression associated with haemocyte polyploidy in the soft-shell clam, Mya arenaria.

The molecular mechanisms by which haemocytes of clams are transformed in the course of haemic neoplasia remain by far unknown. The aim of this study was to quantify the expression of p53/p73 and mortalin genes, in relation with the ploidy status of clam haemocytes and to correlate the p53 expression with mortalin expression. For this purpose, soft-shell clams, Mya arenaria, were collected from an endemic zone for neoplasia. The ploidy of haemocytes was assessed for each individual clam by flow cytometry using a propidium iodide protocol, while p53/p73 and mortalin gene expressions were quantified by real-time RT-PCR. Results show that haemocytes of some clams with a moderate percentage (15-50%) of tetraploid cells have a significantly high level of p53 and p73 in comparison with clams belonging to categories with low (<15%) or high levels (>50%) of tetraploid cells, where low levels of expression of these genes were observed. Furthermore, mortalin gene expression is strongly correlated (r(2)=0.68, p<0.01) with p53 gene expression level. This reinforces the hypothesis of a cytoplasmic p53 sequestration mechanism in clam haemic neoplasia. Further studies are needed to confirm these preliminary results and further unravel the molecular pathways involved in this process. Our results are believed to provide phenotypic foundation for such studies to be undertaken.

Delta de l'Ebre is a natural bay model for Marteilia spp. (Paramyxea) dynamics and life-cycle studies.

Marteilia spp. are paramyxean parasites that affect several bivalve species of economic interest, such as Ostrea edulis and Mytilus galloprovincialis. Certain aspects of Marteilia spp., such as their life cycle and host affinity and infection dynamics, still remain unknown. The 'Delta de l'Ebre' constitutes a natural model for the study of the life cycle of the parasite Marteilia, since uninfected mussels and flat oysters immersed in the bays can become infected. This, along with the geographical and ecological characteristics of the bays, make it a very interesting location to study the Marteilia life cycle. Preliminary results concerning marteiliosis, mainly in mussels, such as prevalence dynamics, infectious periods, host affinity and host intermediate candidates are reported in the present paper. This information will be required for further, more exhaustive, studies in the bays of the Ebre delta.

Comparative experimental infection of the copepod Paracartia grani with Marteilia refringens and Marteilia maurini.

Paracartia grani (Copepoda) has been identified as a potential intermediate host in the life cycle of Marteilia refringens, a paramyxean parasite infecting flat oysters. However, no intermediate host has yet been identified for Marteilia maurini that infects mussels. A better understanding of the life cycle of these two Marteilia types would clarify their taxonomic relationship and hypothesized co-specificity. For this purpose, experimental infections of copepods, P. grani, were performed using naturally infected flat oysters and mussels. Infection patterns were different depending whether copepods were infected from oysters or mussels. M. maurini did not proliferate in copepods while M. refringens rapidly proliferated in infected copepods. Previously unrecognized developmental stages of M. refringens were found during this study.

Validation of in situ hybridisation and histology assays for the detection of the oyster parasite Marteilia refringens.

An in situ hybridisation technique has been developed for the detection of infection in oysters with Marteilia refringens with particular emphasis on light infections or confirmation of suspected cases by means of histology. Although validation of new diagnostic methods is usually achieved by comparison with standard techniques, in our case the sensitivity and specificity of the standard (histology) had not previously been established. Another point to consider is that surveillance and monitoring frequently target populations displaying different levels of prevalence under different field conditions. The objective of our study was to evaluate the sensitivity and specificity values of in situ hybridisation and histology for the detection of M. refringens, based on 3 populations of flat oysters, free of the disease and with mild and high levels of prevalence. A blind assay of 200 individuals from each population was performed using both techniques. Results were analysed by means of the classical approach and latent models (maximum likehood and Bayesian approach). Assumptions and results were found to vary slightly with the different statistical approaches. The more realistic estimate by the Bayesian approach shows a link between the level of prevalence and the sensitivity of the techniques. Values of sensitivity and specificity for histology were 0.7 and 0.99 respectively, and 0.9 and 0.99 respectively in the case of in situ hybridisation. Some uncertainty remains regarding these values because the study does not take into account the severity of infection or the developmental stages of the parasite actually present in each individual. This work provides valuable information with regard to the choice and potential use of those 2 diagnostic methods currently recommended by international standards.

Mikrocytos roughleyi taxonomic affiliation leads to the genus Bonamia (Haplosporidia).

Microcell-type parasites of oysters are associated with a complex of diseases in different oyster species around the world. The etiological agents are protists of very small size that are very difficult to characterize taxonomically. Associated lesions may vary according to the host species, and their occurrence may be related to variations in tissue structure. Lesion morphology cannot be used to distinguish the different agents involved. Ultrastructural observations on Mikrocytos roughleyi revealed similarities with Bonamia spp., particularly in regard to the presence of electron-dense haplosporosomes and mitochondria, whose absence from M. mackini also indicate that M. roughleyi and M. mackini are not congeneric. A partial small subunit (ssu) rRNA gene sequence of M. roughleyi was determined. This partial sequence, 951 nucleotides in length, has 95.2 and 98.4% sequence similarities with B. ostreae and B. exitiosus ssu rDNA sequences, respectively. Polymorphisms among the ssu rDNA sequences of B. ostreae, B. exitiosus and M. roughleyi allowed identification of restriction enzyme digestion patterns diagnostic for each species. Phylogenetic analysis based on the ssu rDNA data suggested that M. roughleyi belongs in the phylum Haplosporidia and that it is closely related to Bonamia spp. On the basis of ultrastructural and molecular considerations, M. roughleyi should be considered a putative member of the genus Bonamia.

Claire ponds as an experimental model for Marteilia refringens life-cycle studies: new perspectives.

Since its first description, the paramyxean parasite Marteilia refringens (Grizel et al.) has been recognised as a significant pathogen of the European flat oyster Ostrea edulis L. The existence of a complex life-cycle involving several hosts was postulated early on by many authors, although it remains unsolved. Recent developments in the DNA-based diagnosis of M. refringens provides new prospects for the detection of the parasite in potential hosts. However, this screening remains impeded by the number of species living in the vicinity of oyster beds. We report here on the use of semi-closed oyster ponds (so called 'claire' in Marennes-Oléron Bay) as a study model for the life-cycle of M. refringens. Claires are located in an endemic area for M. refringens and transmission of the disease to healthy oysters has been shown to be effective during the course of this study. The environmental characteristics of the claires strongly limit the number of species compared with intertidal areas and oyster beds. Consequently, extensive sampling of a limited number of species cohabiting with oysters was possible. These were preserved for future screening of M. refringens. The experimental model should bring new insights to the life-cycle of M. refringens, as it enables us to propose new conceptual schemes of M. refringens transmission. The role of species as potential hosts is discussed regarding their biology and geographical distribution.

Expression of RAS-like family members, c-jun and c-myc mRNA levels in neoplastic hemocytes of soft-shell clams Mya arenaria using microsphere-based 8-plex branched DNA assay.

The molecular mechanisms by which disseminated neoplasia (DN) is developed in soft shell clams Mya arenaria remain largely unknown. This study aims at quantifying Rho-like GTPase, RAS-Rho, RAS-related C3 botulinum (RAS C3), c-jun as well as c-myc transcript levels in clams sampled at North River (Charlottetown, Prince Edward Island, Canada). The transcripts were quantified using multiplex gene analysis (Quantigene(®) 2 Plex, Affymetrix) in 3 groups of clams: (1) Group C (healthy clams considered as control) with a low percentage of tetraploid hemocytes (<10%); (2) Group D (disease in development): individuals presenting a percentage of tetraploid cells ranging between 10% and 50%; (3) Group E (established disease): clams with a high percentage of tetraploid hemocytes (>50%). Data showed a down-regulation of Rho-like GTPase, Rho-like subfamily, RAS C3, c-jun and an up-regulation of c-myc gene expression. It is believed that a deregulation of the expression of these genes could partly unravel the molecular mechanisms involved in the development of DN in soft shell clams Mya arenaria. Further investigations should be pursued to determine the role of these gene products in clams' hemocytes.

Epizootic haematopoietic necrosis virus--an assessment of the likelihood of introduction and establishment in England and Wales.

Epizootic haematopoietic necrosis virus (EHNV) is an iridovirus that affects perch (Perca fluviatilis) and rainbow trout (Oncorhynchus mykiss). It emerged in Australia in the 1980s and has not been discovered elsewhere. It causes a high level of mortality in perch resulting in steep population declines. The main possible routes of introduction of the virus to England and Wales are the importation of infected live fish or carcasses. However, no trade in live susceptible species is permitted under current legislation, and no importation of carcasses currently takes place. The virus is hardy and low levels of challenge can infect perch. Therefore, mechanical transmission through the importation of non-susceptible fish species should be considered as a potential route of introduction and establishment. Carp (Cyprinus carpio) have been imported to the UK from Australia for release into still-water fisheries. A qualitative risk assessment concluded that the likelihood of EHNV introduction and establishment in England and Wales with the importation of a consignment of carp was very low. The level of uncertainty at a number of steps in the risk assessment scenario tree was high, notably the likelihood that carp become contaminated with the virus and whether effective contact (resulting in pathogen transmission) is made between the introduced carp and susceptible species in England and Wales. The virus would only establish when the water temperature is greater than 12 degrees C. Analysis of 10 years of data from two rivers in south-west England indicated that establishment could occur over a period of at least 14 weeks a year in southern England (when average water temperature exceed 12 degrees C). Imports of live fish from Australia need to be evaluated on a case-by-case basis to determine which, if any, sanitary measures are required to reduce the assessed risk to an acceptable level.

Selecting a set of housekeeping genes for quantitative real-time PCR in normal and tetraploid haemocytes of soft-shell clams, Mya arenaria.

The transcripts involved in the molecular mechanisms of haemic neoplasia in relation to the haemocyte ploidy status of the soft-shell clam, Mya arenaria, have yet to be identified. For this purpose, real-time quantitative RT-PCR constitutes a sensitive and efficient technique, which can help determine the gene expression involved in haemocyte tetraploid status in clams affected by haemic neoplasia. One of the critical steps in comparing transcription profiles is the stability of selected housekeeping genes, as well as an accurate normalization. In this study, we selected five reference genes, S18, L37, EF1, EF2 and actin, generally used as single control genes. Their expression was analyzed by real-time quantitative RT-PCR at different levels of haemocyte ploidy status in order to select the most stable genes. Using the geNorm software, our results showed that L37, EF1 and S18 represent the most stable gene expressions related to various ploidy status ranging from 0 to 78% of tetraploid haemocytes in clams sampled in North River (Prince Edward Island, Canada). However, actin gene expression appeared to be highly regulated. Hence, using it as a housekeeping gene in tetraploid haemocytes can result in inaccurate data. To compare gene expression levels related to haemocyte ploidy status in Mya arenaria, using L37, EF1 and S18 as housekeeping genes for accurate normalization is therefore recommended.

Dynamics of the parasite Marteilia refringens (Paramyxea) in Mytilus galloprovincialis and zooplankton populations in Alfacs Bay (Catalonia, Spain).

Since the first description of Marteilia refringens (Paramyxea) in flat oysters Ostrea edulis in 1968 in the Aber Wrach, Brittany (France), the life-cycle of this parasite has remained unknown. However, recent studies, conducted in the 'claire' system, have proposed the planktonic copepod Acartia grani as a potential intermediate host for the parasite. Nevertheless, experimental transmission of the parasite through the copepod has failed. Recent studies in this field have reported the presence of the parasite in zooplankton from the bays of the Delta de l'Ebre, a more complex and natural estuarine environment than that of the claire. As a result, 2 new Marteilia host species were proposed: the copepods Oithona sp. (Cyclopoida) and an indeterminate Harpaticoida. Consequently, the objective of the present work was to study the dynamics of Marteilia in the zooplankton community from one of the bays, Alfacs Bay, as well as the dynamics of the parasite in cultivated mussels during 1 complete year. Six different zooplankton taxa appeared to be parasitized by M. refringens, including copepods (3 Calanoida, Acartia discaudata, A. clausi and A. italica; 1 Cyclopoida, Oithona sp.; and 1 Harpacticoida, Euterpina acutifrons), and larval stages of decapod crustaceans (zoea larvae of Brachyura, probably Portumnus sp.). These taxa are thus proposed as new subjects for study, since they could be intermediate hosts in the infection process of mussels by Marteilia.