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The commercially significant Foveaux Strait Ostrea chilensis fishery has been typified by consistently low brooding rates and recruitment failures since recurrent mass mortalities from Bonamia exit...

There is a growing effort throughout Europe to restore populations of native oysters (Ostrea edulis), with the ecological objective of enhancing ecosystem biodiversity and resilience.The introduced parasite, Bonamia ostreae, caused catastrophic mortalities during the 1980s, furthering the decline of this species, and is now present throughout much of the natural range of O. edulis. It is therefore important that restoration attempts avoid further introduction and spread of this parasite, which can cause lethal infections of O. edulis.This article presents a comprehensive overview of the scale and distribution of current infection, transmission pathways, and preventive measure guidelines, focusing on the seas, inlets, and estuaries of north‐west Europe, where most ecological restoration attempts for the native European oyster have occurred so far.This is critical information for restoration project planning in which the risk of Bonamia infection must be taken into account.

The annual production of European flat oysters (Ostrea edulis) in Croatia is about 50 to 65 tons, and it has a long tradition. All Croatian oyster farms are subjected to the national surveillance program aiming to detect the presence of Bonamia ostreae and Marteilia refringens according to the Council Directive 2006/88/EC. Within the surveillance program, the first findings of the parasite Bonamia spp. occurred in 2016 in two production areas in the north and south of the Eastern Adriatic coast. The repeated findings of the parasite were noted up to 2020 but also on two additional sites in the north. The parasite was detected by cytological analysis of stained heart smears, histological examination, and PCR. PCR positive samples were sequenced for SSU rDNA gene, and BLAST analysis confirmed infection with Bonamia exitiosa. Attempts to prove the Pacific oyster as a putative vector of the parasite failed. The infection prevalence from 2016 until 2020 ranged from 3.3 to 20% in different sites. No mortalities were reported from the infected sites, and it seemed that infection of flat oysters with B. exitiosa did not affect their health. The study has not shown the source and way of infection spread, which imposes the need for more comprehensive molecular and epidemiological studies.

The fan mussel, Pinna nobilis (Linnaeus 1758), is an endemic bivalve of the Mediterranean basin, protected by international legislation as an endangered species. In the early summer of 2018, a mass mortality event (MME) of P. nobilis was recorded in the Gulf of Taranto (Southern Italy, Ionian Sea). Moribund specimens of P. nobilis were collected by scuba divers and processed by bacteriological, parasitological, histopathological and molecular analyses to investigate the causes of this MME. Different developmental stages (i.e., plasmodia, spores and sporocysts) of a presumptive haplosporidian parasite were observed during the histological analysis in the epithelium and in the lumen of the digestive tubules, where mature spores occurred either free or in sporocysts. The spores presented an operculum and an ovoid shape measuring 4.4 µm (±0.232) in length and 3.6 µm (±0.233) in width. BLAST analysis of an 18SrRNA sequence revealed a high nucleotide similarity (99%) with the reference sequence of Haplosporidium pinnae available in GenBank database. Phylogenetic analysis clustered the sequence of the pathogen in a paraphyletic clade with the reference sequence of H. pinnae, excluding other haplosporidians (i.e., Bonamia and Minchinia genera). Based on data reported, H. pinnae was the causative agent of MME in the populations of P. nobilis sampled in the Ionian Sea, where the conservation of this endangered species is heavily threatened by such a protozoan infection. Further investigations should contribute to knowledge about the life cycle of H. pinnae in order to reduce spread of the pathogen and to mitigate the burden of the disease where P. nobilis is facing the risk of extinction.

Since 2017, the European flat oyster (Ostrea edulis) reappeared in the Wadden Sea after decades of absence. In several countries, restoration and reinforcement projects have been initiated. Better insight in Ostrea behaviour is needed to ensure sustainable conservation in the future. In order to acquire substantial numbers of oysters for eco-physiological and transplant experiments without threatening the small local population, indoor rearing facilities were set-up at Royal NIOZ. Here, a broodstock of 38 adults originating from the western Wadden Sea, produced over 21 million larvae in 10 batches within 3 months (Sept-Nov 2018). Release of larvae and rearing took place at a water temperature of 19.7 ± 0.6 °C and a salinity of 30.5 ± 0.5 PSU. Outbreaks of Vibrio bacteria did not occur. The growth rate before settlement was 1.5–1.7 μm per day, which was at the low end of growth rates reported in previous studies. The first competent larvae were observed 17–22 days after release. Size at settlement varied between 255−325 μm. Survival until settlement was 0.96−0.98 per day. After settlement, growth rates increased to 30−47 μm per day, with batches that were released in September having a significantly higher growth rate and initial size compared to larvae that were released later. Mortality from release to the end of the experiment (Feb 2019) was 0.04−0.05 per day. A cryopreservation trial did not result in living larvae after thawing, most likely due to their advanced development when preserved. The larvae and juveniles were negatively tested for the parasite Bonamia ostrea and can thus be used in oyster conservation and restoration projects. Recommendations for future breeding attempts include the use of a higher water temperature and slightly higher food concentrations.

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 (R) 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 (intralaboratory 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.

This paper reviews current knowledge of the structure, genesis, cytochemistry and putative functions of the haplosporosomes of haplosporidians (Urosporidium, Haplosporidium, Bonamia, Minchinia) and paramyxids (Paramyxa, Paramyxoides, Marteilia, Marteilioides, Paramarteilia), and the sporoplasmosomes of myxozoans (Myxozoa – Malacosporea, Myxosporea). In all 3 groups, these bodies occur in plasmodial trophic stages, disappear at the onset of sporogony, and reappear in the spore. Some haplosporidian haplosporosomes lack the internal membrane regarded as characteristic of these bodies and that phylum. Haplosporidian haplosporogenesis is through the Golgi (spherulosome in the spore), either to form haplosporosomes at the trans-Golgi network, or for the Golgi to produce formative bodies from which membranous vesicles bud, thus acquiring the external membrane. The former method also forms sporoplasmosomes in malacosporeans, while the latter is the common method of haplosporogenesis in paramyxids. Sporoplasmogenesis in myxosporeans is largely unknown. The haplosporosomes of Haplosporidium nelsoni and sporoplasmosomes of malacosporeans are similar in arraying themselves beneath the plasmodial plasma membrane with their internal membranes pointing to the exterior, possibly to secrete their contents to lyse host cells or repel haemocytes. It is concluded that these bodies are probably multifunctional within and between groups, their internal membranes separating different functional compartments, and their origin may be from common ancestors in the Neoproterozoic.

Bonamiosis has developed as a problem in Australian native oysters Ostrea angasi since the parasite Bonamia spp. was first detected in Port Phillip Bay, Victoria, in the early 1990s. At that time, large-scale mortalities in both farmed and wild oysters saw the demise of the pilot native oyster culture industry. More recent attempts to farm the species resulted in subclinical infections that progressed over time to clinical disease. The aim of this work was to establish what environmental factors result in the clinical manifestation of disease; determine the diagnostic sensitivity and diagnostic specificity of histopathological examination and a quantitative polymerase chain reaction (qPCR) test for the diagnosis of B. exitiosa infection in clinically diseased farmed native oysters; and calculate the optimal qPCR threshold cycle (CT) epidemiological cut-point for classification of positive and negative cases. After applying a range of stressors to tank-held oysters, results indicated a 58% increased risk (95% CI: 16%, 99%) of a Bonamia-infected oyster dying if the oyster was held at a higher temperature (p = 0.048). Starving and tumbling oysters, in isolation, was not significantly associated with clinical bonamiosis, but a Bonamia-infected oyster was at the greatest risk of death when increased water temperature was combined with both starvation and increased motion (p = 0.02; odds ratio = 3.47). The diagnostic sensitivity and specificity of the World Organisation for Animal Health qPCR protocol were calculated for increasing CT value cut-points from ≤25 to ≤40, with an optimal cut-point identified at ≤34.5 (specificity: 92.2; 95% posterior credible intervals [PCI]: 76.2, 99.8; Sensitivity: 93.5; 95% PCI: 84.7, 99.1).

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.

Background and aims: Bonamia Thoaurs (Convolvulaceae) comprises about 70 species with a tropical and subtropical distribution. Morphological and molecular studies in Bonamia were carried out to clarify relationships in and out of the genus and throughout this work it was detected the need for re-limit the circumscription of some taxa. This paper contributes to the circumscription and lectotypification of Bonamia sericea (Griseb.) Hallier f., an endemic species from northern Argentina. M&M: Morphological analysis of Bonamia sericea, were made based on a study of specimens of herbaria, including the type-material, digital type images and other herbaria website. Results: A new synonym for B. sericea is proposed. The lectotype of B. sericea is here designated and a complete description and illustrations are presented.

Raw-bivalves consumption is a wide trend in Mediterranean countries. Despite the unambiguous nutritional value of seafood, raw consumption of bivalves may involve risks that could pose a significant threat to consumers’ health. Their filter-feeding behavior is responsible for the potential hosting of a wide variety of microorganisms, either pathogenic for the bivalves or public health threats. Under this prism, the current study was conducted in an effort to evaluate the risk of eating raw bivalves originating from the two biggest seafood markets in Thessaloniki, the largest production area of bivalves in Greece. Both microbiological and molecular methodologies were applied in order to assess the presence of various harmful microbes, including noroviruses, Bonamia, Marteilia, Esherichia coli, Salmonella, and Vibrio. Results indicated the presence of several Vibrio strains in the analyzed samples, of which the halophilic Vibrio harveyi was verified by 16S rRNA sequencing, other than this, no enteropathogenic Vibrio spp. was detected. Furthermore, although Esherichia coli was detected in several samples, it was mostly below the European Union (EU) legislation thresholds. Interestingly, the non-target Photobacterium damselae was also detected, which is associated with both wound infections in human and aquatic animals. Regarding host pathogenic microorganisms, apart from Vibrio harveyi, the protozoan parasite Marteilia refrigens was identified in oysters, highlighting the continuous infection of this bivalve in Greece. In conclusion, bivalves can be generally characterized as a safe-to-eat raw food, hosting more bivalve pathogenic microbes than those of public health concern.

Este artículo contiene 11 páginas, 4 figuras, 1 tabla., A mass mortality event is devastating the populations of the endemic bivalve Pinna nobilis in the Mediterranean Sea from early autumn 2016. A newly described Haplosporidian endoparasite (Haplosporidium pinnae) is the most probable cause of this ecological catastrophe placing one of the largest bivalves of the world on the brink of extinction. As a pivotal step towards Pinna nobilis conservation, this contribution combines scientists and citizens’ data to address the fast- and vastdispersion and prevalence outbreaks of the pathogen. Therefore, the potential role of currents on parasite expansion was addressed by means of drift simulations of virtual particles in a high-resolution regional currents model. A generalized additive model was implemented to test if environmental factors could modulate the infection of Pinna nobilis populations. The results strongly suggest that the parasite has probably dispersed regionally by surface currents, and that the disease expression seems to be closely related to temperatures above 13.5 °C and to a salinity range between 36.5–39.7 psu. The most likely spread of the disease along the Mediterranean basin associated with scattered survival spots and very few survivors (potentially resistant individuals), point to a challenging scenario for conservation of the emblematic Pinna nobilis, which will require fast and strategic management measures and should make use of the essential role citizen science projects can play., This study was financed by the special action project “NACRAS – Evaluación de la magnitud de un evento de mortalidad combinando censos científicos ampliados con observaciones de ciencia ciudadana” cofunded by the Regional Government and Direcció General d’Innovació i Recerca of the Balearic Islands and the European Social Fund 2014–2020. Salinity and temperature data at Alfacs Bay was provided from the “Programa de Seguimiento de la calidad de las aguas, moluscos y fitoplancton tóxico en las zonas de producción de marisco del litoral catalán (PSQAM) de la DGPAM.” The authors acknowledge the MEDCLIC project, funded by “La Caixa” Foundation, contributing to the development of the WMOP hydrodynamic model. Maite Vázquez- Luis and Miguel Cabanellas-Reboredo were supported by a postdoctoral contract co-funded by the Regional Government of the Balearic Islands and the European Social Fund 2014–2020. Also, Vázquez-Luis was also supported by a postdoctoral contract Juan de la Cierva-Incorporación (IJCI-2016-29329) of Ministerio de Ciencia, Innovación y Universidades. Elvira Álvarez was supported by a Personal Técnico de Apoyo contract (PTA2015-10829-I) funded by the Spanish Ministry of Economy and Competiveness. Iris E. Hendriks was supported by Ramon y Cajal Fellowship RYC-2014-14970, cofunded by the Conselleria d’Innovació, Recerca i Turisme of the Balearic Government (Pla de ciència, tecnologia, innovació i emprenedoria 2013-2017) and the Spanish Ministry of Economy, Industry and Competitiveness. Jose Rafael Garcia-March and Jose Tena were supported by the project BF/HEM 15-1662

The pustulose ark, Anadara tuberculosa , is considered as an emblematic species of the east Pacific mangrove ecosystem. The decline of its populations is of increasing preoccupation, in particular in Peru. The use of hatchery-produced larvae for stock enhancement or aquaculture is attractive but raises strong criticisms from an ecological point of view, as it could conduce to loss of genetic diversity in wild populations and favor the disease spreading from hatcheries to the environment. In this works, spat production through traditional aquaculture/biotechnology technologies was associated with pathogen molecular diagnostics and with genetic diversity estimation to produce specific pathogen-free (SPF) larvae from genetically characterized populations. All PCR and nested-PCR diagnostics of common bivalve pathogens, such as Bonamia , Martelia , Perkinsus and OsHV1, led to negative results for healthy or moribund adults and spat samples. Microbiological and molecular analyses realized during mortalities led to identifying a pathogenic Pseudomonas strain present on culture tank walls, in moribund spats and adults´ blood. Population genetic variation was assessed by mitochondrial COI gene sequences which showed 36.7% haplotype diversity. This study with hatchery production, pathology and genetic components for an overexploited native bivalve represents an original model for successful mollusk stock enhancement or/and innovative aquaculture programs. Normal 0 21 false false false ES-EC X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Tabla normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:10.0pt; mso-para-margin-left:0cm; line-height:115%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri",sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-ansi-language:ES-MX; mso-fareast-language:ES-MX;}

14 páginas, 1 figura, 3 tablas.-- Simone Sühnel ... et al., The protozoans Perkinsus marinus, Perkinsus olseni, Bonamia ostreae, Bonamia exitiosa, and Marteilia refringens are responsible for some of the most detrimental diseases in the production of cultivated shellfish worldwide and are classified as notifiable diseases by the World Organization for Animal Health (OIE). This study examined the general health status of wild and cultured bivalves from southern Brazil and included diagnostic tests for the presence of Perkinsus sp., Bonamia sp., andMarteilia sp. Cultured bivalves included the mangrove oyster Crassostrea gasar (syn. Crassostrea brasiliana), the brown mussel Perna perna, the lion's paw scallop Nodipecten nodosus, andwing pearl oyster Pteria hirundo. Themangrove oyster Crassostrea rhizophorae and the carib pointed venus clam Anomalocardia brasiliana (syn. Anomalocardia flexuosa) were collected from wild populations. A variety of parasitic or commensal organisms were detected by histology including Ancistrocoma-like and Spenophrya-like organisms; Bucephalus genus, Nematopsis sp., Steinhausia sp., and Tylocephalum sp.; unidentified trematode; unknown protozoan and metazoans; and an amoeba parasite. Hemocytic infiltration was most commonly associated with parasitized animals. Histological, culture and molecular diagnostic tests did not find any evidence of the presence of OIE-listed pathogens or related species in this study. Although the current survey did not identify any pathogens or diseases of concern, it provides baseline health assessment data for these species against which any future disease developments or significant changes in population health can be compared. These data are also valuable with respect to the development and implementation of public policies related to aquatic animal health, We would like to thank to the Ministry of Fisheries and Aquaculture, Brazil, for the research support provided to the Fundaçao de Amparo à Pesquisa do Estado de Santa Catarina (FAPESC), Brazil; the National Council of Technological and ScientificDevelopment, Brazil (CNPq-558222/2008-0), for supporting themacro- andmicroscopical analysis and for providing a grant (CNPq-301072/2007-8); the Coordenaçao de Aperfeiçoamento de Pessoal de Nivel Superior (CAPES), Brazil, for post-doctoral scholarship support for SS; the Pacific Biologic Station and Vancouver Island University (VIU) in Canada for supporting the molecular studies

The Phylum Haplosporidia consists of four genera (Minchinia, Haplosporidium, Urosporidium and Bonamia) that are endoparasitic protists of a wide range of marine invertebrates including commercial bivalve species. Characterization of haplosporidian species remains a challenge due to their patchy spatial and temporal distributions, host-restricted occurrence, and poorly known life cycles. However, they are commonly associated with significant mortality events in bivalves. Due to the recent sporadic mortality events that have occurred in cockles in Europe, the objectives of this study were to determine the diversity, distribution and seasonality of haplosporidian species in Cerastoderma edule populations at several Irish sites. The role of abiotic (temperature, salinity and dissolved oxygen in water) and biotic (cockle size and age) factors as drivers or inhibitors of haplosporidian infection were also assessed. Cockles (n = 998) from the intertidal were sampled from April/July 2018 to April 2019 at three sites with no commercial fishing activity on the south coast (Celtic Sea) and one site on the northeast coast (Irish Sea) with an active commercial fishery. Screening of the cockles by molecular techniques (PCR, Sanger sequencing) and by histopathology was carried out. Two species were identified and confirmed in Irish C. edule for the first time, Minchinia mercenariae -like (14.8%) and Minchinia tapetis (29.6%). Similar to other haplosporidian parasites, the Minchinia spp. detected in our study were present year-round at all sites, except for M. tapetis in Youghal Bay (Celtic Sea). Coinfection of both Minchinia species was only observed in Cork Harbour (Celtic Sea) and Dundalk Bay (Irish Sea), where Minchinia spp. showed a higher presence compared to Youghal Bay and Dungarvan Harbour (Celtic Sea). Moreover, haplosporidians detected with generic primers, were present at all of the sample sites throughout the year but had a higher occurrence during the winter months and were positively correlated with dissolved oxygen. Likewise, smaller and older C.edule seemed to be more vulnerable to the haplosporidian infection. Furthermore, haplosporidian distribution displayed spatial variability between and within sample sites, with the highest presence being observed in cockles at one of the commercially fished Dundalk beds, while the lowest presence was observed in cockles at the second Dundalk bed that was more influenced by freshwater runoff when the tide was out. Findings from this study provide additional information on the distribution and seasonal presence of novel haplosporidian species and their potential abiotic and biotic drivers/inhibitors of infection.

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.

This study provides morphological and molecular characterization of a new species, Haplosporidium pinnae), very likely responsible for mass mortality of fan mussels, Pinna nobilis, in the Western Mediterranean Sea. The parasite was found in dead or moribund P. nobilis but did not occur in healthy fan mussels from locations that were not affected by abnormal mortality. Histological examination of infected fan mussels showed uninucleate cells of a haplosporidan parasite throughout the connective tissue and hemolymph sinuses of the visceral mass and binucleate cells and, rarely, multinucleate plasmodia were also detected in the connective tissue. Additionally, stages of sporulation occurred in the epithelium of the host digestive gland tubules. Spores were slightly ellipsoidal with a hinged operculum in one pole. Typical haplosporosomes were not found with TEM but vesicles with two concentric membranes resembling haplosporosomes were abundant in the cytoplasm of the multinucleate plasmodia occurring in host digestive gland tubules. SEM analysis showed multiple structures on the spore surface; some spores had two or four long tape-like filaments attached to the spore wall. Phylogenetic analysis based on the SSU rDNA sequence placed this parasite within a large clade including species of the order Haplosporida, not in the Bonamia/Minchinia subclade or the subclade containing most Haplosporidium species, but within a subclade of Haplosporidium sp. from Penaeus vannamei. Our results suggested that H. pinnae and the parasite of P. vannamei may represent a distinct new genus within the order Haplosporida.

10 pages, 3 tables, 7 figures, The purpose of this study was to compare the parasites of two coexisting bivalves, the edible Puelche oyster (Ostrea puelchana) and the false oyster (Pododesmus rudis) that lives attached to O. puelchana shells, and to investigate their host specificity. Samples from wild populations, 465 O. puelchana and 131 P. rudis, were collected seasonally during two years in the San José Gulf (northern Patagonia, Argentina) and were processed using standard histological techniques. To increase the natural low prevalences of Bonamia spp. and Perkinsus spp. that are present in wild populations, an in situ experiment was performed by maintaining captive sentinel bivalves at high densities inside a plastic mesh bag to enhance parasite transmission. Polymerase chain reaction (PCR) assays were used to test for apparent Bonamia sp. infections among captive sentinel O. puelchana specimens (n = 80), and Ray’s fluid thioglycollate medium (RFTM) assays and histological immunoassays tested for apparent Perkinsus sp. infections among captive sentinel P. rudis specimens (n = 100). Despite histological observations that revealed the presence of microcells resembling Bonamia sp. infecting hemocytes of some Puelche oysters, PCR assays did not confirm that parasite identification. Among captive sentinel P. rudis that showed histological evidence of Perkinsus sp. infections, neither RFTM nor immunoassays confirmed such parasites. Ostrea puelchana from wild populations were occasional hosts for both Rickettsia-like organism (RLOs) and Urastoma-like turbellarians. In contrast, six parasite taxa infected P. rudis from coexisting populations, including RLOs, Urastoma-like turbellarians, an intracellular gregarine species, Nematopsis-like oocysts, an unidentified coccidian and a Perkinsus qugwadi-like protozoan. These results demonstrated specific infection patterns of the identified parasites in relation to their hosts, Financial support was provided by the Agencia Nacional de Promoción Científica y Tecnológica (Préstamo BID PICT 2013- 1702 and 2582) and by Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) (PIP 0670/14)

SUMMARYBonamia ostreaeis a protozoan parasite that infects the European flat oysterOstrea edulis, causing systemic infections and resulting in massive mortalities in populations of this valuable bivalve species. In this work, we have characterizedB. ostreaeactin genes and used their sequences for a phylogenetic analysis. Design of different primer sets was necessary to amplify the central coding region of actin genes ofB. ostreae. Characterization of the sequences and their amplification in different samples demonstrated the presence of 2 intragenomic actin genes inB. ostreae, without any intron. The phylogenetic analysis placedB. ostreaein a clade withMinchinia tapetis,Minchinia teredinisandHaplosporidium costaleas its closest relatives, and demonstrated that the paralogous actin genes found inBonamiaresulted from a duplication of the original actin gene after theBonamiaorigin.

The ultrastructure of Bonamia from Ostrea angasi from Australia, Crassostrea ariakensis from the USA, O. puelchana from Argentina and O. edulis from Spain was compared with described Bonamia spp. All appear conspecific with B. exitiosa. The Bonamia sp. from Chile had similarities to the type B. exitiosa from New Zealand (NZ), but less so than the other forms recognized as B. exitiosa. Two groups of ultrastructural features were identified; those associated with metabolism (mitochondrial profiles, lipid droplets and endoplasmic reticulum), and those associated with haplosporogenesis (Golgi, indentations in the nuclear surface, the putative trans-Golgi network, perinuclear granular material and haplosporosome-like bodies). Metabolic features were regarded as having little taxonomic value, and as the process of haplosporogenesis is not understood, only haplosporosome shape and size may be of taxonomic value. However, the uni-nucleate stages of spore-forming haplosporidians are poorly known and may be confused with Bonamia spp. uni-nucleate stages. The many forms of NZ B. exitiosa have not been observed in other hosts, which may indicate that it has a plastic life cycle. Although there are similarities between NZ B. exitiosa and Chilean Bonamia in the development of a larger uni-nucleate stage and the occurrence of cylindrical confronting cisternae, the clarification of the identity of Chilean Bonamia must await molecular studies.

Winter mortality (WM) is a poorly studied disease affecting Sydney rock oysters Saccostrea glomerata in estuaries in New South Wales, Australia, where it can cause significant losses. WM is more severe in oysters cultured deeper in the water column and appears linked to higher salinities. Current dogma is that WM is caused by the microcell parasite Bonamia roughleyi, but evidence linking clinical signs and histopathology to molecular data identifying bonamiasis is lacking. We conducted a longitudinal study between February and November 2010 in 2 estuaries where WM has occurred (Georges and Shoalhaven Rivers). Results from molecular testing of experimental oysters for Bonamia spp. were compared to clinical disease signs and histopathology. Available environmental data from the study sites were also collated and compared. Oyster condition declined over the study period, coinciding with decreasing water temperatures, and was inversely correlated with the presence of histological lesions. While mortalities occurred in both estuaries, only oysters from the Georges River study site showed gross clinical signs and histological changes characteristic of WM (lesions were prevalent and intralesional microcell-like structures were sometimes noted). PCR testing for Bonamia spp. revealed the presence of an organism belonging to the B. exitiosa-B. roughleyi clade in some samples; however, the very low prevalence of this organism relative to histological changes and the lack of reactivity of affected oysters in subsequent in situ hybridisation experiments led us to conclude that this Bonamia sp. is not responsible for WM. Another aetiological agent and a confluence of environmental factors are a more likely explanation for the disease.

Bonamiosis constrains the flat oyster industry worldwide. The protistan species Bonamia ostreae had been considered solely responsible for this disease in Europe, but the report of B. exitiosa infecting Ostrea edulis 5 yr ago in Galicia (NW Spain), and subsequently in other European countries, raised the question of the relevance of each species in bonamiosis. The spatial distribution of B. exitiosa and B. ostreae in Galicia was addressed by sampling 7 natural O. edulis beds and 3 culture raft areas, up to 3 times in the period 2009 to 2010. B. ostreae infected flat oysters in every natural bed and every raft culture area. True B. exitiosa infections (histological diagnosis) were detected in every raft culture area but only in 2 natural beds, i.e. in 4 rías. PCR-positive results for B. exitiosa were recorded in 4 out of 5 beds where true infections were not found, thus the occurrence of B. exitiosa in those 4 beds cannot be ruled out. Additionally, 4 cohorts of hatchery-produced oyster spat were transferred to a raft to analyse Bonamia spp. infection dynamics through oyster on-growing. The highest percentages of oysters PCR-positive for both Bonamia spp. were recorded in the first months of on-growing; other peaks of PCR-positive diagnosis were successively lower. Differences in the percentage of PCR-positive cases and in the prevalence of true infection between B. exitiosa and B. ostreae through on-growing were not significant. Our results support that B. exitiosa is adapted to infect O. edulis in the Galician marine ecosystem.

The genus Bonamia (Haplosporidia) includes economically significant oyster parasites. Described species were thought to have fairly circumscribed host and geographic ranges: B. ostreae infecting Ostrea edulis in Europe and North America, B. exitiosa infecting O. chilensis in New Zealand, and B. roughleyi infecting Saccostrea glomerata in Australia. The discovery of B. exitiosa-like parasites in new locations and the observation of a novel species, B. perspora, in non-commercial O. stentina altered this perception and prompted our wider evaluation of the global diversity of Bonamia parasites. Samples of 13 oyster species from 21 locations were screened for Bonamia spp. by PCR, and small subunit and internal transcribed spacer regions of Bonamia sp. ribosomal DNA were sequenced from PCR-positive individuals. Infections were confirmed histologically. Phylogenetic analyses using parsimony and Bayesian methods revealed one species, B. exitiosa, to be widely distributed, infecting 7 oyster species from Australia, New Zealand, Argentina, eastern and western USA, and Tunisia. More limited host and geographic distributions of B. ostreae and B. perspora were confirmed, but nothing genetically identifiable as B. roughleyi was found in Australia or elsewhere. Newly discovered diversity included a Bonamia sp. in Dendostrea sandvicensis from Hawaii, USA, that is basal to the other Bonamia species and a Bonamia sp. in O. edulis from Tomales Bay, California, USA, that is closely related to both B. exitiosa and the previously observed Bonamia sp. from O. chilensis in Chile.

First discovered decades ago, microcell protistan parasites of the genera Bonamia and Mikrocytos remain relevant today for their economic impacts on growing molluscan aquaculture industries and fisheries. Bonamia parasites have received more attention over the years in part because they are more widespread and thus of wider concern, but there has been renewed interest in Mikrocytos recently with the generation of important new findings. Among these has been the surprising observation that Mikrocytos has phylogenetic affinities to the Rhizaria, which includes the haplosporidian protists and the genus Bonamia. This Diseases of Aquatic Organisms Special, emerging from the 5th Meeting of the Microcell Working Group held at the Central Veterinary Institute, Lelystad, the Netherlands, in February 2012, presents new insights into Mikrocytos and Bonamia diversity, distributions, diagnostics, ultrastructure, and infection dynamics, and captures major developments in the field since the last review of these genera in 2004.

The protozoan parasite Bonamia ostreae is a destructive pathogen of flat oysters and has been reported to be widespread in Europe and North America. The biological characteristics of this unicellular parasite are still not fully understood. In this study, 104 Ostrea edulis imported from the USA to the Guangdong province of China for consumption were examined for Bonamia infection. PCR assay, combined with restriction fragment length polymorphism, sequencing and BLAST analysis, showed that B. ostreae DNA could be detected in 1 of the 104 oyster samples. Light microscopy revealed Bonamia-like organisms in the oyster. PCR assay and fluorescent in situ hybridization showed that B. ostreae organisms were present and retained their integrity after 4 wk in culture. Acridine orange-ethidium bromide staining indicated that the B. ostreae were still alive. In conclusion, B. ostreae was present in oysters imported to China. More importantly, the parasite was able to survive for at least 4 wk of in vitro culture at 4 degrees C, which further implied a long-term transmission risk of B. ostreae. Considering the wide culture beds of Crassostrea ariakensis and C. gigas in China, and that C. ariakensis and C. gigas are susceptible hosts or reservoirs of B. ostreae, our study highlights the potential risk of introducing B. ostreae by importing O. edulis from a Bonamia endemic area.

Legume-infecting phytoplasmas identified in symptomatic tree medic (Medicago arborea), lucerne (M. sativa) and pigeonpea (Cajanus cajan) growing at a single trial site at Urrbrae, South Australia, were all shown to belong to the 16SrII phytoplasma taxonomic group, but each host species was found to be infected with a different genotype. Within one species (M. sativa) the genotypes were more uniform as demonstrated by comparing five isolates from lucerne plots. These lucerne genotypes differed from the Australian lucerne yellows phytoplasma genotype previously described in New South Wales, and the pigeonpea phytoplasma isolate was more similar to the non-legume infecting Bonamia little leaf (BoLL) than to pigeonpea little leaf phytoplasma. This diversity supports the view that these legumes were infected from different reservoir phytoplasma hosts rather than from a single perennial host such as tree medic.

An account of the genus Bonamia in Bolivia is presented. Seven species are recognised, of which three (B. rosiewiseae J. R. I. Wood, B. riograndina J. R. I. Wood and B. cerradoensis J. R. I Wood) are described as new and illustrated with line drawings. Taxonomic notes and a key to the identification of the Bolivian species are provided. The typification of B. agrostopolis (Vell.) Hallier f. is discussed and an epitype is selected. A full synonomy for B. agrostopolis, B. balansae Hallier f. and B. subsessilis Hassl. is provided. The distribution and conservation status of all seven species is detailed with the help of a map and attention is drawn to the Chaco as a local centre of diversity in this genus in South America.

Los protozoos parásitos de los géneros Bonamia, Perkinsus, Marteilia y Mikrocytos son responsables de elevadas mortalidades en poblaciones de moluscos bivalvos a lo largo de todo el mundo, causando cuantiosas pérdidas económicas. Algunas de las especies de estos géneros están incluidas en la legislación europea como parásitos que requieren especial vigilancia y control y recogidas en la lista de enfermedades de declaración obligatoria de la Organización Mundial de la Sanidad Animal (OIE). El objetivo general de esta tesis doctoral fue desarrollar nuevas herramientas moleculares que permitan un control sanitario más eficaz de estas enfermedades. Para ello, se han desarrollado y validado ensayos de PCR convencional y PCR en tiempo real para el diagnóstico de Bonamia ostreae y Bonamia exitiosa con una elevada especificidad y sensibilidad, que podrían sustituir a los métodos actuales recomendados por la OIE. Además, se han desarrollado otros métodos de diagnóstico como la PCR multiplex para la detección simultánea de ambas especies (ensayo patentado), los primeros ensayos de hibridación in situ específicos para B. exitiosa y Perkinsus mediterraneus y un ensayo PCR-DGGE capaz de discriminar especies de Perkinsus. Estas herramientas moleculares han servido para realizar un estudio epidemiológico de la bonamiosis y perkinsosis en Galicia y de la perkinsosis en la costa mediterránea, aumentando la distribución geográfica y hospedadora de las especies Perkinsus olseni, Perkinsus mediterraneus y Perkinsus chesapeaki. Asimismo, se han identificado y caracterizado molecularmente dos parásitos emergentes en aguas gallegas: Mikrocytos sp. relacionado con un episodio de mortalidad elevada de almeja japonesa Ruditapes philippinarum, y Marteilia cochillia, una especie descrita recientemente parasitando el berberecho Cerastoderma edule y que ha causado el colapso de la pesquería de este molusco en la Ría de Arousa, principal zona productora de este bivalvo en Galicia.

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%).

The new species Bonamia jiviorum J. R. Grande (Convolvuloideae, Cresseae) is described and illustrated from northwestern Amazonas in Venezuela. This new taxon is morphologically similar to B. apurensis D. F. Austin. It is characterized by its relatively small, ovate-orbicular leaves that are densely rough-tomentulose over both surfaces and have a truncate to subcordate base and rounded to obtuse, mucronulate or minutely retuse apex; relatively lax, aphyllous infructescences with relatively short peduncles and relatively short, adpressed bracts; orbicular sepals; glabrous fruits crowned by copper-colored setose hairs; and densely pustulate, irregularly striate, conspicuously winged seeds. Bonamia jiviorum is known only from the vicinity of Puerto Ayacucho, over low-altitude granitic boulders known locally as lajas.

Production of European flat oysters Ostrea edulis in the Netherlands has been hampered by the presence of the haplosporidian parasite Bonamia ostreae, which is now an enzootic species following its establishment after 1980. We analyzed histopathological data from annual shell- fish disease monitoring from 1988 to 2006 to quantify prevalence of B. ostreae in flat oyster stocks of the marine Lake Grevelingen. In addition, we estimated prevalence of B. ostreae on a monthly basis with field surveys in 2003. The parasite was detected with PCR, using specific primers for B. ostreae. Prevalence of B. ostreae was analyzed relative to O. edulis density, biomass and a range of environ- mental parameters. B. ostreae was detected in flat oysters throughout the year with a higher preva- lence in spring than in autumn, possibly due to termination of spawning and the onset of oyster growth in autumn. Although B. ostreae was detected in all oyster weight classes, prevalence was highest in the largest oysters in spring and declined disproportionately in autumn, possibly due to high mortality of large oysters before autumn, suggesting that prevalence depends on oyster age. Parasite prevalence was independent of oyster density and total biomass, but appeared to be higher after a warm autumn. Abundance of the flat oyster (infected or non-infected with B. ostreae) was neg- atively related to the temperature of the preceding period, suggesting that mortality in flat oysters increased at higher water temperatures. Furthermore, O. edulis appeared to be more susceptible to B. ostreae after years with lower food availability and lower salinities (

SUMMARYA Minchinia sp. (Haplosporidia: Haplosporidiidae) parasite was identified infecting rock oysters and morphologically described by Hine and Thorne (2002) using light microscopy and transmission electron microscopy (TEM). The parasite was associated with up to 80% mortality in the host species and it is suspected that the parasite would be a major impediment to the development of a tropical rock oyster aquaculture industry in northern Western Australia. However, attempts to identify the parasite following the development of a specific probe for Haplosporidium nelsoni were unsuccessful. The SSU region of the parasite's rRNA gene was later characterized in our laboratory and an in situ hybridization assay for the parasite was developed. This study names the parasite as Minchinia occulta n sp. and morphologically describes the parasite using histology, scanning electron microscopy and transmission electron microscopy. The non-spore stages were unusual in that they consisted primarily of uninucleate stages reminiscent of Bonamia spp. The parasite's spores were ovoid to circular shaped and measured 4·5 μm–5·0 μm×3·5–4·1 μm in size. The nucleus of the sporoplasm measured 1·5–2·3 μm and was centrally located. The spores were covered in a branching network of microtubule-like structures that may degrade as the spore matures.

SUMMARYBonamia exitiosais an intracellular parasite (Haplosporidia) that has been associated with mass mortalities in oyster populations in the Southern hemisphere. This parasite was recently detected in the Northern hemisphere including Europe. Some representatives of theBonamiagenus have not been well categorized yet due to the lack of genomic information. In the present work, we have applied Whole-Genome Amplification (WGA) technique in order to characterize the actin gene in the unculturable protozoanB. exitiosa. This is the first protein coding gene described in this species. Molecular analysis revealed thatB. exitiosaactin is more similar toBonamia ostreaeactin gene-1. Actin phylogeny placed theBonamiasp. infected oysters in the same clade where the herein describedB. exitiosaactin resolved, offering novel information about the classification of the genus. Our results showed that WGA methodology is a promising and valuable technique to be applied to unculturable protozoans whose genomic material is limited.

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.

Culture of native flat oysters Ostrea puelchana d'Orbigny in San Antonio Bay (San Matías Gulf, Argentina) began in 1995. After elevated mortality (33%) occurred in September 1996, 18 mo after immersion, histopathological analysis and evaluation of parasitic prevalence was carried out. In October 1997, after 31 mo of cultivation, cumulative mortality was 80%, and in December of the same year, when individuals reached marketable size, mortality was 95% and culture was discontinued. The present study describes the haemocytic parasitism that affected O. puelchana, and suggests that a Bonamia sp. was the etiological agent. This parasite should be considered as a different species from Bonamia sp. detected in Australia and New Zealand until more studies are made to determine the correct taxonomy. This work constitutes the first record of this haemocyte parasite in flat oysters from the Argentinean coast.

Olympia oysters Ostrea conchaphila have declined markedly during the last century and are a focus of restoration in many embayments, including the San Francisco Bay (SFB) estuary. Oysters were collected from 17 sites in this estuary and nearby Tomales Bay in an effort to charac- terize diseases that may impact recovery of this species and captive rearing programs. Three diseases/disease agents including a Mikrocytos-like protist (microcell), a haplosporidian and hemic neoplasia were observed from several sites along the western margins of the SFB estuary suggesting a geographic localization of disease presence. Based on fluoresecent in situ hybridization (FISH) assays, the microcell is distinct from M. mackini and Bonamia spp. These data highlight the need for further elucidation of the haplosporidian and for careful health management of a declining species destined for captive rearing and supplementation.

The current status of the Haplosporidia is reviewed as well as recent information on Haplosporidium nel- soni, the causative agent of MSX disease in oysters. Recent molecular phylogenetic analyses with greatly increased taxon sampling support monophyly of the Haplosporidia and hypothesize placement of the group as sister taxon to the phylum Cercozoa. Oyster pathogens in the genus Bonamia should be considered haplosporidians based on molecular sequence data. Thus, the group contains 4 genera: Uropsoridium, Haplosporidium, Bonamia and Minchinia. Molecular phylogenetic analyses support monophyly of Urosporidium, Bonamia and Minchinia ,b utHaplosporidium forms a pa- raphyletic clade. Reports of haplosporidia worldwide are reviewed. Molecular detection assays have greatly increased our ability to rapidly and specifically diagnose important pathogens in the phylum and have also improved our under- standing of the distribution and biology of H. nelsoni and H. costale. Much of the data available for H. nelsoni has been integrated into a mathematical model of host/parasite/environment interactions. Model simulations support hypotheses that recent H. nelsoni outbreaks in the NE United States are related to increased winter temperatures, and that a host other than oysters is involved in the life cycle. Evidence is presented that natural resistance to H. nelsoni has developed in oysters in Delaware Bay, USA. However, in Chesapeake Bay, USA H. nelsoni has intensified in historically low salinity areas where salinities have increased because of recent drought conditions. Efforts to mitigate the impact of H. nelsoni involve selective breeding programs for disease resistance and the evaluation of disease resistant non-native oysters.

In the Austral summer and autumn of 2000 and 2001, mortalities of black-footed abalone Haliotis iris (Martyn, 1784) occurred in a commercial facility in New Zealand. Histological analyses suggested that infection by a haplosporidian parasite was responsible. To confirm identification as a haplosporidian and to help determine if this parasite represented a new, undescribed species, DNA was extracted from infected host tissues scored as positive for infection by histological examination. Small-subunit rRNA (SSU rRNA) gene sequences from both the host abalone and a parasitic organism were amplified by PCR and characterized. Although the sequence for this parasite was novel, not matching any known SSU rRNA gene sequences, phylogenetic analyses strongly supported grouping this parasite with the haplosporidians. Parsimony analyses placed the parasite at the base of the phylum Haplosporidia, ancestral to Urosporidium crescens and the Haplosporidium, Bonamia, and Minchinia species. Sequencing of multiple parasite DNA clones revealed a single polymorphic site in the haplosporidian SSU rRNA gene sequence.

'Present IJddre55: IfREMER, Laboratoire d'Aquaculture Tropicale, SP 7004, 98719 Tuavao, french Polynesla ABSTRACT: Microcell·type parasites of oysters are assodated with a complex of diseases in different oyster species around the world. The etiologicaJ agents are prolists of very small sue that are very dif· ficult to characterize taxonomieaUy. Assodaled lesions rnay vary according to the hasl spedes, and their occurrence rnay be relaled to variations in tissue structure. Lesion morphology eannot be used 10 distinguish the different agents involved. UltrastructuraJ observations on Mikrocytos rOlJghleyi reveaJed similarities with Bonamiaspp., particularly in regard la the presence of electron-dense hap. losporosomes and mitochondria, wh05e absence from M. mackini also indieate thal M. rougbleyi and M. macldnj are not congeneric. A partial small subunit Issu) rRNA gene sequenee of M. roughJeyi was delennined. This partiaJ sequence, 951 nudeotides in length. has 95.2 and 98.4 % sequence sim· ilarities with B. ostreaeand B. e1dUoslJs ssu rONA sequences, respectively. Polymorpblsms among the 55U rDNA sequences of B. ostreae, B. e1dUosus and M. roughleyi allowed identification 01 restriction enzyme digestion patterns diagnostic for each species. Pbylogenetie anaJysis based on the ssu rONA data suggested thal M. roughleyi belongs in the phylum Hapl05poridia and thal il is dosely relaled to Bonamia spp. On the basis of ultrastructural and molecular considerations, M. roughleyi should be considered a putative member of the genus Bonamja.

Histological examination of 6455 oysters Ostrea chilensis from Foveaux Strait south of New Zealand over a 5 yr period showed > 85 % contained apicomplexan zoites, irrespective of season. Zoites occurred around the haemolymph sinuses and the digestive diverticulae at all intensities of infection; occurrence in the sub-epithelium, Leydig tissue and gills/mantle increased with increasing intensity of infection. Many (>35%) oysters were heavily infected, and most of them had severely damaged tissues. Heavy infections affected gametogenesis; 1 % of lightly infected oysters had empty gonad follicles lacking germinal epithelium compared with 2 % of moderately infected oysters and 9 % of heavily infected oysters. Of oysters with empty gonad follicles, 75 % were heavily infected with zoites. The parasite spread from the haemolymph sinuses and moved between Leydig cells, causing their dissociation and lysis. Some zoites were intracellular in Leydig cells. Lesions contained many haemocytes phagocytosing zoites, leading to haemocyte lysis and causing a haemocytosis. Fibrosis occurred to repair lesions in a few oysters. The zoites had a typical apical complex with 2 polar rings and 84 sub-pellicular microtubules. Prevalence and intensity of concurrent Bonamia exitiosus infection was related to the intensity of zoite infection, with only 3.8 % of B. exitiosus infections occurring in the absence of zoites, 20.0% occurring in light zoite infections, 30.9% in moderate zoite infections, and 45.4% when oysters were heavily infected with zoites. The converse was not the case, as 75.3% of zoite infections occurred in the absence of B. exitiosus infection, including 51.1% of moderate to heavy zoite infections. There was a statistically significant association between intensities of B. exitiosus and of zoites (p < 0.0001). Zoites may increase the susceptibility of oysters to B. exitiosus by occupying and destroying haemocytes, and by destroying connective tissue cells and utilising host glycogen reserves. The parasite may be heteroxenous, with other stages in the terebellid polychaete Pseudopista rostrata.

Bonamiosis is a disease affecting various oyster species and causing oyster mass mortalities worldwide. The protozoans Bonamia exitiosa and B. ostreae (Haplosporidia) are included in the list of notifiable diseases of the World Organisation for Animal Health as the causative agents of this disease. Although the geographic range of both species was considered different for years, both species are now known to co-occur in some European areas affecting the same host, Ostrea edulis, which strengthens the need of species-specific methods to unequivocally identify the species of Bonamia. An oligonucleotide probe for specific detection of B. exitiosa (BEX_ITS) was designed to be used in in situ hybridisation (ISH) assays. ISH assay with BEX_ITS probe showed species-specificity and more sensitivity than traditional histology to visualise the parasite inside host tissue. ISH assay showed that the oyster gonad was the area where the parasite was most frequently located, and was the exclusive organ of infection in some oysters. A recommendation arising from the study is that more than 1 organ (including gonad and gills) should be used for PCR-based diagnosis of B. exitiosa, to maximise the sensitivity.

The name 'microcells' is frequently used to refer to small-sized unicellular stages of molluscan parasites of the genera Bonamia (Rhizaria, Haplosporidia) and Mikrocytos (Rhizaria). Histological examination of Manila clams Ruditapes philippinarum revealed microcells in the connective tissue of adductor muscle, foot, mantle, gills, siphon and visceral mass. The clams had been collected from 4 beds on the coast of Galicia, Spain. The prevalence of these microcells ranged from 73 to 93% in surface clams and from 3 to 33% in buried clams. However, the detection of brown ring disease signs in clams from every bed prevented us from making the assumption that the microcells alone were responsible for clam mortality. PCR assays using primer pairs designed to detect Bonamia spp. and haplosporidians gave negative results, whereas positive results were obtained with primers for the genus Mikrocytos. A consensus sequence of 1670 bp of the ribosomal gene complex of the microcells was obtained. It contained a section of the 18S region, the whole first internal transcribed spacer, the 5.8S region, the second internal transcribed spacer and a section of the 28S region. Comparison of this sequence with those of M. mackini infecting Crassostrea gigas and Mikrocytos sp. infecting Ostrea edulis showed that the microcells of Galician clams were the most divergent among the compared parasites. This is the first report of a Mikrocytos-like parasite infecting Manila clams. Care must be taken to avoid the spread of this parasite through Manila clam transfers.