Detection of haplosporidian protistan parasites supports an increase to their known diversity, geographic range and bivalve host specificity.

Haplosporidian protist parasites are a major concern for aquatic animal health, as they have been responsible for some of the most significant marine epizootics on record. Despite their impact on food security, aquaculture and ecosystem health, characterizing haplosporidian diversity, distributions and host range remains challenging. In this study, water filtering bivalve species, cockles Cerastoderma edule, mussels Mytilus spp. and Pacific oysters Crassostrea gigas, were screened using molecular genetic assays using deoxyribonucleic acid (DNA) markers for the Haplosporidia small subunit ribosomal deoxyribonucleic acid region. Two Haplosporidia species, both belonging to the Minchinia clade, were detected in C. edule and in the blue mussel Mytilus edulis in a new geographic range for the first time. No haplosporidians were detected in the C. gigas, Mediterranean mussel Mytilus galloprovincialis or Mytilus hybrids. These findings indicate that host selection and partitioning are occurring amongst cohabiting bivalve species. The detection of these Haplosporidia spp. raises questions as to whether they were always present, were introduced unintentionally via aquaculture and or shipping or were naturally introduced via water currents. These findings support an increase in the known diversity of a significant parasite group and highlight that parasite species may be present in marine environments but remain undetected, even in well-studied host species.

Diagnosis and prevalence of two new species of haplosporidians infecting shore crabs Carcinus maenas: Haplosporidium carcini n. sp., and H. cranc n. sp.

This study provides a morphological and phylogenetic characterization of two novel species of the order Haplosporida (Haplosporidium carcini n. sp., and H. cranc n. sp.) infecting the common shore crab Carcinus maenas collected at one location in Swansea Bay, South Wales, UK. Both parasites were observed in the haemolymph, gills and hepatopancreas. The prevalence of clinical infections (i.e. parasites seen directly in fresh haemolymph preparations) was low, at ~1%, whereas subclinical levels, detected by polymerase chain reaction, were slightly higher at ~2%. Although no spores were found in any of the infected crabs examined histologically (n = 334), the morphology of monokaryotic and dikaryotic unicellular stages of the parasites enabled differentiation between the two new species. Phylogenetic analyses of the new species based on the small subunit (SSU) rDNA gene placed H. cranc in a clade of otherwise uncharacterized environmental sequences from marine samples, and H. carcini in a clade with other crustacean-associated lineages.

Temporal dynamics of infection of cockles Cerastoderma edule with the protistan parasite Minchinia tapetis (Rhizaria: Haplosporida) in Galicia (NW Spain).

Uninucleate and binucleate cells and multinucleate plasmodia of a haplosporidan-like protist associated with heavy haemocytic infiltration were observed in histological sections of cockles, Cerastoderma edule, from the Ría de Noia (Galicia, NW Spain) in the course of a cockle health surveillance programme. Molecular assays provided identification of this protist as Minchinia tapetis, which we thus record from a new host. Prevalence of M. tapetis as high as 93% was recorded but infection intensity was low to moderate, never heavy, and abnormally high cockle mortality was not observed in the ria by shellfishers. A significant positive correlation was found between M. tapetis prevalence and sea water temperature. Sea water temperature increase associated with climate change might contribute to increase the prevalence of this infection in cockles and, as a consequence, this parasite may be considered a threat for cockle production.

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

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

Haplosporidium pinnae associated with mass mortality in endangered Pinna nobilis (Linnaeus 1758) fan mussels.

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.

Richness and distribution of tropical oyster parasites in two oceans.

Parasites can exert strong effects on population to ecosystem level processes, but data on parasites are limited for many global regions, especially tropical marine systems. Characterizing parasite diversity and distributions are the first steps towards understanding the potential impacts of parasites. The Panama Canal serves as an interesting location to examine tropical parasite diversity and distribution, as it is a conduit between two oceans and a hub for international trade. We examined metazoan and protistan parasites associated with ten oyster species collected from both Panamanian coasts, including the Panama Canal and Bocas del Toro. We found multiple metazoan taxa (pea crabs, Stylochus spp., Urastoma cyrinae). Our molecular screening for protistan parasites detected four species of Perkinsus (Perkinsus marinus, Perkinsus chesapeaki, Perkinsus olseni, Perkinsus beihaiensis) and several haplosporidians, including two genera (Minchinia, Haplosporidium). Species richness was higher for the protistan parasites than for the metazoans, with haplosporidian richness being higher than Perkinsus richness. Perkinsus species were the most frequently detected and most geographically widespread among parasite groups. Parasite richness and overlap differed between regions, locations and oyster hosts. These results have important implications for tropical parasite richness and the dispersal of parasites due to shipping associated with the Panama Canal.

Whole-genome amplification: a useful approach to characterize new genes in unculturable protozoan parasites such as Bonamia exitiosa.

Bonamia exitiosa is 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 the Bonamia genus 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 protozoan B. exitiosa. This is the first protein coding gene described in this species. Molecular analysis revealed that B. exitiosa actin is more similar to Bonamia ostreae actin gene-1. Actin phylogeny placed the Bonamia sp. infected oysters in the same clade where the herein described B. exitiosa actin 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.

Species-specific oligonucleotide probe for detection of Bonamia exitiosa (Haplosporidia) using in situ hybridisation assay.

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.

Interlaboratory variability in screening for Bonamia ostreae, a protistan parasite of the European flat oyster Ostrea edulis.

The spread of the protozoan parasite Bonamia ostreae is of major concern to the European flat oyster Ostrea edulis industry. Many studies have looked at the sensitivity of individual methods available to screen for B. ostreae, but in this study, 3 separate laboratories examined 4 methods of diagnosis currently used routinely in laboratories: heart imprints, histology, polymerase chain reaction (PCR) and in situ hybridisation (ISH). The results were compared to estimate interlaboratory variability. Heart imprints and histology had the highest reproducibility amongst the 3 laboratories, with greatest agreement between detection of infected and uninfected individuals. PCR had the highest detection level in every laboratory. These positives were related to the presence of confirmed infections but also in unconfirmed infections, possibly due to the presence of traces of B. ostreae DNA in oysters where clinical infections were not observed. PCR, in combination with histology or ISH, provided the most reliable detection levels in every laboratory. Variation in results for PCR and ISH observed between laboratories may be due to the different protocols used by each laboratory for both methods. Overall, the findings from the 3 laboratories indicated that at least 2 methods, with fixed protocols, should be used for the accurate detection and determination of infection prevalence within a sample. This combination of methods would allow for a clearer and more precise diagnosis of B. ostreae, preventing further spread of the disease and providing more accurate detection levels and epidemiological information.

Infection of juvenile edible crabs, Cancer pagurus by a haplosporidian-like parasite.

This study aimed to examine the pathobiology of a haplosporidian-like infection in juvenile (pre-recruit) edible crabs (Cancer pagurus) from two locations in South West Wales, UK. Infected crabs showed no external symptoms of the disease but dissection revealed an infected and hypertrophic antennal gland. Histological examination showed extensive parasitisation of the antennal gland overlying the hepatopancreas. Heavily infected crabs also showed the presence of parasites with morphological similarities to Haplosporidia in the labyrinth of the antennal gland and in the gills. The spread of the infection from the antennal gland to the gills suggests that these parasites are released into the haemolymph. Attempts to characterise the haplosporidian-like organism using several primers previously shown to amplify members of the phylum Haplosporidia failed. The prevalence of infection in juvenile edible crabs varied throughout the sampling period of November 2011 to July 2012 with the lowest level of ca. 15% in November peaking at 70% in March. This parasite may represent a threat to the sustainability of edible crab fisheries in this region if the damage observed in the antennal gland and gills results in host mortality. The identification of these parasites as members of the phylum Haplosporidia based on morphology alone must be seen as tentative in the absence of sequence data.

Haplosporidium littoralis sp. nov.: a crustacean pathogen within the Haplosporida (Cercozoa, Ascetosporea).

Previously, we described the pathology and ultrastructure of an apparently asporous haplosporidian-like parasite infecting the common shore crab Carcinus maenas from the European shoreline. In the current study, extraction of genomic DNA from the haemolymph, gill or hepatopancreas of infected C. maenas was carried out and the small subunit ribosomal DNA (SSU rDNA) of the pathogen was amplified by PCR before cloning and sequencing. All 4 crabs yielded an identical 1736 bp parasite sequence. BLAST analysis against the NCBI GenBank database identified the sequence as most similar to the protistan pathogen group comprising the order Haplosporida within the class Ascetosporea of the phylum Cercozoa Cavalier-Smith, 1998. Parsimony analysis placed the crab pathogen within the genus Haplosporidium, sister to the molluscan parasites H. montforti, H. pickfordi and H. lusitanicum. The parasite infecting C. maenas is hereby named as Haplosporidium littoralis sp. nov. The presence of a haplosporidian parasite infecting decapod crustaceans from the European shoreline with close phylogenetic affinity to previously described haplosporidians infecting molluscs is intriguing. The study provides important phylogenetic data for this relatively understudied, but commercially significant, pathogen group.

Species-specific diagnostic assays for Bonamia ostreae and B. exitiosa in European flat oyster Ostrea edulis: conventional, real-time and multiplex PCR.

Bonamia ostreae and B. exitiosa have caused mass mortalities of various oyster species around the world and co-occur in some European areas. The World Organisation for Animal Health (OIE) has included infections with both species in the list of notifiable diseases. However, official methods for species-specific diagnosis of either parasite have certain limitations. In this study, new species-specific conventional PCR (cPCR) and real-time PCR techniques were developed to diagnose each parasite species. Moreover, a multiplex PCR method was designed to detect both parasites in a single assay. The analytical sensitivity and specificity of each new method were evaluated. These new procedures were compared with 2 OIE-recommended methods, viz. standard histology and PCR-RFLP. The new procedures showed higher sensitivity than the OIE recommended ones for the diagnosis of both species. The sensitivity of tests with the new primers was higher using oyster gills and gonad tissue, rather than gills alone. The lack of a 'gold standard' prevented accurate estimation of sensitivity and specificity of the new methods. The implementation of statistical tools (maximum likelihood method) for the comparison of the diagnostic tests showed the possibility of false positives with the new procedures, although the absence of a gold standard precluded certainty. Nevertheless, all procedures showed negative results when used for the analysis of oysters from a Bonamia-free area.

A duplex quantitative real-time PCR assay for the detection of Haplosporidium and Perkinsus species in shellfish.

A duplex quantitative real-time polymerase chain reaction (dq-PCR) assay was optimized to simultaneously detect Haplosporidium spp. and Perkinsus spp. of shellfish in one reaction. Two sets of specific oligonucleotide primers for Haplosporidium spp. and Perkinsus spp., along with two hydrolysis probes specific for each parasite group, were used in the assay. The dq-PCR results were detected and analyzed using the Light Cycler 2.0 software system. The dq-PCR identified and differentiated the two protozoan parasite groups. The sensitivity of the dq-PCR assay was 200 template copies for both Haplosporidium spp. and Perkinsus spp. No DNA product was amplified when known DNA from Marteilia refringens, Toxoplasma gondii, Bonamia ostreae, Escherichia coli, Cymndinium spp., Mykrocytos mackini, Vibrio parahaemolyticus, and shellfish tissue were used as templates. A total of 840 oyster samples from commercial cultivated shellfish farms from two coastal areas in China were randomly collected and tested by dq-PCR. The detection rate of Haplosporidium spp. was 8.6% in the Qindao, Shandong coastal area, whereas Perkinsus spp. was 8.3% coastal oysters cultivated from shellfish farms of Beihai, Guangxi. The dqPCR results suggested that Haplosporidium spp. was prevalent in oysters from Qindao, Shandong, while Perkinsus spp. was prevalent in oysters from the coastal areas of Beihai, Guangxi. This dq-PCR could be used as a diagnostic tool to detect Haplosporidium spp. and Perkinsus spp. in cultivated shellfish.

Haplosporidium raabei n. sp. (Haplosporidia): a parasite of zebra mussels, Dreissena polymorpha (Pallas, 1771).

Extensive connective tissue lysis is a common outcome of haplosporidian infection. Although such infections in marine invertebrates are well documented, they are relatively rarely observed in freshwater invertebrates. Herein, we report a field study using a comprehensive series of methodologies (histology, dissection, electron microscopy, gene sequence analysis, and molecular phylogenetics) to investigate the morphology, taxonomy, systematics, geographical distribution, pathogenicity, and seasonal and annual prevalence of a haplosporidian observed in zebra mussels, Dreissena polymorpha. Based on its genetic sequence, morphology, and host, we describe Haplosporidium raabei n. sp. from D. polymorpha - the first haplosporidian species from a freshwater bivalve. Haplosporidium raabei is rare as we observed it in histological sections in only 0·7% of the zebra mussels collected from 43 water bodies across 11 European countries and in none that were collected from 10 water bodies in the United States. In contrast to its low prevalences, disease intensities were quite high with 79·5% of infections advanced to sporogenesis.

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.

Quantitative PCR assay to determine prevalence and intensity of MSX (Haplosporidium nelsoni) in North Carolina and Rhode Island oysters Crassostrea virginica.

The continuing challenges to the management of both wild and cultured eastern oyster Crassostrea virginica populations resulting from protozoan parasites has stimulated interest in the development of molecular assays for their detection and quantification. For Haplosporidium nelsoni, the causative agent of multinucleated sphere unknown (MSX) disease, diagnostic evaluations depend extensively on traditional but laborious histological approaches and more recently on rapid and sensitive (but not quantitative) end-point polymerase chain reaction (PCR) assays. Here, we describe the development and application of a quantitative PCR (qPCR) assay for H. nelsoni using an Applied Biosystems TaqMan® assay designed with minor groove binder (MGB) probes. The assay was highly sensitive, detecting as few as 20 copies of cloned target DNA. Histologically evaluated parasite density was significantly correlated with the quantification cycle (Cq), regardless of whether quantification was categorical (r2 = 0.696, p < 0.0001) or quantitative (r2 = 0.797, p < 0.0001). Application in field studies conducted in North Carolina, USA (7 locations), revealed widespread occurrence of the parasite with moderate to high intensities noted in some locations. In Rhode Island, USA, application of the assay on oysters from 2 locations resulted in no positives.

Diseases of oysters Crassostrea ariakensis and C. virginica reared in ambient waters from the Choptank River, Maryland and the Indian River Lagoon, Florida.

To assess potential benefits and liabilities from a proposed introduction of Asian Suminoe oysters, susceptibilities of exotic Crassostrea ariakensis and native C. virginica oysters were compared during exposures to pathogens endemic in temperate, mesohaline waters of Chesapeake Bay and sub-tropical, polyhaline Atlantic waters of southern Florida, USA. Cohorts of diploid, sibling oysters of both species were periodically tested for diseases while reared in mesocosms receiving ambient waters from the Choptank River, Maryland (>3 yr) or the Indian River Lagoon, Florida (10 to 11 mo). Haplosporidium sp. infections (e.g. MSX disease) were not detected in oysters from either site. Perkinsus sp. infections (dermo disease) occurred among members of both oyster species at both sites, but infections were generally of low or moderate intensities. A Bonamia sp. was detected by PCR of DNAs from tissues of both oyster species following exposure to Florida waters, with maximum PCR prevalences of 44 and 15% among C. ariakensis and C. virginica oysters respectively during June 2007. Among C. ariakensis oysters sampled during April to July 2007, a Bonamia sp. was detected in 31% of oysters by PCR (range 11 to 35%) and confirmed histologically in 10% (range 0 to 15%). Among simultaneously sampled C. virginica oysters, a Bonamia sp. was detected in 7% by PCR (range 0 to 15%), but histological lesions were absent. Although this is the first report of a Bonamia sp. from Florida waters, sequences of small subunit (SSU) rDNA and in situ hybridization (ISH) assays both identified the Florida pathogen as Bonamia exitiosa, which also infects oysters in the proximate waters of North Carolina, USA.

Haplosporidium nelsoni (MSX) in Japanese scallops Patinopecten yessoensis (Jay, 1857) from Dalian along the northern coast of the Yellow Sea, China.

The protozoan parasite Haplosporidium nelsoni (MSX) was identified in Japanese scallops Patinopecten yessoensis (Jay, 1857) from Dalian along the northern coast of the Yellow Sea, China by histopathologic examination, polymerase chain reaction (PCR) amplification, and in situ hybridization (ISH) assay. H. nelsoni plasmodia-like structures were identified in the digestive glands of scallops by histologic examination, but no parasite spores were observed. PCR using the Hap-F2, R2 primer pair produced a sequence with 100% homology with the corresponding small subunit rDNA region of H. nelsoni. An ISH assay using the oligonucleotide probe MSX1347 produced a positive reaction with the Japanese scallop parasite. This is the first report of H. nelsoni in P. yessoensis in China.

Observations raise the question if the Pacific oyster, Crassostrea gigas, can act as either a carrier or a reservoir for Bonamia ostreae or Bonamia exitiosa.

This study investigated the ability of the Pacific oyster, Crassostrea gigas, to act as a carrier or reservoir of the protistan Bonamia ostreae. Studies were carried out independently in Ireland and in Spain. Naïve C. gigas were exposed to B. ostreae both in the field and in the laboratory via natural exposure or experimental injection. Naïve flat oysters, Ostrea edulis, were placed in tanks with previously exposed C. gigas. Oysters were screened for B. ostreae by examination of ventricular heart smears and by polymerase chain reaction (PCR) screening of tissue samples (gill and/or heart) and shell cavity fluid. PCR-positive oysters were further screened using histology and in situ hybridization (ISH). B. ostreae DNA was detected in the tissues and/or shell cavity fluid of a small number of C. gigas in the field and in the laboratory. B. ostreae-like cells were visualized in the haemocytes of 1 C. gigas and B. ostreae-like cells were observed extracellularly in the connective tissues of 1 other C. gigas. When C. gigas naturally exposed to B. ostreae were held with naïve O. edulis, B. ostreae DNA was detected in O. edulis; however, B. ostreae cells were not visualized. In Spain, B. exitiosa DNA was also detected in Pacific oyster tissues. The results of this study have important implications for C. gigas transfers from B. ostreae-endemic areas to uninfected areas and highlight B. ostreae and B. exitiosa's ability to survive extracellularly and in other non-typical hosts.

Haplosporidium nelsoni and H. costale in the Pacific oyster Crassostrea gigas from China's coasts.

We examined 220 Pacific oysters Crassostrea gigas obtained from 11 locations along China's coasts for the presence of the 2 protistan parasites Haplosporidium nelsoni (MSX; multinucleated sphere X) and H. costale (SSO; seaside organism). Haplosporidium-like plasmodia were histologically observed in 9 oysters (4.09%) from 7 locations. Five oysters had mixed infections, and 4 oysters were infected only with H. nelsoni as determined by in situ hybridization (ISH) and polymerase chain reaction (PCR). This is the first report of H. nelsoni and H. costale infection in bivalves in Chinese waters.