Molecular analysis of a haplosporidian parasite from cultured New Zealand abalone Haliotis iris

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.     

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.     

Lineage-specific molecular probing reveals novel diversity and ecological partitioning of haplosporidians

Haplosporidians are rhizarian parasites of mostly marine invertebrates. They include the causative agents of diseases of commercially important molluscs, including MSX disease in oysters. Despite their importance for food security, their diversity and distributions are poorly known. We used a combination of group-specific PCR primers to probe environmental DNA samples from planktonic and benthic environments in Europe, South Africa and Panama. This revealed several highly distinct novel clades, novel lineages within known clades and seasonal (spring vs autumn) and habitat-related (brackish vs littoral) variation in assemblage composition. High frequencies of haplosporidian lineages in the water column provide the first evidence for life cycles involving planktonic hosts, host-free stages or both. The general absence of haplosporidian lineages from all large online sequence data sets emphasises the importance of lineage-specific approaches for studying these highly divergent and diverse lineages. Combined with host-based field surveys, environmental sampling for pathogens will enhance future detection of known and novel pathogens and the assessment of disease risk.     

Ultrastructure of a haplosporidian containing Rickettsiae,associated with mortalities among cultured paua Haliotis iris

Uninucleate and multinucleate stages of a protozoan parasite are described from cultured abalone Haliotis iris Martyn, 1784 in New Zealand. The parasite is identified as a haplosporidian by the occurrence of multinucleate plasmodia, mitochondria with tubular cristae, lipid droplets, anastomosing endoplasmic reticulum (aER), multivesicular bodies (MVBs), haplosporogenesis by the production of haplosporosome-like bodies from nuclear membrane-bound Golgi, and their maturation to haplosporosomes. Coated pits occurred in the plasma membrane and coated vesicles were scattered in the cytoplasm, particularly in association with the Golgi face away from the nucleus, and aER. It is concluded that the outward face of the Golgi may be the trans face, and that aER is the trans-Golgi network. Coated pits and bristle-coated vesicles are reported from a haplosporidian for the first time. The vesicles in the MVBs resembled the cores and inner membranes of haplosporosomes, without the outer layer. The possible inter-relationships of these features are discussed. The abalone parasite differs from previously described haplosporidians in the apparent absence of a persistent mitotic spindle, and the presence of intracytoplasmic coccoid to rod-shaped bacteria resembling Rickettsiales-like prokaryotes. Phylogenetic analysis of the 16S rRNA gene sequence of the Rickettsiales-like prokaryotes indicated that these organisms belong to the Rickettsia cluster. The prokaryotes have a high (7%) sequence divergence from known Rickettsieae, with Rickettsia sp. and R. massiliae being the closest relatives. The lack of non-molecular evidence prevents us from proposing a new rickettsial genus at this time.     

Haplosporidiosis in the Pacific oyster Crassostrea gigas from the French Atlantic coast

Two cases of haplosporidian infection occurred during 1993 in Pacific oysters Crassostrea gigas from the French Atlantic coast. The localization and ultrastructure of the plasmodia are described. In situ hybridization of infected tissue sections was conducted with DNA probes for oyster-infecting haplosporidians. The Haplosporidium nelsoni-specific DNA probe MSX1347 hybridized with the C. gigas parasite, and the H. costale-specific probe SSO1318 did not hybridize. Total genomic DNA was extracted from the infected tissue sections for polymerase chain reaction (PCR) amplification of the haplosporidian. PCR amplifications with H. nelsoni-specific primers and with 'universal' actin primers did not yield the expected products of 573 and 700 bp, respectively. A series of primers was designed to amplify short regions of small subunit ribosomal DNA (SSU rDNA) from most haplosporidians. The primers encompass a highly variable region of the SSU rDNA and did not amplify oyster DNA. PCR amplification of the infected C. gigas genomic DNA with these primers yielded the expected-sized product from the primer pair targeting the shortest region (94 bp). This PCR product was sequenced and it was identical to the corresponding SSU rDNA region of H. nelsoni.     

Pathology of cultured paua Haliotis iris infected with a novel haplosporidian parasite,with some observations on the course of disease

Mortalities among juvenile paua Haliotis iris Martyn 1784 in a commercial culture facility were reported in April 2000. Histology of moribund paua showed heavy systemic infections of a uni- to multi-nucleate stage of a novel organism later confirmed by transmission electron microscopy (TEM) and molecular studies to be a haplosporidian. Multinucleate plasmodia up to 25 microm diameter with up to 17 nuclei were detectable in wet preparations of hemolymph from heavily infected paua. The presence of the haplosporidian in the affected facility was associated with mortalities of slow growing 'runt' paua during the summer months. Total mortalities in affected raceways 6 mo after mortalities began were between 82.5 and 90%. Heavily infected paua exhibited behavioural abnormalities including lethargy, loss of righting reflex, and were easily detached from surfaces. Some heavily infected paua exhibited oedema and pale lesions in the foot and mantle, but no reliable gross signs of disease were noted. Light infections of the haplosporidian were also found in apparently healthy paua from the facility. Histology indicated that the early stages of infection were characterised by small numbers of plasmodia in the connective tissue surrounding the gut, amongst glial cells adjacent to nerves in the mantle and foot and within gill lamellae. In heavy infections, large numbers of small plasmodia (mean size 5.5 x 7 microm in histological sections) were present in the hemolymph, gills, heart, kidneys, mantle, foot, epipodium and connective tissue of the digestive gland. Infections were not transferred horizontally at 14 and 19 degrees C after cohabiting heavily infected paua with uninfected paua for 3 mo in aquaria, or 3 mo after injecting healthy paua with hemolymph containing haplosporidian plasmodia. This may indicate that the prepatent period for disease is longer than 3 mo, that disease is not expressed below 20 degrees C, or that an intermediate host is required for transmission. Spore formation was not observed in juvenile paua but sporocyst-like bodies containing putative spores were observed amongst haplosporidian plasmodia in the right kidney of poorly performing adult paua collected from the wild.     

First report of three protozoan parasites (a haplosporidian, Marteilia sp. and Marteilioides sp.) from the Manila clam, Venerupis (=Ruditapes) philippinarum in Japan

Recently, natural stocks of the Manila clam, Venerupis (=Ruditapes) philippinarum, have been drastically reduced in Japan. To clarify the reason for this decline in number, clams were sampled monthly from Yamaguchi and processed for histological observations, during which three protozoan parasites were discovered. Transmission electron microscopy revealed that these parasites were unidentified haplosporidian in the connective tissues, Marteilia sp. in the digestive gland and Marteilioides sp. in the oocytes. Histopathological observations suggest that Marteilia sp. and Marteilioides sp. were not pathogenic to the host. However, infection with a haplosporidian may have a negative impact on the clams. The prevalence of these parasites was low and further investigations should be undertaken to clarify their taxonomic status and establish any pathogenicity to clams.     

Ichthyosporidium sp. Schwartz, 1963, Parasite of the Fish Leiostomus xanthurus,Is a Microsporidian*

SYNOPSIS. Schwartz identified a protozoan, parasite of the marine fish Leiostomus xanthurus as a haplosporidian belonging to genus Ichthyosporidium Caullery and Mesnil, 1905. Now, a coiled polar filament in the spore of that organism has been clearly recognized in certain PAS preparations which were used in the original study. The parasite is, therefore, a microsporidian rather than a haplosporidian. The generic characters of this and other protozoans still in genus Ichthyosporidium need further study.     

Ultrastructural description of new Rickettsia-like organisms in the commercial abalone Haliotis tuberculata (Gastropoda: Haliotidae) from the NW of Spain

Rickettsia-like organisms (RLOs) were found in the commercially farmed abalone Haliotis tuberculata in the northwestern region of the Atlantic Coast of Spain and are described from light and transmission electron microscopy observations. The RLOs measured approximately 1.6 x 0.9 microm and were found in intracytoplasmic, spherical to ellipsoidal vacuoles (up to 8 microm) in the epithelial cells of the digestive diverticulae. The morphological ultrastructure of these organisms was typically prokaryotic, including a plasmalemma and a thin Gram-negative type cell wall. Several ultrastructural changes were observed in the epithelial cells of the host containing the RLOs. The nuclei became pycnotic and several basophilic dense inclusions appeared in the cytoplasm. In addition, the host cell appeared lysed and was ruptured in advanced stages of infection. It was impossible to ascertain whether the RLOs are responsible for this disease, as a haplosporidian infection was also present. We can only conclude that the presence of RLOs simultaneously with a haplosporidian parasite may contribute to the mortality of the abalone host.     

Pilot study of the Olympia oyster Ostrea conchaphila in the San Francisco Bay estuary: description and distribution of diseases

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 characterize 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.     

Prevalence of parasites in amphipods Diporeia spp. from Lakes Michigan and Huron, USA

Amphipods of Diporeia spp. have declined considerably during the last decade in the Great Lakes. We examined the possibility that disease may be affecting these populations. A histological survey assessed the parasites in species of Diporeia within Lakes Huron and Michigan, USA, and the host response to some of them and to unknown factors. Amphipods were found to have an intranuclear inclusion body, and were hosts to a rickettsia-like organism, fungi, a haplosporidian, a microsporidian, epibiotic ciliates, a gregarine, a cestode, acanthocephalans and nodule formations. Epibiotic ciliates were most common (37% prevalence of infection), but a microsporidian (3.8%), a rickettsia-like organism (1.6%), fungi, including a yeast-like organism (1.3%), worms (1.3%), and a haplosporidian (0.7%) are likely associated with mortalities or detrimental effects on the host. The role these agents may have played in the decline of Diporeia spp. in the Great Lakes over the last decade is not clear. Interrelationships with the dynamics of various physical and biological factors such as high sedimentation, diminished food supplies, and virulent parasites could synergistically cause the decline in Diporeia spp. populations in Lakes Michigan and Huron.     

Minchinia mercenariae n. sp. (Haplosporidia) in the Hard Clam Mercenaria mercenaria: Implications of a Rare Parasite in a Commercially Important Host

ABSTRACT. During routine histopathology of 180 juvenile hard clams, Mercenaria mercenaria , from a site in Virginia, USA, in 2007, we discovered a single individual heavily infected with a parasite resembling a haplosporidian, some members of which cause lethal bivalve diseases. Scanning electron microscopy of spores and sequencing of small subunit ribosomal DNA confirmed a new species: Minchinia mercenariae n. sp. Further sampling of clams at the site found prevalences up to 38% using polymerase chain reaction (PCR). No parasites were found in routine histological screening of the same individuals, but re-examination of clams judged positive by in situ hybridization (ISH) revealed very faintly staining plasmodia. No unusual mortalities have occurred among the sampled groups. Analysis of clams from Massachusetts to Florida by PCR failed to detect the parasite, but a haplosporidian found in a clam from New Jersey in 2001 was subsequently identified by ISH as M. mercenariae . No other haplosporidians have been reported in thousands of hard clams from the US east coast examined histologically since the mid-1980s. The discovery underscores critical questions about how to assess the risks associated with parasites in groups known to be lethal, but that themselves are not considered a problem.     

Widespread survey finds no evidence of Haplosporidium nelsoni (MSX) in Gulf of Mexico oysters

The advent of molecular detection assays has provided a set of very sensitive tools for the detection of pathogens in marine organisms, but it has also raised problems of how to interpret positive signals that are not accompanied by visual confirmation. PCR-positive results have recently been reported for Haplosporidium nelsoni (MSX), a pathogen of the oyster Crassostrea virginica in 31 of 40 oysters from 6 sites in the Gulf of Mexico and the Caribbean Sea. Histological confirmation of the PCR results was not undertaken, and no haplosporidian has been reported from the numerous histological studies and surveys of oysters in the region. To further investigate the possibility that H. nelsoni is present in this region, we sampled 210 oysters from 40 sites around the Gulf of Mexico and Puerto Rico using PCR and 180 of these using tissue-section histology also. None of the oysters showed evidence of H. nelsoni by PCR or of any haplosporidian by histology. We cannot, therefore, confirm that H. nelsoni is present and widespread in the Gulf of Mexico and the Caribbean Sea. Our results do not prove that H. nelsoni is absent from the region, but taken together with results from previous histological surveys, they suggest that for the purposes of controlling oyster importation, the region should continue to be considered free of the parasite.     

A Sensitive and Specific DNA Probe for the Oyster Pathogen Haplosporidium nelsoni

ABSTRACT. Haplosporidium nelsoni is a pathogen of the eastern oyster, Crassostrea virginica , along the middle Atlantic coast of the U.S. Genomic DNA was extracted from H. nelsoni plasmodia and small subunit (SSU) rDNA was amplified by PCR, cloned and sequenced. The sequence of H. nelsoni SSU rDNA was aligned with that of another haplosporidian, Minchinia teredinis , and with SSU rDNA data of C. virginica and various protists in GenBank. A 21-base oligonucleotide unique to H. nelsoni , designated MSX1347, was commercially synthesized and tested for sensitivity and specificity. In dot blot hybridizations the probe detected 100 pg of cloned H. nelsoni rDNA and the presence of H. nelsoni in 1 μg of genomic DNA from an infected oyster. It did not hybridize with 1 μg of genomic DNA from uninfected C. virginica or with cloned SSU rDNA of M. teredinis. The probe was further tested for specificity with in situ hybridizations on AFA-fixed, paraffin-embedded tissue sections. The probe hybridized well with H. nelsoni plasmodia and immature spores, but poorly with mature spores. The probe did not hybridize with oyster tissue, with other common oyster parasites such as P. marinus or Nematopsis sp., or with the haplosporidians Haplosporidium louisiana from mud crabs ( Panopeus spp.), Haplosporidium costale from C. virginica or M. teredinis from shipworms ( Teredo spp.).     

Molecular phylogenetic evidence that the phylum Haplosporidia has an alveolate ancestry

The phylogenetic position of the phylum Haplosporidia among other protists was investigated with the complete 16S-like rRNA gene sequences from two species in the phylum: Haplosporidium nelsoni, a parasite of oysters, and Minchinia teredinis, a parasite of shipworms. Because the lack of obvious morphological homologies with other protists hampered decisions regarding taxonomic composition for sequence alignment and phylogenetic analysis, the complete sequences for these two haplosporidians were directed as search queries to the blast/ncbi.nlm.nih.gov electronic mail server. The results of this heuristic similarity search provided a basis for constructing a preliminary higher-taxonomic-level analysis comparing the haplosporidians with species from the slime molds, fungi, algae, amoebae, ciliates, dinoflagellates, and apicomplexans. Maximum parsimony yielded equivocal results, whereas transversionally weighted parsimony suggested an affinity with the alveolates (i.e., the ciliates, dinoflagellates, and apicomplexans). Multiple alignment of the two haplosporidian sequences against 17 taxa in a secondary analysis focusing on the alveolates and subsequent parsimony analysis placed the phylum Haplosporidia as a monophyletic group within the Alveolata and as a taxon of equal rank with the other three alveolate phyla. The precise placement within the Alveolata was sensitive to weighting.      

Transmission of the haplosporidian parasite MSX Haplosporidium nelsoni to the eastern oyster Crassostrea virginica in an upweller system

The haplosporidian oyster parasite MSX (Multinucleated Sphere X) Haplosporidium nelsoni was transmitted to eastern oysters Crassostrea virginica. Hatchery-raised, MSX-free juvenile oysters were placed in upweller tanks. Water to the tanks was filtered through a screen with 1 mm2 openings and originated from the water column overlaying naturally infected oysters beds (MSX prevalence 17 to 57%). MSX was diagnosed by histopathological analysis. MSX-disease (57% prevalence) with increased mortality (19%) was observed 11 wk after the beginning of the exposure and mortality of 80% after 16 wk. The study demonstrates transmission of MSX via water-borne infectious agents capable of passing through a 1 mm filter.     

Urosporidium sp. hyperparasite of the turbellarian Paravortex cardii in the cockle Cerastoderma edule

Cysts with spores showing different degree of maturity and a single plasmodium were observed in the connective tissue of the turbellarian Paravortex cardii located in the digestive lumen of the cockle Cerastoderma edule. The study of spore morphology by transmission electron microscopy revealed that they correspond to an haplosporidian belonging to the genus Urosporidium. Spore ornaments were similar to those described from Urosporidium spisuli, infecting a nematode parasite of the Atlantic surf clam, Spisula solidissima.     

Effects of a haplosporidian parasite,Haplosporidium sp., on species of the molluscan woodborer Teredo in Barnegat Bay,New Jersey

Abundance of the molluscan woodborer, Teredo navalis, in Barnegat Bay, New Jersey, was reduced in breeding seasons following levels of infection of 40% or more by a haplosporidian parasite of the genus Haplosporidium. Infections also occurred in T. bartschi and T. furcifera, subtropical species which have been introduced into Barnegat Bay, and which may have originally introduced the parasite. Infections were not observed in Bankia gouldi, the most common molluscan woodborer in the bay.     

Detection of Minchinia sp., in rock oysters Saccostrea cuccullata (Born, 1778) using DNA probes

Haplosporidian parasites infect various invertebrate hosts including some commercially important shellfish. Haplosporidium nelsoni (along with Perkinsus marinus) has severely affected Eastern oyster production on the eastern seaboard of the United States and flat oyster production in Europe has been severely impacted by Bonamia ostreae. These parasites are also often present at a very low prevalence and there are a variety of morphologically similar species that can be difficult to differentiate during cytological or histological diagnosis hence the need to develop specific tests. Recently, a Minchinia sp. was described affecting rock oysters (Saccostrea cuccullata) in north Western Australia. In this study, two in situ hybridisation (ISH) assays and a PCR assay have been developed and optimised for use in investigating these parasites. The first ISH assay used a 166bp polynucleotide probe while the second used a 30bp oligonucleotide probe. The specificity of each ISH assay was assessed by applying each probe to a variety of haplosporidian (5), a paramyxian (1) or ciliophora (1) parasites. The polynucleotide probe produced strong hybridisation signals against all of the haplosporidian parasites tested (Minchinia sp., Minchinia teredinis, Bonamia roughleyi, H. nelsoni and Haplosporidium costale) while the oligonucleotide probe recognised only the Minchinia sp. Both probes failed to detect the paramyxian (Marteilia sp.) or the Rhynchodid-like ciliate. The PCR assay amplifies a 220bp region and detected Minchinia sp. DNA from 50ng of genomic DNA extracted from the tissues of infected oysters and 10fg of amplified Minchinia sp. DNA. The assay did not react to oysters infected with H. nelsoni or H. costale. The ability of the PCR and oligonucleotide ISH assay to diagnose Minchinia sp. infected oysters was compared to histological examination from a sample of 56 oysters. The PCR assay revealed 26 infections while histological examination detected 14 infections. The oligonucleotide ISH assay detected 29 infections. The oligonucleotide ISH and PCR assays were found to be significantly more sensitive than histology for detecting the parasite.