Haplosporidian host:parasite interactions.

The host:parasite interactions of the 3 serious haplosporidian pathogens of oysters, on which most information exists, are reviewed. They are Bonamia ostreae in Ostrea spp. and Crassostrea gigas; Bonamia exitiosa in Ostrea spp.; and Haplosporidium nelsoni in Crassostrea spp. Understanding the haemocytic response to pathogens is constrained by lack of information on haematopoiesis, haemocyte identity and development. Basal haplospridians in spot prawns are probably facultative parasites. H. nelsoni and a species infecting Haliotis iris in New Zealand (NZAP), which have large extracellular plasmodia that eject haplosporosomes or their contents, lyse surrounding cells and are essentially extracellular parasites. Bonamia spp. have small plasmodia that are phagocytosed, haplosporosomes are not ejected and they are intracellular obligate parasites. Phagocytosis by haemocytes is followed by formation of a parasitophorous vacuole, blocking of haemocyte lysosomal enzymes and the endolysosomal pathway. Reactive oxygen species (ROS) are blocked by antioxidants, and host cell apoptosis may occur. Unlike susceptible O. edulis, the destruction of B. ostreae by C. gigas may be due to higher haemolymph proteins, higher rates of granulocyte binding and phagocytosis, production of ROS, the presence of plasma ß-glucosidase, antimicrobial peptides and higher levels of haemolymph and haemocyte enzymes. In B.exitiosa infection of Ostrea chilensis, cytoplasmic lipid bodies (LBs) containing lysosomal enzymes accumulate in host granulocytes and in B. exitiosa following phagocytosis. Their genesis and role in innate immunity and inflammation appears to be the same as in vertebrate granulocytes and macrophages, and other invertebrates. If so, they are probably the site of eicosanoid synthesis from arachidonic acid, and elevated numbers of LBs are probably indicative of haemocyte activation. It is probable that the molecular interaction, and role of LBs in the synthesis and storage of eicosanoids from arachidonic acid, is conserved in innate immunity in vertebrates and invertebrates. However, it seems likely that haplosporidians are more diverse than realized, and that there are many variations in host parasite interactions and life cycles.

Ultrastructure, phylogeny and histopathology of two novel haplosporidians parasitising amphipods, and importance of crustaceans as hosts.

This study provides morphological, ultrastructural and phylogenetic characterization of 2 novel species of Haplosporidia (Haplosporidium echinogammari n. sp. and H. orchestiae n. sp.) infecting amphipods of the genera Echinogammarus and Orchestia collected in southwestern England. Both parasites infect the connective tissues associated with the digestive gland and the tegument, and eventually infect other organs causing disruption of host tissues with associated motor impairment and fitness reduction. Prevalence of infection varied with host species, provenance and season, being as high as 75% for individuals of E. marinus infected with H. echinogammari in June (n = 50). Although no spores were found in any of the infected amphipods examined (n = 82), the morphology of monokaryotic and dikaryotic unicellular stages of the parasites enabled differentiation between the 2 new species. Phylogenetic analysis of the new species based on the small subunit (SSU) rDNA gene placed H. echinogammari close to H. diporeiae in haplosporidian lineage C, and H. orchestiae in a novel branch within Haplosporidium. Genetic diversity of the haplosporidians infecting these and other amphipod species was evaluated and compared to morphological and ultrastructural changes to host tissues. The phylogenetic relationship of haplosporidian infections in other crustacean hosts is discussed after inclusion into the analysis of 25 novel SSU rDNA sequences obtained from crabs, isopods and crayfish.

Molecular and ultrastructural characterization of Haplosporidium diporeiae n. sp., a parasite of Diporeia sp. (Amphipoda, Gammaridea) in the Laurentian Great Lakes (USA).

BACKGROUND: The phylum Haplosporidia contains coelozoic and histozoic, spore-forming, obligate protozoan endoparasites that infect a number of freshwater and marine invertebrates including bivalves, crustaceans, and polychaetes. In amphipods, haplosporidians cause systemic infection resulting in a range of pathologies. While amphipods belonging to the genus Diporeia (Gammarideae) have been shown to host haplosporidians, the taxonomic relationship of the Diporeia haplosporidian(s) is largely unknown due to the lack of phylogenetic and detailed ultrastructural studies. METHODS: The infection characteristics and taxonomic identity of a haplosporidian infecting Diporeia spp. (Diporeia) were based on microscopical investigation, electron microscopy, and Bayesian phylogenetic inference using haplosporidian 16S rRNA gene sequences. RESULTS: In stained sections, the haplosporidian was observed to cause systemic infections in Diporeia that were often accompanied with host tissue degeneration. The haplosporidian appeared as binucleate plasmodia and sporocysts containing different spore maturation stages in the coelom, connective tissue, digestive tissue, and muscle. All of the observed systemic infections progressed to sporogenesis. Transmission electron microscopy revealed that fixed mature spores were slightly ellipsoidal and had a mean spore length X width of 5.34 ± 0.17 × 4.09 ± 0.15 µm. A hinged opercular lid with a length of 3.1 ± 0.17 µm was observed for a number of developing spores. The average thickness of the cell wall was 90.0 ± 8.33 nm. Thin filaments (70 nm) composed of spore wall material were observed projecting from an abopercular thickening of the spore wall. Phylogenetic analysis showed that the haplosporidian is novel bearing some similarities with the oyster pathogen Haplosporidium nelsoni, yet distinctly different. CONCLUSIONS: Based on its morphology, genetic sequence, and host, it became evident that the Diporeia haplosporidian is taxonomically novel and we propose its nomenclature as Haplosporidium diporeiae. This is the first report of a haplosporidian infecting Diporeia in Lake Superior.

Ultrastructural comparison of Bonamia spp. (Haplosporidia) infecting ostreid oysters.

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.

A new species of Haplosporidium Caullery & Mesnil, 1899 in the marine false limpet Siphonaria lessonii (Gastropoda: Siphonariidae) from Patagonia.

A new species of Haplosporidium Caullery & Mesnil, 1899 parasitising the pulmonate gastropod Siphonaria lessonii Blainville in Patagonia, Argentina, is described based on morphological (scanning and transmission electron microscopy) and sequence (small subunit ribosomal RNA gene) data. Different stages of sporulation were observed as infections disseminated in the digestive gland. Haplosporidium patagon n. sp. is characterised by oval or slightly subquadrate spores with an operculum that is ornamented with numerous short digitiform projections of regular height, perpendicular to and covering its outer surface. The operculum diameter is slightly larger than the apical diameter of the spore. Neither the immature nor mature spores showed any kind of projections of the exosporoplasm or of the spore wall. Regarding phylogenetic affinities, the new species was recovered as sister to an undescribed species of Haplosporidium Caullery & Mesnil, 1899 from the polychaete family Syllidae Grube from Japanese waters. The morphological characters (ornamentation of the operculum, spore wall structure, shape and size of spores, and the lack of spore wall projections) corroborate it as an as yet undescribed species of Haplosporidium and the first for the phylum in marine gastropods of South America. Siphonaria lessonii is the only known host to date.

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.

Scanning electron microscopy and molecular characterization of a new Haplosporidium species (Haplosporidia), a parasite of the marine gastropod Siphonaria pectinata (Mollusca: Gastropoda: Siphonariidae) in the Gulf of Mexico.

Based on scanning electron microscopy and the small subunit ribosomal RNA (SSU rRNA), Haplosporidium tuxtlensis n. sp. (Haplosporidia), a parasite found in the visceral tissues of the false limpet Siphonaria pectinata (Linnaeus, 1758), is described. The spores are ellipsoidal (3.61 ± 0.15 µm × 2.69 ± 0.19 µm), with a circular lid (2.94 ± 0.5 µm) representing the operculum. The spore wall bears filaments occurring singly, or in clusters, of 2 to 8, fusing distally. Phylogenetic relationships of H. tuxtlensis n. sp. were assessed with other described species using the SSU rRNA sequence. Haplosporidium tuxtlensis n. sp. is sister taxon to Haplosporidium pickfordi Barrow, 1961. The morphological characteristics (spore wall structure, shape, size, and filament structure) and the unique host identity corroborate it as a new species. Additionally, this is the first record of Haplosporidia infecting striped false limpets in the Gulf of Mexico.

Minchinia mercenariae n. sp. (Haplosporidia) in the hard clam Mercenaria mercenaria: implications of a rare parasite in a commercially important host.

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.

Ultrastructure of Bonamia sp. in Ostrea chilensis in Chile.

Oyster Ostrea chilensis samples were collected from Quihua Island, Chile, in December 2003 and February 2005, and examined in May 2004, and March, April and July 2005, for an ultrastructural comparison of the Chilean Bonamia sp. with other Bonamia spp. Only uni-nucleate stages were encountered, except in the July sample. The Chilean parasite differs from B. perspora in the apparent lack of a plasmodial stage and of sporulation. It resembles B. ostreae in size, the low number of mitochondrial profiles, and the prevalence and mean number of lipid droplets. It differs from B. ostreae in the greater prevalence of nuclear membrane-bound Golgi (NM-BG), associated haplosporogenesis, and smaller size of haplosporosomes. The Chilean Bonamia sp. resembles B. exitiosa in the number of haplosporosomes, prevalence of lipid droplets, anastomosing endoplasmic reticulum and NM-BG, presence of circles of smooth endoplasmic reticulum (sER), confronting cisternae (CC), and cylindrical CC (CCC). It also appears to have a similar developmental cycle to B. exitiosa with larger forms occurring in winter (August). The circles of sER, CC, and CCC have only been reported from B. exitiosa, and it appears that Chilean Bonamia sp. and B. exitiosa are more closely related than they are to B. perspora or B. ostreae. Similarities in ultrastructure and developmental stages between New Zealand and Chilean parasites suggest that the 2 species are related, and that the Chilean Bonamia sp. is either B. exitiosa, a sub-species of B. exitiosa, or a separate species closely related to B. exitiosa.

Inter-relationships of haplosporidians deduced from ultrastructural studies.

We reviewed papers reporting haplosporidian ultrastructure to compare inter-relationships based on ultrastructure with those based on molecular data, to identify features that may be important in haplosporidian taxonomy, and to consider parasite taxonomy in relation to host taxonomy. There were links between the following: (1) the plasmodia of an abalone parasite, Haplosporidium nelsoni and Urosporidium crescens in the release of haplosporosomes; (2) H. costale and H. armoricanum in haplosporosome shape and presence and shape of Golgi in spores; (3) basal asporous crustacean haplosporidians which form haplosporosomes from formative bodies (FBs) in vegetative stages--H. nelsoni, which forms haplosporosomes from FBs in plasmodial cytoplasm, and H. louisiana, Minchinia spp. and Bonamia perspora, which form haplosporosomes from FBs in spores; (4) crustacean haplosporidians, Bonamia spp. and M. occulta in the predominance of uni- and binucleate stages; and (5) lipid-like vesicles in sporoplasms of H. costale, H. armoricanum, H. lusitanicum, H. pickfordi, H. montforti, and B. perspora. In general, these relationships reflect phylogenies based on molecular studies. As well as spore form and ornamentation, haplosporogenesis in spores appears to be taxonomically important. Parasite and host taxonomy were linked in the infection of lower invertebrates by Urosporidium spp., the infection of oysters by Bonamia spp., and of molluscs by Minchinia spp. Haplosporidium spp. are patently an artificial, paraphyletic group probably comprising many taxa. Consequently, the taxonomy of haplosporidians needs a thorough revision.

Ultrastructural developmental cycle of Haplosporidium montforti (phylum Haplosporidia) in its farmed abalone host, Haliotis tuberculata (Gastropoda).

The sequential developmental cycle of Haplosporidium montforti, a recently described species from farmed abalone Haliotis tuberculata (Gastropoda), was studied. Ornamented and operculated mature spores were electron dense. The nucleus of the uninucleated free cell divided successively, giving rise to multinucleate plasmodia, containing up to 100-120 nuclei. Later, the plasmodia developed into sporonts inside sporocysts with irregular contours. Each of their nuclei gave rise to uninucleate sporoblasts. At the next phase of development, a very irregular membranous group of cisternae began to differentiate in the cytoplasm of each sporoblast, surrounding each nucleus and the adjacent cytoplasm. Each sporoblast differentiated into a spore. This process was characterized by the appearance of dense blisters of amorphous material at the periphery that gradually formed the prespore wall and pre-operculum. Simultaneously, in the endosporoplasm, the spherulosome and several haplosporosomes were formed. During the final phase of the maturation process, the spores became gradually denser, and the endosporoplasmic structures were barely visible.

Spore ornamentation of Haplosporidium hinei n. sp. (Haplosporidia) in pearl oysters Pinctada maxima (Jameson, 1901).

An infection of pearl oysters, Pinctada maxima, attributed to a Haplosporidium sp. by Hine and Thorne (1998) has been detected on 3 occasions and is considered to represent a serious concern to the pearling industry in Australia. The spore ornamentation of the parasite was determined by scanning electron microscopy and transmission electron microscopy. Spores of the parasite were pleomorphic, or elongated 3.5-4 microm x 2.5-3.0 microm in size. Two filaments were wound around the spore and originated from 2 'knob-like' posterior thickenings. Both filaments passed up one side of the spore together until just below the operculum whereupon each split and passed obliquely under the lip of the opercula lid. Each filament wrapped around the spore 4 times. The posterior thickenings seem to appear late in the development of the spore and were composed of spore wall material. A second set of branching tubular filaments composed of a different material was observed on the spore body although not on mature spores possessing a 'knob-like' posterior thickening. The ornamentation on the spores of the pearl oyster parasite was unique amongst described haplosporidian species where spore ornamentation is known. The parasite is named in this manuscript as Haplosporidium hinei n. sp.

Ultrastructure of sporulation in Haplosporidium armoricanum.

An ultrastructural study was carried out on the tissues of an oyster (Ostrea edulis), heavily infected with Haplosporidium armoricanum, that had been fixed in Carson's fixative. The well-fixed tissues revealed details of sporulation and of the spores, which had not been previously reported from H. armoricanum. These include the initial presence of sparse haplosporosomes after thickening of the plasma membrane in early sporonts, division of sporont nuclei by multiple fission, cup-like indentations in the nuclear surface associated with putative nuclear material in both the sporonts and spores, and cytoplasmic multi-vesicular bodies in the cytoplasm of sporonts and spores. The spore wall and operculum were formed from a light matrix that occurred in short cisternae of smooth endoplasmic reticulum in the episporoplasm, and parallel bundles of microfibrils were present in some spores. Spores were rarely bi-nucleate with the nuclei occurring as a diplokaryon, with putative nuclear material at the junction of the 2 nuclei. Nuclear membrane-bound Golgi (NM-BG) cisternae were common in spores, and they appeared to synthesise a light granular material into lysosome-like granules. Dense bodies similar to those reported from H. lusitanicum, H. pickfordi and H. monforti occurred in, or outside, the peripheral endosporoplasm, which was closely apposed to the spore wall. Spore haplosporosomes were frequently axehead-shaped, more like those of H. costale than those previously reported from H. armoricanum, and in some haplosporosomes there was a small round lucent patch with a dark point near the centre of the lucent patch. Overall, H. armoricanum appears to be closely related to H. costale and Bonamia spp. Although the endosporoplasm of H. armoricanum has NM-BG and it resembles the uni-nucleate stage, it appears to be unlikely that they are the same, as the axehead-shaped haplosporosomes of the spore differ considerably from the spherical haplosporosomes of vegetative stages.

Characterization of a rediscovered haplosporidian parasite from cultured Penaeus vannamei.

Mortalities of Penaeus vannamei, cultured in ponds in Belize, Central America, began during the last part of the grow-out cycle during the cold weather months from September 2004 through February 2005. Tissue squashes of infected hepatopancreata and histological examination of infected shrimp revealed that the mortalities might have been caused by an endoparasite. To confirm the diagnosis, DNA was extracted from ethanol preserved hepatopancreata and the small-subunit rRNA gene was sequenced. The 1838 bp sequence was novel and phylogenetic analysis placed the P. vannamei parasite within the phylum Haplosporidia as a sister taxon to a clade that includes Bonamia and Minchinia species. In situ hybridization was performed using anti-sense DNA probes that were designed to hybridize specifically with the parasite's nucleic acid. This organism presents similar characteristics to those of a haplosporidian that infected cultured P. vannamei imported from Nicaragua into Cuba, as described by Dyková et al. (1988; Fish Dis 11:15-22).

Ultrastructural and molecular characterization of Haplosporidium montforti n. sp., parasite of the European abalone Haliotis tuberculata.

A new member of the parasitic phylum Haplosporidia, which was found infecting the connective tissue, gill, digestive gland, and foot muscle of Haliotis tuberculata imported from Ireland and experimentally grown in Galicia (NW Spain), is described. Scanning electron microscopy, transmission electron microscopy, and molecular characterization of the small subunit ribosomal RNA (SSU rRNA) gene were carried out to confirm the description of this species. The ultrastructural morphology of the spores and their surrounding ornaments attached to the spore wall was described from light, scanning, and transmission electron microscopy observations. Systemic infection with uninucleated and multinucleated plasmodia containing spherical nuclei was observed among several sporocysts containing the different spore maturation stages. The spores were spherical to slightly ellipsoidal (2.42 +/- 0.5 x 2.31 +/- 0.6 microm). The apical zone of the spore wall was modified into a complex opercular system covering a circular orifice that measured about 0.5 microm in diameter. The operculum was connected to the spore wall by a hinge. The spore wall was about 110 nm thick, with 4 filaments (20-28 microm long). The filaments were composed of the same material that formed the wall. The cross-sections through the base of these filaments showed T-like and X-like sections. Internally, the uninucleated endosporoplasm contained typical haplosporidian structures, such as, haplosporosomes, a spherulosome, and mitochondria with vesicular cristae. The SSU rRNA gene sequence was different from previously reported haplosporidian SSU rRNA gene sequences, corroborating morphological data that this was an undescribed species. Based on differences from previously described haplosporidians in ultrastructural characteristics of the spore and SSU rRNA gene sequence, we describe the abalone haplosporidian as Haplosporidium montforti n. sp.

Spore ornamentation of Haplosporidium nelsoni and Haplosporidium costale (Haplosporidia), and incongruence of molecular phylogeny and spore ornamentation in the Haplosporidia.

Spore ornamentation of Haplosporidium nelsoni and Haplosporidium costale was determined by scanning electron microscopy. For H. nelsoni, the spore surface was covered with individual ribbons that were tightly bound together and occurred as a single sheet. In some spores, this layer was overlaid with a network of branching fibers, about 0.05 microm in diameter, which often was dislodged from the spore at the aboral pole. For H. costale, ornamentation consisted of a sparse network of branching fibers on the spore surface. Molecular phylogenetic analysis of the phylum Haplosporidia revealed that Urosporidium, Bonamia, and Minchinia were monophyletic but that Haplosporidium was paraphyletic. All species of Minchinia have ornamentation composed of epispore cytoplasm, supporting the monophyly of this genus. The presence of spores with a hinged operculum and spore wall-derived ornamentation in Bonamia perspora confounds the distinction between Bonamia and Haplosporidium. Species with ornamentation composed of outer spore wall material and attached to the spore wall do not form a monophyletic group in the molecular phylogenetic analysis. These results suggest that the widely accepted practice of assigning all species with spore wall-derived ornamentation to Haplospordium cannot be supported and that additional genera are needed in which to place some species presently assigned to Haplosporidium.

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.

Haemolymph parasite of the shore crab Carcinus maenas: pathology, ultrastructure and observations on crustacean haplosporidians.

A protozoan parasite with some features of haplosporidians is described from the European shore crab Carcinus maenas. The parasite establishes a systemic infection through the haemal sinuses and connective tissues. Intracellular stages of the parasite were found within reserve inclusion, connective tissue, and muscle cells, while free forms were present in all haemal spaces. A uninucleate stage appeared to develop to a multinucleate plasmodial stage following multiple mitotic divisions of the nucleus. Histopathology also indicated that nuclear division may occur to form multinucleate plasmodia, in connective tissue, reserve inclusion and muscle cells, the multinucleate plasmodium being enclosed in the host-cell plasma membrane. It appears that the multinucleate plasmodium may then undergo internal cleavages which result in plasmodial fragmentation to form many uninucleate stages. Both stages, but particularly the uninucleate stage, contained cytoplasmic, large, ovoid, dense vesicles (DVs), some of which contained an internal membrane separating the medulla from the cortex, as in haplosporosomes. Golgi-like cisternae, closely associated with the nuclear membrane, formed DVs and haplosporosome-like bodies (HLBs), superficially resembling viruses. Infrequently, HLBs may condense to form haplosporosomes. The DVs, as in spores of some Haplosporidium spp. and paramyxeans, may give rise to, and are homologous with, haplosporosomes. Other features, such as the presence of an intranuclear mitotic spindle, lipid droplets, and attachment of DVs and haplosporosomes to the nuclear membrane, indicate that the C. maenas parasite is a haplosporidian. A similar organism reported from the haemolymph of spot prawns Pandalus spp., and haplosporidians reported from prawns Penaeus vannamei and crabs Callinectes sapidus may belong to this group. It is concluded that the well-characterised haplosporidians of molluscs and some other invertebrates may not be characteristic of the whole phylum, and that morphologically and developmentally similar organisms may also be haplosporidians, whether they have haplosporosomes or not.