Resolution of a taxonomic conundrum: an ultrastructural and molecular description of the life cycle of Pleistophora mulleri (Pfeiffer 1895; Georgevitch 1929)

The classification of a microsporidian parasite observed in the abdominal muscles of amphipod hosts has been repeatedly revised but still remains inconclusive. This parasite has variable spore numbers within a sporophorous vesicle and has been assigned to the genera Glugea, Pleistophora, Stempellia, and Thelohania. We used electron microscopy and molecular evidence to resolve the previous taxonomic confusion and confirm its identification as Pleistophora mulleri. The life cycle of P. mulleri is described from the freshwater amphipod host Gammarus duebeni celticus. Infection appeared as white tubular masses within the abdominal muscle of the host. Light and transmission electron microscope examination revealed the presence of an active microsporidian infection that was diffuse within the muscle block with no evidence of xenoma formation. Paucinucleate merogonial plasmodia were surrounded by an amorphous coat immediately external to the plasmalemma. The amorphous coat developed into a merontogenetic sporophorous vesicle that was present throughout sporulation. Sporogony was polysporous resulting in uninucleate spores, with a bipartite polaroplast, an anisofilar polar filament and a large posterior vacuole. SSU rDNA analysis supported the ultrastructural evidence clearly placing this parasite within the genus Pleistophora. This paper indicates that Pleistophora species are not restricted to vertebrate hosts.     

Phylogenetic relationships of Pleistophora-like microsporidia based on small subunit ribosomal DNA sequences and implications for the source of trachipleistophora hominis infections

The microsporidian Trachipleistophora hominis was isolated in vitro from the skeletal muscle of an AIDS patient. Since its discovery several more cases of myositis due to Trachipleistophora have been diagnosed but the source of infection is unknown. Morphologically, T. hominis most closely resembles Pleistophora and Vavraia, which undergo polysporous sporogony in sporophorous vesicles, but differs from these genera in the mode of formation of sporoblasts and in the morphology of the sporophorous vesicles. Alignment and analyses of the small subunit ribosomal DNA sequences of T. hominis and several other polysporoblastic genera indicated that its closest phylogenetic relationships were with species of the genera Pleistophora and Vavraia, in line with morphological predictions. The type species of the latter two genera are Pleistophora typicalis and Vavraia culicis; these are parasites of fish and mosquitoes, respectively. These results suggest two possible routes and sources of infection to AIDS patients, these being perorally by ingestion of inadequately cooked fish or crustaceans or percutaneously during a bloodmeal taken by a haematophagous insect. Support for an insect source has been provided by recent detection of a microsporidium from mosquitoes in human corneal tissue.     

Distribution and host range of the microsporidian Pleistophora mulleri

ABSTRACT. Microsporidia of the genus Pleistophora are important parasites of fish and crustacea. Pleistophora mulleri has been described previously as a parasite of the gammarid amphipod crustacean Gammarus duebeni celticus in Irish freshwater habitats. Through a survey of European G. duebeni populations, P. mulleri was found to be widely distributed in the western British Isles (Wales, Scotland, and the Isle of Man), and populations of the subspecies Gammarus duebeni duebeni as well as G. d. celticus were infected. Pleistophora infections were also detected in G. d. duebeni sampled from the Bay of Gdansk on Poland's Baltic coast, indicating a wide distribution of Pleistophora in European G. duebeni . Sequencing and phylogenetic analysis of the 16S rRNA , 18S rRNA , and Rpb1 genes of P. mulleri suggest that this species may be synonymous with P. typicalis , a parasite of fish. These findings suggest that amphipod crustaceans may act as intermediate or reservoir hosts for microsporidian parasites of fish.     

Relationships of microsporidian genera, with emphasis on the polysporous genera, revealed by sequences of the largest subunit of RNA polymerase II (RPB1)

Molecular data have proved useful as an alternative to morphological data in showing the relationships of genera within the phylum Microsporidia, but until now have been available only for ribosomal genes. In previous studies protein-coding genes of microsporidia have been used only to assess their position in the evolution of eukaryotes. For the first time we report on the use of a protein-coding gene, the A-G region of the largest subunit of RNA polymerase II (RPB1) from 14 mainly polysporous species, to generate an alternative phylogeny for microsporidia. Using the amino acid sequences, the genera and species fell into the same main groupings as had been obtained with 16S rDNA sequences, but the RPB1 data provided better resolution within these groups. The results supported the pairings of Trachipleistophora hominis with Vavraia culicis and Pleistophora hippoglossoideos with Pleistophora typicalis. They also confirmed that the genus Pleistophora is not monophyletic and that it will be necessary to transfer Pleistophora ovariae and Pleistophora mirandellae into one or more other genera, as has already been effected for Pleistophora anguillarum.     

Single and nested polymerase chain reaction assays for the detection of Microsporidium seriolae (Microspora), the causative agent of 'Beko' disease in yellowtail Seriola quinqueradiata.

Single and nested polymerase chain reaction (PCR) assays were developed for the detection of the microsporidian parasite Microsporidium seriolae, which is responsible for emaciation and even death in farmed Japanese yellowtail. Extremely high rDNA identities exist between this parasite and other members of the as yet unclassified genus, necessitating the design of generic, rather than species-specific primer sets. The nested PCR was several orders of magnitude more sensitive than the standard single PCRs, with visible target product amplified from as little as 0.01 pg of parasite DNA (equivalent to that extracted from a single spore). The specificity of the assays was tested against a range of potential host fishes and 6 other microsporidians infecting either fish or the musculature of their hosts. Single PCRs were found to be specific to the target genus, but the nested PCR replicated rDNA from several different microsporidian genera, limiting its utility. This study highlights problems associated with the use of the rRNA gene for PCR assays of certain microsporidians, but nevertheless provides a rapid and sensitive means for the detection of pre-spore forms not possible by current staining methods. Consequently, these assays may be employed for further studies on the portals of entry, migration to the musculature and transmission of this economically important pathogen.     

Ultrastructure and development of Pleistophora ronneafiei n. sp., a microsporidium (Protista) in the skeletal muscle of an immune-compromised individual

This report provides a detailed ultrastructural study of the life cycle, including proliferative and sporogonic developmental stages, of the first Pleistophora species (microsporidium) obtained from an immune-incompetent patient. In 1985, the organism obtained from a muscle biopsy was initially identified as belonging to the genus Pleistophora, based on spore morphology and its location in a sporophorous vesicle. Since that initial report, at least two new microsporidial genera, Trachipleistophora and Brachiola, have been reported to infect the muscle tissue of immunologically compromised patients. Because Trachipleistophora development is similar to Pleistophora, and as Pleistophora was only known to occur in cold-blooded hosts, the question of the proper classification of this microsporidium arose. The information acquired in this study makes it possible to compare Pleistophora sp. (Ledford et al. 1985) to the known human infections and properly determine its correct taxonomic position. Our ultrastructural data have revealed the formation of multinucleate sporogonial plasmodia, a developmental characteristic of the genus Pleistophora and not Trachipleistophora. A comparison with other species of the genus supports the establishment of a new species. This parasite is given the name Pleistophora ronneafiei n. sp.     

Small subunit ribosomal DNA phylogeny of microsporidia with particular reference to genera that infect fish

Molecular data have proved useful in the study of microsporidia phylogeny. Previous studies have shown that there are several important differences between phylogenies based on rRNA and morphological data. In the present study, small subunit (SSU) rDNA sequences were obtained from 7 different fish-infecting microsporidia from 4 different genera (Glugea Thélohan, 1891, Loma Morrison and Sprague, 1981, Pleistophora Gurley, 1893, and Spraguea Weissenberg, 1976). The lengths of the SSU rDNA genes in these species were between 1,332 and 1,343 base pairs. Phylogenetic analysis was performed using parsimony, maximum likelihood, and Kimura 2-parameter with neighbor joining. The analyses revealed that the microsporidia could be divided into 3 major groups. With the exception of Nucleospora salmonis Hedrick, Groff, and Baxa, 1991, all the microsporidia infecting fishes occurred in the same group. The analysis showed that Pleistophora mirandellae Vaney and Conte, 1901 and Pleistophora aguillarum Hoshina, 1951 are not species of Pleistophora. Furthermore, the analysis showed that Loma is not a member of Glugeidae Thélohan, 1892.     

Characterization and phylogenetic relationships among microsporidia infecting silkworm, Bombyx mori, using inter simple sequence repeat (ISSR) and small subunit rRNA (SSU-rRNA) sequence analysis.

This study is the first report on the genetic characterization and relationships among different microsporidia infecting the silkworm, Bombyx mori, using inter simple sequence repeat PCR (ISSR-PCR) analysis. Six different microsporidians were distinguished through molecular DNA typing using ISSR-PCR. Thus, ISSR-PCR analysis can be a powerful tool to detect polymorphisms and identify microsporidians, which are difficult to study with microscopy because of their extremely small size. Of the 100 ISSR primers tested, only 28 primers had reproducibility and high polymorphism (93%). A total of 24 ISSR primers produced 55 unique genetic markers, which could be used to differentiate the microsporidians from each other. Among the 28 SSRs tested, the most abundant were (CA)n, (GA)n, and (GT)n repeats. The degree of band sharing was used to evaluate genetic similarity between different microsporidian isolates and to construct a phylogenetic tree using Jaccard's similarity coefficient. The results indicate that the DNA profiles based on ISSR markers can be used as diagnostic tools to identify different microsporidia with considerable accuracy. In addition, the small subunit ribosomal RNA (SSU-rRNA) sequence gene was amplified, cloned, and sequenced from each of the 6 microsporidian isolates. These sequences were compared with 20 other microsporidian SSU-rRNA sequences to develop a phylogenetic tree for the microsporidia isolated from the silkworms. This method was found to be useful in establishing the phylogenetic relationships among the different microsporidians isolated from silkworms. Of the 6 microsporidian isolates, NIK-1s revealed an SSU-rRNA gene sequence similar to Nosema bombycis, indicating that NIK-1s is similar to N. bombycis; the remaining 5 isolates, which differed from each other and from N. bombycis, were considered to be different variants belonging to the species N. bombycis.     

Fish microsporidia: fine structural diversity and phylogeny

Structural diversity of fish microsporidian life cycle stages and of the host-parasite interface is reviewed. In the infected cell of the fish host, microsporidia may either cause serious degradation of the cytoplasm and demise of the cell, or they may elicit host cell hypertrophy, producing a parasite-hypertrophic host cell complex, the xenoma. The structure of the xenoma and of its cell wall may differ according to the genus of the parasite, and seems to express properties of the parasite rather than those of the host. In merogony, the parasite cell surface interacts with the host cell in diverse ways, the most conspicuous being the production of thick envelopes of different types. Sporogony stages reveal different types of walls or membranes encasing the sporoblasts and later the spores and these envelopes may be of host or parasite origin. Nucleospora differs from all other fish microsporidia by its unique process of sporogony. Except for the formation of conspicuous xenomas, there are no essentially different structures in fish-infecting microsporidia compared with microsporidia from other hosts. Although the structures associated with the development of fish microsporidia cannot be attributed importance in tracing the phylogeny, they are relevant for practical determination and assessing the relation to the host. The possibility of the existence of an intermediate host is discussed. Higher-level classification of Microsporidia is briefly discussed and structure and evolutionary rates in microsporidian rDNA are reviewed. Discussion of rDNA molecular phylogeny of fish-infecting microsporidia is followed by classification of these parasites. Most form a rather cohesive clade. Outside this clade is the genus Nucleospora, separated at least at the level of Order. Within the main clade, however, there are six species infecting hosts other than fish. Based on data available for analysis, a tentative classification of fish-infecting microsporidia into five groups is proposed. Morphologically defined groups represent families, others are referred to as clades. Group 1, represented by family Pleistophoridae, includes Pleistophora, Ovipleistophora and Heterosporis; Vavraia and Trachipleistophora infect non-fish hosts. Group 2, represented by family Glugeidae, is restricted to genus Glugea and Tuzetia weidneri from crustaceans. Group 3 comprises three clades: Loma and a hyperparasitic microsporidian from a myxosporean; Ichthyosporidium and Pseudoloma clade and the Loma acerinae clade. For the latter species a new genus has to be established. Group 4 contains two families, Spragueidae with the genus Spraguea and Tetramicridae with genera Microgemma and Tetramicra, and the Kabatana and Microsporidium seriolae clade. Group 5 is represented by the family Enterocytozoonidae with the genus Nucleospora and mammal-infecting genus Enterocytozoon.     

An analysis of the microsporidian genus Brachiola, with comparisons of human and insect isolates of Brachiola algerae

The genus Brachiola is the newest microsporidian genus established for a human infection with the type species being B. vesicularum in skeletal muscle. Subsequently, the microsporidium, Nosema algerae, identified from mosquitoes, was added to this genus because of morphological and physiological similarities. The present report illustrates a confirmed case of Brachiola algerae infecting skeletal muscle in a 56-year-old woman who was being treated for rheumatoid arthritis with immunosuppressive drugs. In the following study, these two human-infecting microsporidian species are ultrastructurally compared from human biopsy tissue. Additionally, Brachiola algerae from mosquitoes as reference B. algerae, was grown in athymic mice and compared to the human isolate in vivo, and in culture. B. algerae is morphologically identical in the host situations presented and different from B. vesicularum in human skeletal muscle. B. algerae has a consistently, slightly longer spore that typically contains one row of polar filament coils, while B. vesicularum typically contains two rows of polar filament coils and occasionally, one or three rows. In proliferative development, B. vesicularum forms protoplasmic extensions which do not occur on B. algerae, nor have they been reported on any other microsporidium. This report demonstrates that B. vesicularum and B. algerae are two different species of Brachiola that infect human skeletal muscle.     

Organization of 5S rDNA in species of the fish Leporinus: two different genomic locations are characterized by distinct nontranscribed spacers.

To address understanding the organization of the 5S rRNA multigene family in the fish genome, the nucleotide sequence and organization array of 5S rDNA were investigated in the genus Leporinus, a representative freshwater fish group of South American fauna. PCR, subgenomic library screening, genomic blotting, fluorescence in situ hybridization, and DNA sequencing were employed in this study. Two arrays of 5S rDNA were identified for all species investigated, one consisting of monomeric repeat units of around 200 bp and another one with monomers of 900 bp. These 5S rDNA arrays were characterized by distinct NTS sequences (designated NTS-I and NTS-II for the 200- and 900-bp monomers, respectively); however, their coding sequences were nearly identical. The 5S rRNA genes were clustered in two chromosome loci, a major one corresponding to the NTS-I sites and a minor one corresponding to the NTS-II sites. The NTS-I sequence was variable among Leporinus spp., whereas the NTS-II was conserved among them and even in the related genus Schizodon. The distinct 5S rDNA arrays might characterize two 5S rRNA gene subfamilies that have been evolving independently in the genome.     

Phylogeny of the eelpout genus Lycodes (Pisces,Zoarcidae) as inferred from mitochondrial cytochrome b and 12S rDNA

The bottom-dwelling and species-rich eelpout genus Lycodes Reinhardt has a great potential for the study of Arctic marine speciation. Subdivision of the genus has been based on single or few morphological characters (e.g., lateral line configuration) with contradicting results and phylogenetic approaches have not been attended. Here we present the first phylogenetic analysis of the genus employing DNA sequences of the mitochondrial genes cytochrome b and 12S rDNA (714 bp). The analysis with the two genes combined resulted in two equally parsimonious trees. In both cladograms most of the previously suggested subgroups are para- or polyphyletic, except for the so-called short-tailed Lycodes spp., with a short tail, a single mediolateral lateral line configuration and a shallow or filled otolith sulcus. The group of long-tailed Lycodes spp., with ventral or ventro-medio-lateral types of lateral line configuration and a deep otolith sulcus, appears to be paraphyletic, since Pacific and Atlantic species in this group are not each other's closest relatives. Thus, the short-tailed species are placed in a derived clade, indicating a secondary shortening of the tail, and a "slope to shore" type of evolution. This is not in accordance with earlier assumptions of the more elongate, deeper living species being the more derived. The basal position of long-tailed Pacific species supports earlier theories of Pacific origin of the genus/family. Small genetic differences between Arctic/Atlantic species indicate a rather recent radiation in these areas after the opening of the Bering Strait 3.0-3.5 million years ago.     

A new microsporidian infecting the musculature of the endangered tidewater goby (Gobiidae)

A previously unrecognized microsporidian (Kabatana newberryi n. sp.) is described from the musculature of Eucyclogobius newberryi (Gobiidae) in Big Lagoon, Humboldt County, California. Spores are ovoid, ranging in size from 2.8 +/- 0.3 microm in total length and 1.9 +/- 0.4 microm in width (measurements of 30 spores made by calculation from micrograph). The polar filament has 9-10 coils in 1-2 rows. Development occurs in direct contact with host muscle cell cytoplasm, without xenoma or sporophorous vesicle. Phylogenetic analysis of the new species and of 35 other microsporidians known to infect fish using 1115 base pairs of aligned 16S rRNA gene indicate the new species is most closely related to Kabatana takedai. However, the new species differs by 11% sequence divergence from K. takedai. Divergence in morphology and genetic data allow for diagnosis from all other fish-infecting microsporidia and supports recognition of a new species of microsporidian, Kabatana newberryi n. sp., presently known only from a suspected specific host, the endangered tidewater goby Eucyclogobius newberryi.     

A taxonomic study of phlebioid fungi (Basidiomycota)

The genus Phlebia has long been regarded as a polyphyletic or paraphyletic taxon, including distinct groups of more closely related species. Consequently, the delimitation of the genus has been given different interpretations and several rearrangements have been proposed by various authors. In the present study, DNA sequences (25S, rDNA) were obtained for twenty species of the genus Phlebia and a phylogenetic analysis was performed. Because of the presumably paraphyletic nature of the genus, different outgroups were used for different sets of taxa. A core group of species, including the type Ph. radiata , is well distinguished. For the delimitation of the genus, however, the wider scope of a more weakly supported clade is proposed. This clade also includes Phlebiopsis gigantea and its combination in Phlebia is reaffirmed. Two species, Ph. griseoflavescens and Ph. tristis , are distinctly separated from this clade and should be removed from the genus. Morphological characters were used in a separate phylogenetic analysis but the result did not conform with analysis from sequence data.     

Pseudoloma neurophilia n. g., n. sp., a new microsporidium from the central nervous system of the zebrafish (Danio rerio)

An unusual xenoma-forming microsporidium was discovered in the central nervous system of moribund zebrafish from a laboratory colony in Eugene, Oregon. Infected fish were often emaciated and lethargic, and histological examination commonly revealed severe myelitis and myositis associated with the infection. Based on its structure, development, and small subunit ribosomal DNA sequence it is unique among fish microsporidia. Spores are uninucleate, ovoid to pyriform, with a prominent posterior vacuole. Spores average 5.4 x 2.7 microm with 13-16 coils of the polar filament. The microsporidium produces xenomas within the spinal cord and hindbrain of fish, and xenomas contained sporophorous vesicles with up to 16 spores. Sporoblasts and presporoblast stages (probably sporonts) are found occasionally in small aggregates dispersed randomly throughout xenomas. It clustered in the "Ichthyosporidium group" along with other fish microsporidian genera based on rDNA sequence analysis. The rDNA sequence of the zebrafish microsporidium was most similar to that of Ichthyosporidium, but showed only 12.1% similarity and therefore this microsporidium can be considered a distinct genus and species, which we have named Pseudoloma neurophilia n. g., n. sp.     

Ovipleistophora gen. n., a new genus for Pleistophora mirandellae-like microsporidia

Based on ultrastructural study and molecular analysis, a new genus, Ovipleistophora, is established for Pleistophora mirandellae-like microsporidia from roach and ruff oocytes. Unlike Pleistophora, Ovipleistophora has a thick additional envelope around the meront. This envelope breaks open to release the cells into the host cell cytoplasm. The cells, becoming multinuclear sporogonic plasmodia, already have a surface coat that transforms into the sporont wall and eventually into the sporophorous vesicle wall. The surface coat and its transformation differ from those of Pleistophora, but bear some resemblance to those of Trachipleistophora. In Trachipleistophora the sporonts, however, do not form plasmodia, as they do in Ovipleistophora and Pleistophora. Small subunit ribosomal DNA analysis supports the establishment of the new genus and assignment of P. mirandellae from 2 different fish hosts to the same species. The same small subunit ribosomal DNA analysis lends support for transferring P. ovariae into the genus Ovipleistophora.     

Phylogenetic position of Octosporea muscaedomesticae (Microsporidia) and its relationship to Octosporea bayeri based on small subunit rDNA analysis

Comparative phylogenetic analysis of the small subunit rDNA sequence of Octosporea muscaedomesticae (Flu, 1911) (type species) (Microsporidia) isolated from the blowfly Phormia regina (Diptera:Calliphoridae) is presented. Neighbor Joining bootstrap, Maximum Parsimony and Maximum Likelihood analyses with 38 microsporidian taxa representing five major clades of Microsporidia placed O. muscaedomesticae on a separate branch within a clade containing parasites of freshwater hosts. O. muscaedomesticae differed from Octosporea bayeri, a parasite of the microcrustacean, Daphnia magna (Cladocera:Daphniidae) by 29% demonstrating that the latter microsporidium is not closely related to the type species at the generic level, and should not be placed within the genus Octosporea, a conclusion that is further supported by morphological and developmental differences. Considering the number of disparately related hosts from which Octosporea species have been previously described based mostly on developmental and morphological characters it is likely that many will not fit the current definition of the genus, and it is possible that molecular analysis of these species will show that this genus as defined represents a polyphyletic grouping of unrelated taxa.