Protein phylogeny of translation elongation factor EF-1α suggests microsporidians are extremely ancient eukaryotes

Partial regions of the mRNA encoding a major part of translation elongation factor 1 (EF-1) from a mitochondrion-lacking protozoan,Glugea plecoglossi, that belongs to microsporidians, were amplified by polymerase chain reaction (PCR) and their primary structures were analyzed. The deduced amino acid sequence was highly divergent from typical EF-1's of eukaryotes, although it clearly showed a eukaryotic feature when aligned with homologs of the three primary kingdoms. Maximum likelihood (ML) analyses on the basis of six different stochastic models of amino acid substitutions and a maximum parsimony (MP) analysis consistently suggest that among eukaryotic species being analyzed,G. plecoglossi is likely to represent the earliest offshoot of eukaryotes. Microsporidians might be the extremely ancient eukaryotes which have diverged before an occurrence of mitochondrial symbiosis. Sequence availability: The nucleotide sequence data reported here appear in the GSDB, DDBJ, EMBL, and NCBI databases with the accession number D32139     

Comparative Study of Microsporidian Spores by Flow Cytometric Analysis

ABSTRACT. Spore suspensions of microsporidian parasites of fish (Microsporidium ovoideum, Glugea stephani, Glugea atherinae and Spraguea lophii ) have been analyzed by flow cytometry. Spore nuclei were dyed either by propidium iodide or bis-benzimide (Hoechst 33342). By observation of forward light scatter and fluorescence the four species could be distinguished and the mono- and diplokaryotic populations of S. lophii identified. Staining of DNA by bis-benzimide was better and easier than propidium iodide. Forward light scatter and fluorescence values were characteristic of each species and remained unchanged throughout the year, so flow cytometry can be used for distinction of spores of some microsporidian parasites once their flow cytometric parameters are known. However, special care has to be taken in tool calibration and material preparation for analysis because of the high precision of the technique.     

Identification and Phylogenetic Relationships of Microsporidia by Riboprinting

ABSTRACT. The SSUrDNA and the ITS of different microsporidia from eight fishes, four insects and a shrimp were amplified and digested with restriction enzymes. The generated riboprints suggest a close evolutionary relationship between Glugea americanus and Spraguea lophii suggesting that Glugea americanus should be renamed Spraguea americanus and that the tissue infected and host origin should be considered of greater taxonomic importance for defining a genus than previously considered. Phylogenetic analysis of the riboprints demonstrates an unidentified microsporidium from a bumper fish ( Chloroscombrus chrysurus ) is related although not identical to Microgemma oviodea , a parasite from red band fish. We were also able to distinguish between Glugea anomala and Glugea atherinae and Glugea stephani but were not able to differenciate among the latter two. Insects isolates, Nosema costelytrae, N. bombycis, N. trichoplusiae, Nosema sp. and a shrimp isolate, Agmasoma penaei , are not related to the fish isolates.     

Molecular Phylogeny of Microsporidians with Particular Reference to Species that Infect the Muscles of Fish

Ribosomal DNA from eight species of microsporidians infecting fish have been sequenced. Seven of these species infect the skeletal muscle of fish ( Pleistophora spp.) and one species infects migratory mesenchyma cells ( Glugea anomala ). These sequences, in addition to other available microsporidian rDNA sequences from a broad range of host taxa, have been used in phylogenetic analysis. This analysis revealed that muscle-infecting microsporidians from fish are a polyphyletic group, indicating that characters supposed to be important in the classification of the genus Pleistophora have to be re-evaluated. One character that probably has a polyphyletic origin is the amorphous coat, which has been extensively used in the definition of this genus. Furthermore, our results showed that the insect parasitizing Pleistophora spp. are not related to the true pleistophorans parasitic in skeletal muscle of fish. Phylogenetic analysis of small subunit rDNA sequences revealed disagreements between the molecular phylogeny and classifications based upon ultrastruclure. Many of the morphological characters claimed to be important in microsporidian classifications appeared to have arisen several times during evolution: for example, the diplokaryon and sporophorous vesicles.     

Encephalitozoon cuniculi: Light and Electron Microscopic Evidence for Di-, Tetra-, and Octosporous Sporogony and a Note on the Molecular Phylogeny of Encephalitozoonidae

We demonstrate, based on the light, electron microscopic, and immunofluorescence studies carried out on two isolates of Encephalitozoon cuniculi established in culture, that E. cuniculi exhibits di-, tri-, tetra- and octosporous sporogony. We therefore propose that the generic characters of Encephalitozoon should be amended to include tetra-sporous sporogony as generic features. Additionally, the molecular phylogenetic analysis indicates that E. cuniculi, E. hellem, and E. (Septata) intestinalis form a cohesive group.     

Ultrastructure of the Peripheral Zone of a Glugea-Induced Xenoma*

SYNOPSIS. The Glugea stephani-induced xenoma in the winter flounder, Pseudopleuronectes americanus, is a large spherical host-parasite complex, up to 4.0 mm in diameter, with the host and parasite components of the xenoma being most active in the peripheral zone. The xenoma has an extensive periodic acid-silver methenamine-positive surface coat covering the plasma membrane. The surface of this membrane is amplified by the presence of numerous folds and fine tubular extensions. The peripheral zone of the xenoma contains many host-cell mitochondria in addition to numerous microsporidan parasites. At the ultrastructural level, the peripheral zone of the host-cell cytoplasm appears normal. Inside the peripheral region of the 0.4–1.0 mm xenoma, the host-cell component largely disintegrates in the presence of microsporidan parasites undergoing sporogenesis.     

Laboratory and Field Studies on Glugea stephani (Hagenmuller), a Microsporidan Parasite of Pleuronectid Flatfishes*

SYNOPSIS. The microsporidan Glugea stephani is a common parasite of juvenile English sole (Parophrys vetulus) in Yaquina Bay, Oregon. Field observations indicated that fish became infected only in the upper estuary where summer temperatures were above 15 C and the incidence of infection reached 79.8% in the late fall. Laboratory infections developed and parasite growth occurred only at or above 15 C. The parasite was successfully transmitted to juvenile English sole by brine shrimp (Artemia salina) and amphipod (Corophium spinicorne) vectors as well as by direct ingestion of spores by the host. Infections that resulted from ingestion of spore-carrying vectors were much heavier than those resulting from the direct ingestion of spores. The speckled sanddab (Citharichthys stigmaeus), a nonpleuronectid flatfish, and chum salmon (Oncorhynchus keta) were refractory to G. stephani infection in the laboratory. Results of this study suggest that G. stephani is potentially lethal to young pleuronectid flatfishes when heavy infections involve the entire intestine and reduce the capacity to absorb nutrients. Under these circumstances, starvation is probably the direct or indirect cause of death. The restriction of infection to fish that reach the upper estuary very likely mitigates the impact of G. stephani caused mortality on the entire English sole population on the Yaquina Bay nursery ground.     

Fatty acid composition of four microsporidian species compared to that of their host fishes

ABSTRACT. The fatty acid composition of four microsporidian species (Glugea atherinae, Spraguea lophii, Glugea americanus , and Pleistophora mirandellae) and their host fishes has been determined using gas chromatography. Twenty-four fatty acids were identified with differences in relative abundance of fatty acids among the four parasites. Certain even-saturated fatty acids were found in a very high proportion: palmitic acid (16:0) represented one-third of total fatty acids in Pleistophora mirandellae. The level of docosahexaenoic acid (22:6ω3) attained 26–28% in Glugea atherinae, Spraguea lophii , and Glugea americanus , but only 8–9% in P. mirandellae. With respect to fatty acid compositions of host organs, some significant differences were evident between marine and freshwater fishes. Palmitic acid was prevalent in the marine fishes, Atherinae boyeri and Lophius piscatorius , and oleic acid (18:1ω9) in the freshwater fish Leuciscus cephalus. The proportion of docosahexaenoic acid in marine fishes was two or three times as great as in freshwater fish Leuciscus. The high polyunsaturated fatty acid content in both parasites and host fishes may be related to the scavenging of these fatty acids by the parasites rather than a microsporidia-specific fatty acid biosynthesis pathway.     

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.     

Sex-specific survival and parasitism in three-spined sticklebacks: seasonal patterns revealed by molecular analysis

A molecular method of identifying sex in three-spined sticklebacks Gasterosteus aculeatus showed that adult males had a higher prevalence of dermal Glugea anomala cysts than adult females, and young-of-the-year had more than adults. At the end of the breeding season, as predicted, the adult sex ratio became female biased and there was a disproportionate increase in G. anomala parasitism in adult males.