Morphological and molecular characterization of a new freshwater microsporidium, Jirovecia sinensis sp. n. (Microsporidia) infecting the coelomocytes of Branchiura sowerbyi (Oligochaeta: Naididae) in China.

We report a new microsporidium Jirovecia sinensis sp. n. from a freshwater oligochaete, Branchiura sowerbyi collected in Hongze city, Jiangsu province, East China. Numerous whitish hypertrophied coelomocytes of 0.33-0.59 mm in diameter indicated infection. Transmission electron microscopy observations revealed that all developmental stages were diplokaryotic. The earliest life stages observed were meronts that were in direct contact with host cytoplasm, accumulated peripherally in the hypertrophied coelomocytes and connected with host cytoplasm through many pinocytotic canals. Mature spores are rod-shaped with a blunt end, measuring 17.0 ± 0.1 (14.9-18.5) µm long and 2.0 ± 0.2 (1.7-2.2) µm wide. The most conspicuous character of the novel microsporidian parasite is the tail-like posterior prolongations, with a length of 29.6-40.8 µm. Mature spores have a manubrium with a diameter of 447-485 nm which consist of six density-discontinuous concentric circles. Spores possess a collar-shaped anchoring disk and a bipartite polarplast with an anterior lamellar region and a posterior tubular section. SSU rDNA-based phylogenetic analysis indicated with high support values that the new species clustered with two Bacillidium species (B. vesiculoformis and Bacillidium sp.) infecting the freshwater oligochaetes and Janacekia debaisieuxi infecting the insect Simulium maculatum. Based on the ultrastructural features and molecular characteristics, a new species in the genus Jirovecia, Jirovecia sinensis sp. n., is designated.

[Analysis of expression of vesicular transport genes in avesicular cells of the microsporidium Paranosema (Antonospora) locustae].

Long adaptation of microsporidia, a large group fungi-related protozoa, to intracellular lifestyle has resulted in a drastic minimization of parasite cell. Ultrastructural analysis has shown that the Golgi complex of the microsporidia Paranosema (Antonospora) grylli and P. locustae appears as branching or varicose networks of thin tubules. These tubular networks are connected to endoplasmic reticulum, plasma membrane and forming polar tube but have no vesicles. Vesicles were not found even if ultra-fast cryofixation and membrane fusion/uncoating inhibition were used. However, a limited number of genes involved in vesicular transport were found in microsporidia genomes. In this study we used RT-PCR to analyze the content of mRNA transcripts encoding beta and beta' subunits COPI coatomer complex, Sec13 and Sec31 subunits COPII, SNARE-proteins synaptobrevin and syntaxin-like member of SFT family in P. locustae intracellular stages. The level of expression of studied genes was comparable with that of gene encoding alternative oxidase, enzyme envolved in microsporidia core metabolism. Moreover, polyclonal antibodies raised against recombinant Sec13 subunit COPII, expressed in B Escherichia coli, has shown accumulation of the protein is spores and stages of intracellular development as well as its association with membranes. The presence of components of vesicular transport machinery in avesicular microsporidia cells requires their functional analysis.

Chromosomal composition of the genome in the monomorphic diplokaryotic microsporidium Paranosema grylli: analysis by two-dimensional pulsed-field gel electrophoresis.

The molecular karyotype of Paranosema grylli Sokolova, Seleznev, Dolgikh et Issi, 1994, a monomorphic diplokaryotic microsporidium, comprises numerous bright and faint bands of nonstoichiometric staining intensity. Restriction analysis of chromosomal DNAs by "karyotype and restriction display" 2-D PFGE has demonstrated that the complexity of molecular karyotype of P. grylli is related to the pronounced length polymorphism of-homologous chromosomes. The background of this phenomenon is discussed in the context of ploidy state, reproductive strategy and population structure in this microsporidium. We propose that the remarkable size variation between homologous chromosomes in P. grylli may be a consequence of ectopic recombination at the chromosome extremities.

Spraguea lophii (Microsporidia) parasite of the teleost fish, Lophius piscatorius from Tunisian coasts: evidence for an extensive chromosome length polymorphism.

A microsporidian of the genus Spraguea was found parasitizing the nervous tissues of Lophius piscatorius collected from various localities in the Mediterranean coastal areas of Tunisia. The tissue localization, the infection focus aspect and sporal dimorphism are characteristics of Spraguea lophii species. Molecular data based on partial sequence of SSUrRNA encoding gene shows few nucleotide polymorphisms, compared to all described Spraguea isolates. Molecular karyotype obtained on pulsed field gel electrophoresis (1D-PFGE) shows a profile with 14 stained bands in the range of 230-880 kbp and a genome size estimated to 6.700 kbp. The rare cutter endonuclease MluI KARD 2-D-PFGE fingerprint shows an extensive chromosome length polymorphism, but the number of chromosome is unchanged and consists of 15 different molecules. The extensive chromosome length polymorphism is associated to a reduced number of genetic events.

In vitro growth of microsporidia Anncaliia algerae in cell lines from warm water fish.

Anncaliia algerae is an aquatic microsporidium that most commonly infects mosquitoes but can be grown on the rabbit kidney cell line, RK-13. Spores were purified from RK-13 cultures and added to cell lines from warm water fish and from an insect. The cell lines were GFSK-S1 and GFB3C-W1 from goldfish skin and brain respectively, ZEB2J from zebrafish embryos, FHMT-W1 from fathead minnow testis, and Sf9 from ovaries of a fall armyworm moth. All cultures were maintained at 27°C. Infection was judged to have taken place by the appearance of sporonts and/or spores in cells and occurred in all cell lines. Spores were also isolated from ZEB2J cultures and used to successfully infect new cultures of ZEB2J, RK-13 and Sf9. These results suggest that cells of a wide range of vertebrates support A. algerae growth in vitro and fish cells can produce spores infectious to cells of mammals, fish, and insects.