Ultrastructure, molecular phylogeny, and prevalence rates of Alternosema bostrichidis gen. nov. sp. nov. (Microsporidia, Terresporidia), a parasite of Prostephanus truncatus and Dinoderus spp. (Coleoptera, Bostrichidae).

A new species and a new genus of a microsporidium Alternosema bostrichidis isolated from an adult Prostephanus truncatus in Mexico and from three species of the genus Dinoderus in Nigeria are described. The microsporidium is monomorphic, monoxenic, and develops in direct contact with host cell cytoplasm. The infection first appears with thoracic muscles, followed by a generalized invasion of the host. All developmental stages are diplokaryotic. Sporogony is disporoblastic. Mature spores are ovoid. Unfixed spores measure 3.7-4.2 × 2.0-2.6 µm, fixed and stained spores 3.5-5.0 × 2.4-2.8 µm. The polaroplast consists of dense lamellae and rare lamellae. The polar tube is slightly anisofilar, consisting of 11-17 coils, with 9-14 proximal (130 nm in diameter) and 2-3 distal coils (120 nm in diameter) arranged in one layer. Molecular phylogenetic analysis based upon a short portion of small-subunit ribosomal RNA gene (Genbank accession # KP455651) placed the new microsporidium within Liebermannia-Orthosomella lineage, which contains multiple undescribed parasites. In particular, A. bostrichidis showed maximal sequence similarity of 95% to Microsporidium sp. BBRE2 (# FJ755987) from Baikalian Diplacanthus brevispinus (Amphipoda: Acanthogammaridae) and Microsporidium sp. Comp CD Van 2 (# KC111784) from compost and soil in Canada. Frequent, devastating epizootics of laboratory cultures of A. bostrichidis support its potential as a biological control agent of grain borers.

Correction to: A new microsporidium Fibrillaspora daphniae g. n. sp. n. infecting Daphnia magna (Crustacea: Cladocera) in Siberia and its taxonomic placing within a new family Fibrillasporidae and new superfamily Tubulinosematoidea (Opisthosporidia: Microsporidia).

Ultrastructure of Fibrillaspora is highly similar to that of Berwaldia but the former is phylogenetically distant from the taxon annotated as Berwaldia schaefernai (Genbank accession # AY090042). It was shown recently, however, that the latter sequence was not derived from Berwaldia, and correct nucleotide sequence data were provided for Berwaldia. This allowed recondideration of Fibrillaspora as a junior synonym of Berwaldia and redefinition of Fibrillaspora daphniae as a new combination, Berwaldia daphniae comb. nov.

A new microsporidium Fibrillaspora daphniae g. n. sp. n. infecting Daphnia magna (Crustacea: Cladocera) in Siberia and its taxonomic placing within a new family Fibrillasporidae and new superfamily Tubulinosematoidea (Opisthosporidia: Microsporidia).

Infection with a new microsporidium, Fibrillaspora daphniae g. n. sp. n., was found in a local Daphnia magna population in Tomsk region (Western Siberia, Russia) at the prevalence rate of 52%. Histological sections showed parasite cells entirely encompassing the host haemocoel. Methanol-fixed spores were elongate, oval, 4.8 ± 0.3 µm × 2.3 ± 0.2 µm in size. All developmental stages were in direct contact with the host cell cytoplasm, with single nuclei, and division by binary fission. The sporont surface was covered with an additional outer layer composed of fine tubules. The spores possessed a thick endospore, large posterior vacuole filled with electron-dense granules, and a bipartite polaroplast composed of anterior lamellar and posterior globular elements. The polar tube was slightly anisofilar, with 13-19 coils arranged in one row; the two posterior coils were of lesser diameter. The small subunit ribosomal RNA gene sequence was deposited in Genbank under accession # MF278272. Considering the sister relationship between Fibrillanosema crangonycis and our new isolate described here as Fibrillaspora daphniae, we propose a new family Fibrillasporidae fam. n. to contain these two genera and the descendants of their common ancestor. A new superfamily Tubulinosematoidea superfam. n. is proposed as a monophyletic assemblage of Fibrillasporidae fam. n. and Tubulinosematidae.

Molecular and Morphological Characterization of Anncaliia azovica sp. n. (Microsporidia) Infecting Niphargogammarus intermedius (Crustacea, Amphipoda) from the Azov Sea.

Five out of one hundred adults of Niphargogammarus intermedius caught at the Azov sea shore were found to be infected with microsporidia. The infection was found in the subcuticular fat body and myocytes. Parasites developed in direct contact with host cells, displayed a disporoblastic sporogony and diplokaryotic arrangement of nuclei at all stages. Spores were oval, 4.6-5.8 × 2.6-3.0 µm. Exospore appendages, vesicular-tubular secretions, and the anisofilar polar filament indicated a similarity to Anncaliia species. Sporont surfaces displayed ridges of amorphous material. Meronts and sporonts formed protoplastic extensions, similar to A. vesicularum and A. meligheti. Mature spores possessed a bipartite polaroplast. The polar tube was arranged in one row of 13-18 coils including 0-3 distal coils of lesser diameter. Partial sequencing of SSU, ITS, and LSU regions of rRNA gene (GenBank accessions: KY288064-KY288065) confirmed this new species to be congeneric with A. algerae (#AF069063) and A. meligheti (#AY894423). The SSU gene of this novel microsporidium shared 99.4% sequence similarity to A. algerae and 98.9% to A. meligheti. Genes for HSP70 and RPB1 amplified with primers designed for A. algerae orthologs displayed 99.7% and 97.4% similarity, respectively, between A. algerae and the novel microsporidium. A new species, Anncaliia azovica, is described based on morphological and molecular characterization.

Redefinition of Nosema pyrausta (Perezia pyraustae Paillot 1927) basing upon ultrastructural and molecular phylogenetic studies.

Populations of European corn borer (Ostrinia nubilalis Hübner) from Krasnodar Territory (Southwestern Russia) become regularly infected with Nosema-like microsporidia. To identify the parasite, it was subjected to electron microscopy and small subunit ribosomal RNA (SSU rRNA) gene sequencing. The spore ultrastructure of the parasite was highly similar to Nosema bombycis from China and Nosema pyrausta from the USA. The nucleotide sequence of SSU rRNA gene was identical to a microsporidium isolated from O. nubilalis in southern France (GenBank accession no. HM566196) and closely related to Nosema bombycis (no. AY209011, 99.7 % sequence similarity) from Bombyx mori of Chinese origin and N. pyrausta (no. AY958071) from O. nubilalis of North American origin. As the molecular haplotype of SSU rRNA is fixed for the parasite infecting O. nubilalis across Europe and N. pyrausta was initially described in France as Perezia pyraustae (Paillot CR Acad Sci Paris 185: 673-675, 1927), we conclude that the parasite examined under the present study correspond to the type isolate of N. pyrausta. The microsporidium from O. nubilalis in North America (no. AY958071) corresponds therefore to a closely related, yet distinct haplotype.

Ultrastructure and molecular phylogenetics of Helmichia lacustris, a microsporidium with an uncoiled isofilar polar filament.

The description of Helmichia lacustris Voronin (Parazitologiya 34:327-331 1998) is supplemented with morphogenesis and ultrastructure of the extrusion apparatus. Formation of the anterior (made up by rare short lamellae) and posterior (made up by spongy matter or small vesicles) regions of the polaroplast is preceded by granulated spheres and agglomerations of bean-like bodies, respectively. The anchoring disc is formed by an oval structure of moderate electron density, sometimes possessing a granular texture. The parasite development occurs within the cisterns of granular endoplasmatic reticulum (ER) of the host cell. Each group of spores is enclosed within a two-layered sheath, including the smooth inner membrane of the sporophorous vesicle and the outer ribosome-encrusted membrane (which originates from the host cell ER) of the parasitophorous vacuole. Two microsporidia, H. lacustris (GenBank accession number GU130406) and Euplotespora binucleata (GenBank accession number DQ675604) share 78.1% of 16S rRNA gene sequence similarity. Both parasites are characterized by an uncoiled isofilar polar filament. They form a cluster nested among terrestrial and aquatic microsporidia with well-developed coiled polar filaments, suggesting that an uncoiled polar filament in this species is a result of reduction, rather than a "primitive" character.

Taxonomy of Neoperezia chironomi and Neoperezia semenovaiae comb. nov. (Microsporidia, Aquasporidia): Lessons from ultrastructure and ribosomal DNA sequence data.

We did a comparative analysis of the small subunit ribosomal DNA (rDNA) for two species of Microsporidia, Semenovaia chironomi and Neoperezia chironomi, both parasites of Chironomus plumosus (Diptera, Chironomidae). These two microsporidial species have been described previously on the basis of light and electron microscopic studies. The former species is dimorphic, producing both single diplokaryotic spores and uninucleate spores in sporophorous vesicles (SPVs) in packets of 16, while the latter species is monomorphic, disporoblastic, producing only uninucleate spores in SPVs. Based on their life cycles, S. chironomi and N. chironomi were assigned to two different families, Burenellidae and Neopereziidae. However, molecular analysis shows 96.7% sequence similarity for the small subunit rDNA between these two species. Remarkable similarities of the spore ultrastructure (mainly of the extrusion apparatus) justify a transfer of S. chironomi to Neoperezia, establishing a new combination, Neoperezia semenovaiae. Neoperezia belongs to Clade V, Class Aquasporidia sensu Vossbrinck and Debrunner-Vossbrinck (2005), and is in its spore ultrastructure similar to its closest relatives, namely Bryonosema, Schroedera, Pseudonosema, Trichonosema and Janacekia. We therefore conclude that similarities in spore ultrastructure reflect the phylogenetic relatedness of these Microsporidia, as opposed to the strikingly diverse life cycles.

Life cycle, ultrastructure, and molecular phylogeny of Crispospora chironomi g.n. sp.n. (Microsporidia: Terresporidia), a parasite of Chironomus plumosus L. (Diptera: Chironomidae).

The life cycle, ultrastructure, and molecular phylogeny of a new microsporidium Crispospora chironomi g.n. sp.n., a parasite of the midge Chironomus plumosus, are described. The parasite infects the gut epithelium of the host larvae and possesses sporogonies of two types, polysporoblastic and disporoblastic, respectively, proceeding within the same host cell. In the sporogonial sequence of the first type, dozens of spherical monokaryotic spores within a thick-walled capsule are formed. The spores are 1.5-2.0 µm in diameter; the exospore possesses two to three bundles of tubular protrusions. In the sporogonial sequence of the second type, diplokaryotic oval spores, 2.5 × 1.5 µm in size, are formed within a compartment, partially surrounded with multilayered membranes. Spores of both types are similar in respect to inner structure, possessing a well-developed extrusion apparatus with (a) the anterior vesicular part of the polaroplast covering the lamellar posterior one and (b) isofilar polar filament with several coils in one row. Small subunit ribosomal DNA phylogeny showed position of the new microsporidium in a cluster uniting microsporidia of terrestrial origin infecting diverse hosts, nested within Clade IV, corresponding to Class Terresporidia sensu Vossbrinck and Debrunner-Vossbrinck (Folia Parasitol 52:131-142, 2005).

Ultrastructure and molecular phylogeny of Anisofilariata chironomi g.n. sp.n. (Microsporidia: Terresporidia) from Chironomus plumosus L. (Diptera: Chironomidae).

Larvae of Chironomus plumosus, collected in North-Western Russia in September 2008, were infected with a microsporidium possessing broadly oval uninucleate spores in sporophorous vesicles. Sporogony and spore ultrastructure of this microsporidium differed from that of known microsporidian species, suggesting establishment of a new species, Anisofilariata chironomi, being a type species of a new genus. Sporogony di-, tetra-, octo-, and 16-sporoblastic. Fixed and stained spores are 4.7-6.8 x 3.4-5.4 microm in size, the spore measurements varying depending upon the number of spores in the sporophorous vesicle. The polaroplast is bipartite, with anterior and posterior parts composed of very thin and thick lamellae, respectively, and occupies the major volume of the spore. The polar filament is anisofilar, with two broad proximal and 10-13 narrow distal coils arranged in 2-4 layers. The sporophorous vesicle is bounded by a thin membrane and contains multiple tubular structures. Small subunit ribosomal DNA phylogeny showed basal position of the new microsporidium to a cluster uniting microsporidia infecting ciliates (Euplotespora binucleata), microcrustaceans (Glugoides intestinalis, Mrazekia macrocyclopis), lepidopteran insects (Cystosporogenes spp., Endoreticulatus spp.) and human (Vittaforma corneae), nested within Clade IV sensu Vossbrinck and Debrunner-Vossbrinck (2005 Folia Parasitol 52:131-142). No close phylogenetic relationships were found between A. chironomi and microsporidia from other dipteran hosts.

Pankovaia semitubulata gen. et sp. n. (Microsporidia: Tuzetiidae) from nymphs of mayflies Cloeon dipterum L. (Insecta: Ephemeroptera) in Western Siberia.

The ultrastructure of a new microsporidian, Pankovaia semitubulata gen. et sp. n. (Microsporidia: Tuzetiidae), from the fat body of Cloeon dipterum (L.) (Ephemeroptera: Baetidae) is described. The species is monokaryotic throughout the life cycle, developing in direct contact with the host cell cytoplasm. Sporogonial plasmodium divides into 2-8 sporoblasts. Each sporoblast, then spore, is enclosed in an individual sporophorous vesicle. Fixed and stained spores of the type species P. semitubulata are 3.4 x 1.9microm in size. The polaroplast is bipartite (lamellar and vesicular). The polar filament is isofilar, possessing 6 coils in one row. The following features distinguish the genus Pankovaia from other monokaryotic genera of Tuzetiidae: (a) exospore is composed of multiple irregularly laid tubules with a lengthwise opening, referred to as "semitubules"; (b) episporontal space of sporophorous vesicle (SPV) is devoid of secretory formations; (c) SPV envelope is represented by a thin fragile membrane.

Transfer of the members of the genus Brachiola (microsporidia) to the genus Anncaliia based on ultrastructural and molecular data.

Two microsporidian genera, AnncaliiaIssi, Krylova, & Nicolaeva 1993 and BrachiolaCali et al. 1998, possess a Nosema-type life cycle and unique cell surface ornamentations, which include precocious electron-dense coating of the plasmalemma and a variety of secretory structures deposited on the parasite surface and scattered in the host cell cytoplasm. Comparative analysis of ultrastructure of Anncaliia meligethi (the type species of the genus Anncaliia) and of B. vesicularum and B. algerae (the best-studied members of the genus Brachiola) clearly demonstrated that these microsporidia share many distinctive morphological features. The comparison of small subunit ribosomal DNA sequences showed high sequence identity of A. meligethi and B. algerae. Phylogenetic analyses indicated that the rDNA sequences of A. meligethi clustered with those of B. algerae suggesting a close relatedness of these microsporidia. The combination of molecular and morphological data provided clear evidence that these microsporidia belong to the same genus and therefore, warranted emendation of the genus Anncaliia and establishments of the following new combinations: Anncaliia vesicularum nov. comb., Anncaliia algerae nov. comb., Anncaliia connori nov. comb., and Anncaliia gambiae nov. comb. The generic name Brachiola is submerged according to the rule of priority.

Ultrastructural analysis supports transferring Nosema whitei Weiser 1953 to the genus Paranosema and creation a new combination, Paranosema whitei.

The current ultrastructural description of Nosema whitei is in agreement with the genus definition of Paranosema [Sokolova, Y.Y., Dolgikh, V.V., Morzhina, E.V., Nassonova, E.S., Issi, I.V., Terry, R.S., Ironside, J.E., Smith, J.E., Vossbrinck, C.R., 2003. Establishment of the new genus Paranosema based on the ultrastructure and molecular phylogeny of the type species Paranosema grylli Gen. Nov., Comb. Nov (Sokolova, Selezniov, Dolgikh, Issi 1994), from the cricket Gryllus bimaculatus Deg. Journal of Invertebrate Pathology 84, 159-172]. In addition to exhibiting similar spore morphology and sporogony type, N. whitei, like P. grylli and Paranosema locustae, possesses a distinct meront-sporont transitional stage in the life cycle; develops in the host fat body, and produces secretory material arranged in "tubular structures" during sporogony. Ultrastructural analysis supports the similarity of N. whitei to P. grylli and to P. locustae as predicted on the basis SSrDNA sequence data (GenBank Accession Nos. AY305323, AY305325, and AY305324). Comparative studies of these three related species provide a good example of the consistency of morphological and sequence data, and support both the placement of N. whitei inside the genus Paranosema and the validity of the new combination Paranosema whitei (Weiser).