Cytological and molecular description of Hamiltosporidium tvaerminnensis gen. et sp. nov., a microsporidian parasite of Daphnia magna, and establishment of Hamiltosporidium magnivora comb. nov.

We describe the new microsporidium Hamiltosporidium tvaerminnensis gen. et sp. nov. with an emphasis on its ultrastructural characteristics and phylogenetic position as inferred from the sequence data of SSU rDNA, alpha- and beta-tubulin. This parasite was previously identified as Octosporea bayeri Jírovec, 1936 and has become a model system to study the ecology, epidemiology, evolution and genomics of microsporidia - host interactions. Here, we present evidence that shows its differences from O. bayeri. Hamiltosporidium tvaerminnensis exclusively infects the adipose tissue, the ovaries and the hypodermis of Daphnia magna and is found only in host populations located in coastal rock pool populations in Finland and Sweden. Merogonial stages of H. tvaerminnensis have isolated nuclei; merozoites are formed by binary fission or by the cleaving of a plasmodium with a small number of nuclei. A sporogonial plasmodium with isolated nuclei yields 8 sporoblasts. Elongated spores are generated by the most finger-like plasmodia. The mature spores are polymorphic in shape and size. Most spores are pyriform (4·9-5·6×2·2-2·3 µm) and have their polar filament arranged in 12-13 coils. A second, elongated spore type (6·8-12·0×1·6-2·1 µm) is rod-shaped with blunt ends and measures 6·8-12·0×1·6-2·1 µm. The envelope of the sporophorous vesicle is thin and fragile, formed at the beginning of the sporogony. Cytological and molecular comparisons with Flabelliforma magnivora, a parasite infecting the same tissues in the same host species, reveal that these two species are very closely related, yet distinct. Moreover, both cytological and molecular data indicate that these species are quite distant from F. montana, the type species of the genus Flabelliforma. We therefore propose that F. magnivora also be placed in Hamiltosporidium gen. nov.

Cytological variation and pathogenicity of the bumble bee parasite Nosema bombi (Microspora, Nosematidae).

In three field seasons, 2003-2005, bumble bees were collected in southern Sweden and eastern Denmark in search of microsporidian parasites. Of the 16 bumble bee species studied, microsporidia were found in Bombus hortorum, Bombus hypnorum, Bombus lapidarius, Bombus lucorum, Bombus pascuorum, Bombus pratorum, Bombus ruderarius, Bombus subterraneus and Bombus terrestris. Only one microsporidian species, Nosema bombi, was recorded. A microsporidium found in B. pratorum differed cytologically from microsporidia of the other host species. In the most frequently infected host, B. terrestris, the prevalence was 20.6%. Totally 1049 specimens were dissected. The light microscopic and ultrastructural cytology and pathology of N. bombi is described with focus on the variation recorded. Variation was especially prominent in the shape, size and coupling of spores, and in the length and arrangement of the polar filament. In four host species microsporidian infection was restricted to peripheral fat cells.

The ultrastructure and reproduction of Amphiamblys capitellides (Microspora, Metchnikovellidae), a parasite of the gregarine Ancora sagittata (Apicomplexa, Lecudinidae), with redescription of the species and comments on the taxonomy.

The ultrastructural cytology and reproduction of the hyperparasitic microsporidium Amphiamblys capitellides (Caullery and Mesnil, 1897) is described. Merogonial reproduction was not observed. The sporogony comprises two sequences: a sac-bound sporogony in close contact with the cytoplasm of the host and a free sporogony in parasitophorous vacuoles. The free sporogony, which probably precedes the sac-bound, yields a small number of rounded spores. The sac-bound sporogony is polysporoblastic, generating two rows of elongated spores. All stages have isolated nuclei. Both spore types have an extrusion apparatus of the metchnikovellidean type, with a polar sac devoid of anchoring disc, a polar filament with one manubroid and one bulbous part, and a posterior semicircular membrane fold enclosing rounded or tubular structures. Hosts are gregarines of the species Ancora sagittata living in the intestine of polychaetes of the genus Capitella, probably the species Capitella giardi. The cytology, life cycle and classification are discussed. The species is redescribed and the diagnosis of the genus Amphiamblys Caullery and Mesnil, 1914 is emended.

Fixation of microsporidian spores for electron microscopy.

Fresh and frozen spores of the microsporidia Nosema apis and Nosema bombi were fixed using various fixatives at different times and temperatures. Paraformaldehyde and technical formaldehyde gave results comparable to or better than glutaraldehyde. Increased fixation temperature improved the fixation of spores from terrestrial hosts. Freezing did not destroy the cytology of the spore.

The hyperparasitic microsporidium Amphiacantha longa Caullery et Mesnil, 1914 (Microspora: Metchnikovellidae) - description of the cytology, redescription of the species, emended diagnosis of the genus Amphiacantha and establishment of the new family Amphiacanthidae.

The ultrastructural cytology and reproduction of Amphiacantha longa Caullery et Mesnil, 1914 is described. Merogonial reproduction was not observed. The sporogony follows two lines: free disporoblastic and enveloped, polysporoblastic, involving sporoblast mother cells. The enveloped sporogony is endogenous in spore sacs of sporont origin, daughter cells are formed by vacuolation. Probably all stages have coupled nuclei. Both free and enveloped spores are equipped with an extrusion apparatus composed of a flat polar sac, a straight polar filament of manubrium type, and a posterior globular appendix. Manubrium and appendix are enclosed in a membraneous coat. Circular elements of coat material occur in the proximity of the extrusion apparatus. The membraneous coat and the surface layer of the manubrium penetrate the polar sac. The extrusion apparatus is located at the wide pole of the spore, the nuclei at the narrower pole. Hosts are gregarines of the genus Lecudina Mingazzini, living in the gut of the polychaete Lumbrinereis fragilis (O. F. Müller). The cytology and reproduction are discussed and compared to other genera of metchnikovellideans, to the chytridiopsid genera, and to microsporidia expressing the typical cytology for the group. Metchnikovellideans and chytridiopsids exhibit cytological and reproductive similarities. The species is redescribed, the diagnosis of the genus Amphiacantha Caullery et Mesnil, 1914 is emended, and the new family Amphiacanthidae, comprising the genera Amphiacantha and Amphiamblys Caullery et Mesnil, 1914, is established.

Bacillidium cyclopis Vávra, 1962 - Description of the ultrastructural cytology and transfer to the genus Mrazekia Léger and Hesse, 1916 (Microspora, Mrazekiidae).

The ultrastructural cytology of the microsporidium Bacillidium cyclopisVávra, 1962, a parasite of the copepod Acantho cyclop s americanus in Czechoslovakia, is described, with emphasis on the mature spore. All life cycle stages are diplokaryotic. The three-layered spore wall has a stratified exospore. The polaroplast has two distinctly separated lamellar parts where the lamellae are folded concentrically around the anterior part of the polar filament. Anterior lamellae are narrow and closely packed; posterior lamellae are wider and more loosely arranged. The polar filament has an anterior straight and wide section, the "manubrium", where the posterior third is widened in a characteristic manner, and a final section, arranged in 2-3 narrow, isofilar coils. The attachment of the polar filament is more or less oblique in relation to the long axis of the spore. The ejected filament leaves the spore in a distinct angle, and the narrow part is characteristically and regularly coiled. Glycoconjugates are present in the layers of the anchoring disc, in one of the external layers of the manubroid part of the polar filament, and in two distinct layers of the coils. The diplokaryon partially encloses the manubrium in a mantle-like fashion. B. cyclopis is compared to re-examined preparations of Mrazekia argoisiiLéger and Hesse, 1916. These two species are the only Bacillidium-like microsporidia of Crustacea, and they are distinguished from all the other Bacillidium-like microsporidia by their swollen posterior end of the manubrium. This character, which is easily seen also using light microscopy, is considered diagnostic for the genus Mrazekia, and B. cyclopis is transferred to this genus. Mrazekiidae Léger and Hesse, 1922 is the valid family name, with Bacillidiidae Larsson, 1986 as a junior synonym.

Tardivesicula duplicata gen. et sp. nov. (microspora, duboscqiidae), a microsporidian parasite of the caddis fly Limnephilus centralis (trichoptera, limnephilidae) in Sweden.

The microsporidium Tardivesicula duplicata gen. et sp. nov., a parasite of the adipose tissue of larvae of the caddis fly Limnephilus centralis in Sweden, is described based on light microscopic and ultrastructural characteristics. All life cycle stages have isolated nuclei. The sporogony is polysporoblastic, yielding 16-32 spores. The spores are rod-shaped, 1.3-1.4 × 3.7-4.2 µm in living condition. The spore wall has a 34-40 nm thick, layered exospore, with a median layer resembling a unit membrane. The endospore is thicker than the exospore. The polaroplast has two lamellar parts, where the posterior lamellae are the widest. The isofilar, 128-149 nm thick polar filament is arranged in 9-10 coils in a single layer close to the spore wall in the posterior 2/3 of the spore. The angle of tilt is c. 45°. The transversely sectioned polar filament has distinct concentrical layers of variable thickness and electron density. Sporophorous vesicles are ellipsoid and fragile. The thin electron-dense envelope is initiated as narrow tubulus-like promordia, appearing when the nucleus of the sporont has divided once. The episporontal space of vesicles with sporogonial plasmodia is devoid of inclusions. Vesicles with sporoblasts and spores have tubular exospore-derived inclusions of two dimensions. The new genus is compared to genera with polysporoblastic sporogony in sporophorous vesicles, primarily to Cystosporogenes and Flabelliforma. The placing of the new genus in the family Duboscqiidae is discussed.

The ultrastructural cytology of Bacillidium strictum (Léger and Hesse, 1916) Jírovec, 1936 (Microspora, Bacillidiidae).

The ultrastructural cytology of a microsporidium identified as Bacillidium strictum (Léger and Hesse, 1916) is described. The host was one of 28 investigated specimens of the oligochaete Limnodrilus hoffmeisteri, collected from a small stream in southern Sweden, not the type host Lumbriculus variegatus. The microsporidium occurred together with an eugregarine of the genus Rhynchocystis and an actinomyxidian of the genus Echinactinomyxidium. The 2.0-2.2 × 11.6-13.3 µ great spores (fixed and stained) were slightly smaller than the size indicated in the description. They occurred in spherical, hypertrophic haemocytes. The exospore, which is approximately as thick as the endospore, has a wide, electron-dense basal layer, an external layer resembling a double-membrane, and fibrous, periodically arranged surface projections. The double-layer remains attached to the basal layer. The polaroplast has an anterior region with closely packed lamellae and a posterior section with wider lamellae or tubules. The polar filament has a wide, straight anterior part, a zone with successively reduced diameter, and a posterior, narrow section, which turns in anterior direction, forms one or two coils around the diplokaryon and the wide filament, and ends in the proximity of the polaroplast. Approximately 100 nm wide tubules, exhibiting exospore construction, are initiated at the onset of sporogony, and persist also in the proximity of mature spores. The species status of the microsporidium and the ultrastructural characteristics of the Bacillidium-like microsporidia are discussed.

On the cytology of Jirovecia caudata (Léger and Hesse, 1916) (microspora, bacillidiidae).

The cytology of a microsporidium identified as Jirovecia (Mrazekia) caudate (Léger & Hesse, 1916), the type species of the genus Jirovecia Weiser, 1977, is described with emphasis on the ultrastructure of the mature spore. The hosts were Limnodrilus hoffmeisteri and an unidentified species of Tubificidae (Oligochaeta), collected from a brook in southern Sweden, not Tubifex tubifex, the type host. The dimensions of the spore and the caudal spore projection varied more than the size range reported in the description of the species. The exospore is characteristic, with a surface layer resembling a unit membrane and a wide, dense, basal layer. The surface layer remains attached also to mature spores. The caudal projection is formed only from exospore material, and it is ornamented with longitudinal ridges. The polaroplast has an anterior region with closely packed lamellae and a posterior section with wider lamellae or tubules. The polar filament has a wide, straight, anterior part, a zone with successively reduced diameter, and a posterior, narrow section, arranged as an incomplete coil. The filament is easily extruded, and the length and size difference between the wide and narrow parts is apparent also using light microscopy. Membrane-lined compartments and aggregates of granular material are present close to the posterior pole. The species status of the microsporidium and the cytological characteristics of Bacillidium-Jirovecia species are discussed.

Rectispora reticulata gen. et sp. nov. (Microspora, Bacillidiidae), a new microsporidian parasite of Pomatothrix hammoniensis (Michaelsen, 1901) (oligochaeta, tubificidae).

The microsporidium Rectispora reticulata gen. et sp. nov., a parasite of male gonads of the oligochaete Pomatothrix hammoniensis in Sweden, is described based on light microscopic and ultrastructural characteristics. Merogonial and sporogonial stages are diplokaryotic. Sporogony is disporoblastic. The spores are cylindrical, often slightly curved and wider in the mid-region, 1.8-2.1 × 8.1-14.2 um long in fixed and stained condition. The spore wall has a ca. 43 mm thick, uniform exospore. The polaroplast has an anterior part with compressed lamellae and a posterior section with 53-128 nm wide tubules. The polar filament has a 181-283 nm wide, straight, short, anterior section in the centre of the spore, an oblique section with tapering diameter, and a long, posterior, 119-178 nm wide section, arranged in (10-)12(-13) isofilar coils, close to the spore wall in the posterior half of the spore. The most anterior and most posterior coils are irregularly arranged, the median coils form a close, regular group. The angle of tilt of the most anterior, regularly arranged coil is ca. 55°. The transversely sectioned filament has distinct concentrical layers of variable thickness and electron density. The diplokaryon occupies the centre of the spore. Sporophorous vesicle absent, but a folded, ca. 16 nm thick envelope, resembling a sporophorous vesicle, is formed by delamination of exospore material. Teratological sporogony was observed. The microsporidium is compared to microsporidia of the genera Bacillidium, Jirovecia and Hrabyeia. The placing of the new genus in the family Bacillidiidae, the distinction of the families Bacillidiidae and Nosematidae, and the various origins of spore-containing envelopes are discussed.

On the cytology and life cycle of Cougourdella polycentropi weiser, 1965, with comments on the taxonomy of the genus Cougourdella hesse, 1935 (microspora, cougourdellidae).

The cytology and life cycle of a microsporidium identified as Cougourdella polycentropi, with provenance from southern Sweden, is described using light and electron microscopy methods. The life cycle is comprised of two merogonial cycles followed by tetrasporoblastic sporogony in sporophorous vesicles. The lageniform, 7.0-7.7 µm long spores have an anisofilar polar filament arranged in 6-7 coils, a layered exospore and chambered/lamellar polaroplast of unusual construction. The sporophorous vesicles have fibrillar, and temporarily, tubular inclusions. The vesicles were fragile and the tetrasporoblastic condition was not prominent in squash preparations. The microsporidium is compared to microsporidia with lageniform spores from caddis fly hosts, and the identification is discussed. The taxonomic problems with the genera Pyrotheca and Cougourdella are discussed.

Light and electron microscope studies on Jirovecia involuta sp. nov. (microspora, bacillidiidae), a new microsporidian parasite of oligochaetes in Sweden.

The new microsporidium Jirovecia involuta is described, based on light and electron microscopy preparations. The host was the oligochaete Limnodrilus hoffmeisteri, collected from a stream in southern Sweden. All developmental stages are diplokaryotic. Mature spores are cylindrical, with a short tail-like projection of exospore material. The spore wall has a uniform exospore. The polaroplast has two regions, anteriorly with closely packed lamellae, posteriorly with tubules. The polar filament has a wide, straight anterior portion, and an approximately equally long, narrow posterior section, forming one coil. Each spore is enclosed in a double-layered sac-like structure, formed by the sporoblast from exospore material. The identity of the species and some traits on the ultrastructural cytology are discussed.