INTERACTIONS OF MICROSPORIDIA WITH INFECTED HOST CELL.

Microsporidia comprise a group of fungi-related obligate intracellular eukaryotic pathogens with extremely wide host range: from protists to mammals. Adaptation to intracellular parasitism drives these parasites towards significant reduction and modification of the genome and functional apparatus, which causes extreme dependence on the host cell, as well as sophisticated host-parasite relationships. In this review we summarize our results and recent literature data about microsporidian interactions with the host at the cellular level. The impacts of these pathogens to infected cells include induction of hypertrophy, restructuring and modification of the cytoskeleton and the vesicular transport system of the host cells. Microsporidians also able to stimulate the metabolic processes in the infected cells and inhibit their defensive reactions. The main tool of the direct regulatory impact of microsporidia on the host cell apparently is the secretion of the special protein effectors capable to interfere to regulatory and signaling pathways of the host cell.

ULTRASTRUCTURAL DIFFERENCES OF PRESPORES DEVELOPMENTAL STAGES AND MEIOSPORES OF SPECIES OF CLOSELY RELATED MICROSPORIDIA OF GENERA AMBLYOSPORA AND TRICHOCTOSPOREA (AMBLYOSPORIDAE: MICROSPORIDIA) FROM BLOOD-SUKING MOSQUITOES OF THE GENUS AEDES (DIPTERA: CULICIDAE).

Ultrastructure of prespore developmental stages, with emphasis on meiospores, has been examined in closely related genera of Microsporidia, Amblyospora and Trichoctosporea, isolated from larvae adipose tissue of mosquitoes Aedes in order to compare morphology of these parasites. Ultrastructural differences between Amblyospora and Trichoctosporea have been found to concern the secret filling episporal space of sporophorous vesicle, wall of mature spores, the polaroplast and membranes of thin coils of polar tubes. The episporal secret contributes to formation of meiospore wall and its structure is one of key systematic features to be considered in the identification of microsporidia.

INFESTATION OF HONEYBEE (APIS MELLIFERA) FAMILIES BY MICROSPORIDIANS OF THE GENUS NOSEMA IN TOMSK PROVINCE

Infestation of bee colonies and apiaries by representatives of the genus Nosema, microsporidian protozoans of European honeybees (Apis mellifera L.), causing nosemosis, in Tomsk Province was investigated. In 2012­2015, nosemosis was detected in 32 out of 124 honeybee colonies (31.3 %) and in 20 out of 64 studied apiaries (25.8 %). The maximal infestation rate of bee colonies and apiaries constituted more than 40 % in 2014­2015. N. apis pathogen was registered in 84.4 % of infected bee colonies (16 apiaries); N. ceranae was identified in 9.4 % of infected bee colonies (2 apiaries); and co-infection (N. apis and N. ceranae) was detected in 6.3 % of infected bee colonies (2 apiaries). The reasons of the spreading of the nosemosis, such as climatic conditions, control of imported bee colonies on the presence of Nosema infection, and some others are discussed.

[Comparison of taxonomic importance of morphological and molecular-genetic characters in systematics of Microsporidia (Microsporidia) of blood-sucking mosquitoes (Diptera: Culicidae)].

Comparative analysis of the taxonomic position of microsporidians from mosquitoes of the family Culicidae, for which SSU rDNA sequences data were obtained, demonstrates partial contradiction of systems based on morphological and phylogenetic characteristics. Representatives of the genera Anncaliia Issi. et al., 1993 and Vavraia Weiser, 1977 constitute separate evolutionary branches of the phylogenetic tree of microsporidians of blood-sucking mosquitoes. Representatives of other genera, for which the SSU rRNA sequences data were obtained (Amblyospora Hazard, Oldacre, 1975, Andreanna Simakova et al., 2008, Culicospora (Kudo, 1921), Weiser, 1977, Culicosporella Hazard, Savage, 1970, Edhazardia (Kudo, 1930), Sprague, Fucuda, 1989, Hazardia Weiser, 1977, Hyalinocysta Hazard, Oldacre, 1975, Novothelohania Andreadis et al., 2012, Parathelohania Codreanu, 1966, Senoma Simakova et al., 2005, and Trichoctosporea Larsson, 1994), form a separate, monophyletic group in the tree of Microsporidia. They are closely related and probably possess a common ancestor. The genera Amblyospora, Culicospora, Edhazardia, and Trichoctosporea were placed in the family Amblyosporidae with the revision of the diagnosis. On the basis of the obtained data on morphology and molecular phylogeny we placed Intrapredatorus barri Chen et al:, 1998 into the genus Amblyospora as Amblyospora barri, comb. nov., and also the species Amblyospora bakcharia Andreadis et al., 2012, A. kazankia Andreadis et al., 2012, A. mocrushinia Andreadis et al., 2012, and A. rugosa Simakova, Pankova, 2005 into the genus Trichoctosporea as Trichoctosporea bakcharia comb. nov., T. kazankia comb. nov., T. mocrushinia comb. nov., and T. rugosa comb. nov. Microsporidians of blood-sucking mosquitoes originally possessed complicated life cycles with transovarial and oral transmissions and with the presence of intermediate hosts (lower crustaceans). Later, some microsporidians had lost a part of their life cycle, either during disorganization of habitats of main and intermediate hosts, or as a result of adaptation to environmental and (or) physiological characteristics of hosts. Changes in the life cycle occurred rather rapidly in comparison with the duration of evolutionary development and had an adaptive character. Differences in the life cycle of parasites of the genera Amblyospora, Culicospora, Culicosporella, Edhazardia and Hyalinocysta possibly represent an adaptation increasing the probability of host-parasite meeting. In the process of evolution, spores formed stronger spore wall with thick exospores and endospores. The appearance of thick spore walls in mature spores promoted the development of the apparatus of extrusion process, namely the flattening of the polaroplast chambers and the emergence of long anisofilar polar filament.

[Infestation of lower crustaceans (Copepoda, Cladocera) with microsporidians (Microsporidia) in Western Siberia].

The search for intermediate hosts of microsporidians of bloodsucking mosquitoes of the family Culicidae with complicated two-host developmental cycles in Western Siberia resulted in revealing of 19 microsporidian species in crustaceans. Crustacean microsporidians are represented as by specialized parasites of crustacean, being or being not related to microsporidians parasitizing mosquitoes, and by parasites of mosquitoes having only a part of their complicate life cycle in crustaceans. Sequencing of ssrDNA of microsporidins from copepods had demonstrated that Acanthocyclops venustus Norman et Scott can be an intermediate host of Amblyospora rugosa Simakova et Pankova, 2005 .from mosquitoes Oc. cataphylla Dyar., and Acantocyclops reductus (Chappuis) can be an intermediate host of Trichoctosporea pygopellita Larsson, 1994, a parasite of the mosquito Oc. excrucians (Walker). According to their fine structure, microsporidians from Daphnia Muller belong to the genera Bervaldia Larsson, 1981 and Agglomerata Larrson et Yan, 1988. The infestation rate in natural population of crustaceans was low, constituting about 2%. The maximal infestation rate was observed in temporary reservoirs since late April till early May.

[Identification of mosquito-parasitic microsporidia, Amblyospora rugosa and Trichoctosporea pygopellita (Microsporidia: Amblysporidae), from Acanthocyclops venustus and Acanthocyclops reductus (Copepoda: Cyclopidae), based on small subunit rDNA analysis].

Identical small subunit rDNA sequences were obtained for microsporidia Amblyospora rugosa from blood-sucking mosquitoes larvae Ochlerotatus cantans, O. cataphylla and copepods Acanthocyclops venustus, as well as for Trichoctosporea pygopellita from mosquitoes larvae Ochlerotatus cyprius, O. excrucians and copepods Acanthocyclops reductus. The data on molecular phylogeny and ecological researches show that in Siberia mosquito-parasitic microsporidia of the genera Amblyospora and Trichoctosporea have complex life cycle involving likely intermediate hosts, Acanthocyclops copepods. Life cycle of parasites is synchronized with phenology of their hosts. The phylogenetic analyses shows, that genus Trichoctosporea should be transferred from the family Thelohaniidae to the family Amblyosporidae.

[Use of the OTE-staining method for ultrathin sections on the example of microsporidia (Protozoa: Microsporidia)].

A novel method for staining ultrathin sections and examining organelles of taxonomic importance in microsporidian parasites was evaluated using oolong tea extract (OTE) and compared with traditional staining with uranyl acetate (UA). All basic intracellular structures of taxonomic significance were effectively stained with the OTE-staining method and additional layers of the polar filament with more clear boundaries between them were revealed. However, greater resolution and higher general contrast of several structures including membranes, layers of the envelope of mature spores, the structure of rough endoplasmic reticulum, Golgi complex, and nuclear chromatin were achieved with traditional UA-staining. The OTE-staining method has the advantage of being safe and preparations can be stored in light at room temperature with no loss in staining properties. However, greater staining time is required. We conclude that the OTE-staining method may be used as an alternative to traditional staining with UA with successful results.

[Ecology and epizootology of Microsporidia in malarial mosquitoes (diptera: culicidae) from the south of Western Siberia].

The ecology and epizootology of Microsporidia were studied in the natural population of malarial mosquitoes from Western Siberia over a 30-year period. Symptoms of the disease, host specificity and character of parasite localization in host tissues were investigated. Microsporidia of 9 species from 4 genera, namely Amblyospora, Crepidulospora, Senoma, and Parathelohania, were found in the malarial mosquito larvae from the territory examined. The mosquito species Anopheles messeae was infested by larger number of the microsporidian species, than A. beklemishevi. Spores and active stages of the microsporidian Senoma sp. from mosquito larvae are localized in epithelial cells of the host's intestine. There are no external signs of the infestation. The infested larvae do not die, and the infection proceeds to the pupal stage. Microsporidians of the genus Parathelohania infest larvae of both male and female mosquitoes. The parasites localized in the fat body cells. Body of the infested host gets an opaque white color in the period of spore maturation. The infested larvae perish at 4th stage. Microsporidian spores are formed in the mosquito larvae of both sexes. However, infection rate is much higher in the male larvae (77.8%), than that in the female larvae (22.2%). Symptoms of the disease under the infestation by the genera Crepidulospora and Amblyospora are similar to those under the infestation by Parathelohania sp. There are no external signs of the infestation in the adult mosquito females. In these hosts microsporidians form large two-nuclear spores with a thin capsule accounted for transovarial transmission. An about two times decrease of fertility in the Anopheles messeae females infested by the Parathelohania microsporidians is revealed. Mean number of eggs laid by the infested females was 121, while that of the noninfested ones was 232. Thus, the cause of the decrease in the mosquito abundance is both the elimination of male larvae and the decrease of fertility in females. In Siberia malarial mosquitoes yield 2 or 3 generations per year. Infested larvae are rarely occurred in the beginning of summer. Maximal extensiveness of the invasion may be observed in the end of summer, because of the accumulation of the infection in water bodies. Number of the peaks of infestation coincides with the peaks of abundance of mosquito larvae of 4th stage. Peaks of infestation are delayed as compared with the peaks of mosquito abundance. It is an evidence of the development delay in the infested larvae. Long-term dynamics of the infestation by microsporidians is studied. The epizooty caused by Parathelohania was observed in the malarial mosquitoes in the eighties (with the infestation rate up to 62 %). In the last ten years the infestation rate was low (from 0.1 to 2.6%).

[Description of the new type of polaroplast of Microsporidia from Mesocyclops leuckartii (Copepoda: Cyclopidae)].

The polaroplast of microsporidium from copepod Mesocyclops leuckartii was studied by transmission electron microscopy. The polaroplast differs from all previously described types by complex structure.

[Six new species of microsporidia of the genus Amblyospora (Microspora: Amblyosporidae) from blood sucking mosquitoes (Diptera: Culicidae) from the west Siberia].

Microsporidia of the genus Amblyospora parasiting the adipose body of mosquito larvae of the genus Aedes and Culex has been studied with both light and electron microscopy. Six new species of microsporidia are described based on ultrastructural characteristics of spores and sporogony stages. Amblyospora flavescens sp. n. Mature spores are egg-shaped. The spore wall with three layers, about 165 nm. Exospore is two-membranous. Subexospore is absent. Endospore is electron-translucent. Polaroplast consists of three parts: lamellar, large vesicular, lamellar. The anisofilar polar filament with 10--11 coils (3 1/2 + 2 1/2 + 4-5). Fixed spores are 6.3 +/- 0.1 x 4.24 +/- 0.1 microm. Amblyospora kolarovi sp. n. Mature spores are egg-shaped. The spore wall with three layers, about 265-315 nm. Exospore shapes tucks on the surface of spore. It is two-membranous. Subexospore is quagge, structural. Endospore is electron-translucent. Polaroplast consists of two parts: lamellar and large vesicular. The anisofilar polar filament with 11-13 coils (3 + 8-10). Fixed spores are 5.4-5.6 x 3.5-4.2 microm. Amblyospora orbiculata sp. n. Mature spores are widely egg-shaped. On a back pole there is a small concavity. The spore wall with three layers, about 155 nm. Exospore is shapes tucks on a surface of spore. It is two-membranous. Subexospore is absent. Endospore is electron-translucent. Polaroplast consists of three parts: lamellar, vesicular, lamellar. Polar filament is anisofilar, with 11 1/2 coils (4 1/2 + 1 + 6). Fixed spores are 6.3 +/- 0.1 x x 4.0 +/- 0.1 microm. Amblyospora rugosa sp. n. Mature spores are egg-shaped. On a back pole there is a small concavity. The spore wall with three layers, about 225 nm. Exospore is shapes tucks on a surface of spore. It is two-membranous. Subexospore is quaggy, structural. Endospore is electron-translucent. Polaroplast lamellate. Polar filament is anisofilar, with 17 1/2 coils (3 1/2 + 1 + 13). Fixed spores are 5.3 +/- 0.1 x 3.7 +/- 0.1 microm. Amblyospora undata sp. n. Mature spores are egg-shaped. The spore wall is three-layered, about 220 nm. Exospore is shapes tucks on a surface of spore. It is two-membranous. Subexospore is quaggy, structural. Endospore is electron-translucent. Polaroplast lamellate. The anisofilar polar filament with 8 coils (3 + 5). Fixed spores are 5.0 +/- 0.1 x 3.0 +/- 0.1 microm. Amblyospora urski sp. n. Mature spores have widely oval form. The back pole is concave. The spore wall with three layers, about 280 nm. Exospore is shapes tucks on a surface of spore. It is two-membranous. Subexospore is quaggy, structural. Endospore is electron-translucent. Polaroplast lamellate. Polar filament is anisofilar, with 6 coils (2 + 4). Fixed spores are 4.4 +/- 0.1 x 2.9 +/- 0.1 microm.

[Crepidulospora--nomen novum for the junior generic homonym (preoccupied generic name) Crepidula]. / Crepidulospora--novoe nazvanie dlia mladshego gomonima, (preokkupirovannogo rodovogo imeni) Crepidula.

Crepidulospora nom. nov. is a replacement generic name for the genus Crepidula Simakova, Pankova et Issi, 2003 based on the type species Crepidula beklemishevi Simakova, Pankova et Issi, 2003 (Microsporida) from Anopheles beklemishevi. The name proposed by Simakova et al., 2003 is a preoccupied name, because it was already used for the gastropode Crepidula Lamarque 1899 (Echinospirida, Calibraeidae), a parasite of Mytilus. A valid name of the type species of the genus is now Crepidulospora beklemishevi (Simakova, Pankova, Issi, 2003) comb. n. Crepidulospora Simakova, Pankova et Issi nom. non. Type species: Crepidula beklemishevi Simakova, Pankova, Issi, 2003. Diagnosis. Sporogony is octosporoblastic. Sporogonal stages are in direct contact with host cell cytoplasm. 8 uninucleate spores, 4.2 x 2.2 mkm, are sandals-like. Polar tube is anisofilar, with 6-7 coils (2 + 4-5). Polaroplast is three-partite, with broad vesicular, vesicular and lamellar compartments. Microsporidia ilnfects larval adipose tissues. Type host: Anopheles beklemishevi (Diptera, Culicidae).

[Microsporidia of the genus Parahtelohania (Microspora: Amblyosporidae) from blood-sucking mosquitoes of the genus Anopeles (Diptera: Culicidae) from the South of the West Siberia]. / Mikrosporidii roda Parathelohania (Microspora: Amblyosporidae) iz komarov roda Anopheles (Diptera: Culicidae) iuga Zapadnoi Sibiri.

Five new microsporidian species of the genus Parathelohania have been found in the fat body of blood-sucking mosquitoes collected in various water basins in the South of West Siberia: Parathelohania divulgata sp. n., P. formosa sp. n., P. sibirika sp. n., P. teguldeti sp. n., and P. tomski sp. n. Processes of merogony, sporogony, and ultrastructure of spores was were investigated by means light and electronic microscopy.

[Crepidula beklemishevi gen. et sp. n. and Dimeiospora palustris gen. et sp. n. (Microspora: Amblyosporidae)--new microsporidian genera and species from blood-sucking mosquitoes (Diptera: Culicidae) from the south of the western Siberia]. / Crepidula beklemishevi gen. et sp. n. i Dimeiospora palustris gen et sp. n. (Microspora: Amblyosporidae)--novye mikrosporidii iz krovososushchikh komarov (Diptera: Culicidae) iuga zapadnoi Sibiri.

Microsporidia parasitizing the adipose body of mosquito larvae of Anopheles beklemishevi and Aedes punctor has been studied. Two new genera of microsporidia are described based on lightmicroscopic and ultrastructural characteristics of spores and sporogony stages. The spore wall of Crepidula beklemishevi gen. n. et sp. n. is formed by two-membrane exospore, thick exospore, bilayer endospore and thin plasmolemma. Spores with single nucleus, polar filament anisofilar, with 6-7 coils (2+ 4-5), polaroplast consisting of three parts: macrochelicoidal, microhelicoidal and lamellar. Fixed spores 4.2 +/- 0.22 x 2 +/- 0.01 microns. The sporogony of Dimeiospora palustris gen. et. n. results in spore formation of two different types. Spores of the first type are oviform, with thick wall, single-nuclear, 6.1 x 4.9 microns. Spore wall with three layers, about 370 nm. Exospore electron-dense, subexospore moderately electrondense. Exospore and subexospore irregularly pleated on the almost spore surface and slightly thinner on anterior end only. Endospore electron-translucent. Polar filament anisofilar, with 9 coils (3 + 6). Polaroplas consists of three parts: lamellar, fine bubbled, and coarse bubbled. Spores of the second type broad-ovate, with apical pole narrower, distal pole concave, 4.6 x 3.7 microns. Spore wall with three layer, 355 nm. Exospore on the apical end irregularly pleated, consists of thin electrondense exospore, subexospore of variable electron density, endospore electron-translucent. Polar filament anisofilar, with 13 coils (3 + 10). Polaroplast has two parts: lamellar and vesicular.

[New species of Microsporidia in the genus Amblyospora from blood-sucking mosquitoes in the family Culicidae]. / Novye vidy mikrosporidii roda Amblyospora iz krovososushchikh komarov semeistva Culicidae.

Two microsporidians of the genus Amblyospora from mosquito larvae of Aedes and Culex were studied in Tomsk region (Siberia). Peculiarities of the sporogony and the meiospore ultrastructure were studied by light and electron microscopy. The meiospores of A. caspius sp. n. from Aedes caspius and Ae. communis measured on smears, were c. 4.8 x 3.6 microns in size. A polar filament was anisofilar with 5 wide (290 nm) and 9 narrow (190 nm) coils arranged in a single layer. A polaroplast of both species was composed of two parts: anterior lamellae and posterior fibrillae. Meiospores of A. burlaki sp. n. from Culex pipiens measured c. 4.8 x 3.5 microns. A polar filament was anisofilar with 3 wide (260 nm) and 6 narrow (110 nm) coils. The meiospore of this species had a 260 nm thick multilayered exospore.