[On the use of FTA technology for collection, archieving, and molecular analysis of microsporidia dna from clinical stool samples].

The FTA technology was applied for sampling, archiving, and molecular analysis of the DNA isolated from stool samples to diagnose and identify microsporidia, the intracellular opportunistic parasites which induce malabsortion syndrome in immunosuppressed humans, particularly in patients with AIDS. Microsporidia DNA was successfully amplified in 6 of 50 stool samples of HIV-positive patients of the S. P. Botkin Memorial Infectious Disease Hospital (St. Petersburg) applied to FTA cards (FTA-Cars, Whatman Inc. Florham Park, NJ, USA). Amplicons (the fragments of rDNA) were directly sequenced, and microsporidia species--Encephalitozoon intestinalis, E. cuniculi, E. hellem, and Enterocytozoon bieneusi--were identified in Genbank by NCBI BLAST program. The FTA method of DNA immobilization is especially promising for epidemiological and field population studies which involve genotyping of microsporidia species and isolates.

[Golgi apparatus in parasitic protists (review of the literature)].

This review summarizes modem data on Golgi apparatus of parasitic protists and demonstrates how the parasitic lifestyle determines functional and structural peculiarities of secretory systems in unrelated groups of unicellular parasites, in comparison to ones of "model systems", mammalian and yeast cells. The review covers the most well-studied protists, predominantly of high medical importance, belonging to following taxons: Parabasalia (Trichomonas), Diplomonada (Giardia), Entamoebidae (Entamoeba), parasitic Alveolata of the phyllum Apicomplexa (Toxoplasma and Plasmodium), and Kinetoplastida (Trypanosoma and Leishmania). Numerous recent publications demonstrated that studies on intracellular traffic in the mentioned above parasites essentially advanced our knowledge of Golgi function, traditionally based on research of cultured mammalian and yeast cells. Morphology of Golgi organelle in eukaryotes from various taxonomic groups has been compared. Within three of total six the highest taxons of Eukatyota (Adl et al., 2005) there exist at minimum eight groups represented by species lacking Golgi dictiosomes. However, biochemical and (or) molecular (genomic) evidences indicate that the organelle with functions of Golgi was present in every studied so far lineage of eukaryotes. Loss of Golgi organelle is a secondary event, which has been proven by identification of Golgi genes in the genomes of Golgi-lacking lineages. This loss might have occurred independently several times in the course of evolution. Neither the number of stacks, nor the size of the organelle correlates with intensity of secretion, or the position of the species on the evolutionary tree (in terms of presumably early/lately diverged lineages).

[Structural-functional organization of Golgi apparatus].

This review is dedicated to the structure and function of Golgi apparatus (GA). It summarizes contemporary data published in numerous experimental papers and in several reviews. Possible ways of intra-Golgi transport of proteins, existent models of structural and functional organization of Golgi organelle, as well as the issues of its biogenesis, posttranslational modification and sorting of proteins and lipids, and mechanisms of their traffic-king are discussed. Special attention is paid to the role of coatomer proteins (COPI, COPII and clathrin), fusion proteins (SNAREs), and small GTPases (ARF, SARI) in the secretory pathway. In addition, the phenomena of ultrastructural alterations of GA due to various functional conditions and physiological stimuli are specifically accented. We included in this review our original data on a probable involvement of GA in water transport, and on the organization of atypical GA in microsporidia--intracellular parasitic protists.

[Microsporidiosis of the dragonfly Aeshna viridis larvae (Odonata: Aeshnidae) caused by Systenostrema alba Larsson, 1988 (Microsporida: Thelohaniidae)].

A microsporidian species producing octospores in sporophorous vesicles is found in Aeshna viridis larvae from intermittent streams situated in the vicinity of Novosibirsk City. Size of the spores measured on fresh smears was 6.9 +/- 0.09 microm x 4.1 +/- 0.08 microm (6.0-7.6 x 3.5-4.9). Each spore have single elongated nucleus and an anisofilar polar filament composed of 10-11 anterior and 10-11 posterior coils. The infection was restricted to adipose tissue. According to spore morphology the Siberian isolate can be attributed to the species Systenostrema alba described from Aeshna grandis in Sweden (Larsson, 1988). This is the first description of Microsporidia infecting Odonata from Siberia.

[Structural-functional organization of Golgi apparatus].

This review is dedicated to the structure and function of Golgi apparatus (GA). It summarizes contemporary data published in numerous experimental papers and in several reviews. Possible ways of intra-Golgi transport of proteins, existent models of structural and functional organization of Golgi organelle, as well as the issues of its biogenesis, posttranslational modification and sorting of proteins and lipids, and mechanisms of their trafficking are discussed. Special attention is paid to the role of coatomer proteins (COPI, COPII and clathrin), fusion proteins (SNAREs), and small GTPases (ARF, SARI) in the secretory pathway. In addition, the phenomena of ultrastructural alterations of GA due to various functional conditions and physiological stimuli are specifically accented. We included in this review our original data on a probable involvement of GA in water transport, and on the organization of atypical GA in microsporidia--intracellular parasitic protists.

[Microsporidiosis in the wax moth Galleria mellonella (Lepidoptera: Pyralidae) caused by Vairimorpha ephestiae (Microsporidia: Burenellidae)]. / Mikrosporidioz pchelinoi ognevki Galleria mellonella (Lepidoptera: Pyralidae), vyzyvaemyi Vairimorpha ephistiae (Microsporidia: Burenellidae).

An experimental microsporidiosis of the wax moth caterpillars from laboratory population had been caused by oral infecting of early stages larvae and by intracavity injections of the spores of the microsporidian species Vairimorpha ephestiae. Peculiarities of microsporidiosis proceeding, manifestations of host defence reactions, and also an effect of the temperature of caterpillars cultivation and conditions of spores keeping on liability of the insects to the infection were studied. The effect of the microsporidia on the host organism was the early death or the delay of larvae development, but in several cases external manifestations of the effect of the parasite on the host were absent. The development of the parasites from the moment of infecting to the appearing of the mature spores congestions in the host organism proceeded 6 days. Microsporidia invaded insect fat body and caused its hypertrophy and disappearance of lipid granules. In the intestine and salivary glands microsporidia were not observed in the period from 6 to 16 day of the development. On the final stage of microsporidiosis the all contents of fatty tissue cells were replaced by spores of microsporidia. Under microscope only diplocaryotic spores of the Nozema type had been found in infected and died specimens, but not octospores. The spores threw out polar tubes under the change of pH in incubating solution from neutral to alkaline. The effects of microsporidiosis on the wax moth haemolymph were the increased rate of prohaemocytes, appearing of multinuclear free-circulating cells at 6 day after infection, and suppression of the reaction of haemolymph melanization with the mass sporogenesis of the parasite. The characteristic symptom of the wax moth microsporidiosis had been revealed, accumulation of black points and small spots of irregular form under cuticle ("reaction of attretization"). Increase of the temperature of insect cultivation up to 32 degrees C during 3 days after infection contributed to the full deliverance of the insects from the infection in first and second generations. It can be considered as a method of treatment of wax moth laboratory colonies from microsporidiosis. Oral infection of III and IV stage caterpillars by the spores being kept during 3-6 months under 4 degrees C in form of water suspension caused the death of 63.0-61.5 and 91% of caterpillars being cultivated under 25 and 21 degrees C respectively. Under the temperature of cultivation equal 30 degrees C the mortality did not differ from the control sample (8-10%). The spores extracted from dried bodies of caterpillars lost their vitality. It was demonstrated by the test on infectious ability in vivo and by acridine orange staining. This host-parasite system appears to be perspective in investigations of resistance mechanisms in insects and immunosuppressive features of entomopathogen microsporidia.

[Refinement of the diagnosis of Issia globifera (Microsporidia: nosematidae) based on ultrastructural data]. / Utochnenie diagnoza Issia globulifera (Microsporidea, nosematidae) na osnove dannykh po ul'trastrukture mikrosporidii.

An ultrastructure of a microsporidian belonging to the aberrant and poorly studied genus Issia is discribed in this paper. The first description of Issia globulifera (Issi, Pankova, 1983) was made on the basis of light microscopic analysis of midgut smears of the infected insects. Both late merogonic and sporogonic stages can be met on the sections from midguts of Anopheles messae. Meronts are ameba-like cells (3.0-3.5 x 2.0-2.5 microns) forming conglomerates of cells without any regular organization. Meronts and sporonts directly contact with the host cell cytoplasm; they are surrounded by numerous mitochondria. Sporonts divide into 2 sporoblasts, but remain connected with each other by posterial ends. In the vicinity of the contact the paramural bodies (structures, presumably participating in the formation of the exospore) can be seen. Sporoblast morphogenesis is accompanied by the rapid grow of electron dense layer in the region of the contact of two sister sporoblasts. Due to such intensive growth the sporoblasts are finally located at sharp angle to each other. Envelopes of sporonts and sporoblasts possess typical 2-membrane structure. Their nuclei lay in pairs like in Nosema species. Sporoblasts (unlike meronts and sporonts) are surrounded by the electron lucid zone of the cytoplasm with numerous tubule-like and spherical inclusions. Host mitochondria are closely adjacent to the periphery of this zone. The formation of characteristic spherical structures (globules), in which the posterior ends of two (in rare cases of 1 or 4) sister spores are downsinked, starts immediately after the sporont division. Globules rapidly increase in size during the sporogenesis. The homogenous globule (6-8 microns), which is essentially larger than a spore, seems to represent the modified exospore; it is not limited by any visible membrane. Fine structure of spores (2.5-3.0 x 2.0-2.3 microns) is typical for the nosems: spherical polar disk; lamellas polaroplast; nuclei located along the long spore axis; basal part of a polar tube forming a fork. A thin (25-40 nm) heterophilar polar filament forms 12 rows of various structure (8 + 4) packed in two rows. The posterior vacuole was no revealed. Endospore is very thick up to 250-400 nm in mature spores. The exospore covering the 2/3-4/5 of the spore length passes into the globule on the posterior end. The refined diagnosis of Issia globulifera: type host species--Anopheles messae (larvae, pupae); tissue localization: mitgut epithelium; terra typica: Siberia, Tomsk region, Ob river basin; developmental stages: multicellular merogonial plasmodium, sporont gives rise to 2 (rarely 1 or 4) sporoblasts connected with each other by a "globule", a specific formation homologous to an exospore of other microsporidians; globule (6-8 microns) possesses a homogenous inner structure of average electron density; sporophorous vesicle not formed; spore structure is typical for Nosematidae. Embeddings, negatives (## 2301, 2303, 2308, 2311-2313, 2532, 2533) and photos of type material in the collection of All-Russian Institute for Plant Protection.

[Parasitophorous vacuole of microsporidia]. / O parazitofornoi vakuoli mikrosporidii.

The comparative analysis of the ultrastructure of various types of parasitophorous vacuoles (PV) induced by microsporidians is given. The data on the occurrence of PV in the hosts belonging to different systematic phyla are summarised. It is concluded, that the formation of PV around microsporidians might take place either in certain parasite species or in the special type of the invaded cells, or could be connected with the development in the unspecific host. The variety of fine structure of PV might be explained by an extremely broad range of hosts (from protists to mammalians), with different level of development of their immune system (defence reactions). Three basic types of PV are proposed according the organization of their envelopes (walls): (1) a single membrane originated from the host cell plasmalemma (hosts: Aves and Mammalia); (2) a single membrane derived from the host ER (hosts: Pisces); (3) a single- or double-membrane host ER (hosts: protists, invertebrates and animals of other systematic groups). It was assumed that the formation of the PV around microsporidians reflects more primitive host-parasite interactions, than the development of the parasite in a direct contact with the host cell cytoplasm.

[A morphofunctional analysis of the hemocytes in the cricket Gryllus bimaculatus (Orthoptera: Gryllidae) normally and in acute microsporidiosis due to Nosema grylli]. / Morfofunktsional'nyi analiz gemotsitov sverchka Gryllus bimaculatus (Orthoptera: Gryllidae) v norme i pri ostrom mikrosporidioze, vyzyvaemom Nosema grylli.

Microsporidians (M) are supposed to be ancient eukaryotic parasites with a broad range of animal hosts, being especially abundant in Arthropoda. They are supposed to pass a long way of adaptation to parasitism, that usually means inhibiting or avoiding host immune reactions alongside with the reduction of pathogenicity. However M, unlike other eukaryotic obligate parasites, preserved a high pathogenicity, comparable with one of viruses, and thus they could be expected to possess a unique mode of interactions with their hosts. The goal of the present work is to assess how M influence the cellular immune response of an insect host. Experiments were performed on the host-parasite system Gryllus bimaculatus (Orthoptera, Gryllidae)--Nosema grylli (Microsporidia, Nosematidae); coccidia Adelina grylli--infected crickets were used to compare the host cellular response against two pathogens. Haemocytes (H) were observed using phase contrast and electron microscope. H smears were stained for a phenoloxidase (PO), esterase activities and "respiratory burst" reaction. Five H type can be distinguished in the cricket haemolymph. (1) Prohaemocytes, relatively small (13-30 microns) cells with large nuclei, are observed both in control and infected insects. (2) Plasmatocytes, round (30-35 microns in diameter) or fusiod (40-63 x 13-38 microns) cells, can hardly be distinguished from (1) ultrastructurally; during the coccidian infection of the cricket fat body these H infiltrate the infected organ and turn into amebocytes with laciniate nuclei, they usually contain electron dense granules, that release during the formation of a capsule around the coccidian oocytes. (3) Granulocytes (Gr, 20-33 microns in diameter) are cells with the extremely refractive cytoplasm when observed in phase contrast microscope, they contain vacuoles with typical crystal needle-like inclusions. The transitional forms between the mentioned above three cell types can be observed. The next two H types also observed on H monolayers are supposed to be the specialized forms of Gr: (4) coagulocytes, cells with the fragile cytoplasm that are easily disintegrated after a contact with a pathogen; they have been described in Orthoptera for the first time now; (5) spherulocytes, giant cells filled with electron lucid granules and small, often eccentrically located nucleus. Both H types were observed only after infection with A. gryllus in the vicinity of encapsulated oocysts. Infection with M does not cause such intensive concentration of haemocytes near the infected organ, or so abundant nodule formation, until the acute stage of the disease when M spores are liberated from the destroyed cells and contact the insect haemolymph. Thereafter, the number of granulocytes significantly increases. In the presence of M spores, haemocytes produce long cytoplasm protrusions and form clapms. Some spores adhere to the haemocyte surface and are phagocytized. Giant round cells loaded with spores, can be observed in the host lymph. They are surrounded by a sheath composed of flattened cells and resemble xenomas, described for fish microsporidiosis. A. grylli caused the increase in the quote of PO-positively stained cells up to 80% from 40-50% in control, that well corresponds to the host immune reactions activation and melanization of infected tissue, while microsporidiosis significantly reduced quote of PO+ cells. Carboxyl esterase activity expressed as quote of positively stained cells was 40-60% in naive and coccidia-parasitized samples, M decrease this number to 10-20%. "Respiratory burst" reaction, detected by reducing of NBT, did not alter significantly in microsporidia-infected insects. From the presented data in can be concluded: 1) M do not suppress such cellular reactions as a clamp formation and phagocytosis of spores, liberated from the infected tissue; 2) at the same time they suppress activities of enzymes involved in immune response.

[Ameson hybomitrae sp. n. (Microsporidia, Pereziidae) from gadflies of Karelia]. / Ameson hybomitrae sp. n. (Microsporidia, Pereziidae) iz slepnei Karelii.

Microsporidia of the genus Ameson were recorded from larvae of horseflies of the genus Hybomitra in Karelia. Earlier these Microsporidia were recorded from crustaceans. The infection extensiveness ranges from 5.1 to 10.5%. The parasites develop in musculature, fat body and salivary glands of the host. The new species has uninucleate, single-located egg- and pear-shaped spores. The ultrafine structure of developmental stages and spores is studied.

[A rapid method for staining semithin tissue sections embedded in araldite]. / Uskorennyi metod okraski polutonkikh srezov tkanei, zalitykh v araldit.

A new rapid method is proposed for staining of semithin sections. The method involves the treatment of sections with a methylene blue solution with a slight heating for 1-3 minutes. The method allows to receive polychromatic contrast preparations, to save time and reagents. It also permits to avoid restaining effect. The preparations can be preserved for a long time in plastic media (e.g. in polystyrene) for light microscopy. A comparative analysis of the staining methods used in the electron microscopic practice is given.