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14УДК 57.086:57.063.8:582.282
ЦИТОЛОГИЧЕСКОЕ ИЗУЧЕНИЕ КЛЕТОК ВЕГЕТАТИВНОГО МИЦЕЛИЯ SCEDOSPORIUM APIOSPERMUM, РАСТУЩИХ IN VITRO
Степанова А.А. (зав. лаб.)*, Васильева Н.В. (директор института, зав. кафедрой), Богомолова Т.С. (зав. лаб.), Чилина Г.А. (зав. лаб.)
НИИ медицинской микологии им. П.Н. Кашкина, СевероЗападный государственный медицинский университет им. И.И. Мечникова, Санкт-Петербург, Россия
©Коллектив авторов, 2017
В статье представлены данные световой и электронной микроскопии по цитологической организации растущих in vitro клеток гиф культур двух штаммов Scedosporium apiospermum. Показано, что клетки мицелия двух изученных штаммов S. apiospermum содержали по одному ядру, локализованному в центральной части. С помощью трансмиссионной электронной микроскопии (ТЭМ) установлено, что морфогенез клеток воздушного и погруженного мицелия протекает однотипно и заключается в увеличении уровня вакуолизации, формировании одной гигантской митохондрии, синтезе разнообразных запасных веществ, компонентов эндомембранной системы и разных типов внеклеточных веществ. Выявлена корреляция между характером спороношения и способностью сравниваемых штаммов продуцировать внеклеточные липофильные субстанции.
Ключевые слова: in vitro, клетки гиф, Scedosporium apiospermum, сравнительный анализ, ультраструктура, штаммы
CYTOLOGICAL STUDY OF THE IN VITRO GROWING CELLS OF VEGETATIVE MYCELIUM OF SCEDOSPORIUM APIOSPERMUM
Stepanova A.A. (head of the laboratory), Vasilyeva N.V. (director of institute, head of the chair), Bogomolova T.S. (head of the laboratory), Chilina G.A. (head of the laboratory)
Kashkin Research Institute of Medical Mycology of NorthWestern State Medical University named after I.I. Mechnikov, St. Petersburg, Russia
©Collective of authors, 2017
Light- and electron microscopic data about the cytological organization of in vitro growing hyphal cells of two strains of Scedosporium apiospermum are presented. The cells of the mycelium of both strains contained a single nucleus, which is localized in the central part of the cells and contained a moderate level of condensed chromatin. Transmission electron microscopic (TEM) investigations demonstrated that morphogenesis of cells of aerial and submerged mycelium pass an uniformity and consists in increasing of vacuolization level, development of a single, giant mitochondrion, synthesis of various types of storage compounds, component of endomembrane system and different types of extracellular substances. A correlation was observed between the topography of sporulation and the ability of external lipophilic substance production.
Key words: comparative analysis, hyphal cells, in vitro, Scedosporium apiospermum, strains, ultrastructure
Контактное лицо: Степанова Амалия Аркадьевна: тел. (812) 303--51-40
INTRODUCTION
Previously the peculiarity of conidial morphogenesis and septal pore apparatus ultrastructure on example of several Scedosporium / Pseudallescheria species was investigated under TEM [1], [Campbell C.K., Smith M.D. // Mycopathologia. - 1982. - Vol. 78; Salkin I.F., et al. // J. Clin. Microbiol. - 1988. - Vol. 26; Dykstra M, et al // Mycologia. - 1989. - Vol. 86; Huang H.J., et al// J. Tongii Med. Univ. - 1990. - Vol. 10]. Data about the cytological aspects of hyphal cell morphogenesis of these species on example of different strains culture in the literature were absent. Thus, the goal of the present work was the comparative ultrastructural analysis of the pattern of hyphal cells morphogenesis on the example of two strains of in vitro growing cells of S. apiospermum.
MATERIAL AND METHODS
Two strains of S. apiospermum from Russian Collection of Pathogenic Fungi, Kashkin Research Institute of Medical Mycology, St. Petersburg, Russia (PCPF-1491/1057, PCPF-1490/712,) were studied. The origin of the strains was a follows: PCPF-1491/1057 was isolated from sputum of a cystic fibrosis patient, PCPF-1490/712 - from washings of accessory sinuses of nose of a patient with sinusitis. Species identification was verified by rDNA ITS sequencing. The strains were cultured on Potato Dextrose Agar (PDA) at 28 °C. For scanning electron microscopy (SEM) the part of fungal colonies with nutrient medium after 7 and 20 days of growing were transferred in 3% glutaraldehyde (on 0,1 M cacodylate buffer) for 3 hours, post-fixed overnight in 1% osmium tetroxide in the same buffer, dehydrated by ethanol series (30°^70°), critical-point dried (HCP-2) for 15 min, coated with gold and observed in JSM 35 (JEOL, Tokyo, Japan). For TEM blocks of nutrient medium with different parts of fungal colonies were fixed during the 3 h in 3% glutaraldehyde and post-fixed for 10 h in 1% osmium tetroxide. Then samples were treated according the standard method [Stepanova A.A., Sinitskaya I.A. // Problems in medical mycology. - 2003. - Vol. 5]. Prior to TEM observation, light microscopic investigations of semi-thin epoxy sections (3-5 ^m) were performed on Pyramitome 1180 (LKB, Bromma, Sweden) with glass knives. Epoxy section were stained with toluidine blue and investigated under the light microscope Leica LB2 (Leica Microsystem Inc, Germany). Ultrathin sections were cut with an Ultratome V 2088 (LKB, Bromma, Sweden), stained with uranyl acetate and lead citrate and examined under a JEM-100 CX II transmission electron microscope (Jeol, Tokyo, Japan).
RESULTS AND DISCUSSION
Differentiation of vegetative mycelium cells within the colonies of studied S. apiospermum strains passed in the centripetal direction, and the processes of senescence and death - in the centrifugal, what was typical for filamentous fungi growing in vitro [2-6], [Stepanova A.A., Sinitskaya I.A. // Problems in medical mycology. - 2003. - Vol. 5; Stepanova A.A. u dp. // Problems in medical mycology. -2004. - Vol. 6].
Under SEM the 20 days cultures of investigated strains differ one from another by the topography of sporulation places. The moderate number of conidia were typical for upper part of aerial mycelium of strain culture PCPF-1491/1057 (Fig. 1 a), but in the surfaces of another strain
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(PCPF-490/712) - they presented very rare or absent (Fig. 1 b). As it was visible under SEM, the hyphal cells in upper part of colony have porous orientation (Fig. 1 a, b).
Longitudinal semi-thin epoxide section through the mature part of colony (strain PCPF-1491/1057) demonstrated the presence of two layers in composition of aerial mycelium (Fig. 1 c, e, layers 1, 2). The hyphal cells in lower (Fig. 1, layer 2) part of mycelium localized more frequently and have dark coloring. The submerged mycelium was also composed of two layers: more wide upper (Fig. 1, layer 3) and thin lower (Fig. 1 c, e, layer 3 a) with more tightly oriented hyphal cells. In the colony of strain PCPF-1491/1057 conidia were concentrated in aerial mycelium (Fig. 1 c, f) and absent in the submerged one. On semi-thin longitudinal sections of another strain colony (PCPF-490/712) we revealed another uncommon situation: sporulation in aerial mycelium practically absent and in general conidia were concentrated in submerged mycelium (Fig. 1 e, d, table). The quantity of conidia increased in centripetal direction.
Diameter of the mature hyphal cells of aerial and submerged mycelium in 7 days cultures of S. apiospermum varied from 3,0 to 4,0 ^m. The cells of aerial and submerged mycelium contain one ellipsoidal (1,2 x 2,0 ^m) nucleus (Fig. 1 g, h, i, Fig. 2 a-d, Fig. 3 a-d, table) with irregular envelope. The nucleus was localized in central part of cell, occupied all its lumen and contained low level of randomly distributed condensed chromatin (chromocentric nucleus). Nucleolus was large (0,5 ^m), spherical (Fig. 1 l), localized near nuclear envelope and composed of granular and fibrillar components, the first component dominated. The topography, number, size and level of chromatization of inter-phase nucleus in hyphal cells of both strains on this and in more advanced developmental stages (for exception the stage of senescence) were constant.
According to TEM observation morphogenesis of the cells of aerial and submerged mycelium in analyzed strains of S. apiospermum passed uniformly, what was not typical for in vitro growing cells of Aspergillus [2-4], [Stepanova А.А., Sinitskaya I^. // Problems in medical mycology. -2003. - Vol. 5; Stepanova А.А. u др. // Problems in medical mycology. - 2004. - Vol. 6]. For comparison, the pattern of in vitro development of hyphal cells of the species from genus Trichophyton [5, 6] also passed uniformly on hyphal level similar with the analyzed strains S. apiospermum, but with specific ultrastructural peculiarity, typical for dermatomycetes.
In the young growing cells of mycelium vacuoles were small, localized uniformly in cytosol, with the light content or thin-fibrillar material (Fig. 1 h).
Mitochondria were distributed in the periphery of cells and, as a rule, localized near cell wall (Fig. 1 h, i, Fig. 3 a). Their number on median sections of cells varied from 5 to 7. They were single or in small groups, spherical (0,6 ^m) or ellipsoidal (0,5 x 0,6 ^m) and often localized in tight contact. The matrix of this component was darker, in comparison with electron density of cytosol.
The components of endomembrane system were good developed. The two types of cisterns of endoplasmic reticulum (ER) were revealed: granular (Fig. 1 o) and smooth (Fig. 1 n, Fig. 2 b). The granular ER occurred in moderate number and was presented with long, straight or slightly curved cisterns, which were localized in internal part of cell or near cell wall. We revealed much curved and
tightly oriented tubular smooth ER in the content of hyphal cells of second layer of cultures in both strains. The tubules formed the large (1,0-1,2 ^m) spherical single aggregations and closely related with the cell wall (Fig. 1 n). Rare single horseshoe-shaped Golgi cisterns (Fig. 1 m) were found in this developmental stage. Small number (from 3 to 6 on median cell section) single or in small groups (Fig. 1 n) light secretory vesicles (60-70 nm) with thin-fibrillar content were distributed in cytosol. Several number (from 1 to 3 on median section) of large (0,3-0,4 ^m) microbodies with thin-fibrillar content were often revealed near cell wall (Fig. 1 i, o).
Fig. 1. SEM (a, b), light- (c-f) and TEM (g-o) micrographs of the hyphal cells of S. apiospermum growing in vitro strains. a, b, g, l, n - cells of aerial mycelium, h - j, k, m, o - cells of submerged mycelium. a, c, f - j, m, o - PKnrF 1491/1057; b, d, e, k, l, n -PKnrF 490/712. Explanation for this and another figures: C - conidia; EM - extracellular matrix; G - rosettes of a - glycogen, GC - Golgi cistern; GER - granular endoplasmic reticulum; HC - hyphal cells; LI - lipid inclusion; LS - lipophilic substance; M - mitochondrium(a); Mb - microbody; Me - medium; N - nucleus; Nu - nucleolus; PG - protein globule, PfG - polyphosphate granules; S - septum; SER - smooth endoplasmic reticulum; Sl - slime; V - vacuole; Vs - vesicles. Scale: a, b - 10 |m, c, e - x 400, d,
f - x 1000, g, h, k - 1 |m, i, l, n, o - 0,5 |m, j, m - 0,2 |m.
Fig. 2. Ultrastructure of S. apiospermum hyphal cells: d-h, l, n - cells of aerial mycelium; a-c, k - cells of submerged mycelium; a, c, d, e, k - PKnrF 1491/1057; b, f-j - PKnrF 490/712. Scale: a - 0,25 |m; b, d, i, k - 1 |m; c, h, j - 2 |m; e - 4 |m; f - 5 |m; g - 15 |m.
Fig. 3. Diagram showing the structure of growing (a, b), mature (c) and senescent cells of aerial and submerged mycelium in cultures of S. apiospermum strains. The extracellular matrix, micro-bodies and vesicles not demonstrated on diagram
Table
The main cytological characteristics of in vitro growing hyphal cells of S. apiospermum
Strains, PKnrF The area of sporulation in mycelium The number of interphase nucleus Diameter of interphase, nucleus, pm The level of chomatization The contour of nuclear envelope The presence of mitochondrial reticulum in aerial and submerged mycelium The types of storage substances in mature cells The presence ofGERand SER Presence of extracellular lipophylic substances The presence of extracellular slime The presence of micro-bodies
1491/1057 Aerial 1 2,0 x 1,2 Low Irregular ++ G, GV, LI, PG ++ + + +
490/712 Submerged 1 2,0 x 1,2 Low Irregular ++ G, GV, LI, PG ++ + + +
Notice: G - rosettes of a - glycogen; GER - granular endoplasmic reticulum; GV - protein granule in vacuole; LI - lipid inclusion; PG polyphosphate granules.
The moderate electron density cytosol was rich with free ribosomes. Plasma membrane was three-layered, closely contacted with the electron-transparent thin (0,05 ^m) light cell wall (Fig. 1 m, arrow). Detailed data about the ultrastructure of lateral cell wall, septum and septal pore apparatus of investigated S. apiospermum strains were presented previously [1].
During the subsequent stage of hyphal cells development the vacuoles were increased in sizes. Synthesis of storage substances started. In the hyphal cells of aerial and submerged mycelium the four types of storage substances were revealed: polyphosphate granules, lipid inclusions, rosettes of a-glycogen and protein globules (Table). The small (0,01-0,02 ^m) spherical dark or with central light area polyphosphate granules were observed in the nucleoplasm, cytosol, vacuolar content (Fig. 2 b, c, Fig. 3 c,) and in mitochondrial matrix. Lipid inclusions uniformly distributed on cell section, variable in size (0,30,6 ^m, Fig. 1 i, o, Fig. 3 a, b). As a rule, lipid inclusions were surrounded with rosettes of a-glycogen (Fig. 2 a). The rosettes of glycogen revealed on sections as spherical light zones (Fig. 2 a, b, d). The light color of last, perhaps, may be result of its elution during sample preparation for
TEM. Large (0,5-0,6 ^m) single dark protein globule were localized in vacuolar content near tonoplast (Fig. 2 b, c, Fig. 3 b).
The numbers of mitochondria were increased to 11-18 on median section of hyphal cell of aerial and submerged mycelium. Often the long (3,0-4,0 ^m) straight (Fig. 1 j) or curved (Fig. 1 k) profiles of this organelles were revealed. They were localized near cell wall and in space between the last and nucleus (Fig. 1 h, i). The increasing number of mitochondria and peculiarity of its morphology on serial sections provide evidence of development the one giant organelle, so called «mitochondrial reticulum» (Table). It is a first report according the presence of giant mitochondria in the cells of aerial mycelium of pathogenic filamentous fungi. As a rule, formation of giant organelle and its orientation near nucleus was revealed for in vitro growing cells of submerged mycelium of several species from genus Aspergillus [2-4], [Stepanova А.А., Sinitskaya I.A. // Problems in medical mycology. - 2003. - Vol. 5; Stepanova A.A. u др. // Problems in medical mycology. - 2004. - Vol. 6], in vitro germinated conidia [7] and hyphal cells in the human lung [8] of this species. This type of organelle was also specific for growing in vivo (mouse brain after 7
days of beginning experiments) strong virulent strains of Cryptococcus neoformans [9] and budding cells of C. albidus [10]. It is important to note, that during the morphogenesis of the cells of submerged mycelium of T. rubrum [5] and T. violaceum [6] the number of mitochondria also increased, but the giant organelle did not develop.
Under TEM the cells of second layer of aerial mycelium of both strains produced different amounts of homogenous median electron density external substance. Thus, in the mycelium of the strain PCPF-490/712 this external fungal metabolite produce irregular clustering with different thickness around the hyphal cells (Fig. 2 e). Hyphal cells of the aerial mycelium in another strain (PCPF-1491/1057) produced the same external product in more large amount (Fig. 2 f, 7 days young part of colony and 2 g - mature part) which often fully filled the inter hyphal space. We suggest that this external substance according its morphology has lipophilic nature. Also we revealed the special rare type of tubular smooth ER typical for the cells of second layer of aerial mycelium of both strains, which was found in plants [Васильев А.Е. Функциональная морфология секреторных клеток растений. - Л., 1977] and fungal secretory cells, like cystidium in certain species of agaricalean fungi [Stepanova A.A., Vasilyev A.E. Ultrastructural bases of mushroom morphogenesis. - Ashghabat, 1994] typical participation in the synthesis of lipophilic substances. We suppose, that this lipophilic substance contain pigment, which presence in extracellular space of aerial mycelium determinates its color.
The cells of aerial mycelium significantly differ according the amount of synthesized extracellular metabolites. In cultures of both strains the cells of submerged mycelium developed wide so-called «extracellular matrix» [1], but the cells of aerial mycelium - only slime and lipophilic substance.
Another type of external metabolite, which is also typical for aerial mycelium of both strains, was external slime, which formed the variable in size and form deposits of thin-fibrillar material on the hyphal cell surfaces (Fig. 2 d, h, i). Very often slime was present between adjacent hyphae (Fig. 2 h) and played role of adhesive material. The cells of mycelium of the strain PCPF-490/712 produced the slime more abundantly, so that under SEM we may see its aggregations in cords (Fig. 1 b, arrows). The production of slime was typical for the cultures of the Scedos-porium species [11] in which it was in the slimy conidial heads.
The large central vacuole was developed (Fig. 2 e, j) when the process of hyphal cell senescence started. The number of storage substance and components of cytosol was significantly reduced in this stage. In one cells the first destructive changes started in mitochondria (Fig. 2 k), but in other - in nucleus. The final stages of senescence continue in succession, which was revealed for another species of filamentous fungi [5, 12, 13], [Stepanova A.A., Vasilyev A.E. Ultrastructural bases of mushroom morphogenesis. -Ashghabat, 1994].
The hyphal cells of aerial and submerged mycelium of investigated S. apiospermum strains cultures demonstrated the similarity in the pattern of its morphogenesis, which consist in increasing the level of vacuolization, development one giant mitochondrion, synthesis of various types of storage substance, component of endomembrane system and extracellular substances. The presence of extracellular matrix, extracellular slime and lipophilic substance are
very important for biofilm formation in vitro and in vivo, what was important for protective role and, perhaps, some from this extracellular metabolites may play role as food nutrition for morphogenetic processes of new developed cells.
The ability to accumulated storage resources gives our objects «carte blanche» for a high level of sporulation. In mature mycelial cells of S. apiospermum we find practically all types of storage substances, which are typical for fungi. Its variety and ability possibly consider as indicator of its higher and diverse physiological activity and ability to decompose components of nutrient medium. More abundant number of storage compounds was present in cells of second layer of strain colony PCPF-1491/1057 and in the submerged part of another one (PCPF-490/712) directly correlated with the process of sporulation in these zones. It was apparent that the presence of one giant mitochondrion determinates the higher physiological activity of the hyphal cells and may be considered as «cytological indicator» of the level of its virulence and pathogenic properties. For comparison, the giant mitochondrion was developed in the cells of strong virulent strains of C. neoformans, when they infected the mouse brain [9]. Another cell component, which may be indicator of its higher functional activity
- were microbodies. For example, the last components appear in the cells of C. neoformans [9], which infected mouse brain, but absent in cultural form of this fungi.
The hyphal cells of S. apiospermum posses one nucleus. Gilgado F. with co-authors [14] demonstrated that preponderantly the Scedosporium species were heterothallic. The interphase nucleus in the intact cells from different part of mycelium (aerial, submerged) characterized by existence of the small number of condensed chromatin and may be interpreted as chromocentric type [Nagl W. // Protoplasma. - 1979. - Vol. 100]. The last type dominate in fungi [Stepanova A.A., Vasilyev A.E. Ultrastructural bases of mushroom morphogenesis. - Ashghabat, 1994; Kamaletdinova F.I., Vasilyev A.E. Cytology of Discomycetes.
- Alma-Ata, 1982], what correlated with lower amount of DNA in its content. The active condition of nucleolus in growing cells of mycelium correlated with synthesis of the extracellular metabolites, cell wall, cytosol, free ribosomes, storage substance, mitochondrial and another cell components proliferation in this stage.
As it was obvious on the example of a strain PCPF-1491/1057, the abundant presence of lipophilic external metabolite in lower part of aerial mycelium limited sporulation in this area. Perhaps, correlation between the pattern of sporulation and physiological ability of compared strains to produce the secondary metabolites was present.
Thus, mycelium of in vitro growing S. apiospermum culture present the higher organized space construction and composed with layers, which differs according functional activity and its specialization. It may be actually to investigate, how may be change the hyphal cell «role» and its ultrastructural «portrait» during in vitro^in vivo transformation.
RESUME
1. Under SEM the investigated strains of S. apiospermum differ one from another by the ability of the sporulation. Light-microscopic investigations demonstrated differences in pattern of sporulation among investigated strains: in colony of strain PCPF-1491/1057 conidia developed in
aerial mycelium, but in PCPF-490/712 - sporulation occur in the lower part of submerged mycelium, when the number of mature conidia increased in centripetal direction. Correlation between the topography of the sporulation and physiological ability of compared strains in production of external lipophilic substance was revealed.
2. The cells of the mycelium of two S. apiospermum strains contain one chromocentric nucleus, which is localized in the central part.
3. TEM investigations demonstrated that morphogenesis of the cells of aerial and submerged mycelium in S.
apiospermum strains passed uniformity and consisted in increasing the level of vacuolization, development of one giant mitochondrion, synthesis of various types of storage substances, proliferation of the components of endomembrane system and producing the different types and amount of the external substances.
4. The cells of aerial mycelium in cultures of S. apiospermum strains, in comparison with submerged, produce external slime and lipophilic substance. The wide extracellular matrix was typical only for hyphal cells of submerged mycelium.
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Поступила в редакцию журнала 30.01.2017
Рецензент: Л.Е. Сергеева
