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Light-microscopic morphology and ultrastructure of Polychaos annulatum (Penard, 1902) Smirnov et Goodkov, 1998 (Amoebozoa, Tubulinea, Euamo-ebida), re-isolated from the surroundings of St. Petersburg (Russia)
Oksana Kamyshatskaya12, Yelisei Mesentsev1, Ludmila Chistyakova2 and Alexey Smirnov1
1 Department of Invertebrate Zoology, Faculty of Biology, St. Petersburg State University, Universitetskaya nab. 7/9, 199034 St. Petersburg, Russia
2 Core Facility Center “Culturing of microorganisms”, Research park of St. Petersburg State Univeristy, St. Petersburg State University, Botanicheskaya St., 17A, 198504, Peterhof, St. Petersburg, Russia
| Submitted December 15, 2018 | Accepted January 21, 2019 |
Summary
We isolated the species Polychaos annulatum (Penard, 1902) Smirnov et Goodkov, 1998 from a freshwater habitat in the surrounding of Saint-Petersburg. The previous re-isolation of this species took place in 1998; at that time the studies of its light-microscopic morphology were limited with the phase contrast optics, and the electron-microscopic data were obtained using the traditional glutaraldehyde fixation, preceded with prefixation and followed by postfixation with osmium tetroxide. In the present paper, we provide modern DIC images of P. annulatum. Using the fixation protocol that includes a mixture of the glutaraldehyde and paraformaldehyde we were able to obtain better fixation quality for this species. We provide some novel details of its locomotive morphology, nuclear morphology, and ultrastructure. The present finding evidence that P. annulatum is a widely distributed species that could be isolated from a variety of freshwater habitats.
Key words: amoebae, Polychaos, ultrastructure, Amoebozoa
Introduction
The largest species of naked lobose amoebae (gymnamoebae) — members of the family Amoe-bidae Ehrenberg, 1838 sensu Page (1987), belonging to the order Euamoebida Lep§i, 1960 sensu Smirnov et al. (2011) are often called “proteus-type” organisms (named so after their similarity with
Amoeba proteus). These amoebae usually have a relatively large size, exceeding hundred of microns, they produce broad, thick pseudopodia with smooth outlines (lobopodia). Locomotive cells show clear differentiation of the cytoplasm into the granuloplasm and the hyaloplasm and usually have the nucleus of granular type or the nucleus with a complex arrangement ofthe nucleolar material, but
doi:10.21685/1680-0826-2019-13-1-4 © 2019 The Author(s)
Protistology © 2019 Protozoological Society Affiliated with RAS
never — a vesicular nucleus (Page, 1986, 1988, 1991; Smirnov, 2008, 2012). The species identification among these amoebae, despite their large size is often difficult and is based on the light-microscopic characters, size data and details of their ultrastructure (Page, 1986, 1988; Siemensma and Page, 1986; Page and Baldock, 1980; Page and Robson, 1983; Page and Kalinina, 1984; Smirnov and Goodkov, 1997; 1998; Goodkov et al., 1999; Smirnov and Brown, 2004). The amount of molecular data on this group of amoebae remains surprisingly limited. The SSU 18S rRNA gene of four species belonging to the genera Amoeba and Chaos was sequenced in early 2000th (Bolivar et al., 2001; Fahrni et al., 2003) and one species belonging to the genus Deuteramoeba — recently (Kamyshatskaya et al., 2017). A transcriptome of the strain identified as Amoeba proteus was sequenced by Kang et al. (2017). Hence, among the seven genera comprising the family Amoebidae, molecular data are available for three genera only, which is an unusual situation nowadays in amoebae studies. So we still have to rely on light microscopy and electron microscopy in the identification of most of the strains of amoebae belonging to this family.
The primary reason for that limited amount of data is the lack of strains. Many species are known only from the initial description, while type cultures were lost (or never existed). Despite they are widely known, it is hard to find these organisms in the environment, and every finding requires careful study in order to associate the strain with already known species or describe it as a new one (Smirnov and Goodkov, 1998; Smirnov, 1999, 2002; Michel and Smirnov, 1999; Kudryavtsev et al., 2004).
An amoeba species Polychaos annulatum was described by Penard (1902) as “Amoeba annulata” and re-isolated by Smirnov and Goodkov (1998). This species has a very peculiar nuclear structure, so it was reliably identified and studied using phase-contrast microscopy and transmission electron microscopy. However, nowadays technical facilities allow for obtaining much better images, first of all — differential interference contrast (DIC) images, which are getting standard in amoebae studies. In the present study, we isolated an amoeba strain, identical to our year 1998 strain in light-microscopic and ultrastructural characters, and provide modern images of this organism. Using combined glutaraldehyde — paraformaldehyde fixation for electron-microscopic studies, we obtained better transmission electron microscopic
images of this strain, showing novel characters of its ultrastructure.
Material and methods
The studied strain of Polychaos annulatum was isolated in the surroundings ofSt. Petersburg, Push-kinsky district, Aleksandrovskiy park, “Detskiy” Pond, grid reference 59.721086, 30.389882. Cells were maintained in 90 mm plastic Petri dishes in PJ medium (Prescott and James, 1955) with two rice grains per dish. The culture contained bacteria, ciliates, and other eukaryotes, serving as food objects. Attempts to clone and/or purify culture failed, so mixed cultures were maintained and used. We were confident that all amoebae in these cultures belong to the same species; this was checked many times with the light-microscopic examination of these very characteristic cells.
Live cells were studied, measured and photographed on object slides (wet mounts in PJ medium) using a Leica DM2500 microscope equipped with DIC. Special attention was paid not to press the cell with the coverslip as described by Mesentsev and Smirnov (2019).
For electron microscopy, cells were collected individually with the tapering glass Pasteur pipette and fixed in glass wells using the mixture of 2.5% glutaraldehyde and 1.6% formaldehyde prepared in 0.1M phosphate buffer (PH 7.4) for 1.5 hours under room temperature (rt). Further cells were washed for 3x5 min in the same buffer (rt) and postfixed with 1% osmium tetroxide (final concentration) for one hour at +4 °C. Amoebae were washed in the same buffer 3x10 min prior to dehydration (rt) and embedded in 2% LMP agarose (Amresco) before dehydration. Small pieces of agarose (about 1 mm3) containing amoebae were cut out and dehydrated in graded ethanol series followed by 100% acetone. Blocks were embedded in SPI-PON 812 resin (SPI, an analog of Epon 812) according to the manufacturers instructions. Sections were cut using a Leica Ultracut 7 ultramicrotome and double-stained using 2% aqueous solution of uranyl acetate and Reynolds’ lead citrate.
Results
Light microscopy
Locomotive cells varied in shape from palmate, polytactic to oblong, orthotactic or even monotactic (Fig. 1 E-H). Normally, when a cell started the
Fig. 1. Light-microscopic images of Polychaos annulatum. A — Floating cell; B-C — cells soon after settling on the glass, still showing remnants of the pseudopodia of the floating form; D — stationary, rounded cell with numerous peripheral hyaline lobs; E — subsequent stages of the locomotion of the same cell — from palmate, expanded, with fascisculate uroid, slowly moving cell to the one with pronounced leading pseudopodium (the background of the image is natural and was not edited, this is just a mosaic of original photographs); F-H — another cell in locomotion (the cell subsequently changes shape from orthotactic to almost monotactic, with the pronounced hyaline cap). Abbreviations: n — nucleus, cv — contractile vacuole, u — uroid, h — hyaloplasm. Scale bars: A-E — 50 pm, F-H — 20 pm.
movement from stationary form, it became pronouncedly polytactic, palmate and had fasciculate uroidal lobes (Fig. 1 E). The cell could move like this for quite a time, but with the increment of the rate of locomotion it started to elongate and became orthotactic or, in the most rapidly moving cells, monotactic (Fig. 1 F-H). In rapidly moving, orthotactic cells the uroid was morulate. Cells had pronounced frontal hyaline cap, the contractile vacuole was often located in the uroid (Fig. 1 F), while the nucleus was always located in the middle part of the cell.
The length of the locomotive form was 60—188 pm (average 126 pm, n=88), breadth was 48—160 pm (average 86 pm, n=88). Length/Breath ration (L/B) was 0.85—3.4 (average 1.67). The maximal length and minimal breadth, as well as maximal L/B ratio, reflect those of the elongate, orthotactic forms, while the dimensions of palmate forms were close to the average ones and their L/B ratio often was close to 1.0 or only slightly exceeded it.
Stationary cells were rounded, with numerous peripheral hyaline lobes (Fig. 1 D). Floating cells had several uneven thick tapering pseudopodia, radiating from the central cell mass. Pseudopodia contained both the granuloplasm and the hyaloplasm (Fig. 1 A-B), when cell settled down it kept remnants of these pseudopodia for some time and slowly retracted them when started to move (Fig. 1 C).
The nucleus had a complex structure of the nucleolus. In most ofthe cells, it resembled a hollow perforated sphere, located inside the nucleus at some distance from the nuclear envelope (Fig. 2 A, C-E). The nucleolar material was arranged within the walls of this sphere, varying in thickness from a very fine, hardly distinguishable layer to crescent-shape lobes ofvariable thickness. Inside this sphere, it was possible to distinguish a loose body ofgranular material and a spherical central body, which had a central lacuna (Fig. 2 A, B). The size of the nucleus in maximal dimension was 11—22 pm (average 16.7 pm, n=23), the size of the nucleolus, measured according to the external border of the sphere of the nucleolar material was 10-16 pm (average 13.8 pm, n=23).
In older cells, the nucleolar material tended to concentrate on one side of this sphere, making it asymmetrical (Fig. 2 B), or even forming a hemispherical body, in the latter case the central body was eccentrically located (Fig. 2 F). In one cell we have observed a horseshoe-like nucleolus, it had two central bodies symmetrically located on both sides of the nucleus (Fig. 2 G-H).
The granuloplasm was filled with the small opaque granules; it contained food vacuoles, trun-
cate bipyramidal crystals (Fig. 2 I) and rounded bodies enclosed in vacuoles (Fig. 2 J).
Transmission electron microscopy
The nucleus in our sections was spherical or nearly spherical. The nucleolar material in sections was concentrated in a ring varying in thickness from almost invisible layer to thick, crescentshaped bodies (Fig. 3 A-D). The material forming these bodies was not entirely homogeneous, it was possible to distinguish denser areas consisting of better-condensed material (Fig. 3A, G). Inside the material forming the lobes it was possible to see small lacunas (Fig. 3 D). The karyoplasm outside the nucleolus (“external karyoplasm” sensu Smirnov and Goodkov, 1998) looked denser and more finely granulated rather than that inside it (“internal karyoplasm”) (Fig. 3 E-G). In the central area of the nucleus, it was possible to distinguish a spherical body formed by the small patches of the nucleolar material. The karyoplasm inside this body appeared to be denser than outside (Fig. 3 A-B). A patch of nucleolar material probably representing a tangential section of the central body is visible in Fig. 3 D. This structure of the nucleus perfectly correlates with the pattern seen by LM (Fig. 1 A). Nuclear pore complexes were well visible in tangential sections of the nucleus (Fig. 3 F). They had a typical structure, showing the peripheral spoke ring assembly and the central plug. Both tangential and sagittal sections showed no evident fibrous lamina inside the nucleus, however, arrangements of microfilaments under the nuclear membrane and in the external karyoplasm were frequently seen (Fig. 3 E, G).
The cell surface was covered with a layer of filamentous glycocalyx. Very fine filaments radiated from the amorphous basal layer (Fig. 4 A-B). The length of these filaments reached 250 nm. The mitochondria were spherical, ovoid or (rarely) elongate in sections (Fig. 4 C, F). They had cristae of the tubular type and an electron-dense matrix. Dictyosomes of the Golgi complex were not numerous and were represented by stacks, consisting of
3-8 cisternae (Fig. 4 C). No MTOCs were seen in the surrounding of the dictyosomes and no evident microtubules were found in the cytoplasm. Bacteria, apparently located freely in the cytoplasm were seen in all cells (Fig. 4 D). Food vacuoles were usually rather numerous and contained bacteria and/or eukaryotic food (Fig. 4 E). A remarkable thing, found in all studied cells, were rounded bodies bounded by a multilayered envelope (Fig. 4 F-H). This envelope was ca. 7 nm in thickness, double layered and resembled the cell membrane.
Fig. 2. Light-microscopic images of the nucleus and cytoplasmic inclusions of Polychaos annulatum. A — Overall view of the nucleus, showing its typical appearance, seen in the majority of cells — nuclear membrane showing no evidence of the nuclear lamina, the inner perforated sphere of the nucleolar material, considered as a complex nucleolus. Inside the nucleolus, there is a “cloud” of granular material and a small “central body with lacuna”; B — another nucleus, showing better-elaborated nucleolus with thick walls. There are holes (and/ or lacunas) inside the thicker part of the nucleolus (arrowed). The central body in this nucleus is larger and has large lacuna inside; C-E — three optical sections through the same nucleus; F — the nucleus of a cell, probably maintained long in culture. The nucleolar material formed a “hemisphere” in one part of the nucleus, it contains holes and/or lacunas (arrowed). The central body is eccentrically located; G-H — the nucleus with horseshoelike nucleolus, this nucleus contains two central bodies, symmetrically located on both sides of the nucleolus;
I — truncate bipyramidal crystals in the cytoplasm; J — spherical bodies enclosed in vacuoles. Abbreviations: n — nucleus, nm — nuclear membrane, nu — nucleolus, g — “cloud” of granular material inside the nucleus, cb — central body, rb — rounded bodies enclosed in the vacuoles, cr — crystals. Scale bars: A-H — 10 pm, I-J — 1 pm.
The outmost layer of this envelope was significantly seen tubular structures, apparently radiating from denser, looking like a fine black line in our TEM the center of these bodies (Fig. 4 F). The nature of images (Fig. 4H). The inner content of these bodies these bodies remains not yet clear.
was finely granulated, in some sections we have
Fig. 3. Ultrastructure of the nucleus of Polychaos annulatum. A-B — Cross-sections through the same nucleus, showing the typical appearance of the nucleolar material, forming two crescent-shaped bodies in section. Dense patches located inside the nucleolar material are arrowed. The structure of this nucleus corresponds to the LM in Fig 2A; C -D — two sections through the nucleus with almost semispherical nucleolus, corresponding in LM to the Fig. 2B. The nucleolus contains lacunas (white arrow), the dense structure which probably is a tangential section of the central body is visible in D; E — the fragment of the nucleus, showing nuclear membrane with no evidence of the nuclear lamina, filament in the external karyoplasm and a fragment of the nucleolus; F — nuclear membrane showing nuclear pores; G — the fragment of the nucleus showing segregation of the external karyoplasm, located outside the nucleolus and the internal karyoplasm, located inside, with the filamentous layer. Abbreviations: nm — nuclear membrane, nu — nucleolus, g — “cloud” of granular material inside the nucleus, cb — central body, f — filaments in the space between the nucleolus and the nuclear membrane, np — nuclear pores, ec — external karyoplasm containing microfilaments, ic — internal karyoplasm, located inside the nucleolus. Scale bars: A-D — 2 pm, E-F — 500 nm, G — 1 pm.
Fig. 4. General ultrastructure of Polychaos annulatum. A-B — Filamentous cell coat. Filaments are based on the tiny amorphous layer (arrowed) located on the surface of the cell membrane; C — mitochondria with dense matrix and dictyosomes of the Golgi complex; D — bacteria located freely in the cytoplasm; E — food vacuole, containing eukaryotic food object: F-H — bounded bodies with homogeneous inner content. They are located freely in the cytoplasm; radial filamentous structures may be seen in some of them (arrow in F). These bodies are coated with the electron-dense multilayered envelope (arrowed in H). There is always an empty halo between the body and the surrounding cytoplasm (well-visible in H). Abbreviations: f — filaments, m — mitochondria, g — dictyosome of the Golgi complex, b — bacteria, bb — bounded body, fo — food object, fv — food vacuole. Scale bars: 200 nm in A, B, H — 200 nm, C-D — 500 nm, F-G — 1 pm.
Fig. 5. A-C — Nuclei in haematoxylin-stained preparations of Polychaos annulatum made in 1998 by A. Smirnov. Abbreviations: n — nucleus, cb — central body, nl — nucleolus. Scale bar : 10 pm.
Discussion
The present isolate was identified as Polychaos annulatum basing on the locomotive morphology and a very characteristic structure of the cell coat and the nucleus, identical to that described in 1998 for our Valamo Island isolate of P. annulatum (Smirnov and Goodkov, 1998). The present strain shows absolutely the same inner sphere of the nucleolar material, segregation of the karyoplasm to the inner one, located inside the nucleolus and a denser outer one, located outside the nucleolus and filled with numerous filaments. It shows the same characteristic arrangements of the nucleolar material — two crescent-shaped lobes or one larger hemisphere in older cultures. The only difference against the description made in 1998 is the presence of the central body in the nucleus. However, we reexamined permanent stained preparations made in 1999 from the same strain described by Smirnov and Goodkov (1998) and found that at that time we misinterpreted the central body, considering it as a top view on one of the peripheral lobes. The central body is visible in all nuclei in these preparations (Fig. 5 A-C). The surface coat shows the same pattern of filaments. The present isolate has the same characteristic truncate bipyramidal crystals with an average size of about 3 pm, as the Valamo island isolate.
The size of the nucleus in our present isolate (1122 pm) is generally in the same range as indicated for Valamo Island isolate (16-25 pm). The nucleus of the smallest size (11 pm) was the single exception in our present observations. There is a considerable difference in the cell size, which originated because in 1999 we observed mostly orthotactic and monotactic locomotive forms in our cultures and measured them as the fasted moving and this best representing
the locomotive morphology. Hence we had the length ranging from 220 to 325 pm and breadth 3543 pm with L/B about 9. In contrast, in the present isolate monotactic forms were rarely seen, and measured cells were mostly polytactic or palmate. The measurement of the palmate cells of Valamo island isolate done in the available photographic images made in 1998 gives approximately the same sizes (150-200 pm in length). Anyway, the present isolate is generally smaller than the Valamo Island one, but this difference is within the range of the size variability of amoebae species (Page, 1988; Smirnov et al., 2002, 2007). Basing on all this, we believe that we re-isolated the species Polychaos annulatum (Penard, 1902) Smirnov et Goodkov, 1998.
The present study is based on the observation of numerous (several hundred) cells, which is much more than we studied in 1998 (according to our records, ca. 30 cells were found and studied at that time). Non-surprisingly, the present study, performed using much better technical facilities, bring new findings. First of all, this is the structure of the nucleus, which besides the already described hollow sphere ofthe nucleolar material contains the arrangement of the nucleolar material in the centre of this sphere and the central body. A noticeable detail is the presence of membrane-bounded bodies of unclear nature, which were not seen in 1998.
Smirnov and Goodkov (1998) provided a detailed comparison of Polychaos annulatum with all similar amoebae species. Among them, the most similar is P. fasciculatum (Penard, 1902) Schaeffer, 1926. However, in 2000 and 2002 we studied the type strain of P. fasciculatum obtained from CCAP, as 1564/1 strain. These data indicate that the nucleus in P. fasciculatum clearly differs in structure. The nucleolus in this species either represents a ribbon, packed inside the nucleus (Page 1988) or several (2-4) compact bodies, often with large lacunas
inside, arrange as a ring inside the nucleus (Page and Baldock, 1980). Our observation fully confirmed these descriptions. Page and Baldock (1980) show the central body in the nucleus of P. fasciculatum, similar to that found in our species. However, in our studies of the same isolate, we never observed it, which may indicate that its presence may be optional. TEM data provided in this paper shows different nuclear structure — nucleolar bodies in P. fasciculatum are more rounded, they are larger and never form a structure like to crescent-shaped bodies, so typical for P. annulatum. The cell coat in P. fasciculatum appears to have a different organization.
Other species that may be considered as similar to those two is Metachaos gratum Schaeffer, 1926, which is larger than both these species. It possesses a similar nuclear structure but has a number of differences (Smirnov and Goodkov, 1998) and cannot be associated with the present strain.
Amoebae of the Polychaos fasciculatum-like species group all are similar to each other and it is hard to distinguish them. To the moment the most parsimonious solution would be to keep two properly described species with established type material — P.fasciculatum (Penard, 1902) Schaeffer, 1926 and Polychaos annulatum (Penard, 1902) Smirnov et Goodkov, 1998. We provide an emended diagnosis of P. annulatum to incorporate new data obtained in the present study. The studied strain is deposited with the culture collection ofthe Core facility Center “Culturing ofMicroorganisms”: ofSPSU under the number RCCMAm0456, which makes it available for further studies.
Polychaos annulatum (Penard, 1902) Smirnov et Goodkov, 1998, emend.
Monopodial, sometimes slightly clavate in continuous locomotion and polypodial, with many short pseudopodia (“palmate”) in slower movement. Bulbous uroid, separated from the rest of the body with a distinct neck in locomotive form and fasciculate uroid during non-directed movement and when started to move. Length in locomotion 220-325 qm, breadth 35-43 qm. Length/Breadth ratio (L/B) is about 9. Nucleolar material is arranged in a perforated hollow sphere at some distance from the nuclear envelope. No nuclear lamina. No cysts are known.
Observed habitats: Freshwater; reported from Switzerland, North-Western Russia (Valamo Island and surroundings of St.Petersburg), possibly, the Netherlands.
Type material: permanent stained preparations deposited with British Natural History Museum:
number 1995:9:6:11 (holotype) and 1995:9:6:12 (paratype) by Smirnov and Goodkov (1998).
Acknowledgements
Supported by RFBR 16-04-01454 grant (isolation and LM studies) and RSF 17-14-01391 grant (studies of the cell ultrastructure). The present study utilized equipment of the Core facility centers “Development of molecular and cell technologies” and “Culture Collection of microorganisms” of St. Petersburg State University.
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Address for correspondence: Oksana Kamyshatskaya. Department of Invertebrate Zoology, Faculty of Biology, St. Petersburg State University, Universitetskaya nab. 7/9, 199034 St. Petersburg, Russia; e-mail: oksana.kamyshatskaya@gmail.com.
