Protistology 13 (1), 26–35 (2019) Protistology

Light-microscopic morphology and ultrastructure of annulatum (Penard, 1902) Smirnov et Goodkov, 1998 (, , Euamo- ebida), re-isolated from the surroundings of St. Petersburg (Russia)

Oksana Kamyshatskaya1,2, Yelisei Mesentsev1, Ludmila Chistyakova2 and Alexey Smirnov1

1 Department of 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 proteus). These amoebae usually have a relatively large size, exceeding hundred of microns, The largest species of naked lobose amoebae they produce broad, thick with (gymnamoebae) – members of the family Amoe- smooth outlines (lobopodia). Locomotive cells bidae Ehrenberg, 1838 sensu Page (1987), belonging show clear differentiation of the into the to the order Euamoebida Lepşi, 1960 sensu Smirnov granuloplasm and the hyaloplasm and usually have et al. (2011) are often called “proteus-type” the nucleus of granular type or the nucleus with a (named so after their similarity with complex arrangement of the nucleolar material, but

doi:10.21685/1680-0826-2019-13-1-4 © 2019 The Author(s) Protistology © 2019 Protozoological Society Affiliated with RAS Protistology · 27 never – a vesicular nucleus (Page, 1986, 1988, 1991; images of this strain, showing novel characters of Smirnov, 2008, 2012). The species identification its ultrastructure. among these amoebae, despite their large size is often difficult and is based on the light-microscopic characters, size data and details of their ultrastruc- Material and methods ture (Page, 1986, 1988; Siemensma and Page, 1986; Page and Baldock, 1980; Page and Robson, 1983; The studied strain of Polychaos annulatum was Page and Kalinina, 1984; Smirnov and Goodkov, isolated in the surroundings of St. Petersburg, Push- 1997; 1998; Goodkov et al., 1999; Smirnov and kinsky district, Aleksandrovskiy park, “Detskiy” Brown, 2004). The amount of molecular data on Pond, grid reference 59.721086, 30.389882. Cells this group of amoebae remains surprisingly limited. were maintained in 90 mm plastic Petri dishes in PJ medium (Prescott and James, 1955) with two The SSU 18S rRNA gene of four species belonging rice grains per dish. The culture contained bacteria, to the genera and was sequenced Amoeba , and other , serving as food in early 2000th (Bolivar et al., 2001; Fahrni et objects. Attempts to clone and/or purify culture al., 2003) and one species belonging to the failed, so mixed cultures were maintained and used. Deuteramoeba – recently (Kamyshatskaya et al., We were confident that all amoebae in these cultures 2017). A transcriptome of the strain identified as belong to the same species; this was checked many was sequenced by Kang et al. (2017). times with the light-microscopic examination of Hence, among the seven genera comprising the these very characteristic cells. family , molecular data are available Live cells were studied, measured and photo- for three genera only, which is an unusual situation graphed on object slides (wet mounts in PJ medium) nowadays in amoebae studies. So we still have to using a Leica DM2500 microscope equipped with rely on light microscopy and electron microscopy in DIC. Special attention was paid not to press the cell the identification of most of the strains of amoebae with the coverslip as described by Mesentsev and belonging to this family. Smirnov (2019). For electron microscopy, cells were collected The primary reason for that limited amount of individually with the tapering glass Pasteur pipette data is the lack of strains. Many species are known and fixed in glass wells using the mixture of 2.5% only from the initial description, while type cultures glutaraldehyde and 1.6% formaldehyde prepared in were lost (or never existed). Despite they are widely 0.1M phosphate buffer (PH 7.4) for 1.5 hours under known, it is hard to find these organisms in the room temperature (rt). Further cells were washed environment, and every finding requires careful for 3×5 min in the same buffer (rt) and postfixed study in order to associate the strain with already with 1% osmium tetroxide (final concentration) known species or describe it as a new one (Smirnov for one hour at +4 °C. Amoebae were washed in and Goodkov, 1998; Smirnov, 1999, 2002; Michel the same buffer 3×10 min prior to dehydration and Smirnov, 1999; Kudryavtsev et al., 2004). (rt) and embedded in 2% LMP agarose (Amresco) An amoeba species Polychaos annulatum was before dehydration. Small pieces of agarose (about described by Penard (1902) as “Amoeba annulata” 1 mm3) containing amoebae were cut out and and re-isolated by Smirnov and Goodkov (1998). dehydrated in graded ethanol series followed by This species has a very peculiar nuclear structure, 100% acetone. Blocks were embedded in SPI-PON so it was reliably identified and studied using 812 resin (SPI, an analog of Epon 812) according to the manufacturer‘s instructions. Sections were phase-contrast microscopy and transmission cut using a Leica Ultracut 7 ultramicrotome and electron microscopy. However, nowadays technical double-stained using 2% aqueous solution of uranyl facilities allow for obtaining much better images, acetate and Reynolds’ lead citrate. first of all – differential interference contrast (DIC) images, which are getting standard in amoebae studies. In the present study, we isolated an amoeba Results strain, identical to our year 1998 strain in light- microscopic and ultrastructural characters, and LIGHT MICROSCOPY provide modern images of this . Using combined glutaraldehyde – paraformaldehyde Locomotive cells varied in shape from palmate, fixation for electron-microscopic studies, we polytactic to oblong, orthotactic or even monotactic obtained better transmission electron microscopic (Fig. 1 E-H). Normally, when a cell started the 28 · Oksana Kamyshatskaya, Yelisei Mesentsev et al.

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 , u – uroid, h – hyaloplasm. Scale bars: A-E – 50 µm, F-H – 20 µm. Protistology · 29 movement from stationary form, it became pro- cate bipyramidal crystals (Fig. 2 I) and rounded nouncedly polytactic, palmate and had fasciculate bodies enclosed in (Fig. 2 J). uroidal lobes (Fig. 1 E). The cell could move like this for quite a time, but with the increment of TRANSMISSION ELECTRON MICROSCOPY the rate of locomotion it started to elongate and became orthotactic or, in the most rapidly moving The nucleus in our sections was spherical or cells, monotactic (Fig. 1 F-H). In rapidly moving, nearly spherical. The nucleolar material in sections orthotactic cells the uroid was morulate. Cells had was concentrated in a ring varying in thickness pronounced frontal hyaline cap, the contractile from almost invisible layer to thick, crescent- vacuole was often located in the uroid (Fig. 1 F), shaped bodies (Fig. 3 A-D). The material forming while the nucleus was always located in the middle these bodies was not entirely homogeneous, it was part of the cell. possible to distinguish denser areas consisting of The length of the locomotive form was 60–188 better-condensed material (Fig. 3A, G). Inside the µm (average 126 µm, n=88), breadth was 48–160 material forming the lobes it was possible to see µm (average 86 µm, n=88). Length/Breath ration small lacunas (Fig. 3 D). The karyoplasm outside (L/B) was 0.85–3.4 (average 1.67). The maximal the nucleolus (“external karyoplasm” sensu Smirnov length and minimal breadth, as well as maximal and Goodkov, 1998) looked denser and more finely L/B ratio, reflect those of the elongate, orthotactic granulated rather than that inside it (“internal ka- forms, while the dimensions of palmate forms were ryoplasm”) (Fig. 3 E-G). In the central area of the close to the average ones and their L/B ratio often nucleus, it was possible to distinguish a spherical was close to 1.0 or only slightly exceeded it. body formed by the small patches of the nucleolar Stationary cells were rounded, with numerous material. The karyoplasm inside this body appeared peripheral hyaline lobes (Fig. 1 D). Floating cells had to be denser than outside (Fig. 3 A-B). A patch of several uneven thick tapering pseudopodia, radiating nucleolar material probably representing a tangen- from the central cell mass. Pseudopodia contained tial section of the central body is visible in Fig. 3 D. both the granuloplasm and the hyaloplasm (Fig. This structure of the nucleus perfectly correlates 1 A-B), when cell settled down it kept remnants with the pattern seen by LM (Fig. 1 A). Nuclear pore of these pseudopodia for some time and slowly complexes were well visible in tangential sections of retracted them when started to move (Fig. 1 C). the nucleus (Fig. 3 F). They had a typical structure, The nucleus had a complex structure of the showing the peripheral spoke ring assembly and the nucleolus. In most of the cells, it resembled a hollow central plug. Both tangential and sagittal sections perforated sphere, located inside the nucleus at showed no evident fibrous lamina inside the nucleus, some distance from the nuclear envelope (Fig. 2 A, however, arrangements of microfilaments under the C-E). The nucleolar material was arranged within nuclear membrane and in the external karyoplasm the walls of this sphere, varying in thickness from a were frequently seen (Fig. 3 E, G). very fine, hardly distinguishable layer to crescent- The cell surface was covered with a layer of shape lobes of variable thickness. Inside this sphere, filamentous glycocalyx. Very fine filaments radiated it was possible to distinguish a loose body of granular from the amorphous basal layer (Fig. 4 A-B). The material and a spherical central body, which had a length of these filaments reached 250 nm. The central lacuna (Fig. 2 A, B). The size of the nucleus mitochondria were spherical, ovoid or (rarely) in maximal dimension was 11–22 µm (average 16.7 elongate in sections (Fig. 4 C, F). They had cristae µm, n=23), the size of the nucleolus, measured of the tubular type and an electron-dense matrix. according to the external border of the sphere of Dictyosomes of the Golgi complex were not nume- the nucleolar material was 10-16 µm (average 13.8 rous and were represented by stacks, consisting of µm, n=23). 3-8 cisternae (Fig. 4 C). No MTOCs were seen in In older cells, the nucleolar material tended to the surrounding of the dictyosomes and no evident concentrate on one side of this sphere, making it microtubules were found in the cytoplasm. Bacteria, asymmetrical (Fig. 2 B), or even forming a hemi- apparently located freely in the cytoplasm were spherical body, in the latter case the central body seen in all cells (Fig. 4 D). Food vacuoles were was eccentrically located (Fig. 2 F). In one cell we usually rather numerous and contained bacteria have observed a horseshoe-like nucleolus, it had two and/or eukaryotic food (Fig. 4 E). A remarkable central bodies symmetrically located on both sides thing, found in all studied cells, were rounded of the nucleus (Fig. 2 G-H). bodies bounded by a multilayered envelope (Fig. The granuloplasm was filled with the small 4 F-H). This envelope was ca. 7 nm in thickness, opaque granules; it contained food vacuoles, trun- double layered and resembled the . 30 · Oksana Kamyshatskaya, Yelisei Mesentsev et al.

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 horseshoe- like 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 µm, I-J – 1 µm.

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 Protistology · 31

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 µm, E-F – 500 nm, G – 1 µm. 32 · Oksana Kamyshatskaya, Yelisei Mesentsev et al.

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 µm. Protistology · 33

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 µm.

Discussion the locomotive morphology. Hence we had the length ranging from 220 to 325 µm and breadth 35- The present isolate was identified as Polychaos 43 µm with L/B about 9. In contrast, in the present annulatum basing on the locomotive morphology isolate monotactic forms were rarely seen, and and a very characteristic structure of the cell coat measured cells were mostly polytactic or palmate. and the nucleus, identical to that described in The measurement of the palmate cells of Valamo 1998 for our Valamo Island isolate of P. annulatum island isolate done in the available photographic (Smirnov and Goodkov, 1998). The present strain images made in 1998 gives approximately the same shows absolutely the same inner sphere of the sizes (150-200 µm in length). Anyway, the present nucleolar material, segregation of the karyoplasm isolate is generally smaller than the Valamo Island to the inner one, located inside the nucleolus and one, but this difference is within the range of the size a denser outer one, located outside the nucleolus variability of amoebae species (Page, 1988; Smirnov and filled with numerous filaments. It shows the et al., 2002, 2007). Basing on all this, we believe same characteristic arrangements of the nucleolar that we re-isolated the species Polychaos annulatum material – two crescent-shaped lobes or one larger (Penard, 1902) Smirnov et Goodkov, 1998. hemisphere in older cultures. The only difference The present study is based on the observation against the description made in 1998 is the presence of numerous (several hundred) cells, which is of the central body in the nucleus. However, we re- much more than we studied in 1998 (according to examined permanent stained preparations made our records, ca. 30 cells were found and studied at in 1999 from the same strain described by Smirnov that time). Non-surprisingly, the present study, and Goodkov (1998) and found that at that time we performed using much better technical facilities, misinterpreted the central body, considering it as a bring new findings. First of all, this is the structure top view on one of the peripheral lobes. The central of the nucleus, which besides the already described body is visible in all nuclei in these preparations hollow sphere of the nucleolar material contains the (Fig. 5 A-C). The surface coat shows the same arrangement of the nucleolar material in the centre pattern of filaments. The present isolate has the of this sphere and the central body. A noticeable same characteristic truncate bipyramidal crystals detail is the presence of membrane-bounded bodies with an average size of about 3 µm, as the Valamo of unclear nature, which were not seen in 1998. island isolate. Smirnov and Goodkov (1998) provided a de- The size of the nucleus in our present isolate (11- tailed comparison of Polychaos annulatum with all 22 µm) is generally in the same range as indicated similar amoebae species. Among them, the most for Valamo Island isolate (16-25 µm). The nucleus similar is P. fasciculatum (Penard, 1902) Schaeffer, of the smallest size (11 µm) was the single exception 1926. However, in 2000 and 2002 we studied the in our present observations. There is a considerable type strain of P. fasciculatum obtained from CCAP, difference in the cell size, which originated because as 1564/1 strain. These data indicate that the nucleus in 1999 we observed mostly orthotactic and mono- in P. fasciculatum clearly differs in structure. The tactic locomotive forms in our cultures and measured nucleolus in this species either represents a ribbon, them as the fasted moving and this best representing packed inside the nucleus (Page 1988) or several (2-4) compact bodies, often with large lacunas 34 · Oksana Kamyshatskaya, Yelisei Mesentsev et al. inside, arrange as a ring inside the nucleus (Page and number 1995:9:6:11 (holotype) and 1995:9:6:12 Baldock, 1980). Our observation fully confirmed (paratype) by Smirnov and Goodkov (1998). 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 Acknowledgements our studies of the same isolate, we never observed it, which may indicate that its presence may be Supported by RFBR 16-04-01454 grant (isola- optional. TEM data provided in this paper shows tion and LM studies) and RSF 17-14-01391 grant different nuclear structure – nucleolar bodies in (studies of the cell ultrastructure). The present stu- P. fasciculatum are more rounded, they are larger dy utilized equipment of the Core facility centers and never form a structure like to crescent-shaped “Development of molecular and cell technologies” bodies, so typical for P. annulatum. The cell coat in and “Culture Collection of microorganisms” of St. P. fasciculatum appears to have a different organiza- Petersburg State University. tion. Other species that may be considered as similar to those two is Metachaos gratum Schaeffer, 1926, References which is larger than both these species. It possesses a similar nuclear structure but has a number of Bolivar I., Fahrni J.F., Smirnov A. and Paw- differences (Smirnov and Goodkov, 1998) and lowski J. 2001. SSU rRNA-based phylogenetic cannot be associated with the present strain. position of the genera Amoeba and Chaos (Lobosea, Amoebae of the Polychaos fasciculatum-like Gymnamoebia): the origin of gymnamoebae re- species group all are similar to each other and it is visited. Mol. Biol. Evol. 18, 2306–2314. hard to distinguish them. To the moment the most Fahrni J.F., Bolivar I., Berney C., Nassonova parsimonious solution would be to keep two pro- E., Smirnov A. and Pawlowski J. 2003. Phylogeny perly described species with established type mate- of lobose amoebae based on actin and small- rial – P. fasciculatum (Penard, 1902) Schaeffer, 1926 subunit ribosomal RNA genes. Mol. Biol. Evol. 20, and Polychaos annulatum (Penard, 1902) Smirnov et 1881–1886. Goodkov, 1998. We provide an emended diagnosis Goodkov A.V., Smirnov A.V. and Skovorodkin of P. annulatum to incorporate new data obtained I.N. 1999. Study of a rediscovered large freshwater in the present study. The studied strain is deposited amoeba Chaos illinoisense (Kudo, with the culture collection of the Core facility Center 1950). Protistology. 1, 55–61. “Culturing of Microorganisms”: of SPSU under the Kamyshatskaya O., Nassonova E. and Smir- number RCCMAm0456, which makes it available nov A. 2017. Phylogenetic position of amoeba for further studies. genus Deuteramoeba (Amoebozoa, Tubulinea). Protistology. 11, 231–237. Polychaos annulatum (Penard, 1902) Smirnov et Kang S., Tice A.K., Spiegel F.W., Silberman Goodkov, 1998, emend. J.D., Pánek T., Čepička I., Kostka M., Kosakyan Monopodial, sometimes slightly clavate in A., Alcântara D.M.C., Roger A.J., Shadwick L.L., continuous locomotion and polypodial, with many Smirnov A., Kudryavtsev A., Lahr D.J.G. and short pseudopodia (“palmate”) in slower movement. Brown M.W. 2017. Between a pod and a hard test: Bulbous uroid, separated from the rest of the the deep evolution of amoebae. Mol. Biol. Evol. 34, body with a distinct neck in locomotive form and 2258–2270. fasciculate uroid during non-directed movement Kudryavtsev A., Brown S., Smirnov A. 2004. and when started to move. Length in locomotion barki n. sp. (Rhizopoda, Himatis- 220-325 µm, breadth 35-43 µm. Length/Breadth menida) re-isolated from soil 30 years after its initial ratio (L/B) is about 9. Nucleolar material is arranged description. Eur. J. Protistol. 40, 283–287. in a perforated hollow sphere at some distance from Mesentsev Y.S. and Smirnov A.V. 2019. Thec- the nuclear envelope. No nuclear lamina. No cysts amoeba cosmophorea n. sp. (Amoebozoa, , are known. Thecamoebida) – an example of sibling species Observed habitats: Freshwater; reported from within the genus . Eur. J. Protistol. 67, Switzerland, North-Western Russia (Valamo Island 132–141. and surroundings of St.Petersburg), possibly, the Michel R. and Smirnov A.V. 1999. The genus Netherlands. Flamella Schaeffer, 1926 (Lobosea, Gymnamoebia), Type material: permanent stained preparations with description of two new species. Eur. J. Protistol. deposited with British Natural History Museum: 35, 403–410. Protistology · 35

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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: [email protected].