Protistology 13 (1), 26–35 (2019) Protistology 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 Kamyshatskaya1,2, 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 Amoeba proteus). These amoebae usually have a relatively large size, exceeding hundred of microns, The largest species of naked lobose amoebae they produce broad, thick pseudopodia with (gymnamoebae) – members of the family Amoe- smooth outlines (lobopodia). Locomotive cells bidae Ehrenberg, 1838 sensu Page (1987), belonging show clear differentiation of the cytoplasm 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 organisms (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 Chaos ciliates, and other eukaryotes, 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 genus 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 Amoeba proteus 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 Amoebidae, 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 organism. 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 vacuole, 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 vacuoles (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.