Protistology 14 (4) 219–226 (2020)

Protistology 14 (4), 219–226 (2020) Protistology

Molecular phylogeny of Paradermamoeba valamo (Amoebozoa, Discosea, Dermamoebida)

Alexey V. Smirnov1, Nikita S. Kulishkin1, Alina A. Surkova1, Yelisei S. Mesentsev1, Natalia I. Bondarenko1, Elena S. Nassonova1,2 and Yuri A. Mazei3

1 Department of Invertebrate Zoology, Faculty of Biology, St. Petersburg State University, 199034 St. Petersburg, Russia 2 Laboratory of Cytology of Unicellular Organisms, Institute of Cytology RAS, 194064 St. Petersburg, Russia 3 Department of General Ecology and Hydrobiology, Lomonosov Moscow State University, 119991 Moscow, Russia

| Submitted November 12, 2020 | Accepted December 2, 2020 |

Summary

A remarkable amoeba species – Paradermamoeba valamo Smirnov et Goodkov,1993 was re-isolated from the bottom sediment in Izmailovo Park pond (Moscow, Russia). For the first time, we obtained modern DIC images of this species. High-quality light-microscopy allowed us to resolve some subcellular structures, including trichocyst-like bodies, and show congruence of the present optical images with the results of earlier electron-microscopic studies. We applied single-cell PCR to obtain full sequence of the 18S rRNA gene of this species. The analysis of genetic distances, as well as the result of phylogenetic analysis, shows that P. valamo is a valid species, related to P. levis but showing clear morphological and molecular differences.

Key words: Amoebozoa, Discosea, morphology, phylogeny, SSU gene, light microscopy

Introduction similar in appearance to P. valamo, was smaller in size and had almost twice thinner cell coat, hence An amoebae genus Paradermamoeba was estab- of the same structure. At the time of description, lished by Smirnov and Goodkov (1993) to accom- both these species were found to be difficult objects modate P. valamo – a remarkable species of flat- for TEM studies, so only the images of the cell tened, lanceolate amoebae possessing thick cell coat were published in both above-cited paper, coat consisting of spiral glycostyles. One year later, also this was sufficient for morphological species another species of this genus, P. levis was described distinction. The comprehensive ultrastructure of (Smirnov and Goodkov, 1994). This organism, being P. valamo and P. levis was published ten years later

(Smirnov and Goodkov, 2004). This publication ly belong to the same species (further molecular stu- highlighted differences in the ultrastructure of the dies confirmed this). Live cells in culture were nucleus between these two species and confirmed observed using Leica M205C dissection microscope their status of individual species. The same paper equipped with Rottermann contrast and Leica showed that while P. levis was found to be widely DMI3000 inverted microscope equipped with distributed in Europe and Asia, the only confirmed phase-contrast and IMC (Integrated Modulation finding of P. valamo remained the original finding Contrast) optics. Cells were studied, measured and at Valamo Island (North-West Russia). Mrva (2005; photographed on the glass object slides using 2006) listed P. valamo (as well as P. levis) among Leica DM2500 microscope equipped with DIC species recovered in Slovakia (from mosses of the optics on PL-fluotar and plan-apochromatic Malé Karpaty and rainwater pool in Bratislava, lenses. Amoebae were photographed and video- respectively), but the taxonomic identification was recorded using DS-Fi3 Nikon camera powered by based only on the light-microscopic morphology. NisElements – AR software (Nikon). The SSU sequence of P. levis was obtained by For DNA isolation, individual cells were trans- Smirnov et al. (2011) and the phylogenetic analysis ferred in sterile 40 mm Petri dishes containing a performed in this study resulted in the establishment layer of NN agar (see Page, 1988) covered with of the order Dermamoebida, unifying the genera Millipore-filtered (0.22 µm) PJ medium. Cells were Mayorella, Paradermamoeba and Dermamoeba. left to starve for three days, every day they were High-quality light microscopic images of P. levis and transferred to the fresh dish with sterile medium. further data on its ultrastructure became available To isolate DNA, individual amoeba cells were from Kamyshatskaya et al. (2016), while the re-iso- collected manually with the tapered-end Pasteur lation of P. valamo was found to be a problem. This pipette, washed in two subsequent changes of the species appeared in the area of our attention several same medium and placed with 1–2 µl of the medium times, but we were never successful in culturing it. In in a 200 µl PCR tube. DNA was extracted using the the year 2020, we isolated P. valamo from a sample, Arcturus PicoPure DNA Extraction Kit (Thermo collected in Izmailovo Park pond (Moscow), and Fischer Scientific, USA). The extraction mixture managed to obtain SSU sequence of this species. was prepared according to the manufacturer’s inst- The results of phylogenetic analysis and the first ructions; 12 µl of the mixture was added to the tube high-quality DIC images of this species are reported containing the single cell. in the present paper. The SSU sequence was obtained in two ways. First, PCR amplification was performed using RibA (forward, 5’> ac ctg gtt gat cct dcc agt <3’) Material and methods and reverse S12.2R ( 5’> gac tac gac ggt atc tra tc <3’) primers (Pawlowski, 2000). PCR products were The strain of Paradermamoeba valamo designa- sequenced directly, without purification, using the ted as strain MSK1, which is the subject of the pre- Big Dye Terminator Cycle sequencing kit and an sent paper was isolated from the sample, containing ABI PRISM automatic sequencer using the same material from the top 5 cm layer of the sandy bottom primers. In total, 12 different cells were treated and sediments of a freshwater pond in Izmailovo park, sequenced, one sequence was found to belong to a Moscow, Russia (55°46’46.8”N, 37°46’09.2”E). contaminant, while all other were almost identical. Amoebae were found in the dish, inoculated with The consensus sequence was assembled using the sampled sediments diluted 1:1000 with 0.25% CodoneCode software (https://www.codoncode. WG infusion (see Geisen et al., 2014 for protocol), com) on the base of six sequences showing the best made on PJ medium (Prescott and James, 1955). trace quality. After two weeks of incubation, the initial culture The second way included the whole genome contained about a hundred of P. valamo cells. amplification followed by NGS sequencing. We Species identification was performed basing on the performed the Multiple Displacement Amplifica- unique locomotive morphology, nuclear structure tion (MDA) of the DNA from the sample showing and size data on this species. good trace quality in Sanger sequencing (cell #2), All attempts to maintain or to clone culture using the REPLI-g Single Cell DNA Amplification failed, so observations and study were done in ini- Kit (Qiagen, Hilden, Germany) according to the tial enrichment culture. All cells in this culture clear- manufacturer’s instructions. The resulting MDA Protistology · 221 products were sequenced, using Illumina HiSeq (Miller et al., 2010). Sequence identity level was 2500 system at the core facility center “Development calculated using an online tool “Ident and Sim” of Molecular and Cell Technologies” of the SPSU provided at http://www.bioinformatics.org/sms2/ Research Park according to the manufacturer’s ident_sim.html website. protocols; as a result, 20M paired reads with the The obtained sequence was deposited with the length about 150 bp were obtained. Quality control GenBank under the number MW293873 (Para- check of raw sequence data was performed, using dermamoeba valamo strain MSK1, length 2096 bp). FastQC (http://www.bioinformatics.babraham. ac.uk/projects/fastqc). SPAdes assembler was used for de novo genome assembly (Bankevich et al. Results and discussion 2012). The contig containing the SSU rRNA gene fragment was identified, using BLAST (Altschul et Light-microscopic observations made in culture, al. 1990) and the sequence fragment of this isolate as well as those made on the surface of the object obtained by Sanger sequencing as a query. slide show almost identical cell morphology and The alignment constructed for this study was locomotive behaviour. Amoebae were flattened based on one used by Glotova et al. (2018) for Mayo- and oblong in locomotion. They demonstrated rella analysis. It contained all named culture-derived lanceolate morphotype (Smirnov and Goodkov, 18S rRNA gene sequences of Dermamoebida, two 1999; Smirnov and Brown, 2004) and show clear full-length sequences belonging to unnamed strains lateral flatness in moving cells, characteristic to of Mayorella and a number of discosean sequences this species (Smirnov and Goodkov, 1993, 2004) used to form a proper set of outgroups. Other (Fig. 1, A-E). Rapidly moving cells could be more available Mayorella sequences were not used because flattened and often adopted oblong, lingulate shape of their short length. Sequences were aligned using without prominent lateral flatness (Fig. 1, F). When the Muscle algorithm as implemented in SeaView the cell moved more slowly and appeared to be 4.0 (Gouy et al., 2010); the alignment was further “uncertain” with the direction of locomotion, it polished manually. The phylogenetic analysis was could temporarily form short prominent folds and performed using maximum likelihood method as wrinkles (Fig. 1, G-H). These structures were never implemented in PhyML program (Guindon and stable and could be observed for a short time, often Gascuel, 2003) with GTR (eight rate categories) counted by seconds. When the cell moved slower, + γ + I model; 1651 sites were selected for the without the single, clear direction of movement it analysis. The initial selection was made using the could form short triangular pseudopodia (Fig. 1, I-J). g-blocks algorithm as implemented in SeaView Amoebae like this resembled small representatives 4.0, further the set was manually polished. The of the genus Mayorella and could be even missed number of invariant sites, alpha parameter, and with them, especially in mixed culture. However, tree topology was optimized by PhyML. The much thicker cell coat of Paradermamoeba helped program generated 25 random starting trees; the to distinguish these two genera. best tree was further optimized. To test the stability We have not observed a differentiated floating of branching, 1000 bootstrap pseudoreplicates form in our strain; few amoebae cells detached from were used. Bayesian analysis of the same dataset the substrate only for a short time, and soon settled was performed using MrBayes 3.2.7, GTR model down and continue the movement. This might be a with gamma correction for intersite rate variation physiological thing because to get proper locomotive (8 categories) and the covarion model (Ronquist forms we worked with actively growing culture while and Huelsenbeck, 2003). Trees were run as two floating forms are usually observed in older cultures, separate chains (default heating parameters) for 10 which are on the stationary phase. million generations, by which time they had ceased The cell coat of P. valamo was well visible with converging (final average standard deviation of the DIC microscope as a thick line bordering the cell split frequencies was less than 0.01). The quality of (Fig. 1, K). The nucleus was of the vesicular type and chains was estimated using built-in MrBayes tools had a central nucleolus showing non-homogenous and additionally – using the software Tracer 1.6 structure (Fig. 1, L). In the best DIC images, it was (Rambaut et al., 2014); based on the estimates by possible to see the “cords” of nucleolar material and Tracer, the first 25 % of generations were discarded eccentrically located homogeneous area looking as burn-in. MrBayes was run at Cipres V.3.3 website as a lacuna inside the nucleolus. The observed 222 · Alexey V. Smirnov, Nikita S. Kulishkin, Alina A. Surkova, et al.

Fig. 1. Light-microscopic morphology of Paradermamoeba valamo strain MSK1. A-E – The most typical lanceolate locomotive form of this species with pronounced lateral flatness (arrowed in A); the nucleus (n) is usually located in the central part of the cell; the contractile vacuole (cv) is situated in the posterior part of the cell (B) or right in the uroid (D); uroidal structure of bulbous type is arrowed in C; F – flattened, elongate locomotive cell without pronounced lateral flatness; G-H –irregularly moving cell, showing temporary lateral folds and short ridges (arrowed in H); I-J – cells in non-directed locomotion, showing short triangular pseudopodia (amoebae like this may even be missed with Mayorella, especially in mixed culture); K – thick cell coat, forming a well- visible line in DIC images; L – nucleus (n) with central nucleolus (arrowed), consisting of tightly pack cords of nucleolar material (an eccentrically located lacuna is visible in the nucleolus); M – contractile vacuole (cv), surrounded by a vesicular spongiome; N-O – “trichocyst-like” bodies in transversal and longitudinal sections (the central axial core is arrowed in both images). Scale bars: A-J – 10 µm, K-O – 1 µm. Protistology · 223

Fig. 2. Phylogenetic tree based on the 18S rRNA gene, showing the position of Paradermamoeba valamo. 1651 sites are used for the analysis; GTR + γ + I was used for ML analysis and GTR + γ with covarion – for Bayesian analysis. Labelling of nodes: PP/ML support. Black-filled circles are used to label fully supported nodes (1.0/100 support); white-filled circles are used to recognize highly supported nodes (PP > 0.95 and BS > 95). structure is congruent with the TEM data obtained -section, ca 0.5 µm across. The central core pene- by Smirnov and Goodkov (2004); it is possible to trating the body was distinguishable in the best suggest that the “lacuna” observed in DIC images DIC photographs (Fig. 1, N-O). This observation (which could look as elevation depending on the confirms the structure of these bodies, earlier reve- side of the interference spectrum selected with the aled by TEM and further show that the presence of upper DIC prism) correspond to the central body these structures is characteristic for the entire genus of the nucleolar material visible with TEM in this Paradermamoeba. They are still not known in any species (op. cit.). No trace of the nuclear lamina other organism outside this genus. was seen in DIC optics, this is congruent with TEM Molecular phylogeny groups the present sequ- data as well. ence with that of P. levis within fully supported The contractile vacuole was surrounded with Dermamoebida (Fig. 2). The nearest neighbour numerous small bodies (0.3-0.75 µm across), to the genusParadermamoeba in our tree was the rounded or oblong, which probably represented genus Dermamoeba, outgroup to this assemblage mitochondria, located around the vacuole. was formed by sequences of amoebae belonging DIC microscopy allowed us to observe myste- to the genus Mayorella. All other groupings in the rious trichocyst-like bodies mentioned in all studied tree reflected usual molecular relationships between strains of P. valamo and P. levis (Smirnov and amoebae genera within the Discosea clade (e.g. Goodkov, 1993, 1994, 2004; Kamyshatskaya et al., Kudryavtsev et al., 2014; Cavalier-Smith et al., 2016). These bodies were oblong in longitudinal 2016; Udalov et al., 2017, 2020). The artificial section (0.75-1 µm in length) and circular in cross- grouping of Acanthopodida with Thecamoebida 224 · Alexey V. Smirnov, Nikita S. Kulishkin, Alina A. Surkova, et al. was caused with the usage of Pellitida as an outgroup Acknowledgements clade. Before the present sequence was obtained, a little Supported with RFBR project 20-54-53017 to doubt on the validity of P. valamo as an individual YM (sampling, treatment and LM study of cultures) species anyway remained. The major difference and RSF project 20-14-00195 to AS (molecular between these two species is the size, the thickness studies). of the cell coat and the structure of the nucleus. Each of these characters is believed to be reliable, and in combination, they form a rather clear pattern of References differences. However, there are amoebae species, showing a wide size range (Page, 1983, 1988; see Altschul S.F., Gish W., Miller W., Myers E.W. for example size data in Page, 1985). For example, and Lipman D.J. 1990. Basic local alignment search the smallest known specimens of Flabellula baltica tool. 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Address for correspondence: Alexey Smirnov. Department of Invertebrate Zoology, Faculty of Biology, St. Petersburg State University, Universitetskaya Emb. 7/9, 199034 St. Petersburg, Russia; e-mail: [email protected]