Redescriptions of Euplotes encysticus and E. rariseta (Protist: Ciliophora: Euplotida)

Abstract

Two euplotid ciliates, Euplotes encysticus Yonezawa, 1985 and E. rariseta Curds et al., 1974, were isolated from a freshwater pond called Mulgol in Dokdo of the East Sea and from Masan Bay/Jeju Island, Korea, respectively. Both species are redescribed based on live observations and protargol impregnation. Cells of Euplotes encysticus are asymmetrically oval, $63-79{\times}41-61{\mu}m$ in vivo and capable of encystment. The cells have 31-36 adoral zone of membranelles(AZM), 9 fronto-ventral cirri (FVC), 5 transverse cirri (TC), 2-3 caudal cirri (CC), 2 marginal cirri (MC), 7 dorsal kineties (DK), and 19-22 dorsal cilia in middle DK. The cells of Euplotes rariseta has a small ovoid form and are $32-44{\times}23-35{\mu}m$ in vivo, 18-22 AZM, 10 FVC, 5 TC, 2 CC, 1 MC and 6 DK.

Introduction

Euplotes is the species-rich genus, and has a wide distribution and high adaptive potentialities (Curds, 1975; Lobban et al., 2005; Schwarz et al., 2007; Wilbert and Song, 2008). The identification of Euplotes species is difficult because (1) many of the distinguishing characters are known to vary considerably even within clonal cultures, (2) certain common species are morphologically confused, and (3) there is the added difficulty of having to search through a considerable body of literature before an identification can be attempted (Curds, 1975; Yonezawa, 1985; Song et al., 1998; Lobban et al., 2005; Schwarz and Stoeck, 2007).

About 100 Euplotes species have been reported from various habitats (e.g. Tuffrau, 1960; Curd, 1975; Yonezawa, 1985; Song et al., 1998; Berger, 2001; Lobban et al., 2005; Schwarz and Stoeck, 2007; Wilbert and Song, 2008; Fan et al., 2010; Jiang et al., 2010a; 2010b), but 18S rRNA sequences of about 34 species have been submitted to GenBank of the NCBI(15 Sep. 2018).

To date, 11 species of Euplotes have been reported in Korea: E. aediculatus, E. charon, E. cristatus, E. eurystomus, E. minuta, E. muscicola, E. muscorum, E. parawoodruffi, E. patella, E. rariseta and E. vannus (Shin and Kim, 1988; Shin et al., 1992; Shin and Kim, 1995; 1996; Jo and Shin, 2003; Kwon and Shin, 2006; Kwon et al., 2007; Jung et al., 2017; Park et al., 2017).

Here we redescribe two euplotid species, Euplotes encysticus and E. rariseta, based on their living morphology and protargol impregnation.

Materials and Methods

Sample collection, observation and identification

The specimens of Euplotes encysticus and E. rariseta were collected at a freshwater pond (Mulgol) in Dokdo (island; ~87 km from Ulleungdo) of the East Sea and at marine intertidal sediments of Masan Bay/Jeju Island, Korea, respectively. A single Euplotes cell was isolated by micropipetting from a well plate containing sterile seawater or freshwater, supplemented at 1% v/v with lysogeny broth (LB), inoculated into a well plate and maintained at 21℃. For mass and routine cultures, Euplotes rariseta inoculated into a well plate containing sterile seawater with a barley grain, and E. encysticus inoculated into a well plate containing sterile seawater (15 psu and 30 psu) and freshwater with a barley grain. For routine maintenance, subculturing was performed every two weeks.

Live and protargol impregnated specimens were observed with a Leica DMR microscope (Germany) equipped with a Zeiss Axiocam HR digital camera and its associated software (Axiovision 4.6). The infraciliature was revealed using the protargol method (Wilbert, 1975). Measurements and counts were performed at magnifications of 400-1000. Classification follows Lynn (2008), while terminology follows Curds(1975) and Song et al. (2009); Adoral zone of membranelles (AZM, hereafter), adoral membranelles (AM), contractile vacuole (CV), dorsal kineties (DK), macronucleus (Ma), micronucleus (Mi), paroral membrane (PM), fronto-ventral cirri(FVC), transverse cirri(TC), caudal cirri(CC), marginal cirri(MC).

DNA extraction, PCR amplification and sequencing

A 10-mL sample of about one-week-old culture was centrifuged at 5,900 g for 5 min, and then the DNA was extracted using a Qiagen Blood & Cell Culture Extraction Kit(Qiagen, Hilden, Germany) following the manufacturer’s protocol. 18S ribosomal DNA(18S rDNA) sequences were obtained by PCR amplification using a combination of the eukaryote primers EukA (5ʹ-AACCTGGTTGATCCTGCCAGT-3ʹ) and EukB (5ʹ-TGATCCTTCTGCAGGTTCACCTAC-3ʹ) (Medlin et al., 1988). For the PCR amplification, a reaction volume of about 20 μL was used that included 1.5 μL of 10 μM stocks of the primers EukA and EukB, 2 μL of 0.25 mM dNTP-mix, 1.8 μL of 25 mM MgCl2, 0.7 μL total of 5U/μL Taq DNA polymerase (Bioneer, Korea), and 5 μL of DNA template. The cycling conditions started with a denaturing step at 94°C for 5 min, followed by 35 cycles of 30 s at 94°C, 1 min of annealing at 55°C, and extension at 72°C for 2 min (10 min at 72°C for the final cycle only). A PCR product corresponding to the expected size was gel-isolated, directly purified, then sequenced by Sanger sequencing using PCR primers along with internal sequencing primers (514F, 5ʹ-GGTGCCAGCASCCGCGGTAA-3ʹand Euk1209R 5ʹ-GGGCATCACAGACCTG-3ʹ). Individual reads were assembled using Geneious ver. 8.1.5 (Kearse et al., 2012).

Results and dIscussIon

Phylum Ciliophora Doflein, 1901

Class Spirotrichea Bütschli, 1889

Subclass Hypotrichia Stein, 1859

Order Euplotida Small and Lynn, 1985

Family Euplotidae Ehrenberg, 1838

Genus Euplotes Müller, 1786

1. Euplotes encysticus Yonezawa, 1985(Figs. 1a-d, 2, 3)

Fig. 1. Drawings of Euplotes encysticus (a-d) and E. rariseta (e-h). (a) E. encysticus and (e) E. rariseta in vivo, E. encysticus (b-d) and E. rariseta (f-h) after protargol impregnation. (b) and (f) ventral views, (c) and (g) dorsal views, (d) and (h) different shapes of macronucleus. AZM, adoral zone of membranelles; PM, paroral membrane; FVC, fronto-ventral cirri; TC, transverse cirri; CC, caudal cirri; MC, marginal cirrus; DK, dorsal kineties; Ma, macronucleus; Mi, micronucleus. Scale bars in (b) for (a-c) and in (d) represents 20 μm, in (f) for (e-g) and in (h) represent 10 μm.

Fig. 2. Micrographs of Euplotes encysticus strain KF403 (a-c) in vivo and (d-f) after protargol impregnation. (a) Dorsal views showing contractile vacuole (CV) and adoral membranelles, (b) dorsal ridges (arrowheads), (c) ventral side showing FVC (fronto-ventral cirri) and TC (transvers cirri), (d) ventral view showing AZM (adoral zone of membranelles), PM (paroral membrane), FVC, TC, CC (caudal cirri), MC (marginal cirri), Ma (macronucleus), Mi (micronucleus), (e) dorsal side showing DK (dorsal kineties), (f) note 3 CC. Scale bars in (c) for (a-c) and in (f) for (d-f) represent 20 μm.

Fig. 3. Micrographs of excystment of Euplotes encysticus. (a) Cyst of cell 1, (b-j) time series lasting 6 minutes 52 seconds documenting excystment of cell 2, (i) empty cyst and trophozoit, (j) trophozoit. All images are DIC images. Scale bar in (j)=25 μm.

Material examined. Korea: Dokdo(37°14ʹ22ʺN, 131°52ʹ08ʺE), 27 September 2017, from a freshwater pond(Mulgol), collected by Jong Soo Park. Type strain, live cells are kept with the Korean Culture Collection of Protists, Kyungnam University, Korea, reference ‘KF403’.

Description. Cells are 63-79 ×41-61 μm in vivo and 44-56 ×28-38 μm in protargol, asymmetrically oval with posterior portion slightly wider than anterior, and dorso-ventrally flattened. The peristome is narrow and extremely long, extending approximately 72% of cell length and surrounded by 31-36 adoral membranelles. Adoral zone of membranelles (AZM) curved (Figs. 1a, 1b, 2a, 2d, 2f). Cytoplasm colourless, often filled with many light reflecting granules and few food vacuoles (Fig. 2a). Single contractile vacuole located to right of transverse cirri (TC) (Fig. 2a). Seven dorso-lateral ridges (Figs. 2b, arrowheads; 2e). Dorsal kineties (DK) inserted in concaves down the ridges (Figs. 1c, 2e). The kinetosome number of the mid-dorsal kinety is 19- 22. Cirri usually strong; 9 fronto-ventral, 5 transverse, 2-3 (mostly 2) caudal and 2 left marginal cirri. Paroral membrane (PM) about 10 μm long. Macronucleus (Ma) usually C-shaped, with the anterior and posterior ends sometimes curving (Figs. 1d, 2d, 2f). Micronucleus (Mi) small, nearly spherical and located in the upper right half of the macronucleus(Figs. 1b, 1d, 2d). The cells are capable of encystment and excystment (Fig. 3). Move by crawling on crumb or substrate.

Remarks. Euplotes encysticus was originally reported by Yonezawa (1985) as a freshwater form. We performed the inoculation of our strain KF403 into well plates containing seawater (15 and 30 psu) with barley grains. The strain grew well in the seawater, thus this species is adaptable to both seawater and freshwater. Our observations are in accord with those of Yonezawa (1985) and Fan et al. (2010). Euplotes encysticus is very similar to E. muscorum in general appearance, but according to several researchers (e.g., Dragesco, 1970; Dragesco and Dragesco Kernéis, 1986; Jo and Shin, 2003) they can be distinguished from each other because (1) no cyst has been observed so far in E. muscorum, (2) the obvious dorsal-lateral ridges and the granules surrounding the dorsal cirri have not observed in E. muscorum, (3) the AZM of E. encysticus outspreads more broadly than that of E. muscorum, and (4) the AZM covers 3/4 body length in E. encysticus, and 2/3 in E. muscorum. Our strain has all of these characters specific to E. encysticus. Additionally, these two species are clearly distinguished by the differences in their 18S rRNA gene sequences, of which E. encysticus differs from E. muscorum by 2.04% (Fan et al., 2010). Euplotes encysticus is similar to E. petzi, E. roscoffensis and E. sinicus in cell length, but the numbers of AM, DK and dikinetids in mid-DK in E. encysticus are higher than those in E. petzi. Euplotes roscoffensis and E. sinicus have higher number of AM than that of E. encysticus (Tables 1, 2). Euplotes sinicus has 11-16 dikinetids in mid-DK (vs 5-7)(Tables 1, 2).

Table 1. Morphometric data of Euplotes encysticus and E. rariseta. AZM, adoral zone of membranelles; CL, cell length; AM, adoral membranelles; FVC, frontoventral cirri; TC, transverse cirri; MC, marginal cirri; CC, caudal cirri; DK, dorsal kinety; DDK, dikinetids in mid-DK; PM, paroral membrane; n, number.

Table 2. Comparison of morphologically similar Euplotes species. AM, adoral membranelles; FVC, frontoventral cirri; TC, transverse cirri; MC, marginal cirri; CC, caudal cirri; DK, dorsal kinety; DDK, dikinetids in mid-DK; n, number.

NCBI BLASTN search result shows that the closest strains to our strain KF403 are E. encysticus stains [GenBank Accession Nos. FJ346569, EF535728, LN864512; Identity 99%]. The 18S rRNA sequence of our stain is 1,663 bp in length.

Habitat. Marine and Freshwater

Distribution. Freshwater habitats: Korea(Dokdo, this study), Japan(Yonezawa, 1985) Marine habitats: China(Daya Bay, Fan et al., 2010)

Deposition. National Institute of Biological Resources, Korea (NIBRPR0000109440)

NCBI GenBank Accession Number: MK026950

2. Euplotes rariseta Curds, West and Dorahy, 1974(Figs. 1e-h, 4a-f)

Fig. 4. Micrographs of Euplotes rariseta strain KM401 (a-c) in vivo and strain KM444 (d-f) after protargol impregnation. (a) Dorsal ridges (arrowheads) of E. rariseta, (b) ventral view showing longitudinal ridges (arrowheads), (c) ventral view showing FVC and TC, (d) ventral view showing AZM (adoral zone of membranelles), FVC (fronto-ventral cirri), TC (transvers cirri), PM (paroral membrane), (e) dorsal view showing dorsal kineties, (f) ventral view showing ventral kineties and Ma (macronucleus). Scale bars in (c) for (a-c) represent 20 μm and in (f) for (d-f) represent 10 μm.

Material examined. Korea: Masan Bay (35°10ʹ08ʺN, 128°35ʹ58ʺE), 31 May 2016 and Jongdal-ri Beach, Jeju Island (33°30ʹ47ʺN, 126°53ʹ56ʺE), 14 July 2018, from marine intertidal sediments, collected by Won Je Lee. Type strains, live cells are kept with the Korean Culture Collection of Protists, Kyungnam University, Korea, reference ‘KM401’ and ‘KM444’.

Description. Cells are 32-44×23-35 μm in vivo and 23- 30×17-20 μm in protargol, ovoid and dorso-ventrally flattened. The peristome is wide, spindle-shaped and surrounded by 18-22 adoral membranelles, which extend for about 72% of the cell length (Figs. 1e, 1f, 4a, 4d-f). Cytoplasm colorless. Few food vacuoles and single contractile vacuole located in the middle of the cell (Fig. 4a, 4b). Cirri usually strong; 10 fronto-ventral, 5 transverse, and 2 caudal cirri(Fig. 4b, 4c). Caudal cirrus below AZM is slightly stout. Ventral surface heavily sculptured with 6 longitudinal ridges (Fig. 4b, 4c, arrowheads). Dorsal surface with 6 double-edged longitudinal ridges (Fig. 4a, arrowheads). Dorsal bristles sparse; 6 kinetics (Figs. 1g, 4e). The macronucleus(Ma) is horseshoe-shaped, and the micronucleus (Mi) is small and situated anteriorly (Figs. 1f-h, 4e). The cells are capable of encystment. Move by crawling on substrate. This species was found at Masan Bay (Korea; Strain KM401) and Jongdal-ri Beach (Jeju, Korea; Strain KM444).

Remarks. Euplotes rariseta was originally reported by Curds et al. (1974) from Aberystwyth, Wales (UK). Our observations are in good agreement with those of Curds et al. (1974), Ma et al. (2007) and Song and Packoff (1997). Euplotes rariseta is similar to E. parabalteatus and E. trisulcatus in cell length, but can be distinguished because E. parabalteatus has 8-11 dikinetids in mid-DK (vs 5-7) and inconspicuous dorsal ridges, and E. trisulcatus has 25-36 AM(vs 18-22) and 7-11 dikinetids in mid-DK, and (Tables 1, 2). This species is mostly similar to E. orientalis in cell length and in the numbers of AM, DK and dikinetids in mid-DK, but they can be distinguished because E. rariseta has 10 FVC (vs 8) and 1 MC (vs 2) (Tables 1, 2). In addition, the 18S rRNA sequences of E. rariseta and E. orientalis have 93% similarity.

NCBI BLASTN search result shows that the closest strains to our strains(KM401, KM444) are E. rariseta stains(GenBank Accession No. AJ305248, FJ423449; Identity 99%). The 18S rRNA sequences of stain KM401 and KM444 are 1,742 bp and 1,738 bp in length, respectively.

Habitat. Marine

Distribution. Korea (Inchon, Park et al., 2017; Masan Bay and Jeju Island, this study), Antarctica (Ross Sea, Valbonesi and Luporini, 1990; Albergoni et al., 2000), Bellings-hausen Sea (Thompson, 1972), China (Song and Packroff, 1997; Jiang et al., 2010b), Indian Ocean (Dallai et al., 1987), Red Sea (Solar lake; Wilbert and Kahan, 1981), Somalia (Gesira; Dallai et al., 1987), UK (Aberystwyth, Wales, Curds et al., 1974), Weddell Sea (Agatha et al., 1990; Petz et al., 1995).

Deposition. National Institute of Biological Resources, Korea(NIBRPR0000107971) for KM401 strain.

NCBI GenBank Accession Numbers: Strain KM 401: MK028833, Strain KM444: MK050525