Characterization of a Novel Hypotrich Ciliate From Heavy Metal-Contaminated Industrial Outlet in Onsan, Ulsan, South Korea

Very few studies exist on the description of protozoan ciliates from industrially contaminated sites. In this study, we report a description of a novel hypotrich ciliate isolated from water samples collected from an industrially contaminated outlet in Onsan, Ulsan, South Korea. The oxytrichid ciliate, Histriculus tolerans n. sp., was investigated using live observation and protargol impregnation. The morphology, morphogenesis, and molecular phylogeny inferred from small-subunit (SSU) rRNA gene sequences were studied. The new species is mainly characterized by a cell size of about 70 × 40 μm in vivo, two elongate ellipsoidal macronuclear nodules and one or two micronuclei, adoral zone of about 51% of body length with 32 membranelles on average, about 34 cirri in the right and 24 cirri in the left marginal row, 18 frontoventral transverse cirri, six dorsal kineties including two dorsomarginal rows, and dorsal kinety 1 with 26 bristles. Morphogenesis is similar to that of the type species, i.e., Histriculus histrio, except that oral primordium does not contribute to anlage II of the proter. Phylogenetic analyses, based on small-subunit rRNA gene sequences, consistently place the new species within the family Oxytrichidae, clustering with H. histrio.


INTRODUCTION
The genus Histriculus Corliss, 1960, is largely confined to freshwater and marine habitats with type species, i.e., Histriculus histrio, having a cosmopolitan distribution. The genus is mainly characterized by undulating membranes in Oxytricha pattern, one right and one left marginal cirral row, confluent marginal rows at the posterior end, six dorsal kineties, and absence of caudal cirri. Berger (1999), in his detailed revision of the oxytrichids, assigned six species to the genus; however, detailed morphology and morphogenesis, as well as gene sequences, have been reported mainly for the type species, i.e., H. histrio (Müller, 1773), Corliss, 1960. The morphology, though poorly described, and morphogenesis with endogenous bud formation have been reported for Histriculus vorax (Stokes, 1891) Corliss, 1960. Berger (1999 recommended detailed reinvestigations of the remaining species of the genus. Limited studies have been performed on the diversity of ciliates inhabiting industrially contaminated soil and water . Recently, Kumar et al. (2017) discovered a novel soil ciliate isolated from contaminated soil of the petroleum industry. The present paper describes a new species of the genus Histriculus isolated from the heavy metal-contaminated waters, collected from the industrial outlet in Onsan, Ulsan, South Korea. A detailed description of the morphology, morphogenesis, and phylogenetic analyses based on the SSU rRNA gene sequences of the new species, i.e., H. tolerans, has been presented.

Sampling and Sample Processing
Water samples were collected on April 8, 2016 from the outlet of an industrial company in Onsan, Ulsan, South Korea (35 • 25 55.9 N 129 • 21 07.2 E). Ciliates were isolated and fed with green algae Chlorogonium elongatum as a food organism (Ammermann et al., 1974). Live observations were made using a microscope with differential interference contrast illuminations at a magnification range of × 100-1,000. The protargol staining method described by Kamra and Sapra (1990) was used, with some modifications, to reveal the ciliature. Measurements of impregnated specimens were performed at a magnification of × 1,000 using an ocular micrometer. A Zeiss microscope camera was employed for photomicrography. The illustration of the live specimen was prepared using free-hand sketches, while those of impregnated specimens were made with a drawing device. The terminology is according to Wallengren (1900) and Berger (1999).

DNA Extraction, PCR Amplification, and Sequencing
Four clonal cultures were established, and the same were used for live observation and protargol impregnation to study morphology and morphogenesis. Three cells of H. tolerans were isolated and collected in 1.5-mL tubes for DNA extraction from a clonal culture with the help of glass micropipettes. Thus, the morphology, morphogenesis, and molecular phylogeny reported in the present study deal with the same species. The cells were washed at least three times with autoclaved distilled water for genomic DNA extraction using the REDExtract-N-Amp Tissue PCR Kit (Sigma, St. Louis, MO), following the instruction of the manufacturer, except for the reduction of each reaction volume to one-tenth (Gong et al., 2007). Amplifications of the extracted DNA were carried out using the TaKaRa ExTaq DNA polymerase Kit (TaKaRa Bio-medicals, Otsu, Japan) using the universal eukaryotic primers Euk A (FW 5 -AAC CTG GTT GAT CCT GCC AG-3 ) and Euk B (RV 5 -CAC TTG GAC GTC TTC CTA GT-3 ) (Medlin et al., 1988). The PCR program for SSU rRNA gene amplification included an initial denaturation at 94 • C for 3 min, followed by 35 cycles of 94 • C for 1 min, 56 • C for 45 s, and 72 • C for 80 s, with a final extension step at 72 • C for 10 min. After confirmation of the appropriate size, the purified PCR products were directly sequenced on both strands on an ABI 3730 automatic sequencer at the Cosmo Genetech, Seoul, South Korea.

Phylogenetic Analyses
For phylogenetic analyses, the SSU rRNA gene sequence of H. tolerans n. sp. was aligned with 54 SSU rRNA gene sequences of hypotrich ciliates from GenBank using the MAFFT software v. 7 1 and choosing the iterative refinement methods Q-INS-I that consider the secondary structure of the SSU rRNA molecules (Katoh and Standley, 2013).
Ambiguously aligned regions were identified and excluded from the phylogenetic analyses with Gblocks v. 0.91b (Castresana, 2000) using parameters optimized for rRNA gene alignments, leaving 1,562 unambiguous positions. Maximum likelihood (ML) analyses were carried out using RAxML-HPC2 v. 8.0.24 (Stamatakis, 2014) on the CIPRES Science Gateway (Miller et al., 2010) with bootstrapping of 1,000 replicates. A Bayesian inference (BI) analysis was performed using Mr. Bayes v. 3.2.1 (Ronquist et al., 2012) and the TIM2 + I + G model, as selected by the jModel test v. 2.1.3 software (Posada, 2008) under the Akaike Information Criterion. Markov chain Monte Carlo simulations were run, with two sets of four chains using the default settings, for 1,000,000 generations with trees sampled every 100 generations and discarding the first 25% of the sampled trees as burn-in. The remaining trees were used to generate a consensus tree and to calculate the posterior probabilities of all branches using the majority rule consensus approach. Phylogenetic trees were visualized using the free software package FigTree v. 1.4 by A. Rambaut. 2

Diagnosis
Body size about 70 × 40 µm in vivo. Body outline elliptical. Two elongate ellipsoidal macronuclear nodules and one or two micronuclei. Cytoplasm colorless. Buccal cirrus at the anterior end of undulating membranes. Adoral zone about 51% of body length, with 32 membranelles on average. About 34 cirri in the right and 24 cirri in the left marginal row. Six dorsal kineties with 26 bristles in kinety 1.

Type Locality and Habitat
Water sample was collected from a small stream having polluted water from the outlet of an industrial company in Onsan, Ulsan, South Korea (35 • 25 55.9 N 129 • 21 07.2 E). At the time of collection, the water temperature was 15 • C, pH 7.2, and salinity (psu) 0 (freshwater).

Type Material
A protargol slide with the holotype specimen ( Figures 2C, 3A) circled in black ink is deposited at the National Institute of Biological Resources, Incheon, South Korea, with registration number NIBRPR0000111041. Two paratype slides are also deposited with registration numbers NIBRPR0000111039 and NIBRPR0000111040.

Etymology
The species group name tolerans (tolerating) refers to its ability to tolerate heavy metals present at the sampling site, i.e., outlet of the industrial waste.
Adoral zone about 51% of body length, composed of, on average, 32 membranelles, with cilia about 15 µm long in vivo. Paroral and endoral membranes of about equal length, slightly curved and nearly parallel (Figures 1C,D Table  1). Pharyngeal fibers of ordinary length and structure extend obliquely toward the right body margin (Figure 2A).

Resting Cyst
Two-week-old resting cysts about 40 µm in diameter in vivo; cyst surface with hyaline ridges, about 1.5-2.5 µm high (Figures 1F-H). Cyst wall about 1.5 µm thick. Cyst content attached to the wall, composed of lipid droplets and separate macronuclear nodules (Figures 1F-H).

Divisional Morphogenesis
Divisional morphogenesis resembles the type species H. histrio, except that the oral primordium does not contribute to the anlage Frontiers in Microbiology | www.frontiersin.org II of the proter (for a review, see Berger, 1999;Gupta et al., 2006). The parental adoral zone is retained unchanged for the proter, while that of opisthe is formed from the oral primordium that originates close to the transverse cirrus II/1 (Figures 4A-G, 5A-H). Five parental cirri (II/2, III/2, IV/2, IV/3, and V/4) and the parental undulating membranes are involved in the formation of six anlagen each for proter and opisthe. The proter anlage I generate from partial reorganization of the paroral and endoral, anlage II from disaggregation of cirrus II/2, anlage III from cirrus III/2, and anlagen IV-VI from cirrus IV/3. The involvement of cirrus V/4 in the formation of proter anlagen IV-VI was not clear in our observation. The opisthe anlagen I-III originate from oral primordium, anlagen IV from cirrus IV/2, and anlagen V and VI from cirrus V/4. The paroral and endoral and the first frontal cirrus I/1 are formed from anlage I. The postoral ventral cirrus V/3 is not involved in anlagen formation. The 18 frontal-ventraltransverse cirri arise from six anlagen by splitting in a 1:3:3:3:4:4 pattern (Figures 4A-G, 5A-H).
The marginal primordia arise at two levels by within-row anlagen formation by utilizing one to three of the parental cirri. The marginal primordia elongate utilizing three to five parental cirri and differentiate into new marginal rows. The remaining parental cirri are resorbed (Figures 4E-G, 5E-H).
On the dorsal surface, three anlagen are formed within row from dorsal kineties 1, 2, and 3 at two levels (one set each for the proter and the opisthe). The third dorsal anlage fragments in the middle and gives rise to the third and fourth dorsal kineties of equal length. The two dorsomarginal rows arise near the anterior end of two newly formed right marginal anlagen and move from the lateral to the dorsal surface (Figures 4H, 6A-C).  The nuclear division proceeds as usual, i.e., the macronuclear nodules fuse to form a single mass in middle dividers, which divides two times to produce the typical four nodules in late dividers. The micronuclei undergo mitotic division as usual (Figures 4E-H, 5E-H, 6A-C).

Occurrence and Ecology
As yet found only at the type locality, i.e., heavy metalcontaminated water from an industrial outlet in Onsan, Korea.
The main physico-chemical parameters during the sampling month, i.e., April 2016, were the following: water temperature, 15 • C; pH, 7.2; DO, 9.9 mg/L; BOD, 5.4 mg/L; COD, 8.7 mg/L; SS, 12.5 mg/L; T-N, 9.1 mg/L; T-P, 0.27 mg/L; TOC, 3.6 mg/L; EC, 813 µS/cm. The heavy metal concentrations were the following: Cd, 0.0132 mg/L; Pb, 0.0117 mg/L; As, 0.0808 mg/L; Hg, 0.0038 mg/L; Cu, 0.6945 mg/L; Zn, 3.3035 mg/L; Cr, 0.0674; and Ni 0.1513 mg/L. The concentrations of a nearby lesscontaminated site were Cd, less than the limit of quantification (LLQ); Pb, LLQ; As, LLQ; Hg, LLQ; Cu, 0.0495 mg/L; Zn, 0.1230 mg/L; Cr, 0.0222 mg/L; and Ni, 0.0167 mg/L. The heavy metal concentrations in most non-contaminated sites have not been measured so far. However, the concentrations of heavy metals in some of the measured ones were mostly LLQ. The coliform count was 11,000/100 mL (data retrieved from the water quality monitoring system of the Korean Ministry of Environment 3 ).

SSU rRNA Gene Sequence and Phylogeny
The SSU rRNA gene sequence of H. tolerans is 1,663 bp in length and has a GC content of 44.5%. Phylogenetic analyses inferred from the SSU rRNA gene sequences using ML and BI present similar topologies; therefore, only the ML tree is shown here (Figure 7). Phylogenetic trees consistently place the new species within the stylonychine oxytrichids, clustering in a clade with H. histrio.
The new species can be separated from H. complanatus in the arrangement of transverse cirri, i.e., five transverse cirri arranged in a more or less tick mark shape (vs. arranged in rows) of which four leftmost (vs. three rightmost) reaching up to the cell margin (vs. protrudes beyond the posterior end) of the cell, buccal cirrus at the anterior right (vs. distinctly posterior) of undulating membranes, and marginal cirri of the same length [vs. increase in length posteriorly; for which Berger (1999) mentioned that inconspicuous caudal cirri could be present] throughout the body (data from illustrations of Stokes, 1887;Vuxanovici, 1961).
H. vorax has been reported with rather variable shapes, i.e., about 170 µm (Stokes, 1891), small adult cells 70-140 × 50-60 µm, and large adult cells 190-250 × 100-120 µm (Curds, 1966). The body margin is often indented, with the left margin Anterior body end to distal end of adoral zone, distance 7.9 8.0  CV, coefficient of variation (%); M, median; Max, maximum; Mean, arithmetic mean; Min, minimum; n, number of individuals investigated; SD, standard deviation; SE, standard error of arithmetic mean. a Data based on mounted, protargol-impregnated, and randomly selected specimens from several clonal cultures fed with Chlorogonium elongatum (measurements in micrometer, µm). b Distal end of the adoral zone (Berger, 2006). concave and the right convex, which was not observed in the present species. The new species can be distinguished from H. vorax in having a smaller body length, i.e., 60-75 × 40-55 µm. Further differences include widely spaced and fewer marginal cirri (vs. narrowly spaced with 31-37 cirri in the right marginal row) in H. vorax. Berger (1999) mentioned the possibility that H. vorax may possess caudal cirri due to the longer and prominently projecting cirri beyond the posterior end of the cell.
H. similis can be distinguished from H. tolerans in having a long body in vivo, i.e., 110-130 µm (vs. 60-75 µm), body about three (vs. two) times as long as broad, and the adoral zone of membranelles was about 38% (vs. 51%) of body length.
The divisional morphogenesis of H. tolerans resembles that of H. histrio as described by Gupta et al. (2006). The only significant difference observed was that the oral primordium in H. tolerans does not contribute to the formation of anlage II of the proter (for a review, see Berger, 1999;Gupta et al., 2006). For the remaining Histriculus species, no morphogenetic data are known. However, Curds (1965Curds ( , 1966 reported morphogenesis in H. vorax by the formation of an endogenous bud that also needs to be reinvestigated since Pang and Zhang (1981) assumed it to be the cannibalism induced by overfeeding. No endogenous bud formation was observed in H. tolerans.

Phylogenetic Analyses
The SSU rRNA gene sequence of H. tolerans matches well with H. histrio (99% similarity and four base pair difference), the only species within the genus for which gene sequence is available. In the phylogenetic analyses, the new species clustered with the type species with the full support of 100/1.00 (ML/BI). The detailed analyses are restricted due to the limited sequences available within the genus; however, Histriculus is well fitted in the subfamily Stylonychinae clustering in a clade with Tetmemena, Stylonychia, and Sterkiella species (Figure 7).
In our analyses, the genus Sterkiella appeared to be a polyphyletic group, possibly due to the differences in the morphogenetic pattern (Kumar et al., , 2017Bharti et al., 2018). Similarly, a slight variation in morphogenesis was observed in the present study, i.e., no contribution of the oral primordium toward the formation of anlage II of the proter in contrast to the type species where such contribution was reported by Gupta et al. (2006). Furthermore, recent observations on the resting cyst structures have resulted in the identification of the cryptic ciliate species (Foissner, 2016;Kumar et al., 2016;Bharti et al., 2019). We believe that the detailed observations, i.e., future addition of cyst data along with morphology and morphogenesis, as well as molecular sequences, for the remaining Histriculus species will clarify whether the genus Histriculus stays monophyletic or polyphyletic.

Heavy Metal Toxicity in Ciliates
Ciliates have been reported to play a significant role in the bioaccumulation or bio-concentration since they come directly in contact with chemicals in contrast to other single-cell microbes, such as bacteria, yeast, and algae, that possess a cell wall and may affect the uptake of chemicals (Cooley et al., 1972;Carter and Cameron, 1973). Furthermore, several studies have highlighted the potential of protozoan ciliates as a model organism for various in vitro metal toxicological assessments as well as their role in the self-purification of natural aquatic ecosystems (Cooley et al., 1972;Carter and Cameron, 1973;Larsen et al., 1997;Pauli and Berger, 1997;Sauvant et al., 1999;Bogaerts et al., 2001;Schultz et al., 2005;Vilas-Boas et al., 2020).
The heavy metal concentration during the year (2015-2018) from the sampling site ranges significantly and on many occasions rose higher than the permissible levels, e.g., Cd concentration reached up to 0.252 mg/L, Pb up to 0.44 mg/L, and As up to 0.456 mg/L (data retrieved from the water quality monitoring system of the Korean Ministry of Environment, see text footnote 3). Thus, it is believed that the species present in this habitat must possess some defense mechanism to overcome the stress of the heavy metal. The present study reports a novel species that might have the potential to survive heavy metal stress and that it can be used as a model organism for evaluating heavy metal toxicity. However, further studies are required for generating reference data that can be used in future ecotoxicological analyses using H. tolerans.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are publicly available. This data can be found here: National Center for Biotechnology Information (NCBI) BioProject database under accession number OK324152.