**1. Introduction**

The ability of ciliates to form symbiotic associations with other microorganisms is well known [1–5]. Phagocytosis makes these protists predisposed to various infections, especially bacterial ones [6]. With intensification of sampling worldwide, the number of novel endosymbiotic bacteria discovered in ciliates is growing year by year [7–13]. Endosymbiotic systems in ciliates have long been studied in light of their importance in ecological systems and evolutionary impact [14–19]. Besides that, presumably the ancient origin of such associations enables using ciliate symbiotic systems as models for studying the evolution of early endosymbiosis of a protoeukaryote and the mitochondrial ancestor, which lay at the core of modern biodiversity [20]. Moreover, some recent findings support the hypothesis that ciliates, like some other protists, can harbor microbes potentially pathogenic for humans or economically significant animals [21–25]. Interestingly, endosymbiotic bacteria seem to be more frequent in the ciliate

species found in brackish water habitats, which might be connected with the ability of endosymbiotic bacteria to promote expansion of salinity tolerance in ciliates [26–28].

Extensive search for new endosymbiotic systems in ciliates occasionally reverts us to the endosymbionts described in the pre-molecular biology era, which lacked molecular techniques. Consequently, many of about 60 endosymbionts registered in *Paramecium* and beyond [29] had been described only morphologically and, therefore, are out of the scope of modern molecular phylogenetics. A pool of these endosymbionts, which are nowadays being rediscovered, for example, *Lyticum flagellatum* and *L. sinuosum* [30], as well as "*Candidatus* Trichorickettsia mobilis" [18], has been referred to as a hidden bacterial biodiversity from the past [7] (p. 1017).

For a long time, *Paramecium nephridiatum* has been considered an uncertain species [31]. It was rediscovered and described as a true morphospecies only in 1999 [32]. A number of bacterial endosymbionts have been found in the population of *P. nephridiatum*, at the time misidentified as *P. woodru*ffi, isolated from a tidepool on the Sredniy Island (Chupa Inlet, the White Sea) [29,33]. The morphology of three types of cytoplasmic bacteria has been described; however, their phylogenetic position has remained unclear, and two of them have not been given binomial names. The third one was described as *Pseudolyticum minutus*[33]. Nearly 30 years later, sampling in the same tidepool, we isolated a population of *P. nephridiatum* seemingly bearing the same set of endosymbionts. Here we provide a morphological and molecular characterization of two of these previously described cytoplasmic endosymbionts of *P. nephridiatum,* one of which we propose to name "*Ca.* Mystax nordicus", gen. nov., sp. nov., based on its appearance and geographic origin of the isolate.

#### **2. Materials and Methods**

#### *2.1. Cell Cultures and Live-Cell Experiments*

*Paramecium* strains BMS16-23, BMS16-34 and BMS17-1 were isolated in 2016 and 2017, respectively, from a population of *Paramecium* cells found in a brackish water pool in the littoral zone of the Sredniy island the White Sea, Russia (66◦1659.2" N 33◦4229.2" E). Cells of all three strains carried infection in their cytoplasm and were subjected to further investigation. A naïve *P. nephridiatum* strain, ETu5-1, maintained at RC CCM Culture Collection (Core Facility Center for Cultivation of Microorganisms) and kindly provided by N. A. Lebedeva, was used in experimental infections as well as in killer trait assessments. The cells were grown at 16 ◦C in boiled lettuce medium inoculated with *Klebsiella aerogenes*. Live-cell observations were made with a compression device [34] and a Leica 6000B microscope (Leica Microsystems, GmbH, Wetzlar, Germany) equipped with differential interference contrast (DIC) and a digital camera DFC500.

#### *2.2. Killer-Trait Assessment and Experimental Infection*

Killer tests were carried out as follows: 20 cells of the infected BMS17-1 strain were mixed with 10 *Paramecium* cells of ETu5-1 strain in a depression slide containing approximately 500 mL of cultivation medium. The number of living and motile cells was counted after 30 min, 60 min and 24 h.

The experimental infections were performed with homogenate made of infected BMS17-1 cells. The infected cells were washed several times with sterile lettuce medium and concentrated. Then the cells were homogenized using a syringe with a thin needle. The amount of the homogenate added to 20 naive ETu5-1 cells was calculated as a 1:10 ratio of naive to infected cells. A drop of medium containing food bacteria was added to stimulate phagocytosis. The efficiency of the infection was checked in 1.5 and 72 h after the start of the experiment with FISH.

#### *2.3. Transmission Electron Microscopy (TEM)*

Paramecia were fixed in phosphate buffer (0.1 M, pH 7.4) containing 1.6% paraformaldehyde and 2.5% glutaraldehyde for 1.5 h at room temperature as described by Szokoli and colleagues [35]. Then the cells were washed in the same buffer containing 12.5% sucrose and postfixed in 1.6% OsO4

(1 h at 4 ◦C). After that, the cells were dehydrated in ethanol gradient followed by ethanol/acetone mixture (1:1), 100% acetone and embedded in Epoxy embedding medium (FlukaChemie AG, St. Gallen, Switzerland) according to the manufacturer's protocol. The blocks were sectioned with a Leica EM UC6 Ultracut, and ultrathin sections were stained with aqueous 1% uranyl acetate followed by 1% lead citrate. All samples were examined with a JEM-1400 electron microscope (JEOL Ltd., Tokyo, Japan) at 90 kV.

Negative staining was performed to test for the presence of flagella on the symbiont surface. For this purpose, the host cells were washed several times with sterile lettuce medium, incubated in it for 24 h and then washed again before the sample preparation. Several cells were squashed, and a drop of the resulting suspension was transferred to a Formvar coated grid. The bacteria were allowed to precipitate for about 1 min, and then the grid was covered with 1% uranyl acetate in water for about 1 min. Then the liquid was removed with filter paper, and the grid was air-dried.

#### *2.4. Atomic Force Microscopy (AFM)*

A few live *Paramecium* cells were washed several times with sterile lettuce medium, incubated in it for 24 h and then washed again before the sample preparation. Then they were placed on a coverslip in a small drop of the sterile medium and ruptured with a needle. The contents of the ruptured cell were air-dried. The material was examined using NTEGRA Aura (NT MDT, Russia) in a semi-contact mode.

## *2.5. Molecular Characterization*

For total DNA extraction, approximately 100 cells were washed several times with sterile lettuce medium and fixed in 70% ethanol. The cells were homogenized with a syringe in 50 mM EDTA solution to inhibit DNA degradation by DNases in cell lysate. Total DNA was extracted with NucleoSpin®Plant DNA Extraction Kit (Macherey-Nagel GmbH & Co., Düren NRW, Germany) using the protocol for fungal DNA extraction.

The host species were preliminarily identified using morphological features [36], and their attribution was subsequently confirmed by sequencing 18S rRNA gene following the approach suggested by Petroni and colleagues [37]. The amplification of the host 18S rRNA genes from total DNA was performed with the 18S\_F9 [38] and 18S R1513Hypo primers (Table S1) [37]. PCR products were directly sequenced with 18S\_F9 and R1513Hypo or 18S\_R536, 18S\_F783, 18S\_F919 and 18S\_R1052 (Table S1) [39].

For molecular characterization of bacterial endosymbionts, almost the full 16S rRNA gene was amplified with the 16S\_αF19b and 16S\_R1522a primers (Table S1) [40]. The touchdown PCR protocol described by Szokoli and colleagues [35] was employed with modifications, and Thermo Scientific InsTAclone PCR Cloning Kit (Thermo Scientific, USA) was used for molecular cloning. Amplification products were cloned into the pTZ57R/<sup>T</sup> vector and transformed into competent JM107 *Escherichia coli* cells following the manufacturer's instructions. Bacterial colonies were used for colony PCR with M13 primers. PCR products were sequenced using M13 primers and 16S\_R1488\_Holo [30], 16S\_F343ND, 16S\_R515ND and 16S\_F785ND (Table S1) [40].

#### *2.6. Fluorescence in Situ Hybridization (FISH)*

To verify that the obtained 16S rRNA gene sequences were derived from the endosymbiotic bacteria, specific fluorescent oligonucleotide probes were designed: 16S\_Myst965 (5-CCT GTA CTA AAT CGG CCG AAC CG-3) and 16S\_MegVen1226 (5-CCG AAC TGA GAT GCC TTT TGAG-3) which were labeled with either Cy3 or FAM at the 5 end. The probes were synthesized and labeled with fluorescent dyes by Evrogen (Moscow, Russia). The newly designed probes were tested at 0%, 15% and 30% formamide (FA) concentrations. Their specificity was determined in silico using the tools TestProbe 3.0 (SILVA rRNA database project) [41] and the ProbeMatch of the Ribosomal Database Project (RDP) [42], allowing 0 mismatches. All FISH experiments were performed as described by Manz and colleagues [43]. The designed probes were used in combination with the almost universal eubacterial

probe Eub338 (5-FAM-GCT GCC TCC CGT AGG AGT-3) labeled with FAM at the 5 end [44]. The slides were mounted in Mowiol (Calbiochem, Germany) containing PPD (p-phenylenediamine) and DAPI prepared according to manufacturer's protocol. The slides were analyzed with a Leica TCS SPE Confocal Laser Scanning Microscope (CLSM).

## *2.7. Phylogenetic Analysis*

A total of 63 sequences of 16S rRNA genes were used for reconstruction of phylogeny. Among them, 5 sequences of distant relatives of Alphaproteobacteria and 8 sequences originating from 4 different classes of Proteobacteria (Epsilon-, Delta-, Beta- and Gamma-) were used as an outgroup. All the sequences were aligned with SSU-ALIGN 0.1.1 software and masked using default settings [45]. For the selection of model of nucleotide substitution, jModelTest2.1 was employed, and GTR+I+G model was chosen [46]. Phylogenetic analysis was performed with PhyML 3.1 with 1000 pseudo-replicates [47]. The final tree was visualized with FigTree v1.4.3 [48].

## *2.8. Accession Numbers*

The sequences of 16S and 18S rRNA genes were submitted to NCBI GenBank and were assigned accession numbers MK764889 and MK764894 (*P. nephridiatum*); MK775140 and MK775139 ("*Ca.* Megaira venefica"); MK673804 ("*Ca.* Mystax nordicus").
