Algal Diversity in Paramecium bursaria: Species Identification, Detection of Choricystis parasitica, and Assessment of the Interaction Specificity

The ‘green’ ciliate Paramecium bursaria lives in mutualistic symbiosis with green algae belonging to the species Chlorella variabilis or Micractinium conductrix. We analysed the diversity of algal endosymbionts and their P. bursaria hosts in nine strains from geographically diverse origins. Therefore, their phylogenies using different molecular markers were inferred. The green paramecia belong to different syngens of P. bursaria. The intracellular algae were assigned to Chl. variabilis, M. conductrix or, surprisingly, Choricystis parasitica. This usually free-living alga co-occurs with M. conductrix in the host’s cytoplasm. Addressing the potential status of Chor. parasitica as second additional endosymbiont, we determined if it is capable of symbiosis establishment and replication within a host cell. Symbiont-free P. bursaria were generated by cycloheximid treatment. Those aposymbiotic P. bursaria were used for experimental infections to investigate the symbiosis specificity not only between P. bursaria and Chor. parasitica but including also Chl. variabilis and M. conductrix. For each algae we observed the uptake and incorporation in individual perialgal vacuoles. These host-symbiont associations are stable since more than five months. Thus, Chor. parasitica and P. bursaria can form an intimate and long-term interaction. This study provides new insights into the diversity of P. bursaria algal symbionts.

This study addresses the diversity of intracellular algae in P. bursaria. Therefore, ciliate hosts and algal endosymbionts were identified using different molecular markers. In order to analyse the interaction between the three different algal species detected and P. bursaria, experimental re-and cross-infections were performed and the fate of the intracellular algae was monitored. Differences in the establishment of stable associations will allow to draw conclusions regarding the compatibility of the different algae as endosymbionts of P. bursaria and the specificity of such interactions.

Observation and Size Determination of Intracellular Algae
The intracellular algae were investigated by detecting the autofluorescence of the chlorophyll by fluorescence microscopy (Axio Imager.M2, Zeiss, Jena, Germany). Therefore, Paramecium cells were fixed with Bouin's fixative solution (Sigma-Aldrich) and transferred to a microscope slide. Cells were examined using the Cy5 channel (EX BP 640/30, BS FT 660, EM BP 690/50) and an exposure time ranging from 3000 to 7000 ms in order to discriminate between the individual intracellular algae.
To determine the size of the intracellular micro-and picoalgae, the diameter of 15 cells per strain was measured.

DNA Extraction and Amplification of Molecular Markers
All polymerase chain reactions (PCR) were performed with a PeqStar 2× thermocycler (Peqlab, VWR International, Darmstadt, Germany) using TaKaRa reagents and TaKaRa ExTaq polymerase (TaKaRa Bio, Otsu, Japan). Primer sequences ( Table 2) and amplification protocols (Supplementary Tables S1 and S2) are provided. The SSU-ITS1-5.8S-ITS2-5'LSU rRNA gene region of P. bursaria was amplified using the primer combinations Penic_F82 and Penic_R1280 and Penic_F661 and 28S_R457. Primers Penic_F82, Penic_F661 and 28S_R457 were used for sequencing. For the intracellular algae, the nuclear encoded SSU gene and the ITS region were amplified using a semi-nested PCR approach to obtain sufficient DNA amounts for direct sequencing. For the initial amplification (primer combination: AF and ITS055R) as well as for the two semi-nested amplification reactions (primer combinations: AF and Chlo_R841 and Chlo_G800F and ITS055R) the protocol described by Pröschold and colleagues [10] was used. AF, Chlo_F238, Chlo_G800F, Chlo_R841 and ITS055R were used as sequencing primers. For strains Scot and Old-Pf amplification was carried out with primer combination Chlo_G800F and ITS055R (Supplementary Tables S1 and S2) and both primers for sequencing. Additionally, a diagnostic PCR allowing to discriminate between Chl. variabilis and M. conductrix was carried out as described elsewhere [50].
To amplify the SSU gene of Choricystis from strain Frieds the primer combination Chori_F238 and Chori_R841 was used. Those primers were designed to specifically match Choricystis-like algae based on preliminary sequence data. Chori_F238 was used for sequencing.
Purified PCR products (QIAquick PCR Purification Kit, Qiagen, Hilden, Germany) were sequenced at GATC Biotech (Konstanz, Germany). Accession numbers of obtained sequences are provided in Table 1.

Molecular Characterization of Paramecium bursaria
In order to confirm our morphological identification of the ciliates as members of P. bursaria, we sequenced the SSU rRNA gene sequence and the ITS region of our newly obtained strains. Both markers are commonly applied for characterization at generic and species level [5,[42][43][44]47,50].
The obtained sequences were imported into two datasets: (i) The SSU sequences were included into a dataset of representatives of most Paramecium species received from the SILVA SSU Ref NR 99 release 138 database [64]. In addition, nine other members of Peniculidae were included as outgroup resulting in a dataset of 59 sequences with a length of 1493 bp. (ii) The ITS sequences were included into an alignment of 32 P. bursaria strains (506 bp) as described in previous studies [47,50]. For syngen assignment, we added sequences of additional strains (Ard10, Bob2, Ek), which were tested for their mating behaviour by RC CCM to increase the number of sequenced strains per syngen. The ITS sequences were aligned according to their secondary structures.
The best fitting evolutionary models for these datasets were determined using the automated model selection tool implemented in PAUP (version 4.0a, built 167) [65]. The best model (SSU: GTR+I+G; ITS: K81uf+I) was chosen following the Akaike Information Criterion (AIC). For the Bayesian analyses, Bayesian Information Criteria (BIC) were chosen (SSU: GTR+I+G; ITS: GTR+I) for calculation in MrBayes (version 3.2.6 x64; [66]). Non-paramectric Maximum Likelihood (ML) analysis was estimated on 1000 pseudoreplicates (PHYML 2.4.5 from the ARB software package; [67]). Bayesian Interference (BI) analysis was carried out running three runs with one cold and three heated Markov Chain Monte Carlo chains for 1,000,000 generations and sampling the first 25% of the generations as burn-in.

Molecular Characterization of Green Algal Endosymbionts in Paramecium bursaria
For the molecular characterization of the algal endosymbionts we used sequences covering the SSU-ITS1-5.8S-ITS2 region for discrimination at species level in accordance with previous studies [10,55,56,62,68,69]. Especially the ITS2 region of this nuclear encoded ribosomal operon is often used for species discrimination between members of the Chlorellaceae [53,55,56,70]. The here performed amplification of the ITS2 region covered helices I-III but only partial helix IV. The analysed dataset comprised 50 sequences of representative members of the Chlorellaceae, which were aligned according to the secondary structures predicted by the software mfold [71], following the approach of Pröschold and colleagues [10,55]. This software used the thermodynamic model (minimal energy) for RNA folding. The best fitting evolutionary model for this dataset was predicted and ML and BI analyses were conducted as described above except that BI analysis was run for 5,000,000 generations. Additionally, the Neighbour Joining method (implemented in PAUP) was used. The analysis was run with 1000 bootstrap replicates and a 50% majority rule consensus tree was calculated.
Furthermore, we compared the algal endosymbionts' secondary structures of ITS2 helices I-III to that of the type strains of Chl. variabilis and M. conductrix. In case of the symbionts of strains Bob2, Ek, and RanNy these algae were identified by means of diagnostic PCR (Supplementary Figure S1).
To identify the intracellular picoalgae of P. bursaria observed in strain Frieds, we used the obtained partial SSU gene sequence and compared it with 41 representative sequences for the Trebouxia lineage (Trebouxiophyceae; following Darienko and colleagues [62]) with the Prasiola clade (four species) and the genus Neocystis (two species) as outgroup. The alignment was obtained as described for Paramecium SSU and comprised 948 positions. Model selection (TIM+I+G) for this dataset was performed as detailed above. Since this model is not implemented in MrBayes, it was substituted with GTR+I+G. ML and BI analyses were performed as described above for P. bursaria. The settings of all used evolutionary models are provided in Supplementary Table S3.

Establishment of Aposymbiotic Paramecium bursaria
Algal-free P. bursaria were generated by treatment with cycloheximid (Roth, Karlsruhe, Germany). Therefore, symbiotic P. bursaria cultures not fed for at least seven days were incubated in 10 µg/mL cycloheximid solution and kept under constant light conditions for approximately three days, then the cells were washed with Dryl's solution by filtering over a 10 µm membrane and fed with bacterized CM. This filtering step was repeated three to five times over the course of two weeks to assure complete elimination of the algae. If necessary, a second consecutive cycloheximid treatment was performed seven to ten days after the initial exposure. Microscopic inspection via fluorescence microscopy confirmed the elimination of intracellular algae. Aposymbiotic status was confirmed if after 10 seconds of exposure time no autofluorescence signal of the algae's chloroplasts was detected.

Pulse-Chase Infection Experiments
To investigate the symbiosis specificity between P. bursaria and three different symbiotic algal species, we exposed aposymbiotic cells (strains JPN, RanNy, Scot; affiliated to different syngens, see below) to isolated Chl. variabilis, M. conductrix, or Chor. parasitica (Figure 1). Aposymbiotic P. bursaria generated by treatment with cycloheximid were exposed (recipient) to isolated algae obtained from symbiotic P. bursaria cultures (donor; Supplementary Table S4). In re-infection assays, the supplied algae belonged to the same algal species as the recipient strain originally harboured, while in cross-infection experiments a different species was provided. We followed the fate of the algae after their uptake and differentiated between digestion, expulsion, and endosymbiotic maintenance by fluorescence microscopy using the above listed parameters. Each experiment comprised three replicates. In total, 20 aposymbiotic Paramecium cells were exposed to a suspension of the respective alga for 5 min with a ratio of 3 × 10 3 algae per ciliate cell. Then the paramecia were washed and transferred to 200 µL Dryl's solution inoculated with 20 µL of bacterized CM. 20 µL bacterized CM were added every second day for seven to ten days. Afterwards, the amount of supplied food was adjusted with respect to cell densities. A regular weekly feeding schedule was implemented after two to three weeks post infection (p.i.). The establishment of the symbiosis was monitored using the fluorescence signal of the algae's chlorophyll as described above. Instead of Bouin's solution, the Paramecium cells were fixed with 2% paraformaldehyde (PFA), which resulted in lower background signal and thus an improved signal to noise ratio. To monitor the process of re-establishment of symbiosis, P. bursaria cells were microscopically screened after two, four, seven, ten, and 14 days. Successful infection was confirmed when individually enclosed algae were localized beneath the host's cell cortex.  Table S4. Algal culture of Choricystis parasitica was obtained via mechanical lysis of P. bursaria strain Frieds for subsequent extracellular cultivation. This algal culture was then used in infection experiments with aposymbiotic P. bursaria cells (recipient). After 5 min of exposure (pulse), the paramecia were washed to remove extracellular algae and placed into fresh medium inoculated with food bacteria (chase). Status of symbiosis was monitored regularly via autofluorescence microscopy.

Microscopic Investigation of the Intracellular Algae
Microscopic observations of the nine P. bursaria strains (Table 1) confirmed the presence of intracellular green algae (data not shown). In case of strain Frieds, algal cells belonging to two size categories were detected (Figure 2). Algae belonging to the larger category showed cell diameters of 4.08 ± 0.02 µm (referred to as microalgae) while the smaller algae had a diameter of 1.40 ± 0.20 µm (picoalgae). The picoalgae were localized individually throughout the cytoplasm, especially near the host's cell cortex. In addition, several of these picoalgae were observed aggregated in digestive vacuoles ( Figure 2B). In a single occasion we even followed the fate of a digestive vacuole comprising more than 20 algal cells being emptied into the surrounding medium and thus Chor. parasitica were expelled.

Paramecium bursaria and the Five Syngens
The phylogenetic inference of Paramecium species based on SSU rRNA gene sequences (Figure 3) recovers the expected topology and composition of the five subgenera of this genus. All here obtained sequences cluster within the monophyletic and maximal supported subgenus Chloroparamecium. Subgroups within the P. bursaria clade can be observed (Figure 3), but the achieved resolution does not allow unambiguous identification of syngen affiliations. In order to discriminate between the five syngens of P. bursaria, we analysed the ITS1-5.8S-ITS2-5'LSU region alone (Figure 4). While the here characterized sequences span from SSU till the beginning of the LSU (except for strain Ard10), the majority of publicly available sequences of P. bursaria strains with known syngen affiliation are limited to the ITS region. Sequences obtained here cluster either with (Figure 4) P. bursaria syngen R1 (Old-Pf and Scot), R2 (Bob2, Ek, Frieds, and RanNy), R3 (JPN and Tue2015), or R4 (Ard10). A potential correlation between the host's syngen affiliation and the present algal symbiont can be observed. Strains affiliated to syngens R3, R4, and R5 harbour Chl. variabilis while those assigned to syngen R2 contain M. conductrix. Only in case of syngen R1 both algae have been described as symbionts.  The five syngens are highlighted as R1 to R5, a plus marks a strain whose mating behaviour was previously experimentally determined. The identity of the algal symbiont if known is provided. Sequences marked in bold were obtained in this study.

Chlorella variabilis, Micractinium conductrix, and Choricystis parasitica as Paramecium's Endosymbionts
Phylogenetic inference of the intracellular microalgae of P. bursaria based on sequences spanning the SSU-ITS1-5.8S-ITS2 region ( Figure 5) reveals the genera Chlorella and Micractinium as two distinct monophyletic clades. The intracellular microalgal strains JPN and Tue2015 cluster with other Chl. variabilis strains with maximum support in the phylogenetic analyses. Frieds, Old-Pf, and Scot affiliate with sequences belonging to M. conductrix with maximum support.
The sequence of the second intracellular alga detected in strain Frieds clusters with other sequences of the genus Choricystis as monophyletic sister group to the Elliptochloris clade with high support in both analyses ( Figure 6). The obtained sequence is identical to that of several other Chor. parasitica strains.

Establishment of Symbioses
We followed the fate of isolated algae after their uptake and differentiated between digestion, expulsion, and endosymbiotic maintenance by aposymbiotic P. bursaria cells via fluorescence microscopy. Within five days after exposure of aposymbiotic P. bursaria cells ( Figure 7A,D), digestive vacuoles were observed in all paramecia predominantly occurring in the central part of the Paramecium cell with more than one enclosed alga regardless of the supplied species. Multiple perialgal vacuoles enclosing single algal cells were observed throughout the entire host cytoplasm. After 12 to 15 days p.i., numerous perialgal vacuoles were localized in the cytoplasm near the host's cell cortex in each examined Paramecium cell ( Figure 7B,E). Successful re-establishment of symbiosis was achieved for all tested combinations (Supplementary Table S4) regardless of host background (former Chlorella or Micractinium host), syngen affiliation, and algal identity (Chl. variabilis, M. conductrix, or Chor. parasitica). Thus, we observed no differences between re-versus cross-infection and no preference of certain P. bursaria syngens for specific algae. After four months of cultivation, infected P. bursaria cells harbour numerous (200-500) intracellular algal symbionts. The observed symbiotic conditions are comparable to the natural state ( Figure 7C,F). These cultures are stable since over five months and are maintained to date in the laboratory.

Symbiotic Relationships and Specificity of Paramecium bursaria and Its Green Algal Endosymbionts
Previous studies showed that aposymbiotic P. bursaria are able to re-establish symbiosis with their intracellular microalgae [19,72]. After uptake via phagocytosis, most of the algae are incorporated in digestive vacuoles. Some manage to escape the host's lysosomal fusion by 'budding off' into the cytoplasm being enclosed in perialgal vacuoles. Subsequently, those horizontally transmitted algae are distributed throughout the host cell as well as beneath the cell cortex [73]. Based on the characterization of natural P. bursaria-algae symbioses, it was speculated that the occurrence of certain intracellular algae species might depend on the geographic origin of P. bursaria. The observed endosymbionts separated into two groups [10,12,13,[74][75][76], i.e., the "American/Southern" (later on described as Chl. variabilis) and the "European/Northern" (M. conductrix) group. Thus, a correlation between endosymbiont and host population was assumed. Another recent study presents contradicting results reporting the first Chl. variabilis from European P. bursaria [50].
Actual testing the specificity of symbiotic partners accepted by P. bursaria ( [77], this study) revealed that all tested intracellular algae derived from this ciliate were accepted as endosymbionts. On the other hand, free-living algae and those obtained from Hydra were digested [77]. A competition experiment revealed a clear preference of the aposymbiotic P. bursaria strain for its native Chlorella strain [77]. In the here conducted re-and cross-infection experiments without of a competitive setting, algae-free P. bursaria did not show a preference for their original symbiont or symbiont species. Aposymbiotic strains formerly harbouring Chl. variabilis maintained M. conductrix as endosymbiont, and vice versa. The picoalga Chor. parasitica, naturally sharing the cytoplasm of its host with M. conductrix, was able to form a stable symbiosis independently of co-occurring microalgae and with cells naturally harbouring Chl. variabilis. Even though P. bursaria establishes an intimate and long-term stable symbiotic association with Chor. parasitica, the role of the picoalga for P. bursaria remains unclear. An evaluation of the symbiotic relationship between P. bursaria and the intracellular picoalga Chor. parasitica awaits further detailed analyses as current findings do not allow a placement of this interaction on the mutualism-parasitism continuum.

Molecular Characterization of Paramecium bursaria and Its Green Algal Endosymbionts
Unsurprisingly, the novel isolates of 'green' ciliates were unambiguously confirmed as P. bursaria. The phylogenetic tree inferred from SSU sequences is in overall good agreement with those previously published [5,42,44]. It already indicates intraspecific diversity, which becomes obvious in the analysis of the ITS region whose results reflect previous findings [47,50]. The analysis of the currently available ITS data might be interpreted that certain P. bursaria syngens harbour preferentially certain algal symbiont species. On the other hand, the number of symbiotic systems with both partners fully characterized is small, therefore this result should be considered very cautiously. It is likely that the picture will change with an increased amount of characterized strains. A revision of the P. bursaria species complex and conclusions about the suitability of certain syngens as hosts for specific algae await future studies.
The here characterized algal symbionts Chl. variabilis and M. conductrix are morphologically nearly identical [50]. However, they clearly differ in size from the additional algal symbiont detected in strain Frieds. The observed sizes in case of the microalgae were at the minimal range described for M. conductrix (5.0-12.0 µm, [10]) and in the typical size range for intracellular Chor. parasitica (1.5-3.0 µm) in case of the picoalgae. In the phylogenetic inference based on SSU-ITS sequences, the genera Chlorella and Micractinium were recovered as monophyletic and maximum support was obtained for the associations of the here studied microalgae with either Chl. variabilis or M. conductrix. The tree topology is nearly identical to that of previous studies [62,68] except for small differences lacking support in each analysis. The recovery of both algal species as endosymbiont of P. bursaria was not surprising, the detection of picoalgae in strain Frieds and their identification as Chor. parasitica more so. This is the first report of these picoalgae co-occurring with M. conductrix in the cytoplasm of P. bursaria and only the third report of Chor. parasitica as potential endosymbiont of P. bursaria. In previous observations the co-occuring primary symbiont was identified as Chlorella [31,40].

Conclusions
Paramecium bursaria is well adapted to harbour green algae as symbionts. Almost exclusively, either Chl. variabilis or M. conductrix are found in high abundances in the cytoplasm of this ciliate. One route to address the specificity of these associations is the molecular characterizations of host and symbionts. This approach provides information which symbioses are ecologically and evolutionary successful in the studied habitat. It is important to stress that therefore host and symbiont should be characterized in parallel, which is not always the case. Molecular approaches need to be combined with microscopical observations to ensure that indeed all partners are accounted for and none is overlooked or dismissed as environmental contamination. This might easily happen with minor symbionts in multiple infections or with much smaller and intracellularly less abundant picoalgae. Experiments to test the ability for symbiosis formation provide an additional perspective to the observed occurrence of associations in the environment. Infection assays can examine which interactions can be formed at e.g., species or genotype level. Thus, they provide insights into the genetic broadness of potentially realizable symbioses under constant laboratory conditions and allow to entangle the impact of additional biotic or abiotic factors shaping the formation and occurrence of symbioses. The present work can serve as a roadmap how such analyses can be conducted in regard to molecular as well as physiological characterization.