Photobiont Flexibility in Paramecium bursaria : Double and Triple Photobiont CoHabitation

The green ciliate, Paramecium bursaria, has evolved a mutualistic relationship with endosymbiotic green algae (photobionts). Under culture conditions, photobionts are usually unified (to be single species) within each P. bursaria strain. In most cases, the algal partners are restricted to either Chlorella variabilis or Micractinium reisseri (Chlorellaceae, Trebouxiophyceae). Both species are characterized by particular physiology and atypical group I intron insertions, although they are morphologically indistinguishable from each other or from other Chlorella-related species. Both algae are exclusive species that are viable only within P. bursaria cells, and therefore their symbiotic relationship can be considered persistent. In a few cases, the other algal species have been reported as P. bursaria photobionts. Namely, P. bursaria have occasionally replaced their photobiont partner. This paper introduces some P. bursaria strains that maintain more than one species of algae for a long period. This situation prompts speculations about flexibility of host-photobiont relationships, how P. bursaria replaced these photobionts, and the infection theory of the group I introns.


Introduction
The green ciliate Paramecium bursaria is one of the most studied protists due to its observable endosymbiosis.Their symbiotic relationship is able to start over, i.e., artificially algae-removed P. bursaria can absorb again and fix the algae as new photobionts [1].Despite the re-symbiosis ability of P. bursaria, there are unusual characteristics in terms of the small diversity of their photobionts.Although almost 50 strains of photobionts (partly directly gained sequences from P. bursaria extracts) have been genetically identified, most belong to either Chlorella variabilis or Micractinium reisseri (Chlorellaceae, Trebouxiophyceae) [2][3][4][5][6][7][8][9][10][11].Neither species has ever been collected as a free-living species from natural water sources.This is possibly due to following reasons.Both species are essentially nutritionally fastidious [e.g., 12,13].Additionally, they are very sensitive to the Paramecium bursaria Chlorella virus (PBCV), which is abundant in natural water sources [14][15][16].Escaped photobionts from P. bursaria cell would be attacked by PBCV immediately.Both species appear to be highly dependent on their host refuge.Chlorella variabilis and M. reisseri are therefore thought to have adapted to exclusively dwell in P. bursaria.In a few cases, P. bursaria is associated with other species of Chlorella or Scenedesmus (Chlorophyceae) [3,9,11].Namely, P. bursaria has replaced its photobionts on several occasions.
Lichens and corals, representatives of symbiotic associations with algal symbionts, experience a period without symbionts in their life cycle and must acquire fresh algae as symbionts to complete their life cycle.There is no such symbiont-less period for P. bursaria; the algae are retained through cell division as well as sexual reproduction [17].Consequently, the symbiotic relationship with P. bursaria appears to be permanent.However, diversity in the photobionts, as mentioned above, does exist.Thus, it is not understood how P. bursaria gain such algal diversity.
Group I intron evidence shows that C. variabilis and M. reisseri have co-habited in a P. bursaria cell.Group I introns are a distinct RNA group that function as enzymes, splicing themselves out of precursor RNA transcripts and ligating exons.A distinctive character of group I introns is their mobility.Phylogenetic analyses have indicated that introns at homologous gene sites are related (position family), even among distantly related host organisms.This phenomenon is linked to intron spreading mechanisms.Namely, when an intron at a locus of a gene infects a different organism, the new intron will be inserted into the same locus of the gene in which it was originally located (For general characters on group I introns, see Cech [18]; Haugen et al. [19] and references therein).Group I introns are classified into subgroups IA through IE based on their structural diversity and phylogeny, and nuclear encoding introns belong to subgroup IC or IE [20].Chlorella variabilis is particularly intron-rich, containing eight group I introns (four IC and four IE) in the nuclear rDNA [21], and M. reisseri also has two IE introns [22] (Figure 1).Due to the intron spreading mechanisms, IE introns have strong insertion bias to limited positions.In green algae, nearly all IE introns are located at S516 (S = SSU rRNA; the numbering reflects their homologous position in the Escherichia coli rRNA gene) [20,23].However, four (S943, L1688, L2184, and L2449; L = LSU rRNA) out of six introns of P. bursaria photobionts occupied novel insertion sites.Structural and phylogenetic analyses of these IE introns led to the following extremely bizarre findings: 1) these IE introns are monophyletic and independent of those from other green algae; 2) one intron (L2449) of M. reisseri has an archaic state and the other introns are assumed to originate from this intron; 3) the completion of the hereditary line includes two transfer events beyond the species barrier (Figure 1).Explaining these intron transmissions requires a special situation in which two algal species have frequently been in contact with each other [22].It is just as conceivable that there was a long period during which C. variabilis and M. reisseri lived sympatrically and simultaneously in the P. bursaria cell, where cell-cell contact within a small space may accelerate the lateral transfer of group I introns [24,25].The present study will introduce some P. bursaria strains that maintain more than one photobiont species in a long period of culture.These strains encourage the above intron transfer theory, and possibly indicate the way to photobiont switch of P. bursaria.

Paramecium bursaria Culture
Particular kind of Paramecium bursaria strains were maintained in lettuce juice medium [26] under LED illumination (12 h L:12 h D) at 15˚C (Table 1).These strains were once collected by Dr. T. Kosaka (Hiroshima University) in 1992 in the United States and have been maintained in a laboratory at the University.The stock cultures were kindly donated by Prof. H. Hosoya (Hiroshima University) to RH in December 2008 and have been cultured for more than four years; therefore symbiotic conditions should be regarded as stable.

Algal Isolation
Individuals of P. bursaria were carefully picked from the surface of the culture medium (to avoid picking up the coccoids on the bottom of the flask, though there were not many), the cells were disrupted and suspended in pure water then spread onto an oligotrophic agar plate (1/5-concentration Gamborg's B-5 Basal Medium with Minimal Organics, Sigma Aldrich, St Louis).Observed colonies were picked and transferred to 1/5 Gamborg liquid medium.These were maintained under LED illumination (12 h L:12 h D) at 15˚C.

Microscopy
Cells of P. bursaria and their symbiotic algae were observed under light microscopy CX31 (Olympus, Tokyo) and photos were taken with an HDCE-31 digital camera (AS ONE, Osaka).

DNA Extraction, Amplification, and Sequencing
Paramecium bursaria strains with photobionts in each cell were directly used to extract both host and photobiont DNAs.A photobiont strain AG-35_ZF1 isolated from P. bursaria AG-35 (Table 1) was also used in DNA extraction.DNA extractions were performed using the DNeasy plant mini kit (Qiagen, Düsseldorf).Whole P. bursaria DNA extracts were used to amplify both host SSU rDNA and photobiont SSU rDNA.Host targeting PCR was performed with the primer pair SR-1 (universal [27])/Paramecium800R (host specific [6]), and algae targeting PCR was also performed with CHspeR-maeF (trebouxiophyte specific [6])/INT-5R (trebouxio- phyte specific [5]) for the photobiont.The PCR product of the host DNA was purified via Quantum Prep PCR Kleen Spin Columns (Bio-Rad, CA) and directly sequenced.The amplified fragments of photobiont DNA were confirmed by agarose gel electrophoresis, collected by excising the fluorescent band, purified using the Qiaex II Gel Extraction Kit (Qiagen), and directly sequenced.SSU rDNA to internal transcribed spacer 2 of the isolated alga, AG-35_ZF1, was amplified with the primer pairs SR-1/SR-9, SR-6/SR12k, and INT4F/HLR3R (primers are described in Hoshina et al. [6]).These were purified via Quantum Prep PCR Kleen Spin Columns, and directly sequenced.

DNA Sequence Comparisons
Paramecium bursaria and its photobiont DNA sequences were deposited to Standard Nucleotide BLAST (http:// blast.ncbi.nlm.nih.gov/) to find identical or close sequence data.

Results
The P. bursaria strains here were obviously different from the ordinary ones.Under the microscope, green coccoids of different sizes (ca. 5 µm or 1.5 µm) were clearly seen in P. bursaria (strains AG-35, BP-11, and OLG-3) (Figure 2).Note that ordinary P. bursaria retains a cloned single-species of alga (ca. 5 µm).The number of small balls was much larger than the large ones except DC-3 (small balls were remarkably few).The small coccoids were inserted in between the trichocysts (Figure 2(c)).
Next, we conducted algae-targeting PCR (18).Three P. bursaria strains (BP-11, DC-3, and OLG-3) produced two length-polymorphic bands, whereas strain AG-35 produced three bands (Figure 3).The length polymorphisms were due to the variations in intron insertions.BLAST search indicated the sequences were identical with C. variabilis (large bands: AB699101-104), Chori-cystis minor (medium bands: AB699105-110), and some Chlorella and Micractinium species (small band: AB699111) (Table 1).The amplified region (exon) was fairly conservative among Chlorella-related species and we were unable to identify the small band to the species level.
On the oligotrophic agar plates with the P. bursaria extract, only small coccoids were obtained from BP-11, DC-3, and OLG-3 extracts.Because C. variabilis requires organic nitrogen sources to grow [13], this oligotrophic condition might have prevented its growth.Both sizes of green coccoids were present on the plate from the AG-35 extract.We picked up several green colonies from this plate and transferred each to liquid medium.One of the algal strains, an approximately 5 µm coccoid, was named AG-35_ZF1 and we sequenced its SSU-ITS1-5.8S-ITS2rDNA.The sequence (AB-699112) of the isolated alga, AG-35_ZF1, was very close to those of C. vulgaris.An NCBI BLAST Search indicated the closest taxon, C. vulgaris CCAP 211/80 (FM205853, covering ITS1-5.8S-ITS2rDNA), where only two transitions and one indel were found within the ITS1 region.It is likely that this photobiont is C. vulgaris.
SSU rDNAs for P. bursaria (host) were determined.Of these, three (AG-35, BP-11, and DC3: AB699097-099) were identical and matched some previous sequences we refer to as genotype D [28], whereas OLG-3 (AB699100) differed from the others and matched what we refer to as genotype B.

Discussion
Paramecium bursaria usually gains energy by feeding as well as by the photosynthates of photobionts.In general, P. bursaria collected from nature may contain more than one green alga.Photobiont and feed are difficult to determine.These algae will be unifiedin the cell of P. bursaria during several days of culture conditions.In most cases, the remaining algae (namely, natural photobionts) are either C. variabilis or M. reisseri.However, the algae  of P. bursaria strains cited here have not been unified during 20 years of culture conditions.The mother stock of OLG-3 also has shown the same feature [29].Microscopic observations showed C. minor (small coccoids) were inserted in between the trichocysts.This phenomenon can be thought of as the advanced symbiosis stage rather than feed in the food vacuole [30].Therefore, it is regarded that these P. bursaria strains deal both C. variabilis and C. minor as their steady photobionts.This situation, namely, stable symbiotic relationships between P. bursaria and multiple photobionts, will encourage the hypothetical theory for the group I intron transmitions between C. variabilis and M. reisseri (Figure 1).
The present study also focused on the genotype of the host P. bursaria.We reported that P. bursaria is separated into A through D genotypes based on SSU rDNA [28].Genotype D seems to be the first diverged genotype, with differences of at least 12 substitutions and four indels from the others.For these circumstances, Pröschold et al. [9] suggested that P. bursaria is a complex of several species, similar to the P. aurelia complex.We previously found that host genotypes and these photobiont types are closely linked [6], and proposed a P. bursaria evolutionary scenario concerning genotype diversification and photobiont choice [31] (Figure 4).The strain OLG-3 is the first P. bursaria that retains C. variabilis among genotypes A to C and this characteristic negates the evolutionary scenario.Instead, the flexibility of the combination of hosts and photobionts becomes apparent.It is possible that P. bursaria groups exchange their photobionts in some way.In most cases, the most preferred photobiont is either C. variabilis or M. reisseri.Geographic or climatic conditions probably influence the photobiont choice of P. bursaria.
Our findings also give us an imagination about how P. bursaria have replaced their photobionts.Compared to other protozoa that carry coccoid green photobionts, the host-symbiont relationship is much stronger in P. bursaria.In many of the other protozoa, the species of photobionts depends on environment (e.g., lake, pond) rather than host species [10].P. bursaria carries the symbiotic algae throughout its life cycle even during cell division and sexual reproduction [17].Paramecium bursaria has lost none of its ability to take in algae as new symbionts.Consequently, algal switching can occur in two ways.The first mechanism is "new gain after symbiont loss" (Figure 5).Alternatively, we propose another process for algal switching: "choice after co-symbiosis" (Figure 5).Because P. bursaria maintains its re-symbiosis ability, it is possible that it routinely tries to achieve other symbionts through feeding and temporary symbiosis.As mentioned above, C. variabilis and M.  reisseri are heavily dependent on P. bursaria.However, the photobionts are not indispensable to P. bursaria.In other words, P. bursaria is in a position to be selective about photobionts.The P. bursaria strains shown here (Figure 2) can be thought of a process to choose for a better partner.Although C. variabilis and C. vulgaris are not distinguishable under microscopic observation, P. bursaria AG-35 showed the triple photobiont status of C. variabilis, C. vulgaris, and small Choricystis (Figure 3).Chlorella vulgaris is a well-known cosmopolitan coccoid and Choricystis minor is the most common eukaryotic picoalgae in freshwater environments[e.g., 32,33]; therefore, P. bursaria can ingest them at any time.Photosynthate contributions from C. vulgaris or Choricystis in P. bursaria are not known, however this must have occurred in the event of symbiont switching given that P. bursaria possessing C. vulgaris have been found [3,9].
In a single host individual, multiple symbiont species performing similar-functions often have negative effects on the host's growth [34].However, if it is regarded as a phase for determining a more optimal partner, it can be an advantage for survival in the long term.

Figure 1 .
Figure 1.Group I introns intervening in rDNAs of Chlorella variabilis and Micractinium reisseri.Subgroup IE introns are in bold.Numbering reflects their homologous position in the Escherichia coli rRNA gene: S = SSU rRNA, L = LSU rRNA.The transmission contexts of IE introns are indicated by thick (inter specific) and narrow (intra specific) arrows.For details of intron transmission, see Hoshina and Imamura [22].

Figure 3 .
Figure 3. PCR results for four Paramecium bursaria strains.This PCR targeted only green algae using the specific primers CHspeRmaeF/INT-5R.Each P. bursaria strain produced differently sized bands attributed to intron insertions, which indicate that Paramecium maintains two or three kinds of green algal species.Fluorescent values of the bands are presumably influenced by cell wall disruption (C.variabilis is indestructible) or primer matching (four out of 20 nucleotides in the forward primer do not match Choricystis = medium-size band).

Figure 5 .
Figure 5. Two possible contexts in which Paramecium bursaria may switch photobionts.

Table 1 . Paramecium bursaria strains used in this study and their sequence data.
*Offspring of original strain.