Advances in Microbiology, 2012, 2, 227-233
http://dx.doi.org/10.4236/aim.2012.23027 Published Online September 2012 (http://www.SciRP.org/journal/aim)
Photobiont Flexibility in Paramecium bursaria: Double
and Triple Photobiont Co-Habitation
Ryo Hoshina1*, Yuko Fujiwara2
1Department of Biomedical Science, College of Life Sciences, Ritsumeikan University, Kusatsu, Japan
2Department of Bioscience, Nagahama Institute of Bio-Science and Technology, Nagahama, Japan
Received July 3, 2012; revised August 5, 2012; accepted August 15, 2012
ABSTRACT
The green ciliate, Paramecium bursa ria, has evolved a mutualistic relationship with endosymbiotic green algae (photo-
bionts). 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, al-
though 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 symbio tic relation ship can b e con-
sidered 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 main-
tain more than one species of algae for a long period. This situation prompts speculations about flexibility of host-photo-
biont relationships, how P. bur saria repl aced these photobionts, and the infection theory of the group I i ntrons.
Keywords: Parame cium bursaria; Photobiont; Symbiosis
1. 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 ex-
tracts) have been genetically identified, most belong
to either Chlorella variabilis or Micractinium reisseri
(Chlorellaceae, Trebouxiophyceae) [2-11]. Neither spe-
cies 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 fas-
tidious [e.g., 12,13 ]. Additionally, they are very sensitive
to the Paramecium bursaria Chlorella virus (PBCV),
which is abundant in natural water sources [14-16]. Es-
caped photobionts from P. bursaria cell would be at-
tacked by PBCV immediately. Both species appear to be
highly dependent on their host refuge. Chlorella variabi-
lis 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. bur-
saria has replaced its photobionts on several occasions.
Lichens and corals, representatives of symbiotic asso-
ciations with algal symbionts, experience a period with-
out 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 re-
production [17]. Consequently, the symbiotic relation-
ship 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 en-
zymes, splicing themselves out of precursor RNA tran-
scripts 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 lo-
cus of a gene infects a different organism, the new intron
will be inserted into the same locus of the gene in which
*Corresponding a uthor.
C
opyright © 2012 SciRes. AiM

R. HOSHINA, Y. FUJIWARA 228
it was originally located (For general characters on group
I introns, see Cech [18]; Haugen et al. [19] and refer-
ences therein). Group I introns are classified into sub-
groups IA through IE based on their structural diversity
and phylogeny, and nuclear encoding introns belong to
subgroup IC or IE [20]. Chlorella variabilis is particu-
larly intron- rich, co ntain ing eigh t group I intron s (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 number-
ing 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. re-
isseri has an archaic state and the other introns are as-
sumed to originate fro m 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 acceler-
ate the lateral transfer of group I introns [24,25].
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 indi-
cated by thick (inter specific) and narrow (intra specific)
arrows. For details of intron transmission, see Hoshina and
Imamura [22].
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.
2. Materials and Methods
2.1. Paramecium bursaria Culture
Particular kind of Paramecium bursaria strains were
maintained in lettuce juice medium [26] under LED il-
lumination (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 (Hi-
roshima University) to RH in December 2008 and have
been cultured for more than four years; therefore symbi-
otic conditions shou ld be regarded as stable.
2.2. 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 illu-
mination (12 h L:12 h D) at 15˚C.
2.3. Microscopy
Cells of P. bursaria and their symbiotic algae were ob-
served under light microscopy CX31 (Olympus, Tokyo)
and photos were taken with an HDCE-31 digital camera
(AS ONE, Osaka).
2.4. DNA Extraction, Amplification, and
Sequencing
Paramecium bursaria strains with photobionts in each
cell were directly used to extract both host and photobi-
ont DNAs. A photobiont strain AG-35_ZF1 isolated
from P. bursaria AG-35 (Ta bl e 1) was also used in DNA
extraction. DNA extractions were performed using the
DNeasy plant m i ni kit (Qiagen, Düsseldo rf ).
Whole P. bu rsaria DNA ex tracts 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-

R. HOSHINA, Y. FUJIWARA 229
Table 1. Paramecium bursaria strains used in this study and the ir sequence data.
DNA accession
Strain
name Syngen Mating type Collection date Collection site Host Large bandMedium band Small band Isolated alga
AG-35 1 Ⅲ Aug. 1992
Aquatic Garden,
Washington AB699097AB699101AB699105 AB699111 AB699112
BP-11 1 Ⅳ Jun. 1992 Catonsville,
Maryland AB699098AB699102AB699106/107 — —
DC-3 1 Ⅰ Aug. 1992
Delaware River,
New York AB699099AB699103AB699108 — —
OLG-3* 2 ? Dec. 1992 (Orlando, Florida)AB699100AB699104AB699109/110 — —
*Offspring of original strain.
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 se-
quenced. The amplified fragments of photobiont DNA
were confirmed by agarose gel electrophoresis, collected
by excising the fluorescent band, purified using the Qi-
aex II Gel Extraction Kit (Qiagen), and directly se-
quenced.
SSU rDNA to internal transcribed spacer 2 of the iso-
lated alga, AG-35_ZF1, was amplified with the primer
pairs SR-1/SR-9, SR-6/SR12k, and INT4F/HLR3R (pri-
mers are described in Hoshina et al. [6]). These were
purified via Quantum Prep PCR Kleen Spin Columns,
and directly sequenced.
2.5. 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 se-
quence data.
3. 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 re-
tains 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 tricho-
cysts (Figure 2(c)).
Next, we conducted algae-targ eting 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 polymor-
phisms 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 re-
quires organic nitrogen sources to grow [13], this oli-
gotrophic 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-ITS2 rDNA. 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-ITS2 rDNA), 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 se-
quences we refer to as genotype D [28], whereas OLG-3
(AB699100) differed from the others and matched what
we refer to as genotype B.
4. 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 de-
termine. These algae will be unifiedin the cell of P. bur-
saria 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

R. HOSHINA, Y. FUJIWARA 230
Figure 2. Microscopic images of Paramecium bursaria and its contents. a: AG-35; b: OLG-3; c: Head of OLG-3. Both large
(Chlorella variabilis or Chlorella vulgaris) and small (Choricystis minor) coccids in between the trichocysts can be seen. d:
Sample from AG-35 leakage.
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 inser-
tions, 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 Chori-
cystis = medium-size band).
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]. Micro-
scopic observations showed C. minor (small coccoids)
were inserted in between the trichocysts. This phenome-
non 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. vari-
abilis and C. minor as their steady photobionts. This
situation, namely, stable symbiotic relation ships 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 sepa-
rated 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 in-
dels from the others. For these circumstances, Pröschold
et al. [9] suggested that P. bursaria is a complex of sev-
eral species, similar to the P. aurelia complex. We pre-
viously found that host genotypes and these photobiont
types are closely linked [6], and proposed a P. bursaria
evolutionary scenario concerning genotype diversifica-
tion 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 pre-
ferred photobiont is either C. variabilis or M. reisseri.
Geographic or climatic conditions probably influence the
photobiont choice of P. bursaria.

R. HOSHINA, Y. FUJIWARA 231
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. bur-
saria. 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 throughou t its life cycle even during cell
division and sexual reproduction [17]. Paramecium bur-
saria 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 sym-
biont loss” (Figure 5). Alternatively, we propose another
process for algal switching: “choice after co-symbiosis”
(Figure 5). Because P. bursaria maintains its re-sym-
biosis ability, it is possible that it routinely tries to
achieve other symbionts through feeding and temporary
symbiosis. As mentioned above, C. variabilis and M.
Figure 4. An evolutionary scenario for P. bursaria concern-
ing genotype diversification and photobiont choice pro-
posed by Hoshina and Imamura [31].
Figure 5. Two possible contexts in which Paramecium bur-
saria may switch photobionts.
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 ar e
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]; there-
fore, P. bursaria can ingest them at any time. Photosyn-
thate contributions from C. vulgaris or Choricystis in P.
bursaria are not known, however this must have oc-
curred 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 regard ed as a
phase for determining a more optimal partner, it can be
an advantage for survival in the long term.
5. Acknowledgements
We are grateful to Prof. Hiroshi Hosoya (Hiroshima Uni-
versity) for kindly providing Paramecium strains. This
study was supported by The Sumitomo Foundation and
Showa Seitoku Memorial Foundation.
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