Jpn. J. Protozool. Vol. 44, No. 1. (2011)

1

Review

Four important cytological events needed to establishG
endosymbiosis of symbiotic Chlorella sp. to the algafree Paramecium bursaria
Yuuki KODAMA1* and Masahiro FUJISHIMA2
1

Research and Education Faculty, Natural Sciences Cluster, Science Unit, Kochi University,
Kochi 780-8520, Japan2
Department of Environmental Science and Engineering, Graduate School of Science andEngineering, Yamaguchi University, Yamaguchi 753-8512, JapanGG

SUMMARY
Each symbiotic Chlorella of the ciliate Paramecium bursaria is enclosed in a perialgal vacuole(PV) derived from the host digestive vacuole (DV) to protect from lysosomal fusion. Irrespective of themutual relationships between P. bursaria and their symbiotic algae, the alga-free paramecia andsymbiotic algae still have an ability to grow independently and can be experimentally reinfected bymixing them. This phenomenon provides an excellent opportunity to elucidate cell-to-cell interactions
between protozoa and algae during establishment of the secondary endosymbiosis. However, the detailed
algal reinfection process had been unclear. Therefore, using pulse-label of the alga-free paramecia with
the isolated symbiotic algae and chase method, we found four important cytological events needed toestablish endosymbiosis. (1) 3 min after mixing, some algae show resistance to the host lysosomal
enzymes in the DVs even if the digested ones are coexisted. (2) 30 min after mixing, the alga starts toleave from the DV to appear in the cytoplasm by budding of the DV membrane. (3) Within 15 min after
the algal appearance in the cytoplasm, the vacuole enclosing a single green alga differentiates into the PV
from the DV, which gives protection from the host lysosomal fusion. (4) After that, the alga localizes
beneath the host cell cortex. At about 24 h after mixing, the alga increases by cell division andestablishes endosymbiosis. In this review article, we describe our recent studies on the four events during
the algal reinfection process.
Key words: Chlorella spp., Digestive vacuole, Endosymbiosis, Paramecium bursaria, Perialgal vacuole,
Reinfection*Corresponding author
Tel/Fax: +81-88-844-8310G
E-mail: ykodama@kochi-u.ac.jpGG
Received: 10 February 2011; Accepted: 18 March
2011.

2

Secondary symbiosis between Paramecium and Chlorella

INTRODUCTION
Endosymbiosis acts as a primary force forthe biodiversity. Recent studies on algal evolution
have shown that endosymbiosis has occurred several times and has brought a diversity of eukaryotic cells. Despite the importance of this phenomenon, molecular mechanisms of the establishmentof endosymbiosis between eukaryotic cells are still
not well known.
Ciliate Paramecium bursaria cells harbor
several hundreds of symbiotic Chlorella spp. intheir cytoplasmG (Fig. 1). Each symbiotic alga is
enclosed in a special membrane called perialgal
vacuole (PV) membrane derived from the hostdigestive vacuole (DV) membrane (Fig. 2). PV
membrane has a function to protect the symbiotic
alga from host lysosomal fusion (Gu et al., 2002;
Karakashian and Rudzinska, 1981; Kodama andFujishima, 2009b).
Various benefits are induced in both the hostP. bursaria and the algae by algal infection. For
the symbiotic algae, the host can supply the algaewith nitrogen components and CO2 (Albers andWiessner, 1985; Albers et al., 1982; Reisser, 1976;
Reisser, 1980). The algae are protected from infection of the Chlorella virus when the alga is
wrapped with the PV membrane (Kawakami and
Kawakami, 1978; Reisser et al., 1988; Van Etten etal., 1991; Yamada et al., 2006). Algal carbon dioxide fixation is enhanced by the host extracts. Recently, the active substances are identified as threecations (Ca2+, K+, and Mg2+) (Kamako and Imamura, 2006; Kato and Imamura, 2009). On the other
hand, for the host paramecia, the algae can supply
the host with a photosynthetic product, mainly
maltose (Brown and Nielsen, 1974; Reisser, 1976,1986). Inside the host cell, the algae show a higher
rate of photosynthetic oxygen production than in
the isolated condition, thereby guaranteeing an
oxygen supply for their host (Reisser, 1980). Recently, Kato and Imamura (2009) updated a schematic representation of materials that are suppos-

Fig. 1. Light (A) and fluorescence (B) micrographs of alga-bearing P. bursaria. Hundreds of symbiotic Chlorella sp.
are observed in the Paramecium cytoplasm (A). Fluorescence of chlorophyll within chloroplast is red color. Macronucleus (Ma) and micronucleus (Mi) are stained withDAPI (blue) (B). Cy, cytopharynx. From Kodama andFujishima (2010c).

Fig. 2. Transmission electron micrograph of PV membrane
enclosing dividing symbiotic alga. From Kodama andFujishima (2010c).

edly transported, and metabolic controls between
the host and the symbiotic algae. Furthermore, alga-bearing P. bursaria cells can divide better thanJpn. J. Protozool. Vol. 44, No. 1. (2011)

the alga-free cells (Görtz, 1982; Karakashian,1963, 1975). Alga-bearing cells show a higher
survival rate under various stressful conditions
than the alga-free cells. For example, under 0.5
mM nickel chloride (NiCl2), or at high temperatures (42°C), or in 150 mM hydrogen peroxide(H2O2) (Kinoshita et al., 2009; Miwa, 2009). Recently, Summerer et al. (2009) found that the hostparamecia can receive protection against UV damage by their symbiotic algae. Furthermore, because
the timing of the cell divisions of both algae andthe host paramecia is well coordinated, the symbiotic algae can be distributed to the daughter cells(Kadono et al., 2004; Takahashi et al., 2007).
As described above, though the relationshipbetween P. bursaria and Chlorella spp. is a mutualism, the alga-free cells and the symbiotic algae
are still keeping an ability to grow without a partner. Alga-removed P. bursaria cells can be produced easily from alga-bearing cells using one of
the following methods: rapid cell division(Jennings, 1938); cultivation under the constantdark condition (Karakashian, 1963; Pado, 1965;
Weis, 1969); X-ray irradiation (Wichterman,
1948); treatment with 3-(3,4-dichlorophenyl)-1,1dimethylurea (DCMU), a blocker of electron flowin photosystem II (Reisser, 1976); treatment withthe herbicide paraquat (Hosoya et al., 1995) ortreatment with a eukaryotic protein synthesis inhibitor, cycloheximide (Kodama and Fujishima,
2008; Kodama et al., 2007, 2011; Weis, 1984). On
the other hand, symbiotic algae can be isolated
from alga-bearing P. bursaria cells by sonication,
homogenization (Kodama and Fujishima, 2005), or
treatment with some detergents (M. Yasuda andM. Fujishima, unpublished data). Furthermore, by
mixing the alga-removed P. bursaria and the isolated symbiotic algae, endosymbiosis can be easily
established again (Karakashian, 1975; Siegel and
Karakashian, 1959; Kodama and Fujishima, 2005).
In addition to these features, P. bursaria havesome usefulness as follows: P. bursaria can becultivated easily and can get mass culture; the rein-

3

fection process of the algae can be observed easily
under a light microscope; an alga-free mutant
strain of P. bursaria has been collected (Tonooka
and Watanabe, 2002, 2007). For these reasons, P.
bursaria and their symbiotic Chlorella spp. areconsidered an excellent model for studying cell-tocell interaction and the biodiversity through thesecondary endosymbiosis. However, the algal infection process to the alga-free P. bursaria hadremained unknown for a long time. Recently, byusing pulse label and chase method, we clarified
the algal reinfection process and found four important cytological events needed for establishment of endosymbiosis and their timings during
the algal infection process (Kodama and Fujishima, 2005, 2007, 2008, 2009a, 2009b, 2009c,
2010a, 2010b, 2010c, 2010d; Kodama et al., 2007,2011).
DIFFERENTIATION STAGE OF DVS OF
P. BURSARIA
Infection of algae to the alga-free P. bursaria
is conducted through the host’s phagocytosis. To
investigate the algal infection process, we firstclassified the differentiation stages of DVs inphagocytosis by using pulse label and chase method (Kodama and Fujishima, 2005).
Pulse label and chase method
Cells from a 60 ml culture of alga-free P.
bursaria strain Yad1w or OS1w at the early stationary phase of growth were strained througheight layers of fine gauze, washed twice with modified Dryl’s solution (MDS) (Dryl, 1959) (KH2PO4
was used instead of NaH2PO4·2H2O), by handoperated centrifugation, and suspended in 1 ml of
MDS. The cells were mixed at a density of 5 × 103
paramecia/ml with Chlorella sp. isolated from thealga-bearing P. bursaria cells at 5 × 107 algae/ml
in a centrifuge tube (volume, 10 ml) under a fluorescent light (20–30 ȝmol photon/m2s) for 1.5 min
at 25 ± 1°C. The paramecia-algae mixture was4Secondary symbiosis between Paramecium and Chlorella

Fig. 3. GA centrifuge tube with nylon mesh for removal of
algae from Paramecium-Chlorella mixture. By pouring 30
ml of MDS, the paramecia are washed, and algae are simultaneously removed through the nylon mesh.G

transferred to a centrifuge tube equipped with a 15
ȝm pore size nylon mesh (Fig.3) and filtered. By
pouring 30 ml of fresh MDS into this tube, theparamecia were washed and algal cells outside theparamecia were simultaneously removed throughthe mesh. The paramecia retained on the meshwere harvested and transferred to a centrifuge tube
(volume, 10 ml) and resuspended in 1 ml of MDS,
and then chased for various times under a fluorescent light at 25 ± 1°C. Then the cells were observed with or without fixation under a differential
interference contrast (DIC) microscope. In a separate procedure, the paramecia-algae mixture was
fixed without washing with MDS.
The DV stages of P. bursaria
To classify the DV stages of P. bursaria thatappear during the algal infection process and to
determine the timing of the appearance of eachstage, algae-containing P. bursaria’s DVs wereobserved under a DIC microscope. The DVs containing several algae were classified into four stages based on their morphological features and color
changes of the algae accompanied by algal digestion. The first stage, DV-I is characterized by its
round shape and its visible vacuolar membrane.

The color of the algae is green. The second stage,
DV-II is characterized by its condensed vacuolar
membrane. The color of the algae is still green.
The third stage, DV-III is characterized by its
round shape and its visible vacuolar membrane
like DV-I. In this stage, the color of the some algae
becomes faintly yellow accompanied by their digestion. Color of the undigested alga is green. So,
we classified DV-III into three substages. DV-IIIa
contains undigested green algae only. DV-IIIb
contains both digested yellow and undigested
green algae. DV-IIIc contains digested yellow algae only. In the final stage, DV-IV is characterized
by its condensed vacuolar membrane like DV-II.
In this stage, the color of digested algae becomesbrown and algal diameter becomes small. So, we
also classified DV-IV into three substages. DVIVa contains undigested green algae only. DV-IVb
contains both undigested green and digested brownalgae. DV-IVc contains digested brown algae only.
Unlike DV-II, DV-IV was not observed in cells
fixed before 3 min, but was observed 20–30 min
after mixing. The algae in each DV of nonfixedparamecia showed the same characters in those of
fixed paramecia.G
To determine the timing of the appearance ofeach stage of DVs, alga-free P. bursaria cells weremixed with isolated symbiotic algae. After that, theparamecia-algae mixture was fixed at every 10 sintervals for 60 s after the mixing. The fixed cells
were classified into four stages according to themost advanced stage of DV in the cell (i.e., if both
DV-II and DV-III were present in a cell, the cell
was classified as a cell with DV-III). As a result,
DV-II started to appear in the cells fixed at 30 s
after mixing. Furthermore, to determine the timingof the appearance of DV-III, alga-free cells weremixed with isolated symbiotic algae for 1.5 min,washed with MDS, chased, and fixed at every 1
min interval for 10 min after the mixing. As a result, DV-III started to appear in cells fixed at 3 minafter mixing.

Jpn. J. Protozool. Vol. 44, No. 1. (2011)

Timing of the fusion of acidosomes and lysosomes to the DVs
In P. multimicronucleatum, it has been reported that intravacuolar pH was changed accompanied by acidosomal and lysosomal fusion to theDVs (Fok et al., 1982). To determine the timing of
the acidosomal and lysosomal fusion to the P. bursaria’s DVs, we examined the change of the intravacuolar pH of P. bursaria’s DVs with the useof yeast Saccharomyces cerevisiae cells stainedwith pH indicator dyes. Yeast cells were stainedwith three kinds of pH indicator dyes, Congo red,
bromocresol green (BCG), and bromophenol blue
(BPB). Then, the yeast cells were mixed with algafree P. bursaria. Then the color changes of theyeast cells inside the DVs were monitored under a
DIC microscope. The color changes of each dyes
occurring in phthalic acid and citric acid buffers
are confirmed beforehand. These changes did not
differ between the buffers, but they were dependent on the pH. Within 0.5 min after mixing, thecolor of the BCG-labeled yeast cells was blue.
This means that the intravacuolar pH was 6.4–7.0.
At 1–2 min after mixing, the color of the yeast
cells became yellow-green. This means that the
intravacuolar pH decreases to 2.4–3.0. Morphological differentiation of DV-II from DV-I also occurred at 0.5–1 min after mixing. These results
show that acidosomal fusion to the DV occurs at
0.5–1 min after ingestion. Using Congo-red labeled yeast, identical results were obtained(Kodama and Fujishima, 2005).
Partially digested yellow algae started toappear in DV-IIIb at 2–3 min after mixing. In thelate stage of DV-II, the color of the BCG-labeled
yeast cells became blue, indicated that the intravacuolar pH was already 6.4–7.0. These results
suggest that lysosomal fusion might start before 2–3 min after mixing. Gomori’s staining (Gomori,1952), which is used to detect acid phosphatase(AcPase) activity in the DVs showed that both DV-I and DV-II are AcPase-activity negative, but all
substages of DV-III and DV-IV are AcPase-

5

Fig. 4. Schematic representation of DV stages of P. bursaria. When Chlorella sp. and alga-free paramecia are
mixed, one or several algae pass through the host cytopharynx and are pinched off as DV-I. The pH inside DV-I
is 6.4–7.0. Condensed DV-II appears 0.5–1.0 min after
mixing. The pH inside DV-II reduces to 2.4–3.0. Lysosomal fusion occurs at 2.0–3.0 min, leading to swollen DVIIIa to DV-IIIc. Color of some algae becomes yellow because of the algal digestion. Digested algae are observed in
DV-IIIb and DV-IIIc. The pH inside DV-III increases to
6.4–7.0. Condensed DV-IVa, DV-IVb, and DV-IVc areobserved at 20–30 min. The pH inside DV-IV remains at
6.4–7.0. The digested algae inside the DV-IVb and DVIVc become brown by an additional digestion. From Kodama and Fujishima (2010c).

activity positive (Kodama and Fujishima, 2009a)(see Timing of the PV membrane differentiation inthis review). These morphological and histochemical results show that the lysosomal fusion occurs
before 2–3 min after mixing. A schematic representation of DV differentiation of P. bursaria is
depicted inGFig.4.
FATES OF CHRORELLA SP. DURING THE
ALGAL REINFECTION PROCESSAs described above, single green Chlorella(SGC) is enclosed in a PV membrane as shown in
Fig. 2. This fact indicates that timing of the appearance of the SGC can be considered as almost
the same as that of the appearance of the PV mem-

6Secondary symbiosis between Paramecium and Chlorellabrane from DV membrane during the algal infection process. To determine the timing of the appearance of the SGC, alga-free paramecia werepulse labeled with isolated symbiotic algae for 1.5
min, chased, then fixed at 0.05, 0.5, 1, 1.5, 2, 3, 6,
9, 24, 48, and 72 h after mixing. The percentagesof paramecia with DV-IIIa or DV-IIIb, DV-IVa or
DV-IVb, and SGC were monitored. As a result, all
SGCs that were present in the host cytoplasm before 0.5 h after mixing were all digested. However,1 h after mixing, SGCs, which appeared from either DV-IVa or DV-IVb were observed again in
the host cytoplasm. At 24 h, the SGCs began tomultiply by cell division (see INITIATION OF
THE ALGAL CELL DIVISION FOR ESTABLISHMENT ENDOSYMBIOSIS in this review),
indicating that these algae could establish endosymbiosis with alga-free P. bursaria cells. This
result shows that the SGCs to establish endosymbiosis appear from the DV-IVa or the DV-IVb occurs after acidosomal and lysosomal fusion to theDV.
As mentioned above, the SGCs, which could
establish endosymbiosis appeared from either DVIVa or DV-IVb. To clarify the origin of SGCs, thepercentages of the paramecia with DV-IVa, DVIVb, and SGC were monitored by observation of
fixed cells at 0.5, 1, 1.5, 2, 3, 6, 9, 24, 48, and 72 h
after 1.5-min pulse label with isolated Chlorella
sp. The percentage of the paramecia with SGC was
0% at 0.5 h and increased as time elapsed and became to 35% at 72 h after mixing. In contrast, thepercentage of paramecia with DV-IVb was decreased. On the other hand, less than 5% of cells
contained DV-IVa, if any throughout the whole
infection process. This indicates that the majorityof the SGCs established endosymbiosis with algafree P. bursaria cells originated from DV-IVb(Kodama and Fujishima, 2005).
In contrast to results of an earlier study ofMeier and Wiessner (1989), our observations suggest a new escape mechanism from host lysosomal
digestion by Chlorella sp. during the algal infec-

Fig. 5. G Algal reinfection process to the alga-free P. bursaria and four important cytological events needed toestablish endosymbiosis. Using pulse label and chasemethod, algal reinfection process and four important cytological events necessary to establish endosymbiosis wereclarified. First, from 3 min after mixing, some algae acquire temporary resistance to the host lysosomal enzymes
in the DVs. Second, from 30 min after mixing, the algae
start to leave from the DVs to appear in the host cytoplasm. Third, within 15 min after the algal appearance, theDV membrane enclosing a SGC differentiated into the PV
membrane, which provides protection from lysosomal
fusion. Finally, the alga localizes beneath the host cellcortex. At about 24 h after mixing, the alga increases bycell division to establish endosymbiosis.G

Jpn. J. Protozool. Vol. 44, No. 1. (2011)

7

tion process. First, some alga shows temporary
resistance to the host lysosomal enzymes in both
DV-III and DV-IV. Second, the SGCs to establish
endosymbiosis appear from the DV-IVb into thehost cytoplasm. Finally, the alga loses their temporary resistance to the host lysosomal enzymes but
is protected from lysosomal fusion by wrappingwith the PV membrane (Kodama and Fujishima,2005). Schematic representation of the infection
process by Chlorella sp. is shown inGFig.5.

mecia (Kodama and Fujishima, 2005). Furthermore, all isolated symbiotic algae fixed with 2.5%(w/v) glutaraldehyde or with 5.0% (w/v) formaldehyde are also digested in DV-III (Kodama andFujishima, 2005). Therefore, it appears that only
living algae can show temporary resistance to thelysosomal enzymes in the DVs, which were fusedwith host lysosomes. To date, the factor determining whether the algal fates survive or are digested
in DV-IVb has remained unclear.G

FOUR CYTOLOGICAL EVENTS NEEDEDTO ESTABLISH ENDOSYMBIOSISLight microscopy of Chlorella variabilis cells in
DV-IVb
It is known that different types of algae infecting P. bursaria clones show different infection
ratio and host dependencies (Nakahara et el., 2003;
Nishihara et el., 1999). This suggests a possibility
that when different Chlorella species or strainsused together for the infection experiment, it may
yield different algal fates in DV-IVb as shown inG
Fig. 6. To examine this possibility, alga-bearing P.
bursaria cells strain OS1g1N that was produced by
infection of alga-free P. bursaria OS1w cells withsymbiotic algal clone 1 N cells was used for symbiotic algal isolation (Kodama et al., 2007). This
algal clone 1 N was identified as C. variabilis(Hoshina et al., 2010) (formerly C. vulgaris). Although genetically identical symbiotic algal cells
were used for the infection, DV-IVb was appeared(Kodama et al., 2007).
Light microscopy showed that both digested
brown and nondigested green algae presented in
DV-IVb did not show a specific location, indicating that their different fates are not decided bytheir positions in the DV. To determine whether
the survived algae in the DV-IVb had been
wrapped with their original PV membrane, theisolated symbiotic algae were treated with Triton
X-100 or Tween-20 detergents before mixing withalga-free P. bursaria cells. There were no significant differences in the percentage of the cells with
DV-IVb between paramecia mixed with nontreated
and detergent-treated algae. Furthermore, weDuring the algal reinfection process, wefound four cytological events needed to establish
endosymbiosis (Kodama and Fujishima, 2009c,
2010a, 2010c) (Fig. 5). First, from 3 min after mixing, some algae acquire temporary resistance to thehost lysosomal enzymes in the DVs, even when
the digested ones coexist. Second, from 30 min
after mixing, the algae start to leave from the DV
to appear in the cytoplasm. Third, within 15 minafter the algal appearance, the DV membrane enclosing SGC differentiate into the PV membrane,
which provides protection from lysosomal fusion.
Finally, the algae localize beneath the host cellcortex. From next section, some results related toeach event will be described.
Algal resistance to the host lysosomal enzymes inthe DVs
The origin of SGCs that can establish endosymbiosis was DV-IVb, as described above. Approximately 50% of paramecia at 0.5 h after mixing had DV-IVb (Kodama and Fujishima, 2005);
most of the DVs at this time were AcPase activitypositive (Kodama and Fujishima, 2009a), whichshows that some of the algae in DV-IV acquire
resistance to the lysosomal enzyme resistance inthe DVs, although the remaining algae are digested
in the same DV. Boiled algae are all digested in
DV-III when they are added to the alga-free para-

8

Secondary symbiosis between Paramecium and Chlorella

Fig. 6. GPhotomicrographs of P. bursaria 1 h after mixing with algae. B shows a highly magnified image of the squareenclosed area in A. As shown by arrowhead in B, some algae were not digested even if they are coexisted with the digested ones (small arrow in B) in the DV-IVb. Ma, macronucleus. From Kodama and Fujishima (2010c).G

Fig. 7. Photomicrographs of P. bursaria 2.5 h after mixing with algae. B shows highly magnified images of the squareenclosed area in A. Single green Chlorella (SGC, white arrowhead) and single digested Chlorella (SDC, black arrow)appear as a result of the budding of the DV-IVb membrane. Note that this algal budding is observed notwithstanding thealga is nondigested or partially digested. Ma, macronucleus. From Kodama and Fujishima (2010c).G

found no relationships between algal stage in thecell cycle and their fates in DV-IVb (Kodama etal., 2007).
Transmission electron microscopy of DV-IVbG
To confirm the possibility that some algae
might have acquired a PV membrane in the DVIVb prior to their budding from the DV-IVb into
the host cytoplasm, the DV-IVb was observed bytransmission electron microscope (TEM). TEM
observation showed that, in the DV-IVb, nondigested algae appear as electron-dense cells anddigested algae appear as electron-translucent cellswith empty spaces between the cell wall and theircytoplasm. Gu et al. (2002) reported that the space
between the PV membrane and the algal cell wall
is about 25–100 nm in width. Any membrane resembling a PV membrane was not observed
around the nondigested algae. This indicates thatthe algal fates are not determined by PV membrane acquisition in the host DV-IVb, and that the
PV membrane is formed during or after algal budding from the DV-IVb (Kodama et al., 2007).
Effects of protein synthesis inhibitors, cycloheximide and puromycin on algal survival in DVIV during infection
To determine whether algal or host protein
synthesis is needed for the survival of the alga in
the host DV-IV, isolated symbiotic C. variabilis
and alga-free P. bursaria were pretreated withcycloheximide or puromycin. As a result, most of
the paramecia formed DVs enclosing algae 3 minafter mixing with or without cycloheximide treatment. The percentage of cycloheximide-treatedcells with DV-IVb 3 h after mixing was not significantly different from that of the control experiment.

Jpn. J. Protozool. Vol. 44, No. 1. (2011)

9

Fig. 8. Schematic diagram of timing of PV membrane differentiation from DV membrane during the algal infection process. AcPase-activity positive area is shown by a gray color in the diagram. Both inside the DV-I and DV-II are AcPaseactivity negative. After lysosomal fusion, AcPase-activity appears in DV-III. In the DV-IIIb, some algae show resistanceto the lysosomal enzymes and maintain green color and original morphology. Remaining algae are partially digested andshow yellow color in the same DV. DV-IIIb differentiates into DV-IVb which containing both nondigested green anddigested brown algae. Appearance of the SGCs from DV-IVb begins from 30 min by budding of the DV membrane into
the cytoplasm. In the budded membrane, each alga is surrounded by an AcPase activity-positive thin layer. At about 45
min after mixing with algae, the vacuoles enclosing a SGC move quickly and attach just behind the host cell cortex. Suchvacuoles are AcPase activity-negative. This indicates that the PV membrane differentiates soon after the appearance of
the SGCs from the host DV (updated Kodama and Fujishima, 2009a).

On the other hand, number of formed DVs
was significantly reduced to about half by puromycin treatment, although DV-IVb appeared 3 h after
mixing. These results show that neither algal nor
host protein synthesis are not needed for algal acquisition of lysosomal enzyme resistance in thehost DV (Kodama et el., 2007).

se observations raise a possibility that P. bursaria
can recognize the diameter of the content of theDVs, and content with a similar size to algae canbud from the DVs into the cytoplasm. To date, themolecular mechanisms for the budding from theDVs are remained unknown.G


Differentiation into the PV membrane from theAlgal budding from the host DV membrane
DV membrane
Some algae start to leave from the DV-IVb Timing of the PV membrane differentiation
30 min after mixing with the paramecia. This pheTo determine the timing of differentiation ofnomenon can be observed even in cases where the the PV membrane from the DV membrane during
alga was nondigested or partially digested (Fig. 7).G the algal infection process, alga-free P. bursaria
Furthermore, this budding was induced not only by cells were mixed with isolated symbiotic algae forliving algae, but also by the boiled or fixed algae 1.5 min, washed with MDS, chased, and fixed at(Kodama and Fujishima, 2005). Intact or boiled various times after mixing. Then we clarified theyeast S. cerevisiae cells also budded from the DVs timing of the disappearance of AcPase activity(Y. Kodama and M. Fujishima, unpublished data). during the algal reinfection process using
Inorganic polystyrene latex spheres of 3.0 ȝm in Gomori’s staining method because the PV memdiameter budded from the DVs, too (Y. Kodama brane shows no AcPase activity.G Fig. 8 shows
and M. Fujishima, unpublished data). However, presence or absence of AcPase activity inside thethis budding was not induced when India ink, 0.81 DV membrane during the algal infection process.
ȝm in diameter of polystyrene latex spheres, or AcPase activity started to appear in 2–3-min-old
food bacteria Klebsiella pneumoniae were ingested vacuoles, DV-III. Almost all DV-IVs containing
into the DVs (Kodama and Fujishima, 2005). The- algae showed AcPase activity at 30 min. Appear-10

Secondary symbiosis between Paramecium and Chlorella

ance of the SGCs from the DV-IVb begins at 30
min as described before. In the budded membrane,
the alga was surrounded by an AcPase activitypositive thin layer. At about 45 min after mixing,
the vacuoles enclosing a SGC moved quickly andbecame attached beneath the host cell cortex. Inside such vacuoles is AcPase activity-negative.
These results show that the PV membrane differentiates soon after the appearance of the SGCs
from the host DV and is completed prior to thealgal localization beneath the host cell cortex.
Properties of the PV membrane
To date, the following qualitative differences
are reported between the DV and the PV membrane: 1. The PV membrane encloses only one
symbiotic alga (Gu et al., 2002; Karakashian andRudzinska, 1981); 2. The space between the algal
cell wall and the PV membrane is about 0.05 ȝm,
so the PV membrane is hardly observable under alight microscope (Reisser, 1986); 3. The diameterof the PV does not vary much, except during thealgal cell division of the enclosed symbiotic alga
(Reisser, 1992); 4. The PV does not involve incytoplasmic streaming, but attaches beneath thehost cell cortex (Kodama and Fujishima, 2005;
Reisser, 1986); 5. Particle density and its distribution of the PV membrane show few signs hinting
at any endocytotic or exocytotic activity (Meier etal., 1984).
The ability of the PV membrane to protect the algafrom host lysosomal fusion
The timing of differentiation of the PV membrane from the host DV membrane is estimated tooccur soon after the algal budding from the host
DV membrane and before the localization of the
PV membrane beneath the host cell cortex, as described above. However, there was no direct evidence demonstrates that the PV membrane does
not allow lysosomal fusion to the membrane. The
PVs containing SGC of about 3–4-ȝm in diameterare embedded between the trichocysts beneath thehost cell cortex. We clarified that the cortex areaof 5–10-ȝm depth was AcPase activity-negative byGomori’s staining (Kodama and Fujishima, 2008,2009a). These observations raise a possibility that
the PV membrane does not have an ability to protect the alga from lysosomal fusion, but can avoid
lysosomal fusion by localization at the primarylysosome-less area of the host cell cortex. This
AcPase activity-negative area could be reduced toless than 3-ȝm depth at the dorsal surface when thetrichocysts are removed by the treatment with lysozyme. In such cells, some of the PVs were exposed to the AcPase activity-positive area. However, such PVs were not stained with Gomori’s staining, and the algae in each PV were not digested(Kodama and Fujishima, 2009b). These results
demonstrated that the PV membrane does not require the trichocysts for the localization beneaththe host cell cortex, and that the PV membrane,
unlike the DV membrane, provides protection
from host lysosomal fusion, although molecularmechanisms of the PV membrane to give protection from lysosomal fusion are still unknown.
Synchronous swelling of PVs
Cycloheximide is known to inhibit the protein synthesis of the symbiotic algae of P. bursaria
preferentially, but to inhibit host protein synthesis
only slightly (Kodama and Fujishima, 2008; Kodama et al., 2007; Weis, 1984). Previously, we foundthat the PV membrane swells synchronously when
the alga-bearing paramecia are treated with cycloheximide for 24 h (Fig. 9A, Kodama and Fujishima, 2008). This phenomenon was tentatively designated as “synchronous PV swelling (SPVS)”.
The gap between the symbiotic algal cell wall and
the PV membrane expanded to about 25 times thanits general width after the 24 h treatment. Then, thevacuoles left from beneath the host cell cortex andbecame stained with Gomori’s staining, and thealgae in the vacuoles were digested (Fig. 9A, arrow). The SPVS was induced only when the algabearing P. bursaria are treated with cycloheximideJpn. J. Protozool. Vol. 44, No. 1. (2011)

11

Fig. 9. Light (A) and transmission electron (B) micrographs of cycloheximide-treated alga-bearing P. bursaria cells.
Twenty-four h after the treatment, all PVs containing green algae swell synchronously (SPVS, Fig. 9A). After the SPVS,
some algae start to digest and become brown color by the host lysosomal fusion to the PV membrane (A, arrow). Transmission electron microscopy reveals that the PV membrane expands and the algal ultrastructure is degenerated completely. Ma, macronucleus; cw, algal cell wall; mt, paramecium mitochondrion.G

under a constant light (LL) condition, but not under a constant dark (DD) condition. Even under a
LL condition, this phenomenon was not induced inalga-bearing P. bursaria cells treated with cycloheximide in the presence of the photosynthesis
inhibitor DCMU. These results indicate that thealgal proteins synthesized in the presence of thealgal photosynthesis have some important function
to prevent expansion of the PV and to maintain theability of the PV membrane for localization beneath the host cell cortex and for protection fromhost lysosomal digestion (Kodama and Fujishima,2008).
The ultrastructure of the alga inside theswelled PV membrane was observed using TEM.
Before cycloheximide treatment, the color of thesymbiotic algae was green, and the PV membrane
was hardly observable under a DIC microscope.
Using TEM, a rippled PV membrane could be observed and a chloroplast with a pyrenoid and otherultrastructural components of the alga were clearlyvisible. The space between the PV membrane andthe algal cell wall was very narrow. At 15 h after
treatment, observation by DIC revealed that morphological differences were not occurred in thesymbiotic algae, and the PV membrane was notobservable. At the same time, though TEM observation showed that most of the PV membrane islocated very closely to the alga, part of the PV
membrane had started to swell. Furthermore, chlo-roplasts and other ultrastructural components including the algal nucleus had been completely
destroyed. At the same time, many small vesicles
appeared around the PV membrane. Although these small vesicles have a similar size to that of lysosomes, these were electron-translucent, unlike thelysosomes reported by Fok et al (1979). Fusion ofthese small vesicles to the PV membrane was notobserved. Ultrastructural changes showing degradation of mitochondria or trichocysts of the paramecia were not observed by treatment with cycloheximide. At 24 h after treatment, the PV membrane started to swell, so the PV membrane were
easily visible by DIC microscope. Simultaneously,
the algae were started to digest and the color of thealgae changed to brown. As shown in Fig. 9B,
observation using TEM revealed a wide space between the PV membrane and the algal cell walland the degeneration of the algal ultrastructure. By
48 h after treatment, the PV membrane had contracted again, and became hardly visible by DIC
microscope. Observation using TEM also showedthat the PV membrane was contracted again by
unknown mechanism (Kodama et al., 2011).G
To examine the timing of the onset of thedegeneration of the algal ultrastructure, algabearing cells treated with cycloheximide were
fixed for TEM observation at 1, 3, 6, and 9 h after
the treatment under the LL condition. At 1, 3, and6 h after the treatment, no morphological change12Secondary symbiosis between Paramecium and Chlorella

Fig. 10. Photomicrographs of alga-free P. bursaria 4 h after mixing with algae. B shows highly magnified image of thesquare enclosed area in A. As shown by arrowheads in B, SGCs, which were wrapped with PV membrane translocate
beneath the host cell cortex and are embedded among the trichocysts. Ma, macronucleus. From Kodama and Fujishima(2010c).G

was observed in algal ultrastructure. However, at 9h, the ultrastructure in many algae had alreadydegenerated. These observations suggest that algalultrastructure begins to degenerate between 6 h
and 9 h after the treatment (Kodama et al., 2011).
The SPVS and subsequent algal digestion
induced by treatment with cycloheximide under
the LL conditions were also induced in otherstrains of alga-bearing P. bursaria cells: SKS-4-5,
HG1g, HG5g, HG18g, and HG24g. These results
show that SPVS and subsequent algal digestion by
the treatment with cycloheximide are general phenomena that occur in various strains of algabearing P. bursaria cells (Kodama et al., 2011).
Effects of other protein synthesis inhibitors, chloramphenicol, emetin, and puromycin on the PV
membrane
Effects of chloramphenicol, an inhibitor ofbacterial, chloroplastic, and mitochondrial protein
synthesis, and effects of emetine and puromycin,
inhibitors of the paramecia’s protein synthesis on
the SPVS and subsequent algal digestion were
examined. Firstly, to determine appropriate concentrations of these inhibitors, effects of these inhibitors on the growth rate of the paramecia were
examined. In the case of P. caudatum, 3 ȝg/ml ofemetine completely inhibited their cell division(Dohra and Fujishima, 1999). In the case of P.
bursaria, 0.1, and 0.25 mg/ml of chloramphenicol,
1 ȝg/ml of emetine, and 60 ȝg/ml of puromycin

inhibited their cell division. However, 0.5% ethanol, which concentration corresponded to that of asolvent of 0.25 mg/ml of chloramphenicol did notinhibit. The effects of these inhibitors on the SPVS
and following algal digestion were observed using
DIC microscope. All concentrations of chloramphenicol, emetin, or puromycin did not induce
both SPVS and subsequent algal digestion, irrespective of LL and DD conditions. These results
show that, among the protein synthesis inhibitors
examined in our study, only cycloheximide induces SPVS and subsequent algal digestion (Kodama
and Fujishima, 2008).
To date, presence of the PV are reported inendosymbiosis between P. bursaria, Climacostomum virens, Stentor polymorphus, Vorticella sp.,Spongilla fluviatilis, and Hydra viridis with Chlorella sp. (Graham and Graham, 1978, 1980; Karakashian et al., 1968; Oschman, 1967; Reisser,
1981; Reisser et al., 1984). Notwithstanding theimportance and generality of the PV membrane,
little is known about qualitative characteristics of
the PV membrane. Identification of SPVS will be
helpful to clarify the PV membrane properties.
Algal translocation and attachment beneath thehost cell cortex
The SGCs, which appeared from DV-IVb by
budding of the DV membrane translocate beneaththe host cell cortex and are embedded among thetrichocysts as shown inGFig. 10. This phenomenonJpn. J. Protozool. Vol. 44, No. 1. (2011)

13

Fig. 11. Light and transmission electron photomicrographs of alga-free trichocysts-removed (D)–(F) or nonremoved (A)–(C) P. bursaria cells that were pulse labeled with symbiotic algae. B and E show dorsal surfaces of A and D, respectively. A, B, D and E show DIC images of living paramecia at 3 h after mixing with the algae. C and F show TEM images ofparamecia at 3 h after mixing. In a trichocysts-nonremoved cell, the algae move trichocysts aside (A)–(C). Note that thealga can attach beneath the host cell cortex of the trichocysts-removed cell (D)–(F). Arrowheads in B and E show symbiotic algae localized close to the host cell cortex. Ma, macronucleus; a, symbiotic alga localized beneath the host cellcortex; PV, perialgal vacuole membrane; tc, trichocyst; c, cilia; Bars, 20 µm (D and E) and 1 µm (F). From Kodama andFujishima (2009b).G

is observed only with living algae but not withboiled algae (Kodama and Fujishima, 2005). Localization of symbiotic algae has been observed invarious protists (Reisser, 1986). These facts indicate that the PV membrane functions not only forprotection of the symbiont from lysosomal fusion,
but also for stable attachment of the symbiont near
the host cell cortex. Tonooka and Watanabe (2002,2007) reported of a natural alga-free strain of P.
bursaria. Infection experiments using this strain
revealed that this strain couldn’t maintain stableendosymbiosis with algae. The algae ingested by
the paramecia aggregated at the posterior region of
the cell, and then alga-free paramecium was produced after cell division. Crossbreeding analyses
between this strain and a normal strain showed that
all F1 progenies could establish stable endosymbiosis with the algae, but some F2 progenies,
through sibling crosses between symbiotic F1
progenies could not. Their results indicate that thetranslocation and attachment of the PVs beneaththe host cell cortex is genetically controlled byunknown host factor.G
Thousands of trichocysts are embedded under the Paramecium cell cortex, as defensive organelles against predators (Harumoto and Miyake,1991). Using lysozyme, discharge of trichocysts in
Paramecium can be induced without seriously
injuring it (Harumoto and Miyake, 1991). Because
the symbiotic algae seem to push the trichocysts
aside to become localized beneath the host cellcortex after appearance from the host DV, we examined the necessity of trichocysts for the algal
localization. To examine the possibility, alga-freeparamecia and isolated symbiotic algae weremixed for 1.5 min, washed with MDS, and thenresuspended with the same concentration in MDS.
Because the SGCs start to appear from DVs at 0.5
h after mixing with algae (Kodama and Fujishima,
2005, 2009b), the pulse-labeled cells were treatedwith 1 mg/ml lysozyme 0.5 h after mixing. This
concentration of lysozyme could induce full dis-

14

Secondary symbiosis between Paramecium and Chlorella

charge of trichocysts of alga-free P. bursaria cells.
These paramecia were fixed at 3 and 24 h after
mixing with algae in the presence of lysozyme,
and observed to determine whether the SGCs that
had appeared from the DV-IV were able to attachbeneath the host cell cortex of the trichocystremoved paramecia. At 3 h, in the control experiment, SGCs attached beneath the host cell cortex
were observed (Figs. 11A–C). These algae looked
to move the trichocysts aside. On the other hand,
SGCs could localize beneath the host cell cortex,
even in cases where the trichocysts had been removed by the treatment of lysozyme (Figs. 11DF). Interestingly, in the trichocysts-removed cells,
the SGCs localized beneath the host cell cortex
were more numerous than in the non-removed
cells (Figs. 11D and F). With respect to the percentage of the trichocysts-bearing cells with SGCs
localized beneath the host cell cortex was about
65%. However, it increased to about 95% in thetrichocyst-removed cells by 24 h after mixing.
These results demonstrate that the PV membrane
does not require the trichocysts for the algal intracellular localization (Kodama and Fujishima,
2010a).
Besides our results, Omura and Suzaki(2003) reported that the density of trichocysts inthe alga-bearing P. bursaria cells was less thanthat in the alga-free cells. These observations raisea possibility that the symbiotic algae and the hosttrichocysts compete for the host cell cortex to attach themselves there. To examine the possibility,
we developed a monoclonal antibody (mAb)against the trichocysts and observed the changeof trichocysts caused by the algal reinfection to thealga-free cells. Indirect immunofluorescence microscopy shows that an epitope for the mAb localizes in both trichocysts before (Fig. 12A) and after(Fig. 12B) discharge.
The changes in the trichocysts during thealgal reinfection process were observed using indirect immunofluorescence microscopy. Three minafter mixing with algae, Paramecium cells had

Fig. 12. Indirect immunofluorescence micrographs of
Paramecium trichocysts with a monoclonal antibody
against the trichocysts. Immunofluorescence shows that an
epitope for the monoclonal antibody localizes in bothtrichocysts before (A) and after (B) discharge.G

Fig. 13. Indirect immunofluorescence micrograph of algabearing P. bursaria. A shows a DIC micrograph. B shows
an indirect immunofluorescence micrograph. Immunofluorescence shows that the trichocysts are not present in thearea where the symbiotic alga localizes. Ma, macronucleus.G

Jpn. J. Protozool. Vol. 44, No. 1. (2011)

many trichocysts under the cell cortex. Three hafter mixing, the alga localized beneath the hostcell cortex after escaping from the host DVs. At
that time, the trichocysts changed their arrangement to make a space for the algae and form a ringsurrounding each alga localized beneath the hostcell cortex. At about 24 h after mixing, the numberof the algae located beneath the host cell cortex
has increased (Fig. 13A), because the algae started
cell division. The ring-like arrangement of trichocysts increased according to the increase in thenumber of algae as shown in Fig. 13B. These results indicate that the symbiotic algae compete for
their attachment sites with preexisting trichocysts
and the algae have the ability to ensure algal attachment sites beneath the host cell cortex.
The changes in the trichocysts when the
boiled algae were mixed with alga-free P. bursaria
cells were also examined. When the boiled algae
were mixed with alga-free cells, the algae wereingested into the host DVs just as living ones had
been, but all boiled algae in the DVs began to bedigested immediately, and the color of the digested
algae became yellow. Boiled algae also budded
from the DV-IV, though they failed to localize
beneath the host cell cortex. The all ingested
boiled algae were digested and exocytized fromthe cytoproct within 72 h after mixing (Kodama
and Fujishima, 2005). It is noteworthy that thechange of the trichocysts was not observed when
the boiled algae were ingested into the alga-free P.
bursaria cells (Kodama and Fujishima, 2010b).
Therefore, the ring-shaped rearrangement of trichocysts is a phenomenon induced by the algal localization beneath the host cell cortex.
Furthermore, the changes in the trichocysts
when the living symbiotic algae were mixed with
P. caudatum cells were examined. When living
symbiotic algae isolated from alga-bearing P. bursaria cells were added to P. caudatum cells, thealgae were ingested into the DVs. Within 6 h after
mixing, all algae were digested and the color of thealgae became brown. Finally, all digested algae

15

were discharged from a cytoproct. During thisprocess, no change was observed in the trichocyst’s arrangement. These results show that arrangement of the trichocysts in a ring-like shape is
only induced when the living symbiotic algae wereingested by alga-free P. bursaria (Kodama andFujishima, 2010b). Results of these observations
showed that symbiotic algae and the P. bursaria’s
trichocysts are competing for attachment to thehost's unknown structures beneath the host cellcortex.
Because trichocysts are attached at predictable sites of the cell cortex to prevent the dislocation (Hausmann, 1978), the trichocysts may not be
simply pushed away by the attached algae. Using
Gomori’s staining method, we clarified that part of
the trichocysts beneath the host cell cortex are
digested by host lysosomal fusion to reserve thealgal attachment sites during the algal reinfection
process (Kodama and Fujishima, 2010b). Although, the mechanism of replacement of trichocysts by infection of the symbiotic algae remains
unclear, we proposed a hypothesis that trichocysts
are not simply pushed away by SGCs, but thetrichocysts close to the putative algal attachment
site were selectively digested by an unknown
mechanism to ensure the algal attachment sites
beneath the host cell cortex during algal infection
process (Kodama and Fujishima, 2010b).
INITIATION OF THE ALGAL CELL DIVISIONFOR ESTABLISHMENT ENDOSYMBIOSISThe timing of the initiation of the algal cell
division during the algal infection process had
been remained unknown for a long time. Therefore, alga-free P. bursaria cells at the stationary
phase of growth were pulse-labeled with isolated
symbiotic algae for 1.5 min, washed with MDS,
chased, and fixed at 1, 3, 6, 9, 24, and 48 h after
mixing. Then, the mean number of green algae pera P. bursaria cell was counted (Kodama and Fujishima, 2005). At 1 h, the mean number of green

16Secondary symbiosis between Paramecium and Chlorella

algae was 9.4 algae/cell. It decreased to 4.0 algae/
cell at 3 h, and remained constant until 9 h. However, the algae began to increase to 5.3 algae/cellat 24 h. In addition, dividing algae were frequently
observed at 24 h under a DIC microscopy. Thus,
algal cell division initiates at about 24 h after mixing with the alga-free P. bursaria cells for establishment endosymbiosis (Kodama and Fujishima,2005).
CONCLUSION
More than fifty years ago, Siegel and Karakashian (1959) reported that P. bursaria and their
symbiotic Chlorella can separate and then growindependently, and that reinfection of Chlorellafree paramecia is inducible. Since that time, P.
bursaria and its symbiotic Chlorella spp. havebeen considered as a model for studying endosymbiosis by many researchers. Nevertheless, the detailed algal infection process has not been revealed
over those many years. Recently, through pulselabeling with isolated symbiotic algae from algabearing P. bursaria for 1.5 min and chasing forvarious times by our study (Kodama and Fujishima, 2005), classification of P. bursaria’s DVs,
timing of acidosomal and lysosomal fusion to theDVs, the route of algal infection to the alga-free P.
bursaria cells, and four important cytologicalevents needed to establish endosymbiosis were
found as summarized in Fig. 5.
To establish endosymbiosis with the host, anendosymbiont has to meet the following requirements: (1) An endosymbiont invades the host cytoplasm. (2) The endosymbiont avoids digestion by
the host’s lysosomal fusion. (3) The endosymbiont
grows synchronously with the host cell. (4) The
endosymbionts distribute to daughter cells when
the host cell divides. (5) The endosymbiont has tocontribute to the host’s survival. Various phenomena observed in the algal reinfection process provide an excellent opportunity to elucidate theserequirements.

Mixotrophy and endosymbiosis are widespread phenomena among protists than previously
believed (Esteban et al., 2010). Besides P. bursaria, freshwater protozoa Climacostomum virens(Reisser, 1984, 1986), Euplotes daidaleos(Heckmann et al., 1983), Vorticella spp. (Graham
and Graham, 1978, 1980), Stentor polymorphus(Reisser, 1984, 1986), and Mayorella viridis(Cann, 1981) harbour Chlorella spp. as their endosymbionts. Research on the endosymbiosis between P. bursaria and Chlorella spp. may lead tofind a universal mechanism for establishing algaeprotozoa endosymbiosis.
ACKNOWLEDGEMENTS
This work was supported by a grant-in-aid
for Research Activity Start-up (No. 22870023)
from the JSPS to Y. Kodama, by a grant from Inoue Foundation for Science (Inoue ResearchAward for Young Scientists) to Y. Kodama, and by
a grant-in-aid for Scientific Research (B)(No.17405020) from the JSPS to M. Fujishima.
With kind permissions of Elsevier, this original
data could be reused in this review.
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