induction of secondary symbiosis between the ciliate paramecium...

Induction of secondary symbiosis between the ciliate Paramecium and the

green alga Chlorella

Y. Kodama1, and M. Fujishima

2

1 Research and Education Faculty, Natural Sciences Cluster, Sciences Unit, Kochi University, Kochi 780-8520, Japan 2 Department of Environmental Science and Engineering, Graduate School of Science and Engineering, Yamaguchi

University, Yoshida 1677-1, Yamaguchi 753-8512, Japan

The ciliate Paramecium bursaria and the green alga Chlorella species can establish endosymbiosis by mixing them

through the host digestive vacuoles. Pulse-labeling of alga-free Paramecium cells with isolated symbiotic algae for 1.5

min and chasing for various times showed presence of four cytological phenomena which are indispensable for the algal

reinfection. (1) Some algae acquire temporal resistance to lysosomal enzymes in the digestive vacuoles. (2) Not only the

resistant green algae but also partially digested brown algae begin to escape from the digestive vacuoles by budding of the

vacuole membrane at 30 min after mixing. (3) Then each small vacuole enclosing a green alga differentiates to a perialgal

vacuole, which gives protection from the host lysosomal fusion, (4) and the perialgal vacuole translocates and attaches

beneath the host cell cortex. Algal cell division in the perialgal vacuole begins at about 24 h after mixing. Algal proteins

synthesized during photosynthesis serve some important functions to prevent expansion of the perialgal vacuole and to

attach under the host cell cortex, and to protect the perialgal vacuoles from the host lysosomal fusion. The finding of these

phenomena for the reestablishment of the endosymbiosis reveals that P. bursaria is an excellent model for studying the

infection process of the algae and the evolution of eukaryotic cells through secondary endosymbiosis.

Keywords Chlorella; evolution; infection; Paramecium; perialgal vacuole membrane; secondary endosymbiosis

1. Introduction

Endosymbiosis is a primary force in eukaryotic cell evolution, and yields a wide diversity of eukaryotic cells. Despite

the importance of this phenomenon, however, molecular mechanisms for induction of endosymbiosis between different

eukaryotic cells are not so well known. A cell of the ciliate Paramecium bursaria harbors several hundred symbiotic

algae in the cytoplasm (Fig. 1A). Each alga is enclosed in a perialgal vacuole (PV) membrane and localizes beneath the

host cell cortex near trichocysts (Fig. 1B). Irrespective of the mutual relationships between P. bursaria and symbiotic

algae, the alga-free cell and the symbiotic alga keep the ability to grow without a partner. Alga-free P. bursaria can be

easily prepared from alga-bearing paramecia by several methods: rapid cell division [1], cultivation under constant dark

condition [2–4], X-ray irradiation [5], treatment with photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea

(DCMU), a blocker of electron flow in photosystem II [6], protein synthesis inhibitor, cycloheximide [7–9] and

herbicide paraquat [10]. On the basis of these reasons, the symbiotic associations between P. bursaria and Chlorella

spp. are regarded as an excellent model for studying cell-to-cell interaction and the evolution of eukaryotic cells through

secondary endosymbiosis [11, 12]. However, the mechanisms and timings used by the algae to escape from the host

digestive vacuole (DV) to the cytoplasm and to protect themselves from host lysosomal fusion have long remained

unknown.

Fig. 1 Light and transmission electron micrographs of

Paramecium bursaria: A DIC image; B TEM image; Ma,

macronucleus; Cy, cytopharynx; A, symbiotic alga; PVM,

perialgal vacuole membrane; Tc, trichocyst; TcM, trichocyst

membrane; c, cilia.

Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology A. Méndez-Vilas (Ed.)

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2. Algal reinfection process

Infection of algae to alga-free P. bursaria is conducted through the host’s phagocytosis. Although differentiationprocess of P. bursaria’s DVs in phagocytosis and infection process have been unclear, these processes have beenrecently revealed by Kodama and Fujishima [9, 11–16] and Kodama et al. [8].

2.1 Timing of acidosomal and lysosomal fusion to DVs

The DVs of P. multimicronucleatum have been classified into four different stages [17]. To classify the DV stages of P.bursaria that appear during reinfection by symbiotic algae and to determine the timing of the appearance of each stage,isolated symbiotic algae and alga-free P. bursaria strain OS1w cells were mixed at densities of 5 × 107 algae/ml and 5 ×103 paramecia/ml under fluorescent lighting (1500 lux) at 25 ± 1˚C and they were fixed with 4% (w/v)paraformaldehyde at various times after mixing. To synchronize the DV’s differentiation and to observe fates of thealgae ingested in the host DVs later than 2.5 min after mixing, the symbiotic algae and alga-free P. bursaria cells weremixed as described above for 1.5 min at 25 ± 1˚C and the paramecia were washed with modified Dryl’s solution (MDS)[18] (KH2PO4 was used instead of NaH2PO4•2H2O) to removed uningested algae. Then they were resuspended in theoriginal cell density, and fixed with paraformaldehyde at various times after mixing. We named this method as a “pulse-label and chase method” [13]. The experimental procedure is presented in Fig. 2. The DVs containing several algaewere classified into four stages based on their morphological characters and changes of the algal color (Fig. 3): DV-I, atwhich stage the rounded vacuole membrane is clearly observable under a differential-interference-contrast (DIC)microscope and the algal color is green; DV-II, where the vacuole is condensed so that the vacuole membrane is hardlyobservable under a DIC microscope and the algal color is still green; DV-III, where the vacuole has enlarged in size andthe vacuole membrane is observable again and the algal colors are discolored as faintly yellow, green, or both. DV-IIIincludes three substages: DV-IIIa contains green algae only; DV- IIIb contains both discolored yellow and green algae;and DV-IIIc contains discolored yellow algae only. In the final stage, DV-IV, the membrane is condensed again similarto DV-II, making it difficult to observe under a DIC microscope. The algal colors are brown, green or both. Unlike DV-II, this vacuole is not observed in cells fixed before 3 min, but is observed 20–30 min after mixing. DV-IV includesthree substages: DV-IVa contains green algae only; DV-IVb contains both green and brown algae; and DV-IVccontains brown algae only [13].

Fig. 2 Pulse label and chase method. Alga-free P. bursaria cells were pulse labeled with isolated symbiotic C. vulgaris strain 1Ncells under a fluorescent light (1500 lux) at 25 ± 1°C. Paramecia were then washed using nylon mesh filter. It was then resuspendedin Dryl’s solution, and fixed at various times after mixing, and the fates of the algae ingested in the host DVs were observed.

To determine the timing of the appearance of each stage of DVs, alga-free P. bursaria cells were mixed with isolatedsymbiotic algae and fixed at 10 s intervals for 60 s without washing. The fixed cells were classified into four stagesaccording to the most advanced stage of DV in the host cell (i.e., if both DV-I and DV-II were present in a host cell,then the DV was classified as DV-II). Results show that DV-II started to appear in cells fixed at 30 s after mixing. Todetermine the timing of DV-III appearance, alga-free cells were pulsed with isolated symbiotic algae for 1.5 min, thenwashed, chased, and fixed at every 1 min interval for 10 min after mixing. Subsequently, the cells were classified intoeight stages including substages of DV-III and IV based on their morphological characters. As a result, themorphological differentiation of DV-III started to appear in cells fixed at 3 min after mixing with algae [13].

For P. multimicronucleatum, it has been reported that inside pH of the DVs was changed by the followingacidosomal and lysosomal fusion [17]. To determine the timing of the acidosomal and lysosomal fusion to P. bursariaDVs, yeast Saccharomyces cerevisiae cells were stained with three kinds of pH indicator dyes, Congo red, bromocresolgreen and bromophenol blue and mixed with alga-free P. bursaria cells. Then the color changes of the ingested yeastcells in the DVs were observed.

At 1–2 min after mixing, the intravacuolar pH declines to 2.4–3.0. Morphological differentiation of DV-II from DV-Ioccurred at 0.5–1 min after mixing. These results show that acidosomal fusion to the DV occurs at 0.5–1 min aftermixing [13].

Partially digested yellow algae were observed first in DV-IIIb at 2–3 min after mixing. In the late stage of DV-II, theintravacuolar pH was 6.4–7.0. These results suggest that lysosomal fusion might occur before 2–3 min after mixing.Furthermore, Gomori’s staining [19], which is used to detect intravacuolar acid phosphatase (AcPase) activity, showed


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that both DV-I and DV-II are AcPase-activity negative, but all substages of DV-III and DV-IV were AcPase-activitypositive [15]. These results indicate that lysosomal fusion occurs before 2–3 min after mixing. A schematicrepresentation of DV differentiation of P. bursaria is depicted in Fig. 3

Fig. 3. Algal reinfection process to P. bursaria. Four cytological phenomena needed for the algal reinfection were discovered.(Updated from [8, 11–13, 15])

2.2 Four cytological phenomena needed for establishment of secondary symbiosis

Timings for algal escape from the host DV and for protection of themselves from the host’s lysosomal fusion wereclarified. Furthermore, four cytological events that are necessary to establish secondary endosymbiosis between P.bursaria and Chlorella cells were found.

2.2.1 Algal temporal resistance to lysosomal enzymes in DVs

In the host cytoplasm, single green Chlorella (SGC) is wrapped with a PV membrane derived from the host DVmembrane, which gives protection from host lysosomal fusion, as shown in Fig. 1 and 3. This indicates that timing ofthe appearance of SGC should be considered as almost the same as that of the appearance of the PV membrane duringalgal reinfection process. To determine the timing of the appearance of the SGCs, alga-free P. bursaria cells weremixed with isolated symbiotic algae for 1.5 min, washed, chased, then fixed at 0.05, 0.5, 1, 1.5, 2, 3, 6, 9, 24, 48, and 72h after mixing. The percentages of cells with SGC, single digested Chlorella (SDC), DV-IIIa or DV-IIIb, and DV-IVaor DV-IVb showed that all SGCs existed in the host cytoplasm before 0.5 h after mixing were digested. One hour aftermixing, however, SGCs appeared again in the host cytoplasm. We found that the SGCs that appeared after 0.5 h werederived from DV-IVa or DV-IVb because no green algae were present in other DVs. At 24 h, the SGCs began tomultiply by cell division, indicating that these algae had established endosymbiosis [13]. In contrast to results of anearlier study [20], our result shows that the algal escape from the host DV-IVa or DV-IVb occurs after acidosomal andlysosomal fusion to the DV. When boiled algae are added to alga-free paramecia, they are all digested in DV-III and aredischarged from the host cytoproct. This shows that only the living Chlorella have an ability to avoid lysosomaldigestion in the host DVs.

What determines the algal fate to survive in DV-IV fused with lysosomes? At first, symbiotic algae isolated from a P.bursaria were cloned, mixed with the alga-free P. bursaria and established a P. bursaria strain OS1g1N that bears acloned algae. Thereafter, all reinfection experiments were done using this genetically identical algae isolated from P.bursaria strain OS1g1N cells. This cloned symbiotic alga was identified as Chlorella vulgaris and named as strain 1N[8]. Alga-free P. bursaria cells were pulsed with the isolated symbiotic C. vulgaris 1N cells for 1.5 min, washed andchased for 3 h after mixing. Though genetically identical, a few of the algae were not digested but coexisted with thedigested ones in the same DV-IVb vacuole after lysosomal fusion. Furthermore, light microscopy showed that algal fatedid not depend on cell cycle stage of the algae or location in the host DVs. Electron microscopy showed that the non-digested algae were not protected by a PV membrane in the DV-IVb. Moreover, this phenomenon was also observed inthe presence of protein synthesis inhibitors, cycloheximide and puromycin, which are known to inhibit algal and hostprotein synthesis, respectively. These observations suggest that a few algae can acquire temporary resistance to hostlysosomal enzymes in order to establish endosymbiosis without algal protein synthesis [8].

2.2.2 Algal escape from host DV by budding of DV membrane

At 30 min after mixing with symbiotic algae, some algae start to escape from the DV-IVb by budding of the DVmembrane into the cytoplasm. This budding is induced not only by living algae but also by the algae that are boiled,


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fixed with 2.5% (v/v) glutaraldehyde, or fixed with 5.0% (v/v) formaldehyde [13] (Y. Kodama and M. Fujishima,

unpubl. data). Inorganic polystyrene latex spheres of 3 µm in diameter bud too (Y. Kodama and M. Fujishima, unpubl.

data). However, this budding is not induced when India ink, polystyrene latex spheres of 0.81 µm in diameter, or food

bacteria Klebsiella pneumoniae were ingested into the DVs [13] (Y. Kodama and M. Fujishima, unpubl. data). These

results suggest that large DVs of P. bursaria may differentiate to small DVs by the budding of DV membrane but

minimum diameter for the budding is about 3 µm.

2.2.3 Differentiation of PV membrane from DV membrane

To elucidate the timing of the PV from the host DV, alga-free cells were mixed with isolated symbiotic algae for 1.5

min, washed, chased, fixed at various times after mixing, and observed after Gomori’s staining. Figure 4A shows DV-

IVb at 30 min after mixing in which all DVs show AcPase-activity and SGSs attached beneath the host cell cortex

cannot be observed. At 3 h after mixing (Fig. 4B), however, single green algae are just escaping as the result of the

budding of the vacuole membrane, and such escaping algae from the DV are surrounded by a AcPase activity-positive

thin layer (white arrowhead). Inset of Fig. 4B also shows that green algae escaped from the host DV-IVb adhere just

under the host cell cortex (arrows). Such SGCs are not covered by the black thin layer. This important difference

indicates that the inside of the budded DV membrane enclosing green algae is still AcPase-activity positive, although

the inside of the vacuole enclosing an SGC, which is derived from the DV-IVb and localized beneath the host cell

cortex, is AcPase-activity negative. These observations show that differentiation from the host DV membrane to the PV

membrane occurs after the appearance of SGCs as the result of budding of the host DV membrane and before

translocation of the SGCs beneath the host cell cortex. That the first appearance of the SGC and the first localization of

the SGC beneath the host cell cortex, respectively, occur at 30 and 45 min after mixing suggests that differentiation of

the PV membrane occurs within 15 min after the algal escape from the host DV [15].

Fig. 4 Gomori’s staining of P. bursaria in infection process. Alga-free P. bursaria were pulse labeled with isolated symbiotic

algae for 1.5 min, and fixed at 30 min (A) and 3 h (B) after mixing. Then, paramecia were stained with Gomori’s solution to detect

acid AcPase activity. AcPase activity is shown as black granules. D, DV involving algae. Inset B shows enlarged photomicrograph of

broken square area. At 30 min after mixing (A), all DVs are AcPase activity-positive. At 3 h after mixing (B), escaping algae from

the DVs are surrounded by a AcPase activity-positive thin layer (white arrowhead). Note that single green algae, which are enclosed

by PV membrane and attached immediately beneath the host cell cortex are AcPase activity-negative (arrows).

In the host cytoplasm, each symbiotic algal cell enclosed within a PV membrane localizes beneath the host cell

cortex, where thousands of trichocysts are embedded as defensive organelles against predators [21]. The symbiotic

algae exist among the trichocysts. This area has been shown to be an AcPase activity-negative area to 5–10 µm depth

by Gomori’s staining [9, 16], indicating that few if any primary lysosomes are present. These observations raise the

possibility that the PV membrane might have no ability of protection from lysosomal fusion, but can avoid lysosomal

fusion by localization at the primary lysosome-less area of the host cell. To elucidate whether algal protection from the

host lysosomal fusion is controlled by localization of the PV membrane to the AcPase activity-negative area or by the

capability of the PV membrane to give protection from lysosomal fusion, trichocysts were removed from P. bursaria

cells through treatment with 1 mg/ml lysozyme, thereby reducing the AcPase activity-negative area and exposing the

PVs to the AcPase-positive area, and examined whether the PV’s protection from the lysosomal fusion is still achieved

or not. The trichocyst-free cell reduced the AcPase activity-negative area to less than 3 µm depth at the dorsal cortex.

However, even though a part of the algal cell had been exposed in the AcPase activity-positive area, the algae were able

to attach beneath the host cell cortex and the algae were not digested despite the fact that the algae were exposed to the

AcPase activity-positive area. These results show that the PV membrane, unlike the DV membrane, can give protection

from the host lysosomal fusion [16].


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2.2.4 Attachment of PV beneath host cell cortex

The symbiotic algae are looked to push the trichocysts aside to become settled near the host cell cortex after escaping

from the host DVs. To examine the requirement of trichocysts for algal intracellular localization, alga-free cells and

isolated symbiotic algae were mixed for 1.5 min, washed, and then resuspended with the same concentration in MDS.

Algal escape from the host DVs begins at 30 min after mixing with algae [13, 15]. The pulse-labeled cells were treated

with 1 mg/ml lysozyme 30 min after mixing with algae. This concentration of lysozyme is enough to induce full

discharge of trichocysts. These cells were fixed at 3 and 24 h after mixing with algae in the presence of the lysozyme.

They were then observed to determine whether the algae that had escaped from the host DVs were able to attach

beneath the host cell cortex even if trichocysts were removed. In the control experiment, at 3 h, green algae attached

beneath the host cell cortex were observed. On the other hand, green algae, which localized beneath the host cell cortex

were observed, even in cases where the trichocysts had been removed. In such cells, the density of the algae attached

near the host cell surface was higher than that in the trichocyst-bearing control cells. These results demonstrate that the

PV membrane does not require trichocysts for the intracellular localization [16]. Our results suggest that trichocysts are

obstacles to rather than prerequisites for algal localization beneath the host cell cortex. How and where the symbiotic

algae attach to the host cell cortex remains unclear [16].

It is known that only C. vulgaris, C. sorokiniana, and Parachlorella kessleri can establish endosymbiosis with P.

bursaria [22]. We compared behaviors of various Chlorella species or Parachlorella in infection process, and the

nature of their cell wall components. Alga-free P. bursaria cells were mixed with 15 strains of cultivated Chlorella

species and Parachlorella and observed at 1 h and 3 weeks after mixing. Only 2 free-living algal strains, C. sorokiniana

C-212 and P. kessleri C-531, were maintained in the host cells, whereas free-living C. sorokiniana C-43, P. kessleri C-

208, C. vulgaris C-27, C. ellipsoidea C-87 and C-542, C. saccharophila C-183 and C-169, C. fusca var. vacuolata C-

104 and C-28, C. zofingiensis C-111, and C. protothecoides C-150 and C-206 and the cultivated symbiotic Chlorella sp.

strain C-201 derived from Spongilla fluviatilis were not maintained. These infection-incapable strains could escape

from the host DVs but failed to localize beneath the host cell cortex and were eventually discharged from the host

cytoproct. Labeling of their cell walls with Alexa Fluor 488-conjugated wheat germ agglutinin (WGA), GS-II, or

concanavalin A (Con A) showed no relationship between their infectivity and the stainability with these lectins. These

results indicate that the infectivity of Chlorella species and Parachlorella for P. bursaria is not based on the sugar

residues on their cell wall, but on their ability to localize just beneath the host cell cortex after escaping from the host

DVs [14].

3. Property of PV membrane

The DV and PV membranes are known to show the following qualitative differences: (1) The PV membrane encloses a

single algal cell [23, 24]; (2) Because the gap separating the algal cell wall and the PV membrane is about 0.05 µm, the

PV membrane is hardly visible under a light microscope. However, it can be observed readily using a transmission

electron microscope (TEM) [25]; (3) The PV diameter does not change greatly (2.5–4.5 µm), except during the cell

division of the enclosed symbiotic alga [26]; (4) The PV does not participate in cytoplasmic streaming, but it localizes

immediately beneath the host cell cortex [13, 25]; (5) Particle density and its distribution of the PV membrane show few

signs indicating any endocytotic or exocytotic activity, which can be observed in the DV membrane [27]; (6) The PV

can attach beneath the host cell cortex and to protect it from lysosomal fusion whereas the DV cannot [16]. (7)

Treatment with a carboxylic ionophore, monensin in the presence of the algal photosynthesis induces synchronous

swelling of all PVs [28]. (8) Inhibition of algal protein synthesis in the presence of the algal photosynthesis induces

synchronous swelling of all PVs and digestion of the algae [9].

3.1 Effect of cycloheximide to PV membrane

Cycloheximide is known to inhibit protein synthesis of symbiotic Chlorella of the ciliate P. bursaria preferentially, but

it only slightly inhibits host protein synthesis [29]. Therefore, alga-free paramecia can be prepared within 4–6 days after

the treatment of cycloheximide [8, 30]. This phenomenon is expected to be a useful model to examine the features or

functions of PV membrane. Therefore, alga-bearing P. bursaria cells were treated with 10 µg/ml of cycloheximide and

observed under a light microscope. Figure 5 shows an alga-bearing cell treated with cycloheximide for 24 h. As shown

in Fig. 1A, without treatment with cycloheximide, the PV membrane enclosing each symbiotic alga cannot be observed

under a light microscope. However, as depicted in Fig. 5, almost the entire PVs inside the alga-bearing cells began to

swell synchronously at 24 h after the treatment. This indicates that inhibition of the algal protein synthesis induces

swelling of the PV membrane. We named this phenomenon as “synchronous PV-swelling (SPVS)”. It was induced in

all PVs enclosing symbiotic green algae in the host cell [9]. The space between the symbiotic algal cell wall and the PV

membrane widened to about 25 times its normal width 24 h after treatment with cycloheximide. Then, the vacuoles

detached from beneath the host’s cell cortex, were condensed and became stained with Gomori’s solution, and the algae

in the vacuoles were eventually digested. Algal digestion was induced soon after the SPVS. Such synchronized


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symbiotic algal digestion is not observed before SPVS. Therefore, this digestion is inferred as a phenomenon that is

closely related to SPVS.

Fig. 5 Photomicrograph of synchronous PV-swelling (SPVS).

Algae-bearing OS1g1N cells were suspended in fresh culture

medium containing 10 µg/ml of cycloheximide at 25 ± 1°C under

constant light conditions for 24 h. All PVs, which contain green

algae swelled synchronously. Following by SPVS, algal digestion

was also induced. Ma, macronucleus; Cy, cytopharynx.

Although this SPVS is induced only under a fluorescent light condition, and not under a constant dark condition, this

phenomenon was not induced in paramecia treated with cycloheximide even in the light in the presence of

photosynthesis inhibitor, DCMU [9]. These results indicate that algal proteins synthesized in the presence of algal

photosynthesis serve some important function to prevent expansion of the PV and to maintain the ability of the PV

membrane to protect itself from host lysosomal fusion. Schüßler and Schnepf [28] reported that PV swelling could be

induced by treatment of the alga-bearing P. bursaria cells with monensin, and that the PV swelling was observed only

under constant light condition, but inhibited by DCMU. However, unlike SPVS induced by cycloheximide, the PV

swelling induced by monensin does not lead to algal digestion in the PVs.

We observed that the digested algae were discharged from the host cytoproct. By 24 h after the treatment with

cycloheximide, the mean number of symbiotic green algae per host cell had decreased to about one-sixth of its former

value. Three days after the treatment, all green algae in some Paramecium cells had disappeared. At day 7 after the

treatment, all paramecia have lost their symbiotic algae completely and they became alga-free cells.

4. Initiation of algal cell division in reinfection and after establishment of stable

endosymbiosis

To determine the timing of initiation of algal cell division in reinfection process, alga-free P. bursaria cells in early

stationary phase of growth were mixed with isolated symbiotic algae for 1.5 min, chased, and fixed at various times

after mixing. Subsequently, mean numbers of the green algae per a Paramecium cell were counted [13]. The average

was 9.4 algae/cell at 1 h, decreasing to 4.0 algae/cell at 3 h, and keeping constant thereafter until 9 h. However, the

average began to increase to 5.3 algae/cell at 24 h. In addition, dividing algae were frequently observed beneath the host

cell cortex at 24 h. On the other hand, the host cells did not multiply by cell division until 72 h after mixing.

P. bursaria cell shows microtubule-based cytoplasmic streaming. This cytoplasmic streaming is arrested in a dividing

host cell and its symbiotic algae proliferate only during the arrest of cytoplasmic streaming [31]. Thus, unlike in

reinfection, the symbiotic alga initiates its cell division just before the host cell division. Interestingly, arrest of

cytoplasmic streaming with pressure or a microtubule drug also induced initiation of the algal cell division

independently of host cell cycle. In growing host cell, cytoplasmic streaming may control the algal proliferation.

5. Conclusion

In symbiotic or parasitic organisms, various escape mechanisms from the host lysosomal digestion are known: (1)

prevention of acidosomal fusion or lysosomal fusion to the DV, as has been shown for Legionella pneumophila [32],

Plasmodium falciparum [33], Salmonella enterica [34], Mycobacterium tuberculosis [35], Chlamydia trachomatia [36],

and X-bacteria in Amoeba proteus [37]; (2) escape from the DV into the host cytoplasm before lysosomal fusion, as has

been shown for Listeria monocytogenes [38], Tripanosoma cruzi [39], Shigella flexneri [40], and Holospora obtusa

[41], and (3) resistance to or inactivation of lysosomal enzymes after lysosomal fusion to the DV, as has been shown

for infection by Coxiella burnetii [42, 43] and Leismania [44].

Our data indicate that symbiotic Chlorella species has a novel mechanism to avoid digestion by the host lysosomal

enzymes. First, some algae obtain temporary resistance to lysosomal enzymes in DV. Second, the alga escapes from the

DV into the cytoplasm by budding of the DV membrane. Third, the alga is enwrapped with a PV membrane, which

gives protection from the host’s lysosomal fusion. Finally, the alga settles immediately beneath the host cell cortex.

How can the symbiotic algae obtain temporal resistance to lysosomal enzymes in the host DVs? How can algae escape

from the host DV as a result of budding of the DV membrane? What is a mechanism of PV membrane for prevention of


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host lysosomal fusion? What is the moving force of the PV membrane immediately beneath the host cell cortex? What

is the signal for initiation of algal cell division after localization beneath the host cell cortex? Can endosymbiosis be

established when the symbiotic algae are not taken up through DVs, but are instead inserted by microinjection? Why,

among Paramecium species, can only P. bursaria establish endosymbiosis with Chlorella species? How can the PV

membrane separate an old cell wall debris that is discarded after algal cell division, and daughter algal cells? Can a

single Paramecium cell maintain multiple Chlorella species in a single P. bursaria cell? These problems remain to be

clarified to understand the establishment of the secondary symbiosis between Paramecium and Chlorella cells.

Endosymbiosis of Chlorella spp. is widespread among freshwater protozoa: Climacostomum virens [25, 45],

Euplotes daidaleos [46], Vorticella spp. [47, 48], Stentor polymorphus [25, 45], Spongilla lacustris [49–51], Hydra

viridis ([52, 53], and Mayorella viridis [54]. Whether the symbiotic algae in these protozoa have identical mechanisms

for avoiding host lysosomal digestion of symbiotic algae of P. bursaria or not must be examined.

Acknowledgements This work was supported by a Japan Society for the Promotion of Science (JSPS) Research Fellowship for

Young Scientists granted to Y. Kodama, and by a Narishige Zoological Science Award granted to M. Fujishima.

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