Abstracts of the JSP meeting 2013
Oral presentations
1) Effect of cell density on the formation of cannibal giants in Blepharisma
Yuna ONO, Mayumi SUGIURA and Terue HARUMOTO
2) Do the nuclear changes occur in interspecific conjugation of Blepharisma?
Mao YAMADA, Mayumi KOBAYASHI, Mayumi SUGIURA and Terue HARUMOTO
3) Microtubule dynamics during rapid axopodial contraction in heliozoon Raphidiophrys
contractilis revealed by immunoelectron microscopy
Risa INOUE and Motonori ANDO
4) Real-time phenomenological analysis of dielectric behavior of Euglena cells during theircell-shape changes
Ken HANAHARA and Motonori ANDO
5) γH2AX formation in haploid nuclei of Tetrahymena thermophila: A possible involvement of anovel type of DNA double-strand break
Takahiko AKEMATSU, Yasuhiro FUKUDA, Chika TADA, Yutaka NAKAI and
Ronald E. PEARLMAN
6) Detection and functional analysis of plant hormones in malaria parasite
Ryuma MATSUBARA, Mikiko KOJIMA, Michiru TAHARA, Syed Bilal Ahmad ANDRABI,Michika FKSHI, Fumiya KAWAHARA, Akinori YAMANO, Hitoshi SAKAKIBARA andKisaburo NAGAMUNE
7) Analysis of an early stage of the evolution of endosymbiosis between an alga and a ciliate inan experimental microcosm composed of 3 species
Toshiyuki NAKAJIMA
8) The evolution of interspecific relationship between an alga and a bacterium in the long-term
culture of experimental microcosm
Masayuki MATSUURA, Yosuke HUJII and Toshiyuki NAKAJIMA
9) Changes in the algal ingestion rate of ciliate cells in an early stage of evolution of algal-ciliate
endosymbiosis in the long-term culture of experimental microcosm
Yoichiro OHNISHI, Akiko SANO and Toshiyuki NAKAJIMA
10) Life style and evolution of 'Protozoa' and 'Algae', viewed from freshwater microscopic
ecosystem
Masashi M. HAYAKAWA and Toshinobu SUZAKI
11) A study on a rolling structure of plasma membrane isolated from Amoeba proteus
Yukinori NISHIGAMI, Atsushi TANIGUCHI, Shigenori NONAKA, Masatoshi ICHIKAWA and
Seiji SONOBE
12) The observation of ultrastructure in Hypophrya sp.
Go KOBASHIGAWA, Atsushi TANIGUCHI, Tohru YOSHIHISA and Seiji SONOBE
13) Experimental evolution of Eecherichia coli toward constructing a novel intracellularendosymbiosis with Dictyostelium discoideum
Kayo YAMAMOTO, Kazufumi HOSODA, Makoto SUEYOSHI, Isao KUBO and
Tetsuya YOMO
14) Construction of a chloroplast-like novel endosymbiosis between a cyanobacterium and aciliateYuhki AZUMA, Kazufumi HOSODA, Masumi HABUCHI, Kayo YAMAMOTO, Risa TAKAMI
and Tetsuya YOMO
15) Theoretical study about evolution of stalk/spore ratio in a social amoeba
Kouki UCHINOMIYA and Yoh IWASA

16) Maltose release mechanism of symbiotic Chlorella isolated from Japanese Paramecium
bursaria
Aika SHIBATA, Masahiro KASAHARA and Nobutaka IMAMURA
17) Construction of a novel endosymbiosis between Escherichia coli and algae-free Paramecium
bursaria
Risa TAKAMI, Kazuhumi HOSODA, Itsuka KUMANO, Kayo YAMAMOTO and
Tetsuya YOMO18) Effects of the endosymbiosis on phagocytic activities of Paramecium bursaria
Takashi MIURA and Sosuke IWAI
19) The stress tolerance of Paramecium bursaria
Yuka ADACHI, Ayumi SHIMOMURA, Kozue HAMAO and Hiroshi HOSOYA20) Infectivity of the symbiotic Chlorella spp. of the ciliate Paramecium bursaria
Sotaro ARAKI and Yuuki KODAMA21) Cell signaling pathways and analysis of expressed proteins during the resting cyst formationof ciliated protozoan Colpoda cucullus
Yoichiro SOGAME, Katsuhiko KOJIMA, Toshikazu TAKESHITA, Eiji KINOSHITA and
Tatsuomi MATSUOKA
22) Roles of kinesin14A in micronuclear division in Tetrahymena
Yasuharu KUSHIDA, Masak TAKAINE, Kentaro NAKANO, Toshiro SUGAI, Krishna Kumar
VASUDEVAN, Mayukh GUHA, Yu-yang JIANG, Jacek GAERTIG and Osamu NUMATA
23) Tetrahymena as a production plant of foreign proteins
Kohei MASUDA and Hiroshi ENDOH+

24) FAP82 regulates activation of K leak channel in Paramecium
Shinobu IZUTANI, Takashi TOMINAGA and Manabu HORI
25) Development of nickel inducible expression system in Paramecium caudatum
Yasuhiro TAKENAKA, Nobuyuki HAGA and Ikuo INOUE
26) Molecular composition and the function of plant-vacuole like organelle in extracellular
Toxoplasma gondii
Michika FKSHI, Kazuhide YAHATA, Takaya SAKURA, Michiru TAHARA,
Ryuma MATSUBARA, Osamu KANEKO and Kisa NAGAMUNE
27) Megavirus-infecting Acanthamoeba sp. isolated from a keratitis patient
Kenji YAGITA and Yoshitsugu INOUE28) Species composition change in the intestinal protist communities after community mixing
Osamu KITADE and Yoshihiro KURUSU29) Development of microscopic system for precise investigation of mobile protists
Ikuko SHIHIRA-ISHIKAWA, Hiroyuki KAWANO, Yasutaka HANADA and Atsushi MIYAWAKI
30) Growth analysis of Paramecium busaria and the algal endosymbionts
Sosuke IWAI and Takuro TAMURA

#1

Effect of cell density on the formation of cannibal giants in Blepharisma
Yuna ONO1, Mayumi SUGIURA2 and Terue HARUMOTO2(1Dept. Biol. Sci., Fac. Sci., 2Div. Nat. Sci., Nara Women’s Univ.)

SUMMARY
Heterotrich ciliate Blepharisma usually feeds on bacteria. When cells are deprived of food, Blepharisma often
undergoes either “conjugation”, “cyst formation” or “cannibalism” as a strategy for surviving starvation. In this study, tocharacterize giant cells which were generated by cannibalism, we compared the cell size, size of macronucleus and thenumber of micronuclei of giants, with ordinary (normal sized) cells. Major cell axis, minor cell axis and the size of themacronucleus of giants were significantly larger than the ordinary cells in WK-IV strain of Blepharisma japonicum. The
volume of the giant was 5–30 times more than the ordinary ones. Most of the giants had more micronuclei than the ordinary
ones. We also studied the experimental condition to induce giant formation. The number of giant cells was counted in the
different cell densities (800, 1,600, 4,800 and 10,000 cells/ml) for several days. We found that the higher cell density usuallyinduced more giant cells. The observation of giant size and the number of giant cells for successive days suggest that thecannibalism is a transient state for surviving starvation in life cycle of Blepharisma.

#2

Do the nuclear changes occur in interspecific conjugation of Blepharisma?
Mao YAMADA1, Mayumi KOBAYASHI2,3, Mayumi SUGIURA4 and Terue HARUMOTO4(1Dept. Biol. Sci., Fac. Sci., 2Grad. Sch. Nat. Sci. Ecol. Awareness, Nara Women’s Univ.,
3
JSPS Res. Fellow (DC), 4Div. Nat. Sci., Nara Women’s Univ.)

SUMMARY
In the conjugation of Blepharisma, mating type I and mating type II cells secrete conjugation-inducing substances,gamone1 and gamone 2, respectively. Cells are stimulated by complementary gamones and induced pair formation. Twenty
species so far reported in the genus Blepharisma are classified into four groups according to the macronuclear shape. The
previous study revealed that gamone 2 is effective to type I cells of any species between the macronuclear group. On theother hand, gamone 1 was not effective to the species which belongs to the different macronuclear group. Recent studies
revealed that at least between B. japonicum and B. stoltei, both belong to the macronuclear group IV, gamone 1 was
effective in these species. In this work, we studied whether the pair formation is possible between B. japonicum and B.
stoltei, and whether the nuclear changes characteristic to the conjugation occur in the conjugating pair between these two
species. As the results, conjugating pairs formed between these species, and meiosis of micronucleus, subsequent nuclear
changes and the formation of new macronuclear anlagen also occurred in the interspecific conjugants almost same as in theintraspecific conjugants.

#3

Microtubule dynamics during rapid axopodial contraction in heliozoon Raphidiophrys
contractilis revealed by immunoelectron microscopy
Risa INOUE and Motonori ANDO
(Lab. Cell Physiol., Grad. Sch. Educ., Okayama Univ.)

SUMMARY
The centrohelid heliozoon Raphidiophrys contractilis (R. contractilis) has many radiating axopodia, each containing
a bundle of axonemal microtubules as a cytoskeleton. R. contractilis shows rapid axopodial contraction induced bymechanical stimulation. The microtubule-containing axopodia were retracted into the cell body within less than video rate.
However, the contraction mechanism in R. contractilis still remains unresolved. In this study, we investigated themicrotubule dynamics during rapid axopodial contraction in R. contractilis by conventional electron microscopy andimmunoelectron microscopy. Conventional electron microscopy showed that microtubules were well preserved in the cellbody in spite of the induction of axopodial contraction. We did not find the distal parts of the axonemal microtubules moved
into the axopodial base, and any structures like the contractile tubules which have been reported in actinophryid heliozoon
Actinophrys sol. Immunoelectron microscopy showed gold particles on the bundles of microtubules radiating from thecentrosome in the cell body, and those on the cell surface regions of cytosol, where microtubules were not identified. These
results suggest that the axonemal microtubules in R. contractilis may be accompanied by microtubule depolymerization
during the rapid axopodial contraction, and that a novel disassemble manner of microtubules is involved in R. contractilis.

#4

Real-time phenomenological analysis of dielectric behavior of Euglena cells
during their cell-shape changes
Ken HANAHARA and Motonori ANDO
(Lab. Cell Physiol., Grad. Sch. Educ., Okayama Univ.)

SUMMARY
Euglena cells show high sensitivity towards toxicants and can thus reveal any change in environmental water quality.
Dielectric measurements can examine cell-shape dynamics in the flagellate Euglena gracilis. The aim of this study was todevelop an algorithm for the dielectric analysis of Euglena cell suspensions, and was to apply the algorithm to thedescription of cell-shape dynamics in real-time. Equivalent parallel capacitances and conductances were measured between
100 Hz and 100 MHz by Agilent 4294A impedance analyzer. Data acquisition and analysis were performed simultaneously
by our developed software, and then phenomenological parameters were semi-automatically obtained. Curve fitting was
made on the basis of a two-term Cole-Cole equation as modified to include electrode polarization capacitances. The mainalgorithm of this analysis evaluates the goodness of fitting in terms of the minimized residual between experimental andtheoretical curves. The real-time dielectric analysis was performed to monitor cell-shape changes by using Euglena cellsuspensions. Among phenomenological parameters, permittivity increments, characteristic frequencies, and the Cole-Coleparameters allowed sensitive detection of cell-shape changes. This study indicates that our developed bioassay system canbe applied to monitor for aquatic toxicants in environmental water.

#5

γH2AX formation in haploid nuclei of Tetrahymena thermophila: A possible
involvement of a novel type of DNA double-strand break
Takahiko AKEMATSU1, Yasuhiro FUKUDA2, Chika TADA2, Yutaka NAKAI2
and Ronald E. PEARLMAN1(1Dept. Biol., York Univ., 2Dept. Biodiversity Sci., Div. Biol. Resource Sci., Grad. Sch. Agr. Sci.,
Tohoku Univ.)

SUMMARY
γH2AX, the phosphorylated form of histone H2AX, is a well-established marker for double-strand DNA breaks(DSB). In Tetrahymena thermophila, γH2AX is observed not only in crescent-shaped micronuclei characteristic of meiotic
homologous recombination, but also in the developing macronuclear anlagen during genome rearrangement. Here, we
demonstrate that Tetrahymena conjugation involved alternate γH2AX formation in the haploid micronuclei. This
modification was observed in all four nuclei after the completion of meiosis. Subsequently, dephosphorylation occurred in
one of the nuclei that localized near the conjugating junction formed between two complementary mating type cells. This
nucleus attached to the junction and induced elongation for division, while the other three nuclei with γH2AX migrated tothe posterior region of the cytoplasm and eventually disappeared. The somatic knockout mutant (DNA-PKΔ) failed to form
γH2AX and was incapable of attachment and elongation of the nucleus. Consequently, these cells did not undergo
conjugation and reverted to the vegetative stage. These results suggest that γH2AX formation is critical for conjugation. A
novel type of DSB is likely involved in the nuclei in which DNA-PK exerts its function through the formation of γH2AX.

#6

Detection and functional analysis of plant hormones in malaria parasite
Ryuma MATSUBARA1,2, Mikiko KOJIMA3, Michiru TAHARA2, Syed Bilal Ahmad ANDRABI2,4,
Michika FKSHI1,2, Fumiya KAWAHARA5, Akinori YAMANO1,2, Hitoshi SAKAKIBARA3
and Kisaburo NAGAMUNE2,6
3(1Grad. Sch. Life Environ. Sci., Univ. Tsukuba, 2Dept. Parasitol, Natl. Inst. Infect. Dis.,
Plant Sci. Ctr., RIKEN, 4.Ctr. Integr. Med. Res., Sch. Med., Keio Univ., 5Nippon Inst. Biol. Sci.,
6
Fac. Life Environ. Sci., Univ. Tsukuba)

SUMMARY
Plasmodium is an Apicomplexan parasite that causes lethal febrile symptoms called malaria. This parasite belongs
to Alveolata superphylum and has extremely different metabolic systems from its host. Recently, we found that Plasmodium
spp. produced several plant hormones, and in this study, we focused on a dominant one. We succeeded to establish thehormone-deficient mutant genetically. The neural pathogenicity of the mutant to C57BL/6 mice was severer than wild-type
parasite. We will discuss the role of this plant hormone from the immunologic and pathologic viewpoints.

#7

Analysis of an early stage of the evolution of endosymbiosis between an alga anda ciliate in an experimental microcosm composed of 3 species
Toshiyuki NAKAJIMA(Dept. Biol., Ehime Univ.)

SUMMARY
The early stage of the evolution of symbiotic relationships was analyzed by conducting a long-term culture of anexperimental ecosystem model composed of a green alga (Micractinium sp.), a bacterium (Escherichia coli), and a ciliate(Tetrahymena thermophila). The population dynamics of component species was investigated during three years from its
initiation, and interspecific interactions by using isolates from the culture. The results revealed that an early form ofalgal-ciliate endosymbiotic association and algal-bacterial ectosymbiotic association were evolved, and that theseevolutionary changes were closely related in their selection processes in this ecosystem.

#8

The evolution of interspecific relationship between an alga and a bacteriumin the long-term culture of experimental microcosm
Masayuki MATSUURA, Yosuke HUJII and Toshiyuki NAKAJIMA(Nakajima Lab., Ehime Univ.)

SUMMARY
An experimental model ecosystem composed of three species, a green alga (Micractinium sp.), a bacterium(Escherichia coli), and a ciliate (Tetrahymena thermophila) was cultured for about 6 years. During this culture, cellaggregates consisting of Micractinium and E. coli (called the CE-aggregates) appeared. In order to investigate therelationship between the alga and the bacterium in CE-aggregates, 30 clones of the alga and 30 clones of the bacterium wereisolated each from CE-aggregates obtained from the long-term culture of the model ecosystem. The results revealed thatphenotypic variation concerning physiological characteristics existed in the E. coli population as well as in the Micractinium
population, and that one type of E. coli clones required an amino acid (i.e., isoleucine) for growth, indicating a symbiotic
association with Micractinium sp. in CE-aggregates.

#9

Changes in the algal ingestion rate of ciliate cells in an early stage of evolution ofalgal-ciliate endosymbiosis in the long-term culture of experimental microcosm
Yoichiro OHNISHI, Akiko SANO and Toshiyuki NAKAJIMA(Nakajima Lab., Ehime Univ.)

SUMMARY
An experimental model ecosystem composed of three species, a green alga (Micractinium sp.), a bacterium(Escherichia coli), and a ciliate (Tetrahymena thermophila) was cultured for 5–8 years. Ciliate cells that harbored algal cells
emerged during the long-term culture of this system. Our previous studies demonstrated that this algal-harboring ciliate
indicated an initial stage of algal-ciliate endosymbiosis. In this study, we investigated the algal ingestion rates of ciliate
isolates by using algal and ciliate clones isolated from the model ecosystem in comparison with their ancestral strains. The
result revealed that the isolated ciliate strains exhibited higher algal ingestion rates than the rate of the ancestral ciliate strain.

#10

Life style and evolution of 'Protozoa' and 'Algae', viewed from freshwater
microscopic ecosystem
Masashi M. HAYAKAWA1,2 and Toshinobu SUZAKI1(1Dept. Biol., Grad. Sch. Sci., Kobe Univ., 2JSPS Res. Fellow (DC1))

SUMMARY
Protozoa and algae have been classified according to their lifestyles. Both groups are in close relationship with eachother, because algae are considered to have appeared through multiple events of secondary endosymbiosis in protozoa. Thisevolutionary phenomenon is observed in many mixotrophic microorganisms, and probably have played a central role increating protist diversity. In this talk, we are going to discuss the conceptual confusion in science education concerning“protozoa” and “protists”, and then marshal the evolutionary positions of protozoa and algae in Eukaryota, by using dataobtained from freshwater sampling and culturing experiments.

#11

A study on a rolling structure of plasma membrane isolated from Amoeba proteus
Yukinori NISHIGAMI1, Atsushi TANIGUCHI2, Shigenori NONAKA2, Masatoshi ICHIKAWA1
and Seiji SONOBE3(1Grad. Sch. Sci., Kyoto Univ., 2Natl. Inst. Basic Biol., 3Grad. Sch. Life Sci., Univ. Hyogo)

SUMMARY
A plasmalemma is complex of cell coat and cell membrane. The Plasmalemma isolated form Amoeba proteus forms
rolling structure, unlike general structure of isolated plasma membrane. However, the rolling structure, which is made bylipid bilayer, is theoretically unstable. To resolve the confliction, we tried to identify the factor contribute to the stability of
the rolling structure. As a result, we isolated a novel protein, which molecular weight was 26 kDa, from the plasmalemma of
Amoeba proteus. Proteinase K treatment of the isolated plasmalemma abolished the rolling structure and made vesicle
structure. In addition, the protein localize in cell coat, suggesting that the protein controls a curvature of the plasmalemma
from outside of cell membrane. In this talk, we will discuss about the relation between the rolling structure and behavior ofcell membrane in cell locomotion.

#12

The observation of ultrastructure in Hypophrya sp.
Go KOBASHIGAWA1, Atsushi TANIGUCHI2, Tohru YOSHIHISA3 and Seiji SONOBE3(1Dept. Life Sci., Fac. Sci., Univ. Hyogo, 2Lab. Spatiotemporal Regulations, Natl. Inst. Basic Biol.,3
Grad. Sch. Life Sci., Univ. Hyogo)

SUMMARY
A suctorian, a kind of ciliate, has several thin projections radiating from the cell body, called tentacles. The
suctorian catches a prey with in tentacles and sucks endoplasm of the prey. To approach mechanism of predation of thesuctorian, we first examined celluar structure of Hypophrya sp., forcusing on its microtubule organization in and aroundtentacles. Immunofluorescence revealed that microtubules extended from a limited area near the nucleus to tentacles, We
confirmed microtubule organization by electron microscopy. These results suggested that the microtubules in tentacle of
Hypophrya emanated from a limited number of microtubule organizing centers. Microtubules in a tentacle were aligned in ahighly ordered array forming “MT-tube” and cross-bridges were observed between these MTs. A sleeve-like structure
consisting of two MT-tubes was observed near the base of the tentacle, suggesting that tentacle contraction may depend on
this structure. At the tip of the tentacle, the cell membrane was folded, and “missile-like bodies,” described in Tokophrya,
were accumulated. The cell body but not the tentacle was surrounded with a low electron dense layer. Concanavalin
A-staining revealed existence of mannoside only around the cell body, so that the low electron dense layer may be a cellbody-specific wall-like structure.

#13

Experimental evolution of Eecherichia coli toward constructing a novel intracellularendosymbiosis with Dictyostelium discoideum
Kayo YAMAMOTO1, Kazufumi HOSODA2, Makoto SUEYOSHI1, Isao KUBO1 and Tetsuya YOMO1,3(1Grad. Sch. Inform. Sci. Technol., 2Inst. Acad. Initiatives, 3Grad. Sch. Front. Biosci., Osaka Univ.)

SUMMARY
We want to understand a process toward intracellular endosymbiosis from two organisms by direct observation of
the process. Therefore, we are trying to construct a novel intracellular endosymbiosis of a previously non-interacting pair byexperimental evolution. In this study, Dictyostelium discoideum and E. coli are used. Both D. discoideum and E. coli areprominent model organisms, and they have a predator-prey relationship. If E. colis evolve to live within D. discoideum cells
by getting the digestion tolerance, a novel intracellular endosymbiosis of D. discoideum and E. coli would be established.
Thus we are trying to make E. coli become digestion tolerant by using experimental evolution. In this meeting, we will show
you the results of the experimental evolution.

#14

Construction of a chloroplast-like novel endosymbiosis between a cyanobacterium
and a ciliate
Yuhki AZUMA1, Kazufumi HOSODA2, Masumi HABUCHI3, Kayo YAMAMOTO4, Risa TAKAMI4
and Tetsuya YOMO3,4(1Grad. Sch. Engin., 2Inst. Acad. Initiatives, 3Grad. Sch. Front. Biosci., 4Grad. Sch. Inform. Sci. Technol.,
Osaka Univ.)

SUMMARY
Our goal is to construct a chloroplast-like novel endosymbiosis by making a cyanobacterium live together
artificially in a cell of the ciliated protozoan Tetrahymena living independently in the nature. Specifically, we plan to makethe two organisms coevolve under an experimental environment where Tetrahymena depends on the cyanobacterium forsurvival. Thus, it is first necessary to stably cocultivate the two organisms. Here we investigated the condition for the stablecoculture and found some requirements for the stable coculture.

#15

Theoretical study about evolution of stalk/spore ratio in a social amoeba
Kouki UCHINOMIYA and Yoh IWASA(Kyushu Univ.)

SUMMARY
The social amoeba (or cellular slime mold) lives like a single cell organism while there is enough food. However,when food is depleted from the environment, many social amoebas aggregate and differentiate into either spore cells or stalkcells. These cells make a structure called fruiting body. The stalk cells help dispersal of spore cells, which in turn can
produce offspring. Self-generated signaling chemical such as DIF-1 stimulates the differentiation into stalk cell. By
considering the mechanism of cell differentiation with a simple mathematical model, we investigate the effect of thecapacity to produce a signaling chemical and of the sensitivity to the chemical when making fruiting body. We also discuss
the relation between the evolution of stalk/spore ratio and the chemical production.

#16

Maltose release mechanism of symbiotic Chlorella isolated from JapaneseParamecium bursaria
Aika SHIBATA1, Masahiro KASAHARA1 and Nobutaka IMAMURA1,2(1Coll. Biosci. Biotechnol., 2Coll. Pharm. Sci., Ritsumeikan Univ.)

SUMMARY
The endosymbiotic Chlorella in Paramecium bursaria provides a photosynthate, maltose, to maintain the symbiotic
relationship and the maltose release is induced by lowering pH. We studied the mechanism of maltose release using
Japanese endosymbiont Chlorella F36-ZK. We found its maltose release did not occur without light even in an acidic
condition and the effect of light was investigated at first. Blue or red light was effective for the stimulation of the maltose
release. These wavelengths were the most important ones for photosynthesis. However, the photosynthesis should not
directly promote the maltose release because maltose release was observed under weak light condition and the release wasbarely affected by light intensity. Furthermore, we studied F36-ZK maltose transport system. Radioactive photosynthate wasreleased even in a buffer with a high maltose concentration, the transport should be an active. The proton gradient was
required for maltose transport because uncouplers inhibited the maltose release.

#17

Construction of a novel endosymbiosis between Escherichia coli and algae-free
Paramecium bursaria
Risa TAKAMI1, Kazuhumi HOSODA2, Itsuka KUMANO1, Kayo YAMAMOTO1 and Tetsuya YOMO1,3(1Grad. Sch. Inform. Sci. Technol., 2Inst. Acad. Initiatives, 3Grad. Sch. Front. Biosci., Osaka Univ.)

SUMMARY
A lot of changes must have happened to a host and a symbiont in the evolutionary process toward an endosymbiosis
from two independent organisms. The purpose of our study is to observe the evolutionary changes that happen to thesymbiont. Therefore we try to construct a novel endosymbiosis using Escherichia coli and algae-free Paramecium bursaria,
which do not have symbiotic relationship in the nature. Specifically, we aim to evolve E. coli to stay in algae-free P.
bursaria by using experimental evolution. Here, as an initial state before the experimental evolution, we investigated theengulfment and digestion of E. coli by algae-free P. bursaria, comparing them with the case of a natural symbiont of P.
bursaria as a goal of the experimental evolution.

#18

Effects of the endosymbiosis on phagocytic activities of Paramecium bursaria
Takashi MIURA1 and Sosuke IWAI2(1Grad. Sch. Educ., 2Fac. Educ., Hirosaki Univ.)

SUMMARY
To elucidate whether endosymbiosis affect phagocytosis of the hosts, we have evaluated phagocytic activities of aciliate, Paramecium bursaria, with or without the algal endosymbionts. To assess the phagocytic activities, P. bursaria cells
were fed with yeast cells that constitutively express GFP. The fluorescent yeast cells enabled us to readily distinguishbetween the algal endosymbionts and the ingested prey in P. bursaria cells, and also to determine whether the ingested prey
was eventually digested or egested. The aposymbiotic P. bursaria cells showed significantly enhanced ingestion activity,
suggesting that the presence of the endosymbionts affected the phagocytic activities of their host cells. The engulfed yeast
cells were mostly digested within 3–4 hours in the food vacuoles and slightly egested. The digestion activity was only
slightly affected by the presence of the endosymbionts, suggesting that the digestive activity was not specifically suppressed
even under the presence of the endosymbionts. P. bursaria cells in stationary phase showed lower ingestion activity thancells in log growth phase, presumably because of the lower expression level of the proteins involved in phagocytosis.

#19

The stress tolerance of Paramecium bursaria
Yuka ADACHI1, Ayumi SHIMOMURA1, Kozue HAMAO1 and Hiroshi HOSOYA1,2(1Dept. Biol. Sci., 2Marine Biol. Lab., Grad. Sch. Sci., Hiroshima Univ.)

SUMMARY
A single cell of the green paramecia (Paramecium bursaria) harbors several hundreds of endo-symbiotic
Chlorella-like algae in its cytoplasm. The symbiotic association of algae is only established with P. bursaria but not withother kinds of paramecium. However, it is not clear how the specific interaction between alga and green paramecia is
accomplished. The green paramecium might be resistant against reactive oxygen produced by endosymbiotic algae in thecytoplasm. Then, we elucidated the stress tolerance of P. bursaria. Our results obtained revealed that P. bursaria with orwithout algae showed the highest tolerance against H2O2 or high temperature in the several kinds of paramecium.

#20

Infectivity of the symbiotic Chlorella spp. of the ciliate Paramecium bursaria
Sotaro ARAKI1 and Yuuki KODAMA2(1Course of Biol. Sci. Biotechnol., Grad. Sch. Life Environ. Sci., 2Dept. Biol. Sci., Fac. Life Environ. Sci.,
Shimane Univ.)

SUMMARY
Paramecium bursaria cells harbor hundreds of Chlorella spp. in their cytoplasm. Irrespective of the mutual
relationships between P. bursaria and Chlorella spp., both cells retain the ability without their partner. Although theirrelationship is separable and flexible, it is reported that isolated symbiotic Chlorella spp. from European P. bursaria cells
could not be established endosymbiosis to American alga-free cells. In this study, we examined the infectivity of thesymbiotic algae isolated from 6 strains of alga-bearing P. bursaria cells to a different strain of alga-removed P. bursaria
cells which are collected from different areas. As a result, all isolated symbiotic algae examined in this study could localize
beneath the host cell cortex and established endosymbiosis with alga-free P. bursaria cells. Although these strains could bemaintained in the host cells for more than 1 month after mixing, there were some differences in the each algal growth rate in
the host paramecium during cultivation under constant light condition. Our study suggests that the host-symbiont
compatibility occurs after the establishment of endosymbiosis.

#21

Cell signaling pathways and analysis of expressed proteins during the resting cyst
formation of ciliated protozoan Colpoda cucullus
Yoichiro SOGAME1, Katsuhiko KOJIMA2, Toshikazu TAKESHITA2, Eiji KINOSHITA3
and Tatsuomi MATSUOKA1(1Dept. Biol. Sci., Fac. Sci., Kochi Univ., 2Dept. Microbiol. Immunol., Sch. Med., Shinshu Univ.,
3
Dept. Funct. Mol. Sci., Inst. Biomed. Health Sci., Hiroshima Univ.)

SUMMARY
The molecular events during the resting cyst formation (encystment) of free-living unicellular organisms have notbeen elucidated, despite a century’s research into this biological phenomenon. Recently, our research group has explored themolecular events of encystment including the signal transduction pathways of the ciliate Colpoda cucullus by fura-2 (Ca2+indicator) ratiometry, a cAMP enzyme immunoassay, phos-tag/electrochemiluminescence, and two-dimensional
electrophoresis prior to a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay. Cell-to-cell mechanicalstimulation promotes an inflow of Ca2+, which activates intracellular signaling pathways for encystment, in which several
proteins (histone H4, ribosomal protein P0, ribosomal protein S5, etc.) are cAMP-dependently phosphorylated, and theexpression levels of several proteins (actin, elongation factor 1a, heat shock protein, etc.) were enhanced, whereas
mitochondria ATP synthase b chain disappeared. Among these proteins, mitochondria ATP synthase b chain was suggested
to be involved in the maintenance of the mitochondrial membrane potential, because the knockdown of this protein in
Trypanosoma reduced mitochondrial membrane potential (Brown S.V. et al. 2006; Eukaryot. Cell 5: 45–53), and inencystment-induced C. cucullus, the mitochondrial membrane potential disappeared concomitantly with the disappearanceof ATP synthase b chain. HSP60 may be involved in the protection of proteins under stressed conditions.

#22

Roles of kinesin14A in micronuclear division in Tetrahymena
Yasuharu KUSHIDA1, Masak TAKAINE1, Kentaro NAKANO1, Toshiro SUGAI1,
Krishna Kumar VASUDEVAN2, Mayukh GUHA2, Yu-yang JIANG2, Jacek GAERTIG2
and Osamu NUMATA1(1Grad. Sch. Life Environ. Sci., Univ. Tsukuba, 2Dept. Cell. Biol., Univ. Georgia, USA)SUMMARY
Ciliates possess two functionally differentiated nuclei; the micronucleus (Mic; germline nucleus) and themacronucleus (Mac; somatic nucleus). Mic contains single genome and is transcriptionally silent. Mac is far-polyploid andactively transcribed. Our final aim is to understand how these two nuclei can be maintained through nuclear division during
vegetative growth. Here we report the function of microtubule (MT) minus-end motor, kinesin-14s on Mic mitosis andmeiosis. One of two homologues of Tetrahymena kinesin-14s (KIN14A) plays essential role on spindle formation duringmitosis and meiosis in dividing Mic. Plus, over-produced EGFP-KIN14A accumulates at meiotic spindle poles. These
results suggest that KIN14A is essential for the formation of spindle pole in Tetrahymena. On the other hand, KIN14A is not
required during Mac amitosis. Some more descriptive research is important to identify the polarity of MTs in dividing Mic
and Mac, and other key MT motors governing the arrangement of MT in nucleoplasm. Through these intensive futurestudies, we believe that one can explain the evolutional process of establishment of ‘nuclear dualism’ of ciliates.

#23

Tetrahymena as a production plant of foreign proteins
Kohei MASUDA and Hiroshi ENDOH(Grad. Sch. Nat. Sci. Technol., Kanazawa Univ.)

SUMMARY
Recent genome analyses of ciliates have revealed that relatively more genes are encoded in their genomes than thoseof other eukaryotes. For instance, Tetrahymena thermophila have more than 27,000 protein-coding genes, and Paramecium
tetraurelia also retain approximately 40,000 genes in their genomes. These observations suggest that ciliates haveoutstanding abilities such as efficient protein folding and assembly of complicated molecules such as monoclonal antibodies(with the latter, see Cilian AG homepage of German company). In addition, surface display of antigens of viral or parasite
on cell membrane of T. thermophila was successfully reported. Here we discuss a potential of Tetrahymena as a production
plant of foreign proteins, based on expression and secretion of some cellulases from symbiotic flagellates of the termites.

#24

FAP82 regulates activation of K+ leak channel in Paramecium
Shinobu IZUTANI1, Takashi TOMINAGA2 and Manabu HORI1(1Dept. Biosci., Fac. Sci., Yamaguchi Univ., 2Kagawa Sch. Pharm., Tokushima Bunri Univ.)

SUMMARY
Chlamydomonas FAP82, a flagellar protein, is highly conserved from protists to mammals. In Paramecium, theortholog of Chlamydomonas FAP82 is highly expressed in cilia. However, the function of FAP82 ortholog in cilia remains
unclear. For greater insight into the ciliary function in Paramecium, we have studied the function of FAP82 ortholog using
RNAi by the feeding method and through analysis of phenotypes. Pt_FAP82 knockdown cells showed no typical escape
reaction against mechano-stimulation and cGMP. Moreover, their knockdown enhanced the swimming speed by cAMP.
Consequently, our results suggest Pt_FAP82 regulates the activation of cyclic nucleotide dependent K+-leak channel.

#25

Development of nickel inducible expression system in Paramecium caudatum
Yasuhiro TAKENAKA1, Nobuyuki HAGA2 and Ikuo INOUE1(1Saitama Med. Univ., 2Ishinomaki Senshu Univ.)

SUMMARY
Inducible gene expression technology is potentially effective for the better expression of proteins that are toxic to ahost cell or require a temporal control. In Tetrahymena, the Cd2+-inducible metallothionein promoter has been successfully
used to control expression of introduced genes. Here we report the isolation of two nickel-induced genes, Ni46 and Ni66,
from the subtracted cDNA library of Paramecium caudatum. Ni46 and Ni66 mRNAs encode for approximately 16 and 25
kDa proteins, respectively. Both Ni46 and Ni66 mRNAs were remarkably induced by adding nickel sulfate into the culture
medium. Cobalt chloride also enhanced slightly the expression of both mRNAs. Inductions of both mRNAs by Ni2+ andCo2+ ions were dose-dependent, and their levels were continued to increase up to 3 days after the addition of nickel sulfate.
We also cloned ~0.45 kb of 5' flanking region of Ni66 gene, and constructed the Ni66 promoter-driven reporter vector
harboring secreted luciferase isolated from the marine copepods. Functions of nickel induced genes and future research will
be discussed in the oral presentation.

#26

Molecular composition and the function of plant-vacuole like organelle in extracellular
Toxoplasma gondii
Michika FKSHI1,2, Kazuhide YAHATA3, Takaya SAKURA1, Michiru TAHARA3,
Ryuma MATSUBARA1,2, Osamu KANEKO3 and Kisa NAGAMUNE1,4
3(1Dept. Parasitol., Natl. Inst. Infect. Dis, 2Grad. Sch. Life Environ. Sci., Univ. Tsukuba,
Dept. Protzool. Inst. Trop. Med., Nagasaki Univ., 4Fac. Life Environ. Sci., Univ. Tsukuba)

SUMMARY
It was previously assumed that Toxoplasma gondii did not contain lysosomes, although recent research has revealed
that extracellular T. gondii form acidic lysosome-like organelle called plant-like vacuoles (PLV) or vacuolar compartments(VAC). To confirm the acidicity of this organelle, we used LysoTracker, which stains acidic organelles like lysosomes. We
found a LysoTracker-positive organelle specific for extracellular T. gondii. The LysoTracker signal corresponded to thelocation of a previously described PLV marker whereas the signal did not correspond to the location of a VAC marker. We
also found that the PLV marker–positive organelle was divided into two sub-compartment types, i.e., PLV1 and PLV2, by
incubation in an extracellular environment. LysoTracker stained only the PLV2 compartment. We also found that PLV
stored H+ and Ca2+. Disrupting PLV-function using primaquine caused decreased tolerance to high NaCl stress; therefore,
PLV was inferred to be involved in regulating ionic balance in T. gondii. H+ and Ca2+ in PLV2 were released into thecytoplasm after the primaquine treatment, and this treatment also inhibited gliding motility. These results suggest that PLV
is a vacuole-like organelle with a formation process similar to that observed for plant vacuoles and a function involving
ionic balance regulation in the cytoplasm.

#27

Megavirus-infecting Acanthamoeba sp. isolated from a keratitis patient
Kenji YAGITA1 and Yoshitsugu INOUE2(1Dept. Parasitol., Natl. Inst. Infect. Dis., 2Div. Ophthalmol. Visual Sci., Fac. Med., Tottori Univ.)

SUMMARY
Recently unique and giant nucleocytoplasmic large DNA viruses (NCLDV), such as “mimivirus” have been
discovered in Acanthamoeba sp., a common free-living amoeba. These giant viruses are beyond the biological concept of“virus” because they are almost the same size with bacteria and have a very large genome (1.0 Mb~). These characteristicsof the giant viruses place them at the boundary of living and non-living. The presence of the giant viruses have added to thedebate over the origins of life. Beside the impact on biology and evolution, mimivirus has been isolated from a pneumonia
patient, suggesting viruses in amoeba are potentially pathogenic to humans. In the study of a molecular epidemiology on
Acanthamoeba keratitis, we isolated an NCLDV-infecting Acanthamoeba sp. from corena of a keratitis patient. An electron
microscopy (TEM) for morphological study and preliminary genetic analysis revealed that the virus was a member of
megavirus which is bigger in size with larger genome than mimivirus. Megavirus kills the host amoeba and may have acytopathic effect on human corneal somatic cell culture. Megavirus infecting Acanthamoeba is now axenically cultured andmaintained by a conventional procedure.

#28

Species composition change in the intestinal protist communities after
community mixing
Osamu KITADE and Yoshihiro KURUSU(Coll. Sci., Ibaraki Univ.)

SUMMARY
Termites in the genus Reticulitermes harbor host species-specific symbiotic protist communities in their digestive
tracts composed of about 10 excavate species. Using winged reproductives (future kings and queens) of three Reticulitermes
spp., we made hybrid incipient colonies and investigated the transition patterns of the symbiont species compositions of thecolony members. After 800 days of culture, the symbiont compositions of all the hybrid colonies between R. speratus and R.
kanmonensis and those between R. speratus and R. amamianus became very close to the original symbiont composition of R.
speratus. Similarly, the symbiont compositions of the hybrid colonies between R. kanmonensis and R. amamianus
approached a novel, specific composition. These results suggest that the symbiotic protist communities are not random
assemblages of the element species. The presence of the specific stable compositions may explain the community
replacements suggested to have occurred in some termite linages.

#29

Development of microscopic system for precise investigation of mobile protists
Ikuko SHIHIRA-ISHIKAWA1, Hiroyuki KAWANO2, Yasutaka HANADA3
and Atsushi MIYAWAKI1,2(1Biotechnol. Optics Res. Team, Ctr. Adv. Photonics, RIKEN, 2Lab. Cell Funct. Dynamics,
Brain Sci. Inst., RIKEN, 3Grad. Sch. Sci. Technol., Hirosaki Univ.)

SUMMARY
The flagellated microalgae are difficult to catch the clear images under ordinary microscope because of theirextremely rapid movements. We developed a super high speed video-microscope for the visual analysis of their movement,
especially of their flagella movement. Based on this microscope, we repeated the improvement of the system with thecollaboration of Olympus Corporation and Photoron Limited, searching for better method for visual observation of theoutside world of human eyesight. We introduce the various movies of enlarged cell images on screen.

#30

Growth analysis of Paramecium busaria and the algal endosymbionts
Sosuke IWAI and Takuro TAMURA(Fac. Educ., Hirosaki Univ.)

SUMMARY
We have investigated growth of Paramecium bursaria and its algal endosymbionts. For quantitative analysis of thegrowth, P. bursaria cells were grown in a simple, bacteria-free monoxenic culture system, in which yeast cells were the soleorganic nutrients. The population growth analysis using this system showed that the specific growth rate of aposymbiotic(Chlorella-free) cells was comparable to that of wild-type cells hosting the endosymbionts, while the growth yield of theaposymbiotic ones was significantly lower than that of the wild type. Single-cell level growth and survival analysis
demonstrated that, under no-food condition, the wild-type cells survived for 30 days, while the aposymbiotic ones for at
most 7 days. These results suggest that the benefits of acquiring phototrophy for P. bursaria by hosting the algal
endosymbionts involve 1) at least in part, obtaining maintenance energy and 2) probably, obtaining carbon sources for cell
growth. Furthermore, the simultaneous growth analysis of both the hosts and the endosymbionts within the host cells
suggested that the growth of these two species might not be tightly coupled.