Abstracts of the JSP meeting 2012
Oral presentations
1) Mitochondrial nuclease: a major executor of the programmed nuclear degradation in
Tetrahymena thermophila
Eriko OSADA
2) Division and death of mitochondria in conjugation of Tetrahymena thermophila
Mai NAGASHIMA and Hiroshi ENDOH
3) Studies on the mating-type substances in Paramecium caudatum: determination of
DNA sequence for Hp-1 which is a ciliary membrane specific protein associated withmating reactivity in P. caudatum
Yuta CHIBA and Nobuyuki HAGA
4) Excystment-dependent alteration of protein expression in ciliated protozoan
Colpoda cucullus
Yoichiro SOGAME, Katsuhiko KOJIMA, Toshikazu TAKESHITA, Eiji KINOSHITA and
Tatsuomi MATSUOKA
5) Protein analyses in mucopolysaccharide-containing ciliated protozoan Colpoda cucullus:
two-dimensional electrophoresis of SDS-containing samples in encystment-induced cells
Ken MOCHIDUKI, Yoichiro SOGAME and Tatsuomi MATSUOKA
6) Analysis of growth controlling activity in the culture medium of green paramecia
Paramecium bursaria
Yoshihiko YAMASHIN, Eiji HIRAKI, Koyo TETSUKAWA, Yuka ADACHI,
Kosuke MATSUBARA, Kozue HAMAO and Hiroshi HOSOYA
7) Electrical responses of Euglena gracilis in the electromagnetic field at different electrodedistances
Lin CHEN and Toshinobu SUZAKI
8) Feeding habit of Bodo saltans
Yurie SHIRAKAWA, Osamu NISHIMURA and Ryuichi SUDO
9) Morphological observation of a novel green Raphidiophrys sp.
Masashi M. HAYAKAWA, Rina MATSUMOTO and Toshinobu SUZAKI
10) Relationships between termite reproductives and symbiotic protists: gut flagellates are
not harbored in mature queens and kings
Keisuke SHIMADA, Osamu KITADE and Kiyoto MAEKAWA
11) Extension and contraction mechanism of the proboscis in Lacrymaria sp.
Ryuji YANASE, Yukinori NISHIGAMI, Teruo SHIMMEN and Seiji SONOBE
12) A representative model of the gliding motion of a diatom, Bacillaria paradoxa, predictedfrom the action of elastic fibrils
Nozomi YAMAOKA, Yasutaka SUETOMO, Teruo SHIMMEN and Seiji SONOBE
13) Three-dimensional analysis of the plasmalemma and cytoplasm dynamics duringlocomotion of Amoeba proteus
Atsushi TANIGUCHI, Yukinori NISHIGAMI, Hiroko KOBAYASHI, Daisuke TAKAO,
Teruo SHIMMEN, Shigenori NONAKA and Seiji SONOBE
14) Localization and properties of Tetrahymena formin
Fumihide BUNAI and Osamu NUMATA

15) Novel features in the formation and function of the “9 + 2” axoneme revealed by a basal
body-deficient mutant of Chlamydomonas
Yuki NAKAZAWA, Tetsuro ARIYOSHI, Ritsu KAMIYA and Masafumi HIRONO
16) Phosphoinositide-specific phospholipase C (PI-PLC) of Amoeba proteus
Hitoshi YAGISAWA, Ryoko SHINKI-HORIE, Kayoko MAEDA-AKAHANE, Teruo SHIMMEN
and Seiji SONOBE
17) Determination of Immaturin gene sequence: an intragenic structure responsible for sexual
rejuvenescence in Paramecium
Nobuyuki HAGA, Toshinori USUI and Tomoaki ABE18) Comprehensive search for the nuclear pore complex proteins of ciliate
Tetrahymena thermophila
Masaaki IWAMOTO, Hiroko OSAKADA, Chie MORI, Koji NAGAO, Chikashi OBUSE,
Yasushi HIRAOKA and Tokuko HARAGUCHI19) Soil testate amoebae fauna in Japan, re-evaluation
Anatoly BOBROV, Satoshi SHIMANO and Yuri MAZEI20) Analysis of photosynthetic products in green ciliate Paramecium bursaria
Hiroshi HOSOYA, Eiji HIRAKI, Koyo TETSUKAWA, Yuka ADACHI, Kosuke MATSUBARA
and Kozue HAMAO21) Specific connections between symchlorosomes and mitochondria in
Paramecium bursaria as verified by electron tomography and 3D reconstruction fromserial sections
Chihong SONG and Toshinobu SUZAKI
22) New method to eliminate germinal chromosome in Tetrahymena and observationof the arrangement of meiotic chromosome using nullisomics and unisomics obtainedby the method
Toshiro SUGAI and Chie KAJIWARA
23) Prevalence of Sarcocystis in slaughtered horses and imported horse meat
Kenji YAGITA
24) Transfer mechanism of spasmin–Ca2+ binding energy to novel elastic protein
spaconnectin
Hiroshi ASAI and Yasushige MORIYAMA
Poster presentations
P1) Function of glycocalyx in cell adhesion and cell locomotion on Amoeba proteus
Yukinori NISHIGAMI, Hisato IKEGAYA, Rika SHIBATA, Atsushi TANIGUCHI,
Teruo SHIMMEN and Seiji SONOBE
P2) Isolation of actin filaments from a diatom, Craticura sp.
Yuuka KISHIMOTO, Nozomi YAMAOKA, Teruo SHIMMEN and Seiji SONOBE
P3) Ultrastructural changes during rapid axopodial contraction in heliozoon
Raphidiophrys contractilis
Risa INOUE, Midori EDAMATSU and Motonori ANDO
P4) Screening of microbial anti-trypanosomal compounds using Bodo saltans
Koichiro SUMITANI and Nobutaka IMAMURA
P5) Gametogenesis in Noctiluca scintillans under light-dark cycle
Takumi KITAMURA and Hiroshi ENDOH

P6) Construction of foreign protein secretion system in Tetrahymena thermophila
Kohei MASUDA and Hiroshi ENDOH
P7) Measurements of phagocytic activities of Paramecium bursaria using GFP-labeled yeast
Takashi MIURA and Sosuke IWAI
P8) Role of the two-headed inner dynein arm in the ciliary movement inParamecium tetraurelia
Takuya KATOH, Manabu HORI, Kanako INOUE, Osamu KUTOMI and
Munenori NOGUCHI
P9) Maltose release mechanism of symbiotic Chlorella from Japanese Paramecium bursaria
Aika SHIBATA and Nobutaka IMAMURA
P10) Culturing condition and isolation of micronuclei in Blepharisma japonicum
Ryota KOKUTANI and Hiroshi ENDOH
P11) Do the mating pheromones of genus Blepharisma play an important role in speciation?
Mayumi KOBAYASHI, Kiyono SHINOHARA, Mari TAKUSAGAWA and Terue HARUMOTO
P12) Study on the amino acid residues which are involved in the stop codon recognitionin ciliate eRF1s
Ying LI, Oanh KIM T.P., Do Thi HONG, Adriana VALLESI and Terue HARUMOTO
P13) Species identification of wild strains and molecular phylogenetic analysis of COI genein the genus Blepharisma
Kiyono SHINOHARA, Mayumi KOBAYASHI and Terue HARUMOTO
P14) Nickel-induced genes in Paramecium caudatum
Yasuhiro TAKENAKA, Nobuyuki HAGA and Ikuo INOUE
P15) Properties of Paramecium bursaria cells in the stationary phase
Sosuke IWAI and Takashi MIURA
P16) Species composition and structure of symbiotic protist community in the termite
Coptotermes formosanus
Osamu KITADE
P17) New bio-remediation technique for radioactive cesium in contaminated soil using
Paramecium bursaria
Chisato YOSHIMURA and Toshinobu SUZAKI

#1

Mitochondrial nuclease: a major executor of the programmed nuclear degradationin Tetrahymena thermophila
Eriko OSADA(Div. Life Sci., Grad. Sch. Nat. Sci. Technol., Kanazawa Univ.)
SUMMARY
During conjugation of Tetrahymena thermophila, three meiotic products out of four and an old macronucleus areprogrammed to degrade in different stages, following to differentiation of new macronuclei. Mitochondria are involvedin this programmed nuclear death (PND) as a main player. Prior to the nuclear degradation, a lot of mitochondria lose
their membrane potential and are incorporated into autophagosome. The sequestered mitochondria move to the old macronucleus, where they release many apoptotic molecules, such as apoptosis-inducing factor (AIF) and yet-unidentified
nucleases. To identify these nucleases, I previously searched multiple mitochondrial proteins from Tetrahymena thathave nuclease activity using SDS-DNA-PAGE. Especially, a protein of approximately 15 kDa showed the highest andpredominant activity. Mass spectrometry analysis revealed three candidate genes of the nuclease (TTHERM_00426240:
called Tn1 provisionally here, TTHERM_00666370: Tn2 and TTHERM_00825290: Tn3). All of these proteins wereconfirmed to localize in mitochondria. Gene disruption of Tn1 resulted in 1) inhibition of degradation of the meiotic
products and 2) delay of degradation and resorption of the old macronucleus. Actually, Tn1-deficient mitochondria
showed a drastically decreased nuclease activity, when compared with wild type mitochondria. These observations
strongly suggest that Tn1 is one of the major executors of PND.

#2

Division and death of mitochondria in conjugation of Tetrahymena thermophila
Mai NAGASHIMA1 and Hiroshi ENDOH2(1Dept. Sci. Engn., 2Grad. Sch. Nat. Sci. Technol., Kanazawa Univ.)
SUMMARY
Tetrahymena thermophila have a unique apoptotic event in conjugation, in which only parental macronucleus
is selectively degraded, but newly differentiated micro- and macronuclei are unaffected. Therefore, this is called
programmed nuclear degradation (PND). Mitochondria are involved in this process as a major death executor which
carries various death effectors in its inside. During PND, many mitochondria lose membrane potential, and then migrate
to the parental macronucleus destined to die, after incorporated into autophagosomes. It is unknown that mitochondrial
death is accompanied with their division or not. To ask if mitochondria are programmed to divide prior to death, we
treated conjugating cells with ethidium bromide (EtBr), which is known an inhibitor of mitochondrial DNA polymerase“gamma”, at certain time points. Addition of EtBr before meiosis resulted in inhibition of meiosis I, but the macronuclear
condensation occurred, irrespective of no new macronuclear differentiation. When EtBr was added at fertilization stage,
postzygotic nuclear divisions (PZD) were disturbed. Addition after PZD, EtBr inhibited the degradation, where theparental macronucleus suspended before the stage of degradation. The former 2 inhibitions might be due to those of
nuclear divisions directly, whereas the latter is likely to have resulted from inhibition of mitochondrial divisions anddeath.

#3

Studies on the mating-type substances in Paramecium caudatum: determination of
DNA sequence for Hp-1 which is a ciliary membrane specific protein associated withmating reactivity in P. caudatum
Yuta CHIBA and Nobuyuki HAGA
(Dept. Biol. Engn., Fac. Sci. Engn., Ishinomaki Senshu Univ.)
SUMMARY
In Paramecium, mating type substances play a role to recognize mating types in mating reaction. Previous
studies suggested that mating type substances are the integral ciliary membrane proteins (Kitamura et al. 1988). However, no one has so far identified these substances. In this study, we prepared ciliary membrane fractions (CMFs) withtreatment of Triton X-114. SDS-PAGE analysis indicated that a polypeptide with molecular weight of about 52 kDa was
specifically detected in CMFs prepared from mating reactive cilia, but not in ones from mating non-reactive cilia. We
named this polypeptide as Hp-1 (hypothetical protein #1), and have attempted to analyze its function. Based on threepartial amino acid sequences from mass spectrometric data, Hp-1 DNA sequence was determined. The DNA sequence of
Hp-1 was found to be a novel gene, which contains one serine/threonine kinase c domain and four EF hand motifs. Semiquantitative RT-PCR showed that Hp-1 mRNA was detected in mating reactive cells under the conditions which cannot
detect mRNA in non-reactive cells. Western blotting analysis using anti-Hp-1 polyclonal antibody showed that Hp-1 wasstrongly detected in mating reactive CMFs, but was not so strong in non-reactive ones. These results suggest that the
PKC domain with four EF hands in Hp-1 may has an essential role in the expression of mating reactivity.

#4

Excystment-dependent alteration of protein expression in ciliated protozoan
Colpoda cucullus
Yoichiro SOGAME1, Katsuhiko KOJIMA2, Toshikazu TAKESHITA2, Eiji KINOSHITA3 andTatsuomi MATSUOKA1(1Dept. Biol. Sci., Fac. Sci., Kochi Univ., 2Dept. Microbiol. Immunol., Shinshu Univ. Sch. Med.,
3
Dept. Funct. Mol. Sci., Grad. Sch. Biomed. Sci., Hiroshima Univ.)

SUMMARY
SDS-PAGE of the total proteins contained in excystment-induced Colpoda cucullus showed enhancement of theexpression levels of 65-kDa, 60-kDa and 44-kDa proteins at 10 min after the onset of excystment induction. Liquid
chromatography tandem mass spectrometry (LC-MS/MS) analysis showed that the p60 was DEAD-box RNA helicaselike protein. In the later stage (30–60 min after the onset of excystment induction), alterations in the expressions of
50-kDa and 49-kDa proteins (mitochondrial ATP synthase β chain and elongation factor 1α, respectively) were observed;
p50, which had disappeared during encystment, was expressed again, while p49, which is expressed during encystment,
disappeared.

#5

Protein analyses in mucopolysaccharide-containing ciliated protozoan
Colpoda cucullus: two-dimensional electrophoresis of SDS-containing samples
in encystment-induced cells
Ken MOCHIDUKI, Yoichiro SOGAME and Tatsuomi MATSUOKA(Dept. Biol. Sci., Fac. Sci., Kochi Univ.)
SUMMARY
When the samples obtained by a direct solubilization of Colpoda cucullus cells with an IEF sample buffer
without SDS were analyzed on 2-D PAGE, any clear protein spots were not obtained. The 2-D PAGE of a bacterial(Klebsiella pneumoniae) protein sample mixed with Colpoda cell components also failed to obtain the clear spots. On theother hand, 2-D PAGE of bacterial proteins mixed with lysozyme-treated C. cucullus components was successful at
protein focusing. These results suggest that protein aggregation through mucopolysaccharides may prevent proteins fromisoelectric focusing. When C. cucullus samples were pre-treated with a SDS sample buffer for SDS-PAGE and mixed
with an IEF sample buffer, the spot resolution improved in 2-D PAGE. We showed, using the above-mentioned method,
alteration of the expression level of the several proteins during encystment of C. cucullus.

#6

Analysis of growth controlling activity in the culture medium of green paramecia
Paramecium bursaria
Yoshihiko YAMASHIN, Eiji HIRAKI, Koyo TETSUKAWA, Yuka ADACHI,
Kosuke MATSUBARA, Kozue HAMAO and Hiroshi HOSOYA(Dept. Biol. Sci., Grad. Sch. Sci., Hiroshima Univ.)

SUMMARY
A medium for the culturing of green paramecia Paramecium bursaria has been developed by Weis. It is now
used consisting of lettuce infusion inoculated with bacteria. Sometimes, green paramecia are cultured in a mixture of thelettuce infusion and CA medium, which is a widely employed medium used for the culturing of green algae. However, in
those culture media, the initial growth rate of paramecia or final concentration of paramecia in the stationary phase are
not coincided with each experiments. Here, to elucidate the stable experimental conditions for the culture of green
paramecia, we tried to culture green paramecia in several kind of media with or without bacteria. In this meeting, ourresults obtained will be discussed.

#7

Electrical responses of Euglena gracilis in the electromagnetic field at different
electrode distances
Lin CHEN and Toshinobu SUZAKI(Dept. Biol., Grad. Sch. Sci., Kobe Univ.)
SUMMARY
The non-invasive technique of dielectric analysis can be used to monitor changes in electrical characteristics of
biological cells or tissues, which is influenced by environmental factors such as toxic substances in the surrounding
water. In this study we compared the dielectric behavior of Euglena gracilis cells that were confined in parallel-capacitor-like measuring chambers with different electrode distances (8.4 and 7.8 mm). From the observed data set, we obtained
characteristic parameters for simulating the observed dielectric behavior of the cell suspension with “dielectric ellipsoidal-shell” models. For the model fitting, the Euglena cell was considered as an ellipsoid with a surrounding cell membraneand some cytoplasmic inclusions such as nucleus, chloroplasts and mitochondria, being considered as solid ellipsoids.
From the fitting curves, we could obtain a new group of characteristic parameters, which is to be used for real-time
biological monitoring of water quality with Euglena.

#8

Feeding habit of Bodo saltans
Yurie SHIRAKAWA, Osamu NISHIMURA and Ryuichi SUDO
(Grad. Sch. Engn., Tohoku Univ.)
SUMMARY
In this study, we tried to inspect the bacterivory of B. saltans isolated from activated sludge. Using with different
kinds of fluorescent labeled bacteria, we determined the in situ bacterial abundance those were preyed, and alsomeasured specific growth rate of B. saltans. The results of specific growth rate of B. saltans were 6.35/day with
Pseudomonas fluorescens, 4.85 with Escherichia coli, 3.08 with Bacillus subtulis, 3.28 with Bacillus cereus,
3.74 with Alcalignes faecalis, 2.93 with Flavobacterium aquatile, 2.17 with Enteroccocus faecalis, and 1.20/day with
Kocuria varians. According to the fluorescence labeled bacterial uptake ratio between P. fluorescens and K. varians,
we could not find significant differences within 3 hours. It was concluded that B. saltans is able to prey many kinds ofbacteria, and compared with other protozoan specific growth rate, it is suggested that B. saltans might survive in divers
environment. And more, specific growth rate has been changed by bacterial species, some components contained in
specific bacteria may increase the growth of B. saltans.

#9Morphological observation of a novel green Raphidiophrys sp.
Masashi M. HAYAKAWA1,2, Rina MATSUMOTO3 and Toshinobu SUZAKI1(1Dept. Biol., Grad. Sch. Sci., Kobe Univ., 2JSPS Res. Fellow (DC1), 3Dept. Biol., Fac. Sci.,
Kobe Univ.)

SUMMARY
Protozoans bearing zoochlorellae (endosymbiotic green algae) have been reported in various protozoan groups.
Several centrohelid heliozoan species (e.g. Acanthocystis turfacea, Raphidiophrys viridis) were reported as “green” hosts
for zoochlorellae. We newly found and cultured a strain of unicellular green Raphidiophrys from a high moor marsh in
Nasu-Shiobara, Japan, last year. While no unicellular strain of green Raphidiophrys has been reported so far, this strain is
likely to be a new species. Light and electron microscopic observations of this novel strain were carried out. Ultrastructure of the siliceous scales of this strain was examined in details, which resembled those of R. elegans (multicellular but
not green species) with characteristic parallel striations on the surface of the scale, but different from those of R. viridis,
a multicellular species of zoochlorellae-bearing Raphidiophrys.

#10

Relationships between termite reproductives and symbiotic protists: gut flagellates
are not harbored in mature queens and kings
Keisuke SHIMADA1, Osamu KITADE2 and Kiyoto MAEKAWA3(1Ishikawa Museum of Natural History, 2Coll. Sci., Ibaraki Univ., 3Grad. Sch. Sci. Engn.,
Univ. Toyama)

SUMMARY
The symbiotic relationship between lower termites and cellulolytic flagellate protists in their hindguts is one of
the most well-known examples of mutualism. Although the symbiosis in worker termites has attracted much attention,
there have been very few studies of protists in other castes. We have performed the first examination of protist population dynamics in queens and kings during termite colony foundation. Protist numbers and reproductive tissue sizes were
measured at five time points over 400 days in incipient colonies of Reticulitermes speratus, as well as in other castes ofmature colonies. The results showed that queens and kings with slightly developed ovaries and testes had large amountof protists from 30 to 100 days compared with alates and workers. On the other hand, they lost protists by day 400, anddeveloped their ovaries and testes. Unlike workers and soldiers, our study showed that symbiotic protists are notharbored in mature queens and kings.

#11

Extension and contraction mechanism of the proboscis in Lacrymaria sp.
Ryuji YANASE1, Yukinori NISHIGAMI2, Teruo SHIMMEN2 and Seiji SONOBE2(1Dept. Life Sci., Fac. Sci., 2Grad. Sch. Life Sci., Univ. Hyogo)
SUMMARY
A ciliate, Lacrymaria, has a long proboscis which is highly elastic. It‟s considered that the extension of theproboscis is due to both microtubule bundles under the pellicle and the long cilia at the oral region of the proboscis.
While, the contraction of the proboscis is thought to be due to the myoneme, which contract depending on a calcium ion.
However, any direct proofs for these hypothesis have not presented. We examined the relationship between the
movement of the proboscis and the cilia by observing the movement of cilia in the presence or absence of the oral region.
We also analysed the movement of proboscis by using a high-speed camera in detail and the movement of the proboscis
separated from the body of Lacrymaria. Moreover, we prepared a detergent-extracted cell models and added a solution
containing ATP or a calcium ion. In a solution containing ATP, the movement of cilia occurred but the proboscis didn‟textend. In a solution containing a calcium ion, Lacrymaria showed the gradual contraction. These results suggested thepossibility that the movement of oral cilia decides a direction of the extension of proboscis and the contraction of proboscis is performed by the calcium ion-dependent contractile structure.

#12

A representative model of the gliding motion of a diatom, Bacillaria paradoxa,
predicted from the action of elastic fibrils
Nozomi YAMAOKA1, Yasutaka SUETOMO2, Teruo SHIMMEN1 and Seiji SONOBE1(1Grad. Sch. Life Sci., Univ. Hyogo, 2Iwakuni City Microlite Museum)
SUMMARY
Bacillaria paradoxa belongs to pinnate diatom and forms a colony consisting of 2–30 cells. Adjacent cells show
active gliding each other, but its mechanism and physiological meanings are not understood. Alexa 488-phalloidin
staining revealed that two actin bundles are present along with the raphe, elongated slit in the frustule. Both latrunculin
B, an actin polymerization inhibitor, and 2,3-butanedione monoxime, a myosin inhibitor, inhibited gliding motion,suggesting an essential role of actomyosin system. Western blotting using antibodies against tobacco BY-2 actin andagainst Amoeba myosin shows the presence of actin and myosin in Bacillaria paradoxa. We found that a single cell
separated from a colony shows a motion in back and forth at a point attaching to the substratum, and succeeded inobserving a motion of plastic beads attached to the cell surface at the rapha. We believe that this motion reflect the
motion of the elastic fibrils extending from raphe. Here, we present a curtain hook model for the gliding motion of
Bacillaria paradoxa.

#13

Three-dimensional analysis of the plasmalemma and cytoplasm dynamics duringlocomotion of Amoeba proteus
Atsushi TANIGUCHI1, Yukinori NISHIGAMI1, Hiroko KOBAYASHI2, Daisuke TAKAO2,
Teruo SHIMMEN1, Shigenori NONAKA2 and Seiji SONOBE1(1Grad. Sch. Life Sci., Univ. Hyogo, 2Lab. Spatiotemporal Regulations, Natl. Inst. Basic Biol.)

SUMMARY
During locomotion of Amoeba proteus, the plasmalemma is considered essential for amoeboid movement, but its
dynamics remains to be explained. To solve this problem, we carried out two-dimensional simultaneous observations ofboth the plasmalemma and the cytoplasm from a side by a Digital Scanned Light-sheet Microscopy (DSLM). A. proteus
were stained with 1,1„-dioctadecyl-3,3,3‟,3„-tetramethylindocarbo-cyanine perchlorate (DiI) for the plasmalemma andwith MitoTracker DeepRed for mitochondria. Consequently, both ventral and dorsal plasmalemma showed forward flow,
suggesting that the plasmalemma dynamics seemed to be explained by the total folding and unfolding model (TFU
model; the plasmalemma has many folds, and the folds are stretched along with locomotion). In addition, the gap
between the plasmalemma and ectoplasmic gel was suggested, because mitochondria in the ectoplasmic gel did not moveagainst the substratum. Observation by high-speed imaging DSLM (ezDSLM) also showed that the plasmalemma
dynamics of A. proteus is basically consistent with TFU model. Interestingly, revolving movement of the plasmalemma
at the uroid region was observed.

#14

Localization and properties of Tetrahymena formin
Fumihide BUNAI and Osamu NUMATA(Grad. Sch. Life Environ. Sci., Univ. Tsukuba)
SUMMARY
We suggested that cytokinesis of Tetrahymena is occurred by constriction of actin ring just under the division
furrow. In animal cells, Adf/cofilin and profilin regulate actin depolymerization and polymerization respectively, andthose are essential components of contractile ring. However knockout of Tetrahymena Adf/cofilin and profilin genes
showed no effects on cytokinesis. So we focused on formins which are involved in the polymerization of actin and are
Rho-GTPase effector proteins. Formins regulate the actin and microtubule cytoskeleton and are involved in various
cellular functions such as cell polarity, cytokinesis, and cell migration. We found 2 formin-like genes, BNI1 and BNI2,
in the Tetrahymena gene expression database (http://tged.ihb.ac.cn/Default.aspx). The pull down assay showed that BNI1
directly bound to profilin. Immunofluorescence microscopy with anti-BNI1 and anti-actin antibodies revealed thecolocalization of those in the actin ring and BNI1 is involved in the formation of the actin ring. BNI2 was localized in
the cell surface structures, contractile vacuole pores and the dividing micronucleus. In the dividing micronucleus, BNI2
colocalized with chromatin, suggesting that BNI2 has roles in chromatin condensation and chromatin segregation.

#15

Novel features in the formation and function of the “9 + 2” axoneme revealed bya basal body-deficient mutant of Chlamydomonas
Yuki NAKAZAWA, Tetsuro ARIYOSHI, Ritsu KAMIYA and Masafumi HIRONO(Dept. Biol. Sci., Grad. Sch. Sci., Univ. Tokyo)
SUMMARY
The “9 + 2” structure of ciliary or flagellar axoneme consists of the nine outer doublet microtubules and thecentral pair microtubules (CP). How this conserved structure assembles and functions is poorly understood. We investigated this question by examining aberrant axonemes with 8, 9, 10, or 11 doublets produced by a basal body-deficient
Chlamydomonas mutant, bld12. Most of the 8-doublet axonemes contained no CP, but those without radial spokes
contained CP. Thus the CP formation apparently depends on the central space in the axoneme. In 10- or 11-doublet
axonemes, 4 to 6 radial spokes were detached from the CP, resulting in a distorted arrangement of the doublets. The
detachments were found predominantly on the C2 side of the two CP microtubules, named C1 and C2. However, when
CP projections on C1 were removed, radial spokes frequently attached to a projection on C2. From these observations,
we conclude that the radial spokes actually bind to the CP, especially more strongly to projections on C1 than those on
C2, while multiple projections on CP interact with the radial spoke. These results thus provide invaluable insights into
how the central pair microtubules are formed and how they interact with radial spokes.

#16

Phosphoinositide-specific phospholipase C (PI-PLC) of Amoeba proteus
Hitoshi YAGISAWA, Ryoko SHINKI-HORIE, Kayoko MAEDA-AKAHANE, Teruo SHIMMEN and
Seiji SONOBE(Grad. Sch. Life Sci., Univ. Hyogo)

SUMMARY
A free-living amoeba, Amoeba proteus, has been used as a model to study locomotion of cells exhibiting theamoeboid movement such as neutrophils and cancer cells. Although mechanisms of regulation of actin-based cell motility by phosphoinositides (PI) in mammalian cells and Dictyostelium discoideum have been revealed to some extent, thoseof free-living protists remain poorly understood. We have examined roles of PI and PI-PLC in the cell movement of
Amoeba proteus. The movement of the Amoeba was reversibly inhibited by U73122, an “in vivo” inhibitor of PI-PLCs.
Conversely, microinjection of Ins(1,4,5)P3 into resting cells increased the cell motility and pseudopod extension.
We therefore examined whether Amoeba proteus have PI-PLC. Lysates from the Amoebae show the PtdIns(4,5)P2hydrolysing activity. Moreover, we cloned a cDNA encoding a mammalian PLCδ-like protein from a cDNA library ofAmoeba proteus by RT-PCR method and designate it as Applc. Interestingly, unlike mammalian PI-PLCs, Applc has a C2
domain at the N-terminus instead of the pleckstrin homology (PH) domain that generally targets PLCs to the plasma
membrane. We have established a bacterial expression system of GST-Applc. Partially-purified GST-Applc required
Ca2+ for its activity. Nuclear localization of Applc was resolved using an antibody raised against the N-terminal Applc.

#17

Determination of Immaturin gene sequence: an intragenic structure responsible forsexual rejuvenescence in Paramecium
Nobuyuki HAGA, Toshinori USUI and Tomoaki ABE(Dept. Biol. Engn., Fac. Sci. Engn., Ishinomaki Senshu Univ.)
SUMMARY
Sexual rejuvenescence is performed in Paramecium by two ways. One is conjugation in natural and the othercytoplasmic transfer by microinjection. Sexuality of this organism is defined as the expression of the ability to recognize
the cells of complementary mating-types. The life cycle stages are classified in four major phases, such as conjugatingprocesses, immaturity, maturity and senility. We have previously demonstrated that a cytoplasmic polypeptide, Immaturin, induces sexual rejuvenescence in both mature and senescent cells by microinjection. In this study, we have cloned,
sequenced and identified the gene which encodes Immaturin. Immaturin gene is composed of two parts those are
domains of thioredoxin and GST genes. Transformed-cells of YFP-Immaturin fusion gene indicate that localization of
Immaturin is restricted in cytoplasm, but not in cilia. The presence of Immaturin in the macro- or micronucleus is not
fully demonstrated, yet. Indirect immunofluoresent signals using anti-Immaturin antibody indicate that Immaturin
appears in the cytoplasm of conjugating cells at several hours after the initiation of mating reaction, and the signals aremaintained during conjugating processes and immaturity. Our study suggests that Paramecium produces Immaturin geneby combining two parts of old genes to perform the molecular regulation of sexuality in whole life cycle phases.

#18

Comprehensive search for the nuclear pore complex proteins of ciliate
Tetrahymena thermophila
Masaaki IWAMOTO1, Hiroko OSAKADA1, Chie MORI1, Koji NAGAO2, Chikashi OBUSE2,
Yasushi HIRAOKA3 and Tokuko HARAGUCHI1(1Adv. ICT Res. Inst., NICT, 2Grad. Sch. Life Sci., Hokkaido Univ., 3Grad. Sch. Front. Biosci.,
Osaka Univ.)

SUMMARY
In ciliate Tetrahymena thermophila, it is known that some of the nuclear pore complex (NPC) components are
distinct between macronucleus and micronucleus: among 13 kinds of the identified components, 9 components are common and 4 paralogs of Nup98 are distinct. In order to address how these two distinct NPCs are autonomously assembled
in the same cytoplasm, we first examine yet uncovered NPC components of both nuclei of T. thermophila, and second
understand causative of generating structural difference between macronuclear and micronuclear NPCs. Using mass
spectrometry analysis, we identified additional 15 candidates of the NPC components. Among them, we found 2 pairs ofplural components localized exclusively to either nucleus by expressing these candidates tagged with GFP. In addition,some scaffold components of the NPC showed biased localization between macronuclear and micronuclear NPCs. These
components probably make an important structural contribution in generating fundamentals of distinct NPCs to cause theexclusive localization of nucleus-specific components.

#19

Soil testate amoebae fauna in Japan, re-evaluation
Anatoly BOBROV1, Satoshi SHIMANO2 and Yuri MAZEI3(1Fac. Soil Sci., Moscow State Univ., Russia, 2Miyagi Univ. Educ., 3Penza State Pedagogical Univ.,
Russia)SUMMARY
Analyses of 100 samples of forest soil each from the following islands: Mt. Tateyama of Honshu, Yaku,
Kitadaitou, Minamidaitou and Kume were done. The territory of Japan is an interesting example in protistology. It is
made up of large and small islands with a unique island biogeography composed of the subarctic zone, the temperatezone and the subtropics. We identified about 250 taxa of testate amoebae in the samples. More over 100 species of theidentified species were new record to Japan. We hereby describe two new species: Deharvegia japonica andAssulina discoides. Most of fauna belonged to cosmopolitan group of testate amoebae. The following species are notspecific to species of the Holarctic, usually from the Indo-Malayan and Neotropical regions: Centropyxis deflandriana,
C. latideflandriana, C. stenodeflandriana, C. sacciformis, Cyclopyxis lithostoma, Distomatopyxis couillardi,
Hoogenraadia humicola, Planhoogenraadia daurica, Assulina discoides, Deharvengia japonica, Quadrulella
quadrigera, Q. quadrigera v. africana, Wailesella sp.

#20

Analysis of photosynthetic products in green ciliate Paramecium bursaria
Hiroshi HOSOYA, Eiji HIRAKI, Koyo TETSUKAWA, Yuka ADACHI, Kosuke MATSUBARA andKozue HAMAO(Dept. Biol. Sci., Grad. Sch. Sci., Hiroshima Univ.)

SUMMARY
A green paramecium has several hundreds of endosymbiotic algae. It has been known that those symbiotic algae
produce a lot of photosynthetic products, such as glucose and maltose, et al. The algal endosymbionts excrete large
amounts of sugars, which might be consumed by the host green paramecia. Interestingly, it has been found that
free-living Chlorella species, which are believed not to excrete sugars, such as maltose, can experimentally achieve
endocytobiosis in the algae-free paramecia. Thus, roles of such photosynthetic products in green paramecia are not wellunderstood. Here, we analyzed the species and concentrations of sugars contained in the green and algae-free paramecia.
Further, culture conditions to control the excretion of sugars into the paramecia cytoplasm were elucidated. In thismeeting, our results obtained will be discussed.

#21

Specific connections between symchlorosomes and mitochondria
in Paramecium bursaria as verified by electron tomography and 3D reconstruction
from serial sections
Chihong SONG and Toshinobu SUZAKI(Dept. Biol., Grad. Sch. Sci., Kobe Univ.)
SUMMARY
The spatial relationship between symchlorosomes (symbiotic zoochlorella cells enclosed by peri-algal vacuole
membranes, PVMs) and surrounding organelles was carefully examined by transmission electron microscopy after fixing
Paramecium bursaria cells by a metal-contact quick-freezing procedure in situ, followed by freeze substitution. In
contrast to the morphology reported so far by chemical fixation, the PVM was found to be closely apposed to the cellwall of the zoochlorella. Interaction between PVM and other organelles was further investigated by 3-D reconstruction
from serial sections and electron tomography by using a high-voltage electron microscope. The PVM was not in directcontact with the host cell cortex, but was associated with trichocysts, mitochondria, and small vesicles in the vicinity ofciliary basal bodies. As trichocysts and small vesicles were directly attached to the inner surface of the cell cortex, all ofthese organelles constituted a sub-cortical network with symchlorozomes. Mitochondria were always in close contact
with the outer surface of the PVM, providing structural scaffold for symchlorosomes to be anchored at the sub-corticalregion in the host‟s cytoplasm. Moreover, the outer membranes of mitochondria were occasionally found to fuse with the
PVM, and also with various other membranous organelles including trichocysts and food vacuoles.

#22

New method to eliminate germinal chromosome in Tetrahymena
and observation of the arrangement of meiotic chromosome using nullisomics
and unisomics obtained by the method
Toshiro SUGAI1 and Chie KAJIWARA2(1Life Environ. Sci., Univ. Tsukuba, 2Dept. Biol., Ibaraki Univ.)
SUMMARY
In a ciliate Tetrahymena thermophila, as the macronucleus plays vegetative role, micronuclear chromosomes(2n = 10) can be eliminated, although not all of them. Nullisomic which lacks both copies of homologous chromosomes
and unisomic which has only one kind of chromosome were isolated, but the procedure needs long time and a special
strain. We developed a new simple and quick method for isolation of nullisomic and unisomic: conjugating pair was
fused by hypotonic shock and nuclear change was arrested after meiosis to change diploid to haploid. After division of
the fusant, haploid meiosis was induced by conjugation and the nuclear change was arrested again after meiosis to givenullisomic. The procedure was repeated on the nullisomic to give unisomic.
Using nullisomic, chromosomal arrangement in elongated micronucleus (crescent) during meiosis was observed
directly. At stage II, both ends of chromosome located to one end of spindle shaped micronucleus. At stage V of triple
nullisomic which contains two pairs of homologous chromosomes, two loops were observed at one end of the crescent,suggesting bouquet structure of chromosomes. But two parallel chromatin threads and chromomere-like pattern on themwere similar to single pachytene chromosome of other organism.

#23

Prevalence of Sarcocystis in slaughtered horses and imported horse meat
Kenji YAGITA(Dept. Parasitol., Natl. Inst. Infect. Dis.)SUMMARY
In recent years, the food borne illness through eating raw horsemeat (Basashi) became a growing problem offood hygiene in Japan. Sarcocystis fayeri, a coccidian parasite has been identified in the horsemeat collected fromthe cases of food borne illness. S. fayeri cause a gastrointestinal dysfunction experimentally in animal, suggesting thisparasite as a causative agent of the food borne illness. The study of the prevalence of Sarcocystis in horsemeat will
provide indispensable information for the food poisoning risk assessment in the near future. Thus we determined theamount of S. fayeri DNA in 41 meat samples mostly from light horses slaughtered in Japan and 9 samples of dressedhorsemeat imported from Canada, using a quantitative PCR assay. The results showed that the amount of S. fayeri DNA
in domestic horsemeat samples was lower than that of meat samples collected from the cases of food borne illness, while
the amount of S. fayeri DNA in imported meat samples was almost equivalent to that of the cases of food borne illness.
To be noted, the imported meat from Canada is associated with the incidence of the illness in the reported cases.

#24

Transfer mechanism of spasmin–Ca2+ binding energy to novel elastic protein
spaconnectin
Hiroshi ASAI1 and Yasushige MORIYAMA2(1Adv. Res. Ctr. Sci. Engn., Waseda Univ., 2Osyu Corporation)
SUMMARY
In the previous paper (Biophys. J. 1999, 76, 993–1000), we have achieved to measure tension-displacement
relationship of isolated giant spasmoneme in Zoothamnium arbuscula strain Kawagoe. The tension development of thespasmoneme in the presence of enough Ca2+ is due to random coil contraction of the novel protein, spaconnectin. The
tension of the spasmoneme in the absence of free Ca 2+ is due to partial un-folding of α-helix structure of spaconnectin.
We found that, at a high tension, the Ca2+ binding affinity of spasmin is significantly decreased with increase of tension.
The relationship is obeyed to the Le Chatelier-Braun‟s law. Dimeric spasmins in spasmoneme binds the random coil part
and/or α-helix structure in spaconnectin. To confirm this presumption, the various metal ions and lanthanide ions must
use to measure tension-displacement relationship of giant spasmoneme.

#P1

Function of glycocalyx in cell adhesion and cell locomotion on Amoeba proteus
Yukinori NISHIGAMI1,2, Hisato IKEGAYA1, Rika SHIBATA3, Atsushi TANIGUCHI1,
Teruo SHIMMEN1 and Seiji SONOBE1(1Dept. Life Sci., Grad. Sch. Life Sci., Univ. Hyogo, 2JSPS Res. Fellow (DC), 3Fac. Sci. Univ. Hyogo)

SUMMARY
In Amoeba proteus, plasma membrane (PM) is coated by glycocalyx, which was mainly composed of polysaccharide. From electron microscopic analysis amoeba glycocalyx was composed of two-layer structure, called amorphouslayer (about 0.1 mm thick) and filamentous layer (about 2 mm thick). The amorphous layer was directly attached to
PM and filamentous layer was on the amorphous layer. The chemical components of PM-glycocalyx complex were
investigated in detail, and most major constituent sugar was mannose. Indicating participation of glycocalyx in endocytosis, however, other physiological significance of glycocalyx has not been clear. Since the glycocalyx is the most externallayer, involvement of glycocalyx in cell adhesion and amoeboid movement was predicted. To investigate these possibilities we digested glycocalyx of living A. proteus using alpha-mannosidase and measured activity of amoeboid movement
and cell adhesion. Treatment with alpha-mannosidase, which deleted of filamentous layer, decreased activity of cell
locomotion and cell adhesion. Our results indicated importance of filamentous layer of glycocalyx in cell adhesion andcell locomotion.

#P2

Isolation of actin filaments from a diatom, Craticura sp.
Yuuka KISHIMOTO1, Nozomi YAMAOKA2, Teruo SHIMMEN2 and Seiji SONOBE2(1Dept. Life Sci., Fac. Sci., 2Grad. Life Sci., Univ. Hyogo)
SUMMARY
Pennate diatoms have bilateral symmetry and many genera among them possess an elongated slit in the frustulecalled a raphe, at which cells adhere to the substratum and glide. Although a model explaining gliding has beenpresented, the molecular mechanism remained unsolved. It is reported that actin bundles running parallel to the raphe andactomyosin system plays an essential role in gliding. In order to explain the mechanism of bidirectional gliding, determining polarity of actin bundle is essential. Generally, polarity of actin filaments has been determined by myosin S1
fragment to make the arrowhead structure. In application of this method for diatom cells, there are some difficulties;
penetration of S1 fragment because of it‟s large molecular size, many steps required for sample preparation for EM, andsectioning paralleled to actin filament. To avoid these difficulties, we decided to isolate actin filaments from diatoms.
Cells of Craticura sp., a fresh water diatom, were treated with a buffer containing Triton X-100 and rhodaminephalloidin and ruptured by voltex with 0.4 mm glass beads. Homogenate was centrifuged with 30% glycerol cushion toremove frustule. Bundles of rhodamine-labeled filaments were collected in the resultant precipitate. In EM, bundles
consisting of thin filaments approx. 6 nm in diameter were observed, suggesting that we succeeded in isolating actin
filaments from diatoms.

#P3

Ultrastructural changes during rapid axopodial contraction in heliozoon
Raphidiophrys contractilis
Risa INOUE1, Midori EDAMATSU1,2 and Motonori ANDO1,3(1Lab. Cell Physiol., Grad. Sch. Educ., 2Lab. Animal Physiol. Pharm., Grad. Sch. Nat. Sci. Technol.,3
Lab. Animal Physiol. Pharm., Grad. Sch. Environ. Life Sci., Okayama Univ.)

SUMMARY
The centrohelid heliozoon Raphidiophrys contractilis has many radiating axopodia, each containing a bundle ofaxonemal microtubules as a cytoskeleton. Each axopodium has granular kinetocysts which lie beneath the plasma
membrane and have been implicated in the process of food uptake. In this study, we investigated the cell morphology
and fine structure of R. contractilis during rapid axopodial contraction by light and electron microscopy. Changes in the
diameter of axopodia were analyzed using video microscopy. The diameter of axopodia at proximal regions showeda 2-fold increase before and just after induction of rapid axopodial contraction evoked by mechanical stimulation.
Electron microscopy revealed that the kinetocysts accumulated into the proximal regions of axopodia after rapid
axopodial contraction. To prevent axopodial shortening during glutaraldehyde fixation, we examined the effects of theantitumor-drug paclitaxel (including fluorescence-conjugated drugs) on the microtubular axonemes. The sensitivity of
R. contractilis to the drug was lower than that of the actinophryid heliozoon Actinophrys sol as already reported. These
results suggest that a novel mechanism is involved in the rapid axopodial contraction in R. contractilis, and that theproperties of microtubules and/or microtubule-associated proteins in R. contractilis might be different from those in
A. sol.

#P4

Screening of microbial anti-trypanosomal compounds using Bodo saltans
Koichiro SUMITANI1 and Nobutaka IMAMURA2(1Grad. Sch. Life Sci., 2Coll. Pharm. Sci., Ritsumeikan Univ.)
SUMMARY
Trypanosoma is a parasitic protozoan that belongs to kinetoplastida: trypanosomatidae. They are parasitic onhumans and other animals, and cause various diseases. Bodo saltans and trypanosomes are closely related and both ofthem have an organelle called kinetoplast. B. saltans is a free-living flagellate, not parasitic. So, we use B. saltans asa model organism to find a new drug for trypanosomiasis. We searched active ingredient from microbial secondary
metabolites, and tested its toxicity for HeLa cells. As a result, 468 samples out of 2,231 samples showed activity against
B. saltans. Further screening using HeLa cells to test toxicity, we selected 3 strains. We will report on the process ofsearch and purification of the active ingredient.

#P5

Gametogenesis in Noctiluca scintillans under light-dark cycle
Takumi KITAMURA and Hiroshi ENDOH(Grad. Sch. Nat. Sci. Technol., Kanazawa Univ.)
SUMMARY
Noctiluca scintillans is a heterotrophic dinoflagellate. N. scintillans trophonts usually reproduce by binary
fission. In such trophont population, a small fraction of cells spontaneously transform into gametogenic cells, which
undergo two successive nuclear divisions without cellular divisions. The products of nuclear division are called“progametes” and migrate to cell surface accompanied with a small amount of cytoplasm, and then further divide 6–8
times synchronously, finally reaching 256–1,024 progametes. Thus, numerous mature gametes with two flagella(zoospore) are released from a mother cell ghost. At present, it unknown what triggers differentiation of the gametogenic
cells. Here, we report factors/conditions to affect the onset of gametogenesis by successive observations for two weeks
after feeding. 1) Culture stage: frequency of the appearance of the gametogenic cells drastically increased in earlystationary phase, indicating that starvation is a prerequisite for the differentiation. 2) Light-dark cycle: at the beginning oflight period (L0), cells already entered in middle stages (16–64 progamete stages), and then development successively
progressed (L4–L12). At the end of light period, cells, which began differentiation anew, were observed, suggesting thatdifferentiation would have begun around at the end of light period (L8–L10). When light-dark cycle was shifted
for 4 hour forward or backward, developmental timing followed the cycle within 2 days, suggesting gametogenesis
regulation by circadian clock.

#P6

Construction of foreign protein secretion system in Tetrahymena thermophila
Kohei MASUDA and Hiroshi ENDOH(Grad. Sch. Nat. Sci. Technol., Kanazawa Univ.)
SUMMARY
Various heterologous expression and/or secretion systems of foreign protein, e.g. bacteria or fungi, have beenused for mass production. However, these systems do not always express correctly and efficiently such proteins dueto the difficulties of appropriately folding proteins with complex spatial structures. Now we expect that ciliates havepotential to produce foreign proteins, because of high density culture, inexpensive culture media, and remarkably highfolding and assembly abilities of foreign proteins. For instance, Tetrahymena thermophila is shown to produce a monoclonal antibody efficiently (see Cilian AG Homepage in Germany). In this study, we constructed a transformation
plasmid for convenient expression and secretion systems of foreign proteins in T. thermophila, utilizing a pre-propeptideof one of cysteine proteases for extracellular secretion and Neo4 for a selection marker. After introduction of this plasmid
into the macronucleus and subsequent selection under Paromomycin, we succeeded in establishing several clones andconfirming actual protein expression, secretion into culture medium of two anonymous foreign proteins, and higher
enzyme activity of one foreign protein. These results show that T. thermophila is one of the most ideal model systems forexpression and secretion of foreign proteins in researches and for industrial mass production of useful proteins.

#P7

Measurements of phagocytic activities of Paramecium bursaria using
GFP-labeled yeast
Takashi MIURA and Sosuke IWAI
(Fac. Educ., Hirosaki Univ.)

SUMMARY
Paramecium bursaria is a ciliate that contains several hundreds of Chlorella-like algae in its cytoplasm as endosymbionts. To elucidate whether the presence of these algal endosymbionts affect phagocytosis of the host P. bursaria,
we have quantitatively estimated the phagocytic activities of P. bursaria by using the yeast cells that constitutively
express GFP. The GFP-labeled yeast cells were fed to P. bursaria cells and the fluorescent yeast cells that remained in
the P. bursaria cells after various periods were detected by fluorescence microscopy. The use of the GFP-labeled yeast
enabled us to readily distinguish between the endosymbiont and the engulfed yeast cells in P. bursaria cells, and also
to determine whether the engulfed cells were digested or egested. Our observation suggested that once engulfed by
P. bursaria, yeast cells were mostly digested within 3 hours. Comparison of the phagocytic activities between theaposymbiotic (Chlorella-free) and the symbiotic P. bursaria cells showed that the aposymbiotic ones engulfed anddigested much more yeast cells than the symbiotic ones. These results suggest that the presence of algal endosymbionts
may cause reduction in the phagocytic activities of host ciliates.

#P8

Role of the two-headed inner dynein arm in the ciliary movement inParamecium tetraurelia
Takuya KATOH1, Manabu HORI2, Kanako INOUE2, Osamu KUTOMI1,3 and Munenori NOGUCHI1(1Dept. Environ. Biol. Chem., Grad. Sch. Sci. Engn., Univ. Toyama, 2Grad. Sch. Environ. Sci. Engn.,
Yamaguchi Univ., 3Shimoda Marine Res. Ctr., Univ. Tsukuba)

SUMMARY
The molecular mechanism of inner dynein arms in the ciliary movement in Paramecium tetraurelia is still
unclear. Our previous study has shown that the two-headed inner arm dynein heavy chains- and intermediate chainssilenced cells by RNA interference (RNAi) never exhibit backward swimming. To clarify the role of the two-headed
inner dynein arm in the ciliary movement in P. tetraurelia, we examined the effects of gene silencing of intermediatechain 1 and two heavy chains by RNAi using the feeding method. These three kinds of silenced cells exhibited almost
the same characteristic swimming behavior and ciliary response. They swam slowly and meanderingly. The cilia of thesesilenced cells did not show Ca2+-induced ciliary reversal. To clarify the reason for the defect of ciliary response by thetwo-headed inner dynein arm related gene silencing, we analyzed the effects of gene silencing on the composition ofaxonemal proteins. The results indicate that integrity of the two-headed inner dynein arm is essential for creating theeffective stroke of cilia.

#P9

Maltose release mechanism of symbiotic Chlorella from JapaneseParamecium bursaria
Aika SHIBATA1 and Nobutaka IMAMURA1,2(1Grad. Sch. Sci. Engn., 2Coll. Pharm. Sci., Ritsumeikan Univ.)
SUMMARY
The endosymbiotic Chlorella in Paramecium bursaria has been reported to provide a photosynthate, maltose,which is the material to maintain the symbiotic relationship between Chlorella and its host. We have studied on the effectof light on maltose release by Chlorella F36-ZK from P. bursaria F36. It is well known that maltose release of symbiotic
Chlorella is induced by an acidic condition. F36-ZK also released maltose in pH5 buffer in light condition but did not in
dark. However, its maltose release was not inhibited by a photosynthetic inhibitor DCMU and thus it was suggested thatphotosynthesis did not directly induce maltose release but light stimulated it. From the results using single-wavelength
light, it was indicated that blue (450 nm) and red (around 600 nm) light stimulated maltose release in an acidic condition.
F36-ZK seemed to release maltose as a sole photosynthate, and it should possess a maltose-specific transport system.
Since the amount of released maltose was not affected by the environmental maltose concentration, the maltose shouldbe transported by an active transporter. The maltose release was potently inhibited by the treatment with ionophores,
such as CCCP, nigericine and monencine. Therefore, the proton gradient might regulate maltose release on plasma
membrane.

#P10

Culturing condition and isolation of micronuclei in Blepharisma japonicum
Ryota KOKUTANI1 and Hiroshi ENDOH2(1Div. Biol. Sci., 2Div. Life Sci., Grad. Sch. Nat. Sci. Technol., Kanazawa Univ.)
SUMMARY
Ciliates are presently classified into two large assemblages, Intramacronucleata and Postciliodesmatophora. Mostof ciliates represented by Tetrahymena and Paramecium belong to the former group. The latter group is further divided
into two distinct groups, karyorelictids and heterotrichs, which are the most ancestral ciliates that diverged early from themain line of other ciliates. During macronuclear differentiation, ciliates undergo a large-scale genome reorganization,
including precise removal of germline-specific sequences (internal eliminated sequences: IES) from the macronucleus.
To date, identification of IESs has been restricted to a few representative ciliates, belonging to Intramacronucleata.
In contrast, there is no report on any IESs from Postciliodesmatophora. Therefore, they are important groups, when
origin of the macronucleus and evolution of IESs are considered. Here we report a new isolation method of micronuclei
from the heterotrichous ciliate, Blepharisma japonicum, by a combination of a new cell disruption method and the use of
nuclepore membrane filter.
Based on sequences of randomly-cloned macronuclear DNA, paired primers were designed on both termini.
Using these primer sets, PCR was carried out on total micronuclear DNA to detect products with different length fromthose on macronuclear DNA. So far, one micronuclear DNA fragment carrying IES has been obtained.

#P11

Do the mating pheromones of genus Blepharisma play an important rolein speciation?
Mayumi KOBAYASHI1, Kiyono SHINOHARA2, Mari TAKUSAGAWA3 and Terue HARUMOTO4(1Grad. Sch. Nat. Sci. Ecol. Awareness, 2Dept. Biol. Sci., Grad. Sch. Humanities Sci., 3Dept. Biol. Sci.,
Fac. Sci., 4Res. Gr. Biol. Sci., Div. Nat. Sci., Nara Women‟s Univ.)

SUMMARY
In genus Blepharisma, 20 species have been reported, and these species are classified to four groups according tomacronuclear shape. We attempt to reveal a role of mating pheromones in speciation. Blepharisma has mating
pheromones, gamone1 and gamone2 secreted by cells of mating type I and II, respectively. Gamone1 is a glycoprotein,
and gamone2 is a derivative of tryptophan. Blepharisma forms heterotypic and homotypic pairs by the mating
pheromones. In this study, we revealed that gamone1 obtained from macronuclear group II and IV induced mating pairs
in strains which belong to the same group. Gamone1 amino acid homology within group II or IV is higher than thatbetween group II and IV. On the other hand, gamone2 induced pairs of any species irrespective of the macronuclear
group. Hence, in reproductive isolation, gamone1 plays more important role than gamone2. We speculate that gamone1
promoted macronuclear group differentiation, which is initial stage of speciation in genus Blepharisma. Furthermore,
type I of macronuclear group II and IV, both stimulated by gamone2, did not form homotypic pairs between group II and
IV. Therefore, it is suggested that factors other than gamone1 are also involved in speciation of Blepharisma.

#P12

Study on the amino acid residues which are involved in the stop codon recognitionin ciliate eRF1s
Ying LI1, Oanh KIM T.P.2, Do Thi HONG1, Adriana VALLESI3 and Terue HARUMOTO4(1Grad. Sch. Nat. Sci. Ecol. Awareness, Nara Women‟s Univ., 2Inst. Genome Res., Vietnam Acad. Sci.
Technol., 3Dept. Mol., Cell. Animal Biol., Univ. Camerino, Italy, 4Div. Nat. Sci., Res. Gr. Biol. Sci.,
Nara Women‟s Univ.)

SUMMARY
The genetic code of nuclear genes in some ciliates was found to differ from that of other organisms in theassignment of UAA, UAG, and UGA codons, which are normally assigned as stop codons. Eukaryotic release factor 1(eRF1) is a key protein in stop codon recognition, thus, the protein is believed to play an important role in the stop codon
reassignment in ciliates. eRF1 is composed of three domains, and the stop codon recognition site is located in domain1.
In this study, we examined the release activity of Euplotes raikovi eRF1 by in vivo complementation test in yeast anddual-luciferase assay. Our result suggests that E. raikovi eRF1 recognizes the stop codons UAA and UAG, but not UGA.
We also examined the amino acid I128 in E. raikovi (R128 in Dileptus) which was previously concluded to play animportant role in omnipotent recognition of Dileptus eRF1. Our result showed that single amino-acid substitution of
I128R slightly increased recognition of UGA codon. The result supports our previous idea that R128 is a key residue
important for recognition of the UGA codon in Dileptus eRF1.

#P13

Species identification of wild strains and molecular phylogenetic analysis of
COI gene in the genus Blepharisma
Kiyono SHINOHARA1, Mayumi KOBAYASHI2 and Terue HARUMOTO3(1Dept. Biol. Sci., Grad. Sch. Humanities Sci., 2Grad. Sch. Nat. Sci. Ecol. Awareness,
3
Res. Gr. Biol. Sci., Div. Nat. Sci., Nara Women‟s Univ.)

SUMMARY
Genus Blepharisma is classified into 20 species according to morphological characteristics, and these species are
divided into four groups by macronuclear shape. However, the traditional definition to distinguish species is often
obscure, and only few studies were engaged in molecular phylogenetic analysis so far. Our group previously showed thatgamone1, which is a mating pheromone of Blepharisma, had an effect on different species in the same group. This result
conflicts with the species definition that the strains with reproductive isolation are regarded as different species. In thisstudy, wild strains as well as the laboratory strains were reexamined to identify species in detail, and cell characteristics
were expressed as numerical data. We also analyzed COI gene and 18S rRNA gene base sequences, and compared theresults with those by cell morphology. Homology in the COI gene between strains was obviously higher in the same
macronuclear group than in different group, and the strains which belong to the same group form a cluster in phylogenetic tree. From these data, it is suggested that grouping by macronuclear shape is appropriate also in molecular level,
whereas the traditional species identification by morphological characteristics needs reexamination in further study.

#P14

Nickel-induced genes 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 toa host 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 andNi66, from the subtracted cDNA library of P. 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+ and Co2+ were dose-dependent, and their levels were continued to increase up to 3 days after the addition ofnickel sulfate. We also cloned ~0.45 kb of 5' flanking region of Ni66 gene, and made the Ni66 promoter-driven reporter
construct harboring secreted luciferase coding region. Functions of nickel induced genes and future research will be
discussed in the poster session.

#P15

Properties of Paramecium bursaria cells in the stationary phase
Sosuke IWAI and Takashi MIURA(Fac. Educ., Hirosaki Univ.)
SUMMARY
Paramecium bursaria is a ciliate that contains several hundreds of Chlorella-like algae in its cytoplasm as
endosymbionts. It is well known that P. bursaria cells can survive for an extremely long period even after they enter thestationary and/or starvation phase (Karakashian, 1963; Nishihara et al., 1996; Omura et al., 2004). Generally, nonphotosynthetic protists withstand starvation by lowering their respiration rate (Fenchel, 1982), and also by forming
dormant cysts or spores in some cases. Nevertheless, little is known about the stationary phase of P. bursaria bearing
algal endosymbionts. Here we describe various activities of P. busaria cells that have consumed food yeast cells and thus
are in the starvation and stationary phase. The swimming speed and the phagocytic activities of the stationary-phase cells
were somehow lower than those of the log-phase cells. Long-term observations of single cells in the stationary phasedemonstrated that these cells showed almost no proliferation for at least 1–2 weeks. However, when yeast cells were fed,
they resumed growth after a lag of about 24 h. These results suggest that despite of their lower activities, P. bursaria
cells in the stationary phase still retain fundamental cellular functions, unlike other non-photosynthetic protists.

#P16

Species composition and structure of symbiotic protist community in the termite
Coptotermes formosanus
Osamu KITADE(Coll. Sci., Ibaraki Univ.)
SUMMARY
Termites in the family Rhinotermitidae possess wood-degrading symbiotic protists in their digestive tracts. Threetrichonymphid protists have been described from Coptotermes formosanus, a notorious pest termite in Western Japan. On
the other hand, difference of the symbiont community structures between different termite castes (e.g., workers andsoldiers) is not known. In this study we collected ten colonies of this termite from five localities and examined symbiotic
protist species composition. For workers and soldiers of two colonies, community structure was investigated by direct
counting of the symbionts using a counting chamber. The three trichonymphid species were found from each of five
workers in all examined colonies. In Iriomote Is. and Okinawa Is., an undescribed small trichomonad was found. The
mean protist number of the soldier was about 2.5% of that of the worker and some individuals lack one of the threetrichonyphid species. A principal component analysis exhibited that the community structure of the soldier digestive tract
was more variable than that of the worker. Probably the difference in the „local‟ protist community size between castes
affected its stability.

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New bio-remediation technique for radioactive cesium in contaminated soil using
Paramecium bursaria
Chisato YOSHIMURA1,2 and Toshinobu SUZAKI2(1Ctr. Environ. Management, 2Dept. Biol., Grad. Sch. Sci., Kobe Univ.)
SUMMARY
We have developed a novel bioremediation system for radioactive cesium using the symbiosis between
Paramecium bursaria and endosymbiotic zoochlorellae. After adsorbed on kaolin particles as a model soil, cesium wasadded to the cell suspension of P. bursaria with or without containing endosymbiotic zoochlorella cells. Cesium wastaken up and accumulated up to 30 times higher than the surrounding medium by paramecia with zoochlorellae, whereas
those without endosymbionts showed little accumulation. As P. bursaria cells can be easily and efficiently separated
from soil particles by galvanotaxis, bioremediation of radioactive cesium-contaminated soil with P. bursaria is shown to
be a promising method that can be operated on-site at individual farms, without need for costly transportation and time
consuming process of decontamination.