Jpn. J. Protozool. Vol. 43, No. 2. (2010)

95

Short Communication㻌

Timing of commitment to autogamy in Paramecium
bursaria
Akira YANAGI
Department of Biotechnology, Senshu University of Ishinomaki, Ishinomaki 986-8580, JapanSUMMARY
To elucidate the timing of commitment to autogamy in Paramecium bursaria, cells were treatedwith an autogamy-inducing agent (1.25% methyl cellulose) for 0, 1, 2, 3, 5, and 24 h, and the duration oftreatment required for the induction of autogamy was examined 24-25.5 h after the onset of treatment.
Many cells treated for 0-2 h were at the vegetative stage, but many cells treated for 3-24 h were at stages
from the third pre-zygotic division to the third post-zygotic division and normally underwent autogamy.
Therefore, treatment with methyl cellulose for 3 h or more is considered necessary for the induction ofautogamy. However, some of the cells treated for 3 h were at the stage of developing either a swollen
and spindle-shaped micronucleus or an early round-shaped micronucleus, which is indicative of the premeiotic S phase. This suggests that if cells proceed beyond the stage of an early round-shaped
micronucleus, they normally undergo autogamy, but if not, they terminate at the stage of either a swollen
and spindle-shaped micronucleus or an early round-shaped micronucleus. Consequently, cells of P.
bursaria are committed to autogamy just after the stage of an early round-shaped micronucleus (the premeiotic S phase).
Key words: Autogamy, Commitment, Meiosis, Methyl cellulose, Paramecium bursaria, Pre-meiotic S
phase

INTRODUCTION
In ciliates, the major developmental pathways consist of the sexual (conjugation orautogamy) and asexual (cell division) pathways
that can be easily switched each other, providing

Tel: ＋81-225-22-7716
Fax: ＋81-225-22-7746
E-mail: yanagi@isenshu-u.ac.jp㻌㻌
Received: 27 November 2009; Accepted: 2 June
2010.㻌

us a useful experimental system to understand themechanism of commitment to the sexual pathway.
In studies of Paramecium, there are some reports
concerning commitment to the sexual pathway inP. caudatum (Fujishima, 1983b, 1988), P. multimicronucleatum (Shimomura and Takagi, 1985)
and P. tetraurelia (Berger, 1986). These threespecies of Paramecium belong to the “aurelia”
group, though the species of Paramecium fall into
two defined groups based on cell shape: the“aurelia” group and the “bursaria” group. Thus,

96

Commitment to autogamy in Paramecium bursaria㻌㻌

in the “bursaria” group, there are no reports about
the commitment to the sexual pathway.
In the present study, in order to determine
the timing of commitment to autogamy in a species of the “bursaria” group, the period of methyl
cellulose treatment required for the induction ofautogamy in P. bursaria was examined.
Autogamy is a sexual reproduction process occurring in single cells without the direct associationof cells, and in P. bursaria autogamy can be artificially induced by the treatment with methyl cellulose (Yanagi, 2004); methyl cellulose-induced
autogamous cells normally undergo nuclear processes.
MATERIALS AND METHODS
Strains and culture condition
Paramecium bursaria, syngen 1, stock Dd1(mating type II) was used. The culture medium
was 1.25% (w/v) fresh lettuce juice diluted withmodified Dryl’s solution (Dryl, 1959), pH 7.0, andinoculated with Klebsiella pneumoniae one day
before use (Hiwatashi, 1968). In the modified
Dryl’s solution (K-DS), KH2PO4 was substitutedfor NaH2PO4. The cells were incubated at 2426°C.
Treatment with methyl cellulose
Methyl cellulose (100 centipoises, Wako
Chemicals Co., Ltd., Japan) was dissolved in deionized water (Yanagi and Haga, 1996) as 2×stock
solution containing 2.5% (w/v) methyl cellulose.
The 2×stock solution (0.5 ml) was mixed with 0.5
ml of cell suspension (approximately 5,000 cells/ml) in depression slides (Toushinrikou Co., Ltd.,Japan). Therefore, cells were treated with 1.25%
methyl cellulose of the final concentration.
In order to examine the duration of methyl
cellulose treatment required for the induction ofautogamy in P. bursaria, cells were treated with1.25% methyl cellulose for 0, 1, 2, 3, 5, and 24 h.
The cells treated with methyl cellulose for 0 h

were isolated into half the original concentration ofK-DS for 24 h in the wells of a 60-well micro test
plate (Nalge Nunc International, U.S.A.). The
reason why half the original concentration of KDS was used for 0 h was due to the fact, for subsequent time periods (1, 2, 3, 5, and 24 h), whencells were treated with methyl cellulose, equalvolumes of cell suspension in the original concentration of K-DS and 2.5% methyl cellulose weremixed; therefore, the concentration of K-DS in themixed solution was half the original concentration.
The cells treated for 1, 2, 3, and 5 h were
washed with K-DS following treatment withmethyl cellulose and isolated into half the original
concentration of K-DS in the wells of a microplate. The cells to be treated for 24 h (incubated in
1.25% methyl cellulose solution) were isolated
into the wells of a micro test plate 1-3 h after theonset of treatment. Finally, all cells treated for 0,
1, 2, 3, 5, and 24 h were stained with aceto-orcein
24-25.5 h after the onset of treatment. The induction of autogamy was detected by the observationof morphological changes of micronuclei in isolated single cells.
RESULTS AND DISCUSSION
Temporal sequence of micronuclear events incells treated with methyl cellulose
To know the time course of micronuclear
events in the methyl cellulose-induced autogamyof P. bursaria, the time course of nuclear processes in cells treated with methyl cellulose, which
undergo either autogamy or conjugation, was examined. Because it is suggested that the timecourse of nuclear processes in the methyl cellulose-induced autogamy of P. bursaria is substantiallysimilar to that in the methyl cellulose-induced conjugation (Yanagi and Haga, 1998; Yanagi, 2004).
At the vegetative stage, cells had a thin andspindle-shaped micronucleus (Fig. 1A). The micronucleus was swollen within 3 h of the treatmentwith methyl cellulose (Fig. 1B) and then began toJpn. J. Protozool. Vol. 43, No. 2. (2010)

become round-shaped around 3 h after the onset oftreatment (Fig. 1C). The round-shaped micronucleus gradually enlarged until 8 h after the onset oftreatment (prophase of the first meiotic division)and elongated 10-12.5 h after the onset of treat-97

ment (prophase of the first meiotic division). The
micronucleus enlarged around 15 h after the onsetof treatment (prophase and metaphase of the firstmeiotic division) and underwent the anaphase andtelophase of the first meiotic division about 17.5 hFig. 1. Micronuclear changes induced by the treatment with methyl cellulose. Cells at micronuclear stages observed inTable 1 are shown. A, thin and spindle-shaped (vegetative stage); B, swollen and spindle-shaped; C, early round-shaped;
D, 3rd pre-zygotic division; E, synkaryon; F, 1st post-zygotic division; G, pycnosis of one of two micronuclei produced by
1st post-zygotic division; H, 2nd post-zygotic division; I, 3rd post-zygotic division. Arrows and arrowheads indicate micronuclei and pycnotic micronuclei, respectively. Bar, 50 m.

98Commitment to autogamy in Paramecium bursaria㻌㻌

after the onset of treatment. Thereafter the second
meiotic division and the third pre-zygotic division(Fig 1D) occurred and a synkaryon was formed
about 20 h after the onset of treatment (Fig. 1E).
The synkaryon divided two times mitotically (thefirst and second post-zygotic division) 22-24 h
after the onset of treatment (Fig. 1F, G and H) andthe third post-zygotic division began to occur
around 24 h after the onset of treatment (Fig. 1I).
Timing of commitment to autogamy
The duration of methyl cellulose treatment

required for the induction of autogamy in P. bursaria was examined (Table 1). All of the 0 htreated cells and most of the 1 h and 2 h treatedcells had a thin and spindle-shaped micronucleus
at the vegetative stage (Table 1 and Fig. 1A).
Some of the cells treated for 3, 5 and 24 h werealso at the vegetative stage. The percentage of thevegetative cells decreased with the length of theperiod of methyl cellulose treatment. The vegetative cells are not activated for autogamy.
Some of the 1 h and 2 h treated cells were at
the stage of a swollen and spindle-shaped micronu-

Table 1. Duration of methyl cellulose treatment required for the induction of micronuclear events inautogamy
Number of cells
Stages of micronuclei#0h*

1h*

2h*

3h*

5h*

24h*

Thin and spindle-shaped

1519

18

105

1

Swollen andspindle-shaped-

151-

-

Early round-shaped-

--

6-

-

Meiosis------

3rd pre-zygotic division

---1-

2

Synkaryon

-----

2

1st and 2nd post-zygoticdivision

---

3

214

3rd post-zygotic division

---

4

8

3

Total number of cells
examined

1520

2325

1522

#Stages of micronuclei were classified on the basis of Figure 7.9 in R. Wichterman’s The Biology of Paramecium(1986).
*Time indicates the duration of methyl cellulose treatment. Cells were treated with 1.25% methyl cellulose for 0, 1, 2,3, 5, and 24 h, and stained with aceto-orcein 24-25.5 h after the onset of treatment.

Jpn. J. Protozool. Vol. 43, No. 2. (2010)

cleus (Table 1 and Fig. 1B). The stage is normallyobserved 1-3 h after the onset of treatment withmethyl cellulose. Some of the 3 h treated cells
were at the stage of either a swollen and spindleshaped micronucleus or an early round-shaped
micronucleus (Table 1 and Fig. 1C). The stage ofan early round-shaped micronucleus is normallyobserved around 3 h after the onset of treatment.
The cells at the stages of a swollen and spindleshaped micronucleus and an early round-shapedmicronucleus are activated for the stages but could
not proceed to the meiosis stage. Thus the cells
are considered activated for autogamy, but notcommitted to autogamy.
On the other hand, one third of the 3 htreated cells and most of the 5 h and 24 h treatedcells proceeded to later stages from the third prezygotic division to the third post-zygotic division(Table 1 and Fig. 1D-I), when they were stainedwith aceto-orcein 24-25.5 h after the onset of treatment with methyl cellulose. The stages are normally observed around 19-25 h after the onset oftreatment with methyl cellulose and the time issimilar to that of the staining with aceto-orcein (24-25.5 h after the onset of treatment). This showsthat the cells at the stages from the third prezygotic division to the third post-zygotic divisionnormally undergo nuclear events in autogamy.
The cells are thought to complete the whole process of autogamy. Therefore, the cells are considered committed to autogamy. Moreover, none of
the 3 h treated cells was during meiosis, thoughmeiosis occurs between the stages of an earlyround-shaped micronucleus and the third prezygotic division and lasts more than 10 h. In the 5h and 24 h treated cells, no cells were at the stagesof a swollen and spindle-shaped micronucleus, anearly round-shaped micronucleus and meiosis.
In brief, the present results show that, except
for vegetative cells, all of the 1 h and 2 h treatedcells and one half of the 3 h treated cells terminate
at the stage of either a swollen and spindle-shaped
micronucleus or an early round-shaped micronu-99

cleus, but the remaining 3 h treated cells and all of
the 5 h and 24 h treated cells undergo the normal
process of autogamy. Therefore, the treatment
with methyl cellulose for 3 h or more is considerednecessary for the induction of autogamy. Furthermore, the results suggest that if cells proceed beyond the stage of an early round-shaped micronucleus, they normally undergo nuclear changes inautogamy, but if not, they terminate at the stage ofeither a swollen and spindle-shaped micronucleus
or an early round-shaped micronucleus. This leads
to the conclusion that cells are committed toautogamy just after the stage of an early roundshaped micronucleus.
The stages of a swollen and spindle-shaped
micronucleus and an early round-shaped micronucleus are suggested to be in the pre-meiotic Sphase undergoing pre-meiotic DNA synthesis,
because the pre-meiotic S phase begins with themicronuclear swelling and ends immediately before meiotic prophase in conjugation of P. caudatum (Fujishima, 1983a). In P. caudatum, a roundshaped micronucleus, called a stage II micronucleus, is mainly in the pre-meiotic S phase(Fujishima, 1983a). Thus, it appears that cells ofP. bursaria are committed to autogamy just after
the pre-meiotic S phase, namely immediately prior
to initiation of meiosis or at the beginning of meiosis.
In conclusion, it is thought that commitment
to the sexual pathway (autogamy and conjugation)
occurs immediately after the pre-meiotic S phase(immediately prior to initiation of meiosis or at thebeginning of meiosis) not only in species of the“aurelia” group including P. tetraurelia (Berger,1986), P. multimicronucleatum (Shimomura andTakagi, 1985) and P. caudatum (Fujishima, 1983band 1988), but also in a species of the “bursaria”
group, P. bursaria (the present study).
ACKNOWLEDGMENTS
The author thanks Professor Isoji Miwa of100

Commitment to autogamy in Paramecium bursaria㻌㻌

Ibaraki University for providing a stock Dd1 of P.
bursaria.
REFERENCES
Berger, J. D. (1986) Autogamy in Paramecium
cell cycle stage-specific commitment tomeiosis. Exp. Cell Res., 166, 475-485.
Dryl, S. (1959) Antigenic transformation in Paramecium aurelia after homologous antiserum
treatment during autogamy and conjugation.
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Fujishima, M. (1983a) Microspectrophotometricand autoradiographic study of the timing andduration of pre-meiotic DNA synthesis inParamecium caudatum. J. Cell Sci., 60, 5165.
Fujishima, M. (1983b) Transition from mitosis tomeiosis in Paramecium in relation to conjugation period. Zool. Mag., 92, 603. (in Japanese).
Fujishima, M. (1988) Conjugation. In: Paramecium. Göertz, H.-D. (ed.). Springer-Verlag,

Berlin Heidelberg New York, pp 70-84.
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