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A simple method of induction of autogamy by methyl cellulose in Paramecium caudatum
Europ. J. Protisto!' 32, Supp!. I: 183 - 186 (1996) October 31, 1996
European Journal of
PROTISTOLOGY
A Simple Method of Induction of Autogamy by Methyl Cellulose in Paramecium caudatum Akira Yanagi and Nobuyuki Haga Department of Biotechnology, Senshu University of Ishinomaki, Ishinomaki 986-80, Japan
SUMMARY We found that methyl cellulose induced autogamy in Paramecium caudatum. When isolated single cells were treated with 1.25 and 2.5% methyl cellulose, most of the single cells showed autogamy-specific nuclear changes. This suggests that the cells undergo autogamy. The simple method, which we have developed, for artificial induction of autogamy is that cells at low population density are simply treated with 2.5% methyl cellulose in plastic Petri dish. In this method, most of the treated cells underwent nuclear changes without cell-cell adhesion, indicating autogamy. The present method for the induction of autogamy will facilitate genetic studies in P. caudatum .
Introduction Autogamy in Paramecium is a sexual process occurring in a single cell without association of cells of complementary mating types. Autogamy brings about complete homozygosity in the progeny. If a mutagenized population is induced to undergo autogamy, the exautogamous population is screened directly for mutants. Natural autogamy is known to occur in species of the P. aurelia complex, P. jenningsi and some other ciliates. Autogamy has not been found to occur naturally in P. caudatum, so artificial induction of autogamy would be useful for genetic studies. So far, there are two reports about artificial induction of autogamy in P. caudatum [3, 6]. Mikami and Koizumi's method [3] is that cells were normally conjugated and prematurely separated by treatment with trypsin. Tsukii and Hiwatashi's method [6] is that cells were cultured in Ca-poor medium and then treated with some chemical agents. We have reported that methyl cellulose induces selfing conjugation in P. caudatum [8]. In the present study, we found that methyl cellulose induced autogamy in P. caudatum. We report a simple method of artificial induction of autogamy in P. caudatum. The present method is that cells at low population density are simply © 1996 by Gustav Fischer Verlag
treated with methyl cellulose in plastic Petri dish. Cytological observations of autogamy-specific nuclear changes suggest that most of the treated cells undergo autogamy. Material and Methods
Strains and Culture Condition Cells of stock 27aG3 (mating type V) syngen 3 of Parameciun caudatum were mainly used. Also used were stocks Kyk201S1 (V) and cI03 (VI) syngen 3 of P. caudatum. The culture medium was 1.25% (w/v) fresh lettuce juice diluted with modified Dryl's solution [1], pH 7.0, and inoculated with Klebsiella pneumoniae one day before use [2]. The modified Dryl's solution substituted KH 2P0 4 for NaH 2P0 4 • Cells were grown at 24-26°C.
Treatment with Methyl Cellulose Methyl cellulose (100 centipoises, Wako Chemicals Ltd, Japan) was dissolved in deionized water. Cells one or two days after the last feeding and with strong mating reactivity were used. The cells were treated with methyl cellulose as follows. Treatment method I. Cell suspension (0.5 ml) was mixed with 0.5 ml of the methyl cellulose solution of twice the final 0932-4739-96-0032-0183$3.50-0
184 . A. Yanagi and N. Haga concentration in slide depressions. The cells were then isolated into each well of a 60-well micro test plate (Terasaki plate) (Nunc) with a micropipette. The isolated cells were left in the methyl cellulose solution for 24 h and then stained by acetoorcein to observe the morphological changes of both micro- and macronucleus . Treatment method II. Cells were mildly centrifuged in a hand-operated centrifuge and diluted with their own culture medium to adjust to an appropriate cell density. The cell suspension (1.0 ml) was mixed with 1.0 ml of the methyl cellulose solution of twice the final concentration in plastic Petri dish (6 em in diameter, Corning) . The cells were treated with methyl cellulose for 8 h and the percentage of paired cells was examined in the plastic dish. The cells were then stained by acetoorcein .
Table 1. Induction of nuclear changes in isolated cells by methyl cellulose % of cells undergoing nuclear changes
Strains
0% M.e.*
0.5% M.e.
1.25 % M.e.
2.5% M. e.
27aG3
0.0 (21#)
0.0 (21)
64.7 (17)
50.0 (18)
d03
0.0 (27)
0.0 (13)
50.0 (20)
57.1 (7)
Kyk201S1 0.0 (24)
10.3 (29)
63.2 (19)
69.2 (13)
* M.e., methyl cellulose. # Figures in parentheses show number of cells examined. Cells were stained 24 h after the treatment of methyl cellulose.
Results and Discussion
Induction of Autogamy in Isolated Single Cells by Methyl Cellulose To examine induction of autogamy in P. caudatum by methyl cellulose, cells were suspended in 0, 0.5, 1.25 and 2.5% (w/v) methyl cellulose solutions and then isolated into each well of a 60-well micro test plate (Treatment method I in Material and Methods). The isolated cells were left in each methyl cellulose solution for 24 h (Table 1). In 0% methyl cellulose, no cells underwent autogamy-specific nuclear changes. In 0.5% methyl cellulose, a few isolated cells underwent the nuclear changes (macronuclear fragmentation). In 1.25 and 2.5% methyl cellulose, at least 50% of the isolated cells underwent nuclear changes (mainly macronuclear fragmentation). This indicates that the methyl cellulose induces nuclear changes in single cells without cell-cell contact, suggesting the induction of autogamy in this species.
Table 2, more than 50% of the methyl cellulose-treated cells underwent nuclear changes in both 1.25 and 2.5% methyl cellulose solutions. The percentage of paired cells induced by 2.5% methyl cellulose was less than that of cells induced by 1.25% methyl cellulose (Table 2). Since the goal was induction of autogamy, not conjugation, 2.5% methyl cellulose is probably a better concentration to use than 1.25 % . Cells were treated with 2.5% methyl cellulose in various cell densities (500, 1,000 and 2,500 cells/m!) and percentages of paired cells were examined. The percentage of paired cells increased from 2 % to 12% in pro portion to the cell density (Fig. 1). Then, cells at lower population densities (50-500 cells/m!) were treated with 2.5% methyl cellulose . In this experiment, there 15 A
n
Simple Method of Artificial Induction of Autogamy by Methyl Cellulose Since cells can hardly swim in highly viscous methyl cellulose solutions, cells at low population density might rarely contact one another and so might be induced to undergo autogamy. If this is the case, we could develop a simple method for inducton of autogamy by methyl cellulose. To examine this possibility, cells at low population densities were treated with methy cellulose in plastic Petri dishes (Treatment meth od II in Material and Methods) . When cells were treated in depression slides in the previous studies of induction of conjugation by methyl collulose [8], they tended to gather in the bottom of slide depressions. However, the plastic Petri dish has a flat bottom, so that cells do not gather in the center of the bottom. First of all, the effect of concentration of methyl cellulose for the induction of autogamy was examined. As shown in Table 1, most cells were induced to undergo nuclear changes in 1.25 and 2.5% methyl cellulose, while a few cells were induced in 0.5% methyl cellulose. Thus, 1.25 and 2.5% methyl cellulose were used to induce autogamy in the following experiment. In
..!!!
"ii 10 lJ
.
i
'ji
c.
"-
0
5
;I. 0
0 0
1000 Cell
density
2000
3000
(cells/ml)
Fig. 1. Percentage of paired cells in methyl cellulose solution . Cells (27aG3 (V))of various densities were treated with 2.5% methyl cellulose for 8 h in plastic Petri dishes and then percentages of paired cells were examined . The mean values of the percentages in three separate exper iments were used to draw the line.
Chemical Induction of Autogamy in Paramecium· 185 Table 2. Induction of nuclear changes by methyl cellulose
% of paired cells
% of cells undergoing nuclear changes *
Experiment number
Concentration of methyl cellulose (%)
Exp. 1
1.25
6.7 (315#)
57.0
2.5
1.2 (341)
88.1 (84)
1.25
14.2 (212)
86.0 (93)
2.5
1.0 (391)
87.8 (123)
Exp. 2
A
(86)
* Around crescent stage of the first meiotic division or earlier stages. # Figuers in parantheses show number of cells examined. Cells (250 cells/ml) were treated with methyl cellulose for 8 h in plastic Petri dish. Percentage of paired cells examined and then cells were stained by acetoorcein to know nuclear conditions.
Table 3. Induction of nuclear changes by methyl cellulose in various cell densities Experiment number
Exp.1
Exp. 2
Cell density (cells/ml)
% of paired cells
% of cells undergoing nuclear changes "
50
0.0 (81)
92.9 (42)
100
0.0 (190)
83.1 (59)
250
1.6 (376)
85.0 (167)
500
1.6 (376)
85.5 (200)
50
0.0 (125)
76.5 (34)
100
1.1 (186)
83.3 (18)
250
0.0 (327)
62.5 (88)
500
3.5 (454)
75.9 (253)
* Around crescent stage of the first meiotic division or earlier stages. # Figures in parentheses show number of cells examined. Cells were treated with 2.5% methyl cellulose for 8 h in plastic Petri dish. Percentage of paired cells was examin ed and then cells were stained by acetoorcein to know nuclear conditions.
Fig. 2. Nuclear processes in autogamy induced by methyl cellulose. Cells (27aG3 (V), 100 cells/m!) were treated with 2.5% methyl cellulose and stained by acetoorcein 8 (A), 12 (B) and 16 h (C and D) after the treatment. A, crescent stage of the first meiotic division; B, the stage after the first meiotic division; C, the stage after the second meiotic division; D, the stage after the first postzygotic division. Arrowheads indicate micronuclei. Bar, 50 ,um.
Cytological Oberservation were no significant differences in percentage of cells undergoing nuclear changes and paired cells rarely appeared in all cell densities examined (Table 3). In Exp. 1 of Table 3, 83-93% of the methyl cellulose-treated cells underwent nuclear changes and most of the cells (more than 98%) did not pair. Thus, autogamy can be easily induced by simply treating cells at low population density (less than 500 cells/m\) with 2.5% methyl cellulose in a Petri dish.
Nuclear processes in autogamy induced by methyl cellulose were observed. Cells 8 h after the treatment with methyl cellulose had a crescent-shaped micronucleus of the first meiotic division (Fig.2A), and 12 h after the treatment underwent the first meiotic division (Fig. 2B). Then cells underwent the second meiotic division (Fig. 2C). Sixteen hours after the treatment, cells underwent the first postzygotic division (Fig. 2D). Twenty-four hours after the treatment, most of cells
186 . A. Yanagi and N . Haga
underwent macronuclear fragmentation. This nuclear process was substantially identical to that in natural conjugation induced by mating reaction. These observations indicate that the treatment of methyl cellulose induces autogamy wih normal nuclear process in the treated cells. Chemically induced autogamous cells of P. caudatum degenerate ventral cilia [7], though no ciliary degeneration was observed in natural autgarny in P. tetraure/ia. To investigate whether the ciliary degenera tion occurs in methyl cellulose-induced autogamous cells of P. caudatum, the autogamous cells were observed microscopically. The observation confirmed that the ventral cilia in the autogamous cells induced by methyl cellulose degenerated. Autogamy brings about complete homozygosity in the progeny and makes mass screening of recessive mutants possible. The present method induces autogamy by treating cells at low population density with 2.5% methyl cellulose in a plastic Petri dish. Since the present method is simpler than the previously reported methods to induce autogamy in P. caudatum [3, 6] and P. multimicronucleatum [4, 5], the present method will be useful for genetical studies including the mass screening of recessive mutants. We are now trying to examine the segregation of marker genes, such as tnds and cnrs, in the exautogamous clones produced by the present method.
Acknowledgements The authors thank Dr. Donald Cronkite for critical reading of the manuscript. This work was partly supported by a grant from the Ministry of Education, Science and Culture of Japan (#06780617 and 07780663) and the Narishige Zoological Science Award to A. Yanagi.
References
2
3 4 5 6 7 8
Dryl S. ( 1959): Antigenic transformation in Paramecium aurelia after homologous antiserum treatment during autogamy and conjugation. J. Protozool., 6, 25. H iwatashi K. ( 1968): Determination and inheritance of mat ing type in Paramecium caudatum . Genetics, 58, 373-386. Mikami K. and Koizumi S. ( 1979): Induction of autogamy by treatment with trypsin in Paramecium caudatum . J. Cell Sci., 35, 177-184. Miyake A. (1968): Artificial induction of autogamy by chemical agents in Paramecium multimicronucleatum, syngen 2. (Abstr.) Jpn. J. Dev. BioI., 22, 62-63. (in Japanese) Shimomura F. and Takagi Y. (1984): Chemical induction of autogamy in Paramecium multimicronucleatum. J. Protozool., 31, 360-362. Tsukii Y. and Hiwatashi K. (1979): Artificial induction of autogamy in Paramecium caudatum. Genet. Res., 34, 163-172. Watanabe T. (1978): A scanning electron -microscopic study of the local degeneration of cilia during sexual reproduction in Paramecium. J. Cell Sci., 32, 55 -66. Yanagi A. and Haga N. (1995): New method for the induction of selfing conjugation in Paramecium caudatum . (Abstr.) Jpn. J. Protozool., 28, 44-45. (in Japanese)
Key words: Artificial induction - Autogamy - Chemical induction - Methyl cellulose - Paramecium caudatum Akira Yanagi, Department of Biotechnology, Senshu University of Ishinomaki, Ishinomaki 986-80, Japan
European Journal of
PROTISTOLOGY
A Simple Method of Induction of Autogamy by Methyl Cellulose in Paramecium caudatum Akira Yanagi and Nobuyuki Haga Department of Biotechnology, Senshu University of Ishinomaki, Ishinomaki 986-80, Japan
SUMMARY We found that methyl cellulose induced autogamy in Paramecium caudatum. When isolated single cells were treated with 1.25 and 2.5% methyl cellulose, most of the single cells showed autogamy-specific nuclear changes. This suggests that the cells undergo autogamy. The simple method, which we have developed, for artificial induction of autogamy is that cells at low population density are simply treated with 2.5% methyl cellulose in plastic Petri dish. In this method, most of the treated cells underwent nuclear changes without cell-cell adhesion, indicating autogamy. The present method for the induction of autogamy will facilitate genetic studies in P. caudatum .
Introduction Autogamy in Paramecium is a sexual process occurring in a single cell without association of cells of complementary mating types. Autogamy brings about complete homozygosity in the progeny. If a mutagenized population is induced to undergo autogamy, the exautogamous population is screened directly for mutants. Natural autogamy is known to occur in species of the P. aurelia complex, P. jenningsi and some other ciliates. Autogamy has not been found to occur naturally in P. caudatum, so artificial induction of autogamy would be useful for genetic studies. So far, there are two reports about artificial induction of autogamy in P. caudatum [3, 6]. Mikami and Koizumi's method [3] is that cells were normally conjugated and prematurely separated by treatment with trypsin. Tsukii and Hiwatashi's method [6] is that cells were cultured in Ca-poor medium and then treated with some chemical agents. We have reported that methyl cellulose induces selfing conjugation in P. caudatum [8]. In the present study, we found that methyl cellulose induced autogamy in P. caudatum. We report a simple method of artificial induction of autogamy in P. caudatum. The present method is that cells at low population density are simply © 1996 by Gustav Fischer Verlag
treated with methyl cellulose in plastic Petri dish. Cytological observations of autogamy-specific nuclear changes suggest that most of the treated cells undergo autogamy. Material and Methods
Strains and Culture Condition Cells of stock 27aG3 (mating type V) syngen 3 of Parameciun caudatum were mainly used. Also used were stocks Kyk201S1 (V) and cI03 (VI) syngen 3 of P. caudatum. The culture medium was 1.25% (w/v) fresh lettuce juice diluted with modified Dryl's solution [1], pH 7.0, and inoculated with Klebsiella pneumoniae one day before use [2]. The modified Dryl's solution substituted KH 2P0 4 for NaH 2P0 4 • Cells were grown at 24-26°C.
Treatment with Methyl Cellulose Methyl cellulose (100 centipoises, Wako Chemicals Ltd, Japan) was dissolved in deionized water. Cells one or two days after the last feeding and with strong mating reactivity were used. The cells were treated with methyl cellulose as follows. Treatment method I. Cell suspension (0.5 ml) was mixed with 0.5 ml of the methyl cellulose solution of twice the final 0932-4739-96-0032-0183$3.50-0
184 . A. Yanagi and N. Haga concentration in slide depressions. The cells were then isolated into each well of a 60-well micro test plate (Terasaki plate) (Nunc) with a micropipette. The isolated cells were left in the methyl cellulose solution for 24 h and then stained by acetoorcein to observe the morphological changes of both micro- and macronucleus . Treatment method II. Cells were mildly centrifuged in a hand-operated centrifuge and diluted with their own culture medium to adjust to an appropriate cell density. The cell suspension (1.0 ml) was mixed with 1.0 ml of the methyl cellulose solution of twice the final concentration in plastic Petri dish (6 em in diameter, Corning) . The cells were treated with methyl cellulose for 8 h and the percentage of paired cells was examined in the plastic dish. The cells were then stained by acetoorcein .
Table 1. Induction of nuclear changes in isolated cells by methyl cellulose % of cells undergoing nuclear changes
Strains
0% M.e.*
0.5% M.e.
1.25 % M.e.
2.5% M. e.
27aG3
0.0 (21#)
0.0 (21)
64.7 (17)
50.0 (18)
d03
0.0 (27)
0.0 (13)
50.0 (20)
57.1 (7)
Kyk201S1 0.0 (24)
10.3 (29)
63.2 (19)
69.2 (13)
* M.e., methyl cellulose. # Figures in parentheses show number of cells examined. Cells were stained 24 h after the treatment of methyl cellulose.
Results and Discussion
Induction of Autogamy in Isolated Single Cells by Methyl Cellulose To examine induction of autogamy in P. caudatum by methyl cellulose, cells were suspended in 0, 0.5, 1.25 and 2.5% (w/v) methyl cellulose solutions and then isolated into each well of a 60-well micro test plate (Treatment method I in Material and Methods). The isolated cells were left in each methyl cellulose solution for 24 h (Table 1). In 0% methyl cellulose, no cells underwent autogamy-specific nuclear changes. In 0.5% methyl cellulose, a few isolated cells underwent the nuclear changes (macronuclear fragmentation). In 1.25 and 2.5% methyl cellulose, at least 50% of the isolated cells underwent nuclear changes (mainly macronuclear fragmentation). This indicates that the methyl cellulose induces nuclear changes in single cells without cell-cell contact, suggesting the induction of autogamy in this species.
Table 2, more than 50% of the methyl cellulose-treated cells underwent nuclear changes in both 1.25 and 2.5% methyl cellulose solutions. The percentage of paired cells induced by 2.5% methyl cellulose was less than that of cells induced by 1.25% methyl cellulose (Table 2). Since the goal was induction of autogamy, not conjugation, 2.5% methyl cellulose is probably a better concentration to use than 1.25 % . Cells were treated with 2.5% methyl cellulose in various cell densities (500, 1,000 and 2,500 cells/m!) and percentages of paired cells were examined. The percentage of paired cells increased from 2 % to 12% in pro portion to the cell density (Fig. 1). Then, cells at lower population densities (50-500 cells/m!) were treated with 2.5% methyl cellulose . In this experiment, there 15 A
n
Simple Method of Artificial Induction of Autogamy by Methyl Cellulose Since cells can hardly swim in highly viscous methyl cellulose solutions, cells at low population density might rarely contact one another and so might be induced to undergo autogamy. If this is the case, we could develop a simple method for inducton of autogamy by methyl cellulose. To examine this possibility, cells at low population densities were treated with methy cellulose in plastic Petri dishes (Treatment meth od II in Material and Methods) . When cells were treated in depression slides in the previous studies of induction of conjugation by methyl collulose [8], they tended to gather in the bottom of slide depressions. However, the plastic Petri dish has a flat bottom, so that cells do not gather in the center of the bottom. First of all, the effect of concentration of methyl cellulose for the induction of autogamy was examined. As shown in Table 1, most cells were induced to undergo nuclear changes in 1.25 and 2.5% methyl cellulose, while a few cells were induced in 0.5% methyl cellulose. Thus, 1.25 and 2.5% methyl cellulose were used to induce autogamy in the following experiment. In
..!!!
"ii 10 lJ
.
i
'ji
c.
"-
0
5
;I. 0
0 0
1000 Cell
density
2000
3000
(cells/ml)
Fig. 1. Percentage of paired cells in methyl cellulose solution . Cells (27aG3 (V))of various densities were treated with 2.5% methyl cellulose for 8 h in plastic Petri dishes and then percentages of paired cells were examined . The mean values of the percentages in three separate exper iments were used to draw the line.
Chemical Induction of Autogamy in Paramecium· 185 Table 2. Induction of nuclear changes by methyl cellulose
% of paired cells
% of cells undergoing nuclear changes *
Experiment number
Concentration of methyl cellulose (%)
Exp. 1
1.25
6.7 (315#)
57.0
2.5
1.2 (341)
88.1 (84)
1.25
14.2 (212)
86.0 (93)
2.5
1.0 (391)
87.8 (123)
Exp. 2
A
(86)
* Around crescent stage of the first meiotic division or earlier stages. # Figuers in parantheses show number of cells examined. Cells (250 cells/ml) were treated with methyl cellulose for 8 h in plastic Petri dish. Percentage of paired cells examined and then cells were stained by acetoorcein to know nuclear conditions.
Table 3. Induction of nuclear changes by methyl cellulose in various cell densities Experiment number
Exp.1
Exp. 2
Cell density (cells/ml)
% of paired cells
% of cells undergoing nuclear changes "
50
0.0 (81)
92.9 (42)
100
0.0 (190)
83.1 (59)
250
1.6 (376)
85.0 (167)
500
1.6 (376)
85.5 (200)
50
0.0 (125)
76.5 (34)
100
1.1 (186)
83.3 (18)
250
0.0 (327)
62.5 (88)
500
3.5 (454)
75.9 (253)
* Around crescent stage of the first meiotic division or earlier stages. # Figures in parentheses show number of cells examined. Cells were treated with 2.5% methyl cellulose for 8 h in plastic Petri dish. Percentage of paired cells was examin ed and then cells were stained by acetoorcein to know nuclear conditions.
Fig. 2. Nuclear processes in autogamy induced by methyl cellulose. Cells (27aG3 (V), 100 cells/m!) were treated with 2.5% methyl cellulose and stained by acetoorcein 8 (A), 12 (B) and 16 h (C and D) after the treatment. A, crescent stage of the first meiotic division; B, the stage after the first meiotic division; C, the stage after the second meiotic division; D, the stage after the first postzygotic division. Arrowheads indicate micronuclei. Bar, 50 ,um.
Cytological Oberservation were no significant differences in percentage of cells undergoing nuclear changes and paired cells rarely appeared in all cell densities examined (Table 3). In Exp. 1 of Table 3, 83-93% of the methyl cellulose-treated cells underwent nuclear changes and most of the cells (more than 98%) did not pair. Thus, autogamy can be easily induced by simply treating cells at low population density (less than 500 cells/m\) with 2.5% methyl cellulose in a Petri dish.
Nuclear processes in autogamy induced by methyl cellulose were observed. Cells 8 h after the treatment with methyl cellulose had a crescent-shaped micronucleus of the first meiotic division (Fig.2A), and 12 h after the treatment underwent the first meiotic division (Fig. 2B). Then cells underwent the second meiotic division (Fig. 2C). Sixteen hours after the treatment, cells underwent the first postzygotic division (Fig. 2D). Twenty-four hours after the treatment, most of cells
186 . A. Yanagi and N . Haga
underwent macronuclear fragmentation. This nuclear process was substantially identical to that in natural conjugation induced by mating reaction. These observations indicate that the treatment of methyl cellulose induces autogamy wih normal nuclear process in the treated cells. Chemically induced autogamous cells of P. caudatum degenerate ventral cilia [7], though no ciliary degeneration was observed in natural autgarny in P. tetraure/ia. To investigate whether the ciliary degenera tion occurs in methyl cellulose-induced autogamous cells of P. caudatum, the autogamous cells were observed microscopically. The observation confirmed that the ventral cilia in the autogamous cells induced by methyl cellulose degenerated. Autogamy brings about complete homozygosity in the progeny and makes mass screening of recessive mutants possible. The present method induces autogamy by treating cells at low population density with 2.5% methyl cellulose in a plastic Petri dish. Since the present method is simpler than the previously reported methods to induce autogamy in P. caudatum [3, 6] and P. multimicronucleatum [4, 5], the present method will be useful for genetical studies including the mass screening of recessive mutants. We are now trying to examine the segregation of marker genes, such as tnds and cnrs, in the exautogamous clones produced by the present method.
Acknowledgements The authors thank Dr. Donald Cronkite for critical reading of the manuscript. This work was partly supported by a grant from the Ministry of Education, Science and Culture of Japan (#06780617 and 07780663) and the Narishige Zoological Science Award to A. Yanagi.
References
2
3 4 5 6 7 8
Dryl S. ( 1959): Antigenic transformation in Paramecium aurelia after homologous antiserum treatment during autogamy and conjugation. J. Protozool., 6, 25. H iwatashi K. ( 1968): Determination and inheritance of mat ing type in Paramecium caudatum . Genetics, 58, 373-386. Mikami K. and Koizumi S. ( 1979): Induction of autogamy by treatment with trypsin in Paramecium caudatum . J. Cell Sci., 35, 177-184. Miyake A. (1968): Artificial induction of autogamy by chemical agents in Paramecium multimicronucleatum, syngen 2. (Abstr.) Jpn. J. Dev. BioI., 22, 62-63. (in Japanese) Shimomura F. and Takagi Y. (1984): Chemical induction of autogamy in Paramecium multimicronucleatum. J. Protozool., 31, 360-362. Tsukii Y. and Hiwatashi K. (1979): Artificial induction of autogamy in Paramecium caudatum. Genet. Res., 34, 163-172. Watanabe T. (1978): A scanning electron -microscopic study of the local degeneration of cilia during sexual reproduction in Paramecium. J. Cell Sci., 32, 55 -66. Yanagi A. and Haga N. (1995): New method for the induction of selfing conjugation in Paramecium caudatum . (Abstr.) Jpn. J. Protozool., 28, 44-45. (in Japanese)
Key words: Artificial induction - Autogamy - Chemical induction - Methyl cellulose - Paramecium caudatum Akira Yanagi, Department of Biotechnology, Senshu University of Ishinomaki, Ishinomaki 986-80, Japan