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Autogamy is induced in Paramecium bursaria by methyl cellulose
ARTICLE IN PRESS European Journal of
PROTISTOLOGY European Journal of Protistology 40 (2004) 313–315 www.elsevier.de/ejop
Autogamy is induced in Paramecium bursaria by methyl cellulose Akira Yanagi Department of Biotechnology, Senshu University of Ishinomaki, Ishinomaki 986-8580, Japan Received 10 March 2004; accepted 20 May 2004
Abstract When isolated single cells of Paramecium bursaria were treated with 1.25% methyl cellulose, most of the single cells showed nuclear changes which were substantially similar to those seen in conjugation, suggesting that the cells undergo autogamy. Additional cytological observations indicated that the cells underwent normal nuclear processes during autogamy. The ability to induce autogamy will facilitate genetic studies in P. bursaria. r 2004 Elsevier GmbH. All rights reserved. Keywords: Autogamy; Chemical induction of autogamy; Methyl cellulose; Paramecium bursaria
Introduction Autogamy in Paramecium is a sexual process occurring within a single cell which brings about complete homozygosity in the progeny. Therefore, autogamy can be very useful for genetic studies. Natural autogamy is known to occur in species of the P. aurelia complex, P. jenningsi and some other ciliates, but has not been found to occur in P. bursaria. Artificial induction of autogamy has been reported in P. caudatum (Mikami and Koizumi 1979; Tsukii and Hiwatashi 1979; Yanagi and Haga 1996) and P. multimicronucleatum (Miyake 1968; Shimomura and Takagi 1984), but has never been reported in P. bursaria. Attempts were made to induce autogamy in P. bursaria by treatment with methyl cellulose, because methyl cellulose has been reported to induce not only autogamy in P. caudatum (Yanagi and Haga 1996) but also selfing conjugation in P. bursaria and some other species of Paramecium (P. caudatum, P. tetraurelia Tel.:+81-225-22-7716; fax:+81-225-22-7746.
E-mail address: [email protected] (A. Yanagi). 0932-4739/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejop.2004.05.007
and P. multimicronucleatum) (Yanagi and Haga 1998). Cytological observations of nuclear changes in P. bursaria exposed to methyl cellulose indicated that autogamy was taking place.
Material and methods Strains and culture conditions Stocks used were KM2 (mating type I) and Dd1 (II) of Paramecium bursaria syngen 1. The culture medium was 1.25% (w/v) fresh lettuce juice diluted with modified Dryl’s solution (Dryl 1959), pH 7.0, and inoculated with Klebsiella pneumoniae one day before use (Hiwatashi 1968). In the modified Dryl’s solution (K-DS) KH2PO4 was substituted for NaH2PO4. Cells were grown at 24–26 1C.
Treatment with methyl cellulose Methyl cellulose (100 centipoises grade, Wako Chemicals Ltd, Japan) was dissolved in deionized water (Yanagi and Haga 1996) as 2 stock solutions
ARTICLE IN PRESS 314
A. Yanagi / European Journal of Protistology 40 (2004) 313–315
Table 1. Induction of nuclear changes in isolated cells by methyl cellulose Strains
Dd1 KM2 a
% of cells undergoing nuclear changes 0% M.C.a
0.75% M.C.
1.25% M.C.
0.0 (22)b 0.0 (26)
6.7 (30) 21.7 (23)
42.9 (21) 65.6 (32)
M.C., methyl cellulose. Figures in parentheses show number of cells examined. Cells were stained 24 h after the treatment of methyl cellulose. b
containing 1.5% and 2.5% (w/v) methyl cellulose. 0.5 ml of cell suspension (about 5000 cells/ml) was mixed with 0.5 ml of the 2 stock solution of methyl cellulose in depression slides. Cells were isolated in the same solution into the wells of a 60-well micro-test plate (Terasaki plate) (Nunc) about 3 h after starting treatment with methyl cellulose. The cells were left in the methyl cellulose solution for 24 h and then stained by acetoorcein to observe morphological changes of the nuclei. Other cells isolated into K-DS were stained in the same way after the same time.
Fig. 1. Nuclear processes in autogamy induced by methyl cellulose. Cells of stock KM2 were treated with 1.25% methyl cellulose and stained by acetoorcein 0 (A), 4 (B), 7 (C) and 24 h (D) after the treatment. A, a vegetative cell; B, the first meiotic division; C, the first meiotic division; D, the second postzygotic division. Arrows indicate micronuclei. Bar, 50 mm.
ARTICLE IN PRESS A. Yanagi / European Journal of Protistology 40 (2004) 313–315
Results and discussion Induction of autogamy in isolated cells Isolated single cells were treated with 0, 0.75 and 1.25% (w/v) methyl cellulose for 24 h (Table 1). In 0% methyl cellulose, none of the isolated cells underwent nuclear changes. In 0.75% methyl cellulose, 6.7% (stock Dd1) and 21.7% (stock KM2) of the isolated cells underwent the nuclear changes (mainly first or second postzygotic division). In 1.25% methyl cellulose, 42.9% (stock Dd1) and 65.6% (stock KM2) of the isolated cells underwent the nuclear changes (mainly first or second postzygotic division). These results show that the methyl cellulose induces nuclear changes in single cells without cell–cell contact. This suggests that methyl cellulose induces autogamy in P. bursaria.
Cytological observation Nuclear processes typical of autogamy were observed in P. bursaria after treatment with methyl cellulose. Without methyl cellulose treatment, cells had a small spindle-shaped micronucleus (Fig. 1A). But when cells were treated with 1.25% methyl cellulose, they underwent the first meiotic division 4–7 h after treatment started (Figs. 1B, C). Cell nuclei showed features of the first or second postzygotic division after 24 h of treatment (Fig. 1D). These nuclear processes in the methyl cellulose-induced autogamy are substantially similar to those in natural conjugation following the mating reaction. This shows that methyl cellulose induces autogamy with normal nuclear processes in P. bursaria. Microscopical examination revealed that methyl cellulose-induced autogamous cells of P. bursaria underwent degeneration of ventral cilia. Degeneration of ventral cilia was also observed in chemically induced autogamy of P. caudatum (Watanabe 1978; Yanagi and Haga 1996), but no degeneration of cilia has been observed in natural autogamy of P. tetraurelia. Autogamy brings about complete homozygosity in the progeny. If a mutagenized population is induced to
315
undergo autogamy, the exautogamous population can be screened directly for recessive mutants. Therefore, the present method to induce autogamy will be useful for genetic studies including the mass screening of recessive mutants in P. bursaria. Attempts are now being made to obtain exautogamous offspring by the present method, though this is difficult because of low fertility in P. bursaria.
Acknowledgments The author thanks Professor Isoji Miwa of Ibaraki University for providing stocks KM2 and Dd1 of P. bursaria.
References Dryl, S., 1959. Antigenic transformation in Paramecium aurelia after homologous antiserum treatment during autogamy and conjugation. J. Protozool. 6, 25. Hiwatashi, K., 1968. Determination and inheritance of mating type in Paramecium caudatum. Genetics 58, 373–386. Mikami, K., 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 (Abstract). Jpn. J. Dev. Biol. 22, 62–63 (in Japanese). Shimomura, F., Takagi, Y., 1984. Chemical induction of autogamy in Paramecium multimicronucleatum. J. Protozool. 31, 360–362. Tsukii, Y., 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., Haga, N., 1996. A simple method of induction of autogamy by methyl cellulose in Paramecium caudatum. Eur. J. Protistol. 32 (Suppl. I), 183–186. Yanagi, A., Haga, N., 1998. Induction of conjugation by methyl cellulose in Paramecium. J. Euk. Microbiol. 45, 87–90.
PROTISTOLOGY European Journal of Protistology 40 (2004) 313–315 www.elsevier.de/ejop
Autogamy is induced in Paramecium bursaria by methyl cellulose Akira Yanagi Department of Biotechnology, Senshu University of Ishinomaki, Ishinomaki 986-8580, Japan Received 10 March 2004; accepted 20 May 2004
Abstract When isolated single cells of Paramecium bursaria were treated with 1.25% methyl cellulose, most of the single cells showed nuclear changes which were substantially similar to those seen in conjugation, suggesting that the cells undergo autogamy. Additional cytological observations indicated that the cells underwent normal nuclear processes during autogamy. The ability to induce autogamy will facilitate genetic studies in P. bursaria. r 2004 Elsevier GmbH. All rights reserved. Keywords: Autogamy; Chemical induction of autogamy; Methyl cellulose; Paramecium bursaria
Introduction Autogamy in Paramecium is a sexual process occurring within a single cell which brings about complete homozygosity in the progeny. Therefore, autogamy can be very useful for genetic studies. Natural autogamy is known to occur in species of the P. aurelia complex, P. jenningsi and some other ciliates, but has not been found to occur in P. bursaria. Artificial induction of autogamy has been reported in P. caudatum (Mikami and Koizumi 1979; Tsukii and Hiwatashi 1979; Yanagi and Haga 1996) and P. multimicronucleatum (Miyake 1968; Shimomura and Takagi 1984), but has never been reported in P. bursaria. Attempts were made to induce autogamy in P. bursaria by treatment with methyl cellulose, because methyl cellulose has been reported to induce not only autogamy in P. caudatum (Yanagi and Haga 1996) but also selfing conjugation in P. bursaria and some other species of Paramecium (P. caudatum, P. tetraurelia Tel.:+81-225-22-7716; fax:+81-225-22-7746.
E-mail address: [email protected] (A. Yanagi). 0932-4739/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejop.2004.05.007
and P. multimicronucleatum) (Yanagi and Haga 1998). Cytological observations of nuclear changes in P. bursaria exposed to methyl cellulose indicated that autogamy was taking place.
Material and methods Strains and culture conditions Stocks used were KM2 (mating type I) and Dd1 (II) of Paramecium bursaria syngen 1. The culture medium was 1.25% (w/v) fresh lettuce juice diluted with modified Dryl’s solution (Dryl 1959), pH 7.0, and inoculated with Klebsiella pneumoniae one day before use (Hiwatashi 1968). In the modified Dryl’s solution (K-DS) KH2PO4 was substituted for NaH2PO4. Cells were grown at 24–26 1C.
Treatment with methyl cellulose Methyl cellulose (100 centipoises grade, Wako Chemicals Ltd, Japan) was dissolved in deionized water (Yanagi and Haga 1996) as 2 stock solutions
ARTICLE IN PRESS 314
A. Yanagi / European Journal of Protistology 40 (2004) 313–315
Table 1. Induction of nuclear changes in isolated cells by methyl cellulose Strains
Dd1 KM2 a
% of cells undergoing nuclear changes 0% M.C.a
0.75% M.C.
1.25% M.C.
0.0 (22)b 0.0 (26)
6.7 (30) 21.7 (23)
42.9 (21) 65.6 (32)
M.C., methyl cellulose. Figures in parentheses show number of cells examined. Cells were stained 24 h after the treatment of methyl cellulose. b
containing 1.5% and 2.5% (w/v) methyl cellulose. 0.5 ml of cell suspension (about 5000 cells/ml) was mixed with 0.5 ml of the 2 stock solution of methyl cellulose in depression slides. Cells were isolated in the same solution into the wells of a 60-well micro-test plate (Terasaki plate) (Nunc) about 3 h after starting treatment with methyl cellulose. The cells were left in the methyl cellulose solution for 24 h and then stained by acetoorcein to observe morphological changes of the nuclei. Other cells isolated into K-DS were stained in the same way after the same time.
Fig. 1. Nuclear processes in autogamy induced by methyl cellulose. Cells of stock KM2 were treated with 1.25% methyl cellulose and stained by acetoorcein 0 (A), 4 (B), 7 (C) and 24 h (D) after the treatment. A, a vegetative cell; B, the first meiotic division; C, the first meiotic division; D, the second postzygotic division. Arrows indicate micronuclei. Bar, 50 mm.
ARTICLE IN PRESS A. Yanagi / European Journal of Protistology 40 (2004) 313–315
Results and discussion Induction of autogamy in isolated cells Isolated single cells were treated with 0, 0.75 and 1.25% (w/v) methyl cellulose for 24 h (Table 1). In 0% methyl cellulose, none of the isolated cells underwent nuclear changes. In 0.75% methyl cellulose, 6.7% (stock Dd1) and 21.7% (stock KM2) of the isolated cells underwent the nuclear changes (mainly first or second postzygotic division). In 1.25% methyl cellulose, 42.9% (stock Dd1) and 65.6% (stock KM2) of the isolated cells underwent the nuclear changes (mainly first or second postzygotic division). These results show that the methyl cellulose induces nuclear changes in single cells without cell–cell contact. This suggests that methyl cellulose induces autogamy in P. bursaria.
Cytological observation Nuclear processes typical of autogamy were observed in P. bursaria after treatment with methyl cellulose. Without methyl cellulose treatment, cells had a small spindle-shaped micronucleus (Fig. 1A). But when cells were treated with 1.25% methyl cellulose, they underwent the first meiotic division 4–7 h after treatment started (Figs. 1B, C). Cell nuclei showed features of the first or second postzygotic division after 24 h of treatment (Fig. 1D). These nuclear processes in the methyl cellulose-induced autogamy are substantially similar to those in natural conjugation following the mating reaction. This shows that methyl cellulose induces autogamy with normal nuclear processes in P. bursaria. Microscopical examination revealed that methyl cellulose-induced autogamous cells of P. bursaria underwent degeneration of ventral cilia. Degeneration of ventral cilia was also observed in chemically induced autogamy of P. caudatum (Watanabe 1978; Yanagi and Haga 1996), but no degeneration of cilia has been observed in natural autogamy of P. tetraurelia. Autogamy brings about complete homozygosity in the progeny. If a mutagenized population is induced to
315
undergo autogamy, the exautogamous population can be screened directly for recessive mutants. Therefore, the present method to induce autogamy will be useful for genetic studies including the mass screening of recessive mutants in P. bursaria. Attempts are now being made to obtain exautogamous offspring by the present method, though this is difficult because of low fertility in P. bursaria.
Acknowledgments The author thanks Professor Isoji Miwa of Ibaraki University for providing stocks KM2 and Dd1 of P. bursaria.
References Dryl, S., 1959. Antigenic transformation in Paramecium aurelia after homologous antiserum treatment during autogamy and conjugation. J. Protozool. 6, 25. Hiwatashi, K., 1968. Determination and inheritance of mating type in Paramecium caudatum. Genetics 58, 373–386. Mikami, K., 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 (Abstract). Jpn. J. Dev. Biol. 22, 62–63 (in Japanese). Shimomura, F., Takagi, Y., 1984. Chemical induction of autogamy in Paramecium multimicronucleatum. J. Protozool. 31, 360–362. Tsukii, Y., 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., Haga, N., 1996. A simple method of induction of autogamy by methyl cellulose in Paramecium caudatum. Eur. J. Protistol. 32 (Suppl. I), 183–186. Yanagi, A., Haga, N., 1998. Induction of conjugation by methyl cellulose in Paramecium. J. Euk. Microbiol. 45, 87–90.