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A natural strain of Paramecium bursaria lacking symbiotic algae
Europ. J. Protistol. 38, 55–58 (2002) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/ejp
A natural strain of Paramecium bursaria lacking symbiotic algae Yuki Tonooka and Tsuyoshi Watanabe* Biological Institute, Graduate school of Science, Tohoku University, Aramaki-aza Aoba, Aoba-ku, Sendai 980-8578, Japan; e-mail: [email protected] Received: 11 July 2001; 14 January 2002. Accepted: 16 January 2002
Paramecium bursaria contains hundreds of algae in its cytoplasm as symbionts and retains them over many generations. Recently, aposymbiotic cells of P. bursaria were collected from a pond. To know whether the natural aposymbiotic strain (Ysa2) lost ability to associate with algae, infection experiments were performed using symbiotic algae isolated from green paramecia. Results showed that Ysa2 establish symbiotic association in the same way as artificially obtained aposymbiotic cells (control). However, clustered algae appeared in some Ysa2 cells after infection. Algal clusters differ in number and size from cell to cell. Isolation line culture was performed to pursue the fate of cells having algal clusters. Within several cell divisions, some descendants retained large clusters and such cells were sometimes misshapen, ceasing proliferation and eventually dying. At cell division of cluster-bearing cells, unequal distribution of clustered algae occurred because many algal clusters tend to locate in the posterior of the host cell. Most extremely, all algae were distributed to the posterior daughter cell, whereas the anterior daughter cell contained no algae. This is one possible mechanism of production of Ysa2 in nature. From these results, we conclude that Ysa2 can make symbiotic association with potentially symbiotic algae, but they have some incompatibilities with the algae. Key words: Paramecium bursaria; symbiosis; infection; algae.
Introduction The green paramecium, Paramecium bursaria, harbours hundreds of symbiotic algae (Chlorella sp.) in the cytoplasm. It is known that mutual benefits are exchanged between the host cell and symbiotic algae as described by many investigators (Karakashian 1963; Weis 1969; Karakashian and Karakashian 1973; Brown and Nielsen 1974; Karakashian and Rudzinska 1981; Schussler and Schnepf 1992). Algae are retained through cell divisions and sexual reproduction of paramecia (Siegel 1960). Thus, the symbiotic relationship in P. bursaria seems to continue permanently. *corresponding author
On the other hand, it is also known that both paramecia and algae can be grown separately. Symbiotic algae isolated from paramecia can be cultured under appropriate conditions (Siegel 1960; Weis 1978; Nishihara et al. 1998; Takeda et al. 1998). Algae-free paramecia are obtained by various methods, such as long-term cultivation in darkness (Siegel 1960), X-ray irradiation (Wichterman 1948), and chemical treatment (Reisser 1976; Hosoya et al. 1995). However, spontaneous production of aposymbiotic paramecia is unlikely to occur in the field since regulatory conditions, which paramecia scarcely face in the field, are necessary to produce artificial aposymbiotic cells. It has been shown by 0932-4739/02/38/01-55 $ 15.00/0
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many investigators that artificially obtained aposymbiotic paramecia can re-establish a symbiotic relationship with isolated algae which are taken into the cytoplasm through the cytostome (Karakashian and Karakashian 1973; Weis and Ayara 1979; Reisser et al. 1985; Meier and Wiessner 1988). Recently, aposymbiotic cells of P. bursaria were unexpectedly collected together with normal green cells from a pond. Because of the stability of the symbiotic relationship, it is very rare for aposymbiotic P. bursaria to be collected from the field. To clarify whether a natural aposymbiotic cell can establish a symbiotic relationship with algae derived from green paramecia or whether they show any defects in keeping infected symbiotic algae, infection experiments were performed using natural aposymbiotic cells.
Results and discussion Ysa2 is an aposymbiotic strain of P. bursaria First, we examined whether Ysa2 is really a white strain of P. bursaria or not. All characteristics, such as cell shape, size, macro- and micronuclear features, and swimming tracks are identical with those of P. bursaria (Wichterman 1953). Moreover, Ysa2 mate with cells of mating type I, II, and IV in P. bursaria syngen 1 but not with mating type III cells. From these facts, we concluded that Ysa2 is a natural aposymbiotic strain of mating type III, in syngen 1 of P. bursaria.
Materials and methods Cells and culture Some symbiotic (green) and aposymbiotic (white) strains of Paramecium bursaria, syngen 1, were used in this study. Ysa2 is a natural aposymbiotic clone collected from a pond in Sagae City, Yamagata, Japan. An artificially produced white strain Sj2w was also used as a control for infection experiments. Green strains, Gr3 and T316, were used as a source of symbiotic algae for infection. Cells were cultured in Hiwatashi’s lettuce juice medium (Hiwatashi 1968) inoculated with Klebsiella pneumoniae, at 24 °C, under continuous light (17.6 µeinstein m–2s–1).
Infection experiments Symbiotic algae were isolated from green paramecia by a modified McAuley's method (McAuley 1993). Algae were finally suspended in the K-DS, a modification of Dryl's solution (Dryl 1959), in which sodium dihydrogen phosphate was replaced with potassium dihydrogen phosphate (Yanagi 1992). Approximately one thousand white paramecia were exposed to 2 × 106 algal cells in 1 ml of total volume and incubated for 5 hours (Weis and Ayara 1979). Then paramecia were washed well in K-DS to remove external algae and transferred to a test tube containing fresh culture medium. Paramecia were cultured by daily addition of 1–2 ml of fresh culture medium for 40 days. A small number of cells were removed from the culture and examined with a fluorescence microscope to determine whether they retained symbiotic algae. In some infection experiments, daily isolation-line cultures (Sonneborn 1950) were established to determine the fate of infected paramecia and algae.
Fig. 1. Cells of infected Ysa2, a natural aposymbiotic strain of P. bursaria. Clusters of algae of different size and number are seen in these cells (a, b). Algal clusters tend to accumulate in the posterior part of the cell (c). At cell division, extremely unequal distribution of algal clusters causes formation of an aposymbiotic daughter cell (d). Magnification: a–c, ×340; d, ×250.
Natural aposymbiotic strain of P. bursaria
Infection experiments During 5-hour exposure to algal suspension, all white cells ingested many algae. Thereafter, the proportion of cells retaining algae in the cytoplasm (retention rate) was counted every 4 days up to 40 days. The retention rate decreased in both Sj2w (control) and Ysa2 cells until 8 days; thereafter it settled until the end of the experiment. The final Ysa2 retention rate was somewhat lower (40-70%) than that of Sj2w (60-80%). Infection rates have been shown to be affected by physiological conditions of both algae and paramecia or by specificity between them (Siegel 1960; Hirshon 1969; Karakashian and Karakashian 1973; Weis 1978, 1979; Weis and Ayara 1979; Meier and Wiessner 1988, 1989; Tanaka and Miwa 1996; Nishihara et al. 1998). Although the retention rate was slightly lower than Sj2w, many Ysa2 cells retained algae for 40 days through many cell divisions. Therefore, it is concluded that Ysa2 can establish a symbiotic relationship with algae isolated from green paramecia.
57
tion period, some descendants produced normal cells with dispersed algae and others showed various types of abnormality. Misshapen cells due to the presence of a large cluster were found frequently. Such clusters sometimes contained approximately 1,000 algae. At cell division of the misshapen cells, unequal cluster distribution was often observed. In the most excessive case, that type of cell division produced one white cell with no algae and one cell heavily loaded with algae (Fig. 1d). Some posterior daughter cells containing large clusters occasionally ceased cell growth and eventually died, while white anterior daughter cells thus produced were able to grow in the same way as the original Ysa2 cells. This suggests that this type of cell division may be one mechanism for producing natural aposymbiotic Ysa2 cells. To our knowledge, this is the first report of aposymbiotic cells being obtained from the field. This strain may be useful for molecular analysis in establishing an understanding of symbiosis in P. bursaria.
Cluster formation of algae after infection A couple of days after commencement of infection experiments, clustering algae were observed in some Ysa2 cells. Many, but not all, of the algae aggregated to various degrees in the cytoplasm of infected Ysa2 (Fig. 1). In these cells, algae tended to locate in the posterior half of the cell. The ratio of cells containing algal clusters ranged from 40 to 60% in the algae-bearing paramecia. These clusters have never been found in infected Sj2w (control) cells or green paramecia. Karakashian (1963) reported that when algae isolated from a hypotrichous ciliate were ingested by white P. bursaria, they were frequently confined to vacuoles of the host that were much larger than the food vacuoles. Similar findings were reported by Pringsheim (1928) in infection of P. bursaria with the free-living algae, Stichococcus and Hormidium, and by Rahat and Reich (1984) in infection of aposymbiotic green hydra with free-living algae. These reports suggest there are some incompatibilities between the host and the foreign algae. Present results also indicate that Ysa2 cells are incompatible with infecting algae although they are potentially symbiotic. To determine the fate of the cluster-bearing cells, daily isolation culture was carried out for several days. Successive descendants of each cell line were examined by light-microscopy. During the cultiva-
Acknowledgements. The authours express gratitude to Ms. Takako Araki and Dr. Kazuyuki Mikami, Miyagi University of Education, for their kind permission to use stocks of Ysa2. We also thank Dr. Isoji Miwa, Ibaraki University, for other stocks of P. bursaria.
References Brown J. A. and Nielsen P. J. (1974): Transfer of photosynthetically produced carbohydrate from endosymbiotic chlorellae to Paramecium bursaria. J. Protozool. 21, 569–570. Dryl S. (1959): Antigenic transformation in Paramecium aurelia after homologous antiserum treatment during autogamy and conjugation. J. Protozool. 6(suppl.), 25. Hirshon J. B. (1969): The response of Paramecium bursaria to potential endocellular symbionts. Biol. Bull. 136, 33–42. Hiwatashi K. (1968): Determination and inheritance of mating type in Paramecium caudatum. Genetics. 58, 373–386. Hosoya H., Kimura K., Matsuda S., Kitaura M., Takahashi T. and Kosaka T. (1995): Symbiotic algae-free strains of the green paramecium Paramecium bursaria produced by herbicide Paraquat. Zool. Sci. 12, 807–810. Karakashian M. W. and Karakashian S. J. (1973): Intracellular digestion and symbiosis in Paramecium bursaria. Exp. Cell Res. 81, 111–119. Karakashian S. J. (1963): Growth of Paramecium bursaria as influenced by the presence of algal symbionts. Physiol. Zool. 36, 52–68. Karakashian S. J. and Rudzinska M. A. (1981): Inhibition of lysosomal fusion with symbiont-containing vacuoles in Paramecium bursaria. Exp. Cell Res. 131, 387–393.
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McAuley P. J. (1993): Interactions between hosts and symbionts in algal invertebrate intracellular symbioses. Bot. J. Scotl. 47, 97–112. Meier R. and Wiessner W. (1988): Infection of algae-free Paramecium bursaria with symbiotic Chlorella sp. isolated from green paramecia. I. Effect of the incubation period. Europ. J. Protist. 24, 69–74. Meier R. and Wiessner W. (1989): Infection of algae-free Paramecium bursaria with symbiotic Chlorella sp. isolated from green paramecia. II. A timed study. J. Cell Sci. 43, 571–579. Nishihara N., Horiike S., Takahashi, T., Kosaka, T., Shigenaka, Y. and Hosoya H. (1998): Cloning and characterization of endosymbiotic algae isolated from Paramecium bursaria. Protoplasma 203, 91–99. Pringsheim E. G. (1928): Physiologische Untersuchungen an Paramecium bursaria. Ein Beitrag zur Symbioseforschung. Arch. Protist. 64, 289–418. Rahat M. and Reich V. (1984): Intracellular infection of aposymbiotic Hydra viridis by a foreign free-living Chlorella sp.: initiation of a stable symbiosis. J. Cell Sci. 65, 265–277. Reisser W. (1976): Die stoffwechselphysiologischen Beziehungen zwischen Paramecium bursaria Ehrbg. und Chlorella spec. in der Paramecium bursaria-Symbiose. II. Symbiose-spezifische Markmale der Stoffwechselphysiologie und der Cytologie des Symbioseverbandes und ihre Regulation. Arch. Microbiol. 111, 161–170. Reisser W., Meier R., Goertz H-D. and Jeon K. (1985): Establishment, maintenance and integration mechanisms of endosymbionts in protozoa. J. Protozool. 32, 383–390. Schussler A. and Schnepf E. (1992): Photosynthesis dependent acidification of perialgal vacuoles in the Paramecium bursaria/Chlorella symbiosis: visualization by monensin. Protoplasma. 166, 218–222.
Siegel R. W. (1960): Hereditary endosymbiosis in Paramecium bursaria. Exp. Cell Res. 19, 239–252. Sonneborn T. M. (1950): Methods in the general biology and genetics of Paramecium aurelia. J. Exp. Zool. 113, 87–148. Takeda H., Sekiguchi T., Nunokawa S. and Usuki I. (1998): Species-specificity of Chlorella for establishment of symbiotic association with Paramecium bursaria. – Does infectivity depend upon sugar components of cell wall? Europ. J. Protistol. 34, 133–137. Tanaka M. and Miwa I. (1996): Significance of Photosynthetic products of symbiotic Chlorella to establish the endosymbiosis and to express the mating reactivity rhythm in Paramecium bursaria. Zool. Sci. 13, 685–692. Weis D. S. (1969): Regulation of host and symbiont population size in Paramecium bursaria. Experientia 25, 664–666. Weis D. S. (1978): Correlation of infectivity and Concanavalin A agglutinability of algae exsymbiotic from Paramecium bursaria. J. Protozool. 25, 366–370. Weis D. S. (1979): Correlation of sugar release and Concanavain A agglutinability with infectivity of symbiotic algae from Paramecium bursaria of aposymbiotic P. bursaria. J. Protozool. 26, 117–119. Weis D. S. and Ayara A. (1979): Effect of exposure period and algal concentration on the frequency of infection of aposymbiotic ciliates by symbiotic algae from Paramecium bursaria. J. Protozool. 26, 245–248. Wichterman R. (1948): The biological effects of X-rays on mating types and conjugation of Paramecium bursaria. Biol. Bull. 94, 113–127. Wichterman R. (1953): The biology of Paramecium. Blakiston and McGraw-Hill, New York. Yanagi A. (1992): Behavior and DNA synthesis of the nuclei produced after meiosis in Paramecium caudatum. Europ. J. Protistol. 28, 37–42
A natural strain of Paramecium bursaria lacking symbiotic algae Yuki Tonooka and Tsuyoshi Watanabe* Biological Institute, Graduate school of Science, Tohoku University, Aramaki-aza Aoba, Aoba-ku, Sendai 980-8578, Japan; e-mail: [email protected] Received: 11 July 2001; 14 January 2002. Accepted: 16 January 2002
Paramecium bursaria contains hundreds of algae in its cytoplasm as symbionts and retains them over many generations. Recently, aposymbiotic cells of P. bursaria were collected from a pond. To know whether the natural aposymbiotic strain (Ysa2) lost ability to associate with algae, infection experiments were performed using symbiotic algae isolated from green paramecia. Results showed that Ysa2 establish symbiotic association in the same way as artificially obtained aposymbiotic cells (control). However, clustered algae appeared in some Ysa2 cells after infection. Algal clusters differ in number and size from cell to cell. Isolation line culture was performed to pursue the fate of cells having algal clusters. Within several cell divisions, some descendants retained large clusters and such cells were sometimes misshapen, ceasing proliferation and eventually dying. At cell division of cluster-bearing cells, unequal distribution of clustered algae occurred because many algal clusters tend to locate in the posterior of the host cell. Most extremely, all algae were distributed to the posterior daughter cell, whereas the anterior daughter cell contained no algae. This is one possible mechanism of production of Ysa2 in nature. From these results, we conclude that Ysa2 can make symbiotic association with potentially symbiotic algae, but they have some incompatibilities with the algae. Key words: Paramecium bursaria; symbiosis; infection; algae.
Introduction The green paramecium, Paramecium bursaria, harbours hundreds of symbiotic algae (Chlorella sp.) in the cytoplasm. It is known that mutual benefits are exchanged between the host cell and symbiotic algae as described by many investigators (Karakashian 1963; Weis 1969; Karakashian and Karakashian 1973; Brown and Nielsen 1974; Karakashian and Rudzinska 1981; Schussler and Schnepf 1992). Algae are retained through cell divisions and sexual reproduction of paramecia (Siegel 1960). Thus, the symbiotic relationship in P. bursaria seems to continue permanently. *corresponding author
On the other hand, it is also known that both paramecia and algae can be grown separately. Symbiotic algae isolated from paramecia can be cultured under appropriate conditions (Siegel 1960; Weis 1978; Nishihara et al. 1998; Takeda et al. 1998). Algae-free paramecia are obtained by various methods, such as long-term cultivation in darkness (Siegel 1960), X-ray irradiation (Wichterman 1948), and chemical treatment (Reisser 1976; Hosoya et al. 1995). However, spontaneous production of aposymbiotic paramecia is unlikely to occur in the field since regulatory conditions, which paramecia scarcely face in the field, are necessary to produce artificial aposymbiotic cells. It has been shown by 0932-4739/02/38/01-55 $ 15.00/0
56
Y. Tonooka and T. Watanabe
many investigators that artificially obtained aposymbiotic paramecia can re-establish a symbiotic relationship with isolated algae which are taken into the cytoplasm through the cytostome (Karakashian and Karakashian 1973; Weis and Ayara 1979; Reisser et al. 1985; Meier and Wiessner 1988). Recently, aposymbiotic cells of P. bursaria were unexpectedly collected together with normal green cells from a pond. Because of the stability of the symbiotic relationship, it is very rare for aposymbiotic P. bursaria to be collected from the field. To clarify whether a natural aposymbiotic cell can establish a symbiotic relationship with algae derived from green paramecia or whether they show any defects in keeping infected symbiotic algae, infection experiments were performed using natural aposymbiotic cells.
Results and discussion Ysa2 is an aposymbiotic strain of P. bursaria First, we examined whether Ysa2 is really a white strain of P. bursaria or not. All characteristics, such as cell shape, size, macro- and micronuclear features, and swimming tracks are identical with those of P. bursaria (Wichterman 1953). Moreover, Ysa2 mate with cells of mating type I, II, and IV in P. bursaria syngen 1 but not with mating type III cells. From these facts, we concluded that Ysa2 is a natural aposymbiotic strain of mating type III, in syngen 1 of P. bursaria.
Materials and methods Cells and culture Some symbiotic (green) and aposymbiotic (white) strains of Paramecium bursaria, syngen 1, were used in this study. Ysa2 is a natural aposymbiotic clone collected from a pond in Sagae City, Yamagata, Japan. An artificially produced white strain Sj2w was also used as a control for infection experiments. Green strains, Gr3 and T316, were used as a source of symbiotic algae for infection. Cells were cultured in Hiwatashi’s lettuce juice medium (Hiwatashi 1968) inoculated with Klebsiella pneumoniae, at 24 °C, under continuous light (17.6 µeinstein m–2s–1).
Infection experiments Symbiotic algae were isolated from green paramecia by a modified McAuley's method (McAuley 1993). Algae were finally suspended in the K-DS, a modification of Dryl's solution (Dryl 1959), in which sodium dihydrogen phosphate was replaced with potassium dihydrogen phosphate (Yanagi 1992). Approximately one thousand white paramecia were exposed to 2 × 106 algal cells in 1 ml of total volume and incubated for 5 hours (Weis and Ayara 1979). Then paramecia were washed well in K-DS to remove external algae and transferred to a test tube containing fresh culture medium. Paramecia were cultured by daily addition of 1–2 ml of fresh culture medium for 40 days. A small number of cells were removed from the culture and examined with a fluorescence microscope to determine whether they retained symbiotic algae. In some infection experiments, daily isolation-line cultures (Sonneborn 1950) were established to determine the fate of infected paramecia and algae.
Fig. 1. Cells of infected Ysa2, a natural aposymbiotic strain of P. bursaria. Clusters of algae of different size and number are seen in these cells (a, b). Algal clusters tend to accumulate in the posterior part of the cell (c). At cell division, extremely unequal distribution of algal clusters causes formation of an aposymbiotic daughter cell (d). Magnification: a–c, ×340; d, ×250.
Natural aposymbiotic strain of P. bursaria
Infection experiments During 5-hour exposure to algal suspension, all white cells ingested many algae. Thereafter, the proportion of cells retaining algae in the cytoplasm (retention rate) was counted every 4 days up to 40 days. The retention rate decreased in both Sj2w (control) and Ysa2 cells until 8 days; thereafter it settled until the end of the experiment. The final Ysa2 retention rate was somewhat lower (40-70%) than that of Sj2w (60-80%). Infection rates have been shown to be affected by physiological conditions of both algae and paramecia or by specificity between them (Siegel 1960; Hirshon 1969; Karakashian and Karakashian 1973; Weis 1978, 1979; Weis and Ayara 1979; Meier and Wiessner 1988, 1989; Tanaka and Miwa 1996; Nishihara et al. 1998). Although the retention rate was slightly lower than Sj2w, many Ysa2 cells retained algae for 40 days through many cell divisions. Therefore, it is concluded that Ysa2 can establish a symbiotic relationship with algae isolated from green paramecia.
57
tion period, some descendants produced normal cells with dispersed algae and others showed various types of abnormality. Misshapen cells due to the presence of a large cluster were found frequently. Such clusters sometimes contained approximately 1,000 algae. At cell division of the misshapen cells, unequal cluster distribution was often observed. In the most excessive case, that type of cell division produced one white cell with no algae and one cell heavily loaded with algae (Fig. 1d). Some posterior daughter cells containing large clusters occasionally ceased cell growth and eventually died, while white anterior daughter cells thus produced were able to grow in the same way as the original Ysa2 cells. This suggests that this type of cell division may be one mechanism for producing natural aposymbiotic Ysa2 cells. To our knowledge, this is the first report of aposymbiotic cells being obtained from the field. This strain may be useful for molecular analysis in establishing an understanding of symbiosis in P. bursaria.
Cluster formation of algae after infection A couple of days after commencement of infection experiments, clustering algae were observed in some Ysa2 cells. Many, but not all, of the algae aggregated to various degrees in the cytoplasm of infected Ysa2 (Fig. 1). In these cells, algae tended to locate in the posterior half of the cell. The ratio of cells containing algal clusters ranged from 40 to 60% in the algae-bearing paramecia. These clusters have never been found in infected Sj2w (control) cells or green paramecia. Karakashian (1963) reported that when algae isolated from a hypotrichous ciliate were ingested by white P. bursaria, they were frequently confined to vacuoles of the host that were much larger than the food vacuoles. Similar findings were reported by Pringsheim (1928) in infection of P. bursaria with the free-living algae, Stichococcus and Hormidium, and by Rahat and Reich (1984) in infection of aposymbiotic green hydra with free-living algae. These reports suggest there are some incompatibilities between the host and the foreign algae. Present results also indicate that Ysa2 cells are incompatible with infecting algae although they are potentially symbiotic. To determine the fate of the cluster-bearing cells, daily isolation culture was carried out for several days. Successive descendants of each cell line were examined by light-microscopy. During the cultiva-
Acknowledgements. The authours express gratitude to Ms. Takako Araki and Dr. Kazuyuki Mikami, Miyagi University of Education, for their kind permission to use stocks of Ysa2. We also thank Dr. Isoji Miwa, Ibaraki University, for other stocks of P. bursaria.
References Brown J. A. and Nielsen P. J. (1974): Transfer of photosynthetically produced carbohydrate from endosymbiotic chlorellae to Paramecium bursaria. J. Protozool. 21, 569–570. Dryl S. (1959): Antigenic transformation in Paramecium aurelia after homologous antiserum treatment during autogamy and conjugation. J. Protozool. 6(suppl.), 25. Hirshon J. B. (1969): The response of Paramecium bursaria to potential endocellular symbionts. Biol. Bull. 136, 33–42. Hiwatashi K. (1968): Determination and inheritance of mating type in Paramecium caudatum. Genetics. 58, 373–386. Hosoya H., Kimura K., Matsuda S., Kitaura M., Takahashi T. and Kosaka T. (1995): Symbiotic algae-free strains of the green paramecium Paramecium bursaria produced by herbicide Paraquat. Zool. Sci. 12, 807–810. Karakashian M. W. and Karakashian S. J. (1973): Intracellular digestion and symbiosis in Paramecium bursaria. Exp. Cell Res. 81, 111–119. Karakashian S. J. (1963): Growth of Paramecium bursaria as influenced by the presence of algal symbionts. Physiol. Zool. 36, 52–68. Karakashian S. J. and Rudzinska M. A. (1981): Inhibition of lysosomal fusion with symbiont-containing vacuoles in Paramecium bursaria. Exp. Cell Res. 131, 387–393.
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McAuley P. J. (1993): Interactions between hosts and symbionts in algal invertebrate intracellular symbioses. Bot. J. Scotl. 47, 97–112. Meier R. and Wiessner W. (1988): Infection of algae-free Paramecium bursaria with symbiotic Chlorella sp. isolated from green paramecia. I. Effect of the incubation period. Europ. J. Protist. 24, 69–74. Meier R. and Wiessner W. (1989): Infection of algae-free Paramecium bursaria with symbiotic Chlorella sp. isolated from green paramecia. II. A timed study. J. Cell Sci. 43, 571–579. Nishihara N., Horiike S., Takahashi, T., Kosaka, T., Shigenaka, Y. and Hosoya H. (1998): Cloning and characterization of endosymbiotic algae isolated from Paramecium bursaria. Protoplasma 203, 91–99. Pringsheim E. G. (1928): Physiologische Untersuchungen an Paramecium bursaria. Ein Beitrag zur Symbioseforschung. Arch. Protist. 64, 289–418. Rahat M. and Reich V. (1984): Intracellular infection of aposymbiotic Hydra viridis by a foreign free-living Chlorella sp.: initiation of a stable symbiosis. J. Cell Sci. 65, 265–277. Reisser W. (1976): Die stoffwechselphysiologischen Beziehungen zwischen Paramecium bursaria Ehrbg. und Chlorella spec. in der Paramecium bursaria-Symbiose. II. Symbiose-spezifische Markmale der Stoffwechselphysiologie und der Cytologie des Symbioseverbandes und ihre Regulation. Arch. Microbiol. 111, 161–170. Reisser W., Meier R., Goertz H-D. and Jeon K. (1985): Establishment, maintenance and integration mechanisms of endosymbionts in protozoa. J. Protozool. 32, 383–390. Schussler A. and Schnepf E. (1992): Photosynthesis dependent acidification of perialgal vacuoles in the Paramecium bursaria/Chlorella symbiosis: visualization by monensin. Protoplasma. 166, 218–222.
Siegel R. W. (1960): Hereditary endosymbiosis in Paramecium bursaria. Exp. Cell Res. 19, 239–252. Sonneborn T. M. (1950): Methods in the general biology and genetics of Paramecium aurelia. J. Exp. Zool. 113, 87–148. Takeda H., Sekiguchi T., Nunokawa S. and Usuki I. (1998): Species-specificity of Chlorella for establishment of symbiotic association with Paramecium bursaria. – Does infectivity depend upon sugar components of cell wall? Europ. J. Protistol. 34, 133–137. Tanaka M. and Miwa I. (1996): Significance of Photosynthetic products of symbiotic Chlorella to establish the endosymbiosis and to express the mating reactivity rhythm in Paramecium bursaria. Zool. Sci. 13, 685–692. Weis D. S. (1969): Regulation of host and symbiont population size in Paramecium bursaria. Experientia 25, 664–666. Weis D. S. (1978): Correlation of infectivity and Concanavalin A agglutinability of algae exsymbiotic from Paramecium bursaria. J. Protozool. 25, 366–370. Weis D. S. (1979): Correlation of sugar release and Concanavain A agglutinability with infectivity of symbiotic algae from Paramecium bursaria of aposymbiotic P. bursaria. J. Protozool. 26, 117–119. Weis D. S. and Ayara A. (1979): Effect of exposure period and algal concentration on the frequency of infection of aposymbiotic ciliates by symbiotic algae from Paramecium bursaria. J. Protozool. 26, 245–248. Wichterman R. (1948): The biological effects of X-rays on mating types and conjugation of Paramecium bursaria. Biol. Bull. 94, 113–127. Wichterman R. (1953): The biology of Paramecium. Blakiston and McGraw-Hill, New York. Yanagi A. (1992): Behavior and DNA synthesis of the nuclei produced after meiosis in Paramecium caudatum. Europ. J. Protistol. 28, 37–42