J. Cell Sri. 58, 445-453 (1982) 445 Printed in Great Britain © Company of Biologists Limited 1982 INFECTIONS OF PARAMECIUM BURS ARIA WITH BACTERIA AND YEASTS HANS-DIETER GORTZ Zoological Institute, University of MUnster, Badestr. 9, D-4400 MOnster, F.R.G. SUMMARY Infections of Paramecium bursaria with bacteria and yeasts are reported. Bacteria and yeasts multiply in the algae-free ciliate and are transmitted at various conditions as are symbiotic chlorellae. Like chlorellae, the bacteria and the yeast cells are situated in perisymbiont vacuoles. Both bacteria and yeasts maintain their capability for independent existence and can be grown on standard nutrient agar. Infection experiments show that aposymbiotic P. bursaria can be infected with Chlorella, bacteria and yeast. Chlorella-bearing P. bursaria cannot be infected with bacteria or yeast. Bacteria-bearing paramecia can be infected with Chlorella but not with yeast. Yeast-bearing paramecia can be infected with Chlorella but not with bacteria. Following infections with Chlorella the paramecia lose their bacteria or yeast symbionts. The bacteria found in P. bursaria probably belong to the genus Pseudomonas; the yeast has been identified as Rodutorula rubra. INTRODUCTION The symbiosis of Paramecium bursaria and Chlorella is known to be mutually beneficial (M. W. Karakashian, 1975). Paramecia bearing Chlorella are better adapted to suboptimal conditions with little food (Pringsheim, 1928; S. J. Karakashian, 1963), and the ciliates are supported by carbohydrates, chiefly maltose, released by the algae (Muscatine, Karakashian & Karakashian, 1967; Brown & Nielsen, 1974). Chlorella gains motility, and the capability of Chlorella-bearing paramecia for photo-accumulation (Iwatsuki & Naitoh, 1981; Niess, Reisser & Wiessner, 1981; Engelmann, 1882) shows the advantage of symbiotic algae over free-living chlorellae that are immotile. To my knowledge, Chlorella-lree P. bursaria have not been reported from natural environ- ments. Chlorella-bearing paramecia can be freed from the symbiotic algae by various methods (S. J. Karakashian, 1963; Reisser, 1976; see also Tsukii & Hiwatashi, cited by Iwatsuki & Naitoh, 1981). The symbiont-free cells are then called aposymbionts. Aposymbionts can be reinfected with Chlorella taken from homogenized Chlorella- bearing paramecia or with Chlorella that have been cultured outside P. bursaria (Pringsheim, 1928; S. J. Karakashian, 1963). Aposymbiotic P. bursaria can also be infected with various free-living Chlorella species (Bomford, 1965; Karakashian & Karakashian, 1965; Hirshon, 1969) and even infections with algae of other genera, and even yeasts, have been successful (Oehler, 1922; Bomford, 1965). Paramecium aurelia often bears bacterial endosymbionts (Preer, Preer & Jurand, 1974). In the cytoplasm of P. bursaria, however, bacteria have not been observed, to 446 H.-D. Gortz my knowledge. In this paper the occurrence of bacteria and yeast in the cytoplasm of P. bursaria is reported. It has been possible to cultivate the symbiotic bacteriaand yeasts on nutrient agar. The results of infection experiments with Chlorella, bacteria and yeasts show a kind of dominancy of Chlorella over bacteria and yeasts. The first observations of the infection of P. bursaria with bacteria and yeast were presented in a short abstract (Gortz & Dieckmann, 1982). MATERIALS AND METHODS Strain b 103 of P. bursaria used in this study was isolated from a pond in Munster (Germany) by J. Dieckmann, who first observed additional bacteria in the cytoplasm of some cells of this strain. For infection experiments chlorellae of a second strain, So 11 G, were also used. This strain was kindly supplied by Dr M. Fujishima, Yamaguchi, Japan. Cells were cultured either in cerophyl medium (GSrtz & Dieckmann, 1980) or in sterile earth solution with Chlorogonium elongatum as the prey organism. The cultures were kept at 20-22 °C in the light (about 2000 lux) with 8 h in the dark if not otherwise noted. Earth solution was prepared as described by Ruthmann & Heckmann (1961) and autoclaved. Chlorogomum was grown axenically (Ammer- mann, Steinbruck, von Berger & Hennig, 1974), centrifuged and added to the sterile earth solution in a clean bench. This was necessary to avoid contaminations causing infections of the aposymbionts. Infection experiments were done in the wells of depression slides or test tubes. Homogenates of paramecia-bearing chlorellae, bacteria or yeast were added to the cultures of P. bursaria (about 200 paramecia/ml) for infection. The densities of organisms per ml used for infections were about 5 x 10* chlorellae, io8 bacteria, and 2 x io7 yeasts. The cells were washed after 2 days and transferred into fresh culture medium supplemented with Chlorogomum. Infection experiments were also done with bacteria and yeast isolated from paramecium and grown on nutrient agar (Standard-I-Nahragar, Merck, Darmstadt). For light microscopy paramecia were fixed with OsO4 vapour and stained with lacto orcein (Beale & Jurand, 1966). For electron microscopy cells were fixed with 1-5 % glutaraldehyde in 50 mM-Na phosphate buffer (pH 7-2) and postfixed in 1 % OsO,. Samples were embedded in Epon 812. Sections were stained with uranyl acetate and lead citrate and examined with a Siemens-Elmiskop 101 at 60 kV. Bacteria were negatively stained with KOH/phosphotungstic acid (1 %). RESULTS In a Chlorella-bearing strain of P. bursaria some cells were observed with additional bacteria in the cytoplasm. After rapid growth in the dark some paramecia lost the algae but retained the bacteria (Fig. 2). In the original culture grown in the light the para- mecia retained the symbiotic algae, and bacteria have not been observed any more in the cytoplasm of these cells, in later investigations. Cultures of the paramecia now bearing bacteria (1000 to 2000 per cell) were treated with antibiotics. Penicillin (penicillin G.K-salt, Serva) did not kill the bacteria even at 1000 units per ml. Kanamycin (Serva) or streptomycin (Serva) both freed the Figs. 1-3. P. bursaria with different symbionts. OsO4 vapour, lacto orcein. mi, micro- nucleus ; ma, macronucleus. 2000 x . Fig. 1. P. bursaria bearing Chlorella (arrows). Fig. 2. P. bursaria bearing bacteria (arrows). Fig. 3. P. bursaria bearing yeasts (arrows). Bacteria and yeasts in P. bursaria 447 ^—^ -•» — - II 448 H.-D. Gortz ciliates from bacteria when added at a final concentration of 50 /ig/ml for 1 day followed by daily doubling of the cultures with freshly bacteria-treated medium for the next 4 days. The cells had then become aposymbiotic. One culture of these apo- symbiotic paramecia accidentally became infected with a contaminating yeast. The yeast cells, like the bacteria, multiplied in the paramecia and budding stages were observed regularly (Fig. 3). The number of yeast cells per paramecium was 50-200. Another aposymbiotic line was accidentally infected by a different yeast. No further experiments were done with this line. Like the algal symbionts the bacteria and yeasts are chiefly situated around the outer regions of the host cell. Electron microscopic observations showed that bacteria as well as yeasts are harboured in perisymbiotic vacuoles (Figs. 4, 5, 6) as has been described for Chlorella (Karakashian, Karakashian & Rudzinska, 1968). Chlorellae are always situated singly in the vacuoles (Karakashian et al. 1968). This is often also observed with bacteria or yeast. Sometimes, however, two to three yeast cells and up to 10 bacteria are found in one vacuole. The bacteria are monopolarly flagellated, with one or two flagella (Fig. 7). At optimal conditions with excess of food at 20-22 °C the fission rates of the four lines of aposymbiotic, Chlorella-bearing, bacteria-bearing and yeast-bearing cells did not differ significantly in the dark or the light. At suboptimal conditions with little food Chlorella-bearing cells had a higher fission rate than the other three lines. In these experiments the CA/oro^om'ttm-containing culture medium was diluted with sterile earth medium (1:2o) and 45 cells of each line were cultured singly in daily isolation lines at 20 °C in depression slides. The fission rates were determined accord- ing to Sonneborn (1950). The mean fission rates over 10 days were 1-12 fissions/day (Chlorella-bearing cells), o-6o fissions/day (bacteria-bearing cells), 0-89 fissions/day (yeast-bearing cells), and 0-73 fissions/day (aposymbiotic cells). In another experiment equal numbers of cells of the four lines were cultured together in the wells of depres- sion slides with very little food. In these cultures the relative number of Chlorella- bearing cells increased. However, throughout the duration of the experiment (14 days) all four types of cells containing the different symbionts, or being aposymbiotic, were observed in the daily test samples. In starvation experiments with no prey organisms added to the sterile earth medium, all except the Chlorella-bearing cells stopped dividing. When kept in the dark the Chlorella-bearing cells also stopped dividing. Starved cells that had stopped dividing started to divide again when refed 2-3 days after addition of food. In these experiments it was found that aposymbiotic paramecia regularly started to divide about one day earlier than the paramecia bearing any of the symbiont species. The yeast and the Fig. 4. Chlorella in a perialgal vacuole. c, Chlorella. x 24000. Fig. 5. Yeasts in perisymbiont vacuoles. v, yeast, x 24000. Fig. 6. Bacteria in perisymbiont vacuoles. b, bacteria, x 32000. Fig. 7. Isolated symbiotic bacterium negatively stained. Note two flagellae (arrows), x 20000. Bacteria and yeasts in P. bursana w 45° H.-D. Gortz bacterium described in this study were classified by Dr I. Reiff, Microbiological Institute, University of Miinster. The yeast was identified as Rodutorula rubra and the bacterium probably belongs to the genus Pseudomonas. Infection experiments Aposymbionts of strain b 103 could be reinfected with any of the symbionts (chlorellae, bacteria and yeasts) by adding an homogenate of symbiont-bearing cells to the culture medium. It was also possible to reinfect aposymbionts with bacteria or yeast that had been isolated and grown on agar.