J. Cell Sri. 58, 445-453 (1982) 445 Printed in Great Britain © Company of Biologists Limited 1982

INFECTIONS OF BURS ARIA WITH 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 -free and are transmitted at various conditions as are symbiotic chlorellae. Like chlorellae, the bacteria and the yeast cells are situated in perisymbiont . 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 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 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 . 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 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, . 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 . 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. We then tried experiments to bring about double infections with different symbiont species, since some cells of strain b 103 had originally contained both Chlorella and bacteria.

Table 1. Experimental infection of symbiont-bearing and aposymbiont P. bursaria with Chlorella, bacteria and yeasts

Infection tested with

Acceptors Chlorella Yeasts Bacteria Chlorella-bearing P. bursaria o — — Yeast-bearing P. bursaria + o — Bacteria-bearing P. bursaria + — o Aposymbiotic P. bursaria + + + + , successfully infected; —, no infection observed; o, not tested.

The results of the infection experiments are summarized in Table 1. Bacteria- bearing cells could be infected with Chlorella from both donor strains and after 3 days bacteria could no longer be detected in these cells that now contained Chlorella. Yeast-bearing cells could also be infected with Chlorella. After 3 days in most of the paramecia less than 10 yeast cells remained and after 5 days all yeast cells had dis- appeared. Neither Chlorella-bearing nor bacteria-bearing paramecia could be infected with yeast. Yeast-bearing paramecia could not be infected with bacteria. Also, Chlorella-bearing paramecia could not be infected with bacteria in the light. How- ever, when grown in the dark an infection of Chlorella-bearing paramecia with bacteria was successful in almost 10% of the cells. In these cells the bacteria then appeared in groups of more than 10 and some bacteria always appeared closely associated with chlorellae, and were perhaps situated within the perialgal vacuoles. When cultured in the light again the bacteria were lost while the chlorellae remained.

DISCUSSION The bacteria and yeasts described in this study are obviously capable of inducing aposymbiotic P. bursaria to form perisymbiont vacuoles when taken up into food vacuoles. It is known from infection experiments with symbiotic chlorellae that surface sugars of the algae are important for the recognition by P. bursaria: Weis (1978, 1979) has shown that the concanavalin A (Con A) agglutinability of infective Bacteria and yeasts in P. bursaria 451 strains is different from that of non-infective strains of Chlorella. Infective chlorellae are digested after treatment with cellulase or pectinase (Reisser, 1981) and after being coated with antibodies or Con A (Weis, 1978, 1980; Reisser, 1981). It must be assumed that the bacteria and yeasts described in this study have certain surface factors similar to those of symbiotic chlorellae. Maltose released by infective chlorellae also seems to act as a recognition signal and as a signal inducing the recognition capability cf aposymbiotic P. bursaria (Weis, 1979, 1980). However, free-living Chlorella species do not release maltose (Muscatine et al. 1967), but are still infective (S. J. Karakashian, 1963; Bomford, 1965; Hirshon, 1969). So maltose may be favourable but not really necessary for the infection process and the capability of releasing maltose does not need to be postulated for the infective yeast or bacteria. It must be assumed that the recognition of potential symbionts by P. bursaria is not based on factors limited to Chlorella: the infections with two different yeasts and a bacterium described in this study, and the infections with a yeast and a Scenedesmus described by Bomford (1965), show that a variety of organisms may be recognized as potential symbionts, no matter whether they are retained or not. The establishment of a symbiont in the host is certainly a further step. Bacteria and yeasts occupy the same regions of the host cells as the symbiotic alga'e and are maintained in the light and the dark. They inhibit a secondary infection with yeast or bacteria, respectively. This shows that the association is stable and must be regarded as symbiosis, even though it is not necessarily mutually beneficial. However, - the loss of bacteria and yeasts after secondary infections with symbiotic chlorellae shows that the association with the algae is closer. How the bacteria or yeasts are lost is unknown. The loss of yeasts may be simply due to some kind of between yeasts and chlorellae for food substrates provided by the host. Some substrates necessary for the symbionts may be limiting and the algae of course have the advantage of autotrophic growth. The result may be that the yeasts starve and die out, while the algae may even multiply. Accordingly, three days after an infection of yeast-bearing paramecia with chlorellae only a few yeasts are left. The loss of bacteria after an infection with Chlorella is more rapid than the loss of yeasts. Three days after a secondary infection with chlorellae, that is after a maximum of six divisions of the host cell, all of the 1000-2000 bacteria have disappeared. After staining with orcein even one bacterium in the cytoplasm of a paramecium can be detected. So, the bacteria are either digested or excreted. Membranes of perialgal vacuoles containing chlorellae differ from food vacuole membranes (Meier, Reisser, Wiessner & Lefort-Tran, 1980). Perialgal vacuoles containing chlorellae do not fuse with lysosomes (S. J. Karakashian & Rudzinska, 1981). It might be suggested that the membranes of the perisymbiont vacuoles containing bacteria differ from the membranes of perialgal vacuoles, perhaps permitting limited fusion with lysosomes. Then the rapid loss of bacteria after a secondary infection with chlorellae would be the result of digestion. Symbiotic bacteria and jieasts appeared intact in light- and electron microscopic observations. This suggests that the symbionts are not digested. How- ever, to detect a possible digestion of bacteria or yeasts cytochemical methods will be necessary. 452 H.-D. Gortz Also, an antibacterial effect of the symbiotic algae might be possible. Graf & Baier (1981) have observed a strong antibacterial effect of the free-living algae Hydradictyon reticulatum and Aphanothece nidulans. The antibacterial action is linked with the assimilation activity of the algal cultures. Chlorella vulgaris, however, does not show a bacterial growth-inhibiting effect. It would now be interesting to look for other microorganisms capable of infecting P. bursaria. This could help to dissect the infection process further and to discover the nature of the recognition signals. Moreover, using auxotrophic mutants of bacteria or yeasts may help in identifying the substrates provided by the host cells for the symbionts.

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{Received 26 May 1982)