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

INFECTIONS OF PARAMECIUM BURS ARIA

WITH BACTERIA AND YEASTS

HANS-DIETER GORTZZoological 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 yeastsmultiply in the algae-free ciliate and are transmitted at various conditions as are symbioticchlorellae. 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 grownon 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-bearingparamecia can be infected with Chlorella but not with bacteria. Following infections withChlorella the paramecia lose their bacteria or yeast symbionts. The bacteria found in P. bursariaprobably 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 mutuallybeneficial (M. W. Karakashian, 1975). Paramecia bearing Chlorella are better adaptedto 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). Chlorellagains motility, and the capability of Chlorella-bearing paramecia for photo-accumulation(Iwatsuki & Naitoh, 1981; Niess, Reisser & Wiessner, 1981; Engelmann, 1882) showsthe advantage of symbiotic algae over free-living chlorellae that are immotile. To myknowledge, Chlorella-lree P. bursaria have not been reported from natural environ-ments.

Chlorella-bearing paramecia can be freed from the symbiotic algae by variousmethods (S. J. Karakashian, 1963; Reisser, 1976; see also Tsukii & Hiwatashi, citedby 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 beinfected 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 ofP. bursaria is reported. It has been possible to cultivate the symbiotic bacteriaand yeastson nutrient agar. The results of infection experiments with Chlorella, bacteria andyeasts show a kind of dominancy of Chlorella over bacteria and yeasts. The firstobservations of the infection of P. bursaria with bacteria and yeast were presented in ashort 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 thisstrain. For infection experiments chlorellae of a second strain, So 11 G, were also used. Thisstrain was kindly supplied by Dr M. Fujishima, Yamaguchi, Japan. Cells were cultured eitherin cerophyl medium (GSrtz & Dieckmann, 1980) or in sterile earth solution with Chlorogoniumelongatum 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 byRuthmann & Heckmann (1961) and autoclaved. Chlorogomum was grown axenically (Ammer-mann, Steinbruck, von Berger & Hennig, 1974), centrifuged and added to the sterile earthsolution in a clean bench. This was necessary to avoid contaminations causing infections of theaposymbionts.

Infection experiments were done in the wells of depression slides or test tubes. Homogenatesof 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 infectionswere about 5 x 10* chlorellae, io8 bacteria, and 2 x io7 yeasts. The cells were washed after2 days and transferred into fresh culture medium supplemented with Chlorogomum. Infectionexperiments were also done with bacteria and yeast isolated from paramecium and grown onnutrient agar (Standard-I-Nahragar, Merck, Darmstadt). For light microscopy parameciawere 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 phosphatebuffer (pH 7-2) and postfixed in 1 % OsO,. Samples were embedded in Epon 812. Sections werestained with uranyl acetate and lead citrate and examined with a Siemens-Elmiskop 101 at60 kV. Bacteria were negatively stained with KOH/phosphotungstic acid (1 %).

RESULTS

In a Chlorella-bearing strain of P. bursaria some cells were observed with additionalbacteria in the cytoplasm. After rapid growth in the dark some paramecia lost the algaebut 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 inthe cytoplasm of these cells, in later investigations.

Cultures of the paramecia now bearing bacteria (1000 to 2000 per cell) were treatedwith antibiotics. Penicillin (penicillin G.K-salt, Serva) did not kill the bacteria evenat 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 dayfollowed by daily doubling of the cultures with freshly bacteria-treated medium forthe next 4 days. The cells had then become aposymbiotic. One culture of these apo-symbiotic paramecia accidentally became infected with a contaminating yeast. Theyeast cells, like the bacteria, multiplied in the paramecia and budding stages wereobserved regularly (Fig. 3). The number of yeast cells per paramecium was 50-200.Another aposymbiotic line was accidentally infected by a different yeast. No furtherexperiments were done with this line.

Like the algal symbionts the bacteria and yeasts are chiefly situated around theouter regions of the host cell. Electron microscopic observations showed that bacteriaas well as yeasts are harboured in perisymbiotic vacuoles (Figs. 4, 5, 6) as has beendescribed for Chlorella (Karakashian, Karakashian & Rudzinska, 1968). Chlorellae arealways situated singly in the vacuoles (Karakashian et al. 1968). This is often alsoobserved with bacteria or yeast. Sometimes, however, two to three yeast cells and up to10 bacteria are found in one vacuole. The bacteria are monopolarly flagellated, withone or two flagella (Fig. 7).

At optimal conditions with excess of food at 20-22 °C the fission rates of the fourlines of aposymbiotic, Chlorella-bearing, bacteria-bearing and yeast-bearing cells didnot differ significantly in the dark or the light. At suboptimal conditions with littlefood Chlorella-bearing cells had a higher fission rate than the other three lines. Inthese experiments the CA/oro^om'ttm-containing culture medium was diluted withsterile earth medium (1:2o) and 45 cells of each line were cultured singly in dailyisolation 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 experimentequal 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, wereobserved 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 theChlorella-bearing cells also stopped dividing. Starved cells that had stopped dividingstarted to divide again when refed 2-3 days after addition of food. In these experimentsit was found that aposymbiotic paramecia regularly started to divide about one dayearlier 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, MicrobiologicalInstitute, University of Miinster. The yeast was identified as Rodutorula rubra andthe 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 cellsto the culture medium. It was also possible to reinfect aposymbionts with bacteriaor yeast that had been isolated and grown on agar. We then tried experiments to bringabout double infections with different symbiont species, since some cells of strainb 103 had originally contained both Chlorella and bacteria.

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

Infection tested with

Acceptors Chlorella Yeasts Bacteria

Chlorella-bearing P. bursaria o — —Yeast-bearing P. bursaria + o —Bacteria-bearing P. bursaria + — oAposymbiotic 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 daysbacteria 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 theparamecia 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 infectedwith 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 bacteriawas successful in almost 10% of the cells. In these cells the bacteria then appeared ingroups of more than 10 and some bacteria always appeared closely associated withchlorellae, and were perhaps situated within the perialgal vacuoles. When cultured inthe light again the bacteria were lost while the chlorellae remained.

DISCUSSION

The bacteria and yeasts described in this study are obviously capable of inducingaposymbiotic P. bursaria to form perisymbiont vacuoles when taken up into foodvacuoles. It is known from infection experiments with symbiotic chlorellae thatsurface 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 chlorellaeare digested after treatment with cellulase or pectinase (Reisser, 1981) and after beingcoated with antibodies or Con A (Weis, 1978, 1980; Reisser, 1981). It must be assumedthat the bacteria and yeasts described in this study have certain surface factors similarto those of symbiotic chlorellae. Maltose released by infective chlorellae also seems toact as a recognition signal and as a signal inducing the recognition capability cfaposymbiotic P. bursaria (Weis, 1979, 1980). However, free-living Chlorella speciesdo 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 reallynecessary for the infection process and the capability of releasing maltose does notneed to be postulated for the infective yeast or bacteria.

It must be assumed that the recognition of potential symbionts by P. bursaria is notbased on factors limited to Chlorella: the infections with two different yeasts and abacterium described in this study, and the infections with a yeast and a Scenedesmusdescribed by Bomford (1965), show that a variety of organisms may be recognized aspotential symbionts, no matter whether they are retained or not. The establishment ofa symbiont in the host is certainly a further step.

Bacteria and yeasts occupy the same regions of the host cells as the symbiotic alga'eand are maintained in the light and the dark. They inhibit a secondary infection withyeast or bacteria, respectively. This shows that the association is stable and must beregarded as symbiosis, even though it is not necessarily mutually beneficial. However,

- the loss of bacteria and yeasts after secondary infections with symbiotic chlorellaeshows that the association with the algae is closer. How the bacteria or yeasts are lostis unknown. The loss of yeasts may be simply due to some kind of competition betweenyeasts and chlorellae for food substrates provided by the host. Some substratesnecessary for the symbionts may be limiting and the algae of course have the advantageof autotrophic growth. The result may be that the yeasts starve and die out, while thealgae may even multiply. Accordingly, three days after an infection of yeast-bearingparamecia with chlorellae only a few yeasts are left.

The loss of bacteria after an infection with Chlorella is more rapid than the loss ofyeasts. Three days after a secondary infection with chlorellae, that is after a maximumof six divisions of the host cell, all of the 1000-2000 bacteria have disappeared. Afterstaining with orcein even one bacterium in the cytoplasm of a paramecium can bedetected. So, the bacteria are either digested or excreted. Membranes of perialgalvacuoles containing chlorellae differ from food vacuole membranes (Meier, Reisser,Wiessner & Lefort-Tran, 1980). Perialgal vacuoles containing chlorellae do not fusewith lysosomes (S. J. Karakashian & Rudzinska, 1981). It might be suggested thatthe membranes of the perisymbiont vacuoles containing bacteria differ from themembranes of perialgal vacuoles, perhaps permitting limited fusion with lysosomes.Then the rapid loss of bacteria after a secondary infection with chlorellae would be theresult of digestion. Symbiotic bacteria and jieasts appeared intact in light- and electronmicroscopic observations. This suggests that the symbionts are not digested. How-ever, to detect a possible digestion of bacteria or yeasts cytochemical methods will benecessary.

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 Hydradictyonreticulatum and Aphanothece nidulans. The antibacterial action is linked with theassimilation activity of the algal cultures. Chlorella vulgaris, however, does not show abacterial growth-inhibiting effect.

It would now be interesting to look for other microorganisms capable of infectingP. bursaria. This could help to dissect the infection process further and to discoverthe nature of the recognition signals. Moreover, using auxotrophic mutants of bacteriaor yeasts may help in identifying the substrates provided by the host cells for thesymbionts.

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{Received 26 May 1982)