Intracellular digestion and symbiosis in Paramecium bursaria

Printed in Sweden Copyright Q 1973 by Academic Press, Inc. All rights of reproducfion in any form resemed

Experimental

INTRACELLULAR

Cell Research 81 (1973) 111-l 19

DIGESTION

IN PARAMECIUM MARLENE

AND

SYMBIOSIS

BURSARIA

W. KARAKASHIAN

and S. J. KARAKASHIAN

Department of Biology, Queens College, Flushing, N. Y. 11367, and Department of Health Sciences, State University of New York, College at Old Westbury, Old Westbury, N. Y. 11568, USA

SUMMARY Electron microscopic cytochemical methods reveal that acid phosphatase activity appears exclusively in vacuoles containing recently ingested bacteria or inert particles such as carmine, Celkate or latex spheres, and not in the vacuoles surrounding established symbionts. Although newly ingested symbiotic algae are digested in large numbers, some remain to reestablish the symbiosis. Since symbiotic algae are able to delay the digestion of heat-killed algae when they coexist in a phagosome, we propose that symbiotic Chlorella actively interfere with an early event in the host digestive process.

Paramecium bursaria normally harbors several hundred symbiotic Chlorella. Previous studies

of the association have shown that it is hereditary [l], that it is of mutual benefit to both partners [2], and that it depends upon specificities resident in both the host and algal strains involved [3]. Electron microscopic investigation [4] revealed that the algae are located in vacuoles dispersed throughout the host cytoplasm. There is a single alga per vacuole except for a brief time

immediately

following

division

of the

algae. At that time the membrane of the original perialgal vacuole constricts and separates the young algae into new discrete vacuoles. In their apparent immunity to digestion by the host cell, the algae and their surrounding perialgal vacuoles resemble organelles. They are not organelles, however, for the algae may be removed from the host cell and cultured separately. Similarly, aposymbiotic S-731805

host cells grow and divide normally when provided ample food. A stable endocellular association is readily reestablished whenever aposymbiotic paramecia ingest large numbers of formerly symbiotic algae. The intracellular digestive system of the host cell must constitute a formidable barrier to the establishment and maintenance of the symbiotic association. The present study was initiated in order to clarify some of the early events in host cell digestion relevant to an eventual understanding of the specificity and stability of the endocellular symbiotic relationship. MATERIALS AND METHODS Cultures, Media A single stock (478A) of Paramecium bursaria. svnrten 1, was used in this- study. The algae used’ were a clone of Chlorella (NC64A) originally isolated from another stock of P. bursaria, syngen 1. Aposymbiotic cultures of paramecia were obtained initially by Exptl Cell Res 81 (1973)

112

Marlene W. Karakashian & S. J. Karakashian

prolonged starvation in darkness. Methods for culturing infected and aposymbiotic paramecia and formerly symbiotic algae were those previously described [2, 31. Cultures were grown at 25°C under diurnal illumination (12 h light, 12 h darkness) provided by banks of cool white fluorescent tubes (average light intensity 500 foot candles).

For ingestion experiments with algae, groups of 15-20 washed paramecia were placed in 0.3 ml aliquots of a suspension of algae in mPS solution. Cells were exposed to algae for 24 min, after which they were washed and set aside for later observations of the fate of the ingested algae. Intracellular algal numbers were determined by counting the algae visible in squashed paramecia.

Ingestion studies

Electron microscopy, cytochemistry

An effort was made to bring the aposymbiotic paramecia into as uniform a nutritional condition as possible by allowing cultures to starve slightly in exhausted lettuce infusion for several days. Then just prior to an experiment an equal volume of fresh bacterized lettuce infusion was added. Four to 6 h later, individual paramecia were washed by repeated transfers through fresh aliquots of sterile salt solution (mPS) with a micropipette as described previously [2] or paramecia were washed in bulk in the same salt solution in a low speed zonal rotor [5]. Washed cells were left in the salt solution overnight and then washed once more before use in an experiment. Uniform latex particles (1.099 ,um in diameter) were obtained from Dow Chemical Company. Clay particles (Celkate) were obtained from Johns Mansville Corporation. Both Celkate and carmine were swirled vigorously in the mPS salt solution in a vortex mixer and the heavier particles were allowed to settle out before use. Paramecia were allowed to ingest the particles for 30 min. They were then washed and fixed 30 min later. C/&r&r were isolated from infected paramecia which had been broken open by rapid expulsion through a hypodermic needle. The algae were washed several times in mPS solution and then diluted with mPS solution to an appropriate optical density measured with a Klett calorimeter using a 440 nm filter. Algal suspensions with optical densities greater than 500 Klett units were always used since even appreciable differences in optical density in this range had no evident effect on the numbers of algae ingested and retained. Congo Red stained algae were prepared by recentrifuging a measured sample of washed algae and resuspending them in 0.5 % Congo Red (in distilled water). The suspension was then placed in a boilingwater bath for 13 min, cooled, and the algae washed until all excess dye was gone. Finally, the stained killed algae were resuspended in their original volume of mPS solution and used either at that density or diluted by half with a suspension of live algae. Stained algae appeared deep red inside of the paramecia.

Cells were fixed at room temperature in 2.3 % glutaraldehyde in 0.67 M cacodylate buffer for 30-40 min, followed by three 15 min washes in fresh buffer containing 5.8 % glucose. Gomori stain for acid nhosohatase was freshlv prepared to the following concentrations: 0.004 M Pb(NO,),: 0.05 M acetate buffer. uH 5.0: 0.01 M Na$-giycerophosphate. The stain was incubated for 1 h at 58”C, cooled to room temperature and filtered prior to use. ,!I-Glycero phosphate was omitted for controls lacking substrate. Sodium fluoride was added to other controls at a concentration of 0.01 M. In one experiment 4.5 % glucose was added to the Gomori stain with no appreciable effect. Paramecia were incubated in the Gomori stain for l-14 h and were then post fixed in 1 % 0~0, in M/35 veronalacetate buffer. DH 7.2-7.4. for 45-60 min. Cells were rapidly dehydrated in a graded series of ethanols, treated with propylene oxide and impregnated with an Epon-Araldite mixture overnight [4]. Sections were cut with diamond knives, mounted on uncoated grids and viewed with a RCA EMU 3F electron microscope. For light microscopy, thick sections of Gomori-stained material were floated on 6 % (NH&S before examination. RESULTS

Relationship between perialgal vacuoles and the host digestive system [4] indicated Paramecium digest their symbionts as long as there is another source of food. Electron microscopic cytochemical investigation (fig. 1 a) of feeding P. bursaria confirm these earlier observations. Acid phosphatase reaction product is visible only in food vacuoles containing recently ingested Previous

studies

bursaria do not normally

Fig. I. Acid phosphatase reaction product can be seen in food vacuole (FV) containing bacteria and in nearby cytoplasmic vesicles. Note closeness of one cytoplasmic vesicle (arrow) to perialgal membrane and lack of enzyme activity in perialgal vacuole (PV). x 15 200; (b) Cell stained for acid phosphatase activity in the presence of NaF. Note great decrease of reaction product in food vacuole (FV) and cytoplasm. x 15 200. (c) Vacuole

containing alga undergoing digestion (DA) and large amounts of acid phosphatase reaction product. Portions of two adjacent perialgal vacuoles (PV) surrounding healthy algae are shown as well as a vacuole containing carmine particles (C), a discarded cell wall fragment (CW) and reaction product. x 19 000.

Digestion and symbiosis in P. bursaria

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Exptl Ceil Res 81 (1973)

114 Marlene W. Karakashian & S. J. Karakashian bacteria and is absent in perialgal vacuoles. Large amounts of reaction product are also visible in scattered cisternae and vesicles in the cytoplasm. Similar observations on ciliates [6, 71 have been interpreted as evidence for the presenceof acid phosphatase in the endoplasmic reticulum. Fluoride-treated control sections (fig. 1 b) demonstrate that nonspecific electron-dense deposits are slight, thus validating the cytochemical method for this material. Examination of many sections reveals rare vacuoles containing a deteriorating alga [4]. Similar vacuoles encountered in the present study (fig. 1 c) contained acid phosphatase reaction product. It is not known whether an alga within such an exceptional vacuole was ingested from the culture fluid just before the cells were isolated and washed or whether this is an instance of a change in the normal status of a perialgal vacuole due perhaps to the death of its inhabitant. Host response to recently ingestedparticles

The absence of digestive enzyme activity in perialgal vacuoles led to an examination of the host’s digestive response to other kinds of particles lodged in vacuoles. Studies of Paramecium multimicronucleatum and Tetrahymena pyriformis [8] indicated that acid hydrolases are releasedinto protozoan phagosomes regardless of their contents, but the procedures used did not satisfactorily rule out the possibility that bacteria triggered the release of digestive enzymes. In the studies described here, samples of the extracellular salt solutions in which the paramecia were maintained (final washes) were plated on nutrient agar. The bacterial density was less than 3.5 x lo4 cells/ml. The feeding apparatus of the paramecia does not appear to function effectively at this low food concentration, as demonstrated by the complete absence of images of bacteria from Exptl Cell Res 81 (1973)

numerous electron micrographs of phagosomes. Light microscopic investigation of P. bursaria fed carmine particles, latex spheres or clay particles (Celkate) revealed large amounts of acid phosphatase reaction product in phagosomes containing any of these inert materials (fig. 2a, b, c), but not in the many perialgal vacuoles present. Photographs of cells fed bacteria (fig. 2d) emphasize the similarity of the response to nutritive and non-nutritive materials. The single fluoride control picture shown (of latex-fed cells (fig. 2e)) is representative of the results obtained for each type of particle. Copious amounts of acid phosphatase reaction product are clearly visible between and around carmine particles in an electron micrograph of a preparation similar to those described above (fig. 3~). The amount of product is greatly reduced in fluoride-treated material (fig. 3 b). Similar results were obtained for cells fed latex spheres (fig. 3 c). The uneven density of the sectioned latex particles is caused by their partial dissolution during the embedding procedure, as has been described by others [9]. Fluoride-treated control sections (not shown) and preparations from which the Gomori substrate was withheld (fig. 3d) again verified the absence of non-specific electron-dense material. Host response to recently ingested algae

When potential algal symbionts are ingested by aposymbiotic paramecia, many of them are rather quickly digested. Repeated efforts to establish the kinetics of the intracellular algal population during the first few hours. following ingestion have been plagued with variability among host cells. Nevertheless, a pattern has emerged which is described here. Fig. 4 depicts the results of a typical experiment. As shown, approx. 20% of the algae taken in by the paramecia during 2; min

Digestion and symbiosis in P. bursaria

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Fig. 2. (a) Light micrograph of sectioned carmine-fed cell stained for acid phosphatase activity (arrows). Note absence of reaction product around symbiotic algae (A); (b) large amounts of reaction product (mows) are seen in vacuoles of two sectioned cells which had been fed latex spheres; (c) reaction product visible in vacuoles of a cell fed Celkate (clay); (d) acid phosphatase activity in cells fed bacteria; (e) fluoride-treated cells which had been fed latex spheres. Note absence of reaction product. Individual latex particles are visible in ingestion vacuoles (arrow). All micrographs approx. x 400.

exposures to extracellular algae survive for 24 h. Only apparently normal green algae were counted as ‘healthy’ in this study. Many of the paramecia examined, especially at 3 and 9 h after ingestion, contained large numbers of visibly discolored and degenerating algae in addition to the ‘healthy’ cells noted. An examination of the data for individual paramecia examined near the end of the 24 h period (plotted at the right edge of fig. 4), reveals a clear dichotomy in infectivity. Most paramecia either contain large numbers of healthy-looking algae or they are

virtually free of algae. The actual percentage of heavily infected host cells present in a sample varies from a few per cent in some experiments to as many as 30% in others; the basis for these differences is not presently known. These results were obtained with very brief exposure times. The marked heterogeneity of the host cells’ response is not evident if paramecia are allowed to ingest large numbers of symbiotic algae over a period of hours or days. Under these conditions, all aposymbiotic paramecia exposed to formerly symbiotExptl Cell Res 81 (1973)

116 Marlene W. Karakashian & S. J. KCwakashian

Fin. 3. (a) Acid nhosnhatase reaction nroduct in vacuole containing carmine particles (C) and adjace:nt cyto-

plasrnic vesicles.-A portion of the macronucleus (M) can be seen above the vacuole. x 22 000; (b) CJarminefed cell stained for enzyme activity in the nresencr: of NaF. Traces of reaction product are still visible in vacuole (arrow). x 19 000; (c) Portion of -Gomori-stained I cell which had been fed latex spheres (L). Reaction product is present only in phagosome and cytoplasmic Iresicles and not in the perialgal vacuoles (PV). x 15 200. (d) Absence of reaction product in latex-fed cells wh en Gomori substrate is omitted. x 22 000. Exptl Cell Res 81 (1973)

Digestion and symbiosis in P. bursaria

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ic algae are observed to have large numbers of seemingly established symbionts when they are removed from the ingestion suspension [3,

101.

Host reponse to mixtures of live and dead algae

Heat-killed algae are as readily ingested by aposymbiotic P. bursaria as live ones, a fact which prompted an inquiry into whether the presence of live algae in a phagosome together with dead ones would alter a host cell’s disposition of the dead ones. In the experiment reported here, paramecia were exposed for 24 min to a mixture of half-live and half-dead algae or to a suspension of dead algae only. In each case the total concentration of algae was similar.

0r,

Fig. 4. Abscissa: time since algae ingestion (hours); ordinate (left): percentage of initial number of algae

ingested which remain undigested (healthy) at time of examination: (right) number of healthy algae left 23& h post-ingestion. Retention of symbiotic algae by aposymbiotic paramecia. Groups of approx. 15 washed and starved paramecia were placed in suspensions of washed symbiotic algae for 2& min, following which they were washed and squashed or set aside for later examination. The optical density of the algal suspension used in this experiment was 750 Klett units. The average number of ‘healthy’ intracellular algae in samples of 15-17 paramecia decreased from 285 at 0 to 60 at 234 h. Range bars denote S. E.

Fig. 5. Abscissa: time since algae ingested (hours); ordinnte: percentage of initial number of dead algae

ingested which are left at time of examination. Retention of heat-killed algae by aposymbiotic paramecia when ingested with and without live algae. Groups of approx. 15 washed and starved paramecia were placed in suspensions of heat-killed algae alone or in mixed suspensions of heat-killed and live algae for 2: min, following which they were washed and squashed or set aside for later examination. In this experiment, the optical density of the live algal suspension from which the heat-killed algae were prepared (see Methods) was 1 000 Klett units. When dead algae were ingested alone ( l ), the average number per cell decreased from 192 to 0 by 24 h. When dead and live algae were ingested simultaneously ( 0), the average number of dead algae per cell decreased from 126 to 55 at 43 h. At 0 time, the dead algae represented slightly more than half of the algae present in each of the squashed cells. Range bars denote S. E.

The dead algae were stained with Congo Red, a vital stain which made them recognizable throughout the experiment and which had no toxic effect on the paramecia. Microscopic examination revealed that host cells exposed to mixtures of live and dead algae formed phagosomes containing approximately equal numbers of each type. As indicated in fig. 5, those paramecia fed only dead algae had completely digested them all within 24 h. However, dead algae fared very differently when they were ingested together with live ones; under these condiExptl Cell Res 81 (1973)

118 Marlene W. Karakashian & 5’. J. Karakashian tions, only about 50% of the dead cells disappeared after 43 h in host cell vacuoles. This is a clear indication that live algae are able to influence the host cell’s digestive processes so as to prevent their own digestion and delay the digestion of the dead algae ingested with them. The continued slow digestion of the killed algae in the presence of the live ones is probably due to the gradual segregation of the live algae into perialgal vacuoles where they could no longer exert their protective effect over the dead cells.

DISCUSSION A number of light and electron microscopic investigations of intracellular digestion have been undertaken in ciliates [6-8, 11-151. Results presented here and in one early study by Hunter [16] indicate that the digestive process in P. bursaria, like that of other ciliates, involves the release of digestive enzymes into food vacuoles following an appropriate stimulus. In P. bursaria, the enzyme-releasing stimulus is not simply the presence of nutritive material within a phagosome, since neither soluble nor particulate nutrients were present when enzymes were released into phagosomes containing either of several non-nutritive particles. Perhaps the mere formation of a phagosome triggers an eventual discharge of digestive enzymes into it. A similar conclusion was reached by Miiller et al. [S] following their study of latex particle ingestion by P. multimicronucleatum and Tetrahymena pyriformis; however, under their experimental conditions stray bacteria might have stimulated enzyme deposition. Whatever the signal for the conversion of a phagosome into a digestive vacuole, it is clear that symbiotic algae, if present in the vacuole, actively interfere with the transforExptl

Cd2 Res 81 (1973)

mation. Throughout the post-ingestion period they alter the normal course of host cell digestion, as was shown by their ability to delay the digestion of dead algae ingested with them. Possibly this interference is effected by their secretion of an inhibitor of the digestive enzymes, or the algae may induce a change in the phagosome membrane so that it can no longer fuse with enzymecontaining lysosomes. The inhibitor hypothesis implies that there would be a continual production of an excess of an agent or agents which block the activity of any digestive enzymes discharged into the phagosome. According to the alternate hypothesis, digestive enzymes would never gain access to the phagosome. Either hypothesis could account for the lack of detectable acid phosphatase in perialgal vacuoles. Both deDuve [17] and Dingle [18] have suggested that alterations in the fusion compatibilities of diverse membranous cell compartments are responsible for the control of lysosomal activity in cells. Further analyses of the basis for symbiont sparing in P. bursaria should contribute significantly to this discussion. The fate of ingested algae can be viewed as the consequence of a rate competition between the digestive processes of the host on the one hand and the digestion-suppressing activity of the algae on the other. This suggests that the greater the number of algae within a single phagosome, the more likely they are to prevent the digestion of the vacuole contents. Paramecia do occasionally form very large phagosomes containing 50 or more algae; the algae in these vacuoles invariably remain healthy. The observed bimodal pattern of variability with respect to the host cells’ retention of newly ingested algae becomes understandable if the fates of the algae in the several phagosomes in each cell are linked. Any cir-

Digestion and symbiosis in P. bursaria cumstance which affects the outcome of the competition between the host and algal processes in a certain proportion of host cells following their ingestion of algae could account for the results. For example, some host cells may form large phagosomes or some may be deficient in their supply of digestive enzymes. Obviously, even large variations in the digestion-suppressing activity of individual algae would most likely not be expressed coordinately among the phagosomes of a single cell. Data at hand are consistent with the view that the host cell population is subject to large shifts in its susceptibility to infection. In studies to be reported elsewhere, entire clones lost their susceptibility to infection by free-living algae as they aged. These changes must have entailed alterations in some aspect of host cell digestion. By contrast, evidence for marked variations in the digestion-suppressing activity of symbiotic algae is still lacking. Indeed, it appears that symbiotic algae may undergo considerable morphologic change, such as is induced by axenic culture [19], without there being any effect on their digestion-suppressing capacities. This is deduced from the fact that algae taken from axenic cultures exhibit the same pattern of loss through digestion as do symbiotic algae freshly isolated from paramecia when their retention is compared in parallel samples of aposymbiotic paramecia [IO]. The ability of cultured symbiotic algae to protect heat-killed algae ingested with them has not yet been studied. CONCLUSION Light and electron microscopic cytochemical examination of Paramecium bursaria containing symbiotic Chlorella demonstrate the ab-

119

sence of acid phosphatase reaction product in perialgal vacuoles even when it is present in adjacent food vacuoles and cytoplasmic vesicles. The enzyme is deposited in phagosomes containing recently ingested particles regardless of the nutritive value of the contents. Host digestive inefficiencies could account for part of the paramecium’s susceptibility to infection by symbionts, but live algae also actively suppress the digestive capacity of the host cell. This study was supported Foundation grant GB 7362,

by National

Science

REFERENCES 1. Siegel, R W, Exptl cell res 19 (1960) 239. 2. Karakashian, S J, Physiol zoo1 36 (1963) 52. 3. Karakashian, S J & Karakashian, M W, Evolution 19 (1965) 368. 4. Karakashian, S J, Karakashian, M W & Rudzinska, M A, J protozool 15 (1968) 113. 5. Martz, E & Ehret, C F, Argonne natl lab biol med res rept (1965) 228. 6. Carasso, N, Favard, P & Goldfischer, S, J microsc 3 (1964) 297. 7. Elliott, A M & Clemmons, G L, J protozool 13 (1966) 311. 8. Mueller, M, Rohlich, P & TGro, I, J protozool 12 (1965) 27. 9. Korn, E D & Weisman, R A, J cell biol 34 (1967) 219. 10. Karakashian, M W. Unpublished data. 11. Schneider, L, Z Zellforsch mikrosk Anat 62 (1964) 225. 12. Jurand, A, J protozool 8 (1961) 125. 13. Mueller, M & Tot-o, 1, J protozool 9 (1962) 98. 14. Rosenbaum, R N & Wittner, M, Arch protistenk 106 (1962) 223. 15. Rudzinska, M A, Jackson, G J & Tuffrau, M, J protozool 13 (1966) 440. 16. Hunter, N, Am microsc sot trans 82 (1963) 54. 17. deDuve, C, Lysosomes in biology and pathology (ed J T Dingle & H B Fell) vol. 1, p. 3. NorthHolland, Amsterdam (1969). 18. Dingle, J T, Brit med bull 24 (1968) 141. 19. Karakashian, S, Ann NY acad sci 175 (1970) 474.

Received March 19, 1973

Exptl Cell Res 81 (1973)