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Bacterial endocytobionts of the ciliate Paramecium calkinsi
Europ.J.Protistol. 29, 390-395 (1993) November 12, 1993
European Journal of
PROTISTOLOGY
Bacterial Endocytobionts of the Ciliate Paramecium calkinsi Sergei Fokin and Elena Sabaneyeva Biological Research Institute, St. Petersburg State University, Russia
SUMMARY Five species of gram-negative bacteria inhabiting the cytoplasm and the macronucleus of the ciliate Paramecium calkinsi are described. Two species of endocytobionts have been found in the cytoplasm. Both of them were represented by only a vegetative form. In the case of the first species, numerous endocytobiotic bacteria (length, 2 urn; diameter, 0.3-0.4 urn) were enclosed in individual endobiontophoral vacuoles. The second one was not as abundant in its host cells and the bacteria lie freely in the host cytoplasm. These bacteria (length, 2-3 urn; diameter, 0.6-0.9 um) often contained viral particles. Three species of endocytobionts have been discovered in the macronucleus. The first one (1-2 urn long, 0.3-0.4 urn in diameter) resembled Nonospora macronucleata. However, it differed from N. macronucleata in having specific electron dense zones in its cytoplasm and stained differently by Ag-NOR. The second one represents a new species of the Holospora genus: H. curvata sp. nov. The infectious form of this endocytobiont (15-20 !-tm long, 0.7-0.9 !-tm in diameter) is curved, the vegetative form (2-3 urn long, the same diameter) was rod-like or slightly curved. The host specificity of these endocytobionts is well expressed. The third species of intranuclear bacteria, H. bacillata, had been previously described in P. woodruffi. The infective capacity of H. curvata and two "stocks" of H. bacillata from different hosts was compared.
Introduction Bacterial endocytobionts have been described in different ciliates [19,22]. In this respect representatives of the genus Paramecium are of particular interest: more than 40 species of bacteria have been found in different compartments of the paramecium cell. Apparently, for these microorganisms endocytobiosis is obligatory. At the same time the paramecium-hosts can exist without endocytobionts. The significance of microbial infections of ciliates in nature is not really understood. In some species of the genus no bacterial infection has been found at all [7, 19]. For a long time this was also true for P. calkinsi - a euryhaline species, mostly inhabiting sea shores [26]. Two other salt-water paramecia, P. woodruffi and P. duboscqui, live under similar ecological conditions, some endocytobionts of which have been described recently [1-6, 13, 14]. Detailed cytological investigation of cells of numerous stocks of P. calkinsi, isolated from brackish water areas on the shores of the White Sea and the Barents Sea (splashes, 0932-4739/93/0029-0390$3.50/0
rivers and stream estuaries) has revealed endocytobionts in this ciliate, as well [16]. The present study deals with the description of bacteria inhabiting the cytoplasm and the macronucleus of P. calkinsi.
Material and Methods The following stocks were used in the study: Br 15-7, OP-32, MG-10, isolated in the summer of 1989, on the coasts of Ryajkov and Pej islands and the Bear gulf of the Kandalaksha Bay, respectively; OK-6-2 and OK-6-1O, isolated the same year on the coast of Kolguev island (Barents Sea). All of the enumerated stocks were already infected when they were isolated from nature. Besides, numerous endocytobiont-free stocks of P. calkinsi and P. woodruffi of different age and geographical origin and laboratory stocks of P. bursaria, P. trichium, and P. duboscqui were used in the study. The ciliates were maintained according to Sonneborn [25], at 20°C. The infective and killer capacities were determined using homogenates of the infected cells prepared by the technique of Preer [21]. © 1993 by Gustav Fischer Verlag, Stuttgart
Endocytobionts of Paramecium calkinsi . 391 Permanent preparations were stained by the Feulgen techniq ue. Besides, isolated infected macronuclei were stained with silver nitrate [15]. The Gram-reaction was carried out on smashed cells. Living cells were observed and their photographs taken under a Polyvar microscope (Reichert-jung, Austria). The cells were immobilized using a device for controlling the cover slide [24]. For electron microscopy, cells were processed according to a technique described previously [3]. Thin sections were examined under Tesla 500, Jem 100 ex and Siemens Elmiskop 101 electron microscopes.
Results The study of the cells of P. calkinsi stocks OK-6-2 and OK-6-10 revealed two species of cytoplasmic bacteria, one in each stock (Plate I). These endocytobionts are prominent in smashed preparations of living cells. However, their identification as two species became possible only after a fine structural examination.
Plate I. Endocytobionts of the cytoplasm of P. calkinsi.a, b. Electron micrographs of M I-bacteria. Arrows = hostmembranes tightly surrounding individual endobiobionts. - c-e. Electron micrographs of MIl-bacteria. - d. The bacterial cell with specific body of homogeneous material. - e. Possibly the beginning of the bacterial lysis. sb = specific body of homogeneous material. Arrowheads = hexaedrical viral capsids. Bar = 0.5 urn,
The first species of cytoplasmic microorganisms (M I) is represented by rod-like bacteria (Plate Ia, b) 2 urn long, 0.3-0.4 urn in diameter. Each endocytobiont is enclosed in an individual vacuole, its cell wall structure corresponds to that of Gram-negative bacteria; there is no distinct nucleoid, however, the electron density of different parts of cytoplasm is different (Plate Ib). All M l-endocytobionts in a host cell were vegetative cells. An experimental infection of endocytobiont-free stocks of P. calkinsi by these endocytobionts was unsuccessful. No killer effect was revealed when a homogenate of the infected cells was added to free cells of P. calkinsi (data not shown). At the same time in the main part of the cells of this stock a bacterial infection of the macronucleus was found (see below). The second bacterial species (M II) was revealed in the cytoplasm of the cells of stock OK-6-10. The bacteria were not encircled by host membranes. These endocytobionts were 1-3 urn long, 0.6-0.9 urn in diameter (Plate Ie-e). M II were also Gram-negative without any distinct
392 . S. Fokin and E. Sabaneyeva
nucleoid. The number of M II-endocytobionts per host cell was less than M I. Only vegetative forms were observed, many of them carrying hexaedrical viral capsids which were 60 nm in diameter and which had specific bodies of homogeneous material of average electron density, 0.2-0.5 urn wide (Plate Id), in their cytoplasm. Homogen-
Plate II. M III-endocytobionts in the macronucleus of P. calkinsi. a, b: Electron micrographs. Arrows = zones of higher electrondensity in the bacteria. Bar = 1 urn.
ate of the infected cells did not have a killer effect on bacterium-free P. calkinsi. No experimental infection could be obtained in the three stocks tested (data not shown). Macronuclear endocytobionts were found in P. calkinsi by observing living cells under the light microscope using differential interference contrast. The first type of macronuclear microorganisms (stock OK-6-2) could be revealed under the light microscope only in smashed preparations or in silver-stained isolated nuclei. The microorganisms of this type (M III) were evenly distributed in the whole nucleus very often arranged in regular patches oriented parallel to each other. The M III-endocytobiont population consisted only of vegetative forms (Plate II). The M III-bacteria are 1-2 urn long and 0.3-0.4 urn in diameter. Zones of higher electrondensity that were as a rule located at the tips of the cells and undistinguishable under the light microscope were revealed in some of them. After staining with silver nitrate M Ill-endocyrobionrs were brown. The macronuclear infection did not prevent the ordinary macronuclear fission and the fission rate of infected cells was normal (1.7-2 fissions per 24 h). In the stock studied, the macronuclear infection coexisted with the cytoplasmic infection of M I-bacteria (see above ). Attempts to experimentally infect bacteria-free clones by a homogenate of stock OK-6-2 cells did not show any infectious or killer capacity of the MIll-bacteria. The second type of bacteria inhabiting the macronucleus was found in the cells of stock Br 15-7. Two forms of this bacterium, reproductive and infectious forms differing in size, morphology, and their role in the life cycle, were present in the population (Plate III, IV). Both ends of these endocytobionts were rounded; the structure of the cell wall corresponded to that of Gram-negative bacteria. The reproductive cells (length, 2-3 urn, diameter, 0.7-0.9 urn) were rod-like or curved. The infectious form (length, 15-20 urn, diameter, 0.7-0.9 urn) is slightly undulated. A longitudinal cell differentiation into three zones with different electron densities - "acrosornic", periplasmic and cytoplasmic zones - was characteristic of the latter form (Plate IVa). On the other hand, a homogeneous cytoplasm with no traces of any differentiation was typical of the reproductive cells. In addition, the size and the morphology of some bacteria were intermediate between those of the infectious and the reproductive forms. A number of experimentally infected stocks were obtained using the homogenate of cells infected with these bacteria (table). Only the infectious forms of endocytobionts penetrated into the macronucleus, the first ones appearing in the macronucleus within 1 h after the beginning of the experiment. Vegetative cells appeared in the macronucleus within 24 h as a result of the fragmentation of the infectious cells. The number of reproductive cells increased rapidly in the following days. 5-7 days after the experimental infection, new infectious forms were registered in the macronucleus (Plate IlIa). As a rule, a stably infected nucleus contained many endocytobionts of both forms (Plate I1Ib). Bacteria often came in close contact with chromatin aggregates. Ag-NOR
Endocytobionts of Paramecium calkinsi . 393
Plate III. Holospora curvata in the macronucleus of P. calkinsi. a. Macronucleus of P. calkinsi after the host cell was crushed with the cover glass. DIe. Bar = 10 urn. b. Bacterial cells after the macronucleus of the host cell was crushed with the cover glass. DIe. RF = reproductive forms of the bacteria, IF = infecrious forms of the bacteria. Bar = 5 urn.
stammg of isolated infected nuclei showed numerous nucleolar organizers located along the bacterial cell wall, especially of the infectious forms. The fission rate of infected cells did not significantly differ from control cells; infected macronuclei underwent normal fissions without producing any residual body containing infectious forms. Such residual bodies with infectious forms are characteristic of some other Holospora species [3, 8, 17]. A decrease in the number of micronuclei in the cell (from 2-3 in bacteria-free cells to 1 in infected paramecia) was registered in 1 of 3 experimentally infected stocks studied in this respect. Thus, the morphology and the life cycle of the endocytobiont corresponds to that of other Holospora species [3,
Plate IV. Electron micrographs of Holospora curvata. - a, b. Longitudinal sections of the IF and RF, a = "acrosomic", p = periplasmic, c = cytoplasmic zornes of IF. - c. Cross-sections of the IF and RF Cells. Bar = 0.5 urn.
8,17,18]. The results, including the determination of host specificity (see below), permit the identification of the described endocytobiont as a new species of this genus, H. curuata. We managed to infect the cells of stock OK-6-2 by H. curuata found in stock Br 15-7. In this case H. curvata replaced M III-bacteria in the nucleus within one month. When we used homogenates containing two bacterial species (M III and H. curvata) to obtain double infections, we found that all M III-bacteria were completely lysed within 24 h after penetration into the macronucleus.
394 · 5. Fokin and E. Sabaneyeva
Along with the endocytobionts of P. calkinsi described above, in the macronuclei of cells of stock OP-32, we found bacteria which correspond in their morphology to H. bacillata, an endocytobiont previously described in P. woodruffi [3] . In order to determine the infective capacity and the host specificity of these endocytobionts, we carried out experimental infections (3 repetitions) with the new Holospora species and H. bacil/ata isolated from P. calkinsi and P. woodruffi (see table). The results revealeda host specificity of H. curvata. The infectious forms of this endocytobiont did not penetrate into the macronucleus of P. duboscqui, P. trichium and P. bursaria. The bacteria penetrated into the macronucleus in ailS stocks of P. woodruffi which were investigated, but the infection was not maintained for a long time. Only in two stocks of P. woodruffi did the bacteria remain for 9 days. One of the stocks of P. calkinsi also .faile~ to maintain H. curvata (see table). At the same time, In 4 other stocks of this ciliate the endocytobionts underwent the complete life cycle and could be maintained for more than 3 months. The bacteria identified as H. bacil/ata but isolated from different hosts (P. woodruffiand P. calkinsi) behaved differently during the infection. H. bacil/ata from P. calkinsi penetrated into the macronucleus and was maintained in three bacteria-free stocks of P. calhinsi, but in two cases it failed even to penetrate into the macronucleus. In the experimental infection, these bacteria penetrated into the macronucleus of two stocks of P. woodruffi, but were not maintained. One stock of P. woodruffi appeared to be absolutely resistant ~o the.infecti.on, .and two stocks could be infected and the infection maintained for a long time (more than two months). At the same time H. bacil/ata from P. woodruffi was not maintained in the cells of P. calkinsi stocks, although they Tab le 1. Effectiveness of the infection of paramecia by H. curuata and H. bacillata stock
Bacteria H. curuata
OP 1-14 DZ 59-2 2 KB-6 KA-l DV 12-13
53 45 38 15 30
m m m n m
P. woodruffi
DZ 59-7 Br 13-3 DZ 59-11 Br 19-1 KP 2-1
50 30 45 45 50
n" n n" n n
P. duboscqui
DZ 59-25 DZ 59-16 BK-51
0 0
Paramecia species P. calkinsi
P. trichium
P. bursaria
a
bacillata-Y
H.
H. badllata-L
42 m 100 m 100 m
IO n 0 23 n
a a
21 25 35 10 0 0 0 0
n n m m
a
13 m 80 m 40m 0 0 5n 0 0
H . bacillata-1 = "strain " of H. bacillata mainta ined in P. calkinsi; H . bacillata-2 = "strain" of H. bacillata maintained in P. iooodruffi; 53 = average percentage (for 3 repet itions) of the infected cells in two hours after the beginning of the infection; m = endocytobionts can be maintained; n = endocytobionts cann ot be ma inta ined; * = endocytobionts can be maintained for less th an 10 days.
penetrated into the macronucleus in two of four cases. As we found previously [3] even in P. woodruffi there are stocks resistant to a H. bacil/ata infection. In the present study we have obtained similar results: two of five stocks appeared to be resistant to the infection. However, H. bacil/ata isolated from P. woodruffi penetrated into the macronucleus of P. duboscqui. Discussion A significant bacterial flora has been found in the cytoplasm of some ciliates [5, 19,22]. First o! all, th.ese are ciliates belonging to the complex of P. aurelia species and P. woodruffi. A comparison of the morphology of ~he. endocytobionts discovered in the cytoplasm of P. calkins! with bacteria described previously in the various speciesof Paramecium [19,22,23] leads to the conclusion that M I and M II-bacteria are new species. The complex of morphological characteristics of M I differs from that of other Paramecium endocytobionts. The morphology of M II slightly resembles that of the bacteria we have described previously in the cytoplasm of P. woodruffi [5, 6, 13]. However, these bacteria differ in the size and structure of their specific inclusions in their cytoplasms (paracrystalloid bodies in the bacteria from P. woodruffi and homogeneous bodies in the bacteria from P. calkinsi) . Unfortunately, nothing yet can be said concerning the capacity of M II to cause a mate-killer effect, since we had no stocks of P. calkinsi at our disposal that were of complementary mating type. The endocytobionts from P. woodruffi had been shown to cause mate-killing [6]. The morphology of M III-bacteria has features in common with N. macronucleata from the macronucleus of P. caudatum [15]. The different coloring by AgNO and the presence of the terminal electron-dense zones in the cytoplasm of M III, however, is a significant difference between these bacteria. In combination with the Holospora infection they also behave differently: N. macronucleata replaces H. obtusa [1], while M III-bacteria are lost as a result of the development of H. curvata in the macronucleus. The study permits us to consider the representatives of the Holospora genus found in the macronucleus of P. calkinsi to be different species. The bacteria found in the cells of the stock Br 15-7 differ from H. bacil/ata in size: average length of the infectious form of the latter is 12.7 fim , whereas in H. curvata it is 18.5 urn, Infectious H. bacil/ata are never curved, whereas in H. curvata the infectious form is always undulated. The two bacteria differ also in their host specificities: H. curvata cannot be maintained for a long time in P. woodruffi whereas H. bacil/ata infects and can be maintained for a long time in the macronucleusof both of these paramecium species in the laboratory as well as in nature. The observations of the graded infectivity of H. curvata in the paramecia tested indicate that the invasion into the nucleus affords various steps of recognition and communication between host cell and bacterium. Similar results
Endocytobionts of Paramecium calkinsi . 395
have been obtained earlier by the author for H. obtusa and H. undulata [9, 10], and observations on a graded infectivity of H. obtusa and different species of Paramecium were also made by Fujishima and Fujita [12] and Fujishima [11]. Acknowledgements We are grateful to Drs H.-D. Cortz and T. J. Kosco for critically reading the manuscript and for some technical advice.
References 1 Fokin S. I. (1987): Paramecium caudatum: morphological analysis of consequences of infection with different nuclear symbionts. Cytologia, 30, 471-477 (in Russian with English summary). 2 Fokin S. I. (1988): A bacterial symbiont of the macronucleus perinuclear space in the ciliate Paramecium duboscqui. Cytologia, 30, 632-635 (in Russian with English summary). 3 Fokin S. I. (1989a): Bacterial endobionts of the ciliate Paramecium woodruffi. I. Endobionts of the macronucleus. Cytologia, 31, 839-844 (in Russian with English summary). 4 Fokin S. I. (1989b): Bacterial endobionts of the ciliate Paramecium woodruffi. II. Endobionts of the perinuclear space. Cytologia, 31, 845-850 (in Russian with English summary). 5 Fokin S. I. (1989c): Bacterial endobionts in the ciliate Paramecium woodruffi. III. Endobionts of the cytoplasm. Cytologia, 31, 964-970 (in Russian with English summary). 6 Fokin S. I. (1989d): Mate-killer effect induced by endobionts of the ciliate Paramecium woodruffi. Cytologia, 31, 1085-1089 (in Russian with English summary). 7 Fokin S. I. (198ge): Employment of endobionts in taxonomy of Paramecium species, p.75. In: Ecology of marine and freshwater protozoans. Abstr. of the II symposium, Yaroslavl (in Russian with English title). 8 Fokin S. I. (1991): Holospora recta sp. nov. - a micronucleusspecific endobiont of the ciliate Paramecium caudatum. Cytologia, 33, 135-141 (in Russian with English summary). 9 Fokin S. I. and Skovorodkin I. N. (1991): Holospora obtusaendonucleobiont of the ciliate Paramecium caudatum in search for the macronucleus. Cytologia, 33, 64-75 (in Russian with English summary). 10 Fokin S. I. and Skovorodkin I. N. (1991): Holospora undulata - an endobiont of the ciliate Paramecium caudatum in search ofthe micronucleus. Cytologia, 33, 101-115 (in Russian with English summary). 11 Fujishima M. (1986): Further study of the infectivity of Holospora obtusa, a macronucleus-specific bacterium of the
ciliate Paramecium caudatum. Acta Protozool., 25, 345-350. 12 Fujishima M. and Fujita M. (1985): Infection and maintenance of Holospora obtusa, a macronucleus-specific bacterium of the ciliate Paramecium caudatum. J. Cell Sci., 76, 179-187. 13 Fokin S. I., Boss A. O.-L. and Ossipov D. V. (1987a): A virus-containing cytoplasmic symbiont of the ciliate Paramecium woodruffi. Cytologia, 29, 1303-1306 (in Russian with English summary). 14 Fokin S. I. and Cortz H.-D. (1993): Caedibacter macronucleorum sp. nov., a bacterium inhabiting the macronucleus of Paramecium duboscqui. Arch. Protistenkd. 143, 319-324. 15 Fokin S. I., Ossipov D. V., Skoblo I. I., Rautian M. S. and Sabaneyeva E. V. (1987): Nonosporamacronucleata g.n., sp. n. - A vegetative nucleus symbiont of the ciliate Paramecium caudatum. Cytologia, 29, 963-970 (in Russian with English summary). 16 Fokin S. I. and Sabaneyeva E. V. (1990): Paramecium (Ciliophora, Peniculina) from water with low salinity of the Barents Sea and White Sea coasts and their endobionts, pp. 139-140. In: Ecology, reproduction and guarding of the bioresources of the North Europe seas. Abstr. of the III conference, Murmansk (in Russian). 17 Cortz H.-D. (1986): Endonucleobiosis in ciliates. Int. Rev. Cytol., 102, 169-213. 18 Gromov B. V. and Ossipov D. V. (1981): Holospora (ex Hafkine, 1890) nom. rev., a genus of bacteria inhabiting the nuclei of Paramecium. Int. J. Syst. Bacteriol., 31, 348-352. 19 Heckmann K. and Gortz H.-D. (1990): Prokaryotic syrnbionts of ciliates. In: Balows A., Truper H. G., Dworkin M., Harder W. and Shleifer K. H. (eds.): The prokaryotes, 2nd ed., pp. 3865-3890. Springer Verlag, Berlin and New York. 20 Ossipov D. V., Fohn S. I., Borchsenius O. N., Lebedeva N. A., Rautian M. S. and Skoblo I. I. (1989): Diversity and distribution of the intranuclear symbionts of ciliates of the Soviet Union, p. 53. In: Ecology of marine and freshwater protozoans. Abstr. of the II symposium, Yaroslavl (in Russian with English title). 21 Preer L. B. (1969): Alpha, an infectious macronuclear symbiont of Paramecium aurelia. J. Protozool., 16, 570-578. 22 Preer J. R. Jr. and Preer L. B. (1984): Endosymbionts of protozoa, pp.795-813. In: Krieg N. R. (ed.): Bergey's manual of systematic bacteriology, vol. 1. Williams and Wilkins, Baltimore and London. 23 Skoblo I. I., Borchsenius O. N., Lebedeva N. A. and Ossipov D. V. (1985): New species of bacterial cytoplasmic symbiont of ciliate Paramecium bursaria (Ciliophora, Protozoa). Cytologia, 27, 1292-1297 (in Russian with English summary). 24 Skovorodkin I. N. (1990): A device for immobilization biological objects in the light microscope studies. Cytologia, 32,301-302 (in Russian with English summary). 25 Sonneborn T. M. (1970): Methods in Paramecium research, pp. 241-339. In: Methods in cell physiology, vol. 4. Acad. Press, New York. 26 Wichterman R. (1986): The biology of Paramecium, 2nd ed. Plenum Press, New York.
Key words: Bacteria - Endocytobionts - Paramecium - Cytoplasm - Nucleus
Sergei Fokin, S. Kovalewskoj 1411,93, St. Petersburg, 195249 Russia
European Journal of
PROTISTOLOGY
Bacterial Endocytobionts of the Ciliate Paramecium calkinsi Sergei Fokin and Elena Sabaneyeva Biological Research Institute, St. Petersburg State University, Russia
SUMMARY Five species of gram-negative bacteria inhabiting the cytoplasm and the macronucleus of the ciliate Paramecium calkinsi are described. Two species of endocytobionts have been found in the cytoplasm. Both of them were represented by only a vegetative form. In the case of the first species, numerous endocytobiotic bacteria (length, 2 urn; diameter, 0.3-0.4 urn) were enclosed in individual endobiontophoral vacuoles. The second one was not as abundant in its host cells and the bacteria lie freely in the host cytoplasm. These bacteria (length, 2-3 urn; diameter, 0.6-0.9 um) often contained viral particles. Three species of endocytobionts have been discovered in the macronucleus. The first one (1-2 urn long, 0.3-0.4 urn in diameter) resembled Nonospora macronucleata. However, it differed from N. macronucleata in having specific electron dense zones in its cytoplasm and stained differently by Ag-NOR. The second one represents a new species of the Holospora genus: H. curvata sp. nov. The infectious form of this endocytobiont (15-20 !-tm long, 0.7-0.9 !-tm in diameter) is curved, the vegetative form (2-3 urn long, the same diameter) was rod-like or slightly curved. The host specificity of these endocytobionts is well expressed. The third species of intranuclear bacteria, H. bacillata, had been previously described in P. woodruffi. The infective capacity of H. curvata and two "stocks" of H. bacillata from different hosts was compared.
Introduction Bacterial endocytobionts have been described in different ciliates [19,22]. In this respect representatives of the genus Paramecium are of particular interest: more than 40 species of bacteria have been found in different compartments of the paramecium cell. Apparently, for these microorganisms endocytobiosis is obligatory. At the same time the paramecium-hosts can exist without endocytobionts. The significance of microbial infections of ciliates in nature is not really understood. In some species of the genus no bacterial infection has been found at all [7, 19]. For a long time this was also true for P. calkinsi - a euryhaline species, mostly inhabiting sea shores [26]. Two other salt-water paramecia, P. woodruffi and P. duboscqui, live under similar ecological conditions, some endocytobionts of which have been described recently [1-6, 13, 14]. Detailed cytological investigation of cells of numerous stocks of P. calkinsi, isolated from brackish water areas on the shores of the White Sea and the Barents Sea (splashes, 0932-4739/93/0029-0390$3.50/0
rivers and stream estuaries) has revealed endocytobionts in this ciliate, as well [16]. The present study deals with the description of bacteria inhabiting the cytoplasm and the macronucleus of P. calkinsi.
Material and Methods The following stocks were used in the study: Br 15-7, OP-32, MG-10, isolated in the summer of 1989, on the coasts of Ryajkov and Pej islands and the Bear gulf of the Kandalaksha Bay, respectively; OK-6-2 and OK-6-1O, isolated the same year on the coast of Kolguev island (Barents Sea). All of the enumerated stocks were already infected when they were isolated from nature. Besides, numerous endocytobiont-free stocks of P. calkinsi and P. woodruffi of different age and geographical origin and laboratory stocks of P. bursaria, P. trichium, and P. duboscqui were used in the study. The ciliates were maintained according to Sonneborn [25], at 20°C. The infective and killer capacities were determined using homogenates of the infected cells prepared by the technique of Preer [21]. © 1993 by Gustav Fischer Verlag, Stuttgart
Endocytobionts of Paramecium calkinsi . 391 Permanent preparations were stained by the Feulgen techniq ue. Besides, isolated infected macronuclei were stained with silver nitrate [15]. The Gram-reaction was carried out on smashed cells. Living cells were observed and their photographs taken under a Polyvar microscope (Reichert-jung, Austria). The cells were immobilized using a device for controlling the cover slide [24]. For electron microscopy, cells were processed according to a technique described previously [3]. Thin sections were examined under Tesla 500, Jem 100 ex and Siemens Elmiskop 101 electron microscopes.
Results The study of the cells of P. calkinsi stocks OK-6-2 and OK-6-10 revealed two species of cytoplasmic bacteria, one in each stock (Plate I). These endocytobionts are prominent in smashed preparations of living cells. However, their identification as two species became possible only after a fine structural examination.
Plate I. Endocytobionts of the cytoplasm of P. calkinsi.a, b. Electron micrographs of M I-bacteria. Arrows = hostmembranes tightly surrounding individual endobiobionts. - c-e. Electron micrographs of MIl-bacteria. - d. The bacterial cell with specific body of homogeneous material. - e. Possibly the beginning of the bacterial lysis. sb = specific body of homogeneous material. Arrowheads = hexaedrical viral capsids. Bar = 0.5 urn,
The first species of cytoplasmic microorganisms (M I) is represented by rod-like bacteria (Plate Ia, b) 2 urn long, 0.3-0.4 urn in diameter. Each endocytobiont is enclosed in an individual vacuole, its cell wall structure corresponds to that of Gram-negative bacteria; there is no distinct nucleoid, however, the electron density of different parts of cytoplasm is different (Plate Ib). All M l-endocytobionts in a host cell were vegetative cells. An experimental infection of endocytobiont-free stocks of P. calkinsi by these endocytobionts was unsuccessful. No killer effect was revealed when a homogenate of the infected cells was added to free cells of P. calkinsi (data not shown). At the same time in the main part of the cells of this stock a bacterial infection of the macronucleus was found (see below). The second bacterial species (M II) was revealed in the cytoplasm of the cells of stock OK-6-10. The bacteria were not encircled by host membranes. These endocytobionts were 1-3 urn long, 0.6-0.9 urn in diameter (Plate Ie-e). M II were also Gram-negative without any distinct
392 . S. Fokin and E. Sabaneyeva
nucleoid. The number of M II-endocytobionts per host cell was less than M I. Only vegetative forms were observed, many of them carrying hexaedrical viral capsids which were 60 nm in diameter and which had specific bodies of homogeneous material of average electron density, 0.2-0.5 urn wide (Plate Id), in their cytoplasm. Homogen-
Plate II. M III-endocytobionts in the macronucleus of P. calkinsi. a, b: Electron micrographs. Arrows = zones of higher electrondensity in the bacteria. Bar = 1 urn.
ate of the infected cells did not have a killer effect on bacterium-free P. calkinsi. No experimental infection could be obtained in the three stocks tested (data not shown). Macronuclear endocytobionts were found in P. calkinsi by observing living cells under the light microscope using differential interference contrast. The first type of macronuclear microorganisms (stock OK-6-2) could be revealed under the light microscope only in smashed preparations or in silver-stained isolated nuclei. The microorganisms of this type (M III) were evenly distributed in the whole nucleus very often arranged in regular patches oriented parallel to each other. The M III-endocytobiont population consisted only of vegetative forms (Plate II). The M III-bacteria are 1-2 urn long and 0.3-0.4 urn in diameter. Zones of higher electrondensity that were as a rule located at the tips of the cells and undistinguishable under the light microscope were revealed in some of them. After staining with silver nitrate M Ill-endocyrobionrs were brown. The macronuclear infection did not prevent the ordinary macronuclear fission and the fission rate of infected cells was normal (1.7-2 fissions per 24 h). In the stock studied, the macronuclear infection coexisted with the cytoplasmic infection of M I-bacteria (see above ). Attempts to experimentally infect bacteria-free clones by a homogenate of stock OK-6-2 cells did not show any infectious or killer capacity of the MIll-bacteria. The second type of bacteria inhabiting the macronucleus was found in the cells of stock Br 15-7. Two forms of this bacterium, reproductive and infectious forms differing in size, morphology, and their role in the life cycle, were present in the population (Plate III, IV). Both ends of these endocytobionts were rounded; the structure of the cell wall corresponded to that of Gram-negative bacteria. The reproductive cells (length, 2-3 urn, diameter, 0.7-0.9 urn) were rod-like or curved. The infectious form (length, 15-20 urn, diameter, 0.7-0.9 urn) is slightly undulated. A longitudinal cell differentiation into three zones with different electron densities - "acrosornic", periplasmic and cytoplasmic zones - was characteristic of the latter form (Plate IVa). On the other hand, a homogeneous cytoplasm with no traces of any differentiation was typical of the reproductive cells. In addition, the size and the morphology of some bacteria were intermediate between those of the infectious and the reproductive forms. A number of experimentally infected stocks were obtained using the homogenate of cells infected with these bacteria (table). Only the infectious forms of endocytobionts penetrated into the macronucleus, the first ones appearing in the macronucleus within 1 h after the beginning of the experiment. Vegetative cells appeared in the macronucleus within 24 h as a result of the fragmentation of the infectious cells. The number of reproductive cells increased rapidly in the following days. 5-7 days after the experimental infection, new infectious forms were registered in the macronucleus (Plate IlIa). As a rule, a stably infected nucleus contained many endocytobionts of both forms (Plate I1Ib). Bacteria often came in close contact with chromatin aggregates. Ag-NOR
Endocytobionts of Paramecium calkinsi . 393
Plate III. Holospora curvata in the macronucleus of P. calkinsi. a. Macronucleus of P. calkinsi after the host cell was crushed with the cover glass. DIe. Bar = 10 urn. b. Bacterial cells after the macronucleus of the host cell was crushed with the cover glass. DIe. RF = reproductive forms of the bacteria, IF = infecrious forms of the bacteria. Bar = 5 urn.
stammg of isolated infected nuclei showed numerous nucleolar organizers located along the bacterial cell wall, especially of the infectious forms. The fission rate of infected cells did not significantly differ from control cells; infected macronuclei underwent normal fissions without producing any residual body containing infectious forms. Such residual bodies with infectious forms are characteristic of some other Holospora species [3, 8, 17]. A decrease in the number of micronuclei in the cell (from 2-3 in bacteria-free cells to 1 in infected paramecia) was registered in 1 of 3 experimentally infected stocks studied in this respect. Thus, the morphology and the life cycle of the endocytobiont corresponds to that of other Holospora species [3,
Plate IV. Electron micrographs of Holospora curvata. - a, b. Longitudinal sections of the IF and RF, a = "acrosomic", p = periplasmic, c = cytoplasmic zornes of IF. - c. Cross-sections of the IF and RF Cells. Bar = 0.5 urn.
8,17,18]. The results, including the determination of host specificity (see below), permit the identification of the described endocytobiont as a new species of this genus, H. curuata. We managed to infect the cells of stock OK-6-2 by H. curuata found in stock Br 15-7. In this case H. curvata replaced M III-bacteria in the nucleus within one month. When we used homogenates containing two bacterial species (M III and H. curvata) to obtain double infections, we found that all M III-bacteria were completely lysed within 24 h after penetration into the macronucleus.
394 · 5. Fokin and E. Sabaneyeva
Along with the endocytobionts of P. calkinsi described above, in the macronuclei of cells of stock OP-32, we found bacteria which correspond in their morphology to H. bacillata, an endocytobiont previously described in P. woodruffi [3] . In order to determine the infective capacity and the host specificity of these endocytobionts, we carried out experimental infections (3 repetitions) with the new Holospora species and H. bacil/ata isolated from P. calkinsi and P. woodruffi (see table). The results revealeda host specificity of H. curvata. The infectious forms of this endocytobiont did not penetrate into the macronucleus of P. duboscqui, P. trichium and P. bursaria. The bacteria penetrated into the macronucleus in ailS stocks of P. woodruffi which were investigated, but the infection was not maintained for a long time. Only in two stocks of P. woodruffi did the bacteria remain for 9 days. One of the stocks of P. calkinsi also .faile~ to maintain H. curvata (see table). At the same time, In 4 other stocks of this ciliate the endocytobionts underwent the complete life cycle and could be maintained for more than 3 months. The bacteria identified as H. bacil/ata but isolated from different hosts (P. woodruffiand P. calkinsi) behaved differently during the infection. H. bacil/ata from P. calkinsi penetrated into the macronucleus and was maintained in three bacteria-free stocks of P. calhinsi, but in two cases it failed even to penetrate into the macronucleus. In the experimental infection, these bacteria penetrated into the macronucleus of two stocks of P. woodruffi, but were not maintained. One stock of P. woodruffi appeared to be absolutely resistant ~o the.infecti.on, .and two stocks could be infected and the infection maintained for a long time (more than two months). At the same time H. bacil/ata from P. woodruffi was not maintained in the cells of P. calkinsi stocks, although they Tab le 1. Effectiveness of the infection of paramecia by H. curuata and H. bacillata stock
Bacteria H. curuata
OP 1-14 DZ 59-2 2 KB-6 KA-l DV 12-13
53 45 38 15 30
m m m n m
P. woodruffi
DZ 59-7 Br 13-3 DZ 59-11 Br 19-1 KP 2-1
50 30 45 45 50
n" n n" n n
P. duboscqui
DZ 59-25 DZ 59-16 BK-51
0 0
Paramecia species P. calkinsi
P. trichium
P. bursaria
a
bacillata-Y
H.
H. badllata-L
42 m 100 m 100 m
IO n 0 23 n
a a
21 25 35 10 0 0 0 0
n n m m
a
13 m 80 m 40m 0 0 5n 0 0
H . bacillata-1 = "strain " of H. bacillata mainta ined in P. calkinsi; H . bacillata-2 = "strain" of H. bacillata maintained in P. iooodruffi; 53 = average percentage (for 3 repet itions) of the infected cells in two hours after the beginning of the infection; m = endocytobionts can be maintained; n = endocytobionts cann ot be ma inta ined; * = endocytobionts can be maintained for less th an 10 days.
penetrated into the macronucleus in two of four cases. As we found previously [3] even in P. woodruffi there are stocks resistant to a H. bacil/ata infection. In the present study we have obtained similar results: two of five stocks appeared to be resistant to the infection. However, H. bacil/ata isolated from P. woodruffi penetrated into the macronucleus of P. duboscqui. Discussion A significant bacterial flora has been found in the cytoplasm of some ciliates [5, 19,22]. First o! all, th.ese are ciliates belonging to the complex of P. aurelia species and P. woodruffi. A comparison of the morphology of ~he. endocytobionts discovered in the cytoplasm of P. calkins! with bacteria described previously in the various speciesof Paramecium [19,22,23] leads to the conclusion that M I and M II-bacteria are new species. The complex of morphological characteristics of M I differs from that of other Paramecium endocytobionts. The morphology of M II slightly resembles that of the bacteria we have described previously in the cytoplasm of P. woodruffi [5, 6, 13]. However, these bacteria differ in the size and structure of their specific inclusions in their cytoplasms (paracrystalloid bodies in the bacteria from P. woodruffi and homogeneous bodies in the bacteria from P. calkinsi) . Unfortunately, nothing yet can be said concerning the capacity of M II to cause a mate-killer effect, since we had no stocks of P. calkinsi at our disposal that were of complementary mating type. The endocytobionts from P. woodruffi had been shown to cause mate-killing [6]. The morphology of M III-bacteria has features in common with N. macronucleata from the macronucleus of P. caudatum [15]. The different coloring by AgNO and the presence of the terminal electron-dense zones in the cytoplasm of M III, however, is a significant difference between these bacteria. In combination with the Holospora infection they also behave differently: N. macronucleata replaces H. obtusa [1], while M III-bacteria are lost as a result of the development of H. curvata in the macronucleus. The study permits us to consider the representatives of the Holospora genus found in the macronucleus of P. calkinsi to be different species. The bacteria found in the cells of the stock Br 15-7 differ from H. bacil/ata in size: average length of the infectious form of the latter is 12.7 fim , whereas in H. curvata it is 18.5 urn, Infectious H. bacil/ata are never curved, whereas in H. curvata the infectious form is always undulated. The two bacteria differ also in their host specificities: H. curvata cannot be maintained for a long time in P. woodruffi whereas H. bacil/ata infects and can be maintained for a long time in the macronucleusof both of these paramecium species in the laboratory as well as in nature. The observations of the graded infectivity of H. curvata in the paramecia tested indicate that the invasion into the nucleus affords various steps of recognition and communication between host cell and bacterium. Similar results
Endocytobionts of Paramecium calkinsi . 395
have been obtained earlier by the author for H. obtusa and H. undulata [9, 10], and observations on a graded infectivity of H. obtusa and different species of Paramecium were also made by Fujishima and Fujita [12] and Fujishima [11]. Acknowledgements We are grateful to Drs H.-D. Cortz and T. J. Kosco for critically reading the manuscript and for some technical advice.
References 1 Fokin S. I. (1987): Paramecium caudatum: morphological analysis of consequences of infection with different nuclear symbionts. Cytologia, 30, 471-477 (in Russian with English summary). 2 Fokin S. I. (1988): A bacterial symbiont of the macronucleus perinuclear space in the ciliate Paramecium duboscqui. Cytologia, 30, 632-635 (in Russian with English summary). 3 Fokin S. I. (1989a): Bacterial endobionts of the ciliate Paramecium woodruffi. I. Endobionts of the macronucleus. Cytologia, 31, 839-844 (in Russian with English summary). 4 Fokin S. I. (1989b): Bacterial endobionts of the ciliate Paramecium woodruffi. II. Endobionts of the perinuclear space. Cytologia, 31, 845-850 (in Russian with English summary). 5 Fokin S. I. (1989c): Bacterial endobionts in the ciliate Paramecium woodruffi. III. Endobionts of the cytoplasm. Cytologia, 31, 964-970 (in Russian with English summary). 6 Fokin S. I. (1989d): Mate-killer effect induced by endobionts of the ciliate Paramecium woodruffi. Cytologia, 31, 1085-1089 (in Russian with English summary). 7 Fokin S. I. (198ge): Employment of endobionts in taxonomy of Paramecium species, p.75. In: Ecology of marine and freshwater protozoans. Abstr. of the II symposium, Yaroslavl (in Russian with English title). 8 Fokin S. I. (1991): Holospora recta sp. nov. - a micronucleusspecific endobiont of the ciliate Paramecium caudatum. Cytologia, 33, 135-141 (in Russian with English summary). 9 Fokin S. I. and Skovorodkin I. N. (1991): Holospora obtusaendonucleobiont of the ciliate Paramecium caudatum in search for the macronucleus. Cytologia, 33, 64-75 (in Russian with English summary). 10 Fokin S. I. and Skovorodkin I. N. (1991): Holospora undulata - an endobiont of the ciliate Paramecium caudatum in search ofthe micronucleus. Cytologia, 33, 101-115 (in Russian with English summary). 11 Fujishima M. (1986): Further study of the infectivity of Holospora obtusa, a macronucleus-specific bacterium of the
ciliate Paramecium caudatum. Acta Protozool., 25, 345-350. 12 Fujishima M. and Fujita M. (1985): Infection and maintenance of Holospora obtusa, a macronucleus-specific bacterium of the ciliate Paramecium caudatum. J. Cell Sci., 76, 179-187. 13 Fokin S. I., Boss A. O.-L. and Ossipov D. V. (1987a): A virus-containing cytoplasmic symbiont of the ciliate Paramecium woodruffi. Cytologia, 29, 1303-1306 (in Russian with English summary). 14 Fokin S. I. and Cortz H.-D. (1993): Caedibacter macronucleorum sp. nov., a bacterium inhabiting the macronucleus of Paramecium duboscqui. Arch. Protistenkd. 143, 319-324. 15 Fokin S. I., Ossipov D. V., Skoblo I. I., Rautian M. S. and Sabaneyeva E. V. (1987): Nonosporamacronucleata g.n., sp. n. - A vegetative nucleus symbiont of the ciliate Paramecium caudatum. Cytologia, 29, 963-970 (in Russian with English summary). 16 Fokin S. I. and Sabaneyeva E. V. (1990): Paramecium (Ciliophora, Peniculina) from water with low salinity of the Barents Sea and White Sea coasts and their endobionts, pp. 139-140. In: Ecology, reproduction and guarding of the bioresources of the North Europe seas. Abstr. of the III conference, Murmansk (in Russian). 17 Cortz H.-D. (1986): Endonucleobiosis in ciliates. Int. Rev. Cytol., 102, 169-213. 18 Gromov B. V. and Ossipov D. V. (1981): Holospora (ex Hafkine, 1890) nom. rev., a genus of bacteria inhabiting the nuclei of Paramecium. Int. J. Syst. Bacteriol., 31, 348-352. 19 Heckmann K. and Gortz H.-D. (1990): Prokaryotic syrnbionts of ciliates. In: Balows A., Truper H. G., Dworkin M., Harder W. and Shleifer K. H. (eds.): The prokaryotes, 2nd ed., pp. 3865-3890. Springer Verlag, Berlin and New York. 20 Ossipov D. V., Fohn S. I., Borchsenius O. N., Lebedeva N. A., Rautian M. S. and Skoblo I. I. (1989): Diversity and distribution of the intranuclear symbionts of ciliates of the Soviet Union, p. 53. In: Ecology of marine and freshwater protozoans. Abstr. of the II symposium, Yaroslavl (in Russian with English title). 21 Preer L. B. (1969): Alpha, an infectious macronuclear symbiont of Paramecium aurelia. J. Protozool., 16, 570-578. 22 Preer J. R. Jr. and Preer L. B. (1984): Endosymbionts of protozoa, pp.795-813. In: Krieg N. R. (ed.): Bergey's manual of systematic bacteriology, vol. 1. Williams and Wilkins, Baltimore and London. 23 Skoblo I. I., Borchsenius O. N., Lebedeva N. A. and Ossipov D. V. (1985): New species of bacterial cytoplasmic symbiont of ciliate Paramecium bursaria (Ciliophora, Protozoa). Cytologia, 27, 1292-1297 (in Russian with English summary). 24 Skovorodkin I. N. (1990): A device for immobilization biological objects in the light microscope studies. Cytologia, 32,301-302 (in Russian with English summary). 25 Sonneborn T. M. (1970): Methods in Paramecium research, pp. 241-339. In: Methods in cell physiology, vol. 4. Acad. Press, New York. 26 Wichterman R. (1986): The biology of Paramecium, 2nd ed. Plenum Press, New York.
Key words: Bacteria - Endocytobionts - Paramecium - Cytoplasm - Nucleus
Sergei Fokin, S. Kovalewskoj 1411,93, St. Petersburg, 195249 Russia