Spirostomum spp. (Ciliophora, Protista), a suitable system for endocytobiosis research

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Protoplasma (2005) 225: 93–102 DOI 10.1007/s00709-004-0078-y

PROTOPLASMA Printed in Austria

Spirostomum spp. (Ciliophora, Protista), a suitable system for endocytobiosis research S. I. Fokin1,2,*, M. Schweikert 2, F. Brümmer 2, and H.-D. Görtz 2 1 2

Biological Research Institute, St. Petersburg State University, St. Petersburg Biological Institute, University of Stuttgart, Stuttgart

Received April 1, 2004; accepted July 14, 2004; published online May 4, 2005 © Springer-Verlag 2005

Summary. Among ciliate genera, only Paramecium and Euplotes species have been studied extensively as host organisms of bacterial endocytobionts. In this article, we show that members of the genus Spirostomum may also serve as a suitable system for endocytobiosis research. Two strains of Spirostomum minus (Heterotrichea, Ciliophora) collected in Germany and Italy, respectively, were found to harbor different types of bacterial infections. Bacteria of various sizes and shapes were observed in the cytoplasm or in the nuclei of the ciliates. The bacteria in the cytoplasm were either surrounded by a peribacterial membrane or lay naked. One of the bacterial species was found in the vicinity of the contractile fibrillar system (myonemes) of the ciliates. In rare cases, another type of bacteria was observed associated with mitochondria. The macronuclei of both the Italian and the German strains were crowded with endocytobionts. The endonuclear bacteria in the two S. minus strains differed with respect to their cytoplasmic structures but they were of similar size and both were rod shaped. According to the results of in situ hybridization, the endonuclear bacteria of the Italian strain belong to the subgroup of alphaproteobacteria, whereas the bacteria associated with the fibrillar system appeared to be gram-positive bacteria with high GC content. While both the German and the Italian strains were found to permanently maintain their endocytobionts, they were at least partly colonized by different bacteria. This is taken as an indication that geographically separated populations of ciliates may be stably infected by different endocytobionts, possibly due to different ecological conditions. For S. minus and S. ambiguum a total of 7 different bacterial endocytobionts have now been recorded. We recommend the members of the genus Spirostomum as a suitable system for endocytobiosis research. Keywords: Endocytobiosis; In situ hybridization; Intracellular bacteria; Intranuclear bacteria; Spirostomum; Ultrastructure.

Introduction Ciliates appear to be ideal hosts for numerous microorganisms that have developed various adaptations for the intra* Correspondence and reprints (present address): Laboratorio di Protistologia, Dipartimento di Etologia, Ecologia ed Evoluzione, Università di Pisa, Via A. Volta 4, 56126 Pisa, Italy. E-mail: [email protected]

cellular way of life (Preer and Preer 1984; Görtz 1988, 1996; Heckmann and Görtz 1991; Fokin 1993). Endocytobioses in ciliates show a broad spectrum of intercellular communication of prokaryotes and other microorganisms with their eukaryotic hosts. This diversity of symbiosis may range from parasitic to mutualistic, but few cases have yet been investigated. In this paper, we therefore use the term endocytobiosis. When using the term symbiosis, we use the definition given by De Bary (1879) of individuals of different species living in a close association. Ciliates certainly have some significant preadaptations for becoming hosts of other organisms (Görtz 1996). One of the most important features may be the mode of nourishment by phagocytosis in the majority of ciliates. However, even among the bacterivorous representatives of the phylum, the number of such bacterial endocytobioses seems to differ dramatically from one group to another (even within a genus) (Fokin 1993; Fokin et al. 1996, 2000). At present, bacterial symbionts have been recorded for about 200 ciliate species (Görtz 1983; Fokin 1993, 2004; Ossipov et al. 1997), which is likely to be only a small part of their true number. It should be emphasized that there have been no diversity-directed investigations of “bacteria-ciliate” systems. Whereas most cases of bacterial endocytobiosis in ciliates should be regarded as being facultative, in some cases, such as in the symbiosis of Polynucleobacter necessarius with Euplotes aediculatus, the host may even depend upon its symbionts. In other cases, e.g., in ciliates of anaerobic environments that are always associated with methanogenic bacteria, the symbionts appear to be extremely favorable for the host (for reviews, see Heckmann et al. 1983, Fenchel and


S. I. Fokin et al.: Endocytobiosis research in Spirostomum spp.

Finlay 1991, Görtz and Schmidt 2004). In previous studies, symbiotic systems with bacteria have been intensively investigated, but almost exclusively in species of the genera Paramecium, Euplotes, and Parauronema (Preer et al. 1974, Ossipov 1981, Heckmann et al. 1983, Görtz 1983, Soldo 1987, Fokin 1993). Other ciliates have not attracted particular attention as possible hosts for bacteria, despite many occasional observations of such infections (see Görtz 1983, Fokin 1993, Görtz and Schmidt 2004). During our field excursions (1999–2002) to different locations we have collected several stocks of two Spirostomum species infected with bacteria (Fokin et al. 2003b) showing a considerable variety in morphology and a whole spectrum of localization within the host cells. On the basis of our observations, we recommend the members of the genus Spirostomum as a system suitable for endocytobiosis research. Here Spirostomum minus Roux, 1901 is described as a bacterial microcosm. Some results of the study were presented during the 22nd annual meeting of the German Society of Protozoology. Material and methods The following Spirostomum minus cultures, bearing different bacteria, were used: strain IEA2-1, isolated from a puddle on the coastline of Elba Island (near Azzuro city), Italy, 1999; strain GBF-1, isolated from a small pond near the village of Oberhaugstedt, Black Forest, Germany, 2000; a strain of S. minus, isolated from a small pond near the Federsee, Germany, 2001. Several uninfected laboratory stocks of S. minus, S. teres, and S. ambiguum of different clonal age and geographical origin were used for infection and “killer” experiments. Cells were cultivated at 18–20 C in lettuce medium inoculated with Enterobacter aerogenes or in a decoction of cereal leaves and wheat straw in-

oculated with Pseudomonas putida, which apparently did not interfere with the infections. Living cells were immobilized for observation with the help of a compression device (Skovorodkin 1990). Living and fixed cells were examined by phase contrast or Nomarski interference contrast (DIC) microscopy with a Zeiss Axioskop. For the investigation of intracellular bacteria, the material was fixed for electron microscopy according to a protocol used before (Fokin 1989). For in situ hybridization, cells were fixed with 4% (w/v) formaldehyde in phosphate-buffered saline solution, pH 7.2, for 2 h in a reaction vial and washed with phosphate-buffered saline. Cells were then incubated with oligonucleotide probes in hybridization buffer (750 mM NaCl, 75 mM sodium citrate, 0.1% sodium dodecylsulfate, pH 7.2, with and without 30–35% formamide) at 46 C for 1.5 h. Then, specimens were washed twice with the same buffer at 48 C. Cells were mounted on slides with Citifluor (PLANO). A fluorescein isothiocyanate-labeled eubacteriaspecific probe (5-GCTGCCTCCCGTAGG-AGT-3) (Amann et al. 1990, 1991) and additional tetramethylrhodamine isothiocyanate-labeled probes specific for alphaproteobacteria (5-GCGTTCGCTCTGAGCCAG-3), betaproteobacteria (5-GCCTTCCCACTTCGTTT-3), gammaproteobacteria (5-CTCTATCGGGCAGATTCCTAT-3), and deltaproteobacteria (5-CGGCGTCGCTGCGTCAGG-3), as well as gram-positive bacteria (5-TATAGTTACCACCGCCGT-3), were manufactured by MWG Biotech (Ebersberg, Federal Republic of Germany) and used at 1% of commercial strength. Specimens were investigated with a Zeiss LSM 410 confocal laser scanning microscope with a plan neofluar 100 oil immersion objective (numerical aperture, 1.3). For detection of fluorescein and tetramethylrhodamine isothiocyanate, an argon-ion laser (488 nm) and a helium-neon laser (543 nm) with appropriate emission filters, BP 510–525 nm and 575–640 nm, respectively, were used. The optical section in Z-axis corresponded to 1.5 m. Color pictures were taken from optical sections by a digital film recorder (Polaroid).

Results Different types of endocytobionts were found in the cytoplasm and the macronucleus (Ma) of S. minus from different locations (see Figs.1–19 and Table 1). All cells of the ciliates collected in a puddle near Azzuro city, Elba Island

Table 1. Characterization of bacterial endosymbionts recorded in S. minus a Host strain and bacterium

Data for bacterial endosymbiont Localization

IEA2-1 CB1a NB1 GBF-1 CB1b CB2 CB3

CB4 NB2 a b

cytoplasm, surrounded by membrane macronucleus cytoplasm, surrounded by membrane cytoplasm, free cytoplasm, free, associated with mitochondria mitochondrial matrix macronucleus

Reference or source

Size (m)

0.6  0.25

In situ hybridization

gram-positive, high GC alphaproteobacteria

Fokin et al. 2003, this study this study

ND b

this study

5–20  0.9–1.0 1.2–1.5  0.5

gammaproteobacteria ND

this study this study

0.6  0.2 1.5–3.0  0.4–0.5


Fokin et al. 2003 this study

1.5–3.0  0.4–0.5 0.6  0.25

CB1a and CB1b are probably the same species of bacterium In situ hybridization was not done

S. I. Fokin et al.: Endocytobiosis research in Spirostomum spp.


Figs. 1–5. Bacteria (NB-1 and CB1a) in the Ma and the cytoplasm of S. minus strain IEA2-1 investigated with DIC and fluorescence microscopy. Figs. 1–3, DIC, living cell. Figs. 4 and 5, in situ hybridization with two oligonucleotide probes; fluorescent microscopy. Green excitation, eubacteria; yellow excitation, alphaproteobacteria. Bars: Fig. 1, 20 m; Fig. 2, 5 m; Fig. 3, 3 m; Figs. 4 and 5, 25 m Fig. 1. General view of an infected ciliate in the contractile phase. OM Oral membranelles, CV contractile vacuole; the position of the Ma is indicated by arrows Fig. 2. Section of the Ma infected with numerous bacteria (arrows) Fig. 3. Crushed section of the Ma. Many bacteria can be seen (arrows) Fig. 4. Central part of the ciliate body. Bacterial infection in the Ma (arrows) and in the cytoplasm (asterisk) Fig. 5. Posterior part of the ciliate body. A number for cytoplasmic bacteria can be seen (asterisk)


S. I. Fokin et al.: Endocytobiosis research in Spirostomum spp.

Figs. 6–8. Cytoplasmic bacteria (CB1a) of S. minus strain IEA2-1 investigated by transmission electron microscopy (TEM). Bars: Fig. 6, 2 m; Fig. 7, 0.5 m; Fig. 8, 0.1 m Fig. 6. Part of the cytoplasm. The bacteria (CB1a) (asterisk) are preferably located along longitudinal fibrillar bundles. M Mitochondria; arrows, fibrillar bundles; star, area of scattered microtubules and endomembranes Fig. 7. Part of the cytoplasm close to myonemes. A group of bacteria with distinctive nucleoids (N) located to elements of the contractile system of the ciliate (arrows) can be seen Fig. 8. Dividing bacterium surrounded by a host membrane (arrows). N Nucleoid

S. I. Fokin et al.: Endocytobiosis research in Spirostomum spp.


Figs. 9–12. Macronuclear bacterium (NB-1) of S. minus strain IEA2-1 investigated by TEM. Bars: Fig. 1, 2 m; Fig. 10, 0.8 m; Figs. 11 and 12, 0.4 m Fig. 9. Two fragments of the Ma infected with bacteria (arrows). MI Micronucleus Fig. 10. Part of the infected Ma with a cluster of bacteria (arrow) Fig. 11. A group of bacteria in the Ma, cross section Fig. 12. Two bacteria surrounded by macronuclear chromatin bodies, one bacterium is dividing (arrow)


(Italy), were infected (Fig.1). Endocytoplasmic bacteria (CB1a) in this S. minus stock, IEA2-1, had some tendency to localize in the posterior end of the host cell (Figs. 4 and 5). They had a very restricted location inside of the cytoplasm, close to the contractile fibrillar system underneath the cortex of the host cell (Figs. 6–8). A number of these very small bacteria are located along longitudinal fibrillar bundles (“myonemes’’). The size of the CB1a endocytobionts was quite constant at 0.6 by 0.25 m (Table 1). The bacteria showed an inner structure regarded as a distinctive

S. I. Fokin et al.: Endocytobiosis research in Spirostomum spp.

bacterial nucleoid. The bacteria were surrounded by host membranes (Figs. 7 and 8). The phylogenetic position of CB1a, as tested by in situ hybridization with several oligonucleotide probes specific for different bacterial groups, was found to be within the gram-positive bacteria with high GC content (Table 1). The Ma of all individuals investigated from the same ciliate stock (IEA2-1) were densely populated with a second type of bacteria (NB1): rod-shaped particles with a size of 1.5–3.0 by 0.4–0.5 m (Figs. 2–5 and 9–12). They

Figs. 13–15. Cytoplasmic bacteria (CB2) of S. minus strain GBF-1 investigated with DIC and phase contrast microscopy. In Figs. 14 and 15, infections of the macronucleus can also be seen. Bars: 5 m Fig. 13. Part of a fixed infected cell stained with orceinlactate; bacteria are visible as long dark rods (arrows) Fig. 14. Part of the infected stained cell; DIC; refractile spots are indicated with arrows Fig. 15. Part of the infected stained cell; phase contrast; refractile spots are indicated with arrows

S. I. Fokin et al.: Endocytobiosis research in Spirostomum spp.


Figs. 16–19. Cytoplasmic and macronuclear bacteria of S. minus strain GBF-1 investigated with TEM. Bars: Fig. 16, 1 m; Fig. 17, 0.75 m; Fig. 18, 1.5 m; Fig. 19, 0.5 m Fig. 16. Cytoplasmic bacterium (CB2) located close to the Ma which is infected with different microorganisms (NB-2) (arrow) Fig. 17. Two types of cytoplasmic bacteria: the first associated with mitochondria (CB3) (star) and the second (CB1b) located close to the contractile system of host cell (asterisk) Fig. 18. Part of the infected Ma Fig. 19. Bacteria (NB-2) inside the Ma. Some contain longitudinal fibrillar (paracrystalline?) structures (arrow)


were present only as vegetative forms showing a homogeneous cytoplasm (Figs.11 and 12) and lying “naked” in the karyoplasm. Usually, these microorganisms formed clusters inside the nucleus (Figs. 9 and 10); sometimes occupying most of the macronuclear space (Figs. 2–5). According to the results of in situ hybridization, these macronuclear symbionts are related to alphaproteobacteria (Figs. 4 and 5 and Table 1). No infectivity and no ability to produce a killer effect were revealed for either of these (cytoplasmic and endomacronuclear) bacteria. The macronuclear infection was very stable and has now been maintained in culture under different conditions (10–25 C) for up to 5 years. Investigation of the German S. minus stock GBF-1 revealed an even higher variety of symbionts (Figs.13–19 and Table 1). Large bacterial rods (CB2) with dimensions of 5–20 by 0.9–1.0 m overcrowded the cytoplasm of the ciliates (Figs.13–15). As a distinctive trait, they showed a number of refractile spots underneath their surfaces (Figs.14 and 15). The cell walls of these bacteria had a wavy surface (Fig.16) which, to some degree, appeared to be recognizable with the light microscope. In the bacterial cytoplasm, a number of small electron-transparent spots were always visible (Fig.16). This type of endocytobiont (CB2) was found in the cytoplasm of the ciliates, not within vacuoles made by host membranes. A positive signal was obtained by in situ hybridization with the probe specific for the gammaproteobacteria (Table 1). In the cytoplasm of the stock GBF-1, small bacteria (CB1b), similar to CB1a in the stock IEA2-1, were also found associated with the myoneme system of the host cell. Moreover, a different type of bacteria (CB3) was recorded close to mitochondria in some sections (Fig.17 and Table 1). Another type of microorganisms inside the mitochondrial matrix (CB4) has been briefly described previously (Fokin et al. 2003b). However, comparison of their size and cytoplasmic structure suggests that these are two different bacteria. The ultrastructural aspect of this tight association indicates that the CB3 bacteria located in mitochondrial invaginations may be interacting with these organelles (Fig.17). The Ma of ciliates in the German stock GBF-1 were infected with symbionts (NB2) which are apparently different from those (NB1) of the Italian stock (IEA2-1). Despite similarities in size and shape (rod particles with dimensions of 1.5–3.0 by 0.4–0.5 m), NB2 bacteria possessed cytoplasmic vacuoles. Some of them also contained fine fibrillar (paracrystal?) structures (Figs.18 and 19). A very similar infection in the Ma was found in the population of S. minus collected near the Federsee, Germany. It is, therefore, possible that infection with NB2 bacteria may not be rare for

S. I. Fokin et al.: Endocytobiosis research in Spirostomum spp.

S. minus. Unfortunately, the infections in the German stock GBF-1 became unstable and the bacteria were lost from the cells during laboratory cultivation. Therefore, almost no in situ hybridization could be carried out. Discussion Our data support the idea that endocytobiosis with bacteria is widely distributed among members of the subphylum Ciliophora. Also, the variety of intracellular bacteria appears to be great (Fokin 1993, 2004; Görtz and Schmidt 2004). Since endocytobionts observed in protists include pathogens and agents of opportunistic infections, their study seems all the more important (Görtz 1996, Görtz and Schmidt 2004). Bacterial infections in the macronucleus of S. ambiguum were found in 1960 in Japan and then, repeatedly, in the U.S.A. (Inaba 1960; Harrison et al. 1976a, b). While Harrison and co-authors could grow the endocytobionts of their strain of S. ambiguum on a blood agar, which is extremely rare for intracellular bacteria, our attempts to grow the different bacteria on blood agar or other media failed (data not shown). However, the bacterium observed by Harrison et al. (1976a, b) was not investigated in detail. Since that time, no bacteria in Spirostomum spp. were recorded, despite the wide distribution and frequency of some members of the genus, namely S. ambiguum, S. minus, and S. teres (Repak and Isquith 1974, Foissner et al. 1992). However, task-oriented search has recently revealed the presence of such endocytobioses in Spirostomum spp. in Europe as well (Fokin et al. 2003b). Though the diversity of microorganisms in the cytoplasm of ciliates is very high (Fokin 1993, Görtz and Schmidt 2004), no observations of bacteria associated with the contractile system of host cells have been reported (Fokin et al. 2003b). The association of endocytobionts with the fibrillar system is, thus, a rare peculiarity. On the other hand, it is known that some endocytobionts are confined to particular areas of the cytoplasm (Fokin et al. 2000, 2003a; Fokin 2004). We suggest that a specific bacterial distribution might be due in part to the heterogeneous distribution of key metabolites or nutrients in the host cell or be connected somehow with a particular relationship between the bacteria and certain structures or cell organelles of the host. The presence of a gram-positive bacterium is a further significant peculiarity, as gram-positive bacteria are rare among endocytobionts in ciliates (Fokin 2004). Examples of special relationships between organelles and endocytobionts are provided by the discovery of bacteria in the mitochondria of protists. These are extremely rare but involve phylogenetically different organisms: the

S. I. Fokin et al.: Endocytobiosis research in Spirostomum spp.

dinoflagellate Woloszynskia pascheri (Wilcox 1986) and the ciliates Halteria geleiana (Yamataka and Hayashi 1970), Urotricha ovata (Puytorac and Grain 1972) and, finally, S. minus (Fokin et al. 2003b). In U. ovata the bacteria were observed between the mitochondrial membranes, whereas in all other cases they were reliably demonstrated to occupy the matrix of these respiratory organelles. Bacteria associated with the surface of these organelles are, to our knowledge, unknown. However, one example of a siteassociated relationship between ectobiotic bacteria and mitochondria was found in Cyclidium sp. (Beams and Kessel 1973). These bacteria were located on the surface of the ciliate, exactly following the pattern of mitochondria arranged underneath the pellicle. An intimate association between bacteria and secondary plastids inside commonly specialized cisterns of the endoplasmic reticulum was found in the diatom Pinnularia sp. (Schmid 2003a). It was suggested that, in this case, bacteria and plastids form a functional unit (Schmid 2003a, b). The observation of bacteria within invaginations of mitochondrial membranes in our study and the presence of vesicles between bacterial and mitochondrial surfaces are taken as indications for a functional relationship between the endocytobionts and mitochondria. Apparently, some bacterial infections are widely distributed among at least two Spirostomum species (S. ambiguum and S. minus). It may be surprising that the two S. minus strains, Italian and German, show different bacteria, except perhaps for the bacteria associated with the fibrillar system, which may be identical. Two most likely explanations for the colonization of the two strains by different bacteria, all of which appear to be maintained in a stable endocytobiosis, are (1) that different ecological conditions (temperature, light intensity, etc.) favor different types of endocytobiosis, and (2) that infections of certain areas in a cell (nucleus, certain sites in the cytoplasm) may no longer be invaded by microorganisms once these sites are occupied, according to the “first come, first served” principle. Further investigations may clarify which explanation is more likely. Altogether, we have now recorded seven different endocytobionts for these ciliates. Among them are a killer bacterium in the macronucleus (Fokin et al. 2003b), a bacterium intimately associated with mitochondria, and another one associated with the contractile system of the host cell. This diversity of endocytobionts in Spirostomum spp. and their advantages as laboratory organisms make the ciliates a suitable system for endocytobiosis research. Cultivation of Spirostomum species should be easy under laboratory conditions, such as those used for Paramecium spp. using the same food bacteria. Spirostomum spp. are much larger in size than the majority of ciliates (the


medium length of S. minus being 800 m; that of S. ambiguum, 1500 m). Therefore, the biomass of bacteria populating the cells of Spirostomum spp. (if any) can be significantly greater than that in other ciliates, which is important for biochemical and molecular biological investigations. Although no genetic investigations have been carried out in Spirostomum spp., ciliates in general offer good possibilities for molecular genetics. The large and very DNA-rich macronuclei of Spirostomum species provide an excellent source for DNA isolation. They also offer good access for microinjection of host DNA and endosymbionts. Mitochondria can be purified from the large cells in high quantities. PCR techniques combined with a microinjection tool would allow fascinating studies of putative horizontal gene transfer between endocytobionts and hosts and of other molecular interactions – an extremely interesting topic, as yet uninvestigated in ciliate-bacteria systems. Acknowledgments We are grateful to M. T. Ghiselin for correcting our English. This work was supported by a grant from DAAD (325/trilateral project.), Germany, to H.-D. Görtz. The excellent technical assistance by I. Polle is acknowledged.

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