Protistology 2 (1), 48–53 (2001) April, 2001 Flagellated endosymbiotic in a marine sp. (, Peniculida)

Giovanna Rosati, Letizia Modeo, Giulio Petroni and Susanna Bertolini Dipartimento di Etologia, Ecologia, Evoluzione via A. Volta 4–6, 56126 Pisa, Italy

Summary

Frontonia sp. was repeatedly found in sea water samples collected from a shore near Leghorn (Ligurian sea). Endosymbiotic rod shaped bacteria 5–6 µm long were found in the of each Frontonia sp. specimen examined at the fluorescent following DAPI staining procedure. These bacteria, as revealed by electron , are contained in (one or two symbionts are present in each ) and are covered by numerous flagella which are in close contact with the vacuolar membrane. Flagellated symbiotic bacteria in have been seldom described. All the cases referred in the literature concern , a genus that mainly consists of fresh-water species. This is the first record of flagellated bacteria living in a marine species belonging to a different taxon. The fact that these symbionts have been constantly observed in just collected Frontonia and are maintained in specimens grown in the lab for at least one month, suggests that we are not dealing with an occasional relationship. The abundance of cytoplasmic vesicles and glycogen particles, around and sometimes within the vacuoles, suggests a movement of metabolites between the host cytoplasm and the symbionts. The bacteria maintained their motility once outside the host cell.

Key words: ciliates, endosymbiosis, flagellated bacteria, Frontonia, symbiosis, ultra- structure.

Introduction production of sulfide in close proximity to a source of oxygen. In some cases these bacteria are vital for the host Bacterial symbionts have been observed in a variety (Bauer-Nebelsick et al., 1996). of ciliate species. Many reports deal with occasional or As concern endosymbiosis, well known is the mutual- not understood associations. Nevertheless a certain num- istic association between ciliates adapted to an anaerobic ber of well established relationships between ciliates and stile, particularly those possessing , bacteria have been described and studied in detail. Fresh- and methanogenic bacteria. The symbionts use intracellu- water species of a major group of the lar hydrogen as a substrate for methane formation: in this depend upon symbionts (Heckmann, 1975; Schmidt and way they guarantee to their hosts the partial low pressure Heckmann, 1980). The best known species is of hydrogen required for the functioning of necessarius (Heckmann and Schmidt, hydrogenosomes (Fenchel and Finlay, 1995). It has been 1987): it cannot be grown outside its host nor can Euplotes suggested a multiple acquisition and replacement of en- grow and multiply without the bacterium. Both organisms dosymbiotic methanogenic bacteria during their host form an evolutionary entity. In other permanent associa- adaptation to the various anaerobic ecological niches (van tions, it has been demonstrated that the symbiotic bacteria, Hoek et al., 2000): this represents a further indication that although not essential, provide true benefits to their ciliate we are dealing with an association truly favorable for both host. Epixenosomes (Rosati, 1999), ectosymbiotic bacte- partners. ria related to (Petroni et al., 2000), defend A number of different endosymbiotic bacteria confer their host, namely ciliates of Euplotidium genus, from killer traits upon their ciliate host. This trait has been re- predators (Rosati et al., 1999). involving ported in different species of Paramecium (for review see sulphate reducers bacteria are widespread in ciliates liv- Preer et al., 1974), in Parauronema acutum (Soldo and ing in the anaerobic environment (Fenchel and Finlay, Brickson, 1978) and in some Euplotes species (Heckmann 1995), while sulfur-oxidizer bacteria can be found on cili- et al., 1967; Nobili et al., 1976). In every case cells bear- ates living in habitats whose common denominator is the ing these symbionts may kill cells of other strains (or even © 2001 by Russia, Protistology. Protistology · 49 other species) that do not bear symbionts. While the sym- Observations by the fluorescence bionts that confer the killer trait to Euplotes have not been following DAPI staining procedure were made either on identified, those of Paramecium have been diffusely stud- specimens just collected from the sea at different times or ied and included in different genera and species (for review on specimens of various cultures grown in the lab for at see Quackembush, 1988). The most known are bacteria of least one month. Hundreds of Frontonia sp.. individuals Caedibacter genus, most of which are able to produce R were examined on the whole. In every case rod shaped bodies (refractile bodies). Very likely the presence of killer bacteria 5–6 mm long were observed in the cytoplasm, symbionts means a selective advantage in intraspecific always with an apparently similar density (Figs 2, 3). In competition for food. some cases a few curved or spiral bacteria were also iden- In the present paper the constant association between tified (Fig. 3). flagellated bacteria and the marine ciliate Frontonia sp. is Electron microscopical observation showed (Figs 4, described for the first time. 5) that the bacteria are delimited by two membranes, have a diameter of about 0.5 mm and a wrinkled surface from which numerous flagella emerge. The flagella, that do not Material and Methods have appendages, completely surround the cell. The bac- teria are enclosed in cytoplasmic vacuoles bounded by Samples of sand and seawater were repeatedly col- smooth membranes, not regularly lined up by ribosomes lected during two years (1997–1998) from tidal pools in a along the host cytoplasm side. They are separated from rocky shore near Leghorn (Ligurian Sea). The samples the host by a wide space, the vacuolar space. In limited were transferred in the lab and checked for the presence zones the bacterial and host membranes are however in of Frontonia. Once identified, specimens of Frontonia close contact with each other (Figs 4, 5). One or two (Figs were singly picked up with a micropipette and used in dif- 6, 7) symbionts are present in the same vacuole. Probably, ferent ways: a) transferred in artificial sea water enriched in many cases dividing individuals have been found (Fig. with the green Dunaliella salina and the 8). The flagella extend from the bacterial surface into the Pheodactilum tricornutum to start cultures; b) fixed with vacuolar space, reaching the vacuolar membrane (Figs 4– 15% formaldehyde in distilled water added with 0.25M 7). In the host cytoplasm surrounding the vacuoles NaCl for DAPI staining procedure; c) processed for scan- numerous membrane bounded vesicles of various size, ning electron microscopy; d) processed for transmission some of which contain an amorphous material, are present electron microscopy as described below. The latter three (Figs 4–7). methods were successively applied to specimens picked Thiéry procedure, specific for polysaccharidic sub- up from cultures grown in the lab for at least one month. stances, revealed that also glycogen granules, in the form of small granules (b particles), are particularly abundant Electron microscopical techniques in these cytoplasmic regions (Fig. 9). Occasionally glyco- For observation at the scanning electron microscope gen granules are present even in the vacuolar space (Fig. (SEM) specimens of Frontonia sp. were fixed with 2% 10). In the same pictures cytoplasmic vesicles can be seen OsO in sea water, placed on coverslips coated with poly- 4 in close contact with the vacuole membrane. Their mem- L-lysine hydrobromide, dehydrated in ethanol and, after branes, at variance with the vacuolar one, positively reacted critical point drying, coated with gold and examined at a to Thiéry procedure. Only rarely the curved bacteria could Jeol/JSM-5410. be identified on thin sections; anyway they too possess For transmission electron microscopy (TEM) the flagella (Fig. 11). specimens were fixed with 1:1 mixture of 5% glutaralde- The endosymbiotic bacteria of Frontonia can be seen hyde in 0.2 M cacodylate buffer (pH 7.4) and 4% OsO in 4 moving for a while under the interferential contrast micro- distilled water. Then after ethanol dehydration, they were scope only once the host cell is broken (for example by a embedded in epon-araldite mixture. The sections were pressure on the coverslip or for an altered salinity). This is contrasted with uranyl acetate and lead citrate prior to ex- a strong argument that they are really flagellated. amination in a SX 100 Jeol electron microscope. Some sections were picked up with mylar rings and treated ac- cording Thiéry procedure for polysaccharides (Thiéry, Discussion 1967). Endosymbiotic flagellated bacteria were present in all the specimens of Frontonia sp. examined either soon after Results their collection (they were collected repeatedly, at irregu- lar interval time, during two years) or after one month of A specimen of Frontonia sp. as it appears at SEM is permanence in the lab. The constancy of the finding, the shown in figure 1. observation that the flagellated bacteria are able to repro- 50 · Giovanna Rosati, Letizia Modeo, Giulio Petroni and Susanna Bertolini

Figs 1–5. Frontonia sp. and its symbionts: 1 – ventral view of the ciliate at the scanning electron microscope (arrow points to the ); 2, 3 – photographs printed down from colored slides of DAPI stained specimens (2 – the nuclear apparatus and rod shaped bacteria in the cytoplasm are evidenced, 3 – at higher magnification some curved bacteria (arrows) can be distinguished among the rod shaped ones); 4, 5 – different sections of rod shaped bacteria (arrows point to the flagella, asterisks indicate the bacterial and vacuolar membranes in close contact). CV – cytoplasmic vesicles, Ma – , Mi – micronuclei, VM – vacuolar membrane, VS – vacuolar space. Scale bars: 1, 2 – 50 µm, 3 – 10 µm, 4, 5 – 1 µm. Protistology · 51

Figs 6–11. Electron microscopy of the bacteria: 6, 7 – two symbionts are present in the same vacuole; 8 – a bacterium during binary ; 9, 10 – sections stained according Thiéry procedure for polysaccharides (glycogen granules are visible in the cytoplasm surrounding the vacuole and inside the vacuole itself; arrows indicate cytoplasmic vesicles whose membrane positively reacted to the staining procedure.); 11 – a section of a curved bacterium. DV – digestive vacuole. Scale bars: 1 µm throughout. 52 · Giovanna Rosati, Letizia Modeo, Giulio Petroni and Susanna Bertolini duce within the host cytoplasm and that they are main- forms are rare: the difficulty to identify them in thin sec- tained in the stocks grown in an artificial environment, tions rendered it impossible to determine whether they suggest that we are not dealing with an occasional rela- represent a particular stage of the life cycle or they are a tionship but with a well established association, very likely different species. advantageous for both partners. The presence of cytoplas- As far as we know none of the flagellated bacteria mic vesicles and glycogen granules in close association living in Paramecium species have been identified: they with the vacuolar membrane suggests movements of me- have been distinguished and classified on the basis of their tabolites between the host cytoplasm and the symbionts morphological characteristics and their relationship with (Bigliardi et al., 1989). So, whatever the nature of the as- the host. An indication obtained by in situ hybridization sociation between the ciliate host and the flagellated with labeled group-specific oligonucleotides concerns the bacteria, apparently this symbiosis involves a highly inte- motile intranuclear symbionts of P. multimicronucleatum grate system. (Vishnyakov and Rodionova, 1999): they are Eubacteria, Ultrastructural studies on Frontonia species are scanty but a probe specific for Holospora (Amman, 1996), a well and limited to some features such as the cortex of F. leucas. studied genus of infectious intranuclear symbionts in Para- (Gil, 1981) and the of F. vesiculosa (Yusa, mecium (Görtz, 1988; Fokin et al., 1996), did not show 1965). So, although not reported in those studies, the pres- any positive signal. It would be of a great interest to deter- ence of cannot be excluded. Some kind of mine the possible phylogenetic relationships among the bacteria were reported in Frontonia sp. by Fokin (1993). flagellated bacterial endosymbionts found in various Para- Anyway, considering also that Frontonia sp. specimens mecium species and in Frontonia. here studied were all collected along the same shore, fur- ther investigations are needed to consider this association constant and widespread in nature. Acknowledgements Endosymbiotic, flagellated bacteria have been de- scribed in a certain number of Paramecium species. In The authors thank Simone Gabrielli for photographic work some cases these bacteria inhabit the macronuleus. Flag- and are grateful to Prof. F. Giorgi for the kind hospi- ellated intranuclear symbionts, able to move in caryoplasm, tality in his lab of fluorescence microscopy. have been found in P. multimicronucleatum collected in two very remote geographical areas (Vishnyakov and Rodionova, 1999). Cytoplasmic flagellated bacteria in cili- References ates were firstly described in P. aurelia as lambda and sigma particles (Beale et al., 1969). They are among the bacteria Amman R. 1996. In situ visualization of high genetic di- able to confer the killer trait of the “rapid lysis” type to versity in a natural microbial community. J. Bacteriol. their host and are now designated Lyticum flagellatum and 178, 34–96. L. sinuosum respectively (Görtz, 1988). They do not pro- Bauer-Nebelsick M., Bardele C.F. and Ott J.A. 1996. Re- duce R bodies. Cytoplasmic flagellated bacteria have been description of niveum (Heprich et also reported in stocks of P. caudatum (Boss et al., 1987) Ehrenberg, 1831) Ehrenberg, 1838 (Oligohy- and P. woodruffi (Fokin, 1989) and were named menophora, Peritrichida) a ciliate with ectosymbiotic, Pseudolyticum multiflagellatum and Ps. minutus respec- chemoautotrophic bacteria. J. Protistol. 32, 18–30. tively; they both do not produce real R bodies but different Beal G.H., Jurand A. and Preer J.R. 1969. The classes of structures, do not confer killer trait to their host and do not of . J. Cell. Sci. 5, infect the endosymbiont-free cell lines. They were never 65–91. observed to move either in the cytoplasm of the ciliate or Bigliardi E., Selmi M.G., Baccetti B., Sacchi L., Grigolo outside the cell. Some motile infectious bacteria were re- A. and Laudani U. 1989. Membrane Systems in ported in the cytoplasm of P. caudatum by Dieckmann Endocytobiosis. I. Specialization of the vacuolar mem- (1981). brane in bacteriocytes of Blattella germanica (L.) Here we describe for the first time flagellated bacte- (Dictyoptera Battellidae). J. Ultrastruct. Mol. Struct. ria in the cytoplasm of a ciliate species that do not belong Res. 102, 66–70. to Paramecium genus and that, differently from most Para- Boss A.O.-L., Borchesenius, N.O. and Ossipov D.V. 1987. mecium species, is marine. These bacteria maintained their Pseudolyticum multiflagellatum N.G.,N. Sp. – A new motility once outside the host cell. Most of them are rod symbiotic bacterium in the cytoplasm of Paramecium shaped and, except for a major length, they are very simi- caudatum (, ). Tsitologiya (S.-Peters- lar to L. flagellatum in morphology and localization. The burg). 29, 94–99 (in Russian with English summary). possibility that, like the latter symbionts, they confer the Dieckmann J. 1981. An infectious bacterium in the cyto- killer capacity to their host could not be tested, as sym- plasm of . Abstr. Int. Congr. biont-free Frontonia sp. were not available. The curved Protozool., 6th, Warsaw. ð.77. Protistology · 53

Fenchel T. and Finlay B.J. 1995. Symbiosis with prokary- structures are bacteria related to Verrucomicrobia. otes: intracellular syntrophy. In: Ecology and evolution Proc. Nat. Acad. Sci USA 97, 1813–1817. in anoxic worlds (Eds. May R.M. and Harvey H.P.). Preer J.R., Preer L.B. and Jurand A. 1974. Kappa and other Oxford University Press, Oxford. pp. 135–160. endosymbionts in Paramecium aurelia. Bacteriol. Rev. Fokin S.I. 1989. Bacterial endobionts in the ciliate Para- 38, 113–163. mecium woodruffi. Endobionts of the cytoplasm. Quackembush R.L. 1988. Endosymbionts of killer Para- Tsitologiya (S.-Petersburg). 31, 960–970 (in Russian mecia. In: Paramecium (Ed. Görtz H.-D.) Springer- with English summary). Verlag, Heidelberg. pp. 406–417. Fokin S.I. 1993. Bacterial endobionts of ciliates and their Rosati G. 1999. Epixenosomes: symbionts of the hypotrich employment in experimental protozoology. Tsitologiya ciliate Euplotidium itoi. Symbiosis 26, 1–23. (S.-Petersburg). 35, 59–91 (in Russian with English Rosati G., Petroni G., Quochi S., Modeo L. and Verni F. summary). 1999. Epixenosomes peculiar epibionts of the Fokin S.I., Brigge T., Brenner J. and Görtz H.D. 1996. Hypotrich Ciliate Euplotidium itoi defend their host Holospora species infecting the nuclei of Paramecium against predators. J. Eukaryotic Microbiol. 46, 278– appear to belong in two groups of bacteria. Europ. J. 282. Protistol. 32, 19–24. Schmidt H.J. and Heckmann K. 1980. DNA of Omikron. Gil R. 1981. Cortical fine structure of Frontonia leucas In: Endocytobiology Endosymbiosis and cell Biology (Ciliata: ). Trans Am. Microsc. Soc. 100, (Eds. Schwemmler W. and Schenk H.E.A.). Walter 373–383. de Gruyter and Co, Berlin – New York. pp 401–407. Görtz H.D. 1988. Endocytobiosis. In: Paramecium (Ed. Soldo A.T. and Brickson S.A. 1978. Observations on the Görtz H.-D.). Springer-Verlag, Heidelberg. pp. 393– the ultrastructure, mode of infectivity, and host range 402. of xenosomes. and Cell. 10, 284–286. Heckmann K. 1975. Omikron, ein essentieller Endosym- Thiéry J.P. 1967. Mise en évidence des polysaccharides biont von Euplotes aediculatus. J. Protozool. 22, sur coupes fines en microscopie électronique. J. 97–104. Microscopie 6, 987–1018. Heckmann K. and Schmidt H.J. 1987. Polynucleobacter Van Hoek A.H.A.M., van Alen T.A., Sprakel V.S.I., necessarius gen. nov., sp. nov., an obligately endo- Leunissen J.A.M., Brigge T., Vogels G.D. and symbiotic bacterium living in the cytoplasm of Hackstein J.H.P. 2000. Multiple acquisition of Euplotes aediculatus. Inter. J. Syst. Bact. 37, 456– methanogenic archael symbionts by anaerobic ciliates. 457. Mol. Biol. Evol. 17, 251–258. Heckmann K., Preer J.R. and Straetling W.M. 1967. Cy- Vishnyakov A. and Rodionova G. 1999. Motile intra- toplasmic particles in the killers Euplotes minuta and nuclear symbionts of ciliate Paramecium their relationship to the killer substance. J. Protozool. multimicronucleatum. In: Endocytobiology VII (Eds. 14, 360–363. Wagner E., Normann J.,Greppin H., Hackstein R.G., Nobili R., Rosati G. and Verni F. 1976. The killer traits in Kowallik K.V., Shenk H.E.A., Seckbach J.). Univer- Euplotes crassus. Boll. Zool. 43, 251–258. sity of Geneva, Genève. pp. 169–177. Petroni G., Spring S., Schleifer K.-H., Verni F. and Rosati Yusa A. 1965. Fine structure of developing and mature G. 2000. Defensive extrusive ectosymbionts of trichocysts in Frontonia vesiculosa. J. Protozool. 12, Euplotidium (Ciliophora) that contain -like 51–60.

Address for correspondence: Giovanna Rosati, Dipartimento di Etologia, Ecologia, Evoluzione via A.Volta 4–6, 56126 Pisa, Italy. E-mail: [email protected].

The manuscript is presented by S.I.Fokin