1 LS Mikrobielle Ökologie, Fachbereich Biologie, Universität Konstanz, 78457 Konstanz, Germany
2 Institut für Biologie/Zoologie, AG Protozoologie, Freie Universität Berlin, Königin-Luise-Str. 13, 14195 Berlin, Germany
3 Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch-Strasse, 35043 Marburg, Germany
Correspondence
Andreas Brune
brune{at}staff.uni-marburg.de
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Flagellates of the genus Staurojoenina are found only among the dry-wood termites' (family Kalotermitidae) (Yamin, 1979; Dolan & Margulis, 1997
). In their paper, Dolan & Margulis (1997)
published excellent electron micrographs prepared by the late David Chase, which show that the surface of Staurojoenina assimilis from Incisitermes minor is covered with unusual epibiotic bacteria. The ultrastructure of the junctional complexes is completely different from that of the motility symbionts of Caduceia versatilis (Tamm, 1980
; d'Ambrosio et al., 1999
), and only remotely resembles the situation of the second epibiont of M. paradoxa (Cleveland & Grimstone, 1964
; Wenzel et al., 2003
). It is likely that also the nature of the association between Staurojoenina species and their epibiotic bacteria differs from those examples. In this study, we present a detailed analysis of the identity, phylogenetic position and ultrastructure of the epibionts associated with a hitherto undescribed Staurojoenina species colonizing the gut of Neotermes cubanus, and classify them in a provisional Candidatus taxon.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Scanning and transmission electron microscopy.
For scanning electron microscopy, worker larvae of N. cubanus were dissected, and the contents of the hindgut paunch were released into 0·2 M sodium phosphate buffer (pH 7·0) containing 2·5 % glutaraldehyde and 4 % OsO4. The samples were fixed for 1 h on ice, washed three times in the same buffer, dehydrated in a series of ethanol, and critical-point dried with a Bal-Tec CPD 030. Prior to the investigation with a Philips SEM 515 or a Fei Quanta scanning electron microscope, the samples were sputtered with gold in a Balzers Union SCD 040. For transmission electron microscopy, the flagellates were pre-fixed for 1 h in 0·05 M sodium cacodylate buffer (pH 7·0) containing 2·5 % glutaraldehyde, washed three times in the same buffer, and post-fixed in reduced OsO4 (a fresh 1 : 1 mixture of 2 % OsO4 and 3 % K4[Fe(CN)6]) for 1 h on ice. After several further rinses in buffer, the cells were embedded in 3 % agar, dehydrated in a series of ethanol, and embedded in Spurr's resin. Ultrathin sections were stained with saturated aqueous uranyl acetate for 30 min, followed by lead-citrate staining according to Reynolds (1963), and observed using a Philips EM 208 electron microscope.
Preparation of flagellates for DNA extraction.
Termite hindguts were carefully ruptured, and a suspension of gut flagellates was prepared as described previously (Stingl & Brune, 2003), except that the cells were not fixed with formaldehyde. An aliquot (15 µl) of the suspension was spotted into the first well of a 10-well Teflon slide (Roth). Using an inverted microscope, 5060 unambiguously identified flagellates of the genus Staurojoenina, which were identified by their characteristic morphology (Fig. 1A
), were aspirated from the suspension with a micropipette and transferred to a well containing 15 µl sterile PBS (Stingl & Brune, 2003
). This procedure was repeated twice to minimize the amount of loosely attached bacteria in the sample. Finally, 3040 flagellates were transferred to a sterile Eppendorf tube with 200 µl PBS. DNA was extracted with the NucleoSpin kit (Macherey-Nagel), according to the manufacturer's instructions, and eluted in 50 µl sterile water.
|
Sequencing and phylogenetic analysis.
The inserts of three randomly chosen clones were sequenced on both strands using primers 27F (Edwards et al., 1989), 533F, 907R (Lane et al., 1985
; used also reverse complementary), and 1492R (Weisburg et al., 1991
) by GATC (Konstanz, Germany). Sequences were checked and assembled using DNAStar software (http://www.dnastar.com). Phylogenetic analysis was done as described elsewhere (Stingl & Brune, 2003
), using the ARB program package (http://www.arb-home.de; Ludwig & Strunk, 1996
).
Whole-cell in situ hybridization.
Gut contents of five termites were suspended in 900 µl PBS and fixed for 12 h using 3·7 % formaldehyde. Hybridization procedure and conditions were as described in Stingl & Brune (2003). The oligonucleotide probe Bac303, designed to detect the Bacteroides/Porphyromonas subgroup of the CFB phylum (Manz et al., 1996
), was modified to achieve specificity for the sequences obtained in this study. Probe Sym_Stau_303: 5'-CCG GTG TGG GGG ACC TTC-3' had at least one mismatch to all other sequences available in GenBank (www.ncbi.nlm.nih.gov). Maximal formamide concentration for successful hybridization was 15 %, which was used routinely. Non-specific binding of the probes was excluded by checking every sample also with a nonsense probe (Wallner et al., 1993
). The information on probe Sym_Stau_303 has been submitted to probeBase (http://www.microbial-ecology.de/probebase; Loy et al., 2003
).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Transmission electron microscopy (TEM) of ultrathin sections of the flagellates revealed the Gram-negative cell wall of the epibiotic rods (Fig. 1C). In addition to the inner and outer membranes, the cells were surrounded by a diffuse outer layer (capsule). A plaque of electron-dense extracellular material connected the bacteria to the flagellate surface. The attachment sites of the flagellates had the form of elongated ridges, and electron-dense material below the attachment sites apparently serves to support the plasma membrane. Bacterial rods were found not only attached to the surface of the flagellates, but also in vacuoles (Fig. 1D
). The vacuoles often showed remnants of the attachment structures, which confirmed that the bacteria were taken up by phagocytosis. Although phagocytosis apparently occurs all over the non-flagellated surface, the highest density of vacuoles was observed in the posterior region of the host cell. While the bacteria in the tightly fitting vacuoles located close to the flagellate surface appeared fairly intact, those in loosely fitting vacuoles located deeper within the flagellate cell had a changed morphology and were apparently subject to degradation (not shown). Fusion with lysosomes remains to be demonstrated.
Cloning and phylogenetic analysis
PCR amplification and cloning of 16S rRNA genes from DNA extracted from a suspension of hand-picked Staurojoenina cells and subsequent RFLP analysis indicated that the resulting clone library contained only a single ribotype. Almost full-length sequences (14171418 bp) were obtained for three randomly chosen clones; since the sequences were virtually identical (>99·5 % sequence identity), the clone library was considered homogeneous, and only one sequence was submitted to GenBank. The preliminary BLAST searches (Altschul et al., 1997) had already indicated an affiliation of the clones with the Bacteroidales. In a detailed phylogenetic analysis (Fig. 2
), the clone from Staurojoenina sp. always fell within a cluster of clones comprising Bacteroides-related sequences from other termites (Cluster 4; Ohkuma et al., 2002
), which also included the rod-shaped symbiont of the trichomonad flagellate Mixotricha paradoxa (Wenzel et al., 2003
). The closest cultivated relative was the human oral bacterium Tannerella forsythensis, although the sequence similarity was rather low (<85 %).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although the 16S rRNA genes of bacteria related to the Bacteroidales are regularly encountered in termite guts (Schultz & Breznak, 1978; Ohkuma & Kudo, 1996
; Kudo et al., 1998
; Berchtold et al., 1999
), most of them belong to lineages containing no cultured representatives (Ohkuma et al., 2002
; Hongoh et al., 2003
; Schmitt-Wagner et al., 2003
). Considering the results of the present study, one reason for these cultivation problems might lie in the symbiotic association with flagellates and a possibly obligate need for a specific host. Interestingly, the epibionts of Staurojoenina sp. fall into the same cluster of the Bacteroidales as one of the two epibionts of the trichomonad flagellate Mixotricha paradoxa from Mastotermes darwiniensis (Wenzel et al., 2003
).
There are two preliminary reports on the ectosymbionts of the hypermastigid flagellate Barbulanympha spp. from the gut of the wood-eating roach Cryptocercus punctulatus (Merritt et al., 1996) and a devescovinid flagellate Caduceia species from the gut of Cryptotermes cavifrons (Goss et al., 2000
), which were reportedly identified as members of the Bacteroides/Porphyromonas subgroup. Although details were never published, it seems likely that many of the Bacteroides-related 16S rRNA genes recovered from the guts of lower termites (Ohkuma et al., 2002
; Hongoh et al., 2003
) represent epibionts of gut flagellates. A larger dataset might allow an excellent case study of co-evolution between the bacterial symbionts and their flagellate hosts.
The function of the epibionts of Staurojoenina species is not yet clear, but in view of the enormous numbers of bacteria present on each flagellate, it is suggestive that they play an important role for the host. Cleveland & Grimstone (1964) provided an elegant description of the fascinating motility symbiosis between Mixotricha paradoxa and its epibiotic spirochaetes, which were recently identified as members of the Treponema cluster by 16S rRNA gene sequence analysis (Wenzel et al., 2003
). Mixotricha paradoxa, which occurs only in the gut of Mastotermes darwiniensis, is propelled by the helical undulations of the spirochaetes adhering to the host membrane through specialized cell junctions. The spirochaetes are attached to projecting brackets of the cell surface in a manner that allows the helical movement of the individual cells to travel in metachronal waves along the cell surface of the host, resulting in locomotion (Cleveland & Grimstone, 1964
).
Also the devescovinid flagellate Rubberneckia, recently described as Caduceia versatilis (d'Ambrosio et al., 1999), is densely colonized by two different bacterial epibionts (Tamm, 1982
). In this case, the rod-shaped bacteria (20003000 per flagellate) are deeply embedded into the cell surface of the host, which is propelled by the self-synchronizing movement of the bacterial flagella (Tamm, 1982
). Although the ultrastructure of the junctional complex is completely different, the morphology of these symbionts resembles that of the epibionts of Staurojoenina. This agrees with the preliminary report that Caduceia spp. from Cryptotermes cavifrons are associated with members of the Bacteroides/Porphyromonas subgroup (Goss et al., 2000
). However, our light-microscopy observation of live preparations of Staurojoenina flagellates, which are highly motile and possess four large tufts of eukaryotic flagella (Fig. 1
), did not yield any evidence for motility of the attached bacteria, and a motility symbiosis seems unlikely.
It is more feasible that the interactions between epibionts and hosts are of a metabolic nature. Many bacteria among the Bacteroidales are polysaccharide-fermenting anaerobes, some of them producing cellulases and other fibre-degrading enzymes, which might complement enzyme activities lacking in the host. Although the large phylogenetic distance to the closest cultivated relative (<85 % sequence identity) allows no safe predictions of the physiological properties of the epibionts, the epibionts might benefit from the reduced products of the flagellate's fermentative metabolism. Again, nothing is known about the fermentation products of Staurojoenina species, but there is circumstantial evidence that the flagellates in the hindgut of Reticulitremes flavipes form lactate as a major product (Tholen & Brune, 2000). The possibility of a cross feeding of lactate between lactic-acid bacteria and a Bacteroides isolate from this termite has been previously demonstrated (Schultz & Breznak, 1979
).
Another equally plausible function of the epibionts of benefit to the flagellate host might lie in the observation that phagocytized cells of the epibionts were regularly encountered in digestive vacuoles (see Fig. 1D). Since wood is an extremely nitrogen-poor diet, it may be that the protein-rich bacteria on the cell membrane are an excellent nitrogen source for the host. Although it is not known whether the intestinal flagellates require an organic nitrogen source, the epibionts are likely to assimilate ammonia from the gut fluid, and might even fix dinitrogen (Breznak, 2000
). Obviously, more work is necessary to understand the nature of this symbiosis and the respective functions of the symbiotic partners.
Description of Candidatus Vestibaculum illigatum
Vestibaculum illigatum (Ves.ti.ba'cu.lum. L. fem. n. vestis the covering for the body, clothing, L. neut. n. baculum a (walking) stick, N.L. neut. n. vestibaculum a stick-shaped part of the body cover, L. perf. part. pass. illigatum bound, fastened, attached).
Rod-shaped bacterium of constant diameter (0·3 µm) and variable length (2·56 µm). Gram-negative cell-wall structure with outer membrane. Colonizes the cell surface of Staurojoenina sp., to which it is connected by electron-dense extracellular material. Basis of assignment: 16S rRNA gene sequence (accession number AY540335), hybridization with 16S rRNA-targeted oligonucleotide probe (5'-CCG GTG TGG GGG ACC TTC-3'). Source: epibiont of Staurojoenina flagellates from the gut of the termite Neotermes cubanus (Freytaud); so far uncultured.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Amann, R. I., Krumholz, L. & Stahl, D. A. (1990). Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J Bacteriol 172, 762770.[Medline]
Ball, G. H. (1969). Organisms living on and in protozoa. In Research in Protozoology, vol. 3, pp. 565718. Edited by T. T. Chen. New York: Pergamon Press.
Berchtold, M., Chatzinotas, A., Schönhuber, W., Brune, A., Amann, R., Hahn, D. & König, H. (1999). Differential enumeration and in situ localization of micro-organisms in the hindgut of the lower termite Mastotermes darwiniensis by hybridization with rRNA-targeted probes. Arch Microbiol 172, 407416.[CrossRef][Medline]
Breznak, J. A. (2000). Ecology of prokaryotic microbes in the guts of wood- and litter-feeding termites. In Termites: Evolution, Sociality, Symbiosis, Ecology, pp. 209231. Edited by T. Abe, D. E. Bignell & M. Higashi. Dordrecht: Kluwer Academic Publishers.
Breznak, J. A. & Brune, A. (1994). Role of microorganisms in the digestion of lignocellulose by termites. Annu Rev Entomol 39, 453487.[CrossRef]
Brune, A. (2003). Symbionts aiding digestion. In Encyclopedia of Insects, pp. 11021107. Edited by R. T. Cardé & V. H. Resh. New York: Academic Press.
Cleveland, L. R. (1926). Symbiosis among animals with special reference to termites and their intestinal flagellates. Q Rev Biol 1, 5164.[CrossRef]
Cleveland, L. R. & Grimstone, A. V. (1964). The fine structure of the flagellate Mixotricha paradoxa and its associated micro-organisms. Proc R Soc Lond B Biol Sci 159, 668686.
d'Ambrosio, U., Dolan, M., Wier, A. M. & Margulis, L. (1999). Devescovinid trichomonad with axostyle-based rotary motor (Rubberneckia): taxonomic assignment as Caduceia versatilis sp. nov. Eur J Protistol 35, 327337.[Medline]
Dolan, M. F. (2001). Speciation of termite gut protists: the role of bacterial symbionts. Int Microbiol 4, 203208.[CrossRef][Medline]
Dolan, M. & Margulis, L. (1997). Staurojoenina and other symbionts in Neotermes from San Salvador Island, Bahamas. Symbiosis 22, 229239.[Medline]
Edwards, U., Rogall, T., Blöcker, H., Emde, M. & Böttger, E. C. (1989). Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA. Nucleic Acids Res 17, 78437853.[Abstract]
Goss, S. H. & Gunderson, J. H. (2000). Identity of an ectosymbiont of a devescovinid. Abstr. 100th Gen. Meet. Am. Soc. Microbiol. 2000, abstr. N-149.
Henckel, T., Friedrich, M. & Conrad, R. (1999). Molecular analyses of the methane-oxidizing microbial community in rice field soil by targeting the genes of the 16S rRNA, particulate methane monooxygenase, and methanol dehydrogenase. Appl Environ Microbiol 65, 19801990.
Hongoh, Y., Ohkuma, M. & Kudo, T. (2003). Molecular analysis of bacterial microbiota in the gut of the termite Reticulitermes speratus (Isoptera, Rhinotermitidae). FEMS Microbiol Ecol 44, 231242.[CrossRef]
Hungate, R. E. (1955). Mutualistic intestinal protists. In Biochemistry and Physiology of Protists, vol. 2, pp. 159199. Edited by S. H. Hutner & A. Lwoff. New York: Academic Press.
Iida, T., Ohkuma, M., Ohtoko, K. & Kudo, T. (2000). Symbiotic spirochetes in the termite hindgut: phylogenetic identification of ectosymbiotic spirochetes of oxymonad protists. FEMS Microbiol Ecol 34, 1726.[CrossRef][Medline]
Inoue, T., Kitade, O., Yoshimura, T. & Yamaoka, I. (2000). Symbiotic associations with protists. In Termites: Evolution, Sociality, Symbiosis, Ecology, pp. 275288. Edited by T. Abe, D. E. Bignell & M. Higashi. Dordrecht: Kluwer Academic Publishers.
Kudo, T., Ohkuma, M., Moriya, S., Noda, S. & Ohtoko, K. (1998). Molecular phylogenetic identification of the intestinal anaerobic microbial community in the hindgut of the termite, Reticulitermes speratus, without cultivation. Extremophiles 2, 155161.[CrossRef][Medline]
Lane, D. J., Pace, B., Olsen, G. J., Stahl, D. A., Sogin, M. L. & Pace, N. R. (1985). Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc Natl Acad Sci U S A 82, 69556959.[Abstract]
Lavette, A. (1969). Sur la vêture schizophytique des flagellés symbiotes de termites. C R Acad Sci Paris 268, 25852587.
Loy, A., Horn, M. & Wagner, M. (2003). probeBase an online resource for rRNA-targeted oligonucleotide probes. Nucleic Acids Res 31, 514516.
Ludwig, W. & Strunk, O. (1996). ARB: a Software Environment for Sequence Data. http://www.mikro.biologie.tu-muenchen.de/pub/ARB/documentation/arb.ps.
Maiden, M. F. J., Cohee, P. & Tanner, A. C. R. (2003). Proposal to conserve the adjectival form of the specific epithet in the reclassification of Bacteroides forsythus Tanner et al., 1986 to the genus Tannerella Sakamoto et al., 2002 as Tannerella forsythia corrig., gen. nov., comb. nov. Request for an Opinion. Int J Syst Evol Microbiol 53, 21112112.
Manz, W., Amann, R., Ludwig, W., Vancanneyt, M. & Schleifer, K. H. (1996). Application of a suite of 16S rRNA-specific oligonucleotide probes designed to investigate bacteria of the phylum Cytophaga-Flavobacter-Bacteroides in the natural environment. Microbiology 142, 10971106.[Abstract]
Merritt, P., Goss, S. & Gunderson, J. (1996). Identification of the ectosymbiotic eubacteria of Barbulanympha spp. from the wood-eating roach Cryptocercus punctulatus. Abstr. 96th Gen. Meet. Am. Soc. Microbiol. 1996, abstr. N-180.
Noda, S., Ohkuma, M., Yamada, A., Hongoh, Y. & Kudo, T. (2003). Phylogenetic position and in situ identification of ectosymbiotic spirochetes on protists in the termite gut. Appl Environ Microbiol 69, 625633.
Ohkuma, M. & Kudo, T. (1996). Phylogenetic diversity of the intestinal bacterial community in the termite Reticulitermes speratus. Appl Environ Microbiol 62, 461468.[Abstract]
Ohkuma, M., Noda, S., Hongoh, Y. & Kudo, T. (2002). Diverse bacteria related to the Bacteroides subgroup of the CFB phylum within the gut symbiotic communities of various termites. Biosci Biotechnol Biochem 66, 7884.[CrossRef][Medline]
Olsen, G. J., Matsuda, H., Hagstrom, R. & Overbeek, R. (1994). fastDNAmL: a tool for construction of phylogenetic trees of DNA sequences using maximum likelihood. Comput Appl Biosci 10, 4148.[Abstract]
Radek, R. (1999). Flagellates, bacteria, and fungi associated with termites: diversity and function in nutrition a review. Ecotropica 5, 183196.
Radek, R., Hausmann, K. & Breunig, A. (1992). Ectobiotic and endocytobiotic bacteria associated with the termite flagellate Joenia annectens. Acta Protozool 31, 93107.
Radek, R., Rösel, J. & Hausmann, K. (1996). Light and electron microscopic study of the bacterial adhesion to termite flagellates applying lectin cytochemistry. Protoplasma 193, 105122.
Reynolds, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 17, 208212.
Schmitt-Wagner, D., Friedrich, M. W., Wagner, B. & Brune, A. (2003). Phylogenetic diversity, abundance, and axial distribution of bacteria in the intestinal tract of two soil-feeding termites (Cubitermes spp.). Appl Environ Microbiol 69, 60076017.
Schultz, J. E. & Breznak, J. A. (1978). Heterotrophic bacteria present in hindguts of wood-eating termites [Reticulitermes flavipes (Kollar)]. Appl Environ Microbiol 35, 930936.[Medline]
Schultz, J. E. & Breznak, J. A. (1979). Cross-feeding of lactate between Streptococcus lactis and Bacteroides sp. isolated from termite hindguts. Appl Environ Microbiol 37, 12061210.
Smith, H. E. & Arnott, H. J. (1974). Epi- and endobiotic bacteria associated with Pyrsonympha vertens, a symbiotic protozoon of the termite Reticulitermes flavipes. Trans Am Microsc Soc 93, 180194.[Medline]
Starr, M. P. (1975). A generalized scheme for classifying organismic interactions. Symp Soc Exp Biol 29, 120.[Medline]
Stingl, U. & Brune, A. (2003). Phylogenetic diversity and whole-cell hybridization of oxymonad flagellates from the hindgut of the wood-feeding lower termite Reticulitermes flavipes. Protist 154, 147155.[CrossRef][Medline]
Tamm, S. L. (1980). The ultrastructure of prokaryoticeukaryotic cell junctions. J Cell Sci 44, 335352.[Abstract]
Tamm, S. L. (1982). Flagellated epibiotic bacteria propel a eucaryotic cell. J Cell Biol 94, 697709.[Abstract]
Tanner, A. C. R., Listgarten, M. A., Ebersole, J. L. & Strzempko, M. N. (1986). Bacteroides forsythus sp. nov., a slow-growing, fusiform Bacteroides sp. from the human oral cavity. Int J Syst Bacteriol 36, 213221.
Tholen, A. & Brune, A. (2000). Impact of oxygen on metabolic fluxes and in situ rates of reductive acetogenesis in the hindgut of the wood-feeding termite Reticulitermes flavipes. Environ Microbiol 2, 436449.[CrossRef][Medline]
Wallner, G., Amann, R. & Beisker, W. (1993). Optimizing fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes for flow cytometric identification of microorganisms. Cytometry 14, 136143.[Medline]
Weisburg, W. G., Barns, S. M., Pelletier, D. A. & Lane, D. J. (1991). 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173, 697703.[Medline]
Wenzel, M., Radek, R., Brugerolle, G. & König, H. (2003). Identification of the ectosymbiotic bacteria of Mixotricha paradoxa involved in movement symbiosis. Eur J Protistol 39, 1124.
Yamin, M. A. (1979). Flagellates of the orders Trichomonadida Kirby, Oxymonadida Grassé, and Hypermastigida Grassi & Foà reported from lower termites (Isoptera families Mastotermitidae, Kalotermitidae, Hodotermitidae, Termopsidae, Rhinotermitidae, and Serritermitidae) and from the wood-feeding roach Cryptocercus (Dictyoptera: Cryptocercidae). Sociobiology 4, 3117.
Received 4 March 2004;
revised 21 April 2004;
accepted 27 April 2004.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |