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Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch-Strasse, 35043 Marburg, Germany
Correspondence
Andreas Brune
brune{at}mpi-marburg.mpg.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Oregon State University, Department of Microbiology, Nash Hall 220, Corvallis, OR 97331, USA.
The GenBank/EMBL/DDBJ accession numbers for the nucleotide sequence data reported in this paper are AB297984–AB298082, AB326107, AB326370–AB326383, AM747388 and AM747389.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Stingl et al., 2005). Originally discovered as members of the hindgut community of Reticulitermes speratus (Ohkuma & Kudo, 1996; Hongoh et al., 2003), their occurrence seems to be restricted to the guts of phylogenetically lower termites and wood-feeding cockroaches (Cryptocercus punctulatus) (Hongoh et al., 2005; Stingl et al., 2005; Yang et al., 2005). We have demonstrated previously that the ‘Endomicrobia’ clones retrieved from Reticulitermes santonensis represent intracellular symbionts of flagellate protists (as previously proposed by Ohkuma et al., 2001), and documented that the two major gut flagellates of this termite, Trichonympha agilis and Pyrsonympha vertens, each harbour a phylogenetically distinct lineage of ‘Endomicrobia’ (Stingl et al., 2005).
Termite gut flagellates are a unique group of protists consisting of more than 430 species, which have been described mostly on a morphological basis (Brugerolle & Lee, 2000; Yamin, 1979). Phylogenetic studies using small-subunit (SSU) rRNA and other molecular markers have confirmed the presence of two distinct phylogenetic lineages, i.e. Oxymonadida and Parabasalidea (Dacks et al., 2001; Gerbod et al., 2002; Stingl & Brune, 2003; Ohkuma et al., 2005). Although little is known about the metabolic functions of termite gut flagellates (Brune & Stingl, 2005) – the majority of which are uncultivated – they are generally considered to play a major role in the cellulose metabolism of the hindgut (Yamin, 1980; Odelson & Breznak, 1985).
Most termite gut flagellates are associated with prokaryotic symbionts, which colonize the cell surface, the cytoplasm or sometimes the nucleus of their hosts (Kirby, 1941; Berchtold et al., 1999; Brune & Stingl, 2005; Brune, 2006). The high frequency of such associations and an apparent specificity of the symbionts for their host flagellate (Noda et al., 2005, 2006; Stingl et al., 2004) are indicative of a functional significance of such symbioses for the termite gut ecosystem.
The symbiosis between ‘Endomicrobia’ and termite gut flagellates might also represent such an intimate relationship, which has been supported by evidence that some ‘Endomicrobia’ form host-specific associations with their host flagellate (Stingl et al., 2005). Furthermore, the wide distribution and phylogenetic heterogeneity of ‘Endomicrobia’ among lower termites harbouring various gut flagellates (Stingl et al., 2005) collectively suggest a strong connection between the phylogenetic diversity of the symbionts and their flagellate hosts. We hypothesize here that the phylogenetic diversity of ‘Endomicrobia’ in the gut of lower termites reflects the diversity of their flagellate hosts. To test this hypothesis, we phylogenetically analysed SSU rRNA genes of the major flagellates and their symbionts in the termite Hodotermopsis sjoestedti and in selected flagellates of five other termite species.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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DNA extraction from whole hindguts.
Ten hindguts were dissected using sterile forceps and pooled in 750 µl filter-sterilized sodium phosphate buffer (pH 8.0) in a polyethylene tube. The entire content of the tube was transferred into a polyethylene screw-cap tube containing 250 µl TNS solution (500 mM Tris/HCl, 100 mM NaCl, 10 % SDS, pH 8.0) and 0.7 g zirconium beads, and then homogenized in a FastPrep FP120 (Bio 101, Savant Instruments) for 45 s at 6.5 m s–1. The homogenates were centrifuged, and DNA in the supernatant was purified by phenol/chloroform extraction and ethanol precipitation.
DNA extraction from flagellates.
The contents of three to seven hindguts were suspended in Solution U (Trager, 1934) and diluted to a density of approximately 10 flagellate cells µl–1. Aliquots (20 µl) of the diluted suspension were placed in the wells of a 10-well Teflon-coated glass slide (Erie Scientific Company). Flagellate cells were sorted by morphology (Radek et al., 1992; Tamm, 1999; Brugerolle & Bordereau, 2004), and 150–200 flagellate cells of each morphotype were collected by micropipette using an inverted microscope. The cells were collected into a well containing 15 µl sterile PBS and washed by at least three transfers into fresh PBS-containing wells. Approximately 100 cells were finally resuspended in 200 µl sterile PBS. Cells were disrupted by three cycles of freeze–thawing, and DNA was extracted from each sample using the NucleoSpin kit (Macherey-Nagel), following the manufacturer's instructions. The extracted DNA was finally eluted with 30 µl distilled water and used as a template for PCR reactions.
PCR amplification.
Flagellate SSU rRNA genes were amplified using universal eukaryotic primers as described by Keeling et al. (1998). Bacterial SSU rRNA genes were amplified using 27F (Edwards et al., 1989) and 1492R (Weisburg et al., 1991) as described by Henckel et al. (1999). ‘Endomicrobia’ SSU rRNA genes were amplified as previously described, using the forward primer TG1-209F (Stingl et al., 2005) and a slightly modified reverse primer TG1-1325R' (5'-GATTCCTACTTCATGTG-3').
Cloning and sequencing.
PCR products were ligated into plasmid pCR2.1-TOPO and introduced into E. coli TOP10F' by transformation using the TOPO TA cloning kit (Invitrogen), following the manufacturer's instructions. Clones with a flagellate SSU rRNA gene insert and clones with an ‘Endomicrobia’ SSU rRNA gene insert (
1070 bp) were screened by direct PCR using M13 primers. To obtain the almost-full-length ‘Endomicrobia’ SSU rRNA genes, bacterial SSU rRNA gene libraries (1500 bp) were screened with ‘Endomicrobia’-specific primers (see above). PCR products of the expected size were digested separately with the restriction enzymes MspI and AluI, and subjected to electrophoresis on a 3 % agarose gel. The clones were sorted according to their restriction patterns, and two to ten representatives of each ribotype were sequenced using M13 primer sets. For each phylotype (sequence clusters with more than 1 % sequence divergence) obtained in this study, several representative SSU rRNA gene sequences have been submitted to GenBank under accession numbers AB297984–AB298082, AB326107, AB326370–AB326383 and AM747388–AM747389.
Phylogenetic analysis.
The SSU rRNA gene sequences were imported into the database implemented in the ARB software package (Ludwig et al., 2004). The sequences were automatically aligned with the other closely related SSU rRNA sequences using the ARB Fast_Aligner, followed by manual refinement. Phylogenetic trees were constructed using almost-full-length SSU rRNA sequences (>1300 bases) by maximum-likelihood methods (AxML and fastDNAml), and the stability of the tree topology was tested by the neighbour-joining and maximum-parsimony methods implemented in ARB. Shorter sequences were added using the ARB parsimony tool. Chimaeric sequences were identified using the Bellerophon server (Huber et al., 2004; http://foo.maths.uq.edu.au/huber/bellerophon.pl) and by carefully checking for signature sequences in the alignment, and were subsequently removed from the dataset.
Whole-cell in situ hybridization.
Fixed gut contents were prepared and in situ hybridization was performed as described previously (Stingl & Brune, 2003). Probe EUB338 (Amann et al., 1990) and the nonsense probe NON338 (Wallner et al., 1993) were used to identify bacterial cells and to distinguish non-specific probe binding in the same suspension. For each probe, hybridization stringency was optimized by testing formamide concentrations over a range of 0–50 %.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), Trichonympha and Eucomonympha (both Parabasalidea; Fig. 1b, c). DNA extracted from the respective suspensions yielded PCR products of the expected length with eukaryotic (1500–1800 bp), bacterial (1500 bp) and ‘Endomicrobia’-specific (1100 bp) SSU rRNA primers.
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). In the library from the Eucomonympha suspension, the obtained sequence was virtually identical to that of clone HsL3 recovered in a clone library of a mixed flagellate population of H. sjoestedti (Ohkuma et al., 2000), corroborating the tentative assignment of this clone to the genus Eucomonympha. In the case of the Dinenympha suspension, the sequence showed 94 % identity to that of a Dinenympha species from Reticulitermes hesperus (Moriya et al., 2003) and probably represents a new, hitherto unrecognized species of Dinenympha. The Trichonympha suspension yielded three different phylotypes of eukaryotic SSU rRNA genes, whose sequences were virtually identical to those of the three Trichonympha phylotypes previously obtained from this termite by Ohkuma et al. (2000).
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). Additional clusters consisting only of clones from whole-gut preparations were present (WG1–WG9, Fig. 2), suggesting that ‘Endomicrobia’ might be present also in other flagellate species in H. sjoestedti other than those investigated. However, we do not completely preclude the possibility that these unidentified clones are derived from free-living ‘Endomicrobia’ species.
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). Again, we were able to assign the eukaryotic SSU rRNA gene sequences obtained from each flagellate suspension to the identical or similar respective genera, whose SSU rRNA gene sequences have been published. A notable exception was the SSU rRNA gene recovered from the suspension of Joenia sp. of K. flavicollis, which showed the highest identity (98 %) to clone Kf5 (AF215857) obtained from a clone library of the same termite and assigned to the flagellate genus Foaina by other authors (Gerbod et al., 2000).
Each of the flagellate suspensions yielded a single and unique host-specific phylotype of ‘Endomicrobia’ in the corresponding SSU rRNA libraries. The phylogenetic tree of all almost-full-length (>1400 bp) SSU rRNA gene sequences obtained in this and previous studies clearly showed that the ‘Endomicrobia’ sequences from each flagellate host always represent distinct phylotypes (Fig. 2). The ‘Endomicrobia’ of flagellates originating from the same termite did not cluster with each other. Instead, the ‘Endomicrobia’ from the Trichonympha species of H. sjoestedti and Z. nevadensis clustered together with those previously obtained from the Trichonympha species of R. santonensis and R. speratus, and collectively constitute a monophyletic cluster that forms a sister group of the ‘Endomicrobia’ clones recovered from all other flagellates.
Localization of ‘Endomicrobia’ by fluorescence in situ hybridization (FISH)
For selected termites, we conducted FISH to confirm the intracellular location of the ‘Endomicrobia’ phylotypes obtained from the respective flagellate suspensions by the specific PCR amplification. It was not possible to design a specific probe for all ‘Endomicrobia’. Moreover, the limited number of variable regions among different ‘Endomicrobia’ did not allow the design of specific probes covering each phylotype. Therefore, we designed a set of oligonucleotide probes that covered the phylotypes in question (Table 2).
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shows representative examples in which the ‘Endomicrobia’-specific signal is exclusively localized within the corresponding host cells, whereas the Bacteria-specific probe also stained bacteria associated with the surface or content of these and other flagellate species (Fig. 3). In no case did we see evidence for the location of ‘Endomicrobia’ on the cell surface or within the nucleus of the host.
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; details not shown). In the case of Eucomonympha cells, it was not possible to visualize single cells of ‘Endomicrobia’ because of a high affinity of both the Bacteria-specific and nonsense probe to the dense cell content of the host flagellate. Fluorescence signals outside of the flagellate cells observed in dry-wood termites (see Fig. 3f) were present also in non-stained preparations and were caused by autofluorescence of wood particles in the gut content. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Each of the termite gut flagellates analysed in this study invariably harboured ‘Endomicrobia’. Together with the phylotypes that remain to be assigned to a particular host, ‘Endomicrobia’ represent the symbionts of up to 24 parabasalid and oxymonadid species, and probably more in view of the presence of ‘Endomicrobia’ phylotypes retrieved from whole-gut homogenates of H. sjoestedti in addition to those retrieved from the flagellate suspensions. The wide host range and their consistent occurrence within the host indicate a broad host spectrum of ‘Endomicrobia’ as symbionts of termite gut flagellates.
The ‘Endomicrobia’ of each flagellate species form a unique phylogenetic lineage. The case of H. sjoestedti, in which the Trichonympha suspension contained three phylotypes of Trichonympha, but from which only two distinct phylotypes of ‘Endomicrobia’ were recovered, does not necessarily contradict the proposed host specificity. It is possible that the third phylotype of ‘Endomicrobia’ was missed in this study because it had been under-represented in the sample, or that one of the three phylotypes of Trichonympha in H. sjoestedti lacks ‘Endomicrobia’. The first explanation is supported by the presence of another ‘Endomicrobia’ lineage (WG1) recovered from total-gut DNA that clusters with the two other lineages from the Trichonympha suspension (Fig. 2).
All ‘Endomicrobia’ phylotypes associated with Trichonympha species collectively constitute a monophyletic group that is phylogenetically distinct from the phylotypes recovered from all other flagellates. The evidence that host-specificity is present also at the species level is indicative of co-speciation between the partners (Page & Charleston, 1998). This would imply that each of the extant Trichonympha flagellates harbours a specific lineage of ‘Endomicrobia’ inherited by vertical transmission from their common ancestor – an issue that cannot be resolved based on the current dataset. Conversely, it is possible that at one point in time ‘Endomicrobia’ have been horizontally transferred from one flagellate species to another within the same termite gut. This would explain why oxymonads (Dinenympha, Oxymonas) harbour ‘Endomicrobia’ that are relatively closely related to the symbionts of parabasalids, i.e. flagellates of a different phylum.
This study corroborates that ‘Endomicrobia’ form a separate line of descent in the bacterial tree (Stingl et al., 2005). They are part of the TG-1 phylum, which consists of numerous diverse and deep-branching lineages (Herlemann et al., 2007). While ‘Endomicrobia’ seem to be restricted to termites and wood-feeding cockroaches, other representatives of the TG-1 phylum occur in a wide range of chemically and geographically distinct habitats, including soils, sediment and intestinal tracts.
Although nothing is known about the metabolic function of ‘Endomicrobia’, their constant occurrence as intracellular symbionts with a broad host range suggests that their nutritional requirements may be met by substances commonly available in the cytoplasm of gut flagellates. The host flagellates may also benefit from their endosymbionts, e.g. by the provision of nutrients otherwise lacking in the diet of the termites (see also: Stingl et al., 2005). Although certain termite gut flagellates have been shown to ferment cellulose to hydrogen and acetate (Hungate, 1955; Yamin, 1980, 1981; Odelson & Breznak, 1985), the physiology of most termite gut flagellates is still completely unknown. This makes elucidation of the biology of ‘Endomicrobia’ and their function in the symbiosis a most intriguing, but very challenging subject.
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ACKNOWLEDGEMENTS |
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Edited by: H. L. Drake
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Received 19 April 2007;
revised 22 June 2007;
accepted 27 June 2007.
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) and >50 % (
), nodes not supported by all analyses are shown as multifurcations. WG1–WG9: clusters formed by shorter (