• symbiosis;
  • termite;
  • endosymbiont of protist;
  • candidate phylum;
  • coevolution


  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Concluding remarks
  7. Acknowledgements
  8. References

The candidate phylum ‘Termite Group 1’ (TG1) of bacteria, which is abundant in termite guts but has no culturable representative, was investigated with respect to the in situ localization, distribution, and diversity. Based on the 16S rRNA gene sequence analyses and FISH in termite guts, a number of lineages of TG1 members were identified as endosymbionts of a variety of gut flagellated protists from the orders Trichonymphida, Cristamonadida, and Oxymonadida that are mostly unique to termites. However, the survey in various environments using specific PCR primers revealed that TG1 members were also present in termites, a cockroach, and the bovine rumen that typically lack these protist orders. Most of the TG1 members from gut flagellates, termites, cockroaches, and the rumen formed a monophyletic subcluster that showed a shallow branching pattern in the phylogenetic tree, suggesting their recent diversification. Although endosymbionts of the same protist genera tended to be closely related, the endosymbiont lineages were often independent of the higher level classifications of their host protist and were dispersed in the phylogenetic tree. It appears that their cospeciation is not the sole rule for the diversification of TG1 members of endosymbionts.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Concluding remarks
  7. Acknowledgements
  8. References

Termites (Isoptera) harbor a dense and diverse microbial community in their guts, which is responsible for the efficient decomposition of plant litter (Ohkuma, 2003). In evolutionarily lower termites, the gut microbial community consists of both flagellated protists (single-cell eukaryotes) and prokaryotes. Most of these gut protists are unique to termites, and they are affiliated to the orders Trichonymphida, Cristamonadida, Spirotrichonymphida, Trichomonadida, and Oxymonadida. The former four belong to the phylum Parabasalia, and the last to Preaxostyla (Adl et al., 2005). However, higher termites typically lack these flagellated protists. Yeasts are isolated from both lower and higher termites (Prillinger & König, 2006). Recent culture-independent molecular studies have successfully identified the protists (e.g. Ohkuma et al., 2005) and prokaryotes (e.g. Hongoh et al., 2005) in termite guts. A remarkable finding of these studies is that a great majority of the gut bacteria comprise numerous yet-uncultured lineages unique to termites, including novel phylum-level phylogenetic clusters. In our previous study, ‘Termite Group I’ (TG1) was first described as the gut bacteria of a lower termite; it is a new deep-branching lineage that dominates the gut community (Ohkuma & Kudo, 1996). Later, it was defined as a candidate phylum along with members found in several other environments (Hugenholtz et al., 1998). To date, there are a growing number of such candidate phyla or divisions reported from natural environments, but only a few of them have been characterized fully (Hugenholtz et al., 2001; Fieseler et al., 2004; Chouari et al., 2005). The candidate novel phylum TG3, comprising bacteria that are abundant in higher termites, has been characterized with respect to its ecological aspects (Hongoh et al., 2005, 2006a).

In addition to the novelty of the members, another characteristic of the gut microbial community of termites is its highly structured nature, particularly in terms of the spatial distribution of individual species (Berchtold et al., 1999). The gut epithelium is a niche for unique microbial populations, although resident bacteria account for only a small portion of the entire gut community (Nakajima et al., 2005). Physical associations of prokaryotes with flagellated protists in the gut of lower termites are frequently observed (Dolan, 2001), and the protist-associated bacteria form a large portion of the gut community. The associations of surface-attached (ectosymbiotic) spirochetes (Iida et al., 2000; Noda et al., 2003; Wenzel et al., 2003), ectosymbiotic and intracellular endosymbiotic Bacteroidales members (Wenzel et al., 2003; Stingl et al., 2004; Noda et al., 2005, 2006a, b), and endosymbiotic methanogens (Fröhlich & König, 1999; Tokura et al., 2000; Hara et al., 2004) with various gut protist species have been identified on the basis of their molecular sequences. However, given that a large number of gut protist species are present in diverse lower termites, the prokaryotes that have been identified to be associated with these gut protists may still represent a limited fraction of the yet uncharacterized diversity of the endo- and ectosymbiotic populations.

In this study, we characterized the TG1 phylum of bacteria in terms of in situ localization, distribution, diversity, and host specificity. There have been preliminary reports that members of the TG1 phylum are endosymbionts of the large trichonymphid genus Trichonympha (Eldridge et al., 1997; Ohkuma et al., 2001). Additionally, an endosymbiont member of the oxymonad genus Pyrsonympha (Stingl et al., 2005) was recently reported. Here, we investigated TG1 members that are endosymbionts of various protist species in the gut of lower termites. We also examined the distribution and diversity of the TG1 members present in the gut of higher termites and other environments.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Concluding remarks
  7. Acknowledgements
  8. References

Protist species, termites, and other samples

The protist species examined in this study are listed in Table 1. The protist cells were identified by their morphology and manually isolated as described previously (Iida et al., 2000). Specimens preserved in acetone were used after reconstitution as described earlier (Noda et al., 2005; Deevong et al., 2006). The enriched fraction of Trichonympha spp. in the termite Hodotermopsis sjoestedti was obtained by low-speed centrifugation as described previously (Ohkuma et al., 2000). The termite and cockroach samples used for the specific PCR of TG1 subphylum 1 members are shown in Table 2. Samples from the bovine rumen, soils, sediments, and sewage sludges were also used. The collection of these samples and extraction of DNA are described in previous studies (Hongoh et al., 2005, 2006a, b).

Table 1.   Gut protist species and the phylotype identified in the TG1 phylum
  • *

    Abbreviations of the host termite species are as follows: Rs, Reticulitermes speratus; Hs, Hodotermopsis sjoestedti; Cd, Cryptotermes domesticus; Im, Incisitermes minor; Gf, Glyptotermes fuscus; Hm, Hodotermes mossambicus; and Kf, Kalotermes flavicollis.

  • The closely related phylotypes reported from the entire gut community of Reticulitermes speratus (Hongoh et al., 2003) are indicated in parentheses.

  • Two morphotypes (oval and long forms) of Pyrsonympha grandis in this termite that can be also discriminated genetically (Moriya et al., 2003) were independently examined.

 Trichonympha agilisRsRsTG1 (Rs-D17)
 Trichonympha spp.HsHsTG1, HsTG3
 Dinenympha rugosaRsRsDr18 (Rs-D95)
 Dinenympha exilisRsRsDe11 (Rs-D95)
 Pyrsonympha grandis (oval)RsRsPgO2-18 (Rs-D43)
 Pyrsonympha grandis (long)RsRsPgL2-11 (Rs-D43)
 Macrotrichomonas sp.GfGfMT5-8
 Devescovina lemniscataCdCdDl3-24
 Stephanonympha nelumbiumCdCdSn3-1
 Metadevescovina cuspidataImImMc2-4
 Gigantomonas herculeaHmHmGh7
 Joenoides intermediaHmHmJi24
 Joenia annectensKfKfJa16
Table 2.   Specific detection of TG1 subphylum 1 members from the gut of lower and higher termites, cockroach gut, and bovine rumen
  • No amplification was detected in the samples from activated sludge, anaerobic digester, orchard soil, rice paddy soils, lake sediment, coastal sea sediment, and deep sea sediments.

  • *

    Sample codes used to discriminate the origin of the clones in clone names are shown in parentheses.

  • The number of phylotypes obtained by clone analyses is indicated in parentheses.

Lower termites
 Reticulitermes speratus RsTz+
 Reticulitermes amamianus+
 Reticulitermes okinawanus+
 Reticulitermes sp. RPK (RPK)+
 Coptotermes formosanus (Cf)+ (1)
 Neotermes koshunensis+
 Archotermopsis sp. APK (APK)+ (2)
 Zootermopsis nevadensis (Zn)+
 Hodotermopsis sjoestedti+
 Mastotermes darwiniensis (Md)+
Higher termites
 Macrotermes gilvus 2MjD
 Speculitermes sp. (Spe)+ (1)
 Termes comis+
 Pericapritermes nitobei (Pn)+ (2)
 Pericapritermes latignathus (Pl)+ (4)
 Nasutitermes takasagoensis (Nt)+ (1)
 Nasutitermes dimorphus (Nd)+ (2)
 Microcerotermes sp. M1PT4 (M1)+ (3)
 Microcerotermes sp. M2PB4 (M2)+ (1)
 Microcerotermes crassus McPP3+
 Cryptocercus punctulatus (Cp)+
 Panesthia angustipennis (Pta)+ (5)
Bovine rumen (RM)+ (4)

PCR amplification, cloning, sequencing, and phylogeny

PCR using the bacterial universal primers 27F (Hongoh et al., 2003) and 1390R (Thongaram et al., 2005) for the near-full-length 16S rRNA gene was performed with a pool of 1–20 isolated or enriched protist cells under previously described conditions (Noda et al., 2006b). For detecting members of TG1 subphylum 1, the PCR products obtained after 20–24 cycles with the 27F and 1390R primers were used as the template for the nested PCR with specific primers (Table 3) after diluting the sample to approximately the same concentration. An annealing temperature of 60°C was optimized for these primers, as described previously (Hongoh et al., 2006a). Twenty-five PCR cycles for the detection and 10–20 cycles for cloning were carried out at this annealing temperature.

Table 3.   FISH probes and PCR primers designed in this study
Probe or primerSequence (5′–3′)Target
  1. The oligonucleotide probe sequences have been deposited at probeBase (

FISH probe
 comp-TG1-TACCCTCTCAGGCCGGATA(Competitor of TG1-T-287)
PCR primer

The PCR products were cloned using a TOPO TA cloning kit (Invitrogen). Clones randomly chosen from the constructed libraries (15–48 clones from the protist cells and 8–24 clones from the sample DNAs) were subjected to DNA sequencing, as described previously (Hongoh et al., 2003). Artificially produced chimeric sequences were analyzed with multiple methods as described earlier (Hongoh et al., 2005) and excluded from the following analyses. Using the dotur version 1.5 program (Schloss & Handelsman, 2005), the clone sequences that were affiliated to the TG1 phylum were sorted into phylotypes with the criterion of 99.0% sequence identity. The 16S rRNA gene sequences generated in this study have been deposited with DDBJ under accession numbers AB188146AB188148 and AB282956AB282992.

The arb software (Ludwig et al., 2004), the database of which was modified in our previous study (Hongoh et al., 2006b), was used for sequence alignment and preliminary phylogenetic affiliations. A maximum likelihood (ML) tree was inferred using the phyml version 2.4.4 program (Guindon & Gascuel, 2003) with a general time-reversible (GTR) model with gamma-distributed rate variation (G) and a proportion of invariable sites (I), the parameters of which were estimated from the data. Bootstrap analysis was performed with 100 resamplings. Bayesian posterior probabilities were estimated using the mrbayes 3.1 program (Huelsenbeck & Ronquist, 2001), which was run for 500 000 generations with the GTR+G+I model with the first 100 000 generations discarded.


Oligonucleotide probes specific for 16S rRNA gene phylotypes (Table 3) were designed using the probe-designing function of arb. A more universal probe was also designed to enable detection of most members of TG1 subphylum 1. It was simultaneously used with its competitor oligonucleotide (Table 3) as described previously (Hongoh et al., 2006a). The general bacterial probe (Iida et al., 2000) was also used. These probes were labeled with either 6-carboxyfluorescein (FAM) or Texas Red at their 5′ ends. FISH was performed as described previously (Noda et al., 2006a). The endosymbionts were released from 100 isolated Trichonympha agilis cells by adding the Nonidet P-40 (Nacalai Tesque) detergent at a final concentration of 1%, and enumerated after stained with 4,6-diamidino-2-phenyl indole (DAPI). Transmission electron microscopy (TEM) was conducted as described previously (Noda et al., 2006a).

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Concluding remarks
  7. Acknowledgements
  8. References

Endosymbiont members of TG1

The sequence-specific probe for the phylotype Rs-D17 that represented one of the most abundant clones in the TG1 phylum from the gut microbial community of Reticulitermes speratus (Hongoh et al., 2003) was designed and used for detecting the corresponding cells by FISH. The positive signal was obtained in the endosymbiotic bacteria of the gut protist T. agilis (Fig. 1a). No signal was observed in other protist-associated and nonassociated bacteria, indicating the specific detection. The association was further confirmed by analysis of PCR-amplified bacterial 16S rRNA gene sequences (represented by RsTG1) from the isolated T. agilis cells, which showed 99.9% nucleotide identity to the sequence of Rs-D17. Terminal-restriction fragment length polymorphism analysis of the gut microbial community (data not shown) failed to detect the corresponding signal for the TG1 phylum of the 16S rRNA gene sequences when the termites were fed on starch, a condition under which most of the protists had disappeared from the gut. TEM (Fig. 1b) revealed the abundance of rod-shaped endosymbionts of size 1.05±0.49 × 0.29±0.05 μm (mean±SD; n=39). All observed T. agilis cells harbored the FISH-positive endosymbionts. A single T. agilis cell was associated with c. 5500 DAPI-stained prokaryotic cells, and FISH could detect approximately three-fourths of these. Almost all the endosymbionts were detected by the general bacterial probe, suggesting the presence of bacterial taxa other than TG1. Based on the cell numbers of the protist species and prokaryotes present in an average R. speratus gut (Inoue et al., 1997; Nakajima et al., 2005), the T. agilis endosymbiont of the TG1 member was estimated to make up 4–5% of the total prokaryotic population in the gut.


Figure 1. In situ detection of endosymbionts of gut flagellated protists of termites as members of the TG1 phylum. (a) Endosymbionts of Trichonympha agilis in the Reticulitermes speratus gut. The gut content shown in the phase-contrast image (right panel) was simultaneously hybridized with the sequence-specific probe (left) and the general bacterial probe (middle). (b) The TEM image of a thin section of Trichonympha agilis showing endosymbiotic bacteria that are surrounded by two membranes and contain numerous ribosomes. The endosymbionts often but not always coexist with hydrogenosome-like organelles (indicated by H) of the host flagellates that are surrounded by a single membrane. (c) Endosymbionts corresponding to two phylotype sequences of Trichonympha spp. in Hodotermopsis sjoestedti. A mixture of the Trichonympha spp. cells (right, phase contrast) was simultaneously hybridized with the probes TG1-HsTG1-248 labeled with Texas Red (red) and TG1-HsTG3-193 labeled with FAM (green), and the images were merged (middle). The panel on the left side exhibits the DAPI-stained image. (d–h) detection of endosymbionts of Dinenympha rugosa (d) and Pyrsonympha grandis (e) in Reticulitermes speratus, Macrotrichomonas sp. (f) in Glyptotermes fuscus, and Devescovina lemniscata (g) and Stephanonympha nelumbium (h) in Cryptotermes domesticus. Phase-contrast images of these protists are shown in the panels on the right side. The results obtained with sequence-specific (d–f) or TG1 subphylum 1-specific probes (g, h) are shown in the panels on the left side. The panels in the middle show the images obtained with the general bacterial probe. These probes were labeled with either Texas Red (red) or FAM (green). Insets in the left side panels are magnifications of the images of endosymbionts indicated by arrowheads. Note that the endosymbionts of Pyrsonympha grandis frequently form an aggregation (e). Bars, 20 μm in (a, d–h), 0.5 μm in (b), and 100 μm in (c).

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Each of the two phylotype sequences in the TG1 phylum obtained from the enrichment fraction of Trichonympha spp. in the gut of H. sjoestedti (HsTG1 and HsTG3) was specifically identified as an endosymbiont by FISH using the sequence-specific probes (Fig. 1c). Most but not all of the endosymbionts in a single Trichonympha cell corresponded to the TG1 members. Almost all the Trichonympha cells in the H. sjoestedti gut harbored endosymbionts corresponding to either of the two phylotypes, and the two phylotypes did not coexist in a single Trichonympha cell. The ratio of Trichonympha cells harboring HsTG1 to HsTG3 endosymbionts was approximately one to three. As the presence of at least three species of Trichonympha was reported (Kitade et al., 1997; Ohkuma et al., 2000), the results suggested that closely related endosymbionts were shared between Trichonympha species.

Oxymonad species of the genera Dinenympha and Pyrsonympha existed in a high cell number in the R. speratus gut. We could identify TG1 members of the sequences from the isolated oxymonad cells (Table 1). The phylotypes from Dinenympha and Pyrsonympha grandis were closely related to the Rs-D95 and Rs-D43 phylotypes from the gut community, respectively (each showed >98% identity). Rs-D95 and Rs-D43 as well as Rs-D17 represent clones that are abundant in the TG1 phylum and, in total, they make up c. 10% of the clones of the bacterial 16S rRNA genes from the gut community (Ohkuma & Kudo, 1996; Hongoh et al., 2003).

The probe for the phylotypes from Dinenympha specifically detected the rod-shaped endosymbionts by FISH (Fig. 1d). The FISH signals of the endosymbionts were observed in c. 90% of the Dinenympha rugosa cells, 80% of the Dinenympha exilis cells, and 50% of the entire Dinenympha population. The FISH signal has never been observed in Dinenympha porteri and Dinenympha parva that are reported to harbor ectosymbiotic spirochetes and endosymbiotic methanogens, respectively (Iida et al., 2000; Tokura et al., 2000). The numbers of corresponding endosymbionts in single cells of D. rugosa and D. exilis were 29±7 (n=12) and 13±6 (n=34), respectively. The TG1 members of the endosymbionts in Dinenympha spp. were estimated to form up to 3% of the total prokaryotic population in the gut. The probe for the phylotypes from P. grandis specifically detected the endosymbionts in almost all the P. grandis cells, regardless of their morphotypes. An aggregation of the endosymbionts was often observed (Fig. 1e), although random dispersion within the P. grandis cell was also observed as in the case of Trichonympha and Dinenympha. Although the aggregation hampered enumeration of the endosymbionts, several tens to hundreds cells of the TG1 member were present in a single P. grandis cell.

The bacterial 16S rRNA gene sequences belonging to the TG1 phylum were identified from the isolated cells of seven flagellate species of the order Cristamonadida (Table 1). The rod-shaped endosymbionts of Macrotrichomonas were detected by FISH using a sequence-specific probe (Fig. 1f). The endosymbionts of Devescovina lemniscata and Stephanonympha nelumbium in the gut of Cryptotermes domesticus were also confirmed by FISH as members of the TG1 phylum using a probe that covered many of the sequences in the TG1 phylum (Fig. 1g and h). Several to a few hundreds of TG1 endosymbionts were detected in a single cell of these protists. No FISH signal with this probe could be observed from the other protist species in C. domesticus [e.g. Foaina nana (Cristamonadida) and Oxymonas spp. (Oxymonadida)]. Although we did not apply FISH, the protist species of Gigantomonas and Joenia harbor endosymbiotic bacteria that morphologically resemble the endosymbiont of T. agilis (Radek et al., 1992; Brugerolle, 2005). The results indicated that a variety of protist species in the gut of termites harbored endosymbiotic members of the TG1 phylum.

Distribution among termites and other environments

To evaluate the distribution of TG1 members in various environments, PCR screenings were performed using newly designed specific primers that covered almost all the 16S rRNA gene sequences in TG1 subphylum 1 (see below). PCR amplifications (c. 1.1 kbp) were detected from the gut DNA of all 10 lower termites examined (Table 3). Although previous studies failed to detect TG1 members in higher termites (Tokuda et al., 2000; Schmitt-Wagner et al., 2003; Hongoh et al., 2005, 2006a, b; Stingl et al., 2005; Thongaram et al., 2005; Deevong et al., 2006), specific amplifications were successfully obtained from the gut DNA of all higher termites with the exception of one (Table 3). Probably, these are in low abundance in the gut of higher termites.

Additionally, TG1 subphylum 1 was detected from the gut of wood-feeding cockroaches and the bovine rumen. Whereas the Cryptocercus cockroach is known to harbor the related protists to those in lower termites (Yamin, 1979), the Panesthia cockroach and the rumen as well as higher termites do not. The results suggest that the presence of TG1 subphylum 1 is independent of the protist species unique in lower termites and Cryptocercus. Nevertheless, no specific amplification was detected from the other environments examined thus far (sewage disposal plants, soils, and lake and sea sediments). Cockroach guts and rumens in general are known to harbor ciliate protists, although some protist species inhabit other environments to various degrees. It would be interesting to investigate in future whether TG1 members associate with ciliates in cockroach guts and rumens.

The specific amplifications were completely confirmed by clonal analyses of the PCR products in two lower and seven higher termites, the cockroach Panesthia angustipennis, and the rumen (a total of 26 new phylotypes; in three cases, the phylotypes overlapped between related higher termites).

Phylogenetic diversity

Along with the sequences reported previously from lower termites (Hongoh et al., 2003, 2005; Stingl et al., 2005), the new phylotype sequences identified as endosymbionts of the gut protists and obtained from termites, a cockroach, and the rumen formed a subphylum-level monophyletic cluster in the phylogenetic tree of the TG1 phylum (Fig. 2). We tentatively designated this cluster as ‘subphylum 1’. TG1 subphylum 1 showed a shallow branching pattern and shared <10% sequence divergence in most pairwise comparisons, suggesting their recent diversification. The other sequences in the TG1 phylum were more diverse and comprised sequences from various environments including two sequences from the gut wall of the termite R. speratus (Nakajima et al., 2005).


Figure 2.  A phylogenetic tree showing the relationship of phylotypes of the 16S rRNA genes affiliated to the candidate phylum TG1. A total of 1059 unambiguously aligned nucleotide positions were used to infer the tree. Nodes supported by an ML-bootstrap value of >70% and a Bayesian posterior probability of >95% are indicated by filled circles. Those of >50% supports on the basis of either the ML-bootstrap value or the Bayesian posterior probability are represented with open circles at nodes. The phylotypes obtained in this study are shown in bold letters. The host protist genera and species for the endosymbiont members and the origins of phylotypes are shown near their respective phylotypes. The origin of phylotypes is also indicated in the clone codes using the abbreviations listed in Tables 2 and 3. The other clone-code abbreviations for representing termite origins are as follows: Rsa, Reticulitermes santonensis; Nc, Neotermes cubanus; Cs, Cryptotermes secundus; Sl, Schedorhinotermes lamanianus. The horizontal bar represents 0.1 nucleotide substitutions per site.

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The endosymbiotic phylotypes of Trichonympha were included in a monophyletic cluster. The sequences from the termite genera Zootermopsis (Zn7) and Kalotermes (Kf33) were included in this cluster. The former harbors Trichonympha spp. in the gut, but the latter does not harbor the related protist species (Yamin, 1979). The endosymbionts of T. agilis in R. speratus and Reticulitermes santonensis were rather distantly related in this cluster. A distant relationship between T. agilis in R. speratus and Reticulitermes flavipes (synonym of R. santonensis; Jenkins et al., 2001) has been reported based on their small subunit rRNA gene sequences (Ohkuma et al., 2000).

At least two species of Dinenympha protists harbored closely related endosymbionts of TG1 subphylum 1 (represented by Rs-D95). Similarly, two morphologically and genetically distinct P. grandis species in the gut of R. speratus (Moriya et al., 2003) also harbored a closely related TG1 member (represented by Rs-D43). The results appeared to show tight relationships between TG1 endosymbionts and host protists. However, the endosymbiont of Pyrsonympha vertens in the gut of R. santonensis was distantly related to the endosymbionts of P. grandis. The TG1 members identified in the cristamonad protists occurred in at least three polyphyletic lineages. All together, the TG1 endosymbiont members seemed to be dispersed in the phylogenetic tree, suggesting that they have not always cospeciated with their host protists, at least at the level of the protist genera. Similar relationships have also been reported between ectosymbiotic Bacteroidales members and their host protists in termite guts (Noda et al., 2006a, b).

Most of the phylotypes from higher termites were assigned to two clusters; however, each cluster contained one sequence from a lower termite and another from the Panesthia cockroach. Only the sequences from the bovine rumen formed an exclusive monophyletic cluster. As higher termites lack protists from the orders Trichonymphida, Cristamonadida, and Oxymonadida, the TG1 members in higher termites are not endosymbionts of these protists, although the endosymbiotic nature of other inhabiting protists, if present, cannot be denied. We attempted to detect the cells of TG1 members in several higher termites by FISH using a probe for TG1 subphylum 1. However, we failed to obtain specific signals, probably due to the low abundance of the cells in the gut content as well as the high background autofluorescence derived from ingested heterogeneous materials.

Concluding remarks

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Concluding remarks
  7. Acknowledgements
  8. References

In the present study, a variety of protist species that are unique in the gut of lower termites are found to harbor endosymbiotic members of TG1 subphylum 1, although they constitute only a small fraction of the great diversity in termite guts. Considering that there are various associations with termite gut protists involving spirochetes, Bacteroidales members, methanogens, and TG1 members, and given that a great diversity of gut protists remain to be examined with respect to molecular identification of associated bacteria, they may provide rich and attractive examples of cellular symbioses. As the endosymbiotic TG1 members account for a considerable portion of the gut microbial population, at least in the R. speratus termite, their functions and roles in digestion are also important for understanding the impressive triplex symbiotic system comprising termites, gut protists, and their associated bacteria.

Another salient point of this study is the finding that diverse members of TG1 subphylum 1 are present in higher termites, the Panesthia cockroach, and the rumen. The finding implies that TG1 subphylum 1 is widely distributed in the digestive tract of animals, although members of the TG1 phylum have not yet been discovered in comprehensive analyses of intestinal microbial communities of mammals and fish (Leser et al., 2002; Rawls et al., 2006). The distribution and in situ detection of the TG1 phylum of bacteria in the digestive tract of various animals should be a topic of future studies. The primers and probes designed in the present study would be useful in these studies.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Concluding remarks
  7. Acknowledgements
  8. References

We thank M. Kawai, T. Iida, and F. Ohnuma for assistance with the experiments and C. Bordereau, E. Viscogliosi, W. Ohmura, and others for the samples. Y.H. is a recipient of a Special Postdoctoral Research Fellowship from RIKEN. This work was partially supported by grants for the Bioarchitect Research and the EcoMolecular Science Research programs from RIKEN, by a grant from the Noda Institute for Scientific Research to M.O., and by Grants-in-Aid for Scientific Researches from JSPS (Nos. 18687002 and 16380065 to Y.H. and M.O., respectively).


  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Concluding remarks
  7. Acknowledgements
  8. References
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