Endosymbiotic Methanobrevibacter Species Living in Symbiotic Protists of the Termite Reticulitermes Speratus Detected by Fluorescent in Situ Hybridization

Microbes Environ. Vol. 19, No. 2, 120–127, 2004 http://wwwsoc.nii.ac.jp/jsme2/

Endosymbiotic Methanobrevibacter species Living in Symbiotic Protists of the Termite Reticulitermes speratus Detected by Fluorescent In Situ Hybridization

KURT HARA1, NAOYA SHINZATO2, TAIRO OSHIMA1 and AKIHIKO YAMAGISHI1*

1 Department of Molecular Biology, School of Life Science, Tokyo University of Pharmacy and Life Science, 1432–1 Horinouchi, Hachioji, Tokyo 192–0392, Japan 2 Research Institute of Biological Resources, National Institute of Advanced Industrial Science and Technology (AIST), 1–1 Higashi, Tsukuba, Ibaraki 305–8566, Japan

(Received November 12, 2003—Accepted February 18, 2004)

Two species of cellulolytic protist, Dinenympha parva and Microjoenia, living in the guts of the lower termite Reticulitermes speratus are known to harbor endosymbiotic methanogens detectable with an epifluorescent microscope. DNA isolated from the guts of worker termites in a colony of R. speratus was amplified using archaea- specific primers and cloned, and partial 16S rRNA gene sequences were obtained. Archaeal PCR clones obtained from the guts of xylophagous insects in this and previous works formed four subgroups within the Methano- brevibacter branch of a phylogenetic tree; the sequences of the clones obtained in this report belonged to subgroups, designated XSAT1A and XSAT1D. Using a probe specific to each of the subgroups, 50 and 10 endosymbiotic Methanobrevibacter cells per protist respectively were detected with a probe specific to the subgroup XSAT1A by fluorescent in situ hybridization analysis in D. parva and Microjoenia. There was no observed hybridization to the endosymbiont with other subtype-specific probes including XSAT1D. Based on these results, endosymbionts in D. parva and Microjoenia sp. are proposed to belong to the Methanobrevibacter subgroup XSAT1A.

Key words: endosymbiotic Methanobrevibacter, fluorescence in situ hybridization, Reticulitermes speratus, Dinenympha parva, Microjoenia sp.

Termites digest cellulose efficiently using a highly duce methane from H2 and CO2. The free-living archaea evolved symbiotic microbial system3) and are claimed to be Methanobrevibacter curvatus, M. cuticularis, and M. one of the most significant methane gas producers on filiformis are the only methanogens to be isolated from Earth14). Termites are divided into 2 groups: lower and high- lower termites10,11). Some protist-associated methanogens er termites. Protists in the guts of lower termites take up and have been identified in the gut of the lower termite by 12,21) digest cellulose, and produce acetate, fatty acids, H2, and microscopic observation . CO2. The complex symbiotic microflora consists of metha- Previously, we investigated archaeal communities using nogens, acetogens, and fermentative bacteria in the gut of PCR of the 16S rRNA gene prepared from whole gut lower termites9). On the other hand, higher termites rely on samples of various termite species18,19) and xylophagous their own cellulase for the digestion of cellulose and no cockroaches6). We identified several clones of methanogens symbiotic protists have been reported. Symbiotic metha- in these xylophagous insects and found that these clones nogens exist in both lower and higher termites, and pro- formed three groups in the phylogenetic tree of methanogens. These clone groups were named XSAT (Xylophagous 6) * Corresponding author; E-mail: [email protected], Tel: insect Symbiotic Archaeal Type) 1, 2, and 3 . 81–426–76–7139, Fax: 81–426–76–7145 The clones in the XSAT1 group belong to the genus Endosymbiotic Methanogen in Protists 121

Methanobrevibacter and are the most common among ra et al.21) have found that 16S rRNA gene clones from methanogens in the lower termite species18,19). Clones be- Microjoenia sp. belong to the XSAT1A subgroup and the longing to the group XSAT1 are further separated into sub- clones from Dinenympha parva belong to the XSAT1A and groups XSAT1A, 1B, 1C, and 1D (Fig. 1). Frohlich et al. 1D subgroups which are the symbiotic protists of a lower and Tokura et al. have amplified, cloned and sequenced termite, Reticulitermes speratus. However, clones in groups DNA of methanogens associated with gut walls and single XSAT1A and 1D were also recovered from the gut wall of cells of protists isolated with a micromanipulator5,21). Toku- R. speratus. Accordingly, endosymbionts in these protists

Fig. 1. Phylogenetic tree of 16S rRNA sequences of isolated species and PCR clones in the genus Methanobrevibacter. The clones obtained in this study are shown in red letters. Protist-associated PCR clones from R. speratus21), termite gut wall-attached symbiotic PCR clones from R. speratus21) and clones isolated from other lower termites10,11) are shown in dark blue, light blue, and green letters, respectively. The tree was produced by the neighbor-joining method with Thermococcus celer as an outgroup. The scale bar represents 0.1 substitutions per nucleotide position. Genbank, DDBJ and RDP accession numbers are indicated in the parentheses. 122 HARA et al. are likely to belong to one or two of the Methanobrevi- bacter subgroups. However, protists isolated using a micro- Phylogenetic analysis manipulator may contain ambient free-living microbial Sequence data were aligned with the other archaeal se- cells, so that the endosymbiotic methanogens in two species quences using the CLUSTAL X program20). A phylogenetic of protist, D. parva and Microjoenia, remain unclear. In this tree was constructed by the neighbor-joining distance ma- study, we have investigated the endosymbiotic methano- trix method17) using PHILIP version 3.573 (J. Felsenstein, gens in the protists living in the hindgut of R. speratus by the University of Washington). Bootstrap values were fluorescent in situ hybridization using probes specific to estimated by calculating after 1000 resamplings. The maxi- each of these subgroups. mum likelihood method was also used to confirm the tree topology. Materials and Methods Oligonucleotide probes Sample To design subgroup-specific probes, representative se- One colony of R. speratus wood-feeding termites quences of respective subgroups were aligned with the (RS1400) was collected from wood in Hachioji, Tokyo, sequences of the genus Methanobrevibacter. All probes Japan. They were maintained with wood flakes in plastic were designed targeting the same region, 1001–1019 in the containers at room temperature, until use. Worker termites E. coli 16S rRNA gene (see Results and Table 1). Four Cy3- were rinsed with 70% ethanol to reduce contamination from labeled probes specific to each of the subgroups XSAT1A, bacteria adhering to the surface. The guts were pulled out B, C, and D were synthesized by Amersham Bioscience using sterilized forceps, and the symbiotic microbes were (Tokyo Japan). suspended in a 0.4% NaCl solution. In situ hybridization DNA extraction and PCR amplification Termite gut samples were fixed with 4% paraformalde- DNA extraction and PCR amplification were performed hyde in PBS (137 mM NaCl, 2.68 mM KCl, 8.1 mM 6,18,19) as reported previously . In brief, total DNA was extract- Na2HPO4, 1.47 mM KH2PO4, pH 7.2) at 4LC for 16 h. ed from the whole gut of ten individual R. speratus workers Teflon-coated glass slides (Cel-Line/Erie Scientific, Ports- using a Fast DNA kit (BIO101, Calsbad, CA, USA) and a mouth, NH, USA) were coated with 0.1% gelatin, contain- bead beater Fast Prep system (BIO 101, USA), then purified ing 0.01% KCr (SO4)2. Samples were spotted and air dried, with a Qiagen Blood & Cell Culture DNA Mini kit (Qiagen and dehydrated in a graded ethanol series (50, 80, 100%) for Inc., Hilden, Germany). Symbiotic archaeal 16S rRNA 3 min each, then digested with 0.01 mg/ml of Proteinase K genes were amplified by PCR using a pair of archaea- (in 10 mM Tris/HCl, pH 7.5, 1 mM EDTA) at room temper- specific primers6,18,19), ARC856F (5’-TAAAGGAATTG- ature for 3 min, and dehydrated again in a graded ethanol GCGGGGGA-3’) and ARC1354R (5’-TGACGGGCGGT- series (50, 80, 100%) for 3 min each. Samples were hybrid- GTGTGCAAG-3’), with 40 cycles of the following thermal ized with 2 pmol of Cy3-labeled oligonucleotide probe in program: 94LC for 30 s, 60LC for 30 s, and 72LC for 90 s. 10 l of hybridization buffer (0.9 M NaCl, 5 mM EDTA, The amplified DNA fragments were separated by agarose 0.1% SDS, 0.05PPBS, 0.5 mg/ml Poly (A), 10PDenhardt’s) gel electrophoresis and recovered from the gel. The at 30LC for 16 h. Formamide was added at the final concen- fragments were cloned with a TOPO TA Cloning kit trations listed in Table 1. The slides were washed with 10 (Invitrogen, Carlsbad, CA, USA). ml of 6PSSC (0.9 M NaCl, 90 mM Na acetate, pH 7.0) at 35LC for 20 min. The slides were air dried and stained with Sequence analysis 10 l of 10 g/ml DAPI (4',6-diamidino-2-phenylindole) in The nucleotide sequences were determined with a Big distilled water at room temperature for 5 min, and observed Dye Terminator Cycle Sequence kit (Applied Biosystems with an Olympus BX60 microscope (Olympus, Tokyo Japan, Tokyo, Japan) using primers M13pM4 and M13pRV Japan) equipped with a BX-FLA epifluorescence system (Amersham Bioscience Corp., Piscataway, NJ, USA) on with the filter set U-MWU or U-MWIG for DAPI and Cy3 an ABI PRISM 3100 DNA Sequencer (Applied Biosys- fluorescence, respectively. Photographs were taken with tems Japan). The sequences were examined with the an Olympus PM-20 auto photograph system. CHECK CHIMERA program in the ribosomal database project to check for chimeric artifacts13). Endosymbiotic Methanogen in Protists 123

and MD105 amplified from the gut of the Australian Nucleotide sequence accession numbers termite Mastotermes darwiniensis19) were also included in The sequences obtained in this study (RS801 and RS802) the XSAT1D subgroup, although they may form another are available in DDBJ under accession numbers AB096062 subcluster within the subgroup. and AB096063, respectively. In situ hybridization Results R. speratus was found to have clones predominantly belonging to group XSAT118,19). We found only the subgroups Phylogeny of archaeal PCR clones XSAT1A and 1D in R. speratus in this study. Probes were In this study, 110 clones were obtained following PCR designed based on aligned sequences of the clones and spe- amplification of archaeal 16S rRNA genes from gut DNA cies in the genus Methanobrevibacter in the targeting region extracts of 10 individuals of a R. speratus colony and between 1001 and 1019 (E. coli 16S rRNA gene number- sequenced. Six sequences with chimera artifacts were iden- ing). Although we designed and tested other probes target- tified by the program CHECK CHIMERA13) and were not ing other regions of the 16S rRNA gene (1115–1133 and used in the following analyses. Related sequences were 1285–1306), no hybridization could be detected (data not identified through searches of public databases and a phylo- shown). The XSAT1A subgroup-specific probe (1A1001) genetic tree was constructed with the neighbor-joining has no mismatches to sequences of the subgroup HW2 or M. method (Fig. 1). A tree of essentially the same topology was cuticularis. The PCR clone HW2 was obtained from the gut obtained using the maximum likelihood method. In a previ- wall of a lower termite, Hodotermopsis sjoestedti21). How- ous study, we surveyed the symbiotic methanogenic com- ever, we could identify no sequence belonging to subgroup munity in the hindgut of R. speratus by PCR techniques18,19), HW2 or M. cuticularis out of the 110 clones from the R. and showed that the dominant methanogenic clones form speratus RS1400 colony. Sequences belonging to subgroup four subgroups, XSAT1A-D, scattered among the genus HW2 or subgroup M. cuticularis have not been reported for Methanobrevibacter (Fig. 1). Symbiotic methanogenic R. speratus by other authors either (see Fig. 1). These clones in R. speratus from this study belonged to the probes were used for in situ hybridization. Hybridized cells previously defined subgroups XSAT1A and 1D (Fig. 1). were inspected with an epifluorescent microscope. Forma- Thirty-three clones represented by clone RS801 and 71 clones mide concentrations in the hybridization buffer were adjust- represented by RS802 belonged to subgroups XSAT1A ed for each probe to obtain optimum specificity (Table 1). and 1D, respectively18,19). No cross hybridization was detected for these probes when All of the clones (33 clones) belonging to subgroup the representative clones of subgroups (RS801, RS104, XSAT1A were related to the representative clone RS801 RS404, and RS802) were used as target sequences for (98% identity) and formed a cluster with the PCR Southern hybridization (data not shown). clones previously obtained from the guts of lower D. parva cells observed with Nomarsky differential inter- termites15,16,18,19,21). Clone RS801 was closely related ference contrast optics are shown in Fig. 2A, D, and G. The (99.6%) to clone RS208 previously isolated from R. same cells observed with DAPI fluorescence are shown in speratus18). Clone RS801 showed 98.4 and 99.8% identity Fig. 2B, E, and H, and the same fields observed with the fil- to clone Cd30 isolated from the lower termite Cryptotermes ter set for the XSAT1A subgroup-specific probe labeled domesticus16) and clone M4 isolated from R. speratus15), re- with Cy3 are shown in Fig. 2C and F. All of the endosymbi- spectively. Subgroup XSAT1A also includes clone LRsM1, otic cells in D. parva were hybridized with the XSAT1A amplified as a methanogenic clone associated with the subgroup-specific probe. However, these endosymbionts symbiotic protist Microjoenia from the gut of R. speratus were not detected with the XSAT1D subgroup-specific and LRsD3, a clone associated with D. parva from R. probe (N in Fig. 2I). Likewise, the XSAT1B and 1C sub- speratus21). group-specific probes did not hybridize with these endo- On the other hand, all of the clones (71 clones) belonging symbionts (data not shown). Very weak hybridization of the to the XSAT1D subgroup isolated from R. speratus were XSAT1D subgroup-specific probe was observed with all of related to the representative clone RS802 (98% identity). the endosymbiotic cells at lower hybridization stringency They formed a cluster with the species M. curvatus isolated (0, 10 and 20% formamide concentration). These cells were from a lower termite5). The methanogenic clones LRsD2 detected clearly with the XSAT1A subgroup-specific probe associated with D. parva from R. speratus21) and MD101 at higher stringency (20% formamide concentration). It 124 HARA et al.

Fig. 2. Whole cell hybridization of endosymbiotic methanogens living in the hindgut of R. speratus. A, D, G, and J: Photographs taken with a Nomarsky prism. Panels B, E, and H are the photographs taken with DAPI fluorescence in the same field as A, D, and G. Panel K shows the DAPI fluorescence photograph of the same species as that shown in Panel J. Panels C and F show the endosymbionts of D. parva hybridized with the Cy3-labeled probe specific to XSAT1A methanogens in the same field as B and E. Panel L shows the endosymbionts of Microjoenia detected with the XSAT1A-specific probe in the same field as K. Panel I shows the negative signal of the endosymbionts of D. parva with the XSAT1D-specific probe. Scale bars represent 10 m. (P); positive signals. (N); negative signals. Endosymbiotic Methanogen in Protists 125

should be noted that F420 fluorescence of methanogens could not be detected after hybridization because of the destruc- tion of the chromophore during the hybridization procedure. Figure 2J shows a Microjoenia sp. cell observed using a Nomarsky prism. Some endosymbionts were detected with DAPI fluorescence (Fig. 2K is a different field to Fig. 2J). These endosymbionts were also detected with the XSAT1A subgroup-specific probe in Fig. 2L. Our data indicate that endosymbiotic methanogens living in these two species of symbiotic protists, D. parva and Microjoenia sp., are a single species or at least closely related species that belongs to the subgroup Methanobrevibacter XSAT1A. D. parva and Microjoenia sp. constantly harbor about 50 and 10 endosymbiotic methanogens, respectively (Fig. 2B, E, H, and K). Similar numbers of endosymbiotic cells were detected in D. parva and Microjoenia sp. cells with F420 fluorescence (Fig. 3B, C). The size and morphology of the endosymbiotic Fig. 3. Methanogens living in the hindgut of R. speratus observed methanogens in these protists are also similar; they are short by F420 epifluorescence without hybridization Samples were prepared from R. speratus individuals of another colony. A. Gut- straight rods about 1.0 m long and 0.5 m wide in both wall-attached methanogens. B. Methanogens in a cell of D. par- protists. va. C. Methanogens in a cell of Microjoenia. In Fig. 2D and E, another Dinenympha sp. can be seen beside the D. parva cell. This Dinenympha sp. harbors unknown species of endosymbiotic microbes that were stained (RS1400) from Hachioji, Tokyo, Japan. We amplified and by DAPI (Fig. 2E). None of our probes hybridized with the isolated 34 XSAT1A subgroup clones and 72 XSAT1D endosymbionts in this species of Dinenympha (N in Fig. 2F clones (Fig. 1). The clones from the termite colony grouped and data not shown). exclusively in the subgroups XSAT1A and 1D and clones within each subgroup were closely related to each other. Discussion While PCR clone analysis is a powerful tool for investi- gating microbial communities, it can not be used to examine Recently, we investigated the archaeal community in the the morphology or location of microbes. Fluorescence in gut of lower termites by PCR clone analysis18,19) and showed situ hybridization analysis allows one to fill this gap in that most of the PCR clones from several species of lower knowledge1,18). Previously, we analyzed the free-living (gut termites collected in various regions of Japan including R. content was centrifuged, could not differentiate between speratus belong to the subgroups XSAT1A and 1B (origi- free and gut-wall attached) symbiotic methanogens by fluo- nally TYPE1A and 1B) in the genus Methanobrevibacter. rescent in situ hybridization and showed that they belong to However, the dominant archaeal PCR clones obtained from the XSAT1 group18); morphologically, they are short rods R. speratus RS3 belonged to the subgroups XSAT1A and and some are attached to each other (see Fig. 3A). In this 1D18). In this study, we sampled a R. speratus colony study, we designed probes specific to the subgroups of the

Table 1. Cy3-labeled probes used in this study

Probea Specificity Sequence (5’–3’)b Fc 1A1001 XSAT1A, M. cuticularis gp., HW2 gp. TCAACCTGGCCATCATACT 20% 1B1001 XSAT1B TCAATCTGACCATCATACT 0% 1C1001 XSAT1C TCAAACTGGCTATCATAC 0% 1D1001 XSAT1D TTAACCTGGCCATCATACT 0% a Target sites are bases 1001 to 1019 in the E. coli 16S rRNA gene. b Bold letters indicate the nucleotides that differ between the 4 probes. c Formamide concentration used in the hybridization solution. 126 HARA et al. clones amplified from the gut of R. speratus (Table 1). Two supports our conclusion. species of symbiotic protists, Microjoenia sp. and D. parva, Tokura et al.21) used single-cell PCR clone analysis to in the hindgut of R. speratus possess associated methano- show that methanogens associated with D. parva are not a gens detected by epifluorescence microscopy21). In this re- single species, but two species: clone LRsD2 belongs to the port, we showed that endosymbiotic methanogens in both M. curvatus group (XSAT1D in this study) and LRsD3 be- host protists belong to the subgroup XSAT1A. Accordingly, longs to the M4 group (XSAT1A). However, a single type the endosymbiotic methanogens are a single or a group of of clone (LRsM1) from group M4 was also amplified from closely related species in the genus Methanobrevibacter another protist, Microjoenia sp. Our results are compatible (Fig. 2). with the latter result but not with the result that two Based on the PCR clone analysis, methanogenic sym- sequences of methanogens were recovered from D. parva. bionts and anaerobic free-living ciliates have been ana- We detected hybridization of endosymbiotic microbes with lyzed7,8). Based on the finding of incongruence between the XSAT1A-specific probe in both protists. Investigations host and symbiont phylogenies, multiple acquisitions of using a micromanipulator and PCR clone analysis are useful, methanogenic symbionts by anaerobic ciliates have been however, there is a risk of contamination during the ma- proposed8). Embley and Finlay7) have reported that endo- nipulation process. We could not detect any cells with the symbiont sequences are distributed throughout the metha- XSAT1D subgroup-specific probe in any part of the gut nogen tree and some endosymbionts are closely related to content. Therefore, the morphology and the population of free-living methanogens, and proposed that symbioses have the XSAT1D subgroup in the gut of R. speratus (RS1400) formed repeatedly and independently. D. parva and Micro- remain unknown. However, we observed gut-attached non- joenia sp. belong to different taxa, and were even classified endosymbiotic methanogens by epifluorescence micro- in different phyla according to Cavalier-Smith: Meta- scopy. Sequence RS802 of subgroup XSAT1D has 99.0% monada and Trichozoa, respectively4). The methanogen identity to strain M. curvatus isolated from the lower ter- XSAT1A may have been acquired by different Protista mite R. flavipes10). The phylogenetic position of clone living in the same environment. RS802 in Fig. 1 suggests that the subgroup XSAT1D may These methanogens are of rod-shaped morphology, and be a nonendosymbiont. Further study is needed to detect there were about 50 cells and 10 cells per host in D. parva those methanogens with type-specific probes. and Microjoenia sp., respectively. The numbers of cells In this study, we succeeded in the fluorescent in situ hy- per host protist are compatible with those observed using bridization of the endosymbiotic Methanobrevibacter but 21) F420 fluorescence. As shown previously , D. parva and failed in hybridization with gut-attached non-endosymbiotic Microjoenia sp. exist in numbers of 2.95P103 cells and (free living) Methanobrevibacter. Due to the difficulty in 6.81P102 cells per gut of R. speratus, respectively. Accord- detecting Methanobrevibacter2,21), our previous paper is the ingly, the number of cells of the endosymbiotic XSAT1A only report of the detection of free-living Methanobrevi- methanogens in D. parva and Microjoenia sp. is about bacter by fluorescent in situ hybridization18). In situ hy- 1.5P105 and 6.8P103 cells per R. speratus gut, respectively. bridization with Methanobrevibacter is problematic due to Our results suggest that the XSAT1A subgroup is a probe accessibility2,21). Endosymbiotic Methanobrevibacter cluster of endosymbiotic methanogens (Fig. 1). The other living in the gut of R. speratus may have been detected by PCR clones that belong to the XSAT1A subgroup have fluorescence in situ hybridization in this report due in been found in the Australian termite M. darwiniensis as part to their accessibility, in addition to the trials of probes endosymbionts in the protist of another genus, Penta- targeting different regions of the 16S rRNA sequence. trichomonoides scroa5). In that report, the endosymbiotic methanogen was obtained by using a micromanipulator, and the 16S rRNA gene sequence was amplified using single- References cell PCR. The sequence is related to Cd30 isolated from the 1) Amann, R.I. and W. Ludwig. 2000. Ribosomal RNA-targeted 16) lower termite C. domesticus (Fig. 1) , which belongs to the nucleic acid probes for studies in microbial ecology. FEMS XSAT1A subgroup. This result is consistent with our result Microbiol. Rev. 24: 555–565. in the sense that symbiotic protists living in the hindgut of 2) Amann, R.I., W. 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