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 mi- croscope. 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 sub- groups, designated XSAT1A and XSAT1D. Using a probe specific to each of the subgroups, 50 and 10 endo- symbiotic 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 methano- gens. 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.