Methanogenic Symbionts and the Locality of their Host Lower Termites

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1 Vol. 16, No. 1, 43 47, 2001 Methanogenic Symbionts and the Locality of their Host Lower Termites NAOYA SHINZATO 1, 2, TADAO MATSUMOTO 1, IKUO YAMAOKA 3, TAIRO OSHIMA 2 and AKIHIKO YAMAGISHI 2 * 1 Department of Biology, School of Arts and Science, University of Tokyo, Komaba, Meguro-ku, Tokyo , Japan 2 Department of Molecular Biology, School of Life Science, Tokyo University of Pharmacy & Life Science, Horinouchi, Hachioji, Tokyo , Japan 3 Biological Institute, Faculty of Science, Yamaguchi University, Yoshida, Yamaguchi , Japan (Received August 9, 2000 Accepted September 22, 2000) SSU rrna genes of symbiotic methanogens in the hindgut of four lower Japanese termites and one Australian lower termite Mastotermes darwiniensis as well as a soil sample collected near a nest of Reticulitermes speratus were amplified, cloned and phylogenetically analyzed. Most of the clones found in the lower termites were of the genus Methanobrevibacter. The symbiotic methanogens in the Japanese termites and in the soil sample were related to each other. The clones obtained from M. darwiniensis grouped separately from the clones of Japanese termites. These results suggest that the methanogen community of lower termites reflects host locality rather than phylogeny. Key words: symbiosis, termite, methanogen, phylogeny, Thermoplasma Termites harbor methanogenic archaea in the hindgut 9), where methane is formed by the reduction of carbon-dioxide with hydrogen. Methanogens and acetogens in the termite hindgut play a role as a hydrogen-sink. The hydrogenconsuming process facilitates the anaerobic degradation of lignocellulose 14). The microecology of the termite gut was recently reviewed 1). A phylogenetic analysis of methanogens in the hindgut of the lower termite Reticulitermes speratus by Ohkuma et al. 11) found only one type of sequence in PCR clones. We have also analyzed symbiotic methanogens in the hindgut of R. speratus 15). PCR clones of SSU rdna sequences were grouped into three types. Type 1 clones were further classified into subtypes 1A, 1B, 1C and 1D. Most of the PCR clones of methanogen in R. speratus collected around the Japan Archipelago belonged to subtypes 1A and 1B in the genus Methanobrevibacter. Fluorescence in situ hybridization analysis showed that at least some of the type 1 species live on the hindgut epithelial surface. Ohkuma et al. 10,12) * Corresponding author; yamagish@ls.toyaku.ac.jp, Tel: , Fax: have reported two other types of sequences, Cd30 and MHj4, from Cryptotermes domesticus and Hodotermopsis sjostedti, respectively. Leadbetter et al. 5,6) have isolated three species of Methanobrevibacter, M. curvatus, M. cuticularis, and M. filiformis from R. flavipes. In the present study, we examined the molecular phylogeny of symbiotic methanogens of five species that belong to four families of lower termites. Phylogenetic relationships between methanogens and host termites were also discussed. Four lower termite species of three families, Neotermes koshunensis, Hodotermopsis sjostedti, Reticulitermes kanmonensis, and Coptotermes formosanus, were collected in the Japan Archipelago. Sampling points of the Japanese termites are listed in Table 1. Another lower termite Mastotermes darwiniensis was collected in Australia and kindly donated by Dr. O. Kitade. Ten individuals of pseudergate (worker) from each colony of termite were employed for DNA extraction. Total genomic DNA of the digestive tracts of the termites was prepared as described previously 15). PCR primers and reaction conditions were also described in a previous report 15). The primers used were ME855F and ME1354R, which were designed to se-

2 44 SHINZATO et. al. Table 1. Number and type of clones isolated from Japanese lower termites. Number of clones Species Locality Colony Clone type a and affiliation Reference bac b mic b T b 1A Cd30 1B MHj4 1C 1D 2 3 Rhinotermitidae Reticulitermes speratus Tokyo RS [15] RS2 5 5 [15] RS [15] Kobe RS5 8 2 [15] Yamaguchi RS3 7 3 [15] Okinawa Is. RS6 6 4 [15] Wako 6 [11] Reticulitermes kanmonensis Shimonoseki RK1 1 4 This study Coptotermes formosanus Okinawa Is. CF1 1 4 This study Kalotermitidae Neotermes koshunensis Okinawa Is. NK1 2 3 This study Cryptotermes domesticus Iriomote Is. 23 [10] Hodotermitidae Hodotermopsis sjostedti Amamioshima Is. HJ1 5 This study Yakushima Is. 5 [12] a The clone types were reported in the previous studies: 1A D, 2, and 3[15]; Cd30[10]; MHj4[12]. b Abbreviations for orders of archaea: bac, Methanobacteriales; mic, Methanomicrobiales; T, Thermoplasmales. lectively amplify archaeal rdna. DNA fragments of expected length were amplified from the DNA isolated from all the termites used in this study. The amplified DNA fragments were ligated with a plasmid vector using a T-A cloning kit (Invitrogen, The Netherlands), and E. coli strain JM105 was transformed with the ligate. Nucleotide sequences of isolated clones were determined with an automated DNA sequencer ABI model 377 (PE Applied Biosystems) according to the manufacturer s protocol. Sequences of five clones per termite species were determined. The sequences were examined with the CHECK-CHIMERA program in the ribosomal database project to test for chimerical artifacts 7) and then phylogenetically analyzed by the neighbor-joining method 13). The program package and the analysis conditions employed were described previously in detail 15). Phylogenetic analysis of these sequences was also performed on members of Euryarchaeota. Sequences of isolates from the American lower termite R. flavipes, M. curvatus, M. cuticularis and M. filiformis 5,6), and the PCR clones reported for several Japanese lower termites 10 12,15) were also included. In Table 1, the number of clones related to each respective type is listed. In a previous study, we analyzed PCR clones of archaea amplified from R. speratus. The clones were grouped into three types with four subtypes of type 1 15). Analysis of PCR clones of four other species of lower termite revealed that all of the clones are of subtype 1A or 1B. Evolutionary distances of these clones from the respective type clones of subtype 1A or 1B were within 0.02 substitutions per nucleotide position (sequence similarity 98%). Though the subtype 1A sequence was not obtained from H. sjostedti, both subtypes 1A and 1B were detected in all the other termite species examined. A phylogenetic tree of the clones in Methanobacteriales is shown in Fig. 1. The type 1 clones isolated in this study are represented by the type clones obtained from R. speratus as shown in Fig. 1. Though clones Cd30 and MHj4, which were isolated from C. domesticus and H. sjostedti, respectively 10,12), were not of these subtypes, they branched in the vicinity of subtypes 1A and 1B, respectively. To compare the phylogeny of the methanogens in the hindgut microflora between Japanese termites and termites of other regions, the lower termite M. darwiniensis collected in Australia was included in this analysis (Fig. 1). All of the five clones isolated from the hindgut of M. darwiniensis belonged to the genus Methanobrevibacter. Methanobrevibacter species have been also isolated from the American lower termite R. flavipes as mentioned above 5,6). The meth-

3 Methanogenic Symbionts in Termites 45 Fig. 1. Phylogenetic positions of the cloned sequences derived from Japanese termites, the Australian termite M. darwiniensis (MD), and humic soil (XT) within the members of Methanobrevibacter. Bold letters indicate the sequences determined in this study. See Table 1 for the clones grouped with subtypes 1A and 1B of Reticulitermes speratus. The phylogenetic tree was constructed from 485 unambiguously aligned nucleotide positions in the SSU rrna gene using the neighbor-joining method. The tree topology was tested by 1000 repetitions of bootstrap analysis. The accession numbers of the sequence are indicated in parentheses. The standard bar represents 0.01 substitutions per nucleotide position. Genbank and RDP accession numbers are indicated in parentheses. anogen community in the lower termite hindgut appeared to be dominated by the genus Methanobrevibacter irrespective of the host termite species and the host locality. However, the phylogenetic positions of clones isolated from the Australian termite were significantly different from those of the Japanese termites. The clones MD101, 104 and 105 clustered near M. curvatus and the symbiont subtype 1D of R. speratus. The clone MD103 was related to M. arboriphilicus. The last clone MD102 branched deeply in the Methanobrevibacter and was distantly related to the other known sequences. In our previous study, most of the clones isolated from R. speratus were affiliated with subtype 1A or 1B (Table 1 and Fig. 1). All of the clones isolated from the lower termite species collected in Japan were related to subtype 1A or 1B (Table 1). These results suggest that the symbiotic methanogen communities in Japanese lower termites are related to one another. On the other hand, rdna clones of methanogens obtained from M. darwiniensis were substantially different from those of the Japanese termites. The three species isolated from R. flavipes reported by Leadbetter et al. were also distantly related to subtype 1A or 1B and these species seemed to be the dominant methanogens in several termite colonies 5,6). The methanogen community seems to be related to the locality of the host termite. Leadbetter et al. have reported an intraspecies difference in clone phylogeny 6). They have isolated the three species of Methanobrevibacter, M. curvatus, M. cuticularis, and M. filiformis from R. flavipes. Among these isolates, M. filiformis was easily distinguished from the other Methanobrevibacter by its filamentous cell morphology. They reported that though the filamentous M. filiformis was one of the dominant methanogens in R. flavipes collected from Woods Hole in Massachusetts and Janesville in Wisconsin, cells of similar morphology were not observed in R. flavipes collected from Dansville and Spring Arbor in Michigan 6). They have pointed out the possibility that subtle differences in the food consumed by the termites or unknown genetic differences between the termites affected their methanogen composition. The symbiotic flagellate species of lower termites are known to be characteristic to the host species 3,4). The specificity seems to be caused by transmission of the flagellates via proctodeal feeding. Our results, however, showed that the species and lineage of the symbiotic methanogens were related to host locality rather than host phylogeny. In the

4 46 methanogen community, horizontal transfer from the environment via the feeding action of the host termite has to be taken into consideration in addition to vertical transmission. In order to compare the phylogenetic composition of the methanogen community between the termite hindgut and habitat environment, humic soil was sampled near a nest of R. speratus in a forest near Tokyo, and the archaeal community was phylogenetically analyzed. The humic soil was collected at a depth of 15 cm with 1m of the nest. This colony had been used as colony RS1 of R. speratus in a previous study of the methanogen community in termite hindgut 15). The soil sample was used for DNA extraction immediately. Five hundred grams of the soil sample was suspended in 1 L of distilled water. After standing for 10 min, 500 ml of the supernatant was centrifuged at 7,500 g for 30 min at 4 C. The pellet was resuspended in 50 ml of an extraction buffer that consisted of 100 mm Tris-HCl (ph 8.0), 10 mm EDTA, and 0.1% SDS. DNA was prepared by the same procedure as was used for the insect materials. The PCR using the archaeal SSU rrna gene-specific primers resulted in amplification of 0.5 kbp products from the DNA extracted from the humic soil sample. Seven cloned sequences were analyzed. These clones were grouped into two different clusters, one belonging to Methanobrevibacter and the other to Thermoplasmaceae. The results were similar to those obtained from the DNA isolated from Japanese lower termites. Two clones, XT106 and 108, were closely related to subtype 1A and Cd30 with a relatively high bootstrap value (85%). The clone XT109 was clustered with subtype 1B and MHj4 with a bootstrap value of 100% (Fig. 1). Clones of subtypes 1A and 1B were the most abundant in Japanese lower termites (Table 1). The SHINZATO et. al. remaining four clones were affiliated with the order Thermoplasmales. Fig. 2 shows the phylogenetic tree for these clones: type 3 clones of R. speratus, and two constituents of this order, Thermoplasma acidophilum and Picrophilus oshimae. The clones XT101, 102, 103, and 107 formed a cluster within the substitutions per nucleotide position (sequence similarity 99%) near a type 3 R. speratus symbiont. The bootstrap confidence of this relation was 100%. These results show that the methanogen communities of Japanese termites relate more to the communities in the environment than to methanogens found in foreign species. The results suggest that methanogen communities of lower termites reflect host locality rather than host phylogeny. Characteristics of environmental factors such as temperature and humidity or of food may affect the methanogen community found in termite hindgut. Alternatively, horizontal transfer of methanogens through the outside environment via feeding action of the host termite may have contributed to the methanogen community. Infection by Bacillus species of termites has been reported by Margulis et al. 8), who found that the presence of Arthromitus filaments in intestines of arthropods including termites characteristic of Bacillus cereus infection by spores or cells from animal feces or soil particles 8). However, the locality of a methanogen community may not be absolute. The subtypes 1C and 1D obtained from R. speratus as minor clones were closely related to M. filiformis and M. curvatus, respectively. Accordingly, locality of the host may be related to the relative abundance of methanogen species. Recently, an rdna sequence derived from endosymbiotic methanogens of a flagellate living in Fig. 2. Phylogenetic relationships among the Thermoplasma-related clones obtained from the termites and humic soil (XT). Bold letters indicate the sequences determined in this study. The tree was constructed from 482 unambiguously aligned nucleotide positions in the SSU rrna gene using the neighbor-joining method and including the members of Thermoplasmaceae. The tree topology was tested by 1000 repetitions of bootstrap analysis. Accession numbers of the sequence are shown in parentheses. The standard bar represents 0.1 substitutions per nucleotide position. Genbank and RDP accession numbers are indicated in parentheses.

5 Methanogenic Symbionts in Termites 47 M. darwiniensis was reported 2). Though this sequence was not included in the phylogenetic analysis due to its short length (295 bp), a close phylogenetic relationship between the endosymbiont and clone Cd30 obtained from C. domesticus has been reported. Accordingly, endosymbiotic methanogen may have to be considered separately. Endosymbionts must have a close relationship to the host flagellates of termites. We thank Dr. O. Kitade (Ibaraki University) for kind donation of M. darwiniensis. This work was partially supported by grants in aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan (no and ). References 1) Brune, A. and M. Friedrich Microecology of the termite gut: structure and function on a microscale. Curr. Opin. Microbiol. 3: ) Fröhlich, J. and H. König Rapid isolation of single microbial cells from mixed natural and laboratory populations with the aid of a micromanipulator. Syst. Appl. Microbiol. 22: ) Honigberg, B.M Protozoa associated with termites and their role in digestion, p In K. Krishna and F.M. Weesner (ed.), Biology of Termites, Vol. 2, Academic Press, New York. 4) Kirby, H Systematic differentiation and evolution of flagellates in termites. Rev. Soc. Mex. Hist. Nat. 10: ) Leadbetter, J.R. and J.A. Breznak Physiological ecology of Methanobrevibacter cuticularis sp. nov. and Methanobrevibacter curvatus sp. nov., isolated from the hindgut of the termite Reticulitermes flavipes. Appl. Environ. Microbiol. 62: ) Leadbetter, J.R., L.D. Crosby and J.A. Breznak Methanobrevibacter filiformis sp. nov., A filamentous methanogen from termite hindguts. Arch. Microbiol. 169: ) Maidak, B.L., N. Larsen, M.J. McCaughey, R. Overbeek, G.J. Olsen, K. Fogel, J. Blandy and C.R. Woese The ribosomal database project. Nucleic Acid Res. 22: ) Margulis, L., J.Z. Jorgensen, S. Dolan, R. Kolchinsky, F.A. Rainey and S.C. Lo The Arthromitus stage of Bacillus cereus: intestinal symbionts of animals. Proc. Natl. Acad. Sci. USA 95: ) Martius, C., R. Wassmann, U. Thein, A. Bandeira, H. Rennenberg, W. Junk, and W. Seiler Methane emission from wood-feeding termites in Amazonia. Chemosphere 26: ) Ohkuma, M. and T. Kudo Phylogenetic analysis of the symbiotic intestinal microflora of the termite Cryptotermes domesticus. FEMS Microbiol. Lett. 164: ) Ohkuma, M., S. Noda, K. Horikoshi and T. Kudo Phylogeny of symbiotic methanogens in the gut of the termite Reticulitermes speratus. FEMS Microbiol. Lett. 134: ) Ohkuma, M., S. Noda and T. Kudo Phylogenetic relationships of symbiotic methanogens in diverse termites. FEMS Microbiol. Lett. 171: ) Saitou, N. and M. Nei The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: ) Schink, B Syntrophism among prokaryotes, p In A. Balows, H.G. Trüper, M. Dworkin, W. Harder and K.-H. Schleifer (ed.), The Prokaryotes, Vol. 1, Springer-Verlag, New York. 15) Shinzato, N., T. Matsumoto, I. Yamaoka, T. Oshima and A. Yamagishi Phylogenetic diversity of symbiotic methanogens living in the hindgut of the lower termite Reticulitermes speratus analyzed by PCR and in situ hybridization. Appl. Environ. Microbiol. 65: