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Sequencing reactions were determined on an automatic sequence analyzer (ABI model 373). Plasmid DNA was purified from each clone with a Wizard mini preps DNA purification system (Promega) and used as a template for sequencing with the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit with Ampli Taq DNA Polymerase, FS (Perkin Elmer). 2.3 Nucleotide sequencing and phylogenetic analysis The insertion of the appropriated size was determined by PCR-amplification with the universal and reverse primers (TAKARA) which corresponded to the both sides of the cloning site on the vector. The purified PCR-products were ligated into a pGEM-T vector (Promega) according to manufacturer's directions and then introduced into E. PCR-products corresponding to the expected size of the prokaryotic rRNA gene (0.9 kb) were purified on a low melting agarose gel using a Wizard PCR preps DNA purification system (Promega). The reaction conditions were for 35 cycles at 94☌ for 30 s, 45☌ for 45 s, 72☌ for 2 min. The PCR primers used were described previously and corresponded to nucleotide positions 519–5–1405 of E. Ribosomal RNA genes were amplified from the purified DNA by PCR using an ExTaq DNA polymerase (TAKARA) according to manufacturer's directions. Since many termite gut microbes tightly adhere to the gut wall, these populations may not have been included in the analysis described here.
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DNA from the intestinal mixed population were extracted as described previously. The intestinal contents were gently squeezed and the gut debris was removed. 2.2 DNA extraction, PCR amplification, and cloningĪpproximately 100 termites were collected and, after their exterior surfaces were washed with distilled water, their entire guts were removed with forceps. Termite-infested wood moistened with distilled water was kept in plastic boxes at ambient temperature. domesticus (Isoptera, Kalotermitidae), were collected in the Iriomote island, Japan, in July 1996. speratus, for which the phylogenetic diversity of the symbiotic system has already reported, belongs to the family Rhinotermitidae. domesticus belongs to the family Kalotermitidae, while R. In this work, on the basis of PCR-amplified 16S rRNA sequences, we report the phylogeny of members of the intestinal microbial community of the lower termite Cryptotermes domesticus. A comparison of the constituents of the microbial communities between termite species may be beneficial to understand the nature of the termite symbiotic systems.
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Termites (order Isoptera) are divided into seven families, showing considerable variation in life-style, ecology and types of symbiosis. These studies have shown that the termite symbiotic system includes many species yet-uncultured in the laboratory. This approach has already been applied to investigate the biodiversity of the symbiotic microbial community in the gut of a lower termite, Reticulitermes speratus.
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These sequences can then be compared with each other as well as against databases of rRNA sequences from well-characterized microorganisms in order to determine the identity of organisms present in the natural microbial communities. In this approach, genes encoding small subunit ribosomal RNA (16S-like rRNA) derived from the extracted nucleic acids of mixed microbial populations are cloned and sequenced. The application of molecular phylogenetic analysis to ecological studies has enhanced our ability to assess naturally occurring biodiversity in mixed microbial assemblages ( and reviewed in ). Despite the isolation and cultivation of several bacteria and protists from within this community, our understanding of the biology and the physiology of intestinal microbiota is poor because many of the predominant species within the community, such as the spirochete-like bacteria, have not yet been cultured and characterized. The mutualistic relationship between xylophagous termites and microorganisms inhabiting their gut is one of the most fascinating examples of symbiosis, a relationship that enables termites to live by xylophagy. Termite, Symbiosis, 16S rRNA, Methanogen, Bacteroides, Spirochete 1 Introduction