African Journal of Biotechnology Vol. 6 (6), pp. 658-667, 19 March 2007 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB06.253 ISSN 1684–5315 © 2007 Academic Journals Full Length Research Paper Bacterial diversity in the intestinal tract of the fungus- cultivating termite Macrotermes michaelseni (Sjöstedt) Lucy Mwende Mackenzie 1, 2 , Anne Thairu Muigai 1, Ellie Onyango Osir 2, Wilber Lwande 2, Martin Keller 3, Gerardo Toledo 3 and Hamadi Iddi Boga 1* 1Botany Department, Jomo Kenyatta University of Agriculture and Technology (JKUAT), P.O. Box 62000, Nairobi, 00200, Kenya. 2International Center of Insect Physiology and Ecology (ICIPE), P.O. Box 30772, Nairobi, Kenya. 3Diversa Corporation, 4955 Directors Place, San Diego, CA 92121 USA. Accepted 20 March, 2007 Microorganisms in the intestinal tracts of termites play a crucial role in the nutritional physiology of termites. The bacterial diversity in the fungus-cultivating Macrotermes michaelseni was examined using both molecular and culture dependent methods. Total DNA was extracted from the gut of the termite and 16S rRNA genes were amplified using bacterial specific primers. Representatives from forty-one (41) RFLP patterns from a total of one hundred and two (102) clones were sequenced. Most of the clones were affiliated with 3 main groups of the domain Bacteria: Cytophaga-Flexibacter-Bacteriodes (73), Proteobacteria (13), and the low G+C content Gram-positive bacteria (9). Two RFLPs related to planctomycetes, but deeper branching than known members of the phylum, were detected. In addition, 1 and 2 RFLPs represented the spirochetes and TM7-OP11 groups, respectively. In studies using culture dependent techniques, most of the isolates obtained belonged to the Gram-positive bacteria with a high G+C content. However, only one of the clones represented Gram-positive bacteria with High G+C content. These results show a high bacterial diversity in the intestinal microbiota of M. michaelsen i, which continues to escape cultivation. As is the case in other termites many of the clones represent previously uncultured bacteria. The fact that most of the clones clustered with clones from Macrotermes gilvus provides further support for the hypothesis that microorganisms in intestinal tracts of termites have co-evolved with their hosts. Key words: Fungus-cultivating termites, bacterial diversity, intestinal tract, 16S rRNA gene, RFLP. INTRODUCTION Termites are terrestrial social insects, which belong to the vores (0.01-17.5 gm -2) (Lee and Wood, 1971, Collins and order Isoptera (Nutting, 1990). The order Isoptera con- Wood, 1984). Because of their apparent ecological tains 5 families of lower termites and 1 family of higher importance in tropical environments, termites have been termites (Noirot, 1992) and contains over 2600 described the subject of many studies looking at: their role as species (Ohkuma et al., 2001). Termites are important ecosystem engineers and influence on soil properties arthropods involved in the biodegradation of complex (Anderson et al., 1991; Dangerfield et al., 1998; Donovan substances like cellulose and hemicellulose found in et al. 2001), their contribution to trace gas emission plant materials (Wood and Sands, 1978; Brune, 1998), (Khalil et al., 1990; Eggleton et al., 1999) and their role in and their biomass densities in tropical and sub-tropical > the degradation of lignocellulose (Amund et al., 1988; 50 gm -2 often surpass those of grazing mammalian herbi- Breznak et al., 1994; Hinze et al., 2002). Recently termites have also attracted interest through the architecture of the mounds of M. michaelseni (Turner, 2000), which are being viewed as a possible model for *Corresponding author. E-mail: [email protected], Tel: cheap and energy efficient buildings. With the current 002546752711. Fax: 002546752164. energy crisis, the ability of termites to degrade lignocellu- Mackenzie et al. 659 lose within 24 h is increasingly being investigated for mitomyces spp. that aids in digesting cellulose (Rouland et al., production of cellulosic ethanol from biomass. 1991; Turner, 2001) and lignin (Brune 2003). Diverse microorganisms inhabit the intestinal tracts of Termites were collected once from one mound in the compound of Jomo Kenyatta University of Agriculture and Technology at Juja, all termite feeding groups (Brune, 1998; Brauman et al., 6 7 in Thika District, Kenya. The sampling site, Juja is located in the 2001) at a high density of 10 –10 cells per µl of gut transitional zone between the high rainfall area to the north and volume (Schultz and Breznak, 1978; Bignell et al., 1980; northwest and the low rainfall area to the east. The mean annual Brauman et al., 2001). The ability of these Isoptera to dig- rainfall ranges from 782 -933 mm. The vegetation is grassland with est complex molecules is mostly attributed to the action scattered bushes, dominated by Digitaria scalarum (Schweinf.) Chiov, Eragrostis atrovirens (Desf.) Steud and a herb Ocinum sp.; of these intestinal microbes. The diversity of microbes in Acacia seyal is the dominant bush. The soils are generally poorly the guts of diverse termites includes bacteria, fungi and drained and the texture of the topsoil is clay (Muchena et al., 1978) . protozoa (Brune, 1998), which have been characterized During sampling, the termites together with nest fragments were and shown to include many uncultured species (Ohkuma brought to the laboratory; adult major worker caste termites were and Kudo, 1996; Ohkuma and Kudo, 1998; Schmitt- used in experiments, within one day of collection. The Invertebrate Wagner et al., 2003; Hongoh et al., 2005; Shinzato et al., Zoology section of the National Museums of Kenya identified the termites. 2005; Hongoh et al., 2006). In the case of the fungus- cultivating termites, the symbiosis between the termite and fungi of the genus Termitomyces is crucial to the Molecular characterization of bacterial community digestive process (Anklin-Mühlemann et al., 1995). Des- pite the symbiosis with the fungi, the fungus-cultivating Termites were degutted using fine tipped forceps as previously termites are also reported to harbor dense populations of described (Schmitt-Wagner et al., 2003) and 10 guts (70 mg) were pooled and total DNA was extracted using GENECLEAN® Bio101 bacteria and archaea (Anklin-Mühlemann et al., 1995; soil DNA extraction kit according to manufactures instructions. DNA Brauman et al., 2001). Fungus-cultivating termites, play was also extracted from isolates obtained in cultivation experiments an important role in degradation processes in the tropics (see below) using the GENECLEAN® Bio101 soil DNA extraction (Buxton, 1981) but their symbiotic microbial community, kit. Purified total DNA from each sample was used as a template for which plays a role in this process has hardly been amplification of 16S rRNA genes using HotStarTaq Master Mix kit (Qiagen®) according to the manufacturer’s instructions. studied. Earlier studies involved enumeration of bacterial Bacterial 16S rRNA genes were amplified using universal primers cells and quantification of metabolite pools in the gut 27F 5’-TAG AGT TTG ATC CTG GCT CAG-3’ forward primer and compartments of Macrotermes subhyalinus (Anklin-Mü- 1392R 5’-GAC GGG CGG TGT GTA CA-3’ reverse primer hlemann et al ., 1995). In another study using groups according to the position in relation to Escherichia coli gene specific probes M. subhyalinus and Macrotermes bellico- sequence (Lane, 1991). PCR reactions were started by an initial sus were also reported to harbor dense populations of activation of the enzyme at 95ºC for 10 min followed by 25 cycles consisting of denaturation (30 sec at 94ºC), annealing (1 min 45 sec bacteria and a smaller population of archaea (Brauman et at 55ºC) and extension (2 min at 68ºC) and a final extension at al., 2001). Extensive studies have also tried to unravel 68ºC for 10 min. The PCR product was visualized on a 1% agarose the symbiosis between the fungus and the termite gel stained with ethidium bromide. (Bignell et al., 1994; Crosland et al., 1996, Aenen et al., For the construction of the clone library of the 16S rRNA genes, 2002, Hyodo et al., 2003). Recently, Hongoh et al. (2006) the PCR product was excised and eluted using the QIAquick® gel published a comprehensive study of bacterial diversity in extraction kit protocol according to the manufacturer’s instructions and used for cloning. Cloning was done using Invitrogene TOPO- Macrotermes gilvus from Thailand, which showed that TA® cloning kit according to the manufacturer’s instructions, and most of the major bacterial phyla were represented. The introduced into E. coli cells for transformation. One hundred and microbial diversity in M. michaelseni , the dominant four clones were picked and an EcoRI digestion and vector targeted fungus-cultivating termite in tropical and sub-topical PCR was conducted. Restriction fragment length polymorphism Africa has yet to be studied. Apart from their importance (RFLP) analysis of PCR products from clones was conducted using a cocktail of the enzymes AvaI, EcoRI, BamHI, HindIII, KpnI, and in the nutrition of the termites, the microorganisms in the XbaI. A representative clone for each unique RFLP patterns was termite may also include novel species with potential for sequenced. The PCR products of isolates were also purified and exploitation in biotechnology. The current study reports sequenced. on the diversity of the intestinal bacterial community of Sequence data were analyzed with the