The intestinal microbiota of soil-feeding termites Microbial diversity, community structure, and metabolic activities in the highly compartmentalized gut of Cubitermes spp. Dissertation zur Erlangung des Grades eines Doktors der Naturwissenschaften im Fachbereich Biologie der naturwissenschaftlichen Sektion der Universität Konstanz vorgelegt von Dirk Schmitt-Wagner Tag der mündlichen Prüfung: 17. Juni 2003 Referent: Priv.-Doz. Dr. Andreas Brune Referent: Prof. Dr. Bernhard Schink Konstanz 2003 Contents 1 Introduction _________________________________________________________________________ 3 2 Hydrogen profiles and localization of methanogenic activities in the highly compartmentalized hindgut of soil-feeding higher termites (Cubitermes spp.) ________________________________________________ 11 3 Axial differences in community structure of Crenarchaeota and Euryarchaeota in the highly compartmentalized gut of the soil- feeding termite Cubitermes orthognathus ____________________________________ 29 4 Phylogenetic diversity, abundance, and axial distribution of microorganisms in the intestinal tract of soil-feeding termites (Cubitermes spp.)__________________________________________________________________ 53 5 Axial dynamics, stability, and inter-species similarity of bacterial community structure in the highly compartmentalized gut of soil-feeding termites (Cubitermes spp.) ______________________________________ 77 6 Axial distribution and phylogenetic diversity of spirochetes in the hindgut of the soil-feeding termite Cubitermes ugandensis __________ 91 7 Discussion _______________________________________________________________________ 107 Summary ________________________________________________________________________________ 119 Zusammenfassung _____________________________________________________________________ 121 Curriculum vitae _______________________________________________________________________ 123 Abgrenzung der Eigenleistung ______________________________________________________ 124 1 Introduction Significance of termites in the ecosystem Termites, which belong to the order Isoptera, are terrestrial, social insects, phylogenetically related to cockroaches and mantids. Termites comprise 281 genera and over 2600 described species. At present, seven families and 14 subfamilies are recognized, but a large majority (ca. 85%) of genera described to date are included in only one family, the Termitidae. (Krishna, 1970; Wood and Johnson, 1986; Noirot, 1992; Abe et al., 2000; Kambhampati and Eggleton, 2000; see Fig. 1). Cockroaches 1/1 Mastotermitidae 21/411 Kalotermitidae Isoptera (termites) 3/15 Hodotermitidae Lower termites 5/20 Termopsidae 16/305 Rhinotermitidae 1/1 Serritermitidae 236/1895 Termitidae Higher termites Fig. 1. Phylogenetic scheme of termite evolution showing the presumed relationship of the seven termite families, adapted from Higashi and Abe (1997). The numbers on the lines represent the number of genera/species in the different families (Abe et al., 2000). The majority of termites live in tropical and subtropical regions, but they spread also into the temperate zone. About two-thirds of the Earth's land surface between the latitudes 48° N and 45° S is inhabited by termites (Lee and Wood, 1971). In tropical and subtropical regions, their number exceeds 6000 individuals m–2, and their biomass densities range between 5 and 50 g m–2, often surpassing biomass densities of mammalian herbivores (0.01–17.5 g m–2; Lee and Wood, 1971; Collins, 1989). Termites can be classified in phylogenetically lower and higher termites. Lower termites comprise six families and are restricted to a diet of wood or grass (Noirot 1992), whereas higher termites (Termitidae) include soil-feeding (humivorous), wood- and grass-feeding (xylophagous) and fungus-cultivating species (Noirot 1992). It is widely accepted that termites have a major impact on the decomposition of plant material, humification, and soil conditioning (Lee and Wood, 1971; Wood and Sands, 1978; Wood, 1988; Collins, 1989; Martius, 1994; Brussaard and Juma, 1996). 4 Introduction Therefore, termites take actively part in the permanent alteration of their habitat, especially in tropical ecosystems. Approximately 136 × 1015 g of carbon dioxide is fixed annually by photosynthesis in form of plant biomass. The major constituents of plant biomass are cellulose and lignin. About two-thirds of this biomass production occurs in terrestrial ecosystems (Breznak and Brune, 1994 and references therein). The decomposition of plant material is carried out primarily by fungi and bacteria, but termites, as the most abundant and important soil macro-invertebrates, also play an important role in this process. Like shredder organisms, termites can mechanically chop up the plant material with their mandibles and their gizzard, thereby increasing the surface area accessible to soil microorganisms. Furthermore, termites can also directly dissimilate the structural polymers of lignocellulose with the help of their gut symbionts (Lee and Wood, 1971; Breznak and Brune, 1994). Due to the high biomass densities, termites significantly contribute to the emission of atmospheric trace gases like methane and carbon dioxide. The first studies had estimated the contribution of termites to the global methane emission up to 45% (Zimmerman et al., 1982), but more recent estimations based on a larger data set, and the consideration of severe differences in methane emission rates between different termite species, reduced their contribution to less than 5% (Sanderson, 1996; Bignell et al., 1997; Sugimoto et al., 1998). Soil feeding termites Humivorous termites represent one of the most abundant and ecologically important groups of soil macroinvertebrates in tropical ecosystems. In contrast to the phylogenetically lower termites, which are restricted to a diet of wood, the majority of species of the higher termites (family Termitidae) consume soil organic matter in various stages of humification. True soil feeders occur in all subfamilies accept the Macrotermitinae (Abe et al., 2000). The humivorous mode of nutrition rendered the Termitidae independent of the necessity to harbor cellulolytic flagellates as symbionts, and thereby probably removed important evolutionary constraints, allowing further diversification of the gut (Noirot, 1992). Especially in the true soil feeders, the increase in length, volume, and compartmentalization transformed the hindgut into a complex microecosystem with pronounced axial dynamics of the intestinal pH, ranging from slightly acidic conditions in the crop and the P5 segment to extremely alkaline conditions in the P1 and the P3 segment (Bignell and Eggleton, 1995; Brune and Kühl, 1996; see Fig. 3.). Soil-feeding termites ingest large amounts of soil (Wood, 1978a; Wood, 1978b; Okwakol, 1980), and due to their high biomass densities, their feeding activity is important for the biomass turnover in tropical and subtropical ecosystems (Wood and Johnson, 1986; Wood, 1988; Collins, 1989; Martius, 1994; Bignell et al., 1997; Abe et al., 2000). The food ingested by soil-feeding termites is quite heterogeneous. Introduction 5 15/332 Macrotermitinae 236/1895 43/196 Termitidae Apicotermitinae 90/659 Higher termites Nasutitermitinae 88/708 Termitinae Fig. 2. Phylogenetic scheme of the presumed relationship of the four subfamilies of the higher termites (Termitidae), adapted from Bignell and Eggleton (1995). The number on the lines represent the number of genera/species in the respective branch (Abe et al., 2000). The subfamily Termitinae includes the species of the genus Cubitermes which were the objects of this study. The gut contains predominantly soil minerals and humus, but also plant tissue fragments, plant roots, fungal mycelia, and macerated organic material (Sleaford et al., 1996; Donovan et al., 2001). However, the identity of the components used as electron and carbon sources was obscure for a long time (Bignell et al., 1997). Some authors assumed that the aromatic components of the humic substance fraction could contribute to feed the humivorous termites (Bignell, 1994). In a recent study it was shown, that the extreme alkalinity in the anterior hindgut facilitates not only the desorption of humic substances from the mineral matrix, but also decreases their molecular weight and increases their solubility (Kappler and Brune, 1999). Fig. 3. Gut morphology of a Cubitermes sp. worker termite, representative also for other soil- feeding Termitinae. The gut was drawn in its unraveled state to illustrate the various segments: C (crop), M (midgut), ms (mixed segment), P1–5 (proctodeal segments). The average luminal pH was determined for the indicated gut regions in Cubitermes speciosus using intact guts and glass pH microelectrodes (Brune and Kühl, 1996). In feeding experiments with chemically identical synthetic humic acids, radioactively labeled in their proteinaceous or aromatic building blocks, the mineralization rate of the peptidic components of the humic substances increased dramatically in the presence of termites, whereas the mineralization rate of the aromatic components increased only weakly (Ji et al., 2000). The results of this study 6 Introduction were a first indication of the importance of peptidic components of humic acids as substrates for soil-feeding termites. In soil inoculated with radioactively labeled preparations of cellulose, peptidoglycan, protein and whole bacterial cells, the mineralization rate of all these compounds was strongly increased when