Termite gut flagellates and their bacterial symbionts: Phylogenetic analysis and localization in situ Dissertation zur Erlangung des Grades eines Doktors der Naturwissenschaften im Fachbereich Biologie der naturwissenschaftlichen Sektion der Universität Konstanz vorgelegt von Ulrich Stingl Tag der mündlichen Prüfung: 10. Dezember 2004 Referent: Priv.-Doz. Dr. Andreas Brune Referent: Prof. Dr. Bernhard Schink Referent: Prof. Dr. Ralf Conrad Konstanz 2004 Danksagung Die vorliegende Arbeit wurde in der Zeit von April 2001 bis Oktober 2004 am Lehrstuhl für Mikrobielle Ökologie von Prof. Dr. Bernhard Schink unter der Betreuung von PD Dr. Andreas Brune durchgeführt. Dabei gilt mein besonderer Dank: PD Dr. Andreas Brune für das entgegengebrachte Vertrauen, die stetige Hilfsbereitschaft bei Fragen und Diskussionen, sowie die Überlassung des Themas. Prof. Dr. Bernhard Schink für die Möglichkeit, diese Arbeit an seinem Lehrstuhl durchzuführen, für die Übernahme des Koreferats, und für das Interesse und Diskussionsbereitschaft. Dr. Markus Egert, Dr. Michael Friedrich, Annelie Maass, Dr. Renate Radek, Dr. Vladimir Sustr, Yongie Wang und Dr. Martin Zimmer für nette und erfolgreiche Zusammenarbeit in verschiedenen Kooperationsprojekten. Allen jetzigen und ehemaligen Mitarbeitern der Termitengruppe sowie des Lehrstuhls Schink für konstruktive Diskussionen und die angenehme Arbeitsatmosphäre. Mein besonderer Dank geht an die „Ehemaligen“ für das Überlassen zahlreicher Stammkulturen, welche die Arbeit deutlich erleichterten. List of publications The following publications are integrated in this thesis Stingl U, Brune A (2003) Phylogenetic diversity and whole-cell hybridization of oxymonad flagellates from the hindgut of the wood-feeding lower termite Reticulitermes flavipes. Protist 154: 147–155 (Chapter 2) Yang H, Schmitt-Wagner D, Stingl U, Brune A (2004) Niche heterogeneity determines bacterial community structure in the termite gut. Environ Microbiol, in revision (Chapter 3) Stingl U, Radek R, Yang H, Brune A (2004) The “Endomicrobia”: Cytoplasmic symbionts of termite gut protozoa form a separate phylum of prokaryotes. Applied and Environmental Microbiology, in press (Chapter 4) Stingl U, Maass A, Radek R, Brune A (2004) Symbionts of the gut flagellate Staurojoenina sp. from Neotermes cubanus represent a novel, termite-associated lineage of Bacteroidales: Description of 'Candidatus Vestibaculum illigatum'. Microbiol 150: 2229–2235 (Chapter 5) Brune A, Stingl U (2004) Prokaryotic symbionts of termite gut flagellates: phylogenetic and metabolic implications of a tripartite symbiosis. In: Overmann, J. (ed.), Molecular basis of symbiosis. Springer Verlag, in press (Chapter 6) Further publications from cooperation projects, which are not in the main focus of this thesis, can be found in the appendix Lemke T, Stingl U, Egert M, Friedrich MW, Brune A (2003) Physicochemical conditions and microbial activities in the highly alkaline gut of the humus- feeding larva of Pachnoda ephippiata (Coleoptera: Scarabaeidae). Appl Environ Microbiol 69: 6650–6658 Egert M, Stingl U, Dyhrberg Bruun L, Pommerenke B, Brune A, Friedrich MW Physicochemical conditions and microbial diversity in the intestinal tract of the phytophagous larva of Melolontha melolontha (Coleoptera: Scarabaeidae). Applied and Environmental Microbiology, in revision Wang Y, Stingl U, Anton-Erxleben F, Zimmer M, Brune A (2004) ‘Candidatus Hepatincola porcellionum’ gen. nov., sp. nov., a new, stalk-forming lineage of Rickettsiales colonizing the midgut glands of a terrestrial isopod. Arch Microbiol 181: 299–304 Wang Y, Stingl U, Anton-Erxleben F, Geisler S, Brune A, Zimmer M (2004) 'Candidatus Hepatoplasma crinochetorum', a new, stalk-forming lineage of Mollicutes colonizing the midgut glands of a terrestrial isopod. Appl Environ Microbiol 70: 6166–6172 Table of contents 1 Introduction 1 Importance of termites 1 Phylogeny of termites 1 The gut system of termites 2 Microorganisms 2 Associations of termite flagellates with prokaryotes 5 The aims of the study 6 2 Phylogenetic analysis and whole-cell hybridization of oxymonad flagellates from the hindgut of the wood-feeding lower termite Reticulitermes flavipes 7 Abstract 7 Introduction 8 Results 9 Discussion 14 Materials and Methods 16 3 Niche heterogeneity determines bacterial community structure in the termite gut (Reticulitermes santonensis) 21 Summary 21 Introduction 22 Results 23 Discussion 41 Experimental procedures 45 4 The ‘Endomicrobia’: Cytoplasmic symbionts of termite gut flagellates form a separate phylum of prokaryotes 49 Abstract 49 Introduction 50 Materials and Methods 50 Results 52 Discussion 58 5 Symbionts of the gut flagellate Staurojoenina sp. from Neotermes cubanus represent a novel, termite-associated lineage of Bacteroidales: Description of ‘Candidatus Vestibaculum illigatum’ 64 Summary 64 Introduction 65 Methods 66 Results 68 Discussion 71 6 Prokayotic symbionts of termite gut flagellates: Phylogenetic and metabolic implications of a tripartite symbiosis 75 Introduction 75 Hindgut protozoa 78 Symbiotic associations with prokaryotes 81 Conclusions 91 7 General discussion 93 The astonishing diversity of termite gut flagellates 93 Epibionts and endobionts: the uncultivated majority 95 8 Summary 98 9 Zusammenfassung 100 References 103 Abgrenzung der Eigenleistung 121 Appendix 122 Appendix A: Physicochemical conditions and microbial activities in the highly alkaline gut of the humus-feeding larva of Pachnoda ephippiata (Coleoptera: Scarabaeidae) 123 Appendix B: Physicochemical conditions and microbial diversity in the intestinal tract of the phytophagous larva of Melolontha melolontha (Coleoptera: Scarabaeidae) 150 Appendix C: ‘Candidatus Hepatincola porcellionum’, a new, stalk-forming lineage of Rickettsiales colonizing the midgut glands of a terrestrial isopod 173 Appendix D: ‘Candidatus Hepatoplasma crinochetarum’, a new, stalk-forming lineage of Mollicutes colonizing the midgut glands of a terrestrial isopod 188 1 Introduction Importance of termites Termites are terrestrial, social insects that inhabit nearly two-thirds of the Earth’s land surface between 45° N and S latitude (Wood and Sands, 1978), but are also present in temperate zones (e.g., Marini and Mantovani, 2002). Their numbers can exceed 6000 individuals per m2, and their biomass often surpasses that of mammalian herbivores (Lee and Wood, 1971; Collins, 1989), which already indicates their enormous ecological impact. Besides their ecological importance, also the commercial relevance of termites is immense: Damages caused by termites make them a worldwide pest; e.g., solely the Formosan termite Coptotermes formosanus is responsible for more than one billion US$ per year in the USA for preventive and remedial treatment (Lax and Osbrink, 2003). Phylogeny of termites Termites (order: Isoptera) make up, together with mantids and cockroaches, the most primitive winged terrestrial insect taxon, the Dictyoptera (Deitz et al., 2003). The Isoptera are divided, dependent on the reference, in six or seven families (Noirot, 1992; Abe et al., 2000), which can be further separated into evolutionary lower and higher termites (Fig. 1). Higher termites are differentiated from lower termites mainly by the absence of symbiotic flagellates in their enlarged hindgut. The loss of these symbiotic microorganisms in the course of evolution is most likely due to the changed feeding- habit of these species, most of which feed on soil or cultivate fungi in their mounds as symbiotic partner for the digestion of wood (Noirot, 1992). Phylogenetically lower termites, in contrast, depend on symbiotic protozoa in their intestinal tract for the degradation of lignocellulose (Cleveland, 1926). 2 Introduction (Protozoa) (No protozoa) Figure 1: Schematic outline of the phylogenetic relationships among the different termite families and the closely related cockroaches. The numbers on the lines represent the number of genera/species in the different families (modified from Abe et al., 2000). The gut system of lower termites In the last 20 years much work was done to describe the termite gut micro ecosystem (for review, see Breznak, 2000; Brune, 2004). The digestive processes in the rather small midgut are most probably largely controlled by the activity of host enzymes, whereas the digestion in the enlarged hindgut involves symbiotic microorganisms. Microorganisms Termite guts are densely colonized by symbiotic microorganisms, most of which are presumably involved in different steps of the fermentation of lignocellulose. The midgut contains a purely prokaryotic microbiota, whereas the hindgut is colonized by prokaryotes and special single-celled eukaryotes. As a result of the microbial activities, high concentrations of fermentation products accumulate in this system (for review, see Breznak, 2000). Prokaryotes Emphasis of the research on this micro ecosystem in the last years was concentrated on the function of the prokaryotic microorganisms. Important metabolic functions attributed to the prokaryotic community include reductive acetogenesis, Introduction 3 methanogenesis, nitrogen fixation, and uric acid fermentation (for review, see Breznak, 2000). The identification of the microbial community in termite guts is based mainly on molecular tools (Berchtold et al., 1999; Lillburn et al., 1999; Schmitt-Wagner et al., 2003a). Phylogenetic analysis of the 16S rRNA genes of bacteria in the hindgut of Reticulitermes speratus revealed that two-thirds of the clones bore less than 90% sequence similarity