Insect gut microbiology and symbiosis

Termite guts are tiny bioreactors that convert lignocellulosic matter to microbial fermentation products that fuel the metabolism of the host. My research group studies the role of the termite gut microbiota in the symbiotic digestion of wood. Our studies focus on the biology of the prokaryotic and eukaryotic symbionts and their interactions, the structure and functions of the intestinal ecosystem, and the evolution of its microbiota. Other aspects include the microbial processes in the guts of humivorous soil macrofauna, such as soil-feeding termites and scarab beetle larvae.

Bacterial symbionts of termite gut flagellates

In the gut metabolism of lower termites, molecular hydrogen plays a central role. Hydrogen is a major product of lignocellulose digestion by a dense community of anaerobic flagellates, which are found exclusively in termites and occupy the bulk of the hindgut volume. During the past years, we have been studying the prokaryotic symbionts that colonize both surface and cytoplasm of these unique protists (Brune and Ohkuma, 2011; Ohkuma and Brune, 2011).

For a number of years, we have been concentrating on the Endomicrobia—a unique lineage of bacterial endosymbionts in a novel bacterial phylum (Stingl et al., 2005; Ikeda et al., 2007), whose first representative has been cultivated and genome-sequenced in our lab (Geissinger et al., 2009; Herlemann et al., 2009). We have shown that Endomicrobia were acquired by flagellates of the genus Trichonympha long after the establishment of the flagellate–termite symbiosis and that Endomicrobia since then have cospeciated with their flagellate host (Ikeda-Ohtsubo and Brune, 2009). A study of artificially defaunated termites identified basal lineages of Endomicrobia that represent putatively free-living ancestors of the intracellular symbionts (Ikeda-Ohtsubo et al., 2010).

In a collaborative project with the group of Renate Radek at the Free University of Berlin, we have studied the molecular phylogeny and ultrastructure of different lineages of uncultivated Bacteroidales that colonize the cell surface of certain flagellates in dry-wood termites. We have shown that the ectosymbionts of the flagellate genus Devescovina are strictly cospeciating with their host (Desai et al., 2010). This cospeciation indicates an intimate metabolic relationship between the bacteria and the flagellates and an important role of the bacteria in the symbiosis. Further investigation revealed that these symbionts possess—in addition to a nifH homolog probably present in all Cluster-V Bacteroidales—-also a second homolog (anfH) that is preferentially expressed in all termite species that harbor flagellates with such symbionts (Desai and Brune, 2012). In another dry-wood termite, in which the conventional nifH homolog was expressed, flagellates of the genus Joenia are associated with different lineages of Bacteroidales (Strassert et al., 2010).

Diversity and evolution of the termite gut microbiota

The intestinal microbiota of termites comprises numerous bacterial lineages that seem to occur exclusively in termite guts. However, evidence is increasing that representatives of these lineages are present also in other insects, including the closely related cockroaches (e.g., Andert et al., 2010; Ikeda-Ohtsubo et al., 2010; Thompson et al., 2012). Our detailed analysis of Shelfordella lateralis—the first comprehensive study of bacterial diversity in the cockroach gut—documents that community structure reflects not only the close relationship between cockroaches and termites but also indicates the existence of habitat-specific bacterial lineages(Schauer et al., 2012). We found evidence for habitat selection in another system in a collaborative project with the MPI for Ornithology, which also revealed that the bacterial microbiota in the ceca of wild and captive grouse fundamentally differ (Tetrao urogallus) (Wienemann et al., 2011).

Microbial processes in soil-feeding insects

Little is known about the digestive processes of soil- and humus-feeding arthropods and the involvement of their microbial symbionts. Humivorous insects are hot spots of carbon and nitrogen mineralization particularly in tropical soils and globally significant sources of the greenhouse gas methane (Brune, 2010a). In the hindgut of humivorous scarab beetle larvae (Pachnoda spp.), the presence of many insect-specific lineages of uncultivated Clostridiales and Bacteroidales suggests that soil organic matter is metabolized through fermentation (Andert et al., 2010). Fermenting bacteria that use humic substances as external electron acceptor seem to be responsible also for the efficient reduction of Fe(III) minerals during gut passage (Hobbie et al., 2012).

We have followed the microbial mineralization and transformation of nitrogenous soil components in the gut of soil-feeding termites using 15N tracers (Ngugi et al., 2011; Ngugi and Brune, 2012). Both denitrification and nitrate ammonification are important processes in the gut of soil-feeding termites, and the digestion of peptides and the subsequent fermentation of amino acids give rise to an enormous accumulation of ammonia, which is partially oxidized and denitrified by a hitherto unknown process. In a collaborative study, the group of Rong Ji at Nanjing University extended our findings of a preferential mineralization of peptidic soil components also to the gut of humivorous earthworms (Shan et al., 2010).

Current research activities

Several ongoing projects concern the microbial ecology of the termite gut and the metabolic capacities of the microorganisms colonizing this habitat. Projects focusing on the bacterial symbionts of termite gut flagellates (the majority of the prokaryotes in the hindgut of lower, wood-feeding termites) deal with the putative roles of particular symbionts in nitrogen metabolism and use cultivation-independent metagenomic approaches. Since many bacterial lineages are shared among a wide range of termites and cockroaches, we are using high-throughput sequencing techniques to refine our understanding of the structure and evolution of the gut microbial community in cockroaches and termites and to differentiate between mechanisms of cospeciation and habitat selection.

Methanogenesis is of particular relevance in higher termites, and the archaeal communities in the different gut regions are astonishingly diverse (Brune, 2010b). We have enriched a member of a novel, deep-branching lineage of methanogenic archaea that is particularly common in soil-feeding species; we are sequencing the genome and characterizing the physiology of this archaeon. Moreover, we want to identify the drivers of methanogenic community structure in the highly compartmented hindgut, such as the availability of and competition for methanogenic substrates, and differences in adaptation to oxidative stress and other factors imposed by the environment.

In a project affiliated with the Center for Synthetic Microbiology at the Philipps-Universität Marburg, we have successfully established a new research direction using germ-free cockroaches as a model system for gnotobiotic studies. Preliminary results indicated that germ-free larvae develop poorly but are rescued by infection with conventional or foreign gut microbiota. We are presently constructing synthetic gut microbial communities from pure cultures of gut bacteria representing different microbial guilds. Future work will include the experimental testing of ecological principles and—in the long run—the study of potential host–microbe interactions.