Diversity, phylogeny, and metabolism of methanogens in the arthropod digestive tract

Methanogens are obligate anaerobic archaea responsible for 70% of the annual emission of methane to the atmosphere. They are present in various environments such as wetlands, soil, sediments, anaerobic digesters, and animal digestive tracts. Most of the studies of the methanogenic gut microbiota have concentrated on ruminants and humans. Arthropods – specifically termites – are responsible for up to 4% of global methane emission to the atmosphere annually. In my doctoral thesis, I investigated the archaeal diversity in methane-emitting arthropods (termites, cockroaches, millipedes, scarab beetle larvae). Based on several isolates and metagenome-assembled genomes, I reconstructed their metabolism and demonstrated the evolutionary history of two arthropod-associated lineages of methanogens. The archaeal diversity of methane-emitting arthropods was previously poorly characterized based on a few 16S rRNA clone library studies. Using high-throughput PacBio sequencing of full-length 16S rRNA genes, I obtained a detailed inventory of the archaeal diversity in various groups of arthropods. Combining this dataset with a robust phylogenomic-based taxonomy, I demonstrated the distribution of methanogenic lineages among the arthropods, with some present in almost all hosts, whereas others are specific for particular groups of animals. Also, I proposed several novel genera and species of so far uncultivated lineages based on their genomes. I isolated and described six new species of methanogens from the order Methanosarcinales and three from the order Methanomicrobiales. Taxonomic characterization and genome analysis provide evidence of adaptations to the nutrient-rich gut environment in representatives from both orders. Intestinal Methanosarcinales have highly reduced genomes when compared to non-intestinal sister taxa and restricted methyl-reducing type of methanogenesis due to the lack of upper part of Wood-Ljungdahl pathway. They are also devoid of many biosynthetic pathways, for instance for amino acids and vitamins biosynthesis. Remarkably, all genomes encode high-affinity bd-oxidases that point to the ability of those methanogens to detoxify oxygen or even utilize it for ATP production. Isolates from the order Methanomicrobiales (Methanorbis gen. nov. and Methanofrux gen. nov) are the first formate-utilizing methanogens from the arthropod guts. They are also the first gut-associated methanogens from the order. The results of my research revealed a high diversity of methanogens in arthropod guts and their adaptation to the nutrient-rich environment. It demonstrates the differences in evolutionary paths for various lineages of gut-associated methanogens and serves as an example of the challenges of isolation and taxonomic characterization of those anaerobic archaea.