Final Report Summary - METAXYLO (Metagenomic Analysis of Microbial Communities Involved in Wood Degradation in a Xylophagic Catfish) Xylophagy can be defined as the ability to eat and digest wood; this is a rare dietary strategy in vertebrates, due to the recalcitrance of wood. Panaque spp. belong to the diverse family of Lorricaridae catfish found predominantly in freshwater ecosystems of the Neotropics. A subset of the Panaque spp. are xylivorous with robust, fully mineralized, spoon-shaped teeth and unique musculature around the suckermouth; both adaptations are thought to enable adherence to and ingestion of submerged woody materials. Furthermore, wood has relatively low nitrogen to carbon ratio in comparison to animal tissues; therefore the diet of xylophagic organisms often needs to be supplemented. For most xylophagic species (i.e. shipworms and termites) this occurs through the direct interaction between the host and nitrogen fixing endosymbionts. These bacterial endosymbionts contain the enzyme nitrogenase which is able to reduce atmospheric dinitrogen to ammonia providing an accessible nitrogen source to the host. The major goal of this study was to examine microbial communities associated with xylophagy in P. nigrolineatus and to identify novel microbial metabolic genes, enhancing our understanding of the carbon and nitrogen cycles in the environment. Specifically the primary research objectives of this project are to: define the microbial diversity in the different regions of the gastrointestinal (GI) tract within the P. nigrolineatus; identify novel cellulose degradation and nitrogen fixation genes within this GI tract system; determine if nitrogen fixing bacteria are active in the GI tract.To enable the characterisation of the microbial communities associated with xylophagy, P. nigrolineatus were acquired from Peru, through aquarium wholesale and reared on either a mixed diet of wood, palm hearts, and algae, or wood exclusively. After four weeks the foregut, midgut, hindgut, whole auxiliary lobe (a structure attached to the midgut with no assigned function) and tank water were sampled in triplicate, for both dietary regimes. Total DNA and RNA were extracted from each tissue sample from all replicates, for each dietary regime. The DNA extractions from the different regions of the GI tract were examined by 16S rRNA gene clone libraries and pyrosequencing to examine microbial community diversity. Sequence analysis and phylogenetic reconstruction has revealed distinct microbial communities in each tissue region. The foregut community shared many phylotypes in common with aquarium tank water and included Legionella and Hyphomicrobium spp. As the analysis moved further along the GI tract, phylotypes with high levels of 16S rRNA sequence similarity to nitrogen-fixing Rhizobium and Agrobacterium spp. and Clostridium xylanovorans and Clostridium saccharolyticum, were found to dominate midgut and auxillary lobe communities. However, the hindgut was dominated almost exclusively by phylotypes with the highest 16S rRNA sequence similarity to the Cytophaga-Flavobacterium-Bacteroides phylum. Species richness was highest in the foregut, decreased distally through the midgut and hindgut, with the lowest diversity detected in the auxillary lobe, indicating the presence of a specialized microbial community. Using 16S rRNA gene phylogeny, we report that the P. nigrolineatus GI tract possesses a microbial community comprising close relatives of microorganisms capable of cellulose degradation and nitrogen fixation (McDonald, Schreier and Watts 2012). To better understand cellulose degradation pathways present within the microbial community of the fish GI tract, a number of novel bacterial isolates have been described during this study. Analysis of GI tract communities generated from anaerobic microcrystalline cellulose enrichment cultures by 16S rRNA gene analysis revealed phylotypes sharing high sequence similarity to known cellulolytic bacteria including Clostridium, Cellulomonas, Bacteroides, Eubacterium and Aeromonas spp. Related bacteria were identified using a 13C-labeled cellulose DNA stable-isotope probing (SIP) approach DNA-SIP), which also included nitrogen-fixing Azospirillum spp. Our ability to enrich for specialized cellulose-degrading communities suggests that the P. nigrolineatus GI tract provides a favourable environment for this activity and these communities may be involved in providing assimilable carbon under challenging dietary conditions. These bacteria have been characterised using traditional microbiological techniques including microscopy, metabolic profiling, and different carbon source utilisation (Watts et al., 2013). Furthermore, to better understand the nitrogen metabolism with the fish microbiome a number of techniques have been employed to examine the activities of these populations within the fish GI tract. Total RNA was extracted from all tissue regions and reverse transcription reactions were performed using specific primers for the nitrogenase gene which proved to be positive in the hindgut region. After reverse transcription, nifH genes were ampliﬁed from cDNA samples using a nested PCR approach and cloned and found to have high sequence similarity to other known nitrogen fixers. To detect this activity within the GI tract, semi-thin sections (3-5 µm) were visualized through catalyzed reporter deposition with anti-nitrogenase antibodies conjugated with horseradish peroxidase. Cells were counterstained with SYBR green. Green colour denoted positively stained SYBR green tissues and cells, while red represents regions of catalyzed reporter deposition indicating presence of nitrogenase positive cells. This polyphasic approach has allowed the first description of nitrogen fixation within a vertebrate GI tract (McDonald et al., 2015).The METAXYLO project has provided a clear description of the microbial community present within the GI tract of P. nigrolineatus. Focusing upon specific cellulolytic and diazotrophic microbial populations has enabled a better understanding of carbon and nitrogen metabolism pathways in this fish GI tract and the environment. This funding has enabled the detailed analysis of the cellulose and lignin degradation pathways present and due to the importance of cellulose in the global carbon cycle and its potential for biomass fuel generation; these results may have wide ranging socio-economic implications. Importantly, this funding has enabled the first description of nitrogen fixation activity within a vertebrate GI tract. This finding may have interesting implications to supplying dietary nitrogen to fish in aquaculture and other applied implications. Furthermore, the fish microbial community microbiome is a complex environment and insight into inter-organism relationships may enable a better understanding of symbiosis with many practical implications in microbiome and aquaculture research.