Nitrogen Metabolism and Composition of Fish Hindgut Symbiotic Communities

Abstract

The aim of this work was to test the hypothesis that gut microbial symbionts of herbivorous reef fish fix atmospheric nitrogen. This process represents a possible mechanism by which these microbes could contribute to their hosts’ protein supply. Fish were sampled from the temperate reefs of New Zealand and tropical Great Barrier Reef of Australia. The hypothesis was tested in an iterative fashion via three steps. First, the gene coding the dinitrogenase reductase component of the nitrogenase enzyme complex (nifH) was identified in the genomic DNA of the fish gut microbial communities. This was accomplished by amplification by PCR followed by the establishment of a phylogenetic context for the cloned and sequenced genes by comparison with nifH sequences from known diazotrophs. nifH sequences obtained were most similar to those of Firmicutes such as Cellulosilyticum lentocellum and Desulfovibrio salexigens. Next, expression of the nifH genes in the gut communities were investigated by RT-Q-PCR. Thirdly, the activity of nitrogen fixation occurring in the fish guts was investigated using both Acetylene Reduction (AR) and 15N2 incorporation methods. Rates of nitrogen fixation determined by AR in the most distal section of the herbivorous fish guts of the Great Barrier Reef were comparable with those measured in the termite species Neotermes koshunensis when fed on wood. Finally, surveys of the gut microbial communities of the four temperate and seven tropical reef fish were conducted by sequencing of 16S rDNA amplicons by high-throughput sequencing. The study revealed a highly conserved phylum-level composition across species, gut segments, host feeding preferences, and latitudinal range. This result is significant given the phylogenetic disparity of the host species. The work presented in this thesis shows that in addition to their role in providing energy, microbial communities of marine herbivorous fishes may provide a de novo source of protein for the host, via nitrogen fixation. Given that gastrointestinal nitrogen fixation has not been previously demonstrated in vertebrates, the herbivorous fish gut systems studied here appear to be highly novel.