The bacterial gut microbiota of wood- and humus-feeding termites: Diazotrophic populations and compartment-specific response of bacterial communities to environmental factors
The subject of this thesis is the influence of the microenvironment on the symbiosis between higher termites and their intestinal bacteria. The gut environmental factors pH, hydrogen partial pressure, redox potential and nitrogen pool size were measured. Bacterial gut community structure from each h...
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|The subject of this thesis is the influence of the microenvironment on the symbiosis between higher termites and their intestinal bacteria. The gut environmental factors pH, hydrogen partial pressure, redox potential and nitrogen pool size were measured. Bacterial gut community structure from each highly compartmentalized gut section was investigated. Furthermore, one specific function, nitrogen fixation, was comparatively analyzed in lower termites, higher termites and cockroaches.
Hydrogen partial pressure, pH and redox potential in the gut compartments of humus- and soil-feeding termites were measured using microsensors. The size of the entire bacterial communities in each compartment was determined by 16S rRNA gene copies in qPCR. The diets of humus- and soil-feeders are nitrogen-rich, so the pool size of ammonia, nitrite and nitrate were also quantified by colorimetric assay.
Higher termites have adapted to utilize diverse lignocellulosic diets in various stages of humification, like wood, humus and soil. The high alkalinity in the anterior hindgut of humus- and soil-feeding termites may play an important role in the digestion of proteins and polypeptides. Our comprehensive determination of physicochemical parameters reinforce the hypothesis that intestinal microenvironments are evolutionarily adapted to diet-related differences.
The analysis of bacterial diversity by amplicon sequencing (Miseq) of 16S rRNA genes underscored that the community structure of intestinal bacteria in each gut section is influenced by multiple environmental factors like pH, hydrogen and host dietary substrate. The gut bacteria in homologous compartments of hindguts of humus- and soil-feeders showed similarity even when the hosts were from different subfamilies. In wood- and grass- feeding termites, dominating gut microbiota were from Actinobacteria, Fibrobacteres and Spirochaetes. On the other hand, abundant genera were from Bacteroidetes, Spirochaetes and Firmicutes in humus- and litter-feeding termites. This suggests that they make essential contributions to the digestive processes.
Nitrogen supply should also influences the composition of the microbiota in termite guts, especially in wood-feeding termites, where diazotrophy is of major importance. From the study of nitrogen metabolism in different gut sections, the high concentrations of ammonia, nitrite and nitrate were found in the gut of humus- and soil-feeding termites not in wood-feeding termites. This phenomenon associated with the intake of the termites. For the wood feeders, they rely on a nitrogen-limiting diet with a high carbon to nitrogen ratio. They need some strategies to overcome this difficulty. Nitrogen fixation of symbiotic gut bacteria helps them in nitrogen nutrition supply.
Quantification of nitrogen fixing populations was carried at DNA level by qPCR, using the nifH gene as a molecular marker. After normalized by 16S rRNA gene copy numbers, the ratio of nifH to 16S rRNA gene copy numbers was less than 0.15 in all termite species studied. Nevertheless, this surprisingly low proportion of diazotrophs is sufficient to account for the nitrogen fixation rate of the termites. It is supported by the nitrogen fixation ability measured by acetylene reduction assay of Treponema isolates from Zootermopsis angusticollis and live Zootermopsis sp.
The bacterial symbionts of flagellate protists contribute to the nitrogen fixation in lower termites. Especially in Kalotermitidae, the abundant nifH genes which clustered with nifH genes from flagellate symbionts are consistent with the cospeciation of flagellates and lower termites. Nitrogen fixed by the endosymbiont can be converted to more valuable nitrogenous compounds such as amino acids and supplied directly for protein synthesis of the protist. This asset allows the protist to grow stably and independently, and ensures that the host termite maintains the essential cellulolytic protists. In wood-feeding higher termites, flagellates are lost and the diazotrophs in the gut link with fiber-associated bacteria. This was verified by comparative analysis of nifH genes in amplicon libraries and annotated metagenomes.
Apart from flagellate symbionts, another interesting nifH subcluster is in Group IV. The verified diazotroph with only nif genes encoding Group IV nitrogenase revealed potential functional nifH subgroup in previously unfunctional Group IV. Endomicrobium cluster is abundant in Kalotermitidae, Termopsidae and Cryptoceridae. This is the first analysis of the diazotrophic communities in termite gut which take into account the potential diazotrophs with functional nifH in Group IV.