Termite gut flagellates and their bacterial symbionts: Phylogenetic analysis and localization in situ

Abstract

This thesis summarizes a series of studies concerning the tripartite symbioses of bacteria with protozoa in the gut of wood-feeding lower termites. These associations presumably play a key role for the digestion of lignocellulose and the function of the termite gut.A prerequisite for the understanding of the specificity of these symbioses is the identification of the respective protozoan species. Whereas the parabasalid flagellates in Reticulitermes flavipes are well characterized and clearly defined, the case for the morphological variable oxymonad protists in this termite was controversially discussed in the literature. By using molecular tools, it was shown that the morphological variety of these flagellates in Reticulitermes flavipes also reflects a genetical variety, and is not due to different developmental stages of the same organism.Protozoan cells represent the most important habitat for bacteria in the highly structured termite gut. By using T-RFLP analysis and a comparison of 16S rRNA gene libraries from different microniches within the gut of Reticulitermes santonensis, it was shown that the bacterial community associated with the flagellates differs considerably from those in the surrounding gut fluid or located at the gut wall. The most abundant sequences in the flagellate fraction stemmed from the so-called Termite group 1 bacteria (TG-1). These results demonstrate the presence of a specific microbiota colonizing the flagellates.For the exact identification of the bacteria associated with specific flagellates, a technique for isolation of single protozoan species and the subsequent phylogenetic analysis of the bacterial DNA of these fractions was established and used for investigating the symbioses of the two largest flagellates in Reticulitermes santonensis (Trichonympha agilis, Hypermastigida; Pyrsonympha vertens, Oxymonadida) with endosymbiotic bacteria. These two flagellates, while being only distantly related, harbor numerous morphologically similar bacteria in the cytoplasm. It was shown that these endosymbionts are also phylogenetically closely related and represent two distinct groups of TG-1, thus corroborating the results obtained with flagellate suspensions. Now, for the first time, the exact location and the morphotype of the bacteria in this new bacterial phylum is clarified. By designing a specific PCR-assay for TG-1, the distribution of these bacteria in termite species from nearly all famillies and in the flagellates-harboring cockroach Cyptocercus punctulatus was determined. Interestingly, TG-1 were not present in the evolutionary higher termites, which also have lost their flagellates, but were present in all lower termites and the flagellates--harboring cockroach Cyptocercus punctulatus, indicating a co-evolution of symbiotic bacterium and protozoan host. Based on these results, the classification of the Termite Group 1 as new candidate phylum Endomicrobia containing two candidate species Candidatus Endomicrobium pyrsonymphae and Candidatus Endomicrobium trichonymphae was proposed.Except for attached spirochetes on oxymonad protists, surface-associated bacteria are not reported for the flagellates in Reticulitermes spp. Therefore, the symbiosis of bacteria attached to the hypermastigid flagellate Staurojoenina sp. from the dry-wood termite Neotermes cubanus was investigated. Interestingly, the 16S rRNA gene sequence of this symbiont forms together with other termite-derived sequences a termite-specific cluster among the Bacteroidales, which is hypothesized to consist of sequences of epibiotic bacteria of other gut flagellates.The results obtained in these studies together with a critical literature survey on the topic and the current standing are summarized in a review. The established methods were also used in other projects to investigate symbiotic bacteria and microbial activities in intestinal tracts of beetle larvae and isopods. The results of these studies can be found in the appendix of the thesis.