The gut microbiota of the cockroach Shelfordella lateralis Primary colonization, succession, and metabolicresponse to microenvironmental conditions

Cockroaches and their closest relatives, the termites, have a highly complex gut microbiota consisting of different microbial guilds, e.g. fermenting bacteria, methanogenic archaea and protists, which form a metabolic network within the gut. While the microbiota of termites has been studied for deca...

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Bibliographic Details
Main Author: Tegtmeier, Dorothee
Contributors: Brune, Andreas (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2016
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Summary:Cockroaches and their closest relatives, the termites, have a highly complex gut microbiota consisting of different microbial guilds, e.g. fermenting bacteria, methanogenic archaea and protists, which form a metabolic network within the gut. While the microbiota of termites has been studied for decades, little is known about the cockroach gut microbiota, especially about the primary colonization, the succession and metabolic interactions of gut microorganisms. In order to investigate successive changes of the cockroach gut microbiota during host development, I analyzed the bacterial community composition (using high throughput sequencing of the 16S rRNA gene) and major metabolites in different instars of Shelfordella lateralis. First instar nymphs were often colonized by aerobic or oxygen-tolerant, lactate-producing taxa (e.g. Enterobacteriaceae, Enterococcaceae and Lactobacillales), which was associated with a strong lactate accumulation in the gut. By contrast, cockroaches from second instar to adulthood where consistently colonized by anaerobic taxa (e.g. Ruminococcaceae, Lachnospiraceae and Rikenellaceae). Methane emission of living cockroaches was absent in the first instar, relatively low in all other instars but high in adults. For hydrogen emission I found an opposite trend: Emission rates decreased during cockroach development, suggesting an increase in hydrogen consumption by methanogens. Cockroaches that were reared in isolation showed no methane emission but a similar gut microbiota than conventional ones, indicating that the majority of the microorganisms are acquired from the environment, whereas methanogens are likely transferred via coprophagy. Microsensor measurements revealed anoxic conditions in the hindguts of all but the first instar, which suggests that oxygen is an important factor shaping the microbiota of the first instar. The strong differences in gut microbiota and metabolism between first and later instars indicate that the microbiota is assembled by stochastic events and altered depending on the oxygen status of the gut. The results further show that those microorganisms colonize the gut that are picked up from the environment and are favored by the conditions in the gut. While the composition of the gut microbiota has been studied in detail for many insects, a mechanistic understanding of the metabolic activities and interactions among individual microbes is still lacking. A detailed characterization of gut microorganisms requires pure cultures. However, bacteria are exposed to unique microenvironmental conditions in the gut that differ fundamentally from those in pure culture. Therefore, studies that investigate how far the metabolic properties of pure cultures (in vitro) reflect their activities in their natural environment (in situ) are of particular interest. We established the cockroach Shelfordella lateralis as a gnotobiotic model in order to examine metabolic activities and biotic interactions of individual bacterial populations under in situ conditions. Germ-free cockroaches were successfully colonized with the autochthonous strains EbSL (a facultatively anaerobic species of Enterobacteriaceae) and FuSL (an obligately anaerobic Fusobacterium sp.). When monoassociated, both strains grew to high density, but population sizes of strain EbSL were higher than that of strain FuSL. In diassociation however, the population size of strain FuSL was even smaller than in monoassociation. Although microsensor measurements showed that strain EbSL completely consumed the oxygen in the gut, precolonization with the facultative anaerobe did not favor colonization by the obligate anaerobe. The results showed that strain FuSL is outcompeted by strain EbSL, possibly due to a better colonization success of the facultative anaerobe in the oxic zones of the gut. Comparison of the fermentation products of the cultures formed in vitro with those accumulated in situ indicated that the gut environment strongly affected metabolic activities of both strains. Pure cultures of strains EbSL and FuSL formed the typical products of mixed-acid or butyrate fermentation, whereas guts of gnotobiotic cockroaches accumulated mostly lactate and acetate. Shifts towards acetate or lactate were also confirmed in vitro when pure cultures were exposed to oxygen or high glucose concentrations (respectively), conditions similar to those present in the hindguts of germ-free cockroaches. In order to gather more information about hitherto uncultivated representatives of the obligately anaerobic bacterial community in the guts of S. lateralis and other insects, we isolated further members of their gut microbiota and described the ultrastructure, physiology and metabolism of the strains in detail. The novel isolates are strictly anaerobic or slightly aerotolerant representatives of new genera of Erysipelotrichaceae (Firmicutes) and Opitutaceae (Verrucomicrobia) and have a purely fermentative metabolism. While former (strains ErySL and Pei061) showed products of a typical mixed acid fermentation, the latter (strain Ho45) fermented glucose to propionate and acetate. Growth and fermentations of the novel isolates were strongly influenced by oxygen and glucose concentrations in the medium. Since the closest relatives of the novel isolates were incorrectly described as aerobic or microaerophilic in the literature, we provide an emended description of the family Erysipelotrichaceae and the genus Diplosphaera (Opitutaceae).