ANALYSIS OF GUT FLORA FROM DAMP WOOD TERMITES (TRINERVITERMES SPP.) AND EXTRACTION, CHARACTERIZATION OF CELLULASE FROM THE ISOLATE

Objective: The objective of the study was to isolate, characterize, identify, and compare the potentials of cellulolytic strains isolated from the gut of damp wood termites (Trinervitermes species) collected from Chennai, Tamil Nadu, India.Methods: Termites were collected and used as hoard of cellulase producers and screened for the cellulase producers using carboxymethyl cellulose as the sole source of carbon and were authenticated using Congo red plate screening method. The isolates showing a significant zone of clearance were further confirmed by biochemically characterized.Results: Nine effective isolates were characterized and three strained were used for analysis. The organisms were subjected with substrate, temperature (25°C, 37°C, and 45°C), and pH to optimize the cultural condition. The enzyme activity was estimated using endoglucanase, FPase after incubating at appropriate conditions. Five isolates showing a significant zone of clearance were selected, out of which three belonged to Bacillus and one each to Staphylococcus. Optimization of media and genetic modification of the strains can further convalesce their competence. All the isolates have ensuring application in view of use in future.Conclusion: An effective strain of bacteria was isolated from the gut of termites can be used as a potential candidate for the production of cellulase in industries

Comparative study of humic acids of the mound of a wood-feeding termite and of the litter directly below in the Amazon river delta

In order to determine the role of termites in the recycling of organic matter and in humification processes, organic matter from the mound of a wood-feeding termite (Nasutitermes sp.) and from the litter directly below has been studied in secondary forest on the campus of Belem University, in Brazil. The carbon content was slightly lower in the litter ( just beneath the mound) than in the mound, but nitrogen was much more abundant in the mound. As a consequence, the C/N ratio of fragmented litter total organic matter is very high, which shows that the humification process is not complete. Therefore, plant debris seemed to be more degraded in the mound than in the litter, indicating a humification gradient from mound to litter. Humic acid extracted from the mound and from the litterwas compared by using elemental, E4/E6 ratio, spectroscopic (FTIR) analyses, and Sephadex gel chromatography. First, humic acids were more abundant in the mound than in the litter, showing that humification processes were more advanced in the mound than in the litter. Gel-permeation chromatography showed that the humic acids of the mound contained more low-molecular-weight fractions than those of the litter. In addition, the results of infrared spectra, E4/E6 ratio and elemental composition can confirm the fulvic character of mound material and the humic character of litter material. Therefore, the plant debris seems to follow two different humification pathways in the two environments, as long as the mound is alive

Transformation and mineralization of nitrogenous soil components in the gut of soil-feeding termites

This thesis consists of several studies that focused on the role of soil-feeding termites and termite gut microbiota in the transformation and mineralization of nitrogenous soil components. The results can be summarized into four subject matters, namely: 1. N mineralization and transformation during soil gut passage In order to better understand the role of soil-feeding termites in the dynamics of N in tropical soils, soil microcosms that received 15N tracers were incubated with termites. Here, our results demonstrated the importance of nitrogenous soil components (peptides) in the diet of soilfeeding termites, providing close to 50% of the termite’s carbon flux. The mineralization process, also results in the formation of enormous amounts of ammonia both in the gut (~150 mM) and the nest material. Additionally, we provided the first evidence for a termite-associated nitrification activity during the feeding activities of termites, which is coupled to denitrification and dissimilatory nitrate reduction to ammonia. At the ecosystem level, soil-feeding termites are estimated to contribute more towards N retention than to N loss in tropical soils. 2. Roles of termite gut microbiota in peptide breakdown and amino acids turnover Using gut homogenates, our studies revealed that termite gut microbiota play major roles in the hydrolysis and mineralization of peptidic components of soil organic matter. Both proteolytic and lysozyme activities were associated with termite tissues (i.e., salivary glands) and also the particulate fraction of the gut content. Together with the high alkalinity of the gut, soil peptides and microbial biomass are sequentially subjected to hydrolysis, solubilization, and extraction in the intestinal tract. Amino acids, which accumulate, are either directly absorbed by the insect or turned over by the dense hindgut microbiota, preferably by anaerobic amino-acid-fermenting bacteria. This underscores theimportant role of termite gut microbiota and the in situ physiological gut conditions, in enhancing the mineralization and utilization of peptidic components of soil organic matter by the termite. 3. Intestinal nitrate reduction leads to N2O and N2 emission Nitrate, a product of the nitrification activities in the gut, is reduced by the intestinal microbiota either to N2O and N2, or to ammonia. The reduction of nitrate to ammonia takes place mainly in the anterior gut region whereas denitrification occurs in the posterior hindgut. Virtually, no nitrate-reducing activities were present in the alkaline gut sections. Living termites emit both N2O and N2, but the emission of N2 rather than N2O seems to be the prevalent nitrogenous gas produced by soil-feeding termites. Nitrate reduction via denitrification represents ~26% of the total electrons flowing through methanogenesis in the intestinal tracts of soil-feeding termites. This study documents the first report on intestinal nitrate reduction to N2 and also provides the first evidence of soilfeeding termites as a source of the greenhouse gas N2O. 4. Excretion of ammonia via an “acid-trap” mechanism Soil-feeding termites preferentially utilize the peptidic components of soil organic matter. Consequently, ammonia levels in the hemolymph (~300 μM) and in the gut fluid accumulate to enormous concentrations. Using 15N tracers, we demonstrate that the alkalinity of the gut plays an important role removing ammonia by volatilizing NH4 + to NH3, which then diffuses into the hemolymph. Subsequently, NH3 is entrapped in the posterior hindgut with a circumneutral pH, most likely via an active transport mechanism. Finally, ammonia is egested through feces into the nest material. Also, preliminary evidence alludes to the role of Malpighian tubules in the excretion of ammonia as uric acid, a hitherto unknown function in soil-feeding termites

Struktur und räumliche Verteilung mikrobieller Gemeinschaften im Verdauungstrakt ausgewählter Boden-Invertebraten

Boden-Makroinvertebraten sind entscheidend an der Transformation organischer Substanz beteiligt, die für viele Schlüsselfunktionen des Bodens verantwortlich ist. An den Transformationsprozessen, die während der Darmpassage ingestierter organischer Substanz ablaufen, sind intestinale Mikroorganismen beteiligt, die auch für die Ernährung ihrer Wirte von entscheidender Bedeutung sind. Über die Zusammensetzung der mikrobiellen Gemeinschaften im Verdauungstrakt der meisten Boden-Invertebraten sowie ihre räumliche Verteilung innerhalb verschiedener Darmabschnitte ist allerdings nur wenig bekannt. Gerade die Topologie mikrobieller Gemeinschaften wird aber als eine wichtige Voraussetzung zum tieferen Verständnis ihrer Funktion in Verdauungstrakten von Invertebraten angesehen, die von ausgeprägten axialen und radialen Gradienten physiko-chemischer Parameter geprägt sind. Im Rahmen dieser Arbeit wurden daher Struktur und räumliche Verteilung mikrobieller Gemeinschaften im Verdauungstrakt der Larven zweier Scarabaeiden (Pachnoda ephippiata [Kongo-Rosenkäfer] und Melolontha melolontha [Feldmaikäfer]) und von Regenwürmern (Lumbricus terrestris) mit Methoden der molekularen mikrobiellen Ökologie untersucht. Während der Untersuchungen wurde zudem ein neuartiger PCR-Artefakt, die Bildung sog. pseudo-T-RFs, mit bedeutenden Auswirkungen für die T-RFLP-Analyse mikrobieller Gemeinschaften entdeckt, beschrieben und mögliche Ansätze zu seiner Vermeidung aufgezeigt. In einer T-RFLP-Studie mit Regenwürmern wurde gezeigt, dass sich die Darmmikrobiota dieser Tiere aus der Nahrung rekrutiert, d.h. ihnen im Gegensatz zu vielen anderen Boden-Invertebraten eine spezifische Darmflora fehlt. Es wurde deutlich, dass die mit dem gefressenen Boden aufgenommene mikrobielle Gemeinschaft während der Darmpassage signifikante Veränderungen ihrer relativen Zusammensetzung erfährt und dass die Unterschiede zwischen den mikrobiellen Gemeinschaften von Futter, Darm und Losung stark von der Diät der Regenwürmer beeinflusst werden. Die hier präsentierten Ergebnisse zur Intestinalmikrobiologie von Scarabaeidenlarven stellen die ersten ihrer Art für Käferlarven und mit die ersten für andere Boden-Arthropoden als Termiten dar. Es konnte gezeigt werden, dass sich die ausgeprägten Unterschiede physiko-chemischer Parameter (pH-Wert, Redoxpotential, Fettsäurespektren), die zwischen den Haupt-Darmabschnitten (Mittel- und Enddarm) der Larven herrschen, in einer deutlich unter-schiedlichen Besiedlung mit Mikroorganismen widerspiegeln. Im Gegensatz zu den untersuchten Regenwürmern ist die Darmmikrobiota der Scarabaeidenlarven als spezifische Darmflora anzusehen, da sie deutlich verschieden zur Mikrobiota der aufgenommenen Nahrung war. Bei beiden Larven war der Mitteldarmabschnitt weniger dicht besiedelt als der als Gärkammer angesehene Enddarm. Die Methanogenese war stets auf den Enddarm beschränkt; bei den Maikäferlarven wurden Methanobrevibacter-Arten, bei den Rosenkäferlarven zusätzlich Methanomicrococcus-Arten als verantwortliche Methanogene identifiziert. Im Vergleich zu den Bacteria war aber sowohl die Diversität als auch die relative Häufigkeit der Archaea sehr gering. Die phylogenetische Analyse der Bakteriengemeinschaften zeigte eine sehr große Diversität auf, die offensichtlich viele bislang unkultivierte Arten umfasst. Die überwiegende Mehrheit aller Sequenzen ließ sich den Actinobacteria, Bacillales, Bacteroidetes, Clostridiales, Lactobacillales und Proteobacteria zuordnen; viele Klone gruppierten mit Klonen und Isolaten aus anderen Intestinalsystemen, ein weiterer Beleg für die Darmspezifität der Scarabaeiden-Mikrobiota. Die Verwandtschaft vieler Klone zu hydrolytischen, cellulolytischen und gärenden Isolaten stand in Einklang mit den Fettsäureprofilen der Darmabschnitte (v.a. Acetat und Lactat) und deutet die Beteiligung von Mikroorganismen an der Transformation organischer Substanz nach dem Modell einer anaeroben Nahrungskette zumindest im Enddarm an. Ob dies auch für den Mitteldarmabschnitt gilt, ist noch unklar, da bei den Maikäferlarven keine stabile Darmmikrobiota in diesem Kompartiment nachgewiesen werden konnte. Am Enddarm der Maikäferlarven wurde erstmals für Arthropoden eine umfassende Analyse der mikrobiellen Gemeinschaften in den Unterfraktionen Wand und Lumen eines Darmabschnittes durchgeführt. Hierbei wurden ausgeprägte Unterschiede in der Besiedlung dieser beiden Fraktionen festgestellt, die als Anpassungen an morphologische (Chitinbäumchen an der Enddarmwand) und eventuell auch physiko-chemische Unterschiede (Gradient eindringenden Sauerstoffs) zwischen Darmwand und -lumen interpretiert werden können. Der auffälligste Unterschied war eine hohe Abundanz (10 - 15% aller Bakterien) Desulfovibrio-verwandter Bakterien an der Enddarmwand, die sowohl mit PCR-abhängigen als auch PCR-unabhängigen Methoden abgesichert werden konnte. In seiner Eindeutigkeit ist dieser Befund für Arthropoden bislang einmalig

Struktur und räumliche Verteilung mikrobieller Gemeinschaften im Verdauungstrakt ausgewählter Boden-Invertebraten

Boden-Makroinvertebraten sind entscheidend an der Transformation organischer Substanz beteiligt, die für viele Schlüsselfunktionen des Bodens verantwortlich ist. An den Transformationsprozessen, die während der Darmpassage ingestierter organischer Substanz ablaufen, sind intestinale Mikroorganismen beteiligt, die auch für die Ernährung ihrer Wirte von entscheidender Bedeutung sind. Über die Zusammensetzung der mikrobiellen Gemeinschaften im Verdauungstrakt der meisten Boden-Invertebraten sowie ihre räumliche Verteilung innerhalb verschiedener Darmabschnitte ist allerdings nur wenig bekannt. Gerade die Topologie mikrobieller Gemeinschaften wird aber als eine wichtige Voraussetzung zum tieferen Verständnis ihrer Funktion in Verdauungstrakten von Invertebraten angesehen, die von ausgeprägten axialen und radialen Gradienten physiko-chemischer Parameter geprägt sind. Im Rahmen dieser Arbeit wurden daher Struktur und räumliche Verteilung mikrobieller Gemeinschaften im Verdauungstrakt der Larven zweier Scarabaeiden (Pachnoda ephippiata [Kongo-Rosenkäfer] und Melolontha melolontha [Feldmaikäfer]) und von Regenwürmern (Lumbricus terrestris) mit Methoden der molekularen mikrobiellen Ökologie untersucht. Während der Untersuchungen wurde zudem ein neuartiger PCR-Artefakt, die Bildung sog. pseudo-T-RFs, mit bedeutenden Auswirkungen für die T-RFLP-Analyse mikrobieller Gemeinschaften entdeckt, beschrieben und mögliche Ansätze zu seiner Vermeidung aufgezeigt. In einer T-RFLP-Studie mit Regenwürmern wurde gezeigt, dass sich die Darmmikrobiota dieser Tiere aus der Nahrung rekrutiert, d.h. ihnen im Gegensatz zu vielen anderen Boden-Invertebraten eine spezifische Darmflora fehlt. Es wurde deutlich, dass die mit dem gefressenen Boden aufgenommene mikrobielle Gemeinschaft während der Darmpassage signifikante Veränderungen ihrer relativen Zusammensetzung erfährt und dass die Unterschiede zwischen den mikrobiellen Gemeinschaften von Futter, Darm und Losung stark von der Diät der Regenwürmer beeinflusst werden. Die hier präsentierten Ergebnisse zur Intestinalmikrobiologie von Scarabaeidenlarven stellen die ersten ihrer Art für Käferlarven und mit die ersten für andere Boden-Arthropoden als Termiten dar. Es konnte gezeigt werden, dass sich die ausgeprägten Unterschiede physiko-chemischer Parameter (pH-Wert, Redoxpotential, Fettsäurespektren), die zwischen den Haupt-Darmabschnitten (Mittel- und Enddarm) der Larven herrschen, in einer deutlich unter-schiedlichen Besiedlung mit Mikroorganismen widerspiegeln. Im Gegensatz zu den untersuchten Regenwürmern ist die Darmmikrobiota der Scarabaeidenlarven als spezifische Darmflora anzusehen, da sie deutlich verschieden zur Mikrobiota der aufgenommenen Nahrung war. Bei beiden Larven war der Mitteldarmabschnitt weniger dicht besiedelt als der als Gärkammer angesehene Enddarm. Die Methanogenese war stets auf den Enddarm beschränkt; bei den Maikäferlarven wurden Methanobrevibacter-Arten, bei den Rosenkäferlarven zusätzlich Methanomicrococcus-Arten als verantwortliche Methanogene identifiziert. Im Vergleich zu den Bacteria war aber sowohl die Diversität als auch die relative Häufigkeit der Archaea sehr gering. Die phylogenetische Analyse der Bakteriengemeinschaften zeigte eine sehr große Diversität auf, die offensichtlich viele bislang unkultivierte Arten umfasst. Die überwiegende Mehrheit aller Sequenzen ließ sich den Actinobacteria, Bacillales, Bacteroidetes, Clostridiales, Lactobacillales und Proteobacteria zuordnen; viele Klone gruppierten mit Klonen und Isolaten aus anderen Intestinalsystemen, ein weiterer Beleg für die Darmspezifität der Scarabaeiden-Mikrobiota. Die Verwandtschaft vieler Klone zu hydrolytischen, cellulolytischen und gärenden Isolaten stand in Einklang mit den Fettsäureprofilen der Darmabschnitte (v.a. Acetat und Lactat) und deutet die Beteiligung von Mikroorganismen an der Transformation organischer Substanz nach dem Modell einer anaeroben Nahrungskette zumindest im Enddarm an. Ob dies auch für den Mitteldarmabschnitt gilt, ist noch unklar, da bei den Maikäferlarven keine stabile Darmmikrobiota in diesem Kompartiment nachgewiesen werden konnte. Am Enddarm der Maikäferlarven wurde erstmals für Arthropoden eine umfassende Analyse der mikrobiellen Gemeinschaften in den Unterfraktionen Wand und Lumen eines Darmabschnittes durchgeführt. Hierbei wurden ausgeprägte Unterschiede in der Besiedlung dieser beiden Fraktionen festgestellt, die als Anpassungen an morphologische (Chitinbäumchen an der Enddarmwand) und eventuell auch physiko-chemische Unterschiede (Gradient eindringenden Sauerstoffs) zwischen Darmwand und -lumen interpretiert werden können. Der auffälligste Unterschied war eine hohe Abundanz (10 - 15% aller Bakterien) Desulfovibrio-verwandter Bakterien an der Enddarmwand, die sowohl mit PCR-abhängigen als auch PCR-unabhängigen Methoden abgesichert werden konnte. In seiner Eindeutigkeit ist dieser Befund für Arthropoden bislang einmalig

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 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

A review of the microbiome associated with human decomposition

The decomposition of human remains involves a complex microbial ecology that few studies have examined in depth. This review investigated the microbiome of human decomposition to further understand their functions within the decomposition process and their potential to increase the accuracy of post-mortem interval (PMI) estimations in forensic applications. The aims of the literature review were to (1) identify the external microbiome responsible for human decomposition, focusing on insect, soil and skin sources, (2) determine the roles of external bacteria in the various stages of human decomposition and (3) to analyse and compare the current contributions of literature in furthering the understanding of the ecological mosaic of decomposition. The current literature was reviewed and their contributions to necrobiome research was analysed using qualitative and contemporary research techniques. Bacteria were found to play a significant role in each stage of human decomposition with multiple studies demonstrating an observable successive shift in microbial communities through time. This change in community profile was found to be an important biomarker for the estimation of the PMI and potential substitute for entomological techniques currently utilised in forensic investigations. High interpersonal variation between decomposition events, in addition to narrow geographic specificity, represented limitations in the studies which may be remedied by increasing sample size while focusing on different geographic regions and environmental conditions

Transformation of nitrogenous soil components by humivorous beetle larvae

The humivorous scarab beetles of the genus Pachnoda are indigenous to the African continent. The larvae of these insects preferably live in humic soils and feed on plant material and soil organic matter. However, the nature of the soil components used as carbon and energy sources are so far not understood. Previous studies indicated that peptides stabilized in humic acid model compounds are released and mineralized during gut passage. The present thesis documents extent of transformation and mineralization of peptidic compounds of the food soil. Further, the role of peptides in the nutrition of the larvae was estimated. The major site of proteolytic activity was located in the midgut. Also in the hindgut minor proteolytic activities were found. We postulate that the former activities are associated with host-secreted proteinases while the latter are mainly of microbial origin. As a consequence of high proteolytic activities in the midgut considerably high concentration of amino acids are accumulating. Relatively low turnover rates of amino acids suggest amino acids fermentation is the rate-limiting step in the midgut. In vivo, the insect host presumably takes up part of these amino acids. The high ammonia concentration in the hindgut is associated with high a turnover rate of amino acids. Addition of amino acids even stimulated the fermentative activity, suggesting that the supply of free amino acids by proteolysis is the rate-limiting step in this process. Thus, the hindgut is the main site of amino acid fermentation in the gut. Further, nitrification was indirectly observed by the accumulation of nitrite and nitrate. In high dilution steps of MPNs nitrite is subject to denitrification associated with gas production, most probably N2, supported by detectable losses of nitrogen from the food soil during gut passage. With the help of stable isotope probing, for several environments and diverse substrates the function of specific microbial populations was elucidated. However, previous experiments addressing the functions of the insect gut microbiota difficulties were observed using this method. In stable isotope probing experiments with whole guts of the wood-feeding termite Reticulitermes santonensis and hindgut of the humivorous beetle larvae of Pachnoda ephippiata using H13CO3– or 13C-labeled glucose it was confirmed that no isotope effect in the RNA of the gut microbiota can be detected. Also in incubations with 14C-labeled substrates, generally a more sensitive tracer, very little incorporation of substrate carbon into RNA was detected. We suggest several explanations as to why stable isotope probing in the gut of insects is not feasible. Since free nucleotides accumulate in the hindgut of Pachnoda marginata to high concentrations, we hypothesize that salvage pathways, known for many microorganisms as alternative for nucleotide biosynthesis, reduce the de novo formation of nucleic acids from substrate carbon. This hypothesis is supported by considerable inhibition of the incorporation of substrate carbon into RNA in incubations with Bacillus subtilis and Pseudomonas putida in the presence of ribonucleotides. In the insect gut environment, we reached the limits of the otherwise very convenient and successful application of stable isotope probing for the above-mentioned substrates. The actual reason still remains to be elucidated. The allopatric scarab beetle larvae of Pachnoda ephippiata and Pachnoda marginata are closely related and both feeding on soil organic matter. In the present thesis, the question is answered, whether the microbial community in the gut is similar, due to the same food source, or reveals differences as a consequence of the spatial separation. The composition of the bacterial communities of the midgut and the hindgut was studied with the help of terminal restriction fragment length polymorphism (T-RFLP) analysis, representing a PCR-based cultivation-independent technique. In both larvae, the bacterial midgut community is less diverse than the microbial hindgut community. In the T-RFLP profiles from the midgut of the two species only few T-RFs are shared with the hindgut profiles. By contrast, the community fingerprints of the hindguts appeared to be very similar, although the relative abundance of the single T-RF varied between the two beetle species. However, correspondence analysis of the T-RFLP dataset, we demonstrated significant species-dependent differences in the bacterial communities of both, the midgut and the hindgut, respectively. The feeding of larvae of Pachnoda marginata on different food soils did not result in significant differences in the bacterial hindgut community. Together, these results indicate that the differences in the gut microbiota are rather due spatial separation of the species than to differences in the diet

The earthworm microbiome

Background: Host-associated microbial communities play a significant role in a species’ environmental interactions, often performing functions unachievable by the eukaryotic host, and is essential in developing a comprehensive understanding of the species and its impact on the local and global ecosystem. Earthworms (Lumbricina) habituate almost every type of soil environment globally, including sites of severe environmental stress and is an essential ecosystem engineer, central to healthy natural and agricultural soils. To date, only a singular symbiotic species (Verminephrobacter sp.) has been identified, but the earthworm impact on transient microbial communities and the surrounding soil microbiome is profound. Methods: Previous culture and molecular based studies found earthworm-associated microbiota unlikely however, this has not been explored using High Throughput Sequencing. Utilisation of Illumina, 454 and Ion Torrent sequencing has enabled production of the highest resolution microbial analysis of host-associated bacteria of any single eukaryotic species to date, including spatial bacterial localisation of the entire Lumbricus rubellus organism and impact analysis of a wide range of anthropogenic contaminants and environmental stressors on the basal microbiomic community. Results: A core bacterial community has been described which is distinct from the surrounding soil. A number of novel species have been associated with the earthworm crop, body wall and hindgut, contravening claims that the earthworm has limited or no impact on ingested soil bacteria. This demonstrate that the host properties impart significant effects on the transient population, demanding further analysis to determine potential symbiotic functionality. However, while a biologically important community has been described, the significant impact of anthropogenic contamination on the host microbiome must be considered given the observed eradication of the Verminephrobacter symbiont during the host’s exposure to arsenic and the potential subsequent implications on host health