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