Interaction of Daphnia magna with cyanobacteria: New insights from using high-resolution LC-MS

In most freshwater ecosystems the unselectively filter feeding cladoceran Daphnia is of major importance for the transfer of energy, carbon and nutrients across the plant - herbivore interface. Especially in freshwaters with a high abundance of cyanobacteria this transfer is often constrained by food quality rather than food quantity, since cyanobacteria are considered as low quality food for daphnids. Cyanobacteria lack several nutritional components that are important for daphnids, they can interfere mechanically with the filter apparatus of daphnids due to colony formation and they contain numerous cyanotoxins. The importance of the Daphnia - cyanobacteria interaction is additionally emphasized by an increasing anthropogenic influence on ecosystems, which includes artificial nutrient input into lakes and ponds as well as increasing temperatures due to global warming. These human impacts contribute to the extent and frequency of cyanobacterial blooms, which in return constitute a serious hazard for public health and ecosystem services. Studies concerned with this crucial interaction reveal contradictory results, in which sometimes cyanobacteria suppress Daphnia populations whereas in other cases the opposite situation can be observed, namely the suppression of cyanobacteria by daphnids. Daphnia species display an enormous ability to adapt to cyanobacteria. As a result, huge differences among Daphnia species and genotypes with regard to their sensitivity towards cyanobacterial diets can be found. Despite the importance of the Daphnia - cyanobacteria interaction for the structure of freshwater ecosystems, the knowledge about the physiological basis of this interaction is scarce. In order to elucidate important aspects of the physiology that underlies the Daphnia - cyanobacteria interaction the present study focused mainly on toxic cyanobacterial secondary metabolites. To detect and quantify cyanobacterial compounds in exposure experiments with daphnids and toxin containing cyanobacteria, high resolution LC-MS technology was applied. This approach, in combination with other techniques, such as enzyme activity measurements and molecular biological methods, revealed several new insights into the physiology of the interaction of Daphnia with cyanobacteria. It is argued that one major reason explaining the huge differences among daphnids with regard to their sensitivity towards cyanobacteria might lie in their ability to cope with cyanotoxins such as microcystin-LR (MCLR). The widely accepted hypothesis regarding dietary MCLR in Daphnia includes the involvement of glutathione-S-transferase acting as a first step of detoxification by conjugating the toxin with glutathione (GSH). I was able to reject this hypothesis by measuring MCLR amounts in all parts of an exposure experiment (incubation medium, cyanobacteria and Daphnia tissue) with Daphnia magna and the MCLR containing cyanobacterium Microcystis aeruginosa. Since the presence of daphnids neither led to a decrease of overall MCLR nor to an increase of the MCLR-GSH conjugate significant processes of biotransformation of any kind could be ruled out. Instead the data revealed that MCLR is exported out of Daphnia tissue with a high efficiency. These transport processes had not been considered with regard to dietary MCLR in Daphnia yet. Furthermore, the study reveals that these transport mechanisms should not be considered as a special case for MCLR alone but that transport is of general importance with regard to dietary cyanotoxins in Daphnia, since a whole set of other structurally diverse cyanobacterial components were similarly exported out of Daphnia and not subjected to any forms of biotransformation. Additionally, I could identify two types of trans membrane transporters, namely multi xenobiotic resistance (MXR) proteins and organic anion transporting polypeptides (Oatps) being responsible for the export of MCLR out of Daphnia tissue. The application of specific inhibitors of these transporters resulted in significantly increased MCLR amounts within the Daphnia tissue. Although a qPCR analysis demonstrated that a 24 h exposure of daphnids to a MCLR containing diet is not enough to cause significant up-regulation of respective transporter genes, the results in this dissertation strongly emphasize the importance of transport mechanisms for the understanding of the Daphnia - cyanobacteria interaction. In the last part of my thesis I successfully developed a new method in order to unravel seasonal succession patterns of cyanobacterial chemotypes within a pond. Metabolomic fingerprints of a time series of phytoplankton samples were combined with the potential of these samples to inhibit grazer proteases by using multivariate statistics. This new approach revealed rapid changes within the cyanobacterial chemotype composition in a lake, which in return strongly reflect on Daphnia populations and emphasize the need for fast adaptation processes on the Daphnia side. In summary, the results of this dissertation reveal several new insights in the physiology of the Daphnia - cyanobacteria interaction, which elucidate the importance, necessity and nature of transport processes for detrimental cyanotoxins in Daphnia