Molecular mechanisms of adaptive life-history changes in Daphnia magna induced by predator kairomones

Planktonic crustaceans of the genus Daphnia play an important role as keystone species in almost all standing freshwater ecosystems. As unselective filter feeders, Daphnia are typically the major herbivorous grazers of phytoplankton and are major prey for predators at higher trophic levels. Daphnia magna has become a textbook example for inducible defences in response to chemical signals released by predators. Daphnia as important prey organism for both invertebrate predators and vertebrate predators like planktivorous fish, show a multitude of defences against these predators including changes in life-history (LHC), morphology and diel vertical migration (DVM), which have been shown to be induced or enhanced by chemical cues released by predators, so-called kairomones. Inducible anti-fish defences like LHC and DVM are associated with substantial costs. There is evidence for a coadaptation of both defences (LHC and DVM) in Daphnia that hints to a plastic coupling of LHC and DVM and due to the substantial costs it has been argued that it would be maladaptive to exhibit all available defences at the same time, as selecting one or the most efficient defences against a predator. Daphnia appear to be plastic with respect to the degree of response with either defence and it is still an open question, which additional factor besides the fish kairomones might control a possible coupling of anti-predator defences in Daphnia. Furthermore, although the Daphnia genome and the molecular tools are available, there is not much known about the molecular mechanisms that may underlie the anti-predator defences induced by fish kairomones in Daphnia. The present study covers presents novel and deep insights into the mechanisms underlying a well-known inducible anti-predator response in Daphnia on a broad range: besides classical ecological approaches I conducted qPCR analyses, LC-MS analyses and a holistic LC-MS/MS based proteomics approach. I showed for the first time that the presence of light is crucial for the induction of LHC in Daphnia exposed to fish kairomones and the same effect was observed for the fish kairomonemediated expression of candidate genes. These observations point at a plastic coupling of anti-predator defences modulated by the environmental factor light in Daphnia. Under natural conditions animals are subjected to alternating periods of light and dark and different light intensities. I further explored the role of the ambient light intensity for the induction of LHC in Daphnia and the animals were exposed to a gradient of light intensities that represents naturally occurring light intensities in the water column of a lake. The results revealed that LHC in Daphnia are inversely coupled to the ambient light intensity and that a certain thresholds for the induction of LHC exists. A similar light intensity dependence was found for the fish kairomonemediated expression of a candidate gene in Daphnia. I argue that the DVM amplitude alters the ambient light intensity an individual is exposed to and thus inversely determines the degree of LHC. My findings provide strong evidence for a light intensity-mediated plastic coupling of migration behaviour (DVM) and life-history changes (LHC) in Daphnia, which allows for plastic adjustment to fluctuating environments and simultaneously minimizes the associated costs of multiple deployable defences. Such a proposed inverse coupling of DVM and LHC would be especially adaptive in habitats that allow for DVM only during parts in the season, i.e. spring; later in the season, when a deep-water refuge is no longer available owing to hypolimnetic anoxia, alternative defences like LHC (as is frequently observed in eutrophic shallow systems) may be required. The last part of my thesis describes for the first time a LC-MS/MS based proteomics approach with the aim to identify functional proteins involved in the phenotypic changes on the protein level for the fish kairomone-induced LHC in Daphnia. In line with my findings described above, the experimental animals exposed to fish kairomones showed the expected LHC in the presence of light and this LHC response was not observed in the absence of light. In addition, due to a long series of experiments to purify the fish kairomone extract (e.g. fractionation via LC-MS) I was able to confine the biological activity to one highly specific fraction, which was then used in the life-history experiments for the proteomics approach. Only daphnids that showed the expected LHC response were used for the high throughput proteomics. The state-of-the-art proteomics approach leads to the identification of functional proteins in Daphnia that are affected by fish kairomones and light intensity. My results revealed for the first time a metabolic pathway that yields a mechanistic understanding of the observed phenotypic changes