Inducible anti-fish defenses in Daphnia: DVM-induction by 5α-cyprinol sulfate under quasi-natural conditions and identification of the life-history changes inducing kairomone

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Microcrustaceans of the genus Daphnia play a key role in lakes and ponds, as they are major consumers of planktonic primary producers and are important prey for higher trophic levels. Daphnia have become text book examples for the induction of defenses by kairomones released by predators. In response to kairomones released by fish, the major predator of daphnids, Daphnia deploy anti-predator defenses as life-history changes (LHCs) and diel vertical migration behavior (DVM), which reduce the efficiency of the predator and impact many other ecosystem-wide processes. We have recently identified the DVM-inducing kairomone released by fish to be 5α-cyprinol sulfate (5α-CPS) and determined that it induces DVM in Daphnia magna in an indoor-setup at picomolar concentrations. Methods for the preparative purification of 5α-CPS and for the quantification of 5α-CPS by ultra-high-pressure liquid chromatograph coupled to a high-resolution mass spectrometer are well established in the group.Building on this earlier work I here propose to apply 5α-CPS for DVM-induction in mesocosm experiments with a natural Daphnia community and in parallel to determine in-situ concentrations of 5α-CPS with the aim to establish threshold concentrations for DVM-induction under quasi-natural conditions. In nature, in-situ concentrations of 5α-CPS are the result of release by fish and simultaneous bacterial degradation. To account for both processes I propose to determine at different temperatures release rates of 5α-CPS of individual fish differing in size and feeding status. Bacterial degradation rates at different temperatures will be established by incubating lake water that has been spiked with known concentrations of 5α-CPS and by quantifying 5α-CPS over time. Besides DVM, life-history changes (LHCs) are another very important inducible defense against fish in Daphnia. However, the LHC-inducing kairomone is not known yet. Here I propose as the most straightforward approach to test, if 5α-CPS induces LHCs. If not, extract of fish incubation water will be subjected to bioassay-guided purification in several rounds of increasing purification by HPLC coupled to a high-resolution mass spectrometer. Fractions produced by HPLC will be tested in well-established bioassays for LHCs. Subsequent non-targeted metabolomics and fragmentation experiments will aim at identification of the kairomone. The overall aim is to (i) more precisely understand DVM-induction in Daphnia by 5α-CPS under quasi-natural conditions and to estimate production and degredation rates of 5α-CPS and (ii) to identify the LHC-inducing fish kairomone.