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

水蚤属微型甲壳动物在湖泊和池塘中发挥着关键作用，因为它们是浮游初级生产者的主要消费者，也是较高营养水平的重要猎物。水蚤已经成为捕食者释放的凯洛蒙诱导防御的教科书例子。作为水蚤的主要捕食者，水蚤对鱼类释放的凯洛蒙作出反应，随着生活史的变化(LHC)和昼夜垂直迁移行为(DVM)，水蚤部署了反捕食者防御系统，这降低了捕食者的效率，并影响了许多其他生态系统范围的过程。我们最近鉴定了FISH释放的诱导Dvm的凯龙酮为5-α-α-CP，并在皮摩尔浓度的室内实验中确定了它能诱导大型水蚤Dvm。5α-CP的制备纯化和5α-CP的超高压液相色谱与高分辨率质谱仪联用的定量方法在本研究的基础上，提出将5α-CP应用于水蚤群落的中游实验中，并平行测定5α-CP的原位浓度，目的是建立在准自然条件下DVM诱导的阈值浓度。在自然界中，5α-CP的原位浓度是鱼类释放和细菌同时降解的结果。为了解释这两个过程，我建议确定在不同温度下不同大小和摄食状态的鱼的5种α-CP的释放率。不同温度下的细菌降解率将通过孵化添加了已知浓度的5α-CP的湖水并通过随着时间的推移对5α-CP进行量化来确定。除了DVM，生活史变化(LHC)也是水蚤对鱼类的另一种非常重要的诱导防御。然而，诱导LHC的凯龙酮尚不清楚。在这里，我建议作为最直接的测试方法，如果5个α-CP诱导LHC。如果不是，鱼孵化水萃取物将在生物测定的指导下，通过与高分辨率质谱仪耦合的高效液相色谱进行几轮递增净化。高效液相色谱产生的馏分将在成熟的低碳氢化合物生物检测中进行测试。随后的非靶向代谢组学和碎裂实验将以鉴定凯罗酮为目标。总的目的是(I)更准确地了解5-α-CP在准自然条件下对水蚤的Dvm诱导，并估计5-α-CP的产量和脱落率；(Ii)鉴定诱导鱼类Kaironone。

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.