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

水蚤属的小甲壳类动物在湖泊和池塘中起着关键作用，因为它们是浮游生物初级生产者的主要消费者，也是更高营养水平的重要猎物。水蚤已经成为捕食者释放的激素诱导防御的教科书范例。作为水蚤的主要捕食者，水蚤通过生活史变化（life-history changes, lhc）和垂直迁移行为（vertical migration behavior， DVM）来应对鱼类释放的kairomones，从而降低了捕食者的效率，并影响了生态系统的许多其他过程。我们最近鉴定了鱼类释放的诱导DVM的kairomone是5α-cyprinol sulfate (5α-CPS)，并确定它在室内设置中以皮摩尔浓度诱导大水蚤DVM。建立了5α-CPS的制备纯化和超高压液相色谱仪-高分辨率质谱联用定量方法。在此基础上，本文提出将5α-CPS应用于天然水蚤群落的中生态实验，并平行测定5α-CPS的原位浓度，以建立准自然条件下诱发dvm的阈值浓度。在自然界中，5α-CPS的原位浓度是鱼类释放和细菌降解的结果。为了解释这两个过程，我建议确定不同大小和摄食状态的个体鱼在不同温度下的5α-CPS释放率。细菌在不同温度下的降解率将通过培养加入已知浓度的5α-CPS的湖水，并随着时间的推移对5α-CPS进行量化来确定。除DVM外，生活史变化（life-history change, lhc）是水蚤另一种重要的诱导防御机制。然而，诱导lhc的激素尚不清楚。这里我提出最直接的测试方法，如果5α-CPS诱导lhc。如果没有，鱼孵育水的提取物将通过HPLC和高分辨率质谱仪进行多轮的生物测定指导纯化。高效液相色谱法生产的馏分将在成熟的lhc生物测定法中进行测试。随后的非靶向代谢组学和碎片化实验将旨在鉴定凯罗酮。总体目标是：(i)更准确地了解在准自然条件下5α-CPS对水蚤的诱导作用，并估计5α-CPS的产生和降解速率；（ii）鉴定诱导lhc的鱼酮。

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.