Prey are under selection to minimize predation losses. In aquatic environments, many prey use chemical cues released by predators, which initiate predator avoidance. A prominent example of behavioral predator-avoidance constitutes diel vertical migration (DVM) in the freshwater microcrustacean Daphnia spp., which is induced by chemical cues (kairomones) released by planktivorous fish. In a bioassay-guided approach using liquid chromatography and mass spectrometry, we identified the kairomone from fish incubation water as 5α-cyprinol sulfate inducing DVM in Daphnia at picomolar concentrations. The role of 5α-cyprinol sulfate in lipid digestion in fish explains why from an evolutionary perspective fish has not stopped releasing 5α-cyprinol sulfate despite the disadvantages for the releaser. The identification of the DVM-inducing kairomone enables investigating its spatial and temporal distribution and the underlying molecular mechanism of its perception. Furthermore, it allows to test if fish-mediated inducible defenses in other aquatic invertebrates are triggered by the same compound.

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Recently, the first chemical cues, which induce defenses in freshwater zooplankton of the genus Daphnia have been identified. Still it is unknown how general these so-called kairomones are and if they are perceived by and thereby benefitting invasive species. We here report the chemical identity of the fish-derived chemical signal that induces morphological defensive structures in the invasive species Daphnia lumholtzi. We used bioassay-guided isolation (LC-MS) of incubation water of fish and identified the bile salt 5α-cyprinol sulfate (5α-CPS) as the kairomone. We confirmed this finding by bioassays, in which D. lumholtzi was exposed to purified 5α-CPS sulfate from fish bile and showed that 10 pM 5α-CPS induces significantly elongated helmets and spines in relation to body length. These results identify on one hand another Daphnia species besides Daphnia magna, which is responding to this particular fish-borne kairomone, and on the other hand they reveal another defense that the kairomone induces besides diel vertical migration (DVM). Taken together a high evolutionary conservancy of the molecular mechanism behind inducible defenses in Daphnia spp. against fish can be deduced. It is reasonable to expect that 5α-CPS is involved in further predator—prey communication between Daphnia and fish, which may be of special ecological relevance with regard to invasive species as D. lumholtzi.

In freshwater zooplankton diel vertical migration (DVM) is a widespread predator-avoidance behavior that is induced by kairomones released from fish. Thereby zooplankton reduces predation by fish by staying in deep and dark colder strata during daytime and migrating into warmer layers during night, and thus experiences diel alterations in temperature. Constantly lower temperatures have been shown to increase the relative abundance of polyunsaturated fatty acids (PUFAs) in Daphnia sp. Furthermore, a low dietary supply of the ω3-PUFA eicosapentaenoic acid (EPA) has been shown to limit the induction of DVM in Daphnia magna and the performance of D. magna under fluctuating temperatures, as experienced during DVM. In nature DVM of D. magna in response to fish is accompanied by the presence of fish-borne kairomone and diel fluctuations of depth dependent-parameters like temperature, food, and oxygen supply. Here we investigated the effect of factors, which are differing between Daphnia that perform DVM and those which do not. We selected to examine the effect of changing temperature and light conditions and of the presence/absence of fish kairomones on D. magna. For this purpose, we conducted a full factorial experimental design in which we grew D. magna under constantly warm temperatures in a diel light-dark regime or under alternating temperatures in darkness crossed with the presence or absence of fish kairomones. We analyzed the fatty acid composition of mature animals and of their offspring in each treatment. Simulation of the light and temperature regime of migrating animals in presence of the fish kairomone resulted in an increased relative allocation of the ω3-PUFA EPA, from adult animals to their offspring, manifesting as decreased EPA concentrations in mothers and increased EPA concentrations in their offspring in response to simulated DVM (mothers). Additionally, EPA concentrations in the offspring were affected by the interaction of simulated DVM and the fish cue. The presence of the fish kairomone alone increased the EPA concentration in the offspring, that was not experiencing simulated DVM. These findings lead to the conclusion that the temperature and light regime associated with DVM alone, as well as in combination with the DVM-inducing fish kairomones, alter the allocation of fatty acids to the offspring in a manner, which is beneficial for the offspring under the decreased average temperatures, which migrating animals are exposed to. A low dietary supply of ω3-PUFAs may constrain D. magna’s amplitude of DVM, but our results suggest that the next generation of animals may be capable of regaining the full DVM amplitude due to the effect of the fish kairomone and the experienced temperature fluctuations (and darkness) on tissue fatty acid composition. These findings suggest that fatty acid limitation in DVM performing Daphnia may be more severe for the maternal than for the offspring generation.

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