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5α-cyprinol sulfate, a bile salt from fish, induces diel vertical migration in Daphnia

  1. Meike Anika Hahn  Is a corresponding author
  2. Christoph Effertz
  3. Laurent Bigler
  4. Eric von Elert
  1. University of Koeln, Germany
  2. University of Zurich, Switzerland
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Cite this article as: eLife 2019;8:e44791 doi: 10.7554/eLife.44791

Abstract

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.

https://doi.org/10.7554/eLife.44791.001

Introduction

Predation is recognized as an important selective force which has been shown to drive the shape of trophic cascades and numerous aspects of ecosystem ecology (Gliwicz, 1986; Lima, 1998; Werner and Peacor, 2003). Such effects of predators are caused by predation (direct consumptive effect; Preisser et al., 2005) or by effects on plastic behavioral, physiological or morphological traits of prey seeking to avoid predation (indirect, non-consumptive effect, (Heithaus et al., 2008; Lima, 1998; Moll et al., 2017; Werner and Peacor, 2003). The concept has been introduced that prey species must effectively balance consuming their resources against becoming resources for their predators (Abrams, 1984; Sih, 1980) in order to maximize their own fitness. It has become clear that non-consumptive predator effects strongly affect prey distribution, demography and behavior (Heithaus et al., 2008; Lima, 1998; Werner and Peacor, 2003) and the strength of top-down and bottom-up control in communities (Ford et al., 2014). Hence, non-consumptive predator effects are mediated by shifts in plastic traits of prey, and in aquatic food webs such non-consumptive effects exceed direct consumptive effects (Preisser et al., 2005; Turner et al., 2000).

Such adaptive shifts in prey traits require an accurate assessment of the predation risk, which may be accomplished by physical contact, vision or by chemical cues. Chemical cues are superior cues in turbid or dark environments (Kats and Dill, 1998) or in cases in which the escape capability upon attack is low in prey, for example due to low escape velocity. In line with this, the induction of defenses by chemical cues from predators is widespread in aquatic systems (Bjærke et al., 2014; Brönmark and Hansson, 2012), and recently progress has been made by identification of chemical cues involved in aquatic predator-prey chemical communication (Poulin et al., 2018; Selander et al., 2015; Weiss et al., 2018).

One classical example of behavioral predator avoidance is diel vertical migration (DVM) in the freshwater microcrustacean Daphnia, which play a key role in lakes and ponds, as they are major consumers of planktonic primary producers and important prey for higher trophic levels (Miner et al., 2012). DVM is a widespread predator avoidance behavior (Hays, 2003; Williamson et al., 2011), in which the exposure to UV (Rhode et al., 2001) and the risk of predation by visually oriented predators, such as fish, is reduced by daytime residence in the dark, deep layer of the water column (Hays, 2003; Stich and Lampert, 1981). At night, zooplankton emerges from the depth into the upper water layer to minimize demographic costs associated with residence in the cold, food-depleted deep-water refuge (Loose and Dawidowicz, 1994; Stich and Lampert, 1981). Hence, DVM of Daphnia negatively affects the foraging success of planktivorous fish. This behavioral anti-predator defense affects the control of planktonic primary producers by zooplankton in the open water and thus impacts many other ecosystem-wide processes (Haupt et al., 2010; Reichwaldt and Stibor, 2005).

In lakes and ponds, DVM in zooplankton is triggered by changes in light intensity (Effertz and von Elert, 2014; Ringelberg, 1999) and by chemical cues released by predators (Dodson, 1988; Lampert, 1993; Neill, 1990). Such chemical cues are termed kairomones, if they mediate a transfer of information among species that imparts a benefit to the receiving organism while not being beneficial for the producer (Pohnert et al., 2007). The finding that the amplitude of DVM increases with fish density indicates that kairomones provide a reliable indicator for the risk of Daphnia of being preyed upon by fish (Van Gool and Ringelberg, 2002; von Elert and Pohnert, 2000). Thus, fish - Daphnia interactions are to a large degree determined by indirect, non-consumptive effects of fish that are mediated by kairomones inducing predator avoidance by DVM.

Lakes and ponds are the best available freshwater source providing ecosystem services like domestic, industrial and recreational usage of water bodies (Rudman et al., 2017). Global warming and ongoing nutrient input are predicted to deteriorate water quality and negatively affect ecosystem services of lakes and ponds (Huisman et al., 2018; Rudman et al., 2017), which increases the need for succesful lake management. One tool frequently used in lake management is food chain manipulation, which aims at manipulating the interaction of Daphnia and fish (Jeppesen et al., 2017; Peretyatko et al., 2012; Søndergaard et al., 2017). However, at the molecular level this interaction is not fully understood unless the chemical nature of the fish kairomone is disclosed. Earlier we have shown that the release of the DVM-inducing kairomone does not depend on prior feeding of fish (Loose et al., 1993). The kairomone can be extracted from fish incubation water by lipophilic solid phase extraction, and we have characterized it as a low molecular weight compound carrying a hydroxyl group and a negative charge (Loose et al., 1993; von Elert and Pohnert, 2000; von Elert and Loose, 1996). However, the exact chemical identity of the fish kairomone remained unknown. Here, we use the microcrustean Daphnia, which has become a model organism for the induction of DVM in response to fish (Lampert, 2006; Miner et al., 2012), to identify the yet unknown kairomone.

Results

In order to ensure identification of the major infochemical, we used a bioassay-guided approach. DVM was assessed using a well-established indoor setup in which daytime residence depth in a temperature stratified water column is determined as a proxy for DVM of Daphnia (Brzeziński and von Elert, 2015; Loose et al., 1993von Elert and Pohnert, 2000). It has earlier been shown that the DVM-inducing activity of fish incubation water can be efficiently extracted by lipophilic solid-phase extraction, whereas solid-phase extracts of control water were not active (von Elert and Loose, 1996). We followed the activity in such an extract of fish incubation water (EFI) of roach (Rutilus rutilus, Cyprinidae) through separation by HPLC and by investigating fractions that cover the whole chromatogram. EFI was found to be active, as it induced a significant shift in the daytime residence depth of Daphnia. Fractionation of EFI by HPLC yielded six fractions (Figure 1A), of which only fraction three proved to induce DVM (Figure 1B, Figure 1—source data 1).

Figure 1 with 1 supplement see all
Chromatogram and biological activity of extracted fish incubation water (EFI).

(A) Chromatogram of EFI after separation on a 250 mm x 4 mm reversed phase column (Nucleosil 100–5 C18, Macherey-Nagel, Düren, Germany) using the ammonium acetate buffered mobile phases A H2O and B acetonitrile/MeOH (13:6, (v/v) with the portion of B increasing over time. Vertical lines and numbering indicate collection of fractions 1–6. (B) Behavioral response of Daphnia magna to extract of fish incubation water (EFI) and fractions thereof. The control contains the same volume of pure organic solvent as tested from EFI and its fractions. Mean Daphnia daytime residence depth (± SD, n = 4). Different capital letters indicate significant differences among treatments after one-way ANOVA followed by Tukey’s HSD test. Statistical results are summarized in Figure 1—source data 1.

https://doi.org/10.7554/eLife.44791.002

Since the kairomone has been shown to be an anion (von Elert and Loose, 1996), LC high-resolution electrospray mass spectrometry (HR-ESI-MS) was performed in the negative ionization mode. Mass data extracted from the time window corresponding to the active fraction 3 (11–14 min), indicated that in this active fraction the most abundant ion had a m/z of 531.29993 corresponding to the deprotonated molecule [M– H], which led to Mcalc. = m/z 532.3070 (Figure 2A). The collision-induced fragmentation of [M– H] resulted in the detection of the precursor ion and a product ion with a mass of m/z 96.95996 (Figure 2B) corresponding to the monoisotopic mass of hydrogen sulfate (HSO4calc=m/z 96.96010, ∆ = –1.44 ppm), thus indicating the presence of a sulfate group. The tool sCLIPS included in the software MassWorks (cerno BIOSCIENCE) was used to predict the corresponding sum formula of the detected molecule based on the detected peak of its monoisotopic mass as well as its isotope pattern. The predicted sum formula for [M-H]- was C27H48O8S. A data base search using the platform PubChem yielded as best hit cyprinol sulfate (CPS), a known bile compound of fish (Hagey et al., 2010), as a candidate compound for the DVM-inducing activity. This finding was well in accordance with the detected sulfate group.

MS data of the biologically active HPLC fraction.

(A) ESI-MS spectrum extracted from the time window corresponding to the active fraction three in Figure 1 (11 - 14 min) measured in the negative ionization mode. m/z 531.29986 [M–H] indicates the presence of 5α-cyprinol sulfate (α-CPS), and m/z 498.28981 [M–H] (see insert) indicates the presence of the conjugated bile acid taurochenodeoxycholic acid (TCDCA). (B) Negative ionization MS-MS of m/z 531.29986 [M–H]: 96.95996 (HSO4calc=m/z 96.96010, ∆ = –1.44 ppm), which indicated the presence of a sulfate group in m/z 531.29986 [M–H].

https://doi.org/10.7554/eLife.44791.006

Subsequent search for other known bile compounds of fish in the active fraction three revealed the presence of taurochenodeoxycholic acid (TCDCA, Figure 2A, insert, [M-H]-=m/z 498.28981, C24H44NO6S, Mcalc = m/z 499.28981, ∆=1.8 ppm).

As CPS and TCDCA are known constituents of fish bile (Hagey et al., 2010), we tested bile, which was obtained from the gallbladder of common carp (Cyprinus carpio, Cyprinidae) and rainbow trout (Oncorhynchus mykiss, Salmonidae) for DVM induction in Daphnia. Dilutions of extracted bile of both species corresponding to similar CPS concentrations as found in EFI (carp: 1 and 2 nM, trout: 0.4 and 3.9 nM, EFI: 1 nM) proved to induce DVM with amplitudes that were statistically not distinguishable from those induced by EFI (Figure 3—figure supplement 2 and Figure 3—figure supplement 2—source data 1).

When we compared chromatograms of EFI and the fish bile samples, identical retention times, high-resolution m/z ratios and fragmentation patterns after MS-MS confirmed that α-CPS and TCDCA were also present in carp bile and trout bile (Figure 3, Figure 3—figure supplements 1 and 2). It is well known, that 5α-cyprinol sulfate (α-CPS) constitutes the major bile compound in cyprinid fish (Hagey et al., 2010). Since it is not commercially available, we have isolated α-CPS on a semi-preparative scale from gallbladder of common carp (Cyprinidae) and determined its structure by NMR to be α-CPS in a previous study (Hahn et al., 2018). Hence we conclude that m/z 531.29986 indicates the presence of α-CPS in EFI. The chemical identity of α-CPS and TCDCA was further confirmed by chromatography of reference compounds, which showed retention times and high-resolution m/z values that matched those in the chromatogram of EFI (Figure 3). We conclude that the major constituent of the only active fraction in EFI was the bile salt α-CPS. One further abundant bile salt in this fraction was TCDCA. While the ion detected at 2,04 min is of unknown identity (Figure 3C), we speculate that the second peak in the EIC of m/z = 531.29986 at 2.38 min (Figure 3D) is the isomer β-cyprinol sulfate.

Figure 3 with 2 supplements see all
Retention times of selected bile salts in extracted fish incubation water and fish bile.

Extracted ion chromatograms of cyprinol sulfate ([M]calc=m/z 532.3070; black line) and the conjugated bile acid taurochenodeoxycholic acid (TCDCA) ([M]calc=m/z 498.2889; dashed line) after ESI-MS in the negative ionization mode, which were present in the active fraction of extracted fish incubation water (Figure 2A&B). (A) pure TCDCA and (B) reference substances for 5α-cyprinol sulfate from Hahn et al. (2018) (5α-CPS). Based on identical retention time with B, α-CPS purified from carp bile and due to MS/MS (Figure 3—figure supplement 1) 5α-CPS is present in extracted fish incubation water (EFI) from Rutilus rutilus (C) and in extracted bile from Oncorhynchus mykiss (D) and from Cyprinus carpio (E). Relative intensities, with the highest set to 100%, are depicted.

https://doi.org/10.7554/eLife.44791.007

α-CPS isolated from bile of common carp was used for dose response experiments for the induction of DVM (Figure 4) after checking its purity (Figure 4—figure supplements 1 and 2). α-CPS induced the same predator-avoidance behavior as extracted fish incubation water (EFI) at concentrations ≥ 100 pM (Figur 4A, Figure 4—source data 2). TCDCA proved to be active at ≥20 µM only (Figure 4B, Figure 4—source data 3). As a possible microbial degradation product of TCDCA , we further tested chenodeoxycholic acid (Shimada et al., 1969) which proved to be inactive with respect to DVM-induction in concentrations ≤ 25 µM (Figure 4C, Figure 4—source data 4). In other words, Daphnia were five orders of magnitude more sensitive to α-CPS than to TCDCA, while no DVM induction was found in response to chenodeoxycholic acid (CDCA), the deconjugated form of TCDCA.

Figure 4 with 3 supplements see all
Effects of extracted fish incubation water (EFI) and of increasing concentrations of selected bile salts.

(A) 5α-cyprinol sulfate (5α-CPS), (B) taurochenodeoxycholic acid (TCDCA), and. (C) chenodeoxycholic acid (CDCA) on behavioral responses of Daphnia. Controls contain equivalent volumes of pure organic solvent. Mean Daphnia daytime residence depth (± SD, n = 3–4) is depicted. The day:night cycle was 16:8 hr. Different capital letters indicate significant differences among treatments (one-way ANOVA followed by Tuke’s HSD test). Statistical results are summarized in Figure 4—source data 24.

https://doi.org/10.7554/eLife.44791.012

We quantified α-CPS and TCDCA in extracted fish incubation water (EFI, Figure 4—figure supplement 3). As 20 µl of EFI are derived from 1 L of fish incubation water, this volume was dissolved per liter to obtain biologically active water in the bioassay. The actual concentration of α-CPS in biologically active water was 1.03 ± 0.08 nM (mean ± SD, n = 3) and that of TCDCA was 0.14 ± 0.01 nM (mean ± SD, n = 3). Hence the concentration of α-CPS was 7.5-fold higher than that of TCDCA. This concentration of α-CPS in biologically active water clearly exceeded the threshold concentration for biological activity of 100 pM, whereas TCDCA was present in concentrations that were more than five orders of magnitude below the activity threshold concentration of 20 µM. We therefore conclude that α-CPS is the only tested bile salt that is relevant for the induction of DVM in Daphnia in response to fish.

Discussion

We identified an infochemical that Daphnia uses to perceive the risk of being preyed upon by planktivorous fish. We show that the induction of DVM in Daphnia is dependent on a particular bile compound from fish, that is 5α-cyprinol sulfate (α-CPS), which is active already at a concentration of 100 pM. The identification of α-CPS is the result of a bioassay-guided approach in which we have targeted the main activity throughout all purification steps. Hence, α-CPS constitutes the major activity in fish incubation water. Although another bile compound (TCDCA) was found in the active fraction obtained by HPLC, only α-CPS was present in sufficiently high concentrations to be biologically active. This is due to two factors: (i) Daphnia is five orders of magnitude more sensitive to α-CPS than to TCDCA and (ii) the concentration of α-CPS in extracted fish incubation water is approximately sevenfold higher than that of TCDCA. This substantially higher concentration of α-CPS is well in accordance with the findings that α-CPS constitutes the major bile salt in cyprinid bile (Hofmann et al., 2010).

Concerning the discussion of the estimated threshold concentration of α-CPS for DVM induction one methodological detail has to be considered. α-CPS present in EFI was quantified as equivalents of cholesteryl sulfate, since there has not been enough material of α-CPS to conduct an absolute gravimetric quantification. The quantification of α-CPS by means of a calibration curve of cholesteryl sulfate can however be considered quite accurate since both molecules propably result in comparable detector responses of the ESI-MS for the following reasons: i) both α-CPS and cholesteryl sulfate carry a sulfate group and, without prior ionization, are already negatively charged when reaching the ESI source. Different ionizabilities of both analytes can thus be excluded as source of inaccuracy. ii) The m/z ratios of α-CPS and cholesteryl sulfate are so close to each other (m/z = 465.3039 vs. m/z = 531.2992), that tuning of the mass spectrometer’s ion optics will not differently affect the detectability of either compound. iii) Just like the m/z ratios, the molecular masses of the molecules are comparable as well, so that differences with respect to spray formation in the ESI source are probably of minor importance. In conclusion, it is reasonable to assume that quantification of α-CPS as equivalents of cholesteryl sulfate results in minor inaccuracies only.

α-CPS matches perfectly the previously published chemical characteristics for the DVM-inducing fish kairomone of roach (Rutilus rutilus) and other cyprinid fishes (Loose et al., 1993; von Elert and Loose, 1996). Its molecular mass is close to 500 Dalton, it contains hydroxyl and no amino groups, has a strong negative charge and, as predicted, α-CPS is free of olefinic bonds. The anionic character and the hydroxyl groups make α-CPS a good infochemical in water, as they render the molecule water soluble so that it can well diffuse after release by the predator.

α-CPS, a sulfated bile alcohol, belongs to the bile salts, which are metabolites of cholesterol in vertebrates with the primary function to emulsify dietary fats and to facilitate their intestinal absorption (Maldonado-Valderrama et al., 2011). Although bile salts are an essential component of the enterohepatic circulation, fish excrete bile salts via the intestine, the gills and the urinary tract (Buchinger et al., 2014). Excreted fish bile salts are often highly water soluble due to conjugation with a sulfate (in case of bile alcohols), or with taurine (in case of bile acids). Furthermore, bile salts are generally stable as they must be resistant to digestion within an organism, which makes conjugated bile salts ideal infochemicals. Corroborating results for other bile salts (Zhang et al., 2001), we here demonstrate the release of α-CPS by starved fish. Hence, the induction of DVM by fish does not require successful hunting of fish. The fact that bile salts are essential in fish metabolism explains why, from an evolutionary perspective, fish have not stopped to release bile salts despite its disadvantage for the emitter organism.

The DVM-inducing kairomone that we report here is to our knowledge the first identification of an aquatic kairomone that mediates predator-prey interactions between zooplankton and fish. The other few cases in zooplankton, for which chemical cues have been identified, are interactions of herbivorous zooplankton with its phytoplankton prey (Selander et al., 2015; Uchida et al., 2008) and with an invertebrate predator (Weiss et al., 2018): Copepods were shown to release copepodamides, a class of taurine containing polar lipids, which leads to an increased toxicity in their dinoflagellate prey (Selander et al., 2015); Daphnia have been reported to release an alkylsulfate into the water that induces the formation of protective colonies in the green alga Acutodesmus obliquus (Uchida et al., 2008), and larvae of Chaoborus sp. release fatty acids bound to a particular amino acid, which lead to changes in morphology in Daphnia pulex (Weiss et al., 2018). Among benthic animals a predatory crab has been shown to release two pyridin-metabolites that induce defensive behavior in a prey crab (Poulin et al., 2018). The sterane core of α-CPS renders it partly lipophilic, and causes together with the sulfate group the overall amphipathic nature of α-CPS. Sulfate or sulfonium groups are present in several of the compounds known in chemical communication of zooplankton (Selander et al., 2015; Uchida et al., 2008) and as well in the sex pheromone of lamprey (Li et al., 2002).

We accomplished the identification of α-CPS as the DVM-inducing kairomone using incubation water from Rutilus rutilus (Cyprinidae). The family of Cyprinidae is with more than 2000 species the largest family of fishes, and dominates freshwater habitats around the world with the exception of South America and Australia, and a considerable number of species is found in brackish water (Hastings et al., 2015). In line with the high diversity of cyprinid fish, incubation water of many cyprinid species has been demonstrated to induce DVM in Daphnia (Lass and Spaak, 2003), and we have shown that the chemical characteristics of the kairomone are identical among different cyprinid species (von Elert and Loose, 1996) which matches the finding that α-CPS is the dominant bile salt in the Cypriniformes (Hagey et al., 2010).

We further demonstrate that the induction of DVM by TCDCA, a representative for taurine-conjugated C24 bile acids, required substantially higher concentrations (≥20 µM, which equals ≥10 mg dissolved organic carbon/L) than induction by α-CPS. The observation that free bile acids occur in fish feces suggests a low degree of bacterial deconjugation of conjugated bile acids by gut bacteria (Philipp, 2011). However, such bacterial deconjugation of TCDCA would not release DVM-inducing activity, as the resulting free C24 bile acid CDCA was even less active than TCDCA. Neither this finding nor the fact that α-CPS, a fish metabolite that has not been microbially modified, constitutes the major kairomone in fish incubation water support the earlier suggestion that bacteria are involved in the production of the kairomone (Ringelberg and Van Gool, 1998).

As well non-cyprinid fish species like perch, stickleback (Perciformes) and pike (Esociformes) induce DVM in Daphnia (von Elert and Pohnert, 2000), and the chemical characteristics of kairomones from pike and stickleback match those of cyprinids (von Elert and Pohnert, 2000), which suggests that the kairomones of non-cyprinid and cyprinid species are similar and perhaps identical. However, a general pattern that emerges from a comprehensive analysis of bile salt patterns in fish indicates that α-CPS is confined to Cypriniformes and absent in the fish orders Perciformes, Esociformes and Salmoniformes. Yet, we here demonstrate the presence of α-CPS in bile of rainbow trout (Oncorhynchus mykiss), a representative of Salmoniformes. Still, this finding is in accordance with the general bile salt pattern in fish, as this pattern considers only bile salt types present at 5% or greater of the bile salt pool (Hagey et al., 2010). Furthermore, the authors argue, that the production of bile alcohols such as α-cyprinol (a C27 bile alcohol) and their conjugation to sulfate is regarded as an evolutionarily ancient bile salt profile (Hagey et al., 2010). All evolutionarily more recent bile salt patterns of fish seem to have evolved from the putatively ancestral pattern of C27 bile alcohol production by subsequent enzymatic modifications of them (Hagey et al., 2010; Hofmann et al., 2010). In line with this bile of many ray-finned fish species still contains traces of the biosynthetic precursor C27 bile alcohol though their bile is dominated by conjugated C24 bile acids (Hagey et al., 2010). We deduce that even in fish orders with a more recent bile acid pattern, α-CPS may be present in traces (i.e. <5%) as we have demonstrated for salmon, although this remains to be demonstrated for example for stickleback and Perciformes.

Our findings thus suggest that Daphnia evolution has selected for a high sensitivity to the evolutionarily oldest bile product of fish, that is α-CPS, and that this has proven to be an evolutionarily stable strategy as evolutionarily more recent fish bile patterns still contain traces of α-CPS. Thus, by sensing a single bile compound only, Daphnia are able to perceive the presence of fish across very different orders (Hagey et al., 2010). Future research will have to prove that 5α-CPS or other, hithertounidentified compounds, serve as the DVM-inducing kairomone as well in non-cyprinid fish orders.

The identification of α-CPS as the DVM-inducing kairomone in Daphnia allows for the assessment of its in situ concentration in space and time and thus to understand trait-variation in Daphnia and within-lake variations of DVM-amplitudes. The finding that a bile salt from fish serves as an infochemical might stimulate testing for a role of these compounds in the marine environment, where similar patterns of DVM of zooplankton are common. Still, although cessation of DVM in marine copepods in the absence of fish has been shown (Bollens and Frost, 1991), its induction in the presence of fish could not be demonstrated (Bollens and Frost, 1991). Similarly, a potential role of bile salts from fish in other anti-fish defenses in freshwater invertebrates remains to be tested, for example in changes in Daphnia life-history (Stibor, 1992) or morphology (Tollrian, 1994), in the reduced pigmentation of copepods in the presence of fish (Hansson, 2000) or in mediating effects of fish on the oviposition site of female Chaoborus (Berendonk, 1999). Knowing the kairomone now allows for identification of the α-CPS binding receptor in Daphnia in order to understand the rapid evolution of DVM in response to the absence/presence of fish (Cousyn et al., 2001).

Materials and methods

Bioassay

We utilized a plankton organ (Loose et al., 1993) for the assessment of the vertical daytime distribution of daphnids with a setup according to Brzeziński and von Elert (2015). The standardized experimental design has been shown to be a reliable indicator for the assessment of the kairomone, as the daytime depth determined on day 3 of the bioassay is a function of kairomone concentrations (Loose et al., 1993). Briefly, plexiglas tubes (length: 1 m long, volume: 200 mL, stoppered at the bottom) were placed vertically in a thermally stratified water bath and illuminated from the top (photoperiod 16L:8D). The thermal gradient in the organ (Figure 1—figure supplement 1) simulated a thermally stratified lake in summer. The tubes were either filled with control tap water to which the respective volumes of solvents had been added ('Control') or with control tap water to which different extracts had been added. The green alga Acutodesmus obliquus was used as food at saturating concentrations. Each treatment was replicated four- or fivefold with replicates and treatments being randomized.

Cohorts of second, third or fourth clutch neonates of Daphnia magna clone B, which has been isolated from a lake where it coexisted with fish (Lampert, 1991), were raised in tap water at saturating concentrations of A. obliquus until day 5 and then used to initiate the experiments. Five randomly chosen individuals of D. magna were transferred to each tube of the plankton organ and were fed with A. obliquus to a concentration of 2 mg C L−1 every day. The vertical position of the animals in the tubes was determined by visual inspection with 5 cm accuracy. The mean daytime residential depth of the animals on the third or fourth day of incubation were taken as the measure of activity (for further details see Loose et al., 1993).

Statistics

Data on daytime residential depth of Daphnia were analyzed for treatment effects by one-way ANOVA and Tukey's HSD posthoc test, if data met the assumption of homoscedasticity (Levene's test). Differences between treatments were considered to be significant at p<0.05. Data from independent experiments were pooled if daytime depth for control and for EFI did not differ. Statistical analyses were computed by using the software R version 3.3.3 (R Core Team 2017) and the packages agricolae version 1.2–8 (2017) and car (2011).

Kairomone extraction

The enrichment of fish incubation water was carried out for incubation water of Rutilus rutilus. Six pre-starved (24 hr) individuals (body size 10–20 cm) were incubated in 16 L of tap water at 18°C for 24 hr. The water was removed and filtered <0.65 μm. The kairomone was enriched from the water by C18 solid-phase extraction (SPE) (Mega Bond Elut, C18-bonded silica, mass: 75 g, Agilent Technologies) as according to von Elert and Loose (1996). Briefly, 4 liters of the incubation water were adjusted to 1% methanol and passed through the cartridge. After a washing step with 1% methanol, the cartridge was eluted with 200 mL of methanol, and this eluate was evaporated to dryness and re-dissolved in methanol to yield 80 µL of extracted fish incubation water (EFI). It has been shown earlier that controls for C18-SPE do not affect the daytime position of D. magna (von Elert and Loose, 1996).

Extraction of fish bile and purification of 5α-cyprinol sulfate

Gall bladder of Oncorhynchus mykiss (rainbow trout) was homogenized in methanol with a pestle and then centrifuged. After quantification of the α-CPS content in the particle-free supernatant by LC-MS, aliquots were used in the bioassay that resulted in 0.4 and 4 nM α-CPS. These concentrations were close to the α-CPS concentration in active EFI (1 nM). Carp bile was extracted from the gall bladder, and bile salts were extracted with modifications as according to Denton et al. (1974). 0.5 mL of bile were extracted by adding 10 mL of a hot 95:5 (v/v) mixture of ethanol and methanol and subsequent vortexing. After cooling, the supernatant was separated by centrifugation and diluted with ultrapure water to an alcoholic concentration of 10% and then subjected to a C18 solid-phase cartridge (Bond Elut, C18, Agilent Technologies). The activated solid phase was preconditioned with 10% methanol, and the processed bile sample (corresponding to 0.5 mL of bile) was passed through the cartridge. After washing (10% methanol), the cartridge was eluted with methanol. The eluate was evaporated to dryness and dissolved in 1.5 mL methanol (extracted carp bile) corresponding to a 1:3 dilution of the raw bile. α-CPS was purified from this extract by HPLC fractionation as according to Hahn et al. (2018). While 20% of the solvent leaving the LC column was transferred to the ESI-MS, the remaining 80% were at the same time collected by a fraction collector. In this way a fraction which contained α-CPS only was obtained. To eliminate co-eluting taurine-conjugated bile acids, the extracted carp bile was digested by choloylglycine hydrolase prior to chromatography. The collected fraction containing purified α-CPS was subjected to solid-phase extraction to remove the mobile HPLC phase. The identity of the isolated α-CPS was confirmed by NMR (Hahn et al., 2018), and its purity was demonstrated by liquid chromatography coupled to ESI-MS. LC-MS was performed as presented below in ‘Identification of bile salts’ using a reversed-phase (12.5 × 2 mm Nucleosil C18, 100–3, Macherey-Nagel, Düren, Germany).

Fractionation of extracted fish incubation water

EFI was separated into six fractions by HPLC using the mobile phases water (A) and a mixture of acetonitrile (VWR, Radnor, Pennsylvania) and methanol (VWR, Radnor, Pennsylvania) (13:6, v/v) (B). Both solvents were made up to 0.015% (v/v) formic acid (Honeywell, Morristown, USA) and 10 mM ammonium acetate (Merck, Darmstadt, Germany). The following solvent gradient was applied to a 250 mm x 4 mm column (Nucleosil 100–5 C18, Macherey-Nagel, Düren, Germany) at a flow rate of 1 mL/min with the column oven set to 30°C: 0 min: 35% B, 1.5 min: 35% B, 2.1 min: 40% B, 9 min: 45% B, 9.6 min: 55% B, 16.5 min: 65% B, 19.5 min: 100% B, 25.5 min: 100% B, 25.8 min: 35% B, 33 min: 35% B. 80% of the HPLC-derived eluate were collected by a fraction collector, while 20% were analyzed by MS. Chromatography and high-resolution mass spectrometry were conducted using an ultra-high pressure liquid chromatography (UHPLC) system (Thermo Fisher) with an Accela 1250 psi pump coupled with an ExactiveOrbitrap mass spectrometer (MS). Analytes were ionized by ESI in the negative mode. Ions within a mass range from m/z 120 to 1500, were detected at a frequency of one scan per second. The flow rate of the sheath and the aux gas nitrogen were set to 35 and 5 arbitrary units of the device. Spray voltage: 4.3 kV, capillary voltage −60 V, tube lens voltage −200 V, skimmer voltage −20 V, capillary temperature 325°C. The ExactiveOrbitrap mass spectrometer was calibrated once a day with the Pierce LTQ ESI negative calibration mixture. The instruments were controlled using the Xcalibur software (Thermo Scientific).

Quantification of bile salts

Bile salts were quantified by HPLC coupled to MS as described above (Fractionation of EFI). Taurochenodeoxycholic acid (TCDCA) was quantified by the standard addition method (Figure 4—figure supplement 3A), while the concentrations of α-CPS were derived from a calibration curve using the purchasable cholesteryl sulfate (Figure 4—figure supplement 3B), whose molecular structure suggests a similar detectability by ESI-MS. The detected MS signals of all analytes were normalized to the detected signal of glycocholic acid, which was added to the samples as standards prior to the LC-MS analysis to account for variability of the mass spectrometer's sensitivity. All analytes were measured in the matrix of EFI in triplicates to compensate for its effects on MS measurements. The reference compounds sodium taurochenodeoxycholate (CAS:6009-98-9), sodium cholesteryl sulfate (CAS:2864-50-8), chenodeoxycholic acid (CAS: 474-25-9) and glycocholic acid hydrate (CAS:1192657-83-2) were of >95% purity and obtained from Sigma-Aldrich. The evaluation of the chromatograms was performed using the Xcalibur software (Thermo Scientific).

Identification of bile salts

Bile salts were identified by LC-MS taking into account their exact m/z ratios and retention times. For the identification of CPS, additional MS/MS analyses were performed. Chromatography was performed on an UltiMate 3000 UHPLC system (Thermo Fisher). Analytes were separated on a reversed-phase column (BEH C8, 100mmx2.1mm, Acquity), with the column oven adjusted to 60°C. The mobile phase consisted of two solvents: (A) ultrapure water and (B) a mixture of acetonitrile and 2-propanol (both VWR, Radnor, Pennsylvania) (7/3, v/v). Both solvents contained ammonium acetate (Merck, Darmstadt, Germany) at a concentration of 10 mM added in form of a 1 M stock solution (1%, v/v) and acetic acid (VWR, Radnor, Pennsylvania) at a concentration of 1% (v/v). For chromatography, the following gradient was applied: 0 min: 40% B, 1 min: 40% B, 8 min: 75% B, 9 min: 100% B, 12.1 min: 40% B, 14 min: 40% B, 16 min: 40% B. Mass analysis was conducted on a Q Exactive Hybrid Quadrupole-Orbitrap (Thermo Fisher) equipped with an ESI source. Negative ionization was achieved by application of 2.5 kV. Sheath, auxiliary and sweep gas flow rates were adjusted to 50, 13 and 3 arbitrary units of the device. The capillary temperature was set to 263°C and the auxiliary gas heater temperature to 425°C. For the MS/MS measurements, negatively charged ions of the exact m/z of CPS (531.2997 m/z) were selectively fragmented applying collision energy of 50 eV. The evaluation of the chromatograms was performed using the Xcalibur software (Thermo Scientific).

Database research

For the identification of the most likely candidates of the chemical identity of the kairomone, we used the software MassWorks (Cerno Bioscience) to extrapolate from the measured exact masses and isotope patterns in the active HPLC fraction of EFI to sum formulas of the unknown molecules. A database search for molecules with these sum formulas was performed using the online platform ChemSpider.

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Decision letter

  1. Georg Pohnert
    Reviewing Editor; University of Jena, Germany
  2. Ian T Baldwin
    Senior Editor; Max Planck Institute for Chemical Ecology, Germany
  3. Georg Pohnert
    Reviewer; University of Jena, Germany

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "A bile salt from fish induces diel vertical migration in zooplankton" for consideration by eLife. Your article has been reviewed by three peer reviewers, including Georg Pohnert as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Ian Baldwin as the Senior Editor.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

The concept of a compound that has to be released by fish due to their metabolic activity and that signals their presence to few millimeter sized Daphnia who make avoidance reactions is intriguing. The manuscript describes therefore a breakthrough in aquatic kairomone research and clearly demonstrates the activity of a fish bile salt to induce diel vertical migration in Daphnia. It answers a long-standing question and opens up new perspectives in kairomone research. The knowledge about the active principle will further open up new perspectives in environmental and ecological research.

Essential revisions:

All reviewers agreed that the manuscript describes an important progress in the field and a solid piece of chemical ecology. However, several concerns about the presentation of the work were raised.

As can be seen in the detailed comments below all reviewers recommend a substantial focus of the discussion on core-results of the paper. Further, two reviewers recommend that the quantification of the kairomone should be better documented.

The title could be more precise; the compound that was elucidated could be mentioned – especially since a specific activity was observed.

The manuscript is already in itself convincing, but the discussion about anthropogenic infodisruption (Abstract and Discussion) seems to be farfetched. The specificity of the response and the discussion about amounts of potential info disruptors required to triggers a response makes it a quite speculative discussion. The core result is very strong; there is no need to extend it into speculation. I would suggest deleting the part of the Discussion paragraphs five and seven. Also, reviewer 2 states clearly "The infodisruption part is a bit overstated. If TCDCA from anthropogenic sources is really released with sewage in such high concentrations, it might induce DVM in the vicinity of the inflow. However usually it will be diluted and probably most lakes where sewage is flowing in contain fish anyhow. Thus, the proposed ‘far-reaching implications for ecosystem functioning and conservation management’ are not obvious."

Further in many places the authors discuss DVM in general, leading to the impression that 5α-CPS is, or might be, responsible for DVM worldwide. This is most probably not true. There is no indication that DVM in marine environments is chemically induced at all. Bollens and Frost tested chemical cues from fish in enclosures and did not find an induction of DVM. While it is widely accepted that DVM is an anti-predator strategy, behavioral plasticity has not been shown in the marine environment. Furthermore, there are no cyprinids in most marine environments. To emphasize my point, this does not mean that DVM in the marine environment is not induced by 5α-CPS – it simply needs to be shown.

The authors suggest that 5α-CPS is the only relevant fish kairomone and their results indeed indicate that also rainbow trout produce this kairomone. However, Hagey et al., 2010, presented a number of orders which do not produce 5α-CPS, including Perciformes and Esociformes, but also Salmoniformes. Perciformes and Esociformes have been used to induce depth selection behavior, e.g., Ringelberg and van Gool, 1998, and the authors induced with Esox lucius in an earlier study. Thus, there seems to be some deviating results which require some discussion. It might simply indicate that some clarification is necessary, or it might call for future studies. The authors discuss effects on the marine carbon pump and suggest that 5α-CPS is the responsible cue. This is overstating.

Quantification is certainly the weakest point of the study: First normalization to cholesteryl sulfate and CPS would require a response factor of both analytes or better a calibration curve. The sentence “As 20 µL […]” suggests that the concentration of DPS was determined by bioassays not be analytical methods? Given the experimental I thought quantification with normalization to cholesterol sulfate was done. Also, reviewer 2 states: “It is necessary to present the concentrations clearer. E.g., the size of the fish should be mention as the release of the kairomone might be correlated to fish size. How large where the fish for gall bladder extraction? How much bile has been extracted in Rainbow trout?”

The reviewers also discussed the validity of the term bioassay guided fractionation: The approach used is known as "bioassay-guided fractionation", by which compounds purified from fish exudates were tested for escape-inducing behavior, resulting in the eventual identification of a single molecule responsible for the observed effect on prey. The manuscript is largely focused on the process of identifying this molecule via a combination of chemical and behavioral experiments. The molecule, 5α-cyprinol sulfate, is a known product of the digestive systems of fish – a logical candidate for a cue that prey might use to sense their predators and respond defensively. Given the fact that the authors just published a paper on this compound and its NMR data makes us wonder if we are not rather dealing with a targeted study confirming the activity of a logical candidate. This would twist the whole presentation but maybe leave a better feeling after reading. The current manuscript represents one such study. The fact that the molecule the authors identified was previously known (albeit present and active at low nanomolar concentrations), makes the chemistry of the study a little less exciting, but the biological implications are still important.

Reviewer 3 is quite critical in this point: The paper is solid and clear, but it lacks the kind of general appeal that expected for publication in eLife. In particular, the Introduction is quite basic, written in a way that is unlikely to draw in strangers to chemical ecology. The Discussion is similarly restricted to similar systems (fish and plankton). The description of the impacts of predation, chemical cues, and predator escape strategies would need broadening and enriching to be appropriate for this kind of general journal. The chemistry-oriented results are not particularly accessible to a general biology readership. As currently presented, the work is more appropriate for a more specialized journal like J. Chem. Ecol. or one committed to publishing works about freshwater ecology such as Limnology and Oceanography. The section of the Discussion related to movement of zooplankton (and thus effects on their own prey, phytoplankton) and resulting impacts on nutrient cycling would be of particular interest to a limnological and oceanographic audience. After a discussion among the reviewers the study was considered to be important and appealing to warrant further consideration of a revised version of the manuscript but the concerns of reviewer 3 should be kept in mind during revision and re-drafting of the Introduction and Discussion.

Introduction section / Discussion paragraph two: The statement “virtually nothing is known about the chemical nature […]" might have been true 10 years ago, however there is substantial progress in the field and while only few compounds have been fully investigated (these should be cited) several studies address the compound classes, functional modes of action etc. involved. This part of the Introduction should be expanded. This is a common notion among the reviewers "The authors suggest that virtually no aquatic kairomone has been identified, ignoring the recent publication of the Chaoborus kairomone – another relevant kairomone for Daphnia with some similarities (Weiss et al., 2018)."

Results second paragraph: The calculation is incorrect. The value 532.29993 would suggest that H has a mass of 1. It is however 1.007 will lead to a calculated mass of 532.30693.

Results second paragraph: Why is a data base search required to determine the sum formula from a high res mass? The software of the instrument can simply calculate this.

In the same paragraph: The step from the high res mass to the structure is not clear. Is the entire process only based on this one MS?

Figure 3: Are there explanations for the peaks at 2.04 and 2.38?

Figure 4: The structures are the major result of the paper – they could be plotted bigger. The stereocenter on the C/D ring carbon of CPS should be indicated. Is there any information about the stereochemistry in the side chain?

Results paragraph five: The negative ionization ESI MS in Figure 4—figure supplement 1 can by no means be seen as a proof of purity. In fact, negative ionization only picks up a fraction of compounds in a mixture and in addition, the MS is not clearly indicating a pure compound. Show at least chromatograms in positive and negative ionization mode if no NMR data are available.

Discussion paragraph six and onwards: The Discussion loses focus. Results from freshwater systems (this paper) are mixed with marine/oceanographic facts and become very speculative. The paper should focus more. Constructing e.g. a story about ocean acidification from the results is not justified.

Also: Salmon louse responses to CPS have to be proven, deduction from an uncharacterized receptor sequence to a DVM response are not warranted. Basically, a speculation is built on a speculation.

Reviewer #2

Some studies are ignored that proved that UV-radiation is also inducing DVM in Daphnia, showing that it is additionally a strategy leading to avoidance of harmful irradiation (see. Rhode et al., 2001 or Leech and Williamson (2001, L and O).

Ringelberg and van Gool, 1998, suggest that bacteria are responsible at a certain step of the kairomone production as addition of an antibiotic prevented depth selection behavior. This might be addressed in the Discussion.

Specific comments:

I suggest changing the title to indicate that currently 5α-CPS has been shown to induce DVM in Daphnia magna and most likely in all daphnids. There is no indication that other genera respond to this kairomone.

"A bile salt from fish induces diel vertical migration in Daphnia"

https://doi.org/10.7554/eLife.44791.025

Author response

Essential revisions:

All reviewers agreed that the manuscript describes an important progress in the field and a solid piece of chemical ecology. However, several concerns about the presentation of the work were raised.

As can be seen in the detailed comments below all reviewers recommend a substantial focus of the discussion on core-results of the paper. Further, two reviewers recommend that the quantification of the kairomone should be better documented.

The title could be more precise; the compound that was elucidated could be mentioned – especially since a specific activity was observed.

We have accordingly extended/changed the title into: “5α-cyprinol sulfate, a bile salt from fish induces diel vertical migration in Daphnia” (see also below, response to specific comments from #2).

The manuscript is already in itself convincing, but the discussion about anthropogenic infodisruption (Abstract and Discussion) seems to be farfetched. The specificity of the response and the discussion about amounts of potential info disruptors required to triggers a response makes it a quite speculative discussion. The core result is very strong; there is no need to extend it into speculation. I would suggest deleting the part of the Discussion paragraphs five and seven. Also, reviewer 2 states clearly "The infodisruption part is a bit overstated. If TCDCA from anthropogenic sources is really released with sewage in such high concentrations, it might induce DVM in the vicinity of the inflow. However usually it will be diluted and probably most lakes where sewage is flowing in contain fish anyhow. Thus, the proposed ‘far-reaching implications for ecosystem functioning and conservation management’ are not obvious."

As suggested, we now have removed this aspect from the Discussion and also from the Abstract.

Further in many places the authors discuss DVM in general, leading to the impression that 5α-CPS is, or might be, responsible for DVM worldwide. This is most probably not true. There is no indication that DVM in marine environments is chemically induced at all. Bollens and Frost tested chemical cues from fish in enclosures and did not find an induction of DVM. While it is widely accepted that DVM is an anti-predator strategy, behavioral plasticity has not been shown in the marine environment. Furthermore, there are no cyprinids in most marine environments. To emphasize my point, this does not mean that DVM in the marine environment is not induced by 5α-CPS – it simply needs to be shown.

We fully agree, except for the very minor point that in Bollens and Frost, 1991, behavioural plasticity of the copepods was shown (the cessation of DVM after removal of predators), but, of course, no induction by fish-cues. We are no more referring to DVM on a global scale, but confine the introduction to DVM in Daphnia. In the Introduction we are now stating: “One classical example of behavioural predator avoidance is diel vertical migration (DVM) in the freshwater microcrustacean Daphnia, which play a key role in lakes and ponds, as they are major consumers of planktonic primary producers and important prey for higher trophic levels. DVM is a widespread predator avoidance behavior […]”.

The authors suggest that 5α-CPS is the only relevant fish kairomone and their results indeed indicate that also rainbow trout produce this kairomone. However, Hagey et al., 2010, presented a number of orders which do not produce 5α-CPS, including Perciformes and Esociformes, but also Salmoniformes. Perciformes and Esociformes have been used to induce depth selection behavior, e.g., Ringelberg and van Gool, 1998, and the authors induced with Esox lucius in an earlier study. Thus, there seems to be some deviating results which require some discussion. It might simply indicate that some clarification is necessary, or it might call for future studies. The authors discuss effects on the marine carbon pump and suggest that 5α-CPS is the responsible cue. This is overstating.

We have now excluded any reference to the marine carbon pump. We are now addressing this issue of potential deviations in the Discussion in detail. In short, we argue (i) that the general phylogenetic pattern did not consider bile acids with <5% abundance, (ii) that we have shown that salmon has traces of a-CPS though it should not have according to the general phylogenetic pattern, which (iii) renders it possible that a-CPS serves as DVM-inducing kairomone as well in non-cyprinid fish oders, which (iv) remains to be confirmed by future research (Discussion paragraph eight).

Quantification is certainly the weakest point of the study: First normalization to cholesteryl sulfate and CPS would require a response factor of both analytes or better a calibration curve. The sentence “As 20 µL […]” suggests that the concentration of DPS was determined by bioassays not be analytical methods? Given the experimental I thought quantification with normalization to cholesterol sulfate was done.

α-CPS was enriched from fish incubation water by solid-phase extraction. The extract (EFI) corresponded (concerning the volume) to 50,000 times concentrated fish incubation water (density: 3 fish in 8 L water). Thus dissolving 20µL of this 50,000 times concentrated fish incubation water extract in 1 L corresponded to 1 L of the original non-processed incubation water (ignoring losses during extraction) (subsection “Statistics”). The concentration of α-CPS in the extract (EFI) and (thereby also in the artificially produced fish incubation water: EFI + aged and filtered tap water) have not been determined by bioassay. Instead, α-CPS in EFI was determined analytically by LC-MS using a calibration curve for cholesteryl sulfate in the background/matrix of EFI to exclude effects of ion suppression that might be caused by the complex matrix of EFI. We now point out that we only quantified α-CPS as equivalents of cholesteryl sulfate:

“[…] the concentrations of α-CPS were derived from a calibration curve using the purchasable cholesteryl sulfate (Figure 4—figure supplement 6), whose molecular structure suggests a similar detectability by ESI-MS.”

We further discuss the accuracy of this approach:

“Concerning the discussion of the estimated threshold concentration of α-CPS for DVM induction one methodological detail has to be considered. α-CPS present in EFI was quantified as equivalents of cholesteryl sulfate, since there has not been enough material of α-CPS to conduct an absolute gravimetric quantification. The quantification of α-CPS by means of a calibration curve of cholesteryl sulfate can however be considered quite accurate since both molecules probably result in comparable detector responses of the ESI-MS for the following reasons: i) both α-CPS and cholesteryl sulfate carry a sulfate group and, without prior ionisation, are already negatively charged when reaching the ESI source. Different ionizabilities of both analytes can thus be excluded as source of inaccuracy. ii) The m/z ratios of α-CPS and cholesteryl sulfate are so close to each other (m/z=465.3039 vs. m/z=531.2992), that tuning of the mass spectrometer’s ion optics will not differently affect the detectability of either compound. iii) Just like the m/z ratios, the molecular masses of the molecules are comparable as well, so that differences with respect to spray formation in the ESI source are probably of minor importance. In conclusion it is reasonable to assume that quantification of α-CPS as equivalents of cholesteryl sulfate results in minor inaccuracies only.”

Also, reviewer 2 states: It is necessary to present the concentrations clearer. E.g., the size of the fish should be mention as the release of the kairomone might be correlated to fish size. How large where the fish for gall bladder extraction? How much bile has been extracted in Rainbow trout?

We acknowledge the point that size of fish might be related to release of the kairomone and have now included this information in Material and Methods “body size 10– 20 cm”. We do have information about the size of the fish used for gall bladder extraction (2 mL of bile from a trout of 1 kg weight). Please note, that not all bile extracted from a single fish but instead only aliquots of this were used. We therefore felt that neither information on body mass nor on extracted bile volume would make sense and have therefore not included this information. However, if the reviewers require it, we are of course willing to include the above give information on weight and total bile extracted from an individual fish.

The reviewers also discussed the validity of the term bioassay guided fractionation: The approach used is known as "bioassay-guided fractionation", by which compounds purified from fish exudates were tested for escape-inducing behavior, resulting in the eventual identification of a single molecule responsible for the observed effect on prey. The manuscript is largely focused on the process of identifying this molecule via a combination of chemical and behavioral experiments. The molecule, 5α -cyprinol sulfate, is a known product of the digestive systems of fish – a logical candidate for a cue that prey might use to sense their predators and respond defensively. Given the fact that the authors just published a paper on this compound and its NMR data makes us wonder if we are not rather dealing with a targeted study confirming the activity of a logical candidate. This would twist the whole presentation but maybe leave a better feeling after reading. The current manuscript represents one such study. The fact that the molecule the authors identified was previously known (albeit present and active at low nanomolar concentrations), makes the chemistry of the study a little less exciting, but the biological implications are still important.

We acknowledge this point of concern. Below we give a detailed chronology of our approach hoping to convince the reviewers that we have performed a full bioassay-guided approach; it has by no means been a targeted-approach! Briefly, earlier chromatography of the extracted fish incubation water (EFI;using water-MeOH only) resulted in not well-focused biological activity spreading across fractions “Pre”, “8” and “9” (Author response image 1), so that going for less steep gradients with even less focused activity was no option.

Author response image 1
Chromatogram and biological activity of extracted fish incubation water (EFI).

(a) Chromatogram of EFI after separation on a 250 mm x 4 mm reversed phase column (Nucleosil 100-5-C18, Macherey-Nagel, Düren, Germany) using the mobile phases A H2O and B MeOH with the portion of B increasing over time. The total ion current of ESI-MS measurements in the negative ionization mode are depicted. (b) Behavioral response of Daphnia magna to extract of fish incubation water (EFI) and fractions thereof. Fraction numbering corresponds to the time of the chromatogram [min], while fraction “Pre” contains the collected eluate from 0-8 min and fraction “Post” that from 13-25 min. The control contains the same volume of pure organic solvent as tested from EFI and its fractions. Mean Daphnia daytime residence depth (+/- SD, n=4). Different capital letters indicate significant differences among treatments one-way ANOVA followed by Tukey post-hoc). Statistical results are summarized in Supplementary file 1.

We therefore investigated mass-scans spanning these three fractions “Pre”, “8” and “9”, which yielded one common prominent ion with m/z = 531.2941, (see Author response image 2, 4, 6). Comparisons of these three mass scans to corresponding mass scans from a previous blank run confirm that two masses can be neglected since they are constitutively detected in the blank run and that of EFI: 1) m/z=68.9942 (highlighted in red) and 2) m/z=112.9836 (highlighted in green). Excluding these contaminants after blank subtraction resulted in m/z=531.2941 to be the most prominent detected ion in fractions “Pre” (Author response image 2) and “8” (Author response image 4) and the one with the third highest signal detected in fraction “9” (Author response image 6).

Author response image 2
ESI-MS data of the HPLC fractions leading to daytime depths of Daphnia magna which do neither statistically differ from the negative nor from the postive control and the corresponding spectra of a blank run.

ESI-MS spectrum extracted from the time window corresponding to fraction: Author response image 2“Pre”, Author response image 4“8”, Author response image 6“9” in Author response image 1 and Author response image 3, 5, 7 the corresponding mass spectra from a previous blank run measured in the negative ionization mode. MS/MS of m/z=531.3 lead to the appearance of a product ion of m/z=96.9598 which indicated the presence of a sulfate in the precursor ion (Author response image 8).

Author response image 3
Author response image 4
Author response image 5
Author response image 6
Author response image 7
Author response image 8
MS/MS of m/z =531.3 in fraction “8”.

Negative ionization MS/MS of m/z 531 [M– H]–:96.95979 (HSO4calc=m/z 96.96010, ∆ = –3.2ppm), which indicated the presence of a sulfate group in m/z 531.30048 [M– H].

Both pieces of information, the accurate mass and the presence of sulfate, yielded 5α-CPS as a candidate. Since 5α-CPS is not commercially available, we asked Dr. Hagey who kindly provided reference material, which actually was material that he had obtained from his predecessor, Dr. Haslewood, and which was without any information concerning its purity or even its structural elucidation. We received too little material for NMR of this putative reference compound. Although this material from Dr. Hagey showed identical HR-mass and retention time with our compound (see Figure 3B,C in the manuscript) we still wanted to be sure about its chemical identity. Therefore, we isolated new material form carp bile and confirmed its structure by NMR. At that time, we decided to publish this structural elucidation as a separate paper, since it was a confirmation of an already known structure. Then, when being sure, that m/z=531.2941 represents 5α-CPS, we screened for further bile salts in fractions “Pre”, “8” and “9” and discovered that additional bile salts were present.

From there on we optimized our chromatography for the analysis of bile compounds, then using a different solvent system. This new mobile phase allowed for much more focused biological activity and much better resolution of bile compounds than with water-MeOH as mobile phases. It is these results that we now present in the manuscript. We would like to emphasize that at all stages of the research (as well in the part depicted in the paper) all fractions were always subjected to bioassays and the major activity was always considered/investigated further. We strongly regard this as a bioassay-guided approach, and, in line with this, we claim to have identified the major DVM-inducing compound. At no stage this approach has been a targeted approach.

Reviewer 3 is quite critical in this point: The paper is solid and clear, but it lacks the kind of general appeal that expected for publication in eLife. In particular, the Introduction is quite basic, written in a way that is unlikely to draw in strangers to chemical ecology. The Discussion is similarly restricted to similar systems (fish and plankton). The description of the impacts of predation, chemical cues, and predator escape strategies would need broadening and enriching to be appropriate for this kind of general journal. The chemistry-oriented results are not particularly accessible to a general biology readership. As currently presented, the work is more appropriate for a more specialized journal like J. Chem. Ecol. or one committed to publishing works about freshwater ecology such as Limnology and Oceanography. The section of the Discussion related to movement of zooplankton (and thus effects on their own prey, phytoplankton) and resulting impacts on nutrient cycling would be of particular interest to a limnological and oceanographic audience. After a discussion among the reviewers the study was considered to be important and appealing to warrant further consideration of a revised version of the manuscript but the concerns of reviewer 3 should be kept in mind during revision and re-drafting of the Introduction and Discussion.

We appreciate this remark and have thoroughly incorporated the issues raised. However, we admit, that it is not entirely clear what is meant by ‘lacks the kind of general appeal that is expected for publication in eLife’. Lakes and ponds are the best available freshwater source on the Earth's surface. Lakes are valued as water sources and for fishing, water transport, recreation, and tourism and, due to their outstanding importance, issues of management of lakes and ponds are of pivotal importance. One tool for lake management is food chain manipulation, which aims at manipulating the interaction of Daphnia and fish. Our paper provides a fundamentally new understanding of the interaction of Daphnia and fish, which is key to the functioning and ecosystem services of lakes and ponds, and we therefore would like to emphasize that we regard our findings as highly appealing for more general journals as eLife. In response to concerns raised we have provided two new first paragraphs in the Introduction, in which we introduce the general importance of predation, the distinction of direct and indirect effects of predators and the importance of chemical signalling in predator-prey interactions. We hope that this will more appropriate to draw in general biologists into an issue of chemical communication. We have further condensed statements about the chemistry of known kairomones in aquatic chemical communication from formerly three different paragraphs to a single paragraph starting with “The DVM-inducing kairomone that we report here is to […]”. We have further removed all aspects of nutrient recycling from the Discussion in order to be more focused on the main contribution of this manuscript. And we have now included an introductory definition for kairomone: “Such chemical cues are termed kairomones, if they mediate a transfer of information among species that impart a benefit to the receiving organism while not being beneficial for the producer”.

Introduction section / Discussion paragraph two: The statement “virtually nothing is known about the chemical nature […]" might have been true 10 years ago, however there is substantial progress in the field and while only few compounds have been fully investigated (these should be cited) several studies address the compound classes, functional modes of action etc. involved. This part of the Introduction should be expanded. This is a common notion among the reviewers "The authors suggest that virtually no aquatic kairomone has been identified, ignoring the recent publication of the Chaoborus kairomone – another relevant kairomone for Daphnia with some similarities (Weiss et al., 2018)."

We acknowledge this point. First, we would like to add that Weiss et al., 2018 had already been mentioned in the letter to the editor, but unfortunately not been included in the manuscript. We apologize for this. In response to this issue we have now included the few chemically identified kairomones in aquatic plankton and briefly discuss their chemical features. We are now stating in the Introduction “[…] recently progress has been made by identification of infochemicals involved in predator-prey chemical communication (Selander et al., 2015 Weiss et al., 2018, Poulin et al., 2018)”

In the Discussion we are now referring to the few identified compounds in more detail (the papers of Selander et al., 2015, Weiss et al., 2018, Poulin et al., 2018 and Uchida et al., 2008) and discuss chemical similarities starting with “The other few cases in zooplankton, for which chemical cues have been identified […]”.

Results second paragraph: The calculation is incorrect. The value 532.29993 would suggest that H has a mass of 1. It is however 1.007 will lead to a calculated mass of 532.30693.

Yes, we agree. We changed the m/z from 532.29992 to 532.3070.

Results second paragraph: Why is a data base search required to determine the sum formula from a high res mass? The software of the instrument can simply calculate this.

Yes, indeed we calculated it using the tool sCLIPS included in the software MassWorks (cerno BIOSCIENCE). We mention the procedure in the Results: “The tool sCLIPS included in the software MassWorks (cerno BIOSCIENCE) was used to predict the corresponding sum formula of the detected molecule based on the detected peak of its monoisotopic mass as well as its isotope pattern. The predicted sum formula for [M-H]- was C27H48O8S. A data base search using the platform PubChem yielded as best hit cyprinol sulfate (CPS), a known bile compound of fish as a candidate compound for the DVM-inducing activity. This finding was well in accordance with the detected sulfate group.”

In the same paragraph: The step from the high res mass to the structure is not clear. Is the entire process only based on this one MS?

We agree. The structure was elucidated not only using the high res mass. Now, we present the procedure step by step in the Results (see the point above).

Figure 3: Are there explanations for the peaks at 2.04 and 2.38?

The identity of the ion detected at 2.04 min is unknown. The peak at 2.38 min is most probably derived from the isomer β-cyprinol sulfate, since Oncorrhynchos mykiss is known to synthesize β-bile alcohols (Hagey et al., 2010). There was no reference compound available to confirm its identity by retention times. We mention these peaks as follows:

“While the ion detected at 2.04 min is of unknown identity (Figure 3 D), we speculate that the second peak in the EIC of m/z=531.29986 at 2.38 min (Figure 3 E) is the isomoer β-cyprinol sulfate.”

Figure 4: The structures are the major result of the paper – they could be plotted bigger. The stereocenter on the C/D ring carbon of CPS should be indicated. Is there any information about the stereochemistry in the side chain?

Yes, we agree. We now also depict the missing protons, indicating the stereocenter on the C/D ring Carbon of CPS and enlarged the plots of all structures. (Figure 4) Unfortunately, there is no information available on the stereochemistry of the side chain.

Results paragraph five: The negative ionization ESI MS in Figure 4—figure supplement 1 can by no means be seen as a proof of purity. In fact, negative ionization only picks up a fraction of compounds in a mixture and in addition, the MS is not clearly indicating a pure compound. Show at least chromatograms in positive and negative ionization mode if no NMR data are available.

Yes, we agree. Following your request, we now replaced Figure 4—figure supplement 1 by chromatograms of the purified α-CPS in positive and negative ionization mode, as well as chromatograms of blank runs for comparison (Figure 4—figure supplement 1). Additionally, we inserted mass spectra spanning the chromatogram’s time window which is exhibiting peaks in the α-CPS but not in the blank runs to seek for impurities (Figure 4—figure supplement 2).

Discussion paragraph six and onwards: The discussion loses focus. Results from freshwater systems (this paper) are mixed with marine / oceanographic facts and become very speculative. The paper should focus more. Constructing e.g. a story about ocean acidification from the results is not justified.

Also: Salmon louse responses to CPS have to be proven, deduction from an uncharacterized receptor sequence to a DVM response are not warranted. Basically, a speculation is built on a speculation.

We agree and have removed both aspects from the Discussion.

Reviewer #2

Some studies are ignored that proved that UV-radiation is also inducing DVM in Daphnia, showing that it is additionally a strategy leading to avoidance of harmful irradiation (see. Rhode et al., 2001 or Leech and Williamson (2001, L and O).

Ringelberg and van Gool, 1998, suggest that bacteria are responsible at a certain step of the kairomone production as addition of an antibiotic prevented depth selection behavior. This might be addressed in the Discussion.

We are now addressing this aspect in the Discussion by stating:

“Neither this finding nor the fact that α-CPS, a fish metabolite that has not been microbially modified, constitutes the major kairomone in fish incubation water support the suggestion that bacteria are involved in the production of the kairomone (Ringelberg and van Gool, 1998).”

Specific comments:

I suggest changing the title to indicate that currently 5α-CPS has been shown to induce DVM in Daphnia magna and most likely in all daphnids. There is no indication that other genera respond to this kairomone.

"A bile salt from fish induces diel vertical migration in Daphnia"

Accordingly, we have changed the title into: “5α-cyprinol sulfate, a bile salt from fish, induces diel vertical migration in Daphnia”.

https://doi.org/10.7554/eLife.44791.026

Article and author information

Author details

  1. Meike Anika Hahn

    Aquatic Chemical Ecology, Department of Biology, University of Koeln, Koeln, Germany
    Contribution
    Data curation, Software, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing
    For correspondence
    meike.hahn@uni-koeln.de
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5090-0849
  2. Christoph Effertz

    Aquatic Chemical Ecology, Department of Biology, University of Koeln, Koeln, Germany
    Contribution
    Investigation, Methodology
    Competing interests
    No competing interests declared
  3. Laurent Bigler

    Department of Chemistry, University of Zurich, Zurich, Switzerland
    Contribution
    Resources, Methodology
    Competing interests
    No competing interests declared
  4. Eric von Elert

    Aquatic Chemical Ecology, Department of Biology, University of Koeln, Koeln, Germany
    Contribution
    Conceptualization, Resources, Data curation, Supervision, Investigation, Methodology, Project administration, Writing—review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7758-716X

Funding

No external funding was received for this work.

Acknowledgements

We are very thankful to L Hagey for providing α-CPS as a reference compound used in preliminary experiments and to P Fink for valuable comments on an earlier version of this manuscript. The authors acknowledge the significant contributions by the Biocenter-MS facility, University of Cologne.

Senior Editor

  1. Ian T Baldwin, Max Planck Institute for Chemical Ecology, Germany

Reviewing Editor

  1. Georg Pohnert, University of Jena, Germany

Reviewer

  1. Georg Pohnert, University of Jena, Germany

Publication history

  1. Received: December 29, 2018
  2. Accepted: May 2, 2019
  3. Accepted Manuscript published: May 2, 2019 (version 1)
  4. Version of Record published: June 11, 2019 (version 2)

Copyright

© 2019, Hahn et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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