OIKOS 88: 119–128. Copenhagen 2000

Predator specificity of kairomones in diel vertical migration ofDaphnia : a chemical approach

Eric von Elert and Georg Pohnert

Von Elert, E. and Pohnert, G. 2000. Predator specificity of kairomones in dielvertical migration of Daphnia : a chemical approach. – Oikos 88: 119–128.

Daphnia responds to chemical cues released by fish with diel vertical migration(DVM) as a behavioural predator avoidance. We used a bioassay to characterize thechemical nature of the kairomone. Cues released from stickleback (Gasterosteusaculeatus, Gasterosteidae) and a piscivorous pike (Esox lucius, Esocidae) wereenriched from incubation water by reversed-phase sorbent extraction and werereversibly inactivated by acetylation. HPLC yielded only one active fraction withidentical retention times for the kairomones of both species. Chemical features didnot differ from those previously reported for Cyprinidae, indicating that the activecompounds are very similar if not identical. From further investigation of thekairomone released by crucian carp (Carassius carassius, Cyprinidae), glucuronicacids and carboxy-, sulphate- and phosphate-groups can be excluded as essential forbiological activity. The response of Daphnia increased with increasing concentrationsof extracted kairomone. The kairomone was not released from mucus by digestionwith hyaluronidase. Adsorption of the kairomone to food particles seems to be ofminor importance suggesting that the cue is perceived as a freely dissolved molecule.

E. 6on Elert, Max Planck Inst. for Limnology, Postfach 165, D-24302 Plon, Germany(present address: Limnological Inst., Uni6. of Constance, D-78457 Konstanz, Germany[eric.6onelert@uni-konstanz.de]). – G. Pohnert, Max Planck Inst. for Chemical Ecol-ogy, Tatzendpromenade 1a, D-07745 Jena, Germany.

Prey organisms defend themselves by a variety of adap-tations against predators (Edmunds 1974, Endler 1986).These adaptations may be changes in morphology,physiology, life history or behaviour. Since changes inmany of these plastic traits are associated with costs,expression of inducible defences should rely on anaccurate assessment of the risk of predation and thus ofthe benefits of being defended. One way to assess therisk of predation is to trace chemical cues (kairomones,for terminology see Dicke and Sabelis (1988)) releasedby active predators, which function as proximate fac-tors for the onset of these defences. There is a rapidlygrowing number of reports of chemical communicationin pelagic environments (Larsson and Dodson 1993,Verity and Smetacek 1996, Kats and Dill 1998), inparticular on predator-prey systems. In pelagic freshwa-ter systems Daphnia is an important food source for fish

and the abundance of these herbivorous crustaceanshas major effects on structure and dynamics of thewhole food web (Lampert 1987). The effects of fishkairomones on Daphnia involve changes in life-historytraits (Machacek 1993, Stibor and Luning 1994), mor-phology (Tollrian 1994) and behaviour (Dodson 1988,Ringelberg et al. 1991).

Diel vertical migration (DVM) is a widespreadpredator-avoidance behaviour in zooplankton: the riskof daytime predation by visually orientated predators,namely fish, is reduced by residence of prey in the deeplayer of the water column during day. During nightanimals reappear at the surface to escape from lowtemperatures and low food levels in the hypolimnion.DVM can be chemically induced in Daphnia by preda-tor-associated cues (Dodson 1988). For the evolution ofa chemically inducible defence an unpredictably chang-

Accepted 15 July 1999

Copyright © OIKOS 2000ISSN 0030-1299Printed in Ireland – all rights reserved

OIKOS 88:1 (2000) 119

ing predation risk and a reliable chemical cue arerequired (Harvell 1986, Pijanowska 1993). For preyrelying on chemical assessment of predation risk thechemical cue is crucial in avoiding dispensable costs:DVM has inherent costs (Loose and Dawidowicz 1994)and being preyed upon is itself costly. The cue’s reliabil-ity is affected by how it behaves in water, which in turnis determined by the chemical nature of the molecule.The chemical nature of the DVM-inducing cue releasedby three cyprinid species (Leucaspius delineatus, Caras-sius carassius, Rutilus rutilus) has preliminarily beencharacterized as a low-molecular weight compound ofintermediate lipophilicity (Loose et al. 1993, Von Elertand Loose 1996). Separation by HPLC yielded only oneactive fraction with identical retention times for allthree cyprinid species, suggesting that the cues are verysimilar if not identical.

Here we investigate whether these chemical featuresare typical for kairomones released from Cyprinidaeonly or whether they are valid for DVM-inducing cuesreleased from fish of different families as well. Wetherefore preliminarily chemically characterized thecues released from pike (Esox lucius, Esocidae) andstickleback (Gasterosteus aculeatus, Gasterosteidae). Asan important step towards the identification of thekairomone we report further chemical features of thecyprinid-borne cue.

The DVM-inducing kairomone of three cyprinid spe-cies is readily dissolved in water due to the hydrophilicfeatures of the cue; extractability with C18-SPE indi-cated the presence of additional lipophilic groups in thecue (Von Elert and Loose 1996). It is imaginable thatthis lipophilic nature leads to adsorption of thekairomone to membranes, e.g. food particles, so thatthe cue is surface-associated rather than freely dis-solved. Since it is not known how the fish-borne cuesare perceived by Daphnia, we test the hypothesis thatthe cue becomes adsorbed to food particles and issensed via ingested food items.

The specificity of kairomones and alarm substancesin predator-prey interactions is reviewed briefly.

Methods

Bioassay

The characterization of the kairomone was performedby using a bioassay based upon the DVM behaviour ofsmall cohorts of Daphnia. The standardized experimen-tal design has been shown to be a reliable indicator forthe assessment of the kairomone: during night timeanimals reside in the epilimnion and daytime depthdetermined on day 3 and 4 of the bioassay is a functionof kairomone concentrations (Loose et al. 1993). Aclone of Daphnia magna was used as test animals. Itwas isolated from Großer Binnensee (Northern Ger-

many), where it coexists with fish (Lampert 1991). Theanimals were kept in filtered lake water (0.45 mm poresized membrane filters) and five 4-d-old animals wereused per treatment. They were incubated in Perspextubes (1 m length, 200 ml volume) in ‘DVM-medium’.DVM-medium was based on ultrapure water and con-tained CaCl2 (787 mM), NaNO3 (590 mM), MgSO4 (81mM) and KHCO3 (135 mM). The Perspex tubes wereplaced in a water bath with a defined temperaturestratification (20°C at the top, 10°C at the bottom). Thetubes were illuminated from the top (16:8 h day:nightcycle) and Scenedesmus acutus was added as food (2mg/l particulate organic carbon, initial concentration).The mean day-depth of the animals on the third day ofincubation was taken as the measure of activity (forfurther details see Loose et al. (1993)). Mean depthsdeeper than 60 cm (animals in the cold hypolimnion)indicated the presence of kairomone, whereas a meandepth above 30 cm indicated the absence of the cue.

Enrichment and chemical characterization

Artificial DVM-medium was used for control treat-ments. Water containing the kairomone was preparedas follows: an adult pike (Esox lucius, Esocidae, 40 cm)was pre-fed three adult Leucaspius delineatus once aweek and then put into 50 l of artificial medium for 24h. Two sticklebacks (Gasterosteus aculeatus, Gas-terosteidae) or two crucian carp (Carassius carassius,Cyprinidae) were fed with frozen chironomids once aweek and kept for 24 h in 10 l of artificial DVM-medium. Then the water was filtered through mem-brane filters (0.45 mm pore size) and used as ‘fish water’.It has previously been shown that the production of thekairomone does not depend on the feeding history ofthe fish (Loose et al. 1993).

The kairomone was enriched from holding water bysolid-phase extraction (SPE). C18-SPE cartridges (500mg, Analytichem Int.) were preconditioned withmethanol and ultrapure water 10 ml each prior toadding the sample. The pH of the sample was adjustedto 7.0 with 2 M HCl. Methanol was added to achieve a1% concentration and the resultant solution was passedthrough the cartridge. The eluate was collected. Theloaded cartridge was washed with 5 ml of ultrapurewater prior to elution of the isolate with 10 ml ofmethanol (eluent). Both the eluate and the eluent wereevaporated separately to dryness with a rotatory evapo-rator in order to remove organic solvents, resuspendedin artificial medium and tested for biological activity.Kairomone was enriched from 10 l of crucian carpincubation water using two C18-SPE cartridges (10 g),the cartridges were eluted with 50 ml of methanol each.The pooled eluate was evaporated to dryness and resus-pended in 1 ml of methanol.

120 OIKOS 88:1 (2000)

We used base-catalysed acetylation similar to Wattsand Kekwick (1974). An aliquot of C18-SPE enrichedactivity was gently evaporated to dryness. Then 800 mlof dried pyridine and 200 ml of acetic anhydrid wereadded and allowed to react for 6 h at 60°C. Thereaction was stopped by evaporating to dryness undernitrogen. The sample was resuspended in 1 ml ofmethanol, diluted with ultrapure water to a volume of100 ml, and extracted by C18-SPE using the eluent forthe bioassay. In order to make sure that the kairomonewas inactivated but not destroyed by acetylation, re-versibility of the derivatization was verified by alkalinesaponification. The acetylated cue was evaporated todryness, resuspended in 1 ml of 1 M NaOH, andincubated for 45 min at 80°C. Then the solution wasdiluted with ultrapure water to a volume of 50 ml,neutralized, and salts were removed by reextraction ofthe kairomone by C18-SPE, using the methanolic eluatefor the biotest.

EsterificationAn aliquot of C18-SPE enriched activity was deriva-tized using an excess of freshly prepared diazomethanein ether at room temperature (Blau and Halket 1993).After 30 min, the excess reagent and the solvent wereremoved under reduced pressure and the resulting oilyresidue was taken up with methanol.

20 ml of kairomone enriched by C18-SPE from 200ml of holding water was subjected to reversed-phaseHPLC (Knauer, modul 64) on a 250 mm×4.6 mmcolumn packed with Biosil (C18, 5 mm, Biorad, pre-column 20 mm×4 mm) and eluted with a linear gradi-ent of ultrapure water and methanol (0 min: 0%; 3 min:0%; 28 min: 100%; 36 min: 100% methanol) at a flowrate of 1 ml min−1. The chromatogram was recorded at254 nm (LKB-Pharmacia, model 2141) and fractions of4 ml were collected, evaporated and resuspended in 200ml of methanol. Aliquots of 20 ml were tested forbiological activity.

Enzymatic treatmentsAll enzymes were obtained from Sigman. 4.25 mg oflyophilized Driselase were suspended in 6 ml of 50 mMsodium-acetate buffer (pH 5.0) and 25 ml of C18-SPEenriched kairomone were added and incubated for 36 hat 30°C. Mucus from one crucian carp (5 cm length)was ultrasonicated in 40 ml of 300 mM sodiumphos-phate buffer. Six ml of this suspension were added to 60units of Hyaluronidase suspended in 6 ml buffer (75mM sodiumchloride, 20 mM sodiumphosphate pH 7.0,0.01% BSA) and incubated for 36 h at 30°C. AlkalinePhosphatase: 10 ml of enzyme suspension (80 units)were dissolved in 10 ml of a 50 mM ammonium sul-phate solution, pH 8.5 and 10 ml of C18-SPE enrichedkairomone were added and incubated for 4 h at 25°C.Sulphatase: 10 ml (25 units) of enzyme suspension weredissolved in 10 ml of a 50 mM solution of ammonium

sulphate, pH 5.0, and 10 ml of C18-SPE enrichedkairomone were added and incubated for 4 h at 25°C.b-Glucuronidase: 1000 units were suspended in 10 ml of4 mM phosphate buffer (pH 6.8). 10 ml of C18-SPEenriched kairomone were added and incubated for 4hat 25°C. After incubation with b-Glucuronidase andsulfatase the solutions were extracted with diethyletherby adding 5 ml of the organic solvent to the test tube,mixing (Vortex, 30 s) and subsequently removing theorganic layer. The extraction was repeated once. Allenzyme treatments were stopped by reextracting thekairomone by C18-SPE.

Treatment of food particles with kairomone

We tested for adsorption of highly concentratedkairomone to alga cells: volumes of 2 and 20 ml ofC18-SPE enriched kairomone were added to 2 ml of asuspension of S. acutus (1 mg particulate organic car-bon, POC) and incubated on a rotating shaking tablefor 3 h at 20°C. In order to remove non-adsorbedkairomone from the cells, the suspensions were dilutedin 100 ml of filtered lake water each and cells wereconcentrated by centrifugation (4000 g, 5 min) and thesupernatants discarded. The cells were resuspended in 2ml of filtered lake water and assayed for DVM-induc-ing kairomone. We investigated whether dissolved bio-logical activity is removed from water by adsorption toalga cells: 2 ml of C18-SPE enriched kairomone weredissolved in 100 ml of DVM-medium and S. acutus (1mg POC) was added. The suspension was stirred for 3h. After removal of the alga cells by centrifugation thesupernatant was assayed for DVM-inducing activity.

Concentration dependence

The effects of different concentrations of C18-SPE en-riched kairomone on the mean day depth was examinedby adding aliquots of kairomone enriched from incuba-tion water of Carassius carassius to 200 ml of controlwater (artificial DVM-medium). A no-fish control andseven different concentrations of kairomone were usedwhich corresponded to a range of densities from onefish per 0.4 l to one fish per 2000 l. We ran fivereplicates of all treatments and controls simultaneouslyin the same bioassay.

Results

Chemical characterization

Incubation water of Esox lucius and Gasterosteus ac-uleatus was biologically active and induced a mean daydepth of D. magna that was significantly deeper than

OIKOS 88:1 (2000) 121

Table 1. Comparison of biological activity of kairomones released from Esox lucius and Gasterosteus aculeatus after variouschemical treatments. Activity is given as mean day depth of Daphnia magna, maximum mean day depth (95 cm) indicatesmaximum biological activity (N=5). Incubation water or C18-SPE enriched kairomone was used for kairomone treatments,non-fish water was used for controls. Overall ANOVA: F11,48=47.1; PB0.0005. Asterisks indicate biological activity oftreatments which differ significantly from control (Tukeys’ test, **: pB0.005; ***: pB0.0005; ns: not significantly different).

Treatment Mean day depth [cm (1 SE)] and kairomone released by

Control Esox lucius Gasterosteus aculeatus

Incubation water 11.4 (2.91) ***80.8 (2.27) *** 93.6 (0.4)C18-SPE, methanol ***6.4 (2.36) 80.25 (7.75) *** 88 (0)Acetylation 7.8 (3.26) 12 (2) nsns 13.2 (7.09)Acetylation and saponification 27.2 (5.33) ***60 (8.94) ** 67.4 (9.55)

that of non-fish water (control) (Table 1). Whenaliquots of incubation water from pike and sticklebackwere subjected to non-polar C18-solid-phase extraction,biological activity was fully recovered from the C18-cartridges by elution with methanol (Table 1). Thisindicates quantitative extraction of the cue by thelipophilic sorbent in both cases. We therefore usedC18-SPE as a standard method for bulk enrichment ofthe DVM-inducing kairomone. Aliquots of this crudeextract were used for further investigations. The cuesare well dissolved in water which might be due to polarionic or nonionic groups. We tested by acetylation forpolar groups essential for activity. Acetylation ofaliquots of C18-SPE enriched activity from pike andstickleback led to a complete loss of activity (Table 1).Full activity was recovered after subsequent saponifica-tion, indicating that the cue had not been damaged bythe chemical procedure of acetylation itself. Hence apolar group that could be acetylated was essential forbiological activity. Since neither of the kairomones weredestroyed by the strong alkaline conditions of saponifi-cation, esters can be excluded as essential constituentsof the biological activity. When an aliquot of theC18-SPE enriched kairomone from pike and stickle-back was subjected to HPLC on a linear water-methanol gradient and fractions were tested forinduction of DVM, biological activity was confined tothe same fraction in both cases (Fig. 1). The activity ofthe active HPLC fraction corresponded to that of thealiquot prior to injection indicating that total activitywas recovered.

The investigated chemical features of the kairomonesreleased by E. lucius and G. aculeatus did not differ.Furthermore they were identical with the recently pre-liminarily characterized DVM-inducing kairomonesfrom three cyprinid fish species (Von Elert and Loose1996). On the basis of these findings we focused on oneof the cyprinid species, Carassius carassius, for furthercharacterizations of the kairomone, since this specieswas easy to keep at high densities for bulk productionof incubation water. Not only polar functional groupsbut also ionic groups can contribute to the polar natureof the DVM-inducing kairomones from pike, stickle-back and the three Cyprinidae. The kairomones from

the cyprinids were insensitive to proteases (Loose et al.1993) and showed no cationic character, but interactedwith anion exchangers (Von Elert and Loose 1996). Wetested for the presence of carboxy-, phosphate- orsulphate-groups by digestion of C18-SPE enrichedkairomone from crucian carp with different enzymes.After incubation with alkaline phosphatase, the activityremained significantly higher than controls (Table 2)indicating that a phosphate group was not an essentialconstituent of biological activity. Since conjugationwith sulphate or glucuronic acid are major metabolicpathways of formation of excretory metabolites in ver-tebrates (Urich 1990), we digested enriched kairomonewith sulphatase and glucuronidase. Digestion did notaffect activity (Table 2); hence, sulphate and glucuronicacid were no functional group of the cue that is essen-tial for biological activity. Still both functional groups

Fig. 1. Reversed-phase HPLC of enriched kairomones fromholding water of different fish species. Bars indicate biologicalactivity (error bars: 1 SE). The broken line indicates themethanol gradient. TA: total activity prior to HPLC. Frac-tionation of TA from A: Esox lucius, B: Gasterosteus aculea-tus. HPLC of kairomones from the two fish species yielded oneactive fraction with identical retention time. Main activity ofthe HPLC equals total activity.

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Table 2. Effects of enzymatic hydrolysis of anionic groups in C18-SPE enriched kairomone from Carassius carassius onbiological activity. C18-SPE enriched kairomone was used for kairomone treatments, nonfish water was used for controls.Activity is given as mean day depth of Daphnia magna, maximum mean day depth (95 cm) indicates maximum biological activity(N=5). Overall ANOVA: F14,56=24.1; PB0.0005. Asterisks indicate biological activity of treatments which differ significantlyfrom control (Tukey’s test, **: pB0.005; ***: pB0.0005; ns: not significantly different).

Treatment Mean day depth [cm(1 SE)]

Control Kairomone

Enriched kairomone 88.2 (2.46)***Alkaline phosphatase 17.4 (9.12) 68 (11.68)

Sulphatase 15.4 (4.43) 76.25 (9.44) ***after extraction with diethylether: aqueous layer 12.5 (4.79) **55 (8.8)after extraction with diethylether: organic layer ns15 (4.74) 19 (5.10)

Glucuronidase 7.5 (2.5) 79.6 (9.5) ***after extraction with diethylether: aqueous layer 7.5 (2.5) ***87 (3.74)after extraction with diethylether: organic layer 8.4 (2.11) 20.8 (4.36) ns

might be a constituent of the kairomone which isdispensable for biological activity. We therefore re-peated the digestion with sulphatase and glucuronidase,but subsequently partitioned the activity between waterand diethylether. Neither sulphatase nor glucuronidasereleased DVM-inducing compounds into diethylether(Table 2), indicating that no unpolar compounds conju-gated to sulphate or glucuronic acid were inducingDVM.

If sugar acids or other caboxylic acids were a con-stituent of the kairomone, carboxy groups would bepresent that are sensitive to esterification with dia-zomethan. However, after methylation biological activ-ity was still present and significantly different from thatof the control treatment (Table 3), indicating that car-boxy groups are not an essential constituent of biologi-cal activity. Carbohydrates as polar functional groupsmight contribute to the polar nature of the kairomone.We therefore tested for the presence of different carbo-hydrates as essential components of biological activityin kairomone from Carassius carassius (Table 3). Drise-lase, which has cellulase, laminarinase and xylanaseactivity, did not reduce the activity of the kairomone,indicating that xylan (b-1-4 xylose), cellulose (b-1-4glucose) and laminarin (b-1-3 glucose) are not con-stituents of the biological activity. However, these car-bohydrates are common plant constituents. Wetherefore tested the hypothesis that carbohydrates re-leased from mucus of fish are the DVM-inducing cue.Mucus itself had no activity (Table 3). As mucopolysac-charides are a major constituent of mucus, we digestedfreshly taken mucus with hyaluronidase which did notlead to a release of activity from mucus.

Interaction of food particles with kairomone

We hypothesized that the observed lipophilicity of thekairomone as is indicated by binding to C18-SPE mightlead to adsorption of the cue to food particles. The cuewould then be surface-associated rather than freely

dissolved, which would link the perception of thekairomone to the presence of food particles. We investi-gated this by incubating C18-SPE enriched kairomonefrom C. carassius with S. acutus. When S. acutus cellswere removed by centrifugation, we did not find areduction of dissolved activity in the supernatant (Table4). When S. acutus cells that had been incubated evenwith the tenfold concentration of crude kairomone wereconcentrated by centrifugation and resuspended in con-trol water, no biological activity was found indicatingthat adsorption of the kairomone to cells of S. acutus isof minor importance compared to the dissolvedfraction.

Concentration dependence

Daphnia responded in a concentration-dependent man-ner to crude extract of kairomone. In the no-fish con-

Table 3. Effects of esterification, hydrogenation and enzy-matic hydrolysis of carbohydrate groups in C18-SPE enrichedkairomone from Carassius carassius on biological activity.Activity is given as mean day depth of Daphnia magna,maximum mean day depth (95 cm) indicates maximum bio-logical activity (N=5). C18-SPE enriched kairomone wasused for treatments, nonfish water was used for controls.Mucus as a possible source of kairomone was investigated bydigestion with hyaluronidase: enzyme without mucus was usedfor control. Overall ANOVA: F8,36=23.0; PB0.0005. Aster-isks indicate biological activity of treatments which differsignificantly from control (Tukey’s test, **: pB0.005; ***:pB0.0005; ns: not significantly different).

Treatment Mean day depth [cm (1 SE)]

Control Kairomone

Enriched kairomone 74 (8.57)82 (3)17 (3)Digestion with driselase ***

***73 (6.63)34 (5.01)Esterification withdiazomethan

Mucus: incubation in ns29 (2.49) 34 (6)buffer

Mucus: digestion with ns20 (3.54) 40 (5)hyaluronidase

OIKOS 88:1 (2000) 123

Table 4. Interaction of C18-SPE enriched kairomone from Carassius carassius with the planktonic chlorophyte Scenedesmusacutus. Activity is given as mean day depth of Daphnia magna, maximum mean day depth (95 cm) indicates maximum biologicalactivity (N=5). Overall ANOVA: F3,15=52.6; PB0.0005. Asterisks sharing the same vertical column indicate treatments thatare not significantly different at the 95% level (Tukeys’ test).

Treatment HomogeneousMean day depth[cm (1SE)] groups

Enriched kairomone 92 (1) *Enriched kairomone after incubation with S. acutus, alga cells removed 90 (2) *S. acutus incubated in kairomone 22 (9) *S. acutus incubated in tenfold concentrated kairomone 20 (8) *

trol the animals stayed in the epilimnetic part of thetubes during day (Fig. 2). Mean day depth was notsignificantly different from control in treatments corre-sponding to one fish per 2000, 400 and 200 l. However,with increasing kairomone concentrations the mean daydepth of Daphnia increased until, with highest concen-trations, animals stayed at the bottom. We determineda threshold kairomone concentration for the onset ofDVM by linear regression with log10 transformedkairomone concentrations. The threshold concentrationwas determined according to (Sommer 1997) by calcu-lating linear regressions for the lowest concentrations(2000 to 200 l/fish) and sequentially adding data pointsin ascending order of fish density. The regression be-came significant (p=0.0203) when a concentration cor-responding to 40 l/fish was included, indicating thatbetween kairomone concentrations corresponding to200 to 40 l/fish a significant change in mean day depthwas observed, so that the threshold kairomone concen-tration in our experimental setting corresponds to fishdensities of one fish in 40 to 200 l. The amplitude ofDVM was concentration-dependent over a range ofthree orders of magnitude of kairomone concentration(Fig. 2).

Discussion

In general two conditions are required to make chemi-cally induced defences an evolutionarily stable strategy:an unpredictable floating risk of predation and a reli-able cue to predict predation (Harvell 1990, Pijanowska1993). Abundances of fish indeed vary strongly withinand between seasons making it hard to predict preda-tion risks (Jachner 1988) without a predator-associatedchemical signal (Vijverberg et al. 1990). The chemicalnature of the DVM-inducing kairomone is still un-known although several characteristics have been re-vealed (Von Elert and Loose 1996). Here we show thatDVM-inducing kairomones from a planktivorous and apiscivorous fish of the orders Gasterosteidae and Eso-cidae can be quantitatively enriched by reversed phasesorbents indicating at least a moderate lipophilic natureof the cues. Presumably hydroxy groups which can bereversibly acetylated seem to be responsible for the

intermediate polarity of the compound as indicated byreversed-phase HPLC (elution at methanol/water 60/40). Fractionation by HPLC yielded only one activefraction with retention times identical for kairomonesfrom pike and stickleback. The kairomones investigatedin this study show the same chemical features and thesame chromatographic retention as the previously char-acterized kairomones (Von Elert and Loose 1996) fromthree cyprinid species (Table 5). This suggests no or arather low predator specificity of the kairomone evenacross families. Since even a piscivorous pike is chemi-cally recognized by Daphnia, it is tempting to imaginethat evolution has selected for a common metabolite offish. Even the response to a piscivore would be adaptiveto Daphnia given that piscivores always coexist withplanktivores as their prey.

As, by definition, the production of kairomones isdisadvantageous to planktivorous fish, selection againstthe release of these cues might be expected. Askairomone production is widespread and of low speciesspecificity in freshwater fish two explanations are possi-ble: (1) the kairomones themselves or the metabolism

Fig. 2. Effect of no-fish control and different concentrationsof crude kairomone extract from incubation water of Caras-sius carassius on mean day depth of cohorts of Daphniamagna. Concentrations of enriched kairomone are expressed asfish densities. Linear regression for log10 transformed concen-trations corresponding to the range from one fish in 200 to2500 l was not significant, but became significant when re-sponses to higher kairomone concentrations were included inthe regression analysis.

124 OIKOS 88:1 (2000)

Table 5. Comparison of the chemical characteristics of DVM-inducing kairomones released by Gasterosteus aculeatus, Esoxlucius and three cyprinid fish species (Leucaspius delineatus, Carassius carassius, Rutilus rutilus).

Fish species Incubation water C-18 SPE methanol Acetylation Acetylation and saponification

Gasterosteus aculeatus + + − +Esox lucius + ++ −Leucaspius delineatus + + − +Carassius carassius + ++ −

+Rutilus rutilus + + −

ogy of fish; (2) the kairomones are associated with butnot produced by fish themselves. (Mckelvey 1996) pre-liminarily characterized the kairomone from marsh kil-lifish, Fundulus heteroclitus, inducing a photoresponsein shrimp nauplii. In accordance with our study theyfound a low-molecular-weight compound with highthermal and pH stability. The authors could generatebiological activity from mucus by mucopolysaccharideenzyme degradation of fish mucus, suggesting that mu-cus was the source for the kairomone. However, wewere not able to release DVM-inducing activity frommucus of crucian carp by digestion with hyaluronidasenor to inactivate crude kairomone with the carbohy-drate hydrolysing enzyme driselase. This suggests thatthe metabolic source of the cue in the five freshwaterfish species investigated so far differs from that of F.heteroclitus.

It has recently been shown that treatment of perchwith an antibiotic resulted in significantly reducedDVM-inducing activity of the incubation water(Ringelberg and van Gool 1998). The authors con-cluded that bacteria are involved in the production ofthe kairomone. However, after antibiotic treatment bio-logical activity of the incubation water was still half asactive as incubation water of the non-treated fish, indi-cating that bacteria-mediated release of kairomone isonly partially responsible for the avoidance reaction ofDaphnia. It can further be hypothesized that incubatinga perch in 8 l of water for 48 h probably leads toelevated bacterial metabolism and hence probably anoverestimation of the bacterial contribution to the re-lease of the kairomone.

Recently (Boriss et al. 1999) have shown thattrimethylamine (TMA) induces DVM in Daphnia andthe authors reported that TMA is released by thefreshwater fish Leuciscus idus in amounts sufficient tomake TMA an active component of the ‘fish-factor’.However, the spectrophotometric technique (Kakacand Vejdelek 1974) referenced by the authors for theirdetermination of TMA lacks the specificity for thedetermination of particular tertiary alkylamines in sam-ples of unknown composition and further, the presenceof alcohols is known to interfere with the assay (Kakacand Vejdelek 1974). This led to an overestimation ofthe TMA release by fish by more than four orders ofmagnitude (Von Elert and Pohnert unpubl.). Further,removal of TMA from fish incubation water did not

reduce DVM-inducing activity, suggesting that TMAmay not be an ecologically relevant signal mediatingDVM (Von Elert and Pohnert unpubl.).

The chemical nature of molecules determines howthey perform in the respective environment. The DVM-inducing kairomone is non-volatile which enhances per-sistence and a low-molecular-weight compound ofmedium polarity (Von Elert and Loose 1996). Thesechemical features make it spread fast in the water andindeed the onset of DVM seems to be triggered withina few days after the emergence of young-of-the-year fish(Ringelberg et al. 1991). When the cue is persisting,chemical detection of a predator indicates to prey thata given space was risky at some point in time, but doesnot necessarily indicate present risk. Hence a persistingkairomone might induce prey to carry costs for preda-tor avoidance that are not traded off by increasedprotection; it is reported that Daphnia, Chaoborus andmarine copepods exhibited diel migrations even a fewdays after predators had been removed (Bollens andFrost 1989, Dawidowicz et al. 1990, Loose 1993). Al-though it seems that these are cases of non-adaptivechemical recognition, they are probably not found innature: only in experimental setups predators disappearfrom one day to the next, whereas in lakes abundancesof planktivorous fish only change moderately with time.

Though temporal aspects of cue detection have beenshown (Gomez and Atema 1996) for lobsters, it has notbeen investigated for Daphnia, which is so small thatthe maximum distance of putative receptors involved inthe perception of planktivorous fish is in the range of afew millimetres, which is too small to resolve spatialgradients by receptor differentiation. Daphnia ’s inabil-ity to chemically locate planktivorous fish is in fact ofminor importance since short-distance avoidance wouldnot be efficient against a predator being superior withregard to swimming speed and visual sensitivity.Whenever short-distance predator avoidance is inade-quate the persistence of the cue in the absence of thepredator becomes advantageous: it conveys informationon the likelihood of predator presence. The inability ofDaphnia to explore possible spatial gradients ofkairomones does not preclude increasing response ofprey to increasing concentrations of predator chemicalcues (Loose and Dawidowicz 1994) and hence of in-creased likelihood of predation.

OIKOS 88:1 (2000) 125

In predator-prey systems in which predators are uni-formly risky to prey one should expect (1) low specific-ity of kairomones from predators and (2) minorimportance of alarm substances produced by injured orkilled conspecifics. Daphnia responds behaviourally dif-ferent to different types of predators; each of theseshifts appear to be specific, adaptive responses to theparticular foraging habitat of the predator: in the ab-sence of fish Daphnia show an ‘inverse’ pattern of DVMin response to cues from Chaoborus (Dodson 1988) andalter their horizontal migration when they are presentedwith chemicals from invertebrate predators (Notonecta)(Watt and Young 1994). This is contrasted by theobvious absence of species specificity of kairomonesreleased by the uniformly risky predator fish. In caseswhen predators are differently risky or when the preda-tion risk associated with the same predator varies, e.g.with the availability of alternative food, alarm sub-stances alone or in association with kairomones fromthe predator seem to be an adaptive proximate cue forthe onset of predator avoidance. Brook stickleback(Culaea inconstans), allopatric to predatory pike (Esoxlucius), decrease activity when exposed to chemical cuesonly from pike that had eaten conspecific sticklebacks(Gelowitz et al. 1993). Naive fathead minnows(Pimephales promelas) responded with fright reactionsto unfamiliar pike that had eaten conspecific minnowsbut did not respond to pike that had eaten heterospe-cifics (Mathis and Smith 1993). Frog tadpoles (Ranaaurora) showed reduced movement when exposed tochemical cues from predatory newts (Taricha granulosa)fed conspecific frog, but did not respond to newts thathad been fed insects (Wilson and Lefcort 1993). Pul-monate snails (Physella gyrina) increased their refugeuse upon being preyed by molluscivorous pumpkinseedsunfish (Lepomis gibbosus), a specialized snail predator.Turner (1996) showed that this response was due tocrushed conspecifics but not to the fish cue itself. Sinceonly a few fish species are molluscivorous and thosewhich are able do so only seasonally when alternativeprey is scarce (Jansen 1995), it would be maladaptive torely only on chemical cues from the predator to changehabitat use. However, in accordance with the uniformpredation risk for zooplankton exerted by fish no suchcues from injured conspecifics are involved in the onsetof DVM: it has previously been shown that the produc-tion of the kairomone does not depend on the feedinghistory of the fish (Loose et al. 1993).

The reliability of the cue to accurately assess the riskof predation is crucial in balancing benefits and trade-offs of DVM. The costs of this behavioural predatoravoidance can be expressed as a reduction in somaticgrowth and reproduction. The sources of these costs areretarded metabolic processes due to low temperaturesand lower food quality and/or quantity in the hypolim-netic day-time refuge (Loose and Dawidowicz 1994). Inorder to investigate the quantitative assessment of pre-

dation risk, we simulated increasing predator abun-dance by increasing concentration of C18-SPE enrichedkairomone from incubation water of crucian carp. Pre-vious experiments using dilution series of incubationwater had shown that Daphnia respond to increasingpredator abundances by increasing day-time depth(Loose 1993, Loose and Dawidowicz 1994). Here weused partially purified kairomone and show that over arange of several magnitudes of kairomone concentra-tion the animals respond in a dose-dependent wayindicating that the crude kairomone mimics the pres-ence of fish in an adequate way. The determinedthreshold concentration of one fish in 200 l is ratherhigh compared to field data (Jachner 1988). However,four lines of reasons might explain the apparent differ-ence of thresholds: (1) fish abundances in lakes areaverage values, but fish tend to be very patchily dis-tributed (e.g. shoals, residence mainly in the littoral orepilimnion). Hence local fish densities and kairomoneconcentrations might well exceed the threshold concen-tration determined in our small-scale system. (2) Withincreasing scale of the bioassay system threshold con-centrations seem to be lowered. In a mesocosm,threshold concentrations of as low as one fish per 1600l were determined (Loose 1993). This suggests thatincreasing surface to volume ratios in small-scale sys-tems lead to increased threshold levels. The reason forthis might be increased adsorption to surfaces or en-hanced degradation by surface-associated bacteria insmall-scale systems. The kairomone can be degraded bybacteria (Loose et al. 1993), but no rates of turnoverhave been determined. (3) Increased stress by smallcontainers has been reported for fish (Magurran et al.1996) and might as well affect Daphnia thereby increas-ing or decreasing the threshold. (4) It is still not clearwhether Daphnia can assess risk of predation from thesafety of their refuge in the hypolimnion or whether thenocturnal visits to the epilimnion are the periods theanimals are presented with fish kairomones resultingfrom higher fish densities in the epi- than in the hy-polimnion. It is imaginable that it is the increase inconcentration rather than the absolute concentration ofkairomone that the ascending animals detect and thatdetermines the dose-dependent response. The xanthiidcrab Leptodius sangineus responds to the chemical pres-ence of its predator (the swimming crab Thalamitacrennata) with delaying its moult: it seems to be espe-cially responsive to increases in cue concentrationrather than to absolute level of cue per se (Harvey1993). A similar mechanism might explain why thehomogeneously distributed kairomone in our systemleads to higher threshold levels than in the above-men-tioned mesocosm study (Loose 1993) when thekairomone was restricted to the epilimnion.

Another proximate cue that modulates the amplitudeof DVM is the availability of epilimnetic food (Johnsenand Jakobsen 1987): in cases of low food availability

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the DVM amplitude decreases, which is explained bythe increased starvation of animals migrating comparedto those not migrating. An alternative explanationcould be that, due to its lipophilic character, the DVM-inducing kairomone adsorbs to membranes of particles,such as food, and is sensed via ingested particles ratherthan as a freely dissolved cue. Low food availabilitywould then mean less kairomone perceived by thedaphniids and hence lead to a smaller amplitude. Ad-sorption of other lipophilic substances to lipophilicsurfaces in aquatic environments has been reported(Klapes 1990, Kulovaara 1993). However, our resultsclearly demonstrate that DVM-inducing information isnot mediated via food particles indicating that the cueremains well dissolved in the presence of alga cells andprobably is sensed as a dissolved molecule.

Concluding remarks

The DVM-inducing kairomone seems to be chemicallyvery similar if not identical within and across freshwa-ter fish families. Though different fish species probablyrelease the same cue they might differ substantially inthe production of this compound. We are not aware ofany attempts to link the production of kairomone tonumbers, body mass, surface area or physiological ac-tivity of fish. The idea that the kairomone is a metabo-lite of fish-associated bacteria represents such anattempt to investigate the physiology of the kairomoneproduction in order to understand what determines thekairomone level in different lakes. Similarly, morephysiological research is needed for the prey: evenwithin a single clone of Daphnia more than one defen-sive trait may be induced. However, ‘overprotection’ ismaladaptive and the physiology of multiple inducibledefences needs further attraction. Even under compara-ble predator abundances the response in the prey differswith the trophic status of the lake. As this is not due toadsorption of the kairomone to food particles it istempting to speculate that light (in other words: visibil-ity of prey) determines the inducibility of a defence in agiven prey under given kairomone concentrations.

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