# To flee or not to flee: detection, avoidance and attraction of profitable resources by Daphnia magna studied with olfactometer

Johann P. Müller, David Laloi, Claude Yéprémian, Cécile Bernard, Florence D. Hulot

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## To cite this version:

Johann P. Müller, David Laloi, Claude Yéprémian, Cécile Bernard, Florence D. Hulot. To flee or not to flee: detection, avoidance and attraction of profitable resources by Daphnia magna studied with olfactometer. Journal of Limnology, 2013, 72 (3), pp.464-472. 10.4081/jlimnol.2013.e37. hal-01583948

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# To flee or not to flee: detection, avoidance of and attraction to food resources by *Daphnia magna* studied with an olfactometer

Johann P. MÜLLER,<sup>1,2*</sup> David LALOI,<sup>3</sup> Claude YÉPRÉMIAN,<sup>4</sup> Cécile BERNARD,<sup>4</sup> Florence D. HULOT<sup>5</sup>

## ABSTRACT

The cladoceran herbivore *Daphnia magna* is a major consumer of phytoplankton in lakes. Therefore, this organism may control the phytoplankton community and the proliferation of some algae or cyanobacteria. Cladoceran behaviour and migration in relation to temperature, light or presence of planktivorous fishes have been well studied. In particular, it is known that the detection of kairomones produced by predators may induce avoidance. Avoidance could also occur with other semiochemicals such as cyanotoxins. In order to explore this hypothesis, we used an olfactometer to observe and measure the exploratory behaviour of D. magna individuals based on the motivation for food. Daphnids were allowed to choose between different compounds: water, a pure cyanotoxin, i.e. the microcystin-RR [(MC)-RR], extracts of one MC-producing strain (PMC 75.02) and one MC-free strain (PMC 87.02) of Planktothrix agardhii, or a green algae Scenedesmus obliquus. With this experimental design, we observed that i) cladocerans are able to detect resources with different qualities, ii) they can explore before exhibiting preferences, and iii) daphnids are able to avoid compounds that are potentially toxic (e.g., microcystins). First, daphnids explored the environment, subsequently (after about 1.5 h), they showed a significant tendency to stay where there is a profitable resource such as S. obliquus. These results also suggest that specimens of D. magna cannot detect MC compounds from P. agardhii, but they respond to it as a food resource. The study of zooplankton ability to explore the environment when exposed to semiochemicals needs further investigation.

Key words: Daphnia magna, Planktothrix agardhii, olfactometer, migration, secondary metabolites, cyanotoxins.

Received: August 2012. Accepted: March 2013.

## INTRODUCTION

Relationships between cyanobacteria and zooplankton are complex and affect aquatic ecosystems acting as positive or negative controls in microorganism communities (Ghadouani et al., 2006; Oberhaus et al., 2007). Cyanobacteria are considered as inadequate food for zooplankton such as daphnids (Wilson et al., 2006). Indeed, i) cyanobacteria may be deficient in essential nutrients for zooplankton (De Bernardi and Giussani, 1990; Müller-Navarra et al., 2000; Von Elert and Wolfram, 2001), and ii) some cyanobacteria are known to produce toxic metabolites (e.g., cyanotoxins) that may harm or kill not only animals and humans (Carmichael, 1992; Hitzfield et al., 2000; Codd et al., 2005; Dittmann and Wiegand, 2006; Purdie et al., 2009) but also daphnids and other zooplankton (Rohrlack et al., 1999). Cyanotoxins may have various effects on zooplankton: decrease of swimming activity of freshwater cladocerans (Ferrao-Filho et al., 2007), inhibition of digestive enzymes (Arnold, 1971; Nogueira et al., 2006), inhibition of feeding rate, growth

and reproduction (Arnold, 1971; Hawkins and Lampert, 1989; DeMott, 1999), decrease of weight (Gliwicz and Lampert, 1990) or even lethally toxic effect (Nogueira et al., 2004). These results, however, vary with experimental conditions (Lampert, 1987; Tillmanns et al., 2008). Among the many parameters which may influence these impacts we found: i) density of the food provided to *Daphnia* (Kurmayer, 2001), ii) temperature and viscosity of the water (Abrusán, 2004), iii) size of the zooplankton (Hawkins and Lampert, 1989; Kirk and Gilbert, 1992), iv) species of the grazer (Kirk and Gilbert, 1992), v) species and strains of cyanobacteria (Nogueira et al., 2006; Wilson et al., 2006; Tillmanns et al., 2008), or vi) type of metabolites provided (Nogueira et al., 2006).

Among toxic secondary metabolites, cyanotoxins microcystins (MC) are the most abundant ones. Still, several studies have shown that other compounds produced by cyanobacteria can have a negative impact on zooplankton as well (Kurmayer, 2001; Reinikainen et al., 2002; Lürling, 2003a; Rohrlack et al., 2004; Wilson and Hay, 2007; Hulot

<footnote n="1"><sup>1</sup>UMR 7618 Biogéochimie et écologie des milieux continentaux, UPMC, ENS, CNRS, IRD, U, PEC, AgroParisTech, Paris Cedex 05;
<sup>2</sup>UFR Sciences de la Vie, UPMC Université Paris 06, Paris;
<sup>3</sup>UMR 7625 Ecologie et Evolution, UPMC Université Paris 06, Ecole Nor-
male Supérieure, AgroParisTech, CNRS, 7 quai Saint Bernard, 75005 Paris;
<sup>4</sup>UMR 7245 CNRS, MNHN Molécules de Communication
et Adaptation des Microorganismes, Muséum National d'Histoire Naturelle, 57 rue Cuvier, 75231 Paris Cedex 05;
<sup>5</sup>UMR 8079 Ecologie,
Systématique et Evolution, CNRS, Université Paris Sud, Orsay Cedex 91405, France</footnote>

<footnote>*Corresponding author: jmuller@biologie.ens.fr</footnote>
et al., 2012). Although the biological role of these com-
pounds is debated (Schatz et al., 2007), one hypothesis is
their negative effect on planktonic herbivores (Lürling,
2003b). Indeed, through its predatory activity, herbivorous
zooplankton, and particularly *Daphnia* sp., has a direct im-
pact on phytoplankton (Vanni, 1987; Vanni and Findlay,
1990; Hansson and Carpenter, 1993; Brett and Goldman,
1996; Oberhaus et al., 2007). *Daphnia* sp. is a major pred-
ator of algae (Kerfoot and Sih, 1987) and, in the absence
of fish, zooplankton increases and phytoplankton decreases
as a result of cascading trophic interactions (Christoffersen
et al., 1993). Therefore, the cyanobacterial dominance may
be partly due to a low degree of control from large gener-
alist zooplankton, as suggested by the negative relationship
between cyanobacteria and cladoceran (Ghadouani et al.,
2006; Catherine et al., 2008). Cyanobacteria secondary
metabolites production may help to explain food selection
and avoidance in freshwater ecosystems (Jüttner, 2005).
Thus, studying *Daphnia* sp. behaviour facing different nu-
trition choices may contribute to explain why some phyto-
plankton species are favoured and may dominate the
phytoplankton community.

Diel vertical migration of zooplankton is a daily pat-
tern of movement in relation to temperature (Lampert and
Grey, 2003), light (Stearns, 1975) and predator-prey in-
teractions (De Meester et al., 1995). Usually, the pattern
is an evening ascent to surface waters and a morning de-
scent to deep waters. The diel vertical migration of daph-
nids is associated with considerable metabolic cost (Stich
and Lampert, 1984), but plays a key role in predator
avoidance. This migration may be motivated by fish semi-
ochemicals (Dodson, 1988; Loose, 1993; Lampert, 1993;
De Meester et al., 1995). Thus, daphnids are able to detect
kairomones released by fishes (Beklioglu et al., 2006).
Other compounds may also modify the behaviour of zoo-
plankton. For instance, organic compounds produced by
algae and cyanobacteria can play an attractive and defen-
sive role in aquatic ecosystems (Lass and Bittner, 2002;
Fink et al., 2006). Jüttner (2005) showed that diatoms and
other phytoplanktonic species damaged by grazers may
produce compounds that are toxic and repellent to herbiv-
orous zooplankton (Jüttner, 2005). In a more recent arti-
cle, Jüttner et al. (2010) showed that the cell disruption
of the cyanobacterium *Microcystis* produces a defense sig-
nal, the b-Cyclocitral, that induces an increase in swim-
ming velocity of *D. magna*. As a consequence, the signal
induces the escape of the daphnids. Thus, the presence of
fish or algae and the production of organic compounds
such as kairomones influence Daphnia migration (Bekli-
oglu et al., 2008; Rinke and Petzoldt, 2008; Jüttner et al.,
2010; Slusarczyk and Pinel-Alloul, 2010).

The relationships between cyanobacteria and daphnids
reflect the typical complexity of prey-predator interac-
tions and much is still unknown. In particular, to our

knowledge, the influence of the prey on the spatial dis-
placement of the predator has been poorly studied. Since
Daphnia's predators may elicit its migration, one might
ask whether its prey (cyanobacteria) may also induce mi-
gration of the grazer (daphnids). To address this question,
Van Gool and Ringelberg (1996) were the first to use an
Y-tube olfactometer with an inflows of water. Their results
showed that Daphnia may discriminate between clean
water and water that previously contained either
*Scenedesmus acuminatus* or *Planktothrix limnetica* but
not *Planktothrix agardhii* producing MC. Olfactometers
were rarely used in studies on aquatic invertebrates, but
such devices were validated by numerous behavioural
studies of semiochemical recognition in terrestrial species
(Janssen *et al.*, 1990; Scutareanu *et al.*, 1996).

Based on studies on the effects of chemical compounds
on terrestrial insects displacements (Dornhaus and Chittka,
2001; Meinwald and Eisner, 2008), of chemical defences
against predation (Lass and Spaak, 2003), and on migration
of zooplankton stimulated by the presence of kairomones
(Rinke and Petzoldt, 2008; Beklioglu et al., 2008; Slusar-
czyk and Pinel-Alloul, 2010), we explored – with olfac-
tometers adapted to aquatic species – the migration of
starved daphnids when exposed to different signals. Unlike
other studies (Van Gool and Ringelberg, 1996; Roozen and
Lürling, 2001), our design without inflow allowed move-
ment of daphnids in the absence of rheotaxis influence.
Thus, the main aim of our study was to observe not only
daphnids' first choices, but also daphnids' behaviour over
a long time, and to test assumptions on their exploratory
ability. The second aim was to explore daphnids' behaviour
facing different resources extracted from green algae or
cyanobacteria that are potentially attractive or repulsive
with a particular attention to MC.

METHODS

Experimental design

Origin and culture of organisms

We used two monoclonal and non-axenic strains of
*Planktothrix (P.)* *agardhii* isolated from the *Base Nautique*
*de Viry*, France (Yéprémian *et al.*, 2007) and maintained in
the Paris museum collection (PMC). The *P. agardhii* strain
PMC 75.02 produces mainly three variants of microcystins
(m/z 981.6 [D-Asp³]MC-LR; m/z 1024.8 [D-Asp³]MC-RR;
and m/z 1045.6 [D-Asp³]MC-HtyR). The *P. agardhii* strain
PMC 87.02 is a MC-free strain (Yéprémian *et al.*, 2007). We
refer to these strains in the following as the MC-strain and
the MC-free strain respectively. However, both strains are
able to produce other secondary metabolites that may affect
*Daphnia magna* population dynamics (Hulot *et al.*, 2012).
Thus, these two strains will allow a comparison among the
effects of cyanobacterial compounds. Each strain was cul-
turated in modified 2 L Duran bottles containing 1 L of Z8 medium (Kotai, 1972) and inoculated with 100 mL of an exponential pre-culture. The bottles were placed in growth chambers at 20±1°C, and illuminated with cool white fluorescent tubes (L18W/21-840, Osram lumilux Plus Eco; OSRAM GmbH, Munich, Germany) with a 16:8 light:dark cycle, under 10±2 µmol photons m⁻² s⁻¹ (Yéprémian et al., 2007). Growth of the cyanobacteria cultures was monitored by measuring optical density at 436 and 750 nm every two days with a spectrophotometer (Foy and Smith, 1980). The cultures were stopped at the plateau phase, and the MC-strain and MC-free strain were kept at -80°C for conservation, and thawed 24 h before use as a treatment. Microcystin-RR (Alexis® Biochemicals, San Diego, CA, USA) was selected as pure toxin whose toxicity mimics an exposure to a less toxic MC such as the demethyl forms (e.g., LD₅₀ of [D-Asp³]MC-LR ranged from 160 to 300 on mouse) (Sivonen and Jones, 1999).

*Daphnia magna* has a big size and is easily cultured in laboratory. Individuals were isolated from a pond situated at the École Normale Supérieure (Paris, France), and kept in an aquarium with Volvic water (Société des Eaux de Volvic, Volvic, France) and fed on *Scenedesmus*

*obliquus* (CCAP 276/6A). This alga, which is known to be a good resource for Daphnia (Lampert, 1987), was cultured at 22±1°C and under 14:10 h light-dark cycle in COMBO medium (Kilham *et al.*, 1998).

Our experiments in olfactometers were based on feeding stimulation, so daphnids were starved prior to the experiment. To do so, we isolated adults *D. magna* from stock culture 24 h prior to the experiments in vials containing pure Volvic (Société des Eaux de Volvic) water.

## Experimental system

The olfactometer consists of three glass jars connected *via* a Y-junction and corridors made of silicone tubes (Fig. 1). In one jar, *i.e.* the introduction jar, daphnids are introduced at the beginning of the experiment and they may eventually return in it. The two other jars are the migration jars containing the treatments and the individuals which can migrate into it. We used a Y-junction to have no asymmetry in our olfactometer. All corridors have the same diameter (5 mm) and the same length (50 mm), and the jars have a diameter of 50 mm and a volume of 100 mL. After filling the whole system with 100 mL of water, tubes were closed with clips adjoining the Y-junction to

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Olfactometer. Individuals are introduced in an introduction jar (1) and may migrate to the migration jars (2 and 3) that contained the treatment. Clips are represented by black bars at the end of each Y-junction.</figcaption></figure>
isolate the three parts of the olfactometer (Fig. 1). After
removing equivalent volumes of water, we added the cor-
responding treatments in migration jars. One single
starved daphnid was added to the introduction jar, the
clips were removed and the movements of the individual
were recorded during 1.5 h. This device thus allowed in-
dividual daphnids leaving the jar of introduction to choose
between the migration jars depending on different treat-
ments. Two 36 watt fluorescent tubes were suspended
above the olfactometers and the light intensity arriving on
experimental systems was 1500-2000 lux»21-28 µmol
photons m⁻² s⁻¹ (PAR). This ensured homogeneous light
for each olfactometer. Eight olfactometers were used at
the same time to replicate all the experiments. To avoid
any bias due to the relative position of the systems, olfac-
tometers rotated at each repetition.

Experiments

Preliminary experiment

Our goal was to observe the behaviour of zooplankton
in response to olfactory stimuli: we planned to measure
the first choice as well as the choice made after some time,
allowing exploration. To assess whether the observation
lasted long enough (movements of daphnids became rare
after 1.5 h) to give reliable data, and to ensure that there
was no bias in the movements of daphnids due to the de-
vice, a preliminary test was conducted. In this test, the mi-
gration jars contained pure Volvic water (Société des Eaux
de Volvic) and one adult daphnid starved for 24 h was in-
troduced in the jar of insertion. Seventeen repetitions with
eight systems at the same time were conducted. For this
preliminary experiment, we obtained 136 observations of
different daphnids during 17 sessions of 90 min each.

Main experiment

We recorded daphnids' behaviour when they had the
choice between i) MC-strain vs water, ii) MC-free strain
vs water, iii) pure MC-RR vs water, iv) S. obliquus vs
water, v) MC-strain vs S. obliquus, vi) MC-free strain vs
S. obliquus vii) MC-free strain vs MC-strain, and viii)
water vs water as a control. These eight treatments were
tested in parallel with eight olfactometers and the tests
were repeated 100 times strictly in the same conditions.
Thus, we obtained 100 observations of different starved
daphnids for each of the eight oppositions of treatments
(Fig. 2) and our dataset was composed of 800 observa-
tions of displacements during 1.5 h. The water treatment
contained pure Volvic (Société des Eaux de Volvic) water.
The density of resource in the treatments with MC-strain,
MC-free strain and S. obliquus was calculated so that the
final density of phytoplankton was 10 times Daphnia daily
carbon requirement (0.2 mg.day⁻¹) (Sim et al., 1994; Lam-
pert, 1987). Treatments were kept at -80°C and thawed 24

hours before use, which disrupted cells. Unlike previous
studies (Van Gool and Ringelberg, 1996; Roozen and Lür-
ling, 2001), the present design devoid of filtering step, al-
lowed to have the whole S. obliquus and P. agardhii
cellular contents directly available to grazers.

The MC-RR treatment volume was calculated to get the
same microcystin concentration as in the MC-strain treat-
ment. The MC concentration in MC-strain treatment was
9.6 µg eq. MC-RR.L⁻¹ assessed with a PP2A phosphatase
assay (Rivasseau, 1999; Yepremian et al., 2007; Müller et
al., in preparation). In the pure MC-RR treatment, we used
purified MC-RR (Alexis® Biochemicals) solubilised with
MeOH diluted at 1‰ and conserved at -20°C.

We analysed the first choice recorded for all individ-
uals regardless of the migration time (between 0 and 1.5
h) and the position at the end of the observation period
(after 1.5 h). We refer thereafter to these choices as the
first choice and the last choice respectively. For the first
choice, daphnids could choose between both treatments.
For the last choice, daphnids could either rest in the first
choice jar, move to the second treatment jar or come back
to the introduction jar. These observations allowed us to
study Daphnia's behaviour (exploration, detection,
choice) according to the presence or absence of com-
pounds, as well as to test the attractive, repellent or neutral
effects of compounds studied.

Statistical analysis

Two types of analysis were done: i) comparisons of the
distribution of daphnids between the treatments for each
choice, and ii) comparisons of the distributions of daphnids
between the first and the last choices. We used Fisher's exact
test with R software. Fisher's exact test is used in the analy-
sis of contingency tables when sample sizes are small. For
the first and last choices, we tested the significance of the
deviation from a null hypothesis of equal distribution. For
the comparison between the first and last choices, we tested
the significance of the deviation of the last choice from the
first choice. We set significant differences at a threshold of
α=5% and marginal significance at a threshold of α=10%.

RESULTS

The results of the preliminary experiment performed
with 135 replicates show that there is no bias in the ex-
perimental design (Tab. 1). One replicate was lost because
of daphnid's death during the experiment. Tests on the
first and last choices did not show significant difference
between the number of daphnids present in the two mi-
gration jars (P=0.57). In addition, we did not observe any
difference between the distribution of first and last choices
(after 1.5 h). We observed that 71.8% of the individuals
remained in the introduction jar; 7.4% of the individuals
that left introduction jar returned to it; only 20.7% of the
individuals migrated definitely to either of the two treatment jars (Tab. 1).

In the main experiment, according to the treatments, between 66 and 79% of the individuals stayed in the introduction jar (Tab. 2). Between 0 to 6% of the individuals left this jar and returned to it later (Tab. 2). Again, there were no significant differences in the treatment water vs water at the first (P=0.885) and last choices (P=0.88) (Fig. 2a, Tab. 3) confirming the absence of bias from the device. Similarly, there are no significant differences when daphnids had to choose between MC-RR and water (Fig. 2b, Tab. 3), MC-strain and water (Fig. 2c, Tab. 3), MC-free strain and *S. obliquus* (Fig. 2e, Tab. 3), and MC-free strain and MC-strain (Fig. 2h, Tab. 3).

When submitted to the treatments MC-strain and *S. obliquus*, at the last choice, there were more daphnids in the jar with *S. obliquus* than in the jar with MC-strain (P=0.0007) (Fig. 2f, Tab. 3). The change between the first and last daphnids choices is significant (P=0.002). Indeed, daphnids seem to prefer *S. obliquus* after an exploratory phase. A marginal difference is revealed in the treatment *S. obliquus* vs water at the first choice (Fig. 2g, Tab. 3) with more daphnids in the jar containing *S. obliquus* than in the jar with water (P=0.09). The difference in daphnids choice for *S. obliquus* vs water is significant at the last choice (P=0.02). In the treatment where *D. magna* have the choice between MC-free strain and water, we also observed a significant effect of the treatment, with a prefer-

<table><caption>**Tab. 1.** Preliminary experiment. Daphnids' choice in olfactometers with pure Volvic water (Société des Eaux de Volvic, Volvic, France).</caption><thead><tr><th></th><th>Motionless<sup>a</sup></th><th>Jar 2<sup>b</sup></th><th>No. of daphnids<br>Jar 3<sup>b</sup></th><th>Return<sup>c</sup></th><th>Total</th></tr></thead><tbody><tr><th>First choice</th><td>97</td><td>21</td><td>17</td><td>-</td><td>135</td></tr><tr><th>Choice after 1.5 h</th><td>97</td><td>14</td><td>14</td><td>10</td><td>135</td></tr></tbody></table>

<table><caption>**Tab. 2.** Main experiment. Distribution of individual daphnids after 1.5 h.</caption><thead><tr><th>Treatments (%)</th><th>Water<br>vs water</th><th>MC-RR<br>vs water</th><th>MC-Pa<br>vs water</th><th>MC-free Pa<br>vs water</th><th>MC-free Pa<br>vs Sc</th><th>MC-Pa<br>vs Sc</th><th>Sc<br>vs water</th><th>MC-Pa<br>vs MC-free Pa</th></tr></thead><tbody><tr><th>Motionless<sup>a</sup></th><td>71</td><td>66</td><td>76</td><td>73</td><td>68</td><td>79</td><td>73</td><td>66</td></tr><tr><th>With motion<sup>#</sup></th><td>29</td><td>28</td><td>23</td><td>25</td><td>31</td><td>19</td><td>24</td><td>30</td></tr><tr><th>Return<sup>§</sup></th><td>0</td><td>6</td><td>1</td><td>2</td><td>1</td><td>2</td><td>3</td><td>4</td></tr></tbody></table>

MC-RR, treatment with pure microcystin-RR; MC-Pa, treatment with Planktothrix agardhii microcystin-strain (PMC 75.02); MC-free Pa, treatment with Planktothrix agardhii microcystin-free strain (PMC 87.02); Sc, treatment with Scenedesmus obliquus extracts.

*Daphnids that did not move; ‡daphnids that moved; §daphnids that returned into the introduction jar.

One hundred replicates were realised.

<table><caption>**Tab. 3.** P values of the Fisher's exact test on the distribution of daphnids among the treatments for each choice, and on the distributions of daphnids between the first and the last choice.</caption><thead><tr><th>Treatment</th><th>Water<br>vs water</th><th>MC-RR<br>vs water</th><th>MC-Pa<br>vs water</th><th>MC-free Pa<br>vs water</th><th>MC-free Pa<br>vs Sc</th><th>MC-Pa<br>vs Sc</th><th>Sc<br>vs water</th><th>MC-Pa<br>vs MC-free Pa</th></tr></thead><tbody><tr><th>Distribution<br>for first choice</th><td>0.88</td><td>0.77</td><td>0.32</td><td>0.02</td><td>0.77</td><td>0.88</td><td>0.09</td><td>0.77</td></tr><tr><th>Distribution<br>after 1.5 h</th><td>0.88</td><td>0.39</td><td>0.39</td><td>0.06</td><td>0.88</td><td>0.0007</td><td>0.02</td><td>1</td></tr><tr><th>Distribution for first<br>choice vs distribution<br>after 1.5 h</th><td>1</td><td>0.2</td><td>1</td><td>&lt;0.0001</td><td>1</td><td>0.002</td><td>0.65</td><td>0.77</td></tr></tbody></table>

MC-RR, treatment with pure microcystin-RR; MC-Pa, treatment with Planktothrix agardhii microcystin-strain (PMC 75.02); MC-free Pa, treatment with Planktothrix agardhii microcystin-free strain (PMC 87.02); Sc, treatment with Scenedesmus obliquus extracts.

<footnote><sup>a</sup>Number of individuals that stayed in introduction jar during the experiment; <sup>b</sup>number of individuals that migrated to the jar of migration 2; <sup>c</sup>number of individuals that migrated to the jar of migration 3; <sup>d</sup>number of individuals that returned to jar 1 after leaving it.</footnote>
ence for water at the first choice (P=0.02) and a marginal effect with a preference for the MC-free strain at the last choice (P=0.06) (Fig. 2d, Tab. 3). Interestingly, we observed a highly significant inversion between the first and last choices (P<0.0001): daphnids migrated preferably at the beginning of the experiment to the jar containing water and finally in the jar with the MC-free strain treatment (Fig. 2d, Tab. 3).

DISCUSSION

Inspired by olfactometers used with terrestrial and aerial species and by previous studies with aquatic species, we used an aquatic Y-tube olfactometer to study the daphnids' behaviour towards semiochemicals. Preliminary tests of our device showed the absence of bias in our experimental system. Therefore, the differences in distribution observed in the main experiment when daphnids were subjected to different treatments are only due to their choices. Our results showed that *D. magna* did not exhibit preferences in four situations: MC-RR vs water, MC-strain vs water, MC-free strain vs *S. obliquus*, and MC-strain vs MC-free strain. Yet, *D. magna* showed preferences for *S. obliquus* and MC-free strain as an alternative from water, and *S. obliquus* as an alternative to MC-strain.

These results lead to several conclusions. First, the pure cyanotoxin MC-RR has no positive or repellent effects on daphnids. Therefore, the preference for *S. obliquus* as an alternative for the MC-strain cannot be attributed solely to the production of MC by the *P. agardhii* strain. Second, thanks to our design without inflows, we showed that *D. magna* is able to explore its environment and can modify its choice as a result. At their last choice, daphnids chose the treatment with *S. obliquus* when they had the choice between this green alga and the MC-strain, and they tended to choose the treatment with *S. obliquus* extracts when they had the choice between this alga and water. Daphnids being starved, this result shows that, after an exploration phase, they migrated to a nutritious source whatever the species. Indeed, when daphnids had the choice between MC-strain and *S. obliquus*, the analysis of the movements shows first a homogeneous distribution in both jars of migration but, after 1.5 h, they significantly moved to the jar containing *S. obliquus*, the resource they were fed on before the experiments. When daphnids had the choice between MC-free strain and water, *D. magna* preference changed between first and last choice, from water to the MC-free strain. These results show that these organisms might express displacement tropism as suggested by other studies (Van Gool and Ringelberg, 1996; Roozen and Lürling, 2001) and highlight the ability of *D. magna* individuals to explore their environment before making choices.

Van Gool and Ringelberg (1996) concluded from their

experiments that *D. galeata* × *hyalina* may be attracted by odours associated with edible algae and not by non-edible algae. In contrast, Roozen and Lürling (2001) did not observe preference of *D. galeata* × *hyalina*, nor of *D. pulex* or *D. magna* for the edible alga *S. obliquus*. However, they showed that the swimming speed of *D. magna* decreased with increased algal concentration. In the present

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Distribution of daphnids in the different treatments. In each panel, the first two bars give the first choice and last two bars the last choice. Legends as in Tab. 2. Results of statistical analyses are given as NS when not significant, \* when significant (α=5%) and \# when marginally significant (α=10%).</figcaption></figure>
study, we confirmed attraction by nutritious sources, but we also showed that the choice strategy of daphnids might be more complex than those measured in a *first-choice* test, as they might explore their environment and adapt their response accordingly.

Daphnids do not discriminate between the MC-strain and MC-free strain of *P. agardhii*. During blooms, the resource selectivity of major herbivorous zooplankton has an important control effect on the dynamic development of cyanobacteria (Catherine *et al.*, 2008). The daphnids' capacity to metabolise MC is of great importance. Thostrup and Christoffersen (1999) showed that microcystin can accumulate in daphnids. This observation and the absence of effects of MC on *D. magna* migration or discrimination between cyanobacterial strains that produce or not MC suggest that daphnids may play a role of vectors for the transfer of microcystins to higher trophic levels in the aquatic food web.

Besides the results obtained in this study, the experimental design presented here opens a way for further experiments. Indeed, daphnids migrate vertically in the water column according to light intensity (Stearns, 1975), temperature and food concentration (Lampert, 2003; Kessler and Lampert, 2004), presence of predators (De Meester *et al.*, 1995), but they may be confronted to variation of these factors at different depths. Their ability to detect kairomones raises the question of their behaviour in presence of multiple semiochemicals. More specifically, in our main experiment we used disrupted cells. With this procedure, *S. obliquus* and *P. agardhii* intracellular contents were directly available to grazers. We explicitly manipulated only one compound, MC-RR, but many other compounds in different treatments can influence daphnids. Thus, for a better understanding of daphnids' behaviour and of the effects of cyanobacterial secondary products on the relationship between cyanobacteria and herbivorous zooplankton, ideally we should study the metabolome of both *P. agardhii* strains and test daphnids' preference for cyanobacterial compounds or mixture of compounds with these new aquatic olfactometers.

Pawlik-Skowrońska *et al.* (2008) showed that the vertical distribution of *P. agardhii* was almost homogeneous in a shallow lake, whereas the MC distribution was not. In this way, potential signals for zooplankton were heterogeneously distributed in water. Horizontal gradients of abiotic conditions, such as light, temperature, dissolved oxygen and pH, are weaker in shallow lakes relative to vertical gradients in deep lakes and are less likely to influence horizontal migration (Burks *et al.*, 2002) than semiochemicals (Kvam and Kleiven, 1995). Heterogeneity in signals emerges also from the presence of predators and compounds produced by macrophytes and this heterogeneity can elicit different responses of zooplankton (Trochine *et al.*, 2009). However, during our experiments,

a big number of daphnids did not move and stayed in the introduction jar. This might be due to the absence of water inflows in our experimental device or because daphnids lost their rheotaxis with starvation as suggested by Roozen and Lürling (2001). Our experiments were based on feeding motivation: daphnids were starved with the aim of forcing them to look for food. This stimulation proved to be not enough. Further experiments could manipulate light that attracts daphnids in the absence of predators (Ringelberg, 1987).

## CONCLUSIONS

Our results show that the olfactometer is a promising experimental device for understanding the relationship between phytoplankton and zooplankton and, more generally, the signals that could modify the spatial distribution of zooplankton in aquatic ecosystems. Moreover, our results indicate that daphnids are able to explore their environment in a first phase and express preference for profitable resources in a second phase.

## ACKNOWLEDGMENTS

We thank Selma Maloufi for her help with the protein phosphatase 2A (PP2A) assay, David Carmignac for his assistance during our main experiment, and Minus van Baalen for helpful comments on the manuscript.

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