Marine Biology (2003) 143: 845850 DOI 10.

1007/s00227-003-1149-x

E. Linden M. Lehtiniemi M. Viitasalo

Predator avoidance behaviour of Baltic littoral mysids Neomysis integer and Praunus exuosus

Received: 28 April 2003 / Accepted: 20 June 2003 / Published online: 16 July 2003 Springer-Verlag 2003

Abstract The predator avoidance behaviours of two littoral mysid species, Neomysis integer (Leach) and Praunus exuosus (Muller), were studied experimentally. In ingestion experiments, mysids responded to a combination of chemical and visual signals of perch (Perca uviatilis), but not to each stimulus alone. In the presence of the combined visual and chemical predator signal the swimming activity and choice of habitat (open vs. articial vegetation, the Charophyte Chara tomentosa or the brown alga Fucus vesiculosus) were also inuenced. The two mysid species behaved dierently when perceiving predation risk: N. integer reduced swimming activity, whereas P. exuosus increased their use of the vegetation. The dierent antipredator strategies of the two mysid species reect their dierent lifestyles, N. integer being a swarm-forming species and P. exuosus living in association with aquatic macrophyte vegetation.

Introduction
For small aquatic animals, predation is an important source of mortality (Aneer 1980). Thus the ability to detect the presence of a predator and to adjust behaviour accordingly is a major advantage. Zooplankton and
Communicated by L. Hagerman, Helsingr E. Linden (&) Department of Ecology and Systematics, Division of Hydrobiology, University of Helsinki, P.O. Box 65, 00014 Helsinki, Finland E. Linden M. Lehtiniemi Tvarminne Zoological Station, 10900 Hanko, Finland E. Linden M. Lehtiniemi M. Viitasalo Finnish Institute of Marine Research, P.O. Box 33, 00931 Helsinki, Finland E-mail: eveliina.linden@mr. Tel.: +358-9-61394505 Fax: +358-9-61394494

other aquatic crustaceans have many behavioural responses that reduce the probability of being detected and captured by a predator (reviewed in Ohman 1988). Avoidance behaviour can aect dierent phases of the predation cycle, i.e. encounter, attack, capture and ingestion. Prey with developed vision can detect a predator by visual signals (Milinski and Heller 1978), but in dark and turbid environments chemical signalling often plays a key role (reviewed by Larsson and Dodson 1993; Bronmark and Hansson 2000) along with hydrome chanical signalling (Visser 2001). The phenomenon of zooplankton responding to chemical cues released by sh has been thoroughly studied (e.g. van Duren and Videler 1996; Burks et al. 2001), and the chemical substances in question have been analysed (Loose et al. 1993). After detecting a predator, prey may reduce their motility (Pijanowska and Kowalczewski 1997; Ejdung 1998), feeding rate (Milinski and Heller 1978; Ejdung 1998), or other activity (Savino and Stein 1982; Gotceitas and Colgan 1987), ee by escape reactions (Pijanowska and Kowalczewski 1997), engage in diel vertical migration (Loose et al. 1993; Stirling 1995), or react in various other ways (van Duren and Videler 1996; Pijanowska and Kowalczewski 1997). Many sh species have diculties in feeding amongst macrophytes (e.g. Coull and Wells 1983; Rooker et al. 1998) and, in the littoral environments, it may be useful for prey to seek refuge amongst the aquatic vegetation. On the other hand, planktivorous sh may also hide within vegetation to avoid their own predators, and predation pressure on zooplankton may consequently be high (e.g. Wineld 1986; Stanseld et al. 1997). Predation eciency within vegetation is aected by macrophyte density (Stoner 1982; Savino and Stein 1989) and morphology (Coull and Wells 1983; Ryer 1988). Also, the size (Wineld 1986; Ryer 1988) and predation strategy (Flynn and Ritz 1999; Burks et al. 2001) of the predator, as well as the size (Ryer 1988; Rooker et al. 1998) and defensive behaviours (Savino and Stein 1989; Flynn and Ritz 1999) of the prey play a role in the outcome of the interaction.

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Mysids are an important link between trophic levels in various aquatic ecosystems. They are omnivorous, feeding on phytoplankton, zooplankton and detritus (Mauchline 1980; Viherluoto et al. 2000). They are also important prey for a number of sh species, such as Baltic herring (Aneer 1980) and perch (Lappalainen et al. 2001). Factors aecting predatory interactions between mysids and their predators have, however, not been extensively studied. Hamren and Hansson (1999) found that the pelagic mysid Mysis mixta (Lilljeborg) reduces its food consumption when mysids were incubated in water where herring had been kept. This was suggested to be a predation avoidance strategy, since reduced feeding and activity probably make it more dicult for herring to detect the mysid. On the other hand, this behaviour has its downside, since it results in reduced feeding and mating opportunities and, hence, tness, of the prey (reviewed in Magurran 1990; Jeppesen et al. 1997). The aim of the present study was to investigate the predator detection and avoidance behaviours of two mysid species, Neomysis integer (Leach) and Praunus exuosus (Muller). Both species occupy the littoral zone, but N. integer is a swarm-forming species, while P. exuosus has a comparatively more solitary lifestyle (Mauchline 1971) and is strongly phytophilous (Kotta and Kotta 1999), being most often associated with F. vesiculosus. We performed two kinds of laboratory experiments. First, we tested which types of predator signals aect the behaviour of mysid species. We performed experiments to nd out if chemical or visual signals from perch (Perca uviatilis) inuence mysid feeding rates. Second, we studied the antipredator behaviour of the mysids to see if the detection of a predator aects their motility or hiding behaviour. Thus we monitored the swimming activity and habitat choice of the mysids in the presence of the predator signal in various vegetation types by videolming. We hypothesised that the behaviours will be adaptable to the ways of life of these two mysid species.

Ingestion rate experiments The ingestion rate experiments were made to test the eects of various predator signals on mysid feeding rates. The experiments were performed in 2-l aquaria at ambient water temperature (9 16C) in normal indoor light conditions. The chemical signal treatments (experiment I) were made in June and July 2001, and visual signal and combined chemical and visual signal treatments (experiment II) were made in May and June 2002. A total of 100 brine shrimp nauplii were picked under a binocular microscope with a pipette to the experimental aquaria, and one mysid was added to each aquarium using a small sieve. Method controls were conducted without a mysid present to estimate the recovery rate of brine shrimp. In control treatments the aquaria were lled with 0.2-lm-ltered seawater, without any predator signal. In chemical signal treatments the aquaria were lled with water from the perch container. In visual signal treatments an aquarium containing a perch was placed next to the experimental aquarium so that the perch was clearly visible to the mysid. Each set of experiments had ten replicates, and they lasted ca. 3 h (mean=3.031 h, SD=0.017). The experiments were ended by removing the mysids from the aquaria, after which the aquaria were emptied through a sieve. The perch were released back to the sea after the experiments. The remaining brine shrimps were preserved using acid Lugols solution and counted under a binocular microscope to estimate the feeding rate of mysids. The carbon content of brine shrimps was taken from Gorokhova (1999), and the ingestion rate of mysids was calculated as micrograms of carbon per hour. The mysids were measured (total length) to the nearest millimetre and dried at 60C for ca. 12 h, after which their dry weight was measured. The length (meanSD) of N. integer individuals was 1.820.19 cm and dry weight 5.821.84 mg, and the length of P. exuosus individuals was 2.410.16 cm and dry weight 13.533.20 mg. The data were tested for statistical signicance with two-way analysis of variance (ANOVA), after log(x+1) transformations of the data of experiment I.

Vegetation experiments The vegetation experiments (experiment III) were made to reveal the behaviour of mysids in the presence of predator signals. The experiments were conducted in July 2002 under similar conditions to the ingestion rate experiments described above. Three types of macrophytes were used: C. tomentosa, F. vesiculosus and articial vegetation (green plastic cords). The stems and cords were cut to t the height of the aquaria and placed through holes in metal plates that were placed on the bottom of the aquaria. There were 13 stems/ cords in each aquarium, covering one-half of the area. One mysid was added to each aquarium using a small sieve, and its behaviour was studied for 15 min by videolming. The mysids were put in the aquaria ca. 2 min prior to the beginning of the lming. The time spent swimming and hiding within the vegetation of N. integer and P. exuosus were determined from the lms. Any movement from one place to another was considered swimming activity. There were ten replicates of each mysid species in each vegetation type in both the predator signal treatment and in the control. The predator signal causing the strongest response, combined chemical and visual signal, was used as described above. After the experiments, the mysids were measured (total length) to the nearest millimetre and dried at 60C for ca. 12 h, after which their dry weight was measured. The length (meanSD) of N. integer individuals was 1.740.16 cm and dry weight 5.99 1.43 mg, and the length of P. exuosus individuals was 2.360.18 cm and dry weight 13.924.54 mg. The data were tested for statistical signicance with t-test and two-way analysis of variance (ANOVA), after arcsine-transformations of the data. There were slight deviances from normality in the data for time spent hiding in the vegetation (Wilk Shapiro: 0.86410.8707), which was taken into account in the interpretation of the results. Otherwise the data fullled the assumptions of parametric tests.

Materials and methods

Sampling The experiments were conducted in Tvarminne, on the south western coast of Finland. The mysids (Neomysis integer and Praunus exuosus) were collected from the littoral zone at daytime before each experiment using hand nets. They were transported to the laboratory within 30 min from collection in 10-l open containers lled with seawater taken from the collection site. Mysids were then starved at ambient water temperature (916C) in 0.2lm-ltered seawater in aerated containers in 16 h light:8 h dark photoperiods for at least 8 h before the beginning of the experiments. Brine shrimps (Artemia sp.) were hatched at 26C in 20& salinity. The predators, perch (Perca uviatilis), were caught with a sh trap in a shallow bay and kept in ltered seawater in a 30-l aerated container at ambient water temperature for at least 8 h before the beginning of the experiments. They were fed with littoral mysids. C. tomentosa was collected with a rake from a shallow bay, and F. vesiculosus from the rocky littoral.

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Results
Ingestion rate experiments In experiment I (Fig. 1a), Neomysis integer had higher feeding rates than Praunus exuosus (ANOVA: df=1,F=35.624,P<0.001) in both control and chemical signal treatments, but chemical predator signal had no statistically signicant eect on either of the species (ANOVA: df=1, F=1.530, P=0.224). The ingestion rate (lg C h)1, SE) of N. integer on Artemia sp. was 16.572.37 in the chemical signal treatments and 16.261.81 in the control, and the respective rates of P. exuosus were 4.301.55 and 6.561.50. In experiment II (Fig. 1b), there was a signicant dierence between the treatments (ANOVA: df=2, F=3.420, P=0.040), with the lowest feeding rate in the combined chemical and visual treatment. In contrast to experiment I, there was no statistically signicant difference between the mysid species (ANOVA: df=1, F=0.056,P=0.814). The ingestion rate (lg C h)1, SE)

of N. integer on Artemia sp. was 11.241.92 in the visual signal treatments, 9.081.64 in the combined chemical and visual treatments, and 11.622.11 in the control, and the respective rates of P. exuosus were 12.402.42, 5.721.79 and 12.652.18. Vegetation experiments Hiding within vegetation N. integer spent signicantly dierent amounts of time in the dierent macrophyte species (ANOVA: df=2, F= 6.479, P=0.003) (Fig. 2a), but the presence of the predator signal did not cause N. integer to signicantly change their behaviour in general (ANOVA: df=1, F= 1.204, P=0.277), except in articial vegetation (t-test: df=18, t=)2.292, P=0.034). As for P. exuosus (Fig. 2b), there were clear dierences between the use of dierent macrophyte species (ANOVA: df=2, F=3.321, P=0.044), F. vesiculosus being the most favoured one, with more than half of the time spent within it in both control and predator signal treatments. P. exuosus

Fig. 1 Ingestion rate (lg C h)1) (mean+SE) of Neomysis integer and Praunus exuosus on Artemia sp.: a experiment I: control and chemical signal treatments (n=10); b experiment II: control, visual signal and combined visual and chemical signal treatments (n=10). The aquaria did not contain vegetation

Fig. 2 Hiding time (%) in three dierent vegetation types (mean+SE) of: a Neomysis integer and b Praunus exuosus in control and visual and chemical signal treatments (n=10)

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spent more time hiding in the articial vegetation and in C. tomentosa when exposed to predator signals (ANOVA:df=1, F=5.065, P=0.031). P. exuosus individuals spent more time hiding within the vegetation than N. integer (t-test:df=118, t=2.362, P=0.020). The interaction between vegetation and predator signal was not statistically signicant for either mysid species (ANOVA: N. integer, df=2, F=2.272, P=0.113; P. exuosus, df=2, F=1.737, P=0.186). Swimming activity N. integer decreased their swimming activity when the predator signal was present (ANOVA: df=1, F=6.117, P=0.017) (Fig. 3a), without signicant dierences between the macrophyte species (ANOVA: df=2, F= 1.388, P=0.258). The presence of the predator signal did not cause P. exuosus to signicantly change their behaviour in respect to swimming activity (ANOVA: df=1, F=3.342, P=0.073) (Fig. 3b), nor were there clear dierences between the macrophyte species (ANOVA: df=2, F=2.903, P=0.063). The interaction

between vegetation and predator signal was not statistically signicant for either mysid species (ANOVA: N. integer, df=2, F=2.126, P=0.129; P. exuosus, df=2, F=2.818, P=0.069).

Discussion
The littoral mysids (Neomysis integer and Praunus exuosus) did not respond to either chemical or visual predator signals alone, but when combined, these signals reduced the ingestion rate signicantly. These results dier from the observations of Hamren and Hansson (1999), where the pelagic mysid Mysis mixta responded to chemical signals from predatory sh by reducing feeding rate. The habitats of pelagic and littoral mysids dier crucially from each other. Therefore, it is not surprising to nd dierent responses to similar cues in these two mysid groups. Pelagic mysids live in near darkness; they exhibit diel vertical migration, descending to the deep bottom layers at daytime to avoid predators and rising to the thermocline to feed at nighttime (Mauchline 1980). Littoral mysids, in contrast, can utilise vision in the well-lit shallow water they inhabit. Mysids have well-developed compound eyes, and are known to use vision in various situations (Fulton 1982; Lindstrom 2000). Variations in light level do not aect their ingestion rate (Viherluoto and Viitasalo 2001). Thus, the dierences in habitats seem to aect the way in which mysids detect predators; the chemical signals are relatively more important for mysids living in darker waters than for those living in habitats with more light. When only chemical predator signals were provided, mysids showed no response. There may be several explanations to this. First, the detection of a predator does not necessarily lead to major changes in behaviour (Magurran 1990). If the mysids are hungry enough, they may be willing to take the chance of being preyed upon and continue feeding despite the presence of a predator or its odour (Milinski and Heller 1978; van Duren and Videler 1996). In the present study, the mysids were starved before the experiments to assure that feeding took place and to standardise the hunger state, which may have weakened their response to the predator signals. Second, female mysids, which were used in this study, may have less developed olfaction capabilities than males. Male N. integer and P. exuosus have more elaborate chemosensory structures in their antennae and brain than females (Johansson and Hallberg 1992), which have been suggested to function in location of mates by detection of pheromones. Also, variation between individuals was large, and the feeding rate of P. exuosus was lower in experiment I than in experiment II. On the other hand, the values reported in this study are well within the range of earlier results (e.g. Viitasalo and Rautio 1998; Viherluoto and Viitasalo 2001). Despite the eorts to standardise the hunger state of the mysids, their previous feeding history remains unknown and may have aected the results. Large

Fig. 3 Swimming activity (%) in the presence of three dierent vegetation types (mean+SE) of: a Neomysis integer and bPraunus exuosus in control and visual and chemical signal treatments (n=10)

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variation is a common phenomenon in studies dealing with ingestion of individual mysids (e.g. Viitasalo and Rautio 1998; Viherluoto and Viitasalo 2001). Although both inhabit shallow water areas, N.integer and P. exuosus are far from being identical in their lifestyles and behaviours. Our results show that when the predator signal was present, N. integer responded by reducing swimming activity rather than by hiding within the vegetation, whereas P. exuosus did the opposite; it increased time spent hiding rather than reducing motility. In fact, P. exuosus even seemed to increase swimming activity in the presence of the predator signal in articial vegetation and F. vesiculosus, its most natural habitat. According to Stein and Magnuson (1976), predation pressure may in some cases result in increased activity. In addition, N. integer did utilise the articial vegetation as a refuge, unlike the two natural vegetation types. The appearance of the green plastic cords may have resembled lamentous algae of the uppermost littoral zone, which N. integer often inhabit. N. integer lives in swarms, while P. exuosus does not (Mauchline 1971; Arndt and Jansen 1986). In this study only one mysid at a time was tested, and the responses of N. integer reported here may dier somewhat from the responses of individuals in a swarm. Swarming is considered to be an eective antipredator mechanism (OBrien and Ritz 1988; Flynn and Ritz 1999). In addition, unlike many other sh species, perch is capable of successfully capturing prey among aquatic vegetation (Diehl 1988; Burks et al. 2001), which may decrease the protability of the hiding strategy of P. exuosus. Perch is a visual predator (Diehl 1988), and thus the strategy of N. integer to decrease motility, and thus conspicuousness, may prove to be the more eective one against this particular predator. In summary, the littoral mysids N. integer and P. exuosus respond most strongly to a predator when both chemical and visual signals of the predator are present. The mysid species behave dierently when perceiving predation risk: N. integer reduces swimming activity, whereas P. exuosus goes into hiding within aquatic macrophyte vegetation.
Acknowledgements We thank R. Munsterhjelm for his help in the determination of macrophytes, and T. Hakala, A. Hirvonen, S. Londesborough and T. Merilainen for their help in the collection of macrophytes. The Tvarminne Zoological Station, University of Helsinki, provided the eld and laboratory facilities. This study was nanced by the Walter and Andree de Nottbeck Foundation and the Academy of Finland. The experiments comply with the current laws of Finland.

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