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The role of latent inhibition in acquired predator recognition by fathead minnows Maud C.O. Ferrari and Douglas P. Chivers

Abstract: The ability of prey animals to recognize and respond to potential predators has important survival consequences. In many predator–prey systems, prey need to learn which species are potential predators. Consequently, selection should favour efficient learning mechanisms. For aquatic organisms, a very effective way to learn to identify potential predators is by associating cues of injured conspecifics with cues of an unknown predator. To our knowledge, no studies of fishes have failed to show successful acquisition of predator recognition using this learning method. The goal of our study was to begin to address the limits of this learning paradigm. Specifically, we tested whether pre-exposure to a novel predator would prevent the associative learning from occurring. In the first treatment, we pre-exposed minnows to distilled water for 1 h on 5 consecutive days and then conditioned them with conspecific skin extract paired with charr odour. In the second treatment, minnows were pre-exposed to charr odour and conditioned with conspecific skin extract paired with charr odour. In the last treatment, minnows were pre-exposed to charr odour but ‘‘conditioned’’ with distilled water paired with charr odour. When tested for recognition of the charr odour alone, only the fish that were not pre-exposed to charr odour showed responses to the predators. We conclude that latent inhibition affects the efficiency of associative learning of the predator. Re´sume´ : La capacite´ des proies animales de reconnaıˆtre des pre´dateurs potentiels et de re´agir a des conse´quences importantes sur la survie. Dans plusieurs syste`mes pre´dateur–proie, les proies doivent apprendre quelles espe`ces sont des pre´dateurs potentiels. La se´lection devrait donc favoriser les me´canismes efficaces d’apprentissage. Chez les organismes aquatiques, une me´thode tre`s efficace d’apprentissage de la reconnaissance des proies potentielles est l’association des sig` notre connaissance, naux provenant d’un individu blesse´ de meˆme espe`ce avec les signaux d’un pre´dateur inconnu. A toutes les e´tudes utilisant cette me´thode d’apprentissage chez les poissons ont montre´ une acquisition re´ussie de la reconnaissance du pre´dateur. L’objectif de notre travail est de de´terminer, au moins de fac¸on pre´liminaire, les limites de ce paradigme d’apprentissage. Spe´cifiquement, nous de´terminons si une exposition pre´alable a` un nouveau pre´dateur empeˆche la re´alisation d’un apprentissage par association. Dans une premie`re expe´rience, nous avons expose´ pre´alablement des cyprins a` de l’eau distille´e pendant une heure durant cinq jours conse´cutifs et les avons ensuite conditionne´s avec un extrait de peau de leur espe`ce associe´ a` de l’odeur d’omble. Dans une seconde expe´rience, les cyprins ont e´te´ expose´s pre´alablement a` de l’odeur d’omble, puis conditionne´s avec un extrait de peau de leur espe`ce associe´ a` de l’odeur d’omble. Dans une dernie`re expe´rience, les cyprins ont e´te´ expose´s pre´alablement a` l’odeur d’omble, puis ‘‘conditionne´s’’ avec de l’eau distille´e associe´e a` de l’odeur d’omble. Lors d’e´valuations de la reconnaissance de l’odeur d’omble isole´e, seuls les poissons qui n’avaient pas e´te´ expose´s pre´alablement a` l’odeur d’omble ont re´agi aux pre´dateurs. Nous concluons qu’il existe une inhibition latente qui affecte l’efficacite´ de l’apprentissage par association du pre´dateur. [Traduit par la Re´daction]

Introduction Predation is a major selective force acting on both morphology and behaviour of prey. Failing to respond to a predation threat might cost the prey its life (Lima and Dill 1990). However, over-responding to a threat may cost the prey potential foraging and reproductive opportunities. Consequently, selection favours prey that display adaptive antipredator responses. Many prey species do not show innate recognition of their potential predators (invertebrates (Rochette et al. 1998), birds (Curio et al. 1978), mammals Received 16 September 2005. Accepted 13 February 2006. Published on the NRC Research Press Web site at http://cjz.nrc.ca on 31 March 2006. M.C.O. Ferrari1 and D.P. Chivers. Department of Biology, University of Saskatchewan, 112 Science Place, Saskatoon, SK S7N 5E2, Canada. 1Corresponding

author (e-mail: [email protected]).

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(McLean et al. 1996; Griffin et al. 2001), fishes (Mathis et al. 1993; Chivers and Smith 1994a; however, see Berejikian et al. 2003; Vilhunen and Hirvonen 2003)); hence, learning is necessary for them to acquire this information. Learned recognition of novel predators has been shown to significantly increase an individual’s survival during subsequent predator encounters (Mirza and Chivers 2000, 2001; Gazdewich and Chivers 2002). One way of learning to recognize unknown predators is to associate predator cues with direct evidence of predation. Chemical alarm cues released by prey that have been captured by predators provide such direct evidence of predation. For aquatic organisms, it has been demonstrated that pairing the sight or odour of an unknown predator with the odour of damaged conspecifics results in a learned association between the two cues. For example, Chivers et al. (1996) demonstrated that damselfly larvae learned to recognize predators from conspecific chemical cues in the predator diet. Hazlett and Schoolmaster (1998) showed that naı¨ve

doi:10.1139/Z06-027

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crayfish did not show innate avoidance of the odour of predatory snapping turtles but learned to avoid the predator after having experienced a pairing between the turtle and cues of crushed conspecifics. Acquired predator recognition through the pairing of conspecific alarm cues and cues of novel predators is most well documented in fishes (for review see Chivers and Smith 1998; Brown 2003). For example, Chivers and Smith (1994a, 1994b) showed that pike-naı¨ve fathead minnows (Pimephales promelas Rafinesque, 1820) can learn to recognize the sight or odour of a pike when it is associated with alarm cues. A single exposure to the chemical alarm cues paired with the novel predator cue seems enough for learning to occur. Moreover, it has been shown that once minnows learn to recognize the predator, they retain the response for at least 2 months. Acquired predator recognition after a single exposure to paired alarm cues and predator cues has also been demonstrated in the field with Atlantic salmon (Salmo salar L., 1758) (A.O.H.C. Leduc and G.E. Brown, personal communication). To our knowledge, no studies have failed to document learned predator recognition through pairing with alarm cues. Despite the incredible utility of acquired predator recognition through conditioning with alarm cues, very little work has addressed the limits of such learning. In one study, Hazlett (2003) demonstrated learned irrelevance in virile crayfish (Orconectes virilis (Hagen, 1870)). When predatornaı¨ve crayfish were given uncorrelated presentations of crushed conspecifics and the odour of a novel predator, associative learning failed to occur. In a second study, Acquistapace et al. (2003) demonstrated latent inhibition in crayfish. They showed that pre-exposure to the odour of a novel predator for 2 h per day during 3 consecutive days prevented subsequently learned association of risk with that cue. In this experiment, we investigated whether latent inhibition (i.e., pre-exposure to a novel predator odour) inhibits the subsequent associative learning of the predator odour. We exposed naı¨ve fathead minnows to distilled water or brook charr (Salvelinus fontinalis (Mitchill, 1814)) odour for 1 h per day during 5 consecutive days before conditioning them with conspecific skin extract paired with predator odour. The next day we tested the fish for recognition of brook charr odour alone.

Methods Test fish In September 2004, adult fathead minnows were captured using minnow traps at Feedlot pond, located on the University of Saskatchewan campus. This population of minnows originated from the South Saskatchewan River when the pond was filled in 1959 to provide water for agricultural purposes. Intensive trapping and gillnetting over the past 10 years revealed the presence of only one other fish species in the pond, brook stickleback (Culaea inconstans (Kirtland, 1840)), and the absence of any predatory fish. Moreover, previous experiments have demonstrated that minnows from this pond and nearby ponds do not show innate recognition of predatory fish cues (e.g., Chivers and Smith 1994a, 1995; Brown et al. 1997; Ferrari et al. 2005). The minnows were

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housed in an 18 000 L flow-through pool filled with dechlorinated tap water and containing a sandy substrate. The water temperature was maintained at 12 8C and the photoperiod was adjusted to a 14 h light : 10 h dark cycle. Minnows were fed ad libitum once a day with commercial flakes. Brook charr were obtained from the Fort Qu’Appelle fish hatchery, Saskatchewan, in October 2003. They were housed in a 6000 L flow-through pool filled with dechlorinated tap water at 12 8C. Charr were fed ad libitum once a day with commercial charr pellets. Stimulus collection Minnow skin extract We collected skin from four arbitrarily chosen minnows (mean fork length = 5.35 ± 0.30 (SD) cm). Minnows were killed with a blow to the head (in accordance with guidelines of the Canadian Council on Animal Care) and skin fillets were removed from both sides of the body and placed in 100 mL of chilled distilled water. Skin fillets were then homogenized and filtered through glass wool to remove any remaining tissues. We collected a total of 14.1 cm2 of skin in a total of 281 mL of distilled water, which constituted our stock solution containing ~1 cm2 of skin per 20 mL of distilled water. This solution was diluted 2000 times (~1 cm2 of skin per 40 L) to make the experimental solution of alarm cues and was then frozen in 20 mL aliquots at –20 8C until required. Charr odour Prey animals often exhibit antipredator responses to chemical cues of predators fed conspecifics of the prey, or closely related heterospecifics, but not those fed another diet (Chivers and Mirza 2001). Thus, two arbitrarily chosen brook charr (23.5 and 25 cm fork length) were kept in a 115 L tank and fed brine shrimp (Artemia spp.) for 5 days prior to stimulus collection. Both charr were then transferred to a 72 L tank containing 60 L of dechlorinated tap water and an air stone but no filter. The charr remained in the stimulus-collection tank for 24 h (the charr were not fed during this period) and were then transferred back to their initial holding pool. Water containing charr odour was frozen at –20 8C in 400 mL aliquots until required. Experimental protocol The experiment was conducted in three phases. During a 5-day pre-exposure period, groups of three minnows were exposed to the odour of charr (CO) or distilled water (DW) for 60 min each day. This phase was followed by a conditioning phase, which took place the next day, during which groups of minnows were exposed simultaneously to charr odour and conspecific skin extract (SE) or charr odour and distilled water. The next day, recognition testing was performed on each group of minnows by exposing them to charr odour only. During this phase, antipredator behaviours were recorded. We tested 20 groups of three minnows in each of three treatments: (1) 5 days of pre-exposure to distilled water followed by conditioning with skin extract and charr odour (5DW + SE*CO); (2) 5 days of pre-exposure to charr odour followed by conditioning with skin extract and charr odour (5CO + SE*CO); and (3) 5 days of pre-exposure #

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Conditioning phase An hour after the last pre-exposure stimulus injection, the fish were moved to identical 37 L tanks filled with clean dechlorinated tap water. One day later, the same protocol used for pre-exposure injections was repeated, but 5 mL of skin extract or distilled water followed by 20 mL of charr odour was injected into the tanks. Recognition testing phase An hour after the conditioning injections, fish were moved into identical 37 L tanks filled with clean dechlorinated tap water. After 24 h, recognition trials were performed. All trials were conducted between 1300 and 1700. Observations took place during an 8 min pre-stimulus-injection period and an 8 min post-stimulus-injection period. Following the pre-stimulus period, we injected 20 mL of charr odour into the tank, using the same injection protocol described above. Once the stimulus was fully injected, we began the post-stimulus observation period. As measures of antipredator response, we recorded the shoaling index of the three fish every 15 s (1, no fish within a body length of another; 2, two fish within a body length of each other; 3, all fish within a body length of each other). During the first 8 s of the 15 s period (a stop watch was set to beep after 8 s), the number of line crosses (using a 3  3 grid pattern drawn on the side of the tank) was also recorded for one of the three minnows (randomly chosen; the same fish was observed until the end of the observation period). An increase in shoaling index and a decrease in activity are two typical antipredator responses in minnows (for a review see Chivers and Smith 1998).

Change in shoaling index

Pre-exposure phase Twenty-four hours prior to the start of the experiment, groups of three minnows were placed in 37 L tanks (50 cm  25 cm  30 cm) filled with dechlorinated tap water (~13 8C, same photoperiod as mentioned before) for acclimation. Tanks were covered on three sides with black plastic to ensure visual isolation from neighbouring tanks. Fish were fed twice a day: 1 h after being transferred into their tank and 1 h prior to exposure (to reduce the potential trade-off between foraging and predator avoidance; Brown and Smith 1996). Each tank contained a gravel substrate, an air stone, and a 2 m piece of plastic tubing used to inject stimuli into the tank. All exposures were conducted between 1300 and 1500. Prior to stimulus injection, we withdrew and discarded 60 mL of water from the injection tubes (to remove any stagnant water) and then withdrew and retained an additional 60 mL. Twenty millilitres of charr odour or distilled water was then injected into the tank. We used the retained tank water to slowly flush the stimulus into the tank. An hour after the stimulus injection, the fish were moved to an identical 37 L tank filled with clean dechlorinated tap water. After 24 h, the second pre-exposure was performed, following the same procedure described above. This procedure was conducted for a total of 5 days.

Fig. 1. Mean (± SE) change in shoaling index (a) and line crosses (b) for fathead minnows (Pimephales promelas) exposed to charr (Salvelinus fontinalis) odour only during recognition trials. For 5 days prior to conditioning, each group of fish was pre-exposed to charr odour (5CO) or distilled water (5DW). Fish were then conditioned with skin extract paired with charr odour (SE*CO) or distilled water paired with charr odour (DW*CO) (control). 1 (a) 0.8 0.6 0.4 0.2 0 5DW+(SE*CO) 10 Change in line crosses

to charr odour followed by conditioning with distilled water and charr odour (5CO + DW*CO) (control). The mean fork length of the test fish was 5.50 ± 0.40 (SD) cm.

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5CO+(SE*CO)

5CO+(DW*CO)

(b)

0 -10 -20 -30 -40 -50 -60 -70 5DW + (SE*CO) 5CO + (SE*CO) 5CO + (DW*CO)

Statistical analysis We calculated the change in shoaling index and the change in line crosses from the pre-stimulus baseline. Due to the non-normality of the line cross data, we conducted nonparametric Mann–Whitney U tests to compare the effects of the different treatments. The family-wise error rate was assessed and controlled using the modified Bonferroni test following Keppel (1991). The modified Bonferroni test specifies that corrections to the family-wise error rate are introduced only when the number of comparisons exceeds k – 1, where k is the number of treatments (Keppel 1991). The modified alpha value is obtained by the following formula: alpha (0.05) times the degrees of freedom associated with treatment conditions (3 – 1 = 2), divided by the number of comparisons (3). Thus, the rejection probability was set at 0.033 for each comparison.

Results For both shoaling index (U = 24.5, P < 0.001; Fig. 1a) and line crosses (U = 4.5, P < 0.001; Fig. 1b), fish from the 5DW + SE*CO treatment exhibited stronger antipredator responses when exposed to charr odour alone than the control fish (5CO + DW*CO). Interestingly, fish in the 5CO + SE*CO treatment did not respond differently to charr odour #

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than the control fish (5CO + DW*CO) for both shoaling index (U = 84.5, P = 0.250; Fig. 1a) and line crosses (U = 84.0, P = 0.250; Fig. 1b). Moreover, fish in the 5DW + SE*CO treatment exhibited stronger antipredator responses to charr odour than fish in the 5CO + SE*CO treatment for both shoaling index (U = 18.0, P < 0.001) and line crosses (U = 8.5, P < 0.001).

Discussion Previous work has shown that under certain conditions, crayfish fail to learn predator recognition through conditioning with alarm cues (Acquistapace et al. 2003): repeated exposures to the novel predator odours for 3 consecutive days prevented the learned association. Likewise, our results show that under certain conditions, learned predator recognition through conditioning with alarm cues does not occur in fish. Five successive 60 min daily exposures to the odour of the novel predator were enough to stop the learned association from occurring. Indeed, fish pre-exposed to charr odour and then conditioned with charr odour paired with skin extract did not respond differently than control fish, which were pre-exposed to charr odour and conditioned with charr odour paired with distilled water, but their response was statistically different from that of fish pre-exposed to distilled water and conditioned with charr odour paired with skin extract. This is the first study documenting latent inhibition of predator recognition in fish. Until now, we thought that any odour paired with conspecific alarm cues would result in predator learning. In fact, Suboski et al. (1990) even showed that zebra danios (Danio rerio (Hamilton, 1822)) could learn to display an antipredator response to morpholine, a synthetic non-biological odour, paired with alarm cues. The results of our experiment indicate that in order for a fish to learn to recognize an odour, the odour likely needs to be relatively novel to the fish. Odours previously encountered in the absence of predation (i.e., background odours) seem not to be learned. From an ecological perspective, latent inhibition can be seen as an adaptive way to avoid learning irrelevant information. Indeed, aquatic organisms are exposed to a great variety of visual and chemical information present in their environment. When a predation event occurs, alarm cues will be released and spread throughout the area. The advantage of chemical cues over visual cues is that chemical cues travel farther and persist longer in the environment, increasing the chance that they will be detected by conspecifics. However, as they persist longer, there is also a greater probability that the alarm cues will be associated with non-relevant stimuli. Responding to non-threatening cues can be costly. Indeed, time spent hiding, dashing, or freezing is time that cannot be allocated to essential activities such as feeding, reproducing, or defending territories. While failing to learn predator cues to which they have been pre-exposed could serve to prevent prey from learning irrelevant information, it could also limit the ability of prey to learn potential predators. For example, prey exposed to predators that are opportunistic may fail to learn to recognize the predator because the low chance of being attacked means there is a low probability of alarm cues being associated with predator cues. Less efficient learning as a result of

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pre-exposure could also be problematic if predator specialization is such that only a portion of a predator population represents a threat to the prey. There are likely other ecological scenarios that would also result in less efficient learning. For example, depending on the active space of different cues, prey species living in flowing water, such as rivers, might fail to detect alarm cues from an upstream predation event but might still detect the odour of the ‘‘novel’’ predator. According to our results, this would also lead to inefficient learning of the predator identity. From this perspective, pre-exposure may be costly, as failure to learn to recognize a predator (even a low-risk cue) may result in death. Predation usually does not allow for a second chance. Future work should be undertaken to understand more completely the conditions under which predator learning occurs and how such information is used in mediating predator–prey interactions. In one study, Wisenden and Harter (2001) showed that minnows learned to recognize moving objects that were associated with alarm cues but not stationary objects. This seems adaptive, as predators need to move to capture and consume prey. Ferrari and Chivers (2006) showed that in the case of successive conditionings, recently learned information plays a major role in determining the intensity of antipredator responses displayed by fathead minnows exposed to predator odours. Learning occurs continuously and information is updated. For that reason, we speculate that pre-exposed minnows would eventually learn to respond to predation cues if they were conditioned repeatedly. At the time of this writing, it is not known how many exposures are needed to override the inhibitive effect of pre-exposure on recognition learning. Another interesting aspect would be to study the effect of a time delay between pre-exposure and conditioning. Timing likely has an important effect on prey’s perception of the novelty of the predator and hence the ability of the prey to learn to recognize the predator.

Acknowledgments The Natural Sciences and Engineering Research Council of Canada and the University of Saskatchewan provided financial support to D.P. Chivers. All work reported herein was conducted in accordance with University of Saskatchewan Committee of Animal Care and Supply protocol No. 19920077.

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Ferrari and Chivers pike by fathead minnows in a natural habitat. Environ. Biol. Fishes, 49: 89–96. doi:10.1023/A:1007302614292. Chivers, D.P., and Mirza, R.S. 2001. Predator diet cues and the assessment of predation risk by aquatic vertebrates: a review and prospectus. In Chemical signals in vertebrates. Vol. 9. Edited by D.A. Marchlewska-Koj, J.J. Lepri, and D. Mu¨ller-Schwarze, Plenum Press, New York. pp. 277–284. Chivers, D.P., and Smith, R.J.F. 1994a. The role of experience and chemical alarm signalling in predator recognition by fathead minnows, Pimephales promelas. J. Fish Biol. 44: 273–285. doi:10.1111/j.1095-8649.1994.tb01205.x. Chivers, D.P., and Smith, R.J.F. 1994b. Fathead minnows, Pimephales promelas, acquire predator recognition when alarm substance is associated with the sight of unfamiliar fish. Anim. Behav. 48: 597–605. doi:10.1006/anbe.1994.1279. Chivers, D.P., and Smith, R.J.F. 1995. Free-living fathead minnows rapidly learn to recognize pike as predators. J. Fish Biol. 46: 949–954. doi:10.1111/j.1095-8649.1995.tb01399.x. Chivers, D.P., and Smith, R.J.F. 1998. Chemical alarm signaling in aquatic predator/prey interactions: a review and prospectus. Ecoscience, 5: 338–352. Chivers, D.P., Wisenden, B.D., and Smith, R.J.F. 1996. Damselfly larvae learn to recognize predators from chemical cues in the predator’s diet. Anim. Behav. 52: 315–320. doi:10.1006/anbe. 1996.0177. Curio, E., Ernst, U., and Vieth, W. 1978. Cultural transmission of enemy recognition: one function of mobbing. Science (Washington, D.C.), 202: 899–901. Ferrari, M.C.O., and Chivers, D.P. 2006. The role of learning in the development of threat-sensitive predator avoidance: how do fathead minnows incorporate conflicting information? Anim. Behav. 71: 19–26. doi:10.1016/j.anbehav.2005.02.016. Ferrari, M.C.O., Trowell, J.J., Brown, G.E., and Chivers, D.P. 2005. The role of learning in the development of threat-sensitive predator avoidance by fathead minnows. Anim. Behav. 70: 777– 784. doi:10.1016/j.anbehav.2005.01.009. Gazdewich, K.J., and Chivers, D.P. 2002. Acquired predator recognition by fathead minnows: influence of habitat characteristics on survival. J. Chem. Ecol. 28: 439–445. doi:10.1023/ A:1017902712355. PMID: 11925078. Griffin, A.S., Evans, C.S., and Blumstein, D.T. 2001. Learning specificity in acquired predator recognition. Anim. Behav. 62: 577– 589. doi:10.1006/anbe.2001.1781.

509 Hazlett, B.A. 2003. Predator recognition and learned irrelevance in the crayfish Orconectes virilis. Ethology, 109: 765–780. doi:10. 1046/j.1439-0310.2003.00916.x. Hazlett, B.A., and Schoolmaster, D.R. 1998. Responses of cambarid crayfish to predator odor. J. Chem. Ecol. 24: 1757–1770. doi:10.1023/A:1022347214559. Keppel, G. 1991. Design and analysis: a researcher’s handbook. 3rd ed. Prentice-Hall, Engelwood Cliffs, N.J. Lima, S.L., and Dill, L.M. 1990. Behavioral decisions made under the risk of predation: a review and prospectus. Can. J. Zool. 68: 619–640. Mathis, A., Chivers, D.P., and Smith, R.J.F. 1993. Population differences in responses of fathead minnows (Pimephales promelas) to visual and chemical stimuli from predators. Ethology, 93: 31–40. McLean, I.G., Lundie-Jenkins, G., and Jarman, P.J. 1996. Teaching an endangered mammal to recognize predators. Biol. Conserv. 56: 51–62. Mirza, R.S., and Chivers, D.P. 2000. Predator-recognition training enhances survival of brook charr: evidence from laboratory and field enclosure studies. Can. J. Zool. 78: 2198–2208. doi:10. 1139/cjz-78-12-2198. Mirza, R.S., and Chivers, D.P. 2001. Chemical alarm signals enhance survival of brook charr (Salvelinus fontinalis) during encounters with chain pickerel (Esox niger). Ethology, 107: 989– 1006. doi:10.1046/j.1439-0310.2001.00729.x. Rochette, R., Arsenault, D.J., Justome, B., and Himmelman, J.H. 1998. Chemically-mediated predator recognition learning in a marine gastropod. Ecoscience, 5: 353–360. Suboski, M.D., Bain, S., Carty, A.E., McQuoid, L.M., Seelen, M.I., and Seifert, M. 1990. Alarm reaction in acquisition and social transmission of simulated predator recognition by zebra danio fish (Brachydanio rerio). J. Comp. Psychol. 104: 101–112. doi:10.1037/0735-7036.104.1.101. Vilhunen, S., and Hirvonen, H. 2003. Innate antipredator responses of Arctic charr (Salvelinus alpinus) depend on predator species and their diet. Behav. Ecol. Sociobiol. 55: 1–10. doi:10.1007/ s00265-003-0670-8. Wisenden, B.D., and Harter, K.R. 2001. Motion, not shape, facilitates association of predation risk with novel objects by fathead minnows (Pimephales promelas). Ethology, 107: 357–364. doi:10.1046/j.1439-0310.2001.00667.x.

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