UNSUCCESSFUL PREDATION AND LEARNING OF PREDATOR CUES BY CRAYFISH. Patrizia Acquistapace, Brian A. Hazlett, and Francesca Gherardi

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1 JOURNAL OF CRUSTACEAN BIOLOGY, 23(2): , 2003 UNSUCCESSFUL PREDATION AND LEARNING OF PREDATOR CUES BY CRAYFISH Patrizia Acquistapace, Brian A. Hazlett, and Francesca Gherardi (PA, BAH) Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, Michigan U.S.A. (corresponding author (BAH) (PA, FG) Dipartimento di Biologia Animale e Genetica Leo Pardi, Università di Firenze, Via Romana 17, 50125, Firenze, Italy ABSTRACT The effect of latent inhibition on learning about cues related to increased predation risk was studied in a native crayfish, Orconectes virilis, and in an invasive species, Orconectes rusticus. Individuals of both species either were exposed or not exposed to the conditioned stimulus (potential predator odor 5 goldfish odor) prior to simultaneous exposure to conditioned (potential predator odor) and unconditioned (crushed conspecific 5 alarm odor) stimuli. Crayfish were then tested one day and one week later for inhibition of feeding responses following introduction of goldfish odor. Both species were clearly inhibited in their formation of an association between the goldfish odor and a predator risk stimulus after a training period during which they were exposed only to the goldfish odor. Thus, the effect of latent inhibition was demonstrated in both species. The implications for the learning of cues associated with predation risk are discussed. Prey animals often need to learn to recognize cues from potential predators by associating those cues with direct evidence of predation such as haemolymph released from conspecifics during predation. The formation of a learned association has been demonstrated in fish (Chivers et al., 1995), damselflies (Chivers et al., 1996), and crayfish (Hazlett and Schoolmaster, 1998). Prey can even learn to show predation-avoidance behaviors to nonpredators such as goldfish, Carassius auratus, when goldfish cues are paired with alarm odors (Chivers and Smith, 1994; Hazlett et al., 2002). The ability to form associations may be especially well developed in invasive species because the introduction into new habitats may well expose individuals to new predators. The faster they can learn (Hazlett et al., 2002), the more efficient their predator avoidance behaviors will be in decreasing the risk of predation (Mathis and Smith, 1993). Crayfish can learn to associate the smell of potential predators and predation risk by the simultaneous detection of predator odor and the odor of crushed conspecific individuals (Hazlett and Schoolmaster, 1998). Invasive species such as Orconectes rusticus and Procambarus clarkii remember such learned associations longer than native species that are not expanding their ranges (Hazlett et al., 2002). In the behavioral ecology literature, predation has received considerable attention from both theoretical and empirical viewpoints. The importance of unsuccessful predation (Dietl, 2000) has received less attention. Unsuccessful predation has been recognized as a driving force in the evolution of prey traits (Abrams, 1989) because prey with traits that lead to unsuccessful predation will leave more offspring. In addition, unsuccessful predation that inflicts sublethal damage to prey can increase their vulnerability to future predation (Semlitsch, 1990). The presence of predators that prey recognize can also strongly influence prey behavior (Werner and Anholt, 1993). However, a largely unexplored aspect of predator-prey interactions is the role of unsuccessful predation on the learning of predator-related cues. The psychological literature has elucidated a multitude of factors that affect learning. Two processes seem particularly relevant to the role of unsuccessful predation in prey learning. One process is latent inhibition, where prior exposure to a stimulus (without any obvious reinforcement at the time of the exposure) results in a reduction of the strength of a learned association that could be formed later (Ferguson et al., 2001). Thus, the presence of stimuli from a predator without associated stimuli indicating predation (damaged/crushed conspecifics) 364

2 ACQUISTAPACE ET AL.: LATENT INHIBITION AND LEARNING OF CUES 365 should inhibit later learning that would occur normally from the pairing of predator cues and successful predation. A similar phenomenon is learned irrelevance (Bennet et al., 2000), in which random exposure to two cues prior to the simultaneous exposure to the two cues results in a reduction of the strength of the learned association that would otherwise follow from simultaneous exposure. In both of these phenomena, detection of a cue from a potential predator is not temporally linked to cues indicating increased predation risk, and, thus, when the two cues are detected at the same time, the formation of an association is inhibited. In this study, we examined the possible effects of latent inhibition on the formation of a learned association. Crayfish were either exposed or not exposed to the conditioned stimulus (potential predator odor) prior to simultaneous exposure to conditioned (potential predator odor) and unconditioned (crushed conspecific odor or alarm odor) stimuli, and the strengths of the resulting learned associations were compared. We predicted that crayfish exposed only to conditioned and unconditioned stimuli simultaneously would learn to associate the two odors better than those exposed first to the conditioned stimulus alone followed by a simultaneous exposure to both odors. Experiments were conducted on both an indigenous and an invasive species to see whether any differences exist that may allow alien species to survive better in a new environment with new predators. If latent inhibition has a reduced effect on alien species, they would be at an advantage in a new habitat where they would encounter unknown predator species. MATERIALS AND METHODS Experiments were conducted on two species of crayfish: Orconectes virilis (Hagen, 1870), a native species of the Midwest U.S.A. and Canada, and Orconectes rusticus (Girard, 1852), an Appalachian species that invaded many freshwater ecosystems originally occupied by O. virilis (Garvey et al., 1994; Hill and Lodge, 1999). Orconectes virilis individuals were collected from ponds in Saline, Michigan, and experiments were carried out at the University of Michigan, Ann Arbor, during January Orconectes rusticus were collected from Burt Lake near Pellston, Michigan, and studied at the University of Michigan Biological Station (UMBS), Pellston, Michigan, during July August Individuals of both species used in the experiments were intermoult adults. Most of the O. virilis individuals were Form II males with a cephalothorax ranging between 35 and 41 mm. Most of the O. rusticus specimens were females with a cephalothorax ranging between 34 and 44 mm. All crayfish were fed codfish before the beginning of the training and testing periods to familiarize them with the food odor source used during the experiments (Hazlett, 1994). Goldfish odor (Carassius auratus) was used as the source of potential predator odor. Crayfish should not treat this fish as a predator for two reasons: (a) it is an Asiatic species, and, thus, crayfish should not have experienced its odor before; (b) the goldfish is a herbivorous species. Previous experiments on both crayfish species have shown that they do not treat the goldfish as a predator without training (Hazlett et al., 2002). Individuals of both crayfish species were subjected to one of two training protocols as follows. No-experience Training. Twenty crayfish of each species were placed in one of two communal tanks for three days, 10 crayfish per tank; on the fourth day, they were exposed to the combination of goldfish and a predation-associated cue, conspecific alarm odor. The goldfish odor was introduced by placing two medium-sized (30 mm standard length) goldfish in a plastic opaque container with holes drilled in its sides that permitted the water containing their odor to diffuse into the crayfish water. This ensured that the crayfish would experience the odor but not the sight of the goldfish. A container was placed in each communal tank. The alarm odor was produced by crushing a medium-sized (20 25 mm cephalothorax length) conspecific individual in 150 ml of clean water and filtering the solution with coarse filter paper. Previous studies have shown that crayfish treat crushed conspecific odors as danger signals (Hazlett, 1999; 2000a; Hazlett and Schoolmaster, 1998). Two solutions were prepared; one was slowly poured into the water at the beginning of the two-hour goldfish exposure, and the second was added 30 min before the end of the training period. Hazlett et al. (2002) have shown that both species were able to learn to associate the conditioned and unconditioned stimuli after a two-hour exposure alone. Goldfish-experience Training. Twenty crayfish of each species were placed in one of two communal tanks (one for each species, 10 crayfish per tank) and exposed to goldfish odor two hours a day for three consecutive days. On the fourth day, crayfish were exposed to a combination of goldfish and alarm odors for two hours, as in the case of the no-experience training. At the end of each type of training, crayfish were placed in individual aquaria visually isolated from the others and from any possible source of disturbance. Aquaria were filled with 13 liters of lake water, constantly aerated, and provided with a clay pot used as shelter by the crayfish. Animals were tested during the day after a 24-hour acclimation period. Using a personal computer with an event program, we recorded the following behaviors and postures: (a) time spent in shelter; (b) time spent in locomotion by movement of the ambulatory legs; (c) time spent in a specific feeding movement (scraping: crayfish scratched the substrate using chelipeds and pereiopods); (d) time spent in each of the three postures: raised, intermediate, and lowered. In the raised posture, the body was elevated off the substrate, with the chelipeds held off the substrate parallel to the substrate or higher, and the abdomen and tail fan were extended. In the intermediate posture, the body was held just off the substrate, with the tips of the chelae lightly touching the substrate, and the tail fan was nearly perpendicular to the substrate. In the lowered posture, the body was in contact with the substrate, with the chelipeds drawn in towards the body, and the tail fan was curled under the abdomen. Because a crayfish had to be

3 366 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 23, NO. 2, 2003 in one of the three postures, the times in those postures are not independent, and only the time spent in the intermediate and raised postures was analyzed. For each crayfish, the number of seconds spent in all behaviors and postures were recorded for two minutes during three time periods as follows: (1) a 2-min control period following the injection of 5 ml of clean water; (2) a 2-min period following the injection of 5 ml of food odor; and (3) a 2-min period following the injection of 5 ml of goldfish odor. All solutions were introduced via a syringe. Food odor was prepared by macerating 2 g of codfish in 150 ml of clean water and filtering the solution with coarse filter paper. Goldfish odor was generated by placing two medium-size specimens in two liters of clean water for 24 h before testing. Previous work showed that presentation of two, potentially conflicting cues resulted in stronger changes in crayfish behaviors and postures (Hazlett, 1999; Bouwma and Hazlett, 2001); thus, the response to a potential predation risk was tested in the presence of a combination of food and goldfish odors. Previous work (Hazlett et al., 2002) showed that without experiencing goldfish and alarm odors simultaneously, individual crayfish showed no decrease in feeding-related behaviors upon introduction of goldfish odor. When the addition of goldfish odor caused a change in responses related to general feeding movements (in or out the shelter and different postures) and a decrease in the scraping behavior and in locomotion, crayfish were judged to give a positive response to goldfish odor and thus to treat this stimulus as a cue related to potential predation risk. In the cases of a significant response to goldfish odor, animals were observed one week after the initial testing to determine how long the learned association lasted. The Friedman two-way analysis of variance by ranks was used to compare the differences in responses among the three treatments (control, food odor, and goldfish odor); a Multiple Comparisons between Conditions procedure was used for pairwise comparisons of these treatments for behaviors and postures with a significant treatment effect (P, 0.05). RESULTS Locomotion and the scraping behavior were judged to be good measures of the crayfish response to the different treatments in both species. The raised posture was considered as a further measure of a positive response in the alien species. Orconectes virilis was studied during the winter period and appeared to be less active than O. rusticus. As a result, this species did not show the raised posture, and the intermediate posture was judged a good measure. The time spent in shelter did not differ among treatments and was not used to detect a potential predation risk response associated with the goldfish odor. Tests One Day After Training When crayfish had not experienced the goldfish odor during the training period, both species responded to the goldfish odor as a predator cue (Figs. 1, 2; Tables 1, 2). Orconectes virilis individuals showed a reduction in time spent in locomotion and the scraping behavior with the introduction of goldfish odor compared to the food-alone treatment. The intermediate posture increased significantly with the introduction of the food odor and tended to return to control values with the introduction of the goldfish odor (Fig. 1; Table 1). In the alien species Orconectes rusticus, locomotion and the scraping behavior were affected by the introduction of the goldfish odor, and the levels were reduced to those shown in the control treatment. In the presence of goldfish odor, the raised posture was not significantly different from the other two treatments (Fig. 2; Table 2). In both species, when crayfish had experienced the goldfish odor during the training period, there was no indication of the formation of an association between this cue and a potential predation risk (Figs. 1, 2; Tables 1, 2). In Orconectes virilis, the time spent in locomotion, the scraping behavior, and the intermediate posture increased significantly with the introduction of the food solution compared to the control period and did not change with the sequential injection of the goldfish odor (Fig. 1; Table 1). Similar results were obtained in Orconectes rusticus, where the time spent in locomotion, the specific-food related behavior, and the raised posture increased significantly when food odor was presented (Fig. 2; Table 2). Because of the lack of a positive response to goldfish odor, individuals of both species that were experienced with the goldfish odor were not tested further. Tests One Week After Training When tested one week later, individuals of both species seemed to have forgotten the association between the goldfish odor and a potential predation risk. The time spent in all behaviors and postures was not affected by the introduction of goldfish odor compared to that shown with the injection of food solution alone. In Orconectes rusticus, locomotion did not differ significantly among treatments (Tables 1, 2). DISCUSSION When individuals of the two species were subjected just to a training period of two-hour exposure to goldfish and alarm odors, individuals of both O. virilis and O. rusticus were able

4 ACQUISTAPACE ET AL.: LATENT INHIBITION AND LEARNING OF CUES 367 Fig. 1. Number of seconds (mean 1 SE) spent by Orconectes virilis in three feeding-related behaviors under three different treatments, when crayfish either were not experienced (no experience) or experienced (goldfish experience) with goldfish odor during the training period. Crayfish were observed one day after the training period. Different letters indicate significant differences (P, 0.05) among treatments (Multiple Comparisons between Conditions). to learn the association between the two cues and, consequently, treated the fish odor as a cue related to predation risk. When trained only once, the association was retained for less than one week in both species. This result differs quantitatively but not qualitatively from that reported in an earlier study (Hazlett et al., 2002), where the same pair of species was trained and tested with similar methods. In the earlier study, the alien species O. rusticus retained the memory for two weeks after the training period. Because both Orconectes rus- Fig. 2. Number of seconds (mean 1 SE) spent by Orconectes rusticus in three feeding-related behaviors under three different treatments, when crayfish either were not experienced (no experience) or experienced (goldfish experience) with goldfish odor during the training period. Crayfish were observed one day after the training period. Different letters indicate significant differences (P, 0.05) among treatments (Multiple Comparisons between Conditions).

5 368 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 23, NO. 2, 2003 Table 1. Orconectes virilis: comparison between inexperienced and experienced individuals. Comparisons among treatments (ctrl 5 control, food, gold 5 goldfish), used the Multiple Comparisons between Conditions procedure. Results are reported only for those behaviors and postures in which the Friedman two-way analysis of variance by ranks (F r ) showed a significant difference (P, 0.05). Results are presented for crayfish that have not (no experience) or have (goldfish experience) experienced the goldfish odor during the training period. No experience O. virilis Goldfish experience O. virilis 1 day after training F r F r Locomotion 26 food. ctrl 5 gold food 5 gold. ctrl Scraping food. ctrl 5 gold food 5 gold. ctrl Intermediate food. ctrl food 5 gold. ctrl 1 week after training F r Locomotion 38 food 5 gold. ctrl Scraping food 5 gold. ctrl Intermediate food 5 gold. ctrl ticus samples tested in the two experiments were collected from the same environment, the discrepancy in the length of the retained memory could be explained by the different goldfish varieties used. Odors released by the goldfish in the two experiments could be slightly different and/or more or less concentrated, thus to have a different effect on crayfish detection and memory retention. The duration of memory retention varys with the behaviors involved. Crayfish remember a food association for at least three weeks (Hazlett, 1994), whereas lobsters, Homarus americanus, remember social interactions for at least two weeks (Karavanich and Atema, 1998). Hazlett (1999) reported that Orconectes virilis showed an intermediate level of food-related activities when subjected to both predator and food cues but displayed a complete shut-down of feeding behaviors when tested with both alarm and food stimuli. In this case, the two stimuli worked in a hierarchical organization, with the alarm cue completely dominating the food odor. Alarm odors had a stronger effect than those of predator stimuli, probably because crayfish associate alarm odors with an immediate danger, represented both by a conspecific individual that has just been injured and the jeopardy of a potential attraction that this can be to other nearby predators. In this study, in the presence of both food and goldfish odors, both species displayed a complete shut-down of those behaviors closely associated with feeding, such as locomotion, the scraping behavior, and the raised posture. Similar results were obtained by Hazlett et al. (2002) for both species presented with the same combination of odors. The hierarchical organization of response shown here could be related to the training period to both alarm and predator odors one day before testing them with food and predator cues. The effect of latent inhibition was demonstrated in both species. When exposed to the unconditioned stimulus alone for three days before the unconditioned and the conditioned cues were presented together, individuals of both the native and the alien species continued to show normal feeding responses when Table 2. Orconectes rusticus: comparison between inexperienced and experienced individuals. Comparisons among treatments (ctrl 5 control, food, gold 5 goldfish), used the Multiple Comparisons between Conditions procedure. Results are reported only for those behaviors and postures in which the Friedman two-way analysis of variance by ranks (F r ) showed a significant difference (P, 0.01). Results are presented for crayfish that have not (no experience) or have (goldfish experience) experienced the goldfish odor during the training period. No experience O. rusticus Goldfish experience O. rusticus 1 day after training F r F r Locomotion food. ctrl 5 gold food 5 gold. ctrl Scraping food. ctrl 5 gold food 5 gold. ctrl Raised food. ctrl food 5 gold. ctrl 1 week after training F r Locomotion food 5 gold 5 ctrl Scraping food 5 gold. ctrl Raised food 5 gold. ctrl

6 ACQUISTAPACE ET AL.: LATENT INHIBITION AND LEARNING OF CUES 369 goldfish stimulus was added to the food odor. Individuals of these species that experienced the preassociation exposure to goldfish odor were clearly not treating the goldfish as a predator. Because the responses shown in this paper were the same for crayfish tested in winter (O. virilis) and summer (O. rusticus), season apparently did not affect the responses strongly. Following Kaplan and Lubow (2001), there are two general theories explaining latent inhibition. Both propose that the capacity to associate the unconditioned and the conditioned stimuli is altered as a result of a stimulus-noconsequence and/or a stimulus-context association during the exposure to the unconditioned stimulus. The attentional theories state that a decline in attention to the pre-exposed stimulus lead to a loss of association during the test phase. Applied to the current study, the tested animals are not able to detect the goldfish odor to the same degree. The retrieval theories do not invoke a loss of detection of the preexposed stimulus but rather propose that animals learn two conflicting associations during the two experimental phases that vary in retrievability. In the current study, crayfish learn to associate goldfish odor first to a nonpredatory condition, and then to a predatory situation. It would be of interest to study which, between the attentional and the retrieval theories, best explains the latent inhibition phenomenon that occurs in crayfish. The role of unsuccessful predation has been discussed by Abrams (1989). Predator-prey interactions are the driving forces of the adaptive coevolution between predator and prey. Unsuccessful predation may cause the predator to increase its adaptations for prey detection and can determine evolutionarily favored traits in predators. Abrams (1986) also suggested that prey always increase their investment in escape in response to increased predator investment in capture, and that analysis is not changed by adding a cost suffered by the prey in unsuccessful predation attempts (minor injuries, loss of foraging time, etc.). In this study, the hypothesis that latent inhibition would have a different effect on the native and the alien species was not upheld. While invasive species of crayfish use a broader range of danger signals (Gherardi et al., 2002; Hazlett, 2000b) and retain a learned association longer than do native species (Hazlett et al., 2002), no difference in resistance to latent inhibition was found. It would be interesting to test whether longer and/or multiple exposures to both alarm and goldfish odors (which would represent an increased predator success in capture) after a training period to only the unconditioned stimulus would change the latent inhibition effect on both the native and the invasive species. In these conditions, it may be that the alien species has a faster recovery in the associative learning between the two cues. ACKNOWLEDGEMENTS We acknowledge the Fulbright Foundation, whose support allowed PA to study as a Fulbright Scholar, and support from the University of Michigan Biological Station. LITERATURE CITED Abrams, P. A Adaptive response of predators to prey and prey to predators; the failure of the arms race analogy. Evolution 40: The evolution of rates of successful and unsuccessful predation. Evolutionary Ecology 3: Bennet, C. H., S. J. Wills, S. M. Oaakeshott, and N. J. Mackintosh Is the context specificity of latent inhibition a sufficient explanation of learned irrelevance? Quarterly Journal of Experimental Psychology. B. Comparative and Physiological Psychology 53B: Bouwma, P., and B. A. Hazlett Integration of multiple predator cues by the crayfish Orconectes propinquus. Animal Behaviour 61: Chivers, D. P., G. E. Brown, and R. J. F. Smith Acquired recognition of chemical stimuli from pike, Esox lucius, by brook stickleback, Culaea inconstans (Osteichthyes, Gasteroisteidae). Ethology 99: , and R. J. F. Smith Fathead minnows, Pimephales promelas, acquire predator recognition when alarm substance is associated with the sight of unfamiliar fish. Animal Behaviour 48: , B. D. Wisende, and R. J. F. Smith Damselfly larvae learn to recognize predators from chemical cues in the predator s diet. Animal Behaviour 52: Dietl, G. P Successful and unsuccessful predation of the gastropod Nucella lapillus (Muricidae) on the mussel Mytilus edulis from Maine. 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7 370 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 23, NO. 2, a. Responses to single and multiple sources of chemical cues by New Zealand crustaceans. Marine and Freshwater Behaviour and Physiology 34: b. Information use by an invading species: do invaders respond more to alarm odors than native species? Biological Invasions 2: , and D. R. Schoolmaster Responses of cambarid crayfish to predator odor. Journal of Chemical Ecology 24: , P. Acquistapace, and F. Gherardi Differences in memory capabilities in invasive and native crayfish. Journal of Crustacean Biology 22: Hill, A. M., and D. M. Lodge Replacement of resident crayfish by an exotic crayfish: the roles of competition and predation. Ecological Applications 9: Kaplan, O., and R. E. Lubow Context and reminder effects in a visual search analog of latent inhibition. Learning and Motivation 32: Karavanich, C., and J. Atema Individual recognition and memory in lobster dominance. Animal Behaviour 56: Mathis, A., and R. J. F. Smith Chemical alarm signals increase the survival time of fathead minnows (Pimephales promelas) during encounters with northern pike (Esox lucius). Behavioral Ecology 4: Semlitsch, R. D Effects of body size, sibship, and tail injury on the susceptibility of tadpoles to dragonfly predation. Canadian Journal of Zoology 68: Werner, E. E., and B. R. Anholt Ecological consequences of the trade-off between growth and mortality rates mediated by foraging activity. American Naturalist 142: RECEIVED: 4 April ACCEPTED: 16 September 2002.