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Embryonic visual learning in the cuttlefish, Sepia officinalis
ANIMAL BEHAVIOUR, 2008, 76, 131e134 doi:10.1016/j.anbehav.2008.02.006
Available online at www.sciencedirect.com
Embryonic visual learning in the cuttlefish, Sepia officinalis ANNE-SOP HI E D ARMAILLACQ, CLE´ M ENC E LESIM PLE & LUDO VIC D ICK EL
Groupe Me´moire et Plasticite´ comportementale and CREC, Universite´ de Caen Basse-Normandie, Caen (Received 14 November 2007; initial acceptance 11 January 2008; final acceptance 13 February 2008; published online 5 May 2008; MS. number: 9591)
The ability to learn about chemosensory stimuli in the prenatal period is now well established in a wide variety of vertebrate species. This may help to shape development and behaviour. Evidence for early visual learning has already been shown in newly hatched cuttlefish. We exposed cuttlefish embryos to crabs for at least a week before hatching. This crab exposure induced a subsequent visual preference for crabs in 7day-old juveniles. The results show for the first time embryonic visual learning in animals. Such cognitive abilities in embryos that can perceive visual stimulation could confer important adaptive advantages in processing and acquiring information about foods likely to be available after hatching. Ó 2008 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
Keywords: cuttlefish; exposure; embryonic cognition; invertebrate; prenatal learning; Sepia officinalis
Since the 17th century philosopher John Locke’s idea that newborn children can be considered to be blank slates at birth, research has repeatedly confirmed the crucial role of fetal sensory experience in behavioural and cognitive development (Smotherman & Robinson 1992; Alberts & Ronca 1993; Chapillon et al. 2002). Extensive studies of fetal behaviour have shown learning after a single exposure to chemosensory stimuli (humans: DeCasper & Spence 1986; Schaal et al. 2000; Mennella et al. 2001; rats, Rattus norwegicus: Hepper 1988, 1990; dogs, Canis familiaris: Wells & Hepper 2006) and both associative and nonassociative learning (Rudy & Cheatle 1977; Smotherman 1982; Smotherman & Robinson 1992; Hepper 1997; Gruest et al. 2004; Kawai et al. 2004). Overall, these studies suggest that prenatal sensory experience influences postnatal behaviour. Besides mammals, prenatal learning has been recorded in a wide variety of species (auditory imprinting: birds: Gottlieb 1976; Impekoven 1976; Shindler 1984; chemosensory learning: birds: Sneddon et al. 1998; reptiles: Sneddon et al. 2001; fish: Brannon 1972; amphibians: Hepper & Waldman 1992; insects: Isingrini et al. 1985; Caubet et al. 1992). Most of the studies mentioned above investigated prenatal exposure learning based on chemical or auditory stimulation, the most likely cues to pervade the embryo’s
Correspondence: L. Dickel, GMPc, Universite´ de Caen Basse Normandie, BP 5186, 14032 Caen cedex, France (email: ludovic.dickel@ unicaen.fr). 0003e 3472/08/$34.00/0
environment. They suggest that individuals prefer the stimulus to which they have been exposed before birth to an unfamiliar one. The widespread occurrence of prenatal sensory learning means that it may be critical to the individual’s survival in the perinatal period, for example for mother and kin recognition or feeding behaviour (Hepper 1996; Coureaud et al. 2002). This also emphasizes the importance of prenatal external influences in the development of behaviour (either the mother’s influence in mammals or the stimuli present within an ecological niche). As for prenatal visual stimulation, investigations in reptiles and birds showed that stimulation such as patterned light presented during the late prenatal period alters incubation times and postnatal auditory preferences (leopard geckos, Eublepharis macularius: Sleigh & Birchard 2001; bobwhite quails, Colinus virginianus: Honeycutt & Lickliter 2002). However, no studies of prenatal visual learning per se have been conducted in any vertebrate models so far, because visual cues are unlikely to be experienced by the embryos of mammals and birds. Among invertebrates, cuttlefish (a cephalopod mollusc) are suitable models for the study of development (reviewed in Dickel et al. 2006), and they rely strongly on vision in several aspects of their life history. For example, their predatory behaviour (Messenger 1968) and their defensive behaviour (e.g. body patterning) are mediated by the visual system (Hanlon & Messenger 1988; Chiao & Hanlon 2001). The eggs and the newly hatched young do not benefit from parental care so that the latter have to search autonomously for their own food after hatching.
131 Ó 2008 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
132
ANIMAL BEHAVIOUR, 76, 1
Last, the spirally coiled envelopes of the eggs are stained in black with ink. During late embryonic development, however, the elastic envelope is dilated by the increase in osmotic pressure of the perivitelline fluid (von Boletzky 1983) so that it becomes more transparent. Examination of the late embryonic stages showed that the structure of the eyes is by then fully developed (Lemaire 1970). There is evidence that the cuttlefish, Sepia officinalis, is able to learn in the immediate posthatch period. Postnatal visual exposure to crabs (a naturally nonpreferred prey) induced a significant change in the juvenile’s initial food preference (Darmaillacq et al. 2006a). This learning was subsequently characterized as being food imprinting (Darmaillacq et al. 2006b). Then, one question arises: are cuttlefish embryos able to learn about the visual characteristics of the prey present in the environment before hatching? We examined the effect of early embryonic exposure to crabs on subsequent prey preferences in S. officinalis juveniles and then addressed to what extent the envelope of the egg is a barrier to the perception of the immediate environment of the eggs.
METHODS
Study Animals We used eggs laid by wild-caught females obtained from trawling off Luc-sur-Mer, Calvados, France and kept in large tanks (1500 litres) at the CREC (Centre de Recherches en ˆ tier), Luc-sur-Mer, France. Females were Environnement Co fertilized either before they were caught or after mating in the tanks at the CREC. Eggs, initially laid in clusters that females attached to plastic meshes available in the tanks, were separated to ensure optimum development conditions and were put in shallow tanks. All tanks were supplied with running oxygenated sea water (18 1 C). As prey, we used crabs, carcinus sp. (carapace width 2e3 mm) and shrimps, Crangon crangon (1 cm long).
Apparatus: Crab Exposure to Embryos The experimental apparatus allowed controlled exposure to the crabs (Fig. 1). The experimental tanks (each 21 4 cm and 8 cm high) were made of opaque plastic. Each contained two internal, closed, elevated compartments running along each of the long sides into which we placed the crabs. The bottom and top of each elevated compartment were opaque and the inward faces were of clear glass to prevent the crabs escaping into the water but to allow their exposure to the embryos. This was also designed to prevent the embryos or the newly hatched cuttlefish perceiving chemical cues from the crabs. The side walls of each tank, below the elevated compartments, were perforated so that the sea water could circulate freely without allowing hatchlings to escape. In each experimental tank a maximum of 10 eggs were suspended from a nylon thread running along the centre line between the side compartments, the distal tips of the eggs being aligned with the lower edge of the raised
Water surface
2 1
1
3 Figure 1. Cross-section of the experimental apparatus to allow controlled exposure of crabs to the embryos. (1) Crabs were placed in opaque-bottomed, glass-fronted compartments with closed sides separated from the cuttlefish eggs. (2) The eggs were suspended lengthwise between the side compartments. (3) Newly hatched cuttlefish fell into the area, below the elevated compartments, from which the crabs were no longer visible. The apparatus was bottomless and placed in a larger tank to allow us to collect the newly hatched cuttlefish.
compartments so that the embryos (positioned head down in the eggs) could see the crabs. Once prepared, the experimental tanks were placed in a larger sea water tank (100 150 cm and 10 cm high; Fig. 1).
Procedure Eggs were assigned to one of three groups and arranged in the experimental apparatus described above. Eggs of group 1 (G1; N ¼ 24) were exposed to crabs (25 crabs in each of the two side compartments). Eggs of group 2 (G2; N ¼ 24) received the same treatment as G1 except that we removed the outer layers to make the envelope more transparent. As a control, eggs of group 3 (G3; N ¼ 20) were arranged in the same experimental apparatus but were not exposed to crabs. When they hatched, the cuttlefish fell below the side compartments so that they were no longer exposed to the crabs (Fig. 1). Since cuttlefish usually hatch at night (Paulij et al. 1991) they were collected in the morning (day 0) and isolated in individual, perforated, opaque plastic tanks (7 8 cm and 8 cm high) supplied with running sea water at 18 1 C. Juveniles were not fed and did not see or ‘smell’ any prey until day 7, the day of testing; they can endure a week of fasting because they still have inner nutritive reserves (von Boletzky 1975, 2003; Dickel et al. 1997). On day 7, we assessed their visual prey preference in a two way choice between crabs and sand shrimps (apparatus and procedure detailed in Darmaillacq et al. 2004). We recorded a preference for a prey when the cuttlefish carried out the visual attack sequence, ending in the cuttlefish being positioned in front of the corresponding compartment and about to attack the prey (Messenger 1968). Since cuttlefish usually attack prey within 1 min, we allowed the juvenile 5 min to make a choice. Beyond this time, we considered that the cuttlefish did not choose. We then recorded whether cuttlefish in each group preferred crabs or shrimps or made no choice. For half of the trials, crabs were presented in the left-hand compartment and for half in the right-hand one, alternately with shrimps. Each cuttlefish was tested individually and used only once.
DARMAILLACQ ET AL.: EMBRYONIC VISUAL LEARNING
After the study, the juvenile cuttlefish were released at low tide, in pools where prey were abundant.
Statistical Analysis Data were analysed with two-tailed nonparametric tests (Siegel & Castellan 1988) using StatXact-6 (Cytel Software Corporation, Cambridge, MA, U.S.A.). In the statistical analysis we considered only juveniles that made a choice. We used a permutation test to examine whether the length of exposure to crabs had an effect on the cuttlefish’s subsequent preference for shrimp or crab. The preference for crabs or shrimps within groups was analysed with the chisquare exact test. The Fisher’s exact test was used to examine whether prey preferences differed between groups.
RESULTS In G1, three eggs failed to hatch so that the final sample was 21. On day 7, 15 of the 21 juveniles chose crabs, three chose shrimps and three made no choice: there was a significant preference for crabs (chi-square exact test: c21 ¼ 8, P ¼ 0.006; Fig. 2). In G2, 21 of 24 cuttlefish hatched. Among them, 15 chose crabs, one chose shrimps and five made no choice: there was a significant preference for crabs (c21 ¼ 12:25, P ¼ 0.0005; Fig. 2). In the control group G3, 16 cuttlefish chose shrimps, two chose crabs and two made no choice: there was a significant preference for shrimps (c21 ¼ 10:89, P ¼ 0.001; Fig. 2). Counting back from the day of hatching (day 0), embryos had an average of 11.1 days of exposure to crabs (range 7e13 days; G1: X SEM ¼ 11:1 0:4 days; G2: 10.9 0.5 days). We observed no significant effect of the length of exposure to crabs on the juveniles’ subsequent
25 ° °
Number of cuttlefish
20 *
**
G1
G2
*
15
10
5
0
G3
Figure 2. Preference between crabs (-) and shrimps (,) by 7-dayold cuttlefish. In G1 and G2, eggs (i.e. embryos) were exposed to the sight of crabs for at least 1 week, except that in G2 the outer layers of the eggs had been removed. In G3, the embryos had no crab exposure. *Significant preference for one of the two prey items within groups (chi-square exact test: *P < 0.01; **P < 0.001); Bsignificant difference for prey choice between groups (Fisher’s exact test: P < 0.05).
food preference (permutation test: for G1 and G2: NS). Overall, 7-day-old juveniles that were exposed to crabs as embryos significantly preferred crabs to shrimps, compared to cuttlefish that were not (Fisher’s exact test, Bonferroni sequential method: G1/G2: NS; G1/G3: P ¼ 0.0003; G2/G3: P < 0.0001; Fig. 2).
DISCUSSION Our results show that exposure to crabs during the last stages of their embryonic development induces a preference for this prey in ingestively na€ıve 7-day-old juvenile cuttlefish. This is the first evidence of prehatch visual learning in invertebrates. Our results are the first behavioural evidence that the visual system is functional and efficient enough to perceive the shape of a potential future prey through the egg envelope. One of the biological roles of the black-stained envelope is to protect the embryos from predation (A. Guerra, personal communication cited in Dickel et al. 2006). Here we showed that cuttlefish from eggs exposed to crabs subsequently preferred crabs whether the egg envelope was intact or not, which means that the envelope does not prevent the embryos from perceiving the immediate surrounding environment. Darmaillacq et al. (2006b) reported that one exposure to crabs was effective in inducing a visual preference for crabs when it occurred shortly after hatching during a 2e4 h window. We propose that this window may be open even before hatching. More precisely, since we observed a postnatal preference for crabs when embryos were exposed to crabs for as little as 7 days, the sensitive period for exposure could take place within the last week of the embryonic phase. The ability of cuttlefish embryos to perceive visually and to process and learn particular characteristics of prey present in the vicinity of the eggs may confer important adaptive advantages. In mammals, prenatal olfactory learning of the maternal diet may provide the opportunity to learn about a safe and natural diet (Hudson & Distel 1999). In the absence of parental care, neonates have to avoid harmful food on their own. Female cuttlefish generally lay their eggs in shallow water (von Boletzky 1983), and may choose places where newly hatched juveniles can easily find potential prey. The ability to learn the visual characteristics of the prey in ovo would facilitate postnatal imprinting on this kind of prey. The importance of cognitive processes and the plasticity of food preferences in early juveniles are now well established (reviewed in Dickel et al. 2006). Here we have shown for the first time that cognitive processes occur in cuttlefish embryos and we emphasize a continuity of the interactions between the organism and its environment during the perinatal period. Acknowledgments We thank the staff of INSU-CNRS and the CREC. We also thank the referees for helpful comments and suggestions on the manuscript.
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References Alberts, J. R. & Ronca, A. E. 1993. Fetal experience revealed by rats: psychobiological insights. Early Human Development, 35, 153e 166. von Boletzky, S. 1975. A contribution to the study of yolk absorption in the Cephalopoda. Zeitschrift fu¨r Morphologie der Tiere, 80, 229e246. von Boletzky, S. 1983. Sepia officinalis. In: Cephalopod Life Cycles, Species Accounts, Vol. 1 (Ed. by P. R. Boyle), pp. 31e52. New York: Academic Press. von Boletzky, S. 2003. Biology of early life stages in cephalopod molluscs. Advances in Marine Biology, 44, 143e203. Brannon, E. L. 1972. Mechanisms controlling migration of sockeye salmon fry. International Pacific Salmon Fisheries Commission Bulletin, 21, 1e86. Caubet, Y., Jaisson, P. & Lenoir, A. 1992. Preimaginal induction of adult behaviour in insects. Quarterly Journal of Experimental Psychology, 44B, 165e178. Chapillon, P., Patin, V., Roy, V., Vincent, A. & Caston, J. 2002. Effects of pre- and postnatal stimulation on developmental, emotional, and cognitive aspects in rodents: a review. Developmental Psychobiology, 41, 373e387. doi:10.1002/dev.10066. Chiao, C. C. & Hanlon, R. T. 2001. Cuttlefish camouflage: visual perception of size, contrast and number of white squares on artificial checkerboard substrata initiates disruptive coloration. Journal of Experimental Biology, 204, 2119e2125. Coureaud, G., Schaal, B., Hudson, R., Orgeur, P. & Coudert, P. 2002. Transnatal olfactory continuity in the rabbit: behavioural evidence and short-term consequence of its disruption. Developmental Psychobiology, 40, 372e390. doi:10.1002/dev.10038. Darmaillacq, A.-S., Chichery, R., Poirier, R. & Dickel, L. 2004. The effect of early feeding experience on subsequent prey preference by cuttlefish, Sepia officinalis. Developmental Psychobiology, 45, 239e244. doi:10.1002/dev.20034. Darmaillacq, A.-S., Chichery, R., Shashar, N. & Dickel, L. 2006. Early familiarization overrides innate feeding preference in newly hatched Sepia officinalis cuttlefish. Animal Behaviour, 71, 511e514. doi:10.1016/j.anbehav.2005.04.019. Darmaillacq, A.-S., Chichery, R. & Dickel, L. 2006. Food imprinting, new evidence from the cuttlefish Sepia officinalis. Biology Letters, 2, 345e347. doi:10.1098/rsbl.2006.0477. DeCasper, A. J. & Spence, M. J. 1986. Prenatal maternal speech influences newborns’ perception of speech sounds. Infant Behavior & Development, 9, 133e150. Dickel, L., Chichery, M.-P. & Chichery, R. 1997. Postembryonic maturation of the vertical lobe complex and early development of predatory behaviour in the cuttlefish (Sepia officinalis). Neurobiology of Learning and Memory, 67, 150e160. Dickel, L., Darmaillacq, A.-S., Poirier, R., Agin, V., Bellanger, C. & Chichery, R. 2006. Behavioural and neural maturation in the cuttlefish Sepia officinalis. Vie Milieu, 56, 89e97. Gottlieb, G. 1976. Conceptions of prenatal development. Psychological Review, 83, 215e234. Gruest, N., Richer, P. & Hars, B. 2004. Emergence of long-term memory for conditioned aversion in the rat fetus. Developmental Psychobiology, 44, 189e198. doi:10.1002/dev.20004. Hanlon, R. T. & Messenger, J. B. 1988. Adaptive coloration in young cuttlefish (Sepia officinalis): the morphology and development of body patterns and their relation to behaviour. Philosophical Transactions of the Royal Society of London, Series B, 320, 437e487. Hepper, P. G. 1988. Adaptive fetal learning: prenatal exposure to garlic affects postnatal preferences. Animal Behaviour, 36, 935e936. Hepper, P. G. 1990. Foetal olfaction. In: Chemical Signals in Vertebrates 5 (Ed. by D. W. Macdonald, D. Mu ¨ ller-Schwarze & S. E. Natynczuk), pp. 282e288. Oxford: Oxford University Press.
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Available online at www.sciencedirect.com
Embryonic visual learning in the cuttlefish, Sepia officinalis ANNE-SOP HI E D ARMAILLACQ, CLE´ M ENC E LESIM PLE & LUDO VIC D ICK EL
Groupe Me´moire et Plasticite´ comportementale and CREC, Universite´ de Caen Basse-Normandie, Caen (Received 14 November 2007; initial acceptance 11 January 2008; final acceptance 13 February 2008; published online 5 May 2008; MS. number: 9591)
The ability to learn about chemosensory stimuli in the prenatal period is now well established in a wide variety of vertebrate species. This may help to shape development and behaviour. Evidence for early visual learning has already been shown in newly hatched cuttlefish. We exposed cuttlefish embryos to crabs for at least a week before hatching. This crab exposure induced a subsequent visual preference for crabs in 7day-old juveniles. The results show for the first time embryonic visual learning in animals. Such cognitive abilities in embryos that can perceive visual stimulation could confer important adaptive advantages in processing and acquiring information about foods likely to be available after hatching. Ó 2008 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
Keywords: cuttlefish; exposure; embryonic cognition; invertebrate; prenatal learning; Sepia officinalis
Since the 17th century philosopher John Locke’s idea that newborn children can be considered to be blank slates at birth, research has repeatedly confirmed the crucial role of fetal sensory experience in behavioural and cognitive development (Smotherman & Robinson 1992; Alberts & Ronca 1993; Chapillon et al. 2002). Extensive studies of fetal behaviour have shown learning after a single exposure to chemosensory stimuli (humans: DeCasper & Spence 1986; Schaal et al. 2000; Mennella et al. 2001; rats, Rattus norwegicus: Hepper 1988, 1990; dogs, Canis familiaris: Wells & Hepper 2006) and both associative and nonassociative learning (Rudy & Cheatle 1977; Smotherman 1982; Smotherman & Robinson 1992; Hepper 1997; Gruest et al. 2004; Kawai et al. 2004). Overall, these studies suggest that prenatal sensory experience influences postnatal behaviour. Besides mammals, prenatal learning has been recorded in a wide variety of species (auditory imprinting: birds: Gottlieb 1976; Impekoven 1976; Shindler 1984; chemosensory learning: birds: Sneddon et al. 1998; reptiles: Sneddon et al. 2001; fish: Brannon 1972; amphibians: Hepper & Waldman 1992; insects: Isingrini et al. 1985; Caubet et al. 1992). Most of the studies mentioned above investigated prenatal exposure learning based on chemical or auditory stimulation, the most likely cues to pervade the embryo’s
Correspondence: L. Dickel, GMPc, Universite´ de Caen Basse Normandie, BP 5186, 14032 Caen cedex, France (email: ludovic.dickel@ unicaen.fr). 0003e 3472/08/$34.00/0
environment. They suggest that individuals prefer the stimulus to which they have been exposed before birth to an unfamiliar one. The widespread occurrence of prenatal sensory learning means that it may be critical to the individual’s survival in the perinatal period, for example for mother and kin recognition or feeding behaviour (Hepper 1996; Coureaud et al. 2002). This also emphasizes the importance of prenatal external influences in the development of behaviour (either the mother’s influence in mammals or the stimuli present within an ecological niche). As for prenatal visual stimulation, investigations in reptiles and birds showed that stimulation such as patterned light presented during the late prenatal period alters incubation times and postnatal auditory preferences (leopard geckos, Eublepharis macularius: Sleigh & Birchard 2001; bobwhite quails, Colinus virginianus: Honeycutt & Lickliter 2002). However, no studies of prenatal visual learning per se have been conducted in any vertebrate models so far, because visual cues are unlikely to be experienced by the embryos of mammals and birds. Among invertebrates, cuttlefish (a cephalopod mollusc) are suitable models for the study of development (reviewed in Dickel et al. 2006), and they rely strongly on vision in several aspects of their life history. For example, their predatory behaviour (Messenger 1968) and their defensive behaviour (e.g. body patterning) are mediated by the visual system (Hanlon & Messenger 1988; Chiao & Hanlon 2001). The eggs and the newly hatched young do not benefit from parental care so that the latter have to search autonomously for their own food after hatching.
131 Ó 2008 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
132
ANIMAL BEHAVIOUR, 76, 1
Last, the spirally coiled envelopes of the eggs are stained in black with ink. During late embryonic development, however, the elastic envelope is dilated by the increase in osmotic pressure of the perivitelline fluid (von Boletzky 1983) so that it becomes more transparent. Examination of the late embryonic stages showed that the structure of the eyes is by then fully developed (Lemaire 1970). There is evidence that the cuttlefish, Sepia officinalis, is able to learn in the immediate posthatch period. Postnatal visual exposure to crabs (a naturally nonpreferred prey) induced a significant change in the juvenile’s initial food preference (Darmaillacq et al. 2006a). This learning was subsequently characterized as being food imprinting (Darmaillacq et al. 2006b). Then, one question arises: are cuttlefish embryos able to learn about the visual characteristics of the prey present in the environment before hatching? We examined the effect of early embryonic exposure to crabs on subsequent prey preferences in S. officinalis juveniles and then addressed to what extent the envelope of the egg is a barrier to the perception of the immediate environment of the eggs.
METHODS
Study Animals We used eggs laid by wild-caught females obtained from trawling off Luc-sur-Mer, Calvados, France and kept in large tanks (1500 litres) at the CREC (Centre de Recherches en ˆ tier), Luc-sur-Mer, France. Females were Environnement Co fertilized either before they were caught or after mating in the tanks at the CREC. Eggs, initially laid in clusters that females attached to plastic meshes available in the tanks, were separated to ensure optimum development conditions and were put in shallow tanks. All tanks were supplied with running oxygenated sea water (18 1 C). As prey, we used crabs, carcinus sp. (carapace width 2e3 mm) and shrimps, Crangon crangon (1 cm long).
Apparatus: Crab Exposure to Embryos The experimental apparatus allowed controlled exposure to the crabs (Fig. 1). The experimental tanks (each 21 4 cm and 8 cm high) were made of opaque plastic. Each contained two internal, closed, elevated compartments running along each of the long sides into which we placed the crabs. The bottom and top of each elevated compartment were opaque and the inward faces were of clear glass to prevent the crabs escaping into the water but to allow their exposure to the embryos. This was also designed to prevent the embryos or the newly hatched cuttlefish perceiving chemical cues from the crabs. The side walls of each tank, below the elevated compartments, were perforated so that the sea water could circulate freely without allowing hatchlings to escape. In each experimental tank a maximum of 10 eggs were suspended from a nylon thread running along the centre line between the side compartments, the distal tips of the eggs being aligned with the lower edge of the raised
Water surface
2 1
1
3 Figure 1. Cross-section of the experimental apparatus to allow controlled exposure of crabs to the embryos. (1) Crabs were placed in opaque-bottomed, glass-fronted compartments with closed sides separated from the cuttlefish eggs. (2) The eggs were suspended lengthwise between the side compartments. (3) Newly hatched cuttlefish fell into the area, below the elevated compartments, from which the crabs were no longer visible. The apparatus was bottomless and placed in a larger tank to allow us to collect the newly hatched cuttlefish.
compartments so that the embryos (positioned head down in the eggs) could see the crabs. Once prepared, the experimental tanks were placed in a larger sea water tank (100 150 cm and 10 cm high; Fig. 1).
Procedure Eggs were assigned to one of three groups and arranged in the experimental apparatus described above. Eggs of group 1 (G1; N ¼ 24) were exposed to crabs (25 crabs in each of the two side compartments). Eggs of group 2 (G2; N ¼ 24) received the same treatment as G1 except that we removed the outer layers to make the envelope more transparent. As a control, eggs of group 3 (G3; N ¼ 20) were arranged in the same experimental apparatus but were not exposed to crabs. When they hatched, the cuttlefish fell below the side compartments so that they were no longer exposed to the crabs (Fig. 1). Since cuttlefish usually hatch at night (Paulij et al. 1991) they were collected in the morning (day 0) and isolated in individual, perforated, opaque plastic tanks (7 8 cm and 8 cm high) supplied with running sea water at 18 1 C. Juveniles were not fed and did not see or ‘smell’ any prey until day 7, the day of testing; they can endure a week of fasting because they still have inner nutritive reserves (von Boletzky 1975, 2003; Dickel et al. 1997). On day 7, we assessed their visual prey preference in a two way choice between crabs and sand shrimps (apparatus and procedure detailed in Darmaillacq et al. 2004). We recorded a preference for a prey when the cuttlefish carried out the visual attack sequence, ending in the cuttlefish being positioned in front of the corresponding compartment and about to attack the prey (Messenger 1968). Since cuttlefish usually attack prey within 1 min, we allowed the juvenile 5 min to make a choice. Beyond this time, we considered that the cuttlefish did not choose. We then recorded whether cuttlefish in each group preferred crabs or shrimps or made no choice. For half of the trials, crabs were presented in the left-hand compartment and for half in the right-hand one, alternately with shrimps. Each cuttlefish was tested individually and used only once.
DARMAILLACQ ET AL.: EMBRYONIC VISUAL LEARNING
After the study, the juvenile cuttlefish were released at low tide, in pools where prey were abundant.
Statistical Analysis Data were analysed with two-tailed nonparametric tests (Siegel & Castellan 1988) using StatXact-6 (Cytel Software Corporation, Cambridge, MA, U.S.A.). In the statistical analysis we considered only juveniles that made a choice. We used a permutation test to examine whether the length of exposure to crabs had an effect on the cuttlefish’s subsequent preference for shrimp or crab. The preference for crabs or shrimps within groups was analysed with the chisquare exact test. The Fisher’s exact test was used to examine whether prey preferences differed between groups.
RESULTS In G1, three eggs failed to hatch so that the final sample was 21. On day 7, 15 of the 21 juveniles chose crabs, three chose shrimps and three made no choice: there was a significant preference for crabs (chi-square exact test: c21 ¼ 8, P ¼ 0.006; Fig. 2). In G2, 21 of 24 cuttlefish hatched. Among them, 15 chose crabs, one chose shrimps and five made no choice: there was a significant preference for crabs (c21 ¼ 12:25, P ¼ 0.0005; Fig. 2). In the control group G3, 16 cuttlefish chose shrimps, two chose crabs and two made no choice: there was a significant preference for shrimps (c21 ¼ 10:89, P ¼ 0.001; Fig. 2). Counting back from the day of hatching (day 0), embryos had an average of 11.1 days of exposure to crabs (range 7e13 days; G1: X SEM ¼ 11:1 0:4 days; G2: 10.9 0.5 days). We observed no significant effect of the length of exposure to crabs on the juveniles’ subsequent
25 ° °
Number of cuttlefish
20 *
**
G1
G2
*
15
10
5
0
G3
Figure 2. Preference between crabs (-) and shrimps (,) by 7-dayold cuttlefish. In G1 and G2, eggs (i.e. embryos) were exposed to the sight of crabs for at least 1 week, except that in G2 the outer layers of the eggs had been removed. In G3, the embryos had no crab exposure. *Significant preference for one of the two prey items within groups (chi-square exact test: *P < 0.01; **P < 0.001); Bsignificant difference for prey choice between groups (Fisher’s exact test: P < 0.05).
food preference (permutation test: for G1 and G2: NS). Overall, 7-day-old juveniles that were exposed to crabs as embryos significantly preferred crabs to shrimps, compared to cuttlefish that were not (Fisher’s exact test, Bonferroni sequential method: G1/G2: NS; G1/G3: P ¼ 0.0003; G2/G3: P < 0.0001; Fig. 2).
DISCUSSION Our results show that exposure to crabs during the last stages of their embryonic development induces a preference for this prey in ingestively na€ıve 7-day-old juvenile cuttlefish. This is the first evidence of prehatch visual learning in invertebrates. Our results are the first behavioural evidence that the visual system is functional and efficient enough to perceive the shape of a potential future prey through the egg envelope. One of the biological roles of the black-stained envelope is to protect the embryos from predation (A. Guerra, personal communication cited in Dickel et al. 2006). Here we showed that cuttlefish from eggs exposed to crabs subsequently preferred crabs whether the egg envelope was intact or not, which means that the envelope does not prevent the embryos from perceiving the immediate surrounding environment. Darmaillacq et al. (2006b) reported that one exposure to crabs was effective in inducing a visual preference for crabs when it occurred shortly after hatching during a 2e4 h window. We propose that this window may be open even before hatching. More precisely, since we observed a postnatal preference for crabs when embryos were exposed to crabs for as little as 7 days, the sensitive period for exposure could take place within the last week of the embryonic phase. The ability of cuttlefish embryos to perceive visually and to process and learn particular characteristics of prey present in the vicinity of the eggs may confer important adaptive advantages. In mammals, prenatal olfactory learning of the maternal diet may provide the opportunity to learn about a safe and natural diet (Hudson & Distel 1999). In the absence of parental care, neonates have to avoid harmful food on their own. Female cuttlefish generally lay their eggs in shallow water (von Boletzky 1983), and may choose places where newly hatched juveniles can easily find potential prey. The ability to learn the visual characteristics of the prey in ovo would facilitate postnatal imprinting on this kind of prey. The importance of cognitive processes and the plasticity of food preferences in early juveniles are now well established (reviewed in Dickel et al. 2006). Here we have shown for the first time that cognitive processes occur in cuttlefish embryos and we emphasize a continuity of the interactions between the organism and its environment during the perinatal period. Acknowledgments We thank the staff of INSU-CNRS and the CREC. We also thank the referees for helpful comments and suggestions on the manuscript.
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