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Effects of rearing conditions on sand digging efficiency in juvenile cuttlefish
Behavioural Processes 67 (2004) 273–279
Effects of rearing conditions on sand digging efficiency in juvenile cuttlefish R. Poirier a,b , R. Chichery a,b , L. Dickel a,b,∗ a
Laboratoire de Physiologie du Comportement des Cephalopodes, EA 3211, Université de Caen, Esplanade de la Paix, 14032 Caen Cedex, France b Centre Régional d’Études Cˆ otières, 54 Rue du Dr Charcot, 14530 Luc sur Mer, France Received 6 June 2003; received in revised form 21 April 2004; accepted 22 April 2004
Abstract The effect of environment on the maturation of sand digging behaviour in cuttlefish was studied. Sand digging behaviour of cuttlefish individually reared on sand was daily observed in their rearing tanks (first study). Other cuttlefish were individually reared from hatching to 2 weeks of life in different conditions (Group A, on a sandy substrate and group B, without sand). At days 0, 3, 6, 9, 12 and 15, cuttlefish from Groups A and B were placed in a novel tank, the bottom of which was covered by sand (second study). The first study shows that more and more cuttlefish sand dig in their rearing tank during the first 6 days of life. The second study shows that, confronted with a novel sand bottom, cuttlefish from Group A show shorter latencies of sand digging and they cover more completely than do cuttlefish from Group B. This indicates that the developmental changes in sand digging appear not totally pre-programmed, but at least partially experience-dependent. Presence of sand in rearing tanks may allow cuttlefish to acquire experience of digging to make this behaviour more efficient. © 2004 Elsevier B.V. All rights reserved. Keywords: Behavioural development; Cephalopod; Early experience; Sand digging; Sepia officinalis
1. Introduction In cuttlefish Sepia officinalis, the development of predatory behaviour has been the subject of several studies (Wells, 1958, 1962; Messenger, 1973; Dickel et al., 1997, 2000, 2001), but very few works concern the maturation of defensive strategies (Hanlon and Messenger, 1988). Sepia officinalis has two general defensive strategies: primary defence and secondary defence (Hanlon and Messenger, 1996). Whereas secondary defences (i.e. flight, deimatic behaviours, etc.) are brought into play when a cuttlefish has been de∗ Corresponding author. Tel.: +33-231-565583; fax: +33-231-565600. E-mail address: [email protected] (L. Dickel).
tected by a predator, primary defences (i.e. crypsis, involving general background resemblance or disruptive colouration) serve to reduce the risk of detection by a potential predator (Hanlon and Messenger, 1996; Messenger, 2001). Among cryptic behaviours reported (Hanlon and Messenger, 1996), cuttlefish can bury into the sand to conceal themselves from eventual predators or prey. To bury, cuttlefish settle on the sand, and squirt a series of water jets from the flexible funnel underneath the body followed by a lateral wiggling motion of the dorsal mantle (Boletzky, 1987; Mather, 1986). In several Sepiola and Sepietta species, the burying behaviour is a two-stage process (Boletzky and Boletzky, 1970), the first corresponding to what is known in Sepia. In the second process, the animal stretches out its dorso-lateral arms and, with a series
0376-6357/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.beproc.2004.04.006
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of sweeping movements, gathers sand all around to cover its head follows the water-jet stage. This second process is known only in sepiolids. In Sepia, digging is a relatively short and fixed behavioural sequence (lasting less than 5 s, Mather, 1986). Placing subadult cuttlefish on different substrates (fine sand, medium sand, gravel or beads), Mather (1986) has shown that latency to dig is unaffected by the type of substrate but that duration of digging was longer in beads and gravel than in medium and fine sand. More, change in the grain size of the sand into which the cuttlefish may dig (from fine sand to gravel) modifies slightly the amount of cover they may achieve. On the other hand, a large proportion of cuttlefish refused to bury in gravel (Mather, 1986). Mather therefore suggests that sand digging is stereotyped but does not seem rigidly fixed. In the literature, only occasional observations of buried hatchlings Sepia were reported (Boletzky, 1983; Hanlon and Messenger, 1988). There are also no data on the potential influence of a juvenile’s previous experience in the expression of sand digging. This is why, in the present study, we determined first at what age sand digging appears in cuttlefish reared in tanks with a sandy substrate. Then, we studied the maturation of sand digging behaviour in response to a stressor (in the context of this investigation, to a novel environment). The aim of this last study being, on both cuttlefish previously reared with or without sand, to determine whether sand digging maturation is rigidly fixed (i.e. independent of the previous experience of the juveniles), or not.
2. Materials and methods 2.1. Animals All individuals of S. officinalis used in these experiments came from a large number of eggs laid at the Laboratory of the “Centre Régional d’Études Cˆotières” (Luc sur Mer, France) and maintained (in large PVC tanks, 80 cm × 60 cm × 40 cm) under same conditions of water temperature (20 ± 1 ◦ C) and without sand, until hatching. For these studies, in order not to include prematurely hatched cuttlefish, hatchlings were taken on the day with the highest hatching proportion. We collected hatchlings (N = 265) at
8.00 a.m. from cuttlefish that had hatched during the night (i.e. within the previous 10 h).The mean dorsal mantle length (dml) of these hatchlings was dml = 8.6 ± 0.8 mm. In rearing conditions, all cuttlefish were placed individually in black plastic tanks (7.5 cm×8 cm×7 cm) and were maintained in the same system of circulating oxygenated seawater (T = 20 ± 1 ◦ C). All tanks were daylight illuminated. Particular care was taken to avoid any disturbance of cuttlefish during the period of rearing (e.g. sudden changes, vibrations or in lighting, abrupt movements of experimenters during feeding or tank cleaning, etc.). For the first study, 45 cuttlefish were placed individually in black plastic tanks containing fine sand (250 m in diameter) to a depth of 2 cm. For the second study, 220 cuttlefish were used. Twenty cuttlefish tested on the day of hatching (day 0) and 200 cuttlefish were kept in two rearing groups designated A and B groups: • In Group A, 100 cuttlefish were raised in the same conditions as those used for the first study. • In Group B, 100 cuttlefish were placed in the same conditions as Group A, without sand. Cuttlefish of each group were fed each day (between 6.00 and 7.00 p.m.) with mysid shrimp during the first week of life, and thereafter with shrimp and crabs of suitable size. 2.2. First study: daily observation on sand digging efficiency The 45 cuttlefish were daily observed between 9.00 and 10.00 a.m. in their rearing tanks. A Canon® digital video camera connected to a Panasonic® control monitor permanently mounted 50 cm above the tanks and could be moved from one tank to the next without disturbing the cuttlefish. In the course of these observations, we recorded the percentage of buried cuttlefish. All the cuttlefish sitting on the sand surface were considered to be “non-buried”. For the buried cuttlefish, we discriminated the completely buried cuttlefish from partially buried ones. When all the cuttlefish’s body except the eyes was covered by sand, the cuttlefish was considered to be “completely buried”, and when it was buried leaving a part of its mantle visible, the cuttlefish was considered to be “partially buried”.
R. Poirier et al. / Behavioural Processes 67 (2004) 273–279
2.3. Second study: effects of previous experience on sand digging efficiency Hatchlings (day 0) and cuttlefish from the different rearing conditions at days 3, 6, 9, 12 and 15, were placed in a novel tank different in form, dimension and colour (a round grey PVC tank, 30 cm diameter × 10 cm). The bottom of which was uniformly covered by 2 cm depth of fine sand (250 m in diameter). The sand was deep enough to enable them to bury completely. At hatching (day 0), two batches of 10 cuttlefish were tested, this procedure was also used at days 3, 6, 9, 12 and 15, for both Groups A and B. All tests were done under artificial light and were recorded by using a Canon® digital video camera mounted 50 cm above the water surface and connected to a Panasonic® VCR and a Panasonic® control monitor. All cuttlefish were tested only once. To prevent any of the cuttlefish burying before the beginning of the test, a glass cone (base diameter 7 cm, height 6 cm) was placed on the sand in the centre of the bottom of the test tank. A 5 cm diameter cylinder of dark grey opaque PVC was placed on the glass cone and 10 cuttlefish were introduced into the cylinder (Fig. 1). By this means, the cuttlefish were held above the sand level for a period of 1 min until the test began. When the cylinder was removed (t = 0), the cuttlefish were allowed to slide to the sand. At t = 90 s, we recorded the percentages of completely and partially buried cuttlefish. 2.4. Statistical analysis In the first study, to compare the frequencies of partially and completely buried cuttlefish in their rearing tanks between two ages, we used the Mac Nemar test for the significance of changes (Siegel and
275
Castellan, 1988). When the expected frequencies were very small, the binomial test was used rather than the Mac Nemar test (Siegel and Castellan, 1988). The alpha criterion was adjusted according to the number of comparisons, i.e. five. Thus, in that case α was 0.01. In the second study, at days 3, 6, 9, 12 and 15, we compared the frequencies of partially buried cuttlefish between Groups A and B and on the other hand, the frequencies of completely buried cuttlefish in the test tank between Groups A and B using the χ2 test for two independent samples (Siegel and Castellan, 1988). When the smallest expected frequencies were less than 5, we used the Fisher exact probability test (Siegel and Castellan, 1988). Statistics were performed with Statview© .
3. Results 3.1. First study: daily observation of sand digging efficiency One hour after being placed in their rearing tanks, only 8.9% of newly hatched cuttlefish (n = 45) were buried (Fig. 2A). At this stage, none of the cuttlefish was completely buried (Fig. 2B). During the first 2 weeks of post-embryonic life, the percentage of partially and completely buried cuttlefish changed: the percentage of partially buried cuttlefish significantly increased between days 0 and 3 (Fig. 2; Mac Nemar test, χ12 = 12, P < 0.001), and continued to increase up to day 9. After, it decreased to reach less than 45% at day 15 (Fig. 2A). Finally, even if the majority of cuttlefish remained partially buried in their rearing conditions (Fig. 2A), the percentage of completely buried ones progressively increased to reach 40% at day 15 (Fig. 2B). 3.2. Second study: effects of previous substrate on sand digging efficiency
Fig. 1. Schematic representation of the experimental apparatus (not drawn to scale).The PVC cylinder is removed from the cone at the beginning of the test.
When cuttlefish were placed in the test tank within the 10 h after hatching (day 0), 20% of them were partially buried and only 5% were completely buried (Fig. 3A and B). Thereafter, between days 0 and 9, the percentage of cuttlefish that partially buried when placed in the test tank progressively increased whatever their previous
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% of partially buried cuttlefish 100 90 80 70 60
*
50 40 30 20 10 (A)
0 0
3
6
3
6
9
12
15
age (days)
% of completely buried cuttlefish 100
90 80 70 60 50 40 30 20 10 0 (B)
0
9
12
15
age (days)
Fig. 2. (A) Percentage of cuttlefish which were found partially buried in their rearing tanks at days 0, 3, 6, 9, 12 and 15. (B) Percentage of cuttlefish which were found completely buried in their rearing tanks at days 0, 3, 6, 9, 12 and 15. Asterisk (∗) indicates significant differences between age groups (Mc Nemar test, α = 0.01).
rearing conditions (Fig. 3A). However, we observed some differences between cuttlefish from Groups A and B. At days 3 and 6, Fig. 3A shows that the percentage of partially buried cuttlefish from Group A in the test tank was more important that those of cuttlefish from Group B. At days 9 and 12, there was no significant differences between the two rearing groups. Finally at day 15, cuttlefish from Group B were significantly more partially buried than cuttlefish from Group A (Fig. 3A; χ2 test: χ12 = 6.4, P < 0.05).
Fig. 3B shows that the percentage of completely buried cuttlefish from Group A significantly increased between days 9 and 12: from 20% at day 9 to 65% at day 12 and 70% at day 15 (Fig. 3B; χ2 test: χ12 = 8.28, P < 0.01). At days 3, 6 and 9, Fig. 3B shows no difference between Groups A and B in the percentages of completely buried cuttlefish. However, at days 12 and 15, there were significantly more completely buried cuttlefish in Group A than in Group B (Fig. 3B; χ2 test
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Fig. 3. (A) Percentage of partially buried cuttlefish from Groups A and B, at days 0, 3, 6, 9, 12 and 15. (B) Percentage of completely buried cuttlefish at days 0, 3, 6, 9, 12 and 15. Asterisk (∗) indicates significant differences between Groups A (reared with sand) and B (reared without sand). The symbol (#) indicates significant differences between days 9 and 12 in Group A.
at day 12: χ12 = 4.91, P < 0.05; χ2 test at day 15: χ12 = 8.12, P < 0.005). 4. Discussion 4.1. Appearance of sand digging behaviour and changing in sand digging efficiency during early development According to our data, we can confirm preliminary observations (Boletzky, 1987; Hanlon and Messenger, 1988) about the capacity of hatchlings to produce the
behavioural pattern of sand digging (Fig. 2A and B). However, in the earliest post-embryonic development, we can now state that very few of them express the sand digging behaviour. However, the percentage of completely buried cuttlefish increased in their rearing tanks during the post-embryonic development (Fig. 2B). This observation confirms those made by Boletzky and Boletzky (1970) in several Sepiola and Sepietta species. One could argue that the low number of buried cuttlefish observed at hatching may be due to the size of the newly hatched cuttlefish in relation to that of the sand particles (the fine sand particles for hatch-
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lings may correspond to gravel for subadult cuttlefish). However, we can discard this hypothesis. Indeed, during the first 9 days of post-embryonic life, the proportion of buried cuttlefish drastically increases from 8.9 to 80% (Fig. 2A and B). This proportion increases much faster than their growth in size (from dml = 8.6 ± 0.8 mm at day 0 to dml = 11.4 ± 0.9 mm at day 9), the proportion of digging in 9-day-old cuttlefish reaching those found in subadult cuttlefish (dml = 50–80 mm) in fine sand (450 m in diameter, Mather, 1986). The appearance of sand digging during the first 2 weeks of life is therefore an outcome of behavioural maturation. 4.2. Maturation of sand digging efficiency and previous experience of the juveniles Whatever the rearing group, the percentage partially and completely buried cuttlefish in the test conditions increased during early post-embryonic development (Fig. 3A and B). Since the majority of cuttlefish from Group B (which had never seen sand before the test) dig in the sand from the ninth day, the development of this behaviour may appear relatively “pre-programmed”. However, the percentage of 3- and 6-day-old partially buried cuttlefish from Group A, in the test conditions, is higher than that of cuttlefish from Group B. This indicates that the presence of sand in rearing tanks is an efficient “facilitating” factor in the emergence of sand digging during the first days of post-embryonic development, indicating an obvious developmental flexibility in the appearance of this behaviour. Fig. 3B shows that the percentage of completely buried cuttlefish, which remains low (between 15 and 30%) in cuttlefish from Group B (rearing without sand) during the first 2 weeks of life, noticeably increases after day 9 in cuttlefish from Group A (reared with sand). Here again, to explain this behavioural change in the course of post-embryonic development, a hypothesis involving the growth in size of cuttlefish can be discarded: cuttlefish from Group A are not bigger than those of Group B at days 12 and 15 (dml at day 12: Group A = 12.1 ± 0.7 mm, Group B = 11.9 ± 0.8 mm; dml at day 15: Group A = 12.4 ± 0.8 mm, Group B = 12.2 ± 0.9 mm). An other hypothesis may be that cuttlefish which were kept on sandy substrates (Group A) were in-
evitably more practised in burying than those of Group B. The fact that, the majority of 6-, 9-, 12- and 15-day-old cuttlefish spends the daytime buried in the sand in their rearing tanks (first study) supports this hypothesis. In absence of potential predator, we can state that, in cuttlefish from Group A, sand digging may facilitate prey hunting by rendering cuttlefish less conspicuous towards their prey. Thus, comparison of sand-digging efficiency between cuttlefish coming from Groups A and B shows that these developmental changes are, at least, partially experience-dependent. 4.3. Sand digging and behavioural adaptation in early juveniles It is known that, placed on sandy substrate, nearly all adults sand dig in daytime (Mather, 1986). The fact that very few hatchlings bury may be due to a change of preference in concealment strategy during development. Our studies showing that some hatchlings are perfectly able to produce a sand-digging behaviour, particularly in stressful conditions (Fig. 3) may argue for this possibility. In this way, newly hatched cuttlefish could preferentially use their body patterns (which are mainly disruptive at hatching, Hanlon and Messenger, 1988) to conceal themselves in the field. They can choose to stay on a contrasted substrate on which the disruptive colouration is efficient (Hanlon and Messenger, 1988). In fact, we frequently observed in laboratory tanks that newly hatched cuttlefish remained among the eggs of the spawn for some days, being able to attach themselves to smooth surfaces, like those of the egg surface. For this, they can use the ventral surface of the mantle and the ventral arms as suckers (Boletzky, 1983). In this way, the spawn may constitute a shelter where hatchlings can efficiently hide from any potential predator. The proportion of buried cuttlefish noticeably increases during the first days of post-embryonic life, in their rearing tanks (Fig. 2) or in test conditions (Fig. 3). This phenomenon occurs in parallel with both maturation of prey-hunting behaviour and resorption of yolk reserves (Boletzky, 1983; Dickel et al., 1997). Then, during the first days of life, through being constrained to move out from the spawn to feed, cuttlefish become particularly vulnerable to predation. This could explain why sand digging occurs progressively in the course of development when cuttlefish are placed in
R. Poirier et al. / Behavioural Processes 67 (2004) 273–279
an open environment (as the rearing and the testing tanks in our experiments; Figs. 2 and 3). In conclusion, Mather (1986) suggested that sand digging behaviour is relatively stereotyped but not rigidly fixed. In the same way, this study showed that this behaviour is partially stereotyped (it progressively appears during the first days of development, independently of environmental conditions) but its qualitative expression, at least during the second week of life, is experience-dependent.
Acknowledgements We thank T. Lemoine for her help in these studies, and we are particularly grateful to Professor J. Lejeune (Biostatistician, University of Caen) for his help in statistics and J. Harris and C. Harris for the English corrections. References Boletzky, S.V., Boletzky, M.V.V., 1970. Das Eingraben in Sand bei Sepiola und Sepietta (Mollusca, Cephalopoda). Rev. Zool. 77, 536–548. Boletzky, S.V., 1983. Sepia officinalis. In: Boyle, P.R. (Ed.), Cephalopod Life Cycles: Species Accounts, vol. I. Academic Press, London, pp. 31–52.
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Boletzky, S.V., 1987. Juvenile behaviour. In: Boyle, P.R. (Ed.), Cephalopod Life Cycles: Comp. Rev., vol. II. Academic Press, London, pp. 45–60. Dickel, L., Chichery, M.P., Chichery, R., 1997. Postembryonic maturation of the vertical lobe complex and early Development of predatory behavior in the cuttlefish (Sepia officinalis). Neurob. Learn Mem. 67, 150–160. Dickel, L., Boal, J.G., Budelmann, B.U., 2000. The effect of early experience on learning and memory in cuttlefish. Dev. Psychobiol. 36, 101–110. Dickel, L., Chichery, M.P., Chichery, R., 2001. Increase of learning abilities and maturation of the vertical lobe complex during postembryonic development in the cuttlefish Sepia. Dev. Psychobiol. 39, 92–98. Hanlon, R.T., Messenger, J.B., 1988. Adaptative coloration in young cuttlefish (Sepia officinalis): the morphology and development of body patterns and their relation to behaviour. Phil. Trans. R. Soc. Lond. B 320, 437–487. Hanlon, R.T., Messenger, J.B., 1996. Cephalopod Behaviour. University Press, Cambridge. Mather, J.A., 1986. Sand burying in Sepia officinalis: assessment of a cephalopod mollusc’s ‘fixed’ behavior pattern. J. Comp. Psychol. 100, 315–320. Messenger, J.B., 1973. Learning performance and brain structure: a study in development. Brain Res. 58, 519–523. Messenger, J.B., 2001. Cephalopod chromatophores: neurobiology and natural history. Biol. Rev. 76, 473–528. Siegel, S., Castellan, N.J., 1988. Nonparametric Statistics, 2nd ed. McGraw-Hill, New York. Wells, M.J., 1962. Early learning in Sepia. Symp. Zool. Soc. Lond. 8, 149–169. Wells, M.J., 1958. Factors affecting reactions to Mysis by newly hatched Sepia. Behavior 13, 96–111.
Effects of rearing conditions on sand digging efficiency in juvenile cuttlefish R. Poirier a,b , R. Chichery a,b , L. Dickel a,b,∗ a
Laboratoire de Physiologie du Comportement des Cephalopodes, EA 3211, Université de Caen, Esplanade de la Paix, 14032 Caen Cedex, France b Centre Régional d’Études Cˆ otières, 54 Rue du Dr Charcot, 14530 Luc sur Mer, France Received 6 June 2003; received in revised form 21 April 2004; accepted 22 April 2004
Abstract The effect of environment on the maturation of sand digging behaviour in cuttlefish was studied. Sand digging behaviour of cuttlefish individually reared on sand was daily observed in their rearing tanks (first study). Other cuttlefish were individually reared from hatching to 2 weeks of life in different conditions (Group A, on a sandy substrate and group B, without sand). At days 0, 3, 6, 9, 12 and 15, cuttlefish from Groups A and B were placed in a novel tank, the bottom of which was covered by sand (second study). The first study shows that more and more cuttlefish sand dig in their rearing tank during the first 6 days of life. The second study shows that, confronted with a novel sand bottom, cuttlefish from Group A show shorter latencies of sand digging and they cover more completely than do cuttlefish from Group B. This indicates that the developmental changes in sand digging appear not totally pre-programmed, but at least partially experience-dependent. Presence of sand in rearing tanks may allow cuttlefish to acquire experience of digging to make this behaviour more efficient. © 2004 Elsevier B.V. All rights reserved. Keywords: Behavioural development; Cephalopod; Early experience; Sand digging; Sepia officinalis
1. Introduction In cuttlefish Sepia officinalis, the development of predatory behaviour has been the subject of several studies (Wells, 1958, 1962; Messenger, 1973; Dickel et al., 1997, 2000, 2001), but very few works concern the maturation of defensive strategies (Hanlon and Messenger, 1988). Sepia officinalis has two general defensive strategies: primary defence and secondary defence (Hanlon and Messenger, 1996). Whereas secondary defences (i.e. flight, deimatic behaviours, etc.) are brought into play when a cuttlefish has been de∗ Corresponding author. Tel.: +33-231-565583; fax: +33-231-565600. E-mail address: [email protected] (L. Dickel).
tected by a predator, primary defences (i.e. crypsis, involving general background resemblance or disruptive colouration) serve to reduce the risk of detection by a potential predator (Hanlon and Messenger, 1996; Messenger, 2001). Among cryptic behaviours reported (Hanlon and Messenger, 1996), cuttlefish can bury into the sand to conceal themselves from eventual predators or prey. To bury, cuttlefish settle on the sand, and squirt a series of water jets from the flexible funnel underneath the body followed by a lateral wiggling motion of the dorsal mantle (Boletzky, 1987; Mather, 1986). In several Sepiola and Sepietta species, the burying behaviour is a two-stage process (Boletzky and Boletzky, 1970), the first corresponding to what is known in Sepia. In the second process, the animal stretches out its dorso-lateral arms and, with a series
0376-6357/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.beproc.2004.04.006
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of sweeping movements, gathers sand all around to cover its head follows the water-jet stage. This second process is known only in sepiolids. In Sepia, digging is a relatively short and fixed behavioural sequence (lasting less than 5 s, Mather, 1986). Placing subadult cuttlefish on different substrates (fine sand, medium sand, gravel or beads), Mather (1986) has shown that latency to dig is unaffected by the type of substrate but that duration of digging was longer in beads and gravel than in medium and fine sand. More, change in the grain size of the sand into which the cuttlefish may dig (from fine sand to gravel) modifies slightly the amount of cover they may achieve. On the other hand, a large proportion of cuttlefish refused to bury in gravel (Mather, 1986). Mather therefore suggests that sand digging is stereotyped but does not seem rigidly fixed. In the literature, only occasional observations of buried hatchlings Sepia were reported (Boletzky, 1983; Hanlon and Messenger, 1988). There are also no data on the potential influence of a juvenile’s previous experience in the expression of sand digging. This is why, in the present study, we determined first at what age sand digging appears in cuttlefish reared in tanks with a sandy substrate. Then, we studied the maturation of sand digging behaviour in response to a stressor (in the context of this investigation, to a novel environment). The aim of this last study being, on both cuttlefish previously reared with or without sand, to determine whether sand digging maturation is rigidly fixed (i.e. independent of the previous experience of the juveniles), or not.
2. Materials and methods 2.1. Animals All individuals of S. officinalis used in these experiments came from a large number of eggs laid at the Laboratory of the “Centre Régional d’Études Cˆotières” (Luc sur Mer, France) and maintained (in large PVC tanks, 80 cm × 60 cm × 40 cm) under same conditions of water temperature (20 ± 1 ◦ C) and without sand, until hatching. For these studies, in order not to include prematurely hatched cuttlefish, hatchlings were taken on the day with the highest hatching proportion. We collected hatchlings (N = 265) at
8.00 a.m. from cuttlefish that had hatched during the night (i.e. within the previous 10 h).The mean dorsal mantle length (dml) of these hatchlings was dml = 8.6 ± 0.8 mm. In rearing conditions, all cuttlefish were placed individually in black plastic tanks (7.5 cm×8 cm×7 cm) and were maintained in the same system of circulating oxygenated seawater (T = 20 ± 1 ◦ C). All tanks were daylight illuminated. Particular care was taken to avoid any disturbance of cuttlefish during the period of rearing (e.g. sudden changes, vibrations or in lighting, abrupt movements of experimenters during feeding or tank cleaning, etc.). For the first study, 45 cuttlefish were placed individually in black plastic tanks containing fine sand (250 m in diameter) to a depth of 2 cm. For the second study, 220 cuttlefish were used. Twenty cuttlefish tested on the day of hatching (day 0) and 200 cuttlefish were kept in two rearing groups designated A and B groups: • In Group A, 100 cuttlefish were raised in the same conditions as those used for the first study. • In Group B, 100 cuttlefish were placed in the same conditions as Group A, without sand. Cuttlefish of each group were fed each day (between 6.00 and 7.00 p.m.) with mysid shrimp during the first week of life, and thereafter with shrimp and crabs of suitable size. 2.2. First study: daily observation on sand digging efficiency The 45 cuttlefish were daily observed between 9.00 and 10.00 a.m. in their rearing tanks. A Canon® digital video camera connected to a Panasonic® control monitor permanently mounted 50 cm above the tanks and could be moved from one tank to the next without disturbing the cuttlefish. In the course of these observations, we recorded the percentage of buried cuttlefish. All the cuttlefish sitting on the sand surface were considered to be “non-buried”. For the buried cuttlefish, we discriminated the completely buried cuttlefish from partially buried ones. When all the cuttlefish’s body except the eyes was covered by sand, the cuttlefish was considered to be “completely buried”, and when it was buried leaving a part of its mantle visible, the cuttlefish was considered to be “partially buried”.
R. Poirier et al. / Behavioural Processes 67 (2004) 273–279
2.3. Second study: effects of previous experience on sand digging efficiency Hatchlings (day 0) and cuttlefish from the different rearing conditions at days 3, 6, 9, 12 and 15, were placed in a novel tank different in form, dimension and colour (a round grey PVC tank, 30 cm diameter × 10 cm). The bottom of which was uniformly covered by 2 cm depth of fine sand (250 m in diameter). The sand was deep enough to enable them to bury completely. At hatching (day 0), two batches of 10 cuttlefish were tested, this procedure was also used at days 3, 6, 9, 12 and 15, for both Groups A and B. All tests were done under artificial light and were recorded by using a Canon® digital video camera mounted 50 cm above the water surface and connected to a Panasonic® VCR and a Panasonic® control monitor. All cuttlefish were tested only once. To prevent any of the cuttlefish burying before the beginning of the test, a glass cone (base diameter 7 cm, height 6 cm) was placed on the sand in the centre of the bottom of the test tank. A 5 cm diameter cylinder of dark grey opaque PVC was placed on the glass cone and 10 cuttlefish were introduced into the cylinder (Fig. 1). By this means, the cuttlefish were held above the sand level for a period of 1 min until the test began. When the cylinder was removed (t = 0), the cuttlefish were allowed to slide to the sand. At t = 90 s, we recorded the percentages of completely and partially buried cuttlefish. 2.4. Statistical analysis In the first study, to compare the frequencies of partially and completely buried cuttlefish in their rearing tanks between two ages, we used the Mac Nemar test for the significance of changes (Siegel and
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Castellan, 1988). When the expected frequencies were very small, the binomial test was used rather than the Mac Nemar test (Siegel and Castellan, 1988). The alpha criterion was adjusted according to the number of comparisons, i.e. five. Thus, in that case α was 0.01. In the second study, at days 3, 6, 9, 12 and 15, we compared the frequencies of partially buried cuttlefish between Groups A and B and on the other hand, the frequencies of completely buried cuttlefish in the test tank between Groups A and B using the χ2 test for two independent samples (Siegel and Castellan, 1988). When the smallest expected frequencies were less than 5, we used the Fisher exact probability test (Siegel and Castellan, 1988). Statistics were performed with Statview© .
3. Results 3.1. First study: daily observation of sand digging efficiency One hour after being placed in their rearing tanks, only 8.9% of newly hatched cuttlefish (n = 45) were buried (Fig. 2A). At this stage, none of the cuttlefish was completely buried (Fig. 2B). During the first 2 weeks of post-embryonic life, the percentage of partially and completely buried cuttlefish changed: the percentage of partially buried cuttlefish significantly increased between days 0 and 3 (Fig. 2; Mac Nemar test, χ12 = 12, P < 0.001), and continued to increase up to day 9. After, it decreased to reach less than 45% at day 15 (Fig. 2A). Finally, even if the majority of cuttlefish remained partially buried in their rearing conditions (Fig. 2A), the percentage of completely buried ones progressively increased to reach 40% at day 15 (Fig. 2B). 3.2. Second study: effects of previous substrate on sand digging efficiency
Fig. 1. Schematic representation of the experimental apparatus (not drawn to scale).The PVC cylinder is removed from the cone at the beginning of the test.
When cuttlefish were placed in the test tank within the 10 h after hatching (day 0), 20% of them were partially buried and only 5% were completely buried (Fig. 3A and B). Thereafter, between days 0 and 9, the percentage of cuttlefish that partially buried when placed in the test tank progressively increased whatever their previous
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% of partially buried cuttlefish 100 90 80 70 60
*
50 40 30 20 10 (A)
0 0
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6
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6
9
12
15
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% of completely buried cuttlefish 100
90 80 70 60 50 40 30 20 10 0 (B)
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age (days)
Fig. 2. (A) Percentage of cuttlefish which were found partially buried in their rearing tanks at days 0, 3, 6, 9, 12 and 15. (B) Percentage of cuttlefish which were found completely buried in their rearing tanks at days 0, 3, 6, 9, 12 and 15. Asterisk (∗) indicates significant differences between age groups (Mc Nemar test, α = 0.01).
rearing conditions (Fig. 3A). However, we observed some differences between cuttlefish from Groups A and B. At days 3 and 6, Fig. 3A shows that the percentage of partially buried cuttlefish from Group A in the test tank was more important that those of cuttlefish from Group B. At days 9 and 12, there was no significant differences between the two rearing groups. Finally at day 15, cuttlefish from Group B were significantly more partially buried than cuttlefish from Group A (Fig. 3A; χ2 test: χ12 = 6.4, P < 0.05).
Fig. 3B shows that the percentage of completely buried cuttlefish from Group A significantly increased between days 9 and 12: from 20% at day 9 to 65% at day 12 and 70% at day 15 (Fig. 3B; χ2 test: χ12 = 8.28, P < 0.01). At days 3, 6 and 9, Fig. 3B shows no difference between Groups A and B in the percentages of completely buried cuttlefish. However, at days 12 and 15, there were significantly more completely buried cuttlefish in Group A than in Group B (Fig. 3B; χ2 test
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Fig. 3. (A) Percentage of partially buried cuttlefish from Groups A and B, at days 0, 3, 6, 9, 12 and 15. (B) Percentage of completely buried cuttlefish at days 0, 3, 6, 9, 12 and 15. Asterisk (∗) indicates significant differences between Groups A (reared with sand) and B (reared without sand). The symbol (#) indicates significant differences between days 9 and 12 in Group A.
at day 12: χ12 = 4.91, P < 0.05; χ2 test at day 15: χ12 = 8.12, P < 0.005). 4. Discussion 4.1. Appearance of sand digging behaviour and changing in sand digging efficiency during early development According to our data, we can confirm preliminary observations (Boletzky, 1987; Hanlon and Messenger, 1988) about the capacity of hatchlings to produce the
behavioural pattern of sand digging (Fig. 2A and B). However, in the earliest post-embryonic development, we can now state that very few of them express the sand digging behaviour. However, the percentage of completely buried cuttlefish increased in their rearing tanks during the post-embryonic development (Fig. 2B). This observation confirms those made by Boletzky and Boletzky (1970) in several Sepiola and Sepietta species. One could argue that the low number of buried cuttlefish observed at hatching may be due to the size of the newly hatched cuttlefish in relation to that of the sand particles (the fine sand particles for hatch-
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lings may correspond to gravel for subadult cuttlefish). However, we can discard this hypothesis. Indeed, during the first 9 days of post-embryonic life, the proportion of buried cuttlefish drastically increases from 8.9 to 80% (Fig. 2A and B). This proportion increases much faster than their growth in size (from dml = 8.6 ± 0.8 mm at day 0 to dml = 11.4 ± 0.9 mm at day 9), the proportion of digging in 9-day-old cuttlefish reaching those found in subadult cuttlefish (dml = 50–80 mm) in fine sand (450 m in diameter, Mather, 1986). The appearance of sand digging during the first 2 weeks of life is therefore an outcome of behavioural maturation. 4.2. Maturation of sand digging efficiency and previous experience of the juveniles Whatever the rearing group, the percentage partially and completely buried cuttlefish in the test conditions increased during early post-embryonic development (Fig. 3A and B). Since the majority of cuttlefish from Group B (which had never seen sand before the test) dig in the sand from the ninth day, the development of this behaviour may appear relatively “pre-programmed”. However, the percentage of 3- and 6-day-old partially buried cuttlefish from Group A, in the test conditions, is higher than that of cuttlefish from Group B. This indicates that the presence of sand in rearing tanks is an efficient “facilitating” factor in the emergence of sand digging during the first days of post-embryonic development, indicating an obvious developmental flexibility in the appearance of this behaviour. Fig. 3B shows that the percentage of completely buried cuttlefish, which remains low (between 15 and 30%) in cuttlefish from Group B (rearing without sand) during the first 2 weeks of life, noticeably increases after day 9 in cuttlefish from Group A (reared with sand). Here again, to explain this behavioural change in the course of post-embryonic development, a hypothesis involving the growth in size of cuttlefish can be discarded: cuttlefish from Group A are not bigger than those of Group B at days 12 and 15 (dml at day 12: Group A = 12.1 ± 0.7 mm, Group B = 11.9 ± 0.8 mm; dml at day 15: Group A = 12.4 ± 0.8 mm, Group B = 12.2 ± 0.9 mm). An other hypothesis may be that cuttlefish which were kept on sandy substrates (Group A) were in-
evitably more practised in burying than those of Group B. The fact that, the majority of 6-, 9-, 12- and 15-day-old cuttlefish spends the daytime buried in the sand in their rearing tanks (first study) supports this hypothesis. In absence of potential predator, we can state that, in cuttlefish from Group A, sand digging may facilitate prey hunting by rendering cuttlefish less conspicuous towards their prey. Thus, comparison of sand-digging efficiency between cuttlefish coming from Groups A and B shows that these developmental changes are, at least, partially experience-dependent. 4.3. Sand digging and behavioural adaptation in early juveniles It is known that, placed on sandy substrate, nearly all adults sand dig in daytime (Mather, 1986). The fact that very few hatchlings bury may be due to a change of preference in concealment strategy during development. Our studies showing that some hatchlings are perfectly able to produce a sand-digging behaviour, particularly in stressful conditions (Fig. 3) may argue for this possibility. In this way, newly hatched cuttlefish could preferentially use their body patterns (which are mainly disruptive at hatching, Hanlon and Messenger, 1988) to conceal themselves in the field. They can choose to stay on a contrasted substrate on which the disruptive colouration is efficient (Hanlon and Messenger, 1988). In fact, we frequently observed in laboratory tanks that newly hatched cuttlefish remained among the eggs of the spawn for some days, being able to attach themselves to smooth surfaces, like those of the egg surface. For this, they can use the ventral surface of the mantle and the ventral arms as suckers (Boletzky, 1983). In this way, the spawn may constitute a shelter where hatchlings can efficiently hide from any potential predator. The proportion of buried cuttlefish noticeably increases during the first days of post-embryonic life, in their rearing tanks (Fig. 2) or in test conditions (Fig. 3). This phenomenon occurs in parallel with both maturation of prey-hunting behaviour and resorption of yolk reserves (Boletzky, 1983; Dickel et al., 1997). Then, during the first days of life, through being constrained to move out from the spawn to feed, cuttlefish become particularly vulnerable to predation. This could explain why sand digging occurs progressively in the course of development when cuttlefish are placed in
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an open environment (as the rearing and the testing tanks in our experiments; Figs. 2 and 3). In conclusion, Mather (1986) suggested that sand digging behaviour is relatively stereotyped but not rigidly fixed. In the same way, this study showed that this behaviour is partially stereotyped (it progressively appears during the first days of development, independently of environmental conditions) but its qualitative expression, at least during the second week of life, is experience-dependent.
Acknowledgements We thank T. Lemoine for her help in these studies, and we are particularly grateful to Professor J. Lejeune (Biostatistician, University of Caen) for his help in statistics and J. Harris and C. Harris for the English corrections. References Boletzky, S.V., Boletzky, M.V.V., 1970. Das Eingraben in Sand bei Sepiola und Sepietta (Mollusca, Cephalopoda). Rev. Zool. 77, 536–548. Boletzky, S.V., 1983. Sepia officinalis. In: Boyle, P.R. (Ed.), Cephalopod Life Cycles: Species Accounts, vol. I. Academic Press, London, pp. 31–52.
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