Visual Contrast Modulates Maturation of Camouflage Body Patterning in ( pharaonis)

Yi-Hsin Lee National Tsing Hua University

Hong Young Yan Academia Sinica

Chuan-Chin Chiao National Tsing Hua University

Abstract

Camouflage is the primary defense behavior in . It is known that cuttlefish immediately after hatching are capable of showing various body patterns for concealing themselves, however recent studies suggest that maturation of camouflage body patterns is faster for cuttlefish (Sepia officinalis) reared in enriched environments than those reared in impoverished environments. Since camouflage patterning in cephalopods is predominately visually driven, this study specifically investigates effects of the rearing background contrast on the maturation of body patterns in cuttlefish (Sepia pharaonis). Newly hatched were separated into two cohorts, one reared in a uniform-gray background (low-contrast, or L group) and the other raised in a black/white checkerboard background (high-contrast, or H group). At Weeks 2, 4, 6, 8, 10, and 12, cuttlefish were placed individually either on uniform or checkerboard substrates to examine their body patterns. Animals from both L and H groups appear to show moderate disruptive patterns on the checkerboard and less disruptive on the uniform background at Week 2. Throughout development, however, cuttlefish from the H group showed stronger disruptive patterns than that of the L group on the checkerboard background at Weeks 10 and 12. In interesting findings, cuttlefish from both L and H groups showed similar strength but different disruptive components on the uniform background in later postembryonic stages. These results suggest that the maturation of camouflage body patterns in S. pharaonis is at least in part affected by visual contrast of their rearing backgrounds, although environmental complexity or social interaction is also likely to be involved in this process. This also implies that early visual experience could exert its effect on the seemingly preprogrammed behaviors such as camouflage body patterning in cephalopods.

Yi-Hsin Lee, Institute of Molecular Medicine, National Tsing Hua University; Hong Young Yan, Institute of Cellular and Organismic Biology, Academia Sinica; and Chuan-Chin Chiao, Institute of Molecular Medicine, Institute of Systems Neuroscience, and Department of Life Science, National Tsing Hua University. This work is supported by the National Science Council of Taiwan (NSC-95–2621-B-007–001-MY3 and NSC-98–2628-B-007– 001-MY3 to CCC; NSC-94–2313-B-0z01–010, NSC-95–2313-B-001–024, and NSC-96–2313-B-001–006 to HYY), and a Thematic Research Grant from Academia Sinica (AS-95-TP-B05 to HYY). Correspondence concerning this article should be addressed to Chuan-Chin Chiao, PhD, Department of Life Science, National Tsing Hua University, 101, Section 2, Kuang Fu Road, Hsinchu, 30013, Taiwan. E-mail: [email protected] 1

Experience-dependent plasticity is an important discovery in modern neuroscience (Hooks & Chen, 2007; Karmarkar & Dan, 2006). In late 1940s, Donald Hebb first proposed the concept of use-dependent plasticity of the nervous system, and emphasized the influence of enriched environment in improving learning and behavior (Hebb, 1949). Subsequently, many studies in rodents have demonstrated that environmental complexity can significantly enhance brain structures and functions (review: Rosenzweig & Bennett, 1996; van Praag, Kempermann, & Gage, 2000). In invertebrates, it has been shown that the neurogenesis of mushroom bodies which are essential for learning and memory is influenced by experience and environmental stimulation in adult worker honey bees (Apis mellifera) and house crickets (Acheta domesticus) (Farris, Robinson, & Fahrbach, 2001; Lomassese et al., 2000). Similarly, in fruit flies (Drosophila melanogaster) and spiders (Hogna carolinensis), the enrichment of rearing environments is known to exert direct impacts on many of their behaviors (Barth, Hirsch, & Heisenberg, 1997; Barth, Hirsch, Meinertzhagen, & Heisenberg, 1997; Carducci & Jakob, 2000; Dukas & Mooers, 2003; Hirsch & Tompkins, 1994).

Coleoid cephalopods (octopus, squid, and cuttlefish), a group of animals in the phylum of , have highly organized brains and their behaviors are sophisticated among all invertebrates (Hanlon & Messenger, 1996). In a series of experiments, it has been reported that the development of associative learning and memory of cuttlefish (Sepia officinalis, the European common cuttlefish) in enriched environments is significantly faster than in impoverished conditions (Dickel, Boal, & Budelmann, 2000). The same research group also showed that the maturation of sand digging behavior in S. officinalis is affected by rearing environments (Poirier, Chichery, & Dickel, 2004), and early feeding experience or visual familiarization can influence the subsequent prey preference (Darmaillacq, Chichery, & Dickel, 2006; Darmaillacq, Chichery, Poirier, & Dickel, 2004; Darmaillacq, Chichery, Shashar, & Dickel, 2006). All these studies demonstrated that cuttlefish behaviors are not strictly preprogrammed and are subject to environmental influence.

Perhaps the most surprising finding of experience-dependent plasticity in cuttlefish is that the maturation of camouflage body patterns is faster for animals reared in enriched environments than those reared in impoverished environments (Poirier, Chichery, & Dickel, 2005). Camouflage is the primary defense behavior in cephalopods (Hanlon & Messenger, 1996). It has been known that hatchlings of cuttlefish are capable of showing various body patterns for concealing themselves on various backgrounds (Barbosa et al., 2007; Hanlon & Messenger, 1988), although individual components responsible for different body patterns mature gradually after hatching from eggs (Barbosa et al., 2007; Poirier et al., 2005). It is often thought that camouflage body patterning is essential for the survival of hatchlings, thus this defensive behavior must be a preprogrammed behavior (or fixed action pattern, FAP) (Ruxton, Sherratt, & Speed, 2004). The fact that rearing environments can affect the maturation of camouflage body patterns suggests that as fundamental as camouflage is subject to experience-dependent plasticity.

While the study of Poirier et al. (2005) in S. officinalis indicates that the enriched environment (group reared on variegated backgrounds; varied-social conditions) facilitates the development of body patterning, and the impoverished environment (individually reared on a uniform background; uniformsolitary conditions) retards the maturation of camouflage patterns, it is not clear if other Sepia species also shows this experience-dependent plasticity, and more importantly, to what extent visual stimulation alone (as opposed to social interaction) plays a key role in shaping this developmental process. It is known from previous studies that background contrast is a crucial visual feature of substrates for evoking three 2

main categories of camouflage body patterns, namely Uniform, Mottle, and Disruptive in cuttlefish (review: Hanlon et al., 2009). Visual contrast has also been shown to be an important factor in the development of the visual system (Daw, 2005). In the present study, we used Sepia pharaonis (the ), a widespread Indo-Pacific cuttlefish species, to test the hypothesis that visual experience, specifically the rearing background contrast, is essential for the maturation of camouflage body patterns. By group rearing animals in either low-contrast (uniform substrate) or high-contrast (black/white checkerboard) backgrounds after hatching, and testing the expression of their camouflage body patterns repeatedly on uniform and checkerboard substrates every 2 weeks (up to 12 weeks of age), we showed that rearing background contrast is in part responsible for the maturation of individual components of camouflage patterns in S. pharaonis.

Method

Subject

Individuals of Sepia pharaonis were hatched from eggs that were collected from the by-catch of bottom trawls in southwestern Taiwan. The species identification was based on the sucker patterns on the club, and later confirmed by the characters of in postmortem (Jereb & Roper, 2005). The hatchlings were reared and maintained in the laboratory with two close-circulation aquarium systems (700 L each; water temperature = 22 °C) in the National Tsing Hua University (Hsinchu, Taiwan). Animals were separated into two cohorts at around hatching. One cohort was reared in a uniform-gray background (Figure 1A; designated as a low contrast group, or L group), and the other was raised in a black/white checkerboard background with its check size equivalent to approximately 4–10% of the body area, or 40–200% of the White square area (Figure 1B; designated as a high contrast group, or H group). Note that the uniform-gray and the black/white checkerboard were made to have an equal mean intensity (i.e., the percent reflectance of the uniform-gray surface is roughly half of that of the white surface on the black/white checkerboard), thereby the only difference is the contrast (but see below for some caveats). The choice of these two extreme contrast backgrounds was due to lack of contrast sensitivity functions in developing S. pharaonis, thus this binary approach represents the simplified version of two distinct visual backgrounds. Nevertheless, the visual difference between these two environments is not strictly contrast. Many other visual cues (e.g., the presence of object, the object area and edge, etc.) may also contribute to the distinctness of the H and L rearing backgrounds. All background substrates (laminated to be waterproof) were presented on both the floor and wall outside the Plexiglas containers. Both groups were kept in artificial lighting conditions with a normal light/dark cycle (12h/12h). Each group has eight individuals initially, but as they grew, the number of animals in a group was adjusted (either two or four individuals depending on the tank size) to maintain similar rearing spaces (see supplementary Table S1 for details). Accordingly, the check size of the checkerboard background for the H group was increased proportionally as the animals grew up (Table S1). Cuttlefish of each group were fed daily ad libitum with white shrimps throughout the experiment. Note that the prey remains were regularly cleaned to keep visual backgrounds simple. The mantle length (ML) of the cuttlefish from L and H groups was measured every two weeks.

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Experimental Procedures

Experiment 1

To provide a stable visual environment and minimize stress to the animals, the experimental trials were conducted inside a tent made of black plastic sheeting. Animals from either the H group or the L group were placed at random in an apparatus composed of four round compartments (see Figure 1C), one per compartment, where either black/white checkerboard (the check size is about 20–120% of the White square area, see Table S1) or uniform-gray background (same as their rearing background) was presented on both the floor and wall of the arena (coded as C test vs. U test). Note that the U test always preceded the C test in the experiment to minimize the contrast priming effect. To accommodate the animal growth, the diameter of the compartment as well as the check size of the checkerboard background were increased accordingly (see Table S1 for the compartment size and the check size of each age group). Cuttlefish were repeatedly used for behavioral experiments every 2 weeks (at postembryonic 2, 4, 6, 8, 10, and 12 weeks). Fluorescent light sources (Mitsubishi, FCL30EX-D/28 or Philips, TLE30W/54–765) were used to illuminate the arenas from above. Once the animals had been moved into the round compartment, a 1-hr trial was recorded immediately using a digital video camera (Sony DSR-PD150P) mounted above the arena. A small window on the tent was cut opened for observing the animals from the camera’s view finder, so that the animals’ movements could be followed from outside the chamber without disturbing them. The camera was set to record for 2 s every 30 s, thus yielding 240 s of footage per animal per substrate. Since different individuals acclimated (i.e., ceased swimming and hovering movements and expressed stable body patterns) at different rates, only the last 30 min (120 s of footage) of the trial were used for analysis. Most of animals acclimated within the first 30 min. It was interesting that we noticed that the acclimation rate of individuals on different substrates correlate with their rearing environments (see supplementary Figure S1 for details). From the 30 min video recordings, we took five still images (one in each 6 min segment) per animal, and graded them using the grading scheme described below (see Figure 2). Only the images in which animals showed stable body patterns (i.e., skin coloration remained unchanged for at least 1 min or two sampling points within each 6-min recording time) were used in grading. Scores from the five selected images were then averaged to obtain a mean score to represent an individual’s body pattern response in the analysis.

Experiment 2

To examine whether the repeated exposures of the L group animals in the high contrast 4

background during the C test early in life affect the camouflage body patterning in later developmental stages, in a separate experiment, we exposed nonrepeated L group cuttlefish in a black/white checkerboard environment for either 0 or 3 hr at the fourth week, and subjected these animals for both the U test and C test at the eighth week. Note that these L group cuttlefish have never experienced the high contrast substrates before the fourth week, and each animal was tested only twice (U test and C test) at the eighth week.

Quantification of Camouflage Body Patterning

In S. officinalis, the chromatic components have been clearly defined (Hanlon & Messenger, 1988), and the grading method for quantifying disruptive body patterns has been well established (Chiao, Chubb, & Hanlon, 2007; Chiao, Kelman, & Hanlon, 2005; Mathger, Barbosa, Miner, & Hanlon, 2006). However, in S. pharaonis, individual chromatic skin components responsible for disruptive patterning have not been systematically characterized,̈ although this species has been used previously in several camouflage experiments (Chiao & Hanlon, 2001a, 2001b; Shohet, Baddeley, Anderson, & Osorio, 2007). Since the major chromatic components of the disruptive body patterns of Sepia spp. are similar (Hanlon & Messenger, 1996), we modified the grading scheme developed for S. officinalis to quantify disruptive patterning in S. pharaonis (Mahger et al., 2006). Similar quantitative approach has been used to characterize the development of disruptive body patterns in S. officinalis (Barbosa et al., 2007; Poirier et al., 2005).̈ These components are produced by selective expansion (dark components) and retraction (light components) of chromatophores, which either cover or expose underlying white reflectors (Messenger, 2001). When expressed, components can be shown with varying intensities. The most commonly shown nine dark and five light components selected from thousands of cuttlefish pictures taken in the laboratory were used for grading (Figure 2A). Nine dark components are: (1) Dark arm (DA), (2) Anterior head bar (AHB), (3) Posterior head bar (PHB), (4) Anterior transverse mantle line (ATML), (5) Posterior transverse mantle line (PTML), (6) Anterior paired mantle spots (APMS), (7) Middle paired mantle spots (MPMS), (8) Posterior paired mantle spots (PPMS), and (9) Central annulus (CA); and five light components are: (10) White head bar (WHB), (11) White square (WS), (12) White mantle bar (WMB), (13) White posterior triangle (WPT), and (14) White central dot (WCD). Each component was assigned a score ranging from 0 (not expressed), 1 (weakly expressed), 2 (moderately expressed) to 3 (strongly expressed), and was graded separately and with equal weight. Grades for all 14 components were summed to yield the final scores. Thus, using this grading scheme, an animal can be assigned a total grade ranging from 0 (no expression of any disruptive components) to 42 (maximum expression of all 14 disruptive components), resulting in a continuum of disruptive body patterning scores (see Figure 2B, e.g., of grading). A similar grading scheme has been used recently in studying camouflage of plaice (Pleuronectes platessa), in which two body patterns can be well separated by this qualitative scoring scheme (Kelman, Tiptus, & Osorio, 2006).

To ensure that the experimenter grading the images was not influenced by the background on which the animals were placed, scores from a subset of images in which backgrounds were removed using Photoshop CS (Adobe Systems, Inc.) before grading were compared with scores of the same set of images without removing backgrounds. The Pearson product–moment correlation coefficient of these two sets of measurements was 0.93 ( p < .01), which suggests that the presence of backgrounds during grading has little influence on the scores. Each picture of five captured images within the trial was graded sequentially. 5

Grading was done by one of the authors, and the repeatability of the grading method within the scorer was reliable based on the high correlation between two repeats of randomly selected image sets (r = .90, p < .01).

Statistical Analyses

The unpaired one-tailed t test was used to evaluate (1) if two different rearing conditions resulted in different disruptive scores for animals tested on the checkerboard and uniform backgrounds at various postembryonic stages (Experiment 1: H group vs. L group); (2) if two different testing backgrounds resulted in different responses for animals reared in the high contrast and low contrast environments at various postembryonic stages (Experiment 1: C test vs. U test); and (3) if two different testing backgrounds resulted in different responses at the eighth week for L group animals either repeatedly used or exposed to a high contrast environment for a brief period of time (0 or 3 hr) at the fourth week (Experiment 2: C test vs. U test). The one-way analysis of variance (ANOVA) test was also used to evaluate if there is a response difference among three groups of cuttlefish in Experiment 2 (both in the C and U tests). Only the p values less than 0.05 were considered statistically significant in the present study. Statistical analyses were performed using the add-on functions in the Microsoft Office Excel, 2004.

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Results

Growth Rates Are Similar in Cuttlefish From Both the L and H Groups

It has been previously reported that the mantle length of S. officinalis reared in the enriched environment is significantly greater than that of the animals reared in the impoverished environment at Days 30 and 60 (Dickel et al., 2000; Poirier et al., 2005). We also compared the mantle length of S. pharaonis in both the L and H groups every two weeks after hatching (see Figure 3), and found that there was no significant difference in the mantle length between these two groups at all ages examined ( p = .42, 0.20, 0.86, 0.61, 0.47, and 0.45 for weeks 2, 4, 6, 8, 10, and 12, respectively). This result is apparently different from the finding of growth rate difference in S. officinalis reared in enriched and impoverished environments.

Body Patterns of Cuttlefish on the Checkerboard and Uniform Backgrounds Are Different Between the L and H Groups in Experiment 1

To examine whether rearing background contrast would influence camouflage body patterns throughout various postembryonic stages, we first compare the disruptive scores of cuttlefish reared in different contrast backgrounds (the H group vs. the L group) on either the black/white checkerboard or uniformgray substrate (the C or U test). When cuttlefish were tested on the checkerboard, the H group animals generally had higher disruptive scores than the L group at most of developmental stages, although only the differences at weeks 10 and 12 were significant ( p < .05; Figure 4A). This suggests that cuttlefish exposed in high contrast environments during the first 12 weeks tend to enhance their disruptive camouflage body patterns, a result that is consistent with the finding of Poirier et al. (2005), in which they showed the varied-social reared animals (S. officinalis) expressed more chromatic components than the uniform-solitary reared ones on the variegated background. In contrast, 7 both the H and L groups showed similar disruptive scores in the U test at all postembryonic stages, except at week 6 (Figure 4B). This indicates that lacking visual contrast during the development does not appear to affect cuttlefish’s body patterning on the uniform substrates, a result that is somewhat different from the finding of Poirier et al. (2005), in which they showed the uniform-solitary reared animals (S. officinalis) expressed more chromatic components than the varied-social reared ones on the uniform background. Note that the disruptive scores of animals from both the H and L groups were lower in the first 4 weeks than in other developmental stages. We found interesting that cuttlefish from both the H and L groups showed more disruptive components on the uniform background in the later stages than those in the earlier stages, suggesting that the genuine uniform body pattern is not a norm on the uniform background for more developed animals. Further analysis of activation of individual disruptive components in these tests revealed that where the differences between the disruptive scores of the H and L group animals were not significant, some components were differentially activated either in the C test or in the U test (Table 1, supplementary Figure S2). These results indicate that the maturation of different disruptive components may be modulated differentially by visual contrast of the rearing environments.

To compare body patterning of cuttlefish on two different substrates (the C test vs. the U test) for the H and L groups separately, we analyzed the same data described above in an alternative way. As expected, the H group animals typically showed more disruptive components in the C test than in the U test (Figure 5A). Similarly, the L group animals also evoked more disruptive components in the C test than in the U test during early development (Figure 5B). Although the reason that cuttlefish showed similar disruptive scores in the C and U tests at some developmental stages is unknown, further analysis revealed that animals in the C and U tests elicited significantly different sets of disruptive components (Table 2, supplementary Figure S3). This may explain the apparent mismatch of camouflage body patterning in some of the U tests (i.e., cuttlefish did not show uniform body pattern on the uniform-gray background). As pointed out by Shohet et al. (2007), S. pharaonis, unlike S.

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officinalis, do not show distinct disruptive and uniform body patterns on nature substrates, rather they have mixed body patterns with differential expression of skin components on different backgrounds.

Repeated Exposure to the High Contrast Background During Experiment 1 Does Not Significantly Alter Body Patterning of Cuttlefish in Experiment 2

Since our animals were repeatedly used in every 2 weeks for both the C and U tests, it is possible that briefly exposing the L group cuttlefish to a high contrast background (the C test) early in life (e.g., 2–4 weeks) might have a profound effect on their camouflage body patterning later in the development (e.g., 8–12 weeks). We compared body patterning of cuttlefish at the eighth week on two different substrates (the C test vs. the U test) from the repeated L group animals (the same eighth week data analyzed above) and from animals that have only been exposed briefly to the black/white checkerboard for 0 or 3 hr at the fourth

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week. The result showed that there was no significant difference for these three groups of cuttlefish in both the C and U tests (see Figure 6). This suggests that repeated exposure to the high contrast background during earlier tests does not affect camouflage body patterning of cuttlefish tested in later developmental stages, although different sets of disruptive components elicited in the C and U tests varied among these groups of animals (Table 3, supplementary Figure S4).

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Discussion

Following the recent work of Poirier et al. (2005) showing that early experience affects postembryonic maturation of body patterns in S. officinalis, the present study extends this finding to a specific visual experience, namely the background visual contrast of the rearing environment, and its effect on camouflage body patterning in another species of cuttlefish (S. pharaonis). While our results suggest that animals reared in high contrast background express more disruptive body components than the ones reared in low contrast background when tested on the checkerboard substrate, the overall maturation difference of body patterns between S. pharaonis from the high and low contrast rearing groups is not as dramatic as shown in the difference between S. officinalis reared in uniform-solitary and varied-social conditions (Poirier et al., 2005).

Social Interaction and Environmental Complexity Is Essential for Normal Growth of Cuttlefish

In contrast to previous results that the mantle length of S. officinalis reared in the enriched environment is significantly greater than the animals reared in the impoverished environment at Days 30 and 60 (Dickel et al., 2000; Poirier et al., 2005), we found that the growth rates of S. pharaonis raised in either high or low contrast backgrounds were similar at Weeks 2, 4, 6, 8, 10, and 12 (see Figure 3). This discrepancy may be attributed to the factors of social interaction and environmental complexity in different experimental designs. In our study, all cuttlefish were kept in socially interacting environments throughout development and the visual background was relatively simple (checkerboard and uniform-gray backgrounds, but see below for concerns of additional visual information from cohabited animals). Thus, our results indicate that the difference in substrate visual features of the rearing environments alone does not exert the effect on cuttlefish’s growth rate. It has been hypothesized that the presence of conspecifics could increase the alimentary motivation, thus enhance the food intake (Dickel et al., 2000; Warnke, 1994). Our finding also suggests that group rearing and other nonvisual components of complex substrates (Correia, Domingues, Sykes, & Andrade, 2005; Forsythe, Lee, Walsh, & Clark, 2002) contribute significantly on the growth of cuttlefish.

Background Contrast Is One of the Visual Features in Affecting Maturation of Body Patterns in Cuttlefish

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Previous experiments in S. officinalis have shown that the enriched environment is crucial for the development of associative learning and memory, and maturation of sand digging behavior and body patterns (Dickel et al., 2000; Poirier et al., 2004, 2005). Although it is not easy to specify the enriched environment, the standard definition given by Rosenzweig et al. (1978) is “a combination of complex inanimate and social stimulation.” While some controls for the importance of single variables on the effects of enriched environment (e.g., the effects of socialization and general activity) have been tested in rodents (Bernstein, 1973), it has thus far no attempt in isolating the visual components of the enriched environment (van Praag et al., 2000). In the development of mammalian visual system, rearing background contrast is known to be an important factor (Daw, 2005). Thus, visual contrast of the rearing environment may be an essential variable for behavioral development.

In cuttlefish experiments, researchers typically placed a group of animals in a variegated substrate (i.e., differently colored rocks and shells on fine yellow sand with green plastic seaweeds) to approximate the enriched environment (Dickel et al., 2000; Poirier et al., 2004, 2005). In these experiments, it is difficult to separate the importance of social interaction and/or general activity from visual/tactile sensory inputs. In our study, we focused on the effects of background contrast on the maturation of body patterns. To keep other factors similar, animals were group reared and their tactile inputs from substrates were identical. High-contrast black/white checkerboard (H group) and low-contrast uniform-gray background (L group) were chosen to represent two different early visual experiences for cuttlefish. However, this inevitably makes both rearing environments visually impoverished, which may explain some of the unexpected body patterns of cuttlefish shown on the uniform background (see Discussion below).

The fact that the disruptive scores of the H group animals are generally higher than those of the L group animals on the checkerboard substrate (Figure 4A) suggests that background contrast alone can affect maturation of body patterns in S. pharaonis, even though animals were not reared in visually enriched environments. It is more important that when the expression of individual disruptive components was compared between the H and L group animals, several distinct skin components were more strongly evoked in the H group than in the L group (Table 1 and supplementary Figure S2). This suggests that high contrast visual information is required for maturation of certain disruptive components and their expression for camouflage body patterns. However, as pointed out in the Method section, the visual difference between the H and L rearing environments is not merely the contrast. Other visual features such as the presence of objects, the object area, and the presence of edges (all have been shown to influence camouflage body patterns in cuttlefish) may also play roles in the body pattern development of S. pharaonis. Furthermore, it should be noted that besides the aforementioned static visual background differences, our group rearing protocol could also contribute to dynamic visual background differences. Since we reared 8, 4, or 2 cuttlefish in the same tank (see Table S1), and each animal displayed various body patterns, thus the L group animals may also experience some contrast visual cues from other cohabited animals, which could potentially decrease the background contrast difference between the H and L rearing environments. Nevertheless, this result is consistent with the finding of Poirier et al. (2005), in which they showed that the enriched rearing environment with moderate contrast enhances the expression of disruptive body patterns of S. officinalis on a variegated substrate, although the method of body pattern quantification in their study is slightly different from ours in the present study.

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It is known that hatchlings of S. officinalis can show disruptive body patterns on the checkerboard background (Barbosa et al., 2007) and on natural substrates (Boletzky, 1983; Hanlon & Messenger, 1988; Poirier et al., 2005). Barbosa et al. (2007) also suggested that not all disruptive components are expressed right after hatching, and some postembryonic time is required for disruptive components to be fully matured. In S. pharaonis, we also observed a similar trend of maturation process of distinct disruptive components. This postembryonic development of disruptive components in Sepia species provides a useful model for studying how experience-dependent neural plasticity exerts its effect on maturation of body patterns. Furthermore, this finding also has an implication in the development of camouflage behavior in natural environments. When cuttlefish hatch from eggs, they are miniature adults. Without going through the pelagic stage, hatchlings settle down and grow up in the same environment. Their visual background and social interaction thus provide environmental cues to shape their camouflage body patterning. Since the natural habitats of Sepia species are likely to be diverse, visual environment complexity may refine individual’s body pattern maturation.

In the Method section, we noted that the acclimation rate of individuals on different substrates correlates with their rearing environments (Figure S1). The fact that the H group but not L group cuttlefish took significantly longer time to settle down in the first 30 min of the U test than in that of the C test suggests that substrate contrast may also affect animal’s background preference during the testing period. This observation raises the possibility that the early visual experience could influence cuttlefish’s later habitat preference. Further investigation of the rearing background contrast on the impact of the testing background preference should provide evidences to support this suggestion.

Visually Impoverished Environment Is Responsible for Developmentally Increased Expression of Disruptive Body Components on the Uniform Background in S. Pharaonis

Perhaps the most paradoxical result in our findings is that cuttlefish from both the H and L groups tend to show significantly more disruptive components on the uniform-gray background after Week 6 (Figure 4B), although both groups of animals generally showed slightly higher disruptive scores on the checkerboard than on the uniform background (see Figure 5). For cuttlefish to camouflage on the uniform substrate, it has been shown that a uniform body pattern would achieve the best background matching (Barbosa et al., 2008; Chiao & Hanlon, 2001a; Hanlon, 2007; Ma¨thger et al., 2007; Shohet et al., 2007). The fact that S. pharaonis elicited moderate disruptive body patterns on the uniform background from Week 6 to Week 12 regardless their rearing environments in our experiments raises the concern of their camouflage patterning development. This observation is also inconsistent with the finding of Poirier et al. (2005), in which they showed that the enriched rearing environment facilitates the reduction of the disruptive component expression in S. officinalis when tested on a uniform substrate.

One possible cause to account for this discrepancy is the species difference. It has been reported that juvenile S. officinalis showed uniform/stipple body patterns on natural substrates with small particle size, while juvenile S. pharaonis expressed mixed disruptive and mottle body patterns on the same background (Shohet et al., 2007). This species dependent camouflage patterning has been attributed to the habitat difference as well as effectiveness against predators from different distances (Shohet et al., 2007). It is known that temperate waters where S. officinalis inhabit are often more turbid than tropical waters where 13

S. pharaonis were typically found. The less turbid environment for S. pharaonis would make them rely more on background resemblance for effective camouflage. Alternatively, the visually impoverished rearing environments in both H and L groups may be responsible for the mismatched body patterns of S. pharaonis when tested on the uniform-gray background after Week 6. It is conceivable that our H group animals have much reduced visual complexity compared to the enriched environment reared animals in Poirier et al. (2005), even though the background contrast in our H group is significantly higher than in their enriched group. In rodents, numerous papers have demonstrated that visually impoverished environments can affect brain development and behavior (Rosenzweig & Bennett, 1996; van Praag et al., 2000). In invertebrates, many complex behaviors such as learning/ memory and mating are also known to be affected by early experience and environmental complexity (Barth, Hirsch, & Heisenberg, 1997; Barth, Hirsch, Meinertzhagen et al., 1997; Carducci & Jakob, 2000; Dukas & Mooers, 2003; Farris et al., 2001; Hirsch & Tompkins, 1994; Lomassese et al., 2000). Thus, it is plausible that cuttlefish reared in the visually impoverished environment result in deficient camouflage body patterning on the uniform background. This also suggests that the background contrast of the rearing environment has little effect on uniform body pattern development for background matching camouflage.

In conclusion, we demonstrate that background contrast of the rearing environment is in part responsible for postembryonic maturation of body patterns in S. pharaonis. Since body patterning in cephalopods is neurally controlled and mainly mediated by the elaborate visual system (Hanlon & Messenger, 1996; Messenger, 2001; Packard, 1995), it is possible that brain areas such as the optic lobe and lateral basal lobe involved in sensorimotor control (Boycott, 1961; Chichery & Chanelet, 1976) could be altered by early visual experience. Future studies on examining specific circuit maturation in ’s brain influenced by rearing visual inputs could provide neural basis of this experience-dependent plasticity in camouflage behavior development.

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