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CUTTLEFISH CAMOUFLAGE VISUAL PERCEPTION OF SIZE, CONTRAST AND NUMBER OF WHITE SQUARES ON AR

Cephalopod camou?age (or crypsis) is among the most sophisticated in the animal kingdom because the neurally controlled chromatophores permit a diverse repertoire of body patterning that can be changed instantly (e.g. Hanlon and Messenger, 1996). The extraordinary patterns in the skin are mainly under visual control (Hanlon and Messenger, 1988; Marshall and Messenger, 1996). While the body patterns of cuttle?sh such as Sepia officinalis have been well characterized (Hanlon and Messenger, 1988), it is not yet understood how the cuttle?sh brain controls this patterning (see Boycott, 1961; Packard, 1995). Since cephalopods change their body patterning almost entirely as a result of visual input, applying a de?ned visual stimulus and recording the corresponding body patterning (i.e. motor output) allows aspects of visual perception to be examined (Marshall and Messenger, 1996; see also Saidel, 1988; Ramachandran et al., 1996). Using a behavioral approach, we report here the kinds of potential visual information that young cuttle?sh, Sepia pharaonis, use to control body patterning for camou?age on diverse backgrounds. Cuttle?sh use tens of skin patterns for camou?age on benthic substrata, yet the repertoire can be grouped into three general categories of pattern: uniformly stippled, mottled and disruptive (Hanlon and Messenger, 1988). We measured only the extremes of this continuum, looking particularly for visual features of the substratum that would produce a change from uniformly stippled to the disruptive body patterns that can be so effective for camou?age (Fig.1). Cephalopod body patterns can be analyzed into their ‘components’, which may be postural, locomotor, textural or chromatic (Packard and Hochberg, 1977). The chromatic components, which result mostly from direct neural control of skin patterns, are discrete entities that can number 15–45 in different species. There are several chromatic components that are particularly well de?ned and are major contributors to the disruptive patterns; these are the components White square and White mantle bar on the mantle, and the transverse White head bar (outlined in Fig.2). These chromatic components can be thought of not only as morphological units in the skin but also as physiological units in the brain (Packard, 1982). That is, expression of these morphological units is determined by the physiological units in the brain of cuttle?sh that control the skin through the pathway: visual input → eyes →optical lobes →lateral basal lobes →chromatophore lobes →skin. Thus, the appearance of certain chromatic components against well-de?ned backgrounds may give us clues about the

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The Journal of Experimental Biology 204, 2119–2125 (2001) Printed in Great Britain ?The Company of Biologists Limited 2001 JEB3276

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visual perception and neural processing of body patterning. Since the major chromatic components of the disruptive body patterns of Sepia spp. are white rectangles, splotches and bars, we decided to test various features of dark and light substrata whose features we could control with some precision. A similar approach has been applied in ?at?sh (Ramachandran et al., 1996) to examine basic components of the skin patterns. In the present study, black/white checkerboard patterns with various sizes (experiment 1) and contrasts (experiment 2) were used to determine which visual features cuttle?sh use to select disruptive patterns in the skin. Then, in experiment 3, various numbers of regularly spaced white squares in the black background were used to examine more closely the effects of background features on the expression of disruptive body patterns.

Materials and methods

Six young cuttle?sh, Sepia pharaonis(8–12cm mantle length), were reared from eggs in the laboratory of the National Research Center for Cephalopods (University of Texas Medical Branch, Galveston, TX, USA) and were maintained in the Marine Resources Center at the Marine Biological Laboratory. Cuttle?sh were 10 weeks old at the beginning of the experiments and 20 weeks old at the end. Each animal was placed in a running seawater tank (25cm×40cm×10cm) and was restricted by a four-wall divider to an area (20cm×26cm) where various computer-generated backgrounds (laminated to be waterproof) were presented as the substratum. A digital video camera was used to record the body patterning of S. pharaonis over a period of 30min (i.e. to record for 2s in every 1min period; to give a total of 60s recorded for each cuttle?sh on each substratum). In nature, it has been observed that cuttle?sh can match their body pattern to the background within a second or so (Hanlon and Messenger, 1988). However, cuttle?sh cannot perfectly match backgrounds that are completely arti?cial (e.g. checkerboard patterns). Furthermore, it was imperative to wait until the cuttle?sh were acclimated to the tank, and this could sometimes take several hours. Acclimation was gauged by the cessation of excessive swimming and hovering movements and by the chronic expression of a stable body pattern. Nevertheless, once acclimated, S. pharaonis showed various grades of disruptive patterns based on certain features of these well-de?ned backgrounds. Therefore, we sought to quantify the corresponding body patterns as functions of the known and controlled features of the checkerboard (i.e. the size and the contrast of each square and the number of white squares).

A simple grading scheme of patterning (Fig.2) was used to determine the responses of the animals to different substrata. The assigned grades were: 1, uniformly stippled pattern; 2, indistinct pattern; 3, disruptive pattern. We graded ‘1’ if the animal was uniformly stippled, ‘3’ if it clearly and distinctively showed the White square or White mantle bar on its mantle, and ‘2’ if it showed anything in between. For example, in grade 2, there were elements of both a uniform stipple and a partial disruptive pattern (see Fig.3, Fig.4, Fig.5). Fig.2

B illustrates some details of grading the White square. Often only a part of the White square on the animal’s mantle was expressed unilaterally (e.g. Fig.3B) or the White square itself was not uniformly white (e.g. Fig.2B, Fig.3D). We did not grade the White head bar, nor did we assess features such as the dark contrast of skin on other parts of the mantle. Grading was conducted by playing the video tape back and assigning a grade (whole integer, 1–3) every 10s. Thus, since all tapes lasted 60s, six grades were determined for each animal on each substratum. The mean values (and overall standard deviation) of all animals combined were plotted in the ?gures.

For testing different checker sizes (experiment 1), we chose a variety of square sizes that ranged from slightly larger than the White square on the mantle to much smaller than the White square on the mantle (note that the White square is not truly square, but rectangular; see Fig.1). To test contrast (experiment 2), we chose the checker size that gave the highest grade of disruptive pattern in experiment 1, and we varied only the percentage of contrast while keeping the mean intensity the

C.-C. C HIAO AND R. T. H ANLON

Fig.1. Disruptive body patterning by a young cuttle?sh on a rocky substratum. Notice the strongly expressed ‘White square’ chromatic component on the mantle of the animal that resembles other randomly scattered white rocks amidst the substratum. Scale bar, 9mm.

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Cuttle?sh camou?age same for all checkerboards. The contrast here is de?ned as (I white ?I black

)/(I white +I black ), where I

is the intensity value of black/white squares. For testing the number of white checker squares (experiment 3), we again chose the checker size that gave the highest grade of disruptive pattern in experiment 1,and varied only the number of white squares in the black background. Note that we distributed the white squares regularly (not randomly) across the space, so that there was a

greater likelihood of both eyes of the cuttle?sh seeing a similar number (or proportion) of them. To be consistent with the existing literature on cephalopod skin patterning (e.g. Hanlon and Messenger, 1988; Hanlon and Messenger, 1996; Packard,1995), we capitalize the ?rst word of the formal name of the chromatic components in the skin (e.g. White square).Results

Thirteen trials were conducted over the course of 10 weeks:four cuttle?sh were run through experiment 1, ?ve through experiment 2 and four through experiment 3. Video examples are available on the Journal of Experimental Biology website to support the data and ?gures presented herein (https://www.doczj.com/doc/9e12480922.html,/JEB/movies/jeb3276.html).

In experiment 1, we tested substratum checker sizes of 2.6,6.5, 13.0, 19.5 and 26.0mm. The White square component on the dorsal mantle of the cuttle?sh was approximately 18–22mm (long dimension). Cuttle?sh generally showed uniformly stippled body patterns when the checker size was 2.6 or 26.0mm (Fig.3A,E). Checker sizes of 6.5 and 19.5mm produced mixtures of patterning in which the White square was partially expressed (e.g. Fig.3B,D; see also Fig.2B, grade 2).With a checker size of 13.0mm, the cuttle?sh almost always showed a consistent and clear expression of White square (Fig.3C,F; see also Fig.2B, grade 3). The white checkers were approximately half the size of the White square component (Fig.3C). The same trend of expression was seen in the White head bar, which is not present in Fig.3A,E, but is slightly expressed in Fig.3B,D and most strongly expressed in Fig.3C with 13.0mm squares.In experiment 2, we altered the contrast between black and white squares by presenting checkerboards at different contrast values: 10, 20, 30, 50 and 100% (note that no unit value can be assigned since this is a relative measurement). The White square was expressed occasionally at 10% contrast (Fig.4A),but its incidence and clarity of expression increased at 20 and 30% contrast (Fig.4B,C). The clearest responses occurred at 50 and 100% contrast (Fig.4D,E). There was no clear threshold at which visual contrast suddenly induced all of the cuttle?sh to produce the White square (Fig.4F). It is evident from Fig.4B–D that White square could be shown in different gradations, particularly within the square. For example,Fig.4B,C were scored as ‘2’ because there are dark stipples within the square, whereas in Fig.4D,E, a score of ‘3’ was assigned because fewer or no stipples were present within the square, resulting in a whiter and thus stronger disruptive effect.Furthermore, note that the White head bar was not expressed at 10% contrast, slightly expressed at 20 and 30% contrast and expressed most strongly at 50 and 100% contrast.

In experiment 3, we tested various numbers of regularly spaced white squares on a black background, all with 100%contrast (note that our standard checkerboard pattern had 160white squares and 160 black squares, each with 13.0mm sides).First, on a completely black background (i.e. no white squares;Fig.5A), no cuttle?sh showed the White square component on its mantle. Second, when only four white squares were present (out of a total of 320 black and white squares, or 1.25%), some cuttle?sh showed partial White squares (Fig.5B). Third, when 20 white squares (or 6.25% of the 320) were present, the White square on the mantle was shown often, albeit in various forms with other chromatic components (Fig.5C). Fourth, with increased numbers (i.e. proportions) of white squares, the frequency and clarity of expression of the White square chromatic component did not increase substantially (Fig.5D,E),as indicated by the fairly large amount of variation (note the standard deviations in Fig.5F). Note also that the White head bar was absent when no white squares were present, that it was expressed vaguely with four white squares, yet it was expressed strongly with 20 or more white squares on the substratum.

Fig.2. Grading criteria for judging disruptive coloration in young cuttle?sh. (A) Cuttle?sh outline showing (i) the White square, (ii) the White mantle bar (which includes the White square) and (iii) the White head bar. (B) Actual close-up images of the White square on the cuttle?sh shown in Fig.3 to illustrate different expressions that were used to assign grades 1, 2 and 3. Compare Figs 3–5. Images are life-size and exactly the same region of skin.

(a)

Gr a de 1 Gr a de 2 Gr a de 3

(a)i

ii

iii A B

2122 C.-C. C HIAO AND R. T. H ANLON

A B C

D E

2123

Cuttle?sh camou?age

A B C

D E

2124 C.-C. C HIAO AND R. T. H ANLON

A B C

D E

2125 Cuttle?sh camou?age

stippled patterns depending on the checker size. Although cuttle?sh possess a large repertoire of patterns, including mottled patterns, they did not use mottling because this does not camou?age as well on checkerboards as do disruptive patterns. In this respect, the more re?ned skin of cuttle?sh (with its neural correlates) imparts a more ?exible and adaptive system for camou?age than that of ?at?shes. Nevertheless, both organisms provide behavioral assays (manifest through adaptive skin) that provide insights into visual perception. From knowledge of the natural habitat of Sepia spp. (e.g. Boletzky, 1983; Hanlon and Messenger, 1988; Hanlon and Messenger, 1996), one would predict that checkerboard substrata would be extremely unnatural and challenging for cuttle?sh to adapt to. Nevertheless, the cuttle?sh did attempt to match these arti?cial substrata and respond to changes in their features. Our use of checkerboards as the substratum was appropriate only insofar as we were testing for the presence/absence of the White square component in cuttle?sh. Our results revealed two ideas worthy of future investigation. First, it is probably not the shape per se(i.e. square) that is most important to the cuttle?sh for producing disruptive patterns, but the contrast and size of an object in the substratum background. Second, Fig.5B–E (which shows some mottled skin patterns) provides clues about how to test for mottled patterns using various combinations of different sizes, contrasts and numbers of light objects against dark backgrounds. Further analyses of the spatial frequency components of the experimental substrata may also shed light on the mechanism of skin patterning (e.g. principal component analysis in ?at?sh, Ramachandran et al., 1996; independent component analysis in both ?at?sh and cuttle?sh, J.

C. Anderson, R. J. Baddeley,

D. Osorio, N. Shashar, C. W. Tyler, V. S. Ramachadran, A. C. Crook and R. T. Hanlon, in preparation). Sepia spp. may provide a particularly good model of visual perception because the rigid mantle (due to the presence of the cuttlebone) presents an immovable and non-?exible body part that relies on ?ne-tuned skin patterning to achieve a wide range of optical illusions. As pointed out previously (Hanlon and Messenger, 1988), the wide range of disruptive patterns shown by Sepia spp. are carried out with ?ve light chromatic and six dark chromatic components of patterning; we concentrated on only two of these: the White square and White head bar. Perhaps most useful for future studies is the fact that several optical principles of disruption (?rst mentioned by Cott, 1940) are found in Sepia spp. and are illustrated in Fig.1. These include ?rst the principle of differential blending, achieved when some chromatic components blend with the substratum while others contrast sharply with it, thus allowing some body parts to stand out and others to fade away. In addition, the principle of maximum disruptive contrast operates when adjacent components of the pattern have great tonal contrast and, thus, provide a strong disruptive function. In another principle, that of adjacent contrast, a broken visual pattern made up of sudden transitions of color, sharply contrasted passages of tone and of irregular shapes of all kinds results in an image of multiple objects rather than parts of one form. Finally, gradations of tone within individual components such as the White square can also produce the visual illusion of relief to a human observer, giving the impression that the square is elevated or depressed, making it seem even more separate from the body. Thus, it is clear that aspects of grading the resultant skin patterning can be re?ned and correlated with increasingly sophisticated substrata, both of which could be quanti?ed in a manner not attempted in this initial experiment. This work was supported by a Grass Foundation Fellowship to C.-C.C. We thank Janice Hanley, Bill Mebane, Jim Carroll and Hazel Richmond for help with cuttle?sh rearing and maintenance and Gabrielle Santore for manuscript preparation. We are also grateful for discussions with Tom Cronin, Nadav Shashar, John Messenger, Daniel Osorio, Bill Saidel, Roland Baddeley and Richard Masland.

References

Boletzky, S. v.(1983). Sepia officinalis. In Cephalopod Life Cycles, vol. I, Species Accounts(ed. P. R. Boyle), pp. 31–52. London: Academic Press. Boycott, B. B.(1961). The functional organization of the brain of the cuttle?sh Sepia officinalis. Proc. R. Soc. Lond. B153, 503–534.

Burton, D.(1981). Physiological responses of melanophores and xanthophores of hypophysectomized and spinal winter ?ounder, Pseudopleuronectes americanus Walbaum. Proc. R. Soc. Lond. B 213, 217–231.

Cott, H. B.(1940). Adaptive Coloration in Animals. London: Methuen & Co., Ltd.

Edmunds, M.(1974). Defence in Animals. A Survey of Anti-Predator Defences.New York: Longman Group, Ltd.

Endler, J. A.(1991). Interactions between predator and prey. In Behavioural Ecology. An Evolutionary Approach(ed. J. R. Krebs and N. B. Davies), pp. 169–196. Oxford: Blackwell Scienti?c Publications.

Hanlon, R. T., Forsythe, J. W. and Joneschild, D. E.(1999). Crypsis, conspicuousness, mimicry and polyphenism as antipredator defences of foraging octopuses on Indo-Paci?c coral reefs, with a method of quantifying crypsis from video tapes. Biol. J. Linn. Soc.66, 1–22.

Hanlon, R. T. and Messenger, J. B.(1988). Adaptive coloration in young cuttle?sh (Sepia officinalis L.): The morphology and development of body patterns and their relation to behaviour. Phil. Trans. R. Soc. Lond. B320, 437–487.

Hanlon, R. T. and Messenger, J. B.(1996). Cephalopod Behaviour. Cambridge: Cambridge University Press.

Holmes, W.(1940). The colour changes and colour patterns of Sepia officinalis L. Proc. Zool. Soc., Lond.110, 2–35.

Marshall, N. J. and Messenger, J. B.(1996). Colour-blind camou?age. Nature382, 408–409.

Mast, S. O.(1916). Changes in shape, color and pattern in ?shes and their bearing on the problems of adaptation and behavior, with special reference to the ?ounders, Paralichthys and Ancylopsetta. Bull. U.S. Bur. Fish.34, 173–238. Messenger, J. B.(1991). Photoreception and vision in molluscs. In Evolution of the Eye and Visual System(ed. J. R. Cronly-Dillon and R. L. Gregory), pp. 364–367. London: Macmillan Press.

Packard, A.(1982). Morphogenesis of chromatophore patterns in cephalopods: Are morphological and physiological ‘units’ the same? Malacologia23, 193–201.

Packard, A.(1995). Organization of cephalopod chromatophore systems: A neuromuscular image-generator. In Cephalopod Neurobiology(ed. N. J. Abbott, R. Williamson and L. Maddock), pp. 331–367. New York: Oxford University Press.

Packard, A. and Hochberg, F. G.(1977). Skin patterning in Octopus and other genera. Symp. Zool. Soc. Lond.38, 191–231.

Ramachandran, V. S., Tyler, C. W., Gregory, R. L., Rogers-Ramachandran, D., Duensing, S., Pillsbury, C. and Ramachandran, C.(1996). Rapid adaptive camou?age in tropical ?ounders. Nature379, 815–818.

Saidel, W. M.(1988). How to be unseen: An essay in obscurity. In Sensory Biology of Aquatic Animals(ed. J. Atema, R. R. Fay, A. N. Popper and W. N. Tavolga), pp. 487–513. New York: Springer-Verlag.

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