Role of vision in behavior, visual field, and visual acuity of

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1 FISHERIES SCIENCE 2000; 66: Original Article Role of vision in behavior, visual field, and visual acuity of cuttlefish Sepia esculenta Naohiko WATANUKI, 1, *,a Gunzo KAWAMURA, 1 Shohei KANEUCHI 1 AND Toru IWASHITA 2 1 Faculty of Fisheries, Kagoshima University, Shimoarata, Kagoshima , and 2 Kumamoto Prefectural Fisheries Research Center, Oyano, Amakusa, Kumamoto , Japan SUMMARY: The role of vision in the entry of the cuttlefish Sepia esculenta into basket traps was examined in laboratory experiments and by histological examination of the retina. Both entry into the trap and feeding on shore crabs stopped when the tank was completely darkened. The eyes of cuttlefish have a high sensitivity to light. The visual field of cuttlefish was determined by the optical method, based on the assumption that incident light on the pupil from any direction reaches the retina through a refractive lens. The uniocular visual field was found to be 253 on the horizontal plane, and the anterior and posterior binocular visual fields were 86 and 60 respectively. On the retina, areas with especially high visual cell density formed a visual equator slightly above the optical equator. The distribution of the visual cell density indicates no specific visual axis. The visual acuity is 0.36 when estimated from the bait recognition distance and the size of bait during feeding, and 0.89 when determined from the visual cell density at the visual equator and the focal length of the lens. Cuttlefish have far superior visual acuity than fish. KEY WORDS: basket trap, behavior, cuttlefish, Sepia esculeta, visual acuity, visual field. INTRODUCTION The authors previously reported that the behavior of cuttlefish Sepia esculenta around basket traps relies on vision. 1,2 However, this reliance on vision must ultimately be confirmed by the vision blockage test. Knowledge of the visual field, shape recognition and visual acuity is also essential to understand visual recognition which is the first stage of the reaction of cuttlefish to a trap. Basic research has been conducted in Japan and elsewhere on the vision of cephalopods and the relationship between vision and ecology. The relation between vision and capture techniques has been described for the Japanese common squid Todarodes pacificus and neonflying squid Ommastrephes bartrami caught by jigging with fishing lights. 3-5 The light conditions in the habitat of these two pelagic species of squid differ from those of demersal cuttlefish. Thus, cuttlefish must have a visual function suitable for its bottom or sea-bed *Corresponding author: Tel: Fax: watanuki@oafic.co.jp a Present address: Overseas Agro-Fisheries Consultants Co. Ltd, Toranomon, Minato, Tokyo Received 24 August Accepted 1 October habitat. It has been argued that the visual cells of cephalopods are evenly distributed over the entire retina 6 8 but the density distribution in cuttlefish must be uneven, because a lateral thick belt is observed slightly above the center of the eye. The present study examined the role of vision in the entry of cuttlefish into a trap and in feeding. The behavior of cuttlefish in a completely darkened water tank was compared with that of others in an illuminated water tank. This experiment was followed by determination of the visual field, the density distribution of the visual cells, and the visual acuity. 1 MATERIALS AND METHODS Behavior test in darkness and in the light The test was conducted for 10 days from 20 to 29 April 1992 at the Kumamoto Prefectural Fisheries Research Center using two gray-colored indoor water tanks: a 30 t rectangular water tank (6 5 m: water depth of 1.3 m) and a 25 t rectangular water tank (5 5 m: water depth of 1.3 m). The 30 t tank was used as the dark tank under two types of dark conditions created by covering the tank

2 418 FISHERIES SCIENCE N Watanuki et al. with: (i) a single layer of 0.33 mm thick black vinyl sheet (first 5 days); and (ii) a double layer of the same sheet (second 5 days). Hereafter, the tank is referred to as dark tank I and dark tank II, respectively. These dark tanks were placed under natural light conditions. Since this tank was connected to the outside via a drainpipe, a high-sensitivity film (Neo-pan 1600; Fuji, Tokyo, Japan) was exposed in the empty drainpipe for different time durations to check light-leak via the drainpipe. Light transmittance through the developed film was measured with a spectrophotometer (UW-200S; Shimazu, Tokyo, Japan). Indeed, light leaked through the drainpipe (Fig. 1), but this was negligible as the transmittance difference between non-exposure and 100 min exposure was only 3.2% at 490 nm (l max for S. esculenta 8 ). The 25 t tank was used as the control tank illuminated by fluorescent lamps and natural light during the day (illuminance in water: lx, Yu Fung-1065; Minolta T-1m, Tokyo, Japan). A flashlight was used for observations of the cuttlefish and bait animals at night, and these observation times lasted only several seconds. The cuttlefish used for the experiment were mature individuals (mantle length: mm) caught by jigging on 17 April, Males and females were identified and tagged individually. The control tank had eight males and eight females and the dark tank had 10 males and 10 females. The resulting density of cuttlefish per unit area was 0.64/m 2 in the control tank and 0.67/m 2 in the dark tank. The water in both tanks was constantly aerated and changed at a rate of 0.5 m 3 /min. The water temperature ranged C during the test. Entry into trap One foldable cuttlefish trap was placed in the center of each tank and was lifted twice a day (1 2 h before sunrise and 1 2 h after sunset). Male and female cuttlefish inside the trap were counted, then released back into the tank. The trap was returned to its original position. The trap entry ratio (Ri) was computed as the number of cuttlefish in the trap/number in the tanks, and Ri was compared between light and dark tanks and between the sexes. Feeding behavior Live shore crabs Hemigrapsus sanguineus (carapace width: mm), caught on the beach near the Center, were used as bait and fed once a day (1 2 h after sunset). The number of shore crabs at the start of the test and the number remaining just before the feeding and after 12 h were counted to calculate the feeding ratio (Rf) as the number of shore crabs eaten/number at start of test. Measurement of visual field Fig. 1 Spectral transmittance of the film exposed to a drainpipe for different periods of time. The visual field of vertebrates, including fish, is limited by the anatomical structure of the cranial bones and is not determined by the optical characteristics of the eyes. 9 Thus, the visual field may be determined by eliminating the directions in which vision is anatomically blocked in all directions around the cornea. In this study, the visual field of cuttlefish, whose eyes protrude from the body, was established as the range in which beams, radiated from various directions towards the eyes, reach the pupil. The test was conducted inside a temporary dark room constructed at the quay next to the Izumi Fisheries Co-operative Association building in Kagoshima Prefecture. The room was illuminated by a 20 W incandescent lamp and the light intensity at the water surface was 10-6 mmm -2 s -1. When an individual cuttlefish placed in a glass tank ( cm) stopped moving, a HeNe laser beam (NEO ARK NAL-6F; Japan Sci. Eng. Inc., Tokyo, Japan) was radiated from outside the tank to one pupil. The pass of the beam reached at the pupil was plotted on a transparent glass ball and the angle of incidence of the beam was measured to establish the uniocular visual field.

3 Role of vision in behavior of cuttlefish FISHERIES SCIENCE 419 Visual acuity Histological determination The head of a female cuttlefish with a mantle length of 123 mm was dissected and the eyes removed, fixed in Bouin solution, transferred to a 70% alcohol solution, and processed for histological examination of the retina. In order to establish the density distribution of the visual cells, the retina was divided into 22 small pieces for processing. Since a thick belt-like area was observed across the retina, smaller pieces of this area were dissected. Cross-sections (8 mm thick) of the retina were stained with hematoxylin-eosin. To obtain the visual cell density, visual cell nuclei were counted within a 100 mm width in each cross-section of the retina. The median of five counts was taken as the density of the visual cells in that part of the retina. Using the visual cell density (n) and the focal length of the cuttlefish lens (F), the minimum separable angle (a radian) was calculated with Tamura s formula 9 modified as follows: sin a = a = 1 F{ ( ) 2 ns F} where 0.25 is a coefficient of contraction by fixation. 9 Behavioral determination Upon visually recognizing bait, cuttlefish position themselves at some distance from the bait, with the arms extended in the direction of the bait, and then move straight towards the bait. This distance between the stationary cuttlefish and the bait was considered the visual recognition distance. This distance and the size of the bait were used to calculate the visual acuity. The test was conducted on 30 cuttlefish in a 30 t water tank at the Center. Shore crabs without the crusher claws were used as the bait. The shore crabs were given one at a time and the visual recognition distance (L) of the farthest cuttlefish that reacted to the bait was measured to the nearest 5 cm. The stationary position of the cuttlefish at the visual recognition of the bait was up to 30 cm off the bottom of the tank, and thus the shore crabs were seen sideways on. The position of the cuttlefish and the narrowest section of a visual target gives a measure of the visual acuity. Thus, the recognition distance (L) and the body width of the shore crab (w) were used to calculate the minimum separable angle (a): sina = a = wl ( radian) The light intensity in the tank during the test was mmm -2 s -1 at 14:25 15:25 h under natural light on a cloudy day and mmm -2 s -1 at night under mercury lamps (both measured with light quantum meter: LI-192SA, LI-1000, Tokyo, Japan). RESULTS Behavior in control tank and dark tanks In the control tank, the cuttlefish spawned both during the day and at night. In the dark tanks, no eggs were found attached to the trap but more than 10 eggs were found on the rope (7 mm in diameter) fixing the trap, several eggs on the spawning substrate outside the trap, and more than 10 eggs dropped onto the bottom of the tank. Entry into trap In the control tank, cuttlefish entered the trap during the day and at night, indicating that cuttlefish under natural lighting enter a trap regardless of the time being day or night. The trapped ratio during the day was significantly higher than that at night (Table 1). Males and females did not differ in the trapped ratio. In the dark tanks, only two females were trapped in the dark tank I during the day, four males and two females were trapped in the dark tank II during the day and at night, respectively. Thus, fewer cuttlefish were trapped in the dark tanks (Table 2). Feeding behavior In the control tank, during the day, all of the 20 shore crabs given were eaten within 3 5 min (Rf: 1.00). At night, the crabs were all eaten except for 2 nights (Rf: 0.94) (Table 3). In the dark tanks, both during the day and at night, uneaten crusher claws and shells were left at the bottom of the tank. In dark tank I, all crabs were eaten during the day (Rf: 1.00) and a lot of crab parts remained at night (Rf: 0.27). In dark tank II, the feeding ratio was 0.61 in the daytime and 0.06 at night (Table 4). These differences in the feeding ratio between dark I and II was due to the double black vinyl sheet blocking vision in dark tank II. Visual field As the visual field was measured when cuttlefish were stationary at the bottom of the tank, the measurement scope was restricted to above the horizontal plane of the body axis. The uniocular visual field was as wide as 253 on the horizontal plane and the binocular visual field was 86 anterior and 60 posterior (Fig. 2). The pupil of a cuttlefish has a W shape and is wide in the sideways direction. The shape of the eye is maintained by the cartilaginoid sclera that covers the dorsal half of the eye. The ratio between the horizontal axis and

4 420 FISHERIES SCIENCE N Watanuki et al. Table 1 Entry of Sepia esculenta into a basket trap in a control tank Date Day Night No. in tank No. in trap No. in tank No. in trap Female Male Female Male Female Male Female Male April Total Ri ±m 0.95 by sex 0.60 ± ± ± ± Ri ±m 0.95 for total 0.52 ± ± m 0.95, 95% confidence interval. Table 2 Entry of Sepia esculenta into a basket trap in darkened tanks Tank Date Day Night No. in tank No. in trap No. in tank No. in trap Female Male Female Male Dark I April Total Dark II April Total Table 3 Feeding of Sepia esculenta on shore crabs in a control tank Date No. of cuttlefish No. of crab Rf in tank Given Eaten April Mean 0.94 ± the vertical axis is 1:0.75 (measured in six specimens). Thus, the eye is flat in the direction of the body axis. Consistent with this anatomical feature of the eye, the uniocular visual field is wide in the horizontal plane. Histology of retina The transverse section of the eye is shown in Fig. 3. The retina of cuttlefish is formed by visual cells and nerve fibers. The visual cell layer, in fact, consists of the rhabdome layer, black pigment layer and visual cell nucleus layer. The density distribution of the visual cells is uneven, being higher at a belt-like area slightly above the optical equator (Figs 3,4). The top width of the trapezoidal distribution through the visual equator is

5 Role of vision in behavior of cuttlefish FISHERIES SCIENCE 421 Table 4 Feeding of Sepia esculenta on shore crabs in darkened tanks Tank Date Day Night No. of crab No. of crab No. of cuttlefish Given Eaten Rf No. of cuttlefish Given Eaten Rf in tank in tank Dark I April Mean 1.00 ± ± Dark II April Mean 0.61 ± ± Fig. 2 Dorsal view of the visual fields of Sepia esculenta. The uniocular visual field (dotted area) is 253 in arc. The anterior binocular field is 86 and the posterior binocular field is 60 wide. The center of the circle represents the position of the eye. Fig. 3 Transverse section through the eye of Sepia esculenta. The thickest area of the retina forms the visual equator. approximately 2 mm (Fig. 5). Incident light passing through the slit-shaped pupil (when the cuttlefish is in a bright place) is projected onto the visual equator. Visual acuity The minimum separable angle (a) calculated from the recognition distance (L = cm) and the body width of the shore crab (w = cm) ranged The highest visual acuity of 0.36 was obtained for cuttlefish reacting to the smallest shore crab when L was 340 cm and w was 0.28 cm. The highest visual cell density was 90 in 100 mm at the visual equator, and the minimum separable angle calculated was radian (0.89 in visual acuity).

6 422 FISHERIES SCIENCE N Watanuki et al. Fig. 4 The visual cell density in different parts of the retina of Sepia esculenta. Cell density is highest at the visual equator above the optical equator. DISCUSSION Since the frequency of feeding and entry to the trap were lower in the dark tanks, there is no doubt that vision plays a major part in the behavior of S. esculenta. The significantly lower feeding rate for dark tank II compared to dark tank I indicates that the vision of cuttlefish was not completely blocked in dark tank I. The cuttlefish ate all of the shore crabs in dark tank I during the day although the tank appeared pitch black to human eyes. This result indicates the astonishing sensitivity of the eyes of S. esculenta. This species are assumed to inhabit water depths of m, 10,11 where the light is limited and, therefore, their vision is adapted to dim light. For most fish species, the minimum brightness required to form a school or to feed is lx. 12 But the vision of S. esculenta is assumed to effectively function in a darker environment. The maximum spectral sensitivity of the retina of S. esculenta at 490 nm 8 matches the wavelength of best transmittance in the sea with high transparency. 13 The squid T. pacificus can see down to lx. 14 The vision of S. esculenta functioned excellently in a dark environment and the W-shaped pupil narrowed to a slit under strong light. The regulation of incident light by the pupil is a special feature of coastal species 15 and is a function suited to a relatively shallow and bright environment. Narrowing of the pupil also allows a visual target to be more clearly seen. Thus, the eyes of S. esculenta function well over a wide range of light intensities. That the cuttlefish fed in dark tank II meant that feeding behavior was entirely dependent on vision. Messenger 16 reports that the European common cuttlefish Sepia officinalis cannot feed in total darkness. But other behavioral tests have shown that chemoreception is involved in the feeding behavior of S. esculenta (K. Anraku, Kagoshima University, Japan pers. comm. 1998). In addition, the tactile sense of the arms may be Fig. 5 The density distribution of the visual cell nuclei through the visual equator of the retina of two Sepia esculenta. involved. 17 It is also possible that shore crabs moving around in the dark accidentally touched the cuttlefish and were then eaten. The entry of cuttlefish into the trap can not be explained by a chemoreception or tactile sense. The entry in dark tank II indicates that the cuttlefish somehow senses the existence of the trap, but the spawning of eggs on the bottom of the tank indicates that they failed to sense the existence of the trap. These two results are contradictory. In order to solve this contradiction, it is necessary to observe the entire behavioral process of the cuttlefish in dark tank II rather than simply relying on such behavioral results as the number of cuttlefish and eggs in the trap. For the purposes of the present study, it is sufficient to emphasize the importance of vision in the behavior of cuttlefish. In S. officinalis, the uniocular visual field is 177 and the anterior binocular visual fields is These are quite different from those of S. esculenta and are assumed to be due mainly to different measurement methods. Messenger 16 measured the visual field based on the relative positions between the visual target and individuals responding to the target. In this way, it is difficult to accurately distinguish between the uniocular visual field and binocular visual field. Cuttlefish are capable of rotational movements by bending the head or changing the direction of the funnel, even though they still have a wide visual field without such movements. The squids T. pacificus 18 and Loligo forbesi 19 are said to be incapable of posterior vision. When they are kept in tanks, vertical stripes for the visual recognition of obstacles are drawn

7 Role of vision in behavior of cuttlefish FISHERIES SCIENCE 423 on the walls to avoid collision damage. None of the cuttlefish in the present study collided with the tank walls, except a few that were agitated. Visual acuity data may differ depending on the measurement method. In those methods relying on behavior, the visual recognition distance is affected by the water conditions (light, turbulence etc.) and by the physiological condition of the subject animal, including its movements. Moreover, it is difficult to place a visual target on the visual axis of a test animal. 20 In contrast, the visual acuity determined from visual cell density probably represents the optimal conditions. Since this method is most frequently used to determine the visual acuity of fish, it is a convenient way of comparing cuttlefish with fish. Compared to tuna with a visual acuity of 0.49, 21 the best among fishes, the cuttlefish S. esculenta is far more sensitive. REFERENCES 1. Watanuki N, Iwashita T, Kawamura G. Cuttlefish spawning and visually mediated entry into basket traps. Fisheries Sci. 2000; 66: Watanuki N, Hirayama I, Kawamura G. Why do cuttlefish Sepia esculenta enter basket traps? Space occupation habit hypothesis. Fisheries Sci. 2000; 66: Flores EEC. Studies on the maintenance and behaviour of the japanese common squid Todarodes pacificus Steenstrup in relation to fishing. PhD thesis, University of Hokkaido, Hakodate, 1979, pp Inada H. Studies on effects of fishing lights used for the jigging of Japanese common squid Todarodes pacificus. PhD thesis, University of Hokkaido, Hakodate, 1988, pp (in Japanese). 5. Watanabe T, Inada H, Takayama T, Yamasaki S, Kitamura M. Retinal adaptation of neon flying squid Ommastrephes bartrami at capture with jigs and fishing lights. Nippon Suisan Gakkaishi 1997; 63: Duke-Elder S. System of ophthalmology. In: The Eye in Evolution Vol. 1. Henry Kimpton, London. 1958; Hara T. Photosensitive pigments in the cephalopod retina. Zool. Mag. 1968; 77: (in Japanese). 8. Kito Y, Sugawara M, Nashima K. Photoreceptor membranes. In: Shibatani K (ed.). Biological and Chemical Utilization of Solar Energy II. Gakkai Press, Tokyo. 1979; (in Japanese). 9. Tamura T. Vision. In: Kawamoto N (ed.). Fish Physiology. Koseishakoseikaku, Tokyo. 1977; (in Japanese). 10. Chikuni S. Cephalopod resources in the Indo-Pacific region. FAO Fisheries Technical Paper 1983; 231: Okutani T, Tagawa M, Horikawa M. Cephalopods from Continental Shelf and Slope Around Japan. Japan Fisheries Resource Conservation Association, Tokyo Blaxter JHS. Light, animals, fishes. In: Kinne O (ed.). Marine Ecology Vol. 1, Part 1. Wiley Interscience, London. 1970; Hishida K. Transfer of light through seawater. In: Marine Physics 1. Tokai University Press, Tokyo. 1970; (in Japanese). 14. Suzuki T. Culture and live transportation of Japanese common squid. In: Nasu K, Okutani T, Ogura M (eds). Cuttlefish and Squids from Catch to Consumption. Seizando, Tokyo. 1991; (in Japanese). 15. Muntz WRA. Pupillary response of cephalopods. Symp. Zool. Soc. Lond. 1977; 38: Messenger JB. The visual attack of the cuttlefish, Sepia officinalis. Anim. Behav. 1968; 16: Wells MJ. Cephalopod sense organ. In: Wilbur KM, Yong CM (eds). Physiology of Mollusca Vol. 2. Academic Press, New York. 1966; Sakurai Y, Ikeda Y. Laboratory rearing methods of Todarodes pacificus for the ecological study of life cycle. In: Reports of the 1993 Annual Meeting on Squid Resources, Fishing and Oceanographical Conditions, National Research Institute of Far Seas Fisheries. 1994; (in Japanese). 19. Yang WT, Hanlon RT, Lee PG, Turk PE. Design and function of closed seawater systems for culturing loliginid squids. Aquaculture Eng. 1989; 8: Nakamura EL. Visual acuity of two tunas, Katsuwonus pelamis and Euthynnus affinis. FAO Conference on fish behaviour in relation to fishing technology and tactics, Bergen. 1967; FR:FB/67/ E/19, Kawamura G, Nishimura W, Ueda S, Nishi T. Vision in tunas and marlins. Mem. Kagoshima Univ. Res. South Pac. 1981; 2: 3 47.