Vision in the grass goby, Zosterisessor ophiocephalus (Teleostei, Gobiidae): A morphological and behavioural study

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1 Italian Journal of Zoology ISSN: (Print) (Online) Journal homepage: Vision in the grass goby, Zosterisessor ophiocephalus (Teleostei, Gobiidae): A morphological and behavioural study Damijana Ota, Marco Francese & Enrico A. Ferrero To cite this article: Damijana Ota, Marco Francese & Enrico A. Ferrero (1999) Vision in the grass goby, Zosterisessorophiocephalus (Teleostei, Gobiidae): A morphological and behavioural study, Italian Journal of Zoology, 66:2, , DOI: / To link to this article: Copyright Taylor and Francis Group, LLC Published online: 28 Jan Submit your article to this journal Article views: 331 Citing articles: 7 View citing articles Full Terms & Conditions of access and use can be found at

2 Ital. J. Zool., (1999) Vision in the grass goby, Zosterisessor ophiocephalus (Teleostei, Gobiidae): a morphological and behavioural study DAMIJANA OTA National Institute of Biology, University of Ljubljana, 1000 Ljubljana (Slovenia) and BRAIN - Centro Interdipartimentale per le Neuroscienze Dipartimento di Biologia, Universita di Trieste, via Giorgieri 7, Trieste (Italy) MARCO FRANCESE* ENRICO A. FERRERO BRAIN - Centro Interdipartimentale per le Neuroscienze Dipartimento di Biologia, Università di Trieste, via Giorgieri 7, Trieste (Italy) ABSTRACT The visual abilities of Zosterisessor ophiocephalus, an ambushing predator inhabiting the intertidal seagrass meadows of lagoons, were studied by morphological and behavioural methods. In its retina, a high number of rods, double and single cones packed in a square mosaic were found, together with the retinomotor response involving the pigment granules and both kinds of photoreceptors. The retinal topography was made by analysing the cone density and density of cells in the ganglion cell layer, and it revealed the area centralis in the dorso-temporal region of the retina. No influence of different light intensities (2.4 pimol m -2 s -1, 0.7 µmol m -2 s -1 and 0.1 µmol m -2 s -1 ) on its visual abilities for prey detection was found by comparing the detection and capture efficiencies and the detection and capture distance. A preference for larger food targets was observed. Two capture strategies are described: fast direct and slower hopping capture, the latter exhibited with higher frequency under dim light. Histological visual acuity was 7' and behavioural visual acuity was 15'. The morphological and behavioural studies showed that Z. ophiocephalus can use vision for feeding in bright and dim light conditions, but it probably feeds more at dusk to avoid predators. KEY WORDS: Retina - Retinal topography - Light intensity and behaviour - Visual acuity - Lagoon. ACKNOWLEDGEMENTS We are grateful to Prof. M. A. Ali, Prof. P. Stusek and Dr. J. E. G. Downing for helpful discussions and working facilities provided in their laboratories. We thank Prof. K. Draslar, Dr. S. P. Collin, S. Perrault, M. Balestra, P. G. Giulianini, M. Marchesan, G. Milani and F. Micali for technical assistance, and P. Pasqual for drawings. The revision of the English text by S. Fabrizio is gratefully acknowledged. This research was supported by the Research Community of Slovenia to D. O. and by Italian MURST (40%) "Biologia deu'ambiente neritico" to E. A. F.. * Present address: Riserva Naturale Marina di Miramare - Centro di Eto-ecologia ' Viale Miramare 349, Trieste (Italy) (Received 30 September Accepted 25 February 1999) INTRODUCTION Vision in fish depends on: size and position of the eyes, structure of the cornea and lens, and filters present in them, on the morphology of the retina and its neurophysiological activity, and visual pathways and integrating areas in the brain (reviews in Lythgoe, 1979; Nicol, 1989; Douglas & Djamgoz, 1990). Retinal morphology shows a great diversity in teleosts and is determined by ecological and ethological factors of the living environment for a given species rather than by its taxonomic position (Ali & Anctil, 1976; Ali, 1981; Pankhurst, 1989; Wagner, 1990). The ratios between the thickness of the outer retina (photoreceptor layer including the pigment epithelium) and of the neural retina (all the other layers together) (Nicol, 1989) have been found to be smaller among diurnal fishes and larger in crepuscular or nocturnal ones (Wagner, 1990). In species inhabiting low illuminated environments, a lower number of pigment granules is found in their pigment epithelium; they have a tapetum lucidum made by layers of guanine crystals, and rods with long outer segments and in a higher number than cones to enhance retinal sensitivity, as well as a higher number of rows of horizontal cells in the inner nuclear layer and a thinner inner plexiform layer (Ali, 1981; Wagner, 1990). In fish, vision under different light conditions is provided by the retinomotor response that involves positional changes of the pigment granules in the pigment epithelium as well as of the photoreceptors. Fish with a well-developed retinomotor response in their retina have a good vision both in bright light and dark conditions (Ali, 1975; Wagner, 1990). A fovea, the small depression in the retina with the highest cell density, is reported in some fish species only (Nicol, 1989; Wagner, 1990; Collin, 1997). In most fish species, a region with a higher cell density called area centralis is present. The distribution of different cell densities, or retinal topography, was studied by analysing the cone density or cell density in the layer of ganglion cells (reviews in Browman et al, 1990; Beaudet et al., 1997; Collin, 1997). The studies show that the shape and position of the area centralis differ among the species and depend on their foraging ecology. The position of the area centralis in the retina indicates the direction of the visual axis, i.e. the direction of the highest visual acuity and main visual interest (Tamura, 1957; Lythgoe, 1979). Light is one among the factors that influence activity and prey detection in fish (review in O'Brien, 1987). In planktivorous and benthic fish species studied up to now, it has been found that under decreasing light intensity their feeding activity, feeding rate, and reaction distance to the prey decrease (Ali, 1959; Winslade, 1974; review in O'Brien, 1987; Bergman, 1988; Pankhurst & Montgomery, 1989; Beers & Culp, 1990; James & Heck, 1994; Utne, 1997). Reduced feeding activity and reduced reaction distance have been correlated with a worser visual detection under low light conditions. Published online 28 Jan 2009

3 126 D. OTA, M. FRANCESE, E. A. FERRERO The measure of the ability to distinguish details is visual acuity, expressed in min of arc. Visual acuity is determined by the size of the image on the retina and by the cell density in the retina. The more cells covered by the image, the better is its resolution (Tamura, 1957). In teleosts, visual acuity has been determined by histological, electrophysiological and behavioural measures (reviews in Collin & Pettigrew, 1989; Douglas & Hawrhyshyn, 1990; Browman et al, 1990; Wanzenbock et al, 1996). The differences in visual acuity found among the species are due to feeding ecology: a lower visual acuity was calculated in herbivorous and nocturnal species of fish, while a higher acuity was calculated in predatory and piscivorous fish. In some species, the histological and behavioural measures of acuity were compared but did not coincide in all cases (review in Douglas & Hawryshyn, 1990). The aim of the present work was to describe the eye and retinal morphology and retinal topography of the grass goby Zosterisessor ophiocephalus (Pallas, 1811), as well as to observe the influence of light intensity on its feeding behaviour under laboratory conditions and to calculate its histological and behavioural visual acuities. This approach was used in order to relate the goby's morphological and behavioural adaptations to its living environment, i.e. the intertidal seagrass meadows of the lagoon. Zosterisessor ophiocephalus excavates burrows in the muddy lagoon bottom to reproduce and to hide during the low tide periods when the meadows emerge from the water (Ninni, 1938; Ota et al, 1999b, in press). Analyses of stomach contents showed that it feeds on small invertebrates and small fish (Pagotto & Campesan, 1980). MATERIALS AND METHODS Grass gobies Z. opbiocephalus were obtained from local fishermen in Grado near Trieste. The larvae used in the present study hatched in the laboratory from eggs collected in the nests of Z. ophiocephalus in the lagoon of Grado and kept under controlled conditions. Eye and retinal morphology To calculate the allometric growth curve, the total body length and the eye diameter were measured in 136 adults of both sexes (total body length, TL = 9-23 cm), deeply anaesthetized with 3- aminobenzoic acid ethyl ester (MS-222), and then sacrificed and dissected for further morphological, histological, or biochemical studies. The total body length and eye diameter were measured also in 28 hatched larvae anaesthetized as above. These measurements were made under a Wild M3 dissecting microscope, fitted with a calibrated eyepiece grid. Corneal iridescence and its colour change was monitored in four alive gobies before and after a 3-h period of dark adaptation. Fish were illuminated from above and the iridescent reflections viewed from the side at 2-min intervals for 2 h after the onset of light (Lythgoe, 1975). To observe the retinal morphology, the eyes were enucleated from seven light adapted gobies (TL = 16 ± 1 cm) and fixed in Karnovski solution (2.5% glutaraldehyde, 4% formaldehyde in 0.15 M cacodylate buffer, ph 7.2). Six enucleated eyes were embedded in toto in paraffin and sections cut at 10 lm on a Leitz 1207 microtome and stained with hematoxylin and eosin. From the other enucleated eyes, the cornea and the lens were removed and the lens diameter measured under the dissecting microscope. The isolated retinae were divided into 16 pieces (eight peripheral and eight central), post fixed in 1% osmium tetroxide and, after dehydration, embedded in Spurr or Epon - Araldite resins. Thin transverse and tangential sections of the retinae were cut on a LKB Ultrotome III and Reichert OM-U3 and stained with 0.5% methylene blue in 1% borax. The rods: cones ratio was determined by counting the rod and cone nuclei in the outer nuclear layer on transversal sections from all retinal regions. The single cones: double cones ratio was determined by counting the single and double cones on tangential sections from all retinal regions. Both countings were performed at 40x magnification objective lens. To observe the retinomotor response, four dark adapted (3 h in total darkness after 6 p.m.) gobies (TL = 16 ± 1 cm) were anaesthetized, and their eyes were enucleated and fixed as above. Thin transverse sections were made and observed at 40x magnification objective lens. Retinal topography Retinal topography was determined on retinal wholemounts by analysing the cell densities in the cone outer segment layer and in the ganglion cell layer. Whole retinae were isolated from fish as above. Cone density was analysed on retinal wholemounts incubated for the NADPH-diaphorase reaction that stains the cone ellipsoids dark blue (Ota & Downing, 1996), while the density in the ganglion cell layer was assessed by Nissl staining. For NADPH-diaphorase reaction, three light adapted retinae were isolated, fixed for 15 min in 4% paraformaldehyde in 0.1 M phosphate buffer (ph 7.4), and then stored in the same buffer at 4 C. The pigment epithelium was easily detached and gently brushed away. The retinae were then rinsed in 0.1 M Tris (ph 8.0), for 30 min and incubated as wholemounts for 24 h at 37 C in a reaction mixture containing 0.2 mm nitro blue tetrazolium (NBT), 1 mm P-nicotinamide adenine dinucleotide phosphate ((3- NADP), 30 mm malic acid dissolved, 1 mm manganese chloride, 0.5% Triton X-100, all in 0.1 M Tris HC1 buffer (ph 8.0), after the method of Downing (1994). All chemicals were purchased from Sigma. After incubation, the retinal wholemounts were rinsed in 0.1 M phosphate buffer (ph 7.4), mounted in glycerol and coverslipped. They were examined under a microscope and the outline of each retinal wholemount was first traced onto a 1-cm 2 grid paper at a 20x magnification objective lens. With the NADPH-diaphorase reaction, the double cones stained in dark blue, while single cones were, for some unknown reasons, stained only in some regions. To determine the cone density in the retinal regions, double cones only were counted. The countings were performed at a 40x magnification objective lens on a TV screen using a video camera on the microscope. The image transferred on the TV screen corresponded to a surface of 28 x 10 3 lm 2. The countings were made every 1.0 mm of the retina. The numbers were then converted to double cones per square millimetre. In this way, from 37 to 81 areas per retina were sampled. Iso-density contours were drawn by interpolation between the values of double cone density. The intent was to indicate the relative cone densities, rather than to determine the absolute number of cones present. Topography of cells in the ganglion cell layer was analysed on Nissl stained retinal wholemounts. Six retinae were isolated from dark adapted eyes and fixed for 40 min in 4% paraformaldehyde. Then, the retinae were rinsed in 0.1 M phosphate buffer (ph 7.2) and stored at 4 C. For Nissl staining, retinae were transferred on gelatine coated slides with the ganglion cell layer uppermost and dried for 12 h. Each retinal wholemout was then stained for 8-12 min in cold 0.05% cresyl violet for Nissl susbstance, dehydrated, cleared, mounted in D.P.X. and coverslipped. Under the microscope, the outline of each retinal wholemount was first traced onto a 1 cm 2 grid paper at a 20x magnification objective lens. The eyepiece was fitted with a square grid of 7 x 10 3 (im 2 of surface

4 VISION IN GRASS GOBY FISH 127 at a loox magnification objective lens and all the cells within the square grid were counted. The countings were made every 0.5 mm of the retina. The frequencies were then converted to cells per square millimetre. In this way, up to 150 areas per retina were sampled. Iso-density contours were drawn by interpolation between the values of retinal ganglion cell density. Cell soma size of cells in the ganglion cell layer were calculated with the Vidas video analyser (Zeiss) on black and white pictures of different retinal regions. Each cell outline was traced using an electronic stylus, and the area of each soma was directly calculated. A sample of cells was measured in each density region, and histograms of soma area ( im 2 ) versus frequency were made. Retinal sections and retinal wholemounts were examined, photographed, and analysed on Leitz Dialux 20 and Olympus BX 50 microscopes and with video image analyser system (Image PRO PLUS 30; Media Cybernetics). Behavioural observations Grass gobies (TL = 16 ± 1 cm) were kept in individual tanks (60 x 40 X 23 cm) with well-aerated and filtered sea water (T = C; salinity 34%o). The bottom was covered with sand and a plastic tube cut in half served as a shelter. The fish were fed with small pieces of fish meat every day ad libitum. For experiments, one goby at a time was placed the evening before the start of the experimental session in a 190-litre test tank (120 x 40 x 40 cm), filled with sea water to a depth of 30 cm. The bottom was covered with fine sand. A shelter of the same size and shape as in the individual tanks was placed near the shorter wall of the tank with the entrance oriented toward the longitudinal axis of the tank. Seven plastic tubes extending to the observer were placed overhanging along the midline of the tank at increasing distances from the shelter (from 17 to 87 cm) and were held in place by a ribbon tape. The tips of the tubes were filled with a few millilitres of water containing a piece of food (fish meat) which was released in the tank by the remote observer during the experiments. The test tank was surrounded with a black plastic screen to avoid any disturbance of the gobies during the trials. The behaviour of the fish was observed and videorecorded (Hitachi VM-S7200E) through holes in the screen. Zosterisessor ophiocephalus is a sit and wait predator able to detect a living prey at a longer distance, but it attacks only when the prey is close to its shelter (personal observations). To determine its visual and detection abilities and its reaction distance, a standard prey stimulus was used, i.e. pieces of fish, released at increasing distances from the shelter. A pre-experimental two-day training was given to the gobies in order for them to adapt to the experimental setup. During the experiments, the food targets, one at a time, were released from the tubes and the reactions of the goby recorded. Each experiment ended when all the food targets were released from the tubes, and the next started when the food pieces were replaced. During replacement, a plastic opaque screen was placed in the front of the shelter to avoid the exit of the fish from it. In each experiment, targets of both 3 and 6 mm were used and released in a random order. Each target was released when the goby was in the shelter facing the longitudinal axis of the tank and with head and eyes slightly protruding. Each subsequent release was made when the fish was again in this starting posture. As a control, food conditioned water only was released from one or two tubes in each experiment. In our experiments, the gobies reacted to the visual stimuli of the food by exiting from the shelter and swimming towards the target within 15 s from their release. Preliminary observations on chemoreceptive abilities of the gobies showed that the reaction to a chemical stimulus released by food has a longer latency (over 30 s) and consists of some rapid chewing movements of the goby that sometimes collects sand from the bottom and ejects it after some chews. Sometimes the fish also exits from the shelter and swims randomly across the tank. The experiments were performed at three light-intensities simulating bright light (120 lx umol nr 2 s' 1 ), dusk light (35 lx = 0.7 Xmol m- 2 s" 1 ), and dim light (5 lx = 0.1 u.mol nr 2 s" 1 ) measured at bottom level with CLIMALUX, Laboratori di Strumentazione Industriale S.p.A. The light intensities were provided by a bank of type 94 Philips tubes matching the solar spectrum placed at different distances from the tank. Only one light intensity was used in one day, and the gobies were adapted to the given light for at least 3 h before the experiments. Each fish was tested at each light intensity for one or two days. All the experiments were videorecorded and the experiments performed under dim light were, in addition, commented on tape simultaneously by two observers, one reporting the vertical position of the food target and the other reporting the behaviour of the goby. In one day, three or four experiments were performed with the same goby. In total, seven gobies of both sexes were used that underwent an average of 12 experiments each. The data for seven gobies were grouped and a total % of their reactions to the food targets at each distance was calculated. The videotapes were analysed by slow motion and single frame (Panasonic NV- FS90EG). The detection distance (the distance between the eye of the goby and the food target at the moment when the goby first notices it and dashes forward or exits from the shelter) was measured on the monitor screen, and the time to capture calculated. Detection distance and time to capture were recorded for each light intensity and for both food targets. Statistical tests (t-test, chi square) were used to compare the data. Occasional reactions of the fish to the movements of the water were excluded from analysis. To determine the dorso-ventral extent of the visual field and the reaction angle, the positions of all food targets at the moment when the goby noticed them were plotted in a diagram. Visual acuity The histological visual acuity can be calculated by dividing the linear distance between the centres of adjacent cones (the reciprocal of the number of cones per millimetre) or the density of cells from the ganglion cell layer by the focal length of the lens (Tamura, 1957; Neave, 1984; Williamson & Keast, 1988; Collin & Pettigrew, 1989; Wanzenbock et al., 1996). The focal length of the lens is calculated by multiplying its radius (r) by 2.34, the Matthiessen ratio for gobies (Hansen, 1988). This yields the following expression: sin a 1.11 / o/ N x 2.34 r) where a is the minimum separable angle in degrees, 1.11 represents an 11% correction factor to account for histological shrinkage of the retina prior to cone density measurements, N is the number of cones (double and single) per mm 2. This equation assumes that to discriminate two objects, their images must be separated by at least 1 photoreceptor. To determine the behavioural visual acuity, measurements of the maximum capture distance on prey of known size can be expressed as an angle subtended on the retina because the image of the prey is formed by straight lines from the ends of its longest visible dimension to a point on the fish's retina. For a prey target of known size at a given distance from the eye, this visual angle in degrees is calculated as (Hairston et al., 1982; Browman et al, 1990): a = 2 arctan (0.5 H / MCD) where H is the visible size of the prey and MCD is the maximum capture distance observed. RESULTS Eye and retinal morphology In adult Zosterisessor ophiocephalus, the eye diameter increases with the total body length (r 2 = 0.74, P < 0.001,

5 128 D. OTA, M. FRANCESE, E. A. FERRERO 9 _ 8 i'< Q) E 4 CO >. larvae adults total body length (mm) 250 Fig. 1 - Eye diameter and total body length in larvae and adults of Zosterisessor ophiocephalus. n = 136) (Fig. 1). The average eye size in the hatched larvae (TL = mm) is 0.31 ± 0.01 mm SD (n = 28). The calculated relative average eye size expressed in % of the total body length is 4.2 ± 0.35% SD in the adults and 7.8 ± 0.8% SD in the larvae. The higher % of the relative eye size in the larvae indicates a negative allometric growth of the eye of Z. ophiocephalus in relation to its body growth. The cornea of Z. ophiocephalus shows a green-yellow iridescence in its dorsal region and retains it after the dark adaptation. The corneal iridescence of a dark adapted fish does not change in colour during the 2 h of observations after the onset of light. The lens is spheric and transparent. Its average diameter is 2.4 ± 0.0 mm SD (n = 10, TL = 16 ± 1 cm). The retina of Z. ophiocephalus is 220 um thick at the optic papilla and around 160 urn in the periphery. The average ratio between the outer and neural retina is 1:1 (Fig. 2A, B). The retinal layers are well-defined. In the pigment epithelium, many dark pigment granules are present and a tapetum lucidum is lacking (Fig. 2A, B, C). The processes of the pigment epithelium cells extend to the cone outer segments in the periphery (Fig. 2A, C), whereas in the central retina they are shorter and extend to the scleral third of the photoreceptor layer envelopping part of the rod outer segments (Fig. 2B). The photoreceptors are rods, single and double cones (Fig. 2C, D). Double cones are made by two equal cones (Fig. 2D). The double cones: single cones ratio is 2:1 in all retinal regions. Rods are more numerous than cones in all retinal regions (the rods: cones ratio is 2.5-3:1). Single and double cones are densely packed and arranged in a regular square mosaic, where four double cones surround one single cone (Fig. 2D). No intervening accessory single cones occur. The square mosaic is observed in all retinal regions. In the inner nuclear layer, only one row of horizontal cells is observed (Fig. 2E), densely packed (Fig. 2F), and bipolar and amacrine cells predominate (Fig. 2E). At the vitreal border of the inner nuclear layer (Fig. 2E) and in the inner plexiform layer, single large cells are found. At the retinal vitreal border, the ganglion cell layer and the nerve fibre layer are observed (Fig. 2A, B, G). In the central retina, axons of the nerve fibre layer are in large bundles and penetrate the ganglion cell layer where columns of cells alternate with bundles of fibres (Fig. 2G, H). Among axon bundles, fibres and endfeet of Miiller cells are visible (Fig. 2G). The Nissl stained wholemounts (Fig. 2H) revealed that the bundles of axons extend radially from the optic disc. Their width decreases at increasing distance from the optic disc, and in the periphery they are no longer evident. Blood vessels are observed on the retinal vitreal surface. The retinomotor response in the retina of Z. ophiocephalus involves the displacement of pigment granules in the pigment epithelium and of all types of photoreceptors (Fig. 3A, B). In the dark adapted retina, the pigment granules are sclerally concentrated, rods are aligned along the outer limiting membrane and double and single cones are sclerally located, close to the pigment epithelium cells (Fig. 3A, B). The average displacement distance of rods is um SD (n = 23), and that of cones is 44.5 ± 4.4 um SD (n = 24). Retinal topography Both analyses of the retinal topography, based on the density of double cones and density of cells in the ganglion cell layer, reveal an area centralis, the region with the highest cell density, in the temporo-dorsal region of the retina (Fig. 4A, B). In the area centralis, the calculated average density of double cones is above per mm 2, while the average cell density of the cells in the ganglion cell layer is above per mm 2. Regions with lower cell density surround the area centralis and are elongated across the horizontal axis (Fig. 4A, B). The comparison of soma size within the ganglion cell layer between cells of the highest cell density region (Fig. 4C) and peripheral region (Fig. 4D) reveal significant differences in soma area (t-test, P < 0.001). However, the distribution of cell size frequency is unimodal in both regions (Fig. 4E, F) and in the intermediate density regions. Influence of light intensity on behaviour The grass gobies in the experimental tank spent most of the time in their shelters and slightly protruded their heads and eyes from it. In the shelter, the gobies dug and made a sloping bottom. They stopped digging when a plastic inclined bottom was provided inside the shelter. They left the shelter spontaneously from time to time, alternating short distance swims (20-30 cm) with periods of laying on the bottom raised on their ventral fins for around 15 with respect to the bottom surface (Fig. 5A). The gobies left their shelter spontaneously most often at low light intensity, and the time spent outside the shelter significantly increased with decreasing light intensity (Table I).

6 VISION IN GRASS GOBY FISH 129 Fig. 2 - Transverse and tangential sections of light adapted retina of Zosterisessor ophiocephaliis. A: retina in periphery. B: central retina. C: photoreceptor layer. D: square mosaic of double and single cones (tangential section). E: inner nuclear layer. F: horizontal cells (tangential section). G: bundles of axons in the nerve fibre layer. H: alternation of columns of cells in the ganglion cell layer with bundles of nerve fibres (transparent) in the central region of Nissl stained retinal wholemount. Abbreviations: B, bundles of axons; C, cone ellipsoids; DG, displaced ganglion cell; GC, ganglion cells; GL, ganglion cell layer; H, horizontal cells; IN, inner nuclear layer; IP, inner plexiform layer; M, Muller cells endfeet; NF, nerve fibre layer; ON, outer nuclear layer; PE, pigment epithelium; PL, photoreceptor layer; R, rod ellipsoids; V, blood vessel. Scale bar, 10 M.m.

7 130 D. OTA, M. FRANCESE, E. A. FERRERO ft ' n- '!/ / ' Jf v, /' J Fig. 3 - Transverse section of the dark adapted retina of Zosterisessor ophiocephalus. A: photoreceptor layer and outer nuclear layer. B: cones close to the perikarya of pigment epithelium cells. Abbreviations: C, cones; DC, double cones; ON, outer nuclear layer; OM, outer limiting membrane; PE, pigment epithelium; R, rods. Scale bar, 10 um. TABLE I - Frequencies (n) of the spontaneous exits from the shelter, mean time in s (x) spent outside the shelter, and the relative t-test under the three light intensities. t-test n X SD 120 lx 35 lx 51x 120 lx lx 5.74 P = lx lx 3.36 P x k 2.02 P = TABLE II - Percent detections and captures of targets of 6 and 3 mm under the three light intensities. Light intensity 120 lx 35 lx 5k * P < 0.05; *' P < 0.01 Detections (%) 6 mm 3 mm " 77 55' Captures (%) 6 mm 3 mm * The seven individuals of Z. ophiocephalus used in the experiments exhibited four different responses to the food released in the tank (Fig. 6): 1) direct capture of the food target (DC); 2) hopping capture with one or more stops (HC); 3) forward dashing without leaving the shelter (R); 4) no reaction (NR). In the direct capture, the fish started a fast swim from the shelter and approached and snapped the food target from below. In the hopping capture, the fish slowed down or stopped its swim toward the food target once or twice. In the forward dash, the fish jerked forward for a few centimetres, but did not swim out of the shelter. These reactions were performed under all the three light intensities used in our experiments. During control releases (ji = 162) the gobies reacted, in an average of 10% trials, with forward dashing or exiting from the shelter with a random slow swim alternated with laying on the tank bottom. In the experiments, reactions 1, 2, and 3 all together were considered as detections of the food target, and reactions 1 and 2 all together as captures of the food targets. Data on the % of detections and captures, and for both food targets are presented separately (Table II). For targets of 6 mm, the % of detections was higher than the % of captures, while for targets of 3 mm, this % was higher for detections at bright light only. To see if the light influences the detection and capture of the food targets, the number of detections and captures of both food targets were compared (chi square test). For food targets of 6 mm, no significant differences were found in detection and capture efficiency among the three light intensities. However, for the food target of 3 mm, a significantly higher detection was found at the highest intensity, while no significant differences were found in the number of captures among the three light

8 VISION IN GRASS GOBY FISH u» "33 60 o area centralis n = jn "53 60 u L JjJ periphery n = 552 Fig. 4 - Retinal topography and ganglion cell soma analyses. A: iso-density contour map of double cone densities in the retinal wholemount stained with the NADPH-diaphorase. B: iso-density contour map of densities of cells in the ganglion cell layer in a Nissl stained retinal wholemount. Cell density x Abbreviations: D, dorsal; T, temporal; V, ventral; N, nasal; OD, optic disc. C, D: Nissl stained cell somata in area centralis (C) and in the periphery (D). Scale bar, 10 Jim. E, F: histograms of frequency versus soma area ( im 2 ) in area centralis (E) and in the periphery (F). Arrows indicate the mean values.

9 132 D. OTA, M. FRANCESE, E. A. FERRERO TABLE HI - Frequencies (n) and percent of direct (DC) and hopping captures (HC) under the three light intensities. Light intensity u %DC %HC B 120 lx 35 lx 51x Fig. 5 - Head posture of Zosterisessor ophiocephalus. A: raised on ventral fins. Dotted line shows the direction of its body axis. B: head and the visual axis. Scale bar, 1 cm. intensities. These data indicate that because of a better detection of the 3-mm food targets at the highest intensity, the light level may improve target visibility, but it does not influence its capture. To assess whether the target size influences detection or capture efficiency, the number of detections and captures of both food target sizes were compared at the same light level (chi square test) (Table II). Consistently higher detection and capture rates of food targets of 6 mm over the 3 mm ones were recorded, and significantly so at the intermediate and at low light levels. These data indicate a better detection of targets of 6 mm under lower light intensities and a significant preference to capture the larger food target size under the intermediate light intensity. The gobies exhibited two different strategies to capture a food target: direct capture (DC) and hopping capture (HC) (Fig. 6). The average capture time during DC was significantly shorter (2.8 ± 1.3 s SD, n = 122) than the average capture time during HC (4.3 ± 1.9 s SD, n = 61; t-test = 6.06, P < 0.01). Under all the three light intensities and with increasing distance from the shelter, the % of DC decreased, while the % of HC increased (Fig. 7, Table III). The cross point between DC and HC was recorded at the shortest distance at 5 lx. The frequencies of DC and HC exhibited at 5 lx significantly differed from those exhibited at 120 lx (% 2 = 10.69, P < 0.01) and 35 lx (% 2 = 6.2, P = 0.016), while no significant difference was observed comparing the two strategies between 120 and 35 lx (x 2 = 0.62, P = 0.45). These data indicate that at the lowest light intensity the gobies use the slower approach to the food targets; that implies a longer capture time and a longer period outside the shelter. The gobies captured, or at least detected, targets of both sizes up to the maximum distance available in the experimental setup (Fig. 6). A decreasing light intensity does not reduce the maximum detection distance to targets of both sizes, however the % starts decreasing at about 40 cm from the shelter under each light intensity. To see whether the light level affects the distance for detection and capture, the distance at which the probability of 50% of reactions may occur was calculated by linear regression. In all cases, the 50% detection distance was longer than the 50% capture distance (Fig. 6). Under all light intensities both the 50% detection and capture distances were longer for the 6-mm target compared with the 3-mm one. However, for a food target of 6 mm, the distances of 50% probability of detection and capture were shortest at the highest light intensity. For food targets of 3 mm, the distances of 50% probability of detection and capture were similar among the three light intensities. These data indicate that light level does not influence detection and capture distance for food targets of 3 mm. Visual field and visual axis The gobies detected the food targets overhead, in front and close to the tank bottom (Fig. 8). In the shelter, they lay on an inclined bottom and raised on their ventral fin, and the body axis was tilted upward by 15 with regard to the tank bottom surface. On distances up to 40 cm, detections occurred above and below the body axis and in the dorsal, frontal, and ventral portion of the visual field. On longer distances, detection occurred below the body axis and in the fronto-ventral portion of the visual field. Under our experimental conditions, these data indicate a scanning area of 38 where dorso-temporal, temporal, and ventro-temporal regions of the retina are used for detection. The eyes on the head of Z. ophiocephalus are located laterally, parallel to each other, slightly oriented upward and immobile. The pupil in the eye is eccentric (pearshaped) (Fig. 5B) with an aphakic aperture directed fronto-ventrally at an angle of 15 with regard to the body axis. Since in the tank the goby's body axis is oriented 15 upward, the visual axis looks almost parallel to the bottom line. Visual acuity The histological visual acuity of Z. ophiocephalus was calculated using the cone density and the density of

10 VISION IN GRASS GOBY FISH 133 A 120 lux (12) (18) (8) (15) (11) (10) (11) 100% T B 120 lux (18) (6) (9) (14) (6) (5) (7) 100% i 80% 20% 20% 0% cm 0% C 100% i 35 lux (10) (14) (9) (11) (12) (14) (15) D 35 lux (10) (11) (3) (14) (13) (10) (11) 100% T 20%- 0% % cm E 100% i 5 lux (6) (8) (4) (5) (6) (6) (12) F 5 lux (8) (6) (9) (4) (5) (5) 100% T I 1 i (10) 20% 0% cm 0% DC I HC R NR DC ihc lr Fig. 6 - Reactions of Zosterisessor ophiocephalus to food targets of 6 mm (A, C, and E) and 3 mm (B, D, and F) released at increasing distances (central values) from the shelter under the three light intensities. White arrows on abscissa indicate the distance of 50% probability of detection; black arrows indicate the distance of 50% probability of capture. Abbreviations: DC, direct captures; HC, hopping captures; R, forward dash; NR, no reaction. D NR cells in the ganglion cell layer in the area centralis. Cone density was 3.7 x 10 4 mm" 2, and the calculated acuity was 7' of arc. The density of cells within the ganglion cell layer was above 4.5 x 10^ mm" 2, and the cal- culated visual acuity was 6' of arc. The difference in the two calculated histological acuities is due to the higher number of cells in the ganglion cell layer. But in this layer not all cells are ganglion cells involved in the sig-

11 134 D. OTA, M. FRANCESE, E. A. FERRERO lux 5 lux Fig. 7 - Frequencies in percent of direct and hopping captures of Zosterisessor ophiocephalus at increasing distances (central values) under the three light intensities. Filled squares indicate direct captures, open squares indicate hopping captures. nal transmission to the higher integrating areas, since also a population of displaced amacrine cells is present, representing 20% of all cells in the ganglion cell layer (Collin & Pettigrew, 1989), that do not contribute to visual acuity, and therefore this visual acuity is overestimated. To calculate the behavioural visual acuity the maximum capture distance performed by direct captures only was considered. The maximum direct capture distance (MCD) recorded in our experimental setup was 70 cm for a food target of 3 mm (Fig. 6) resulting in the best calculated behavioural visual acuity of 15'. DISCUSSION Eye and retinal morphology In fishes, eye growth with respect to body growth shows in general a negative allometric increase (Moskal'kova, 1971; Williamson & Keast, 1988; Pankhurst & Montgomery, 1990) as also shown in Z. ophiocephalus. In Gobius melanostomus (Moskal'kova, 1971) the relatively smaller eye size in adults was correlated with their reduced use of vision in feeding in comparison with the larvae, which use only vision in feeding. The larvae of Z. ophiocephalus feed on plankton and use vision for prey detection (Privileggi et al., 1997) and have relatively larger eyes than the adults, which use, beside vision, also mechano- and chemoreception for feeding (Pagotto & Campesan, 1980; personal observations) and for interindividual communication (Marchesan et al., in prep.). Corneal iridescence is common in teleost fish that live in shallow and well-illuminated waters (Lythgoe, 1975; Shand, 1988). This adaptation is thought to reduce the intra-ocular flare caused by bright downwelling light by reflecting it out of the eye, while having little effect on horizontal lateral rays. The abolition of iridescent reflections in dark is reported for the sand goby Pomatoschistus minutus (Shand, 1988) and the subsequent increase in corneal transparency to downwelling light will have visual significance. In Z. ophiocephalus, the corneal iridescence does not disappear in the dark and does not change in colour after dark, indicating that the reflection of the downwelling light occurs at bright and dim light conditions and provides a good lateral vision in both situations. The retinal morphology described in Z. ophiocephalus is in agreement with previous observations on the same species made by Verrier (1928) and confirms the predatory habit of Z. ophiocephalus reported by stomach content studies (Pagotto & Campesan, 1980). Its retina shows well-defined layers, and an average ratio of 1:1 between outer and neural retina; there is no tapetum lucidum, but long processes of the pigment epithelium cells containing many pigment granules. Both kinds of cones occur, single and double, packed in a square mosaic present in all retinal regions, bipolar and amacrine cells predominate over the horizontal ones in the inner nuclear layer. These features are typical of retinae of fishes inhabiting clear, shallow and well-illuminated waters that prey upon small crustaceans, insect larvae, and fish (Ali & Anctil, 1976; Ali, 1981; Wagner, 1990). Its vision is good also under lower light intensities, subserved by the higher number of rods with long outer segments, that indicate a good retinal sensitivity, and support its suggested crepuscular feeding habits (Pagotto & Campesan, 1980). The well-developed retinomotor response with displacement of the pigment granules in the pigment epithelium, and alternative alignment of cones and rods along the outer limiting membrane, where the photon capture is optimal, also provides

12 VISION IN GRASS GOBY FISH 135 [cm) n» water column hei 30 i E -5 J L tank bottom * distance from shelter (cm) t body axis water surface visual ax Fig. 8 - Positions of 6- and 3-mm food targets in the experimental tank at the moment of their detection, with body and visual axis of Zosterisessor ophiocephalus. E, eye position of the goby in the shelter. 90 good vision in both low and high light conditions. Vision in both bright and dim light conditions may allow feeding and social activities at different times of the day, relevant for a species, such as Z. ophiocephalus, living in intertidal seagrass meadows, where the activities are limited to the high tide period. Axon fasciculation and ganglion cells The axon fasciculation and the radial arrangement of cells in the ganglion cell layer found in Z. ophiocephalus is a common characteristic of retinae in primitive groups of fishes like Agnatha and Chondrostea, but it has been reported also in other teleosts and found also in amphibia and mammalia (reviews in Collin & Collin, 1993; Collin & Northcutt, 1993). The function of this arrangement is unknown, but according to Collin & Northcutt (1993), this character is probably primitive for all vertebrates, being secondarily lost (no fasciculation and a separate axon layer) in some groups. The axon fasciculation is usually accompanied by vitreal vascularization, as found also in Z. ophiocephalus (Ota & Lahnsteiner, 1996), that was also proposed as a primitive feature (Collin & Collin, 1993). In the case of Z. ophiocephalus, the well-developed vitreal vasculature may be an adaptation to avoid retinal damages caused by hypoxic conditions of the muddy lagoon bottom (Ota & Lahnsteiner, 1996). In many fish, studies on cell soma size of retinal ganglion cells revealed specific size populations of cells that probably have different functions (review in Collin & Northcutt, 1993). Large ganglion cells have been found in the layer of the ganglion cells and displaced in the inner nuclear layer, belonging to alpha-ganglion cells and biplexiform cells (reviews in Collin & Northcutt, 1993; Cook & Sharma, 1995; Cook et al, 1996). In Z. ophiocephalus, no great differences in cell soma sizes in the layer of ganglion cells were found, but single large cells were observed in the inner nuclear layer and in the inner plexiform layer. Detailed subsequent studies on retinae using the NADPH-diaphorase reaction revealed axons leading from these cells to the nerve fibre layer, identifying them as displaced ganglion cells (Ota & Downing, 1996; Ota et al, 1999a, in press). Topography The terrain theory (Hughes, 1977) suggests that the distribution of density of cells across the retina reflects the symmetry of the perceived world. Species inhabiting open environments have a prominent visual streak with a high density of cells that allows scanning of the horizon. This visual streak is lacking in species living in enclosed environments, where the vision of the horizon is disturbed by plants, rocks, or corals. It has been postulated that the horizontal streak provides a lower threshold for the perception of movement (Munk, 1970; Rowe & Stone, 1977) and that a temporal area centralis subserves binocular vision in feeding situations (Tamura, 1957; Collin & Pettigrew, 1988, 1989). In Z. ophiocephalus, a dorso-temporal area centralis and a nonprominent visual streak across the horizontal meridian of the retina have been found. The area centralis is the region with the highest visual acuity and its dorso-temporal position in the retina of Z. ophiocephalus provides the best, and probably also binocular, vision in the fronto-ventral portion of the visual field, where the prey of a benthic predator is usually located and captured. The weak horizontal streak could provide some ability of scanning across the horizon when out of the burrow for feeding or social interactions, but being exposed to predators, like sea basses, and birds, such as herons and gulls (personal observations), some predator detection is thus required. The topography described in Z. ophiocephalus is similar to that described for Aulostoma chinensis, a benthic

13 136 D. OTA, M. FRANCESE, E. A. FERRERO species which periodically ventures out into open waters (Collin & Pettigrew, 1988) and in the blenny Dasson variabilis that inhabits the seagrass meadows in Australia and ambushes small invertebrates (Collin & Pettigrew, 1989). Both these species and Z. ophiocephalus have similar habits and behaviours and show similar retinal topographies, that reflect their living environment, enclosed benthic spaces and occasionally open waters, which is in agreement with and supports the terrain theory (Hughes, 1977). The present study shows that the analyses of cell densities at different retinal levels reveal similar topographies, as already shown in goldfish (Mednick & Springer, 1988), where the increase in cell density in the area centralis was found also in the inner nuclear layer. Light intensity and behaviour In Z. ophiocephalus, a decreasing light intensity does not reduce its maximum detection distance and its overall food detection and capture efficiencies. This indicates that its vision abilities are good at high as well as at low light conditions, confirming the retinal morphology observations and the occurrence of the retinomotor response (see above). However, the light can influence its feeding behaviour. A higher spontaneous activity and slower, hopping captures were observed at the lowest light intensity, while lower activity and fast captures were observed at the highest light intensity. A higher activity at low light conditions was observed also in the black goby Gobius niger (Hesthagen, 1976) and it has been suggested that the goby is more active at low light conditions because its prey is more active at dusk, and in addition the goby is less vulnerable to predators such as birds. Similar conclusions have been reported also for the lotic minnow Rhinichtys cataractae (Beers & Culp, 1990) that feeds during the night probably to avoid predators, even if its visual abilities are better during the day. In gobies Gobius niger, Pomatoschistus minutus, and Gobiusculus Jlavescens, a decreasing activity in the presence of their predator the cod, Gadus morhua, has been reported (Magnhagen, 1988; Utne et al., 1993; Utne & Bacchi, 1997). Predators of grass gobies are sea basses and birds as herons and gulls, daily visual predators that ambush the gobies at their burrow entrance during the day (personal observations). In the tank, the grass gobies may correlate the high light intensity with daytime, i.e. with higher chances of predator presence and therefore at this light level they used faster attacks and spent less time outside the shelter, while at low light, being less vulnerable, they spent more time outside the shelter. Behavioural plasticity in foraging develops as an adaptive mechanism under several environmental pressures. Shifts in foraging strategies are reported depending on light, habitat complexity, cover density, prey habits, predator presence (Ali, 1959; Bergman, 1988; Savino & Stein, 1989a, b; Beers & Culp, 1990; James & Heck, 1994). Similar conditional strategies enable a predator to maintain a relatively constant prey capture level, whereas purely attack strategists in a similar habitat do not. More reactions and also at longer distances were recorded to larger targets than to the smaller ones. The preference to a larger prey over a smaller one is related with a better detection and with a positive cost-benefit balance (O'Brien, 1987; Magnhagen, 1988, 1993; Utne, 1997). Z. ophiocephalus, like other benthic species, is relatively heavy; it is a territorial species (Ota et al., 1999b, in press), that ambushes from its shelter rather than actively chasing the prey, since for such species, swimming is a costly activity from the energetic point of view. It is possible that the grass gobies, because of a negative cost-benefit balance, sometimes disregarded small targets at longer distances even if they had detected them. In effect, under predation prone conditions (bright light), the detection efficiency of 3-mm targets was significantly higher than at low light levels. In contrast, capture efficiency was not significantly different. An even more extreme case is the reduced 50% detection distance for 6-mm targets under high light intensity compared with the dim ones. The conflicting motivations of fear of predators vs hunger may bias the attentional processes towards sensory neglect of small (food) objects at a distance where only large (predator) objects are scanned for. Visualfield Eye position is related to feeding vectors and determines the extent and the direction of the visual field (Lythgoe, 1979; Nicol, 1989; Pankhurst & Montgomery, 1989). Benthic fish with dorsolaterally placed eyes on the head, as the case of Z. ophiocephalus, feed mainly on benthic prey but also from the water column overhead (Pankhurst & Montgomery, 1989), as also confirmed by our observations on the dorso-ventral extent of the visual field. Aphakic apertures in the pupil are commonly found in shallow water teleosts (Nicol, 1989). They allow accomodation of the lens along a visual axis (Sivak, 1973) and/or increase peripheral vision by greatest admissions of light coming from the objects placed peripherally to the visual axis (Munk & Frederiksen, 1974) and indicate the direction of the visual axis (Lythgoe, 1979; Pankhurst, 1989). In Z. ophiocephalus the fronto-ventral position of the aphakic aperture indicates a visual axis directed in the ventro-frontal region of the visual field, confirmed also by the dorso-temporal position of its area centralis in the retina (see above). These observations of the visual morphology are confirmed by stomach content studies of Z. ophiocephalus (Pagotto & Campesan, 1980), where small fish have been found as well as benthic preys like crustaceans, molluscs and worms, indicating its ability to locate prey from the water column overhead and prey on the bottom. In the tank, grass gobies ambush food while raised on their pectoral fin. In this position, the visual axis, the direction of the highest visual acuity, and the fronto-

14 VISION IN GRASS GOBY FISH 137 ventral binocular field used during prey capture, are moved upward. In this raised position, the overhead vision is improved, which could be an adaptation to our experimental setup, where the food was coming from above, as well as an adaptation to the seagrass meadow, where vision ahead parallel to the bottom is limited by leaves of the sea grass and therefore a vision directed slightly upward is more appropriate. The observations on the extent of the visual field in Z. ophiocephalus indicate its ability to detect food in different directions, that are related to different regions of the retina. This ability of detection in different portions of its visual field is supported by the occurrence of the square mosaic of cones found in all retinal regions. Although the functional significance of the square mosaic is still unclear, among its hypothesized functions such as optimization of spatial-frequency sensitivity, more uniform spectral sampling or detection of polarized light, also a motion detection was proposed (reviews in Wagner, 1990; Beaudet et al, 1997). Visual acuity Variations in histological visual acuities in fish reflect their various feeding strategies (Tamura, 1957; Williamson & Keast, 1988; Collin & Pettigrew, 1989; Pankhurst, 1989; Zaunreiter & Goldschmid, 1989; Zaunreiter et al., 1991). The calculated histological visual acuity of Z. ophiocephalus is better than that calculated for nocturnal, herbivorous species or species that graze encrusting organisms, but worse than the one calculated for fast swimming piscivorous species. Its histological visual acuity is in good agreement with that calculated for the blenny Dasson variabilis (Collin & Pettigrew, 1989), that like Z. ophiocephalus, inhabits seagrass meadows and feeds on slow benthic invertebrates for which a high visual acuity is not necessary. In addition, the leaves of the sea grass limit visibility on longer distances, and therefore a very good visual acuity is not required in these species. In Z. ophiocephalus, the calculated histological visual acuity was better than the behavioural one, as reported in other fish species (review in Douglas & Hawryshyn, 1990; Browman et al, 1990; van der Meer, 1995; Wanzenbock et al., 1996). Estimates of visual acuity based upon inter-cone spacing could be higher than those observed behaviourally because counts of cell density alone do not take signal convergence and processing into account (Browman et al, 1990). This occurs in the retina of lower vertebrates before the ganglion cells are reached, and it is quite conceivable that the relevant information has been extracted before ganglion-cell level (in Douglas & Hawryshyn, 1990). For example, in goldfish, electrophysiological estimates of the minimum resolvable angle in its retinal ganglion cells (Schwassman, 1975), that would take convergence into account, are much closer to behavioural estimates of visual acuity in this species (Northmore & Dvorak, 1979) than histological calculations (Stell & Harosi, 1976). According to Douglas & Hawryshyn (1990) "the acuity determined by behavioural means is almost certainly a reflection of the fish's true visual abilities, since the results approximate the limits imposed by the physiology and anatomy of the visual system". In Z. ophiocephalus, a large difference has been found between histological and behavioural visual acuity. 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