Marine Biology (2002) 141: 549–559 DOI 10.1007/s00227-002-0840-7
A. Bozzano Æ I.A. Catala´n
Ontogenetic changes in the retinal topography of the European hake, Merluccius merluccius : implications for feeding and depth distribution
Received: 1 December 2001 / Accepted: 10 March 2002 / Published online: 8 May 2002 Springer-Verlag 2002
Abstract Changes in the distribution pattern of cells in the ganglion cell layer were studied in the retina of the European hake, Merluccius merluccius (L.), to identify the possible adaptations of visual capabilities to different bathymetric distributions and feeding habits. From early juveniles to adults, the eye diameter increased eightfold; thus, retinal surface increased dramatically with size also. In early juveniles the retinal topography of the cells in the ganglion cell layer showed a concentric arrangement with respect to the centre of the retina. Two specialised areas were found, located at the ventral and dorso-rostral periphery, where the cell density reached 47,900 cells mm–2, which corresponds to a theoretical visual acuity of 21¢ (minutes of arc). The visual axes were located upwards and downwards at around 80 from the geometric centre of the retina. In juveniles, the retina underwent important changes as the concentric topographic pattern transformed: the ventral specialised area progressively disappeared, the dorso-rostral area relocated to a rostral position and a new specialised area formed in the temporal retinal region. The visual axes were directed forward and backward. For fish with a total length of 12 cm or more, a horizontal visual streak formed along the rostro-temporal axis of the retina and a new specialised area was formed in the temporo-central region of the visual streak. In adults, acute vision could be identified with the two specialised areas at the temporal and rostral periphery, where the ganglion cell density peak decreased to 3,200–3,600 cells mm–2 and the resolving power increased to 10¢. As visual acuity is partially dependent on the cell types in the ganglion cell
Communicated by R. Cattaneo-Vietti, Genova A. Bozzano (&) Æ I.A. Catala´n Institut de Cie`ncies del Mar (CMIMA-CSIC), Passeig Marı´ tim de la Barceloneta 37–49, 08003 Barcelona, Spain E-mail:
[email protected] Tel.: +34-93-2309500 Fax: +34-93-2309555
layer, cell populations in this layer were distinguished into either ganglion cells or displaced amacrine cells, using morphometric and histological criteria. The proportion of displaced amacrine cells was fairly uniform throughout the retinal surface, always representing between 32% and 39% of all cells in early and advanced juveniles. Only in adults did their density increase to 50%, probably as an adaptation to low light levels, which fish encounter as their distribution increases in depth. A small population of giant ganglion cells was also present in the retina. In the young and adult retinae, they represented 1.2% and 2.7% of the total cell population, respectively. Therefore, it has been shown how in M. merluccius the retinal topography undergoes important changes in relation to varying environmental demands.
Introduction Actively swimming predators depend heavily on sensory organs for successful feeding behaviour. The European hake, Merluccius merluccius, is an active predator that occupies a high level in the marine trophic web, playing an important role in the energy transference between species (Sartor 1993; Guichet 1995; Bozzano et al. 1997; Velasco and Olaso 1998). The bathymetric distribution of M. merluccius ranges from 50 to 750 m in depth (Campillo et al. 1991; Recasens et al. 1998). Early juveniles and juveniles are found mainly between 100 and 200 m, while adults have a wider bathymetric distribution, and move up the trophic web throughout life. Small individuals feed primarily on small crustaceans living on the bottom, while adults are mostly piscivorous, looking for food both near the bottom and in the water column (Froglia 1973; Papaconstantinou and Caragitsou 1987; Du Buit 1996; Bozzano et al. 1997). Therefore, during development, M. merluccius experiences a variety of light levels and its visual system must be adaptable to the different requirements imposed by
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habitat, type of prey and/or vulnerability to predators. It has been found in another gadiform, the cod Gadus morhua, that the primary sense involved in midwater feeding is vision, even under low light conditions (Brawn 1969; Anthony 1981). Few studies have dealt with ontogenetic changes in visual parameters in M. merluccius, and even fewer in relation to prey detection. Only MasRiera (1991) pointed out that changes in hake feeding behaviour during growth might depend primarily on vision. In addition, Orsi-Relini et al. (1997) provided information about possible light-dependent behaviour in young hake. On the other hand, Brawn (1969) and Anthony (1981) hypothesised that light intensity could be correlated with successful feeding in some gadiforms. In fact, in order to catch a prey, the predator needs to know both the shape and size of the object and its location. This information is provided by the visual acuity, the visual axis direction and the field of best vision (Heffner and Heffner 1992; Gutherie and Muntz 1993; Murayama and Somiya 1998). A good estimate of theoretical visual acuity is given by the analysis of retinal ganglion cell density and distribution (Collin and Pettigrew 1988c). This procedure allows for the examination of the retina as a whole, and for the location of areas of highly discriminating power (Collin and Pettigrew 1989), from which the visual axis direction and the field of best vision can be determined. It is generally accepted that retinal topography is a highly species-specific characteristic and that density gradients show little variation within species (Hughes 1977). However, Shand et al. (2000) found a correlation between the preferred mode of feeding and the position of the area of best vision on the retina in a small sparid fish, Acanthopargus butcheri. As hake show pronounced changes in trophic spectrum and depth distribution throughout life, the aim of the present work was to analyse the changes in visual capability throughout ontogeny, interpreting them in the framework of functional adaptation.
Materials and methods
primarily at the retinal edge and along the radial cuts (Hughes 1975; Collin 1988; Mednich and Springer 1988; Heffner and Heffner 1992). Cell counts were not taken when shrinkage was noticeable. Retinal topography Cell densities within the ganglion cell layer, stained with cresyl violet, were determined and counted for each retina, employing a technique described by Collin and Pettigrew (1989). Using a slide projector and a graduated slide, the retinal amplification was drawn on a paper grid. After matching the retina drawing to the vernier scale of a microscope, a graticule of 100 squares was placed into the eyepiece and was used to choose the areas for cell counting at overall magnifications of ·400, ·600 and ·1,000. Cell number within each microscope field was counted every 0.05 mm across the retina. In high-density areas, counting frequency was reduced to 0.02 mm. These numbers were then converted to cells per square millimetre. Between 150 and 800 areas per retina (depending on retinal size) were sampled, thus allowing for detection of small fluctuations in cell densities. Elongated cells that contained densely stained nuclei, presumed to be astroglia, were excluded from the counts. A population of displaced amacrine cells, known to exist within the ganglion cell layer of fish (Collin and Pettigrew 1988c; Collin and Partridge 1997) and elasmobranchs (Collin 1988; Bozzano and Collin 2000), as well as in reptiles, birds and mammals (Hinds and Hinds 1978; Ehrlich and Morgan 1980; Hayes and Holden 1980; Perry et al. 1983; Wong and Hughes 1987) was included in the count. Therefore, the density values presented herein might be overestimated and should be revised by retrograde labelling analysis (Collin and Pettigrew 1988c). Isodensity contour maps were constructed following the technique of Collin and Pettigrew (1988a,b). The retinal area and the total number of cells within the ganglion cell layer over the entire retina were calculated by means of an image analyser (OPTIMAS 6.0), after scanning the retina drawings. Theoretical visual acuity Theoretical visual acuity was obtained through transformation of the maximum ganglion cell density (GCD) into its discriminating power, expressed as the minimum separable angle (MSA), according to Collin and Pettigrew (1989). The MSA (in minutes of arc) was calculated as: MSA=60·(2tana·G–1), where a is the angle subtending 1 mm on the retina, which is, in turn, given by tana=1 mm/f. The focal length (f) is calculated by m·r where r is the lens radius and m is the distance from the centre of the lens to the retina, assumed to be 2.55 in fishes (Matthiensen’s ratio, Matthiessen 1880). G is the GCD in the rostral-specialised area.
Collection of material
Field of best vision and visual axis
Twelve individuals of Merluccius merluccius (L. 1758) from 4 to 38 cm total length (TL) were collected off Barcelona (NW Mediterranean Sea) in 1999–2000, during the scientific cruises Lluc¸et I– IV. Three large individuals (45–60 cm TL) were obtained from commercial bottom-trawl landings. The right eye of each individual was excised (fresh) and measured to the nearest 0.5 mm. The lens was then removed and measured to the nearest 0.05 mm. Each eye was fixed in 4% paraformaldehyde in 0.1 M phosphate buffer for 1 h, transferred to 0.1 M phosphate buffer and left overnight. The retina was then extricated from the optic capsule, and the pigment epithelium removed. The position of the falciform process allowed deduction of the orientation of the retina throughout the process. Peripheral slits were made, and the retinae were wholemounted using the technique developed by Stone (1981). Each retina was stained for 3–7 min in 0.5% cresyl violet, dehydrated, cleared and coverslipped with DPX, and the ganglion cell layer was examined for cells containing Nissl substance. With this method, retinal shrinkage is thought to be between 2% and 10%, and occurs
The field of best vision was calculated according to the method of Heffner and Heffner (1992). Based on the distribution of the cells in the ganglion cell layer, the field of best vision was determined as the area containing cell densities of at least 75% of the maximum. The width of this field was measured in millimetres on the isodensity map according to Murayama et al. (1995). Using the angle a subtending 1 mm on the retina, the linear measurement was converted into spherical co-ordinates according to Mass and Supin (1986). The visual axes, corresponding to the fixation point on the retina, were determined by measuring the location of the specialised areas on the map, and converting these values into degrees in the same way as for the field of best vision (Murayama et al. 1995). Retinal cell size variation during fish growth Classification of cell types in the ganglion cell layer (GCL) was based on morphometric and histological criteria. For each cell-
551 counting considered for the retinal topography, small, roundshaped, darkly stained cells were identified as displaced amacrine cells (DAC), as confirmed by Collin and Pettigrew (1989) through the retrograde labelling method in reef teleosts. Cells, usually larger than DACs, tending to be more irregular in shape and containing clumped rather than wispy Nissl substance, were identified as ganglion cells (GC). Percentages of DAC and GC in each counting were calculated and converted to cells per square millimetre. Cell soma size and distribution of both cell types were analysed within retinal regions along with growth. With this aim, the retinae from 5, 12, 25 and 45 cm TL individuals were chosen. Cell soma areas were randomly measured at the retinal periphery, centre and in the specialised areas. The variation of cell soma size between different retinal regions was analysed by comparing the frequency histograms of the cell soma size within and between the four retinae using a non-parametric, two-sample test (Kolmogorov–Smirnov). To determine if fish size had any effect on cell size, linear regression analysis was used (Sokal and Rohlf 1986). Table 1. Merluccius merluccius. Summary of the retinal specialisations. Peak of retinal cells (RC) in the ganglion cell layer refers to the highest value found in the specialised areas (SA). Visual acuity Fish Eye Lens Retinal Total length diameter diameter surface RC (N) (cm) (mm) (mm) (mm)2)
4
3
1
7.5
5
3.1
1.2
10.2
5.5
3.2
1.2
16.4
6.5
3.8
1.4
15.4
8
4.7
1.9
30.4
9.5
5.2
2.1
42.4
12
6.7
2.4
60.1
13
6.8
2.6
69.1
24
11.2
4.1
115.5
25
12.0
4.6
168.7
34.5
15.3
7.6
267.4
38
19.0
7.4
317.8
45
20.3
7.9
596.9
45.5
20.4
7.6
436.3
60
24.0
9.0
1040.9
165,538
Results Merluccius merluccius has lateral eyes. The optic nerve is situated in the centre of the retina. It is joined by a long falciform process that extends to the rostro-ventral border of the retina. A summary of the ontogenetic changes in eye and lens size, retinal ganglion cell density, visual acuity and visual axis direction is given in Table 1. Retinal topography From early juveniles to adults, the diameter of hake eyes increased eightfold, and retinal surface increased is expressed as the minimum separable angle (MSA), and a is the angle subtending 1 mm on the retina (D dorsal; R rostral; V ventral; T temporal; C central)
Position Peak Peak of the RC density density SA (cells mm–2) gradient
D–R V 200,040 D–R V 230,408 R V 242,942 R V T 377,990 R V T 414,765 R T V 488,341 R T V 412,256 R T C 525,286 R T C 561,597 R T C 842,585 R C T 459,874 R C T 1,033,872 R C T 1,166,140 R C T 1,689,390 R C T
47,933 47,603 45,123 42,314 27,685 27,685 30,578 29,256 28,925 22,975 22,809 20,330 21,983 21,074 21,002 22,789 22,491 17,726 15,206 13,636 10,909 13,373 13,356 10,484 12,066 9,917 8,181 13,305 12,314 12,148 4,184 3,753 3,492 4,584 3,938 3,846 5,259 4,394 3,952 3,636 3,553 3,223
2.3:1 2.3:1 1.8:1 1.8:1 1.8:1 1.8:1 1.9:1 1.9:1 1.9:1 2.2:1 2.2:1 2.1:1 2.2:1 2.2:1 1.8:1 1.8:1 2.2:1 2.2:1 3.0:1 3.0:1 2.0:1 4.0:1 3.3:1 2.6:1 2.1:1 2.0:1 1.8:1 2.6:1 2.0:1 2.0:1 2.6:1 2.3:1 2.0:1 3.0:1 2.3:1 2.3:1 2.3:1 2.8:1 2.8:1 2.4:1 2.4:1 2.0:1
Visual acuity a
Cycles per degree
Visual axis MSA (¢) position
Field of best vision ()
21 11 21 12 40 24 31 20 28 28 11 27 28 26 9 18 20 11 21 24 19 17 11 11 19 19 10 22 11 9 23 16 6 22 14 9 18 11 9 22 14 10
38.1
2.8
21.1
34.6
3.0
19.2
33.2
3.1
19.9
28.4
3.0
19.2
82.1 83.0 83.5 83.5 81.7 83.5 83.5
21.9
3.5
19.9
84.1 82.9
20
3.7
17.3
18.1
3.7
14.9
83.3 84.7
16.6
3.7
16.1
75.1 84.6
10.8
4.8
12.4
72.9 81.4
9.7
5.7
10.6
63.1 78.8
7.1
6.3
9.5
60.3
6.1
5.3
11.3
71.1 61.8
5.6
6.1
10.1
76.2 62.6
5.7
5.7
10.4
75.9 58.6
4.9
6.1
9.9
71.7 64.9
83.9 83.0 83.8
77.4
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allometrically with size (Fig. 1A). During growth, the retinae revealed an unstable topographic pattern of retinal ganglion cell distribution. In the retinae of early juveniles (4–5 cm TL), the ganglion cell distribution showed a concentric arrangement with respect to the centre of the retina. Also, two specialised areas, where the ganglion cell density reached highest values, were found at the ventral and dorso-rostral periphery (Fig. 2A). With growth, the concentric topography pattern transforms into two large low-density patches above
Fig. 2A–D. Merluccius merluccius. Isodensity contour maps showing the topography of the retinal ganglion cell distribution in individuals of 4 (A), 5 (B), 6.5 (C) and 9.5 cm TL (D), respectively. All densities are to be multiplied by 103 cells mm–2. All figures depict the right retina, except the C one that is left (R rostral; V ventral; T temporal; arrows retinal orientation; black area optic nerve head; asterisks falciform process position)
Fig. 1A–C. Merluccius merluccius. A Growth of the retinal surface during hake development, B changes in the density of retinal ganglion cells (RGC) in the rostral specialised area, and C variations in retinal resolving power expressed as the minimum separable angle (MSA) are shown. The power equations and the coefficients of correlation are: y=0.77x1.69, r2=0.98 (A); y=163,488x–0.89, r2=0.91 (B); y=34.3x–0.32, r2=0.94 (C)
and underneath the horizontal retinal plane (Fig. 2B– D). In the ventral specialised areas, cell density progressively declines, while a new specialised area appears at the temporal retinal periphery. At the same time, the dorso-rostral specialised area ‘‘relocates’’ to a more rostral position. Thus, during this transitional phase hake have triple retinal specialisations, at the temporal, ventral and rostral retinal periphery (Fig. 2B–D). At 12–13 cm TL (Fig. 3A, B), the retina shows a more pronounced visual streak, with two retinal specialisations in the temporal and rostral areas. The ventral specialised area disappears, and a new specialised area in the centre of the visual streak progressively arises. From this size on (Fig. 3B–F), the retina maintains this topographic pattern, with the horizontal streak across the central meridian of the eye, and the rostral, temporal and central specialised areas. In larger individuals (Fig. 3E, F), the visual streak becomes more dorsotemporally–rostro-ventrally orientated. To our knowledge, this is the first record of horizontal streak forming in a teleost. The density gradient between nonspecialised and specialised areas remained constant during hake growth, showing a mean value of 2.3 (Table 1).
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Fig. 4. Merluccius merluccius. Trend in the best visual field along with growth in the rostral, caudal, temporal and ventral retina
Fig. 3A–F. Merluccius merluccius. Isodensity contour maps showing the topography of the retinal ganglion cell distribution in individuals of 12 (A), 13 (B), 24 (C), 34.5 (D), 45 (E) and 60 cm TL (F). All densities have to be multiplied by 103 cells mm–2 (R rostral; V ventral; T temporal; arrows retinal orientation; black area optic nerve head; asterisks falciform process position)
Theoretical visual acuity Although cell density in the ganglion cell layer decreases during hake growth (Fig. 1B), the increase in eye size and lens diameter leads to an increase in visual acuity. Changes in theoretical visual acuity, presented as an MSA, are shown in Fig. 1C and Table 1. In early juvenile and juvenile individuals, resolving power increased steeply (from 21 to 10.6¢) due to a pronounced enlargement of the eye diameter, which almost tripled its value (Table 1). In advanced juveniles and adults (>24–25 cm TL), resolving power is maintained at around 10¢. Field of best vision and visual axis In early juveniles and juveniles, the width of the field of best vision quickly increases in the dorso-rostral and ventral areas to a maximum value of 40 and 24, respectively, then progressively decreases (Table 1; Fig. 4). The ventral visual field is present only in individuals
