Abstract Relatively little is known about the physical

structure and ecological adaptations of elasmobranch

sensory systems. In particular, elasmobranch vision has

been poorly studied compared to the other senses.

Virtually nothing is known about whether elasmo-

branchs possess multiple cone types, and therefore the

potential for colour vision, or how the spectral tuning

of their visual pigments is adapted to their different

lifestyles. In this study, we measured the spectral

absorption of the rod and cone visual pigments of the

blue-spotted maskray, Dasyatis kuhlii, using micro-

spectrophotometry. D. kuhlii possesses a rod visual

pigment with a wavelength of maximum absorbance

(kmax) at 497 nm and three spectrally distinct cone

types with kmax values at 476, 498 and 552 nm. Mea-

surements of the spectral transmittance of the ocular

media reveal that wavelengths below 380 nm do not

reach the retina, indicating that D. kuhlii is relatively

insensitive to ultraviolet radiation. Topographic anal-

ysis of retinal ganglion cell distribution reveals an area

of increased neuronal density in the dorsal retina.

Based on peak cell densities and using measurements

of lens focal length made using laser ray tracing and

sections of frozen eyes, the estimated spatial resolving

power of D. kuhlii is 4.10 cycles per degree.

Keywords Elasmobranch �Microspectrophotometry �Visual pigment � Ocular media � Ganglion cell

topography

AbbreviationsDW Disc width

IR Infra-red

LED Light emitting diode

MS222 Tricaine methanesulphonate salt

OLM Outer limiting membrane

T0.5 Wavelength at 0.5 normalized transmittance

UV Ultraviolet

kmax Wavelength of maximum absorbance

Introduction

Elasmobranchs (sharks, skates and rays) are an ancient

group of vertebrates that first appeared over 400 mil-

lion years ago. Traditionally, these fishes were thought

to have poorly developed visual systems and were be-

lieved to rely heavily on non-visual senses such as

olfaction and electroreception for prey detection,

predator avoidance, mating and communication

(Cohen 1989). Indeed, until the discovery of cones in

the retina of the lemon shark, Negaprion brevirostris

(Gruber et al. 1963), and despite some early reports of

cones in the elasmobranch retina (Neumayer 1897;

Schaper 1899; Walls 1942), it was commonly assumed

that the elasmobranch eye contained an all-rod retina

and was specialised only for dim light (scotopic) vision

(Walls 1942). While a few species examined with

modern microscopic techniques are reported to be

cone free, namely the skates Raja erinacea and Raja

S. M. Theiss (&) � T. J. Lisney � S. P. Collin �N. S. HartVision, Touch and Hearing Research Centre,School of Biomedical Sciences, University of Queensland,St Lucia, Brisbane, QLD 4072, Australiae-mail: s.theiss@uq.edu.au

J Comp Physiol A (2007) 193:67–79

DOI 10.1007/s00359-006-0171-0

123

ORIGINAL PAPER

Colour vision and visual ecology of the blue-spotted maskray,Dasyatis kuhlii Muller & Henle, 1814

Susan M. Theiss Æ Thomas J. Lisney ÆShaun P. Collin Æ Nathan S. Hart

Received: 30 June 2006 / Revised: 29 August 2006 / Accepted: 2 September 2006 / Published online: 26 September 2006� Springer-Verlag 2006

ocellata, the blackmouth catshark, Galeus melastomus,

and the Portuguese dogfish, Centroscymnus coelolepis

(Dowling and Ripps 1970; Bozzano et al. 2001; Bozzano

2004), the majority of over 40 species investigated to

date have duplex retinae with varying rod to cone

ratios (Hart et al. 2006). However, while the spectral

properties of elasmobranch rods have been studied

extensively (Denton and Shaw 1963; Crescitelli et al.

1985; Cohen et al. 1990), almost nothing is known of

the spectral absorption characteristics of the visual

pigments contained within the cones.

A single spectral class of photoreceptor can only

code for the brightness of an image and not its colour.

Thus, for an animal to have colour vision, it must

possess at least two different photoreceptor types with

spectrally distinct but overlapping spectral sensitivities

and the neural machinery to compare their outputs

(Jacobs 1981). Few studies have investigated the

physiological basis for, or behavioural manifestation

of, colour discrimination in elasmobranchs, and at

present it is unknown whether elasmobranchs have

colour vision. Past behavioural experiments on the

blacktip reef shark, Carcharhinus melanopterus, grey

reef shark, Carcharhinus menisorrah = amblyrhynchos,

and the lemon shark, N. brevirostris have proved

inconclusive (Tester and Kato 1966; Gruber 1975),

mostly because these tests did not control for the

effects of perceived stimulus intensity, making it impos-

sible to distinguish whether the animal was responding

to the hue of the stimulus colour or its effective

brightness. The most encouraging results on the

potential for colour vision in elasmobranchs have come

from a recent study by Hart et al. (2004), where three

spectrally distinct cone visual pigments were found

using microspectrophotometry in two species of shov-

elnose ray, the giant shovelnose ray, Rhinobatos typus,

and the eastern shovelnose ray, Aptychotrema rostrata.

This was the first study to demonstrate that some

species of elasmobranchs have multiple cone types and,

therefore, the potential for colour vision. Moreover,

the discovery of five cone photoreceptor types, each

containing a different visual pigment (opsin) gene, in a

jawless vertebrate, the southern hemisphere lamprey,

Geotria australis, (Collin et al. 2003a, b), suggests that

the retinal prerequisites for colour vision existed in

ancestral vertebrates, prior to the divergence of the

elasmobranchs. Consequently, one would anticipate

that multiple spectrally distinct cone pigments are

widespread in other cartilaginous fish.

The range of wavelengths to which an animal is

sensitive is dependent not only on the spectral sensi-

tivity of its visual pigments, but also upon the wave-

lengths of light that reach the retina. Therefore, any

consideration of colour vision must take into account

the transmission of the ocular media (Douglas and

McGuigan 1989). Other aspects of the visual system,

such as spatial resolving power, are equally important.

The peak density and topographic distribution of retinal

neurons can often be correlated with the visual ecology

of a species (Hughes 1977; Collin and Pettigrew 1988).

Topographic analyses can reveal the presence of areae

of increased photoreceptor and ganglion cell density

that mediate increased spatial resolving power in a

particular region of the visual field. Several elasmo-

branchs, for example, possess a horizontal visual ‘streak’

of increased ganglion cell density. This retinal organi-

sation is thought to be an adaptation to open, relatively

featureless environments dominated by a clear visual

horizon, such as the sand–water interface, or the hori-

zontal gradient of light in the water column (Hueter

1991; Bozzano and Collin 2000; Hueter et al. 2004).

The blue-spotted maskray, Dasyatis kuhlii, is a

tropical benthic marine ray usually found on sandy

bottoms near rocky or coral reefs (Last and Stevens

1994). This species is widely distributed throughout the

Indo-Pacific region (Last and Stevens 1994; Last and

Compagno 1999) and derives its name from the bright

blue spots found on its grey to brownish coloured disc.

The retina of Dasyatis kuhlii is already known to

possess cones (Kohbara et al. 1987), and it inhabits a

similar, brightly-lit habitat to both Rhinobatos typus

and Aptychotrema rostrata, the two species of shovel-

nose ray previously shown to have multiple cone types

(Hart et al. 2004). Although they share similar habitats,

D. kuhlii and the two shovelnose rays, R. typus and A.

rostrata, are not closely related and belong to different

elasmobranch orders (Myliobatiformes and Rajafor-

mes respectively) (McEachran and Aschliman 2004) D.

kuhlii also has a number of different morphological

and behavioural adaptations, suggesting that their

ecology may be quite different. For example, D. kuhlii

has an angular disc and feeds mainly on polychaete

worms (S. Pierce and S. Pardo, personal communica-

tion), while shovelnose rays have a distinct wedge-

shaped disc and feed on sand-dwelling crustaceans and

shellfish (Last and Stevens 1994). One significant dif-

ference between D. kuhlii and the vast majority of

other rays is the blue spots found on the disc, which

could be beneficial to this species for camouflage and/

or visual communication, although their function is

presently unknown. Here, we describe the visual sys-

tem of D. kuhlii in terms of the morphology and

spectral absorption properties of its retinal photore-

ceptors, the spectral transmittance of the ocular media,

the topographic distribution of ganglion cells in the

retina and the optics of the lens.

68 J Comp Physiol A (2007) 193:67–79

123

Materials and methods

Animal collection and handling

All procedures were approved by the University of

Queensland Animal Ethics Committee and conducted

under the ethical guidelines of the National Health and

Medical Research Council of Australia. A total of 27

specimens of Dasyatis kuhlii were collected using seine

nets from shallow coastal waters around Moreton Bay,

Queensland under a Queensland General Fisheries

Permit (PRM02910F). Animals ranged in size from

19.0 to 39.4 cm disc width (DW) and were housed in

circulating seawater aquaria under fluorescent strip

lighting on a 12 h:12 h light:dark cycle.

Light and electron microscopy

One animal (23.5 cm DW) was euthanased with an

overdose of tricaine methanesulphonate salt (MS222;

1:2,000 in seawater) for light microscopical examina-

tion of the retina and ultrastructural examination of

rod and cone photoreceptors following the methods of

Collin et al. (1999) and Hart et al. (2004). For both

light and electron microscopy, hemisected eyes were

fixed in 2% paraformaldehyde, 2.5% glutaraldehyde in

0.1 M cacodylate buffer (pH 7.4) at 4�C for 2 days and

post-fixed in 2% osmium tetroxide. For light micros-

copy, retinal tissue was dehydrated and embedded in

EPON before semi-thin sections (1 lm) were cut on an

LKB ultramicrotome, mounted on slides and stained

with toluidine blue. Following permanent mounting

and coverslipping in DEPEX (Aldrich Chemical

Company Inc., USA), stained retinal sections were

photographed using an Olympus DP30 digital camera

mounted on a Leitz Dialux 20 compound microscope.

For electron microscopy, sections were also cut on an

LKB rotary ultramicrotome using glass knives. Sec-

tions were placed on grids and stained with lead citrate

and uranyl acetate, and examined on either a Phillips

410 or a Phillips CM10 transmission electron micro-

scope set at 80 kV (Phillips Inc., Eindhoven, The

Netherlands). Measurements of individual (unfixed)

photoreceptors were undertaken during microspectro-

photometric analysis and taken from the television

screen used to view the preparations, using a piece of

transparent acetate marked with a scale to the cali-

brated magnification of the microscope.

Microspectrophotometry

Sixteen animals (20.5–35.5 cm DW) were dark-adapted

for 1.5–2.5 h to allow for visual pigment regeneration

and euthanased with an overdose of MS222 (1:2,000 in

seawater) followed by spinal pithing. Following the

methods of Hart (2004) and Hart et al. (2004), eye

removal and retinal dissection was performed under

the illumination provided by a bank of 24 infra-red

(IR) light emitting diodes (LED) and visualized using

an IR image converter (FJW Industries, USA) attached

to one ocular of a stereo dissection microscope. After

enucleation, one eye was immersed in elasmobranch

Ringer solution (330 mmol l–1 urea, 350 mmol l–1 NaCl,

4 mmol l–1 KCl, 5 mmol l–1 CaCl2, 2 mmol l–1 MgCl2;

approximate osmolality 1,050 mOsm kg–1; pH 7.4),

while the second eye was sealed in a light-tight container

and stored at 4�C for use the following day. The

immersed eye was then hemisected and the lens

removed. To facilitate microdissection of the retina, a

quarter of the eyecup was cut away and the pigment

epithelium, tapetum, and retina were gently pulled away

from the sclera. The vitreous humour was then cut away

from the retina as much as possible. Using fine forceps,

the retina was carefully teased away from the pigment

epithelium and small pieces of retinal tissue (approxi-

mately 1–3 mm2) were dissected free and transferred to

a drop of elasmobranch Ringer solution containing 5%

dextran (MW 200,000–300,000; ICN Biomedicals Inc.,

USA) on a 24 · 64 mm No. 1 glass coverslip. The retina

was teased apart using fine forceps, covered with an

18 · 18 mm No. 0 coverslip and then gently compressed

beneath filter paper to soak up any excess liquid around

the edges of the coverslip. The edges of the top coverslip

were then sealed with nail varnish to prevent dehydra-

tion of the preparation and slides were stored in a light-

tight box at 4�C until use. The following day the same

procedure was carried out for the second eye.

Transverse absorbance spectra (330–800 nm) of

visual pigments within both rod and cone outer

segments were made using a computer-controlled,

single-beam, wavelength-scanning microspectropho-

tometer described in detail elsewhere (Hart 2004). A

sample scan was made by aligning the measuring

beam within the outer segment and recording the

amount of light transmitted at each wavelength.

Measuring beam dimensions varied from 1 · 1 lm

for small cone outer segments to 2 · 12 lm for rod

outer segments. A baseline scan was then made in an

identical way from a cell-free area of the preparation

as close to the cell as possible. The baseline trans-

mittance was subtracted from the sample scan at each

corresponding wavelength to give a ‘pre-bleach’ spec-

trum for that cell. Subsequently, the cell was bleached

with full spectrum ‘white’ light from the monochroma-

tor for three minutes and identical sample and baseline

scans were made to give a ‘post-bleach’ spectrum. The

J Comp Physiol A (2007) 193:67–79 69

123

post-bleach spectrum was deducted from the pre-bleach

spectrum to create a difference spectrum that was

analysed along with the pre-bleach spectra as described

elsewhere (MacNichol 1986; Govardovskii et al. 2000;

Hart 2002). Briefly, sample and baseline data were

converted to absorbance values at 1 nm intervals and

the upward and downward scans averaged together by

fitting a weighted three-point running average to the

absorbance data (Hart 2002; Shand et al. 2002). Pre-

and post-bleach absorbance spectra were normalized to

the peak and long-wavelength offset absorbances

determined by fitting a variable-point unweighted

running average to the data (Hart 2002, 2004; Shand

et al. 2002,). A regression line was fitted to the long-

wavelength limb of the normalized absorbance data

between 30 and 70% of the normalized maximum

absorbance (MacNichol 1986), and the regression

equation was used to estimate the wavelength of max-

imum absorbance (kmax) with a corresponding template

overlayed for display (Govardovskii et al. 2000).

Ocular media spectral transmittance

Spectral transmittance measurements (300–800 nm) of

the ocular media were made using a calibrated, com-

puter-controlled Ocean Optics S2000 spectroradiome-

ter (Ocean Optics, Florida, USA), with a pulsed xenon

light source (PX-2 Ocean Optics), following methods

similar to those used by Siebeck and Marshall (2000)

and Hart (2004). One individual (25.1 cm DW) was

euthanased with an overdose of MS222 (1:2,000 in

seawater) and both eyes quickly removed. A small

piece of the sclera (approximately 2–3 mm2) was cut

out of the back of the eye, in line with the optic axis,

and the underlying choroid, pigment epithelium and

retina were removed to create an aperture for the

incident measuring beam to leave the eye. Each eye

was then placed cornea-side down on a metal specimen

holder, with a 50 lm diameter fibre optic cable posi-

tioned near the cornea in order to pass light through

the entire ocular media (cornea, aqueous humour, lens,

and vitreous humour). A 1,000 lm diameter fibre optic

cable was positioned above the eye, in line with the

hole in the sclera, to collect light transmitted through

the ocular media. The integration time was set at 20 ms

with 20 scans averaged for each measurement. Four

measurements were taken from each eye with the

baseline measurement consisting of an identical

arrangement but with the eye removed from the light

path. Transmittance data for both eyes were averaged,

linearly interpolated to 1 nm intervals from 300 to

800 nm, and fitted with a 31-point unweighted running

average. The curve was normalized and the wavelength

at which there was 0.5 of the normalized transmittance

(T0.5) was determined (Douglas and McGuigan 1989).

Results were largely qualitative as absolute measure-

ments of reflectance and transmittance are not possible

using this technique.

Ganglion cell topography

Three animals (21.2, 22.8 and 23.7 cm DW) were eu-

thanased with an overdose of MS222 (1:2,000 in sea-

water) prior to enucleation. Following the methods of

Stone (1981), a ventral slit was made in the eye in order

to establish orientation of the retina. Eyes were

hemisected and fixed in 4% paraformaldehyde

in 0.1 M phosphate buffer solution (pH 7.4) at 4�C for

2–7 days and then stored in 0.1 M phosphate buffer

(pH 7.4) at 4�C. The retina was dissected away from

the sclera and separated from the tapetum and

underlying pigment epithelium, with care taken to

maintain the orientation of the retina. Once the vitre-

ous humour was carefully cut away from the retina,

peripheral slits were made in order to flatten the retina

onto a gelatinised glass slide. The retinae were fixed to

the slide using 16% paraformaldehyde, rinsed in dis-

tilled water and air dried. The five retinae examined

were first defatted in xylene, and then rehydrated

through a descending alcohol series followed by a rinse

in distilled water. The retinae were then stained for

Nissl substance using a 0.05% cresyl violet solution

(pH 4.3) for 5–9 min. Once staining was complete, the

retinae were rinsed in distilled water, dehydrated

through an ascending alcohol series and cleared in

xylene. Stained slides were then coverslipped in

DEPEX (Aldrich Chemical Company Inc., USA).

Retinae were placed on a visualiser (ELMO Visual

Presenter EV-550) and the enlarged outline was traced

onto transparent graph paper, with care taken to match

the calibrations of the graph paper to the projected

image of a calibrated micrometer slide. Retinae were

then examined using a compound microscope (Leitz

Dialux 20), fitted with an eyepiece graticule, at a mag-

nification of either ·630 or ·1,000. A scale was deter-

mined by matching obvious landmarks on the retina

with the relative position of the microscope stage. Ret-

inal cells located within the ganglion cell layer were

visualized using a 10 · 10 square eyepiece graticule and

sampled every 1 mm across the retina. In elasmo-

branchs, ganglion cells, in addition to amacrine and glial

cells, lie within both the inner plexiform and ganglion

cell layers (Walls 1942; Anctil and Ali 1974; Gruber and

Cohen 1978; Peterson and Rowe 1980; Collin 1988). All

neural elements in these two layers were counted and

no attempt was made to distinguish ganglion cells from

70 J Comp Physiol A (2007) 193:67–79

123

amacrine cells. Glial cells were easily identified by their

dense staining and elongated shape (Hughes 1977) and

were excluded from cell counts. Retinal shrinkage is

known to vary between 2 and 10% using the whole-

mount method (Hart 2002) but was not taken into ac-

count here. However, since the artefactually high cell

packing associated with retinal shrinkage occurs pre-

dominantly at the periphery and along the radial cuts

placed into the retina prior to wholemounting (Mednick

and Springer 1988), these areas were avoided in the

assessment of cell density. The number of cells counted

was converted to cells mm–2 by dividing by the sampled

area (15.1 · 10–3 mm2 at ·630 magnification and

7.2 · 10–3 mm2 at ·1,000 magnification). Iso-density

contour maps were then constructed by joining areas of

similar cell density and creating smooth curves.

Lens focal length–ray tracing and cryosectioning

To establish the focal length of the lens, we used laser

ray tracing and direct measurements from transverse

sections of frozen eyes. Four eyes from three individ-

uals (21.1, 25.1, and 30.2 DW) were used for laser ray

tracing. Following euthanasia with an overdose of

MS222 (1:2,000 in seawater), the eyes were quickly

removed. A minute orientation mark was made in the

lens to ensure that ray tracing measurements were

made along the optic axis. The lens was then excised

and stabilised on a small washer situated on top of a

glass block within a container filled with elasmobranch

Ringer solution. A moveable, collimated red laser

diode (k = 655 nm) was mounted on a rack and pinion

assembly at one end of the container, and the lens

positioned such that the laser beam would pass through

the horizontal meridian. A digital camera (Optio 555,

Pentax, Japan) was mounted directly above the lens.

For each eye, five photographs were taken with the

laser beam passing through the lens at five different

points; one directly through the centre of the lens, one

on each edge of the lens, and one between the edge

and the centre of the lens on either side. To improve

visualisation of the laser beam, two drops of milk were

added to the Ringer solution. The five digital images

were then downloaded to a PC and overlayed using

Photoshop 7.0 (Adobe) to determine where the beams

converged. The distance from the lens centre to the

point of convergence was considered to be the focal

length. Matthiessen’s ratio was calculated by dividing

this distance by the lens radius (Matthiessen 1880).

For cryosectioning, the right eye from a single indi-

vidual (24.0 cm DW) was frozen and sectioned to obtain

intraocular dimensions in the axial plane. The eye was

removed and frozen in a mixture of dry ice and ethanol

(Sivak 1978) and stored at –80�C. Having first noted the

orientation, the eye was then embedded in Tissue-Tek

OCT compound (Sakura Finetechnical Co., Ltd, Tokyo,

Japan) containing a few drops of black Indian ink to

distinguish the outline of the cornea. The resultant block

was sectioned at –25�C on a cryostat (American Optical

Corporation, New York, USA). Scaled photographs of

the block face were taken every 80 lm. The photograph

showing the greatest lens thickness was identified and

taken to represent a section through the geometric axis

of the eye (Sivak 1976, 1978) and used for optical

measurements. Measurements of lens to retina distances

were made from the lens centre to the choroid–retina

border (Sivak 1976; Hueter 1980) at a range of angles

(from +45� to –60�) on either side of the geometric axis,

which is the line joining the centre of the lens to the

centre of the pupil (Sivak 1976; Collin et al. 1999).

Calculating spatial resolving power assuming

a hexagonal mosaic

Focal lengths (f) of lenses from eyes used to measure

ganglion cell topography were calculated using the

averaged values measured using laser ray tracing.

Estimates of the theoretical peak spatial resolving

power were calculated using the maximum ganglion

cell density found in the retina and the focal length.

Following the methods of Hart (2002) and assuming a

hexagonal retinal mosaic, the distance (d) subtended

by one degree on the retina was calculated where:

d ¼ 2pf

360: ð1Þ

Assuming that ganglion cells are the limiting factor

for spatial resolving power and that they are packed in

a hexagonal array, the cell-to-cell spacing (S) was

determined using the formula:

S2 ¼ 2

Dffiffiffi

3p

� � ; ð2Þ

where D is the peak density of ganglion cells mm–2.

The maximum spatial (Nyquist) frequency (v) of a

grating resolvable by this arrangement (Snyder and

Miller 1977) was calculated as:

v ¼ 1

Sffiffiffi

3p

� � : ð3Þ

To express spatial resolving power in cycles per

degree, the value of v was multiplied by d.

J Comp Physiol A (2007) 193:67–79 71

123

Results

The eyes of Dasyatis kuhlii are located on the dorsal

surface of the head and face laterally, as seen in other

batoid elasmobranchs (Fig. 1a). Each eye has a near-

spherical lens and possesses a highly reflective blue–

green tapetum, extending across the retinal meridian,

but which is absent from the dorsal and ventral retinal

margins. The cornea is clear and continuous with a

hard, fibrous sclera. The cornea overlies a golden-

brown iris, which forms an oval-shaped pupil in the

dark-adapted state and a crescent-shaped pupil in the

light-adapted state (Fig. 1b).

Photoreceptor morphology

The retina of D. kuhlii possesses both rod and cone

photoreceptors (Fig. 1c, d). Photoreceptor morphology

and characterisation is based on light and electron

microscopy as well as observations and measurements

of unfixed receptors during microspectrophotometry

(Table 1). Rods are characterized by their long, cylin-

drical outer segments, varying in diameter from 2 to

3.5 lm and from 13 to 34 lm in length (Fig. 2a). Cones

are more difficult to locate within the retinal prepara-

tion, but are easily distinguishable from the rods by

their shorter, conical outer segments (Fig. 2b–d). Cone

outer segments were typically 2–10 lm in length, and

tapered from 2 to 5 lm at the base closest to the inner

segment to 1–2 lm at the tip. Three different cone

types can be distinguished based solely on the diameter

of the inner segment and the location of the nucleus

(vitread to the outer limiting membrane (OLM),

sclerad to the OLM or straddling the OLM) (Fig. 2).

Microspectrophotometry

Microspectrophotometric data for all photoreceptors

are summarized in Table 2 and Fig. 3. All visual pig-

ment absorbance spectra are considered to represent

vitamin A1-based visual pigments, or rhodopsins, based

on the fit of the data to A1 visual pigment templates

(Govardovskii et al. 2000). The retina of D. kuhlii

contains a single type of rod, the outer segment of

Fig. 1 Dorsal (a) and lateral (b) views of the blue-spottedmaskray, Dasyatis kuhlii, showing the elevated eyes andcrescent-shaped pupil in the light-adapted state. c Lightmicrograph of the retina in transverse section showing the thicklayer of photoreceptors (p). Note the myelinated axons of theganglion cells situated in the ganglion cell layer (gcl) and thelarge horizontal cells tiered into at least three sublaminae. d Low

power electron micrograph showing the slender outer segmentsof the rod photoreceptors (rp) abutting the retinal pigmentepithelium (rpe) and the layer of cone photoreceptors (cp)adjacent to the outer nuclear layer (onl). Four cones are depictedby asterisks. ct choroidal tapetum, inl inner nuclear layer, iplinner plexiform layer. Scale bars 50 mm in a; 30 mm in b; 25 lmin c; 20 lm in d

72 J Comp Physiol A (2007) 193:67–79

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Table 1 Morphological characteristics of photoreceptor outer segments from the retina of Dasyatis kuhlii measured duringmicrospectrophotometry

Rod SWS cone MWS cone LWS cone

Mean outer segment base width (lm) 2.4 ± 0.4 2.6 ± 0.7 2.5 ± 0.7 2.6 ± 0.6Mean outer segment length (lm) 24.4 ± 4.0 5.9 ± 1.7 5.3 ± 2.1 4.6 ± 1.3Mean outer segment tip width (lm) 2.4 ± 0.4 1.2 ± 0.4 1.3 ± 0.4 1.2 ± 0.3Number of cells used in morphology analysis 63 22 12 29

SWS short-wavelength sensitive, MWS medium-wavelength sensitive, LWS long-wavelength sensitive. Mean values are given withstandard deviations

Fig. 2 Morphologicaldifferentiation ofphotoreceptors in Dasyatiskuhlii. The rod nuclei (rn) areosmiophillic and stain moredarkly than cone nuclei (cn).Three types of cones could bedifferentiated on the size ofthe inner segment and therelative position of thenucleus with respect to theouter limiting membrane(olm, located at the level ofthe dashed lines). The nucleusmay lie vitread to the olm(a, d), sclerad of the olm(b), and/or bisect the olm(c, d). m mitochondria of coneinner segments, r rod myoids.Scale bars 5 lm in a–d

Table 2 Spectral characteristics of rod and cone visual pigments in the retina of Dasyatis kuhlii measured usingmicrospectrophotometry

Rod SWS cone MWS cone LWS cone

Mean kmax of pre-bleach absorbance spectra (nm) 496.7 ± 1.7 475.9 ± 4.2 498.4 ± 3.6 552.1 ± 8.4kmax of mean pre-bleach absorbance spectrum (nm) 496.7 476.7 500.1 552.6Absorbance at mean kmax of pre-bleach spectra 0.032 ± 0.006 0.017 ± 0.005 0.015 ± 0.007 0.013 ± 0.003Absorbance at kmax of mean pre-bleach spectrum 0.032 0.017 0.015 0.012Number of cells used in pre-bleach analysis 37 13 8 11Mean kmax of difference spectra (nm) 499.6 ± 2.4 475.0 ± 6.2 500.2 ± 3.1 551.1 ± 9.3kmax of mean difference spectrum (nm) 500.0 476.0 504.6 552.1Absorbance at mean kmax of difference spectra 0.024 ± 0.006 0.017 ± 0.005 0.013 ± 0.006 0.013 ± 0.003Absorbance at kmax of mean difference spectrum 0.023 0.016 0.013 0.012Number of cells used in difference analysis 37 13 8 11

SWS short-wavelength sensitive, MWS medium-wavelength sensitive, LWS long-wavelength sensitive, kmax wavelength of maximumabsorbance. Mean values are given with standard deviations

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123

which contains a medium-wavelength (‘green’) sensi-

tive visual pigment with a mean wavelength of maxi-

mum absorbance (kmax) of the pre-bleach spectra at

496.7 ± 1.7 nm (n = 37). Cone outer segments contain

visual pigments that could be characterised into three

spectrally different types: a short-wavelength (‘blue’)

sensitive pigment with a mean pre-bleach kmax of

475.9 ± 4.2 nm (n = 13), a medium-wavelength

(‘green’) sensitive pigment with a mean pre-bleach

kmax of 498.4 ± 3.6 nm (n = 8), and a long-wavelength

(‘red’) sensitive pigment with a mean pre-bleach kmax

of 552.1 ± 8.4 nm (n = 11). Green cones were encoun-

tered much less frequently than the other cone types.

Only records from short, distinctly tapered outer

segments were included for analysis to avoid the possi-

bility of measuring a rod outer segment by mistake, as

the kmax values are almost identical.

Ocular media spectral transmittance

Ocular media spectral transmittance measurements

were averaged and normalized for the two eyes and are

Fig. 3 a Normalized meanphotoreceptor visual pigmentabsorbance spectra fromDasyatis kuhlii. The pre-bleach spectra (circles) areoverlayed with rhodopsinvisual pigment templates ofthe appropriate kmax

(Govardovskii et al. 2000);the post-bleach spectra(squares) are fitted with avariable point unweightedrunning average. bNormalized meanphotoreceptor visual pigmentbleaching difference spectraoverlayed with rhodopsinvisual pigment templates ofthe appropriate kmax

(Govardovskii et al. 2000).SWS, MWS and LWS refer toshort-, medium- and long-wavelength sensitive visualpigments, respectively

74 J Comp Physiol A (2007) 193:67–79

123

shown in Fig. 4. Ocular media measurements were ta-

ken from whole eyes and not for individual compo-

nents such as the cornea or lens. The T0.5 value of the

mean spectrum is 412 nm, and no light is transmitted

through the ocular media below approximately

380 nm.

Ganglion cell topography

The retina of D. kuhlii shows an elongated horizontal

streak of increased cell density extending across the

retinal meridian, dorsal to the optic nerve head

(Fig. 5). Cell densities within the horizontal streak

range from 2,000 to 4,000 cells mm–2. The retina of D.

kuhlii also possesses multiple areae of increased cell

density within the horizontal streak located both na-

sally and temporally, although the temporal area

(subtending frontal visual space) is larger and better

developed with an average peak of 4,251 ± 720 cells

mm–2 (n = 5). There is also an increase in cell density

around the centro-ventral located optic nerve head

(2,000–3,000 cells mm–2).

Lens focal length and spatial resolving power

Ray tracing was performed on four lenses of both right

and left eyes of D. kuhlii (Fig. 6a). Measurements of

focal length and Matthiessen’s ratio ranged from 5.63

to 10.41 mm and 2.17–2.67, respectively, with an

average Matthiessen’s ratio of 2.49 ± 0.20 (n = 4). The

lens is close to spherical with a dorso-ventral diameter

that is slightly longer than the axial diameter (ratio of

dorso-ventral diameter to axial diameter = 1:0.98).

From measurements made on the frozen, hemisected

eye, the distance from the centre of the lens to the

retina for a range of incident angles (from +30� to –60�about the visual axis, 0�) is essentially the same, with

an on-axis focal length of 8.00 mm (Fig. 6b). There is

no evidence of a ramped retina, which is seen in some

other stingrays (Sivak 1976). Using the peak ganglion

cell density from the topographical maps and the

averaged Matthiessen’s ratio from the ray tracing,

spatial resolving power values were calculated assum-

ing a hexagonal retinal mosaic for the five eyes used for

ganglion cell topography and range from 3.13 to 4.95

cycles per degree, with an average of 4.10 ± 0.69 cycles

per degree (n = 5).

Discussion

Dasyatis kuhlii used in this study were caught in water

less than 2 m deep, mainly off North Stradbroke Island

in Moreton Bay, Queensland, Australia. Although the

spectral characteristics of the water at this location

were not measured as part of this study, previous

radiance measurements taken nearby reveal that there

is a predominance of medium (‘green’) wavelengths at

depths of up to 5 m (Hart et al. 2004). A predominance

of medium wavelength light is typical of most coastal

waters, where the concentration of dissolved sub-

stances is higher than offshore waters, and as the depth

increases, the spectrum of light shifts to shorter

wavelengths since longer wavelengths are filtered out

(McFarland 1991).

Multiple cone types and the potential for colour

vision

The photoreceptors of D. kuhlii are typical of those

found in other elasmobranch species (Collin 1988;

Braekevelt 1994); rods were easily identifiable by their

long cylindrical outer segments, while cones were dis-

tinguished by their shorter, conical outer segments.

The presence of cones in the retina of D. kuhlii was

reported previously by Kohbara et al. (1987); however,

a quantitative estimate of the rod to cone ratio for D.

kuhlii has not been determined thus far. Peak rod to

cone ratios for other members of the Dasyatidae

studied range from 3:1 in the Atlantic stingray, D. sa-

bina (Logiudice and Laird 1994), to 10:1 in the short-

tailed stingray, D. brevicaudata (Braekevelt 1994). It is

expected that D. kuhlii will possess a rod to cone ratio

within this range. Although the size of the cone inner

segments and the relative position of their nuclei with

respect to the outer limiting membrane suggest three

morphological cone subtypes, there was no attempt to

correlate cone morphology with spectral sensitivity.

Fig. 4 Normalized mean (n = 8) transmittance spectrum of theocular media of Dasyatis kuhlii smoothed with a 31 nmunweighted running average. The wavelength at 0.5 of thenormalized transmittance (T0.5) is 412 nm

J Comp Physiol A (2007) 193:67–79 75

123

The rod visual pigment of D. kuhlii is a vitamin A1-

based rhodopsin with a kmax at 497 nm and is similar to

the rods of most other species of elasmobranch studied

to date, with the exception of deep-sea elasmobranchs,

which tend to have blue-shifted rod visual pigments

(Crescitelli et al. 1985). Although many elasmobranchs

are known to be active and feed during both day and

night, many are most active during dawn and dusk and

D. kuhlii is no exception. Light intensity is dramatically

decreased during these crepuscular periods, and the

spectral distribution of light varies at different times of

the day (McFarland 1986). In particular, a reduction of

yellow-orange light in the atmospheric spectrum re-

sults in an underwater spectrum dominated by short

wavelength photons and under these conditions, it

would be presumed that a rod visual pigment with a

shorter kmax would be optimal for enhancing scotopic

vision (McFarland 1991). These findings suggest that

the spectral position of the rod visual pigment of D.

kuhlii is a compromise that optimizes photon capture

under a variety of scotopic conditions, a hypothesis first

presented for tropical marine fishes that assumes that

the rod system is primarily used for the detection of

contrasting objects (Munz and McFarland 1973).

Dasyatis kuhlii possesses at least three spectrally-

distinct cone visual pigments and, therefore, is the third

recorded elasmobranch species to possess the potential

for trichromatic colour vision. While the rod and green

cone pigments had a similar maximum absorbance

value, every care was taken to distinguish between rods

and cone-like photoreceptors similar to the other cones

with different pigments. Rod and green cone pigments

are genetically distinct in a variety of vertebrates but

often show a similar kmax value (Heath et al. 1997;

Vihtelic et al. 1999). Using microspectrophotometry,

Gruber (1991) found only one cone type in the retina

of the Atlantic guitarfish, Rhinobatos lentiginosus, with

a kmax (499 nm) nearly identical to that of the rod vi-

sual pigment (496 nm). In contrast, Sillman et al.

(1996) were unable to measure the spectral sensitivity

of any cones known to be present in either the leopard

shark, Triakis semifasciata, or the brown smoothhound

Fig. 5 Topography of allneurons within the retinalganglion cell layer of the lefteye of a single specimen ofDasyatis kuhlii. (Discwidth = 228 mm; Equatorialeye diameter = 16.8 mm) Nnasal, V ventral, ON opticnerve head. Scale bar 2 mm

Fig. 6 a Laser ray tracing of the right lens of Dasyatis kuhlii. Thelens is immersed in elasmobranch Ringer solution containing twodrops of milk to aid in the visualisation of the laser beam, whichis directed through the axial meridian. (Lens axial diame-ter = 5.20 mm, lens focal length = 5.63 mm). b Cryosection of

the right eye in the axial plane along the optical axis (greatestlens diameter) showing the distance from the lens (l) to theretina (r). (Lens axial diameter = 6.50 mm, lens focallength = 8.00 mm). M medial, V ventral, c cornea. Scalebar 3 mm

76 J Comp Physiol A (2007) 193:67–79

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shark, Mustelus henlei, using the same technique. In the

latter study, it is possible that cones were missed due to

the relatively low abundance and fragility of cone outer

segments and the low number of individuals examined.

To date, the only other species of elasmobranchs

known to have multiple cone visual pigments are the

reef-dwelling giant shovelnose ray, Rhinobatos typus,

and the coastal eastern shovelnose ray, Aptychotrema

rostrata. These two species have cone visual pigments

with kmax values at 477, 502 and 561 nm, and 459, 492

and 553 nm, respectively (Hart et al. 2004). An inter-

esting outcome of that study was that the visual pig-

ments of A. rostrata all had shorter kmax values than

those of R. typus. This was unexpected as the coral

habitat of R. typus is richer in shorter wavelengths than

the coastal waters in which A. rostrata live. The blue

and green cone pigments of D. kuhlii are more spec-

trally similar to those of R. typus, while the red cone

pigment is spectrally similar to that of A. rostrata.

Colour vision would be a significant advantage for D.

kuhlii and beneficial in prey detection, predator

avoidance, and possibly social behaviours and intra-

specific communication.

Spectral transmittance measurements showed that

wavelengths below about 380 nm are blocked by the

ocular media. The T0.5 value at 412 nm is comparable

to the spectral transmittance of the ocular media of

other dasyatids, such as the cowtail stingray, Pastina-

chus sephen (T0.5 402 nm) and the blue-spotted fantail

ray, Taeniura lymma (T0.5 437 nm), and the rhinobatid

ray Rhinobatos typus, which has a T0.5 value at 408 nm

(Siebeck and Marshall 2001). Many teleost fish are

known to have the potential for UV vision (Bowmaker

and Kunz 1987; Bowmaker et al. 1991; Hawryshyn and

Harosi 1994; McFarland and Loew 1994; Losey et al.

2003), and the genetic basis for ultraviolet-sensitive

visual pigments in the common ancestor of all extant

vertebrates has been established through molecular

studies on the southern hemisphere lamprey, Geotria

australis (Collin et al. 2003b; Collin and Trezise 2004).

It is possible that UV cones were missed using micro-

spectrophotometry, especially if they were present in

low numbers, but the ocular media transmittance data

suggests that UV wavelengths are not transmitted to

the retina and that D. kuhlii probably does not use UV

for vision.

Visual ecology and spatial resolving power

Dasyatis kuhlii possesses a dorsal horizontal streak of

increased ganglion cell density within which lie multi-

ple areae of higher cell density, located nasally, cen-

trally and temporally. Other species of ray possess a

very similar topographic arrangement, including the

giant shovelnose, ray Rhinobatos typus (Collin 1988),

the Atlantic stingray, Dasyatis sabina (Logiudice and

Laird 1994), Bigelow’s ray, Raja bigelowi (Bozzano

and Collin 2000), and the eastern shovelnose ray,

Aptychotrema rostrata (Litherland 2001). Many of

these bottom-dwelling rays, like D. kuhlii, lie on the

bottom or bury themselves in the substrate (often with

only their eyes and tail protruding), only swimming off

the bottom to forage for food or mates, or to escape

from predators (Tricas et al. 1997). Therefore, the

dorsal horizontal streak prevalent in most rays may be

important for the panoramic surveillance of the hori-

zon for potential predators, without the need for

extensive eye or body movements, which could reveal

the animals’ location (Bozzano and Collin 2000). The

temporal area in D. kuhlii is the largest of the three

areae and contains the highest cell density (mean

4,251 cells mm–2), suggesting that the lower frontal

region of the visual field is especially important. The

nasal, central and temporal areae found within the

streak would allow regions of the caudal, lateral and

nasal visual fields to be observed, respectively, with

higher spatial resolving power.

Essentially nothing is known of accommodation in

elasmobranchs, but it is assumed that the protractor

lentis muscle, which contracts to move the lens forward

for near vision, would remain relaxed post mortem.

This would then yield an altered focal length and

Matthiessen’s ratio when compared to a live animal or

the ray tracing, but it should be noted that the values

obtained for Matthiessen’s ratio were very similar

using both ray tracing and frozen section methods. A

hexagonal retinal mosaic optimises the spatial packing

of ganglion cells with circular receptive field profiles

and is probably a more accurate representation of

ganglion cell distribution in the retina than a square

mosaic (Snyder and Miller 1977; Wassle and Riemann

1978; Hart 2002), but nothing is known of retinal

packing in elasmobranchs and all previous studies

concerning elasmobranchs have calculated spatial

resolving powers assuming a square mosaic and a

Matthiessen’s ratio of 2.55. For comparison, values of

spatial resolving power for different species varied

from 3.98 to 7.64 cycles per degree, when corrected for

a hexagonal array (Hueter 1991; Bozzano and Collin

2000; Bozzano 2004). Pelagic species such as the tiger

shark, Galeocerdo cuvier, tend to have higher spatial

resolving power (7.19 cycles per degree), when com-

pared with benthic species such as the small-spotted

catshark, Scyliorhinus canicula (3.98 cycles per degree)

(Bozzano and Collin 2000). The calculated spatial

resolving power of D. kuhlii (4.12 cycles per degree) is

J Comp Physiol A (2007) 193:67–79 77

123

moderate when compared with the previous elasmo-

branchs studied and conforms to the trend that benthic

species have lower spatial resolving power than pelagic

species. This suggests that D. kuhlii is not as heavily

dependent on acute distance vision for such behaviours

as prey detection and predator avoidance and that in-

creased visual sensitivity, in conjunction with other

sensory modalities, may be more important.

Specific dietary information is lacking for most rays,

including D. kuhlii, but the majority of dasyatid species

feed on benthic or demersal invertebrates and fish

(Last and Stevens 1994; Tricas et al. 1997). The posi-

tion of the ventral mouth in the centre of the disc

dictates that in the final stages of a prey-directed

movement before a feeding strike, vision is obscured

by the animal itself. Therefore, the mechanoreceptive

lateral line and the electroreceptors (ampullae of

Lorenzini) are thought to play a large and important

role in foraging behaviour in rays, especially during the

final strike (Raschi 1986; Maruska and Tricas 1998;

Maruska 2001; Collin and Whitehead 2004). Vision,

however, may be useful in the initial detection of

epifaunal prey on the surface of the benthos, or the

detection of infaunal prey as they venture from holes

or burrows. Future studies should investigate the

relative sensitivity of the other sensory systems of

D. kuhlii, such as olfaction and electroreception, in

order to gain a more comprehensive analysis of how all

the sensory modalities function together.

Acknowledgments The authors would like to first thank twoanonymous reviewers for helpful comments on improving themanuscript. The authors would also like to thank Mike Bennett,Simon Pierce, Scott Cutmore, Peter Kyne, Joanna Stead, TinaChua, Justin Marshall and the staff of the University ofQueensland Moreton Bay Research Station. This work wasfunded by ARC Discovery Grants DP0558681 and DP0558844,NHMRC Project Grant 179837, the American ElasmobranchSociety (Donald R. Nelson Behaviour Research Award), andSigma Xi.

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