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THE ANATOMICAL RECORD 295:1727–1735 (2012)
Ocular Anatomy and Retinal
Photoreceptors in a Skink, the Sleepy
Lizard (Tiliqua rugosa)
SHAUN T.D. NEW,1,2,3* JAN M. HEMMI,2,4 GREGORY D. KERR,1
1
AND C. MICHAEL BULL
1
School of Biological Sciences, Flinders University, Adelaide, Australia
2
ARC Centre of Excellence in Vision Science, The Australian National University,
Canberra, Australia
3
Evolution, Ecology and Genetics, Research School of Biology, The Australian National
University, Canberra, Australia
4
School of Animal Biology and The UWA Oceans Institute, The University of Western
Australia, Crawley, Australia
ABSTRACT
The Australian sleepy lizard (Tiliqua rugosa) is a large day-active
skink which occupies stable overlapping home ranges and maintains
long-term monogamous relationships. Its behavioral ecology has been
extensively studied, making the sleepy lizard an ideal model for investigation of the lizard visual system and its specializations, for which relatively little is known. We examine the morphology, density, and
distribution of retinal photoreceptors and describe the anatomy of the
sleepy lizard eye. The sleepy lizard retina is composed solely of photoreceptors containing oil droplets, a characteristic of cones. Two groups could
be distinguished; single cones and double cones, consistent with morphological descriptions of photoreceptors in other diurnal lizards. Although
all photoreceptors were cone-like in morphology, a subset of photoreceptors displayed immunoreactivity to rhodopsin—the visual pigment of
rods. This finding suggests that while the morphological properties of rod
photoreceptors have been lost, photopigment protein composition has
been conserved during evolutionary history. Anat Rec, 295:1727–1735,
C 2012 Wiley Periodicals, Inc.
2012. V
Key words: vision; retina; photoreceptor; rhodopsin; lizard
INTRODUCTION
During their evolutionary history, obligate diurnal
lizards lost the classical vertebrate duplex retina containing both rods and cones [Walls, 1942]. Rod
photoreceptors were replaced in favor of single and double cones, an adaptation for the demands of a brightly lit
environment. Little is known of when this change took
place, or whether corroborating physiological limitations
led to associated specializations of the lizard eye.
Much of our understanding of the lizard visual system
has come from research on the Iguania (comprising iguanids, chameleons, and agamid lizards). These lizards
possess four functionally distinct visual pigments, and
colored oil droplets positioned at the scleral margin of
the inner segment of each photoreceptor [Fleishman
et al., 1997; Loew et al., 2002; Bowmaker et al., 2005], a
C 2012 WILEY PERIODICALS, INC.
V
specialization shared only by birds and other reptiles.
Gekkonid lizards have a suite of visual specializations
which accompanied their shift to nocturnal activity [Citron and Pinto, 1973; R€oll, 2000, 2001a; Roth et al.,
Grant sponsor: Australian Research Council under the ARC
Centres of Excellence Program.
*Correspondence to: Shaun T.D. New, Research School of
Biology, RN Robertson Building, Sullivans Creek Road, The
Australian National University, Canberra ACT 0200, Australia.
Tel: þ61 2 6125 8273; Fax: þ61 2 6125 3808.
E-mail: [email protected]
Received 14 February 2012; Accepted 7 July 2012.
DOI 10.1002/ar.22546
Published online 31 July 2012 in Wiley Online Library
(wileyonlinelibrary.com).
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NEW ET AL.
2009]. There are few descriptions, however, of the visual
system among other groups of lizards.
In the late 1980s, rod photoreceptors were described
using cytology in the Australian sleepy lizard (Tiliqua
rugosa), a day-active member of the Scincidae [Braekevelt, 1989]. This finding, in contrast to the ‘‘typical"
cone-only retina of diurnal lizards, was questioned [R€oll,
2001b]. However, immunohistochemical assays have
revealed the presence of rhodopsin (the visual pigment
of rods) in the retina of the American anolid (Anolis carolinensis) and the chameleon (Chamaeleo chamaeleon)
[Mcdevitt et al., 1993; Bennis et al., 2005]. This raises
questions about the classification of lizard photoreceptors. Here, we reexamine the photoreceptors present in
the sleepy lizard retina.
The sleepy lizard has a region of heightened ganglion
cell density within the central retina [New and Bull,
2011]. Elongated horizontally to form a weak visual
streak, this specialization is thought to provide a wide
field of distinct vision in its exposed environment. We
extend these findings, describing the ocular anatomy of
the sleepy lizard eye and the density, distribution and
types of retinal photoreceptors, which are the first interface with the visual environment. As the sleepy lizard
belongs to the basal Scleroglossa clade of lizards [Vidal
and Hedges, 2009], under represented in comparative
visual anatomy, this study has the potential to offer new
insights into the evolution of the lizard visual system.
MATERIALS AND METHODS
Subjects
Nine adult sleepy lizards (Tiliqua rugosa aspera)
(three #, three $, and three unknown) were collected
from Kadina and Bundy Bore Station, South Australia.
They were housed individually in outdoor pens (3.5 3.0 m) and fed once every 3 days with sliced fruit, vegetables, and tinned cat food. Water was supplied ad
libitum. Animal care and experimental procedures were
carried out in accordance with guidelines provided by
Flinders University of South Australia Animal Welfare
Committee in compliance with the Australian Code of
Practice for the use of animals for scientific purposes.
Head and Eye Morphology
The length of the head (rostrum to the tip of the most
caudal head scale) and snout (rostrum to nasal eye margin), width of the base of the snout and rostrum (at the
position of the nasal eye margin and nares, respectively),
and the snout-vent length of each lizard were measured
in triplicate using vernier calipers to an accuracy of 0.1
mm. These measurements allowed an estimate of cranial
limitations on the breadth of the frontal binocular overlap. Ocular morphology was examined in the eyes of one
lizard using an Oculus Pentacam (Oculus, Optikger€
ate
GmbH). Several scans were taken for each eye under
dark conditions and mydriatic agents were not used. Pupil size was measured in two lizards individually placed
in an observation chamber of known, stable light intensity. Experiments were conducted at luminances of 0.005
and 430 cd/m2, approximating dim moonlight and typical
indoor light levels, respectively. After waiting several
minutes to allow lizards to adapt to light levels, subject’s
eyes were filmed using an infrared sensitive digital HD
video camera (Sony Handycam HDR-CX550). Video
recordings were converted to jpeg files and four representative images were chosen for each lizard at each
light level. Pupil size was measured by fitting an ellipse
to the edge of the pupil using Adobe Illustrator CS3 software, and mean pupil area was calculated.
Two specimens were dark adapted, euthanized with
Nembutal injected intraperitoneally ( 2 cc.) and the
eyes excised. Two eyes were paraffin embedded for examination of the anatomy of the eyeball and retina, and
two eyes plastic embedded for investigation of photoreceptor distribution. Details of the techniques used are
described below.
Anatomy of the Eye
Following fixation in 10% buffered formalin for 48 hr,
two eyes were dehydrated in graded ethanols, cleared in
chloroform, and embedded in histological paraffin wax.
Radial sections of 5 lm thickness were deparaffinized,
rehydrated, and stained with Hematoxylin (Harris type)
for 10 min and Eosin for 1 min. Images of the eyeball
and its components were taken using Q-capture software
on an Olympus 5 mega pixel micropublisher digital camera attached to an Olympus BX50 microscope.
Retinal Photoreceptors
Two eyes were hemisectioned at the equator and the
lens and vitreous carefully removed. Each eyecup was
orientated with a dorsal stitch, fixed in 2% paraformaldehyde for 30 min, and post fixed in 1% osmium
tetroxide for 1 hr. Eyecups were cut into individual
regions, dehydrated, passed through propylene oxide,
and embedded in epoxy resin. Sections were tangentially
cut (parallel to retinal layers) at a thickness of 1 lm,
double labeled with methylene blue and basic fuchsin,
and mounted with Depex mounting medium.
The density, distribution, and packing arrangement of
retinal photoreceptors were analyzed using ImageJTM
1.36b software. Photoreceptors were counted within a
40,000 lm2 field at a depth approximately equal to the
ellipsoid body, and double cones were counted as a single
entity. Cells overlapping the upper or right edges were
recorded, but those overlapping the left or bottom borders of sampling fields were excluded from counts. Cell
counts were converted to cells/mm2. No corrections were
applied for the slight shrinkage that occurred during the
embedding process. Ultrathin tangential sections (100
nm) were stained with 1% uranyl acetate and 0.1% lead
citrate for closer examination of photoreceptors on a
JEOL 1200EX transmission electron microscope.
To investigate the functional characteristics of sleepy
lizard photoreceptors, expressed visual opsins were
examined using immunohistochemical labeling. Radially
cut paraffin sections (perpendicular to retinal layers, see
above) were rehydrated in graded alcohols, rinsed in 0.1
M phosphate buffered saline (PBS), and incubated in
10% normal goat serum (NGS) in PBS for 1 hr at 37 C
to limit nonspecific binding of antibodies. Preparations
were then incubated overnight at 4 C in a 1:200 dilution
of anti-rhodopsin (Rho-4d2) and a 1:200 dilution of either rabbit anti-opsin blue or red-green polyclonal
antibody in PBS containing 1% NGS. Rho-4D2 binds to
the N-terminal part (amino acids 2–39) of bovine
SKINK OCULAR ANATOMY AND RETINAL RECEPTORS
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rhodopsin [Hicks and Molday, 1986], the photopigment
of rods. The Rho-4D2 antibody is a reliable marker of
rods in fish [Knight and Raymond, 1990], amphibians
[Hicks and Molday, 1986; Bugra et al., 1992], birds
(Hicks, unpublished observation), and mammals [Hicks
and Molday, 1986; Hicks et al., 1989], and its specificity
has been verified in the retina of an anolid lizard by immunoblotting [Mcdevitt et al., 1993]. Preparations were
incubated at 37 C for 1 hr in goat anti-rabbit 594 and
goat anti-rabbit 488 secondary antibodies to 1:1,000 dilution in PBS containing 1% NGS, and mounted in
glycerol gelatin. Omission of the primary antibody gave
no apparent immunolabeling. Consecutive serial sections
reacted with different antibodies were photographed
with a Zeiss LSM 5 Pascal confocal microscope system
and Pascal version 4.0 software (Jena, Germany).
RESULTS
The sleepy lizard has a short, broad head tapering to
a rotund snout (Fig. 1). Eyes are placed laterally and
anteriorly within the skull, and bordered dorsally by a
prominent row of scales forming a distinct brow. This
cranial morphology and eye placement provides the
potential for a wide visual field and up to 25 of frontal
binocular overlap. The eye was globular in shape and
the pupil round and dynamic, measuring 3.7 mm2
(St. Dev. ¼ 0.06) in area under low-light levels and
constricting to 2.6 mm2 (St. Dev. ¼ 0.15) on exposure to
bright light (Fig. 2).
Cornea
The cornea formed a distinct spectacle, was 270 lm
thick and consisted of five layers; epithelium, Bowman’s
layer, stroma, Descemet’s membrane, and endothelium
(Fig. 3a). The fibrous stroma comprised 75% of the total
corneal thickness and was protected by the thick noncellular Bowman’s layer and corneal epithelium. The thin,
noncellular Descemet’s membrane was discernable
between the stroma and corneal endothelium (arrow in
Fig. 3A).
Lens
Oculus Pentacam imaging allowed examination of the
anterior chamber of the eye in vivo. The lens was
revealed to be distinctly flat and positioned 900 lm
from the posterior corneal surface at the optic axis (Fig.
3B). Although fixation and dehydration resulted in
shrinkage and distortion of the lens, its anatomy was
well preserved (see inset Fig. 4). The lens was composed
of thin concentrically arranged fibers encapsulated by an
epithelial layer (Fig. 4), which was equatorially thickened as an annular pad. Zonular fibers stretching from
the equator of the eyes to the ciliary body held the lens
in place.
Iris
The iris bordered the anterior surface of the lens (Fig.
3B). Dark pigment cells enclosed the iridial stroma, with
the double-layered inner epithelium more heavily pigmented than the capillary-rich outer layer (Fig. 4). The
sphincter pupillae (SM) contained prominent circular
muscle fibers and scattered melanin. Radial muscle
Fig. 1. Schematic head morphology. The nasolateral position of the
eyes provides a broad field of vision. Head length (HL), snout length
(SL), width between nares (NW), and snout base with (SBW) measured
in nine lizards (three #, three $, and three unknown). Head morphology
limits the binocular visual field to a maximum potential breadth of 25
degree. Scale ¼ 10 mm.
fibers of the dilator pupillae (DM) followed the anterior
margin of the inner epithelium.
Retina
The retina was 250 lm thick adjacent the optic disc
and tapered slightly peripherally. It had no distinguishable landmarks and it lacked a fovea. The photoreceptor
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NEW ET AL.
Fig. 2. Pupil size under varying light intensity. Digitized video
images of the pupillary response in the same eye of the same subject
following exposure to 0.005 cd/m2 (left panel) and 430 cd/m2 (right
panel) white light. Top and bottom row show identical pictures. The
broken circle in the lower right panel demarks pupil size at 0.005 cd/
m2. Scale ¼ 5 mm.
layer contained densely packed, elongated processes
embraced by the retinal pigment epithelium (Fig. 5).
The outer nuclear layer was 20 lm thick, composed of
two rows of rigidly organized receptor perikarya. The
first row of cells was lightly stained and ovoid, the second, bordering the thin outer plexiform layer, round and
densely stained. The inner nuclear layer was much
thicker than its outer counterpart (50 lm), consisting of
loosely organized cells up to 12 cells deep in the central
retina. Further vitreal, the inner plexiform layer formed
a meshwork of neuronal processes connecting the inner
nuclear layer with the ganglion cell layer.
Blood Supply
The completely avascular retina was bordered by a welldeveloped choroid containing numerous, broad venous
sinuses intermingled within melanin pigmentation. In
addition, the conus papillaris, considered to be a nutritive
device for the inner retina [Rodieck, 1973], projected toward the centre of the vitreous chamber from the optic
disc. The appearance of a histological section of the conus
papillaris cut along its length is shown in Fig. 6. Internally,
the conus consisted of an extensive array of capillaries and
larger blood vessels separated by a matrix of melanocytes
and connective tissue. The optic nerve extended from the
eyecup at the base of the conus papillaris.
Retinal Photoreceptors
Photoreceptors projected through the external limiting
membrane for up to 30 lm in the dark adapted state
(Fig. 7A). The inner segment consisted of a nucleus, a
large paraboloid, and an ellipsoid body bordered further
Fig. 3. Corneal structure. (A) Photomicrograph of a sectioned and
H&E-stained cornea. The fibrous stroma contained keratocyte nuclei
(arrowheads) and was bordered anteriorly by the Bowman’s layer (Bl) and
a defined epithelium (Ep). Descemet’s membrane (arrow) was evident,
though less distinct, lining the endothelium (En). The concave shape of the
cornea is an artifact of histology. No manipulation of images was undertaken, apart from contrast and brightness enhancement to maximize
detail. Scale ¼ 100 lm. (B) Oculus Pentacam Scheimpflug image of the
anterior segment of the eye. The broken line defines the anterior surface of
the lens which was bordered by the iris (arrowheads). Scale ¼ 1 mm.
scleral by an oil droplet. Outer segments were small (5–
7 lm) and conical. Pairs of cone cells were observed with
their inner segments in broad contiguity though kept
separate by their cell membranes (Fig. 7B). The two
members of these pairings were morphologically different; one member short and broad containing a
prominent paraboloid body and the second member long
and narrow with a small or absent paraboloid body but
containing an oil droplet. These cells resembled the
accessory and principal members, respectively, of double
cones [Fleishman et al., 1997]. Oil droplets were pale
yellow in color and located in single cones and within
the principal member of double cones.
Photoreceptors were ovoid in tangential cross section,
and isolated from those around it by processes of pigment epithelium (Fig. 7B). Cell counts integrated from
two eyes revealed that photoreceptors were heterogeneously distributed throughout the retina. Photoreceptor
density peaked at 76,600 cells/mm2 (Fig. 8) within the
central retina (mean ¼ 50,050 6 SE 6,800 cells/mm2),
and decreased peripherally to 30,275 6 4,370 cells/mm2
SKINK OCULAR ANATOMY AND RETINAL RECEPTORS
Fig. 4. Iris and lens stained with H&E. Capillaries (arrowheads) surrounded by pigmented epithelia bordered the anterior margin of the
iris. The posterior iris margin was heavily pigmented and the iridial
stroma (IS), iris sphincter (SM), and dilator (DM) muscles prominent.
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The lens consisted of a fibrous inner region bordered by an epithelial
layer (LE) containing a prominent row of nuclei, and a lens capsule
(arrow). Scale ¼ 50 lm. A section through the anterior eye is shown
inset (scale ¼ 250 lm).
Fig. 6. Conus papillaris. The conus papillaris contained an extensive array of capillaries and larger blood vessels separated by a matrix
of melanocytes and connective tissue. Scale ¼ 25 lm.
Fig. 5. Photomicrographs of the retina. Laminar organization of the
retina at approximately 2 mm dorsal eccentricity from the optic disc.
Scale ¼ 50 lm.
(unpaired t test; t ¼ 2.45, df ¼ 14, P ¼ 0.028). The ventral hemisphere contained a significantly higher density
of photoreceptors than the dorsal retina (t ¼ 4.45, df ¼
22, P < 0.001) (Fig. 9A), while there was no significant
difference in receptor density between the temporal and
nasal retina (t ¼ 0.42, df ¼ 22, P ¼ 0.68) (Fig. 9B).
Double cones comprised 19% of all photoreceptors
throughout the retina (Table 1). Their density strongly
correlated with single cone abundance (r2 ¼ 0.73; twotailed test for significance of correlation coefficient, t ¼
6.835, df ¼ 17, P < 0.005; Fig. 10). Double cones were
interspersed between single cones such that two double
cones were rarely adjacent to one another (Fig. 7B). A
significant population of photoreceptors exhibited strong
immunofluorescence for Rho-4D2 (green labeling in Fig.
7C,D). Double labeling with either rabbit anti-opsin medium-long wavelength or short-wavelength-sensitive
polyclonal antibodies (red labeling in Fig. 7C,D, respectively) revealed that Rho-4D2 labeled a different
population of photoreceptors not recognized by any of
the other antibodies.
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DISCUSSION
The Australian sleepy lizard occupies stable overlapping home ranges [Bull, 1987; Bull and Freake, 1999] and
maintains long-term monogamous relationships [Bull,
1988, 1990] that require accurate interindividual recognition. Although olfactory cues play a major role in social
recognition [Bull et al., 1993], visual cues are also thought
to be used [Zuri and Bull, 2000a] and play a significant
role in food choice [Wohlfeil, 2008], refuge selection [Kerr
et al., 2003; Auburn et al., 2009], spatial orientation [Zuri
and Bull, 2000b; Freake, 2001], and the detection of
approaching threats [Murray and Bull, 2004]. Clearly, the
sleepy lizard is a visual animal, and its eyes are adapted
for this lifestyle. The eyeball is globular in shape [New
and Bull, 2011], and the lens distinctly flat and positioned
forward within the eye, creating a relatively shallow anterior segment. This optical configuration favors visual
acuity over sensitivity, maximizing focal length and producing a relatively larger retinal image spread over a
greater number of photoreceptors [Land and Nilsson,
2001; Hall, 2008]. The iris dilator and sphincter muscles
are well developed, enabling changes in pupil aperture. A
Fig. 8. Photoreceptor density throughout the retina. Values indicate
mean density (cells mm2) obtained from cell counts of two retinas,
except those regions marked with asterisks where density was
obtained from a single retina. D, dorsal; T, temporal; V, ventral, and N,
nasal retina; OD, optic disc. Sampling regions above the horizontal
line adjoining T and N are defined here as the dorsal retina, while
those below comprise the ventral retina. Similarly, points D and V separate the temporal from the nasal retina.
Fig. 7. Photoreceptor morphology. (A) Radial cross section of retinal
photoreceptors stained with H&E. A double cone photoreceptor is
indicated (arrow). (B) Transmission electron micrograph of photoreceptor inner segments tangentially sectioned at the level of single cone oil
droplets (*). The principle member (PM) and accessory member (AM)
of each closely connected double cone was clearly evident. Accessory member oil droplets are not visible here, however, due to the
slightly longer length of double cones. Photoreceptors were isolated
from each other by the processes of pigment epithelial cells (arrowheads). (C) Immunofluorescence for Rho-4D2 rhodopsin antibody
(green) and medium-long wavelength opsin antibody (red). (D) Immunofluorescence for rhodopsin (green) and short wavelength opsin antibody (red). Immunhistochemistry was conducted on 5 lm retinal
sections. Scale ¼ 10 lm.
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SKINK OCULAR ANATOMY AND RETINAL RECEPTORS
TABLE 1. Regional variation in photoreceptor
density and double cone percentage throughout
the sleepy lizard retina
Retinal region
Central
Outer
Peripheral
Mean
Receptor
density (mm2)
SE
Double
cones (%)
50,050
41,775
30,275
40,700
6,800
5,790
4,370
3,590
19.8
20.4
16.6
18.9
Cell counts were taken at approximately the ellipsoid body
of cone photoreceptor cells and double cones were counted
as a single entity.
Fig. 9. Spatial variation in photoreceptor density. Mean cell density
and standard errors for the central, outer, and peripheral retinal
regions as defined in Fig. 8 are shown. (A) The ventral retina contained
a significantly greater photoreceptor density than the dorsal retina. (B)
In contrast, the density of photoreceptors was consistent between the
nasal and temporal halves of the retina.
dynamic pupil facilitates more rapid retinal adaptation,
likely aiding visually controlled movements between
exposed areas and lower light levels experienced beneath
bushes or within burrows.
Paradox of the Rods
The retina of the sleepy lizard contains only cone photoreceptors which could be separated into two distinct
classes; single cones and double cones. The cones are characterized by an oil droplet and small conical outer
segments, and their morphology is consistent with descriptions from other diurnal lizards [Peterson, 1992; R€oll,
2001a; Barbour et al., 2002; Bowmaker et al., 2005]. In contrast to Braekevelt (1989), no morphological evidence of
rods was observed. R€oll (2001a) noted that Braekevelt’s
(1989) description of rods—joining closely with the neighboring visual cell at the level of the ellipsoid—suggested he
may have erroneously ascribed the accessory member of
double cone photoreceptors as rods. This is plausible given
that Braekevelt did not identify double cone photoreceptors, and that the accessory member of sleepy lizard double
cones, like in other diurnal lizards, lacks an oil droplet.
However, Braekevelt described ‘‘rods" as narrow and containing no paraboloid body, while the accessory member
Fig. 10. Cone and double cone density. Circles demark cell density
within the peripheral retina, triangles outer retina, and squares the
central retina. A strong correlation between double cone and single
cone density is evident.
inner segment was observed here to be broad and containing a prominent paraboloid body.
Although the sleepy lizard retina contains only cone
photoreceptors based on morphological criteria, a subset
of photoreceptors display immunoreactivity to Rho-4D2,
a monoclonal antibody raised against the N-terminal
part of rhodopsin. This is not the first evidence of rhodopsin within a pure-cone retina [Mcdevitt et al., 1993].
Kawamura and Yokoyama (1997) identified a gene
orthologous to rhodopsin in other vertebrates in the
pure-cone retina of an anolid lizard (Anolis carolinensis)
(see references therein). This gene encodes the RH1Ac
opsin, and is expressed in addition to the SWS1Ac,
SWS2Ac, RH2Ac, and LWSAc visual opsins, and labeled
by the Rho-4D2 antibody [Mcdevitt et al., 1993]. Rhodopsin labeling has also been demonstrated in the rod-free
retina of the chameleon (Chamaeleo chamaeleon) [Bennis et al., 2005]. Chamaeleo and Anolis both belong to
the suborder Iguania [Vidal and Hedges, 2009]. The
presence of rhodopsin in a representative species of the
Scincidae, which belong to the more basal suborder Scleroglossa, suggests occurrence of the RH1Ac opsin
represents the ancestral pattern.
Rhodopsin may enable photoreceptors to lower their
threshold to light levels well below that required to stimulate cones, thus affording vision in lower conditions.
Cones, are often defined according to the amino acid
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NEW ET AL.
sequence of their photopigments and in turn the spectral
wavelength in which pigments are most sensitive. The
spectral and biochemical characteristics of lizard retinal
visual pigments are not well understood, and have not
been examined at all in the skinks, though lizards
appear tetrachromatic. In those diurnal lizards examined, four functionally distinct retinal pigments have
been characterized as follows; ultraviolet-sensitive (maximum absorbance kmax range ¼ 365–385 nm; opsin
protein ¼ SWS1Ac), short-wavelength-sensitive (440–455
nm; SWS2Ac), medium-wavelength-sensitive (480–505
nm; RH2Ac), and long-wavelength-sensitive (555–625
nm; LWSAc) [Kawamura and Yokoyama, 1997, 1998;
Loew et al., 2002; Fleishman et al., 2011]. The discrepancy in the number of opsin genes and functionally
distinct pigments is curious. Where is the RH1Ac opsin?
Sleepy lizard photoreceptors immunoreactive to Rho-4D2
did not co-label with either the short wavelength or medium-long wavelength opsin antibodies, which likely
targeted the SWS1Ac and LWSAc opsins, respectively.
The RH1Ac opsin is thus not expressed within these photoreceptors. It has been posited that the RH1Ac pigment
is expressed in a medium-wavelength-sensitive photoreceptor together with the RH2Ac opsin pigment
[Kawamura and Yokoyama, 1998], but further examination is required. Moreover, cone-like sensitivity to
hydroxylamine [Kawamura and Yokoyama, 1998] suggests that the RH1Ac pigment may even be cone-like in
sensitivity. Characterization of receptor phototransduction and regeneration rates [e.g., Pugh and Lamb, 2000;
Lamb and Pugh, 2006], including among the skinks, will
provide a clearer understanding of the functional and
adaptive properties of lizard vision.
Photoreceptor Density and Ecology
Specimens were wild caught sexually mature adults,
though their exact age was unknown. Age-related differences in photoreceptor numbers have not been reported
among lizards, but such differences cannot be discounted
as sleepy lizards are long lived [Bull, 1995] and inhabit
brightly lit environments. We provide here an assessment
of retinal photoreceptors based on two retinas. Although
not permitting the description of retinal specializations
afforded by topographical retinal maps, it does allow us to
examine regional differences in receptor density and their
correspondence with the topographic distribution of ganglion cells in this species [New and Bull, 2011].
Photoreceptors
are heterogeneously distributed
throughout the sleepy lizard retina, peaking centrally at
76,000 cells/mm2 and decreasing peripherally. This is
substantially lower than maximum densities observed
within foveal specializations, such as the 290,000 photoreceptors/mm2 reported for the central fovea of Anolis
carolinensis [Makaretz and Levine, 1980]. That species
actively hunts and pursues fast moving insect prey. In
contrast, the sleepy lizard moves slowly through its habitat, foraging opportunistically and predominantly on
plant matter. This lifestyle would place less demand on
a foveal specialization.
Photoreceptor density in Anolis carolinensis drops to
3,200 cells/mm2 adjacent the fovea and to 1,600 receptors/mm2 at the retinal periphery [Makaretz and Levine,
1980], and thus must rely on head and eye movements
to center objects of interest onto their central fovea for
distinct vision. In contrast, the lowest photoreceptor density we observed in the sleepy lizard eye is an order of
magnitude higher than in Anolis carolinensis, 15,900
cells/mm2. The more uniform distribution of photoreceptors in the sleepy lizard retina, together with the
nasolateral position of the eyes within the skull, affords
a wide field of distinct vision without the need for
directed eye movements. The sleepy lizard occupies fully
terrestrial open environments, where much of the biologically relevant visual cues, including predators, will
come from above. The significantly higher density of
photoreceptors within the ventral retina, in correspondence with the topographic distribution of ganglion cells
[New and Bull, 2011], supports greater resolving power
and sensitivity in the dorsal visual field into which the
majority of visual targets might appear.
CONCLUSIONS
The sleepy lizard (Tiliqua rugosa) has several visual
specializations related to the demands of its diurnal lifestyle in an exposed environment. The eyes are laterally
placed and a relatively high density of photoreceptors
within the peripheral retina affords a wide field of distinct vision. A large focal length provides a wide image
throw onto the retina, and the dynamic pupil enables
adjustment of retinal illumination assisting photoreceptor adaptation. Although all retinal photoreceptors
contain an oil droplet, a subset of cones also contains
rhodopsin. Our results provide further evidence that the
traditional categorization of vertebrate photoreceptors
into two distinct ‘‘morphs" possessing exclusive functional and optical properties is too simplistich.
ACKNOWLEDGEMENTS
We are grateful to Michelle Lewis, Kerry Gascoigne, Bill
Stell, and Krisztina Valter for providing technical assistance. Thank you also to Dale Burzacott for measurement of sleepy lizard head morphology and Robert
Molday for his kind gift of Rho-4D2 antibody used here
in immunohistochemistry. Comments from Jochen Zeil
and three anonymous reviewers on earlier versions of
this manuscript were greatly appreciated.
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