Available via license: CC BY 4.0
Content may be subject to copyright.
RESEARCH ARTICLE
Pupil dilation and constriction in the skate Leucoraja erinacea
in a simulated natural light field
Lydia M. Ma
thger
1,
*
,‡
, Michael J. Bok
2
, Jan Liebich
3
, Lucia Sicius
1,4
and Dan-Eric Nilsson
2,
*
ABSTRACT
The skate Leucoraja erinacea has an elaborately shaped pupil,
whose characteristics and functions have received little attention. The
goal of our study was to investigate the pupil response in relation to
natural ambient light intensities. First, we took a recently developed
sensory–ecological approach, which gave us a tool for creating a
controlled light environment for behavioural work: during a field
survey, we collected a series of calibrated natural habitat images from
the perspective of the skates’eyes. From these images, we derived a
vertical illumination profile using custom-written software for
quantification of the environmental light field (ELF). After collecting
and analysing these natural light field data, we created an illumination
set-up in the laboratory, which closely simulated the natural vertical
light gradient that skates experience in the wild and tested the light
responsiveness –in particular the extent of dilation –of the skate pupil
to controlled changes in this simulated light field. Additionally, we
measured pupillary dilation and constriction speeds. Our results
confirm that the skate pupil changes from nearly circular under low
light to a series of small triangular apertures under bright light. A linear
regression analysis showed a trend towards smaller skates having a
smaller dynamic range of pupil area (dilation versus constriction ratio
around 4-fold), and larger skates showing larger ranges (around 10-
to 20-fold). Dilation took longer than constriction (between 30 and
45 min for dilation; less than 20 min for constriction), and there was
considerable individual variation in dilation/constriction time. We
discuss our findings in terms of the visual ecology of L. erinacea and
consider the importance of accurately simulating natural light fields in
the laboratory.
KEY WORDS: Environmental light field, ELF, Batoid, Elasmobranch,
Vision
INTRODUCTION
Pupils are found in the eyes of vertebrates and invertebrates, and
most pupils generally respond to light by constricting. This limits
the amount of light entering the eye (Denton, 1956; Wilcox
and Barlow, 1975; Hammond and Mouat, 1985; Douglas, 2018;
Land and Nilsson, 2012). In terms of visual function, we have a
good understanding of circular, horizontal and vertical slit-shaped
pupils (Nilsson et al., 2005; Malmström and Kröger, 2006;
Lind et al., 2008; Land and Nilsson, 2012; Douglas, 2018) but
there are several other pupil shapes that have not been studied very
much at all, including the multiple-pinhole pupil found in geckos
(Denton, 1956; Roth et al., 2009), the elaborate W-shaped pupil of
cuttlefish Sepia officinalis (Muntz, 1977; Murphy and Howland,
1991; Douglas et al., 2005; Mäthger et al., 2013) and the frill-like
pupil of the little skate, Leucoraja erinacea, which we studied here
(Fig. 1; Kuchnow, 1971; Youn et al., 2019).
There is still much to learn about the full extent of pupil dilation
and constriction in elasmobranchs. Earlier work suggests that, with
some exceptions, mobile light-sensitive pupils are common in
batoids (skates and rays), including Raja species. In selachians
(sharks), pupils were generally assumed not to be mobile, although
we now know that many shark species certainly have mobile
pupils (Beer, 1894; Franz, 1906, 1931; Young, 1933; Walls, 1963;
Kuchnow, 1971; Collin, 1988; Nicol, 1989; Douglas, 2018).
However, systematic studies are lacking.
Leucoraja erinacea is an excellent batoid species to study these
kinds of questions. It is easy to maintain in the laboratory and its
comparatively small size makes experimental logistics more
manageable. Leucoraja erinacea has a cathemeral lifestyle: it is
irregularly active throughout night and day. However, some
evidence looking at metabolic rate at different times of night/day
suggests that it is slightly more active during crepuscular and
nocturnal times of day (Hove and Moss, 1997). Other skate species
follow similar patterns. Activity has been recorded day and night,
with a slight crepuscular/nocturnal increase (Wearmouth and Sims,
2009). As has been shown for several other elasmobranch species,
L. erinacea has monochromatic vision. Its retina has been reported
to contain only rods with an absorbance peak at 500 nm (Dowling
and Ripps, 1970; Green and Siegel, 1975).
More recently, while testing the visual field characteristics of
four batoid elasmobranch species, McComb and Kajiura (2008)
reported a wide-open pupil during dark adaptation but did not further
test pupil light responsiveness or dilation/constriction rates. Similarly,
pupil dilation and visual field were studied in dogfish (Kajiura, 2010)
but no details regarding the full extent of dilation or constriction were
given. McComb and Kajiura (2008) and Kajiura (2010) anaesthetized
their animals using MS222 to facilitate handling and prevent animals
from moving during experimentation. Although the authors discuss
that anaesthesia may have negatively influenced the natural light
response of the batoids’eyes, they deemed it unlikely that the
anaesthetic dramatically affected the response. In contrast, Hove and
Moss (1997) report that MS222 does indeed disrupt the metabolism
associated with vision. Unfortunately, thorough studies are lacking,
but we can say with certainty that anaesthesia negatively impacts
behavioural and physiological studies, including visually related
physiological responses (e.g. Charng et al., 2013).
Received 22 July 2021; Accepted 17 January 2022
1
Marine Biological Laboratory, Bell Center, Woods Hole, MA 02543, USA.
2
Lund
Vision Group, Department of Biology, University of Lund, 223 62 Lund, Sweden.
3
Westphalian Institute for Biomimetics, Westphalian University of Applied Sciences,
Bocholt 43697, Germany.
4
Florida State University, Tallahassee, FL 32306, USA.
*Joint senior authors
‡
Author for correspondence (lmathger@mbl.edu)
M.J.B., 0000-0002-3169-8523; J.L., 0000-0002-9621-3777; D.-E.N., 0000-0003-
1028-9314
This is an Open Access article distributed under the terms of the Creative Commons Attribution
License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use,
distribution and reproduction in any medium provided that the original work is properly attributed.
1
© 2022. Published by The Company of Biologists Ltd
|
Journal of Experimental Biology (2022) 225, jeb243221. doi:10.1242/jeb.243221
Journal of Experimental Biology
Given the likelihood that drugs affect vision, the pupillary
response should ideally be studied in unmedicated animals.
Provided that skates were handled carefully, and given size-
appropriate tanks and sufficient acclimation time, we found that
skates voluntarily settled in our experimental tanks and remained
calm for experimental trials to be conducted without anaesthesia.
In live animal experimentation concerned with physiology and
behaviour, researchers are generally aware of the potential negative
effects of experimental/laboratory techniques on an animal’s
response. Studies of animal behaviour and/or physiological
responses are difficult and time consuming, requiring intense
planning, testing and trouble shooting of experimental methods, so
that results are accurate. Still, even the most carefully conducted
experiment may leave doubt whether a given method negatively
affects the very response one intends to quantify. One area that has
received scant attention is the ambient lighting in which animals are
kept, and in which experiments are conducted. Traditionally,
laboratories are equipped with white-light fluorescent, incandescent
or LED lighting, paying little attention to what an animal’s natural
light field may be. Similarly, experiments are usually designed around
the recording cameras: the brighter the light, the better the recording
quality. Luckily, with the advancement of technology, more and more
low-light sensitive and night-vision cameras have become available,
finally making high-wattage photographic lights for animal
behavioural work obsolete. In the present study, we have taken a
new sensory–ecological approach to performing a physiological
experiment in a freely behaving animal. Importantly, this new tool can
be applied to any animal behavioural experiment. We conducted a
field survey to obtain data on the natural light field of skates (L.
erinacea), and created an ambientlight field using recently developed
software (environmental light field, ELF; Nilsson and Smolka, 2021).
For our laboratory experiments, we then used these field data to re-
create, as best we could, a laboratory light field that closely simulated
the natural vertical light gradient that skates experience in the wild.
The advantage of using a tool of this kind is far reaching in that it
allows us to correlate real-world situations at different times of day
and different depths with the animal’spupilstate.
Using this improved sensory–ecological approach, the goal of our
study was to document the pupil dynamics of unrestrained and
unmedicated L. erinacea, under naturalistic lighting conditions. In
particular, we wanted to quantify visually important parameters such
as the extent of dilation and constriction and the speeds involved.
MATERIALS AND METHODS
Animals
The little skate Leucoraja erinacea (Mitchill 1825) (Fig. 1A) is
common off the coast of Woods Hole, MA, USA. Animals are
regularly collected, as well as raised in captivity at the Marine
Resources Center of the Marine Biological Laboratory (MBL). All
animals used were wild-caught (within less than a year). Skates were
kept at 12–15°C in a large holding tank (1.2 m×3.6 m; 40 cm
height) surrounded by large windows. During the weeks of these
experiments, the animals’dark cycle lasted from approximately
20:00 h to approximately 05:00 h. Skates were identified by sex and
length, as well as individual physical characteristics (e.g. identifying
scars, lines or spots, etc.), and fed 5 times a week (variation of squid,
butterfish and capelin). All animals were cared for and experiments
were conducted in accordance with the regulations of the MBL
Institutional Animal Care and Use Committees.
Natural ELF data
Using SCUBA, we collected light field data in the natural habitats
of L. erinacea from 18 to 20 October 2016 according to the ELF
method described by Nilsson and Smolka (2021).The data
collecting apparatus consisted of a SLR camera (Nikon D810),
equipped with a Sigma 8 mm f/3.5 EX DG circular fisheye lens,
placed in an underwater housing that was custom-made to allow the
full 180 deg field of view of the fisheye lens to be used (The Sexton
Corporation, Salem, OR, USA). We collected data at two typical
local skate habitats (all approximately 3–5 m depth): site 1 was a
rubble, sand and sargassum habitat off Stony Beach in Woods Hole,
MA, USA (41.516537, −70.659976); site 2 was a sandy habitat off
A
B
C
D
E
Fig. 1. The frill-like pupil of the little skate, Leucoraja erinacea. (A) Whole-
animal image, approximate length: 35 cm.(B–E) The light-responsive pupil
changes from near circular under low light to an elaborate shape under bright
light. (B) Lowest light setting, 0.00018 lx; (C) 0.12 lx; (D) 7 lx; (E) brightest light
setting, 500 lx. Scale bar in B–E: 5 mm.
2
RESEARCH ARTICLE Journal of Experimental Biology (2022) 225, jeb243221. doi:10.1242/jeb.243221
Journal of Experimental Biology
Nobska Beach in Woods Hole, MA, USA (41.529946,
−70.674377). Data were collected at four times throughout the
day: at solar noon, mid-afternoon, and just before and after sunset.
For all datasets, the weather was clear and sunny with few clouds.
Images were collected with the camera housing resting on the
substrate. Between 12 and 21 scenes were imaged at each site
depending on the structural visual complexity of the sites, each with
the camera levelled to the horizon. Sites without algae or rocks
required fewer scene imagesto create a homogenous average image.
Each scene was imaged at randomly selected horizontal directions
and utilized three bracketed exposures each to produce high
dynamic range images (Fig. 2A,B). The scenes imaged at each
site were then analysed according to the ELF method (Nilsson and
Smolka, 2021). Briefly, average images were generated for each set
of scenes (Fig. 2C). From these averaged images, calibrated spectral
photon radiance information was extracted along the vertical axis
from straight up (90 deg), through the horizon (0 deg), to straight
down (−90 deg) (Fig. 2D). These data gave us a powerful tool for re-
creating a natural light field in the laboratory.
Experimental set-up
Our experimental set-up (Fig. 3) consisted of a glass tank that was
placed inside an improvised dark room constructed from a black ice
fishing tent surrounded by black plastic sheeting to block out all
exterior light. The glass tank dimensions were: 61 cm length, 61 cm
width, 46 cm height. To give skates a more natural bottom, we
lowered a Plexiglas sheet onto the floor, which consisted of glued-
on natural sand and small pebbles (glued on using silicone
adhesive). The overhead light source consisted of three side-by-
side custom-made light boxes, lined with LE Flexible Strip, SMD
2835 Daylight White LEDs to provide even illumination across the
entire tank. The light boxes were constructed in such a way that
neutral density filters (a combination of 0.15 ND and 0.6 ND, Lee
Filters) could be inserted to decrease the light intensity to the desired
level. The outside of the experimental tank was lined with a white
diffuser (Lee Filters half white diffusion) as well as a blue–green
filter (Roscolux #374 sea green) (note: the ‘outside’refers to all four
tank sides and a tight-fitting lid that was placed on top of the tank
during experiments to completely ‘seal’the experimental tank,
except for a small opening for filming). Around the outside of the
experimental tank, secured to the inside of the ice fishing tent, we
placed sheets of 2 mm reflective Mylar (Virtual Sun Hydroponics,
Inc.), which effectively channelled light downwards and increased
the relative light intensity coming into the experimental tank from
the sides. Our goal was to design the experimental light field to
match the average natural light field characterized using the ELF
method as closely as possible (Fig. 2E,F). This took some time to
achieve, and filters, light sources and tank had to be adjusted
multiple times. During the set-up phase, an Extech EasyView 30
light meter (irradiance, measured in lux) was used to guide us in the
selection and placement of filters, locations of light sources, and
lighting intensities. This type of light meter is convenient because
15 16 17 0.5 1 1.5
log Spectral photon radiance
log Spectral photon radiance
Relative colour
Remap
16 randomly captured HDR scenes Average scene
Average scenes
Computed radiometric data
Average
Horizon Horizon Horizon
180 deg HDR
scene
Sand
bottom
Optimized
experimental
tank
Rock and
algae
bottom
Noon Mid-afternoon Sunset After sunset
AB C D
i
i
0
90
60
30
–30
–60
–90
0
90
60
30
–30
–60
–90
0 deg 180 degAzimuth
Elevation (deg)
141312 15 16 17 18
Horizon
FE
0
90
60
30
–30
–60
–90
Elevation (deg)
Example of a
standard
un-optimized tank
Elevation (deg)
Fig. 2. Environmental light field (ELF) analysis of natural L. erinacea habitats and laboratory set-ups. The ELF method uses 180 deg high dynamic range
(HDR) images taken in a specific natural habitat levelled to the horizon (A). Multiple individual scenes are captured at random orientations throughout the habitat.
They are remapped (B) and averaged (C) to create a mean profile of the vertical light gradient. From this, radiometric data (spectral photon radiance in
photons m
–2
s
−1
sr
−1
nm
−1
) are computed along the vertical light gradient (D). We used this method to analyse the natural habitats of skates at varioustimes of the
day and aid in the design of an optimized experimental tank (E,F). An example of an un-optimized tank set-up using a black and white checkerboard substrate is
given. Line formats (solid or dotted) and shades (greys, yellow, teal) of the radiometric plots in F refer to the averaged scenes in E as indicated. FullELF analysis
plots are available in Fig. S1.
3
RESEARCH ARTICLE Journal of Experimental Biology (2022) 225, jeb243221. doi:10.1242/jeb.243221
Journal of Experimental Biology
light data are immediately available and progress is not delayed by
the time-consuming computation that is often required for obtaining
radiometric light data. However, as lux meters are based on the
human visual system, they have drawbacks when used in the context
of animal vision. Despite this, the use of these instruments may be
justified when working with monochromatic animals (e.g. skates)
because lux meters are most sensitive in the green part of the
spectrum. In addition to using a lux meter, we also measured the
radiance inside the tank at different vertical angles using an Ocean
Optics QE65000 spectrometer (Ocean Optics, Dunedin, FL, USA)
and a 400 µm fibre (QP400-2-UV/VIS) with an attached Gershun
tube (Gershun tube kit, Ocean Optics). The fibre was attached to a
goniometer, which allowed measurements to be taken in 10 deg
steps. A calibration light source (LS-1-CAL, Ocean Optics) was
used to convert data into absolute radiance. Radiance data were
converted into photons m
−2
s
−1
sr
−1
nm
−1
. We then used the same
underwater camera system and ELF method described above to
analyse the light field of the experimental set-up. Final set-up
adjustments were made to obtain an experimental light field that fell
well into the types of light fields measured in the natural habitats of
skates (Fig. 2B).
Experimental procedure
Experiment 1: speed of dilation/constriction of skate pupil
For this experiment, 11 skates (body length 33–38 cm) were used.
All experiments were conducted between 08:00 h and 17:00 h (their
‘dark cycle’lasted from approximately 20:00 h to 05:00 h). Skates
were placed in the experimental tank at full light intensity (500 lx,
measured with the Extech EasyView 30 light meter, placed on the
bottom of the tank at the level of the skates’eyes), and given
approximately 30 min acclimation time. After acclimation, the
lights were turned off and the skate’s pupil dilation was filmed for
10–20 s every 3–5 min. When no further changes in pupil dilation
were observed, the light was turned on, and pupil constriction was
filmed for 10–20 s every 3–5 min until no further changes in pupil
constriction were observed. It was important to allow sufficient time
for the skate pupil to fully adjust. Skates were given approximately
5–10 min after the experimenter had decided that full constriction/
dilation had been achieved to ensure there were no further subtle
pupillary changes.
All video recording was done using a Sony HDR-XR550VEB
video camera, which was placed perpendicular to the skate’seye,so
that the entire pupillary area could be monitored. During dark
adaptation, the night-shot setting of the camera was used.
Skates generally settled quickly and positioned themselves in
such a way that one eye was perpendicular to the camera; however,
on the occasion that a skate shifted, we were able to move it gently to
position it perpendicular to the camera. Usually, the skates did not
react to being moved and remained settled. Occasionally, if a skate
did react by swimming around, it was given more acclimation time
until it was settled. The skates that were available to us had a range
of temperaments, and when we initially selected animals for these
trials, we avoided selecting animals that would not settle in the
experimental tank within an hour.
Analysis
From each video segment, a single image, showing the eye in a
position perpendicular to the camera, was extracted. For each
animal, we had 11–26 images for the entire constriction/dilation
speed trial. These images were then used to measure the effective
pupil area using ImageJ (NIH). We used the polygon selection tool
to trace the pupil margins, so that the effective pupil area could be
measured.
Experiment 2: response of skate pupil to changes in intensity
Using neutral density filters (0.6 ND and 0.15 ND), we created 10
light intensities. The highest light intensity, without any neutral
density filters, was 500 lx (less than 5% variation across the
experimental tank, verified by Extech EasyView 30 light meter).
The next lower light intensity was obtained by adding one 0.6 ND
filter, which reduced the light intensity to 125 lx. The next lower
light intensity was obtained by adding a second 0.6 ND filter, which
reduced the light intensity to 31.25 lx, and so on. The lowest light
intensity, 0.00018 lx, was obtained by adding eight 0.6 ND filters
and one 0.15 ND filter.
Before experiments started, all neutral density filters were placed
in front of the light sources, resulting in the lowest light intensity
(0.00018 lx). Filters were progressively removed to proceed to the
next brighter light field. As before, the video camera was placed
perpendicular to the skate’s eye. The night-shot setting of the
camera was used for all but the brightest light intensity setting.
Ten skates (body length 33–38 cm) were used for this experiment
(these were the same skates as used for experiment 1 but we lost one
skate; in the results and figures, these skates are referred to as group
1). Additionally, we found that a linear regression analysis was
useful in interpreting our data, so data from a pilot experiment were
added, referred to as group 2. Skates in group 1 were overall larger
and slightly older than the skates in group 2, although the sizes
overlapped to some degree. This added six smaller skates (body
length 25–33 cm) to the dataset. All methods were identical to
those described above; the only difference was the size of the
experimental tank (50 cm length, 40 cm width, 30 cm height) and a
slightly higher light intensity at the brighter light setting (at the
highest setting, a difference between 500 lx versus 700 lx), which
caused no noticeable difference in pupil constriction (note, the
reason for the slight discrepancy in the set-up was that larger
animals needed a larger tank, which also meant a larger light source
had to be employed).
Light boxes
ND filter slot
Glass tank
Blue–green filter
Mylar sheet
White diffuser
*
* No filter here for filming
Fig. 3. Experimental set-up. The set-up that was designed and built to
simulate the natural light field of L. erinacea. An ice fishing tent (indicated by
the dark grey box surrounding the tank) was used to house a glass tank,
surrounded by blue–green and white diffuser filters. Reflective Mylar material
was placed along the sides of the tank. At the front of the tank, the filters were
lifted to provide a small window for filming. See Materials and Methods for more
details.
4
RESEARCH ARTICLE Journal of Experimental Biology (2022) 225, jeb243221. doi:10.1242/jeb.243221
Journal of Experimental Biology
Each skate was taken out of its holding tank and placed in the
experimental tank, where it was given approximately 30 min
adaptation time. During this time, the pupil dilation process
was monitored, and experiments began when full dilation had
been reached and no further dilation movements were detected.
When full dilation was reached, the skate was filmed for
approximately 1 min. We then removed neutral density filters to
proceed to the next brighter light setting and allowed another 30 min
adaptation, during which the pupil dilation was recorded as
described before. After 30 min, the skate was again video-
recorded for approximately 1 min. This procedure was repeated
for each light intensity.
Analysis
As before, only images showing the eye in a position perpendicular
to the camera were selected. From the 1 min video clips taken
at each light intensity, we extracted 4 images at intervals of
approximately 5 s. The pupil margin and effective pupil area
were determined in the same way as described for experiment
1. Data obtained for the four images were averaged. In the
Results, we show dilation/constriction of all 10 skates. Additionally,
to test the relationship between eye size and dilation/constriction
state, we used Microsoft Excel to perform a linear regression
analysis.
RESULTS
ELF analysis of skate habitats
We performed ELF analysis at two natural study sites of L. erinacea
to develop the optimal light conditions for our experimental use. In
Fig. 2, we show the steps that led to our final dataset. Individual
images were first re-mapped to correct for the camera’s equisolid
image projection. The resulting images were square, consisting of a
new array of pixels with equal angular span in the vertical and
horizontal fields of view. Using these images, pixels were averaged
(Fig. 2C), revealing a vertical gradient profile, from which
radiometric data were computed (Fig. 2D). As every natural light
field undergoes circadian changes owing to the position of the sun/
moon, cloud cover, etc., we collected data at different times of day
(Fig. 2E,F; Fig. S1). We found that vertical intensity gradients of
light in both natural habitats presented a similar profile that
decreased in intensity uniformly from noon to sunset, with only
some subtle angular changes. Snell’s window, which occupies
angles of elevation between 41.5 and 90 deg, provided the brightest
light (above 17 log
10
spectral photon radiance units at noon in our
sandy bottom site). Beyond Snell’s window, the intensity decreased
sharply towards the horizon. Below the horizon, the intensity
continued to decrease gradually, which is partially caused by the
camera’s shadow. Green and blue wavelengths dominated above the
horizon but decreased below the horizon, while red wavelengths
increased slightly (Fig. 2D; Fig. S1).
With these data in mind, we set out to create an artificial light field
that simulated the natural light field as closely as possible. We
made use of a variety of filters (see Materials and Methods) and
repeatedly adjusted these, as well as the overhead light sources,
until we succeeded in obtaining a light field with an intensity and
relative colour gradient similar to the ELF averages (teal line in
Fig. 2F; Fig. S1). Importantly, it became obvious that the use of
artificial backgrounds, which are often employed in behavioural
experimentation because of the ease with which spatial frequency
and intensity can be modulated, created an artificial light field
that did not resemble the natural light field at all (yellow line in
Fig. 2E,F; Fig. S1).
Speed of dilation/constriction of skate pupil
Dilation took longer than constriction, and there was individual
variation in the time it took for the skates’pupils to fully dilate and
constrict (Fig. 4). For all skates, dilation took from 30 min to over
50 min; constriction was usually achieved in under 25 min (Fig. 4,
Table 1). Interestingly, in the first 8–10 min following the onset of
dark adaptation (i.e. after lights were turned off ), there was very
little dilation. Most of the dilation occurred after approximately
10 min, and approximately 5 min before full dilation was achieved.
Conversely, during light adaptation (i.e. after lights were turned on),
most constriction occurred in the first 10 min; within the final
minutes, constriction was slower. As Table 1 shows, pupil
constriction area was relatively similar across all animals
(mean±s.d. 1.3±0.37 mm
2
, range 0.8–1.7 mm
2
). Dilation area
varied more (mean±s.d. 16.3±4.56 mm
2
, or 17.4±2.88 mm
2
for the
three skates that were tested a second time).
Response of skate pupil to changes in intensity
There was individual variation in the degree to which the pupil
constricted in response to light but all skates followed the same
pattern: during the darkest light setting, the pupil took on an almost
circular shape; as light intensity increased, the pupil became more
elaborate: the frilled dorsal portion lengthened from dorsal to
ventral, resulting in a crescent shape with frilled elongations
dropping down from the top of the eye. At the highest light intensity,
the resulting series of small triangular apertures (e.g. Fig. 1E)
caused a drastic reduction in effective pupil area (Fig. 5A). Notably,
the overall pupil area (dilated and constricted) of smaller (and
younger) skates (mostly in group 2) was smaller than that of larger
(and older) skates (mostly in group 1). To determine the extent of the
pupil area change (dilation:constriction ratio), we combined the data
shown in Fig. 5A in a linear regression analysis (see Fig. 5B).
Looking at this combined dataset, we found that skates with smaller
eyes (i.e. younger skates) achieved a smaller dilation:constriction
range (i.e. the area change from completely dilated to completely
constricted was lower) compared with that of skates with larger eyes
(i.e. older skates). The constriction state, however, was similar
across all animals, irrespective of size: When constricted, the mean
(±s.d.) pupil area was 2.36±1.44 mm
2
. However, dilation state
varied dramatically: when fully dilated, the average pupil area was
18.69±5.96 mm
2
(note the much higher standard deviation,
indicating a larger variation). This shows clearly that, as the eye
Max. constriction
Max. dilation
1.00
0.75
Normalized pupil area
0.50
0.25
0
0 1020304050
Time (min)
60 70 80 90
Fig. 4. Dilation and constriction speeds for 11 skates (L. erinacea). For
comparative reasons, data were normalized; i.e. each skate’s maximum
dilation area was given a value of 1. In the initial 8–10 min, little dilation was
observed. Most dilation occurred after 10 min following the onset of dark
adaptation. There was considerable variation in dilation time between skates.
Constriction was generally faster; it was achieved in under 20 min. At time 0,
lights were turned off. Lights were turned on at different times for each skate,
determined by when maximum dilation was achieved.
5
RESEARCH ARTICLE Journal of Experimental Biology (2022) 225, jeb243221. doi:10.1242/jeb.243221
Journal of Experimental Biology
grows, skates are able to dilate their pupils more. The linear
regression analysis that tested the relationship between eye size
(here, we used eye length as an indicator of eye size) and
dilation:constriction ratio showed that dilation:constriction
ratios were smaller in smaller skates and greater in larger skates.
Although not statistically significant, the regression equation
(F
1,13
=4.33, P=0.057, with an R
2
of 0.2498; see Fig. 5B)
demonstrated a trend.
DISCUSSION
It is important to provide experimental settings that allow natural
behaviours to ensue as much as possible. Field studies are
Table 1. Dilation and constriction times and corresponding pupil area for 11 skates (Leucoraja erinacea)
Skate ID
Eye length
(mm)
Dilation time
(min)
Dilation area
(mm
2
)
Constriction time
(min)
Constriction area
(mm
2
)
Dilation:constriction area
ratio
M1 11.8 35.3 18 16.1 0.8 22.5
M2 10.2 43.5 21 22.0 1.0 21
M3 11.0 54.0 16.6 22.5 0.9 18.4
M4 10.3 49.5 16.1 21.25 1.9 8.5
M5 9.6 38.0 13.6 23 1.2 11.3
M6 11.0 28.0 24.9 11.5 1.6 15.6
M7 10.9 49.5 22.9 23.5 1.7 13.5
F2 8.7 44.5 15.9 16.0 1.0 15.9
F3 9.7 28.3 17.1 10.2 1.7 10
F4 8.0 31.3 11.9 20.5 1.3 9.2
F5 9.2 40.2 15.2 12.2 1.25 12.2
Average 40.2 17.6 18.1 1.3 14.4
M, male; F, female. See also Fig. 4. Note, these are the same animals that were used in the intensity experiment (experiment 2; Fig. 5A).
A
B
35
30
25
Pupil area (mm2)
Dilation:constriction ratio
20
15
10
5
0
25
20
15
10
5
0
678910
Eye length (mm)
11 12 13
0.0001 0.001 0.01 0.1
Light intensity (lx)
1 10 100 1000
y=1.6194x–5.4809
R2=0.2498
Fig. 5. Changes in skate pupil area under different light
intensities. (A) Response of 10 larger/older skates (black
lines; group 1, 33–38 cm body length) and six smaller/
younger skates (grey lines; group 2, 25–33 cm body length)
to 10 light settings inside an experimental set-up that
simulated a natural underwater light environment. The x-axis
is presented on a log scale (note, lux values are given for
ease of replicating this set-up; the equivalent radiometric
value at maximum light intensity, measured with Ocean
Optics equipment, was around
2e+17 photons m
−2
s
−1
sr
−1
nm
−1
). (B) The ratio of dilation to
constriction (maximum:minimum dilation) for skates of
different sizes from group 1 (black circles) and group 2 (grey
circles). A linear regression analysis shows that skates with
smaller eyes (i.e. younger skates) achieved a smaller
dilation:constriction range compared with skates with larger
eyes (i.e. older skates); n=15. One outlier (with a value of 1.5
times the interquartile range) was removed from this
analysis. The regression equation was not statistically
significant (F
1,13
=4.33, P=0.057); however, there was a clear
trend.
6
RESEARCH ARTICLE Journal of Experimental Biology (2022) 225, jeb243221. doi:10.1242/jeb.243221
Journal of Experimental Biology
challenging owing to a huge array of variables that are impossible to
control. This often makes the laboratory the only place where natural
behaviours can be quantified. When conducting behavioural
experiments in the laboratory, it is important to consider the
effects of the laboratory environment (e.g. artificial tanks, lighting,
etc.) on the outcome of the behaviours that are under investigation.
Conventionally, researchers choose methods that optimize and
maximize data collection, e.g. sufficient high-wattage lighting for
clear video recording; small experimental chambers, harnesses or
anaesthesia to prevent unwanted movement, etc. It is difficult to
determine the potential negative impact of such methods on the
behaviour that is being quantified, and is often impossible to design
control experiments to test these potential effects. Here, we show
how a comparatively simple method introduced by Nilsson and
Smolka (2021) yields data on natural light fields that can
subsequently guide laboratory experimental methods much better
than lux meters or spectrometers, which do not take into account the
structures and objects that a given visual scene contains. While the
light data obtained by lux meters/spectrometers can be useful in
guiding an experimental design, ultimately, we found that the ELF
method provided us with the most accurate tool for simulating a
natural light field in the laboratory.
Interestingly, while the skate L. erinacea has been a study
organism for a variety of research topics (visual and non-visual)
(Dowling and Ripps, 1970, 1972, 1990; Koester and Spirito,
2003; Gillis et al., 2013), the extent and rate of pupillary movement
have not been documented. In our study, we aimed to give
skates a laboratory setting that was as natural as possible, so that we
could make inferences about their natural pupillary responses to
light.
Not all fish have light-sensitive pupils. Although pupil mobility
has been documented in several teleost species, the pupils of most
teleost fish do not vary in response to ambient light changes
(Nilsson, 1980; Somiya, 1987; Fujimoto et al., 1995; Douglas,
2018; Douglas et al., 1998, 2002). Within the elasmobranchs, there
appears to be a similarly diverse distribution of mobile pupils (Beer,
1894; Franz, 1906, 1931; Young, 1933; Walls, 1963; Kuchnow,
1971; Collin, 1988; Nicol, 1989; Douglas, 2018). Kuchnow (1971)
performed an investigation of a range of elasmobranch species,
testing members of three selachian orders (Carcharhiniformes,
Heterodontiformes, Squaliformes) and two batoid orders
(Myliobatiformes, Rajiformes), and found mobile pupils in all but
the deep-sea species (two spp. from the Squaliformes order). In the
species Kuchnow (1971) worked on, the dilation/constriction ratios
ranged from 1.2:1 to 50:1, demonstrating clearly the wide variety of
pupil area changes in this animal group (see Table 1 for dilation:
constriction ratios in L. erinacea). Pupil dilation:constriction speed
also varied: some dilated and constricted in a matter of a minute or
two, whereas others took half an hour or longer. The low sample
size in Kuchnow’s (1971) study makes it difficult to gain a more
solid understanding of dilation and constriction extent and speed.
Another important aspect to consider is that low-light video
equipment has improved enormously since the 1970s and it is likely
that Kuchnow (1971) would not have been able to track head
movements as carefully as is possible now. Thus, the measured
pupil areas in Kuchnow’s (1971) work may not be as exact as
necessary to fully characterize these pupil movements. Some of
Kuchnow’s (1971) data were obtained during cruises, making
steady experimental conditions difficult.
Because of the large sizes of many elasmobranch species,
studying their pupil light responses is a challenge. Furthermore,
pelagic species (e.g. selachians) are even more difficult to study than
benthic batoids, and it is therefore not surprising that the current
literature on pupil mobility in elasmobranchs is so patchy. Clearly,
there has been a need for more work in this area, particularly with
regard to the diversity of body and head shapes, as well as lifestyles
of elasmobranchs.
Our results undoubtedly show that light intensity causes
significant changes in the pupil area of L. erinacea, with the pupil
going from nearly circular to an elaborate crescent shape with
multiple triangular apertures. As we can see in Fig. 5B, this change
in area from constricted to dilated varied from nearly 4-fold in
smaller (and younger) animals to 10- and up to 20-fold in larger (and
older) animals (see also Table 1). While not statistically significant
in our study, a trend was seen. A larger sample size may have added
statistical significance to this finding.
An important consideration of this experiment is to ensure that
skates are given sufficient time for full dilation/constriction to
occur. Each animal has a different response time and data
inaccuracies can easily occur. In the experiments that are shown
in Table 1, several animals appeared initially to have completed the
full constriction/dilation cycle but upon data analysis we suspected
that the experimenter had determined too early that full dilation/
constriction had been reached. After repeating the trials for these
animals, it became apparent that these individuals did indeed need
more time (an additional 5–10 min) until full dilation/constriction
was achieved. Why individual skates (and possibly even the same
individual) may have different dilation and constriction times
remains to be investigated. This result could be due to numerous
factors, such as circadian rhythm, seasonal aspects (e.g. breeding,
etc.), hunger status, etc., none of which were controlled for in this
study. However, it can be stated with confidence that we can rule out
unintended light intensity variations, which were carefully
controlled for with our sensory–ecological approach.
Why pupil dilation and constriction are so slow in skates is an
interesting question that remains to be fully explored. Certainly, the
light intensities that these cathmeral (day and night active) skates are
exposed to on a daily basis do not change as fast or as drastically as
they do for some other aquatic species, or compared with terrestrial
organisms, suggesting that fast dilation and constriction are really
not needed. Sun exposure, cloud coverage, etc., have a considerable
impact on light intensity, both on land and underwater, and while
underwater light fields may not be subject to such changes as fast or
as drastically as terrestrial light fields, these changes are certainly
noticeable, particularly at shallow depths (Jerlov, 1976; Cronin and
Shashar, 2001). However, the most dramatic light intensity changes
are associated with an animal’s lifestyle. Moving in and out of
crevices, caves and other forms of light-reducing/eliminating
coverage can present light intensity differences of several orders
of magnitude. Skates do not have this kind of an active lifestyle.
They are benthic sit-and-wait predators that prefer open sandy sea
floors, near shore to depths of about 90 m (Robins and Ray, 1986).
From that perspective, skates are unlikely to experience light
intensity changes beyond the natural circadian changes and those
resulting from clouds covering the sun.
Interestingly, at the morphological level, the skate retina contains
only rods, which, in general, are more light sensitive than cones but
they do not function during photopic light intensities. Skate rods
have solved this problem by taking on cone-like physiological
properties under bright light (Dowling and Ripps, 1970), which
supports the skate’s cathemeral lifestyle. Additionally, their
temporal response times are slower compared with those of other
species (Dowling, 2012); thus, to support a cathemeral existence,
skates may not need a fast-acting pupil.
7
RESEARCH ARTICLE Journal of Experimental Biology (2022) 225, jeb243221. doi:10.1242/jeb.243221
Journal of Experimental Biology
Surprisingly, we found that larger skates had greater pupil
dilation:constriction ratios than younger animals. The pupil area at
constriction was relatively similar across all skate sizes/ages;
however, the dilated pupil area was greater in larger (i.e. older)
skates. To the best of our knowledge, this has so far not been
observed in any animal. Interestingly, in humans, pupil size changes
with age: it is smaller in younger children, maximum at an age of
around 20, but then decreases with age to be smaller than that of a
young child, which in turn reduces the amount of light that reaches
the retina (Weale, 1961; Winn et al., 1994). While we used skates of
different size ranges in our study, we cannot make any inferences
regarding age beyond suggesting that the smaller animals were
likely younger, based on their husbandry/collection history. A
carefully controlled study on different skate age groups, including
hatchlings, juveniles and late adult stages, would be extremely
interesting. The reason for the variation in the dilation:constriction
ratios remains to be investigated. There are obvious visual
advantages to a larger pupil size, such as increased sensitivity in
low light, although a large pupil size comes at a potential cost in that
it may contribute to optical aberrations, which result in poorer
resolution. Future studies should look at the interactions between
lifestyle and ontogeny (retinal development, as well as overall body
design) to determine the reason for a larger dilation:constriction
ratio in older skates.
It is unclear why the speed of dilation and constriction in skates
varied so much between individuals. In cephalopods, a similar
variation has been reported (Douglas et al., 2005; Soto et al., 2020),
although the cephalopod pupillary light reflex is much faster than
that in skates, and also responds to stimuli other than light (e.g.
during accommodation for hunting, as well as mating; Muntz, 1977;
Messenger, 1981), which can confound light-induced dilation and
constriction speeds. In vertebrates, there are several species for
which non-light-induced pupillary movements have been described.
For example, in humans, pupil dilation is affected by many factors,
including hormones, emotions and behaviours, as well as visual and
auditory perception, and it has been shown that the human pupillary
light response is neither fully reflexive nor under complete
voluntary control (Lowenfeld, 1993; Barbur et al., 2002;
Einhäuser et al., 2007; Mathôt et al., 2013). In birds, pupil
movements have been called ‘eye pinning’, and it has been
suggested that they are used as a form of communication (Gregory
and Hopkins, 1974). All of these non-light-induced pupillary
changes are fast and subtle, and reflect a particular short-term
physiological condition or have a particular short-term optical
function. In our study, we did not test any factors, other than light
intensity, that influence pupil size in skates. Furthermore, as
pupillary movement is so slow in skates, it seems doubtful that
skates have the ability to vary their pupil dilation/constriction to the
degree that is reported in cephalopod or human subjects. It therefore
seems unlikely that the variation in individual dilation/constriction
speed reported here is due to non-light-related factors. However, in a
separate study (Youn et al., 2019), we found that the skate pupil also
changes in response to the spatial frequency of the background, so
skates certainly appear to have the ability to change pupil dilation/
constriction irrespective of light intensity, although at a much
slower rate.
Another aspect that adds complexity to the pupillary response in
skates is the connection between the two eyes. In many animals, the
pupil response of each eye is independent of the other eye [e.g. some
teleost fish (Steinach, 1890; Young, 1931; Nilsson, 1980); some
sharks (Franz, 1931; von Studnitz, 1933; Kuchnow, 1971); some
amphibians, reptiles and birds (Denton, 1956; Werner, 1972;
Henning et al., 1991; Schaeffel and Wagner, 1992)], but there are
also many species that have consensual response; that is, one eye
responds when the other eye is stimulated [e.g. some teleosts
(Douglas et al., 1998); some rays (Bateson, 1890; Franz, 1931; von
Studnitz, 1933); some amphibians (von Campenhausen, 1963); and
probably most mammals, including humans (Lowenfeld, 1993)].
According to Bateson (1890) and Franz (1931), skates (including L.
erinacea) have a consensual pupillary reflex. Therefore, when
studying animals that have a consensual pupillary light reflex, this is
an added challenge that should be considered, and would be
interesting to investigate in these skates.
Although beyond the scope of this study, the functional reasons
for the elaborate pupil shape found in many skates deserves some
comment. The circular shape of the fully dilated pupil makes perfect
sense for maximizing visual sensitivity in dim light because it
exploits the full aperture of the lens over the full visual field.
Certainly, large eyes and circular pupils are common in nocturnal
animals (Land and Nilsson, 2012). The adaptive value of the
constricted pupil with multiple triangular pinholes is more obscure.
The row of small apertures will generate specific blur patterns for
objects that are out of focus because they are too close or too far
away to be sharply imaged on the retina. This may be used as a
mechanism for distance estimation. Another possibility is that wave-
optics phenomena in the row of minute apertures reduces diffraction
blur to improve the detection of high spatial frequencies in bright
light. It is also possible that the elaborate pupil contributes to even
out retinal illumination, as was demonstrated for the elaborate pupil
of cuttlefish (Mäthger et al., 2013). A role in camouflaging
conspicuous eyes can of course not be ruled out. It is likely that
several of these functional reasons have contributed to the evolution
of elaborate pupils in skates.
Acknowledgements
We would like to thank the staff of the Marine Resources Center at the MBL for
their assistance. Furthermore, we would like to thank Cynthia Tedore and Alan
Kuzirian for valuable input, and Bill Grossman, Sean Youn and Corey Okinaka for
assistance. Co-authors J.L. and L.S. were undergraduate students at the time of this
project.
Competing interests
The authors declare no competing or financial interests.
Author contributions
Conceptualization: L.M.M., D.-E.N.; Methodology: L.M.M., M.J.B., D.-E.N.;
Software: M.J.B., D.-E.N..; Validation: L.M.M., M.J.B., D.-E.N.; Formal analysis:
L.M.M., M.J.B., J.L., D.-E.N.; Investigation: L.M.M., M.J.B., J.L., L.S., D.-E.N.;
Resources: L.M.M., D.-E.N.; Data curation: L.M.M., M.J.B., J.L., L.S., D.-E.N.;
Writing - original draft: L.M.M., M.J.B.; Writing - review & editing: L.M.M., M.J.B., J.L.,
L.S., D.-E.N.; Visualization: L.M.M., M.J.B., D.-E.N.; Supervision: L.M.M.; Project
administration: L.M.M.; Funding acquisition: L.M.M., D.-E.N.
Funding
This work was funded by a Joan Ruderman Award from the Marine Biological
Laboratory to L.M.M. and a National Science Foundation REU fellowship to L.S.
M.J.B. and D.-E.N. were supported by grants from the Human Frontier Science
Program and the Swedish Research Council (Vetenskapsrådet). Open access
funding provided by Marine Biological Laboratory. Deposited in PMC for immediate
release.
References
Barbur, J. L., Prescott, N. B., Douglas, R. H., Jarvis, J. R. and Wathes, C. M.
(2002). A comparative study of stimulus-specific pupil responses in the domestic
fowl (Gallus gallus domesticus) and the human. Vision Res. 42, 249-255.
doi:10.1016/S0042-6989(01)00279-6
Bateson, W. (1890). Contractility of the iris in fishes and cephalopods. J. Mar. Biol.
Ass. UK 1, 225-256. doi:10.1017/S0025315400072118
Beer, T. (1894). Die Akkommodation des Fischauges. Pflu
g. Arch. Gesammt.
Physiol. 58, 523-650. doi:10.1007/BF01671307
8
RESEARCH ARTICLE Journal of Experimental Biology (2022) 225, jeb243221. doi:10.1242/jeb.243221
Journal of Experimental Biology
Charng, J., Nguyen, C. T., He, Z., Dang, T. M., Vingrys, A. J., Fish, R. L.,
Gurrell, R., Brain, P. and Bui, B. V. (2013). Conscious wireless electroretinogram
and visual evoked potentials in rats. PLoS ONE 8, e74172. doi:10.1371/journal.
pone.0074172
Collin, S. P. (1988). The retina of the shovel-nose ray, Rhinobatos batillum
(Rhinobatidae): morphology and quantitative analysis of the ganglion, amacrine
and bipolar cell populations. Exp. Biol. 47, 195-207.
Cronin, T. W. and Shashar, N. (2001). The linearly polarized light field in clear,
tropical marine waters: spatial and temporal variation of light intensity, degree of
polarization and e-vector angle. J. Exp. Biol. 204, 2461-2467. doi:10.1242/jeb.
204.14.2461
Denton, E. J. (1956). The responses of the pupil of Gekko gekko to external light
stimulus. J. Gen. Physiol. 40, 201-216. doi:10.1085/jgp.40.2.201
Douglas, R. H. (2018). The pupillary light responses of animals; a review of their
distribution, dynamics, mechanisms and functions. Prog. Retin. Eye Res. 66,
17-48. doi:10.1016/j.preteyeres.2018.04.005
Douglas, R. H., Harper, R. D. and Case, J. F. (1998). The pupil response of a
teleost fish, Porichthys notatus: description and comparison to other species.
Vision Res. 38, 2697-2710. doi:10.1016/S0042-6989(98)00021-2
Douglas, R. H., Collin, S. P. and Corrigan, J. (2002). The eyes of suckermouth
armoured catfish (Loricariidae, subfamily Hypostomus): pupil response, lenticular
longitudinal spherical aberration and retinal topography. J. Exp. Biol. 205,
3425-3433. doi:10.1242/jeb.205.22.3425
Douglas, R. H., Williamson, R. and Wagner, H.-J. (2005). The pupillary response
of cephalopods. J. Exp. Biol. 208, 261-265. doi:10.1242/jeb.01395
Dowling, J. E. (2012). The Retina. The Belknap Press of Harvard University Press.
Dowling, J. E. and Ripps, H. (1970). Visual adaptation in the retina of the skate.
J. Gen. Physiol. 56, 491-520. doi:10.1085/jgp.56.4.491
Dowling, J. E. and Ripps, H. (1972). Adaptations in skate photoreceptors. J. Gen.
Physiol. 60, 698-719. doi:10.1085/jgp.60.6.698
Dowling, J. E. and Ripps, H. (1990). On the duplex nature of the skate retina.
J. Exp. Zool. 256, 55-65. doi:10.1002/jez.1402560509
Einha
user, W., Stout, J., Koch, C. and Carter, O. (2007). Pupil dilation reflects
perceptual selection and predicts subsequent stability in perceptual rivalry. Proc.
Natl. Acad. Sci. USA 105, 1704-1709. doi:10.1073/pnas.0707727105
Franz, V. (1906). Beobachtungen am lebenden Selachierauge. Jenaische
Z. Naturwiss. 41, 429-471.
Franz, V. (1931). Die Akkommodation des Selachierauges und seine
Abblendungsapparate, nebst Befunden an der Retina. Zool. Jahrb. Abt. Allg.
Zool. Physiol. Tiere 49, 323-462.
Fujimoto, M., Kunioka, H. and Katayama, H. (1995). Pharmacology of the retractor
lentis muscle of the bluegill (Lepomis macrochirus). J. Comp. Physiol. A 176,
673-677. doi:10.1007/BF01021587
Gillis, J. A., Modrell, M. S. and Baker, C. V. H. (2013). Developmental evidence for
serial homology of the vertebrate jaw and gill arch skeleton. Nat. Commun. 4,
1436. doi:10.1038/ncomms2429
Green, D. G. and Siegel, I. M. (1975). Double branched flicker fusion curves from
the all-rod skate retina. Science 188, 1120-1122. doi:10.1126/science.1215989
Gregory, R. and Hopkins, P. (1974). Pupils of a talking parrot. Nature 252, 637-638.
doi:10.1038/252637a0
Hammond, P. and Mouat, G. S. V. (1985). The relationship between feline pupil
size and luminance. Exp. Brain Res. 59, 485-490. doi:10.1007/BF00261338
Henning, J., Henning, P. A. and Himstedt, W. (1991). Peripheral and central
contribution to the pupillary reflex control in amphibians: pupillographic and
theoretical considerations. Biol. Cybernet. 64, 511-518. doi:10.1007/BF00202616
Hove, J. R. and Moss, S. A. (1997). Effect of MS-222 on response to light and rateof
metabolism of the little skate Raja erinacea.Mar. Biol. 128, 579-583. doi:10.1007/
s002270050124
Jerlov, N. G. (1976). Marine Optics. Amsterdam: Elsevier.
Kajiura, S. M. (2010). Pupil dilation and visual field in the piked dogfish, Squalus
acanthias.Environ. Biol. Fishes 88, 133-141. doi:10.1007/s10641-010-9623-z
Koester, D. M. and Spirito, C. P. (2003). Punting: an unusual mode of locomotion in
the little skate, Leucoraja erinacea (Chondrichthyes: Rajidae). Copeia 2003,
553-561. doi:10.1643/CG-02-153R1
Kuchnow, K. P. (1971). The elasmobranch pupillary response. Vision Res. 11,
1395-1406. doi:10.1016/0042-6989(71)90062-9
Land, M. F. and Nilsson, D.-E. (2012). Animal Eyes. Oxford: Oxford University
Press.
Lind, O. E., Kelber, A. and Kro
ger, R. H. H. (2008). Multifocal optical systems and
pupil dynamics in birds. J. Exp. Biol. 211, 2752-2758. doi:10.1242/jeb.018630
Lowenfeld, I. E. (1993). The Pupil: Anatomy, Physiology and Clinical Applications.
Detroit: Wayne State University Press.
Malmstro
m, T. and Kro
ger, R. H. H. (2006). Pupil shapes and lens optics in the
eyes of terrestrial vertebrates. J. Exp. Biol. 209, 18-25. doi:10.1242/jeb.01959
Ma
thger, L. M., Hanlon, R. T., Håkansson, J. and Nilsson, D.-E. (2013). The W-
shaped pupil in cuttlefish (Sepia officinalis): a function in reducing vertically
uneven illumination. Vision Res. 83, 19-24. doi:10.1016/j.visres.2013.02.016
Matho
t, S., van der Linden, L., Grainger, J. and Vitu, F. (2013). The pupillary light
response reveals the focus of covert visual attention. PLoS ONE 8, e78168.
doi:10.1371/journal.pone.0078168
McComb, D. M. and Kajiura, S. M. (2008). Visual fields of four batoid fishes: a
comparative study. J. Exp. Biol. 211, 482-490. doi:10.1242/jeb.014506
Messenger, J. B. (1981). Comparative physiology of vision in molluscs. In
Comparative Physiology and Evolution of Vision in Invertebrates. C:
Invertebrate Visual Centers and Behavior II (ed. H. Autrum), pp. 93-200.
Springer Verlag.
Muntz, W. R. A. (1977). Pupillary response of cephalopods. Symp. Zool. Soc. Lond.
38, 277-285.
Murphy, C. J. and Howland, H. C. (1991). The functional significance of crescent-
shaped pupils and multiple pupillary apertures. J. Exp. Zool. 5, 22-28.
doi:10.1002/jez.1402560505
Nicol, J. A. C. (1989). The Eyes of Fishes. Oxford: Oxford University Press.
Nilsson, S. (1980). Sympathetic nervous control of the iris sphincter of the Atlantic
cod, Gadus morhua.J. Comp. Physiol. A 138, 149-155. doi:10.1007/BF00680438
Nilsson, D.-E. and Smolka, J. (2021). Quantifying biologically essential aspects of
environmental light. J. R. Soc. Interface 18. doi:10.1098/rsif.2021.0184
Nilsson, D.-E., Gislen, L., Coates, M. M., Skogh, C. and Garm, A. (2005).
Advanced optics in a jellyfish eye. Nature 435, 201-205. doi:10.1038/nature03484
Robins, C. R. and Ray, G. C. (1986). A Field Guide to Atlantic Coast Fishes of North
America. Boston: Houghton Mifflin Company.
Roth, L. S. V., Lundstro
m, L., Kelber, A. and Kro
ger, R. H. H. (2009). The pupils
and optical systems of gecko eyes. J. Vis. 9, 27. doi:10.1167/9.3.27
Schaeffel, F. and Wagner, H. (1992). Barn Owls have symmetrical accommodation
in both eyes, but independent pupillary responses to light. Vision Res. 32,
1149-1155. doi:10.1016/0042-6989(92)90016-C
Somiya, H. (1987). Dynamic mechanism of the visual accommodation in teleosts:
structure of the lens muscle and its nerve control. Proc. R. Soc. B 230, 77-91.
doi:10.1098/rspb.1987.0010
Soto, C., Kelber, A. and Hanke, F. D. (2020). The pupillary response of the
common octopus (Octopus vulgaris). Front. Physiol. 11, 1112. doi:10.3389/fphys.
2020.01112
Steinach, E. (1890). Untersuchungen zur vergleichenden Physiologie der Iris.
Pflu
g. Arch. Gesammt. Physiol. 47, 289-340. doi:10.1007/BF01789819
von Campenhausen, C. (1963). Quantitative Beziehungen zwischen Lichtreiz und
Kontraktion des Musculus sphincter pupillae vom Scheibenzu
ngler (Discoglossus
pictus). Kybernetik 1, 249-267.
von Studnitz, G. (1933). Studien zur vergleichenden Physiologie der Iris. III.
Selachier. Z. Vgl. Physiol. 19, 619-631. doi:10.1007/BF00395644
Walls, G. L. (1963). The Vertebrate Eye and Its Adaptive Radiation. New York:
Hafner Publishing Co.
Weale, R. A. (1961). Retinal illumination and age. Trans. Illum. Eng. Soc. 26,
95-100. doi:10.1177/147715356102600204
Wearmouth, V. J. and Sims, D. W. (2009). Movement and behaviour patterns of the
critically endangered common skate Dipturus batis revealed by electronic tagging.
J. Exp. Mar. Bio. Ecol. 380, 77-87. doi:10.1016/j.jembe.2009.07.035
Werner, D. (1972). Beobachtungen an Ptyodactylus hasselquistii guttatus
(Geckonidae). Verh Naturforsch Ges Basel 82, 54-87.
Wilcox, J. G. and Barlow, H. B. (1975). The size and shape of the pupil in lightly
anaesthetized cats as a function of luminance. Vision Res. 15, 1363-1365.
doi:10.1016/0042-6989(75)90191-1
Winn, B., Whitaker, S., Elliott, D. B. and Phillips, N. J. (1994). Factors affecting
light-adapted pupil size in normal human subjects. Invest. Ophthalmol. Vis. Sci.
35, 1132-1137.
Youn, S., Okinaka, C. and Ma
thger, L. M. (2019). Elaborate pupils in skates may
help camouflage the eye. J. Exp. Biol. 222, jeb195966. doi:10.1242/jeb.195966
Young, J. Z. (1931). The pupillary mechanism of the teleostean fish, Uranoscopus
scaber.Proc. R. Soc. B 107, 464-485. doi:10.1098/rspb.1931.0009
Young, J. Z. (1933). Comparative studies of the physiology of the iris. I. Selachians.
Proc. R. Soc. B 112, 228-241. doi:10.1098/rspb.1933.0006
9
RESEARCH ARTICLE Journal of Experimental Biology (2022) 225, jeb243221. doi:10.1242/jeb.243221
Journal of Experimental Biology
