Occlusable corneas in toadfishes: light transmission, movement and ultrastruture of pigment during light- and dark-adaptation
School of Biomedical Sciences, University of Queensland, Brisbane QLD 4072 Australia
* Author for correspondence (e-mail: u.siebeck{at}uq.edu.au)
Accepted 24 March 2003
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Summary |
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Key words: occlusable cornea, ocular media transmission, ultrastructure, pigment movement, chromatophores, toadfish, Torquigener pleurogramma, Tetractenos hamiltoni
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Introduction |
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The cells containing the coloured pigment are specialised chromatophores.
These chromatophores consist of a cell body situated in the periphery and a
single long process that extends into the centre of the cornea
(Gnyubkina and Levin, 1987).
The width of these processes has not been investigated. In some fish, these
corneal colouration cells (CCCs;
Gamburtseva et al., 1980
), or
corneal staining cells (CSCs; Kondrashev
and Khodtsev, 1984
), contain two different pigment types of either
yellow or orange appearance (Orlov and
Gamburtseva, 1976
). Both types of pigment are present in the
cornea of Hexagrammos octogrammus, and the yellow pigment differs
from the orange pigment in its response to illumination. When the dark-adapted
fish is exposed to light, the yellow pigment expands first, and does so
irrespective of light composition. The orange pigment, on the other hand,
migrates more slowly and does not disperse when illuminated in the
420560 nm waveband (Kondrashev and
Khodtsev, 1984
).
Three different pigmentation patterns have been described
(Gamburtseva et al., 1980). In
most fishes with occlusable corneas, the cell bodies of the pigment cells are
found in two sickle-shaped aggregations located in the dorsal and ventral
periphery (e.g. in Hexagrammos octogrammus;
Orlov and Gamburtseva, 1976
).
During light-adaptation, the pigment is shifted from the cell bodies into the
processes, which extend into the centre of the cornea, so that after 12
h the entire cornea is covered with pigment. In other species, the pigment
cell bodies are found mainly dorsally, where they form one sickle-shaped
reservoir (e.g. in Chirolophis japonicus;
Gamburtseva et al., 1980
).
During light-adaptation, the pigment is shifted into the cell processes, which
extend into the centre of the cornea just covering the pupil zone. In
Bathymaster derjugini, the pigment cell bodies lie in a ring around
the perimeter of the cornea (Gamburtseva et
al., 1980
). During light-adaptation, the pigment is shifted into
the cell processes progressively, covering the entire cornea within 12
h of light-adaptation. All of the experiments to date have been performed
during the day.
The mechanisms underlying pigment expansion and retraction have not yet
been resolved, but possibilities include nervous, humoral or autonomous
control of the pigment migration. Central control (neural or humoral) seems
unlikely, because unilateral illumination only induced pigment movement in the
illuminated eye, and optic nerve sectioning (in one eye) has no effect, i.e.
no response difference was found between two illuminated eyes
(Appleby and Muntz, 1979).
Kondrachev and Khodtsev (1984
)
found differential changes in corneal colouration when left and right eyes
were exposed to different illumination conditions in the same fish, and
therefore agree that central control is unlikely.
Local (retinal) humoral control has been proposed by Kondrachev and
Khodtsev (1984), who suggest
that the illumination of the photoreceptors in each eye induces release of a
hormone that triggers pigment expansion. In darkness, the retina releases
acetylcholine, which reaches the pigment cell bodies in the cornea
via the choroid, where it stimulates pigment aggregation. The rate of
pigmentation change is slow, which supports the hypothesis that pigment
migration is under humoral rather than neural control
(Appleby and Muntz, 1979
;
Kondrashev and Khodtsev,
1984
).
Another possibility is that pigment migration is under autonomous control
within the pigment cells. Kondrachev and Khodtsev
(1984) believe this
possibility is unlikely, because the response of the pigment is dependant on
the stimulating wavelength, i.e. the red pigment only expanded when
illuminated with wavelengths between 560 nm and 620 nm. Also, they found that
pigment migration was disturbed in isolated corneas (Kondrachev and Khodtsev,
1984).
Recently, novel opsins localised in extraretinal tissues have been
implicated in the control of skin pigmentation, pupillary aperture, and
circadian and photoperiodic physiology
(Provencio et al., 1998;
Philp et al., 2000
).
Melanopsin, which possesses an invertebrate opsin character with a stable
metastate that retains the chromophore, is converted from one of the two
possible stable states into the other, independently of the supply of the
11-cis chromophore (Provencio et
al., 1998
). Provencio et al. conclude that this independence would
permit melanopsin to act in a large variety of tissues. It is, therefore,
possible that the corneal pigment cells contain such novel opsins, which may
regulate the pigment response in a wavelength-dependent manner as proposed by
Kondrashev and Khodtsev
(1984
).
In addition to CCCs, fish corneas contain other types of chromatophores,
such as erythrophores, melanophores and xanthophores
(Gamburtseva et al., 1980).
None of these pigment cell types possess long processes or play a role in the
changeable filter properties of fish corneas
(Orlov and Gamburtseva, 1976
;
Gamburtseva et al., 1980
).
The chemical composition of the yellow pigment is believed to be of
carotenoid origin, based on the shape of its absorption curve
(Moreland and Lythgoe, 1968;
Bridges, 1969
). The orange
pigment is believed to be different from the yellow pigment as its absorption
curve does not show the three intermediate peaks, but rather displays a single
broad absorption band (half-band about 400550 nm;
Orlov and Gamburtseva,
1976
).
Several functions have been proposed for yellow ocular filters, including
protection from potentially deleterious UV wavelengths
(Zigman, 1971) and the
elimination of scattered light. The reduction of chromatic aberration has also
been suggested for these yellow filters to improve visual acuity and contrast
discrimination (Walls and Judd,
1933a
,
b
). In contrast to these
functional advantages yellow filters decrease sensitivity during low light
conditions. However, fish with occlusable yellow corneas can take advantage of
coloured filters during high intensity illumination conditions while avoiding
the disadvantage of losing sensitivity during low light conditions by
retracting their pigment (Gamburtseva et
al., 1980
; Heinermann,
1984
).
In this study, we compare pigment movement induced by light- and dark-adaptation during the day with that caused by light- and dark-adaptation during the night to test the influence of a possible underlying diurnal pigment migration rhythm on the observed pigment migration pattern. We also determine the temporal properties of pigment retraction and expansion during the day and measure the changes in transmission properties across the cornea as the pigment moves. Ultrastructural examination of the corneas of the two toadfish species in the light- and dark-adapted states reveals that the pigment migration within large sheet-like processes causes concomitant changes in corneal thickness.
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Materials and methods |
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Dark- and light-adaptation
14 specimens of each species were used for experiments during the day,
while the other 14 were used during the night. Six fish of each species were
kept in a glass aquarium that was illuminated from above by sunlight on a
bright day to ensure that the pigment was maximally extended and the remaining
two fish of each species were dark-adapted by placing them into an aquarium
kept in a dark room for at least 60 min. The pigment distribution of one of
the day-adapted fish was used as a control for the maximal pigment cover of
the cornea. The five remaining light-adapted fish were dark-adapted for
different lengths of time (1070 min) by placing them into a second
aquarium in the darkroom. In the second part of the daytime experiment one of
the fish of each species that had been placed into the dark aquarium was used
as a dark reference while the second fish of each species was light-adapted
again (for 70 min).
The 14 night-adapted fish were kept in an aquarium outside the station in an area that was shaded from street illumination. One specimen of each species was used to determine the minimal pigment cover prior to the light-adaptation experiment. For 1070 min, five fish of each species were exposed to a cold-light halogen lamp (KL1500, 150 W, Schott, Mainz, Germany) in combination with the normal room lighting (Fig. 2). The lamp was placed above the tank so that the entire area was illuminated uniformly and the walls of the tank were lined with Teflon to maximise brightness. The remaining two fish of each species were light-adapted for 60 min. One specimen of each was used as a light control while the other one was dark-adapted again.
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For the analysis of pigment movement patterns, transmission properties and ultrastructure we present results for the dark-adaptation during the day and the light-adaptation during the night, simulating the natural direction of pigment movement at dusk and dawn. The results of the light-adaptation following dark-adaptation during the day and of the dark-adaptation following light-adaptation during the night were used to test whether pigment movement could be induced in both directions during the night and day.
Spectral transmission analysis
At the end of each experimental period, the fish were anaesthetised with
methane tricaine sulfonate salt (MS222, 1:2000) and killed by decapitation in
the dark (dark-adapted fish) or light (light-adapted fish). The eyes were
enucleated and a window was cut into the back of the scleral eyecup so that
the lens and vitreous could be removed without destroying the cornea. The eyes
were then placed on white filter paper and photographed with a SLR camera
(Canon EOS 1000). Remains of the retina and iris were then removed, and the
cornea was rinsed in saltwater to remove traces of blood. Another photograph
was taken of the isolated cornea before light transmission analysis. Spectral
transmission spectra (350800 nm) were obtained using `Sub-Spec', a
portable spectrophotometer (modified version of Oriel Instruments Intraspec IV
system; described in Marshall,
1996). All samples were measured in air
(Douglas and McGuigan, 1989
). A
`Spectralon' white tablet was used as a 99% reflection standard. The
instrument beam was aimed at the white tablet through different areas of the
cornea under visual control. The cornea was mounted on a holder with which the
position of the cornea could be controlled. Xenon illumination was provided
with a camera flash with the front UV filter removed. Transmission spectra
were normalised with respect to the transmission level at 700 nm
(Douglas and McGuigan, 1989
;
Siebeck and Marshall, 2000
,
2001
).
During preliminary experiments, it was observed that the pigment of a dark-adapted isolated cornea, kept in a dish with salt water, dispersed under illumination. This pigment migration ceased if the isolated cornea was placed on filter paper that absorbed the excess water. All test corneas were therefore placed on filter paper as soon as the dissection was finished. Additionally, the overall illumination surrounding the experimental set up was matched to the adaptation condition to negate any post-enucleation pigment migration. We cannot completely rule out that anaesthesia and handling during the dissection and isolation of the corneas may also influence the pigment dispersal. We therefore took care that the preparations were made as quickly as possible. Approximately 5 min were needed for anaesthesia and preparation of the first cornea of each fish. We found that pigment migration from maximal to minimal (and vice versa) pigment cover takes about 60 min. It therefore seems that any changes caused by handling etc during the 5 min cannot be very large. Also, we compared the pigmentation pattern of the first cornea of each fish with that of the second cornea, which was left in the fish until the measurements of the first cornea were completed. A time difference of 5 min did not appear to affect our results. All corneas were treated exactly the same and any artefact due to handling should therefore affect all corneas in the same way. Differences between the different preparations must therefore be due to different stages of light- and dark-adaptation.
The locus of each transmission measurement was noted on both detailed drawings and photographs of the corneal pigmentation pattern. For each cornea, seven locations were scanned. Since the direction of the pigment movement was from the dorsal and ventral rims into the centre and back, five positions were selected along a vertical line through the centre of the cornea; dorsal rim, halfway point between the centre and the dorsal rim, centre, half-way point between the centre and the ventral rim, and the ventral rim. Also, two measurements were made along a horizontal line through the centre, one at the halfway point between the nasal rim and the centre and one at the halfway point between the temporal rim and the centre. At each of the positions three measurements were made and averaged.
Characterisation of corneal pigment distribution
To characterise the pigment distribution after the different light- and
dark-adapted treatments, corneal photographs were scanned using a Canon slide
scanner (Nikon LS-1000, Tokyo, Japan). Images were then imported into Adobe
Photoshop and a grid was superimposed over the cornea. The area of the
isolated cornea, the pupil zone and the different regions of pigment cover
were determined by counting the grid cells. The pupil zone was defined as the
area of the cornea that was not covered by the immovable iris.
Temporal analysis of pigment migration
The pigment cover of the whole cornea, the pupil zone and the ventral and
dorsal corneal hemifields of each species were determined during the day
(light-adapted) and night (dark-adapted) as described above. Various
measurements of pigment cover were performed after 10, 20, 30, 40, and 60 min
of dark-adaptation of the light-adapted fish during the day and at the same
time points of light-adaptation of the dark-adapted fish during the night. The
two corneas of each fish were analysed and their results averaged. The two
corneas of each fish were analysed as described above and their results were
averaged (mean).
Ultrastructural analysis
For each species, both eyes of two specimens were fixed in 0.1 mol
l-1 phosphate buffer containing 3.5% paraformaldehyde, 0.25%
glutaraldehyde and 2% sucrose. Two eyes were fixed in the light-adapted
condition and two in the dark-adapted condition. The corneas were postfixed in
2% osmium tetroxide with 1.5% potassium ferrocyanide in 0.1 mol l-1
sodium cacodylate buffer (as described in
Collin and Collin, 1996). The
tissue was then dehydrated in acetone and embedded in resin (polybed/812,
Polysciences Inc.). For light microscopy thick sections (1 µm) were cut,
stained with Toluidine Blue and examined with a Zeiss compound microscope
(Axioskop, Jena, Germany) fitted with a SPOT digital camera (Diagnostic
Instruments Inc., Sterling Heights, USA). Thin sections (60 nm) were prepared
for transmission electron microscopy, stained with lead citrate and uranyl
acetate and examined using a JEOL 1010 Transmission Electron Microscope
(Peabody, USA). Negatives of all micrographs were digitised with a LeafScan 45
Negative Digitiser (Tel Aviv, Israel).
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Results |
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During the night, the dark-adapted corneas of T. hamiltoni and T. pleurogramma both show a 53% and 42% level of corneal pigmentation. In both cases, the pigment is retracted to the ventral and dorsal rims of the cornea and within these areas, two zones can be distinguished on the basis of a difference in colouration (Fig. 1CF). The outermost zone appears orange, while the more central zone appears yellow. The centre of the corneas of both species is clear, i.e. the pupil zone contains no pigment.
After dark-adaptation during the day, the pigment is retracted so that the pupil zone is almost free of pigment (Fig. 1). Pigment retraction is not as strong as after dark-adaptation at night. Similarly, after light-adaptation during the night, the pigment expansion is not as strong as during light-adaptation during the day (Fig. 1).
Temporal changes in pigment migration
A change in the pigment cover of the cornea was induced by dark-adapting
fish during the day, as well as by light-adapting fish during the night
(Fig. 1). After 6070 min
illumination at night, the pigment of both species of toadfish appeared to be
extended almost as far as it was during the day in bright sunshine. The
pigment cover found in fish that were dark-adapted during the day was similar
to that found in night-adapted fish (Fig.
1). However, the distribution pattern of the yellow and orange
pigments appeared to be different in both species. During light-adaptation at
night, the yellow pigment extended as far as it did during the day while the
coverage of the orange pigment did not change
(Fig. 1).
In 60 min of light-adaptation at night, pigmentation of the whole cornea changed from an average of 42% cover to 87% cover in T. pleurogramma and from 53% cover to 96% in T. hamiltoni (Fig. 3A). The time course in both species is very similar. After 10 min of exposure to light, pigment cover had already reached 79% and 80% of the entire cornea, respectively. In the first 10 min of illumination, the pigment cover within the pupil zone changed from 0% to over 30% in T. hamiltoni and from 0% to over 60% in T. pleurogramma (Fig. 3B). After 30 min, the pigment cover within the pupil zone had reached 84% and 91%, respectively. The ventral and dorsal hemifields showed a similar symmetrical pigmentation change in both species (Fig. 3C,D). The pigmentation change in T. hamiltoni was found to be slightly slower than that in T. pleurogramma.
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In 60 min of dark-adaptation during the day, the pigmentation decreased from 83% to 37% cover in T. pleurogramma and from 100% to 42% in T. hamiltoni (Fig. 3A). However, the time course in the two species is quite different. T. pleurogramma shows an abrupt change from 83% to 53% pigment cover between 10 and 20 min of dark-adaptation, whereas T. hamiltoni reaches 54% pigment cover after only 60 min of darkness (Fig. 3A). The pigmentation of the pupil area also shows the difference in the speed of the pigmentation change. After 10 min dark-adaptation during the day, the pigmentation of T. pleurogramma does not change while T. hamiltoni reduces its pigment cover from 100% to 96% (Fig. 3B). The pigment in the pupil zone of T. hamiltoni retracts at a slow but relatively constant rate until it reaches 0% after 70 min. In T. pleurogramma, on the other hand, the pigment movement increases after a slow start so that after 20 min, only 30% of the pupil zone contains pigment, and after 60 min the pupil zone is completely clear. The overall change in dorsal and ventral hemifield pigmentation is symmetrical in both species (Fig. 3C,D).
Spectral changes in light transmission
Different classes of transmission curves can be distinguished by the slope
and the shape of the function (Siebeck and
Marshall, 2001). Class I consists of curves with a very steep
slope (<30 nm between 0% and 100% transmission) and a sharp cut-off. Class
II consists of curves with a less steep slope and a gradual onset of the
cut-off and Class III is characterised by the three intermediate maxima
between maximal and minimal transmission. Transmission curves of the corneas
of the two toadfish investigated here showed class I, II or III
characteristics. Class I and II curves found here could be divided into two
sub-groups that contained curves with 50% transmission values below 400 nm and
curves with 50% transmission values above 550 nm. Transmission curves in the
latter group typically show less than 10% transmission at wavelengths below
530 nm (Fig. 4).
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The transmission characteristics of the corneas of both species changed during light- and dark-adaptation. In each adaptation condition a continuous spectrum of transmission curves was found across the cornea. Individual transmission curves differed in the amount light transmitted in the 400500 nm wavelength band (Fig. 4). Here are shown the transmission curves of five selected areas of each cornea: the rim of the cornea, the area halfway between rim and centre of the cornea, the centre of the cornea, and areas nasal and temporal of the central cornea (Fig. 4). The transmission values for dorsal and ventral hemifields of the cornea were averaged as the pigment change was found to be symmetrical in both hemifields.
The largest transmission change was observed in the central cornea (Fig. 4C, Area 3), where the dark-adapted cornea transmits wavelength above 400 nm to more than 80% while in the light-adapted cornea the transmission for wavelengths between 400500 nm is reduced to less than 40%. The nasal part of the central cornea (location of the pseudopupil in T. pleurogramma) shows the same general pattern of transmission change, with the difference that relatively more light of the 400500 nm wavelength band is transmitted through the light-adapted cornea (Fig. 4D, Area 4). The temporal part of the central cornea also shows a similar trend (Fig. 4E, Area 5).
The dorsal and ventral rims of the cornea (Fig. 4A, Area 1) show no differences in transmission properties during light- and dark-adaptation. The 50% transmission cut-off of this area lies around 550 nm. The transmission properties of the area halfway between the centre and the rim of the cornea (Fig. 4B, Area 2) change depending on the adaptation condition; the transmission in the 400500 nm wavelength band decreases with increasing dark-adaptation.
Corneal ultrastructure and changes in corneal pigmentation
The structure of the corneas of both species of toadfish is similar. In
both species, the cornea comprises a dermal (secondary spectacle) and a
scleral cornea loosely joined by a mucoid layer
(Fig. 5A). The dermal cornea
comprises an epithelium (23 cell layers thick), a basement membrane
(0.40.5 µm thick) and a stroma consisting of many layers of collagen
fibre bundles, which are arranged perpendicular to each other
(Fig. 5Bi). The thickness of
the dermal stroma of T. hamiltoni and of T. pleurogramma
ranges between 96.5 µm and 151.9 µm (central to peripheral) and 58.7
µm and 55.5 µm (central to peripheral), respectively. A mucoid layer
separates the dermal stroma from the anterior scleral stroma. Within the
anterior scleral stroma of T. hamiltoni, two areas with a different
affinity for osmium can be distinguished. The thickness of the scleral stroma
ranges from 113.4 µm to 157.3 µm in T. hamiltoni and from 54.3
µm to 73.1 µm in T. pleurogramma (central to peripheral). An
iridescent layer underlies the anterior scleral stroma
(Fig. 5Bii,iii). The iridescent
layer of both species consists of a series of aligned rough endoplasmic
reticuli oriented approximately perpendicular to the incident light striking
the cornea from above. The layer extends across the entire cornea and
terminates just beyond the iris (Fig.
6). The iridescent layer ranges from 34.9 µm to 14 µm
(central to peripheral) thickness in T. hamiltoni and from 29.3 µm
to 23.6 µm in thickness in T. pleurogramma.
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The pigment layer is located within the scleral cornea adjacent to the iridescent layer. In T. pleurogramma, the pigment layer is split and comprises two layers of processes situated on either side of the iridescent layer, while in T. hamiltoni a single pigment layer is situated beneath the iridescent layer (Fig. 6). The cell bodies of the pigment cells are found in the periphery of the cornea, outside the pupil zone (Fig. 6). The average diameter of the cell bodies in the pigment cell reservoir is 2.5 µm during light-adaptation and 13.8 µm during dark-adaptation (T. pleurogramma) and 8.7 µm during light-adaptation and 29 µm during dark-adaptation (T. hamiltoni). Long processes extend from each cell body into the center of the cornea. It appears that the processes are organised in thin sheets that terminate in the center of the cornea (Figs 7A, 8A). As the pigment processes originate in both the ventral and dorsal cornea, the processes extend throughout the entire cornea.
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The chromatophores contain a variety of organelles and structures
(Fig. 8B,C). The cytoplasm is
granular and surrounds a large number of both lightly (<1 µm diameter)
and darkly- (approximately 1 µm in diameter) stained vacuoles, which may
contain lipid and contribute to at least part of the corneal pigmentation
(Murphy and Tilney, 1974).
In the light-adapted state, the pigment cell processes are filled with granular cytoplasm and lipid-filled vacuoles (Fig. 7A,C). The width of the processes is 1.4±0.4 µm (mean ± S.D.) (T. hamiltoni) and 1.1±0.6 µm (T. pleurogramma). The cytoplasm appears to be closely associated with microtubules (0.2 nm in diameter), which lie parallel to the cell membrane (Fig. 8C). The pigment layer contains between 710 processes and, on average, reaches a width of 10.3±0.5 µm and 9.4±2.9 µm (T. hamiltoni, T. pleurogramma, respectively).
In the dark-adapted state, only the pigment reservoir contains the granular component of the cytoplasm and the lipid granules while the processes are empty, collapsing to less than half of their light-adapted diameter (T. hamiltoni 0.4±0.2 µm and T. pleurogramma 0.42±0.26 µm; Fig. 7B,C). As the pigment reservoir is situated outside the borders of the iris (pupil zone), the part of the cornea overlying the pupil is devoid of granules and lipid-filled vacuoles, providing at least circumstantial evidence in support of the structural identification of the pigment when considered together with the transmission data.
The corneas of both species of toadfish contain a posterior scleral stroma ranging from 4.3 µm to 10 µm in T. hamiltoni and from 2.4 to 6.2 µm in T. pleurogramma, which lies anterior to a monolayer of cells, Desçemets membrane (0.89 µm in T. hamiltoni and 1 µm in T. pleurogramma) and an endothelium (0.8 µm in both T. hamiltoni and T. pleurogramma, Fig. 5Biv).
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Discussion |
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Slight differences were observed in the responses of the yellow and orange
components of the pigmentation pattern during the night and day. One
possibility is that these differences are due to an underlying diurnal rhythm.
Alternatively, they could be caused by spectral differences between the
illumination used for light-adaptation during the day and night. During the
day, the broad spectrum of the sun was used for light-adaptation, while the
short-wavelength-deprived spectrum of the halogen lamp was used during the
night. Kondrashev and Khodtsev
(1984) report that the orange
pigment in the corneas of two greenlings (Hexagrammos octogrammus and
H. stelleri) did not disperse when illuminated with short wavelengths
only, whereas the yellow pigment seemed equally sensitive to all wavelengths
between 420650 nm.
The pattern of pigmentation change found here is very similar to that
described for other toadfish (Appleby and
Muntz, 1979; Gamburtseva et
al., 1980
). However, in the light-adapted condition, the cornea of
Torquigener pleurogramma is not uniformly coloured but shows less
densely pigmented areas situated temporally and nasally, forming a pattern
similar to that of species with constant pigment patterns
(Kondrashev et al., 1986
;
Siebeck and Marshall, 2000
).
The function of these pseudopupils has not yet been resolved, but it is
possible that they form a window through which the fish can look forward with
maximal sensitivity, while still protecting the retina from downwelling
(dorsal sunshield) and upwelling (ventral sunshield) light
(Kondrashev et al., 1986
).
The pigment of the toadfish Torquigener pleurogramma and
Tetractenos hamiltoni takes approximately 60 min to expand fully when
transferred from the dark into bright sunlight, which is similar to
descriptions for the toadfish Tetraodon steindachneri
(Appleby and Muntz, 1979). In
all three species, pigment expansion follows a similar time course while
pigment retraction seems more variable. All three species reach maximal
pigment retraction at the same time; however, this is achieved by two
different strategies. In T. hamiltoni the rate of pigment retraction
remains constant, while in T. pleurogramma and T.
steindachneri the pigment initially retracts slowly, followed by a rapid
and a final slow phase.
During dark-adaptation, the minimal pigment cover (3742%) in both
species of toadfish investigated here differs from that in T.
steindachneri (15%). Despite these differences in the degree of coverage,
the pupil zone is still pigment-free in the species investigated here and
presumably also in T. steindachneri. Therefore, there should be no
functional differences between the various amounts of corneal pigment cover
during dark-adaptation. The situation changes, however, when the fish are
exposed to bright light, where there may be some advantage in screening their
corneas as quickly as possible. Within 10 min, both T. pleurogramma
and T. hamiltoni achieve a much larger corneal coverage than T.
steindachneri (Appleby and Muntz,
1979), due to their larger initial coverage. It appears that the
two species studied here have a time advantage over T. steindachneri
as a result of the pigment `standing by' just outside the pupil zone.
The relatively slow change of pigmentation cover found here is similar to
what occurs in many species within at least nine teleost families, described
by Gamburtseva et al. (1980).
This slow response has been used as evidence that the response mechanism is
unlikely to be under central control
(Appleby and Muntz, 1979
;
Kondrachev and Khodtsev, 1984).
General structure of the cornea
The structure of the corneas of both toadfish species is very similar and
shows the features typical of the corneas of some other teleosts
(Collin and Collin, 2001). The
division of the cornea into dermal (continuous with the underlying epithelium
of the conjunctiva and skin) and scleral (continuous with the sclera
surrounding the globe) components has previously been described for a range of
shallow-water species by Walls
(1942
). However, a `secondary
spectacle' has recently been found in the pipefish, Corythoichthyes
paxtoni (Collin and Collin,
1995
), a range of gadiform deep-sea fish
(Collin, 1997
) and the
salamanderfish Lepidogalaxias salamandroides
(Collin and Collin, 1996
),
where the separation of the cornea by a mucoid layer has been attributed to
the need to rotate the eye while maintaining a protective goggle, to reduce
abrasion or to reduce the friction associated with eyes that project beyond
the contour of the head (Collin,
1997
).
The subdivision of the scleral stroma into an anterior and a posterior
stroma was previously thought to be unique to deep-sea gadiforms
(Collin and Collin, 1998). It
is not clear why the shallow-water toadfish have three stromas, which, in the
deep-sea teleosts examined, are thought to be an adaptation for strengthening
the cornea and maintaining a robust intraocular pressure when subjected to the
increased pressures at depth (Collin and
Collin, 1998
).
Iridescent layers have been found in many shallow-water marine teleosts
(Lythgoe, 1974;
Collin, 1997
). Various types
exist and can be situated in different layers of the cornea. The iridescent
layer found in the toadfish as described here is situated between the scleral
anterior stroma and the cellular processes containing pigment. The same
position has been described for another toadfish, Tetraodon
samphongsi (Lythgoe,
1974
). In all three cases, the multi-layered stacks are comprised
of rough endoplasmic reticulum. The orientation of the stacks is perpendicular
to the incident light striking the cornea from above and therefore changes in
relation to the orientation of the other layers in the cornea from dorsal to
ventral cornea.
The iridescent layers of the two toadfish species investigated here do not
seem to act as colour filters, as the transmission of the cornea in
unpigmented areas is between 70% and 90%, which is similar to the findings of
Lythgoe (1974). It is
possible, however, that the transmission properties change when the cornea is
separated from the rest of the eye in preparation for the transmission
measurements. By cutting into the eye, the natural tension on the cornea
caused by the intraocular pressure is destroyed and the cornea flattens. The
iridescence that was clearly visible in the intact eye becomes invisible in
the isolated cornea.
Transmission properties and pigment location
The cornea of Hexagrammos octogrammus, the first fish for which
changeable corneal colouration was described, contains two types of pigment of
yellow and orange appearance (Orlov and
Gamburtseva, 1976). The authors conclude, from differences in
their absorption spectra, that these colours are not a function of differences
in pigment density and propose that there are two different kinds of corneal
chromatophores, one containing the yellow pigment and the other containing the
orange pigment. The corneas of the two species of toadfish described here also
have areas with orange and yellow pigmentation, and the transmission spectra
of these areas resemble those of H. octogrammus. However,
measurements taken in different areas of the cornea reveal that there is a
gradual change in the transmission spectrum from the more typical spectrum
(with three intermediate maxima) to a spectrum with a cut-off at wavelengths
above 570 nm. It is, therefore, possible that the yellow and orange appearance
of some areas of the corneas of T. hamiltoni and T.
pleurogramma is due to different concentrations of the same pigment.
The transmission change in the different areas of the cornea can be
explained with the pigment migration during the different adaptation
conditions. During dark-adaptation, the long sheet-like processes extending
from the pigment reservoir into the center of the cornea are empty and the
transmission in the center of the cornea is maximal. The structures containing
the pigment (presumably the granular cytoplasm and/or the lipid containing
vacuoles) are concentrated inside the pigment reservoir, which is situated
outside the pupil zone along the dorsal and ventral rim of the cornea. The
transmission in that area is minimal and stays relatively constant during all
adaptation conditions, indicating a continuously high concentration of pigment
in the reservoir. In the process of light-adaptation, pigment is shifted from
the reservoir along the sheet-like processes towards the center of the cornea,
resulting in a swelling of the processes and in less shortwave light being
transmitted through the central cornea. In the process of dark-adaptation, the
pigment is moved back towards the pigment reservoir until the processes are
empty and the transmission in the central cornea reaches maximal values. This
pigment migration may be aided by the microtubules identified within the
corneal chromatophores in both species of toadfish
(Murphy and Tilney, 1974;
Murphy, 1975
).
Function of occlusable corneas
Several advantages of yellow corneas have been discussed in the literature.
As early as 1933, Walls and Judd state that yellow filters increase visual
acuity by `reducing chromatic aberration, promote comfort by reducing
glare and dazzle, enhance detail by the absorption of blue haze [scattered
light] and also enhance contrast' (Walls and Judd,
1933a,
b
). These are all theoretical
benefits of short-wavelength filters that have not yet been confirmed with
behavioural experiments. It is obvious, however, that the disadvantage of
having short-wavelength filters is the loss of sensitivity during periods of
low light intensity such as dusk and dawn and also at depth. This disadvantage
no longer applies if the filter can be removed. Appleby and Muntz
(1979
) demonstrated this by
calculating the effect of such filters on the sensitivity of T.
steindachneri in different water types and at different depths
(Jerlov, 1976
). In clear water
(Jerlov type 1A), they found a large sensitivity loss that increased with
depth (i.e. a 0.45 log unit loss at 20 m for a corneal pigment density of
0.73). In order to absorb the same number of quanta, a fish with this corneal
density would have to be 30 m shallower than a fish without a yellow filter
(Appleby and Muntz, 1979
).
Appleby and Muntz (1979)
also evaluated the impact of occlusable versus permanent yellow
corneas on vision at dusk and dawn, demonstrating that, while there is no
advantage during sunset as the pigment moves as fast as the light diminishes,
there might be a considerable advantage during sunrise. They concluded that
T. steindachneri would possess a 35 min advantage over fish
with permanent yellow corneas, which is considerable in the short tropical
twilight when predation is highest. Since the two toadfish species studied
here had slightly faster pigment movement rates, a greater advantage may be
expected.
In all stages of pigment retraction or expansion, the ventral and dorsal
hemifields of the cornea were equally covered with pigment. The area of
highest pigment density in the corneas of fish with fixed colouration patterns
was found in the dorsal hemifield or overlying the pupil zone, which is where
it is also found in other species
(Kondrashev et al., 1986). One
of the reasons suggested for this pattern is that the yellow pigment acts as a
protective sunshield from the intense downwelling light
(Heinermann, 1984
). The two
toadfish species described here possess changeable `sunglasses' that have
their fastest and highest degree of protection in the dorsal and ventral
corneal hemifields. Both species live in shallow water over a sandy bottom,
which strongly reflects the downwelling light. With a symmetrical dorsal and
ventral pigmentation, they achieve protection from downwelling and upwelling
light simultaneously.
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References |
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