Rapid colour changes in multilayer reflecting stripes in the paradise whiptail, Pentapodus paradiseus
1 Vision, Touch and Hearing Research Centre, School of Biomedical Sciences,
The University of Queensland, Brisbane, Queensland 4072, Australia
2 Sussex Centre for Neuroscience, School of Biological Sciences, University
of Sussex, Brighton BN1 9QG, UK
* Author for correspondence (e-mail: l.mathger{at}uq.edu.au)
Accepted 11 July 2003
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Summary |
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Key words: fish reflector, iridophore, paradise whiptail, Pentapodus, paradiseus, multilayer reflector, rapid colour change, colour vision
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Introduction |
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Paradise whiptails have very distinct reflective stripes on their head and
body (Fig. 1A). When we first
observed these fish in tanks, we noticed that the colour of these stripes
changes from blue to red within less than one second. These changes are
unusually fast for fish iridophores, which have been reported to take several
seconds or minutes (Kasukawa et al.,
1987; Lythgoe and Shand,
1982
).
Iridophores, both `active' and `passive', are common in many fish, and
their likely functions in camouflage and communication (e.g. schooling and
mating) have been discussed by several authors (see e.g.
Denton, 1970;
Denton and Rowe, 1994
;
Fujii et al., 1989
;
Herring, 1994
). The blue
damselfish (Chrysiptera cyanea), for example, normally displays a
characteristic blue colouration, which is produced mainly by iridophores in
the skin (Kasukawa et al.,
1987
). During stressful conditions, the fish changes its hue
rapidly to ultraviolet (UV), seen as black by humans. These changes have been
associated with simultaneous changes in the distance between adjoining
reflecting plates that make up these iridophores (Kasukawa et al.,
1986
,
1987
;
Oshima and Fujii, 1987
).
Active iridophores have also been reported in the blue-green damselfish
(Chromis viridis; Fujii et al.,
1989
), the common surgeonfish (Paracanthurus hepatus;
Goda and Fujii, 1998
), the neon
tetra (Paracheirodon innesi;
Lythgoe and Shand, 1982
;
Nagaishi and Oshima, 1989
),
the dark sleeper (Odontobutis obscura;
Fujii et al., 1991
) and the
domino damsel (Dascillus trimaculatus;
Goda and Fujii, 2001
).
Many fish iridophores have been shown to be multilayer reflectors. Light
reflected from a reflector of this kind is almost always coloured. Multilayer
reflectors are characterised by the fact that they contain thin plates of a
higher refractive index than the spaces separating them. In fish, it is
assumed that the plates are made of guanine, which has a refractive index
(n) of 1.83, while the spaces are believed to be made of cytoplasm,
which has n=1.33 (Land,
1972). In an ideal multilayer reflector, the plates and spaces
both have an optical thickness (actual thickness multiplied by refractive
index) of a quarter of the wavelength reflected by the stack at normal
incidence. `Ideal' here means that such a stack has the highest reflectivity
in comparison with `non-ideal' reflectors, for which the plates and spaces
differ in optical thickness. It thus becomes obvious that by varying the
thickness of the plates and/or spaces, the wavelengths reflected from the
stack can be changed. Another characteristic feature of these types of
reflectors is that, as the angle of the incident light becomes more oblique,
the reflected light shifts towards the shorter (blue/UV) end of the spectrum
(Huxley, 1968
;
Land, 1972
).
It seems quite clear from the evidence presented by Oshima and Fujii
(1987) and Kasukawa et al.
(1987
) that the reflective
changes in the blue damselfish are brought about by a change in the distance
between the plates that are contained within the reflective cells of the skin.
Furthermore, Oshima and Fujii
(1987
) showed that
microtubular structures connect adjacent plates and that their assembly and
disassembly may provide the mechanism for the reflective changes in the blue
damselfish. Kasukawa et al.
(1987
) showed that the
reflective changes are controlled by the sympathetic nervous system. Whilst
noradrenaline (norepinephrine) causes the reflected wavelengths to change to
the longer end of the spectrum, adenosine causes the reverse effect.
In the present study, we report reflective changes in the paradise whiptail and show that this unusually fast colour change is mediated by a change in the spacing between the plates of multilayer reflectors.
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Materials and methods |
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Spectral reflectivity measurements
For measurements of spectral reflectivity changes in a living paradise
whiptail, one fish was placed in a small glass tank. Spectral reflectance
measurements (300-800 nm) were obtained using a fibre optic spectrometer
(S-2000; Ocean Optics Inc., Dunedin, FL, USA) and a pulsed xenon light source
(220-750 nm; PX-2; Ocean Optics). The flash rate (up to 220 Hz) was controlled
by a laptop running the S-2000 and was synchronised with the detector. A
bifurcated fibre optic cable (1 mm diameter) provided illumination with 50% of
its fibres, while the other 50% acted as detectors of the reflected light. A
`Spectralon' white tablet was used as a 99% reflection standard. All
measurements were made in a dark room to prevent the influence of stray light.
The fibre optic cable was held by a small stage, enabling the angle of
incidence to be determined. Measurements were made at approximately normal
incidence to the skin surface of the reflective stripes on the head.
The same methods were used on skin preparation to investigate the effects of osmotic changes and drugs on the wavelengths of the reflected light. For measurements at oblique angles of incidence, two fibre optic cables were used (illumination, 200 µm; detector, 100 µm). The fibres were held by a small stage, and the angle of incidence was measured using a protractor.
Transmission electron microscopy
One paradise whiptail was killed by an overdose of clove oil (UQ AEC number
Phys/Ph/289/02/VTHRC; Munday and Wilson,
1997) and fixed in 3.5% paraformaldehyde and 0.25% glutaraldehyde
in 0.1 mol l-1 phosphate buffer. The reflective stripes were in a
blue-reflective phase (resting phase) after fixation. Small pieces of skin
containing the reflective areas were cut out and post-fixed in 2% osmium
tetraoxide in 0.1 mol l-1 phosphate buffer. Specimens were washed
and dehydrated in a graded series of acetone and embedded in Spurr's resin
(Spurr, 1969
). Sections were
cut on a Reichert Jung Ultracut microtome and viewed on a JEOL JEM 1010
transmission electron microscope.
Perfusion of drugs and osmotic effects
Three paradise whiptails were killed by an overdose of clove oil. The
tissue on the head and caudal peduncle containing the reflective stripes was
cut out using a scalpel blade and placed in a petri dish. The tissue was
equilibrated in a physiological saline, made according to Kasukawa et al.
(1987): 125.3 mmol
l-1 NaCl, 2.7 mmol l-1 KCl, 1.8 mmol l-1
CaCl2, 1.8 mmol l-1 MgCl2, 5.6 mmol
l-1 D-glucose, 5.0 mmol l-1 Tris-HCl buffer,
pH 7.2 (osmolality: 272 mOsm kg-1). The osmotic effects on the
reflective properties were tested by making saline solutions of different
osmotic strengths. A hyposmotic solution was made by diluting the saline in
ultrapure water to concentrations varying from 10% to 75% (with osmolalities
varying from 28 mOsm kg-1 to 194 mOsm kg-1,
respectively). A hyperosmotic solution was made by adding 200 mol
l-1 sucrose to the saline (460 mOsm kg-1). Noradrenaline
(norepinephrine) and adenosine (Sigma; 100 mmol l-1) were diluted
in the saline before experiments. The volume of the dish (approximately 10 ml)
was then replaced with the experimental solution, and the reflective changes
were recorded with the spectrometer (S-2000, Ocean Optics) described
earlier.
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Results |
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Cycles of colour change
Fig. 1B shows a typical
cycle of colour changes observed in the nose stripes of a paradise whiptail.
Each reflective cycle can be divided into four phases: (1) `resting phase',
which is dull blue (Fig. 1B,
panel a), (2) `green flash phase' (Fig.
1B, panel b), (3) `red phase'
(Fig. 1B, panel c) and (4)
`recovery phase', during which the peak wavelength shifts from red through
yellow and green back to blue (resting phase;
Fig. 1B, panels d-f). The cycle
shown in Fig. 1B can occur
continuously for several minutes, although more commonly an interval from
seconds to minutes may separate successive cycles. The change from the resting
phase to the red phase (Fig.
1B, panels a-c) is accomplished within approximately 0.25 s. The
reflective stripes may then remain in the red phase
(Fig. 1B, panel c) for several
seconds (typically 3 s). The recovery phase from red through green to blue
(Fig. 1B,panels c-f) may last a
few seconds (typically around 2 s), after which another cycle may occur. The
initiation of this reflective cycle as well as the duration of any of its
phases is entirely under the control of the animal. The colour change appears
to occur nearly instantaneously across all reflective stripes of the body.
The body colouration also changes during a reflective cycle, becoming redder as the reflective stripes change from blue to red. This is, however, very weak compared with the change of the reflective stripes. The body colouration is probably caused by iridophores and pigment-containing chromatophores.
Spectral changes in living fish
The spectral measurements of the reflective changes shown by living fish
are shown in Fig. 2A. In this
figure, spectral curves are shown for all wavelengths reflected by these
stripes during one cycle. It can be seen that the reflected light undergoes a
change from blue (465 nm) through the green and yellow parts of the spectrum
to red (650 nm).
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Fig. 2B shows the ratio of
reflectance half-width to peak wavelength
[/
max, where
is the bandwidth at 50% reflectivity and
max is the peak wavelength
(Land, 1972
)]. The reflectance
half-widths increase with wavelength (see
Fig. 2A). The regression line
in Fig. 2B shows that, as a
fraction of the peak wavelength, they increase slightly. This increase was
significantly different from zero (ANOVA F1,63=8.83,
P=0.0042). This is in contrast to what we expected to find. The
predicted reflectance half-widths are also shown in
Fig. 2B (blue line; calculated
from Land, 1972
). The possible
significance of this is outlined in the Discussion.
Changes in reflected wavelength with angle of incidence
Fig. 2C shows the effect of
varying the angle of the incident light source on the peak wavelength of the
reflected light. Here, we show measurements of a group of iridophores
reflecting yellow-green light at normal incidence. It can be seen that as the
angle of the incident light becomes more oblique, the wavelength of the light
reflected from the same group of iridophores moves towards the shorter
(blue/UV) end of the spectrum. This behaviour is typical of multilayer
interference reflectors.
Transmission electron microscopy (TEM)
In Fig. 3, we show electron
micrographs of the skin from the reflective stripes on the head. Sections were
cut perpendicular to the skin surface. Fig.
3A is a low-power micrograph that shows the arrangement of the
iridophores and chromatophores in the skin of these fish. It can be seen that
the plates contained within an iridophore are arranged circularly around the
pigment-containing chromatophores. Note that the plates have broken away
during the process of sectioning and that the white areas are those that
normally contain the plates. Some of the areas that contained the plates have
expanded considerably during examination with the electron microscope. Only
specimens preserved as well as those shown in
Fig. 3B,C,showing iridophores
with intact plates, were used to measure plate and space thicknesses.
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The plates have a mean thickness of 51.4±3.5 nm (mean ± S.E.M.; N=24), while the spaces varied from 26.3 nm to163.6 nm (mean=83.5±11.64 nm; N=9) (see Fig. 3B,C). The number of plates making up a stack within an iridophore varied between individual cells. On average, we counted 9.7 plates (N=31).
Physiological experiments
The results of the physiological experiments are shown in
Fig. 4. Fig. 4A shows that reducing the
osmotic pressure of the external solution by diluting the saline resulted in a
wavelength shift to the longer (red) end of the spectrum. This wavelength
shift could be reversed by returning the preparation to 100% saline
(Fig. 4B). Increasing the
osmotic pressure by adding 200 mmol l-1 sucrose to the saline
shifted the reflected wavelengths to the shorter end of the spectrum by a
further 50 nm (Fig. 4B). These
results are consistent with the swelling of the spaces between the plates in
hyposmotic solutions and shrinkage in hyperosmotic solutions.
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The effects of norepinephrine and adenosine are shown in Fig. 4C,D. From Fig. 4C, it can be seen that topical applications of 100 µmol l-1 norepinephrine resulted in a shift in the peak wavelength to the longer (red) end of the spectrum, whilst applications of adenosine (100 µmol l-1) reversed the reflected peak to the shorter (blue) end of the spectrum (Fig. 4D). It took approximately 20 min before the wavelength shifts (blue to red, as well as red to blue) could be observed. Washing in saline following norepinephrine application resulted in the same spectral shift as during adenosine applications. However, it took approximately twice as long for the blue reflection to return.
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Discussion |
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The evidence presented here is in strong support of the theory that the
reflectors that make up these stripes act as multilayer reflectors. With
increasing angle of incidence, the reflected wavelengths shifted towards the
shorter (blue/UV) end of the spectrum, a feature typical of multilayer
reflectors. As has been shown by Huxley
(1968) and Land
(1972
), the light reflected
from reflectors of this kind is invariably coloured if the thickness of the
plates and the spaces separating them are comparable with the wavelength of
light. In the ideal configuration, for which highest reflectivity is achieved
for the minimum number of plates in the stack, each space and plate has an
optical thickness of a quarter of the wavelength reflected by the stack at
normal incidence.
Structural inferences from optical measurements
The change in peak reflectance during a colour change cycle, from 465 nm to
650 nm, in conjunction with the mean plate thickness of 51.4 nm, can tell us
the extent of the change in the spacing of the plates (we assume that only the
spacing changes, not the plate thickness). For a given peak wavelength, the
sum of the optical thicknesses (actual thickness x refractive index) of
each plate and space must equal half a wavelength, i.e. 232.5 nm for the 465
nm maximum and 325 nm for the 650 nm maximum. If we assume that the plates are
made of guanine (n=1.83), as is common in fish, then their optical
thickness is 51.4x1.83=94.1 nm. This means that the optical
thickness of the spaces must increase from 138.4 nm (i.e. 232.5-94.1 nm) to
230.9 nm (i.e. 325-94.1 nm). Assuming the spaces are made of cytoplasm
(n1.33), the change in the actual thickness of the spaces is from
103.3 nm to 172.3 nm, an increase of 67%.
Another calculation that can be made is how closely the stack approaches
the `ideal' situation where
n1t1=n2t2=/4
(where n is the refractive index and t is the actual
thickness; n1t1 refers to the plates
and n2t2 to the spaces). An ideal
reflector has the highest reflectance for a given number of plates and also
the widest reflected spectral waveband. For an ideal multilayer
n1t1/(n1t1+n2t2)=0.5,
and lower values indicate a departure from this condition. When the whiptail
stack reflects blue (465 nm),
n1t1/(n1t1+n2t2)=94.1/232.5,
i.e. 0.405, which is close to ideal. When the stack reflects red light,
however,
n1t1/(n1t1+n2t2)=94.1/325,
i.e. 0.290, which is non-ideal. For a guanine-cytoplasm multilayer with 10
plates, these figures predict that the half-width of the reflected waveband
should be 0.24
max and 0.21
max for blue
light and red light, respectively (fig. 7b from
Land, 1972
), whereas
half-widths measured from Fig.
2A are both narrower than these values:
0.185
max and 0.194
max, respectively (see
Fig. 2B). Possible explanations
are that the measured curves are truncated on the short-wavelength side by the
red screening pigment of the chromatophores or that the plates are not
actually made of guanine but rather a substance of lower refractive index. The
fracturing of the plates during preparation for electron microscopy suggests
that they are crystalline, so the screening explanation is the more
likely.
The shift in max towards the blue with increasing angle
of incidence is consistent with the behaviour of multilayers. However, the
shift (Fig. 2C) is less than
expected. If
max (0° incidence) is 515 nm, then for a
10-plate guanine-cytoplasm multilayer one would expect a shift to 420 nm
(40°) and 360 nm (55°) (fig. 6 from
Land, 1972
). The measured
values were 450 nm and 415 nm, respectively. The most likely explanation here
is that the overall layout of the stacks in the iridophores is not flat but
curved, so that even at high angles the incident beam meets some stacks at
close to normal incidence. Another possible explanation is that the refractive
index difference between the plates and the spaces is actually higher than
guanine and cytoplasm would provide (i.e. the opposite of the change needed to
fit the half-width data above). The curvature explanation is quite consistent
with the known anatomy.
Osmotic and pharmacological changes
The data presented here suggest that the wavelength changes are elicited by
a change in the distance between adjacent iridophore plates. This is supported
by the finding that altering the osmolarity of the external solution resulted
in a change in the reflected wavelength. Hyposmotic saline shifted the
reflected wavelengths towards the longer (red) end of the spectrum, presumably
caused by water diffusing into the cells and pushing the iridophore plates
further apart. Removing intracellular water by returning the preparations to
100% saline, or even adding hyperosmotic saline, shifted the reflected
wavelengths towards the shorter (blue/UV) end of the spectrum.
Some simple physiological experiments were conducted to investigate
mechanisms of control of the reflective changes. We found that the reflective
changes in the paradise whiptail are under the control of the sympathetic
nervous system, which is in agreement with the findings of Kasukawa et al.
(1986,
1987
), Fujii et al.
(1989
) and Nagaishi and Oshima
(1989
) on other fish species.
Applications of norepinephrine shifted the reflections of blue-reflecting
iridophores towards the longer (red) end of the spectrum, whilst washing in
saline reversed this effect, shifting the reflections back towards the blue
parts. The shift from red to blue was speeded up by adenosine. In our study,
we have not, however, investigated the controlling mechanisms in any more
detail. Oshima and Fuji (1987
)
showed that in the blue damselfish the mechanism of reflective changes is
probably based on a microtubular system.
In comparison with the studies of Kasukawa et al.
(1986), we used very high
concentrations of norepinephrine. The reflective stripes of the paradise
whiptail were difficult to dissect intact and, as a consequence, we had to cut
thick slices of muscle and cartilage containing the reflective stripes.
Therefore, we had to increase the concentrations of norepinephrine in order to
ensure that the drug diffused into the tissue.
When looking at the wavelengths of the different phases of a colour change
cycle it becomes obvious that the green phases ('green flash' and green during
`recovery phase') may be the most visible reflections of the entire cycle.
Light in the sea is absorbed preferentially with depth, the blue-green
spectral region (around 500 nm) being transmitted best, while the red, deep
blue and UV parts are absorbed in the first 5-10 m of the water column
(Jerlov, 1976;
Tyler and Smith, 1970
). The
wavelengths of light best transmitted in coastal waters of Moreton Bay, where
paradise whiptails occur, are those of the green parts of the spectrum
(N.J.M., unpublished). There are a number of accounts in the literature
presenting evidence for a strong correlation between spectral absorption of
cone visual pigments of fish and the spectral composition of their
environments (Levine and MacNicol,
1979
). Many coastal fish, for example, have visual pigments
absorbing in the green and yellow regions of the spectrum, compared with many
off-shore species that have spectral sensitivities in the green and blue parts
(Lythgoe et al., 1994
). The
visual pigments of paradise whiptails have, to our knowledge, not been studied
to date; however, as coastal fish, their spectral sensitivities may be
expected to be best in the longer-wavelength regions of the spectrum.
Therefore, the contrast between the red-brown body colour and the green
reflections may appear stronger to them than the contrast between the body
colour and the other reflected wavelengths, which may make the green
reflections much easier to detect.
It is remarkable to observe the rapidity with which the paradise whiptail
can change the colour of its reflective stripes. This colour change is much
faster than that reported for other fish (see e.g.
Kasukawa et al., 1987;
Lythgoe and Shand, 1982
);
however, it appears to be controlled by the same mechanisms (see e.g.
Kasukawa et al., 1986
). To
date, we can only speculate about the functions of these colour changes. We
observed that the paradise whiptail changes the reflections from blue to red,
especially when excited by external stimuli, and so it appears likely that
colour change in this fish plays some role in communication.
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Acknowledgments |
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References |
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