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First published online March 17, 2006
Journal of Experimental Biology 209, 1245-1250 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02115
Significance of a basal melanin layer to production of non-iridescent structural plumage color: evidence from an amelanotic Steller's jay (Cyanocitta stelleri)
Department of Biological Sciences, 331 Funchess Hall, Auburn University, Auburn, AL 36849, USA
* Author for correspondence at present address: Department of Environmental Science, Policy and Management, Ecosystem Science Division, 137 Mulford Hall #3114, University of California, Berkeley, CA 94720-3114 (e-mail: mshawkey{at}nature.berkeley.edu)
Accepted 19 January 2006
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Key words: melanism, sexual selection, Fourier analysis, feather, Steller's jay, Cyanocitta stelleri
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; Shawkey and Hill,
2005; Hill and McGraw,
2006). The latter form of coloration is referred to as structural
coloration, and is typically classified as either iridescent (i.e. varying in
hue at different angles of observation) or non-iridescent. Non-iridescent
coloration is produced in many cases by coherent scattering of light by highly
organized matrices of keratin and air within feather barbs
(Dyck, 1971;
Dyck, 1976;
Prum, 1999;
Prum, 2006;
Shawkey et al., 2003). This
medullary `spongy layer' lies beneath a keratin cortex and above a layer of
melanin granules surrounding large central vacuoles. Whereas this spongy layer
has been studied in some detail, the functions of other anatomical features of
feather barbs in color production are less well understood. In particular, the
role of the melanin layer that underlies the spongy layer in barbs is still
unclear.
Two functions have been hypothesized for this melanin layer. The first
hypothesis (hereafter referred to as the "absorbance" hypothesis)
posits that melanin absorbs incoherently backscattered white light from the
vacuoles and thereby lowers reflectance `noise' to increase the purity of the
color reflected by the spongy layer (Prum,
2006). The second hypothesis (hereafter referred to as the
`backdrop' hypothesis) posits that the melanin layer serves as a black
backdrop that darkens the color of the spongy layer, an effect proposed to
explain color differences between dark and light blue morphs of Budgerigars
Melopsittacus undulatus (Simon,
1971). Alternatively, melanin may serve no purpose in color
production, existing in feathers to enhance rigidity
(Burtt, 1979) or resistance to
degradation (Goldstein et al.,
2004; Shawkey and Hill,
2004) or for some other function.
The amelanotic feathers from individuals of species with structural
coloration provide a unique opportunity to test the function of the basal
melanin layer in structural color production. Amelanotic individuals lack
melanin because of a disruption in the pathway of melanin synthesis that
typically has no effect on other mechanisms in the body (e.g. keratin
deposition) (Majerus, 1998).
Thus, by comparing amelanotic to normal feathers, we can determine the effect
of melanin on the color of these feathers.
We have used full-spectrum spectrometry and transmission electron
microscopy (TEM) to compare the color and nanostructure of the feathers of
amelanotic white and normal blue Steller's jays (Cyanocitta stelleri
Gmelin), and the white feathers from a domestic chicken (Gallus
gallus Linnaeus). We wanted to determine if loss of melanin could explain
the shift from blue to white color in the amelanotic feather or if other
anatomical differences such as loss of spongy layer were also involved. Thus,
we compared the nanostructure of the three feathers using TEM and the Fourier
tool for biological nano-optics (Prum and
Torres, 2003). This tool allowed us to predict the hue of feathers
through analysis of the nanostructural arrangement of the spongy layer. If
loss of melanin alone explained the observed color differences, then both the
normal and amelanotic feathers should have well-defined spongy layers with
predicted hue values in the blue wavelengths. This would suggest that both the
amelanotic and blue feathers could produce blue color, but that the lack of
melanin granules in the amelanotic feathers prevents the color from being
expressed. Alternatively, the barbs of the amelanotic feather could lack a
spongy layer and resemble barbs from a normal white feather. The white color
of the amelanotic feather could then be explained as a result of a loss of
color-producing structures, rather than as a loss of melanin granules per
se.
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20 mm2
patches of color approximately three-quarters of the feather's length away
from the proximal end of each feather were chosen for analysis. Using a block
sheath that excluded ambient light, a bifurcated micron fiber optic probe was
held at a 90° angle 5 mm from the feather surface, creating a measurement
area of 2 mm in diameter. This measurement area was illuminated by both a UV
(deuterium bulb) and a visible (tungsten–halogen bulb) light source. All
data were expressed relative to a white standard (WS-1, Ocean Optics). OOIbase
software was used to record and average 20 spectra sequentially, and these
spectra were recorded and averaged from five arbitrarily chosen points within
the selected locations on each feather.
From these reflectance curves several different color variables were
calculated. These indices were restricted to wavelengths between 320 and 700
nm, as evidence suggests that passerine birds are sensitive to ultraviolet
(UV) wavelengths (Cuthill et al.,
2000), and that 700 nm is the upper limit of the vertebrate visual
system (Jacobs, 1981). The
wavelength of maximum reflectance was used as an index of hue, the principal
color reflected by the feathers (Keyser
and Hill, 1999). Brightness, the sum of reflectance from
320–700 nm, is a measure of the total amount of light reflected by the
feathers (Andersson, 1999).
Ultraviolet (UV) and blue chromas are the summed reflectances of light in the
ranges of 320–400, and 435–500 nm, respectively, divided by
brightness, and are indices of color purity
(Andersson et al., 1998).
Feather barbs from the amelanotic and the blue Steller's Jay and the white
chicken were prepared for transmission electron microscopy (TEM) following the
methods of Shawkey et al. (Shawkey et al.,
2003) and viewed on a Phillips EM301 TEM (Veeco FEI Inc,
Hillsboro, OR, USA). Micrographs of feather barbs and a waffle-pattern
diffraction grating (Ted Pella, Redding, CA, USA) accurate to 1 nm ±5%
were taken at the same magnifications for calibration of the images.
TEM micrograph negatives were scanned at 400 d.p.i. using an Epson
PerfectionTM 1240U flatbed scanner. These micrographs were then analyzed
using Prum and Torres' Fourier analysis program for biological nano-optics
(Prum and Torres, 2003). This
MATLAB-based program uses Fourier analysis to determine whether the spongy
layer of feather barbs is sufficiently organized, and at an appropriate scale,
to produce color by coherent light scattering alone
(Prum et al., 1999;
Prum et al., 1998). Subsequent
radial analyses incorporating the estimated refractive indices of keratin
(RI=1.56) and air (RI=1.00) allow the user to obtain a predicted hue. For all
analyses, the largest available square portion of spongy layer (>500
pixels) uninterrupted by melanin granules, cell boundaries or keratin cortex
was selected. Because the barbs of white chicken feather lacked spongy layers,
the central vacuoles and the keratin surrounding them were selected for
analysis.
Because other microanatomical features of barb morphology other than the
spongy layer may affect color production
(Shawkey et al., 2005) the
program NIH Image version 1.62 (available for download at
http://rsb.info.nih.gov/nih-image)
was used to measure additional structural components of the two colored barbs.
The thickness of the keratin cortex and spongy layer was measured at six
different, evenly spaced points around the barb. Barbs from the white chicken
and jay feathers contained no melanin, so we could not measure density or size
of melanin granules for these feathers.
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;
Shawkey and Hill, 2005) with
uniformly high reflectance across all wavelengths tailing off at short
wavelengths and with no discrete peaks. The reflectance spectrum of the
amelanotic feather was similar to that of the white feather
(Fig. 2). Unlike the white
feather of a chicken or chickadee (Mennill
et al., 2003), however, the amelanotic feather had a single
shallow peak in the blue/green range followed by gradually decreasing
reflectance. The reflectance spectrum of the blue portion of the typical
Steller's jay feather was similar to that of other species with non-iridescent
blue coloration (Dyck, 1971;
Prum et al., 1999;
Shawkey et al., 2003), with a
bell shape and a peak in the UV/violet range
(Fig. 2).
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The microanatomy of the blue feather barbs was similar to that of barbs of
other species producing non-iridescent blue structural color
(Fig. 3A) (e.g.
Dyck, 1971;
Shawkey et al., 2003). The
medullary spongy layer sat beneath a fairly thick keratin cortex and above a
single layer of melanin granules surrounding hollow central vacuoles. This
spongy layer was composed of a matrix of irregularly shaped keratin and air
`bars', resembling the structure observed in the blue feathers of peach-faced
lovebirds Agapornis roseicollis
(Dyck, 1971), Eastern bluebirds
Sialia sialis (Shawkey et al.,
2003) and others (see Prum,
2006). The amelanotic feather differed from this blue feather in
two ways (Fig. 3A,B). First,
the basal layer of melanin granules was absent. Second, the keratin cortex of
the amelanotic feather was considerably thicker than that of the blue feather
(Fig. 3A,B;
Table 1). In the white chicken
feather, the spongy layer and melanin layer were completely absent, but the
cortex was about as thick as that of the blue feather
(Fig. 3C;
Table 1).
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Fourier analysis revealed that the spongy layers of the blue and amelanotic
feather barbs were sufficiently organized and at the correct scale to produce
color by coherent light scattering alone. The discrete rings in the Fourier
power spectrums (Fig. 3D,E)
indicate high levels of nanostructural organization
(Prum et al., 1998;
Prum et al., 1999). Fourier
analysis of the spongy layer of these two feathers predicted hue values close
to measured values (Fig. 3G,H;
Table 1). The predicted hue for
the amelanotic feather was 21 nm shorter than the measured hue and the
predicted hue for the blue feather was 28 nm longer than the measured hue
(Table 1). This degree of error
is comparable to that seen in other studies using this tool (Prum et al.,
2003). By contrast, the Fourier power spectrum of the chicken feather showed
no discrete shape and very low power, indicating a lack of nanostructural
organization (Fig. 3F). This
lack of organization results in a lack of discrete peaks in the radial
analysis (Fig. 3I).
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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), but as
far as we are aware, this is the first study to examine the effects of loss of
melanin on the production of blue structural color in feather barbs. Whereas
the role of melanin in non-iridescent avian structural color production has
been the subject of speculation (Simon,
1971; Prum, 1999;
Prum, 2006), no tests of the
function for this melanin layer have been conducted. Our visual assessment,
spectrometric measurements and nanostructural analyses of the white feathers
of an amelanotic Steller's jay all support the hypothesis that melanin
primarily functions to absorb incoherently scattered white light from feather
barbs. First, the amelanotic feather was white with faint blue overtones
suggesting that loss of melanin has a clear and dramatic effect on structural
color production, refuting the predictions of the null hypotheses. Our
spectrometric analyses provide more quantitative data on this effect. If loss
of melanin simply lightened the blue color of the feather, as predicted by the
backdrop hypothesis, we would expect the reflectance curve of the amelanotic
feather to have a peak but have higher reflectance values than the blue
feather. Instead, the reflectance spectra of the blue feather has a peak,
whereas that of the amelanotic curve is fairly saturated across all
wavelengths and has only the suggestion of a peak in the blue/green
wavelengths. The higher UV chroma value of the blue feather further suggests
that it reflects a more pure, saturated color. However, these observations
leave open the possibility that additional changes in the microstructure of
the amelanotic feather barbs, such as loss of spongy layer, could explain its
washed-out appearance. Our microstructural analyses, however, indicate that the amelanotic feather has a well-defined spongy layer that is organized at the proper scale to produce a blue/green color. The fact that this amelanotic feather lacks blue/green coloration and appears white to the human eye suggests that the loss of melanin from the barb allows non-specific white reflectance to swamp out the blue color. The absorption of light by the underlying melanin granules in the blue feather thus appears to be essential for expression of blue coloration.
The thicker keratin cortex of the amelanotic barb may also contribute to
the observed differences in reflectance. Previous research suggests that the
cortex primarily absorbs light in non-iridescent structural plumage color
(Finger, 1995). The thicker
cortex of the amelanotic feather could therefore reduce the amount of white
light reflected. However, the estimated extinction coefficient (a measure of
light absorption properties) of melanin is about 20 times higher than that of
keratin (Brink and van der Berg,
2004), and thus any absorption by the thicker keratin cortex would
be negligible compared to that of melanin.
The low brightness of blue feathers may also largely be caused by
absorption of light by the melanin layer within blue barbs, as predicted by
both the backdrop and absorbance hypotheses. Although it may seem obvious that
barbs containing melanin will reflect less light than barbs without melanin,
the absorption of light by melanin has been rarely considered in mechanistic
studies of structural plumage color
(Greenewalt et al., 1960;
Land, 1972). Interspecific
differences in brightness and other color variables of both iridescent and
non-iridescent structural ornaments may be affected by the presence and
concentration of melanin within barbs. Indeed, in a recent study
(Brink and van der Berg, 2004),
it was shown that the coppery iridescence of feathers of the Hadeda ibis
Bostrychia hagedash could not be properly predicted by thin-film
models without taking the absorbance of melanin into account. Here, we present
data suggesting that this absorbance may also play an important role in the
production of non-iridescent structural plumage color.
Our results also suggest that melanin density affects the brightness of
individual birds; however, in another study we found that density of melanin
granules was not correlated with brightness among individual eastern bluebirds
(Shawkey et al., 2005).
Further studies are needed to determine whether variation in melanin density
among individuals affects brightness of structurally colored feathers.
Our small sample size (N=1 for each group) clearly warrants
caution in the interpretation of our results. However, because we were
observing the effects of complete removal of melanin, many of our conclusions
are inescapable. Melanin absorbs incoherently scattered light, increasing
color purity, and also darkens feathers. Similar comparisons of normal and
amelanotic individuals of other vertebrate taxa (e.g. fish, frogs) with
three-layer dermal chromatophore units
(Grether et al., 2004) would
provide significant insight into the role that melanin plays in this type of
structural color. More theoretical and empirical work on the mechanisms that
create structural color display is needed. In birds, the development of
explicit physical models incorporating all aspects of barb structure will
greatly improve our understanding of the mechanics of structural color
production. Understanding the proximate role of melanin in structural color
production will help us understand how the basic components of almost every
feather (keratin, air and melanin) have been modified over evolutionary time
to create the amazing diversity of structural color found in birds.
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Acknowledgments |
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|
References |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Andersson, S. (1999). Morphology of UV reflectance in a whistling-thrush: implications for the study of structural colour signalling in birds. J. Avian Biol. 30,193 -204.
Andersson, S., Ornborg, J. and Andersson, M. (1998). Ultraviolet sexual dimorphism and assortative mating in blue tits. Proc. R. Soc. Lond. B Biol. Sci. 265,445 -450.[CrossRef]
Brink, D. J. and van der Berg, N. G. (2004). Structural colours from feathers of the bird Bostrychia hagedash. J. Phys. D App. Phys. 37,813 -818.[CrossRef]
Burtt, E. H., Jr (ed.) (1979). Tips on wings and other things. In The Behavioral Significance of Color (ed. E. H. Burtt, Jr), pp. 70-123. New York: Garland STPM Press.
Cuthill, I. C., Partridge, J. C., Bennett, A. T. D., Church, S. C., Hart, N. S. and Hunt, S. (2000). Ultraviolet vision in birds. Adv. Stud. Behav. 29,159 -214.
Dyck, J. (1971). Structure and colour-production of the blue barbs of Agapornis roseicollis and Cotinga maynana. Z. Zellforsch. 115, 17-29.[Medline]
Dyck, J. (1976). Structural colours. Proc. Intern. Ornithol. Cong. 16,426 -437.
Finger, E. (1995). Visible and UV coloration in birds: mie scattering as the basis of color in many bird feathers. Naturwissenschaften 82,570 -573.[CrossRef]
Gill, F. (1995). Ornithology. New York: W. H. Freeman and Co.
Goldstein, G., Flory, K. R., Browne, B. A., Majid, S., Ichida, J. M. and Burtt, E. H., Jr (2004). Bacterial degradation of black and white feathers. Auk 121,656 -659.
Greenewalt, C. H., Brandt, W. and Friel, D. (1960). The iridescent colors of hummingbird feathers. J. Opt. Soc. Am. 50,1005 -1013.
Grether, G. F., Kolluru, G. R. and Nersissian, K. (2004). Individual color patches as multicomponent signals. Biol. Rev. 79,583 -610.[Medline]
Hill, G. E. and McGraw, K. J. (2006).Bird Coloration, vol. 1. Mechanisms and Measurements . Boston, USA: Havard University Press.
Jacobs, G. H. (1981). Comparative Color Vision. New York: Academic Press.
Keyser, A. J. and Hill, G. E. (1999). Condition-dependent variation in the blue-ultraviolet coloration of a structurally based plumage ornament. Proc. R. Soc. Lond. B Biol. Sci. 266,771 -777.[CrossRef]
Land, M. F. (1972). The physics and biology of animal reflectors. Prog. Biophys. Mol. Biol. 24, 77-106.
Majerus, M. E. N. (1998). Melanism: Evolution in Action. New York, USA: Oxford University Press.
Mennill, D. J., Doucet, S. M., Montgomerie, R. and Ratcliffe, L. M. (2003). Achromatic color variation in black-capped chickadees, Poecile atricapilla: black and white signals of sex and rank. Behav. Ecol. Sociobiol. 53,350 -357.
Prum, R. O. (1999). The anatomy and physics of avian structural colours. In Proceedings of the 22nd International Ornithological Congress (ed. N. J. Adams and R. H. Slotow). Durban: Bird Life South Africa.
Prum, R. O. (2006). Anatomy, physics and evolution of avian structural colors. In Bird Coloration, vol. 1. Mechanisms and Measurements (ed. G. E. Hill and K. J. McGraw). Boston, USA: Harvard University Press.
Prum, R. O. and Torres, R. H. (2003). A Fourier tool for the analysis of coherent light scattering by bio-optical nanostructures. Integrat. Comp. Biol. 43,591 -602.
Prum, R. O., Torres, R. H., Williamson, S. and Dyck, J. (1998). Coherent light scattering by blue feather barbs. Nature 396,28 -29.[CrossRef]
Prum, R. O., Torres, R. H., Williamson, S. and Dyck, J. (1999). Two-dimensional Fourier analysis of the spongy medullary keratin of structurally coloured feather barbs. Proc. R. Soc. Lond. B Biol. Sci. 266,13 -22.[CrossRef]
Shawkey, M. D. and Hill, G. E. (2004). Feathers at a fine scale. Auk 141,652 -655.
Shawkey, M. D. and Hill, G. E. (2005). Carotenoids need structural colours to shine. Biol. Lett. 1,121 -124.[CrossRef][Medline]
Shawkey, M. D., Estes, A. M., Siefferman, L. M. and Hill, G. E. (2003). Nanostructure predicts intraspecific variation in ultraviolet–blue plumage colours. Proc. R. Soc. Lond. B Biol. Sci. 270,1455 -1460.[Medline]
Shawkey, M. D., Estes, A. M., Siefferman, L. and Hill, G. E. (2005). The anatomical basis of sexual dichromatism in non-iridescent, ultraviolet–blue structural coloration of feathers. Biol. J. Linn. Soc. 84,259 -271.[CrossRef]
Simon, H. (1971). The Splendor of Iridescence. New York, USA: Dodd.
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