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First published online March 27, 2009
Journal of Experimental Biology 212, 1225-1233 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.026963
A multifactorial test of the effects of carotenoid access, food intake and parasite load on the production of ornamental feathers and bill coloration in American goldfinches
Department of Biological Sciences, Auburn University, Auburn, AL 36830, USA
* Author for correspondence (e-mail: ghill{at}auburn.edu)
Accepted 28 January 2009
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: carotenoids, melanins, plumage color, body composition, indicator mechanism
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). These so-called indicator traits are proposed to reveal
specific aspects of individual condition, knowledge of which benefits a
receiver such as a female choosing a mate
(Andersson, 1994;
Hill, 2002). While a general
link between condition and ornament expression has been established for many
traits, the relative importance of and the interaction between different
environmental challenges in shaping the expression of ornamental traits
remains essentially unstudied.
Integumentary coloration is a complex trait that seems to encode a variety
of information about the condition of individuals
(Hill and McGraw, 2006). Two
classes of pigments are responsible for much of the coloration in plumage,
bills and the bare parts of birds. Melanins produce the black, brown and rusty
coloration of feathers (McGraw,
2006b) whereas carotenoid pigments are responsible for most of the
yellow, orange and red coloration
(Goodwin, 1984). The
mechanisms of production of these different types of color displays are likely
to affect the manner in which each responds to specific environmental
challenges. Carotenoid pigments cannot be synthesized by birds or any
vertebrates and must be ingested (McGraw,
2006a; Völker,
1934). Thus, carotenoid coloration can potentially vary with
access to appropriate dietary pigments needed for coloration
(Hill, 2002;
Hill, 2006). Within the bodies
of birds, carotenoids must be absorbed, transported and deposited, and these
processes of utilization require energy and might be disrupted by various
environmental perturbations (Hill,
2002; Hill, 2006).
Finally, while carotenoids cannot be synthesized, they can be biochemically
modified by birds once they are ingested
(McGraw, 2006a). For instance,
some species can convert yellow dietary pigments to red pigments before they
deposit them in feathers (McGraw et al.,
2001; Stradi et al.,
1997).
Melanin pigments are synthesized within the bodies of birds from the amino
acid tyrosine (McGraw, 2006b).
Dietary tyrosine can be used directly to synthesize melanin, or phenylalanine
can be converted into tyrosine, which can then be used to synthesize melanin
(McGraw, 2006b). Thus, while
melanin pigmentation is not dependent on dietary pigments as is carotenoid
pigmentation, individuals must ingest enough of the right type of amino acids
to produce maximum color expression, so nutrition has the potential to affect
pigmentation. The need to ingest specific minerals during molt might also
affect the expression of melanin coloration
(McGraw, 2003;
McGraw, 2007;
McGraw, 2008).
To date, studies on the signal content of melanin and carotenoid
pigmentation have focused on the singular effects of specific environmental
factors on the expression of these different types of pigment coloration.
These studies clearly show that the environment in which feather and bill
coloration are produced can have a large impact on color expression. Access to
quantities of specific types of dietary carotenoid pigments at the time of
molt has been shown to have a significant effect on the expression of red,
orange and yellow coloration in many species of birds in captivity (reviewed
by Hill, 2006). In a study of
wild house finches (Carpodacus mexicanus), the concentration of
carotenoids in the diet of males was positively correlated with the redness of
the feathers that they were growing (Hill
et al., 2002). Infection by various parasites has also been shown
to depress the expression of carotenoid coloration. Male house finches and
American goldfinches (Carduelis tristis) infected with coccidia
(Isospora spp.) had less red and less saturated plumage coloration
than males kept free of coccidiosis
(Brawner et al., 2000;
McGraw and Hill, 2000). In
addition, infection with the bacterium Mycoplasma gallicepticum at
the time of molt caused male house finches to grow less red and less saturated
plumage compared with control males (Hill
et al., 2004). The same bacterial and coccidial infections that
depressed the expression of carotenoid coloration had no effect on the color
quality of either ornamental or non-ornamental melanin pigmentation of
American goldfinches and house finches, respectively, or on the size of
melanin crown patches in American goldfinches
(Hill and Brawner, 1998;
McGraw and Hill, 2000;
McGraw et al., 2005). And
finally, restricting food access during molt caused male house finches and
American goldfinches to grow less red and less saturated plumage
(Hill, 2000;
McGraw et al., 2001) but the
same food restriction had no effect on color quality of non-ornamental tail
feathers of house finches (Hill,
2000; McGraw et al.,
2001) or the size or color quality of house sparrow (Passer
domesticus) badges (McGraw et al.,
2002). Restricting the availability of specific amino acids in the
diets of house sparrows, however, caused males to grow badges with lower
achromatic brightness than controls
(Poston et al., 2005). Amino
acid restriction had no effect on the badge size of male house sparrows
(Poston et al., 2005).
These single-factor experiments identified specific environmental factors that can affect the expression of ornamental coloration. From these single-factor experiments, however, it is impossible to know the relative importance of pigment access, parasite load and nutrition in determining color expression. Moreover, these environmental challenges do not act on individuals independently but rather they interact in complex manners. To gain a more comprehensive understanding of how environmental factors shape color expression, a multifactorial design is needed.
We simultaneously tested the effects of pigment access, parasite load and
food access on ornamental yellow carotenoid and black eumelanin pigmentation
in male American goldfinches (Carduelis tristis Linnaeus 1758). A
primary goal of this experiment was to test for links between the expression
of ornamental coloration and condition; however, condition is often loosely
defined in studies of ornamental traits. Chemical analyses of body composition
are the most accurate and direct measures of an individual's stored resources
and muscle and organ development. Therefore, we directly measured the body
composition of birds in our treatment groups and used body composition as an
index of condition. We focused on both feathers, in which pigment coloration
is fixed at the completion of molt, and bill color, which can respond to
factors such as stress and carotenoid supplementation within days
(McGraw, 2006a).
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). We
retained only first-year males for this study, releasing females and older
males. In this way, we standardized for the effect of sex and age on
coloration. Birds were collected and handled according to federal (# 21661),
state (#97181) and IACUC (2005–0825) permits.
General design
Within one week of capture, we placed the birds in small cages
(0.5x0.5x0.5 m) in rooms with large windows that emitted abundant
natural light, allowing the birds in the present study to molt on a natural
light regime. We randomly assigned two birds to each cage, and we assigned
each cage to a carotenoid, disease and nutritional treatment as follows. Half
of the males were provided with a high dose of carotenoids in their water and
half of the males were provided with a moderate dose of carotenoids in their
water. Half of the males were given continuous treatment with the
anti-coccidial drug sulfadimethoxine and half were given sulfadimethoxine two
out of every three days. Half the males were provided with ad libitum
food and half had the food removed from their cages periodically during molt.
A total of eight treatment combinations were possible and our design called
for eight replicates of each treatment combination, so we maintained 32 cages
of birds housing 64 individuals (Table
1).
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Carotenoid treatment
Carotenoids were provided as a 70:30 mix of lutein and zeaxanthin following
Navara and Hill (Navara and Hill,
2003). Males in half of the cages were provided with carotenoids
in the form of starch gel beadlets dissolved in water at a concentration of
1000 mg of beadlets per liter of drinking water, which was the high-carotenoid
supplementation and half were provided with 10 mg of beadlets per liter of
drinking water, which was the low-carotenoid treatment. High- and
low-carotenoid levels were chosen based on the response of male goldfinches to
various doses of supplemental carotenoids in Navara and Hill
(Navara and Hill, 2003).
Parasite treatment
As a means to manipulate the degree to which male goldfinches were
parasitized, we added to the drinking water either a constant or pulsed dose
of sulfadimethoxine (0.26 g l–1), a broad-spectrum
antimicrobial drug that depresses a wide range of parasitic microbes
(Cates, 1986). For the pulsed
dose, sulfadimethoxine was withheld every third day. Our target parasite was
isoporan coccidia, which we knew from previous studies depresses feather
coloration in American goldfinches (McGraw
and Hill, 2000) and responds to sulfadimethoxine; however, we
expected treatment with sulfadimethoxine to potentially affect a range of
parasites. Sulfadimethoxine is broad-spectrum and microbiostatic rather than
microbiosidal (Chambers and Jawetz,
1998), meaning that this class of drugs depresses the biological
activity of microbial pathogens but does not kill them. We assumed that the
pulsed dose would allow parasites to persist at higher levels than at the
constant dose. To check the effect of sulfadimethoxine treatment on coccidial
infection, one month after males were assigned to a treatment, when all
individuals were undergoing molt, we collected a fecal sample from each male
after 15:00 h and screened the collected fecal samples for coccidial oocysts
following Brawner and colleagues (Brawner
et al., 2000).
Food treatment
Males were either given unlimited access to food or had all food removed
from their cages during mornings or afternoons. We staggered food removal
between mornings and evenings following Hill
(Hill, 2000), such that birds
in the food-restricted treatment group had no access to food for 38% of
daylight hours during molt. On no-food mornings, food dishes were removed just
before dark on the evening before and returned at the midpoint of daylight the
following day. Alternatively, for afternoons with no food, food was removed at
the midpoint of daylight and returned to cages just before sunrise the
following morning.
Body composition and feather collection
After all of the birds had completed growth of yellow and black feathers,
all food was removed from the cages on 29 April 2005 at sunset to ensure that
birds would be post-absorptive the next morning. On 30 April 2005, males were
removed from the cages and killed. We immediately weighed each bird and took a
digital image of the bill with color references in the image. We pulled
approximately 20 feathers from the crown and 20 feathers from the upper breast
of each male and measured the cap size as the length of black feathering from
the bill to the back of the cap. Carcasses were then placed in airtight
plastic bags and frozen.
At a later date, carcasses were thawed to determine the fat and lean mass. Birds were weighed and then homogenized in a Waring Laboratory Blender (Torrington, CT, USA), dried to a constant mass for approximately 76 h in a laboratory oven at 60°C and then blended with a Braun coffee grinder (Proctor and Gamble, South Boston, MA, USA) to improve homogeneity. Carcasses were then dried again for an additional 76 h to determine the final dry mass. Care was taken to account for all tissue lost during homogenization. The fat content of homogenized samples (1.00±0.15 g each) (±s.e.m.) was determined in duplicate in a soxhlet apparatus (Pyrex Brand, Corning, Lowell, MA, USA). Samples were sealed in paper tea bags, with the top of the bag folded and stapled to reduce fine particulate loss. The full tea bag was then placed in an alundum extraction thimble within the soxhlet extraction tube, and lipids were extracted with petroleum ether for approximately 12 h. After all of the ether had evaporated, the bagged sample was removed from the soxhlet, the sample was air-dried overnight and dried for approximately 3 h at 60°C before determining the final fat-free dry mass. We calculated percentage fat (fat mass/body massx100) as an indicator of relative energy reserves and lean dry mass [(total dry mass–fat mass)/(dry massx100)] as a measure of total muscle and organ mass.
Color measurements
We measured the color quality of yellow feathers using a reflectance
spectrophotometer following standard techniques as described in Shawkey and
colleagues (Shawkey et al.,
2006). Briefly, we took reflectance measurements with an Ocean
Optics S2000 spectrometer (range 250–880 nm: Dunedin, FL, USA) using a
bi-furcated micron fiber optic probe at a 90 deg. angle 5 mm from the feather
surface. A 2 mm area was illuminated with both UV (deuterium bulb) and visible
(tungsten–halogen bulb) light sources. Reflectance data were generated
relative to a white standard (Labsphere, North Sutton, NH, USA).
We calculated color variables from spectral reflectance data between 320 and 700 nm. We calculated hue as the point of maximum inflection of the curve, brightness as the mean reflectance between 320 and 700 nm and UV chroma and yellow chroma as the percentages of total light reflected in the range of 320–400 and 575–600 nm, respectively.
We were unable to take bill color measurements with the spectrometer when birds were killed at the end of the experiment, and we suspected that bill coloration would fade in frozen birds. Therefore, we used digital images with a color standard taken of the right side of each bird within 5 min of death for color analysis. We used Adobe Photoshop color sampler tool (Adobe Photoshop CS3 extended, v. 10.0, Adobe Systems, San Jose, CA, USA) to quantify yellow hue, saturation and brightness at three points on the lateral lower mandible and medium gray to black (hereafter, black) pigmentation at three points at the tip of the upper mandible. The assistant who made these measurements did not know the treatment grouping of any of the males and was instructed to sample the most intense areas of yellow pigmentation and the darkest areas of melanin pigmentation, thereby eliminating the possibility of quantifying scuffed portions of the bill or regions with glare as may have happened if the points were chosen randomly. We averaged the color measurements from each bill to arrive at single yellow hue, chroma (saturation) and brightness and a single black brightness for each bird. For melanin pigmentation of bills, we were interested in achromatic brightness so we excluded hue and chroma measurements. We included the same yellow and gray color swatches in each image, measured the hue, chroma and brightness of each and used these measurements to standardize all photographs based on the deviation of each standard from the mean hue, chroma and brightness of each swatch. Because we used standardized lighting for all digital images, only minor adjustments between images were necessary.
Units generated with Photoshop differ from those generated from reflectance spectrophotometry values and thus spectrophotometry and Photoshop values should not be considered comparable. In Photoshop, yellow–orange hue is a measure of the rotation around a color wheel (0–360 deg.); high values are closer to green wavelengths and lower values are closer to red wavelengths. Saturation is scaled from 0 to 100% with 0% dull and 100% fully saturated. Brightness is also scaled from 0 to 100% with 0% black and 100% white (Adobe Photoshop CS3 extended, v. 10.0 Manual). Digital photographs record only human visible coloration, so no analysis of UV coloration was possible for bills.
Digital photographs were also used to compare the relative area of bills with dark melanin pigmentation. Extent of melanin pigmentation was based on the right profile of the bird. Each photograph was opened in `ImageJ' software (National Institutes of Health, Bethesda, MD, USA); measurements were standardized to a 1 cm ruler in each photograph. The polygon tool was then used to determine the area of the bill and the area that had conspicuous melanin pigmentation.
Statistics
All statistical analyses were completed using SAS 9.1.3 (SAS Institute,
Cary, NC, USA). We used analysis of variance [ANOVA (proc GLM)] to determine
if there were differences in the number of coccidia oocytes between treatment
groups. We used factorial ANOVA (proc GLM) to examine the effect of treatment
(high or low carotenoids, continuous or pulsed sulfadimethoxine and ad
libitum or restricted food access) on breast coloration, bill coloration,
cap brightness, cap size and body composition variables, with an independent
test run for each dependent variable. Because birds were held as pairs in
cages, we tested for a cage effect by including cage as a covariate in initial
analyses. We found no significant effect of cage in any comparison
(P>0.17), so we removed cage from our final analyses. All
proportional data were arcsine transformed, including all feather chroma
measurements, body fat and lean body mass measurements and bill coloration
chroma and saturation measurements. We then used an a posteriori Eta-squared
test, which quantifies the proportion of total variation within the model
explained by each treatment (Olejnik and
Algina, 2000; Olejnik and
Algina, 2003). The relationship between body mass and body
composition variables and breast coloration and bill coloration variables were
examined using multiple regressions. Single-variable regressions were used to
examine the relationship between body mass and body composition variables and
cap size.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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).
Sulfadimethoxine is a broad-spectrum antimicrobial that might affect a wide
range of microbial pathogens in American goldfinches
(Cates, 1986), so we retained
drug treatment groupings in our statistical analyses. Hereafter, we refer to
the sulfadimethoxine treatment as the `drug treatment' due to the non-specific
nature of this drug.
Treatment effects on yellow feathers
Carotenoid access had significant positive effects on the yellow hue and
yellow chroma of carotenoid-based breast feathers
(Fig. 1,
Table 2). Based on the results
of the Eta-squared analyses, carotenoid treatment accounted for 44.4% of the
variation in yellow hue and 28.7% of the variation in yellow chroma
(Fig. 2). Food access and drug
treatment did not independently influence yellow body variables. There was,
however, a significant interaction between carotenoid access and drug
treatment and yellow hue (Table
2). In this interaction and in other treatment interactions that
had significant effects on color expression, we observed that multiple
environmental challenges do not necessarily have simple additive effects on
color. For example, we predicted that the low carotenoid and low drug
treatment birds would have the lowest hue among the four treatments in this
interaction and that the high carotenoid and high drug treatment group would
have the highest hue values. What we observed, however, was a ranking of
treatment combinations from the lowest color expression to the highest as
follows: (1) low carotenoids, low drug treatment
[
=490.4±0.1 (±s.e.m.)],
(2) low carotenoids, low drug treatment
[=491.4±0.4 (±s.e.m.)],
(3) high carotenoids, high drug treatment
[=493.3±0.6 (±s.e.m.)]
and (4) high carotenoids and low drug treatment
[=493.8±0.2
(±s.e.m.)].
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For the UV component of breast coloration, the UV hue did not vary with
treatment but treatment did significantly affect UV chroma with a significant
interaction between carotenoids and food access
(Table 2) {interactions ranked
from lowest to highest color expression: (1) high carotenoids, ad
libitum food [=18.7±0.9
(±s.e.m.)], (2) low carotenoids, restricted food
[=19.7±0.6 (±s.e.m.)],
(3) low carotenoids, ad libitum food
[=20.4±0.8 (±s.e.m.)]
and (4) high carotenoids, restricted food
[=21.2±0.5 (±s.e.m.)]}.
Brightness of body feathers was not impacted by carotenoids, food access or
drug treatment independently; however, interactions between carotenoids and
drug treatment {ranked from lowest to highest: (1) high carotenoids, low drug
treatment [=12,104±745
(±s.e.m.)], (2) low carotenoids, high drug treatment
[=14,072±765
(±s.e.m.)], (3) high carotenoids, high drug treatment
[=14,910±1242
(±s.e.m.)] and (4) low carotenoids and low drug treatment
[=15,825±1219
(±s.e.m.)]}, carotenoids and food access {ranked from lowest to
highest: (1) high carotenoids, ad libitum food
[=12,459±863
(±s.e.m.)], (2) low carotenoids, restricted food
[=14,232±862
(±s.e.m.)], (3) high carotenoids, restricted food
[=15,010±1320
(±s.e.m.)] and (4) low carotenoids, ad libitum food
[=15,948±1308
(±s.e.m.)]} and drug treatment and food access were significant
(Table 1) {ranked from lowest
to highest: (1) high drug treatment, ad libitum food
[=13,196±803
(±s.e.m.)], (2) low drug treatment, restricted food
[=13,285±862
(±s.e.m.)], (3) low drug treatment, ad libitum food
[=15,378±1542
(±s.e.m.)] and (4) high drug treatment, restricted food
[=16,863±1151
(±s.e.m.)]}.
All significant interactions account for no more than 10.9% of the variation in color (Fig. 2).
Treatment effects on bill coloration
Treatment influenced all measures of bill coloration including yellow hue
and yellow chroma and both yellow and black brightness
(Table 3); however, treatment
did not influence the proportion of the bill with black pigmentation
[=4.86±0.41% (±s.e.m.);
factorial ANOVA, F7,40=1.13, P=0.363]. Drug
treatment significantly impacted yellow hue
(Table 3;
Fig. 3A), with birds receiving
a continuous dose of sulfadimethoxine displaying a greater hue than those
receiving a pulsed dose; drug treatment accounted for 18.0% of the variation
in yellow hue (Fig. 5A). Food
access also significantly impacted yellow hue and both yellow and black
brightness with animals on restricted food access, displaying a bill with low
yellow hue and greater yellow and black brightness
(Table 3;
Fig. 3B,
Fig. 4B,D). This treatment
accounted for 6.4% of the variation in bill hue and a large proportion of the
variation in yellow (39.9%) and black brightness (27.3%)
(Fig. 5A,C,D). Carotenoid
access influenced the yellow chroma of the bill; animals on the high
carotenoid treatment displaying greater chroma than animals on the low
carotenoid treatment (Table 3;
Fig. 3C); carotenoid treatment
accounted for 31.2% of the variation in chroma
(Fig. 5B). There were also
significant interactions between brightness and carotenoid access and food
access {ranked from lowest to highest: (1) high carotenoids, ad
libitum food [=12,459±863
(±s.e.m.)], (2) low carotenoids, restricted food
[=14,232±862
(±s.e.m.)], (3) high carotenoids, restricted food
[=15,010±1320
(±s.e.m.)] and (4) low carotenoids, ad libitum food
[=15,948±1308
(±s.e.m.)]} and the three-way interaction between carotenoid, food
access and drug treatment {ranked from lowest to highest: (1) high
carotenoids, low drug treatment, ad libitum food
[=42.9±7.1 (±s.e.m.)],
(2) low carotenoids, high drug treatment, ad libitum food
[=52.0±2.6 (±s.e.m.)],
(3) low carotenoids, low drug treatment, ad libitum food
[=57.1±4.1 (±s.e.m.)],
(4) high carotenoids, high drug treatment, restricted food
[=59.8±16.2 (±s.e.m.)],
(5) high carotenoids, high drug treatment, ad libitum food
[=62.4±5.3 (±s.e.m.)]
and (6) high carotenoids, low drug treatment, restricted food
[=76.4±2.7 (±s.e.m.)]}.
Significant interaction terms accounted for no more than 6.35% of the
variation in bill coloration (Fig.
5).
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Treatment effects on black feathers and condition
There was no significant effect of treatment on cap brightness
[=2135±100 (±s.e.m.);
factorial ANOVA, F7,40=2.09, P=0.067] or cap size
[=14.15±0.44 (±s.e.m.);
factorial ANOVA, F7,40=0.74, P=0.638]. Likewise,
we found no significant effect of treatment on body mass
[=14.30±0.23 (±s.e.m.);
factorial ANOVA, F7,40=0.84, P=0.558], percentage
body fat [=10.1±0.5
(±s.e.m.); factorial ANOVA, F7,37=1.05,
P=0.415] or percentage lean dry mass
[=78.7±1.0 (±s.e.m.);
factorial ANOVA, F7,37=0.95, P=0.481].
Relationships among color and condition variables
Linear regressions examining the relationship between breast feather
coloration and bill yellow coloration indicate that the hue and brightness of
these structures are independent (hue: F1,44=0.21,
P=0.648; brightness: F1,44=0.26,
P=0.610) whereas there is a significant positive relationship between
breast feather coloration and bill yellow chroma
(F1,44=27.6, P<0.001,
R2=0.386, bill chroma=(20.72xfeather
chroma)–1.63). Breast coloration, bill coloration and cap size were
independent of body mass, percentage body fat or percentage lean dry mass
(breast coloration: body mass: F5,38=1.54,
P=0.201; percentage body fat: F5,38=0.48,
P=0.792; percentage lean dry mass: F5,38=0.40,
P=0.849; bill coloration: body mass: F7,36=0.47,
P=0.853; percentage body fat: F7,36=0.96,
P=0.477; percentage lean dry mass: F7,36=1.34,
P=0.260; cap size: body mass: F1,42=1.20,
P=0.279; percentage body fat: F1,42=1.27,
P=0.267; percentage lean dry mass: F1,42=0.57,
P=0.455).
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Hill, 2006). In wild
populations, we would expect most individual birds to be subjected to all
three environmental challenges during feather growth. Yet the relative
importance of these factors to feather and bill coloration had not been
previously studied. The three-factor experiment that we report in the present
study is the first attempt to assess both the relative importance and
interactive effects of pigment access, food access and parasite load on
feather and bill coloration. In addition, we determined whether coloration was
correlated with body condition variables, including body mass, body fat (used
as an indicator of energy reserves) and total body lean mass (indicative of
cumulative muscle and organ condition). Our results confirm some central
themes of prior research but also reveal new patterns.
Multiple factors and feather color
In our three-treatment experiment, the amount of carotenoid pigment
ingested during molt had an overriding effect on the hue and chroma of yellow
feathers of male American goldfinches; males that ingested more carotenoids
grew feathers that were more intensely pigmented and had hues shifted toward
orange. Contrary to the results of single-variable studies, we found that
access to food and protection from pathogens had no significant effect on any
aspect of yellow feather coloration. Various interactions among treatments had
small effects on the coloration of yellow feathers accounting for less than
11% of the variation in any color parameter. In previous single-factor
studies, carotenoid access had a large effect on the expression of
carotenoid-based feather coloration (Hill,
2002; Hill, 2006),
and in the present study we show that the effects of pigment access on yellow
feather coloration can swamp the effects of nutrition and drug treatment.
These observations indicate that, at least under some conditions, pigment
access can be the most important environmental factor in determining
expression of carotenoid-based plumage coloration.
Interaction effects were much smaller than the effects of carotenoid supplementation alone but the interactions that we observed among treatments indicate that the relationships among environmental variables are complex. Because the response to multiple variables is not additive in predicted directions, multiple challenges in some cases may act to dampen rather than enhance coloration variation between treated and untreated individuals.
Multiple ornaments, multiple signals
The response of bill coloration to treatments was distinctly different from
the response of yellow feather coloration, revealing a complex interplay among
the three treatments. As with feathers, access to carotenoid pigments had a
significant effect on the color of bills but the effect was essentially
restricted to yellow chroma; there was only a small effect of carotenoid
pigments on brightness and none on hue. Yellow hue was affected primarily by
drug treatment and secondarily by food access. Food access had the biggest
effect on both yellow and black brightness with small additional effects of
carotenoid pigment access. In contrast to the conclusions regarding plumage
coloration, these observations indicate that carotenoid intake, food access
and likely parasite load all shape expression of bill coloration in American
goldfinches.
These effects of carotenoid supplementation and food access on bill
coloration in American goldfinches are the opposite of the patterns found in a
study of the coloration of the feathers of great tits (Parus major)
by Senar and colleagues (Senar et al.,
2008). In the tit study, intake of lutein affected the hue but not
the chroma of feathers and body condition affected the chroma but not the hue.
The observations of Senar and colleagues
(Senar et al., 2008) concern
feathers rather than bills and in the great tit, dietary pigments are
deposited in feathers unchanged. In American goldfinches, lutein and
zeaxanthin are converted into canary xanthophylls before being deposited.
These differences in carotenoid processing may account for the differences in
the response of coloration in the two studies but it may be a general feature
of carotenoid systems that the importance of specific environmental factors to
color expression differs by circumstance.
The different responses of feather and bill coloration support the idea
that bill and feather coloration are fundamentally different traits in
songbirds, even if both are produced through carotenoid pigmentation
(Hill, 2002;
Hill, 2006). Indeed, not only
did bill and feather coloration of male goldfinches respond differently to
treatments but within individuals, bill coloration was a poor predictor of
feather coloration. There was no significant relationship between either the
hues or brightnesses of bills and feathers. Only yellow chroma was
significantly correlated between feathers and bills. The very different
response of feathers and bills supports the idea that birds have multiple
ornaments like colored bills and feathers because the different ornaments
signal different aspects of condition
(Møller and Pomiankowski,
1993).
It is particularly interesting that the hue of bill coloration was
significantly affected by drug treatment (presumably through the action of
some unmeasured pathogen) whereas yellow feather coloration was not. It has
been proposed that birds like American goldfinches trade-off the use of
carotenoid pigments for enhancement of the immune system versus for
color display (Lozano, 1994;
Moller et al., 1999;
von Schantz et al., 1999). To
date, all attempts to link feather coloration with oxidative stress have
failed (Fitze et al., 2007;
Isaksson et al., 2005;
Navara and Hill, 2003). By
contrast, several studies have shown that activating the immune system or
inducing oxidative stress depresses bill coloration
(Bertrand et al., 2006;
Blount et al., 2003;
Faivre et al., 2003;
McGraw and Ardia, 2003;
Perez et al., 2008). It is
interesting in this regard that drug treatment affected the hue of goldfinch
bills but not feather coloration.
Melanins versus carotenoids
In contrast to the strong effects of treatment on carotenoid coloration of
both feathers and bills, we found no effect of our treatments on the
brightness or size of the bold black melanin caps of the male goldfinches.
These observations are consistent with a previous studies of the effects of
coccidiosis on feather coloration in American goldfinches in which severe
coccidial infection depressed the hue and chroma of feathers but did not
affect cap size or blackness (McGraw and
Hill, 2000) as well as other experimental studies on other
songbirds showing the melanin pigmentation is not directly affected by food
access or parasite load (reviewed by Hill,
2006). A study of house sparrows
(McGraw et al., 2002) showed
that the size of melanin badges is mediated by social interactions, a variable
not manipulated or recorded in our study.
The black cap plumage of male American goldfinches was not affected by any
of the variables that we manipulated in this experiment but black melanin
pigmentation in the bill was. In the spring, male American goldfinches replace
dark melanin pigmentation of bills with yellow and orange carotenoid
pigmentation as they come into breeding condition
(Mundinger, 1972). By the end
of our experiment, all birds in our study had mostly orange bills with the
majority of melanin withdrawn but most birds retained some black melanin
pigmentation at the tip. We found that the amount of melanin pigmentation at
the tip of the bill was not related to any treatment but the brightness (best
thought of as a measure of blackness in this context) of the bill, which
reflects melanin pigment concentration, was significantly affected by food
access. The mechanisms by which food affected bill blackness is unknown, but
it seems probable that better nutrition accelerated the transition to nuptial
condition in some males causing more melanin to be withdrawn.
Body condition
In response to a challenging environment, birds may change how energy and
other nutritional resources are partitioned between the maintenance and the
development of yellow- and black-pigmented feathers. A change in energy
partitioning can affect overall body composition
(Lopez and Leeson, 2008) but
the interaction between body condition and coloration has largely been
overlooked. Although relative carotenoid intake is unlikely to have an effect
on body condition, both parasite load and food intake can independently affect
body composition (Daan et al.,
1990; Delahay et al.,
1995; Lopez and Leeson,
2008). Interestingly, in all prior studies on the effect of food
access on coloration, body mass did not differ between groups
(Hill, 2000;
McGraw et al., 2001;
McGraw et al., 2002). This
suggests that animals with restricted intake were able to metabolically
compensate for periods without access to food. Body mass was only reported in
one prior study on parasites and coloration. McGraw and colleagues
(McGraw et al., 2005) found
that parasite treatment reduced body mass in the American goldfinch,
suggesting that variation in color expression may be a secondary effect of
change in body condition, rather than a direct effect of the parasites.
In our experiment, the body mass and composition of birds (percentage body fat and percentage lean dry mass) did not vary with treatment and was not correlated with breast or bill coloration or cap patch size. These observations suggest that food access and drug treatment are affecting carotenoid coloration through mechanisms other than body condition. One caveat to these conclusions is that all animals were sacrificed at the end of the molt of feathers after the majority of the feathers had been replaced. It remains possible that body condition earlier in molt impacted coloration.
Aviary versus field studies
In studies of color production in captive animals, environmental challenges
must be manipulated in an artificial and somewhat contrived manner. Parasite
and food manipulations were demonstrably mild in this study. We could find no
effect of our variable drug treatment on degree of coccidiosis, our target
effect. We have to assume that the higher dose of sulfa drugs in one treatment
reduced one or a suite of unmeasured parasites leading to the effect that we
observed but we cannot rule out the possibility that the drug itself caused
the effect. Our food manipulation treatment caused no significant change in
body composition between birds in the two groups, showing that it was a very
mild nutritional stress. In previous studies, this same food removal technique
had a significant effect on carotenoid feather coloration in house finches
(Hill, 2000). The greater
effects of food restriction in this previous study were probably a consequence
of it being conducted in outdoor aviaries where weather subjected birds to
greater thermal stress. Despite what appears to have been a modest
manipulation of parasite exposure and food intake, parasite exposure was the
treatment with the largest effect on bill hue and food intake was the
treatment with the largest effect on bill brightness. These observations
underscore the value to birds of multiple ornaments and to ornaments such as
bill coloration that can reflect small differences in the condition of
individuals.
The insights from the present study are important but are necessarily limited to the context of birds in cages. The obvious next step in this line of investigation is to conduct a similar multifactorial study on wild birds in natural habitats. Such a study will require special circumstances because individual birds will need to be tracked over months. Some means will have to be found to sample the food intake, carotenoid intake and parasite loads of the birds during molt. Such a study would be difficult on many of the birds traditionally studied with regard to carotenoid and melanin pigmentation but there do exist populations of other species that can be more easily tracked and repeatedly sampled, and it is to these species that future studies should look.
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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