Content uploaded by Matthew B Toomey
Author content
All content in this area was uploaded by Matthew B Toomey on Nov 03, 2015
Content may be subject to copyright.
ORIGINAL PAPER
Effects of carotenoid supplementation and oxidative challenges
on physiological parameters and carotenoid-based
coloration in an urbanization context
Mathieu Giraudeau
1,3
&Afton Chavez
1
&Matthew B. Toomey
1,2
&Kevin J. McGraw
1
Received: 23 July 2014 /Revised: 15 March 2015/Accepted: 16 March 2015 / Published online: 15 April 2015
#Springer-Verlag Berlin Heidelberg 2015
Abstract Worldwide urbanization continues to present new
selection pressures on organisms. Carotenoid pigmentation of
animals provides an ideal study system for identifying the
source and significance of urban impacts because it is an en-
vironmentally derived trait and carotenoid molecules have
widespread physiological, phenotypic, and fitness functions.
Prior work indicates that in some bird species, urban individ-
uals display less colorful carotenoid ornaments than rural
birds. However, few studies have experimentally identified
the causal factors that drive such a pattern of reduced
Bsexiness in the city^. We performed two common-garden
experiments with house finches, in which we manipulated
carotenoid access and exposure to oxidative stress to under-
stand how urban and desert birds respond to these drivers of
carotenoid utilization. Urban finches were less colorful than
desert birds at capture, but we found no differences between
urban and desert finches in how carotenoid provisioning or
oxidative stress affected plumage coloration. The only notable
site differences in our experiments were that (a) the oxidative
challenge caused a larger mass loss in urban compared to
desert birds (experiment 1), (b) urban birds circulated higher
levels of carotenoids thandesert birds after receiving the same
diet for 4 months (experiment 2), suggesting that, compared to
desert birds, urban finches can better assimilate carotenoids
from food or do not deplete as many carotenoids for use in
free-radical scavenging. Overall, our results fail to reveal key
carotenoid-specific physiological differences in urban and de-
sert finches, and instead implicate other ecophysiological fac-
tors that drive urban/desert differences in carotenoid
ornamentation.
Keywords Urbanization .Carotenoids .House finch .
Oxidative stress .Plumage coloration
Introduction
Recent anthropogenic activity hashad a range of environmen-
tal consequences due to, among other things, rapid urbaniza-
tion. Such consequences include higher temperature, elevated
levels of carbon dioxide and other pollutants (Shocat et al.
2006), and reduced habitat availability for wildlife (Marzluff
2001). These effects challenge the ability of organisms to per-
sist in urban environments and have served as selection pres-
sures on animals and plants that have led to changes in mor-
phology, physiology, behavior, and genetics (Shocat et al.
2006). For example, the physiological stress response is lower
and timing of reproduction is advanced in urban blackbirds
(Turdus merula) from Europe compared to rural ones
(Partecke et al. 2004,2006).
One means by which to assess the impact of these human-
induced environmental changes on organisms is to track the
expression of condition-dependent signals like songs or colors
Communicated by E. Fernandez-Juricic
Mathieu Giraudeau and Afton Chavez have participated equally in this
study.
Electronic supplementary material The online version of this article
(doi:10.1007/s00265-015-1908-y) contains supplementary material,
which is available to authorized users.
*Mathieu Giraudeau
giraudeau.mathieu@gmail.com
1
School of Life Sciences, Arizona State University,
Tempe, AZ 85287-4501, USA
2
Department of Pathology and Immunology, Washington University
in St. Louis, School of Medicine, St. Louis, MO 63110, USA
3
Present address: School of Biological Sciences A08, University of
Sydney, Sydney, NSW 2006, Australia
Behav Ecol Sociobiol (2015) 69:957–970
DOI 10.1007/s00265-015-1908-y
(Hill 1995; Hõrak et al. 2000). Carotenoid-based color is a
classic example of a condition-dependent signal of sexual at-
tractiveness and competitive ability because animals must ac-
quire carotenoid pigments from foods (i.e., a limiting re-
source) and because these pigments are also used to boost
health, as antioxidants and immunomodulators (Blount et al.
2003;McGrawandArdia2003;Alonso-Alvarezetal.2004).
Thus, animals displaying the most intense coloration are those
in the best nutritional and health condition or oxidative status
(i.e., imbalance between the levels of reactive oxygen species
and the status of the antioxidant machinery) and hence can
afford to devote substantial amounts of pigment to their plum-
age or integument (Møller et al. 2000;Petersetal.2004;
McGraw 2006).
There is evidence that carotenoid-based plumage color in
great tits (Parus major;Eevaetal.1998; Hõrak et al. 2000,
2001;Isakssonetal.2005) and house finches (Haemorhous
mexicanus,seeBResults^section) is paler in animals living in
cities than in those from natural surroundings, suggesting that
carotenoid-based plumage coloration can be used to monitor
environmental quality. To date, however, most studies on this
subject have been observational, and the abiotic or biotic
mechanism(s) driving urban–rural differences in plumage col-
oration are unclear.
There are several possible proximate explanations for why
carotenoid-based plumage color differs between urban and
rural environments. Differences in the dietary availability of
carotenoids (e.g., types and amounts in insects, seeds, etc.)
between urban and rural environments may explain urban
decreases in plumage coloration (Slagsvold and Lifjeld
1985;Eevaetal.1998,2005; Isaksson et al. 2007).
Alternatively, animals in cities may be exposed to more dis-
ease or oxidative stress (Schilderman et al. 1997; Isaksson
et al. 2005) and, due to their role as immunomodulators and
antioxidants (Edge et al. 1997;vonSchantzetal.1999;Møller
et al. 2000), carotenoids in the body may be drained and thus
comparatively less available to urban birds for use in ornate
coloration. At present, however, there is considerable debate
over the potency of carotenoids as antioxidants in birds
(Alonso-Alvarez et al. 2004; Isaksson et al. 2005,2007;
Tummelehtetal.2006; Costantini et al. 2007; Costantini
and Møller 2008). Thirdly, exposure to urban pollutants may
affect carotenoid-based coloration through an alteration of the
endocrine system and especially on steroid hormones
(Bortolotti et al. 2003). A fourth possibility is that plumage
color differences between urban and rural birds could arise
from a differential ability to physiologically assimilate carot-
enoids from food (e.g., uptake, storage, conversion of precur-
sors to those deposited into feathers; Blount et al. 2003;
Koutsos et al. 2003; McGraw and Parker 2006).
To begin to isolate causal factors behind this geographic
variation in color, we performed two common-garden exper-
iments (experiment 1 in winter and experiment 2 during the
late summer/early fall) in which we exposed urban- and
desert-caught male house finches (H. mexicanus)tovariation
in carotenoid availability [supplementation with zeaxanthin
(experiment 1) and canthaxanthin (experiment 2)] and oxida-
tive stress (paraquat treatment) under controlled captive con-
ditions. Our specific objective was to determine if and/or how
differential carotenoid intake and use abilities (i.e., in oxida-
tive challenges) explains observed urban ecological patterns
of carotenoid color expression. House finches are a model
system for studying sexual selection and condition-
dependent color signaling (Hill 2002), with males showing
striking plumage variation from brilliant red through drab yel-
low, and females preferring to mate with the reddest males
(including in our Arizona population; Toomey and McGraw
2012). House finches inhabit both natural desert and urban
environments in their native range (southwestern United
States), and desert house finches have redder plumage than
those living around humans in our study population in the
Phoenix metropolitan area (Hasegawa et al. 2014; also see
below). We predicted that if natural, local environmental fea-
tures (e.g., diet) drive geographic variation in carotenoid sta-
tus, we would see few differences in carotenoid circulation,
oxidative stress susceptibility, and plumage coloration be-
tween captive urban and desert birds fed standardized diets.
However, if genetic, developmental, or physiological varia-
tion is behind observed site differences in carotenoid status,
then we expected desert birds to show superior carotenoid
circulation and/or resistance to oxidative stress in our experi-
ments. As in our prior study of carotenoids, oxidative stress,
and immunity in house finches (McGraw et al. 2011;
Giraudeau et al. 2013), we ran this experiment two times, in
November/December (outside of the breeding or molt pe-
riods) and August (during molt), to assess whether or not there
are special seasonally restricted differential carotenoid intake
and use abilities between urban and rural birds.
Methods
Experimental procedure
Experiment 1—winter
We captured 80 male house finches from 2 to 13 November
2009 using basket and Potter traps baited with sunflower
seeds; trapping sessions lasted from 0700 to 1100 h each
morning. Urban birds were trapped on the campus of
Arizona State University (ASU) in Tempe, AZ (33°25′N,
112°55′W), which is an area that has had considerable human
presence (current city population size >160,000) for at least
50 years (Fig. 1). Desert (which we use interchangeably with
Brural^throughout) birds were captured at South Mountain
Park/Preserve (SMP, 33°21′N, 112°4′W), which consists of
958 Behav Ecol Sociobiol (2015) 69:957–970
over 16,000 acres of undisturbed Sonoran desert habitat locat-
ed 11.5 kmsouth of the large urban center of Phoenix, AZ and
21.5 km from our urban capture site (Fig. 1, see Giraudeau
et al. 2014 for a detailed description of the sites). We are
confident that these represent distinct sites and samples of
adult birds because, in a larger project involving the trapping
of approximately 2000 adult house finches during an 18-
month period in the Phoenix metropolitan area along a gradi-
ent of urbanization (with some sites separated by less than
3 km), we never trapped the same bird in two different sites.
Thus, we believe that the adult home range is limited to
<3 km.
At capture, each bird was leg banded with a numbered
United States Fish and Wildlife Service metal tag for individ-
ual identification. We determined body mass with a digital
scale (to the nearest 0.1 g) and tarsus length (to the nearest
0.1 mm) and drew a sample of blood (∼80 μL) from the alar
vein into heparinized microcapillary tubes. Plasma samples
were stored at −80 °C until laboratory analyses were per-
formed (see below).
Wild-caught finches were randomly divided into four
groups (20 birds for each treatment group, comprised of ten
desert and ten urban finches) that each received a different
carotenoid and/or oxidative stress treatment for a period of
4 weeks (24 November–21 December 2009): control (CO;
nothing added to drinking water), carotenoid-supplemented
(CA), paraquat-supplemented (PA), or both carotenoid- and
paraquat-supplemented (BO). To the drinking water of CA
finches, we added 0.756 g L
−1
of Optisharp™zeaxanthin
beadlets (DSM Nutritional Products, Belvidere, NJ).
Zeaxanthin is a common and concentrated xanthophyll carot-
enoid found in finch food, plasma, and yellow feathers
and among other body sites (e.g., liver, retina; McGraw et al.
2006; Toomey and McGraw 2009). We chose this carotenoid
dose based on a pilot study (MBT and KJM, unpublished data)
so that we could elevate plasma carotenoid levels over the
course of a month to the high end of the physiological range
of wild birds. To the drinking water of PA finches, we added
0.1gL
−1
methyl viologen dichloride (i.e., paraquat dichloride;
Sigma-Aldrich Corporation, St. Louis, MO). Paraquat is an
herbicide that has been commonly used to induce oxidative
stress in animals (Galvani et al. 2000; Salmon et al. 2001;
Wang et al. 2001; Isaksson and Andersson 2008; Almroth
et al. 2010; Meitern et al. 2013) and may have other physio-
logical side effects (e.g., gastro-intestinal, renal—Berny 2007;
on insulin—Kimura et al. 2010). Isaksson and Andersson
(2008) previously used this chemical at a dose of 0.09 g L
−1
in a study of oxidative stress in great tits. We chose a slightly
higher dosage (0.1 g L
−1
) to account for the fact that, on
average, house finches (17–22 g) are slightly larger than great
tits (16–20 g). This concentration was tested on three pilot
finches for 8 days, who showed no declines in activity level
Fig. 1 Land Use Land Cover
(LULC) within the 1-km radius
around each of our trapping sites
Behav Ecol Sociobiol (2015) 69:957–970 959
or body mass. Finally, to the water of BO birds, we added both
zeaxanthin and paraquat at the aforementioned concentra-
tions. All of the water treatments were kept refrigerated in
plastic gallon jugs, and any solution was discarded and
renewed no later than 1 week after preparation.
We measured body mass and drew blood from captive
finches as above at the beginning (day 1), middle (day 15),
and end (day 28) of the experiment. We were also interested to
test if the paraquat treatment influenced water intake (since the
paraquat treatment was provided through the drinking water),
food intake, and activity because a potential effect of the para-
quat treatment on these behaviors may influence our results on
body mass and oxidative stress levels. From days 12–27 of the
experiment, we videotaped each bird for one randomly select-
ed afternoon hour (range of observation times of day=1500–
1800 h). Later we scored these for time spent eating from the
food dish (time spent with seed in beak or chewing) and
drinking water (time spent with the beak in the water cup,
all in seconds), and the number of hops per minute (hops on
the same perch or from the first to the second perch of the cage
were counted). Ideally, we would have gathered continuous
feeding and behavioral data, but because this was not the focus
of the study, we only took a snapshot of individual activities
during the experiment.
Experiment 2—fall molt
We again used basket traps to capture 44 adult male house
finches from 28 June to 29 July 2010. Urban birds were again
trapped on the ASU campus in Tempe, AZ, but for this exper-
iment, desert birds were captured at Estrella Mountain
Regional Park (33°25′N, 112°25′W), which consists of 19,
840 acres of desert habitat located 53 km southwest of ASU
(Fig. 1). We used a different desert site in this experiment
because the trapping rate was really low in July 2010 at
South Mountain; the Estrella Mountain site provided the
added advantage of sampling birds from an even more distant,
rural area than South Mountain. As above, each bird was
banded and weighed at capture. Tarsus length was measured
and a blood sample was collected and stored at −80 °C until
laboratory analyses were performed.
Because of our smaller sample size in this study compared
to experiment 1, we limited the experiment to two treatment
groups: carotenoid-supplemented (CA, 11 desert and 12 urban
birds) and carotenoid- and paraquat-supplemented (BO, nine
desert and 12 urban birds). We avoided using carotenoid-
unsupplemented groups in this study because it was run dur-
ing the molt period and we wanted to provide sufficient carot-
enoids (above those low levels found in seed provided in
captivity; McGraw et al. 2001) for all birds to become color-
ful. Thus, this second experiment allows us to examine the
potential detrimental effects of paraquat treatment in urban
and desert birds that receive carotenoid supplementation.
Treatments were administered for a period of 15 weeks (27
July–10 November 2010, spanning the full molt period), and
to better control dosage in this experiment we hand-fed birds
known amounts of each substance, using a plastic syringe and
a feeding needle that was inserted into the bill, twice per week.
Carotenoid-supplemented birds received 0.4 mL of water con-
taining canthaxanthin (70 μg), while carotenoid- and
paraquat-supplemented birds received 0.4 mL of water con-
taining both this carotenoid amount and paraquat (0.5 μg). We
chose a slightly lower dosage of paraquat than in the first
experiment knowing that house finches in this study would
receive the treatment for 3 months, compared to 4 weeks in
experiment 1. In addition, we decided to supplement birds
with canthaxanthin during molt instead of zeaxanthin because
canthaxanthin is a component of the sexually attractive red
coloration of house finch plumage (Inouye et al. 2001;by
comparison, zeaxanthin is a yellow plumage colorant). We
measured body mass and drew blood from captive finches
as above every month, starting on the first day of the experi-
ment. At the end of the experiment, birds were euthanized by
rapid decapitation and liver samples were collected and frozen
at −80 °C for later carotenoid analyses (see more below).
Housing and husbandry
All birds were held in captivity in an animal-approved indoor
room on the ASU campus. Finches were housed individually
in wire cages (35.5 cm tall×27.5 cm wide×20 cm deep) and
received an ad libitum diet of black oil sunflower seeds (which
have low carotenoid content; McGraw et al. 2001) and water.
Room temperature was held constant at 24 °C and lighting
conditions were set to the outdoor day/night cycle.
Throughout the study, food was replenished daily and water
was replaced every other day to ensure freshness.
Plumage coloration
Plumage coloration was quantified at the start and end of the
second experiment using digital photography, following stan-
dard published methods for this species (Oh and Badyaev
2006; Giraudeau et al. 2012) and others (e.g., McGraw et al.
2003). Because house finch plumage does not reflect signifi-
cantly in the UV (McGraw and Hill 2000), techniques that
rely on visible light are sufficient to capture variation in
carotenoid-based coloration. Using a Canon PowerShot
SD1200S (Lake Success, NY), we took two separate pho-
tographs of the head, breast, and rump of each bird against
a gray-board, using identical distance from camera to ob-
ject, shutter, exposure, and flash settings for each photo-
graph and including a color/size standard in each photo to
control for any slight variations in object illumination.
Ambient lighting was kept constant by photographing
finches inside a room with no external light. Digital images
960 Behav Ecol Sociobiol (2015) 69:957–970
(JPEG, 3648×2736 pixels) were imported into Adobe
Photoshop to extract hue values for each of the three body
regions photographed. Values for the two pictures of each
bird were averaged for statistical analyses (repeatability= 0.99,
Lessells and Boag 1987) to obtain an overall plumage hue
score for each bird. Because Photoshop assigns hue values
around a 360° color wheel, with red set at 0, lower hue scores
denote redder birds.
Carotenoids and vitamin E in plasma and tissues
We chose to measure plasma levels of vitamin E, in addition to
carotenoids, because of its similar lipid solubility to caroten-
oids and because of its positive effects on carotenoid colora-
tion in another bird species (yellow-legged gull, Larus
michahellis; Pérez-Rodriguez et al. 2008). For carotenoid
and vitamin E analyses, we vortexed 20 μL thawed plasma
with 200 μL ethanol in a 1.5-mL Eppendorf snap-cap tube and
then added 200 μL 1:1 hexane/tert-butyl methyl ether
(MTBE). The mixture was then vortexed briefly again and
centrifuged for 3 min at 10,000 rpm, at which time we trans-
ferred the supernatant to a 1.5-mL screw-cap Eppendorf tube
and evaporated it to dryness under a stream of nitrogen. We
resuspended the residue in 200 μL HPLC mobile phase (meth-
anol/acetonitrile/dichloromethane, 46:46:8, v/v/v) and injected
50 μL into a Waters Alliance autosampler HPLC (Waters
Corporation, Milford, MA) fitted with a C30, 5 μm Waters
Carotenoid column (250×4.6 mm inner diameter; Waters
Corporation) and a column heater (set at 30 °C). A gradient
solvent system was run as previously described (McGraw
et al. 2006), and carotenoids were identified and quantified
by comparing their retention times and absorbance spectra to
external purified standards. In our field samples, we detected a
more complex and variable mixture of carotenoids (e.g., lu-
tein, zeaxanthin, β-cryptoxanthin, 3-hydroxy-echinenone)
than in our experiments, when birds circulated only lutein
and zeaxanthin in experiment 1 (which were highly positively
intercorrelated within samples; data not shown) as well as the
canthaxanthin we supplemented in experiment 2.
For liver carotenoid analyses in experiment 2, we weighed
a thawed, excised portion of the tissue to the nearest 0.0001 g,
ground the tissue in the presence of 1:1 hexane/MTBE using a
ball grinder (Retsch MM200), and followed the chemical ex-
traction procedures described in Butler and McGraw (2010)
prior to HPLC analyses (as above).
Oxidative stress analysis
Similar to previous studies of songbirds (Pérez-Rodriguez
et al. 2008; Isaksson et al. 2009;Alonso-Alvarezetal.2010;
Giraudeau et al. 2013) and other animals (e.g., Hunnisett et al.
1995;Callawayetal.1998; Oakes and Van Der Kraak 2003;
Almroth et al. 2005), we measured thiobarbituric acid-reactive
substances (TBARS) in plasma as an index of oxidative stress.
We used a miniaturized TBARS assay modified from a com-
mercially available kit (Oxi-Tek TBARS assay kit;
ZeptoMetrix Corp., Buffalo, NY). The TBARS assay quan-
tifies oxidative stress by mainly measuring levels of lipid per-
oxidation (even if other products such as the sialic acid present
in glycoproteins may lead to an overestimation of the lipid
peroxidation Devasagayam et al. 2003), which is a major bio-
marker of oxidative stress in animal tissues. Specifically, this
assay involves the reaction of malondialdehyde (MDA), a
naturally occurring product of lipid peroxidation, with thio-
barbituric acid (TBA) under conditions of high temperature
and acidity to generate an adduct that can be measured by
spectrophotometry. Briefly, 20 μL thawed plasma was mixed
with 20 μL 8.1 % sodium dodecyl sulfate (SDS) and 500 μL
TBA buffer reagent. The TBA buffer reagent was prepared by
mixing 50 mg thiobarbituric acid with 10 mL acetic acid and
10 mL NaOH. Samples were then vortexed and incubated at
95 °C in capped tubes for 60 min. Thereafter, the sample was
placed on ice for 10 min and centrifuged at 3000 rpm for
15 min. After centrifugation, the supernatant was removed
and absorbance of the sample was measured in duplicates at
532 nm (Bio-Tek μQuant microplate spectrophotometer).
Sample concentrations from the absorbance averages were
calculated by interpolation from a standard curve of MDA in
concentration from 0 to 100 nmol mL
−1
and are expressed in
nanomoles per milliliter of MDA equivalents.
Statistics
We used ANOVAs to test or the effects of treatment(s), geo-
graphic location, and their interactions before the start of the
experiment. We used general linear mixed models to test for
the effects of treatment(s), geographic location, time, and their
interactions on all of our variables. We have also included the
individual identity as a random factor in all of these models.
We reduced each full model stepwise by excluding the vari-
able with the highest Pvalue in each step until only P<0.05
predictors remained. Oxidative stress data were log trans-
formed to achieve normality; no transformation could normal-
ize plasma carotenoid, hue, liver carotenoid, and behavioral
data, so we rankerized them (Conover and Iman 1981)before
analyses. Data were analyzed using STATISTICA 6.0 soft-
ware (Statsoft, Tulsa, OK, USA), SYSTAT 12, and R 3.01.
Results
Experiment 1
At capture Urban house finches had significantly higher hue
scores (i.e., were less red; F
1,73
=19.60, P<0.0001), higher
body mass (F
1,80
=5.40, P= 0.02), and higher concentrations
Behav Ecol Sociobiol (2015) 69:957–970 961
of plasma zeaxanthin (Z=−3.49, P=0.0005) and β-
cryptoxanthin (Z=−2.91, P=0.004) than desert birds. Desert
males had a higher concentration of plasma 3-hydroxy-
echinenone (the main red plumage carotenoid in house
finches; Inouye et al. 2001) than urban males (Z=3.17, P=
0.02), but similar levels of lutein (P>0.5). Urban birds had
higher levels of oxidative stress (OS) than desert birds (Fig. 2,
F
1,52
=6.36, P=0.01). Urban and desert finches had similar
tarsus lengths (F
1,74
=2.71, P=0.104).
Plasmatic levels of carotenoids During the experiment, total
carotenoid levels differed by site (significant site×time inter-
action), with urban birds having higher levels of carotenoids
(Table 1, Fig. 2a). As expected, our carotenoid (CA) treatment
significantly increased total levels of plasma carotenoids dur-
ing the experiment (Table 1,Fig.2a). The carotenoid treat-
ment equally affected birds from the two sites, but the para-
quat treatment differentially impacted desert and urban birds
(Table 1,Fig.2a). Desert birds circulated lower levels of ca-
rotenoids compared to urban birds when they were given para-
quat+carotenoids whereas that was not the case in any other
groups (Table 1,Fig.2a). Finally, we did not find any signif-
icant interaction effects of the two treatments (paraquat and
carotenoid) on total plasma carotenoid concentrations
(Fig. 2a; see online supplementary material and
Supplementary material Fig. 1for the plasma zeaxanthin
results).
Body mass At the start of the experiment, body mass did not
differ between urban and desert birds (F
1,73
=0.76, P=0.39)
nor among treatments groups (F
3,73
=0.45, P=0.72). Body
mass significantly decreased over time in our experiment
(Table 1, Fig. 2b), though these changes were very small in
magnitude [e.g., CO birds lost only 0.1 g and CA birds lost
0.3 g (CA); on average, this is less than 2 % of total body
mass]. Body mass was not influenced by site or the ca-
rotenoid treatment but was affected by the paraquat treat-
ment (Table 1,Fig.2b). Birds that received paraquat lost
more mass than those from the other treatment groups
(Table 1,Fig.2b); the interactions between site and the
two treatments were not significant. Finally, the time ×
site × treatment carotenoid interaction was significant.
Within the PA group, urban birds lost significantly more
mass than desert birds (F
2,32
=7.88, P=0.0016), while in
the BO group, desert birds tended to lose more mass than
urban birds (F
2,32
=3.15, P=0.057). Lost more mass than
desert birds when they received Paraquat while the oppo-
site pattern emerged when birds received the Paraquat+
Carotenoid treatment (Table 1).
Oxidative stress The paraquat treatment significantly in-
creased OS levels from beginning to end of the experi-
ment in finches that received the paraquat+carotenoids,
while these levels stayed constant in the other treatment
groups, including the treatment group that received only
paraquat (PA) (Table 1,Fig.2c). We found no effect of
Fig. 2 Effects of paraquat (PA) and carotenoid (CA) administration on a
plasma carotenoid concentrations, bbody mass, and coxidative damages
levels during the first experiment (BO carotenoid +paraquat-
supplemented group, CA carotenoid-supplemented group, CO control
group, PA paraquat-supplemented group). Results are presented with
means (+SE)
962 Behav Ecol Sociobiol (2015) 69:957–970
Tabl e 1 Results of statistical tests examining the effects of geographic location, carotenoid, and paraquat supplementation on various physiological,
behavioral, and morphological variables in house finches during two experiments (experiment 1= winter experiment; experiment 2= molting experiment)
Dependent variables Factors Initial model Reduced model
df F P F P
Experiment 1
Plasma carotenoid levels
Time 2,209 34.77 <0.0001 36.07 <0.0001
Site 1,209 2.80 0.10 2.91 0.09
Carotenoid treatment 1,209 16.82 <0.0001 17.47 <0.0001
Paraquat treatment 1,209 0.0062 0.94 0.065 0.93
Time × site 2,209 6.18 0.002 6.42 0.002
Time×carotenoid treatment 2,209 221.70 <0.0001 230.48 <0.0001
Time×paraquat treatment 2,209 0.95 0.91
Carotenoid× paraquat treatment 1,209 0.51 0.60
Site×paraquat treatment 1,209 4.36 0.04 4.53 0.04
Site×carotenoid treatment 1,209 0.11 0.74
Time×site×paraquat treatment 2,209 1.19 0.31
Time×site×carotenoid treatment 2,209 0.0092 0.99
Time×carotenoid×paraquat treatment 2,209 0.23 0.87
Site×carotenoid×paraquat treatment 1,209 0.17 0.68
Time×site×carotenoid×paraquat treatment 2,209 0.75 0.48
Body mass
Time 2,209 38.80 <0.0001 38.80 <0.0001
Site 1,209 1.52 0.22 1.52 0.22
Carotenoid treatment 1,209 0.52 0.47 0.52 0.47
Paraquat treatment 1,209 6.76 0.01 6.76 0.01
Time×site 2,209 1.11 0.33
Time×carotenoid treatment 2,209 1.44 0.24 1.35 0.26
Time×paraquat treatment 2,209 5.90 0.003 6.10 0.003
Carotenoid× paraquat treatment 1,209 0.35 0.55
Site×paraquat treatment 1,209 0.26 0.61
Site×carotenoid treatment 1,209 0.09 0.77
Time×site×paraquat treatment 2,209 0.56 0.57
Time×site×carotenoid treatment 2,209 4.66 0.01 2.88 0.02
Time×carotenoid×paraquat treatment 2,209 2.88 0.06 2.94 0.06
Site×carotenoid×paraquat treatment 1,209 5.12 0.03 1.45 0.23
Time×site×carotenoid×paraquat treatment 2,209 3.06 0.05 1.78 0.13
Eating behavior
Site 1,66 0.01 0.92
Carotenoid treatment 1,66 0.13 0.71
Paraquat treatment 1,66 1.40 0.24
Carotenoid× paraquat treatment 1,66 0.21 0.65
Site×carotenoid treatment 1,66 0.49 0.49
Site×paraquat treatment 1,66 0.73 0.40
Site×carotenoid×paraquat treatment 1,66 0.69 0.41
Drinking behavior
Site 1,66 0.45 0.50
Carotenoid treatment 1,66 2.81 0.10
Paraquat treatment 1,66 0.05 0.81
Carotenoid× paraquat treatment 1,66 0.77 0.38
Behav Ecol Sociobiol (2015) 69:957–970 963
Tabl e 1 (continued)
Dependent variables Factors Initial model Reduced model
df F P F P
Site×carotenoid treatment 1,66 0.35 0.56
Site×paraquat treatment 1,66 1.22 0.27
Site×carotenoid×paraquat treatment 1,66 0.41 0.53
Activity
Site 1,66 1.86 0.18 1.75 0.19
Carotenoid treatment 1,66 0.23 0.64 0.26 0.61
Paraquat treatment 1,66 3.39 0.07 3.58 0.06
Carotenoid× paraquat treatment 1,66 3.92 0.06 3.85 0.052
Site×carotenoid treatment 1,66 0.34 0.56
Site×paraquat treatment 1,66 1.40 0.24
Site×carotenoid×paraquat treatment 1,66 0.19 0.66
Oxidative damage
Time 1,113 0.43 0.52 0.41 0.52
Site 1,113 5.03 0.03 5.14 0.03
Carotenoid treatment 1,113 2.43 0.13
Paraquat treatment 1,113 0.37 0.55 0.37 0.54
Time×site 1,113 0.47 0.23
Time×carotenoid treatment 1,113 1.23 0.27
Time×paraquat treatment 1,113 6.31 0.01 5.36 0.02
Carotenoid× paraquat treatment 1,113 1.14 0.71
Site×paraquat treatment 1,113 0.80 0.37
Site×carotenoid treatment 1,113 0.39 0.53
Time×site×paraquat treatment 1,113 1.56 0.22
Time×site×carotenoid treatment 1,113 2.60 0.11
Time×carotenoid×paraquat treatment 1,113 1.26 0.27
Site×carotenoid×paraquat treatment 1,113 0.005 0.94
Time×site×carotenoid×paraquat treatment 1,113 0.05 0.82
Experiment 2
Body mass
Site 1,297 2.91 0.10 2.94 0.09
Time 7,297 6.42 <0.0001 6.66 <0.0001
Paraquat treatment 1,297 0.77 0.38
Time×site 7,297 0.32 0.94
Time×paraquat treatment 7,297 0.43 0.88
Site×paraquat treatment 1,297 0.01 0.99
Time×site×paraquat treatment 7,297 0.49 0.84
Plasma carotenoid levels
Site 1,178 0.02 0.87 0.03 0.87
Time 4,178 15.39 <0.0001 15.37 <0.0001
Paraquat treatment 1,178 2.88 0.10 2.81 0.10
Time×site 4,178 18.30 <0.0001 17.79 <0.0001
Time×paraquat treatment 4,178 0.78 0.54
Site×paraquat treatment 1,178 1.86 0.18
Time×site×paraquat treatment 4,178 0.83 0.51
Carotenoid titers in liver
Site 1,34 3.39 0.07 3.43 0.07
Paraquat treatment 1,34 0.40 0.53 0.58 0.45
964 Behav Ecol Sociobiol (2015) 69:957–970
time or the carotenoid treatment on OS change during the
experiment (Table 1). We found a significant effect of site
on oxidative stress levels (due to the difference of oxida-
tive stress levels at the start of the experiment), but we did
not find a significant effect of the site on the change of
oxidative stress levels during the experiment (time×site
interaction). All interactions involving site and diet treat-
ments were not significant (Table 1).
Behavior Time spent drinking and eating were not influenced
by site, carotenoid treatment, paraquat treatment, or any inter-
actions (Table 1). These results show that the four groups of
birds that received the paraquat treatment (PA and BO in ur-
ban and desert birds) have drunk comparableamounts of para-
quat during this experiment. In addition, these results show
that potential effects of our treatments on body mass, oxida-
tive stress, or circulating carotenoid levels were not due to an
effect of the paraquat treatment on food/water intake.
However, these results should be considered very cautiously
since a post hoc power analysis (alpha level of 0.05) revealed
that the power of detecting an effect of the paraquat treatment
on the time spent eating and drinking were 0.2 and 0.11,
respectively.
The number of hops per minute was not influenced
by site, carotenoid treatment, and paraquat treatment.
However, among the birds that received carotenoid, the
number of hops tended to be reduced for those who
also received paraquat compared to the birds that did
not receive paraquat, while no effect of the paraquat
treatment was found in birds that have not received
the carotenoid treatment (Table 1).
Experiment 2
At capture Urban and desert house finches had similar hue
scores (F
1,41
=2.66, P= 0.11). Desert birds had a higher body
mass at the time of capture than urban birds (F
1,41
=5.66, P=
0.02). Urban and desert finches had similar tarsus lengths (F
1,
41
=0.65, P=0.42) and plasma vitamin E levels (t=−1.6, P=
0.12). At capture, urban finches had lower concentrations of
zeaxanthin (t=4.3, P=0.0001) and lutein (t=−2.4, P=0.02)
but similar levels of β-cryptoxanthin (Z=−0.61, P=0.54)
and β-carotene (Z=−0.93, P=0.35).
Plasmatic levels of carotenoids Total carotenoid concentra-
tions differed by site at the beginning of the experiment
(F
1,40
=21.40, P<0.0001), with desert finches having a
higher plasma carotenoid concentration than urban birds.
However, total carotenoid levels did not differ among
treatment groups prior to the experiment (F
1,40
<0.0001,
P=0.98; treatment×site: F
1,40
<0.0001, P=0.99). In addi-
tion, lutein and zeaxanthin levels did not differ among
treatment groups prior to the experiment (lutein: treatment:
F
1,40
=0.12, P=0.73; treatment×location: F
1,40
=0.20, P=
0.66; zeaxanthin: treatment: F
1,40
=0.29, P=0.59; treat-
ment×location: F
1, 40
=0.24, P=0.53).
Tabl e 1 (continued)
Dependent variables Factors Initial model Reduced model
df F P F P
Site×paraquat treatment 1,34 0.55 0.47
Oxidative damage
Site 1,143 0.54 0.47 0.54 0.47
Time 5,143 4.91 <0.001 5.04 <0.001
Paraquat treatment 1,143 0.06 0.81 0.06 0.81
Time×site 5,143 0.73 0.60
Time×paraquat treatment 5,143 1.88 0.10 1.83 0.09
Site×paraquat treatment 1,143 5.83 0.02 5.83 0.02
Time×site×paraquat treatment 5,143 0.62 0.68
Plumage hue
Site 1,64 1.66 0.21 1.63 0.21
Time 1,64 223.76 <0.0001 235.81 <0.0001
Paraquat treatment 1,64 1.23 0.28 1.20 0.28
Time× site 1,64 0.52 0.48
Time×paraquat treatment 1,64 0.35 0.56
Site×paraquat treatment 1,64 0.52 0.48
Time×site×paraquat treatment 1,64 0.12 0.73
Values in bold are statistically significant (P<0.05)
Behav Ecol Sociobiol (2015) 69:957–970 965
During the experiment, total carotenoid levels differed by
site (significant site×time interaction) but not treatment
(Table 1, Fig. 3a). Plasma levels of carotenoids decreased in
desert birds during the first month of the experiment, while
these levels increased in urban birds. This difference is ex-
plained by the fact that desert birds started the experiment with
higher levels of carotenoids. Then, at the end of the experi-
ment, carotenoid levels increased for all the birds, but this
increase was higher in urban birds (Table 1, Fig. 3a). The
interaction between site and treatment did not influence total
carotenoids levels during the experiment (Table 1;see
supplementary material and the supplementary material
Fig. 2for the canthaxanthin results).
Carotenoid titers in liver Total liver carotenoid concentra-
tion was not significantly affected by treatment, but urban
birds (in the BO group) tended to have higher liver carotenoid
levels than desert birds (Table 1, Fig. 3b).
Body mass Desert birds had higher body mass at the start of
the experiment than did urban birds (F
1,40
=8.65, P=0.005),
but birds from the different treatment groups did not differ in
mass (F
1,40
=0.1,P=0.75). Body mass significantly decreased
over time during the experiment, but did not differ by site or
treatment group (Table 1, Fig. 3c).
Oxidative stress Oxidative damage levels did not differ by
site, but tended to be affected by the paraquat treatment during
the experiment (paraquat treatment×time interaction, Table 1,
Fig. 3d). Oxidative stress increased during the first month of
experiment for CA birds and then decreased during the rest of
the experiment, whereas these levels stayed constant during
the entire experiment for BO birds. In addition, desert birds
that received the paraquat treatment had higher levels of oxi-
dative damage than birds that only received carotenoid (in
July and October), which was not the case for urban birds
(Table 1, Fig. 3d).
Carotenoid-based coloration Similar to numerous studies
where house finches molt in captivity on a generally low-
carotenoid base diet (Hill et al. 2004; Giraudeau et al. 2013),
we found that ornamental plumage was less colorful at the end
of the experiment than at the beginning.We found no effectof
site, paraquat treatment, or the interaction between these two
factors on carotenoid coloration developed during our exper-
iment (Table 1, Fig. 3e).
Discussion
To better isolate possible carotenoid-specific mechanisms and
differences in urban and desert house finches, we conducted
two common-garden experiments in which we provided
supplemental dietary carotenoid access and performed an oxida-
tive challenge to determine if, under standardized environmental
conditions, urban and desert birds exhibit physiological differ-
ences in carotenoid uptake or use abilities. During experiment 2,
we showed that urban birds circulated higher levels of caroten-
oids and tended to have higher levels of carotenoids in the liver
than deserts birds (when exposed to Paraquat) after 4 months on
the same diet, suggesting that urban birds are better able to extract
carotenoids (canthaxanthin) from food or do not use as many
carotenoids as desert birds for pigmentation, antioxidant action,
or immunomodulation. We also showed during our second ex-
periment that when urban and desert birds are supplemented with
carotenoids, they develop similar plumage color. Taken together,
these results suggest that there are no substantial urban/desert
differences in carotenoid assimilation that could translate into
the site-related plumage color differences we observe locally in
the wild (i.e., desert birds are redder).
We also showed that, compared to urban birds at capture,
desert birds had higher body mass during molt but lower mass
during winter. Similarly, desert finches circulated a lower con-
centration of zeaxanthin and β-cryptoxanthin (but a higher
concentration of 3-hydroxy-echinenone and similar concen-
tration of lutein) in winter but had higher levels of zeaxanthin
and lutein during molt compared to urban birds. Thus, it seems
that desert birds are more colorful than urban birds because of
higher body condition and superior ability to either locate
carotenoid-rich foods or assimilate those carotenoids from
the food during the molt period. These results are in accor-
dance with a recent study showing that rural populations have
higher concentrations of vitamin E and carotenoids in their
liver than urban populations in several bird species (Møller
et al. 2010). Our results on body mass and plasma carotenoids
levels in winter were more unexpected. The dietary availabil-
ity of zeaxanthin and β-cryptoxanthin may decrease in desert
environment in winter while the vegetation present in city
parks or private gardens may provide a higher availability of
these two carotenoids in urban environments. Finally, it
should be noted that the differences of body mass between
urban and desert finches at capture disappeared (before the
start of the first experiment and after 2 weeks during the sec-
ond experiment) when the birds were feeding on the same
food in captivity, strongly suggesting that none of our results
were influenced by these differences in mass at capture.
We also used an oxidative challenge to probe the antioxi-
dant value/use of carotenoids in urban and desert house
finches. Here, patterns of body mass loss during the first ex-
periment revealed one of the few experimental differences we
uncovered between sites, namely that PA treatment induced
more of a mass loss in urban than in desert birds. Because we
failed to find a significant increase in oxidative stress in PA
birds during this experiment, it is difficult to identify the
mechanism behind this difference of mass loss in this treat-
ment group. One possibility is that the urban PA birds may
966 Behav Ecol Sociobiol (2015) 69:957–970
Fig. 3 Effects of paraquat (PA) and carotenoid (CA) administration on a
plasma carotenoid concentrations, bliver carotenoid concentrations, c
body mass, doxidative damages levels in plasma, and ebreast plumage
coloration in urban and desert birds during the second experiment (BO
carotenoid+ paraquat-supplemented group, CA carotenoid-supplemented
group). Results are presented with means (+SE)
Behav Ecol Sociobiol (2015) 69:957–970 967
have been more oxidatively stressed in ways not probed by the
TBARS assay. Another hypothesis is that paraquat treatment
may have had physiological effects other than increasing ox-
idative stress, such as on intestinal absorption (Berny 2007).
However, BO birds did not lose more mass than CO birds,
suggesting that carotenoid supplementation potentially helped
birds avoid any deleterious effect of paraquat treatment on
intestinal absorption.
Our experiment also generally speaks to possible relation-
ships between carotenoids and oxidative stress that are cur-
rently debated in the avian literature (Hartley and Kennedy
2004; Alonso-Alvarez et al. 2004; Isaksson et al. 2005,
2007;Tummelehtetal.2006; Costantini et al. 2007;
Costantini and Møller 2008; Alonso-Alvarez and Galvan
2011). First, the fact that carotenoid levels were consistently
higher in CA birds compared to BO birds during the first
experiment suggests that carotenoids may generally be serv-
ing as antioxidants (hence drained in the system) in animals
subjected to oxidative stress. This is one of the few experi-
mental examples of this phenomenon in a wild-caught bird
(von Schantz et al. 1999;Mølleretal.2000; Krinsky 2001;
Surai 2002;Alonso-Alvarezetal.2004). However, we cannot
rule out the idea that paraquat may have reduced carotenoid
absorption efficiency. The fact that we failed to detect a dif-
ference in plasma carotenoid levels between CA and BO birds
during the second experiment when we gave them canthaxan-
thin suggests that the phenomenon observed during the first
experiment may have a seasonal- and/or carotenoid-specific
component.
Juxtaposed with this result, we found that oxidative
stress levels were significantly increased only by the BO
treatment and not by the PA treatment during the first ex-
periment. It is unclear why the BO treatment would in-
crease oxidative stress more than PA and not have a similar
or increased effect as PA treatment on body mass. At first
glance, one might presume that PA birds showed decreased
food or water intake but our video monitoring data revealed
that there were no significant differences in time spent eat-
ing or drinking across treatment groups. In addition, during
the second experiment, we found that oxidative stress in-
creased during the first month of experiment for CA birds
and then decreased during the rest of the experiment,
whereas these levels stayed constant during the entire ex-
periment for BO birds. One possible idea behind these
contrasting results during both experiments is that caroten-
oids concurrently serve as both antioxidants and pro-oxi-
dants, rescuing some oxidative damage but also serving as
degraded by-products that might also exert direct free-
radical damage. The antioxidant and pro-oxidant actions
of carotenoids have been touted elsewhere (Young and
Lowe 2001; El-Agamey et al. 2004; Siems et al. 2005;
Costantini and Møller 2008), and clearly what is now need-
ed in the avian literature is a good index of carotenoid pro-
oxidant activity to better identify its series of oxidant ef-
fects or more importantly its most dominant effect.
To the best of our knowledge, the idea that carotenoid-
based color reflects individual antioxidant status (von
Schantz et al. 1999) has been experimentally tested only two
times in animals (Isaksson et al. 2007;Alonso-Alvarezand
Galvan 2011). Isaksson et al. (2007) failed to detect any effect
of an oxidative challenge induced by the administration of
paraquat in the drinking water on carotenoid-based color of
great tits. However, Alonso-Alvarez and Galvan (2011)found
that red-legged partridges (Alectoris rufa) exposed to diquat
during development developed paler carotenoid-based orna-
ments. In our molt experiment, we did not find any effect of
paraquat administration on carotenoid-based coloration, con-
sistent with the results of Isaksson et al. (2007). As proposed
by Alonso-Alvarez and Galvan (2011), these contrasting re-
sults on the effect of an oxidative challenge on carotenoid-
based coloration may be explained by the use of different
methodology (adult vs. growing animals, diquat vs. paraquat)
or by species- or tissue-specific (plumage vs. bare parts) re-
sponses to oxidative challenges. We now need to repeat sim-
ilar experiments in free-ranging animals in order to assess how
an oxidative challenge would affect animal coloration in the
field (i.e., with natural molt conditions and carotenoid intake).
In summary, we uncovered little evidence that urban and
desert birds differ in key carotenoid-accumulation or
oxidative-stress physiological parameters that could explain
geographic variation in carotenoid-based coloration observed
in the wild. Thus, our results strongly suggest that local envi-
ronmental factors (e.g., food availability, plant diversity and
abundance) drive urban/desert differences in carotenoid orna-
mentation in house finches. Finally, we see this complex ca-
rotenoid system in house finches as one that is ideal for exam-
ining the spectrum of antioxidant and prooxidant actions and
benefits in wild animals—something that has largely been
addressed under artificial/lab settings or in domesticated ani-
mals to date.
Ethical Standards The study was approved by the Arizona State
UniversityInstitutional Animal Care and Use Committee, and it complies
with the current laws of the USA.
References
Almroth BC, Sturve J, Berglund A, Förlin L (2005) Oxidative damage in
eelpout (Zoarces viviparous), measured as protein carbonyls and
TBARS, as biomarkers. Aquat Toxicol 73:171–180
Almroth BC,Johansson A, Förlin L, Sturve J (2010) Early-age changes in
oxidative stress in brown trout, Salmo trutta. Comp Biochem
Physiol B 155:442–448
Alonso-Alvarez C, Galvan I (2011) Free radical exposure creates paler
carotenoid-based ornaments: a possible interaction in the expression
of black and red traits. PLoS ONE 6:e19403
968 Behav Ecol Sociobiol (2015) 69:957–970
Alonso-Alvarez C, Bertrand S, Devevey G, Maria G, Prost J, Faivre B,
Sorci G (2004) An experimental test of the dose-dependent effect of
carotenoids and immune activation on sexual signals and antioxi-
dant activity. Am Nat 164:651–659
Alonso-Alvarez C, Pérez-Rodriguez L, García JT, Viñuela J, Mateo R
(2010) Age and breedingeffort as sources of individual variability in
oxidative stress markers in a bird species. Physiol Biochem Zool 83:
110–118
Berny P (2007) Pesticides and the intoxication of wild animals. J Vet
Pharmacol Ther 30:93–100
Blount JD, Metcalfe NB, Birkhead TR, Surai PF (2003) Carotenoid mod-
ulation of immune function and sexual attractiveness in zebra
finches. Science 300:125–127
Bortolotti GR, Fernie KJ, Smits JE (2003) Carotenoid concentra-
tion and coloration of American Kestrels (Falco sparverius)
disrupted by experimental exposure to PCBs. Funct Ecol 17:
651–657
Butler MW, McGraw KJ (2010) Relationships between dietary caroten-
oids, body tissue carotenoids, parasite burden, and health state in
wild mallard (Anas platyrhynchos) ducklings. Arch Biochem
Biophys 504:154–160
Callaway JK, Beart PM, Jarrott B (1998) A reliable procedure for com-
parison of antioxidants in rat brain homogenates. J Pharmacol
Toxicol 39:155–162
Conover WJ, Iman RL (1981) Rank transformations as a bridge between
parametric and nonparametric statistics. Am Stat 35:124–129
Costantini D, Møller AP (2008) Carotenoids are minor antioxidants for
birds. Funct Ecol 22:367–370
Costantini D, Coluzza C, Fanfani A, Dell’Omo G (2007) Effects of ca-
rotenoid supplementation on colour expression, oxidative stress and
body mass in rehabilitated captive adult kestrels (Falco
tinnunculus). J Comp Physiol B 177:723–731
Devasagayam TPA, Boloor KK, Ramasarma T (2003) Methods for esti-
mating lipid peroxidation: an analysis of merits and demerits. Indian
J Biochem Biophys 40:300–308
Edge R, McGarvey DJ, Truscott TG (1997) The carotenoids as anti-ox-
idants: a review. J Photochem Photobiol B 41:189–200
Eeva T, Lehikoinen E, Rönkä M (1998) Air pollution fades the plumage
of the great tit. Funct Ecol 12:607–612
Eeva T, Ryömä M, Riihimäki J (2005) Pollution-related changes in diets
of two insectivorous passerines. Oecologia 145:629–639
El-Agamey A, Lowe GM, McGarvey DJ, Mortensen A, Phillip DM,
Truscott TG, Young AJ (2004) Carotenoid radical chemistry and
antioxidant/pro-oxidant properties. Arch Biochem Biophys 430:
37–48
Galvani P, Cassani A, Fumagalli P, Santagostino A (2000) Effect of para-
quat on glutathione activity in Japanese quail. Bull Environ Contam
Toxicol 64:74–80
Giraudeau M, Toomey M, McGraw KJ (2012) Can house finches
(Carpodacus mexicanus) use non-visual cues to discriminate the
carotenoid content of foods? J Ornithol 153:1017–1023
Giraudeau M, Sweazea K, Butler MW, McGraw KJ (2013) Effects
of carotenoid and vitamin E supplementation on oxidative
stress and plumage coloration in house finches (Haemorhous
mexicanus). Comp Biochem Physiol A Mol Integr Physiol 166:
406–413
GiraudeauM,MouselM,EarlS,McGrawK(2014)Parasitesinthe
city: degree of urbanization predicts poxvirus and coccidian
infections in house finches (Haemorhous mexicanus). PLoS
ONE 9:e86747
Hartley RC, Kennedy MW (2004) Are carotenoids a red herring in sexual
display? Trends Ecol Evol 19:353–354
Hasegawa M, Ligon RA, Giraudeau M, Watanabe M, McGraw KJ (2014)
Urban and colorful male house finches are less aggressive. Behav
Ecol 25:641–649
Hill GE (1995) Ornamental traits as indicators of environmental health.
Bioscience 45:25–31
Hill GE (2002) A red bird in a brown bag: the function and evolution of
colorful plumage in the house finch. Oxford University Press, New
York
Hill GE, Farmer KL, Beck ML (2004) The effect of mycoplasmosis on
carotenoid plumage coloration in male house finches. J Exp Biol
207:2095–2099
Hõrak P, Vellau H, Ots I, Møller AP (2000) Growth conditions affect
carotenoid-based plumage coloration of great tit nestlings.
Naturwissenschaften 87:460–464
Hõrak P, Ots I, Vellau H, Spottiswoode C, Møller AP (2001)
Carotenoid-based plumage coloration reflects hemoparasite
infection and local survival in breeding great tits. Oecologia
126:166–173
Hunnisett A, Davies S, McLaren-Howard J, Gravett P, Finn M,
Gueret-Wardle D (1995) Lipoperoxides as an index of free
radical activity in bone marrow transplant recipients. Biol
Trace Elem Res 47:125–132
Inouye CY, Hill GE, Stradi RD, Montgomerie R (2001) Carotenoid pig-
ments in male house finch plumage in relation to age, subspecies,
and ornamental coloration. Auk 118:900–915
Isaksson C, Andersson S (2008) Oxidative stress does not influence ca-
rotenoid mobilization and plumage pigmentation. Proc R Soc Lond
B 275:309–314
Isaksson C, Örnborg J, Stephensen E, Andersson S (2005) Plasma gluta-
thione and carotenoid coloration as potential biomarkers of environ-
mental stress in great tits. EcoHealth 2:138–146
Isaksson C, McLaughlin P, Monaghan P, Andersson S (2007) Carotenoid
pigmentation does not reflect total non-enzymatic antioxidant activ-
ity in plasma of adult and nestling great tits, Parus major.FunctEcol
21:1123–1129
Isaksson C, Sturve J, Almroth BC, Andersson S (2009) The impact of
urban environment on oxidative damage (TBARS) and antioxidant
systems in lungs and liver of great tits, Parus major. Environ Res
109:46-50
Kimura K, Katsumata Y, Ozawa T, Tawara S, Igarashi K, Cho Y, Shibata
N, Hakuno F, Takahashi SI, Takenaka A (2010) Effect of paraquat-
induced oxidative stress on insulin regulation of insulin-like growth
factor-binding protein-1 gene expression. J Clin Biochem Nutr 46:
157–167
Koutsos EA, Clifford AJ, Calvert CC, Klasing KC (2003) Maternal ca-
rotenoid status modifies the incorporation ofdietary carotenoids into
immune tissues of growing chickens (Gallus gallus domesticus). J
Nutr 133:1132–1138
Krinsky NI (2001) Carotenoids as antioxidants. Nutrition 17:815–817
Lessells CM, Boag PT (1987) Unrepeatable repeatabilities: a common
mistake. Auk 104:116–121
Marzluff JM (2001) Worldwide urbanization and its effects on birds. In:
Marzluff JM, Bowman R, Donnelly R (eds) Avian ecology in an
urbanizing world. Kluwer, Norwell, pp 19–47
McGraw KJ (2006) The mechanics of carotenoid coloration in birds. In:
Hill GE, McGraw KJM (eds) Bird coloration. I. Mechanisms and
measurements. Harvard University Press, Cambridge, pp 177–242
McGraw KJ, Ardia DR (2003) Carotenoids, immunocompetence, and the
information content of sexual colors: an experimental test. Am Nat
162:704–712
McGraw KJ, Hill GE (2000) Plumage brightness and breeding-season
dominance in the house finch: a negatively correlated handicap?
Condor 102:456–461
McGraw KJ, Parker RS (2006) A novel lipoprotein-mediated mechanism
controlling sexual attractiveness in a colorful songbird. Physiol
Behav 87:103–108
McGraw KJ, Hill GE, Stradi R, Parker RS (2001) The influence of ca-
rotenoid acquisition and utilization on the maintenance of species-
typical plumage pigmentation in male American goldfinches
Behav Ecol Sociobiol (2015) 69:957–970 969
(Carduelis tristis) and northern cardinals (Cardinalis cardinalis).
Physiol Biochem Zool 74:843–852
McGraw KJ, Dale J, Mackillop EA (2003) Social environment during
molt and the expression of melanin-based plumage pigmentation in
male house sparrows (Passer domesticus). Behav Ecol Sociobiol 53:
116–122
McGraw KJ, Nolan PM, Crino OL (2006) Carotenoid accumulation strat-
egies for becoming a colourful house finch: analyses of plasma and
liver pigments in wild moulting birds. Funct Ecol 20:678–688
McGraw KJ, Crino OL, Nolan PM (2011) Carotenoids boost immunity
during molt in a songbird with sexually selected carotenoid-based
plumage coloration. Biol J Linn Soc 102:560–572
Meitern R, Sild E, Kilk K, Porsok R, Hõrak P (2013) On the methodo-
logical limitations of detecting oxidative stress: effects of paraquat
on measures of oxidative stress in greenfinches. J Exp Biol 216:
2713–2721
Møller AP, Biard C, Blount JD, Houston DC, Ninni P, Saino N (2000)
Carotenoid-dependent signals: indicators of foraging efficiency, im-
munocompetence or detoxification ability? Avian Poult Biol Rev
11:137–159
Møller AP, Erritzøe J, Karadas F (2010) Levels of antioxidants in rural
and urban birds and their consequences. Oecologia 163:35–45
Oakes KD, Van Der Kraak GJ (2003) Utility of the TBARS assay in
detecting oxidative stress in white sucker (Catostomus commersoni)
populations exposed to pulp mill effluent. Aquat Toxicol 63:447–463
Oh KP, Badyaev AV (2006) Adaptive genetic complementarity in mate
choice coexists with preference for elaborate sexual traits. Proc R
Soc Lond B 273:1913–1919
Partecke J, Van’t Hof T, Gwinner E (2004) Differences in the timing of
reproduction between urban and forest European blackbirds (Turd us
merula): result of phenotypic flexibility or genetic differences? Proc
R Soc Lond B 271:1995–2001
Partecke J, Schwabl I, Gwinner E (2006) Stress and the city: urbanization
and its effects on the stress physiology in European Blackbirds.
Ecology 87:1945–1952
Pérez-Rodriguez L, Mougeot F, Alonso-Alvarez C, Blas J, Viñuela J,
Bortolotti GR (2008) Cell-mediated immune activation rapidly de-
creases plasma carotenoids but does not affect oxidative stress in
red-legged partridges (Alectoris rufa). J Exp Biol 211:2155–2161
Peters A, Denk AG, Delhey K, Kempenaers B (2004) Cartenoid-based
bill colour as an indicator of immunocompetence and sperm perfor-
manceinmalemallards.JEvolBiol17:1111–1120
Salmon AB, Marx DB, Harshman LG (2001) A cost of reproduction in
Drosophila melanogaster: stress susceptibility. Evolution 55:1600–
1608
Schilderman PAEL, Hoogewerff JA, van Schooten FJ, Maas LM,
Moonen EJC, van Os BJH, van Wijnen JH, Kleinjans JCS (1997)
Possible relevance of pigeons as an indicator species for monitoring
air pollution. Environ Health Perspect 105:322–330
Shocat E, Warren PS, Faeth SH, McIntyre NE, Hope D (2006) From
patterns to emerging processes in mechanistic urban ecology.
Trends Ecol Evol 21:186–191
Siems W, Wiswedel I, Salerno C, CrifόC, Augustin W, Schild L,
Langhans C, Sommerburg O (2005) β-Carotene breakdown prod-
ucts may impair mitochondrial functions—potential side effects of
high-dose β-carotenesupplementation. J Nutr Biochem 16:385–397
Slagsvold T, Lifjeld JT (1985) Variation in plumage colour of the great tit
Parus major in relation to habitat, season and food.J Zool 206:321–
328
Surai PF (2002) Natural antioxidants in avian nutrition and reproduction.
Nottingham University Press, Nottingham
Toomey MB, McGraw KJ (2009) Seasonal, sexual, and quality related
variation in retinal carotenoid accumulation in the house finch.
Funct Ecol 23:321–329
Toomey MB, McGraw KJ (2012) Mate choice for a male carotenoid-
based ornament is linked to female dietary carotenoid intake and
accumulation. BMC Evol Biol 12:3
Tummeleht L, Mägi M, Kilgas P, Mänd R, Hõrak P (2006) Antioxidant
protection and plasma carotenoids of incubating great tits (Parus
major L.) in relation to health state and breeding conditions. Comp
Biochem Physiol C 144:166–172
von Schantz TS, Bensch S, Grahn M, Hasselquist D, Wittzell H (1999)
Good genes, oxidative stress and condition-dependent sexual sig-
nals. Proc R Soc Lond B 266:1–12
Wang Y, Salmon AB, Harshman LG (2001) A cost of reproduction:
oxidative stress susceptibility is associated with increased egg pro-
duction in Drosophila melanogaster. Exp Gerontol 36:1349–1359
Young AJ, Lowe GM (2001) Antioxidant and prooxidant properties of
carotenoids. Arch Biochem Biophys 385:20–27
970 Behav Ecol Sociobiol (2015) 69:957–970
