environmental pollution affects the plumage color of great tit nestlings through carotenoid...

Environmental Pollution Affects the Plumage Color of GreatTit Nestlings through Carotenoid Availability

Tapio Eeva,1 Saila Sillanpaa,1 Juha-Pekka Salminen,2 Lauri Nikkinen,1 Anu Tuominen,2

Eija Toivonen,1 Kalevi Pihlaja,2 and Esa Lehikoinen1

1Section of Ecology, University of Turku, Turku 20014, Finland2Laboratory of Organic Chemistry and Chemical Biology, University of Turku, Turku 20014, Finland

Abstract: Birds need to acquire carotenoids for their feather pigmentation from their diet, which means that

their plumage color may change as a consequence of human impact on their environment. For example, the

carotenoid-based plumage coloration of Great tit, Parus major, nestlings is associated with the degree of

environmental pollution. Breast feathers of birds in territories exposed to heavy metals are less yellow than

those in unpolluted environments. Here we tested two hypotheses that could explain the observed pattern: (I)

deficiency of carotenoids in diet, and (II) pollution-related changes in transfer of carotenoids to feathers. We

manipulated dietary carotenoid levels of nestlings and measured the responses in plumage color and tissue

concentrations. Our carotenoid supplementation produced the same response in tissue carotenoid concen-

trations and plumage color in polluted and unpolluted environments. Variation in heavy metal levels did not

explain the variation in tissue (yolk, plasma, and feathers) carotenoid concentrations and was not related to

plumage coloration. Instead, the variation in plumage yellowness was associated with the availability of

carotenoid-rich caterpillars in territories. Our results support the hypothesis that the primary reason for

pollution-related variation in plumage color is carotenoid deficiency in the diet.

Keywords: carotenoids, bioindicator, heavy metal pollution, oxidative stress, Parus major, plumage color

INTRODUCTION

Differences in carotenoid-based plumage coloration in

birds may reflect human impact in their environment. For

example, the yellow plumage of Great tit (Parus major)

nestlings may depend on the degree of pollution exposure

and urbanization of territories. The yellow color of breast

feathers tend to be paler at industrialized and urban terri-

tories (Eeva et al., 1998; Horak et al., 2001; Isaksson et al.,

2005). The yellow color in the breast feathers of P. major is

based on dietary carotenoids, primarily lutein (Partali

et al., 1985). Animals are not able to synthesize carotenoids

de novo, so they need to acquire them from their diet

(Brush, 1978). Therefore, a possible reason for the observed

human-related change in plumage color could be dietary

changes in polluted and urban areas (Eeva et al., 2005). For

example, environmental pollution may affect birds’ diet if

the availability of carotenoid-rich food, like caterpillars, is

reduced. As a consequence, the expression of carotenoid-

based signals may be impaired. The proportion of cater-

pillars in nestling diet and caterpillar abundance in a

territory have been shown to be positively related withPublished online: August 13, 2008

Correspondence to: Tapio Eeva, e-mail: [email protected]

EcoHealth 5, 328–337, 2008DOI: 10.1007/s10393-008-0184-y

Original Contribution

� 2008 International Association for Ecology and Health

plumage yellowness in P. major nestlings (Slagsvold and

Lifjeld, 1985; Eeva et al., 1998).

There are, however, alternative mechanisms that could

be responsible for the observed color difference. Besides

being important for pigmentation, carotenoids are also

antioxidants that protect cells and organisms against oxi-

dative stress caused by free radicals (Edge et al., 1997; von

Schantz et al., 1999; Møller et al., 2000). Carotenoids are

known to be involved in two important physiological

functions: modulation of the immune system and detoxi-

fication (Møller et al., 2000). For example, heavy metals are

known to produce oxidative stress in humans and animals,

probably by disturbing the antioxidant-based defense of the

body (Stohs and Bagchi, 1995; Mateo and Hoffman, 2001;

Aykin-Burns et al., 2003). Food-derived antioxidants are

consumed when the defense system is activated, and high

amounts of pollutants in the body may lead to a depletion

of antioxidants (Polidori et al., 2001; Krinsky and Yeum,

2003; Stehbens, 2003). Experimental support for the trade-

off between carotenoid-based traits and immunity has been

found in recent laboratory studies (Blount, 2004). How-

ever, information on the relationship between carotenoids

and detoxification is much more limited, and is lacking for

any free-living population. Some toxins are also known to

disrupt carotenoid uptake. Also, the physical composition

of food, which may differ, e.g., between urban and rural

habitats, may also affect carotenoid retrieval from food

(McGraw, 2006).

We tested two hypotheses to explain the observed

human-related variation in plumage color of P. major

nestlings: (I) deficiency of carotenoids in diet, and (II)

pollution-related changes in transfer of carotenoids from

food to feathers. These hypotheses can be tested by

manipulating dietary carotenoid levels and by measuring

subsequent responses in plumage color. If the responses are

the same in polluted and unpolluted environments, we can

conclude that plumage color is directly related to carot-

enoid availability. If, however, the response is weaker in a

polluted environment than in an unpolluted one, a larger

proportion of the ingested carotenoid is unused for pig-

mentation in a polluted environment, suggesting different

absorption or deposition in two environments. We

manipulated the dietary carotenoid levels of P. major nes-

tlings and measured responses in plumage color and tissue

(plasma, feathers) concentrations in a well-known heavy

metal pollution gradient around a copper smelter (Eeva

et al., 1997). We also tested for the effects of pollution and

carotenoid treatment on nestling body mass, hematocrit

level, and survival. Carotenoid levels in egg yolk (an

important carotenoid source for an embryo; Blount et al.,

2000) were measured to see whether natural carotenoid

levels differ in the beginning of breeding in relation to the

pollution levels. The abundance of caterpillars (main

carotenoid source for tits; Partali et al., 1985) was mea-

sured to account for natural variation in the availability of

carotenoid-rich food. Fecal samples from nestlings were

analyzed for their heavy metal concentrations to get a

measure of exposure to pollutants in the study area.

METHODS

Study Area

The experiment was performed in 2004, in the surround-

ings of a copper smelter in the town of Harjavalta (61�200

N, 22�100 E), SW Finland. Sulfuric oxides and heavy metals

(especially Cu, Zn, Ni, Pb, and As) are common pollutants

in the area due to emissions from the smelter and adjoining

industry (Kiikkila, 2003). Elevated heavy metal concentra-

tions occur in soil, vegetation, insects, and birds (Eeva and

Lehikoinen, 2000) in the polluted area due to current and

historical deposition. Metal contents decrease exponentially

with increasing distance from the smelter, approaching

background levels at sites >5 km from the smelter (Eeva

et al., 1997). Twelve study sites were established along the

air pollution gradient in three main directions (SW, SE,

and NW) away from the copper smelter complex. The sites

closer than 2 km from the smelter are hereafter referred to

as the ‘‘polluted area’’ and sites over 5 km from the smelter

as the ‘‘unpolluted area.’’ To avoid extra variation in results

due to varying habitat quality, we selected study areas of the

same habitat type, i.e., relatively barren pine (Pinus sylves-

tris)-dominated forests typical of the study area. The data

was collected under the licenses of the Animal Care & Use

Committee of Turku University and Regional Environment

Centre.

Egg Samples and Assignment of Treatment Groups

Nest boxes were visited regularly from the beginning of the

breeding season. One egg was collected from each nest

(n = 74 nests; polluted 35 + unpolluted 39) to measure

yolk carotenoid concentration. Only first clutches were

included. Clutch sizes did not differ between polluted and

unpolluted areas (Generalized linear model: n = 74,

v2 = 0.20, P = 0.66). Because yolk carotenoid concentra-

Air Pollution and Plumage Color of Great Tit 329

tion may decrease along the laying sequence (Royle et al.,

1999; Horak et al., 2002), we standardized our sampling by

taking the ninth egg (the modal clutch-size in our popu-

lation) in sequence, or the last egg if clutch-size was less

than nine. We considered that if carotenoid availability is

restricted, the differences among territories should be more

clearly shown in eggs near the end of the laying sequence.

For this purpose, eggs were marked individually on daily

visits to the nests during the laying period. Yolks were

separated from albumen and stored protected from light at

-22�C until the analyses.

To determine the precise hatching date, nests were

checked daily starting from 2 days before the estimated

hatching date (day 0) onwards. At the age of 3 days, nes-

tlings were weighed and, within each brood, divided into

two comparable groups according to their body mass.

These two groups were randomly assigned to the treatment

(water-dispersed carotenoid supplementation) or control

(water supplementation) group. Nestlings were marked

with black ink on their right or left tarsus according to the

treatment.

Carotenoid Supplementation

We started the carotenoid/water supplementation at day 3

for a total of 71 broods (polluted 33 + unpolluted 38). We

avoided disturbing the birds just after hatching, but an

early start was important because carotenoid-based plum-

age coloration in this species is probably primarily deter-

mined during the first 6 days after hatching (Fitze et al.,

2003b). Carotenoid beadlets (Lutein 5% CWS, Roche,

Basel, Switzerland) containing 5% lutein and 0.25% zea-

xanthin were dissolved in distilled water to achieve a lutein

concentration of 5 mg/ml. The supplement was given to

nestlings, by orally dosing 0.1 ml of the supplement per

nestling daily from day 3 to day 8. The supplemented dose

(= 0.5 mg lutein/day/nestling) was approximately 3–89

higher than an estimated natural daily lutein intake (Fitze

et al., 2003b). However, despite the relatively high dosage,

circulating lutein levels were not exceptionally high in our

experiment since only two of the supplemented nestlings

showed plasma lutein concentrations (139 and 149 lg/ml)

above the maximum found among the unsupplemented

nestlings (121 lg/ml). An explanation to this may be that a

substantial part of the ingested carotenoid may not be

absorbed if the given dosage is high (McGraw et al., 2001).

From day 8 onwards, 0.2 ml of supplement was given every

second day until day 14. At the same time that carotenoid

supplementation was taking place, control nestlings re-

ceived the same amount of distilled water.

Measures of Nestling Condition

Nestlings were ringed with individual aluminum rings at day

6, and their body mass was recorded at days 8 and 16.

Nestling body mass and subsequent survival, as measured by

recruitment to breeding populations, are correlated in many

bird species, including P. major (Perrins, 1965). We used

body mass and fledging probability (the probability of a

hatchling to fledge) as overall responses to pollution stress

and carotenoid treatment. Two nestlings (1 treatment + 1

control) per brood were selected for blood sampling at day 9

(n = 60 broods; polluted 26 + unpolluted 34). We avoided

sampling exceptionally small nestlings (runts), but otherwise

the selection was random. Blood samples were collected with

capillary tubes from the brachial vein and centrifuged

immediately for 5 minutes at 4000 r/min. Hematocrit level

(the proportion of packed red blood cell volume) was mea-

sured with a ruler and plasma was separated, preserved in ice

during the transport, and kept protected from light at -22�C

until the carotenoid analyses. Hematocrit level is commonly

used as a general measure of condition in wild birds, though

many factors besides nutritional condition may affect it (Fair

et al., 2007). In our study area, one such factor might be the

heavy metal pollution, which could cause anemia in birds

(Fair et al., 2007). In nestlings, hematocrit also increases with

age due to increased erythropoiesis (Fair et al., 2007). Sam-

pling age, however, was standardized in our experiment.

Assessment of Plumage Color

At day 16, two nestlings (1 treatment + 1 control) per

brood were randomly selected and photographed side by

side on a uniform gray cardboard with a digital camera.

Nestlings were placed in a plastic holder to keep them in

the same position while being photographed (Fig. 1). Pic-

tures were taken from the ventral side, and the same yellow

reference card (C2, M17, Y86, K0) was included in each

picture. Direct sunlight was avoided during photographing,

and the reference card was preserved light-protected. Two

images were taken of each pair of nestlings for assessing the

repeatability of the measurements. After photographing, we

took a sample of the yellow breast feathers from the same

two nestlings for lutein determination (n = 58 broods;

polluted 25 + unpolluted 33). Feathers were kept protected

from light at -22�C until the analyses.

330 Tapio Eeva et al.

Digital imaging has shown to be a sensitive and

repeatable way of measuring color variation, as long as

inter-photograph variation in ambient lightning can be

controlled (Villafuerte and Negro, 1998; Montgomerie,

2006). Digital image analyses are based on color variation

in the range of human color vision and cannot measure

variation in the UV-range, which birds can see. Our main

interest, however, was focused on the intensity of one

pigment (lutein) in the human visible range. A positive

correlation between plumage yellowness and feather lutein

concentrations (see Results) shows that we were measuring

a relevant color parameter. Plumage yellowness was ana-

lyzed from digital images with Corel Photo-Paint 12 soft-

ware. The images were transformed to CMYK color profile.

CMYK is a color scheme for combining four primary

pigments. C, M, Y, and K are the percent values for the

cyan, magenta, yellow, and black values of the color,

respectively. We used the proportion (%) of the yellow

component (Y) as a measure of plumage yellowness. In the

case of yellow coloration of P. major feathers, the variation

in Y primarily describes the variation of chroma or satu-

ration in HSB color space. Average plumage color was

measured with the ‘‘eyedropper’’ tool of the software at

both sides of the abdomen by taking the largest possible

yellow rectangle while avoiding including black parts

(Fig. 1). The average value of these two measurements was

taken and a standard value (Ys) was measured in each

image from the reference color card (Fig. 1). For each

image, the reference color value was used to calculate

corrected plumage color values (Yc) that take into account

variations in ambient light conditions during photo-

graphing: Yc ¼ Y þ ððð�Ys � YsÞ=YsÞÞ � YÞ, where the mean

value of Ys was calculated over the all images. All mea-

surements were repeated over two sets of images to cal-

culate repeatability of the yellowness value (see Lessells and

Boag, 1987). The repeatability of the measurements was

0.91 (F114, 115 = 22.4, P < 0.0001) over the two sides of

nestlings when measured from the same image and 0.83

(F124, 105 = 18.5, P < 0.0001) over the replicated images.

Caterpillar Abundance

Caterpillar abundance in territories was established by

shaking birches (Betula spp.) (dominant deciduous trees in

the study area) and collecting the fallen larvae from the white

plastic sheet (2 m 9 2 m) underneath. Sampling took place

between May 17th– 21st. Because plumage color determi-

nation probably happens early in development, sampling of

larvae was made close to the hatching date of P. major

(median May 22nd). Five trees, 1–5 m high, were randomly

selected in the vicinity (radius of 50 m) of P. major nests, and

the trunk of each tree was vigorously shaken once. Alto-

gether, we sampled 295 trees from 59 territories. Tree height

(m) and leaf biomass (visual scores from 1 to 10, larger scores

denoting larger biomass) were taken into account. As an

index of caterpillar abundance in a territory, we used the

logarithm of their mean biomass (mg/tree).

Lutein Quantification

Lutein concentrations in all samples were determined with

high-performance liquid chromatography (HPLC). Plasma

was analyzed as such, while yolks and feathers were freeze-

dried for 48 hours and ground into fine powder. A known

amount of powder (yolk: 20 mg, feathers: 1–35 mg), or a

known volume of plasma (10–35 ll), was extracted 39 with

100% acetone. The solvent was evaporated from the com-

bined extract under vacuum and the residue was dissolved

into a small volume of 80% acetone. The carotenoid

Figure 1. An example of an image on which color measurements

were taken. Plastic holders keep the nestlings (A, B) in the same

position while being photographed. Rectangles show the places

where measures of yellowness were taken. A yellow reference card

(C) was included in each picture to standardize measurements for

varying light conditions.


composition of the extracts was analyzed with HPLC at

450 nm using a Merck Purospher STAR RP-18

(55 9 2 mm, i.d., 3 lm) column (Darmstadt, Germany).

Lutein was quantified as lutein equivalents. One of the

feather samples was too small for the analysis.

Heavy Metal Analyses

Fresh feces were collected from defecating nestlings at day 7

directly to plastic Eppendorf tubes (2–4 nestlings/brood).

Fecal sacs from the same brood were combined, dried in a

laboratory at 50�C for 72 hours, and weighed to form samples

of 0.15–0.20 g. Two milliliters of Supra-pure HNO3 and

0.5 ml of H2O2 was added to the samples in Teflon bombs for

digestion with a microwave system (Milestone High Perfor-

mance Microwave Digestion Unit mls 1200 mega, Leutkirch,

Germany). After the digestion, the samples were diluted to

50 ml with de-ionized water. The determination of metal

concentrations (As, Cd, Cu, Ni, Pb, and Zn) was done with

ICP-MS (Elan 6100 DRC + from PerkinElmer-Sciex, Boston,

USA). The detection limit for most of the elements was around

ppt (ng/l) level and below. The calibration of the instrument

was done with certified solution (Claritas PPT, Multi element

solution 2A, Metuchen, NJ, USA) from Spex Certiprep.

Statistical Analyses

All the statistical analyses were done with the SAS statistical

system for Windows (SAS Institute, 2001). As a basic model

structure for the analyses of all the response variables, we

used a model with carotenoid treatment (lutein vs. water),

pollution level (polluted vs. unpolluted), and interaction

between these as independent factors.

The variation in nestling body mass was analyzed with a

linear mixed model ANOVA (MIXED procedure in SAS,

type III analysis; hereafter LMM) by using the basic model

with nestling age (8 and 16 days) as a repeated factor and

brood as a random factor. For hematocrit, we used the basic

model with brood as a clustering factor (to take account that

the treatment and control nestlings were not independent

because they were from the same brood). Brood size and

hatching date were included in these models but they were

omitted from the final models as nonsignificant variables.

Fledging probability was analyzed with the basic model

by using a generalized linear model with binomial distri-

bution (GENMOD procedure in SAS, type 3 analysis).

Hatching date was first included in the model but omitted

from the final model as a nonsignificant variable.

Yolk lutein concentrations were analyzed with LMM,

where pollution level was used as an independent factor

and brood as a random factor. Lutein concentrations in

plasma and feathers were analyzed with LMM, by using the

basic model with brood as a clustering factor (see above).

Because plasma lutein concentration may change during

the day, we included time as a covariate in this model.

Brood size and nestling body mass were also first included

in these models but omitted from the final models as

nonsignificant covariates. Log10-transformation was done

for all the lutein concentrations before the analyses.

The natural relationships (i.e., in the control group)

between heavy metal levels and lutein concentrations in

various tissues (yolk, plasma, and feathers) were analyzed

with Pearson correlations. To provide a single measure

describing the variation in pollution levels, we calculated

the first principal component (PC1) from the heavy metal

data (As, Cd, Cu, Ni, Pb, Zn) and correlated it with lutein

concentrations. PC1 had strongly positive loading from all

the measured heavy metals, and it describes the general

level of heavy metal exposure well.

The yellowness of nestling plumage was analyzed with

LMM by using the basic model with brood as a clustering

factor. Body mass was first included in the model as a

covariate but was omitted as a nonsignificant variable.

A log10 transformed biomass (mg/tree) of caterpillars

was compared among sites with LMM, where pollution

level, tree height, and leaf biomass were used as indepen-

dent factors, and brood as a clustering factor. Tree height

was omitted from the final model as a nonsignificant var-

iable. The relationship between plumage color and cater-

pillar abundance was analyzed with LMM with carotenoid

treatment, caterpillar abundance, and their interaction as

explaining factors, and brood as a clustering factor.

In all of the above-mentioned LMMs, degrees of free-

dom were calculated with the Satterthwaite procedure. The

normality of residuals was tested after each ANOVA with

the Kolmogorov-Smirnov test (UNIVARIATE procedure in

SAS). All means in the text are given with their standard

errors.

RESULTS

Nestling Condition and Survival

Nestlings weighed less in the polluted area than in the

unpolluted area, but carotenoid supplementation had no

effect on body mass (Table 1). There was no interaction


between nestling age (8 and 16 days) and location (polluted

and unpolluted), meaning that the pollution effect on body

mass was similar at both ages (Table 1). Neither pollution

nor carotenoid supplementation significantly affected the

hematocrit level (Table 1), although hematocrit was

strongly correlated with nestling body mass (Pearson cor-

relation: r = 0.43, P < 0.001, n = 116 nestlings). In

accordance with the variation in body mass, the probability

of fledging was 19% lower in the polluted area but did not

depend on carotenoid supplementation (Table 1).

Tissue Lutein Concentrations

There was no difference in yolk lutein concentrations

between the two areas (polluted 69.3 ± 4.1 lg/g; unpol-

luted 76.2 ± 5.1 lg/g; LMM: F1, 70 = 0.55, P = 0.46).

Plasma lutein concentration was 2.19 higher in carot-

enoid-supplemented birds compared to control birds

(LMM: F1, 113 = 38.8, P < 0.0001; Fig. 2) and 1.29

higher in the polluted area than in the unpolluted one

(LMM: F1, 113 = 6.0, P = 0.016; Fig. 2). The interaction

between lutein treatment and area was not significant

(LMM: F1, 113 = 0.02, P = 0.90), suggesting that carot-

enoid treatment produced the same response in the two

environments. Plasma lutein concentration was positively

related to time (a covariate in our model), i.e., concen-

trations were lower in the morning and increased during

the day (LMM: F1, 113 = 14.2, P = 0.0003).

Feather lutein concentrations were 2.89 higher in

carotenoid-supplemented nestlings than in control nes-

tlings (LMM: F1, 108 = 56.9, P < 0.0001; Fig. 2), but there

was no difference between the study areas (LMM:

F1, 108 = 0.23, P = 0.64; Fig. 2). The interaction between

carotenoid treatment and area was not significant (LMM:

F1, 108 = 0.44, P = 0.51), again suggesting similar response

between the two areas. Feather lutein concentration cor-

related positively with plasma lutein concentration in the

nonsupplemented birds (Pearson correlation: r = 0.45,

n = 56, P = 0.0006) but not in the lutein-supplemented

birds (Pearson correlation: r = -0.055, n = 56, P = 0.69).

Yolk (Pearson correlation: r = -0.095, n = 60,

P = 0.47), plasma (Pearson correlation: r = 0.020, n = 59,

P = 0.88) and feather (Pearson correlation: r = 0.12,

n = 58, P = 0.38) lutein concentrations were not correlated

Table 1. Averages of brood means for condition measures (body mass, hematocrit) and fledging probability (a probability of a

hatchling to fledge) of Parus major nestlings in four experimental groups (two treatments in two areas; in all the analyses, n = 59 broods)

Group Agea Body massb (g) Hematocritc (%) Fledging probabilityd

n mean SE n mean SE n Prob CL

Polluted, water 8–9 26 10.9 0.30 26 36.3 0.94 26 0.59 0.46–0.70

16 24 15.9 0.48

Polluted, lutein 8–9 26 11.1 0.43 26 38.2 0.77 26 0.62 0.51–0.72

16 25 16.3 0.43

Unpolluted water 8–9 33 12.0 0.39 33 38.7 1.02 33 0.71 0.62–0.79

16 32 17.1 0.40

Unpolluted lutein 8–9 33 12.1 0.41 33 38.9 0.77 33 0.79 0.69–0.86

16 32 16.7 0.43

Source of variation df F P df F P df v2 P

Area 1, 57 5.03 0.029 1, 114 1.85 0.094 1, 114 5.14 0.023

Treatment 1, 605 0.17 0.68 1, 114 1.41 0.26 1, 114 1.92 0.17

Area 9 treatment 1, 605 0.39 0.53 1, 114 0.92 0.34 1, 114 0.48 0.49

Age 1, 603 1119 <0.0001 – – – – – –

aEight days for measurements of body mass, 9 days for hematocrit.bA linear mixed model ANOVA, where age was used as a repeated factor and brood as a random factor.cA linear mixed model ANOVA, where brood was used as a clustering factor.dA generalized linear model, where brood was used as a clustering factor.


with PC1 of fecal heavy metal concentrations (tested for the

control group only). PC1 was significantly higher in the

polluted area than in the unpolluted one (LMM:

F1, 58 = 14.2, P < 0.0001).

Plumage Color

The proportion of the yellow component (Yc) in nestling

plumage color was 13% higher in the carotenoid-supple-

mented group compared to the control group (LMM,

F1, 109 = 39.9, P < 0.0001; Fig. 2). The value was also 4.8%

higher in the unpolluted area than in the polluted one

(LMM, F1, 109 = 4.38, P = 0.039). There was no interaction

between the two treatments (LMM, F1, 109 = 0.52,

P = 0.47), showing that our carotenoid supplementation

produced the same response in birds exposed to pollution

as in those not exposed. Plumage yellowness correlated

positively with feather lutein concentration in the non-

supplemented birds (Pearson correlation: r = 0.45, n = 56,

P = 0.0006) and in the lutein-supplemented birds (Pearson

correlation: r = 0.28, n = 56, P = 0.039). Plumage yellow-

ness did not correlate (Pearson correlation: r = 0.027,

n = 56, P = 0.84) with PC1 of fecal heavy metal concen-

trations (tested for the control group only). The variation

in plumage color was smaller in the carotenoid-supple-

mented group (Levene’s test: F1, 109 = 13.1, P = 0.0005)

but well within the natural range of the nestling plumage

color shown by the control group (Fig. 3).

Caterpillar Abundance

Caterpillar biomass at the time of hatching was 29% higher

in the unpolluted (mean = 9.5 ± 1.9 mg/tree, n = 33 ter-

ritories) than in the polluted (mean = 7.4 ± 3.1 mg/tree,

n = 26 territories) area (LMM: F1, 282 = 5.38, P = 0.021).

Caterpillar biomass was also dependent on leaf biomass (a

covariate in our model), higher scores being associated with

higher biomass (LMM: F1, 282 = 7.53, P = 0.0064). Cater-

pillar biomass in territories was further positively associ-

ated with the proportion of the yellow component (Yc) in

nestling plumage color (LMM: F1, 109 = 10.8, P = 0.0014).

The interaction between carotenoid treatment and cater-

pillar abundance was not significant (LMM: F1, 109 = 0.38,

P = 0.53), meaning that the relationship between caterpil-

lar availability and plumage color was similar for supple-

mented and control nestlings (Fig. 3). There was also a

positive correlation between caterpillar biomass and feather

Figure 2. The mean (±SE) lutein concentrations in plasma (A) and

breast feathers (B) and mean index of yellowness of breast plumage

(C) of P. major nestlings in a polluted area (black bars) and

unpolluted area (gray bars) for lutein treatment (T) and control (C)

groups. Yellowness of the breast plumage was determined from

digital images as a proportion of the yellow component in the CMYK

color profile (see Methods). The sample numbers are shown above

the bars.


lutein concentration in nonsupplemented (Pearson corre-

lation: r = 0.33, n = 56, P = 0.012) and lutein-supple-

mented (Pearson correlation: r = 0.32, n = 56, P = 0.015)

groups. The latter correlation shows that the variation in

natural carotenoid availability is apparent in tissue con-

centrations even after relatively high supplemented lutein

dosage.

DISCUSSION

Our results show that a large amount of variation in the

plumage yellowness of P. major nestlings can be explained

by variation in the availability of lutein rich food, i.e.,

caterpillars, in territories at the time of early development

of nestlings. On the contrary, the variation in fecal heavy

metal levels (a proxy for dietary exposure) did not explain

the variation in yolk, plasma, or feather lutein concentra-

tions, and was not related to the yellowness of nestlings’

breast feathers. Most importantly, our lutein supplemen-

tation experiment produced the same response in tissue

lutein concentrations and plumage color in polluted and

unpolluted environments. Therefore, our results suggest

that birds in areas with heavy metal pollution deposited the

ingested lutein in feathers and other purposes in similar

proportions as birds in unpolluted environments. This

supports the hypothesis that the primary reason for a pale

plumage in a polluted environment is lutein deficiency in

the diet and not carotenoid-depletion or changes in

deposition caused by environmental pollutants. This con-

clusion is further supported by our observational data on

caterpillar abundance and the diet of nestlings. Caterpillar

abundance was lower, and the diet of P. major nestlings

contained less caterpillars, in a polluted area than in an

unpolluted one (Eeva et al., 2005). Some recent studies,

which have measured oxidative stress levels and plumage

yellowness in P. major, further suggest that carotenoid

coloration is not directly associated with individual oxi-

dative stress levels in this species (Isaksson et al., 2005,

2007).

On the basis of an earlier study on plumage color of

P. major, we expected tissue carotenoid concentrations to

be smaller in the polluted area (Eeva et al., 1998). Con-

trary to our expectations, tissue carotenoid concentrations

were not smaller in the polluted area in summer 2004.

However, the result is understandable when one considers

that natural caterpillar availability for P. major seems to

vary considerably among years in our study area (Eeva

et al., 2005). Also, differences in tissue carotenoid con-

centrations between polluted and unpolluted areas are not

constant between years. For example, whereas in 2004,

plasma lutein concentration was 1.29 higher in the pol-

luted area, in 2005, it was 3.09 higher in the unpolluted

area [Eeva T., unpublished data]. This nonsynchronous

variation is likely to be explained by yearly variation in

timing and abundance of caterpillars. Thus, our carot-

enoid supplementation experiment was done in a field

season when the pollution-related differences in caroten-

oid availability and tissue concentrations were small. This

is why we found only small difference in plumage color

and no significant difference in feather lutein concentra-

tions. However, the outcome of our experiment, which is

based on within-brood comparisons in differently pol-

luted territories, is not dependent on the extent of dif-

ferences in natural carotenoid availability between

pollution zones.

Why was nestling plumage still more yellow in the

unpolluted area, even though there was no corresponding

difference in tissue concentrations? One possible explana-

tion could be pollution-related changes in feather micro-

structure. Recent studies have shown that structural

components of feathers contribute to the intensity of yellow

coloration, although its role is supposed to be small when

compared to variation in feather carotenoid levels (Shaw-

key and Hill, 2005; Shawkey et al., 2006). Since nestlings in

Figure 3. Plumage yellowness index of 16-day-old P. major nestlings

in relation to caterpillar biomass index at territories at the time of

hatching in the lutein supplementation (solid circles) and control

(open circles) group. Yellowness of the breast plumage was

determined from digital images as a proportion of the yellow

component in the CMYK color profile (see Methods). N = 113

nestlings.


a polluted area grow more slowly, they might also produce

lower quality feathers than nestlings in an unpolluted area.

However, the discrepancy between tissue concentrations

and color might also be explained by different timing of

caterpillar peaks in the two study areas. In summer 2004,

caterpillar peak seemed to be later in the polluted area than

in the control area [Eeva T., unpublished data]. Even

though there were fewer caterpillars in the polluted area at

the time of hatching, when the effect on plumage color is

supposed to be strongest (Fitze et al., 2003b), their avail-

ability was good in the polluted area at the time of col-

lecting plasma samples. Therefore, plumage color may

reflect caterpillar abundance during early breeding, whereas

plasma concentrations reflect the carotenoid availability at

the time of blood sampling (9 days). Regardless of the

yearly variation (see also Slagsvold and Lifjeld, 1985; Biard

et al., 2005), nestlings in the polluted area have shown

consistently smaller values for their plumage yellowness in

all of the four seasons (1996, 1998, 2004, and 2005) when

plumage color has been assessed in our study area (Eeva

et al., 1998; Ronka, 1999) [Eeva T., unpublished data from

2005].

The expression of carotenoid-based plumage coloration

in birds is generally supposed to be condition-dependent

(Johnsen et al., 2003; Senar et al., 2003; Tschirren et al.,

2003). Because we found that P. major nestlings in the pol-

luted area were less yellow and smaller in size, there remains

the possibility that food-limitation and the resulting nutri-

tional stress (Eeva et al., 2003) as such might cause, e.g.,

oxidative stress in a polluted area, which is not directly re-

lated to heavy metal levels. For example, Hill (2000) showed

that young food-deprived House finches (Carpodacus mex-

icanus) expressed a paler plumage even though they were

supplied with the same amount of carotenoids as well-

nourished ones. In our experiment, however, nestling body

mass and plumage yellowness were not correlated, and our

carotenoid supplementation did not affect nestling body

mass or hematocrit level. Unlike tits, House finches metab-

olize carotenoids before depositing them in the feathers,

which means that there are higher energetic constraints in

House finch coloration. In agreement with our results, Fitze

et al. (2003a) found that the quantity of food provided to

nestlings did not correlate with the plumage color of P. major

nestlings. Tschirren et al. (2005) further showed that nestling

plumage color does not affect parents’ food provisioning

rates. Therefore, we consider it unlikely that paler plumage in

a polluted environment would be a consequence of energetic

limitation.

CONCLUSIONS

Our experiment showed that pollution-related variation in

the availability of carotenoid-rich food is mainly responsible

for the observed variation in the plumage color of P. major

nestlings around a point source of heavy metals. The mea-

surement of yolk lutein concentrations further showed that

pollutant levels were not related to the amount of lutein

available via egg yolk for developing young. Our results do

not mean that physiological factors like oxidative stress could

have no role in color expression under pollution exposure,

because the observed responses are likely to be dose and

pollutant-dependent. At higher levels of heavy metal expo-

sure, or under an exposure to other types of pollutants, the

importance of the latter mechanism might increase.

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