S CIE N CE OF T H E TOT AL E N V I RO N ME N T 4 0 6 ( 2 00 8 ) 2 4 7–2 55
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / s c i t o t e n v
Carotenoids in a food chain along a pollution gradient
Saila Sillanpääa,⁎, Juha-Pekka Salminenb , Esa Lehikoinena , Eija Toivonena , Tapio Eevaa
a
b
Section of Ecology, 20014 University of Turku, Finland
Laboratory of Organic Chemistry and Chemical Biology, 20014 University of Turku, Finland
AR TIC LE I N FO
ABS TR ACT
Article history:
Carotenoids are synthesized by plants, therefore insects and birds must obtain them from
Received 28 April 2008
their diet. They function in pigmentation and as antioxidants. We studied the carotenoid
Received in revised form
profiles in a model food chain (plant–insect–bird) in an air pollution gradient to find out
18 June 2008
whether heavy metal pollution affects the transfer of carotenoids across the trophic levels.
Accepted 30 July 2008
Birch leaves showed higher β-carotene and, one of the birch species (Betula pendula), higher
Available online 13 September 2008
total carotenoids levels in the polluted area. There was no difference in the lutein
concentration of caterpillars’ food source, birch leaves, between the study areas.
Keywords:
Autumnal moth larvae accumulated lutein more efficiently than β-carotene while sawfly
Carotenoids
larvae accumulated β-carotene over lutein. Because of different antioxidant profiles in
Caterpillars
different leaf chewing insects their sensitivity to pollution stress may differ. The lutein
Great tit
concentration of plasma and feathers of Great tit nestlings did not differ along the pollution
Heavy metal pollution
gradient. The lack of difference in lutein concentration of autumnal moth larvae along
Lutein
pollution gradient may partly explain the lutein concentrations of Great tit nestlings, since
Terrestrial food chain
the abundance of autumnal moth larvae peak during the nestling phase of Great tit. The
lutein concentration of autumnal moth larvae was positively associated to circulating
plasma lutein level of Great tit indicating the importance of carotenoid rich diet during the
nestling phase. In addition, the higher the plasma lutein concentration the more lutein was
deposited to feathers, irrespective of the other possible functions of lutein in nestlings. We
found that carotenoid levels differed between the polluted and the unpolluted area
especially at lower levels of food chain: in birches and in caterpillars.
© 2008 Elsevier B.V. All rights reserved.
1.
Introduction
Carotenoids have many important physiological functions at
all trophic levels (Krinsky, 1994; Olson and Owens, 1998; Fraser
and Bramley, 2004; Škerget et al., 2005; Carrol and Berenbaum,
2006). Plants are able to produce carotenoids in chloroplasts,
and also to regulate their amounts better than organisms at
higher trophic levels. Carotenoids affect plant development
and adaptation, which suggest that their synthesis is coordinated with other developmental processes (Young, 1991;
Young and Britton, 1993; Castemiller and West, 1998; Fraser
and Bramley, 2004). Organisms higher in food chain, such as
insects and birds, are unable to synthesize carotenoids de novo,
therefore they have to obtain carotenoids from their diet
(McGraw and Hill, 2001) and thus are dependent of the
carotenoid availability in their environment. The carotenoid
content of an insect is dependent of the carotenoid content of
its diet, but many insect species selectively absorb one
carotenoid, lutein, over the others (Ahmad, 1992). Carotenoids
also act in pigmentation of insects, e.g. protecting them from
predation and light damage (Rothchild, 1978; Carrol and
Berenbaum, 2006). Carotenoid depletion may further delay
hatching of insect larvae (Sakamoto et al., 2003). In plants and
insects carotenoids function as antioxidants against endogenous and exogenous oxidative stress (Ahmad, 1992; Bungard
et al., 1999).
⁎ Corresponding author. Tel.: +358 23335717; fax: +358 23336550.
E-mail address: [email protected] (S. Sillanpää).
0048-9697/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.scitotenv.2008.07.065
248
SC IE N CE OF T H E TOT AL E N V I RO N ME N T 4 0 6 ( 2 00 8 ) 2 4 7–2 55
Similar functions are found in birds, which absorb carotenoids from their diet and transfer it via plasma to tissues (Parker,
1996; McGraw et al., 2003a). In passerine birds carotenoids and
melanins are the most important pigments in feather coloration
(Badyaev and Hill, 2000). In many bird species the most
abundant carotenoid expressed in plumage coloration is lutein,
often associated with its close molecular relative, zeaxanthin, or
zeaxanthin derivative, dehydrolutein (Brush, 1978; McGraw
et al., 2003b). Lutein may be selectively accumulated over
other carotenoids into feathers (McGraw et al., 2003b).
Pollutants cause oxidative destruction of carotenoids (Bačkor
and Vaczi, 2001) and may also increase and/or decrease the
amount of primary (e.g. nutrients) and secondary compounds
(e.g. phenolics) of plants and hence change the nutritive value of
plants for herbivorous insects (Heliövaara and Väisänen, 1993;
Riemer and Whittaker, 1989; Loponen et al., 2001). Insects, on
the other hand, are the primary carotenoid source for many
birds. For example, the Great tit (Parus major L.), an insectivorous
passerine, prefers carotenoid rich caterpillars in its diet (Gosler,
1993). Matching nestling phase to insect larval period is crucial
for caterpillar eating birds (Nager and van Noordwijk, 1995; van
Noordwijk et al., 1995; Isaksson and Andersson, 2007). In
addition to being a source of nutrients, energy and carotenoids,
phytophagous insects are also a source of pollutants to birds,
since they accumulate heavy metals from plants (Heliövaara
et al., 1987; Heliövaara and Väisänen, 1990; Lindqvist, 1992;
Kozlov et al., 2000; Dauwe et al., 2004a).
Heavy metals cause oxidative stress (Kaminski et al., 2007)
and thus affect different physiological functions of birds, for
example the ability of bird to mount an immune response
(Hõrak et al., 2004). Oxidative stress by pollutants is caused
when there are more oxidants than antioxidants present in the
target organism or cell (see Ahmad, 1992). Environmental
pollution and urbanization can also affect the carotenoidbased coloration of feathers in insectivorous birds (Eeva et al.,
1998; Hõrak et al., 2001; Isaksson et al., 2005). One possible
explanation to pale plumage coloration is the scarcity of
carotenoid rich food items in polluted and urban areas.
Alternatively pollution-related oxidative stress might lead to
depletion of carotenoids in the body. Great tits suffer higher
oxidative stress in urban (e.g. higher air pollution) than in rural
populations (Isaksson et al., 2005). However, the association
between carotenoid coloration (which may act as indicator of
pollution stress) and oxidative stress is unclear (Isaksson et al.,
2005). Isaksson et al. (2007) have shown that plumage carotenoid
coloration may not be reliable signal of current oxidative stress
and it is possible that plasma carotenoids are not active as free
radical scavengers.
Heavy metals pass through the food chain from bottom till
top, highest concentrations being usually found at higher
trophic levels (Laersen et al., 1994; Niecke et al., 1999). This
may also cause higher need of antioxidants for detoxification
at higher levels of a food chain. On the other hand,
carotenoids, which function as antioxidants, are synthesized
in the basal part of food chain, in plants (Fraser and Bramley,
2004). Therefore, carotenoids are thought to be scarce and
costly resources for organisms higher in the food chain (see
Olson and Owens, 1998).
We studied the transmission of carotenoids in a model
food chain, plant–insect–bird, in the surroundings of a well
known pollution gradient around a copper smelter. Our
interest was to study carotenoid concentrations in three
different trophic levels. We measured carotenoid profiles
along the whole food chain during the nestling phase of an
insectivorous passerine, the Great tit. Our main interest
among carotenoids was lutein since it is exclusively the
dominant carotenoid in egg yolk, plasma and feathers of many
bird species, including the Great tit (Saino et al., 2002; McGraw
et al., 2003a,b; Hargitai et al., 2006). Accordingly, we studied
lutein and β-carotene concentration of two birch species:
silver birch (Betula pendula) and downy birch (Betula pubescens),
two insect groups, i.e. autumnal moth (Epirrita autumnata,
Geometridae) and phytophagous sawflies (Hymenoptera:
Symphyta). Finally we studied pollution effects on lutein
concentration of Great tit nestling plasma and feathers. With
pollution effect we mean both direct and indirect effects that
result from environmental changes due to pollution. Isaksson
and Andersson (2007) studied carotenoid availability by diet
quantity and quality and feeding behaviour of parent Great
tits. Partali et al. (1985) studied carotenoids in a terrestrial food
chain. However, this is to our knowledge the first study that
takes to account carotenoid profiles along a pollution gradient
in a terrestrial food chain: plant–insect–bird.
2.
Materials and methods
2.1.
Study organisms
The Great tit is a territorial and resident species that is
susceptible to pollution in the study area all the year round
(see Gosler, 1993; Eeva, 1996). Though Great tit is omnivorous,
it prefers insects especially when feeding nestlings. Depending on their availability, phytophagous caterpillars make up to
60–95% of nestling diet (Gosler, 1993; Eeva, 1996).
We concentrated on two important food sources for tits:
sawfly (Hymenoptera: Symphyta) and autumnal moth (Epirrita
autumnata, Geometridae) larvae. Sawflies are a group of
insects, in which larval stage spreads over the growing season
from the burst of leaves of deciduous trees till leaf senescence
depending on the species (Riipi, 2004). We treated all sawfly
species as a group. Two unidentified species of sawflies were
the main species feeding on birch leaves in the polluted and in
the unpolluted area during the nestling phase of Great tit. The
autumnal moth hatches in spring at leaf flush (at our study
area early May). The larval period of autumnal moth lasts
approximately one month in southern Finland and consists of
five distinct instars (Tenow, 1972).
Birch leaves are a food resource for larvae of autumnal
moths and many sawfly species. Phytophagous insects obtain
carotenoids as well as heavy metals from the leaves they feed
upon. Birches are also good objects for studying pollution
effects since they tolerate pollution exposure relatively well
and readily colonize polluted areas (Denny and Wilkins, 1987;
Eltrop et al., 1991). We measured carotenoid profiles along the
pollution gradient in two closely related birch species, downy
birch (Betula pubescens) and silver birch (Betula pendula). In our
study area these two common birch species form mixed stands
with Scotch pine (Pinus sylvestris) and Norway spruce (Picea
abies).
S CIE N CE OF T H E TOT AL E N V I RO N ME N T 4 0 6 ( 2 00 8 ) 2 4 7–2 55
2.2.
Study area
The study was carried out in 2004 in twelve nest box sites at 0.5
to 12 km distance from the point source of pollution in town
Harjavalta (61°20′N, 22°10′E) and surrounding area (see e.g.
Eeva, 1996). The main pollutants in the area are sulphuric
oxides and heavy metals (Kiikkilä, 2003). Heavy metal concentrations diminish exponentially from the pollution source
approaching background levels at the distance of 5 km (Eeva
et al., 1997). Vegetation, insects and birds are known to suffer
from heavy metal contamination (Eeva and Lehikoinen, 2000;
Kiikkilä, 2003). Nest box sites were classified as polluted area (6
sites less than 2 km from the smelter) and unpolluted area (6
sites over 5 km from the smelter). Nest boxes were visited daily
during egg laying period and every second day during the
nestling phase. The study was performed under the licenses of
the Animal care & use Committee of Turku University and
Regional Environment Centre.
2.3.
Collection of samples
2.3.1.
Plasma
tit nestling period. We classified larvae to three different
groups: autumnal moth larvae, sawfly larvae, and other
larvae. Sawfly larvae consisted from early season species.
Larvae were stored in − 20 °C and protected from light till the
analyses. Lutein and β-carotene were analysed from 67
autumnal moth and 48 sawfly larvae. Larvae, whose dry
mass was too small to perform carotenoid analyses, were left
out from the analyses.
2.3.5.
2.3.2.
Feathers
We collected yellow breast feathers from one haphazardly
selected nestling in each nest. Feather samples were collected at
the age of 16 days from 25 nests in the polluted and from 33
nests in the unpolluted area. Samples were stored in −20 °C until
the carotenoid analysis. All collected samples were protected
from heat and light both in the field and during storage, since
temperature and light may affect carotenoid concentrations.
2.3.3.
Faeces
To study the relationship between heavy metals and carotenoid concentrations in Great tit nestlings we collected fresh
faeces of nestlings for heavy metal analyses. Nestling faeces
are considered to be good indicators of heavy metal concentration in the environment (Dauwe et al., 2004b). One sample
per brood was taken, consisting of faecal sacs of 3–4 nestlings.
In all 60 samples were collected from defecating nestlings at
the age of 8 days directly to plastic Eppendorf tubes, which
were sealed and kept frozen until the heavy metal analyses.
2.3.4.
Caterpillars
The amount of caterpillars was estimated from five birch trees
in every Great tit territory (290 trees in 58 territories). Birches of
1–4 m of height were shaken, and fallen larvae were collected
from white plastic sheet (2 m × 2 m) underneath each birch.
Collection of larvae was repeated four times during the Great
Leaves
We collected leaf samples of both silver birch (Betula pendula)
and downy birch (Betula pubescens) in the middle (1st–4th June)
of nestling period of Great tit in every territory. In each
territory two silver (31 territories) and downy birches (78
territories) of same height were sampled, if they were present.
Leaves were collected evenly from different parts of the
canopy. Leaves were cut from branches without damaging
the leaf itself. Leaves were stored in − 20 °C and protected from
light till carotenoid analysis.
2.4.
We took 59 blood samples, one from each nest, from brachial
vein of 9 days old chicks for the analyses of lutein concentration of plasma by using sterile needles and heparinized micro
capillaries. Blood cells were separated from plasma by
standardized centrifuging (5 min, 4000 rpm) immediately
after sample collection. Plasma was separated, preserved in
ice and kept protected from light during the transportation
and stored in − 20 °C until the carotenoid analyses. At the age
of 6 days each nestling was ringed with an individual
aluminum ring. Nestlings were weighed and their wing length
was measured at the ages of 8 days.
249
Extraction and analyses of carotenoids
Feathers, birch leaves and insect larvae were freeze-dried and
ground into fine powder. A known amount of fine powder
(birch leaves 20 mg, feathers 1–35 mg, larvae 0.35–35.87 mg)
and a known volume of Great tit plasma (10–35 μl), were
extracted three times with 100% acetone (3× 500 μl, 3 × 1 h). The
solvent was evaporated from the combined extract under
vacuum and the residue dissolved into a small volume of 80%
aqueous acetone (feathers and plasma) or 100% acetone (birch
leaves and larvae). The carotenoid composition of the
extracts was analysed with high-performance liquid chromatography at 450 nm using either a Merck Purospher STAR RP-18
(55 × 2 mm, i.d., 3 μm; for feathers and plasma) or an YMC C-30
(250× 4 mm, i.d., 5 μm; for birch leaves and larvae) column. βcarotene was quantified as β-carotene equivalents and the
other carotenoids as lutein equivalents. In the total carotenoid
concentration lutein, zeaxanthin and β-carotene were identified. Other carotenoids that were present especially in leaves are
yet to be characterized. However, lutein zeaxanthin and βcarotene were the main components in all samples. Saponification step was not used during the extraction, since birch
leaves or caterpillars were not found to contain any carotenoid
esters. We have found these types of esters e.g. in rose hips
(Salminen J-P, unpublished data) and with those samples
saponification is necessary to quantify the “total lutein”
concentration of rose hips. If the sample (like birch leaves)
contains no carotenoid esters, the saponification is unnecessary. Standards were used in the analyses of carotenoids. One of
the feather samples was too small for the analysis.
2.5.
Heavy metal analyses
Faecal samples were dried in laboratory at 50 °C for 72 h and
weighed in a range of 150–200 mg. Two milliliters of Suprapure HNO3 and 0.5 ml of H2O2 was added to the samples in
Teflon bombs for digestion with microwave system (Milestone
High Performance Microwave Digestion Unit mls 1200 mega).
Samples were diluted to 50 ml with deionized water (Elgastat
Maxima) after the digestion. The determination of metal
250
SC IE N CE OF T H E TOT AL E N V I RO N ME N T 4 0 6 ( 2 00 8 ) 2 4 7–2 55
Table 1 – The mean (95% confidence limits) lutein, β-carotene and total carotenoid concentrations (μg/g d.w.) in birch leaves
(the means are estimates from general linear mixed models shown belowa)
β-Carotene
Lutein
Betula
Betula
Betula
Betula
pubescens, polluted area (N = 25)
pendula, polluted area (N = 13)
pubescens, unpolluted area (N = 53)
pendula, unpolluted area (N = 18)
343
334
333
312
Zone (polluted vs. unpolluted)
Species (B. pendula vs. B. pubescens)
Zone ⁎ species
(313–377)
(293–380)
(312–355)
(279–348)
351
378
336
321
Total carotenoids
(319–384)
(333–426)
(315–358)
(285–358)
1000 (917–1090)
1160 (1029–1307)
990 (933–1050)
961 (868–1063)
Fdf
p
Fdf
p
Fdf
p
0.931,105
0.821,105
0.141,105
0.34
0.37
0.71
4.091,105
0.101,105
1.431,105
0.046
0.78
0.23
4.361,105
1.571,105
3.551,105
0.039
0.21
0.063
a
For the analyses, lutein and total carotenoids were log10 transformed and β-carotene was square root transformed to normalize distributions.
Estimates and their 95% confidence limits are back transformed to original scale.
concentrations (As, Cd, Cu, Ni, Pb and Zn) was done with ICPMS (Elan 6100 DRC, PerkinElmer-Sciex). The detection limit for
most of the metals was around ppt (ng/l) level and below. The
calibration of the instrument was done with certified solution
(Claritas PPT, Multi element solution 2A) from Spex Certiprep.
2.6.
Statistical analyses
All analyses with carotenoid concentrations as response
variables were done with linear mixed models (LMM) by
using SAS Mixed model procedure. Degrees of freedom were
calculated with Satterthwaite’s procedure as recommended
by Littell et al. (1996). All analyses started with the full model
including a number of explanatory variables and their
interactions. During model selection insignificant interactions
and/or explanatory variables were dropped from the model
until the final models were achieved following the parsimony
principal. Random variables were tested with log likelihood
test and their significance was assigned with χ2. To study the
effect of caterpillar biomass on the lutein concentration of
plasma and feathers of Great tit we performed LMMs with a
logarithm of the territory mean of caterpillar biomass in a tree
(mg/tree) as an explaining factor.
Principal components (PC) of faecal heavy metal concentrations (As, Cd, Cu, Ni, Pb and Zn) were calculated since
concentrations were intercorrelated. Only the first principal
component (PC1) had an eigenvalue greater than 1, and it
explained 66% of variation in the data. Other PCs were not thus
included in further analyses. PC1 correlated strongly with the
distance to the pollution source (r = −0.77, p b 0.0001). All heavy
metals correlated positively in PC1. The effect of pollution on
fledgling number was analysed by SAS Genmod procedure
using Poisson distribution and log function. The effects of
plasma and feather lutein concentrations and PC1 on fledgling
number was analysed with SAS Mixed model procedure. The
relationships between lutein concentrations in different
trophic levels were analysed with linear mixed models.
In all the analyses the response variables were appropriately transformed, if needed, to meet the assumptions for
parametric testing. Estimates and their 95% confidence limits
were back transformed to the original scale in tables. Note that
though some of the confidence intervals overlap, there may be
still statistically significant differences between estimates, as
statistical tests show, since the α = 0.05 significance level
equals to 85% confidence intervals (see Payton et al., 2000).
3.
Results
3.1.
Carotenoids in birch leaves
Lutein concentration of birch leaves did not differ between the
study areas but birch leaves contained more β-carotene in the
polluted area than in the background area (Table 1). Lutein and
β-carotene concentrations of birch leaves did not differ between
Table 2 – The mean (95% confidence limits) lutein and β-carotene concentrations (μg/g d.w.) of autumnal moth (N = 24 in the
polluted and N = 43 in the unpolluted area) and sawfly larvae (N = 27 in the polluted and N = 21 in the unpolluted area) (the
means are estimates from general linear mixed models shown belowa)
β-Carotene
Lutein
Autumnal moth
Sawflies
Autumnal moth
Sawflies
82 (68–100)
88 (74–106)
18 (14–23)
15 (11–19)
26 (20–35)
30 (24–38)
48 (39–60)
47 (37–59)
Polluted
Unpolluted
Zone
Larval mass
Zone ⁎ mass
a
Fdf
p
Fdf
p
Fdf
p
Fdf
p
3.601,62.9
6.621,62.3
3.531, 61.7
0.062
0.013
0.065
4.241,43.1
10.31,41
2.321,43.5
0.046
0.0026
0.14
0.821,63
4.321,63
0.261,63
0.36
0.042
0.61
5.051,44
0.081,33.5
7.181,37
0.030
0.030
0.011
Lutein and β-carotene concentration are log10 transformed. Estimates and 95% confidence limits are back transformed.
251
S CIE N CE OF T H E TOT AL E N V I RO N ME N T 4 0 6 ( 2 00 8 ) 2 4 7–2 55
the two birch species (Table 1). Lutein and β-carotene represented up to 70% of total carotenoids of birch leaves (Table 1).
The total carotenoid concentration of birch leaves was higher in
the polluted area than in the unpolluted area. This was due to
total carotenoid concentration of B. pendula (Table 1).
3.2.
Carotenoids in larvae
Autumnal moths contained higher concentration of lutein, but
lower concentration of β-carotene than sawflies (F1,108 = 271.5,
p b 0.0001, F1,113 = 16.01, p = 0.0001 respectively, Table 2). Sawflies
contained higher concentration of lutein in the polluted than in
the unpolluted area, but there was no difference in lutein
concentrations of autumnal moth larvae between the areas
(Table 2). The smaller was the dry mass of autumnal moth and
sawfly larvae, the higher was their lutein concentration
(Table 2). The concentration of β-carotene between the polluted
and the unpolluted area did not differ in autumnal moth larvae,
but sawfly larvae had a higher amount of β-carotene in the
polluted area than in the unpolluted area (Table 2). The higher
was the dry mass of autumnal moths the lower was their level of
β-carotene. In sawflies, the overall association of dry mass and
β-carotene was insignificant, but the interaction between area
and dry mass revealed that in the polluted area the association
was negative, while in the unpolluted area it was positive
(Table 2, Fig. 1). Sampling date (taking to account sampled trees)
had no effect on lutein or β-carotene concentration of larvae
(autumnal moth: lutein: log likelihood = 54.8, χ2 p N 0.05, βcarotene: log likelihood = 0, χ2 p N 0.05; sawflies: lutein: χ2 = 0, χ2
p N 0.05, β-carotene: χ2 = 0.7, χ2 p N 0.05). Also the wet mass of both
autumnal moths and sawflies was higher in the polluted area
compared to unpolluted area (F1,79 = 4.44, P = 0.038) and autumnal
moth larvae had higher wet mass than sawflies (F1,79 = 13.51,
P = 0.0004).
3.3.
Carotenoids in plasma and feathers
There was no difference in lutein concentration of Great tit
plasma between the unpolluted and the polluted area (Table 3).
Faecal heavy metal concentration (PC1) showed no association
Table 3 – The mean (95% confidence limits) lutein
concentrations (μg/g) in plasma (w.w.) and breast
feathers (d.w.) of P. major(the means are estimates from
general linear mixed models shown belowa)
Polluted area
Unpolluted area
Feather
22.3 (14.0–35.5)
22.4 (14.7–34.3)
9.3 (5.2–16.2)
9.3 (5.8–14.5)
Zone (polluted vs. unpolluted)
PC1 of heavy metals
PC1 ⁎ zone
Wing length
Fdf
p
Fdf
p
0.881,54
0.001,54
0.441,54
0.35
0.98
0.51
0.011,50
0.181,50
0.161,50
4.391,50
0.93
0.67
0.69
0.041
a
The lutein concentration of plasma (N = 24 in the polluted and
N = 35 in the unpolluted area) and feathers (N = 23 in the polluted
and N = 33 in the unpolluted area) is log10 transformed respectively.
Estimates and 95% confidence limits are back transformed.
with the lutein concentration of plasma (Table 3). Date or time of
blood sampling had no effect on the lutein concentration of
plasma (date: F1,52 = 1.51, p = 0.22; time: F1,52 = 0.09, p = 0.76). Nestling body mass (F1,52 = 0.65, p = 0.43), wing length (F1,53 = 1.66,
p = 0.20) number of hatched chicks (F1,53 = 0.25, p = 0.62) or the
amount of chicks at sampling date (F1,53 = 1.24, p = 0.27) had no
effect on the lutein concentration of plasma and were dropped
from the final model. The biomass of larvae on great tit
territories showed no association with lutein concentration of
plasma (F1,54 = 0.20, p = 0.65).
There was also no difference in lutein concentration of
feathers between the unpolluted and the polluted area
(Table 3). Heavy metal concentrations (PC1) showed no
association with lutein concentration of feathers (Table 3).
Number of hatchlings (F1,49 = 0.00, p = 0.97) or the number of
chicks at sampling date (F1,49 = 0.25, p = 0.62) or sampling date
(F1,49 = 0.25, p = 0.62) had no effect on the lutein concentration
of feathers and were dropped from the final model. There was
no association between body mass and feather lutein concentration of nestlings (F1,49 = 2.28, p = 0.14), but the association
of wing length and feather lutein concentration was negative
(F1,49 = 4.39, p = 0.0413). There was a positive association
between the biomass of larvae and lutein concentration of
feathers (F1,53 = 5.2, p = 0.027).
3.4.
Fig. 1 – β-Carotene concentration of sawflies vs. dry mass of
sawflies. Black triangle = polluted area, open
triangle = unpolluted area.
Plasma
Lutein transportation in the food chain
The lutein concentrations of autumnal moth or sawfly larvae
were not related to lutein concentration of birch leaves at
territory level (F1,57 = 0.06, p = 0.81, F1,55 = 1.89, p = 0.18, respectively). Note, however, that the leaf samples and larvae were not
collected simultaneously and temporal variations may affect
this relationship. Instead, concentrations in Great tit was
positively related to concentration in autumnal moth larvae
the higher was the lutein concentration of autumnal moths the
higher was the lutein concentration of nestling plasma
(F1,33 = 5.11, p = 0.033, Fig. 2). There was, however, no significant
relationship between autumnal moth lutein concentration and
Great tit feather lutein concentrations (F1,33 = 2.11, p = 0.15). The
lutein concentration of sawfly larvae had no effect on Great tit’s
lutein concentration of plasma (F1,22 = 0.04, p = 0.84) or feathers
(F1,21 = 1.53, p = 0.23). The higher was the lutein concentration of
252
SC IE N CE OF T H E TOT AL E N V I RO N ME N T 4 0 6 ( 2 00 8 ) 2 4 7–2 55
Fig. 2 – Lutein concentration of autumnal moth larvae vs.
lutein concentration of Great tit plasma.
Great tit nestlings’ plasma the higher was lutein concentration
of feathers (F1,56 = 17.6, p = 0.0001).
3.5.
Fledgling number
The clutch size of Great tit did not differ between study areas
(GLMM; χ21,56 = 1.74, p = 0.19). The fledgling number, however,
was smaller in the polluted area (mean = 4.08, SD = 1.68) than in
the unpolluted area (mean = 5.64, SD = 1.52) (GLMM; χ21,56 = 7.9,
p = 0.0050). The higher was the heavy metal concentration of
faeces (PC1), the lower was fledging success (F1,56 = 6.36,
p = 0.015). The lutein concentration of plasma or feathers was
not associated with fledgling number (F1,56 = 0.61, p = 0.44,
F1,56 = 0.97, p = 0.33 respectively) and were dropped from the
final model. The larval biomass in territories during the whole
nestling period affected positively fledging number (GLMM;
χ21,56 = 4.16, p = 0.041).
4.
Discussion
4.1.
Carotenoids in birches
We expected lower carotenoid concentrations in birches near
the pollution source (see Ekmekcki and Terzioglu, 2005).
Surprisingly, however, we found higher carotenoid concentrations in birches in the polluted area. Leaf β-carotene concentration was higher in the polluted than in the unpolluted area in
both birch species studied (4.5% in B. pubescens, 18% in B.
pendula). The total carotenoid concentration was also 21% higher
in the polluted area than in the unpolluted area in B. pendula.
The higher concentrations of total carotenoids and β-carotene
in birch leaves in the polluted area may suggest an adaptive
response of birches near the pollution source. Increased
antioxidant level might protect birches under potentially
stressful conditions. Increased β-carotene content could probably be explained by the importance of β-carotene as an
antioxidant. β-Carotene is known to have a protective role in
cells against oxidative stress and it is relatively resistant to
degradation (see e.g. Young and Britton, 1993; Choudhury and
Behera, 2001; Wisniewski and Dickinson, 2003). Kozlov (2005)
found that mountain birch is relatively resistant to pollution
and also shows phenotypic acclimation to pollution. In our
study, the elevation of birch leaf carotenoid levels in the
polluted area might also be explained by the same phenomenon. Contrary to β-carotene, leaf lutein concentration was not
higher in the polluted area. This may suggest that lutein has a
less important role as an antioxidant and its production is not
induced in birch leaves as a response to pollution stress.
The levels of lutein and β-carotene also increase with
increasing lightness (McKinnon and Mitchell, 2003), as does
also photo-oxidative stress, when light is combined with
environmental stress (Ekmekci and Terzioglu, 2005). In our
study area the canopy in the polluted area is more open than the
canopy in the unpolluted area, which increases the amount of
light for birches in the polluted environment. Carotenoids
function in photoprotection (Demming-Adams et al., 1996) and
we may thus expect that birches in the polluted area need more
photoprotection than birches in the unpolluted area. In plants
β-carotene is further metabolized to zeaxanthin, which also has
a protective role against light stress (Davison et al., 2002). Higher
plants may differ in their ability to respond to light-mediated
environmental stress by producing zeaxanthin (Young, 1991).
This may affect the plant performance in habitats with
environmental stress, e.g. sun, shade (Young, 1991) and/or
pollution. Both birches studied are shade intolerant, but B.
pubescens is more shade intolerant than B. pendula (Portsmuth
and Niimets, 2007). Shading affects many traits in birches,
including net assimilation rate, relative growth rate, leaf mass
and leaf nitrogen and phosphorus concentration (Portsmuth
and Niimets, 2006) and it might well be that also carotenoid
concentrations are affected by shading. In the unpolluted area
coniferous trees (Pinus sylvestris and Picea abies) shade birches
more than in the polluted area.
4.2.
Carotenoids in autumnal moth and sawflies
Since lutein concentrations of birches did not differ between
the polluted and the unpolluted area, there was a similar
amount of lutein available for herbivorous larvae in the next
trophic level in both areas. We found no difference in lutein
concentrations of autumnal moth larvae between the areas.
However, sawflies accumulated more lutein in the polluted
area than in the unpolluted area, though there was no
difference in the lutein concentration of their main food
source. This may suggest that sawfly larvae are more sensitive
to pollution and thus need to accumulate lutein more
efficiently than autumnal moths. However, sawfly species
composition, which was not known in our sample, may
confound this result. The association between body mass
and lutein concentration of autumnal moth and sawfly larvae
was negative. This could be due to different allometric
relationships in different sized larvae. The relative proportion
of leaf mass in the intestine of larva per mass unit is probably
higher in small than in larger larvae. In the polluted area there
is more β-carotene available for leaf chewing insects, but this
does not show up as higher β-carotene content of autumnal
moth larvae. Again, sawfly larvae accumulated more βcarotene in the polluted area than in the unpolluted area.
With respect to lutein and β-carotene concentration there
S CIE N CE OF T H E TOT AL E N V I RO N ME N T 4 0 6 ( 2 00 8 ) 2 4 7–2 55
253
seems to be difference between autumnal moth and sawfly
larvae in the efficiency and/or need to accumulate these
carotenoids in the polluted area. Contrary to Isaksson and
Andersson (2007), who found lower concentrations of carotenoids in urban than in rural environment, in our study the
carotenoid concentrations either had no association with
pollution or carotenoid concentrations were higher in the
polluted area depending the insect species in question.
Lutein and β-carotene have the same quenching constant for
free radicals but insects often selectively accumulate lutein over
other carotenoids (see Ahmad, 1992). Our data suggest that this
preference is not ubiquitous, since sawflies accumulated βcarotene more than lutein. The difference between sawfly and
autumnal moth larvae cannot be explained by diet, since both
used birch leaves as their food resource. In addition to pollution
stress, leaf chewing insects have to cope with plant derived
allelochemicals which may also function as pro-oxidants and
the sensitivity of autumnal moths and sawflies to these prooxidants may differ (see Ahmad, 1992). Different allelochemicals may increase, decrease and/or stay constant in birches due
to pollution (Loponen et al., 2001). Secondary chemicals, such as
phenolics, also vary during the growing season (see Haukioja
et al., 2002). This temporal variation may affect the carotenoid
profiles, since the later occurring sawflies may be exposed to
larger amounts of secondary chemicals than the earlier
occurring moths.
variation in both parameters. The fledgling number in nests did
not affect the carotenoid levels in plasma. This suggests that
other aspects than lutein in food items plays more important
role for nestling survival. Such factors may be the overall
amount, nutritive value and/or heavy metal content of food
consumed. Fledgling number was indeed dependent on heavy
metal levels. The more there were heavy metals in faeces the
smaller was the fledgling number. As expected, heavy metals
were present in higher concentration in nestling faeces near the
smelter than further away.
Also growth introduces stress to nestlings by free radical
bursts (Hõrak et al., 2000). We found that larger nestlings (in
wing length) showed lower feather lutein concentrations. This
might suggest that growth stress was met by allocation of
lutein to free radical scavenging. However, plasma lutein
levels were not associated with nestling size. Isaksson and
Andersson (2008) suggested that carotenoid antioxidant functions are not traded off against e.g. feather pigmentation, i.e.
carotenoids important in pigmentation may have a minor role
as antioxidants in birds. It is possible that the lower feather
lutein concentration of large nestlings could be due to dilution
effect in growing feathers, i.e. smaller and less developed
feathers might contain more pigment per mass unit than
larger ones. Also carotenoid concentration may affect feather
growth (see Badyaev and Landeen, 2007) and thus also the
wing length of growing P. major nestlings.
4.3.
4.4.
Carotenoids in Great tit
Lutein is the main pigment determining the yellow coloration in
P. major (Partali et al., 1985). The yellow coloration of P. major
nestlings has been found to be paler in urban and polluted areas
than in rural or unpolluted areas (Eeva et al., 1998; Hõrak et al.,
2001; Isaksson et al., 2005). Therefore we expected plasma and
feather carotenoid concentration to be smaller in the polluted
area (Eeva et al., 1998). Contrary to our expectation, tissue
carotenoid concentrations were not smaller in the polluted area
in the summer 2004. The result is, however, understandable
when considered that natural caterpillar availability for P. major
seems to vary considerably among years in our study area (Eeva
et al., 2005) and also the difference in tissue carotenoid
concentrations between the polluted and unpolluted area is
not constant over years (Eeva T., unpublished data). In 2004 the
availability of larvae was fairly good in both areas which could
explain the lack of differences in tissue concentrations. As
shown by the positive correlation between plasma levels and
feather concentrations, the carotenoid concentration of plasma
predicts the plumage concentration of carotenoids (see also
McGraw et al., 2006).
The biomass of caterpillars showed a positive association
with feather lutein concentration, suggesting that caterpillar
abundance is an important determinant of tissue carotenoid
content in Great tit nestlings. We did not find an association
between the availability of larvae and plasma lutein concentration. However, at some level such association is likely to exist
since caterpillar biomass correlated positively with feather
lutein concentrations and there was a positive correlation
between plasma and feather concentrations. We may have
been unable to detect a direct association between plasma
lutein levels and caterpillar biomass because of relatively high
Carotenoids in a food chain
We found no association between lutein concentrations of
birches and sawflies or autumnal moths. Leaf chewing insects
ingest all compounds present in leaves and these may interact
with accumulation of lutein. The positive association between
lutein concentration of autumnal moth larvae and that of P.
major nestlings’ plasma indicates the importance of carotenoid
rich caterpillars as determinants of plasma circulation of lutein.
In accordance with this, caterpillar abundance was positively
associated with feather lutein concentration, though we were
not able to confirm an association between caterpillar abundance and plasma lutein levels. On the contrary, there was no
association between lutein concentration of sawfly larvae and
nestling plasma lutein level. This is most probably explained by
the low overall lutein concentration of sawflies and the timing
of their larval period so that their availability tends to peak too
late for Great tit nestlings. There was also a positive association
between the plasma and feather lutein concentrations which
suggest that the more plasma circulates lutein the more lutein is
deposited to feathers irrespective of the other functions of
carotenoids in nestlings.
Acknowledgements
We thank Mikhail V. Kozlov and Toni Laaksonen and three
anonymous reviewers for invaluable comments on the manuscript. We thank Lauri Nikkinen for field assistance and Anu
Tuominen for assistance in the laboratory. The study was
financed by Maj and Tor Nessling foundation, Satakunta
Cultural foundation (grants to SS), Emil Aaltonen foundation
(grant to TE), Academy of Finland (project 8119367) and heavy
254
SC IE N CE OF T H E TOT AL E N V I RO N ME N T 4 0 6 ( 2 00 8 ) 2 4 7–2 55
metal analyses by Boliden Company. The study was performed under the licenses of the Animal care & use
Committee of Turku University and Regional Environment
Centre.
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