Seasonal variation in diet quality - Oxford Journals

Biological Journal of the Linnean Society, 2010, 99, 708–717. With 4 figures

Seasonal variation in diet quality: antioxidants, invertebrates and blue tits Cyanistes caeruleus KATHRYN E. ARNOLD*, SCOT L. RAMSAY†, LINDSAY HENDERSON and STEPHEN D. LARCOMBE‡ Division of Ecology and Evolutionary Biology, Faculty of Biomedical and Life Sciences, Graham Kerr Building, University of Glasgow, Glasgow G12 8QQ, UK Received 23 July 2009; accepted for publication 1 October 2009

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708..717

Breeding success is often dictated by the degree to which parents can synchronize the maximum food requirements of offspring to the peak in abundance of invertebrate prey. Less studied is how the nutritional quality of individual diet items impacts on breeding. In the present study, we assessed the abundance and antioxidant concentrations of arboreal arthropods from oak woodland and provisioning behaviour of the blue tit Cyanistes caeruleus. Dietary antioxidants are important during development because they defend against oxidative stress. Operophtera caterpillars, Erannis caterpillars, and spiders contained significantly different levels of individual carotenoids and a-tocopherol. Concentrations of lutein and b-carotene in Operophtera caterpillars did not vary seasonally, although concentrations of zeaxanthin declined and a-tocopherol increased with date. Blue tit broods hatched later in the season received significantly fewer caterpillars and more spiders per chick compared to earlier broods. Reflecting changes in prey composition, blue tit nestling plasma showed decreases in zeaxanthin and increases in a-tocopherol with date. Thus, processes that shift the timing of breeding in birds and/or prey composition are likely to alter antioxidant intake and thus potentially influence the oxidative stress status of animals. The data obtained in the present study suggest a mechanism by which environmental change as a result of human activities could influence the health and fitness of individuals in natural populations. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99, 708–717.

ADDITIONAL KEYWORDS: a-tocopherol – carotenoids – caterpillars – neonatal nutrition – oxidative stress – spiders.

INTRODUCTION Increasingly, it is recognized that diet during early development not only dictates survival, but also impacts upon many fitness related traits in offspring, such as fecundity, sexual ornamentation, and cognitive ability (Olson & Owens, 1999; Arnold et al., 2007; Catoni, Peters & Schaefer, 2008). Thus, parents should try to optimize the diet that they provide to their offspring to maximize their own fitness. In temperate regions, the food available to insectivorous birds increases enormously in spring. For many *Corresponding author. E-mail: [email protected] †Current address: Macaulay Institute, Craigiebuckler, Aberdeen AB15 8QH, UK. ‡Current address: The EGI, Department of Zoology, South Parks Road, Oxford, OX1 3PS, UK.

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species, it is the degree to which parents synchronize their breeding with the peak in this food bonanza that dictates reproductive success (Noordwijk, McCleery & Perrins, 1995; Naef-Daenzer & Keller, 1999). Ecologists generally focus on the quantity of food or sometimes the energy or crude protein levels available, but rarely assess the variability in nutritional quality over the season or between prey items (Ramsay & Houston, 2003). During the chick rearing phase, when energy demands are being satisfied, poor quality foods may fail to provide essential nutrients at the time when they are required for growth, development, and therefore fitness (Arnold et al., 2007). In Parids, the main food source for chicks is Lepidopteran larvae, which can vary hugely and unpredictably in abundance between and within areas and years (Perrins, 1991; Noordwijk et al., 1995). Spiders,

© 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99, 708–717

ANTIOXIDANTS IN INVERTEBRATES AND CHICKS meanwhile, are a relatively small component of the diet of nestling blue tits Cyanistes caeruleus, across a range of habitats and laying dates (Naef-Daenzer, Naef-Daenzer & Nager, 2000; Arnold et al., 2007). However, if the nutritional quality of the diet is considered, then relatively infrequently provisioned prey items can be important because they may be sources of rare but limiting nutrients (Arnold et al., 2007). Thus, the quality (i.e. independent of the quantity) of food can impact upon offspring development (Catoni et al., 2008). However, little is known about nutrient availability in natural systems. The lack of information about the nutritional quality of avian diets in the wild is particularly surprising in relation to a group of nutrients called antioxidants. Antioxidants are known to play an important role in quenching reactive oxygen species (ROS), thus ameliorating oxidative stress. ROS are produced as a natural byproduct of metabolism. Thus, physiological processes that increase metabolism, such as growth (Alonso-Alvarez, Bertrand, Faivre & Sorci, 2007), may cause a rise in oxidative stress and therefore damage to lipids, proteins, DNA, and other biological complexes (Diplock, 1994). ROS may therefore limit the growth potential of young unless antioxidant systems in the body can mop them up. Dietary-derived antioxidants play a role in protecting against oxidative damage. There has been a great deal of interest in the roles of lipophilic antioxidants (Catoni et al., 2008), in particular carotenoids (xanthophylls and carotenes), with respect to offspring development (Horak et al., 2000; Larcombe, Arnold & Alexander, 2006; Isaksson et al., 2007; Isaksson, Johansson & Andersson, 2008) and adult sexual signalling (Olson & Owens, 1999; Saks, McGraw & Hõrak, 2003; Senar, Figuerola & Domenech, 2003). In birds, there have been a number of studies on the honesty of carotenoid-pigmented ornaments in signalling individual ‘quality’ in terms of immune function and more generally antioxidant status (Olson & Owens, 1999; Schantz et al., 1999). However, the effectiveness of carotenoids as antioxidants in vivo remains a matter for debate (Costantini & Moller, 2008). An abundant and powerful antioxidant in birds is a-tocopherol (vitamin E). a-tocopherol is integrated into the lipid bilayer of cellular membranes and protects them from oxidative damage by terminating the chain reactions of ROS. Tissue a-tocopherol content increases proportionally with dietary intake in poultry and maternal deficiencies result in late embryonic mortality in eggs (Surai, 2002). However, a-tocopherol has been less well studied than carotenoids, at least in noncommercial species of birds (de Ayala, Martinelli & Saino, 2006; Larcombe et al., 2006, 2008). a-tocopherol, similar to carotenoids, is synthesized only by plants and is

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found at highest levels in food derived from plants. Many animals, including invertebrates, consume and further metabolize a-tocopherol and carotenoids from plants, potentially providing a rich source for avian predators. In the present study, we investigated the seasonal variation in the availability of antioxidants to breeding birds and how this might impact upon fitnessrelated traits. This study aimed to combine ecological and biochemical approaches to assess: (1) the seasonal change in the availability of invertebrates in mixed oak woodland; (2) differences in antioxidant levels between prey types; (3) seasonal variation in antioxidant concentrations in caterpillars; (4) variation in invertebrate provisioning by parental blue tits; and (5) seasonal changes in the antioxidant intake and blood plasma concentrations of nestling blue tits.

MATERIAL AND METHODS A population of blue tits in Scotland was studied from April to June 2005 (Arnold et al., 2007). The study site was Ross Woods, an oak-dominated woodland on the east side of Loch Lomond (56°08′N, 04°39′W). Nestboxes were monitored to record dates of egg laying, the start of incubation, and hatching. Invertebrates were collected weekly during chick rearing; when a brood was 13 days old, parental provisioning was recorded; at 14 days of age, biometric measures and blood samples were taken from nestlings; finally, high-performance liquid chromatography (HPLC) analyses of the antioxidant concentrations in invertebrates and blue tit plasma samples were performed.

ARTHROPOD

PREY COLLECTION

Throughout the period of chick rearing, arboreal invertebrates were sampled weekly from the leaves, twigs, and thinner branches of oak trees, where blue tits tend to forage. Sampling began in the week that the first blue tit chicks hatched and ceased when approximately 90% of blue tit broods had fledged. Each week, eight trees from across the site were chosen at random for sampling. All samples for a given week were collected over a period of 2 days. Sampling was only carried out on dry days. A rope was thrown over a large branch approximately 2–3 m high and used to shake the branch for 2 min. Invertebrates were collected from cotton sheets spread out on the ground below. Although this method cannot be used to accurately quantify differences between dates, the relative numbers of individual invertebrates, range of taxa, and the ratios between prey types should be broadly representative of the arboreal prey available to blue tits at that point in the


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season. The dislodged invertebrates were counted, collected into plastic vials, and immediately placed on ice packs, to slow down digestion and prevent expulsion of the gut contents. Within 1 h of collection, all samples were stored at -20 °C and later -70 °C until HPLC analysis. The invertebrates were later weighed when frozen.

INVERTEBRATE

INTAKE BY BLUE TIT NESTLINGS

To assess the number and type of prey items brought to chicks aged 13 days, we filmed 20 families. When a brood was 12 days old, we put a small infrared video camera opposite the entrance hole of the nestbox so that we could observe the adults entering. The camera started recording at 06.00 h the following morning for 4 h. Video tapes were later studied, and each time an adult entered the box and the type of prey being carried (i.e. caterpillar or spider) were noted. When assessing the provisioning patterns, we first calculated the hourly rate of intake of spiders or caterpillars per chick within a brood based on the video data. Next, we multiplied each of these values by the mean mass of each prey type in the week of the season that brood was 13 days old, aiming to calculate the hourly rate of biomass eaten per chick. Finally, we multiplied the mean mass of spiders, for example, consumed per hour per chick by the concentration of each antioxidant (mg g-1) in spiders in that week of the breeding season (see below). Thus, we could compare seasonal changes in the rate of antioxidant intake from spiders throughout the season. This was repeated for caterpillars and then a rate of hourly total antioxidants consumption per chick per brood was calculated.

BLOOD

SAMPLING AND MORPHOMETRIC

MEASUREMENTS OF NESTLINGS

When the chicks were 14 days old, half of each brood was taken from the nestbox to the SCENE laboratory in a heated bag. For each bird, a British Trust for Ornithology leg ring was applied and a small volume of blood was taken by venipuncture from a wing vein. One drop of blood was put in ethanol for subsequent molecular sexing (Griffiths, Double, Orr & Dawson, 1998; Arnold et al., 2007). The remaining blood was collected in 75-mL capillary tubes which were then centrifuged at 14 000 g for 5 min. Blood plasma was stored at -20 °C and later at -70 °C. After blood sampling, each bird was weighed and had its wing and tarsus measured. All work was conducted under UK Home Office Licence, and no chicks died as a result of our experiment.

ANTIOXIDANT

ANALYSES OF INVERTEBRATES AND AVIAN BLOOD PLASMA

Samples of invertebrates were prepared: 100–500 mg (wet weight) of sample, composed of multiple individual invertebrates (10–30 individuals depending on their size), was accurately weighed into a glass sample tube. One millilitre of 5% NaCl solution was immediately added and the mixture briefly vortexed. One millilitre of ethanol was then added and the mixture homogenized for 2 min using an Ultra-Turrox T25 homogenizer (IKA) at the same time as adding 3 mL of hexane. The sample was then centrifuged for 10 min at 11 000 g. Then the top layer containing hexane and lipid soluble components was transferred into a glass tube and stored in the dark at 4 °C. This process was repeated so the original sample was then homogenized again for 1 min at the same time as adding 2 mL of hexane, centrifuged as before and the top layer was transferred to small tube containing hexane/extract mixture removed earlier. Subsequently this tube was placed in a ‘Speed Vac’ at 30 °C for 8 min or until all solvent had evaporated and only the sample extract remained. The extract in tube was redissolved in 100 mL of methanol and 100 mL of dichloromethane. Avian plasma samples were prepared (Larcombe et al., 2008): 40 mL of ethanol was added to 20 mL of plasma and vortexed thoroughly. Fifty microlitres of hexane was then added and the mixture vortexed again. The sample was then centrifuged for 10 min at 11 000 g, after which the hexane layer, containing the antioxidants, was drawn off, transferred to a small glass tube and stored in the dark at 4 °C. This process was then repeated with an additional 40 mL of hexane, before the combined hexane extract was placed in a SpeedVac as before. The antioxidant extract remaining in each tube was then re-dissolved in 20 mL of methanol. A Spectra Model 8800 HPLC pump system with a Phenomenex 250 mm ¥ 2 mm inner diameter column was used to determine antioxidant composition of each invertebrate or plasma sample. We used HPLC at a flow rate of 0.2 mL min-1 with a mobile phase of water/acetonitrile (2.5 : 97.5), and water/ethyl acetate (2.5 : 97.5) in a gradient elution. Using a Diode array absorbance detector type Thermo model UV6000, chromatograms were subsequently plotted at 323– 327 nm for retinoids, 449.5–450.5 nm for carotenoids, and 290–295 nm for tocopherols. Peak areas were calculated using the software XCALIBUR (Thermo Finnigan). Concentrations of identified components in original samples were calculated using appropriate dilution coefficients and calibration equations derived from HPLC analysis of prepared standards. The HPLC outputs indicated that there were consistently seven distinguishable peaks for caterpillar


ANTIOXIDANTS IN INVERTEBRATES AND CHICKS extracts, with an additional six peaks for spider extracts. By comparison with standards, we identified four of the major peaks in both caterpillars and spiders as lutein, zeaxanthin, b-carotene, and a-tocopherol. Other small peaks appeared to be unidentified carotenoids. Retinol was either absent from our invertebrate samples, or at concentrations below our detection limit. Finally, we tested the repeatability of our antioxidant analyses. For each defined HPLC peak the variation between different samples was significantly higher (analysis of variance: P < 0.0001 in all cases) than the variation within replicate samples (i.e. between repeat runs of the same sample). Variances were then partitioned. Across all peaks, the mean ± SD variance between replicates of the same sample, expressed as a percentage of total variation, was 7.8% ± 5.6.

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Table 1. Seasonal variation in the numbers of Operophtera spp.larvae, Erannis spp. larvae, and spiders collected Number of invertebrates

Week

Operophtera larvae

Erannis larvae

Spiders

1 2 3 4 5

167 296 417 394 52

15 14 18 13 18

45 27 23 30 26

Start of week 1 = 11 May 2005, when the first chicks hatched.

STATISTICAL

ANALYSIS

The numbers, masses and antioxidant concentrations of invertebrates were analysed using general linear models (GLM) in SPSS, version 15 (SPSS Inc.). Proportional data were transformed prior to analysis. Data from chicks were analysed using general linear mixed models (GLMM) in SAS, version 9.1 (SAS Institute) with a normal error distribution and with nest identity as a random factor in the model to control for the non-independence of data from siblings. Data are shown as the mean ± SD.

RESULTS SEASONAL

CHANGE IN THE AVAILABILITY OF INVERTEBRATES

A range of arboreal arthropods were collected including shield bugs (Hemiptera), various beetles (Coleoptera), millipedes (Diplopoda), and leaf hoppers (Hemiptera). The main groups that we identified were ‘Operophtera spp. caterpillars’ (which included Winter Moth Operophtera brumata larvae and other caterpillar species), ‘Erannis spp. caterpillars’ (including larvae of the mottled umber moth Erannis defoliaria and some other species), and spiders (wolf spiders Lycosidae were locally abundant but smaller spiders remain unidentified). The mean number of Operophtera spp. caterpillars collected differed significantly between weeks (GLM: F = 4.22, d.f. = 4,19, P = 0.013), increasing then reaching a peak on week 3 and dropping sharply by week 5 (Table 1). Mean Operophtera spp. mass varied significantly throughout the breeding season (GLM: F = 8.44, d.f. = 4,15, P = 0.002) (Fig. 1). Mean mass increased progressively, reaching a peak at week 4 before declining. Erannis spp. numbers did not vary significantly over the season

Mean mass of invertebrates (g)

0.05

Operophtera caterpillars 0.04

0.03

Erannis caterpillars 0.02

Spiders

0.01

0.00 1

2

3

4

5

Week

Figure 1. Seasonal variation in mean ± SE mass of individual Operophtera spp. caterpillars, Erannis spp. caterpillars, and spiders. Start of week 1 = 11 May 2005, which coincided with hatching of the first chick.

(GLM: F = 1.25, d.f. = 4,18, P > 0.3) (Table 1), neither did mean mass (GLM: F = 1.30, d.f. = 4,18, P > 0.3) (Fig. 1) but sample size was low and resolution of any seasonal trends may be low. Spider numbers (F = 0.90, d.f. = 2,18, P > 0.4) (Table 1) and mean individual spider mass (GLM: F = 0.74, d.f. = 4,15, P > 0.5) (Fig. 1) did not vary significantly or appear to show any pattern through the breeding season, but the sample size was relatively low.

DIFFERENCES

IN ANTIOXIDANT LEVELS BETWEEN PREY TYPES

Operophtera spp. caterpillars, Erannis spp. caterpillars and spiders were found to have significantly different concentrations of lutein (GLM: F = 9.15, d.f. = 2,17, P = 0.002) (Fig. 2A), b-cartotene (GLM: F = 6.39, d.f. = 2,19, P = 0.009) (Fig. 2B) and a-tocopherol (GLM: F = 10.68, d.f. = 2,17, P = 0.001) (Fig. 2D).


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K. E. ARNOLD ET AL. A)

Lutein (microgram/g)

30 25 20 15 10

B)

50

Beta-carotene (microgram/g)

35

5

40

30

20

10

Operophtera

Erannis

Spiders

Operophtera

Erannis

Spiders

D) C)

Alpha-tocopherol (microgram/g)

Zeaxanthin (microgram/g)

10

8

6

4

2

0

120 100 80 60 40 20 0

Operophtera

Erannis

Spiders

Operophtera

Erannis

Spiders

Figure 2. Differences in the mean ± SE concentrations of lipophilic antioxidants in Operophtera spp. caterpillars, Erannis spp. caterpillars, and spiders. A, lutein; B, b-carotene; C, zeaxanthin; D, a-tocopherol. Means were calculated from samples collected over the whole season. Note the different scales on the y-axes.

Operophtera spp. had higher levels of both lutein (Fig. 2A) and lutein-like carotenoids than other invertebrates (total lutein-type carotenoids: Operophtera spp. mean = 97.77 mg g-1 ± 29.45; Erannis spp. mean = 65.52 mg g-1 ± 12.18 and spiders mean = 15.44 mg g-1 ± 9.08; GLM F = 11.98, d.f. = 2,17, P = 0.001). Erannis spp. had significantly higher levels of b-carotene (Fig. 2B) than other prey types. As might be expected, spiders differed more from either caterpillar genera than caterpillar genera did from each other. Spiders had much lower levels of both lutein, luteinlike carotenoids, and b-carotene than caterpillars. None of the invertebrates analysed were found to contain any detectable retinol. Both groups of caterpillars contained a-tocopherol, with levels being highest in Operophtera spp., although spiders did not appear to contain tocopherols. Zeaxanthin levels did not differ significantly between invertebrate groups (GLM: F = 0.9, d.f. = 2,17, P > 0.4) (Fig. 2C). However, HPLC analysis of spider extracts revealed two peaks of zeaxanthin-like

carotenoids which were not present in caterpillar extracts. If these are included, then spiders had six- to eight-fold higher levels of ‘zeaxanthin & zeaxanthin-like carotenoids’ than caterpillars (total of all zeaxanthin-type carotenoids: Operophtera spp. mean = 5.687 mg g-1 ± 3.65; Erannis spp. mean = 7.91 mg g-1 ± 0.74 and spiders mean = 43.24 mg g-1 ± 3.70; GLM: F = 109.28, d.f. = 2,17, P < 0.0001). These carotenoid-like compounds, which could not be conclusively identified based on our standards, are potentially metabolites of carotenoids, and require further investigation. The concentrations of all carotenoids and carotenoid-like compounds were totalled for each sample and did not differ between invertebrate types (GLM: F = 2.36, d.f. = 2,17, P > 0.1). Next, we looked at the relative concentrations of antioxidants compared with the concentration of total carotenoids to allow comparison with a previous analysis of Parid prey (Partali, Liaaenjensen, Slagsvold & Lifjeld, 1987). Partali et al. (1987) analysed a single sample of 15 caterpillars and reported that


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SEASONAL

VARIATION IN ANTIOXIDANT

CONCENTRATIONS IN

OPEROPHTERA

CATERPILLARS

For Operophtera spp. caterpillars only, we had enough samples to assess seasonal changes in antioxidant levels. Lutein (Spearman’s rho = -0.44, N = 14, P > 0.1) (Fig. 3A) and b-carotene (Spearman’s rho = -0.20, N = 14, P > 0.4) (Fig. 3B) concentrations did not vary significantly with date. However, zeaxanthin concentrations in Operophtera spp. were found to significantly decrease (Spearman’s rho = -0.68, N = 14, P = 0.006) (Fig. 3C) and a-tocopherol concentrations increased significantly with date (Spearman’s rho = 0.76, N = 14, P = 0.001) (Fig. 3D). Lutein, zeaxanthin, and b-carotene were strongly correlated with one another (P < 0.01 in all cases). a-tocopherol concentrations were not correlated with individual carotenoids (P > 0.2 in all cases) or total carotenoids (P > 0.5).

VARIATION

IN INVERTEBRATE PROVISIONING BY PARENTAL BLUE TITS

A)

50

40

30

20

10 1

2

3

4

5

4

5

4

5

4

5

Week

Beta-carotene (microgram/g)

40

B)

30

20

10

0 1

2

3

Week

C) Zeaxanthin (microgram/g)

total carotenoids constituted 0.05mg of sample and that these were composed of 17% b-carotene, 80% lutein, and 3% zeaxanthin. Relative to total carotenoids, we found that spiders had a significantly lower proportion of lutein (9.2% ± 2.2) than Operophtera spp. (23.5% ± 2.0) and Erannis spp. (19.8% ± 0.2) caterpillars (GLM: F = 80.9, d.f. = 2,17, P < 0.0001). By contrast, Erannis spp. caterpillars had approximately double the percentage of b-carotene (33.3% ± 8.4) than either Operophtera spp. caterpillars (16.9% ± 3.4) or spiders (15.5% ± 0.9; GLM: F = 17.1, d.f. = 2,17, P < 0.0001). No statistically significant difference in proportion of zeaxanthin between invertebrate groups was found (GLM: F = 2.4, d.f. = 2,17, P > 0.05), although Erannis spp. appeared to have a somewhat higher percentage of zeaxanthin (7.2% ± 0.3) than Operophtera spp. (4.2% ± 1.8) and spiders (5.0% ± 2.0). Relative to total carotenoids, Operophtera spp. contained a higher percentage of a-tocopherol (47% ± 10.6; GLM: F = 73.9; d.f. = 1,14, P < 0.0001) than Erannis spp. (27.7% ± 6.9).

12

8

4

0 1

2

3

Week

Alpha-tocopherol (microgram/g)

Figure 3. Seasonal changes in antioxidant concentrations in Operophtera spp. caterpillars. A, lutein (not significant); B, b-carotene (not significant); C, zeaxanthin (P = 0.006); D, a-tocopherol (P = 0.001). Start of week 1 = 11 May 2005 (i.e. the date the first brood hatched). 䉴

Lutein concentration (microgram/g)

ANTIOXIDANTS IN INVERTEBRATES AND CHICKS

D)

250

200

150

100

50 1

2

3

Week

The numbers of spiders per 13-day-old chick per hour significantly increased (Spearman’s rho = 0.517, N = 20, P = 0.020) and the number of caterpillars per chick © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99, 708–717

K. E. ARNOLD ET AL.

CHANGES IN THE ANTIOXIDANT INTAKE

AND BLOOD PLASMA CONCENTRATIONS

Next, we estimated the hourly intake of antioxidants per chick in a specific brood. The genus of prey could not be identified from the video, so caterpillar antioxidant values are based on Operophtera spp. The mean hourly intake of lutein per chick from both spiders (Spearman’s rho = -0.320, N = 20, P > 0.1) and caterpillars (Spearman’s rho = 0.303, N = 20, P > 0.1) did not vary with date. The levels of zeaxanthin consumed by 13-day-old chicks via caterpillars (Spearman’s rho = -0.071, N = 20, P > 0.7) did not vary with date. Zeaxanthin intake from spiders tended to decline throughout the season (Spearman’s rho = -0.410, N = 20, P = 0.073). a-tocopherol intake from caterpillars was estimated to increase significantly throughout the season (Spearman’s rho = 0.597, N = 20, P = 0.005). Consequently, the total intake of lipophilic antioxidants measured was estimated to have increased significantly from the start of the season (Spearman’s rho = 0.544, N = 20, P = 0.013). However, it should be noted that we stopped recording videos before the latest 10% of broods had fledged, which might have been particularly poorly nourished. In 14-day-old nestlings, blood plasma concentrations of lutein did not vary with date or sex (Fig. 4). Blood plasma concentrations of zeaxanthin declined significantly over the course of the season (GLMM: F = 20.43, d.f. = 1,310, P < 0.0001) (Fig. 4A), although this did not vary between the sexes. a-tocopherol concentrations in 14-day-old chicks increased significantly over the season (GLMM: F = 24.31, d.f. = 1, 314, P < 0.0001). This rate of increase tended to differ between the sexes (GLMM: date ¥ sex, F = 3.67, d.f. = 1, 310, P = 0.056), with the seasonal rate of increase in a-tocopherol being higher in females than males. The random factor (i.e. nest identity) did not contribute significantly to the models, suggesting that environmental factors, such as the composition of available prey, rather than differences between parental abilities, affected the antioxidant status of nestlings (Fig. 4). When looking at the antioxidant intake data in relation to the invertebrate availability and also the blood plasma values, it should be noted that they are not fully coincident. The first nests to be filmed were in week 3 and the last in week 5, with the majority in week 3; thus, not only were most filmed

Plasma lutein (micrograms/ml)

SEASONAL

A)

300 250 200 150 100 50 0 16

18

20

22

24

26

28

30

Julian date

Plasma zeaxanthin (micrograms/ml)

per hour tended to decrease (Spearman’s rho = -0.442, N = 20, P = 0.051) with hatching date. The total number of prey per blue tit nestling did not vary with hatching date (Spearman’s rho = -0.347, N = 20, P = 0.13).

B)

250

200

150

100

50

0 16

18

20

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24

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Julian date

Plasma tocopherol (micrograms/ml)

714

C)

100

80

60

40

20

0 16

18

20

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30

Julian date

Figure 4. Seasonal changes in blood plasma concentrations of antioxidant in 14-day-old blue tit nestlings. A, lutein concentrations did not vary with date; B, zeaxanthin concentrations declined significantly with hatching date; C, a-tocopherol concentrations increased significantly with date. Julian day 1 = 11 May (i.e. the date of the first brood hatching). Week 3 started on Julian day 14. The appropriate statistics are detailed in the text.


ANTIOXIDANTS IN INVERTEBRATES AND CHICKS nests synchronous with each other, reflecting the pattern in the population, but also they were largely in synchrony with the peaks in invertebrate availability (Fig. 1).

DISCUSSION In the present study, we provide evidence that the timing of breeding by insectivorous birds will impact upon both the quantity and nutritional quality of food available during breeding. At our study site in Scotland, we found the predicted patterns of seasonal variation in prey abundance. Both the numbers and individual body mass of arboreal caterpillars, but not spiders, increased from tree leaf emergence, peaked, and then declined. In line with these patterns of prey abundance, blue tit nestling were more likely to be fed caterpillars at the start of the breeding season and spiders at the end. Seasonal patterns of antioxidant concentrations in caterpillars differed from prey abundance patterns: lutein and b-carotene did not vary, zeaxanthin declined, and a-tocopherol increased in concentration over the season. Interestingly, these seasonal changes in nutritional profiles of caterpillars were reflected in the blood plasma concentrations of dietary antioxidants in blue tit nestlings. Our observed patterns of antioxidant availability and acquisition are predicted to influence fitness related traits in both parents and offspring, by altering the susceptibility of individuals to oxidative damage and potentially by influencing the quality of colourful, carotenoid mediated, signals (Olson & Owens 1999; Catoni et al., 2008). Although food can be present in sufficient amounts in nature, parents may have difficulties in finding sources of limiting nutrients, particularly when provisioning demand is high. Single parents or parents feeding experimentally enlarged broods often have to feed at higher rates than under normal conditions. Accordingly, they may bring food items that are usually ignored because of poor nutritional profiles, even if they are common in the environment (Banbura et al., 1994; Sasvári et al., 2000). In the present study, caterpillars were fed to nestlings at a level that approximately reflected their overall abundance, with fewer caterpillars and more spiders entering the diets of late broods compared to earlier broods. Because spiders were found to contain no a-tocopherol as well as significantly lower levels of carotenoids than caterpillars, individuals hatched later in the breeding season are predicted to have poorer antioxidant defences and higher levels of oxidative damage than early laid birds. However, this relationship may be further complicated because, although Operophtera spp. caterpillars are less abundant later in the season, they contain higher concentrations of a-tocopherol

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than those available to earlier nesting birds. a-tocopherol is an important nutrient in birds and is a powerful antioxidant (i.e. more efficacious than carotenoids), protecting cell membranes against ROS damage (Surai, 2002). Because growth is a period of high ROS production in tissues, nestlings can be exposed to particularly high levels of ROS and therefore oxidative damage (Larcombe et al., 2006). The absolute and relative supply of different dietary antioxidants to developing chicks is therefore predicted to influence fitness. However, to date, we know little about the impacts of individual antioxidants for overall oxidative stress. For example, although the plasma carotenoid levels that we recorded in blue tit nestlings are up to three-fold higher than those of breeding adult great tits (Isaksson, Von Post & Andersson, 2007), we do not know what the requirements of nestling Parids are compared to adults. In addition, to identify biochemical mechanisms, studies of oxidative damage in relation to antioxidant intake are needed to establish whether antioxidants provide a currency by which evolutionary and developmental trade-offs can operate. The seasonal changes in the concentrations of nutrients in caterpillars are driven by changes in the biochemical composition of the leaves upon which they feed. The very low concentrations or absence of retinol in invertebrates, particularly larvae and earthworms (Barker, Fitzpatrick & Dierenfeld, 1998), are probably a result of the absence of retinol in plants. The leaves of oaks and other tree species increase in a-tocopherol concentrations from spring through to late-summer (Hansen et al., 2003), which is reflected by a corresponding change in the a-tocopherol levels in the midguts of folivorous lepidopteran larvae (Barbehenn, Walker & Uddin, 2003). Total carotenoid levels in leaves are known to decline at the end of summer but more fine scaled patterns of seasonal changes in individual carotenoids of Quercus are little known. b-carotene concentrations in Quercus spp. leaves are known to vary in relation to soil quality and also light levels (Corcuera et al., 2005), which might explain why we found different levels compared with other studies (Partali et al., 1987; Isaksson et al., 2008). Environmental factors, particularly those that are changing rapidly as a result of human activities, such as temperature, light levels reaching leaves covered in particulate pollution, soil quality, and moisture levels in disturbed forests, will determine the levels of secondary metabolites in primary producers. Thus, environmental change will alter the concentrations of antioxidants in the invertebrates consumed by many animals. This provides a mechanistic link by which anthropogenic environmental changes can alter the fitness of individuals and thus dynamics within natural populations.


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In summary, we have shown that key prey species show differing patterns of antioxidant availability. Furthermore, different nutritional components can vary independently of both each other and the overall abundance of the prey over the avian breeding season. Thus, it is possible that certain antioxidants, particularly a-tocopherol, which is known to have profound effects on embryo development (at least in commercial bird species) and chick development (Surai, 2002; De Ayala et al., 2006), might be in short supply during key stages of breeding. Thus, the quality, independent of the quantity, of prey can impact upon fitness-related traits of insectivorous birds (Arnold et al., 2007; Isaksson & Andersson, 2007; Catoni et al., 2008). Manipulative experiments are required to investigate this further. In addition, more studies are needed to identify the natural patterns of dietary antioxidant availability and oxidative stress in wild systems, as well as their associated food webs.

ACKNOWLEDGEMENTS We thank Gabrielle Roy, Patrick White, Clare Toner, Chris Donaldson, and many others for their assistance in the field. This work was supported by the Leverhulme Trust, Natural Environment Research Council, Glasgow Natural History Society and University of Glasgow. K.A. was funded by a Royal Society University Research Fellowship and S.L. by a BBSRC industrial CASE studentship.

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