Effect of human presence and handling on circulating corticosterone levels in breeding blue tits (Parus caeruleus)

General and Comparative Endocrinology 148 (2006) 163–171 www.elsevier.com/locate/ygcen

EVect of human presence and handling on circulating corticosterone levels in breeding blue tits (Parus caeruleus) Claudia Müller a, Susanne Jenni-Eiermann a, Jacques Blondel b, Philippe Perret b, Samuel P. Caro b, Marcel Lambrechts b, Lukas Jenni a,¤ b

a Swiss Ornithological Institute, CH-6204 Sempach, Switzerland CEFE (UMR 5175 du CNRS), 1919 route de Mende, F-34293 Montpellier Cedex 5, France

Received 27 July 2005; revised 10 February 2006; accepted 24 February 2006 Available online 11 April 2006

Abstract Birds may react to the presence of humans with an immediate primary behavioural reaction and with physiological responses, such as the activation of the hypothalamo–pituitary–adrenal axis. This study investigates the suite of behavioural and adrenocortical responses to the presence of humans and to handling in two subspecies of blue tits Parus caeruleus, a small hole-nesting passerine, during the period of feeding their nestlings. The Wrst aim was to investigate whether the presence of humans near their nests elicits an adrenocortical response and whether the increase in circulating corticosterone is correlated with the behavioural reaction of the birds. The second aim was to determine the time-lag between the onset of capture and handling stress and the increase in circulating corticosterone levels. The third aim was to try to explain individual variation in the adrenocortical response to handling with 9 intrinsic and extrinsic factors (sex, age, body size, measures of body condition, time of day, and date). One half of the parents showed a behavioural reaction to our presence near the nest, such as alarming, and hesitating to enter the nestbox. However, the degree of behavioural reaction before handling was not related to circulating corticosterone levels which remained low. The results conWrm that primary behavioural and adrenocortical reactions to the presence of predators are independent of each other. A comparison with published Wndings supports the hypothesis that birds react to predators with an adrenocortical response only in a situation that is imminently life-threatening. Hence, the primary behavioural response of the bird to a predator may determine whether or not an adrenocortical response is elicited. An adrenocortical response to handling started 3 min after capture in the nest box. Individual variation in baseline corticosterone levels could be explained by subspecies and body condition (fat stores), variation in handling-induced corticosterone levels by subspecies, body condition, body size, and time of day. © 2006 Elsevier Inc. All rights reserved. Keywords: Human disturbance; Glucocorticoid; Aves; Stress; Handling

1. Introduction Birds, like other vertebrates, react to the presence of predators with behavioural and physiological responses. Behavioural reactions are e.g., vigilance, alarming, Xeeing, and changes in habitat use (Frid and Dill, 2002). Physiological reactions include cardiovascular changes (e.g., increase

*

Corresponding author. Fax: +41 41 462 97 10. E-mail address: [email protected] (L. Jenni).

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in heart rate; Nephew et al., 2003) and endocrine responses. Among the latter, the activation of the hypothalamo–pituitary–adrenal (HPA) axis, a universal reaction of vertebrates to a wide variety of unpredictable environmental events (Vellucci, 1997; WingWeld and Romero, 1999), results in the release of glucocorticoids (corticosterone in birds), putting the animal into an emergency life-history stage (sensu WingWeld et al., 1998). The increase in corticosterone may inXuence many physiological functions and elicit a change in behaviour (e.g. Buckingham et al., 1997; WingWeld and Romero, 1999).

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In many respects, human-caused disturbance is thought to be analogous to mammal predation risk and evokes very similar behavioural and physiological reactions (risk-disturbance hypothesis; Frid and Dill, 2002). Thus, the reaction of birds to the presence of mammalian predators or humans may comprise a variety of behavioural and physiological responses. Among them a primary behavioural reaction is elicited by the central nervous system upon detecting a predator. An adrenocortical response may ensue that may, by the release of glucocorticoids, trigger a secondary behavioural response. Indeed, birds have been found to react to the presence of a predator or human either with only a behavioural response, or only with an adrenocortical response or with both (e.g., Cockrem and Silverin, 2002b; Fowler, 1999; Nephew et al., 2003; Silverin, 1998). In small birds feeding their nestlings, the presence of humans near the nest normally provokes strong behavioural responses. The parents emit alarm calls, hesitate to, or do not, approach the nest, presumably to not divulge its position or not to be surprised while feeding the nestlings inside a cavity. If the disturbance persists for a long time, nestlings may die from starvation or hypothermia. To our knowledge, no study investigated so far whether the presence of humans near nests elicits an adrenocortical response in small birds, i.e., whether the temporary reduction in feeding or the abandonment of the brood is governed by an increased secretion of glucocorticoids. The Wrst aim of this study was to investigate whether the presence of humans near the nest of blue tits Parus caeruleus elicits an adrenocortical response and whether this response is correlated with some behavioural reaction of the birds. The blue tit is a small passerine bird, widely distributed and abundant in broad-leaved forests, woods, parks, and gardens. It frequently breeds in nestboxes in the vicinity of humans. In birds, baseline corticosterone levels may depend on a variety of factors, such as sex, time of day, size (related to social dominance; Braillet et al., 2002), amount of body energy stores (e.g. Jenni et al., 2000; Smith et al., 1994; WingWeld et al., 1994a). These factors, if correlated with the behaviour of the birds against human presence, may confound a possible relationship between corticosterone levels and behaviour. Therefore, we tested for relationships with these factors in a multivariate analysis. When birds are physically threatened (handled by humans), there is usually a clear behavioural (struggling, alarming, pecking, head feathers raised, or Xattened) and adrenocortical reaction. Because the activation of the HPA axis involves several steps, the increase of circulating corticosterone in birds occurs only some minutes after the onset of the stressor (e.g., Sapolsky et al., 2000). When the aim is to measure baseline corticosterone levels (i.e., not inXuenced by capture and handling) in birds caught, the exact onset of the rise in circulating corticosterone after capture needs to be known (Romero and Romero, 2002). Only a few studies investigated the initial increase in corticosterone levels, and indications about the magnitude of the time lag

between capture and a measurable increase in circulating corticosterone vary as much as between 1 and 5 min (Dawson and Howe, 1983; Hood et al., 1998; Le Maho et al., 1992; Romero and Reed, 2005; Schwabl et al., 1991; WingWeld et al., 1982). Therefore, the second aim of this study was to determine the time-lag between the onset of capture and handling and the increase in plasma corticosterone levels in blue tits. Because such small birds can hardly be blood-sampled several times at very short time intervals without causing harm, we used a cross-sectional design and sampled blood only once or twice per individual at intervals between 1.5 and 8 min. The so-called capture-stress protocol in which repeated blood samples of individual birds are taken makes use of the usual increase in corticosterone to measure a bird’s ability to physiologically react to threats (WingWeld et al., 1997; Silverin, 1998). The increase in circulating corticosterone during handling and its time course varies between individuals (Cockrem and Silverin, 2002a; Schwabl, 1995), and birds with high baseline corticosterone levels cannot mount an adrenocortical response to handling (e.g., Jenni et al., 2000; Romero and Romero, 2002; Smith et al., 1994). The variation in handling-induced stress responses between individuals could be explained by a series of factors including body condition (Hood et al., 1998; Smith et al., 1994; WingWeld et al., 1994a; WingWeld et al., 1994b), sex, age, and morph (Schwabl, 1995), time of day (Breuner et al., 1999), or genetics (e.g., Edens and Siegel, 1975). Therefore, the third aim of this study was to examine various factors simultaneously in a multivariate analysis for correlations with the steepness of the individual increase in corticosterone as a response to handling. 2. Materials and methods 2.1. Study site and study species Blue tits were investigated in the scope of a long-term study on population biology in two Mediterranean areas of France: on mainland Southern France near Montpellier and on the island of Corsica near Calvi (Fig. 1). The sedentary breeding birds of mainland Southern France belong to the nominate subspecies P.c. caeruleus, while the birds from Corsica are assigned to the subspecies P.c. ogliastrae which is about 15% smaller and of darker plumage. Only the female incubates the eggs and broods the nestlings. Both parents feed the nestlings. In both areas, populations nesting in boxes were studied in woods dominated by the deciduous downy oak Quercus humilis (Rouvière 43°40⬘N, 3°40⬘E and Muro 42°36⬘N, 8°58⬘E), or by the evergreen holm oak Quercus ilex (Vic-le-Fesq 43°52N, 4°14E and Pirio 42°24⬘N, 8°44⬘E; see Blondel, 1985; Blondel et al., 2001; Dias and Blondel, 1996; Lambrechts et al., 2004, for further information). On Corsica, the two study areas were subdivided in several plots. Blue tits in all these woods are regularly experiencing the presence of humans (recreational activities, farmers, hunters, mushroom collectors, ornithologists, etc.). Their nests were routinely checked at least once a week by researchers and most adults were captured each year.

2.2. Capture and blood sampling During the entire breeding season, from the beginning of May to the end of June in 2001 and 2003, breeding adults tending 9–15-day-old nestlings

C. Müller et al. / General and Comparative Endocrinology 148 (2006) 163–171

165

0.5 mm and body mass to the nearest 0.1 g. Because body mass and wing length diVer between the two subspecies, sexes, and age classes, we used the diVerence from the means of each subspecies–sex–age class as a measure of relative wing length and relative body mass. Fat stores were estimated by assigning the visible amount of subcutaneous fat between the furcula and on the abdomen to one of 31 fat scores, ranging from 0 to 8 (Kaiser, 1993). These scores correlate well with the amount of fat extracted from whole birds (Kaiser, 1993). As is usual in brood-rearing small passerines, birds were rather lean and only fat scores 0–3 were found. An estimate of breast muscle mass was obtained by assigning the shape of the breast muscle to four muscle score classes (Bairlein, 1995) ranging from class 0 (muscle emaciated, sternum keel prominent, and sharp) to 3 (muscle bulging and shaped convexly). Blue tits in this study had muscle scores 1–3.

2.4. Hormone assay

Fig. 1. The four study sites on mainland Southern France and on the Mediterranean island Corsica. were caught in their nestboxes with one of three diVerent methods: (a) A plug (pine cone, small apple, or ball) was installed hanging about 1 m below the nestbox from a nylon thread, that was led through the entrance hole and to the observer 20–30 m away. By pulling the thread, the entrance hole could be closed with the plug. Plugs were installed some hours before capture or the day before. (b) A wire trap was installed inside the nestbox and barricaded the entrance hole when released by the bird at entering. (c) A wooden stick was used to close the entrance hole when running to the nestbox after the bird had entered it. 145 blue tits were caught using a plug, 58 using a trap and 41 using a stick. The three methods needed roughly the same amount of time to catch a bird. We tried to catch both parents within about 1 h. With all capture methods, we were 20–30 m from the nestbox behind some vegetation. We thus were visible to the birds approaching the nestbox, but screened oV from birds at the nestbox itself. We observed the box continuously until the bird was trapped. We recorded the time diVerence between our arrival in the nestbox area and capture, time of day of capture and the order of capture when both parents of a brood were caught during the same capture session. The behaviour of the blue tits before capture was assigned to four behaviour classes. (1) The bird entered the nestbox immediately without calling. (2) The bird emitted scolding or churring calls (Cramp, 1993), but entered the nestbox within a few minutes. (3) The bird emitted churring calls and hesitated several times before entering the nestbox, but for less than 20 min. (4) The bird produced churring calls and hesitated for more than 20 min before entering the box. As soon as possible after capture, a blood sample was taken by puncturing the brachial vein and collecting the blood with heparinised capillaries or (more rarely) by collecting the blood with a heparinised syringe from the jugular vein. The time diVerence between capture (closing the entrance of the nestbox) and blood sampling was measured with a stopwatch. In 31 birds, a second blood sample was taken within a few min. Within 2 h at most, the blood was centrifuged and the plasma stored in liquid nitrogen or on dry ice. After the transfer to the laboratory, the samples were stored at ¡20 °C until analysis. In total, blood samples from 244 breeding blue tits (54% females) were obtained, 127 from the mainland and 117 from Corsica. From 60 birds (24 from the mainland, 36 from Corsica), blood samples were obtained within 3 min after capture, the remaining between 3 and 11 min after capture.

Plasma corticosterone concentration was determined using an enzyme immuno assay (Munro and Stabenfeldt, 1984; Munro and Lasley, 1988). Corticosterone in 5 l of plasma and 195 l water was extracted with 4 ml dichloromethane, re-dissolved in phosphate buVer and given in triplicates in the enzyme immuno assay. The dilution of the corticosterone antibody (Chemicon; cross-reactivity: 11-dehydrocorticosterone 0.35%, progesterone 0.004%, 18-OH-DOC 0.01%, cortisol 0.12%, 18-OH-B 0.02%, and aldosterone 0.06%) was 1:8⬘000. HRP (1:400⬘000) linked to corticosterone served as enzyme label and ABTS as substrate. The concentration of corticosterone in plasma samples was calculated by using the standard curve run in duplicate on each plate. Plasma pools from chickens with two diVerent corticosterone concentrations were used as internal controls on each plate. Whenever the amount of plasma allowed, all samples were determined twice, and the mean used for data analysis. If the concentration was below the detection threshold, the value of the lowest detectable concentration (2.15 ng ml¡1) was assigned. Intra-assay variation ranged from 5.2 to 12.5%, inter-assay variation from 7.7 to 19.2%, depending on the concentration of the internal control and the year of determination.

2.5. Statistical analysis The eVects of various factors (see Tables 1 and 2 and above) were tested on baseline corticosterone levels (within 3 min after capture) and for Table 1 Dependence of basal corticosterone levels (ln-transformed, n D 60) on various factors analysed in a multivariate linear REML-model (see Methods, deviance 24.13, df D 38) Independent variables

EVect § SE

Wald statistic

df

P

— — — +0.031 § 0.05 ¡0.095 § 0.34 +0.003 § 0.01 ¡0.186 § 0.11 — ¡0.050 § 0.16 ¡0.004 § 0.01 +0.119 § 0.31

23.09 0.41 0.23 0.44 2.14 0.46 5.06 4.37 0.40 3.38 0.08

1 1 1 1 1 1 1 2 1 1 1

<0.001 0.523 0.632 0.507 0.143 0.499 0.025 0.113 0.527 0.066 0.778

2.3. Body size and fat stores

Area (mainland, Corsica) Sex Age Relative wing length Time of day Time of day (squared) Fat score Muscle score Relative body mass Date Time between arrival and capture Capture order within pair Behaviour before capture Capture method

0.96 0.80 3.50

1 3 2

0.326 0.849 0.174

Sex was determined from the presence of the brood patch in females. Second calendar year birds (yearlings) were distinguished from older birds after Jenni and Winkler (1994). Wing length was measured to the nearest

Interaction terms were not signiWcant and, therefore, omitted from this model. The signiWcant terms (in bold) remained signiWcant when eliminating backwards non-signiWcant variables from the model. The eVect sizes of only the continuous variables are shown.

— — —

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Table 2 Dependence of corticosterone levels (ln-transformed, n D 244) taken within 8 min after capture on various factors analysed in a multivariate linear REML-model (see Methods, deviance ¡76.07, df D 221) EVect § SE

Wald statistic

df

P

+0.236 § 0.02 — — — +0.053 § 0.02 ¡0.394 § 0.11 +0.016 § 0.00 ¡0.183 § 0.05 — ¡0.040 § 0.06 ¡0.005 § 0.00 ¡0.040 § 0.11

174.86 47.33 0.45 0.01 6.13 7.00 13.39 13.62 3.97 0.63 7.31 0.13

1 1 1 1 1 1 1 1 2 1 1 1

<0.001 <0.001 0.50 0.91 0.013 0.008 <0.001 <0.001 0.14 0.43 0.007 0.71

0.05 0.92 3.47

1 3 2

0.83 0.82 0.18

— — —

No interaction term was signiWcant and, therefore, omitted from this model. The signiWcant terms (in bold) remained signiWcant when eliminating backwards non-signiWcant variables from the model. The eVect sizes of only the continuous variables are shown. corticosterone levels over the Wrst 8 min after capture (including the Wrst 3 min). If two blood samples were taken from the same bird, the Wrst sample was used for the analysis of baseline levels and the second for the analysis of levels until 8 min after capture. Years were pooled. Mixed models were Wtted with the method of Residual Maximum Likelihood Analysis REML (Patterson and Thompson, 1971). REML allows the analysis of unbalanced data sets, but otherwise, is analogous to General Linear Models. Study plot and nestbox were introduced as random variables, thus accounting for any possible common eVects of study plot and pair on corticosterone levels. To obtain normally distributed residuals, corticosterone concentrations were ln-transformed. The time diVerence between capture and blood sample was introduced as the Wrst variable into the analysis of corticosterone levels up to 8 min after capture (Table 2). To test for eVects of various factors on the adrenocortical response to handling, interactions between time after capture and region, sex, age, fat score, muscle score, body mass, wing length, time of day, and the four variables characterising capture were also introduced into the model, but none proved to be signiWcant.

3. Results 3.1. Behaviour of blue tits in the presence of humans The behaviour of blue tits before capture varied between individuals. Despite our presence near the nestbox, 52% of the birds (of a total of n D 244) entered the nestbox quickly and without churring calls to feed their nestlings (Fig. 2). The others showed behaviours which we interpreted as elicited by our presence near the nestbox. 31% emitted churring calls but did not hesitate noticeably before entering the nestbox, 14% churred and hesitated for less than 20 min before entering the nestbox, and 3% churred and hesitated for more than 20 min. The frequencies of the behaviours did not diVer signiWcantly between the mainland and Corsica (2 D 7.0, df D 3, P D 0.07), between the sexes (2 D 0.1, df D 3, P D 0.99), between Wrst- and second-caught individuals (2 D 2.0, df D 3, P D 0.57), or between the capture methods used (2 D 5.6,

Southern France 50 Corsica Frequency [%]

Independent variables Time after capture Area (mainland, Corsica) Sex Age Relative wing length Time of day Time of day (squared) Fat score Muscle score Relative body mass Date Time between arrival and capture Capture order within pair Behaviour before capture Capture method

60

40 30 20 10 0 1

2 3 Behaviour before capture

4

Fig. 2. Behaviour of blue tits before being caught in their nestboxes on the mainland (n D 127) and on Corsica (n D 117). Behaviour class: 1, bird enters the nestbox immediately without warning; 2, bird emits alarm calls, but enters the nestbox within a few minutes; 3, bird emits alarm calls and hesitates several times before entering the nestbox within less than 20 min; 4, bird alarms and hesitates for more than 20 min before entering the nestbox.

df D 6, P D 0.53). We also did not Wnd any signiWcant diVerences in behaviour of the parents with the age of nestlings (up to 11-day-old versus from 12-day-old onwards, 2 D 4.0, df D 3, P D 0.27) or the fat score of the parents (fat score 0–1 versus more than 1, 2 D 6.0, df D 3, P D 0.11). 3.2. Baseline corticosterone levels We regarded plasma corticosterone levels of birds bled within 3 min after capture as representing baseline levels (see below). From the 13 variables examined in a multivariate analysis, only the area (mainland versus Corsica) and fat score were signiWcantly related to baseline corticosterone levels (Table 1). Introducing interaction terms between the main variables of interest (region, sex, relative wing length, fat score, muscle score, and the 4 parameters characterising capture) did not reveal any further signiWcant relationships. Blue tits from the mainland had higher baseline corticosterone levels (means § SE: 8.93 § 0.69 ng ml¡1, n D 25) than Corsican blue tits (5.23 § 0.36 ng ml¡1, n D 35). Baseline corticosterone levels decreased with increasing fat score (Fig. 3A; the signiWcance of this relationship relied on the two fattest individuals). All parameters characterising capture were not signiWcantly related to baseline corticosterone levels (Table 1). Baseline corticosterone levels did not show any signs to increase with time between our arrival near the nestbox and capture (Fig. 3B). Blue tits caught second among a pair did not have higher corticosterone levels than their partners caught Wrst (Fig. 4A). Birds churring and hesitating before entering the nestbox did not have signiWcantly higher baseline corticosterone levels than birds not showing signs of a behavioural reaction to our presence (Fig. 4B). Capture method used also was unrelated to corticosterone levels (Table 1). Parameters characterising the individual bird, such as sex, age, size (relative wing length), body mass, and size of

C. Müller et al. / General and Comparative Endocrinology 148 (2006) 163–171

A 20

167

Southern France

Southern France

A 12

B 12

10

10

10

5

0 0

0.5

1

1.5 2 Fat score

2.5

3

3.5

Corticosterone [ng/ml]

15

Corticosterone [ng/ml]

Corticosterone [ng/ml]

Corsica

Corsica

8 6 4

Corticosterone [ng/ml]

Corsica

15

1 2 Capture order within pair

5

0

70.00

1.2

Fig. 3. Baseline corticosterone levels of blue tits from the mainland (n D 25) and Corsica (n D 35) (A) against fat score and (B) against the time diVerence between our arrival near the nestbox and capture. Corticosterone levels decreased signiWcantly with fat score, but were unrelated to time to capture (see Table 1 for statistics).

the breast muscle were not signiWcantly correlated with baseline corticosterone levels (Table 1). Similarly, baseline corticosterone levels did not vary signiWcantly with time of day, but there were only two samples taken before 8:00 h, when corticosterone levels were expected to be high (e.g., Romero and Remage-Healey, 2000). 3.3. Adrenocortical response to capture and handling In individuals sampled twice within a few min after capture, plasma corticosterone levels remained stable during the Wrst 3 min and started to rise only about 3 min after capture (Fig. 5). The Wrst substantial increase in corticosterone (from 4.22 to 13.63 ng ml¡1) occurred in a bird sampled 2.75 and 4 min after capture. The mean increase in birds sampled twice within 3 min after capture was 0.18 § 0.69 ng ml¡1 min¡1 (SE, n D 5, range ¡1.45–+2.14) which was not signiWcantly diVerent from zero (t D 0.27, P D 0.80), while birds sampled twice between 3 and 5 min after capture signiWcantly increased their corticosterone levels by 2.14 § 0.91 ng ml¡1 min¡1(SE, n D 14, range ¡6.05–+7.53, 2 negative values, signiWcantly diVerent from 0: t D 2.35, P D 0.035).

Corticosterone [ng/ml]

0.2 0.4 0.6 0.8 1 Time between arrival and capture [h]

1 2 3 4 Behaviour before capture

Fig. 4. (A) Mean baseline corticosterone levels (§SE) of blue tits captured Wrst and second among a pair within the same capture session. There was no signiWcant diVerence between the two groups within the mainland (n D 14 and 11, respectively) and Corsica (n D 22 and 13). (B) Mean baseline corticosterone concentration (§SE) for the four classes of behaviour before capture (see Fig. 2) on the mainland and on Corsica. There was no signiWcant variation between behaviour classes (Table 1) and the interaction term between area and behaviour was not signiWcant (P D 0.20) when introduced into the model.

10

0

4

0

0

Southern France

6

2

2

B 20

8

60.00 50.00 40.00 30.00 20.00 10.00 0.00 0.00

2.00

4.00 6.00 8.00 10.00 Time after capture [min]

12.00

Fig. 5. Plasma corticosterone concentrations of blue tits sampled twice within a few minutes after capture showing the onset of the adrenocortical stress response to capture and handling. Lines connect the two values of 31 individuals.

In birds sampled once, the Wrst high plasma corticosterone concentrations (>20 ng ml¡1) occurred shortly after 3 min after capture. Within 3 min after capture, plasma corticosterone concentrations increased only slightly and nonsigniWcantly (Fig. 6; slope 1.45, P D 0.10, eVect of area P < 0.001, n D 60, interaction area £ time after capture not signiWcant P D 0.14). From 3 to 7 min after capture, circulating corticosterone increased from 10.19 to 22.84 ng ml¡1 (by 124 %) on the mainland and from 5.65 to 19.47 ng ml¡1 (by 245 %) in Corsica (Fig. 7). The eVect of various factors to explain individual variation in the rate of increase in corticosterone was explored in a mixed model analysis with the data up to 8 min after

168

C. Müller et al. / General and Comparative Endocrinology 148 (2006) 163–171

Residuals corticosterone [ng/ml]

20

Corticosterone [ng/ml]

Southern France Corsica 15

10

5

6

Mainland

4

Corsica

2 0 -2 -4 -6

7

0 0

1

2 3 Time after capture [min]

40 Corticosterone [ng/ml]

Mainland Corsica 30

20

10

0 1

2

3 4 5 6 Time after capture [min]

11 13 15 Time of day [h]

17

4

Fig. 6. Plasma corticosterone concentrations of blue tits sampled once within 3 min after capture (n D 60). Within this time period, corticosterone levels did not show a signiWcant increase.

0

9

7

8

Fig. 7. Mean corticosterone levels § SE up to 8 min after capture, grouped by min (mainland n D 127, Corsica n D 117).

capture (Table 2). Not surprisingly, time after capture aVected corticosterone levels strongly. However, there was no signiWcant diVerence in the rate of corticosterone increase with handling time between areas, sexes, and behaviour classes before capture or with any other variable examined (interactions between time after capture and any other variable not signiWcant and thus omitted from Table 2). This indicates that the rate of increase did not diVer signiWcantly between groups of birds. Consequently, residuals from the relationship between corticosterone and time after capture can be analysed for individual variation, reXecting whether a bird has a higher or lower plasma corticosterone concentration than expected from its handling time. As with baseline levels, residual corticosterone concentrations diVered between the mainland and Corsica (Table 2) with birds from the mainland having consistently higher values over the Wrst 8 min after capture. Relative wing length was positively associated with residual corticosterone levels. Large blue tits had higher corticosterone levels than small individuals. Residual corticosterone levels decreased signiWcantly in the early

Fig. 8. Corticosterone levels during the course of the day on the mainland (n D 127) and Corsica (n D 117). Presented are mean residuals (corrected for time after capture) of corticosterone levels § SE for 2 h-intervals.

morning and remained stable over the rest of the day or increased slightly in the afternoon (Fig. 8), so that a quadratic relationship described this relationship better than a linear one (Table 2). After accounting for this diurnal eVect, fat score was still signiWcantly related to corticosterone levels. Blue tits with larger fat stores had lower residual corticosterone levels than lean ones. Fat stores increased during the day (by 0.078 fat scores per hour, linear least-squares regression, F D 23.8, P < 0.001, n D 244), but did not follow a curved relationship (time of day squared not signiWcant, P D 0.44). If fat score was introduced into the model before time of day, time of day (squared) remained signiWcant (Wald statistic 9.31, P D 0.002). This indicates that eVects of fat stores and time of day on corticosterone levels were additional. All other variables introduced into the model had no signiWcant relationship with residual corticosterone levels, except date. This eVect can be explained Wrstly by diVerences in the timing of breeding between the two regions (on average, birds on Corsica with their lower corticosterone levels breed later in the year than on the mainland), and secondly by a slight trend to decreasing corticosterone levels with ongoing breeding season. 4. Discussion 4.1. Behavioural and adrenocortical response to humans and predators Blue tits did not show any noticeable adrenocortical response to our presence near the nest for up to 1 h, but half of the individuals showed a behavioural reaction. Thus, blue tits did not enter an emergency life-history stage (sensu WingWeld et al., 1998) as a response to our presence near the nest, although they were capable of mounting an adrenocortical stress response, as shown by the increasing corticosterone levels after capture. Our study demonstrates that the temporary reduction in feeding, caused by our presence near the nestbox, was not governed by an adrenocortical stress response. Our study is another example showing that a behavioural reaction to a potential stressor is not

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necessarily an indicator of an adrenocortical stress response, and vice versa (Eilam et al., 1999; Nephew et al., 2003; Silverin, 1998). Obviously, the behavioural reaction to our presence near the nest was induced independently from an adrenocortical response, thus it was a primary behavioural response probably elicited by the central nervous system upon detecting the human. Normally, churring calls are emitted immediately after discovering a potential predator and, thus, cannot be triggered by an adrenocortical stress response that is developing only some min after the onset of stress. Why did the blue tits not show an adrenocortical response to our presence near the nestbox? A comparison of Wndings from the literature supports the hypothesis that birds react to predators with an adrenocortical response only in a situation that is imminently life-threatening to themselves, as suggested by Cockrem and Silverin (2002b). Free-living great tits Parus major exposed to an artiWcial predator at a feeder showed only a behavioural reaction, while captive individuals, unable to escape, showed behavioural and adrenocortical reactions (Cockrem and Silverin, 2002b). Breeding pied Xycatchers Ficedula hypoleuca feeding nestlings showed an adrenocortical response to a weasel model near the nestbox, presumably because the weasel predates on adults and nestlings, but these birds did not elevate corticosterone levels when confronted with a moving woodpecker model, presumably because woodpeckers predate only on nestlings (Silverin, 1998). In caged European starlings, unable to escape, visual or auditory stressors or the presence of humans provoke both an adrenocortical and behavioural reaction (Nephew et al., 2003). At least some bird species are known to show a diVerent behavioural reaction to a mammalian (usually less threatening) than to an avian predator (usually more threatening) and, hence, are clearly able to distinguish between diVerent predators (e.g., Clemmons and Lambrechts, 1992; Templeton et al., 2005). Following this hypothesis, it appears that birds are able to distinguish between situations in which predators are imminently life-threatening and not life-threatening. A human being at a distance from the nest box is not imminently life-threatening to a parent blue tit. Blue tits, including those of our study populations, regularly encounter humans. At least one study shows that birds may adapt to the presence of humans. Free-living breeding Magellanic penguins Spheniscus magellanicus not habituated to the presence of humans near their nests showed elevated corticosterone levels, while frequently visited ones did not (Fowler, 1999). For a Xightless penguin, a human is obviously a potentially life-threatening predator. Only with experience, humans may be recognized as not lifethreatening. It is advantageous for birds not to mount an adrenocortical response to any potential predator, particularly when feeding nestlings. An acute adrenocortical stress response is costly and triggers behaviours of self-maintenance and survival. Birds given corticosterone implants reduce parental

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care or abandon their brood (Silverin, 1986; Silverin, 1998; WingWeld and Silverin, 1986). Therefore, in a situation of parents feeding nestlings the trade-oV between avoiding predators and foraging seems not to be mediated by glucocorticoids, as would be proposed by the chronic stress hypothesis (Clinchy et al., 2004), or only in the case of immediately life-threatening predators. An open question is how abandonment of a brood is triggered when disturbance persists. Is it the lack of signals from weak or dead nestlings or is it a secondary behavioural response triggered by glucocorticoids which eventually increase when disturbance persists over longer time periods than investigated in this study? Following this hypothesis, birds may escape an adrenocortical response by keeping a safe distance from a predator. Hence, the primary behavioural response of the bird to a predator may determine whether or not an adrenocortical response is elicited. 4.2. Onset of the adrenocortical response to capture and handling As observed in many other bird species, blue tits reacted to capture and handling with a clear increase in circulating corticosterone. Birds sampled twice within short intervals and birds sampled once at various intervals after capture indicated that corticosterone levels started to increase 3 min after capture. This is in agreement with the general Wnding that corticosterone and other steroids are released only within several min (e.g., Sapolsky et al., 2000). It also agrees in general with Wndings in Wve bird species sampled at diVerent times of year that showed an increase only after 2 or 3 min (Romero and Reed, 2005; and literature cited therein). Also captive garden warblers Sylvia borin (Schwabl et al., 1991) and free-living Magellanic penguins (Hood et al., 1998) showed increasing levels of corticosterone between 2 and 3 min after capture, respectively. Slight diVerences in the time until corticosterone release (e.g., 2 or 3 min) may also be due to a varying deWnition of handling time. We deWned handling time to start when the nestbox entry was closed. This was 0.5–1 min before we grabbed the bird. It is possible, that locking up the bird in its nestbox did not trigger an increase in corticosterone, similar to birds caught in potter traps (Romero and Romero, 2002). In wild European starlings (Dawson and Howe, 1983) and in captive greylag geese Anser anser (Le Maho et al., 1992), corticosterone levels were observed to increase already 1 min after the onset of the stressor. Whether this is due to diVering blood sampling methods remains an open question. 4.3. Individual variation of plasma corticosterone levels As found in other studies (e.g., Cockrem and Silverin, 2002a), levels of circulating corticosterone varied considerably between individuals. This was true for baseline levels (Table 1) and for capture-induced corticosterone (residuals,

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accounting for the eVect of handling time during the Wrst 8 min; Table 2). Part of this variation could be explained by three factors: size, time of day, and body fat stores, while sex, age and date were not signiWcantly related with corticosterone levels during handling. In addition, the two subspecies diVered in corticosterone levels, a Wnding that will be discussed elsewhere. Male and female blue tits did not diVer in baseline or capture-induced corticosterone plasma levels. This result agrees with Wndings in other bird species sharing parental care for feeding nestlings equally. This contrasts with species with unequal parental investment in which a reduced acute stress response is found in the sex that cares for the young (Meddle et al., 2003; O’Reilly and WingWeld, 2001; Silverin, 1990). Large blue tits (for their sex and age) had higher capture-induced residual corticosterone levels than small individuals. It may be that large individuals are dominant and that dominants are able to mount a stronger stress response, as indicated in other studies (e.g., Poisbleau et al., 2005; Pravosudov et al., 2003). Plasma corticosterone levels of blue tits changed with time of day in a similar manner as described previously in captive white-crowned sparrows Zonotrichia leucophrys (Breuner et al., 1999), European starlings (Romero and Remage-Healey, 2000) and House sparrows Passer domesticus (Rich and Romero, 2001). In these studies, corticosterone levels peaked during the dark phase and remained relatively constant during the day-light period. Levels measured in the early morning in blue tits were highest, indicating a possible maximum before the onset of the day. Towards the evening, corticosterone levels seemed to rise again, similarly as shown in white-crowned sparrows (Breuner et al., 1999). Baseline and capture-induced residual corticosterone levels were negatively correlated with body fat stores. Our multivariate analysis showed that this was not an eVect of time of day being correlated with fat stores, but a genuine eVect of body fat stores. Negative relationships between body mass or fat stores and baseline or stress-induced corticosterone levels have been found in several other studies (e.g., Jenni et al., 2000; WingWeld et al., 1994a), but not in others (e.g., Romero et al., 2000). Acknowledgments We thank Paula Dias, Jonas Örnborg, Valérie Roy, and others for their help in the Weld and Michael Schaub for his statistical assistance. We are grateful to Bill Lasley, Davies USA, who kindly introduced us into the determination of steroids with enzyme immuno assay to adopt in our lab and provided us with the HRP-linked corticosterone. References Bairlein, F., 1995. Manual of Field Methods. European-African Songbird Migration Network. Institut für Vogelkunde, Wilhemshaven.

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