General and Comparative Endocrinology xxx (2015) xxx–xxx
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General and Comparative Endocrinology journal homepage: www.elsevier.com/locate/ygcen
Baseline corticosterone levels are higher in migrating than sedentary common blackbirds in autumn, but not in spring Cas Eikenaar ⇑, Florian Müller, Thomas Klinner, Franz Bairlein Institute of Avian Research, An der Vogelwarte 21, 26386 Wilhelmshaven, Germany
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Article history: Received 6 March 2015 Revised 15 June 2015 Accepted 6 July 2015 Available online xxxx Keywords: Corticosterone Stopover Migrant Resident Season Blackbird
a b s t r a c t Corticosterone at baseline levels is thought to be mainly involved in the regulation of uptake, storage and release of energy, processes central to avian migration. Consequently, corticosterone levels are thought to be upregulated during migration, but the temporal pattern of its secretion during migration is not well defined. For example, although it appears that corticosterone levels decrease from flight to stopover, it is unknown if levels at stopover are still elevated and it is largely unclear how these levels compare to non-migratory life-history stages. Furthermore, what role corticosterone plays in crucial migratory processes, such as refueling and departure from stopover, is far from understood. We here determined baseline corticosterone levels in migrating and resident common blackbirds (Turdus merula), sampled simultaneously on Helgoland, a stopover site that also supports a sedentary breeding population. In autumn, migrants had higher corticosterone levels than residents, but in spring levels did not differ between the two groups. Corticosterone levels of migrants were very similar in spring and autumn, whereas in residents levels tended to be higher in spring than autumn. Higher levels in residents in spring than autumn most likely reflect the higher daily workload faced by birds during the pre-breeding than the post-breeding period. Our study thus indicates that, relative to the levels observed in residents in autumn, in spring baseline corticosterone levels were moderately elevated in both migrants and residents and that in autumn levels were moderately elevated in migrants only. Currently, corticosterone’s main function at stopover is thought to lie in the regulation of departure. Because most migrant blackbirds stay only one or two days on Helgoland, our results are in line with this idea and suggest that migrating blackbirds up-regulated their corticosterone level in anticipation of an oncoming flight bout. Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction Corticosterone at baseline and moderately elevated levels (i.e. not the levels observed in response to a stressor) is thought to mainly function in the regulation of food intake, locomotor activity and energy metabolism (Sapolsky et al., 2000; Landys et al., 2006). During the migration life-history (LH) stage in birds, these processes either increase or decrease from one to the next sub-stage of migration (Ramenofsky, 2010). Prior to the initial onset of migratory flight, birds engage in pre-migratory fat deposition (fueling) through over-eating (Gwinner, 1996; Berthold, 1996). This stage is followed by the actual migration consisting of migratory flight bouts, characterized by enhanced metabolism and locomotor activity, alternated with stopover periods that serve to rest and to replenish or augment fuel stores through over-eating (refueling) (Jenni and Jenni-Eiermann, 1992). Hence, corticosterone is thought ⇑ Corresponding author. E-mail address: [email protected] (C. Eikenaar).
to play an important role in the regulation of avian migratory behavior (Cornelius et al., 2013). However, the temporal pattern of corticosterone secretion over the migratory sequence (pre-migratory fueling, flight bouts and stopovers) and its exact role during migration are far from understood. In captive migrants, corticosterone levels have repeatedly been observed to increase during pre-migratory fueling (Piersma et al., 2000; Landys et al., 2004a; Holberton et al., 2008). Levels seem to remain elevated above baseline to support the high energetic demands of migratory flights (Falsone et al., 2009; but see Jenni-Eiermann et al., 2009). After landing for stopover, levels appear to decrease (Landys-Cianelli et al., 2002), but if levels reach baseline and how they compare to levels in non-migratory LH stages is unknown. When migrants are ready to depart from stopover, i.e. have adequate fuel stores, levels seem to increase again (Landys-Cianelli et al., 2002; Eikenaar et al., 2013). Although derived from species with different migratory strategies, and a mixture of laboratory and field studies, the pattern of corticosterone secretion over the migratory LH cycle suggests that
http://dx.doi.org/10.1016/j.ygcen.2015.07.003 0016-6480/Ó 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: Eikenaar, C., et al. Baseline corticosterone levels are higher in migrating than sedentary common blackbirds in autumn, but not in spring. Gen. Comp. Endocrinol. (2015), http://dx.doi.org/10.1016/j.ygcen.2015.07.003
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C. Eikenaar et al. / General and Comparative Endocrinology xxx (2015) xxx–xxx
corticosterone mainly functions in the regulation of departure on, and support of, migratory flights. In line with this idea is the observation that, similar to other LH stages (e.g. Astheimer et al., 1992; Breuner et al., 1998), corticosterone is positively associated with locomotor activity; in migrants caught and temporarily caged at stopover, corticosterone levels were positively correlated with nocturnal migratory restlessness (Eikenaar et al., 2014a). Migratory restlessness occurs when birds in migratory disposition are confined to a limited space (Berthold, 1996 and references therein), and is an accurate proxy for individual migrants’ likelihood of departure (Eikenaar et al., 2014b). Correlative and experimental studies indicate that the effect of corticosterone on food intake and (re)fueling during migration is merely permissive, i.e. corticosterone does not stimulate food intake and (re)fueling (Landys et al., 2004b; Holberton et al., 2007; Eikenaar et al., 2013, 2014c). To investigate the temporal pattern of corticosterone secretion and its role during migration, we focused on the stopover period and compared corticosterone levels between resident and migrating common blackbirds (Turdus merula, blackbird hereafter). Data were collected on the island of Helgoland during both the spring and autumn migration seasons. Helgoland supports a small (ca. 80 pairs) breeding population of blackbirds (Dierschke et al., 2011), and a radio-telemetry study showed that the large majority (91%) of blackbirds breeding on Helgoland are sedentary (Sacher, 2009). Birds were sampled close to, or at the start of the blackbird breeding season on Helgoland in spring, and after the breeding season and molt in autumn (Sacher, 2009; TK pers. obs.). Because both types of blackbirds were sampled during the same time period and at the same location, our dataset is the first to allow a direct and proper comparison of corticosterone levels of migratory vs. sedentary birds. Most migrating blackbirds caught on Helgoland stay only one or two days (Raiss, 1979; Ommo Hüppop, unpubl. data). Therefore, if corticosterone is up-regulated close to departure from stopover, we expected corticosterone levels to be higher in migrating than sedentary blackbirds.
This assumption is probably valid as all but one of the 22 birds that fell into this category were re-sighted on Helgoland after we color-ringed them. Second, 18 newly caught birds were considered resident because they were re-sighted more than nine days after initial trapping. We chose nine days as a cut-off point, because 95% of 1307 re-traps of supposedly migrant blackbirds on Helgoland occurred within nine days from first trapping (Ommo Hüppop, unpubl. data). Newly caught birds that were not re-sighted were considered migrants (n = 72). To reduce misassignment of status, eight newly caught birds that were re-sighted only within 9 days of color-ringing (range: 1–6 days) were not considered in the current study. In both seasons, searches for color-ringed birds were made almost daily from the start of trapping until at least three weeks after the last bird had been color-ringed. Given the small size of Helgoland (1 km2) and the fact that nearly all residents were re-sighted multiple times, we are confident that we re-sighted practically all resident color-ringed blackbirds. 2.2. Corticosterone assay
2. Methods
Corticosterone levels in plasma were determined using enzyme immunoassay kits (Enzo Life Sciences, Inc., former Assay Designs). Corticosterone in 15 ll of plasma (diluted in 200 ll H2O bidest) was extracted with 4 ml dichloromethane. The dichloromethane containing the extracted corticosterone was aspirated with a disposable Pasteur pipette and evaporated in a water bath at 48 °C. The remaining corticosterone was re-dissolved in 250 ll assay buffer and analyzed in duplicates following the kit manufacturer’s protocol (with the only difference that we used a 6 point standard curve with a range of 20,000–15.63 pg/ml). An external standard was run in duplicate on each of the four plates for calculation of intra-and inter-assay variation. The intra-assay variation ranged from 1.46% to 2.33%, and the inter-assay variation was 2.62%. The lower limit of detection in our assay was 0.62 ng/ml. To determine extraction efficiency, pooled wheatear plasma samples were spiked with corticosterone standard from the kit. Recoveries of the low (1 ng/ml), intermediate (2.5 ng/ml) and high (10 ng/ml) spikes were 87%, 93% and 70%, respectively.
2.1. Field procedures
2.3. Data analysis
The study was conducted on Helgoland (54°110 N, 07°550 E), a small island ca. 50 km off the German North Sea coastline. Blackbirds were caught from mid-March to mid-April and throughout October, periods representative of the spring and autumn blackbird migration seasons on Helgoland (Dierschke et al., 2011). Most blackbirds caught during stopover on Helgoland winter in the UK and breed in Scandinavia (Dierschke et al., 2011). Within 3 min. from capture birds were blood-sampled from the wing vein. The plasma was separated within 4 h of capture and frozen at 20 °C until hormone assaying. Birds were sexed and aged (1st year or adult) on plumage (after Svensson, 1992), ringed, and fitted with a unique combination of four color-rings for later identification in the field. Fat stores were scored after Kaiser (1993) on a scale from 0 (no fat) to 8 (furcular and abdomen bulging, and breast covered with fat). All procedures were approved by the Ministry for Energy, Agriculture, the Environment and Rural Areas, Schleswig–Holstein, Germany. Migrants were separated from residents combining two approaches. First, we made use of the ongoing, intensive ringing scheme of the Institute of Avian Research on Helgoland, during which many resident blackbirds were ringed, part as nestlings, in the breeding seasons preceding our corticosterone sampling. We assumed that birds that were ringed on Helgoland in previous breeding seasons and re-trapped by us were Helgoland residents.
We used T-tests to determine whether corticosterone level differed between resident and migrant blackbirds. Because both for migrants and residents, spring and autumn represent different LH-stages, seasons were tested separately. To explore the variation in corticosterone level within migrants and within residents we employed general linear modeling. Explanatory variables were: season, sex, fat score, and the time of sampling. We used a T-test to determine whether age affected corticosterone levels, because only for autumn migrants there was an adequate sample of both 1st year and adult birds. Corticosterone levels were log10-transformed prior to all analyses, which resulted in a normal distribution of corticosterone data (Kolmogorov–Smirnov Test: p = 0.2). 3. Results Sampling time (time from trapping until end of blood-sampling) did not affect corticosterone level in either season (spring: Spearman’s rho = 0.17, p = 0.30, n = 41; autumn: Spearman’s rho = 0.17, p = 0.17, n = 71). In spring, corticosterone levels did not differ between resident and migrating blackbirds (t = 0.88, p = 0.38, n = 41, Fig. 1). In autumn, corticosterone levels were higher in migrating than resident blackbirds (t = 2.88, p = 0.005, n = 71, Fig. 1). Excluding the extreme outlier from the
Please cite this article in press as: Eikenaar, C., et al. Baseline corticosterone levels are higher in migrating than sedentary common blackbirds in autumn, but not in spring. Gen. Comp. Endocrinol. (2015), http://dx.doi.org/10.1016/j.ygcen.2015.07.003
C. Eikenaar et al. / General and Comparative Endocrinology xxx (2015) xxx–xxx
Fig. 1. Boxplots of baseline corticosterone levels in migrating and resident common blackbirds, sampled on Helgoland in spring and autumn. Plotted are the median, 10th, 25th, 75th and 90th percentiles, and SE. Black dots indicate outliers. Numbers above boxes are sample sizes. The star indicates a significant difference between the groups.
autumn residents dataset (with a corticosterone level of 16 ng/ml, Fig. 1) enhanced this difference (t = 3.51, p = 0.001, n = 70). Corticosterone levels of migrating blackbirds were comparable in spring and autumn (Fig. 1, Table 1). Levels in residents also did not differ significantly, but there was a trend for levels to be higher in spring than in autumn (Table 1). This trend became significant when the extreme autumn outlier was excluded (b ± SE = 0.24 ± 0.10, t = 2.40, p = 0.022, n = 39). Sex, fat score and time of sampling did not explain much of the variation in corticosterone levels, neither in migrants nor in residents (Table 1). Blackbirds on their first autumn migration did not have different corticosterone levels from adult autumn migrants (t = 0.97, p = 0.75, n = 27 first year birds and 19 adults). 4. Discussion By studying a partial migrant, we were able to show, for the first time, that baseline corticosterone levels are higher in migrants making a stopover than in resident conspecifics sampled at the same location, during the same time period. This difference, however, was apparent only in the autumn migration season and not during spring migration. Corticosterone levels of migrants were very comparable in spring and autumn, while in residents levels tended to be higher in spring than autumn (Fig. 1). Hence, the observation that in spring, migrants and residents did not have different corticosterone levels was attributable to a seasonal Table 1 The effects of season, sex, fat score and time of sampling on baseline corticosterone level in migrating and resident common blackbirds sampled on Helgoland in spring and autumn. N = 72 for migrants and n = 40 for residents. Variable
b ± SE
t
p
Migrants
Season Sex Fat score Time of sampling
0.035 ± 0.079 0.094 ± 0.081 0.028 ± 0.037 0.007 ± 0.108
0.44 1.16 0.78 0.07
0.66 0.25 0.44 0.95
Residents
Season Sex Fat score Time of sampling
0.175 ± 0.111 0.023 ± 0.099 0.001 ± 0.055 0.082 ± 0.184
1.58 0.23 0.01 0.45
0.12 0.82 0.99 0.66
Reference categories are autumn for season, and female for sex.
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difference in corticosterone levels in residents. During spring migration, resident blackbirds were sampled close to, or at the start of the breeding season of the Helgoland blackbird population (Sacher, 2009). This was also indicated by the presence of a brood patch in some of the resident females. Our sampling during autumn migration fell after the residents’ breeding season and molt (Sacher, 2009; TK pers. obs.). In birds, corticosterone levels are generally highest during energetically demanding periods (Romero, 2002), and levels increase with workload (Harvey and Phillips, 1982; Rees et al., 1984). Pre-breeding and early breeding behaviour and processes likely are energetically more demanding than merely foraging in autumn. Therefore, spring and autumn corticosterone levels of residents most likely differed because the birds’ energetic requirements were higher in spring. To summarize, it thus appears that in autumn baseline corticosterone levels were moderately elevated in migrants only and that in spring levels were moderately elevated in both migrants and residents. In this study we measured total corticosterone, i.e. the sum of corticosterone bound to corticosteroid binding globulin (CBG) and free (unbound) corticosterone. In some, but not all bird species CBG levels vary seasonally, being highest during the breeding season (reviewed in Malisch and Breuner, 2010). As the fraction of corticosterone bound to CBGs affects the hormone’s clearance rate (Malisch and Breuner, 2010), it is possible that part of the variation in corticosterone levels observed in our study is explained by seasonal variation in CBG level. For example, lower CBG level in resident blackbirds in autumn (when none were breeding) than in spring (when some were breeding) could, in theory, have lowered corticosterone levels in autumn relative to levels in spring. Corticosterone is closely associated with locomotor activity (Landys et al., 2006), and correspondingly, thought to be involved in take-off from stopover sites (Landys-Cianelli et al., 2002; Lõhmus et al., 2003; Eikenaar et al., 2013, 2014a). Because on Helgoland most migrant blackbirds stay only one or two days (Raiss, 1979; Ommo Hüppop, unpubl. data), in our study the migrant blackbirds probably had elevated corticosterone levels in anticipation of an oncoming flight bout. A role for corticosterone in migratory departures is further supported by the observation that in blackbirds, corticosterone level just prior to autumn migration tended to be higher in individuals that eventually migrated than in individuals that remained sedentary (Fudickar et al., 2013). In the current study, however, we also made an observation that is not in line with the idea that corticosterone is involved in the regulation of migratory departures. At stopover, fat birds are generally more likely to resume migration than lean birds (e.g. Bairlein, 1985; Goymann et al., 2010; Eikenaar and Schläfke, 2013). Therefore, if corticosterone is up-regulated in migrants that are ready to depart, one would expect fat birds to have higher corticosterone levels than lean birds. This relationship was apparent in several long-distance migrants (e.g. bar-tailed godwit (Limosa lapponica), Landys-Cianelli et al., 2002; northern wheatear (Oenanthe oenanthe), Eikenaar et al., 2013), but not in the blackbirds sampled in the current study. A possible explanation is that because blackbirds are short-distance migrants, the need to refuel at stopover may not be very strong. Consequently, lean blackbirds may be almost as likely to depart as fat blackbirds. It is also possible that migrant blackbirds had elevated corticosterone levels for reasons other than being close to departure. First, corticosterone levels appear to be elevated during migratory flight (Falsone et al., 2009; but see Jenni-Eiermann et al., 2009). The elevated levels we observed in migrant blackbirds could therefore also (in part) be due to recent flight, a possibility indirectly supported by the lack of a relationship between corticosterone level and fat score. It is, however, unclear if corticosterone levels during the day still reflect elevated levels from when birds landed at night (a carry-over effect from night into day). If such a carry-over effect
Please cite this article in press as: Eikenaar, C., et al. Baseline corticosterone levels are higher in migrating than sedentary common blackbirds in autumn, but not in spring. Gen. Comp. Endocrinol. (2015), http://dx.doi.org/10.1016/j.ygcen.2015.07.003
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exists, one would expect this to gradually weaken over the course of the day, something which was not apparent in our data (no effect of time of sampling on corticosterone level). This suggests that if a carry-over effect exists, it was not very large. Second, migrants and residents come from different populations, which, if corticosterone levels are population specific, may have affected our data. A likely population-specific difference between migrant and resident blackbirds in our sample is the degree of urbanization, with migrants originating from less urbanized areas than the Helgoland residents. However, the relationship between urbanization and baseline corticosterone levels is currently unclear, with some studies observing higher levels in urban than rural areas (Bonier et al., 2007; Zhang et al., 2011), while others observed the opposite pattern (Schoech et al., 2004), or no difference (Partecke et al., 2006; Fodokis et al., 2009). Third, Silverin et al. (1989) compared corticosterone levels in resident and floating juvenile willow tits (Parus montanus) and found that levels were higher in the latter. Silverin et al. (1989) suggested that the difference is explained by floating juveniles being forced to leave their natal area and consequently being exposed to food restriction, which may have increased their corticosterone levels. Regarding our study, it is possible that the best foraging areas on Helgoland are permanently occupied by the resident blackbirds and that, consequently, migrating blackbirds making a stopover on Helgoland have to settle for inferior foraging habitat. This in theory could have increased corticosterone levels in migrants relative to residents. However, body condition (fat score) was not associated with corticosterone levels, neither in migrants nor residents (Table 1). Also, both migrants and residents had a median fat score of 2. Finally, sedentary and migratory blackbirds were seen foraging at the same locations (FM and TK, pers. obs.). It therefore seems unlikely that differential access to food explains the elevated corticosterone levels we observed in migrants. Considering above arguments, we presently favor the hypothesis that migrant blackbirds had elevated corticosterone levels because they were close to departing from stopover. Only a handful of stopover studies report baseline corticosterone levels of migrants for both spring and autumn. In white-crowned sparrows (Zonotrichia leucophrys gambelli) corticosterone levels were higher in spring than in autumn (Romero et al., 1997), whereas the opposite pattern was found in western sandpipers (Calidris mauri, O’Reilly and Wingfield, 2003). Corticosterone levels were not different between the seasons in semipalmated sandpipers (Calidris pussila, Tsipoura et al., 1999), nor in northern wheatears (Eikenaar et al., 2013, 2014a). Similarly, in the current study corticosterone levels in migrating blackbirds were not different in spring and autumn. Although much more studies are needed to draw any conclusions, the data available so far tentatively suggest that during stopover, corticosterone secretion is not higher in one particular migration season. Acknowledgments We are much obliged to Sinja Werner and the staff of the IfV Helgoland station for color-band reading. Ommo Hüppop kindly provided and analyzed Helgoland blackbird trapping data. Thanks also go to Jochen Dierschke and Klaus Müller for logistical support on Helgoland. Jan Engler gave useful tips for trapping resident blackbirds on Helgoland. Two anonymous reviewers provided useful comments. References Astheimer, L.B., Buttemer, B.A., Wingfield, J.C., 1992. Interactions of corticosterone with feeding, activity and metabolism in passerine birds. Ornis Scand. 23, 355–365.
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General and Comparative Endocrinology journal homepage: www.elsevier.com/locate/ygcen
Baseline corticosterone levels are higher in migrating than sedentary common blackbirds in autumn, but not in spring Cas Eikenaar ⇑, Florian Müller, Thomas Klinner, Franz Bairlein Institute of Avian Research, An der Vogelwarte 21, 26386 Wilhelmshaven, Germany
a r t i c l e
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Article history: Received 6 March 2015 Revised 15 June 2015 Accepted 6 July 2015 Available online xxxx Keywords: Corticosterone Stopover Migrant Resident Season Blackbird
a b s t r a c t Corticosterone at baseline levels is thought to be mainly involved in the regulation of uptake, storage and release of energy, processes central to avian migration. Consequently, corticosterone levels are thought to be upregulated during migration, but the temporal pattern of its secretion during migration is not well defined. For example, although it appears that corticosterone levels decrease from flight to stopover, it is unknown if levels at stopover are still elevated and it is largely unclear how these levels compare to non-migratory life-history stages. Furthermore, what role corticosterone plays in crucial migratory processes, such as refueling and departure from stopover, is far from understood. We here determined baseline corticosterone levels in migrating and resident common blackbirds (Turdus merula), sampled simultaneously on Helgoland, a stopover site that also supports a sedentary breeding population. In autumn, migrants had higher corticosterone levels than residents, but in spring levels did not differ between the two groups. Corticosterone levels of migrants were very similar in spring and autumn, whereas in residents levels tended to be higher in spring than autumn. Higher levels in residents in spring than autumn most likely reflect the higher daily workload faced by birds during the pre-breeding than the post-breeding period. Our study thus indicates that, relative to the levels observed in residents in autumn, in spring baseline corticosterone levels were moderately elevated in both migrants and residents and that in autumn levels were moderately elevated in migrants only. Currently, corticosterone’s main function at stopover is thought to lie in the regulation of departure. Because most migrant blackbirds stay only one or two days on Helgoland, our results are in line with this idea and suggest that migrating blackbirds up-regulated their corticosterone level in anticipation of an oncoming flight bout. Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction Corticosterone at baseline and moderately elevated levels (i.e. not the levels observed in response to a stressor) is thought to mainly function in the regulation of food intake, locomotor activity and energy metabolism (Sapolsky et al., 2000; Landys et al., 2006). During the migration life-history (LH) stage in birds, these processes either increase or decrease from one to the next sub-stage of migration (Ramenofsky, 2010). Prior to the initial onset of migratory flight, birds engage in pre-migratory fat deposition (fueling) through over-eating (Gwinner, 1996; Berthold, 1996). This stage is followed by the actual migration consisting of migratory flight bouts, characterized by enhanced metabolism and locomotor activity, alternated with stopover periods that serve to rest and to replenish or augment fuel stores through over-eating (refueling) (Jenni and Jenni-Eiermann, 1992). Hence, corticosterone is thought ⇑ Corresponding author. E-mail address: [email protected] (C. Eikenaar).
to play an important role in the regulation of avian migratory behavior (Cornelius et al., 2013). However, the temporal pattern of corticosterone secretion over the migratory sequence (pre-migratory fueling, flight bouts and stopovers) and its exact role during migration are far from understood. In captive migrants, corticosterone levels have repeatedly been observed to increase during pre-migratory fueling (Piersma et al., 2000; Landys et al., 2004a; Holberton et al., 2008). Levels seem to remain elevated above baseline to support the high energetic demands of migratory flights (Falsone et al., 2009; but see Jenni-Eiermann et al., 2009). After landing for stopover, levels appear to decrease (Landys-Cianelli et al., 2002), but if levels reach baseline and how they compare to levels in non-migratory LH stages is unknown. When migrants are ready to depart from stopover, i.e. have adequate fuel stores, levels seem to increase again (Landys-Cianelli et al., 2002; Eikenaar et al., 2013). Although derived from species with different migratory strategies, and a mixture of laboratory and field studies, the pattern of corticosterone secretion over the migratory LH cycle suggests that
http://dx.doi.org/10.1016/j.ygcen.2015.07.003 0016-6480/Ó 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: Eikenaar, C., et al. Baseline corticosterone levels are higher in migrating than sedentary common blackbirds in autumn, but not in spring. Gen. Comp. Endocrinol. (2015), http://dx.doi.org/10.1016/j.ygcen.2015.07.003
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corticosterone mainly functions in the regulation of departure on, and support of, migratory flights. In line with this idea is the observation that, similar to other LH stages (e.g. Astheimer et al., 1992; Breuner et al., 1998), corticosterone is positively associated with locomotor activity; in migrants caught and temporarily caged at stopover, corticosterone levels were positively correlated with nocturnal migratory restlessness (Eikenaar et al., 2014a). Migratory restlessness occurs when birds in migratory disposition are confined to a limited space (Berthold, 1996 and references therein), and is an accurate proxy for individual migrants’ likelihood of departure (Eikenaar et al., 2014b). Correlative and experimental studies indicate that the effect of corticosterone on food intake and (re)fueling during migration is merely permissive, i.e. corticosterone does not stimulate food intake and (re)fueling (Landys et al., 2004b; Holberton et al., 2007; Eikenaar et al., 2013, 2014c). To investigate the temporal pattern of corticosterone secretion and its role during migration, we focused on the stopover period and compared corticosterone levels between resident and migrating common blackbirds (Turdus merula, blackbird hereafter). Data were collected on the island of Helgoland during both the spring and autumn migration seasons. Helgoland supports a small (ca. 80 pairs) breeding population of blackbirds (Dierschke et al., 2011), and a radio-telemetry study showed that the large majority (91%) of blackbirds breeding on Helgoland are sedentary (Sacher, 2009). Birds were sampled close to, or at the start of the blackbird breeding season on Helgoland in spring, and after the breeding season and molt in autumn (Sacher, 2009; TK pers. obs.). Because both types of blackbirds were sampled during the same time period and at the same location, our dataset is the first to allow a direct and proper comparison of corticosterone levels of migratory vs. sedentary birds. Most migrating blackbirds caught on Helgoland stay only one or two days (Raiss, 1979; Ommo Hüppop, unpubl. data). Therefore, if corticosterone is up-regulated close to departure from stopover, we expected corticosterone levels to be higher in migrating than sedentary blackbirds.
This assumption is probably valid as all but one of the 22 birds that fell into this category were re-sighted on Helgoland after we color-ringed them. Second, 18 newly caught birds were considered resident because they were re-sighted more than nine days after initial trapping. We chose nine days as a cut-off point, because 95% of 1307 re-traps of supposedly migrant blackbirds on Helgoland occurred within nine days from first trapping (Ommo Hüppop, unpubl. data). Newly caught birds that were not re-sighted were considered migrants (n = 72). To reduce misassignment of status, eight newly caught birds that were re-sighted only within 9 days of color-ringing (range: 1–6 days) were not considered in the current study. In both seasons, searches for color-ringed birds were made almost daily from the start of trapping until at least three weeks after the last bird had been color-ringed. Given the small size of Helgoland (1 km2) and the fact that nearly all residents were re-sighted multiple times, we are confident that we re-sighted practically all resident color-ringed blackbirds. 2.2. Corticosterone assay
2. Methods
Corticosterone levels in plasma were determined using enzyme immunoassay kits (Enzo Life Sciences, Inc., former Assay Designs). Corticosterone in 15 ll of plasma (diluted in 200 ll H2O bidest) was extracted with 4 ml dichloromethane. The dichloromethane containing the extracted corticosterone was aspirated with a disposable Pasteur pipette and evaporated in a water bath at 48 °C. The remaining corticosterone was re-dissolved in 250 ll assay buffer and analyzed in duplicates following the kit manufacturer’s protocol (with the only difference that we used a 6 point standard curve with a range of 20,000–15.63 pg/ml). An external standard was run in duplicate on each of the four plates for calculation of intra-and inter-assay variation. The intra-assay variation ranged from 1.46% to 2.33%, and the inter-assay variation was 2.62%. The lower limit of detection in our assay was 0.62 ng/ml. To determine extraction efficiency, pooled wheatear plasma samples were spiked with corticosterone standard from the kit. Recoveries of the low (1 ng/ml), intermediate (2.5 ng/ml) and high (10 ng/ml) spikes were 87%, 93% and 70%, respectively.
2.1. Field procedures
2.3. Data analysis
The study was conducted on Helgoland (54°110 N, 07°550 E), a small island ca. 50 km off the German North Sea coastline. Blackbirds were caught from mid-March to mid-April and throughout October, periods representative of the spring and autumn blackbird migration seasons on Helgoland (Dierschke et al., 2011). Most blackbirds caught during stopover on Helgoland winter in the UK and breed in Scandinavia (Dierschke et al., 2011). Within 3 min. from capture birds were blood-sampled from the wing vein. The plasma was separated within 4 h of capture and frozen at 20 °C until hormone assaying. Birds were sexed and aged (1st year or adult) on plumage (after Svensson, 1992), ringed, and fitted with a unique combination of four color-rings for later identification in the field. Fat stores were scored after Kaiser (1993) on a scale from 0 (no fat) to 8 (furcular and abdomen bulging, and breast covered with fat). All procedures were approved by the Ministry for Energy, Agriculture, the Environment and Rural Areas, Schleswig–Holstein, Germany. Migrants were separated from residents combining two approaches. First, we made use of the ongoing, intensive ringing scheme of the Institute of Avian Research on Helgoland, during which many resident blackbirds were ringed, part as nestlings, in the breeding seasons preceding our corticosterone sampling. We assumed that birds that were ringed on Helgoland in previous breeding seasons and re-trapped by us were Helgoland residents.
We used T-tests to determine whether corticosterone level differed between resident and migrant blackbirds. Because both for migrants and residents, spring and autumn represent different LH-stages, seasons were tested separately. To explore the variation in corticosterone level within migrants and within residents we employed general linear modeling. Explanatory variables were: season, sex, fat score, and the time of sampling. We used a T-test to determine whether age affected corticosterone levels, because only for autumn migrants there was an adequate sample of both 1st year and adult birds. Corticosterone levels were log10-transformed prior to all analyses, which resulted in a normal distribution of corticosterone data (Kolmogorov–Smirnov Test: p = 0.2). 3. Results Sampling time (time from trapping until end of blood-sampling) did not affect corticosterone level in either season (spring: Spearman’s rho = 0.17, p = 0.30, n = 41; autumn: Spearman’s rho = 0.17, p = 0.17, n = 71). In spring, corticosterone levels did not differ between resident and migrating blackbirds (t = 0.88, p = 0.38, n = 41, Fig. 1). In autumn, corticosterone levels were higher in migrating than resident blackbirds (t = 2.88, p = 0.005, n = 71, Fig. 1). Excluding the extreme outlier from the
Please cite this article in press as: Eikenaar, C., et al. Baseline corticosterone levels are higher in migrating than sedentary common blackbirds in autumn, but not in spring. Gen. Comp. Endocrinol. (2015), http://dx.doi.org/10.1016/j.ygcen.2015.07.003
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Fig. 1. Boxplots of baseline corticosterone levels in migrating and resident common blackbirds, sampled on Helgoland in spring and autumn. Plotted are the median, 10th, 25th, 75th and 90th percentiles, and SE. Black dots indicate outliers. Numbers above boxes are sample sizes. The star indicates a significant difference between the groups.
autumn residents dataset (with a corticosterone level of 16 ng/ml, Fig. 1) enhanced this difference (t = 3.51, p = 0.001, n = 70). Corticosterone levels of migrating blackbirds were comparable in spring and autumn (Fig. 1, Table 1). Levels in residents also did not differ significantly, but there was a trend for levels to be higher in spring than in autumn (Table 1). This trend became significant when the extreme autumn outlier was excluded (b ± SE = 0.24 ± 0.10, t = 2.40, p = 0.022, n = 39). Sex, fat score and time of sampling did not explain much of the variation in corticosterone levels, neither in migrants nor in residents (Table 1). Blackbirds on their first autumn migration did not have different corticosterone levels from adult autumn migrants (t = 0.97, p = 0.75, n = 27 first year birds and 19 adults). 4. Discussion By studying a partial migrant, we were able to show, for the first time, that baseline corticosterone levels are higher in migrants making a stopover than in resident conspecifics sampled at the same location, during the same time period. This difference, however, was apparent only in the autumn migration season and not during spring migration. Corticosterone levels of migrants were very comparable in spring and autumn, while in residents levels tended to be higher in spring than autumn (Fig. 1). Hence, the observation that in spring, migrants and residents did not have different corticosterone levels was attributable to a seasonal Table 1 The effects of season, sex, fat score and time of sampling on baseline corticosterone level in migrating and resident common blackbirds sampled on Helgoland in spring and autumn. N = 72 for migrants and n = 40 for residents. Variable
b ± SE
t
p
Migrants
Season Sex Fat score Time of sampling
0.035 ± 0.079 0.094 ± 0.081 0.028 ± 0.037 0.007 ± 0.108
0.44 1.16 0.78 0.07
0.66 0.25 0.44 0.95
Residents
Season Sex Fat score Time of sampling
0.175 ± 0.111 0.023 ± 0.099 0.001 ± 0.055 0.082 ± 0.184
1.58 0.23 0.01 0.45
0.12 0.82 0.99 0.66
Reference categories are autumn for season, and female for sex.
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difference in corticosterone levels in residents. During spring migration, resident blackbirds were sampled close to, or at the start of the breeding season of the Helgoland blackbird population (Sacher, 2009). This was also indicated by the presence of a brood patch in some of the resident females. Our sampling during autumn migration fell after the residents’ breeding season and molt (Sacher, 2009; TK pers. obs.). In birds, corticosterone levels are generally highest during energetically demanding periods (Romero, 2002), and levels increase with workload (Harvey and Phillips, 1982; Rees et al., 1984). Pre-breeding and early breeding behaviour and processes likely are energetically more demanding than merely foraging in autumn. Therefore, spring and autumn corticosterone levels of residents most likely differed because the birds’ energetic requirements were higher in spring. To summarize, it thus appears that in autumn baseline corticosterone levels were moderately elevated in migrants only and that in spring levels were moderately elevated in both migrants and residents. In this study we measured total corticosterone, i.e. the sum of corticosterone bound to corticosteroid binding globulin (CBG) and free (unbound) corticosterone. In some, but not all bird species CBG levels vary seasonally, being highest during the breeding season (reviewed in Malisch and Breuner, 2010). As the fraction of corticosterone bound to CBGs affects the hormone’s clearance rate (Malisch and Breuner, 2010), it is possible that part of the variation in corticosterone levels observed in our study is explained by seasonal variation in CBG level. For example, lower CBG level in resident blackbirds in autumn (when none were breeding) than in spring (when some were breeding) could, in theory, have lowered corticosterone levels in autumn relative to levels in spring. Corticosterone is closely associated with locomotor activity (Landys et al., 2006), and correspondingly, thought to be involved in take-off from stopover sites (Landys-Cianelli et al., 2002; Lõhmus et al., 2003; Eikenaar et al., 2013, 2014a). Because on Helgoland most migrant blackbirds stay only one or two days (Raiss, 1979; Ommo Hüppop, unpubl. data), in our study the migrant blackbirds probably had elevated corticosterone levels in anticipation of an oncoming flight bout. A role for corticosterone in migratory departures is further supported by the observation that in blackbirds, corticosterone level just prior to autumn migration tended to be higher in individuals that eventually migrated than in individuals that remained sedentary (Fudickar et al., 2013). In the current study, however, we also made an observation that is not in line with the idea that corticosterone is involved in the regulation of migratory departures. At stopover, fat birds are generally more likely to resume migration than lean birds (e.g. Bairlein, 1985; Goymann et al., 2010; Eikenaar and Schläfke, 2013). Therefore, if corticosterone is up-regulated in migrants that are ready to depart, one would expect fat birds to have higher corticosterone levels than lean birds. This relationship was apparent in several long-distance migrants (e.g. bar-tailed godwit (Limosa lapponica), Landys-Cianelli et al., 2002; northern wheatear (Oenanthe oenanthe), Eikenaar et al., 2013), but not in the blackbirds sampled in the current study. A possible explanation is that because blackbirds are short-distance migrants, the need to refuel at stopover may not be very strong. Consequently, lean blackbirds may be almost as likely to depart as fat blackbirds. It is also possible that migrant blackbirds had elevated corticosterone levels for reasons other than being close to departure. First, corticosterone levels appear to be elevated during migratory flight (Falsone et al., 2009; but see Jenni-Eiermann et al., 2009). The elevated levels we observed in migrant blackbirds could therefore also (in part) be due to recent flight, a possibility indirectly supported by the lack of a relationship between corticosterone level and fat score. It is, however, unclear if corticosterone levels during the day still reflect elevated levels from when birds landed at night (a carry-over effect from night into day). If such a carry-over effect
Please cite this article in press as: Eikenaar, C., et al. Baseline corticosterone levels are higher in migrating than sedentary common blackbirds in autumn, but not in spring. Gen. Comp. Endocrinol. (2015), http://dx.doi.org/10.1016/j.ygcen.2015.07.003
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exists, one would expect this to gradually weaken over the course of the day, something which was not apparent in our data (no effect of time of sampling on corticosterone level). This suggests that if a carry-over effect exists, it was not very large. Second, migrants and residents come from different populations, which, if corticosterone levels are population specific, may have affected our data. A likely population-specific difference between migrant and resident blackbirds in our sample is the degree of urbanization, with migrants originating from less urbanized areas than the Helgoland residents. However, the relationship between urbanization and baseline corticosterone levels is currently unclear, with some studies observing higher levels in urban than rural areas (Bonier et al., 2007; Zhang et al., 2011), while others observed the opposite pattern (Schoech et al., 2004), or no difference (Partecke et al., 2006; Fodokis et al., 2009). Third, Silverin et al. (1989) compared corticosterone levels in resident and floating juvenile willow tits (Parus montanus) and found that levels were higher in the latter. Silverin et al. (1989) suggested that the difference is explained by floating juveniles being forced to leave their natal area and consequently being exposed to food restriction, which may have increased their corticosterone levels. Regarding our study, it is possible that the best foraging areas on Helgoland are permanently occupied by the resident blackbirds and that, consequently, migrating blackbirds making a stopover on Helgoland have to settle for inferior foraging habitat. This in theory could have increased corticosterone levels in migrants relative to residents. However, body condition (fat score) was not associated with corticosterone levels, neither in migrants nor residents (Table 1). Also, both migrants and residents had a median fat score of 2. Finally, sedentary and migratory blackbirds were seen foraging at the same locations (FM and TK, pers. obs.). It therefore seems unlikely that differential access to food explains the elevated corticosterone levels we observed in migrants. Considering above arguments, we presently favor the hypothesis that migrant blackbirds had elevated corticosterone levels because they were close to departing from stopover. Only a handful of stopover studies report baseline corticosterone levels of migrants for both spring and autumn. In white-crowned sparrows (Zonotrichia leucophrys gambelli) corticosterone levels were higher in spring than in autumn (Romero et al., 1997), whereas the opposite pattern was found in western sandpipers (Calidris mauri, O’Reilly and Wingfield, 2003). Corticosterone levels were not different between the seasons in semipalmated sandpipers (Calidris pussila, Tsipoura et al., 1999), nor in northern wheatears (Eikenaar et al., 2013, 2014a). Similarly, in the current study corticosterone levels in migrating blackbirds were not different in spring and autumn. Although much more studies are needed to draw any conclusions, the data available so far tentatively suggest that during stopover, corticosterone secretion is not higher in one particular migration season. Acknowledgments We are much obliged to Sinja Werner and the staff of the IfV Helgoland station for color-band reading. Ommo Hüppop kindly provided and analyzed Helgoland blackbird trapping data. Thanks also go to Jochen Dierschke and Klaus Müller for logistical support on Helgoland. Jan Engler gave useful tips for trapping resident blackbirds on Helgoland. Two anonymous reviewers provided useful comments. References Astheimer, L.B., Buttemer, B.A., Wingfield, J.C., 1992. Interactions of corticosterone with feeding, activity and metabolism in passerine birds. Ornis Scand. 23, 355–365.
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