Endocrine regulation of migratory departure from stopover: Evidence from a longitudinal migratory restlessness study on northern wheatears.

Most migrating birds make stopovers to replenish fuel stores. The decision to resume migration from stopover to a large extent shapes the temporal organization of migration. This decision is known to be shaped by a suite of intrinsic and extrinsic factors such as the bird's fuel stores and current weather conditions. However, how departures from stopover are physiologically regulated is largely unknown. We here present data that strongly indicate that corticosterone, a hormone with a stimulatory effect on locomotion, acts as a mediator between fuel stores and departure from stopover. In migrating northern wheatears (Oenanthe oenanthe) temporarily caged at stopover, we observed a positive relationship between the change in fuel stores and the concurrent change in glucocorticoid metabolite (GCM) levels measured in the birds' droppings. We also found a positive relationship between the change in GCM levels and the change in the intensity of nocturnal migratory restlessness. As in northern wheatears nocturnal migratory restlessness is an accurate proxy for stopover departure likelihood, our results indicate that corticosterone mediates between fuel stores and the decision to resume migration. Our unique longitudinal study represents a considerable advance in our understanding of the endocrine regulation of avian migration.

Loner or socializer? Ravens' adrenocortical response to individual separation depends on social integration.

Non-breeding common ravens (Corvus corax) live in complex social groups with a high degree of fission-fusion dynamics. They form valuable relationships and alliances with some conspecifics, while taking coordinated action against others. In ravens, affiliates reconcile their conflicts, console each other after conflicts with a third party, and provide each other with social support - all behaviors that presumably reduce corticosterone levels and alleviate stress. However, how well an individual is socially integrated in a (sub)group might vary substantially. This raises the question whether the social integration of a raven affects its stress responses to fission-fusion dynamics. The present study aims to investigate this effect experimentally by separating single ravens (n=16) individually from their group for four days and subsequently reintroducing them. To determine stress response patterns in the separated individuals we measured the amounts of immunoreactive corticosterone metabolites (CM) in droppings. We compared two enzyme immunoassays, which we validated by conducting an ACTH challenge, and finally decided to apply an 11-oxoetiocholanolone enzyme immunoassay. Additionally, we determined levels of social integration using focal observations. Our findings suggest that a strong social integration is related to low CM levels when the individuals are within the group and high levels during separations, implying that separation leads to stress in these birds. In contrast, poorly socially integrated ravens seem to exhibit the opposite pattern, indicating that to them group living is more stressful than being temporarily separated. We, therefore, conclude that the birds' adrenocortical activity is modulated by their social integration.

Corticosterone predicts nocturnal restlessness in a long-distance migrant.

The decision made by migrating birds to stop refueling and to depart from stopover depends on cues from innate rhythms, intrinsic factors such as fuel reserves, and extrinsic factors such as weather conditions. The physiological mechanism behind this decision, however, is largely unexplored. The transition from refueling to flight involves an increase in both locomotion and energetic demands. Because, at baseline levels, corticosterone stimulates locomotion and is involved in the mobilization of energy, this hormone could encourage departure of migrants. We collected field data on baseline corticosterone, migratory restlessness, and actual departure in northern wheatears at stopover. Additionally, in refueling long-term captive conspecifics, we measured migratory restlessness while simultaneously collecting droppings to determine glucocorticoid metabolite (GCM) levels. We found that migratory restlessness at stopover was positively correlated with corticosterone level. Similarly, in refueling long-term captive birds, migratory restlessness was positively correlated with concurrently measured GCM levels in droppings. To our best knowledge, our study is the first to simultaneously measure a hormonal signal and migratory restlessness. In conclusion, our results are consistent with the hypothesis that, by increasing locomotor activity, baseline corticosterone is involved in the regulation of departure of migrants at stopover. Future studies could reveal how corticosterone is up-regulated in migrants that are ready to depart.

Corticosterone, food intake and refueling in a long-distance migrant.

Elevated baseline corticosterone levels function to mobilize energy in predictable life-history stages, such as bird migration. At the same time, baseline corticosterone has a permissive effect on the accumulation of fat stores (fueling) needed for migratory flight. Most migrants alternate flight bouts with stopovers, during which they replenish the fuel used during the preceding flight (refueling). The role of corticosterone in refueling is currently unclear. In a fasting-re-feeding experiment on northern wheatears (Oenanthe oenanthe) in autumn we found that baseline total and free corticosterone levels were negatively related with both food intake and the rate of fuel deposition after fasting. This confirms our earlier findings in wild conspecifics in spring and indicates that corticosterone does not stimulate stopover refueling. Whether the negative relationship between baseline corticosterone level and fuel deposition rate is causal is questionable, because within-individual comparison of corticosterone metabolite levels in droppings did not reveal differences between refueling and control periods. In other words, corticosterone does not appear to be down-regulated during refueling, which would be expected if it directly hampers refueling. We discuss possible correlates of corticosterone level that may explain the negative association between corticosterone and stopover refueling. Additionally, we found that fasting decreases total corticosterone level, which contrasts with previous studies. We propose that the difference is due to the other studies being conducted outside of the migration life-history stage, and provide a possible explanation for the decrease in corticosterone during fasting in migrating birds.

Experimental manipulation of dietary lead levels in great tit nestlings: limited effects on growth, physiology and survival.

We manipulated dietary lead (Pb) levels of nestlings in wild populations of the great tit (Parus major L) to find out if environmentally relevant Pb levels would affect some physiological biomarkers (haematocrit [HT], fecal corticosterone metabolites [CORT], heat shock proteins [HSPs], erythrocyte delta-aminolevulinic acid dehydratase activity [ALAd]), growth (body mass, wing length), phenotype (plumage coloration) or survival of nestlings. The responses to three experimental manipulation (control, low and high: 0, 1 and 4 µg/g body mass/day) are compared with those in a P. major population breeding in the vicinity of a heavy metal source, a copper smelter. Our Pb supplementation was successful in raising the fecal concentrations to the levels found in polluted environments (high: 8.0 µg/g d.w.). Despite relatively high range of exposure levels we found only few effects on growth rates or physiology. The lack of blood ALAd inhibition suggests that the circulating Pb levels were generally below the toxic level despite that marked accumulation of Pb in femur (high: 27.8 µg/g d.w.) was observed. Instead, birds in the metal polluted environment around the smelter showed decreased growth rates, lower HT, higher CORT, less colorful plumage and lower survival probabilities than any of the Pb treated groups. These effects are likely related to decreased food quality/quantity for these insectivorous birds at the smelter site. In general, the responses of nestlings to metal exposure and/or associated resource limitation were not gender specific. One of the stress proteins (HSP60), however, was more strongly induced in Pb exposed males and further studies are needed to explore if this was due to higher accumulation of Pb or higher sensitivity of males. In all, our results emphasize the importance of secondary pollution effects (e.g. via food chain disruption) on reproductive output of birds.

Selection for fast and slow exploration affects baseline and stress-induced corticosterone excretion in Great tit nestlings, Parus major.

In nestlings, glucocorticoid (GC) secretion has short-term and long-term fitness consequences. For example, short-time elevations trigger begging activity, whereas chronically elevated GC levels impair body condition, growth and cognitive abilities. Despite a growing body of literature on personality traits, the effects of selection for fast and slow exploration on GC secretion have received little attention. We compared baseline and stress-induced hypothalamic-pituitary-adrenal (HPA) axis activity of hand-reared great tit nestlings of lines selected for fast and slow exploration. Nestling droppings were collected under three conditions: control, test (following handling stress, day 14 after hatching) and the following day. The concentrations of excreted immunoreactive corticosterone metabolites (CM) were determined via an enzyme immunoassay. We also observed nestlings' begging behaviour. CM differed significantly between the lines. Nestlings of the fast line excreted lower CM than slow-line birds. In response to handling stress, nestlings excreted significantly higher concentrations of CM than during the control and on the day after handling. Sex and begging activity were not related to CM levels. Under the control condition, but not after handling, males begged significantly more often than females. In both lines, adults excreted significantly less CM compared to nestlings. Both nestlings and adults of the slow line produced higher baseline CM values than fast-line birds. Fast-line nestlings excreted lower baseline CM than nestlings of a wild population not selected for fast or slow exploration. Slow-line nestlings did not. Our results show that selection on the basis of exploratory behaviour affected HPA axis reactivity.

Behavioural and Hormonal Stress Responses to Social Separation in Ravens, Corvus corax.

Social life is profitable, but it facilitates conflicts over resources and creates interdependence between individuals. Separating highly social animals triggers intense reactions aimed at re-establishing lost connections. Less is known, however, about behavioural and physiological responses to separation in socially facultative species, where individuals temporarily form groups and may subsequently leave them. Non-breeding common ravens (Corvus corax) gather in large numbers at feeding and roosting sites, but otherwise spend time seemingly solitary or in small subgroups. We here studied how ravens cope with being socially isolated, and investigated the life characteristics that might explain potential individual differences. For this, we individually separated captive subadult ravens (n = 25) and housed them in physical and visual isolation from their group members across 4 d. During the separation period, we collected behavioural data and measured the amount of immunoreactive corticosterone metabolites from bird droppings to assess the ravens' physiological stress response. We found behavioural indicators of stress at the start of the separation, when ravens showed higher levels of tension than of comfort - a pattern that reversed at the end of the separation. Furthermore, we found that the upbringing of ravens affected their behaviour during separation. Hand-raised birds produced more vocalisations in the beginning of the separation, and were less active at the end, while the reverse pattern occurred with parent-raised ravens. Contrary to our predictions, we did not find differences in hormonal responses between the beginning and end of the separation period or any link between hormonal responses and behaviours. Ravens' behavioural responses to social separation stress seem to be dependent on their arousal states, although possible links with hormonal reactions remain unclear. Our results show that behavioural reactions are not always linked with hormonal responses to stress, and further emphasise the importance of investigating effects of early-life experiences.

Novel object exploration in ravens (Corvus corax): effects of social relationships.

Social context has been shown to encourage, or to delay object exploration and learning. This ambiguity might be due to factors such as social relationships and personality of the individuals involved. Here, we investigated in ravens (Corvus corax) individuals' consistency in response to novel objects over development and across contexts: alone versus social. In the social setting we focussed on the effects of social relationships on social facilitation during the approach to novel objects. We tested 11 hand-raised ravens with novel objects individually at three and six months of age and in dyadic combinations at six months of age. Individuals were consistent over development and contexts in their response to different novel objects. Birds joined siblings faster to approach novel objects than non-siblings. They also spent more time sitting close to siblings than to non-siblings. In male-male dyads but not in female-female dyads, subordinates approached the novel objects significantly faster than dominant birds. In contrast, dominant males were the first to approach the novel objects in mixed-sex combinations. Hence, the effect of social context seems to depend on the social relationships towards the companions and on the combination of the sexes.

Ravens, Corvus corax, follow gaze direction of humans around obstacles.

The ability to follow gaze (i.e. head and eye direction) has recently been shown for social mammals, particularly primates. In most studies, individuals could use gaze direction as a behavioural cue without understanding that the view of others may be different from their own. Here, we show that hand-raised ravens not only visually co-orient with the look-ups of a human experimenter but also reposition themselves to follow the experimenter's gaze around a visual barrier. Birds were capable of visual co-orientation already as fledglings but consistently tracked gaze direction behind obstacles not before six months of age. These results raise the possibility that sub-adult and adult ravens can project a line of sight for the other person into the distance. To what extent ravens may attribute mental significance to the visual behaviour of others is discussed.

Corticosterone excretion patterns and affiliative behavior over development in ravens (Corvus corax).

Averse effects of social stress may be buffered by the presence of social allies, which mainly has been demonstrated in mammals and recently also in birds. However, effects of socio-positive behavior prior to fledging in relation to corticosterone excretion in altricial birds have not been investigated yet. We here monitored corticosterone excretion patterns in three groups of hand raised juvenile ravens (n=5, 6 and 11) in the nest, post-fledging (May-July) and when ravens would be independent from their parents (September-November). We related these corticosterone excretion patterns to socio-positive behavior. Behavioral data were collected via focal sampling in each developmental period considered. We analyzed amounts of excreted immunoreactive corticosterone metabolites (CM) using enzyme immuno assays. We collected fecal samples in each developmental period considered and evaluated the most appropriate assay via an isolation stress experiment. Basal CM was significantly higher during the nestling period than post-fledging or when birds were independent. The time nestlings spent allopreening correlated negatively with mean CM. Post-fledging, individuals with higher CM levels sat close to (distance <50 cm) conspecifics more frequently and tended to preen them longer. When birds were independent and a stable rank hierarchy was established, dominant individuals were preened significantly longer than subordinates. These patterns observed in ravens parallel those described for primates, which could indicate that animal species living in a complex social environment may deal with social problems in a similar way that is not restricted to mammals or primates.