Tuul Sepp

Oxidative stress and information content of black and yellow plumage coloration: An experiment with greenfinches

SUMMARY Carotenoid and melanin pigments in the plumage of birds are hypothesized to be sensitive to oxidative stress. We manipulated oxidative status of captive greenfinches (Carduelis chloris L.) by the administration of buthionine sulfoximine (BSO), a selective inhibitor of the synthesis of glutathione (GSH), an intracellular antioxidant. Half of the birds in the treated group, as well as in the control group, also received dietary carotenoid (lutein) supplementation. BSO treatment reduced erythrocyte GSH levels and caused oxidative damage as indicated by the increased concentration of plasma malondialdehyde (MDA), an end product of lipid peroxidation. BSO treatment also reduced the brightness (i.e. increased blackness) of the tips of tail feathers grown during the experiment. These results show that a low systemic GSH level is required for development of eumelanin plumage coloration and that such a low GSH level is also potentially dangerous for the organism. Carotenoid supplementation increased plasma carotenoid levels and chroma of the yellow parts of the feathers grown during the experiment. However, carotenoid supplementation did not reduce plasma MDA levels. Manipulation of GSH did not affect plasma carotenoids or carotenoid-based plumage coloration. These findings argue against the antioxidant function of lutein in vivo and carotenoid signaling of antioxidant status.

Hematological Condition Indexes in Greenfinches: Effects of Captivity and Diurnal Variation

Ecophysiological research aiming at explaining the causes and consequences of variation in individual condition, health state, and allostasis is traditionally performed on captive animals under controlled laboratory conditions. The question about how captivity per se affects studied parameters is therefore of central importance for generalizing the information gained from such studies. We addressed this question by comparing various indexes of physiological condition of wintering greenfinches sampled in the wild and kept in captivity for different time periods. Bringing wild greenfinches into captivity did not result in systematic alteration in nine of 12 physiological parameters studied. Captive birds had consistently lower plasma carotenoid and uric acid levels than wild ones. Variation in differential leukocyte counts did not reveal any signs of elevated stress of birds kept in captivity. These results indicate that for a number of physiological parameters, information obtained from captive animals can be generalized to natural situations. Variance in traits most closely related to physical exercise capacity (body mass and hematocrit) were much lower in the wild than in captivity. These findings suggest that under harsh environmental conditions experienced by wild birds (i.e., predation threat, scarce resources), traits such as hematocrit and body mass are fine tuned by physiological trade‐offs.

Individual Consistency and Covariation of Measures of Oxidative Status in Greenfinches

Oxidative stress results from a mismatch between production of reactive oxygen species (ROS) and the organisms capacity to mitigate their damaging effects by building up sufficient antioxidant protection and/or repair mechanisms. Because ROS production is a universal consequence of cellular metabolism and immune responses, evolutionary animal ecologists have become increasingly interested in involvement of oxidative stress as a proximate mechanism responsible for the emergence of trade-offs related to the evolution of life-history and signal traits. Among the most practical problems pertinent to ecological research on oxidative stress is finding a combination of biomarkers of oxidative status that can be applied to typical wild animal models such as small birds, mammals, and reptiles. This study describes covariation and individual consistency of eight parameters of oxidative status in a small passerine bird, wild-caught captive greenfinch (Carduelis chloris). We measured two markers of plasma antioxidant potential--total antioxidant capacity (TAC) and oxygen radical absorbance (OXY)--and concentrations of one lipophilic (carotenoids) and two hydrophilic (uric acid and ascorbate) antioxidants in plasma. We also measured total glutathione (GSH) concentration and superoxide dismutase (SOD) activity in erythrocytes. Oxidative damage was assessed on the basis of plasma malondialdehyde (MDA) levels, measured by high-performance liquid chromatography. Plasma carotenoids, TAC, and erythrocyte GSH showed significant individual consistency over an 8-d period, indicating that those variables reflected more persistent differences between individuals than plasma OXY, MDA, and uric acid. We did not detect any strong or moderate correlations between the studied parameters, which suggests that all of these biomarkers contain potentially unique information. Injection of a synthetic mimetic of SOD and catalase--EUK-134--did not affect any of the parameters of oxidative status. Capability of phagocytes to produce oxidative burst was not associated with MDA, indicating that under our experimental conditions, ROS production by phagocytes was not a strong determinant of oxidative damage. Altogether these findings suggest that attempts to characterize oxidative balance should use a wide range of biomarkers, and further studies of oxidative status in wild animals may benefit from the experimental induction of oxidative stress.

Coccidian infection causes oxidative damage in greenfinches.

The main tenet of immunoecology is that individual variation in immune responsiveness is caused by the costs of immune responses to the hosts. Oxidative damage resulting from the excessive production of reactive oxygen species during immune response is hypothesized to form one of such costs. We tested this hypothesis in experimental coccidian infection model in greenfinches Carduelis chloris. Administration of isosporan coccidians to experimental birds did not affect indices of antioxidant protection (TAC and OXY), plasma triglyceride and carotenoid levels or body mass, indicating that pathological consequences of infection were generally mild. Infected birds had on average 8% higher levels of plasma malondialdehyde (MDA, a toxic end-product of lipid peroxidation) than un-infected birds. The birds that had highest MDA levels subsequent to experimental infection experienced the highest decrease in infection intensity. This observation is consistent with the idea that oxidative stress is a causative agent in the control of coccidiosis and supports the concept of oxidative costs of immune responses and parasite resistance. The finding that oxidative damage accompanies even the mild infection with a common parasite highlights the relevance of oxidative stress biology for the immunoecological research.

Dexamethasone inhibits corticosterone deposition in feathers of greenfinches

Corticosterone (CORT) content of feathers is a potent source of information about activation of hypothalamus-pituitary-adrenal (HPA) axis during feather growth, which is used for assessment of well-being and stress history of individuals and populations in avian studies. However, little is known about factors affecting deposition of CORT into feathers and how feather CORT covaries with other markers of stress imposed upon individuals during feather growth. We addressed these questions by measuring CORT levels in feathers of wild-caught greenfinches (Carduelis chloris) brought into captivity. One tail feather was removed from all the birds upon arrival to the laboratory and the CORT levels of replacement feathers, grown in captivity were recorded. The birds were subjected to treatments of immune activation (by injection of phytohaemagglutinin) and synthetic glucocorticoid (dexamethasone, DEX) administration. Only DEX injection affected feather CORT levels. DEX-injected birds deposited on average 37% less of CORT in their feathers than saline-injected birds. Despite significant effects of DEX and immune activation treatments on differential leukocyte counts, we did not find any correlations between CORT and leukocyte hemoconcentrations or heterophil/lymphocyte ratios (a haematological index of stress), measured at three stages of feather growth. Our findings provide novel evidence that feather CORT levels are sensitive to manipulation of hormonal balance of birds, thereby supporting the diagnostic value of feather CORT measurements. However, we did not find any evidence about covariation between feather CORT and other markers of stress perceived during the period of feather growth. This calls for further research on information content of feather CORT, preferably in experiments manipulating more diverse array of psychological, immunological and abiotic stressors.

Carotenoid intake does not affect immune-stimulated oxidative burst in greenfinches

SUMMARY Carotenoid-based integument colouration is extremely widespread in the animal kingdom. It has been hypothesized that carotenoid colouration is used for communicating the health status of the bearers because carotenoids are efficient immunomodulators or antioxidants. However, the latter argument has been recently debated and the mechanisms by which carotenoids modulate immunity or oxidative balance are poorly known. We performed an experiment on wild-caught captive greenfinches, passerine birds with carotenoid-based plumage colouration, in order to test whether dietary carotenoid supplementation affects immune-stimulated oxidative burst of phagocytes in the whole blood and humoral immune response to a novel antigen, Brucella abortus (BA). Additionally, we tested whether immune stimulation with bacterial lipopolysaccharide (LPS) affects blood carotenoid levels. We thus tested the effects of carotenoids on the oxidative burst of phagocytes under neutral conditions and during in vivo immune challenge. LPS injection depleted plasma carotenoids, indicating involvement of these phytochemicals in the immune response. However, we did not find any evidence that manipulation of carotenoid intake had modulated anti-BA antibody production, LPS-stimulated oxidative burst of phagocytes, or basal levels of circulating reactive oxygen species. This indicates that carotenoid intake does not affect endogenous production of reactive oxygen species by immune cells. This finding is consistent with the view that carotenoids are unlikely to provide a direct link between oxidative stress and colouration. However, it remains to be tested whether the oxidative burst of phagocytes induced in our experiment actually inflicts oxidative damage and whether carotenoids play a role in the attenuation of such potential damages.

Effects of Endotoxin and Psychological Stress on Redox Physiology, Immunity and Feather Corticosterone in Greenfinches

Assessment of costs accompanying activation of immune system and related neuroendocrine pathways is essential for understanding the selective forces operating on these systems. Here we attempted to detect such costs in terms of disruption to redox balance and interference between different immune system components in captive wild-caught greenfinches (Carduelis chloris). Study birds were subjected to an endotoxin-induced inflammatory challenge and temporary exposure to a psychological stressor (an image of a predator) in a 2*2 factorial experiment. Injection of bacterial endotoxin resulted in up-regulation of two markers of antioxidant protection – erythrocyte glutathione, and plasma oxygen radical absorbance (OXY). These findings suggest that inflammatory responses alter redox homeostasis. However, no effect on markers of oxidative damage to proteins or DNA in erythrocytes could be detected. We found no evidence that the endotoxin injection interfered with antibody production against Brucella abortus antigen or the intensity of chronic coccidiosis. The hypothesis of within-immune system trade-offs as a cost of immunity was thus not supported in our model system. We showed for the first time that administration of endotoxin can reduce the level of corticosterone deposited into feathers. This finding suggests a down-regulation of the corticosterone secretion cascade due to an endotoxin-induced immune response, a phenomenon that has not been reported previously. Exposure to the predator image did not affect any of the measured physiological parameters.

Multidimensionality of fear in captive greenfinches ( Carduelis chloris )

Covariation of individual responses to different fear-eliciting situations is expected to manifest in general “fearfulness syndrome.” We tested for the existence of such a syndrome in wild-caught captive greenfinches. We assessed the propensity to give distress calls at handling, latency to feed at the presence of a predator image, and changes in locomotor activity in response to distress calls of conspecifics. Additionally, we measured the frequency of flapping flight movements against cage bars and tendency to damage tail feathers in captivity as indicators of the ability of birds to cope with captive conditions. As a proxy of neuroendocrine activity (an expected covariate of fearfulness), we measured the amount of stress hormone corticosterone deposited into feathers grown during captivity. All the behavioral traits were individually repeatable in time, but there were no correlations between them. Lack of the behavioral syndrome for fearfulness was also revealed by structural equation modelling. The findings of this experiment challenge the concept of a single internal variable responsible for fearfulness and support the proposed multidimensional nature of fear responses.

Investment in a sexual signal results in reduced survival under extreme conditions in the male great tit (Parus major)

Here, we tested for an ecologically valid cost to secondary sexual trait expression in the male great tit (Parus major). We compared the breast stripe area of a sample of males who survived extremely low night time temperatures with a sample who did not survive. Breast stripe area was significantly smaller in surviving males, suggesting a real cost of signalling in terms of survival. The relationship between breast stripe area and survival was moderated by the effects of body condition: Males with larger breast stripes were in poorer condition and hence suffered increased mortality. Finally, we tested for relationships between breast stripe area and body condition, and tarsus asymmetry and immunological parameters (Brucella abortus antibody count, heterophil to lymphocyte ratio, eosinophil count, monocyte count) in surviving males. Only eosinophil count was related to breast stripe area. Our results suggest a survival cost to investment in sexual signals, such that males who invest somatic resources in social signalling risk increased mortality under extreme environmental conditions.

Variation in the Markers of Nutritional and Oxidative State in a Long-Lived Seabird: Associations with Age and Longevity

Age-related declines in life-history traits have been widely observed in free-living animals. Several theories link senescence to oxidative stress. The aim of this study was to measure several widely used markers of oxidative and nutritional state in a long-lived seabird, the common gull (Larus canus), in order to assess the suitability of these markers for describing deterioration in physiological condition associated with chronological age and survival. Associations with longevity and individual consistency of these parameters over the years (repeatability) were also assessed. Senescence in fitness parameters was observed during the study period: in females, laying date and clutch mass were related to bird age in a curvilinear manner, with middle-aged birds breeding earlier and laying heavier eggs. The only parameter associated with aging processes was glutathione concentration in erythrocytes, which was lower in female birds with longer life spans. Of indexes of nutritional state, plasma triglyceride concentration showed a between-individual increase with age, suggesting selective mortality of birds with low levels. Additionally, total plasma protein levels of individual males increased with age. The mostly negative results of this study hint that the commonly used parameters of physiological condition and oxidative state used in this study do not adequately reflect an individual’s long-term health condition. Alternatively, it is possible that in common gulls, senescence occurs in reproductive mechanisms but not in mechanisms responsible for maintaining an organism’s redox balance, consistent with the idea that different aspects of an organism’s physiological condition age at different rates. Significant interannual repeatability was detected in three plasma constituents—carotenoids, uric acid, and total protein—all of which can possibly be linked to variation in dietary habits.