Elin Sild

Oxidative stress and life histories: unresolved issues and current needs

Abstract Life‐history theory concerns the trade‐offs that mold the patterns of investment by animals between reproduction, growth, and survival. It is widely recognized that physiology plays a role in the mediation of life‐history trade‐offs, but the details remain obscure. As life‐history theory concerns aspects of investment in the soma that influence survival, understanding the physiological basis of life histories is related, but not identical, to understanding the process of aging. One idea from the field of aging that has gained considerable traction in the area of life histories is that life‐history trade‐offs may be mediated by free radical production and oxidative stress. We outline here developments in this field and summarize a number of important unresolved issues that may guide future research efforts. The issues are as follows. First, different tissues and macromolecular targets of oxidative stress respond differently during reproduction. The functional significance of these changes, however, remains uncertain. Consequently there is a need for studies that link oxidative stress measurements to functional outcomes, such as survival. Second, measurements of oxidative stress are often highly invasive or terminal. Terminal studies of oxidative stress in wild animals, where detailed life‐history information is available, cannot generally be performed without compromising the aims of the studies that generated the life‐history data. There is a need therefore for novel non‐invasive measurements of multi‐tissue oxidative stress. Third, laboratory studies provide unrivaled opportunities for experimental manipulation but may fail to expose the physiology underpinning life‐history effects, because of the benign laboratory environment. Fourth, the idea that oxidative stress might underlie life‐history trade‐offs does not make specific enough predictions that are amenable to testing. Moreover, there is a paucity of good alternative theoretical models on which contrasting predictions might be based. Fifth, there is an enormous diversity of life‐history variation to test the idea that oxidative stress may be a key mediator. So far we have only scratched the surface. Broadening the scope may reveal new strategies linked to the processes of oxidative damage and repair. Finally, understanding the trade‐offs in life histories and understanding the process of aging are related but not identical questions. Scientists inhabiting these two spheres of activity seldom collide, yet they have much to learn from each other.

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.

Heterophil/lymphocyte ratios predict the magnitude of humoral immune response to a novel antigen in great tits (Parus major).

Animals display remarkable individual variation in their capacity to mount immune responses against novel antigens. According to the life-history theory, this variation is caused by the costs of immune responses to the hosts. We studied one of such potential costs, depletion of somatic resources in wintering wild-caught captive passerines, the great tits (Parus major) by immune challenging the birds with a novel antigen, killed Brucella abortus (BA) suspension. We found that despite mild temperature conditions in captivity and ad libitum availability of food, immune challenge depleted somatic resources (as indicated by a body mass loss) and elevated relative proportion of heterophils to lymphocytes (H/L ratio) in the peripheral blood of birds. However, body mass loss did not covary with an increase in H/L ratios between two sampling events, which indicates that these two markers of health state describe different aspects of individual physiological condition. Antibody titres were not associated with the extent of body mass loss during the development of immune response, which shows that the somatic cost of immune response was not proportional to the amount of antibody produced. Birds with high pre-immunisation H/L ratios mounted weaker antibody response, which is indicative of stress-induced suppression of humoral immune response and is consistent with the concept of an antagonistic cross-regulation between different components of the immune system. The latter finding suggests a novel diagnostic value of H/L ratios, which reinforces the utility of this simple haematological index for prediction of the outcomes of complicated immune processes.

On the methodological limitations of detecting oxidative stress: effects of paraquat on measures of oxidative status in greenfinches

SUMMARY Oxidative stress (OS) is widely believed to be responsible for the generation of trade-offs in evolutionary ecology by means of constraining investment into a number of components of fitness. Yet, progress in understanding the true role of OS in ecology and evolution has remained elusive. Interpretation of current findings is particularly hampered by the scarcity of experiments demonstrating which of the many available parameters of oxidative status respond most sensitively to and are relevant for measuring OS. We addressed these questions in wild-caught captive greenfinches (Carduelis chloris) by experimental induction of OS by administration of the pro-oxidant compound paraquat with drinking water. Treatment induced 50% mortality, a significant drop in body mass and an increase in oxidative DNA damage and glutathione levels in erythrocytes among the survivors of the high paraquat (0.2 g l−1 over 7 days) group. Samples taken 3 days after the end of paraquat treatment showed no effect on the peroxidation of lipids (plasma malondialdehyde), carbonylation of proteins (in erythrocytes), parameters of plasma antioxidant protection (total antioxidant capacity and oxygen radical absorbance), uric acid or carotenoids. Our findings of an increase in one marker of damage and one marker of protection from the multitude of measured variables indicate that detection of OS is difficult even under the most stringent experimental induction of oxidative insult. We hope that this study highlights the need for reconsideration of over-simplistic models of OS and draws attention to the limitations of detection of OS due to time-lagged and hormetic upregulation of protective mechanisms. This study also underpins the diagnostic value of measurement of oxidative damage to DNA bases and assessment of erythrocyte glutathione levels.

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.