Corticosterone and glucose are correlated and show similar response patterns to temperature and stress in a free-living bird.

Glucocorticoid (GC) hormones have traditionally been interpreted as indicators of stress, but the extent to which they provide information on physiological state remains debated. GCs are metabolic hormones that amongst other functions ensure increasing fuel (i.e. glucose) supply on the face of fluctuating energetic demands, a role often overlooked by ecological studies investigating the consequences of GC variation. Furthermore, because energy budget is limited, in natural contexts where multiple stimuli coexist, the organisms ability to respond physiologically may be constrained when multiple triggers of metabolic responses overlap in time. Using free-living spotless starling (Sturnus unicolor) chicks, we experimentally tested whether two stimuli of different nature known to trigger a metabolic or GC response respectively cause a comparable increase in plasma GCs and glucose. We further tested whether response patterns differed when both stimuli occurred consecutively. We found that both experimental treatments caused increases in GCs and glucose of similar magnitude, suggesting that both variables fluctuate along with variation in energy expenditure, independently of the trigger. Exposure to the two stimuli occurring subsequently did not cause a difference in GC or glucose responses compared to exposure to a single stimulus, suggesting a limited capacity to respond to an additional stimulus during an ongoing acute response. Lastly, we found a positive and significant correlation between plasma GCs and glucose after the experimental treatments. Our results add-up to the increasing research on the role of energy expenditure on GC variation, by providing experimental evidence on the association between plasma GCs and energy metabolism.

Male starling floaters preferentially visit nests of males with reduced resource holding potential.

Floaters are sexually mature individuals that are not able to reproduce by defending breeding resources. Floaters often visit active nests, probably to gather public information or to compete for a nesting site. We tested the hypothesis that floaters preferentially prospect nests in which they have a better chance of taking over, and that they do so by assessing the owners' resource holding potential (RHP). We manipulated the flight capacity of male and female breeders in a population of spotless starlings (Sturnus unicolor) by clipping two flight feathers per wing before egg laying, thus increasing their wing-load and likely impairing their condition. We subsequently monitored breeder and floater activity by means of transponder readers during the nestling period. We found that nests owned by wing-clipped males were visited by a greater number of male floaters than control nests. This effect was absent in the case of wing-clipped females. The number of male floaters also increased with increasing nestling age and number of parental visits. The experiment shows that male floaters preferentially prospect nests in which the owner shows a reduced RHP, a strategy that likely allows them to evict weak owners and take over their nests for future reproductive attempts.

Increased male-induced harm in response to female-limited selection: interactive effects between intra- and interlocus sexual conflict?

Interlocus sexual conflict (IRSC) occurs because of shared interactions that have opposite effects on male and female fitness. Typically, it is assumed that loci involved in IRSC have sex-limited expression and are thus not directly affected by selective pressures acting on the other sex. However, if loci involved in IRSC have pleiotropic effects in the other sex, intersexual selection can shape the evolutionary dynamics of conflict escalation and resolution, as well as the evolution of reproductive traits linked to IRSC loci, and vice versa. Here we used an artificial selection approach in Japanese quail (Coturnix japonica) to test if female-limited selection on reproductive investment affects the amount of harm caused by males during mating. We found that males originating from lines selected for high female reproductive investment caused more oxidative damage in the female reproductive tract than males originating from lines selected for low female reproductive investment. This male-induced damage was specific to the oviduct and not found in other female tissues, suggesting that it was ejaculate-mediated. Our results suggest that intersexual selection shapes the evolution of IRSC and that male-induced harm may contribute to the maintenance of variation in female reproductive investment.

Sexual differences in phenotypical predictors of floating status: body condition influences male but not female reproductive status in a wild passerine.

Floaters constitute the sexually mature but non-breeding part of populations. Despite being ubiquitous in most species, knowledge about floaters is scarce. Ignoring this significant number of individuals may strongly bias our understanding of population dynamics and sexual selection processes. We used the spotless starling (Sturnus unicolor) to examine whether phenotypical and non-phenotypical variables from early and adult life predict reproductive status, focusing on the earliest age at which most individuals start to breed, when the percentage of floaters is the highest. We compiled data from a long-term study involving eight female and seven male cohorts of individuals PIT-tagged at birth. We compared a suite of nestling (condition, hatching date and brood size) and adult variables (condition, size and ornamentation) between floaters and breeders. We found that adult and nestling body condition strongly and positively influenced the likelihood of breeding in males. Path analysis revealed that male reproductive status could only be predicted by considering nestling body condition-the influence of this variable superseded adult body condition. Female reproductive status was only negatively associated with hatching date. Ornamentation was not associated with reproductive status in any of the sexes, although path analyses revealed that body condition was positively associated with throat feather length. We conclude that predictors of reproductive status are sex-specific in the spotless starling, suggesting an important role of body condition in access to breeding resources in males. Our results also highlight the long-term influence of early life on life trajectories and their potential implications on floating status.

Harsh conditions during early development influence telomere length in an altricial passerine: Links with oxidative stress and corticosteroids.

Stress during early development can induce substantial long-term effects in organisms. In the case of birds, despite growth compensations, nestlings reared under harsh conditions typically show reduced survival chances in adulthood. It has been proposed that environmental early-life stressors could affect longevity via effects on telomere length, possibly mediated through oxidative stress. However, the link between these processes is not clear. In this study, we experimentally manipulated brood size in spotless starlings (Sturnus unicolor) to test the causal relationship between early stress, oxidative and corticosterone-mediated stress and telomere shortening. Our results show that experimentally enlarged brood sizes led to a reduction in morphometric development on nestlings, the effect being stronger for females than males. Additionally, basal corticosterone levels increased with increasing brood size in female nestlings. Neither plasma antioxidant status nor malondialdehyde levels (a marker of lipid peroxidation) were affected by experimental brood size, although the levels of a key intracellular antioxidant (glutathione) decreased with increasing brood size. We found that the treatment showed a quadratic effect on nestling telomere lengths: these were shortened either by increases or by decreases in the original brood size. Our study provides experimental evidence for a link between developmental stress and telomere length, but does not support a direct causal link of this reduction with corticosterone or oxidative stress. We suggest that future studies should focus on how telomere length responds to additional markers of allostatic load.

Shape matters: animal colour patterns as signals of individual quality.

Colour patterns (e.g. irregular, spotted or barred forms) are widespread in the animal kingdom, yet their potential role as signals of quality has been mostly neglected. However, a review of the published literature reveals that pattern itself (irrespective of its size or colour intensity) is a promising signal of individual quality across species of many different taxa. We propose at least four main pathways whereby patterns may reliably reflect individual quality: (i) as conventional signals of status, (ii) as indices of developmental homeostasis, (iii) by amplifying cues of somatic integrity and (iv) by amplifying individual investment in maintenance activities. Methodological constraints have traditionally hampered research on the signalling potential of colour patterns. To overcome this, we report a series of tools (e.g. colour adjacency and pattern regularity analyses, Fourier and granularity approaches, fractal geometry, geometric morphometrics) that allow objective quantification of pattern variability. We discuss how information provided by these methods should consider the visual system of the model species and behavioural responses to pattern metrics, in order to allow biologically meaningful conclusions. Finally, we propose future challenges in this research area that will require a multidisciplinary approach, bringing together inputs from genetics, physiology, behavioural ecology and evolutionary-developmental biology.

The role of the mating system and intraspecific brood parasitism in the costs of reproduction in a passerine bird.

Limited resources trigger trade-offs in resource allocation. Reproduction is one of the most demanding activities in terms of energy, and costs related to reproduction can be apparent either as reduced future reproduction or as survival. However, costs are not always detected, because individual heterogeneity in quality and resource acquisition may explain variation in costs among individuals. We conducted a comprehensive study in spotless starlings (Sturnus unicolor) nesting in Central Spain to determine whether costs of reproduction could be detectable in the short and/or long term and whether prenatal or postnatal investment were driving these costs. We took into account the life history of the species, where egg volume is highly repeatable, males are facultatively polygynous, and intraspecific brood parasitism occurs. Females with a high reproductive effort in a breeding event also had a high reproductive effort in the second event of the season. The mating system and maternal age were reliable predictors of breeding success within a breeding event: monogamous and primary females, as well as older mothers, raised more nestlings than secondary females and 1-year-old females, respectively. However, when high-investing females suffered intra-brood parasitism in 1 year, we found a negative correlation between current and future reproduction. These results suggest that, under some circumstances, females are able to skip the short-term costs of reproduction, but when extra effort is added, trade-offs arise. While most studies have focused on different aspects of individual quality on costs, the ecological context seems to represent an important component that should be taken into account.

Diverse dose-response effects of yolk androgens on embryo development and nestling growth in a wild passerine.

Avian egg yolks contain various amounts of maternally derived androgens that can modify offspring phenotype and adjust their development to the post-hatching environment. Seemingly adaptive variation in yolk androgen levels with respect to breeding density conditions or male attractiveness has been found in numerous studies. One important consideration that has been overlooked in previous research is the likely non-linear nature of hormone effects. To examine possible complex dose-response effects of maternal androgens on chick development, we experimentally administered three different androgen doses of the naturally occurring mixture of yolk testosterone and androstenedione to spotless starling eggs (Sturnus unicolor). We found that yolk androgens induce a non-linear dose-response pattern in several traits. Androgens had a stimulatory effect on hatchling body mass and nestling skeletal growth, but maximum values were found at intermediate doses, whereas our highest dose resulted in a decrease. However, the opposite U-shaped effect was found on nestling body mass. We also detected linear negative and positive effects on embryonic development period and nestling gape width, respectively. Our results suggest differential tissue responsiveness to yolk androgens, which may result in compromises in maternal allocation to produce adapted phenotypes. Because of the non-linear dose-response pattern, future investigations should carefully consider a wide range of concentrations, as the balance of costs and benefits may strongly differ depending on concentration.

Nest-dwelling ectoparasites reduce antioxidant defences in females and nestlings of a passerine: a field experiment.

Ectoparasites may imply a cost in terms of oxidative stress provoked by inflammatory responses in hosts. Ectoparasites may also result in costs for nestlings and brooding females because of the direct loss of nutrients and reduced metabolic capacity resulting from parasite feeding activities. These responses may involve the production of reactive oxygen and nitrogen species that may induce oxidative damage in host tissues. Our goal was to examine the effect of ectoparasites in terms of oxidative stress for nestlings and adult females in a population of pied flycatchers Ficedula hypoleuca. We manipulated the entire nest ectoparasite community by reducing ectoparasite loads in some nests through a heating treatment and compared them with a control group of nests with natural loads. A marker of total antioxidant capacity (TAS) in plasma and total levels of glutathione (tGSH) in red blood cells as well as a marker of oxidative damage in plasma lipids (malondialdehyde; MDA) were assessed simultaneously. Levels of tGSH were higher in heat-treated nests than in controls for both females and nestlings. Higher TAS values were observed in females from heat-treated nests. In nestlings there was a negative correlation between TAS and MDA. Our study supports the hypothesis that ectoparasites expose cavity-nesting birds to an oxidative challenge. This could be paid for in the long term, ultimately compromising individual fitness.

Fractal geometry of a complex plumage trait reveals bird's quality.

Animal coloration is key in natural and sexual selection, playing significant roles in intra- and interspecific communication because of its linkage to individual behaviour, genetics and physiology. Simple animal traits such as the area or the colour intensity of homogeneous patches have been profusely studied. More complex patterns are widespread in nature, but they escape our understanding because their variation is difficult to capture effectively by standard, simple measures. Here, we used fractal geometry to quantify inter-individual variation in the expression of a complex plumage trait, the heterogeneous black bib of the red-legged partridge (Alectoris rufa). We show that a higher bib fractal dimension (FD) predicted better individual body condition, as well as immune responsiveness, which is condition-dependent in our study species. Moreover, when food intake was experimentally reduced during moult as a means to reduce body condition, the bib's FD significantly decreased. Fractal geometry therefore provides new opportunities for the study of complex animal colour patterns and their roles in animal communication.

Differential effects of yolk testosterone and androstenedione in embryo development and nestling growth in the spotless starling (Sturnus unicolor).

Yolk androgens in avian eggs play a significant role in embryo and nestling development. However, few studies have examined the differential effect of two of the main yolk androgens, testosterone (T) and androstenedione (A4). Here, we injected eggs of spotless starlings with physiological levels of either T, A4, the combination T+A4 or vehicle substance (control), to examine the differential ability of these steroids to influence nestling development. We found that the duration of the embryonic period was increased by T, and less so by A4, but not by the combination T+A4. Body condition was reduced in all experimental treatments where A4 was present, particularly so in the combination T+A4. Tarsus length was increased in males by A4, and in a lower degree by T, whereas the combination T+A4 inhibited growth. However, these differences in tarsus length between groups disappeared at the end of the nestling period. Cell-mediated immune responsiveness was marginally affected by the interaction between treatment and sex. These patterns suggest that in this species, T has a stronger influence during embryo development than A4, whereas during nestling development the capacities of both androgens to influence growth are similar. The combination T+A4 showed non-additive effects, suggesting either some kind of inhibition between the two androgens, or else an excessive effect due to a bell-shaped pattern of response. Our results suggest a complex picture of sex and age-dependent effects of T and A4, and underline the necessity of further research in the metabolism and action of egg androgens.

Passive and active parental food allocation in a songbird.

Parent-offspring conflict over food allocation can be modeled using two theoretical frameworks: passive (scramble competition) and active choice (signaling) resolution models. However, differentiating between these models empirically can be challenging. One possibility involves investigating details of decision-making by feeding parents. Different nestling traits, related to competitive prowess or signaling cryptic condition, may interact additively or non-additively as predictors of parental feeding responses. To explore this, we experimentally created even-sized, small broods of pied flycatchers and manipulated nestling cryptic quality, independently of size, by vitamin E supplementation. We explored how interactions between nestling cryptic condition, size, signals, and spatial location predicted food allocation and prey-testing by parents. Parents created the potential for spatial scramble competition between nestlings by feeding from and to a narrow range of nest locations. Heavier supplemented nestlings grew faster and were more likely to access profitable nest locations. However, the most profitable locations were not more contested, and nestling turnover did not vary in relation to spatial predictability or food supply. Postural begging was only predicted by nestling hunger and body mass, but parents did not favor heavier nestlings. This suggests that size-mediated and spatial competition in experimental broods was mild. Pied flycatcher fathers allocated food in response to nestling position and begging order, while mothers seemingly followed an active choice mechanism involving assessment of more complex traits, including postural intensity interacting with order, position, and treatment, and perhaps other stimuli when performing prey-testings. Differences in time constraints may underlie sex differences in food allocation rules.

Intergenerational Costs of Oxidative Stress: Reduced Fitness in Daughters of Mothers That Experienced High Levels of Oxidative Damage during Reproduction.

AbstractParental condition transfer effects occur when the parents' physiological state during reproduction affects offspring performance. Oxidative damage may mediate such effects, yet evidence that oxidative damage experienced by parents during reproduction negatively affects offspring fitness is scarce and limited to early life stages. We show in Japanese quail (Coturnix japonica) that maternal levels of oxidative damage, measured during reproduction, negatively predict the number of offspring produced by daughters. This maternal effect on daughters' reproductive success was mediated by an effect on hatching success rather than on the number of eggs laid by daughters. We also observed a negative association between fathers' oxidative damage levels and the number of eggs laid by daughters but a positive association between fathers' oxidative damage levels and the hatching success of those eggs. These opposing paternal effects canceled each other out, resulting in no overall effect on the number of offspring produced by daughters. No significant association between a female's own level of oxidative damage during reproduction and her reproductive success was observed. Our results suggest that oxidative damage experienced by parents is a better predictor of an individual's reproductive performance than oxidative damage experienced by the individual itself. Although the mechanisms underlying these parental condition transfer effects are currently unknown, changes in egg composition or (epi)genetic alterations of gametes may play a role. These findings highlight the importance of an intergenerational perspective when quantifying costs of physiological stress.

Carotenoids in evolutionary ecology: re-evaluating the antioxidant role.

The antioxidant role of carotenoids in the living organism was proposed as a possible basis for the honesty of carotenoid-based signals. However, recent studies have questioned the relevance of carotenoids as powerful antioxidants in vivo. Current evidence does not seem to support the "antioxidant role" hypothesis, but it does not allow us to reject it either. This paper proposes some steps to solve this controversy, such as taking a dynamic approach to antioxidant responses, designing protocols that expose individuals to oxidative challenges, analyzing tissues other than blood, and obtaining measures of antioxidant capacity and oxidative damage simultaneously. However, it should be considered that, irrespective of their antioxidant potential, carotenoids might still give information on oxidative stress levels if they are particularly sensitive to free radicals. Finally, lumping together the immunostimulatory and antioxidant roles of carotenoids should be avoided as these functions are not necessarily associated.

Ontogeny of leukocyte profiles in a wild altricial passerine.

Ecophysiological studies have highlighted the relevance of the avian immune system in individual fitness prospects in the wild. However, studies on the ontogeny of avian immunity are scarce. We analyse age-related changes in the cellular constitutive immunity throughout nestling development, as well as its relationship with sex and brood size. We found that cellular constitutive immunity could be affected by age, sex, brood size, or daily rhythm. Early-stage nestlings relied more on cells of the innate immunity rather than on cells linked to the adaptive immune system. Cellular immunity may not be fully mature in fledglings, as reflected by differences in phagocytic cell counts with regard to adults. Beyond the age-dependent effects, agranulocyte cell counts were affected by sibling competition while granulocyte cell counts showed a daily rhythm. We also show that the heterophil to lymphocyte ratio was negatively related to body weight when nestlings become more independent. Our study contributes knowledge to the fields of developmental immunology and ecological immunology based on essential components of the cellular immune system.

Carotenoid-based coloration predicts resistance to oxidative damage during immune challenge.

Many animal ornaments may have evolved as signals advertising the quality of the bearer. The honesty of the information content of these signals would rely on the costs associated with their expression, these being relatively greater for low-quality than for high-quality individuals. Given the physiological functions of carotenoids, carotenoid-based ornaments could indicate individual immunocompetence, and possibly the ability to mount an immune response at a lower cost. We evaluated whether the red carotenoid-based coloration of male red-legged partridges (Alectoris rufa) predicts the capacity of the individual to counteract the oxidative stress generated by a cell-mediated immune response. Individuals were subcutaneously injected with phytohaemagglutinin (PHA) or phosphate buffer solution (PBS) as a control. We found that eye ring pigmentation predicted the change in the amount of peroxidized lipids (TBARS) in blood after the PHA-induced inflammatory challenge. The degree of pigmentation of this carotenoid-based ornament was also negatively related to individual changes in gamma-glutamyl transferase (GGT), another biomarker of oxidative stress involved in antioxidant metabolism (i.e. glutathione recycling). However, changes in circulating carotenoids did not significantly explain changes in lipid peroxidation or GGT levels, suggesting that the higher resistance to oxidative stress of those individuals with more pigmented eye rings was not directly mediated by their greater circulating levels of carotenoids. Our results indicate that carotenoid-based coloration can predict not only immune responsiveness (more coloured males mount greater responses) but also an individual's ability to counter the oxidative stress generated during immune challenge (more coloured males experience less oxidative damage when mounting an immune response).

Does egg colouration reflect male condition in birds?

How colouration provides information about individuals in birds has been a central issue in recent decades. Although much information has been derived, little is known about the adaptive significance of egg colouration in birds. A recent idea suggests that biliverdin- and porphyrin-pigmented eggs may act as a post-mating sexual signal for males to assess female quality. In birds, it is common for males to influence prelaying female condition by courtship feeding. Using Eurasian kestrels, a species that lays protoporphyrin-pigmented eggs, we descriptively assessed the influence of male feeding on egg pigmentation by considering female phenotype, condition, breeding parameters and male body condition. We found that older females and females with greyer tails (an index of individual quality) produce highly pigmented eggs. However, male body condition was the only variable that explained egg colouration when considered together with the female-related variables. Therefore, females that mated with males in better condition laid highly pigmented eggs. With the same species, we also explored the cost of producing protoporphyrin-pigmented eggs using a food-supply experiment before the laying period. Food supplementation did not increase egg pigmentation, but hatching success and egg mass were positively related to egg colouration only in food supplied pairs. We suggest that egg colouration might be costly to produce and probably suggests egg quality. However, this cost cannot be explained by female quality, but by male condition instead. In general, our results do not support the theory that egg colouration is a post-mating sexual signal in species where males determine female condition at the time of laying.

DNA Integrity Estimated via the Comet Assay Reflects Oxidative Stress and Competitive Disadvantage in Developing Birds.

AbstractIncreases in DNA degradation have been detected in numerous situations in which organisms are exposed to pollutants. However, outside of the ecotoxicological literature, few studies have investigated whether there exists important variation in DNA integrity in free-living, healthy animals. Using the alkaline version of the comet assay to estimate DNA integrity in blood samples, we aimed to evaluate whether DNA integrity during early life is associated with nestlings' age, body mass, within-brood status, and oxidative stress using nestlings from a wild population of spotless starlings (Sturnus unicolor) as a model. We found important levels of variation in DNA integrity, suggesting the possibility that DNA integrity may have implications for offspring fitness. DNA integrity was dependent on the developmental stage, being lower at hatching than at the end of the nestling period. DNA integrity was also negatively related to the levels of oxidative damage at hatching and positively associated with wing length at fledging. In addition, position within the size hierarchy of the brood at fledging explained differences in DNA integrity, with higher levels in core than in marginal nestlings. Finally, despite extensive within-individual variation along nestling's age, we found DNA integrity during early life to be moderately repeatable within broods. Hence, DNA integrity in early life appears to be mainly affected by environmental factors, such as natural stressors. Our results suggest that measuring the variation in DNA integrity may be a fruitful approach for the assessment of individual fitness in natural populations and can be applied to studies in developmental biology and ecology.

Testosterone-mediated trade-offs in the old age: a new approach to the immunocompetence handicap and carotenoid-based sexual signalling.

The immunocompetence handicap hypothesis proposes that testosterone mediates a trade-off between sexual signalling and immunocompetence in males. Such a trade-off could favour the reliability of sexual signals on the basis that testosterone required for signal expression also promotes immunosuppression. However, the immunosuppressive activity of testosterone has not been convincingly demonstrated. We propose that the optimal solution to the testosterone-mediated trade-off should change with age, explaining ambiguous results in the past. Testosterone and ageing would promote two simultaneous immunosuppressive challenges unaffordable for low-quality males. Oxidative stress, as intimately related to ageing and immunosenescence, could contribute to enhance signal reliability. In this context, traits coloured by carotenoids (yellow-red traits) could play a crucial role due to the immunostimulatory and antioxidant properties of these pigments. Here, old and middle-aged male red-legged partridges were treated with testosterone or manipulated as controls. In the presence of high-testosterone levels, middle-aged males increased both circulating carotenoid levels and colour expression, whereas their cell-mediated immunity was not significantly altered. However, in old males, neither circulating carotenoids nor sexual signalling increased when treated with testosterone, but immunosuppression was detected. The link between testosterone and carotenoids could favour the reliability of sexual signals throughout the life.

Honest sexual signalling mediated by parasite and testosterone effects on oxidative balance.

Extravagant ornaments evolved to advertise their bearers' quality, the honesty of the signal being ensured by the cost paid to produce or maintain it. The oxidation handicap hypothesis (OHH) proposes that a main cost of testosterone-dependent ornamentation is oxidative stress, a condition whereby the production of reactive oxygen and nitrogen species (ROS/RNS) overwhelms the capacity of antioxidant defences. ROS/RNS are unstable, very reactive by-products of normal metabolic processes that can cause extensive damage to key biomolecules (cellular proteins, lipids and DNA). Oxidative stress has been implicated in the aetiology of many diseases and could link ornamentation and genetic variation in fitness-related traits. We tested the OHH in a free-living bird, the red grouse. We show that elevated testosterone enhanced ornamentation and increased circulating antioxidant levels, but caused oxidative damage. Males with smaller ornaments suffered more oxidative damage than those with larger ornaments when forced to increase testosterone levels, consistent with a handicap mechanism. Parasites depleted antioxidant defences, caused oxidative damage and reduced ornament expression. Oxidative damage extent and the ability of males to increase antioxidant defences also explained the impacts of testosterone and parasites on ornamentation within treatment groups. Because oxidative stress is intimately linked to immune function, parasite resistance and fitness, it provides a reliable currency in the trade-off between individual health and ornamentation. The costs induced by oxidative stress can apply to a wide range of signals, which are testosterone-dependent or coloured by pigments with antioxidant properties.