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.

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.

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.

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.

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.

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.

Carotenoid-based coloration predicts both longevity and lifetime fecundity in male birds, but testosterone disrupts signal reliability.

Sexual selection promotes the evolution of conspicuous animal ornaments. To evolve as signals, these traits must reliably express the "quality" of the bearer, an indicator of individual fitness. Direct estimates of individual fitness may include the contribution of longevity and fecundity. However, evidence of a correlation between the level of signal expression and these two fitness components are scarce, at least among vertebrates. Relative fitness is difficult to assess in the wild as age at death and extra-pair paternity rates are often unknown. Here, in captive male red-legged partridges, we show that carotenoid-based ornament expression, i.e., redness of the bill and eye rings, at the beginning of reproductive life predicts both longevity (1-7 years) and lifetime breeding output (offspring number and hatching success). The recently proposed link between the individual capacity to produce red (keto) carotenoid pigments and the efficiency of cell respiration could, ultimately, explain the correlation with lifespan and, indirectly, fecundity. Nonetheless, in males of avian species, carotenoid-based coloration in bare parts is also partially controlled by testosterone. We also manipulated androgen levels throughout life by treating males with testosterone or antiandrogen compounds. Treatments caused correlations between signal levels and both fitness components to disappear, thus making the signals unreliable. This suggests that the evolution of carotenoid-based sexual signals requires a tightly-controlled steroid metabolism.

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.

Sex-Specific Associations between Telomere Dynamics and Oxidative Status in Adult and Nestling Pied Flycatchers.

Oxidative stress can contribute to an acceleration of telomere erosion, leading to cellular senescence and aging. Increased investment in reproduction is known to accelerate senescence, generally resulting in reduced future reproductive potential and survival. To better understand the role played by oxidative status and telomere dynamics in the conflict between maintenance and reproduction, it is important to determine how these factors are related in parents and their offspring. We investigated the relationship between oxidative status and telomere measurements in pied flycatchers (Ficedula hypoleuca). Total antioxidant status (TAS) in plasma, total levels of glutathione in red blood cells (RBCs), and oxidative damage in plasma lipids (malondialdehyde [MDA]) were assessed in both parents and nestlings. Telomeres were measured in RBCs in adults. Our results showed sex differences in oxidative variables in adults that are likely to be mediated by sex steroids, with testosterone and estrogens increasing and reducing, respectively, the production of reactive oxygen and nitrogen species. We found a negative association between telomere length (TL) and MDA in adults in the previous season. Moreover, TL was positively associated with TAS in females, while telomere shortening (ΔTL) correlated positively with MDA in males in the current year. These associations could be reflecting differences between sexes in reproductive physiology. We found a positive correlation between parental ΔTL and nestling MDA, an example of how parental physiological aging could affect offspring quality in terms of oxidative stress that highlights the constraints imposed by higher rates of ΔTL during reproduction and rearing.

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.

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.

Sex-Specific Effects of High Yolk Androgen Levels on Constitutive and Cell-Mediated Immune Responses in Nestlings of an Altricial Passerine.

Avian embryos are exposed to yolk androgens that are incorporated into the egg by the ovulating female. These steroids can affect several aspects of embryo development, often resulting in increases in overall size or the speed of growth of different traits. However, several studies suggest that they also entail immune costs to the offspring. In this study, we explored whether variation in yolk androgen concentration affected several measures of the constitutive and cell-mediated immune axes in the spotless starling (Sturnus unicolor). Using a within-brood design, we injected different doses of androgens (testosterone and androstenedione) into the eggs. Our study showed that experimentally increased yolk androgens led to sex-specific immunosuppression in both the innate and adaptive axes of the immune system. Both cell-mediated immune response (CMI) and lysozyme activity decreased with increasing androgen levels injected into the egg in the case of male nestlings, whereas there were no effects on females. The effects that we found were always linear: no quadratic or threshold patterns were detected. We found no effects of the experimental treatment in hemolysis or agglutination capacity, but these measures were negatively correlated with CMI, suggesting negative correlation among different branches of the immune system. Blood (trypanosomes and hemosporidians) and intestinal (coccidia) parasites were not affected by the experimental increase of yolk androgen levels. Our results show that in our study species yolk androgens induce immunosuppression in some axes of the male nestling immune system. Further studies should analyze the proximate causes for these contrasting effects in different axes of the immune system and the reason for the differential impact on males and females.

Oxidative Stress in Early Life: Associations with Sex, Rearing Conditions, and Parental Physiological Traits in Nestling Pied Flycatchers.

Conditions experienced during juvenile development can affect the fitness of an organism. During early life, oxidative stress levels can be particularly high as a result of the increased metabolism and the relatively immature antioxidant system of the individual, and this may have medium- and long-term fitness consequences. Here we explore variation in levels of oxidative stress measured during early life in relation to sex, rearing conditions (hatching date and brood size), and parental condition and levels of oxidative markers in a wild population of the pied flycatcher (Ficedula hypoleuca) followed for 2 yr. A marker of total antioxidant status (TAS) in plasma and total levels of glutathione (GSH) in red blood cells, as well as a marker of oxidative damage in plasma lipids (malondialdehyde [MDA]), were assessed simultaneously. Our results show that nestling total GSH levels were associated with parental oxidative status, correlating negatively with maternal MDA and positively with total GSH levels of both parents, with a high estimated heritability. This suggests that parental physiology and genes could be determinants for endogenous components of the antioxidant system of the offspring. Moreover, we found that total GSH levels were higher in female than in male nestlings and that hatching date was positively associated with antioxidant defenses (higher TAS and total GSH levels). These results suggest that different components of oxidative balance are related to a variety of environmental and intrinsic--including parental--influencing factors. Future experimental studies must disentangle the relative contribution of each of these on nestling oxidative status and how the resulting oxidative stress at early phases shape adult phenotype and fitness.

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.

Measuring oxidative stress: the confounding effect of lipid concentration in measures of lipid peroxidation.

Lipid peroxidation products are widely used as markers of oxidative damage in the organism. To properly interpret the information provided by these markers, it is necessary to know potential sources of bias and control confounding factors. Here, we investigated the relationship between two indicators of lipid mobilization (circulating levels of triglycerides and cholesterol) and two common markers of oxidative damage (plasma levels of malondialdehyde and hydroperoxides; the latter estimated from the d-ROMs assay kit). The following five avian species were studied: red-legged partridge (Alectoris rufa), zebra finch (Taeniopygia guttata), spotless starling (Sturnus unicolor), marsh harrier (Circus aeroginosus), and Montagu's harrier (Circus pygargus). In all cases, plasma triglyceride levels positively and significantly correlated with lipid peroxidation markers, explaining between 8% and 34% of their variability. Plasma cholesterol, in contrast, showed a significant positive relationship only among spotless starling nestlings and a marginally significant association in zebra finches. These results indicate that lipid peroxidation marker levels covary with circulating lipid levels. We discuss the potential causes and implications of this covariation and recommend that future studies that measure oxidative damage using lipid peroxidation markers report both raw and relative levels (i.e., corrected for circulating triglycerides). Whether the observed pattern also holds for other tissues and in other taxa would deserve further research.

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.

Free and esterified carotenoids in ornaments of an avian species: the relationship to color expression and sources of variability.

Many animal species show ornaments with yellow-orange-red colors produced by carotenoid pigments. Such traits have evolved as reliable signals of individual quality because of the costs inherent to their production or maintenance. In animal tissues, carotenoids are often found combined with free fatty acids, as carotenoid esters, which may confer more stability to coloration than free carotenoids. Surprisingly, the potential relevance of carotenoid esterification in the expression of animal sexual signals has been virtually ignored. Moreover, the sources of variability of esterified carotenoid levels are barely known, because most studies have not quantified their concentrations. Here, carotenoids in the ornaments (bill, eye rings, and legs) of red-legged partridges Alectoris rufa were quantified in their free and esterified forms. Carotenoid ester levels were the best predictors of leg color, whereas the redness of the other traits was better explained by free carotenoids. Nonetheless, total carotenoid levels (the sum of free and esterified forms) were always significantly correlated to redness. Young partridges had lower levels of free and esterified carotenids in the legs than did older individuals. Also, wild animals had higher ester levels and a higher proportion of carotenoids in esterified forms in all traits than did captive partridges. Probable physiological mechanisms explaining these patterns are discussed.