Cristina Daniela Possenti

Polymorphism at the Clock gene predicts phenology of long-distance migration in birds

Dissecting phenotypic variance in life history traits into its genetic and environmental components is at the focus of evolutionary studies and of pivotal importance to identify the mechanisms and predict the consequences of human‐driven environmental change. The timing of recurrent life history events (phenology) is under strong selection, but the study of the genes that control potential environmental canalization in phenological traits is at its infancy. Candidate genes for circadian behaviour entrained by photoperiod have been screened as potential controllers of phenological variation of breeding and moult in birds, with inconsistent results. Despite photoperiodic control of migration is well established, no study has reported on migration phenology in relation to polymorphism at candidate genes in birds. We analysed variation in spring migration dates within four trans‐Saharan migratory species (Luscinia megarhynchos; Ficedula hypoleuca; Anthus trivialis; Saxicola rubetra) at a Mediterranean island in relation to Clock and Adcyap1 polymorphism. Individuals with larger number of glutamine residues in the poly‐Q region of Clock gene migrated significantly later in one or, respectively, two species depending on sex and whether the within‐individual mean length or the length of the longer Clock allele was considered. The results hinted at dominance of the longer Clock allele. No significant evidence for migration date to covary with Adcyap1 polymorphism emerged. This is the first evidence that migration phenology is associated with Clock in birds. This finding is important for evolutionary studies of migration and sheds light on the mechanisms that drive bird phenological changes and population trends in response to climate change.

Brood size, telomere length, and parent-offspring color signaling in barn swallows

Trade-offs select for optimal allocation of resources among competing functions. Parents are selected to maximize production of viable offspring by balancing between progeny number and “quality.” Telomeres are nucleoproteins, at the ends of eukaryotic chromosomes, that shorten when cells divide. Because shortening below a certain threshold depresses organismal functioning and rate of shortening depends on environmental conditions, telomeres are good candidates as mediators of trade-offs. We altered brood size of barn swallow Hirundo rustica and found that brood enlargement caused a reduction in relative telomere length (RTL). Reliable signals of offspring quality should evolve that mediate adaptive parental care allocation. Because nestlings with darker coloration receive more care, we analyzed the covariation between RTL and coloration and found that RTL increased with plumage darkness, both within and between broods. Hence, we provide unprecedented evidence that signals relevant to parent-offspring communication reflect telomere length and thus offspring reproductive value.

Yolk vitamin E prevents oxidative damage in gull hatchlings

Oxidative stress experienced during early development can negatively affect diverse life-history traits, and organisms have evolved complex defence systems against its detrimental effects. Bird eggs contain maternally derived exogenous antioxidants that play a major role in embryo protection from oxidative damage, including the negative effects on telomere dynamics. In this study on the yellow-legged gull (Larus michahellis), we manipulated the concentration of vitamin E (VE) in the egg yolk and analysed the consequences on oxidative status markers and telomere length in the hatchlings. This study provides the first experimental evidence that, contrary to the expectation, a physiological increase in yolk VE concentration boosted total antioxidant capacity and reduced the concentration of pro-oxidant molecules in the plasma, but did not reduce telomere attrition or ameliorate oxidative damage to proteins and lipids in the early postnatal period.

Clock gene polymorphism, migratory behaviour and geographic distribution: a comparative study of trans‐Saharan migratory birds

Migratory behaviour is controlled by endogenous circannual rhythms that are synchronized by external cues, such as photoperiod. Investigations on the genetic basis of circannual rhythmicity in vertebrates have highlighted that variation at candidate ‘circadian clock’ genes may play a major role in regulating photoperiodic responses and timing of life cycle events, such as reproduction and migration. In this comparative study of 23 trans‐Saharan migratory bird species, we investigated the relationships between species‐level genetic variation at two candidate genes, Clock and Adcyap1, and species’ traits related to migration and geographic distribution, including timing of spring migration across the Mediterranean Sea, migration distance and breeding latitude. Consistently with previous evidence showing latitudinal clines in ‘circadian clock’ genotype frequencies, Clock allele size increased with breeding latitude across species. However, early‐ and late‐migrating species had similar Clock allele size. Species migrating over longer distances, showing delayed spring migration and smaller phenotypic variance in spring migration timing, had significantly reduced Clock (but not Adcyap1) gene diversity. Phylogenetic confirmatory path analysis suggested that migration date and distance were the most important variables directly affecting Clock gene diversity. Hence, our study supports the hypothesis that Clock allele size increases poleward as a consequence of adaptation to the photoperiodic regime of the breeding areas. Moreover, we show that long‐distance migration is associated with lower Clock diversity, coherently with strong stabilizing selection acting on timing of life cycle events in long‐distance migratory species, likely resulting from the time constraints imposed by late spring migration.

Contrasting effects of increased yolk testosterone content on development and oxidative status in gull embryos

ABSTRACT Hormone-mediated maternal effects generate variation in offspring phenotype. In birds, maternal egg testosterone (T) exerts differential effects on offspring traits after hatching, suggesting that mothers experience a trade-off between contrasting T effects. However, there is very little information on T pre-natal effects. In the yellow-legged gull (Larus michahellis), we increased yolk T concentration within physiological limits and measured the effects on development and oxidative status of late-stage embryos. T-treated embryos had a larger body size but a smaller brain than controls. Males had a larger brain than females, controlling for overall size. T treatment differentially affected brain mass and total amount of pro-oxidants in the brain depending on laying order. T-treatment effects were not sex dependent. For the first time in the wild, we show contrasting T pre-natal effects on body mass and brain size. Hence, T may enforce trade-offs between different embryonic traits, but also within the same trait during different developmental periods. Summary: Increased yolk testosterone concentration in yellow-legged gulls promotes body mass growth of embryos but reduces brain size. Moreover, sex differences in brain size exist among control embryos.

Antioxidants and embryo phenotype: is there experimental evidence for strong integration of the antioxidant system?

ABSTRACT Organisms have evolved complex defense systems against oxidative stress. Bird eggs contain maternally derived antioxidants that protect embryos from oxidative damage. The antioxidant system components are thought to be integrated, but few studies have analyzed the covariation between antioxidant concentrations, embryo ‘oxidative status’ and morphology. In addition, no study has tested the effects of experimental change in yolk antioxidant concentration on other antioxidants, on their reciprocal relationships and on their relationships with embryo oxidative status or growth, which are expected if antioxidants defenses are integrated. In yellow-legged gull (Larus michahellis) embryos, we analyzed the covariation between several antioxidants, markers of ‘oxidative status’ [total antioxidant capacity (TAC), concentration of pro-oxidants (TOS), lipid peroxidation (LPO) and protein carbonylation (PC)] in the yolk, liver and brain, and morphology. Yolk and liver antioxidant concentrations were positively correlated reciprocally and with embryo size, and positively predicted TAC but not oxidative status. TOS and LPO were positively correlated in the liver, while TAC and LPO were negatively correlated in the brain. Weak relationships existed between antioxidants and TOS, PC and LPO. The effects of antioxidants on oxidative status and morphology were non-synergistic. An experimental physiological increase in yolk vitamin E had very weak effects on the relationships between other antioxidants or oxidative status and vitamin E concentration, the concentration of other antioxidants or oxidative status; the covariation between other antioxidants and oxidative status, and relationships between morphology or oxidative status and other antioxidants, challenging the common wisdom of strong functional relationships among antioxidants, at least for embryos in the wild. Summary: Weak relationships exist between several yolk antioxidants, oxidative status markers in the yolk, liver and brain, and embryo morphology in the yellow-legged gull.

Effect of yolk corticosterone on begging in the yellow-legged gull

ABSTRACT Behavioral lateralization is widespread across vertebrates. The development of lateralization is affected by both genetic and environmental factors. In birds, maternal substances in the egg can affect offspring lateralization via activational and/or organizational effects. Corticosterone affects the development of brain asymmetry, suggesting that variation in yolk corticosterone concentration may also influence post‐natal behavioral lateralization, a hypothesis that has never been tested so far. In the yellow‐legged gull (Larus michahellis), we increased yolk corticosterone concentration within physiological limits and analyzed the direction of lateralization of hatchlings in reverting from supine to prone position (‘RTP’ response) and in pecking at dummy parental bills to solicit food provisioning (‘begging’ response). We found that corticosterone treatment negatively affected the frequency of begging and it may cause a slight leftward lateralization. However, the direction of lateralization of the RTP response was not affected by corticosterone administration. Thus, our study shows a maternal effect mediated by corticosterone on a behavioral trait involved in parent‐offspring communication during food provisioning events. The findings on lateralization are not conclusive due to the weak effect size but provide information for further ecological and evolutionary studies, investigating mechanisms underlying the development of lateralization. HighlightsThe effect of corticosterone on behavioral lateralization of gull chicks is analyzed.Begging behavior and tonic immobility are tested.Corticosterone affects lateralization of begging behavior.Corticosterone treatment prolongs the duration of tonic immobility.

Yolk vitamin E positively affects prenatal growth but not oxidative status in yellow-legged gull embryos

Abstract Parental effects occur whenever the phenotype of parents or the environment that they experience influences the phenotype and fitness of their offspring. In birds, parental effects are often mediated by the size and biochemical quality of the eggs in terms of maternally transferred components. Exogenous antioxidants are key egg components that accomplish crucial physiological functions during early life. Among these, vitamin E plays a vital role during prenatal development when the intense metabolism accompanying rapid embryo growth results in overproduction of pro-oxidant molecules. Studies of captive birds have demonstrated the positive effect of vitamin E supplementation on diverse phenotypic traits of hatchling and adult individuals, but its effects on embryo phenotype has never been investigated neither in captivity nor under a natural selection regime. In the present study, we experimentally tested the effect of the in ovo supplementation of vitamin E on morphological traits and oxidative status of yellow-legged gull (Larus michahellis) embryos. The supplementation of vitamin E promoted somatic growth in embryos soon before hatching, but did not affect their oxidative status. Our results suggest that maternally transferred vitamin E concentrations are optimized to prevent imbalances of oxidative status and the consequent raise of oxidative damage in yellow-legged gull embryos during prenatal development.

Independent and combined effects of egg pro- and anti-oxidants on gull chick phenotype

ABSTRACT Oviparous mothers transfer to their eggs components that have both independent and combined effects on offspring phenotype. The functional interaction between egg components, such as antioxidants and hormones, suggests that a change in the concentration of one component will have effects on offspring traits that depend on the concentration of other interacting components. However, the combined effects of variation in different egg components are virtually unknown. Bird eggs contain vitamin E, a major antioxidant, and also maternal corticosterone. The independent consequences of variation in the egg concentrations of these compounds for offspring phenotype are largely unknown and no study has investigated their combined effects. We manipulated the concentration of vitamin E and corticosterone in the eggs of the yellow-legged gull (Larus michahellis) by administering a physiological (2 s.d.) dose both independently and in combination. We tested for an effect on chick post-natal growth, plasma antioxidant capacity (TAC) and oxidative compounds (TOS). Separate administration of vitamin E or corticosterone caused a reduction in body mass relative to controls, whereas the combined administration of the two compounds reversed their negative effects. These results suggest that maternal egg components, such as antioxidants and steroid hormones, interact and mothers must balance their concentrations in order to achieve optimal offspring phenotype. The functional relationship between vitamin E and corticosterone is corroborated by the observation of positive covariation between these compounds. Summary: Supplementation of either vitamin E or corticosterone in the egg reduces body mass of gull hatchlings; combined administration of the two compounds nullifies their independent effects.

Physiological increase of yolk testosterone level does not affect oxidative status and telomere length in gull hatchlings

Conditions experienced during early-life can cause the onset of oxidative stress, resulting in pervasive effects on diverse life-history traits, including lifespan. In birds, maternally-transferred egg substances may exert positive or negative influence over the offspring phenotype. Among these, testosterone can upregulate the bioavailability of certain antioxidants but simultaneously promotes the production of pro-oxidants, leading to an oxidative stress situation, which is one of the main forces causing telomere attrition However, no study has investigated the role of this androgen on telomere dynamics in birds and little is known about the effects of yolk testosterone on oxidative status in early-life of these species. We physiologically increased the levels of yolk testosterone by in ovo injections in yellow-legged gull (Larus michahellis) to evaluate the effects induced by this androgen on hatchlings plasma total antioxidant capacity, amount of pro-oxidant molecules and telomere length at hatching. Testosterone supplementation did not increase hatchling body growth, did not result in the overproduction of pro-oxidant molecules nor a reduction of antioxidant capacity. Accordingly, telomere length at hatching was not affected by testosterone treatment, although hatchlings from the third-laid eggs showed shorter telomeres than their siblings from first- and second-laid eggs, independently of testosterone treatment. Our results suggest that injection of physiological levels of testosterone does not induce oxidative stress to hatchlings and, consequently do not affect telomere dynamics during early post-natal periods.