The evolutionary history of the polyQ tract in huntingtin sheds light on its functional pro-neural activities.

Huntington's disease is caused by a pathologically long (>35) CAG repeat located in the first exon of the Huntingtin gene (HTT). While pathologically expanded CAG repeats are the focus of extensive investigations, non-pathogenic CAG tracts in protein-coding genes are less well characterized. Here, we investigated the function and evolution of the physiological CAG tract in the HTT gene. We show that the poly-glutamine (polyQ) tract encoded by CAGs in the huntingtin protein (HTT) is under purifying selection and subjected to stronger selective pressures than CAG-encoded polyQ tracts in other proteins. For natural selection to operate, the polyQ must perform a function. By combining genome-edited mouse embryonic stem cells and cell assays, we show that small variations in HTT polyQ lengths significantly correlate with cells' neurogenic potential and with changes in the gene transcription network governing neuronal function. We conclude that during evolution natural selection promotes the conservation and purity of the CAG-encoded polyQ tract and that small increases in its physiological length influence neural functions of HTT. We propose that these changes in HTT polyQ length contribute to evolutionary fitness including potentially to the development of a more complex nervous system.

Perinatal variation and covariation of oxidative status and telomere length in yellow-legged gull chicks.

The perinatal period is critical to survival and performance of many organisms. In birds, rapid postnatal growth and sudden exposure to aerial oxygen around hatching markedly affect the chick redox status, with potentially negative consequences on physiology mediated by oxidative stress. In addition, telomere length (TL) undergoes reduction during birds' early life, partly depending on oxidative status. However, relatively few studies have focused specifically on the changes in oxidative status and TL that occur immediately after hatching. In this study of the yellow-legged gull Larus michahellis, we found that chicks undergo a marked increase in plasma total antioxidant capacity and a marked decrease in the concentration of pro-oxidant molecules during the first days after hatching. In addition, TL in erythrocytes decreased by 1 standard deviation over the 4 days post-hatching. Body mass and tarsus length covaried with total antioxidant capacity and concentration of pro-oxidants in a complex way, that partly depended on sex and laying order, suggesting that oxidative status can affect growth. Moreover, TL positively covaried with the concentration of pro-oxidant molecules, possibly because retention of high concentrations of pro-oxidant molecules results from mechanisms of prevention of their negative effects, including reduction in TL. Thus, this study shows that chicks undergo marked variation in oxidative status, which predicts growth and subsequent TL, prompting for more studies of the perinatal changes in the critical post-hatching stages.

Predation risk affects egg mass but not egg steroid hormone concentrations in yellow-legged gulls.

Predators have both direct, consumptive effects on their prey and non-lethal effects on physiology and behavior, including reproductive decisions, with cascading effects on prey ecology and evolution. Here, we experimentally tested such non-lethal effects of exposure to increased predation risk on clutch size, egg mass, and the concentration of yolk steroid hormones in the yellow-legged gull Larus michahellis. We simulated increased predation risk by displaying stuffed predators (adult fox Vulpes vulpes, and adult buzzard Buteo buteo) to breeding adults before egg laying. The concentration of corticosterone, which has been shown to increase under exposure to maternal predation risk in other species, and of testosterone did not differ between eggs from mothers exposed to the predators and eggs from control mothers (i.e., eggs exposed to a novel object of similar size and position to the stuffed predators). The concentration of the two hormones negatively covaried. Clutch size did not vary according to experimental treatment, whereas egg mass was markedly larger in clutches from nests exposed to predators than in clutches from control nests. By increasing egg mass, mothers may reduce the risk of cooling of the eggs when incubation is impeded by predators, boost energy reserves, reduce post-natal detectability caused by food solicitation, and/or enhance development at hatching, thus increasing the chances of offspring survival. In general, our results are inconsistent with most of the few previous studies on similar non-lethal predator effects and suggest that such effects may vary among species according to ecological conditions, social behavior, and developmental mode.

Egg Testosterone Differentially Affects Telomere Length in Somatic Tissues of Yellow-Legged Gull Embryos.

Maternal decisions on egg composition have major consequences for offspring. Maternal egg androgens have diverse, often contrasting, effects depending on offspring trait and life stage, suggesting that mothers face trade-offs in egg hormone transfer. However, the effect of egg androgens on embryonic telomere length, which is a major trait potentially affecting performance, has been never investigated. We administered a physiological dose of testosterone (T) to yellow-legged gull (Larus michahellis) eggs and found that, compared to controls, telomere length shortly before hatching was reduced in the liver but unaffected in the brain, heart, and pectoralis muscle. Telomere length varied across somatic tissues, and, independent of egg treatment, it was not correlated between them, suggesting independent telomere dynamics. Thus, we showed for the first time that increased egg T can increase telomere shortening in the embryo and that maternal T allocation strategies may evolve also in response to such effect. Moreover, contrary to observations in adult birds, at the embryonic stage telomere length in one somatic tissue may not reflect telomere length in other body districts.

Biochemical and behavioral effects induced by cocaine exposure to Daphnia magna.

Illicit drugs and their metabolites have been identified as emerging aquatic pollutants. Cocaine (COC) is one of the most used illicit drug worldwide. After human consumption, COC enters the aquatic ecosystems, where it is commonly detected in ng L-1 concentration range. Although a number of studies have shown that the exposure to environmental concentrations of COC can induce diverse biochemical, molecular and histological effects on aquatic organisms, the information of COC-induced behavioral alterations is scant. Thus, the present study aimed at exploring both biochemical and behavioral effects induced by the exposure to two environmental concentrations (50 ng L-1 and 500 ng L-1) of COC on the freshwater cladoceran Daphnia magna. Specimens were exposed to selected COC concentrations for 21 days and the effects on the oxidative status, including the amount of reactive oxygen species and the activity of antioxidant (SOD, CAT and GPx) and detoxifying (GST) enzymes, and swimming activity were investigated after 7, 14 and 21 days of treatment, while effects on reproductive success was assessed after 21-days only.. Exposure to COC induced an overproduction of reactive oxygen species and a modulation of the activity of defense enzymes. Moreover, COC affected the swimming behavior and altered the reproductive success of treated specimens. Our results highlighted that environmental concentrations of COC can cause adverse effects at different levels of the biological hierarchy in a zooplanktonic species, confirming the potential threat due to this illicit drug for the aquatic community.

Reconstruction of long-distance bird migration routes using advanced machine learning techniques on geolocator data.

Geolocators are a well-established technology to reconstruct migration routes of animals that are too small to carry satellite tags (e.g. passerine birds). These devices record environmental light-level data that enable the reconstruction of daily positions from the time of twilight. However, all current methods for analysing geolocator data require manual pre-processing of raw records to eliminate twilight events showing unnatural variation in light levels, a step that is time-consuming and must be accomplished by a trained expert. Here, we propose and implement advanced machine learning techniques to automate this procedure and we apply them to 108 migration tracks of barn swallows ( Hirundo rustica). We show that routes reconstructed from the automated pre-processing are comparable to those obtained from manual selection accomplished by a human expert. This raises the possibility of fully automating light-level geolocator data analysis and possibly analysing the large amount of data already collected on several species.

Cloacal microbiomes and ecology of individual barn swallows.

Microbiomes can be considered as 'second genomes' for the host, and can deeply affect its physiology, behaviour and fitness. We investigated the cloacal microbiomes (CMs) of adult and nestling barn swallows (Hirundo rustica), a small insectivorous migratory passerine bird, in order to assess whether CM structure was related to major ecological traits of individuals. Illumina sequencing of the V5-V6 hypervariable regions of the bacterial 16S rRNA gene showed that barn swallow CMs were dominated by Proteobacteria, Firmicutes, Actinobacteria, Tenericutes and Bacteroidetes. Nestling CMs were more similar to one another than adult ones, but showed higher alpha diversity. Sibling nestlings had more similar CMs than non-sibling ones. CMs of adult males also differed from those of adult females, but pair members had more similar CMs than expected by chance. In contrast, CMs did not differ between male and female nestlings. Finally, in adults, CMs strongly different from the 'average' CM were associated to lower survival prospects of the host. The CMs of a bird species in the wild are therefore related to important traits of individuals, such as survival, suggesting that microbiomes should be included among the traits examined in ecological studies.

Prenatal independent and combined effects of yolk vitamin E and corticosterone on embryo growth and oxidative status in the yellow-legged gull.

Variation in the concentration of antioxidants and hormones of maternal origin in the eggs of birds can have a profound influence on offspring phenotype both prenatally and postnatally. Egg maternal substances can have interacting effects, but experimental studies of the consequences of the combined variation in the egg concentration of such molecules are extremely rare, particularly as far as prenatal stages are concerned. We manipulated the yolk concentration of vitamin E and corticosterone, which are, respectively, the main antioxidant and the main glucocorticoid hormone in bird eggs, both independently and simultaneously, and we tested their separate and combined effects on growth and oxidative status in the liver and in the brain of yellow-legged gull (Larus michahellis) embryos. Egg supplementation of relatively large physiological doses of corticosterone depressed embryo growth (total body mass, tarsus length and liver mass), whereas administration of vitamin E in association with corticosterone restored normal growth. Vitamin E did not affect embryo growth when administered alone. We further analysed the independent and combined effects of vitamin E and corticosterone on liver and brain total antioxidant capacity, the concentration of reactive oxygen molecules and lipid peroxidation. Vitamin E significantly reduced liver total antioxidant capacity, while corticosterone depressed brain lipid peroxidation. Prenatal exposure to vitamin E and corticosterone appears to have antagonistic effects on body growth, although vitamin E is not limiting in yellow-legged gull eggs. In combination with the results of previous experiments on the same species applying smaller experimental doses or focusing on the postnatal rather than prenatal life stages, our findings indicate that the effects of a physiological increase in the egg concentration of these substances can be life stage and dose specific, implying that generalizing prenatal effects of egg compounds may not be feasible.

Inter-generational resemblance of methylation levels at circadian genes and associations with phenology in the barn swallow.

Regulation of gene expression can occur via epigenetic effects as mediated by DNA methylation. The potential for epigenetic effects to be transmitted across generations, thus modulating phenotypic variation and affecting ecological and evolutionary processes, is increasingly appreciated. However, the study of variation in epigenomes and inter-generational transmission of epigenetic alterations in wild populations is at its very infancy. We studied sex- and age-related variation in DNA methylation and parent-offspring resemblance in methylation profiles in the barn swallows. We focused on a class of highly conserved 'clock' genes (clock, cry1, per2, per3, timeless) relevant in the timing of activities of major ecological importance. In addition, we considerably expanded previous analyses on the relationship between methylation at clock genes and breeding date, a key fitness trait in barn swallows. We found positive assortative mating for methylation at one clock locus. Methylation varied between the nestling and the adult stage, and according to sex. Individuals with relatively high methylation as nestlings also had high methylation levels when adults. Extensive parent-nestling resemblance in methylation levels was observed. Occurrence of extra-pair fertilizations allowed to disclose evidence hinting at a prevalence of paternal germline or sperm quality effects over common environment effects in generating father-offspring resemblance in methylation. Finally, we found an association between methylation at the clock poly-Q region, but not at other loci, and breeding date. We thus provided evidence for sex-dependent variation and the first account of parent-offspring resemblance in methylation in any wild vertebrate. We also showed that epigenetics may influence phenotypic plasticity of timing of life cycle events, thus having a major impact on fitness.

Epistatic mutations under divergent selection govern phenotypic variation in the crow hybrid zone.

The evolution of genetic barriers opposing interspecific gene flow is key to the origin of new species. Drawing from information on over 400 admixed genomes sourced from replicate transects across the European hybrid zone between all-black carrion crows and grey-coated hooded crows, we decipher the interplay between phenotypic divergence and selection at the molecular level. Over 68% of plumage variation was explained by epistasis between the gene NDP and a ~2.8-megabase region on chromosome 18 with suppressed recombination. Both pigmentation loci showed evidence for divergent selection resisting introgression. This study reveals how few, large-effect loci can govern prezygotic isolation and shield phenotypic divergence from gene flow.

Age- and sex-dependent variation in the activity of antioxidant enzymes in the brown trout (Salmo trutta).

Oxidative stress is defined as the imbalance between pro-oxidant and antioxidant molecules in favor of the former and it represents one of the main driving forces of aging. To counteract the harmful effects of oxidative stress, organisms evolved a complex antioxidant system. According to the free radical theory of aging, while the production of reactive oxygen species (ROS) increases with age, the antioxidant defenses decline. Although this relationship has been investigated in diverse vertebrate taxa, the information in fish is scant and inconsistent, particularly for wild populations. Thus, the aim of the present study was the investigation of age- and sex-related changes of the antioxidant enzymes activity in free-living individuals of the brown trout (Salmo trutta). We measured the activity of the main enzymes involved in antioxidant protection, namely superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione S-transferase (GST), as well as lipid peroxidation, in the gills and the liver dissected from brown trout (1+- to 5+-year-old). A significant age-dependent variation in the activity of antioxidant enzymes was noted, with the exception of CAT. GPx activity followed a significant increasing trend with age in both the organs, while SOD decreased in the liver. Increased GST activity was found in the gills only. Lipid peroxidation levels significantly decreased with age in both the organs. SOD and CAT showed sex-dependent differences in the liver of brown trout, with males showing lower enzymatic activity than females. Our data contribute to improve the knowledge on the relationship between antioxidant enzyme activity, aging, and sex in fish.

SMRT long reads and Direct Label and Stain optical maps allow the generation of a high-quality genome assembly for the European barn swallow (Hirundo rustica rustica).

Background: The barn swallow (Hirundo rustica) is a migratory bird that has been the focus of a large number of ecological, behavioral, and genetic studies. To facilitate further population genetics and genomic studies, we present a reference genome assembly for the European subspecies (H. r. rustica). Findings: As part of the Genome10K effort on generating high-quality vertebrate genomes (Vertebrate Genomes Project), we have assembled a highly contiguous genome assembly using single molecule real-time (SMRT) DNA sequencing and several Bionano optical map technologies. We compared and integrated optical maps derived from both the Nick, Label, Repair, and Stain technology and from the Direct Label and Stain (DLS) technology. As proposed by Bionano, DLS more than doubled the scaffold N50 with respect to the nickase. The dual enzyme hybrid scaffold led to a further marginal increase in scaffold N50 and an overall increase of confidence in the scaffolds. After removal of haplotigs, the final assembly is approximately 1.21 Gbp in size, with a scaffold N50 value of more than 25.95 Mbp. Conclusions: This high-quality genome assembly represents a valuable resource for future studies of population genetics and genomics in the barn swallow and for studies concerning the evolution of avian genomes. It also represents one of the very first genomes assembled by combining SMRT long-read sequencing with the new Bionano DLS technology for scaffolding. The quality of this assembly demonstrates the potential of this methodology to substantially increase the contiguity of genome assemblies.

Embryotoxic effects of in-ovo triclosan injection to the yellow-legged gull.

Triclosan (TCS) is an antimicrobial agent used in diverse personal care products that is considered as an emerging contaminant of both aquatic and terrestrial ecosystems. Although TCS aquatic ecotoxicity is well known, information on the presence and effects on terrestrial organisms is still scarce. This study was aimed at exploring the embryotoxicity of TCS to the yellow-legged gull (Larus michahellis) induced by the in-ovo injection of 150 ng TCS/g egg weight. Effects of TCS on embryo morphological traits (i.e. body mass, tarsus length and head size). Moreover, oxidative and genetic effects were assessed in the embryo liver, by measuring the amount of reactive oxygen species (ROS), the activity of antioxidant (superoxide dismutase and catalase) and detoxifying (glutathione S-transferase - GST) enzymes, the levels of lipid peroxidation and DNA fragmentation. After the injection, the concentration of TCS measured in the yolk of unincubated eggs (159 ±â€¯35 ng/g wet weight, ww) was close to the expected concentration. Triclosan was found in residual yolk (2.9 ±â€¯1.1 ng/g ww), liver (2.3 ±â€¯1.1 ng/g ww) and brain (0.2 ±â€¯0.1 ng/g ww) of embryos soon before hatching. Triclosan did not significantly affect embryo morphological traits, while it increased ROS levels and promoted GST activity, inducing the onset of both oxidative and genetic damage. This study demonstrated, for the first time in a wild euriecious bird species with mixed habits, that TCS can be maternally transferred to developing embryos, representing a potential threat for offspring.

Home, dirty home: effect of old nest material on nest-site selection and breeding performance in a cavity-nesting raptor.

The quality of a breeding site may have major fitness consequences. A fundamental step to understanding the process of nest-site selection is the identification of the information individuals use to choose high-quality nest sites. For secondary cavity-nesting bird species that do not add nest lining material, organic remains (faeces, pellets) accumulated inside nest cavities during previous breeding events may be a cue for high-quality nest-sites, as they contain information about past successful breeding and may improve thermal insulation of eggs during incubation. However, cavities in which breeding was successful might also contain more nest-dwelling ectoparasites than unoccupied cavities, offering an incentive for prospective parents to avoid them. We exposed breeding cavity-nesting lesser kestrels (Falco naumanni) to nestbox dyads consisting of a dirty (with a thick layer of organic substrate) and a clean nestbox (without organic material). Dirty nestboxes were strongly preferred, being occupied earlier and more frequently than clean ones. Hatching success in dirty nestboxes was significantly higher than in clean ones, suggesting a positive effect of organic nest material on incubation efficiency, while nestbox dirtiness did not significantly affect clutch and brood size. Nestlings from dirty nestboxes had significantly higher ectoparasite load than those from clean nestboxes soon after egg hatching, but this difference was not evident a few days later. Nest substrate did not significantly affect nestling growth. We concluded that nest substrate is a key driver of nest-site choice in lesser kestrels, although the adaptive value of such a strong preference appears elusive and may be context-dependent.

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

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.

High-ranking alleviates male local competition in lek mating systems.

Territoriality entails demanding social interactions with competing individuals, typically males. Variation in quality of males can be predicted to affect the spatial arrangement of territories. We present a model aimed at understanding the spatial properties of territories on leks, where the presence of a hierarchy in a population of males leads to the clustering of individuals around high-ranking 'hotshot' males. The hierarchy results in a decrease in the number of nearest neighbors interacting directly with high-ranking males, with potential socio-sexual benefits for such males.

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.

Matching geographical assignment by stable isotopes with African non-breeding sites of barn swallows Hirundo rustica tracked by geolocation.

Knowledge on whereabouts within the annual cycle of migratory species is prerequisite for many aspects in ecology and biological conservation. Spatial assignments of stable isotopes archived in tissues allows for later inference on sites where the specific tissue had been grown. It has been rarely tested whether spatial assignments match directly tracked non-breeding residences, especially for migratory songbirds. We here compare assignments of stable isotopes from feathers of Palaearctic Barn swallows Hirundo rustica with their African non-breeding residence sites tracked by geolocation.Assignments based on δ2H, δ13C and δ15N isotope compositions delineate three main non-breeding regions: a main cluster in central Africa, a second in West Africa, and the third cluster in Northern Africa. Using δ13C, δ15N only, non-breeding sites ranged from clusters in West/Southwest Africa to South East Africa with a centre in Central Africa. The non-breeding areas (50% and 75% Kernel density estimates, KDE) of the birds tracked by geolocation stretched from West Africa via central Africa to southern Africa. We found little overlap of 0.3% (assuming a 1:1 odds ratio) to 1.4% (3:1 odds ratio) in the three element assignments and KDEs for only 2 and 13 individuals out of 32 birds. Assignment maps for two elements (δ13C, δ15N) and KDEs showed higher consistencies with an overlap of 3.6 and 8.5% for 12 and 18 birds. We argue that the low matching between stable isotope assignments and non-breeding sites in our study arise from insufficient baseline data for Africa (concerning both isoscapes and specific discrimination functions). However, other factors like aerial foraging habit of the species, and a potential mismatch of non-breeding site location and the spatial origin of aerial plankton might further hamper accurate assignments. Finally we call for concerted analyses of tissues i.e. feathers and claws of birds which are grown at known sites across the continent and from species with various ecological requirements (diverse habitats, foraging behaviours, and diet compositions) to establish isoscapes for general applicability.

Barn swallows long-distance migration occurs between significantly temperature-correlated areas.

Organisms are routinely confronted with crucial decisions on the best time and place to perform fundamental activities. However, unpredictable spatio-temporal variation in ecological factors makes life-history optimization difficult particularly for long-distance migrants, which are putatively blind of conditions thousands of kilometers and weeks ahead along their journey. Here we challenge, on a hierarchy of geographical scales, the common wisdom that migratory birds have no clue to ecological conditions at destination. Using ringing data of the inter-continental migrating barn swallow (Hirundo rustica), we show that temperatures at breeding sites and at times of arrival from migration are more correlated with those at actual wintering sites and at times of departure than with those at other sites and at periods before/after departure. Hence, individual swallows have clues to adjust timing of spring migration based on expected conditions at destination, and they apparently choose wintering sites to increase availability of such information.

Melanin-Based Skin Coloration Predicts Antioxidant Capacity in the Brown Trout (Salmo trutta).

In many vertebrate species, individuals exhibit large variation in the degree of melanin-based coloration on their body. Dark and pale individuals differ in diverse physiological and behavioral traits, suggesting that melanic coloration may reveal individual quality. However, research into the relationships between physiological and skin traits, in terms of melanin-based skin coloration, in wild fish is scant. Our correlative study aimed at investigating the relationships between physiology and melanin-based coloration of the skin of free-living brown trout (Salmo trutta Linnaeus, 1758). We scrutinized the relationships between body condition (body mass and Fulton's K condition factor), oxidative status (plasma total antioxidant capacity and amount of pro-oxidant molecules), and the degree of melanin-based skin coloration assessed by digital photography and image analysis. We found heavier fish to be, on average, darker colored than paler conspecifics. Moreover, a significant covariation between plasma total antioxidant capacity and melanic coloration was noted. Our findings suggest that the melanin-based coloration of brown trout serves as a signal to communicate a better antioxidant defense to conspecifics.