Pre- and postnatal noise directly impairs avian development, with fitness consequences.

Noise pollution is expanding at an unprecedented rate and is increasingly associated with impaired reproduction and development across taxa. However, whether noise sound waves are intrinsically harmful for developing young-or merely disturb parents-and the fitness consequences of early exposure remain unknown. Here, by only manipulating the offspring, we show that sole exposure to noise in early life in zebra finches has fitness consequences and causes embryonic death during exposure. Exposure to pre- and postnatal traffic noise cumulatively impaired nestling growth and physiology and aggravated telomere shortening across life stages until adulthood. Consistent with a long-term somatic impact, early life noise exposure, especially prenatally, decreased individual offspring production throughout adulthood. Our findings suggest that the effects of noise pollution are more pervasive than previously realized.

Neurogenomic insights into the behavioral and vocal development of the zebra finch.

The zebra finch (Taeniopygia guttata) is a socially monogamous and colonial opportunistic breeder with pronounced sexual differences in singing and plumage coloration. Its natural history has led to it becoming a model species for research into sex differences in vocal communication, as well as behavioral, neural and genomic studies of imitative auditory learning. As scientists tap into the genetic and behavioral diversity of both wild and captive lineages, the zebra finch will continue to inform research into culture, learning, and social bonding, as well as adaptability to a changing climate.

Sex identification in embryos and adults of Darwin's finches.

Darwin's finches are an iconic example of adaptive radiation and evolution under natural selection. Comparative genetic studies using embryos of Darwin's finches have shed light on the possible evolutionary processes underlying the speciation of this clade. Molecular identification of the sex of embryonic samples is important for such studies, where this information often cannot be inferred otherwise. We tested a fast and simple chicken embryo protocol to extract DNA from Darwin's finch embryos. In addition, we applied minor modifications to two of the previously reported PCR primer sets for CHD1, a gene used for sexing adult passerine birds. The sex of all 29 tested embryos of six species of Darwin's finches was determined successfully by PCR, using both primer sets. Next to embryos, hatchlings and fledglings are also impossible to distinguish visually. This extends to juveniles of sexually dimorphic species which are yet to moult in adult-like plumage and beak colouration. Furthermore, four species of Darwin's finches are monomorphic, males and females looking alike. Therefore, sex assessment in the field can be a source of error, especially with respect to juveniles and mature monomorphic birds outside of the mating season. We caught 567 juveniles and adults belonging to six species of Darwin's finches and only 44% had unambiguous sex-specific morphology. We sexed 363 birds by PCR: individuals sexed based on marginal sex specific morphological traits; and birds which were impossible to classify in the field. PCR revealed that for birds with marginal sex specific traits, sexing in the field produced a 13% error rate. This demonstrates that PCR based sexing can improve field studies on Darwin's finches, especially when individuals with unclear sex-related morphology are involved. The protocols used here provide an easy and reliable way to sex Darwin's finches throughout ontogeny, from embryos to adults.

Syringeal vocal folds do not have a voice in zebra finch vocal development.

Vocal behavior can be dramatically changed by both neural circuit development and postnatal maturation of the body. During song learning in songbirds, both the song system and syringeal muscles are functionally changing, but it is unknown if maturation of sound generators within the syrinx contributes to vocal development. Here we densely sample the respiratory pressure control space of the zebra finch syrinx in vitro. We show that the syrinx produces sound very efficiently and that key acoustic parameters, minimal fundamental frequency, entropy and source level, do not change over development in both sexes. Thus, our data suggest that the observed acoustic changes in vocal development must be attributed to changes in the motor control pathway, from song system circuitry to muscle force, and not by material property changes in the avian analog of the vocal folds. We propose that in songbirds, muscle use and training driven by the sexually dimorphic song system are the crucial drivers that lead to sexual dimorphism of the syringeal skeleton and musculature. The size and properties of the instrument are thus not changing, while its player is.

Periodic Cooling during Incubation Alters the Adrenocortical Response and Posthatch Growth in Zebra Finches.

AbstractIn birds, incubation temperature is critically deterministic for a range of traits. When parents leave the nest to forage, developing embryos can be exposed to cooling events that represent thermal stress. To investigate the consequences of periodic cooling on offspring development and physiology, we exposed zebra finch embryos to cooling events throughout the incubation period. We then compared embryonic survival, egg mass change, incubation duration, posthatch growth, and adrenocortical response of these individuals with embryos reared at a constant optimal temperature of 37.4°C and embryos reared at a constant suboptimal temperature of 36.4°C, the mean incubation temperature of periodically cooled embryos. There were no differences in embryonic survival or egg mass change during incubation, but individuals exposed to periodic cooling had longer incubation periods than those from the 37.4°C treatment and shorter incubation periods than those from the 36.4°C treatment. Periodically cooled individuals showed slower posthatch growth in comparison with both constant-temperature treatments, but this did not impact adult body size. Treatment groups did not differ in their adrenocortical response, but embryos exposed to periodic cooling and a constant temperature of 37.4°C were able to habituate to repeated capture and restraint stress, while individuals exposed to the constant temperature of 36.4°C were not. These results point to the differential impacts of cooling events versus constant low temperatures during incubation on posthatch growth and physiology and may represent a way for parents to devote less energy toward incubation while still ensuring offspring success.

The potential role of Arhgef33 RhoGEF in foveal development in the zebra finch retina.

The fovea is a pit formed in the center of the retina that enables high-acuity vision in certain vertebrate species. While formation of the fovea fascinates many researchers, the molecular mechanisms underlying foveal development are poorly understood. In the current study, we histologically investigated foveal development in zebra finch (Taeniopygia guttata) and found that foveal pit formation begins just before post-hatch day 14 (P14). We next performed RNA-seq analysis to compare gene expression profiles between the central (foveal and parafoveal) and peripheral retina in zebra finch at P14. We found that the Arhgef33 expression is enriched in the middle layer of the inner nuclear layer at the parafovea, suggesting that Arhgef33 is dominantly expressed in Müller glial cells in the developing parafovea. We then performed a pull-down assay using Rhotekin-RBD and observed GEF activity of Arhgef33 against RhoA. We found that overexpression of Arhgef33 in HEK293 cells induces cell contraction and that Arhgef33 expression inhibits neurite extension in Neuro 2A cells, which is partially recovered by a Rho-kinase (ROCK) inhibitor. Taken together, we used zebra finch as a model animal to investigate foveal development and identified Arhgef33 as a candidate protein possibly involved in foveal development through modulating RhoA activity.

Oxidative Stress Experienced during Early Development Influences the Offspring Phenotype.

AbstractOxidative stress (OS) experienced early in life can affect an individual's phenotype. However, its consequences for the next generation remain largely unexplored. We manipulated the OS level endured by zebra finches (Taeniopygia guttata) during their development by transitorily inhibiting the synthesis of the key antioxidant glutathione ("early-high-OS"). The offspring of these birds and control parents were cross fostered at hatching to enlarge or reduce its brood size. Independent of parents' early-life OS levels, the chicks raised in enlarged broods showed lower erythrocyte glutathione levels, revealing glutathione sensitivity to environmental conditions. Control biological mothers produced females, not males, that attained a higher body mass when raised in a benign environment (i.e., the reduced brood). In contrast, biological mothers exposed to early-life OS produced heavier males, not females, when allocated in reduced broods. Early-life OS also affected the parental rearing capacity because 12-day-old nestlings raised by a foster pair with both early-high-OS members grew shorter legs (tarsus) than chicks from other groups. The results indicate that environmental conditions during development can affect early glutathione levels, which may in turn influence the next generation through both pre- and postnatal parental effects. The results also demonstrate that early-life OS can constrain the offspring phenotype.

Offspring performance is well buffered against stress experienced by ancestors.

Evolution should render individuals resistant to stress and particularly to stress experienced by ancestors. However, many studies report negative effects of stress experienced by one generation on the performance of subsequent generations. To assess the strength of such transgenerational effects we propose a strategy aimed at overcoming the problem of type I errors when testing multiple proxies of stress in multiple ancestors against multiple offspring performance traits, and we apply it to a large observational dataset on captive zebra finches (Taeniopygia guttata). We combine clear one-tailed hypotheses with steps of validation, meta-analytic summary of mean effect sizes, and independent confirmatory testing. We find that drastic differences in early growth conditions (nestling body mass 8 days after hatching varied sevenfold between 1.7 and 12.4 g) had only moderate direct effects on adult morphology (95% confidence interval [CI]: r = 0.19-0.27) and small direct effects on adult fitness traits (r = 0.02-0.12). In contrast, we found no indirect effects of parental or grandparental condition (r = -0.017 to 0.002; meta-analytic summary of 138 effect sizes), and mixed evidence for small benefits of matching environments between parents and offspring, as the latter was not robust to confirmatory testing in independent datasets. This study shows that evolution has led to a remarkable robustness of zebra finches against undernourishment. Our study suggests that transgenerational effects are absent in this species, because CIs exclude all biologically relevant effect sizes.

Developmental polychlorinated biphenyl exposure influences adult zebra finch reproductive behaviour.

Polychlorinated biphenyls (PCBs) are worldwide chemical pollutants that have been linked to disrupted reproduction and altered sexual behaviour in many organisms. However, the effect of developmental PCB-exposure on adult passerine reproductive behaviour remains unknown. A commercial PCB mixture (Aroclor 1242) or an estrogenic congener (PCB 52) were administered in sublethal amounts to nestling zebra finches (Taeniopygia guttata) in the laboratory to identify effects of developmental PCB-exposure on adult zebra finch reproductive parameters. Results indicate that although traditional measures of reproductive success are not altered by this PCB dosage, PCBs do alter sexual behaviours such as male song and nesting behaviour. Males treated with PCB 52 in the nest sang significantly fewer syllables than control males, while females treated with Aroclor 1242 in the nest showed the strongest song preferences. PCB treatment also caused an increase in the number of nesting attempts and abandoned nests in the Aroclor 1242 treatment relative to the PCB 52 treatment, and offspring with control fathers fledged significantly earlier than those with fathers treated with Aroclor 1242. Behavioural differences between males seem to best explain these reproductive effects, most notably aggression. These findings suggest that sublethal PCB-exposure during development can significantly alter key reproductive characteristics of adult zebra finches, likely reducing fitness in the wild.

Developmental conditions modulate DNA methylation at the glucocorticoid receptor gene with cascading effects on expression and corticosterone levels in zebra finches.

Developmental conditions can impact the adult phenotype via epigenetic changes that modulate gene expression. In mammals, methylation of the glucocorticoid receptor gene Nr3c1 has been implicated as mediator of long-term effects of developmental conditions, but this evidence is limited to humans and rodents, and few studies have simultaneously tested for associations between DNA methylation, gene expression and phenotype. Adverse environmental conditions during early life (large natal brood size) or adulthood (high foraging costs) exert multiple long-term phenotypic effects in zebra finches, and we here test for effects of these manipulations on DNA methylation and expression of the Nr3c1 gene in blood. Having been reared in a large brood induced higher DNA methylation of the Nr3c1 regulatory region in adulthood, and this effect persisted over years. Nr3c1 expression was negatively correlated with methylation at 2 out of 8 CpG sites, and was lower in hard foraging conditions, despite foraging conditions having no effect on Nr3c1 methylation at our target region. Nr3c1 expression also correlated with glucocorticoid traits: higher expression level was associated with lower plasma baseline corticosterone concentrations and enhanced corticosterone reactivity. Our results suggest that methylation of the Nr3c1 regulatory region can contribute to the mechanisms underlying the emergence of long-term effects of developmental conditions in birds, but in our system current adversity dominated over early life experiences with respect to receptor expression.

Delta-9-THC exposure during zebra finch sensorimotor vocal learning increases cocaine reinforcement in adulthood.

Zebra finches are songbirds that learn vocal patterns during a sensitive period of development that approximates adolescence. Exposure of these animals to a cannabinoid agonist during their period of sensorimotor vocal learning alters song patterns produced in adulthood. Thus, songbirds have unique value in studying developmental effects of drug exposure on a naturally learned behavior. A missing feature of this animal model has been a method to study drug reinforcement of behavior. To address this gap we have adapted place conditioning methods, used previously to determine that singing behavior is rewarding, to study cocaine reinforcement of behavior. We have found that cocaine dose-dependently reinforces both place conditioning and aversion at potencies consistent with those observed in mammalian species. Use of this place conditioning method has allowed us to determine that, when administered during periods of sensorimotor vocal learning, delta-9-THC, but not nicotine persistently increases sensitivity to cocaine through adulthood. Establishment of this method significantly expands the songbird drug exposure model, and holds promise for better appreciation of mechanisms important to sensorimotor learning that is dependent upon successful progress through sensitive periods of CNS development.

In ovo exposure to brominated flame retardants Part I: Assessment of effects of TBBPA-BDBPE on survival, morphometric and physiological endpoints in zebra finches.

Tetrabromobisphenol A bis(2,3-dibromopropyl) ether (TBBPA-BDBPE) is an additive flame retardant used in polyolefins and polymers. It has been detected in biota, including in avian eggs, yet little is known of its effects. We assessed the pattern of TBBPA-BDBPE concentrations in songbird eggs over the incubation period, and the effects of embryonic exposure to TBBPA-BDBPE in a model songbird species, the zebra finch (Taeniopygia guttata). To assess concentrations during embryo development, eggs were injected on the day they were laid with the vehicle control (safflower oil) or 100 ng TBBPA-BDBPE/g egg, and whole egg contents were collected throughout embryonic development on day 0 (unincubated), 5, 10 and 13. To evaluate effects of embryonic exposure to TBBPA-BDBPE, eggs were injected at Hamburger-Hamilton stage 18 (∼80 h after initiation of incubation) with safflower oil only, 10, 50 or 100 ng TBBPA-BDBPE/g egg (albumin injection volume 1 µl/g). Eggs were monitored for hatching success, and nestlings were monitored for growth and survival. At 15 days post-hatch, tissues were collected to assess physiological effects. TBBPA-BDBPE was incorporated into the egg as the embryo developed, and concentrations started declining in late incubation, suggesting biotransformation by the embryo. There were no effects on hatching success, nestling survival, growth, organ somatic indices, or thyroid hormone homeostasis; however, there was evidence that body condition declined in a dose-dependent manner towards the end of the rapid nestling growth phase. This decreased body condition could be a delayed effect of early developmental exposure, or it may be the result of increased exposure to biotransformation products of TBBPA-BDBPE produced over the nestling period, which are predicted to be more bioaccumulative and toxic than the parent compound.

Early-life social environment predicts social network position in wild zebra finches.

Early-life experience can fundamentally shape individual life-history trajectories. Previous research has suggested that exposure to stress during development causes differences in social behaviour later in life. In captivity, juvenile zebra finches exposed to elevated corticosterone levels were less socially choosy and more central in their social networks when compared to untreated siblings. These differences extended to other aspects of social life, with 'stress-exposed' juveniles switching social learning strategies and juvenile males less faithfully learning their father's song. However, while this body of research suggests that the impacts of early-life stress could be profound, it remains unknown whether such effects are strong enough to be expressed under natural conditions. Here, we collected data on social associations of zebra finches in the Australian desert after experimentally manipulating brood sizes. Juveniles from enlarged broods experienced heightened sibling competition, and we predicted that they would express similar patterns of social associations to stress-treated birds in the captive study by having more, but less differentiated, relationships. We show striking support for the suggested consequences of developmental stress on social network positions, with our data from the wild replicating the same results in 9 out of 10 predictions previously tested in captivity. Chicks raised in enlarged broods foraged with greater numbers of conspecifics but were less 'choosy' and more central in the social network. Our results confirm that the natural range of variation in early-life experience can be sufficient to predict individuals' social trajectories and support theory highlighting the potential importance of developmental conditions on behaviour.

Coupling lifespan and aging? The age at onset of body mass decline associates positively with sex-specific lifespan but negatively with environment-specific lifespan.

Whether lifespan scales to age-associated changes in health and disease is an urgent question in societies with increasing lifespan. Body mass is associated with organismal functioning in many species, and often changes with age. We here tested in zebra finches whether two factors that decreased lifespan, sex and poor environmental quality, accelerated the onset of body mass declines. We subjected 597 birds for nine years to experimentally manipulated foraging costs (harsh = H, benign = B) during development (small vs large brood size) and in adulthood (easy vs hard foraging conditions) in a 2 × 2 design. This yielded four treatment combinations (HH, HB, BH, BB) for each sex. Harsh environments during development and in adulthood decreased average body mass additively. The body mass aging trajectory showed a short steep increase in early adulthood, followed by a plateau and then a decline after 5 years. This decline occurred in all groups except for HB females, which gained mass until death. Surprisingly, the onset of body mass decline was earlier in experimental groups with a longer lifespan. In contrast, the onset of body mass decline was one year earlier in females, which lived two months (4%) shorter than males. Thus, the onset of body mass aging associated positively with the sex-specific differences in lifespan, but negatively with the environmental modulation of lifespan. Thus, body mass aging trajectories did not generally scale to lifespan, and we discuss the possible causes and implications of this finding.

Early Life Stress Strengthens Trait Covariance: A Plastic Response That Results in Reduced Flexibility.

Stress exposure during development can impact both the expression of individual traits and associations between traits, but whether stress results in stronger or weaker associations between traits is unclear. In this study, we examined within- and among-trait associations for morphological and physiological traits in zebra finches (Taeniopygia guttata) exposed to corticosterone (CORT) during the nestling and fledging stages as well as in control birds. Birds exposed to CORT exhibited stronger within-trait correlations over time and stronger associations among traits. We found preliminary evidence that birds that died before the median age of death had stronger within- and among-trait correlations independent of treatment, and among CORT-treated birds, smaller birds were more likely to survive beyond the median age than larger birds. These findings suggest that stress hormone exposure in early life can result in reduced developmental flexibility, with potential fitness ramifications, and that these costs may be greater for larger offspring. Furthermore, our results provide experimental evidence for pleiotropic effects of hormones during development through altered patterns of phenotypic correlation.

A non-coding region near Follistatin controls head colour polymorphism in the Gouldian finch.

Discrete colour morphs coexisting within a single population are common in nature. In a broad range of organisms, sympatric colour morphs often display major differences in other traits, including morphology, physiology or behaviour. Despite the repeated occurrence of this phenomenon, our understanding of the genetics that underlie multi-trait differences and the factors that promote the long-term maintenance of phenotypic variability within a freely interbreeding population are incomplete. Here, we investigated the genetic basis of red and black head colour in the Gouldian finch (Erythrura gouldiae), a classic polymorphic system in which naturally occurring colour morphs also display differences in aggressivity and reproductive success. We show that the candidate locus is a small (approx. 70 kb) non-coding region mapping to the Z chromosome near the Follistatin (FST) gene. Unlike recent findings in other systems where phenotypic morphs are explained by large inversions containing hundreds of genes (so-called supergenes), we did not identify any structural rearrangements between the two haplotypes using linked-read sequencing technology. Nucleotide divergence between the red and black alleles was high when compared to the remainder of the Z chromosome, consistent with their maintenance as balanced polymorphisms over several million years. Our results illustrate how pleiotropic phenotypes can arise from simple genetic variation, probably regulatory in nature.

Influence of early-life nutritional stress on songbird memory formation.

In birds, vocal learning enables the production of sexually selected complex songs, dialects and song copy matching. But stressful conditions during development have been shown to affect song production and complexity, mediated by changes in neural development. However, to date, no studies have tested whether early-life stress affects the neural processes underlying vocal learning, in contrast to song production. Here, we hypothesized that developmental stress alters auditory memory formation and neural processing of song stimuli. We experimentally stressed male nestling zebra finches and, in two separate experiments, tested their neural responses to song playbacks as adults, using either immediate early gene (IEG) expression or electrophysiological response. Once adult, nutritionally stressed males exhibited a reduced response to tutor song playback, as demonstrated by reduced expressions of two IEGs (Arc and ZENK) and reduced neuronal response, in both the caudomedial nidopallium (NCM) and mesopallium (CMM). Furthermore, nutritionally stressed males also showed impaired neuronal memory for novel songs heard in adulthood. These findings demonstrate, for the first time, that developmental conditions affect auditory memories that subserve vocal learning. Although the fitness consequences of such memory impairments remain to be determined, this study highlights the lasting impact early-life experiences can have on cognitive abilities.

Volumetric development of the zebra finch brain throughout the first 200 days of post-hatch life traced by in vivo MRI.

The first months of life are characterized by massive neuroplastic processes that parallel the acquisition of skills and abilities vital for proper functioning in later life. Likewise, juvenile songbirds learn the song sung by their tutor during the first months after hatching. To date, most studies targeting brain development in songbirds exclusively focus on the song control and auditory pathways. To gain a comprehensive insight into structural developmental plasticity of the entire zebra finch brain throughout the different subphases of song learning, we designed a longitudinal study in a group of male (16) and female (19) zebra finches. We collected T2-weighted 3-dimensional anatomical scans at six developmental milestones throughout the process of song learning, i.e. 20, 30, 40, 65, 90 and 120 days post hatching (dph), and one additional time point well after song crystallization, i.e. 200 dph. We observed that the total brain volume initially increases, peaks around 30-40 dph and decreases towards the end of the study. Further, we performed brain-wide voxel-based volumetric analyses to create spatio-temporal maps indicating when specific brain areas increase or decrease in volume, relative to the subphases of song learning. These maps informed (1) that most areas implicated in song control change early, i.e. between 20 and 65 dph, and are embedded in large clusters that cover major subdivisions of the zebra finch brain, (2) that volume changes between consecutive subphases of vocal learning appear highly similar in males and females, and (3) that only more rostrally situated brain regions change in volume towards later ages. Lastly, besides detecting sex differences in local tissue volume that align with previous studies, we uncovered two additional brain loci that are larger in male compared to female zebra finches. These volume differences co-localize with areas related to the song control and auditory pathways and can therefore be associated to the behavioral difference as only male zebra finches sing. In sum, our data point to clear heterochronous patterns of brain development similar to brain development in mammalian species and this work can serve as a reference for future neurodevelopmental imaging studies in zebra finches.

Timing of perineuronal net development in the zebra finch song control system correlates with developmental song learning.

The appearance of perineuronal nets (PNNs) represents one of the mechanisms that contribute to the closing of sensitive periods for neural plasticity. This relationship has mostly been studied in the ocular dominance model in rodents. Previous studies also indicated that PNN might control neural plasticity in the song control system of songbirds. To further elucidate this relationship, we quantified PNN expression and their localization around parvalbumin interneurons at key time-points during ontogeny in both male and female zebra finches, and correlated these data with the well-described development of song in this species. We also extended these analyses to the auditory system. The development of PNN during ontogeny correlated with song crystallization although the timing of PNN appearance in the four main telencephalic song control nuclei slightly varied between nuclei in agreement with the established role these nuclei play during song learning. Our data also indicate that very few PNN develop in the secondary auditory forebrain areas even in adult birds, which may allow constant adaptation to a changing acoustic environment by allowing synaptic reorganization during adulthood.

Early stress priming and the effects on fitness-related traits following an adult stress exposure.

Developmental stressors can have strong effects that persist well into adulthood, and are generally seen as detrimental. However, recent work suggests that a mild developmental stressor can have beneficial effects by preparing the organism to better withstand negative impacts when exposed to high levels of the stressor later in life, also known as a conditioning hormesis. Still, little is known about the influence of such hormetic effects on fitness-related measures. We hypothesized that exposure to a mild stressor during development will protect individuals later in life from the negative effects of a high heat stressor on immune function and reproduction. To test this hypothesis, we subjected zebra finches (Taeniopygia guttata) to a repeated mild heat stressor (38°C) as juveniles for 28 days. As adults, the birds were then exposed to a high heat stressor (42°C) for 3 consecutive days and we examined the effects on immune function via wound healing, and on female reproductive output. We found that females given the mild heat stressor as juveniles healed wounds marginally slower, but also had higher clutch viability than controls. For the adult treatment, we saw that high heat had a stimulatory effect on clutch viability as well. Our findings point toward the occurrence of trade-offs between immune function and reproduction due to a cost of hormetic priming when the adult environment does not match that of early life.