Body fat and circulating leptin levels in the captive short-beaked echidna (Tachyglossus aculeatus).

It is possible that the reproductive strategy of the short-beaked echidna is related to seasonal changes in fat deposition and energy availability, regulated by seasonal changes in endocrine function. We predicted that circulating leptin levels would be directly proportional to adiposity during most of the year, but that a change in this relationship would occur during the pre-breeding season to allow increased fat deposition. To test this hypothesis, we made use of a captive colony of echidnas to describe and quantify changes in fat distribution and the adipostatic hormone leptin. First we assessed seasonal changes in circulating leptin levels, body mass and adiposity for three male and three female adult echidnas maintained on a standard diet. Second, we explored the relationship between circulating leptin levels and increased caloric intake for an additional five adult female echidnas that were provided with supplemented nutrition. Third we visualised fat distribution in male and female adult echidnas using magnetic resonance imaging (MRI) before and after the breeding season, to determine where fat is deposited in this species. For echidnas maintained on the standard diet, there were no seasonal changes in body mass, body fat or plasma leptin levels. However, female echidnas provided with supplemented nutrition had significantly elevated plasma leptin levels during the breeding season, compared to the pre-and post- breeding periods. MRI showed substantial subcutaneous fat depots extending dorso-laterally from the base of the skull to the base of the tail, in both sexes. Pre-breeding season, both sexes had considerable fat deposition in the pelvic/rump region, whilst the female echidna accumulated most fat in the abdominal region. This study shows that male and female echidnas accumulate body fat in the pelvic/rump and the abdominal regions, respectively and that circulating leptin may promote fattening in female echidnas during the breeding season by means of leptin resistance. However, further research is required to evaluate the precise relationship between seasonal changes in leptin and adiposity.

A prenatal acoustic signal of heat reduces a biomarker of chronic stress at adulthood across seasons.

During development, phenotype can be adaptively modulated by environmental conditions, sometimes in the long-term. However, with weather variability increasing under climate change, the potential for maladaptive long-term responses to environmental variations may increase. In the arid-adapted zebra finch, parents emit "heat-calls" when experiencing heat during incubation, which adaptively affects offspring growth in the heat, and adult heat tolerance. This suggests that heat-call exposure may adjust individual phenotype to hot conditions, potentially compromising individual sensitivity to cool weather conditions. To test this hypothesis, we manipulated individual prenatal acoustic and postnatal thermal experiences during development, and sought to assess subsequent chronic responses to thermal fluctuations at adulthood. We thus measured heterophil to lymphocyte (H/L) ratios in adults, when held in outdoor aviaries during two summers and two winters. We found that birds exposed to heat-calls as embryos, had consistently lower H/L ratios than controls at adulthood, indicative of lower chronic stress, irrespective of the season. Nonetheless, in all birds, the H/L ratio did vary with short-term weather fluctuations (2, 5 or 7 days), increasing at more extreme (low and high) air temperatures. In addition, the H/L ratio was higher in males than females. Overall, while H/L ratio may reflect how individuals were being impacted by temperature, heat-call exposed individuals did not show a stronger chronic response in winter, and instead appeared more resilient to thermal variability than control individuals. Our findings therefore suggest that heat-call exposure did not compromise individual sensitivity to low temperatures at adulthood. Our study also reveals that prenatal sound can lead to long-term differences in individual physiology or quality/condition, as reflected by H/L ratios, which are consistent with previously-demonstrated reproductive fitness differences.

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.

Maternally derived avian corticosterone affects offspring genome-wide DNA methylation in a passerine species.

Avian embryos develop in an egg composition which reflects both maternal condition and the recent environment of their mother. In birds, yolk corticosterone (CORT) influences development by impacting pre- and postnatal growth, as well as nestling stress responses and development. One possible mechanism through which maternal CORT may affect offspring development is via changes to offspring DNA methylation. We sought to investigate this, for the first time in birds, by quantifying the impact of manipulations to maternal CORT on offspring DNA methylation. We non-invasively manipulated plasma CORT concentrations of egg-laying female zebra finches (Taeniopygia castanotis) with an acute dose of CORT administered around the time of ovulation and collected their eggs. We then assessed DNA methylation in the resulting embryonic tissue and in their associated vitelline membrane blood vessels, during early development (5 days after lay), using two established methods - liquid chromatography-mass spectrometry (LC-MS) and methylation-sensitive amplification fragment length polymorphism (MS-AFLP). LC-MS analysis showed that global DNA methylation was lower in embryos from CORT-treated mothers, compared to control embryos. In contrast, blood vessel DNA from eggs from CORT-treated mothers showed global methylation increases, compared to control samples. There was a higher proportion of global DNA methylation in the embryonic DNA of second clutches, compared to first clutches. Locus-specific analyses using MS-AFLP did not reveal a treatment effect. Our results indicate that an acute elevation of maternal CORT around ovulation impacts DNA methylation patterns in their offspring. This could provide a mechanistic understanding of how a mother's experience can affect her offspring's phenotype.

Concurrent invasions of European starlings in Australia and North America reveal population-specific differentiation in shared genomic regions.

A species' success during the invasion of new areas hinges on an interplay between the demographic processes common to invasions and the specific ecological context of the novel environment. Evolutionary genetic studies of invasive species can investigate how genetic bottlenecks and ecological conditions shape genetic variation in invasions, and our study pairs two invasive populations that are hypothesized to be from the same source population to compare how each population evolved during and after introduction. Invasive European starlings (Sturnus vulgaris) established populations in both Australia and North America in the 19th century. Here, we compare whole-genome sequences among native and independently introduced European starling populations to determine how demographic processes interact with rapid evolution to generate similar genetic patterns in these recent and replicated invasions. Demographic models indicate that both invasive populations experienced genetic bottlenecks as expected based on invasion history, and we find that specific genomic regions have differentiated even on this short evolutionary timescale. Despite genetic bottlenecks, we suggest that genetic drift alone cannot explain differentiation in at least two of these regions. The demographic boom intrinsic to many invasions as well as potential inversions may have led to high population-specific differentiation, although the patterns of genetic variation are also consistent with the hypothesis that this infamous and highly mobile invader adapted to novel selection (e.g., extrinsic factors). We use targeted sampling of replicated invasions to identify and evaluate support for multiple, interacting evolutionary mechanisms that lead to differentiation during the invasion process.

Longitudinal covariation of testosterone and sperm quality across reproductive stages in the zebra finch.

Birds that breed opportunistically maintain partial activation of reproductive systems to rapidly exploit environmental conditions when they become suitable for breeding. Maintaining reproductive systems outside of a breeding context is costly. For males, these costs are thought to include continual exposure to testosterone. Males of seasonally breeding birds minimise these costs by downregulating testosterone production outside of a breeding context. Opportunistically breeding birds trade off the need to rapidly initiate reproduction with the costs of elevated testosterone production. One way opportunistically breeding males could minimise these costs is through fine scale changes in testosterone production across discrete reproductive stages which have a greater or lesser requirement for active sperm production. Although spermatogenesis broadly depends on testosterone production, whether changes in testosterone levels across the reproductive stages affect sperm quality and production is unknown. Here, we measured testosterone, sperm quality, and body condition in male zebra finches at discrete stages within reproductive bouts (egg laying, incubation, nestling provisioning, and fledging) and across two consecutive reproductive events in captive male zebra finches (Taeniopygia castanotis). We also examined associations between male testosterone, sperm quality/production, body condition, and nestling body condition. We found that testosterone levels varied across discrete reproductive stages with the lowest levels during incubation and the highest following chick fledging. Testosterone levels were positively associated with sperm velocity and the proportion of motile sperm but were not associated with male body condition. We found no associations between paternal body condition, testosterone levels, or sperm traits with nestling body condition (a proxy for the reproductive quality of a male and his partner). This study is the first to show that opportunistically breeding males vary testosterone synthesis and sperm traits at discrete stages within a reproductive event.

Acute exposure to high temperature affects expression of heat shock proteins in altricial avian embryos.

As the world warms, understanding the fundamental mechanisms available to organisms to protect themselves from thermal stress is becoming ever more important. Heat shock proteins are highly conserved molecular chaperones which serve to maintain cellular processes during stress, including thermal extremes. Developing animals may be particularly vulnerable to elevated temperatures, but the relevance of heat shock proteins for developing altricial birds exposed to a thermal stressor has never been investigated. Here, we sought to test whether three stress-induced genes - HSPD1, HSPA2, HSP90AA1 - and two constitutively expressed genes - HSPA8, HSP90B1 - are upregulated in response to acute thermal shock in zebra finch (Taeniopygia guttata) embryos half-way through incubation. Tested on a gradient from 37.5 °C (control) to 45 °C, we found that all genes, except HSPD1, were upregulated. However, not all genes initiated upregulation at the same temperature. For all genes, the best fitting model included a correlate of developmental stage that, although it was never significant after multiple-test correction, hints that heat shock protein upregulation might increase through embryonic development. Together, these results show that altricial avian embryos are capable of upregulating a known protective mechanism against thermal stress, and suggest that these highly conserved cellular mechanisms may be a vital component of early developmental protection under climate change.

Transcript- and annotation-guided genome assembly of the European starling.

The European starling, Sturnus vulgaris, is an ecologically significant, globally invasive avian species that is also suffering from a major decline in its native range. Here, we present the genome assembly and long-read transcriptome of an Australian-sourced European starling (S. vulgaris vAU), and a second, North American, short-read genome assembly (S. vulgaris vNA), as complementary reference genomes for population genetic and evolutionary characterization. S. vulgaris vAU combined 10× genomics linked-reads, low-coverage Nanopore sequencing, and PacBio Iso-Seq full-length transcript scaffolding to generate a 1050 Mb assembly on 6222 scaffolds (7.6 Mb scaffold N50, 94.6% busco completeness). Further scaffolding against the high-quality zebra finch (Taeniopygia guttata) genome assigned 98.6% of the assembly to 32 putative nuclear chromosome scaffolds. Species-specific transcript mapping and gene annotation revealed good gene-level assembly and high functional completeness. Using S. vulgaris vAU, we demonstrate how the multifunctional use of PacBio Iso-Seq transcript data and complementary homology-based annotation of sequential assembly steps (assessed using a new tool, saaga) can be used to assess, inform, and validate assembly workflow decisions. We also highlight some counterintuitive behaviour in traditional busco metrics, and present buscomp, a complementary tool for assembly comparison designed to be robust to differences in assembly size and base-calling quality. This work expands our knowledge of avian genomes and the available toolkit for assessing and improving genome quality. The new genomic resources presented will facilitate further global genomic and transcriptomic analysis on this ecologically important species.

Mitochondria as the powerhouses of sexual selection: Testing mechanistic links between development, cellular respiration, and bird song.

The developmental environment can affect the expression of sexually selected traits in adulthood. The physiological mechanisms that modulate such effects remain a matter of intense debate. Here, we test the role of the developmental environment in shaping adult mitochondrial function and link mitochondrial function to expression of a sexually selected trait in males (bird song). We exposed male zebra finches (Taeniopygia guttata) to corticosterone (CORT) treatment during development. After males reached adulthood, we quantified mitochondrial function from whole red blood cells and measured baseline CORT and testosterone levels, body condition/composition, and song structure. CORT-treated males had mitochondria that were less efficient (FCRL/R) and used a lower proportion of maximum capacity (FCRR/ETS) than control males. Additionally, CORT-treated males had higher baseline levels of CORT as adults compared to control males. Using structural equation modelling, we found that the effects of CORT treatment during development on adult mitochondrial function were indirect and modulated by baseline CORT levels, which are programmed by CORT treatment during development. Developmental treatment also had an indirect effect on song peak frequency. Males treated with CORT during development sang songs with higher peak frequency than control males, but this effect was modulated through increased CORT levels and by a decrease in FCRR/ETS. CORT-treated males had smaller tarsi compared to control males; however, there were no associations between body size and measures of song frequency. Here, we provide the first evidence supporting links between the developmental environment, mitochondrial function, and the expression of a sexually selected trait (bird song).

Prenatal acoustic programming of mitochondrial function for high temperatures in an arid-adapted bird.

Sound is an essential source of information in many taxa and can notably be used by embryos to programme their phenotypes for postnatal environments. While underlying mechanisms are mostly unknown, there is growing evidence for the involvement of mitochondria-main source of cellular energy (i.e. ATP)-in developmental programming processes. Here, we tested whether prenatal sound programmes mitochondrial metabolism. In the arid-adapted zebra finch, prenatal exposure to 'heat-calls'-produced by parents incubating at high temperatures-adaptively alters nestling growth in the heat. We measured red blood cell mitochondrial function, in nestlings exposed prenatally to heat- or control-calls, and reared in contrasting thermal environments. Exposure to high temperatures always reduced mitochondrial ATP production efficiency. However, as expected to reduce heat production, prenatal exposure to heat-calls improved mitochondrial efficiency under mild heat conditions. In addition, when exposed to an acute heat-challenge, LEAK respiration was higher in heat-call nestlings, and mitochondrial efficiency low across temperatures. Consistent with its role in reducing oxidative damage, LEAK under extreme heat was also higher in fast growing nestlings. Our study therefore provides the first demonstration of mitochondrial acoustic sensitivity, and brings us closer to understanding the underpinning of acoustic developmental programming and avian strategies for heat adaptation.

Experimental manipulation of maternal corticosterone: Hormone transfer to the yolk in the zebra finch Taeniopygia guttata.

Maternally-derived hormones affect offspring physiological and behavioural phenotype, plausibly as an adaptive response to maternal environmental conditions. Corticosterone (CORT), the principal avian glucocorticoid produced in response to stress, is recognised as a potential mediator of such maternal reproductive effects. Maternally-derived yolk CORT is implicated in mediating offspring growth and hatchling begging behaviour. However, determining the potential for maternal effects in opportunistic breeders subject to variable environments relies on understanding whether natural variation in maternal circulating hormones may directly impact the embryo during development. Therefore, we tested whether elevated maternal CORT concentrations increase yolk CORT concentrations in zebra finch (Taeniopygia guttata) eggs. We remotely dosed breeding females with biologically-relevant doses of CORT, or the oil vehicle, 0-3 h prior to the predicted time of ovulation, and allowed pairs to produce two clutches, one under each treatment, in a crosswise, balanced design. CORT dosing elevated maternal plasma CORT and increased mean yolk CORT by a factor of 1.75 compared to the egg yolks of control mothers. Importantly, CORT concentrations did not differ between inner and outer layers of yolk. We found no egg lay order effect and maternal CORT dosing did not influence reproductive outputs (clutch initiation date, clutch size or egg mass). Our results confirm the direct impact of biologically-relevant increases in maternal CORT on yolk CORT, providing evidence that maternal CORT concentrations during yolk deposition to the follicle alters embryonic exogenous CORT exposure. Further research is required to determine the impact of maternal CORT on embryonic developmental programming.

Developmental conditions have intergenerational effects on corticosterone levels in a passerine.

The developmental environment can have powerful, canalizing effects that last throughout an animal's life and even across generations. Intergenerational effects of early-life conditions may affect offspring phenotype through changes in the hypothalamic-pituitary-adrenal axis (HPA). However, such effects remain largely untested in altricial birds. Here, we tested the impact of maternal and paternal developmental conditions on offspring physiology and morphology in the zebra finch (Taeniopygia guttata). Specifically, we exposed one generation (F1) to elevated corticosterone (CORT) during development and quantified the impact on offspring (F2) phenotype. We predicted that intergenerational effects would be apparent through effects of parental developmental treatment on offspring body mass, growth, body condition, body composition, and CORT levels. We found an intergenerational impact on CORT levels, such that F2 birds reared by CORT-treated fathers had higher baseline CORT than F2 birds reared by control fathers. This result shows the potential for intergenerational effects on endocrine function, resulting from developmental conditions. We found no effect of parental treatment on F2 body mass, size, or body condition, but we found that the body mass and tarsus length for offspring and parent were correlated. Our study demonstrates the subtle effects of developmental conditions across generations and highlights the importance of distinguishing between maternal and paternal effects when studying intergenerational effects, especially for species with biparental care.

Acoustic developmental programming: a mechanistic and evolutionary framework.

Conditions experienced prenatally, by modulating developmental processes, have lifelong effects on individual phenotypes and fitness, ultimately influencing population dynamics. In addition to maternal biochemical cues, prenatal sound is emerging as a potent alternative source of information to direct embryonic development. Recent evidence suggests that prenatal acoustic signals can program individual phenotypes for predicted postnatal environmental conditions, which improves fitness. Across taxonomic groups, embryos have now been shown to have immediate adaptive responses to external sounds and vibrations, and direct developmental effects of sound and noise are increasingly found. Establishing the full developmental, ecological, and evolutionary impact of early soundscapes will reveal how embryos interact with the external world, and potentially transform our understanding of developmental plasticity and adaptation to changing environments.

Integrating Mitochondrial Aerobic Metabolism into Ecology and Evolution.

Biologists have long appreciated the critical role that energy turnover plays in understanding variation in performance and fitness among individuals. Whole-organism metabolic studies have provided key insights into fundamental ecological and evolutionary processes. However, constraints operating at subcellular levels, such as those operating within the mitochondria, can also play important roles in optimizing metabolism over different energetic demands and time scales. Herein, we explore how mitochondrial aerobic metabolism influences different aspects of organismal performance, such as through changing adenosine triphosphate (ATP) and reactive oxygen species (ROS) production. We consider how such insights have advanced our understanding of the mechanisms underpinning key ecological and evolutionary processes, from variation in life-history traits to adaptation to changing thermal conditions, and we highlight key areas for future research.

Vocal panting: a novel thermoregulatory mechanism for enhancing heat tolerance in a desert-adapted bird.

Animals thriving in hot deserts rely on extraordinary adaptations and thermoregulatory capacities to cope with heat. Uncovering such adaptations, and how they may be favoured by selection, is essential for predicting climate change impacts. Recently, the arid-adapted zebra finch was discovered to program their offspring's development for heat, by producing 'heat-calls' during incubation in hot conditions. Intriguingly, heat-calls always occur during panting; and, strikingly, avian evaporative cooling mechanisms typically involve vibrating an element of the respiratory tract, which could conceivably produce sound. Therefore, we tested whether heat-call emission results from a particular thermoregulatory mechanism increasing the parent's heat tolerance. We repeatedly measured resting metabolic rate, evaporative water loss (EWL) and heat tolerance in adult wild-derived captive zebra finches (n = 44) at increasing air temperatures up to 44 °C. We found high within-individual repeatability in thermoregulatory patterns, with heat-calling triggered at an individual-specific stage of panting. As expected for thermoregulatory mechanisms, both silent panting and heat-calling significantly increased EWL. However, only heat-calling resulted in greater heat tolerance, demonstrating that "vocal panting" brings a thermoregulatory benefit to the emitter. Our findings therefore not only improve our understanding of the evolution of passerine thermal adaptations, but also highlight a novel evolutionary precursor for acoustic signals.

Senescence of song revealed by a long-term study of the Seychelles warbler (Acrocephalus sechellensis).

Senescence is widespread in nature, often resulting in diminishing survival or reproduction with age, but its role in age-dependent variation in sexual traits is often poorly understood. One reason is that few studies of sexual traits consider non-linear relationships with age, or only consider a narrow range of years relative to the life span of the species. Birdsong has evolved to allow assessment of conspecific quality in numerous bird species. Whilst theory and empirical work suggests that song may become more elaborate with age, there are a paucity of long-term studies testing whether song is associated with age or longevity. In particular, the occurrence of song senescence has rarely been demonstrated. Using an exceptional long-term dataset for the Seychelles warbler (Acrocephalus sechellensis), we analysed relationships between male song, age, survival, and longevity. This species is a long-lived songbird with early life increases, followed by senescent declines, in survival and reproduction. The study population (Cousin Island, Seychelles) is a closed population, with no depredation of adults, providing an excellent opportunity to study senescence in free-living animals. We tested whether song traits were related to age at recording, future survival, longevity, and territory quality. We found age-dependent changes in five song traits (duration, maximum frequency, peak frequency of songs, and duration and frequency bandwidth of trills). Relationships with age were quadratic, indicating reversal in the expression of song coinciding with the onset of senescence in reproduction and survival in this species. One song trait (trill bandwidth) had a quadratic relationship with future survival, but no song traits were related to longevity, suggesting age-related patterns were not the result of selective disappearance. Our study provides one of the first examples of functional senescence in song, offering new insights into avian senescence. Late-life declines in avian song, and possibly other sexual traits, may be more common than currently known, and may play a fundamental role in age-dependent changes in reproductive success.

Repeatability of behavior and physiology: No impact of reproductive investment.

Animals have well-documented individual differences in their behaviour, including in their response to stressful stimuli. The physiological bases for the repeatability of these traits has been the focus of much research in recent years, in an attempt to explain the mechanistic drivers for behavioral syndromes. Whilst a range of studies have demonstrated repeatable individual differences in physiological traits, little is known about potential trade-offs between reproductive investment and the physiological responses to subsequent stressors. We therefore sought to test the behavioral and physiological responses of male zebra finches (Taeniopygia guttata) to a novel environment, quantifying a series of repeated "temporal reaction norms" before and after reproduction. Given that reproductive investment is costly both in time and energy, it is likely to affect expression of behavioral and physiological traits. We hypothesised that reproductive investment would impact the consistency of these temporal reaction norms. Specifically, we predicted that individuals which invested more in reproduction would show altered rates of habituation to a stressful stimulus. Therefore, we quantified temporal reaction norm components (i.e., intercept and slope) of two behaviours and metabolic rate (MR) within and among individuals before and after a breeding season. We found that individuals consistently differed in how their locomotor and feeding activity increased upon introduction into a novel environment and also how their MR decreased after being handled and confined within the metabolic chamber. We also found that the slope of the feeding activity reaction norm was negatively correlated with stress-induced corticosterone levels at the within-individual level. Finally, in contrast to our prediction, we found that neither the intercept nor slope of the reaction norms were influenced by the reproductive effort (the number of fledglings produced) displayed by individual males. This suggests that the substantial individual variation in the expression of physiological and behavioural traits is not plastic with respect to the immediate consequences of reproductive investment. This study is the first quantification of metabolic rate reaction norms and their relationships with fitness, which represents an important first step towards understanding the evolutionary significance of instantaneous habituation to stressful and novel situations.

Under the weather: Corticosterone levels in wild nestlings are associated with ambient temperature and wind.

Animals time reproductive events to overlap with periods of favorable environmental conditions. However, weather conditions can be unpredictable. Young animals may be particularly susceptible to extreme weather during sensitive developmental periods. Here, we investigated the effects of adverse weather conditions on corticosterone levels (a hormone linked to the avian stress response) and body condition of wild nestling zebra finches (Taeniopygia guttata). We sought to tease apart the direct versus indirect (i.e. parental) effects of weather on nestling physiology and condition by increasing parental work load with a clutch manipulation experiment. We found that high temperatures were associated with lower levels of restraint-induced corticosterone and high wind speeds were associated with higher levels of baseline corticosterone. We found no associations between weather and nestling body condition. However, clutch manipulation did affect body condition, with nestlings from experimentally enlarged clutches in worse condition compared to nestlings from experimentally reduced clutches. Our findings suggest that weather can directly affect wild nestlings via changes in corticosterone levels. Further research is needed to understand how changes in corticosterone levels affect phenotype and survival in wild nestlings. Understanding how developing animals respond to changes in environmental predictability and extreme weather is vital for understanding the potential for rapid adaptation in the face of changing climatic conditions.