Functional inhibition of deep brain non-visual opsins facilitates acute long day induction of reproductive recrudescence in male Japanese quail.

For nearly a century, we have known that brain photoreceptors regulate avian seasonal biology. Two photopigments, vertebrate ancient opsin (VA) and neuropsin (OPN5), provide possible molecular substrates for these photoreceptor pathways. VA fulfills many criteria for providing light input to the reproductive response, but a functional link has yet to be demonstrated. This study examined the role of VA and OPN5 in the avian photoperiodic response of Japanese quail (Coturnix japonica). Non-breeding male quail were housed under short days (6L:18D) and received an intracerebroventricular infusion of adeno-associated viral vectors with shRNAi that selectively inhibited either VA or OPN5. An empty viral vector acted as a control. Quail were then photostimulated (16L:8D) to stimulate gonadal growth. Two long days significantly increased pituitary thyrotrophin-stimulating hormone ß-subunit (TSHß) and luteinizing hormone ß-subunit (LHß) mRNA of VA shRNAi treated quail compared to controls. Furthermore, at one week there was a significant increase, compared to controls, in both hypothalamic gonadotrophin releasing hormone-I (GnRH-I) mRNA and paired testicular mass in VA shRNAi birds. Opn5 shRNAi facilitated the photoinduced increase in TSHß mRNA at 2 days, but no other differences were identified compared to controls. Contrary to our expectations, the silencing of deep brain photoreceptors enhanced the response of the reproductive axis to photostimulation rather than preventing it. In addition, we show that VA opsin plays a dominant role in the light-dependent neuroendocrine control of seasonal reproduction in birds. Together our findings suggest the photoperiodic response involves at least two photoreceptor types and populations working together with VA opsin playing a dominant role.

Gene expression of sex steroid metabolizing enzymes and receptors in the skeletal muscle of migrant and resident subspecies of white-crowned sparrow (Zonotrichia leucophrys).

Circulating sex steroid concentrations vary dramatically across the year in seasonally breeding animals. The ability of circulating sex steroids to effect muscle function can be modulated by changes in intracellular expression of steroid metabolizing enzymes (e.g., 5α-reductase type 2 and aromatase) and receptors. Together, these combined changes in plasma hormones, metabolizing enzymes and receptors allow for seasonally appropriate changes in skeletal muscle function. We tested the hypothesis that gene expression of sex steroid metabolizing enzymes and receptors would vary seasonally in skeletal muscle and these changes would differ between a migrant and resident life history strategy. We quantified annual changes in plasma testosterone and gene expression in pectoralis and gastrocnemius skeletal muscles using quantitative polymerase chain reaction (qPCR) in free-living migrant (Zonotrichia leucophrys gambelii) and resident (Z. l. nuttalli) subspecies of white-crowned sparrow during breeding, pre-basic molt, and wintering life history stages. Pectoralis muscle profile was largest in migrants during breeding, while residents maintained large muscle profiles year-round. Circulating testosterone peaked during breeding in both subspecies. Pectoralis muscle androgen receptor mRNA expression was lower in females of both subspecies during breeding. Estrogen receptor-α expression was higher in the pectoralis muscle, but not gastrocnemius, of residents throughout the annual cycle when compared to migrants. Pectoralis aromatase expression was higher in resident males compared to migrant males. No differences were observed for 5α-reductase 2. Between these two subspecies, patterns of plasma testosterone and androgen receptors appear to be conserved, however estrogen receptor gene expression appears to have diverged.

Annual regulation of adrenocortical function in migrant and resident subspecies of white-crowned sparrow.

Corticosterone affects physiology and behavior both during normal daily processes but also in response to environmental challenges and is known to mediate life history trade-offs. Many studies have investigated patterns of corticosterone production at targeted times of year, while ignoring underlying annual profiles. We aimed to understand the annual regulation of hypothalamic-pituitary-adrenal (HPA) axis function of both migrant (Zonotrichia leucophrys gambelii; n = 926) and resident (Z. l. nutalli; n = 688) subspecies of white-crowned sparrow and how it is influenced by environmental conditions - wind, precipitation, and temperature. We predicted that more dramatic seasonal changes in baseline and stress-induced corticosterone would occur in migrants to precisely time the onset of breeding and cope with environmental extremes on their arctic breeding grounds, while changes in residents would be muted as they experience a more forgiving breeding schedule and comparatively benign environmental conditions in coastal California. During the course of a year, the harshest conditions were experienced the summer breeding grounds for migrants, at which point they had higher corticosterone levels compared to residents. For residents, the winter months coincided with harshest conditions at which point they had higher corticosterone levels than migrants. For both subspecies, corticosterone tended to rise as environmental conditions became colder and windier. We found that the annual maxima in stress-induced corticosterone occurred prior to egg lay for all birds except resident females. Migrants had much higher baseline and acute stress-induced corticosterone during breeding compared to residents; where in a harsher environment the timing of the onset of reproduction is more critical because the breeding season is shorter. Interestingly, molt was the only stage within the annual cycle in which subspecies differences were absent suggesting that a requisite reduction in corticosterone may have to be met for feather growth. These data suggest that modulation of the HPA axis is largely driven by environmental factors, social cues, and their potential interactions with a genetic program.

Acute restraint stress does not alter corticosteroid receptors or 11ß-hydroxysteroid dehydrogenase gene expression at hypothalamic-pituitary-adrenal axis regulatory sites in captive male white-crowned sparrows (Zonotrichia leucophrys gambelii).

Capture-restraint is often used to investigate the acute hypothalamic-pituitary-adrenal axis (HPA) response to stress in wild and captive animals through the production of glucocorticoids. Although this approach is useful for understanding changes in glucocorticoids, it overlooks potential changes in the complex regulatory systems associated with the glucocorticoid response, including genomic receptors, steroid metabolizing enzymes, carrier proteins, and downstream target proteins (e.g. gonadotropin-inhibitory hormone; GnIH). The present study in captive male white-crowned sparrows (Zonotrichia leucophrys) tests the hypothesis that corticosteroid receptors (mineralocorticoid - MR and glucocorticoid - GR), 11ß-hydroxysteroid dehydrogenase 1 (11ßHSD1) and 2 (11ßHSD2), corticosteroid binding globulin (CBG), and GnIH undergo rapid changes in expression to mediate the glucocorticoid response to acute stress. To determine dynamic changes in gene mRNA expression in the hippocampus, hypothalamus, pituitary gland, and liver, birds were sampled within 3 min of entering the room and after 10, 30, and 60 min of capture restraint stress in a cloth bag. Restraint stress handling increased CBG and decreased GnIH mRNA expression in the liver and hypothalamus, respectively. MR, GR, 11ßHSD1, and 11ßHSD2 mRNA expression in the brain, pituitary gland, and liver did not change. No correlations were found between gene expression and baseline or stress-induced plasma corticosterone levels. No rapid changes of MR, GR, 11ßHSD1, and 11ßHSD2 mRNA expression during a standardized acute restraint protocol suggests that tissue level sensitivity may remain constant during acute stressors. However, the observed rise in CBG mRNA expression could act to facilitate transport to target tissues or buffer the rise in circulating glucocorticoids. Further studies on tissue specific sensitivity are warranted.

Tissue-specific expression of 11ß-HSD and its effects on plasma corticosterone during the stress response.

The hypothalamic-pituitary-adrenal (HPA) axis is under complex regulatory control at multiple levels. Enzymatic regulation plays an important role in both circulating levels of glucocorticoids and target tissue exposure. Three key enzyme pathways are responsible for the immediate control of glucocorticoids. De novo synthesis of glucocorticoid from cholesterol involves a multistep enzymatic cascade. This cascade terminates with 11ß-hydroxylase, responsible for the final conversion of 11-deoxy precursors into active glucocorticoids. Additionally, 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) controls regeneration of glucocorticoids from inactive metabolites, providing a secondary source of active glucocorticoids. Localized inactivation of glucocorticoids is under the control of Type 2 11ß-HSD (11ß-HSD2). The function of these enzymes is largely unexplored in wild species, particularly songbirds. Here, we investigated the contribution of both clearance and generation of glucocorticoids to regulation of the hormonal stress response via the use of pharmacological antagonists. Additionally, we mapped 11ß-HSD gene expression. We found 11ß-HSD1 primarily in liver, kidney and adrenal glands, although it was detectable across all tissue types. 11ß-HSD2 was predominately expressed in the adrenal glands and kidney with moderate gonadal and liver expression. Inhibition of glucocorticoid generation by metyrapone was found to decrease levels peripherally, while both peripheral and central administration of the 11ß-HSD2 inhibitor DETC resulted in elevated concentrations of corticosterone. These data suggest that during the stress response, peripheral antagonism of the 11ß-HSD system has a greater impact on circulating glucocorticoid levels than central control. Further studies should aim to elucidate the respective roles of the 11ß-HSD and 11ß-hydroxylase enzymes.

Contrasting seasonal and aseasonal environments across stages of the annual cycle in the rufous-collared sparrow, Zonotrichia capensis: Differences in endocrine function, proteome and body condition.

The timing and duration of life-history stages (LHSs) within the annual cycle can be affected by local environmental cues which are integrated through endocrine signalling mechanisms and changes in protein function. Most animals express a single LHS within a given period of the year because synchronous expression of LHSs is thought to be too costly energetically. However, in very rare and extremely stable conditions, breeding and moult have been observed to overlap extensively in rufous-collared sparrows (Zonotrichia capensis) living in valleys of the Atacama Desert-one of the most stable and aseasonal environments on Earth. To examine how LHS traits at different levels of organization are affected by environmental variability, we compared the temporal organization and duration of LHSs in populations in the Atacama Desert with those in the semiarid Fray Jorge National Park in the north of Chile-an extremely seasonal climate but with unpredictable droughts and heavy rainy seasons. We studied the effects of environmental variability on morphological variables related to body condition, endocrine traits and proteome. Birds living in the seasonal environment had a strict temporal division of LHSs, while birds living in the aseasonal environment failed to maintain a temporal division of LHSs resulting in direct overlap of breeding and moult. Further, higher circulating glucocorticoids and androgen concentrations were found in birds from seasonal compared to aseasonal populations. Despite these differences, body condition variables and protein expression were not related to the degree of seasonality but rather showed a strong relationship with hormone levels. These results suggest that animals adjust to their environment through changes in behavioural and endocrine traits and may be limited by less labile traits such as morphological variables or expression of specific proteins under certain circumstances. These data on free-living birds shed light on how different levels of life-history organization within an individual are linked to increasing environmental heterogeneity.

Effects of thyroid hormone manipulation on pre-nuptial molt, luteinizing hormone and testicular growth in male white-crowned sparrows (Zonotrichia leuchophrys gambelii).

Most seasonal species rely on the annual change in day length as the primary cue to appropriately time major spring events such as pre-nuptial molt and breeding. Thyroid hormones are thought to be involved in the regulation of both of these spring life history stages. Here we investigated the effects of chemical inhibition of thyroid hormone production using methimazole, subsequently coupled with either triiodothyronine (T3) or thyroxine (T4) replacement, on the photostimulation of pre-nuptial molt and breeding in Gambel's white-crowned sparrows (Zonotrichia leuchophrys gambelii). Suppression of thyroid hormones completely prevented pre-nuptial molt, while both T3 and T4 treatment restored normal patterns of molt in thyroid hormone-suppressed birds. Testicular recrudescence was blocked by methimazole, and restored by T4 but not T3, in contrast to previous findings demonstrating central action of T3 in the photostimulation of breeding. Methimazole and replacement treatments elevated plasma luteinizing hormone levels compared to controls. These data are partially consistent with existing theories on the role of thyroid hormones in the photostimulation of breeding, while highlighting the possibility of additional feedback pathways. Thus we suggest that regulation of the hypothalamic pituitary gonad axis that controls breeding may be more complex than previously considered.

Weathering the storm: Do arctic blizzards cause repeatable changes in stress physiology and body condition in breeding songbirds?

Severe weather events are increasing worldwide because of climate change. To cope with severe weather events, vertebrates rely on the stress response which is activated by the hypothalamic-pituitary adrenal (HPA) axis to adjust physiology and behavior. Previous studies have detailed changes in baseline concentrations of the stress hormone corticosterone during a single storm event, but little data exists on how stress physiology and body condition are adjusted as the storm progresses across multiple days. This represents a serious gap in our understanding of how birds respond physiologically over the duration of a storm. We documented arctic snowstorms that occurred over five consecutive years that were endured by Lapland longspurs (Calcarius lapponicus; 2012-2016) and in three consecutive years by white-crowned sparrows (Zonotrichia leucophrys gambelii; 2014-2016). Data were collected on storm-free days, during snowstorms ranging in length from 1 to 3 days, and the day immediately following a snowstorm. The specific aims were to understand how stress physiology, measured at baseline and in response to restraint handling, and body condition changed over multiple days of the storm, and if these responses were consistent across years. Snowstorms did not affect baseline corticosterone concentrations for either species except for female Lapland longspurs and male white-crowned sparrows in 2014. Lapland longspurs, regardless of sex, increased stress-induced (restraint handling) corticosterone in response to snowstorms in all years but 2013, which was characterized by unusually harsh conditions. Both sexes of White-crowned sparrows showed a significant increase in the stress-induced levels of corticosterone during snowstorms in one of the three years of the study. Stress-induced corticosterone concentrations were only different across each day of the storm in one year of the study for Lapland longspurs. Changes in fat and body mass were not uniform across years, but measurable increases in fat stores and body mass were detected in males of both species during the first day of a snowstorm with declines typically occurring by the second day. Our study showed that severe weather events often caused rapid increases in HPA axis activity and body condition, but these profiles are likely dependent upon ecological and environmental context within the breeding season.

Behavioral and physiological traits of migrant and resident white-crowned sparrows: a common garden approach.

To accommodate a migratory life history, migrants express a greater number of physiological and behavioral stages per annum than residents and are thus considered to have higher finite state diversity (FSD). To investigate the physiological mechanisms and constraints associated with migration, direct comparison of two subspecies of white-crowned sparrow - migrant, Zonotrichia leucophrys gambelii, and resident, Z. l. nuttalli - were made under common garden conditions of photoperiod and housing, as birds progressed from winter through the vernal life history stages. We tested the hypothesis that migrants (higher FSD) respond differently than residents (lower FSD) to the initial predictive cue, photoperiod, to initiate and integrate the progression of vernal stages of prenuptial molt, migration and development of breeding. If differences in vernal phenology were noted, then the basis for the distinctions was considered genetic. Results indicate that (1) residents had a lower threshold to vernal photoperiod with elevations of plasma androgen, growth and development of reproductive structures preceding those of migrants; (2) only migrants displayed prenuptial molt, preparations for migration and migratory restlessness; and (3) neither baseline nor stress-induced plasma corticosterone differed across subspecies, suggesting energetic demands of the common garden were insufficient to induce a differential adrenocortical response in either subspecies, highlighting the impact of environmental conditions on corticosterone secretion. Thus, in a common garden experiment, Z. l. gambelii responds differently to the initial predictive cue, photoperiod, to initiate and execute the vernal stages of molt, migration and development of breeding in comparison to the shared stage of breeding with Z. l. nuttalli, confirming a genetic basis for the subspecies differences.

Extreme spring conditions in the Arctic delay spring phenology of long-distance migratory songbirds.

Arctic regions are warming rapidly, with extreme weather events increasing in frequency, duration, and intensity just as in other regions. Many studies have focused on how shifting seasonality in environmental conditions affects vegetation phenology, while far fewer have examined how the breeding phenology of arctic fauna responds. We studied two species of long-distance migratory songbirds, Lapland longspurs, Calcarius lapponicus, and white-crowned sparrows, Zonotrichia leucophrys gambelii, across five consecutive breeding seasons in northern Alaskan tundra. We aimed to understand how spring environmental conditions affected breeding cycle phenology, including the timing of arrival on breeding grounds, territory establishment, and clutch initiation. Spring temperatures, precipitation, and snow-free dates differed significantly among years, with 2013 characterized by unusually late snow cover. In response, we found a significant delay in breeding-cycle phenology for both study species in 2013 relative to other study years: the first bird observed was delayed by 6-10 days, with mean arrival by 3-6 days, territory establishment by 6-13 days, and clutch initiation by 4-10 days. Further, snow cover, temperature, and precipitation during the territory establishment period were important predictors of clutch initiation dates for both species. These findings suggest that Arctic-breeding passerine communities may have the flexibility required to adjust breeding phenology in response to the increasingly extreme and unpredictable environmental conditions-although future generations may encounter conditions that exceed their current range of phenological flexibility.

Regulation of vernal migration in Gambel's white-crowned sparrows: Role of thyroxine and triiodothyronine.

Appropriate timing of migratory behavior is critical for migrant species. For many temperate zone birds in the spring, lengthening photoperiod is the initial cue leading to morphological, physiological and behavior changes that are necessary for vernal migration and breeding. Strong evidence has emerged in recent years linking thyroid hormone signaling to the photoinduction of breeding in birds while more limited information suggest a potential role in the regulation of vernal migration in photoperiodic songbirds. Here we investigate the development and expression of the vernal migratory life history stage in captive Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelii) in a hypothyroidic state, induced by chemical inhibition of thyroid hormone production. To explore possible variations in the effects of the two thyroid hormones, triiodothyronine and thyroxine, we subsequently performed a thyroid inhibition coupled with replacement therapy. We found that chemical inhibition of thyroid hormones resulted in complete abolishment of mass gain, fattening, and muscle hypertrophy associated with migratory preparation as well as resulting in failure to display nocturnal restlessness behavior. Replacement of thyroxine rescued all of these elements to near control levels while triiodothyronine replacement displayed partial or delayed rescue. Our findings support thyroid hormones as being necessary for the expression of changes in morphology and physiology associated with migration as well as migratory behavior itself.

The stress response is attenuated during inclement weather in parental, but not in pre-parental, Lapland longspurs (Calcarius lapponicus) breeding in the Low Arctic.

Birds breeding at high latitudes can be faced with extreme weather events throughout the breeding season. In response to environmental perturbations, vertebrates activate the hypothalamic-pituitary-adrenal (HPA) axis and synthesize corticosterone, which promotes changes in behavior and physiology to help the animal survive. The parental care hypothesis suggests that the HPA axis activity should be downregulated during the parental stage of breeding to prevent nest abandonment. However, it is unknown what happens to HPA axis activity in response to severe weather at the transition from the pre-parental to parental stages of breeding. We sampled baseline corticosterone levels and the time course of corticosterone elevation over 60min of restraint stress and assessed body condition and fat stores in Lapland longspurs (Calcarius lapponicus) breeding in the Low Arctic in the presence and absence of snowstorms. The results showed that during the pre-parental stage, HPA axis activity was up-regulated in response to snowstorms, with corticosterone levels continuing to increase through 60min of restraint. However, once birds were parental, HPA axis activity was unaffected by snowstorms and levels peaked at 10min. Fat levels and body condition did not change in response to snowstorms but fat levels declined in males during the pre-parental stage. These data suggest that the parental care hypothesis can be applied to severe storm events; parental birds restrained the activity of the HPA axis, likely to focus on the reproductive effort that is already underway, while pre-parental birds greatly upregulated HPA axis activity in response to snowstorms to maximize self-preservation.

Breeding on the leading edge of a northward range expansion: differences in morphology and the stress response in the arctic Gambel's white-crowned sparrow.

Individuals at the forefront of a range shift are likely to exhibit phenotypic traits that distinguish them from the population breeding within the historic range. Recent studies have examined morphological, physiological and behavioral phenotypes of individuals at the edge of their range. Several studies have found differences in the hypothalamic-pituitary-adrenal (HPA) axis activity in response to acute restraint stress in individuals at the range limits. HPA axis activation leads to elevations in glucocorticoids that regulate physiology and behavior. Here we compare the hormonal profiles and morphometrics from Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelii) breeding at the northern limit of the population's range to those birds breeding within the historic population range. Birds breeding at the northern limit experienced a harsher environment with colder temperatures; however, we found no differences in arthropod prey biomass between the northern limit and more southern (historic) sites. Males at the northern limit had higher body condition scores (mass corrected for body size) compared to individuals within the historic range, but no differences were found in beak and tarsus lengths, wing chord, muscle profile or fat stores. In males during the pre-parental stage, before breeding commenced, HPA axis activity was elevated in birds at the northern limit of the range, but no differences were found during the parental or molt stages. Females showed no differences in HPA axis activity during the parental stage. This study suggests that "pioneering" individuals at the limits of their breeding range exhibit physiology and morphology that are distinct from individuals within the historic range.

The effect of extreme spring weather on body condition and stress physiology in Lapland longspurs and white-crowned sparrows breeding in the Arctic.

Climate change is causing rapid shifts in temperature while also increasing the frequency, duration, and intensity of extreme weather. In the northern hemisphere, the spring of 2013 was characterized as extreme due to record high snow cover and low temperatures. Studies that describe the effects of extreme weather on phenology across taxa are limited while morphological and physiological responses remain poorly understood. Stress physiology, as measured through baseline and stress-induced concentrations of cortisol or corticosterone, has often been studied to understand how organisms respond to environmental stressors. We compared body condition and stress physiology of two long-distance migrants breeding in low arctic Alaska - the white-crowned sparrow (Zonotrichia leucophrys) and Lapland longspur (Calcarius lapponicus) - in 2013, an extreme weather year, with three more typical years (2011, 2012, and 2014). The extended snow cover in spring 2013 caused measureable changes in phenology, body condition and physiology. Arrival timing for both species was delayed 4-5days compared to the other three years. Lapland longspurs had reduced fat stores, pectoralis muscle profiles, body mass, and hematocrit levels, while stress-induced concentrations of corticosterone were increased. Similarly, white-crowned sparrows had reduced pectoralis muscle profiles and hematocrit levels, but in contrast to Lapland longspurs, had elevated fat stores and no difference in mass or stress physiology relative to other study years. An understanding of physiological mechanisms that regulate coping strategies is of critical importance for predicting how species will respond to the occurrence of extreme events in the future due to global climate change.

Greater shrub dominance alters breeding habitat and food resources for migratory songbirds in Alaskan arctic tundra.

Climate warming is affecting the Arctic in multiple ways, including via increased dominance of deciduous shrubs. Although many studies have focused on how this vegetation shift is altering nutrient cycling and energy balance, few have explicitly considered effects on tundra fauna, such as the millions of migratory songbirds that breed in northern regions every year. To understand how increasing deciduous shrub dominance may alter breeding songbird habitat, we quantified vegetation and arthropod community characteristics in both graminoid and shrub dominated tundra. We combined measurements of preferred nest site characteristics for Lapland longspurs (Calcarius lapponicus) and Gambel's White-crowned sparrows (Zonotrichia leucophrys gambelii) with modeled predictions for the distribution of plant community types in the Alaskan arctic foothills region for the year 2050. Lapland longspur nests were found in sedge-dominated tussock tundra where shrub height does not exceed 20 cm, whereas White-crowned sparrows nested only under shrubs between 20 cm and 1 m in height, with no preference for shrub species. Shrub canopies had higher canopy-dwelling arthropod availability (i.e. small flies and spiders) but lower ground-dwelling arthropod availability (i.e. large spiders and beetles). Since flies are the birds' preferred prey, increasing shrubs may result in a net enhancement in preferred prey availability. Acknowledging the coarse resolution of existing tundra vegetation models, we predict that by 2050 there will be a northward shift in current White-crowned sparrow habitat range and a 20-60% increase in their preferred habitat extent, while Lapland longspur habitat extent will be equivalently reduced. Our findings can be used to make first approximations of future habitat change for species with similar nesting requirements. However, we contend that as exemplified by this study's findings, existing tundra modeling tools cannot yet simulate the fine-scale habitat characteristics that are critical to accurately predicting future habitat extent for many wildlife species.

A chromosome-level genome assembly of a free-living white-crowned sparrow (Zonotrichia leucophrys gambelii).

The white-crowned sparrow, Zonotrichia leucophrys, is a passerine bird with a wide distribution and it is extensively adapted to environmental changes. It has historically acted as a model species in studies on avian ecology, physiology and behaviour. Here, we present a high-quality chromosome-level genome of Zonotrichia leucophrys using PacBio and OmniC sequencing data. Gene models were constructed by combining RNA-seq and Iso-seq data from liver, hypothalamus, and ovary. In total a 1,123,996,003 bp genome was generated, including 31 chromosomes assembled in complete scaffolds along with other, unplaced scaffolds. This high-quality genome assembly offers an important genomic resource for the research community using the white-crowned sparrow as a model for understanding avian genome biology and development, and provides a genomic basis for future studies, both fundamental and applied.

Comparison of the Phenotypic Flexibility of Muscle and Body Condition of Migrant and Resident White-Crowned Sparrows.

AbstractSeasonally breeding birds express variations of traits (phenotypic flexibility) throughout their life history stages that represent adaptations to environmental conditions. Changes of body condition during migration have been well studied, whereas alterations of skeletal and cardiac muscles, body mass, and fat scores have yet to be characterized throughout the spring or fall migratory stages. Additionally, we examined flexible patterns of muscle, body mass, and fat score in migrant white-crowned sparrows (Zonotrichia leucophrys gambelii) in comparison with those in a resident subspecies (Zonotrichia leucophrys nuttalli) during the stages they share to evaluate the influence of different life histories. Migrants showed hypertrophy of the pectoralis muscle fiber area on the wintering grounds in late prealternate molt, yet increased pectoralis muscle mass was not detected until birds readied for spring departure. While pectoralis profile and fat scores enlarged at predeparture in spring and fall, pectoralis, cardiac, and body masses were greater only in spring stages, suggesting seasonal differences for migratory preparation. Gastrocnemius mass showed little change throughout all stages, whereas gastrocnemius fiber area declined steadily but rebounded in fall on the wintering grounds, where migrants become more sedentary. In general, residents are heavier birds with larger leg structures, while migrants sport longer wings and greater heart mass. Phenotypic flexibility was most prominent among residents with peaks of pectoralis, gastrocnemius, and body masses during the winter stage, when local weather is most severe. Thus, the subspecies express specific patterns of phenotypic flexibility with peaks coinciding with the stages of heightened energy demands: the winter stage for residents and the spring stages for migrants.

Revisiting TSHß's Role in Avian Seasonal Reproduction, Insights, and Challenges from Mammalian Models.

The core neuroendocrine pathways regulating seasonal reproduction in vertebrates were characterized over a decade ago. This has led to the development of a "consensus" model of seasonal reproduction that appears to be largely conserved across mammals, birds, amphibians, reptile and fish. This model centers around the photoinduced increase in TSHß expression in the pars tuberalis of the anterior pituitary gland as the key transducer of photic information from sensory cells to the critical switch in hypothalamic deiodinase enzyme expression that drives changes in localized thyroid hormone signaling. These changes in localized thyroid hormone signaling in the medial basal hypothalamus ultimately activate the reproductive axis. This model has in turn, been consistently supported by studies in a variety of taxa. As such, it has become the standard against which subsequent work is compared, particularly in the non-mammalian literature. However, as new studies move away from the handful of canonical model systems and begin to explore the effects of naturalistic rather than artificial photoperiod manipulations, a more nuanced picture has begun to emerge. Yet, progress in elucidating the detailed events of reproductive photostimulation has been uneven across the research community. In this perspective, I draw on emerging data from studies in free living animals that challenges some of the established assumptions of the avian consensus model of reproduction. Specifically, the role of TSHß and its dissociation from deiodinase signaling. I then discuss how these apparently surprising findings can be contextualized within the context of the mammalian seasonal literature. In turn, this ability to contextualize from the mammalian literature highlights the breadth of the current gap versus our understanding of the molecular neuroendocrine mechanisms of seasonality in mammals versus birds and other non-mammalian seasonal breeders.

Light receptors in the avian brain and seasonal reproduction.

Detection and transduction of photic cues by nonvisual photoreceptors, located in the deep brain, is a critical component of timing seasonal reproduction in birds. However, the precise identity of the photoreceptors responsible for detection of salient photic cues remains uncertain and debated. Here I review of the existing evidence for each of the three candidate photoreceptive opsins: Vertebrate Ancient Opsin, Melanopsin, and Neuropsin, including localization, action spectrum, and data from experimental manipulation of opsin expression. These findings are compared to an updated list of key criteria established in the literature as a litmus for classifying an opsin as the "breeding photoreceptor." Integrating evidence for each of the candidate photoreceptors with respect to these criteria reveals support for all three opsins in regulation of seasonal reproduction. Taken together these findings strongly suggest that transduction of seasonal photoperiodic information involves the activity of multiple photoreceptor types and populations functioning in concert. This review also highlights the need to shift attention from simply identifying "the breeding photoreceptor" to a more integrative approach aiming to parse the contribution of specific photoreceptor populations within the brain.

Seasonal differences in hypothalamic thyroid-stimulating hormone ß, gonadotropin-releasing hormone-I and deiodinase expression between migrant and resident subspecies of white-crowned sparrow (Zonotrichia leucophrys).

Across taxa, the seasonal transition between non-breeding and breeding states is controlled by localised thyroid hormone signalling in the deep brain via reciprocal switching of deiodinase enzyme expression from type 3 (DIO3) to type 2 (DIO2). This reciprocal switch is considered to be mediated by increasing thyroid-stimulating hormone ß (TSHß) release from the pars tuberalis, which occurs in response to a change in photoperiod. Although well characterised in a handful of model organisms in controlled laboratory settings, this pathway remains largely unexplored in free-living animals under natural environmental conditions. In this comparative gene expression study, we investigated hypothalamic thyroid hormone signalling in two seasonally breeding subspecies of white-crowned sparrow (Zonotrichia leucophrys), across the entirety of their annual cycles. The migratory Gambel's (Z. l. gambelii) and resident Nuttall's (Z. l. nuttalii) subspecies differ with respect to timing of reproduction, as well as life history stage and migratory strategies. Although DIO3 mRNA expression was elevated and DIO2 mRNA expression was reduced in the wintering period in both subspecies, DIO2 peaked in both subspecies prior to the onset of reproduction. However, there was differential timing between subspecies in peak DIO2 expression. Intriguingly, seasonal modulation of TSHß mRNA was only observed in migrants, where expression was elevated at the start of breeding, consistent with observations from other highly photoperiodic species. There was no correlation between TSHß, DIO2 and gonadotropin-releasing hormone-I mRNA or reproductive metrics in residents. Based on these observed differences, we discuss potential implications for our understanding of how changes in medial basal hypothalamic gene expression mediates initiation of seasonal reproduction.